Micoorganisms in Plant Conservation and Biodiversity

MICROORGANISMS IN PLANT CONSERVATIONAND BIODIVERSITY

Microorganisms in PlantConservation andBiodiversity

Edited by

K. SivasithamparamThe University of Western Australia, Perth

K.W. DixonKings Park & Botanic Garden,Western Australia andThe University of Western Australia, Perth

and

R.L. BarrettKings Park & Botanic Garden, Western Australia

KLUWER ACADEMIC PUBLISHERSNEW YORK, BOSTON, DORDRECHT, LONDON, MOSCOW

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CONTENTS

Contributors

Foreword

Preface

Acknowledgements

Plant conservation and biodiversity: the place of microorganismsDR Given, KW Dixon, RL Barrett and K Sivasithamparam

Conservation of mycorrhizal fungal communities under elevatedatmospheric and anthropogenic nitrogen depositionLM Egerton-Warburton, EB Allen and MF Allen

Symbiotic nitrogen fixation between microorganismsand higher plants of natural ecosystemsJS Pate

Bacterial associations with plants:beneficial, non N-fixing interactionsB Gerhardson and S Wright

Ectomycorrhizas in plant communitiesMC Brundrett and JWG Cairney

Arbuscular mycorrhizas in plant communitiesMC Brundrett and LK Abbott

Orchid conservation and mycorrhizal associationsAL Batty, KW Dixon, MC Brundrett and K Sivasithamparam

Ericoid mycorrhizas in plant communitiesKW Dixon, K Sivasithamparam and DJ Read

The diversity of plant pathogens and conservation:bacteria and fungi sensu latoDS Ingram

Ex situ conservation of microbial diversityW Gams

v

vii

ix

xi

xiii

1

19

45

79

105

151

195

227

241

269

1.

2.

3.

4.

5.

6.

7.

8.

9.

10.

vi

Impact of fungal pathogens in natural forest ecosystems:a focus on EucalyptsT Burgess and MJ Wingfield

11.

12.

13.

Index

Microbial contaminants in plant tissue culture propagationE Bunn and B Tan

Phytosanitary considerations in species recovery programsGEStJ Hardy and K Sivasithamparam

Contents

285

307

337

369

vii

CONTRIBUTORS

Soil Science and Plant Nutr i t ion , Faculty of Natura l and Agricultural Sciences, TheUniversity of Western Australia, Crawley 6009, Western Australia.

Department of Botany and Plant Sciences, The Univers i ty of California, Riverside CA,92521, USA.

Centre for Conservation Biology, The University of California, Riverside CA, 92521, USA.

Kings Park and Botanic Garden, Botanic Gardens and Parks Authority, West Perth 6005,Western Australia.

Kings Park and Botanic Garden, Botanic Gardens and Parks Authority, West Perth 6005,Western Australia; Soil Science and Plant Nutri t ion, Faculty of Natural and AgriculturalSciences, The University of Western Austra l ia , Crawley 6009, Western Australia.

CSIRO Forestry and Forest Products, CSIRO Centre for Mediterranean AgriculturalResearch, Private Bag No 5, Wembley, 6913, Western Australia; Soil Science and PlantNutri t ion, Faculty of Natural and Agricultural Sciences, The University of Western Australia,Crawley 6009, Western Australia (current address).

Kings Park and Botanic Garden, Botanic Gardens and Parks Authority, West Perth 6005,Western Australia.

Forestry and Agr icul ture Biotechnology Ins t i tu te (FABI), University of Pretoria, Pretoria,0002, RSA.

Mycorrhiza Research Group, Centre for Horticulture and Plant Sciences, Parramatta Campus,University of Western Sydney, Locked Bag 1797, Penrith South DC, 1797, New SouthWales, Australia.

Kings Park and Botanic Garden, Botanic Gardens and Parks Authority, West Perth 6005,Western Aus t ra l ia ; Plant Biology, Faculty of Natura l and Agricultural Sciences, TheUniversity of Western Australia, Crawley 6009, Western Australia.

Lynette K. Abbott

Edith B. Allen

Michael F. Allen

Russell L. Barrett

Andrew J. Batty

Mark C. Brundrett

Eric Bunn

Treena Burgess

John W.G. Cairney

Kingsley W. Dixon

viii Contributors

Louise M. Egerton-WarburtonDepartment of Botany and P lan t Sciences, The U n i v e r s i t y of Cal i forn ia , Riverside CA,92521, USA; Chicago Botanic Garden, Lake Cook Rd, Glencoe IL 60022, USA (currentaddress).

Walter GamsCentraalbureau voor Schimmelcultures, P.O. Box 85167, 3508 AD, Utrecht, Netherlands.

Berndt GerhardsonPlant Pathology and Biocontrol Unit, P.O. Box 7035, S-750 07 Uppsala, Sweden.

David R. GivenIsaac Centre for Nature Conservation, P.O. 84, Lincoln Univers i ty , Canterbury 8150, NewZealand.

Giles E. St J. HardySchool of Biological Sciences and Biotechnology, Murdoch Universi ty , Perth 6150, WesternAustralia.

David S. IngramSt Catharine’s College, Cambridge Univers i ty , Cambridge, CB2 1RL, UK.

John S. PatePlant Biology, Faculty of Na tu ra l and Agr i cu l tu ra l Sciences, The University of WesternAustral ia , Crawley, 6009, Western Aust ra l ia .

David J. ReadDepartment of Animal and P lan t Sciences, The Univers i ty of Sheff ie ld, Sheffield S10 2TN,UK.

Soil Science and Plant N u t r i t i o n , Facul ty of N a t u r a l and Agricul tura l Sciences, TheUniversity of Western Austral ia , Crawley 6009, Western Australia.

K. Sivasithamparam

Beng TanDepartment of Biology, Curtin University of Technology, Bentley 6102, Western Australia.

Michael J. WingfieldForestry and Agricul ture Biotechnology I n s t i t u t e (FABI), Univers i ty of Pretoria, Pretoria,0002, RSA.

Sandra WrightPlant Pathology and Biocontrol U n i t , P.O. Box 7035, S-750 07 Uppsala, Sweden.

ix

FOREWORD

Plant conservation is increasingly recognised as an outstanding global priority, notonly by scientists and committed conservationists but also by the global communityand many governments. We know that tens of thousands of plant species throughoutthe world likely face extinction this century if current trends continue. The mostcomprehensive global list of endangered plant species produced to date waspublished by IUCN-The World Conservation Union in 1997 which documentedalmost 34,000 threatened plant species, and that work acknowledged that this is aconsiderable underestimation of the true figure of plant species threatened byextinction. Plant species loss is caused by many diverse factors, but primarilythrough the loss or damage of natural ecosystems and other impacts on wild plantpopulations and diversity caused by humankind, such as unsustainable collectingand uncontrolled invasions by alien species (plants, animals and microorganisms).

Despite considerable efforts made over the last few decades to safeguard theworld’s biodiversity in national parks, nature reserves and other forms of protectedareas, we are today very much aware that despite our best efforts, the number ofthreatened species continues to rise. Innovative multi-disciplinary strategies in plantconservation are increasingly recognised as the best option for saving many species.We recognise that not only must we protect plants growing in the wild but also thatwe must seek recovery for an ever growing number of damaged plant populationsand restore their habitats. Unless we gain a comprehensive insight into the factorsthat sustain these wild populations, our efforts to conserve diversity are ultimatelylikely to fa i l . The development of practical conservation and restoration methods,based on principles determined from the results of conservation biology research, istherefore an urgent priority.

The practice of plant conservation has for too long been a rather hit-or-missmixture of methods. Species recovery work involving cultivation, reintroductionand restoration has often had to be undertaken without adequate knowledge of theunderlying causes of endangerment or of the factors required to successfully recovera threatened species. While we have recognised that microorganisms are often acrucial and essential element in supporting the life-cycles of plant species, we havegenerally had to hope that our effort undertaken at a macro level will be sufficient tofacilitate ecosystem functioning at the micro level. Many of our successful efforts inplant conservation have probably been as a result of good fortune rather than goodscience.

I greatly welcome therefore the preparation of this valuable book. With itsfocus on both the beneficial and detrimental importance of microorganisms (e.g. asmycorrhizas and pathogens), it provides an important review of the current state ofknowledge on the importance and significance of microorganisms for plantconservation. I also hope that it wil l stimulate many more institutions to recognisethat fundamental research on microbiology is an important element of plantconservation programs. The devastating impacts caused by the loss of biodiversityon our global environment and for the future of humanity can only be addressed ifour future plant conservation efforts are based on understanding the complexinteractions of biodiversity with its environment at all levels, rather than having to

Forewordx

rely on guesswork and good luck. This book should act as an extremely usefulcontribution to raising awareness of the importance of such aspects of plantconservation and provide an authoritat ive text to guide many plant conservationpractitioners to the importance of microorganisms for successful plant conservation.

Peter S. Wyse JacksonSecretary GeneralBotanic Gardens Conservation InternationalRichmond, Surrey, U.K.March 2002

PREFACE

If ‘all grass is flesh’ and the productivity of plant systems is underpinned by theactivity of microorganisms, then much of human existence depends upon the presentbiological diversity of microorganisms. Even the foundations of the industrialisedsocieties of today – fossil oil and iron (the major source of the metal is from bandediron formations which formed when oxygen produced by microorganisms some3,000 to 2,500 million years ago precipitated oxides of iron (Schopf 1999)) dependupon the past activities of microorganisms. Microorganisms are therefore not onlythe origin of life itself some 3.5 billion years ago (Shen et al. 2001) but they supportmuch of the biotic, industrial and social fabric of today’s world. As vital andintegral components of the engine of existence, the diversity of microorganisms andthe biological diversity they support are therefore fundamental in the debate to bettermanage the processes of conservation and threat abatement.

This book addresses the role of microorganisms in conservation – both theirsupport functions and deleterious roles in ecosystem function and species survival.Importantly, a number of contributing authors highlight how microbial diversity is,itself, now under threat from the many and pervasive influences of man. What isclear from this volume is that like many contemporary treatments of plant andanimal conservation, the solution to mitigate the erosion of biodiversity is notsimple, made all the more complex by the lack of reliable taxonomic information,particularly for the predicted immense diversity of microorganisms.

The impacts of human activity touch all parts of the biosphere as highlightedby Egerton-Warburton et al. (this volume) and only now are some of the moreadvanced economies of the world coming to grips with the scale and inertia of theproblem. The fate of an estimated two thirds of the plant species on earth nowhangs in the balance (Anon. 2000). As man forges ahead monopolising biodiversityto a mere 100 plant species which represent the major human food and fibre species,another estimated 250,000 other plant species are in peril (Heywood & Watson1995). With microbial diversity conservatively estimated in the millions of species,the impact of the loss of equ i l ib r ium of microbial diversity is daunting andpotentially irreversible. Take for example the ‘knock-on’ effects on plantproduction systems if there is a careless disregard for maintenance of a diverse,healthy and functional microflora. Many agricultural systems do just this and toremain productive, require unsustainably high inputs of energy and chemicals.These inputs themselves further perpetuate the artificiality of the system, ultimatelyleading to a process of agricultural productivity devoid of natural inputs – essentiallybroad-scale hydroponics! Egerton-Warburton et al. (this volume) cite 90% of plantspecies as having some form of symbiotic association with fungi. How surprising isit then that other than a few esoteric examples in forestry, much of our broad-scaleagricultural systems pay little or no attention to the role of helper fungi inmaintaining soil and plant health?

The pivotal role of some microorganisms in maintenance of biodiversity isclassically seen in the multifaceted benefits of ectomycorrhizas (Brundrett andCairney this volume) in supporting a host of other organisms from bacteria andprotists to invertebrates and vertebrates – including the elusive hypogeous fruit

xi

xii Preface

bodies of truffles. If only similar prologues of the level of interaction of moremicroorganisms could be drafted before the loss of biodiversity eliminates taxa tothe point where reconstruction of the intricate and elegant processes of ecologicalequilibrium is impossible. This book represents an attempt to bring to the fore theecological underwriting provided by microorganisms. Let us hope that many morevolumes will ensue as the value of microorganisms in conservation is recognised aspart of the global conservation process.

References

Anonymous (2000) ‘Gran Canaria declaration.’ (Botanic Gardens Conservation International:Kew)

Brundrett MC, Cairney J (2002) Ectomycorrhizas in plant communities. In ‘Microorganismsin plant conservation and biodiversi ty’. (Eds K Sivasithamparam, KW Dixon and RLBarrett) pp. 105–150. (Kluwer Academic Publishers: Dordrecht)

Egerton-Warburton LM, Allen EB, Allen MF (2002) Mycorrhizal fungal communities underelevated atmospheric and anthropogenic nitrogen deposition. In ‘Microorganisms inplant conservation and biodiversity’. (Eds K Sivasithamparam, KW Dixon and RLBarrett) pp. 19–43. (Kluwer Academic Publishers: Dordrecht)

Heywood VH, Watson RT (1995) (eds) ‘Global biodiversity assessment.’ (CambridgeUniversity Press: Cambridge)

Shen Y, Buick R, Canfield DE (2001) Isotopic evidence for microbial sulphate reduction inthe early Archaean era. Nature 410, 77–81.

Schopf JW (1999) ‘Cradle of l i fe . The discovery of ear th’s earliest fossils.’ (PrincetonUniversity Press: Princeton)

The editorsPerth, Western AustraliaMarch 2002

xiii

ACKNOWLEDGEMENTS

The editors would like to express their gratitude for the work of all the contributorsto this volume. We would like to especially thank those who kindly agreed toreview chapters: Craig Atkins. Mark Brundrett, John Cairney, Brett Gaskell, JanetGorst, Roger Finlay, Stephen Hopper, Maarten Ryder and Sally Smith. We wouldalso like to thank our colleagues at Kings Park & Botanic Garden and TheUniversity of Western Australia for their assistance with this project.

K.Sivasithamparam, K.W.Dixon & R.L.Barrett (eds) 2002. Microorganisms inPlant Conservation and Biodiversity. pp. 1–18. © Kluwer Academic Publishers.

Chapter 1

PLANT CONSERVATION AND BIODIVERSITY:THE PLACE OF MICROORGANISMS

David R. Given

Isaac Centre for Nature Conservation, P.O. 84, Lincoln University, Canterbury 8150, NewZealand.

Kingsley W. Dixon

Kings Park & Botanic Garden, Botanic Gardens & Parks Authori ty, West Perth 6005,Western Australia; Plant Biology, Faculty of Natural & Agricultural Science, The Universityof Western Australia, Crawley 6009, Western Australia.

Russell L. Barrett

Kings Park & Botanic Garden, Botanic Gardens & Parks Authority, West Perth 6005,Western Australia.

K. Sivasithamparam

Soil Science and Plant Nutr i t ion , Faculty of Natural & Agricultural Science, The Universityof Western Australia, Crawley 6009, Western Australia.

1. IntroductionThe total number of organisms that make up the array of living species thatcharacterise the th in ecosphere on Earth sti l l remains unaccounted.Estimates range from eleven million species (of which 1.5 million have beendescribed) to over a bil l ion (Table 1). Only now is the scientific communitybeginning to understand the monumental task of cataloguing life on earth.What this exercise is showing is that it is the unseen and forgotten world ofinvertebrates, fungi , non-flowering plants, marine organisms and

2 Microorganisms in Plant Conservation and Biodiversity

microorganisms that makes up the vast bulk of the unaccounted species. Inany part of the world, the numbers of fungal species are higher than those ofgreen plants (Gams, this volume). Indeed, much of the essential primaryproduction (oceanic algae) and organic recycling to sustain life is a result ofmicroorganisms i.e. life begins and ends with microorganisms.

Fungal diversity appears less localised than that of green plants, butmuch still remains to be explored, particularly in tropical regions, and in agreat diversity of ecological niches (Hawksworth 2001). Fungi play manydecisive roles in the health and well-being of all ecosystems (Ingram, thisvolume).

Diversity of bacteria and archaea has recently been recognised as beingpotentially far greater than that of all Eukaryotes and represents the greatestchallenge for scientists documenting the diversity of life (Figure 1; Barns etal. 1996; Whitman et al. 1998; Patterson 1999; Cohan 2001; DeLong andPace 2001; Dunlap 2001). The task of refining the estimates, by the processof cataloguing biodiversity, wi l l take a great deal of time and expense.Pimm et al. (2001) have estimated that $25 bi l l ion annually is required forthe protection and management of global biodiversity hotspots identified byMyers et al. (2000) with James et al. (2001) suggesting a similar figure of$21.5 billion, rising to $317 bil l ion for global biodiversity conservation. TheAll Species Foundation estimate that the description of the remainingbiodivers i ty on earth w i l l take over 650 years at current rates(http://www.all-species.com), by which time a great deal of our biologicaldiversity may have been lost (Myers and Knoll 2001).

Microorganisms e x h i b i t a great range of adaptation to extremeenvironments, from deep ocean trenches, to high temperature and pressuredeep wi thin the earth, to thermal springs, to ice sheets and exhibit a range of

Plant conservation and biodiversity 3

metabolic processes to survive in this environment (Horikoshi and Grant1998; Nealson and Conrad 1999). Plant surfaces, from root-tip to shoot-tip,provide a myriad of habitats for microorganisms, many of which sustainhealthy growth, while others are pathogenic (Andrews and Harris 2000). Inaddition to plant surfaces, another suit of microorganisms live within theplant (as endophytes) with a range of antagonistic to mutualistic roles withinthe host plant (Saikkonen et al. 1998). These mutualistic associations canlead to sometimes startling outcomes such as the spectacular flowering in theholomycotrophic underground orchid (Batty et al., this volume).

In a world of explosive scientific discovery and rapidly changingdefinitions, it is necessary to begin with an explanation of the focus of thisbook on microorganisms and their role in plant conservation andbiodiversity. An extensive review of microbial diversity and ecosystemfunction is given by Allsopp et al. (1995). Also, since completion ofmanuscripts for this book, a further volume on fungal conservation has beenpublished (Moore et al. 2001) which is recommended reading as acompanion to this volume. This chapter provides an overview of aspects ofmicrobial function affecting plant biodiversity and conservation, i.e. the roleof microorganisms in the survival of the earth’s biota. Microorganismsshape plant biodiversity and conservation by either suppressing or enhancingplant development and establishment as expanded in the chapters of thisvolume.

Defining microorganisms is a continually evolving process. As ourunderstanding of microbial ecology and phylogeny grows, so our taxonomicand functional group concepts continue to change. Figure 1 illustrates whatare now recognised as three Domains, with the requirement to completely re-think our concept of Kingdoms, which, if retained in the traditional sense,must increase in number to reflect the genetic diversity now evident thoughanalyses such as rDNA sequencing (Barns et al. 1996; Nealson and Conrad1999; DeLong and Pace 2001; Stackebrandt 2001).

Colwell et al. (1995) and Zavarzin (1995) discuss the issuessurrounding the concept and definition of the term ‘microorganisms.’Bacteria, as a Domain, represent a distinct lineage of microorganisms.Within bacteria, further lineages such as Actinobacteria, Cyanobacteria andProteobacteria are recognised (more readily from sequence data thanphysiological characteristics), however definition of bacterial ‘species’,generally accepted as “a collection of strains showing a high degree ofoverall similarity, compared to other, related groups of stains” (Colwell et al.1995) is relatively vague. A bacterial ‘species’ may refer to a ‘taxospecies’(a phenetic cluster), a ‘genospecies’ (a group capable of genetic exchange)or a ‘nomenspecies’ (a group given a binomial name) (Sneath and Sokal1973; Sneath 1984; Colwell et al. 1995). As evident in figure 1, “the


concept of ‘microorganisms’ is not essentially phylogenetic” (Zavarzin1995), encompassing bacteria, archaea and much of the eucarya. As a termin broad usage (in both application and definition), the concept of‘microorganisms’ is entrenched in our thinking, and indeed remains a usefulterm when making general statements. Zavarzin (1995) concludes with ageneralistic definition: “Microorganisms are living beings invisible to thenaked eye (except when developing in large masses). Most microorganismsare osmotrophic, few are holozoic. Diffusion is the main limitation to


dimensions and thus the most essential feature.” This definition captures theessence of microorganisms as those organisms too small to see, and thususually overlooked or ignored completely.

Microorganisms are basal to the very foundations of life on earth. The firstevidence of l ife from 3.5 Byr in the archaean rocks from north westernAustralia are represented by cyanobacteria (Shen et al. 2001). From thesebeginnings, microorganisms have focussed the evolution of all life. Theintegration of microbial components provided the very being for thecomplex eukaryotes we know today. The chloroplasts and mitochondriawhich represent both the energy capturing and energy production system ofall eukaryotes is a result of microbial integration on a sophisticated andunparalleled scale (Martin et al. 1998; Gray et al. 1999; McFadden 1999).Secondary encounters with microorganisms has resulted in symbioses whichmay have paved the way for the movement of plants onto land including therole of mycorrhizal agents as defacto roots in early land plants such asRhynia (Bateman et al. 1998; Brundrett 2002). Symbioses continue to play arole in the evolutionary success of plants in particular as outlined in thechapters of this volume. For example, the Ericales clade is a remarkablyancient and monophyletic group (APG 1998). Since all modern ericadsinvestigated possess ericoid mycorrhiza it is tempting to speculate that thissymbiosis provided the necessary resilience for this order to colonise thefour corners of the globe and comprise a remarkable level of diversity(Dixon et al., th is volume). As pathogens, microorganisms could,conceivably, have provided important drivers for the evolution of adaptivetraits and speciation events (Ingram, this volume).

The global catalogue of life is also starting to reveal the extent of theextinction crisis for biological diversity. A tragedy being played out againstthe backdrop of current changes to nature is that future generations may notsee certain life forms and landscapes that today we take for granted.

“As many as two-thirds of the world’s plant species are in danger ofextinction in nature during the course of the 21st century, threatened bypopulation growth, deforestation, habitat loss, destructive development,over consumption of resources, the spread of alien invasive species andagricultural expansion. ” (Anon. 2000).

Catastrophic or localised extinctions can occur without humanintervention, but in many parts of the world and for vulnerable ecosystemsgenerally, humans are primarily responsible for current extirpations ofspecies and ecosystems. May et al. (1995) have estimated the mean

2. Conservation hypotheses

3. Biodiversity conservation


background rate of ext inc t ion in the geological record as about one speciesper year. The same authors, using three independent analyses based ondifferent scientific approaches, concluded that impending extinction rates areat least four orders-of-magnitude faster than the background rates seen in thefossil record. This figure does not take into account the observation byHawksworth (1998) that the extinction of an obvious, large organism such asa forest tree probably resul ts in the loss of at least fifteen organismsdependent on or confined to that single species.

Do i n d i v i d u a l microorganisms become endangered or extinct in theirnatural env i ronments? There is l i t t l e or no evidence to indicate thatdisturbance, large or small, could lead to the extinction of specific strains ofa microorganism, although several reports exist of numbers of selectedgroups of culturable soil microorganisms dropping to non-detectable levels(Alexander 1971). From an ecological point of view it is possible to arguethat compositional changes may not be very critical if activities of functionalgroups of organisms are maintained, even at the expense of rare taxa.

Raven (quoted in Josephson 2000) cites three principle factors thataccelerate the rate of ex t inc t ion of p lan t species: habitat destruction,ecosystem fragmentat ion and invas ion of wi ld habitats by exotic species.There is, moreover, a finality to extinction and the best way to avoid it in thewords of Raven is to “save real estate. If a rain forest is destroyed, 19 or 20species there will remain unknown to science”. A second-best strategy is toreconstruct ecological communit ies , but few if any examples exist of thesuccess of this process. Species can be restored to a system, but it is muchharder and sometimes impossible to restore processes, interspeciesrelationships and the pre-disturbance microflora. A third-best strategy is topreserve species as germplasm. As Raven concludes, “we would rather havegermplasm than not have the species at all.”

The spectrum of biological diversi ty ranges from genetic to ecosystemand biome diversi ty. The task of conserving as much of the biosphere ofEarth as possible, is made more d i f f i c u l t by our poor knowledge of geneticvariation w i t h i n species and the signif icance of these variations on thesurvival of the threatened taxa. Erosion of biological diversity is occurringthroughout the whole spectrum of diversity from small to large, and from themicroscopic to landscape level . According to Myers and Knoll (2001) “Wehave only a rudimentary unders tanding of how we are altering theevolutionary future. As a result of our ignorance, conservation policies failto reflect long-term evolutionary aspects of biodiversity loss”.

Should biod ivers i ty be determined only by classical taxonomy?Literature is mount ing on the var ia t ion within species. Although therecognition of in t raspeci f ic differences in the pathogenicity of microbialpathogens of plants and an imal s ( inc lud ing humans) have lead to the creation


of subspecific taxonomic separations based on their chemistry, suchseparations of non-pathogenic microorganisms (which greatly outnumberparasitic forms) is not widely accepted. The role of infra-specific variation,par t icular ly in symbiot ic relat ionships, is now being more clearlyunderstood, particularly in orchids (Batty et al., this volume) and ericads(Dixon et al., this volume).

Myers (1988) identified ten globally important centres of diversity(mostly areas of tropical moist forest) and later a further eight (mostlymediterranean-climate regions). This work has been followed up by morerigorous research, and the recent publication of a monumental analysis(Myers et al. 2000) identifying 25 ‘hot spots’ – those places on earth wherethere are concentrations of species with numerous endemics and where thereare often considerable threats to biodiversity. Resources cannot be madeavailable everywhere for all species, genes and ecosystems. One suggestedapproach to prioritisation is to first of all consider the conservation prioritiesfor biological ‘hot spots.’ We cannot escape the fact that these are the placeson Earth where there is greatest responsibility for stewardship, protected areasystems, environmental education, community conservation initiatives, andsustainable resource use. Recent reassessment of the ‘hot spot’ conceptshows that the whole of New Zealand and parts of Australia and SouthAfrica are among the most important focal points on Earth for animals andplants, each with significant threats to biodiversity conservation (Burbidge1994; Cowling et al. 1996; Craig et al. 2000).

Myers et al. (2000) and most other systems for analysis of biologicaldiversity pay scant regard for the collateral diversity of microorganisms thatcoexist, support, rely upon or are pathogens of plants. Whether globalhotspots of vascular plant d ivers i ty adequately represent the diversityhotspots for microorganisms remains a virtually unknown yet vital area forresearch.

Wholly unrelated microorganisms may play a critical role in theestablishment and survival of native flora. These include mycorrhizal forms(see Egerton-Warburton et al.; Brundrett and Cairney; Brundrett and Abbott;Batty et al. and Dixon et al., this volume), rhizobia (Pate, this volume) andbeneficial bacteria that enhance plant growth or suppress parasitic activitiesof pathogens through their antagonistic activities (Gerhardson and Wright,this volume). This diversity allows the evolution of ecosystems and/or ofcolonisat ion of env i ronments which would otherwise restrict theestablishment of the plant taxa that now occupy them (see Cairney 2000).


Human activities have undoubtedly had highly significant impacts on thediversity and efficacy of microorganisms, although the effects are frequentlyquite subtle and crypt ic . Such changes may be manifest as minorfluctuations in species composition through to more extreme shifts thatencompass the loss of changes in species dominance and even loss of taxa(e.g. from natural woodland to p l an ta t ion forestry as in the case ofmycorrhizal taxa). Al though s i m i l a r shif ts in plant communities areacknowledged as being cr i t ical in understanding ecosystem processes theinfluence of comparable shifts in mycobiont diversity on the mycorrhizaland, in turn, the plant communi ty has yet to be ful ly appreciated (Egerton-Warburton et al., this volume).

How can we conserve these organisms? The first step is to study thebiology, including survival mechanisms in nature, of microorganisms. Ourgreatest knowledge is generally of the fungi , bacteria and microorganismswhich have a significant impact on human life. These are not only the usefulmicroorganisms, but also include such rogues as the nasty “killer bugs”, ofwhich Ebola virus is one of the most notorious. Much research is underwayto eradicate such v i ru len t pathogens. Although it is desirable to totallyeradicate them, it is also important to preserve (of course under strictest ofcare) l ive cultures of the pathogens for future research to combat re-emergence of the same or the evolut ion of similar parasites (Babiuk et al.2000). This situation indeed arose recently (Bryan 1999; Ertem et al. 2000)when the World Health Organisation was faced with the dilemma ofdestroying the last known laboratory cultures of the small-pox virus.Understanding the biology of microorganisms is a challenge because weknow less than 5% of the taxonomic diversity and knowledge of their role inecosystems, whether benef ic ia l or not, is generally rudimentary. In thissituation, how do we know what benefits accorded to microorganisms arebeing lost from ecosystems?

Several niches rich in microflora remain under-explored, the mostexciting of which are marine environments such as deep-sea troughs (Levinet al. 2001) and deep subsurface environments (Haldeman et al. 1994;Chandler et al. 1998). Recent interest in marine microflora, especially thoseassociated with sponges have resulted not only in the description of newtaxa, but the discovery of a whole host of organic metabolites new to scienceand valuable for pharmaceutical and extractive industries (Grassle 2001;Fenchel 2001; Watson et al. 2001).

Ecosystem ma in t enance depends h e a v i l y on the func t ion ofmicroorganisms. Microorganisms play a decisive role in nutrient cycling(McKenzie 2000; de Boer and Kowalchuk 2001; Jobbagy and Jackson2001), decomposition (HyeongTae 2000; Monreal and Bergstrom 2000;

4. Microorganism conservation


Fenchel 2001), mineralisation (Puri and Ashman 1998; Saetre et al. 1999;Fenchel 2001), nu t r i en t and mineral accession by plants and animals(Marschner 1995; Geeta Singh and Tilak 1998; van Vuuren et al. 1999).

Egerton-Warburton et al. (this volume) point out that mycorrhizal fungiare a functional group of organisms that form symbiotic associations withover 90 % of plant species and in most biomes. This indicates that nine outof ten plants we see around us have and use mycorrhizas, not ‘just roots.’Such associations have been linked to the enhanced growth, survival,drought tolerance, pathogen resistance and nutrient status of the host plant.In return, the mycobiont gains a receptive host and an energy source. Bydirectly utilising C acquired by plants, mycorrhizal fungi process from 10 to85% of the net primary productivity. Hyphal networks, especially those inroots colonised by two or more fungal links, may provide pathways for themovement of P and N among plants, and C-sharing among fungi. Therefore,mycorrhizas may influence the structure, diversity and productivity of plantcommunities, and their conservation is critical for maintaining ecosystemstability and function.

Gerhardson and Wright ( th is volume) propose that during evolution,these close contacts between plants and the microorganisms infecting orinvariably sur rounding them have developed into various dependencies onboth sides. These in turn have in many cases led to specific biologicalinteractions, or symbioses, presumably resulting from a long co-evolutionaryprocess, and in other cases to more or less loose, or even chanceassociations. We now find these dependencies as host-pathogen interactions,which may be biotrophic or necrotrophic, clear symbiotic interactions (e.g.with certain N-fixing microbes, and the mycorrhiza) and as a variety oflooser, probably facultative associations.

5. Ecosystem dynamicsConservation of plants ultimately requires conservation of their associatedmicroorganisms, a task poorly addressed and subject to a far greater degreeof complexity and subtlety than most plant conservation initiatives consider.Brundrett (2002) has suggested that plant roots may have evolved as habitatsfor mycorrhizal fungi, and in any case, there is evidence to suggest thatmycorrhiza were a key factor in the colonisation of land by plants (Batemanet al. 1998; Selosse and LeTacon 1998).

The interactions between microorganisms and plants are complex,including those related to scale. Whereas an area of high plant diversity mayhave up to 63 species in a 1 X 1 m quadrant (Kull and Zobel 1991), Torsviket al. (1990) found that up to 5,000 species of bacteria may occur in a singlegram of soil, and Dykhuizen (1998) estimated that a 30 g sample of forestsoil may contain over half a million species of microorganisms. Generally


only 0.1 to 1% of these microorganisms are culturable using current methodsand our knowledge of th is diversi ty is reliant on various DNA analyses(Torsvik et al. 1998). If we struggle to understand the dynamics responsiblefor such plant diversity, how much less do we understand bacterial diversityand population dynamics (which probably exert great influence on dynamicsof plant growth).

While it is necessary to consider both species diversity and ecosystemfunction in conservation programs, these two components are not directlyrelated, as functional groups generally consist of multiple taxa of varyingabundance (Bengtsson 1998). Changes in species diversity can impact thefunctional diversity of an ecosystem in that significant changes in abundanceor removal of al l taxa in a specific functional group can have strong effectson the ecosystem as a whole (Chapin et al. 1997).

Species rich heathlands of mediterranean climates, often with over 100species in a 100 m x 100 m quadrat, depend utterly on the nutrientimpoverishment of the site, i.e. add nutrients and the system begins todecline (Egerton-Warburton et al., this volume). Many plant communitiesare h igh ly dependant on mycorrhizal associations (Hawksworth 1991).Disturbance events such as mining can remove all mycorrhiza from the soiland restoration efforts can be hampered unless sites are inoculated withmycorrhizas, as ericoid mycorrhizas can take up to 12 years to re-colonise,with lesser time for other mycorrhiza types (Hutton et al. 1997; Dixon et al.,this volume).

The role of microorganisms in the maintenance of the ‘integrity’ ofnature has essentially been underestimated as it affects all existing biotic andabiotic components of earth’s environments. For instance, morphology andgrowth of plant roots appear not to be ‘normal’ when the plants are grown inthe absence of microorganisms naturally resident on or around roots (Rovira1972). Thus all large life forms have evolved with microorganisms andtherefore are natural ly dependent on them and/or are affected by them.

An example of the under-appreciation of microorganism activity isprovided by agrarian systems which, as diversity of the crop species isreduced, require increasing reliance on inorganic inputs of nutrients alongwith increasing amounts of external energy. Feedback results in decline inthe diversi ty of microorganisms wi th enhancement of the reliance ofartificial inputs. This can resul t in increased disease loading in croppingsystems – but increase the overall diversity of species and the organicallybased nut r ien t loading, and the abi l i ty of non-desirable organisms todominate is naturally decreased.


Biodiversity in regional hot spots can often be threatened by naturaldisasters such as diseases and pests (see Burgess and Wingfield, thisvolume). Containment of diseases in their environments can be difficult anda bigger challenge is to rehabilitate affected areas (see Hardy andSivasithamparam, this volume). The acceleration of disease impacts innatural ecosystems is highl ighted in the case of the onslaught ofPhytophthora spp. in the floristically diverse south-western Australia. Here,introduced pathogens have selectively removed key understorey andoverstorey elements on a scale not recorded in any other natural biodiverseecosystems (Wil ls 1993). The result ing melt-down in in situ conservationmeasures has resulted in a range of emergency measures being put in place.

Plant pathogens may have played a pivotal role in shaping current plantdiversity and ecosystem dynamics (see Ingram, this volume). Growingawareness of plant pathogen effects and observations of changes they causehas provided numerous modern examples of this. Phellinus weirii, a nativeplant pathogen in western North America has been identified as a keydeterminant of forest structure and processes (Hansen and Goheen 2000).The effects of Ceratocystis fagacearum on community structure of oakwoodlands in Texas and Wisconsin has been monitored, particularly inrelation to an epidemic in central Texas which caused mass tree death andsubsequent changes in community composition (Collada and Haney 1998;McDonald et al. 1998). Dutch elm disease (caused by Ophiostoma ulmi, O.novo-ulmi and O. himal-ulmi) has a varying effect on trees of the genusUlmus, in some cases having minimal effect (see Siebrecht 2000), while inothers, decimating populations (Hubbes 1999). In ecosystems previouslydominated by species susceptible to such devastating pathogens, this canresult in the replacement of species communities by less competitive speciesresistant to the pathogen (Hansen and Goheen 2000). Ristaino andGumpertz (2000) examine the spatial dynamics of Phytophthora epidemicswhich can be responsible for significant changes in ecosystem structure anddivers i ty , largely due to the broad host range and multiple distributionmechanisms of propagules.

It is highly likely that indigenous pathogens have been key determinantsof plant community structure throughout history, and that this should be seenas a natural process. Greater concern has rightly been raised over thispotential for change of community structure and composition where thepathogen has been introduced. This is further compounded by the potentialfor interspecific hybridisation to result in the rapid evolution of introducedpathogenic species resulting in the development of new strains or species ofpathogens (Brasier 2001).

6. The role of pathogens in shaping plant diversity


Disturbance events can be as obvious as land clearing, or as subtle aspollut ion of water from upstream flow. In a microcosm experimentinvestigating the effects of disturbance (freezing and heating) on microbialbiomass, and specifically the role of oribatid mites in facilitating recovery ofthe microbial populations, s igni f icant benefits of the mites were evident,including dispersal of fungal spores and stimulation of microbial metabolism(Maraun et al. 1998).

Agricultural ecosystems often represent some of the most modifiedsystems where plants are grown with ini t ial cultivation having a significantimpact on microbial communi t ies (Calderon et al. 2000). Even in suchmodified env i ronments , exper iments comparing organic and conventionalfanning systems consistently show significant benefits of increased organic-content in the soil ( i . e . higher diversi ty of microorganisms and increaseact ivi ty) (Fliessbach et al. 2000; Kushwaha et al. 2000). Comparison oft i l led fields of va ry ing age w i t h undisturbed native sites in Colorado founddecreasing fungal a c t i v i t y with length of time since disturbance was initiated(Kle in et al. 1996). Microbial diversity in such environments changes withvariations in agr icu l tu ra l practices and is therefore constantly in a state offlux.

Microorganisms permeate all aspects of human life – from the bread we eatto antibiotics. Microorganisms are actively utilised in the mining industryfor processes such as acid mine leaching to dissolve ores using acidsproduced by bacterial processes (Muller et al. 1995; Groudev et al. 1996;Krebs et al. 1997) as well as detoxification of hazardous wastes in soils(McGrath et al. 1998; Fein et al. 1999). A quick scan of relevant journalsshows that pharmaceutical and industrial research into the roles and benefitsof microorganisms dominates al l other microbial research areas. Recentpharmaceutical advances include the potential use of polyhydroxylatedalkaloids as anticancer, antidiabetic and antiviral agents (Watson et al.2001). Indus t r ia l advances inc lude biopolymer production bymicroorganisms (Daniell and Guda 1997) and ethanol production by thebacterium Zymomonas mobils (Gunasekaran and Raj (1999).

Bacteria and viruses are considered to have constituted the first life-forms toevolve on planet Earth (DeLong and Pace 2001). They were not only theprecursors of more complex life forms, but those more complex life forms(including our own species) have evolved from them (figure 1; Bateman etal. 1998). Their fossil remains provide our iron ore and much of our oil, gas

9. Conclusions

8. Social and scientific perceptions of microorganisms

7. Disturbance, conservation and land management


and breathable atmosphere. Depleting our planet of the rich, cryptic andelusive diversity of microorganisms is therefore at our own peril as wemislay and even deliberately destroy this planet’s unique library of evolution(Myers and Knoll 2001).

Is it possible to introduce cultured microorganisms into native or exoticenvironments? Such introductions into soil environments could beextremely diff icul t in microbiologically buffered habitats, especially for theestablishment of microorganisms that have been cultured in nutrient rich(and unnatural) laboratory media (Bunn and Tan this volume). Much of thenutr ient substrates (e.g. glucose) on which several generations of thosemicroorganisms have been raised in the laboratory are likely to be limitingor absent in the natural envi ronments . In addition, it is also likely thatseveral enzymes and metabolic pathways required for nutrition in nutrient-impoverished natural environments can be shut-down in laboratory cultures.This d i f f i cu l ty is further complicated by the limitations of labour andmethodology involved in tracing the introduced organism and in maintainingthe extraordinary complexity of genetic variation found in single species innature.

Plants and microorganisms have evolved together and have in manyinstances developed a certain level of mutual dependency on each other.Their r e la t ionsh ips range from obligate mycorrhizal associations toopportunist ic interact ions that may hinder or favour plant growth andestablishment. Conservation of either of these partners can therefore bepossible only in the presence of each other. This is clearly evident for theassociations described in this volume.

We would like to thank Steve Hopper for comments on the manuscript.

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Chapter 2

CONSERVATION OF MYCORRHIZAL FUNGALCOMMUNITIES UNDER ELEVATEDATMOSPHERIC AND ANTHROPOGENICNITROGEN DEPOSITION

Louise M. Egerton-Warburton

Chicago Botanic Garden, Lake Cook Rd, Glencoe IL 60022, USA; Department of Botanyand Plant Sciences, The University of California, Riverside CA, 92521, USA.

Edith B. Allen

Department of Botany and Plant Sciences, The University of California, Riverside CA,92521, USA.

Michael F. Allen

Centre for Conservation Biology, The University of California, Riverside CA, 92521,USA.

1. IntroductionOver the last century, the combination of rapid population growth,consumption of fossil fuels and industrial expansion has resulted in asteady increase in the quantities of pollutants discharged into theatmosphere on a daily basis. During the early 20th century, sulphurdioxide emissions formed the bulk of the pollutants. However, the latterhalf of the 20th century has seen a shift in the fundamental nature, as wellas quantity, of the air pollutants. The most ubiquitous air pollutants arecurrently carbon dioxide particulate matter, and nitrogenousemissions and their subsequent photochemical oxidants such as ozone.Despite the introduction of legislation and management strategies,monitoring has shown that emissions of and nitrogenous pollutantsregularly attain atmospheric levels that exceed acceptable thresholds fordamage to the biota. In this review, we address the responses ofarbuscular mycorrhizal communities to elevated anthropogenicnitrogen (N) eutrophication or x N interactions, and the predictedglobal climate change that is correlated with increasing atmospheric


concentrations of trace gases. Since arbuscular mycorrhiza are a commonand widespread phenomenon, the impacts of altered atmosphericcomponents on these organisms is indicative of the wider implications ofglobal pollutants on many other micro-symbiotic associations.

Increasing atmospheric concentrations of and nitrogenousemissions are the major determinants of global change (Bazzaz 1990;Galloway et al. 1995; Houghton et al. 1996; Norby 1998; IPCC 2000).Consumption of fossil fuels and deforestation has resulted in animbalance in the global carbon (C) cycle by increasing the atmosphericloading of In turn, altered C cycling is accompanied byphysiological modifications in plants, changes in the rates of C cyclingand N transformation in terrestrial ecosystems, and allied alterations in C-dependent systems, such as soil microbes (Zak et al. 1993). Globalatmospheric concentrations of have risen from 280 ppm in the pre-industrial era to ambient levels of almost 370 ppm in the year 2000(IPCC 2001). Current models predict that atmospheric loads willaverage 700 ppm by the end of the century (IPCC 1994, 2001; Bazzaz1990; Indermuhle et al. 1999).

Anthropogenic sources also dominate the global N budget. Over 80percent of oxides of nitrogen and 70% of ammonia emissions worldwideare generated by human activities (Vitousek et al. 1997; Matson et al.1999). Industrial fixation of N for use as a synthetic fertilizer currentlycontributes up to 80 Tg of new N into the global cycle each year.Additional new anthropogenic inputs into the N cycles can be traceddirectly to nitrogenous emissions derived from the combustion of fossilfuels by vehicles and industry (> 20 Tg NO-N per annum; Dignon andHameed 1989), domestic animal wastes (32 Tg the burning offorests (15 Tg NO- and and nitric oxide emissions from soils (5-20 Tg; Vitousek et al. 1997). Such increases in the emissions of airborneN have resulted in enhanced deposition of anthropogenic N intoterrestrial ecosystems. Increasing the N input modifies the N cycle bothdirectly, by increasing soil N pool, and indirectly, by altering C andphosphorous (P) cycling (Zak et al. 1993). Both and N depositioncan bring about change in terrestrial ecosystems. Because anthropogenicrelease of and N are inextr icably-l inked at the source and the gasesrelate mutually with one another in their reactive forms, the (im)balancesof C and N fluxes on the biota need to be considered in concert (Norby1998).

Almost invariably, global change has been evaluated by source-sinkanalyses of C allocation in plants (Bazzaz 1990; Wedin and Tilman1996). The contr ibution of the below-ground biota, especiallymycorrhizal fungi, as a feedback mechanism has been largely ignored(Zak et al. 1993; Berntson and Bazzaz 1996; Hu et al. 1999, 2001).Mycorrhizas, plant-fungus mutualisms, are critical for understandingecosystem dynamics in changing environments. Plant and mycorrhizalgrowth are t ight ly coupled due to the reciprocal nature of their C and Ncycles (Zak et al. 1993), and thus the symbiosis may impact the rate atwhich C and N are cycled within terrestrial ecosystems (Allen 1991).

Conservation of mycorrhizas under global change 21

Mycorrhizal fungi are a functional group of organisms that areestimated to form symbiotic associations with over 90 % of plant speciesand in most biomes (Smith and Read 1997). Such associations have beenlinked to the enhanced growth, survival, drought tolerance, pathogenresistance and nutrient status of the host plant. In return, the mycobiontgains C (Smith and Read 1997, and references therein). By directlyutilizing C acquired by plants, mycorrhizal fungi are responsible forprocesses that account for 10-85% of the net primary productivity (Vogtel al. 1991; Allen 1991). Hyphal networks (sensu Robinson and Fitter1999), especially those in roots colonised by two or more fungal links,may provide pathways for the movement of P and N among plants(Eissenstat 1990; Johansen et al. 1992), and C-sharing among fungi(Simard et al. 1997). Therefore, mycorrhizas may influence thestructure, diversity and productivity of plant communities (Allen andAllen 1986, 1990; Perry et al. 1989; Hartnett et al. 1993; Allen et al.1995; van der Heijden et al. 1998), and their conservation is critical formaintaining ecosystem stability and function.

Human activities can have important impacts on the diversity andefficacy of mycorrhizal associations. Their effects on mycorrhizae,however, are often chronic and frequently quite subtle (reviewed in Allenet al. 1993). Such changes may be manifest as minor fluctuations inspecies composition (Allen et al. 2000) through to more extreme shiftswhich encompass the loss of genera or changes in species dominance(Johnson 1993; Egerton-Warburton and Allen 2000). Although similarshifts in plant communities are acknowledged as being critical inunderstanding ecosystem processes (Wedin and Tilman 1996; Tilman etal. 1997), the influence of comparable shifts in mycobiont diversity onmycorrhizae and in turn, the plant community, has yet to be ful lyappreciated.

In this review, we focus on the functional diversity and conservationof mycorrhizal communities in the Californian floristic province asindicative of the way in which mycorrhizal relationships in plantcommunities are affected by increases in anthropogenically-linkedpollution, elevated or N eutrophication. California is a highlyurbanized region (est. population 40 million) and a recently designatedglobal hotspot of biodiversity and priority for conservation (Myers et al.2000). Southern California, and in particular the Los Angeles area, has ahistory of the most extreme air pollution in the contiguous United States.Both elevated and N deposition co-occur within this region.However, N eutrophication may currently constitute the biggest threat toecosystem stability. Nitrogen eutrophication in southern Californiacorresponds to an increased input of nitrate and between 10-13 %of all nitrogenous emissions (or 33-38 kg N per annum) are returnedto the soil annually as in southern California. In contrast, Neutrophication events in Europe are largely the result of depositionfrom intensive agriculture and animal husbandry (Schulze 1989;Bobbink 1991). Anthropogenic acidification is not a significant outcomeof atmospheric pollution in California (Fenn and Bytnerowicz 1993;


Bytnerowicz and Fenn 1996). Regardless, the photo-oxidation ofnitrogen oxide emissions in concert with volatile organic compoundsfrequently contribute to elevated ground level ozone concentrations andsevere photochemical smog within much of the Los Angeles Basin andparticularly during summer (Miller et al. 1998).

The California biota is characteristically termed a high diversity plantcommunity (Sawyer and Keeler-Wolf 1995), and one in which the manyplant species are frequently either obligately or facultatively dependenton mycorrhizal fungi, depending on soil nutrient availability, effectiveprecipitation and successional status. In addition, a diversity ofmycorrhizal forms occur in the California biota that include, but are notlimited to: arbuscular mycorrhizal fungi (AMF) in coastal sage scrub andchaparral, ectomycorrhizal fungi (EM) in chaparral (Adenostomaspecies), oak woodlands (species of Quercus) and higher elevation forests(species of Pinus), and ericoid, orchidaceous and arbutoid mycorrhizas.We reviewed the responses of the AMF community following exposure toelevated Neutrophication or x N interactions, and the relevanceof a trace gas-derived climate change on the symbiosis. Arbuscularmycorrhizal fungi are the most ubiquitous mycorrhizal association andthus shifts or responses in AMF dynamics to human impacts may beparticularly widespread and of considerable concern. We use AMFresponse data to answer the following questions:1. How does functional diversity and community dynamics of

mycorrhizas and efficacy of the symbiosis vary with elevated Ndeposition, or following x N interactions?

2. How will the downstream effects of elevated and nitrogen oxides,such as ozone and climate warming, influence mycorrhizalfunctioning?

3. What are the likely outcomes of global change-induced shifts inmycorrhizas on plant community productivity and stability?

4. How may mycorrhizas be best conserved in the climate of globalchange?

2. Mycorrhizal functioning and responses to elevated atmosphericCurrent analyses of the global carbon cycle argue in favor of a vital rolefor terrestrial ecosystems in carbon uptake. Partitioning of the terrestrialsink indicates that enrichment stimulates photosynthesis andsubsequently increases plant primary productivity, litter fall and carboninput into terrestrial ecosystems (Bazzaz 1990; Berntson and Bazzaz1996). As the production of above-ground resources is amplified, thebelow-ground carbon allocation increases correspondingly and promotesa marked increase in specific root length and the deposition of carbonper unit area wi th in the rhizosphere (Rouhier et al. 1996), and, in turn,improved carbon allocation to mycorrhizas (Hodge 1996). Theoretically,such circumstances should favor mycorrhizal proliferation (Figure 1a).

enrichment in California shrublands and grasslands alters carbonallocation to mycorrhizal, but not pathogenic, fungi, and increases thesink strength of the mycobiont (Rillig and Allen 1998, 1999; Rillig et al.


1998a, 1999a,b). With few exceptions, elevated results in an increasein extra-radical hyphal biomass, intra-radical hyphal infection intensityand arbuscular infection (Table 1; Rillig and Allen 1999). In particular,hyphal infection intensity increased significantly in root classes (180-400µm diameter) where the cortex can readily accommodate fungalproliferation (Rillig and Allen 1998, Rillig et al. 1998b). Rygiewicz et al.(1997) also noted an increased turnover of mycorrhizal hyphae withenrichment. However, there is no apparent change (increase or decrease)in the production of vesicles or intra-radical coils, or the abundance ofpathogenic fungi in response to enrichment (Klironomos et al. 1996,1997; Ri l l ig and Allen 1998; Rill ig et al. 1997, 1999a). Thedevelopment and proliferation of mycorrhizal structures implies anincrease in carbon allocation to the mycobiont, especially to the intra-radical structures (hyphae and arbuscules but not vesicles), and anincrease in the carbon sink strength of the mycobiont (Morgan et al.1994). Since the arbuscular interface regulates the directional transfer ofcarbon and nutrients between host and mycobiont, these observationssuggest that the potential for nutrient and carbon transfer may beenhanced under elevated conditions. Parallel studies ofenrichment in other ecosystems indicate a positive relationship betweenarbuscular infection, P inflow and content of the host plant withenrichment (Rouhier and Read 1998). In particular, plants grown inambient conditions failed to access declining soil P reserves (Rouhierand Read 1998).

Strong host interspecific differences exist with respect to mycorrhizalresponse under elevated and habitat; such differences could not berelated to life history or phylogeny (Rillig et al. 1998a, 1999b).Responses of AMF, as measured by arbuscular infection, to elevatedare inconsistent and can be positive (Linanthus, Plantago, Euphorbia),weakly negative (Lolium), or non-significant (Epilobium, Avena)depending on the study system (Rillig et al. 1999b). Notably, arbuscularinfection in Lolium increased in a serpentine soil, but decreased insandstone annual grasslands (Ril l ig et al. 1999a,b). The positive responseto in Euphorbia is of interest since members of the genus maypossess or physiology or Crassulacean Acid Metabolism (CAM),and as atmospheric concentrations rise, will this influence the relativedistributions of and CAM plants in general? Experimental datagenerally demonstrate that elevated levels favour plants due totheir compensation of 30-70 ppm at optimal temperatures (versus<10 ppm in plants; Larcher 1995). On the other hand, CAMplants temporarily segregate the processes of carbon dioxide uptake andfixation when grown under arid conditions but follow thephotosynthetic pathway when water is not limiting. Elevated and awetter climate as predicted by global change analysts may thus also favorCAM plants (0-200 ppm compensation in light; Larcher 1995).These differences illustrate that plant responses to may depend onvariations among mycorrhizal functional groups and substratum.Correspondingly, mycorrhizal and non-mycorrhizal plant species, and



differences in metabolic pathways, such as or CAM, among hostplants may also be expected to differ in their responses to elevated

Striking differences also exist among mycorrhizal groups withrespect to responses (Klironomos et al. 1998). Artemisia tridentataseedlings inoculated with Acaulospora denticulata, Glomus etunicatum,Glomus intraradices or Scutellospora calospora demonstrated thatmycorrhizal taxa differ in their growth allocation strategies in a -enriched environment. Percent infection of roots by arbuscules andhyphae increased in Artemisia inoculated with either Glomus species. Onthe contrary, there was no detectable change in infection in plantsinoculated with Scutellospora or Acaulospora. Hence, AMF taxa mayvary in their ecological specificity and capacity to influence the growth ofthe host plant.

More subtle responses to occur at the AMF community level. Atambient or sub-ambient atmospheric (up to 350 ppm), the AMFcommunity associated with Adenostoma fasciculatum was dominated bythe genus Glomus, with respect to spore bio-volume and extra-radicalAMF hyphal length. Increasing atmospheric (350-650 ppm) wasassociated with an increase in hyphal length within soil aggregates, sporebio-volume and subtle shifts within the AMF community composition.Specifically, the abundance and bio-volume of AMF spores increases with

this outcome was due to the increasing prevalence of the large-spored Scutellospora calospora, and a decline in the abundance of small-spored Glomus species. These findings were paralleled in the AMFhyphal community. An increasing availability of was correlated witha marked increase in the hyphal lengths and abundance of Scutellosporaand Acaulospora (Klironomos et al. 1998; L. Egerton-Warburton,unpublished data). Hence, AMF differ in their growth allocationstrategies and capacity to sequester carbon under elevated asindicated by the increasing hyphal length and abundance of spores. Inconcert with the changes in functional diversity of AMF taxa (see above),shifts in the AMF community may therefore impact the structure, nutrientstatus and productivity of the plant community.

We anticipate that such shifts are also influenced by temporal andseasonal changes in both mycorrhizal and host plant root growth andactivity (e.g. Zak et al. 1993). Although these responses have yet to beevaluated in California, studies in other biomes demonstrate a distinctphenology of mycorrhizal infection. Little or no mycorrhizal responsecan be expected in the two months following initiation ofenrichment. However, s ignif icant increases in mycorrhizal infectionfollows prolonged enrichment in both AM and EM systems (Rouhierand Read 1998). These phenologies may be driven by alterations incarbon allocation to the mycobiont (Rouhier and Read 1998).

The net effect of elevated on AMF appears to be an increase inhyphal biomass or growth, root infection and shifts in communitycomposition and the dominance of AMF taxa. Nevertheless, species-specific responses to individual AMF taxa appear to be the rule ratherthan the exception, and interactions between host plant and mycobiont



most likely determine ecosystem feedback to elevated in a bi-directional feedback loop. Changes in mycorrhizal infection and theextra-radical phase (spores plus hyphae), however, indicate that AMFcould substantially alter nutrient availability to the host plant by up- ordown-regulating nutr ient transfer. Such changes conserve nutrientswithin ecosystems and result in the downstream effects in increased hostplant fecundity and litter production, changes in the quality (C:N) anddecomposition of l i t ter and mycorrhizal activity. The magnitude of theseresponses will dictate long-term ecosystem responses to by alteringthe dynamics of mycobiont-plant species interaction.

3. Mycorrhizal community dynamics following anthropogenic nitrogendepositionThe global upsurge in nitrogen emissions and deposition followingurbanization and industrialization is well documented. In many regions,anthropogenic N deposition is increasing at a faster rate than thecorresponding rise in atmospheric Nitrogen enrichmentdramatically increases aboveground productivity but negatively impactsbiological diversity, composition and functioning (e.g. carbon cycling) ofecosystems (Tilman 1988; Berendse 1995). In addition, feedbacksbetween the below- and above-ground biota can also contribute to thisoutcome. As ecosystems become increasingly N enriched, plants allocatemore C to above- than below-ground structures (Tilman 1988), whichresults in an intense C sink competition between shoots and roots, anddecreased C allocation to mycorrhizas (Smith 1980). Consequently,models exploring the relationship between N enrichment and mycorrhizalstructure and function have demonstrated that N eutrophicationnegatively impacts the mycorrhizal association (Figure 1b; Allen 1991;Smith and Read 1997 and references therein). However, unlike theeffects of elevated on AMF dynamics, the effects of N eutrophicationon AMF community and hence ecosystem dynamics, has received littleattention. Still, the influence of N eutrophication on AMF communitiesin California can be readily identified by a suite of traits that illustrate anegative response to N enrichment.

On a global scale, mycorrhizal diversity is strongly and negativelyinfluenced by chronic N enrichment. Chronic N deposition has beendirectly linked to a decline in EM diversity and productivity elsewhere(reviewed in Wallenda and Kottke 1998). However, AMF communitycomposition and diversity may shift in response to host plant speciesalone (Johnson et al. 1992) or combined host plant-nutrient availabilityinteractions (Johnson et al. 1991). In southern California, there was noapparent effect of host plant species (native shrub species) onmycorrhizal diversity (Egerton-Warburton and Allen 2000; Sigüenza2000; Sigüenza et al. 2000). Arbuscular mycorrhizal communities werecomparable among host plant species, and they responded similarly to Nenrichment (Egerton-Warburton and Allen 2000). Regardless, anincreasing N input in shrublands and grasslands, either via anthropogenicN deposition or N fertilisation, resulted in significant shifts in AMF


community dynamics that were identified by alterations in mycorrhizaldiversity and productivi ty.

Firstly, N eutrophication results in a rapid and marked shift in AMFrichness and diversity (Egerton-Warburton and Allen 2000). Data froman anthropogenic N deposition gradient and N fertilisation studies inCalifornia shrublands demonstrate a reduction in AMF species richnessand diversity with increasing N input. Such changes were accompaniedby displacement of the larger-spored genera Scutellospora andGigaspora due to a failure to sporulate, and concomitant proliferation ofsmall-spored Glomus species, such as Glomus aggregatum, Glomusoccultum and Glomus leptotichum (Egerton-Warburton and Allen 2000),and Glomus tenue (Sigüenza 2000; Sigüenza et al. 2000). These Glomusspecies are considered indicative of N eutrophication and affiliated withcolonization by invasive grasses in southern California. Comparablestudies in ecosystems elsewhere indicate that such changes most likelyinfluence plant community dynamics by altering the functional diversityof the AMF community (Johnson 1993). Specifically, these Glomusspecies tend to be less effective mutualists, particularly in N-enrichedsoils, and negatively influence host plant productivity and nutrient status.

Secondly, N eutrophication may alter the productivity of the AMFcommunity by reducing the carbon sink strength of the mycobiont. Anincreasing input of N can be correlated with a decline in root infection.In particular, N eutrophication is associated with a marked loss of hyphaland vesicular, but not arbuscular, infection (Egerton-Warburton and Allen2000). Such data suggests an overall reduction in the carbon expenditureby the mycobiont but that the potential for nutrient and carbon transferhas not been altered by N enrichment. More simply, the carbon sinkstrength of the mycobiont cannot compete against that of the host at highnutrient loads. Responses of AMF abundance to N eutrophication, asrepresented by extra-radical hyphal abundance and length, are lessconsistent. Hyphal responses have been noted as negative and manifest asa significant reduction in hyphal length following prolonged (> 14months) of N enrichment (Klironomos et al. 1997; Rillig et al. 1998b),or in other cases, non-significant (Rillig et al. 1998b). However, if theabundance of live hyphae declines, then the capacity for plants to acquirewater and sparingly available nutrients does so proportionately. Inaddition, if hyphal turnover is influenced by N enrichment, then soilstructure may be altered because microbial decomposition of labilematerials is enhanced wi th increasing N availabi l i ty (reviewed in Hu et al.1999).

Alterations in the ava i l ab i l i ty of resources invariably impact thestructure of the vegetation (Tilman 1993), either by the direct effect of Non plants or via those mediated by the effects of N on mycorrhizalassociations and, in turn, the host plant (Smith and Read 1997). A recentsynopsis indicates that coastal sage communities in southern Californiaare currently undergoing a shift from native shrublands to one dominatedby exotic annual grasses (Figure 2; Allen et al. 1998; Padgett and Allen1999; Padgett et al. 1999). Native shrub species frequently demonstrate


poor establishment rates due to competition from exotic grasses coupledwith the premature senescence of seedlings. Moreover, native shrubspecies that are obligately mycorrhizal in the field, such as Salviamellifera and Artemisia californica, or facultatively mycorrhizal, such asEriogonum fasciculatum, all demonstrate an inability to regulate growthwith N enrichment (Padgett and Allen 1999). Specifically, N fertilisationpromotes constant and luxuriant shoot growth and high foliar N contentat the expense of root growth and development in native shrub species.These data coupled with the high mortality rates in seedlings grown underN enrichment suggested that high N inputs maycontribute to an acceleration in shrub mortality and the decline ofshrublands (Allen et al. 1998). Contrary to expectations, exotic grassspecies (Avena, Bromus) all demonstrated a depression in productivityfollowing N eutrophication (Padgett and Allen 1999). It is possible thatplant (grass) growth can be increased by N fertilisation, but at some point,growth may become limited by an insufficiency of other essentialresources such as P, calcium or water availability (Tilman 1993).

Some of the most critical effects of N enrichment on plant growthcan be exerted indirectly via interactions with mycorrhizas(Termorshuizen 1993). Currently, little is known about the interactionsbetween N enrichment, mycorrhizal response and plant productivity insouthern California. Yoshida and Allen (2000, 2001) reported thatarbuscular mycorrhizas had a more negative influence on the uptake of

in Artemisia californica than the invasive annual grass, Bromusmadritensis. As a result, Bromus may be a better competitor forthan Artemisia, and in vegetation where high anthropogenicdeposition is increasing, the encroachment of Bromus may be in partrelated to N nutrit ion. Additional studies are currently in progress todetermine the positive and negative contributions of host and mycobiontto the plant community with N enrichment. However, we consider thatthere is sufficient knowledge regarding the effects of N enrichment oneach of plant productivity and mycorrhizas in southern California topredict that N eutrophication most likely negatively impacts thefunctioning of the symbiosis.

We can conclude that N eutrophication has profoundly influencedthe AMF community. We further suggest that N enrichment may initiatea cascade of negative effects on mycorrhizas that consequently maydetermine ecosystem responses to N deposition. Nitrogen eutrophicationpromotes the decline of AMF species richness and diversity that, in turn,initiates a potential loss in plant community diversity, productivity andfunctioning (van der Heijden et al. 1998). In addition, functional shiftsin the mycorrhizal community may be driven by changes in the qualityand quantity of plant C allocation to the mycobiont. As a result, suchchanges may negatively impact host plant productivity, nutrient captureand competitive abil i ty, and potentially alter P cycling and C allocation atthe ecosystem level (Figure 1b). Subsequently, changes in C allocationamong mycobiont, host and rhizosphere may modify the capacity ofterrestrial ecosystems to absorb and sequester C (Figure 1a).


A more variable mycorrhizal response to enrichment occurs in thepresence of N, P or NPK enrichment (Table 1), suggesting thatinteractions between elevated and N deposition may have quitedifferent implications for the terrestrial environment (Figure 3). InGutierrezia sarothrae (Rillig et al. 1997; Rillig and Allen 1998), asynergistic x N interaction regulates mycorrhizal infection. A greaterAMF infection response was noted for the combined treatment thanoccurred with or N alone. Strong x NPK interactions alsooccurred in some mycorrhizal type plant species combinations. Thesemay be manifest in the increased production of fine roots and extra-

4. Interactions between elevated x nitrogen enrichment andinfluences on mycorrhizas


radical hyphae (e.g. Linanthus, Ri l l ig et al. 1998b). In contrast, xNPK enrichment does not el ici t the same response in infection intensity inAMF inoculated plants of Bromus hordeaceus (Rillig et al. 1998a). Inaddition, the level of applied nutrient influences the outcome. Forexample, at low N availability, a high level of stimulates hyphalgrowth in Populus, whereas there was no significant effect of withhigh N availability. These data suggest that AMF communities canrespond quite differently to elevated depending on edaphicparameters such as N or N+P availability, and that strong interactionsbetween and soil nutrients most likely influence mycorrhizalfunct ioning (Klironomos et al. 1997). Thus, the combined effects ofand N may differ wi th in and among ecosystems, and may be driven bylocal edaphic constraints.

5. Nitrogen oxides, their photochemical conversion to ozone and effectson mycorrhizasOzone in both the stratosphere and at ground level has become animportant global air quality issue. In the stratosphere, where acts as afilter to absorb UV, short- and long-wave infrared radiation, ozone isbeing depleted. Conversely, levels at ground level are steadilyincreasing and currently constitute a major component of photochemicalsmog (UNEP 1988). Ground level ozone is formed by the photo-oxidation of volatile organic compounds, such as carbon monoxide,methane and non-methane hydrocarbons, in combination with nitrogenoxides (UNEP 1988). The burn ing of fossil fuels is a majoranthropogenic cause of nitrogen oxides, while the use of motor vehicles,solvents, and indus t r ia l processes in the petrochemical industry constitutethe sources of volati le organic compounds.

Since the mid-1970s, ground level ozone concentrations haveincreased globally by 1% on average. However, increases in ozone levelshave been considerably higher in the Los Angeles Basin (Miller et al.1998). Ozone directly affects plant physiology and metabolism. Suchchanges are manifest in visible foliar injury, reduced reproductivedevelopment, annual productivity, increased susceptibility to insects anddisease, and altered plant physiology, such as photosynthetic capacity,modification in the part i t ioning of carbohydrate allocation andparticularly the delay of photosynthate allocation to below-groundstructures (Davison and Barnes 1998; Tausz et al. 1998).

In California, the adverse effects of ozone alone or in combinationwith other edaphic stressors on plants are well documented (Peterson andArbaugh 1992) but the influences on the mycorrhizal community haveyet to be elucidated. This is surprising given the intensity of thephotochemical smog wi th in the Los Angeles Basin during summer(Miller et al. 1998). However, many species in the coastal sage andchaparral shrublands avoid the impact of by being dormant and/ ordrought deciduous in summer when levels tend to be highest (Sawyerand Keeler-Wolfe 1995). Studies from other ecosystems illustrate theanticipated to impact of on those summer-active species. Because


ozone does not penetrate the soil to any extent (Reich et al. 1986),influences mycorrhizas via changes in carbon allocation from the hostplant (Andersen and Rygiewicz 1995). From a structural perspective, thefrequency of vesicles, hyphal coils and intra-radical hyphae in arbuscularplants increases at heightened ozone levels, while arbuscular colonizationdecreases. Duckmanton and Widden (1994) suggested that mycorrhizasmay respond to stress by increasing the production of less energydemanding and less e f f ic ien t organs for exchange of nutr ients(arbuscules), and by increasing the resources allocated to storage andfuture growth (vesicles). The increase in internal mycelium appeared torepresent an increase in suscept ibi l i ty of the root to infection bysaprophytic fungi . In addition, ozone stress promoted a shift in carbonallocation to the roots of arbuscular mycorrhizal plants that, in turn,reduced the extent and func t ion ing of the mycorrhizas (McCool andMenge 1983). Such effects can be further modified, both positively andnegatively, by the nature of the mycobiont (McCool and Menge 1984).

In summary, ozone stress appears to be associated with negativechanges in the allometric re la t ionship of mycorrhizal occupancy to rootvolume (reduced mycorrhizal colonization among roots), an alteration inthe allocation of carbon s ink strength of the mycobiont (reducedabundance of mycorrhizal structures) and a decline in functioning (as


indicated by the loss of arbuscules). In turn, a reduction in functioningmay influence the growth and mineral nutrition of the host plants (Smithand Read 1997) and impact the sustainabil i ty of ecosystems.

Escalating concentrations of atmospheric nitrogen oxides and ozonecan be correlated with changes in temperature and precipitation patternsthat, in turn, have the capacity to modify the global climate (Houghton etal. 1990, 1996; Schiffer and Unninayar 1991). These trace gases canalso influence the radiative balance of the atmosphere. This so-called‘greenhouse effect’ results from “the dirt on the atmospheric infraredwindow" by trace gases such as that allows incoming solar radiationto reach the surface of the Earth unhindered but restricts the outwardprogress of long-wave infrared radiation. Such gases can also absorb andre-radiate this outgoing radiat ion to promote a net warming within thefirst 10-20 km of the atmosphere; the global mean temperature isexpected to increase by 1°C by 2030 (Bolin et al. 1986; IPCC 1994;Barret 1995).

Model-predicted maps of temperature and rainfall have forecast awarmer summers (up to 6°C on average) and wetter winters along thePacific seaboard in the future (Smith and Tirpak 1988). Such changesmay alter the migration and distribution of plant species (Vitousek 1994)and subsequently the composition and productivity of plant communities,albeit slowly (King and Tingey 1992). It is reasonable to expect that anychanges in plant communi ty composition will directly influencemycorrhizal communities (Johnson et al. 1992; van der Heijden et al.1998; Klironomos et al. 2000). Because mycorrhizas are carbon sinksand amongst the firs t soil biota to receive carbon from plants (Rygiewiczand Andersen 1994), cl imate change may also indirectly influence thedominance and functioning of fungi within a community (Rygiewicz etal. 1997).

Microbial communit ies may respond rapidly to changes (increase ordecrease) in soil moisture and temperature, or the combined effects ofelevated trace gas concentrations, soil moisture and temperature (Schwartz1992; King and Tingey 1992; Hu et al. 2001) but not always (seeWhitbeck 1994). For example, four cycles of simulated climate change(elevated modified temperature and precipitation regimes)influenced mycorrhizal colonization in a grass (Pascopyrum smithii),but not Bouteloua gracilis (Monz et al. 1994). Specifically, increasesin mycorrhizal colonization due to effects in Pascopyrum could beoffset by elevated temperatures (4°C above control), and increasedprecipitation. Hence, any mycorrhizal response may be influenced bythe synergy of edaphic and biotic constraints. At first glance, these directand indirect changes appear modest however any of these shifts will befurther amplified during host plant species variation with progressiveclimate change.

6. Trace gases and the greenhouse effect


7. Scaling up from the fungal to the global communityScaling and modeling from community level to global platform can oftenpredict performance or survival of mycorrhizas, and contribute to anunderstanding of the funct ioning of ecosystems and human impact onthe terrestrial environment. The studies presented herein indicate thatelevated and N deposition were not random in their effects onmycorrhizal community productivity and functioning (Figure 3). Inaddition, alterations in the mycorrhizal fungal community appear to be alikely and potential ly irreversible outcome of global change (but seeOechel et al. 1994).

Human impacts are f requent ly viewed only in terms of the negativeimpact on the affected AMF communities. However, disturbance maybenefit some mycorrhizal species, in, for example, the geographicexpansion of some species (so-called ‘winners’), and a concomitantreduction in others (‘losers’) (McKinney and Lockwood 1999).Evidence from the studies on the AMF communities in Californiaillustrates that exposure to anthropogenic change results in both winnersand losers. The winners are represented by a small number of rapidlyexpanding species that benefit from human alteration of the habitat.Under conditions of elevated these appear to be the genera,Scutellospora and Acaulospora. In contrast, chronic N depositionappears to favor small-spored, weedy Glomus species. These Glomusspecies in particular possess traits that promote survival in disturbedhabitats. For example, they are highly dispersable, possess small sporeswith high fecundity, and display mutualistic to parasitic traits in symbiosis(Johnson 1993; Johnson et al. 1997). The net outcome in both instancesis a change in functional diversity and productivity.

The replacement of formerly diverse AMF communities by winnersmay cause a more widespread homogenization of the mycorrhizalcommunity than is currently appreciated, particularly if AMF species arenot randomly distributed, such as in species or genets that are specializedin function, or explicit to a particular plant species. Selective extinctionmay accelerate the loss of (functional) diversity, and denote the removalof unique genetic or morphological diversity, such as Gigaspora on the Ndeposition gradient (Egerton-Warburton and Allen 2000). Thehomogenisation may be taxonomically and ecologically depressing to theeffect that global change results in simpler AMF communities composedof generalists, the over-representation of some genera and fewerecological specialists. Based on the causal relationship betweenmycorrhizal diversity and plant community diversity and productivity(van der Heijden et al. 1998), such shifts may exert a profound impact onecosystem funct ioning. Specifically, we suggest these may be manifest asa reduction in plant community diversity at the regional and global scalesand changes in plant func t iona l response via mycorrhizal-regulatedalterations in C and N cycling, the movement of water, and the uptakeand/ or exchange of P, N and C.

The ecological consequences of AMF homogeneity are obviouslymultifaceted. However, the question remains as to conservation of


mycorrhizas with global change. Levin (1990) suggested that thepersistence of populations depended on heterogeneity. Thus a decline inheterogeneity, or increase in homogeneity, indicates a decline in thepotential for persistence of an AMF community. All organisms andcommunities demonstrate heterogeneity and diversity and this should beselected for in relation to physiological, morphological and demographicresponses of the organism (Levin 1990). The key is how to determinethe criteria by which to conserve AMF diversity, heterogeneity, andidentify the mycorrhizal genera, species or communities at most risk in aclimate of global change.

The Montréal Process identified seven conceptual criteria andindicators for the conservation and management of ecosystems(http://www.mpci.org/meetings/tac-mexico/tnl-6_e.html#s2). While thesecriteria were originally intended for forest ecosystems, in a modified formthey apply equally well to mycorrhizal communities and their role inecosystem functioning. We suggest the following seven adapted criteria:

1. Conservation of mycorrhizal diversity2. Maintenance of the productivity of mycorrhizas in ecosystems3. Maintenance of mycorrhizal functioning4. Conservation of a mycorrhizal role in maintaining soil and water

resources5. Maintenance of the mycorrhizal contribution to global carbon and

nitrogen cycles6. Maintenance and enhancement of the long-term multiple socio-

economic benefits of mycorrhizas7. Legal, inst i tutional and economic framework for conservation and

sustainable management of mycorrhizas

Criteria 1-3 Conservation of mycorrhizal diversity, productivity andfunctioningDiversity, productivity and functioning of a community, whether plant orfungal, drives ecosystem efficacy and the capacity to respond toanthropogenic disturbance. The variability in AMF species richness andrange of genotypes of each species within a given area may constitutesurrogate descriptors of diversity. However, the full extent of geneticdiversity in AMF is poorly understood (Bentivenga et al. 1997). Thenucleate state of the AM spore may harbor greater levels of geneticdiversity in AMF than is currently appreciated (Sanders et al. 1995;Redecker et al. 1999), and the issue of ecotypes and concepts of plasticityhave yet to be fully realized in these fungi (see Bever and Morton 1999).In addition, keystone species, or AMF that are pivotal to communityfunctioning, remain to be fully documented. Clearly, an understandingof the ecological and evolutionary significance of AMF diversity withinand among functional groups, the expansion of taxonomic surveys andinventories, and the documentation of endemism among AMFcommunities is important. Recent and anticipated advances in moleculargenetic techniques may ensure that AMF surveys will be productive in


many contexts. However, it is equal ly important to perform research onAMF species in symbiosis in order to interpret the significance of geneticdiversity on host plant product iv i ty and functioning, particularly inresponse to anthropogenic change. In the absence of such data, we areforced to assume that AMF species in southern California arephylogenetically similar and exert comparable effects in symbiosis asAMF documented elsewhere.

Criteria 4-5 Conservation of the mycorrhizal contribution to maintainingsoil and water resources and global carbon and nitrogen cyclesMycorrhizas contribute significantly to terrestrial C and N cycling,constitute sources and sinks in this cycle, and soil structure andaggregation with global change (Ri l l ig and Allen 1999). What is missingis quantitative information for integrating AMF species-based responsesinto functional group responses. Mycorrhizal functional groups may bebased on edaphic criteria, such as the turnover of soil N and C, but guidedby quanti tat ive biological descriptions of an individual AMF species and/or genus. The development of realistic models that link AMFcommunities to edaphic constraints also requires exploration of thephysiological responses of AMF and the interactions that drive alterationsin functional diversity with global change. These include, but are notlimited to, factors that stimulate or reduce growth rates, nutrient and wateracquisition, or C transfer via common mycorrhizal networks (Robinsonand Fitter 1999).

Criteria 6-7 Maintaining the socioeconomic benefits of mycorrhizaswithin the frameworks of political and economic institutionsA rapidly expanding population with increasing demands for space andland, subsequent changes in consumption patterns, especially of fossilfuels, and consumer-driven access to natural resources has resulted in anexceptional rate of habitat loss and fragmentation of which threatens mostnative ecosystems as typified by the shrublands in southern California.Historically, the value of the environment in California as elsewhere has

been largely ignored unt i l it has been substantially depleted or lost.Attempts to arouse public and governmental support for the preventionof habitat loss often fails due to a lack of understanding of the l inkbetween natural resources and the economy. In southern California, themost impacted regions are remnant tracts of coastal sage scrub. Unsulliedcoastal sage communities tend to support highly diverse AMFcommunities. However, these remnants are continually sacrificed forurban expansion, or increasingly invaded by plant species that benefitfrom high soil N loads (Padgett et al. 1999). As the weedy speciesinvade, the community becomes dominated by plant species with loweredor negligible mycorrhizal funct ioning (Sigüenza 2000; Sigüenza et al.2000). The remaining tracts of shrublands tend to be adjacent to urbandevelopments and proximity enhances invasion by exotic grass species(Padgett et al. 1999). In th is urban environment as in many others in theworld, maintenance of the mycorrhizal community has broader


environmental values, namely the preservation of remnant shrublands,since many of the native plant species are obligately mycorrhizal. Thus,conservation of the mycorrhizal community requires preservation ofremaining tracts of shrublands, and measures to reduce soil N levels viathe introduction of legislation to further reduction in vehicular emissionsand ameliorative measures, such as mulching, to immobilize inorganic N(Allen et al. 1999).

8. ConclusionsHumans have been extraordinari ly successful in developing industrializednations and permitting the scale of human enterprise to develop sorapidly that their consequences are felt at the scale of individualorganisms, ecosystems, and the biosphere, and more notably in thehuman alteration of the global carbon and nitrogen cycles (D’Arrigo etal. 1987; Keeling et al. 1995). How mycorrhizal fungi respond to thesechanges may well depend on the biogeochemical conditions thatoccurred during their evolution and their genetic ‘memory’ of these pastevents.

The past four glacial-interglacial cycles (up to 420 thousand yearsbefore present; Indermuhle et al. 1999) demonstrate both the extent ofclimate change and low (<180 ppm) during glacial periods andhigher (>300 ppm during interglacial periods (Lorius et al. 1990).Thus, the current and anticipated rise in atmospheric levels is wellwithin those encountered during the evolutionary history of AMF (Allen1996). Consequently, the capacity of mycorrhizal fungi to respond toelevated is most likely within the scope of their genotypic variability.On the other hand, N is usually a limiting nutrient in many ecosystems(Tilman 1993) and there is substantial evidence that N limits net primaryproduction in most terrestrial biomes (Vitousek and Howarth 1991). Anexcess of N in geological time scales was most likely associated withmajor disturbance events whereas human activities have altered thenitrogen cycle deliberately through the production and use of industrialfertilizers, the growth of nitrogen fixing crops and burning fossil fuels(Vitousek and Howarth 1991). The consequences of the anthropogenicchanges in N cycle are important because they affect not only thecomposition of atmosphere, but because N interacts strongly with thecarbon cycle to the point of regulating the flux of carbon in manyecosystems. The current convergence of these two major determinants ofecosystem functioning on mycorrhizas appears to push fungalcommunity and ecosystem dynamics in opposite directions, but for themost part the responses to these stressors may depend on the precisemycobiont-host combination and local edaphic constraints, such as soilmoisture and temperature.

As a corollary, human activities have also impacted upon speciesdiversity at the global scale. While extinction and immigration of speciesare acknowledged as natural ecological processes, the magnitude andscope of these events is faster than any that have occurred over the past20,000 years (IPCC 2001). Because emissions of greenhouse gases such


as and nitrogen oxides are long-lived, they will have a lasting effecton the atmospheric composition, climate and terrestrial ecosystems. Froma mycological perspective, this is of particular concern because thecurrent knowledge base of mycorrhizal fungal diversity and functioningis relatively small and the loss of species diversity is irreversible. Inaddition, humans are also responsible for the introduction of exoticspecies into communit ies . These non-native species often change thebiotic and abiotic environment sufficiently to promote the decline or lossof mycorrhizas from the community. Synergistically, these changes areoccurring so rapidly in both California and on a global basis that anassessment of mycorrhizal fungal diversity and the functionalconsequences for population, community, and ecosystem processes arenow critical for their conservation.

AcknowledgmentsWe thank the National Science Foundation and the United StatesDepartments of Agriculture and Energy Competitive Grants Programmes,and the Australian-American Education Foundation (Fulbright) forresearch funding leading to this synthesis.

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151 , 105–117 .

Chapter 3

SYMBIOTIC NITROGEN FIXATION BETWEENMICROORGANISMS AND HIGHER PLANTS OFNATURAL ECOSYSTEMS


John S. Pate

Plant Biology, Faculty of Natural and Agricultural Sciences, The University of WesternAustralia. Crawley, 6009, Western Australia.

1. IntroductionNitrogen (N) is generally considered to rank next in overall importance forplant growth to the elements carbon, hydrogen and oxygen which greenplants assimilate photosynthetically from carbon dioxide and water. Unlikeother mineral elements essential to life, N is absent from the primarymaterials of the earth’s crust, resul t ing in overwhelmingly great dependenceof essentially all world fauna and flora on atmospheric molecular nitrogen

To a lesser extent, there are also inputs from other atmospheric sourcessuch as nitrate generated during thunderstorms, fallout of ammoniagas from volcanism and and oxides of nitrogen arisingfrom various combustion processes associated with human activity (seeJenkinson 1990, 2001; Galloway et al 1995). Counteracting these inputs,significant net losses of N occur from soils of natural ecosystems, whether as

released through denitrification, lost from decomposing organicmatter, and released to the atmosphere during fire, or leaching of

through surface run-off and deep drainage (Mansfield et al. 1998;Fowler et al. 1998). Inputs of fixed thus remain critical to restoration ofnitrogen capital of ecosystems and to the long and short-term functioning ofconstituent vegetation and dependent biota (Schlesinger and Hartley 1992;Stevenson and Cole 1999).

The capacity to fix is largely confined to microorganisms possessingnitrogenase the enzyme system uniquely capable of reducing to


ammonium A m m o n i u m becomes incorporated by N - f i x i n gorganisms into amino acids, protein and other N-containing molecules, thusproviding the p r imary source of organical ly-bound N for growth.Functioning of in reducing N is costly in terms of energy inputs in theform of reductant and adenosine triphosphate (ATP) (see Pate and Layzell1990), while effective funct ioning of is also compromised by its highsensi t ivi ty to oxygen Accordingly, where microorganisms fix infree-l iving state, the process occurs most e f f ic ien t ly under near anoxiccondit ions - as applies for example, to the obligate anaerobe Clostridiumwhen operating in waterlogged soils or to a range of anaerobic bacteriafixing symbiotically in the guts of various animals such as termites andhigher animals. Where high external levels prevail, as in the case ofenvironments support ing the free-l iving aerobic nitrogen fixers such asAzotobacter, their systems are protected by high rates of cellularrespiration in conjunct ion with a number of other biochemical mechanismsthat increase diffusive resistance to

This chapter w i l l be deal ing exclusively with symbioticassociations in which a particular bacterium (Azospirillum, Bradyrhizobium,Rhizobium), actinomycete (Frankia) or member of the cyanophyta (Nostoc,Anabaena) are housed in specialised nodule-like structures produced onroots or occasionally on stems of a higher plant host. In each of these cases,inhibition by is overcome by anatomical factors which restrict entry ofoxygen into infected tissues and strategic forms of internal packaging of themicrosymbiont which further inh ib i t diffusion of to sites where theis functioning. Additionally, the symbiotic organs in question are providedwith effective phloem transport systems for del ivering photosyntheticproducts of the host to the enclosed microsymbiont. Readily metabolizablesubstrates are thereby c o n t i n u o u s l y avai lab le for ass imila t ion of thepotentially toxic released by the partner to its host. As ageneral rule, recently assimilated nitrogenous solutes are immediatelyexported to above ground parts of the host plant through the xylem andtranspiration stream of the plant. Different host species employ specific setsof N-rich solutes such as ureides, amides or citrull ine to effect such transfer(Pate and Atkins 1983; Pate and Layzell 1990).

Symbiotic N fixation collectively results in high overall efficiency, afeature no doubt contributing to the much greater inputs of N made by suchsystems on a global basis compared to free- l iving fixers (Jenkinson2001). As further tes t imony to the efficiency of operation of symbioticassociations, nodulated annua l grain legumes such as peas, lupins or cowpeagrown totally dependent on the atmosphere for their N supply carry only 5-10% of their total plant fresh weight as nodules, yet can still generate fixedN from these nodules at rates mainta ining near maximum growth over the

47Symbiotic nitrogen fixation

life cycle of the host plant (Layzell et al. 1984; Pate 1999). In so doing,fu l ly functional nodules regularly consume in the order of only 10-15% ofthe current net photosynthate produced by the shoot system.

2.1 Cycad:Cyanophyta symbiosesCycads are frequently referred to as living fossils since the few specieswhich we see today represent sparsely distributed relics of a group whichextended back to the early Permian (Hill 1998b) and clearly comprised adiverse and major component of vegetation during Mesozoic times. Thefossil record (see references listed in Jones 1993; Hill 1998b) depicts cycadsas a highly distinctive monophyletic group with many widely distributed andprobably common genera and species, most of which are not represented inthe cycad floras which we see today. Nevertheless, the scattered present daydistribution of l iv ing forms across the tropics and subtropics of all majorcontinents of both hemispheres speaks clearly of a remarkably ancient andfascinating Gondwanan heritage.

According to recent accounts (Stevenson 1992; Grobbelaar 1993; Jones1993; Hill 1998a, b) 10 genera and about 250 species of living cycads arecurrently known, comprising three families, Cycadaceae, Stangeriaceae andZamiaceae, whose modern distributions probably indicate disjunctionsdating back to fragmentation of Pangaea in the middle of the Mesozoic (Hill1998b). Cycas, with some 80 species and the only genus of the Cycadaceae,is generally regarded as the most primit ive and of Laurasian origin, withmajor lineages or ig inat ing in Asia and certain members subsequentlymigrating to Austral ia (H i l l 1998a). The Stangeriaceae consist of themonospecific genus Stangeria (South Africa) and three species of Bowenia(Australia). Zamiaceae contains all remaining living genera of cycads,namely Ceratozamia (11 species), Chigua (2), Dioon (10), Encephalartos(~50), Lepidozamia (2), Macrozamia (~40), Microcycas (1) and the largestgenus Zamia (~60 species). Encephalartos is strictly South African,Macrozamia and Lepidozamia are confined to Australia, whereas theremaining genera (Dioon, Ceratozamia, Chigua, Zamia and Microcycas) areall essentially central American. Again, it would appear that Laurasian andGondwanan elements contribute to present day distribution (Hill 1998a).

According to l i s t ings provided by Grobbelaar (1993), Lindblad andcolleagues (Pate et al. 1988; Paulsrud et al. 1998, 2000) and anecdotalobservations made by or given to the author, virtually all cycads are capableof regularly forming symbiotic associations with filamentouscyanobacteria. This applies regardless of whether host plants are in naturalhabitat or raised in pot culture.

2. Biodiversity, distribution and general biology of the associates

Microorganisms in Plant Conservation and Biodiversity48

The structure in which nitrogen fixation occurs in cycads is termed a‘coralloid root’ in view of the densely packed, dichotomously-branchedroots of which it is composed. Coralloid roots form on cycad seedlings (e.g.Macrozamia spp.) by infection of a forerunner structure termed a ‘pre-coralloid’ root (see Ahern and Staff 1994). A pair of such roots arisesspontaneously from just below the cotyledonary node and grows upwards(apogeotropically) to the soil surface (McLuckie 1922). Formation of pre-coralloid roots can take place under aseptic conditions (Nathanielsz and Staff1975b; Lamont and Ryan 1977; Webb et al. 1984) and, unless cyanophytesymbionts are present, coralloid root formation fails to take place (J.S. Pateunpubl ished) . The apogeotrophic nature of the roots suggests apneumatophore-like func t ion , possibly reflecting an ancestral situationwhere cycads inhabited swamps and seasonally-waterlogged areas.Acquisition of symbiotic may therefore have arisen secondarily,fo l l owing casual i n v a s i o n of the ‘pneumatophore’ by f ree - l iv ingcyanobacteria.

Replacement sets of coralloid roots form periodically during the life ofa cycad, whether from the base of the main body of the root-stock, or inolder plants, at distance along lateral roots (Grobbelaar 1993). In eithercase, infection wi th cyanobacteria must take place at the soil surfacefollowing emergence of apogeotropic roots.

The cyanobacterial symbiont of coralloid roots is intercellular andharboured in specialised, loosely packed cells in the mid-cortex of the root.The densely packed algal filaments are readily recognisable macroscopicallyby their dark green colour and are surrounded by copious mucilage (seeliterature reviewed by Grobbelaar 1993; Ahern and Staff 1994).Proliferation of new filaments leads to progressive invasion of newly formedcortical tissue close to the apex of the coralloid roots. Young filamentsclosest to the apex are typical ly long and show similar ratios of vegetativecells to thick-walled heterocysts as are encountered in comparablefree-living material of the same cyanobacterium. However, ratios ofvegetative cells to heterocysts and total number of both types of cells perfilament decrease with distance back from the root apex until , in regionswhere is most active, f i laments are relatively few celled and maybe composed of almost as many heterocysts as vegetative cells (Nathanielszand Staff 1975a,b; Lindblad et al. 1991).

Growth of cycads is generally recorded as being very slow, with largespecimens in nature probably 1000 years of age (see Giddy 1974;Grobbelaar 1993; Jones 1993; Pate 1993). Plants are strictly dioecious withleaf production and subsequent reproduction markedly stimulated by fire insome taxa (Grove et al. 1980; Orndorf 1985; Pate 1993). Where populationsof Macrozamia riedlei in jarrah (Eucalyptus marginata) forest are exposed


to frequent fire, mature females fail to form cones following each burnwhereas male counterparts typically cone after even very frequent fires (Pate1993; J.S. Pate unpublished).

Pollination of cycads was ini t ia l ly considered to be wind pollinated (seeHill 1998a) in view of the copious release of pollen (microspores) from malecones and the frequent finding of female cones bearing heavy deposits ofpollen presumably originating from adjacent male plants. However, sincecone scales fail to open fully and ovules awaiting fertilisation are deeplyenclosed, superficial deposits of pollen are unlikely to be effective. It is nowincreasingly evident that insects may be involved in pollination of cycads(Norstog and Fawcett 1989; Jones 1993; Hill 1998a). For example, a snoutweevil has been shown to poll inate Zamia furfuracea (Norstog 1987),species of weevil poll inate Macrozamia communis and Lepidozamiaperoffskyana (Jones 1993), and a weevil and beetle pollinate Zamia pumila(Tang 1987). Cycad cones are generally thermogenic, that is, they achievean intense climacteric in their respiration at their maturity, accompanied by asubstantial rise in cone temperature and emission of chemically-distinctiveodours. These features may be attractive to specific pollinating agents.Starch reserves within female cones provide a food source for developinglarvae or beetles in the case of some of the above cycads.

Cycad seeds t y p i c a l l y carry attractively-coloured edible coatssurrounding their thick-walled seeds. The starchy kernel of the seed isusually highly poisonous and where indigenous human communities ofvarious geographical regions consume seeds of native cycads, varioustreatment procedures are tradi t ional ly employed. Even so, carcinogeniccompounds are unlikely to be removed and long-term effects on health mayensue. A number of cases are on record of poisoning of humans afterconsumption of untreated seeds as emergency sources of food, e.g. Cycasspp. during the Japanese occupation of Guam. Because of their large size,dispersal of cycad seeds is principally implemented by large megafauna suchas hornbills and baboons (Africa) (Grobbelaar 1993), bandicoots and emus(Australia) (Pate 1993) and fru i t bats (USA) (Tang 1989). Germinationtypically occurs on the ground surface and may be delayed for a year ormore in certain cases, presumably due to an obligate after-ripening period.The large reserves of cycad seeds and prompt formation of coralloid roots onseedlings result in high rates of growth of juvenile plants and earlyachievement of N sufficiency, whether in native habitat or pot culture (Jones1993, Pate unpublished data).

The cyanobionts recoverable from coralloid roots of cycads aregenerally classed as Nostoc, or less frequently as Anabaena (Grobbelaar1993). Usually only one strain of microsymbiont predominates in a specificcoralloid root. Recent PCR-based molecular analyses of the genetic


constitution of freshly isolated symbionts show (UAA) sequencessimilar to free-living Nostoc and to corresponding cyanobionts of certainlichens (Paulsrud et al. 2000) and two bryophytes (Costa et al. 2001).Similar analyses have revealed diversity in host specificity even amongisolates of host genera obtained from botanic garden settings (Paulsrud et al.1998). Bearing in mind the difficulty of identifying different strains/speciesof Nostoc purely on the basis of cultural and morphological characteristics,re-examination of earlier work (e.g. Grobbelaar et al. 1988; Marshall et al.1989) indicating taxonomic diversity of symbionts and specificity orpromiscuity of cyanobiont:host relationships is clearly required usingmolecular-based approaches. Nevertheless, there is already sufficientevidence that fu l ly effective symbioses can be established on a single hostusing any of a broad range of Nostoc strains or species. Furthermore,coralloid roots form readily on cycads raised from seed using non-sterile soilbased pot culture. With inoculation rarely practised in such situations, localrooting substrates must have provided cyanobiont strains which can engageas effectively in symbioses as occurs naturally in soils of the habitats fromwhich the cycads were derived.

2.2 Cyanophycean (Nostoc) symbiosis with GunneraThis association provides the only known case of an algal-basedin Angiosperms. Unlike lichen, liverwort, fern and cycad associations, thecyanobiont of Gunnera is intracellular and the mode of infection and cellularfunctioning of the symbiotic structures exhibit an interactive complexityequalling that of nodules of actinorhizal and Rhizobium-based symbioses.The reader is referred to the review of Becking (1977) for early literature onthe geographical distribution and ecology of Gunnera and more specificstudies indicating that symbiotic occurs at high frequency acrossthe 50 or so species of the genus.

Gunnera is the sole genus of the Gunneraceae which is distributedacross the tropical and subtropical Southern Hemisphere, including regionsof South America, New Zealand, SE Asia and South Africa. The genus isassociated with wet conditions, sometimes at high altitudes where the plantoften comprises a pioneer element of disturbed habitats such as landslidesand areas adjacent to volcanic activity.

The Gunnera symbiosis is relatively easy to manipulate underlaboratory conditions and has resulted in a detailed understanding of themorphological basis of infection (see reviews of Bergman et al. 1992, 1996).The microsymbiont is always a Nostoc sp.

Infection occurs through pre-formed pairs of glands at the junction ofeach leaf with the stem. Carbohydrate-rich mucus secreted by the


glands appears to attract a number of microorganisms in addition toinfective cyanobionts.Proliferating first on the gland surface, the cyanobacterium then enters amotile infective stage. The hormogonia involved invade the gland by anintercellular route and eventually accumulate in great quantity within anextracellular cavity at the base of the gland.Presumably due to some form of mitotic stimulus emanating from themicrosymbiont, new host cells are initiated close to the gland and it isthese which become specifically invaded by the Nostoc. Achievementof an in t race l lu lar location for the microsymbiont is clearly hostdetermined, since the same strain of cyanobacterium wil l remainintercellular, yet s t i l l capable of when in symbioticassociation with non-angiosperms such as Azolla and liverworts.Once inside the cells of the ‘nodule’ of Gunnera, filaments of thecyanobiont d iv ide , the i r vegetative cells enlarge, andheterocysts differentiate.

activity achieves a maximum as intracellular infections spreadthrough the nodule, with up to 65 to 80% of the cells of the filaments ofthe symbiont found to consist of heterocysts as opposed to vegetativecells.

2.3 Actinomycete (Frankia) symbioses with non-legume Angiosperm taxaRepresentatives from eight different families encompassing 24 separategenera are current ly classed as actinorhizal . The total species resourceinvolved is over 200 in number (Table 1), although presence of actinorhizalnodules has yet to be confirmed for some genera. A number of instances(e.g. in Casuarina, Dryas, Myrica) are on record where nodulation has beenobserved in certain locations but not others (Bond 1967, 1974; Becking1977; Torrey 1978). Direct proof of activity by feeding oracetylene reducing capacity, has been confirmed for almost all genera, butonly for a relatively small portion of the species known to engage inactinorhizal associations.

Actinorhizal species embody a very diverse range of locations andhabitats, often associated with natural or other types of disturbance. Of thelarger ecologically-important genera, Allocasuarina/Casuarina are foundwidely and commonly in Australia, Gymnostoma occurs in Malaysia throughto the West Pacific, Ceanothus in North America, while species ofElaeagnus are encountered in Europe, Asia and North America, and Alnusand Myrica are broadly spread across the northern hemisphere with somespecies in South America and South Africa respectively.

Various inven tor ies of ecological and physiological features ofactinorhizal species (see Becking 1977; Torrey 1978; Dawson 1990; Benson


and Silvester 1993) suggest a col lect ive global range of habitat associationswhich may be regarded as r i v a l l i n g in breadth that of the hundred-fold largerspecies resource of legumes. Herbaceous and annual forms are essentiallyabsent among actinorhizal plants but members of certain genera (e.g. Alnus,Ceanothus, Comptonia, Dryas, Myrica), act as important pioneercomponents of natural habitats such as mobile sand dunes, deposits left byretreating glaciers and mineral-r ich soils exposed following landslides orfire. These attributes, combined with the abil i ty of certain species to performwell in saline and dry or acid permanently waterlogged environments haveprompted fairly widespread use of act inorhizal taxa for rehabilitation ofmine sites, seaside plant ings and reforestation of eroded soils at high altitude(Wheeler and M i l l e r 1990). Some taxa (e.g. Casuarina, Alnus) are also


exported for t imber or fuel alongside woody legumes in agroforestryenterprises. There are a number of reports of substantial amounts of fixed Nbeing returned under both natural or cultivated conditions by taxa such asCasuarina, Alnus, Hypophae and Ceanothus (see Becking 1977; Benson andSilvester 1993).

Phylogenetic studies, especially recent molecular-based cladisticanalyses, continue to shed light on the possible origin of actinorhizal andother higher plant-based symbioses. The molecular-based (chloroplast generbcL) perspective recently provided by Doyle (1998) indicates that capacityfor nodulation and associated probably originated on severalindependent occasions during evolut ion of angiosperms and that closerrelationships between legumes and actinorhizal taxa are now evident thanwere previously suspected. The scheme presented by Doyle (1998)(incorporated into Figure 1) places all known actinorhizal Angiospermswithin the single relatively small ‘Rosid I’ group of families which alsoincludes rhizobial symbioses between legumes and the non-legume genusParasponia (Ulmaceae). The analysis thus supports a general predispositiontowards nodulation within this group (Soltis et al. 1995; Swensen and Mullin1997). Parallel examination of actinorhizal plants by Swensen (1996), usingrbcL sequence data supports a cladistic tree embracing four distinctgroupings of actinorhizal taxa. One group comprises three families,Betulaceae, Casuarinaceae and Myricaceae alongside three other non-nodulating families, the second group consists of the family Rosaceae whichcontains mostly non-nodulat ing and a few actinorhizal genera, the thirdgroup comprises the actinorhizal families Coriariaceae and Datiscaceaetogether with three non-nodula t ing families. The final group includesElaeagnaceae and Rhamnaceae, both of which contain nodulating and non-nodulating taxa.

Actinorhizal nodules are essentially modified rootlets which typicallydevelop dense aggregate co ra l lo id - l ike structures through repeateddichotomous branching of root apices. Nodules can be long lived andachieve diameters exceeding 5 cm under field conditions (Schwintzer andTjepkema 1990). In waterlogged habitats (e.g. in cases involving species ofMyrica, Casuarina and Alnus) nodules may be located close to the surface oremerge distinctly above the rooting substrate or may even form on trunkswhen trees are flooded (e.g. Casuarina spp., Duhoux et al. 1993). In sharpcontrast, nodules are normally absent from surface soil layers in dry habitats,albeit in some cases recoverable in appreciable amount where excavationsare made deep down a soil profile.

The early literature (see review by Becking 1977) records that invasionof roots of ac t i no rh i za l p lan t s takes place via root hairs, and that

characteristic hair curling precedes infection, just as in the case of therhizobial infection sequence of many legumes. In Alnus spp., invasion ofroot hairs leads to a ‘pre-nodule’ being formed by proliferation of underlying



pericycle tissues, just as occurs during the initiation of regular lateral rootson uninfected plants. This clearly confirms that the symbiotic organ isessentially a modified root. Intracellular infection of cortical cells of thepre-nodule leads to hyphae of the microsymbiont becoming transformed intothick-walled, internally septate ‘vesicles’ and this is where nitrogen fixationis generally assumed to be located. The same early work (see Bond 1974;Becking 1977) describes two types of nodules, one where constituentinfected roots extend slowly and then stop growing (the Alnus type), thesecond type (Myrica, Casuarina type) where continued attenuatedapogeotropic growth of infected roots results in the dense central body of thenodule becoming invested in a sparse outer clothing of thin uninfected roots.

The review of Berry (1994) summarises some of the more recentliterature on the biology of actinorhizal symbioses, including discussion ofthe biochemical interactions of host and microsymbiont likely to be involvedin the nodulation process. Infection of certain hosts (e.g. species ofElaeagnaceae, Rhamnaceae and Rosaceae) typically takes place by anintercellular route, not via root hairs. The type of infection pathway whichoccurs is clearly not determined by the microsymbiont, since when oneFrankia strain infects two different hosts, invasion may be eitherintercellular or via root hairs, depending on the host which is involved inconsummation of the symbiosis (Miller and Baker 1986; Racette and Torrey1989). Secretions of extra-cellular polysaccharide materials by hosts may beinvolved in recognition and sustenance of Frankia where invasion isintercellular.

After entering the cytoplasm of host cells, the microsymbiont becomesenveloped in a host-derived membrane while still continuing for a time todivide and form further containing vesicles. Berry (1994) suggeststhat the multi-lamellate hopanoid external layer of the vesicle protects the

system of the Frankia against inactivation by Analogies can thusbe drawn between this l ip id layer and the thick wall encapsulatingheterocysts of cyanobacteria or the peribacterial membranes envelopingbacteroids of legume nodules (although there is no direct evidence that thismembrane effects diffusion or protects from inactivation byBerry (1994) identifies onset of N starvation in a host as a causative factor inthe initiation of nodules and then in the promotion of activity inthe resulting vesicles.

Vesicles are absent in actinorhizal nodules of Casuarinaceae sofixation must be accomplished in such cases in unspecialised cells of theendophyte. Interestingly, haemoglobin is encountered at relatively highlevels in Casuarina nodules, just as in Rhizobium-based nodules. In therbcL gene sequence analysis conducted by Swensen (1996), detailedanalysis of the four clades of actinorhizal species referred to earlier (Table 1,


Figure 1) provided some interesting correlations between molecular basedattributes and certain morphological and anatomical features of the nodulesconcerned (e.g. mode of infection, presence or absence of uninfectedapogeotropic roots on nodule surfaces, presence or absence of distinctvesicle-like structures and the shape and location of vesicles, if present, ininfected tissues). Although not providing information for many actinorhizaltaxa, the analysis of Swensen (1996) (see Figure 1) further suggests thatact inorhizal- type symbioses probably originated on several independentoccasions during the evolution of Angiosperms.

The Frankia organism associated wi th various ‘actinorhizal’Angiosperms described above is usua l ly classified as a filamentousprokaryote (Actinomycete), as indicated in i t i a l ly from morphological andcultural characteristics and more recently in terms of the high guanosine andcytidine content of its DNA and sequence analysis of its 16S ribosomal RNAgenes (Berry 1994, Swensen and Mullin 1997). Pure cultures of theorganism were not available u n t i l the studies of Callahan et al. (1978) onComptonia, but now include a broad range of isolates from most genera (e.g.see Kohls et al. 1994; Clawson et al. 1998). Substantial progress iscontinuing on the extent of cross inoculation specificity between strains ofFrankia and various groupings of host taxa. However, interpretation of datais difficult where isolates from a nodule are subsequently shown not to re-infect the same host (e.g. Racette and Torrey 1989) or where pure isolatesfail to re-nodulate a plant host whereas inoculations with crushed nodulesuspensions result in normal nodulation (Kohls et al 1994). Cases are alsoreported where nodules contain several strains of Frankia (Bloom et al.1989) or where isolating media appear to have selected symboticallyineffective strains, while f a i l i ng to support growth of effective ones.Furthermore, some Frankia strains infect hosts which are ecologically ortaxonomically very distant from the hosts of origin and 16s rDNA signaturesof certain strains most closely resemble those of strains infecting distantlyrelated families of actinorhizal species.

The recently developed phylogenetic tree for Frankia and phyleticneighbours (Normand and Bousqet 1989) indicates four clusters of genomicspecies based on 16S rDNA. The first is a group effective on Alnus,Casuarina, Allocasuarina and Myrica, the second on Dryas, Coriaria andDatisca, the third on Eleagnaceae and Gymnostoma and the fourth is a groupof strains isolated from a diverse range of host plants, but are geneticallydistinct from the other clusters.

Evidence indicates that a number of Frankia strains can cross nodulatewithin but not outside specific ‘cross inoculation’ groups of hosts (e.g. seestudies of Clawson et al 1998; Akimov et al. 1990; Rodriguez-Barrueco etal. 1993). Thus, certain ac t inorhiza l hosts may have co-evolved with


specific microsymbionts as indicated by the marked compatibility groupingsof certain Frankia strains for specific host plant groupings.

Genes coding for nitrogenase have now been identified byprobing Frankia DNA. In one case (the nif K gene), high sequencehomology is evident against the same gene from cyanobacteria. This resultand topologies recorded for other nif genes, are regarded by Normand andBousquet (1989) as indicative of ‘illegitimate’ gene transfers betweenFrankia, cyanobacteria and other fixers such as Clostridium. Judgingfrom the much more extensive literature on Rhizobium, symbiotic behaviouris likely to become greatly modified by relatively small changes in genomiccomposition and, if applicable to Frankia, anomalous host patterns ofinfectivity and effectivity across actinorhizal species might be expected toarise relatively easily during evolution of the various symbioses which weknow of today.

2.4 Rhizobial:legume symbioses‘Rhizobial’ and ‘Rhizobium-type’ are terms used to refer to that broadly-based group of nodule-forming bacteria which can fix nitrogen symbioticallywith legumes. In one exceptional case, involving such bacteriaextends to the legume-like nodules formed by the non-legume Parasponia(see next section). The bacteria concerned comprise a vast assemblage offorms, some of highly restricted and others of highly promiscuousnodulating capacities in respect of host species. Furthermore, some arecapable of in the free-living state others only in the protectednear anaerobic environment of the nodule.

The Leguminosae (Polhill and Raven 1981) ranks in size with othermajor Angiosperm families and far exceeds in diversity of life and growthforms and ecological preference similarly large families such asOrchidaceae, Gramineae and Asteraceae. Some 640 genera and 18,000species are recognised and the family is usually treated taxonomically asthree sub-families (Papi l ionoideae, Mimosoideae and Caesalpinioideae).These are distinguished from one another mostly on the basis of differencesin floral morphology. The 31 tribes, approximately 420 genera and 12,000species of Papilionoideae are predominantly herbaceous and containvirtually all agriculturally important pasture and crop legumes, whereas thefive tribes, 64 genera and 2,900 species of Mimosoideae and four tribes, 153genera and 2,200 species of Caesalpinioideae are mostly woody and mostlynot under cultivation. Trees, shrubs and lianas are well represented amongthe constituent taxa of all subfamilies (Sprent 1999).

The current state of knowledge of the extent of nodulation amonglegumes is summarised by Sutherland and Sprent (1993), Sprent (1994,personal communication and see Table 2) and the reader is referred to the


classic early work of Al l en and Al l en (1981 ) around which a largeproportion of the current database has been bui l t . Recent studies ofnodulation, especially of Caesalpinioideae in Brazil (see Faria et al. 1989)include a number of previously unstudied Caesalpinioideae and have provedparticularly useful in clar ifying the situation for woody taxa neglected inearlier work. Sprent (2001) has recently provided extensive listings ofrecords for nodulation and in the process, corrected erroneous earlier recordsand discussed issues relating to groupings where certain members nodulatewhile others apparently do not.

Over one fifth of the total species resource of the Leguminosae has beenexamined in respect of nodulat ion. In the Papilionoideae, where almost twothirds of the genera have been examined. 97% of the species examined haveproved to be capable of nodulation. Notable exceptions are the Dipterygeae(all species so far studied are non-nodulat ing) , Sophoreae and Swartzieae( i n c l u d i n g n o d u l a t i n g and n o n - n o d u l a t i n g genera) (Sprent 2000).Nodulation is also very common (90% of species examined) in theMimosaceae for which two thirds of the genera examined are nodulated, fiveare non-nodulated and for some other genera, both positive and apparentlynegative records have been reported. Puzzling cases of non-nodulation havebeen recorded for Acacia, a genus whose species are mostly prolif ical lynodulated (Odee and Sprent 1992). Records of the extent of nodulation ares t i l l relatively incomplete for Caesalpinioideae. However, all species so farexamined in 13 genera of the subfamily were found to be nodulated, whilenodulation was apparently absent in a further 38 genera, and in another 20genera both nodulated and non-nodulated species were encountered. It thusappears that members of the Caesalpinioideae are predominantly notnodulated.

From the earliest observations of nodule morphology (e.g. see Spratt1919; Fred et al. 1932; Allen and Allen 1981), effectively fixing noduleswere shown to vary cons i s ten t ly between genera and large taxonomicgroupings in terms of shape, pattern of growth and longevity of nodules.Corby (1981) provided the first comprehensive account of such variation ina classification of nodules on the basis of whether they were branched,essentially non-branched or exhibited an essentially dimorphic morphology.Taxonomic aff i l ia t ions were implied in designations such as ‘astragaloid’,‘crotalaroid’ and ‘lupinoid’ among the branched types of nodules and‘aeschynomenoid’, ‘desmodioid’ and ‘mucunoid’ among the mostlyunbranched types of nodules. Correlations between host taxonomy andnodules type have subsequently been borne out to a certain extent wheremore extensive groupings of legumes in terms of types of nodules have beenconsidered (see Sprent 1981 and more recent treatments of Sprent et al.1989; Sutherland and Sprent 1993; Doyle 1998; Table 2).


Sprent (1981) first drew attention to the prevalence of legumes whichproduce ureides (allantoic acid and allantoin) as exportable products of fixedN among taxa (most ly Phaseoleae) which develop relatively largeunbranched, determinate nodules . Conversely, taxa with branchedindeterminate nodules and a number with small unbranched nodules arewidely known to produce amides (asparagine and to a lesser extentglutamine) when exporting fixed N (Pate and Atkins 1983; Pate 1986).

Nodule ini t ia t ion may also involve rhizobia invading root hairs, rootepidermis, or sites of emergence of lateral roots. In some unusual cases (e.g.species of Aeschynomene and Sesbania), nodules may form on stems.Again, there is good evidence of taxonomic consistencies in suchcharacteristics (Sprent 2000).

The reasonably substantial fossil records of legumes (see Herendeenand Dilcher 1992) suggest an origin in the Cretaceous with the extant sub-fami l ies already wel l represented by the early Tertiary. Classicmorphologically-based analyses (see review of Polhill and Raven 1981;Crisp and Doyle 1995) delineate most of the orders recognised today, but arenot fu l l y supported by recent cladistic analyses based primarily onmorphological (see articles in Crisp and Doyle 1995) or molecular basedcriteria such as rbcL gene analysis (see Doyle et al. 1997).

Using rbcL sequence data, Doyle (1998; figure 2) suggests threeindependent origins for nodulation and the phylogeny proposes unbranchedintermediate ‘caesalpinioid’, nodules to be the ancestral nodule type, basal toall three or ig ins . Modi f i ca t ion of this basic nodule type in thePapil ionoideae is then suggested to have given rise to indeterminate


branched nodules, some of lupinoid type with a peripheral meristem, or todeterminate nodules of aeschynomenoid type (e.g. Arachis) or to thedesmodioid, ureide-producing nodules of many Phaseoleae (see Table 2).

2.5 The Rhizobium-Parasponia symbiosisThis unusual class of symbiosis is the only one so far known betweenRhizobium (sens. lat.) and a non-legume (Parasponia). It was first describedby Trinick (see reviews of Trinick 1982; Trinick and Hadobas 1988), whoi n i t i a l l y referred to the host concerned as a species of Trema. This closelyrelated sister genus to Parasponia is now considered non-symbiotic.Parasponia is a member of the family Ulmaceae and its five species inhabitthe Malay / West Pacific region (Mabberley 1997). Species of Parasponiaprefer disturbed habitats and nodulate well at low pH (Trinick and Hadobas1988). Nodules of Parasponia resemble those of actinorhizal species inbeing essentially modified roots with a central vascular cylinder surroundedby a cortical region of infected tissues (Trinick 1979).

Swensen (1996), in an evaluation of actinorhizal symbioses based onrbcL sequence analyses of hosts, places Parasponia in a clade together withthe actinorhizal fami l ies Elaeagnaceae and Rhamnaceae and a number ofnon-ac t inorhiza l hamamel id f ami l i e s . In a further comparison withactinorhizal genera, Swensen (1996) suggests that the intercellular pattern ofbacterial invasion and absence of apogeotrophic root extensions fromnodules in Parasponia find counterparts within two of the four clades (I andII) of Frankia-based actinorhizal species. Conversely, the presence in thenodule of Parasponia of an outer anatomical barrier restricting access tothe nodule and appreciable levels of haemoglobin in infected tissues ofnodules of this symbiosis finds parallels in the actinorhizal taxaCasuarina/Allocasuarina, (Clade IV of actinorhizal family representatives).

Trinick (1980) and Trinick and Hadobas (1988) show that both slowand fast growing rhizobia can nodulate Parasponia while also engaging ineffective symbioses with certain groups of tropical legumes. Interestingly,the bacterial phylogeny recent ly proposed by Doyle (1998) provides anexample of a single Rhizobium strain (NGR 234) exhibit ing a very wide hostrange, inc luding a diverse assemblage of legumes and Parasponia.

2.6 Rhizobial associates of legumes and ParasponiaThe literature concerning nodule bacteria isolated from legumes spans morethan a century, although studies up to the 1960’s deal almost exclusivelywith agriculturally-important grain and forage legumes (e.g. see Fred et al.1932). Incentive for such studies related principally to the introduction ofmostly European legumes into farming regions of the world where soils didnot u s u a l l y c o n t a i n c o m p a t i b l e i n d i g e n o u s rhizobia . Alongside



morphological descriptions of isolated bacterial colonies and whetherisolates from nodules were fast or slow growing, early descriptions ofrhizobia were concentrated part icularly on cross inoculation specificity, inthe hope of predicting the likelihood of success when inoculating cultivatedlegumes under f ie ld cond i t ions . Grading of strain effectiveness whenmatched with distantly and closely related host genotypes and persistence ofintroduced strains in competition with indigenous rhizobia were equallyimportant practical research issues, when attempting to fine tune formaximum symbiotic fixation returns under specific situations.

Through use of sophisticated means for characterising rhizobia bymolecular-based techniques such as mapped restriction site polymorphism(MRSP) analysis of 16s rRNA genes, and PCR/DNA fingerprinting usingrepetitive sequences (REP-PCR), concepts concerning the genetic diversityof rhizobial symbionts are being cont inua l ly revised and augmented.Included in such assessments have been an increasing number of studies onsymbionts of nat ive legumes (e.g. see Jordan 1984; Dobereiner 1984;Lieberman et al. 1985; Roughley 1987; Galiana et al. 1990; Sutherland andSprent 1993; Laguerre et al. 1997; Haukka et al. 1998; Lafay and Burdon1998, Burdon et al. 1999; Thrall et al. 2000). Particularly relevant reviewson these issues are provided by Padmanabhan et al. (1990), Young (1991,1996), Sprent and Raven (1992), Sutherland and Sprent (1993), Bryan et al.(1996), Doyle (1998) and Young et al. (2001). The key findings of thesestudies are:

Six ‘genera’ of n i t r o g e n - f i x i n g bacteria are currently recognised(Azorhizobium, Bradyrhizobium, Mesorhizobium , Phyllobacterium,Rhizobium and Sinorhizobium) and earlier ‘species’ concepts have beensubstantially altered (Young et al. 2001).Phylogenetic studies based on 16S ribosomal RNA gene sequencessuggest that rhizobia may have arisen from several major lineages ofproteo-bacteria, with affinit ies in some cases with non-symbiotic formspreviously recognised as belonging to a separate genus, Agrobacteriumbut now included in Rhizob ium (Young et al. 2001). Althoughevaluating only a small fraction of the known taxonomic resource ofrhizobia, the above studies increasingly suggest a poor level ofcorrelation against earlier-established cross inoculation groupings. Thisis not surprising when one remembers that in many cases, the host rangeof a rhizobium is plasmid-borne and therefore potentially interchange-able with other compatible bacterial strains co-existing in free-livingstate outside the host legume and classifications must consider this(Young 2001).There is evidence suggesting that characteristics displayed by manytropical rhizobia, inc luding those associated with woody taxa of native

Symbiotic nitrogen fixation 63

habitats, correlate poorly in terms of culture morphology and growthrates, host ranges and molecular-based attributes when matched againstrhizobia isolated from mostly temperate herbaceous legumes ofagricultural significance. Thus, rhizobia of native taxa showingcharacterist ics in te rmedia te between those of Rhizobium andBradyrhizobium, may exhibit complex grades in degrees of effectivenessacross host groupings (as shown for example for Acacia spp., Burdon etal. 1999), and may also inc lude slow-growing isolates exhibitingunusually narrow host specificity. Molecular and biochemical evidencesuggests that this is most l ikely due to directed mutagenesis alteringmany of these ‘specificities’.

These studies will clearly prove vital to improving our understanding of thedynamics of bacterial populations and their capacity to co-evolve with hosts.

2.7 Nutrient acquisition strategies of symbiotic associations inrespect of limited availability of phosphorusWhile there is still continued debate world-wide as to which of the elementsessential to growth and surv iva l of higher plants are likely to constituteprimary l imitants to productivi ty of natural ecosystems, N or P feature mostprominently in the literature in this respect. Thus, N is frequently identifiedas l imit ing in temperate Northern Hemisphere regions and certain youngsoils of tropical regions (Vitousek and Howarth 1991), whereas P is morecommonly implicated in older heavily leached old landscapes of continentssuch as Australia, South America and Africa (Reddell 1993; Crews 1993,1999; Newman 1995). The situation is further complicated by ecosystemsappearing to switch in time between phases of limitation by P and N, asreported by Raich et al. (1996) for lava flows in Hawaii and referred to laterin respect of post-fire recovery of ecosystems of south west WesternAustralia.

Alongside evidence of P as a regular limitant, one should also considerthe confusing literature concerning the basic nutritional role of the elementin symbiotic associations. Some accounts (e.g. Robson 1983;Israel 1987; Crews 1993; Reddell 1993) indicate that requirements for P bysymbiotically-active legumes are measurably greater than those offixing counterparts or comparable non-legumes. However, as discussed byReddell (1993), this may merely denote a catalytic effect of P on nodulationand early growth, with benefits then flowing automatically under N limitedsi tuat ions where companion non-legumes would be specificallydisadvantaged. In any event, there should be strong selection pressure inboth and non-f ix ing plants for effectiveness in capture of limitingresources of P, as is indeed regularly practised using specialised mechanisms


for accessing intractable pools of the element not available to unspecialisedroot systems of other species.

By far the most widely deployed strategy for combating limitation inrespect of P involves symbiosis with various forms of mycorrhizae (e.g. seeAllen 1992; Smith and Read 1997; Brundrett and Abbott this vol.; Egerton-Warburton et al. this vol.) . Alternatively, improved access to P can beachieved by non-mycorrhizal ‘proteoid’ or ‘cluster’ roots (Purnell 1960; Lee1978; Lamont 1982; 1993 and recent reviews of Dinkelaker et al. 1995;Skene 1998; Pate et al. 2001; Pate and Watt 2001).

Not surprisingly therefore, there is now overwhelming evidence that thevast majority of symbioses regularly employ ‘dual’ symbioticsystems, namely their regular microorganism and some form ofmycorrhizal partner. The mycorrhizal elements most commonly involvedamong herbaceous and woody legumes are endophytic AM (arbuscular) typemycorrhiza but ectomycorrhizas may feature in certain woody legume taxa(e.g. Aziz and Sylv ia 1993; Herrera et al. 1993; Sprent 2001). Amongactinorhizal species, certain taxa. (e.g. Gymnostoma, Casuarinaceae) producemycorrhizal root nodules colonised by arbuscular mycorrhiza (Duhoux et al.2001) addi t ional to regular nodules. Incidentally, similarmycorrhizal structures are known to occur on certain gymnosperm taxa andwere erroneously concluded in the early literature to be capable of fixing(see McLuckie 1923; Bond 1963, 1967; Morrison and English 1967;Becking 1977).

3. Quantification of the likely returns of fixed N by nativeassociations3.1 Assessing symbiotic competenceThe underlying premise of this chapter is that symbiotic associationsbetween microorganisms and higher plants constitute keybeneficial components by providing fixed atmospheric to themselves andeventually to other organisms of their ecosystems. The first step towardstesting this supposition is to demonstrate visually that the putativeof a system do indeed bear symbiotic organs and that these appear likely tobe currently active in Potential symbiotic activity would beindicated, for example, by presence of green, cyanobacteria-filled in mid-cortical tissues of the coralloid roots of cycads, by healthy arbuscule-containing infected tissues in actinorhizal symbioses or by haemoglobin-filled bacterial tissues in nodules of legumes.

The second step would be to attempt to determine what proportion oftotal plant mass is devoted to symbiotic structures during different seasonsor over a whole year. For example, if the complement of nodules on a plantwere to regularly comprise less than 1% of current plant fresh weight, and


nodules were recorded as absent or unhealthy for much of the year, onewould be safe in concluding that the association was only minimallydependent on atmospheric Conversely, heavy reliance on fixation wouldbe expected if proportional mass of healthy symbiotic organs were regularlyin the range 5-15% of p l an t fresh weight, as observed for example in manyagr icu l tu ra l ly - impor tan t legume crops and pasture species under N-limitingconditions (Pate 1977; Sutherland and Sprent 1993). However, it should beremembered that greater proportional weights of symbiotic organs would beexpected of rapidly growing host plants than if plant growth was beingsuppressed by environmental constraints.

The third step would be to determine by suitable chemical assay thespecific activity in of symbiotic organs harvested sequentiallyfrom a population of the species across a season of study. By combiningsuch informat ion on act iv i ty with corresponding data for seasonalchanges in weight of symbiot ic organs, it should then become possible topredict with a reasonable degree of accuracy how much is being fixedover a particular time frame and environmental situation.

The f inal step would be to assess the absolute amounts and proportionsof the total biomass of an ecosystem comprised by the symbiotic associationunder study, and thereby assess the annual inputs of fixed N which it isl ikely to be making per u n i t habitat area per year. One would thus achievethe ult imate goal of i den t i fy ing the likely quantitative importance of suchinputs towards meeting the long-term demands for N by all interacting biotaof the system.

Fu l f i l l i ng this step-wise assessment of returns within nativeecosystems is both time consuming and logistically difficult, even in thoserelatively few cases where un i fo rm populations of the symbiotic associationare present and where re la t ive ly complete recoveries of symbiotic organs canbe made from the host p lants concerned. Not surprisingly therefore, ful lycomprehensive evaluat ions of benefits of symbioses have only been rarelyaccomplished for na t ive ecosystems (see Sutherland and Sprent 1993),despite many reported successes in respect of crops and pasture herbaceouslegumes of agricul tural systems (Pate and Unkovich 1998) and for somewoody leguminous taxa of p lanta t ion and agroforestry systems (seeUnkovich et al. 2000; Peoples et al. 2001).

Access to reliable and easi ly applied assay systems for measuringnitrogenase act ivi ty is obviously pivotal to precise quantification ofinputs of fixed Three contrasting methodologies have been employedfor this purpose over the years and the merits and limitations of each will beaddressed below.


3.2 Acetylene reduction assay techniqueDespite many inadequacies, the reduction assay method remains ofconsiderable value f o r in i t i a l testing of whether a newly discovered system iscurrently active symbiotically and, in broader context, for comparing on asemi-quantitative basis the extent of variation in activity betweenspecies, habitats and seasons (though errors can be 2-3 fold). For example,studies conducted in mediterranean-type ecosystems of southern Australiainvolving shrub legumes of genera such as Acacia, Aotus, Bossiaea,Dillwynia, Jacksonia, Kennedia , Platylobium and Viminaria collectivelyhave shown that nodulation cycles are strongly seasonal, with peak specific

reducing ac t iv i ty in spring, loss of nodules with onset of the summerdrought conditions and ini t ia t ion of new symbiotic organs is normallydelayed until the fol lowing wet season (e.g. Grove et al. 1980; Lawrie 1981;Monk et al. 1981; Hingston et al. 1982; Langkamp and Dalling 1982;Walker et al. 1983; Hansen et al. 1987a,b; Sutherland and Sprent 1993; Pateand Unkovich 1998). Where symbiotic performances of short-livedunderstorey shrub legumes have been followed after recruitment from seedafter fire, greatest dependence on atmospheric (even up to 70-80%) istypical ly evident in j u v e n i l e stages when phosphorus is transiently readilyavailable. Then, as these legume stands approach middle age and resourcesof the element become cr i t i ca l ly l i m i t i n g , symbiotic dependence tends todecline sharply and may even cease ent i re ly (see case studies reviewed byPate and Unkovich 1998). In one notable case (Hansen and Pate 1987a,b),mid aged, mostly non-nodulated legume stands in native south west WesternAustralian eucalypt forest were found to develop prolific, symbiotically-active sets of nodules when phosphate was applied, indicating that innutrient impoverished ecosystems of this type, symbiotic performance isnormally l imited by phosphorus supply. By contrast, growth and fixationcontinued unabated and at much faster rates in comparably aged stands ofthe same species sown on phosphate-fert i l ised rehabilitation plots wi th inmined areas of the same forest.

Turning to cyanophycean:cycad symbioses, acetylene reduction assaysof coralloid roots, combined in certain cases with direct val idat ion offixation by have recorded appreciable nitrogenase activi ty occurs inapproximately one f i f th of the world’s recognised cycad species (Grobbelaar1993), albeit mostly in respect of pot grown plants. A reduction basedstudy of two naturally growing populations of Macrozamia riedlei in nativemixed Banksia, Eucalyptus, Allocasuarina woodland near Perth, WesternAustralia (Halliday and Pate 1976) provided estimates of 18.8 and 18.6 kg N

in situations where the cycads comprised the dominant understoreyelement. Incorporating in fo rma t ion on population densities, mean biomassand nitrogen contents, these cycad populat ions were suggested to be f ix ing


nitrogen at rates capable of doubling the N content of plant biomass roughlyevery 10 years (see further discussion by Pate and Unkovich 1998).

Finally, the acetylene reduction technique has featured prominently inquali tat ive val ida t ions of act ivi ty conducted across a diverseassemblage of genera and angiosperm families known to form actinorhizalnodules (e.g. see reviews of Quispel et al. 1993 and Subba Rao 1993). Inmost of these cases appreciable rates of symbiotic were indicatedunder natural condi t ions or where species were being employed inrehabilitation of degraded areas.

3.3 natural abundance (NA) assays of symbiotic dependenceTo illustrate some of the failures and successes of the assay methodwhich have been obtained in their studies, Pate and Unkovich (1998) cited aset of equivocal results obtained in their study of two populations of thecycad Macrozamia fraseri and companion woody vegetation in heathlandsnear Eneabba, Western Australia. Comparisons of values for nonfixing reference species and the respective cycads then suggested thatsubstantial levels of fixation appeared to be occurring in one population butnot in the other. However, effective symbiosis was strongly suspected forboth populations, since each showed prolific sets of healthy coralloid rootson virtually all cycads excavated.

In contrast to the above, Pate and Unkovich (1998) also refer to asupposedly well validated result obtained by Tennakoon et al. (1997) in astudy of N interrelat ionships of the xylem-tapping root hemiparasiteSantalum acuminatum and its woody hosts in coastal heath near Dongara,Western Australia. The resulting data showed close matchings of theisotopic signatures of the Santalum and the various cohabitingassociations (Acacia spp. and Allocasuarina sp.) at the sites studied, whereasother less parasitised or non-parasitised non N-fixing taxa showedsignificantly more positive values. With values of heavily parasitisedlegumes and of the matching sets of Santalum close to zero of theatmosphere), compared to +1 to +4 %° for other hosts, dependence of thelegumes and the parasite on fixed N was considered highly likely.Excavations examining the intensities of formation of haustoria on thevarious hosts exploited by the Santalum corroborated the above conclusionby demonstrating highly biased parasitism by Santalum, and indeed otherparasitic species, towards the nitrogen-fixing associations.

A second equally definitive study using signatures is that recentlyconducted on Australian mulga ecosystems (Erskine et al. 1996; Pate et al.1998). In this case, a range of Acacia spp. (predominantly A. aneura) in thegeneral mulga were found to be essentially non symbiotic, and this wasattributed to inh ib i t ion of nodule formation and functioning by the high


levels of nitrate in soil and ground water in the region. In contrast, legumesgrowing in the heavi ly leached sand dunes surrounding salt lakes in theregion were heavi ly nodulated and showed signals indicative ofbuoyant symbiotic activity.

To summarise, the quanti tat ive information concerning the nitrogen-f ix ing performances of microorganism:higher plant associations is veryincomplete and mostly of a qual i ta t ive rather than quantitative nature.Technological and logistical di f f icul t ies beset measurement of nitrogenfixation inputs by woody taxa of natural ecosystem and further progress inthis direction w i l l continue to be slow unt i l much greater resources offinance and manpower become available, particularly towards evaluation ofautotrophic inputs of N in survival and functioning of pristine andanthropogenically-modified ecosystems.

4. Conservation issues relating to symbiotic associationsThere is ever increas ing concern among a wide-ranging body ofconservation-minded biologists about the alarming loss worldwide of naturalhabitat and associated attri t ion of flora and fauna. There is every reason forbelieving that the threats to biodiversity and ecosystem structure shouldapply as much to symbiotic partnerships as to all other associatednon plants and microorganisms of a habitat. Dealing in turn withthe higher plant components considered in this chapter one would single outcycads as a particularly threatened group, due to the extraordinary largedemand for a number of cycad taxa in the horticultural trade, combined withthe failure of many of the highly localised populations of certain species toprovide seed for such purposes and the limited success of ex situconservation programs. However, on the posit ive side, appropriatecyanophycean partners to cycads are generally prevalent, judging from thevery high frequency of coralloid root formation in natural habitats, pot,glasshouse and garden culture. However, the purist might well argue thatirreplaceable cyanophycean resources would be lost wherever the naturalpopulations of the host are debilitated to the point of extinction (e.g. in thecase of several Encephalartos species in South Africa).

Symbioses invo lv ing Frankia and actinorhizal higher plants, rhizobium-type symbioses of Parasponia and cyanophycean symbiosis with Gunneraprovide little or no evidence of associations of this kind being significantlymore at risk in an ecosystem than cohabiting taxa. Just aswith cycads, high levels of promiscuity in the abilities of potential microbialpartners to nodulate taxa wi th in each of the above host groupings wouldsuggest that, when cultivated outside its normal range, would bemore or less guaranteed, albeit possibly only after artificial inoculation.


The overall role of nodulated legumes in complex ecosystems is stillpoorly understood and many questions remain unanswered (see Sprent2002). Given the world’s large resource of legumes, many instances of taxafrom this group are to be found in listings of rare and endangered taxa, or inthe gazetted listings of species requiring further evaluation. Woody shruband tree taxa appear to be particularly vulnerable in this connection (e.g.Sophora toromiro (Maunder et al. 1999)). However, it is also not possible toconclude that legumes as a whole are more at risk than other taxonomicgroupings, nor, indeed, to find cases in which demise of a particular legumecan be directly attributed to prior loss of a suitable rhizobial partner.However with the ab i l i t y of rhizobia to interchange being restricted incertain cases to specific cross inoculation groupings, especially amongtemperate herbaceous legumes, a situation may be pictured in which anendangered taxon with increasingly sparsely distributed individuals mightsuffer ‘loss’ of its own specific rhizobial associate. It would then be able toregain symbiotic competency only if able to ‘borrow’ compatible rhizobiafrom other legumes in its habitat. These issues are considered further in arecent article by Parker (2001), where a legume may fail to colonise ifmutualistic partner bacteria are scarce or absent. The same constraints arealso discussed more broadly by Richardson et al. (2000) in relation to othermutualisms as well as those between organisms and their hosts.Mycorrhizal association may be a potential aid in rehabilitation wheremutualistic partner bacteria are scarce (Franco et al. 1997; Sprent 2002).

Considering the effects of anthropogenic pollution, one might argue thatwhere run-off or atmospheric fall-out of a nitrogenous nature is prominent,symbiotic associations might be especially at risk, whetherdirectly from the well authenticated adverse effects of added N on formationand functioning of symbiotic organs, or indirectly by increasing competitivestress on the symbiotically-operating taxa in a community by nontaxa better nourished by the additional input of N.

Finally, one may attempt to forecast possible effects of increasedatmospheric and associated global warming on performance ofsymbiotic associations compared to that of cohabiting nonf ix ing plant taxa. Unfor tuna te ly , judging from the large and oftenconflicting information regarding plant and ecosystem responses to elevated

it would be extremely diff icult to generalise on this issue. However, anattractive scenario is that with rising levels, photosynthetic inputs ofplants would increase relative to water utilised. This might then lead to hostplants of associations becoming less penalised when providingphotosynthate specifically to symbiotic activity, under conditions wherelimitation of water is the overriding factor in species survival andproductivity. This and other possible suggestions clearly need to be


evaluated against the whole range of complex interactions which maycondition competitive responses of plants to long-term climate change.

AcknowledgementsI am extremely grateful to Russell Barrett for his superb assistance inpreparation of earlier versions of the manuscript and in the formatting andchecking of the final version. I am also indebted to Emeritus Professor JanetSprent and Dr Jenny Chappil l for information regarding the phylogeny oflegumes and a current synopsis of records of their nodulation.

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Chapter 4

BACTERIAL ASSOCIATIONS WITH PLANTS:BENEFICIAL, NON N-FIXING INTERACTIONS

Berndt Gerhardson

Sandra Wright

Plant Pathology and Biocontrol Unit, P.O. Box 7035, S-750 07 Uppsala, Sweden.

1. IntroductionLike all other organisms on earth, the higher plants are surrounded bysmaller, mostly harmless creatures, the microorganisms. For plants andother organisms l iv ing outdoors, one could say that they are bathing inmicrobes. The microflora on and around the aerial plant parts mainlyconsists of bacteria and small fungal spores, and is in many aspects similarto the air-borne flora. However, since the green terrestrial plants that are soilanchored by root systems are also soil organisms, like the earthworms forexample, they have to cope with a soil flora and fauna. In comparison to theaerial microflora, the soil microflora usually is much larger, more diverse,and commonly also more aggressive.

During evolut ion, these close contacts between plants and themicroorganisms infecting or invariably surrounding them have developedinto various dependencies on both sides. These in turn have in many casesled to specific biological interactions, or symbioses, presumably resultingfrom a long co-evolutionary process, and in other cases to more or less loose,or even chance associations. We now find these dependencies as host-pathogen interactions, which may be biotrophic or necrotrophic, clearsymbiotic interactions (e.g. wi th certain N-f ix ing microbes, and themycorrhizal fung i ) and as a variety of looser, probably facultativeassociations. In these interactions, many microorganisms are found inassociation either with specific plants or with plants generally. They


commonly induce no typical pathogenic symptoms or morphologicalchanges, even though they may give measurable and significant effects onplant growth and development.

The broader and typical ly non-associated, ecological relations betweenplants and microorganisms, e.g. that saprophytes are dependent on plants ascarbon sources and plants in turn on microbial mineralisation of nutrients,are not treated here. Furthermore, the bacteria that typically infect plants,e.g. plant pathogens and N - f i x i n g , symbiotic bacteria, are treated in otherchapters of this book (3 and 9). We here concentrate on the predominantlynon-pathogenic, plant-associated bacteria that are non N-fixing. Althoughthey are less well investigated than the typically pathogenic microorganisms,these also show many different kinds of spatial as well as functionalassociations with plants. We have the phyllosphere flora on leaves, flowers,fruits and shoots, the spermosphere flora on seeds, the rhizosphere flora onand in the roots, and in addition a possibly fairly specific endophyticbacterial flora within the plant tissues (Hallman et al. 1997). All these florascontain their clearly characteristic groups of bacteria, and within all of themthere are bacterial strains that interfere with plant growth and development,either directly by interacting wi th the plants themselves, or indirectly byinteracting with other organisms that affect the plant.

2. The plant growth affecting bacteriaThe scientific awareness of the existence of beneficial, non N-fixingbacterial interactions with plants is relatively recent. While interactionsinvolving plant pathogenic bacteria received an obvious attention in thedawn of bacteriology (Postgate 1992), and the N-fixing interactions weredescribed at the beginning of the century, the earliest records of otherclearly plant beneficial bacterial-plant interactions are from the middle andsecond part of the century (Tveit and Wood 1955; Bowen and Rovira1961; Baker and Snyder 1965). An often-quoted early study was the reportof growth increase of cereals and carrots obtained after treatment withcertain Bacillus and Streptomyces strains (Merriman et al. 1974), and Brown(1974) wrote one of the earlier reviews in this subject. These and other earlystudies (e.g. Kerr 1972; Howell and Stipanovic 1979; Kloepper et al 1980)were then followed by an increasing number of reports and also reviews(Schroth and Hancock 1981; Suslow 1982; Lynch 1982; Burr and Caesar1984; Schippers et al. 1987; Cook 1993) until we at present have animposing amount of li terature treating this subject. There are also regularinternational conferences covering this topic, especially the plant growthpromoting rhizobacteria (PGPR) (see http://www.ag.auburn.edu/pgpr/).

Since there is no clear or generally accepted delineation of differentfunctional groups of plant growth affecting bacteria, for the purpose of this

Bacterial associations with plants 81

chapter we wil l use the general term ‘plant beneficial bacteria’, which aredivided into two main subgroups: ‘plant growth affecting bacteria’ and‘disease suppressing bacteria’. In the literature terms like PGPR (Kloepperet al. 1980), deleterious rhizobacteria (DRB) (Suslow and Schroth 1982;Åström 1990), biocontrol agents, pathogen antagonists, disease suppressingagents (Cook and Baker 1983), resistance inducers (Kuc 1995) and othershave been used, partly interchangeably. One of the problems in classifyingand naming these bacteria, especially those directly affecting plants, is theirdependency on external conditions for inducing effects. A bacterial strainthat is clearly a plant growth promoter under certain conditions may, thus,have a plant deleterious effect in association with another plant species, orunder other environmental conditions (Schroth and Hancock 1981; Åström1990).

The bacterial strains so far reported as affecting plants usually belong tothe bacterial species commonly found in the soil and in the rhizosphere.Most described isolates have been identified as pseudomonads and withinthe Pseudomonas genus, the fluorescing species such as P. fluorescens, P.putida and P. aeruginosa predominate. The genus Bacillus is also wellrepresented, and the species B. subtilis especially seems to contain manyactive isolates. Other genera of soil or rhizosphere bacteria reported as plantgrowth-affecting are Serratia and Azospirillum (Glick et al. 1999). On theplant leaves we have, among others, the intriguing associations betweenplants and the methanol-utilising bacteria, Methylobacterium spp. (Hollandand Polacco 1994). However, even though these genera are well representedin literature reports, there could well be doubts as to what extent this mirrorsthe actual situation in nature. All of the genera mentioned are fairly easy toisolate from environmental samples including plants, on commonly usedlaboratory media, with free access to oxygen, and after incubation at roomtemperature. Since the bacteria tested for plant affecting abilities have oftenbeen isolated using such standard methods (e.g. Gerhardson et al. 1985;Kloepper et al. 1988a; Glick et al. 1999), many other bacterial species thatare not competitive under these conditions probably do not grow, and thus,have not been isolated and tested. A strong selection already at the isolationstage thereby probably occurs.

The suspicion that we have hitherto had a very unclear picture of thenature of the bacteria that are able to interact with and affect plant growthand development in nature is further substantiated both directly bymicroscopy (Foster 1983) and by using DNA-based methods. Analysis of16S rDNA from environmental samples has given evidence for a muchbroader bacterial diversity than hitherto appreciated (Ward et al. 1995).Based on such studies, we can conclude that only a very small fraction of thebacteria present in nature so far have been cultured, studied and described.


Presumably this applies to the plant-associated bacteria equally well as tobacteria from other habitats. Certain plant-associated bacteria may bespecialised and have a biotrophic association with their plant hosts, andtherefore may contain a higher proportion of bacteria that are difficult orimpossible to culture, than do bacteria isolated from other sources. Thisinability at present to culture and study but a fraction of the bacteria innature is also aggravated by the findings that many common bacterial strainsthat normally are easy to isolate and culture, do enter non-culturable stages(Roszak and Colwell 1978; Ward et al. 1995). Although the fullimplications of these findings in soil and plant bacteriology is still unclear,this ability has been established for certain Pseudomonas strains (Dendurandet al. 1994) and other soil bacteria (Oliver et al. 1995; Bogosian 1998). Inlight of this, what we presently know and what we have so far seen ofbeneficial plant-bacterial associations may be just the tip of the iceberg.

3. Plant reactions to non-pathogenic bacterial associationsA majority of the data that has been gathered on plant reactions to specificnon-pathogenic (non-infecting) bacterial strains derives from inoculationexperiments on various crop plants. Typically, the laboratory producedcultures of specific bacterial isolates have been inoculated on seeds, roots orin the soil matrix, and the effects induced have then been recorded asdifferences in shoot dry weight, after harvesting the treated plants grown inthe field, in the greenhouse or in growth chambers (Merriman et al. 1974;Gerhardson et al. 1985; Kloepper et al. 1980; Kloepper et al. 1988a; Glick1995). Shoot growth increases of 30-50 % or more are not unusual in suchexperiments (Kloepper et al. 1980; Glick 1995), and observations of earlierand faster germination and of greener leaf colour for example in treatedplants than in control plants, are also often reported (Kropp et al. 1996).However, in cases where screenings of randomly chosen bacterial strainsisolated from nature, e.g. from plant rhizospheres are carried out, variouskinds of plant deleterious effects may be as frequent as plant growthpromoting effects (Bolton and Elliott 1989; Åström 1990; Suslow andSchroth 1982), and in these cases, not just weaker shoot growth, but alsovarious plant symptoms like epinasty, dwarfing, leaf spots or stripes, andnecrosis of leaf edges may be induced (Gerhardson et al. 1985).

The significance of these results, plant beneficial as well as plantdeleterious effects, may in certain cases be questioned, and then mainly onthe grounds that they represent ar t i f ic ial and non-natural conditions.Another major objection is that the bacteria tested have often been applied inlarger amounts than they would occur under natural conditions, andfurthermore, that the material applied include not just the bacterial cells, butalso the bacterial culture supernatant, which may contain active metabolites.


The plant effects observed, might thus have been drastically exaggeratedbecause of large bacterial numbers. Such experiments are reasonable from ascientific point of view, but they may tell little about natural conditions.Also, where several millilitres of inoculum per litre of soil are applied, theinoculum may partly act just as a readily available fertiliser supplying extraN and P, or minor elements. However, even though such effects may havebeen at hand in many cases, and results have to be interpreted carefully, theoverall evidence that significant plant growth effects are induced by plantassociated non-infecting bacteria is overwhelming. We have, for example,cases where seed inoculation with only tiny amounts of bacteria givedramatic growth effect on the plant (Kloepper et al. 1980), and the plantgrowth promoting effects also have been amply tested in field experiments(Suslow 1982; Kloepper et al. 1988b, Johnsson et al. 1998).

Specificity is often encountered, where a certain active bacterial strainmay induce effects in one plant species, but not when tested on severalothers, giving us evidence that specific biological, or molecular interactionsare at hand. Such specificity is probably common in these kinds ofinteractions (Lemanceau et al. 1995; Chanway et al. 1988a), but it has beenmost clearly shown for bacteria inducing plant deleterious effects (Åströmand Gerhardson 1988), where the experimental recording of effects may beeasier than the recording of growth promotion. Interestingly, a clearspecificity in such interactions has been shown on a plant species level(Åström 1990), as well as on the cultivar level in wheat (Chanway et al.1988b; Åström and Gerhardson 1989). A strain that induces deleteriouseffects in one plant may also induce growth promotion in another species, orwhen tested on the same species under other conditions (Åström 1990).Specificity on a plant cultivar level, most probably driven by differences inexudates/bacterial nutrient sources, has been shown also for indirectly plant-affecting, pathogen-antagonistic bacteria. One example of this is theexperiments performed on isogenic lines of tomato, where the plantgenotype directly influenced the degree of suppression of the seed pathogenPythium torulosum by Bacillus cereus (Smith et al. 1999). It is intriguingthat the plant may affect bacterial growth and activity not only by exudingspecific metabolizable substances, but also by specific regulating molecules.Most of those studied are various N-acyl homoserine lactone mimiccompounds. By producing and exuding such molecules, the plants may to acertain degree govern the activity and composition of their residentmicroflora (Teplitski et al. 2000). These findings concerning specificity,together with the indications that we probably have a very common naturaloccurrence of bacteria with plant growth–affecting abilities (Gerhardson etal. 1985; Glick et al. 1999), give rise to a number of questions concerningtheir role in nature and in plant ecology.


In the case of plant deleterious effects, the effect-inducing bacteria maybe regarded as soil-borne pathogens, and, if so, there are far more rootpathogens in the soil than we have ever been aware of. The similarity inplant reaction to inocula t ion wi th the typical ly non-infecting, plantdeleterious bacteria, and to weak root pathogens, has also given thesemicroorganisms denotations as exo-pathogens, or non-invading pathogens(Woltz 1978; Timonin 1946; Salt 1979). Regarded as such, they have beensuggested as causes of crop rotation effects (Schippers et al. 1987) (such asthe crop losses almost invariably experienced in narrow rotations of cropplants) and replant diseases (Sewell 1979). Since many clearly plantdeleterious bacterial strains show specificity in relation to plant species, theyhave even been tested and shown efficacy as weed biological control agentsin bioherbicides (Boyetchko 1996; Kennedy 1997).

The plant growth promotion effects have in some cases also beenattributed to competition between growth deleterious and growth promotingstrains in the rhizosphere (Schippers et al. 1987), viz. a biological control ofdeleterious, plant-associated bacteria (Schippers et al. 1986), but so far, theexperimental evidence for such effects is generally lacking. For cropproduction specialists, the occurrence of effective plant-associated, growthpromoting bacteria raises the question of the growth and yield potential ofour crop plants. Has it hitherto been seriously underestimated? The plantecologist could equally question to what extent these plant-bacterialinteractions may interfere with plant development, plant reproduction andplant competition in natural systems.

4. The interaction mechanisms and problems of classificationThe non-pathogenic, plant associated bacteria that convey plant growthpromoting ability will most probably be grouped in a number of differentclasses in the future depending on the manner by which they affect the plant.So far we have too little knowledge to form a reasonable taxonomicframework, but two main classes (as used in this chapter) are immediatelyobvious: i) those interacting more or less directly with the plant, and ii) thoseinteracting indirectly by suppressing diseases caused by plant pathogens, viz.pathogen antagonists (reviewed by Weller 1988; Handelsman and Stabb1996; Thomashow and Weller 1996). There are also good candidates fortwo additional groups, with one containing bacteria that enhance availabilityof plant nutrients. These could be exemplified by the bacteria that are ableto solubilise inorganic or organic phosphorus in the soil. Strong evidence forenhanced growth resulting from such phosphate solubilisation comes fromthe large number of reports that link phosphate solubilisation by rhizospherebacteria to growth promotion in a number of different crop plants (Rodríguezand Fraga 1999). The second additional group consists of various “helper

For the bacterial strains inducing a consistent and significant direct plantgrowth promoting effect in our measuring systems most commonly applied,we can often deduce a bacterial interference with the plant hormone balance(Young et al. 1991; Loper and Schroth 1986). It is well demonstrated thatmany plant-associated bacteria, especially rhizosphere bacteria, are able toproduce secondary metabolites with phytohormonal activity (Dowling andO’Gara 1994; Glick et al. 1999). Most common is auxin (IAA) productionand as much as 80% of rhizosphere bacteria were estimated to producevarious auxins (Glick et al. 1999). Cytokinins, gibberellins and ethylene arealso established as microbially produced (Frankenberger and Arshad 1995).However, production of these substances by bacteria is not sufficientevidence for inferring bacterial hormonal action on plants.

The main evidence that such metabolites are actually involved comesfrom the numerous studies where increases in seed germination, seedlingemergence, growth and yield of various crops is obtained in response to seed

5. Hormonal action and detoxification

bacteria”, e.g. bacteria that are beneficial to plant growth through theirenhancing effect on other plant beneficial microorganisms, like N-fixingsymbionts, or the disease antagonists. These are also treated elsewhere inthis book and are only exemplified here by a brief mentioning of the“mycorrhiza helper bacteria” below.

Any such coarse classifications naturally meet problems when it comesto details. We have, for example, ample reports of bacteria that are primarilyclassified as pathogen antagonists, but which also affect the plant byinducing disease resistance (Liu et al. 1995; Leeman et al. 1995; van Loon etal. 1998). That is, even though they may not directly antagonise thepathogen, they are plant disease suppressers. We also have a group of plant-associated bacteria that may be denoted as pathogen synergists (Vancura andStanek 1976; Huber and McKay-Buis 1993). These increase diseaseseverity, and, thereby, are indirectly plant growth deleterious. They areprobably as common as the pathogen antagonists in nature, but will not(being plant deleterious) be treated further here. It is obvious however, fromsuch findings and from microcosm studies (Gerhardson and Clarholm 1985;Schippers et al. 1987) that under natural conditions, the effect on plantgrowth is, in most cases, exerted by microbial communities, rather than bysingle bacterial s t ra ins , or isolates. Concerted multi-microbial-plantinteractions, often also including soil fungi and animals, are probably asimportant for obtaining plant growth effects as are the direct plant-singlebacterial isolate interactions. Isolating single bacterial strains from theirenvironment and studying their mode of action in simplified systems thusmay easily lead to erroneous conclusions.



or root inoculation (Glick et al. 1999), and where the effects are similar tothose typically induced by plant growth hormones (Oberhänsli et al. 1991;Selvadurai et al. 1991). Evidence to this end is also added from variousother cause-effect studies. Young et al. (1991) screened a collection of plantgrowth promoting bacteria for production of plant growth hormones andfound a correlation between the ability to induce root elongation, or promotegermination and emergence, and a s ignif icant high production of plantgrowth regulating substances. Loper and Schroth, (1986) also screenedseveral bacterial isolates for their production of an auxin, indole-3-aceticacid (IAA), and the accumulat ion of IAA in bacterial supernatantssignificantly correlated with root elongation of sugar beet. Similarly, Nietoand Frankenberger (1991) demonstrated the production of phytohormonesindole acetic acid and cytokinins by Azotobacter species, which are wellknown to affect p lant growth and development. Culture supernatants ofAzospirillum spp. often contain both aux ins , cytokinins and gibberellins(Steenhoudt and Vanderleyden 2000), but the production and role of thesehormones in the rhizosphere has yet to be established. However, the role ofmicrobially produced IAA and the sensi t iv i ty of plants to various levels ofIAA was demonstrated by the inhibi t ion of canola root growth by IAAoverproducing mutants of P. putida (Xie et al. 1996).

A picture is now emerging that despite the fact that plants have tunedmetabolic systems for regulation of hormone production levels, and can alsostore excess amounts of the hormones as conjugate for later use (Glick et al.1999), an excess of a growth regulating substance, or a hormone group,produced by associated bacteria can also result in a plant hormonal response.The plant-associated bacteria here have probably evolved such intimaterelationships with their host plants that they may influence plant physiologyin different ways, possibly to an extent that the plants partly depend on thosepartners for their needs of at least certain growth regulating substances(Freyermuth et al. 1996). The ability of the plant to produce the necessary,although usually very small amount, of growth regulating substances foritself may be impeded, especia l ly under less than ideal climatic andenvironmental condit ions (Nieto and Frankenberger 1991), and it wil l thenbe more dependent on the exogenous sources. The plant-associated bacteriacould here widen the envi ronmenta l range of a plant species. It is alsoevident that uptake of excess hormonal substances, such as cytokinins, in thepresence of an exogenous supply may be converted into storage forms withinthe plant, and these can later be transformed into free-base metabolites foractive utilisation and control of plant growth (Wareing and Phillips 1981).

This conceptual model of m u t u a l benef i t , or symbiosis- l ikeassociations, is further strengthened by the fact that specific plant-associatedbacteria also can affect the endogenous hormonal pattern of the plant by


interaction with the synthesis, translocation or regulation of the existinghormonal level (Frankenberger and Arshad 1995). Certain bacteria may thusmodify the plant’s own pool of hormones and subsequently stimulate plantgrowth. A good example here is the findings of Glick et al. (1998) andJacobson et al. (1994), who demonstrated the ability of beneficial bacteria tolower the production of ethylene in developing seedlings of canola, tomatoand lettuce. High amounts of ethylene may impede an optimal developmentat the seedling stage (Gl ick et al. 1999). They suggested that the plant-associated bacteria regulate ethylene synthesis in plants by binding to theroots and/or seed coats and hydrolysing the ethylene precursor, 1-aminocyclopropane-1-carboxylate (ACC) exuded from the plant seeds orroots through the action of the bacterial-specific enzyme ACC deaminase.

This mode of action shows similarities to detoxification processes,which are probably also important mechanisms for directly bacterial-inducedplant growth promotion. Both the plant itself and its surrounding microflora,especially the rhizosphere flora, exude substances deleterious to plantgrowth (Barber and Martin 1976; Bolton and Elliott 1989; Becker et al.1985) and such substances may also be present in the soil or other growingmedia (Bruehl 1987). By breaking down or inactivating such deleterioussubstances, a significant effect on the plant growth may be found (Lynch1983). Special cases here are the microbes that are able to change the pH,and/or the redox potential in the plant rhizosphere (e.g. Huber and McKay-Buis 1993). An intriguing theory is also that the plant growth promotingbacteria are able to antagonise, or eradicate the effect of plant deleteriousbacteria, or other “minor pathogens” (Schippers et al. 1987), that accordingto this theory, are common inhabitants, especially in plant rhizospheres(Elliot and Lynch 1984; Schippers et al. 1987). However, this mechanismcan equally well belong under the heading “disease suppressing, plantbeneficial bacteria.”

6. Microbial synergists - the example of mycorrhiza helper bacteriaMicrobial synergists may, as mentioned above be as common in nature asthe microbial antagonists that, when antagonising pathogens, are often plantbeneficial by suppressing plant diseases. There is then likewise a group ofbacteria that enhance the activities of plant beneficial microorganisms,whether these are plant growth affecting bacteria, pathogen antagonists, N-fixing symbionts, or mycorrhizal fungi. However, our knowledge aboutthese microbe-plant-associated microbe interactions that ultimately maybecome beneficial to the plants is so far very scanty, and only one example isbriefly mentioned here: the mycorrhiza helper bacteria.

The term “mycorrhiza helper bacteria” refers to certain soil bacteria,mostly Pseudomonas spp., that have shown ability to significantly enhance


mycorrhizal formations in plants (Garbaye and Bowen 1989). Since certainplants, especially those with ectomycorrhiza, are dependent on mycorrhizalformation for normal growth (Harley and Smith 1983), the helper bacteriacould be seen as plant growth promoting. Furthermore, when we considerthe bacterial promotion of plant-beneficial mycorrhizal fungi, these bacteriaare also plant growth affecting in this respect. However, the mode of actionof these bacteria is s t i l l unclear (Garbaye 1994; Frey-Klett et al. 1997) andthey may also have an action whereby they enhance the plants receptivity tomycorrhizal infection (Garbaye 1994; Karabaghli et al. 1998). If true, thispoints to a direct plant growth-affecting abil i ty and, furthermore, tosimilarities between these bacteria and the pathogen synergists.

7. Disease suppressing, plant beneficial bacteriaBacteria that are plant beneficial by suppressing plant diseases may also besubdivided into two categories, as mentioned above: i) a direct antagonismof the pathogens or ii) indirectly an enhancement of plant disease resistance(Davison 1988). Such microbial-induced plant disease or pest resistance isat present further divided into two types, representing distinct pathways ofdisease resistance responses. We have either, systemic acquired resistance(SAR) that is induced by pathogens, or an induced systemic resistance (ISR)that is salicylic acid-independent and typically induced by non-pathogenicbacteria (van Loon et al. 1998). The latter group also includes severalbacteria that by virtue of their promotion of plant growth, help the plantswithstand infection. These direct and indirect modes of disease suppressionvery probably operate in consort. However, due to the definition of inducedresistance: “...operating at a distal plant part” (Kuc 1995), the relativecontribution of induced resistance to disease suppression becomes almostimpossible to measure and determine at the site of interaction betweenpathogen, plant and antagonist. Only when the antagonist is applied at a sitedistal to that of the pathogen, would induced resistance, by definition, beoperating. Moreover, disease suppression by beneficial bacteria is usuallyeasier to measure and enumerate than the direct effect on plant growth.

Disease suppressing bacteria were originally revealed through theirassociation with naturally ‘disease-suppressive soils’, and by the ability ofcertain randomly isolated soil and plant bacteria to protect plants againstdisease after exogenous application to plant parts. Many of these bacteriawere also selected as candidates for disease suppression on the basis of theirproduction of anti-microbial metabolites in laboratory media. Commonlyreported disease suppressing bacterial species include Agrobacteriumradiobacter, Bacillus subtilis, Burkholderia cepacia, Enterobacteragglomerans, E. cloacae, Pseudomonas aureofaciens, P. chlororaphis, P.fluorescens and P. putida (Schippers 1988; Becker and Schwinn 1993;


Bevivino et al. 1998; Kang et al. 1998; Pierson III 1998), and some strainsof P. fluorescens are simultaneously plant growth promoting (Glick et al.1999).

Mechanisms of disease-suppression by bacteria that have direct effectson pathogens include competition for nutrients (and/or space), siderophoreproduction, antibiosis, production of hydrolytic enzymes and other generaltraits that make them successful colonisers of plant parts and competentsurvivors in the phyllo-, rhizo- and/or spermo-sphere. However, with a fewexceptions, the activities related to nutrient competition has generally beenvery difficult to demonstrate in vivo. Efficient uptake and utilisation ofnutrients is a prerequisite for activity in a highly competitive environmentsuch as the soil. Roots and seeds exude a variety of amino acids andcarbohydrates (Rovira 1965; 1969) that are effectively utilised by microbialresidents, including many pathogens. A good example is the sporangia ofthe seedling pathogen Pythium ultimum that germinate in response to thepresence of nutrients in form of seed exudates (Nelson and Hsu 1994). Inthis case, it has been shown that the utilisation of the long chain fatty acidcomponents of cotton seed exudate by the disease suppressing bacteriumEnterobacter cloacae prevented the sporangia from germinating (van Dijkand Nelson 1997). Another demonstration of nutrient competition is theobservation that disease suppressing strains that are isogenic to the samespecies as the pathogen and thus share the same nutritional niche as thepathogen are often very successful competitors. Examples includeAgrobacterium radiobacter controlling A. tumefaciens (Kerr 1989), a non-pathogenic strain of Ralstonia solanacearum controlling R. solanacearum(Sunaina et al. 1997), non-pathogenic Clavibacter xyli that control C. xyli,isogenic strains of P. fluorescens out-competing each other in therhizosphere (Nautiyal 1997), the control of ice nucleation active bacteria bynon-ice nucleating isogenic mutants (Lindemann and Suslow 1987) and thecontrol of vascular wilt fusaria (Fusarium oxysporum) by non-pathogenicstrains of F. oxysporum (Alabouvette et al. 1993). They are often onlyeffective when provided with an additional competitive advantage, such ashigher initial cell numbers (Nautiyal 1997), earlier establishment than thepathogen, or the production of an antibiotic, such as in the example ofagrocin 84 (Kerr 1989).

Certain antagonists also out-compete pathogens by sequestering theferric iron available, which expresses itself through the production ofsiderophores, iron-chelating molecules that facilitate iron uptake for themicroorganisms producing them, but may make it unavailable for thepathogens (Teintze et al. 1981, O’Sullivan and O’Gara 1992). A number ofsuch iron-chelating molecules have been studied, e.g. pyoverdin, pyochelin,ferribactin, ferrichrome, ferroxamine B, phytosiderophores and pseudobactin


(Dowling and O’Gara 1994). Fluorescent siderophores are also what makesfluorescent pseudomonads glow upon exposure to UV light under iron-limiting conditions. The role of siderophores in biological pathogen controldepends on a variety of external factors; the nature of the host plant, thepathogen and the soil environment. Thus, in some cases, siderophores haveproved to play a role (Buysens et al. 1996), whereas in other cases they havenot (Hamdan et al. 1991).

Antibiosis, as caused by specific exuded inhibitory molecules, is one ofthe most extensively studied mechanisms in biological control systems. Thepresence or absence of antibiotics, like e.g. streptomycin, is relatively easy todemonstrate on laboratory media. The contribution of antibiosis to thepathogen suppressing ab i l i ty of an antagonist is often assessed byconstructing mutants that differ only in the augmented presence or totalabsence of antibiotics, and comparing their disease suppressing efficacy tothat of the wild-type strain. In that way, it was established that phenazines(Pierson III et al. 1994, Thomashow and Weller 1988) and 2,4–diacetyl-phloroglucinol (Keel et al. 1992; Vincent et al. 1991) produced byrhizosphere pseudomonads were involved in suppression of take-all diseasein wheat. Pyoluteorin was involved in suppressing Pythium ultimum oncress (Maurhofer et al. 1994), pyrrolnitrin suppressed Pyrenophora tritici-repentis on wheat (Pfender et al. 1993) and DDR (2,3-deepoxy-2,3-didehydro-rhizoxin) produced by Pseudomonas chlororaphis was involvedin suppression of Drechslera teres on wheat (Hökeberg 1998). The role ofantibiosis is in many cases, like that of siderophore production, dependent onthe environmental conditions (Dowling and O’Gara 1994), including thetype of plant species colonised (Maurhofer et al. 1994). Some of theantimicrobial compounds produced have activity against fungi, e.g.phenazines (Thomashow et al. 1990) and some against bacteria, e.g. agrocin84 produced by A. radiobacter (Das et al. 1978), while others have activityagainst both. Also the production of compounds like of hydrogen cyanide(Voisard et al. 1989) and hydrolyt ic enzymes such as chitinases, b-1,3-glucanases, proteases and lipases have been implicated in diseasesuppression (Chet and Inbar 1994).

Mild pathogens or non-pathogens can, as mentioned above, inducedisease resistance in plants (Deacon and Berry 1993). It often acts againstboth pathogenic viruses, fungi and bacteria (Kloepper et al. 1999), with aneffect that is indirect with regard to the interaction of the pathogen and thedisease suppressing agent. The resistance response may be induced byindividual components of bacteria, such as the O-antigenic side chain of theouter membrane lipopoly-saccharide, siderophores and salicylic acidproduced by the bacteria (reviewed by van Loon et al. 1998). This type ofresistance has been induced toward several pathogens, for example vascular


wilt in carnation caused by Fusarium oxysporum (van Peer et al. 1991) andPseudomonas syringae pv. lachrymans in cucumber (Wei et al. 1996). Inthe latter case, it also operated under field conditions. Interestingly, theresistance response can be both bacterial strain- and plant species-specific, asnoted by van Wees et al. (1997).

In cases where directly or indirectly disease suppressing bacteria havebeen tested or commercialised as biological control agent in biopesticides,their exact modes of action often have been difficult to elucidate (Hökeberg1998). It is generally very rare for only one of the mechanisms mentionedabove to operate alone. Usually, the disease-suppressing bacteria insteadutilise a number of biocontrol factors simultaneously, and in order to do so atall it is sometimes crucial that they colonise and establish on the plant partthey are protecting (Cook and Baker 1983). Thus, colonisation ability andgeneral ‘rhizosphere, phyllosphere or spermosphere competence’ also maybe a prerequisite, in order for the above-mentioned mechanisms to takeeffect.

8. Disease suppressive soils – involvement of plant-associated bacteriaThe term “disease suppressive soils” refers to specific soils withcharacteristics that cause disease levels to remain low even though thepathogen is present and the environmental conditions for diseasedevelopment is favourable (Alabouvette 1990; Hornby 1983). Usually,when compared to sterile plant growth media, all unsterilised soils willexhibit some disease suppression, especially those with high biologicalactivity, or biomass, but to classify as disease suppressive, disease ratings onplants grown has to constantly remain low, even after pathogen inoculation.Based on its origin, such soil disease suppressiveness is commonly dividedinto either i) long-standing, natural suppressiveness, or ii) inducedsuppressiveness (Hornby 1983). Both types are usually specific, whichmeans that in each specific case it is acting on one, or a few related soil-borne diseases. Natural suppressiveness is often permanent and it has inmost cases been found to have a microbiological origin, but its occurrence isprobably also dependent on soil physicochemical properties (Höper andAlabouvette 1996). A good example is the early finding of soil diseasesuppressiveness in banana plantations in Central America, where it wasobserved that Fusarium wilt of banana developed much more rapidly incertain areas, and soils, than in others (Stotzky and Martin 1963). Theinduced soil disease suppressiveness may develop after a monoculture of thehost plant for several years, or by soil amendments (Hoitink et al. 1996) andit also has a microbiological background (Shipton 1977). Induction of soildisease suppressiveness was often observed when cultivating virgin soils,e.g. in United States and Austral ia , which commonly involved an initial


increase in diseases and a decline in crop productivity during the first years,but after a continued cul t iva t ion the productivi ty returned (Huber andSchneider 1982) (disease decline soils).

In experiments where a disease suppressive soil is added to and mixedwith a non-suppressive, or conducive soil, the suppressiveness is usuallyeffectively transferred, and often also with low amounts of suppressive soil(Alabouvette et al. 1996), which points to a transfer of specific substances orbiological entities. A common approach for assessing the involvement ofbiotic factors has also been to measure the degree of suppressiveness beforeand after soil biocidal treatments, such as heat, irradiation with X-rays, ortreatment with methyl bromide (Burke 1965; Louvet et al. 1976; Scher andBaker 1980). When such sterilisation treatments result in loss ofsuppressiveness, some part of the soil microbiota is assumed as the agent(s)directly causing the suppression (Persson 1998). For finding the possibleactive agents a number of specific organisms have also been isolated fromsuppressive soils. By reintroducing these into sterilised suppressive soil, orinto sterilised or non-treated conducive soil, it has been possible to assess towhat extent the disease suppression is re-established (Alabouvette 1986;Scher and Baker 1980).

Among the specific organisms isolated from suppressive soils andtested are several Pseudomonas bacteria (Defago et al. 1990; Weller 1988;Alabouvette et al. 1996; Scher and Baker 1982; Raaijmakers and Weller1998) besides a number of fungal species such as Trichoderma spp. (Simon1989; Duffy et al. 1996), Pythium oligandrum (Ribeiro and Butler 1995),and non-pathogenic species of F. oxysporum (Alabouvette et al. 1996; Duffyet al. 1996; McQuilken et al. 1990). Antagonistic activities, and/or inducedresistance (Alabouvette et al. 1996; Liu et al. 1995) as described in thepreceding paragraph, are thought to be the main modes of action, but it isless easy to visualise activities of bacterial-produced antibiotics andsiderophores in the soil than on a plant surface, or in the rhizosphere. Thecompetition for nutrients and the restricted availability of these in soil in theabsence of plant roots is also thought to induce fungistasis, viz. prevention ofgermination of dormant fungal spores (Lockwood 1988). The prevention ofgermination of spores and thereby of infection of the host may also beinduced by a generally high microbial activity, obtained by soil amendmentby composts, as reported for diseases caused by Pythium and Phytophthoraspp. (Hoitink et al. 1996). Various biosurfactants produced by bacteria havealso been shown to act especially on such zoosporic pathogens by destroyingthe membranes of the zoospores (Stanghellini and Miller 1997).

We, thus have reasonable evidence of the involvement of the soilmicroflora and of non-infect ing plant beneficial bacteria in this naturallyoccurring biological disease control . However, since the specific


microorganisms isolated and investigated very seldom give a full effectwhen reintroduced (Alabouvette et al. 1996), they do not tell us the wholestory of the mechanism. The soil physiochemical properties, climateconditions, specific microbial communities, and/or activities, maybe fosteredby a very long cropping, or rather vegetation history, probably also have arole. From a plant conservation point of view, the occurrence of plantdisease suppressiveness may have two main implications: i) that our abilityto grow, or conserve a specific plant may to a great extent depend on the soiland its microbial content, or site chosen, and ii) there may occur specific soilmicroflora-plant interactions that take a very long time to develop and whenand where developed could be well worth conserving because of theiruniqueness.

9. Implications for plant conservation and diversityAlthough they are very common in nature, and often closely associated toplant physiology, plant health and plant growth, the plant beneficial, non-N-fixing bacteria are not considered to be critical for native plant survival andreproduction. This assumption is based on our present knowledge and willpossibly be challenged by new findings to come. There may for example, beobligate plant endophytic bacteria in nature that are highly specialised andpossibly required for normal plant growth. The plant endophytic bacterialcommunity can, like other plant-associated bacteria, influence rootmorphology and structure, and it may also have an impact on the bacteriathat reside on the outside of the plant (reviewed by Sturz and Novak 2000).However, since the endophytic bacteria hitherto found in many plant speciesare regarded as usually originating from the bacterial flora residing on theplant surface (Hallman et al. 1997), they have warranted no special treatmentin this chapter.

It is generally accepted that all plant species are affected to varyingdegrees by microflora that surround them. Specific strains associated with,or especially beneficial to one, or a number of plant species, may occur onlyin certain soils or environments and, if so, this probably substantially affectsthe success of the species concerned, and consequently their distribution.Furthermore, there are probably strong interactions between a plantcommunity and the population and activity the associated, growth beneficialbacteria. By analogy to the conclusions of Newman (1985) working withrhizosphere fungi, a plant community may have a more or less commonrhizosphere flora mediating interactions between plant species that are notpossible to discern by traditional ecological methods. This would againpoint to the advantage of community conservation rather than speciesconservation. The non-invading, plant beneficial bacteria may, in addition,be more or less critical to survival and reproduction of specific plant species


on marginal or less suitable locations where they, much like mycorrhiza onreclamation land (Brundrett and Cairney, this volume), may extend the rangeof plant survival. However at present, these assumptions or theories are notsubstantiated well enough by experimental evidence to be considered asadequate for exp la in ing broader impl icat ions or for developingrecommendations for practical plant conservation.

In certain practices, we could nonetheless gain substantial advantagesfrom considering beneficial , plant-associated bacteria, such as in thetransplanting or growing of specific plants outside their natural habitats, suchas in botanical gardens. Inoculations by, or supplying of, an appropriatecomplement of bacterial flora should be advantageous. A theory that is alsoclearly supported is the empir ica l ly found advantageous practice ofenhancing revegetation by using inoculum of native microbes, e.g. by addinga handful of soil from under nearby exis t ing plants at replanting.Straightforward inocula t ions of cul tures of beneficial , direct growthpromoting bacteria, as is an increasingly used practice in agriculture andhorticulture (Glick et al. 1999), may so far be premature for most wild, non-crop plants from which the experiences of direct beneficial inoculations arecomparatively very scarce. Better experimental data is at hand for theindirect growth-promoting, disease suppressing bacteria (Cook 1993;Whipps 1997). However, as the microbiological competence and equipmentneeded for practical application are usually not freely available, theiruti l isat ion in p lan t conservation is much dependent on commercialpreparations. As more of such products become available, these willnormally be easier to handle and also environmentally very safe, but in otherways similar to apply and to use as most chemical plant protection products.No specific requirement should here be set in plant conservation.

The area where knowledge of plant associated, growth beneficialbacteria presently may have most significant implications for plantconservation and diversity is probably in the appreciation and managing ofthe natural microflora in the soil or in the growth substrate where plantcommunities are to be re-established, or where plants are to be conserved.As mentioned above, it could easily be envisioned how disturbed areas, e.g.mined or cleared land, may become defiant of, or suppressive to, beneficialbacterial associations that are critical for ful l re-establishment. However, thesoils or other growth substrates used in replanting are presently moremanageable and usually carefully conditioned concerning plant nutrients,texture, pH etc. Managing the soil biology may be similarly critical, andgenerally high soil microbial activity may not always be optimal. Ideally,suppressiveness to important soil-borne diseases is a preference, and thepreceding vegetation, or even the vegetation history important fordevelopment of suppression at a site is l ikewise of importance. Well


documented examples from crop plants are the apple replant diseaseproblems (Sewell 1979) and the necessity of crop rotations in agriculture(Cook and Veseth 1991) that partly shape our agricultural landscapes. Inreplanting, there are also, as discussed above, good possibilities forinoculations by specific microbial communities or by selectedmicroorganisms that may have profound influences on the replant results(Hoitink et al. 1996).

10. Implications for microorganism conservation and diversityWhere needed for scientific, mainly taxonomic, or for commercial purposes,specific and identified microorganisms are normally conserved as living, orin some cases, as dead and dried samples in culture collections (Allsopp etal. 1995; Gams, this volume), and we know of no natural reserves, orhabitats that have been protected because of their microorganisms. Nor hasthere been the need for conserving specific strains or their habitats for otherreasons. The non-infecting microorganisms are cosmopolitan in theiroccurrence on a specific host, or hosts, and are usually regarded asubiquitous. They are very easily spread, even at distances, by winds, bymigrating birds and animals (including humans) and in light of this weassume that they have few geographic barriers (Andrews 1991). Theiroccurrence and conservation then is more dependent on availability ofsuitable habitats. Specific plants growing under specific conditions may be arequired niche, and the microbial conservation should in these cases beconcurrent with plant conservation. In this connection the assumption thatold cultivars of crop plants may have microfloras worth preserving alsocould have a bearing, but scientific evidence to this end is still scarce. Ofimportance is here also that we presently have a very inadequate picture ofthe plant-associated microorganisms, and especially the bacteria. Probablyonly a small fraction of the non-infecting microorganisms (1 to 10%) can becultured in synthetic media (Campbell and Greaves 1990) and even less, 1 to5%, are known and described (Allsopp et al. 1995). The plant-associated,growth beneficial bacteria are no exception. Another difficulty in evaluatingthe need and possibilities for bacterial conservation and diversity, especiallyfor soil and plant-associated bacteria, is also their fast genetic change andseemingly constant exchange of genes (Reanney et al. 1983; Sonea andPanisset 1983). In light of this, it may be questioned whether effort shouldbe directed towards maintaining their diversity, or if it is at all possible tomaintain them. After all, they will evolve much faster than the highercreatures in their own way, depending on their genetic make-up andenvironment they encounter.


AcknowledgementsWe thank the Foundation for Strategic Environmental Research (MISTRA)for financial support.

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Chapter 5

ECTOMYCORRHIZAS IN PLANTCOMMUNITIES

Mark C. Brundrett

CSIRO Forestry and Forest Products, CSIRO Centre for Mediterranean Agricultural

Research, Private Bag No 5, Wembley, 6913, Western Australia; Soil Science and Plant

Nutrition, Faculty of Natural and Agricultural Sciences, The University of WesternAustralia, Crawley 6009, Western Australia (current address) and Science Directorate,

Kings Park and Botanic Garden, Botanic Gardens and Parks Authority, West Perth 6005,Western Australia (correspondence).

John W.G. Cairney

Mycorrhiza Research Group, Centre for Horticulture and Plant Sciences, ParramattaCampus, University of Western Sydney, Locked Bag 1797, Penrith South DC, 1797, NewSouth Wales, Australia.

1. Introduction

Ectomycorrhizal associations (abbreviated as ECM) are sometimes calledectotrophic associations or sheathing mycorrhizas. They are mutualisticassociations between higher fungi and Gymnosperm or Angiospermplants belonging to the families listed in Table 1. These associationsconsist of mycorrhizal roots and fungal storage or reproductive structuresthat are interconnected by soil-borne mycelia (Figure 1).

Ectomycorrhizal associations are formed predominantly on the fineroot tips of the host. These ECM roots are defined by the presence of amantle, consisting of interwoven hyphae on the root surface, and a Hartignet, which is a labyrinth of highly branched hyphae between cells of theroot epidermis or cortex. These structures are not always both welldeveloped in the same association. These roots and their associatedfungal hyphae typically are most abundant in topsoil layers containinghumus and are thought to make a substantial contribution to soil biomassand nutrient cycling in many ecosystems (Section 3.1). Detaileddescriptions of the structure and development of ECM are availableelsewhere (e.g. Kottke and Oberwinkler 1986; Massicotte et al. 1987).

1.1. Associations


1.2. Host plantsTrees with ECM associations typically are dominant in coniferous forestsin boreal or alpine regions, but are also important in some temperatedeciduous forest, tropical forest, as well as savannah and mediterraneanplant communities (Meyer 1973; Högberg 1986; Brundrett 1991). Plantfamilies reported to have ECM are listed in Table 1. This table excludes

Ectomycorrhizas in plant communities 107

families with arbutoid or monotropoid ECM associations and those withatypical ECM associations, such as Australian herbaceous plants in thefamilies Goodeniaceae, Asteraceae and Stylidiaceae (Kope and Warcup1986; Brundrett 1999b). The majority of ECM hosts are trees, or shrubs(Table 1), but associations are formed by a few herbaceous plants,including Kobresia, Polygonum and Cassiope species found in arctic oralpine regions (Kohn and Stasovski 1990; Massicotte et al. 1998).

1.3. FungiThe reproductive structures of ECM fungi include epigeous fungi(mushrooms, puffballs, coral fungi, etc.) and subterranean structures(hypogeous fungi which are called truffles or truffle-like fungi). Mostepigeous fungi have a hymenium consisting of gills, pores, teeth, etc.which actively releases spores, but the hypogeous fungi and puffballshave sequestrate fruit bodies which enclose their spores. The majority ofECM fungi are Basidiomycetes, but there also are a number ofAscomycetes and a handful of Zygomycetes (Molina et al. 1992). Mostidentification guides for larger fungi provide information about theirprobable host plants from field observations. Lists of known AustralianECM fungal genera and those which associate with Eucalyptus species areprovided on the Web (Brundrett and Bougher 1999).

Detailed descriptions of the process required to collect, document,store and identify fungal specimens are provided in manuals (e.g.Largent 1986; Brundrett et al. 1996c). However, accurate fungalidentification is a time consuming process that requires much expertise.Fungal identification is most difficult in tropical regions and the southernhemisphere, where many species have yet to be described or illustrated inidentification guides. In particular, the hypogeous ECM fungi have beenneglected in many regions (Bougher and Lebel 2001). Due to thesetaxonomic difficulties, it is probable that many lists of fungi containerrors. Consequently, it is imperative that voucher specimens of all fungireferred to in publications are lodged with an internationally recognisedherbarium, and these specimens contain material of sufficient quality andquantity to allow future taxonomic and DNA-based studies.

2. Fungal biology2.1. Distribution and diversityInformation on the identity and relative abundance of ECM fungi presentin a particular habitat is required to understand the relationship betweentaxonomic and functional diversity (Section 4.1). Approximately 5500species of ECM fungi have been listed world-wide (Molina et al. 1992).However, this list would significantly underestimate the diversity of thesefungi, as the fungal flora in tropical and southern regions is poorlyknown. For example, it has been estimated that there could be as many as6500 species of ECM fungi in Australia alone, but only about 700species from this region have been named so far (Bougher 1995;Brundrett and Bougher 1999). Most estimates of diversity are based on


surveys of obvious epigeous fruiting bodies, which exclude hypogeousand resupinate fungi.


Ectomycorrhizal fungi are normally identified by observations offruiting under their putative hosts, but the relationship between fruitbodies and the activity of mycelia in soils has usually not beenestablished. The recognition of fungi by ECM morphology (colour,texture, structure, size, branching, etc.), has provided a powerful tool forthe identification of fungi in individual root tips (e.g. Agerer 1995;Bradbury et al. 1998; Hagerman et al. 1999; Massicotte et al. 1999).Lipid profiles can also be used to identify ECM fungi in soils (Olsson1999). Methods based on DNA are now often employed to identify ECMroots and provide a much more accurate picture of below-ground fungalactivity than observations of fruit bodies (e.g. Dahlberg and Stenlid1995; Horton and Bruns 1998; Jonsson et al. 1999a,b). A list ofmolecular sequence data that can help to identify ECM fungi is providedby Bruns et al. (1998). Molecular studies of fungi inhabiting ECM roottips in the field have shown that up to 60% of ECM roots are inhabitedby fungi not observed to produce obvious epigeous fruiting bodies (e.g.Gardes and Bruns 1996; Jonsson et al. 1999a,b). These cryptic fungiwould likely include those with hypogeous sequestrate (truffle-like)fruiting bodies (Castellano and Bougher 1994), or inconspicuous,epigeous, resupinate fruit ing bodies (Erland and Taylor 1999), largelyignored in previous diversity surveys. Fungi such as Cenococcum, whichdo not produce macroscopic fruit bodies, also appear to be widespread.However, the observed discrepancies between the fungi which formmycorrhizas and those which fruit at a particular site seem to be due tothe fact that many fungi reproduce very sporadically.

Most genera of ECM fungi are reported to be widely distributedthroughout the world, but there are also genera restricted to certainregions. Individual species of ECM fungi are often restricted to particulargeographic regions. When considering functional aspects of mycorrhizalassociations we should consider isolates of fungi rather than species orgenera, because considerable intraspecific physiological variation isknown to exist in ECM fungi (Trappe 1977; Brundrett 1991; Cairney1999). Thus information about the soil and climatic conditions and hostspecies present where fungi occur may be as important as their accurateidentification. Future taxonomic studies are likely to reveal much finerdifferences between taxa of fungi, than are used in current classificationschemes, especially for the neglected floras of the tropics and southernhemisphere. Molecular investigations are also revealing that taxapreviously regarded as conspecific are complexes of several species. Agood example is the cosmopolitan species Pisolithus tinctorius, for whicha number of putative species have been identified using restrictionfragment polymorphism (RFLP) and sequence analyses of the rDNAinternal transcribed spacer (ITS) region (Anderson et al. 1998; Martin etal. 1998; Chambers and Cairney 1999; Sims et al. 1999). Theseapproaches are likely to result in taxonomic revisions of many othergenera of ECM fungi and provide us with a greater understanding ofcorrelation between the taxonomy and physiological attributes of fungi(Section 1.3).


2.2. Lifecycles and inoculumThere are a number of dist inct stages in the life cycle of a mycorrhizalassociation (Table 2). These stages often occur at particular times of theyear. Mycorrhizal exchange is a short-lived process dependent on rootgrowth (Downes et al. 1992; Cairney and Alexander 1992a,b; Massicotteet al. 1987) – which in turn is regulated by environmental conditions andhost plant phenology. Fungal fruiting, and thus the availability of sporeinoculum, is also regulated by climatic factors and fungal phenology. Itis important to think of ECM associations as dynamic processes as fungiwould utilise mycelia and spores to move through soil while competingwith other fungi to claim new roots. Thus, the size and shape ofindividual fungi is constantly changing with time, due to their foragingbehaviour (Section 3.1), interactions with other soil organisms (Section2.3) and environmental factors (Section 3D). These dynamic processesmay, in part at least, explain discrepancies between the fungi f ru i t ingabove ground and those forming mycorrhizas in a particular location(Section 2.1).

Propagules of ECM fungi include individual hyphae, strands(aggregations of hyphae), spores, sclerotia and probably also mycorrhizalroots (Ogawa 1985; Fries 1987; Ba et al. 1991; Miller et al. 1994; Torresand Honrubia 1997). Most ECM fungi do not produce conidia(Hutchinson 1989). Boreal forest soil and leaf litter often contains sporescapable of ini t ia t ing mycorrhizas (Amaranthus and Perry 1987; Parke etal. 1983b). Localised patterns of ECM fungus proliferation depend onthe production of hyphae or strands by a particular endophyte (Ogawa1985; Agerer 1995; Unestam and Sun 1995). Mycelia of some ECMfungi are thought to require attachment to a living host root to initiatenew mycorrhizas (Fleming et al. 1984). Ectomycorrhizal short roots livefor months (Majdi and Nylund 1996; Rygiewicz et al. 1997) and areoften protected by a thick covering of mantle hyphae, suggesting thatthey may be important fungus survival structures. However, it is notknown how long ECM fungi in these roots can survive after detachmentfrom the host. The implications of ECM fungus dispersal and survival areconsidered in sections 2.3 and 2.4 below.

2.3. Consumption and dispersalEctomycorrhizal fungus structures are a major structural component

of certain soils, and thus are an important food source for many soilorganisms (Table 3). Hyphal grazing by soil organisms can reducemycorrhiza formation and nutrient translocation by hyphae in soils (Hiolet al. 1995; Setälä 1995). Soil organisms which ingest, inhabit, orassociate with hyphae or sporophores of ECM fungi include members ofmost soil trophic levels (Table 3). Dense mats of ECM roots and myceliain forest soils can have substantially higher populations of microbes andmicro-arthropods than other areas (Cromack et al. 1988; Griffiths et al.1991).


Dispersal of fungal propagules is required for colonising newhabitats, increasing fungal diversity, changing fungal populationstructure, or introducing new genes to existing fungi. Most localised

spread is by mycelial growth through the soil, resulting in discretepatches of soil occupied by the hyphal networks of individual fungi, thatcan be distinguished from others of their species using genetic or DNA-based methods (de la Bastide et al. 1994; Dahlberg and Stenlid 1995).Many fungi forming ECM associations have large fruiting structures(mushrooms) that produce abundant wind-borne spores, but survival anddispersal of these spores may be limited. Certain ECM fungi producesclerotia which probably are much more resilient than other propagules(Miller et al. 1994). Fungi with hypogeous fruiting bodies are oftenexcavated and consumed by small mammals or marsupials and thusspread to new locations (Table 3). Spores of ECM fungi contained inanimal faeces are a viable source of inoculum (Claridge et al. 1992;Cázares and Trappe 1994; Reddell et al. 1997).


2.4. DisturbanceSevere disturbance includes situations where vegetation has been lost andtopsoil has been removed or mechanically disrupted, or where plants areintroduced to new substrates resulting from mining, glaciation, orvolcanic activity. Soil disturbance can result in temperature extremes,anaerobic conditions, loss of organic matter, loss of nutrients, structuralchanges and loss of biological components (Abdul-Kareem and McRae1984; Danielson 1985). There is a high degree of spatial variability inECM fungus inoculum in Australian natural habitats (Brundrett andAbbott 1995; Brundrett et al. 1996a), but this variability is even larger indisturbed habitats, where large gaps between patches containing inoculumwould prevent many seedlings from encountering these fungi (Brundrettet al. 1996b). Forestry activities can also result in reductions in ECMfungus inoculum due to the absence of host plants and soil degradation(Amaranthus and Perry 1987; Parke et al. 1983a; Harvey et al. 1997;Perry et al. 1987; Visser et al. 1998; Hagerman et al. 1999). Aftermechanical disturbance of soils, surviving ECM fungal inoculum may beconcentrated in localised soil pockets high in organic materials (Christyet al. 1982; Parke et al. 1983b; McAfee and Fortin 1989). Other formsof severe disturbance known to adversely affect ECM fungi includeerosion and fires (Table 3).

Mycorrhizal inoculum is often limited in recently disturbed habitats(Danielson 1985; Malajczuk et al. 1994; Brundrett et al. 1996b; Reddellet al. 1999). Propagules expected to survive in disturbed soils are thoughtto include mycorrhizal root fragments, hyphae within organic matter,segments of rhizomorphs, sclerotia and perhaps spores and root pieces(Ba et al. 1991; Brundrett 1991). The networks of fungal hyphae whichare the main propagules of ECM fungi would be highly susceptible todisturbance and other more resilient propagules would decline in theabsence of host roots (Figure 2). Reductions in fungal diversity couldresult because fungi have a l imited capacity to adapt to major changes inenvironmental conditions (Table 4). Fungus specificity (Section 3.3) mayalso prevent mycorrhiza formation if surviving fungi are not compatiblewith new host plants (e.g. fungi from Eucalyptus may not form


mycorrhizas if Acacia or Melaleuca are dominant after disturbance). Thetime required for fungi to grow from residual inoculum or re-colonisesites by dispersal, w i l l determine the rate of recovery of fungal diversity.

Spore dispersal by the wind and mycophagous animals (Table 3) areconsidered important in the colonisation of new habitats by ECM fungi(Cázares and Trappe 1994; Johnson 1995; Brundrett et al. 1996b). Theeffectiveness of these natural vectors will depend on the proximity ofdisturbed sites to habitats containing suitable fungi (and their associatedanimals) as well as the phenology of fruiting of fungi. It is not known ifthese fungi are more or less readily dispersed than their host plants(Figure 2). Unfortunately, there is insufficient information about thebiology of mycorrhizal fungi to make robust predictions about thecapacity of particular strains of fungi to survive disturbance, or to adaptto changes in soil conditions following disturbance. Fungal communitiesin disturbed habitats are considered further in Section 4.1.


3. Mycorrhizal plants3.1. Benefits to plantsEctomycorrhizal associations are assumed to have key roles in nutrientcycling processes in ecosystems where their hosts are dominant. Thisassumption is based on the large biomass of their fruit bodies whichappear at certain times (Fogel and Hunt 1979; Vogt et al. 1982) and thepervasiveness of mycelium assumed to belong to ECM fungi in soils.Further evidence is provided by measurements of substantial carbontransfer from host plants to ECM fungi (Rygiewicz and Andersen 1994;Markkola et al. 1995; Setälä et al. 1999) and between interconnectedplants (Simard et al. 1997). The mycelia of ECM fungi also are a majorstructural component of soils (Table 5). Glasshouse experiments haveprovided many demonstrations of substantial benefits from inoculationwith ECM fungi, due to growth responses resulting from enhanced


nutrient uptake, improved disease resistance, etc. (Table 5).Unfortunately, there have been few attempts to measure these parametersin natural ecosystems and it is dangerous to extrapolate results fromhighly simplified experimental systems to the real world.

The “foraging behaviour” of ECM fungus mycelia results inproliferation within nutrient rich patches of soil, lowering nutrientconcentrations in these zones (Bending and Read 1995). Different partsof the mycelial systems of ECM fungi exhibit different physiologicalcapabilities and structural properties (Unestam and Sun 1995; Cairneyand Burke 1996). Only a fraction of the hyphae of an ECM fungus isconsidered to be capable of nutrient uptake at any one time and thisproportion decreases with association age (Taylor and Peterson 1998).Mycelial systems produced by ECM fungi in soils are considered to playa key role in nutrient cycling in many ecosystems and function as theprimary soil-plant interface for their hosts, which include many important


forest trees. Unfortunately, there have been relatively few attempts tostudy ECM fungus systems in-situ. Some knowledge has come fromobservations of large wide) patches dominated by the myceliumof some ECM fungi, where soil physical and chemical properties arealtered (Griffiths et al. 1994, 1996; Unestam and Sun 1995). It isconsidered that changes to soil properties (higher ion concentrations,oxalate accumulation, etc.) in these “hyphal mats” result in increasednutrient availability due to accelerated weathering of soil minerals(Griffiths et al. 1994; Paris et al. 1995). Hyphae of ECM fungi have alsobeen implicated in the weathering of rock fragments in soils (Jongmanset al. 1997; Landeweert et al. 2001).The role of ECM fungi in nutrient cycling depends on their ability toacquire the mineral nutrients required by host plants from inorganic andorganic sources in soils. It is generally assumed that ECM fungi have agreater capacity to acquire organic forms of nutrients than AM fungi(Marschner 1995; Smith and Read 1997). Ectomycorrhizal rootsnormally are more abundant in topsoil layers containing humus, than inunderlying layers of mineral soil (Meyer 1973; Harvey et al. 1978;1997). Evidence for the utilisation of organic materials by ECM fungihas been provided by production of enzymes capable of breaking downorganic N sources in experimental systems (Table 5) and studies ofECM plants and fungi (Michelsen et al. 1998). Some ECM fungispecialise in the breakdown of organic compounds in animal wastes(Sagara 1995; Yamanaka 1999). Observations of substrate utilisation andisotopic composition of fungi have shown that ECM fungi generally havea much lower capacity to degrade complex substrates such as cellulose,lignin or phenolics than saprophytic fungi (Dighton et al. 1987; Bending

resulting in substantial transfer of to the mycorrhizal fungus. We stillhave much to learn about how ECM fungi acquire nutrients directly from

and Read 1997; Kohzu et al. 1999). However, measurements ofcontent of fruit bodies by Gebauer and Taylor (1999) suggested thatsome ECM fungi primarily utilised organic N sources from humus, whileothers depend on inorganic N from the soil. Organic N sources havebeen shown to be important to plants with ECM in arctic tundra,Australian eucalypt forests and boreal forests (Kielland 1994; Turnbull etal. 1995). The impact of different nutrient sources on competitionbetween plants is considered in Section 4.2.

In situ studies of litter decomposition have shown that hyphaeconsidered to belong to ECM fungi were only present in the latter stagesof this complex process and most of the work is done by other types ofsoil microbes and animals (Ponge 1991). The presence of ECM fungimay increase, or reduce the rate of breakdown of soil organic matter(Gadgil and Gadgil 1975; Dighton et al. 1987). Even if some ECM fungihave a role in organic matter breakdown, this would be less importantthan their role in coupling plants into the soil food web. Lindahl et al.(1999) studied interactions between mycelia of several ECM fungi and awood rotting fungus (Hypholoma) in a microcosm experiment. Theyobserved antagonistic interaction by several ECM fungi on Hypholoma,


organic sources or indirectly from other soil organisms responsible fornutrient cycling and how these processes are affected by environmentalfactors. Ectomycorrhizal fungi are the final step in the soil nutrientcycling process over a large portion of the earth’s surface, a role that isessential for ecosystem sustainability.


3.2. Mycorrhizal dependencyDifferent categories of mycorrhizal dependency have been defined forplants with AM associations (Chapter 4, Section 3.2), but it is generallyassumed that all plants with ECM are obligately mycorrhizal (unable tosurvive to reproductive maturity without fungi). Experimentsdemonstrating substantial growth responses to ECM fungus inoculationare too numerous to list here and involve many of the plant genera listedin Table 1. However, these experiments normally use relatively infertilesubstrates that are ini t ia l ly devoid of ECM fungi and favourableenvironmental conditions. Demonstrations of growth responses due tomycorrhizal inoculation in the field have been less common, probablybecause mycorrhizal fungi are already present, soils may be more fertileand environmental conditions are not always suitable for fungal activity(Castellano 1994; Jackson et al. 1995; Brundrett 2000). Practical uses ofECM fungi are considered in section 5.

There normally is a strong positive correlation between a plant’sdependency on mycorrhizas and the degree of mycorrhizal formation inits roots (Janos 1980; Brundrett 1991). Reports of ECM host plants innatural habitats without these structures on most of their fine root tips arerare. However, trees growing in flooded soils (e.g. Salix, Populus andMelaleuca) can have low levels of ECM, relative to trees of the samespecies in drier soils (Lodge 1989; Khan and Belik 1995). Trees withECM even occur naturally in extremely fertile soils, such as Pisonia oncoral islands where nesting birds provide massive fertiliser inputs(Ashford and Allaway 1985). In this case mycorrhizas appear to beinvolved in acquiring transiently-available nitrogenous products of uricacid degradation prior to leaching from the coral cay soils (Sharples andCairney 1997).

Perhaps the best evidence that hosts have an obligate requirement formycorrhizas come from plantation forestry, where the failure of treessuch as pines to establish without mycorrhizal inoculum has been notedwhen they are first planted in exotic locations (Trappe 1977; Smith andRead 1997). Further evidence comes from the nature of the root systemsof host trees such as conifers with thick, slow-growing roots without longroot hairs that contrast starkly with the fine root systems of plants that areable to grow well without mycorrhizas (Brundrett 1991; Marschner1995).

3.3. SpecificityThree different categories of host-fungus specificity were defined byMolina et al. (1992), who provide lists of fungi considered to belong ineach category. Narrow host range fungi are only known to associate withone genus of host plants, intermediate host range fungi associate withdifferent species of hosts wi th in a single plant family or group such as theGymnosperms and broad host range fungi form mycorrhizas with plantsfrom unrelated families. Most knowledge about the host specificity offungi is based on observations of fruit bodies under trees, so it should beassumed that lists of fungal associates for plant taxa contain some errors.


For example, the fungus Boletinellus merulioides and the tree Fraxinusappear in lists as ECM associates, but this tree only has AM in its rootsand this fungus forms a mutualistic relationship with a subterraneanaphid (Brundrett and Kendrick 1987). Further evidence of specificity isprovided by glasshouse and sterile culture synthesis experiments whichconfirm that fungi can form normal looking mycorrhizas with some hostplants but not others (e.g. Malajczuk et al. 1982; Burgess et al. 1993).However, some host-fungus combinations which form mycorrhizas in soilare incapable of doing so in sterile environments and vice versa(Malajczuk et al. 1982; Ba et al. 1994). Baiting experiments, wheredifferent ECM fungi were detected in the same soil using different hostplants, have also demonstrated fungus specificity (Jones et al. 1997;Massicotte et al. 1999). Lists of ECM host-fungus associations are basedon observations of fungal fruiting under certain plants backed up by theknowledge gained from synthesis experiments. Consequently, we mustexpect such lists to contain some errors.

Although some ECM fungi, including Cenococcum geophilum(LoBuglio 1999) and Hebeloma cristuliniforme (Marmeisse et al. 1999)have been reported to form ECM with a diverse array of host generabelonging to different plant families, many have a much narrower hostrange (Molina et al. 1992). These narrow range fungi commonly showspecificity at the host genus level with, for example, ca. 250 taxa thoughtto form ECM only with Douglas fir (Pseudotsuga) in North America(Molina et al. 1992) and a much larger number are likely to be restrictedto Eucalyptus in Australia (Bougher 1995). We must be mindful,however, that host ranges of the majority of ECM fungi have beeninferred from observations of co-occurrence of sporocarps and hosts inthe field (Molina et al. 1992). In many cases it is impossible to separateeffects of geographical d is t r ibut ion from true host specificity. Somereports of fungi with a single host may result from our limited knowledgeof fungal distribution patterns in many regions.

Intermediate host range fungi appear to be most common (Molina etal. 1992: Horton and Bruns 1998). This notwithstanding, individual treesin the field are normally colonised by both narrow and broad host rangemycobionts and this may have important influences on competitiveinteractions between tree species (Section 4.2). The number of species offungi in the broad host range category may well decrease with futuretaxonomic studies, as widely distributed fungi are found to comprise anumber of similar looking species. A good example of this is the speciesPisolithus tinctorius – reported to be one of the most common ECMfungi in the world, with a wide range of reported hosts and habitats.However, recent molecular, and biochemical evidence suggests that thistaxon consists of a number of species with more restricted host andhabitat preferences (Section 2 .1 ) .

Perhaps the best evidence that most ECM fungi are relatively specificcomes from the low diversity of fungi which occur in plantations of pinesand eucalypts grown in exotic locations (Dunstan et al. 1998; Brundrettand Bougher 1999). In many countries only a few genera have been


reported to occur with these hosts, but there are many species foundunder indigenous tree species. The number of species of fungi fruiting inAustralian Eucalyptus plantat ions increases with time in Australia, butremains low, even after many years, in most exotic locations (Lu et al.1999). Newton and Haigh (1998) found that diversity of ECM fungiassociated with particular hosts was positively correlated with the area ofthe UK occupied by these hosts plants. However, exotic trees introducedfrom other continents had a lower diversity of ECM fungi than wassuggested by the importance of these trees in landscapes. Ecologicalimplications of specificity of host fungal relationships are considered insection 5.2.

3.4. Pollution and climateNumerous reports attest to altered levels of ECM inoculum and/ordiversity at field sites affected by anthropogenic pollution from industrialor urban sources (e.g. Danielson and Pruden 1989; Tosh et al. 1993;Kieliszewksa-Roikicka et al. 1997). Because some of these studies wereconducted without quantification of soil pollutant status, and since acomplex range of pollutants is generally present at such sites, it isdifficult to infer any cause and effect relating to ECM fungal diversityand pollutants from these data. A clearer picture has arisen from simpleglasshouse- and field-based experiments that have considered the effectsof pollutants on ECM associations either singly or in combination(Cairney and Meharg 1999). These studies provide strong evidence thatmost forms of pollution can result in decreased ECM infection andaltered below-ground community structure, although such effects mayvary with the level and duration of exposure to the pollutant(s).

Nitrogen deposition can lead to a decrease in percentage total rootcolonisation by ECM fung i (e.g. Tétreault et al. 1978; Taylor andAlexander 1989; Haug et al. 1992; Taylor et al. 2000), although suchdecreases may be rather short-lived, disappearing within a few years ofsoil treatment (Arnebrant and Söderström 1992; Kårén and Nylund1997; Nilsen et al. 1998). Nitrogen fertilisation can also shorten thelifespan of mycorrhizal roots (Majdi and Nylund 1996).

Nitrogen addition can also profoundly affect the below-groundstructure of ECM fungal communities. Taylor and Read (1996) reporteda clear pattern of decreased ECM morphotype richness on Picea spp.hosts was associated with increased nitrogen deposition across Europe.Moreover, they observed a change from those ECM fungi that canreadily utilise organic nitrogen in favour of those which rely largely orsolely upon inorganic nitrogen sources, at sites where nitrogen depositionwas greatest. Smaller-scale studies also support marked shifts in ECMfungal community s t ructure in response to nitrogen deposition, withdecreases in the relative frequency of particular ECM fungi notedfollowing nitrogen fertilisation (Taylor and Alexander 1989; Kårén andNylund; 1997). The form of nitrogen in soil may further differentiallyinfluence below-ground ECM fungal community structure (Arnebrantand Söderström 1992). Significantly, the data of Arnebrant and


Söderström (1992) were collected 13 years following fertilisation andindicate that nitrogen-mediated changes to ECM fungal communitiesmay have more long-term ecological relevance than those observed foroverall ECM colonisation. Arnebrant (1994) has shown that intraspecificdifferences exist in the sensitivity of ECM extramatrical mycelial systemsto nitrogen additions, suggesting that growth of some fungi through soilcan be profoundly affected by nitrogen inputs, while others appearrelatively insensitive. Differences of this nature are likely to stronglyinfluence the relative competitiveness of ECM fungi (Arnebrant 1996)and may underpin nitrogen pollution-related shifts in structure of below-ground communities.

The influence of acid deposition on forest trees and their associatedECM fungi has received considerable attention. Some field andglasshouse investigations indicate that acid deposition may significantlydecrease percentage ECM infection (e.g. Danielson and Visser 1989;Esher et al. 1992; Stroo et al. 1988), while others report no obviouscorrelation between the two (e.g. Nowotny et al. 1998; Adams andO’Neill 1991). Frequently-cited reports of changes in ECM morphotypeassemblages resulting from acidification (e.g. Gronbach and Agerer1986; Roth and Fahey 1998), however, provide convincing evidence thatsoil acidification effects below-ground ECM fungus communities.Notably several studies recorded a decline in ECM fungal taxa thatproduce extensive mycelial systems in soil (Dighton and Skeffington1987; Markkola et al. 1995). Toxic metal pollution may similarly reduceECM infection and alter below-ground community structure (e.g.Chappelka et al. 1991), as may a range of organic chemical pollutants(e.g. Nicolotti and Egli 1998). Interactive effects between pollutants mayfurther influence the structure of ECM fungal communities (see Cairneyand Meharg 1999).

In contrast to other forms of pollution, elevated atmosphericconcentrations can increase percentage ECM infection of coniferous andhardwood hosts (e.g. Norby et al. 1987; Godbold et al. 1997). Thiseffect may be short-lived under some conditions (e.g. Runion et al.1997; Walker and McLaughlin 1997; Walker et al. 1999b) and there maybe strong interactive effects with edaphic conditions such as soil nutrientand/or moisture status and atmospheric temperature (Conroy et al. 1990;Delucia et al. 1997; Tingey et al. 1997). Recent work reveals profoundeffects of enrichment on ECM fungal communities, with some taxabeing positively influenced at the expense of others. Specifically, thesestudies suggest shifts in community structure in favour of taxa thatproduce extensive extramatrical mycelial systems in soil (Godbold andBerntson 1997; Godbold et al. 1997; Rey and Jarvis 1997). Single ECMfungi are also known to produce more substantial mycelial systems underelevated concentrations (Ineichen et al. 1995), the implication beingthat some ECM fungi may be carbon-limited at ambientconcentrations and that increased carbon availability favours taxa thatproduce more substantial mycelia. Ectomycorrhizal fungi are a majorpathway for carbon flow into soils and the magnitude of this pathway can


be increased by elevated at least in some cases (Runion et al. 1997;Walker et al. 1999b).

The distribution and mycorrhizal efficacy of fungi forming ECMassociations is influenced by climatic and edaphic factors (Slankis 1974;Smith and Read 1997). These fungi are generally considered to beacidophilic (preferring a low soil pH) inhabitants of litter layers near thesoil surface (A horizon), but some “early stage fungi” (Section 4.1)prefer mineral soils which may be calcareous. Tyler (1992) found thatfor macrofungi in a European forest (dominated by the ECM treeFagus), the relative importance of ECM fungi increased (and saprobesdecreased) in more-acidic soils. In this study, the distribution of manyfungi was correlated with edaphic factors, such as soil organic matter andmetal ion content.

Isolates of ECM fungi show considerable inter- and intraspecificvariations in responses to the factors listed in Table 4. Isolates of ECMfungi from polluted soils often have higher tolerance to metal ions underexperimental conditions, than isolates of the same species from normalsoils and thus may help their hosts to survive in these conditions (Hartleyet al. 1997; Meharg and Cairney 2000a). However, results obtained fromin vitro experiments are often poorly correlated with responses to similarfactors in soils (Cline et al. 1987; Coleman et al. 1989; Hung and Trappe1983; Hartley et al. 1997). It has been suggested that variations intolerance to edaphic factors may restrict geographic ranges of fungi,influence the outcome of fungal competition, or responses to factors suchas drought (Parke et al. 1983a; Last et al. 1984; McAfee and Fortin1986). It is apparent that a wide range of variation in tolerance to edaphicand climatic factors (such as temperature extremes, drought, soil toxicityetc.) often occurs, both between and within species of mycorrhizal fungiand that this variation likely represents adaptation to specific siteconditions (Trappe 1977; Trappe and Molina 1986).

4. Natural ecosystems4.1. Fungal communitiesForest plant communities that host ECM fungi are often relativelyspecies-poor, however fungal species richness within these forests ischaracteristically high (Malloch et al. 1980; Allen et al. 1995). Goodmanand Trofymow (1998); for example, estimated that up to 100 ECM rootmorphotypes (considered to representing either species or genera) werepresent in four 0.36 ha plots of an old-growth Canadian Douglas firstand. Similar estimates have been derived from ITS-RFLP and /ormorphotype data for stands of a range of other forest types dominatedby coniferous, deciduous, eucalypt, or dipterocarp trees (Pritsch et al.1997; Gehring et al. 1998; Goodman and Trofymow 1998; Ingleby etal. 1998; Glen et al. 1999; Jonsson et al. 1999a). It must be stressed thatthere are also some ECM forest habitats in which fungal diversity is low.These include, Alnus rubra stands in North America which host only ahandful of ECM fungal taxa (Miller et al. 1991), and Pisonia grandis oncoral cays in the western Indian and eastern Pacific oceans – which may


be associated with a single ECM taxon across its entire geographicalrange (Ashford and Allaway 1985; Chambers et al. 1998).

The diversity of ECM fungi in recently-disturbed habitats istypically much lower than in undisturbed sites and there are some specieswhich are characteristically found in disturbed sites (Danielson 1985;Mason et al. 1987; Jumpponen et al. 1999). Reductions in fungaldiversity from disturbance likely result because many fungi areeliminated, do not have resistant propagules, or have a limited capacity toadapt to large changes to their environment (Section 2.4).

Fungal succession occurs under maturing stands of trees as a fewpioneering fungi are gradually replaced by increasing numbers of fungiwhich typically fruit in older habitats (Gardner and Malajczuk 1988;Termorshuizen 1991; Richter and Brun 1993; Keizer and Arnolds 1994;Lu et al. 1999). Visser (1995) examined species richness in regeneratingPinus banksiana stands following fire. For the first six years, roots werecolonised by relatively few fungi, but diversity increased markedly in 41year-old and 65 year-old stands, the latter having a broadly similarcommunity to a 112 year-old stand. Lu et al. (1999) observed that thediversity of ECM fungi fruiting in Eucalyptus globulus plantations inWestern Australia steadily increased by approximately two species peryear (Figure 3). In another study of ECM fungal successions onpreviously cultivated land, ECM species richness increased until canopyclosure and then declined (Dighton and Mason 1985). Fungal diversitygenerally increases until late in succession, when the number of speciespresent may decline when fungi with more specialised host or substratepreferences predominate (Bills et al. 1986; Last et al. 1984).

Observations of fungal succession in aging tree stands have resultedin the designation of two groups of fungi which occupy separate ends ofthis continuum. Fungi which typically associate with young trees indisturbed habitats or plantations have been termed early-stage fungi,while those that associate with old trees are termed late-stage fungi(Dighton and Mason 1985). Most studies of tree monocultures indisturbed habitats have supported this concept (e.g. Chu-Chou and Grace


1982; Gardner and Malajczuk 1988; Cripps and Miller 1993; Visser1995; Lu et al. 1999). However, there are cases where succession inyoung forests does not start with early stage fungi (Newton 1992; Keizerand Arnolds 1994; Helm et al. 1996; Bradbury et al. 1998). Early stageECM fungi generally are easier to introduce into disturbed sites than latestage fungi (Danielson 1985; Lu et al. 1999). Typical examples of earlystage fungi that are often observed in young plantations include membersof the genera Pisolithus, Scleroderma and Laccaria.

The factors underlying ECM fungal species richness in foresthabitats have not been elucidated but, spatial and/or temporal resourcepartitioning, along with patterns of disturbance and competition may allplay a role (Bruns 1995). A number of soil factors, including host plantage and physiological status, soil microbes, litter accumulation, fungalcompetition and inoculum availability could drive mycorrhizal fungussuccession (Danielson 1985; Keizer and Arnolds 1994; Bruns 1995;Smith and Read 1997; Lu et al. 1999). Early stage ECM fungi typicallygrow in disturbed mineral soils with low organic matter with young hostplants, while late stage fungi occur in the litter layer of mature forest soils(Mason et al. 1987; Gardner and Malajczuk 1988; de Vries et al. 1995;Lu et al. 1999). Physiological differences between early and late stagefungi are also apparent in aseptic culture experiments (e.g. Gibson andDeacon 1990). While we do not fully understand the factors influencingchanges in ECM species richness with stand age, it is likely that changingsoil properties and/or altered patterns of carbon allocation associated withtree maturation play a significant role.

Spatial variability in soil properties and fungal population structureare also likely to be important in forests with high ECM fungal diversity.For example, of the 2000 or so fungi which associate with Douglas firthroughout its range, only about 10% of these will be found at any onelocation (Helm et al. 1996). Some of this high beta diversity may be due,in part, to spatial variations in soil properties which influence thecompetitiveness of individual fungi . However, it is likely that localvariations in site histories and fungal dispersal events were responsible forinitiating this spatial variability, which is maintained by processes we donot yet understand.

Although generally species-rich, below-ground communities ofECM fungi are characteristically dominated by a small number ofcommon taxa (e.g. Gehring et al. 1998; Horton and Bruns 1998; Jonssonet al. 1999b). These locally-abundant taxa, which inhabit a largeproportion of available roots and presumably explore a proportionatelylarge soil volume, may be functionally dominant (Horton and Bruns1998), but, this remains to be demonstrated. Indeed, while ECM fungaldiversity is widely regarded as important in ecosystem functioning andforest sustainability (e.g. Jones et al. 1997; Pritsch et al. 1997), the extentof functional diversity within the taxonomically diverse ECM fungalcommunities is currently unknown. It is likely that many ECM fungaltaxa fulfil broadly similar ecological roles and, as such, that a high degreeof functional redundancy exists (Allen et al. 1995). Attempts have been


made to group taxa based on their abilities to utilise various substrates assources of nutrients such as amino acids, which some ECM fungi canutilise but others cannot (Abuzinudah and Read 1986; Gebauer andTaylor 1999). It is known that substantial inter- and intra-specificvariations occur between ECM fungi in their responses to environmentalconditions (Table 4 and see Cairney 1999), growth and survival strategies(Section 2.2), etc. Our relatively poor understanding of ECM funct ioningin natural ecosystems, severely hampers our ability to predict the extentto which disturbance, pollut ion and other stresses will influencefunctioning of ECM-dominated plant communities.

4.2. Plant communitiesMycorrhizal associations could influence plant community structure, byaffecting the richness or evenness of populations of coexisting plants, orby changing the competitive ability of species. Table 7 in Chapter 4,shows 4 categories of intraspecific and interspecific interactions involvingplants with different mycorrhizal requirements and association types.Facultatively mycorrhizal plants are not considered here, as plants withECM generally are h igh ly dependent on these associations (Section 3.2).Interactions between non-mycorrhizal plants are considered in Chapter 4.The impact of ECM associations on competitive interactions betweenplants would dif fer if competitors have ( 1 ) the same type of association,or (2) different types of associations. These cases are consideredseparately below.

4.2.1. Interactions between plants with ECMWhen growing together, plants with the same type of mycorrhizas arelikely to be more equal competitors than plants with different types ofmycorrhizas (Newman 1988). Plants with ECM presumably compete withother ECM plants for the same pools of soil resources (forms ofnutrients), but plants with other nutrient uptake strategies (e.g. AM,ericoid mycorrhizas, non-mycorrhizal roots) may access different formsof nutrients. Competition between ECM hosts is more complex thanbetween plants with AM (Chapter 4, Section 4.2), because ECM fungivary more widely in their capabilities (e.g. to access organic nutrients)than AM fungi . Also, many ECM fungi are specific to certain hosts(Section 3.3), while AM fungi generally are non-specific.

When different species of ECM plants grow together, the nature ofcompetition will differ if they share the same (broad host range) fungiwith a common pool of mycelia, or have separate host-specific ECMfungi that compete for soil resources. Finlay (1989) and Perry et al.(1989b) found that fungi which associate with two competing hosts, allowboth to grow well together, but more specific fungi stimulated the growthof one host to the detriment of the other plant. The presence of host-specific fungi in these experiments shifted the balance of nutrientcompetition in favour of their hosts. However, these experiments wereconducted with only a few fungi , while plants in nature normallyassociate with a diverse mixture of broad and narrow host range fungi, so


the impact of any one fungus on plant competition is likely to be muchless dramatic in the field. Nevertheless, we can postulate that the evolutionof specific host-fungus interactions may have occurred to providebenefits during nutrient competition and is more likely to occur fordominant trees in forest communities.

Broad host range ECM fungi can form mycelial connectionsbetween different host taxa in the field, facilitating bi-directional transferof carbon between them and resulting in a net carbon gain by one host(Simard et al. 1997). Common hyphal networks may assist theestablishment of seedlings which share mycorrhizal partners withdominant trees. It has often been suggested that seedlings growing undermature trees of the same species are supported in this way (Newman1988; Smith and Read 1997). This may explain why Pseudotsuga treesusually become established in patches of the ectendomycorrhizal shrubArctostaphylos, but not under AM plants (Horton et al. 1999). A deltastudy has shown that most of the carbon received by shared ECM fungicomes from the overstorey trees, which help to support understoreyspecies (Högberg et al. 1999). Preservation of ‘guilds’ of interconnectedhost plants (Perry et al. 1989a) and their associated fungal partners mayhelp support the sustainability of ECM plant communities. Molina andTrappe (1982) have suggested that plants such as Alnus rubra andPseudotsuga menziesii, which often form pure stands during earlysuccession, tend to form specific associations with ECM fungi, whilespecies such as Tsuga heterophylla, which become established in theunderstorey of other trees, usually have non-specific ECM associates.Thus, the availability of particular strains specifically required bydifferent hosts could be a regulat ing factor during plant succession insome habitats.

Despite the evidence provided above, the ecological importance ofcarbon and nutrient transfer between interconnected plants in naturalecosystems is not clear. The fact that only a very small proportion ofmycorrhizal seedlings survive to become trees, demonstrates that helpprovided by mycelial interconnections is generally not sufficient to affectthe outcomes of competit ion (Newman 1988; Brundrett 1991). Thegreatest impact of sharing a common type of mycorrhiza appears to bean increase in the functional similarity of the roots systems of differentspecies, so they are more equal competitors for soil nutrients which limitplant growth (Brundrett 1991) . Even if the magnitude of below-groundcarbon movements along hyphal interconnections is not sufficient toinfluence survival and growth of subordinate taxa, these interconnectioncan stil l function as a form of Cupertino where plants help each other bysupporting a common mycelial network.

The most extreme examples of resource transfer between plants withcommon mycorrhizal fungi are non-photosynthetic plants in theOrchidaceae and Monotropoideae (Ericaceae). Plants such as Monotropalive entirely by tapping into mycorrhizal fungus networks supported byconnected autotrophic plants (Björkman 1960; Furman and Trappe1971). Associations of non-photosynthetic plants appear to be more


parasitic (from the fungal perspective) than mutualistic. These plantsapparently have evolved a high degree of specificity in their associationswith ECM fungi (Cul l ings et al. 1996; Taylor and Bruns 1997, 1999).

4.2.2. Interactions between different types of mycorrhizal associationsVegetation dominated by plants with one mycorrhiza type apparently canbe a hostile environment for plants with other associations to grow in. Ithas been reported that trees with ECM often fail to become established insites dominated by plants with ericoid mycorrhizas such as shrublands ofGaultheria, Kalmia, Rhododendron or Calluna heathlands (Robinson1972; Messier 1993; Yamasaki et al. 1998; Walker et al. 1999b).Restricted availability of mineral nutrients and reductions in ECM fungusactivity were reported within these habitats. Robinson (1972) suggestedthat an allelopathic inhibition of ECM fungi by exudates of Callunacontributed to nutrient deficiency problems for tree seedlings inheathlands.

Examination of the world-wide distribution of plant communitiesreveals that most forests are dominated by trees which form ECM or AMassociations and forests where both types of trees are equally dominantare rare (Brundrett 1991; Allen et al. 1995; Smith and Read 1997). It hasbeen proposed that ECM-tree dominated plant communities are morelikely to occur in soils with high organic matter and a predominance oforganic nutrients, while AM-trees are more likely to dominate in mineralsoils (Section 3.1). These relationships may explain the predominance ofECM forests in cooler climates. The situation in tropical regions is morecomplex as there do not appear to be major differences in soil propertiesbetween ECM and AM dominated forests in the same regions (Högberg1986; Högberg and Alexander 1995; Newbery et al. 1997; Moyersoen etal. 1998). In eucalypt-dominated forest in Western Australia, roots ofplants with ECM, AM or non-mycorrhizal cluster roots tended to bedistributed in different soil patches, so may avoid direct competition fornutrients (Brundrett and Abbott 1995).

Changes to soil properties apparently result because host treesproduce leaves which are highly resistant to decomposition, resulting inslower nutrient cycling and a predominance of organic nutrient sourceswhich are more accessible to ECM than AM fungi (Girard and Fortin1985; Allen et al. 1995). It has also been proposed that substances in theleaf litter of ECM plants such as pine trees can inhibit AM fungi(Tobiessen and Werner 1980; Kovacic et al. 1984). Plant communitiesdominated by ECM plants may have a tendency to be self-perpetuating,by producing a soil environment which is hostile to AM fungi.

Some plants with AM have also been reported to adversely affectECM fungi. Hanson and Dixon (1987) found that ECM fungi couldreduce the impact of allelopathy due to fern frond leachates on oak(Quercus rubra) seedlings. The abundance of weeds with AMassociations can influence ECM formation by pine trees in plantations(Sylvia and Jarstfer 1997). Colonisation by AM fungi reduces thelifespan of roots of Populus, a tree which predominantly has ECM


associations (Hooker et al. 1995). Allelopathic interactions betweencompeting plants are considered to be common in plant communities, butthe role of mycorrhizal fungi in these interactions has rarely beeninvestigated.

A second form of competition between different types ofmycorrhizal fungi occurs within the root systems of plants which arehosts to two types of mycorrhizal associations. In Australia, plants withECM associations usually also have some VAM in their roots. Theseplants include major species used in plantation forestry belonging to thegenera Casuarina, Allocasuarina (Casuarinaceae), Eucalyptus, Melaleuca(Myrtaceae) and Acacia (Mimosaceae) (Brundrett 1999). Plants with dualECM/VAM associations are less often reported from other parts of theworld (Brundrett 1991), but there are exceptions such as Alnus, Populus,Salix and Uapaca (Lodge and Wentworth 1990; Arveby and Granhall1998; Moyersoen and Fitter 1998). Most gymnosperms with ECM arehighly resistant to AM fungi, but their roots may contain vesicles andhyphae if grown with a companion AM plant (Hooker et al. 1995; Smithet al. 1998). A succession from VAM to ECM associations in Eucalyptusand Alnus roots often occurs as trees age in field soils (Gardner andMalajczuk 1988; Bellei et al. 1992; Oliveira et al. 1997; Arveby andGranhall 1998). Ectomycorrhizal fungi have also been observed togradually displace VAM in Eucalyptus roots in the glasshouse (Lapeyrieand Chilvers 1985; Chen et al. 2000). Plants which can form both ECMand AM associations occur in some ecosystems. For these plants, it seemsthat AM are most important for their young seedlings, perhaps due to thetime required for ECM fungus dispersal and establishment, while ECMusually becomes dominant when they grow older. There are plants, suchas some Acacia species, which are highly receptive to ECM and AMassociations throughout their lives, but this is rare and most species showa clear preference for a single type of mycorrhiza (Brundrett 1999).

4.3. AnimalsThe roles of animals as consumers and dispersal agents of ECM fungi areconsidered in section 2.3. Tree-mycorrhizal fungus-dispersing animalinterrelationships are important in forests, especially in western NorthAmerica and Australia (Maser and Maser 1988; Claridge et al. 1992).Animals which disperse ECM fungi facilitate ecosystem recovery afterdisturbance (e.g. Claridge et al. 1992; Cázares and Trappe 1994;Johnson 1995; Reddell et al. 1997; Cázares et al. 1999).Ectomycorrhizal fungi and their host plants must also be consideredduring any attempts to manage mycophagous animal species. Forexample, the occurrence of hypogeous ECM fungi in different vegetationtypes is thought to limit the distribution of the Northern Bettong(Bettongia tropica) which needs these fungi for food in the dry season(Johnson 1996).

The importance of animals as vectors for ECM fungi is suggested bythe evolution of truffle-like fruit bodies in most families of ECM fungi inAustralian eucalypt forests (Bougher and Lebel 2001). These


subterranean fruit bodies would only have a selective advantage inhabitats where animal dispersal of fungi is more effective than airbornespore dispersal. Marsupials which consume spores are efficient dispersalagents, because they forage near trees over a wide area and havecommensal dung beetles which bury their spore-laden faeces (Johnson1996). Unfortunately, many Australian mycophagous marsupials are nowextinct throughout most of their former ranges, and this could effectecosystem functions, especially the capacity for recovery afterdisturbance. We must consider secondary symbiotic associations, such asthe animal vectors of ECM fungi, as well as the primary tree-fungusassociations when we monitor the quality of remnant vegetation, orattempt to restore plant communities.

5. Utilising mycorrhizasThis section will focus on plant conservation and ecosystem restoration.However, there also are concerns about the conservation of ECM fungi inregions where their populations are declining due to changes to soilconditions caused by pollution or over-collection by humans (Arnolds1991; Boujon 1997; Hosford et al. 1997). Ectomycorrhizal fungi exhibitfunctional diversity and are adapted to local environmental and soilconditions (Table 4). Consequently, it would be important to conserverepresentatives of all habitats and soils within regions to maintain thefunctional diversity of mycorrhizal fungi and other organisms.Conservation of particular ECM fungi may also be required for non-photosynthetic plants in the Ericaceae (Monotropoideae) which arewholly dependent on a type of ECM fungi and have specific fungalpartners (Cullings et al. 1996; Kretzer et al. 2000).

5.1. Ecosystem restorationMycorrhizal technology can be designated as any artificial means ofintroducing fungi to new habitats or manipulating existing populations offungi. Most work on the introduction of ECM fungi has been forplantation forestry, or for valuable edible fungi such as the black truffle(Tuber melanosporum). Inoculation technologies have been developed tointroduce fungi in the nursery or field, using soil, spores or myceliaproduced in sterile culture as inoculum (Brundrett et al. 1996c). Beforepromoting mycorrhizal technology, it is necessary to evaluate its relativecosts and benefits (Brundrett 2000). Costs arise from the resources andtime required to acquire appropriate fungi, produce and apply inoculumand implement quality control measures to confirm inoculated fungi arepresent. Benefits primarily concern short-term increases in survival and/orgrowth of plants. Some ECM fungi may offer added benefits byinfluencing the activities of other soil microorganisms, or degradation ofpersistent organic pollutants in soil (Meharg and Cairney 2000b). Long-term benefits from increased mycorrhizal fungus biomass orfunctionality are suggested in the literature, but have not been measuredin the field (Section 3.1). Potential environmental costs that may arisefrom introductions of mycorrhizal fungi into different locations should


also be considered (Section 5.2).When ECM fungi are introduced to a site (usually by planting

seedlings inoculated in the nursery), their success depends on their abilityto spread through the soil to new roots and the outcome of competitionwith indigenous fungi (Last et al. 1984; McAfee and Fortin 1986). Host-fungus specificity is also important, as poorly compatible fungal isolateswill often fail to establish. Cases where substantial improvements in treegrowth due to mycorrhizal inoculation were measured are outnumberedby trials where there was no measurable response (Castellano 1994;Jackson et al. 1995; Brundrett 2000). Nevertheless, there have been caseswhere ECM fungus inoculation has resulted in significant growthenhancement in the field, especially where trees have been grown indisturbed habitats such as mine sites, or exotic locations with fewcompatible fungi (Malajczuk et al. 1994; Castellano 1994; Reddell et al.1999). Responses to mycorrhizal inoculation are also highly dependenton soil conditions, especially soil fertility. For example, Cistus incanusfails to establish in infertile calcareous soils without mycorrhizal fungi,but grows well without fungi if fertilised (Berliner et al. 1986).

Mycorrhizal fungi may be an important consideration in rare speciesrecovery programs. They sometimes are included in cultivation attemptsas an insurance policy to eliminate nutritional factors as a cause offailure. Inoculation of tissue cultured plants is especially important forsubsequent plant survival and growth during critical early stages ofestablishment in soil (Martins et al. 1996; Reddy and Satyanarayana1998).

5.2. Potential problems with fungal technologyWhile ECM inoculation programs were required for the successfulestablishment of plantations of trees such as pines and eucalypts at exoticlocations, they have the effect of introducing alien ECM fungal taxaalong with their hosts. It is possible that alien fungi may influence localECM fungal diversity via invasion of native forest systems. The extent towhich this occurs will depend upon the ability of the introduced fungi topersist at the exotic sites and the extent to which they are able to formECM with the native vegetation. There is certainly evidence that someintroduced ECM fungi can persist in plantations at exotic locations formany years following introduction (e.g. Martin et al. 1998; Selosse et al.1999). It has generally been assumed that no environmental costs willarise from introductions of mycorrhizal fungi into different geographiclocations. The host specificity of many fungi often prevents fungi fromassociating with indigenous hosts belonging to other families (Section3.3). However, introducing fungi that are more, or less effectivesymbiotic partners than indigenous strains, may impact on plantcommunity structure, by inf luencing the outcome of competitionbetween plants.

We would expect the potential for broad host range fungi to invadeindigenous vegetation should be greater than for fungi which associatewith few host plants. Fungi associated with Eucalyptus spp. are a useful


example in this context. The geographical isolation of the Australianflora is considered to have enabled co-evolution of highly specific ECMassociations within the genus Eucalyptus, that are poorly compatible withtree species from other continents (Malajczuk et al. 1982, 1990; Bougher1995). Recent investigations in Kenya, for example, indicate thatPisolithus species introduced during the past 100 years into Eucalyptusand Pinus plantations are only found in association with their respectivehosts (Martin et al. 1998). Australian species of Laccaria, Hydnangiumand Hysterangium associated with eucalypt plantations at exotic sites alsoappear to remain confined to that genus (Castellano 1999). None of theECM fungi introduced to Australia with Pinus are known to have spreadinto Eucalyptus forests (Molina et al. 1992; Bougher 1994; Dunstan etal. 1998). However, the pine fungus Amanita muscaria is now foundunder Nothofagus in Tasmania and New Zealand (T. May pers. com., P.Johnson and P. Buchanan pers. com.). Fungi which readily switch to newfamilies of host plants include several Amanita species which havecrossed over to eucalypts introduced to North America and Europe(Brundrett and Bougher 1999). These relatively promiscuous fungiapparently can invade indigenous forests as “weeds” that compete withindigenous species. Perhaps we should be concerned about some feralfungi, such as extremely toxic Amanita species, that have the potential toinvade native forests and ki l l indigenous fungus-feeding animals.

6. ConclusionsThis review attempts to summarise key roles of ECM fungi in naturalecosystems and provide information that should be of value to peoplewho study processes in or help to conserve natural habitats. Plants withmycorrhizas are dominant in most communities (Table 1 in Brundrettand Abbott, this volume). Thus, mycorrhizal fungi typically are theprimary soil interface for plants and must be considered in all studies ofnutrient cycling or impacts of nutrient supply on plant productivity ordiversity. Processes mediated by ECM associations in natural ecosystemsare listed in Table 6.

There is much scope for future ecological work investigating theprocesses listed in Table 6. In these studies, the first challenge is todetermine what type of mycorrhizal associations the plant(s) beingconsidered have. Information about mycorrhizal associations has beensummarised for some locations such as the UK (Harley and Harley 1987)and Australia (Brundrett 1999). Information exists for many otherlocations, but may be harder to find. Published lists must be expected tocontain some errors or misinterpretations and contradictory data existsfor some species. Consequently, it will often be necessary for researchersto examine roots of their plant species using microscopic techniques toconfirm the presence of mycorrhizal associations (Brundrett et al.1996c).

The second challenge is to determine if mycorrhizal fungi arealready present in soils where host plants will be grown. Samplingmethodologies exists for detecting inoculum of these fungi (Brundrett et


al. 1996c). However, we would expect ECM fungi already to be presentin most habitats, provided compatible hosts occur nearby and soils havenot been substantially altered by disturbance, pollution, etc. A thirdchallenge may be to determine if it is appropriate to introduce fungi tosites and when it is unnecessary or even harmful to do so (Section 5.2).Ectomycorrhizal fungi can be dispersed by wind and animals and mayrapidly arrive in new sites. Some host plants may also grow well initiallywithout ECM fungi provided AM fungi are present or soils are relativelyfertile.

We hope that we have provided some guidance about where ECMassociations need to be considered in scientific studies of ecosystems orindividual species. This information would also be relevant to themanagement of plants, plant communities and mycophagous animals.These fungi have many important roles (Table 5) and the fruit bodies ofsome species are important as food sources for animals and humans. Theimportance of ECM associations was first proposed by Frank in 1885.Since then, we have learned how to manipulate these associations inexperiments and have amassed a substantial body of information about


their roles in plant nutrition. However, there is still much to learn aboutECM in natural ecosystems, especially concerning:

How they acquire soil nutrients from organic and inorganic sources inco-operation/competition with other organisms in soil food webs.The magnitude of nutrient and carbon transfer between connectedindividuals of the same and different species and the role of thesetransfers in ecosystems.The impact of ECM associations on the disease resistance, waterrelations, etc. of their hosts.The influence of changes to soil structure and chemistry caused byECM fungus hyphae (e.g. hyphal mats, carbon storage, weatheringof minerals, etc.) on plant nutrient supplies.

The role of taxonomic and functional diversity in these fungi. (i.e.Why there are many species of ECM fungi in some habitats and fewin others?)

(i)

(ii)

(iii)

(iv)

(v)

Perhaps the start of a new millennium is an appropriate time toreconsider where we heading. It is time to stop repeating the same basicexperiments demonstrating benefits provided to yet another plant speciesusing highly simplified experimental conditions. Instead we need to shiftfocus to the roles of ECM in-situ in natural ecosystems, by consideringthe neglected areas of research listed above. Experiments will be moredifficult in complex systems, but results will be much more meaningful.

AcknowledgementsThe authors are grateful to Neale Bougher, Bill Dunstan, Antoni Milewskiand a reviewer for comments on the manuscript.

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Chapter 6

ARBUSCULAR MYCORRHIZAS IN PLANTCOMMUNITIES

Mark C. Brundrett

Soil Science and Plant Nutrition, Faculty of Natural and Agricultural Sciences, TheUniversity of Western Australia, Crawley 6009, Western Australia (current address);

CSIRO Forestry and Forest Products, CSIRO Centre for Mediterranean AgriculturalResearch, Private Bag No 5, Wembley 6913, Western Australia (former address); andScience Directorate, Kings Park and Botanic Garden, Botanic Gardens and ParksAuthority, West Perth 6005, Western Australia (correspondence)

Lynette K. Abbott

Soil Science and Plant Nutrition, Faculty of Natural and Agricultural Sciences, TheUniversity of Western Australia, Crawley 6009, Western Australia

1. General introductionMuch of our understanding of the role of arbuscular mycorrhizas (AM)comes from experiments with individual plants and fungi using simplifiedsoil conditions, but there have been some attempts to study their functionsin natural ecosystems. This review concentrates on the importance of AMassociations in plant communities, their role in competition betweenplants and in plant establishment in disturbed habitats - as these have thegreatest relevance to plant conservation.

1.1. AssociationsArbuscular mycorrhizas are mutualistic associations between soil fungiand plant roots. These associations are also referred to as vesicular-arbuscular mycorrhizas and are abbreviated as AM or VAM (the termendomycorrhiza should no longer be used). Arbuscular mycorrhizasconsist of hyphae, arbuscules and vesicles within roots, as well as hyphae,spores and other structures in the soil (Figure 1). The partners in theseassociations include members of the fungus kingdom in the Zygomyceteorder Glomales and most vascular plants. The host plant usually receivesmineral nutrients obtained from the soil by the fungus while, in exchange,the fungus partners obtain photosynthetically derived carbon compounds


from the plant. Plants may receive other benefits from mycorrhizal fungi,but these are not well understood (see Section 3.1). Mycorrhizas consistof plant roots and mutualistic fungi in their soil environment and thesethree factors must be considered together when studying theseassociations.

Arbuscular mycorrhizas in plant communities 153

1.2. Host plantsIt has often been stated that most plants in terrestrial ecosystems havemycorrhizal associations, but there has only been one recent attempt tocatalogue data supporting this assertion (Brundrett 1991). Informationabout the mycorrhizal status of plants occurring in major ecosystems andedaphic communities is summarised in table 1. Arbuscular mycorrhizasare the most important type of association in most ecosystems. The onlyexceptions are ecosystems or zones within ecosystems dominated by treeswith ectomycorrhizas and habitats where adverse climatic or soilconditions severely limit plant productivity. However, plants with AM arestill also important in most extreme habitats. Summaries of mycorrhizalstrategies by plant taxa (Newman and Reddell 1987; Trappe 1987; Peatand Fitter 1993), or within geographic regions (Harley and Harley 1987;Koske et al. 1992; Brundrett 1999) are also available.


It is common to see statements such as 95% of plants aremycorrhizal in the literature, but this is not accurate. Trappe (1987)provided a more accurate estimate by reviewing data for 6500angiosperm species whose mycorrhizal status is known from the scientificliterature (approx. 3% of angiosperm species). Figure 2 summarises theseresults. The actual proportion of plants with mycorrhizas isapproximately 80% of examined species with about 60% reported to haveAM. However, these conclusions may be somewhat biased because moredata came from the temperate northern-hemisphere than from otherregions. Many gymnosperms and ferns also have AM associations(Brundrett 2001). An understanding of the importance of mycorrhizalassociations at the community level requires data on the relativedominance of plants with different types of mycorrhizal associations innatural ecosystems. These data are only available for a few plantcommunities (Brundrett 1991).

155Arbuscular mycorrhizas in plant communities

1.3. FungiFungi forming AM associations include over 150 species belonging tothe genera Gigaspora and Scutellospora, Glomus, Acaulospora,Entrophospora, Paraglomus and Archaeospora in the Zygomycete orderGlomales (Morton and Benny 1990; Redecker et al. 2000b; Morton andRedecker 2001). They are primitive members of the fungus kingdomwhich are not closely related to any other living group of fungi. Similarlooking associations have been found in fossilised rhizomes of earlyvascular land plants and evidence of their spores extends back to theOrdovician (Stubblefield and Taylor 1988; Pirozynski and Dalpé 1989;Redecker et al. 2000a). Mycorrhizal fungi are thought to live in aparticular habitat for thousands of years with little genetic change(Trappe and Molina 1986). The relatively small number of extant AMfungus species and the lack of sexual reproduction in this group of fungialso suggest that the potential for genetic change within these species islimited (Tommerup 1988; Morton 1990). The hyphae and spores of AMfungi are multinucleate and likely also heterokaryotic, so genetic changesmay occur through hyphal anastomosis or somatic recombinationinvolving different nuclei (Tommerup 1988; Trappe and Molina 1986;Sanders et al. 1996; Bever and Morton 1999; Lanfranco et al. 1999).Careful taxonomic studies (Morton 1988), the use of isoenzymes andDNA-based methods (Hepper et al. 1988; Sanders et al. 1996; Clapp etal. 1999) and differential responses to soil and environmental conditions(Section 3.3) have demonstrated considerable variation within currentlydefined taxa of AM fungi.

2. Biology and ecology of AM fungi2.1. Distribution, abundance and diversityKnowledge of the biology and ecology of AM fungi is limited bytechnical difficult ies in both ident i fying and quantifying species presentin soils. The methods used to identify glomalean fungi in soils or rootsinclude (i) separation of spores from soil, (ii) isolation into living cultureswith a host plant, ( i i i ) recognition of infection patterns in roots and (iv)use of biochemical or DNA-based methods (Brundrett et al. 1996c). Caremust be taken in interpreting results obtained using any of these methodsin population studies, as the apparent diversity of fungi and theirperceived relative dominance will depend on the procedure used(Brundrett et al. 1999a; Douds and Millner 1999).

Most of our knowledge about fungal populations comes fromlooking at spores, because they are relatively easy to separate from soilsand used to identify fungal species. However, the other methods discussedbelow are now considered to provide a more accurate picture of fungaldiversity. Surveys of AM spores have found up to 23 species of fungi inone soil sample (Brundrett 1991; Douds and Millner 1999). Thisrepresents a fairly high taxonomic diversity of these fungi (consideringthat only about 150 species have been named) (Bever et al. 2001).Technical difficult ies with spore separation from soils contribute to theinaccuracy of surveys, especially if soils are fine textured, fungi sporulateinfrequently, spores occur within organic matter, or they are notdistributed uniformly.

DNA methods have recently been used to identify or quantify AMfungi in soils (Clapp et al. 1995; Helgason et al. 1999). However, dataobtained by this means has been limited due to technical difficulties(Sanders et al. 1996; Douds and Millner 1999; Lanfranco et al. 1999).The extraction of lipids and analysis of fatty acid profiles is anotherpromising method for quantifying AM fungi in soils (Graham et al.1995; Olsson 1999).

Glomalean fungi must be grown in association with a living plant toprovide material for research purposes, practical applications andtaxonomic study. Fungi are usually propagated using “pot cultures”where an inoculated plant is grown in a sandy soil supplied with low levelsof phosphorus (Menge 1984; Jarstfer and Sylvia 1993). These fungi canalso be grown using aeroponics or root-organ cultures (Jarstfer andSylvia 1993; Bécard and Piché 1992). Most pot cultures are initiatedusing spores separated from a field soil, or soil from a field site (Jarstferand Sylvia 1993; Bever et al. 1996; Brundrett et al. 1996c). Soil-basedtrap cultures often contain additional species to those found byexamining spores extracted from the same soils (An et al. 1990; Stutzand Morton 1996; Watson and Millner 1996; Koske et al. 1997;Brundrett et al. 1999a). However, trap cultures contain a mixture of fungithat changes over time, so must be further purified before use inexperiments. Production of living cultures of AM fungi is difficult andtime consuming, and consequently, is the main factor limiting researchactivities and practical applications with these fungi.


Microscopic examination of cleared and stained roots revealsstructures produced by AM fungi (Brundrett et al. 1996c). Thesestructures can be used to identify fungi to determine their relativeabundance in natural ecosystems (Abbott 1982; McGee 1989; Thomsonet al. 1992; Brundrett and Abbott 1995; Merryweather and Fitter 1998b),or glasshouse experiments (Abbott and Robson 1984a; Jasper et al. 1991;Lopez-Aguillon and Mosse 1987; Pearson et al. 1993; Brundrett et al.1999b). This method avoids problems with spore-based surveys, becausenon-sporulating fungi are often important in soils and there are largedifferences in spore production between species (Brundrett et al. 1999a).This method allows the relative contributions of individual fungi sharingroots with other fungi to be determined. However, working with rootsfrom natural habitats may be difficult if (i) young roots with activeassociations are hard to obtain due to the seasonality of root growth andlong root life-spans, (ii) roots are thick and have abundant secondarymetabolites, or ( i i i ) roots of different plant species are hard to separate inmixtures (Brundrett et al. 1996c).


2.2. Lifecycles and inoculumThe spread to new roots, long range dispersal and persistence ofmycorrhizal fungi in ecosystems is dependent on the formation ofpropagules which are resistant to soil and environmental conditions.Propagules of AM fungi include asexual spores formed in soil, rootfragments containing hyphae and vesicles (storage structures) and soilhyphae (Figure 1). Mycorrhizal fungus propagules are usuallyconcentrated in the topsoil, but can also occur at greater depths (up to 4m) in arid ecosystems (Virginia et al. 1986; Zajicek et al. 1986). Fungimust be active when root growth activity occurs, since roots have a limitedperiod of receptivity (Brundrett and Kendrick 1990; Hepper 1985; Smithet al. 1992) and efficient colonization of roots is required for an effectiveassociation (Abbott and Robson 1984b; Bowen 1987).

Soil-borne spores have traditionally been thought to be the mostimportant type of inoculum of AM fungi. However, spore numbers areoften poorly related to mycorrhizal formation in soils and fungi which donot produce recognisable spores are important in many soils (Abbott andRobson 1991; Brundrett and Abbott 1995; Stutz and Morton 1996;Merryweather and Fitter 1998a). Living spores of AM fungi in soil maynot function as propagules if they are quiescent, dormant, or have beenparasitised (Tommerup 1992; Lee and Koske 1994). A link betweenmycorrhizal colonisation levels and the timing of sporulation has beenobserved in experiments with some isolates of AM fungi (Table 2).Observations from these experiments suggest that: (i) colonised rootlength can be a good predictor of sporulation (Douds 1994), (ii) aminimum colonisation level is required for sporulation for some species(Gazey et al. 1992), ( i i i ) mycorrhizal formation may decline aftersporulation begins (Pearson and Schweiger 1993) and (iv) soil hyphaemay lose viability after sporulation commences (Jasper et al. 1993). Itshould be noted that these observations apply to particular fungal isolatesas others may behave differently (Table 2).


A pre-existing network of soil hyphae is often considered to be themain source of AM inoculum in ecosystems because spore numbers arenot correlated with mycorrhizal formation and colonisation startsimmediately after root growth commences (Jasper et al. 1989; McGee1989; Brundrett and Abbott 1995). Fragments of dead roots present inthe soil can also initiate AM (Rives et al. 1980; McGee 1987).

Many AM fungi form vesicles within roots which function as storageorgans and/or propagules (Biermann and Lindermann 1983) which canbe structurally and functionally similar to the spores of AM fungi in soil(e.g. some Glomus species), or may be temporary storage structures thatdo not persist in old roots (e.g. most Acaulospora species) (Table 2). Oldroots and coarse soil organic matter colonised by AM fungi are alsothought to contribute to the survival and spread of AM fungi (Warner1984; Brundrett and Kendrick 1988; Koske and Gemma 1990).

Since the most important propagules of AM fungi in soils aregenerally unknown, it is best to measure the total inoculum potential ofthese fungi. This value can be estimated by most probable numbermethods (serial di lut ions using sterilised soil), or bioassays which measurethe degree of colonisation of a plant grown in that soil (see Abbott andRobson 1991; Brundrett et al. 1996c). A study of AM fungi in tropicalsoils found there were two major functional categories of these fungi,which either used spores as an important propagule, or rarely producedviable spores (Brundrett et al. 1999a). AM fungus population studiesbased entirely on spores in soils are likely to be misleading. Majordifferences in life history characteristics between species of AM fungi arelisted in table 2. It should be noted that some pairs of characteristics intable 2 reflect ends of a continuum. Further research is required todetermine how these functional characteristics are correlated with thecapacity of fungi to grow in soils and provide benefits to plants.

2.3. Predators and dispersalA wide variety of mycophyllous soil microorganisms occupy, or associatewith soil hyphae, mycorrhizal roots or spores of AM fungi (Table 3).Spores of AM fungi isolated from soils in natural ecosystems often showsigns of predation, which may be responsible for seasonal fluctuations inspore abundance (Ross and Ruttencutter 1977; Lee and Koske 1994).Interactions between AM fungi and soil microbes include (i) occurrencewithin living hyphae, ( i i) necrotrophic associations with old hyphae orspores, or ( i i i ) parasitism of hyphae and spores which may be detrimentalto associations (Macdonald and Chandler 1981; Sylvia and Schenck1983; Paulitz and Menge 1984; Lee and Koske 1994).

Fungus-feeding nematodes, springtails and mites feed on AM fungushyphae (Table 3). Hyphal grazing by soil arthropods reduced thebenefits provided by AM in some experiments (Ingham 1988; Rabatinand Stinner 1988), however in others, these animals were found to havelittle effect on plant yield (Larsen and Jakobsen 1996), preferentiallygrazing on other types of fungi when given a choice (Klironomos andKendrick 1996), or were only detrimental at high animal densities(Klironomos and Ursic 1998). It is not surprising that a wide diversity of


organisms are known to consume, decompose or associate with AM fungi,as these fungi are a major component of the microflora of most soils.However, most mycophyllous organisms seem to have a limited impact onthe performance of mycorrhizal fungi in natural habitats.


The spread of mycorrhizal fungi (Section 2.2) occurs by activeprocesses (hyphal growth through soil) or passive dispersal mechanisms.Dispersal allows the introduction of mycorrhizal fungi to new geographiclocations and the transfer of genetic information. Growth of hyphae ofAM fungi through soil can slowly spread the association to adjacentplants (e.g. Scheltema et al. 1985). Propagules of AM fungi can besuspended in moving air currents (Tommerup 1982) and wind dispersalhas been observed in ecosystems (Warner et al. 1987). AM fungi areprobably also transported by water erosion and human activities. Koskeand Gemma (1990) observed that rhizome leaf sheaths and old rootscontaining AM fungi could be transported by wind or water in coastalhabitats.

The animals which ingest and disperse AM fungi include smallmammals, marsupials, grasshoppers, worms, ants, wasps and birds (Table3). Soil-dwelling arthropods and earthworms are considered to beimportant vectors of these fungi (Reddell and Spain 1991; Gange 1993;Harinikumar and Bagyaraj 1994). Larger animals that feed on AM fungican transport viable spores for considerable distances (McGee andBaczocha 1994; Janos et al. 1995). The introduction of AM fungi byanimal digging act ivi t ies was considered important during theestablishment of vegetation in areas devastated by the Mt. St. Helensvolcanic eruption (Allen et al. 1992).

2.4. DisturbanceAM fungi can survive soil freezing (Addy et al. 1997), or wetting anddrying cycles (Braunberger et al. 1996). Daft et al. (1987) found thatspores were more resistant to topsoil disturbance and storage than rootfragments and much more resistant than hyphae. Networks of soil hyphaeare an important source of inoculum in natural ecosystems that are h ighlysusceptible to disturbance (Jasper et al. 1989; Evans and Miller 1990;Bellgard 1993). Disturbance is also likely to reduce the effectiveness ofroot inoculum (Evans and Miller 1988; Rives et al. 1980). Numbers ofsurviving propagules of AM fungi in soils decline with time in theabsence of host plants (see Figure 3 in Chapter 3).

Mycorrhizal propagules can be severely influenced by damage tovegetation and soils resulting from natural processes or humanintervention. Destructive processes which adversely affect AM fungiinclude: intense fires, topsoil removal and flooding (Table 4).Mycorrhizal fungi may be absent from soils affected by salinity, aridity,waterlogging, or severe climatic conditions (Brundrett 1991). AMinoculum is in i t i a l ly absent from very young habitats such as sand dunes(Corkidi and Rincón 1997a), volcanic substrates (Allen et al. 1992;Gemma and Koske 1990), or glacial deposits (Helm et al. 1996).Anthropogenic disturbances which impact on AM fungi include min ing(Section 5.2), forestry (Norani 1996) and agriculture (Alexander et al.1992). Agricultural practices such as tillage, long fallow periods, soilcompaction, or growth of non-mycorrhizal crops may also be detrimentalto mycorrhizal associations (Thompson 1987; Wallace 1987; Evans and


Miller 1988; Abbott and Robson 1991). The role of AM fungi indisturbed habitats is considered in Section 5.2.


3. Mycorrhizal plantsMuch research has focussed on the potential for mycorrhizal associationsto increase plant productivity in plantation forestry, or during ecosystemrecovery after severe disturbance, as well as in agriculture andhorticulture. However, it could be argued that we do not know enoughabout the role of mycorrhizal associations in natural, disturbed, ormanaged ecosystems to safely evaluate their potential for applied use.

3.1. Benefits to plantsThe main function of AM associations is considered to be mineralnutrient acquisition from soil, but these fungi can also provide manyother benefits to individual plants or other organisms (Table 5).Knowledge of the importance in natural ecosystems of many of thebenefits listed in table 5 is very limited (Section 4.1).

Plants have demands for mineral nutrients determined by theirinternal requirements and soils have a limited capacity to supply nutrientsdue to their availability and mobility (Russell 1977; Marschner 1995).Phosphorus is normally considered to be the most important plant-growthlimiting factor which can be supplied by mycorrhizal associations,because its restricted mobility in soils causes depletion zones around roots(Bolan 1991; Marschner 1995). Thus hyphae of AM fungi would beprimarily responsible for acquiring phosphorus from outside rootdepletion zones (Marschner and Dell 1994; Smith and Read 1997). Thesehyphae were considered to utilise the same forms of nutrients as roots, butthere now is evidence that they have a greater benefit when phosphorus ispresent in less-soluble forms (Bolan 1991; Schweiger et al. 1995;Kahiluoto and Vestberg 1998). AM fungus hyphae help plants acquire Pfrom some organic compounds, but not others (Tarafdar and Marschner1994; Joner et al. 1995). AM fungus hyphae may also help plantsacquire some nitrogen and trace elements (Table 5).

Hyphae of AM fungi can respond to localised sources of soilnutrients by hyphal proliferation (St John et al. 1983; Warner 1984) andproduction of fine highly-branched “absorptive” hyphae (Mosse 1959;Bago et al. 1998). Thus, AM fungi may access nutrients which arespatially and chronologically separated from roots. AM fungi may alsowork synergistically with organisms which decompose organic materialsin soils, by providing a link between roots and localised sites wherenutrients are made available by saprobic organisms (Table 5).

Because some AM fungi clearly increase the uptake of P and henceplant growth, in glasshouse experiments using P-deficient soils, it isgenerally assumed that the same happens on a larger scale in agriculture,revegetation sites and in natural ecosystems. This assumption underpinsmost research on AM fungi. However, the P uptake and translocation byhyphae of AM fungi associated with perennial plants is expected to differfrom that of annuals such as crop plants (Smith et al. 1994). The greatfunctional diversity of the roots of perennial hosts remains virtuallyunexplored.Attempts to quantify the benefits provided by AM fungi in naturalecosystems have been complicated by the complexity of these systems.For example, associations may be active only for a short part of the year(Lapointe and Molard 1997) and acquired nutrients may not be utilisedimmediately by plants (Sanders and Fitter 1992). The distribution of P is


Benefits provided by AM associations to plants and their interactions withenvironmental factors are considered here.



likely to be patchy in soils of natural ecosystems (Cui and Caldwell 1996)and the distribution of AM fungi in soils can be extremely heterogeneous(Friese and Koske 1991; Brundrett and Abbott 1994; Moyersoen et al.1998). Thus, AM fungi in natural ecosystems may be important forlocating pockets of phosphorus. The lack of information on the role ofAM fungi in natural ecosystems is also due to the difficulty of measuringphosphorus uptake and translocation to plants by hyphae. Methodologydeveloped for use in agricultural soils (e.g. Jakobsen 1994) could be usedto measure nutrient translocation by AM fungi in soils from naturalecosystems.

Some studies have shown that mycorrhizas can provide benefits toplants even when there is little or no short-term growth response. Otherbenefits that have been reported include greater reproductive success,increased disease resistance, changes to water relations and/or nutrientaccumulation (Table 5). It is often difficult to separate indirect effects ofmycorrhizas from those caused by improved nutrition in experiments(Brundrett 1991; Smith and Read 1997). Mycorrhizas can also influencethe outcome of competition between species (see 4B below).

3.2. Mycorrhizal dependencyObservations of plants in natural ecosystems have shown that speciesgenerally have (a) consistently high levels of mycorrhizas, (b)intermediate, or variable levels of mycorrhizas, or (c) are consistently notmycorrhizal (Janos 1980; Brundrett 1991). Plants belonging to thesecategories are often called obligatorily mycorrhizal, facultativelymycorrhizal, or non-mycorrhizal respectively. Obligatorily mycorrhizalplants are defined as those which will not survive to reproductive maturitywithout being associated with mycorrhizal fungi in their natural habitats,while facultatively mycorrhizal plants benefit from mycorrhizas only ininfertile soils and non-mycorrhizal plants have roots that are resistant tocolonisation by mycorrhizal fungi (Janos 1980). Dependence onmycorrhizas can be measured by comparing the growth of plants withmycorrhizas in experiments to plants grown without them at a particularsoil P level (e.g. Koide et al. 1988; Manjunath and Habte 1991; Hetricket al. 1992). Mycorrhizal benefits should be examined across a range ofsoil P levels, by producing nutrient response curves (Abbott and Robson1991; Schweiger et al. 1995).

Growth responses to AM associations are measured by comparingthe growth of plants with and without mycorrhizas in a particular soil(Table 6). In practice, it is very difficult to provide adequate controls inmycorrhizal studies because removal of mycorrhizal fungi causeschanges to the chemical, biological and physical properties of soil andinoculation with fungi is likely to introduce other organisms (Ames et al.1987; Hetrick et al. 1992; Baas et al. 1989; Koide and Li 1989).However, the impact of microbial factors or changes to soil fertility due tosterilisation on plant growth has usually been small relative to the impactof mycorrhizal treatments. Some studies attempting to quantify thebenefits of AM fungi in plant communities have measured the impact


of temporary suppression of these fungi by the application of fungicides(e.g. Gange et al. 1993; Newsham et al. 1995; Lapointe and Molard1997). These studies are complicated by possible non-target effects of


fungicides, but have shown reduced yields for some plant species whenmycorrhizal formation was inhibited.

Table 6 lists examples of plants from natural ecosystems whosemycorrhizal dependency has been measured experimentally, by growingplants with and without mycorrhizas at appropriate soil nutrient levels.Plants which are h ighly dependant on mycorrhizas appear to beimportant in most habitats, but apparently are less dominant in grasslandsand disturbed habitats dominated by herbaceous plants. Non-mycorrhizalplants are generally only prevalent in habitats where plant productivity isseverely limited by soil or climatic conditions, such as very dry, wet,saline, cold, or disturbed soils (Table 1). Typically non-mycorrhizalplants include members of the families Amaranthaceae, Caryophyllaceae,Chenopodiaceae, Brassicaceae, Commelinaceae, Cyperaceae, Juncaceae,Proteaceae, Polygonaceae and Scrophulariaceae (Tester et al. 1987;Brundrett 1991; Peat and Fitter 1993; Brundrett 1999).

Plants can acquire nutrients both directly through their roots and/orvia mycorrhizal associations. Thus, the benefit provided by mycorrhizaswil l depend on the capacity of roots to directly acquire mineral nutrientsfrom the soil (Janos 1980; Brundrett 1991; Marschner 1995). Plants withfacultatively mycorrhizal associations or non-mycorrhizal roots normallyhave much finer roots and longer root hairs than obligately mycorrhizalspecies (Bayliss 1975; Manjunath and Habte 1991; Baon et al. 1994;Schweiger et al. 1995). Thus, the roots of non-mycorrhizal plantstypically would be much more efficient at direct nutrient uptake than theroots of mycorrhizal species.

3.3. Soil factorsLand degradation due to sal ini ty, waterlogging, erosion, etc. are nowrecognised as serious and growing problem in Australia and othercountries. In ecosystem studies and glasshouse experiments soil factorscan influence both the diversity of AM fungi and overall levels ofmycorrhizal root formation and sporulation (Table 4). Observations innatural ecosystems have shown that AM plants are often less commonthan non-mycorrhizal species in soils which are waterlogged or saline, butthat some mycorrhizal plants are normally present in even the mostinhospitable habitats (Table 1). Low soil pH, high aluminium levels orhigh soil phosphorus levels prevent some AM fungi from providingsubstantial benefits to the host plants and can influence the distribution offungi (Table 4). Excessive NaCl levels in soil inhibit mycorrhizalformation and restrict the activity of most mycorrhizal fungi, but somefungi are more tolerant of these conditions than others (Juniper andAbbott 1993).

3.4. Pollution and climateVarious forms of pollution can inhibit mycorrhizal formation inexperimental systems (Table 4). There is evidence that fungi adapted tohigh levels of metals help plants to grow in contaminated soils (Diaz et al.1996; Joner and Leyval 1997). However, the roles of mycorrhizas in soilscontaminated by metals are complex, as they increase the uptake of some


metal ions but not others and their role also varies with the concentrationof ions in soil (Heggo et al. 1990; Guo et al. 1996; Joner and Leyval1997). This suggests that mycorrhizas would increase plant survival insome contaminated soils, but not in others.

Forest decline associated with air and/or precipitation borne pol lu t ionhas become a serious problem in Europe and North America.Mycorrhizal fungi can help plants to survive in soils affected by acidicprecipitation (Malcova et al. 1998). Much recent research has focussedon the impact of elevated levels on mycorrhizal associations.Elevated can result in increased fungal biomass due to higher rootcolonization or increased soil hypha production (Rillig et al. 1998;Sanders et al. 1998). However, direct effects of on fungi can bemuch smaller than indirect effects mediated by changes in plant growth(Staddon et al. 1999).

4. Natural ecosystemsThe many roles of mycorrhizal fung i in ecosystems include providingfood and habitats for other soil organisms and soil-feeding animals(Section 2.3), but only impacts on fungal and plant communities will beconsidered further here.

4.1. Fungal communitiesA succession of AM fungi has been observed in pot cultures (Brundrett etal. 1999b) and seasonal var ia t ions in fungal activit ies (measured by rootcolonisation) also occur in soils (Brundret t and Abbott 1995). Spores ofone fungus in the jarrah forest remain dormant for 11 months and thisleads to its appearance as a dominant member of the community on atwo-year cycle (Jayasundara, pers. comm.). Therefore, for some species,low relative abundance of a fungus can be quickly followed by itsdominance in roots. Fluctuations in the relative abundance of fung iwithin roots depend on the relative abundance of infective hyphae ofdifferent fungi in the original inoculum. Maximum activity of somefungi may not coincide with the optimum period for plant utilisation ofnutrients, as a consequence of AM fungus phenology patterns. Soildisturbance can also alter the relative abundance of AM fungi in roots(Jasper et al. 1991) . AM fungus communi ty dynamics can also beinfluenced by competitive interactions between fungi (Pearson et al.1993).

Plants normally have more than one AM fungus simultaneouslypresent in their roots (Abbott and Robson 1978; Abbott and Robson1981; Merryweather and Fitter 1998a). Indirect evidence shows thatdifferent fungi have different roles in field soils (Merryweather and Fitter1998b). The presence of different host plants influences the diversity offungi in soils (Schenck and Kinloch 1980; Johnson et al. 1992; Hendrixet al. 1995; Bever et al. 1996). The effect of host species on fungalpopulations in roots has been called “ecological specificity” (McGonigleand Fitter 1990; van der Heijden et al. 1998). There also can be seasonalvariation in colonisation by different fungi (Merryweather and Fitter1998b) and different host plants can induce sporulation of a fungus at

different times (Bever et al. 1996). These experiments suggest thatmycorrhizal formation within the roots of two plant species by a singlefungus may be very different, even where both plants are equallyreceptive to the fungus.

The majority of AM fungi used in experiments have increased plantgrowth under particular conditions (Table 6). However, they differ in theextent to which they increase the uptake of phosphorus in relation to theamount of carbon that they remove (Pearson and Jakobsen 1993). Thereare cases where AM fungi reduce the growth of cultivated plants in highlyfertile soils (Graham and Eissenstat 1998). The effectiveness of fungalisolates has been attributed to differences between them in the rate andextent of colonisation (Abbott and Robson 1981), but other factors arealso important (Sylvia et al. 1993; Dickson et al. 1999). AM fungi differin their patterns of colonisation of roots (Abbott 1982; Merryweather andFitter 1998a), characteristics of the plant/fungus interface (Smith et al.1994) and in the architecture and function of the hyphal networks thatthey form in soil (Jakobsen et al. 1992; Smith et al. 2000). Further, theefficacy of different fungi is expected to depend on the host plant species(Ravnskov and Jakobsen 1995).

Evidence of the physiological diversity of AM fungi has beenobtained by comparing responses of different species or isolates to thesoil conditions listed in table 4. These comparisons have demonstratedvariations between taxa and intraspecific variability within species of AMfungi in their ability to promote plant growth (e.g. Lambert et al. 1980;Stahl et al. 1988). Isolates of AM fungi are often most infective whenused in the soil from which they were collected (Molina et al. 1978;Adelman and Morton 1986; Porter et al. 1987; Stahl et al. 1988; Henkelet al. 1989). Unfortunately, AM research has mainly been concerned withplant responses to mycorrhizas with little consideration of differencesbetween specific fungi (Morton 1988; Abbott and Robson 1991;Brundrett 1991).

Correlations between the diversity of AM fungi in microcosms andplant productivity (van der Heijen et al. 1998), may be a consequence ofthe functional diversity of AM fungi. However, in this study plantresponses to mixtures of fungi appear to be similar to responses to themost effective species inoculated individually (see also Wardle 1999).Further research, which determines the relative contribution of differentspecies of AM fungi within roots of competing plant species, is requiredto establish the importance of AM fungus diversity.


4.2. Plant community structureArbuscular mycorrhizal fungi are a key part of almost all plantcommunities (Table 1), so they must be considered in all studies ofnutrient cycling or impacts of nutrient supply on plant productivity ordiversity. Mycorrhizal associations could potentially influence plantcommunity structure by affecting richness or evenness of coexistingplants.


Table 7 shows 4 categories of intraspecific and interspecific interactionsinvolving plants wi th different mycorrhizal requirements with the sametype of association (facultative and obligately mycorrhizal plants areconsidered to be equivalent in interactions with non-mycorrhizal plants).Interactions which only involve non-mycorrhizal plants will not bediscussed (type IV). Interactions between plants with different types ofmycorrhizas are also considered below (type V). The experiments citedbelow examined the roles of mycorrhizas by withholding fungi fromsome experimental treatments, or by suppressing AM fungi in soil withfungicides. These studies measured the productivity and diversity ofplants relative to treatments with AM fungi.

4.2.1. Competition between mycorrhizal plantsInterspecific competition between mycorrhizal plants. The presence ofAM fungi can reduce the strength of competition between two specieswhere one has a competitive advantage due to larger size or fastergrowth (Moora and Zobel 1996). In highly competitive situationssmall plants may benefit from sharing a common network ofmycorrhizal fungus hyphae with larger plants, which may reduce thecost of supporting an association. However, it does not seem likelythat mature plants acquire nutrients from competing mycorrhizalplants through these networks (Newman et al. 1992; Fitter et al.1998; Robinson and Fitter 1999). Most competition experiments haveused plants which differ in their mycorrhizal dependency (see IIbelow).Intraspecific competition. Most studies of the impact of withholdingAM fungi from competing plants of one species have found greatercompetition at high plant densities with AM fungi than without(Eissenstat and Newman 1990; Allsopp and Stock 1992; Shumwayand Koide 1995; Moora and Zobel 1996; 1998; West 1996). Thesestudies all reported greater size differences between large and smallplants when they were mycorrhizal, indicating that mycorrhizasincreased variabil i ty in the capacity of plants to compete forresources. Mycorrhizal benefits were reduced by high plant densities,since fewer resources were available per plant (Allsopp and Stock1992; Moora and Zobel 1998).


4.2.2. Interactions of plants with high and low mycorrhizal dependencyMost studies have shown increased competitive ability of obligatelymycorrhizal plant species growing with facultatively mycorrhizal speciesresulting from the presence of AM fungi (Hall 1978; Grime et al. 1987;Gange 1993; Hartnett et al. 1993; Hetrick et al. 1994; van der Heijden1998). These experiments typically compared a plant with a relativelyfine root system (such as a grass) with a herb with coarser roots, thatnaturally occur together in pastures or meadows. In these cases, the plantwith coarser roots would be less efficient at direct nutrient uptake andthus, mycorrhizas would greatly increase its nutrient uptake andcompetitive ability (Bergelson and Crawley 1987; Brundrett 1991). It isinteresting to note that mycorrhizas can help indigenous plants tocompete with weeds threatening their establishment (Nelson and Allen1993; Smith et al. 1998). The presence of facultatively mycorrhizalspecies can result in increased mycorrhizal inoculum levels in disturbedsoils, which may help obligately mycorrhizal species become establishedlater in succession (Allen and Allen 1988).

4.2.3. Competition between mycorrhizal and non-mycorrhizal plantsCompetition between non-mycorrhizal species and normally mycorrhizaltaxa is similar to competition between obligately and facultativelymycorrhizal plants, as described above (Crowell and Boerner 1988;Boerner and Harris 1991; Sanders and Koide 1994). This results becausenon-mycorrhizal plants typically have similar root systems to facultativespecies (Brundrett 1991). In many natural ecosystems, non-mycorrhizalplants are out-competed by mycorrhizal species during succession, butthe mechanisms involved are not clear (Schmidt and Reeves 1989;Brundrett 1991; Francis and Read 1995).

Most examples of mycorrhizal plants out-competing non-mycorrhizal species (with adequate inoculum of suitable AM fungi)probably result because the mycorrhizal species are more efficient atacquiring limiting soil nutrients such as P. However, AM fungi may alsohave more direct adverse effects on non-host plants. Francis and Read(1995) found that hyphae of AM fungi damaged the roots of non-mycorrhizal plants. Allen et al. (1989) also observed wounding reactioninduced by AM fungi, which apparently resulted in reduced growth andsurvival of the non-host plant. This may have resulted in the suppressionof competing non-host plants, by AM fungi when inoculum levels werehigh in the presence of host plants.

There are also cases where non-mycorrhizal plants adversely affectAM fungi. Non-mycorrhizal plants have poorly understood mechanismswhich keep most fungi out of their roots (Brundrett 1991). Many non-mycorrhizal plants are considered to accumulate secondary metaboliteswhich may have defensive roles (Kumar and Mahadevan 1984; Brundrett1991; Schreiner and Koide 1993). Roots of non-host plants in soils canreduce germination of AM fungus spores or mycorrhizal formation inhosts (Schreiner and Koide 1993; Fontenla et al. 1999). The implicationsof this form of allelopathy in natural ecosystems are worthy of furtherinvestigation.


4.2.4. Plants with different types of mycorrhizasInterrelationships between plants with different mycorrhizal types arecomplex. For example, trees with ectomycorrhizas (ECM) are dominantin some forests and coexist with AM trees in others, while AM trees aredominant in other similar habitats (Högberg and Piearce 1986; Newberyet al. 1988; Brundrett 1991). Climatic factors seem to be important indetermining the outcome of competition between plants with differenttypes of mycorrhizas (e.g. ECM trees dominate boreal and alpine forests,while many forests in tropical regions are dominated by AM trees). It hasbeen suggested that trees with ECM are more likely to be important insoils with low pH, high levels of aluminium or other toxic ions (Högbergand Piearce 1986), or where nutrient cycling occurs slowly because leaflitter is resistant to decomposition (Gebauer and Taylor 1999; Lindahl etal. 1999). However, the relative importance of these factors has not beenestablished.

It is usually considered that the outcome of competition betweenplants with different types of associations will be determined by therelative success of their associated fungi in acquiring different forms ofmineral nutrients which l imi t plant growth. In an Australian forest, directcompetition between plants with ECM, AM or non-mycorrhizal roots maybe avoided because their roots occurred wi th in separate zones within soilsand may have used different forms of nutrients (Brundrett and Abbott1995).

4.2.5. ConclusionsInterpretation of results of competition experiments is difficult becausemycorrhizal effects on plant performance can be counteracted bychanges to the nature of competition (Watkinson and Freckleton 1997).Mycorrhizas can result in higher plant diversity due to increasedintraspecific and reduced interspecific competition (Moora and Zobel1996). Mycorrhizal dependency primarily determines the impact ofmycorrhizas on competitive interactions between plants.

5. Utilising mycorrhizasThis section will focus on plants because we do not know enough aboutthe distribution and abundance of arbuscular mycorrhizal fungi to knowif any are rare. AM fungi exhibit functional diversity (Table 2) and areadapted to local environmental and soil conditions (Table 4).Consequently, it would be important to conserve representatives of allhabitats and soils within regions to maintain the functional diversity ofAM fungi and other organisms. Substantial reductions in fungal diversityoccur when ecosystems are disturbed or converted to agriculture(Schenck and Kinloch 1980; Hetrick and Bloom 1983; Allen et al. 1987;1998; Brundrett et al. 1996a,b), but it is not known if this loss ispermanent, or represents depletion below the level of detection.

5.1. Propagation of rare plantsAttempts to inoculate plants with AM fungi are less common than forectomycorrhizas, because AM fungi are ubiquitous in soils and are


considered not to have host preferences. Thus, it is believed that any AMfungus has the capacity to form mycorrhizas with any host. Mycorrhizalinoculation technology has been developed for forestry and horticulture,especially when soils have been sterilised to remove pathogens, but hasrarely been applied to plants from natural ecosystems (Brundrett et al.1996c). However, the mycorrhizal literature provides many examples thatmay be relevant to the propagation of indigenous plants. For example,AM fungi can greatly reduce the mortality of transplantedmicropropagated plants (Subhan et al. 1997).

Manipulation of mycorrhizal fungi may sometimes be required torehabilitate habitats or conserve rare species. Many plant species arehighly dependent on mycorrhizas (Table 6) and the potting mixes usedto propagate them are likely to be devoid of AM fungi. Koske andGemma (1995) worked with endangered species of Hawaiian plants whichwere difficult to propagate. They found that inoculation of nursery-grown seedlings and cuttings resulted in substantial increases in plantgrowth and survival in certain growth media. Barroetavena et al. (1998)also found that AM fung i were important to the survival of propagatedplants of an endangered Astragalus species. It may be worthwhile toroutinely include mycorrhizal inoculation as a precautionary measure inany programs which attempt to propagate rare species, if AM inoculum iscommercially available and its expense is insignificant relative to othercosts in a species recovery program. However, inoculated fungi maypersist after plants are transplanted in field soils and compete withindigenous fungi which are better adapted to local conditions.

5.2. Ecosystem restorationMycorrhizal inoculation is only likely to be valuable in disturbed habitatswith little or no mycorrhizal inoculum (Section 2.4), because AM fungiare relatively non-specific and already occur in most soils (Abbott andRobson 1991; Brundrett 1991). Consequently, the primary focus of mostpractical research with AM fungi has been the restoration of disturbedhabitats, such as mine sites. Research with AM fungi has also focused onsand-dune stabilising grasses in coastal habitats, where AM colonisationestablishes slowly (Sylvia and Will 1988; Corkidi and Rincón 1997a,b).These studies have found that out-planted grasses inoculated with AMfungi grow better than uninoculated plants in sand dunes initially devoidof vegetation (Sylvia 1989; Gemma and Koske 1997).

Massive destruction of native vegetation is occurring throughout theworld primarily because of land use changes by people. Severedisturbance of ecosystems is likely to result in the loss of somemycorrhizal fungi which have adapted to local conditions. Duringsubsequent attempts at ecosystem reconstruction, the impact of thisreduction in mycorrhizal fungus genetic resources will depend on (i) howfungal genetic and funct ional diversity varies between habitats, (ii) howrapidly surviving fung i adapt to changing soil conditions duringsuccession and (iii) how effectively well-adapted isolates are dispersedfrom any surviving remnant patches of native vegetation.

Most studies of mycorrhizal associations in highly disturbed habitats


such as mine sites have found reduced levels of mycorrhizal propagules(Jasper et al. 1987, 1991; Pfleger et al. 1994; Brundrett et al. 1996b).Topsoil removal and stockpiling during mining have major impacts onAM fungi (Jasper et al. 1987; Rives et al. 1980; Stahl et al. 1988;Bellgard 1993). Soil disturbance may also have an indirect effect throughchanges to soil properties which reduce the efficacy of surviving fungi(Abdul-Kareem and McRae 1984; Stahl et al. 1988; Waaland and Allen1987). Disturbance impacts on AM fungi that have been hypothesisedinclude: (i) a reduction in numbers of viable spores, ( i i) loss of a hyphalnetwork in the soil, or ( i i i ) the prevention of hyphal growth from rootinoculum to new roots (Evans and Miller 1988; Jasper et al. 1989; Riveset al. 1980). The relative importance of these mechanisms in differentsituations is unknown.

6. ConclusionsAM fungal associations must be considered in plant conservationprograms because of their roles in key ecosystem processes (Table 8).The processes listed in table 8 are l ikely to be adversely affected ifmycorrhizal fungi were absent or not functioning due to unsuitable soilconditions.

Despite the scarcity of direct measurements of mycorrhizal benefitsoutside the glasshouse, there is considerable indirect evidence to supportthe assumption that AM fungi have a major role in nutrient uptake innatural ecosystems. This evidence includes the facts that most plantsupport high levels of mycorrhizal colonisation and many plants haveroot systems that otherwise would be inefficient at nutrient uptake(Section 3.2). Most attempts to quantify the value of AM fungi have


focused on shor t - l ived plants growing in relatively fertile soils (e.g.“weeds” of meadows and early successional habitats). These annualspecies are used in most glasshouse and field experiments becausetreatment effects can be analysed after one growing season. However,evidence from growth experiments (Table 6) and studies of mycorrhizalcolonisation levels in natural habitats (Table 1) have shown that annualplants are often less dependent on mycorrhizas than are perennial plantsin undisturbed habitats (Table 6). Studies of annual plants are of littlevalue if they do not consider their reproductive success by measuringsurvival and growth of subsequent generations (e.g. Stanley et al. 1993;Shumway and Koide 1995). Non-nutr i t ional benefits such as antagonismof pathogens and changes to water relations are also suspected to beimportant, but have rarely been investigated (Section 3.1). As AM fungiare an integral component of soils (except in disturbed habitats, sites withhostile soil condi t ions, or a severe climate), experiments wheremycorrhizas are w i t h h e l d are a r t i f i c ia l situations.

Prediction of the benefits resulting from inoculating plants with AMfungi during attempts to restore ecosystems, or for rare species recoveryprograms are hampered by our lack of knowledge about the basicbiology of these fungi. We do not know how many species of these fungioccur in most habitats, or if funct ional differences between isolates aremore important than variations in the distribution of taxa. Thecommunity dynamics of AM fungi have the potential to alter the overallcontribution of mycorrhizas to plant productivity and dominance of plantspecies, by mechanisms that are not known (e.g. Moora and Zobel 1996;van der Heijden et al. 1998). The life cycles of AM fungi in soils are notwell understood (Section 2.2). Litt le is known about the contribution ofAM fungi to nut r ient uptake in natural ecosystems. Such voids in ourunderstanding of AM fungi should be of major concern, because theseorganisms are one of the most important groups of organisms in plantcommunities. Mycorrhizal fungi are a major conduit of carbon into thesoil and a key part of the plant/soil interface. We need to know how thisinterface functions if we are to understand the impact of changes to soilor climatic conditions on plant communities.

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Chapter 7

ORCHID CONSERVATION ANDMYCORRHIZAL ASSOCIATIONS

Andrew L. Batty1,2

Kingsley W. Dixon1,3

Mark C. Brundrett1,2

K. Sivasithamparam2

1Kings Park and Botanic Garden, Botanic Gardens and Parks Authority, West Perth 6005,Western Australia. (correspondence)2Soil Science and Plant Nutrition, Faculty of Natural and Agricultural Sciences, TheUniversity of Western Australia, Crawley 6009, Western Australia.3Plant Biology, Faculty of Natural and Agricultural Sciences, The University of WesternAustralia, Crawley 6009, Western Australia.

1. Introduction1.1. OrchidsOrchids (members of the plant family Orchidaceae) include terrestrialsthat typically occur in temperate regions, and epiphytes, which mostlyoccur in tropical regions where the taxonomic diversity of orchids ishighest (Dressler 1993). The flowers of these highly evolved plants areoften beautiful and sometimes bizarre with complex pollinationmechanisms involving specific insect pollinators (Dressler 1981; Benzing1982; Jones 1993). The Orchidaceae contains more species than anyother flowering plant family, with estimates ranging from 17,500 to35,000 species (Garay and Sweet 1974; Gentry and Dodson 1987;Mabberley 1990). The diversity of epiphytic orchid species increasesalong moisture and latitude gradients and with habitat complexity(Gentry and Dodson 1987). Orchids produce large numbers of minuteseeds that favour the expression of genetic variability and high dispersalrates across geographical and ecological barriers.

Many orchids are now considered to be at risk of extinction as anindirect or direct result of human activities, which include habitatalteration or destruction and extraction of wild plants for trade (Table 1).


These processes and the phenomenal diversity of orchids have resulted inmany threatened orchid species (Hágsater and Dumont 1996). Theimpacts of alteration or habitat destruction on orchid taxa will depend onits geographical distribution, habitat specificity, and population size(Rabinowitz et al. 1986). Generally it can be assumed that rare specieshave more specific habitat preferences than common species. Habitatspecificity is l ikely to be a consequence of distribution of mycorrhizalfungi (see below), pol l ina t ion mechanisms, seed distribution, etc. Rareorchid species are in t r ins i ca l ly prone to extinction due to naturallyoccurring catastrophes (e.g. intense fires, floods, or severe climaticvariations). For example, Epidendrum floridense, a Florida epiphyticorchid species, appears to have nearly become extinct as a consequenceof severe unseasonal frosts (Hágsater 1993).

This review focuses on the ecological importance of orchid mycorrhizalassociations, and readers should consult other works for details of thephysiology and morphology of these associations, and other aspects ofthe biology and ecology of orchids. The body of scientific knowledge toback up much of what is believed about the mycorrhizal biology andecology of orchids is l imi ted , so many topics cannot be covered in greatdepth. Review sections concerning the biology of associations arepresented, before ecological consequences of these associations and thepractical use of mycorrhizas in orchid conservation are discussed.

1.2. MycorrhizasMycorrhizas (fungus-roots) are symbiotic associations betweenspecialised soil fungi and plants involved in nutrient transfer (seeBrundrett 2002). Plants with vesicular-arbuscular mycorrhizas (VAM)are ubiquitous, while those with ectomycorrhizas (ECM) are important inmany ecosystems, several other types of mycorrhizas occur in particularfamilies of plants, and some plants also have roots that remain non-mycorrhizal (Brundrett and Abbott; Brundrett and Cairney, this volume).Orchid mycorrhizas differ from most other types of mycorrhizas, as theyoccur in stems as well as roots, and fungi may re-colonise older cells(Hadley 1982; Smith and Read 1997). Orchids mycorrhizas are

Orchid conservation and mycorrhizal associations 197

morphologically different from other mycorrhizas and involve aphylogenetically distinct group of soil fungi (Rasmussen 1995; Currah etal. 1997). These fungal associates apparently have limited specialisationsas mycorrhizal fungi, and show little evidence of co-evolution with theirhosts (Brundrett 2002). Consequently, knowledge about the physiologyand ecology of other mycorrhizal associations (that are well studied)cannot be safely applied to orchid associations (of which we knowrelatively little).

In 1886, Wahrlich surveyed more than 50 cultivated orchid speciesand found fungal infections in every one of them. Subsequent surveyshave found mycorrhizas to be ubiquitous in terrestrial orchids and alsopresent in many epiphytes (e.g. Hadley and Williamson 1972; Benzingand Friedman 1981; Ramsay et al. 1986; Currah et al. 1997). However,it needs to be remembered that in most cases the roles of these fungi werenot examined and the benefits provided to orchids have rarely beenmeasured (see below).

There are considerable variations in the distribution of mycorrhizaswithin roots or stems of orchids. Mycorrhizal colonisation has beenreported to be sporadic in most epiphytes, but is generally morewidespread consistent in terrestrial orchids (Burgeff 1959; Rasmussen1995). Different genera of terrestrial orchids can have distinctivecolonisation patterns within their roots (Figure 1) or stems (Ramsay et al.1986). These mycorrhizal infection patterns in the whole plant (i.e. root,collar, stem, rhizome, etc) may be associated with particular fungal types(Ramsay et al. 1986). Colonisation patterns of mycorrhizal fungi withinplants are primarily determined by host cell properties, but mycorrhizalmorphological features can also be correlated with the presence of certainfungi (Brundrett 2002).

In the early 1900’s investigators first succeeded in germinatingorchids in vitro and observed mycorrhizal formation in embryos and


seedlings (Bernard 1903; Burgeff 1909). Orchid mycorrhizas arecharacterised by the presence of coils of undifferentiated hyphae incortical cells of the root, stem or protocorms of orchid species (Masuharaand Katsuya 1992; Smith and Read 1997; Peterson et al. 1998). Earlyhistological researchers recognised two types of orchid mycorrhizas (i)tolypophagy, which occurs in most species, and (ii) ptyophagy, found inmyco-heterotrophic species (e.g. Gastrodia) (Rasmussen 1995).Ptyophagy has been interpreted as hyphal lysis through which fungal cellcontents are released into plant cells (Burgeff 1959), but this requiresconfirmation by observations using techniques such as electronmicroscopy.

The collapse of old coils of hyphae also occurs within autotrophicorchid, and is s imi lar to the collapse of older arbuscules in VAMassociations, but the significance of these processes to plant nutrit ion isunknown (Brundrett 2002). Hyphal digestion allows successive waves ofpeloton formation, digestion and re-infection within the same root cells(Burgeff 1959; Smith and Read 1997) and would thus primarily be ameans of increasing the duration of active associations (Brundrett 2002).Otherwise, the fluxes of substances across the host-fungus interface wouldhave to be much higher than occurs in other types of associations becauseof the extremely small volume of plant tissues of some orchidmycorrhizas (see below). Most mycorrhizal scientists now consider activetransport of metabolites across the host-fungus interface of living cellsrather than digestion to be the primary mode of nutrient transfer in alltypes of mycorrhizas (Smith and Smith 1990). However, there is stillmuch to learn about how orchid mycorrhizas function and howassociations of autotrophic and myco-heterotrophic plants differ. Theearly work by Burgeff (1932) and others provides insight into thecomplexity of orchid associations, and this work should now becontinued by modern orchid scientists.

2. Orchid fungi2.1. Identity and specificityMost of the endophytic fungi known to form orchid mycorrhizas areBasidiomycetes, and many belong to the form-genus Rhizoctonia (Snehet al. 1991; Currah et al. 1997). Orchid rhizoctonias are distinguishedby their appearance in culture (the presence of short inflated segmentswhich resemble spores and the formation of loose aggregates of hyphaeregarded as poorly developed sclerotia or resting bodies (Hadley 1982)).Isolates which form sexual stages in culture (a difficult and poorlyrepeatable procedure) belong to the basidiomycete generaCeratobasidium, Ceratorhiza, Epulorhiza, Sebacina, Thanatephorus, andTulasnella (Warcup and Talbot 1967, 1980; Currah et al. 1997). Adiverse assemblage of fungi belonging to other taxonomic groups havealso been isolated from orchid roots, but may not all be beneficial(Currah et al. 1997).

It is not clear if orchid fungi from different regions are more closelyrelated to each other or to other saprophytic or parasitic groups ofRhizoctonia species. The form genus Rhizoctonia contains saprophytes,


pathogens and orchid symbionts (and probably also mycoparasites), butits unclear how much overlap and crossover there is been members ofthese groups (Figure 2). If orchid fungi typically have dual roles as,saprophytes or parasites, they are fundamentally different from thehighly specialised fungi forming other types of mycorrhizas. Orchidfungi could not co-evolve with their hosts like other mycorrhizal fungi, asthey receive few benefits from their associations with plants (Brundrett2002). It seems most likely that the orchid fungi are a disparate groupwith many separate origins and that the recruitment of new fungallineages by orchids probably continues today. Most of the mycorrhizalassociates identified form achlorophyllous orchids are different fromthose of green orchids (see below).

The question of host-fungus specificity within the Orchidaceae hasbeen a point of contention for many years. Knudson (1927) believedthere were low levels of specificity for tropical epiphytic species.Conversely, Burgeff (1909, 1959), who worked with many terrestrialorchids, thought that there was strong specificity in the associationbetween orchid and fungus. The evidence available today suggests thatboth these hypotheses can be correct (Table 2).

Harvais and Hadley (1967) isolated 244 Rhizoctonia strains fromDactylorhiza purpurella and other north British orchids. These belongedto 15 main groups, but most groups were confined to a single habitat,with the exception of R. repens which was widespread. Curtis (1939)argued that ecological distribution of the fungi was related to habitatrather than host. This was supported by Harvais and Hadley (1967) whoalso showed that Dactylorhiza purpurella was symbiotic with most of the


isolates they tested. However, there are other orchids, such as Goodyerarepens that usually associate with a single fungus (Ceratobasidiumcornigerum) as an adult (Hadley 1982). This may explain why G. repenshas a more restricted distribution than D. purpurella. Many other orchidshave been found to have fairly specific fungal associates that vary muchmore between hosts than between habitats (e.g. Warcup 1981; Ramsay etal. 1987; Currah et al. 1997; Sen et al. 1999). We may conclude thatorchids can associate with either a broad or narrow range of fungalisolates (Table 2). This contrasts with the situation in ECM associationswhere the fungi seem to primarily regulate specificity and can associatewith a narrow or broad range of host plants (Chapter 5). We mightexpect that the breadth of fungal specificity of orchid taxa would be oneof the most important factors determining the breadth of their habitatspecificity. However, this hypothesis needs to be tested by obtainingfurther investigation about the change with habitats and between taxa,using DNA-based fungal identification methods to characterise thediversity of orchid fungi.


Most of what we know about orchid fungus specificity comes fromgermination tests conducted under sterile conditions (e.g. Warcup 1981;Ramsay et al. 1986). However, these studies show the importance ofmycorrhizal fungi to germination, but do not confirm that the same fungiare important to the survival of adult plants in natural environments.Seed of some orchids will not germinate with fungi isolated from adultplants, but there are also many examples where these fungi promotesuccessful germination (Warcup 1981; Alexander and Hadley 1983; Muir1989). The establishment of healthy seedlings with leaves or a tuber, notmerely successful germination should be regarded as the decisivecriterion of host-fungus compatibility (Batty et al. 2001a).

Orchids may be able to live in symbiosis with one or several differentfungi, but the relative importance of co-occurring fungi is unknown.Differences in fungi wi thin orchid seedlings and adult plants may equateto the successional trends that occur in other types of mycorrhizal fungi(Brundrett and Abbott; Brundrett and Cairney; Dixon and Read, thisvolume). Co-occurring orchid species with different fungi may minimisecompetition for the same nutrient resource, if different species of fungiaccess different soil resources. Relationships between plant productivityand the diversity of mycorrhizal fungi have been observed in microcosmexperiments with VAM fungi (van der Heijden et al. 1998). It isgenerally assumed that greater taxonomic diversity of fungi equates to agreater function diversity that would benefit plants, but further research isrequired to test this hypothesis. In general, the diversity of orchid fungiassociating with a particular orchid species appears to be much lower thanfor other types of mycorrhizal fungi (i.e. most studies have reported oneor two fungi per species). The greatest implication of high-host fungusspecificity and low fungal diversity to orchids would be to restrict orchidsto certain habitats where these specific fungi occur.

2.2. Distribution in substratesUnderstanding the distribution of orchid mycorrhizal fungi within soil orother substrates is important for attempts to return orchids to the field andin understanding the distribution of orchids. The patchy distribution oforchids may be a result of the presence or absence of the specificmycorrhizal fungi essential for the survival of the orchid. Orchiddispersal may be a function of mycorrhizal distribution, seed dispersaland conditions suitable for the germination of orchid seed and theestablishment of orchid plants. Studies into the in situ germination oforchid seed in field sites have shown that good germination can beobtained (Table 3). A seed burial technique devised by Rasmussen andWhigham (1993) allows the distribution of effective orchid endophytes tobe assessed in situ in natural habitats. In a similar study in WesternAustralia, where seeds remained in the soil throughout the growing seasondemonstrated tuber development from protocorms, confirming thateffective fungi were present (Batty et al. 2001a). In this study thesuccessful germination of orchid seeds was found to be higher in closeproximity to adult plants of the same species.


The spatial d i s t r ibu t ion of orchid fungi wi th in soils is largelyunknown, but we might assume that it is similar to the distributionpatterns of other types of mycorrhizal fungi and is l ikely to be correlatedwith certain soil properties. Organic matter is likely to be an importantresource for orchid fungi , and is unevenly distributed in the soil, as arepopulations of other mycorrhizal fungi which tend to occur in discretepatches (e.g. Brundrett and Abbott 1995).

Some species of orchids tend to grow in clusters, while others tend tobe widely spaced. This depends to a large extent on a differentialtendency to produce vegetative offshoots, but may also be affected byfungus-mediated intraspecif ic competition. Competition between orchidsiblings for available resources supplied by mycelia occurs in vitro(Alexander and Hadley 1983; Rasmussen et al. 1989; Tsutsui and Tomita1989), and is also likely to occur in soils. This form of intraspecificcompetition would affect the number of individuals that can be supportedby a patch of fungi and affect seedling recruitment near parent plants.Subordinate plants sharing the same fungi in ECM and VAM associationsmay receive limited direct or indirect support from dominant plants(Brundrett and Abbott ; Brundrett and Cairney, this volume). However,this may not occur in orchids, as there is no evidence of energy transferto the fungus from the plant .

Orchid fungi are more widespread in the soil than their hosts, as theycan be isolated from soil in areas where orchids were absent (e.g. Curtis1939; Harvais and Hadley 1967; Warcup and Talbot 1967). Orchidfungi such as Ceratobasidium cornigerum and Tulasnella calosporaappear to have world-wide dis t r ibut ion, but this requires fur therinvestigation as substantial genetic heterogeneity has been found in otherwide spread fungal taxa (Chapter 5). Orchid fungi in soils are likely tohave substantial temporal as well as spatial variations in distr ibution(Perkins and McGee 1995).


2.3. PhenologyRasmussen (1995) noted that there is little data on the phenology of thefungi associated with orchids, or the impacts of low winter temperaturesor dry summer weather on the activity of external mycelium. In regionsof the World where the growing season is delimited by periods of cold ordry conditions other mycorrhizal fungi are active in the growing season,but persist as networks of hyphae and propagules in soils at other times(Brundrett and Abbott 1994; Braunberger et al. 1997). If it is assumedthat orchid fungi behave in a similar manner to other mycorrhizal fungithen there may only be a small seasonal window in which soil conditionsand fungal activity are conducive for germination of orchid seedlings(see Batty et al. 2000). In moderate climates, fungi could be active allyear, but may have peak periods of activity, such as cool wet periodswhen the most nutrient inputs from dead plant material occur (Rasmussen1995; Masuhara et al. 1988). Alexander and Alexander (1984) observedthat the terrestrial orchids Bletilla striata and Goodyera repens showedsigns of fungal infection throughout the year with a peak from Decemberto May. It appears that the main limiting factor for endophyte (fungal)activity is the lack of soil moisture and not low temperatures (seeRasmussen 1995).

In mediterranean climates with dry summers, soil microbial activity ismaximised during the cool wet winter months of the year while thisactivity is limited by soil moisture during the dry summer months(Sivasithamparam 1993). Sivasithamparam (1993) indicates that thesaprophytic phase of Rhizoctonia solani (AG8) survives the dry summerboth in colonised stubble and as mycelia (probably in a network) in thesoil. We might assume that orchids with mycorrhizal fungi behave in asimilar manner to plants with VAM fungi, which are known to providereservoirs of fungal inoculum in older roots, as only a fraction of the rootsystem of most perennial plants is replaced each year (Brundrett andKendrick 1988). Many terrestrial orchids perenniate only as rhizomesor tubers, but their roots or stems probably often follow the samechannels through the soil and these remnants of mycorrhizal tissue maybe important reservoirs of fungal inoculum, ensuring early infection.Intact networks of fungal mycelium are key reservoirs of fungalinoculum in many natural habitats for other types of mycorrhizas(Brundrett and Abbott 1995). Propagules of other fungi normally format the end of the growing season then decline due to predation and areactivated during the following growing season. We need to determine theform of saprophytic survival (Sivasithamparam 1993) orchid fungi use topersist in soils and where they are localised in the soil to devise methodsof identifying sites where appropriate fungi are present for orchidrecovery work (see below).

For natural recruitment to occur at a site, a supply of seed mustarrive at the location at the end of the growing season prior to therecruitment period. Orchid seed bank size depends on the level of seedproduction for that season and the life-span of seeds in soil. In WesternAustralian soils, seed survival is limited to a single year (Batty et al.200la) and these t iny seeds are unlikely to survive for much longer in


other habitats with a distinct dry season. Orchid seed germination islikely to be cued to periods of high fungal activity in soil and at the startof a growing period which would enable the pro-embryo to differentiate,germinate, and produce a tuber or plantlet of sufficient size to enablesurvival unt i l the next growing season (Batty et al. 200la). The presenceor absence of an appropriate fungus in an active growth phase at the fieldsite ultimately determines whether seed will convert into an orchidseedling and recruit into the adult orchid population. As seasonal trendsin saprophytic activity of orchid fungi have rarely been measured, we canonly assume that this is limited to periods when root and fungal activityare not limited by drought or temperature extremes, or when soil is notsubjected to excessive dryness or water-logging in seasonal climates.

3. Mycorrhizal associations3.1. Mineral nutrition and mycorrhizal dependencyEarly workers believed that orchids could only acquire nutrients throughtheir mycorrhizal fungi. Knudson’s (1922) experiments firstdemonstrated that seedlings did not have an absolute requirement formycorrhizas, as they could take up nutrients directly in sterile culture.Some investigators have considered the mycorrhizas of adult plants to beinsignificant, except in the chlorophyll-deficient species (Hadley andPegg 1989). However, there have been many demonstrations of thebenefits provided to adult orchids by their fungal partners, and it isprobable that most terrestrial orchid have an obligate requirement formycorrhizas when growing in natural habitats. Experiments withradioactive phosphorus have confirmed that orchid fungi can transportphosphorus into roots (Smith 1966, 1967; Alexander et al. 1984;Alexander and Hadley 1985).

Orchid plants typically have very coarse roots with limited lateralbranching. Extreme examples are provided by several genera of WestAustralian terrestrial orchids that are almost completely without roots andform associations in a h ighly confined space in their stem collars(Ramsay et al. 1986). Thus, orchids typically are not efficient for directabsorption of mineral nutrients from soil, in contrast with the fine highlybranched roots of plants that can grow without mycorrhizas in naturalhabitats (Brundrett 1991). Further evidence of the effectiveness of orchidmycorrhizas is provided by the occurrence of many orchid species inhabitats with extremely infertile soils with low accessibility of minerals, orwith extremely high or low pH, which often have a loose texture and highhumus content (Sheviak 1974).

The degree of mycorrhizal dependency of epiphytic orchids is lessclear, as their protocorms often become photosynthetic at an early stageand the roots of adult plants often have limited and sporadic fungalcolonisation (Arditti 1992). Orchids growing as epiphytes on trees obtainadequate mineral nutrients from dust, organic debris and stem-flow alongthe bark of the host (Arditti 1992). It has recently been suggested thatgerminating epiphytic orchid seeds obtain water through mycorrhizalfungi (Yoder et al. 2000).


Orchid mycorrhizas differ from typical ECM or VAM associations,as orchid fungi can provide a source of energy as well as mineralnutrients to their host plants (Rasmussen 1995). These carboncompounds presumably are derived from the breakdown of organicsubstances in the surrounding substrate. Radioactive carbon has beentraced from fungi to seedlings but not from seedlings to fungi. It hasgenerally been assumed that orchids with chlorophyll provide their fungiwith energy in exchange for fertiliser, as is the case with most ECM orVAM associations. The plant may supply essential vitamins or aminoacids to fungi in some cases (Leake 1994). However, there is no realevidence that fungi receive substantial benefits from any of theirassociations with orchids. These fungi appear to be much less specialisedthan other types of mycorrhizal fungi and presumably are highlyindependent and often grow without any assistance from orchids (Section3.2).

Little is known about the other ecological roles of fungi thatassociate with green orchids and some of these fungi may have an adverseimpact on other plants. For example, epiphytic orchids sometimes appearto have a detrimental effect on trees that harbour them (Ruinen 1953;Johansson 1977), but this could result from a correlation betweenepiphyte abundance and tree decline due to other factors. There aresome cases where Rhizoctonia isolates from orchids have been identifiedas pathogens in roots of other plants (Warcup 1985a; Zelmer et al. 1996),or parasites of VAM fungi (Williams 1985). However, most orchids havefairly specific associations with Rhizoctonia strains that are not known tobe pathogens of other plants (Warcup 1981; Ramsay et al. 1987; Muir1989; Currah et al. 1997; Sen et al. 1999). There currently isinsufficient information to safely say whether autotrophic orchidsnormally have mutualistic or exploitative associations with soil fungi. Weknow very little about the other roles of orchid fungi in soils. Thisknowledge is essential for us to develop an understanding of the biologyand ecology of these beautiful and fascinating plants. If orchid fungi arealso plant pathogens or mycoparasites, then orchids are epiparisites ofother plants in their communities. Green orchids would be epiparasitic toa lesser extent than myco-heterotrophic orchids which are entirelydependent on fungi supported by other plants.

Some terrestrial orchids have a higher degree of shade tolerance thanother plants (McKendrick 1996). Numerous observations of orchids innatural habitats also support the conclusion that many adult greenterrestrial orchids are less dependant on sunlight for energy than otherplants, because of their mycorrhizal fungi, but this requires furtherinvestigation (Burgeff 1959; Rasmussen 1995; Smith and Read 1997).Many orchids require full sun to grow, and typically occur in grasslandsand open woodlands (Case 1990; Rasmussen 1995).

3.2. Myco-heterotrophic orchidsSaprophytic (myco-heterotrophic) orchids without chlorophyll areassumed to have fully-exploitative mycorrhizal associations that supplyboth the energy and nutrient requirements of the host (Leake 1994).


Achlorophyllous plants with VAM or ECM associations such asMonotropa (Ericaceae) have established similar exploitative associationswith fungi (Leake 1994; Brundrett 2002). Mycorrhizal associationswhere fungi do not seem to receive any benefits from plants are alsocalled epiparasitic, myco-heterotrophic, cheating, or exploitativeassociations (Furman and Trappe 1971; Leake 1994; Taylor and Bruns1999; Brundrett 2002). The nature of mycorrhizal associations of manyof these plants has not been investigated and their nutritional dependencyon fungi has been assumed whenever other explanations are lacking.Myco-heterotrophy has evolved independently in approximately 20separate orchid lineages much more often than in any other group ofplants (Molvray et al. 2000). Orchids also appear to evolve more rapidlythan other plants (Molvray et al. 2000).

Myco-heterotrophic associations involve fungi that belong toseparate lineages to those forming mycorrhizas with green orchids,including ECM associates of trees, wood decaying fungi and parasites ofother plants (Table 4). These associations have a high degree of host-fungus specificity and orchids such as Corallorhiza, Gastrodia andGaleola are only known to associate with a single fungal genus (Table 4).

Achlorophyllous myco-heterotrophic orchids species includeRhizanthella gardneri which is ful ly subterranean (Dixon and Pate 1984),but other achlorophyllous orchids emerge from the ground for floweringand seed dispersal. The survival of achlorophyllous orchid mutantswhich are normally green shows that mycorrhizal associations can alsosupply carbon to the plants (Salmia 1988; Rasmussen 1995). Rasmussen,(1995) summarises the reports where chlorophyllous orchid species haveremained underground for a number of seasons. Some Australianspecies, such as Caladenia spp. and Leporella fimbriata, can to carry outseasonal replacement of tubers without producing a shoot above ground(Dixon 1991). This feature has important conservation implications, asorchid populations can remain unnoticed below-ground for a number ofseasons.

Seedlings of most plants must develop leaves before the nutrientreserves in the seed are exhausted, and remain phototrophic throughouttheir life. In contrast orchid seedlings have an option of living forextended periods as myco-heterotrophic organisms, opening habitats toorchids that would not otherwise be accessible. The extremely highdegree of fungus specificity in myco-heterotrophs confines these plantsto habitats where a particular fungus remains viable for long enough forthe orchid to reach reproductive maturity. Many of the fungi listed inTable 4 are vigorous saprophytes or parasites that are dominant in largesubstrates such as tree trunks that take a long time to decompose, thusproviding sufficient time for orchid establishment and reproduction.Myco-heterotrophs will be more difficult to conserve than other orchidsif their fungi are hard to manipulate. However, some fungi listed in Table4 can be grown efficiently (as for mushroom production). Theidentification of associated fungi and a sound understanding of theirbiology is a fundamental requirement for the conservation of theseorchids.


3.3. Orchid habitat preferencesIt can be hypothesised that since orchid seedlings have and obligaterequirement for mycorrhizas their establishment will be determined bythe distribution in substrates of fungi required for germination andgrowth (Figure 3). Some of the many questions about the role ofmycorrhizal fungi in orchid population biology that still need to beanswered are listed below:1. How important is host-fungus specificity and compatibility?2. How do fungi vary in effectiveness for promoting seed germination

and subsequent growth?3. How critical is the soil status (‘soil saprophytic growth’ sensu Garrett

(1970)) of the orchid endophyte for the germination of the orchidseed?

4. Do plants normally contain multiple fungi? If so, are there seasonaltrends in the dominance of individual strains?

5. Are there changes to fungus population in soil during the life ofplants?

6. Is the composition of fungal populations for an orchid speciesdetermined more by habitat factors or historical events?

7. Is knowledge of habitat preferences of orchid fungi required toidentify potential orchid habitats?

These questions cannot be answered without knowledge of thedistribution and diversity of orchid fungi within soils. Reasons for thesuccess or failure of orchid establishment from seeds in soil have beenexamined experimentally in only a few cases (Table 3). In cases whereseedlings fail to become established, it is difficult to separate factors


affecting orchids directly from those affecting their mycorrhizal fungi(Table 5).

The successful establishment of an orchid to a field site is likely onlyto be achieved when conditions are favourable for both the orchid andassociated fungi (Figure 3). In nature the development of a seedlingwould require the arrival of viable seed at a point containing compatiblefungi. Following germination, subsequent seedling development couldonly occur in the presence of the associated fungus. Establishment of anadult plant capable of producing seed is most likely subject to similarconstraints. However, successful establishment occurs in those situationswhere factors remain conducive for both the level of receptivity to anddependence of adult terrestrial orchid plants on fungi.

4. Utilising orchid mycorrhizasThis section focuses on terrestrial orchids, as epiphytes typically arepropagated by asymbiotic means. As discussed below, there is strongevidence for the vital role of orchid fungi in the propagation and growthof terrestrial orchids. This contrasts with epiphytic orchids wheremycorrhizal fungi may have less importance after seed germination.

4.1. Seed germinationThe seeds of terrestrial orchids measure from 0.07 to 0.40 mm across andfrom 0.11 to 1.97 mm in length, including the testa (Figure 4; Arditti andGhani 2000). These minute seeds have very little stored nutrient reservesavailable to support seedling development. These limited reserves and thesubterranean germination of many terrestrial species, result in the generalbelief that mycorrhizal fungi are normally essential for seed germination(Figure 5).

Studies comparing the effectiveness of symbiotic and asymbioticgermination have usually shown that symbiotic germination was more


rapid and effective than asymbiotic germination (Table 6). In someterrestrial orchids successful germination was only achieved by symbioticgermination (Hadley 1982). Muir (1989) screened a wide range offungal isolates for their capacity to promote germination and growth ofEuropean species of Orchis, Ophrys, Dactylorrhiza and Serapias. Hefound that rare species were compatible with fewer isolates of fungi thancommon species in same genus. In some cases, different fungi areresponsible for the growth of mature plants than those responsible forgermination (Section 2.1).

4.2. Germplasm storageEx situ conservation includes studies into the long-term storage of orchidseed and mycorrhiza in liquid nitrogen (-196°C) as a back up of geneticstock in the event that critically endangered species become extinct in thewild. This may give us a second chance for some species and in no wayis meant to replace conserving the species in their natural habitat. Wheresymbiotic seed germination methods are to be used to propagateterrestrial orchids it is important that both orchid seed and associatedfungi are stored successfully. The plunging of orchid seed into liquidnitrogen has also been found to increase germination percentages for arange of orchid species (Batty et al. 2001b). This may be used toincrease germination of seed from endangered orchid species where seedis often in short supply.

4.3. Orchid recovery plansWhen a species is recognised as critically endangered, the first step indeveloping a conservation strategy normally is the preparation of arecovery plan. Through surveys of existing populations, risks areidentified and a list of required actions is drawn up. Actions usuallyinclude research into the species biology, to identify factors essential forits survival. This is especially so for terrestrial orchids due to theircomplex associations with specific mycorrhiza and highly evolved



pollination systems. The preferred way to conserve a species is tomanage the plants in their natural habitat before they become criticallyendangered and in need of intense off-site conservation effort to preventextinction. On-site (in situ) management should be of the highestpriority in any conservation program.

Orchids are well researched taxonomically, but relatively little isknown of their conservation biology and importantly, methods formanagement and translocation to field sites. Unlike other plants,terrestrial orchids are unique in their highly specialised pollinationmechanisms and habitat requirements. They also require advanced



technology for large-scale propagation. These methods include: (i)Collection of seed and fungal symbionts which are effective forgermination, (ii) Development of effective propagation methods for thetarget species; and ( i i i ) Translocation of orchids to safe sites.

There are a number of different protocols available for the isolationof orchid mycorrhizal endophytes. These include the block method andthe peloton isolation (Dixon et al. 1989). Depending on the isolationprotocol being adopted by the researcher and the fate of the isolate(s) thismay affect results. For example, isolation of fungi from surface-sterilisedblocks of orchid tissue may be unsuitable, if the researcher wasattempting to investigate fungal diversity in orchids. This occurs becauseslow growing or more sensitive cultures will be over-run by fungi thatgrow more aggressively on agar. We have also observed problems witheffective surface sterilisation that result in isolation failures from sometissues. Single peloton isolation methods are often best, as they shouldallow any isolates that form mycorrhizas to be obtained (Rasmussen1995). The isolation technique used to obtain orchid fungi may impacton specificity studies, as some methods may select rapidly growingisolates or contaminants.

A survey of the literature suggests that there are several differentapproaches to working with orchid fungi for conservation (Table 3). Insome cases, it is assumed that each orchid has a new or specific fungalpartner and isolate new fungi for each orchid to be conserved, which arethen tested for host-compatibility by a germination assay. In other cases,it is assumed that host specificity is less important than habitat specificityand therefore a wide range of isolates from the same and differentorchids are screened to find the most effective partners. Although bothapproaches have resulted in successful propagation of terrestrial orchids,there may still be regional differences in fungal biology. This maybecome evident when field introductions are attempted. Examples wheresymbiotic seed germination has allowed the successful propagation andreintroduction of orchids are summarised in Table 7.

5. Case studies5.1. The Western Australian Underground Orchid, Rhizanthella gardneriThe majority of achlorophyllous orchids develop an above-ground phasewhich functions in flowering and seed dispersal (e.g. members of thegenera Epipogium, Didymoplexis and Gastrodia). However theAustralian genus Rhizanthella remains below ground, or, at least, belowthe litter layer, even when forming flowers (Figure 6) and seeds. Thiscryptic behaviour has continued to confound observers since accidentaldiscoveries of the orchids half a century ago. Even at present, the easternAustralian species R. slateri has been seen only once in flower in the wildin the past decade, while its western counterpart, R. gardneri, has onlyrecently been rediscovered (George 1980; Dixon and Pate 1984).Although known in several locations, R. gardneri still requiresconsiderable effort to locate specimens non-destructively in its nativehabitat, even where it is currently known to be prolific. Early discoveriesof this mysterious, fu l ly subterranean plant roused great excitement


among the local public and scientific fraternity of the day, and, within sixmonths of its discovery, wax models of the plant had been placed onexhibition at scientific meetings and museums in Western Australia.

5.1.1. The myco-heterotrophic nutrition of Rhizanthella gardneriThe endophyte of Rhizanthella was originally described by Pittman(1929) as being “Rhizoctonia-like” and recent work by Warcup (1985b)has confirmed that at least two strains of the form genus Rhizoctonia maybe involved in symbiosis with the orchid and its host Melaleuca uncinata.The endophyte resembles the basidiomycete genus Thanatephorus, aknown symbiont of orchids (Hadley 1982).

Orchid conservation and Mycorrhizal associations 215

It is widely held that holomycotrophic orchids derive carbon fromtheir fungal partner which breaks down the organic matter ofsurrounding litter or humus. Alternatively or additionally they mayengage in a three-way epiparasitic relationship with an autotrophic tree orshrub species. In the latter case, carbon passes from roots of the woodyspecies to a shared mycorrhizal partner and thence becomes available tothe orchid. Relationships predominantly of an epiparasitic kind havebeen suggested for other achlorophyllous orchids such as: Gastrodia(Table 4), and, outside the Orchidaceae, in achlorophyllousrepresentatives of the subfamily Monotropoideae of the Ericaceae(Pterospora, Sarcodes and Monotropa, Robertson and Robertson 1982).However, with few exceptions (Bjoerkman 1960) definitive tracer studiesof nutrient flow from woody hosts to epiparasitic partner are lacking, andsince the surrounding soil is rich in organic matter, a significantsaprophytic element in nut r i t ion of this orchid is highly likely.

As stated previously, R. gardneri inhabits soils extremely deficient inorganic matter, implying that a purely or predominantly organic matter-dependent nut r i t ional base for the orchid would be extremely doubtful .The recent success of Warcup (1985b) and the authors in cultivating theorchid from a seed in an artificial Melaleuca-Rhizoctonia-Rhizanthellasystem corroborates this view, at least to the extent of demonstrating ahighly effective epiparasitism by the orchid when organic matter is absentor at very low level in the medium.

5.1.2. Seed morphology and structure, germination, and earlyestablishment of symbiosis with a fungal partnerGermination of seeds in laboratory culture occurs more readily in l ightthan dark coloured seeds. It is preceded by swelling of the embryo andcracking of the seed coat. The h i lum corresponds to the point of entry ofthe endophyte into the seed, and adjacent cells of the embryo eventuallyshow intracellular proliferation of the fungus. By three months aftergermination, the body of the protocorm is already some 2-3 times thesize of the seed. Radiating from it are a number of trichomes, each apossible entry site for fungal hyphae. After five months the elongatedshape of the mature tuber is clearly apparent and the growing shoot apexclearly demarcated and flanked by trichome primordia.

Seedlings germinated in mineral agar in the presence of the fungusare ready for t ransplant ing into the rooting medium of soil grownRhizoctonia-inoculated Melaleuca uncinata plants 3-4 months aftergermination. Our observations on seedling growth through perspexwindow in the side of the potted host plant show rapid tuber growth overthe ensuing 9 months. First f lowering was recorded by Warcup (1985b)in 15 month-old plants.

Rhizanthella gardneri possesses several characters consideredprimitive for the Orchidaceae (Dressler 1981). These include spiralarrangement of leaves, a terminal inflorescence, non-resupinate flowers,fleshy fruits, large hard seeds, low seed number, an ‘endosperm-like’embryo, mealy pollen, and stomata lacking subsidiary cells. On the otherhand, by virtue of its totally underground habit and ability to survive


aphotosynthet ical ly in soils extremely low in organic matter, it may wellbe considered to represent the u l t ima te in specialisation among orchids.

5.1.3. Conservation and managementAs far as we know, Rhizanthella gardneri is an extremely rare specieswhich unfortunately occurs pr imari ly in areas suitable for cerealproduction. On these grounds it would appear that all but less than 10%of the habitats where it may have once occurred are now underagriculture. Worse still, applications for release of new lands foragriculture in potential Rhizanthella habitats continue to outstrip surveyefforts to locate the orchid.

The enormous d i f f i c u l t y in locating Rhizanthella, let alone theproblem of effectively monitoring the vigor of existing populations,presents great obstacles in management of the species. Clearly the bestpolicy is to direct conservation and management policies towardsmaintaining adequate healthy stands of Melaleuca uncinata which co-hosts the fungus, in the hope that they will provide large and variedresource of habitats in which the orchid may well st i l l be present, or ontowhich it might be introduced where the species is deemed to be greatlyendangered.

Frequent fires, invasion of habitats by weeds and exotic animals,deliberate or accidental human interference of known habitats of theorchid, and the general deterioration of native bush caused by aerial driftor surface water leaching of agricultural fertilisers, are all potentdeleterious influences (Dixon and Pate 1984). A further, more subtlecause for concern, is the poss ibi l i ty that the few remaining populationswill degenerate through e l imina t ion of pol l inat ing agents, nativemammals which might d is t r ibute seeds, or merely by genetic deteriorationof the species due to lack of input by sexually derived genotypes. Asshown here, natural seed set in our study locations is extremely poor inpresent populations, but can be increased many fold by hand poll inat ion.This may indicate that current maintenance of the species is largely byvegetative multiplication through formation of daughter tubers, givinglittle potential for long-distance spread within a habitat.

The methodology exists for establishing the Rhizanthella-Rhizoctonia-Melaleuca association in glasshouse culture, thereby givingthe opportunity for replenishing stands of the orchid in existing habitatsor even for establishing the species in areas where it currently does notexist. Considerable thought must be given to the relative merits of in situand ex situ conservation for this species, but the fact that this type ofapproach is avai lable for conservation of the species must surely offerconsiderable reassurance.

5.2. Other Western Australian orchidsThe southwestern botanical province of WA is one of the world’s maincentres of biodiversity, where a long period of geographical isolation andhighly infert i le soils have resulted in many plant species that occurnowhere else. This region has one of the world’s most diverse terrestrialorchid floras, wi th over 340 species known. Southwestern WA is a


“living biological laboratory,” where the endemism and diversity of ournative orchid flora provides a unique opportunity to study issues relatingto the conservation and management of terrestrial orchid species. Inparticular, it is possible to contrast closely related species which arecommon and rare, have very general or highly specific habitatrequirements, or have geographically disjunct populations. At present, 34taxa of southwestern orchids are designated as critically endangered(Brown et al. 1998) and a 34 more are only known from a few locationsand require further study (Atkins 1999). Even relatively common speciesare declining in many urban and rural areas due to habitat loss and landdegradation.Increasing urban and industrial development in many plants of the Perthregion threatens the existence of many once common plant communitiesand species. Perth’s native orchids are a unique part of our natural andcultural heritage, but are being severely impacted upon by land usechanges and habitat degradation. There are many endemic species ofterrestrial orchids in WA that are rare because of highly specific habitatrequirements, requirements for specific pollinators and/or ineffectivedispersal mechanisms coupled with extensive land clearing for agricultureand housing. Other threats to orchid habitats include, weed invasion,frequent fires, changes to the water table, grazing by feral animals anddisturbance by humans. No other capital city in Australia has as manythreatened native plants and clearly urgent conservation actions areneeded if species extinctions on the doorstep of Perth are to be averted.

It is suspected that the low recruitment rates from natural seeddispersal observed in WA probably result from the patchy distribution ofmycorrhizal fungi in soils and the scarcity of suitable habitats in thelandscape. Information about the biology of fungi that associate withWA terrestrial orchids is urgently required as a cornerstone forconservation work – to identify habitat requirements and understandfactors that determine the success of seedling establishment.

Where possible seed has been collected from rare orchid species,specific endophytes have been extracted from cortical cells and cultured.Seed and endophyte have been combined to produce seedlings of manyspecies for the first time. New protocols for the transfer of seedlings tonatural bush sites have resulted in the successful translocation of theCinnamon Sun Orchid (Thelymitra manginii) (Figure 7), Dwarf BeeOrchid (Diuris micrantha) and the Swamp Donkey Orchid (Diurispurdiei) (Batty et al. 2001 a).

Diuris purdiei seedlings were translocated to two sites by introducingtissue-cultured plants and mycorrhizal fungi. These plants were still aliveafter one year but a lack of funding prevented further monitoring (ANPC1997). More recently, laboratory produced seedlings of common andrare orchids (Caladenia arenicola, Pterostylis sanguinea, Thelymitramanginii and Diuris micrantha were returned to field sites in attempts tore-establish orchid plants in natural habitats. Plants that were returned tothe field as actively growing seedlings or as dormant tubers showedsurvival beyond the first season where as those initiated from in situ seedgermination failed to survive the first summer. These methods now


require further testing to see if they would be suitable for many other WAterrestrial orchids and to understand further the role of associatedmycorrhiza in establishing orchids to field sites.

6. ConclusionsSymbiotic associations are generally considered to be essentialthroughout the Orchidaceae. However, we have a very limitedunderstanding of the ecology of the orchid, their associated symbionts, orthe interactions between them. The majority of current knowledge onorchid fungus and host plant interactions is based on in vitro studiesusing fungal isolates from mature orchid plants. However, in recent yearsresearchers have demonstrated the ability to unlock some of the mysteriesof orchid mycorrhizas under field (in situ) situations. Most of ourknowledge of the role of orchid mycorrhizal associations is from studiesof terrestrial orchids and relatively little is known about their role in theestablishment and growth of epiphytes.

This type of information is essential if successful large-scale re-introductions are to be carried out on some of the large number ofendangered terrestrial orchid species occurring throughout the world.This review considers the ecological implications of orchid mycorrhizas,especially their implications for the conservation of threatened orchidspecies. This review considers orchid conservation on a world scale, butalso includes case studies which address regional issues.

Orchid mycorrhizas have been studied since the relationship betweenorchids and endophytes was discovered by Noël Bernard (1909) andmuch data has been massed, mostly from in vitro studies (for recentreviews see Arditti 1990; Peterson 1998). Recently, research has begun tofocus on in situ studies. It has become evident that not all knowledgegained from in vitro studies can be applied to field situations and when


dealing with critically endangered taxa we need to understand whathappens at the ground level, not the Petri dish. Field studies have beendifficult in the past unt i l the development of innovative protocols forstudying dust seeds in situ (Rasmussen and Whigham 1993).

AcknowledgementsThe financial support of Western Power is gratefully acknowledged.

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Chapter 8

ERICOID MYCORRHIZAS IN PLANTCOMMUNITIES

Kingsley W. Dixon

Kings Park & Botanic Garden, Botanic Gardens & Parks Authority, West Perth 6005,Western Australia; Plant Biology, Faculty of Natural and Agricultural Sciences, TheUniversity of Western Australia, Crawley 6009, Western Australia.

K. Sivasithamparam

Soil Science and Plant Nutrition, Faculty of Natural and Agricultural Sciences, TheUniversity of Western Australia, Crawley 6009, Western Australia.

David J. Read

Department of Animal and Plant Sciences, The University of Sheffield, Sheffield S10 2TN,UK.

1. IntroductionThe Ericales encompass a large, but monophyletic lineage of taxa, allgenerally recognised as possessing an anatomically and mycologicallyunique association with a mycorrhizal partner (Smith and Read 1997;Cairney 2000; Stevens 2001 onwards). The association with ericoid fungi inthe Ericaceae (sens. APG 1998) has been the basis of considerable researchand debate, issues ranging from the nitrogen (N) and phosphorus (P)impoverishment of soils where these plants grow and the role of the ericoidfungal partner in nutrient acquisition (Read and Kerley 1999), to elegantexperiments demonstrating high levels of diversity of the fungal partners ofericads.

The relationship between ericoid fungi and the Ericales provides aremarkable l ink between a cosmopolitan group of plants and mycorrhizal


fungi based on the unique anatomy of the association where unsuberisedepidermal cells are occupied by a typically dematiaceous fungal partner(Read 1983). The description by Harley (1959) of the structure as an‘ericoid mycorrhiza’ provided reinforcement of the linkage of these fungiwith particular anatomical structures (unique to the Ericaceae:Cassiopoideae, Ericoideae, Epacridoideae, Empetreae, Vaccinioideae). Thefact that these families represent many heathland groups has been used tocontextualise the nutrient uptake benefits of the fungi. For example, theheathlands of the boreal regions with their ombrotrophic status has resultedin many authors concluding that the fungus operates in a environment ofprevailing acidity, leaching (among other things), metal ions into solution(Read and Kerley 1999). In low pH environments, this raises issues ofphytotoxicity for the plant (Jalal and Read 1983a,b). The fungus is thoughtto enable a ‘detoxifying’ system to operate (Bradley et al. 1982; Leake andRead 1991) for establishment of plants in otherwise hostile soil conditions(Cairney 2000).

The variety and diversi ty of habitats in which ericads operate istestament to the adaptability and resilience of the ericoid endophyte. Fromthe moor-humus conditions of Calluna heathlands (Read 1983) in thenorthern hemisphere to the extreme seasonality and nutrient impoverishmentof the hotspots of ericad diversity in the fynbos of Southern Africa and thekwongan of south-western Australia is indeed comparatively unique inmycorrhizal systems (Read 1989; Smith and Read 1997). Understanding therelationship between the diversity of ericoid fungi and their attributes andtheir specificity as a key to the endemism and conservation of Ericales is thesubject of this chapter.

2. Anatomical basis to the ericoid stateMcLennan (1935) illustrated the ericoid mycorrhizal root as comprisinghypertrophied epidermal cells, with attendant endophytic hyphal matrix,surrounding an exodermis and a monarch stele. Ericoid mycorrhizal roots or‘hair roots’ (Beijerinck 1940) are devoid of root hairs and can be producedfrom all orders of roots. Dixon (pers. obs.) has recorded hair roots beingproduced directly on the surface of eight year old roots of Leucopogonconostephioides and Conostephium pendulum in kwongan heathland in southwest Australia. The hair root apparatus and attendant fungal biomass canoccupy up to 80% of the root system of Calluna (Read 1983) and up to 85%in the kwongan ericad Astroloma xerophyllum (Hutton et al. 1994). Thepercentage contribution of annual hair root production to total root biomass(Bell et al. 1996) and the abundance, seasonality and ecological value of hairroot production in the Magellanic tundra of Patagonia (Pisano 1983) andSphagnum bogs of New Zealand (Wardle 1991) represent systems which

Ericoid mycorrhizas in plant communities 229

require further investigation. The production of hair roots is periodic in theseasonally droughted ericads of south western Australia (Figure 1; Hutton etal. 1994). Here, the seasonality of production of hair roots in the wintergrowth season implies that the ericoid fungus may have more to do withnutrient acquisition than water balance.

The unique morphological and anatomical nature of the hair roots andits fungal associates in the Ericaceae offers a basis for attempting tounderstand the cosmopolitan success of the Ericales known to participate inericoid mycorrhizal partnerships. Whereas Straker (1996) details host andecological specificity in ericoid mycorrhizas and Duckett and Read (1991,1995) raise the spectre of ericoid fungi co-associating with hepatics, ericoidmycorrhiza in the Ericales is seen as one of the most specific of mycorrhizas.Linking benefit to the plant with the presence of the endophyte within theunique enlarged epidermal cells of the hair root provides a clear mechanismfor determination of the extent of the ericoid syndrome in plants. Read andKerley (1999) go one step further in providing a more rigorous system to


accurately define not just the ‘occurrence in or on the ericaceous root’. Thissystem, based on the testing of the simplest of Koch’s (1912) postulatesprovides three steps for estimating the root-fungus association asmycorrhizal:1.

2 .

3.

Candidate fungus is isolatable and maintained in pure culture.The fungus is grown w i t h i t s puta t ive host p lant under definedconditions.There needs to be evidence that infection by the fungus leads toenhancement of growth or nutr ient uptake.Read and Kerley (1999) indicate that the dark (rarely pale), slow

growing, sterile and dematiaceous mycelia commonly isolated fromEricaceae have only rarely been thoroughly evaluated using these principles(Leake and Read 1991). Monreal et al. (1999) described a raft of 34mycorrhizal root-associated isolates of Gaultheria shallon (Ericaceae) fromconifer plantations in Western Canada, yet failed to demonstrate that thefungi isolated from root sections (hair roots were not specifically stated)were truly endophytic. The issue of defining endophytism is addressed byStone et al. (2000) who follow Petrini (1991) who suggests “Endophytescolonise symptomlessly the l iving, internal tissues of their host, even thoughthe endophyte may, after an incubation or latency period, cause disease.”This definition covers all microorganisms as well as endophytic vascularplants (Stone et al. 2000). Jansen and Vosátka (2000) screened a wide rangeof fungi from Rhododendron and found that of 200 strains tested, only 10%exhibited positive effects on the growth of Rhododendron micro-cuttings. Inthe absence of isolation from within the specialised plant structures fromwhich the ericoid association was or iginal ly described, fungi isolated fromthe surface of roots or dead cells within active regions of roots remainambiguous in their functional relationship and ecological importance.

The presence of the hair root (Figure 2) as a morphological attribute ofthe ericoid symbiotic system is also unusual compared with many other plantstructures involved in symbiotic relationships. What is most surprising isthat unlike most other mycorrhizal systems, the hair roots and attendanthypertrophic epidermal cells are formed in ‘anticipation’ of an infectionevent. Hutton et al. (1994), in a phenological study of hair root production,found that 40% of the hair root system was devoid of endophyte yet retainedthe anatomical characteristics of the hair root. The hypertrophic collar andstem region of some terrestrial orchids (Ramsay et al. 1986; Dixon 1991;Batty et al 2001a,b) represent other rare instances where plant structures areformed in anticipation of a mycorrhization event.

The chemical composition of the cell walls of hair roots differ from thatof most other higher pants (Albersheim 1976) in the lack of fucose andpolygalacturonic acid residues and the presence of N-acetylglucosamine


(Straker 1996). While providing endorsement of the unique nature of thehair root, the chemistry of the hair root wall may play a key role in therecognition of ericoid fungal strains. The development of the fibrillar sheath(Gianinazzi-Pearson and Bonfante-Fasolo 1986) by some efficacious ericoidendophytes may be associated with cell wall recognition prior to ‘docking’of the fungus (Straker 1996).

The hair root provides a simple morphological criterion for thedetermination of the likelihood of a possible mycorrhizal association. Infriable soils, particularly the loose sands of southern Africa and southwestern Australia, hair roots (Figure 2) are visible in excavated roots asgossamer-like threads, often with fine soil particles adhering to them.Careful clearing and staining either with a general fungal stain or a specificfluorescent dye such as 3,3’-dihexyloxacarbocyamine iodine (Duckett andRead 1991) will help to detect the presence of intracellular hyphae. Thismay indicate that the ericad is l ikely to be involved in a mycorrhizalrelationship. Thus, for the conservation practitioner, it is possible to quicklydetermine if an ericoid mycorrhizal association is present or likely to bepresent. However, to accurately ascribe ecological function, more complexand exhaustive trials are required (Vrålstad et al. 2002). Management ofericad habitats (e.g. nutrient loading, impact of fire or soil disturbance)


would therefore need to consider the impact of such disturbance on thehealth and sustainability of the ericoid mycorrhizal system.

3. Potential value of ericoid mycorrhiza as indicators for conservation ofEricaceae speciesHarley (1959) coined the term ‘ericoid mycorrhiza’ to describe therelationship between the hair root and a fungal endophyte. Ericoidmycorrhiza have been reported to occur in the majority of the genera ofEricoideae (e.g. Calluna, Erica, Gaultheria, Kalmia, Ledum, Phyllodoce,Rhododendron and Vaccinium), Empetreae (e.g. Empetrum) (Read 1996;Read and Kerley 1999) and Epacridoideae (25 genera) (Reed 1987; Huttonet al. 1994; Bell et al. 1994). Cullings (1996) provided a circumscription ofthe Ericaceae and found that ericoid mycorrhiza-forming taxa aremonophyletic, indicating that the Epacridaceae and Empetraceae should beincluded within Ericaceae sens. lat. Subsequent comprehensive study of theEricales reinforces this view (Kron 1996; APG 1998; Stevens 2001onwards).

What is clear is that re-evaluation of the phylogenetic relationship ofthe Ericaceae as proposed by Kron (1996) convincingly shows that theEmpetraceae (Cataula) nests well within the Rhododendron/Calluna clade,sharing with those taxa the dis t inct ive characteristics of the ericoidmycorrhizal association (Smith and Read 1997). The analysis provided byKron (1996) segregates Arbutus as distinctive from the Ericaceae in a basalposition (though included as a subfamily of Ericaceae by APG (1998) andStevens (2001 onwards)) in concurrence with the distinctive arbutoidmycorrhizal system (Smith and Read 1997). With the knowledge of a morenatural and rigorous interpretation of the phylogenetic relationships of theEricaceae, it is of interest to relate the fungal associates with the knownevolutionary history of the Ericaceae (see Cairney 2000).

The remarkable morphological similarity of the hair root, therelatedness of these three families with a restricted array of distinctiveascomycetous fungi adds further support to the inclusion of the Empetraceaeand Epacridaceae within the Ericaceae.

Since the Ericaceae encompasses such a broad and cosmopolitan familyof some 140 genera and 3,800 species (Mabberley 1997) across Arctic tosouthern hemisphere mediterranean-type to cool temperate ecosystems, it istempting to speculate as to the functional attributes and benefits of themycorrhizal association across this broad range of habitats.

4. Characterisation of ericoid endophytic fungiThe identity of mycorrhizal fungi which associate with the Ericaceae hasbeen a source of debate and conjecture (Vrålstad et al. 2002). A variety of


Ascomycetous and imperfect fungi have been associated with ericoid roots(Smith and Read 1997) although not all of them have been established to bemycorrhizal. Much of the work, especially in the United Kingdom, has beencarried out with Hymenoscyphus ericae. Among the fungi imperfectiassociates, Oidiodendron species have featured most commonly (Smith andRead 1997).

The centre of diversity for extant Ericales is in the southern hemisphereand most likely reflects a Gondwanan origin (Cullings 1996). Ericales-likeplants are found in the fossil record from the Cretaceous (Nixon and Crepet1993). Although no mycorrhizal involvement can be discerned, speculationis that a coincident major radiation of ascomycete fungi (Berbee and Taylor1993) may have provided the early Ericaceae with access to a fungalsymbiont (Cairney 2000).

New molecular evidence (Sharples et al. 2000a) points to the similarityof root-associated fungi of Calluna vulgaris from southwest England withendophytes from North America and Australian Ericaceae in contrast to theearlier findings of McLean et al. (1999). Parry et al. (2000) found that therewas a level of sero-relatedness in antipodean and boreal Ericaceaeendophytes, lending further support to a ‘common origin’ theory for ericoidmycorrhiza.

The diversity in the root-inhabiting endophytes of Ericales can be seento operate at three levels:1.

2.

3.

High diversity within plant (Xiao and Berch 1996; Liu et al. 1998;Monreal et al. 1999; Sharpies et al. 2000a).High diversity within species (Hutton et al. 1994; Chambers et al. 2000;Parry et al. 2000; van Leerdam et al. 2001; Whittaker and Cairney2001).High diversity within sites (Hutton et al. 1996).The remarkable levels of diversity of endophyte types (either at a

cultural, enzymatic or molecular level) recorded within plants (see 1 above)is highlighted in the study of Sharples et al. (2000a) where 14 RFLP-typeswere assigned to 107 root-associating fungi of Calluna vulgaris. Evenwithin single root segments the level of diversity of root-associating fungi issurprising. For example, Monreal et al. (1999) found four distinctivemolecular types of fungi in small sections of root of the common Gaultheriashallon in Canada. In a geographically distant sense, Chambers et al. (2000)found up to four distinct fungi in one root segment of the eastern Australianspecies Woollsia pungens, with potentially six fungal taxa in four co-locatedplants.

The biological and ecological implications for the wide diversity offungi that coexist with Ericaceae may be based on an ancestral tolerance asthe early ericads radiated from their centre of origin and encountered an


array of endophytically competent fungi (Straker 1996; Cairney 2000)and/or the development of ‘intense distinctive mutualisms’ (Read 1996).The latter refers to a means whereby more species of Ericaceae may be‘packed’ into a niche (such as the high species richness of ericads insouthern Africa (672 species) and Australia (c. 350 species)) by virtue ofparticipating with a range of ericoid mycorrhizal fungi, which may varyspatially, temporally and taxonomically. The end result remains that a wideand diverse array of ericoid mycorrhiza provide another level of speciationto extract the limited or locked pools of N and P (Gimingham 1972; Pearsonand Read 1975) or to cope with organic extremes, metal toxicity (Burt et al.1986; Turnau et al. 1998) in the soils in which they inhabit, or drought(Hutton et al. 1996).

In the highly endemic flora of south-west Western Australia (with 246species of Ericaceae), Hutton et al. (1996) found that intensive sampling ofendophytic fungi from wet (swampy) to dry habitats resulted in a scale inhomogeneity of endophytic zymo-types from less to more diverserespectively. Since plant diversity in this region resides mostly on relativelydry sites, Hutton et al. (1996) postulated that it is possible that the samepressures which drive mega-diversity in higher plants in such extremeenvironments could also ‘exact similarly diverse levels of specialisationwithin populations of associated soil microorganisms’. It is thereforetempting to consider the co-evolutionary opportunities provided to Ericaceaein these environments through associating with endophytes with competencyfor particular pedological attributes with a niche (see Cairney 2000 forfurther discussion of this aspect).

5. Conservation implications of the ericoid mycorrhizal associationBenefits of the ericoid association have existed for at least 100 Myr (Cairney2000), in which time, extant Ericaceae have occupied all continents and avast array of habitats. The unique attributes of ericoid fungi which enablegrowth in ecosystems often characterised by extreme nutrientimpoverishment relies on the special ability of ericoid fungi to degradepolyphenols and other complex organic materials, attributes which are notavailable in many other non-mycorrhizal plants (Haselwandter et al. 1990).In addition, the production of proteases, siderophores and other chemicalsystems from the ericoid fungus provides a remarkable armoury ofphysiological attributes for the host ericad. The degree to which theseattributes act to ensure plant survival is illustrated in the extreme in the studyof Sharples et al. (2000b) who demonstrated that the ericoid fungusHymenoscyphus ericae had the ability to efflux arsenic from its hyphaewhile providing P to its host, Calluna vulgaris. Similar studies of toleranceto metals by ericoid mycorrhiza (e.g. Bardi et al. 1999; Martino et al.


2000a,b) serve to i l lus t ra te the emerging theme of the possible co-evolutionary patterns involved with the ericoid mycorrhizal system.

Just as the ericoid fungi have the capacity to adapt to extremes, so thefungal associates of Ericaceae appear to be sensitive to habitat alterationparticularly invo lv ing nutr ient accretion. For example, Heil and Diemont(1983) showed that nitrogen saturation may have been a major contributingfactor leading to the replacement of Ericaceae communities by grasslands.Levels of N or indeed P saturation removes the advantages of tight nutrientconservation so typical of the sclerophyllous communities in which manyEricaceae reside and for which the ericoid mycorrhiza provides such benefits(Specht and Rundel 1990; Read 1996). For the biodiverse communities ofSouthern Africa and south western Australia, nutrient fluxes, particularlyassociated with fire, may therefore play a crucial role in the conservation ofrare or threatened Ericaceae species (Stewart et al. 1993). Equally, the lackof competitive abili ty in strains of ericoid fungi may lead to displacement byother microorganisms through changes in biotic balances in ecosystems (e.g.Hutton et al. 1997).

The impact of habitat changes on the performance of the endophyte andsubsequent impacts on the survival of Ericaceae in terms of nutrient uptake,water balance and stress tolerance is vir tual ly unknown. The conservation ofEricaceae and inter alia, ericoid mycorrhizal diversity will initially rely onstudies to determine the degree to which both fungus and host can tolerateand/or adapt to env i ronmenta l changes. In one of the few studies toinvestigate the re-es tabl ishment of measurable abundance of ericoidmycorrhiza in to disturbed sites, Hutton et al. (1997) found that up to 12years elapsed before root associating endophytes returned to a post-minedsite. A caveat on this study was that topsoil was replaced to site and that themin ing operation retained an interface with un-mined native vegetation.How long, if at all, it would take for the return of ericoid fungi to sites wheretopsoil/natural system interfaces did not exist e.g. revegetated farmland,requires urgent attention.

6. Summary and conclusionsThe study of the microbial endophytes of Ericaceae may help us tounderstand the evolution and distr ibution of the taxa within the Ericalesworld-wide. It w i l l also indicate whether the fungal associates moved withtheir plant hosts or whether new associations with resident strains wereformed as the plants spread. This information is also l ikely to tell uswhether the genetic d ivers i ty of the fungal associates could help todetermine the taxonomic relationships within the host order.

Much needs to be done on the determination of the role ericoid fungiplay in the successful establishment of horticulturally important members of


the Ericaceae such as species of Rhododendron (see Jansa and Vosátka2000) and Vaccinium that are di f f icul t to establish in certain environments.The ecological importance of hair roots in certain environments is poorlyunderstood. In the Western Australian Banksia woodlands, for example,their occurrence in the soil profile is often constrained because of excessivecompetition by the persuasiveness of cluster roots in the Proteaceae (Pateand Watt 2001). Studies by Hutton et al. (1994) for instance showed that thedominant activity of hair roots and endophytes is restricted to the cooler wetmonths in the highly seasonal mediterranean-type climate of south westernAustralia. These same mycorrhizas are also unusua l l y sensitive to soildisturbance with long periods elapsing before recolonisation (Hutton et al.1997).

Finally, while horticulturally important Ericaceae are often translocated,little attention is paid to concurrently including the mycorrhizal partner inthe t ranslocat ion or conservat ion process (especially with rare andendangered taxa) as has been done with members of the Orchidaceae (Battyet al. 200la). The importance of the ericoid association for the long termsustainable management and recovery of rare or threatened Ericaceaeremains an important issue for conservation practitioners.

AcknowledgementsThe authors would l i k e to thank Mark Brundrett for comments on themanuscript.

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Chapter 9

THE DIVERSITY OF PLANT PATHOGENS ANDCONSERVATION: BACTERIA AND FUNGISENSU LATO

D S Ingram

St Catharine’s College, Cambridge University, Cambridge, CB2 1RL, UK.

1. IntroductionThe conservation of plant pathogen diversity is counter-intuitive to any plantpathologist dedicated to the prevention or eradication of plant disease. Tothe lay public, I suspect, such a notion would seem at best incomprehensibleand at worst irresponsible (Ingram 1998a,b, 1999). Yet, although plant pestsand diseases cause some 30% of losses of agricultural production world-wide (Agrios 1997) and have sometimes devastated native species (e.g.Newhook and Podger 1972), plant pathogens are now being recognised askey components of many natural and semi-natural ecosystems andpotentially of great benefit to humankind in spheres as diverse as, forexample, basic scientific research, biotechnology and novel drug andpesticide production.

In the pages that follow, based on Ingram (1999), the nature andsignificance of plant pathogen diversity and the threats to it will beadumbrated. Then, some approaches to the conservation of plant pathogendiversity will be reviewed, briefly, with due attention being given to thepotential risks involved. The discussions will largely be confined to bacteriaand fungi, sensu lato.

2. The diversity of plant pathogensIt is important to emphasise at the outset that our current knowledge ofmicrobial diversity is woefully inadequate, only about 5% of fungi, 1% ofviruses and 0.1% of bacteria having so far been described (Ingram 1999;


Table 1). Even where species have been named, our knowledge of their lifecycles and general biology is frequently very limited (see, for example,Helfer 1993; Parbery 1996; Rodriguez and Redman 1997). The greatestareas of ignorance are the diversity and biology of microorganisms,especially plant pathogens, inhabit ing tropical ecosystems (Day 1993) andaquatic ecosystems (Andrews 1976; Canter-Lund and Lund 1995; Smetacek2001). There is a pressing need for a s ignif icantly greater investment insystematics and biological research to rectify such lacunae in the knowledgebase (Hawksworth 1991; Anon. 1992; Day 1993; Anon. 1994a; Ingram1999). That said, the diversity of bacteria and fungi known to be pathogensof plants is immense, and has been classified in a variety of ways:morphologically, ecologically, physiologically, genetically and so on(Hawksworth et al. 1995; Agrios 1997; Holliday 1998).

In recent morphological and molecular classifications, plant pathogensare scattered across two Super-kingdoms, the Prokaryotae and theEukaryotae, and four Kingdoms, MONERA, PROTOZOA, CHROMISTAand FUNGI (Ingram and Robertson 1999; Table 2). They cause disease inplant groups as diverse as angiosperms, gymnosperms, pteridophytes,mosses, liverworts and algae. Within the MONERA (Prokaryotae), six majorgenera of Gram negative bacteria (Agrobacterium, Erwinia, Pseudomonas,Ralstonia, Xanthomonas and Xylella) and two major genera of Grampositive forms (Clavihacter and Streptomyces) include plant pathogens.Most species are un ice l lu la r , reproducing by binary fission, and many areflagellate. Streptomyces forms a rudimentary branching mycelium of narrowseptate filaments. In addition, a number of plant diseases once thought to beof viral origin are now known to be caused by a group of small, prokaryoticorganisms known as mycoplasma-like organisms (MLOs). This groupincludes phytoplasmas and spiroplasmas, which may be regarded as bacteriawhich lack the ability to form a rigid cell wall. They are pleomorphic andunder natural conditions are obligate parasites. Bacteria are generally lessimportant as pathogens of plants in temperate climates, but are of greatsignificance in the warmer regions of the globe, especially the tropics.

Diversity and conservation of plant pathogens 243

The plant pathogenic members of the group referred to colloquially asthe ‘fungi’ (Table 2) are classified in three Kingdoms, PROTOZOA,CHROMISTA and FUNGI (Eukaryotae), with the FUNGI being the largestgroup. (In the rest of this chapter the word ‘fungi’ will be used in its wider,colloquial sense and FUNGI, printed in upper case, wil l be used in the stricttaxonomic sense to refer to the Kingdom of that name.)

The fungi includes the largest group of plant pathogens, although some92% of the approximately 7730 genera described as fungi are said to be


entirely saprophytic (Schäfer 1994). This figure probably needs revision,however, for the fungi as a whole are relatively poorly researched (Parbery1996). Nevertheless, the diversity within the group is considerable, asfollows.

Plant pathogenic members of the Plasmodiophoromycota, in theKingdom PROTOZOA, normally grow in l iv ing host cells as plasmodia,masses of cytoplasm with many nuclei contained within membranes, or aspseudoplasmodia, comprising many smaller masses of cytoplasm, each witha single nucleus. Members of the group reproduce by means of zoosporesand may also produce th ick walled resting spores. In most cases thepositions of meiosis and nuclear fusion in the life cycle is not known withcertainty, but the diplophase is probably very short. Pathogenic members ofthis group are holobiotrophic in their nutrition (see Table 3).

The plant pathogenic fungi with cell walls fall into two Kingdoms, theCHROMISTA and the true FUNGI. The CHROMISTA normally grow asbranched hyphae containing cellulose as the main structural component ofthe walls. There are normally no septa, each hypha containing many nuclei,which are usually diploid. Asexual reproduction is usually by means ofdiploid zoospores produced wi th in a sporangium, although in some phylazoospores are not produced and the multinucleate sporangium is the mainasexual un i t of dispersal. Sexual reproduction is by means of thick walled,diploid resting spores called oospores, formed following a short haplophasein the unequal gametangia prior to fer t i l isat ion and nuclear fusion. Thegroup includes necrotrophs, holobiotrophs and hemibiotrophs (see Table 3).The holobiotrophs and some of the hemibiotrophs form haustoria.

With in the Kingdom FUNGI only the Chytridiomycetes producezoospores. Here the fungal body is a microscopic rounded structure withchitin walls and rhizoids arising from its base. Thick walled resting sporesare sometimes formed, often associated with sexual reproduction. Thepathogenic members of the group are holobiotrophic, the fungal body beingeither embedded in the l iv ing host cell or attached to it by rhizoids.

The three other groups of the FUNGI normal ly grow as branchedhyphae, l ike the CHROMISTA, but all contain chi t inous polymers as majorstructural components of the walls and are haploid for the greater part oftheir life cycles. The hyphae of the Zygomycota are normally aseptate andasexual spores, formed in a sporangium, and are non-motile. The sexualspores, the zygospores, are resting spores and are formed by the fusion oftwo gametangia, followed by nuclear fusion. Meiosis precedes germination.Plant pathogenic members of the group are extreme necrotrophs (Table 3).

The hyphae of the Ascomycota are divided into ‘cells’ by septa, butthese have a central pore through which nuclei and cytoplasm move quitefreely. The naked asexual spores (conidia) are non-motile. There is


immense diversity in the size, shape, form and manner of formation,deployment and dispersal of conidia. Sexual reproduction involves fusion ofhaploid nuclei in pairs, immediately followed by meiosis and then mitosis toproduce a series of (usually) eight haploid ascospores. The products of each


fusion/division are contained in a sac, the ascus. In most genera, the asci areprotected by a complex fruit body, the ascocarp. The group includesnecrotrophs, holobiotrophs and hemibiotrophs. Some of the holobiotrophsform haustoria.

The hyphae of the Basidiomycota are septate, and although the septamay possess pores these are usual ly par t ia l ly blocked by membranes,restricting the movement of the nuclei. The hyphae are, therefore,effectively divided up in to cells. In most species each cell is a dikaryon,containing a complementary pair of haploid nuclei. Clamp connectionsensure in some groups that following cell division, each daughter cellcontains complementary nuclei. Asexual spores are not produced by allspecies, but when present, usual ly take the form of dikaryotic conidia.Sexual reproduction, fusion of complementary nuclei, followed by meiosisto restore the haploid state, occurs in specialised hyphal branches, thebasidia. These each form a group of four, occasionally two, haploiduninucleate basidiospores, the product of one fusion/meiotic d ivis ion.Following dispersal, the basidiospores germinate to form a haploid,monokaryotic mycelium. Hyphal anastomosis restores the dikaryotic state.

Within the Basidiomycota, the Basidiomycetes form their basidiosporesin structures (toadstools or brackets) composed of differentiated hyphae,which bear the basidia on gills, in pores or on teeth. The parasitic membersof this group are mainly necrotrophic (see Table 3) and many are capable ofdegrading lignin as well as other plant cell wall polymers.

There are two other large classes of the Basidiomycota, theTeliomycetes (Rusts) and the Ustomycetes (Smuts and Bunts). These do notform basidiocarps. Instead, teliospores or ustilospores germinate to formhypha-like basidia which bear the basidiospores. Many of the Teliomyceteshave complex l i fe cycles, with several spore stages that may alternatebetween two different hosts. The members of both groups are holobiotrophs(see Table 3), but only the Teliomycetes produce haustoria.

The final group of the mycelial FUNGI, the ‘Deuteromycetes’ ormitosporic fungi, has no formal taxonomic status. The term is widely used,however, as a convenient way of referring to fungi that have either lost theability to reproduce sexually or do so only rarely and cannot therefore beclassified in the usual way. Most, but not all, are probably members of theAscomycota and many are necrotrophic pathogens of plants.

The detailed taxonomy of the plant pathogenic bacteria and fungi isdealt with by Hawksworth et al. (1995), Ell is and Ell is (1997), Holliday(1998) and Bradbury (1999), and more general accounts are provided byAgrios (1997) and Ingram and Robertson (1999). Nevertheless, it is clearfrom this brief survey that the niche of plant pathogenicity has beenexploited by many diverse organisms and has evolved many times


(Pirozynski and Hawks worth 1988). There is, in addition, furthersignificant diversity apparent below the level of the species, as reflected inmolecular analyses (Karp et al. 1997a,b, 1998) or as revealed by moreconventional analyses of host ranges using genetically defined differentialhost species, subspecies and cultivars carrying different genes for resistance(Table 4), or of sens i t iv i ty to natural or synthetic bacteriocides andfungicides (Allen et al. 1999).

Finally, the great taxonomic diversity of the pathogenic bacteria andfungi is reflected in the range of symptoms they cause in infected hosts(Table 5), itself a reflection of the spectrum of ecophysiological (Table 6)and trophic (Table 3) strategies that have resulted from the co-evolution ofso many different pathogens with such a diversity of hosts.

The diversity of pathogenic bacteria and fungi, especially at theinfraspecific level, must be regarded as extremely fluid and subject to rapidchanges in both space and time. This is an inevitable consequence of theoften rapid generation times and dispersal rates of populations ofmicroorganisms, linked with: high rates of mutation; long haplophases inmany groups; the presence of extrachromosomal nucleic acid (plasmids etc),especially among the bacteria; and, in the fungi, complex mating systemsincluding homothallism, secondary homothallism and heterothallism (oftenwith several mating types); and sometimes also, in the fungi, the capacity forhyphal anastomosis with nuclear exchange and parasexual recombination(Fincham 1979; Agrios 1997; Caten 1996; Hartleb et al. 1997; Bradbury1999). The capacity for interspecific hybridisation is now also known to besignificant in pathogen variation (Brasier 2000a). The consequences of suchplasticity are well known to plant breeders attempting to produce durabledisease-resistant cultivars or to those attempting to produce effective, long-lived, bacteriocides and fungicides (Wood and Lenné 1999). This plasticityis of great relevance to the success of plant pathogens in natural ecosystems.

3. The value of plant pathogensThe wholesale destruction that is characteristic of plant disease epidemics ingenetically uniform crop monocultures does not normally occur in naturalecosystems. This is in part due to the fact that the ecosystems themselvesare usually made up of a great diversity of species, with genetically uniformstands being the exception rather than the rule. Moreover, although racespecific resistance does occur in natural ecosystems (Heath 1991; Clarke1997; Burdon 1997), epidemics do not normally follow its breakdownbecause stable equilibria normally exist between the host plant and pathogenpopulations (Prell and Day 2001). These equilibria are dependent upon thenumber of alleles for avirulence and virulence in the pathogen populationsand for resistance and susceptibility in the host populations, and on the




balance of advantage and disadvantage conferred by any new plant andpathogen genotypes. Such stable equilibria ensure the survival of both hostand pathogen populations.

Occasionally, however, massive epidemics of plant disease do occur innatural ecosystems, causing immense damage over wide areas. Recentdramatic examples include: Jarrah die-back of Eucalyptus and other nativespecies in Australia and New Zealand, caused by Phytophthora cinnamomi(Newhook and Podger 1972); chestnut bl ight in North America, caused byCryphonectria (Endothia) parasitica (Agrios 1997); Dutch elm disease inNorth America and Europe caused by Ophiostoma novo-ulmi (Brasier2000b); and, more recently, certain forms of oak decline in central Europecaused by species of Phytophthora (Jung et al. 2000). The reasons for suchepidemics are not always clear, but may include introduction of alien speciesand novel genetic forms, as in the case of chestnut blight and Dutch elmdisease, or major genetic changes and interspecific hybridisation in thepathogen, as wi th Dutch elm disease, complicated by changes inenvironmental condit ions, as with Jarrah die-back. In the future, climaticshif ts may also be expected to trigger the breakdown of equilibria in hostand pathogen popu la t ions leading to fur ther epidemics of and in otherspecies. Even though ecosystem recovery may sometimes be possiblefol lowing such catastrophes (e.g Weste and Kennedy 1997), this is a slowand erratic process and is by no means ful ly understood. The damagealready caused to natural ecosystems by plant pathogens, the threat of furtherepidemics in the future and recognition that disease control in such situationsis rarely if ever possible, must be of great concern to conservationists andmust be taken into account in planning future conservation strategies. In thiscontext, the importance of gaining a better understanding of the biology andgenetics of plant pathogens in natural ecosystems cannot be overemphasised, but more of that below.

But there is another side to the coin. It has already been stated that theconcept of conserving p lan t pathogen d ive rs i ty is counter in tu i t ive , andknowledge of catastrophic epidemics in natural ecosystems fuels suchprejudice. It is important to explore therefore, the positive role of plantpathogens in natural ecosystems and their value in providing scientific,technological and economic benefits to human societies (Table 7).

3.1 The significance of plant pathogens in natural ecosystemsEvidence from model ecosystems and natural grasslands has suggested thathigh biodivers i ty may lead to greater product ivi ty and stabil i ty and,conversely, that reducing divers i ty lowers productivity (Naeem et al. 1994;Tilman et al. 1996). More recent research suggests, however, that therelationship between diversity, energy availability and productivity in


natural ecosystems is far more complex than this simplistic statementimplies (Naeem et al 2000; Morin 2000; Emmerson et al 2001). Withresearch at such an early stage, it is impossible to assess the significance ofplant pathogen diversity in this context.

Any visual survey of a natural ecosystem, however, no matter howsuperficial, wil l reveal the presence of plant pathogens, often in abundance(Ingram and Robertson 1999). Such observations are confirmed by themany checklists of plant pathogens that have been produced for natural and


semi-natural habitats, especially in Europe and North America (e.g.Blackwell et al. 1997). The mere presence of pathogens in a plantcommunity , however, no matter how damaging or debilitating to theirindividual hosts, does not in itself constitute evidence of their contribution toecosystem dynamics, productivity or stability. They may be nothing morethan another level of biological complexity of only limited structuralsignificance (Harper 1990). So, what, if any, is the evidence to the contrary?

The significant role of parasitic fungi that establish mycorrhizalassociations in natural ecosystems is now well documented (Simard et al.1997; Helgason et al. 1998; Read 1998; Chapin and Ruess 2001). A recentstudy of forest diversity in the United States provides dramatic evidence tosupport the hypothesis that fungi that are pathogens are also of majorsignificance (Packer and Clay 2000).

Packer and Clay (2000) examined the problem of why some forests aremore heterogeneous than others. In a black cherry forest near Bloomington,Indiana, USA, they observed that black cherry seedlings growing beneathmature black cherry trees died soon after germination, whereas seedsdispersed to some distance from the parent grew on to produce new trees. Itcould be argued that the cause of seedling mortality was overcrowdingbeneath the parent trees, but Packer and Clay showed a better correlationbetween distance from the parent and survival than between overcrowdingand mortality.

Next, in a series of pot experiments, they found strong evidence that thecause of death of seedlings close to their parent was a species of the soil-borne pathogen Pythium, which they had previously isolated from dyingseedlings. The seedlings of several tree species other than black cherry thatwere able to establish and grow where black cherry seedlings were killedwere apparently resistant to the fungus. Thus the Pythium species seemed toplay a vital role in promoting diversity in the cherry forest in a mannersimilar to that previously attributed to host specific herbivores by Janzen(1970) and Connell (1978).

A flaw in this hypothesis is that Pythium spp., being necrotrophs, areusually regarded by plant pathologists as being non-specific pathogens withwide host ranges. However, although there is no doubt that hosts of suchnecrotrophic pathogens do not normally develop the highly specific gene-for-gene resistance-avirulence systems characteristic of relationshipsinvolving holobiotrophic and hemibiotrophic pathogens (Agrios 1997; Prelland Day 2001), a measure of broad specificity at the level of the species orfamily is not at all unusual. Thus a Pythium species could well exhibit thelevel of specificity ascribed to it by Packer and Clay (2000).

The phenomenon described by Packer and Clay (2000) in black cherryforests is very similar to a syndrome long familiar to horticulturists, called


‘replant disease’ or ‘soil sickness’ (Pollock and Griffiths 1998) in whichroses and fruit trees, when dug up, can only be replaced successfully by ayoung plant of a different species or family. The demise of replants of thesame species is usually associated with the occurrence on the roots of lesionsof Pythium sylvaticum.

van der Putten (2000), in a commentary on Packer and Clay (2000),hypothesises that the existence of soil pathogens may even select for long-distance dispersal traits in their hosts. He cites in support of this hypothesisthe work of Wennström (1994) who demonstrated that, in six vegetativelypropagated plant species, sensitivity to pathogens correlated positively withthe speed and extent of vegetative outgrowth. In further experimentsconducted by D’Hertefeldt and van der Putten (1998), the sedge Car exarenaria exhibited reduced branching and a switch to unidirectional growthof rhizomes when challenged by patches of soil-borne pathogens.

The studies of Packer and Clay (2000), and van der Putten (2000),although excellent, are far from complete: the Pythium species pathogenic toblack cherry has yet to be identified and its specificity needs to be definedmore precisely; the role of the fungus in generating black cherry forestdiversity has yet to be established unequivocally; and the interestinghypothesis that the evolution of long-distance dispersal is a response to apathogen is far from proven. Moreover, although a similar process to thatdescribed for black cherry forests may explain species diversity in Douglasfir forests (Holah et al. 1997), the universality of the process is far fromclear. It is interesting to speculate, however, that some necrotrophic plantpathogens at least may have a significant widespread role in promotingecosystem divers i ty and thus, ind i rec t ly , ecosystem productivity andstability.

This work of Packer and Clay serves to illustrate in a striking way thepoint that the role of plant pathogens in natural ecosystems may in the pasthave been overlooked or underestimated. Nevertheless, some excitingresearch has been carried out and more is now being done, as was evidentfrom the contributions to the Seventh International Congress of PlantPathology (Ingram 1999).

An essential pre-requisite for such studies is a clear understanding ofthe distribution and epidemiology of the pathogens concerned. A majorcontribution to this field has been made by Burdon (1991, 1992, 1993,1997), who has reviewed his own research, especially with wild populationsof flax infected with the rust fungus Melampsora lini, and also the researchof others with a great diversity of host and pathogen combinations. As abasis for analysis , Burdon (1993) classified pathogens of wild hostsaccording to their effect on host survival, fecundity and individual vigour(Table 8).


The broad conclusion of Burdon (1993) is that the biology of aparticular plant pathogen is affected by such a wide variety of factors,including its breeding system, survival strategies, host range and dispersalmechanisms, that the population dynamics and genetics of each pathogenspecies must be regarded as unique. He recognises, however, the need to bepragmatic and goes on to suggest that, despite the significant differencesbetween pathogens, s imilar i t ies in the ecological contexts in which theyoccur impose similar constraints. He lists these constraints as the size,spatial distr ibution and genetic structure of host populations, coupled withthe effective methods of dispersal possessed by most pathogens, andsuggests that they reinforce the need to consider pathogen demography andgenetics in a metapopulation context. Burdon (1993) also points out that in


any one deme, population levels rise and fall, often quite sharply, while thegenetic structure can change rapidly and very significantly. Suchpopulations rarely exist in isolation, however, and only by following thepattern of change over many demes within a particular area can a completepicture of the total population be determined. The development of methodsfor the detection of molecular and other markers is now greatly facilitatingstudies of pathogen epidemiology in natural and semi-natural ecosystems(Lenné et al. 1994; Karp et al. 1997a,b; Wood and Lenné 1999) and isalready resulting in a widening and deepening of the knowledge reviewed byBurdon(1993).

The complementary question of the interaction between disease andplant competi t ion, especial ly in in f luenc ing the structure of plantcommunities, has recently been analysed in depth by, among several others,Dobson and Crawley (1994) and Alexander and Holt (1998). Dobson andCrawley conclude that there may be a direct influence of pathogens on thestructure of plant communities in cases where pathogens reduce thepopulation of adult and seedling plants, as in the extreme case of Jarrah die-back, caused by Phytophthora cinnamomi in Eucalyptus spp. in Australia(Newhook and Podger 1972) or Dutch elm disease caused by Ophiostomanovo-ulmi on elms in Western Europe (Brasier 1986 and 2000b). Theremoval of a dominant tree species by a pathogen has in these cases led toforests and woodlands dominated by less competitive species from earlierstages of the succession or has opened the canopy, allowing colonisation byless competitive species. Less dramatic examples, in which the pathogenreduces the competitive ability of a plant in a succession include the studiesof van der Putten and colleagues (van der Putten 2000) of the succession ofgrasses and sedges on foredunes. Dobson and Crawley (1994) conclude thatsoil-borne diseases may affect both the rate and direction of the successionof plants in specific ecosystems. It is hypothesised that pathogens may bothslow down and speed up successions, depending on circumstances. Theyalso cite examples of pathogens that affect the fecundity of their hosts,thereby influencing plant population dynamics, as in the case, for example,of Atkinsonella hypoxylon, which reduces flower production in the grassDanthonia spicata but increases growth rates and survival of infectedramets.

Dobson and Crawley (1994) also draw attention to ways in whichpathogens may influence seedling recruitment in tropical forests, therebyincreasing species diversity, as in the example of the effect of Pythium spp.on the diversity of black cherry forests cited above. Finally, they outlinehow the effects of plant pathogens on animals exert indirect effects on plantcommunities through a reduction in grazing pressure. For example, someplant pathogens render their hosts toxic to herbivores, as with certain


endophytes of grasses (Parbery 1996). These may in turn lead to reductionsin grazing pressures, wi th concomitant effects on the composition orstructure of grassland ecosystems.

In a comprehensive review, Alexander and Holt (1998) also assess theevidence in support of the hypothesis that plant disease may have as igni f icant effect on the compet i t ive in terac t ion between plants, withecological or evo lu t ionary consequences. They cover some of the sameground as Dobson and Crawley (1994), but also review the more recentresearch. In addressing the effects of disease on intraspecific competitionbetween host plants, they present a simple model that suggests that a varietyof outcomes might be expected. From the evidence available it can beconcluded that pathogens may have a large or small effect on intraspecificpopulation dynamics, depending most notably on the density-dependentability of healthy plants to compensate for loss of diseased individuals, theability of non-infected leaves or shoots to compensate for infected ones onthe same plant and the spatial patterns of infection. Next Alexander andHolt (1998) analyse the effects of disease on the competitive abilities ofi n d i v i d u a l genotypes of the same species and thus on the geneticcomposition of populations. They conclude that such genetic processes feedback on populat ion dynamics, assuming trade-offs between diseaseresistance and other fitness characters. Finally, they show that the effects ofdisease on the interspecific interaction between plants may have significanteffects on the composition of communities. Host-specific pathogens such asholobiotrophs and hemibiotrophs, for example, may change a competitivehierarchy between a host and a non-host species, while relatively non-specific, necrotrophic pathogens may induce indirect competi t iveinteractions between host species.

The evidence reviewed by Alexander and Holt (1998) is limited in thefol lowing ways: it derives m a i n l y from research with a limited range ofpathogens, especial ly f ung i ; most of the conclusions were based onlaboratory rather than field studies; interactions were studied over relativelyfew generations; l i t t le evidence was available concerning the effects ofdensity-dependent processes in both host and pathogen populations; andpathogen population dynamics were largely ignored. Despite the largenumber of empirical studies reviewed, Alexander and Holt (1998) are thusunable to conclude with any certainty that pathogens affect plant populationdynamics, the genetic composition of host populations or plant communitycomposition. Nevertheless, there is strong circumstantial evidence that theydo affect all of these, although significantly more research is required to becertain.

Finally, it should be noted that quite apart from any direct effects onplants in natural ecosystems, necrotrophic pathogens, especially those with


the capacity to degrade cellulose (e.g. Pythium spp.) and l ignin (e.g.Heterobasidion annosum) have a key, although as yet unquantified, role ininducing decay and ensuring the cycling of nutrients, especially carbon, inall natural ecosystems (Andrews 1991; Copley 2000; Naeem et al. 2000).

3.2. The economic and scientific value of plant pathogensThe positive value of the pathogens of wild relatives of crop plants and intraditional agro-ecosystems is widely recognised. Most of the progenitors ofcrops have co-evolved with their pathogens in the centres of origin anddiversity - the Middle East in the case of cereals, for example (Heath 1987,1991; Dinoor and Eshed 1990; Frank 1993; Smartt and Simmonds 1995;Wood and Lenné 1999). This has thrown up a great diversity of diseaseresistance factors in the hosts and these have been a major resource for thefarmer and plant breeder in producing new disease-resistant cultivars (Lennéand Wood 1991; Burden 1997; Wood and Lenné 1997, 1999; ten Kate andLaird 1999). Both existing and newly evolving ‘wild’ sources of resistance(and recent evidence suggests that new plant disease resistance factors mayarise relatively rapidly and frequently) are likely to be required long into thefuture for the production of novel disease-resistant crops (Lenné and Wood1991; Hawtin 1996; Wood and Lenné 1997; Allen et al. 1999; ten Kate andLaird 1999).

Other potential economic benefits are numerous. Genetically definedcollections of plant pathogens are essential to the process of revealingdiversity and in selection for disease resistance in plant breeding. The greatrange of, for example, cereal cultivars resistant to rust and powdery mildewdiseases is testimony to this (Allen et al. 1999). Pathogens may also beimportant as sources of novel drugs. Ergot alkaloids and other compoundsderived from Claviceps purpurea have been widely used in medicine formany years, and ergotamine is current ly of great significance in thetreatment of migraine. A wide-range of antibiotics has been obtained fromboth bacteria and fungi , some of them pathogenic on plants (ten Kate andLaird 1999). Other pathogen-produced or pathogen-induced compounds ofpharmaceutical significance are l ikely to be revealed as the search for novelcompounds from plants and microorganisms intensifies (ten Kate and Laird1999). Similarly, plant pathogens or infected hosts may be good sources ofnovel fungicides, pesticides and herbicides. For example, a protein derivedfrom the necrotrophic pathogen Fusarium oxysporum has recently beenshown to have great potential as a herbicide for broad-leaved weeds(Jennings et al. 2000). Moreover, spores of Ascochyta caulina, a pathogenof fat hen (Chenopodium sp.), have been shown to have excellent potentialas organic weed-killers for this pernicious weed (W. Seel, University ofAberdeen, UK, pers. comm.). F ina l ly , plant pathogens may produce or


cause their hosts to produce chemical molecules which, although not ofdirect significance to the pharmaceutical or agrochemical industries, may beof indirect importance in producing chemical templates for novel bio-activemolecules (ten Kate and Laird 1999).

Plant pathogens have also long been of significance to the food anddrinks industry. For example, Botrytis cinerea, the widespread cause of greymould of mor ibund f ru i t s , f lowers and leaves, as a pathogen of late-harvested grapes imparts both sweetness and flavour to the resulting dessertwines. Maize infected by the smut fungus Ustilago maydis is considered adelicacy if eaten before the black ustilospores form. The potential for futureuse of plant pathogens in food production is, however, probably limited.

In contrast, the role of plant pathogens as models for scientific researchleading to technological innovation cannot be overestimated. Two exampleswi l l suffice to make this point . Plants of rice infected by Gibberellafujikuroi, the cause of bakanae or ‘foolish seedling’ disease, are taller, palergreen and more spindly than uninfected individuals . Study of the causes ofthese symptoms, overproduction by the fungus of gibberellic acid, led to thediscovery of a major group of p lan t hormones, the gibberellins. Thisdiscovery was not only of great scientific significance but was also of greattechnological importance, for gibberel l ins are now widely used in thebrewing industry for the synchronisation of barley malting and in frui tproduction to induce parthenocarpic ripening.

Secondly, studies of the way in which Agrobacterium tumefacienscauses crown gall tumours in infected hosts led to pioneering experiments ingenetic modification of plants and ultimately to the widespread applicationof this technology in the production of GM crops. There is little doubt thatin the future, scientific studies of plant disease organisms will continue tolead to major scientific and technological advances, either by chance, as inthe above two examples, or by design (ten Kate and Laird 1999).

Finally, it is important to end this section by making a point that is soobvious that it is all too easily forgotten or overlooked. For as long as thereis a need to control the devastating effects of pathogens on plants, there is anequal need to study in depth the ways in which pathogens interact with theirhosts and with their environment at every level and the way that epidemicsoccur and develop, especially in natural and semi-natural ecosystems. Thispoint is particularly well exemplified by the works of Burdon (1997), Clarke(1996), Heath (1987, 1991), Thompson and Burdon (1992), Rausher (2001)and Stuiver and Custers (2001). Only as a result of such research will it bepossible to continue to develop effective strategies for the control of diseasesinto the future. For this reason alone, if for no other, it is essential that thediversity of plant pathogens is conserved in appropriate ways.


4. Threats to plant pathogen diversityBut is plant pathogen diversity threatened? The stark conclusion of therecently published IUCN Red List of Threatened Plants (Walter and Gillett1998) is that some 34,000 species of plants, representing 12.5% of theworld’s flora, face extinction. The list includes wild relatives of almostevery major crop and forestry genus or group.

Species, of course, are only one component of the totality of plantdiversity. The IUCN Red List, however, is an indication that erosion isprobably occurring at the genetic, population and ecosystem levels too,although such erosion is not always easy to measure (Groombridge 1992;Day 1993; Watson et al. 1995). The rate of this loss of plant diversity isequally di f f icul t to estimate, but considerable work has been done onrainforests. Evidence suggests that from the rainforests alone 27,000 speciesof all organisms are being lost per annum (Wilson 1993). This is roughlyequivalent to one thousand times the natural rate of extinction of species andis also equivalent to the rate of some of the great extinctions of thegeological past. Other estimates of the current rate of extinction oforganisms vary widely (e.g. Groombridge 1992; Myers 1993; Myers et al.2000; Pimm and Raven 2000), but there is general agreement that it isdangerously high. Plant pathogen diversity is, of course, entirely dependentupon the diversity of host plants.

More importantly, natural ecosystems and traditional agro-ecosystemsare being destroyed at an unprecedented and dangerous rate, including thosein the centres of origin and diversity of major crops (e.g. Wilson 1993;Myers et al. 2000), and with them, no doubt, the associated pathogens. Suchlosses are l ikely to include both species and, perhaps more significantly,infra-specific diversity for characters such as virulence and avirulence (Allenet al. 1999). However, all this is conjecture and the need for further andbetter information on the extent and erosion of pathogen diversity in naturalecosystems and traditional agro-ecosystems, and the potential impact ofclimate change, cannot be over emphasised.

Finally, plant pathologists and crop producers are, quite properly,dedicated to the elimination of plant pathogen diversity, for it is a seriousthreat to production (Agrios 1997). Even though plant pathogens areprobably sufficiently plastic, genetically, to adapt to new bacteriocides,fungicides and resistance genes (Fincham et al. 1979) the resulting year onyear erosion of pathogen diversity is likely to be significant, although nodata are currently available to substantiate this statement (Day 1993).Moreover, the ever widening use of industrial farming and crop productionmethods is itself likely to be a threat to pathogen diversity in adjacent naturaland semi natural ecosystems as well as on agricultural land itself.


5. Conservation of plant pathogen diversityIf plant pathogen diversity has potential value, ecologically, economicallyand scientifically, and if this diversi ty is threatened, it is necessary toconsider what strategies might be available to ensure its conservation.

5.1. Ex situ conservationOne strategy for the conservation of plant pathogenic bacteria and fungi is exsitu in culture collections or spore and gene banks (Hawksworth 1991; Smithand Waller 1992; Sugawara et al. 1993; Anon. 1994b; Suihko 1995; Kirsop1996; Smith and Ryan 2001). This is a most effective approach, and thereare many important collections and banks around the world, their activitiesin part co-ordinated by the World Federation for Culture Collections (Kirsopand Hawksworth 1994; see also Gams, this volume). Ex situ strategies,however, are not devoid of problems.

Firstly, at a time when the emphasis is on short term funding ofscientific endeavours and the commercialisation of science is widespread,culture collections and spore and gene banks, which require secure long termfunding, must be at r isk (e.g. Anon 1994a; Sly 1998). Secondly, plantpathogens are currently poorly represented in most culture collections, whichhold no more than 10% of the species so far identified, while infra-specificdiversity is largely ignored. In this context, high priority should be given inthe future to the conservation of the working collections of individual plantpathologists and plant breeders. These are usually invaluable and are oftenirreplaceable, yet at present are frequently at risk in financially pressedinstitutions. Thirdly , although methods for the long-term storage andmanagement of microbial cultures, spores and nucleic acids are extensive(Hawksworth 1990; Smith 1997), further development is still required (Day1993), especially to ensure genetic stability. Fourthly, the ownership ofmaterial held in culture collections and spore and gene banks is frequentlyfar from clear (Hawtin 1996; ten Kate and Laird 1999). And finally, sinceculture collections and banks facilitate the international movement oforganisms for study, there is the ever present risk of accidental release of apathogen far from its place of collection, with potentially devastatingconsequences. This last point is emphasised by, for example, the widespreaddestruction of elms in Europe and America fol lowing the accidentalinternational transport of Ceratocystis species (Brasier 1986).

The work of Newcombe et al. (2000), Brasier (2000a,b) and Brasier andKirk (2001) has further emphasised the risks associated with such events,suggesting that inter-specific hybridisat ion between related native andintroduced species of pathogens may produce a ‘devastating array’ of novelphenotypes. It is recognised, of course, that these ‘escapes’ were not fromculture collections or spore banks, but they serve as reminders of the need


for care.Despite these caveats, culture collections and gene and spore banks

probably represent the most effective and certainly the most secure way ofconserving plant pathogen diversity. However, being held in suspendedanimation, isolated from their hosts, plant pathogens conserved ex situ haveno opportunity to co-evolve wi th their hosts and generate yet furtherdiversity.

5.2. In situ conservationThe controversial alternative to ex situ conservation is to conserve plantpathogens in situ, together with their hosts (Ingram 1998a, 1998b, 1999).This has the obvious advantage of allowing the evolution of diversity tocontinue unchecked, and might therefore be regarded as more natural than exsitu conservation. At its simplest, in situ conservation of pathogens merelymeans taking account of pathogen biology when devising conservationstrategies for particular ecosystems (Helfer 1993; Burdon 1998). Becausepathogen populations may be unevenly distributed in host populations,especially at the infra-specific level (Burdon 1993, 1998), however, greatcare must be taken to ensure that host populations chosen for conservationinclude all known components of pathogen diversity. And herein lies asignificant diff iculty. It has already been emphasised that knowledge of thegeneral biology and epidemiology of pathogens of wild plants is, with a fewnotable exceptions, very limited (see Helfer (1993), for example, regardingrusts on rare plants in the UK). Thus if the conservation of plant pathogendiversity in natural ecosystems is to be taken seriously, a major drive toincrease knowledge of their biology is urgently required.

A danger with this simple in situ approach is that certain pathogens,especially those that are host specific, such as the rusts and smuts, may posea very real threat to vulnerable populations of an endangered host species(Helfer 1993). In these circumstances, the conservationist is faced with thedilemma of whether to concentrate on conservation of the host at theexpense of the pathogen, or vice versa, or whether to attempt to manage hostand pathogen populations together, thereby running the risk of losing both.The pragmatic solution, of course, would be to conserve the pathogen in aspore or gene bank and the host both in situ and in a seed bank.

A more complex dilemma concerns the proposition that the pathogensof the wild relatives of crop plants might be conserved in situ, in the centresof origin and diversity of those crops. This idea was first proposed for thewild relatives of cereals by Browning (1974), who also suggested the name‘living gene parks’ for such areas of conservation. Dinoor and Eshed (1990)returned to the idea nearly twenty years later and coined the alternative name‘genetic reserve’. At f irst sight such an idea is seductive. The reserves


could provide a pool of act ively evolving host populations, continuallychallenged by evolving populations of key pathogens. There might thereforebe the continued generation and selection of novel genes, thus ensuring anongoing supply of raw material for plant breeders (Wood and Lenné 1999).

The idea merits further consideration (Ingram 1999), but it is importantto emphasise certain caveats. Firstly, despite intensive study of thepathogens of major crops, we still need to know far more of the detailedbiology, epidemiology and population genetics of populations of plantpathogens and their hosts in agro-ecosystems and in natural ecosystems (seeBurdon 1993; Wood and Lenné 1997, 1999) before we shall have sufficientknowledge to manage such reserves effectively. Secondly and moreimportantly, as Lenné (Hall 1998) and Wood and Lenné (1999) haveemphasised, the risks to adjacent crops are very considerable. The centres oforigin of most major crops are regions of the world that although rich inbiodiversity are, to a large extent, extremely poor in resources (Myers et al.2000). Thus the presence of a reservoir of pathogen inoculum close tocultivated crops could have serious economic consequences, and the escapeof a new, virulent line or strain of a pathogen could create a disaster. Thespread of rust from wild to cultivated wheat in India and Pakistan (Joshi1986) is a salutary reminder of the need for extreme caution.

6. Summary and conclusionsThe diversity of plant pathogenic bacteria and fungi is immense, at everytaxonomic level. To conserve such diversity is, however, counter intuitive tomost plant pathologists and conservationists because of the great economicdamage caused by plant diseases of crop plants and the dangers pathogenspose to both common and endangered species in natural ecosystems.Nevertheless, it is suggested that plant pathogen diversity may be of positivevalue as a component of natural ecosystems, although the evidence tosupport this suggestion is incomplete. Moreover, plant pathogens have been,and will probably continue to be, of significant economic value as sources ofnovel drugs and agrochemicals. Pathogen diversity is also essential to theplant breeder in selecting for novel disease resistance factors both in wildpopulations of host plants and in plant breeding trials. And finally, plantpathogens provide model systems for scientific research leading tobiotechnological innovations. In view of all this, it is essential that greaterweight is given in the future to understanding the basic biology, genetics andepidemiology of pathogens in natural ecosystems than is currently the case.Moreover, the importance of plant pathogen diversity and the threatspathogens impose must be considered in planning conservation strategies.

The threats to pathogen diversity are significant, being related to thethreats to host plant diversity, intensified by the agricultural tradition of


destroying plant pathogens whenever possible. Given the desirability ofconserving plant pathogen diversity (by no means universally accepted),both ex situ and in situ approaches are of potential value, but pose the ever-present threat of escape, wi th potentially devastating consequences. Ex situconservation strategies pose fewer risks than in situ strategies, but are lesseffective for conserving a broad spectrum of diversity and suffer from theserious disadvantage of not allowing continuing host-pathogen co-evolution.

A major d i f f i c u l t y faced by a l l those concerned with the role ofpathogens in natural ecosystems and the conservation of plant pathogendiversity is that with the exception of a small number of pathogens of majorcrop plants, research on the biology, genetics and epidemiology of plantpathogens has so far been totally inadequate.

AcknowledgementsI wish to thank Dr P Harris for his help during the preparation of parts of thischapter.

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(CAB International: Wallingford)


Chapter 10

EX SITU CONSERVATION OF MICROBIALDIVERSITY

Walter Gams

Centraalbureau voor Schimmelcultures, P.O. Box 85167, 3508 AD, Utrecht, Netherlands.

1. IntroductionNo organisms lend themselves better to long-term ex-situ preservation thanmicroorganisms (Kirsop and Kurtzman 1988; Hawksworth and Kirsop 1988;Samson et al. 1996b). Microbial culture collections preserve large numbersof pure cultures, viz. strains of bacteria, fungi and algae, over long periods.The strains are maintained either in an active condition on agar media withregular transfers, or anabiot ica l ly under conditions that assure as littlealteration as possible over decades. Provided no mutation or contaminationoccurs, a strain retains its identi ty permanently. The scope of microbialculture collections is not comparable to that of botanical or zoologicalgardens which are mainly concerned with the conservation of largecomponents of the earth’s biodiversity that elsewhere may be threatened insitu. Preserved microorganisms main ly serve as reference material orstandards in comparative research.

Because of my personal l imitations, most of the examples used in thischapter are drawn from mycology and fungal collections. Collections ofl iv ing cultures are complementary to the indispensable documentationpreserved in herbaria and both must be linked as far as possible. Certainforms of sporulation are only seen in nature and rarely in culture; it is crucialto preserve dried specimens of these conjointly with the culture (Agerer etal. 2000). After collecting a fungus in nature, “the availabili ty of culturesalso can mean that other than just naming a specimen, you are naming aspecies in the true sense of the word. Your species concept can now be testedat various levels, and the isolate can also add data to address many other


exciting questions” (Crous 1999). Mycologists studying fungi in pure culturehave a great advantage over those working with fungi in situ only. Factorsdetermining morphology and development can be analysed and conditionsfor growth optimised, concomitant ly y ie ld ing basic ecological information.The correlation of different k i n d s of sporulat ion as components of onefungus (part icularly in es tab l i sh ing anamorph–teleomorph connections) ismostly accomplished through study of isolates obtained from sexual spores(mostly ascospores) in cul ture . In nearly al l discipl ines in which micro-organisms are involved, well-preserved pure cultures are indispensable.

Thanks to lyophi l iza t ion and cryo-techniques, culture collections arenow in the position to preserve large numbers of axenic (or monoxenic)fungal isolates with a m i n i m u m of alteration. The in i t i a l handl ing of thematerial is labour-intensive but, once in an inact ive state, the material isdurable for decades. With older techniques, particularly those requiringserial transfers, degeneration of cultures is a regularly occurring problem.Loss of sporulat ion, loss of enzyme act ivi t ies and, part icular ly, loss ofpathogenicity were disadvantages of old collection strains. To circumventthis, special techniques for particular groups of fungi were often successfullyused, such as a soil tube method (Schneider 1958) particularly suitable forFusarium, and preservation of fungal fragments in distilled water (Castellani1967; Boesewinkel 1976; Ellis 1979). The more permanent techniques haveto a large extent displaced these early improvements. Even with moderntechnologies, some fungal groups, such as mycorrhizal fungi , Oomycetes,and particularly some large-spored fungi , e.g. nematophagous orbiliaceousanamorphs, s t i l l cause problems; the more hypha-l ike the spores are themore d i f f i c u l t is the i r preservat ion (Tan et al. 1994, 1998). Withmodifications of the cryoprotectant or freezing regime such problems canpartly be overcome (Tan 1997; Tan et al. 1994, 1998).

2. Culture collectionsThere are some 480 registered microbial culture collections (Sugawara et al.1993; Sugawara and Ma 1995). Professional culture collections have a full-t ime staff whose pr imary duty consists of the receipt, verif icat ion,preservation, documenta t ion , maintenance and shipping of cultures forsc i en t i f i c and commercia l purposes on a da i l y basis. Large servicecollections, who make their catalogues accessible, include the AmericanType Culture Collection (ATCC), the Belgian Coordinated Collections ofMicroorganisms in Louva in - l a -Neuve , Brussels and Gent (BCCM),Centraalbureau voor Schimmelcultures in Utrecht, Netherlands (CBS), theCzech Collection of Microorganisms, Brno (CCM), Deutsche Sammlungvon Mikroorganismen und Zel lkul turen , Braunschweig, Germany (DSM),the Institute for Fermentation, Osaka, Japan (IFO), CABI Bioscience,

Ex Situ conservation of microbial diversity 271

Egham, UK (IMI), the Japan Collection of Microorganisms RIKEN, Tokyo(JCM), and the Al l -Russian Collection of Microorganisms, Pushchino(VKM).

3. Methods for preservationAn ample literature describes methods used in culture collections. For thepermanent preservation of microbial strains several techniques are available,most of which are summarised by Smith (1988) and Kirsop and Doyle(1991). Lyophilization (freeze-drying) is mainly available for sporulatingcultures, while deep-freezing lends itself also for many other fungi. Detailsof freezing procedures as used in CBS are described by Tan et al. (1994).

4. What is preserved in culture collections?Normally, representative (if possible ex-type and some other) strains of eachspecies should be preserved in one or more culture collections. Also, anysubdivision of species down to formae speciales, anastomosis or vegetativecompatibility groups and isozyme groups must be represented with referencestrains. Principally, it is desirable to maintain strains of all culturable speciesas pure or at least monoxenic cultures (for obligate mycoparasites) incollections, whether they play beneficial (see below) or deleterious roles tohumans, or are neutral. The whole microbial diversity on earth should bedocumented in this way, though it is still quite incompletely explored,particularly in tropical countries (Hawksworth 199la; Hawksworth andMound 1991; Subramanian 1992; Hyde 1997). But also in Australia theexploration of the fungal flora is only in an initial phase (Fungi of Australia1996). The exploration of unusual niches, microhabitats or substrata willundoubtedly yield numerous new species (Subramanian 1992). Depositionof strains of new taxa is in the interest of every taxonomically activemycologist. Culture collections try to acquire strains of all new speciesappearing in the literature if they are not supplied by the authorsspontaneously. Moreover, taxonomists working at culture collectionscontribute to the enrichment of the collection by their own collectingactivities. It is profitable to concentrate search activities in centres of theorigin of certain fungal groups, particularly pathogens. To the benefit ofscientific work, a free exchange of cultures between countries is highlydesirable, with due regard to all necessary safety regulations applying topathogenic organisms. The countries of origin of strains, particularly whendeveloping countries are involved, should share in revenues obtained byindustrial applications as recommended by the Convention on biologicaldiversity, Rio de Janeiro (1992). But this requirement should not preventfree exchange of cultures for research between countries, even though theprocedure is not yet sufficiently regulated.


For bacteria it has been estimated that the approx. 4000 speciesdescribed represent a minor i ty (estimated to be 0.1%) of the predicteddiversity and that the majority may be unculturable with current methods(Stackebrandt 1996; Ward et al. 1995; Hammond 1995). For fungi, less than100,000 species are now described, which also represents only a smallproportion of the total diversi ty; but estimates of about 5% (Hawksworth199la; Hyde and Hawksworth 1997) are probably too low.

The oldest and most commonly preserved fungal strains in culturecollections are those of saprotrophic genera of Mucorales and manyascomycetes and their anamorphs (Penicillium, Aspergillus, Fusarium,Trichoderma, Cladosporium, etc., etc.), isolated from soil, food, dung andvarious plant substrata. These are the ecological niches that are richest inculturable fungal species.

Numerous fungi cannot be cultured, among which are the insect-commensal Laboulbeniomycetes, the Pneumocystidales, many ectomycor-rhizal fungi , the zygomycete order Glomales comprising arbuscularsymbionts of the majority of green land plants, and many biotrophic plant-parasites. These fungi defy axenic culture, but many members of the last twogroups can be propagated together with a host plant and then theirpropagules can be stored, though not quite axenically. A special culturecollection has been established for the Glomales (Morton et al. 1993).

Destructive, necrotrophic plant parasites can usually be cultured readily,though many tend to remain sterile (non-reproductive) in culture. Mostbiotrophic plant parasites cannot be grown axenically and are not preservedover long periods in culture collections. As an exception, many Ustilaginalesare cultured and preserved axenically; but Uredinales, which can growaxenically for limited periods, are not kept in culture collections. Severalprojects are on-going for the permanent preservation of spore material ofnon-culturable plant parasites (e.g. Uredinales) encapsulated in alginatebeads that allow infection experiments to be carried out later.

Al l human-pathogenic species are preserved in culture collectionsexcept for Pneumocystis, Rhinosporidium and Lacazia (de Hoog et al.1992). The preservation of any new additions to this diversity is highlydesirable in order to ascertain their identity (de Hoog and Guého 1985).

Among the basidiomycetes, wood-decomposing species in particularare readily cultured and well represented in culture collections. Only afraction of ectomycorrhizal species can be cultured and they often growextremely slowly; orchid mycorrhizal partners mostly are more easily grownin vitro. As these fungi normally remain sterile in culture, they are amenableonly to deep-freezing, not lyophilization.

Zoosporic fungi , which depend on free water for zoospore formation,are diff icult to maintain over prolonged periods and at least require frequent


transfers unless they are deep-frozen. Plant-pathogenic members of theOomycetes (Chromista, unrelated to true fungi), such as members ofPythium and Phytophthora, grow well on agar media but also require ratherfrequent transfers. Using cryo-techniques, about 50–70% of the cultures canbe successfully preserved. Another ecologically relevant group of aquaticfungi is the Ingoldian fungi (aquatic hyphomycetes), which can be culturedbut tend to remain sterile in culture unless they are aerated in water(Bärlocher 1992).

Extremophilic organisms can often be cultured if special conditions,imitating those of their natural habitat, are applied. They can be of interest,particularly as producers of enzymes under very low or high temperatures(Horikoshi and Grant 1998).

5. Criteria for selecting material to be conservedCulture collections can generally be divided into service collections thatpreserve a wide spectrum of taxa (determined according to relevance tosociety and potential needs of customers), and more or less specialisedresearch collections that, at least for limited periods, preserve larger numbersof strains of particular groups for the sake of research projects. Generalculture collections must have a wider scope for preserving at least singlerepresentatives of all cultured species and, particularly, strains that havebeen extensively characterised with any advanced method.

Staff and space are limiting factors in culture collections and each strainpreserved costs money. Equivalent amounts of money are generally notrecovered by revenue generated from the sale of limited numbers of cultures- culture collections therefore strongly depend on state support. Still, thediversity of cultures preserved can only increase if culture collections do notcharge for preserving offered strains (unless commercial interests areinvolved). Also the free delivery of cultures on an exchange basis is animportant incentive for the enrichment of the culture collection. But culturecollections must determine a policy in deciding what strains can bepreserved. While it would be ideal that all critically examined isolates werepreserved in at least one culture collection, it is essential that a selection ofrepresentative isolates is permanently maintained and made accessible,together with voucher material of the fungus as it occurs on the naturalsubstratum. Full documentation of the origin is essential (Agerer et al.2000). Particularly in the case of genetically variable and poorly definedtaxa, only investigation of large numbers of isolates allows an assessment ofthe population-genetic structure.


6. The importance of living cultures for taxonomyLiving cultures are of prime importance as sources of documentation fordetermination of the identi ty of an organism. Bacterial species are typifiedby living cultures (Lapage et al. 1992). For fungi, cultures are not consideredsufficiently stable to serve as nomenclatural types. Only in 1999 was itrecognised that permanently preserved, inactive fungal material can alsoserve as type of a new species. But when part of the permanently preservedfreeze-dried or deep-frozen material from ampoules is reactivated, thisautomatically becomes ‘ex-type’ (Art. 8.4 of the International Code ofBotanical Nomenclature, Greuter et al. 2000).

The taxonomy of genera l ike Penicillium, Aspergillus, Fusarium,Verticillium, Acremonium, Phialophora and many others is entirely culture-based. Taxonomy and nomenclature themselves, however, are formallybased entirely on immutably preserved (i.e. dried or at least inactive) typematerial. Ex-type cultures are generally much more relevant as referencematerial than the corresponding dried holotype specimens. Referencecultures are needed in all kinds of investigations; this may be official ex-typestrains or other, well-developed representative strains. To build a stabletaxonomy of critical groups a ‘polyphasic approach’ is increasingly adopted:numerous isolates preserved of the same or related taxa from different originare analysed in detail with many available methods to ascertain specieslimits. In many groups of p lan t parasites and saprotrophs which can begrown axenically, cultural work has hitherto been neglected.

Culture collections are the most qualified institutions to uphold a hightaxonomic standard and to offer identification services. Though databasesand other identification software is becoming available at an increasing rateon the Internet and elsewhere, they remain complementary to the humancomponent of the identification process.

7. The use of preserved pure culturesBesides taxonomy, many d isc ip l ines benefi t greatly from culturedmicroorganisms, as exemplified here.

7.1. TeachingMany features of microorganisms are observed best in pure cultures. Themorphology of many fungi is ma in ly studied in cultures. The optimal stageof a culture to be used in classroom demonstration can usually be wellplanned (Emerson 1958). A course on taxonomic mycology, mainly basedon culture work, is given annually at CBS (Gams et al. 1998).


7.2. GeneticsConventional genetics wi th fungi generally is done with pure cultures.Cri t ical comparison and mat ing of single-spore isolates of manybasidiomycetes contribute to the understanding of species structures, thedynamics of genetic segregation and speciation (Burnett 1983; Petersen1997; Petersen and Hughes 1999). Comparative analysis of DNA sequencesallows the reconstruction of phylogeny. For DNA extraction and molecularwork, cultures are the material of choice to provide reproducible results.From a combination of phylogenetic relationships and the geographic originof related taxa, phylogeographic trends can be inferred (O’Donnell et al.1998). In some genera, stable, well-delimited species of great evolutionaryage can be recognised, in others the process of diversification seems in fullprogress (de Hoog and McGinnis 1987). This can be seen in plant pathogenssuch as the causal agents of Dutch elm disease, where new species, in thiscase Ophiostoma novo-ulmi, develop under the eye of the investigator(Brasier 1991). Population genetics becomes relevant in all kinds of fungalgroups. Molecular comparisons among numerous isolates obtained for onetaxon allow important conclusions about the genetic structure of this taxon.Thus the development of, e.g. pathogenic populations of a fungus, can betraced. To render the work reproducible, all these disciplines require stableand well-documented collection strains as a basis for comparison.

7.3. Physiology and ecologyNutritional requirements of fungi are mostly determined from growth underin vitro conditions which rely on pure cultures. A wide and complex array ofsecondary metabolites has also become known from investigations of invitro cultures. Conditions leading to their production and the roles of otherecological factors are often determined first in vitro. However, the results ofsuch studies require careful consideration and detailed experimentationbefore relating in vitro requirements to field conditions.

7.4. BiotechnologyProduction of microbial metabolites has gained tremendous importance inthe pharmaceutical industry (e.g. antibiotics, immuno-suppressants). Variousmicrobial fermentations of food and beverages are performed from with pureculture sources (Beuchat 1987; Samson et al. 1996a). A considerable varietyof edible macromycete species is cultivated all over the world, for whichstandardised strains are available (Stamets and Chilton 1993). On the otherhand, some fungi are deleterious spoiling agents of foods and are oftentoxinogenic (Samson et al. 1996a). Microbially produced enzymes find theirapplication in many unexpected areas of human life. The identity of suchstrains must be fixed by means of stable cultures; moreover, patented


processes in which certain microorganisms are involved, require a stabledeposit of these cultures. The industrial requirements that need to be met byculture collections have been summarised by Gürt ler and Sasa (1996).

7.5. BioremediationNoxious chemicals can be efficiently inactivated by certain microorganisms.As an example, certain white-rot basidiomycetes are able to detoxifypolyaromatic hydrocarbons (May et al. 1997; Boyle et al. 1998; Martens andZadrazil 1998).

7.6. Medically relevant isolatesIt is important to preserve isolates of organisms found to be causal agents ofdisease in view of further comparative studies (de Hoog and Guého 1985).Isolates with well-preserved virulence are necessary for in vitro and animalmodel studies of new drugs, disease mechanisms, natural immunological aswell as vaccine responses and immunodiagnostic or molecular reagentpreparation. There is also a high demand for standardised quality-controlisolates to be used in medical laboratory procedures as well as dedicatedtraining for staff involved in testing strains. Certain beneficial fungi produceknown metabolites, mainly antibiotics (see under Biotechnology), whileothers have various other beneficial actions, e.g. anti-tumor activity (Stametzand Chilton 1993). The importance of preservation of such strains has beenemphasised by Subramanian (1992).

7.7. Plant pathologyPlant pathological investigations rely on inoculation studies which, amongothers, are important for determination of pathogenesis, testing of Koch’spostulates, estimation of resistance and susceptibility of host plants andtesting for control measures such as biocidal chemicals. Highly qualifiedtester strains need stable preservation. Then a constant inoculum can beproduced at any moment.

7.8. Biological controlBacteria and fungi are gaining importance for controlling insects, nematodes,pathogenic and spoilage fungi, and weeds. Seed coatings which incorporatebacteria or fungi can serve to promote growth and inhibit seedlingcolonization with pathogens (McQuilken et al. 1990; Callan et al. 1991;Jensen et al. 2000).

7.9. SymbiosesExperiments of the physiology of ectomycorrhiza have always been carriedout with artificially established 2-partner symbioses. Plants are sometimes


art i f ic ial ly inoculated with their cultured symbionts in order to improvegrowth (Jeffries and Dodd 1991). Inoculation of nursery plants withectomycorrhiza is of great practical importance in reafforestation. Thequality of ectomycorrhiza in forest trees can be crucial for reafforestation inproblematic areas, e.g. h igh-a lp ine regions (Moser 1958). Mycorrhizalsyntheses are now practised on industrial scales using cultured mycelium ofthe most suitable mycorrhizal fungus, for example in alginate encapsulationof mycorrhiza for Eucalyptus plantations in Australia (Thomson et al. 1993;Hardy and Sivasithamparam, this volume); this practice may increase treegrowth by a factor of two. In vitro inoculum production from cultures is thusbecoming very important for the efficient propagation of mycorrhizalsymbionts . Synthes is wi th arbuscular mycorrhizal fungi can becomeimportant in soils with low phosphorus content, particularly if the naturalmicroflora has been reduced by chemical treatments (Jeffries and Dodd1991; Strullu et al. 1991). Inoculation with symbiotic actinomycetes of thegenus Frankia has made it possible to introduce Alnus in a newly reclaimedpolder in the Netherlands, where the tree was able to accumulate nitrogen tothe benefit of subsequent tree species (van Dijk 1979; Houwers andAkkermans 1981). Orchid symbionts show a wider diversity in Australiathan in many other countries and they are becoming important for theartificial propagation of endangered species (Batty et al., this volume). Inthe case of the slow-growing autotrophic lichen symbioses, culture studieshave not resulted in substantial collections of mycobionts. The slow-growingand often sterile fungal partners of lichens are, however, becoming relevantas producers of valuable metabolites (Stocker-Wörgötter 1995; Crittenden etal. 1995).

8. Culture collections and the conservation of biological diversityIn any part of the world, the numbers of fungal species are usually higherthan those of green plants (Hawksworth 1991a). But the fungal diversity isless localised than that of green plants. Therefore local relationships betweenthe ratio of green plants to fungi, ranging between 1:3 and 1:6 for the UK(Hawksworth 1991a), probably do not hold at a world-wide scale. Of thepresently described fungal species (less than 100,000), only about 13,000 areavailable in culture collections (Sugawara et al. 1993; CBS 2002; Rossman1997). Much still remains to be explored, particularly in tropical countries(Hyde 1997), and in the diverse range of ecological niches (Hyde andHawksworth 1997).

In all ecosystems, fungi play many decisive roles (Hawksworth 1992;Carroll and Wicklow 1992; Allsopp et al. 1995). They form a majorcomponent of the earth’s b iod ivers i ty . A rich microbial diversity is animportant component of sus ta inable agr icul ture (Hawksworth 1991b;


Hawksworth and Mound 1991; Edwards et al. 1993; Brussaard and Ferrera-Cerrato 1997). In the interest of ma in t a in ing biological diversity andavoiding collateral damage, pathogens and pests that threaten crops, speciesshould be managed to reduce levels below the critical threshold of damage,while m i n i m i s i n g dis turbance to other competing organisms and thedynamic biological equ i l i b r i um (see Ingram, this volume). In these efforts,integrated control and part icular ly, biological control, play a crucial role.

The application of fung i as control agents has been successfullyemployed against insect pests (Ferron 1978; Clarkson and Charnley 1996),nematodes (Siddiqui and Mahmood 1996), fungi (Vakili 1984; Jeffries andYoung 1994; Lynch 1996) and weeds (Pemberton and Hoover 1980; Hasanand Ayres 1990; Evans 1995). Many virulent strains are already present inculture collections, but the spectrum of species with potential benefits in pestcontrol is st i l l to be exhaustively investigated.

Red lists of endangered species of fungi have been drawn up formacromycetes in many countries, but no such approach has been undertakenfor micromycetes. Nevertheless, there are a few examples. Species of themycoparasitic genus N y c t a l i s have declined in the Netherlands in recentdecades. A microscopic hyperparasite, Pyxidiophora asterophorae (Tul.)Lindau, described as growing on Nyctalis, has not been found in recentdecades. Culture collections of such a species therefore play a vital role inthe conservation of fungal divers i ty . There are many cases of single recordsof a particular fungus where the only known extant material is in a culturecollection. Normally, such observations cannot be taken as proof of rarity,but there are except ions, such as the conspicuous species of thehyphomycete genus Pleurocatena, which through extensive searching ofprobable habitats are now considered rare and of which two species are nowavailable in culture.

Most Red-list macromycetes are ectomycorrhizal fungi (Arnolds 1991,1992). For example, commercial production of truffles (e.g., Pirazzi et al.1990; Jeffries and Dodd 1991) is possible from artificial inoculation ofassociated tree species. The role of microorganisms in the restoration ofdegraded or altered landscapes is an emerging field of research and a numberof recent studies have explored the benefits of introduction of mycorrhizalagents in post-mining restoration (Brundrett et al. 1996; Hutton et al. 1997)and in reintroduction of orchid species (Quay et al. 1995). Microorganismsdefy introduction into a complex ecosystem if the biotope does not suit themand if they are not provided with ample nutrient sources. Unless indigenousfungi have survived adverse conditions, somewhere hidden in their biotopeor in neighbouring areas, they may require artificial inoculation to return tothe site. But many apparently threatened organisms show a remarkably rapidreappearance when environmental changes favour the microorganism. It is


not yet known to what extent the local destruction of biotopes leads to theextinction of certain fungi, but for macromycetes the possibility exists. Alsopollution of biotopes, particularly flowing waters, is a factor that eliminatesmany aquatic fungi, although certain aquatic hyphomycetes are surprisinglyresistant to heavy metals (Krauss et al. 2001).

Not only ectomycorrhiza, but also a diverse population of arbuscularmycorrhizal fungi have been shown to promote plant diversity in plots ofnatural vegetation (van der Heijden et al. 1998). For all the purposesdescribed here, suitable well-preserved strains are needed.

AcknowledgementsMy colleagues Drs D. van der Mei, R. C. Summerbell and J. A. Stalperskindly contributed suggestions to improve this chapter.

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Chapter 11

IMPACT OF FUNGAL PATHOGENS INNATURAL FOREST ECOSYSTEMS: A FOCUSON EUCALYPTS

Treena Burgess

Michael J. Wingfield

Forestry and Agriculture Biotechnology Institute (FABI), University of Pretoria, Pretoria,0002, RSA.

1. IntroductionFungal pathogens are integral components of healthy natural forestecosystems, where they play a major role in eliminating weak and unfit trees(Manion 1981; Burdon 1991; Castello et al. 1995). They also affect speciesoccurrence and distribution, especially in the regeneration layer (Castello etal. 1995). Soil-borne pathogens, in particular, are thought to be important inmaintaining plant species diversity and distribution (Augspurger and Kelly1984; Bever et al. 1997; Mills and Bever 1998; Packer and Clay 2000). It ishypothesised that seedlings close to their parents or other conspecific treesare more likely to be killed by host specific soil-borne pathogens thanseedlings further away. Over time, this results in a shift in the juvenilepopulation away from the adults. This relationship has been demonstratedbetween the temperate tree Prunus serotina and the pathogen Pythium(Packer and Clay 2000) and also for seedling damping off of the tropical treePlatypodium elegans caused by a variety of soil-borne pathogens(Augspurger and Kelly 1984). Interestingly, canker fungi also have beenshown to impart a similar effect on the distribution of the tropical treeOcotea whitei (Gilbert et al. 1994). Higher levels of light intensity, such asthose experienced in light gaps caused by fallen trees, reduce both pathogen


activity and the net impact of pathogens (Augspurger and Kelly 1984;Castello et al. 1995).

The epidemiology and visual impact of indigenous pathogens in naturalforest ecosystems is greatly minimised by genetic diversity. Here, trees andtheir pathogens have co-evolved and the population structure of the hosts ischaracterised by genetic and age diversity (Manion 1981; Hansen 1999).Consequently, individuals in a mature tree population will vary in theirsusceptibility to a particular pathogen. Usually, the tree is not susceptiblethroughout its life cycle, consequently, some individuals in some age classesmay be susceptible, but not the whole population. Large populations ofsusceptible trees do not develop and therefore, widespread disease epidemicscannot occur.

This immuni ty of natural ecosystems to disease epidemics can beovercome in two ways. Firstly, either a natural or human disturbance couldresult in the growth of an even aged stand of a single species that may thenbe susceptible to an indigenous pathogen. Secondly, virulent pathogens, towhich the trees have no resistance, could be introduced to a naturalecosystem. Introduced pathogens often lead to the elimination of entirespecies and result in permanent changes in the species composition of anecosystem. Ineffective quarantine has often led to epidemics in indigenousand exotic tree populations caused by introduced fungal pathogens (Old andDudzinski 1998; Palm 1999). Examples of mass destruction of indigenousforests due to fungal pathogens inc lude Chestnut Blight caused byCryphonectria parasitica (Anagnostakis 1987), Dutch Elm Disease causedby Ophiostoma ulmi and O. novo-ulmi in Europe and North America(Brasier 1991; Hubbes 1999) and Cypress Canker in Europe caused bySeiridium cardinale (Graniti 1998).

Destructive epidemics caused by fungi in natural woody ecosystems,particularly in the Northern Hemisphere, have been covered extensivelyelsewhere (Anagnostakis 1987; Brasier 1991; Graniti 1998; Hubbes 1999).In this chapter, we wil l rather focus on pathogens of eucalypts in their nativerange in Australia and in exotic plantations throughout the world. We alsoconsider the threat to na t ive forests in Australia due to so-called newpathogens that have emerged on exotic plantations elsewhere in the world.Emphasis is placed on the importance of the origin of a pathogen and thestructure of the host population.

2. Eucalypts in AustraliaThere are about 700 species of eucalypts, the majority of which are native toAustralia (Brooker and Kleinig 1994; Ladiges 1997; Potts and Pederick2000). A small number of species are native to Papua New Guinea and afew islands in the Indonesian archipelago (Brooker and Kleinig 1994).

Fungal pathogens and biodiversity 287

Eucalypts occur naturally from latitude 7°N to 40°S and from sea level to1,800 m. They have thus adapted to climatic extremes from the desert gumsgrowing under hot dry conditions to snow gums that can tolerate extremecold (Poynton 1979; Brooker and Kleinig 1994 Ladiges 1997;). In Australia,eucalypts define much of the forest environment and dominate 95% of theforest area. The most impressive examples are the forest eucalypts found inmagnificent stands on the eastern seaboard, Tasmania and WesternAustralia. It is from these large timber trees that species were selected forexotic plantation forestry in the Tropics and the Southern Hemisphere(Poynton 1979; Potts and Pederik 2000).

Indigenous pathogens play an integral role in undisturbed eucalyptforests. Fungi capable of causing leaf diseases and stem cankers suchMycosphaerella spp., Endothia gyrosa, Endothiella spp., Botryosphaeriadothidea and Cytospora eucalypticola are commonly found in nativeeucalypt forests (Davison and Tay 1983; Walker et al. 1985; Old et al. 1986;Barber and Keane 1999; Ivory 1999; Yuan 1999; Old and Davison 2000;Park et al. 2000). There are no reported cases of these fungi causingsignificant disease in undisturbed forests (Park et al. 2000). However, muchof the Australian eucalypt forests has been heavily logged and is nowintensively managed as regrowth forests. This disturbance has resulted inlocalised outbreaks of indigenous pathogens.

Between 1995 and 1998, hardwood plantations in Australia almostdoubled from 160,000 ha to 290,000 (Love et al. 1999; Turnbull 2000). Thisincrease was mainly due to a trebling in the area under plantations(predominantly E. globulus) in Western Australia from 42,000 to 120,000ha. A major concern with this rapid expansion is the danger associated withpests and pathogens. There is an increased disease risk in plantationsbecause of reduced genetic variation and site conditions that are often notsuited to the species being planted (Potts and Pederick 2000). Althougheucalypts are native to Australia, the species used in plantations are often notendemic to the region in which they are planted (e.g. Tasmanian bluegums,E. globulus, in Western Australia). In addition, plantations represent even-aged monocultures and are more susceptible to disease than native forestswhere epidemics are restricted by the age structure and the diversity of theplant community. To date, the plantations have been relatively free frommajor diseases. However, considering the experience in other countries, thissituation seems likely to change (Old and Dudzinski 1998; Wingfield 1999;Old and Davison 2000; Park et al. 2000).

2.1. Indigenous pathogens in logged forestsArmillaria spp. are widely distributed in many forests where they cause littledisturbance besides colonising dead stumps and roots (Kile et al. 1991; Kile


2000). However, Armillaria spp. have been observed as epiphytes of livingroots and as primary root and butt pathogens in disturbed forests (Kile et al.1991). As a primary pathogen, Armillaria spp. cause typical diebacksymptoms, crown decline, root rot, basal lesions and ultimately tree death.Sub-lethal infections reduce tree growth (Kile et al. 1991; Shearer 1995).Armillaria spp. spread by aerial dispersal of basidiospores, by rhizomorphsin the soil or by root contacts. Basidiospores seem not to be a commonmethod of dispersal in Australia, likewise the Australian climate is often notconducive to rhizomorph development (Podger et al. 1978). Thus, the mostcommon method of spread is through direct contact between the tree roots(Kile et al. 1991; Shearer 1994). Consequently, tree death tends to be inpatches with dead trees at the centre and dying trees at the periphery ofpatches. As Armillaria spp. are common saprophytes of dead roots andstumps, the removal of this primary source of inoculum can restrict thespread of the disease.

Armillaria luteobubalina is an unimportant indigenous pathogen insouthern Australian forest communities (Kile et al. 1991). However, thisfungus has emerged as a serious primary pathogen in the dry sclerophyllmixed species eucalypt forests, subjected to heavy logging, in centralVictoria and Western Australia (Shearer and Tippett 1988; Kile et al. 1991;Shearer 1994). Regeneration of E. diversicolor forests following logging isalso impeded by A. luteobubalina because logged stumps provide a source ofinfection for young seedlings (Pearce et al. 1986) (Figure 1). There is astrong positive relationship between proximity to infected stumps anddisease incidence in regenerating E. diversicolor stands. A. luteobubalina ismost active in young stands, but mortality is restricted to weaker trees inolder stands (Pearce et al. 1986).

2.2. Indigenous pathogens in eucalypt plantationsA. luteobubalina has also led to disease outbreaks when native eucalyptforests have been cleared to allow for reafforestation (Kile 2000). InWestern Australia, plantations of E. saligna were established after theclearing of native E. marginata forests. Trees close to infected stumps werekilled by A. luteobubalina (Shearer 1995). Similarly, death and diseasecaused by A. luteobubalina in a fast-growing E. regnans plantation inVictoria was in i t ia ted from an infected stump at the edge of the plantation(Podger et al. 1978).

Endothia gyrosa is a common canker pathogen in eucalypt forests ineastern Australia (Walker et al. 1985; Old et al. 1986; Old et al. 1990; Oldand Davison 2000) and its anamorph Endothiella gyrosa is common oncankered trees in Western Australia (Davison and Tay 1983; Shearer 1994).Endothia gyrosa is an opportunistic pathogen that generally causes


superficial non-lethal cankers, but under experimental conditions it can killstressed trees (Old et al. 1986; Old et al. 1990; Old and Davison 2000). InTasmania, E. gyrosa is also found to cause cankers in stressed trees, and inone plantation, infection resulted in severe disease (Waldlow 1999). Thisoutbreak was apparently not due to a more pathogenic strain of E. gyrosa,but more likely an unknown stress event predisposing the trees to infection(Yuan and Mohammed 2000). In Western Australia, low rainfall in a hybridplantation (E. grandis crossed with E. camaldulensis) led to a severecankering of stems caused mainly by Endothiella gyrosa (Neumeister-Kemp,pers. comm.).

Botryosphaeria ribis is an opportunistic pathogen and is frequentlyisolated from stem cankers throughout Australia (Davison and Tay 1983;Shearer et al. 1987; Old et al. 1990; Old and Davison 2000). Death isuncommon, but cankers girdled and eventually killed individuals ofEucalyptus radiata originating from eastern Australia that had been planted


in a species trial in Western Australia (Shearer et al. 1987). The trees weremost likely not suited to the experimental site and were thus predisposed toinfection by unfavourable climatic conditions.

Numerous Mycosphaerella spp. and their anamorphs have beendescribed from Australia (Carnegie et al. 1994; Crous 1998; Crous et al.1998; Barber and Keane 1999; Ivory 1999; Maxwell et al. 2000; Yuan 1999;Park et al. 2000). These fungi form necrotic lesions on leafs and youngshoots of eucalypts causing a disease commonly referred to asMycosphaerella leaf blotch disease (Figure 2). In the most severe cases,they can reduce the photosynthetic capacity of trees to such an extent thatdeath eventually occurs. This does not happen in forest ecosystems. E.globulus, the plantation species of choice in many parts of Australia, isparticularly susceptible to Mycosphaerella leaf blotch disease (Crous et al.1998). E. globulus plantations in Tasmania have experienced severedefoliation of juvenile leaves due to M. cryptica and also to a lesser degree,M. molleriania (Milgate et al. 1997). Recently, the juvenile foliage of E.globulus grown in plantations in Western Australia has been severelyaffected by M. nubilosa and the adult foliage by M. cryptica (Carnegie et al.1997, Maxwell et al. 2000). M. cryptica is also widely distributed in

Fungal pathogens and biodiversity

Queensland where it causes disease on a wide range of eucalypt species(Ivory 1999).

Cylindrocladium leaf spot and shoot blight caused by the fungusCalonectria quinquiseptatum has damaged to young eucalypt plantations intropical areas of Queensland for approximately two decades (Bolland et al.1985). Lesions cover the foliage and new shoots of susceptible young treesand cause the leaves to be shed and the branches to die-off (Ivory 1999).

There are many other indigenous pathogens reported from eucalyptplantations (Old et al. 1986; Kile et al. 1996; Barber and Keane 1999; Yuanet al. 1999; Park et al. 2000), but they are apparently of relatively minorimportance. Cytospora eucalypticola is one of the most commonly isolatedcanker pathogens (Davison and Tay 1983; Shearer et al. 1987; Yuan et al.1999), but is considered to be less pathogenic than either Botyrosphaeriaspp. or Endothiella spp. (Old et al. 1990). Ceratocystis eucalypti has alsobeen isolated from simulated stem wounds in eucalypt regrowth in Victoria,although it is not thought to be a serious pathogen (Kile et al. 1996).

291

2.3. Introduced pathogensThe forests and near-coastal vegetation of south-western Australia supportone of the richest regions of floristic biodiversity in the world with over7,000 native plant species. Eucalyptus marginata (jarrah) is the dominantoverstorey tree species in much of this region (Figure 3). The introducedroot and collar pathogen Phytophthora cinnamomi has had a major impacton the jarrah forest ecosystem (Shearer and Tippett 1989; Wills 1993;Shearer and Smith 2000). At least 2,000 species in Western Australia aredirectly susceptible to P. cinnamomi and the growth and germination ofmany other species would be affected indirectly through the loss of the over-story and changes in light and moisture conditions.

Infection by P. cinnamomi in the jarrah forest causes dieback of speciesin the overstorey, mid-storey and under-story (Davison and Shearer 1989).This results in changes to the vegetation structure and ultimately speciesbiodiversity. A more open forest with a sedge-dominated understoreyemerges and this in turn affects the endemic fauna (Davison and Shearer1989). This was demonstrated in a detailed study of P. cinnamomi diseasecentres in Banksia woodlands on the Swan coastal plain, Western Australia(Shearer and Dil lon 1996). Infection by P. cinnamomi decreased speciesnumber, changed vegetation structure and totally altered the visual andfloristic characteristics of the ecosystem.

P. cinnamomi infects the roots and collar of trees via motile zoosporesand thus, water plays an important role in their movement. The up-slope inthe jarrah forest is usual ly free of P. cinnamomi while the down-slope andthe valleys and any area where water collects tend to be infected (Davison


1994). In the Swan coastal plain, disease is worst where the water table ishigh (Shearer and Dillon 1996).

The impact of P. cinnamomi on Australian ecosystems is not limited toWestern Australia, but causes problems throughout Australia in regions withmediterranean or temperate climates. Comprehensive studies on vegetationchanges in forests of Victoria following infestation showed a reducedoverstorey and a change in the species composition of the understorey(Weste 1986). P. cinnamomi has also devastated natural ecosystems andirreversibly changed the vegetation structure in Tasmania, the AustralianCapital Territory and New South Wales (Davison and Shearer 1989). Theextent of the disease associated with P. cinnamomi is so great, this pathogenis considered one of the f ive major threats to biodiversity in Australia(Commonwealth of Australia 1992).

3. Eucalypts as exoticsEucalypts were introduced from Australia to the rest of the world in thecentury. They adapted well as exotics and became a part of the landscape in


many regions of the world. From the beginning, eucalypts were found to beuseful for shade, t imber and fuel and in stabilising degraded lands.Commercial plantations were established in many countries by the turn ofthe century, and in the last 40 years there has been a rapid expansion inareas under afforestation, particularly in the tropics and the SouthernHemisphere. In 1990, there were 8 million ha of exotic eucalypt plantations,this increased to over 10 million ha by the year 2000 and is expected toincrease to over 20 million ha by the year 2010 (Turnbull 2000).

Exotic eucalypt plantations have primarily been established to meet theworld’s demand for paper and pulp. Species and provenance trials have beenused to select the best p lan t ing stock for new regions and subsequentlyextensive breeding programs have been established world-wide. Hybridspecies and local land races have emerged from trial plantings of differentspecies and have become the basis of major planting programs (Cotterill andBrolin 1997). In addition, breeding programs and artificial hybridisation ofdifferent species has been actively pursued and vegetative propagation ofsuperior genotypes has led to extensive clonal forestry (Borralho 1997;Cotterill and Brolin 1997).

Ultimately, the ini t ia l success of eucalypts as exotics has been due tothe absence of pests and pathogens naturally associated with them in theirregions of origin (Wingfield 1999). Only a small number of diseases werereported in the early days of eucalypt plantation development. However, asthe planted areas have increased and more plantations have been established‘off-site’, disease problems have also increased (Day 1950; Wingfield 1999;Wingfield et al. 2001).

Pathogens on exotic eucalypts are predominantly those accidentallyintroduced to all eucalypt-growing regions of the world along with thosetrees. The majority of these pathogens infect stems and leaves and havebeen introduced with the germplasm, either on seed or chaff or asendophytes of vegetative material (Burley 1987). No known root pathogenshave been introduced as soil was rarely, if ever, transported with plantingstock.

Interesting examples of soil fungi introduced world-wide are thebeneficial ectomycorrhizal fungi . It was recognised early on that eucalypts,especially from the subgenus Monocalyptus, grew poorly as exotics due tothe absence of ectomycorrhizal fungi (Pyror 1956). As a consequence, soiland spores were distributed world wide and now eucalypt-specificectomycorrhizal fungi such as some species of Pisolithus, Laccaria laccata,Hydnangium carneum and Scleroderma verrucosum are found wherevereucalypts are grown (Dell et al. 2000). These species are common inAustralia, but represent only a few of the thousands of ectomycorrhizal fungiof eucalypts (Bougher 1995).


In many cases, exotic eucalypts are planted in close association withnative plant species that are closely related to eucalypts. The destruction ofthe native forests, coupled with genetically uniform eucalypt plantations,places a very high selection pressure on indigenous pathogens to jump hostsand infect eucalypts. A good example of this is the eucalypt rust, Pucciniapsidii, that has moved from native Myrtaceae to eucalypts in South America(Coutinho et al. 1998). However, there are many other examples of new andemerging pathogens of unknown origin. Some pathogens such asBotyrosphaeria dothidea are found in Australia, but is also found world-wide with many woody plants as hosts. Thus, a population of B. dothidea inone region may include introduced and indigenous individuals. Populationstudies examining allelic distribution of polymorphic loci will be necessaryto determine the origin of such pathogens. Others pathogens such asCryphonectria cubensis seem to be indigenous to some areas and introducedto others (Hodges et al. 1986; Myburgh et al. 1999).

3.1. Stem pathogensCryphonectria cubensis causes a serious and destructive stem canker diseaseon eucalypts in tropical and sub-tropical regions of the world (Figure 4). Itwas first reported in Cuba (Bruner 1916), but has since been recorded in


eucalypt growing countries in the tropics and sub-tropics of Asia, Africa andthe Americas (Boerboom and Maas 1970; Hodges et al. 1979; Gibson 1981;Sharma et al. 1985; Wingfield et al. 1989).

In Australia, C. cubensis has been found only associated with rootcankers on Eucalyptus marginata (Davison and Coates 1991). This nicheand the mediterranean climate of the region are unusual for the fungus. Theimpact of the disease was also minimal and the distribution of the pathogenlocalised. These facts do not support an Australian origin for the fungus.One hypothesis regarding the origin of C. cubensis is that it jumped fromcloves (Svzygium aromaticum, Myrtaceae) to eucalypts, possibly inIndonesia, and from there has spread to other eucalypt growing regions ofthe world (Hodges et al. 1986). This hypothesis is based on the fact thatHodges et al. (1986) showed C. cubensis to be conspecific with the clovepathogen Endothia eugeniae.

Studies on the population diversity of C. cubensis in Brazil (van Zyl etal. 1998) as well as in Venezuela and Indonesia (van Heerden et al. 1997)suggest an equal likelihood of origin in South America and South East Asia.Moreover, phylogenetic studies on the fungus by Myburgh et al. (1999),have revealed two distinct clades, one containing isolates of C. cubensisfrom Asia (including clove isolates and those from Australia) and anothercontaining isolates from South Africa and America. This suggests thepathogen has either crossed over from native plants to eucalypts at leasttwice or the fungus has been geographically isolated in the two regions for along time. Although much research is still needed to resolve the origin of C.cubensis, it is an excellent example of a pathogen adapting to a new host.

Puccinia psidii provides a classic example of a pathogen that hascrossed over to a new host from a known host of known origin. P. psidiicauses eucalypt rust, one of the most serious diseases of eucalypts in Braziland has been found in other South American countries, Central America andFlorida (Laundon and Waterson 1965; Marlatt and Kimbrough 1979;Coutinho et al. 1998). P. psidii is a rust pathogen first reported from guava(Psidium guajava), but it has a wide host range among Myrtaceae in SouthAmerica. To date, this pathogen is unknown in Australia, Africa or Asia.The young leaves and buds of eucalypts under two years of age are the mostsusceptible to infection (Figure 5). Growth is restricted or terminated,resulting in stunted multi-stemmed trees, with the most susceptible speciesand individuals being killed.

A number of Coniothyrium spp. have been described from eucalypts,most of them causing leaf diseases of little economic importance. Incontrast, Coniothyrium zuluense has recently emerged as a serious primarystem pathogen in the sub-tropical regions of South Africa (Wingfield et al.1997). The disease was first observed in 1988 on a single clone of E.


grandis in Zululand. Since its discovery, it has become widespread in SouthAfrica and affects many different eucalypt species, clones and hybrids.Subsequently, C. zuluense has been reported in Uruguay, Argentina,Thailand and Mexico where it is also causing significant disease (Wingfield,unpubl.). This disease is typified by lesions on young green stem tissueduring the early part of growing season. Small lesions then coalesce to formlarge necrotic patches and swellings develop and crack (Figure 6). Insusceptible clones, the stem cankers coalesce and girdle the tree. Mostinterestingly, the fungus is associated with two species of the Pantoeae,which are common soft rot causing bacteria. The fungus alone is not aspathogenic as it is in association with the bacteria (van Zyl 1999). C.zuluense probably also represents a component of a newly emerging diseasecomplex and not a pathogen introduced from Australia.

Ceratocystis fimbriata is a well known wi l t and canker pathogen ofmany economically important woody plants (Kile et al. 1996). The fungusrequires wounds for the in i t ia t ion of infection, but unti l recently was notrecognised as a serious pathogen of eucalypts. It has, however, recentlybeen found to cause a serious disease of Eucalyptus clones in the Congo andBrazil (Roux et al. 2000). This appears to represent another newly emergingdisease, the origin of which is most l ikely outside Australia.


3.2. Leaf pathogensMycosphaerella spp. and their anamorphs are common pathogens ofeucalypts and have become one of the major disease causing agents affectingexotic plantations throughout the world (Crous 1998). Afforestation with E.globulus in South Africa was suspended in the 1930’s due to the severity ofinfection by M. juvensis (Crous 1998). Mycosphearella leaf blotch diseaserestricts the growth of infected trees by reducing the photosynthetic area ofthe leaves (Figure 2). Different species of Mycosphearella infect differentspecies of eucalypts. Many of these (e.g. M. suttoniae) have a broad hostrange, while others such as M. molleriana, only known on E. globulus,appear to be more specific. Numerous species have been recorded fromeucalypts in South Africa and elsewhere (Park and Keane 1984; Carnegieand Keane 1994; Crous 1998; Park et al. 2000). Mycosphaerella spp. foundon eucalypts appear to be eucalypt-specific and are thought to be movedabout via seed. However, many Mycosphaerella spp. known to infecteucalypts have not been found in Australia, indicating that some species mayhave emerged on native plants and adapted to eucalypts outside Australia.


Cylindrocladium spp. include a number of serious pathogens ofeucalypts and other woody hosts (Crous and Wingf ie ld 1994).Cylindrocladium spp. have been found wherever eucalypts are grown, butcause the most severe damage in the tropics and sub-tropics. The causalagent of new outbreaks is usual ly indigenous to the region of the outbreak(Wingfield unpubl.). Unlike Mycosphaerella spp., species ofCylindrocladium found on eucalypts usually have a wide host range (Crouset al. 1991). They cause a range of serious diseases from damping-off ofseedlings, seedling and shoot b l i g h t , leaf spot, stem cankers and in somecountries, even root diseases.

3.3. EndophytesAn intriguing group of eucalypt pathogens are those with an opportunistichabit. In their natural environment , they cause disease only to stressed treesand are considered of minor importance. Exotic trees, however, are exposedto a wide diversity of stresses and these opportunistic pathogens can becomevery important. Their ubiquitous nature has led researchers to propose andeventually demonstrate that they are endophytes (Bettucci and Saravay 1993;Smith et al. 1996). Endophytic fungi are able colonise healthy plant tissuewi thou t causing disease. Many endophytes are seed-borne and areconsequently present in the plant tissue from germination, while others enterthe plant early in development through lenticels and stomata (Carroll 1988;Stone and Petrini 1997).

The most important of these endophytic pathogens on eucalypts isBotryosphaeria rhodina in the tropics and B. dothidea in the sub-tropics andtemperate regions. Since first reports in 1989, B. dothidea has become oneof the most commonly recognised pathogens of exotic eucalypts in SouthAfrica where it is considered an important and widespread threat to eucalyptproduction (Smith et al. 1994, 1996). Symptoms of Botryosphaeriainfection include twig dieback and stem cankers on the current year’s shoots,terminal leader shoots and main stems associated with the extensiveproduction of k ino (Smith et al. 1994). If trees recover, the death of theleader results in a twisted and weak stem susceptible to wind damage (Figure7). The production of k ino veins or pockets in older trees renders themunacceptable for solid wood production. Botryosphaeria dieback is alwaysassociated with physiological stresses including drought, hot or cold winds,nutritional imbalance, waterlogging, hail wounds, insect damage and damageby other pathogens (Smith et al. 1994, 1996). Both B. dothidea and B.rhodina are cosmopolitan fungi having a wide host range and geographicdistr ibution. As such, they could be introduced or indigenous to a regionwhere they are found. Endothia gyrosa and Colletotrichum gloeosporiodes


are other opportunist ic stress pathogens often associated withBotryosphaeria (van der Westhuizen et al. 1993; Smith et al. 1998).

Endophytic pathogens can pose greater threats to plantation forestrythan non-endophytic plant pathogens. Most pathogens only cause disease ata certain stage of their host’s life (for example Mycosphaerella spp. onjuvenile leaves of E. globulus and E. nitens), while endophytic pathogenscan cause disease in response to stress throughout the trees’ life. Thedramatic increase in eucalypt plantations world-wide will result in treesbeing planted outside their natural range. Hence, the risk of stress-induceddiseases is also increasing. Clonal forestry also raises additionalcomplications as clones vary in their tolerance to Botryosphaeria (Smith etal. 1996). In the absence of careful screening, a susceptible clone could beseverely damaged.

4. Quarantine and emerging diseasesHealthy eucalypt forests in Australia are rarely affected by fungal pathogens.Conversely, in disturbed forests and plantations, outbreaks of indigenouspathogens are becoming more common. As the areas of eucalypt plantationsin Australia increase, it is likely infections by indigenous pathogens will


result in more serious disease problems. Of greater concern, however, is theemergence in exotic eucalypt plantations of new diseases not previouslyencountered in Australia (see Hardy and Sivasithamparam this volume). Theintroduction of P. cinnamomi into Australia provides one sobering exampleof the devastating impact a foreign pathogen can have on a naturalecosystem. The accidental introduct ion of pathogens such as P. psidii, C.zuluense or the virulent South African strains of C. cubensis could have anequally devastating effect on forests in Australia (O'Neill, 2000).

In the last 25 years, there have been numerous incursions of exoticforest pests and diseases throughout the southern hemisphere (Eldridge andSimpson 1987; Old and Dudzinski 1998; Wingfield 1999). Introducedpathogens often have no initial impact and are not detected until a sizeablearea is affected (Walker 1987; Bright 1998). By this time, the pathogen willhave generally spread extensively beyond the diseased area and thuseradication becomes almost impossible (Walker 1987).

Currently, global trade agreements and the removal of embargos andtariffs have facilitated the movement of forestry products around the world(Palm 1999). Consequently, the chance of introducing pathogens into newareas has also increased. Some new diseases of eucalypts may already be inAustralia, although their presence may not have been detected. Despite this,the only means to prevent introductions of new and damaging pathogens toeucalypts in Australia is by vigilant quarantine at all points of entry. This isperhaps easier for eucalypts than for other timber species, as there is little, ifany, importation of eucalypt timber into Australia. A more likely source ofinfection would be germplasm. Many other countries have been breedingsuperior quality eucalypt material, especially hybrids and clones. These arevery attractive to commercial forestry in Australia and there are plans tobring selected material back in to Australia. This material, as seed orvegetatively propagated tissue, should clearly be detained in quarantine untilit can be safely released.

5. ConclusionsNatural forest ecosystems have many indigenous pathogens associated withthem, but genetic and age diversity of the host community prevents diseaseepidemics. This is the situation in undisturbed eucalypt forests in Australia.Disturbed ecosystems and plantations, however, are more susceptible tooutbreaks because of a reduction in both genetic and age diversity andbecause of increased external stress. Observations and records of eucalyptpathogens and diseases in Australia are increasing. In addition, many newdiseases are emerging on exotic eucalypt plantations throughout the world,especially in the tropics and sub-tropics. These emerging diseases pose athreat to native eucalypt forests in Australia. Vigilant and strictly applied

301Fungal pathogens and biodiversity

quarantine measures are necessary to prevent the introduction of potentiallydevastating pathogens into Australia.

AcknowledgementsWe are grateful to Bernard Slippers for critically reviewing the manuscript.Financial support was provided by The University of Pretoria and theNational Research Foundation (NRF).

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van Zyl LM (1999) Factors associated with Coniothyrium canker of Eucalyptus in SouthAfrica. In ‘Microbiology and Biochemistry.’ pp. 193. (University of the Orange FreeState: Bloemfontein)

van Zyl LM, Wingfield MJ, Alfenas AC, Crous PW (1998) Population diversity amongstisolates of Cryphonectria cubensis. Forest Ecology and Management 112, 41–47.

Waldlow TJ (1999) Endothia gyrosa associated with severe stem cankers on plantationgrown Eucalyptus nitens in Tasmania, Australia. European Journal of Forest Pathology29, 199–208.

Walker J (1987) Development of contingency plans for use against exotic pests and diseasesof trees and timber. 1. Problems with the detection and identification of exotic plantpathogens of forest trees. Australian Forestry 50, 5–15.

Walker J, Old KM, Murray DIL (1985) Endothia gyrosa on Eucalyptus in Australia withnotes on some other species of Endothia and Cryphonectria. Mycotaxon 23, 353–370.

Weste G (1986) Vegetation changes associated with invasion by Phytophthora cinnamomi ondefined plots in the Brisbane Ranges, Victoria, 1975-1985. Australian Journal of Botany29, 261–276.

Wills RT (1993) The ecological impact of Phytophthora cinnamomi in the Stirling RangeNational Park. Australian Journal of Ecology 171, 145–159.

Wingfield MJ (1999) Pathogens in exotic plantation forestry. International Forestry Review1, 163–168.

Wingfield MJ, Crous PW, Coutinho TA (1997) A serious canker disease of Eucalyptus inSouth Africa caused by a new species of Coniothyrium. Mycopathologia 136, 139–145.


Wingfield MJ, Slippers B, Roux J, Wingfield BD (2001) Worldwide movement of exoticforest fungi especially in the tropics and Southern Hemisphere. Bioscience: 51, 134–140.

Wingfield MJ, Swart WJ, Abear BJ (1989) First record of Cryphonectria canker ofEucalyptus in South Africa. Phytophylactica 21, 311–313.

Yuan ZQ, Mohammed C (2000) The pathogenicity of isolates of Endothia gyrosa toEucalyptus nitens and E. globulus. Australasian Plant Pathology 29, 29–35.

Yuan ZQ (1999) ‘Fungi associated with diseases delected during health surveys of eucalyptplantations in Tasmania. A report on the project funded by the FWPRDC Fellowship.’(School of Agricultural Science, University of Tasmania: Hobart)

K. Sivasithamparam, K.W. Dixon & R.L. Barrett (eds) 2002. Microorganisms inPlant Conservation and Biodiversity. pp. 307–335. © Kluwer Academic Publishers.

Chapter 12

MICROBIAL CONTAMINANTS IN PLANTTISSUE CULTURE PROPAGATION

Eric Bunn

Kings Park & Botanic Garden, Science Directorate, Botanic Gardens and Parks Authority,West Perth 6005, Western Austral ia .

Beng Tan

Department of Env i ronmen ta l Biology, Curtin University of Technology, Bentley 6102,Western Australia.

Bacteria, f u n g i , moulds and yeasts are common contaminatingmicroorganisms in tissue culture. Microorganisms and their reproductivestructures (e.g., spores) are ubiquitous although their relative abundance mayvary considerably with environment and season (Atlas and Bartha 1987).Bacteria, fungi and other microorganisms are found not only in soil, waterand air, but also on and inside plants (and animals). In plants they occur asmicroflora associated with roots (rhizosphere and rhizoplane) leaves(phyllosphere and phylloplane), other plant parts and subliminally asendophytes in plant tissues and vegetative propagules. It would beexceptional for plants growing in nature to be free of both epiphytic andendophytic microorganisms. Whilst some organisms may be pathogens orsymbionts, many microbial epiphytes and endophytes associated with plantsoperate as either saprophytes or asymptomatic parasites.

Micropropagation refers to the rapid cloning of desirable plantgenotypes through in vitro (tissue culture) propagation of shoots, and massproduct ion of whole plants capable of establishment in soil.Micropropagation is an integrated sequence of preparatory and in vitro

1. Introduction


procedures (see Table 1) typical ly involv ing four stages. Stage 1 involvesthe initiation of a “clean” shoot starter culture, from which exponential shootmultiplication is obtained through regular sub-culturing of proliferatingshoots (stage 2). Stage 3 involves root induction in selected well-developedshoots, and finally deflasking and acclimatisation (stage 4). Where somaticembryos or calluses are induced or init iated, germination and shootregeneration are prerequisites within the propagation protocol (see Figure 1).Stage 1 is therefore the primary stage of disinfestation, althoughmicroorganisms can st i l l re-infest cultures at later stages.

Microorganisms become problematic by virtue of their prolific growthunder high nutrient in vitro conditions, hence the need to eliminate themfrom explants before entering the in vitro cycle. Exclusion of

Microbial contaminants in plant tissue culture propagation 309

microorganisms from potential explant material can be approached in anumber of ways. Reducing the abundance of fungi (spores and small hyphalfragments) and bacteria on donor plants before explant material is taken willobviously be beneficial in reducing reliance on harsh surface “sterilisation”(disinfestation) procedures, which often only just avoid killing the explantwhile causing considerable damage to sensitive tissues. Even where theexplant is effectively surface sterilised, tissue injury attributable directly tothe decontamination process often leads to irreversible oxidation andphenolic leakage, and eventual death.

The phenomenon of microbial contamination in plant tissue culture hasbeen reviewed by Cassells (1991), Dodd et al. (1992), George (1993),Leifert et al. (1994), Cole (1996), and Herman (1996). Collectively thesequite recent reviews provide a broad background on the subject. Theintention of this chapter is to focus on practical means of circumvention,recognition and eradication of microbial contaminants in applied plant tissueculture.

An aspect that has not been discussed in previous reviews is the use ofthe broad-spectrum biocide Plant Preservative Mixture (PPM®)(manufactured by Plant Cell Technology Inc., Washington DC. ) in planttissue culture applications. This proprietary mixture has been available to


tissue culture researchers only since late 1997 and may be of benefit in someapplications.

In theory, any microorganism (or its reproductive structures, e. g. spores),that is capable of growing on a plant tissue culture medium or in plant tissuein vitro, is potentially a contaminant. Potential microbial contaminants arethose intimately associated with plant tissues and surfaces, as well as sporesadhering to plant surfaces or present in the laboratory environment. Residentnon-pathogenic bacteria present in the initial explant are a common source ofendogenous contaminants (see ‘endophytic contamination’, next section).However, superficial contaminants may be traced to residual surfacemicroflora on explants that have survived ineffectual surface treatment or, inthe case of clean established cultures, from chance introduction or cross-contamination due to poor aseptic technique. A faulty autoclave orinsufficient autoclaving can be a source of contamination, but this can beidentified from the consistent and even distribution of contaminant fungaland bacterial colonies within the body of the medium rather than as surfacegrowths.

Fungal contamination is visually manifest: a filamentous fungus will carpetthe tissue culture medium with mycelia within days. Bacterial or yeastcontaminants, by contrast, are generally slower growing, developing smalldiscrete pale or coloured colonies. Yeasts can be distinguished from bacteriaby cell size (typically versus for bacteria), as well as bythe larger non-translucent colony size and fermentation odour (mediumfouling).

Cole (1996) gives a practical guide to identification of common fungalcontaminants at the genus level. Fungal contaminants are generally aeriallyor dust-borne (e. g. Alternaria, Aspergillus, Botrytis, Candida (yeast),Cladosporium, Epicoccum, Microsporum, Mucor, Penicillium, Phialophora,Rhizopus, Rhodotorula (yeast), Trichoderma) or associated with soil e. g.Fusarium (George 1993, Leifert et al. 1994, Cole 1996). The spores of some(e.g. Fusarium poae) are specifically carried and spread by dust mites(Leifert and Woodford 1997). An outbreak of mite infestation can bededuced from “signature footprints” of init ial ly discrete mycelial colonies,around the periphery and/or across the surface of the medium (Dodd et al.1992; see also Figure 2).

Over 30 genera of bacteria are known to be associated with plants(Bradbury 1988). In theory, any bacterium that is associated with explant

2.1. External contaminants

2. Source, vectors and types of microbial contaminants found in planttissue culture (PTC)


tissue as an epiphyte, endophyte or pathogen is a potential contaminant inplant tissue culture. Leifert et al. (1994) list in excess of 40 differentbacteria which have been isolated as contaminants in tissue and cell culturesof a wide range of plant species. Overall, Gram-positive and Gram-negativebacteria appear equally common. Bacillus spp. and Pseudomonas spp. arewell represented among the Gram-positive and Gram-negative isolates,respectively.

In a study of contamination pattern in two different commerciallaboratories in England, Leifert et al. (1989) noted a preponderance ofGram-negative bacteria (e. g. Pseudomonas and Enterobacter) when planttissues were indexed in the early stages of micropropagation, but found thatGram-positive ones (e. g. Bacillus, Staphylococcus and Micrococcus) weremore likely to be contaminants of older cultures. This is consistent with thedominance of Gram-negative bacteria in epiphytic bacterial populations inthe natural growing environment. In older cultures, contamination tends tostem from faulty aseptic technique, and Gram-positive bacteria found in


these instances are known to be associated wi th humans (Weller 1997) orlaboratory environments (Leifert et al. 1994).

It may also be possible to infe r the l i k e l y source for the contaminationthrough ident i f ica t ion of the contaminant i tself , at the microorganism andgeneric level (Leifert and Woodford 1997). For instance, the presence ofBacillus spp. is an indication of inefficient sterilisation of media orinstruments. Common grey, black and green moulds (e.g. Botrytis,Aspergillus, Alternaria, and Penicillium) and pink yeasts (Rhodotorula spp.)indicate high laboratory air contaminat ion, suggesting faulty laminar flowcabinets or operator error. However, the appearance of Cladosporium mouldwould suggest i n s u f f i c i e n t protection of the laboratory atmosphere fromcontamination with outside air, since spores of this fungus are very commonin the air outdoors. Positive laboratory air pressure and dual-door entrancessignificantly reduce contaminat ion from Cladosporium.

Plant pathogens are less l ike ly to be introduced into plant tissue cultureif explants are screened for absence of blemishes and disease symptoms or ifpre-treatments (e. g. fungicidal and/or antibiotics) are applied to donor plants.The presence of non- la t en t , pathogenic p lan t viruses, v i ro ids andmycoplasmas are usually predicated by classical symptoms such as leafstreaking, mosaicism, yellowing or plant stunting. However latent virusescan be introduced through infected but symptomless explant tissue.

2.2. Endophytic contaminantsHealthy-looking plants and tissues may be host to non-pathogenic bacterialendophytes - bacteria that are normal ly associated with plants in the in vivoenvironment. I n i t i a l entry may be gained through natural openings (e. g.stomata, lenticels) or wounds (e. g. leaf scars, cortical ruptures of lateralroots), or as a result of tissue damage caused by herbivores and insect pests.Some well-known endophytic bacteria (e.g. Enterobacter asburiae,Pseudomonas fluorescens) are thought to engineer entry into intact root andleaf tissues by producing cel lulase and pectinase (Quadt-Hallmann et al.1997). Distribution of P. fluorescens is confined to intercellular spaces(Hollis 1951, Petit et al. 1968, Quadt-Hal lmann and Kloepper 1996), orsystemically in vascular tissues (Hayward 1974, Gagne et al. 1987, Lamb etal. 1996). Intracellular colonization is generally less common, although ithas been recorded for the underground parts of numerous Australian Orchids(Wilkinson et al. 1989, 1994). Colonization, however, is generally “spatiallylimited and is probably a major factor differentiating endophytic bacteriafrom plant pathogens” (Hal lmann et al. 1997).

Underground vegetative organs are particularly predisposed to internalcolonisation by soil bacteria. Healthy potato tuber, for example, is internallycolonised by non-pathogenic bacteria or ig inat ing from the root zone soil


(Hollis 1951, Sturz 1995). On the basis of bioassays, these bacteria werefound to have promotory, retarding or neutral effects on plant growth (Sturz1995), and it was suggested that these non-pathogenic endophytic bacteriamay play an important role in tuber decay, tuberisation and plant growth.Even without fresh colonization, propagation from vegetative organs ensurestransmission of resident endophytes from mother to clonal progeny plants.

From a micropropagation perspective, endophytes are not eliminated bysurface treatment(s), as they are not exposed to the sterilant during treatment.Although normally introduced into plant tissue cultures throughendogenously contaminated initial explants, they can also appear inestablished, previously clean cultures through the careless use ofinadequately sterilised instruments in the sub-culturing process (Singha et al.1987).

When endophytic contamination is recognised in established plantcultures, it poses a dilemma for the micropropagator. Should a valuableculture be destroyed, or should efforts be expended in cleansing thecontaminated culture (e.g. Taji and Williams 1990). Easy-to-initiate culturesare obviously best disposed of (“when in doubt - out!”) but there is apragmatic view that endophytic microorganisms can be tolerated, even incommercial propagation, if their presence elicits no symptom and the plantsappear to grow and multiply satisfactorily in culture (de Fossard 1990). Onthe other hand, they pose a real financial risk for commercial operationstrading locally or in terna t iona l ly where phytosanitary considerationspreclude the transfer of contaminated cultures. Although the infection maybe benign, reputation is compromised when contamination is discovered byclients before or after plant weaning, or when a whole consignment of invitro cultures has to be returned or destroyed when a single contaminatedculture is discovered during quarantine inspection.

Non-latent bacterial endophytes in surface-treated explants are initiallydetected from a halo or cloudiness around the base of the explant in themedium. When endophytic bacteria are suspected, although no colony orcloudiness is observable, detection is made by incubating tissue macerate ina bacteriological medium or broth. The assay is non-specific but is useful forindexing plant tissue for cultivable bacteria. Bacterial identification requiresbiochemical tests using commercially available diagnostic kits, whichcomplement Gram staining and morphological characterisation. Thedetection of non-bacterial endophytes such as viruses, viroids, mycoplasmasand bacteria-like microorganisms are more involved e.g. ELISA (enzyme-linked immunosorbent assay), serological tests, fatty acid profiling,DNA/RNA fluorochrome staining, etc. (Cassells 1991, George 1993).

Some endophytic bacteria are latent, i. e. producing no visible plantsymptoms or growth in the medium through many subculture cycles. In

314

contrast, fungal and yeast contaminants normally do not remain latent inplant tissue cultures, with the exception of certain obligate fungal pathogens,e.g. Sclerospora sacchari, the fungus causing downy mildew in sugarcane(Leifert et al. 1994). George (1993) attributes the latency of endophyticbacteria to their need to adapt to conditions in vitro; they may not multiplyuntil the host is transferred to a medium more favourable to their growth. Ithas also been suggested that poor growth of latent bacteria may be due toinsufficient release of essential nutrients by the host (Gunson and Spencer-Phillips 1993). Latency ends when the normally latent endophyte becomespathogenic, or when sub-culturing is unduly delayed; tissue death in oldcultures forces endophytic bacteria to survive saprophytically on dyingnecrotic tissues, revealing themselves as isolated colonies on dead tissues.

The physiological effects of bacterial endophytes on in vitro culturedplant tissues appear to depend on the host species, bacteria and strain, andage of the cultures; these can range from severe to mild and non-discernible.For example, potato shoot cultures infected with Clavibacter(Corynebacterium) exhibit leaf tip necrosis, reduced leaf development andthinner stems (Long et al. 1988), and, shoot necrosis and even death wasobserved in apple shoot cul tures infected with a strain of Xanthomonascampestris (Maas et al. 1985). Milder effects include lower plant growthand multiplication rates (Leifert et al. 1989), or reduced rooting (Hanus andRohr 1987). In contrast, latent infection has no discernible adverse effect inRiesling grape; infected cultures grew as well as non-contaminated ones(Reustle et al. 1988).

Whether some endophytic bacteria may confer beneficial effects on thehost in an otherwise sterile in vitro environment remains speculative. It isnow recognised however, that certain bacteria associated with plants in thenatural growing environment can stimulate plant growth indirectly byproducing antibiotics against plant pathogens (antibiosis), by competing withpathogens (antagonism), induced resistance or by enhancing the effect ofuseful microorganisms (synergism) (Hallmann et al. 1997).

The eco-pathogenic behaviour of bacterial endophytes can beanomalous. Bacteria that are normally saprophytic in the epiphytic phasecan be pathogenic in plant tissue cultures (“vitropaths”), and knownpathogens in vivo can become saprophytic in vitro (Herman 1990).Whereas pathogens weaken or kill plants by direct parasitisation, vitropathsreduce growth or kill plants growing in vitro by inducing changes to thegrowth medium, rather than by direct parasitisation of host tissue (Leifertand Waites 1992). It is assumed that the change in pathogenic behaviour isbrought about by a pronounced change in the environmental conditions andthe physiology of the plants. For instance, surface sterilisation may haveremoved natural antagonist ic microflora so that surviving bacteria, which

Microorganisms in Plant Conservation and Biodiversity


were once kept in check, can proliferate in the in vitro environment withoutthe competit ion from other microorganisms, i. e. they can become“vitropathic”. The second factor is nutrient luxuriance of the tissue culturemedium compared with what is available on plant surfaces, especially insugars and organics. Pathogenic bacteria that (in vivo) derive nutrients bykil l ing plant tissues with toxins (or macerating enzymes) may, in a well-nurtured heterotrophic host, switch adaptively to a saprophytic mode.

Endophytic bacteria are, in many respects, intermediate betweensaprophytic bacteria and plant pathogens in their behaviour. This has ledHallmann et al. (1997) to speculate that “they are saprophytes evolvingtoward plant pathogens, or are more highly evolved than plant pathogens (tothe extent that they) conserve protective shelter and nutrient supplies by notkilling their host”.

3. Combating contaminants in vivo and in vitro3.1. In vivo and avoidance strategiesCassells (1991) reports that over 20 species of yeasts, 32 species of bacteriaand 6 species of mycoplasma are found on and in the tissues of somecommon agricultural and or horticultural species. The number and type ofmicroorganisms vary considerably between species, habitats, and seasons,and between individuals according to their age, health and vigour. In thecase of wild species, very little is known of the species of saprophytic and orepiphytic microorganisms associated with them. It is therefore largelyempirical knowledge and observation that guides disinfestation procedureswith wild species. The following strategies represent the basic options opento the tissue culturist in minimising the impact of contaminating organisms.

Avoidance strategies are ult imately dependent on (a) selecting the typeof explant material, (b) selection of the appropriate stage of development and(c) scope for pre-treatment of the starter material in vivo.

3.1.1. Selection of explant material – type of explant

Seed or extracted seed embryos are a good choice if the need for matureclonal material is not crucial. Seeds are generally able to withstand vigoroussterilisation, including sterilisation under vacuum, which usually results inelimination of external contaminants.

With care, embryos can be extracted aseptically from most seedsprovided the surface steril isation procedure is effective. The exteriorsurfaces of some seeds are deeply pitted, striated or hirsute and may haveadhering remnants of f ru i t which can harbour contaminating organisms(particularly with dry seed or seed harvested from the soil). Seeds may haveto be thoroughly cleaned or even scrubbed before surface sterilisation can be

Seed (seedling, seedling tissues, seed embryo)


truly effective. In general, the interior of seeds is naturally aseptic unlessthere is systemic infection, or if seeds are perforated by borers or otherinsects during the early stages of seed development. With some seeds (e.g.native rush (Restionaceae) and sedge (Cyperaceae) species of WesternAustralia), the embryos are located at one end of the seed requiring onlypartial removal or piercing of the testa to facilitate extraction of the embryo(Meney and Dixon 1988). Similarly, with very small-seeded species, asepticembryo extraction presents a technical challenge but this can beaccomplished with adequate practice. Very small or soft seeds may requirebenign sterilisation protocols; hence it may be better not to use vacuumsterilisation for seeds with a thin testa and delicate endosperm.

Shoot tips (and apical meristems)These are the most common explants used to initiate an in vitro culture for awide variety of plant species. Shoot tips are considered to be geneticallystable and where clonal ( i . e. genetic) f ideli ty is required these are thepreferred explants (Dixon and Touchell 1995). New shoots are less likely tobe contaminated with fungal propagules or bacteria and may be free ofinvertebrate predation in early stages of development. Such material is idealfor culture init iat ion as it requires less rigorous surface sterilisation andhence better survival, and a higher percentage of clean explant initials can beobtained compared with older and more heavily contaminated shootmaterial.

The dissection and isolat ion of apical meristems is technicallydemanding; however, apical meristems provide superior explant materialwhich are generally free of exogenous and endogenous microorganisms(particularly viruses).

NodesNodal material may be an alternative where shoots are not available orcannot be used. Nodes are older and therefore potentially morecontaminated than young apical shoots, requiring correspondingly morestringent surface steril isation protocols. However, nodal explants aregenerally more robust and can survive harsher sterilisation treatments,although in woody species oxidation damage and phenolic leakage are stillmajor concerns. Even if external damage occurs, sub-cuticular axillary buds,which generally sustain less damage, are the primary sites of shootregeneration in nodal cultures. After sprouting and vigorous growth, theaxillary shoots can be removed and, if need be, given a precautionarysterilisation treatment before proceeding to induce shoot multiplication. Theoriginal node explant can be discarded as it may harbour latentcontaminants.


Non-apical (stem, leaf, floral, root, bulb scales)(i) Stem tissuesStem culture can be used for in vitro propagation of a number of species(George 1993, 1996). As with apical tissues, the younger the stem material,the lower the incidence of surface and endogenous microbial contamination.Occasionally the inner pi th tissues, which should be largely contaminantfree, can be used providing the epidermis can be effectively sterilised orcarefully excised. However, Leifert et al. (1994) and Bove and Gamier(1998) report that certain types of pathogenic microorganisms have beenlocated in vascular tissues of some species, which adds a certain element ofrisk in using stem explants over apical meristems, for example.(ii) Leaf tissuesLeaves have a large surface area to mass ratio. A thin epidermis is generallyideal for rapid penetration of media supplements, particularly growthregulators that mediate rapid direct adventitious shoot production. Youngshoots are also sites of very active metabolism relative to tissue mass, andwhere many young, highly active cells are available to convert to vegetativeregeneration. Leaves are also easily damaged by surfactants, sterilisingagents and handling. Leaves of many plant taxa accumulate phenoliccompounds to combat predation and disease, which may restrict theirsuitabil i ty in culture in i t i a t i on . Through insect predation, bacteria andviruses may be introduced in the saliva of sucking insects and further entrymay be gained through these wounds by opportunistic fungal and bacterialpathogens. Highly sclerophyllous leaves may also be unsuitable as explantsdue to their thick, waxy epidermis, as well as their relatively high proportionof structural rather than actively metabolising tissues and cells. The use ofleaves as explants is therefore limited to certain plant species, particularlythose with fleshy leaves from which plant regeneration has been successfulin conventional propagation. As with non-leaf tissues, leaf explants shouldbe young, non-predated, and visually disease-free. Bagging of branches (toexclude light but not air) for a short period of time may induce slightetiolation and reduce phenolic content in the shoots and leaves (see below).(iii) Flowers (and all floral parts)Flowers have been used as explant sources in cases where vegetativematerial is in short supply, endogenously contaminated (Tan 1995), or isotherwise unsuitable or too difficult to use (Collins and Dixon 1992; Bunnand Dixon 1996). Unopened flower buds should be aseptic internally,provided there has been no predation, disease or mechanical damage.

3.1.2. Age and stage of development of explant materialSome prior knowledge of the growth habit and life history of the plantspecies to be cultured is useful; these contribute towards sampling of non-


predated explant material in peak physiological condition. Sometimes thechoice may be robust nodal mater ia l if softer exp lan t t issue cannot bedecontaminated success fu l ly due to tissue damage or severe oxidationreaction, e. g. many Eucalyptus species (Le Roux and van Staden 1994).Generally, however, juvenile or new apical growth provides the best materialwi th which to i n i t i a t e aseptic shoot cultures in mature plants, whereasmicroorganisms tend to accumulate in the more mature parts of the plant(George 1993). Shoot material from seedlings or saplings is more likely toretain juveni le characteristics whereas that from mature plants (depending onthe type of material and location on the parent p lan t ) may exhibit adultcharacteristics e. g. precocious flowering. So, while there may be compellingreasons for choosing explant material from mature plants rather than fromseedlings or juveni le plants, there is generally a greater risk of microbialcontamination.

3.1.3. Pre-treatment of explant material in situPreliminary decontamination can be ini t ia ted on the donor plant growing invivo with chemical application or physical treatment.

Fungicides and/or antibioticsA program of spraying of the donor plant with a fungicide(s) may beundertaken several weeks to a few days prior to taking explant material,depending on the severity of fungal contamination, type of fungicide(s) andconcentration(s) used. An t ib io t i c sprays can be applied to plants prior totaking material for tissue culture. George (1993) describes several instanceswhere fungic ides and a n t i b i o t i c s are combined to reduce externalcontaminants of cul t ivated plants prior to sampling for in vitro culture.Whichever anti-microbial compound(s) is used, it should not cause unduestress to the treated plant , or the qual i ty of explant material would otherwisebe compromised. However, widespread use of fungicides, biocides andantibiotics in the natural environment should be used with caution as theimpacts of these compounds in natural ecosystems is not ful ly understood.

Heat treatmentHeat treatment is usual ly applied to plants suspected of harbouring a viral ora systemic bacterial pathogen (George 1993). Many viruses are killed orinactivated at temperatures as low as 30ºC, while others with higher thermaldeath points may require higher temperatures for effective inactivation. Theupper limits of heat treatment are limited by the plant species or cultivartolerance to elevated temperatures for extended periods. Hence a regime ofalternating periods of high and lower temperatures may be beneficial toallow for the plant’s recovery from heat stress. Details on the production ofvirus-free plants through tissue cul ture techniques are reviewed by Horst


(1988) and Walkey (1985), while virus-indexing techniques are described byGeorge (1993).

Irradiation (UV or Gamma-rays)Although used to reduce microbial spoilage of stored fruit and vegetables(Sendra et al. 1996), irradiation requires specialised equipment and facilitiesand highly trained operators. As a result, irradiation is also expensive interms of equipment and safety requirements. Irradiation as a routine methodfor decontamination of plant material is therefore considered impractical.

EtiolationThe strategy of induc ing etiolation in donor plants to obtain lesscontaminated explant material has been successful in a number of cases(Cooper 1987; Murasaki and Tsurushima 1988), even in field-grown plants(Fay et al. 1999). Etiolation is achieved by bagging to exclude light fromends of branches that have been pruned and treated with fungicide to preventmould development. The bagging material should be porous to allowgaseous exchange and prevent excessive humidity (which is conducive tofungi and moulds). The thin-stemmed, fast-growing etiolated shoots are lesslikely to harbour microorganisms; bagging excludes a large number ofairborne spores from landing on the shoots, and insects which may bevectors for plant viruses or bacteria. A further advantage of etiolation maybe a reduction in phenolic compounds in etiolated material (George 1996),which is detrimental to culture initiation.

Growth regulatorsPlant growth regulators can be applied to donor plants to stimulate newshoot growth to provide source material for initiation of tissue cultures.Cytokinin (e. g. k ine t in , 6-benzylaminopurine (BAP)) and/or gibberellic acid(GA) can be sprayed on intact plants, or applied in forcing solutions tostimulate new shoot growth from cuttings (Read and Yang 1987). They canalso be combined with fungicides and/or antibiotic treatment to furtherreduce or prevent microbial contamination. Relatively clean explantmaterial can then be taken prior to re-establishment of surface microflorawhich may result in a reduced need for other sterilisation procedures.

3. 2. In vitroSurface sterilisation treatments for explant material can be applied in anumber of ways i. e. as a l iqu id or (rarely) as a gas and even more rarely asirradiation. The use of gas or irradiation is covered in George (1993) andwill not be dealt with here. In most cases sterilisation is performed withliquid chemical steri lant which may be a single or mixture of generalbiocide(s), fungicides/algicides, or antibiotics. The concentration, durationof treatment, sequential sterilisation, repetition and co-usage of sterilants ishighly variable and there are many factors to consider in arriving at the


optimal sterilising procedure for specific explant material. The effectivenessof a variety of sterilisation protocols employing different chemical sterilantson field-collected nat ive plant material is given in Table 2 and discussedfurther below.

3.2.1. General biocidesGeorge (1993) details the recommended concentrations and exposure timesfor three chemical biocides ( i . e. sodium and calcium hypochlorite, andmercuric chloride) and the species and type of explant material treated.These sterilants are used at relatively low concentrations (typically 0. 1% formercuric chloride 0. 5% for sodium hypochlorite (NaOCl) and 3-5%for calcium hypochlorite ranging from a few minutes of treatmentfor soft shoot material up to 45 minutes for dormant buds and seeds. Severalfactors need to be considered when choosing an appropriate chemicalsterilant, concentration and exposure time. The type of explant availableusually predetermines the sterilisation procedure. New apical shoots fromdisease-free cultivated plants, for example, are soft and relatively microbe-free, requiring only minimal surface sterilisation with a dilute but moderatelytoxic sterilant. At the other extreme, field-collected or older dormantmaterial which is likely to be predated and highly contaminated will requirea more rigorous sterilisation protocol, invariably requiring the use of a moretoxic sterilant. Chlorine compounds should be effective in most instancesbut there is a l imi t to the concentration of NaOCl that can be used onvegetative material before oxida t ive damage becomes problematic.Mercuric chloride by comparison is highly toxic at a relatively lowconcentration. This makes a very effective surface sterilant butdisposal of spent mercury-containing compounds poses occupational healthand environmental hazards. This inevitably constrains the use of tothe most di f f icul t - to-s ter i l i se cases, when other chemical sterilants haveproved ineffective.

The use and efficacy of other surface sterilants are described in theliterature (e. g. hydrogen peroxide, benzalkonium chloride, potassiumpermanganate) but few are reported to be as effective as NaOCl, CaOCl or

A different form of chlorine (sodium dicloroisocyanurate) with a lowphytotoxic effect has been used successfully as a surface sterilant at high orlow concentrations (Parkinson et al. 1996).

Many years of practical experience invested on disinfestation of field-harvested material from a wide range of Western Australian plant species issummarised in Table 2. The data presented illustrates surface sterilisationprotocols for field-collected material of a diverse flora. Accumulatedexperience with local flora is invaluable in the choice of appropriatesterilisation protocols for the explant material. Nearly 70% of the adopted


procedures resulted in a 50% success rate, which must be considered highlysatisfactory for field-collected material. Although it is difficult to makecritical comparisons between varied procedures, appears consistentlyto be the most effective general chemical biocide. Chlorine compounds havealso, in many cases, proved efficacious in yielding viable explants that arevis ibly free of contaminants and these compounds would be preferred.

is recommended only in cases of more severe infestation.

3.2.2. Other in vitro disinfestation techniquesThermosterilisation

Heat treatment of in vitro cultures is usually aimed at virus elimination inexcised shoots and shoot meristems. However it has also been used to ridexplants of other microbial contaminants (George 1993). Heat treatment hasalso recently been combined with salicylic acid treatment (see below).

ASA (acquired resistance)The use of salicylic acid (SA) and its derivative acetyl salicylic acid (ASA)as a spray purportedly enhances plant resistance to pathogen attack (i.e.acquired resistance) (Sticher et al. 1997; Mauchmani and Metraux 1998).SA or ASA treatment of plants in vivo may yield explant material that ismore tolerant of the sterilisation process and be resistant to infection bysurviving contaminating organisms in vitro, but this does not solve theprimary problem of ridding the explant of contaminating microorganisms. Itis possible that SA- or ASA-treated explants may better tolerate repeatedsterilisation before and after establishment in vitro compared with untreatedexplants. ASA treatment confers greater thermotolerance on potatomicroplants (Lopezdelgardo et al. 1998) as part of heat treatment for virusel imination. It is possible that this protocol could be adapted to otherspecies, under in vitro conditions, for management of endogenous viral orbacterial pathogens.

SonicationSonication, i. e. the use of ultrasound, can be applied as a pre-treatment toassist in dislodging contaminants and is generally used in conjunction withchemical sterilants (NaOCl, CaOCl or other sterilant) to give the best result(Herman 1997).

ElectrosterilisationThis is a relatively new method of sterilising plant material for tissue cultureand is achieved by brief exposure of plant material to a low level of electriccurrent and it has been used with some success for the elimination of virusfrom potato (Herman 1997).


3.2.3. AntibioticsAntibiotics are not normally used in plant tissue culture; when they are, theyare used ad hoc to prevent, control, or to eliminate persistent bacterialcontaminat ion. In theory, an effective antibiot ic can be appliedprophylactically or therapeutically in plant tissue culture. However, relianceon antibiotics for prophylaxis, as an alternative to asepsis, is questioned assuch a practice may encourage the development of antibiotic resistance incontaminating bacteria (Falkiner 1997). Exposures of short duration, e.g. inexplant decontamination, appears to be the most appropriate application forantibiotics in plant tissue culture, in instances where surface sterilants aloneare not adequate.

Antibiotics generally permit temporary control (suppression) rather thancomplete eradication of bacterial contaminants in infected cultures. Rarelyis a single antibiotic effective when several contaminating bacteria arepresent. Although sensi t ivi ty of a contaminant isolate(s) can be determinedfrom its antibiogram (growth inhibition of lawn cultures around paper disksimpregnated with different ant ibiot ics) , poor tissue absorption andphytotoxicity often compromise antibiotic efficacy. Endosporous bacteria(e.g. Bacillus) tend to persist even when absorption is systemic, as evidencedby reappearance of the same bacterial contaminant when “cleansed” culturesare transferred to a medium without the antibiotic.

Gentamycin, r i fampic in and cefotaxime are used to treat in vitrocultures of tansy (Tanacetum vulgare L.) infested with several Gramnegative bacteria (Keskitalo et al. 1998) with growth retarding effectsexperienced with both gentamycin and rifampicin, but less so with bothgentamycin and cefotaxime. Some antibiotics may have growth enhancingeffects such as increased root induction with some genotypes of tansy(Keskitalo et al. 1998).

Some endogenous bacteria may have growth enhancing effects, forinstance, some Pseudomonas strains have the capacity to produce IAA(indole acetic acid) which promotes rooting and shoot multiplication inCotoneaster lacteus (Monier et al. 1998), whereas deleterious bacterialstrains produced cyanide. Antibiotics in combination with a broad-spectrumfungicide (e. g. benomyl, miconazole) may be useful for decontaminatingexplants in certain “stubborn” cases. George (1993) and Herman (1996,1997) cite cases of successful disinfestation with antibiotics (e. g. ampicillin,cefotaxime, rifampicin, and tetracycline) applied either sequentially or in amixture.

The widespread resistance to antibiotics and release (accidental orotherwise) of antibiotic resistant organisms into the environment is a causefor concern. In some cases the use of antibiotics or several antibiotics insequence or in unison is the only way to get rid of ubiquitous internal


contaminants in t issue cul tures (Herman 1997). Nevertheless theindiscriminate use of antibiotics is not advisable or warranted. Also, someantibiotics are known to be phytotoxic and need to be used with caution(George 1993; Leifert et al. 1994). A prevailing view is that “antibiotics areno substitute for careful aseptic techniques“ (George 1993), and, “it is poorscience to develop a micropropagation system which relies on the routineincorporation of antibiotics into the culture medium” (Cole 1996).

3.2.4. Plant Preservative Mixture (PPMTM)In 1996, Plant Cell Technology (PCT), an American plant technologycompany in Washington USA, introduced a broad-spectrum proprietarybiocide (‘PPM’) which it claims can prevent or reduce microbialcontamination in plant tissue culture. This biocide contains a mixture of twoisothiazolones, methylchloroisothiazolinone and methylisothiazolinone, twowidely used industrial biocides (Niedz 1998) which may be beneficial for thecontrol of contamination in in vitro cultures.

As the product is relatively new to plant tissue culture applications,much of the technical and scientific information is disseminated throughPCT’s Internet website (PCT 1996 onwards), newsletters or via agents.Information on laboratory experience with PPM is exchanged amongsubscribers to the University of Minnesota’s Plant Tissue Culture Server(PTC 1994 onwards). There has been only one scientific publication onPPM application in plant tissue culture: Niedz (1998) reported on theelimination of contaminating bacteria with PPM “under certain conditionsof low inoculum density in citrus protoplasts, callus culture, shootorganogenesis and seed germination”.

Isothiazolone biocides (PPM) act by removing protein sulphyryl groupsand succinate dehydrogenase, a key respiratory enzyme of bacteria and fungi(Chapman and Diehl 1995). Antibiotics, by contrast, act on microorganismsby inhibiting DNA or protein synthesis, or by altering cell wall or membraneproperties (Russell and Chopra 1990). According to the literature from thePTC website, plant tissues are less sensitive to PPM than microorganismsbecause PPM molecules penetrate bacterial and fungal cells more easily thanplant cells owing to differences in their cell wall composition andcomplexity. Depending on applied concentration and duration of exposure,localised tissue cytotoxicity rather than acute phytotoxicity may be expectedin treated plant tissue. Biocidal efficacy can be improved somewhat byjudicious reduction of salts and organics (sugars and amino acids) in theculture medium to discourage microbial growth.

PCT’s confidence in PPM’s biocidal potency is reflected by itsassurance that “PPM-containing culture media can be dispensed outside thelaminar flow hood, exposed to ambient air” (PCT 1994 onwards) and that




membrane-filtering or autoclaving is superfluous for heat-sensitive liquidmedia containing PPM. However, PCT cautions that PPM is less effectivewhen the explant has a high density of bacteria or fungal spores (i. e. a lowratio of PPM molecules to contaminant microbial cell). A more tangibleproof of PPM’s potency is, ironically, not from treated in vitro cultures butin in vivo situations such as direct seed germination on non-sterile filterpaper, where spore germination and fungal growth is clearly suppressed(Figure 3).

Although many aerobic bacteria, mould and yeast fungi have beenreported to be sensitive to PPM (Table 3), its effectiveness against anaerobicbacteria is unknown. The latter may not be relevant, as the anaerobiccondition is not typical of the in vitro plant or cell culture environment.

The question also arises of whether PPM can become ineffective overtime due to mutations in the bacteria and fungi. PCT maintains that “this isunlikely because PPM targets multiple enzymes” (PCT 1996 onwards). Theprognosis may be disregarding the occurrence of naturally resistantmicrobes. A species of Phoma (Figure 4) and several bacteria that appear tobe insensitive to PPM have been isolated from explants of kangaroo paws(Anigozanthos spp. ) (B. Tan unpubl. ).

The second constraint to PPM’s universal applicability is the sensitivityof some plant species. In Agrobacterium-mediated transformation, forexample, exposure to PPM after co-cultivation of host (Ilex and Capsicum)and the bacterium vector revealed detrimental effects on host explant tissue(PTC 1994 onwards, Hu 30/4/98). Freshly prepared protoplasts, forexample, are extremely sensitive to very low concentrations of mediasupplements (Niedz 1998). However, many plant species appear not to be


adversely affected by PPM at typical recommended concentrations (0. 5-2. 0ml/L); these include Nicotiana tabacum, Arabidopsis thaliana, Betavulgaris, Epidendrum spp., Eucalyptus grandis, Nepenthes spp., sweetpepper, Citrus spp. (PTC 1994 onwards).

Although procedures for eliminating endophytic bacteria with PPMhave been suggested, unpublished results have been mixed or equivocal(PTC 1994 onwards). In kangaroo paws (Anigozanthos spp. ), for instance, itwas noted that two four-weekly continuous subcultures on a mediumcontaining PPM (5 ml/L) failed to eradicate certain endogenous bacteriaalthough treated shoots were visibly healthier after they were returned toroutine medium (B. Tan unpubl. ). For example, leaf tip necrosis haddisappeared and multiplication rate had improved, but leaf tissues werepositive when indexed on nutrient agar. It is interpreted that PPM hassignificantly reduced but not eliminated the endophyte population.

The sterility of explant material after surface sterilisation is critical tosuccessful culture initiation. Residual contamination may be detectablevisually or indirectly through indexing. Index-positive or visuallycontaminated explants are culled at this stage. The prevention of re-infection (including mite infestation) and maintenance of “clean” cultures atall later stages of propagation should be possible through good hygiene andsound aseptic techniques; in special cases, culture sterility can be maintainedby routine incorporation of a prophylactic biocide (such as PPM) in themedium.



3.2.5. Recovery of contaminated culturesIn some cases aseptic material can be rescued from contaminated explants.If the contaminant is external, i. e. has not penetrated into the tissues of theexplant, then disinfestation of at least part of the explant is possible,generally the tissues furthest from the site of contamination. A usefulmethod for “rescuing” material that is contaminated is to induce etiolation ofapices, axillary shoots or buds so that the sprouting shoot grows rapidlyaway from the contaminated basal node or shoot stems. This etiolatedgrowth can then be excised (and briefly sterilised as a precaution if desired)and transferred to fresh medium. This method is relatively simple to apply;however, some species do not respond well to etiolation by dark incubation,so there are some limitations (see George 1993). Another method is tocombine the above techniques with the use of media supplemented withbiocides, fungicides and/or antibiotics or substances like PPM, however,with the removal of the additives the contaminants may arise again. Theefficacy of such methods is not well reported. Generally it is better todiscard all contaminated cultures but there are some instances, i. e. in the caseof rare or extremely valuable clonal material, where attempting to recovercontaminated explants is worthwhile.

3. 2. 6. Mite infestationMite infestation is extremely serious in terms of contamination of previouslysterile cultures. Losses due to mite infestation can be devastating if notdiscovered and appropriate action taken immediately, especially in largecommercial operations. Mites can only crawl or be artificially introduced toculture collections, so all means of preventing mites from entering theculture room in the first place and restricting mites crawling from one site toanother in the culture room once introduced, is advantageous. Regularcleaning (with a mild disinfectant) of floors is very helpful, as is regularcleaning (with mild disinfectant) of culture racks and shelves.

Operators need to ensure all culture containers entering the cultureroom are clean. Storing media in dusty conditions for long periods of timewill risk mite contamination. All contaminated cultures need to be promptlyremoved from culture rooms and if mites are confirmed or suspected, allassociated and adjacent cultures should be quarantined pendingdisinfestation of the culture shelving and the suspect cultures. Ifcontamination via mites is detected early enough, simple transfer of explantspromptly to fresh medium may be sufficient to rescue the explants andmaintain asepsis, but a brief disinfestation with a low concentration ofCaOCl or NaOCl (and rinsed with sterile water) is recommended as aprecaution.


Prevention of mite infestations is very difficult if strict hygienestandards cannot be guaranteed. Smith (1967) reports the use of theacaracide Kelthane® (active ingredient di-(p-chlorophenyl) trichloromethyl-carbonol) on cotton wool plugs of test tubes, to ki l l entering Tarsonemidmites, mite eggs and hatching larvae. However the particular contaminantsintroduced by the mites s t i l l posed a problem, therefore the effectiveness ofsuch chemicals in preventing contaminants from actually entering andproliferating in culture containers must be considered questionable.Nevertheless acaracides could be useful in restricting the spread of mitesunder conditions of severe infestation.

Mites tend to infest the culture containers which offer the leastresistance to their entry, which means that Petri dishes, which need to besealed with plastic films, are generally more likely to acquire miteinfestations than more tightly sealed jars and tubes. Mites also appear tohave definite media preferences, as plates of potato dextrose agar (PDA)even if sealed with thermoplastic film, still become infested with mites(almost exclusively), in the presence of other plates containing MS(Murashige and Skoog 1962) derived media. It is therefore important tokeep plated media separate from other cultures as plates can quickly becomea breeding ground for mites.

Mite infestations can be managed by strict hygiene, good aseptictechniques, regular spraying of the laboratory and culture room with anappropriate acaracide, separate incubation of plated media (particularlynutrient agars which mites seem to prefer) and constant vigilance to dueprocess.

4. ConclusionsEffective disinfestation of plant material for in vitro culture is reliant onmany mitigating factors, i. e. type, age, health and predation history ofexplants, seasonal f luc tua t ions of surface microorganisms, the type,concentration, toxicity, specificity and duration of sterilant application, andthe species and life history of the donor plant. Examples of practicalexperience with a range of species and sterilisation procedures suggests thateffective surface disinfestation of a wide range of field collected material ispossible with accumulated knowledge of local flora to draw on. It wouldappear both from the literature and based on practical experience thatcommon biocides containing sodium or calcium hypochlorite are effective inmost circumstances for disinfestation of shoot material. More toxicsterilants such as mercuric chloride are highly effective in many cases butnot environmentally friendly and should be used sparingly. Antibiotics arevery useful in some cases but not recommended unless absolutely necessary


owing to hazards of uncontrolled selection for antibiotic resistance incommon bacterial and fungal contaminants.

Newer sterilising agents such as PPM are still being evaluated and mayoffer low-cost, easy-to-use preventive measures against accidental infectionof sterile cultures. Other sterilising methods have been reported (e. g.sonica t ion , e l e c t r o s t e r i l i s a t i o n ) bu t these appear to have been usedinfrequently hence a comprehensive assessment of their efficacy is difficult.Pre-sterilisation treatments such as thermal treatment appear to be useful forl imi t ing viral spread prior to shoot tip culture for some species. Theinduction of acquired systemic resistance and stress tolerance by compoundssuch as SA or ASA is an interesting development that may assist with thesuccessful ini t iat ion of pathogen-free cultures or maintenance of symptom-free plant propagation material. Maintenance of sterile cultures also relieson many factors, the most important of which are: ensuring good basicaseptic practices at al l times in the laboratory and culture room – includingpreventative measures such as basic cleanliness and spraying regimes formite control, coupled with vigilance for diagnosing contamination as early aspossible, and taking immediate steps to limit the spread.

In vitro microbial contaminat ion and control is one of the mostimportant phenomena confronting the tissue culturist. With microorganismsnow known to inhabi t the inner tissues and intercellular spaces of a largenumber of plant species, the concept of “sterile culture” appears to beoverstated. Leifert et al. (1994) suggests the terms ‘index negative’ or ‘freeof detectable contaminants’ if cultures have been indexed for contaminants.

Although the number and sens i t iv i ty of methods for screening planttissue cultures for exogenous and endogenous microorganisms is presentlyconsiderable and s t i l l expanding, it is the larger laboratories indulging inextensive exporting of tissue cultured plants that wil l be using such methodson a regular basis, particularly those which require specialist skills. Howwidespread the practice of disease indexing is in the wider world of tissueculture work is more di f f icu l t to quantify. The likelihood is that very littlecomprehensive in vitro disease indexing is practiced. The reasons for thisprobably range from the most common, i. e. the tests are too expensive to use(even if facilities are available) for the average tissue culture laboratory, toocomplicated, or require specialised equipment and technical expertise notreadily available; to the fact that many operators just do not see a need for it,especially if their product is used exclusively within the country (or State) oforigin, or is for research purposes only.

Regardless of the various arguments for or against regular screening/indexing for microbial contaminants and endpoint usage of the product, itcan be argued that unless achievable, practical and uniform standards(national and international) are formulated and that test procedures are truly


affordable for all operators, there wil l be a reluctance to embrace in vitrodisease indexing. In some cases, e. g. domestic markets, less stringentstandards may be acceptable, but exporting tissue cultured plants is verydifferent. Quarantine laws for most countries now encompass requirementsfor tissue cultured plant material to be free of specified disease-causingmicroorganisms, and the likelihood is that these laws will become morestringent in the future as countries try to block all avenues for the accidentalintroduction of exotic plant pathogens. The challenge lies ahead for all planttissue culturists to be aware of the limitat ions of their craft in producing trulypathogen-free material and keep abreast of new methods for in vitro diseaseindexing, part icularly if re lying on exporting and importing tissue culturedplants.

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Chapter 13

PHYTOSANITARY CONSIDERATIONS INSPECIES RECOVERY PROGRAMS

Giles E.St.J. Hardy

School of Biological Sciences and Biotechnology, Murdoch University, Perth 6150, WesternAustralia.

K. Sivasithamparam

Soil Science and Plant Nut r i t ion , Faculty of Natural and Agricultural Sciences, TheUniversity of Western Austral ia , Crawley 6009, Western Australia.

1. IntroductionPlant species recovery programs are enacted when naturally occurringdiversity of a species or population falls close to or below what is consideredto be a sufficient size for the species to continue to exist without humanintervention. The purpose of these plans is to ensure the long-term survivalof the taxon concerned, and where possible, to re-establish self-sustainingpopulations in their natural habitat. In such an endeavour to increase plantnumbers, there exists a risk that seed, soil, machinery and plant material usedin the introduction of plants to native habitats will include the introduction ofphytopathogens capable of destroying the very population that we areattempting to increase. Alternatively, changes to environmental conditionscreated by the activity of the program may favour disease development bynative or naturalised pathogens. For these reasons, phytosanitation (literally,plant-health-process) procedures are employed to minimise the threat ofpathogens already at a site and, more importantly, to prevent infected orinfested material entering a site.

Development of diseases wi th in plant populations is a natural processand an inherent part of dynamics within ecosystems (Ingram, this volume).Under natural conditions within an area, interactions between the pathogensand host plants are mediated by environmental conditions including soil,moisture, temperature, other organisms and commonly occurring


disturbances (such as fire in the Australian biota) (Gi l l et al. 1981; Palti1981). Hazardous levels of disease capable of seriously reducing plantnumbers can arise when the balance w i th in a system is modified byintroducing hosts or changing environmental factors within the range of anindigenous (or na tu ra l i sed ) pathogen or when an exotic pathogen isintroduced into a new area (Chesson 2000; Burgess and Wingfield, thisvolume).

This chapter primarily focuses on the interaction of phytopathogens andhosts in an environment and various methods that can be employed to helpprevent the development of diseases in species recovery programs. Thereare three main scenarios to be considered:

Firstly, a plant host species being introduced into the present distributionof a phytopathogen. For example, the rust disease of eucalypts newlyintroduced into South America (see Burgess and Wingfield, thisvolume). This rust disease does not occur in the region of origin of theeucalypts.Secondly, the in t roduc t ion of an exotic phytopathogen into anenvironment. The two most notorious examples of this being thechestnut blight caused by Cryphonectria parasitica and the destructionof susceptible native f lo ra of south Western Australia by Phytophthoracinnamomi (Cook and Baker 1983). The destruction of west Europeanelms by Ophiostoma novo-ulmi (Brasier 1986) is yet another significantevent in which the disease onslaught resulted in the removal of adominant tree species allowing the emergence and replacement by lesscompetitive species.Thirdly, a change in environmental conditions (often caused by humanactivity) that brings about a disease outbreak in a native plant populationby a native phytopathogen. For example, the Cryptodiaporthe canker ofBanksia coccinia both of which are native to the south west of WesternAustralia (Shearer 1994). This disease is currently threatening all naturalstands of the host.Although the belated quarantine regulations enacted by authorities

world-wide cannot restrain the localised spread of major pathogenspreviously introduced, regulations wi l l however be most useful in reducingthe incidence of new introductions. Equally important are the monitoringand/or avoidance of disturbances that lead to serious disease outbreaks innatural vegetation.

Human endeavours have caused major changes to environments aroundthe world. These include intensive and extensive farming, forestry, mining,and clearing for urbanisat ion and associated transport networks. Thisconstant demand for space has resulted in many detrimental changes to thelandscape. These include sa l in i ty , rising water tables, eutrophication, and

Phytosanitary considerations in species recovery programs 339

pollution. These activities have put many plant species under threat of totalor local extinction both directly and indirectly. Consequently, there is a needfor species recovery programs to ensure the long-term survival of plantspecies that are in decline. In developed areas, it is possible to argue thatany remaining natural bush land, heath land, forests or woodlands should betreated and managed as a recovery program.

Improper management could not only affect the success of suchprograms but also contribute to the destruction of flora outside the managedareas. Consequently, phytosanitat ion should be a major factor in allmanagement activities associated with the long-term maintenance of theselandscapes.

Large-scale conservation programs are not only difficult to manage butare also l ikely to be plagued by limitation of funds. A species recoveryprogram however could be considered at a much more localised level. Theseinclude the (i) protection of a plant or plants in a natural ecosystem that areunder threat from an introduced pathogen or pest, (i i) introduction of plantsby seed or vegetative material into denuded areas such as rehabilitated minesites, ex-farm sites, areas impacted by salinity or water-logging or (i i i )production of plants in production nurseries. Whatever the reason, it isimportant to consider the role of plant disease management to ensureeffective and sustainable recovery programs. Plant diseases must bemanaged to ensure that they do not spread from pathogen infested into non-infested areas. In addition, the ecosystem being managed must be treated insuch a way that environmental factors such as receding or rising watertables, sal ini ty or fire do not predispose plants in that ecosystem to attack byinfectious agents.

Plant diseases pose great threats to plant conservation. In manyins t i tu t ions and enterprises dedicated to the conservation of rare andendangered flora, diseases threaten not only in the propagation of plantmaterial in the laboratory and glasshouse but also in the field introduction ofthe cultured plantlets. Thus methods have to be developed to minimisedisease hazards both during and after recovery of such threatened flora.Importance of these strategies is relevant to horticulture and forestry e.g.propagation of rare p lan t species wi th potential for large-scalecommercialisation of the material for floriculture and tree plantations. Suchrecovery could also be relevant to propagation of native species for therehabilitation of disturbed land such as mined sites.

1.1. Leaf and stem diseasesLeaf and stem diseases of plants cause considerable damage to herbaceousand woody plants. In comparison to agricultural and horticultural cropsrelatively little is known of the diseases of rare and endangered plants. For


instance, the examinat ion of herbarium material of Western Australianorchids revealed the existence of several unrecorded rust diseases ofAustralian orchids, some of which affected certain endangered orchid taxa(Nichol et al. 1988). More recent monographs (e.g. on fungi recorded onEucalyptus spp., see Crous 1998) should enthuse future studies on diseasesof such rare taxa. Recent studies also indicate the existence of severalcanker fungi which have a significant latent (endophytic) phase in woodyplants which could make its diagnosis d i f f icul t at quarantine points worldwide (see Burgess and Wingfield, this volume). These examples indicatethat the lack of accurate disease records from countries of origin and theexistence of asymptomatic infections in plant materials imported for speciesrehabil i tat ion purposes may become sources of invasions by exoticpathogens with undetermined host ranges.

1.2. Historical disastersTwo pathogens that have caused considerable damage to native flora ofWestern Australia are Phytophthora cinnamomi and Cryptodiaporthesemiperda. P. cinnamomi was apparently introduced with horticultural plants(Zentmyer 1980). Phytophthora species in general thrive under conditionsthat prevail in production nurseries (Hardy and Sivasithamparam 1988). Theinfected plants and/or infested soil could be sources of spread of the fungiwhich can then be distributed to a wide range of geographical regions(Shearer and Tippett 1989). The introduction of P. cinnamomi has causedlarge scale destruction of a wide range of susceptible flora as the nativespecies were not naturally selected for resistance to this exotic pathogen.

C. semiperda on the other hand appears to be endemic to WesternAustralia causing major destruction only of Banksia coccinia in naturalstands in the lower South West of the state (Shearer 1994). As it does notappear to have a wide host range, the origin of C. semiperda is not clear, norwhy the epidemic occurred only recently. Since this pathogen spreads byrain splash and wind, it is diff icul t to control. There is some suggestion thatthe use of fire could be used to reduce or eradicate the inoculum potential ofdiseased stands as the inoculum forms in dead branches of the host. This inturn would reduce spread between infested and non-infested stands andpossibly in burnt stands wi th seedlings or resprouting plants (Shearer 1994).

1.3. Factors affecting species recovery – disease and disturbance1.3.1. The disease triangleIn order to consider the development of a disease outbreak in an ecosystemand its subsequent control, it is useful to consider the potential impact ofdisturbance on the role of environment in the plant disease triangle (Figure1). All major disease outbreaks result from an interaction of three factors,


(1) the presence of susceptible hosts, (2) a pathogen that is capable ofinfecting the host, (3) environmental conditions that favour infection andsubsequent reproduction and spread of the pathogen (Agrios 1978). Thedisease is invariably most severe when there is inadequate time for the hostto respond to the pathogen while sufficient for the pathogen to reproduce.

Major disease outbreaks result if all three factors are favourable fordisease development, such as an abundance of susceptible hosts, thepresence of a virulent, aggressive pathogen and environmental conditionsthat favour the rapid multiplication, spread and invasion of the host plant bythe pathogen. Consequently, a disease can often be managed by changingthe dynamics of one or all of these factors. In order to manipulate thetriangle to manage a disease, it is critical that the biology, ecology andpathology of the pathogen and how it interacts with its hosts and theenvironment are well understood (van der Plank 1982). Without this basicand fundamental knowledge, management or control of the disease cannot beundertaken effectively, whether in natural ecosystems or in rehabilitatedsites.

1.3.2. Susceptible hostsSusceptible plants are those that provide a food source for the pathogen. Inrecovery programs, plants are often grown in large numbers in a limitedspace in plant production nurseries. Some plant species are more susceptibleto disease than others. Plants can also vary in their susceptibility to apathogen according to their age and physiological status. For example, newgrowth may be more susceptible to a pathogen than older growth. In a


species recovery program, it is therefore useful to determine what pathogensthe plants are susceptible to at particular growth phases and whether they arel ike ly to come in contact w i th these pathogens in the nursery or onceestablished in the field.

1.3.3. Capable pathogensPathogens are l iv ing organisms that cause disease. A pathogen must bepresent for disease to occur. The quantity of the pathogen and its ability tocause disease (virulence) directly affects the occurrence of a disease and itsseverity. Some pathogens such as Botrytis cinerea (grey mould) orPhytophthora cinnamomi (root and collar rot pathogen) can cause disease inmany different k inds of p lan ts (Cook and Baker 1983; Ristaino andGumpertz 2000). Other pathogens such as powdery mildews tend to be hostspecific (Agrios 1978). Once present, pathogens have to be able to enter thehost, reproduce, and disseminate their infective propagules to other plants.Some pathogens are spread in water splash while others produce spores thatare wind-borne (Agrios 1978). Of note is that some pathogens can be spreadduring propagation in or on plant parts during nursery operations (Baker1962; Cook and Baker 1983).

1.3.4. Favourable environmentThe environment is made up of all the factors and conditions that affect thegrowth and development of l iv ing organisms including the host plants andthe pathogens. Relevant factors include l ight , temperature, nutri t ion,moisture and physical and chemical characteristics of the soil or substrate inwhich the plant is growing. Other factors include insects, animals and themanagement activities of people. All of these factors influence both thepathogen and the host plant and how they interact with each other.However, where a host has no resistance to a particular pathogen, a widerrange of environmental conditions are conducive to the disease than whenthe host has some level of resistance (van der Plank 1982).

Temperature influences the growth and development of both the hostplant and the pathogen. Some pathogens are most pathogenic during coolweather, while others many be active at high temperatures. Many pathogensdevelop dur ing periods conducive for disease when temperatures aremoderate, the occurrence of ra infa l l is frequent and plant growth is rapid(Agrios 1978).

Moisture in the form of humid i ty , rain or irrigation (in the case ofcontainer grown nursery plants) is critical in the development of mostinfectious diseases. There is an optimum amount of water required forhealthy plant development. Increasing or reducing the amount of water


available to the plants tends to predispose them to disease (Handreck andBlack 1989).

1.3.5. Sufficient timeDisease development can be strongly influenced by time. For example, thelonger plants remain in containers in a nursery, the more likely that allconditions required for disease development wi l l occur simultaneously. Allpathogens require time to infect and develop in a host to a stage that allows itto spread. The longer conditions remain favourable for the pathogen (fungalspread us generally seasonal), the more severe will be the disease resultingfrom the progressive build-up of inoculum (Baker 1962).

1.4. DisturbanceDisturbances are processes that change an ecosystem beyond thoseconditions considered normal. For the purposes of this chapter we willconsider those disturbances that tend to leave the ecosystem permanentlychanged.

Disturbances are discrete or gradual processes that change ecosystemcharacteristics beyond what is normal wi th in a human timeframe.Disturbances are commonly classified as natural or anthropogenic althoughthis line can be quite blurred as in the case of forest fires (humanintervention in burning some areas and preventing fires in other areas areboth disturbances). Both natural and anthropogenic disturbances occur on avariety of scales with the overall impact of the disturbance including a timecomponent and whether the disturbed ecosystem will ever fully recover fromthe process. All disturbances change at least one of the three factorsconsidered above in the disease triangle, and thus affect all three factors asthey are inter-linked. Some of the most profound changes to ecosystems thathave occurred wi th in a human timeframe have been due to the introductionof new plant or pathogen species (see Burgess and Wingfield, this volume).

Natural disturbances (which by default illogically assumes humans arenot part of all ecosystems) are those that appear to not be directly attributableto human activity. These occur on a wide range of scales from smallindividual tree falls in a forest to volcanic destruction, with fire (common inAustralia) being somewhere in between. In some cases, ecosystems haveadapted to a relatively frequently occurring natural disturbance (within atimeframe long enough for natural selection to have occurred). An exampleof such an adaptation is the adaptation of flora of mediterraneanenvironments to periodic fire events (Gill et al. 1981). In extreme cases,preventing these disturbances w i l l change the ecosystem beyond what isnormal and can therefore be considered a disturbance in itself! A goodexample of this is the natural / aboriginal fire regime in some Australian


ecosystems, where seed germination requires fires, without which thespecies composition shifts (Bell et al. 1984). As diseases are a natural partof ecosystems, it is possible for major disease outbreaks of naturallyoccurring diseases on native plants to result from natural disturbance.

Anthropogenic disturbances include obvious destructive disturbancessuch as logging, clearing, pollut ion, rising water tables and the like. In asimilar manner to natural disturbances, anthropogenic disturbances occur ona variety of scales, i nc lud ing bush-walking, bee keeping and wildflowerpicking. What is not so obvious in this is that while logging appears moredestructive, in the longer term and with careful management the forest canpotentially regenerate. Where as while hiking appears to be a lower scaledisturbance, it is possible that the introduction of a disease spread on hikingboots can be more destructive in the long term. This is not to say thatlogging is good and h ik ing bad, but rather as an example of the innocuousnature of plant pathogens in a wider vision of disturbance and conservation.It also points out how critical the need for phytosanitation is in preventinglarge scale, permanent disturbances in an environment.

1.5. Species conservation and disturbanceMining operations often cause large shifts in the composition of plantspecies native to the site. This may be an outcome of changes in theenvironment but also in the flora and fauna associated with the environmentprior to the disturbance. Disturbance can shift interactions of host, pathogen,environment or all three. Hutton et al. (1997) found that Western Australianmembers of Ericaceae (Epacridaceae) took about twelve years to re-establishby natural means in the soil environment following mining operations. Thisdelay in reestablishment is considered to be due to the time taken for themycorrhizal associates to re-colonise the site. In Western Australian siteswhere Eucalyptus marginata and associated flora are severely affected byPhytophthora cinnamomi, affected areas are often dominated by rushes andsedges (Websdane et al. 1994) especially where the death of large treesresults in the opening up of the canopy which can further encourage theactivities of the pathogen. In Victoria, Australia, the death of the overstoreyspecies results in the exposure of ground infested by P. cinnamomi tosunlight which render the soil warm and conducive for the saprophyticactivities of the pathogen (Weste and Monks 1987). In either case,ecosystems as a uni t can be remarkably resilient in response to disturbances.This in itself can be a problem as a decline in host numbers can be balancedby an increase in another species tolerant of the changes.

Ecosystem disturbance can be the result of either direct human activityor natural processes. Human instituted disturbances to an ecosystem may beminimal (e.g. bush-walking, bee keeping and wildflower picking) or may be

345Phytosanitary considerations in species recovery programs

considerable such as those resulting from broad-scale agriculture, selectivelogging or clear f e l l i ng of timber, or mining. The effect of such localiseddisturbances can be m i n i m i s e d by developing management plans thatquarantines the affected areas (Shearer and Tippett 1989) and the reducedneed for bu i ld ing roads in and around national parks and reserves. Whateverthe level and source of disturbance, the dynamics of the ecosystem willchange as a result of the disturbance. Disturbances at a minor scale can attimes lead to a major catastrophe. For example, as mentioned above, bushwalkers in the montane vegetation of the Stirling Ranges National Park inthe southwest of Western A u s t r a l i a probably transported the soil-bornePhytophthora cinnamomi on their boots into the upper reaches of the Park.Consequently, over 70% of the park is now infested by the pathogen. Thishas had a profound and devastating affect on the many susceptible plantfamilies such as the Proteaceae, Ericaceae (Epacridaceae), Xanthorrhoeaceaeand others. There is now a change in the flora in this park from woodyperennials to rushes and sedges (Wills 1993). The building of firebreaks andthe use of contaminated water for fire control can also inadvertently spreadP. cinnamoini through vegetation in this region. Stands of many susceptibleplant species such as Banksia brownii that are endemic to the region faceextinction as al l known populations or individuals are only known to occurin infested areas (S. Barrett, pers. comm.). The conservation of such standsrepresents a substantial challenge in recovery programs.

Again, using P. cinnamomi as an example, the erection of a roadthrough the Fitzgerald River National Park, one of the most biodiverseregions in the lower south-west of Western Australia using heavy equipmentis known to have resulted in the contamination with P. cinnamomi into anotherwise disease-free area (Wi l l s 1993). The pathogen is spreading slowlybut passively along this road and into the native vegetation that consists ofmany susceptible plant species. The pathogen can move by root to rootcontacts, by the movement of animals or al ternatively passively with watermovement for very long distances (Shearer and Tippett 1989). Therefore,what may appear ini t ia l ly as a small disturbance with potentially little impacthas the potential to result in major structural changes to a plant community.The need for recovery programs and appropriate phytosanitary practices inthis situation is considerable. Currently, there are no completely effectivetreatments that can stop this spread, and in the long-term large areas of thepark w i l l be impacted. This situation again poses considerable challenge toland managers who wish to conserve those species being impacted directlyand indirectly by the pathogen. In such situations when a pathogen is firstdetected and while the extent of its impact is minimal it may be necessary toconsider extreme measures in attempts to eradicate the pathogen. Forexample, is it feasible to remove and burn all plants in the affected area and


in an extensive buffer zone surrounding the infested area? Could the baresoil possibly be fumigated, and/or solarised, and/or treated with chemicals orbio-fumigants, or kept bare for a number of years? Such questions (probablynever previously addressed) need to be considered as an ini t ial ly extrememeasure over a relat ively small area. Though costly and risky, such astrategy could help the eradication of the pathogen. By making no attempt atall, we would al low the pathogen to eventua l ly move passively over a largeregion and have considerable effect on p lant species, especially those thatare endemic to an area and susceptible to the pathogen.

Although a d is turbance may i n i t i a l l y appear min ima l , i t might underoptimum env i ronmenta l cond i t i ons in the presence of an introduced orendemic plant pathogen result in profound changes to the native plantcommunities. In undisturbed natural ecosystems, there tends to be stabilityin the re la t ionship between the resident pathogens, non-pathogenicmicroorganisms associated with plant surfaces (Andrews and Harris 2000)and their plant hosts and therefore major plant disease outbreaks are rare (seeIngram, this volume). It is not until activities such as logging, mining,intensive agricul ture, or other dis turbances to the environment that diseaserelated problems become ev iden t . The need for a species recovery programis usually the result of one or more of the above.

Development and spread of new strains – an example of humandisturbance creating new disease or severe disease outbreaks

2.

The movement of infested soil or plant material whether knowingly orinadvertently can result in the development of new and virulent species orhybrids. It is d i f f i cu l t to monitor entry of exotic pathogens that may bebought in passively in soil or in asymptomatic plant hosts. Entry of novelstrains of extensive pathogens can also cause serious problems. Newcombinat ions can develop that may create hitherto unrecorded virulencespectrum in common pathogens (see also Burgess & Wingf ie ld , th i svolume). A good example of th is is the alder hybrid Phytophthora whichhas been shown to be comprised of a range of heteroploid species hybrids(Brasier et al. 1999). A common standard hybrid occurs across most ofEurope (Brasier 2000). Evidence based on ITS profiles indicates that theDNA signatures of these isolates are of more than one species, and the likelyparents are P. cambivora and P. fragariae (Brasier 2000). These virulentand aggressive hybrids are capable of attacking and causing disease in theriparian Alder (Alnus glutinosa) and the horticultural and woodland alders,i nc lud ing A. incana and A. cordata in England, Scotland and Europe(Brasier 2000). This pathogen can move rapidly in waterways and willpotential ly have a major impact even on the integri ty of rivers and streamsdue to the loss of the r iparian Alder that holds the river banks together by an


extensive root system. The current loss of Alder along UK rivers isestimated to be c. 2% per annum (Gibbs et al. 1999).

Cryphonectria parasitica, the causal agent of Chestnut blight in theUSA is another interesting example where a strain avirulent on an Asianchestnut species was introduced inadvertently on plant material into the NewYork Botanic Gardens. It spread rapidly along the eastern states of the USdestroying much of the native species of chestnuts (Anagnostakis 1982).

Current molecular methods help in the determination of straincharacteristics of pathogens from various geographical regions of the world.Despite these advances it is likely that massive world-wide movements ofinfected plant material prior to the enforcement of quarantine regulations,especially in the and centuries, would make it difficult to trace theorigin and genetic development of strains of cosmopolitan fungi.

3. Components of species recovery programsRecovery programs require careful planning and cover monitoring andimplementation of a number of steps. Field sites in the program should besecure and protected by fencing or by the choice of an isolated locality. Thesites should be free of other growth limiting factors such as salinity, andhave secure tenure. It should be within a healthy ecosystem withoutsignificant threats from weeds and be large enough to sustain a stable geneticcommunity. These sites could be located either in undisturbed or disturbedlocations. If the recovery in the ini t ial test sites appear to be successful,further propagation and introduction of promising provenances could becarried out.

The aim of these recovery programs should be clear. For instance,recreating a niche is not only expensive, but can be beset by practicalproblems related to environments. This can be particularly challenging inthe rehabilitation of mined sites. Ecological restoration of the flora may beadequate if the level of diversity and sustainability of the ecosystem ismaintained. Therefore, in the choice of planting materials, considerationneeds to be given to maximising the genetic diversity and the plants shouldbe appropriate ecotypes or provenances tolerant of resident pathogens andfree of disease themselves. Following planting out, there is a need formonitoring to follow performances of the various components of the flora.This would help in developing strategies to expand the area in the programor to find alternative ones that may contain components of the introductionsthat failed initially.

4. Management of diseases in species recovery programsManagement practices for minimising disease hazards require surveillanceand application of treatment procedures from the early stages of propagation


in the nursery to the stage where the plants continue to be protected in thefield through to reproductive maturity. Pathogens can be introduced to anatural environment in a variety of ways. At production phase, these includepathogens carried in seed or other propagation material, through entry intonurseries of infested container grown plants, through movement of infestedsoil, sand, infested bark or wood wastes used as potting mixes for mulches,infested water from dams or other water bodies, and other inadvertent humanactivities where suitable hygiene or quarantine procedures break down(Sivasithamparam and Goss 1981).

The majority of nursery diseases are caused by fungi, including speciesof Alternaria, Colletotrichum, Cylindrocladium, Fusarium, Mycosphaerella,Pythium, Phytophthora, Rhizoctonia and Botrytis cinerea. These diseasescan occur any time from germination (damping-off) through to thehardening-off stage, immediately prior to their dispatch for planting out.

Prophylactic sprays can be expensive and sometimes ineffective unlessthe diseases and the l ife cycle of specific pathogens are understood.Protecting large trees can also make effective application of pesticidesdifficult. Low volume aerial sprays with phosphite using light aircraft havebeen successfully used to contain Phytophthora root rot of Banksia coccineain natural stands in Western Australia (Komorek et al. 2001).

4.1. SeedSeeds may be passive carriers of bacterial, viral, fungal and nematodepathogens (Neergaard 1977). Pathogens present in seed can spread withseed into new areas and can be a major source of pathogen introduction.Seed-borne pathogens can cause damping-off, blight, wilt, anthracnose, leafspot, and cankers (Maude 1996). Consequently, it is important to ensure thatseed is pathogen-free when used for recovery programs, whether initially tostart nursery stock or for their in situ establishment. There are many fungithat can destroy seed during its development on the plant, during storage,after sowing or during germination (Anderson and Miller 1989; Maude1996). The site of infection/infestation in a seed is largely determined by thesource of the pathogen. Some are a result of superficial contamination fromsoil, while certain pathogens that are systemic in a host find it easy to lodgethemselves in the embryo. Control of seed-borne pathogens can beconsidered in terms of exclusion and elimination of inoculum (Neergaard1977). Exclusion strategies include the isolation of seed production areasand breeding for resistance to seed-borne infections (such as in the cases ofvirus infections). Application of these two strategies in recovery programsshould be restricted as they limit or modify the genetic integrity of thespecies. Elimination strategies include the control of organisms by treatmentwith fungicides of seeds and other plant propagules. Where there are


responses, systemic or non-systemic fungicides may be applied to the seedprior to sowing.

Observation of appropriate hygiene during harvesting and processingand the use of low temperatures and low humidity environments duringstorage best control seedborne fungi. It is beneficial to collect seed whilst itis still held in capsules or within fruits on the plant, as once on the ground,the seed becomes readily contaminated by soil-borne pathogens. It is alsoadvisable to check the seed before sowing and reject infected seed prior tosowing or treat with seed protectants such as benomyl, carboxin, triforine,chlorothalonil, thiram and captan (Chalermpongse 1987). Hot watertreatments (50°C for 5-20 minutes), surface disinfection (10% sodiumhypochlorite or 33% hydrogen peroxide for 1, 2 or 4 minutes) and fungicideapplication (1% captan) have been used to restrict fungal development oneucalypt seed (Donald and Lundquist 1988). While these methods areadequate to minimise seedling deaths the use of very sensitive detectionmethods such as enzyme-linked immunosorbent assays or PCR basedmethods are required to clear seed or other propagules of the presence ofexotic quarantinable diseases.

4.2. Nursery practices favouring diseasesPlant production nurseries are a key step for the production of seedlings orplants from cuttings in many recovery programs, consequently the control ofplant diseases in the nursery are critical. Cultivated plants are usually moresusceptible to disease outbreaks than their wild relatives. This is partly dueto the large numbers of the same species or clones being grown denselypacked together within propagation areas. Under these conditions,pathogens can often establish themselves, sporulate and spread rapidly. Asalready mentioned (Figure 1), plant diseases are a result of interactionsbetween pathogens, hosts and the environment. In the nursery, severestresses may be imposed on a host because of a space limitation, use ofextensive monocultures, the abili ty of the grower to increase productivitybeyond normal limits through water, temperature and nutrient managementand due to poor or inadequate hygiene. Losses of planting stock in thenursery and subsequently after ‘planting-out’ can be severe if basic hygienepractices and environmental conditions that predispose plants to root orfoliar pathogens are not managed.

Nurseries can provide conditions that are ideal for the development ofmajor disease outbreaks. These include inadequate physical, chemical andbiological characteristics of the container substrate(s) used, the use ofpathogen infested water, inadequate air flow, poor hygiene and poorquarantine practices or from the introduction of pathogens from surroundingareas or from soil, seed or vegetative planting material introduced into the


nursery (Sivasithamparam and Goss 1980). Nursery diseases are bestavoided, however, to do this a thorough understanding of the ecology of thepathogens and their commensal microorganisms is required. Implicit in thisunderstanding is the impact of environmental stresses on both the pathogenand the host, as well as stresses on and imposed by other microorganisms.Intensive nursery production of plants does impose its own set of stresses onall three components of the host-pathogen-environment disease triangle.

5. Integrated disease managementThe disease triangle (Figure 1) illustrates the importance of deploying a widerange of management practices to prevent disease or keep it at minimallevels (Agrios 1978). There is no one practice that controls all plant diseasesin a nursery program. By adopting an integrated approach and utilising anumber of control strategies it is possible to (1) reduce the overall incidenceand severity of diseases, (2) reduce the chances of major disease outbreaks,(3) improve the quali ty of plant material being distributed for the recoveryprogram, (4) minimise the need for chemical treatments, and (5) reduce thechances of developing chemical-resistant pathogens. Implementing theabove also increases the efficiency of the nursery and subsequently itsprofits.

Good sanitation and cultural practices carried out stringently andconsistently wi l l have some effect on diseases of plants grown in thenursery. It is also important to be aware of symptoms and signs of disease inthe nursery. This involves monitoring for disease visually by focusing onplant health and understanding the nature of each of the plant speciesinvolved. Observations should be made on all aspects of plant health. Forexample, general plant vigour and growth rates, leaf colour and size. Thepresence of leaf spots and blights, collar rots and wil t should be noted.Where necessary plant and container substrates should be analysed for pH,cation exchange capacity and nutrient levels to enhance plant growth andvigour necessary for defence against pathogens. Suspect samples of roots orleaf material should be sent to diagnostic laboratories for analysis so thatappropriate treatments can be applied as rapidly as possible. Concurrently,thorough written and photographic records should be maintained for eachspecies. A historical database can be developed where symptoms and signsof plant disease and their causative agents are linked with cultural practicesand with environmental factors such as rainfall, temperature and humidity.It then becomes possible to l i nk predisposing cultural practices such asfertiliser regimes, fungicide and pesticide applications, container substrates,and changes in environmental factors with disease outbreaks. The managerthen becomes aware of the pathogens that cause major problems and theplant species affected. Patterns and t iming of disease development within


the nursery can be established that can be related to the factors that areassociated with severe disease problems.

5. 1. Wood and bark mulchesThe use of wood and bark mulches whether in propagation or forweed/moisture control in p lan t ing out needs to be carefully considered forthe following reasons. Such material can contain a range of soil-borne or air-borne pathogens that could adversely affect recovery programs. Materialthat is not composted or adequately aged can cause nitrogen ‘draw down’ orin the release of phytotoxic compounds that are detrimental to plantestablishment and growth. Wood and bark wastes should be adequatelycomposted to ensure that any potential pathogens such as species ofPhytophthora, Pythium, Armillaria etc are killed. Composting is a complexprocess, that combines thermal eradication of a portion of microflora in theorganic waste wi th certain physical and chemical properties of the compostthat are valuable in disease control (Hoi t ink and Fahy 1986; Hardy andSivasithamparam 1991). Composting involves placing mulched material inwindrows, maintaining adequate moisture levels and applying nitrogen andphosphate to fac i l i ta te microb ia l ac t iv i ty . For rapid and appropriatecomposting, the material should be maintained at between 38°C and 55 °Cand turned-over regularly. In the presence of adequate moisture andtemperatures, the composting process results in the disinfestation of thematerial.

5.2. ExclusionA highly effective way of main ta in ing a disease-free nursery is the exclusionof pathogens. Exclusion is a very effective method of disease control offungi that are not airborne and have a limited host range. It is a very cost-effective practice. The regulatory inspection and certification of disease-freeplants and plant parts (cuttings, tissue cultures, seed, bulbs and corms) are acritical component of a disease management program for fungal diseases.

5.3. Sanitation strategiesAn effective sanitation program should be considered as the most importantmanagement practice in a nursery. It should ideally be effective against allmajor diseases. In particular, it is invaluable for the control of root and stemdiseases. Sanitat ion invo lves a l l practices that are aimed at reducing ore l imina t ing the amount of i nocu lum present in the nursery. Sanitationcannot be observed in a haphazard fashion. This will subsequently preventthe movement of disease out of the nursery into disease-free areas and intorecovery programs. Wi thout a sound sanitation policy, no nursery wil lremain disease-free for long (Baker 1962).


The prevention or minimisation of disease is a much more effectivestrategy than reacting to a disease after it is established, which in any case isoften too late. Any sani tat ion program must be designed to reduce theamount of inoculum in the nursery by removing dead and dying plants orplant parts and to reduce any potential carryover inoculum between crops.There are a number of fungal pathogens such as Phytophthora cinnamomi,Rhizoctonia solani, and Sclerotium spp. that are not airborne and aretherefore unlikely to be introduced into a nursery in air currents. Therefore,their presence in a nursery w i l l be due to poor nursery hygiene practices.Stopping the movement of such pathogens out of the nursery will reduce thedistribution and incidence of the disease (Hardy and Sivasithamparam 1988).Air-borne or splash-borne diseases can be a threat at production or plantationlevels (see Burgess and Wingf ie ld , this volume). Management of thesediseases is economical only through the use of disease resistant hostmaterial. Fungicides are expensive and may be ineffective.

For effective sanitation practices to be undertaken it is critical that thenursery managers and personnel understand the scientific principles behindeffective disease management. Most important is the exclusion of pathogensand in particular root and stem pathogens that tend to be soil-borne. Thesepathogens appear in the nursery from known sources. These include (1)infested container substrates, (2) infested water sources, (3) poor orinadequate hygiene practices during propagation, (4) media and containersbeing placed on the ground in contact with soil and (5) the use of infestedseed, cuttings, bulbs or other plant material. Ultimately, sanitation involvesa series of different but linked functions wi th in the nursery production chain.

5.4. Cultural practicesGrowers must concentrate on providing conditions for the optimal growthand development of their plants and at the same time avoid conditions thatfavour pathogens (Palti 1981). The majority of pathogens can only penetrateplants when their surfaces are wet. Consequently, many diseases, especiallyfoliar diseases caused by pathogens such as Botrytis cinerea andCylindrocladium spp. can be managed by avoiding the use of overheadirrigation, or irrigating at times when the plant foliage can dry rapidly.Many opportunistic pathogens can only infect plants when they are woundedor injured in some way, so avoiding injuries will reduce disease. Preventingthe build-up of humid micro-climates in seedling trays or closely spaced potsas plants grow, by increasing spacing and airflow will dry out wet foliagemore quickly, reduce humid microclimates and reduce the chances of foliarpathogens causing major problems. Finally, the removal and destruction ofdiseased plants or plant parts will reduce inoculum sources and subsequentdisease outbreaks.


5.5. Container substratesThe use of appropriate container substrates is vital (Handreck and Black1989). Container substrates vary in form depending on cost and onavailability. These include sphagnum peat, composted or aged hardwood orsoftwood barks and sawdust, coarse sand, perlite, plastic beads, coconut coirand many other substrates. All of these substrates will perform adequatelyas long as they have the right physical, chemical and biologicalcharacteristics for plant growth for the life of the plant whilst in the pot, andare pathogen free. If any one of these characteristics is less than optimum,plants can be predisposed to disease, through waterlogging, drought orinadequate or excessive supply of nutrients.

From a physical point of view, the substrate must provide anchorage forplant support and it must regulate the supply of oxygen and water to theroots. In nurseries plants tend to be grown in small, shallow multi-celledtrays or containers that create physical constraints to plant growth. Firstly,the volume of substrate and water available to the plant in these containers issmall. Secondly, since the substrate is contained in a shallow layer, a‘perched’ water table is created (Handreck and Black 1989; Bunt 1982).This prevents adequate drainage and makes the substrate wetter than itshould be immediately after irrigation. There is less oxygen available afterevery irrigation or rainfall event. In addition, under dry conditions shortageof water can occur rapidly especially during periods of high evapo-transpiration.

Plants can be predisposed to foliar and root diseases as a result of theserapid wetting and drying events. Consequently, it is important to formulate acontainer substrate that has a total pore space of approximately 85%,airspace between 25-30%, easily available water of 25-30%, and a waterbuffering capacity of around 10% (De Boodt and Verdonk 1972).Consequently, particle size of container substrates is critical. The physicaland chemical characteristics of pine and other bark substrates are affected byage of the material and by how the material is handled during mixing of theingredients. When using wood or bark based products it is important to havea C:N ratio of approximately 30:1 to avoid nitrogen draw-down. Howeverpine bark, even when composted, may not have a C:N ratio this low due to itbeing mainly composed of lignin, not cellulose.

The chemical characteristics of container media are also a criticalconsideration in the healthy production of plants. The optimal range fororganic potting substrates is pH 5.0 to 6.0. Cation exchange capacity shouldbe between 6-15 milli-equivalents (meq) per (Handreck and Black1989).

All handling and storing of each of the ingredients of the containermedia should be undertaken with proper care. The media should be stored


on sloping concrete surfaces above soil level, to prevent surface water“runoff” from surrounding areas contaminating the media. The mediashould never be mixed on the ground, and the machinery used to carry andmix the media should be clean. If containers are recycled they should becleaned and sterilised prior to being reused.

The container growing areas should be constructed in such a way thatcontainers never come into contact with contaminated soil. Above groundbenches, or coarse gravel beds allow for rapid drainage of free water. Theyalso reduce the chances of soil splash and thereby reduce the chances ofdisease spread. Some nurseries use t ight ly woven plastic ground clothdirectly over soil surfaces, which are an adequate surface as long as the soilunderneath rapidly drains. If ponding of water occurs on the surface of thecloth, then the chances of water-borne fungi coming into contact with theplants is greatly increased.

5.6. Disinfestation of pathogens from container substratesThe removal of potential pathogens from substrates that are to be used asingredients in container substrates is essential. Substrates such as washedriver sand, sawdust, and crushed bark all carry a risk of containingpathogenic organisms such as species of Pythium, Phytophthora, Fusariumand Rhizoctonia. The removal of potential pathogens from these substratesis a major cost in nursery production. Common methods involve the use ofsoil fumigants, steam pasteurisation or composting. Microorganisms aremore susceptible to heat or fumigants when they are in an active metabolicstate, however, many pathogens can form resistant resting structures such asoospores, chlamydospores, or sclerotia in dry and cool substrates.Therefore, effective removal of inoculum is best undertaken when thecontainer substrates are warm and moist. Composting of potting materialsmay be useful in the destruction of certain pathogens (Hoitink and Fahy1988).

5.7. Steam pasteurisationHeating soil or container substrates to approximately 60°C with aeratedsteam for 30-60 minutes and held at 60°C for 30 minutes kills mostpathogens without creating a biological vacuum (Baker 1962). Beneficialorganisms that form resistant spores survive the pasteurisation. This leavesthe soil in its naturally suppressive state with its complement of saprophyticmicroflora.

5.8. FumigationFumigation involves infiltrating a substrate with the vapour of a volatilechemical. Chemicals used include metham sodium, methyl bromide, methyl


bromide-chloropicrin mixture, chloropicrin, dazomet, ethylene dibromide,and 1, 3-dichloropropane-dichloropropene mixture (Jarvis 1992). Not all ofthese are registered pesticides in many countries. Methyl bromide is themost widely used fumigant in nurseries, however it is progressively beingbanned from use in many countries due to its adverse effects on the ozonelayer. Fumigation shows varying levels of effectiveness depending on thepathogen(s) being controlled and can leave residues that can have adverseside effects in terms of phytotoxicity and residues that can progress throughthe food chain.

5.9. Soil solarisationSoil solarisation (Katan 1981) u t i l i ses heat from the sun to raise thetemperature of the substrate being treated to levels that are sufficient to killpests, diseases and many weed seeds. The soil or container substrate ismoistened to field capacity and then covered with a thin layer oftransparent polythene. Care is taken to minimise air spaces between thepolythene and substrate to prevent an insulating effect of the air space. Thelonger the substrate is treated at high temperatures, the more effective thecontrol.

5.10. Disinfestation of irrigation waterWater moulds such as Pythium and Phytophthora are readily carried in waterbodies such as dams, water tanks and shallow bore holes. This problem canbe increased substantially if the water is recycled, such as in hydroponicsystems. Water can be effectively disinfested by filtration, irradiation,chlorination, bromination or ozonation.

For effective filtration, filters with pore sizes between mustbe used. If the water contains particulate matter it is necessary to have aseries of larger filters to remove the particulate matter. The filter membranesneed to be cleaned regularly. Irradiation uses ultraviolet (UV) light in the200-280 nm range to disinfect water. However, a number of parameterssuch as flow rate, the duration of exposure, level of particulates in the water,and the strength of the UV lamp must be considered when using this process.Chlorination is the most common and cheapest disinfestation method. Theprocess uses ei ther chor ine gas, sodium hyphochlorite or calciumhypochlorite. To be effective, a minimum concentration of 5 ppm for 20minutes is recommended. It is necessary to filter out organic particulatematter prior to chlor inat ion. Particulates reduce the effectiveness of thetreatment. The water pH should be around pH 6-7.


5.11. FungicidesFungicides are used prophylactically by most container or bare root nurseriesto control a range of soil-borne or foliar plant pathogens. Many fungicidesare fungistatic rather than fungicidal to a range of plant pathogens, especiallyin their dormant or resting stages. These pathogens express themselves afterthe fungistatic effects of the fungicides have disappeared on the cessation oftheir use. It is critical to assess the use of fungicides on plants that are beingused in recovery programs and special attention must be given to soil-bornepathogens such as Phytophthora species. Total reliance on fungicides suchas phosphite (phosphonate) that do not necessarily kill the pathogen butinduce a resistant host response which contains but does not kill thepathogen must be avoided.

It is advisable that plants to be used in recovery programs grown inplant nurseries be produced under stringent hygiene measures. These wouldinclude the use of disease-free container substrates that have been adequatelyand stringently composted, or steam pasteurised, or produced from materialssuch as polystyrene and perlite. The water source must be pathogen-free,containers must be kept off the ground on raised, free-draining benches andenvironmental conditions such as temperature, relative humidity and air flowmust be conducive to plant growth and not to plant pathogens. Theaccreditation of nurseries that adopt strict and regularly monitored hygieneand quarantine practices must be encouraged and preferably legislated for.

5.12. Ecological restorationThe aim of ecological restoration is to reach a sustainable and diverse florawhich may or may not be identical to that which existed prior to disturbance.This approach not only requires a sound knowledge of the environment ofthe affected site and the nature of the suitable flora but also funds to supporta large labour force necessary to carry out the program. Although achallenge, such programs have been successfully carried out. A goodexample is that of Grevillea scapigera, which was at the point of extinctionand has recently been restored ecologically through the efforts of communityand conservation groups in Western Australia (Bunn and Dixon 1992;Touchell et al. 1992; Rossetto et al. 1995; Krauss et al. 2002).

5.13. Planting outPlanting out may not simply be a routine procedure. Timing of planting,land preparation (including soil amendments and irrigation duringestablishment) needs to be considered carefully in addition to prophylacticmeasures to reduce disease hazards.

Hygiene is an important component of any species recovery program.This includes keeping plants disease-free when planted out and minimising


the spread of a pathogen into non-infested areas if a recovery program isbeing undertaken on disease infested sites. For example, the control andmanagement of P. cinnamomi in natural ecosystems raises considerablechallenges in terms of reducing spread and the impact of the pathogen inrecovery programs in diverse plant communities. There are a number ofstrategic control procedures that can be used by managers involved inrecovery programs (Colquhoun and Hardy 2000). These include:

Producing reliable up-to-date maps and field demarcation of diseaseaffected areas. This involves having trained interpreters who have agood understanding of which plants are P. cinnamomi susceptible‘indicator’ species. The interpreters must be able to discount otherfactors that can cause plant death such as drought, insects, fire, and otherplant diseases. The information on disease affected areas to be stored ona Geographical Information System. Once mapped, appropriateoperations planning, scheduling and implementation involving high-riskactivities of infested and adjacent non-infested areas must be undertaken.Planning high-risk operations such as road building, forestry activitiesand mining in diseased areas when conditions optimum for the spread ofP. cinnamomi are minimal, such as during hot and dry periods.Restricting the movement of vehicles from disease affected to disease-free areas. This can be achieved by blocking tracks to stop their use,erecting gates and signs, removing roads and by ensuring roadconstruction through disease-free areas uses non-infested materials.Preventing the movement of infested water moving into disease-freeareas. The zoospores of P. cinnamomi are readily transported in waterso any surface water movement between infested and non-infested areasneeds to be minimised.Thoroughly cleaning vehicles and equipment to remove all adhering soilor plant debris before moving between infested and non-infested areas tominimise the risk of spreading infested soil into disease-free areas.Wash-down stations must be designed so that once washed the vehiclesare unlikely to be re-contaminated by passing through wash water thatcontains propagules of P. cinnamomi.Training all field personnel and planners in good hygiene managementand operations to reduce the spread of the pathogen.Increasing the awareness of the general public of P. cinnamomi. This isachieved by information leaflets, newspaper articles, displays at publicfunctions, television interviews, classroom teaching materials andinformation signs.Similar strategies can be developed for other soil-borne and foliar plant

pathogens. In conclusion, phytosanitary protocols should be established foreach step or process involved in the conservation and propagation of plants.


This requirement needs to be most stringently observed in programs relatingto the conservation of rare and endangered plants.

5.14. RootsMost root pathogens are necrotrophic and tend to have, with few exceptions,wide host range. The most common examples of these are species ofPhytophthora and Pythium, Many of these species occur world-wide,although it is likely that pathogens such as P. cinnamomi were spread world-wide through movements of horticultural plants.

Certain necrotrophs such as the formae speciales of Fusariumoxysporum have specific host ranges, but can easily be spread with non-hostplant species. These pathogens can be effectively managed only by theexploitation of host resistance. With exceptions of diseases caused byoomycetes and certain nematodes, root-disease causing pathogens can onlybe eliminated by pre-planting soil fumigation or drenches.

This means that it is possible to introduce perfectly healthy plants of thespecies you have targeted into an area, with latent infections of fungalpathogens that then cause the loss of other plant species in the area.

5.15. Disease-free planting materialsSeed, transplants, and cuttings can be major sources of inoculum in nurseryproduction of plants. There are three main ways to obtain pathogen-freeplant material. Firstly, it is essential to ensure that the planting material isderived from a disease-free area and stock. Secondly, a culture-indexingprogram whereby multiplicative propagation is done only from healthy plantparts such as stem tips or meristems (or even through micropropagationusing protoplasts), can eradicate pathogens. Thirdly, once healthy materialhas been obtained, basic hygiene and quarantine procedures should bemaintained by the grower to minimise the chances of reinfection.

There are a number of pathogens, particularly fungal pathogens, thatcan be missed by existing quarantine protocols. For example, there are fungithat can exist for extended periods as endophytes (Saikkonen et al. 1998).These do not express themselves until some stress factor or change inenvironmental conditions predisposes the host to damaging pathogenicactivities of the pathogen. It is therefore critical that any plant materialbeing used in recovery programs that has been imported from areas otherthan where the plants will be planted is screened for the presence ofpathogens. It is critical that this material is disease-free. The use ofindexing, serology, ELISA (enzyme linked immuno assorbant assay) andPCR-based molecular tools are critical to ensure the absence of potentialpathogens. The use of fungicides that impose stasis on fungi in planta mustalso be considered. If fungicides have been used prior to plants being


subjected to quarantine then the duration of the quarantine period should beextended to allow time for dormant propagules to germinate once fungistaticeffects of fungicides have dissipated.

It could be argued that in some instances systemic fungicides should notbe allowed for use in nursery production. An example for this would beoomycete fungicides that have the potential to mask the presence of apathogen such as P. cinnamomi. The distribution of outwardly ‘healthy’ butinfected plants previously treated with fungicides should be avoided.Infested plants introduced into disease-free areas can have potentiallycatastrophic effects.

6. Case studies6.1. Phosphite fungicidesPhosphite, the anionic form of phosphonic acid has been used tomanage many plant diseases caused by Phytophthora species in horticultureand in agriculture. It is effective even at concentrations in planta that onlypartially inhib i t pathogen growth in vitro (Guest and Bombeix 1984; Guestand Grant 1991; Wilkinson et al. 2001a). Phosphite is a systemic fungicidethat is translocated in both the xylem and the phloem (Ouimette and Coffey1989). In the phloem, phosphite is trapped and therefore translocatedthrough the plant in association with photoassimilates in a source-sinkrelationship (Saindrenan et al. 1988; Ouimette and Coffey 1990; Guest andGrant 1991). The phosphite concentration in plant tissues is directly relatedto its application rate (Smil l ie et al. 1989). Phosphite treatment induces astrong and rapid defense response in the challenged plant (Guest andBompeix 1990; Smith et al. 1997). These defense responses stop pathogenspread in a large number of hosts. Phosphite exhibits a complex mode ofaction, acting directly on the pathogen and indirectly in stimulating hostdefence responses to u l t imate ly inh ib i t pathogen growth (Guest and Grant1991).

In Western Australia, phosphite is currently applied to native plantspecies as an injection to the trunks of trees or large shrubs, as aconventional foliar application to run-off or as an ultra-low volume mist(Komorek et al. 1997, 2001; Barrett 1999; Hardy 2000; Tynan et al. 2001).The latter is applied by aerial application, usually to communities of highconservation value which contain rare and threatened plant species. It isused routinely in the S t i r l i n g Ranges to protect rare and endangered plantspecies that are growing in infested areas. Without the use of phosphite, anumber of plant species would no longer exist in the wild. Consequently,phosphite is a vital short to medium term ‘tool’ currently utilised in somespecies recovery programs.


6.2. CostsAerial applicat ion of phosphite as an ul tra low-volume mist costsapproximately this includes the cost of the fungicide andaircraft hire. Addi t ional costs are involved in the set-up of targets, inparticular for mountain areas, where personnel must be on site to ensurewind conditions are adequate for application. Rates of phosphite applicationapplied as a low-volume mist range from using 40% phosphitesprayed at The rate is applied in two separate sprays4-6 weeks apart to minimise phytotoxicity. Conventional backpack sprayersor trailer mounted sprayers can be used to apply phosphite to run-off at ratesof between 0.5-1% phosphite. At higher rates, phytotoxicity can become amajor problem (Hardy 2000). Feasibility of conventional spraying bybackpacks and trailers is usually restricted to small areas of approximately 1ha or less. These include spot infestations or small areas of remnant bush-land. Injecting trees is only viable for large trees in areas where their losswould have a visual impact, al though in some instances volunteer groupshave treated whole reserves by injection trees and spraying the understory torun-off (I. Colquhoun pers comm.). It costs approximately AUD$0.50 centsto treat a medium size jarrah (E. marginata) tree by injection. The best timeto inject a tree is during spring and summer in the morning when the tree isactively transpiring.

It is critical to add an adjuvant when applying phosphite as a foliarapplication. In Western Australia, Synertrol Oil (Organic Crop ProtectantsPty Ltd), based on food grade canola oil (832 gL) is used. Synertrolincreases spray coverage by droplet spreading, promotes spray retention,reduces spray drift, evaporation and wash-off (Organic Crop Protectants PtyLtd).

6.2.1. Control of P. cinnamomiInjections using 50, 100 and phosphite have controlled P.cinnamomi in a number of species for up to 5 years (Shearer and Fairman1997a,b). Foliar application to run-off of phosphite increased the timeto 50% mortality of three species of Banksia growing along a P. cinnamomidisease-front by an average of 2-6 years depending on the species treated(Shearer and Fairman 1997a). Aberton et al. (1999) stated that foliarapplication of phosphite to Xanthorrhoea australis prevented deathsfor at least two years in P. cinnamomi infested vegetation. Pilbeam et al.(2000) demonstrated that foliar applications of 2, 5 or phosphiteeffectively restricted the colonisation by P. cinnamomi of stems of threenative species.

In an operational program using ul tra- low volume applications ofphosphite to a native plant community in the Fitzgerald River National Park,


percentage survival of Banksia baxteri and Lambertia inermis plantsgrowing along a dieback front at two years post-spray was 68% and 78%compared with 31% and 54% for non-sprayed plants, respectively (Barrett1999).

Therefore, at the rates of phosphite applied to native plants, P.cinnamomi colonisation is contained or reduced in plant tissue but thepathogen is seldom killed (Ali et al. 1998; Pilbeam et al. 2000; Shearer perscomm). This obviously has implications with regard to the continued spreadof the pathogen under optimum environmental conditions. In a studyconducted on 1-2 year old E. marginata in a rehabilitated mine site,Wilkinson et al. (2001 b) showed that phosphite, when applied as a foliarspray, contained lesions caused by P. cinnamomi, but sporangial productionand zoospore release were not prevented from diseased tissue, although theywere reduced in numbers. Consequently, phosphite may slow down orprevent deaths of plants in natural plant communities but not necessarilyprevent the spread of inoculum into non-infested areas. Trials in native plantcommunities to determine whether applications to affected areas could helpto prevent the spread off the pathogen to unaffected areas are required toconfirm this observation. However, until this is undertaken, good hygienepractices in and around infested areas that have been treated with phosphitemust still be a priority.

6.2.2. PhytotoxicityPhytotoxicity in native plant species has been observed (Komorek et al.1997; Aberton et al. 1999; Fairbanks et al. 2000; Pilbeam et al. 2000; S.Barrett, pers. comm.). In some cases leaf scorching has occurred on plantsspecifically sprayed for protection against P. cinnamomi. Consequently,there is a fine balance between the rates of phosphite applied, phytotoxicitysymptoms and the control of P. cinnamomi. Generally, as the rates ofphosphite applied increase, so do the concentrations of phosphite in planttissue. However, above as a spray to run-off or as a mist orlow-volume application, phytotoxicity symptoms increase substantially in alarge range of species from different genera and families.

It is likely that in whatever natural ecosystem phosphite is applied, itwill, even at recommended rates, cause phytotoxicity in some plant due todifferences in uptake between species and their sensitivity to the fungicide.However, it is necessary to offset this phytotoxicity with the benefits ofphosphite, in terms of P. cinnamomi containment (Hardy et al. 2001).


6.2.3. Detrimental effects (on plant reproduction, plant growth, tolerantisolates and mycorrhizas)There have been limited studies on the effects of phosphite on plantreproduction. However, Fairbanks et al. (2000) showed that recommendedrates of phosphite reduced the reproductive fitness of some annualand perennial understory species from the Eucalyptus marginata (jarrah)forest. Phosphite reduced pollen fertility in a native annual species whensprayed in the vegetative stage and of three others when sprayed at anthesis.Seed germination from treated plants was reduced by phosphite in twospecies when the plants were sprayed in the vegetative stage. Consequently,more work is required, but it does appear that it would be strategic tom i n i m i s e the use of phosphite when annua ls are growing in plantcommunities, or to apply phosphite in early autumn prior to germination.

Phosphite also affected sexual reproduction of three perennial jarrahforest species (Fairbanks et al. 2001). In Pterochaeta paniculata(Asteraceae), pollen fert i l i ty was reduced by phosphite for up to 60 weeksafter spraying in autumn, and 35 weeks in spring. Whilst in Trymaliumledifolium (Rhamnaceae), pollen ferti l i ty was reduced for up to 38 weeksafter spraying with phosphite in spring, and up to 61 weeks after spraying inautumn.

6.3. Effects on mycorrhizal fungiMycorrhizal fungi are symbionts that confer benefits to their host plant andat the same time obtain a niche and nutrients from their host. Many plantscan fail to establish or thrive in the absence of their mycorrhizal symbionts.Consequently, the use of fungicides has the potential to be detrimental tomycorrhizal fungi . Pre l iminary glasshouse studies on the effects ofphosphite on mycorrhizal fungi have shown that phosphite applied as a foliarspray to Eucalyptus globulus, E. marginata and Agonis flexuosa had noeffect on ectomycorrhizal (ECM) formation, whilst vesicular-arbuscularmycorrhizal (VAM) colonisat ion increased four-fold in Agonis flexuosa(Howard et al. 2000).

6.4. Phosphite tolerant P. cinnamomi isolatesWhen using fungicides on plants in recovery programs, it is important toconsider the likelihood of the pathogen becoming resistant or tolerant to thefungicide. There is some evidence of tolerance of P. cinnamomi tophosphite in treated plants among isolates from native vegetation which havenot previously been exposed to phosphite (Hardy et al. (2001). Thisobservation is of concern, especially in native ecosystems that are beingtreated regularly wi th phosphite to save ‘cr i t ica l ly endangered’ species.Regular spraying could provide a selection pressure for these more phosphite


tolerant isolates and could pose additional problems to managers in thefuture.

6.5. Conclusion on phosphite use in recovery programsPhosphite appears to be effective in keeping susceptible plants in naturalecosystems alive in the short to medium term in areas impacted byPhytophthora. It has particular application to plant communities where rareand endangered plant species are threatened with extinction. Phosphite mayhave some detrimental effects such as reduced reproductive capacity in somespecies. However, if a susceptible species is threatened by extinction thenphosphite could applied un t i l some better alternative becomes available. Inthese cases, phosphite provides managers with the time to developalternative control strategies such as placing endangered species into cryo-preservation or developing other conservation strategies. However,appropriate hygiene practices must not be reduced or stopped sincephosphite used as a foliar application or injection often will only contain thepathogen in the plant and not kill it. Therefore, under optimum moist andwarm conditions it is possible for sporulation to occur.

These studies on phosphite in natural plant communities clearly indicatethat fungicides can have a role in protection of natural plant communities.However, there is the need to consider the impact of phosphite or otherfungicides on other components of the plant community in addition to theireffects on the pathogen.

7. ConclusionsPrevention is clearly better than cure. Effective conservation efforts

aimed at avoiding or preventing disease outbreaks can be far more beneficialthan intensive management of affected areas. Options for intensivemanagement of destructive diseases, especially in natural ecosystems, arefew, expensive and may at best be only temporary measures to manage theproblems until more effective and permanent solutions are found.

Recovery programs can succeed only if adequate research is carried outon the ecosystem of the region and the potential for resident and/or exoticpathogens to the restored area. Emphasis must also be made on theemployment of appropriate hygiene practices at production and planting-outphases.

Several major questions remain from this chapter. Are devastationevents such as those of Castanea in north America and other susceptiblenative flora devastated by introduced pathogens rare events in history? Or isit that they happened at a time and in places were researchers were ready tomonitor them? Could they have happened throughout evolution and time?Could they be happening currently in ecosystems in countries too poor or too


distracted to monitor them? Answers to these questions may need extensiveand possible expensive research which is unlikely to be funded.

AcknowledgementsThe authors are grateful to Brett Gaskell for extensive comments andsuggestions on the structure of this chapter; Mark Brundrett for commentson the chapter and Sarah Barrett for data on effects of phosphite on nativeplant species.

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INDEX

369

studies2-partner symbiosesAcacia

melanoxylonAcaulospora

denticulataACC deaminaseAcclimatisationAcetylenereductionassayAchromobacter parvulusAcremonium

loliiActinomyceteActinorhizalAdenostoma fasciculatumAeschynomeneAfrica

AgaricalesAgonis flexuosaAgrobacterium

radiobactertumefaciens

Alcaligenes faecalisAlgaeAllocasuarina

Alnusglutinosarubra

AlternariaAM fungiAmanita muscariaAmaranthaceaeAnabaenaAnigozanthos

viridis ssp. terraspectansAnthericaceaeAnthropogenic acidification

AntibioticAotusAponogeton hexatepalusAponogetonaceaeArabidopsis thalianaArachisArbutusArchaeaArchaeosporaArctostaphylosArgentinaArmillaria

luteobubalinamellea

Artemisia californicatridentata

Ascochyta caulinaAscomycetesAscomycotaAsiaAsian chestnutAspergillus

nigeroryzae

AsteraceaeAsterolasia drummondii

grandifloranivea

Astroloma xerophyllumAtkinsonella hypoxylonAureobasidium pullulansAustralia

Autricularia polytrichaAvenaAzollaAzorhizobiumAzospirillum

116276

58,63,66-7, 108, 113, 128211

32, 155, 15825

87, 99308

66328274245

51, 5651, 53

2559

49, 52, 63, 228, 231,234-5, 295-7

243362

62, 24288-9258328

251-2, 56,60, 66-7,

108, 12851-4, 56, 108, 128, 277

346122, 126

310, 312, 348116, 125, 127-8, 132, Ch 6

106, 131167

46,49326-7

324324

21

31866

32432432760

2322,4

155, 187126

52, 296287, 351

288-9, 3042112925

257107

243-4, 246, 249, 25447, 50-2, 210, 295

347272, 274, 310, 312

328328

57, 107325325325

228-9255328

1, 5, 7, 11,45,47,49, 51-2,63, 66,91, 105, 107, 119-20, 128,131, 151 ,167, 195, 200, 202, 212,214, 217, 227-8, 231, 234-5, 250,255-6, 271, 277, 286-97, 299-300,

337,343-4210

23, 295162

46, 81, 86


AzotobacterBacillus

cereussubtilis

Balaustion microphyllumBanksia

baxteribrowniicoccinia

BasidiomycetesBasidiomycotaBasidiosporesBeilschmiediaBeta vulgarisBettongia tropicaBetulaBetulaceaeBiological controlBletilla striataBoletalesBoletinellus merulioidesBoreal forestsBoronia adamsianaBossiaeaBotryosphaeria dothidea

rhodinaribis

Botrytiscinerea

Botyrosphaeria dothideaBouteloua gracilisBoweniaBradyrhizobiumBrassicaceaeBrazilBrevibacterium ammoniagenesBromus hordeaceus

madritensisBuntsBurkholderia cepacia

reductionor physiology

CaesalpinioideaeCaladenia

arenicolaCalifornia

Callunavulgaris

Calonectria quinquiseptatumCalytrix breviseta ssp. brevisetaCandida

albicansCankerCapsicumCarex arenariaCaryophyllaceaeCassiopeCassiopoideaeCasuarinaCasuarinaceaeCataulaCeanothusCenococcum

geophilumCentral AmericaCephalanthera austiniaeCeratobasidium

cornigerumCeratocystis

fimbriataCeratorhizaCeratozamiaChaetomium globosumChenopodiaceaeChenopodiumChestnut blightChiguaChromistaCryphonectria parasiticaChytridialesChytridiomycetesChytridiomycotaCitrus

46, 8680-1, 88, 311-2, 322

83, 32888

32466, 291, 348, 360

361345

338, 340107, 198, 243, 246

243, 246288211327128

106, 108, 21052-3, 108

27620324311921032566

287298289

310, 312258, 342, 348, 352

2943347

46, 62-3167

58, 295-6328

3129

246886623

57-9, 61202, 206

208-9, 21719, 21-2, 25, 27-31, 34,

36, 52, 193127, 228, 232

233-4291324310328

249, 286326253167

107-8228

51-3, 55-6, 60, 10852-3, 55, 64, 108

23251-3

109119

91, 102, 295210198

200, 20211, 260, 291, 303

296, 304198, 200, 224

47328167257

301, 34747

273250243

243-4243327

Index 371

Cladosporiumresinae

Clamp connectionsClavibacter

xyliClaviceps purpureaClostridium

enrichmentCo-evolutionColchiaceaeColletotrichum

gloeosporioidesCommelinaceaeCompetitionComptoniaConiothyrium

zuluenseConostephium pendulumConostylis misera

wonganensisConvention on biological diversityCoralloidrootCorallorhiza

maculatatrifida

CoriariaceaeCorybas cryptanthusCorynebacteriumCrassulacean Acid MetabolismCryphonectria cubensis

parasiticaCryptodiaporthe

semiperdaCyanobacteriaCyanophytaCycadaceaeCycadsCylindrocladiumCyperaceaeCypress cankerCytokininCytospora eucalypticola

Dactylorhiza

aristatapurpurella

Danthonia spicataDatiscaceaeDaviesia atrophylla

speciosaDebilitatorsDeuteromycetesDidymoplexisDie-backDillwyniaDioonDiplolaena andrewsiiDipterygeaeDisease suppressing bacteria

suppressive soils-free planting materials

DisinfestationDisturbance

Diurismicranthapurdiei

DNA/RNA fluorochrome stainingDothidealesDouglas firDowny mildewDrechslera teresDrummondita ericoidesDryasDutch elm diseaseEbola virusEcological restoration

specificityEcosystem dynamics

restorationEctomycorrhizasElectrosterilisationElaeagnaceaeElaeagnusElythrantheraEmpetraceae

272, 310, 312, 328328246

242, 31489

254-746, 57

22, 25, 30, 121266324

245, 298, 348298167170

52, 56, 10829729522832432427148

202, 206210

200, 21053

21031423

294286, 338, 347

338340

3474747

291, 298, 348, 352167, 316

286319

287, 291

202, 212, 214200199255

52-3324324254246

210, 2132926647

325588891

358354-5

10, 12, 112, 160, 174,212, 343-4

212, 332217

214, 217313243

119, 122, 124, 253, 26524890

32551-2, 56, 108

11, 250, 254-5, 2758

347, 356186

9129, 173

Ch 5321

52-3, 55, 6051-2212232


EmpetreaeEmpetrumEncephalartosEndosporous bacteriaEndothia eugeniae

gyrosaEndothiellaEnglandEnterobacter

aerogenesagglomeransasburiaecloacae

EntrophosporaEpacridaceaeEpacridoideaeEpichloe typhinaEpicoccumEpidendrum floridenseEpigoniumEpilobiumEpipactisEpiphytesEpiphytic orchidsEpipogiumEpulorhizaEremophila resinosaErgotEricaEricaceae

EricalesEricoid fungiEricoid mycorrhizasEricoideaeEriogonum fasciculatumErwinia

amylovaraErysiphalesErythromyces crocicreasErythrorchis ochobiensisEscherichia coli

EtiolationEucalyptus

diversicolordolorosaglobulusgrandisgraniticolaimpensamarginatanitens

EukaryotesEuphorbiaEurope

European forestEx situ conservationExobasidialesExplantFabaceaeFireFlavobacterium suaveolensFluorescent siderophoresFomes

mastoporusForestry activitiesFormae specialesFrankia

-based actinorhizal speciesFraxinusFungal propagules

successionFungi imperfecti.FungicidesFungus-feeding nematodesFusarium 254, 270, 272, 274, 348, 354

oxysporumpoaewilt of banana

Galearis

228232

47, 68322295

287-8, 298287-8, 291

223, 233, 311, 34688-9, 311-2, 328

32888

31289

155232, 281, 344-5

228254310

196, 222, 327210

23202, 212, 223-5

154222213

324257232

108, 129, 206, 215,227-30, 232-5, 344-5

227, 229227

Ch 8228

29242254

243, 245, 249, 254210210328

31966, 107-8, 112, 119, 120,

128, 130, 250, 255, 277,297, 299, 318, 340

289324

106, 123, 362294, 296, 327

324324

48, 291, 295, 344290

223

21, 51-2, 120, 131, 168, 200,202, 212, 250, 252,255, 260, 286, 346

122209, 260243, 245

324-532421232890

210211

112, 115248

46, 51, 55-7, 68, 27760

119111123233

318, 352, 356158, 161

89, 91, 257, 35831091

202

198, 200

Index 373

Galeolaaltissimahydraseptentrionalis

GallingGanodermatalesGastrodia

cunninghamiielatajavanica

Gastrodia minorsesamoides

Gastrolobium hamulosumGaultheria

shallonGentamycinGeographical distributionGeographical Information SystemGermplasmGibberella fujikuroiGigasporaGliocladium fimbriatumGlomalesGlomus

aggregatumetunicatumintraradicesleptotichumoccultumtenue

Genetically modified cropsGondwanaGoodeniaceaeGoodyera

repensGram negative bacteriaGramineaeGrazingGrevillea dryandroides

scapigeraGuignardiaGunnera

GunneraceaeGutierrezia sarothraeGymnospermGymnostomaHabitat destructionHaemodoraceaeHaplophaseHartig netHawaiian plantsHebeloma cristuliniformeHemiandra gardneri

rutilansHemibiotrophsHemigenia exilisHeterobasidion annosumHolobiotrophicHolobiotrophsHomobasidiomycetidaeHormonal actionHorticultural tradeHost-fungusHydnangium

carneumHymenoscyphus ericaeHypersensitive fleckingHyphaeHyphal anastomosis

digestiongrazingnetworksproliferationresponses

HypholomaHypochytrialesHypochytriomycetesHypochytriomycotaHypocrealesHysterangiumIlexIn situ conservationIndonesiaIndonesian archipelagoIngoldian fungi

206210210211249243

198, 206, 213, 215211211211211211324

127, 230, 232230, 233

32252

357209258

28, 34, 155328

151, 15534, 155, 158

282525282828

25847

107, 324202, 212200, 203242, 322

57212324

356, 364, 366-7245

50-1, 54, 68

5030

10551-2196324248

105-6, 111173, 185

119324324245324257248245243

85196

130, 222131293

233-4249

116-7, 163-4246198

110, 1589, 21

11128

116243243243243131326261

210-1, 295286273


Injecting treesInoculationInoculumIntroduced pathogensIrradiation (UV or Gamma-rays)JacksoniaJarrah die-backJuncaceaeKalmiaKennediaKobresiaLaboulbeniomycetesLabrinthulalesLabrinthulomycetesLabrinthulomycotaLacaziaLaccaria

laccataLambertia echinata

inermisorbifolia

LamiaceaeLaurasianLeaf and stem diseasesLechenaultia pulvinarisLedumLegumesLentinulaLeotealesLepidozamia

peroffskyanaLeporella fimbriataLeptospermum scopariumLeucopogon conostephioidesLinanthusLoggingLoliumLycoperdonLyophilizationLysinema ciliatumMacrozamia

communisfraseri

Macrozamia riedleiMaizeMalayMarasmius coniatusMarsupialsMeiosisMelaleuca

uncinataMelampsora liniMeliolalesMenyanthaceaeMercuric chlorideMesozoicMethyl bromideMexicoMicroascalesMicrobial communities

diversitysynergists

MicrocycasMicrococcusMicroorganism conservationMicropropagationMicrosporumMicrotis parvifloraMiddle EastMimosoideaeMineral nutritionMining operationsMitesMoniliopsisMonocalyptusMonotropaMonotropoideaeMouldsMucorMucor rouxiiMucoralesMyceliaMyco-heterotrophic orchidsMycologyMycorrhizal colonisation

community dynamics

360129-30, 277

159, 183, 186286, 291

31966, 108

250, 255167

127, 23266

107-8272243243243272

124, 13129332436132432447

339324232108

210, 225243

4749

206211228

23, 3119623

211271231

47, 664967

48, 66258

60210

129, 161244

108, 113, 118, 128211, 214-6

25324332432047

355214, 296

24333, 335

128747

3118

307, 309310

200, 223257

57, 59, 61204344

161, 311, 329-30200293

126, 206, 215126, 129, 215

328243, 272, 310, 328

328243, 272

110205, 210

30419727

Index 375

Mycorrhizal exchangefunctioningsyntheses

Mycosphaerella

leaf blotch diseaseMyoporaceaeMyricaMyricaceaeMyrtaceaeMyxomycotaN + P availabilityN-containing moleculesN eutrophication

associationN-fixing microbesN-limiting conditionsN starvationN transformationNarrow host range fungiNatural disturbances

suppressivenessNecrotic spottingNecrotrophsNeottiaNepenthesNew Zealand

Nicotiana tabacumNitrogen depositionNitrogen oxidesNorth America

Northern BettongNostocNothofagusNutrient acquisition strategiesNyctalisO-antigenic side chain

Obligate parasitesOcotea whiteiOidiodendron

Old rootsOlpidiopsidalesOomycetesOomycotaOphiostoma himal-ulmi

novo-ulmiulmi

OphrysOrchid rhizoctonias

symbiontsOrchidaceae 57, 126, 195, 199, 215, 218Orchids

OrchisOrdovicianOzonePacific seaboardPangaeaPapilionoideaePapua New GuineaParaglomusParasponiaPascopyrum smithiiPatagoniaPCR/DNA fingerprintingPCR-based molecular analysesPeloton isolationPenicillium

funiculosumvariable (glaucum)

PeronosporalesPestspH

PhaseoleaePhellinus weiriiPhenologyPhialophoraPhilotheca wonganensisPhoma

herbarum (pigmentivora)Phosphite

11022

277245, 287, 290,

297-9, 348290324

51-3, 55-6, 10852-3, 108

128, 294-5, 324243

3146

21-2, 27-946, 63-4, 67-9

9, 79

5565

2011834391

249245, 248

211327

1, 7, 50, 52, 131,210-11, 228, 239, 250

32712031

11, 51, 119, 122, 128,131, 168, 200, 210,233, 250, 252, 286

12846, 49- 51

108, 131, 210-1163

27890

46, 55, 60251285233

158-9243

243, 270, 273243

11250, 254-5, 275, 338

11, 286209, 212

198277

195-6, 200-1, 204, 206,211, 312

209, 212, 214155

31, 1623347

57-9, 61210, 286

15553-4, 57, 60, 68

332286249

213245, 272, 274, 310,

312, 328328328

243, 248, 254264-5

60, 87, 94, 115, 122, 162, 167,172, 204, 228, 350, 353, 355

59-6011

203, 212, 334274, 310

325326328

359, 362-3


Phyllodoce 232Phylogeny 61, 236, 303Phytophthora 11, 92, 254, 273, 346,

348, 351, 354-6, 358-9, 363cambivora 263cinnamomi 250, 255, 291-2, 338,

340, 342, 344-5, 352Phytotoxicity 361Pinus 22, 106, 108, 123, 131, 210

banksiana 123Pisolithus 109, 119, 124, 131, 293

tinctorius 109, 119,Pisonia 108, 118

grandis 122Pithomyces chartarum 245Pityrodia scabra 324Plant pathogen diversity 259Plant Preservative Mixture 309, 323Plantago 23Platylobium 66Plasmodiophora brassicae 254Plasmodiophorales 243Plasmodiophoromycetes 243Plasmodiophoromycota 243-4Platanthera 200, 212, 214Platypodium elegans 285Pleurocatena 278Pneumocystidales 272Pneumocystis 272Pollination 49, 212Pollution 120, 167Polygonaceae 167Polygonum 144Populus 31, 106, 108, 118, 127-8Poriales 243Positive laboratory air pressure 312Powdery mildew 249Pre-coralloid root 48Propagation of rare plants 172Proteaceae 167, 324, 345Proteus vulgaris 328Protosteliales 243Protosteliomycetes 243

Prunus 285Pseudomonas 81-2, 87, 92, 242

aeruginosa 328aureofaciens 88, 103, 311, 322cepacia ‘Gibraltar’ 328chlororaphis 90fluorescens 312, 328oleoverans 328syringae pv. lachrymans 91

Pseudotsuga 108, 119menziesii 126

Psidium guajava 295Pterochaeta paniculata 362Pterospora 215Pterostylis 202

acuminata 200, 223sanguinea 217

Puccinia psidii 294-6Pyrenophora tritici-repentis 90, 101Pythiales 243Pythium 252, 254-5, 257, 273, 285,

305, 348, 351, 354-5, 358oligandrum 92, 282sylvaticum 253torulosum 83ultimum 89-90

Pyxidiophora asterophorae 278Quercus rubra 127Radioactive carbon 205Rainfall 229Ralstonia solanacearum 89Ralstonia 89, 242rDNA internal transcribed spacer (ITS)

109rDNA sequencing 3Recovery of contaminated cultures 329Red-list macromycetes 278Rhamnaceae 52-3, 55, 60, 108, 362Rhinosporidium 272Rhizanthella gardneri 206, 211, 213-6,

221, 223, 225Rhizobium 46, 57, 62-3

Index 377

Rhizobium-based symbioses 50-Parasponia symbiosis 60

Rhizoctonia 198-9, 205, 211, 214,254, 348, 354

solani 200, 203, 352solani (AG8) 203

Rhizopus 310stolonifer 328

Rhododendron 127, 230, 232Rhodotorula 310, 312Rhodotorula rubra 328Rhytismatales 243Riesling grape 314Rosaceae 52-3, 55, 73, 334Rushes 166Russula sp 210Rusts 246Rutaceae 325Saccharomyces cerevisiae 328Salix 108, 118, 128, 143, 210Salmonella typhosa 328Salvia mellifera 29Santalum acuminatum 67Saprotrophs 245Sarcina lutea 328Sarcodes 142, 215Scab 249Scleroderma 124, 148

verrucosum 293Sclerospora sacchari 314Sclerosporales 243, 248Sclerotinia 245Sclerotium 245, 352Scotland 346Scrophulariaceae 167Scutellospora

calospora25, 28, 34, 155

25Sebacina 198Sedges 166Seed coat 276

germination 208-borne pathogens 348

Seiridium cardinale 286Septoria 245Serapias 209, 212Sesbania 59Shigella sonnei 328Shoot growth increases 82Smuts 246Soil-borne spores 157

disturbance 112, 168, 174-dwelling arthropods 160hyphae 117, 159, 164nutrient availability 140organic matter 115

Solanaceae 325Sonication 321South Africa 7, 47, 50-1, 68, 295,

297-8, 300South America 50-1, 63, 294-5, 338South East Asia 295Southern Hemisphere 50, 287, 293Sowerbaea multicaulis 324Spatial variability 220Species conservation 344Specificity 83, 118Sphagnum 228Spiranthes sinensis 200Spores 112, 158-9, 168Sporocarps 117Stangeria 47Stangeriaceae 47Staphylococcus 311

aureus 328epidermidis 328

Starch reserves 49Steam pasteurisation 354Stereales 243Streptococcus pyogenes 328Streptomyces 80, 242, 249Stylidiaceae 107, 325Stylidium scabridum 325Sub-culturing 308Succession 132, 174Swartzieae 58


Symbioses 5, 68, 276Symbiotic associations 218

seed germination 212Symonanthus bancroftii 325Synchytrium endobioticum 254Synertrol Oil 360Tanacetum vulgare 322Taphrinales 243, 245Tarsonemid mites 330Tasmanian bluegum 287Telephoraceae 210Teliomycetes 243, 246Temperature 342Tetratheca deltoidea 325Thailand 296Thanatephorus 198, 211, 214, 224Thelephora tomentella 200Thelymitra manginii 214, 217-8Thermosterilisation 321Thraustochytriales 243Tipularia 202, 211Tissue culture Ch 12Topsoil removal 174Topsoil 162, 174Toxic metal pollution 121Trametes 210Translocation 213Trema 60Tremandraceae 325Trichoderma 92, 272, 310Trichophyton mentagrophytes 328Trophic 245Trymalium ledifolium 362Tsuga heterophylla 137Tuber melanosporum 129Tulasnella 198

calospora 202Uapaca 108, 128Ulmaceae 53, 60United States 21, 91, 252Unopened flower buds 317Uredinales 243, 245, 248, 254, 272Uruguay 296

Ustilaginales 243, 245, 248, 254, 272Ustilago maydis 258Ustomycetes 243, 246UV light 90Vaccinioideae 228Vaccinium 232Vascular wilt 249, 254Venezuela 295Verticillium 254, 274Verticordia albidia 324

jamesonii 324sp 324

Vesicles 55, 159Villarsia calthifolia 324Viminaria 66Wash-down stations 357Waterlogging 115, 162Weeds 166Woollsia pungens 233Wurmbea tubulosa 324Xanthomonas 242

campestris 314Xanthorrhoea australis 360Xanthorrhoeaceae 345Xerotus javanicus 211Xylella 242Yeasts 310, 328Yoania australis 211Zamia 47

furfuracea 49pumila 49

Zamiaceae 47Zoosporic fungi 272Zululand 296Zygomycetes 107, 151, 155, 243Zygomycota 243, 244

Documents

Micoorganisms in Plant Conservation and Biodiversity