VOLUME 16 Supplement 2 Bulletin of the British Mycological Society

ESTABLISHMENT OF INFECTION

Pathogens Group MeetingRoyal Holloway College

7 July 1982

This was the first venture of the Pathogens Group and it was gratifying, there-fore , that more than one hundred people attended the meeting. This was despitethe national rail strike ; clearly , the Falklands Spirit was still fresh . Those luckyenough to stay overnight from the previous day's B.S.P.P. Offered Papers inPhysiological Pathology session were treated to a sherry reception amongst theawe-inspiring collection of Turners and Constables in the College's picturegallery. Our thanks are due to the local secretary, Richard Dixon, for organizingthe viewing and for his other excellent arrangements.

Papers dealt with the initial stages of fungal infection of plants , includingboth pathogenic and mutualistic associations, animals and other fungi . Develop-mental aspects were examined in each system but throughout the day speakersreturned to consider the common problems of fungal specificity and recog-nition phenomena. One objective of the Pathogens Group was to bring togethermycologists (in the broadest sense) interested in apparently disparate systems .It was our belief that this would enable us to recognize and to understand betterthose threads common to different areas. For me, this objective was realized inthe fascinating talk given by Jean-Paul Latge (Pasteur Institute, Paris). Hedemonstrated how similar are the processes of penetration and enzymic disso-lution of insect cuticle by entomogenous fungi and the more familiar processesof invasion of foliar organs by plant pathogens.

Having established the fungal infection, we shall next year (at ChelseaCollege) observe its Progress, in terms of solute exchanges between fungus andinfected organism, toxin and enzyme production and release. In a later meetingwe will consider the Outcome of Infection . Those who contributed to the firstmeeting have set high standards which we will hope to maintain.

Peter Ayres

Abstracts

Chemotaxis of Phytophthora zoospores. M.J. Carlile (Department of Pure andApplied Biology, Imperial College of Science and Technology, London SW72BB).

The zoospores of Phytophthora are capable of prolonged motility in soils. Theyare repelled by hydrogen ions and attracted by root exudates, with the catio-nic, neutral and anion ic fractions all being active . A range of amino acids,alcohols (including ethanol) , aldehydes and organic acids have been shown tobe attractants ; many of these are emitted by plants . It is probable that zoosporemotility and sensory responses , and the chemotropism of germ tubes thatemerge from encysted zoospores , all have a role in plant infection . A fulleraccount is being published - Carlile, M.J. (1982) in Erwin, D.C., Bartnicki-Garcia, S. & Tsao, P.H. ed. Phytophthora: its Biology, Ecology and Pathology.American Phytopathological Society, St . Paul, Minnesota.

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Spores on leaves: endogenous and exogenous regulation of development. I.K.Knights and J .A. Lucas, (R .H.S. Wisley and University of Nottingham BotanyDepartment).

Rust uredospores germinating on leaves undergo a complex developmentalsequence prior to infection, comprising germ tube emergence and extension,location of host stomata, appressorial formation and the differentiation ofassociated infection structures. Each step in this process is regulated by endo-genous checks and exogenous environmental factors prevailing on the leafsurface . This paper will review the various factors implicated in the control ofpre-infectional development, with particular emphasis on the early eventsassociated with germ tube emergence. Possible links between dormancy imposedby self-inhibitors and external .factors, notably light, will be explored, andthe significance of these factors in uredospore survival and subsequent infectionof the host will be assessed.

Ultrastructure of the establishment of the host-parasite interaction: comparisonsbetween mycoparasites and plant parasites. P. Jeffries, (Biological Laboratory,University of Kent).

Comparisons will be drawn between the ultrastructural events occurring duringthe establishment of the mature host-parasite interface during mycoparasitisrnand those occurring during phytoparasitism. Many close structural similaritiesexist and will be discussed from both a necrotrophic and a biotrophic per-spective . In both mycoparasitic and phytoparasitic systems , necrotrophy in-volves contact of host and parasite , the reorganisation and degeneration of hostcytoplasm, and subsequent death of host tissues. Biotrophic parasitism is morecomplex structurally, and a variety of interfaces may be involved during thepenetration phase, dependent both on host -mediated and parasite-mediatedresponses. In mycoparasitism , the host-parasite interface differs within thevarious host-mycoparasite combinations, and closer parallels can often bedrawn between haustoria of some mycoparasites and certain plant parasites,than amongst the mycoparasites themselves. Examples will be illustrated, and itwill be indicated that patterns of haustorial morphology appear to be limited,irrespective of the taxa to which host and parasite belong.

Ultrastructure of infection of animal tissues by animal pathogenic fungi . C.K.Campbell , (Mycological Reference Laboratory, London School of Hygiene andTropical Medicine).

A major group of mycoses begin invasion in the lungs, following passive carriageto the alveoli in inhaled air , and degree of infection has been correlated withaerial spore load on many occasions.

Events of subsequent fungal growth and host reaction have been docu-mented by electron microscope studies in experimentally-infected fowl chicks,in which the success or otherwise of the invading fungus varies considerablyand can be correlated with the immune response of the host .

Early establishment of other types of infection depends upon physicalcontact, either with conidia or, in the case of some ringworm fungi , with vegeta-tive hyphae inside tissue fragments from another host. For many , the simplepresence of fungus on the skin, given the correct environmental conditions,will be enough to allow the fungus to invade. For other, less adapted pathogens,infection only results from traumatic inoculation through the skin into deepertissues.

A third type of mycosis involves the change of yeasts in the gut flora

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from a commensal, harmless life, to active invasion of the tissue, and recentwork has concentrated upon the adhesive properties of yeast cells in earlyestablishment of infection, although here, too, inoculum potential plays animportant role.

Pathogenisity of entomogenous fungi with special reference to spore germina-tion and enzyme production. J.P. Latge , (Pasteur Institute, Paris).

Contrary to other pathogenic microorganisms of insects, such as bacteria andviruses, which infect their host via the digestive tract, entomogenous fungi doso by penetrating the integument of their host. The success of such infectionsdepends upon the presence of physical and chemical conditions favourableto the development of the fungus on the insect cuticle. In spite of the necessityto understand these mechanisms of infection before starting any biologicalcontrol field experiments, studies concerning the insect-fungus interactionsduring infection process have only been developed in the last 10 years with thehelp of natural or artificially produced mutants presenting various degrees ofvirulence.

The first critical event is the germination of the infectious propagule onthe cuticle of the insect. Germ tube formation can be stimulated by nutrients(originally present on the cuticle or resulting from the degradation of the integu-ment by fungal exocellular ensymes) or inhibited by toxic products of theepicuticle (short chain fatty acid, phenols) and/or by the cuticular microflora.Biochemical recognition of the cuticle by the fungus, inducing subsequently itsadherence to the integument, has been poorly studied.

The second or critical phase of the infection is the penetration of thecuticle by mechanical and enzymatic processes, requiring sequential enzymaticactivities. Depending on the species or strain studied, the specific influenceof lipase,glucanase, protease and chitinase on the aggressiveness of fungi has beenrecognized.

The third barrier against invasion is the humoral and/or cellular defencereactions, which the fungal pathogen encounters in the haemocoel of the insect.However, these reactions are only effective in the case of weak pathogens.

Host invasion in entomogenous members of the Entomophthorales., Patricia1. Brobyn ' , T.M. Butt"; A. Beckett", and N. Wilding! . e Rothamsted Experi-mental Station, Harpenden, Herts., AL5 2JQ. 2 Department of Botany, BristolUniversity, Bristol, BS8 lUG).

The primary conidia of the five species of entomophthoralean fungi examinedare enveloped in a sticky coat by which they adhere to the host. They germinateto form a short hyphal tube which usually, though not in Erynia neoaphidis ,develops an appressoriurn-like structure (ALS), terminally. In Neozygites freseniithe ALS forms, probably exclusively, on the germ tube developing from adistinctive type of secondary conidium, the capilloconidium. A penetrationtube develops from the ALS and penetrates the host by enzymic and physicalmeans. At the points of entry, the penetrating hyphae of Entomophthoramuscae make characteristic tri-radiate fissures in the cuticle; those of Conidiobolus obscurus and Entomophthora planchoniana usually produce tetra-radiatefissures. The germ tube of E. neoaphidis either attacks the host adjacent to theconidium or it grows a short distance over the cuticle before turning abruptlyand entering the host.

e. obscurus penetrates the cuticle of houseflies, which are resistant to thisspecies, but fails to develop further, suggesting cellular or humoral resistancemechanisms in the host haemolymph. N. fresenii, however, forms an ALS on

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resistant insects but fails to penetrate the cuticle, demonstrating that the cuticlecan act as a barrier to infection.

The Science Research Council are thanked for a CASE Studentship to oneof the authors (T.M.B.) and for Grant No. GR/B/04914 (to A.B.) for the EM-scope Sputter Cryo Apparatus used in part of this study.

Extracellular fungal enzymes in the infection process. R.M. Cooper (School ofBiological Sciences, University of Bath) .

Consideration will be given to the degradation of structural polymers of plantsduring infection by fungi .

Many pathogens of aerial organs of plants penetrate the cuticle directly .Recent ultrastructural and immunological studies clearly show the participat ionof (constitutive 7) cutinases in this process and also suggest they are a prere-quisite to pathogenicity. Cutinesterase action renders cuticles permeable tomolecules which may act in advance of invasion, such as fungal cell wall-de-grading enzymes (CWDE) as well as smaller molecules which could function astoxins . Penetration of walls containing the related biopolyester , suberin, isachieved by some pathogens of roots and tubers which can utilize it as a solecarbon source in culture. Along with lignin in secondary walls its degradationby plant pathogens is negligible but could function in overcoming rapidly formedwound barriers , e.g, as induced in wheat epidermal cells.

Subsequently, pathogens encounter host cell wall polysaccharides andmost produce a wide range of corresponding inducible , extra-cellular CWDE.This early molecular interaction can modify the type or balance of the host -parasite relationship but is not involved in varietal specificity. Enzymes ran-domly cleaving wall rhamnogalacturonan are especially significant in establish -ment of infection by necrotrophs as they are the first CWDE produced in vitroand in vivo (can exist in non germinated conid ia), rapidly release much of thewall as galacturonide and covalently linked neutral sugars, and cause protoplastdisruption following wall swelling or maceration often in advance of invasion.

In contrast , obligate biotrophs effect minimal wall changes during pene-tration probably due to low activities of partly bound CWDE such as arabina-nase and galactanase but absence of the destructive endo-polygalacturonidases.Facultative biotrophs may depend on limitation of the ir CWDE in host wallsby inhibitors, ionic binding , or molecular sieving.

Recognition and specificity in the plant-parasite interaction. Chris. Lamb" ,Ian Vose", and Frances Dewey' etThe Salk Institute, P.O. Box 85800, SanDiego, California 92138. 2 University of Oxford, Dept. of Biochemistry, SouthParks Rd., Oxford. 3 University of Durham, Science Laboratories, Durham).

Genetic and ultrastructural studies imply molecular interactions at the plant :fungus interface that lead to recognition and specificity in host : parasite inter-actions. Although a number of fungal molecules such as elicitors , suppressorsand specificity factors are apparently active in various bioassays related toplant : pathogen interactions, our understanding of the molecular basis ofrecognition and specificity is still fragmentary. The topic will be criticallyreviewed from a biochemical viewpoint. Recent data from our laboratoryobtained using immunological and high resolution electrophoretic and iso-electric focussing techniques to study fungal cell surface and extracellularmolecules will also be discussed.

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Specificity in mycorrhizal association. J.A. Duddridge (Department of Agri-cultural and Forest Sciences, University of Oxford, South Parks Road, Oxford,OXl3RB).

In nature most plants are susceptible to infection by mycorrhizal fungi but notto pathogens. In mycorrhizal associations selection pressure favours suscepti-bility rather than resistance and thus an important source of specificity is lost.Within each type of mycorrhiza both fungi and hosts lack close specificity.The highest degrees of specificity exhibited are only intergeneric and not in-frageneric.

Specificity requires recognition between symbionts. Recognition inducesa response in the plant which either facilitates or prevents infection by a poten-tial associate. Mycorrhizal symbionts seem to have evolved so that either theyevade being recognised as potential pathogens, or they are recognised as com-patible symbionts or, lastly, recognition only occurs between incompatiblesymbionts,

To understand specificity in mycorrhizal associations it is thereforeimportant to study interactions between mycorrhizal fungi and incompatiblehosts and those conditions which may induce incompatibility between normallycompatible partners. With this in mind I chose to study the interactions betweenSuillus grevillei, which is reputedly highly specific for Larix spp. with Larixkaempferi, Pinus sylvestris and Betula pubescens under different nutrientregimes.

Inoculum, inoculum potential and the biographical control of take-all. J.W.Deacon (Department of Microbiology, School of Agriculture, University ofEdinburgh, EH9 3JG).

The importance of inoculum potential in a range of host-parasite interactionswill be considered, with examples being drawn from leaf pathogens, root patho-gens, mycoparasites and mycorrhizal associations. Special attention will bedevoted to cereal root pathogens, for which we have evidence of an importantrole of natural senescence of the host root cortex in 'boosting' the pathogens'inoculum potential. Competition between parasites on the host root surfaceand especially in the senescing root cortex helps to explain the success of somebiological control agents. The implications of this are discussed for the take-allfungus, Gaeumannomyces graminis, one of the most important cereal rootpathogens.

Ectomycorrhizal synthesis using basidiospores as inocula. M.H. Ivory (Common-wealth Forestry Institute, Oxford).

Brief summaries are given of the reasons for inoculating with ectornycorrhizalfungi and the techniques which can be used for this purpose, followed by amore detailed discussion of the benefits and disadvantages of using spores asinocula.

The use which has been made of spore inocula, and the effectiveness ofthe results, are then reviewed, drawing mainly on published material, but alsoincluding more observations made in preparation for a research project. Threedifferent categories of spores, based on their mode of dispersal, are consideredseparately.

Finally, some problems which arise from the use of spore inocula, such asthe assessment of inoculum quality at the time of inoculation, are discussedin greater detail.