Rhizoctonia -Scots pine interactions: detection, impact onseedling performance and host defence gene response

Henrietta Grönberg

General MicrobiologyDepartment of Biological and Environmental Sciences

Faculty of Biosciencesand

Viikki Graduate School in BiosciencesUniversity of Helsinki

Academic Dissertation in General Microbiology

To be presented, with the permission of the Faculty of Biosciences of the University ofHelsinki, for public criticism in the auditorium 1041 at Viikki Biocenter 2 (Viikinkaari 5,

Helsinki) on October 10th 2008, at 12 noon.

Helsinki 2008

Supervisors: Professor Kielo HaahtelaFaculty of BiosciencesDepartment of Biological and Environmental SciencesGeneral MicrobiologyUniversity of Helsinki

Dr. Robin SenFaculty of BiosciencesDepartment of Biological and Environmental SciencesGeneral MicrobiologyUniversity of Helsinki(Current address: Department of Environmental andGeographical Sciences, Manchester Metropolitan UniversityDepartment of Environmental and Geographical Sciences, SoilEcology, Chester Street, Manchester M1 5 GD, UK)

Reviewers: Professor Frederick AsiegbuFaculty of Agriculture and ForestryDepartment of Forest EcologyUniversity of Helsinki

Dr. Taina PennanenThe Finnish Forest Research Institute,Vantaa Research UnitPL 1801301 Vantaa

Opponent: Professor Jarkko HantulaThe Finnish Forest Research Institute,Vantaa Research UnitPL 1801301 Vantaa

ISSN 1795-7079ISBN 978-952-10-4936-1 (paperback)ISBN 978-952-10-4933-0 (PDF, http://ethesis.helsinki.fi)

Printed: Ylipistopaino 2008, Helsinki, Finland

e-mail: Henrietta.gronberg@helsinki.fi

Front cover picture: Containerized healthy Scots pine seedlings at Röykkä nursery (topleft), tryphan blue stained Scots pine root colonised by BNR highlighting intracellularmonilioid cells (top right) and the apical region of a Scots pine root infected with tryphanblue stained hyphae (bottom left) and sclerotia and hyphae on the root surface (photosHenrietta Grönberg).

Table of Contents

List of original publications………………………………………………………………..4Abbreviations and definitions…………………………………………………………..….5Abstract………………………………………………………………………………….....6Tiivistelmä (Abstract in Finnish)…………………………………………………………..71. Introduction……………………………………………………………………………..8

1.1. Rhizoctonia taxonomy………………………………………………………….....81.2. Internal transcribed spacer (ITS) as a target marker for fungal identification…....91.3. Rhizoctonia spp. identification and molecular detection………………………….91.4. Rhizoctonia spp. as economically important fungal pathogens of plants………...101.5. Binucleate Rhizoctonia as symbiotic fungi and bicontrol agents…………….......11

1.5.1. Biocontrol: Principles, applications and mechanisms.....…………….......111.5.2. Competition as a potential mechanism for biocontrol................................121.5.3. Induced resistance: another potential mechanism for biocontrol...............12

1.6. Defence-gene activity…………………………………………………………….121.6.1. Defence-genes in conifers……………………………………………......121.6.2. Phenylpropanoid pathway……………….…………………………….....13

1.7. Induction of host defence-gene activity by hypovirulent strains………………...142. Aims of the study………………………………………………………………………153. Materials and Methods…………………………………………………………………16

3.1. Strains and methods described in publications…………………………………..163.2. Phylogenetic analysis………………………………………………...…………..18

4. Results………………………………………………………………………………….194.1. ITS probe development for Southern and liquid hybridization………………….19

4.1.1. Primary ITS PCR fragments and sequence alignment…....……...............194.1.2. Phylogenetic analysis…………………………………………………….194.1.3. Probe construction and application in Southern dot-blot- and liquidhybridization……………………………………………………………………20

4.2. Scots pine seedling growth and root architectural responses to binucleateRhizoctonia inoculation…………………………………………………………….....214.3. Bi- and uninucleate Rhizoctonia infection biology: antagonism and microscopystudies……………………………………………………………………………........214.4. Real time PCR…………………………………………………..………………..23

4.4.1. Quantification of fungal genomic DNA in infected roots…………….….234.4.1.1. Experiment 1..................................................................................244.4.1.2. Experiment 2.………………………… …....................................24

4.4.2. Defence-gene transcript levels……………………………………...…....245. Discussion……………………………………………………………………………...26 5.1. Rhizoctonia spp. ITS sequences and phylogenetic analysis ………………..…...26 5.2. ITS as a target for Rhizoctonia spp. probe development …………………..........26 5.3. Binucleate Rhizoctonia linked effects on Scots pine growth………………..…...28 5.4. Scots pine host defence responses to bi- and uninucleate Rhizoctonia and the

interactions in co-inoculation……………………………………………….….…..…296. Conclusions………………………………………………………………………….....327. Acknowledgements……………………………………………………………..……...338. References………………………………………………………………………….......34

List of original publications

The thesis is based on the following articles, which in the text are referred to by theirRoman numerals.

I Grönberg, H., Paulin, L. and Sen, R. (2003). ITS probe development forspecific detection of Rhizoctonia spp. and Suillus bovinus based on Southernblot and liquid hybridization-fragment polymorphism. Mycological Research107 (4): 428-438.

II Grönberg, H., Kaparakis, G. and Sen, R. (2006). Binucleate Rhizoctonia(Ceratorhiza spp.) as non-mycorrhizal endophytes alter Pinus Sylvestris L.seedling root architecture and affect growth of rooted cuttings. ScandinavianJournal of Forest Research, 21: 450-457.

III Grönberg, H., Hietala, A. M. and Haahtela, K. (2008). Analysing Scots pinedefence-related transcripts and fungal DNA levels in seedlings single- ordual-inoculated with endophytic and pathogenic Rhizoctonia speciesSubmitted to Forest Pathology.

The original articles were reprinted with kind permission from the copyright holders:British Mycological Society (I), and Taylor & Francis (II: Ref: RW/SFOR/N5904,http://www.informaworld.com).

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Abbreviations and definitions

AG anastomosis groupAM arbuscular mycorrhizal fungusSp-AMP antimicrobial peptide geneanamorph the asexual form of fungi also known as the vegetative stageavirulent non-pathogenicBNR binucleate RhizoctoniaCt cycle threshold, the PCR cycle at which the fluorescence exceeds that of

backgroundcDNA DNA synthesized from mRNA with reverse transcriptase (contains no introns)CHS chalcone synthasedhy-like dehydrin-like proteind.p.i. days post inoculationDNA deoxyribonucleic acid, the genetic materialGAPDH glyceraldehyde-3-phosphate dehydrogenasePsGER1 germin-like proteinh.p.i. hours post inoculationHR hypersensitive responsehypovirulent from avirulent to low virulent strains, which show no disease symptoms in host

plantsintron non-coding sequences within coding genes in eukaryotic genomeISR induced systemic resistanceITS internal transcribed spacer. The non-coding sequence between coding areas of

rDNA subunitsMCA microbial control agentsMF mycorrhizal fungiMNR multinuncleate Rhizoctoniamonilioid hyphal structures typical for Rhizoctonia infection in host cellNCBI national center for biotechnology informationprimer short oligonucleotide sequence used for specific amplification of target DNA by

PCR/qRT-PCRprobe oligonucleotide sequence used in hybridizations with target DNAqRT-PCR quantitative real time PCRrDNA ribosomal DNAr.l.c. root length colonisedpal1 phenylalanine ammonia lyasePCR polymerase chain reactionPsyp1 peroxidaseRAPD random amplified polymorphic DNA fragmentRFLP restriction fragment length polymorphismPR-proteins pathogenesis related proteinsPsACRE rapidly elicited defence-related geneRNA ribonucleic acidSCAR sequence-characterized amplified regionsclerotia structures formed by fungi typical to RhizoctoniaSOD superoxide dismutaseSp species (singular form)Spp species (plural form)STS stilbene synthaseteleomorph the sexual form of fungi producing spores and sclerotiaTm the specific melting temperature, where respective double stranded DNA denatures

to single strandsUNR uninucleate Rhizoctonia

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Abstract

Rhizoctonia spp. are ubiquitous soil inhabiting fungi that enter into pathogenic orsymbiotic associations with plants. In general Rhizoctonia spp. are regarded as plantpathogenic fungi and many cause root rot and other plant diseases which results inconsiderable economic losses both in agriculture and forestry. Many Rhizoctonia strainsenter into symbiotic mycorrhizal associations with orchids and some hypovirulent strainsare promising biocontrol candidates in preventing host plant infection by pathogenicRhizoctonia strains.

This work focuses on uni- and binucleate Rhizoctonia (respectively UNR and BNR)strains belonging to the teleomorphic genus Ceratobasidium, but multinucleateRhizoctonia (MNR) belonging to teleomorphic genus Thanatephorus and ectomycorrhizalfungal species, such as Suillus bovinus, were also included in DNA probe developmentwork. Strain specific probes were developed to target rDNA ITS (internal transcribedspacer) sequences (ITS1, 5.8S and ITS2) and applied in Southern dot blot and liquidhybridization assays. Liquid hybridization was more sensitive and the size of thehybridized PCR products could be detected simultaneously, but the advantage in Southernhybridization was that sample DNA could be used without additional PCR amplification.

The impacts of four Finnish BNR Ceratorhiza sp. strains 251, 266, 268 and 269 wereinvestigated on Scot pine (Pinus sylvestris) seedling growth, and the infection biology andinfection levels were microscopically examined following tryphan blue staining ofinfected roots. All BNR strains enhanced early seedling growth and affected the rootarchitecture, while the infection levels remained low. The fungal infection was restrictedto the outer cortical regions of long roots and typical monilioid cells detected with strain268.

The interactions of pathogenic UNR Ceratobasidium bicorne strain 1983-111/1N, andendophytic BNR Ceratorhiza sp. strain 268 were studied in single or dual inoculated Scotspine roots. The fungal infection levels and host defence-gene activity of nine transcripts[phenylalanine ammonia lyase (pal1), silbene synthase (STS), chalcone synthase (CHS),short-root specific peroxidase (Psyp1), antimicrobial peptide gene (Sp-AMP), rapidlyelicited defence-related gene (PsACRE), germin-like protein (PsGER1), CuZn- superoxidedismutase (SOD), and dehydrin-like protein (dhy-like)] were measured from differentiallytreated and un-treated control roots by quantitative real time PCR (qRT-PCR). Theinfection level of pathogenic UNR was restricted in BNR- pre-inoculated Scots pine roots,while UNR was more competitive in simultaneous dual infection. The STS transcript washighly up-regulated in all treated roots, while CHS, pal1, and Psyp1 transcripts were moremoderately activated. No significant activity of Sp-AMP, PsACRE, PsGER1, SOD, ordhy-like transcripts were detected compared to control roots.

The integrated experiments presented, provide tools to assist in the future detection ofthese fungi in the environment and to understand the host infection biology and defence,and relationships between these interacting fungi in roots and soils. This study furtherconfirms the complexity of the Rhizoctonia group both phylogenetically and in theirinfection biology and plant host specificity. The knowledge obtained could be applied inintegrated forestry nursery management programmes.

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Tiivistelmä (Abstract in Finnish)

Rhizoctonia-suvun sienet ovat maaperässä yleisesti esiintyviä sieniä, jotka voivat ollatautia aihettavia patogeeneja tai muodostaa hyödyllisiä symbionttisiavuorovaikutussuhteita kasvien kanssa. Yleensä Rhizoctonia spp. sienet ovatkasvipatogeeneja aiheuttaen mm. juurilahotautia niin metsäpuille kuin viljelykasveillekin.Useat Rhizoctonia–suvun sienet muodostavat orchidea-kasveille elintärkeitä symbionttisiasienijuuria (mykorritsa) ja jotkut tauteja aiheuttamattomista Rhizoctonia-kannoista voivatestää patogeenisten kantojen infektion tutkituilla kasveilla.

Tässä työssä keskityttiin yksi- ja kaksitumaisiin Rhizoctonia- sieniin, jotka kuuluvatteleomorfi Ceratobasidium-sukuun, mutta DNA koettimet kehitettiin myösmonitumaiselle Rhizoctonia-sienelle (teleomorfi Thanatephorus) ja nummitatti-ektomykorritsasienelle (Suillus bovinus). Kantaspesifiset koettimet suunniteltiinribosomaalisen DNA:n ”internal transcribed spacer” (ITS) -alueelle (ITS1, 5.8S ja ITS2)ja valmistettuja koettimia käytettiin “Southern dot blot”- ja nestemäisenhybridisaatiomenetelmän vertailuun. Nestemäinen hybridisaatio oli herkempi ja siinävoitiin määrittää myös hybridisoituvan PCR-tuotteen koko, mutta ”Southern”hybridisaation etuna oli, että koettimen hybridisoinnissa voitiin käyttää suoraan sienistäeristettyä kokonais-DNA:ta, jolloin PCR-reaktiota ei tarvittu.

Työssä tutkittiin suomalaisten kaksitumaisten Rhizoctonia-kantojen (Ceratorhiza sp.)251, 266, 268 and 269 vaikutusta männyn (Pinus sylvestris) taimien kasvuun jainfektiobiologiaan. Infektion taso määritettiin mikroskopoimalla trypaanisinellä värjättyjäinfektoituja juuria. Kaikki kaksitumaiset Rhizoctonia-kannat tehostivat nuorten taimienkasvua ja vaikuttivat juurten ulkomuotoon (lyhytjuurien määrä, juurten paksuus jne.),vaikka infektion määrä pysyikin alhaisena. Infektio keskittyi juuren uloimpaankuorikerrokseen ja näille sienille tyypillisiä ”monilioid”-soluja oli nähtävissä 268 kannallainfektoiduissa juurten soluissa.

Patogeenisen yksitumaisen Rhizoctonia-sienen (Ceratobasidium bicorne) 1983-111/1N ja endofyyttisen kaksitumaisen Rhizoctonia-sienen (Ceratorhiza sp.) 268infektiobiologiaa ja vuorovaikutusta tutkittiin kummallakin kannalla yksinään sekämolemmilla kannoilla samanaikaisesti infektoiduissa männyn juurissa. Sieni-infektionmäärä ja männyn transkriptioaktiivisuus määritettiin kaikista käsitellyistä juurista javerrokkijuurista kvantitatiivisella ”real time PCR” –menetelmällä (qRT-PCR) seuraavienentsyymien osalta: fenyylialaniiniammoniumlyaasi (pal1), stilpeenisyntetaasi (STS),kalkonisyntetaasi (CHS), lyhytjuurispesifinen peroksidaasi (Psyp1), mikrobeja hylkiväpeptidi (Sp-AMP), nopean puolustusreaktion aktivaation liittyvä geeni (PsACRE),germiinin kaltainen proteiini (PsGER1), CuZn- superoksididismutaasi (SOD) jadehydriinin kaltainen proteiini (dhy-like). Taimien siirrostus kaksitumaisella Rhizoctonia-sienellä ennen yksitumaisen Rhizoctonia-sienen infektiota rajoitti yksitumaisen kannanmäärää, mutta yksitumainen Rhizoctonia oli tehokkaampi infektoimaan juuria, kun sienetsiirrostettiin samanaikaisesti. Stilpeenisyntetaasin geenituotetta syntetisoitiin erittäintehokkaasti kaikissa käsittelyissä, mutta kalkonisyntaasi, fenyylialaniiniammoniumlyaasija peroksidaasi aktivoituivat vain kohtalaisesti. Geenien Sp-AMP, PsACRE, PsGER1,SOD, ja dhy-like transkriptioaktiivisuuksissa ei havaittu merkittäviä muutoksiaverrokkeihin nähden.

Työssä kehitetyt koettimet mahdollistavat kyseisten sienten osoittamisen juuristosta jamaaperästä. Tehty tutkimus auttaa ymmärtämään Rhizoctonia-sienten infektiobiologiaa,eri Rhizoctonia-kantojen vuorovaikutus-suhteita männyssä, sekä männynpuolustusreaktiota Rhizoctonia-infektion aikana. Tämä työ todisti aiemman käsityksennäiden sienten monimutkaisesta fylogeniasta, monimuotoisesta infektiobiologiasta jaisäntäspesifisyydestä. Tutkimuksesta saatua tietoa patogeenisen ja endofyyttisenRhizoctonia-sienen infektiobiologian eroista ja männyn puolustusreaktiosta voidaanhyödyntää entistä tehokkaampien torjuntamenetelmien kehittämiseksi sienitauteja vastaan.

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1. Introduction

1.1. Rhizoctonia taxonomy

The genus Rhizoctonia is a large, diverse and complex group of fungi (Sneh et a1.1996). In the identification of Rhizoctonia species, the teleomorph is the most importantmorphological character, but fruiting of Rhizoctonia species is difficult to induce underlaboratory conditions. Therefore, characterization and species designation is mainly basedon the vegetative characters of the anamorph that include mycelial colour, hyphaldiameter, number of nuclei, length of cells, shape and size of monilioid cells and sclerotialsize (Hietala 1998).

Rhizoctonia strains are designated as uni-, bi- or multi-nucleate (UNR, BNR andMNR, respectively), with respect to nuclear condition. The determination of nuclearcondition is particularly informative in isolate characterization of vegetative hyphae.Where teleomorph data is available, Rhizoctonia-like anamorphs are distributed in severalfamilies: Platygloeaceae (genus Helicobasidium), Exidiaceae (Sebacina), Atheliaceae(Athelia), Tulasnellaceae (Tulasnella), Ceratobasidiaceae (Thanatephorus,Ceratobasidium), and Botryobasidiaceae (Waitea) (Hawksworth et al. 1995). Overall, thelatter families with multinucleate (Thanatephorus and Waitea) and mostly binucleate(Ceratobasidium) anamorphs have received the most attention (see Sneh et al. 1996).Uninucleate Rhizoctonia strains, isolated from roots of conifer trees in Finland (Hietala etal. 1994) and Norway (Venn et al. 1986), belong to the Ceratobasidium family and areconspecific with a moss parasite Ceratobasidium bicorne, based on rDNA (internalspacer, ITS) sequence analysis (Hietala et al. 2001). More recently, a large number offungi displaying Rhizoctonia anamorphs have been isolated from tropical orchids inPuerto Rico by Otero et al. (2002) who found that 66 of 108 examined were uninucleateand showed highly related anamorphic and ITS phylogenetic relationships with Finnishconifer pathogenic uninucleate Rhizoctonia (Ceratobasidium bicorne) (Hietala et al. 1994,Hietala et al. 2001).

Due to its broad host range and common pathogenic association with importantagricultural crops, probably the most studied Rhizoctonia species is Rhizoctonia solaniKühn. The perfect state of R. solani, Thanatephorus cucumeris, belongs to the familyCeratobasidiaceae (order Ceratobasidiales) of class Basidiomycetes (Hawksworth et al.1995). Based on hyphal interactions in pairing experiments, multinucleate R. solani hasbeen grouped into different anastomosis groups (AGs) and represents a collection ofclosely related isolates grouped together based on their ability to anastomose with oneanother. In the so called “killing reaction” anastomosis results in death of anastomosingand adjacent cells, but no killing reaction occurs in self-anastomosis and in hyphalreactions between clonal isolates (Carling 1996).

Our understanding of Rhizoctonia genetic diversity and taxonomy has been greatlyimproved by this powerful tool, the anastomosis group concept (Vilgalys & Cubeta 1994).At least 12 anastomosis groups of R. solani are reported that include AG 1-11 and AG-B1(Carling et al. 1994, Carling 1996).

The telemorph Ceratobasidium belongs to the family Ceratobasidiaceae (orderCeratobasisiales) of class Basidiomycetes (Hawksworth et al. 1995). The anamorphs ofthis genus are mainly binucleate, except for the uninucleate Ceratobasidium bicorne Eriks.& Ryv. (Hietala et al. 1994, Hietala et al. 2001) mentioned earlier. The binucleateRhizoctonia species are also grouped into different AGs. The genus is divided into 21 AGs(from AG-A to AG-S) (Sneh et al. 1991). Ceratobasidium species commonly cause rootand foliar diseases (Kataria & Hoffmann 1988) but also forms symbiotic mycorrhizalassociations with orchids (Andersen & Ramussen 1996).

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Sen et al. (1999) proved by AG tests and rDNA-RFLP (restriction fragment lengthpolymorphism) fingerprinting a high degree of intraspecific variation within BNRbelonging to teleomorphs of Ceratobasidium cornigerum. These fungi include isolatesable to enter into either mutualistic or pathogenic root association with susceptible hostplants. In the anastomosis grouping analysis of different BNR strains, the Scots pine BNRisolates were found to be related to mycorrhizal BNR (anamorph Ceratorhiza spp.)endophytes of local Finnish and Canadian orchids. This was further confirmed in the ITS-RFLP analysis (Sen et al. 1999). Two of the Scots pine BNR isolates (268 and 251) fellinto the AG-I grouping known to contain members that are plant pathogens (Ogoshi et al.1983, Sneh et al. 1991). The AG-I tester isolate (AV-2) used, is a pathogen of Artemisiavulgaris var. indica (Ogoshi et al. 1979). The type anamorph of AG-I is R. fragariae(Ogoshi et al. 1983), which is known to cause the black root disease in strawberry(Wilhelm et al. 1972, Martin 1998). Interestingly, the Scots pine isolate 251 alsoanastomosed with two Canadian orchid isolates. Two other isolates (266 and 269) werenot members of any of the other 20 described AGs (AG-A - AG-S) of binucleateRhizoctonia (Sen et al. 1999).

1.2. Internal transcribed spacer (ITS) as a target marker for fungalidentification

The ITS region occurs as a part of a multicopy rDNA cistron in the fungal genome (I:Fig. 1), allowing PCR amplification also from degraded DNA samples. The ribosomalRNA genes are generally arranged as a tandemly repeated array with both variable andhighly conserved regions (Gardes et al. 1991). Internal transcribed spacer regions (ITS 1and ITS 2), between conserved rRNA genes (18 S, 5.8 S and 28 S subunits), are non-coding areas and are known to be quite variable within certain species and also tend to behighly variable between distantly related species and related genera (Bruns & Gardes1993).

Universal primers for fungal PCR amplification of the ITS region (ITS1/ITS 4) wereoriginally developed by White et al. (1990) (I: Fig.1). These primers allow fungal specificITS amplification directly from conifer tree associated mycorrhizas as they discriminateagainst most conifer host DNA templates, for example Scots pine (Pinus sylvestris)(Timonen et al. 1997) and Norway spruce (Picea abies) (Erland 1995).

The ITS is the most well-studied DNA marker in fungi and there is an extensive ITSfungal sequence database (cf. Kõljalg et al. 2005). This enables multiple sequencealignments between numerous fungal ITS sequences for robust phylogenetic analyses. Italso allows identification of target rDNA, both variable and conserved regions that couldalso represent suitable sites for molecular probe development. The ITS has been a target inseveral different molecular studies within Rhizoctonia species (see section 1.3) andmolecular approaches have been very fruitful in answering basic questions concerningsystematic relationships and genetic variation in Rhizoctonia spp. (Vilgalys & Cubeta1994). The continued growth of Rhizoctonia ITS sequence data (Gonzalez et al. 2001) hasenabled increasingly robust sequence comparisons and phylogenetic analyses that supportthe earlier anastomosis grouping in Rhizoctonia spp. classification (Sharon et al. 2008).

1.3. Rhizoctonia spp. identification and molecular detection

Because telemorph induction is a major problem within Rhizoctonia species, thecharacterization of isolates has been usually based on anamorphic characteristics. Sincethe vegetative characteristics of many Rhizoctonia species overlap, it is difficult to identify

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these species (Hietala 1998). Anastomosis grouping based on hyphal fusion has been apowerful tool in recognising Rhizoctonia groups within species (Carling 1996), butmolecular methods have improved Rhizoctonia isolate identification. Methods such asDNA/DNA hybridization (Kuninaga & Yokosawa 1984 and 1985), isozyme analysis (Liuet al. 1990), Restriction Fragment Length Polymorphism (RFLP) (Liu & Sinclair 1992),Randomly Amplified Polymorphic DNA (RAPD) analysis (Lilja et al. 1996, Leclerc-Potvin et al. 1999, Grosch et al. 2007) and sequence-characterized amplified region(SCAR) (Grosch et al. 2007) are specific tools used.

Another approach used, is PCR-based identification utilising strain specific rDNA-ITSregion targeted primers (Salazar et al. 2000, Kasiamdari et al. 2002) or taxon-specificprimers, as designed by Taylor and McCormick (2008) for basidiomyceteous orchidmycorrhizas belonging families Tulasnella and Thelephora-Tomentella complex. Lees etal. (2002), on the other hand, applied ITS region for detection and identification ofRhizoctonia solani AG-3 in potato and soil by more sensitive qRT-PCR-method, whichoptimally permits quantification at sensitivity of one gene copy.

1.4. Rhizoctonia spp. as economically important fungalpathogens of plants

Many Rhizoctonia species cause economically important diseases on most of theworld’s important crop plants, such as: cereals, cotton, sugar beet, potato, vegetables, fieldcrops, turf grasses, ornamentals, fruit trees and forest trees (Sneh et al. 1996). The bestknown of these, the multinucleate R. solani Kühn [teleomorph: Thanatephorus cucumeris(Frank) Donk ], causes losses averaging up to 20 % yearly in over 200 crops world-wide(Sneh et al. 1996). It causes damping-off disease on conifer trees and has been detected inforest nurseries in many countries including France (Perrin & Sampangi 1998) and Poland(St pniewska-Jarosz et al. 2006).

In Finland, on the other hand, mainly uni- or binucleate Rhizoctonia strains have beenisolated from diseased conifer seedlings, the most pathogenic strains being uninucleate(Lilja 1994, Hietala et al. 1994). Both in Finland and Norway, a uninucleate Rhizoctonia(Ceratobasidium bicorne) has been identified causing root dieback disease on Norwayspruce (Picea abies) and Scots pine (Pinus sylvestris) seedlings (Hietala et al. 1994;Hietala & Sen 1996, Hietala et al. 2001). These two tree species are economically themost important in Finland and thus form a major proportion of the different tree speciesgrown in nurseries and delivered for planting. Nurseries are involved in cultivation of treeseedling stock (1-3 years) for reforestation. Seedlings can be cultivated as bare-root stockin open nursery beds or in containers made from plastic, paper or peat. In Finland, papermanufacture and wood industries are an important source of national income andRhizoctonia species, that infects young conifer tree seedlings, may result in majoreconomic losses. To date, no effective control treatment against these pathogenic fungi hasemerged. The uninucleate Rhizoctonia attacks conifer roots from root tips and penetratesinto the vascular cylinder (Hietala 1998). The infection results in the root dieback diseasewith symptoms such as needle discoloration, partial death of the root system and stuntedseedling growth (Lilja 1996). Binucleate Rhizoctonia (BNR) are well known plantpathogens and have also been isolated from diseased and healthy looking Scots pine andNorway spruce seedlings (Lilja et al. 1992), but BNR isolates did not negatively influenceseedling growth in nursery peat soil pathogenicity tests (Lilja 1994, Hietala 1995).

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1.5. Binucleate Rhizoctonia as symbiotic fungi and bicontrolagents

Strains of Ceratobasidium spp. commonly exist in mycorrhizal associations withorchids (Sneh et al. 1996). These orchid associated Rhizoctonia strains are reported toform typical morphological structures in host tissues that have been identified as monilioidcells (Zelmer et al. 1996, Sharma et al. 2003).

BNR effects on plant growth promotion have been studied by Villajuan-Abgona et al.(1996) who observed that BNR strain W7 showed plant growth promotion in terms ofsignificant increase in plant height (P = 0.01) and fresh weight (P = 0.05). Harris (1999)presented a study where BNR isolates increased Capsicum shoot and root dry weights.Chang & Chou (2007) showed both a binucleate Rhizoctonia spp. and multinucleateRhizoctonia AG-6 of orchid mycorrhizal fungi (MF) stimulating the orchid Anoectochilusformosanus Hyata growth compared to non-mycorrhizal plants. Plant height, number ofleaves, root length and fresh weights were enhanced by these fungi and they also showedhigher enzymic activities of superoxide dismutase (SOD) in leaves, and acid and alkalinephosphatases in roots, together with higher contents of ascorbic acid, polyphenols, andflavonoids.

1.5.1. Biocontrol: principles, applications and mechanisms

According to a plant dictionary (Allaby 2006) biological control is defined as “human useof natural predators for the control of pests, weeds etc.” or the “total or partial destructionof pathogen populations by other organisms” (Agrios 1997). Biocontrol organisms areusually microbial control agents (MCA) namely bacteria, protozoa, fungi or viruses.Biopestisides, on the other hand, are “mass-produced, biologically based agents used forthe control of plant pests” (Chandler et al. 2008), which can be divided to livingorganisms, naturally occurring substances, and genetically modified plants (Copping &Menn 2000). Biocontrol agents are mainly developed to prevent specific pests orpathogens from infecting the target plant. Examples include hypovirulent or avirulentstrains, which infect the host without causing a disease. Protection is achieved eitherthrough direct biocontrol agent-mediated antagonism against the pathogen, throughcompetition for infection sites, or the biocontrol agent may strengthen the host defencethrough induced resistance (Cardoso & Echandi 1987).

Due to its wide distribution and severity as an economically important pathogen ofcrop plants, many studies have concentrated on investigating promising biocontrolsagainst R. solani Kühn. Both bacteria (Adesina et al. 2007, Yanqui et al. 2008,Scherwinski et al. 2008) and avirulent binucleate Rhizoctonia strains (e.g Escande &Echandi 1991, Villajuan-Abgona et al. 1996, Poromarto et al. 1998, Hwang & Benson2003, Pascual et al. 2004) have shown promise as biocontrol agents against R. solani indifferent plants. BNR (Ceratobasidium albatensis) has been shown to control R. solaniinduced damping-off in Pinus spp. (Rubio et al. 2001 and pers. comm.). The authorssuggested that the timing of the biocontrol inoculation is an important parameter. BNRprotective effects against R. solani are usually obtained following BNR pre-inoculationbefore R. solani attack, either after short periods of preinoculation e.g. 24 h (Poromarto etal. 1998) or 48 h (Xue et al. 1998) and up to 7 or more days of pre-inoculation (Hwang &Benson 2003). Because the biocontrol effect is not activated during simultaneousinfection, direct antagonism is probably not the mechanism involved (Herr 1995). Rathercompetition and induced resistance are suggested as being the controlling mechanism inthese relationships (see 1.5.2. and 1.5.3.).

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1.5.2. Competition as a potential mechanism for biocontrol

Competition signifies competition for nutrients or infection sites, or both. If the hostplant cells are pre-occupied by an avirulent strain, the pathogen tends to be unable toinfect same cells. Additionally, lack of nutrients may reduce the attraction of pathogens.Therefore indirect protection exists despite antimicrobial or defence related moleculesbeing involved. Competition is suggested to be one possible biocontrol mechanism inR.solani suppression in BNR treated plants (Cardoso & Echandi 1987, Herr 1995).

1.5.3. Induced resistance: another potential mechanism for biocontrol

Infection of plant with an avirulent strain or its secreted molecules might elicit a hostdefence reaction, which in subsequent challenge or infection is more rapidly andefficiently activated. This systemic acquired resistance (SAR) (Ryals et al. 1994) orinduced systemic resistance (ISR) (Xue et al. 1998) can prevent further infection of planthost by potentially more severe pathogens. Thus, SAR/ISR can be exploited as abiocontrol mechanism. The SAR includes the activation of host defence genes and oftenalso a plant hypersensitive response (HR). HR is expressed in plant tissues as self-inducedlocalized cell death at the site of infection, which prevents the pathogen from furtherpenetration into adjacent cells, and is associated with generation of activated oxygenradicals (Agrios 1997).

Many authors have suggested induced resistance being the mechanism of protection inBNR-preinoculated plants against pathogenic R. solani (Cardoso & Echandi 1987, Xue etal. 1998, Porommarto et al. 1998, Hwang & Benson 2003) and several defence genes havebeen reported to be involved.

1.6. Defence-gene activity

1.6.1. Defence-genes in conifers

The arsenal of defence genes activated in coniferous trees and the timing of thisactivation depends on the specific elicitor and the pathogenicity of each strain. In Scotspine (Pinus sylvestris), for example, the transcriptional responses were differentiallyactivated in mutualistic, saprotrophic or pathogenic fungi (Adomas et al. 2008), andduring the early stage fewer genes were differentially expressed in pathogenic, as opposedto ectomycorrhizal or saprotrophic interaction. The authors suggested that as the host wasunable to recognise the nature of the attack during the early stages of infection, thisenabled the pathogen to overcome the host’s defence. In the later stages of infection, thehost defence responded more strongly to interaction with the pathogen. Additionally,induction of defence genes in Norway spruce were more rapid in a pathogen resistantclone compared to less resistant one (Karlsson et al. 2007).

Pathogenesis related (PR) proteins are a diverse group of plant proteins possessingantimicrobial properties against invading fungal pathogen. Some of the best known arePR1 proteins, -1,3-glucanases, chitinases, lysozymes, PR 4 proteins, thaumatinelikeproteins, osmotinlike proteins, cysteine-rich proteins, glysine-rich proteins, proteinaseinhibitors, proteinases, chitosanases, and peroxidases (Agrios 1997). Some of these havebeen reported as also being involved in the interactions between coniferous trees anddifferent fungal strains.

13

Chitinases were an important example of differentially expressed genes in Scots pine(Pinus sylvestris) during the infections involving mutualistic, saprotrophic or pathogenicfungi (Adomas et al. 2008). These enzymes degrade the fungal cell wall and have, inmany other studies, been reported to be involved in coniferous plants defence (Hietala etal. 2004, Jøhnk et al. 2005, Fossdal et al. 2007, Karlsson et al. 2007, Adomas et al. 2007).Peroxidases, on the other hand, are activated during both ectomycorrhizal (Laccariabicolor) and pathogenic (Heterobasidion annosum) interaction in Scots pine (Adomas etal. 2008) and in H. annosum infection on Norway spruce [Picea abies (L.)Karst.] (Jøhnket al. 2005, Karlsson et al. 2007). A class III peroxidase activity and thaumatin wereactivated constitutively (1, 5, and 15 d.p.i.) in Pinus sylvestris during Heterobasidionannosum- infection (Adomas et al. 2007) and related transcripts were also up regulated insaprotrophic (Trichoderma aureoviride) infection at 1 d.p.i. (Adomas et al. 2008). Othertranscripts reported to be induced in H. annosum- Scots pine interaction includes forexample glutathione-S-transferase and germin-like protein (Karlsson et al. 2007).

Another pathogen, a uninucleate Rhizoctonia (Certatobasidium bicorne), induced 25transcripts in Norway spruce, including oxidative-process-associated proteins, such asgermin-like isoforms, peroxidase and a glutathione S-transferase (Jøhnk et al. 2005).Some of the sequences used in a related study (Fossdal et al. 2007) for primer design andmany transcripts, including chitinases, and defensin, together with other previouslymentioned enzymes, were activated in the same host by this pathogen.On Scots pine, an antimicrobial peptide gene Sp-AMP (Asiegbu et al. 2003) and adefence-related gene PsACRE (Li & Asiegbu 2004) were associated in Heterobasidionannosum infection. The antimicrobial gene was also induced by the saprotroph andsymbiotic fungi at 1 d.p.i., but up-regulation continued only in the pathogenic interaction 5and 15 d.p.i. (Adomas et al. 2008). This pathogen induced 179 differentially expressedsequence tags in a cDNA microarray study, the most abundant ones being enzymesinvolved in the phenylpropanoid pathway and defence-related proteins with antimicrobialproperties (Adomas et al. 2007). Whether these genes are correspondingly up-regulated inScots pine roots during the Ceratobasidium bicorne infection remains unknown.

1.6.2. Phenylpropanoid pathway

The amino acid L-Phenylalanine is a precursor for phenylalanine ammonia lyase(PAL) catalyzing the first reaction in a phenylpropanoid pathway leading to cinnamicacids. It consists of a C6C3 carbon skeleton, which by further hydroxylation andmethylation yields simple phenylpropanoids such as, p-courmaric, caffeic, ferulic andsinapic acids (Gross 1985). Many other important and more complex secondary metaboliteproducts are also produced in further steps of this pathway that include lignin, flavonoids,and stilbenes. The latter two are respectively synthesized by chalcone synthase (CHS) andstilbene synthase (STS), two closely-related polyketide synthases (PKS), in acondensation of p-coumaroyl-CoA /cinnamoyl-CoA with three molecules of malonyl-CoA(Hahlbrock & Grisebach 1979, Dixon & Paiva 1995). The STS in Pinus sylvestris isclassified as pinosylvin synthase, since it prefers cinnamoyl-CoA to dihydrocinnamoyl-CoA as a substrate, but the substrate preference in expression studies in vitro is influencedby factor(s) co-existing in plant or bacterial extracts (Schannz et al. 1992). In Pinusstrobus (Eastern white pine), on the other hand, both substrates are utilized and thereforetwo stilbene types - pinosylvin and dihydropinosylvin- exist (Raiber et al. 1995). The STSsequences of P. strobus clustered in their own subgenus (STROBUS STS) in the study ofKodan et al. (2001), while Pinus densiflora clustered together with P. sylvestris in anothersubgenus (PINUS STS) and all CHS sequences from these three Pinus species in theirown CHS subgenus. Two P. strobus CHS sequences have been identified by Schröder etal. (1998), one being more closely related to P. sylvestris CHS, while the other usingmethylmalonyl-CoA in a condensation with c-methylated chalcones and suggested to have

14

a special role in the biosynthesis of secondary products. The latter type of CHS is notpresent in P. sylvestris (Schröder et al. 1998). The Scots pine defence-related activation ofstilbenes is elicited by stress factors such fungi, ozone, or mechanical stress (Lange et al.1994, Fliegmann et al. 1992, Zinser et al. 1998, Chiron et al. 2000).

Many enzymes of the phenylpropanoid pathway are coded by multiple genes in manyplants (Dixon et al. 2002). For example, PAL, is encoded by a multigene family in pine(Pinus banksiana) (Butland et al. 1998) and also has multiple isoforms in Arabidopsis(Cochrane et al. 2004). PAL defence-related activation together with many other defence-related transcripts, were recently shown in Heterobasidium annosum elicited conifers(Karlsson et al. 2007, Adomas et al. 2007, 2008) and uninucleate Rhizoctonia(Ceratobasidium bicorne) -infected Norway spruce, where the enzyme activationcompared during the infection and drought stress (Fossdal et al. 2007).

1.7. Induction of host defence-gene activity by hypovirulentstrains

Pre-inoculation of bean seedlings with non-pathogenic BNR have been reported toinduce the expression of host defence enzymes e.g. peroxidases, and 1,3- - glucanases,which positively correlated with induced systemic resistance (ISR) against R.solani andColletotrichum lindemuthianum pathogens (Xue et al. 1998). Seedlings treated with BNRprior to infection elicited significantly more of these host defence enzymes compared tothe diseased and control plants. Similar enzymes were also induced in soybeans pre-treated with Pseudomonas aureofacien bacteria (Jung et al. 2007) and are also generallyconsidered as being involved in plants induced resistance (Agrios 1997). Inducedresistance was also suggested as being the mechanism in a study of Cardoso & Echandi(1987) who showed that BNR filtrates did not inhibit R. solani but BNR treated rootexudates did such that the R. solani protective effect was retained in BNR pre-inoculatedroots after surface sterilization.

Preinoculation of bean seedlings with an arbuscular mycorrhizal fungus (AM) Glomusintraradices Schenck and Smith, on the other hand, did not significantly prevent diseasesymptoms of R. solani, and no change in phenylalanine ammonia lyase, (PAL), chalconesynthase (CHS), or chalcone isomerase were observed. R. solani has been reported toinduce a systemic increase of defence genes, regardless of the AM status of the plant(Guillon et al. 2002). Wen et al. (2005) found that the highest defence-gene activities of1,3- -D- glucanase, PAL, and CHS, were in R. solani infected beans, but were alsoexpressed, though down-regulated in seedlings pretreated with non-pathogenic binucleateRhizoctonia before R. solani infection. Cardinale et al. (2006) investigated the activities ofbiological defence markers PR1, laminarinase, and chitinase to follow the plant-mediatedresistance of hypovirulent R. solani against Botrytis cinerea pathogen in tomato.Hypovirulent strain of R. solani boosted the B. cinerea induced enzyme activities and theauthors suggested a systemic induced resistance being the nature of protection.

15

2. Aims of the study

The aim of this study was to investigate the infection biology and impact of pathogenicuninucleate Rhizoctonia (UNR) (Ceratobasidium bicorne) strains 1983-111/1N and 263,and endophytic binucleate Rhizoctonia (BNR) (Ceratorhiza sp.) strains 268, 251, 266 and269 on Scots pine growth and host defence responses, and to detect these strains frominfected roots. More specific goals in the respective studies were to:

develop DNA probes targeted to ITS region of fungal rRNA to differentRhizoctonia groups (uni-, bi-, or multinucleate) and Suillus bovinus and applyprobes in comparison of Southern- and liquid hybridization (Hendolin et al.2000) against target DNA from relevant fungi (Suillus, Rhizoctonia and otherplant root pathogenic fungi). Probes were designed to be used for strain-specific detection.investigate and to better understand fungal-host interactions - infection biology,morphology, and root and shoot growth responses - of Scots pine seedlinginfected with four binucleate Rhizoctonia strains (268, 251, 266 and 269).quantify Scots pine host defence-gene (pal1, STS, CHS, Psyp1, Sp-AMP,PsGER1, PsACRE, SOD, dhy-like) activities in roots during infection with theroot pathogenic UNR strain 1983-111/1N and endophytic BNR strain 268, byinfecting respective roots either separately on single-inoculations or as dual-inoculations in order to detect possible strain specific responses in Scots pinedefence-reaction.

16

3. Materials and Methods

3.1. Strains and methods described in publications

This study targeted uni- and binucleate Rhizoctonia spp. strains (Table 1), but relevantfungi from the genus Suillus and Rhizoctonia, and other plant root pathogenic fungi wereincluded in hybridizations (I: Table 1). All methods used in the three separate studies arebriefly described in Table 2, except for methods relating to unpublished experiments,which are described separately.

Table 1. Fungal Rhizoctonia spp. strains

Strain Telomorph Nuclear status Reference Study1983-111/1N (Ceratobasidium

bicorne)UNR Hietala et al. (1994) III

263 (Ceratobasidiumbicorne)

UNR Hietala et al. (1994) I

268 (Ceratorhiza sp.) BNR Sen et al. (1999) I, II, III251 (Ceratorhiza sp.) BNR Sen et al. (1999) I, II266 (Ceratorhiza sp.) BNR Sen et al. (1999) I, II269 (Ceratorhiza sp.) BNR Sen et al. (1999) I, II

17

Table 2. Methods used in three separate studies.

Method Reference study Short descriptionSeed sterilization Sen (2001) II, III Seeds: M29-85-2725 and M29-92-0079, Finnish Forest

Research Institute, sterilization: +4ºC o/n, 30% H202

Seedling growth Sen (2001), Heinonsalo etal. (2001)

II, III pots: 20 h day length, >230 µmol m-2 s-1, +20/12ºC,cuttings in work II and seedlings in work III, 19 h daylength, 170-300 µmol m-2 s-1, +19-24/15ºC

Inoculation Sen (2001) II, III II: over 30 days after or in the cuttings at the time of 14-day-old seedling removal, III: one week after or BNR in theexperiment 2 at the time of 10-day-old seedling removal

Fungal growth Marx (1969), Modess,(1941), Sen et al. (1999),Murashige & Skoog(1962)

I, II, III +20ºC, medias: Modified Melin Norkans (MMN), Hagem´sagar (HA), malt-extract agar (MA), potato-dextrose-agar(PDA), Murashige and Skoog nutrient salts medium (MS),Water agar (WA)

DNA extraction Heinonsalo et al. (2001),Qiagen Product Manual

I, III CTAB-method, DNeasy plant mini kit (Qiagen, Hilden,Germany)

RNA extraction Qiagen Product Manual III Plant MiniRNeasy kit (Qiagen, Maryland, USA)PCR Heinonsalo et al. (2001) I, IIICloning Kõljalg et al. (2002) I pGEM®-T easy vector (Promega, Madison,WI )Sequencing I ALF sequencer (Amersham Biosciences, Chalfont) or ABI

Prism 310 capillary sequencer (Applied Biosciences,Foster City, CA)Analysis: GCG Fragment Assembly System (ProgramManual for the Wisconsin Package, Version 8, Wisconsin)

Sequencealignment

Higgins et al. (1992) I Clusteral method

Primer design I, III III: ABI Primer Express® (Applied Biosystems, Foster City,CA, USA)

Liquid- andSouthernhybridization

DIG system’s user guide(Roche, Germany),Karcher (1991), Hendolinet al. (2000).

I DIG (Roche, Germany) or Biotin (Institute ofBiotechnology, UH, Finland) labelled probes

cDNA synthesis III Taqman Reverse Transcription Reagent, (AppliedBiosystems, New Jersey, USA)

Real time PCR Guide to performingRelative Quantitation ofGene Expression UsingReal Time QuantitativePCR. Applied Biosystems

III ABI 7300 quantitative real-time PCR machine, (AppliedBiosystems, Singapore)

Tryphan bluestaining

Koske & Gemma (1989) II, III fungal root-infection visualization, III: 10 min. +60 ºC in 2,5% KOH, rinsed twice with H2O, neutralized in 1% HCl 1 hat RT, stained 10 min. +60 ºC in tryphan blue.

Microscopy II, III II: Olympus BX50 (Olympus, Japan), III:Olympus BX50F-3(Co., Hamburg, Germany) and Image-® PLUS software(version 4).

Fungal infectiondetermination

Giovanetti & Mosse(1980),Hietala et al. (2008)

II, III II: Grid-line intersect method, III: ITS-primer and qRT-PCR-standard curve based quantification

Statistics Paffl (2001) (III) II, III II: one-way ANOVA and pair wise differences by Tukey’sHSD test, SPSS version 8.0 for MS Windows.III: GLMprocedure, SAS (P ‹ 0.05)., III: analysis of variance, GLMprocedure of SAS (P<0.05)

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3.2. Phylogenetic analysis

A nearest neighbor tree based on ITS (ITS1-5,8S-ITS2) sequence (I) analysis wasgenerated using the MEGA4 program (Tamura et al. 2007). Waitea circinata strain TGCC17.1 (de la Cerda et al. 2007) was selected for outgroup in this analysis.

19

4. Results

4.1. ITS probe development for Southern and liquid hybridization

4.1.1. Primary ITS PCR fragments and sequence alignment

The size of the single fragments PCR products amplified with ITS 1 and ITS 2 primersfrom genomic DNA were between 644-703 base pairs ( ). The Rhizoctonia solani HK1PCR product was 703 bp.

As expected the 5.8 S was highly conserved among different Rhizoctonia strains andSuillus species. In the Suillus ITS sequence comparison the most variable region located inITS 1 for the two S. bovinus strains (SBH1 and C3-1-M1) differed only by a single basepair in their ITS sequence (I). The beginning of this ITS 1 region was targeted for Suillusprobe construction (I: Fig. 1). ITS 1 was also the most variable region identified in theRhizoctonia ITS sequence comparison, but the similarly variable ITS 2 region wastargeted for probe development, since suitable optimal primer sequences for PCRamplification were found in this region. The conserved 5.8 S ribosomal gene region wastargeted for generation of a control ‘fungal’ probe (I: Fig. 1)

4.1.2. Phylogenetic analysis

The nearest neighbour phylogenetic analysis of the ITS sequences indicated that BNRAV-2 (AG-I) and 268 strains were closely related in the same Rhizoctonia cluster (Fig. 1.).Strains 251 and 266 were more closely related with each other than these strains to otherbinucleate Rhizoctonia strains. All binucleate Rhizoctonia strains (251, 266, AV-2, 268and 269) were found in a general cluster and as was expected, the uninucleate 263 andmultinucleate Rhizoctonia solani strains were clearly less related to the binucleateRhizoctonia cluster.

20

97

63

72

72

40

0.1

Figure 1.Nearest neighbour tree. Symbols: AV-2, 268, 269, 266, 251, 263, R.solaniHKI, Waitea circinata.

4.1.3. Probe construction and application in Southern dot-blot- and liquidhybridization

Primer sequences designed to amplify particular probe sequence from ITS targetsequence are presented (I: Table 3). The size of the PCR amplified probes were between124 and 151 bp. PCR products were of the correct size, except for a few problematicsamples. When control primers were used to amplify the control probe sequence and whenRhizoctonia solani ITS insert in plasmid vector was used as a template, the PCR productoccasionally included additional faint bands (data not shown). Amplification of Suillusstrain template with Suillus primers also produced a faint smaller band, but DNA from thecorrect sized bands extracted from agarose gel and used in secondary PCR, producedmostly only the expected sized DNA fragment without additional bands.

The restriction analysis of amplified probes gave the expected size restrictionfragments, except the Rhizoctonia solani probe sequence amplified with Rhizoctoniaprimers, where some additional bands were detected (data not shown).

The optimal hybridization temperature for oligonucleotide probes were about 15-30ºC lower than the Tm, while near the Tm in PCR amplified probes (I: Table 2.). Theestimated detection limit to DIG labeled probes in dot blotting were 0.1-0.01ng/ l(dilutions 1:10-1:100). Dot blot hybridizations with DIG-labelled probes targeted totalDNA templates extracted from fungal pure cultures or from Scots pine (Pinus sylvestris)roots and ITS-PCR amplified from this same DNA was used in biotin-probehybridizations. A summary of dot blot hybridization results (I: Table 4) shows the biotinlabeled oligonucleotide probes specificity over PCR amplified DIG-probes. For example,a probe (number 3) developed to strain 268, hybridized only with the respective strain,while the DIG-labelled probe (number 7) developed to the close relative AV-2 hybridizedwith all Finnish binucleate strains (I: Table 4). Biotin labelled S. bovinus probe (number 2)also detected only strains of S. bovinus species, while DIG-labelled probe (number 1) alsodetected S. granulatus.

The same hybridization temperatures optimised for Southern hybridization also gavethe best results in liquid hybridization, indicating the temperatures being dependent onprobe sequences rather than method applied. Low temperature washing (30 or 35ºC)occasionally affected the specificity with 268 or Suillus probe (number 3 and 2 probesrespectively in I: Table 2). The biotin labelled probe in combination with liquidhybridization delivered the highest detection specificity (I: Table 5 and Fig. 4).

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4.2. Scots pine seedling growth and root architectural responsesto binucleate Rhizoctonia inoculation

Compared to uninoculated controls, the shoot and root weights in the BNR-inoculatedseedlings exhibited significant (P=0.05) differences at 87 d.p.i. (II). No strain specificdifferences were detected among the four Finnish BNR isolates (251, 266, 268 and 269).The root biomass and length increased, while the long root width decreased and the shortroot numbers (cm-1 long root) remained of a similar magnitude as uninoculated seedlings.

By the second harvest (240 d.p.i.), the mean short root numbers increasedsignificantly, but no differences in shoot biomass were detected compared to uninoculatedcontrols. The root growth responses were also more attenuated. The root infection levelsremained extremely low (<6% and 1% r.l.c. at 87 and 240 d.p.i., respectively).

4.3. Bi- and uninucleate Rhizoctonia infection biology:antagonism and microscopy studies

No antagonistic effects were detected between BNR 268 and UNR 1983-111/1N in adual inoculation on water-agar Petri-dish (Fig. 3 a). Monilioid cells detected from theroots of tryphan blue stained Scots pine radicals infected with BNR strain 268 nine weeksp.i. (Fig. 2 a) (II: fig 2), and eight d.p.i. in the surface of UNR-infected roots (Fig 2. c)(III). No monilioid cells were developed in BNR infected roots during the monitoredperiod in study III. In single inoculations, BNR and UNR colonization was detected incortical cells throughout the entire root, but BNR colonized more intensively root cellsfrom the basal region, while UNR colonized heavily the root tips and less intensively basalregions in a contrasting manner to BNR (Fig. 3 b, d). Both UNR colonized anduncolonized root tips were detected in dual- infected roots (Fig. 3 c, III).

22

Figure 2. a.) BNR moniloid cells b.) BNR septate hyphae (arrow), c.) UNR monilioid structures inthe root surface and d.) UNR hyphae growing between root cells. Bars 1 µm.

23

Figure 3. a.) UNR and BNR hyphae growing towards each other on WA-plate. Arrows show thehyphal tips, which are not affected by the other strain (Bar 5 µm) b.) Scots pine root tip infectedwith BNR, c.) BNR and UNR, and d.) UNR. (Bars 10 µm).

4.4. Real time PCR

4.4.1. Quantification of fungal genomic DNA in infected roots

Fungal genomic DNA was measured as in Hietala et al. (2008) using strain-specificrDNA ITS-primers and GAPDH primers respectively for detection of fungal genomicDNA from infected roots and host genomic DNA (III: Table 1). The level of colonizationwas estimated from the fungal DNA/ (host DNA +fungal DNA) -ratio in each root sample.

4.4.1.1. Experiment 1.

The BNR infection in dual infected roots in experiment 1 remained low, but thedifference in the BNR DNA content at 96 h.p.i between single and dual inoculation wasnot significant (p = 0.07). In the single inoculations, the mean DNA content of both UNRand BNR reached near 5% 96 h.p.i. (Fig. 4 a and b, III: Fig. 1). The contents of UNR

24

infection were similar both in single and dual inoculated roots (Fig. 4 b., III: Fig. 1 b). TheBNR and UNR contents transiently decreased at 24 and 48 h.p.i., respectively.

a.

-2 %

0 %

2 %

4 %

6 %

8 %

10 %

12 %

14 %

16 %

0h 4h 24h 48h 96h 192h

inoculation time

DN

A

BNR Exp 1BNR Exp 2BNR+ Exp 1BNR+ Exp 2

b.

-2 %0 %2 %4 %6 %8 %

10 %12 %14 %16 %18 %20 %22 %24 %

0h 4h 24h 48h 96h 192h

inoculation time

DN

A

UNR Exp 1UNR Exp 2UNR+ Exp 1UNR+ Exp 2

Figure 4. The mean fungal DNA contents (%) of total (host and fungal DNA) in infected roots atinoculation (0) and 4, 24, 48, 96 and 192 h.p.i. DNA contents in single BNR (a) and UNR (b)infected roots, DNA of respective strains detected from dual- infected roots (+). In experiment 1,both fungi were inoculated simultaneously while in the experiment 2, BNR was inoculated oneweek earlier.

4.4.1.2. Experiment 2.

Experiment 2 was performed similarly to experiment 1, except that BNR waspreinoculated one week earlier in respective roots in order to determine possiblebiocontrol effects of BNR against UNR, and prolonged inoculation times were used. In theBNR-preinoculated dual infected roots, the DNA content of UNR remained significantly(p = 0.02) lower 192 h.p.i. than in single inoculated roots (Fig. 4 b, III: Fig. 1 b). Theoverall BNR DNA contents were similar between single and dual infected roots through-out the experiment. (Fig. 4 a, III: Fig. 1 a).

4.4.2. Defence-gene transcript levels

The STS transcript was highly up-regulated in all treated roots compared to the controlover the experimental period in both experiments (Fig. 5 a, and 6 a, III: Fig. 2 a). Thehighest activities were detected in UNR single infections. BNR and dual inoculated rootsshowed relatively similar STS transcript levels.

25

a.

-180

-130

-80

-30

20

70

120

170

220

4 24 48 96 4 24 48 96 4 24 48 96 4 24 48 96

Control BNR UNR BNR+UNR

norm

aliz

ed tr

ansc

ript

leve

lsPALCHSSTSPRX

b.

-202468

10121416

4 24 48 96 4 24 48 96 4 24 48 96 4 24 48 96

Control BNR UNR BNR+UNR

norm

aliz

ed tr

ansc

ript

leve

ls PALCHSPRX

Figure 5. Defence-gene transcript activities in the experiment 1 (III). a.) STS, PAL, CHS, andPRX. b.) PAL, CHS, and PRX repeated in order visualize differences between the transcripts.

a.

-150-5050

150250350450550650

0 48 96 192 0 48 96 192 0 48 96 192 0 48 96 192

Control BNR UNR BNR+UNR

norm

aliz

ed tr

ansc

ript

leve

ls PALCHSSTSPRX

b.

0

5

10

15

20

25

30

0 48 96 192 0 48 96 192 0 48 96 192 0 48 96 192

Control BNR UNR BNR+UNR

norm

aliz

ed tr

ansc

ript

leve

ls PALCHSPRX

Figure 6. Defence-gene transcript activities in the experiment 2 (III). a.) STS, PAL, CHS, andPRX. b.) PAL, CHS, and PRX.

The mean PRX activity increased in all fungal treatments at 96 h.p.i. in experiment 2(Fig. 6 b, and III: Fig. 2 b), but the standard deviation between replicates was extremelyhigh, and only the activity in dual infected roots in experiment 1, 48 and 96 h.p.i., weresignificant (P ‹ 0.03).

Significant up-regulation of PAL were detected in dual infection 96 h.p.i. in bothexperiments and 192 h.p.i. in the experiment 2. Single UNR infected roots showedsignificant up-regulation at all time points post 48 h.p.i.

The only significant CHS activity was detected in experiment 2 between dualinoculated roots and single UNR inoculated roots 96 (p =0.0075) and 192 h.p.i. (p=0.0438).

AMP, RED, GLP, SOD, DEH transcripts were not significantly activated in anytreatments or incubation times (data not shown.)

26

5. Discussion

5.1. Rhizoctonia spp. ITS sequences and phylogenetic analysis

The binucleate Rhizoctonia (BNR) strains used in this study have earlier beenidentified as anamorphic Ceratorhiza spp., which include binucleate Rhizoctonia that formmycorrhiza with orchids (Sen et al. 1999). A phylogenetic analysis of ITS sequences ofBNR from this study and diverse binucleate Rhizoctonia (including members of allanastomosis groups) strictly confirms that our binucleate Rhizoctonia are the telemorphicspecies Ceratobasidium cornigerum (Gonzalez et al. unpublished data). This also nowconfirms the earlier anastomosis group/ RFLP study of Sen et al. (1999). The ITS-sequences of strains 268 and AV-2 (=AG-I) revealed 99% similarity (I). The uninucleateRhizoctonia (UNR) strains 263 and 1983-111/1N, selected for this study, have beenidentified as Ceratobasidium bicorne based on rDNA (internal spacer, ITS) sequenceanalysis (Hietala et al. 2001).

In Rhizoctonia taxonomy, the faster and more precise ITS sequencing techniquecombined with phylogenetic analyses have now mostly replaced earlier anastomosisgrouping methodology, nevertheless the AG grouping remain valuable tools inidentification. Similarly, in agreement with a phylogenetic analysis of our strains(Gonzalez et al. unpublished data) many authors including Stepniewska-Jarosz et al.(2006) and Sharon et al. (2008) have more recently showed that the ITS sequence analysesdistinguishes the isolates into separate clades according to their AG grouping. Thisconfirms the value of this methodology and that the AG grouping has a clear genetic basis.

5.2. ITS as a target for Rhizoctonia spp. probe development

The ITS sequence within the rDNA cistron represents a suitable target for strainidentification due to the presence of high sequence similarity between related strains andsimultaneously sufficient differences between unrelated strains. We observed that the ITS-1 and ITS-2 regions offered relatively high sequence variation for >100 bp probe, andhighly variable areas for shorter oligonucleotide probe development. Species/taxa specificprobes specific for conifer root pathogenic and endophytic Rhizoctonia spp. and theectomycorrhizal Suillus bovinus were successfully developed. PCR amplified (124-151bp) probes and oligonucleotide (20-25 bp) probes were respectively developed forSouthern and liquid hybridization (Hendolin et al. 2000). Oligonucleotide probes werealso highly specific in Southern hybridization against ITS1/4 amplified-PCR samples.

The ITS sequences of uninucleate Rhizoctonia are highly conserved (Hietala et al.2001), but more variable between binucleate Rhizoctonia. The 5.8 S ribosomal gene isknown to be highly conserved among different Rhizoctonia species (Kuninaga et al. 1997,Hietala et al. 2001), but it also proved to be highly conserved in all fungi used in thesequence alignment. The non-coding ITS1 and ITS 2 regions were more variable, as couldhave been expected. Binucleate Rhizoctonia strains 268 and AV-2 (AG-I), which belongsto the same anastomosis group, were practically identical (99%) having only fewnucleotide differences in ITS 1. Interestingly, two Suillus bovinus strains isolated fromdifferent forest areas in South and central Finland (Leppäkorpi and Hyytiälä), differed by asingle nucleotide substitution; C and T at base position 123, respectively.

The additional PCR amplified bands obtained with control and Suillus primers can beexplained by the fact that the ITS region occurs as a part of a multicopy rDNA cistron inthe fungal genome so that ribosomal RNA genes are arranged as a tandemly repeated array(Gardes et al. 1991). This means that one ITS product obtained in PCR could include

27

several minor ITS sequence length variants. As such, a cloning step has to be includedbefore sequencing, where only one DNA fragment is ligated to the vector and in this wayoverlapping signals can be avoided. However, additional bands with control primersappeared on only a single occasion and there might have been some contamination in theparticular PCR reaction. Suillus primers regularly amplified additional bands in theprimary PCR. Targeted sized PCR product was obtained after re-amplification of extractedDNA of the correct size.

We preferred the approach of direct fungal detection from root material, than usingPCR amplified DNA, because it is faster and eliminate the amplification-related problems(errors or bias) that might occur during the PCR amplification. In this respect, >100 bpDIG labelled probes were targeted specifically against total DNA. All these longer probeswere tested against DNA extracted from Scots pine roots. This plant serves as a host toboth Suillus bovinus forming mycorrhiza and the uni- and binucleate Rhizoctonia strainsin pathogenic and biotrophic interactions (Hietala & Sen 1996). Only the probe developedagainst uninucleate Rhizoctonia strain 263 hybridized with Scots pine root DNA sample.While direct detection would shorten the identification process, the PCR based methodused in liquid hybridization was found to be more sensitive and reliable than Southernhybridization, because the size of the hybridized PCR product is visualized on thecomputer screen simultaneously. Additionally, possible similarities between probe andnon-target of fungal DNA sequences are eliminated through pre-amplification by PCRwith ITS1 and ITS2 primers. These universal primers specifically amplify the ITS regionfrom most fungi (Bruns & Gardes 1993) and in this way, for example bacterial or hostplant DNA could have been excluded in hybridization test.

The Suillus probe was not, however, Suillus bovinus specific, since it hybridized withall other Suillus species except Suillus luteus. Further screening with additional Suillusstrains and strains of other species should be carried out to determine the limits of probespesificity. Conversely, probes developed targeting spesific binucleate Rhizoctonia strainswere highly specific and thus of diagnostic value. The uninucleate 263 probe (number 8, I:Table 2) might need more stringent conditions or 1:100 dilutions should be used to avoidunspecific fungal or root binding.

Another alternative approach for fungal identification is to design specific primers forPCR detection of certain fungal species e.g. Salazar et al. (2000) and Taylor &McCormick (2008), but the possible contamination risk is one disadvantage in such aPCR-based procedure. Groppe et al. (1995), and Groppe & Boller (1997) have usedmicrosatellite containing locus as a target in PCR detection for ecology and diversitystudies of the plant associated fungus Epichloë. Buscot et al. (1996) used microsatellite-primed PCR in morels DNA polymorphism investigations. This is another alternativetarget in fungal DNA based studies, although ITS is a better DNA target for probedevelopment. Microsatellite regions are stretches of tandem mono-, di-, tri-, andtetranucleotide repeats of various lengths and a wide range of repeat lengths can be presentin a single population (Groppe & Boller 1997). The most sensitive method available isperhaps the quantitative real-time PCR (qRT-PCR), successfully used for R. solani AG-3(Lees et al. 2002, Lehtonen et al. 2008) and AG-1 IA detection (Sayler et al. 2007). Thismethod combined with TaqMan chemistry, allows also more reliable quantitativedetection of the target fungi than the conventional PCR, since only a specific PCR productis detected.

Classical morphological methods are still of value, even though molecular methodshave obvious advantages in species identification. Specific probes for direct detection oftarget sequences for example in soil, are almost impossible to obtain, since there are somany different organisms and different species in nature, with uncharacterized DNA andthere is always a possibility that the developed probe give "false" positives in hybridizingto DNA regions from these other organism. However, the qRT-PCR with TaqMan probes,now allows more specific detection also from complicated soil samples, as showed byLees et al. (2002) in comparison to qRT-PCR with conventional PCR. Nevertheless, inorder to reduce errors, morphological characters of isolated fungi (where possible) and

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lesions tissue analysis for example from Rhizoctonia infected plants should be the firststep in species identification and detection with specific probes, in conventionalhybridization methods or qRT-PCR associated, could be used to confirm fungal identity.

The developed Rhizoctonia probes (I) or the use of strain specific primers or probes inqRT-PCR would help us to detect these economically important strains both fromnurseries and the forest site and probably greatly help improve our understanding ofRhizoctonia infection biology. These probes would also help further answer the questionas to whether these different fungal groups or different strains of Rhizoctonia spp.commonly co-colonize the same root system (Hietala 1995).

5.3. Binucleate Rhizoctonia linked effects on Scots pine growth

BNR are typically associated with orchid species (Andersen & Rasmussen 1996, Zelmeret al. 1996, Sharma et al. 2003), but have also been reported to show beneficial effects onother plants. Growth inducing effects had earlier been reported in capsium (Harris 1999)and cucumber (Villajuan-Abgona et al. 1996), and now in Scots pine seedlings (II). SomeBNR strains are pathogenic (Stepniewska-Jarosz et al. 2006) and this was originallythought to be the case with our Finnish binucleate Ceratorhiza spp. (strains 251, 266, 268and 299) when isolated from diseased nursery Scots pine seedlings (Lilja et al. 1996).However, our BNR stains induced early (87 d.p.i.) Scots pine shoot and root growth, butin the second harvest (240 d.p.i.) the differences between inoculated and uninoculatedseedlings were not significant. Instead, the short root number increased and was morenumerous in BNR inoculated than uninoculated seedlings. The low root infection-rate, and–morphology identified by tryphan blue staining, suggests that a true mycorrhizalrelationship does not occur between host and fungus. In forest soil, Scots pine rapidlyenters into mycorrhizal associations with several fungal species (Smith & Read 1997,Timonen et al. 1997), but typical ectomycorrhiza structures have, to our knowledge, notbeen reported in Rhizoctonia colonized conifer roots. Additionally BNR associated Scotspine seedlings showed yellow-green needles in N- and C- limited nursery soil (II),suggesting lack of nutrients, whereas in similar aged mycorrhizal seedlings associatedwith the true mycorrhizal fungus Suillus bovinus, the needles remained a healthy darkgreen in the same nursery soil (Sen 2001). These data indicate that early root inductionwas not related to improved nutrient uptake through a classical mycorrhizal association.However, BNR linked root growth could be explained through possible hormonal linkedstimulation. Plant growth promoting hormonal signaling is crucial for development ofplant-microbe interactions, and indole-acetic acid (IAA) has been detected in R. solanicultures (Furukawa et al. 1996); the amount of IAA was found to increased in R. solanirice suspension cultures (Furukawa & Syono 1998). In a related study, Kaparakis and Sen(2006) showed that similar Scots pine adventitious root inducing effects resulting fromindol-3-butyric acid (IBA) exposure could be obtained in BNR inoculation experimentsand, importantly, that BNR-linked root induction was even more efficient. Whether thereare differences in hormonal communication between pathogenic or non-pathogenicRhizoctonia-host interactions is not known. It is clear that further work is needed in orderto understand the molecular and physiological mechanisms of hormonal interaction inthese associations.

Hwang & Benson (2003) investigated the potential of BNR strains as biocontrolagents against R. solani attack in poinsettia cuttings, but they did not compare the rootingor growth promotion effects of BNR. Their work focused only on the biocontrol aspect,but it would be interesting to find out whether their BNR would stimulate the growthpromoting effects as our results suggests from Scots pine cuttings (Kaparakis & Sen2006). The typical monilioid cells present within the cells of the outer cortex, as visualizedby tryphan blue staining, have been earlier reported in orchid mycorrhiza (Zelmer et al.1996, Sharma et al. 2003) and uninucleate Rhizoctonia (Hietala 1997).

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Seedlings infected by BNR produce more short roots, which in forest soils, wouldrapidly become colonized by mycorrhizal fungi during mycorrhiza development. Innursery soils these nonmycorrhizal short roots could represent sites for UNR entry. AsRhizoctonia spp. have been isolated from fine roots of Scots pine trees in Scottish foreststands (Steve Woodward, pers. comm.) and from S. bovinus mycorrhizas (Sen 2001),more work needs to be carried out using the developed probes in both nursery and forestsites.

5.4. Scots pine host defence responses to bi- and uninucleateRhizoctonia and the interactions in co-inoculation

Preinoculation of binucleate Rhizoctonia (BNR) strain 268 decreased the infectionlevel of pathogenic uninucleate Rhizoctonia (UNR) strain 1983-111-/1N in the Scots pineradicals. However, in in vitro confrontation assays on water agar it neither showed anyantagonistic effects against UNR (Fig. 3a), nor prevented UNR entry into the root cells invivo. The simultaneous coinoculation with UNR decreased the BNR infection levelcompared to BNR single inoculations, indicating UNR being more competitive in theinfection process, despite the slower in vitro growth on WA plate. In BNR preinoculatedroots, on the other hand, the UNR level was restricted compared to single UNRinoculations. This might be due to the root cells being precolonized with BNR.Surprisingly, both BNR and UNR infection levels were temporarily decreased during theinfection process, respectively, 24 and 48 h.p.i., suggesting that the biochemicalcommunication between fungus and host retards the infection.

Absolute quantification of UNR and BNR infection in this study was impossible, sincedata on the size of their respective genomes and ITS copy numbers is not available.However, our results show the relative variation in each treatment and at different timepost inoculation. Regardless of potential differences in the ITS copy number betweenBNR and UNR, it would not alter the fact that the amount of UNR was restricted in BNRpreinoculated roots, compared to simultaneous dual inoculation.

In a study by Cardoso & Echandi (1987), BNR also failed to show antagonisticinteraction in dual cultures of bean (Phaseolus vulgaris L.) with R. solani. Filtrates alonefrom BNR cultures did not, but filtrates from BNR treated root exudates did inhibit R.solani hyphal growth and BNR pretreatment inhibited formation of R. solani infectioncushions. Therefore, the authors suggested that BNR- induced metabolic response was themechanism of R. solani suppression. In our study (III) the UNR infection specific DNAlevels decreased similarly in BNR preinoculated roots, but the impact of host defence-gene activity is not clear.

In the BNR-preinoculated dual infected roots, lower PAL transcript activity wasidentified compared to simultaneous dual infection, although the transcript wassignificantly up-regulated in both experiments compared to control. Significant PALactivity was also obtained in single UNR infected roots in experiment 2 compared tocontrol, but regardless of the respective identically accomplished treatments of singleUNR infections in experiment 1, these later roots showed no significant PAL activation.Wen et al. (2005) detected increased PAL, CHS, and 1,3-ß-D-glucanase activities in R.solani infected beans (Phaseolus vulgaris L.), while in BNR treated, and BNR -pretreatedand R. solani-infected bean seedlings, the activity was lower. The preinoculation with themycorrhizal fungus, Glomus intraradices Schenck and Smith, however, did not alter thebean PAL-, CHS-, chalcone isomerase-, and hydroxyproline-rich glycoprotein activitiesduring R. solani infection (Guillon et al. 2002).

In our study, CHS was activated significantly only in dual infected roots inexperiment 2, compared to UNR inoculated roots 96 and 192 h.p.i. Some activity,although not significant, was already detected at the time of inoculation (T=0). CHS gene-

30

activity is probably not solely induced in Scots pine defence reactions, since it has beenshown to be also expressed in non-stressed plantlets (Fliegmann et al. 1992). In Norwayspruce, on the other hand, Ophiostoma polonicum-infection elicited both CHS and STSactivities, but in contrast to our results, the CHS was more stimulated (Bringolas et al.1995). In our study, the STS activity was highly up-regulated during all treatments, beinghighest in UNR single inoculated roots. The STS responded also to stress in nicked Scotspine hypocotyls more efficiently than CHS (Fliegman et al. 1992). Our study showedrelatively high variations in all transcript activities between replicates, indicating highdegree of genetic variation between seedlings. Thus, the use of clonal material would bethe solution in future experiments.

All three studied phenylpropanoid pathway involved enzymes, phenylalanine ammonialyase, CHS, and STS, have long been known to be induced by different pathogens(Gehlert et al. 1990, Lange et al. 1994, Fliegmann et al. 1992, Zinser et al. 1998, Chironet al. 2000, Yu et al. 2005). Adomas et al. (2007) reported that enzymes from thephenylpropanoid pathway, defence-related proteins with antimicrobial properties, andclass III peroxidases, were the most abundant genes up-regulated during H. annosuminfection in Pinus sylvestris. However, a Pinus sylvestris short root-specific peroxidase(Psyp1), which is also a class III peroxidase, is known to be down-regulated inectomycorrhizas of the Scots pine- Suillus bovinus symbiosis (Tarkka et al. 2001).However, similar down-regulation of this enzyme, was not observed in our BNRtreatments, regardless of their beneficial role in Scots pine roots (II). This might be due tothe non-mycorrhizal role of this fungus in Scots pine. It would be interesting todeterminate the activation of Psyp1-related transcript in BNR-mycorrhizal associations inorchidea plants. In our study, significant peroxidase activities detected only in thesimultaneous dual infected roots suggests this infection type being more effective onPsyp1 activation than either of these strains separately. Rhizoctonia challengedperoxidases have been also detected from bean (Phaseolus vulgaris L.) and Norwayspruce in several studies (Xue et al. 1998, Nagy et al. 2004, Jøhnk et al. 2005, Fossdahl etal. 2007) and selected peroxidase genes most probably affect the results, since for examplein a study of Nagy et al. (2004), 12 peroxidase isoforms are identified from the Norwayspruce (Picea abies) roots. The same fungal strain also induced several other defencetranscripts in Norway spruce, including phenylalanine ammonia lyase, and two germin-like proteins (Fossdal et al. 2007). In our experiment, however, the germin-like protein(Mathieu et al. unpublished) showed no significant up- or down- regulation compared tothe control. Neither did the rest of the selected potential pathogen-induced transcripts:antimicrobial peptide gene Sp-AMP (Asiegbu et al. 2003), a defence related genePsACRE (Li & Asiegbu 2004), CuZn- superoxide dismutase (Karpinski et al. 1992) ordehydrin-like protein (Pyhäjärvi et al. 2007). In addition to plant defence reactions duringpathogen infection (Karlsson et al. 2007, Fossdal et al. 2007), the germin-like proteins arealso involved in somatic and zygotic embryogenesis (Mathieu et al. 2006, Neutelings etal. 1998).

Herr (1995) suggested that the mechanisms of protection, by binucleate Rhizoctoniaor hypovirulent strains of R. solani against pathogenic strains, are via competition andinduced host resistance rather than direct antagonisms or mycoparatism. Our resultssupport this hypothesis of competition, since BNR was not antagonistic against UNR, butrestricted the UNR infection level in BNR-preinoculated roots. Analysis of a subset ofdefence-gene activities, however, showed no clear correlation in their activity anddecreased UNR infection levels in BNR- preinoculated roots suggesting that they are notinvolved as inducing the host resistance in these interactions.

Plant defence reactions activated by the interaction of different micro-organisms arecomplicated and the enzyme activities are most probably differentially regulated inseparate plant tissues and during different developmental stages. Therefore, thecomparison of these different studies that involve a range of experimental designs isdemanding. More detailed and extensive studies similar to Adomas et al. (2007 and 2008)are needed. Additionally, revealing the role of each metabolic pathway in particular host-

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pathogen interaction and the regulation of the enzymes involved in the particularinteractions, would increase our knowledge and the information could be used for futureplant protection for example in agriculture or in forest nurseries.

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6. Conclusions

The molecular detection of Rhizoctonia is essential in order to discriminatepathogenic and non-pathogenic strains, because vegetative characteristics often overlap.Rapid field diagnosis in agriculture or forest nurseries could provide targeted pathogen-control treatments before extensive problems arise and could reduce or eliminate the needfor fungicides in an integrated pest management scenario. Our knowledge regarding hostdefence reactions, on the other hand, provides information for selection of disease resistanthost genotypes as well as development of new more environmental friendly and moreefficient antifungal agents.

This study further confirms the complexity of the Rhizoctonia group not only from aphylogenetical perspective, but also with regard to their infection biology and plant hostspecificity. Root infecting UNR and BNR strains, belonging to the teleomorph genusCeratobasidium, respectively enter into pathogenic or beneficial associations in Scotspine. BNR strains revealed a propensity to early seedling growth enhancement and laterthe induction of short roots, which are vital for symbiotic mycorrhizal formation, in Scotspine. They also proved to enhance the rooting of hypocotyls cuttings in a related study(Kaparakis & Sen 2006).

The pathogenic UNR appeared to be more competitive in infection of Scots pine cellsthan endophytic BNRs, in co-inoculation. This supports the pathogenic role of UNR. Thedecrease in UNR infection level in BNR preinoculated roots indicates that beneficialmicrobes could be directly utilized as inoculants for example in tree seedling nursery soils,or used to supplement fertilizers in planting-process to benefit plant growth andproductivity as such, but also induce the host response against oncoming fast spreadingpathogenic strains. Nevertheless, neither Suillus bovinus (Sen 2001) nor BNR strains fromthis study entirely prevented UNR infection but both could be included in inoculationsystems in Scots pine seedling production. S. bovinus improves the nutrient uptake, whichsupports improved host survival, while BNR strains stimulate the growth and rooting inthe host. The later short-root inducing activity of BNR, on the other hand, could providemore entry points for increased beneficial ectomycorrhiza development in the nursery.

As UNR is not the only disease causing pathogen in nursery or forest soils, no singlebeneficial microbe will control all possible pathogens. Therefore, combinations ofbeneficial microbes could be introduced into the growth substrates of plants. However,little is known about the effects of these soil- or root-inhabiting beneficial microbes oneach other in vitro or in vivo in nature and further work is needed to uncover suchrelationships. The combination of the BNR and S. bovinus and their impacts on the Scotspine defence and UNR infection, although complicated, would be very interesting topicfor future investigation. Additionally more defence genes could be included as newcandidate gene sequences become available in public databases. Another important futureperspective that stems from this and earlier studies by our group members would be toinitiate an applied programme under actual nursery condition.

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7. Acknowledgements

This work was carried out at the division of General Microbiology, in the Departmentof Biological and Environmental sciences, University of Helsinki and was funded byUniversity of Helsinki (Young researcher fund), Viikki Graduate School in Biosciences(VGSB), Finnish Cultural Foundation and Finnish Society of Soil Sciences.

I express my deep gratitude to Prof. Kielo Haahtela, Head of the Department and mostrecent supervisor of this work, for her encouraging attitude and great support, especiallyduring these later years. I am also grateful for the financial support, which made the finalpart of the work possible. I would also like to thank Prof. Timo Korhonen, the Head of thedivision, for providing the facilities for my work.

This work was mainly supervised by Dr. Robin Sen, my warmest thanks for yourenthusiastic support and scientific expertise which carried me all the way from that veryinitial academic start in Ruskeasuo in 1995, as a research assistant, even before mybiology education, to eventually a PhD candidate.

I am very grateful to Dr. Ari Hietala, a co-author of the third article and a member ofmy follow-up group. You generously provided your expertise in the study design, taughtme the real time PCR method and the interpretation of the results both in person, andthrough e-mail communication from Norway.

I also owe deep gratitude to the following persons:- Prof. Fred Asiegbu and Dr. Taina Pennanen for generously and critically reviewing

the thesis.-Co-authors Dr. George Kaparakis and Lars Paulin, and members of the sequencing

lab for all technical help, especially Miia, Paula, and Eeva-Marja.-All the members who shared our former MYCO-group: Sari, Jussi, Hanna, Malin,

Edda, Micce, Vanamo, Sanna, Jarmo, Maarja, Ilpo and all students whose lab work I havesupervised: Irina, Hanna, Outi and Pave. Especially I thank Sari for introducing me to thepractical mycorrhizal lab work and for your careful hands-on training at the verybeginning of my studies, but I am also grateful for the advice of all other members in thelab. Malin and Edda sheared with me many lunch and coffee breaks of cheerfulconversation.

-The personnel in the General Microbiology, some of them I have known since myresearch assistant time in Ruskeasuo. Thank you for all the help you provided.

-Prof. Per Saris, a member of my follow-up group, and his research group for the shortlaboratory visit. I am especially pleased to get to know Shea Beasley and JanettaHakovirta, who became my friends together with Anne Ylinen and Mari Koskinen fromthe Division of Applied Microbiology, whom I am very grateful for sharing knittingevenings etc. with good food and conversation and all the refreshing coffee breaks inViikki. Anne has also provided technical help in real time PCR and Prof. Kaarina Sivonenis acknowledged for letting me use the machine.

-VGSB coordinators Nina Saris and Eeva Sievi for helpfulness and support, and ViikkiBiosciences Doctoral Student Association (VIBA) board members, Pinja, Suvi, Maija,Katrianna, Johanna, Anne, Vassileios and Sylvie for organising outside lab activities toVGSB students and shearing grateful moments in VIBA.

-My family: especially my husband Dick, for loving me all these years despite myfrequent mood swings, and our lovely children Daniel and Minea, you have given meenormous delight. My parents Martti and Hilkka for their tremendous encouragement andmy sisters Eerika and Emilia for being good friends and having you nearby, and for thewhole family for your support. I am also grateful to my mother-in-law Cati for so oftentaking care of our pets and to all the grandparents of our children for taking care of Danieland Minea whenever needed.

Helsinki, October 2008

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