Rhizoctonia -Scots Pine Interactions: Detection, Impact on Seedling Performance and Host Defence Gene Response

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

Henrietta Grönberg

General Microbiology Department of Biological and Environmental Sciences Faculty of Biosciences and Viikki Graduate School in Biosciences University of Helsinki

Academic Dissertation in General Microbiology

To be presented, with the permission of the Faculty of Biosciences of the University of Helsinki, 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 Haahtela Faculty of Biosciences Department of Biological and Environmental Sciences General Microbiology University of Helsinki Dr. Robin Sen Faculty of Biosciences Department of Biological and Environmental Sciences General Microbiology University of Helsinki (Current address: Department of Environmental and Geographical Sciences, Manchester Metropolitan University Department of Environmental and Geographical Sciences, Soil Ecology, Chester Street, Manchester M1 5 GD, UK) Reviewers: Professor Frederick Asiegbu Faculty of Agriculture and Forestry Department of Forest Ecology University of Helsinki Dr. Taina Pennanen The Finnish Forest Research Institute, Vantaa Research Unit PL 18 01301 Vantaa Opponent: Professor Jarkko Hantula The Finnish Forest Research Institute, Vantaa Research Unit PL 18 01301 Vantaa

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

Printed: Ylipistopaino 2008, Helsinki, Finland e-mail: [email protected]

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

List of original publications………………………………………………………………..4 Abbreviations and definitions…………………………………………………………..….5 Abstract………………………………………………………………………………….....6 Tiivistelmä (Abstract in Finnish)…………………………………………………………..7 1. Introduction……………………………………………………………………………..8 1.1. Rhizoctonia taxonomy………………………………………………………….....8 1.2. Internal transcribed spacer (ITS) as a target marker for fungal identification…....9 1.3. Rhizoctonia spp. identification and molecular detection………………………….9 1.4. Rhizoctonia spp. as economically important fungal pathogens of plants………...10 1.5. Binucleate Rhizoctonia as symbiotic fungi and bicontrol agents……………...... 11 1.5.1. Biocontrol: Principles, applications and mechanisms.....……………...... 11 1.5.2. Competition as a potential mechanism for biocontrol...... 12 1.5.3. Induced resistance: another potential mechanism for biocontrol...... 12 1.6. Defence-gene activity…………………………………………………………….12 1.6.1. Defence-genes in conifers……………………………………………...... 12 1.6.2. Phenylpropanoid pathway……………….…………………………….....13 1.7. Induction of host defence-gene activity by hypovirulent strains………………...14 2. Aims of the study………………………………………………………………………15 3. Materials and Methods…………………………………………………………………16 3.1. Strains and methods described in publications…………………………………..16 3.2. Phylogenetic analysis………………………………………………...…………..18 4. Results………………………………………………………………………………….19 4.1. ITS probe development for Southern and liquid hybridization………………….19 4.1.1. Primary ITS PCR fragments and sequence alignment…....……...... 19 4.1.2. Phylogenetic analysis…………………………………………………….19 4.1.3. Probe construction and application in Southern dot-blot- and liquid hybridization……………………………………………………………………20 4.2. Scots pine seedling growth and root architectural responses to binucleate Rhizoctonia inoculation…………………………………………………………….....21 4.3. Bi- and uninucleate Rhizoctonia infection biology: antagonism and microscopy studies……………………………………………………………………………...... 21 4.4. Real time PCR…………………………………………………..………………..23 4.4.1. Quantification of fungal genomic DNA in infected roots…………….….23 4.4.1.1. Experiment 1...... 24 4.4.1.2. Experiment 2.………………………… …...... 24 4.4.2. Defence-gene transcript levels……………………………………...…....24 5. 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……………………………………………….….…..…29 6. Conclusions………………………………………………………………………….....32 7. Acknowledgements……………………………………………………………..……...33 8. References…………………………………………………………………………...... 34 List of original publications

The thesis is based on the following articles, which in the text are referred to by their Roman numerals. I Grönberg, H., Paulin, L. and Sen, R. (2003). ITS probe development for specific detection of Rhizoctonia spp. and Suillus bovinus based on Southern blot and liquid hybridization-fragment polymorphism. Mycological Research 107 (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. Scandinavian Journal of Forest Research, 21: 450-457. III Grönberg, H., Hietala, A. M. and Haahtela, K. (2008). Analysing Scots pine defence-related transcripts and fungal DNA levels in seedlings single- or dual-inoculated with endophytic and pathogenic Rhizoctonia species Submitted 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). Abbreviations and definitions

AG anastomosis group AM arbuscular mycorrhizal fungus Sp-AMP antimicrobial peptide gene anamorph the asexual form of fungi also known as the vegetative stage avirulent non-pathogenic BNR binucleate Rhizoctonia Ct cycle threshold, the PCR cycle at which the fluorescence exceeds that of background cDNA DNA synthesized from mRNA with reverse transcriptase (contains no introns) CHS chalcone synthase dhy-like dehydrin-like protein d.p.i. days post inoculation DNA deoxyribonucleic acid, the genetic material GAPDH glyceraldehyde-3-phosphate dehydrogenase PsGER1 germin-like protein h.p.i. hours post inoculation HR hypersensitive response hypovirulent from avirulent to low virulent strains, which show no disease symptoms in host plants intron non-coding sequences within coding genes in eukaryotic genome ISR induced systemic resistance ITS internal transcribed spacer. The non-coding sequence between coding areas of rDNA subunits MCA microbial control agents MF mycorrhizal fungi MNR multinuncleate Rhizoctonia monilioid hyphal structures typical for Rhizoctonia infection in host cell NCBI national center for biotechnology information primer short oligonucleotide sequence used for specific amplification of target DNA by PCR/qRT-PCR probe oligonucleotide sequence used in hybridizations with target DNA qRT-PCR quantitative real time PCR rDNA ribosomal DNA r.l.c. root length colonised pal1 phenylalanine ammonia lyase PCR polymerase chain reaction Psyp1 peroxidase RAPD random amplified polymorphic DNA fragment RFLP restriction fragment length polymorphism PR-proteins pathogenesis related proteins PsACRE rapidly elicited defence-related gene RNA ribonucleic acid SCAR sequence-characterized amplified region sclerotia structures formed by fungi typical to Rhizoctonia SOD superoxide dismutase Sp species (singular form) Spp species (plural form) STS stilbene synthase teleomorph the sexual form of fungi producing spores and sclerotia Tm the specific melting temperature, where respective double stranded DNA denatures to single strands UNR uninucleate Rhizoctonia 5 Abstract

Rhizoctonia spp. are ubiquitous soil inhabiting fungi that enter into pathogenic or symbiotic associations with plants. In general Rhizoctonia spp. are regarded as plant pathogenic fungi and many cause root rot and other plant diseases which results in considerable economic losses both in agriculture and forestry. Many Rhizoctonia strains enter into symbiotic mycorrhizal associations with orchids and some hypovirulent strains are promising biocontrol candidates in preventing host plant infection by pathogenic Rhizoctonia strains. This work focuses on uni- and binucleate Rhizoctonia (respectively UNR and BNR) strains belonging to the teleomorphic genus Ceratobasidium, but multinucleate Rhizoctonia (MNR) belonging to teleomorphic genus Thanatephorus and ectomycorrhizal fungal species, such as Suillus bovinus, were also included in DNA probe development work. Strain specific probes were developed to target rDNA ITS (internal transcribed spacer) sequences (ITS1, 5.8S and ITS2) and applied in Southern dot blot and liquid hybridization assays. Liquid hybridization was more sensitive and the size of the hybridized PCR products could be detected simultaneously, but the advantage in Southern hybridization 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 were investigated on Scot pine (Pinus sylvestris) seedling growth, and the infection biology and infection levels were microscopically examined following tryphan blue staining of infected roots. All BNR strains enhanced early seedling growth and affected the root architecture, while the infection levels remained low. The fungal infection was restricted to the outer cortical regions of long roots and typical monilioid cells detected with strain 268. The interactions of pathogenic UNR Ceratobasidium bicorne strain 1983-111/1N, and endophytic BNR Ceratorhiza sp. strain 268 were studied in single or dual inoculated Scots pine 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), rapidly elicited defence-related gene (PsACRE), germin-like protein (PsGER1), CuZn- superoxide dismutase (SOD), and dehydrin-like protein (dhy-like)] were measured from differentially treated and un-treated control roots by quantitative real time PCR (qRT-PCR). The infection 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 was highly up-regulated in all treated roots, while CHS, pal1, and Psyp1 transcripts were more moderately activated. No significant activity of Sp-AMP, PsACRE, PsGER1, SOD, or dhy-like transcripts were detected compared to control roots. The integrated experiments presented, provide tools to assist in the future detection of these 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 further confirms the complexity of the Rhizoctonia group both phylogenetically and in their infection biology and plant host specificity. The knowledge obtained could be applied in integrated forestry nursery management programmes.

6 Tiivistelmä (Abstract in Finnish)

Rhizoctonia-suvun sienet ovat maaperässä yleisesti esiintyviä sieniä, jotka voivat olla tautia aihettavia patogeeneja tai muodostaa hyödyllisiä symbionttisia vuorovaikutussuhteita kasvien kanssa. Yleensä Rhizoctonia spp. sienet ovat kasvipatogeeneja aiheuttaen mm. juurilahotautia niin metsäpuille kuin viljelykasveillekin. Useat Rhizoctonia–suvun sienet muodostavat orchidea-kasveille elintärkeitä symbionttisia sienijuuria (mykorritsa) ja jotkut tauteja aiheuttamattomista Rhizoctonia-kannoista voivat estää patogeenisten kantojen infektion tutkituilla kasveilla. Tässä työssä keskityttiin yksi- ja kaksitumaisiin Rhizoctonia- sieniin, jotka kuuluvat teleomorfi Ceratobasidium-sukuun, mutta DNA koettimet kehitettiin myös monitumaiselle Rhizoctonia-sienelle (teleomorfi Thanatephorus) ja nummitatti- ektomykorritsasienelle (Suillus bovinus). Kantaspesifiset koettimet suunniteltiin ribosomaalisen DNA:n ”internal transcribed spacer” (ITS) -alueelle (ITS1, 5.8S ja ITS2) ja valmistettuja koettimia käytettiin “Southern dot blot”- ja nestemäisen hybridisaatiomenetelmän vertailuun. Nestemäinen hybridisaatio oli herkempi ja siinä voitiin määrittää myös hybridisoituvan PCR-tuotteen koko, mutta ”Southern” hybridisaation etuna oli, että koettimen hybridisoinnissa voitiin käyttää suoraan sienistä eristettyä 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 ja infektiobiologiaan. Infektion taso määritettiin mikroskopoimalla trypaanisinellä värjättyjä infektoituja juuria. Kaikki kaksitumaiset Rhizoctonia-kannat tehostivat nuorten taimien kasvua ja vaikuttivat juurten ulkomuotoon (lyhytjuurien määrä, juurten paksuus jne.), vaikka infektion määrä pysyikin alhaisena. Infektio keskittyi juuren uloimpaan kuorikerrokseen ja näille sienille tyypillisiä ”monilioid”-soluja oli nähtävissä 268 kannalla infektoiduissa juurten soluissa. Patogeenisen yksitumaisen Rhizoctonia-sienen (Ceratobasidium bicorne) 1983- 111/1N ja endofyyttisen kaksitumaisen Rhizoctonia-sienen (Ceratorhiza sp.) 268 infektiobiologiaa ja vuorovaikutusta tutkittiin kummallakin kannalla yksinään sekä molemmilla kannoilla samanaikaisesti infektoiduissa männyn juurissa. Sieni-infektion määrä ja männyn transkriptioaktiivisuus määritettiin kaikista käsitellyistä juurista ja verrokkijuurista kvantitatiivisella ”real time PCR” –menetelmällä (qRT-PCR) seuraavien entsyymien osalta: fenyylialaniiniammoniumlyaasi (pal1), stilpeenisyntetaasi (STS), kalkonisyntetaasi (CHS), lyhytjuurispesifinen peroksidaasi (Psyp1), mikrobeja hylkivä peptidi (Sp-AMP), nopean puolustusreaktion aktivaation liittyvä geeni (PsACRE), germiinin kaltainen proteiini (PsGER1), CuZn- superoksididismutaasi (SOD) ja dehydriinin kaltainen proteiini (dhy-like). Taimien siirrostus kaksitumaisella Rhizoctonia- sienellä ennen yksitumaisen Rhizoctonia-sienen infektiota rajoitti yksitumaisen kannan määrää, mutta yksitumainen Rhizoctonia oli tehokkaampi infektoimaan juuria, kun sienet siirrostettiin samanaikaisesti. Stilpeenisyntetaasin geenituotetta syntetisoitiin erittäin tehokkaasti kaikissa käsittelyissä, mutta kalkonisyntaasi, fenyylialaniiniammoniumlyaasi ja peroksidaasi aktivoituivat vain kohtalaisesti. Geenien Sp-AMP, PsACRE, PsGER1, SOD, ja dhy-like transkriptioaktiivisuuksissa ei havaittu merkittäviä muutoksia verrokkeihin nähden. Työssä kehitetyt koettimet mahdollistavat kyseisten sienten osoittamisen juuristosta ja maaperästä. Tehty tutkimus auttaa ymmärtämään Rhizoctonia-sienten infektiobiologiaa, eri Rhizoctonia-kantojen vuorovaikutus-suhteita männyssä, sekä männyn puolustusreaktiota Rhizoctonia-infektion aikana. Tämä työ todisti aiemman käsityksen näiden sienten monimutkaisesta fylogeniasta, monimuotoisesta infektiobiologiasta ja isäntäspesifisyydestä. Tutkimuksesta saatua tietoa patogeenisen ja endofyyttisen Rhizoctonia-sienen infektiobiologian eroista ja männyn puolustusreaktiosta voidaan hyödyntää entistä tehokkaampien torjuntamenetelmien kehittämiseksi sienitauteja vastaan. 7 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 important morphological character, but fruiting of Rhizoctonia species is difficult to induce under laboratory conditions. Therefore, characterization and species designation is mainly based on the vegetative characters of the anamorph that include mycelial colour, hyphal diameter, number of nuclei, length of cells, shape and size of monilioid cells and sclerotial size (Hietala 1998). Rhizoctonia strains are designated as uni-, bi- or multi-nucleate (UNR, BNR and MNR, respectively), with respect to nuclear condition. The determination of nuclear condition is particularly informative in isolate characterization of vegetative hyphae. Where teleomorph data is available, Rhizoctonia-like anamorphs are distributed in several families: Platygloeaceae (genus Helicobasidium), Exidiaceae (Sebacina), Atheliaceae (Athelia), Tulasnellaceae (Tulasnella), Ceratobasidiaceae (Thanatephorus, Ceratobasidium), and Botryobasidiaceae (Waitea) (Hawksworth et al. 1995). Overall, the latter 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 et al. 1994) and Norway (Venn et al. 1986), belong to the Ceratobasidium family and are conspecific with a moss parasite Ceratobasidium bicorne, based on rDNA (internal spacer, ITS) sequence analysis (Hietala et al. 2001). More recently, a large number of fungi displaying Rhizoctonia anamorphs have been isolated from tropical orchids in Puerto Rico by Otero et al. (2002) who found that 66 of 108 examined were uninucleate and showed highly related anamorphic and ITS phylogenetic relationships with Finnish conifer pathogenic uninucleate Rhizoctonia (Ceratobasidium bicorne) (Hietala et al. 1994, Hietala et al. 2001). Due to its broad host range and common pathogenic association with important agricultural crops, probably the most studied Rhizoctonia species is Rhizoctonia solani Kühn. The perfect state of R. solani, Thanatephorus cucumeris, belongs to the family Ceratobasidiaceae (order Ceratobasidiales) of class Basidiomycetes (Hawksworth et al. 1995). Based on hyphal interactions in pairing experiments, multinucleate R. solani has been grouped into different anastomosis groups (AGs) and represents a collection of closely related isolates grouped together based on their ability to anastomose with one another. In the so called “killing reaction” anastomosis results in death of anastomosing and adjacent cells, but no killing reaction occurs in self-anastomosis and in hyphal reactions between clonal isolates (Carling 1996). Our understanding of Rhizoctonia genetic diversity and taxonomy has been greatly improved 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 (order Ceratobasisiales) of class Basidiomycetes (Hawksworth et al. 1995). The anamorphs of this genus are mainly binucleate, except for the uninucleate Ceratobasidium bicorne Eriks. & Ryv. (Hietala et al. 1994, Hietala et al. 2001) mentioned earlier. The binucleate Rhizoctonia 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 root and foliar diseases (Kataria & Hoffmann 1988) but also forms symbiotic mycorrhizal associations with orchids (Andersen & Ramussen 1996).

8 Sen et al. (1999) proved by AG tests and rDNA-RFLP (restriction fragment length polymorphism) fingerprinting a high degree of intraspecific variation within BNR belonging to teleomorphs of Ceratobasidium cornigerum. These fungi include isolates able to enter into either mutualistic or pathogenic root association with susceptible host plants. In the anastomosis grouping analysis of different BNR strains, the Scots pine BNR isolates 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) fell into 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 Artemisia vulgaris 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 also anastomosed with two Canadian orchid isolates. Two other isolates (266 and 269) were not members of any of the other 20 described AGs (AG-A - AG-S) of binucleate Rhizoctonia (Sen et al. 1999).

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

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 ribosomal RNA genes are generally arranged as a tandemly repeated array with both variable and highly conserved regions (Gardes et al. 1991). Internal transcribed spacer regions (ITS 1 and 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 be highly variable between distantly related species and related genera (Bruns & Gardes 1993). Universal primers for fungal PCR amplification of the ITS region (ITS1/ITS 4) were originally developed by White et al. (1990) (I: Fig.1). These primers allow fungal specific ITS amplification directly from conifer tree associated mycorrhizas as they discriminate against 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 ITS fungal sequence database (cf. Kõljalg et al. 2005). This enables multiple sequence alignments between numerous fungal ITS sequences for robust phylogenetic analyses. It also allows identification of target rDNA, both variable and conserved regions that could also represent suitable sites for molecular probe development. The ITS has been a target in several different molecular studies within Rhizoctonia species (see section 1.3) and molecular approaches have been very fruitful in answering basic questions concerning systematic relationships and genetic variation in Rhizoctonia spp. (Vilgalys & Cubeta 1994). The continued growth of Rhizoctonia ITS sequence data (Gonzalez et al. 2001) has enabled increasingly robust sequence comparisons and phylogenetic analyses that support the 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, the characterization of isolates has been usually based on anamorphic characteristics. Since the vegetative characteristics of many Rhizoctonia species overlap, it is difficult to identify 9 these species (Hietala 1998). Anastomosis grouping based on hyphal fusion has been a powerful tool in recognising Rhizoctonia groups within species (Carling 1996), but molecular methods have improved Rhizoctonia isolate identification. Methods such as DNA/DNA hybridization (Kuninaga & Yokosawa 1984 and 1985), isozyme analysis (Liu et 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-ITS region targeted primers (Salazar et al. 2000, Kasiamdari et al. 2002) or taxon-specific primers, as designed by Taylor and McCormick (2008) for basidiomyceteous orchid mycorrhizas belonging families Tulasnella and Thelephora-Tomentella complex. Lees et al. (2002), on the other hand, applied ITS region for detection and identification of Rhizoctonia solani AG-3 in potato and soil by more sensitive qRT-PCR-method, which optimally permits quantification at sensitivity of one gene copy.

1.4. Rhizoctonia spp. as economically important fungal pathogens of plants

Many Rhizoctonia species cause economically important diseases on most of the world’s important crop plants, such as: cereals, cotton, sugar beet, potato, vegetables, field crops, turf grasses, ornamentals, fruit trees and forest trees (Sneh et al. 1996). The best known 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 in forest nurseries in many countries including France (Perrin & Sampangi 1998) and Poland (StĊpniewska-Jarosz et al. 2006). In Finland, on the other hand, mainly uni- or binucleate Rhizoctonia strains have been isolated 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 Norway spruce (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 the most important in Finland and thus form a major proportion of the different tree species grown in nurseries and delivered for planting. Nurseries are involved in cultivation of tree seedling stock (1-3 years) for reforestation. Seedlings can be cultivated as bare-root stock in open nursery beds or in containers made from plastic, paper or peat. In Finland, paper manufacture and wood industries are an important source of national income and Rhizoctonia species, that infects young conifer tree seedlings, may result in major economic losses. To date, no effective control treatment against these pathogenic fungi has emerged. The uninucleate Rhizoctonia attacks conifer roots from root tips and penetrates into the vascular cylinder (Hietala 1998). The infection results in the root dieback disease with symptoms such as needle discoloration, partial death of the root system and stunted seedling growth (Lilja 1996). Binucleate Rhizoctonia (BNR) are well known plant pathogens and have also been isolated from diseased and healthy looking Scots pine and Norway spruce seedlings (Lilja et al. 1992), but BNR isolates did not negatively influence seedling growth in nursery peat soil pathogenicity tests (Lilja 1994, Hietala 1995).

10 1.5. Binucleate Rhizoctonia as symbiotic fungi and bicontrol agents

Strains of Ceratobasidium spp. commonly exist in mycorrhizal associations with orchids (Sneh et al. 1996). These orchid associated Rhizoctonia strains are reported to form typical morphological structures in host tissues that have been identified as monilioid cells (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 of significant 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 multinucleate Rhizoctonia AG-6 of orchid mycorrhizal fungi (MF) stimulating the orchid Anoectochilus formosanus Hyata growth compared to non-mycorrhizal plants. Plant height, number of leaves, root length and fresh weights were enhanced by these fungi and they also showed higher enzymic activities of superoxide dismutase (SOD) in leaves, and acid and alkaline phosphatases in roots, together with higher contents of ascorbic acid, polyphenols, and flavonoids.

1.5.1. Biocontrol: principles, applications and mechanisms

According to a plant dictionary (Allaby 2006) biological control is defined as “human use of natural predators for the control of pests, weeds etc.” or the “total or partial destruction of pathogen populations by other organisms” (Agrios 1997). Biocontrol organisms are usually microbial control agents (MCA) namely bacteria, protozoa, fungi or viruses. Biopestisides, on the other hand, are “mass-produced, biologically based agents used for the control of plant pests” (Chandler et al. 2008), which can be divided to living organisms, naturally occurring substances, and genetically modified plants (Copping & Menn 2000). Biocontrol agents are mainly developed to prevent specific pests or pathogens from infecting the target plant. Examples include hypovirulent or avirulent strains, which infect the host without causing a disease. Protection is achieved either through direct biocontrol agent-mediated antagonism against the pathogen, through competition for infection sites, or the biocontrol agent may strengthen the host defence through induced resistance (Cardoso & Echandi 1987). Due to its wide distribution and severity as an economically important pathogen of crop plants, many studies have concentrated on investigating promising biocontrols against 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 & Benson 2003, Pascual et al. 2004) have shown promise as biocontrol agents against R. solani in different plants. BNR (Ceratobasidium albatensis) has been shown to control R. solani induced damping-off in Pinus spp. (Rubio et al. 2001 and pers. comm.). The authors suggested that the timing of the biocontrol inoculation is an important parameter. BNR protective effects against R. solani are usually obtained following BNR pre-inoculation before R. solani attack, either after short periods of preinoculation e.g. 24 h (Poromarto et al. 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 simultaneous infection, direct antagonism is probably not the mechanism involved (Herr 1995). Rather competition and induced resistance are suggested as being the controlling mechanism in these relationships (see 1.5.2. and 1.5.3.).

11 1.5.2. Competition as a potential mechanism for biocontrol

Competition signifies competition for nutrients or infection sites, or both. If the host plant cells are pre-occupied by an avirulent strain, the pathogen tends to be unable to infect same cells. Additionally, lack of nutrients may reduce the attraction of pathogens. Therefore indirect protection exists despite antimicrobial or defence related molecules being involved. Competition is suggested to be one possible biocontrol mechanism in R.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 host defence reaction, which in subsequent challenge or infection is more rapidly and efficiently activated. This systemic acquired resistance (SAR) (Ryals et al. 1994) or induced systemic resistance (ISR) (Xue et al. 1998) can prevent further infection of plant host by potentially more severe pathogens. Thus, SAR/ISR can be exploited as a biocontrol mechanism. The SAR includes the activation of host defence genes and often also a plant hypersensitive response (HR). HR is expressed in plant tissues as self-induced localized cell death at the site of infection, which prevents the pathogen from further penetration into adjacent cells, and is associated with generation of activated oxygen radicals (Agrios 1997). Many authors have suggested induced resistance being the mechanism of protection in BNR-preinoculated plants against pathogenic R. solani (Cardoso & Echandi 1987, Xue et al. 1998, Porommarto et al. 1998, Hwang & Benson 2003) and several defence genes have been 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 this activation depends on the specific elicitor and the pathogenicity of each strain. In Scots pine (Pinus sylvestris), for example, the transcriptional responses were differentially activated in mutualistic, saprotrophic or pathogenic fungi (Adomas et al. 2008), and during the early stage fewer genes were differentially expressed in pathogenic, as opposed to ectomycorrhizal or saprotrophic interaction. The authors suggested that as the host was unable to recognise the nature of the attack during the early stages of infection, this enabled the pathogen to overcome the host’s defence. In the later stages of infection, the host defence responded more strongly to interaction with the pathogen. Additionally, induction of defence genes in Norway spruce were more rapid in a pathogen resistant clone compared to less resistant one (Karlsson et al. 2007). Pathogenesis related (PR) proteins are a diverse group of plant proteins possessing antimicrobial properties against invading fungal pathogen. Some of the best known are PR1 proteins, ȕ-1,3-glucanases, chitinases, lysozymes, PR 4 proteins, thaumatinelike proteins, osmotinlike proteins, cysteine-rich proteins, glysine-rich proteins, proteinase inhibitors, proteinases, chitosanases, and peroxidases (Agrios 1997). Some of these have been reported as also being involved in the interactions between coniferous trees and different fungal strains.

12 Chitinases were an important example of differentially expressed genes in Scots pine (Pinus sylvestris) during the infections involving mutualistic, saprotrophic or pathogenic fungi (Adomas et al. 2008). These enzymes degrade the fungal cell wall and have, in many other studies, been reported to be involved in coniferous plants defence (Hietala et al. 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 (Laccaria bicolor) and pathogenic (Heterobasidion annosum) interaction in Scots pine (Adomas et al. 2008) and in H. annosum infection on Norway spruce [Picea abies (L.)Karst.] (Jøhnk et al. 2005, Karlsson et al. 2007). A class III peroxidase activity and thaumatin were activated constitutively (1, 5, and 15 d.p.i.) in Pinus sylvestris during Heterobasidion annosum- infection (Adomas et al. 2007) and related transcripts were also up regulated in saprotrophic (Trichoderma aureoviride) infection at 1 d.p.i. (Adomas et al. 2008). Other transcripts reported to be induced in H. annosum- Scots pine interaction includes for example glutathione-S-transferase and germin-like protein (Karlsson et al. 2007). Another pathogen, a uninucleate Rhizoctonia (Certatobasidium bicorne), induced 25 transcripts in Norway spruce, including oxidative-process-associated proteins, such as germin-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 and many transcripts, including chitinases, and defensin, together with other previously mentioned enzymes, were activated in the same host by this pathogen. On Scots pine, an antimicrobial peptide gene Sp-AMP (Asiegbu et al. 2003) and a defence-related gene PsACRE (Li & Asiegbu 2004) were associated in Heterobasidion annosum infection. The antimicrobial gene was also induced by the saprotroph and symbiotic fungi at 1 d.p.i., but up-regulation continued only in the pathogenic interaction 5 and 15 d.p.i. (Adomas et al. 2008). This pathogen induced 179 differentially expressed sequence tags in a cDNA microarray study, the most abundant ones being enzymes involved in the phenylpropanoid pathway and defence-related proteins with antimicrobial properties (Adomas et al. 2007). Whether these genes are correspondingly up-regulated in Scots 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 cinnamic acids. It consists of a C6C3 carbon skeleton, which by further hydroxylation and methylation yields simple phenylpropanoids such as, p-courmaric, caffeic, ferulic and sinapic acids (Gross 1985). Many other important and more complex secondary metabolite products 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) and stilbene synthase (STS), two closely-related polyketide synthases (PKS), in a condensation of p-coumaroyl-CoA /cinnamoyl-CoA with three molecules of malonyl-CoA (Hahlbrock & Grisebach 1979, Dixon & Paiva 1995). The STS in Pinus sylvestris is classified as pinosylvin synthase, since it prefers cinnamoyl-CoA to dihydrocinnamoyl- CoA as a substrate, but the substrate preference in expression studies in vitro is influenced by factor(s) co-existing in plant or bacterial extracts (Schannz et al. 1992). In Pinus strobus (Eastern white pine), on the other hand, both substrates are utilized and therefore two stilbene types - pinosylvin and dihydropinosylvin- exist (Raiber et al. 1995). The STS sequences of P. strobus clustered in their own subgenus (STROBUS STS) in the study of Kodan et al. (2001), while Pinus densiflora clustered together with P. sylvestris in another subgenus (PINUS STS) and all CHS sequences from these three Pinus species in their own CHS subgenus. Two P. strobus CHS sequences have been identified by Schröder et al. (1998), one being more closely related to P. sylvestris CHS, while the other using methylmalonyl-CoA in a condensation with c-methylated chalcones and suggested to have

13 a special role in the biosynthesis of secondary products. The latter type of CHS is not present in P. sylvestris (Schröder et al. 1998). The Scots pine defence-related activation of stilbenes 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 many plants (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 activation compared during the infection and drought stress (Fossdal et al. 2007).

1.7. Induction of host defence-gene activity by hypovirulent strains

Pre-inoculation of bean seedlings with non-pathogenic BNR have been reported to induce the expression of host defence enzymes e.g. peroxidases, and 1,3-ȕ- glucanases, which positively correlated with induced systemic resistance (ISR) against R.solani and Colletotrichum lindemuthianum pathogens (Xue et al. 1998). Seedlings treated with BNR prior to infection elicited significantly more of these host defence enzymes compared to the diseased and control plants. Similar enzymes were also induced in soybeans pre- treated with Pseudomonas aureofacien bacteria (Jung et al. 2007) and are also generally considered as being involved in plants induced resistance (Agrios 1997). Induced resistance 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 root exudates did such that the R. solani protective effect was retained in BNR pre-inoculated roots after surface sterilization. Preinoculation of bean seedlings with an arbuscular mycorrhizal fungus (AM) Glomus intraradices Schenck and Smith, on the other hand, did not significantly prevent disease symptoms of R. solani, and no change in phenylalanine ammonia lyase, (PAL), chalcone synthase (CHS), or chalcone isomerase were observed. R. solani has been reported to induce 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 of 1,3-ȕ-D- glucanase, PAL, and CHS, were in R. solani infected beans, but were also expressed, though down-regulated in seedlings pretreated with non-pathogenic binucleate Rhizoctonia before R. solani infection. Cardinale et al. (2006) investigated the activities of biological defence markers PR1, laminarinase, and chitinase to follow the plant-mediated resistance of hypovirulent R. solani against Botrytis cinerea pathogen in tomato. Hypovirulent strain of R. solani boosted the B. cinerea induced enzyme activities and the authors suggested a systemic induced resistance being the nature of protection.

14 2. Aims of the study

The aim of this study was to investigate the infection biology and impact of pathogenic uninucleate Rhizoctonia (UNR) (Ceratobasidium bicorne) strains 1983-111/1N and 263, and endophytic binucleate Rhizoctonia (BNR) (Ceratorhiza sp.) strains 268, 251, 266 and 269 on Scots pine growth and host defence responses, and to detect these strains from infected roots. More specific goals in the respective studies were to:

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

15 3. Materials and Methods

3.1. Strains and methods described in publications

This study targeted uni- and binucleate Rhizoctonia spp. strains (Table 1), but relevant fungi from the genus Suillus and Rhizoctonia, and other plant root pathogenic fungi were included in hybridizations (I: Table 1). All methods used in the three separate studies are briefly 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 Study 1983-111/1N (Ceratobasidium UNR Hietala et al. (1994) III bicorne) 263 (Ceratobasidium UNR Hietala et al. (1994) I bicorne) 268 (Ceratorhiza sp.) BNR Sen et al. (1999) I, II, III 251 (Ceratorhiza sp.) BNR Sen et al. (1999) I, II 266 (Ceratorhiza sp.) BNR Sen et al. (1999) I, II 269 (Ceratorhiza sp.) BNR Sen et al. (1999) I, II

16 Table 2. Methods used in three separate studies.

Method Reference study Short description Seed 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 et II, III pots: 20 h day length, >230 µmol m-2 s-1, +20/12ºC, al. (2001) cuttings in work II and seedlings in work III, 19 h day length, 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 the experiment 2 at the time of 10-day-old seedling removal Fungal growth Marx (1969), Modess, I, II, III +20ºC, medias: Modified Melin Norkans (MMN), Hagem´s (1941), Sen et al. (1999), agar (HA), malt-extract agar (MA), potato-dextrose-agar Murashige & Skoog (PDA), Murashige and Skoog nutrient salts medium (MS), (1962) Water agar (WA) DNA extraction Heinonsalo et al. (2001), I, III CTAB-method, DNeasy plant mini kit (Qiagen, Hilden, Qiagen Product Manual Germany) RNA extraction Qiagen Product Manual III Plant MiniRNeasy kit (Qiagen, Maryland, USA) PCR Heinonsalo et al. (2001) I, III Cloning 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 (Program Manual for the Wisconsin Package, Version 8, Wisconsin) Sequence Higgins et al. (1992) I Clusteral method alignment Primer design I, III III: ABI Primer Express® (Applied Biosystems, Foster City, CA, USA) Liquid- and DIG system’s user guide I DIG (Roche, Germany) or Biotin (Institute of Southern (Roche, Germany), Biotechnology, UH, Finland) labelled probes hybridization Karcher (1991), Hendolin et al. (2000). cDNA synthesis III Taqman Reverse Transcription Reagent, (Applied Biosystems, New Jersey, USA) Real time PCR Guide to performing III ABI 7300 quantitative real-time PCR machine, (Applied Relative Quantitation of Biosystems, Singapore) Gene Expression Using Real Time Quantitative PCR. Applied Biosystems Tryphan blue Koske & Gemma (1989) II, III fungal root-infection visualization, III: 10 min. +60 ºC in 2,5 staining % KOH, rinsed twice with H2O, neutralized in 1% HCl 1 h at 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 infection Giovanetti & Mosse(1980), II, III II: Grid-line intersect method, III: ITS-primer and qRT- determination Hietala et al. (2008) PCR-standard curve based quantification Statistics Paffl (2001) (III) II, III II: one-way ANOVA and pair wise differences by Tukey’s HSD test, SPSS version 8.0 for MS Windows.III: GLM procedure, SAS (P ‹ 0.05)., III: analysis of variance, GLM procedure of SAS (P<0.05)

17 3.2. Phylogenetic analysis

A nearest neighbor tree based on ITS (ITS1-5,8S-ITS2) sequence (I) analysis was generated using the MEGA4 program (Tamura et al. 2007). Waitea circinata strain TGCC 17.1 (de la Cerda et al. 2007) was selected for outgroup in this analysis.

18 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 primers from genomic DNA were between 644-703 base pairs (ȱ). The Rhizoctonia solani HK1 PCR product was 703 bp. As expected the 5.8 S was highly conserved among different Rhizoctonia strains and Suillus species. In the Suillus ITS sequence comparison the most variable region located in ITS 1 for the two S. bovinus strains (SBH1 and C3-1-M1) differed only by a single base pair in their ITS sequence (I). The beginning of this ITS 1 region was targeted for Suillus probe construction (I: Fig. 1). ITS 1 was also the most variable region identified in the Rhizoctonia ITS sequence comparison, but the similarly variable ITS 2 region was targeted for probe development, since suitable optimal primer sequences for PCR amplification were found in this region. The conserved 5.8 S ribosomal gene region was targeted 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 BNR AV-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 other binucleate Rhizoctonia strains. All binucleate Rhizoctonia strains (251, 266, AV-2, 268 and 269) were found in a general cluster and as was expected, the uninucleate 263 and multinucleate Rhizoctonia solani strains were clearly less related to the binucleate Rhizoctonia cluster.

19 97

72

72 63 40

0.1

Figure 1.Nearest neighbour tree. Symbols: żAV-2, Ɣ268, Ƒ269, Ŷ266, ǻ251, Ÿ263, źR.solani HKI, Ƈ Waitea circinata.

4.1.3. Probe construction and application in Southern dot-blot- and liquid hybridization

Primer sequences designed to amplify particular probe sequence from ITS target sequence are presented (I: Table 3). The size of the PCR amplified probes were between 124 and 151 bp. PCR products were of the correct size, except for a few problematic samples. When control primers were used to amplify the control probe sequence and when Rhizoctonia solani ITS insert in plasmid vector was used as a template, the PCR product occasionally included additional faint bands (data not shown). Amplification of Suillus strain template with Suillus primers also produced a faint smaller band, but DNA from the correct sized bands extracted from agarose gel and used in secondary PCR, produced mostly only the expected sized DNA fragment without additional bands. The restriction analysis of amplified probes gave the expected size restriction fragments, except the Rhizoctonia solani probe sequence amplified with Rhizoctonia primers, where some additional bands were detected (data not shown). The optimal hybridization temperature for oligonucleotide probes were about 15-30 ºC lower than the Tm, while near the Tm in PCR amplified probes (I: Table 2.). The estimated detection limit to DIG labeled probes in dot blotting were 0.1-0.01ng/Pl (dilutions 1:10-1:100). Dot blot hybridizations with DIG-labelled probes targeted total DNA 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-probe hybridizations. A summary of dot blot hybridization results (I: Table 4) shows the biotin labeled 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 hybridized with 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) also detected S. granulatus. The same hybridization temperatures optimised for Southern hybridization also gave the best results in liquid hybridization, indicating the temperatures being dependent on probe sequences rather than method applied. Low temperature washing (30 or 35ºC) occasionally affected the specificity with 268 or Suillus probe (number 3 and 2 probes respectively in I: Table 2). The biotin labelled probe in combination with liquid hybridization delivered the highest detection specificity (I: Table 5 and Fig. 4). 20 4.2. Scots pine seedling growth and root architectural responses to binucleate Rhizoctonia inoculation

Compared to uninoculated controls, the shoot and root weights in the BNR-inoculated seedlings exhibited significant (P=0.05) differences at 87 d.p.i. (II). No strain specific differences 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 short root 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 increased significantly, but no differences in shoot biomass were detected compared to uninoculated controls. The root growth responses were also more attenuated. The root infection levels remained 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 a dual inoculation on water-agar Petri-dish (Fig. 3 a). Monilioid cells detected from the roots of tryphan blue stained Scots pine radicals infected with BNR strain 268 nine weeks p.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 monitored period in study III. In single inoculations, BNR and UNR colonization was detected in cortical cells throughout the entire root, but BNR colonized more intensively root cells from the basal region, while UNR colonized heavily the root tips and less intensively basal regions in a contrasting manner to BNR (Fig. 3 b, d). Both UNR colonized and uncolonized root tips were detected in dual- infected roots (Fig. 3 c, III).

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

22 Figure 3. a.) UNR and BNR hyphae growing towards each other on WA-plate. Arrows show the hyphal tips, which are not affected by the other strain (Bar 5 µm) b.) Scots pine root tip infected with 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-specific rDNA ITS-primers and GAPDH primers respectively for detection of fungal genomic DNA from infected roots and host genomic DNA (III: Table 1). The level of colonization was 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 the difference in the BNR DNA content at 96 h.p.i between single and dual inoculation was not significant (p = 0.07). In the single inoculations, the mean DNA content of both UNR and BNR reached near 5% 96 h.p.i. (Fig. 4 a and b, III: Fig. 1). The contents of UNR

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

16 % b. 24 % a. 22 % 14 % BNR Exp 1 UNR Exp 1 20 % 12 % BNR Exp 2 18 % UNR Exp 2 10 % BNR+ Exp 1 16 % UNR+ Exp 1 BNR+ Exp 2 14 % UNR+ Exp 2 8 % 12 % DNA 10 % DNA 6 % 8 % 4 % 6 % 2 % 4 % 2 % 0 % 0 % -2 % 0h 4h 24h 48h 96h 192h -2 % 0h 4h 24h 48h 96h 192h inoculation time inoculation time

Figure 4. The mean fungal DNA contents (%) of total (host and fungal DNA) in infected roots at inoculation (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 one week earlier.

4.4.1.2. Experiment 2.

Experiment 2 was performed similarly to experiment 1, except that BNR was preinoculated one week earlier in respective roots in order to determine possible biocontrol effects of BNR against UNR, and prolonged inoculation times were used. In the BNR-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). The overall 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 control over the experimental period in both experiments (Fig. 5 a, and 6 a, III: Fig. 2 a). The highest activities were detected in UNR single infections. BNR and dual inoculated roots showed relatively similar STS transcript levels.

24 a. 220 b. 16 PAL 170 14 PAL CHS 12 120 CHS S TS 10 PRX 70 PRX 8 20 6 4 -30 4 24 48 96 4 24 48 96 4 24 48 96 4 244896 2 -80 Control BNR UNR BNR+UNR 0

-130 -2 4 4 4 4 24 48 96 24 48 96 24 48 96 24 48 96 norm alizedlevels transcript normlevels alized transcript -180 Control BNR UNR BNR+UNR

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

a. 650 b. 30 550 PAL 25 PAL CHS CHS 450 20 PRX 350 STS PRX 15 250 10 150 5 50 -50 0 0 0 0 0 0 0 0 0 48 96 48 96 48 96 48 96 48 96 48 96 48 96 48 96 192 192 192 192 192 192 192 192 normalizedlevels transcript normalizedlevels transcript -150 Control BNR UNR BNR+UNR Control BNR UNR BNR+UNR

Figure 6. Defence-gene transcript activities in the experiment 2 (III). a.) STS, PAL, CHS, and PRX. 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 extremely high, and only the activity in dual infected roots in experiment 1, 48 and 96 h.p.i., were significant (P ‹ 0.03). Significant up-regulation of PAL were detected in dual infection 96 h.p.i. in both experiments and 192 h.p.i. in the experiment 2. Single UNR infected roots showed significant up-regulation at all time points post 48 h.p.i. The only significant CHS activity was detected in experiment 2 between dual inoculated 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 any treatments or incubation times (data not shown.)

25 5. Discussion

5.1. Rhizoctonia spp. ITS sequences and phylogenetic analysis

The binucleate Rhizoctonia (BNR) strains used in this study have earlier been identified as anamorphic Ceratorhiza spp., which include binucleate Rhizoctonia that form mycorrhiza with orchids (Sen et al. 1999). A phylogenetic analysis of ITS sequences of BNR from this study and diverse binucleate Rhizoctonia (including members of all anastomosis groups) strictly confirms that our binucleate Rhizoctonia are the telemorphic species Ceratobasidium cornigerum (Gonzalez et al. unpublished data). This also now confirms 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 uninucleate Rhizoctonia (UNR) strains 263 and 1983-111/1N, selected for this study, have been identified as Ceratobasidium bicorne based on rDNA (internal spacer, ITS) sequence analysis (Hietala et al. 2001). In Rhizoctonia taxonomy, the faster and more precise ITS sequencing technique combined with phylogenetic analyses have now mostly replaced earlier anastomosis grouping methodology, nevertheless the AG grouping remain valuable tools in identification. 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 analyses distinguishes the isolates into separate clades according to their AG grouping. This confirms 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 strain identification due to the presence of high sequence similarity between related strains and simultaneously sufficient differences between unrelated strains. We observed that the ITS- 1 and ITS-2 regions offered relatively high sequence variation for >100 bp probe, and highly variable areas for shorter oligonucleotide probe development. Species/taxa specific probes specific for conifer root pathogenic and endophytic Rhizoctonia spp. and the ectomycorrhizal Suillus bovinus were successfully developed. PCR amplified (124-151 bp) probes and oligonucleotide (20-25 bp) probes were respectively developed for Southern and liquid hybridization (Hendolin et al. 2000). Oligonucleotide probes were also 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 is known 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 the sequence alignment. The non-coding ITS1 and ITS 2 regions were more variable, as could have been expected. Binucleate Rhizoctonia strains 268 and AV-2 (AG-I), which belongs to the same anastomosis group, were practically identical (99%) having only few nucleotide differences in ITS 1. Interestingly, two Suillus bovinus strains isolated from different forest areas in South and central Finland (Leppäkorpi and Hyytiälä), differed by a single nucleotide substitution; C and T at base position 123, respectively. The additional PCR amplified bands obtained with control and Suillus primers can be explained by the fact that the ITS region occurs as a part of a multicopy rDNA cistron in the 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 26 several minor ITS sequence length variants. As such, a cloning step has to be included before sequencing, where only one DNA fragment is ligated to the vector and in this way overlapping signals can be avoided. However, additional bands with control primers appeared on only a single occasion and there might have been some contamination in the particular PCR reaction. Suillus primers regularly amplified additional bands in the primary PCR. Targeted sized PCR product was obtained after re-amplification of extracted DNA of the correct size. We preferred the approach of direct fungal detection from root material, than using PCR 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 bp DIG labelled probes were targeted specifically against total DNA. All these longer probes were tested against DNA extracted from Scots pine roots. This plant serves as a host to both Suillus bovinus forming mycorrhiza and the uni- and binucleate Rhizoctonia strains in pathogenic and biotrophic interactions (Hietala & Sen 1996). Only the probe developed against uninucleate Rhizoctonia strain 263 hybridized with Scots pine root DNA sample. While direct detection would shorten the identification process, the PCR based method used in liquid hybridization was found to be more sensitive and reliable than Southern hybridization, because the size of the hybridized PCR product is visualized on the computer screen simultaneously. Additionally, possible similarities between probe and non-target of fungal DNA sequences are eliminated through pre-amplification by PCR with ITS1 and ITS2 primers. These universal primers specifically amplify the ITS region from most fungi (Bruns & Gardes 1993) and in this way, for example bacterial or host plant DNA could have been excluded in hybridization test. The Suillus probe was not, however, Suillus bovinus specific, since it hybridized with all other Suillus species except Suillus luteus. Further screening with additional Suillus strains and strains of other species should be carried out to determine the limits of probe spesificity. Conversely, probes developed targeting spesific binucleate Rhizoctonia strains were 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 avoid unspecific fungal or root binding. Another alternative approach for fungal identification is to design specific primers for PCR 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 a PCR-based procedure. Groppe et al. (1995), and Groppe & Boller (1997) have used microsatellite containing locus as a target in PCR detection for ecology and diversity studies of the plant associated fungus Epichloë. Buscot et al. (1996) used microsatellite- primed PCR in morels DNA polymorphism investigations. This is another alternative target in fungal DNA based studies, although ITS is a better DNA target for probe development. Microsatellite regions are stretches of tandem mono-, di-, tri-, and tetranucleotide repeats of various lengths and a wide range of repeat lengths can be present in a single population (Groppe & Boller 1997). The most sensitive method available is perhaps 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). This method combined with TaqMan chemistry, allows also more reliable quantitative detection of the target fungi than the conventional PCR, since only a specific PCR product is detected. Classical morphological methods are still of value, even though molecular methods have obvious advantages in species identification. Specific probes for direct detection of target sequences for example in soil, are almost impossible to obtain, since there are so many different organisms and different species in nature, with uncharacterized DNA and there is always a possibility that the developed probe give "false" positives in hybridizing to 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 by Lees et al. (2002) in comparison to qRT-PCR with conventional PCR. Nevertheless, in order to reduce errors, morphological characters of isolated fungi (where possible) and

27 lesions tissue analysis for example from Rhizoctonia infected plants should be the first step in species identification and detection with specific probes, in conventional hybridization 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 in qRT-PCR would help us to detect these economically important strains both from nurseries and the forest site and probably greatly help improve our understanding of Rhizoctonia infection biology. These probes would also help further answer the question as 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, Zelmer et al. 1996, Sharma et al. 2003), but have also been reported to show beneficial effects on other 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). Some BNR strains are pathogenic (Stepniewska-Jarosz et al. 2006) and this was originally thought to be the case with our Finnish binucleate Ceratorhiza spp. (strains 251, 266, 268 and 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, but in the second harvest (240 d.p.i.) the differences between inoculated and uninoculated seedlings were not significant. Instead, the short root number increased and was more numerous in BNR inoculated than uninoculated seedlings. The low root infection-rate, and –morphology identified by tryphan blue staining, suggests that a true mycorrhizal relationship does not occur between host and fungus. In forest soil, Scots pine rapidly enters into mycorrhizal associations with several fungal species (Smith & Read 1997, Timonen et al. 1997), but typical ectomycorrhiza structures have, to our knowledge, not been reported in Rhizoctonia colonized conifer roots. Additionally BNR associated Scots pine seedlings showed yellow-green needles in N- and C- limited nursery soil (II), suggesting lack of nutrients, whereas in similar aged mycorrhizal seedlings associated with the true mycorrhizal fungus Suillus bovinus, the needles remained a healthy dark green in the same nursery soil (Sen 2001). These data indicate that early root induction was not related to improved nutrient uptake through a classical mycorrhizal association. However, BNR linked root growth could be explained through possible hormonal linked stimulation. Plant growth promoting hormonal signaling is crucial for development of plant-microbe interactions, and indole-acetic acid (IAA) has been detected in R. solani cultures (Furukawa et al. 1996); the amount of IAA was found to increased in R. solani rice suspension cultures (Furukawa & Syono 1998). In a related study, Kaparakis and Sen (2006) showed that similar Scots pine adventitious root inducing effects resulting from indol-3-butyric acid (IBA) exposure could be obtained in BNR inoculation experiments and, importantly, that BNR-linked root induction was even more efficient. Whether there are differences in hormonal communication between pathogenic or non-pathogenic Rhizoctonia-host interactions is not known. It is clear that further work is needed in order to understand the molecular and physiological mechanisms of hormonal interaction in these associations. Hwang & Benson (2003) investigated the potential of BNR strains as biocontrol agents against R. solani attack in poinsettia cuttings, but they did not compare the rooting or 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 growth promoting effects as our results suggests from Scots pine cuttings (Kaparakis & Sen 2006). The typical monilioid cells present within the cells of the outer cortex, as visualized by tryphan blue staining, have been earlier reported in orchid mycorrhiza (Zelmer et al. 1996, Sharma et al. 2003) and uninucleate Rhizoctonia (Hietala 1997). 28 Seedlings infected by BNR produce more short roots, which in forest soils, would rapidly become colonized by mycorrhizal fungi during mycorrhiza development. In nursery soils these nonmycorrhizal short roots could represent sites for UNR entry. As Rhizoctonia spp. have been isolated from fine roots of Scots pine trees in Scottish forest stands (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 forest sites.

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

Preinoculation of binucleate Rhizoctonia (BNR) strain 268 decreased the infection level of pathogenic uninucleate Rhizoctonia (UNR) strain 1983-111-/1N in the Scots pine radicals. However, in in vitro confrontation assays on water agar it neither showed any antagonistic effects against UNR (Fig. 3a), nor prevented UNR entry into the root cells in vivo. The simultaneous coinoculation with UNR decreased the BNR infection level compared to BNR single inoculations, indicating UNR being more competitive in the infection process, despite the slower in vitro growth on WA plate. In BNR preinoculated roots, on the other hand, the UNR level was restricted compared to single UNR inoculations. This might be due to the root cells being precolonized with BNR. Surprisingly, both BNR and UNR infection levels were temporarily decreased during the infection process, respectively, 24 and 48 h.p.i., suggesting that the biochemical communication between fungus and host retards the infection. Absolute quantification of UNR and BNR infection in this study was impossible, since data 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 time post inoculation. Regardless of potential differences in the ITS copy number between BNR and UNR, it would not alter the fact that the amount of UNR was restricted in BNR preinoculated roots, compared to simultaneous dual inoculation. In a study by Cardoso & Echandi (1987), BNR also failed to show antagonistic interaction in dual cultures of bean (Phaseolus vulgaris L.) with R. solani. Filtrates alone from 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 infection cushions. Therefore, the authors suggested that BNR- induced metabolic response was the mechanism of R. solani suppression. In our study (III) the UNR infection specific DNA levels 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 was identified compared to simultaneous dual infection, although the transcript was significantly up-regulated in both experiments compared to control. Significant PAL activity was also obtained in single UNR infected roots in experiment 2 compared to control, but regardless of the respective identically accomplished treatments of single UNR 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 -pretreated and R. solani-infected bean seedlings, the activity was lower. The preinoculation with the mycorrhizal fungus, Glomus intraradices Schenck and Smith, however, did not alter the bean PAL-, CHS-, chalcone isomerase-, and hydroxyproline-rich glycoprotein activities during R. solani infection (Guillon et al. 2002). In our study, CHS was activated significantly only in dual infected roots in experiment 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-

29 activity is probably not solely induced in Scots pine defence reactions, since it has been shown to be also expressed in non-stressed plantlets (Fliegmann et al. 1992). In Norway spruce, on the other hand, Ophiostoma polonicum-infection elicited both CHS and STS activities, 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, being highest in UNR single inoculated roots. The STS responded also to stress in nicked Scots pine hypocotyls more efficiently than CHS (Fliegman et al. 1992). Our study showed relatively high variations in all transcript activities between replicates, indicating high degree of genetic variation between seedlings. Thus, the use of clonal material would be the solution in future experiments. All three studied phenylpropanoid pathway involved enzymes, phenylalanine ammonia lyase, 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, Chiron et al. 2000, Yu et al. 2005). Adomas et al. (2007) reported that enzymes from the phenylpropanoid pathway, defence-related proteins with antimicrobial properties, and class III peroxidases, were the most abundant genes up-regulated during H. annosum infection 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 in ectomycorrhizas of the Scots pine- Suillus bovinus symbiosis (Tarkka et al. 2001). However, similar down-regulation of this enzyme, was not observed in our BNR treatments, regardless of their beneficial role in Scots pine roots (II). This might be due to the non-mycorrhizal role of this fungus in Scots pine. It would be interesting to determinate the activation of Psyp1-related transcript in BNR-mycorrhizal associations in orchidea plants. In our study, significant peroxidase activities detected only in the simultaneous dual infected roots suggests this infection type being more effective on Psyp1 activation than either of these strains separately. Rhizoctonia challenged peroxidases have been also detected from bean (Phaseolus vulgaris L.) and Norway spruce in several studies (Xue et al. 1998, Nagy et al. 2004, Jøhnk et al. 2005, Fossdahl et al. 2007) and selected peroxidase genes most probably affect the results, since for example in a study of Nagy et al. (2004), 12 peroxidase isoforms are identified from the Norway spruce (Picea abies) roots. The same fungal strain also induced several other defence transcripts 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 to the control. Neither did the rest of the selected potential pathogen-induced transcripts: antimicrobial peptide gene Sp-AMP (Asiegbu et al. 2003), a defence related gene PsACRE (Li & Asiegbu 2004), CuZn- superoxide dismutase (Karpinski et al. 1992) or dehydrin-like protein (Pyhäjärvi et al. 2007). In addition to plant defence reactions during pathogen infection (Karlsson et al. 2007, Fossdal et al. 2007), the germin-like proteins are also involved in somatic and zygotic embryogenesis (Mathieu et al. 2006, Neutelings et al. 1998). Herr (1995) suggested that the mechanisms of protection, by binucleate Rhizoctonia or hypovirulent strains of R. solani against pathogenic strains, are via competition and induced host resistance rather than direct antagonisms or mycoparatism. Our results support this hypothesis of competition, since BNR was not antagonistic against UNR, but restricted the UNR infection level in BNR-preinoculated roots. Analysis of a subset of defence-gene activities, however, showed no clear correlation in their activity and decreased UNR infection levels in BNR- preinoculated roots suggesting that they are not involved as inducing the host resistance in these interactions. Plant defence reactions activated by the interaction of different micro-organisms are complicated and the enzyme activities are most probably differentially regulated in separate plant tissues and during different developmental stages. Therefore, the comparison of these different studies that involve a range of experimental designs is demanding. 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-

30 pathogen interaction and the regulation of the enzymes involved in the particular interactions, would increase our knowledge and the information could be used for future plant protection for example in agriculture or in forest nurseries.

31 6. Conclusions

The molecular detection of Rhizoctonia is essential in order to discriminate pathogenic 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 need for fungicides in an integrated pest management scenario. Our knowledge regarding host defence reactions, on the other hand, provides information for selection of disease resistant host genotypes as well as development of new more environmental friendly and more efficient antifungal agents. This study further confirms the complexity of the Rhizoctonia group not only from a phylogenetical perspective, but also with regard to their infection biology and plant host specificity. Root infecting UNR and BNR strains, belonging to the teleomorph genus Ceratobasidium, respectively enter into pathogenic or beneficial associations in Scots pine. BNR strains revealed a propensity to early seedling growth enhancement and later the induction of short roots, which are vital for symbiotic mycorrhizal formation, in Scots pine. 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 cells than endophytic BNRs, in co-inoculation. This supports the pathogenic role of UNR. The decrease in UNR infection level in BNR preinoculated roots indicates that beneficial microbes 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 and productivity as such, but also induce the host response against oncoming fast spreading pathogenic strains. Nevertheless, neither Suillus bovinus (Sen 2001) nor BNR strains from this study entirely prevented UNR infection but both could be included in inoculation systems in Scots pine seedling production. S. bovinus improves the nutrient uptake, which supports improved host survival, while BNR strains stimulate the growth and rooting in the host. The later short-root inducing activity of BNR, on the other hand, could provide more 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 single beneficial microbe will control all possible pathogens. Therefore, combinations of beneficial 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 on each other in vitro or in vivo in nature and further work is needed to uncover such relationships. The combination of the BNR and S. bovinus and their impacts on the Scots pine defence and UNR infection, although complicated, would be very interesting topic for future investigation. Additionally more defence genes could be included as new candidate gene sequences become available in public databases. Another important future perspective that stems from this and earlier studies by our group members would be to initiate an applied programme under actual nursery condition.

32 7. Acknowledgements

This work was carried out at the division of General Microbiology, in the Department of Biological and Environmental sciences, University of Helsinki and was funded by University 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 most recent supervisor of this work, for her encouraging attitude and great support, especially during these later years. I am also grateful for the financial support, which made the final part of the work possible. I would also like to thank Prof. Timo Korhonen, the Head of the division, for providing the facilities for my work. This work was mainly supervised by Dr. Robin Sen, my warmest thanks for your enthusiastic support and scientific expertise which carried me all the way from that very initial academic start in Ruskeasuo in 1995, as a research assistant, even before my biology education, to eventually a PhD candidate. I am very grateful to Dr. Ari Hietala, a co-author of the third article and a member of my follow-up group. You generously provided your expertise in the study design, taught me the real time PCR method and the interpretation of the results both in person, and through 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 have supervised: Irina, Hanna, Outi and Pave. Especially I thank Sari for introducing me to the practical mycorrhizal lab work and for your careful hands-on training at the very beginning of my studies, but I am also grateful for the advice of all other members in the lab. Malin and Edda sheared with me many lunch and coffee breaks of cheerful conversation. -The personnel in the General Microbiology, some of them I have known since my research 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 short laboratory visit. I am especially pleased to get to know Shea Beasley and Janetta Hakovirta, who became my friends together with Anne Ylinen and Mari Koskinen from the Division of Applied Microbiology, whom I am very grateful for sharing knitting evenings etc. with good food and conversation and all the refreshing coffee breaks in Viikki. Anne has also provided technical help in real time PCR and Prof. Kaarina Sivonen is acknowledged for letting me use the machine. -VGSB coordinators Nina Saris and Eeva Sievi for helpfulness and support, and Viikki Biosciences Doctoral Student Association (VIBA) board members, Pinja, Suvi, Maija, Katrianna, Johanna, Anne, Vassileios and Sylvie for organising outside lab activities to VGSB students and shearing grateful moments in VIBA. -My family: especially my husband Dick, for loving me all these years despite my frequent mood swings, and our lovely children Daniel and Minea, you have given me enormous delight. My parents Martti and Hilkka for their tremendous encouragement and my sisters Eerika and Emilia for being good friends and having you nearby, and for the whole family for your support. I am also grateful to my mother-in-law Cati for so often taking care of our pets and to all the grandparents of our children for taking care of Daniel and Minea whenever needed. Helsinki, October 2008

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