Eesti Maaülikooli Doktoritööd Doctoral Theses of the Estonian University Of

Eesti Maaülikooli doktoritööd Doctoral Theses of the Estonian University of Life Sciences INTERACTION BETWEEN LARGE PINE WEEVIL (HYLOBIUS ABIETIS L.), PATHOGENIC AND SAPROTROPHIC FUNGI AND VIRUSES HARILIKU MÄNNIKÄRSAKA (HYLOBIUS ABIETIS L.) SEOS PATOGEENSETE JA SAPROTROOFSETE SEENTE NING VIIRUSTEGA TIIA DRENKHAN A Thesis for applying for the degree of Doctor of Philosophy in Forestry Väitekiri filosoofiadoktori kraadi taotlemiseks metsanduse erialal Tartu 2017 Institute of Forestry and Rural Engineering Estonian University of Life Sciences According to verdict No 6-14/2-2 of 17 April 2017, the Defence Board of PhD theses in Forestry of the Estonian University of Life Sciences has accepted the thesis for the defence of the degree of Doctor of Philosophy in Forestry. Opponent: Research Professor Isabella Børja, PhD Biotechnology and Plant Health Norwegian Institute of Bioeconomy Research Supervisors: Assoc. Prof. Kaljo Voolma, PhD (Entomology) Institute of Forestry and Rural Engineering Estonian University of Life Sciences Assoc. Prof. Ivar Sibul, Doctor of Science (Forestry) Institute of Forestry and Rural Engineering Estonian University of Life Sciences Assoc. Prof. Risto Kasanen, Doctor of Science (Forestry) Department of Forest Sciences University of Helsinki Defence of the thesis: Estonian University of Life Sciences, room 2A1, Kreutzwaldi 5, Tartu, on 19 June, 2017, at 10:00. The English language was edited by Dr. Ingrid H. Williams and Estonian by Mrs. Urve Ansip. Publication of this dissertation is supported by the Estonian University of Life Sciences © Tiia Drenkhan, 2017 ISSN 2382-7076 ISBN 978-9949-569-81-6 (trükis) ISBN 978-9949-569-82-3 (pdf) CONTENTS LIST OF ORIGINAL PUBLICATIONS ......................7 ABBREVIATIONS.......................................8 1. INTRODUCTION ....................................9 2. REVIEW OF THE LITERATURE .......................12 2.1 Hylobius spp. ......................................12 2.2 The biology of large pine weevil........................13 2.3 Feeding behaviour of Hylobius abietis....................15 2.4 Control of large pine weevil...........................16 2.5 Species complex Heterobasidion annosum sensu lato .........17 2.6 Infection cycle of H. annosum s.l........................18 2.7 Viruses in H. annosum s.l. ............................19 2.8 Diplodia sapinea (Fr.) Fuckel ..........................20 2.9 Phlebiopsis gigantea (Fr.) Jülich.........................21 2.10 Mycoviruses......................................23 2.11 Partitiviridae .....................................25 3. AIMS OF THE STUDY................................26 4. MATERIALS AND METHODS .........................27 4.1 Sample collection ..................................27 4.2 Fungal isolates .....................................27 4.3 Laboratory feeding experiment ........................28 4.4 Genotype determination (I, II) ........................30 4.5 Counting of oidial spores (II) .........................31 4.6 Isolation of hyphal tips and single oidial spores (II) .........31 4.7 Growth rate measurements and statistical testing (II)........32 4.8 Virus detection by RT-PCR...........................32 4.9 DNA extraction and fungal species identification (III).......34 5. RESULTS ...........................................35 5.1 The laboratory feeding experiment (I–III) . .35 5.2 Occurrence of viruses from insect faeces (I–II).............36 5.3 CF11 chromatography (I) ............................37 5.4 Growth rate measurements and oidial counting (II).........37 5 5.5 Field collected insects (III)............................38 6. DISCUSSION .......................................39 6.1 Viruses in pathogenic and saprotrophic fungi .............39 6.2 Viruses and fungi transmitted by insects .................40 6.3 Insects as vectors of fungi.............................42 7. CONCLUSIONS.....................................44 REFERENCES.........................................45 SUMMARY IN ESTONIAN ..............................66 ACKNOWLEDGEMENTS...............................78 ORIGINAL PUBLICATIONS ............................81 CURRICULUM VITAE.................................113 ELULOOKIRJELDUS..................................117 LIST OF PUBLICATIONS ..............................121 6 LIST OF ORIGINAL PUBLICATIONS This thesis is based on the following papers, which are referred to in text by their Roman numerals. I Drenkhan, T., Sibul, I., Kasanen, R., Vainio, E.J. 2013. Viruses of Heterobasidion parviporum persist within their fungal host during pas- sage through the alimentary tract of Hylobius abietis. Forest Pathology, 43: 317–323. II Drenkhan, T., Kasanen, R., Vainio, E.J. 2016. Phlebiopsis gigantea and associated viruses survive passing through the digestive tract of Hylobius abietis. Biocontrol Science and Technology, 26: 320–330. III Drenkhan, T., Voolma, K., Adamson, K., Sibul, I., Drenkhan, R. 2017. The large pine weevil Hylobius abietis (L.) as a potential vector of the pathogenic fungus Diplodia sapinea (Fr.) Fuckel. Agricultural and Forest Entomology, 19: 4–9. The contributions of the authors to the papers were as follows: I II III Original idea RK, EV, TD, IS EV, RK, TD RD, TD Study design TD, EV EV, TD TD, RD Data collection TD TD TD, RD Data analysis EV EV, TD TD Manuscript EV, TD, RK, IS EV, TD, RK TD, RD, IS, KA, preparation KV TD – Tiia Drenkhan, EV – Eeva J. Vainio, RK – Risto Kasanen, IS – Ivar Sibul, KV – Kaljo Voolma, KA – Kalev Adamson, RD – Rein Drenkhan 7 ABBREVIATIONS CF11 cellulose affinity chromatography CP coat protein DNA deoxyribonucleic acid dsRNA double-stranded RNA HetPV2-pa1 Heterobasidion partitivirus 2 HetPV4-pa1 Heterobasidion partitivirus 4 HetRV6-pa17 Heterobasidion RNA virus 6 mt SSU rDNA mitochondrial small subunit ribosomal DNA ORF one long open reading frame PCR polymerase chain reaction PgLV-1 Phlebiopsis gigantea large virus-1 RAPD random amplification of polymorphic DNA RDRP RNA dependent RNA polymerase RNA ribonucleic acid RT reverse transcription RT-PCR reverse transcription - polymerase chain reaction ssDNA single-stranded DNA ssRNA single-stranded RNA SSPP species-specific polymerase chain reaction (PCR) priming 8 1. INTRODUCTION The interaction between insects and fungi to break down woody substrate is crucial in nutrient cycling in forest ecosystems (Mahendrappa et al., 1986). It also has been recognized as one of the most serious risks threatening the health of forests. A striking example of realization of such risk is the unexcpected outbreak of mountain bark beetle, Dendroctonus ponderosae (Coleoptera: Scolytidae) and associated fungus, causing massive forest decline in Northern America (Dhar et al., 2016). The kingdom Fungi is one of the most diverse groups of organisms on earth; the fungi are integral ecosystem agents that govern soil carbon cycling, plant nutrition, and pathology (Tedersoo et al., 2014). In Esto- nia, 7,000–8,000 species of fungi are known. When species identified in environmental samples using molecular methods are also included, the number exceeds 100,000 species (Käärt, 2016). Almost all types of microorganisms (fungi, bacteria, viruses, nematodes, protozoa, phytoplasmas) can be transmitted by insects (Agrios, 2008). The important association between fungi and insects (bark beetles) is especially well known (Nuorteva, Laine, 1972; Viiri, Lieutier, 2004; Harrington, 2005; Linnakoski et al., 2008, 2012). Many of these fungi are pathogens causing diseases on leaves, stems, twigs, branches and roots (Phillips, Burdekin, 1992). Fungi show adaptations for insect dispersal, as they produce asexual or sexual spores in sticky drops at the tips of fungal fruiting structures. Some fungi (e.g. Phlebiopsis gigantea (Fr.) Jülich) have an evolutionary conidial state which is probably an adaptation for insect dissemination that supplements wind dissemination of basidiospores (Harrington, 2005). Insects as vectors can introduce pathogens such as fungi, bacteria or viruses into a plant and cause an infection (D’Arcy, Nault, 1982; Purcell, Almeida, 2005; Hogenhout et al., 2008). Generally, insects transmit pathogens in on of three main ways: (i) by feeding or walking through a plant area infected with bacteria or fungal spores thus carrying the infection to the same or another plant; (ii) by feeding on infected plant tissue and carrying the pathogen on their mouthparts to other plants; (iii) by sucking up the pathogen (viruses, phytoplasmas, protozoa, nematodes, some bacteria) with the plant sap on which they feed (Agrios, 2008; Büttner et al., 2013). 9 Many insects are not only agents of dissemination but also very effective agents of inoculation (Leach, 1935). Insect transmission occurs while wounding plants during oviposition or most frequently, when plants are wounded during feeding (Agrios, 2008). Pathogens and viruses can be transmitted to insects horizontally by contact, ingestion, and by vectors, and vertically from parents to their offspring (Wegensteiner, 2004). Viruses are widespread (Ahn, Lee, 2001; Bao, Roossinck, 2013; Ghabrial, 2013) small, infectious pathogens attacking animals, plants, bacteria, archaea, fungi (Agrios, 2005; Cho et al., 2013) and viruses. The viruses have been detected in different environments, such as woody and herba- ceous plants, soil, surface waters, glacier ice, seawater and clouds (Büttner et al., 2013). Mycoviruses (fungal viruses) are a specific group of viruses which naturally infect and replicate in fungi (Özkan, Coutts, 2015). Mycoviruses are transmitted by cytoplasmic

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