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Molecular Studies on the Taxonomy, Host-Associations and Viruses of the Diplodia-Like Anamorphs of the Botryosphaeriaceae

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Molecular studies on the taxonomy, host-associations and viruses of the Diplodia-like anamorphs of the Botryosphaeriaceae By Juanita de Wet Submitted in partial fulfilment of the requirements for the degree PHILOSOPHIAE DOCTOR In the Faculty of Natural and Agricultural Science Department of Microbiology and Plant Pathology University of Pretoria Pretoria August 2008 Supervisor: Prof. M.J. Wingfield Co-supervisors: Prof. B.D. Wingfield Dr. O. Preisig © University of Pretoria DECLARATION I, the undersigned, hereby declare that the thesis submitted herewith for the degree Philosophiae Doctor (PhD) to the University of Pretoria contains my own independent work and has hitherto not been submitted for any degree at any other University. __________________ Juanita de Wet August 2008 TABLE OF CONTENTS ACKNOWLEDGEMENTS…………………………………………………………. i PREFACE………………………………………………………………………………. ii CHAPTER 1 Diplodia pinea sensu lato as part of the Botryosphaeriaceae and associated mycoviruses. 1. Introduction ……………………………………………………………………………... 1 2. Taxonomy of the Diplodia pinea species complex 2.1 Taxonomic history……………………………………………………………... 4 2.2 Taxonomy of Diplodia pinea and its morphotypes……………………………. 6 3. Pathogen biology 3.1 Distribution and host range………………………………………….………… 9 3.2 Disease symptoms ……………………………………………………………... 9 3.2.1 Die-back and shoot blight……………………………………………. 10 3.2.2 Whorl cankers and crown wilt……………………………………….. 10 3.2.3 Collar rot and a root disease…………………………………………..10 3.2.4 Blue stain…………………………………………………………….. 11 3.3 Spore development, dispersal and infection…………………………………… 11 3.4 Wounding, stress, virulence and host susceptibility…………………………… 13 4. Population genetics……………………………………………………………………… 17 5. Disease management 5.1 Conventional disease management…………………………………………….. 19 5.2 Biological control……………………………………………………………….22 5.2.1 Virus-like particles in fungi…………………..……………………… 23 5.2.2 Hypovirulence-mediated dsRNA elements as biocontrol agents…….. 26 5.2.3 DsRNA elements in Diplodia pinea sensu lato.................................... 28 6. Conclusions..…………………………………………………………………………… 30 7. References……………………………………………………………………………… 32 8. Figures………………………………………………………………………………… 53 CHAPTER 2 Multiple gene genealogies and microsatellite markers reflect relationships between the morphotypes of Sphaeropsis sapinea and identify a new species of Diplodia. Abstract…………………………………………………………………………………… 59 Introduction………………………………………………………………………………… 60 Materials and Methods Fungal isolates…………………………………………………………………………... 61 DNA extractions………………………………………………………………..……….. 62 Amplification of partial protein-coding genes and microsatelite loci…………………... 62 Sequencing……………………………………………………………………………… 62 Phylogenetic analyses…………………………………………………………………... 63 Results Amplification and sequencing protein-coding genes and microsatelite loci ..……….… 63 Phylogenetic analyses…………………………………………………………………... 63 Taxonomy………………………………………………………………………………. 65 Discussion………………………………………………………………………………….. 66 References………………………………………………………………………………….. 70 Table.………………………………………………………………………………………. 74 Figures……………………………………………………………………………………... 75 CHAPTER 3 Molecular and morphological characterization of Dothiorella casuarini sp. nov. and other Botryosphaeriaceae with Diplodia-like conidia. Abstract…………………………………………………………………………….………. 85 Introduction………………………………………………………………………….……... 86 Materials and Methods Fungal isolates and morphological characterization……..…………….……………... 87 DNA extractions…………………………………………………………….………….. 88 DNA amplification and sequencing………………………………………….…………. 89 Phylogenetic analyses……………………………………………………….………….. 89 Results Phylogenetic analyses…………………………………………………………………... 90 Taxonomy………………………………………………………………………………. 92 Discussion………………………………………………………………………………….. 93 References………………………………………………………………………………….. 97 Table.………………………………………………………………………………………. 102 Figures………………………………………………………………………………….….. 104 CHAPTER 4 Phylogeny of the Botryosphaeriaceae reveals patterns of host association. Abstract…………………………………………………………………………………….. 108 Introduction………………………………………………………………………………… 109 Materials and Methods Fungal isolates…………………………………………………………………………... 112 DNA extractions, amplification and sequencing……………………………………….. 113 Phylogenetic analyses…………………………………………………………………... 113 Results Phylogenetic analyses of Botryosphaeriaceae with Diplodia-like anamorphs….…….... 116 Phylogenetic analyses for seven lineages in Botryosphaeriaceae………………………. 117 Discussion………………………………………………………………………………….. 120 References…………………………………………………………………………………. 126 Tables……………………………………………………………………………………..... 133 Figures……………………………………………………………………………………... 135 CHAPTER 5 Patterns of multiple virus infections in the conifer pathogenic fungi, Diplodia pinea and D. scrobiculata. Abstract………………………………………………………………………………….…. 139 Introduction……………………………………………………………………………….... 140 Materials and Methods DsRNA extraction, cDNA synthesis and cloning of a putative RdRp gene…….……… 142 Primer development……………………………………………………………….......... 144 Fungal isolates used for genotyping…………………………………………………….. 145 Total RNA isolations……………………………………………………………………. 145 cDNA synthesis and Real-time PCR genotyping……………………………….………. 146 Amplicon sequence confirmation………………………………………………………. 147 Results Partial characterization of a putative RdRp gene………………………………….......... 148 cDNA synthesis and Real-time PCR genotyping……………………………………….. 148 Amplicon sequence confirmation………………………………………………………. 149 Virus distribution in isolates……………………………………………………..……... 149 Discussion……………………………………………………………………………...…... 150 References………………………………………………………………………………..… 153 Table…………………………………………………………………………………….… 157 CHAPTER 6 Characterization of a novel dsRNA element associated with the pine endophytic fungus, Diplodia scrobiculata. Abstract…………………………………………………………………………………….. 159 Introduction………………………………………………………………………………… 160 Materials and Methods Fungal isolate and dsRNA extraction…………………………………………………... 162 Synthesis and cloning of cDNA using random hexamer primers………………………. 163 Amplification and cloning of the complete viral genome………………………………. 164 Determination of the distal ends of the viral genome..…………………………………. 165 Isolation and amplification of genomic DNA…………………………………………... 165 Sequencing and sequence analysis……………………………………………………… 166 Phylogenetic analysis…………………………………………………………………… 167 Results Synthesis and sequencing of cDNA from D. scrobiculata dsRNA..…………………… 167 Genome organization of DsRV1………………………………………………………... 168 Amplification of genomic DNA………………………………………………………… 169 Phylogenetic analysis…………………………………………………………………… 169 Discussion………………………………………………………………………………….. 170 References………………………………………………………………………………….. 174 Table……………………………………………………………………………………….. 180 Figures……………………………………………………………………………………... 181 SUMMARY………………………………………………………………….………….. v i ACKNOWLEDGEMENTS I would like to express my sincere thanks and gratitude to the following: FABI (Forestry and Agricultural Biotechnology Institute), CTHB (Centre of Excellence in Tree Health Biotechnology), TPCP (Tree Protection Co-operative Programme) and the University of Pretoria for funding, a science-inducing, multidisciplinary work environment and state of the art facilities in which this research was conducted. A special thanks to my promoters, Mike, Brenda and Oliver for their mentorship, guidance and passion, as well as Bernard Slippers for his valuable contribution. I also want to acknowledge The National Research Foundation (NRF), The Technology and Human Resources for Industry Programme (THRIP) and members of the TPCP for funding. A big thank you to all the Fabians but especially: • My colleagues in the Shaw lab for their friendship and valuable discussions. • Rose, Helen, Martha, Eva, Martie and the rest of the Culture Collection staff for never hesitating to assist me with whatever I needed. To those who kept me going outside FABI, my husband, family and friends, thanks for your continued love, support and encouragement through the years. Above all, praise to my Father in Heaven for giving me Faith, perseverance and always listening to my prayers. ii PREFACE The Botryosphaeriaceae represents a family of fungi that includes important pathogens of agricultural and forestry crops. Diplodia pinea is one of the best-known members of this family causing serious die-back and canker diseases mainly on Pinus spp. but also other conifers. The D. pinea species complex includes three morphologically similar forms that have been referred to as the A, B and C morphotypes of the fungus. Management of Diplodia-associated diseases is difficult as various biotic and abiotic factors influence the initiation and severity of the diseases. Hypovirulence (attenuation of virulence)-inducing dsRNA elements could provide an alternative mode of control against this fungus in a manner that has been shown for the chestnut blight fungus, Cryphonectria parasitica. An overall theme during studies conducted as part of this dissertation was to consider the taxonomy, host associations and viruses in isolates of the D. pinea species complex. Associated with this objective, I also considered the phylogenetic relationships of this fungus with other Diplodia-like anamorphs of the Botryosphaeriaceae. The first chapter represents a literature review with a particular focus on members of the D. pinea species complex and their associated viruses. It reflects on various aspects of the taxonomy, pathogen biology, population genetics and disease management of members of this species complex. The potential of using dsRNA elements,
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