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Fungi associated with the mountain pine beetle, Dendroctonus ponderosae Lee, Sangwon 2006

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FUNGI ASSOCIATED WITH THE MOUNTAIN PINE BEETLE,DENDROCTONUS PONDEROSAE by SANGWON LEE B.Sc. Sungkyunkwan University, 1991 M.Sc. Sungkyunkwan University, 1993 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n THE FACULTY OF GRADUATE STUDIES (Forestry) THE UNIVERSITY OF BRITISH COLUMBIA April 2006 © Sangwon Lee, 2006 A B S T R A C T The mountain pine beetle (MPB; Dendroctonusponderosae) and its fungal associates have infested large areas of lodgepole pine {Pinus contorta var. latifolia) forests in British Columbia. In order to understand how the fungi affect the beetle epidemics and tree defenses, in this work we identified the fungal species associated with the MPB, characterized their pathogenicity, and investigated the genetic structure of a fungal species, one of the major pathogens. Identifying ophiostomatoid fungi by a classical morphology approach is often inconclusive. For accurate identification, molecular approaches were developed for Ophiostoma montium and Ophiostoma clavigerum. O. montium could be differentiated from a synonymous species, O. ips, using a single polymerase chain reaction of either the B-tubulin gene or ribosomal DNA. Similarly, restriction fragment length polymorphism analysis of the P-tubulin gene using the enzyme Hinfl could distinguish O. clavigerum from other morphologically related ophiostomatoid fungi. Using molecular and morphological approaches, we characterized the diversity of MPB fungal associates. Fungi were isolated from beetles, beetle galleries and sapwood of infested lodgepole pines in six epidemic sites across British Columbia. A total of 1042 fungi that belong to nine species were recognized. Unexpectedly, an O. minutum-hke species was frequently isolated from the beetle. Unknown Leptographium and Entomocorticium species were also isolated in addition to the known MPB associates O. clavigerum and O. montium. The unknown Leptographium species was reported as Leptographium longiclavatum sp.nov. The pathogenicity of L. longiclavatum to mature lodgepole pines was estimated and compared to the pathogenicity of O. clavigerum, an MPB associate that is known to kill mature pines when inoculated at high density. For this test, foliage colour, phloem lesions, occlusions, and sapwood moisture content of inoculated trees were examined. The data showed that!, longiclavatum was a pathogen, although it seemed slightly less virulent than O. clavigerum. The genetic diversity of 170 O. clavigerum isolates from five sites in Canada and two sites in the USA was characterized using amplified fragment length polymorphism (AFLP). While the genetic diversity was low, and the geographic and genetic distances were not significantly correlated, we identified two genetically distinct groups in the population. ii TABLE OF CONTENTS ABSTRACT ii TABLE OF CONTENTS iii LIST OF TABLES v LIST OF FIGURES vi ACKNOWLEDGEMENTS viii CO-AUTHORSHIP STATEMENT x CHAPTER 1: INTRODUCTION 1 THESIS OBJECTIVES A N D OVERVIEW 12 REFERENCES 13 CHAPTER 2: IDENTIFICATION OF FUNGI 18 2-1: DISTINGUISHING OPHIOSTOMA IPS AND OPHIOSTOMA MONTIUM, TWO BARK BEETLE-ASSOCIATED SAPSTAIN FUNGI 18 INTRODUCTION 18 M A T E R I A L S A N D METHODS 19 RESULTS A N D DISCUSSION 24 REFERENCES 32 2-2: A PCR-RFLP MARKER DISTINGUISHING OPHIOSTOMA CLAVIGERUM FROM MORPHOLOGICALLY SIMILAR LEPTOGRAPHIUM SPECIES ASSOCIATED WITH BARK BEETLES 34 INTRODUCTION 34 M A T E R I A L S A N D METHODS 36 RESULTS 42 DISCUSSION 49 REFERENCES 52 2-3: LEPTOGRAPHIUM LONGICLAVATUM SP. NOV., A NEW SPECIES ASSOCIATED WITH THE MOUNTAIN PINE BEETLE, DENDROCTONUS PONDEROSAE. 54 INTRODUCTION 54 M A T E R I A L S A N D METHODS 55 RESULTS 60 DISCUSSION 67 REFERENCES 73 CHAPTER 3: FUNGAL DIVERSITY ASSOCIATED WITH THE MOUNTAIN PINE BEETLE, DENDROCTONUS PONDEROSAE AND INFESTED LODGEPOLE PINES IN BRITISH COLUMBIA 75 INTRODUCTION 75 M A T E R I A L S A N D METHODS 76 RESULTS 82 DISCUSSION 87 i i i REFERENCES 90 CHAPTER 4: PATHOGENICITY OF LEPTOGRAPHIUM LONGICLAVATUM ASSOCIATED WITH DENDROCTONUS PONDEROSAE TO PINUS CONTORTA 93 INTRODUCTION 93 M A T E R I A L S A N D METHODS 95 RESULTS 100 DISCUSSION 107 REFERENCES 112 CHAPTER 5: GENETIC DIVERSITY AND THE PRESENCE OF TWO DISTINCT GROUPS IN THE OPHIOSTOMA CLAVIGERUM POPULATION ASSOCIATED WITH THE MOUNTAIN PINE BEETLE, DENDROCTONUS PONDEROSAE IN NORTH AMERICA 116 INTRODUCTION 116 M A T E R I A L S A N D METHODS 118 RESULTS 123 DISCUSSION 137 REFERENCES 141 CHAPTER 6: SIGNIFICANCE OF T H E WORK AND CONCLUDING REMARKS 144 R E F E R E N C E S 150 APPENDICES 151 APPENDIX A 151 APPENDIX B 152 iv LIST O F T A B L E S Table 2.1.1. Growth of Ophiostoma isolates, and size of PCR amplicons obtained from their genomic D N A , using primer pairs ITS3-LR3 and T10-BT12 20 Table 2.2.1. Cultures, GenBank accession numbers for sequences, and the results of PCR-RFLP analysis 37 Table 2.3.1. Cultures and GenBank accession numbers for the sequences used in the phylogenetic analysis of L. longiclavatum 58 Table 2.3.2. Morphological comparisons of L. longiclavatum and phylogenetically closely related species 71 Table 3.1. Characteristics of the MPB-infested trees used for samplings 78 Table 3.2. GenBank accession numbers and growth rates of the fungi isolated from D. ponderosae... SO Table 3.3. Number of fungal isolates from D. ponderosae and number of beetles yielding each fungal species at six sites in British Columbia 83 Table 3.4. Number of fungal isolates from larvae, pupae and adults of D. ponderosae 85 Table 3.5. Fungal isolates from gallery and wood at six sites in British Columbia 86 Table 4.1. Characteristics of the lodgepole pines (Pinus contorta Dougl. var. latifolia Engelm.) inoculated with L. longiclavatum, O. clavigerum, or agar control 96 Table 5.1. The sampling sites, sources of isolation, and number of fungal isolates in each location... 120 Table 5.2. The number of monomorphic and polymorphic loci observed in the AFLP profdes of 170 O. clavigerum isolates with the six selected primer combinations 125 Table 5.3. The polymorphic alleles specific to the Rocky Mountain population (BF, HR, and HV) or the BC population (HT, S U , MP, and WL) of O. clavigerum 126 Table 5.4. The genetic diversity indices of Rocky Mountain populations (BF, HR, and HV) and the BC populations (HT, St.J, MP, and WL) of O. clavigerum 127 Table 5.5. Analysis of molecular variance of AFLP markers for 170 isolates of O. clavigerum collected from seven sites in North America 1 129 Table 5.6. Analysis of molecular variance of AFLP markers for two O. clavigerum subpopulations: one subpopulation was sampled from P. contorta and the other from D.ponderosae 130 Table 5.7. Population pairwise Fsi indices among seven O. clavigerum populations from AMOVA.131 v LIST OF FIGURES Fig. 2.1.1. Comparison of the PCR amplicons of the 5.8S-ITS2 rDNA and P-tubulin genes of O. montium and O. ips 26 Fig. 2.1.2. Differentiation of O. montium and O. ips shown in the neighbour-joining tree based on the 5.8S and ITS2 rDNA 28 Fig. 2.1.3. Differences in the exon and intron regions of P-tubulin gene between O. montium and O.ips 30 Figs 2.2.1-2.2.6. The ascocarps and ascospores of O. clavigerum 43 Figs 2.2.7-2.2.12. The conidiophores and conidia of O. clavigerum 45 Figs 2.2.13-2.2.15. Variance in the colony morphology among O. clavigerum strains 45 Fig. 2.2.16. A parsimonious tree of O. clavigerum and its morphologically similar species 47 Fig.2.2.17. The O. clavigerum-specific PCR-RFLP profile of the P-tubulin gene generated with Hinfl 48 Figs 2.3.1-2.3.9. Morphological characteristics of L. longiclavatum 61 Figs 2.3.10-2.3.13. Morphological characteristics of O. clavigerum 62 Fig. 2.3.14. Phylogenetic dendrograms of the four genes showing distinct clades of L. longiclavatum ...65 Fig. 2.3.15. One of the most parsimonious trees based on the combined datasets of four loci showing a distinct clade of L. longiclavatum 68 Fig. 2.3.16. Differentiation of L. longiclavatum from O. clavigerum by PCR-RFLP 69 Fig. 3.1. Map of the sampling sites in British Columbia, Canada 77 Fig. 4.1. Inoculation of trees with fungi or agar plugs using the cork borer technique 97 Fig. 4.2. Lesions on phloem or the surface of xylem caused by L. longiclavatum, O. clavigerum, or agar plugs 101 Fig. 4.3. Comparison of the lengths of lesions on phloem caused by L. longiclavatum, O. clavigerum or agar plugs at an inoculation density of 200 points/m2 103 Fig. 4.4A-4.4E. Occlusions in the sapwood underneath the inoculated points 104 Fig. 4.5. Percentage of occluded areas in the sapwood of lodgepole pines inoculated with either agar plugs, L. longiclavatum, or O. clavigerum, at 200 points/m2 or 800 points/m2 105 Fig. 4.6. Growth of L. longiclavatum and O. clavigerum in aerobic and anaerobic conditions 108 vi Fig. 5.1. Map showing the seven sampling sites of O. clavigerum in Canada and the USA 119 Fig. 5.2. An A F L P profde of O. clavigerum generated with the primer combination £coRI+T/.P.srf+A.124 Fig. 5.3. An U P G M A (Unweighted Paired Group Method) tree based on Nei's unbiased genetic distance among seven O. clavigerum populations 133 Fig. 5.4. A N U P G M A (Unweighted Paired Group Method) tree of 170 O. clavigerum isolates from seven populations showing two distinct groups 134 Fig. 5.5. Two groups in the O. clavigerum populations shown by principal components ('Prin') analysis 136 vii ACKNOWLEDGEMENTS I would like to thank my supervisor, Colette Breuil for her direction throughout the doctoral program. I also would like to thank my committee members, John McLean and Mary Berbee for their precious suggestions and guidance. I would like to express my gratitude to the people listed below, who were willing to help me with samplings of mountain pine beetle infested trees. Especially, I cannot thank Lorraine Maclauchlan in the BC Ministry of Forests and Range enough, because she provided access to the resources for the fieldworks at Kamloops and greatly supported me with her passion and expertise in the entomology. I am grateful to post-doctoral fellows, Jae-Jin Kim and Seong Hwan Kim, for valuable discussion and support and to co-op students, Simon Fung and Ada Man, for their excellent work. I thank all my friends, especially Renata Bura, Jiyoung Park, Sannie Tang, Sepideh Alamouti and Vera Maximenko for their invaluable friendships. Their hearty encouragement and humour made my graduate life more enjoyable. I thank Jeff Keating, my husband, for his support. I am deeply grateful to my family (grandmother, father, mother, sisters and brother) for their profound love and support. I also would like to honour the memory of my grandfather with the completion of the thesis. Acknowledgements for each manuscript follow: Chapter 2 -1 . This work was supported by the Natural Sciences and Engineering Research Council of Canada. J.-J. Kim was supported by a Postdoctoral Fellowship Program from the Korea Science & Engineering Foundation. I thank H. Masuya (Tohoku Research Center of Forestry and Forest Products Research Institute), H. Solheim (Norwegian Forest Research Institute), and A. Uzunovic (Forintek) for providing O. ips and O. montium cultures. Chapter 2-2. This work was supported by the Natural Sciences and Engineering Research Council of Canada. I thank A. Uzunovic, D. Six (Univ. Montana), M . Wingfield (Univ. Pretoria), and T. viii Harrrington (Iowa State Univ.) for providing fungal cultures and L. Maclauchlan (BC Ministry of Forests and Range) and J. Alexander (Lignum Ltd.) for providing lodgepole pine trees attacked by MPB. Chapter 2-3. This work was supported by the Natural Sciences and Engineering Research Council of Canada. We thank L. Maclauchlan, A . Carroll (Pacific Forestry Centre, Natural Resources Canada) and J. Alexander for providing infested trees and D. Six for sharing some isolates from the mycangia of D. ponderosae. I am grateful to M . Berbee (Dept. of Botany at UBC) and C . K . M . Tsui (Dept. of Botany at UBC) for reviewing our manuscript and their valuable comments. Chapter 3. This work was funded by the Natural Sciences and Engineering Research Council of Canada and by Natural Resources Canada through the Mountain Pine Beetle Initiative funds. I would like to thank L. Maclauchlan for her great help with the field work at Kamloops. I am grateful to BC Ministry of Forests and Range, Pacific Forestry Centre in Natural Resources Canada, Lignum Ltd., and U B C Research Forest for providing MPB-infested trees. I thank L. Humble (PFC) for confirming the identity of the beetles. I greatly appreciate the assistance of the co-op students (A. Man, S. Fung, and M . Carof) for their excellent work in fungal isolation, storage and organizing the data and of T. Kozak (UBC) and R. Chedgy (UBC) for their advice on statistical analysis. Chapter 4. This work was funded by the Natural Sciences and Engineering Research Council of Canada and by Natural Resources Canada through the Mountain Pine Beetle Initiative funds. I would like to thank the co-op students, Karen and Monica, for their help with fungal inoculations to trees. I am grateful to L. Maclauchlan for providing the access to the healthy trees. Chapter 5. This work was funded by the Natural Sciences and Engineering Research Council of Canada and by Natural Resources Canada through the Mountain Pine Beetle Initiative funds. I am greatly thankful to C. Liewlaksaneeyanawin (UBC) for his valuable support and discussion. I am also grateful to R.Hamelin (Canadian Forest Service) for reviewing the manuscript. ix CO-AUTHORSHIP STATEMENT This thesis consists of six manuscripts which were written with the intent of publication in peer-reviewed journals. The manuscripts of chapters 2, 3, and 4 have been published or submitted. The contributions to the work by each author are as follows: Chapter 2-1: Distinguishing Ophiostoma ips and Ophiostoma montium, two bark beetle-associated sapstain fungi. J.-J. Kim, S. H. Kim, S. Lee, and C. Breuil. 2003. FEMSMicrobiol. Lett. 222: 187-192. For this manuscript, I initiated the work after noticing a prevalent confusion between the two species. I performed fungal culturing, sequencing, and testing of optimal growth temperature. J.-J. Kim, a post-doctoral fellow, also participated in fungal culturing and sequencing. S.H. Kim, a research associate, analyzed the exon and intron components in the sequences and provided helpful discussion. The work was overseen by my supervisor, C. Breuil. Chapter 2-2: A PCR-RFLP marker distinguishing Ophiostoma clavigerum from morphologically similar Leptographium species associated with bark beetles. S. Lee, J.-J. Kim, S. Fung, and C. Breuil. 2003. Can. J. Bot. 81(11): 1104-1112. I performed fungal culturing and sequencing, and designed the RFLP marker. S. Fung, a co-op student, helped me throughout the process. I also found a teleomorph of O. clavigerum with unknown shape and carried out scanning microscopy on it. J.-J. Kim contributed to the light microscopy pictures. The work was conducted under the supervision of C. Breuil. Chapter 2-3: Leptographium longiclavatum sp. nov., a new species associated with the mountain pine beetle, Dendroctonusponderosae. S. Lee, J.-J. Kim, and C. Breuil. Mycol. Res. 2005. 109 (10): 1162 - 1170.1 isolated the new species and observed its morphological and physiological characteristics. I performed all the sequencing and phylogenetic work. J.-J. Kim helped with the Latin description and a part of the morphological description. J.-J Kim and C. Breuil reviewed the manuscript. x Chapter 3: Fungal diversity associated with the mountain pine beetle, Dendroctonus ponderosae and infested lodgepole pines in British Columbia. S. Lee, J.-J. Kim, and C. Breuil. Fungal diversity. 2006. In press. I did most of the work involved in the survey (fungal isolation and preservation) and identification. J.-J Kim helped with the identification of O. minutum-\\ke species, Entomocorticium species, and Ambrosiella species. C. Breuil supervised the work and reviewed the manuscript. Chapter 4: Pathogenicity of Leptographium longiclavatum associated with Dendroctonus ponderosae to Pinus contorta. S. Lee, J.-J. Kim, and C. Breuil. Submitted to Can. J. For. Res. Apr. 2006.1 designed the experiment and conducted most of work. J.-J. Kim helped with inoculating fungi and cutting trees. C. Breuil reviewed the manuscript. Chapter 5: I carried out all the work, aided by helpful discussions with C. Breuil and R. Hamelin, the latter a research scientist with the Canadian Forest Service in Quebec. x i Chapter 1 Introduction The mountain pine beetle (Dendroctonusponderosae) outbreak in British Columbia, Canada and its impact on lodgepole pine forests One of the most detrimental bark beetles in British Columbia (BC), the mountain pine beetle (MPB; Dendroctonus ponderosae) kills healthy living trees, while most other bark beetles affecting lodgepole pines cause little or no economic damage, as they normally infest dead or severely weakened trees. The MPB is one of the natural elements of the lodgepole pine (Pinus contorta Dougl. var. latifolia Engelm.) ecosystem. In endemic situations, MPB kills and removes over-mature pines and allows other tree species to flourish. However, in epidemic conditions the beetle and its fungal associates cause large-scale economic impacts on industrially productive forests. British Columbia supplies about 50% of Canada's total softwood lumber exports and more than 270,000 British Columbians are employed directly and indirectly by the forest industry (COFI 2005). The major markets for Canadian forest product exports are the USA, Japan and European countries. Lodgepole pine is one of the main tree species in BC, accounting for 24% of the total growing stock in the province and 50% of the growing stock in the central and interior parts of the province. Lumber and boards produced from lodgepole pine are used as construction material, furniture, poles, and railroad ties. The residuals from sawmilling are used for producing pulp and paper (COFI 2005). Several factors contributed to the current MPB epidemic in BC. Usually, cold winters result in significant larval mortality (BC Ministry of Forests 2005), keeping the MPB population under control. However, repeated mild winters since 1994 have been one of the main factors responsible for the current outbreak. Furthermore, successful fire management throughout the past 40 years unintentionally 1 generated a high percentage of mature lodgepole pines, which are more susceptible to MPB attacks. The MPB epidemic has quickly spread across BC, and as of 2004 it covers an area of 7 million hectares (BC Ministry of Forests 2005; Appendix A). Since 1998, 283 million cubic metres of lodgepole pine with a lumber value of 18 billion Canadian dollars have been infested (COFI2005). In addition to this loss, the industry has spent over $100 million on detecting infestation by aerial or ground surveys, cutting and burning the infested trees, and other control methods in order to slow down the beetle epidemic and to minimize timber loss (COFI 2005). Infested lodgepole pine trees are less favoured for several reasons. First, infested trees are devalued because of the stain in their sapwood. Sapstain fungi, which are introduced into trees by MPB, cause blue to black discolouration as they grow inside the sapwood. Even though the stained wood retains its structural strength, its usage becomes limited due to its appearance. Stained wood could also be faced with quarantine regulations. Since MPB only occupy the cambial layer of the tree, directly beneath the bark, they are removed in the debarking process. Thus the risk of spreading insects by shipping wood products made from beetle-infested trees is very low. However, wood products made from sapwood stained by fungi could be subjected to quarantine in international trade. The Japanese market, which accounts for 10% of total export shipments (COFI 2005), rejects the import of stained wood. Second, the wood becomes drier when the trees are left too long in the stand following infestation. Over-dried wood makes manufacturing processes difficult, requiring changes in equipment and procedures at the mill. Over-dryness also lowers the yield of wood chips for pulp and paper products. Infested trees have relatively short shelf lives; therefore, they need to be harvested within 5-10 years. Finally, in addition to the economic loss, dead lodgepole pine stands pose various environmental problems such as risk of fire and blow-down covering wide areas, negative impacts on visual and recreational forest qualities, and increased environmental disturbance when left without management. Managing the infested forests to retain economic, ecological and social values is challenging to both government and industry. 2 The mountain pine beetle Bark beetles are members of the family Curculionidae (subfamily Scolytinae), within the order Coleoptera (Farrell et al. 2001). The adult beetles are black and small, just 5-7 mm long (see Appendix B). The MPB is native to North America and found in areas from B C to western Alberta in Canada and from the Pacific Coast to South Dakota in the USA. The primary hosts of MPB are lodgepole (Pinus contorta) and ponderosa pine (Pinusponderosa). Whitebark (Pinus albicaulis), white (Pinus strobus) and limber pines (Pinus flexilis) are also attacked by MPB in the Rocky Mountain regions of the USA. Occasional hosts may include Scots (Pinus sylvestris) and sugar pines (Pinus lambertiana). MPB can kill mature pine trees within a year. The beetles attack the lower two thirds of the tree trunk (approximately 15 metres high), usually in July or August when trees can often be under stress due to water deficiency. The female beetles bore through the bark to the cambium and attract males and other females by emitting pheromones. Both sexes of MPB carry sapstain fungi and introduce the fungi into the tree during their attack. Once parental beetles get into the trees, they move upwards and produce a long and narrow vertical tunnel called an "egg gallery". Each female lays 60-80 eggs, enabling populations to grow very quickly. The number of the next generation of beetles emerging from a single tree is large enough to attack 15 new trees. The beetles, especially larvae cause mechanical damage to the trees by tunneling horizontally under the bark and thereby cutting off the paths for nutrients (phloem) (Paine et al. 1997). The beetle spends winter in the larval stage underneath the bark. The larva pupates between June and early July, and then develops into an adult. MPB is mycophagous (Harrington 2005) as well as phytophagous (Berryman 1989). Before emerging, newly eclosed adult beetles feed on fungi that line the pupal chamber. Fungi acquired by the beetle will be dispersed to new trees. The beetles bore exit holes and fly out to find another suitable host. As beetles attack the trees, the cycle is repeated with each generation taking generally one year. The beetles can potentially disperse over great distances (thirty kilometres or more) when transported by wind currents (COFI 2005). Natural enemies of MPB include woodpeckers, predaceous and parasitic insects (Enoclerus sphegeus, Xylophagus sp., and Coeloides dendroctoni), and nematodes (Bellows et al. 1998). 3 After a successful beetle attack, the foliage of a tree becomes discoloured, from green to yellow to red in one year post-attack and then to gray in two years post-attack. Pitch tubes, visible symptoms of the MPB attack, are solidified resin around beetle-attacked spots on the bark. The infested trees will also show boring dust in bark crevices and around their base. Once a tree has been infested with MPB, it become vulnerable to infestation by secondary beetles such as Ips and ambrosia species. Interaction among fungi, bark beetles and trees In general, specific fungi are carried by particular bark beetle species. This specific association between the fungi and beetle species suggests a symbiotic interaction. To fully understand the relationship between the bark beetle and the fungi, the influence of the host trees should not be ignored. The beetle, fungi and hosts interact with each other and therefore need to be considered as an integrative complex. Fungal associates of bark beetles The fungi benefit from their association with the beetles in two ways. They are disseminated and transported to new trees where they have access to fresh nutrients. The most commonly reported fungal associates of bark beetles are ascomycetes in the genus Ophiostoma. The Ophiostoma species reproduce both sexually, generating teleomorphs, and asexually, generating anamorphs. Their asexual and sexual spores, conidia and ascospores respectively, are produced in slimy masses and are highly adapted to insect dispersal (Harrington 1993). When the newly eclosed beetle feeds on fungi, the spores produced in the pupal chambers are acquired in the gut or mycangia (specialized structures that carry fungi), or simply attach to the exoskeleton of the beetle. For certain bark beetles, the species or frequencies of fungal associates have been compared between the mycangia and exoskeleton (Klepzig 2001, Six 2003a). Beneficial fungi may have been selected by adapting to mycangia and may have evolved with the beetles (Paine et al. 1997). Some fungal associates, however, can adversely affect beetle fitness. For instance, Ophiostoma minus, which is carried 4 phoretically by Dendroctonus frontalis and likely helps the beetle colonize hosts with its pathogenicity, inhibits beetle brood development. In contrast, Ceratocystis ranaculosus and Entomocorticium sp., found in the mycangia, appear to be good sources of nutrition for the beetles. Entomocorticium sp., furthermore, helps successful brood development by inhibiting the detrimental growth of O. minus (Klepzig 2001). With the exception of D. frontalis, interactions among fungal associates have not yet been characterized. Only some of the fungal associates of bark beetles are pathogenic. Although fungi can contribute to tree mortality, the degree of pathogenicity of fungal associates does not always correspond to the aggressiveness of the beetles. Weak pathogens are often isolated from the most aggressive beetles and vice versa (Harrington 1993, Paine et al. 1997). The flora of fungal associates might be affected by the dynamics of the beetle population. Ips typographus, a destructive bark beetle in Europe, is associated with three Ophiostomatoid fungal species. In an epidemic state, the beetle mainly carries Ceratocystis polonica, the most pathogenic of its known fungal associates, and attacks living trees. In contrast, a less pathogenic species, Ophiostoma bicolor, is commonly found in the endemic state when the beetle attacks dead or dying trees (Solheim 1992). However, it is difficult to draw a general conclusion from this phenomenon, since the change in fungal populations might have resulted from other factors such as host variations (Paine et al. 1997). Benefits to bark beetles from fungi Fungi contribute to beetle fitness in many ways. First, fungi are good food sources for most bark beetles. In many cases, mycelia, yeast cells, and conidia are ingested by the larvae and teneral adults of bark beetles (Six 2003b). It has also been observed that some bark beetles ingest perithecia during maturation feeding (Yearian et al. 1972). Fungi provide essential nutrients which are not found or are not present in sufficient amounts in phloem, or modify nutrients to more available forms for beetles. The beetles feed on the phloem, which is rich in carbon and nitrogen but poor in sterols, vitamins and other growth factors (Norris and Baker 1967, Six 2003b). Sterols are essential for the normal growth, molting and reproduction of beetles (Clayton 1964). Fungal ergosterol can be easily metabolized by insects (Clayton 5 1964, Svoboda et al. 1978), and it has been shown that fungal ergosterol has positive impacts on the weight, size and normal development of beetles (Barras 1973, Kok et al. 1970, Norris et al. 1969, Morales-Ramos et a/.2000, Six and Paine 1998). It appears that fungi also help beetles by concentrating nitrogen from the inner bark, thereby reducing the amount of phloem required for development (Ayres et al. 2000). However, fungi may only be a good supplement rather than a requirement for the MPB diet, since the larvae can develop into normal adults in axenic pine phloem (Whitney 1971). Second, fungal growth in sapwood reduces the moisture content of trees to a level suitable for successful beetle brood development (Reid 1961, Wagner et al. 1979, Webb and Franklin 1978). Third, fungi can modify the metabolites of the tree. For example, yeasts convert MPB aggregation pheromone (/rara-verbenol) to anti-aggregation pheromone (verbenone) and may contribute to successful beetle colonization by regulating the density of attack (Hunt and Borden 1990). Tree defence Fungi can reduce the level of host defence that the beetles have to overcome (Berryman 1972, Nelson 1934, Paine et al. 1997). Fungi inoculated into trees cause extensive resin reactions (Reid et al., 1967). In addition, fungi utilize sugars from the phloem, which are needed for the synthesis of resin, a tree defence component. Therefore, it appears that beetles carrying fungi are more effective in overcoming host resistance. Pinus species have two resin defence mechanisms: a preformed system and an induced system (Berryman 1972). The preformed resin and resin duct systems are the first host defence for the invading beetles and fungi to overcome. The resin duct system of Pinus spp. is highly developed (Balantinecz and Kennedy 1967), unlike other genera of conifers, such as Abies, Tsuga, or Cedrus, which do not have preformed resin ducts. The preformed resin (oleoresin) is terpenoid in nature, with anti-fungal and anti-insect activity. It consists of volatile turpentine (monoterpenes, diterpenes and sesquiterpene) and nonvolatile resin acids (Bridges 1987, Cobb et al. 1968, Himejima et al. 1992, Paine et al. 1994). Often, monoterpene is most important in determining the capabilities of host defences. The preformed resins 6 immediately react to exogenous wounds and clean the wound tissue by resin flushing, and then seal it through resin crystallization (Berryman 1972, Nebeker et al. 1995a). The pressure of the resin outflow can prevent beetles from entering the tree, or block the emission of aggregation pheromones from the entrance hole (Raffa and Berryman 1982). However, a mass attack with a greater number of beetles than the resistance threshold can drain the resin reservoir and thereby colonize trees successfully. The chemical composition and physical pressure of the preformed resin system are under the influence of the genetic elements of trees (Nebeker et al. 1992, Wilkinson et al. 1971), as well as environmental factors such as site, spacing (Brown et al. 1987, Mitchell et al. 1983), root diseases (Nebeker et al. 1995b), physical injuries (Nebeker et al. 1995a), tree age and season (Shrimpton 1973a). Insect invasion or fungal infection of host inner bark tissues induces a secondary resin reaction (Barbara et al. 2005, Berryman 1972, Krekling et al. 2004, Shrimpton 1973b). Induced resistance involves both cellular and biochemical changes in the affected host tissues. It includes division of ray parenchyma and other cells in the cambial zone, accumulation of phenolic inclusions in ray parenchyma cells, cellular necrosis, formation of traumatic resin ducts in the xylem and synthesis of phenolic and oleoresin constituents. These processes tend to confine fungal colonization to a discrete area showing a visible lesion (Reid and Shrimpton 1971, Shrimpton 1978, Shrimpton and Watson 1971). Generally, the length and intensity of lesions are dependent on fungal growth rate and tree vigour (Ross et al, 1992). The induced resin has a detrimental effect on the reproductive capability of beetles as well as on the growth of fungi (Berryman and Ashraf 1970, Raffa and Berryman 1983, Reid et al., 1967, Shrimpton and Whitney 1967). Although preformed and induced defence have been observed in lodgepole pines infested by MPB, the mechanism affecting fungal growth is not yet clear. One hypothesis is that non-polar components of the resin may protect the phloem and sapwood from fungal extracellular enzymes. In lodgepole pine, wounded tissues produce a larger quantity of resins with an increased ratio of monoterpenes, especially P~ phellandrene, than healthy ones (Shrimpton 1973b). It has been reported that fifty to seventy year old 7 lodgepole pines are more resistant to fungi than younger or older trees. The resin response of lodgepole pines was weaker in July than in June during an MPB attack period (Shrimpton 1973a). Fungi associated with MPB Known fungal associates of MPB In 1941, Ophiostoma montium was first isolated from the mountain pine beetle by Rumbold in the USA, and named Ceratocystis montia (Rumbold 1941). In 1968, Ophiostoma clavigerum was found in beetle-attacked lodgepole pine in B C and described by Robinson-Jeffery et a/.(1968). This species has also been found in the USA. It was reported recently that Jeffrey pine beetle (Dendroctonus Jeffreyi Hopkins) also carries O. clavigerum (Six and Paine 1997). In 1970, Whitney and Farris discovered the existence of mycangia in mountain pine beetles and examined the contents of such structures in flying beetles (Whitney and Farris 1970). They found mostly O. clavigerum, O. montium, and yeasts including Pichia capsulata (Wick.) Kurtzman, Pichia holstii (Wick.) Kurtzman and Pichiapini (Hoist) Phaff in the mycangia. Occasionally, they also found Trichoderma and Penicillium species. The fungal flora associated with MPB across the entire infestation area in BC is not yet known, because previous examinations of fungal species have been conducted in a limited area. It would not be appropriate to conclude that the reported fungal species represent the current fungal flora in BC. Since the weather conditions across a broad infestation area vary and the dynamics of the beetle population change, the dominant fungal species associated with the beetles might be affected. Growth and pathogenicity of the MPB fungal associates O. clavigerum and O. montium can overcome host resin defence and grow in sapwood. Most of their spores germinated on plates containing oleoresin derived from lodgepole pine (Whitney and Blauel 1972), although their hyphal growth was inhibited by volatile wood extractive (Shrimpton and Whitney 1967). O. clavigerum and O. montium can kill trees in the absence of the beetle. The strong pathogenicity of O. 8 clavigerum has been shown (Yamaoka et al., 1995), while the pathogenicity of O. montium is debatable (Yamaoka et al., 1995; Strobel et al., 1986). Sapstain fungi appear to interrupt water conduction in sapwood and lead trees to death, even though the mechanism is not yet clear. It has been suggested that fungi damage water conducting cells by creating aspiration of tracheid tori (Nelson 1934), or by secreting an isocoumarin toxin (Hemingway et al. 1977, McGraw and Hemingway 1977, Paine 1984). Dye conduction studies have shown that disruption of water conduction occurs in a wedge shaped zone ahead of the stained sapwood and the fungal colonized area. This fungus-free zone is 0.1 to 5 mm wide and was demonstrated by histological methods (Mathre, 1964). Identification of Ophiostomatoid fungi Difficulty in the identification of Ophiostomatoidfungi Characteristics of the teleomorph are of primary importance in the classification of Ophiostomatoid fungi. However, the teleomorphs often do not or rarely occur in artificial conditions. As a consequence, anamorphs, which are observed much more readily in cultures, have been frequently used for the identification of these genera. However, pleomorphism and the degeneration of anamorphs by repeated subculturing on artificial media have been noticed in many Ophiostomatoid fungi (Michael et al. 1993, Tsunedaand Hiratsuka 1984). With these difficulties, the identity of O. montium has been confused with Ophiostoma ips (= Ceratostomella ips). O. ips was considered to be synonymous with O. montium by Upadhyay (1981), and this had been generally accepted, until we recently suggested that these are two different species based on molecular and physiological characteristics. As such, identification based only on anamorphic characteristics can often be inconclusive. Therefore, in addition to morphological characterization, molecular analysis or diagnostic markers are needed for precise identification. Molecular tools for fungal identification 9 The taxonomic position of a fungal species can be elucidated from its phylogenic relationship with other fungal taxa. The ribosomal D N A (rDNA) cluster has been the most extensively studied region for phylogenetic information. The nuclear large 28S and small 18S subunits have evolved slowly, so the comparison of these sequences can be used for examining distant relationships. In contrast, the internal transcribed spacers (ITS) between the subunits evolved rapidly, generating more variation and making the comparison between genus and species often possible. Extensive rDNA sequence information from many fungal species has been accumulated and can be accessed through D N A databases such as GenBank or E M B L . In addition to rDNA, protein coding genes such as beta-tubulin, glyceraldehyde-3-phosphate dehydrogenase, elongation factor and actin, as well as multi-gene sequences, which are combined sequences of several gene sequences, have been successfully used for investigating fungal phylogeny. Especially for those Ophiostoma species with Leptographium anamorphs, these protein-coding genes often generate phylogenetic trees with higher resolution than those constructed using rDNA (Lim et al. 2004). Species-specific PCR-RFLP (Polymerase Chain Reaction-Restriction Fragment Length Polymorphism) or species-specific primers based on the sequence differences among fungal species can be a reliable method for accurate identification. For instance, two morphologically similar Ophiostoma species, O. piceae and O. quercus, can be readily distinguished using species-specific primers (Kim et al. 1999). In this work, therefore, we developed some molecular diagnostic markers and also implemented a phylogenic analysis to describe a new species. Population structure of O. clavigerum Different strains belonging to one species can be defined based on their genetic diversity. Defined groups can be different in biology or in distribution. The pathogenic fungus O. clavigerum, which is frequently found on MPB, reproduces both asexually and sexually. The mycelia of this species are primarily haploid except for a short diploid phase during sexual reproduction. The mating type of this species, however, is not yet known. Unfortunately, in artificial conditions the mating test for this species has not been 10 successful (Six, personal unpublished data); therefore, it is not clear whether this species is homothallic or heterothallic. The genetic structure of O. clavigerum has only been studied with isolates from the USA using an allozyme method (Six and Paine 1999). In Canada, no information is available on the genetic diversity of this species. The physiological characteristics (including pathogenicity) of O. clavigerum may vary among isolates in different geographic regions. The first step in assessing variations in fungal pathogenicity over large regions would be the characterization of fungal genetic diversity. Questions As aforementioned, fungal associates may play an important role in bark beetle infestation. Since it is not yet clear whether O. clavigerum and O. montium are always associated with MPB, we cannot generalize their functions in the process of infestation. The fungi associated with MPB have not been investigated systematically and the possibility remains that more fungal species are involved in the infestation process. Therefore, in this study, I examined the following questions: Are there any other fungi associated with the mountain pine beetle in addition to the known species described above? Are O. clavigerum and O. montium consistently associated with MPB across their entire geographic range in BC? Are there any subgroups in major fungal species that can be defined by genetic diversity? Are current fungal associates pathogenic? 11 THESIS OBJECTIVES AND OVERVIEW The overall objective of this research is to understand the fungal flora associated with the MPB. The specific objectives of this study were 1) to examine the MPB fungal associates across the entire infestation area in B C , 2) to identify the fungi collected in the survey, 3) to investigate the pathogenicity of L. longiclavatum, and 4) to assess the genetic diversity of a pathogenic fungus, O. clavigerum. In chapter 2,1 present the key molecular, morphological and physiological characteristics of the major fungal associates O. montium and O. clavigerum. The first part of Chapter 2 points out a prevalent confusion between O. montium and O. ips, and suggests a way to distinguish one from the other. In the second part of the chapter, the development of an O. clavigerum-specific PCR-RFLP marker is reported. In the last part of Chapter 2,1 describe a new fungal species, L. longiclavatum, which has been isolated throughout the survey. The information generated in this chapter made it possible to conduct precise and effective identification of a large number of fungal isolates from the survey. In chapter 3,1 report on the fungal diversity from MPB and infested lodgepole pine, which was investigated through a three-year survey. In Chapter 4,1 describe the pathogenicity of L. longiclavatum sp. nov. Its pathogenicity toward lodgepole pine was tested in the field and its virulence was compared with that of O. clavigerum, which has been known as a primary pathogen killing lodgepole pines infested by mountain pine beetle. In Chapter 5,1 investigate the genetic diversity of O. clavigerum, one of the important fungal associates of MPB. 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CRC press, New York, NY. Six, D.L., and Paine, T.D. 1997. Ophiostoma clavigerum is the mycangial fungus of the Jeffrey pine beetle, Dendroctomus jeffreyi. Mycologia. 89 (6): 858-866. Six, D.L., and Paine, T.D. 1998. Effects of mycangial fungi and host tree species on progeny survival and emergence of Dendroctonus ponderosae (Coleoptera: Scolytidae). Environ. Entomol. 27: 1393-1401. Six, D.L., and Paine, T.D. 1999. Allozyme diversity and gene flow in Ophiostoma clavigerum (Ophiostomatales: Ophiostomataceae), the mycangial fungus of the Jeffrey pine beetle, Dendroctonus jeffreyi (Coleoptera: Scolytidae). Can. J. For. Res. 29 (3):324-331. Solheim H. 1992. The early stages of fungal invasion in Norway spruce ingested by the bark beetle Ips typographus. Can. J. Bot. 70: 1-5. Strobel G.A., and Sugawara, F. 1986. The pathogenicity of Ceratocystis montia to lodgepole pine. Can. J. Bot. 64: 113-16. Svoboda, J.A., Thomposn, M.J., Robbins, W.E., and Kaplanis, J.N. 1978. Insect sterol metabolism. Lipids. 13: 742-753. Tsuneda A., and Hiratsuka, Y. 1984. Sympodial and annellic conidiation in Ceratocystis clavigera Can J Bot. 62: 2618-2624. 16 Upadhyay, H.P. 1981. A monograph of Ceratocystis and Ceratocystiopsis. University of Georgia Press, Athens, GA. Wagner, T. L. , Gagne, J.A., Doraiswamy, P.C., Coulson, R.N., and Brown, K.W. 1979. Development time and mortality of Dendroctonus frontalis in relation to changes in tree moisture and xylem water potential. Environ. Entomol. 8: 1129-1138. Webb, J.W., and Franklin, R.T. 1978. Influence of phloem moisture on brood development of the southern pine beetle (Coleoptera: Scolytidae). Environ. Entomol. 7: 405-410. Whitney H.S. 1971. Association of Dendroctonus ponderosae (Coleoptera: Scolytidae) with blue stain fungi and yeasts during brood development in lodgepole pine. Can. Entomol. 103: 1495-1503. Whitney H.S., and Farris, S.H. 1970. Maxillary Mycangium in the Mountain Pine Beetle. Science. 167: 54-55. Whitney H.S., and Blauel, R.A. 1972. Ascospore dispersion in Ceratocystis spp. and Europhium clavigerum in conifer resin. Mycologia. 64: 410-414. Wilkinson R . C , Hanover, J.W., Wright, J.W., and Flake, R.H. 1971. Genetic variation in the monoterpene composition of white spruce. For. Sci. 17:83-90. Yamaoka Y., Hiratsuka, Y. , and Maruyama, P J . 1995. The ability of Ophiostoma clavigerum to kill mature lodgepole pine trees. Eur. J. For. Pathol.25: 401-404. Yearian W.C., Gouger, R.J., and Wilkinson, R.C. 1972. Effects of the blue stain fungus, Ceratocystis ips, on development of Ips bark beetles in pine. 17 Chapter 2. Identification of fungi Chapter 2.1 Distinguishing Ophiostoma ips and Ophiostoma montium two bark beetle-associated sapstain fungi Introduction Ophiostoma ips (Rumbold) Nannfeldt and Ophiostoma montium Rumbold are economically important bark beetle-associated fungi that cause sapstain in coniferous trees, logs, and lumber (Seifert 1993). O. ips has been reported in North America, Europe, Japan, New Zealand, and South Africa, and appears to be vectored by a broad range of insects, including Ips and Dendroctonus species (Benade et al. 1995, Rane and Tartar 1987, Rumbold 1931). O. ips was first described as Ceratostomella ips by Rumbold in 1931 (Rumbold 1931). Because this species produces different anamorphs, its taxonomy, based on cultural and micro-morphological characteristics, has changed several times since then (Seifert et al. 1993). The introduction of conidium development as a taxonomic characteristic for anamorphic fungi did not resolve the O. ips anamorph, because the original author's description was insufficiently precise. Recently, detailed observation of conidium development using scanning electron microscopy suggested that the anamorph form of O. ips is a Hyalorhinocladiella (Benade et al. 1995). O. montium (Rumbold) von Arx, first described as Ceratostomella montium by Rumbold in 1941 (Rumbold 1941), has been reported in pine trees in the USA and Canada (Solheim 1995). Like O. ips, O. montium has been given different names at different times (Seifert et al. 1993). In 1981, Upadhyay synonymized this fungus as Ceratocystis ips (now called Ophiostoma ips) (Upadhyay 1981), since the sizes of the ascocarp bases of the two fungal species overlapped. Cultures called C. montia in Upadhyay's monograph have been preserved as O. ips in reference collections like the American Type Culture Collection (ATCC) and Centraalbureau voor Schimmelcultures (CBS). In 1993 Hausner et al. A version of this chapter has been published. Distinguishing O. ips and 0. montium two bark beetle-associated sapstain fungi. J.-J. Kim, S. H. Kim, S. Lee, and C. Breuil. 2003. FEMS Microbiol. Lett. 222: 187-192. 18 suggested that O. ips and O. montium were distinct species (Hausner et al. 1993). They used four-cutter analysis of rDNA and mtDNA to group these species. However, their two O. montium strains did not produce perithecia; their identity was uncertain, and the taxonomy of these two species was unresolved. While characteristics of the teleomorph are of primary importance in identifying Ophiostomatoid fungi, the ascocarps for O. ips and O. montium are similar in size and shape and are difficult to obtain in artificial media. Consequently, taxonomists working with these two species using artificial media have been forced to differentiate them by morphological characteristics; however, these species are pleomorphic and have distinct anamorphs that include Graphilbum, Hyalorhinocladiella, and Acremonium (Upadhyay 1981). Consequently, their identification remains a challenge and demands an approach that includes molecular analysis. Currently, we are surveying fungi associated with the mountain pine beetle, which has infested 108 million cubic meters of lodgepole pine (Pinus contorta Dougl. var. latifolia Engelm.) in British Columbia (CLMA 2002). In this survey we are finding many isolates morphologically similar to O. montium (Lee et al. 2002). To better understand the ecological role of the fungi isolated in this epidemic, it is necessary to accurately identify the species and to clarify whether O. montium should be synonymized with O. ips. In this work we report two methods that can distinguish O. ips and O. montium: growth ability at 35°C and nucleotide sequences of the rDNA and P-tubulin genes. Our results resolve the uncertainties from more limited taxonomic approaches and indicate that these two fungal species are distinct. Materials and methods Fungal isolates, growth and DNA preparation The hosts and origins of O. ips and O. montium isolates used in this work are listed in Table 2.1.1. Fungal isolates were obtained from the A T C C , CBS, and University of Alberta Microfungus Collection (UAMH) or were provided by Drs. A. Uzunovic (Forintek Canada Corp., Western lab), H. Masuya (Forestry and Forest Products Research Institute, Japan), H. Solheim (Norwegian Forest Research Institute, Norway). CBS 137.36 is a type culture of O. ips, isolated and deposited by the original author. 1 9 Table 2.1.1. Growth of Ophiostoma isolates, and size of PCR amplicons obtained from their genomic DNA, using primer pairs ITS3-LR3 and T10-BT12. Isolate Species (previous name) Isolation source a, origin, identifier Growth a t35°C b Amplicon sizec (P-tubulin gene /rDNA) Newly suggested name CBS 151.54 O. ips Gallery of Op in SP, Sweden, A. M.-Kaarik + 772/981 O. ips MCC 023 O. ips Tp beetle, Japan, H. Masuya + 772/981 O. ips MCC 036 O. ips Tp beetle, Japan, H. Masuya + 772/981 O. ips ATCC 24285 O. ips (O. montium) LP, Canada, H. S. Whitney + 772/981 O. ips UAMH 9962 O. ips RP, Canada, J. Reid + 772/981 O. ips CBS 137.36 O. ips Ips beetle, USA, C. T. Rumbold + 876/984 O. ips SYPT1 O. ips-like SYP, USA, S. H. Kim + 772/981 O. ips SYPT2 O. ips-\ike SYP, USA, S. H. Kim + 772/981 . O. ips SYPT3 O. ips-Mks SYP, USA, S. H. Kim + 772/981 O. ips C345 O. ips SYP, USA, provided by D. McNew + 772/981 O. ips C1307 O. ips SP, USA, provided by D. McNew + 772/981 O. ips ATCC 64697 O. ips Beetle gallery in LP, Canada, Y. Hiratsuka - 607/1007 O. montium CBS 151.78 O. ips {O. montium) Gallery of Dp in PP, USA, R. W. Davidson - 607/1012 O. montium K77 O. montium -like Dp beetle, Canada, S. W. Lee - 607/1007 O. montium Kw413 O. montium -like LP infested with Dp, Canada, S. W. Lee - 607/1007 O. montium Kw 430 O. montium -like LP infested with Dp, Canada, S. W. Lee - 607/1012 O. montium Ww 420 O. montium -like LP infested with Dp, Canada, S. W. Lee - 607/1007 O. montium 92-628/55/4 O. montium LP infested with Dp, Canada, H. Solheim - 607/1007 O. montium 92-630/131/2 O. montium LP infested with Dp, Canada, H. Solheim - 607/1007 O. montium ATCC 64698 O. ips LP, Canada, R. C. Robinson-Jeffrey - 607/1007 O. montium NOF-1399 O. ips Dp beetle, Canada, Y. Yamaoka - 607/1007 O. montium NOF-1420 O. montium Dp beetle, Canada, Y. Hiratsuka - 607/1007 O. montium C-l204 O. ips Dp beetle, Canada, Y. Hiratsuka - 607/1007 O. montium C-l207 0. ips-\\ke LP infested with Dp, Canada, Y. Hiratsuka - 607/1007 O. montium C-l 208 0. z/w-like LP infested with Dp, Canada, Y. Hiratsuka - 607/1007 O. montium C-l 282 0. ips LP infested with Dp, Canada, Y. Hiratsuka _ 607/1007 O. montium 20 Isolate Species (previous name) Isolation source a, origin, identifier Growth a t35°C b Amplicon sizec Newly suggested name C-l 284 0. ips Dp beetle, Canada, Y. Hiratsuka - 607/1007 O. montium 92-628/44/3 O. montium LP infested with Dp, Canada, H. Solheim - 607/1007 O. montium 92-628/45/1 0. montium LP infested with Dp, Canada, H. Solheim - 607/1007 O. montium UAMH 1363 0. ips LP, Canada, R. C. Robinson-Jeffrey - 607/1007 O. montium UAMH 4875 0. ips Beetle gallery in LP, Canada, L. Sigler - 607/1007 O. montium a Op: Orthotomicus proximus, SP: scots pine, Tp: Tomicus piniperda, LP: lodgepole pine, RP: red pine, SYP: southern yellow pine, PP: ponderosa pine, Dp: Dendroctonus ponderosae,. b +: growth, - : no growth. c PCR amplicons sequenced are marked in bold. 21 No type cultures of O. montium isolated and identified by the original author were available. For O. montium isolates we used cultures identified by authors who do not consider O. ips and O. montium as synonymous species. We also included O. montium-\ike and O. ips-\ike cultures that we isolated from lodgepole pine infested with mountain pine beetle and from southern yellow pine sapwood. The fungal cultures were pre-grown on 2 % malt extract agar (MEA) for 4-5 days at 24°C before D N A extraction. For DNA preparation, agar plugs taken from M E A cultures were spread onto sterile sheets of cellophane overlaid on M E A plates. After 3-7 days of growth at 24°C in the dark, about 0.2 g (fresh wt.) of mycelium was harvested from the cellophane sheet by scraping the surface with a scalpel. Fungal genomic DNA was extracted from the mycelium using a drilling method described by Kim et al. (1999). Growth ability at high temperatures was determined at 32.5 and 35 °C. Agar disks (5 mm in diameter) taken from the edge of a freshly grown colony were placed on 2 % M E A media (20 ml) in 90 mm Petri dishes and three replicates were prepared for each isolate. Colony diameters (two perpendicular measurements) on each plate were determined at 3, 5, and 7 days after incubation and growth rates were calculated in mm per day. PCR amplification and DNA sequencing analysis The internal transcribed spacer (ITS) and 5.8S regions of the nuclear rDNA were amplified using the primer pairs ITS1-F-ITS4 (Gardes and Bruns 1993, White et al. 1990); the P-tubulin gene was amplified using the primer pairs T10-T222 (O'Donnell and Cigelnik 1997) or T10-BT12. The primer BT12 (5'-G T T G T C A A T G C A G A A G G T C T C G - 3 ' ) was provided by P. Loppnau (UBC Wood Science Department). Each PCR amplification was performed in a total volume of 50 pi consisting of 40 pmol of each primer, 1 x PCR Buffer (10 mM Tris-Cl [pH 8.0], 1.5 mM MgCl 2 , 50 mM KC1), 50 p M (each) of the four deoxynucleotide triphosphates (dNTPs), 20 pM dimethylsulfoxide and 1 unit Thermostable DNA polymerase (Rose Scientific) overlaid with two drops of mineral oil. 200r|g of fungal DNA from cultures obtained by single spore isolation was added to each reaction. PCR reactions were performed in a Hybaid 22 Touch Down Thermocycler using an initial cycle with denaturation at 94°C for 4 min, 30 cycles with each cycle consisting of a denaturation at 94°C for 30 s, annealing at 55°C for 50 s and primer extension (72°C, 50 s) and one final cycle of a primer extension (72°C, 10 min). Negative controls (excluding DNA template) were included in each reaction to insure against contamination in experimental materials. To determine whether amplification was successful, 10 pi of PCR product was separated on 1% agarose gels in Tris-Acetate-EDTA buffer with ethidium bromide at 100 ng/ml, visualised under U V light, and documented with an Image Analyser System (Bio/Can). PCR amplifications were repeated at least three times. For DNA sequencing, the PCR-amplified DNA fragments were gel purified using a QIAGEN Gel Extraction Kit (Qiagen Inc.), subcloned into pCR 2.1-TOPO T A vectors (Invitrogen) according to the manufacturer's instructions, and sequenced. Sequencing reactions with Big Dye Taq premix (Perkin Elmer Applied Biosystems) were performed in a Hybaid Touch Down Thermocycler using Ml3 reverse and forward primers as sequencing primers. Sequencing was performed on an ABI 373 automated sequencer at the UBC Nucleic Acid and Protein Service unit. All the nucleotide sequences were determined on both sense and antisense strands. The nucleotide sequences determined in the present study have been deposited to the GenBank DNA sequence database. The accession numbers for the rDNA are AY194933-AY194940 for O. ips and A Y 194941-AY 194948 for O. montium. The accession numbers for the B-tubulin gene are A Y 194949-AY194956 for O. ips and AY194957-AY194964 for O. montium. Phylogenetic analysis The sequences generated and reference sequences of Ophiostoma fungi obtained from GenBank (http://www.ncbi.nlm.nih.gov/Genbank/index.html) were aligned with Clustal W program, version 1.8 (Thompson et al. 1994). Sequence relationships were analyzed from the alignment using M E G A version 2.1 (Kumar et al. 2001). Distance was calculated using the Jukes and Cantor model (1969) with the equal 23 rate option, with gaps and missing data handled as complete deletions. Trees were constructed using the neighbour-joining method of Saitou and Nei (1987) or U P G M A (Sneath and Sokal 1973). Character-based analysis was carried out using the minimum evolution method (Rzhetsky and Nei 1992) with the close-neighbor-interchange search option. Tree topology was evaluated by bootstrap analysis using 1000 replicates. Results and discussion Growth test at 35 °C Because conidial states of O. ips are extremely variable or sometimes lacking, ascocarp and ascospore characteristics have been considered the most important for identifying this species. However, mature ascocarps are not easily obtained in artificial media, thus growth comparisons, although rarely reported for the two Ophiostoma species described in this paper, could help differentiate these fungi. Upadhyay compared the growth ability of the two species at different temperatures (Upadhyay 1981). He found that all the strains tested grew well at 22 and 29°C and not at 5 and 35°C, supporting the concept that O. ips and O. montium were synonymous. However, his observation was different from Rumbold's observation that O. ips could grow in a wide range of temperature (10 to 35.5°C) (Rumbold 1941). To clarify this discrepancy, we inoculated the isolates listed in Table 2.1.1 on M E A and measured their growth at 32.5 and 35°C for 7 days. All isolates grew at 32.5°C, while at 35°C only some isolates grew. To confirm the viability of isolates from the group that did not grow at 35°C, we then incubated these culture plates at room temperature. Some of the isolates recovered and grew within a few days, while others had not grown. Except for isolate A T C C 24285, no O. ips isolates from lodgepole pine grew at 35°C, while isolates from scots, red, and southern pines grew at 35°C. The O. ips type (CBS 137.36) described by Rumbold belonged to the group that grew. The average growth rate at 35°C of the isolates that grew was 9.9 ± 1.3 mm/day. The group that did not grow at 35°C included isolates originally named either O. ips or O. 24 montium. Interestingly, no isolates from D. ponderosae or its infested pine trees grew at 35°C, while isolates from other beetles such as D. frontalis (southern pine beetle), Ips, and Tomicus grew at this temperature. These results suggest that a simple growth test at 35°C could tentatively differentiate O. ips and O. montium isolates into two groups. PCR amplification of rDNA regions and B-tubulin gene The genes coding for nuclear ribosomal DNA (rDNA) and P-tubulin contain both conserved and variable regions that are suitable for resolving diverse taxonomic levels such as species, genus, family or subphyla (Bridge and Arora 1998, Lanfranco et al. 1998). The value of these genes as molecular targets for differentiating fungal species has been shown for many groups, including Ophiostoma (Schroeder et al. 2002). Given this, we selected these two genes for PCR amplification in all isolates listed in Table 2.1.1. Initially, to amplify the ITS regions containing ITS1-5.8STTS2 sequences we used the ITS1-F and ITS4 primer pair. PCR amplicons of approximately 700 bp were only produced in some isolates. Furthermore, for unknown reasons, the ITS1 regions were poorly sequenced these amplicons. We had previously encountered similar difficulties in sequencing the ITS1 region of another sapstain fungus, O. piliferum (Schroeder et al. 2001). However, by changing to the DNA region that includes the ITS2 and part of the 28S rDNA and the primer pair ITS3-LR3 (Vilgalys and Hester 1990), we were able to amplify the genomic DNA of all the isolates in Table 2.1.1. Interestingly, all PCR amplicons from the isolates that grew at 35°C showed a single 981 bp band, while those from the isolates that did not grow at 35°C showed a band of 1007 or 1012 bp (Table 2.1.1). Figure 2.1.1 shows some examples of the band patterns for these rDNA amplicons. For the P-tubulin gene, an initial attempt with the primers T10 and T222 that had been used successfully with O. piliferum (Schroeder et al. 2002) failed to amplify many of the isolates. A different primer pair, T10-BT12, produced a PCR product for every isolate. As for rDNA, the sizes of the P-tubulin PCR amplicons permitted separating the isolates into two groups. All isolates that did not grow at 35°C 25 Non-growing group Growing group T 2 3 4 5 6 M 7 8 9 10 11 V2~ 1 2 3 4 5 6 M 7 8 9 10 11 12 0.5 kb Fig. 2.1.1. Examples of the PCR amplicons of the ITS2-28S rDNA (upper) and partial P-tubulin gene (lower), produced with primer pair ITS3-LR3 and T10-BT12, respectively. Lanes: M , 500 bp DNA marker (Bio-Rad); 1, 92-628/55/4; 2, Ww420; 3, K77; 4, Kw413; 5, Kw430; 6, CBS151.78; 7, CBS151.54; 8, MCC036; 9, SYPT1; 10, SYPT2; 11, MCC023; and 12, CBS137.36. Growing group: isolates that grew at 35°C. Non-growing group: isolates that did not grow at 35°C. 26 produced a 607 bp amplicon, while all isolates that grew at 35°C produced a 772 or 876 bp amplicon (Table 2.1.1). Some examples of the band patterns for these P-tubulin gene amplicons are given in Figure 2.1.1. At this point, all isolates tested could be consistently placed into two groups using growth at 35°C and PCR amplicon sizes for the P-tubulin gene and rDNA. Nucleotide sequence analysis To elucidate the genetic relationship between the isolates of the two groups, we sequenced the rDNA and P-tubulin gene PCR amplicons from several isolates of each group. GenBank searches showed that the sequences had high homology with known Ophiostoma rDNA and P-tubulin genes. These results confirmed that the PCR had targeted the rDNA and P-tubulin genes as intended. Pairwise comparison of the rDNA sequences showed that nucleotide sequence identity was between 98 and 100% for isolates that grew at 35°C, and 99 and 100% for isolates that did not grow at 35°C. The two groups shared 97 to 98% sequence identity. For the P-tubulin gene, nucleotide sequence identity was between 92 and 100% for isolates that grew at 35°C, 99 to 100% for isolates that did, and 76 to 87%) between the two groups. The level of sequence identity of both the rDNA and P-tubulin genes was lower between the two groups than between isolates of each group. This suggests that these two groups are genetically different. Genetic separation of these two groups was also supported by phylogenetic analysis of the rDNA and by comparison of structural features of the P-tubulin gene. The rDNA-based phylogram from a neighbour-joining analysis resolved Ophiostoma fungi into two clades (Fig. 2.1.2). All the isolates of O. ips and O. montium were placed in one clade and separated from another clade that containing the sapstain species O. floccosum, O. piceae, O. quercus, and O. piliferum. Consistent with this, the two clades have different anarmorphs: O. floccosum, O. piceae, O. quercus, and O. piliferum have Sporthrix and/or Pesotum anarmorphs, while O. ips or O. montium have Graphilbum, Hyalorhinocladiella, and Acremonium anamorphs (Upadhyay 1981). The isolates in the O. ipslO. montium clade contained two sub-groups that corresponded to isolates able and not able to grow at 35°C. This separation was strongly supported 27 99 57 98 79 Kw413 K77 92-630/131/2 CBS151.78 92-628/55/4 Ww420 Kw430 r ATCC24285 CBS137.36 |— UAMH9962 • MCC023 SYPT2 MCC036 |— CBS151.54 SYPT1 O. piliferum 57 NGG O. quercus ~ O. piceae O. floccosum P. fragrans GG 0.02 Fig. 2.1.2. Phylogenetic tree generated by neighbour-joining analyses using the nucleotide sequences of the ITS and 28S rDNA. Bootstrap values (percentage) are presented at the node. The scale bar indicates one base change per 100 nucleotide positions. GenBank accession numbers: O. floccosum (AF 198231), O. piceae (AF211844), O. piliferum (AF221071), O. quercus (AF081132), and P. fragrans (AF198248). Accession numbers for O. ips and O. montium sequences from this study are given in materials and methods. NGG: non-growing group (isolates that did not grow at 35°C), G G : growing group (isolates that grew at 35°C). 28 by a 99% bootstrap value. The same results were obtained through phylogenetic analyses using U P G M A and minimum evolution methods (data not shown). The analysis of partial P-tubulin genes also showed that the two groups had distinct sequence features. The 607 bp PCR amplicons from the isolates unable to grow at 35°C contained one intron and one exon, while the 772 and 876 bp amplicons from the isolates able to grow at 35°C contained 3 or 4 introns and 3 or 4 exons. The two groups shared only a 7 bp first intron located at the beginning of the sequence (Fig. 2.1.3). Three introns of 7, 106, and 59 bp with nucleotide sequence identity between 98 and 100% were common among all 772 bp amplicons. The type O. ips CBS 137.36 was the only isolate that produced a 876 bp amplicon containing four introns of 7, 105, 109, and 55 bp and four exons. The position of intron and exon junctions in PCR products is indicated in Figure 2.1.3. The location of intron insertion appears to be conserved in the P-tubulin gene of these Ophiostoma fungi. The combined size of the four exons (600 bp) was the same in all the isolates of the two groups. Comparison of these joined exon nucleotide sequences revealed sequence identity between 96 and 100% for isolates that grew at 35°C, 99 to 100% for isolates that did not grow at 35°C, and 91 to 99% between isolates of the two groups. Exon sequences were better conserved within a group than between groups. When we compared the deduced amino acid sequences (200 aa) from the 600 bp exon nucleotide sequences, the two groups shared 98 to 100% sequence identity. The two groups' deduced amino acid sequences showed 92-96%) identity with those of Aspergillus nidulans, Rhynchosporium secalis, and Gibberella fujikuroi (GenBank accession numbers: AAA33328, CAA56936, and AAB18275). An alignment of the deduced amino acid sequences revealed that 2 sites were unique between the two groups. At sites 125 and 185 of the 200 aa, all isolates that grew at 35°C had cysteine and valine residues, while all isolates that did not grow at 35°C had serine and isoleucine residues. The Ophiostoma isolates in this study clearly comprised two physiologically and genetically distinct groups. The groups could be separated by growth at 35°C and by molecular analyses. Therefore, we reject the synonymy of O. ips and O. montium. Based on growing ability at 35.5°, we recommend identifying isolates from the O. ipslO.montium group that grow at 35°C as O. ips. Isolates 29 I Intron site 0 9 17 37 O. piliferum I I I Growing group 1 Non-growing group Fig. 2.1.3. Schematic diagram indicating generic structure of a partial P-tubulin gene amplified with primer pair T10-BT12 and location of introns. Growing group: isolates that grew at 35°C. Non-growing group: isolates that did not grow at 35°C. 30 that do not grow at 35°C should be identified as O. montium. 31 References Benade, E. , Wingfield, M.J., and Van Wyk, P.S. 1995. Conidium development in the Hyalorhinocladiella anamorph of Ophiostoma ips. Mycologia 87, 298-303. Bridge, P.D., and Arora D.K. 1998. Interpretation of PCR methods for species definition. In: Applications of PCR in Mycology (Bridge, P.D, Arora, D.K., Reddy, C A . and Elander, R.P., Eds.), pp. 63-76. C A B International, New York. Cariboo Lumber Manufacturers' Association / Northern Forest Products Association. 2002. http://www.mountainpinebeetle.com/2002_update.htm. Gardes, M . , and Bruns, T.D. 1993. ITS primers with enhanced specificity for basidiomycetes -application to the identification of mycorrhizae and rusts. Mol. Ecol. 2, 113-118. Hausner, G., Reid, J., and Klassen, G.R. 1993. Grouping of isolates and species of Ceratocystis sensu lato on the basis of molecular and morphological characters. In: Ceratocystis and Ophiostoma taxonomy, ecology and pathogenicity (Wingfield, M.J., Seifert, K.A. and Webber, J.F., Eds.), pp. 93-104. The American Phytopathological Society Press, St. Paul, Minnesota. Jukes, T.H., and Cantor, C R . 1969. Evolution of protein molecules, pp. 21-132. In: Munro, H. M . , Ed. Mammalian protein metabolism. Academic Press, New York. Kim, S.H., Uzunovic, A,, and Breuil, C. 1999. Rapid detection of Ophiostoma piceae and O. quercus in stained wood using PCR. Appl. Environ. Microbiol. 65, 287-290. Kumar, S., Tamura, K., Jakobsen, I.B.,and Nei, M . 2001. MEGA2: Molecular Evolutionary Genetics Analysis software, Arizona State University, Tempe, Arizona. Lanfranco, L. , Perotto, S., and Bonfante, P. 1998. Applications of PCR for studying the biodiversity of mycorrhizal fungi. In: Applications of PCR in Mycology (Bridge, P.D, Arora, D.K., Reddy, C A . and Elander, R.P., Eds.), pp. 107-124. C A B International, New York. Lee, S.W., Kim, S.H., Kim, J.-J., and Breuil, C. 2002. Ophiostomatoid fungi associated with mountain pine beetle. Mycological Society of America Annual Meeting 2002, p. 57. Oregon State University, Oregon. O'Donnell, K., and Cigelnik, E . 1997. Two divergent intragenomic rDNA ITS2 types within a monophyletic lineage of the fungus Fusarium are nonorthologous. Mol. Phylogenet. Evol. 7, 103-116. Rane, K.K. , and Tattar, T A . 1987. Pathogenicity of blue-stain fungi associated with Dendroctonus terebrans. Plant Disease 71, 8798-883. Rumbold, C T . 1931. Two blue-staining fungi associated with bark-beetle infestation of pines. J. Agr. Res. 43, 847-873. Rumbold, C T . 1941. A blue stain fungus, Ceratostomella montium n. sp., and some yeasts associated with two species of Dendroctonus. J. Agr. Res. 62, 589-601. Rzhetsky, A., and Nei, M . 1992. A simple method for estimating and testing minimum-evolution trees. Mol. Biol. Evol. 9, 945-967. 32 Saitou, N., and Nei, M . 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4, 406-425. Schroeder, S., Kim, S.H., Cheung, W.T., Sterflinger, K., and Breuil, C. 2001. Phylogenetic relationship of Ophiostoma piliferum to other sapstain fungi based on the nuclear rRNA gene. FEMS Microbiol. Lett. 195, 163-167. Schroeder, S., Kim, S.H., Lee, S.W., Sterflinger, K., and Breuil, C. 2002. The P-tubulin gene is a useful target for PCR-based detection of an albino Ophiostoma piliferum used in biological control of sapstain. Eur. J. Plant Pathol. 108, 793-801. Seifert, K.A. 1993. Sapstain of commercial lumber by species of Ophiostoma and Ceratocystis. In: Ceratocystis and Ophiostoma taxonomy, ecology and pathogenicity (Wingfield, M.J., Seifert, K.A. and Webber, J.F., Eds.), pp. 141-151. The American Phytopathological Society Press, St. Paul, Minnesota. Seifert, K.A. , Wingfield, M.J., and Kendrick, W.B. 1993. A nomenclator for described species of Ceratocystis, Ophiostoma, Ceratocystiopsis, Ceratostomella and Sphaeronaemella. In: Ceratocystis and Ophiostoma taxonomy, ecology and pathogenicity (Wingfield, M.J., Seifert, K .A . and Webber, J.F., Eds.), pp. 269-287. The American Phytopathological Society Press, St. Paul, Minnesota. Sneath, P.H.A., and Sokal, R.R. 1973. Numerical Taxonomy, pp. 230-234. W.H. Freeman and Company, San Francisco. Solheim, H. 1995. Early stages of blue-stain fungus invasion of lodgepole pine sapwood following mountain pine beetle attack. Can. J. Bot. 70, 1-5. Thompson, J.D., Higgins, D . C , and Gibson, T.J. 1994. C L U S T A L W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position specific gap penalties and weight matrix choice. Nucl. Acids Res. 22, 4673-4680. Upadhyay, H.P. 1981. A monograph of Ceratocystis and Ceratocystiopsis. The University of Georgia Press, Athens, Georgia. Vilgalys, R., and Hester, M . 1990. Rapid genetic identification and mapping of enzymatically amplified ribosomal DNA from several Cryptococcus species. J. Bacterid. 172, 4238-4246. White, J.J., Bruns, T., Lee, S., and Taylor, J. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: PCR Protocols, A Guide to Methods and Amplifications (Innis, M . A., Gelfand, D.H., Sninsky, J.J. and White, T.J., Eds.), pp. 315-322. Academic Press, New York. 33 Chapter 2.2 A PCR-RFLP marker distinguishing Ophiostoma clavigerum from morphologically similar Leptographium species associated with bark beetles Introduction Dendroctonus ponderosae Hopkins (mountain pine beetle) and its fungal associates cause extensive economic and environmental loss in North America. In 2002, the infestation spread exponentially to over 9 million hectares and the volume of infested lodgepole pine (Pinus contorta Dougl. var. latifolia Engelm.) grew by 108 million cubic meters in British Columbia alone (CLMA/NFPA 2002). D. ponderosae carries Ophiostomatoid fungi that include several deep stainers, which discolor conifer sapwood. The fungi produce asexual and sexual spores in slimy masses that are well adapted to dispersal by insects (Harrington 1988 and 1993). Some Ophiostomatoid fungi are dispersed by more than one beetle species, while others are associated with only one. For example, O. clavigerum is carried by D. ponderosae and D.jeffreyi, while Leptographium pyrinum is only associated with D. adjunctus (Whitney and Farris 1970; Six and Paine 1997a and 1997b). The beetles and fungi have a mutualistic relationship (Paine et al. 1997). The vectored fungi benefit, because the beetles carry them through the bark of trees making available a fresh, moist, nutrient-rich wood environment where they will not encounter competing microflora. The beetles benefit, because the fungi change wood moisture content (Wagner et al. 1979), weaken tree defense mechanisms, and make nutrients available for growth and reproduction (Paine et al. 1997). Six and Paine (1998) have shown that at least one of O. clavigerum or O. montium is required for the successful brood development of D. ponderosae. A version of this chapter has been published. A PCR-RFLP marker distinguishing O. clavigerum from morphologically similar Leptographium species associated with bark beetles. S. Lee, J.-J. Kim, S. Fung and C Breuil. 2003. Can. J. Bot. 81(11): 1104-1112. 34 Previous reports on the fungi associated with D. ponderosae have suggested that one of the predominant fungi, O. clavigerum, is a strong pathogen (Yamaoka et al. 1995), while the other, O. montium is a weak pathogen (Stobel and Sugawara 1986). In order to respond efficiently to an epidemic outbreak, it is necessary to accurately identify existing pathogens and quickly recognize new ones. However, identification of these two fungal species is not simple and their classification has been continuously revised. O. montium has been synonymized to O. ips because they have almost identical teleomorphs and anamorphs (Upadhyay 1981). We have shown recently that these two species are different physiologically and genetically and can be separated by their growth at 35°C and by molecular analyses (Kim et al. 2003). O. clavigerum was first described by Robinson-Jeffrey and Davidson (1968) under the name of Europhium clavigerum. The genus Europhium was characterized by spherical cleistothecia having no special opening. The authors observed the neckless ascocarps (cleistothecia) of E. clavigerum in insect galleries and in or on the inner bark, and sapwood of lodgepole pine. They also described very similar cleistothecia for Europhium aureum and Europhium robustum (Robinson-Jeffrey and Davidson 1968), which were later renamed as O. aureum and O. robustum, respectively (Harrington 1988). To our knowledge, nobody has isolated or observed these neckless ascocarps since Robinson-Jeffrey and Davidson's early description (1968). Given that they have not been isolated again, it is not surprising that the mating system of O. clavigerum has not been defined. Furthermore the ascoscarps have not been produced under artificial conditions (Six and Paine 1997b). Teleomorphs are fundamental characteristics for the classification of Ophiostomatoid fungi. However, when teleomorphs are not available, identification relies on the morphological characteristics of the anamorphs, which are often unstable. Like many other Ophiostoma species, O. clavigerum is pleoanamorphic, producing several anamorphs. Robinson-Jeffrey and Davidson described its anamorph as a Verticicladiella (1968), and then Upadhyay described it as a Graphiocladiella, which has both Graphium and Leptographium anamorphs (1981). In 1984, Tsuneda and Hiratsuka observed holoblastic-yeast state, annellidic-yeast state, Hyalorhinocladiella (sympodial-mononematous), Leptographium (annellidic-mononematous), Verticicladiella-Wke, 35 (sympodial-mononematous), and sympodial synnematous anamorphs. In addition to being pleoanamorphic, O. clavigerum has a wide range of spore shapes and sizes. As a result, its anamorphs can appear very similar to those of other Leptographium and Ophiostoma species. Therefore, identification based only on anamorphs often can be inconclusive. In this work, we developed a PCR-RFLP marker using the P-tubulin gene in order to precisely and promptly distinguish O. clavigerum from morphologically and phylogenetically similar species, especially when the ascosarps are not available. The P-tubulin gene as well as the ITS1 and ITS2 regions of ribosomal DNA has been widely used in phylogenetic study. The P-tubulin gene was chosen because it was more easily amplified and sequenced than the ITS1 and ITS2. Furthermore its sequence had sufficient variability among the species to develop O. clavigerum specific RFLP profile. We are also reporting O. clavigerum ascocarps with short necks; these were found in lodgepole pines that had been infested by D. ponderosae. Materials and methods The fungal strains The isolates analyzed in this work are listed in Table 2.2.1. The strains were obtained from the American Type Culture Collection (ATCC), the University of Alberta Microfungus Collection (UAMH), the Canadian Collection of Fungal Cultures (DAOM), or provided by A. Uzunovic (Forintek Canada Corp., Western lab), D. L. Six (University of Montana, School of Forestry), M. J. Wingfield (University of Pretoria, Forestry and Agricultural Biotechnology Institute), and P. Loppnau (University of British Columbia, Dept. of Wood Science). We also used strains, which had been isolated from D. ponderosae, or galleries of D. ponderosae, or the stained sapwood of lodgepole pine collected in British Columbia. The holotypes of 0. aureum (ATCC 16936), O. clavigerum (ATCC 18086), and O. robustum (CMW 668 = ATCC 16937) were also included as references. Ascocarps were collected from the inner bark or outer sapwood at the bottom of lodgepole pines at Kamloops in October 2002, one year after D. ponderosae 36 Table 2.2.1. Cultures used in this work, GenBank accession numbers for sequences, and the results of PCR-RFLP analysis. Species Isolate No. Accession No. Hinfl-cui' Host Origin Isolation source Collector O. clavigerum A T C C 18086 AY263194 P Pinus ponderosa Cache Creek, B.C. , Canada Tree attacked by Dendroctonus sp. R.C.R.-Jeffrey/ R.W.Davidson UAMH4818 AY263207 P Pinus contorta Westcastle, Alta., Canada Sapwood between beetle galleries A. Tsuneda UAMH4585 AY263206 P Pinus contorta B.C. , Canada Ascocarps in D. ponderosae-infested tree L. Sigler C842*2 P Pinus contorta Alta., Canada Unknown A. Tsuneda C843*2 AY263196 P Unknown Nevada Mtns., C A , U.S.A. D. jeffreyi D. L. Six L P K G O C - 1 AY267828 P Pinus contorta Kamloops, B.C. , Canada Sapwood infested by D. ponderosae J.-J. Kim L P W R O C - 2 P Pinus contorta Williams Lake, B.C. , Canada Sapwood infested by D. ponderosae J.-J. Kim L P W G O C - 3 P Pinus contorta Williams Lake, B.C. , Canada Sapwood infested by D. ponderosae J.-J. Kim SL-H400 AY263208 P Pinus contorta Houston, B.C. , Canada D. ponderosae S. Lee SL-HglOO AY263197 P Pinus contorta Houston, B.C. , Canada Gallery of D. ponderosae S. Lee SL-K1 AY263210 P Pinus contorta Kamloops, B.C. , Canada D. ponderosae S. Lee SL-K58 AY263199 P Pinus contorta Kamloops, B .C. , Canada D. ponderosae S. Lee SL-K402 AY263198 P Pinus contorta Kamloops, B.C. , Canada D. ponderosae S. Lee SL-Kg4 AY263202 P Pinus contorta Kamloops, B .C. , Canada Gallery of TJ. ponderosae S. Lee SL-Kg602 AY263200 P Pinus contorta Kamloops, B.C. , Canada Gallery of D. ponderosae S. Lee SL-Kp54 AY263193 P Pinus contorta Kamloops, B .C. , Canada Ascocarps in D. ponderosae-infested tree S. Lee SL-Kwl407 AY263195 P Pinus contorta Kamloops, B.C. , Canada Sapwood infested by D. ponderosae S. Lee SL-Kwl414 AY263209 P Pinus contorta Kamloops, B.C. , Canada Sapwood infested by D. ponderosae S. Lee Species Isolate No. Accession No. Hinfl-cut Host Origin Isolation source Collector SL-Kw3827 P Pinus contorta Kamloops, B.C. , Canada Sapwood infested by D. ponderosae S. Lee SL-St.J 1 AY263203 P Pinus contorta Fort St. James, B.C. , Canada D. ponderosae S. Lee SL-St .J l l AY263201 P Pinus contorta Fort St. James, B.C. , Canada D. ponderosae S. Lee SL-Wg004 AY263204 P Pinus contorta Williams Lake, B.C. , Canada Gallery of D. ponderosae S. Lee SL-Wg602 AY263205 P Pinus contorta Williams Lake, B.C. , Canada Gallery of D. ponderosae S. Lee 0. aureum ATCC16936 AY263187 NP Pinus contorta Invermere, B.C. , Canada Ascocarps in bark beetle-infested tree R.C.R.-Jeffrey/ R.W.Davidson CMW714 NP Pinus contorta Canada Unknown R.W. Davidson AU98Pr2-141 AY263186 NP Pinus contorta Princeton, B.C. , Canada Sapwood A. Uzunovic AU98Pr2-169 AY263188 NP Pinus contorta Princeton, B.C. , Canada Sapwood A . Uzunovic SL-Ww402 NP Pinus contorta Williams Lake, B.C. , Canada Sapwood infested by D. ponderosae S. Lee 0. robustum CMW668 AY263190 NP Pinus ponderosa McCall, ID, U.S.A. Ambrosia and Dendroctonus spp. R.C.R.-Jeffrey/ R.W.Davidson CMW2805 AY263189 NP Pinus ponderosa ID, U.S.A. Unknown T. Hinds L. abietinum DAOM60343 AY263182 NP Unknown Unknown Unknown R. W. Davidson DAOM37980A AY263183 NP Picea engelmanni Victoria, B .C. , Canada Unknown A. Molnar EW4-45 AY263180 NP Picea glauca Edson, Alta., Canada Sapwood P. Loppnau AI1-1 (2-10) AY263179 NP Interior spruce Williams Lake, B.C. , Canada D. rufipennis S. Lee AU157-144 AY263181 NP Picea glauca Princeton, B.C. , Canada Sapwood A. Uzunovic Species Isolate No. Accession No. Hinjl-cut'1 Host Origin Isolation source Collector L. lundbergii U A M H 9584 AY263184 NP Pinus sylvestris Uppland, Sweden Board A. Mathiesen-Kaarik DAOM64746 AY267827 NP Pinus stobus Kiosk, Ont., Canada Unknown K. Shields L. pyrinum DLS879 AY263185 NP Pinus arizonica Pinaleno Mtns., A Z , U.S.A. D. adjunctus D. L . Six D L S 1093 NP Unknown Sacramento Mtns., N M , U.S.A. D. adjunctus D. L. Six L. terebrantis UAMH9722 AY263192 NP Pinus contorta Victoria, B.C. , Canada Unknown J. Reid C418*2 AY263191 NP Pinus ponderosa Blodgett, C A , U.S.A. D. brevicomis T. C. Harrington L P W Y L T - 1 AY267826 NP Pinus contorta Williams Lake, B.C. , Canada Sapwood infested by D. ponderosae J.-J. Kim L P W Y L T - 2 NP Pinus contorta Williams Lake, B.C. , Canada Sapwood infested by D. ponderosae J.-J. Kim L P K R L T - 3 NP Pinus contorta Kamloops, B.C., Canada Sapwood infested by D. ponderosae J.-J. Kim L P W Y L T - 4 NP Pinus contorta Williams Lake, B.C., Canada Sapwood infested by D. ponderosae J.-J. Kim *'P and NP, presence and no presence of O. clavigerum-spec\f\c 238 and 490 bp D N A fragments. *2Provided by A. Uzunovic. attack. All strains in this work were derived from single spore isolations of either a conidial mass or a crushed ascocarp. Single ascospore isolations were carried out after sterilizing the outside of the ascocarps as described by Bernier and Hubbes (1989). Morphological observation One to four week-old fungal cultures, grown at room temperature on 2% malt extract agar (MEA, Oxoid/Difco), or 20% lodgepole pine sawdust media (200 g sawdust, 1000 ml distilled water, 15 g agar), were observed with a Zeiss (Axioplan II) Light Microscope. For scanning electron microscopy, ascocarps on the inner bark and conidiophores produced on lodgepole pine sapwood chips (7><7x2 mm) were primarily fixed with 2.5% glutaraldehyde in 0.05% sodium cacodylate (pH 7.1). The samples were held for 1 min, microwaved for 40 s at 212 watts, and maintained under vacuum for 3 min to enhance penetration of the fixation solution into the tissue. The samples were rinsed with a 0.05 M cacodylate buffer (pH 7.1) for 5 min, microwaved under vacuum for 40 s at 115 watts and then post fixed with 2% osmium tetroxide using the same conditions used for primary fixation. After being rinsed with water, the samples were dehydrated in a graded ethanol series (50, 70, 85, 95, 100, and then 100% again). For each step in the ethanol series, the samples were held for 1 min and microwaved under vacuum for 40 s at 115 watts. After fixation, the samples were dried with a Balzers CPD 020 critical point drier using C 0 2 , and then mounted on metal stubs. Specimens were coated with gold-palladium using a Nanotech Semprep II Sputter Coater, and then examined with a Hitachi 4700 scanning electron microscope. DNA extraction, PCR and sequencing of P-tubulin gene, and RFLP analysis For DNA extraction, the fungal isolates were grown for 3 - 5 days on cellophane covered M E A . Using the drilling method described by Kim et al. (1999), genomic DNA was extracted from mycelia. The partial P-tubulin gene was amplified using the primer set T10 (5 - C G A T A G G T T C A C C T C C A G A C -3') (O'Donnell and Cigelnik 1997) and BT12 (5 - G T T G T C A A T G C A G A A G G T C T C G -3') (Kim et al. 40 2003). PCR reactions were carried out in a total volume of 25 ul containing 100 ng of DNA template, 0.5 U of Thermostable D N A Polymerase (Rose Scientific), lx buffer (10 mM Tris-HCl pH 8.0, 1.5 mM MgCI 2, 50 mM KC1), 80 uM of each dNTP, 20 pmol of each primer, 10 uM of dimethyl sulfoxide and overlaid with one drop of mineral oil. Amplifications were performed on a Hybaid Touch Down Thermocycler using an initial denaturation at 94°C for 4 min, 30 cycles of denaturation at 94°C for 30 s, primer annealing at 55°C for 50 s, extension at 72°C for 50 s and one additional extension at 72°C for 10 min. PCR products were observed after being separated on a 1% agarose gel in a lx T A E buffer (0.04 M Tris-acetate, 0.001M EDTA). For sequencing, the PCR-amplified (3-tubulin gene products were cut out of the gel, purified using the Qiaquick Gel Extraction Kit (Qiagen Inc.) and subcloned into pCR® 2.1-TOPO T A vectors using a TOPO™ T A Cloning Kit (Invitogen). Sequencing reactions were performed in a total volume of 20 ul consisting of 4 ul B igDye™ premix (Applied Biosystems), 3.2 pmol of primer, 30 - 60 ng/ul of DNA and one drop of mineral oil on a Hybaid Touch Down Thermocycler using 25 cycles at 96°C for 30 s, 50°C for 15 s, and 60°C for 4 min. Sequencing was carried out on a PRISM 377 D N A sequencer (Applied Biosystems) at the University of British Columbia Nucleic Acid and Protein Service Unit. The sequence data obtained from both strands of DNA were used to determine the nucleotide sequences of the amplicons. These nucleotide sequences have been deposited in the GenBank DNA sequence database under the accession numbers shown in Table 2.2.1. The amplified P-tubulin gene sequences were analyzed using Gene Tool (BioTools Inc.) in order to search for a potential RFLP marker. Restriction maps of the sequences were defined and Hinfl was selected as it generated a unique O. clavigerum profile. Successful PCR-amplifications of the P-tubulin gene were confirmed by visualizing 10 ul of PCR products on 1% gels prior to restriction enzyme digestion. 15 pi of the PCR products were digested with 10 units of Hinfl (Amersham Pharmacia Biotech Inc.) at 37°C for 3 hrs according to the supplier's instructions. The digested DNA fragments were 41 separated on 2% agarose gels and then RFLP patterns were recorded with an Image Analyser System (Bio/Can). Phylogenetic analysis For the phylogenic analysis, 16 sequences were aligned using the ClustalX (Thompson et al. 1997) and then optimized using the PHYDIT program version 3.2 (http://plasza.snu.ac.kr/~jchun/phydit/). Multiple alignment parameters used were a gap open penalty of ten and a gap extension penalty of one. Intron and exon junctions in the (3-tubulin sequences were inferred based on comparison with the known sequences of Epichloe typhina (X52616) (Ayliffe et al. 2001) and O. piliferum (AF221628). Levels of molecular sequence divergence among the data sets were determined by calculating pairwise estimates of nucleotide substitution rates using Hasegawa, Kishino, and Yano's model (1985). The trees based on the P-tubulin gene were constructed by neighbor-joining (Saitou and Nei 1987) and parsimony analyses using PAUP 4.0bl0 (Swofford 2001). A heuristic search option was chosen in parsimony analysis. The stability of branches was evaluated by bootstrap tests with 1000 replications (Felsenstein 1985). Results The teleomorphs and anamorphs of O. clavigerum The ascoscarps of O. clavigerum were observed at the bottom of red trees, which had been attacked by D. ponderosae in the previous summer; the frequency was higher when the trees were in a wet environment. Groups of ascocarps with or without short necks were observed on or inside the inner bark (Figs 2.2.1 and 2.2.2). In contrast to Robinson-Jeffrey and Davidson, who described spherical cleistothecia of O. clavigerum (1968), we noted that many of the ascocarps had very short necks varying from 20 to 65 um long (Figs 2.2.1, 2.2.3, 2.2.4, and 2.2.5). However, sometimes it was very difficult to recognize a neck, and some ascocarps appeared as spherical cleistothecia. Both forms of ascocarps had diameters ranging from 250 to 550 pm. The ascospores obtained from both types of ascocarps were 3.5 - 5.0 pm long, 42 Figs 2.2.1-2.2.6. The ascocarps and ascospores of Ophiostoma clavigerum. Figs 2.2.1, 2.2.2. Stereomicrographs of ascocarps with (bold arrow)/ without necks, shown in or on the inner bark of lodgepole pine. Figs 2.2.3, 2.2.4. Scanning electron micrographs of ascocarps with relatively short necks (arrows). Scale bars: Fig. 2.2.3. = 100 pm; Fig.2.2.4 = 100 pm. Fig. 2.2.5. Light micrograph of perithecium showing a short neck without ostiolar hyphae. Scale bar = 100 pm. Figs 2.2.6. Ascospores. Scale bar = 10 pm. 43 hyaline, one-celled, reniform and appeared cucullate with a hyaline sheath (Fig. 2.2.6). We examined 10 ascocarps and isolated 10-40 ascospores from each perithecium. Al l single spore isolates derived from both types of ascocarps were identified as O. clavigerum based on their morphological characteristics. These observations were further confirmed with the SL-Kp 54 strain by PCR-RFLP analysis (Fig. 2.2.17). The complex mononematous conidiophores, having sympodulae attached in whorls reported by Robinson-Jeffrey and Davidson (1968), and synnematous conidiopores described by Upadhyay (1981), were observed in O. clavigerum grown for approximately 20 - 30 days on M E A (Figs 2.2.7, 2.2.8, and 2.2.9) , and were more abundantly produced when grown on sawdust media. However, they were not present in representatives of other closely related species such as L. lundbergii, L. pyrinum, L. terebrantis, O. aureum, and O. robustum listed in Table 2.2.1. The short mononematous conidiophores having a mean length of 225 u.m (range 80 - 576 um) (Fig. 2.2.10) were much more abundant than the long conidiophores (up to 1150 urn; Upadhyay 1980). The short conidiophores had a variety of spore shapes in 2.5 - 14.8 urn length. They were oblong, ellipsoid, peanut with a truncated end, and subglobose (Figs 2.2.11 and 2.2.12). The clavate spores, which have been considered typical of O. clavigerum were not observed abundantly in the O. clavigerum strains examined in this study. In culture, variations of mycelial appearance were observed among O. clavigerum strains. In comparison to the holotype A T C C 18086, the mycelia of LPKGOC-1, LPWROC-2, and LPWGOC-3 were more darkly pigmented and roughly branched. They also had more conidiophores (Figs 2.2.13, 2.2.14, and 2.2.15). Phylogenetic analysis and O. clavigerum specific RFLP marker The amplified regions of the P-tubulin gene included three introns and four exons. These exons corresponded to the second through sixth exons of Neurospora crassa's P-tubulin gene (M13630). The intraspecies nucleotide variation in this region among nineteen O. clavigerum strains was as low as 0.13% 44 Figs 2.2.7-2.2.12. The conidiophores and conidia of Ophiostoma clavigerum. Fig. 2.2.7. Stereomicrograph of synnematous conidiophores on 2% M E A (Oxoid). Fig. 2.2.8. Light micrograph of synnematous conidiophores with clavate conidia. Scale bar = 100 urn. Fig. 2.2.9. Light micrograph of mononematous conidiophore penicillately branched with clavate conidia (ATCC 18086). Scale bar = 50 um. Fig. 2.2.10. Light micrograph of Leptographium anamorphs. Scale bar = 100 um. Fig. 2.2.11. Light micrograph of conidia. Scale bar = 10 um. Fig. 2.2.12. Scanning electron micrograph of conidia. Scale bar = 5 um. Figs 2.2.13-2.2.15. Variance in colony morphology among O. clavigerum strains. Fig. 2.2.13. A common O. clavigerum (ATCC 18086). Fig. 2.2.14. Unusual O. clavigerum (LPKGOC-1) grown on 2% M E A (Difco) for one week at room temperature. Fig. 2.2.15. Light micrograph of short conidiophores densely grown on the mycelia of O. clavigerum (LPKGOC-1). Scale bar = 100 urn. 4 5 to 0.26%. The variation among interspecies was higher: 0.38% to 0.51% between O. clavigerum and L. terebrantis, 0.51% to 0.77% between O. clavigerum and O. robustum, 1.53%) to 1.92% between O. clavigerum and O. aureum, and 0.89% to 1.02% between O. clavigerum and L. pyrinum. The neighbor-joining trees and most parsimonious trees were constructed with only the exons or with both the exons and introns of the p-tubulin gene, and these showed almost identical topology. The neighbor-joining trees (not shown) were supported by higher bootstrapping values than most parsimonious trees. One of four most parsimonious trees based on both the exons and introns is shown in Fig. 2.2.16. O. clavigerum formed a monophyletic group closely related to L. terebrantis, O. robustum, and L. pyrinum, which had similar shapes of spores. The physical restriction maps using Hinfl were deduced from the p-tubulin gene sequence data. The expected sizes of digested fragments from the 836 bp amplicon of O. clavigerum were 51, 57, 238, and 490 bp. In contrast, the predicted sizes from the 844 bp amplicon of L. lundbergii were 51, 57, and 736 bp, and the fragments of the 838 bp amplicons of four other species (L. pyrinum, L. terebrantis, O. aureum, and O. robustum) were 51, 57, and 730 bp. The two target sites of Hinfl at the first intron and forth exon in the amplicons were common to all strains except L. abietinum, which does not have a target sequence for Hinfl. However, the two nucleotides (GT) deletion located in the third intron (266bp) of O. clavigerum generated O. clavigerum specific fragments, 238 and 490 bp long. The resulting two bands were clearly detectable in all electrophoresis gels while the smaller bands, less than 60 bp in size, were not always visible (Fig. 2.2.17). The specificity of the O. clavigerum RFLP profile was confirmed by testing all of the strains listed in Table 2.2.1. 46 100 98 100 10 changes O. piliferum AF221628 Epichloe typhina X52616 100 63" 65 77 O. robustum CMW2805 • O.robustum CMW668 L. terebrantis C418 E terebrantis UAMH9722 SL-Kp54 A T C C 18086 SL-Kwl407 C843 E pyrinum DLS879 O. clavigerum 98 O. aureum A T C C 16936 - O. aureum AU98Pr2-169 E lundbergii UAMH9584 " E abietinum DAOM60343 E abietinum DAOM37980A Fig. 2.2.16. One of four parsimonious trees of O. clavigerum and its morphologically similar species. The trees is based on the exons and introns of the P-tubulin gene. The tree is 505 steps long and has a consistency index of 0.8970, a retention index of 0.8523, and a rescaled consistency index of 0.7645. The numbers at the branch nodes indicate the confidence values from bootstrap analysis using 1000 replications. Bootstrap values above 50% are presented. The accession numbers for each P-tubulin gene are listed in Table 1. Outgroup: Epichloe typhina. Fig. 2.2.17. The O. clavigerum-specific PCR-RFLP profile of the B-tubulin gene generatedd with Hinfi. Lanes: M l , 1 kb D N A marker; M2, 100 bp D N A marker; 1, O. clavigerum (ATCC 18086); 2, O. clavigerum (C 843); 3, O. clavigerum (SL-Kp 54); 4, O. clavigerum (SL-Kw 1407); 5, O. aureum (ATCC 16936); 6, O. robustum ( C M W 2805); 7, L. lundbergii ( U A M H 9584); 8, L. terebrantis (C 418); 9, L. pyrinum (DLS 879); and 10, L. abietinum ( D A O M 37980A). 48 Discussion For Ophiostomatoid fungi, identification based on anamorph characteristics has been confusing because of their pleoanamorphisms, similarities, and degenerations. In this study, analysis of the P-tubulin gene showed that O. clavigerum is closely related to L. terebrantis, O. robustum, and L. pyrinum phylogenetically. These phylogenetically closely related species also have morphologically similar anamorphs. The most characteristic features of O. clavigerum are long clavate spores (12.5 - 85 pm), and long synnematous conidiophores (500 - 1150 pm) with broom shaped tips (Upadhyay 1981). However, these anamorphs were not consistently observed in all O. clavigerum strains grown on MEA media. In fact, the strains UAMH 4818 and C 843 very rarely produced clavate spores. Short mononematous conidiophores with various shapes and sizes of spores were observed predominantly rather than long synnematous conidiophores. These spores can be easily confused with the spores of the following species: L. terebrantis which has oblong to ellipsoid spores 2.4 - 12.8 pm long, with a truncated end (Barras and Perry 1971), L. pyrinum which has broad ovoid spores 7-10 pm long (Davidson 1978), L. lundbergii which has ellipsoid spores 3-5 pm long, with a truncate end (Lagerberg et al. 1927), O. aureum which has cylindrical to obclavate spores 2.5 - 29 pm long (Robinson-Jeffrey and Davidson 1968), and O. robustum which has cylindrical to globose-ovoid spores 5.3 - 11 pm long (Robinson-Jeffrey and Davidson 1968). It is also important to note that the anamorphs of Ophiostomatoid fungi can easily degenerate on artificial media, and consequently can look like the other closely related species. Tsuneda and Hiratsuka (1984) reported that the anamorphs of O. clavigerum tend to shift from complex to simpler forms as the cultures are repeatedly transferred on artificial media or stored for long periods of time. They have also questioned the identity of O. aureum, O. clavigerum, and O. robustum, because these three species had almost identical ascocarps and their separation by Robinson-Jeffrey and Davidson (1968) was based only on unstable anamorphs. O. aureum (Robinson-Jeffrey and Davidson 1968) has been isolated from lodgepole pine. L. pyrinum (Davidson 1978) and O. robustum (Robinson-Jeffrey and Davidson 1968) have been found on ponderosa 49 pine (Pinus ponderosa). However, O. clavigerum (Robinson-Jeffrey and Davidson 1968) and L. terebrantis (Hausner et al. 2000; Harrington 1988) have been isolated from both lodgepole pine and ponderosa pine. Thus, fungal host specificity cannot be used to distinguish O. clavigerum from these morphologically similar species. The presence of synnematous conidiophores can be useful to differentiate O. clavigerum from the other species, but they are not always produced. In fact, they are formed sparsely on plates and it takes 20-30 days before they can be observed. Furthermore, they are very rarely produced in degenerate cultures. Therefore, it is difficult to accurately diagnose fungal isolates in a reasonable amount of time based only on morphological characteristics, especially when faced with a large number of isolates. To efficiently differentiate O. clavigerum from the other similar species, we developed an O. clavigerum specific PCR-RFLP marker. It utilized Hinfl enzyme, which cut O. clavigerum exclusively at the third intron of the amplified the p-tubulin gene. It was difficult to confirm the identity of UAMH 4818 and C 843 based on their anamorphs, since they were already degenerate when we obtained these cultures. However, the PCR-RFLP marker clearly distinguished these O. clavigerum strains from the other species. This marker was very efficient because the p-tubulin gene was easily amplified from all the strains under the same PCR amplification conditions. And, furthermore, only one enzyme was required to generate the O. clavigerum specific RFLP profile. The neckless ascocarps, described for O. aureum, O. clavigerum, and O. robustum (Robinson-Jeffrey and Davidson 1968) are unusual in the Ophiostoma species (Six et al. 2003). During our research, we observed that many ascocarps of O. clavigerum had short necks while some appeared spherical. It is not clear whether neckless ascocarps were immature, or if O. clavigerum produces two types of ascocarps. More systematic studies on the developmental process of the ascocarps of O. clavigerum could resolve this uncertainty. Further investigation of the ascocarps of O. aureum and O. robustum would also be valuable since they might have necks like those of O. clavigerum but were only overlooked in the past. In this study, a very efficient and reliable O. clavigerum specific PCR-RFLP marker was developed, 50 and O. clavigerum ascocarps with necks were clearly described for the first time. To better understand the biology of this species, further investigation of a mating system using the isolates derived from a single ascocarp would be valuable, even though the production of ascocarps under artificial conditions is a challenge that has yet to be solved. 51 References Ayliffe, M.A. , Dodds, P.N., and Lawrence, G.J. 2001. Characterization of a p-tubulin gene from Melampsora Uni and comparison of fungal P-tubulin genes. Mycol. Res. 105: 818-826. Barras, S.J., and Perry, T. 1971. Leptographium terabrantis sp. nov. association with Dendroctonus terebrans in loblolly pine. Mycopathologia et Mycologia applicata, 43: 1-10. Bernier, L. , and Hubbes, M . 1990. Meiotic analysis of induced mutations in Ophiostoma ulmi. Can. J. Bot. 68: 232-235. Cariboo Lumber Manufacturers' Association / Northern Forest Products Association. 2002. http://www.mountainpinebeetle.com/2002_update.htm. Davidson, R.W. 1978. Staining fungi associated with Dendroctonus adjunctus in pines. Mycologia, 70: 35-40. Felsenstein, J. 1985. Confidence limits on phylogenies: an approach using the bootstrap. Evol. 39: 783-791. Harrington, T.C. 1988. Leptographium species, their distributions, hosts and insect vectors. In Leptographium root disease on conifers. Edited by T.C. Harrington and F.W. Cobb(Jr.). The American Phytopathological Society Press, St. Paul, Minnesota, pp. 1-39. Harrington, T.C. 1993. Biology and taxonomy of fungi associated with bark beetles. In Beetle-pathogen interactions in conifer forests. Edited by T.D. Schowalter and G.M. Filip. Academic Press, San Diego, pp. 37-51. Hasegawa, M . , Kishino, H. , and Yano, T. 1985. Dating the human-ape split by a molecular clock of mitochondrial DNA. J. Mol. Evol. 22: 160-174. Hausner, G., Reid, J., and Klassen., G.R. 2000. On the phylogeny of members of Ceratocystis s.s. and Ophiostoma that possess different anamorphic states, with emphasis on the anamorph genus Leptographium, based on partial ribosomal DNA sequences. Can. J. Bot. 78: 903-916. Kim, J.-J., Kim, S.H., Lee, S., and Breuil, C. 2003. Distinguishing Ophiostoma ips and O. montium two bark beetle-associated sapstain fungi. FEMS Microbiol. Lett. 222: 187-192. Kim, S.H., Uzunovic, A., and Breuil, C. 1999. Rapid detection of Ophiostoma piceae and O. quercus in stained wood using PCR. Appl. Environ. Microbiol. 65: 287-290. Lagerberg, T., Lundberg, G., and Melin, E. 1927. Biological and practical researches into blueing in pine and spruce. Svenska Skogsvardsforeningens Tidskrift, 25: 145-272. O'Donnell, K., and Cigelnik, E. 1997. Two divergent intragenomic rDNA ITS2 types within a monophyletic lineage of the fungus Fusarium are nonorthologous. Mol. Phylogenet. Evol. 7: 103-116. Paine, T.D., Raffa, K.F. , and Harrington, T.C. 1997. Interactions among Scolytidae bark beetles, their associated fungi, and live host conifers. Ann. Rev. Entomol. 42: 179-206. 52 Robinson-Jeffrey, R . C , and Davidson, R.W. 1968. Three new Europhium species with Verticicladiella imperfect states on blue-stained pine. Can. J. Bot. 46: 1523-1527. Saitou, N., and Nei, M . 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4: 406-425 Six, D.L., and Paine, T.D. 1997a. Leptographium pyrinum is a mycangial of Dendroctonus adjunctus. Mycologia, 88: 739-744. Six, D.L., and Paine, T.D. 1997b. Ophiostoma clavigerum is the mycangial fungus of the Jeffrey pine beetle, Dendroctonus jeffreyi. Mycologia, 89: 858-866. Six, D.L., and Paine, T.D. 1998. Effects of mycangial fungi and host tree species on progeny survival and emergence of Dendroctonus ponderosae (Coleoptera: Scolytidae). Environ. Entomol. 27: 1393-1401. Six, D.L., Harrington, T.C. , Steimel, J., McNew, D., and Paine, T.D. 2003. Genetic relationships among Leptographium terebrantis and the mycangial fungi of three western Dendroctonus bark beetles. Mycologia, (in press). Stobel, G.A., and Sugawara, F. 1986. The pathogenicity of Ceratocystis montiato lodgepole pine. Can. J. Bot. 64: 113-116. Swofford, D.L. 2001. PAUP: phylogenetic analysis using parsimony. Version 4.0M0. Sinauer Associates, Inc., Sunderland, Massachusetts. Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F., and Higgins, D.G. 1997. The ClustalX windows interface: fexible strategies for multiple sequence alignment aided by quality analysis tools. Nucl. Acids Res. 24: 4876-4882. Tsuneda, A., and Hiratsuka, Y . 1984. Sympodial and annellidic conidiation in Ceratocystis clavigera. Can. J. Bot. 62: 2618-2624. Upadhyay, H.P. 1981. A monograph of Ceratocystis and Ceratocystiopsis. The University of Georgia Press, Athens, Georgia. Wagner, T.L. , Gagne, J.A., Doraiswamy, P.C., Coulson, R.N., and Brown, K.W. 1979. Development time and mortality of Dendroctonus frontalis in relation to changes in tree moisture and xylem water potential. Environ. Entomol. 8: 1129-1138. Whitney, H.S., and Farris, S.H. 1970. Maxillary mycangium in the Mountain Pine Beetle. Science, 167: 54-55. Yamaoka, Y., Hiratsuka, Y., and Maruyama, P.J. 1995. The ability of Ophiostoma clavigerum to kill mature lodgepole-pine trees. Eur. J. For. Path. 25: 401-404. 53 Chapter 2.3 Leptographium longiclavatum sp. nov., a new species associated with the mountain pine beetle, Dendroctonus ponderosae Introduction For the last twelve years, an epidemic of the mountain pine beetle, Dendroctonus ponderosae Hopkins, has spread over 10.1 million hectares in British Columbia (COFI 2004). This bark beetle has infested 173.5 million m 3 of lodgepole pine (Pinus contorta var. latifolia), one of the most prevalent and commercially valuable tree species. The epidemic is driven by mutually beneficial relationships between the beetle and its fungal associates (Paine et al. 1997). D. ponderosae mechanically damages trees by feeding on the phloem, while the fungi carried by the beetle discolor the sapwood and disrupt the transportation of water to the tree crown (Reid 1961). The infested trees are killed by the simultaneous actions of the beetle and fungi. To better understand the effects of fungi related with D. ponderosae outbreak, it is important to identify existing or new fungal associates. Many bark beetles (Coleoptera, Scolytidae) that infest coniferous trees carry Ophiostomatoid fungi, especially species with Leptographium anamorphs (Harrington 1988, 1993, Wingfield and Gibbs 1991). These fungi are best known as agents that stain the sapwood of conifers (Wingfield et al. 1993, Jacobs and Wingfield 2001), and they are casually or specifically associated with insect vectors (Harrington 1988 ). Leptographium is one of the most common anamorphs of Ophiostoma species. It is typically characterized by mononematous conidiophores with darkly coloured stipes and a series of branches at the apices. Many Leptographium species are weak pathogens; only a few are strong pathogens such as Leptographium wageneri (Kendrick) Wingfield (Harrington and Cobb 1983, Wingfield A v e r s i o n o f th is chap te r has b e e n p u b l i s h e d . Leptographium longiclavatum sp . n o v . , a n e w spec ies a s soc ia t ed w i t h the m o u n t a i n p i n e beet le , Dendroctonus ponderosae. S. L e e , J . - J . K i m , and C . B r e u i l . 2 0 0 5 . Mycol. Res. 109 (10) : 1 1 6 2 - 1170 . 54 et al. 1988). Of the described Leptographium species, only Leptographium clavigerum (Upad.) Harrington, the asexual stage of Ophiostoma clavigerum (Robison-Jeffrey and Davids.) Harrington, has been known as an associate of D. ponderosae (Robison-Jeffrey and Davidson 1968, Whitney and Farris 1970) . Ophiostoma huntii (Robinson) deHoog & Scheffer, a species with a Leptographium anamorph, has also been isolated from old galleries of the trees attacked by D. ponderosae (Solheim 1995, Whitney 1971) . However, this fungal species has not been isolated from the D. ponderosae (Whitney and Farris 1970), and it was suggested that this fungus may have been introduced by other insects such as secondary bark beetles or mites. During our ecological survey in 2001-2004, an unidentified Leptographium species had been isolated from both D. ponderosae and D. ponderosae-infested lodgepole pines in British Columbia (unpublished data). The fungus showed morphological similarity with O. clavigerum, especially in the shapes of conidia. The aim of this work was to describe the unknown Leptographium sp. associated with D. ponderosae, using its distinct morphological and physiological characteristics, and its phylogenetic relationships to closely related species. The phylogenetic trees were constructed using the partial regions of rDNA and three protein-coding genes: P-tubulin, actin, and glyceraldehyde-3-phosphate dehydrogenase (GPD). Material and methods Fungal isolation The fungi were isolated from D. ponderosae, stained sapwood, or bark of lodgepole pines, which had been infested for less than one year. The sampling was conducted at Kamloops, Princeton, and Williams Lake in British Columbia, Canada in 2001. To isolate the fungi from D. ponderosae, the beetles were collected under the bark of infested trees and washed with 1 mL of 0.01% (v/v) Tween 20 (Sigma-Aldrich, Oakville, ON) solution. The diluted suspension was spread on 2% O M E A (33 g Oxoid malt extract agar, 10 g Oxoid agar and distilled water in total volume of 1000 mL) amended with ampicillin at 50 pg/mL 55 and incubated at room temperature for 2-3 days. The separated colonies on the plates were sub-cultured on 2% O M E A individually and incubated at room temperature. The fragments of bark and stained sapwood were also aseptically removed and placed on 2% OMEA/amp. The hypal tip was sub-cultured to 2% O M E A and incubated at room temperature. The purity of the cultures used in this study was ensured through single spore isolation. Al l isolates were maintained at the Culture Collection of the Department of Wood Science, University of British Columbia and also been deposited at the Canadian Collection of Fungal Cultures (DAOM). Cultural and microscopic characteristics Cultural appearances were observed on both 2% O M E A and 2% D M E A (20 g Difco malt extract, 15 g Difco agar, and distilled water in total volume of 1000 mL) plates. The colony colour was described following Methuen handbook of colour (Kornerup and Wanscher 1961). The growth rates were examined at temperatures ranging from 5 to 35 °C, at five-degree intervals. The colony diameters of three replicates on 2% O M E A plates were measured along two perpendicular lines two and four days after inoculation. The radial growth rates were subsequently calculated. The tolerance to cycloheximide was assessed by measuring fungal growth on 2% O M E A containing 0.05, 0.1 and 0.5 % of cycloheximide (Sigma-Aldrich) at 25 °C. For microscopic characteristics, two to three- week old fungal cultures, grown at room temperature on 2% D M E A , were observed with a Zeiss (Axioplan II) light microscope. Each microscopic value was obtained from the average of at least 50 measurements. The conidiophore was also observed using scanning electron microscopy (SEM) as described by Lee et al. (2003). DNA extraction, PCR, sequencing, and PCR-RFLP marker The genomic DNA was extracted from the mycelium of fungi, which were grown for 3-4 d on 2% O M E A plates overlaid with cellophane sheets (Bio-Rad Ltd. Mississauga, ON). The DNA extraction and PCR amplifications of rDNA (ITS2 and partial 28S) and the partial regions of three protein-coding genes (actin, P-tubulin, and GPD) were performed as described Lim et al. (2004). The primer sets used in this study 56 were as follows: ITS3 / LR3 for amplifying rDNA (Vilgalys and Hester 1990, White et al. 1990), Lepact F / Lepact R for actin gene (Lim et al. 2004), T10 (O'Donnell et al. 2000) / BT12 (Kim et al. 2003) for fi-tubulin gene, and GPD5-ex2 (5 - A T T G G C C G Y A T C G T C T T C C G ) / GPD-int (5 - T T G C C G T T A A G C T C T G G A A T ) for GPD gene. PCR amplicons were purified using Qiaquick Gel Extraction Kit (Qiagen Inc., Mississauga, Canada) and sequenced with an ABI 3700 automated sequencer (Perkin-Elmer, Foster City, CA) at Macrogen Inc. (Seoul, Korea). The O. clavigerum specific PCR-RFLP marker was tested on eleven isolates of this new Leptographium species listed in Table 2.3.1, following the method described by Lee et al. (2003). Phylogenetic analysis Twenty-two strains comprising eight species were analyzed phylogenetically (Table 2.3.1). The sequences were aligned using the ClustalW algorithm (Higgins, Bleasby and Fuchs 1991) and optimized using the PHYDIT program version 3.2 (http://plasza.snu.ac.kr/~jchun/phydit/). Multiple alignment parameters were 'gap open penalty of ten' and 'gap extension penalty of one'. The phylogenetic analyses of individual loci and combined sequence of four loci were performed with PAUP*4.0bl0 (Swofford 2002). A partition homogeneity test was carried out with PAUP*4.0blO to evaluate whether rDNA and three protein-coding genes could be combined. The most parsimonious tree was searched with heuristic search option in which all characters were of type 'uncord', all characters had equal weight and gaps were treated as missing. The tree-bisection-reconnecting (TBR) was applied as branch swapping algorithm and 'MulTrees' option was in effect. The stability of branches was evaluated by bootstrap analysis with 1000 replications (Felsenstein 1985). Full heuristic search option was chosen for bootsrapping. Based on the previous studies (Lim et al. 2004), O. huntii and L. lundbergii were assigned as outgroups. 57 Table 2.3.1. Cultures and GenBank accession numbers for the sequences used in the phylogenetic analysis ofZ. longiclavatum. Species Isolate 1 Host*2 Origin Isolation source Collector/Year GenBank accession No. 3 Actin ITS2 & L S U P-tubulin GPD L. longiclavatum SL-K.W1436 PC Kamloops, B.C. , Canada Sapwood infested by D. ponderosae S.Lee, 2001 AY816679 AY816686 AY288934 AY816693 S L - K p l l PC Kamloops, B .C . , Canada Bark infested by D. ponderosae S.Lee, 2001 AY816680 AY816687 AY816712 AY816694 SL-W001 PC Williams Lake, B.C. , Canada D. ponderosae S.Lee, 2001 AY816681 AY816688 AY288936 AY816695 SL-Pw5 PC Princeton, B .C . , Canada Sapwood infested by D. ponderosae S.Lee, 2001 AY816682 AY816689 AY288935 AY816696 C187 PP Yosemite Valley, C A , U S A . Phloem infested by D. ponderosae D. Owen, 1984 AY816683 AY816690 AY816713 AY816697 SL-K215 PC Kamloops D. ponderosae S.Lee, 2001 -- - AY288931 -SL-Kw439 PC Kamloops Sapwood infested by D. ponderosae S. Lee, 2001 -- - AY288932 -SL-Kw442 PC Kamloops Sapwood infested by D. ponderosae S. Lee, 2001 - - AY288933 -SL-Wg403 PC Williams Lake Gallery of D. ponderosae S. Lee, 2001 - -- AY288937 -SL-Ww405 PC Williams Lake Sapwood infested by D. ponderosae S. Lee, 2001 - - AY288938 -SL-Ww407 PC Williams Lake Sapwood infested by D. ponderosae S. Lee, 2001 - - AY288939 -L. lundbergii UAMH9584 PS Uppland, Sweden Board A . Mathiesen-Kaarik AY544585 AY544603 AY263184 AY816698 L. pyrinum DLS879 P A Pinaleno Mtns., A Z , U.S .A. Dendroctonus adjunctus D. L. Six AY544586 AY544604 AY263185 AY816699 CMW3889 PJ C A , U . S A . Unknown D. L. Six AY544587 AY544605 AY544621 AY816700 L. terebrantis UAMH9722 PC Sooke, B.C. , Canada Unknown J. Reid AY544588 AY544606 AY263192 AY816701 C418 PP Blodgett, C A , U.S.A. Associated with D. brevicomis T. C. Harrington AY544589 AY544607 AY263191 DQ082862 AU98Pr2-155 PC Princeton, B .C . , Canada Sapwood A. Uzunovic AY544590 AY544608 AY544622 -O. aureum ATCC16936 PC Invermere, B .C . , Canada Ascocarps in bark beetle-infested tree R. C R.-Jeffrey/ R. W. Davidson AY544592 AY544610 AY263187 AY816702 AU98Pr2-169 PC Princeton, B .C . , Canada Sapwood A . Uzunovic AY544594 AY544612 AY263188 AY816703 0. clavigerum A T C C 18086 PP Cache Creek, B .C . , Canada Tree attacked by Dendroctonus sp. R. C R.-Jeffrey /R. W. Davidson AY544595 AY544613 AY263194 AY816704 C843 U N Nevada Mtns., C A , U.S.A. D. Jeffrey i D. L. Six AY544596 AY544614 AY263196 AY816705 S L - K w l 4 0 7 PC Kamloops, B .C . , Canada Sapwood infested by D. ponderosae S. Lee, 2001 AY544597 AY544615 AY263195 AY816706 SL-SU11 PC Fort St. James, B .C . , Canada D. ponderosae S. Lee, 2002 AY816684 AY816691 AY263201 AY816707 SL-Wg602 PC Williams Lake, B .C . , Canada Gallery of D. ponderosae S. Lee, 2001 AY816685 AY816692 AY263205 AY816708 0. huntii UAMH4997 PC Invermere, B .C . , Canada Bark beetle galleries R. C. R.-Jeffrey AY544599 AY544617 AY349023 DQ082861 Species Isolate Host ' Origin Isolation source Collector/Year GenBank accession No. O. robustum CMW668 PP McCall, ID, U.S.A. CMW2805 PP ID, U.S.A. Ambrosia and Dendroctonus spp. Unknown R. C. R.-Jeffrey/ R. W. Davidson T. Hinds Actin ITS2&LSU p-tubulin GPD AY544601 AY544619 AY263I90 AY816710 AY544602 AY544620 AY263189 AY816711 UAMH, the University of Alberta Microfungus Collection and Herbarium, Devonian Botanic Garden, Canada; DLS, the culture collection of D. L. Six, University of Montana, U. S. A.; CMW, Culture Collection Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, South Africa; C, Culture Collection of T. C. Harrington, Iowa State University, U. S. A.; ATCC, American Type Culture Collection, Manassas, VA, U. S. A.; AU- and SL- isolates, Breuil's Culture Collection, University of British Columbia, Canada. PC, Pinus contorta; PP, Pinus ponderosa; PS, Pinus sylvestris; PA, Pinus arizonica; PJ, Pinus jeffreyi; UN, Unknown. 5 Accession numbers of sequences obtained in this study presented in bold. Results Taxonomy The Leptographium species isolated from D. ponderosae exoskeleton and D. ponderosae-infested lodgepole pines is typical of the genus, having well-developed long conidiophores and masses of conidia carried in moist drops at their apices. The fungus is able to tolerate high concentrations of cycloheximide, a typical feature of the Ophiostoma species and their anamorphs. The most distinct characteristics of this fungus are its clavate conidia and long conidiophores. The shapes of conidia are much more homogenous than in O. clavigerum, which also produces clavate and other shapes of conidia. The Leptographium species (Figs 2.3.1-2.3.9) can be clearly distinguished from the Leptographium anamorph of O. clavigerum by the length of conidiophores (Figs 2.3.10-2.3.11), colony colour and edge, optimal growth rate and the stability of anamorph shape as well as conidia shapes (Figs 2.3.12-2.3.13). Therefore, we suggest that this fungus is a new taxon and provide the following description. Leptographium longiclavatum S. Lee, J.-J. Kim & C. Breuil, sp. nov. (Figs 2.3.1-2.3.9) Etym.: longiclavatum refers to the long clavate conidia. Crescit optime ad 25 °C, turn 7.3 mm per diem in 2% ' O M E A ' . Non crescit infra 5 °C vel supra 35 °C. In ' O M E A ' cum alio 0.05, 0.1 et 0.5% 'cycloheximide', crescit ad 25 °C alium 7.3, 7.7 et 5.8 mm per diem. Coloniae in ' D M E A ' effusae, extendentes, olivaces. Hypharum parietes laeves, hyphae 2-5 pm diametro. Conidiophorae singulae vel aggregatae, mononematae, macronematae, in basibus cum rhizoideis, interdum in hyphis aeriis factae. Stipites erecti, brunnei, simplices, 3-20 septati, 75-1202 (medius = 752 ± 329) pm longi, basi 8-11 (medius = 10 + 1.5) pm lati. Apparatus conidiogenus 40-451 (medius = 280 ± 1 3 7 ) pm longus, massa conidiali exclusa; ramis primariis bisulcus vel ternis, medio-brunneis, ramis centralibus aegre quam aliis maioribus, 25-45 (medius = 34 ± 4) x 2.5-3 pm; ramis secondariis laete brunneis, 25-35 (27 ± 2) x 2.5-3 pm; ramis tertiariis hyalinis, 25-30 (26 ± 2) x 2.5-3 60 Figs 2.3.1-2.3.9. Morphological characteristics of L. longiclavatum (SL-Kwl436). Fig. 2.3.1. Colony grown on 2% D M E A for 5 d at room temperature. Figs 2.3.2-2.3.3. Light micrographs of long conidiophores with clavate conidia. Figs 2.3.4-2.3.5. Scanning electron micrographs of conidiogenous apparatus with clavate conidia. Fig. 2.3.6. Scanning electron micrograph of conidiogenous cells showing annellations (arrows). Fig. 2.3.7. Scanning electron micrographs of clavate conidia. Figs 2.3.8-2.3.9. Light micrographs of conidia. Scale bars: Fig. 2.3.2 = 100 pm; Fig. 2.3.3 = 50 pm; Figs 2.3.4-2.3.5 = 20 pm. Fig. 2.3.6 = 5 pm. Figs 2.3.7-2.3.8 = 10 pm; Fig. 2.3.9 = 20 pm. 61 Figs 2.3.10 - 2.3.13. Morphological characteristics of Ophiostoma clavigerum (ex-holotype, A T C C 18086). Figs 2.3.10-2.3.11. Light micrographs of conidiophores with different shape of conidia: clavate (Fig. 2.3.10); oblong (Fig. 2.3.11). Figs 2.3.12-2.3.13. Light micrographs of various conidia: ovoid to subglobose (Fig. 2.3.12) and oblong, ovoid, or peanut-shaped with a truncated end (Fig. 2.3.13). Scale bars: Figs 2.3.10-2.3.11 = 50 um; Fig. 2.3.12 = 10 um; Fig. 2.3.13 = 20 um. 62 urn. Cellulae conidiogenae discretae, apicem versus angustatae, 35-50 (medius = 39 ± 8) um longae. Evolutio conidii per aedificationem parietis supplementariae ontogenia holoblastica et proliferatione percurrenti. Conidia hyalina, 0-4 septata, clavata vel obclavata, 10-85 (medius = 28 ± 18) x 3-6 (medius = 4 ± 1.5). Conidia alia, parva: hyalina, ovoidea vel clavata 2.5-7 (medius = 5 ± 2.5) x 2-4 (medius = 3 + 1.5) um. Typus: Canada: British Columbia: Kamloops: Opax Mt., Pinus contorta var. latifolia, November 2001, S. Lee, SL-Kwl436 (DAOM 234192-holotypus). Growth. The optimal growth temperature for L. longiclavatum was 25°C with a growth rate of 7.3 mm d"1 on 2% O M E A . No growth was found below 5 °C or above 35 °C. On 2% O M E A amended with 0.05, 0.1, and 0.5 % cycloheximide, the growth rates at 25 °C were 7.3, 7.7 and 5.8 mm d"1, respectively. Cultures. On DMEA, colonies with sinuate edges, effuse, spreading, olive (1E4; Kornerup & Wanscher 1961). Hyphae, submerged in the medium, or aerial, smooth-walled, 2-5 um diam. Conidiophores single or in groups up to seven, mononematous, macronematous without rhizoids at their bases, sometimes produced on aerial hyphae. Stipes erect, brown, simple, 3-20 septate, 75-1202 (mean = 752 ± 329) um long, and 8-11 (mean = 10 ± 1.5) um wide at the base. Conidiogenous apparatus 40-451 (mean = 280 ± 137) um long excluding conidial mass; primary branch medium brown, two to three (mostly two), central branches slightly larger than the others, 25-45 (mean 34 + 4) x 2.5-3 um; secondary branches pale brown, 25-35 (27 ± 2) x 2.5-3 um; tertiary branches hyaline, 25-30 (26 ± 2) x 2.5-3 um; branches in 3-10 series, the ultimate branches being conidiogenous cells. Conidiogenous cells discrete, tapering distally, 35-50 (mean = 39 ± 8) um long. Conidium development in the manner of holoblastic ontogeny and percurrent proliferation with delayed secession giving a false impreosion of sympodial proliferation as described by Wingfield (1993). Conidia accumulating in light cream-coloured mucilaginous masses at the apices of the conidiophores, two types: large conidia, hyaline, 0-4 septate, clavate to obclavate, 10-85 (mean = 28 ± 18) x 3-6 (mean = 4 ± 1.5) um; smaller conidia, hyaline, aseptate, ovoid to clavate, 2.5-7 (mean = 5 ± 63 2.5) x 2-4 (mean = 3 ±1.5) urn, produced on smaller conidiophores on aerial mycelia, also occasionally produced on the large clavate conidia. Additional specimens examined: Canada: British Columbia: Williams Lake: D. ponderosae, August 2001, S. Lee, SL-W001 (DAOM 234191); Kamloops: Pinus contorta, October 2001, S. Lee, S L - K p l 1 (DAOM 234190); Princeton: P. contorta, July 2001, S. Lee, SL-Pw5 ( D A O M 234189). Phylogenetic analysis and PCR-RFLP marker L. longiclavatum was also recognized as a phylogenetic species (Taylor et al. 2000) based on concordance of multiple gene genealogies. Ribosomal DNA (ITS2 and 28S) and partial regions of three protein-coding genes (P-tubulin, actin and GPD) were analyzed. The sequences generated in this work and the reference sequences are listed in Table 2.3.1. In all parsimonious trees generated with the rDNA and protein-coding genes, L. longiclavatum isolates were consistently grouped in a single clade, which was clearly separated from the other species. The ITS2 and partial 28S regions of rDNA generated trees, in which the in-group taxa were separated into four clades: L. longiclavatum, O. clavigerum, L. terebrantis/L. pyrinum, and O. robustumlO. aureum (Fig. 2.3.14A). Clade resolutions were better in the protein-coding gene trees than in the rDNA tree. The aligned partial P-tubulin gene sequences contained four exons and three introns, while partial actin gene region included two exons and one intron. Although a strain (C187) of L. longiclavatum showed sequence variations in these two genes, all isolates of L. longiclavatum formed a single clade, which was resolved to the base of O. clavigerum, L. terebrantis and O. robustum clades (Figs 2.3.14B-C). The tree of the partial GPD gene which contains one intron and one exon, showed that the five species emerging from a polytomy included O. clavigerum, L. terebrantis, O. robustum, L. pyrinum, and L. longiclavatum (Fig. 2.3.14D). Regardless of the differences in the branch resolutions between gene trees, the separation of L. longiclavatum was consistent. To obtain more robust phylogenetic information, the combined sequences of rDNA, P-tubulin, actin, and GPD genes were analyzed. A total of 2892 characters were aligned, in 64 (A) r D N A 75 _6i_ 62 ATCC 18086 C843 SL-K.W1407 SL-Wg602 SL-St.Jl 1 UAMH9722 C418 AU98Pr2-l55 CMW3889 DLS879 _6J_ 59 SL-Pw5 SL-Kpll SL-W001 SL-Kwl436 C187 CMW668 CMW2805 ATCC 16936 AU98Pr2-169 UAMH4997 O. clavigerum J L. terebrantis | L. pyrinum L. longiclavatum | O. robustum | O. aureum UAMH4825 •UAMH9584 L. lundbergii I O. huntii (B) B-tubulin ATCC 18086 SL-Kwl407 r l l j - C843 " SL-St.Jll SL-Wg602 AU98Pr2-155 C418 UAMH9722 CMW2805 621 82 CMW668 SL-Pw5 SL-W001 ^ S L - K p l l SL-Kwl436 — C187 DLS879 1 CMW3889 99 j ATCC16936 99 99 ' 0.5 changes ' 5 changes AU98Pr2-169 UAMH4997 UAMH4825 UAMH9584 L. lundbergii O. clavigerum L. terebrantis O. robustum L. longiclavatum | L. pyrinum | O. aureum | O. huntii (C) Act in 92 100 76 SL-Kwl407 SL-St.Jll SL-Wg602 LSZ] ATCC 18086 1 C843 C418 J M UAMH9722 r ' 691 AU98Pr2-155 |_| CMW668 7? CMW2805 SL-Kwl436 6^ J SL-Kpll SL-Pw5 SL-W001 C187 I DLS879 CMW3889 U5J 98 O. clavigerum L. terebrantis | O. robustum L. longiclavatum _97_r- ATCC 16936 AU98Pr2-169 J UAMH4997 UAMH4825 UAMH9584 L. lundbergii I L. pyrinum | O. aureum | O. huntii 1 change (D) G P D 100 L//-" C843 " ATCC 18086 SL-Kwl407 SL-St.Jll SL-Wg602 C418 UAMH9722 £J T" CMW668 CMW2805 95 I 86 ~ I I SL-Kwl436 SL-Kpll • SL-Pw5 SL-W001 C187 CMW3889 DLS879 O. clavigerum | L. terebrantis O. robustum L. longiclavatum 95 | L. pyrinum AU98Pr2-169 1 ATCC 16936 UAMH4825 UAMH4997 |0. aureum O. huntii -UAMH9584 L. lundbergii "5 changes Fig. 2.3.14. Phylogenetic dendrograms of the four genes showing distinct clades of L. longiclavatum. One of the most parsimonious trees for each of the four gene datasets using Leptographium lundbergii and Ophiostoma huntii as outgroups were shown. Numbers above branches are bootstrap values with 1000 replications and shown when greater than 50%. (A) ITS2 and partial 28S region of rDNA. Among total 714 aligned characters, 699 characters were constant, 10 were variable and 5 were parsimony-informative 65 characters. Three most parsimonious trees were generated with tree length 15, consistency index (CI) 1.0, Homoplasy index (HI) 1.0, retention index (RI) 1.0. (B) P-tubulin. 679 out of 794 aligned characters were constant and 64 were parsimony-informative characters. One most parsimonious tree with a tree length 139, CI 0.9281, HI 0.0719 and RI 0.9206 was generated. (C) Actin. 758 out of a total 841 characters were constant and 56 parsimony-informative. The number of most parsimonious trees was four and they had tree length 97, CI 0.9072, HI 0.0928 and RI 0.9167. (D) GPD. 543 total characters with 461 constant characters and 47 parsimony-informative characters produced five most parsimonious trees, which had tree length 99, CI 0.9293, HI 0.0707 and RI 0.9205. 66 which 2597 characters were constant and 172 were parsimony-informative, and two most parsimonious trees were generated. Both trees showed basically the same topology, in which a distinct clade of L. longiclavatum was basal to of O. clavigerum, L. terebrantis and O. robustum clades. The clades of I. longiclavatum and O. clavigerum were strongly supported with high bootstrap values, 100% and 99%, respectively (Fig. 2.3.15). In previous work, we have reported a PCR-RFLP marker based on the P-tubulin that can differentiate O. clavigerum from other morphologically similar species (Lee et al. 2003). In this work, the marker also distinguished L. longiclavatum from the most morphologically similar species, O. clavigerum (Fig. 2.3.16). The digested fragments of the P-tubulin gene amplicon (838 bp) of L. longiclavatum were 51, 57, and 730 bp. In contrast, the fragments from the 836 bp amplicon of O. clavigerum were 51, 57, 238, and 490 bp. Discussion Leptographium longiclavatum sp. nov. was isolated from D. ponderosae, the bark and the stained sapwood of infested lodgepole pines at several sites in British Columbia. This fungus was more commonly isolated when the trees were not too dry. Its frequency on the beetle body surface was relatively low (unpublished data). We also found this fungus in the mycangia of D. ponderosae collected in the United States; the isolates (TF20-2, FTV7, HR1141) were provided by Dr. Diana Six (University of Montana, USA). It appears that L. longiclavatum is associated with D. ponderosae as specifically as O. clavigerum and O. montium (Whitney and Farris 1970). The most distinct morphological characteristic of L. longiclavatum is its clavate conidia. Among species with the Leptographium anamorph, only O. clavigerum was known to have septated clavate conidia, so this conidium shape has been used as a fast diagnostic key for O. clavigerum. Given this, L. longiclavatum could have been easily misidentified as O. clavigerum; this was the case for C187 which was reported as O. clavigerum before this study. Although clavate conidia are common features of both L. longiclavatum and O. clavigerum, O. clavigerum also produces other shapes of conidia that vary from 67 100 5 changes mr ATCC 18086 52 591 33. PL 6 7 i r C843 SL-Kwl407 SL-StJl l SL-Wg602 UAMH9722 5 9 L C 4 1 8 100 581 65. 96 CMW668 CMW2805 SL-Kwl436 Vr 100 SL-Kpll SL-Pw5 SL-W001 C187 I DLS879 100 CMW3889 ATCC16936 AU98Pr2-169 UAMH4825 - UAMH4997 UAMH9584 O. clavigerum L. terebrantis O. robustum L. longiclavatum L. pyrinum O. aureum O. huntii | L. lundbergii Fig. 2.3.15. One of the most parsimonious tree based on the combined datasets of four loci (rDNA, B-tubulin, actin, and GPD) showing a distinct clade of L. longiclavatum. The tree was rooted with Leptographium lundbergii and Ophiostoma huntii as outgroups. Bootstrap values from 1000 replication were indicated above the branches when greater than 50%. Among total 2892 aligned characters, 2597 characters were constant and 172 were parsimony-informative characters. Two most parsimonious trees (tree length 352, CI 0.9205, HI 0.0795 and RI 0.9157) were generated. 68 M1M2 1 5 6 7 8 750 bp 500 bp 250 bp Fig. 2.3.16. Differentiation of L. longiclavatum from O. clavigerum by PCR-RFLP. The partial p-tubulin gene was digested with Hinfl. Lanes: M l , lkb DNA marker; M2, 100 bp DNA marker; 1, Leptographium longiclavatum (SL-Kwl436); 2, L. longiclavatum (SL-W001); 3, L. longiclavatum (SL-Pw5); 4, L. longiclavatum (C187); 5, Ophiostoma clavigerum (ATCC 18086); 6, O. clavigerum (C843); 7, O. clavigerum (SL-Wg602); and 8, O. clavigerum (SL-Kwl407). 69 oblong to peanut-shaped with a truncated end, ovoid, and subglobose (Lee et al. 2003, Robinson-Jeffrey and Davidson 1968, Tsuneda and Hiratsuka 1984). Non-clavate conidia are more abundant than clavate conidia in O. clavigerum (Lee et al. 2003). The conidiophores of L. longiclavatum are considerably longer than those of O. clavigerum and any other Leptographium spp. (Jacobs and Wingfield 2001). Furthermore, the teleomorphs and synnematous anamorphs observed in O. clavigerum (Upadhyay 1981) were not found in L. longiclavatum. In addition to these microscopic characteristics, L. longiclavatum differs from O. clavigerum in its cultural appearances. The colony color of L. longiclavatum is olive (1E4; Kornerup and Wanscher 1961), while that of O. clavigerum is olive brown (4E4; Kornerup and Wanscher 1961) on 2% D M E A at 7 d. The conidiophores of L. longiclavatum are more abundantly found at the center of the colony, while those of O. clavigerum are spread throughout the colony. L. longiclavatum appears fluffier, especially at the center, and become pigmented earlier than O. clavigerum. The sinuate edge of L. longiclavatum is distinct from the smooth edge of O. clavigerum. These two species are also differentiated physiologically. The optimal growth rate of L. longiclavatum is approximately half that of O. clavigerum (14.3 mm d"1) on 2% O M E A at 25 °C. Furthermore, in contrast to L. longiclavatum, O. clavigerum shows a high degree of plasticity losing complex structures of anamorphs after continuous subculturing (Tsuneda & Hiratsuka 1984). The phylogenetic analysis with rDNA, P-tubulin, actin, and GPD gene sequences also strongly supported the separation of these two fungal species. In all the phylogenetic trees, L. longiclavatum and O. clavigerum consistently formed distinct clades. In the highly resolved combined tree, L. longiclavatum was placed at the base of the O. robustum, L. terebrantis, and O. clavigerum clades. Al l these phylogenetically close species have a common anamorph: Leptographium. However, the shape and the size of their conidia, and the length of their conidiophores differed (Table2.3.2). In O. robustum and L. terebrantis, conidia are obovoid and oblong, with truncated ends and round apices, like those of another related species, O. aureum. None of these species produce long clavate conidia, which are typical of L. longiclavatum and sometimes of O. clavigerum. Furthermore, L. longiclavatum conidiophores are longer than those of the other species, particularly those of O. robustum, L. pyrinum and L. terebrantis. Four of 70 Table 2.3.2. Morphological comparisons ofZ. longiclavatum and phylogenetically closely related species. L. longiclavatum L. pyrinum" L. terebrantis* r^i a b c O. aureum O. clavigerumb,c O. robustum*bQ Region North America U. S. A. North America North America North America North America Host Pinus contorta, P. ponderosa, P. ponderosa, P. contorta, P. contorta, P. contorta, P. monticola, P. ponderosa P. ponderosa Pjeffreyi P. sylvestris, P. taeda, P. banksiana, P. resinosa, P. edulis, P. strobes P. ponderosa, P. edulis P. ponderosa Insect D. ponderosae D. adjunctus D. frontalis, D. terebrans, D. ponderosae, D. ponderosae Dendroctonus association D. valens, Hylobius radicis, H. rhizophagus Dendroctonus sp., H. porosus sp. Conidiophore 110-1650 nm 118-393 nm 143-509 um 100-1350 um mononematous: 100-300 um 31-116 um length synnematous: 500-1150 urn Conidium L: clavate to obclavate oblong (pear-shaped) obovoid with truncate ends oblong with truncate L: clavate to obclavate oblong with shape S: ovoid to clavate and round apices ends and round apices S: cylindrical to clavate or truncate ends (two types: L, ellipsoidal and round apices large; S, small) Conidium size L: 10-85 x 3.0-6.0um 5.0-12 x 4.0-6.0 um 4.0-10 x 2.0-3.0 um 5.5-12.5x2.0-4.0um L: 12.5-85x2.0-6.0 um 3.0-7.0x2.0-6.0 (Iengthxwidth) S: 2.5-7.0 x 2.0-4.0um S: 2.0-4.0 x 1.0-2.5 um um Teleomorph absent absent absent Ophiostoma Ophiostoma Ophiostoma Rhizoids absent present absent absent present absent Optimum 25 °C 25 °C 25 °C 20 °C 25 °C 25 °C temp. for growth Jacobs and Wingfield (2001);b Robinson-Jeffrey and Davidson (1968); and 0 Upadhyay (1981). the species have common hosts; L. longiclavatum, O. clavigerum (Robinson-Jeffrey and Davidson 1968), and L. terebrantis (Hausner, Reid and Klassen 2000, Harrington 1988) have been found on both lodgepole pine and ponderosa pine {Pinusponderosa) infested with D. ponderosae, while O. robustum (Robinson-Jeffrey and Davidson 1968) has been only isolated from ponderosa pine. In conclusion, L. longiclavatum can be readily distinguished from the most similar Leptographium anamorph of O. clavigerum by its longer conidiophores, homogenous spore shapes, slower growth rate, p-tubulin RFLP profdes, and multigene phylogenies. Given such evidences, we describe L. longiclavatum as a new taxon. In order to understand the role of L. longiclavatum in the D. ponderosae-vectored infestation process, we are currently investigating its pathogenicity on mature lodgepole pines. 72 References Council of Forest Industries. 2004. Mountain pine beetle task force. Epidemic expansion facts. Available from http://mountainpinebeetle.com/epidemic_facts.html [cited 8. Nov. 2004]. Felsenstein, J. 1985. Confidence limits on phylogenies: an approach using the bootstrap. Evolution. 39: 783-791. Harrington, T.C. 1988. Leptographim species, their distributions, hosts and insect vectors. In Leptographim root disease on conifers (T.C. Harrington & F.W. Cobb Jr., eds). 1-39. The American Phytopathological Society Press, St. Paul, Minnesota. Harrington, T.C. 1993. Biology and taxonomy of fungi associated with bark beetles. In Beetle- pathogen interactions in conifer forests (T.D. Schowalter & G.M. Filip, eds). 37-51. Academic Press, San Diego. Harrington T . C , and Cobb, F.W. Jr. 1983. Pathogenicity of Leptographium and Verticicladiella spp. isolated from roots of western North American conifers. Phytopathology. 73 (4): 596-599. Hausner, G., Reid, J., and Klassen., G.R. 2000. On the phylogeny of members of Ceratocystis s.s. and Ophiostoma that possess different anamorphic states, with emphasis on the anamorph genus Leptographium, based on partial ribosomal DNA sequences. Can. J. Bot. 78: 903-916. Higgins D.G., Bleasby A.J. , and Fuchs, R. 1991. C L U S T A L W: improved software for multiple sequence alignment. CABIOS. 8: 189-191. Jacobs, K. and Wingfield, M.J. 2001. Leptographium: Tree pathogens, Insect associates, and agent of blue-stain. 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Association of Dendroctonus ponderosae (Coleoptera: Scolytidae) with blue stain fungi and yeasts during brood development in lodgepole pine. Can. Entomol. 103 (11): 1495-1503. Whitney, H.S. & Farris, S.H. 1970. Maxillary mycangium in the mountain pine beetle. Science. 167: 54-55. Wingfield, M.J. 1993. Leptographium species as anamorphs of Ophiostoma: progress in establishing acceptable generic and species concepts. In Ceratocystis and Ophiostoma: taxonomy, ecology, and pathogenicity (M. J. Wingfield, K. A. Seifert, & J. F. Webber, eds): 315-322. American Phytopathological Society Press, St. Paul, Minnesota. Wingfield, M.J., Capretti, P., and Mackenzie, M . 1988. Leptographium spp. as root pathogens of conifers. An International perspective. In Leptographim root disease on conifers (T.C. Harrington & F.W. Cobb Jr., eds): 113-128. The American Phytopathological Society Press, St. Paul, Minnesota. Wingfield, M.J. & Gibbs, J.N. 1991. Leptographium and Graphium species associated with pine-infesting bark beetles in England. Mycol. Res. 95 (11): 1257-1260. Wingfield M.J., Seifert. K.A. , and Webber J.F. 1993. Ceratocystis and Ophiostoma: taxonomy, ecology, and pathogenicity. American Phytopathological Society Press, St. Paul, Minnesota. 74 C h a p t e r 3 Diversity of fungi associated with the mountain pine beetle, Dendroctonus ponderosae, and infested lodgepole pines in British Columbia Introduction Lodgepole pine (Pinus contorta var. latifolia) forests represent 25% of the total growing stock in British Columbia (BC) (COFI, 2005). However, large number of mature lodgepole pine have been killed by the mountain pine beetle (MPB), Dendroctonus ponderosae Hopkins, and its fungal associates. Although outbreaks of the MPB have occurred in BC in the past, the present infestation is the most severe infestation ever recorded. The current MPB epidemic started near Tweedsmuir Park and spread to northern B C (Houston and Prince George) and the Alberta border. As of 2004, it has spread over 7 million ha, infesting 283 million m 3 of lodgepole pine (BC Ministry of Forests 2005). Usually, the MPB attacks its host in July and August. At this time, trees are often stressed by water deficiency. The MPB feeds on fungi and tree tissues (Harrington, 2005). Newly eclosed adults graze on fungi and acquire fungal spores in their mycangia and guts and on their exoskeletons before they emerge from the pupal chambers and attack new hosts. It appears that the MPB and its fungal associates have a mutually beneficial relationship (Whitney, 1982; Paine et al., 1997; Six 2003a; Harrington, 2005). The known fungal associates of the MPB are ascomycetes in the genera Ophiostoma and Leptographium. They produce asexual and sexual spores in slimy masses that attach to insect bodies and are dispersed to new hosts that represent fresh nutrient sources (Harrington, 1993). Two fungal species, Ophiostoma montium and Ophiostoma clavigerum have been consistently isolated from the mycangia and exoskeletons of the MPB as well as from infested pines (Robinson, 1962; Whitney and Farris, 1970; A versioin of this chapter has been accepted. Fungal diversity associated with the mountain pine beetle, D. ponderosae and infested lodgepole pines in British Columbia. S. Lee, J.-J. Kim, and C. Breuil. 2006. Fungal diversity. In press. 75 Solheim, 1995; Six, 2003b; Kim et al., 2005). Recently, Leptographium longiclavatum associated with MPB has also been reported (Lee et al. 2005). In comparison, Entomocorticium dendroctoni, Ophiostoma minutum and Ophiostoma minus have been occasionally found in MPB galleries and their association with the MPB has been suggested (Robinson 1962; Whitney et al. 1987). However,, except for Robinson (1962), most of the previous work was carried out with a limited number of isolation sites, and fungi were isolated only from the beetle or infested trees. Recent outbreaks have occurred over a wide range in BC, in areas which have not been affected by MPB epidemics in the past century and which have different climates from the ones previously studied. Therefore, the mycoflora in the current epidemic area in BC might be different from what has been reported. To obtain accurate information on the fungal diversity involved in the current MPB outbreak, we investigated the fungi collected at six sites in BC. We examined, first, which fungal species were consistently associated with the MPB across the large epidemic area in BC, and second, whether the fungal species and their frequencies as isolated from the beetles, beetle galleries and infested trees were different or not. Materials and methods Sampling strategies Six epidemic sites (Fort St. James, Houston, Kamloops, Princeton, Tweedsmuir Park, and Williams Lake) were selected to cover the current outbreak regions in Canada (Fig. 3.1). Samplings were conducted before the emergence of teneral adult beetles from the hosts. A total of 23 trees (3-5 trees/site), which were attacked by the MPB in the same or previous summer of the sampling year (early green and late green phase of trees, respectively), were harvested in 2001 and 2002 (Table 3.1). One bolt (50-80 cm in length) from each tree was cut at breast height and placed in a plastic bag in the field and then transported to the laboratory. The bolts were kept at 4 °C for 1-3 days before fungal isolations were conducted. 76 77 Table 3.1. Characteristics of the MPB-infested trees used for samplings. Site No. of trees Location (latitude, longitude) Date sampled Tree phase* Kamloops 2 N50° 65', W 120° 36' June 6, 2001 Late green 1 N 50° 45', W 120° 31' Aug 16, 2001 Early green 1 N50° 45', W 120° 31' Aug 31, 2001 Early green 1 N 50° 45', W 120° 31' May 20, 2002 Late green Williams Lake 3 N 52° 26'21", W 122° 03' 15" July 9, 2001 Late green Princeton 3 N 49° 14' 58", W 120° 34' 43" July 13,2001 Late green Tweedsmuir Park 4 N52° 43'41", W 125° 30' 23" July 19, 2001 Late green Houston 4 N 54° 08', W 126° 40' July 17, 2002 Late green Fort St. James 4 N54°38' 66", W 124° 25' 14" August 8, 2002 Late green 'Late green' indicates that trees were attacked in the previous summer and 'early green' means that the trees were attacked in the same summer of the sampling year (Kim et al. 2005). The trees harvested ranged in age from 75 to 140 years, with diameters varying from 20.5-35 cm. Most of the trees were heavily attacked with 70-160 pitch tubes/m2, and we often observed that the MPB larval galleries of adjacent broods overlapped at their ends. Generally, the entire sapwood was stained and secondary beetles such as Ips and ambrosia were commonly found in most of the trees. Isolation offungi from MPB, galleries and sapwood Bolts were debarked, and beetles were collected from galleries that were not intermingled with other galleries. Fungi on body surfaces of larvae, pupae, and adults were isolated using serial dilution with 0.01% (v/v) Tween-20, and were cultured on 2% malt extract agar (MEA) with ampicillin as described by Lee et al. (2005). Fungi were sampled from the same galleries from which the beetles were collected, by aseptically transferring small amounts of frass onto 2% M E A with ampicillin. After 3-30 days of incubation at room temperature, either hyphal tips or the conidial mass on top of conidiophores were sub-cultured and grown on 2% M E A for identification. Fungi were isolated from sapwood as described by Kim et al. (2005). Fungal mycelia removed from the edge of a young colony were placed in micro-tubes (Sarstedt, Montreal, Quebec) with either 1 mL of water for storage at 4 °C, or 1 mL of 20% (v/v) glycerol (Sigma-Aldrich, Oakville, ON) solution for storage at -80 °C. Fungal identification The filamentous fungal isolates were identified by the morphology of asexual structures (Rumbold, 1941; Batra, 1967; Robinson-Jeffrey and Davidson, 1968; Upadhyay, 1981; Jacobs and Wingfield, 2001). Identification was confirmed by sequencing the ribosomal DNA (rDNA) or B-tubulin gene of representative isolates (Table 3.2). Fungal D N A extraction and PCR were conducted following Lee et al. (2003), and sequencing was carried out as described by Lim et al. (2005). The identification of O. clavigerum was confirmed using an O. clavigerum-specific PCR-RFLP marker (Lee et al., 2003). Fungal growth rates were measured at 23 °C on 2% M E A as described by Lee et al. (2005). 79 Table 3.2. GenBank accession numbers and growth rates of the fungi isolated from D. ponderosae. Taxon Strain Site* Primer used for amplification^ Accession no.* Closest match in BLAST Accession of match Identity§ % Growth rate mm/day Entomocorticium dendroctoni Whitney SL-A69 TP ITS5/ITS4 DQ118419 E. dendroctoni AF 119506 99.6 0.7 ±0.2 SL-P44 P ITS5/ITS4 DQ118418 E. dendroctoni AF1 19506 99.6 0.5 ±0.2 Entomocorticium sp. SL-A3 TP ITS5/ITS4 DQ118416 Entomocorticium sp. H AF119512 99.1 3.6 ±0.3 SL-W002 WL ITS5/ITS4 DQ118417 Entomocorticium sp. H AF119512 99.1 3.5 ±0.2 Leptographium longiclavatum Lee et al. SL-K215 K T10/BT12 AY288931 -- ~ - 6.3 ±0.9 SL-W001 WL T10/BT12 AY288936 - - - 7.1 ±0.8 Leptographium terebrantis Barras & Perry SL-A57 TP T10/BT12 DQ118421 L. terebrantis AY263192 100 12.8 ±0.4 Ophiostoma clavigerum (Robins-Jeff. & Davids.) Harrington SL-K1 K T10/BT12 AY263210 O. clavigerum AY263194 100 14.3± 0.7 Ophiostoma minutum-\ike sp. SL-K70 K ITS5/ITS4 DQ128175 0. minutum DQ128173 93.1 1.5 ±0.3 SL-W15 WL ITS5/ITS4 DQ128174 0. minutum DQ128173 92.9 1.9 ±0.3 Ophiostoma montium (Rumbold) von Arx SL-K77 K ITS1-F/ITS4 AY 194942 0. montium AY194941 100 6.7 ±0.3 Ophiostoma nigrocarpum-Mke. sp. SL-A54 TP ITS5/ITS4 DQ118420 0. nigrocarpum-Wke AF484452 99.8 1.6 ±0.2 Pichia capsulate (Wick.) Kurtzman SL-WY2 WL LR0R/LR3 DQ128167 P. capsulata U70178 99.8 -Pichia holstii (Wick.) Kurtzman SL-W2Y4 WL LR0R/LR3 DQ128171 P. holstii U75722 99.2 -Pichia scolyti (Phaff & Yoney.) Kreger SL-W2Y3 WL LR0R/LR3 DQ128172 P. scolyti U45788 99.8 Taxon Strain Site* Primer used for Accession no.{ Closest match in BLAST Accession of Identity§ Growth rate amplification* match % mm/day SL-PY1 P LR0R/LR3 DQ128170 P. scolyti U45788 99.8 Unidentified yeast SL-WY4 WL LR0R/LR3 DQ128168 P. ofunaensis U45829 98.3 SL-W2Y1 WL LR0R/LR3 DQ128169 P. ofunaensis U45829 96.8 K: Kamloops, P: Princeton, TP: Tweedsmuir Park, WL: Williams Lake f ITS1-F (Gardes and Bruns, 1993), ITS4 and ITS5 (White etal, 1990), LROR and LR3 (Vilgalys and Hester, 1990), T10 and BT12 (Lee et al, 2003). * Accession numbers in bold were sequenced during this work. § Identity (%) was derived from the paiwise alignment of each isolate sequence with the closest BLAST match in GenBank or a reference strain (DQ 128173, O. minutum CBS 145.59; AF484452, O. nigrocarpum-like C314). oo Statistical analyses As in our previous work (Kim et al, 2005), the Simpson diversity index (Simpson, 1949) was used to indicate fungal diversity because sample sizes in this study were relatively small (Mouillot and Lepretre, 1999). The index is defined as c = i - f>, 2 , ;'=1 where Pt is the relative abundance of a species /, and S is the species richness, which is defined as the number of competing species present in the community. Fungal dominance was determined by Camargo's index (MS) (Camargo, 1992), where S represents species richness. A species was defined as dominant if/>,-> MS. Results A total of 1042 fungal isolates were obtained from MBP adults, pupae and larvae, galleries, and sapwood from 23 lodgepole pines. Nine fungal species were isolated. These included O. montium, O. clavigerum, an O. minutum-\ike species, an O. nigrocarpum-Wke, species, L. longiclavatum, L. terebrantis, E. dendroctoni, and two unknown fungi: an Entomocorticium species and an Ambrosiella species. Fungal diversity on the MPB From the exoskeletons of MPB adults we obtained 516 fungal isolates comprising eight species (Table 3.3). The most dominant species was O. montium, whose average isolation frequency was 68% (from 44% at Fort St. James to 92% at Williams Lake) of the total number of fungal isolates. In total, 85% of the beetles (from 67% at Tweedsmuir Park to 100% at Princeton and Williams Lake) yielded O. montium. Unexpectedly, the second-most dominant isolate was the O. minutum-\\ke species, which was isolated from 32% of the beetles with an average isolation frequency of 16% (from 0% at Princeton to 38% at Fort St. James). O. clavigerum was also commonly isolated. However, it was isolated from fewer beetles (24%) than the O. minutum-\ike species, and its average frequency was lower (9%). In contrast to 82 Table 3.3. Number of fungal isolates from D. ponderosae and number of beetles yielding each fungal species at six sites in British Columbia. Number of Isolates Taxon Fort St. James Houston Kamloops Princeton Tweedsmuir Park Williams Lake Total isolates Ambrosiella sp. -- 3 (2) -- -- -- -- 3 (2) Entomocorticium dendroctoni Whitney -- -- -- 17 (2) 3 (1) -- 20 (3) Entomocorticium sp. - -- 1 (1) 2 (2) 8 (2) -- 11 (5) Leptographium longiclavatum Lee et al. -- -- 2 (1) -- -- - 2 (1) Leptographium terebrantis Barras & Perry -- -- -- -- 1 (1) -- 1 (1) Ophiostoma clavigerum (Robins-Jeff. & Davids.) 11 (3) 20 (6) 9 (3) -- 2 (2) 2 (2) 44 (16) Harrington Ophiostoma minutum-hke sp. 23a (6) 25 (5) 29a (7) - 2 (2) 5 (2) 84a (22) Ophiostoma montium (Rumbold) von Arx 27a (5) 57a (11) 91" (16) 77a (10) 24 a (4)T 75a (12) 35la (58) No. of total fungal isolates 61 105 132 96 40 82 516 No. of total MPB 7 13 20 10 6 12 68 Species richness (S) 3 4 5 3 6 3 8 Camargo's index (1/5) 0.33 0.25 0.20 0.33 0.17 0.33 0.13 Simpson's index of diversity (C) 0.63 0.58 0.47 0.33 0.59 0.16 0.50 The total number of fungi on the beetle = the number of fungal isolates written in the table x 104. ••"Values in parentheses are the number of MPB yielding each fungal species. a Dominant species. Species was considered as dominant if Pi>l/S, where Pi is the relative abundance of a species and S is the species richness, which is the number of competing species present in the community (Camargo, 1992). the O. minutum-Uke species, which was dominant at two sites, O. clavigerum was not dominant at any site. When the data were pooled, the dominant species on the MPB exoskeletons were O. montium and the O. minutum-Yike species. Entomocorticium sp. and E. dendroctoni were often isolated, while L. longiclavatum, Ambrosiella sp., and L. terebrantis were found occasionally. At the sites where larvae, pupae and adults were found together (Kamloops, Tweedsmuir Park, and Williams Lake), the fungi obtained at each developmental stage were similar (Table 3.4). Often, more than one fdamentous fungal species was isolated from one beetle. Many beetles yielded two species (Tweedsmuir Park 83%, Houston 39%, Kamloops 29%, and Williams Lake 25% of beetles), or even three (Houston 15%, Princeton 10%, and Williams Lake 1% of beetles). Yeasts were present in higher number than fdamentous fungi. At Princeton, Williams Lake, and Kamloops, the average number of yeast colonies per adult beetle was approximately 3 x 105, 5 x 105, and 7x 105, respectively. Yeasts were obtained at all beetle developmental stages, but were more abundant on eggs (data not shown). Similarly to the fdamentous fungi, more than one yeast species was isolated from most beetles. The 26S rDNA of the yeasts isolated in this study had high sequence identity (> 99.2 %) with those of Pichia capsulata, Pichia holstii, and Pichia scolyti (Table 3.2). Fungal diversity in the beetle galleries and the stained sapwood A total of 274 isolates were collected from galleries and sapwood (Table 3.5). 0. montium and O. clavigerum were frequently isolated from both galleries and sapwood. In contrast to the results from the beetle exoskeletons, the O. minutum-Wke. species was isolated at low frequency, while L. longiclavatum was often isolated. Other species, including Entomocorticium sp., Ambrosiella sp., and L. terebrantis, were occasionally isolated in the sapwood. However, E. dendroctoni was not found in sapwood. Yeasts were often found in galleries and occasionally in sapwood (data not shown). 84 Table 3.4. Number of fungal isolates from larvae, pupae and adults of D. ponderosae* Number of Isolates* Fungal species Kamloops Tweedsmuir Park Williams Lake Total Larvae Pupae Adults Larvae Pupae Adults Larvae Pupae Adults Larvae 3upae Adults Entomocorticium dendroctoni Whitney 2 - - - - 3 ~ - - 2 - 3 Entomocorticium sp. 2 - 1 9 9 8 - - - 11 9 9 Ophiostoma clavigerum (Robins-Jeff. &Davids.) Harrington 10 ~ 9 ~ 4 2 - - 2 10 4 13 Ophiostoma minutum-like sp. 8 4 29 3 18 2 - 3 5 11 25 36 Ophiostoma montium (Rumbold) von Arx 96 26 91 5 15 24 14 23 75 115 64 190 Ophiostoma nigrocarpum-\\ke sp. ~ - - - 1 - - - - 1 -Leptographium longiclavatum Lee et al. - ~ 2 - - - - ~ - - - 2 Leptographium terebrantis Barras & Perry - ~ - - - 1 - - - — — 1 No. of D. ponderosae No. of fungi 14 118 4 30 20 132 2 17 5 47 6 40 2 14 3 26 12 82 18 149 12 103 38 254 * The total number of fungi on the beetle = the number of fungal isolates written in the table x 104. Table 3.5. Number of fungal isolates from gallery and wood at six sites in British Columbia. Number of Isolates Fort. St. James Houston Kamloops Princeton Tweedsmuir Williams Total isolate Park Lake Fungal species G* W f G W G W G W G W G W G W Ambrosiella sp. -- -- -- -- -- -- -- 2 - -- - -- -- 2 Entomocorticium sp. -- -- -- 1 -- 1 -- - -- -- - - - 2 Leptographium longiclavatum Lee et al. 3 -- 1 -- 4 7 -- -- -- -- - - 8 7 Leptographium terebrantis Barras & Perry - -- -- -- -- 1 -- - -- -- -- 1 -- 2 Ophiostoma clavigerum (Robins-Jeff. & Davids.) Harrington 8 a 13a 5 a 2 27a 54a 2 6 a 7 a 3 4 8 a 53a 86a Ophiostoma minutum-like sp. 2 -- 1 -- - 1 -- -- -- -- -- 1 3 2 Ophiostoma montium (Rumbold) von Arx 7 a 4 4 a 5 a 25a 32a 4 a 5 a 1 14° 5 a 3 46a 63a No. of total fungal isolates 20 17 11 8 56 96 6 13 8 17 9 13 110 164 Species richness (5) 4 2 4 3 3 6 2 3 2 2 2 4 4 7 Camargo's index (1/5) 0.25 0.50 0.25 0.33 0.33 0.17 0.50 0.33 0.50 0.50 0.50 0.25 0.25 0.14 Simpson's index of diversity (C) 0.69 0.36 0.65 0.53 0.56 0.57 0.44 0.49 0.22 0.29 0.49 0.56 0.59 0.56 * gallery, Vood a Dominant species. Species was considered dominant if Pi>\/S, where Pi is the relative abundance of a species and 5 (Species richness) is the number of competing species present in the community (Camargo, 1992). Discussion Through an extensive survey, we isolated more diverse fungal associates of the MPB than previously reported. While O. montium and O. clavigerum were frequently isolated from the MPB in accordance with previous studies (Robinson 1962; Whitney and Farris 1970; Six 2003b), we also isolated an O. minutum-\ike species, L. longiclavatum, Entomocorticium sp., and E. dendroctoni. The isolation methods in each study could have affected the species found. In this work, the beetles were washed and the diluted washes were plated onto media. In previous research the beetles were either streaked onto or allowed to walk on the surface of the media; with such methods, it is less likely that all spores in the cavities of the beetle exoskeletons would be removed. Since sampling fungi in mycangia without cross contamination from fungi present on the exoskeletons was difficult, we only isolated fungi from beetle body surfaces. Unexpectedly, the frequency of the O. minutum-like species on the MPB exoskeletons was high; often this fungus was isolated more frequently than O. clavigerum. The O. minutum-\ike species appears to belong to a complex phylogenetic group that is often reported as Ophiostoma minutum Siem., or as Ceratocystiopsis minuta (Siem.) Upadhyay & Kendrick (Hausner et al., 1993, 2003). To our knowledge, O. minutum has been isolated occasionally from infested lodgepole pines (Robinson, 1962), but not from the MPB exoskeletons. It has also been isolated from phoretic mites carried by other bark beetles (Moser and Macias-Samano, 2000). We often observed mites in the MPB galleries, but further work needs to be done to determine whether MPB is associated with phoretic mites carrying the O. minutum-like species. Consistent with Robinson's (1962) data, we isolated O. montium more frequently than O. clavigerum from the beetle exoskeletons. This was also the case when O. clavigerum was more prevalent than O. montium in the galleries, even though, for such cases, the beetles had more opportunity to contact O. clavigerum than O. montium. The lower frequency of O. clavigerum on beetles could be due to its large clavate spores, which might not be able to adhere to the beetles as stably as the small conidia of O. montium. Leptographium longiclavatum was isolated from the MPB exoskeletons and infested sapwood. In previous work it has been found in MPB mycangia (Lee et al., 2005). This species appeared to be 87 affected by the moisture content of its environment, as it was isolated more often from early green phase trees than from the drier late green phase trees that had been infested in the previous summer (Kim et al, 2005). Like O. clavigerum, L. longiclavatum has long conidia, and may be more easily grazed by the beetles and carried preferentially in the mycangia rather than on the exoskeleton (Harrington and Zambino, 1990; Hsiau and Harrington, 1997; Six 2003b). In this work we isolated two basidiomycetes. E. dendroctoni was isolated from the MPB exoskeletons, although it has been only reported in beetle galleries (Whitney et al. 1987). The Entomocorticium sp. had a faster growth rate than E. dendroctoni, and its rDNA showed only 97.8 - 98.1% sequence identity to that of E. dendroctoni. Entomocorticium species have been suggested to be good nutritional sources for MPB and other Dendroctonus species (Barras and Perry 1972; Whitney and Cobb 1972; Whitney et al. 1987). In contrast to all the above fungal species, which appear to be specifically associated with MPB, L. terebrantis, Ambrosiella sp., and the O. nigrocarpumAike sp. seemed to be incidental associates. The presence of these fungi on MPB is likely due to cross-contamination with fungal associates of other cohabiting beetles (e.g. Ips and ambrosia beetles), which were frequently observed in sampled trees. At this point, the information available on fungal associates of such other beetles is limited. O. minus and O. huntii, which have been found in old MPB galleries (Robinson, 1962; Robinson-Jeffrey and Grinchenko, 1964), were not isolated in this work. The fungi that we isolated can be grouped into fast growing sapstaining fungi and slow growing non-staining fungi. It is likely that non-staining fungi, such as the O. minutum-\)ke species, Entomocorticium species and yeasts, found mainly on the beetle and rarely in sapwood, might be better nutritional sources for the MPB than staining fungi, which contain melanin, a phenolic derivative. Whitney er al. (1987) has shown that the sapstaining fungi O. montium and O. clavigerum were less efficient in supporting beetle reproduction than E. dendroctoni. Yeasts, commonly found on the MPB in current and previous studies (Whitney 1971, Lim et al. 2005), may also be an important nutritional source. In addition, they may contribute to successful brood development by preventing excess numbers of beetles in individual trees, 88 since some yeasts can convert the MPB aggregation pheromone /raws-verbenol into the anti-aggregation pheromone verbenone (Hunt and Borden, 1990). Similarly, pathogenic species O. clavigerum, O. montium and L. longiclavatum, by invading the sapwood and interrupting water transportation, decrease the moisture content in the tree and may provide a better environment for beetle brood development (Strobel and Sugawara, 1986; Yamaoka et al., 1995; unpublished data for L. longiclavatum, Webb and Franklin, 1978). In conclusion, in contrast to previous work, we showed that more diverse fungal species were associated with MPB. We also found that species dominant on MPB exoskeletons differed from those dominant in galleries and sapwood; with the difference mainly due to the O. minutum-\ike species. Comparison of fungal frequencies from different parts of MPB, such as the exoskeleton, mycangia and gut, would suggest which fungi might be more preferentially consumed by the beetle. Further work will be needed to clarify interactions among fungi and the impact of each fungal species on MPB fitness and on host defence mechanisms. 89 References Barras, S.J., and Perry T. 1972. Fungal symbionts in the prothoractic mycangium of Dendroctonus frontalis (Coleoptera: Scolytidae). Z. Ang. Entomol. 71: 95-104. Batra, L.R. 1967. Ambrosia fungi: a taxonomic revision, and nutritional studies of some species. Mycologia. 59: 976-1017. BC Ministry of Forests. 2005. British Columbia's mountain pine beetle action plan 2005-2010. Available from http://www.for.gov.bc.ca/hfp/mountain_pine_beetle/actionplan/2005/intro.htm [cited 14 July 2005]. Camargo, J.A. 1992. Can dominance influence stability in competitive interactions? Oikos. 64(3): 605-609. Council of Forest Industries. 2005. 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Pathol. 25: 401-404. 92 Chapter 4 Pathogenicity of Leptographium longiclavatum associated with Dendroctonus ponderosae to Pinus contorta Introduction By the fall of 2004, an epidemic of the mountain pine beetle, Dendroctonus ponderosae, had spread over 7 million hectares and infested 283 million m 3 of lodgepole pines in British Columbia, Canada (BC Ministry of Forests 2005). This represents the largest forest insect infestation in Canadian history. Tree mortality results from the damage caused by the beetle and its fungal associates; D. ponderosae feeds on the phloem and mechanically wounds it, while the fungi colonize the sapwood and disrupt the transportation of water to the tree crown (Paine et al. 1997; Reid 1961). The fungi associated with D. ponderosae have been investigated in several studies and two sapstaining species, Ophiostoma montium and Ophiostoma clavigerum, have been shown to be vectored by the beetle (Robinson 1962; Robinson-Jeffrey and Davidson 1968; Rumbold 1941; Six 2003; Solheim 1995; Whitney and Farris 1970). Recently, Leptographium longiclavatum, an Ophiostoma minutum-\\ke sp. and Entomocorticium spp. have also been isolated from the beetles as well as from the sapwood of infested lodgepole pines at several epidemic sites in BC (Kim et al. 2005; Lee et al. 2005b). L. longiclavatum makes a stain in sapwood and is morphologically very similar to O. clavigerum. Both fungi have very distinct long clavate spores and Leptographium anamorphs (Lee et al. 2005a). In contrast, the O. minutum-Wke species and Entomocorticium species do not appear to stain sapwood. They grow primarily in the beetle galleries, which are located just underneath the tree bark; occasionally they are found in the sapwood (Lee et al. 2005b). They grow very slowly compared to the three sapstaining species, which are major colonizers in A version of this chapter has been submitted. Pathogenicity of Leptographium longiclavatum associated with Dendroctonus ponderosae to Pinus contorta. S. Lee, J.-J. Kim, and C. Breuil. Apr. 2006. Can. J. For. Res. 93 the sapwood at the early beetle infestation stage (Kim et al. 2005; Lee et al. 2005b; Solheim 1995). Among the five fungal associates, only the pathogenicity of O. clavigerum and O. montium has been examined. O. clavigerum appears to be a more aggressive pathogen than O. montium. Reid et al. (1967) reported that lodgepole pine inoculated with O. clavigerum showed stronger defence reactions producing more extensive resinosis than the trees inoculated with O. montium. Although strong tree defence response does not always reflect fungal aggressiveness (Paine et al. 1997), similar defence reactions have been observed in both mature trees (Reid and Shrimpton 1971; Shrimpton 1973) and seedling lodgepole pines (Shrimpton and Watson 1971). Using a heat pulse velocity instrument, which can indicate xylem translocation, Yamaoka et al. (1990) showed that O. clavigerum colonized the sapwood of lodgepole pine, and disrupted sap flow within a short period after fungal inoculation. The pathogenicity of O. clavigerum inoculated into mature lodgepole pine was re-examined by Yamaoka et al. (1995); they observed a change in the colour of the tree foliage from green to yellow one year after inoculation. Compared to O. clavigerum, the pathogenicity of O. montium is variable in literature. The degree of its pathogenicity reported in previous studies ranges from weak to strong (Mathre 1964; Strobel and Sugawara 1986; Yamaoka et al. 1995). This inconsistency appears to have resulted from the different inoculation methods employed in each study, as well as the vigour and age of the trees (Raffa and Berryman 1982; Shrimpton 1973). Some of the inoculation methods involved extensive girdling of the phloem; for example, cutting several flaps of bark (each in 10-12x4-6 cm) down to the cambium around the circumference of the trees to insert fungal inoculum (Strobel and Sugawara 1986). In contrast, the cork borer technique that was used in our work has been more widely employed for sapstaining fungal pathogens (Christiansen 1985a; Christiansen and Solheim 1990; Fernandez et al. 2004; Solheim and Safranyik 1997; Solheim et al. 1993; Wright 1933; Yamaoka et al. 2000). The aim of this study was to assess the pathogenicity of the recently discovered mountain pine beetle associate, L. longiclavatum, when inoculated into mature lodgepole pine. Indicators of pathogenicity such as lesion size, sapwood occlusion and moisture contents, and changes in the foliage colour were observed as well as the ability of L. longiclavatum to grow under low oxygen concentrations. The aggressiveness 94 of L. longiclavatum was compared in all these tests with the well known mountain pine beetle associate, O. clavigerum. Materials and methods Inoculation of lodgepole pines Inoculations were conducted on mature lodgepole pines (Pinus contorta) at Kamloops (N50 31' 11", 120 31' 33"), BC, on September 29, 30 and October 1, 2003. The inocula were agar plugs colonized by either L. longiclavatum SL-Kwl436 (DAOM234192) or O. clavigerum SL-Kwl407 (DAOM234193), as well as sterile agar plugs (the control). The fungal isolates SL-Kwl436 and SL-Kwl407, which had been obtained from D. ponderosae-'mfested pines at Kamloops in 2001, were grown on 2% M E A (33g malt extract agar, Oxoid, Nepean, ON; lOg agar Oxoid; IL dE^O) for 7d. The host trees were naturally regenerated trees with a mean D B H (diameter at breast height) of 18.8 (14.5-26) cm and an average age of 116 (98-130) years at D B H (Table 4.1). They were healthy with no apparent disease or insect attack before the inoculation. Thirty pines were randomly assigned to the following treatments: 200 or 800 inoculation points/m2 of each fungal species (6 tree replicates for 200 points/m2 and 6 trees for 800 points/m2) and agar plugs (3 trees for each inoculation density). Agar plugs were included as controls to assess the impact of mechanical damage caused during the inoculation process. Inoculation densities were selected based on previous pathogenicity tests with similar sapstain fungi (Christiansen 1985b; Fernandez et al. 2004; Ross and Solheim 1997; Solheim and Safranyik 1997). Bark plugs were removed from the outer bark down to the cambium using a metal cork borer of 5 mm diameter (Fisher Scientific, Ottawa, ON) in a 60 cm-wide band at breast height on each tree (Fig. 4.1A-1). The inoculation holes were made in an even distribution within the band. The distance between the inoculation points was approximately 3.5 cm at high density and 7.1 cm at low density. Fungal inoculums consisting of 5 mm plugs were inserted into the holes (Fig. 4.1A-2), the bark pieces were replaced to close holes (Fig. 4.1A-3), and then the entire inoculating area was wrapped with duct tape (Fig. 4.IB). 95 Table 4.1. Characteristics of the lodgepole pines (Pinus contorta var. latifolia) inoculated with L. longiclavatum, O. clavigerum or agar control. Treatment Inoculation density No. of trees inoculated Tree age (years) Tree diameter (cm) Tree crown colour^ Sapwood moisture content (%) Agar 200/m2 3 117 ± 2.5 18.1 ± 1.6 3G 109.4 ± 17.4 800/m2 3 114 ±9.2 18.3 ±0.4 3G 98.7 ±5.7 L. longiclavatum 200/m2 6 122 ±5.9 20.7 ±2.8 6G 89.9 ±7.7 800/m2 6 110 ± 8.5 18.1 ± 1.9 5Y, 1G 60.7 ±5.1 0. clavigerum 200/m2 6 119 ±6.3 19.8 ± 1.3 6G 62.8 ±7.8 800/m2 6 115 ±9.7 17.2 ± 1.7 4Y, 1YG, 1G 38.8 ±7.9 * Diameter at breast height. f The number of trees in each foliage colour in July 2004, nine months after inoculation. G: green, YG: yellowish green, Y: yellow. Fig. 4.1. Inoculation with fungi or agar plugs using the cork borer technique. A - l : bark plugs of 5 mm diameter were removed down to the cambium layer by the cork borer. A-2: agar plugs with or without fungi were inserted into the holes. A-3: the inoculum holes were covered with bark plugs. B: the inoculated zone was wrapped with tape. 97 Analysis of symptoms Symptoms were observed on July 7, 2004. At the site, crown colour was recorded with a digital camera. The trees were felled, and logs of approximately 1.4 m in length (containing both the entire inoculated region and the adjacent non-inoculated regions above and below) were cut and transported to the laboratory for detailed examination. Lesion development To measure the length of the lesions on the inner bark (phloem), the outer bark was carefully removed from the entire length of the logs using a chisel. Since the lesions between inoculum points overlapped, the lesion lengths were measured only from the inoculation points in the uppermost and lowermost rows to the ends of the lesion margins (upward and downward, respectively) (Fig. 4.2C). The lesions were measured only from the trees inoculated with a density of 200 points/m2 due to the difficulty associated with peeling the intact bark on trees with a density of 800 points/m2. Occlusions, cell viability and moisture content of sapwood To examine the changes in the sapwood, five discs (approximately 2.5 cm in thickness) were cross-sectioned from debarked logs using a chain saw: three discs from the centre, and two discs at 15 cm above and below the centre of the inoculated region. The centre discs were used to estimate the size and viability of occluded sapwood and to re-isolate fungi. The other two discs were used to measure the sapwood moisture content. Occlusions in sapwood caused by fungal growth have a dry, whitish appearance and are readily distinguishable from areas of wet, healthy sapwood (Fig. 4.4B). Both areas of occluded and healthy sapwood were traced on Mars Vellum® paper (Staedtler Inc., Mississauga, ON.) The drawings were then cut out and weighed. The percentage of occluded sapwood was calculated as a proportion of the total sapwood area following a conventional method, defined as: os = -^—100 o + h 98 where os is the percentage of occluded sapwood, o is the weight of the drawings of occluded area and h is the weight of the drawings of healthy area in sapwood. The living regions in the sapwood were detected by immersing the discs overnight in a 1% (w/v) solution of 2, 3, 5-triphenyltetrazolium chloride (Sigma-Aldrich, Oakville, ON) in the dark. The regions exhibiting red staining were considered to be viable (Towill and Mazur 1975). The sapwood moisture content was measured with the entire sapwood according to the A S T M (D 4442) standard oven dry method (ASTM 2000) and defined as: OTC = _ _ _ _ _ 1 0 0 d where mc is the percentage moisture content, w is the wet weight and d is the dry weight. Statistical analysis Statistical analysis of data was conducted using SAS (version 9.1.3, SAS Institute Inc.). The lesion data were analyzed via one way analysis of variance (ANOVA). The moisture content and occluded sapwood data were analyzed via two-way A N O V A and since we had different numbers of replicated trees for the control and fungal treatments, the General Linear Model procedure was used. The mean values were compared with Bonferroni's multiple comparison test. The lesion data had a normal distribution (Kolmogorov-Smirnov test,/) = 0.15). The moisture content data also met the normality assumption (Kolmogorov-Smirnov test,p = 0.12) and no interaction between density and treatments was found (F ( 2 i 24)= 2.06, p = 0.15). Arcsine transformation was conducted before analysis of the occlusion data, because the data did not meet the normality assumption. Also, in order to meet the assumption of uniform variances, the agar control data had to be eliminated. It is obvious from figure 5 that the means of the two controls are significantly lower than other means. No statistical test is needed to prove it. 99 Re-isolation of fungi Survival of the inoculated fungi was confirmed by re-isolating the fungal species from the trees. In each tree, isolation was attempted at six points in the sapwood (middle and extremity of the occluded area, and stained area if there is any) and at three points in the phloem (around inoculation holes, in the middle and ends of lesions). In agar control trees, isolation was also attempted at three points on the outer sapwood and at three points in the phloem (around inoculation holes). To isolate fungi, small pieces of inner bark or sapwood were aseptically removed and placed on 2% M E A containing ampicillin (50 ug/mL). The plates were incubated at room temperature for one to four weeks and hyphal tips or conidial masses were sub-cultured for identification. Fungal growth under aerobic and anaerobic conditions Plugs of 5 mm diameter were removed from the edge of colonies growing actively on 2% M E A and placed on pre-poured 20 mL 2% M E A in 10 cm diameter petridishes. To measure growth in aerobic condition, the cultures were incubated at 21 °C. To measure growth in anaerobic conditions, the cultures were kept in a BBL GasPak Pouch™ system (Becton, Dickinson and Company, Flanklin Lakes, NJ), which produced a C02-enriched anaerobic environment, at 21 °C. The colony diameter which includes a fungal core of 5 mm was measured daily from three replicate plates. The growth differences between two fungi were analyzed with paired t-test. Results Lesion caused by L. longiclavatum and O. clavigerum Lesions consisting of necrotic tissue on the surface of the phloem were visible around the inoculation holes. The lesions were easily distinguished from the healthy, cream-coloured phloem. The agar controls caused relatively small lesions. At a density of 200 points/m2, most of the control inoculation points were surrounded by oblong lesions (Fig. 4.2A). At the high inoculation density (800 points/m2), the lesions were larger (Fig. 4.2B). Trees inoculated with L. longiclavatum and O. clavigerum at 200 points/m2 100 Fig. 4.2. Lesions on phloem or the surface of xylem caused by L. longiclavatum, O. clavigerum or agar control. The lesions were observed nine months after inoculation. A , B: Lesions on phloem caused by agar control at 200 points/m2 (A), and at 800 points/m2 (B). C, D: Long strip lesions on phloem (C) and xylem (D) caused by L. longiclavatum at 200 points/m2. Double head arrow in (C) indicates length of lesion measured. E, F: Lesions caused by L. longiclavatum at 800 points/m2 on phloem (E), and xylem (F). Most of the phloem within the inoculation zone showed necrosis. G, H: Trees inoculated with O. clavigerum at 800 points/m2, legions on the phloem (G). Open arrows represent the inoculum point on the last row. Bold arrows point to the invasive secondary Ips beetle or Ips galleries. showed long strip lesions along the column of inoculum points both on the phloem (Fig. 4.2C) and on the xylem (Fig. 4.2D). The lesion lengths on the phloem caused by L. longiclavatum or O. clavigerum were significantly greater than the ones caused by the agar controls (Fig. 4.3) (F ( 2 , i2) = 17.58,/? = 0.0003; L. longiclavatum t(i2)= 4.38,/> = 0.0009, O. clavigerum t( ) 2 )= 5.89,/? < 0.0001). O. clavigerum produced longer lesions than L. longiclavatum, but the difference was not statistically significant (t(]2)= 1.85,/? = 0.088). At the higher inoculation density of L. longiclavatum or O. clavigerum, the inoculation points were much closer to each other than at the lower density and the bark was drier and easily broken into small pieces. Most of the phloem within the 60 cm inoculation zone appeared brown and dead (Fig. 4.2E). The lesions were often longer than 40 cm and extended beyond the 60 cm inoculation band. Therefore, the length of lesions at 800 points/m2 inoculation density could not be measured. The margins of the lesions on the xylem were also difficult to distinguish (Figs. 4.2F and 4.2G). Occlusions, cell viability, and reduction in moisture content in sapwood Occlusions were observed in the sapwood underneath both the inoculated agar controls and fungi (Figs. 4.4A-C). The percentage of occlusions in the sapwood caused by L. longiclavatum or O. clavigerum was significantly different from the agar controls (Fig. 4.5). The percentage of occluded sapwood increased with the increasing density of fungal inocula (F(i20) = 200.93, p < 0.0001). Ophiostoma clavigerum produced more occlusion than L. longiclavatum (F ( i 2 0 )= 112.94,/? < 0.0001). The difference in the percentage of occlusions between L. longiclavatum and O. clavigerum was statistically significant at both density (at 200 points/m2, t ( 2 0 ) = 2.94,/? = 0.0081; at 800 points/m2, t ( 2 0 ) = 12.09,/? < 0.0001). The areas of occluded sapwood were found to be non-viable using 1% T T C solution (Figs. 4.4D-F). The moisture content of the sapwood decreased with increasing inoculation density (F (, 2 4 ) = 40.19,/? < 0.0001) (Table 1). Compared to the agar control, both L. longiclavatum and O. clavigerum significantly reduced the moisture content ( F ( 2 i 2 4 ) = 80.83,/? < 0.0001; L. longiclavatum t (24)= 6.99,/? < 0.0001, O. clavigerum t(24)= 12.58,/? < 0.0001). The moisture content reduced by O. clavigerum was significantly lower than that caused by L. longiclavatum (t( 24) = 6.84, p < 0.0001). 102 30 25 Agar L. longiclavatum 0. clavigerum Fig. 4.3. Comparison of the lesion lengths on phloem caused by agar control, L. longiclavatum and O. clavigerum at an inoculation density of 200 points/m2. The length was measured from the lowest and uppermost inoculation points within the inoculation zone nine months after inoculation. Bars represent standard deviations. 103 Fig. 4.4. A - E : Occlusions in sapwood underneath the inoculation points. (A) agar control, 200 points/m2; (B) L. longiclavatum, 200 points/m2, arrow indicates occluded area with whitish, dried appearance; (C) L. longiclavatum, 800 points/m2. D-F: cell viability test via 1% TTC solution; red area indicates that the cells are viable. (D) agar control, 200 points/m2; (E) L. longiclavatum, 200 points/m2, arrow points to viable cells in red; (F) L. longiclavatum, 800 points/m2. Fig. 4.5. Percentage of occluded areas in the sapwood of lodgepole pine following inoculation with agar control (H), L. longiclavatum (SI), O. clavigerum (ES), at two inoculation densities, 200 points/m2 or 800 points/m2. The occluded areas were measured nine months after inoculation. Bars represent standard deviations. Healthy sapwood (• ) . 105 Discoloration of the occluded sapwood was detected only in the trees inoculated with O. clavigerum at 800points/m2 (Fig. 4.4E). The moisture content of the sapwood in these trees was, on average 38.8%, while in the other treatments, without any visible stain, the moisture content was above 60%. External symptoms: changes in foliage colour Five out of six trees inoculated with L. longiclavatum and four of six trees inoculated with O. clavigerum at 800 points/m2 developed yellow to brownish crowns (Table 4.1). The foliage of the trees inoculated with agar controls at 200 points/m2 and 800 points/m2 remained green. Galleries of a secondary bark beetle, a species of Ips, were often found in the un-inoculated regions adjacent to the inoculation bands (Figs. 4.2F-4.2H) where the bark surface had not been covered with tape (Fig. 4. IB). Al l of the trees inoculated with fungi at the density of 800 points/m2 had Ips galleries: 17-68 galleries in trees inoculated with O. clavigerum, and 3-38 galleries in trees inoculated with L. longiclavatum. Most galleries were short parental galleries. At low density, there were galleries in two of the six trees inoculated with O. clavigerum (1-11 galleries per tree), and in one of the six trees inoculated with L. longiclavatum (5 galleries). None of the trees inoculated with agar controls had any Ips galleries. Survival of fungal inocula From the 24 trees inoculated with fungi, a total of 168 isolates of L. longiclavatum and O. clavigerum were successfully cultured out of 216 isolation attempts. Thirty-four isolates were mold (primarily Trichoderma species), which were most likely air-borne contaminants introduced during the isolation process. The proportion of successful re-isolation was similar between the two fungi. No cross-contamination between these fungi was found in any of the trees. From the 6 control trees, no organisms were cultured from 20 isolation attempts, but 16 isolates were mold. 106 Fungal growth in aerobic and anaerobic conditions Leptographium longiclavatum grew more slowly than did O. clavigerum under both aerobic and anaerobic conditions (t ( 3 )= 9.35,p < 0.01; t ( 7 ) =8.37p < 0.01, respectively)(Fig. 4.6). Discussion The results of this work suggest that L. longiclavatum is pathogenic to lodgepole pine. When L. longiclavatum and O. clavigerum were inoculated into trees in September, they survived through the winter and could be re-isolated the following spring, surviving temperatures in Kamloops during December 2003 and January 2004 of -5.8 °C on average, with a minimum of -26.6 °C (Environmental Canada 2005). Although much of Kamloops is infested by D. ponderosae, the trees did not show any sign of bark beetle attack before inoculation. However, Ips beetles that usually attack only severely weakened trees were found in July 2004, when we felled the inoculated trees. The Ips beetles were present outside the inoculated area, which was not protected with tape. The size of the Ips galleries suggests that the beetles were colonized the trees shortly before felling. No distinct lesions around the Ips galleries were found, reflecting weak to absent host resistance to Ips beetles. Therefore, it is possible that the Ips beetles were attracted to the trees stressed by fungal inoculation and did not contribute to tree death. In nature, after the initial D. ponderosae mass attack, weakened lodgepole pines are often colonized by other secondary bark beetles including Ips species (Kim et al. 2005; Paine et al. 1997). Trees have a critical attack density threshold beyond which they cannot survive (Christiansen et al. 1987; Raffa and Berryman 1983a). For D. ponderosae, the density of successful attacks was found to be in the range of 83-160/m2 (mean = 113, n = 6 trees, authors' unpublished data). Therefore, the density of natural attack that causes tree death appears to be lower than the inoculation density which caused foliage chlorosis in the current work (800 points/m2). However, D. ponderosae attacks over the lower two-thirds of the tree stem (usually about 15 m) in nature, while the fungal inoculation in this work was contained within a 60 cm-wide band. Thus, the total amount of fungal inocula in the whole tree was considerably less than that introduced naturally. Furthermore, beetles often introduce several fungal species (O. clavigerum, L. longiclavatum, 107 Fig. 4.6. Growth of the fungi under aerobic (^: L. longiclavatum, SL-Kwl436, o : o. clavigerum, SL-Kwl407), and anaerobic conditions on 2 % M E A at 21 °C (>: L. longiclavatum, SL-Kwl436, • : O. clavigerum, SL-Kwl407). 108 O. montium, O. minutum-like sp., or Entomocorticium spp.), and it is likely that some synergistic or competitive interaction occurs during natural fungal colonization (Bridges and Perry 1985; Parmeter et al. 1989). Due to the massive fire in the Kamloops area in the summer of 2003, the inoculation was inevitably delayed until September. In contrast to July to August, when D. ponderosae usually attacks trees, the defence reaction of P. contorta in September could be different and might affect the measurement of fungal aggressiveness. It has been speculated that the defence response varies depending on the weather, which affects the growth-differentiation balance of trees and consequently alters the resin production (Lorio 1986). Picea abies shows less resin flow and shorter bark necroses in September than in July to August (Horntvedt 1988) while Pinus taeda produces more monoterpenes in cooler and wetter seasons (Conners et al. 2001). P. contorta appears to be less resistant in late July than in June, but no information is available for September (Shrimpton 1973). To assess the pathogenicity of sapstain fungi to pine, spruce or fir, several indicators have been used as a measure of their aggressiveness. The most commonly used indicator is the lesion area which consists of necrotic tissue having accumulated terpenoid compounds (Owen et al. 1987; Solheim and Krokene 1998; Solheim et al. 1993; Wingfield 1986). Another frequently reported symptom is occlusion in sapwood (Christiansen and Solheim 1990; Solheim and Krokene 1998; Solheim and Safranyik 1997), which has a distinct whitish appearance (Mathre 1964; Solheim 1995). It becomes drier through an uncertain mechanism as fungi grow (Hemingway et al. 1977; McGraw and Hemingway 1977; Nelson 1934). Foliage chlorosis, another indicator of pathogenicity, indicates irreversible damage to the inoculated trees. This symptom was more frequently observed six months to one year after inoculation (Yamaoka et al. 1995). In this work we measured all the indicators described above, but also the moisture content and the cell viability of the sapwood. The occlusions often reached the innermost part of the sapwood. They were much wider than the occlusions observed in naturally infested sapwood, which have been reported to be 3-8 mm in width and to surround the wedge-shape stained areas (Solheim 1995). With both the fungal species used in the study, the amount of sapwood occlusion increased with inoculation density; high-109 density inoculations led to the death of trees. Other researchers have also shown that with aggressive fungal pathogens that kill inoculated trees, the amount of occlusion increases with the inoculation density (i.e. Ceratocystispolonica, Christiansen 1985a; Ceratocystis rufipenni, Solheim and Safranyik 1997; Leptographium wingfieldii and Ophiostoma minus, Solheim et al. 1993). However, for weaker pathogens such as Leptographium abietinum and Ophiostomapseudotsugae, increasing inoculation densities do not significantly change the amount of occlusion, and hosts survive even at the highest inoculation density of 800 points/m2 (Ross and Solheim 1997; Solheim and Safranyik 1997). In contrast to naturally infested lodgepole pines that show stain over the entire sapwood shortly after infestation, we usually observed occlusions rather than large stained areas in inoculated trees, confirming similar observations made by Yamaoka et al. (1990). However, despite the absence of stain, both L. longiclavatum and O. clavigerum were re-isolated from the occluded areas. This suggests that either the sapstain fungi growing in occluded areas did not produce the melanin pigment, or the fungal mass was too small to produce visible coloration. Staining of the sapwood might result from an increase of the mycelial mass as the sapwood moisture content decreases after infestation (Reid 1961). In the current work, stain in sapwood was also negatively correlated with moisture content. Stain was observed only in the trees inoculated with O. clavigerum at high density (800 points/m2), in which moisture content was similar to that of stained sapwood in naturally infected trees one year after infestation (Kim et al. 2005). It has been suggested that the fungi found at the front of the leading edge of sapstain penetration are primarily responsible for tree mortality (Solheim 1991, 1992, 1995; Solheim and Krokene 1998). It has been speculated that they may be better adapted to invade fresh sapwood because of their tolerance to low oxygen content (Solheim 1991; Solheim and Krokene 1998). The two fungal species that we used in this work, L. longiclavatum and O. clavigerum, were able to grow and survive under oxygen-deficient conditions. However, L. longiclavatum grew more slowly than O. clavigerum and this might explain the difference in the occluded sapwood caused by the two fungal species. It is also possible that the two fungal species might be affected differently by the host defense metabolites (terpenoid compounds), 110 which can affect fungal spore germination or mycelial growth (Paine and Hanlon 1994; Shrimpton and Whitney 1967). In conclusion, this study shows that L. longiclavatum is pathogenic to lodgepole pine and is almost as virulent as O. clavigerum. Since L. longiclavatum is found at relatively high frequency in the sapwood at the early infestation phase (Kim et al. 2005; Lee et al. 2005b), it may contribute significantly to the mortality of lodgepole pines attacked by D. ponderosae. However, as we used only one isolate of L. longiclavatum in this work, we cannot exclude the possibility of variation in virulence among isolates from different geographic areas. Replicated experiments with more strains would be desirable in the future. I l l References American Society for Testing Materials (ASTM). 2000. 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Entomol. 11: 486-492. Raffa, K.F., and Berryman, A.A. 1983a. The role of host plant resistance in the colonization behaviour and ecology of bark beetles (Coleoptera: Scolytidae). Ecol. Monogr. 53: 27-49. Reid, R.W. 1961. Moisture changes in lodgepole pine before and after attack by mountain pine beetle. For. Chron. 37: 368-375. Reid, R.W., and Shrimpton, D.M. 1971. Resistant response of lodgepole pine to inoculation with Europhium clavigerum in different months and at different heights on stem. Can. J. Bot. 49: 349-351. Reid, R.W., Whitney, H.S., and Watson, J.A. 1967. Reactions of lodgepole pine to attack by Dendroctonus ponderosae Hopkins and blue stain fungi. Can. J. Bot. 45: 1115-1126. Robinson, R.C. 1962. Blue stain fungi in lodgepole pine (Pinus contorta Dougl. var. latifolia Engelm.) infested by the mountain pine beetle (Dendroctonus monticolae Hopk.). Can. J. Bot. 40: 609-614. 113 Robinson-Jeffrey, R . C , and Davidson, R.W. 1968. Three new Europhium species with Verticicladiella imperfect states on blue-stained pine. Can. J. Bot. 46: 1523-1527. Ross, D.W., and Solheim, H. 1997. Pathogenicity to Douglas-fir of Ophiostomapseudotsugae and Leptographium abietinum, fungi associated with the Douglas-fir beetle. Can. J. For. Res. 27(1): 39-43. Rumbold, C T . 1941. A blue stain fungus, Ceratostomella monitum n. sp., and some Yeasts associated with two species of Dendroctonus. J. Agric. Res. 62(10): 589-601. Shrimpton, D .M. 1973. Age- and size-related response of lodgepole pine to inoculation with Europhium clavigerum. Can. J. Bot. 51: 155-1160. Shrimpton, D.M. , and Watson, J.A. 1971. Response of lodgepole pine seedlings to inoculation with Europhium clavigerum, a blue stain fungus. Can. J. Bot. 49:373-375. Shrimpton, D.M. , and Whitney, H.S. 1967. Inhibition of growth of blue stain fungi by wood extractives. Can. J. Bot. 46: 757- 766. Six, D.L. 2003. A comparison of mycangial and phoretic fungi of individual mountain pine beetles. Can. J. For. Res. 33: 1331-1334. Solheim, H. 1991. Oxygen deficiency and spruce resin inhibition of growth of blue stain fungi associated with Ips typographus. Mycol. Res. 95(12): 1387-1392. Solheim, H. 1992. The early stages of fungal invasion in Norway spruce infested by the bark beetle Ips typographus. Can. J. Bot. 70: 1-5. Solheim, H. 1995. Early stages of blue-stain fungus invasion of lodgepole pine sapwood following mountain pine beetle attack. Can. J. Bot. 73: 70-74. Solheim H., and Krokene, P. 1998. Growth and virulence of mountain pine beetle associated blue stain fungi, Ophiostoma clavigerum and Ophiostoma montium. Can. J. Bot. 76: 561-566 Solheim, H., and Safranyik, L . 1997. Pathogenicity to Sitka spruce of Ceratocystis rufipenni and Leptographium abietinum, blue-stain fungi associated with the spruce beetle. Can. J. For. Res. 27(9): 1336-1341. Solheim, H. , Langstrom, B., and Hellqvist, C. 1993. Pathogenicity of the blue-stain fungi Leptographium wingfieldii and Ophiostoma minus to Scots pine: effect of tree pruning and inoculum density. Can. J. For. Res. 23(7): 1438-1443. Strobel, G.A., and Sugawara, F. 1986. The pathogenicity of Ceratocystis montia to lodgepole pine. Can. J. Bot. 64(1): 113-116. Towill, L . E . , and Mazur, P. 1975. Studies on the reduction of T T C as an availability assay for plant tissue cultures: Can. J. Bot. 53: 1097-1102. Whitney, H.S., and Farris, S.H. 1970. Maxillary Mycangium in the Mountain Pine Beetle. Science 167: 54-55. 114 Wingfield, M.J. 1986. Pathogenicity of Leptographium procerum and L. terebrantis on Pinus strobes seedlings and established trees. Eur. J. For. Path. 16: 299-308. Wright, E. 1933. A cork borer mehod for inoculating trees. Phytopathol. 23: 487-488. Yamaoka, Y., Hiratsuka, Y., and Maruyama, P.J. 1995. The ability of Ophiostoma clavigerum to kill mature lodgepole-pine trees. Eur. J. For. Path. 25: 401-404. Yamaoka, Y., Swanson, R.H., and Hiratsuka, Y. 1990. Inoculation of lodgepole pine with four blue-stain fungi associated with mountain pine beetle, monitored by a heat pulse velocity (HPV) instrument. Can. J. For. Res. 20:31-31. Yamaoka, Y., Takahashi, I., and Igushi, K. 2000. Virulence of Ophiostomatoid Fungi associated with the Spruce bark beetle Ips tyographus i.japonicus in Yezo Spruce. J. For. Res. 5: 87-94. 115 C h a p t e r s Genetic diversity and the presence of two distinct groups in the Ophiostoma clavigerum population associated with the mountain pine beetle, Dendroctonus ponderosae in North America Introduction The sapstaining fungus Ophiostoma clavigerum (Robinson-Jeffrey & Davidson) Harrington is a forest pathogen belonging to the Ascomycetes. O. clavigerum is haploid through most of its life cycle, but is transiently diploid when it reproduces sexually. Like many Ophiostomatoid fungi, it produces asexual and sexual spores in a slimy mass that can be easily dispersed by insects (Harrington 1993). O. clavigerum is found in North America where its two insect vectors, Dendroctonus ponderosae and D.jeffreyi, and their respective hosts are distributed (Whitney and Farris 1970, Six 2003a, Six and Paine 1997). The two bark beetle species are morphologically similar but infest different hosts. D. ponderosae mainly infests Pinus contorta and P. ponderosa, and occasionally P. albicaulis and P. lambertiana in Canada (British Columbia and Alberta) and the USA (Washington, Oregon, California, Idaho and Montana). In contrast, D. jeffreyi attacks P. Jeffrey in a relatively limited area in the USA (California) and Mexico (Baja) (BC Ministry of Forests and Range 2005, Six and Paine 1999, USDA 2001). In Canada, three major D. ponderosae outbreaks have occurred during the last century; the current one is the most severe epidemic ever recorded (BC Ministry of Forests and Range, 2005). The epidemic spread to the northern part of BC that had not been affected before and 173 million m 3 of P. contorta in BC forests had been infested as of 2004 (BC Ministry of Forests and Range, 2005). In addition to O. clavigerum, D. ponderosae carries other fungi such as O. montium, Leptographium longiclavatum, O. minutum-Uke species and Entomocorticium species (Kim et al. 2005, Lee et al. 2005, Six 2003a, Solheim 1995, Whitney and Farris 1970). The bark beetle may benefit from its fungal associates in several ways: overcoming tree defences, providing nutrients and reducing sapwood moisture 116 content for successful brood development (Berryman 1972, Paine et al. 1997, Reid 1961, Six 2003b, Web and Franklin 1978, Whitney et al. 1987). Among the fungi associated with D. ponderosae, O. clavigerum appears to be the most virulent pathogen to lodgepole pine. It grows at the leading edge of infested sapwood, disrupts water transportation shortly after infestation, and kills trees when inoculated at high densities (Solheim 1995, Yamaoka et al. 1990, 1995). However, neither the genetic diversity nor the variance in pathogenicity of O. clavigerum strains isolated from D. ponderosae has been characterized. Six and Paine (1999) reported that O. clavigerum isolated from D. jeffreyi in the USA has low genetic diversity. However, the results cannot be extrapolated to the O. clavigerum population associated with D. ponderosae, as both fungi and beetle populations may have co-evolved in host-specific ways. Furthermore, Canadian, O. clavigerum populations have not yet been investigated. Amplified fragment length polymorphism (AFLP) analysis is a powerful analytical tool for population genetic studies (Vos et al. 1995). AFLP is a dominant D N A fingerprinting marker and suitable for genetic studies for haploid fungi. AFLP can produce a large number of markers with few primers and is more reproducible than random amplified polymorphic DNA (RAPD) analysis (McDonald 1997, Mueller and Wolfenbarger 1999). The analysis appears to be more suitable to investigate differentiation within and among populations than allozyme analysis that produces low variation (McDonald and McDermott 1993). Generally, the data generated by the neutral AFLP marker are less biased than selective markers such as pathogenicity phenotype (McDonald and McDermott 1993). In this work, we examined the genetic structure of the O. clavigerum population associated with D. ponderosae and lodgepole pine in seven outbreak regions in Canada and the USA. AFLP markers from 170 isolates were analyzed to 1) investigate genetic diversity in the O. clavigerum population, and 2) determine whether O. clavigerum exists as a single metapopulation across the wide range of the epidemic area or as genetically distinct subpopulations. 117 Materials and methods Sampling and identification of the O. clavigerum isolates Ophiostoma clavigerum was isolated from five sites in Canada (Banff, Fort St. James, Houston, Manning Park, and Williams Lake) and two sites in the USA (Hellroaring and Hidden Valley), in 2002 or in 2003 (Fig 5.1, Table 5.1). The longest distance between sampling sites spanned over 1400 km (between Fort St. James and Hellroaring). The fungi were isolated from either infested phloem or sapwood of P. contorta as described by Kim et al. (2005), or from maxillary cardines containing fungus-bearing mycangia of D. ponderosae as described by Six and Paine (1999) (Table 5.1). The purity of O. clavigerum isolates was ensured by single spore isolation. The identification of O. clavigerum was based on the conidiogenous structures described by Robinson-Jeffrey & Davidson (1968) and Lee et al. (2003), and confirmed by O. clavigerum specific PCR-RFLP markers (Lee etal. 2003). DNA isolation Two plugs were removed from the edge of an actively growing colony and placed on 2% M E A overlaid with cellophane sheath. After 3-4 days of incubation at room temperature, mycelia were harvested aseptically and kept at -80 °C for up to 30 days before DNA extraction. To yield high quality DNA, non-pigmented mycelia were harvested. Approximately 50-60 mg of frozen mycelia per isolate was ground in mortars and pestles with liquid nitrogen. Genomic DNA was isolated following the protocol of Zolan and Pukilla (1986), with a modification as follows: The grounded mycelia were transferred to 0.7 mL C T A B buffer (2.5% cetylmethylammonium bromide, 1% PVP-40, 1.4 mol/L NaCl, 0.02 mol/L EDTA, 0.1 mol/L Tris-HCl, and 0.5%> B-mercaptoethanol freshly added) in a 2 mL microtube (Sarstedt, Montreal, Quebec). The mixture was vortexed for 15 sec, incubated at 60 °C and then treated twice with 700 uL of chloroform and isoamylalcohol (v/v = 24:1). The solution was gently rotated for 10 min at room temperature and then centrifuged at 12000 rpm (Eppendorf Centrifuge 5417R, Hamburg, Germany) for 5 min to collect the top aqueous layer. DNA was precipitated with ice-cold isopropanol and resuspended in 118 Fig. 5.1. Map showing the seven sampling sites of O. clavigerum in Canada (Fort St. James, Houston, Williams Lake, and Manning Park in BC; Banff in Alberta) and the USA (Hellroaring in Idaho; Hidden Valley in Montana). 119 Table 5.1. The sampling sites, sources of isolation, and number of fungal isolates in each location. Site No. Location Longitude, latitude Date sampled Source Number of source Number of fungal isolates 1 Banff, AB, Canada N51° 10', W 115° 34' July, 2003 P. contorta infested by D. ponderosae 10 trees 24 2 Fort St. James, BC, Canada N53°50' 25", W 124° 31'41" July, 2003 P. contorta infested by D. ponderosae 10 trees 28 3 Hellroaring, ID, USA N44° 2', W 114° 51' Summer, 2002 D. ponderosae 29 beetles 28 4 Hidden Valley, MT, USA N47° 23' 15", W 115° 18' 15" Summer, 2003 D. ponderosae 21 beetles 21 5 Houston, BC, Canada N54° 08', W 126° 40' July, 2003 P. contorta infested by D. ponderosae 10 trees 22 6 Manning Park, BC, Canada N49° 11'35", W 120° 35' 05" June, 2003 P. contorta infested by D. ponderosae 10 trees 29 7 Williams Lake, BC, Canada N 52° 01'35", W 122° 31'27" August, 2003 P. contorta infested by D. ponderosae 10 trees 18 300 uL T E buffer (10 mmol/L Tris-HCl, pH 8.0 and 1 mmol/L EDTA). RNA was digested with 3 uL of RNase A (10 mg/mL, Sigma-Aldrich, Oakville, ON) and incubated overnight at room temperature. The chloroform extraction was repeated one more time. DNA was precipitated by adding 100 uE of 5 M sodium acetate (pH 5.2; Sigma-Aldrich) and 725 uL of ice-cold 100% ethanol. The pellet was washed with 500 uL ice-cold 70% ethanol, air dried for 5-25 min avoiding over-drying, dissolved in 20-46 uL of water depending on the pellet size and incubated at room temperature overnight. The quality and concentration of DNA were checked using 1.2% agarose gel and a spectrophotometer (Ultraspec3000, Pharmacia Biotech, Amersham, Piscataway, NJ). Only high quality genomic D N A of which absorbance ratio at 260/280 nm was between 1.8 and 2 was used for AFLP reactions. Amplifiedfragment length polymorphism (AFLP) The AFLP was conducted following a protocol developed by the Genetic Data Centre at the University of British Columbia as described by Wilkin et al. (2005). To screen primers, 30 randomly selected fungal genomic DNA (500 ng/strain) samples were digested with either the enzyme pair of EcoRI/Msel, or EcoKL/Pstl. The digested D N A was then ligated with primer adapters, pre-amplified and amplified with various primer sets. A total of 34 primer sets were screened and the best six primer sets were selected based on the number of clear polymorphic bands generated: EcoRl+T and Pstl+T; EcoRI+T and Pstl+A; £coRI+C and Pstl+G; £coRI+G and Pstl+A; £coRI+G and Pstl+G; £coRI+G and Pstl+T. These six sets were used for generating the AFLP profile of the genomic DNA of 170 isolates. A pre-amplification before the amplification was carried out with the same primer sets. To ensure reproducibility, three independent reactions were repeated with the DNA of 15 randomly chosen isolates and the same six primer sets. The amplified band profiles were visualized with the Li-Cor® 4200 system (Li-Cor Inc. Lincoln, Nebraska, USA) and scored using S A G A - M X for AFLP bands (Li-Cor Inc.). When scoring the AFLP profiles, it was assumed that co-migrating bands on a gel were the same loci with identical sequences. 121 Data analysis Allele frequency, proportions of polymorphic loci, and Nei's (1978) unbiased genetic diversity index (Hs) were calculated with Tools for Population Genetic Analyses (TFPGA, Version 1.3, Miller, Northern Arizona University, Flagstaff, AZ). Haplotypes were determined by pairwise differences using Arlequin (Version 2.000, Schneider et al, University of Geneva, Geneva, Switzerland). For the inference of haplotypes, forty isolates with missing data were excluded from the dataset because they biased pairwise comparisons. An analysis of molecular variance (AMOVA) was conducted with Arlequin to partition variances into hierarchical structures, such as among groups, among populations, among populations within groups, within groups, and within populations (Excoffier et al. 1992). The analysis also estimated the genetic differentiation (cD) among populations with F statistics. With the analysis, a matrix of population pairwise FST values were generated as well (Weir 1996). To estimate the genetic similarity among the seven O. clavigerum populations, Nei's unbiased genetic distances (Nei 1978) between populations were calculated and a dendrogram using the unweighted pair group method with arithmetic averages (UPGMA) was generated with TFPGA. The confidence level of branches in the UPGMA tree was estimated with bootstrapping values (Felsenstein 1985) using a 1000-randomized dataset. To investigate the correlation between genetic and geographic distances, a Mantel test was performed with TFPGA. The distance between sampling sites were estimated using ArcMap (Version 9.1, ESRI Inc., Redlands, CA). Fine level cluster analysis, in which each O. clavigerum isolate was considered as a single unit, was achieved by constructing an UPGMA tree with PAUP* v.4.0b (Swofford, Sinauer Associates, Sunderland, MA) using the Nei and Li coefficient (Nei and Li 1979). To statistically support the branches within the UPGMA tree, bootstrapping values were determined. A spatial distribution of haplotypes was illustrated using principal component analysis (PCA) in SAS (Version 8.0, SAS Institute Inc., Cary, NC). To estimate the level of asexual reproduction versus sexual reproduction, the indices of multilocus association, IA and rd (Agapow and Burt 2001, Maynard et al. 1993), were computed using MultiLocus (Version 1.3, Agapow and Burt, Imperial College, Berkshire, UK). The significance of 7A was 122 tested by comparing the observed value with the expected values from 100 randomized datasets under the null hypothesis of free recombination. Linkage disequilibrium (LD) was also measured with an exact test using Arlequin. The L D (%) was given as the number of disequilibrium values significant at P < 0.05 over the total number of pairwise comparisons between polymorphic loci. Results Genetic polymorphisms, heterozygosity, and haplotypes A total of 175 fungal isolates were collected and were initially identified as O. clavigerum based on morphology. Among them, five isolates did not generate the O. clavigerum-specific bands (238 and 490 bp long) by the PCR-RFLP marker based on the (3-tubulin gene (Lee et al. 2003) and were re-identified as L. longiclavatum or L. terebrantis. Therefore, only 170 isolates with confirmed identification were used to generate AFLP profiles with six sets of primers. Each primer set generated 59-88 scorable loci with sizes varying from 64 to 660 bp (Fig. 5.2). The reproducibility test indicated that the markers were highly reproducible, with 92% replication of the same banding patterns. A total of 469 loci were scored; of these, 243 were monomorphic and 226 were polymorphic. One hundred and ninety three polymorphic loci had allele frequencies between 0.01 and 0.99 and the remaining 33 were rare alleles (Table 5.2). One hundred polymorphic loci with allele frequencies between 0.05 and 0.95 from each of the 170 isolates were used as a dataset for most of the analyses. Some polymorphic alleles were not randomly distributed throughout the geographic area studied. Thirty-eight markers were observed only in the populations of the Rocky Mountain region (Banff, Hellroaring and Hidden Valley) and not in the BC populations (Fort St. James, Houston, Manning Park, and Williams Lake) (Table 5.3). In contrast, six markers were observed exclusively in the BC populations. Nei's unbiased heterozygosity (Hs) values over all loci in seven O. clavigerum populations ranged from 0.040 to 0.075 and averaged 0.053 (Table 5.4). In general, higher values of heterozygosity and percentage of polymorphic loci were observed in the Rocky Mountain populations than in the BC 123 Fig. 5.2. An A F L P profile of O. clavigerum generated with the primer combination EcoBl+T/Pstl+A. Lane 1: marker; lanes 2-17: rill-22, the O. clavigerum isolates collected from Houston, BC ; lanes 18-37: HV1-19, O. clavigerum collected from Hidden Valley, Montana. Note distinct profiles in lane 25 (HV4), lane 28 (HV7) and lane 31 (HV10). Arrows indicate the polymorphic bands. 1 2 4 Table 5.2. The number of monomorphic and polymorphic loci observed in the AFLP profiles of 170 O. clavigerum isolates with the six selected primer combinations. Primer sets Total loci Monomorphic loci Polymorphic loci Polymorphic loci (5 < % <95) EcoRI + C and Pstl + G 88 62 26 12 EcoRI + G and Pstl + A 85 60 25 18 EcoRI + G and Pstl + G 78 44 34 18 £ c o R I + G and Pstl + T 73 17 56 16 £coRI + T and Pstl + A 86 27 59 18 EcoRI + T and Pstl + T 59 33 26 18 Total number 469 243 226 100 125 Table 5.3. The polymorphic alleles specific to the Rocky Mountain population (BF, FIR, and HV) or the BC population (HT, St J , MP, and WL) of O. clavigerum. Allele frequency locus* Total BF HR HV Ht St.J MP WL n = 170 n = 24 n = 28 n = 21 n = 22 n = 28 n = 29 n = 18 C G _ 6 7 2 0.031 0.143 0.000 0.095 0.000 0.000 0.000 0.000 C G _ 5 7 9 0.055 0.238 0.038 0.143 0.000 0.000 0.000 0.000 C G _ 4 4 3 0.055 0.238 0.038 0.143 0.000 0.000 0.000 0.000 C G _ 3 3 5 0.049 0.238 0.038 0.095 0.000 0.000 0.000 0.000 CG_302 0.049 0.238 0.038 0.095 0.000 0.000 0.000 0.000 C G _ 2 6 6 0.055 0.238 0.038 0.143 0.000 0.000 0.000 0.000 C G _ 2 6 0 0.049 0.238 0.038 0.095 0.000 0.000 0.000 0.000 C G _ 2 3 5 0.055 0.238 0.038 0.143 0.000 0.000 0.000 0.000 C G _ 1 5 5 0.055 0.238 0.038 0.143 0.000 0.000 0.000 0.000 CG_135 0.067 0.238 0.038 0.238 0.000 0.000 0.000 0.000 GG_451 0.067 0.333 0.000 0.143 0.000 0.000 0.000 0.000 GG_408 0.146 0.292 0.480 0.238 0.000 0.000 0.000 0.000 G G _ 3 5 7 0.049 0.208 0.000 0.143 0.000 0.000 0.000 0.000 GG_296 0.043 0.208 0.000 0.095 0.000 0.000 0.000 0.000 GG_253 0.055 0.250 0.000 0.143 0.000 0.000 0.000 0.000 GT_651 0.012 0.000 0.000 0.095 0.000 0.000 0.000 0.000 GT_559 0.012 0.000 0.037 0.048 0.000 0.000 0.000 0.000 GT_436 0.055 0.333 0.000 0.048 0.000 0.000 0.000 0.000 GT_284 0.048 0.208 0.037 0.095 0.000 0.000 0.000 0.000 GT_213 0.012 0.000 0.095 0.000 0.000 0.000 0.000 0.000 GT_83 0.012 0.000 0.000 0.095 0.000 0.000 0.000 0.000 GA_642 0.012 0.000 0.000 0.095 0.000 0.000 0.000 0.000 G A _ 5 8 6 0.036 0.167 0.000 0.095 0.000 0.000 0.000 0.000 GA_533 0.054 0.208 0.038 0.143 0.000 0.000 0.000 0.000 TA_587 0.056 0.182 0.038 0.200 0.000 0.000 0.000 0.000 T A _ 5 7 1 0.031 0.136 0.038 0.050 0.000 0.000 0.000 0.000 T A _ 5 4 3 0.043 0.182 0.038 0.100 0.000 0.000 0.000 0.000 TA_530 0.031 0.136 0.038 0.050 0.000 0.000 0.000 0.000 TA_429 0.031 0.136 0.038 0.050 0.000 0.000 0.000 0.000 T A _ 3 7 3 0.049 0.182 0.038 0.150 0.000 0.000 0.000 0.000 TA_304 0.056 0.227 0.038 0.150 0.000 0.000 0.000 0.000 TA_285 0.012 0.000 0.038 0.050 0.000 0.000 0.000 0.000 TA_268 0.068 0.182 0.077 0.250 0.000 0.000 0.000 0.000 T T _ 6 8 5 0.059 0.227 0.038 0.143 0.000 0.000 0.000 0.000 TT_625 0.039 0.045 0.115 0.095 0.000 0.000 0.000 0.000 T T _ 3 1 4 0.059 0.227 0.038 0.000 0.143 0.000 0.000 0.000 TT_168 0.046 0.227 0.038 0.048 0.000 0.000 0.000 0.000 T T 154 0.059 0.227 0.038 0.143 0.000 0.000 0.000 0.000 CG_168 0.037 0.000 0.000 0.000 0.000 0.143 0.000 0.111 CG_106 0.031 0.000 0.000 0.000 0.136 0.071 0.000 0.000 GA_405 0.083 0.000 0.000 0.000 0.227 0.214 0.103 0.000 GA_249 0.054 0.000 0.000 0.000 0.000 0.000 0.172 0.222 TA_251 0.037 0.000 0.000 0.000 0.136 0.074 0.036 0.000 TA 242 0.049 0.000 0.000 0.000 0.000 0.074 0.214 0.000 * The polymorphic loci with allele frequencies between 0.01 and 0.99 are listed. BF, Banff; HR, Hellroaring; HT, Houston; H V , Hidden Valley; MP, Manning Park; St. J, Fort St. James; W L , Williams Lake. * The A F L P markers for the loci written in bold were observed only in O. clavigerum Group 2. 126 Table 5.4. The genetic diversity indices of Rocky Mountain populations (BF, HR, and HV) and the BC populations (HT, St.J, MP, and WL) of O. clavigerum. Population Polymorphic loci (%){ over all loci over polymorphic loci 0.05 < frequency < 0.95 0.01 < frequency < 0.99 BF 0.070 (0.045) 0.270 (0.207) 19.4 22.6 HR 0.053 (0.050) 0.214 (0.234) 13.9 23.9 HV 0.075 (0.064) 0.267 (0.237) 24.6 32.1 HT 0.043 (0.048) 0.182 (0.211) 12.6 15.8 MP 0.045 (0.047) 0.193 (0.225) 12.8 16.2 St.J 0.040 (0.041) 0.173 (0.201) 10.9 13.5 WL 0.046 (0.048) 0.203 (0.236) 14.3 14.3 Average 0.053 (0.049) 0.215 (0.221) 15.5 19.8 * BF, Banff; HR, Hellroaring; HT, Houston; HV, Hidden Valley; MP, Manning Park; St. J, Fort St. James; WL, Williams Lake, t Hs: Nei's (1987) unbiased genetic diversity index for each population over all loci, including monomorphic and rare alleles, or over polymorphic loci with allele frequencies greater than 0.05 and less than 0.95. The numbers in brackets are for Group 1 only (see discussion). J The percentage of polymorphic loci with allele frequencies between 0.05 and 0.95, or between 0.01 and 0.99, over the total 469 loci. populations. The Hidden Valley population, located in the middle-eastern Rocky Mountains (Fig. 5.1), showed the highest level of Hs (0.075), whereas the Fort St. James population collected from the most northern part of the epidemic area had the lowest level of Hs (0.040). The values of Hs over polymorphic loci ranged from 0.173 (Fort St. James) to 0.270 (Banff), with an average of 0.215. The diversity of haplotypes in the O. clavigerum population was high. A total of 128 haplotypes were identified in 130 isolates using polymorphic loci (0.05 < allele frequency < 0.95). Two isolates from Fort St. James had the same haplotype. One haplotype was found at two sites: Banff and Hellroaring. Analysis of molecular variance A M O V A analysis performed on seven O. clavigerum populations showed that 85.7% (P < 0.001) of the genetic variability was due to differences among individual isolates within populations (Table 5.5). The remaining variability resulted from differences among the seven populations (14.3%), 0 = 0.143, P < 0.001). To further investigate the differentiation among the geographical populations, all the isolates from the seven populations were pooled and re-sorted into two distinct geographic groups: Rocky Mountain region and B C plateau. A high proportion of the variance was still attributed to differences among individual isolates within populations (83.6%, P < 0.001), whereas the differences between groups contributed only 5.8% to the total genetic variability (<£ = 0.058, P = 0.031). The genetic differentiation observed between the isolates sampled from P. contorta and D. ponderosae was not significant (O = -0.0059, P = 0.520) (Table 5.6). Pairwise comparison of the populations and cluster analyses The matrix of pairwise F S T indices suggested that the Banff and Fort St. James populations were the most differentiated CF S T = 0.222, P < 0.01), whereas the Houston and Fort St. James populations were the least differentiated CF ST = 0.082, P < 0.01) (Table 5.7). The matrix of population pairwise F^T indices was calculated from the analysis of molecular variance. 128 Table 5.5. Analysis of molecular variance of AFLP markers for 170 isolates of O. clavigerum collected from seven sites in North America.* to V£5 Source of variation df SSD Variance components Percentage of variation (%) F statistics ( O ) P Analysis for seven populations* Among populations 6 293.31 1.62 14.3 0.143 O.001 Within populations 163 1581.49 9.70 85.7 Total 169 1874.80 11.32 Analysis for two groups of populations* Between groups 1 96.73 0.68 5.8 0.058 0.031 Among populations within groups 5 196.57 1.23 10.6 0.112 O.001 Within populations 163 1581.49 9.70 83.6 0.164 <0.001 Total 169 ' 1874.80 11.61 AMOVA was performed on a dataset including all 469 loci, df: degree of freedom; SSD, sums of squared deviations; P, probability of obtaining larger or equal values of variance components and <I> by chance, determined by 1023 permutations. * The seven populations were treated as one group (Banff, Hellroaring, Hidden Valley, Houston, Fort St. James, Williams Lake, and Manning Park). * The two groups were 1: Rocky Mountain region (Banff, Hellroaring, and Hidden Valley), 2: BC (Houston, Fort St. James, Williams Lake, and Manning Park). Table 5.6. Analysis of molecular variance of AFLP markers for two O. clavigerum subpopulations: one subpopulation was sampled from P. contorta and the other from D. ponderosae Source of variation df SSD Variance components Percentage of variation (%) F statistics ( O ) P Analysis for two groups of populations* Among groups 1 46.31 -0.07 -0.6 -0.006 0.520 Among populations within groups 5 247.00 1.65 14.6 0.146 <0.001 Within populations 163 1581.49 9.70 86.0 0.140 O.001 Total 169 1874.80 11.29 * AMOVA was performed on a dataset including all 469 loci, df: degree of freedom; SSD, sums of squared deviations; P, probability of obtaining larger or equal values of variance components and O by chance, determined by 1023 permutations. * The two groups were 1: The populations isolated from D. ponderosae (Hellroaring, and Hidden Valley), 2: The populations isolated from P. contorta (Banff, Houston, Fort St. James, Williams Lake, and Manning Park). Table 5.7. Population pairwise FST indices among seven O. clavigerum populations from A M O V A . Population BF HR HT HV MP St.J WL BF HR 0.090 HT 0.160 0.144 HV 0.085 0.101 0.101 M P 0.205 0.170 0.148 0.105 St. J 0.222 0.193 0.083 0.157 0.171 WL 0.176 0.129 0.181 0.108 0.117 0.110 All the FST values in the table were significant at P < 0.01 (P, probability of obtaining larger or equal values determined by 1023 permutations). 1 BF, Banff; HR, Hellroaring; HT, Houston; HV, Hidden Valley; MP, Manning Park; St. J, Fort St. James; WL, Williams Lake. An U P G M A tree based on Nei's unbiased genetic distances among populations showed a similar trend to that observed in the pairwise FSr analysis (Fig. 5.3). The U P G M A dendrogram showed that the Houston and Fort St. James populations were the most similar and formed one cluster with a strong bootstrapping value (97%). The other two clusters consisted of the Rocky Mountain populations and the central to southern BC populations, and were supported by relatively low bootstrapping values (61% and 42%, respectively). The general association of genetic distance to geographic distance was evaluated with the Mantel test. The test showed that Nei's unbiased genetic distances were not significantly correlated with spatial distances (r = 0.324, P = 0.074). Fine level cluster analysis An U P G M A analysis with all individual isolates showed that the O. clavigerum population has two genetically distinct groups (Figure 5.4). The clearly separated two clusters were supported with strong bootstrapping values; 93% for Group 1 and 100% for Group 2. Most O. clavigerum isolates belonged to Group 1, whereas Group 2 contained only nine isolates, which included five isolates from Banff (B5, B6, B9, B13, and B26), three isolates from Hidden Valley (HV4, HV7, and HV10) and one isolate from Hellroaring (HR25). Twenty markers, which represent 53% of the Rocky Mountain population-specific markers (20/38), were observed only in the isolates belonging to Group 2 (Table 5.3). Similarly, all of the isolates in Group 1 (but none of the isolates in Group 2) had the GG354 allele. Principal component analysis Twenty-five principal components had eigenvalues greater than one and explained 83% of the variability in the dataset. The first two principal components accounted for 24% and 7% of the variation in the dataset and yielded a plot with two clusters (Fig. 5.5), consistent with the two clusters in the U P G M A results (Fig. 5.4). Group 2 contained seven isolates from Banff and Hidden Valley (B5, B6, B9, B26, HV4, HV7, and HV10) and Group 1 contained the rest of the isolates. Isolates B13 and HR25, which had 132 + 0 100 0.075 0 050 0.025 —I 0 000 97 100 52 61 73 42 HT St.J HR HV BF MP WL Fig. 5.3. An U P G M A (Unweighted Paired Group Method) tree based on Nei's unbiased genetic distance among seven O. clavigerum populations. Genetic distance was computed from 100 polymorphic loci with allele frequencies between 0.05 and 0.95. The bootstrapping values above the branches were inferred from 1000 replicates. Bar indicates Nei's unbiased genetic distance. BF, Banff; HR, Hellroaring; HT, Houston; HV, Hidden Valley; MP, Manning Park; St. J, Fort St. James; WL, Williams Lake. 1 3 3 0.005 changes Fig. 5.4. U P G M A (Unweighted Paired Group Method) tree of 170 O. clavigerum isolates from seven populations showing two distinct groups. The bootstrapping values for Group 1 and Group 2 clades were shown at each node. 1000 replicates were used to infer the bootstrapping values. The tree was constructed based on the 100 polymorphic loci with allele frequencies greater than 0.05 and less than 0.95. 135 Group 2 + HV4 BA9 HV10 B5 A £ A B 6 B26 + HV7 Group 1 * 4 • <K>U ft<> -6 -2 0 Prin 2 • HR o HT + HV x MP • St.J A W L A B F Fig. 5.5. Two groups in the O. clavigerum populations shown by principal components ('Prin') analysis. The first two principal components plotted here accounted for 30.5% of the variation. Only polymorphic loci with allele frequencies greater than 0.05 and less than 0.95 were included in the analysis to reduce statistical bias. HR, Hellroaring; HT, Houston; HV, Hidden Valley; MP , Manning Park; St.J, Fort St. James; WL, Williams Lake; BF, Banff. 136 missing data, were not shown in Fig. 5.4 because of their exclusion by PCA. The plot also showed differentiation among populations in Group 1 by clustering individuals from the same population; the Houston and Fort St. James populations tended to have higher positive values of the second principal component, while the Manning Park and Williams Lake populations tended to have smaller positive or negative values of the second component (Fig. 5.5). Linkage disequilibrium and indices of association Linkage disequilibrium estimated for the seven populations was relatively high at 27.5% and the index of association was significantly different from zero, indicating non-random association among loci across the entire populations (IA = 4.20 and rd = 0.044, P < 0.01). The high index of association may indicate reproductive isolation of O. clavigerum Group 1 from Group 2. Indeed, each group had much lower indices of association when considered separately (Group 1: IA = 1.29 and rd = 0.016, P < 0.01; Group 2: IA = 0.94 and rd = 0.018, P < 0.01). Even considered separately, the indices were significantly different from zero for both groups. Discussion All isolates used in the AFLP analysis were morphologically similar to the O. clavigerum holotype (ATCC 18086). We used the molecular PCR-RFLP marker that we developed to differentiate this species from morphologically similar Ophiostoma and Leptographium species to confirm that all these isolates were O. clavigerum. Given this, we assumed that the fungal isolates examined in this analysis were a single species. Our analyses showed that the O. clavigerum population had a low level of genetic diversity (Hs = 0.0531). Low genetic diversity of O. clavigerum has also been reported using allozymes with isolates from Dendroctonus jeffreyi in the USA (HS=0.07, Six and Paine, 1999). Genetic diversities have been reported for a number of other ophiostomatoid species with different mating types: Ceratocystis eucalypti (Hs = 0.249, outcrossing species), O. piceae (#5=0.369, for polymorphic loci, outcrossing), C. resinifera 137 (Hs = 0.045, outcrossing and selfing), C. virescens (Hs = 0.093, selfing) and Chalara australis (7/5=0.002, an asexually reproducing species) (Gagne et al 2001, Harrington et al. 1998, Morin et al. 2004). Although we cannot exclude that different factors may be involved in the diversity of each species, the Hs values of these fungi suggest that selfing and/or outcrossing can occur in the O. clavigerum population. Further work is required to determine the mating system (selfing and/or outcrossing) of O. clavigerum. However, determining this may be challenging, because sexual structures are not produced in artificial media (Six and Paine 1999). The indices of multilocus association shown in this study deviated significantly from zero, leading to us reject the null hypothesis of complete panmixis. Moreover, linkage disequilibrium was relatively high (27.5%). Therefore, asexual reproduction or sexual reproduction by selfing might be also important in shaping the genetic structure of the fungus. This agreed with our observations that most of O. clavigerum isolates from the beetle or in trees were vegetative mycelia or asexual spores. Its sexual structures have been occasionally found in old galleries (Lee et al. 2003, Robinson-Jeffrey and Davidson 1968). Although only the isolates identified as O. clavigerum were used for AFLP, the data strongly suggested that two genetically distinct groups were present in the Rocky Mountain O. clavigerum populations. The Group 1 and Group 2 with clearly different AFLP profiles were further differentiated by U P G M A and PCA analyses. However, it is not clear whether the two groups were simply subpopulations of the same species, or two different sympatric species. Unfortunately, we could not test the mating between Group 1 and Group 2, as we were unable to obtain the sexual structures on malt extract agar or on lodgepole pine blocks after pairing many isolates derived from the ascospores of a single teleomorph of O. clavigerum. Although we have no biological evidence yet that Group 1 and Group 2 are two different species, the following data indicate that O. clavigerum might be a species complex containing reproductively isolated cryptic species. First, a large number of AFLP markers were observed only in Group 2 suggesting that gene flow between Group 1 and Group 2 was limited. When the two groups were treated as two subpopulations of the O. clavigerum population in A M O V A , the differentiation between the two groups was very high (O = 0.587, P < 0.001). In contrast, the seven geographic populations, which were artificial 138 subdivisions based on accessibility of sampling sites, showed moderate differentiation among the populations (O = 0.143). Second, the multilocus association indices of Group 1 and Group 2 (7A = 1.29 and 0.94 respectively) were much lower than the index computed from the total population (YA= 4.20). If populations are genetically isolated, a comparison of individual fungi taken from different populations would be like a comparison of clonal organisms, because they would not recombine (Taylor et al. 1999). If the O. clavigerum population contained cryptic species, we should consider each species separately in the analyses, in order to avoid biased results. Group2 contained only nine individuals. Excluding it from the analysis lowered the heterozygosity of the O. clavigerum population only slightly (Hs = 0.049 vs. Hs = 0.053; Table 5.4), but did not change which were the most and least heterozygous populations (Hidden Valley and Fort St. James, respectively). For Group 1 only, Mantel test showed no significant correlation between the genetic and geographic distances (r = 0.383, P = 0.069), as was the case for the whole population. A U P G M A tree for Group 1 showed a topology similar to that of the tree for the two groups. The northern B C populations (Houston and Fort St. James) formed a separate clade, while the Rocky Mountain and southern BC populations were in the same clade (data not shown). Unfortunately, the number of individuals in Group 2 was too small to conduct a thorough population analyses. More number of individuals belonging to Group 2 need to be isolated to compare the Group 1 and Group 2 populations. D. ponderosae, the vector of O. clavigerum, is a temperate pest native to North America. The beetle is a natural element in P. contorta ecosystems. An endemic D. ponderosae population can expand to an epidemic level when permitted by environmental factors (mild temperatures and abundant mature hosts) (Paine et al. 1997). The current D. ponderosae outbreak is the third such incident in B C in the last hundred years. The first one occurred near Kootenay National Park and the Chilcotin Plateau in the central region of BC in the 1930s. The second, in the late 1970s and early 1980s, was again in the Kootenay National Park and Chilcotin Plateau region. The current outbreak started near Tweedsmuir Park in east-central BC in 1993. Primarily due to continuous warm winters, this epidemic has spread much 139 further north than the previous outbreaks, reaching Houston, Fort St. James and the Rocky Mountain region, where D. ponderosae might have been present at an endemic level. We found both Groups 1 and 2 in the Rocky Mountain region, but only Group 1 in the other BC regions we surveyed. Given that the epidemic is spreading from central B C to northern BC and the Rocky Mountain region, it is possible that Group 2 isolates belong to the original endemic population in the Rocky Mountain region, while the Group 1 isolates have been recently introduced into this region by the epidemic beetles. In conclusion, this work provided strong evidence that two distinct groups were present in the O. clavigerum population. While gene flow between the two groups appeared to be limited, it is not yet clear whether Group 2 is a cryptic species. Characterizing the two groups more fully requires establishing a mating test and expanding the phylogenetic analyses. In order to better understand the current beetle epidemic, further investigations are needed of the two groups' physiological characteristics and pathogenicity. Group 1 may have been more prevalent because it is a stronger pathogen or is more beneficial to the beetles. Group 2, which was found only in the Rocky Mountain region, might have been better adapted to colder conditions. Investigation with more isolates from the expanding epidemic in Canada and from more areas in the USA, as well as isolates from different hosts, would provide a better understanding of the genetic structure of the O. clavigerum population. 140 References Agapow, P.M. and Burt, A. 2001. Indices of multilocus linkage disequilibrium. Mol. Ecol. Notes 1:101-102. Berryman, A.A. 1972. Resistance of conifers to invasion by bark beetle-fungal associations. Bioscience. 22:598-602. BC Ministry of Forests. 2005. British Columbia's mountain pine beetle action plan 2005-2010. Available from http://www.for.gov.bc.ca/hfp/mountain_pine_beetle/actionplan/2005/intro.htm [cited 30. Aug. 2005]. Excoffier, L. , Smouse, P., and Quattro, J. 1992. 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Biol. 6: 195-200. 143 Chapter 6 Significance of the work and concluding remarks Of the MPB epidemics that have occurred in B C over the last hundred years, the current one is the most damaging economically and environmentally to the province's lodgepole pine forests. Efforts by government, industry, academia and public have focused on containing or reducing the outbreak. A significant research effort has been to predict beetle dynamics with respect to weather and hosts, and on early detection of infested areas in order to salvage infested trees. However, the beetle epidemic cannot be controlled without understanding all the factors that affect tree colonization and beetle development and reproduction. The MPB infestation is a complex interaction among the beetle, tree and its fungal associates. In general, bark beetles and fungi have a mutually beneficial relationship: fungi can reduce the level of host defence that beetles have to overcome, contribute to tree mortality and provide nutrient (Paine et al. 1997). However, the fungal impacts on beetles and trees vary depending on the fungal species. For instance, some fungi might be more nutritious, or more pathogenic than others. Therefore, it is essential to know the fungal species associated with the MPB in order to understand their roles in the beetle infestation. The current work made a big step forward by providing comprehensive information on the fungal associates of MPB, which includes diversity, pathogenicity and population genetic structures. In previous studies on MPB, only a few fungal species (O. clavigerum, O. montium and E. dendroctoni) have been recognized and consequently our knowledge on fungal impacts on the beetle development and tree mortality has been limited to these fungi and thus, incomplete (Six and Paine 1998, Strobel and Sugawara 1986, Yamaoka et al. 1995, Whitney and Farris 1970, Whitney et al. 1987). However, results from an extensive three-year survey that I carried out across BC for this thesis and another study conducted in our lab for the MPB Initiative (Kim et al. 2005) showed that the mycoflora were more diverse than reported in the literature (Robinson 1962, Whitney and Farris 1970, Six 2003). In 144 the current work fungi were isolated not only from beetles but also from the beetle galleries and sapwood, which allowed us to have an insight into fungal colonization and dissemination. The MPB-fungal associates found this work included ascomycetes and basidiomycetes. The dominant species varied with the source of isolation; on the exoskeletons of beetles, O. montium and the O. minutum-like species were dominant, while in the sapwood, O. montium and O. clavigerum were prevalent. The association of the O. minutum-like species with the MPB was reported here for the first time. Considering its high frequency on the beetle, it would be worth to examine whether O. minutum-like species affects the beetle development. The Ophiostoma minutum-like species requires additional genetic and morphological work. Although O. minutum has been described in the literature, no holotype is available, as the original one from Europe has been lost during the Second World War. Thus, O. minutum needs to be described again before specimens from other countries, including Canada, can be accurately identified as O. minutum or as different species. In addition, the possibility that O. minutum might be a species complex is further investigated in our lab. The fungi reported here were obtained from different sources and from many sites, and contributed to a more comprehensive description of the mycoflora associated with the MPB-lodgepole pine ecosystem. However, we recognized that the sampling could be improved. First, sampling more trees and beetles at each site would provide statistically stronger data, but would require more resources. Second, this work focused only on the exoskeleton of the MPB due to technical difficulties in aseptically dissecting mycangia and the gut of the beetle. Including more parts of the MPB body (mycangia, exoskeleton and gut) in the fungal sampling would allow us to confirm which fungi are better nutritional sources and which are more selectively taken by the beetle. Third, although endemic sites are more difficult to locate than epidemic ones, including endemic sites in the sampling survey would permit comparing endemic and epidemic flora, which could help clarify the relationship between fungal aggressiveness and the beetle outbreak. In the future, to determine the roles of fungi in the MPB infestation, all fungal associates should ideally be investigated. Similarly, the interaction among different fungal species, either antagonistic or beneficial, also needs to be examined. 145 While accurate identification of species is critical in any research using living organisms, the ophiostomatoid fungi are difficult to identify for several reasons. First, morphological differences between some species are small. Second, key morphological structures, especially sexual ones, may take more than a month to be observed in artificial conditions, and often are not produced under such conditions. Third, many species have more than one asexual structure, and these are not always observed simultaneously, which can lead to confusion in differentiating species that share these structures. Further, asexual structures degenerate easily after repeated subculture in artificial media. Finally, criteria for taxonomic classification are evolving. In ophiostomatoid fungi, genus classification based on anamorphs has been frequently restructured. This is partially related to improved microscopy, which has allowed more precise observation of conidia ontogeny and anamorph structures. Most of the ophiostomatoid fungi have undergone name changes (Seifert et al. 1993, Upadhyay 1981), and the same fungus has often been cited with different names in the scientific literature. Given these difficulties, it is not surprising that misidentification has been frequently reported. Molecular tools have made identification more accurate. As reported in Chapter 2, PCR of either the /^-tubulin gene or rDNA allowed us to differentiate O. montium and O. ips, which have been confused for an extensive period of time, even in worldwide fungal culture collections. Similarly, developing a specific PCR-RFLP marker for O. clavigerum enabled us to differentiate this species from morphologically similar species such as O. aureum, O. robustum, L. terbrantis, L. lundbergii, and L. pyrinum. The marker is simple to use, requiring only one enzyme reaction {Hinfl) following the amplification of a single gene (/?-tubulin). Further, the marker requires only a small amount of fungal mass to yield enough DNA for the reaction. With molecular markers, one can identify fungal species regardless of the production of certain structures, more reliably and quickly than with the conventional morphological method. During the study, molecular markers enabled me to identify a large number of fungal isolates collected through surveys. Chapter 2 described a new fungal species, L. longiclavatum, which was found during the survey. Its distinct morphological characteristics were described in detail. The phylogenetic relationship between L. longiclavatum and other morphologically similar ophiostomatoid fungi was thoroughly investigated using 146 multi-gene sequences from protein-coding genes (/J-tubulin, actin, and glyceraldehyde-3-phosphate dehydrogenase), ribosomal genes, and combined genes. All the parsimony trees indicated that L. longiclavatum was a new taxon. Although rDNA has been most commonly used for phylogenetic analyses (Bruns et al. 1991), the data also suggested that the protein-coding genes were better for investigating phylogenetic relationships among the ophiostomatoid fungi. Some of the MPB fungal associates significantly contribute to the mortality of infested trees. In order to respond efficiently to the MPB epidemic, it is necessary to assess the pathogenicity of recognized pathogens and quickly identify new ones. The information available on the pathogenicity of the beetle's fungal associates was incomplete and limited to two known fungi: O. clavigerum and O. montium (Strobel and Sugawara 1986, Yamaoka et al. 1995). In this study, among the MPB fungal associates, the deep colonizer, O. clavigerum, O. montium, and L. longiclavatum were the major species in the infested sapwood. Fungal growth in sapwood interrupts water transportation in hosts through unknown mechanisms and resulted in tree death. The pathogenicity of O. clavigerum has been confirmed, while the pathogenicity of O. montium is not yet certain. Chapter 4 reported the pathogenicity of L. longiclavatum. The symptoms of the inoculated hosts, foliage colour, lesions in the phloem, and occlusions and moisture content in the sapwood, suggested that L. longiclavatum is pathogenic. In nature, although the frequency of L. longiclavatum in sapwood was lower than that of O. clavigerum, L. longiclavatum may contribute to the mortality of MPB-infested pines. Because only one strain of each fungal species was tested, it is difficult to conclude that the degree of virulence of these two fungal species was similar and more work is required to resolve this issue. The data generated in this study examined a large number of mature pines over an extended period of time. Fungal inoculation methods using extensive girdling of the phloem might be too traumatic for the tree and results could be questionable as in previous study with O. montium (Strobel and Sugawara 1986). In this work a cork borer technique was used in order to minimize the effects of wounding; thus, the data here more accurately showed fungal pathogenicity. Although the pathogenicity of L. longiclavatum was clearly shown in this study, additional inoculation tests in a different year or at a different site could further confirm the results. In addition, the timing of inoculation 147 could be adjusted to late July or early August, when the MPB usually attacks trees in nature. Due to the sudden fire in the Okanagan area (where the experimental site was located) in 2003, the inoculation was delayed until the end of September, 2003. The efficiency of host defence against fungi could be slightly different between these months, and therefore a different fungal inoculum density might be needed to observe similar symptoms. Knowledge of fungal genetic diversity could be important to assess variations in their pathogenicity over large epidemic areas. Depending on the distribution of fungi of varying virulence, discriminating management strategies might be more effectively employed in different regions. However, none of the MPB fungal associates have been studied with respect to genetic structure. Chapter 5 was the first report that provided genetic information on O. clavigerum, which is one of the most virulent fungal associates of the MPB. The A F L P data of O. clavigerum with various analyses showed that two genetically distinct groups existed in the O. clavigerum population. This is an important finding because the two O. clavigerum groups may differently interact with MPB and hosts. However, characterizing differences in physiology and pathogenicity between the two groups was beyond the scope of this work and remains to be determined. The two groups might have evolved independently in BC and in the Rocky Mountain area. As the beetle epidemic in BC spread to Rocky Mountain area, the fungal group originating in BC might have been vectored to the Rocky Mountain area. It is not yet clear whether the two groups are reproductively isolated. More work on the mating system, as well as phylogenetic analyses, would provide more information on the two groups. In addition, although sampling at a sufficient number of endemic sites could be a challenge, fungi isolated at endemic sites would provide more information on the relationship between fungal and beetle populations (endemic or epidemic). Further, host species of the beetle might affect the genetic diversity of the fungal associates. The current MPB epidemic area in the USA spans from Washington, Oregon, and California inland to Colorado and New Mexico, where the major MPB hosts (P. contorta and P. ponderosa) and other pines (e.g. P. lambertiana, P. albicaulis and P. flexilis) have been infested. Sampling of O. clavigerum isolates from a wider range of infestation sites in 148 the USA, especially from California where P. lambertiana grows commonly, could provide more information on the genetic diversity on this fungal species. The current work has increased our knowledge about the fungal associates of the MPB in their diversity, pathogenicity and population genetic structures. Future work to better understand the effects of fungi on the beetle and hosts in the complex MPB-fungi-tree ecosystem should involve tree physiologists, entomologists as well as mycologists. 149 Reference Bruns, T.D., White, T.J., and Taylor J.W. 1991. Fungal molecular systematics. Ann. Rev. Ecol. and Syst. 22:525-564. Kim, J.J., Allen, E.A. , Humble, L . M . , and Breuil, C. 2005. Ophiostomatoid and basidiomycetous fungi associated with green, red and grey lodgepole pines after mountain pine beetle (Dendroctonus ponderosae) infestation. Can. J. For. Res. 35: 274-284. Paine, T.D., Raffa, K.F. , and Harrington, T.C. 1997. Interactions among Solytid bark beetles, their associated fungi, and live host conifers. Ann. Rew. Entomol. 42: 179-206. Robinson, R.C. 1962. Blue stain fungi in lodgepole pine (Pinus contorta Dougl. var. latifolia Engelm.) infested by the mountain pine beetle (Dendroctonus monticolae Hopk.). Can. J. Bot. 40: 609-614. Seifert, K.A. , Wingfield, M.J., and Kendrick, W.B. 1993. A nomenclator for described species of Ceratocystis, Ophiostoma, Ceratocystiopsis, Ceratostomella and Sphaeronaemella. In: Ceratocystis and Ophiostoma taxonomy, ecology and pathogenicity (Wingfield, M.J., Seifert, K . A . and Webber, J.F., Eds.), pp. 269-287. The American Phytopathological Society Press, St. Paul, Minnesota. Six, D.L. 2003. A comparison of mycangial and phoretic fungi of individual mountain pine beetle. Can. J. For. Res. 33: 1331-1334. Six, D.L., and Paine, T.D. 1998. Effects of mycangial fungi and host tree species on progeny survival and emergence of Dendroctonus ponderosae (Coleoptera: Scolytidae). Environ. Entomol. 27: 1393- 1401. Strobel, G.A., and Sugawara, F. 1986. The pathogenicity of Ceratocystis montia to lodgepole pine. Can. J. Bot. 64(1): 113-116. Upadhyay, H.P. 1981. A monograph of Ceratocystis and Ceratocystiopsis. University of Georgia Press, Athens, GA. Whitney H.S., and Farris, S.H. 1970. Maxillary Mycangium in the Mountain Pine Beetle. Science. 167: 54-55. Whitney, H.S., Bandoni, R.J., and Oberwinkler, F. 1987. Entomocorticium dendroctoni gen. et sp. nov. (Basidiomycotina), a possible nutritional symbiote of the mountain pine beetle in lodgepole pine in British Columbia. Can. J. Bot. 65: 95-102. Yamaoka, Y. , Hiratsuka, Y. , and Maruyama, P.J. 1995. The ability of Ophiostoma clavigerum to kill mature lodgepole-pine trees. Eur. J. For. Path. 25: 401-404. 150 Appendix A Cumulative Percentage of Pine Killed 2 0 0 4 Data A map showing the mountain pine beetle-infested area in British Columbia, Canada. : pictures by The BC Ministry of Forest and Range. http://www.for.gov.b.cca/hfp/mountain_pine_beetle 151 Appendix B Stereomicroscopic pictures of the mountain pine beetle, Dendroctonus ponderosae. : pictures by Canadian Forest Service, Natural Resources Canada, available at http://www.pfc.cfs.nrcan.gc.ca/entomology/mpb/index_e.html 152 

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