Antonie van Leeuwenhoek

, Volume 100, Issue 2, pp 231–244 | Cite as

Fungal associates of the lodgepole pine beetle, Dendroctonus murrayanae

  • Diana L. Six
  • Z. Wilhelm de Beer
  • Tuan A. Duong
  • Allan L. Carroll
  • Michael J. Wingfield
Original Paper


Bark beetles are well known vectors of ophiostomatoid fungi including species of Ophiostoma, Grosmannia and Ceratocystis. In this study, the most common ophiostomatoid fungi associated with the lodgepole pine beetle, Dendroctonus murrayanae, were characterized. Pre-emergent and post-attack adult beetles were collected from lodgepole pines at four sites in British Columbia, Canada. Fungi were isolated from these beetles and identified using a combination of morphology and DNA sequence comparisons of five gene regions. In all four populations, Grosmannia aurea was the most common associate (74–100% of all beetles) followed closely by Ophiostoma abietinum (29–75%). Other fungi isolated, in order of their relative prevalence with individual beetles were an undescribed Leptographium sp. (0–13%), Ophiostoma ips (0–15%), Ophiostoma piliferum (0–11%), a Pesotum sp. (0–11%) and Ophiostoma floccosum (0–1%). Comparisons of the DNA sequences of Leptographium strains isolated in this study, with ex-type isolates of G. aurea, Grosmannia robusta, Leptographium longiclavatum, and Leptographium terebrantis, as well as with sequences from GenBank, revealed a novel lineage within the Grosmannia clavigera complex. This lineage included some of the D. murrayane isolates as well as several isolates from previous studies referred to as L. terebrantis. However, the monophyly of this lineage is not well supported and a more comprehensive study will be needed to resolve its taxonomic status as one or more novel taxa.


Bark beetle Symbiosis Leptographium Ophiostoma Grosmannia aurea 



We thank Staffan Lindgren for collections of dispersed D. murrayanae and Kathy Bleiker for her help with isolations. This study was supported by National Science Foundation grant OISE-0434171 awarded to DLS and a Natural Resources Canada, Canadian Forest Service Mountain Pine Beetle Initiative Grant to ALC. We also acknowledge the members of the Tree Co-operative Programme and the THRIP initiative of the Department of Trade and Industry, South Africa for financial support.

Supplementary material

10482_2011_9582_MOESM1_ESM.doc (253 kb)
Supplementary material 1 (DOC 253 kb)


  1. Aghayeva DN, Wingfield MJ, De Beer ZW, Kirisits T (2004) Two new Ophiostoma species with Sporothrix anamorphs from Austria and Azerbaijan. Mycologia 96:866–878PubMedCrossRefGoogle Scholar
  2. Bright DE (1976) The bark beetles of Canada and Alaska: Coleoptera, Scolytidae. Canadian Department of Agriculture Publ. 1576, OttawaGoogle Scholar
  3. Carbone I, Kohn LM (1999) A method for designing primer sets for speciation studies in filamentous Ascomycetes. Mycologia 91:553–556CrossRefGoogle Scholar
  4. De Beer ZW, Harrington TC, Vismer HF, Wingfield BD, Wingfield MJ (2003) Phylogeny of the Ophiostoma stenocerasSporothrix schenckii complex. Mycologia 95:434–441PubMedCrossRefGoogle Scholar
  5. Furniss RL, Carolin VM (1977) Western forest insects. Miscellaneous Publication No. 1339, US Department of Agriculture Forest Service, Washington, DCGoogle Scholar
  6. Furniss RL, Kegley S (2008) Biology of Dendroctonus murrayanae (Coleoptera: Curculionidae: Scolytinae) in Idaho and Montana and comparative taxonomic notes. Ann Entomol Soc Am 101:1010–1016CrossRefGoogle Scholar
  7. Furniss MM, Harvey AE, Solheim H (1995) Transmission of Ophiostoma ips (Ophiostomatales: Ophiostomataceae) by Ips pini (Coleoptera: Scolytidae). Ann Entomol Soc Am 88:653–660Google Scholar
  8. Glass NL, Donaldson GC (1995) Development of primer sets designed for use with the PCR to amplify conserved genes from filamentous Ascomycetes. Appl Environ Microbiol 61:1323–1330PubMedGoogle Scholar
  9. Grylls BT, Seifert KA (1993) A synoptic key to species of Ophiostoma, Ceratocystis, and Ceratocystiopsis. In: Wingfield MJ, Seifert KA, Webber JF (eds) Ceratocystis and Ophiostoma: taxonomy ecology and pathogenicity. APS Press, St. Paul, pp 261–268Google Scholar
  10. Guindon S, Gascuel O (2003) A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst Biol 52:696–704PubMedCrossRefGoogle Scholar
  11. Harrington TC (1982) Verticicladiella wageneri: taxonomy and vector relations. PhD Thesis, University of California, Berkeley, pp 1–113Google Scholar
  12. Harrington TC (1988) Leptographium species, their distributions, hosts and insect vectors. In: Harrington TC, Cobb FW Jr (eds) Leptographium root diseases on conifers. APS Press, St. Paul, pp 1–39Google Scholar
  13. Harrington TC (2005) Ecology and evolution of mycophagous bark beetles and their fungal partners. In: Vega FE, Blackwell M (eds) Insect-fungal associations: ecology and evolution. Oxford University Press, New York, pp 257–291Google Scholar
  14. Harrington TC, Cobb FW Jr (1983) Pathogenicity of Leptographium and Verticicladiella spp. isolated from roots of western North American conifers. Phytopathology 73:596–599CrossRefGoogle Scholar
  15. Jacobs K, Wingfield MJ (2001) Leptographium species: tree pathogens, insect associates, and agents of blue stain. APS Press, St. PaulGoogle Scholar
  16. Jacobs K, Bergdahl DR, Wingfield MJ, Halik S, Seifert KA, Bright DE, Wingfield BD (2004) Leptographium wingfieldii introduced into North America and found associated with exotic Tomicus piniperda and native bark beetles. Mycol Res 108:411–418PubMedCrossRefGoogle Scholar
  17. Katoh K, Toh H (2008) Recent developments in the MAFFT multiple sequence alignment program. Brief Bioinform 9:286–298PubMedCrossRefGoogle Scholar
  18. Kim J-J, Allen EA, Humble LM, 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 35:274–284CrossRefGoogle Scholar
  19. Kirisits T (2004) Fungal associates of European bark beetles with special emphasis on the Ophiostomatoid fungi. In: Lieutier F, Day KR, Battisti A, Gregoire J-C, Evans H (eds) Bark and wood boring insects in living trees in Europe a synthesis. Kluwer Academic Publishers, Dordrecht, pp 181–236CrossRefGoogle Scholar
  20. Lee S, Kim J-J, Fung S, Breuil C (2003) A PCR-RFLP marker distinguishing Ophiostoma clavigerum from morphologically similar Leptographium species associated with bark beetles. Can J Bot 81:1104–1113CrossRefGoogle Scholar
  21. Lee S, Kim J-J, Breuil C (2005) Leptographium longiclavatum sp. nov., a new species associated with the mountain pine beetle, Dendroctonus ponderosae. Mycol Res 109:1162–1170PubMedCrossRefGoogle Scholar
  22. Lim WL, Alamouti SM, Kim J-J, Lee S, Breuil C (2004) Multigene phylogenies of Ophiostoma clavigerum and closely related species from bark beetle-attacked Pinus in North America. FEMS Microbiol Lett 237:89–96PubMedCrossRefGoogle Scholar
  23. Lu M, Zhou XD, De Beer ZW, Wingfield MJ, Sun J-H (2009) Ophiostomatoid fungi associated with the invasive pine-infesting bark beetle, Dendroctonus valens, in China. Fungal Divers 38:133–145Google Scholar
  24. Marmolejo JG, Butin H (1990) New conifer-inhabiting species of Ophiostoma and Ceratocystiopsis (Ascomyctetes: Microascales) from Mexico. Sydowia 42:193–199Google Scholar
  25. O’Donnell K, Cigelnik E (1997) Two divergent intragenomic rDNA ITS2 types within a monophyletic lineage of the fungus Fusarium are nonorthologous. Mol Phylogenet Evol 7:103–116PubMedCrossRefGoogle Scholar
  26. Posada D (2008) jModelTest: phylogenetic model averaging. Mol Biol Evol 25:1253–1256PubMedCrossRefGoogle Scholar
  27. Robinson-Jeffrey RC, Davidson RW (1968) Three new Europhium species with Verticicladiella imperfect states on blue-stained pine. Can J Bot 46:1523–1527CrossRefGoogle Scholar
  28. Roe AD, Rice AV, Bromilow SE, Cooke JEK, Sperling FAH (2010) Multilocus species identification and fungal DNA barcoding: insights from blue stain fungal symbionts of the mountain pine beetle. Mol Ecol Resour 10:946–959PubMedCrossRefGoogle Scholar
  29. Ronquist F, Huelsenbeck JP (2003) MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19:1572–1574PubMedCrossRefGoogle Scholar
  30. Safranyik L, Shore TL, Linton DA (1999) Attack by bark beetles (Coleoptera: Scolytidae) following spacing of mature lodgepole pine (Pinaceae) stands. Can Entomol 131:671–685CrossRefGoogle Scholar
  31. Safranyik L, Shore TL, Carroll AL, Linton DA (2004) Bark beetle (Coleoptera: Scolytidae) diversity in spaced and unmanaged lodgepole pine (Pinaceae) in southeastern British Columbia. For Ecol Manag 200:23–38CrossRefGoogle Scholar
  32. Seifert KA (1993) Sapstain of commercial lumber by species of Ophiostoma and Ceratocystis. In: Wingfield MJ, Seifert KA, Webber J (eds) Ceratocystis and Ophiostoma: taxonomy, ecology and pathogenicity. American Phytopathological Society, St. Paul, pp 141–152Google Scholar
  33. Six DL (2003) Bark beetle-fungus symbioses. In: Bourtzis K, Miller TA (eds) Insect symbiosis. CRC Press, Boca Raton, pp 99–116Google Scholar
  34. Six DL, Klepzig KD (2004) Dendroctonus bark beetles as model systems for studies on symbiosis. Symbiosis 37:207–232Google Scholar
  35. Six DL, Wingfield MJ (2011) The role of phytopathogenicity in bark beetle–fungus symbioses: a challenge to the classic paradigm. Annu Rev Entomol 56:255–272PubMedCrossRefGoogle Scholar
  36. Six DL, Harrington TC, Steimel J, McNew D, Paine TD (2003) Genetic relationships among Leptographium terebrantis and the mycangial fungi of three western Dendroctonus bark beetles. Mycologia 95:781–792PubMedCrossRefGoogle Scholar
  37. Six D, Stone WD, De Beer ZW, Woolfolk S (2009) Ambrosiella beaveri sp. nov., associated with an exotic ambrosia beetle, Xylosandrus mutilatus (Coleoptera: Curculionidae, Scolytinae), in Mississippi, USA. Antonie van Leeuwenhoek 96:17–29PubMedCrossRefGoogle Scholar
  38. Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol 24:1596–1599PubMedCrossRefGoogle Scholar
  39. Upadhyay HP (1981) A monograph of Ceratocystis and Ceratocystiopsis. University of Georgia Press, AthensGoogle Scholar
  40. Vilgalys R, Hester M (1990) Rapid genetic identification and mapping of enzymatically amplified ribosomal DNA from several Cryptococcus species. J Bacteriol 172:4238–4246PubMedGoogle Scholar
  41. White TJ, Bruns T, Lee S, Taylor J (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ (eds) PCR protocols: a guide to methods and application. Academic Press, San Diego, pp 315–322Google Scholar
  42. Wood SL (1982) The bark and ambrosia beetles of North and Central America (Coleoptera: Scolytidae), a taxonomic monograph. Great Basin Nat Mem 6:1–1356Google Scholar
  43. Zhou XD, De Beer ZW, Wingfield MJ (2006) DNA sequence comparisons of Ophiostoma spp., including Ophiostoma aurorae sp. nov., associated with bark beetles in South Africa. Stud Mycol 55:269–277PubMedCrossRefGoogle Scholar
  44. Zipfel RD, De Beer ZW, Jacobs K, Wingfield BD, Wingfield MJ (2006) Multigene phylogenies define Ceratocystiopsis and Grosmannia distinct from Ophiostoma. Stud Mycol 55:75–97PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2011

Authors and Affiliations

  • Diana L. Six
    • 1
  • Z. Wilhelm de Beer
    • 2
  • Tuan A. Duong
    • 3
  • Allan L. Carroll
    • 4
  • Michael J. Wingfield
    • 2
  1. 1.Department of Ecosystem and Conservation SciencesCollege of Forestry and Conservation, The University of MontanaMissoulaUSA
  2. 2.Department of Microbiology and Plant PathologyForestry and Agricultural Biotechnology Institute, University of PretoriaPretoriaSouth Africa
  3. 3.Department of Genetics, Forestry and Agricultural Biotechnology InstituteUniversity of PretoriaPretoriaSouth Africa
  4. 4.Department of Forest SciencesUniversity of British ColumbiaVancouverCanada

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