Antonie van Leeuwenhoek

, Volume 107, Issue 2, pp 547–563 | Cite as

Taxonomy and phylogeny of the Leptographium procerum complex, including Leptographium sinense sp. nov. and Leptographium longiconidiophorum sp. nov.

  • Mingliang Yin
  • Tuan A. Duong
  • Michael J. Wingfield
  • XuDong Zhou
  • Z. Wilhelm de Beer
Original Paper


Leptographium procerum (Ophiostomatales, Ascomycota) is a well-known fungal associate of pine root-infesting bark beetles and weevils, occurring in several countries of the world. The fungus is not a primary pathogen but has been associated with white pine root decline in the USA and with serious damage caused by the introduced red turpentine beetle (RTB) Dendroctonus valens in China. Several species closely related to L. procerum have been described during the past decade. The aim of this study was to reevaluate species boundaries in the L. procerum complex using multigene phylogenetic analyses and morphological comparisons. Phylogenetic analyses of seven gene regions (ITS2-LSU, actin, β-tubulin, calmodulin, translation elongation factor 1-α, and the mating type genes MAT1-1-3 and MAT1-2-1) distinguished between nine species in the complex. These included L. procerum, L. bhutanense, L. gracile, L. profanum, L. pini-densiflorae, L. sibiricum, L. sinoprocerum, as well as two new species described here as Leptographium sinense sp. nov. from Hylobitelus xiaoi on Pinus elliottii in China, and Leptographium longiconidiophorum sp. nov. from Pinus densiflora in Japan. Leptographium latens is reduced to synonymy with L. gracile, and an epitype is designated for L. procerum, because a living culture associated with the holotype of L. procerum did not exist. Amplification patterns of the mating type genes suggest that all known species in the L. procerum complex are heterothallic, although sexual states have not been observed for any of the species. The results also suggest that Eastern Asia is most probably the centre of species diversity for the L. procerum complex.


Bark beetle associates Epitype Leptographium Ophiostomatales Phylogeny Taxonomy 



This study was initiated through the bilateral agreement between the Governments of South Africa and China, and we are grateful for the funding via projects 2012DFG31830 (International Science & Technology Cooperation Program of China), 2010KJCX015-03 (Forestry Science and Technology Innovation Project of Guangdong Province of China). We acknowledge members of Tree Protection and Cooperation Programme (TPCP), the National Research Foundation (NRF), the Department of Science and Technology (DST)/NRF, Center of Excellence in Tree Health Biotechnology (CTHB) and the University of Pretoria, Pretoria, South Africa. We also thank Mr. Runlei Chang for assistance with the fieldwork, and Ms. Yalin Fu for assistance with fungal isolations.

Supplementary material

10482_2014_351_MOESM1_ESM.pdf (450 kb)
Supplementary material 1 (PDF 1050 kb)


  1. Alexander SA, Horner WE, Lewis KJ (1988) Leptographium procerum as a pathogen on pines. In: Harrington TC, Cobb FW Jr (eds) Leptographium root disease on conifers. The American Phytopathological Society Press, St. Paul, pp 97–122Google Scholar
  2. Cognato AI, Sun J-H, Anducho-Reyes MA, Donald RO (2005) Genetic variation and origin of red turpentine beetle (Dendroctonus valens LeConte) introduced to the People’s Republic of China. Agric For Entomol 7:87–94CrossRefGoogle Scholar
  3. De Beer ZW, Wingfield MJ (2013) Emerging lineages in the Ophiostomatales. In: Seifert KA, De Beer ZW, Wingfield MJ (eds) The ophiostomatoid fungi: expanding frontiers, CBS biodiversity series 12. CBS Press, Utrecht, pp 21–46Google Scholar
  4. DiGuistini S, Wang Y, Liao NY, Taylor G, Tanguay P, Feau N, Henrissat B, Chan SK, Hesse-Orce U, Alamouti SM, Tsui CKM, Docking RT, Levasseur A, Haridas S, Robertson G, Birol I, Holt RA, Marra MA, Hamelin RC, Hirst M, Jones SJM, Bohlmann J, Breuil C (2011) Genome and transcriptome analyses of the mountain pine beetle-fungal symbiont Grosmannia clavigera, a lodgepole pine pathogen. PNAS 108:2504–2509PubMedCentralPubMedCrossRefGoogle Scholar
  5. Duong TA, De Beer ZW, Wingfield BD, Wingfield MJ (2012) Phylogeny and taxonomy of species in the Grosmannia serpens complex. Mycologia 104:715–732PubMedCrossRefGoogle Scholar
  6. Duong TA, De Beer ZW, Wingfield BD, Wingfield MJ (2013) Characterization of the mating-type genes in Leptographium procerum and Leptographium profanum. Fungal Biol 117:411–421PubMedCrossRefGoogle Scholar
  7. Duong TA, De Beer ZW, Wingfield BD, Eckhardt LG, Wingfield MJ (2014) Microsatellite and mating type markers reveal unexpected patterns of genetic diversity in the pine root infecting fungus Grosmannia alacris. Plant Pathol. doi: 10.1111/ppa.12231 Google 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 61:1323–1330PubMedCentralPubMedGoogle Scholar
  9. Goidànich (1936) II genere di Ascomiceti ‘Grosmanni’ G. Goid. Boll Stn Patol Veg Roma 16:26–40Google Scholar
  10. Grobbelaar JW, De Beer ZW, Bloomer P, Wingfield MJ, Wingfield BD (2010) Ophiostoma tsotsi sp. nov., a wound-infesting fungus of hardwood trees in Africa. Mycopathol 169:413–423CrossRefGoogle Scholar
  11. Guindon S, Dufayard JF, Lefort V, Anisimova M, Hordijk W, Gascuel O (2010) New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0. Syst Biol 59:307–321PubMedCrossRefGoogle Scholar
  12. Hawksworth DL (2011) A new dawn for the naming of fungi: impacts of decisions made in Melbourne in July 2011 on the future publication and regulation of fungal names. IMA Fungus 2:155–162PubMedCentralPubMedCrossRefGoogle Scholar
  13. Jacobs K, Wingfield MJ (2001) Leptographium species: tree pathogens, insect associates, and agents of blue-stain. The American Phytopathological Society Press, St. PaulGoogle Scholar
  14. Jacobs K, Wingfield MJ, Pashenova NV, Vetrova VP (2000) A new Leptographium species from Russia. Mycol Res 104:1524–1529CrossRefGoogle Scholar
  15. 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
  16. Jacobs K, Eckhardt LG, Wingfield MJ (2006) Leptographium profanum sp. nov., a new species from hardwood roots in North America. Can J Bot 84:759–766CrossRefGoogle Scholar
  17. Jankowiak R (2006) Fungi associated with Tomicus piniperda in Poland and assessment of their virulence using Scots pine seedlings. Ann For Sci 63:801–808CrossRefGoogle Scholar
  18. Jankowiak R (2012) Ophiostomatoid fungi associated with Ips sexdentatus on Pinus sylvestris in Poland. Dendrobiology 68:43–54Google Scholar
  19. Jankowiak R, Bilański P (2013a) Diversity of ophiostomatoid fungi associated with the large pine weevil, Hylobius abietis and infested Scots pine seedlings in Poland. Ann For Sci 70:391–402CrossRefGoogle Scholar
  20. Jankowiak R, Bilański P (2013b) Association of the pine-infesting Pissodes species with ophiostomatoid fungi in Poland. Eur J For Res 132:523–534CrossRefGoogle Scholar
  21. Jankowiak R, Bilański P (2013c) Ophiostomatoid fungi associated with root-feeding bark beetles in Poland. For Pathol 43:422–428Google Scholar
  22. Jankowiak R, Kolařík M (2010) Diversity and pathogenicity of ophiostomatoid fungi associated with Tetropium species colonizing Picea abies in Poland. Folia Microbiol 55:145–154CrossRefGoogle Scholar
  23. Jankowiak R, Bilański P, Kolařík M, Wasiuta D (2012) Root-colonizing ophiostomatoid fungi associated with dying and dead young Scots pine in Poland. For Pathol 42:492–500CrossRefGoogle Scholar
  24. Katoh K, Misawa K, Kuma K, Miyata T (2002) MAFFT: a novel method for rapid multiple sequence alignment based on fast Fourier transform. Nucleic Acids Res 30:3059–3066PubMedCentralPubMedCrossRefGoogle Scholar
  25. Kendrick WB (1962) The Leptographium complex Verticicladiella hughes. Can J Bot 40:772–797CrossRefGoogle Scholar
  26. Kim GH, Kim JJ, Lim YW, Breuil C (2005) Ophiostomatoid fungi isolated from Pinus radiata logs imported from New Zealand to Korea. Can J Bot 83:272–278CrossRefGoogle Scholar
  27. Lackner AL, Alexander SA (1982) Occurrence and pathogenicity of Verticicladiella procera in Christmas tree plantations in Virgina. Plant Dis 66:211–212CrossRefGoogle Scholar
  28. Lagerberg T, Lundberg G, Melin E (1927) Biological and practical researches into blueing in pine and spruce. Sven Skogsvårdsfören Tidskr 25:145–272Google Scholar
  29. Lim YW, Massoumi Alamouti S, Kim JJ, 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
  30. Linnakoski R, De Beer ZW, Duong TA, Niemelä P, Pappinen A, Wingfield MJ (2012) Grosmannia and Leptographium spp. associated with conifer-infesting bark beetles in Finland and Russia, including Leptographium taigense sp. nov. Antonie Van Leeuwenhoek 102:375–399PubMedCrossRefGoogle Scholar
  31. Lu Q, Decock C, Zhang XY, Maraite H (2008) Leptographium sinoprocerum sp. nov., an undescribed species associated with Pinus tabuliformis-Dendroctonus valens in northern China. Mycologia 100:275–290PubMedCrossRefGoogle Scholar
  32. Lu M, Zhou XD, De Beer ZW, Wingfield MJ, Sun JH (2009a) Ophiostomatoid fungi associated with the invasive pine-infesting bark beetle, Dendroctonus valens, in China. Fungal Divers 38:133–145Google Scholar
  33. Lu Q, Decock C, Zhang XY, Maraite H (2009b) Ophiostomatoid fungi (Ascomycota) associated with Pinus tabuliformis infested by Dendroctonus valens (Coleoptera) in northern China and an assessment of their pathogenicity on mature trees. Antonie Van Leeuwenhoek 96:275–293PubMedCrossRefGoogle Scholar
  34. Lu M, Wingfield MJ, Gillette NE, Mori SR, Sun JH (2010) Complex interactions among host pines and fungi vectored by an invasive bark beetle. New Phytol 187:859–866PubMedCrossRefGoogle Scholar
  35. Masuya H, Kaneko S, Yamaoka Y, Ohsawa M (1999) Comparisons of ophiostomatoid fungi associated with Tomicus piniperda and T. minor in Japanese red pine. J For Res 4:131–135CrossRefGoogle Scholar
  36. Masuya H, Wingfield MJ, Kaneko S, Yamaoka Y (2000) Leptographium pini-densiflorae sp. nov. from Japanese red pine. Mycoscience 41:425–430CrossRefGoogle Scholar
  37. Masuya H, Kaneko S, Yamaura Y, Yamaoka Y (2009) Ophiostomatoid fungi isolated from Japanese red pine and their relationships with bark beetles. Mycoscience 50:212–223CrossRefGoogle Scholar
  38. Matusick G, Somers G, Eckhardt L (2012) Root lesions in large loblolly pine (Pinus taeda L.) following inoculation with four root-inhabiting ophiostomatoid fungi. For Pathol 42:37–43CrossRefGoogle Scholar
  39. Miao ZW, Chou WM, Huo FY, Wang XL, Fang JX, Zhao MM (2001) Biology of Dendroctonus valens in Shanxi province. Shanxi For Sci Technol 23:34–37Google Scholar
  40. O’Donnell K, Cigelnik E (1997) Two divergent intragenomic rDNA ITS2 types within a monophyletic lineage of the fungus Fusarium are nonorthologous. Mol Phylogent Evol 7:103–116CrossRefGoogle Scholar
  41. Paciura D, De Beer ZW, Jacobs K, Zhou XD, Ye H, Wingfield MJ (2010) Eight new Leptographium species associated with tree-infesting bark beetles in China. Persoonia 25:94–108PubMedCentralPubMedCrossRefGoogle Scholar
  42. Posada D (2008) jModelTest: phylogenetic model averaging. Mol Biol Evol 25:1253–1256PubMedCrossRefGoogle Scholar
  43. Rambaut A, Drummond AJ (2007) Tracer 1.4. Available at Accessed 12 Dec 2014
  44. Rayner RW (1970) A mycological color chart. CMI and British Mycological Society, KewGoogle Scholar
  45. Robert V, Vu D, Amor AB, van de Wiele N, Brouwer C, Jabas B, Szoke S, Dridi A, Triki M, Ben Daoud S, Chouchen O, Vaas L, de Cock A, Stalpers JA, Stalpers D, Verkley GJ, Groenewald M, Dos Santos FB, Stegehuis G, Li W, Wu L, Zhang R, Ma J, Zhou M, Gorjon SP, Eurwilaichitr L, Ingsriswang S, Hansen K, Schoch C, Robbertse B, Irinyi L, Meyer W, Cardinali G, Hawksworth DL, Taylor JW, Crous PW (2013) MycoBank gearing up for new horizons. IMA Fungus 4:371–379PubMedCentralPubMedCrossRefGoogle Scholar
  46. 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
  47. Ronquist F, Teslenko M, van der Mark P, Ayres DL, Darling A, Höhna S, Larget B, Liu L, Suchard MA, Huelsenbeck JP (2012) MrBayes 3.2: efficient bayesian phylogenetic inference and model choice across a large model space. Syst Biol 61:539–542PubMedCentralPubMedCrossRefGoogle Scholar
  48. Schoch CL, Seifert KA, Huhndorf S, Robert V, Spouge JL, Levesque CA, Chen W, Bolchacova E, Voigt K, Crous PW (2012) Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for fungi. PNAS 109:6241–6246PubMedCentralPubMedCrossRefGoogle Scholar
  49. Shaw C, Dick M (1979) Verticicladiella root disease of Pinus strobus in New Zealand. Plant Dis 64:96–98CrossRefGoogle Scholar
  50. Sun JH, Lu M, Gillette NE, Wingfield MJ (2013) Red turpentine beetle: innocuous native becomes invasive tree killer in China. Annu Rev Entomol 58:293–311PubMedCrossRefGoogle Scholar
  51. Swofford DL (2003) PAUP* 4.0: phylogenetic analysis using parsimony (*and other methods). Sinauer Associates, Sunderland, MassachusettsGoogle Scholar
  52. Taerum SJ, Duong TA, De Beer ZW, Gillette N, Sun JH, Owen DR, Wingfield MJ (2013) Large shift in symbiont assemblage in the invasive red turpentine beetle. PLoS One 8:e78126PubMedCentralPubMedCrossRefGoogle Scholar
  53. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28:2731–2739PubMedCentralPubMedCrossRefGoogle Scholar
  54. Wen X, Kuang Y, Shi M, Li H, Luo Y, Deng R (2004) Biology of Hylobitelus xiaoi (Coleoptera: Curculionidae), a new pest of slash pine, Pinus elliottii. J Econ Entomol 97:1958–1964PubMedCrossRefGoogle Scholar
  55. 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, Snisky JJ, White TJ (eds) PCR protocols: a guide to methods and applications. Academic Press, New York, pp 315–322CrossRefGoogle Scholar
  56. Wingfield MJ (1985) Reclassification of Verticicladiella based on conidial development. Trans Br Mycol Soc 85:81–93CrossRefGoogle Scholar
  57. Wingfield MJ (1986) Pathogenicity of Leptographium procerum and L. terebrantis on Pinus strobus seedlings and established trees. Eur J For Pathol 16:299–308CrossRefGoogle Scholar
  58. Wingfield MJ (1993) Leptographium species as anamorphs of Ophiostoma: Progress in establishing acceptable generic and species concepts. In: Wingfield MJ, Seifert KA, Webber JF (eds) Ceratocystis and Ophiostoma: taxonomy, ecology and pathogenicity. The American Phytopathological Society Press, St. Paul, pp 43–51Google Scholar
  59. Wingfield MJ, Gibbs JN (1991) Leptographium and Graphium species associated with pine-infesting bark beetles in England. Mycol Res 95:1257–1260CrossRefGoogle Scholar
  60. Wingfield MJ, Marasas WFO (1983) Some Verticicladiella species, including V. truncata sp. nov., associated with root diseases of pine in New Zealand and South Africa. Trans Br Mycol Soc 80:231–236CrossRefGoogle Scholar
  61. Wingfield MJ, Capretti P, Mackenzie M (1988) Leptographium spp. as root pathogens of conifers. An international perspective. In: Harrington TC, Cobb FW Jr (eds) Leptographium root disease on conifers. The American Phytopathological Society Press, St. Paul, pp 113–128Google Scholar
  62. Yamaoka Y, Masuya H, Chung WH, Goto H, To-Anun C, Tokumasu S, Zhou XD, Wingfield MJ (2008) The teleomorph of Leptographium yunnanense, discovered in crosses among isolates from Thailand, China, and Japan. Mycoscience 49:233–240CrossRefGoogle Scholar
  63. Zhang R (1997) Description of a new species Hylobitelus xiaoi (Coleoptera: Curculionidae). Sci Silvae Sin 33:541–545Google Scholar
  64. Zhou XD, De Beer ZW, Wingfield BD, Wingfield MJ (2001) Ophiostomatoid fungi associated with three pine-infesting bark beetles in South Africa. Sydowia 53:290–300Google Scholar
  65. Zhou XD, Jacobs K, Kirisits T, Chhetri DB, Wingfield MJ (2008) Leptographium bhutanense sp. nov., associated with the root collar weevil Hylobitelus chenkupdorjii on Pinus wallichiana in Bhutan. Persoonia 21:1–8PubMedCentralPubMedCrossRefGoogle Scholar
  66. Zipfel RD, De Beer ZW, Jacobs K, Wingfield BD, Wingfield MJ (2006) Multi-gene phylogenies define Ceratocystiopsis and Grosmannia distinct from Ophiostoma. Stud Mycol 55:75–97PubMedCentralPubMedCrossRefGoogle Scholar

Copyright information

© Springer International Publishing Switzerland 2014

Authors and Affiliations

  • Mingliang Yin
    • 1
    • 2
  • Tuan A. Duong
    • 3
  • Michael J. Wingfield
    • 1
  • XuDong Zhou
    • 1
    • 2
    • 4
  • Z. Wilhelm de Beer
    • 1
  1. 1.Department of Microbiology and Plant Pathology, Forestry and Agricultural Biotechnology Institute (FABI)University of PretoriaPretoriaSouth Africa
  2. 2.China Eucalypt Research Centre (CERC)Chinese Academy of Forestry (CAF)ZhanjiangChina
  3. 3.Department of Genetics, Forestry and Agricultural Biotechnology Institute (FABI)University of PretoriaPretoriaSouth Africa
  4. 4.FuturaGene Biotechnology (Shanghai) Co., Ltd.ShanghaiChina

Personalised recommendations