Skip to main content Skip to main navigation menu Skip to site footer
Published: 2023-03-16

Three interesting fungi from American bullfrog larvae (Rana catesbeiana) in Yunnan, China

Center for Yunnan Plateau Biological Resources Protection and Utilization, College of Biological Resource and Food Engineering, Qujing Normal University, Qujing, Yunnan 655011, People’s Republic of China; Master of Science Program in Applied Microbiology (International Program), Faculty of Science, Chiang Mai University, Chiang Mai 50200, Thailand
Center for Yunnan Plateau Biological Resources Protection and Utilization, College of Biological Resource and Food Engineering, Qujing Normal University, Qujing, Yunnan 655011, People’s Republic of China
Center for Yunnan Plateau Biological Resources Protection and Utilization, College of Biological Resource and Food Engineering, Qujing Normal University, Qujing, Yunnan 655011, People’s Republic of China
Center for Yunnan Plateau Biological Resources Protection and Utilization, College of Biological Resource and Food Engineering, Qujing Normal University, Qujing, Yunnan 655011, People’s Republic of China
Center of Excellence in Fungal Research, Mae Fah Luang University, Chiang Rai 57100, Thailand
Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai, Thailand
Center for Yunnan Plateau Biological Resources Protection and Utilization, College of Biological Resource and Food Engineering, Qujing Normal University, Qujing, Yunnan 655011, People’s Republic of China; National Institute of Fundamental Studies (NIFS), Sri Lanka
Amphibian Boothiella Microfungi Sporulation Sordaria Trichoderma Fungi


American bullfrog (Rana catesbeiana) is an alien invasive species in southwest China native to the central and eastern United States and southeastern Canada. After the 19th century, they extensively appear in aquaculture and natural environments worldwide as a delicious food but also creating a serious threat to the survival and development of native species. In the early rainy season, dead American bullfrog larvae floating on the water of unnamed ponds in Qujing Normal University, Yunnan Province, China were collected and brought to the mycology laboratory, and three interesting fungal strains were isolated from their intestinal contents. Phylogenetic analyses were carried out on the resultant isolates based on multiple gene sequences (ITS, LSU, rpb2, tub2, tef1-α), and results confirmed that the three strains belong to three species, namely; Boothiella tetraspora, Sordaria macrospora and Trichoderma virens. The morphological characteristics were also used to describe the fungal taxa. Photographic plates, descriptions, and phylogenetic trees that show the placements of the fungal species are reported herein.


  1. Abd-Aziz, S., Fernandez, C.C., Salleh, M.M., Illias, R.M. & Hassan, M. (2008) Effect of agitation and aeration rates on chitinase production using Trichoderma virens UKM1 in 2-l stirred tank reactor. Applied Biochemistry and Biotechnology 150: 193–204.

  2. Adams, M.J. (1999) Correlated factors in amphibian decline: exotic species and habitat change in western Washington. The Journal of Wildlife Management 63: 1162–1171.

  3. Arx, J.A. (1987) Plant pathogenic fungi. Beihefte zur Nova Hedwigia 87: 1–288

  4. Auerswald, B. (1866) Sordaria macrospora. Hedwigia 5: 192.

  5. Brotman, Y., Kapuganti, J.G. & Viterbo, A. (2010) Trichoderma. Current Biology 20: 390–391.

  6. Cai, L., Jeewon, R. & Hyde, K.D. (2006) Phylogenetic investigations of Sordariaceae based on multiple gene sequences and morphology. Mycological Research 110: 137–150.

  7. Chaverri, P., Samuels, G.J. & Stewart, E.L. (2001) Hypocrea virens sp. nov., the teleomorph of Trichoderma virens. Mycologia 93: 1113–1124.

  8. Contreras-Cornejo, H.A., Macías-Rodríguez, L., Cortés-Penagos, C. & López-Bucio, J. (2009) Trichoderma virens, a plant beneficial fungus, enhances biomass production and promotes lateral root growth through an auxin-dependent mechanism in Arabidopsis. Plant Physiology 149: 1579–1592.

  9. Cui, P., Kong, K., Yao, Y., Huang, Z., Shi, S., Liu, P., Huang, Y., Abbas, N., Yu, L. & Zhang, Y. (2022) Community composition, bacterial symbionts, antibacterial and antioxidant activities of honeybee-associated fungi. BMC Microbiology 22: 1–15.

  10. Cunningham, A.A., Langton, T.E., Bennett, P.M., Lewin, J.F., Drury, S.E., Gough, R. & Macgregor, S.K. (1996) Pathological and microbiological findings from incidents of unusual mortality of the common frog (Rana temporaria). Philosophical Transactions of the Royal Society of London. Series B: Biological Sciences 351: 1539–1557.

  11. Eriksson, O.E., Baral, H.O., Currah, R.S., Kurtzman, C.P., Rambold, G. & Læssøe, T. (2004) Outline of Ascomycota—2004. Myconet 10: 1–99.

  12. Farr, D.F. & Rossman, A.Y. (2022) Fungal Databases, U.S. National Fungus Collections, ARS, USDA. Available from: https://nt.ars-grin. gov/fungaldatabases/ (accessed 10 December 2022).

  13. Fisher, P.J., Petrini, O., Petrini, L.E. & Descals, E. (1992) A preliminary study of fungi inhabiting xylem and whole stems of Olea europaea. Sydowia 44: 117–121.

  14. Fisher, P.J., Petrini, O. & Sutton, B.C. (1993) A comparative study of fungal endophytes in leaves, xylem and bark of Eucalyptus in Australia and England. Sydowia 45: 338–345.

  15. Furtado, J.S. (1969) Ascal cytology of Sordaria sclerogenia. Protoplasma 67: 473–478.

  16. Gal-Hemed, I., Atanasova, L., Komon-Zelazowska, M., Druzhinina, I.S., Viterbo, A. & Yarden, O. (2011) Marine isolates of Trichoderma spp. as potential halotolerant agents of biological control for arid-zone agriculture. Applied and Environmental Microbiology 77: 5100–5109.

  17. Garner, T.W., Perkins, M.W., Govindarajulu, P., Seglie, D., Walker, S., Cunningham, A.A. & Fisher, M.C. (2006) The emerging amphibian pathogen Batrachochytrium dendrobatidis globally infects introduced populations of the North American bullfrog, Rana catesbeiana. Biology Letters 2: 455–459.

  18. Garner, T.W., Walker, S., Bosch, J., Hyatt, A.D., Cunningham, A.A. & Fisher, M.C. (2005) Chytrid fungus in Europe. Emerging Infectious Diseases 11: 1639–1641.

  19. Glez-Peña, D., Gómez-Blanco, D., Reboiro-Jato, M., Fdez-Riverola, F. & Posada, D. (2010) ALTER: programoriented conversion of DNA and protein alignments. Nucleic Acids Research 38: 14–18.

  20. Glorioso, J.C., Amborski, R.L., Amborsky, G.F. & Culley, D.D. (1974) Microbiological studies on septicemic bullfrogs (Rana catesbeiana). American Journal of Veterinary Research 35: 1241–1245.

  21. Goraya, J., Wang, Y., Li, Z., O’Flaherty, M., Knoop, F.C., Platz, J.E. & Conlon, J.M. (2000) Peptides with antimicrobial activity from four different families isolated from the skins of the North American frogs Rana luteiventris, Rana berlandieri and Rana pipiens. European Journal of Biochemistry 267: 894–900.

  22. Hall, T.A. (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symposium Series 41: 95–98.

  23. Han, D.B., Lu, Y., Wang, D.R. & Zhang, Z.Z. (1991) Assay of the common nutritional compositions on the bullfrog. Zoology 12: 161–162.

  24. Harman, G.E. (2000) Myths and dogmas of biocontrol. Changes in perceptions derived from research on Trichoderma harzianum T-22. Plant Disease 84: 377–393.

  25. Harman, G.E. (2006) Overview of mechanisms and uses of Trichoderma spp. Phytopathology 96: 190–194.

  26. Huang, S.K., Hyde, K.D., Mapook, A., Maharachchikumbura, S.S.N., Bhat, J.D., McKenzie, E.H.C., Jeewon, R. & Wen, T.C. (2021) Taxonomic studies of some often over-looked Diaporthomycetidae and Sordariomycetidae. Fungal Diversity 111: 443–572.

  27. Ivanova, H., Pristaš, P. & Ondrušková, E.M.Í.L.I.A. (2015) Comparison of two Coniochaeta species (C. ligniaria and C. malacotricha) with a new pathogen of black pine needles–Sordaria macrospora. Plant Protection Science 52: 18–25.

  28. Ivanová, H., Onderková, A. & Pristaš, P. (2018) Sordaria fimicola-like ascomycete isolated from Pinus coulteri needles in Slovakia. Biologia 73: 553–559.

  29. Jacobson, D.J., Powell, A.J., Dettman, J.R., Saenz, G.S., Barton, M.M., Hiltz, M.D., Dvorachek, W.H., Glass, N.L., Taylor, J.W. & Natvig, D.O. (2004) Neurospora in temperate forests of western North America. Mycologia 96: 66–74.

  30. Jaklitsch, W.M., Komon, M., Kubicek, C.P. & Druzhinina, I.S. (2005) Hypocrea voglmayrii sp. nov. from the Austrian Alps represents a new phylogenetic clade in Hypocrea/Trichoderma. Mycologia 97: 1365–1378.

  31. Jancowski, K. & Orchard, S. (2013) Stomach contents from invasive American bullfrogs Rana catesbeiana (= Lithobates catesbeianus) on southern Vancouver Island, British Columbia, Canada. NeoBiota 16: 17–37.

  32. Katoh, K. & Standley, D.M. (2013) MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Molecular Biology and Evolution 30: 772–780.

  33. Kats, L.B. & Ferrer, R.P. (2003) Alien predators and amphibian declines: review of two decades of science and the transition to conservation. Diversity and Distributions 9: 99–110.

  34. Kirk, P.M., Cannon, P.F., Minter, D.W. & Stalpers, J.A. (2008) Ainsworth & Bisby’s Dictionary of the Fungi, 10th ed. CABI, UK. pp. 1–771.

  35. Kirk, P.M. (2023) Index Fungorum. Available from: (accessed 5 January 2023)

  36. Lee, S., Najiah, M., Wendy, W., Nadirah, M. & Faizah, S. (2009) Occurrence of heavy metals and antibiotic resistance in bacteria from internal organs of American bullfrog (Rana catesbeiana) raised in Malaysia. Journal of Venomous Animals and Toxins including Tropical Diseases 15: 353–358.

  37. Leivas, P.T., Leivas, F.W. & Moura, M.O. (2012) Diet and trophic niche of Lithobates catesbeianus (Amphibia: Anura). Zoologia 29: 405–412.

  38. Liu, Y.J., Whelen, S. & Hall, B.D. (1999) Phylogenetic relationships among Ascomycetes: evidence from an RNA polymerase II. subunit. Molecular Biology and Evolution 16: 1799–1808.

  39. Liu, X., Luo, Y., Chen, J., Guo, Y., Bai, C. & Li, Y. (2015) Diet and prey selection of the Invasive American bullfrog (Lithobates catesbeianus) in southwestern China. Asian Herpetological Research 6: 34–44.

  40. Lodhi, S.A. & Mirza, R.F. (1962) A new genus of the Eurotiales. Mycologia 54: 217–219.

  41. López-Bucio, J., Pelagio-Flores, R. & Herrera-Estrella, A. (2015) Trichoderma as biostimulant: exploiting the multilevel properties of a plant beneficial fungus. Scientia Horticulturae 196: 109–123.

  42. Lord, K.M. & Read, N.D. (2011) Perithecium morphogenesis in Sordaria macrospora. Fungal Genetics and Biology 48: 388–399.

  43. Lytvynenko, Y.I. & Hayova, V.P. (2018) New and noteworthy records of coprophilous species of Coniochaeta and Sordaria (Sordariomycetes, Ascomycota) from Ukraine. Ukrainian Botanical Journal 75: 538–551.

  44. Mauel, M.J., Miller, D.L., Frazier, K.S. & Hines, M.E. (2002) Bacterial pathogens isolated from cultured bullfrogs (Rana catesbeiana). Journal of Veterinary Diagnostic Investigation 14: 431–433.

  45. Mazzoni, R. (2000) Diseases in farmed American bull frog (Rana catesbeiana Shaw, 1802) in Uruguay. GETTING THE JUMP! ON AMPHIBIAN DISEASE, 2000, Cairns, Australia. Conference and Workshop Compendium. Rainforest CRC, Cairns, pp. 37–38.

  46. Miaud, C., Dejean, T., Savard, K., Millery-Vigues, A., Valentini, A., Curt Grand Gaudin, N. & Garner, T.W. (2016) Invasive North American bullfrogs transmit lethal fungus Batrachochytrium dendrobatidis infections to native amphibian host species. Biological Invasions 18: 2299–2308.

  47. Miller, M.A., Pfeiffer, W. & Schwartz, T. (2010) Creating the CIPRES Science Gateway for inference of large phylogenetic trees. In: Proceedings of the 2010 Gateway Computing Environments Workshop (GCE). New Orleans, LA, USA, 14 November, pp. 1–8.

  48. Mungai, P.G., Chukeatirote, E., Njogu, J.G. & Hyde, K.D. (2012) Studies of coprophilous ascomycetes in Kenya: Sordariales from wildlife dung. Mycosphere 3: 437–448.

  49. Niederle, M.V., Bosch, J., Ale, C.E., Nader-Macías, M.E., Aristimuno Ficoseco, C., Toledo, L.F., Valenzuela-Sa´nchez, A., Soto-Azat, C. & Pasteris, S.E. (2019) Skin-associated lactic acid bacteria from North American bullfrogs as potential control agents of Batrachochytrium dendrobatidis. PLoS one 14: e0223020.

  50. O’Donnell, K. & Cigelnik, E. (1997) Two divergent intragenomic rDNA ITS2 types within a monophyletic lineage of the fungus Fusarium are nonorthologous. Molecular Phylogenetics and Evolution 7: 103–116.

  51. Olson, D.H., Aanensen, D.M., Ronnenberg, K.L., Powell, C.I., Walker, S.F., Bielby, J., Garner, T.W.J., Weaver, G., The Bd Mapping Group & Fisher, M.C. (2013) Mapping the global emergence of Batrachochytrium dendrobatidis, the amphibian chytrid fungus. PloS one 8: e56802.

  52. Pasteris, S.E., Buhler, M.I. & Nader-Macias, M.E. (2006) Microbiological and histological studies of farmed-bullfrog (Rana catesbeiana) tissues displaying red-leg syndrome. Aquaculture 251: 11–18.

  53. Petrini, O. & Fisher, P.J. (1988) A comparative study of fungal endophytes in xylem and whole stem of Pinus sylvestris and Fagus sylvatica. Transactions of the British Mycological Society 91: 233–238.

  54. Petrini, O. & Fisher, P.J. (1990) Occurrence of fungal endophytes in twigs of Salix fragilis and Quercus robur. Mycology Research 94: 1077–1080.

  55. Phukhamsakda, C., McKenzie, E.H.C., Phillips, A.J.L., Gareth Jones, E.B., Jayarama Bhat, D., Marc, S., Bhunjun, C.S., Wanasinghe, D.N., Thongbai, B., Camporesi, E., Ertz, D., Jayawardena, R.S., Perera, R.H., Ekanayake, A.H., Tibpromma, S., Doilom, M., Xu, J.C. & Hyde, K.D. (2020) Microfungi associated with Clematis (Ranunculaceae) with an integrated approach to delimiting species boundaries. Fungal Diversity 102: 1–203.

  56. Rambaut, A. FigTree v1. 4.0. A Graphical Viewer of Phylogenetic Trees. Available online: (accessed on 1 December 2022).

  57. Ronquist, F., Teslenko, M., van der Mark, P., Ayres, D.L., Darling, A., Höhna, S., Larget, B., Liu, L., Suchard, M.A. & Huelsenbeck, J.P. (2012) MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. Systematic Biology 61: 539–542.

  58. Sanchez Marquez, S., Bills, G.F. & Zabalgogeazcoa, I. (2007) The endophytic mycobiota of the grass Dactylis glomerata. Fungal Diversity 27: 171–195.

  59. Schuster, A. & Schmoll, M. (2010) Biology and biotechnology of Trichoderma. Applied Microbiology and Biotechnology 87: 787–799.

  60. Shirose, L.J. & Brooks, R.J. (1995) Age structure, mortality, and longevity in syntopic populations of three species of ranid frogs in central Ontario. Canadian Journal of Zoology 73: 1878–1886.

  61. Silva, E.T.D., Reis, E.P.D., Feio, R.N. & Filho, O.P.R. (2009) Diet of the invasive frog Lithobates catesbeianus (Shaw, 1802) (Anura: Ranidae) in Viçeosa, Minas Gerais State, Brazil. South American Journal of Herpetology 4: 286–294.

  62. Stamatakis, A. (2014) RAxML Version 8: A tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics 30: 1312–1313.

  63. Teichert, I., Pöggeler, S., Nowrousian, M. (2020) Sordaria macrospora: 25 years as a model organism for studying the molecular mechanisms of fruiting body development. Applied Microbiology and Biotechnology 104: 3691–3704.

  64. Tomah, A.A., Abd Alamer, I.S., Li, B. & Zhang, J.Z. (2020) A new species of Trichoderma and gliotoxin role: A new observation in enhancing biocontrol potential of T. virens against Phytophthora capsici on chili pepper. Biological Control 145: 104261.

  65. Uecker, F.A. (1976) Development and cytology of Sordaria humana. Mycologia 68: 30–46.

  66. Vilgalys, R. & Hester, M. (1990) Rapid genetic identification and mapping of enzymatically amplified ribosomal DNA from several Cryptococcus species. Journal of Bacteriology 172: 4238–4246.

  67. Vu, D., Groenewald, M., de Vries, M.D., Gehrmann, T., Stielow, B., Eberhardt, U., Al-Hatmi, A., Groenewald, J.Z., Cardinali, J. & Houbraken, J. (2019) Large-scale generation and analysis of filamentous fungal DNA barcodes boosts coverage for kingdom fungi and reveals thresholds for fungal species and higher taxon delimitation. Studies in Mycology 92: 135–154.

  68. Walke, J.B., Becker, M.H., Loftus, S.C., House, L.L., Teotonio, T.L., Minbiole, K.P.C. & Belden, L.K. (2015) Community structure and function of amphibian skin microbes: an experiment with bullfrogs exposed to a chytrid fungus. PloS one 10: e0139848.

  69. Wang, Y. & Li, Y. (2009) Habitat selection by the introduced American bullfrog (Lithobates catesbeianus) on Daishan Island, China. Journal of Herpetology 43: 205–211.

  70. Wang, X.W., Bai, F.Y., Bensch, K., Meijer, M., Sun, B.D., Han, Y.F., Crous, P.W., Samson, R.A., Yang, F.Y. & Houbraken, J. (2019) Phylogenetic re-evaluation of Thielavia with the introduction of a new family Podosporaceae. Studies in Mycology 93: 155–252.

  71. Watanabe, T. (1989) Three species of Sordaria, and Eudarluca biconica from cherry seeds. Transactions of the British Mycological Society 30: 395–400.

  72. Weindling, R. (1934) Studies on a lethal principle effective in the parasitic action of Trichoderma lignorum on Rhizoctonia solani and other soil fungi. Phytopathology 24: 1153–1179.

  73. Weindling, R. & Fawcett, H.S. (1936) Experiments in the control of Rhizoctonia damping-off of citrus seedlings. Hilgardia 10: 1–16.

  74. White, T.J., Bruns, T., Lee, S.J.W.T. & Taylor, J. (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. PCR Protocols: a guide to methods and applications 18: 315–322.

  75. Woo, S.L., Ruocco, M., Vinale, F., Nigro, M., Marra, R., Lombardi, N., Pascale, A., Lanzuise, S., Manganiello, G. & Lorito, M. (2014) Trichoderma-based products and their widespread use in agriculture. The Open Mycology Journal 8: 71–126.

  76. Wu, Z.J., Wang, Y.P. & Li, Y.M. (2004) Natural populations of bullfrog (Rana catesbeiana) and their potential threat in the east of Zhejiang Province. Biodiversity Science 12: 441.

  77. Yang, E.F., Tibpromma, S., Dai, D.Q., Promputtha, I., Mortimer, P.E. & Karunarathna, S.C. (2022) Three interesting fungal species associated with the Asian House Gecko in Kunming, China. Phytotaxa 545: 37–56.

  78. Yul, G., Mikos, I.G. & OYu, A. (2010) New records of coprophilous ascomycetes in the Crimea. Chornomorski Botanical Journal 6: 67–83.

  79. Zhang, G.Z., Yang, H.T., Zhang, X.J., Zhou, F.Y., Wu, X.Q., Xie, X.Y., Zhao, X.Y. & Zhou, H.Z. (2022) Five new species of Trichoderma from moist soils in China. MycoKeys 87: 133–157.

  80. Zhaxybayeva, O. & Gogarten, J.P. (2002) Bootstrap, Bayesian probability and maximum likelihood mapping: exploring new tools for comparative genome analyses. MBC Genomics 3: 1–4.

  81. Zin, N.A. & Badaluddin, N.A. (2020) Biological functions of Trichoderma spp. for agriculture applications. Annals of Agricultural Sciences 65: 168–178.