Abstract
Entomopathogenic fungi are considered to be a safe microbiological pesticide alternative to chemical control. Efforts are underway to understand precisely their taxonomy and natural distribution through mycological and biodiversity studies based on molecular markers. Here, we present descriptions of the diversity of the entomopathogenic fungi in the genera Metarhizium and Beauveria found along the elevational gradients of the Qinling subtropical and temperate forests of Shaanxi province in China, using morphological aspects and molecular markers. Molecular characterization using the Mz_IGS3 intergenic region revealed that Metarhizium isolates phylogenetically clustered in the PARB clade with four different distinguishable species, but the 5′-TEF gene allowed only ambiguous delimitation of Metarhizium species. Beauveria isolates were characterized by sequence analyses of the translation elongation factor 1-α and the Bloc region. The richness of Metarhizium species decreased with increasing elevation, with Metarhizium robertsii s.l. being the most abundant species along the elevational gradient. Our bioassay suggests that certain species of Metarhizium are significantly pathogenic to the insect model Tenebrio molitor at both the adult and larvae stages and could potentially serve as a control of insect pests of forests.
Similar content being viewed by others
References
Bidochka MJ, Kasperski JE, Wild GA (1998) Occurrence of the entomopathogenic fungi Metarhizium anisopliae and Beauveria bassiana in soils from temperate and near-northern habitats. Can J Bot 76(7):1198–1204
Bidochka MJ, Kamp AM, Lavender TM, Dekoning J, De Croos JNA (2001) Habitat association in two genetic groups of the insect-pathogenic fungus Metarhizium anisopliae: uncovering cryptic species? Appl Environ Microbiol 67(3):1335–1342. https://doi.org/10.1128/AEM.67.3.1335-1342.2001
Bidochka MJ, Menzies FV, Kamp AM (2002) Genetic groups of the insect-pathogenic fungus Beauveria bassiana are associated with habitat and thermal growth preferences. Arch Microbiol 178(6):531–537. https://doi.org/10.1007/s00203-002-0490-7
Bidochka MJ, Small CLN, Spironello M (2005) Recombination within sympatric cryptic species of the insect pathogenic fungus Metarhizium anisopliae. Environ Microbiol 7(9):1361–1368. https://doi.org/10.1111/j.1462-5822.2005.00823.x
Bischoff JF, Rehner SA, Humber RA (2009) A multilocus phylogeny of the Metarhizium anisopliae lineage. Mycologia 101(4):512–530. https://doi.org/10.3852/07-202
Braga GUL, Flint SD, Miller CD, Anderson AJ, Roberts DW (2001) Variability in response to UV-B among species and strains of Metarhizium isolated from sites at latitudes from 61°N to 54°S. J Invertebr Pathol 78(2):98–108. https://doi.org/10.1006/jipa.2001.5048
Carrillo-Benítez MG, Guzmán-Franco AW, Alatorre-Rosas R, Enríquez-Vara JN (2013) Diversity and genetic population structure of fungal pathogens infecting white grub larvae in agricultural soils. Microb Ecol 65(2):437–449. https://doi.org/10.1007/s00248-012-0124-9
Castrillo LA, Griggs MH, Ranger CM, Reding ME, Vandenberg JD (2011) Virulence of commercial strains of Beauveria bassiana and Metarhizium brunneum (Ascomycota: Hypocreales) against adult Xylosandrus germanus (Coleoptera: Curculionidae) and impact on brood. Biol Control 58(2):121–126. https://doi.org/10.1016/j.biocontrol.2011.04.010
Chandler D, Hay D, Reid AP (1997) Sampling and occurrence of entomopathogenic fungi and nematodes in UK soils. Appl Soil Ecol 5(2):133–141. https://doi.org/10.1016/S0929-1393(96)00144-8
Chen Z-H, Xu L, Yang F-l, Ji G-H, Yang J, Wang J-Y (2014) Efficacy of Metarhizium anisopliae isolate MAX-2 from Shangri-la, China under desiccation stress. BMC Microbiol 14(1):1
Choi Y-W, Hyde KD, Ho W (1999) Single spore isolation of fungi. Fungal Divers 3:29–38
de Faria MR, Wraight SP (2007) Mycoinsecticides and Mycoacaricides: a comprehensive list with worldwide coverage and international classification of formulation types. Biol Control 43(3):237–256. https://doi.org/10.1016/j.biocontrol.2007.08.001
Driver F, Milner RJ, Trueman JW (2000) A taxonomic revision of Metarhizium based on a phylogenetic analysis of rDNA sequence data. Mycol Res 104(2):134–150. https://doi.org/10.1017/S0953756299001756
Du X-C, Ren Y, Dang G-D, Lundholm J (2011) Distribution and plant community associations of the understory bamboo Fargesia qinlingensis in the Foping National Nature Reserve, China. Ann For Sci 68(7):1197–1206. https://doi.org/10.1007/s13595-011-0104-0
Fang W, St Leger RJ (2010) RNA binding proteins mediate the ability of a fungus to adapt to the cold. Environ Microbiol 12(3):810–820. https://doi.org/10.1111/j.1462-2920.2009.02127.x
Felsenstein J (1981) Evolutionary trees from DNA sequences: a maximum likelihood approach. J Mol Evol 17(6):368–376. https://doi.org/10.1007/BF01734359
Fernandes ÉKK, Rangel DEN, Moraes ÁML, Bittencourt VREP, Roberts DW (2008) Cold activity of Beauveria and Metarhizium, and thermotolerance of Beauveria. J Invertebr Pathol 98(1):69–78. https://doi.org/10.1016/j.jip.2007.10.011
Fernandes ÉKK, Keyser CA, Chong JP, Rangel DEN, Miller MP, Roberts DW (2010) Characterization of Metarhizium species and varieties based on molecular analysis, heat tolerance and cold activity. J Appl Microbiol 108(1):115–128. https://doi.org/10.1111/j.1365-2672.2009.04422.x
Gao Q, Jin K, Ying S-H, Zhang Y, Xiao G, Shang Y, Duan Z, Hu X, Xie X-Q, Zhou G, Peng G, Luo Z, Huang W, Wang B, Fang W, Wang S, Zhong Y, Ma L-J, St Leger RJ, Zhao G-P, Pei Y, Feng M-G, Xia Y, Wang C (2011) Genome sequencing and comparative transcriptomics of the model entomopathogenic fungi Metarhizium anisopliae and M. acridum. PLoS Genet 7(1):e1001264. https://doi.org/10.1371/journal.pgen.1001264
Garrido-Jurado I, Fernández-Bravo M, Campos C, Quesada-Moraga E (2015) Diversity of entomopathogenic Hypocreales in soil and phylloplanes of five Mediterranean cropping systems. J Invertebr Pathol 130:97–106. https://doi.org/10.1016/j.jip.2015.06.001
Glare TR, Reay SD, Nelson TL, Moore R (2008) Beauveria caledonica is a naturally occurring pathogen of forest beetles. Mycol Res 112(3):352–360. https://doi.org/10.1016/j.mycres.2007.10.015
Hajek AE (1999) Pathology and epizootiology of Entomophaga maimaiga infections in forest Lepidoptera. Microbiol Mol Biol Rev 63(4):814–835
Hallsworth JE, Magan N (1996) Culture age, temperature, and pH affect the polyol and trehalose contents of fungal propagules. Appl Environ Microbiol 62(7):2435–2442
Han SK, Lee D, Lee H, Kim D, Son HG, Yang J-S, Lee S-JV, Kim S (2016) OASIS 2: online application for survival analysis 2 with features for the analysis of maximal lifespan and healthspan in aging research. Oncotarget 7(35):56147–56152. 10.18632/oncotarget.11269
Hutwimmer S, Wang H, Strasser H, Burgstaller W (2010) Formation of exudate droplets by Metarhizium anisopliae and the presence of destruxins. Mycologia 102(1):1–10. https://doi.org/10.3852/09-079
Inglis G, Johnson D, Goettel M (1997) Field and laboratory evaluation of two conidial batches of Beauveria bassiana (Balsamo) Vuillemin against grasshoppers. Can Entomol 129(01):171–186. https://doi.org/10.4039/Ent129171-1
Ivey FD, Hodge PN, Turner GE, Borkovich KA (1996) The G alpha i homologue gna-1 controls multiple differentiation pathways in Neurospora crassa. Mol Biol Cell 7(8):1283–1297. https://doi.org/10.1091/mbc.7.8.1283
Johnson D, Sung G-H, Hywel-Jones NL, Luangsa-Ard JJ, Bischoff JF, Kepler RM, Spatafora JW (2009) Systematics and evolution of the genus Torrubiella (Hypocreales, Ascomycota). Mycol Res 113(3):279–289. https://doi.org/10.1016/j.mycres.2008.09.008
Kamp AM, Bidochka MJ (2002) Conidium production by insect pathogenic fungi on commercially available agars. Lett Appl Microbiol 35(1):74–77. https://doi.org/10.1046/j.1472-765X.2002.01128.x
Katoh K, Standley DM (2013) MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol Biol Evol 30(4):772–780. https://doi.org/10.1093/molbev/mst010
Kepler RM, Rehner SA (2013) Genome-assisted development of nuclear intergenic sequence markers for entomopathogenic fungi of the Metarhizium anisopliae species complex. Mol Ecol 13(2):210–217. https://doi.org/10.1111/1755-0998.12058
Kepler RM, Humber RA, Bischoff JF, Rehner SA (2014) Clarification of generic and species boundaries for Metarhizium and related fungi through multigene phylogenetics. Mycologia 106(4):811–829. https://doi.org/10.3852/13-319
Kepler RM, Ugine TA, Maul JE, Cavigelli MA, Rehner SA (2015) Community composition and population genetics of insect pathogenic fungi in the genus Metarhizium from soils of a long-term agricultural research system. Environ Microbiol 17(8):2791–2804. https://doi.org/10.1111/1462-2920.12778
Kessler P, Matzke H, Keller S (2003) The effect of application time and soil factors on the occurrence of Beauveria brongniartii applied as a biological control agent in soil. J Invertebr Pathol 84(1):15–23. https://doi.org/10.1016/j.jip.2003.08.003
Keyser CA, De Fine Licht HH, Steinwender BM, Meyling NV (2015) Diversity within the entomopathogenic fungal species Metarhizium flavoviride associated with agricultural crops in Denmark. BMC Microbiol 15(1):249. https://doi.org/10.1186/s12866-015-0589-z
Kimura M (1980) A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 16(2):111–120. https://doi.org/10.1007/BF01731581
Lopes RB, Mesquita ALM, Tigano MS, Souza DA, Martins I, Faria M (2013a) Diversity of indigenous Beauveria and Metarhizium spp. in a commercial banana field and their virulence toward Cosmopolites sordidus (Coleoptera: Curculionidae). Fungal Ecol 6(5):356–364. https://doi.org/10.1016/j.funeco.2013.06.007
Lopes RB, Souza DA, Oliveira CM, Faria M (2013b) Genetic diversity and pathogenicity of Metarhizium spp. associated with the white grub Phyllophaga capillata (Blanchard) (Coleoptera: Melolonthidae) in a soybean field. Neotrop Entomol 42(4):436–438. https://doi.org/10.1007/s13744-013-0146-0
Luan F, Zhang S, Wang B, Huang B, Li Z (2013) Genetic diversity of the fungal pathogen Metarhizium spp., causing epizootics in Chinese burrower bugs in the Jingting Mountains, eastern China. Mol Biol Rep 40(1):515–523. https://doi.org/10.1007/s11033-012-2088-8
Maddison WP, Maddison DR (2015) Mesquite: a modular system for evolutionary analysis. Version 3.04
Medo J, Cagáň Ľ (2011) Factors affecting the occurrence of entomopathogenic fungi in soils of Slovakia as revealed using two methods. Biol Control 59(2):200–208. https://doi.org/10.1016/j.biocontrol.2011.07.020
Meyling NV, Eilenberg J (2006) Occurrence and distribution of soil borne entomopathogenic fungi within a single organic agroecosystem. Agric Ecosyst Environ 113(1–4):336–341. https://doi.org/10.1016/j.agee.2005.10.011
Meyling NV, Eilenberg J (2007) Ecology of the entomopathogenic fungi Beauveria bassiana and Metarhizium anisopliae in temperate agroecosystems: potential for conservation biological control. Biol Control 43(2):145–155. https://doi.org/10.1016/j.biocontrol.2007.07.007
Meyling NV, LuBeck M, Buckley EP, Eilenberg J, Rehner SA (2009) Community composition, host range and genetic structure of the fungal entomopathogen Beauveria in adjoining agricultural and seminatural habitats. Mol Ecol 18(6):1282–1293. https://doi.org/10.1111/j.1365-294X.2009.04095.x
Meyling NV, Thorup-Kristensen K, Eilenberg J (2011) Below- and aboveground abundance and distribution of fungal entomopathogens in experimental conventional and organic cropping systems. Biol Control 59(2):180–186. https://doi.org/10.1016/j.biocontrol.2011.07.017
Miller MA, Pfeiffer W, Schwartz T (2010) Creating the CIPRES Science Gateway for inference of large phylogenetic trees. In: Gateway Computing Environments Workshop (GCE). IEEE, New Orleans, pp 1–8
Milne I, Lindner D, Bayer M, Husmeier D, McGuire G, Marshall DF, Wright F (2009) TOPALi v2: a rich graphical interface for evolutionary analyses of multiple alignments on HPC clusters and multi-core desktops. Bioinformatics 25(1):126–127. https://doi.org/10.1093/bioinformatics/btn575
Muñiz-Reyes E, Guzmán-Franco AW, Sánchez-Escudero J, Nieto-Angel R (2014) Occurrence of entomopathogenic fungi in tejocote (Crataegus mexicana) orchard soils and their pathogenicity against Rhagoletis pomonella. J Appl Microbiol 117(5):1450–1462. https://doi.org/10.1111/jam.12617
Nishi O, Hasegawa K, Iiyama K, Yasunaga-Aoki C, Shimizu S (2011) Phylogenetic analysis of Metarhizium spp. isolated from soil in Japan. Appl Entomol Zool 46(3):301–309. https://doi.org/10.1007/s13355-011-0045-y
Nishi O, Iiyama K, Yasunaga-Aoki C, Shimizu S (2015) Phylogenetic status and pathogenicity of Metarhizium majus isolated from a fruit beetle larva in Japan. Mycol Prog 14(8):58. https://doi.org/10.1007/s11557-015-1082-7
Nunes Rocha LF, Hua Tai MH, dos Santos AH, dos Santos Albernaz DA, Humber RA, Luz C (2009) Occurrence of invertebrate-pathogenic fungi in a Cerrado ecosystem in Central Brazil. Biocontrol Sci Tech 19(5–6):547–553. https://doi.org/10.1080/09583150902789337
Ormond EL, Thomas APM, Pugh PJA, Pell JK, Roy HE (2010) A fungal pathogen in time and space: the population dynamics of Beauveria bassiana in a conifer forest. FEMS Microbiol Ecol 74(1):146–154. https://doi.org/10.1111/j.1574-6941.2010.00939.x
Pérez-González VH, Guzmán-Franco AW, Alatorre-Rosas R, Hernández-López J, Hernández-López A, Carrillo-Benítez MG, Baverstock J (2014) Specific diversity of the entomopathogenic fungi Beauveria and Metarhizium in Mexican agricultural soils. J Invertebr Pathol 119:54–61. https://doi.org/10.1016/j.jip.2014.04.004
Popowska-Nowak E, Skrzecz I, Tumialis D, Pezowics E, Samborska I, Goral K (2016) Entomopathogenic fungi in the soils of forest plantations: towards the control of large pine weevil, Hylobius abietis. Balt For 22(1):8–15
Posada D, Crandall KA (1998) MODELTEST: testing the model of DNA substitution. Bioinformatics 14(9):817–818. https://doi.org/10.1093/bioinformatics/14.9.817
Posadas JB, Comerio RM, Mini JI, Nussenbaum AL, Lecuona RE (2012) A novel dodine-free selective medium based on the use of cetyl trimethyl ammonium bromide (CTAB) to isolate Beauveria bassiana, Metarhizium anisopliae sensu lato and Paecilomyces lilacinus from soil. Mycologia 104(4):974–980. https://doi.org/10.3852/11-234
Qian H, Ricklefs RE (2016) Out of the tropical lowlands: latitude versus elevation. Trends Ecol Evol 31(10):738–741. https://doi.org/10.1016/j.tree.2016.07.012
Rangel DEN, Braga GUL, Fernandes ÉKK, Keyser CA, Hallsworth JE, Roberts DW (2015) Stress tolerance and virulence of insect-pathogenic fungi are determined by environmental conditions during conidial formation. Curr Genet 61(3):383–404. https://doi.org/10.1007/s00294-015-0477-y
Rehner SA, Buckley E (2005) A Beauveria phylogeny inferred from nuclear ITS and EF1-α sequences: evidence for cryptic diversification and links to Cordyceps teleomorphs. Mycologia 97(1):84–98
Rehner SA, Posada F, Buckley EP, Infante F, Castillo A, Vega FE (2006) Phylogenetic origins of African and Neotropical Beauveria bassiana s.l. pathogens of the coffee berry borer, Hypothenemus hampei. J Invertebr Pathol 93(1):11–21. https://doi.org/10.1016/j.jip.2006.04.005
Rehner SA, Minnis AM, Sung G-H, Luangsa-ard JJ, Devotto L, Humber RA (2011) Phylogeny and systematics of the anamorphic, entomopathogenic genus Beauveria. Mycologia 103(5):1055–1073. https://doi.org/10.3852/10-302
Reich PB, Oleksyn J, Modrzynski J, Mrozinski P, Hobbie SE, Eissenstat DM, Chorover J, Chadwick OA, Hale CM, Tjoelker MG (2005) Linking litter calcium, earthworms and soil properties: a common garden test with 14 tree species. Ecol Lett 8(8):811–818. https://doi.org/10.1111/j.1461-0248.2005.00779.x
Rezende JM, Zanardo ABR, da Silva Lopes M, Delalibera I, Rehner SA (2015) Phylogenetic diversity of Brazilian Metarhizium associated with sugarcane agriculture. BioControl 60(4):495–505. https://doi.org/10.1007/s10526-015-9656-5
Rocha LFN, Inglis PW, Humber RA, Kipnis A, Luz C (2013) Occurrence of Metarhizium spp. in Central Brazilian soils. J Basic Microbiol 53(3):251–259. https://doi.org/10.1002/jobm.201100482
Rouxel T, Grandaubert J, Hane JK, Hoede C, van de Wouw AP, Couloux A, Dominguez V, Anthouard V, Bally P, Bourras S, Cozijnsen AJ, Ciuffetti LM, Degrave A, Dilmaghani A, Duret L, Fudal I, Goodwin SB, Gout L, Glaser N, Linglin J, Kema GHJ, Lapalu N, Lawrence CB, May K, Meyer M, Ollivier B, Poulain J, Schoch CL, Simon A, Spatafora JW, Stachowiak A, Turgeon BG, Tyler BM, Vincent D, Weissenbach J, Amselem J, Quesneville H, Oliver RP, Wincker P, Balesdent M-H, Howlett BJ (2011) Effector diversification within compartments of the Leptosphaeria maculans genome affected by repeat-induced point mutations. Nat Commun 2:202. https://doi.org/10.1038/ncomms1189
Santos MP, Dias LP, Ferreira PC, Pasin LAAP, Rangel DEN (2011) Cold activity and tolerance of the entomopathogenic fungus Tolypocladium spp. to UV-B irradiation and heat. J Invertebr Pathol 108(3):209–213. https://doi.org/10.1016/j.jip.2011.09.001
Spatafora JW, Sung GH, Sung JM, Hywel-Jones NL, White JF (2007) Phylogenetic evidence for an animal pathogen origin of ergot and the grass endophytes. Mol Ecol 16(8):1701–1711. https://doi.org/10.1111/j.1365-294X.2007.03225.x
Sun J, Fuxa JR, Henderson G (2002) Sporulation of Metarhizium anisopliae and Beauveria bassiana on Coptotermes formosanus and in vitro. J Invertebr Pathol 81(2):78–85. https://doi.org/10.1016/S0022-2011(02)00152-0
Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6: Molecular Evolutionary Genetics Analysis Version 6.0. Mol Biol Evol 30(12):2725–2729. https://doi.org/10.1093/molbev/mst197
Taylor JW, Jacobson DJ, Kroken S, Kasuga T, Geiser DM, Hibbett DS, Fisher MC (2000) Phylogenetic species recognition and species concepts in fungi. Fungal Genet Biol 31(1):21–32. https://doi.org/10.1006/fgbi.2000.1228
Vanninen I (1996) Distribution and occurrence of four entomopathogenic fungi in Finland: effect of geographical location, habitat type and soil type. Mycol Res 100(1):93–101. https://doi.org/10.1016/S0953-7562(96)80106-7
Wang Y-C, Wang D-X, Liu H-R (2013) Altitudinal distribution of seed plants in Foping and Changqing, Qinling Mountains, China. Ann Bot Fenn 50(1–2):43–49. https://doi.org/10.5735/085.050.0106
Wyrebek M, Huber C, Sasan RK, Bidochka MJ (2011) Three sympatrically occurring species of Metarhizium show plant rhizosphere specificity. Microbiology 157(10):2904–2911. https://doi.org/10.1099/mic.0.051102-0
Acknowledgements
We would like to thank team members from the Lab of Insect Related Resources (LIRR) who assisted during the forest survey. We are also grateful to Prof. Liang-jian Qu from the Chinese Academy of Forestry (CAF), Beijing, China, for providing the gypsy moth eggs and Dr. Lin Lv (Northwest A&F University) for the support using the advanced stereo microscope. The authors would like to thank Prof. John Richard Schrock (Emporia State University, USA) for scientific language editing of the whole manuscript. We gratefully acknowledge the critical reviews by the anonymous reviewers for their constructive comments that helped strengthen the paper.
Funding
This study was supported by NSFC grant (31670659), Special Fund for Forest Scientific Research in the Public Welfare (201404403-09), and Shaanxi Provincial Science and Technology Innovation Project (2014KTCL02-14).
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Conflict of interest
The authors declare that they have no competing interests.
Ethical approval
This article does not contain any studies with human participants or animals performed by any of the authors.
Electronic supplementary material
ESM 1
(PDF 117 kb)
Rights and permissions
About this article
Cite this article
Masoudi, A., Koprowski, J.l., Bhattarai, U.R. et al. Elevational distribution and morphological attributes of the entomopathogenic fungi from forests of the Qinling Mountains in China. Appl Microbiol Biotechnol 102, 1483–1499 (2018). https://doi.org/10.1007/s00253-017-8651-4
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00253-017-8651-4