Abstract
A novel system was developed for efficient transformation of the fungal biocontrol agent Beauveriabassiana. Competent blastospores were prepared and stored in LiAc- and glycerol-inclusive suspension at −76 °C for sequential use in transformation. The system was successfully applied to integrating phosphinothricin resistance gene bar and enhanced green fluorescence protein gene egfp into B. bassiana via blastospore absorption of a plasmid vectoring bar and egfp. A frequency of 24 transformants per microgram of DNA was achieved. The blastospore-based transformation system has proven to be very convenient and would be highly potential for use in genetic manipulation of B. bassiana and other filamentous species.
Similar content being viewed by others
References
Andaluz E, Ciudad T, Larriba G (2002) An evaluation of the role of LIG4 in genomic instability and adaptive mutagenesis in Candida albicans. FEMS Yeast Res 2:341–348
Barreto CC, Alves LC, Aragao FJ, Rech E, Schrank A, Vainstein MH (1997) High frequency gene transfer by microprojectile bombardment of intact conidia from the entomopathogenic fungus Paecilomyces fumosoroseus. FEMS Microbiol Lett 156:95–99
Bidochka MJ, Pfeifer TA, Khachatourians GG (1987) Development of entomopathogenic fungus Beauveria bassiana in liquid culture. Mycopathologia 99:77–83
Braga GUL, Flint SD, Miller CD, Anderson AJ, Roberts DW (2001a) Both solar UVA and UVB radiation impair conidial culturability and delay germination in the entomopathogenic fungus, Metarhizium anisopliae. Photochem Photobiol 74:734–739
Braga GUL, Flint SD, Miller CD, Anderson AJ, Roberts DW (2001b) 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 78:98–108
Brown JS, Aufauvre-Brown A, Holden DW (1998) Insertional mutagenesis of Aspergillus fumigatus. Mol Gen Genet 259:327–335
Daboussi MJ, Djeballi A, Gerlinger C, Blaiseau PL, Bouvier I, Cassan M, Lebrun MH, Parisot D, Brygoo Y (1989) Transformation of seven species of filamentous fungi using the nitrate reductase gene of Aspergillus nidulans. Curr Genet 15:453–456
de Groot MJA, Bundock P, Hooykaas PJJ, Beijersbergen AGM (1998) Agrobacterium tumefaciens-mediated transformation of filamentous fungi. Nat Biotechnol 16:839–842
dos Reis MC, Fungaro MHP, Duarte RTD, Furlaneto L, Furlaneto MC (2004) Agrobacterium tumefaciens-mediated genetic transformation of the entomopathogenic fungus Beauveria bassiana. J Microbiol Methods 58:197–202
Fang WG, Leng B, Xiao YH, Jin K, Ma JC, Fan YH, Feng J, Yang XY, Zhang YJ, Pei Y (2005) Cloning of Beauveria bassiana chitinase gene Bbchit1 and its application to improve fungal strain virulence. Appl Environ Microbiol 71:363–370
Feng MG, Johnson JB, Kish LP (1990) Survey of entomopathogenic fungi naturally infecting cereal aphids in irrigated cereal crops in southwestern Idaho. Environ Entomol 19:1534–1542
Feng MG, Poprawski TJ, Khachatourians GG (1994) Production, formulation and application of the entomopathogenic fungus Beauveria bassiana for insect control: current status. Biocontrol Sci Technol 4:3–34
Fincham JRS (1989) Transformation in fungi. Microbiol Rev 53:148–170
Fungaro MHP, Rech E, Mohlen GS, Vainstein MH, Pascon RC, de Queiroz MV, Pizzirani-Kleiner AA, de Azevedo JL (1995) Transformation of Aspergillus nidulans by microprojectile bombardment on intact conidia. FEMS Microbiol Lett 125:293–298
Koukaki M, Giannoutsou E, Karagouni A, Diallinas G (2003) A novel improved method for Aspergillus nidulans transformation. J Microbiol Methods 55:687–695
Pfeifer TA, Khachatourians GG (1992) Beauveria bassiana protoplast regeneration and transformation using electroporation. Appl Microbiol Biotechnol 38:376–381
Pribylova L, Sychrova H (2003) Efficient transformation of the osmotolerant yeast Zygosaccharomyces rouxii by electroporation. J Microbiol Methods 55:481–484
Rangel DEN, Braga GUL, Anderson AJ, Roberts DW (2005) Variability in conidial thermotolerance of Metarhizium anisopliae, isolates from different geographic origins. J Invertebr Pathol 88:116–125
Reader U, Broda P (1985) Rapid preparation of DNA from filamentous fungi. Lett Appl Microbiol 1:17–20
Roberts DW, St. Leger RJ (2004) Metarhizium spp., cosmopolitan insect-pathogenic fungi: mycological aspects. Adv Appl Microbiol 54:1–70
Sandhu SS, Unkles SE, Rajak RC, Kinghorn JR (2001) Generation of benomyl resistant Beauveria bassiana strains and their infectivity against Helicoverpa armigera. Biocontrol Sci Technol 11:250–254
Schiestl RH, Gietz RD (1989) High efficiency transformation of intact yeast cells using single stranded nucleic acids as a carrier. Curr Genet 16:339–346
Wraight SP, Jackson MA, de Kock SL (2001) Production, stabilization and formulation of fungal biological agents. In: Butt TM, Jackson C, Magan N (eds) Fungal biocontrol agents—progress, problems and potential. CAB International, Wallingford, pp 253–287
Ye SD, Dun YH, Feng MG (2005) Time and concentration dependent interactions of Beauveria bassiana with sublethal rates of imidacloprid against the aphid pests Macrosiphoniella sanborni and Myzus persicae. Ann Appl Biol 146:459–468
Ying SH, Feng MG (2004) Relationship between thermotolerance and hydrophobin-like proteins in aerial conidia of Beauveria bassiana and Paecilomyces fumosoroseus as fungal biocontrol agents. J Appl Microbiol 97:323–331
Ypsilos IK, Magan N (2005) Characterisation of optimum cultural environmental conditions for the production of high numbers of Metarhizium anisopliae blastospores with enhanced ecological fitness. Biocontrol Sci Technol 15:683–699
Acknowledgements
Punt PJ (TNO Nutrition and Food Research Institute, Zeist, The Netherlands) and Sun ZX (China National Rice Research Institute, Hangzhou, China) are thanked for providing plasmids pAN52-1N and pDS-1, respectively. Funding of this study was provided by the National ‘Program 973’ of the Ministry of Science and Technology of China (2003CB114203), the Natural Science Foundation of China (30571250), and Zhejiang Provincial R&D Program (2003G10038).
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Ying, SH., Feng, MG. Novel blastospore-based transformation system for integration of phosphinothricin resistance and green fluorescence protein genes into Beauveria bassiana . Appl Microbiol Biotechnol 72, 206–210 (2006). https://doi.org/10.1007/s00253-006-0447-x
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00253-006-0447-x