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The application of entomopathogenic fungi such as Isaria fumosorosea to combat insect pests on plants is complicated by their sensitivity to commonly used fungicides. In this study, I. fumosorosea mutants with enhanced resistance to the fungicide benomyl were induced by irradiation using either ion beams or gamma rays, or a combination of the two. When grown on agar containing benomyl, mycelial growth was observed for five of the six mutant isolates at benomyl concentrations that were more than 2000-fold those observed for the wild-type isolate (EC50 : > 5000 mg L(-1) c.f. EC50 : 2.5 mg L(-1) for the wild-type isolate). The mutant isolates evaluated also showed enhanced resistance to other fungicides at recommended field application rates. No differences were observed at the β-tubulin locus between the wild-type and the mutant isolates, suggesting that the enhanced benomyl resistance was not attributable to mutations in that gene. Ion beams and gamma rays are thus potentially useful tools for inducing beneficial fungal mutations and thereby improving the potential for application of entomopathogenic fungi as microbial control agents., (© 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.)