Phytopathogenic and mycotoxigenic characterization of laboratory mutant strains of Aspergillus carbonarius and Penicillium expansum resistant to phenylpyrrole fungicides

Mutants of Aspergillus carbonarius and Penicillium expansum highly resistant (Rf: 100-130) to phenylpyrroles were isolated at high mutation frequency after UV-mutagenesis and selection on media containing fludioxonil. Cross resistance studies with other fungicides showed that the mutation(s) for resistance to fludioxonil also reduced the sensitivity of mutant strains to the aromatic hydrocarbon (tecnazene, tolclofos-methyl) and dicarboximide (iprodione, vinclozolin) fungicides. No effect of phenylpyrroles resistance mutation(s) on fungitoxicity of triazoles, anilinopyrimidines, phenylpyridinamines and to the non-site specific inhibitor chlorothalonil was observed. However, an increased sensitivity (Rf: 0.06-0.1) of fludioxonil-resistant strains to the strobilurin-type fungicides azoxystrobin and pyraclostrobin was observed in all mutant strains tested. Tests for the evaluation of osmosensitivity of isolates showed that most fludioxonil-resistant isolates were more sensitive to high osmotic pressure than the wild-type parent strains. Study of other fitness determining parameters showed that the mutation(s) for resistance to phenylpyrroles may or may not affect the mycelial growth rate, sporulation, conidial germination and pathogenicity. However, in a few mutant strains these fitness parameters were unaffected or only slightly affected. Analysis of mycelial extracts from the wild-types and mutant strains, using thin layer chromatography (TLC) and high performance liquid chromatography (HPLC), showed that most A. carbonarius and all P. expansum mutant strains produced mycotoxins (ochratoxins and patulin, respectively) at significantly lower concentrations than the wild-type parent strains. However, in few A. carbonarius mutant strains the ochratoxin (OTA and OTB) production was much higher (up to 4-fold) than the wild-type strain. All P. expansum mutants produced citrinin at concentrations much higher than the wild-type strain. Interestingly, in all mutant strains tested the mycotoxigenic ability was further increased when they were grown on nudioxonil-amended medium. Similar results were also found in tests with artificially inoculated grapes and apples. The data of the present study indicate, for the first time, the potential risk of increased mycotoxin contamination of grapes and apples after intensive use of dicarboximides and/or phenylpyrroles.