Characterization of boscalid-resistance conferring mutations in the SdhB subunit of respiratory complex II and impact on fitness and mycotoxin production in Penicillium expansum laboratory strains.

Laboratory mutants of Penicillium expansum highly resistant (Rfs: 90 to >500, based on EC50s) to Succinate Dehydrogenase Inhibitors (SDHIs) were isolated after UV-mutagenesis and selection on media containing boscalid. A positive correlation was found between sensitivity of isolates to boscalid and other SDHIs such as isopyrazam and carboxin but not to fungicides affecting other cellular pathways or processes, such as the triazole flusilazole, the phenylpyrrole fludioxonil, the anilinopyrimidine cyprodinil and the benzimidazole benomyl. Most of the boscalid-resistant strains were more sensitive to the SDHI fluopyram and the QoI pyraclostrobin. In order to investigate the mechanism responsible for the observed resistance profiles, part of the SdhB subunit isolated the wild type and boscalid-resistant isolates, was genetically characterized. Comparison of the deduced amino-acid sequence between resistant and wild-type isolates revealed two point mutations at a position corresponding to codon 272 of the respective SdhB protein in Botrytis cinerea. The substitution of histidine by arginine was found in boscalid-resistant isolates which were equally sensitive to fluopyram compared with the wild-type whereas the replacement of histidine by tyrosine was found in strains with increased sensitivity to fluopyram. No adverse effects of resistance mutations were observed on fitness determining parameters such as osmotic sensitivity, sporulation and pathogenicity, while mycelial growth rate and spore germination was negatively affected in some of the mutants studied. P. expansum mutant strains displayed significantly perturbed patulin and citrinin levels as compared to the wild-type parent strain both in vitro and in vivo as revealed by thin layer (TLC) and high performance liquid chromatography (HPLC).

Effect of phenylpyrrole-resistance on fitness parameters and ochratoxin production in Aspergillus carbonarius.

The risk of resistance development to fludioxonil and the potential implications of resistance mutations to ochratoxin production in Aspergillus carbonarius were investigated. Mutants of A. carbonarius highly resistant to phenylpyrroles were isolated at a high mutation frequency after N-MNTG-mutagenesis and selection on media containing fludioxonil. A highly reduced sensitivity to fungicides belonging to the same cross-resistance group (AHDs and phenylpyrroles) such as the aromatic hydrocarbon tolclofos-methyl and the dicarboximide fungicides iprodione and vinclozolin was also observed. No cross-resistance relationships were found between fludioxonil and the triazole epoxiconazole, the anilinopyrimidine cyprodinil and the chloronitrile chlorothalonil. Interestingly, fludioxonil-resistant isolates were highly sensitive to the QoI fungicide pyraclostrobin compared to the wild-type parental strain. Fitness studies revealed that resistance mutation(s) had a negative effect on mycelial growth, resistance to osmotic stress and pathogenicity of the fludioxonil-resistant strains. Mycotoxin analysis showed that most fludioxonil-resistant strains produce less quantities of ochratoxin A (OTA) than the wild-type strain both when grown on artificial medium and on grapes. Increased osmotic sensitivity and reduced pathogenicity of the mutant strains were significantly correlated with reduced ochratoxin production in vivo but not in vitro. The above-mentioned data indicate that fludioxonil is an excellent fungicide for the control of A. carbonarius in grapes and a valuable asset for farmers in terms of resistance management and ochratoxin contamination of grapes, vine products and wines.