Molecular and biochemical study of resistance to zoxamide and other oomycete fungicides in Phytophthora infestans

Mutants of Phytophthora infestans with high resistance to the benzamide fungicide zoxamide were isolated from a wild-type strain after UV-mutagenesis and selection on medium containing zoxamide. Cross-resistance studies showed that the mutation(s) for resistance to zoxamide also greatly reduced the sensitivity of mutant strains to other oomycete fungicides from different chemical groups. Three possible mechanisms of fungicide resistance were studied to elucidate the resistance of P. infestans mutant strains: (a) The mechanism of target site modification with isolation and sequencing of b-tubulin gene from the wild type and mutant strains. (b) The mechanism of increased secretion with inhibitors of energy production, with the study of intracellular fungicide concentrationby LC-MS chromatography and with molecular analysis of a part of ABC-transporters gene. (c) The mechanism of detoxification with the use of synergists. The sequency of b-tubulin gene, the site of action ofbenzamide zoxamide andbenzimidazoles, showed common mutations in all resistant strains studied at the position 200 from methionine to phenylalanine and in other sites close to the positions of 200, 198 and 167, which are known mutation sites for benzimidazole resistance in other fungal species. From the above data, it seems that the target site modification explains the resistance of P. infestans to zoxamide. However, the resistance to other oomycete fungicides is not explained by a modification of b-tubulin gene. Study of the other mentioned above mechanisms did not provide positive results and more efforts are needed for that. An over expression of an ABC transporter gene using real time PCR, possibly, will give an explanation of multi drug resistance phenomenon in P. infestans.