Effect of DMI-resistance mechanisms on cross-resistance patterns, fitness parameters and aflatoxin production in Aspergillus parasiticus Speare.

Aspergillus parasiticus mutant strains resistant to DMIs were isolated in a high mutation frequency after UV-mutagenesis and selection on media containing flusilazole. Two different resistant phenotypes, R(1) and R(2), on the basis of their aflatoxigenic ability were identified. All R(1) mutant strains produced aflatoxins at concentrations significantly higher (up to 3-fold) than the wild-type parent strain on yeast extract sucrose medium, whereas the majority of mutant strains (R(2) phenotype) lost their aflatoxigenic ability. Real-time PCR analysis of the expression levels of the aflR gene, a pathway transcriptional regulatory gene in aflatoxin biosynthesis, showed that this gene was not expressed in R(2) mutant strains tested. Study of fitness determining parameters showed that most flusilazole-resistant mutant strains had mycelial growth rate, sporulation and spore germination lower that the sensitive one. Cross-resistance studies with other fungicides showed that all R(1) mutant strains were also resistant to the DMIs imazalil and tebuconazole, but retained their parental sensitivity to fungicides affecting other metabolic pathways and/or cellular processes. Contrary to the above, all R(2) mutant strains exhibited a low to moderate multi-drug resistance to DMIs and to several other fungicide classes. Two different homologous genes, cyp51A and cyp51B, encoding C-14 alpha sterol demethylase (Cyp51) and an mdr gene encoding an ATP-binding cassette protein which may be involved in multidrug resistance were cloned and characterized. Sequence comparison of cyp51A gene revealed an amino acid substitution from glycine (GGG) to tryptophan (TGG) at position 54 (G54W) in two out of three of R(1) mutant strains. Analysis of deduced amino acid sequence of cyp51B showed that no mutations were associated with DMI resistance. Study for the transcriptional levels of cyp51A showed that this gene was over-expressed in the third aflatoxigenic mutant strain. Neither amino acid substitutions nor an overexpression of the cyp51A gene were found in the R(2) mutant strains tested. Real-time PCR analysis showed high levels (up to 25-fold higher) of the mdr transcript in all R(2) mutant strains tested. This is the first report describing the existence of two cyp51 genes and a potential mdr gene coding for an ATP binding cassette protein in A. parasiticus. These results also indicate that multiple biochemical mechanisms, including target-site modification due to mutation at cyp51A gene, overexpression of cyp51A gene and the function of an ABC transporter protein, are responsible for DMI-resistance in A. parasiticus. Our findings suggest that A. parasiticus have the genetic and biochemical potential for the appearance of highly aflatoxigenic DMI-resistant isolates in the field.