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.

Molecular characterization, fitness and mycotoxin production of benzimidazole-resistant isolates of Penicillium expansum.

Penicillium expansum field-strains resistant to benzimidazole fungicides were isolated in high frequency from decayed apple fruit collected from packinghouses and processing industries located in the region of Imathia, N. Greece. In vitro fungitoxicity tests resulted in the identification of two different resistant phenotypes: highly (BEN-HR) and moderately (BEN-MR) carbendazim-resistant. Thirty seven percent of the isolated P. expansum strains belonged to the BEN-HR phenotype, carried no apparent fitness penalties and exhibited resistance levels higher than 60 based on EC50 values. Cross resistance studies with other benzimidazole fungicides showed that all BEN-HR and BEN-MR isolates were also less sensitive to benomyl and thiabendazole. Fungitoxicity tests on the response of BEN-HR isolates to fungicides belonging to other chemical classes revealed no cross-resistance relationships between benzimidazoles and the phenylpyrrole fludioxonil, the dicarboximide iprodione, the anilinopyrimidine cyprodinil, the QoI pyraclostrobin, the imidazole imazalil and the triazole tebuconazole, indicating that a target-site modification is probably responsible for the BEN-HR phenotype observed. Contrary to the above, some BEN-MR isolates exhibited an increased sensitivity to cyprodinil compared to benzimidazole-sensitive ones. BEN-MR isolates had fitness parameters similar to the benzimidazole-sensitive isolates except for conidia production which appeared significantly decreased. Analysis of mycotoxin production (patulin and citrinin) showed that all benzimidazole-resistant isolates produced mycotoxins at concentrations significantly higher than sensitive isolates both on culture medium and on artificially inoculated apple fruit. Comparison of the ß-tubulin gene DNA sequence between resistant and sensitive isolates revealed a point mutation resulting from the E198A substitution of the corresponding protein in most but not all HR isolates tested. Molecular analysis of the ß-tubulin gene in moderately resistant isolates did not reveal any amino acid substitution. This is the first report on the existence and distribution of highly mycotoxigenic field isolates of P. expansum resistant to the benzimidazoles indicating a high potential risk of increased mycotoxin contamination of pome fruit and by-products.

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.

Effect of anilinopyrimidine resistance on aflatoxin production and fitness parameters in Aspergillus parasiticus Speare.

Mutants of Aspergillus parasiticus resistant to the anilinopyrimidine fungicides were isolated at a high mutation frequency after UV-mutagenesis and selection on media containing cyprodinil. In vitro fungitoxicity tests resulted in the identification of two predominant resistant phenotypes that were highly (R(1)-phenotype) and moderately (R(2)-phenotype) resistant to the anilinopyrimidines cyprodinil, pyrimethanil and mepanipyrim. Cross-resistance studies with fungicides from other chemical groups showed that the highly resistance mutation(s) did not affect the sensitivity of R(1)-mutant strains to fungicides affecting other cellular pathways. Contrary to that, a reduction in the sensitivity to the triazoles epoxiconazole and flusilazole, the benzimidazole carbendazim, the phenylpyrrole fludioxonil, the dicarboximide iprodione and to the strobilurin-type fungicide pyraclostrobin was observed in R(2)-mutant strains. Study of fitness parameters of anilinopyrimidine-resistant strains of both phenotypic classes showed that all R(1) mutant strains had mycelial growth rate, sporulation and conidial germination similar to or even higher than the wild-type parent strain, while these fitness parameters were negatively affected in R(2) mutant strains. Analysis of the aflatoxin production showed that most R(1) mutant strains produced aflatoxins at concentrations markedly higher than the wild-type parent strain. A considerable reduction in the aflatoxin production was observed on cultured medium and on wheat grains by all R(2) mutant strains, indicating a possible correlation between fitness penalties and aflatoxigenic ability of A. parasiticus. The potential risk of increased aflatoxin contamination of agricultural products and their byproducts by the appearance and predominance of highly aflatoxigenic mutant strains of A. parasiticus resistant to the anilinopyrimidines is discussed.

Sensitivity of Penicillium expansum field isolates to tebuconazole, iprodione, fludioxonil and cyprodinil and characterization of fitness parameters and patulin production.

A total of 236 Penicillium expansum field isolates from decayed apple fruit collected from packinghouses and processing industries located in the region of Imathia, Northern Greece were tested for their sensitivity to tebuconazole, fludioxonil, iprodione and cyprodinil. Preliminary fungitoxicity tests on the response of the isolates showed several phenotypes, distinguished according to their sensitivity to fungicides tested. The EC(50) values ranged from 0.64 to 5 (average = 0.98) µg/ml for iprodione, 0.9 to 7.3 (average = 2.66) µg/ml for tebuconazole, 0.008 to 1.28 (average = 0.55) µg/ml for cyprodinil and from 0.013 to 0.47 (average = 0.08) µg/ml for fludioxonil. A bimodal distribution of the EC(50) values of isolates with distinct sensitive and resistant populations to fludioxonil and tebuconazole were observed. In the case of cyprodinil, a much broader, hundred-fold, range of sensitivity was found, probably indicating that some isolates are relatively insensitive to cyprodinil compared to the most sensitive ones. Isolates exhibiting simultaneously reduced sensitivity to tebuconazole and fludioxonil or tebuconazole and iprodione or to tebuconazole and cyprodinil were also observed at low frequencies. A small portion of the population (7.5%) showed multiple resistance to tebuconazole, fludioxonil and iprodione. Study of fitness determining parameters showed that the resistance to tebuconazole, fludioxonil and iprodione had a significant adverse effect on mycelial growth rate and pathogenicity. Contrary to that, these fitness parameters were not affected in the isolates showing reduced sensitivity to cyprodinil. Analysis of patulin production on YES-agar growth medium and on artificially inoculated apple fruit showed that all isolates were mycotoxigenic. Most of the cyprodinil-insensitive isolates produced patulin at concentrations similar to or relatively higher (up to 1.5-fold on growth medium) than the sensitive ones. In contrast, a significant reduction (up to 98% of multiple resistant isolates) in patulin production was observed in all other phenotypes, indicating an adverse effect of fitness penalties on the mycotoxigenic ability of resistant isolates. The above mentioned data clearly show a considerable risk for the selection of P. expansum isolates resistant to fludioxonil, iprodione, tebuconazole and cyprodinil. The potential risk of increased patulin contamination of apples and their byproducts by the appearance and predominance of highly mycotoxigenic isolates of P. expansum resistant to the anilinopyrimidines is discussed.

Phenylpyrrole-resistance and aflatoxin production in Aspergillus parasiticus Speare.

Mutants of Aspergillus parasiticus highly resistant to phenylpyrroles were isolated at a high mutation frequency, after UV-mutagenesis and selection on media containing fludioxonil. Studies on the effect of mutation(s) on the aflatoxin production resulted in the identification of two fludioxonil-resistant phenotypes: aflatoxigenic (FLD(afl)(+)) and non-aflatoxigenic (FLD(afl)(-)) mutant strains. Most of the FLD(afl)(+) mutant strains produced the aflatoxin B(1) at similar or even higher (up to 2.5-fold) concentrations than the wild-type parent strain on yeast extract sucrose medium. Interestingly, in most of these mutant strains the aflatoxigenic ability significantly increased (up to 4-fold) when the mutants were grown on fungicide-amended medium. However, a significant reduction in the aflatoxin production was observed in wheat grains by all FLD(afl)(+) mutant strains. Tests on the response of mutant strains to high osmotic pressure showed that most fludioxonil-resistant mutants were more sensitive to high osmolarity than the wild-type parent strain. 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 and conidial germination. However, in a number of aflatoxigenic-mutant strains these fitness parameters were unaffected or only slightly affected. Cross resistance studies with fungicides from different chemical groups showed that the mutation(s) for resistance to fludioxonil also highly reduced the sensitivity of mutant strains to the aromatic hydrocarbon and dicarboximide fungicides. No effect of phenylpyrroles resistance mutation(s) on fungitoxicity of triazoles, benzimidazoles, anilinopyrimidines, phenylpyridinamines, strobilurin-type fungicides and to the non site-specific inhibitors chlorothalonil and maneb was observed. The above mentioned data indicate, for the first time, the potential risk of increased aflatoxin contamination of agricultural products by the appearance and predominance of highly aflatoxigenic mutant strains of A. parasiticus resistant to aromatic hydrocarbon, dicarboximide and phenylpyrrole fungicides.