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.

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.

Differential Effect of SdhB Gene Mutations on the Sensitivity to SDHI Fungicides in Botrytis cinerea.

Succinate dehydrogenase inhibiting (SDHI) fungicides constitute a relatively novel fungicide group used for gray mold control caused mainly by Botrytis cinerea. Shortly after registration, resistance was observed in fungal populations that correlated with several mutations in the succinate dehydrogenase complex (complex II). In the current study, 30 B. cinerea isolates possessing five different mutations at three different codons of SdhB (P225F, N230I, and H272L/R/Y) were characterized for their sensitivities to eight SDHI fungicides. The results show different sensitivities and cross-resistance patterns between structurally different SDHIs. P225F mutants were resistant in vitro to all SDHIs tested. Similarly, isolates possessing the H272L mutation were highly resistant to boscalid but showed low to moderate levels of resistance to other SDHIs. The N230I mutants were moderately resistant to boscalid, fluopyram, and fluxapyroxad and showed low resistance levels to isopyrazam, bixafen, fenfuram, benodanil, and carboxin. The H272R mutants showed moderate levels of resistance to boscalid and low resistance levels to isopyrazam, fenfuram, and carboxin but remained sensitive to fluopyram, bixafen, fluxapyroxad, and benodanil. Similarly, the H272Y showed moderate levels of resistance to boscalid and very low resistance levels to isopyrazam, bixafen, fenfuram, and carboxin but showed increased sensitivity to benodanil and fluopyram. Boscalid provided moderate to high control of H272R/Y and N230I mutants in detached fruit assays but provided little control against the H272L and P225F mutants. In contrast, fluopyram controlled H272R/Y mutants and provided moderate levels of control toward H272L, N230I, and P225F mutants. Our findings suggest that sensitivity to SDHIs may vary greatly, dependent on the point mutation in the sdhb subunit.

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.

Determination of benzoylurea insecticide residues in tomatoes by high-performance liquid chromatography with ultraviolet-diode array and atmospheric pressure chemical ionization-mass spectrometry detection.

A simple and sensitive method using high-performance liquid chromatography/ mass spectrometry (LC/MS) was developed and validated for simultaneous determination of 5 benzoylurea insecticides-diflubenzuron, triflumuron, teflubenzuron, lufenuron, and flufenoxuron-in tomatoes. Residues were successfully separated on a C18 column by methanol-water isocratic elution. Detection was carried out by an ultraviolet diode array detector (UV-DAD) coupled with a quadrupole mass spectrometer, using atmospheric pressure chemical ionization (APCI) in negative-ion mode. The main ions were the deprotonated molecules [M-H]- for triflumuron, and the anions formed by elimination of hydrofluoric acid [M-H-HF]- for diflubenzuron and flufenoxuron, and [M-2H-HF] for lufenuron and teflubenzuron. The calibration plots were linear for both detectors over the range 0.05 to 10 microg/mL, and the method presented good quality parameters. The limits of detection for standard solutions were 0.008-0.01 mg/L (equivalent to 0.08-0.1 ng injected) for both detectors, and the limits of quantification (LOQs) were approximately 10 times lower than national maximum residue levels (MRLs). Depending on the compound and the detector, the LOQ values ranged from 0.2 to 0.4 ng injected. The optimum LC-UV-DAD/APCI-MS conditions were applied to the analysis of benzoylureas in tomatoes. The obtained recoveries from fortified tomato samples (50 g), extracted with ethyl acetate and purified by solid-phase extraction on silica sorbent, were 88-100 and 92.9-105% for the UV-DAD and MS detectors, respectively, with precision values (relative standard deviations) of 2.9-11 and 3.7-14%, respectively. The method was applied to 12 tomato samples from local markets, and diflubenzuron and lufenuron were detected in only one sample at concentrations lower than the MRLs. The results indicate that the developed LC/MS method is accurate, precise, and sensitive for quantitative and qualitative analysis at low levels of benzoylureas required by legislation.

A non-Mendelian inheritance of resistance to strobilurin fungicides in Ustilago maydis.

Mutants of Ustilago maydis (DC) Corda with high resistance to azoxystrobin (RF 164 to 4714, based on EC50 values), an inhibitor of mitochondrial electron transport at the cytochrome bc1 complex, were isolated in a mutation frequency of 2.3 x 10(-7) after nitrosoguanidine mutagenesis and selection on media containing 1 microgram ml-1 azoxystrobin in addition to 0.5 mM salicylhydroxamate (SHAM), a specific inhibitor of cyanide-resistant (alternative) respiration. Oxygen uptake in whole cells was strongly inhibited in the wild-type strains by azoxystrobin (1.5 micrograms ml-1) in addition to SHAM (1 mM), but not in the mutant isolates. Genetic analysis with nine such mutant isolates resulted in progeny phenotypes which did not follow Mendelian segregation, but satisfied the criteria of non-Mendelian (cytoplasmic) heredity. In crosses between three mutant isolates with the compatible wild-type strains, the sensitivity was inherited by progeny maternally from the wild-type parent strain (criterion of uniparental inheritance). In crosses between wild-type strains and remaining mutant isolates, a continuous distribution of sensitivity in the progeny was found (criterion of vegetative segregation). The third criterion of cytoplasmic resistance (criterion of intracellular selection) was fulfilled by experiments on the stability of resistance phenotypes. With two exceptions, a reduction of resistance was observed in the mutant strains when they were grown on inhibitor-free medium. Recovery of the high resistance level was observed after they were returned to the selection medium. Cross-resistance studies with other fungicides, which also inhibit electron transport through complex III of respiratory chain, showed that mutations for resistance to azoxystrobin were also responsible for reduced sensitivity to kresoxim-methyl (RF 18 to 1199) and to antimycin-A (RF 20 to 305), which act at the Qo and Qi sites of the cytochrome bc1 complex, respectively. Studies of the fitness of azoxystrobin-resistant isolates showed that these mutations appeared to be pleiotropic, having significant adverse effects on growth in liquid culture and pathogenicity on young corn plants.