Pesticide Science Laboratory, Agricultural University of Athens, 75 Iera Odos, 118 55 Athens, Greece.
Int J Food Microbiol. 2013 Apr 1;162(3):237-44. doi: 10.1016/j.ijfoodmicro.2013.01.014. Epub 2013 Jan 31.
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 β-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 β-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.
从希腊北部伊马夏地区包装厂和加工厂采集的腐烂苹果中,高频分离到对苯并咪唑类杀真菌剂有抗性的扩展青霉田间菌株。体外杀真菌毒性试验鉴定出两种不同的抗性表型:高度(BEN-HR)和中度(BEN-MR)多菌灵抗性。分离的 37%扩展青霉菌株属于 BEN-HR 表型,没有明显的适应性代价,且根据 EC50 值,其抗性水平高于 60。对其他苯并咪唑类杀真菌剂的交叉抗性研究表明,所有 BEN-HR 和 BEN-MR 分离株对苯菌灵和噻菌灵的敏感性也较低。对 BEN-HR 分离株对属于其他化学类别的杀真菌剂的反应进行杀真菌毒性试验,发现苯并咪唑类与苯并吡咯氟啶酮、二羧酰亚胺异菌脲、苯胺嘧啶嘧菌酯、QoI 吡唑醚菌酯、咪唑类抑霉唑和三唑类戊唑醇之间不存在交叉抗性关系,表明可能是靶标位点的改变导致了观察到的 BEN-HR 表型。与上述情况相反,一些 BEN-MR 分离株对环丙啶的敏感性高于苯并咪唑敏感分离株。BEN-MR 分离株的适应度参数与苯并咪唑敏感分离株相似,但产孢量明显下降。对产毒素(棒曲霉素和桔霉素)的分析表明,所有苯并咪唑抗性分离株在培养基和人工接种的苹果果实上产生的真菌毒素浓度均明显高于敏感分离株。对抗性和敏感分离株的β-微管蛋白基因 DNA 序列进行比较,发现大多数但并非所有 HR 分离株中,E198A 取代导致相应蛋白的点突变。对中度抗性分离株的β-微管蛋白基因进行分子分析,未发现任何氨基酸取代。这是第一个关于对苯并咪唑类具有高度产毒能力的扩展青霉田间分离株存在和分布的报告,表明高度潜在的风险增加了苹果和副产品中真菌毒素的污染。