Pesticide Science Laboratory, Agricultural University of Athens, 75 Iera Odos Str., Votanikos, 11855 Athens, Greece.
Fungal Genet Biol. 2012 Oct;49(10):792-801. doi: 10.1016/j.fgb.2012.07.008. Epub 2012 Aug 11.
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.
经紫外线诱变和含氟硅唑的培养基选择，从烟曲霉突变株中分离出对 DMIs 具有抗性的突变株，其在高突变频率下。根据产黄曲霉毒素的能力，确定了两种不同的抗性表型，R(1)和 R(2)。所有 R(1)突变株在酵母提取物蔗糖培养基上产生的黄曲霉毒素浓度显著高于（高达 3 倍）野生型亲本菌株，而大多数突变株（R(2)表型）失去了产黄曲霉毒素的能力。实时 PCR 分析黄曲霉毒素生物合成途径转录调节基因 aflR 的表达水平表明，在所测试的 R(2)突变株中未检测到该基因的表达。对决定适应性的参数的研究表明，大多数氟硅唑抗性突变株的菌丝生长速度、产孢和孢子萌发率低于敏感株。与其他杀菌剂的交叉抗性研究表明，所有 R(1)突变株对 DMIs 中的咪唑菌和戊唑醇也具有抗性，但对影响其他代谢途径和/或细胞过程的杀菌剂保持其亲本敏感性。与此相反，所有 R(2)突变株对 DMIs 和几种其他杀菌剂类别的多药耐药性均较低至中度。克隆并表征了两个不同的同源基因 cyp51A 和 cyp51B，它们分别编码 C-14α 甾醇去甲基化酶（Cyp51）和 mdr 基因，该基因编码一种可能参与多药耐药的 ATP 结合盒蛋白。cyp51A 基因序列比较显示，在三个 R(1)突变株中的两个中，甘氨酸（GGG）突变为色氨酸（TGG）在第 54 位（G54W）。cyp51B 推导的氨基酸序列分析表明，与 DMI 抗性相关的突变不存在。对 cyp51A 基因转录水平的研究表明，该基因在第三个产黄曲霉毒素的突变株中过度表达。在测试的 R(2)突变株中未发现 cyp51A 基因的氨基酸取代或过度表达。实时 PCR 分析显示，所有测试的 R(2)突变株中的 mdr 转录物水平均很高（高达 25 倍）。这是首次描述烟曲霉中存在两个 cyp51 基因和一个可能编码 ATP 结合盒蛋白的 mdr 基因。这些结果还表明，包括由于 cyp51A 基因突变导致靶位修饰、cyp51A 基因过度表达和 ABC 转运蛋白功能在内的多种生化机制，导致烟曲霉对 DMIs 的抗性。我们的研究结果表明，烟曲霉具有在田间出现高产黄曲霉毒素 DMIs 抗性分离株的遗传和生化潜力。
