Phytopathology. 2001 Jan;91(1):102-10. doi: 10.1094/PHYTO.2001.91.1.102.
ABSTRACT We identified the cytochrome P450 sterol 14alpha-demethylase (CYP51A1) gene from Venturia inaequalis and optional insertions located upstream from CYP51A1 and evaluated their potential role in conferring resistance to the sterol demethylation-inhibitor (DMI) fungicide my-clobutanil. The CYP51A1 gene was completely sequenced from one my-clobutanil sensitive (S) and two myclobutanil-resistant (R) strains. No nucleotide variation was found when the three sequences were aligned. Allele-specific polymerase chain reaction (PCR) analysis indicated that a previously described single base pair mutation that correlated with resistance to DMI fungicides in strains of other filamentous fungi was absent in 19 S and 32 R strains of V. inaequalis from Michigan and elsewhere. The sequencing results and PCR analyses suggest that resistance in these strains was not due to a mutation in the sterol demethylase target site for DMI fungicides. Expression of CYP51A1 was determined for strains from an orchard that had never been sprayed with DMI fungicides (baseline orchard), and the data provided a reference for evaluating the expression of strains collected from a research orchard and from three commercial Michigan apple orchards with a long history of DMI use and a high frequency of R strains. Overexpression of CYP51A1 was significantly higher in 9 of 11 R strains from the research orchard than in S strains from the baseline orchard. The high expression was correlated with the presence of a 553-bp insertion located upstream of CYP51A1. Overexpression of the CYP51A1 gene was also detected in eight of eight, five of nine, and nine of nine R strains from three commercial orchards, but the insertion was not detected in the majority of these strains. The results suggest that overexpression of the target-site CYP51A1 gene is an important mechanism of resistance in some field resistant strains of V. inaequalis, but other mechanisms of resistance also appear to exist.
摘要 我们从梨形孢（Venturia inaequalis）中鉴定出细胞色素 P450 固醇 14α-脱甲基酶（CYP51A1）基因，并对其上游的选择性插入序列进行了评估，以研究它们在赋予对甾醇脱甲基抑制剂（DMI）杀真菌剂丙环唑抗性方面的潜在作用。我们对来自一个丙环唑敏感（S）菌株和两个丙环唑抗性（R）菌株的 CYP51A1 基因进行了完全测序。当将这三个序列进行比对时，未发现核苷酸变异。等位基因特异性聚合酶链反应（PCR）分析表明，在其他丝状真菌的菌株中，与 DMI 杀真菌剂抗性相关的一个先前描述的碱基对突变在来自密歇根州和其他地区的 19 个 S 和 32 个 R 菌株中并不存在。测序结果和 PCR 分析表明，这些菌株的抗性不是由于 DMI 杀真菌剂靶位固醇脱甲基酶的突变所致。我们对从未喷洒过 DMI 杀真菌剂的果园（基线果园）的菌株进行了 CYP51A1 的表达测定，并为评估从研究果园和三个具有长期 DMI 使用史和高 R 菌株频率的密歇根商业苹果园采集的菌株的表达提供了参考。在来自研究果园的 11 个 R 菌株中，有 9 个的 CYP51A1 过表达水平显著高于基线果园的 S 菌株。高表达与 CYP51A1 上游 553bp 插入的存在有关。在来自三个商业果园的 8/8、5/9 和 9/9 的 R 菌株中也检测到 CYP51A1 基因的过表达，但在大多数这些菌株中未检测到插入。结果表明，CYP51A1 靶位基因的过表达是一些田间抗性菌株对梨形孢抗性的重要机制，但也存在其他抗性机制。
