College of Plant Protection, Anhui Agricultural University, 130 West of Changjiang Road, Hefei 230036, Anhui, China; School of Life Sciences, Anhui Agricultural University, 130 West of Changjiang Road, Hefei 230036, Anhui, China.
College of Plant Protection, Anhui Agricultural University, 130 West of Changjiang Road, Hefei 230036, Anhui, China.
Gene. 2021 Jan 5;764:145081. doi: 10.1016/j.gene.2020.145081. Epub 2020 Aug 26.
Metalaxyl is one of the main fungicides used to control pepper blight caused by Phytophthora capsici. Metalaxyl resistance of P. capsici, caused by the long-term intense use of this fungicide, has become one of the most serious challenges facing pest management. In this study, a conserved domain RPOLA-N of the RPA190 gene of P. capsici (RPA190-pc) was identified from the P. capsici SD1-9 strain. The role of the RPA190-pc underlying the metalaxyl resistance of P. capsici was investigated. Three P. capsici mutants, two with downregulated RPA190-pc (SD1-9C-3 and C-4) expression and one showing upregulation (OESD1-9-1), were obtained by Polyethylene Glycol (PEG) mediated protoplast transformations of P. capsici SD1-9. Quantitative real-time reverse transcription PCR results showed that RPA190-pc was downregulated by more than 60% in SD1-9C-3/C-4 and upregulated 3-fold in OESD1-9-1 compared with that of the control strain SD1-9. Evaluation of the metalaxyl resistance of these three transformants showed that the EC values of metalaxyl against SD1-9C-3, SD1-9C-4, and OESD1-9-1 were 120.0 µg·mL, 24.4 µg·mL, and 15573.0 µg·mL, respectively, corresponding to 63.3% decrease, 92.5% decrease, and 47.7-fold increase relative to the EC value in SD1-9. Compared with SD1-9, the mycelia of transformants SD1-9C-3, SD1-9C-4, and OESD1-9-1 showed more branches and shorter branches; and the transformants had different pathogenicity to different hosts plants. The expression of the candidate gene RPA190-pc during 10 life-history stages was further studied, the results showed that expression level reached a maximum at the zoospores stage, and it gradually increased with the increase of SD1 and SD1-9 infection time of pepper leaves, indicated that RPA190-pc may be related to the growth and pathogenicity of P. capsici. These results indicate that the expression of RPA190-pc is involved in the regulation of P. capsici resistance to metalaxyl.
甲霜灵是防治辣椒疫病的主要杀菌剂之一。由于长期大量使用该杀菌剂，辣椒疫霉菌对甲霜灵的抗性已成为害虫管理面临的最严重挑战之一。本研究从辣椒疫霉菌 SD1-9 菌株中鉴定出辣椒疫霉菌 RPA190 基因的保守结构域 RPOLA-N（RPA190-pc）。研究了 RPA190-pc 在辣椒疫霉菌抗甲霜灵中的作用。通过聚乙二醇（PEG）介导的辣椒疫霉菌 SD1-9 原生质体转化，获得了两个下调 RPA190-pc 表达的 SD1-9C-3 和 C-4 突变体和一个上调 RPA190-pc 表达的 OESD1-9-1 突变体。实时荧光定量 RT-PCR 结果表明，与对照菌株 SD1-9 相比，SD1-9C-3/C-4 中 RPA190-pc 的表达下调了 60%以上，而 OESD1-9-1 中则上调了 3 倍。对这三种转化体的抗甲霜灵活性评价表明，SD1-9C-3、SD1-9C-4 和 OESD1-9-1 对甲霜灵的 EC 值分别为 120.0 μg·mL、24.4 μg·mL 和 15573.0 μg·mL，分别比 SD1-9 的 EC 值降低了 63.3%、92.5%和 47.7 倍。与 SD1-9 相比，转化体 SD1-9C-3、SD1-9C-4 和 OESD1-9-1 的菌丝分枝更多，分枝更短；并且这些转化体对不同的宿主植物具有不同的致病性。进一步研究候选基因 RPA190-pc 在 10 个生活史阶段的表达情况，结果表明，在游动孢子阶段表达水平达到最大值，随着 SD1 和 SD1-9 感染辣椒叶片时间的增加，表达水平逐渐升高，表明 RPA190-pc 可能与辣椒疫霉菌的生长和致病性有关。这些结果表明，RPA190-pc 的表达参与了辣椒疫霉菌对甲霜灵的抗性调节。
