| 殷丽华,张金鹏,章国庆,刘国辉,柯希望,左豫虎.小豆锈病分子检测体系的构建及应用[J].干旱地区农业研究,2023,(6):238~244 |
| 小豆锈病分子检测体系的构建及应用 |
| Construction and application of molecular detection system for adzuki bean rust |
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| DOI：10.7606/j.issn.1000-7601.2023.06.25 |
| 中文关键词: 小豆 锈病 豇豆单胞锈菌 分子检测 巢式PCR |
| 英文关键词:Vigna angularis rust disease Uromyces vignae molecular detection nested PCR |
| 基金项目:国家自然科学基金项目（32102173）；黑龙江省自然科学基金项目（YQ2020C034） |
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| 中文摘要: |
| 由豇豆单胞锈菌（Uromyces vignae Barclay）引起的小豆锈病是小豆生产中危害最为严重的病害之一，建立早期分子检测技术体系对病害早期诊断和及时防控具有重要意义。本研究以豇豆单胞锈菌ITS序列为依据设计引物，结合已报道的单胞锈菌属特异检测引物，以豇豆单胞锈菌为目标菌，以茄链格孢、茄丝核菌、禾谷镰刀菌、稻瘟菌、苹果轮纹病菌、半裸镰刀菌、玉米链格孢菌、尖孢镰刀菌和禾顶囊壳等9种常见植物病原真菌为参照菌，采用CTAB法提取上述各菌株的基因组DNA，应用普通PCR技术筛选出豇豆单胞锈菌的特异性检测引物UV-ITS，在此基础上设计巢式PCR引物UV-MX，构建了基于巢式PCR的高灵敏度小豆锈病分子检测体系。该巢式PCR体系在基因组DNA浓度仅为4×10-6 ng·μL-1时仍可实现有效扩增，其灵敏度是普通PCR的10万倍。以感病小豆品种‘宝清红’接种锈菌后不同时间的叶片为材料检验检测体系的应用效果发现，接种后12 h的小豆叶片样本即可扩增出豇豆单胞锈菌的特异性条带。本研究建立的小豆锈病分子检测体系特异性强、灵敏度高，具备对潜伏期病害进行早期诊断的应用潜力，可为小豆锈病的早期快速诊断和及时防控提供重要的技术支撑。 |
| 英文摘要: |
| Adzuki bean rust, caused by the fungus Uromyces vignae Barclay, is one of the most serious diseases in adzuki bean production. Estabishing early molecular detection system can play important roles in the disease early diagnosis and timely prevention. Primers designed based on the ITS sequence of U. vignae and the specific primers of the genus Uromyces were used for specific primer screening in this study. A specific primer pair UV-ITS of U. vignae was screened based on the normal PCR by amplifying the genomic DNA, in which the fungi such as Alternaria solani, Rhizoctonia solanii, Fusarium graminearum, Magnaporthe oryzae, Botryosphaeria dothidea, Fusarium semitectum, Alternaria tenuis, Fusarium oxysporum, and Gaeumanomyces graminis were used as control. Accordingly, a pair of nested PCR primers UV-MX was designed, and a high sensitivity molecular detection system for adzuki bean rust based on nested PCR was established. The nested PCR system still achieved effective amplification when the genomic DNA concentration was only 4×10-6 ng·μL-1, and the detection efficiency was 100,000 times higher than the normal PCR. The application effect of this detection system was tested for U. vignae detection in the leaves of susceptible cultivar ‘Baoqinghong’ that inoculated with U. vignae at different time points. The results showed that a specific band of U. vignae was amplified at 12 h after inoculation. The molecular detection system of adzuki bean rust established in this study has high specificity and sensitivity and has the potential for early diagnosis of latent disease. This nested PCR detection system can provide important technical support for early diagnosis and timely prevention of adzuki bean rust. |
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