| 官 鑫，涂 敏，蔡海滨，王云月，曾 霞，胡彦师.橡胶树白粉病 PMA-qPCR 检测体系及应用[J].广东农业科学,2022,49(5):102-109 |
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橡胶树白粉病 PMA-qPCR 检测体系及应用 |
| PMA-qPCR Detection System for Powdery Mildew of Rubber Tree and Its Application |
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| DOI：10.16768/j.issn.1004-874X.2022.05.012 |
| 中文关键词: 橡胶树 白粉病 PMA-qPCR 技术 分生孢子 丙环唑 |
| 英文关键词: rubber tree powdery mildew PMA-qPCR conidium propiconazole |
| 基金项目:国家重点研发计划项目（2019YFD10005002-0303）；农业农村部农垦局物种资源保护项目（18220019） |
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| 中文摘要: |
| 【目的】橡胶树白粉病是迄今为止我国橡胶树最重要的叶部病害，通过将叠氮溴化丙啶（PMA）
与实时荧光定量 PCR 技术（qPCR）相结合，构建橡胶树白粉病 PMA-qPCR 检测体系，为及时高效检测橡胶
树白粉菌分生孢子活菌量提供技术支撑。【方法】根据橡胶树白粉菌核糖体内转录间隔区（ITS）序列保守区
设计引物，验证其特异性，以该菌重组质粒 pMD-19T-Eq 绘制标准曲线，建立橡胶树白粉病 PMA-qPCR 检测
体系，对其重复性和灵敏度进行评价，并运用该体系对丙环唑处理橡胶树白粉病病叶后的活菌数进行检测。
【结果】研究设计的引物特异性良好，构建的橡胶树白粉菌 qPCR 标准曲线循环阈值（Ct）与模板浓度呈良
好的线性关系，相关系数为 0.9981，扩增效率为 107.97%。PMA-qPCR 对活孢子悬浮液扩增几乎无影响，检
测灵敏度为 4.77×102
copies/μL。在丙环唑处理橡胶树白粉病 15 min 后，活菌比例从 66.13% 下降至 23.18%。
【结论】PMA-qPCR 检测体系能快速定量检测橡胶树白粉病活菌量，具有灵敏、准确和高效的优点，为该病害
的预测预报及防治效果提供理论依据和技术手段。 |
| 英文摘要: |
| 【Objective】Powdery mildew of rubber tree is the most serious leaf disease of rubber tree in China so far.
The PMA-qPCR detection system for powdery mildew of rubber tree was constructed by combining propidium monoazide
(PMA) with real-time fluorescence quantitative PCR (qPCR), which provided technical support for the timely and efficient
detection of viable conidia of powdery mildew in rubber tree.【Method】Primers were designed according to the conserved
region of ribosomal internal transcribed spacer (ITS) sequence of Erysiphe quercicola and its specificity was verified.
The standard curve was drawn with the recombinant plasmid pMD-19T-Eq. A PMA-qPCR detection system for powdery
mildew of rubber tree was established to evaluate its repeatability and sensitivity. The system was used to detect the viable
count of powdery mildew after propiconazole treatment.【Result】The designed primers had good specificity. A good linear
relationship between cycle threshold (Ct) and template concentration was found in the constructed qPCR standard curve for Erysiphe quercicola, the correlation coefficient was 0.9981, and the amplification efficiency was 107.97%. PMA-qPCR had
little effect on the amplification of live spore suspension, and the detection sensitivity was 4.77×102
copies/μL. After treatment
with propiconazole for 15 min, the proportion of live bacteria decreased from 66.13% to 23.18%.【Conclusion】The PMAqPCR system could rapidly and quantitatively detect the number of live bacteria of powdery mildew in rubber tree, which had
the advantages of sensitivity, accuracy and efficiency, and provided theoretical basis and technical means for the prediction and
control of the disease. |
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