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

【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4.77×10^(2) 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。

【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 4.77×10^(2) copies/μL.After treatment with propiconazole for 15 min,the proportion of live bacteria decreased from 66.13% to 23.18%.【Conclusion】The PMA-qPCR 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.