PMA-qPCR Detection System for Powdery Mildew of Rubber Tree and Its Application
2022
Xin GUAN | Min TU | Haibin CAI | Yunyue WANG | Xia ENG | Yanshi HU
【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 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.
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