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| Establishment and Application of Real-time Quantitative PCR Detection System for Black Scurf Pathogen in Potato (Solanum tuberosum) |
| LI Lei CHEN Li-Da Huang Yi-Shuo XIE Xue-Wen SHI Yan-Xia CHAI A-Li LI Bao-Ju* |
| Insititute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 100081, China |
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Abstract Rhizoctonia solani, the pathogen of potato black scurf, has been a threatening factor to potato (Solanum tuberosum) production. R. solani in the soil mainly uses the sclerotium structure as the initial source of infection for latent dormancy, and its density is positively correlated with the incidence of potato black scurf disease. In order to quickly and accurately detect the density of R. solani and sclerotia in soil, TEF3/TEF7 specific primers were designed based on the conservative region of translation elongation factor (TEF) of R. solani anastomosis group 3 (AG3). A real-time quantitative PCR (qPCR) detection system for quantification of R. solani AG3 was developed using recombinant plasmid of 191-bp amplification product as the standard, and the sensitivity of qPCR (19.5 fg/µL) was 1 000 fold higher than that of common PCR. Common PCR and qPCR tests were carried out on suspected potato moles (n=18) and diseased soil (n=41) in 4 regions. The detection rate of potato moles was 100%, but common PCR (65.85%) failed to detect all DNA in the diseased soil, while the detection rate of the diseased soil by qPCR was 97.56%. In order to determine the pathogen quantity in the early detection of diseased soil in the field, the soil with different amount of sclerotium was artificially simulated, and qPCR detection was carried out by wet-sieving method. The curve relationship between cycle threshold (Ct) and sclerotium content in soil was established. The results showed that regression equation was y=-3.982x+12.687, and correlation coefficient R2 was 0.986, showing good linear relationship, and the detection limit of artificially simulated pathogen quantity was 5×10-6 g/g soil. The detection system developed in present study could detect sclerotium density of AG3 in soil rapidly and accurately. Detection of sclerotium content of R. solani in soil before planting could provide technical support for early warning of potato black scurf.
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Received: 09 November 2020
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Corresponding Authors:
*libaoju@caas.cn
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