Abstract:
Objective Fusarium wilt and tobacco black shank, caused by Fusarium oxysporum and Phytophthora parasitica var. Nicotianae, are common soil-borne diseases in tobacco planting soil, leading to severe damages. Therefore, a rapid detection method for the two pathogens was established to provide technical support for disease epidemic and treatment in the process of tobacco planting and ensure the stable production of tobacco industry.
Method Taking the soil with soil-borne disease and healthy soil in Baise area of Guangxi were used as test materials, and the standard curve was established with the standard pathogen as reference template. The primers were designed by the conservative regions of the two pathogens, and the specific primers were screened according to the target bands and the dissolution curve and Ct value of fluorescence quantitative PCR. At the same time, the extraction method of DNA kit of microbial in soil was improved, and the detection systems of PCR and qPCR were established.
Result A pair of specific primers PN3 and JBR for F. oxysporum and P. parasitica var. nicotianae were screened respectively, and the template concentration ranged from 1×102 to 1×10-4 ng, with good linear relationship. The number of two pathogens in diseased soil was higher than that in healthy soil. The method of combination of soil drying, grinding and eluting for twice was helpful to extract the DNA of pathogens in the soil. A qPCR detection method with the products obtained by ordinary PCR pre-amplification reaction as a template was established.
Conclusion The combination of PCR and qPCR can quickly distinguish and detect F. oxysporum and P. parasitica var. nicotianae, which plays an important role in real-time monitoring and early warning of fusarium wilt and tobacco black shank.
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