Study On Rapid Molecular Detection Technique Of Verticillium Dahliae Of Cotton

Cotton verticillium wilt, caused by Verticillium dahliae, is a worldwide soil spread fungus disease which can cause a serious threat to the cotton production. The fungus can survive in soil for many years, and it’s an important source of infection of the following cotton disease. Rapidly and timely detect V.dahliae in soil or the diseased cotton tissues, and the results will have important significance for early detection and later control of cotton verticillium wilt.Soil is the main place of V.dahliae to survive, but there are countless and a wide variety of microbes in the soil, therefore it is very difficult to directly isolate V.dahliae from the soil; On the other hand, inhibiting factors such as humic substances that exist in the soil, thus it is also difficult to directly extract pathogen DNA from soil. In this study, we improved the extraction method of soil fungus DNA, and established a rapid molecular detection system of V.dahliae in the soil using nested PCR technology.The detection system has a high specificity and sensitivity and lays a foundation for rapid detection technology based on microfluidic PCR technology of V.dahliae.The main research results are as follows:1. The specific primers VDS-F/VDS-R of V.dahliae is designed. In the optimization of reaction conditions and amplification, only a single band of PCR product about 520 bp was amplified from V. dahliae genomic DNA. The sensitivity limit of the primers VDS-F / VDS-R was 10 pg of the template DNA in the reaction mixture. Three different strains of VD, V991, and V250 of V. dahliae all can be accurately detected, and the fungus also can be specifically detected in the diseased cotton tissues and soil.2. Comparing five methods for soil DNA extraction, the standardized procedures, using liquid nitrogen grinding based extraction of V.dahliae genome followed by soil DNA extracted kit, was established. Yielding DNA of superior quality can be used for PCR amplification directly.3. Nested PCR detection technology of V. dahliae in the soil was established. The first round of PCR primers were ITS1 / ITS4, and the second round of PCR primers were VDS-F/VDS-R. The nested PCR can accurately detect the fungus in the soil with the soil DNA extracted by the improved soil extraction method and the detection sensitivity reached 10 spores in per gram soil. Nested PCR detection technology provides the technical support of detecting V. dahliae in soil. At the same time, the establishment of this method also lays a foundation for rapid detection technology based on microfluidic PCR technology and provides reference and basis for other pathogenic fungus in the soil.