| Verticillium dahliae is a worldwide silborne fungal disease of cotton. It can survive in the soil for several years, making the output reduced and the quality of cotton fiber debased. In this study,a positive plasmid was used as a reference included a highly conserved regions of 16s rDNA of Verticillium dahliae. And a SYBR Green I real time quantitative PCR method was established for the detection of Verticillium dahliae of cotton in naturally infested soil. The main results are as follows:1.Various fungal genomic DNA extraction method were comprehensive compared and finally the improved CTAB method was adopted. A strong pathogenicity strain from Shaanxi Jingyang were used to study. The results showed that the concentration of DNA samples were 1500 ng/μL or so. A260/A280 and A260/A230 values also indicate that the higher quality DNA was extracted.2.Collecting five samples from five areas at rhizosphere soil in the field where the Verticillium dahliae are especially rich. Genomic DNA was obtained from the soil using CTAB-SDS-frozen-thawing method and purified with Ultra Clean Soil DNA Purification Kit. According to electrophoresis and PCR results, a highly quality DNA was extracted which can be used for subsequent molecular biological research.3.The 16s rDNA ITS sequences was chosen as research object. The fragment amplified with primers dllz1 and dllz2 was cloned, sequenced, and transferred to pGEM-T easy vector. Sequence alignment indicated that a 342bp PCR product was cloned into the vector. According to the 100% of sequence of homology of ITS of Verticillium dahliae and database in GenBank, finally the recombinant was obtained and could be used as a reference for the SYBR Green I real time quantitative PCR.4. The method of quantitatively detecting the number of Verticillium dahliae in soil was developed by the SYBR Green I real time quantitative PCR. The specificity, sensitivity and reproducibility of the curve were evaluated and compared with conventional PCR techniques. The result showed that the real time RT-PCR assay was rapid, highly sensitive and specific, and had a broad linear detecting range (3.8×10~3copies/μL-3.8×10~8copies/μL, R2=0.996) with 101.5% PCR amplification efficiency and 102 more sensitive than that of the conventional PCR. And the Verticillium dahliae from soil samples were quantified successfully in accordance with that calibration curve. According to the average of Ct value, the amount of pathogen in the soil can be calculated.Therefore, the establishment of SYBR Green I real time quantitative detecting system can provide a technical support and theoretic foundation for quantitatively detecting the number of Verticillium dahliae of cotton in soil and as an aid in disease risk prediction, as well as establishing the integrated disease control measures. Moreover,this method can also provides theoretical and technical reference for the detection of other pathogens in soil. |
