Quantitative Detection And Dynamic Analysis Of Rhizoctonia Cerealis Of Wheat In Soil By Real-time PCR

Wheat sharp eyespot is a soil-borne fungal disease infected by Rhizoctonia cerealis, which is one of the most destructive diseases occurring wherever wheat was grown throughout the world. In China, the disease occurred mainly in winter wheat regions in the Huaihe River and the Yellow River and happened in the provinces of Jiangsu, Zhejiang, Anhui, Shandong, Henan, Shanxi, Hunan, Hubei, Sichuan and so on and was becoming more and more harmful. R. cerealis is a stubborn living soil fungus, through summer and winter in the soil by mycelium or sclerotia in soil or on plant debris. Wheat was infected throughout the wheat growing season. Currently traditional detection methods of R. cerealis are difficult to achieve the quantitative detection in soil. In this study, we succeeded in designing a pair of specific primers for R. cerealis and established a real-time PCR system for rapid detection of R. cerealis in soil. The population of the pathogen was detected every10days from3th March in spring in2012and2013, which could be useful to understand the population dynamic of R. cerealis in the soil. Meanwhile the relationship among the dynamic occurrence of the pathogen in soil, effective accumulative temperature of10d gained from meteorological data of local weather station and disease index of wheat sharp eyespot investigation from the sampling point was analyzed. Follows are the main conclusions:1. Based on the sequence of5.8S and internal transcribed spacers (ITS) of R. cerealis, we designed specific primer pairs WKF-8/WKR-8. There was a fragment of about200bp from the PCR amplification of DNA from wk-206(.R. cerealis) gained from CTAB method. DNA of wk-206, Rhizoctonia solani in cotton(AG4-HG-I, AG4-HG-Ⅲ), potato (AG3, AG4) and corn (AG4-HG-Ⅲ, AG1-IB), Fusarium proliferum, Helminthosporium sorokiniana and other binuclear Rhizoctonia strains were used as templates to detect the specific of the primer pairs further. Conventional PCR test results showed that the specific primers designed could effectively amplify the DNA of R. cerealis and gained about200bp fragment, on the contrary the DNA of other detective fungal could not get the specific fragments. Test results showed that the primer pairs WKF-8/WKR-8on R. cerealis with high specificity.2. Using the designed specific primers, we optimized annealing temperature from58℃ to62℃and chose58.4℃as the optimal annealing temperature. A real-time PCR reaction system was established based on the fluorescent dye S YBR Green I. Concentration of R. cerealis DNA (wk-206) was2.02×102ng/μl and serially diluted to obtain10-fold serial dilutions of the wk-206. The sensitivity of the primer pairs was detected with DNA of R. cerealis after10-fold serial dilutions, using conventional PCR and real-time PCR. The results showed that the detection limit of conventional PCR was1.0×10-3ng/μl and that of real-time PCR was1.0×10-5ng/μl. As a result, the sensitivity of real-time PCR is100times higher than conventional PCR.3. A standard curve was established by serial dilutions DNA of wk-206(10-fold dilution series froml.O×1O-1ng to1.0×103ng in reaction volumes of25μl) from cultured mycelia. The amplification results showed that the simple linear regression line was normative when the template concentration was from1.0×10-1ng/μl to1.0×103ng/μl (the qPCR reaction efficiency was99.7%, standard deviation of each DNA concentration was0.995) and the linear range was up to5magnitudes. The melting temperature of the melting curve for the desired products was (83±0.5)℃, a single product without amplification primer dimmers, which proved the method could be used to detect the DNA in the soil.4. The result showed that the dynamic of the growth of R. cerealis in soil was similar during the growth period of wheat of the two years. There are two obvious growing peaks of R. cerealis during the wheat growth period in late April (elongation stage) and late May (dough stage) according to the graph of population of mycelia. Aging organizations also affected the expansion of R. cerealis in the late. Differences in test of the results between the two years was that the performance of the biomass peak of R. cerealis in soil in2012appeared on April12th, however the performance of the biomass peak of R. cerealis in soil in2013appeared on April22th, which had great influence on the difference of effective accumulated temperature of ten days.5. According to comprehensive analysis of the curves of disease index of wheat sharp eyespot and curves of biomass of R. cerealis in soil, the rapid growth period of the disease index of wheat sharp eyespot was the same as the period of the biomass of R. cerealis in the soil occuring elevated-peak-fell-the second peak. The stage is also the transfection period of R. cerealis from soil to the wheat plant and the rapid expansion period of the pathogen. The second peak of biomass of R. cerealis in soils was also an important sign on transfer of R. cerealis from wheat plants to the soil. The biomass changes of R cerealis in soil and the changes of disease index of R. cerealis reflected the tendency of R. cerealis in soil-wheat plant-soil.