Quantitative Assay Rhizoctonia Solani AG1-IA In Soil Via Real-time PCR

Isolates 1,2,4,6 ,four plant pathogens isolated from different rice plants infected by rice sheath blight., were classified into Rhizoctonia solani AG1- I A, respectively, by the combination of analyzing the ITS+5.8S region sequences and observation of hyphal fusion, culture characteristics and microscopic characteristics. Real-time PCR can differentiate one from others by identifying the difference of sequences even if as low as1 bp and can make it possible that pathogens could be quantified correctly without being affected by soil condition. This research is to quantify rice sheath blight pathogen in soil by Real-time PCR.Firstly, the specific primers (RsFx/ RsRn) and Taqman-MGB probe were designed (RsP 1 a1)- Then the reagent configuration was optimized and the specificity and conservation of the probe were confirmed. The result shows that both specificity and conservation are very good.Secondly, we use the primers and probe to quantify Rice sheath blight pathogen added to natural soil in ladder to test the feasibility of this quantitative method. It is found that 4 replicate samples' variation of the same treatment can be detected. These variations are likely due to variation in the amount of R. solani contained in the replicate soil samples and variation in the amount of soil DNA extraction. But we have obtained a curve with R2=0.93, which is generated by logarithm of 4 replicate samples' average and logarithm of added hyphal concentration of each treatment. So we conclude that this method has some feasibility in soilborn plant pathogens quantification.Finally, we quantify rice sheath blight pathogen in natural soil and plot the graph of rice sheath blight pathogen population dynamics in the period of one year. It shows that the population dynamics is closely associated with soil temperature, soil moisture, soilO2 ventilate and farming behavior.