Establishment Of Seminested-PCR And Real-time Quantitative PCR For The Detection Of Alternaria Solani In Soil

Potato early blight caused by Alternaria solani is an important foliar disease. Arelationship was observed between the number of pathogens in soil and the prevalence ofdisease. This is one of the most importantest prerequisite for predicting the occurrence ofearly blight. In this study, we established the seminested-PCR and real-time quantitativePCR assays for the detection of Alternaria solani in soil. The main results aresummarized as follows:1. A low-cost, rapid and highly sensitive method for detection A. solani of soil wasdeveloped. A pair of specific primers ptAsQ-F/ptAs-R was designed to distinguish A.solani from other potato pathogens and other relative Alternaria species, which couldobtain a unique251bp specific fragment. The seminested-PCR was performed using theprimer of ptAs-F/ptAs-R for the first run and the ptAsQ-F/ptAs-R sets for the second runby using the first PCR product. This method can detect as low as10fg genomic DNA,which is equivalent to one percent content of conidium genomic DNA. The sample of0.5g soil containing one spore also could be accurately tested. Three naturally infested soilsamples from Hebei were detected, the detection rate of A.solani was60%,60%and80%,respectively.2. The pair of primers ptAsQ-F/ptAs-R was selected to establish the real-timequantitative PCR. The positive plasmid included a251bp PCR product was used as thereference and the specification curve was created on that basis. The specificity,sensitivity and reproducibility of the curve were evaluated and compared withconventional PCR techniques. The result showed that the real time PCR assay was rapid,highly sensitive and specific. A linear relationship was observed between the amount ofinput plasmid DNA and cycle threshold (Ct) values over a range of6.4×101copies/μL to1×106copies/μL, and correlation coefficient was0.992. PCR amplification efficiency was94.4%, which was625more sensitive than that of the conventional PCR. The intra-assayvariations and Inter-assay variations was0.11%-1.15%and1.08%-2.51%, respectively.3. Using the real-time quantitative PCR method, we detected A.solani in the infectedpotato leaves and soil. The results showed that the susceptible cultivars contained morepathogen than the resistant cultivars. A linear relationship was observed between theamount of A. solani conidium and cycle threshold (Ct) values. The regression equation was y=-0.6748x+32.189, the correlation coefficient (R2) was0.903. Three naturallyinfested soil samples from Hebei were detected, and the detection rate of A.solani was40%,60%and80%, respectively. The test results were basically consistent with theresults of the semi-nested PCR.