A Method For Molecluar Detection Of Alternaria Solani And Its Applicability To Early Diagnosis Potato Early Blight In Field

Potato early blight is caused by Alternaria solani. However, traditional morphologymethod is hard to carry out for its low sporation ability. Meanwhile, it is hard to observesymptoms in fields at early infection stage. So a rapid and reliable detection method of A.solani is necessary for determination whether the pathogen has infected potato leaves. Inthis study, a PCR-based and primer-specific method was developed and applied for bothdetection of A. solani and diagnosis of early blight at latent infection stage. It is importantto rapid and early detect early blight for disease control.The main results are summarized as follows:1. An accurate method for detection A. solani was developed. A pair of specificprimers (i. e. ptAs-F:5’-CACCTCCCGGGGTGGCCA-3’ and ptAs-R:5’-CCCGCAAGGGGAGACAAA-3’) were designed, based on sequence divergence within the internaltranscribed spacer (ITS) region of nuclear ribosomal DNA. We optimizated PCR cyclingconditions, which the annealing temperature was50°C for30s, extension at72°C for23s.An expected band of358bp was observed by PCR amplification in samples of purified A.solani isolates or infected potato leaves. It is easy to distinguish A. solani from other potatofungal pathogens, bacterial pathogens and other relative Alternaria species. It wasconfirmed that this PCR-based and primer-specific method was available and accurate todetect the pathogen of A. solani. The sensitivity of the specific primers was evaluated by10-folded serial dilution of genomic DNA and spores of A. solani. The result illustratedthat the PCR detection by ptAs-F and ptAs-R primer pairs was sensitive to as low as1.0ng/μl genomic DNA and105CFU/mL spores of A. solani. Meanwhile, this method alsocould be detected the diffusion level of A. solani. The DNA bands intensity were more andmore brighter with the lesion bigger.2. A new method for early detection of potato early blight was developed. We usedmodified glass beads oscillation method to extract the pathogen DNA from potato leaves,and then PCR detection by ptAs-F and ptAs-R primer pairs. It only needs2.5hours toobtain the results of the potato leaves which were infected by A. solani. A. solani can bespecifically detected from infected leaves by using the species-specific primers, even the symptomless leaves from diseased plant. The PCR-based method provided a valuable toolfor early and rapid detection of A. solani from early blight in potato.3. Using the PCR-based method, we detected the infection level of potato leaves atearly stage and symptomless leaves from diseased plant in three fields of Zhangzhai,Jiangtun, Tengzhou City at April16th,2013. The results showed that early infection ratesof bottom leaves in three fields were90.3%,61.7%and48.3%, respectively. The resultslater infection rates of diseased potato leaves in three fields at May3rd,2013were91.4%,62.8%and49.6%, and the results of disease index in three fields were24.8,10.9and7.5,respectively, which is consistant with the early detection results. It was confirmed that thisearly and rapid detection method is available and accurate to detect potato early blight infield.