Molecular Detection Of Pathogens On Cucumber And Strawberry

Cucumber bacterial wilt, cucumber anthracnose and cucumber sclerotinia rot are serious diseases on cucumber, which can badly reduce the cucumber field yield. Verticillium wilt, gray mold and anthracnose are major diseases on strawberry. The symptoms of these diseases are very similar, so it is very hard to distinguish by analyzing early symptom. To establish a rapid and accurate method of diagnosing these diseases has become a pressing problem.Optimized, by quartz sand grinding, TENP and PBS washing, SDS high-salt lyses and isopropanol precipitation the DNA extraction method which is suitable for agricultrual soil could significantly increase the sensibility of detection. Based on the amplification of Erwinia tracheiphila, the 16S rDNA phylogenetic tree was constructed, and the primer ET-P1/ET-P2 was designed which could amplify the specific band of 794bp in length, and sensitivity to 100fg/μL. The triplex PCR system for detection of E. tracheiphila, Colletotrichum orbiculare and Sclerotinia sclerotiorum was successfully established by orthogonal design method. The reaction system volume was 25μL and the annealing temperature was 63℃. The reaction solution contained 0.336μmol/L ET-P1/ET-P2, 0.24μmol/L CY1/CY2,0.72μmol/L SSFWD/SSREV,1U Taq DNA polymerase,0.15mmol/L dNTP and 1mmol/L MgCl2. This triplex PCR system could detect E. tracheiphila, C. orbicular and S. sclerotiorum in infected plant tissues and soil rapidly, and the sensitivity of this method can reach to 10pg/μL.Sequence analysis on the ITS of Verticillium dahliae which were isolated from strawberry, cotton and eggplant, showed that the sequence homology was accounted for 99.6%. The three host source strains may be the same one, as they couldn't be distinguished from each other by RAPD of 69 random primers. The triplex PCR system for detection of these three pathogens on strawberry was successfully established. The reaction system volume was 25μL and the annealing temperature was 50℃. The reaction solution contained 0.32μmol/L C729+/-,0.032μmol/L DB19/DB22,0.32μmol/L CgInt/ITS4,1.5 U Taq DNA polymerase,0.15 mmol/L dNTP and 1.6 mmol/L Mg2+. The triplex PCR system could detect B. cinerea, C. gloeosporioides and V. dahliae in infected plant tissues and soil rapidly and the sensitivity of this method can reach to 10pg/μL.Both of the triplex PCR systems can be used for accurate and rapid identification of diseases on cucumber and strawberry simultaneously in the early stage.