Detection Of Fusarium Oxysporum F.sp.cucumarinum By PCR-RFLP And Nested-PCR

The 59 pure cultural isolates, including 3 Fusarium oxysporum f.sp.cucumarinum, 26 other Fusarium isolates and 20 fungi, 6 bacteria, 7 actinomycetes from cucumber rhizosphere, have been detected by PCR-RFLP and Nested-PCR. It shows that PCR-RFLP is special for the pathogen of Cucurbits Fusarium Wilt, and Nested-PCR is special for Fusarium oxysporum f.sp.cucumarinum. Nested-PCR is more special than PCR-RFLP in detection of Fusarium oxysporum f.sp.cucumarinum.The primers and the detection programs have been used to detect the pathogens in the inoculated-diseased cucumber plants .The details are reported as follows:1. By using the primers ITS1/ITS4, only Fusarium isolates could be amplified to produce a special fragment of 750bp. This product generated 480bp, 200bp and 100bp fragments after digested by restriction enzyme Hinfâ… only from the pathogen of Cucurbits Fusarium Wilt. By using the primers BIK1/BIK4 and BIK2/BIK3 of Nested-PCR, only Fusarium oxysporum f.sp.cucumarinum could be amplified to form a special fragment of 800bp, other isolates have no this fragment. That indicates that Nested-PCR is more special than PCR-RFLP in detection of Fusarium oxysporum f.sp.cucumarinum.2. PCR-RFLP and Nested-PCR detected successfully the pathogens in the diseased cucumber plants inoculated with Fusarium oxysporum f.sp.cucumarinum, the characteristic 750bp fragment was obtained by PCR-RFLP and also the characteristic 480bp, 200bp and 100bp fragments after digested by restriction enzyme Hinfâ… . The special 800bp fragment which represents Fusarium oxysporum f.sp.cucumarinum generated by using Nested-PCR. It indicates that there exists Fusarium oxysporum f.sp.cucumarnum in the detected inoculated tissues of cucumber, and this PCR-RFLP and Nested-PCR program could exactly and sensitively detect Fusarium oxysporum f.sp.cucumarinum in the inoculated tissues of cucumber.3. PCR-RFLP could detect Fusarium oxysporum f.sp.cucumarinum in the inoculated tissues of susceptible cucumber plants on the 3th day. Nested-PCR could detect the pathogens on the 5th day. It indicates that PCR-RFLP is more sensitive than Nested-PCR in detection of Fusarium oxysporum f.sp.cucumarinum.4. Nested-PCR could detect Fusarium oxysporum f.sp.cucumarinum in the inoculated tissues of susceptible cucumber plants on the 5th day, however resistant cucumber plants were on the 7th day. It indicates that pathogens invade more earlierly in susceptible plants than in resistant plants.