应用PCR-RFLP和巢式PCR检测黄瓜尖镰孢菌

Detection of Fusarium oxysporum f. sp. cucumarinum by PCR-RFLP and Nested-PCR

摘要:

以3株黄瓜尖镰孢菌(Fusarium oxysporum f. sp. cucumarinum)、23株镰孢菌属(Fusarium spp.)真菌和分离自土壤的20株真菌、6株细菌和7株放线菌为材料, 采用化学裂解法提取总DNA, 进行PCR-RFLP和巢式PCR检测, 试验证明PCR-RFLP程序不能完全区分Fusarium属内不同种, 而巢式PCR对黄瓜尖镰孢菌具有特异性。运用优化的PCR-RFLP和巢式PCR检测程序对染病黄瓜组织进行了检测, 结果表明, 两种方法均可在接种发病早期(未显症时)检测出黄瓜枯萎病菌, PCR-RFLP在感病品种接种后3d即可检测到病原菌, 而巢式PCR在接种后5d才能检测到病原菌。

Abstract:

Using three strains of Fusarium oxysporum f.sp.cucumarinum, twenty three other Fusarium isolates, thirty three comparison strains from cucumber rhizosphere as materials, we used PCR-RFLP and Nested-PCR for detecting Fusarium oxysporum f.sp.cucumarinum on base of total DNA chemical cracking method. It showed that PCR-RFLP could not differenciate different species of Fusarium. Nested-PCR is special for Fusarium oxysporum f.sp.cucumarinum. PCR-RFLP and Nested-PCR detected successfully the pathogen in diseased cucumber plants inoculated with Fusarium oxysporum f.sp.cucumarinum. The two programs both can be used to detect Fusarium oxysporum f.sp.cucumarinum in cucumber tissues without eyeable symptoms. PCR-RFLP could detect Fusarium oxysporum f.sp.cucumarinum in the inoculated tissues of susceptible cucumber plants on 3rd day after inoculation, but Nested-PCR could detect the pathogens on the 5th day after inoculation. It indicates that PCR-RFLP is more sensitive than Nested-PCR in detection of Fusarium oxysporum f.sp.cucumarinum.