建兰胶孢炭疽菌ITS序列分析及其PCR快速检测

Sequence analysis of ITS and PCR rapid-detection of Colletotrichum gloeosporioides from Cymbidium ensifolium

由胶孢炭疽菌Colletotrichum gloeosporioides引起的炭疽病是建兰的重要病害。为建立快速检测该病原菌的方法,以ITS1/ITS4为引物,对15个建兰胶孢炭疽菌的ITS进行PCR扩增及测序,将测定的序列与炭疽菌属其它种的ITS序列进行比对分析,设计特异性引物CF1/CR1,并通过常规和巢式PCR对建兰胶孢炭疽菌进行检测。结果显示,15个菌株中有13个菌株ITS序列与该菌的模式种序列相似性高达99%以上,而另外2个菌株相似性则为86%;供试菌株在系统发育树上聚为2个不同的分支;引物CF1/CR1通过常规PCR可从1 ng的建兰胶孢炭疽菌基因组DNA中扩增到目的条带,而利用引物ITS1/ITS4和CF1/CR1通过巢式PCR可从1 pg的基因组DNA中扩增到目的条带,即巢式PCR反应检测灵敏度较常规PCR至少高1000倍。研究表明建立的巢式PCR法可从自然感病的建兰叶片组织中检测到胶孢炭疽菌。

Anthracnose caused by Colletotrichum gloeosporioides is a destructive disease of Cymbidium ensifolium. To develop rapid techniques for the detection of C. gloeosporioides, the universal primers of ITS1/ITS4 were used to amplify the internal transcribed spacer （ITS） sequences of 15 C. gloeosporioides isolates, and based on the sequence analysis of these isolates and other C.spp., a pair of species-specific primers CF1/CR1 were designed, and then, the detection methods of conventional PCR and nest-PCR for C. gloeosporioides were developed. The result demonstrated that 13 out of the 15 tested C. gloeosporioides isolates showed 99% similarities, and the other two isolates showed 86% similarities, compared with the wild-type species. Meanwhile, phylogenetic analysis displayed that the tested isolates were classified into two different groups. Furthermore, conventional PCR using the species-specific primers CF1/CR1 could detect the specific fragment from 1 ng of genomic DNA, but the sensitivity increased 1 000-fold to 1 pg by developing nested PCR procedure that used ITS1/ITS4 as the first-round primers combined with CF1/CR1 as the second-round primers. The results suggested that the nested PCR technique developed here could be used to detect C. gloeosporioides from C. ensifolium tissues infected naturally.