寄生隐丛赤壳菌 ITS 分子检测技术研究

摘要

Cryphonectria parasitica ,an important pathogenic fungus,causes chestnut blight in Castanea mollis-sima .The objective of this study was to set up a PCR-based technique for detection of C .parasitica .The internal spacer(ITS)sequences of C .parasitica and other strains isolated from Ya’an,Luzhou and Chongqing region were obtained by PCR amplification using fungal universal primers ITS1 and ITS4.Based on different ITS sequences of Cryphonectria genus,a pair of species-specific primers ITSP1 and ITSP2 were designed.PCR results showed that primers ITSP1/ITSP2 amplified a single product of 462 bp from DNA prepared from C .parasitica ,but not from other strains.The detection sensitivity was 30 pg of genomic DNA.Nested PCR using primers ITS1/ITS4 and ITSP1/ITSP2 could detect 30 fg of genomic DNA and the detection sensitivity was 1 000 fold higher than that of regular PCR.This nested PCR could stably and quickly detect C .parasitica from natural diseased and infected plant tissues.%寄生隐丛赤壳菌是引起板栗疫病的致病菌。为建立该菌的分子检测技术，本研究首先采用通用引物 ITS1／ITS4对分离自四川雅安、泸州及重庆的寄生隐丛赤壳菌及其他参试菌株的 ITS 区进行 PCR 扩增和测序比对。根据该片段与 GenBank 中隐丛赤壳属其他种的 ITS 序列差异，设计了寄生隐丛赤壳菌的特异性引物 ITSP1／ITSP2，片段扩增大小为462 bp。利用该引物对菌株基因组 DNA 进行扩增，可以将寄生隐丛赤壳菌与其他参试菌区分开，检测灵敏度达30 pg。而以引物 ITS1／ITS4和 ITSP1／ITSP2进行的巢氏 PCR，可检测到30 fg 基因组 DNA，其灵敏度较常规 PCR 提高了1000倍。利用巢氏 PCR 检测体系对发病程度不同的组织和携菌组织进行检测，均能快速稳定地检测出寄生隐丛赤壳菌。