番石榴焦腐病菌的ITS分析及PCR检测

Analysis of ITS sequence and PCR detection of Botryosphaeria rhodina

番石榴焦腐病是台湾入境大陆水果的重要植物病害,由葡萄座腔菌Botryosphaeria rhodina引起。为建立该病原菌快速、灵敏的检测技术,比较分析了葡萄座腔菌和葡萄座腔菌属其它种的ITS序列,在此基础上设计了1对检测番石榴焦腐病菌的特异性引物BF1/BR1,利用此引物从葡萄座腔菌中特异性扩增出287 bp条带,而其余参试的菌株未能获得扩增条带。将真菌通用引物ITS1/ITS4和BF1/BR1进行巢式PCR扩增后,检测灵敏度提高1 000倍,可检测到葡萄座腔菌1 pg的基因组DNA。结合快速碱裂解法提取发病组织的DNA,采用该PCR检测技术可从自然感染焦腐病果实中检测到葡萄座腔菌。

Black-rotten disease in Psidium guajava,caused by Botryosphaeria rhodina,is one of the most destructive plant diseases.A rapid and accurate method for the specific detection of B.rhodina is essential to prevent widespread devastation in mainland of China.Based on differences in internal transcribed spacer（ITS） sequences of B.rhodina and other Botryosphaeria spp.,a pair of species-specific primers BF1/BR1 was designed.The primer pairs amplified a single 287bp product from all isolates of B.rhodina that was not amplified from any other isolates tested.The sensitivity increased by 1000-fold to 1 pg by developing a nested PCR procedure that used ITS1/ITS4 as the first-round primers combined with BF1/BR1 specificity was confirmed by using the PCR assay to detect B.rhodina in plant tissues infected by the pathogen.The PCR-based detection methods developed here could simplify both plant disease diagnosis and pathogen detection,as well as guide plant disease management.In addition,the classification implication of ITS sequence homology was found in fungi by comparing sequences from Botryosphaeria spp.