摘要： 为了快速区分柑橘黑斑病菌、亚洲柑橘叶点霉菌、首都叶点霉菌和中国柑橘叶点霉菌，在ITS1和18S区域设计了针对柑橘黑斑病菌、首都叶点霉菌和中国柑橘叶点霉菌的上游引物，以ITS4作为下游引物，并对所设计引物的特异性和退火温度进行了筛选，对引物的灵敏度进行了验证，并进行了果园疑似病斑的检测。结果表明，在退火温度为60℃时，引物Pc1/ITS4仅能从柑橘黑斑病菌中扩增出593 bp的特异性条带，引物Pct4/ITS4仅能从首都叶点霉菌中扩增出551 bp的条带，引物Pcc1/ITS4仅能从中国柑橘叶点霉菌中扩增出706 bp的条带，不能从柑橘常见病害病原菌中扩增出任何条带。特异性引物的灵敏度检测结果表明，引物Pc1/ITS4和Pct4/ITS4的检测灵敏度为200 pg，引物Pcc1/ITS4的检测灵敏度为20 pg。筛选的特异性引物Pc1/ITS4可以对果园疑似柑橘黑斑病病斑进行检测。因此，利用设计的特异性引物Pc1/ITS4、Pct4/ITS4和Pcc1/ITS4，结合简单的病原菌基因组DNA提取方法，可以在短时间内完成对病原菌的分子检测。

关键词: 柑橘黑斑病, 柑橘黑斑病菌, 中国柑橘叶点霉菌, 首都叶点霉菌, 特异性引物

Abstract: In order to distinguish P.citricarpa, P.citriasiana, P.capitalensis and P.citrichinaensis,upstream primers for P.citricarpa, P.capitalensis and P.citrichinaensis were designed in ITS1 and 18S regions,and these primers were used together with the ITS4 primer.Then,the specificity and annealing temperature of primers were screened,and the sensitivity of the specific primers were verified.Screened primer was used to detect P.citricarpa when DNA was extracted from black/tan spot lesions on fruits or leaves.The results showed that 593 bp specific fragments were only obtained from P.citricarpa by specific primer Pc1/ITS4,551 bp specific fragments were only obtained from P.capitalensis by specific primer Pct4/ITS4,and 706 bp specific fragments were only obtained from P.citrichinaensis by specific primer Pcc1/ITS4,when annealing temperature at 60℃.These specific primers were never amplified any fragment from the common Citrus pathogens.The sensitivity test results showed that the sensitivity of specific primers Pc1/ITS4 and Pct4/ITS4 was 200 pg,and the sensitivity of specific primer Pcc1/ITS4 was 20 pg.The screened specific primer Pc1/ITS4 could detect P.citricarpa from DNA of black spot lesions on fruit.Therefore,using the specific primers,combined with the simple method of DNA extraction,the molecular detection for the pathogen of Citrus black spot could be completed in a short time.

Key words: Citrus black spot, Phyllosticta citricarpa, Phyllosticta citrichinaensis, Phyllosticta capitalensis, Specific primers

中图分类号: 

• S436.66