猕猴桃溃疡病菌的分子检测技术研究-植物病理学报.pdfVIP

植物病理学报 ACTA PHYTOPATHOLOGICA SINICA　 43(5):458-466(2013) 猕猴桃溃疡病菌的分子检测技术研究 1,2 1 1* 1 邵宝林 , 刘 瑶 , 朱天辉 , 李姝江 1 2 ( 四川农业大学林学院, 雅安625014; 四川出入境检验检疫局, 成都610000) 摘要:猕猴桃溃疡病是猕猴桃生产上的主要病害,为建立该病的快速诊断技术,本实验通过RAPD分析获得一条1300 bp 左右的致病菌的特异片段,对该片段进行克隆测序,在测序的基础上设计并合成一对特异引物F7/ R7,优化特异引物扩增 条件,并验证引物的特异性和灵敏性。 利用该特异引物对包括猕猴桃溃疡病菌在内的14个菌株基因组DNA进行PCR扩 增表明,只有猕猴桃溃疡病菌能扩增出1条约为950 bp 的特异条带,其他菌株及对照均未扩增出特异条带。 对采自果园的 染病枝干组织和接种致病菌的枝干组织的检测表明,该特异引物能特异性地检测到猕猴桃溃疡病菌的存在,其在组织中的 检测灵敏度为100 fg/ μL。 因此,利用设计合成的特异引物F7/ R7,参考优化的体系和程序,结合简单的试剂盒法提取猕猴 桃溃疡病菌或植物组织DNA,可以在短时间内完成对该病原菌的分子检测。 关键词:猕猴桃;溃疡病;RAPD;特异引物;丁香假单胞杆菌猕猴桃致病变种 Moleculardetectionof Pseudomonas syringae pv. actinidiae on bacterialcanker of kiwifruit 1,2 1 1 1 1 　 SHAO Bao-lin ,LIU Yao ,ZHU Tian-hui ,LI Shu-jiang 　 ( College of Forestry,Sichuan Agri- 2 cultural University,Ya’an625014,Sichuan,China; Sichuan Entry-Exit Inspection and Quarantine Bureau,Chengdu610000, China) Abstract: Bacterialcanker ofkiwifruitisthemaindiseaseinkiwifruitproduction. In ordertoestablishtherap- id diagnostic technology,a 1300 bp specific fragment wasobtained and sequenced frompathogenicbacteria by RAPD analysis. Based on the sequences of the specific fragments,a pair of specific primers(F7/ R7)was de- signed and synthesized. Then,the amplification conditionswere optimized and the specificity and sensitivity of the specificprimerswereverified. AfterthatthegenomicDNAof14bacterial strainswereamplifiedbythe spe- cific primer set. The results showed that a950bp band wasonly amplifiedfromthe strainsofPseudomonassy- ringaepv. actinidiae. In addition,detectionsfor diseased kiwifruit trunk tissuescollected from orchard and in- oculated kiwifruit trunk indicated that the prime