2009 - 04在河南省中牟县番茄保护地内发现疑似番茄溃疡病病株,对来自发病植株及其根围土壤的20种细菌性分离物进行了生理生化特征、致病性测定及形态学特征分析,初步确定其中出现最频繁的一种分离物J16为番茄溃疡病菌,将其人工接种时能够诱发出溃疡病症状.利用番茄溃疡病菌的16S-23S rDNA ITS序列特异性引物( ITS1/ITS2)和毒性基因特异性引物(CMM5/CMM6)进行的PCR联合分子鉴定表明,分离物J16的基因组中能扩增出与预期一致的420 bp与614 bp的目标片段.对扩增出的ITS序列进行克隆、测序、并进行ITS序列的系统学分析,结果表明J16菌株属于CMM亚种,而与同种的其它4个亚种亲缘关系较远,但与已知CMM菌株又有所区别,至此,确定该保护地番茄病害为番茄溃疡病,在河南省是首次采用分子方法鉴定该病害.研究还表明,基于ITS和毒性基因的PCR方法能够快速、准确、特异地鉴定番茄溃疡病菌,检测灵敏度可以达到1×103 cfu·mL-1菌体.%In April, 2009, a bacterial canker-like symptom on tomato plants was found in Zhongmou county, Henan Province. After morphology analysis, physiological & biochemical identification and pathogenecity determination, only isolate J16 of 20 bacterial isolates from diseased tomato tissues and the rhizosphere soil was pathogenic, causing the same canker-like symptom and the tomato bacterial canker disease when inoculated artificially. Using sequence-specific primers targeting to 16S-23S rDNA ITS and virulence-related genes of C. M. Subsp. Michiganensis, the PCR joint identification revealed that a 420 bp ITS product and a 614 bp product could be amplified only from genome DNA of J16 and CMM reference strains. Cloning, sequencing and phylogenetic analysis of the ITS amplified product showed that the bacterial isolate J16 belongs to CMM subspecies. All the results revealed that this disease found in greenhouse tomato is confirmed to be tomato bacterial canker, and this was the first report and identification by molecular method in Henan Province, suggesting that the PCR retrial based on ITS and virulence gene was rapid, believable and specific to identify tomato bacterial canker disease, the detection limitation can reach 1 ×103 cfu · mL-1 strains.
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