基于16S-rDNA序列的核桃细菌性疫病病原菌遗传多态性分析

Investigation of walnut bacterial blight pathogens based on 16S-rDNA sequences and RFLP

核桃细菌性疫病(Xanthomonas arboricola pv.juglandis)是世界范围内核桃产区最主要的细菌病害,对其病原菌16S-rDNA的序列进行分析,为阐明其遗传多样性提供参考。本研究从湖北省保康县和丹江口市多个核桃园采集病样,对不同核桃病组织样进行分离纯化,得到77个病原菌类似菌株,对部分菌株进行了接种和分子鉴定。用通用引物27F和1492R对其中有代表性的22株进行16S-rDNA的PCR扩增和测序。用软件MEGA7.0对所有测序结果进行遗传发育树分析,菌株之间平均遗传距离为0.244。用3种限制性内切酶MspI、AfeI和HinfI对16S-rDNA的PCR产物进行酶切,酶切结果用软件NTSYSpc2.1进行聚类分析。所有菌株之间相似系数大于52%,在76%的相似水平,可以将22个菌株划分为6个类群。用软件ApE对所有序列进行同样的3个酶切,RFLP分析结果与试验酶切结果相似。不同方法构建的聚类和发育树分析结果表明,湖北省不同地理来源和病组织的菌株存在一定的遗传多态性。

Walnut bacterial blight caused by Xanthomonas arboricola pv.juglandis is the most important disease in walnut plant area all around the world.To elucidate the genetic diversity of the pathogen,the 16S-rDNA sequences and its RFLP were analyzed in this paper.Total 77 single colony strains were isolated from diseased walnut tissues which were collected from walnut orchard in Baokang and Danjiangkou county,Hubei province,and portion of them accomplished pathogenicity test and molecular identification.The 16S-rDNA sequences of twenty-two strains were amplified by PCR using universal primers 27 Fand 1492R.The molecular phylogenetic tree was constructed by MEGA 7.0based on 16S-rDNA sequences,and the aver-age genetic distance was 0.244 among 22strains.The PCR products were digested by multiple restriction enzymes MspI,AfeI and HinfI,and then the digested profiles were clustered by NTSYSpc2.1software.The results showed that 22 strains could be clustered into 6groups with a similarity coefficient of 76%,and the coefficient value was above 52% among all tested strains.Furthermore,using the software ApE to digest the sequences with the same 3restriction enzymes showed the similar clustering results.In conclusion,the walnut bacterial blight pathogens isolated from different areas and tissues in Hubei province showed genetic diversity with different analyzing methods.