湖南猕猴桃溃疡病菌16S rDNA和16S-23S rDNA间隔区序列的克隆与分析  

Sequence analysis of bacterial canker of kiwifruit based on the 16S rDNA and 16S-23S rDNA intertranscribed spacer region

作　　者：尹春峰[1] 林文力[2] 肖伏莲[2] 卜范文[2] 段科平 王运生[1] 

机构地区：[1]湖南农业大学植物保护学院,湖南长沙410128 [2]湖南省园艺研究所农业部华中地区果树科学观测实验站 [3]吉首市农业植物保护检疫局

出　　处：《植物检疫》2015年第3期57-61,共5页Plant Quarantine

基　　金："十二五"科技支撑课题(2012BAK11B02)

摘　　要：为明确湖南省猕猴桃溃疡病其致病菌的种类与特征,以猕猴桃主栽品种红阳、米良1号的溃疡病感病枝条为材料,采用BPA培养基、平板划线和梯度稀释法分离病原菌。利用引物27F/1492R和Psa F1/Psa R2对湖南省猕猴桃溃疡病菌16S r DNA和16S-23S r DNA间隔区序列(ITS)进行PCR扩增并进行核苷酸序列测定及相似性分析。获得Psa-JSHY株系、Psa-LXHY株系以及Psa-LXML株系的16S r DNA基因片段1 383 bp及16S-23S r DNA间隔区序列280 bp,且序列一致。经序列相似性比较表明:所分离株系的16S r DNA与法国181、新西兰ABAC9、意大利IKB4等株系的16S r DNA基因序列一致,与日本的KW11等株系存在3个碱基的差异,相似性为99.78%,与国内分离的11-830-1株系存在4个碱基的差异,相似性为99.71%;16S-23S r DNA-ITS序列与中国SXHY11-1、西班牙EFA131.1、葡萄牙PA838、韩国KBE9等株系的ITS序列一致。构建16S r DNA及16S-23S r DNA-ITS序列的进化树,可以看出所分离的株系与已报道的P.syringae pv.actinidiae各菌株聚在同一个进化枝上。以上结果表明,湖南地区分离的3个猕猴桃溃疡病菌致病株系均属于P.syringae pv.actinidiae。To identify the type and characteristics of bacteria canker of kiwifruit, the infected kiwifruit branches and barks of Hongyang and Miliang One from Hunan Province were collected as the experiment materials. Plate streaking, gradient dilution and beef extract peptone agar （BPA） media cultivation were used for bacteria isolation. By the use of PCR, the 16S rDNA and 16S-23S rDNA intertranscribed spacer region （ITS） of bacterial canker associated with kiwifruit in Hunan was amplified with primer pairs 27F/1492R and PsaF1/PsaR2. we obtained 16S rDNA gene and 16S-23S rDNA-ITS in length of 1 383 bp and 280 bp respectively from the diseased sample named Psa-JSHY,Psa-LXHY and Psa-LXML. The 3 strains of 16S rDNA and 16S-23S rDNA-ITS sequences were identical. The blast sequential analysis showed that the 16S rDNA of isolations were identical to that of the foreign reports in France 181, New Zealand ABAC9, Italy 1KB4 strains. Three bases were different from the Japanese KWll strains, and the similarity was 99.78%. The difference between our isolations and the strain 11-830-1 which is isolated in China is 4 base pairs and the similarity was 99.71%.The result of the Blast searching of 16S-23S rDNA-ITS se- quence indicated that it is identical with the strains reported SXHYll-1 in China, EFA131.1 in Spain, PA838 in Portugal and KBE9 in South Korea. Based on the phylogenetic tree of 16S rDNA and ITS, the isolations were in the same clade and belong to P. syringae pv. actinidiae. It is concluded that the isolates from the canker infected kiwifruit branches and barks in Hunan Province were P. syringae pv. actinidiae.

关 键 词：猕猴桃溃疡病 16S RDNA ITS间隔区 序列分析 

分 类 号：S436.634[农业科学—农业昆虫与害虫防治]