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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。
In order to clarify the species and characteristics of pathogenic bacteria in the kiwifruit disease in Hunan Province, BPA culture medium was used as the susceptible shoots of the kiwifruit cultivars Hongyang and MiLiang No.1, and the pathogenic bacteria . The 16S rDNA and 16S-23S r DNA spacer sequences (ITS) of Actinidia ulmoides were amplified by PCR and sequenced. The nucleotide sequence and similarity of 27S rDNA sequences were analyzed by primers 27F / 1492R and Psa F1 / Psa R2. The sequences of 1 383 bp and 16S-23S r DNA spacer sequences of Psa-JSHY, Psa-LXHY and Psa-LXML were 280 bp and the sequences were consistent. The sequence similarity analysis showed that the 16S r DNA of the isolates was consistent with the 16S r DNA sequence of France 181, ABAC9 of New Zealand and IKB4 of Italy, The similarity of the 16S-23S rDNA-ITS sequence to that of the Chinese SXHY11-1, the Spanish EFA131.1, and the Chinese isolate 11-830-1 was 99.78% Portugal PA838, Korea KBE9 other strains of the same ITS sequence. The phylogenetic tree of 16S r DNA and 16S-23S r DNA-ITS sequences was constructed. It can be seen that the isolated strains were clustered with the same strain of P. syringae pv. Actinidiae. The above results show that the three kiwifruit canker pathogenic strains isolated in Hunan belong to P. syringae pv.actinidiae.