云南烟草靶斑病(Rhizoctonia solani Kühn)病原鉴定及其融合群研究

摘要

2016年云南省普洱市和临沧市烟草种植区大面积发生叶部病害,经鉴定为烟草靶斑病(Rhizoctonia solani Kühn),此病为云南烟区首次发现.广泛采集2个市的烟草靶斑病病叶标本59份,采用常规组织分离法获得58个菌株,将所获菌株分别与立枯丝核菌标准融合群菌株AG-1-IA、AG-2-1、AG-3、AG-4-HGⅡ、AG-5、AG-6GV、AG-8和AG-9进行载玻片对峙培养,并进行菌丝融合观察,结果表明:58个菌株均属于立枯丝核菌AG-3标准融合群,且不与其他标准融合群发生融合反应.随机选取云南省6个地区各1个代表性菌株,以待测菌株的基因组DNA为模板,利用真菌核糖体基因转录间隔区(ITS) 通用引物ITS1和ITS4对病菌rDNA ITS序列进行PCR 扩增,扩增产物序列在GenBank中进行BLAST同源性检索比对.应用MEGA 6.06软件和Neighbor-Joining法,分别计算遗传距离及构建系统发育进化树分析亲缘关系.基于5.8S rDNA-ITS区序列的系统发育树进一步分析,结果表明,Rhizoctonia solani菌株的ITS序列可明显的分为2个支系,同一菌株的不同ITS序列可分别存在不同的分支中,但所有菌株的序列均隶属相同的融合群即AG-3,且隶属相同融合群的不同菌株之间其序列的一致性可高达99%~100%.%A kind of tobacco leaf disease occurred in a large area in Puer and Lincang of Yunnan Province in 2016, which was identified as tobacco target spot disease caused by Rhizoctonia solani Kühn, the first discovery in Yunnan tobacco planting areas. A total of 58 isolates of R. solani were obtained from 59 samples of tobacco leaf spot disease samples from Yunnan Province. Furthermore, they were respectively cultured on slide with R. solani standard strains, AG-1-IA, AG-2-1, AG-3, AG-4-HGⅡ, AG-5, AG-6GV, AG-8 and AG-9. Observation for the mycelium fusion showed that all the 58 isolates belonged to the anastomosis group 3 (AG-3). Six representative strains from 58 isolates were randomly selected, and the rDNA ITS sequences of the R.solani were amplified by PCR using the fungal ribosome gene transcribed spacer (ITS) universal primers ITS1 and ITS4, and compared by nucleotide BLAST with other nucleotides in GenBank.The MEGA 6.06 software and the Neighbor-Joining method were applied separately to calculate the genetic distance and construct phylogenetic tree for analyzing the genetic relationship. Phylogenetic tree analyzing based on the 5.8S rDNA-ITS region sequence showed that the ITS sequences of Rhizoctonia solani strains could be clearly divided into two branch lines, and different ITS sequences of the same strain could exist in different branches, and the sequence identity of different strains belonged to the same fusion group with similarity as 99%-100%,which showed that all the strains belonged to the same fusion group of AG-3. This is the first report of tobacco target spot disease caused by Rhizoctonia solani (AG-3) in Yunnan Province, China.