青海地区萝卜黑心病病原

Pathogen causing root internal discoloration of radish(Raphanus sativus)in Qinghai,Northwest China

2018-2021年自青海西宁及其周边地区采集萝卜黑心病样品66份,采用组织分离法对样品进行分离并纯化,获得80株轮枝菌属菌株,编号为VL1-VL80。供试菌株在PDA上生长十分缓慢,培养后产生微菌核和轮枝状分生孢子梗。微菌核不规则形,53.91-164.42×8.91-29.81μm,平均长宽比大于2。分生孢子透明且较长,5.35-9.31×2.12-4.78μm,不产生厚垣孢子和休眠菌丝。ITS和ACT序列测定分析表明,供试菌株均属于长孢轮枝菌A1/D1株系。测定4株代表性菌株的ITS、ACT、EF、GPD、OX和TS的片段序列,分别构建ITS片段和多基因系统发育树,ITS发育树表明供试菌株与长孢轮枝菌位于同一个分支,多基因系统发育分析表明供试菌株与长孢轮枝菌A1和D1株系位于同一个分支。致病性测定结果表明,可侵染萝卜引起黑心病症状。以上研究结果表明,青海地区萝卜黑心病菌病原为长孢轮枝菌A1/D1株系,这是我国首次报道长孢轮枝菌A1/D1株系。

From 2018 to 2021,66 samples of radish(Raphanus sativus)with visible symptoms of root internal discoloration were collected from Xining,Qinghai Province and its surrounding areas.Eighty strains of Verticillium were isolated and purified by conventional plate separation method,numbered VL1-VL80.The strains grew slowly on potato dextrose agar,and could produce microsclerotia and verticillate-arranged conidiophores bearing conidia.Microsclerotia are elongated and irregularly-shaped,53.91-164.42×8.91-29.81μm in size and the average ratio of length to width is greater than 2.Conidia are hyaline and elongate,5.35-9.31×2.12-4.78μm.Chlamydospores and dormant hyphae were not observed.Direct sequencing of PCR products using the primers ITS1/ITS4,ActFa1/VactR,and ActF2d1/VactR were conducted to identify these isolates,then four isolates were chosen for direct sequencing of four protein coding genes EF,GPD,OX and TS.Phylogenetic analyses based on the ITS region indicated that these strains were located in the same branch of Verticillium longisporum clade.Phylogenetic analyses using five protein coding genes showed the four isolates belonged to the V.longisporum lineage A1/D1.The pathogenicity test showed that the strains could infect radish and cause the symptoms of root internal discoloration,indicating the causal agent of radish root internal discoloration in Qinghai,China was V.longisporum lineage A1/D1.To our knowledge,this is the first report of V.longisporum lineage A1/D1 in China.