杨树水泡型溃疡病菌培养性状、无性型与分子系统学分析

Cultural Characteristics,Anamorph and DNA Phylogeny of Botryosphaeria dothidea Causing Poplar Canker

对采自陕西、甘肃等地的杨树水泡型溃疡病菌培养特征、无性型形态等进行观察鉴定,并利用真菌通用引物ITS1/ITS4,EF1-728F/EF1-986R和Bt2a/Bt2b对供试菌系的rDNA内部转录间隔区ITS序列、β-微管蛋白序列和转录延伸因子EF-1α基因序列分别进行PCR扩增与测序,比对分析后构建系统发育树。结果表明:供试62个溃疡病菌菌系可按培养特征划分为2个菌落类群:类群Ⅰ为典型喷泉状菌落,类群Ⅱ为平伏状菌落。依据该病菌培养物所产生的分生孢子形态特征,该菌系被鉴定为七叶树壳梭孢,即葡萄座腔菌的无性型。同时,供试菌系在rDNA-ITS基因、β-tubulin基因和EF-1α基因序列的NJ系统发育树中分别被分为2、3和3个类群。在构建的3个NJ系统发育树中,包含绝大多数参试菌系的类群Ⅰ均与GenBank上报道的B.dothidea聚在一起,证实形态学分类鉴定结果正确;来自宝鸡陇县的菌系Fldh1在rDNA-ITS系统树中独立成群;来自陕西杨凌区的菌系Fyxn,Fyrg和Fyxj在β-tubulin及EF-1α基因序列系统树中均聚为一类;在EF-1α基因序列构建的系统树中,采自陕西咸阳的Fxfh,Fxdh1和Fxdh2 3菌系被聚为一支,并显示与B.dothidea种内其他地域菌系亲缘关系较远。总体来看,供试菌系在培养性状、产孢情况等方面的差异与其分子系统发育不存在一一对应关系。

The cultural characteristics and anamorph morphology of poplar canker fungus isolates collecting from Shaanxi and Gansu were observed,the rDNA-ITS,β-tubulin and EF-1α gene sequences of these fungal isolates were sequenced using universal primer ITS1 / ITS4, EF1-728F / EF1-986R and Bt2a / Bt2b respectively, and the phylogenetic trees were constructed after sequence alignment. The results showed that the 62 isolates could be divided into two colonial groups based on the cultural characteristics on PDA medium： groupⅠ showed typical fountain colony,group Ⅱ showed flat colony. Acconding to characteristics of conidium and culture,these isolates are identified as Fusicoccum aesculi,that is anamorph of Botryosphaeria dothidea. Meanwhile tested isolates were clustered into two,three and three groups based on neighbor-joining（ NJ） analysis of the rDNA-ITS,β-tubulin and EF-1α gene sequence data respectively. The isolates of groupⅠin three NJ trees,included most tested isolates,were clustered together with B. dothidea retrieved from GenBank,which confirmed the morphological identification of these isolates; the isolate Fldh1 from Long county was classified as one group in the rDNA-ITS gene phylogenetic trees; the isolates Fyxn,Fyrg and Fyxj from Yangling of Shaanxi were all classified as one separate group in the β-tubulin and EF-1α gene phylogenetic trees; three isolates Fxfh, Fxdh1 and Fxdh2 from Xianyang of Shaanxi were classified as group Ⅲ in the EF-1α gene phylogenetic trees,displaying far away phylogenetic relationship from other isolates of B. dothidea. Overall there was no one-to-one relationship among cultural characteristics,sporulation and molecular phylogeny of the tested isolates.