广西地区葡萄黑痘病病原菌的分离与鉴定

Isolation and identification of the pathogens causing grape anthracnose in Guangxi

采用常规组织分离法对广西南宁、河池两地的葡萄黑痘病菌进行分离、纯化,分别得到37和31株分离菌。经菌落形态观察及r DNA ITS序列分析,南宁37株分离菌为同一菌株,河池31株分离菌为同一菌株,以NN和HC分别代表两地菌株,对它们进行形态学、致病性鉴定及r DNA ITS区域序列分析。结果显示,两地菌株形态学与致病性存在较大差异,但都符合黑痘病菌生长形态。NN菌落为红棕色、近圆形,边缘光滑。菌落中心位置丘状凸起,表面有白色菌丝和透明粘稠的小液滴,周围边缘有较规则的褶皱。HC菌落呈浅橙色、近圆形,边缘光滑。菌落中心位置丘状凸起,表面有少量白色菌丝,无液滴,周围边缘有不规则褶皱隆起。人工接种葡萄后均能引起典型的黑痘病症状,NN致病性强于HC。使用r DNA ITS区域通用引物ITS1F/ITS4进行PCR扩增后,NN和HC分别得到1 124 bp和818 bp的片段。比对结果显示1 124 bp与Elsinoe ampelina(AY826763.1)序列覆盖率达92%,序列一致性达99%;818 bp与Elsinoe ampelina(AY826762.1)序列覆盖率达75%,序列一致性达99%。因此,NN和HC均是引起广西葡萄黑痘病的病原菌。

Through the conventional tissue isolation approach from the infected tissues of grapevines with anthracnose symptoms,a total of 37 and 31 isolates from Nanning and Hechi,Guangxi was isolated and purified,respectively.Based on the morphology and rDNA ITS sequences analysis,37 isolates from Nanning was the same strain as well as 31 isolates from Hechi,which were named NN and HC strains,respectively.Even though NN and HC strains both fit the description of Elsinoe ampelina,the two strains showed big differences on morphology and pathogenicity.NN colonies were red brown,suborbicular with smooth edges,white mycelium and transparent sticky droplets on the hummocky raised surface,regular folds on the surrounding edge.HC colonies were light orange,suborbicular with smooth edges,few white mycelium on the hummocky raised surface,but no droplets,irregular folds on the surrounding edge.By inoculation of young grapevine shoots,both strains could cause typical symptom of grape anthracnose disease,while the NN strain showed stronger pathogenicity than HC.PCR amplification using a pair of rDNA ITS primers ITS1F/ITS4 resulted in 1 124 bp and 818 bp fragments from NN and HC,respectively.The 1 124 bp fragment showed 92%coverage and 99%similarity to the sequence of Elsinoe ampelina（GenBank AY826763.1）.The 818 bp fragment showed 75%coverage and 99%similarity to the sequence of Elsinoe ampelina（GenBank AY826762.1）.Therefore,NN and HC are both pathogens causing grape anthracnose in Guangxi.