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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。
Isolation and purification of grape black pox in Nanning and Hechi of Guangxi were carried out by conventional tissue separation method, and 37 and 31 isolates were obtained respectively. After colony morphology observation and r DNA ITS sequence analysis, 37 isolates of Nanning were the same strain, 31 isolates of Hechi were the same strain, and NN and HC were respectively representative of the two strains. Their morphological and pathogenicity were identified r DNA ITS sequence analysis. The results showed that there was a great difference between the morphological and pathogenicity of the two strains, but all of them were in accordance with the growth pattern of P. acnes. NN colony is reddish brown, nearly circular, smooth edges. Colony at the center of the hill-shaped bulge, the surface of white mycelium and transparent sticky droplets, around the edge of a more regular folds. The HC colonies are light orange, nearly round, with smooth edges. Colony at the center of the hill-shaped bulge, the surface of a small amount of white mycelium, no droplets, around the edge of irregular fold bulge. Artificially inoculated grapes can cause typical symptoms of acne disease, NN pathogenicity stronger than HC. After PCR amplification using the ITS1F / ITS4 universal primer of rDNA ITS region, fragments of 1 124 bp and 818 bp were obtained respectively by NN and HC. The alignment results showed that the sequence coverage of 1 124 bp and Elsinoe ampelina (AY826763.1) was 92% and the sequence identity was 99%. The sequence coverage of 818 bp and Elsinoe ampelina (AY826762.1) was 75% 99%. Therefore, both NN and HC are pathogenic bacteria that cause anthracnose in Guangxi grape.