喷施菌剂后葡萄叶片真菌多样性的PCR-DGGE分析

Analysis of Fungal Diversity on Grape Leaves after Spraying Bacterial Agents by PCR-DGGE

为探讨葡萄叶片喷施砖红微杆菌Y24菌剂和解淀粉芽胞杆菌N22菌剂后其表面真菌多样性的变化,本研究采用PCR-DGGE技术,以样品的基因组DNA为模板,扩增出大小约为400 bp的真菌18S rDNA高变区,DGGE电泳后进行图谱分析,并对标记条带进行切胶、回收、克隆和测序以研究真菌多样性相关的特征数和菌群组成。DGGE图谱分析表明,喷施砖红微杆菌Y24菌剂的叶片样品的丰富度指数S最高,香农-维纳指数H′最大,喷施解淀粉芽胞杆菌N22菌剂后的这两个指标与对照相比无明显变化。对DGGE图谱上标记出的25个条带进行测序比对,发现葡萄叶片上的真菌主要为非可培养链格孢(Uncultured Alternaria alternata)、枯叶格孢腔菌Pleospora herbarum、近平滑假丝酵母Candida parapsilosis和非可培养真核生物等。喷施砖红微杆菌Y24菌剂的特有的真菌类群包括非可培养真菌、隐球菌属Cryptococcussp.和非可培养真核生物。葡萄叶片喷施砖红微杆菌Y24菌剂后附生真菌种类变得更丰富,喷施解淀粉芽胞杆菌N22菌剂后与对照无显著差别。

PCR-DGGE technology was used to explore the variation of fungal diversity on the surface of grape leaves after spraying Microbacterium testaceum Y24and Bacillus amyloliquefaciens N22 agents.The 18S rDNA high variation area of the fungus with the size of 400 bp was amplified with the sample genome DNA as the template After DGGE,the map and the marker strips were analyzed.The bands were cut,reclaimed,cloned and sequenced to determine the characteristic value and composition of fungal diversity.The DGGE map analysis showed that the species richness,Shannon Wiener index of the leaf samples spraying Y24 was the highest,and the two indexes of samples spraying N22 was not significantly changed compared with those of the control.The sequence alignment of 25 bands marked on the DGGE map showed that the fungi on the grape leaves were mainly unculturable Alternaria alternata,Pleospora herbarum,Candida parapsilosis and unculturable eukaryotes.The specific fungal groups of leaf spraying Y24 were unculturable fungi,Cryptococcus sp.and unculturable eukaryotes.The fungal diversity on leaves treated with Y24 was more abundant than the control and N22agent,and there was no difference between N22and control.