【作者】 杨波；

【导师】 郭泽建； 方敦煌；

【作者基本信息】 浙江大学 ， 植物病理学， 2005， 硕士

【摘要】 烟草赤星病是烟草的一类重要叶部病害,其主要病原菌为链格孢属真菌(Alternaria alterrrata (Fties) Keisslar),在烟草成熟后期危害十分严重,造成重大经济损失。目前防治烟草赤星病的化学药剂对环境和烟叶的污染日益严重,采用生物防治势在必行。本论文通过拮抗细菌的分离、平皿拮抗试验进行了拮抗菌的筛选研究,为此病害的生物防治奠定了基础。 本研究通过平皿拮抗试验分离测定了2,846株细菌菌株对烟草赤星病的离体防治效果。在平皿拮抗试验中,筛选得到B2、B4等22个对烟草赤星病表现出较强拮抗作用的菌株,B1、B3等114个菌株则对烟草赤星病表现出一定的拮抗作用。 上述136株拮抗赤星病的菌株经过16S rDNA PCR—RFLP分析,结果表明这些菌株具有遗传多样性,归于3个系统发育分支。通过对B2、B4、B39、B44、B45、B58、B84、B98、B108、B109、B120等11株拮抗菌进行16S rDNA全序列测定分析,结果表明1l株拮抗茵可以分为3个类群。16S rDNA分类结果和16S rDNA PCR—RFLP分析结果一致,但在个别菌株的归群上也存在一些差异。 菌株B75在温室盆栽测定和大田防效试验中,对烟草赤星病都有显著防效。因此利用传统的形态学特征观察B75的特征,发现此菌株革兰氏反应呈阳性,在NA培养基平皿上菌落呈土黄色,近圆形,表面干燥、起皱、不透明。进一步对葡萄糖产酸等10个生理生化指标进行测定,并结合形态学观察结果,初步鉴定B75菌株为枯草芽孢杆菌(Bacillus subtilis)。对菌株B75进行16S rDNA全序列分析,测序结果在GenBank中进行Blast同源序列检索,利用DNAStar软件与芽孢杆菌属的其它种进行同源性比对。结果表明B75与已报道的B. subtilis 16SrDNA序列(登录号:AY030331)有100%的相似性,且两者在系统发育树中处于同一个分支。结合形态学特征、生理生化特征及16S rDNA全序列分析,确定菌株B75为B. subtilis的一个菌株。更多还原

【Abstract】 Tobacco brown spot disease has been an important leaf disease in tobacco. In tobacco maturation period, this disease causes tobacco brown spot disease seriously, and makes large economic losses. Its main pathogen is Alternaria alterrrata (Fries) Keisslar. In order to avoid the pollution of environment and tobacco leaf, it is necessary to use biological control. In this dissertation, antagonistic bacterium that can control tobacco brown spot disease are screened by dual cultures on PDA.2,846 bacterial strains were isolated from the soil and leaf samples. These strains were screened for antagonists against A. alterrrata by dual cultures on PDA. The result showed that 22 strains were strongly antagonistic to the pathogen, and one hundred and 114 strains were antagonistic to the pathogen too.136 antagonistic strains were analysed by 16S rDNA PCR-RFLP. The result showed that all tested strains were divided into 3 groups. 11 antagonistic strains wered analysed by the sequence analysis of 16S rDNA. The result showed that these tested strains were also divided into 3 groups.Strain B75 was strongly antagonistic to tobacco brown spot disease in pot test and field test. According to its morphological characteristics, strain B75 was considered to be the member of genus Bacillus. According to its physiological and biochemistrical characteristics, the strain was confirmed to be one strain of B. subtilis. Strain B75 shared 100% homology with published sequence of B. subtilis(accession number: AY03031) by this means of sequence analysis of 16S rDNA. In the phylogenetic tree, both strain B75 and B. subtilis were on the same branch, which showed that strain B75 was closely related with B. subtilis. So we can make a conclusion that the isolate B75 was one strain of B. subtilis.更多还原