【作者】 王珍；

【导师】 肖炎农；

【作者基本信息】 华中农业大学 ， 植物病理学， 2010， 硕士

【摘要】 油菜是我国主要油料作物,油菜菌核病[Sclerotinia sclerotiorum]是油菜的常发性病害,对油菜生产危害极大。一般造成减产10%～30%,严重时可达80%。本研究从湖北油菜产区广泛采集油菜样品,以健康的油菜植株作为材料,采用平皿稀释和对峙培养法等分离筛选到一株对油菜菌核病菌具有强拮抗作用的生防菌株。其16SrDNA序列与Genbank中枯草芽孢杆菌(Bacillus subtilis)的序列同源性高达99%,进一步对其形态学和生理生化特性分析,将该菌株确认为枯草芽孢杆菌,命名为Bacillus subtilis K-106。Bacillus subtilisK-106在PDA平板上对核盘菌菌丝的抑制作用明显,显微镜下观察到被抑制的菌丝内细胞质分部不均匀,菌丝体中的原生质颗粒状且中间膨大。用该菌株的发酵菌体悬浮液处理正常的核盘菌菌核,推迟了菌核的萌发,且萌发后再次形成菌核的数量与对照相比减少了85.7%,而上清液无明显抑制作用。Bacillus subtilisK-106的菌体悬浮液(108个细胞/ml)在离体叶片条件下对菌核病病斑扩展的抑制率为71.87%,但其发酵上清液的抑病效果不明显。经田间防治试验表明,Bacillus subtilisK-106菌体悬浮液(108个细胞/ml)的防治效果为52.21%.利用该菌的菌体悬浮液(108个细胞/ml)进行根际拌种,处理后,油菜幼苗株高较对照增长了47.3%,且能有效促进油菜苗期根部的生长,增长率达到56.7%。BsK-106对其地下部分的干重与湿重有一定的促进作用,分别为30.2%和12.6%。菌株Bacillus subtilisK-106对油菜植株有一定的诱导抗性作用。选择苯丙氨酸解氨酶(PAL)、超氧化物歧化酶(SOD)、过氧化物酶(PO)和多酚氧化酶(PPO)作为油菜植株抗病性反应的指标,对接种油菜菌核病和生防拮抗细菌BsK-106后油菜植株叶片内这些酶的活性水平进行比较,测定结果表明,PO酶的活性在48h后达到最大值,PPO、PAL、SOD三种酶的活性都是在24h后活性达到最高峰,这四种酶的活性达到最高峰的时间可能与生防菌株在油菜植株体内所起的作用有关。各种处理后这四种酶的活性与对照相比都有不同程度的提高,可能是由于此原因从而增强了油菜抵御菌核病的侵染及蔓延能力。大田防治实验表明,经菌株Bacillus subtilisK-106 104个细胞/ml、106个细胞/ml、108个细胞/ml三种不同浓度的发酵液处理后,防治效果分别为25.71%、41.56%、52.21%。更多还原

【Abstract】 Rapeseed is Chinese major oil crops, Sclerotinia sclerotiorum is the constant disease of the rapeseed which caused a great harm on rapeseed production. Generally result in cuts of 10% to 30%, severe 80%.In this study, producing a broad collection healthy rapeseed plant of Hubei, using agar dilution and confrontational culture isolated one strain which has a strong antagonistic effective. Its 16SrDNA sequence sequence 99% homology with Bacillus subtilis in Genbank, further to its morphological, physiological and biochemical characteristics, the strain identified as Bacillus subtilis, named for the Bacillus subtilis K-106.Bacillus subtilis K-106 has clear inhibition with Sclerotinia sclerotiorum on the PDA media. Under the microscopic, the inhibited mycelia cytoplasm uneven, granular protoplasm in the mycelium and the middle of the mycelia is enlarged. Using the suspensions of the BsK-106 dealing with the normal sclerotium, delayed the germination of sclerotia, and the number of formation sclerotia germination once again decreased of 85.71% compared to the control, while no significant inhibition of the supernatant.The rate of cell suspensions(108 cells/ml) of Bacillus subtilis K-106 inhibition of Sclerotinia sclerotiorum lesion expansion is 71.87% on detached leaves, but the suppression effect is not obvious. The field test showed that the control efficacy of Bacillus subtilis K-106 cell suspension (108 cells/ml) is 52.21% compare with the control.Using the cell suspension (108 cells/ml)of BsK-106 deal with rapeseeds, rapeseed seedling height increased by 47.3% compared with the control, and its underground parts increased 56.7%. Its aboveground and underground parts of the dry weight and wet weight is 30.2% and 12.6% higher compared with the control.Bacillus subtilis K-106 have some induction resistance to the rapeseed plants. Phenylalanine ammonialyase (PAL), superoxide dismutase (SOD), peroxidase (PO) and polyphenoase (PPO) of rapeseed plants were different responses which analyzed as indicators of rapeseed resistance to diseases after inoculated with Sclerotinia sclerotiorum and Bacillus subtilis K-106. The results showed that PO activity peaks reached at 48hr after inoculation, and PPO、PAL、SOD activity peak reached at 24hr. These four enzyme activity peak reached at different time may be related to the biocontrol strain functioning in rapeseed plant. These four enzymes activity were increased compared with the control, may be due to this reason increasing the ability of rapeseed plant resistance infection and spread of the Sclerotinia sclerotiorum.Field control experiment showed that after 104 cells/ml、106 cells/ml and 108 cells/ ml of BsK-106’s liquid culture, the control efficacy was 25.71%、41.56%and52.21%.更多还原