【作者】 李衫衫；

【导师】 梁五生；

【作者基本信息】 浙江大学 ， 植物保护， 2019， 硕士

【摘要】 稻曲病菌(Villosiclava virens)能够在水稻孕穗期特异侵染水稻花丝产生稻曲球,造成大量相邻的小花不结实,导致水稻严重减产;同时稻曲球含有大量真菌毒素,严重影响稻米的食用安全。稻曲球的外围可产生大量的厚垣孢子,有时还会产生一至数个菌核。最近的研究表明稻曲病菌菌核可安全越冬,并成为来年主要的初侵染源。因此,菌核应作为防治稻曲病的重要作用对象之一。本实验对稻曲病菌菌核的萌发过程进行了仔细的观察,并分别筛选了抑制稻曲病菌菌丝生长及降解菌核的生防菌,对作用机制也进行了一些分析,取得了以下主要结果。1.在实验中筛选到的一株对稻曲病菌具有潜在抑制作用的细菌,对其16s rDNA序列进行克隆、测序及blast分析的结果显示该菌为芽孢杆菌。对峙培养实验结果证实该芽孢杆菌及其无菌滤液对稻曲病菌菌丝的生长具有显著的抑制作用。随着芽孢杆菌无菌滤液的浓度增大,对稻曲病菌的抑制作用也不断增强;但随着时间的延长,抑制效果会逐渐减弱。对田间防效进行了初步测定,但仍需要进一步验证。2.通过室内菌核的萌发及室外田间环境的模拟,发现菌核的萌发需要一定的光照,如果将萌发的菌核长期放在黑暗条件下,萌发的菌丝会出现消解的情况。但后续补充光照仍可萌发产生子实体。在合适的条件下,菌核可以多次萌发。田间越冬的菌核只要没有被完全降解,越冬后在合适的条件下仍然可以萌发。3.通过稻曲病菌和生防菌的平板对峙培养,及稻曲病菌的液体培养,发现稻曲病菌在营养缺乏、生长环境不利等不良条件下,会主动形成休眠状态来抵抗不良的环境。4.对来自稻田土壤中降解稻曲病菌菌核的真菌进行分离、筛选和鉴定,共筛选到3株具有生防潜力的真菌。对它们的rDNA-ITS序列进行克隆、测序及blast分析的结果显示这3株真菌分别属于附球菌属真菌、木霉属真菌和镰刀菌属真菌.将筛选到的菌株回接到稻曲病菌菌核上,发现上述生防真菌可以在菌核上定殖,5 d后基本覆盖住整个菌核,16 d的时候有的菌核开始在生防菌的作用下质地变得柔软,轻触即碎;30 d左右菌核开始变形;60 d的时候已经基本完全腐烂,看不出原来的形状。菌核在条件适合的状态下均不能再萌发。与之前筛选到的烟曲霉相比,新筛选的菌株更容易在菌核上长期定殖,降解效果更好。选择附球菌属真菌和木霉菌属真菌进行固体发酵,对田间防治稻曲病的效果进行了初步测定。更多还原

【Abstract】 The Villosiclava virens can specifically infect rice filaments at the booting stage,and form false smut balls,which caused a large number of adjacent florets not fructified,resulting in reduced rice yield.Meanwhile,rice false smut balls contain a lot of mycotoxins,which seriously affect the edible safety of rice.A large number of chlamydospores can be produced on the surface of rice false smut ball,and sometimes one to several sclerotia are also produced.Recent studies have shown that the sclerotia of the rice false smut can be safely overwintered and become the main source of primary infection in the next year.Therefore,sclerotia should be one of the important action targets for prevention and control of rice false smut.In this experiment,the germination process of sclerotia was carefully observed,and several biocontrol microorganisms with inhibition and degradation effects on V.virens and its sclerotia were screened.The mechanism of effect was also analyzed.The following main results were obtained.1.A bacterial isolate with potential biocontrol activity against V.virens was obtained.Blast results of the sequence of its 16s rDNA indicted that it belonged to Bacillus.Both dual culture and metabolites tests were carried out in this experiment.Results showed that both the Bacillus culture and its sterile filtrate could obviously inhibit the growth of V.virens hyphae.The inhibitory effects of the Bacillus sterile filtrate of increased along with the increase of its concentration,but the inhibitory effect decreased when time increased.A preliminary determination of its control effects on V.virens in field was carried out,but further verification was needed.2.The sclerotia could germinate under laboratory conditions and simulated field conditions,but the germination required a certain amount of illumination.When the germinated sclerotia were placed in the dark for a long time,the germinated hyphae were degradated.However,the sclerotia still germinated to produce fruiting bodies when the illumination was supplemented subsequently.The sclerotia could germinate multiple times under appropriate conditions.Therefore,as long as the sclerotia in fields have not been completely degraded,the overwintering ones can still germinate under appropriate conditions.3.Through the dual culture of V.virens with biocontrol bacteria and the liquid culture of V.virens,it was found that under the unfavorable conditions such as lack of nutrients and unfavorable growth environment,the V.virens formed a dormant state to resist the bad environment.4.Three strains of biocontrol fungi were screened,isolated and identified from paddy soil.Blast results of their sequences of rDNA-ITS indicted that they belonged to Epicoccum spp.,Trichoderma spp.and Fusarium spp.,respectively.Inoculation test to V.virens sclerotia showed that the isolated fungi could grow on the sclerotia.The sclerotia were fully covered by the biocontrol fungi after 5 days.After 16 days some sclerotia became soft under the action of biocontrol fungi,and could be broken by light touching.After about 30 days the sclerotia began to deform.After 60 days all the sclerotia nearly completely rotted and the original shapes could not be seen.The sclerotia could no longer germinate under suitable environmental conditions.The isolates of Epicoccum spp.and Trichoderma spp,were set to solid ferment.Preliminary tests of their controlling effects on V.virens in fields were carried out,but further verifications were needed.更多还原