【作者】 梁志怀；

【导师】 肖启明；

【作者基本信息】 湖南农业大学 ， 植物病理学， 2008， 博士

【摘要】 植物可通过根部与土壤中的微生物,建立植物—微生物系统,进行代谢产物的交换,激活植物的防御机理,影响植物的生长。本论文为探明生防木霉菌与水稻互作关系及作用,通过对木霉菌敏感药剂的筛选,确定了三唑类化学杀菌剂腈菌唑作为木霉菌抗药性遗传标记筛选药剂,同时,确定了木霉菌抗药性遗传改良的紫外线诱变剂量与腈菌唑复筛药剂浓度。在此基础上,利用紫外线与腈菌唑协同诱变技术对本实验室自行筛选的优良生防木霉菌T_2-16进行遗传改良,通过三年多时间的诱变改造,共筛选出106株抗药性木霉菌突变体,利用对水稻主要病原菌的拮抗作用,从中选育出既对腈菌唑药物具有较强的抗性又保持优良拮抗性的木霉菌突变体TUV-13。经与出发菌株的培养性状、乙酸乙酯提取物成分与含量、在水稻植株定殖能力、遗传稳定性等生物学特征的比较,证明TUV-13是具有明显双抗遗传标记木霉突变型菌株,可作为研究木霉菌大田条件下与水稻互作共生体关系的标记菌株。此外,利用RAPD-PCR分子标记技术检测了突变体与其出发菌株的DNA多态性,佐证了紫外线与腈菌唑协同诱变主要是通过其遗传物质DNA位点突变而实现的。为生防木霉菌抗药性改良提供一套稳定的技术,拓宽了木霉菌大田推广应用范围。为探明木霉菌TUV-13浸种处理水稻种子后,在大田生产条件下能否在水稻根际与其他器官定殖,与水稻形成共生体,通过对两地不同品种不同栽培模式的有关纹枯病发生发展、抗病相关酶活力、水稻生长势等因素的变化动态调查,结果确定了木霉菌与水稻形成机遇性共生体过程及动态,即通过浸种接种方式,木霉菌可随着水稻生长发育,定殖于水稻植株整个器官的表面及皮层细胞间隙。在秧苗期可增殖,但随着水稻进一步发育生长,定殖的木霉菌数量却越来越少。到了水稻生殖生长阶段,急剧降低。整个水稻生育期,木霉菌定殖动态呈现出前升后降,后期消失的动态规律;木霉菌与水稻的这种共生体关系,可诱导水稻植株抗病相关的PPO、POD、PAL酶活力提高,增强水稻对其主要病害的抗病性。尤其明显的是能够增强水稻秧苗素质、促进分蘖抽穗,最终明显提高水稻产量。此外,田间秧苗抗寒性与水稻纹枯病发生发展调查表明,木霉菌与水稻共生体关系还能增强秧苗的抗寒能力,对水稻纹枯病防治效果呈现前高中低后高的马鞍型动态规律。为进一步探明木霉菌与水稻形成共生体关系的机制,在室内盆栽仅有木霉菌与水稻的条件下,采用木霉菌孢悬液浸种、沾根和叶面接种等3种方法,观察木霉菌在水稻植株上如何侵入、定殖与转移动态。结果表明,浸种处理是木霉菌与水稻形成系统共生体关系的最有效方法,而蘸根、叶部接种处理只能使木霉菌与水稻形成局部的共生体关系。不同木霉菌株浸种处理结果确定了木霉菌与水稻形成共生体的机制为:木霉菌可吸附在水稻颖壳表面,随着种子萌发,通过产生附着孢、缠绕等方式吸附在水稻胚根、胚芽以及两者之间的隆起组织,并克服水稻的防御机制,主动侵入水稻各组织器官皮层细胞间隙,并随着植株的生长而扩展。其中,水温、浸种时间的长短可影响木霉菌在水稻植株中定殖。木霉菌与水稻共生体中存在游离氨基酸的交换。还能诱导水稻秧苗对纹枯病抗病性。系统溶剂法提取试验结果表明,氯仿是木霉菌TUV-13水稻体外模拟培养物抗菌物质提取的最适溶剂。GC-MS检测到TUV-13氯仿提取物有40多种化学成分,其中烷烃类数量最多,为13种,其他成分为有机酸类、酯类、酮类、类固醇类等有机化合物。大孔吸附树脂HP-20与硅胶柱层析分离试验结果确定石油醚乙酸乙酯作洗脱剂的硅胶柱层析系统是木霉菌YUV-13抗菌物质的最适分离方法,能有效地分离出3种结构不同的抗菌物质。其中2号流分抗菌活性最强,鉴定为十八碳二烯酸甲酯,9号流分次之,鉴定为类固醇类抗生素。室内体外细胞毒力测定,木霉菌TUV-13代谢产生的十八碳二烯酸甲酯对不同病原菌抑制效果具有明显差异,其中对水稻纹枯病菌最为敏感,EC₅₀值9.27μg/mL;对花生白绢病EC₅₀值为21.53μg/mL;对辣椒疫病EC₅₀为131.48μg/mL;对水稻稻瘟病EC₅₀为428μg/mL。同时对不同水稻品种种子活力还具有促进作用更多还原

【Abstract】 The plants-microbiology system combining with plants and soil microorganisms is possessed of many functions, such as exchanging metabolic product, activing plant defense behaviour, and the effect of plants growth. The Trichoderma mutant TUV-13 was obtained by means of the UV-induced in this paper to study the interaction with rice. The strain TUV-13 was labeled of myclobutanil and from the origin of strain T_2-16 with the strongest antagonistic activity to fungus pathogens in our lab. The comprison on the the culture traits, ethyl acetate extract composition and content, colonization capacity in the rice plant, genetic stability, and other biological characteristics between TUV-13 and T_2-16, has proved that TUV-13 is a clear genetic markers of anti-double-mutant Trichoderma Strain, which can be used as research marked strain to study the symbiotic relationship with rice under open field conditions. In addition, the biocontrol agent of TUV-13 mutant and the original strain were subjected to a random amplified polymorphic DNAs （RAPD） study. Furthermore, based on NTSYS cluster analysis, the results showed that the inheritable character of induced mutant TUV-13 was stable. The DNA homology between TUV-13 mutant and the original strain shows significance of difference.Then the colonization trends of stain TUV-13 in rice was analyzed in the field test.The results showed that TUV-13 could be subsist and develop in tissues by presoaking the seeds with TUV-13 fermented production.The symbiotic relation between the Trichoderma mutant TUV-13 and rice could establish. The establishment and dynamic ecology of the symbiont studied through the surveying the growth rates and the resistance under two different cultivated varieties and different cultivated type in past two years. The results showed that soaking, through the inoculation method Trichoderma can colonize in the surface of rice organs and the cortex gap in cells, as the growth and development of rice. Proliferation in the seedlings period, but with the further development of the growth of rice plants, the number of the colonization of Trichoderma is less and less, to the reproductive phase of growth, the number of Trichoderma sharply reduced. The symbiotic relationship between Trichoderma with Rice body can induce the defensive enzyme activities of peroxidase（POD）, polyphenol oxidase （PPO） and phenylalanine ammonia-lyase（PAL） in rice seedling were increased by various level, enhancing resistance to its main rice disease. The most obvious is to enhance the quality of rice seedlings to promote the tiller heading, eventually improve rice production. In addition, the investigation of the seedlings in the field on chilling injury and the occurrence and development of rice sheath blight show that Trichoderma-symbiotic relationship can enhance the ability of cold resistance, control of rice sheath blight.Further, the best way to establish the symbiotic was studied. The spore suspension of TUV-13 was applied in rice by soaking, pruned-root dip and inoculations of daubing leave. The results indicated that each method could led to colonization of TUV-13 strain in rice tissues, and among them, the soaking is the most effective way to establishing symbiotic relationship between Trichoderma strain and rice. Because the TUV-13 strain could systematic distribution in rice when it used by soaking, and the others inocula-tions methods could only be the local symbiotic relationship between the rice body and Trichoderma.The mechanism of the symbiotic formation also obtains. Trichoderma strains could be absorbed on the surface of rice chaff when the rice seeds soaked with the spore suspension of Trichoderma.Follow seed germining, Trichoderma spore began to germinate and form primary germtube.Then the appressoria began to form at the tips of germtube and attached on the surface of host. Further, it coule be microscopically observed the hyphae and conidiophores to existdetween mesophyllic cells. And the target strain TUV-13 could to internal stem ,leaf and root stably .It was confirmed that the strain was endophytes in rice and could develop, transfer in rice. Observation to the macroscopical and microscopical process indicated that the rice and Trichoderma strain were compatible.The temperature, soaking time could affect the colonization of Trichoderma in the rice plants. The symbiont of Trichoderma and rice existed exchange of the free amino acids. Meanwhile, the symbiont were induced resistance to the sheath blight.The test results of Solvent Extraction System showed that chloroform is the most solvent for Trichoderma TUV-13 anti-bacterial substance extracted from culture in rice vitro. GC-MS detected TUV-13 chloroform extract included more than 40 kinds of chemical substances, of which number of alkanes,13 kinds, is the largest.The others contains organic acids, esters, ketones, steroids, and other types of organic compounds. The comprison on macroporous resin HP-20 and silica gel column chromatography, which the outcome of the trial of oil ethyl ether-eluting agent for the silica gel column chromatography system was Trichoderma TUV-13 anti-bacterial substances, determined that the optimum separation methods are effectiveto isolate the 3 different structure of anti-bacterial substance. Sub flow 2, the strongest anti-bacterial activity, identified as 18 carbon methyl diene, Sub flow 9, followed by sub-stream, identified as steroid antibiotics. Indoor cells in vitro Toxicity show Trichoderma TUV-13 metabolites of 18-carbon diene methyl inhibiting effect of different pathogens have significant differences, of which rice sheath blight was the most sensitive, its EC₅₀ was 9.27μg/mL; to peanuts southern blight EC50 value was 21.53μg/mL; to phytophthora EC50 was 131.48μg/mL; torice blast EC₅₀ was 428μg/mL. At the same time, the seeds vigor of different rice varieties were improved by the substance.更多还原