【作者】 李毅；

【导师】 王国平； 张克诚；

【作者基本信息】 湖南农业大学 ， 农药学， 2004， 硕士

【摘要】 从3种不同的蔬菜地根际土壤中筛选出对棉花黄萎病病原菌（Verticillium dahliae Klebahn）具有拮抗作用的放线菌130株，经初步分类和鉴定，这130株放线菌分为6个属，其中链霉菌属114株，为优势种群。拮抗性试验结果表明，放线菌的拮抗菌虽然广泛分布于植物根际土壤中，但具有强烈抑菌作用的拮抗性菌株是非常稀少的。本试验中所选用的LJ50和MJ52分别分离自健康辣椒土壤和健康马铃薯土壤，经鉴定均属链霉菌属（Streptomyces spp．）的白孢类群（Albosporus），种待定。 初步测定拮抗菌LJ50和MJ52的最佳培养条件和培养方案。根据试验结果，确定了最佳发酵条件： LJ50产生抗菌物质的最佳培养液方案是D培养液，最佳培养时间是48小时。最佳培养温度为27±1℃，产生抗菌物质的发酵液的最适pH值为6.0，最佳培养方式为全天振荡培养。 MJ52产生抗菌物质的最佳培养液疗案足C培养液，最佳培养时间是60小时。最佳培养温度为27±1℃，产生抗菌物质的发酵液的最适pH值为6.0，最佳培养方式为全天振荡培养。 初步测定LJ50和ML52发酵液的效价为1000倍，确定其EC₅₀为40倍。实验结果表明LJ50和MJ52这两个拮抗菌株发酵液的抗菌谱具有广谱性，对8种病原真菌均有抑制作用。两菌株连续转管培养10代的过程中，其抗菌效果有所下降，出现菌株退化现象。 作用机制的研究表明LJ50和MJ52的发酵液能抑制棉花黄萎病病原菌（Verticillium dahliae Klebahn）菌丝生长和分生孢子的萌发，且导致菌丝体膨大、畸形，分生孢子不萌发或萌发异常。LJ50和MJ52的发酵液能诱导棉苗体内的抗性相关酶如过氧化物酶（POD）、苯丙氨酸解氨酶（PAL）、多酚氧化酶（PPO）酶活性的增加，从而可能对棉花产生诱导抗性。室内促生长试验表明，不论是发酵液原液，还是发酵液稀释液均具有促进棉花种子萌发的作用。 通过盆栽温室试验表明供试拮抗菌不同浓度的发酵液均可以不同程度地降低棉花黄萎病的发病率和病情指数。LJ50和MJ52的最佳相对防效分别达到57.57％和57.02％，接近多菌灵的相对防效（63.63％）。室内无菌条件下采用琼脂平板法证实LJSO和MJ52能够在棉花种子的表面和胚根上进行定殖，同时在棉苗根部的定殖还具有可转移的特性，能够随着棉苗根系的延伸而不断定殖于新形成的根组织_匕。更多还原

【Abstract】 130 Actinomyces strains that have antagonistic activity against Verticillium dahliae Klebahn from 3 vegetable fields rhizosphere soil were screened. Preliminary identification and classification, these Actinomyces could be classified into 6 genus with dominative Streptomyces. In antibiotic test, the datum showed that the antagonistic Actinomyces extensive distribute in the plant rhizosphere soil, but the strongly antagonistic Actinomyces were few. LJ50 and MJ52 isolated from the healthy pepper soil and the healthy potato soil, respectively, they were identified as Slreptomyces.Optimum fermention conditions for LJ50 and MJ52 were studied. According to the results, the best fermention condition as fellows: LJ50 is in the D-liquid medium shaked 48 hours; MJ52 is in the C-liquid medium shaked 60 hours. Both of two antagonistic Streptomyces optimum temperature and pH value are 27 ±1 ℃ and 6.0, respectively. Meanwile, the optimum foster way was shaked all day.Preliminary determined the potency of the fermentation extract of LJ50 and MJ52 against the pathogen was 1000 times and their EC50 was diluted 40 times. The fermentation extract of LJ50 and MJ52 exhibited an inhibitory ability to 8 species of phytopathogenic fungi, this showed that the fermentation extract had a broad antibiotic spectrum. By the experiment of stablity of an antifungal substances showed that continuous culturing up to 10 generations, LJ50 and MJ52 had a negative influences on inhibitory activity of their antifungal substances against the pathogen.Mechanism of control Verticillium dahliae Klebahn with the fermentation extract of LJ50 and MJ52, microscopic observation showed that they could interfer the normal development of hyphae and conidiospores, leading to hyphae malformed and contdispores abnormal germination or failure to germination. The incresed activities of POD, PAL, PPO in cotton seedlings upon the treatment by the fermentation extract of LJ50 and MJ52 were detected. This stuggsted the fermentation extract could trigger the defense reactions of cotton plants. The germination of cotton seeds was promoted by the fermentation extract and its diluent in the indoor environment test.Results from greenhouse trial showed that the fermentation extract could drop down the disease percentage and disease index of cotton verticillium wilt. LJ50 and MJ52 their optimun control effect were 57.57% and 57.02%, close to the control effect of carbendazol (63.63%). Sterilizing chamber test showed that applied agar plate method LJ50 and MJ52 had colonization ability in the surface of cotton seeds and radicle, they could continuously be transported along with elongating root.更多还原