木霉菌对大豆根腐病菌的生防机制及其制剂的初步研究
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摘要
木霉菌(Trichoderma spp.)是被认为最有希望的生物防治因子之一,其对大豆根腐病的病原菌有较好的抑制作用。为了进一步开发利用木霉菌,使其早日用于生产,本文对6株木霉菌进行了对大豆根腐病菌拮抗机理,木霉菌的分类鉴定,选出3株菌进行了颗粒剂研制的初步探索以及木霉菌颗粒剂对大豆根腐病田间防效的试验等研究,结果如下:
1.采用对峙培养、对扣培养和圆盘滤膜法,研究木霉菌对大豆根腐病菌的拮抗机理,结果表明,木霉菌可以通过空间竞争、寄生和抗生作用抑制大豆根腐病菌的生长。对峙培养结果表明,木霉菌对大豆根腐病菌有较强的空间竞争优势;6株木霉菌对立枯丝核菌、终极腐霉菌、尖孢镰孢菌和燕麦镰孢菌都有明显的寄生作用。木霉菌通过螺旋缠绕、贴附生长并同时产生细小菌丝分支环绕病原菌菌丝的方式寄生于病原菌菌丝上,最终导致病原菌解体死亡;木霉菌的代谢产物中,难挥发性代谢产物是重要的抑菌物质,对大豆根腐病菌的抑制效果明显。
2.采用形态学和分子生物学方法鉴定6株木霉菌的种类。通过对扩增出木霉菌的ITS序列测序,得到6个不同的ITS序列,通过ITSH网站TrichOKEY和TrichBLAST录入,进行序列的比对分析,同时结合木霉菌形态学鉴定,确定木霉菌T20A、T20G、T38D、T61F和T78E为Trichoderma koningiopsis种的不同菌株;木霉菌T115D为Trichoderma longibrachiaum。
3.采用正交试验对木霉菌T38D、T78E和T115D的液体和固体发酵条件进行优化,确定了利于木霉菌快速大量产孢的培养方法、培养基质和条件,制备出木霉菌颗粒剂。
4.木霉菌颗粒剂田间防治大豆根腐病试验结果表明,木霉菌剂均可降低根腐病的发生,混合木霉菌颗粒剂对大豆根腐病防效好于单一木霉菌剂,防效达42.79%,小于化学防治(55.63%),混合木霉菌颗粒剂与1/2药量的化学药剂共同使用的防治效果为48.18%。不同时期大豆的形态指标调查结果和测产结果表明,木霉菌颗粒剂对大豆有促生作用,可提高大豆产量。
Trichoderma spp. is considered one of the most promising biological control factors, and has well inhibition to pathogens of soybean root rot. To make early for the production of Trichoderma spp. by further developping and utilizing, six strains of Trichoderma spp. were indentified and studied to the pathogens of soybean root rot in the study. Three strains which were selected from six were initially used for exploring the development of preparation. As the same time, the field efficiency of Trichoderma preparation to soybean root rot were also studied in the paper. The experimental results were as follows:
1. The inhibition of Trichoderma spp. to pathogens of soybean root rot were studied by the space competition, parasitism and antibiosis. The results showed that Trichoderma spp. can inhibite the growth of pathogens of soybean root rot through confronting each other, cultivating face to face, cultivating in cellophane film. Confront culture results showed that Trichoderma had stronger advantages of spatial competion to the pathogens of soybean root rot. The microscopic observation showed that six strains of Trichoderma spp. had the significant parasitic effect against Rhizoctonia solani, Pythium ultimum, Fusarium oxysporum and Fusarium avenaceum. Trichoderma spp. was parasitized on the mycelia by the way of spiral wound, adhering, as while, surrounding the mycelia through producing small hyphae, which could finally cause the disintegration and death of pathogens. The hard volatile metabolite in the metabolites of Trichoderma spp was an important antibacterial substance which had the significant antibacterial effect on the pathogens of soybean root rot.
2. Six strains of Trichoderma spp were identified by the methods of molecular biology and morphological. Six different ITS sequences have been got by PCR and sequencing. Through inputting TrichOKEY and TrichBLAST in the ITSH website, the comparative analysis of sequence and combination with morphological identification of Trichoderma spp, T20A, T20G, T38D, T61F, and T78E have been determined to be different strains of Trichoderma koningiopsis s and T115D to be Trichoderma longibrachiaum.
3. Liquid and Solid Fermentation conditions of Trichoderma T38D, T78E, and T115D have been selected and optimized by using orthogonal test. The favorable training matrix and conditions that Trichoderma spp quickly produced large number of spores have been determined. Trichoderma granules had been prepared.
4. The field pathogens control results of Trichoderma preparation to the soybean root rot showed that the occurrence of soybean root rot can be reduced by Trichoderma preparation. And the effect of mix Trichoderma preparation was better than single Trichoderma preparation; the controlling effect was 42.79%, lower than the Chemica Control (55.63%). The control effect was 48.18% by used mix Trichoderma preparation mixed with 1/2 dose of the chemicals. In different periods, the results of soybean morphological index survey and the yield estimation showed that the growth of soybean could be promoted by Trichoderma preparation, and its yield also improved.
1.采用对峙培养、对扣培养和圆盘滤膜法,研究木霉菌对大豆根腐病菌的拮抗机理,结果表明,木霉菌可以通过空间竞争、寄生和抗生作用抑制大豆根腐病菌的生长。对峙培养结果表明,木霉菌对大豆根腐病菌有较强的空间竞争优势;6株木霉菌对立枯丝核菌、终极腐霉菌、尖孢镰孢菌和燕麦镰孢菌都有明显的寄生作用。木霉菌通过螺旋缠绕、贴附生长并同时产生细小菌丝分支环绕病原菌菌丝的方式寄生于病原菌菌丝上,最终导致病原菌解体死亡;木霉菌的代谢产物中,难挥发性代谢产物是重要的抑菌物质,对大豆根腐病菌的抑制效果明显。
2.采用形态学和分子生物学方法鉴定6株木霉菌的种类。通过对扩增出木霉菌的ITS序列测序,得到6个不同的ITS序列,通过ITSH网站TrichOKEY和TrichBLAST录入,进行序列的比对分析,同时结合木霉菌形态学鉴定,确定木霉菌T20A、T20G、T38D、T61F和T78E为Trichoderma koningiopsis种的不同菌株;木霉菌T115D为Trichoderma longibrachiaum。
3.采用正交试验对木霉菌T38D、T78E和T115D的液体和固体发酵条件进行优化,确定了利于木霉菌快速大量产孢的培养方法、培养基质和条件,制备出木霉菌颗粒剂。
4.木霉菌颗粒剂田间防治大豆根腐病试验结果表明,木霉菌剂均可降低根腐病的发生,混合木霉菌颗粒剂对大豆根腐病防效好于单一木霉菌剂,防效达42.79%,小于化学防治(55.63%),混合木霉菌颗粒剂与1/2药量的化学药剂共同使用的防治效果为48.18%。不同时期大豆的形态指标调查结果和测产结果表明,木霉菌颗粒剂对大豆有促生作用,可提高大豆产量。
Trichoderma spp. is considered one of the most promising biological control factors, and has well inhibition to pathogens of soybean root rot. To make early for the production of Trichoderma spp. by further developping and utilizing, six strains of Trichoderma spp. were indentified and studied to the pathogens of soybean root rot in the study. Three strains which were selected from six were initially used for exploring the development of preparation. As the same time, the field efficiency of Trichoderma preparation to soybean root rot were also studied in the paper. The experimental results were as follows:
1. The inhibition of Trichoderma spp. to pathogens of soybean root rot were studied by the space competition, parasitism and antibiosis. The results showed that Trichoderma spp. can inhibite the growth of pathogens of soybean root rot through confronting each other, cultivating face to face, cultivating in cellophane film. Confront culture results showed that Trichoderma had stronger advantages of spatial competion to the pathogens of soybean root rot. The microscopic observation showed that six strains of Trichoderma spp. had the significant parasitic effect against Rhizoctonia solani, Pythium ultimum, Fusarium oxysporum and Fusarium avenaceum. Trichoderma spp. was parasitized on the mycelia by the way of spiral wound, adhering, as while, surrounding the mycelia through producing small hyphae, which could finally cause the disintegration and death of pathogens. The hard volatile metabolite in the metabolites of Trichoderma spp was an important antibacterial substance which had the significant antibacterial effect on the pathogens of soybean root rot.
2. Six strains of Trichoderma spp were identified by the methods of molecular biology and morphological. Six different ITS sequences have been got by PCR and sequencing. Through inputting TrichOKEY and TrichBLAST in the ITSH website, the comparative analysis of sequence and combination with morphological identification of Trichoderma spp, T20A, T20G, T38D, T61F, and T78E have been determined to be different strains of Trichoderma koningiopsis s and T115D to be Trichoderma longibrachiaum.
3. Liquid and Solid Fermentation conditions of Trichoderma T38D, T78E, and T115D have been selected and optimized by using orthogonal test. The favorable training matrix and conditions that Trichoderma spp quickly produced large number of spores have been determined. Trichoderma granules had been prepared.
4. The field pathogens control results of Trichoderma preparation to the soybean root rot showed that the occurrence of soybean root rot can be reduced by Trichoderma preparation. And the effect of mix Trichoderma preparation was better than single Trichoderma preparation; the controlling effect was 42.79%, lower than the Chemica Control (55.63%). The control effect was 48.18% by used mix Trichoderma preparation mixed with 1/2 dose of the chemicals. In different periods, the results of soybean morphological index survey and the yield estimation showed that the growth of soybean could be promoted by Trichoderma preparation, and its yield also improved.
引文
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