广谱高效生物农药木霉菌的诱变及其拮抗和生防作用的研究
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摘要
木霉(Trichoderma)常分离于根际、土壤、叶围等环境，生存范围广、适应性强、对植物病原菌具有广谱的拮抗作用，而且还能产生多种酶类分解土壤中的动植物残体，产生使植物能够吸收利用的有机物。
本研究从山东省多种生态区取得不同土样、利用微生物分离方法，从852份采集样品中，分离到野生木霉菌株255株，利用常规形态鉴定法和凝胶电泳分类的聚类分析鉴定，隶属木霉属的8个种，它们分别是绿色木霉(T. viride)、哈茨木霉(T. harziamum)、钩状木霉(T. hamatum)、黄绿木霉(T. aureoviride)、桔绿木霉(T. citrinoviride)、长枝木霉(T. longibrachiarum)、康氏木霉(T. koningii)、拟康氏木霉(T. pseudokoning)。
进一步与植物土传病原真菌进行拮抗作用实验，选出了拮抗作用较好的菌株T32，分析了木霉的拮抗作用机理。对部分植物病菌拮抗作用的方式主要有重寄生作用、营养竞争作用和空间位点竞争作用等几种。其中对立枯丝核菌(Rhizoctonia solani)(小麦纹枯病)的作用是重寄生作用：而对大丽轮枝菌(Verticillium dahliae)(棉花黄萎病)、棉蔫萎镰孢霉(Fusarium vasinfectum)(棉花枯萎病)、玉米黑粉菌(Ustilago maydis)(玉米黑粉病)主要是营养竞争作用：对瓜蔫萎座镰孢霉(F. bulbigenum)(西瓜枯萎病)、茄腐皮镰孢霉(F. solani)(大豆根腐病)等是空间位点竞争作用。
利用核辐射等诱变技术，~(60)Co-γ射线最佳辐射剂量为0.8kGy-1kGy，紫外线照射为5-7分钟，结合突变菌株对部分杀菌剂化学农药耐药性及对6种植物土传病菌的拮抗作用大小、生长速度快慢、产孢量多少等指标，选出高拮抗性、高产孢量、生长速度快的突变菌株T1010。
对突变菌株T1010进行发酵条件进行实验，结果表明其生长所需碳氮比为24：1，葡萄糖为最佳碳源，甘氨酸为最佳氮源，磷、钾、硫是生长必须元素。培养菌丝最适温度为30℃，pH值6，高温培养3天后转入低温20℃培养产孢。大量培养时麦麸作培养基既方便又省钱，大量生产木霉制剂0.5吨，制剂木霉孢子含量达
Trichoderma spp. are fungi that are highly interactive in root, soil and foliar environments. They are frequently dominant components of the microflora in widely varying habitats. They can biocontrol against a wide range of plant-pathogen. Furthermore, they are potent producers of a variety of plant polysaccharide degrading enzymes and capable of degrading a variety of xenobiotic materials. Trichoderma frequently enhances plant growth, the uptake and use of these nutrients.
Out of 255 isolates of Trichoderma from 852 soil samples in Shandong Province, eight Trichoderma species were identified by conidiophore morphology and cluster analysis for soluble protein. With optical microscope and polyacrylamide gel eletrophoresis, they are Trichoderma harzianum, Trichoderma citrinoviride, Trichoderma longibrachiatum, Trichoderma viride, Trichoderma hametum, Trichoderma aureoviride, Trichoderma konningii and Trichoderma pseudokong.
T32 was efficient for controlling some soil-borne plant-pathogenic fungi than others. The main mechanisms are as follows: mycoparasition, competition for nutrients and space. In dual cultures with 6 soil-borne plant-pathogenic fungi, T32 hyphae attached to the host Rhizoctonia solani and coiling around the pathogen hyphae and parasitied into pathogenic hyphae. The phenomenon of competition for nutrients was illustrated in dual cultures with Verticillium dabliae, Fusarium var vasinfectum, Ustilago maydic. T32 competed space against Fusarium bulbigenum, Fusarium solani in dual cultures.
Mutations were conducted under certain condition, UV light and ~(60)Co-γ rays. T32 was treated with an appropriate ~(60)Co- γ radiation dose (0.8 kGy-1 kGy) and UV radiation time was 5-7 minites. Mutants tolerant of chemical fungicides were selected. The stable mutants T1010 with high production of spores and high growth rate were selected against soil-borne plant-pathogenic fungi in dual cultures in comparison with
本研究从山东省多种生态区取得不同土样、利用微生物分离方法，从852份采集样品中，分离到野生木霉菌株255株，利用常规形态鉴定法和凝胶电泳分类的聚类分析鉴定，隶属木霉属的8个种，它们分别是绿色木霉(T. viride)、哈茨木霉(T. harziamum)、钩状木霉(T. hamatum)、黄绿木霉(T. aureoviride)、桔绿木霉(T. citrinoviride)、长枝木霉(T. longibrachiarum)、康氏木霉(T. koningii)、拟康氏木霉(T. pseudokoning)。
进一步与植物土传病原真菌进行拮抗作用实验，选出了拮抗作用较好的菌株T32，分析了木霉的拮抗作用机理。对部分植物病菌拮抗作用的方式主要有重寄生作用、营养竞争作用和空间位点竞争作用等几种。其中对立枯丝核菌(Rhizoctonia solani)(小麦纹枯病)的作用是重寄生作用：而对大丽轮枝菌(Verticillium dahliae)(棉花黄萎病)、棉蔫萎镰孢霉(Fusarium vasinfectum)(棉花枯萎病)、玉米黑粉菌(Ustilago maydis)(玉米黑粉病)主要是营养竞争作用：对瓜蔫萎座镰孢霉(F. bulbigenum)(西瓜枯萎病)、茄腐皮镰孢霉(F. solani)(大豆根腐病)等是空间位点竞争作用。
利用核辐射等诱变技术，~(60)Co-γ射线最佳辐射剂量为0.8kGy-1kGy，紫外线照射为5-7分钟，结合突变菌株对部分杀菌剂化学农药耐药性及对6种植物土传病菌的拮抗作用大小、生长速度快慢、产孢量多少等指标，选出高拮抗性、高产孢量、生长速度快的突变菌株T1010。
对突变菌株T1010进行发酵条件进行实验，结果表明其生长所需碳氮比为24：1，葡萄糖为最佳碳源，甘氨酸为最佳氮源，磷、钾、硫是生长必须元素。培养菌丝最适温度为30℃，pH值6，高温培养3天后转入低温20℃培养产孢。大量培养时麦麸作培养基既方便又省钱，大量生产木霉制剂0.5吨，制剂木霉孢子含量达
Trichoderma spp. are fungi that are highly interactive in root, soil and foliar environments. They are frequently dominant components of the microflora in widely varying habitats. They can biocontrol against a wide range of plant-pathogen. Furthermore, they are potent producers of a variety of plant polysaccharide degrading enzymes and capable of degrading a variety of xenobiotic materials. Trichoderma frequently enhances plant growth, the uptake and use of these nutrients.
Out of 255 isolates of Trichoderma from 852 soil samples in Shandong Province, eight Trichoderma species were identified by conidiophore morphology and cluster analysis for soluble protein. With optical microscope and polyacrylamide gel eletrophoresis, they are Trichoderma harzianum, Trichoderma citrinoviride, Trichoderma longibrachiatum, Trichoderma viride, Trichoderma hametum, Trichoderma aureoviride, Trichoderma konningii and Trichoderma pseudokong.
T32 was efficient for controlling some soil-borne plant-pathogenic fungi than others. The main mechanisms are as follows: mycoparasition, competition for nutrients and space. In dual cultures with 6 soil-borne plant-pathogenic fungi, T32 hyphae attached to the host Rhizoctonia solani and coiling around the pathogen hyphae and parasitied into pathogenic hyphae. The phenomenon of competition for nutrients was illustrated in dual cultures with Verticillium dabliae, Fusarium var vasinfectum, Ustilago maydic. T32 competed space against Fusarium bulbigenum, Fusarium solani in dual cultures.
Mutations were conducted under certain condition, UV light and ~(60)Co-γ rays. T32 was treated with an appropriate ~(60)Co- γ radiation dose (0.8 kGy-1 kGy) and UV radiation time was 5-7 minites. Mutants tolerant of chemical fungicides were selected. The stable mutants T1010 with high production of spores and high growth rate were selected against soil-borne plant-pathogenic fungi in dual cultures in comparison with
引文
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