深绿木霉的拮抗机理、促生作用及制剂加工研究
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摘要
本文利用已筛选的具有拮抗作用的深绿木霉菌(Trichoderma aureoviride)作为试材,进行深绿木霉对百合疫霉菌(Phytophthora nicotianae van Brede de Haan)抑制机理、深绿木霉制剂的制备及其对不同植物促生作用等方面的研究。
通过对峙培养、菌丝降解、抗生物质测定、对扣培养、圆盘滤膜法、酶活性测定等一系列方法,研究深绿木霉对百合疫霉菌的多种拮抗机理,确定其具有竞争营养与重寄生作用,并发现有抗生物质产生。两者在对峙培养60h时,生长速率比达到3.68,且在其发酵液中测定出较高的β-1,3-葡聚糖酶和几丁质酶的活性。另外,还观察到深绿木霉发酵液对百合疫霉菌丝有降解作用,进一步证实了重寄生作用的存在。
在探明拮抗机理的基础上,探讨液固两相发酵的培养成份和条件,确定最佳培养基和培养条件,获得深绿木霉制剂。先将1.0%(V/V)深绿木霉接种于液体麸皮培养基,置于全光照,25℃,pH5.0,摇瓶转速150r/min的条件下进行5d二级扩大培养,得到1.91×1010个/ml的孢子量。再以30%(V/V)的孢子量接种于含水量1:0.8的麦麸-泥土(1:1)固料中,进行三级固体浅盘发酵。采用前期20℃,中期24℃,后期28℃的变温处理,固料厚度1.0cm,每12h搅拌一次,发酵10d,获得孢子浓度为3.41×1010个/g的深绿木霉制剂。试验还确定深绿木霉制剂的干燥温度在30～35℃范围内,干燥48h为宜(活孢率90%以上),且最佳贮存组合为4℃-牛皮纸包装,测定其六个月后的孢子萌发率为32.8%。
研究还发现深绿木霉制剂对小麦、苜蓿、黄瓜的发芽和生长均有促进作用。浸种试验表明,深绿木霉剂200X稀释液均能促进小麦、苜蓿发芽,小麦尤其明显(发芽指数和活力指数分别为11.8和0.437)。但其高浓度(10X、20X、50X)却抑制发芽。而盆栽试验显示,对小麦、苜蓿的出苗率,浸种法促进效果比施入法明显。小麦最佳浸种倍数为500,出苗率83%;苜蓿最佳浸种倍数为200,出苗率达87.50%。此外,适当的施入组合(施入量+施入深度)更利于小麦、苜蓿的生长,其苗干重、相对生长率及植株干物质积累等指标均高于对照,促生作用明显。对黄瓜,则喷洒法促生效果优于施入法,最佳喷洒浓度为106个/ml。
The antagonistic mechanism of Trichoderma aureoviride to the pathogen causing Lily blights (Phytophthora nicotianae van Brede de Haan), promotion of growth to various plants and the preparation of T. aureoviride were studied in this paper.
The results showed that the biocontrol mechanism of T. aureoviride restraining P. nicotianae was mycoparasitism and competition of nutrition,and antibiotic substances were detected by a series of investigation means,such as confronting each other,degrading hypha,mensurating antibiosis substances,cultivating face to face, cultivating in sieve film of disc, mensurating activities of enzyme.The ratio of growth speed was 3.68 in confronting each other after 60h,and the activities of chitinase andβ-1,3-glucanase were detected in the ferment filtrate. In addition, the pathogenic hyphae could be degraded by T. aureoviride filtrate, which showed existence of parasitism.
The medium and culture conditions were tested in experiment.In second-grade liquid culture, the optimum medium was wheat bran decoction.The optimum conditions were the inoculum 1.0%, continuous light, 25℃,pH5.0, and rotate speed 150r/min.The harvest period was five days.The conidial concentration reached 1.91×1010 spores/ml. In third-grade solid culture in plate, the optimum medium was wheat bran-clay(1:1).The optimum conditions were the proportion between water and medium 1:0.8, the inoculum 30%, the variational temperature(prophase 20℃,metaphase 24℃a nd anaphase 28℃),the thickness of medium 1.0cm and agitating medium every 12h. The fermentation period was ten days.The conidial concentration reached 3.41×1010spores/g.To T. aureoviride preparation ,the optimum temperature and time of desiccation were 30～35℃,48h respectively.Under these conditions the rate of alive spore reached 90%.And the preparation was packed with kraft at 4℃.The rate of bud was 32.8% after six months.
The bud and growth of wheat, alfalfa and cucumber was promoted by T. aureoviride preparation. In soaking seed experiment, the growth of wheat and alfalfa was promoted by 200X preparation,especially wheat (index of bud and energy were 11.8 and 0.437).But high concentration(10X、20X、50X) restrained the growth of plants.In soil experiment, it was helpful to soak seed to germination of wheat(500X) and alfalfa
通过对峙培养、菌丝降解、抗生物质测定、对扣培养、圆盘滤膜法、酶活性测定等一系列方法,研究深绿木霉对百合疫霉菌的多种拮抗机理,确定其具有竞争营养与重寄生作用,并发现有抗生物质产生。两者在对峙培养60h时,生长速率比达到3.68,且在其发酵液中测定出较高的β-1,3-葡聚糖酶和几丁质酶的活性。另外,还观察到深绿木霉发酵液对百合疫霉菌丝有降解作用,进一步证实了重寄生作用的存在。
在探明拮抗机理的基础上,探讨液固两相发酵的培养成份和条件,确定最佳培养基和培养条件,获得深绿木霉制剂。先将1.0%(V/V)深绿木霉接种于液体麸皮培养基,置于全光照,25℃,pH5.0,摇瓶转速150r/min的条件下进行5d二级扩大培养,得到1.91×1010个/ml的孢子量。再以30%(V/V)的孢子量接种于含水量1:0.8的麦麸-泥土(1:1)固料中,进行三级固体浅盘发酵。采用前期20℃,中期24℃,后期28℃的变温处理,固料厚度1.0cm,每12h搅拌一次,发酵10d,获得孢子浓度为3.41×1010个/g的深绿木霉制剂。试验还确定深绿木霉制剂的干燥温度在30～35℃范围内,干燥48h为宜(活孢率90%以上),且最佳贮存组合为4℃-牛皮纸包装,测定其六个月后的孢子萌发率为32.8%。
研究还发现深绿木霉制剂对小麦、苜蓿、黄瓜的发芽和生长均有促进作用。浸种试验表明,深绿木霉剂200X稀释液均能促进小麦、苜蓿发芽,小麦尤其明显(发芽指数和活力指数分别为11.8和0.437)。但其高浓度(10X、20X、50X)却抑制发芽。而盆栽试验显示,对小麦、苜蓿的出苗率,浸种法促进效果比施入法明显。小麦最佳浸种倍数为500,出苗率83%;苜蓿最佳浸种倍数为200,出苗率达87.50%。此外,适当的施入组合(施入量+施入深度)更利于小麦、苜蓿的生长,其苗干重、相对生长率及植株干物质积累等指标均高于对照,促生作用明显。对黄瓜,则喷洒法促生效果优于施入法,最佳喷洒浓度为106个/ml。
The antagonistic mechanism of Trichoderma aureoviride to the pathogen causing Lily blights (Phytophthora nicotianae van Brede de Haan), promotion of growth to various plants and the preparation of T. aureoviride were studied in this paper.
The results showed that the biocontrol mechanism of T. aureoviride restraining P. nicotianae was mycoparasitism and competition of nutrition,and antibiotic substances were detected by a series of investigation means,such as confronting each other,degrading hypha,mensurating antibiosis substances,cultivating face to face, cultivating in sieve film of disc, mensurating activities of enzyme.The ratio of growth speed was 3.68 in confronting each other after 60h,and the activities of chitinase andβ-1,3-glucanase were detected in the ferment filtrate. In addition, the pathogenic hyphae could be degraded by T. aureoviride filtrate, which showed existence of parasitism.
The medium and culture conditions were tested in experiment.In second-grade liquid culture, the optimum medium was wheat bran decoction.The optimum conditions were the inoculum 1.0%, continuous light, 25℃,pH5.0, and rotate speed 150r/min.The harvest period was five days.The conidial concentration reached 1.91×1010 spores/ml. In third-grade solid culture in plate, the optimum medium was wheat bran-clay(1:1).The optimum conditions were the proportion between water and medium 1:0.8, the inoculum 30%, the variational temperature(prophase 20℃,metaphase 24℃a nd anaphase 28℃),the thickness of medium 1.0cm and agitating medium every 12h. The fermentation period was ten days.The conidial concentration reached 3.41×1010spores/g.To T. aureoviride preparation ,the optimum temperature and time of desiccation were 30～35℃,48h respectively.Under these conditions the rate of alive spore reached 90%.And the preparation was packed with kraft at 4℃.The rate of bud was 32.8% after six months.
The bud and growth of wheat, alfalfa and cucumber was promoted by T. aureoviride preparation. In soaking seed experiment, the growth of wheat and alfalfa was promoted by 200X preparation,especially wheat (index of bud and energy were 11.8 and 0.437).But high concentration(10X、20X、50X) restrained the growth of plants.In soil experiment, it was helpful to soak seed to germination of wheat(500X) and alfalfa
引文
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