防治根结线虫的原核生防菌的筛选及作用
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摘要
根结线虫（Meloidogyne spp.）是一种主要的植物寄生性线虫，每年给农业造成严重的损失。根结线虫的原核生防菌（细菌及放线菌），具有多重防治根结线虫机制，防治效果高，周期长，对环境、人畜及益虫安全，适合于绿色环保型农业发展的需要。根际细菌及放线菌已经成为防治根结线虫的研究热点。
在研究中，首先进行供试线虫培养，在室内用灰葡萄孢菌（Borytis cinerea）培养松材线虫 (Bursaphelenchus xylophilus )，在28℃下恒温培养5d线虫数量可以达到1400条/cm2。在清水中松材线虫可以存活7～12 d（1条／ul），且12 h内线虫的自然死亡率小于5％。采用液滴法，以Abamectin(1.8%AVM. B1)的1000倍稀释液的处理供试线虫，确定以处理30～45 min线虫的击倒率达到57.7%～76.9％为室内的筛选指标。在温室中，采用盆栽法确定418＃菌株为温室筛选对照。
从全国采集32个土壤样品，分别为植物寄生性线虫病害土壤和自然土壤,用等比稀释平板法，以高氏1号培养基和牛肉汁蛋白胨培养基分离出放线菌960株，细菌346株。通过在室内以松材线虫为供试对象，采用液滴法进行初步筛选试验，温室中以根结线虫为供试对象，采用盆栽方法进行复选，共筛选出六个菌株，它们可以有效的杀死线虫，明显的减少根结数量。B24-14菌株、G21-31菌株培养液在45min 时对线虫的击倒率分别为95.4％、84.5％，在温室试验中分别使根结减少75.0％、67.3％。B24-14菌株、G21-31菌株在防治根结线虫,促进植物生长方面效果显著优于418＃菌株及abamectin(1.8%AVM. B1)。
通过经典的分类方法、化学分类方法和16SrDNA方法相结合，对筛选出优秀的细菌和放线菌菌株进行分类鉴定。经鉴定菌株G10-16为链霉菌属弗氏亚群亚群（Streptomyces Fradiae）；菌株G21-31为链霉菌属蓝色亚群（Streptomyces Gyaneus）；菌株G21-33为链霉菌属黄色亚群（Streptomyces Flavus）；菌株B22-3、菌株B24-14为芽孢杆菌属苏云金杆菌（Bacillus thuringiensis）。
对B24-14菌株的防治作用机制研究中，采用乙醇分级沉淀法并结合紫外分光检测，从B24-14菌株的培养液中分离出β－外毒素，并通过液滴法及细胞培养板法试验，证明β－外毒素（4mg/ml）在8h时线虫的杀死率可以达到93.75％。采用离心法制得伴孢晶体，并用蛋白晶体碱解液将其降解为可溶性的小分子蛋白，经试验伴孢晶体蛋白（1.6mg/ml）在16h时对线虫的杀死率为66.4%。采用AgNO3检测方法,证明B24-14菌株在培养过程中可以产生氨和其水溶性前体，且在室内有很高的杀线虫活性，可以在45min内100％的杀死线虫。通过室内的液体栽培方法,证明了B24-14菌株可以产生影响植物生长的物质，这种物质可以在高浓度下明显抑制植株生长，在低浓度下显著的促进植株的生长。
The root knot nematode (Meloidogynidae spp.) is an important kind of plant parasitic nematodes, which harm agriculture badly every year. The prokaryotic Organisms (bacteria and actinomycete) have multiply mechanisms to control the root-knot nematodes, the effects are significant, the period long. In addition, the biocontrol agents are safety to human, mammal and beneficial insects, and adapt to the development of green agriculture. Nowadays, the research of bacteria and actinomycete control the root knot nematodes have became a hot field.
In laboratory, we get a large number of target nematodes (Bursaphelenchus xylophilus), which are cultured with the Borytis cinerea. When the nematodes are cultured 5 days in 28℃, the density is about 1400 individual/cm2. In water, the nematodes can live 7-12 days (the density is about 1 individual /ul), their mortalities are below 5% in 12 hours. With the method of drops, treat the nematodes with Abamectin 30-45 minutes, the knockdown rate of nematodes is between 57.7% and 76.9%. In green house, through the examination in the pots, the 418# strain was selected as CK.
We collected 32 samples of the plant parasitic nematodes disease soil and natural soil from china diluted solution method, 346 strains of bacteria and 960 strains of actinomycete were isolated from all soil samples. Through the bioassay in laboratory and greenhouse, six strains were selected, which can kill the nematodes and decrease the root knots significantly. Among them, the strain B24-14 and the strain G21-31 are the excellent. In laboratory, their knockdown rates to the nematodes are 95.4%, 84.5%. In greenhouse, the effects of control nematodes are 75.0% and 67.6%. They can promote plant growth .The strains B24-14 and G21-31 are better than the 418# strain and Abamectin.
Combined the classical, chemical and 16SrDNA sequence methods, the five selected strains were identified. The strain G10-16 is Streptomyces Fradiae）；the strain G21-31 is Streptomyces Gyaneus；the strain G21-33 is Streptomyces Flavus；Both of the strains B22-3 and B24-14 are Bacillus thuringiensis.
With the methods of the ethanol sediment and ultraviolet ray scan, the β-exotoxin was extracted and identified from the fermentation broth of the
strain B24-14, it can kill the nematodes 93.6% at 8 hours. The resolvable protein was get from the crystal protein solution, the solution (1.6mg/ml) can kill the nematodes 66.4% at 16 hours. Through the test of AgNO3, the B24-14strain can produce the NH3 and resolvable raw, which show the strong activity to the nematodes in laboratory and greenhouse, and can kill the nematodes 100% in 30-45 min. In addition, with the method of solution culture, showed that the B24-14 strain produce some substance, which promote plant growth in diluted solution and restrain in concentrated solution.
在研究中，首先进行供试线虫培养，在室内用灰葡萄孢菌（Borytis cinerea）培养松材线虫 (Bursaphelenchus xylophilus )，在28℃下恒温培养5d线虫数量可以达到1400条/cm2。在清水中松材线虫可以存活7～12 d（1条／ul），且12 h内线虫的自然死亡率小于5％。采用液滴法，以Abamectin(1.8%AVM. B1)的1000倍稀释液的处理供试线虫，确定以处理30～45 min线虫的击倒率达到57.7%～76.9％为室内的筛选指标。在温室中，采用盆栽法确定418＃菌株为温室筛选对照。
从全国采集32个土壤样品，分别为植物寄生性线虫病害土壤和自然土壤,用等比稀释平板法，以高氏1号培养基和牛肉汁蛋白胨培养基分离出放线菌960株，细菌346株。通过在室内以松材线虫为供试对象，采用液滴法进行初步筛选试验，温室中以根结线虫为供试对象，采用盆栽方法进行复选，共筛选出六个菌株，它们可以有效的杀死线虫，明显的减少根结数量。B24-14菌株、G21-31菌株培养液在45min 时对线虫的击倒率分别为95.4％、84.5％，在温室试验中分别使根结减少75.0％、67.3％。B24-14菌株、G21-31菌株在防治根结线虫,促进植物生长方面效果显著优于418＃菌株及abamectin(1.8%AVM. B1)。
通过经典的分类方法、化学分类方法和16SrDNA方法相结合，对筛选出优秀的细菌和放线菌菌株进行分类鉴定。经鉴定菌株G10-16为链霉菌属弗氏亚群亚群（Streptomyces Fradiae）；菌株G21-31为链霉菌属蓝色亚群（Streptomyces Gyaneus）；菌株G21-33为链霉菌属黄色亚群（Streptomyces Flavus）；菌株B22-3、菌株B24-14为芽孢杆菌属苏云金杆菌（Bacillus thuringiensis）。
对B24-14菌株的防治作用机制研究中，采用乙醇分级沉淀法并结合紫外分光检测，从B24-14菌株的培养液中分离出β－外毒素，并通过液滴法及细胞培养板法试验，证明β－外毒素（4mg/ml）在8h时线虫的杀死率可以达到93.75％。采用离心法制得伴孢晶体，并用蛋白晶体碱解液将其降解为可溶性的小分子蛋白，经试验伴孢晶体蛋白（1.6mg/ml）在16h时对线虫的杀死率为66.4%。采用AgNO3检测方法,证明B24-14菌株在培养过程中可以产生氨和其水溶性前体，且在室内有很高的杀线虫活性，可以在45min内100％的杀死线虫。通过室内的液体栽培方法,证明了B24-14菌株可以产生影响植物生长的物质，这种物质可以在高浓度下明显抑制植株生长，在低浓度下显著的促进植株的生长。
The root knot nematode (Meloidogynidae spp.) is an important kind of plant parasitic nematodes, which harm agriculture badly every year. The prokaryotic Organisms (bacteria and actinomycete) have multiply mechanisms to control the root-knot nematodes, the effects are significant, the period long. In addition, the biocontrol agents are safety to human, mammal and beneficial insects, and adapt to the development of green agriculture. Nowadays, the research of bacteria and actinomycete control the root knot nematodes have became a hot field.
In laboratory, we get a large number of target nematodes (Bursaphelenchus xylophilus), which are cultured with the Borytis cinerea. When the nematodes are cultured 5 days in 28℃, the density is about 1400 individual/cm2. In water, the nematodes can live 7-12 days (the density is about 1 individual /ul), their mortalities are below 5% in 12 hours. With the method of drops, treat the nematodes with Abamectin 30-45 minutes, the knockdown rate of nematodes is between 57.7% and 76.9%. In green house, through the examination in the pots, the 418# strain was selected as CK.
We collected 32 samples of the plant parasitic nematodes disease soil and natural soil from china diluted solution method, 346 strains of bacteria and 960 strains of actinomycete were isolated from all soil samples. Through the bioassay in laboratory and greenhouse, six strains were selected, which can kill the nematodes and decrease the root knots significantly. Among them, the strain B24-14 and the strain G21-31 are the excellent. In laboratory, their knockdown rates to the nematodes are 95.4%, 84.5%. In greenhouse, the effects of control nematodes are 75.0% and 67.6%. They can promote plant growth .The strains B24-14 and G21-31 are better than the 418# strain and Abamectin.
Combined the classical, chemical and 16SrDNA sequence methods, the five selected strains were identified. The strain G10-16 is Streptomyces Fradiae）；the strain G21-31 is Streptomyces Gyaneus；the strain G21-33 is Streptomyces Flavus；Both of the strains B22-3 and B24-14 are Bacillus thuringiensis.
With the methods of the ethanol sediment and ultraviolet ray scan, the β-exotoxin was extracted and identified from the fermentation broth of the
strain B24-14, it can kill the nematodes 93.6% at 8 hours. The resolvable protein was get from the crystal protein solution, the solution (1.6mg/ml) can kill the nematodes 66.4% at 16 hours. Through the test of AgNO3, the B24-14strain can produce the NH3 and resolvable raw, which show the strong activity to the nematodes in laboratory and greenhouse, and can kill the nematodes 100% in 30-45 min. In addition, with the method of solution culture, showed that the B24-14 strain produce some substance, which promote plant growth in diluted solution and restrain in concentrated solution.
引文
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