松苗猝倒病生防细菌活性物质及抑病效果研究
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摘要
本文对从湖北不同苗圃松苗根际土壤中分离得到的生防菌株进行了筛选鉴定,并测定了对松苗猝倒病主要病原菌的室内抑菌效果;在此基础上,进一步与已知生防菌株C23和美国农业部根病生物防治实验室(USDA-ARS,RootDisease Biological Control Unit)提供的优良生防细菌菌株进行了室内和田间防病效果比较试验;并对生防菌株C23抗菌活性物质进行了检测和定性分析。主要结果如下:
1.从湖北省不同林区松苗根际筛选获得了21株具有生防效果的细菌菌株。通过对松苗猝倒病菌拮抗效果比较测定,进一步筛选得到12株具有较强拮抗作用的生防菌株。根据其培养特性和相关生物学性状,鉴定出其中3株(L-2,L-5,L-8)为芽孢杆菌。同时对生防菌株C23测序鉴定,结果显示该菌株为洋葱伯克霍尔德菌(Burkholderia cepacia)。
2.供试9种生防菌对松苗猝倒病主要病原菌室内抑菌效果表明,不同生防菌株对不同病原菌抑制指数有所差异。对丝核菌A,MVP1-4与C23的抑菌效果较好,T=0.7时,抑制指数分别为35.11,33.24;T=1时,抑制指数为22.23,21.67。对于丝核菌B,T=0.7时Q37-87拮抗效果较好,抑制指数为34.59;T=1时,C23拮抗效果较好,抑制指数为21.16。对于镰刀菌,T=0.7时,FFL1R8的拮抗效果最好,抑制指数为39.06;T=1时,Q2-87抑制指数为21.21,拮抗效果最好。对于链格孢菌,T=0.7时拮抗效果最好的为Pf-5,抑制指数为30.50。T=1时,拮抗效果较好的为1M1-96,抑制指数为17.12.
3.室内和田间防病效果比较试验显示,室内盆栽湿地松苗36d后调查,采用浸根方法施用5种供试菌1M1-96,HT5-1,Q37-87,FFL1R8,C23表现出较好的防病效果,存活率平均为44.06%,而对照为28.5%。使用灌根处理的供试菌的防病效果没有很大差异,只有C23和1M1-96的防病效果高于对照。田间试验,供试生防菌株对猝倒病均有明显防效。
4.研究摸清了C23菌株发酵粗提液最适培养基,其粗提液中抗菌物质基本特性表现为对热稳定,具有一定的耐酸、碱能力,能够被蛋白酶K酶解,采用80%饱和度的硫酸铵盐析,沉淀物具有抑菌活性,表现出蛋白质的某些特性,据此认为该抗菌物质可能是蛋白质或肽类物质。
本研究初步证实了相关生防菌对松苗猝倒病主要病原菌的抑菌作用和室内、田间防病效果,并明确了生防菌株C23发酵粗提液抗菌活性物质的相关特性,为防治松苗猝倒病生防产品的开发应用奠定了基础。
Slash pine(Pinus elliottii) is an important tree species for foresting in the south China.On the base that the slash pine disease is caused by Rhizoctonia sp.,Fusarium oxysporum and Alternaria sp..We measuring the inhibiting disease efficacy of C23 and the bacteria,including Q2-87,Ps2-79,Pf-5,1M1-96,Q37-87, HT5-1,MVP1-4 and FFL1R8 afforded by the lab of roots diseases biocontrol department of agriculture USA in the greenhouse and the field.Screening the new bacteria which have antagonistic effects on the pathogens of slash pine damping-off.Studying the characterization of an antifungal substance produced by C23.The research demonstrate that:
1.21 isolates of the bioncontrol bacteria are gained by new screening,we decide the number W1-13、L1-8、Q1-8、B1-4、J1-4 as the names.there are 12 strong isolates of the biocontrol ones.We have base biology assay,maybe having 3 isolates (L-2,L-5,L-8) as Bacillus subtilis.
2.The inhibiting disease efficacy of nine biocontrol bacteria demonstrate that:for Rhizoctonia sp.,MVP1-4 and C231M1-96 show very good antagonistic ability on the plate against the pathogens;for Fusarium oxysporum,FFL1R8 and Q2-87 show good antagonistic ability;for Alternaria sp.Pf-5 shows good antagonistic ability.
3.1M1-96,HT5-1,Q37-87,FFL1R8,C23 show very good antagonistic ability in the greenhouse test by the method of dipping the roots with bacteria liquid.1M1-96 and C23 show good antagonistic ability beyond CK by the method of watering the roots with bacteria liquid.All of the strains show very good antagonistic ability in the field test.
4.Media screening experiment disclosed that the optimum media for C23 producing the antifungal substance is 1/2PDA~+.Treated at 20-40℃for 30 min,the crude extract maintained more than 80%of its antifungal activity.Treated at about pH6,the crude extract have the best antifungal activity.Treatment with proteinase K,there has no antifungal activity of the crude extract.The antifungal substance can precipitate full with 80%(NH_4)SO_4.The substance can not be extracted by the nonpolarity solvent(methenyl trichloride,ethyl acetate,ethyl oxide).These demonstrate that the antifungal substance must be protein or peptide.
The research show diseases biocontrol in the greenhouse and the field,screens new bioncontrol strains,Studying the characterization of an antifungal substance of C23.This will provide the theoretical reference for further utilizing the bioncontrol bacteria to control the slash pine damping-off.
1.从湖北省不同林区松苗根际筛选获得了21株具有生防效果的细菌菌株。通过对松苗猝倒病菌拮抗效果比较测定,进一步筛选得到12株具有较强拮抗作用的生防菌株。根据其培养特性和相关生物学性状,鉴定出其中3株(L-2,L-5,L-8)为芽孢杆菌。同时对生防菌株C23测序鉴定,结果显示该菌株为洋葱伯克霍尔德菌(Burkholderia cepacia)。
2.供试9种生防菌对松苗猝倒病主要病原菌室内抑菌效果表明,不同生防菌株对不同病原菌抑制指数有所差异。对丝核菌A,MVP1-4与C23的抑菌效果较好,T=0.7时,抑制指数分别为35.11,33.24;T=1时,抑制指数为22.23,21.67。对于丝核菌B,T=0.7时Q37-87拮抗效果较好,抑制指数为34.59;T=1时,C23拮抗效果较好,抑制指数为21.16。对于镰刀菌,T=0.7时,FFL1R8的拮抗效果最好,抑制指数为39.06;T=1时,Q2-87抑制指数为21.21,拮抗效果最好。对于链格孢菌,T=0.7时拮抗效果最好的为Pf-5,抑制指数为30.50。T=1时,拮抗效果较好的为1M1-96,抑制指数为17.12.
3.室内和田间防病效果比较试验显示,室内盆栽湿地松苗36d后调查,采用浸根方法施用5种供试菌1M1-96,HT5-1,Q37-87,FFL1R8,C23表现出较好的防病效果,存活率平均为44.06%,而对照为28.5%。使用灌根处理的供试菌的防病效果没有很大差异,只有C23和1M1-96的防病效果高于对照。田间试验,供试生防菌株对猝倒病均有明显防效。
4.研究摸清了C23菌株发酵粗提液最适培养基,其粗提液中抗菌物质基本特性表现为对热稳定,具有一定的耐酸、碱能力,能够被蛋白酶K酶解,采用80%饱和度的硫酸铵盐析,沉淀物具有抑菌活性,表现出蛋白质的某些特性,据此认为该抗菌物质可能是蛋白质或肽类物质。
本研究初步证实了相关生防菌对松苗猝倒病主要病原菌的抑菌作用和室内、田间防病效果,并明确了生防菌株C23发酵粗提液抗菌活性物质的相关特性,为防治松苗猝倒病生防产品的开发应用奠定了基础。
Slash pine(Pinus elliottii) is an important tree species for foresting in the south China.On the base that the slash pine disease is caused by Rhizoctonia sp.,Fusarium oxysporum and Alternaria sp..We measuring the inhibiting disease efficacy of C23 and the bacteria,including Q2-87,Ps2-79,Pf-5,1M1-96,Q37-87, HT5-1,MVP1-4 and FFL1R8 afforded by the lab of roots diseases biocontrol department of agriculture USA in the greenhouse and the field.Screening the new bacteria which have antagonistic effects on the pathogens of slash pine damping-off.Studying the characterization of an antifungal substance produced by C23.The research demonstrate that:
1.21 isolates of the bioncontrol bacteria are gained by new screening,we decide the number W1-13、L1-8、Q1-8、B1-4、J1-4 as the names.there are 12 strong isolates of the biocontrol ones.We have base biology assay,maybe having 3 isolates (L-2,L-5,L-8) as Bacillus subtilis.
2.The inhibiting disease efficacy of nine biocontrol bacteria demonstrate that:for Rhizoctonia sp.,MVP1-4 and C231M1-96 show very good antagonistic ability on the plate against the pathogens;for Fusarium oxysporum,FFL1R8 and Q2-87 show good antagonistic ability;for Alternaria sp.Pf-5 shows good antagonistic ability.
3.1M1-96,HT5-1,Q37-87,FFL1R8,C23 show very good antagonistic ability in the greenhouse test by the method of dipping the roots with bacteria liquid.1M1-96 and C23 show good antagonistic ability beyond CK by the method of watering the roots with bacteria liquid.All of the strains show very good antagonistic ability in the field test.
4.Media screening experiment disclosed that the optimum media for C23 producing the antifungal substance is 1/2PDA~+.Treated at 20-40℃for 30 min,the crude extract maintained more than 80%of its antifungal activity.Treated at about pH6,the crude extract have the best antifungal activity.Treatment with proteinase K,there has no antifungal activity of the crude extract.The antifungal substance can precipitate full with 80%(NH_4)SO_4.The substance can not be extracted by the nonpolarity solvent(methenyl trichloride,ethyl acetate,ethyl oxide).These demonstrate that the antifungal substance must be protein or peptide.
The research show diseases biocontrol in the greenhouse and the field,screens new bioncontrol strains,Studying the characterization of an antifungal substance of C23.This will provide the theoretical reference for further utilizing the bioncontrol bacteria to control the slash pine damping-off.
引文
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