烟草青枯病（Ralstonia solanacearum）内生细菌及根围拮抗细菌研究
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摘要
烟草青枯病是烟草上的一大毁灭性病害，至今还没有一种有效药剂能够防治它，因此找到一种有效的生防菌进行生物防治，意义重大。
本研究于2001～2002年，从湖南永州、郴州、衡阳和宁乡等地采集病株及健株共分离到青枯菌菌株19株、内生细菌菌株160株，经鉴定青枯菌菌株2316为强致病力菌株，32株内生菌菌株在室内对烟青枯病菌株有拮抗作用。根据形态特征和生理生化性状，初步鉴定了001、009、011三个内生菌株的分类，认为内生菌株001为枯草芽孢杆菌(Bacillus subtilis)，内生菌株009与011为短芽孢杆菌(Bacillus brevis)，其田间初步防效试验证明：内生芽孢菌001、009、011对烟草青枯病都有很好的防治效果，防效分别达到了82.5％、100％、84.5％。
烟青枯病根围拮抗菌菌株湘2—3和2—轻—9在多年的田间试验中，具有较好的生防效果，为了使这两种菌株能够大规模用于工业化生产，对菌株湘2—3和2—轻—9的发酵条件进行了系统的研究。试验结果表明，菌株湘2—3的最佳发酵培养基配方为：豆饼粉20‰，鱼粉3‰，CaCO_33‰，KH_2PO_40.8‰，最佳发酵条件为：发酵温度28℃、发酵时间24h、接种量4％、容氧量120ml；菌株2—轻—9最佳发酵培养基配方为：豆饼粉25‰，鱼粉3‰，CaCO_34‰，KH_2PO_40.7‰；最佳发酵条件为：发酵温度30℃、发酵时间26h、接种量3％、容氧量80ml。
Tobacco bacterial wilt is a very important quarantine bacterium on the tobac -co,which has not yet been inhibited effectively .so it is significance to find a bio -controlled bacterial strain to inhibit the disease.
Nineteen pathogen were isolated from inflected plant and one hundred sixty endophytic bacterial strains were isolated in health tobacco from Yongzhou , chen zhou,Hengyang and Ningxiang region in the research. By identifying, the pathogen strain 2316 was most strong one on inflecting capability. Thirty-two endophytic strain had antagonistic action to tobacco bacterial wilt. According tomorphologi -cal ,physiological and biochemical characteries ,strain 001,009,011 were identified and the strain 001 belong to Bacillus subtilis .strain 009 and 011 belong to Bacillus brevis. By strain 001,009,011 used as bio-control agent against tobacco bacterial wilt in frield, the results show that the three strains had strong inhibitive effect. The inhibitive efficacy was respectively 82.5%, 100%, 84.5%.
The inhibitive strain xiang2-3 and 2-qing-9 have good controlled effect for many years in the field .The fermentation conditions are studied in order to enable the two strains to be used for industry product. The result showed that the optimum culture medium for the strain xiang2-3 is : soybean cake meal 20 %, fish mea13% KH2PO40.8%,CaCO3 3%; the optimum fermentation conditions is : fermentation temperature 28C,fermentation time 24h , inoculum size 4%,dissolved oxygen120ml; The optimum culture medium for the strain 2-qing-9 is : soybean cake meal 25 %, fish mea13% KH2PO4 0.7%,CaCO3 4%; the optimum fermentation conditions is : fermentation temperature 30C .fermentation time 26h ,inoculum size 3%,dissolved oxygen80ml.
本研究于2001～2002年，从湖南永州、郴州、衡阳和宁乡等地采集病株及健株共分离到青枯菌菌株19株、内生细菌菌株160株，经鉴定青枯菌菌株2316为强致病力菌株，32株内生菌菌株在室内对烟青枯病菌株有拮抗作用。根据形态特征和生理生化性状，初步鉴定了001、009、011三个内生菌株的分类，认为内生菌株001为枯草芽孢杆菌(Bacillus subtilis)，内生菌株009与011为短芽孢杆菌(Bacillus brevis)，其田间初步防效试验证明：内生芽孢菌001、009、011对烟草青枯病都有很好的防治效果，防效分别达到了82.5％、100％、84.5％。
烟青枯病根围拮抗菌菌株湘2—3和2—轻—9在多年的田间试验中，具有较好的生防效果，为了使这两种菌株能够大规模用于工业化生产，对菌株湘2—3和2—轻—9的发酵条件进行了系统的研究。试验结果表明，菌株湘2—3的最佳发酵培养基配方为：豆饼粉20‰，鱼粉3‰，CaCO_33‰，KH_2PO_40.8‰，最佳发酵条件为：发酵温度28℃、发酵时间24h、接种量4％、容氧量120ml；菌株2—轻—9最佳发酵培养基配方为：豆饼粉25‰，鱼粉3‰，CaCO_34‰，KH_2PO_40.7‰；最佳发酵条件为：发酵温度30℃、发酵时间26h、接种量3％、容氧量80ml。
Tobacco bacterial wilt is a very important quarantine bacterium on the tobac -co,which has not yet been inhibited effectively .so it is significance to find a bio -controlled bacterial strain to inhibit the disease.
Nineteen pathogen were isolated from inflected plant and one hundred sixty endophytic bacterial strains were isolated in health tobacco from Yongzhou , chen zhou,Hengyang and Ningxiang region in the research. By identifying, the pathogen strain 2316 was most strong one on inflecting capability. Thirty-two endophytic strain had antagonistic action to tobacco bacterial wilt. According tomorphologi -cal ,physiological and biochemical characteries ,strain 001,009,011 were identified and the strain 001 belong to Bacillus subtilis .strain 009 and 011 belong to Bacillus brevis. By strain 001,009,011 used as bio-control agent against tobacco bacterial wilt in frield, the results show that the three strains had strong inhibitive effect. The inhibitive efficacy was respectively 82.5%, 100%, 84.5%.
The inhibitive strain xiang2-3 and 2-qing-9 have good controlled effect for many years in the field .The fermentation conditions are studied in order to enable the two strains to be used for industry product. The result showed that the optimum culture medium for the strain xiang2-3 is : soybean cake meal 20 %, fish mea13% KH2PO40.8%,CaCO3 3%; the optimum fermentation conditions is : fermentation temperature 28C,fermentation time 24h , inoculum size 4%,dissolved oxygen120ml; The optimum culture medium for the strain 2-qing-9 is : soybean cake meal 25 %, fish mea13% KH2PO4 0.7%,CaCO3 4%; the optimum fermentation conditions is : fermentation temperature 30C .fermentation time 26h ,inoculum size 3%,dissolved oxygen80ml.
引文
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10. Kao C, Sequeira L.A gene cluster required for the coordinated biosynthsis of lipopolysacctaride also affects virulence of pseudomonas solanaccaru J Bacteriol, 1991,173:7841～7847
11. Schell M A, Roberts D P. Denny T P. Analysis of the pseudomonas solanacearum polygalaoturonase encode J by pglA And its involvement in pathogenicity. J Bacteriol, 1988,170:4501～4508
12. Boucher A A, Barberis P A, Arlat M. Acridime orange selects for deletion of hrp genes in all races of pseudomonas solanacearum. Mol Plant microbe Interact, 1988, 1: 282～288
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2. Smith Burkholderia, solanacearun Validanion of publication ofnewnames and new combinations previosity effectively pubished outside the USB. Apr, 1996, 625～626
3. PALLERONI N J, DOUDOROFF M. Phenotypic characterization and deoxyniontypic acid homologics of Pseudomonas
4. S E SEAL, M TAGHAVI, N FEGAN, et al, Detemination of Ralstonia solanacearum rDNA subgroups by PCR tests. Plant pathology, 1999,48,115～120。
5．刘焕利，何礼远，毛国漳，等．植物青枯病原细菌胞外蛋白相关基因的克隆．植物病理学报，1999，29(2)：110～119．
6. Hayward A C. Characteristics of pseudomonas solanacearum. Journal of Applied Bacteriology, 1964,27:265～277;
7. Schmit J. Microscopic study of early stages of infection by pseudomonas solanacearum on in vitro grown tomato seedlings, in Proceedings of 4th ICPPB. 1978,841～856
8. Wallis F M. Truter S J. Histopathology of tomato plants infected with Pseudomonas solanacearum with emphasis on ultrastucture. Physilo Plant Pathol, 1978,13:307～315
9. Brumbley S M, Denny T P. Cloning of the wild type pseudomonas solanacearum phcA, a gene that when mutated alters expression of multiple traits that contribute to virulence. J Bacteriology, 1990,172:5677～5685
10. Kao C, Sequeira L.A gene cluster required for the coordinated biosynthsis of lipopolysacctaride also affects virulence of pseudomonas solanaccaru J Bacteriol, 1991,173:7841～7847
11. Schell M A, Roberts D P. Denny T P. Analysis of the pseudomonas solanacearum polygalaoturonase encode J by pglA And its involvement in pathogenicity. J Bacteriol, 1988,170:4501～4508
12. Boucher A A, Barberis P A, Arlat M. Acridime orange selects for deletion of hrp genes in all races of pseudomonas solanacearum. Mol Plant microbe Interact, 1988, 1: 282～288
13．孙光军，林代福，刘呈义，等．烟草根结线虫病与黑胫病、青枯病的发生关系及品种抗性研究初报．烟草科技，1999，(5)：48
14. KAZUHARU KOGA HIDEKI HARA, HIROSHI TANAKA. Suppressive to Bacterial Wilt of Tobacco in Japan and Population Dynamics of Pseudomonas solanacearum in These siols. Ann. Phytopathol Soc. Jpn, 1997, 301～308
15．黄福新，陈水惠，周兴华等．烟草青枯病综合防治研究．广西农业科学，1997，(1)：32～35
16．孙光军，林代福，刘呈义等．烟草根结线虫病与黑胫病、青枯病的发生关系及品种抗性研究初报．烟草科技，1999，(5)：48
17．卢洪兴，曾军，邱志丹等．烟草青枯病发生与药剂防治研究．福建省农科院学报，1996．11(3)：41～45．
18．罗宽，王庄．利用桔抗的Pseudomonas spp．无致病力的P．Solanacearum防治青枯病的研究[J]．植物病理学报，1983，13(1)：51～55．
19．汪中一．枯草杆菌对青枯菌的拮抗作用．福建农业科技，1990．
20．任欣正．番茄青枯病的生物防治，南京农业大学学报。1993，16(1)：45～49
21．董春，董成刚，赵青峰等．利用拮抗细菌防治烟草青枯病初步研究．广西农业科学，1996，(5)：28～30
22．张竹青，罗宽，高必达等．七株抗青枯病菌尘防菌的初步鉴定．湖南农业大学学报，2002，28(6)：512～513
23．袁立和．江西省番茄青枯病抑病土壤研究初报．植物保护学报，1995，22(1)：93～94
24. De. Bary A Molecular biology an international series of monographs and textbooks. The molecular biology of the BacilIi. 1993, 6:1～316
25．张明朗．土壤添加剂防治细菌性青枯病
26．王金文．烟草青枯病药剂防治试验．中国烟草，1989，(4)：12～13．
27．陆思瑚．烟草青枯病防治试验简报．广西农业科学，1991，(2)：87～88
28．陈永惠，黄福新．烟草青枯病药剂防治试验．广西植保，1996，(4)：23～25．
29．我国植物青枯菌的遗传多样性和马铃薯青枯菌的PCR检测技术研究．中国农业科学院研究生院．北京：2000
30. Black R et al. Developing appropriate detection methods for developing countries. In Bacterial wilt disease: Molecular and Ecological Aspects, 1998, 128～132;
31. Cook D. The use of subtractive hybridizrion to obtain a DNA probe specific for Pseudomonas solanacearum race3. Molecular and General Genetics. 1991, 227, 401～410
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