番茄防病内生细菌ZB-6的初步研究
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摘要
2002-2004年，对番茄根、茎、叶等组织可培养内生细菌进行分离培养，获得了大量的内生细菌菌株。通过拮抗作用测定，从分离自番茄叶片的菌株中，筛选出对主要作物病原微生物具有较强拮抗作用的番茄内生细菌菌株ZB-6。在种类鉴定的基础上，对ZB-6在番茄体内的内生定殖能力、培养过滤液的抑菌活性与机制，进行了较为系统地研究，同时，评估了该菌株对番茄青枯病的生防作用。通过三年的实验，取得以下实验结果：
1、番茄组织中存在大量的内生细菌。采用平板分离法，对番茄植株内生细菌进行分离培养，结果表明，番茄根、茎、叶等组织中存在大量的可培养内生细菌，各组织内生细菌数量约为：1.78×10~3～2.75×10~4CFU／克(FW)，根中内生细菌含量最多，叶中次之，茎中最少。
2、部分番茄内生细菌对病原菌具较为明显的拮抗活性。对分离所得的93个可培养内生细菌株进行拮抗作用测定，38个菌株对待测病原真菌表现出拮抗活性，12个菌株对番茄青枯雷尔氏菌(Ralstonia solanacearum)具有拮抗作用，拮抗菌频率为40.86％，其中从番茄叶片中分离获得的菌株ZB-6，对待测病原真菌与细菌的拮抗作用最强。
3、经形态学和生理生化特征测定，确定菌株ZB-6为枯草芽孢杆菌(Bacillus subtilis)。
4、菌株ZB-6可在番茄体内成功定殖并迅速传导。对ZB-6进行利福平(500μg／ml)抗性标记，采用灌根和叶片喷雾接种，测定ZB-6在番茄体内的定殖与传导，结果表明，标记菌株可进入番茄体内并成功增殖，35d后在番茄植株内各器官均可分离到大量的标记菌株；标记菌株能在番
茄植株内自由转移，其转移速率因接种部位不同而略有差异。
5、菌株ZB一6培养滤液具有较强的抑菌活性，可引起病原物发育异
常，甚至死亡。对菌株zB一6培养滤液的抑菌活性进行测定，结果表明，
培养滤液对多种病原菌有较强的抑制作用，根据测试病原菌的不同，培
养滤液的拮抗能力呈现较大差别。该培养滤液可使番茄早疫病菌(茄链
格抱，Alternarl’asoZanl’)分生抱子萌发异常，芽管畸形膨大，呈泡囊状，
24h后，泡囊消融、破裂，分生抱子萌发受到完全抑制。
6、菌株ZB一6对番茄青枯病具有良好的控制效果。盆栽试验表明，
菌株ZB一6对番茄青枯病具有良好的防效，7d时防效达63.7%，27d时平
均防效仍高达58.0%。田间防效略低。据认为，菌株ZB一6以拮抗和竞争
二种方式协同作用，来控制番茄青枯病的危害，其接种时间对病害防治
效果影响显著。
A number of endophytic culturable bacterial strains were obtained from roots, stems and leaves of tomato plants by plate culture during 2002-2004. Among all culturable endophytic bacterial strains, ZB-6, a strain from tomato leaf, showed the strongest inhibitory activity to plant fungal pathogens. Main attention was paid to taxonomy of ZB-6, colonization and movement of strain ZB-6 in tomato plants. Inhibition of the filtrate from ZB-6 was evaluated systematically. Meanwhile, control effects of ZB-6 on tomato bacterial wilt (Ralstonia solanacearum) were assessed. The main results are as follows:
1. There are a large number of endophytic bacteria in tomato plants. With the surface sterilized with 0.1% mercuric chloride, culturable endophytic bacteria were counted by plate culture. The results showed that culturable endophytic bacteria in tomato plants ranged from 1.78?03 to 2.75?04 CFU (colony formation unit) per gram (FW) . There are more culturable endophytic bacteria in roots than in stems and leaves.
2. The endophytic bacteria from tomato plants were with strong inhibition to fungal phytopathogens. Among the total of 93 endophytic bacteria isolates tested, 38, or 40.86%, had in vitro inhibitory activity to fungal phytopathogens, such as Colletotrichum dematium and Alternaria solani,etc. and 12 isolates had in vitro inhibitory activity to Ralstonia solanacearum. The strain ZB-6, isolated from tomato leaf, was with the strongest inhibitory activity.
3. The strain ZB-6 was identified as Bacillus subtilis, based on the taxonomic results including cell shape, Gram's stain, sporulation, production of catalase and oxidase ect.
3
4. The strain ZB-6 was able to colonize successfully and move frequently between different parts in tomato plants. ZB-6Rif500, the mutant strain of ZB-6, was resistant to rifampicin. After inoculated with ZB-6Rif500 by spraying leaves and watering roots, a number of ZB0 were found in all parts of tomato plants at 35d after inoculation. The results implied that ZB-6 was able to colonize successfully in tomato and move frequently between different parts of plants.
5. Filtrate from ZB-6 cultivation in medium gave strong inhibition to the fungal phytopathogens tested. However, there was obvious difference in inhibitory activity to various fungal phytopathogens. The deformation and disintegration of the spore germinal tube from Alternaria solani were observed 24h after treatment with the filtrate.
6. The strain ZB-6 showed effective control to tomato bacterial wilt (Ralstonia solanacearum) in both outdoor pots and field. The control effects of ZB-6 on tomato bacterial wilt were 63.7% at 7d and 58.0% at 27d, respectively in outdoor experiments. Preventive inoculation of strain ZB-6 improved greatly its control effect to tomato bacterial wilt.
1、番茄组织中存在大量的内生细菌。采用平板分离法，对番茄植株内生细菌进行分离培养，结果表明，番茄根、茎、叶等组织中存在大量的可培养内生细菌，各组织内生细菌数量约为：1.78×10~3～2.75×10~4CFU／克(FW)，根中内生细菌含量最多，叶中次之，茎中最少。
2、部分番茄内生细菌对病原菌具较为明显的拮抗活性。对分离所得的93个可培养内生细菌株进行拮抗作用测定，38个菌株对待测病原真菌表现出拮抗活性，12个菌株对番茄青枯雷尔氏菌(Ralstonia solanacearum)具有拮抗作用，拮抗菌频率为40.86％，其中从番茄叶片中分离获得的菌株ZB-6，对待测病原真菌与细菌的拮抗作用最强。
3、经形态学和生理生化特征测定，确定菌株ZB-6为枯草芽孢杆菌(Bacillus subtilis)。
4、菌株ZB-6可在番茄体内成功定殖并迅速传导。对ZB-6进行利福平(500μg／ml)抗性标记，采用灌根和叶片喷雾接种，测定ZB-6在番茄体内的定殖与传导，结果表明，标记菌株可进入番茄体内并成功增殖，35d后在番茄植株内各器官均可分离到大量的标记菌株；标记菌株能在番
茄植株内自由转移，其转移速率因接种部位不同而略有差异。
5、菌株ZB一6培养滤液具有较强的抑菌活性，可引起病原物发育异
常，甚至死亡。对菌株zB一6培养滤液的抑菌活性进行测定，结果表明，
培养滤液对多种病原菌有较强的抑制作用，根据测试病原菌的不同，培
养滤液的拮抗能力呈现较大差别。该培养滤液可使番茄早疫病菌(茄链
格抱，Alternarl’asoZanl’)分生抱子萌发异常，芽管畸形膨大，呈泡囊状，
24h后，泡囊消融、破裂，分生抱子萌发受到完全抑制。
6、菌株ZB一6对番茄青枯病具有良好的控制效果。盆栽试验表明，
菌株ZB一6对番茄青枯病具有良好的防效，7d时防效达63.7%，27d时平
均防效仍高达58.0%。田间防效略低。据认为，菌株ZB一6以拮抗和竞争
二种方式协同作用，来控制番茄青枯病的危害，其接种时间对病害防治
效果影响显著。
A number of endophytic culturable bacterial strains were obtained from roots, stems and leaves of tomato plants by plate culture during 2002-2004. Among all culturable endophytic bacterial strains, ZB-6, a strain from tomato leaf, showed the strongest inhibitory activity to plant fungal pathogens. Main attention was paid to taxonomy of ZB-6, colonization and movement of strain ZB-6 in tomato plants. Inhibition of the filtrate from ZB-6 was evaluated systematically. Meanwhile, control effects of ZB-6 on tomato bacterial wilt (Ralstonia solanacearum) were assessed. The main results are as follows:
1. There are a large number of endophytic bacteria in tomato plants. With the surface sterilized with 0.1% mercuric chloride, culturable endophytic bacteria were counted by plate culture. The results showed that culturable endophytic bacteria in tomato plants ranged from 1.78?03 to 2.75?04 CFU (colony formation unit) per gram (FW) . There are more culturable endophytic bacteria in roots than in stems and leaves.
2. The endophytic bacteria from tomato plants were with strong inhibition to fungal phytopathogens. Among the total of 93 endophytic bacteria isolates tested, 38, or 40.86%, had in vitro inhibitory activity to fungal phytopathogens, such as Colletotrichum dematium and Alternaria solani,etc. and 12 isolates had in vitro inhibitory activity to Ralstonia solanacearum. The strain ZB-6, isolated from tomato leaf, was with the strongest inhibitory activity.
3. The strain ZB-6 was identified as Bacillus subtilis, based on the taxonomic results including cell shape, Gram's stain, sporulation, production of catalase and oxidase ect.
3
4. The strain ZB-6 was able to colonize successfully and move frequently between different parts in tomato plants. ZB-6Rif500, the mutant strain of ZB-6, was resistant to rifampicin. After inoculated with ZB-6Rif500 by spraying leaves and watering roots, a number of ZB0 were found in all parts of tomato plants at 35d after inoculation. The results implied that ZB-6 was able to colonize successfully in tomato and move frequently between different parts of plants.
5. Filtrate from ZB-6 cultivation in medium gave strong inhibition to the fungal phytopathogens tested. However, there was obvious difference in inhibitory activity to various fungal phytopathogens. The deformation and disintegration of the spore germinal tube from Alternaria solani were observed 24h after treatment with the filtrate.
6. The strain ZB-6 showed effective control to tomato bacterial wilt (Ralstonia solanacearum) in both outdoor pots and field. The control effects of ZB-6 on tomato bacterial wilt were 63.7% at 7d and 58.0% at 27d, respectively in outdoor experiments. Preventive inoculation of strain ZB-6 improved greatly its control effect to tomato bacterial wilt.
引文
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75. Yanni Y G, Rizk R Y, Corich V, et al. Natural endophytic association between Rhizobium leguminosarum bv. Trifolii and rice roots and assessment of its potential to promote rice growth. Plant Soil. 1997,194:99-114.
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84. MahaffeeW F, Kloepper J W, et al. In Improving Plant Productivity with Rhizosphere Bacteria. Ryder,M,H., Stephenes,P.M., Bowen, G.D., (eds). CSIRO, Australia, 1994:180
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86. Tomasino S F, Leister R T, Dimock M B, et al. Field performance of Clavibacter xyli subsp. Cynodontis expressing the insecticidal protein gene crylA (c) of Bacillus thuringiensis against European corn borer in field corn. Biological Control 1995,5:442-448.
87. Wilhelm E, Arthofer W and Sehafleitner R. Bacillus subtilis, an endophyte of chestnut (Castanea saliva), as antagonist against chestnut blight (Cryphonectria parasitica), In A. C. Cassells (ed.), Pathogen and microbial contamination management in micropropagation. Kluwer Academic Publishers, Dortrecht, The Netherlands. 1997:331-337
88. Vargas C, Lopes A and Hemerly A. Variability and interactions between endophytie bacteria and
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89．林东，徐庆．枯草芽孢杆菌S0113分泌蛋白的抑菌作用及抗菌蛋白的分离纯化．农业生物技术学报2001，9(1)．-77-80
90．东秀珠，蔡妙英等．常见细菌鉴定手册．科学出版社．2001
91．唐启义，冯明光．实用统计分析及其DPS数据处理系统．科学出版社．2002
92．任欣正，谢贻格．番茄青枯病的生物防治．南京农业大学学报1993，16(1)．45-49
93．龙良鲲，肖崇刚．内生细菌叭01-144在番茄根茎内定殖的初步研究．微生物学通报2003，30(5)：53-56
94．孔建，王同贵．枯草芽孢杆菌B—903菌株抗菌物质对植物病原真菌的抑制作用．植物病理学报1995，25(1)：69-72
95．郭坚华，孙平华，吴云波等．植物细菌性青枯病的生物防治机制和途径．中国生物防治1997，13(1)：42-46