烟草内生与附生细菌互作对赤星病的控病及机理研究
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摘要
烟草赤星病病[Alternaria alternata(Fries)Keissler]是烟草生产上重要病害，严重威胁烟叶的生产，对烟草赤星病的防治，目前主要采用化学防治为主的防治技术。烟草是叶用经济作物，对烟叶的外观和内在质量有特殊的要求，农药残留、病原菌抗药性以及对卷烟卫生的影响，限制了农药的大规模利用，因此有必要寻找新的防治途径以克服农药残留和病菌抗药性等问题；生物防治安全、无污染、无公害，是烟草上有害生物进行综合治理的发展方向。本研究从烟草体内、外分别寻找赤星病的内生和附生生防菌，并进一步明确其互作的控病作用及作用机理。
1．采用表面消毒研磨分离法，从健康烟草体内获得细菌分离物302个，室内平板拮抗试验表明，11个菌株在PDA平板上可明显抑制赤星病菌Tbs3的生长，在温室控病试验中，获得了对烟草赤星病有较好防效的内生细菌Itb162，烟苗经菌液处理3d后接种赤星病菌的控病效果达52.0％。
2．通过水洗法、印像法，从健康烟叶上分离获得236株附生细菌，室内平板拮抗测试表明，5个菌株在PDA平板上可明显抑制赤星病菌Tbs3的生长，温室控病试验表明，附生细菌Ata28对烟草赤星病有较好防治效果，烟苗经菌液处理3d后接种赤星病菌的控病效果达50.4％。
3．内生细菌Itb162和附生细菌Ata28无菌滤液对赤星病的孢子萌发和菌丝生长均有明显的抑制作用，表现为孢子萌发减少，即使萌发的孢子也不能正常生长，呈畸形；赤星病菌菌丝形成膨胀泡，破裂、消解，致使菌丝停止生长，且浓度越高，抑制能力越强。内生细菌Itb162和附生细菌Ata28无菌滤液混合液对赤星病病菌孢子及菌丝的抑制作用同单一内生细菌或附生细菌无菌滤液的处理无明显差异。
4．在互作控病试验中，内生细菌Itb162和附生细菌Ata28菌株提前3d以1：1(V/V)混合处理烟草后，接种赤星病菌Tbs3，控病效果达58.4％，比单一使用内生细菌或附生细菌处理的防效有所提高，且差异明显。测定混合菌液、内生细菌及附生细菌分别处理3d后的烟苗，其中经内生菌和混合菌液处理的烟苗其体内POD、PPO、PAL活性变化同对照相比有明显提高，病程相关蛋白(PRP)也有量的积累。
Tobacco brown spot caused by Alternaria alternata (Fries) Keissler is an important disease on the crop worldwide. Presently pesticides application is the major measure of controlling the disease, but the use of pesticides is restricted because this may induce problems as fungicide resistance, product quality decrease and chemical residues in the environment. Biocontrol may be an alternative to avoid these problems in tobacco disease control. In the present study, antagonistic bacteria against A. alternata were screened from epiphytic and endophytic isolates of tobacco leaves, and the biocontrol efficiency and mechanisms of the antagonistic bacteria against the pathogen was analyzed.
1. A total number of 302 endophytic bacterial isolates were obtained from healthy tobacco leaves through surface sterilization and molar-mortar grinding. From these, 11 isolates were shown antagonistic against Tbs3 strain of A. alternata by antagonism test on PDA plates. The endophytic isolate Itb162 was found most effective in brown spot control and a rate of disease control reached 52.0% on tobacco plants three days after bacteria spraying.
2. Surface washing and printing methods were applied to isolate epiphytic bacteria and 236 isolates were obtained. Five of these epiphytic isolates exhibited
marked suppression of A. alternata strain Tbs3 on PDA plates and the isolate Ata28 had the highest brown spot control effect, with a disease reduction of 50.4% three days after treatment.
3. The filtrates of Itb162 and Ata28 bacteria were shown separately to suppress mycelium growth and spore germination of A. alternata. Spore germination rate was significantly reduced and malformation of the spore germlings and rupture or dissolving of mycelium were observed. The combination to the two bacterial filtrates did not modify the fungal suppression effect compared with the application of individual filtrates.
4. The suspension mixed with Itbl62 and Ata28 bacteria (1:1, v/v) was sprayed and the pathogen Tbs3 was inoculated on to tobacco seedlings in greenhouse three days afterwards. The disease control effect was 58.4%, which was significantly higher than spraying suspension of individual bacteria isolates. Tests and analyses showed that the enzyme activity of POD, PPO and PAL markedly increased in tobacco seedlings three days after spraying Itb 162 and the mixed suspension, and the pathogenesis-related protein (PRP) content was also accumulated.
1．采用表面消毒研磨分离法，从健康烟草体内获得细菌分离物302个，室内平板拮抗试验表明，11个菌株在PDA平板上可明显抑制赤星病菌Tbs3的生长，在温室控病试验中，获得了对烟草赤星病有较好防效的内生细菌Itb162，烟苗经菌液处理3d后接种赤星病菌的控病效果达52.0％。
2．通过水洗法、印像法，从健康烟叶上分离获得236株附生细菌，室内平板拮抗测试表明，5个菌株在PDA平板上可明显抑制赤星病菌Tbs3的生长，温室控病试验表明，附生细菌Ata28对烟草赤星病有较好防治效果，烟苗经菌液处理3d后接种赤星病菌的控病效果达50.4％。
3．内生细菌Itb162和附生细菌Ata28无菌滤液对赤星病的孢子萌发和菌丝生长均有明显的抑制作用，表现为孢子萌发减少，即使萌发的孢子也不能正常生长，呈畸形；赤星病菌菌丝形成膨胀泡，破裂、消解，致使菌丝停止生长，且浓度越高，抑制能力越强。内生细菌Itb162和附生细菌Ata28无菌滤液混合液对赤星病病菌孢子及菌丝的抑制作用同单一内生细菌或附生细菌无菌滤液的处理无明显差异。
4．在互作控病试验中，内生细菌Itb162和附生细菌Ata28菌株提前3d以1：1(V/V)混合处理烟草后，接种赤星病菌Tbs3，控病效果达58.4％，比单一使用内生细菌或附生细菌处理的防效有所提高，且差异明显。测定混合菌液、内生细菌及附生细菌分别处理3d后的烟苗，其中经内生菌和混合菌液处理的烟苗其体内POD、PPO、PAL活性变化同对照相比有明显提高，病程相关蛋白(PRP)也有量的积累。
Tobacco brown spot caused by Alternaria alternata (Fries) Keissler is an important disease on the crop worldwide. Presently pesticides application is the major measure of controlling the disease, but the use of pesticides is restricted because this may induce problems as fungicide resistance, product quality decrease and chemical residues in the environment. Biocontrol may be an alternative to avoid these problems in tobacco disease control. In the present study, antagonistic bacteria against A. alternata were screened from epiphytic and endophytic isolates of tobacco leaves, and the biocontrol efficiency and mechanisms of the antagonistic bacteria against the pathogen was analyzed.
1. A total number of 302 endophytic bacterial isolates were obtained from healthy tobacco leaves through surface sterilization and molar-mortar grinding. From these, 11 isolates were shown antagonistic against Tbs3 strain of A. alternata by antagonism test on PDA plates. The endophytic isolate Itb162 was found most effective in brown spot control and a rate of disease control reached 52.0% on tobacco plants three days after bacteria spraying.
2. Surface washing and printing methods were applied to isolate epiphytic bacteria and 236 isolates were obtained. Five of these epiphytic isolates exhibited
marked suppression of A. alternata strain Tbs3 on PDA plates and the isolate Ata28 had the highest brown spot control effect, with a disease reduction of 50.4% three days after treatment.
3. The filtrates of Itb162 and Ata28 bacteria were shown separately to suppress mycelium growth and spore germination of A. alternata. Spore germination rate was significantly reduced and malformation of the spore germlings and rupture or dissolving of mycelium were observed. The combination to the two bacterial filtrates did not modify the fungal suppression effect compared with the application of individual filtrates.
4. The suspension mixed with Itbl62 and Ata28 bacteria (1:1, v/v) was sprayed and the pathogen Tbs3 was inoculated on to tobacco seedlings in greenhouse three days afterwards. The disease control effect was 58.4%, which was significantly higher than spraying suspension of individual bacteria isolates. Tests and analyses showed that the enzyme activity of POD, PPO and PAL markedly increased in tobacco seedlings three days after spraying Itb 162 and the mixed suspension, and the pathogenesis-related protein (PRP) content was also accumulated.
引文
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