烟草野火病菌拮抗菌的筛选、鉴定及控病研究
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摘要
烟草是一种重要的经济作物,烟草野火病是世界烟草生产上的重要病害之一。在我国大部分烟草种植区均有发生,且多个省市危害严重。由于生产上所种植的烟草品种抗性程度不高,防治上仍然以化学药剂为主要控病手段。长期使用化学农药易导致抗(耐)药性菌株的产生、环境污染和烟叶重金属含量超标等影响防治效果、生态安全和人类健康等问题出现。为此,本文针对烟草野火病生防控制设计试验,试验内容主要是拮抗菌株室内筛选,拮抗菌温室控病研究以及拮抗菌代谢产物分析,拮抗菌抑菌谱测定,拮抗菌生理生化特性以及16S rDNA序列分析。研究结果如下：
1、烟草野火病菌拮抗活性菌株初步筛选
利用实验室保存的120株菌株分别对烟草野火病菌进行抑菌测定,得到有拮抗活性的菌株35株,占待测菌总数的29.17%,其中放线菌21株、细菌14株。
2、拮抗菌株对烟草野火病的温室控制效果
利用平板拮抗效果较好的16个拮抗菌株进行温室盆栽试验,喷雾法接种20天后调查发现,有14个菌株对烟草野火病的相对防效大于60%；对初筛中有较好防效的菌株在接种病菌前后进行施菌试验,结果表明,有6株先喷施拮抗菌的相对防效好于后施拮抗菌的相对防效；喷施拮抗菌菌悬液和无菌发酵滤液及农用链霉素对野火病都有很明显的防控作用,其相对防效均在68%以上,其中菌株YCD2-7-2的防效最好,相对防效都在90%以上。
3、抑菌谱测定
抑菌谱测定结果表明,4个菌株除了对烟草野火菌具有抑制作用外,对供试的25种植物病原菌也有不同程度的抑菌作用,抑菌带宽度在2.0-26.6mm之间。菌株Itb80的抑菌范围最广,对烟草灰霉病菌、番茄圆纹病菌和烟草根黑腐病菌等19种供试的植物病原真菌有抑菌作用,对其中14株和7株抑菌带宽度分别在12～22mm和5～12mm之间,仅对番茄酸腐病菌、油菜菌核菌没有抑制作用。对供试的3种植物病原细菌的抑菌圈在5.6～15.6mmm。其次为菌株Itb162,该菌株对番茄酸腐病菌、油菜菌核菌则表现出一定的抑制作用。两株放线菌YCD1-7-1和YCD2-7-2菌株对病原细菌柑桔溃疡病菌和烟草角斑病菌表现出较强的抑菌作用,抑菌圈半径能达20.0mm以上。
4、拮抗菌株的分类地位
结合菌株菌落培养性状、形态特征和生理生化性状以及16S rDNA序列分析,确定菌株Itb80和Itb162为枯草芽孢杆菌(Bacillus subtilis),菌株YCD2-7-2和YCD1-7-1为灰产色链霉菌(Streptomyces griseochromogenes)
5、拮抗菌代谢产物分析
对4株温室防效较好的菌株进行拮抗代谢产物分析,结果表明,4株菌株均能产生纤维素酶、蛋白酶以及嗜铁素等生防活性物质,不产生几丁质酶。
The tobacco is a kind of important economic crops. Tobacco wildfire caused by Pseudomonas syringae pv. tabaci is a worldwide important disease. It is widely distributed throughout the tobacco planting area of china, and the disease severity is high in some provinces. For the resistance of planting varieties to tobacco wildfire is low, the main way to control the disease still keeps taking chemical pesticide. Thus, it causes the drug-resistant development, environmental pollution, and the exceeded heavy metals contents in tobacco leaves, and so on, finally impacts the disease control effect, ecological safety and mankind health. In this paper, trials for searching biological control of tobacco wildfire disease are designed, mainly for screening antagonistic strains, evaluation of resistance in greenhouse and antagonistic metabolic products analysis, determination of antifungal or antibacterial spectrum, physiological and biochemical characteristics of antagonistic and 16S rDNA sequence analysis. Major findings of the study are as follows.
1. The preliminary screening of antagonistic strains against P. syringae pv. tabaci
120 isolated laboratory strains have been screened by plate spraying method. Preliminary screening,29.17 percent of total number of test strains are screened out, showed that 35 strains have antagonistic effcet on P. syringae pv. tabaci,14 strains of which are actinomycetes and 21 strains of which are bacteria.
2. Evaluation of resistance to tobacco wildfire disease in greenhouse
16 strains antagonists whose resistant effect is good in agar plate confrontation test were chosen to use for greenhouse pot experimental. Spraying antagonist 20 days later,14 strains were found with a good antagonistic effect on tobacco wildfire disease, and the relative efficiency over 60%; Farther test were carried out by spraying antagonist vaccinating before and after vaccinating. The results showed that,6 strains had prevention effect better than cure effect. Greenhouse control disease test indicated that, bacterial suspension, sterile fermentation filtrate of antagonists and agricultural streptomycin kept good control effect on tobacco wildfire disease with relative efficiency over 68%, The strain YCD2-7-2 had better effect and more stable than others in both treatments, with relative efficiency over 90%.
3. The antimicrobial effect of antagonists
Antimicrobial spectrum test indicated that the antagonistic spectrum of the four strains was much broad, with a good antagonistic effect on 25 tested plant pathogens and the inhibition zone radius ranged from 2.0 to 26.6 mm. Itb80 was the best, which had good antagonistic effect on 19 pathogenic fungi, such as Botrytis cinerea, Phomopsis vexans, Thielaviopsis basicola, Sclerotium rolfsii., and so on. The best inhibition on the pathogens Botrytis cinerea was up to 22.0 mm. And the strain had antagonistic effect on 3 of plant pathogenic bacteria and the inhibition zone ranged from 5.6 to 15.6 mm. Followed by Itb162 strains, which have better antagonistic effect on Oospora lactis Fr. var. parasitica and Sclerotinia sclerotiorum, on Xathomonas campestris pv. citri., Pseudomonas angulata and Ralstonia solanacearum with the inhibition zone ranged from 5.0 to 12.6 mm. Two anti-actinomycetes strains (YCD1-7-1 and YCD2-7-2) showed good antimicrobial activity on Xathomonas campestris pv. citri and Pseudomona angulata with the inhibition zone over 20mm;
4. The taxonomy of antagonistic strains
Based on colonies'properties, morphological, physiological and biochemical characteristics, and 16S rDNA phylogenetic analysis, strains Itb80 and Itb 162 were identified as Bacillus subtilis. Strains YCD1-7-1 and YCD2-7-2 were identified as Streptomyces griseus.
5. Analysis of the metabolic products of the four strains
The results showed all of the strains had good biocontrol effects, and all of them could produce active substance for biocontrol, such as proteases, siderophores and so on. But it was found that they were unable to produce cellulases.
1、烟草野火病菌拮抗活性菌株初步筛选
利用实验室保存的120株菌株分别对烟草野火病菌进行抑菌测定,得到有拮抗活性的菌株35株,占待测菌总数的29.17%,其中放线菌21株、细菌14株。
2、拮抗菌株对烟草野火病的温室控制效果
利用平板拮抗效果较好的16个拮抗菌株进行温室盆栽试验,喷雾法接种20天后调查发现,有14个菌株对烟草野火病的相对防效大于60%；对初筛中有较好防效的菌株在接种病菌前后进行施菌试验,结果表明,有6株先喷施拮抗菌的相对防效好于后施拮抗菌的相对防效；喷施拮抗菌菌悬液和无菌发酵滤液及农用链霉素对野火病都有很明显的防控作用,其相对防效均在68%以上,其中菌株YCD2-7-2的防效最好,相对防效都在90%以上。
3、抑菌谱测定
抑菌谱测定结果表明,4个菌株除了对烟草野火菌具有抑制作用外,对供试的25种植物病原菌也有不同程度的抑菌作用,抑菌带宽度在2.0-26.6mm之间。菌株Itb80的抑菌范围最广,对烟草灰霉病菌、番茄圆纹病菌和烟草根黑腐病菌等19种供试的植物病原真菌有抑菌作用,对其中14株和7株抑菌带宽度分别在12～22mm和5～12mm之间,仅对番茄酸腐病菌、油菜菌核菌没有抑制作用。对供试的3种植物病原细菌的抑菌圈在5.6～15.6mmm。其次为菌株Itb162,该菌株对番茄酸腐病菌、油菜菌核菌则表现出一定的抑制作用。两株放线菌YCD1-7-1和YCD2-7-2菌株对病原细菌柑桔溃疡病菌和烟草角斑病菌表现出较强的抑菌作用,抑菌圈半径能达20.0mm以上。
4、拮抗菌株的分类地位
结合菌株菌落培养性状、形态特征和生理生化性状以及16S rDNA序列分析,确定菌株Itb80和Itb162为枯草芽孢杆菌(Bacillus subtilis),菌株YCD2-7-2和YCD1-7-1为灰产色链霉菌(Streptomyces griseochromogenes)
5、拮抗菌代谢产物分析
对4株温室防效较好的菌株进行拮抗代谢产物分析,结果表明,4株菌株均能产生纤维素酶、蛋白酶以及嗜铁素等生防活性物质,不产生几丁质酶。
The tobacco is a kind of important economic crops. Tobacco wildfire caused by Pseudomonas syringae pv. tabaci is a worldwide important disease. It is widely distributed throughout the tobacco planting area of china, and the disease severity is high in some provinces. For the resistance of planting varieties to tobacco wildfire is low, the main way to control the disease still keeps taking chemical pesticide. Thus, it causes the drug-resistant development, environmental pollution, and the exceeded heavy metals contents in tobacco leaves, and so on, finally impacts the disease control effect, ecological safety and mankind health. In this paper, trials for searching biological control of tobacco wildfire disease are designed, mainly for screening antagonistic strains, evaluation of resistance in greenhouse and antagonistic metabolic products analysis, determination of antifungal or antibacterial spectrum, physiological and biochemical characteristics of antagonistic and 16S rDNA sequence analysis. Major findings of the study are as follows.
1. The preliminary screening of antagonistic strains against P. syringae pv. tabaci
120 isolated laboratory strains have been screened by plate spraying method. Preliminary screening,29.17 percent of total number of test strains are screened out, showed that 35 strains have antagonistic effcet on P. syringae pv. tabaci,14 strains of which are actinomycetes and 21 strains of which are bacteria.
2. Evaluation of resistance to tobacco wildfire disease in greenhouse
16 strains antagonists whose resistant effect is good in agar plate confrontation test were chosen to use for greenhouse pot experimental. Spraying antagonist 20 days later,14 strains were found with a good antagonistic effect on tobacco wildfire disease, and the relative efficiency over 60%; Farther test were carried out by spraying antagonist vaccinating before and after vaccinating. The results showed that,6 strains had prevention effect better than cure effect. Greenhouse control disease test indicated that, bacterial suspension, sterile fermentation filtrate of antagonists and agricultural streptomycin kept good control effect on tobacco wildfire disease with relative efficiency over 68%, The strain YCD2-7-2 had better effect and more stable than others in both treatments, with relative efficiency over 90%.
3. The antimicrobial effect of antagonists
Antimicrobial spectrum test indicated that the antagonistic spectrum of the four strains was much broad, with a good antagonistic effect on 25 tested plant pathogens and the inhibition zone radius ranged from 2.0 to 26.6 mm. Itb80 was the best, which had good antagonistic effect on 19 pathogenic fungi, such as Botrytis cinerea, Phomopsis vexans, Thielaviopsis basicola, Sclerotium rolfsii., and so on. The best inhibition on the pathogens Botrytis cinerea was up to 22.0 mm. And the strain had antagonistic effect on 3 of plant pathogenic bacteria and the inhibition zone ranged from 5.6 to 15.6 mm. Followed by Itb162 strains, which have better antagonistic effect on Oospora lactis Fr. var. parasitica and Sclerotinia sclerotiorum, on Xathomonas campestris pv. citri., Pseudomonas angulata and Ralstonia solanacearum with the inhibition zone ranged from 5.0 to 12.6 mm. Two anti-actinomycetes strains (YCD1-7-1 and YCD2-7-2) showed good antimicrobial activity on Xathomonas campestris pv. citri and Pseudomona angulata with the inhibition zone over 20mm;
4. The taxonomy of antagonistic strains
Based on colonies'properties, morphological, physiological and biochemical characteristics, and 16S rDNA phylogenetic analysis, strains Itb80 and Itb 162 were identified as Bacillus subtilis. Strains YCD1-7-1 and YCD2-7-2 were identified as Streptomyces griseus.
5. Analysis of the metabolic products of the four strains
The results showed all of the strains had good biocontrol effects, and all of them could produce active substance for biocontrol, such as proteases, siderophores and so on. But it was found that they were unable to produce cellulases.
引文
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