草莓灰霉病菌生防细菌的筛选及其抗菌机制的研究
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摘要
草莓灰霉病是导致草莓产量和质量降低的主要限制因素之一,传统的化学防治导致了严重的病菌抗药性与环境污染问题。安全、低毒的微生物防治是控制草莓灰霉病的一条重要而有效的途径,本研究旨在从草莓根际土壤、草莓植株及其它植物内分离、筛选能够抑制草莓灰霉病的拮抗细菌,对菌株进行鉴定、发酵优化,并研究其抑菌机理。主要结果如下:
1、从草莓根际土壤、草莓植株及其它植物内分离、筛选到6株对草莓灰霉病菌有强拮抗作用的菌株,来自草莓叶的SL_6菌株抑菌效果最佳,抑菌半径达15 mm,无菌发酵液对草莓灰霉菌丝的抑制率为97.6%。经形态、生理生化和16S rDNA等分析测定,并通过MEGA4方法构建其16S rDNA系统发育树,将SL_6菌株鉴定为枯草芽孢杆菌(Bacillus subtilis)。
2、菌株抑菌机理初步研究表明:SL_6代谢产物能强烈抑制病原菌菌丝的生长并对病菌菌丝形态明显的破坏作用。滤液对菌丝形态的破坏作用主要表现为菌丝原生质收缩、菌丝部分膨大变形、细胞壁破裂和原生质溢出等,使得菌丝不能继续生长,甚至发生断裂。培养滤液用70%硫酸铵沉淀,上清没有抑菌活性。可以初步确定该抗菌物质为可溶性蛋白质。其拮抗物质产生的最佳条件为:接种于100 mL pH 7.0的PSB液体培养基中,置于37℃恒温振荡器中培养72 h。
3、SL_6培养滤液用70%硫酸铵进行盐析,沉淀物经透析后得粗蛋白。粗蛋白进行DEAE Sepharose Fast Flow离子交换层析得到抗菌活性蛋白,活性物质中具有很高的蛋白酶活力,进行SDS-PAGE及非变性凝胶电泳,均出现三条分子量大小相近蛋白带,通过质谱分析,得三条蛋白分别为30S核糖体蛋白、核酸酶和脱羧酶。测定了温度和pH对抗菌粗蛋白发挥抑菌作用的影响,结果表明,抗菌粗蛋白具有热稳定性,100℃处理30 min后,抑菌活性仍能达到60%以上,pH 2.0-9.0之间均有较强抑菌活性作用。
4、通过田间试验,可以验证生防枯草芽孢杆菌SL_6发酵液对于草莓灰霉病的发生具有预防和治疗的效果,3次处理后的防效为86.6%,有效地抑制了草莓灰霉病情的扩散,验证了其可在自然环境中长期稳定地发挥作用,可用于开发草莓灰霉病害生防制剂研究证明生物制剂如能阻止或减少灰霉对于草莓花朵的侵染,就能有效地控制灰霉病害,而内生细菌能发挥该项作用。草莓内生细菌具有通过产生吲哚乙酸和溶解有机磷来促进植株生长的作用,但对其生防作用的研究国内外尚未有报道。本文筛选出的高效菌株来源于草莓叶片,其发酵液可在田间试验中发挥很好的防效。研究其生防机理及相关的蛋白并使之能应用于生产,具有重要的理论和实际意义。
Botrytis cinerea in strawberry is one of the major constraints which made the yield and quality reduced. The traditional chemical control has led to the formation of serious resistance and environmental problems. The microbial control of Botrytis cinerea in strawberry with safety and low toxicity is an important and effective way. In this research, we hope to get the antagonistic strain by screening the bacteria isolated from rhizosphere soil of strawberry, strawberry plants and other plants and study identification of the strains, fermentation optimization, and its antagonistic reaction mechanism.The main results are as follows:
1.Six strong antagonistic strains were isolated from the rhizosphere soil of strawberry, in them, strain SL_6 isolated from the leaf of strawberry was found to have the best control efficacy, the inhibition zone diameter was 15 mm and the inhibition rate could reach 97.6% with the fermentation filtrate, revealing potential in the aspect of antagonistic action. Based on the morphological,physiological,biochemical characteristics and 16S rDNA sequence analysis,through its 16S rDNA phylogenetic tree constructed with MEGA4, SL_6 was identified as Bacillus subtilis.
2. Study on inhibition mechanism of strain SL_6 demonstrated that metabolites produced by antagonistic strain SL_6 could do great damage to the mycelia and had inhibitory effects on the growth of Botrytis cinerea. Microscopic observation indicated that the mycelium inhibited by strain SL_6 mostly twisted, inflated, deformed, the mycelia wall broke, the protoplasm leaked from the wound and even hyphae lysed. The bacterial metabolites in the culture filtrate could be precipitated by 70% ammonium sulfate and its inhibition activity was similar to the damage of mycelium by strain SL_6, while the supernate couldn’t inhibit the pathogen growth, thereby indicating that the antagonistic substance was protein. The optimal culture condition of SL_6 was shaking culture at 37℃with initial pH 7.0 in 100mL PSB liquid medium for 3 days.
3. The bacterial metabolites in the culture filtrate could be precipitated by 70% ammonium sulfate. The crude protein was obtained by the dialysis of the precipitate and purified by using ammonium sulfate precipitation following DEAE Sepharose Fast Flow chromatography. There were three bands with similar molecular weight in SDS-PAGE and PAGE of the active substances which had high protease activity. By mass spectrometry, three proteins were identified respectively as 30S ribosomal protein, nuclease subunit and uncharacterized protein. The crude protein was rather stable, its antibacterial activity remained above 60% after treated for 30 min at 100℃, and its pH ranged from 2.0 to 9.0.
4. The fermentation filtrate of SL_6 showed effective control to Botrytis cinerea in strawberry in field experiments. After three treatments, the effect of fermentation broth was 86.6% and effectively inhibited the spread of disease. It could arrive to the conclusion that it could play a stable role in the long-term natural environment.
Studies had shown that biological agents, which could prevent or reduce gray mold of strawberry flowers from infection, can effectively control gray mold damage, and endophytic bacteria can play the role. The endophytic bacteria of strawberry could promote the plant growth by producing indole acetic acid or dissolving organic phosphorus. But their roles in biological control have not yet been reported at domestic and overseas reports. This highly effective strain was selected from strawberry leaves, whose fermentation liquid could play a very good role in the field test. Studying the biological control mechanisms and their associated proteins which were expected to be applied to production had important theoretical and practical significance.
1、从草莓根际土壤、草莓植株及其它植物内分离、筛选到6株对草莓灰霉病菌有强拮抗作用的菌株,来自草莓叶的SL_6菌株抑菌效果最佳,抑菌半径达15 mm,无菌发酵液对草莓灰霉菌丝的抑制率为97.6%。经形态、生理生化和16S rDNA等分析测定,并通过MEGA4方法构建其16S rDNA系统发育树,将SL_6菌株鉴定为枯草芽孢杆菌(Bacillus subtilis)。
2、菌株抑菌机理初步研究表明:SL_6代谢产物能强烈抑制病原菌菌丝的生长并对病菌菌丝形态明显的破坏作用。滤液对菌丝形态的破坏作用主要表现为菌丝原生质收缩、菌丝部分膨大变形、细胞壁破裂和原生质溢出等,使得菌丝不能继续生长,甚至发生断裂。培养滤液用70%硫酸铵沉淀,上清没有抑菌活性。可以初步确定该抗菌物质为可溶性蛋白质。其拮抗物质产生的最佳条件为:接种于100 mL pH 7.0的PSB液体培养基中,置于37℃恒温振荡器中培养72 h。
3、SL_6培养滤液用70%硫酸铵进行盐析,沉淀物经透析后得粗蛋白。粗蛋白进行DEAE Sepharose Fast Flow离子交换层析得到抗菌活性蛋白,活性物质中具有很高的蛋白酶活力,进行SDS-PAGE及非变性凝胶电泳,均出现三条分子量大小相近蛋白带,通过质谱分析,得三条蛋白分别为30S核糖体蛋白、核酸酶和脱羧酶。测定了温度和pH对抗菌粗蛋白发挥抑菌作用的影响,结果表明,抗菌粗蛋白具有热稳定性,100℃处理30 min后,抑菌活性仍能达到60%以上,pH 2.0-9.0之间均有较强抑菌活性作用。
4、通过田间试验,可以验证生防枯草芽孢杆菌SL_6发酵液对于草莓灰霉病的发生具有预防和治疗的效果,3次处理后的防效为86.6%,有效地抑制了草莓灰霉病情的扩散,验证了其可在自然环境中长期稳定地发挥作用,可用于开发草莓灰霉病害生防制剂研究证明生物制剂如能阻止或减少灰霉对于草莓花朵的侵染,就能有效地控制灰霉病害,而内生细菌能发挥该项作用。草莓内生细菌具有通过产生吲哚乙酸和溶解有机磷来促进植株生长的作用,但对其生防作用的研究国内外尚未有报道。本文筛选出的高效菌株来源于草莓叶片,其发酵液可在田间试验中发挥很好的防效。研究其生防机理及相关的蛋白并使之能应用于生产,具有重要的理论和实际意义。
Botrytis cinerea in strawberry is one of the major constraints which made the yield and quality reduced. The traditional chemical control has led to the formation of serious resistance and environmental problems. The microbial control of Botrytis cinerea in strawberry with safety and low toxicity is an important and effective way. In this research, we hope to get the antagonistic strain by screening the bacteria isolated from rhizosphere soil of strawberry, strawberry plants and other plants and study identification of the strains, fermentation optimization, and its antagonistic reaction mechanism.The main results are as follows:
1.Six strong antagonistic strains were isolated from the rhizosphere soil of strawberry, in them, strain SL_6 isolated from the leaf of strawberry was found to have the best control efficacy, the inhibition zone diameter was 15 mm and the inhibition rate could reach 97.6% with the fermentation filtrate, revealing potential in the aspect of antagonistic action. Based on the morphological,physiological,biochemical characteristics and 16S rDNA sequence analysis,through its 16S rDNA phylogenetic tree constructed with MEGA4, SL_6 was identified as Bacillus subtilis.
2. Study on inhibition mechanism of strain SL_6 demonstrated that metabolites produced by antagonistic strain SL_6 could do great damage to the mycelia and had inhibitory effects on the growth of Botrytis cinerea. Microscopic observation indicated that the mycelium inhibited by strain SL_6 mostly twisted, inflated, deformed, the mycelia wall broke, the protoplasm leaked from the wound and even hyphae lysed. The bacterial metabolites in the culture filtrate could be precipitated by 70% ammonium sulfate and its inhibition activity was similar to the damage of mycelium by strain SL_6, while the supernate couldn’t inhibit the pathogen growth, thereby indicating that the antagonistic substance was protein. The optimal culture condition of SL_6 was shaking culture at 37℃with initial pH 7.0 in 100mL PSB liquid medium for 3 days.
3. The bacterial metabolites in the culture filtrate could be precipitated by 70% ammonium sulfate. The crude protein was obtained by the dialysis of the precipitate and purified by using ammonium sulfate precipitation following DEAE Sepharose Fast Flow chromatography. There were three bands with similar molecular weight in SDS-PAGE and PAGE of the active substances which had high protease activity. By mass spectrometry, three proteins were identified respectively as 30S ribosomal protein, nuclease subunit and uncharacterized protein. The crude protein was rather stable, its antibacterial activity remained above 60% after treated for 30 min at 100℃, and its pH ranged from 2.0 to 9.0.
4. The fermentation filtrate of SL_6 showed effective control to Botrytis cinerea in strawberry in field experiments. After three treatments, the effect of fermentation broth was 86.6% and effectively inhibited the spread of disease. It could arrive to the conclusion that it could play a stable role in the long-term natural environment.
Studies had shown that biological agents, which could prevent or reduce gray mold of strawberry flowers from infection, can effectively control gray mold damage, and endophytic bacteria can play the role. The endophytic bacteria of strawberry could promote the plant growth by producing indole acetic acid or dissolving organic phosphorus. But their roles in biological control have not yet been reported at domestic and overseas reports. This highly effective strain was selected from strawberry leaves, whose fermentation liquid could play a very good role in the field test. Studying the biological control mechanisms and their associated proteins which were expected to be applied to production had important theoretical and practical significance.
引文
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