多功能菌剂的组合效应研究及其抑菌物质的特性和分离
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摘要
棉花和加工番茄是新疆重要的经济作物,针对早春气温变化大、苗期烂根病发病重和单一菌株生防效果不稳定等问题,开展了多功能细菌组合的筛选及其效应研究。研究结果如下:
根据菌株生长特性、相互间拮抗和组合抑菌活性等试验结果,筛选了23个抑菌活性优于单一芽孢杆菌的组合,发现芽孢杆菌组合BCL-8具有显著的抗病促生效果。室内促生试验表明:组合BCL-8可促进加工番茄发芽和苗期生长,发芽率可提高10%,使苗期株高、鲜重和干重分别增加66.58%、176.19%和143.75%。在温室和田间条件下,组合BCL-8对加工番茄立枯病的防治效果分别达40.08%和55.42%。对构成组合BCL-8的3株芽孢杆菌SL-13、SL-14和SL-44进行了常规生理生化特征和16S rDNA的分析,结果表明SL-13、SL-14和SL-44均为枯草芽孢杆菌(Bacillus subtilis),Genbank登录号分别为:FJ788426,FJ788427和FJ788428。
通过研究BCL-8发酵后各组分的抑菌活性,确定了盐析粗提物为该组合的主要抑菌物质,并初步确定为蛋白类物质。BCL-8抑菌粗蛋白理化性质研究表明,该抑菌蛋白热稳定性好,在120℃处理30min后仍保持较高的抑菌活性;活性pH范围在4.0~10.0之间;能耐受乙醇、甲醇、乙酸乙酯、丙酮、乙腈、石油醚、氯仿多种有机溶剂,且稳定性好;紫外线照射不影响其抑菌活性;对蛋白酶K作用不敏感;粗蛋白能有效抑制Rhizoctonia solani Kuhn、Fusarium oxysporum f. sp. lycopersici、Phytophthora capsici、Sclerotinia sclerotiorum、Acidovorax avenae sub sp. citrulli、Alternaria solani Sorauer等多种病原菌。
枯草芽孢杆菌SL-13,SL-14和SL-44的无菌发酵液可分别通过30%-60%硫酸铵、40%-100%硫酸铵和30%-50%硫酸铵盐析沉淀获得活性粗蛋白。经DEAE -Sepharose Fast Flow离子交换层析、Sephadex G-75凝胶过滤和二次离子交换层析后,从菌株SL-13、SL-14和SL-44的活性粗蛋白中分别获得一个活性峰,经检测其蛋白含量分别为0.0052 mg/mL、0.0087 mg/mL和0.0021 mg/mL,蛋白回收率分别为6.1%、8.7%和4.2%,纯化倍数分别为60.1、56.7和75.1,经SDS-PAGE电泳分析其分子量大小分别为30kD,24kD和17.8kD。
通过对菌株SL-13,SL-14和SL-44抑菌物质基本性质的检测和含有几丁质和酪蛋白的平板实验发现SL-13菌株的抑菌蛋白具有较高的几丁质酶活性。在合成培养基条件下研究了菌体生长、抑菌活性以及几丁质酶活性三者的关系,发现抑菌活性与几丁质酶活性成正相关,说明几丁质酶是枯草芽孢杆菌SL-13产生的主要抑菌物质。使用纯化的几丁质酶处理立枯丝核菌能够水解破坏立枯丝核菌,使菌丝断裂,内溶物泄露,具有抑菌杀菌的作用;SL-13产几丁质酶的最适反应温度为50℃;在pH3～9的范围内都具有良好的活性,酶与底物反应的最适pH为7.0。
将抗病促生芽孢杆菌和缓解盐胁迫促生细菌进行组合筛选,根据体外抑菌活性初筛、盆栽和小区试验,筛选出组合多功能菌剂BCL-8+Rs-5,该菌剂在盆栽和小区条件下对棉花立枯病的防效分别达54.41%和51.06%,在盐胁迫条件下使棉苗干重提高了40.85%,具有显著的抗病促生和缓解盐胁迫效能。多功能菌剂的大田试验结果表明,菌剂包衣处理对棉花苗期立枯病的防治效果达16.24%,滴灌处理对棉花苗期生长有明显的促进作用,增产率可达10.51%;菌剂滴灌处理对加工番茄苗期立枯病的防治效果达26.34%,使开花数提高30.31%,结果数提高29.07%,增产率为5.04%。
经多功能菌剂处理后,棉花和加工番茄植株根部的抗性酶类(POD,PPO,PAL,SOD和CAT)活性均有不同程度的提高,即多功能菌剂能诱导棉花和加工番茄产生系统抗病性。多功能菌剂对作物根际土壤可培养微生物组成和数量的影响不同,滴灌处理可有效提高土壤中细菌和放线菌数量,减少真菌数量,符合健康土壤的微生态分布趋势。土壤细菌16S-rDNA DGGE指纹分析与平板计数分离方法得到的结果一致。
The cotton and the processing tomato are the main industrial crops in Xinjiang province. The root rot diseases happened severely because of temperature changes in spring in xinjiang and the biological control effect of single strain was unstable. So we carried out the research of screen and efficacy of multifunctional strains formulation. The major results of this study are as follows:
According to the results of growth characterization, antagonistic experiments between single strain and antagonistic examination of combinations in vitro, 23 combinations with superior suppressive effect than mono antagonistic strains were obtained. By further in vivo inoculation test, the combination BCL-8 was shown to have remarkable disease control and growth-promoting effect. The combination BCL-8 could promote seed sprouting of processing tomato. When treated with BCL-8, the seeds germination rate increased 10%, and the seedling height, fresh weight and dry weight of processing tomato seedlings increased 66.58%, 176.19% and 143.75%, respectively. The control efficacy of the combination BCL-8 to seedling Rhizoctonia rot of processing tomato were 40.08% and 55.53% under greenhouse and field conditions, respectively. The combination BCL-8 also increased production of processing tomato 24.32% in the field. The combination BCL-8 was composed of Bacillus sp. SL-13, SL-14 and SL-44.These three strains have been identified as Bacillus subtilis,GenBank accession number are FJ788426,FJ788427 and FJ788428.
It was showed that the crude antagonistic substance was determined as a kind of protein by the reactions of triketohydrindene hydrate and biure. This protein was thermostable for it had strong inhibitory activities after treated at 120℃for 30min. It was proved that the protein was still active in buffer with pH from 4.0 to 10.0. The antagonistic effect of protein treated with various kinds of organic solvent was displayed that it was stable in ethnol, methanol, acetone, chloroform and so on. The inhibitory titer of protein exposed under ultraviolet radiation was similar to visible light. Proteinase K had no effect on its activity. Several pathogens were strongly inhibited by the protein, such as Fusarium oxysporum f.sp. lycopersici,Rhizoctonia solani Kuhn, Phytophthora capsici,Sclerotinia sclerotiorum, Acidovorax avenae sub sp Citrulli and Alternaria solani Sorauer.
Antibiotic substances produced by B. subtilis SL-13, SL-14 and SL-44 were extracted by 30%-60%,40%-100% and 30%-50% ammonium sulfate-fractionation, respectively. Then antibiotic substances were partially separated by means of DEAE-Sepharose Fast Flow column chromatography, sephadex G-75 column and second round DEAE-Sepharose Fast Flow column chromatography. One active peak of B. subtilis SL-13, SL-14 and SL-44 were yielded during step elution. The antifungal proteins recycling rate were 6.1%、8.7% and 4.2%, respectively. The concentration were 0.0052 mg/mL、0.0087 mg/mL and 0.0021 mg/mL, respectively. The molecular weights of the purified protein were confirmed as 33kD, 24kD and 17.8kD by SDS-PAGE, respectively.
Through examination of antifungal substances to B.subtilis SL-13, SL-14 and SL-44 and the plate experiments contain the chitin and the casein. We found that the B. subtilis strain SL-13 secretes proteins have high chitinase activity. The relations of B. subtilis SL-13 growth, chitinase activity and antifungal activity were studied in synthesized medium condition. With the extension of incubation the chitinase production had a positive correlation with the changing of antifungal activity. That is the chitinase was the main antifungal substances of strain SL-13. When detecting antifungal activity, the Rhizoctonia solani Kuhn treated by purified enzyme could not prolong, the cell wall broke, protoplasm leaked out, and the mycelia cracked. The microscopic analysis showed that the main effect of the purified enzyme was to degrade cell wall. The optimum temperature of the purified chitinase was 50?C. The enzyme was stable in the pH range of 5～9 for 1h at 50?C. It had an optimal pH of 7.0 when assayed with colloidal chitin as a substrate.
The growth promotion and disease control strains and salt-relieving and growth promotion strains were combined to screening multifunctional strains formulation. According to the results of antagonistic examination in vitro and greenhouse and plot experiment in vivo, The combination BCL-8+Rs-5 was found have remarkable growth promotion, salt-relieving and disease control effects. The control efficacy of the combination BCL-8+Rs-5 to seedling Rhizoctonia rot of cotton were 54.41% and 51.06% under greenhouse and plot conditions, respectively. The cotton seedling dry weight was increased by 40.85%. The study result indicated the control efficacy of BCL-8+Rs-5 to seedling Rhizoctonia root rot of cotton was 16.24% in field. The treatment of drip irrigation with BCL-8+Rs-5 could obvious promote growth of cotton seedling. The effect of increase production of cotton reached 10.51%. The control efficacy of treatment of drip irrigation with BCL-8+Rs-5 to seedling Rhizoctonia root rot of processing tomato was 26.34%. The number of blossoms was enhanced 30.31%, and the number of fruits was enhanced 29.07%. The effect of increase production of processing tomato reached 5.04%.
The activity of resistant enzyme (POD,PPO,PAL,SOD and CAT) increased after treated with BCL-8+Rs-5 in roots of cotton and processing tomato. It was obvious that the multifunctional strains formulation BCL-8+ Rs-5 have induction of systemic resistance. The influence of multifunctional strains formulation BCL-8+ Rs-5 is different to composition and quantity of plant rhizosphere microorganism. BCL-8+ Rs-5 can effectively raise the amount of the bacterial and actinomycete in soil and decrease the amount of the fungi. It conforms to the distributed tendency of healthy soil microecology. The result of 16S-rDNA DGGE fingerprint analysis showed consistent.
根据菌株生长特性、相互间拮抗和组合抑菌活性等试验结果,筛选了23个抑菌活性优于单一芽孢杆菌的组合,发现芽孢杆菌组合BCL-8具有显著的抗病促生效果。室内促生试验表明:组合BCL-8可促进加工番茄发芽和苗期生长,发芽率可提高10%,使苗期株高、鲜重和干重分别增加66.58%、176.19%和143.75%。在温室和田间条件下,组合BCL-8对加工番茄立枯病的防治效果分别达40.08%和55.42%。对构成组合BCL-8的3株芽孢杆菌SL-13、SL-14和SL-44进行了常规生理生化特征和16S rDNA的分析,结果表明SL-13、SL-14和SL-44均为枯草芽孢杆菌(Bacillus subtilis),Genbank登录号分别为:FJ788426,FJ788427和FJ788428。
通过研究BCL-8发酵后各组分的抑菌活性,确定了盐析粗提物为该组合的主要抑菌物质,并初步确定为蛋白类物质。BCL-8抑菌粗蛋白理化性质研究表明,该抑菌蛋白热稳定性好,在120℃处理30min后仍保持较高的抑菌活性;活性pH范围在4.0~10.0之间;能耐受乙醇、甲醇、乙酸乙酯、丙酮、乙腈、石油醚、氯仿多种有机溶剂,且稳定性好;紫外线照射不影响其抑菌活性;对蛋白酶K作用不敏感;粗蛋白能有效抑制Rhizoctonia solani Kuhn、Fusarium oxysporum f. sp. lycopersici、Phytophthora capsici、Sclerotinia sclerotiorum、Acidovorax avenae sub sp. citrulli、Alternaria solani Sorauer等多种病原菌。
枯草芽孢杆菌SL-13,SL-14和SL-44的无菌发酵液可分别通过30%-60%硫酸铵、40%-100%硫酸铵和30%-50%硫酸铵盐析沉淀获得活性粗蛋白。经DEAE -Sepharose Fast Flow离子交换层析、Sephadex G-75凝胶过滤和二次离子交换层析后,从菌株SL-13、SL-14和SL-44的活性粗蛋白中分别获得一个活性峰,经检测其蛋白含量分别为0.0052 mg/mL、0.0087 mg/mL和0.0021 mg/mL,蛋白回收率分别为6.1%、8.7%和4.2%,纯化倍数分别为60.1、56.7和75.1,经SDS-PAGE电泳分析其分子量大小分别为30kD,24kD和17.8kD。
通过对菌株SL-13,SL-14和SL-44抑菌物质基本性质的检测和含有几丁质和酪蛋白的平板实验发现SL-13菌株的抑菌蛋白具有较高的几丁质酶活性。在合成培养基条件下研究了菌体生长、抑菌活性以及几丁质酶活性三者的关系,发现抑菌活性与几丁质酶活性成正相关,说明几丁质酶是枯草芽孢杆菌SL-13产生的主要抑菌物质。使用纯化的几丁质酶处理立枯丝核菌能够水解破坏立枯丝核菌,使菌丝断裂,内溶物泄露,具有抑菌杀菌的作用;SL-13产几丁质酶的最适反应温度为50℃;在pH3～9的范围内都具有良好的活性,酶与底物反应的最适pH为7.0。
将抗病促生芽孢杆菌和缓解盐胁迫促生细菌进行组合筛选,根据体外抑菌活性初筛、盆栽和小区试验,筛选出组合多功能菌剂BCL-8+Rs-5,该菌剂在盆栽和小区条件下对棉花立枯病的防效分别达54.41%和51.06%,在盐胁迫条件下使棉苗干重提高了40.85%,具有显著的抗病促生和缓解盐胁迫效能。多功能菌剂的大田试验结果表明,菌剂包衣处理对棉花苗期立枯病的防治效果达16.24%,滴灌处理对棉花苗期生长有明显的促进作用,增产率可达10.51%;菌剂滴灌处理对加工番茄苗期立枯病的防治效果达26.34%,使开花数提高30.31%,结果数提高29.07%,增产率为5.04%。
经多功能菌剂处理后,棉花和加工番茄植株根部的抗性酶类(POD,PPO,PAL,SOD和CAT)活性均有不同程度的提高,即多功能菌剂能诱导棉花和加工番茄产生系统抗病性。多功能菌剂对作物根际土壤可培养微生物组成和数量的影响不同,滴灌处理可有效提高土壤中细菌和放线菌数量,减少真菌数量,符合健康土壤的微生态分布趋势。土壤细菌16S-rDNA DGGE指纹分析与平板计数分离方法得到的结果一致。
The cotton and the processing tomato are the main industrial crops in Xinjiang province. The root rot diseases happened severely because of temperature changes in spring in xinjiang and the biological control effect of single strain was unstable. So we carried out the research of screen and efficacy of multifunctional strains formulation. The major results of this study are as follows:
According to the results of growth characterization, antagonistic experiments between single strain and antagonistic examination of combinations in vitro, 23 combinations with superior suppressive effect than mono antagonistic strains were obtained. By further in vivo inoculation test, the combination BCL-8 was shown to have remarkable disease control and growth-promoting effect. The combination BCL-8 could promote seed sprouting of processing tomato. When treated with BCL-8, the seeds germination rate increased 10%, and the seedling height, fresh weight and dry weight of processing tomato seedlings increased 66.58%, 176.19% and 143.75%, respectively. The control efficacy of the combination BCL-8 to seedling Rhizoctonia rot of processing tomato were 40.08% and 55.53% under greenhouse and field conditions, respectively. The combination BCL-8 also increased production of processing tomato 24.32% in the field. The combination BCL-8 was composed of Bacillus sp. SL-13, SL-14 and SL-44.These three strains have been identified as Bacillus subtilis,GenBank accession number are FJ788426,FJ788427 and FJ788428.
It was showed that the crude antagonistic substance was determined as a kind of protein by the reactions of triketohydrindene hydrate and biure. This protein was thermostable for it had strong inhibitory activities after treated at 120℃for 30min. It was proved that the protein was still active in buffer with pH from 4.0 to 10.0. The antagonistic effect of protein treated with various kinds of organic solvent was displayed that it was stable in ethnol, methanol, acetone, chloroform and so on. The inhibitory titer of protein exposed under ultraviolet radiation was similar to visible light. Proteinase K had no effect on its activity. Several pathogens were strongly inhibited by the protein, such as Fusarium oxysporum f.sp. lycopersici,Rhizoctonia solani Kuhn, Phytophthora capsici,Sclerotinia sclerotiorum, Acidovorax avenae sub sp Citrulli and Alternaria solani Sorauer.
Antibiotic substances produced by B. subtilis SL-13, SL-14 and SL-44 were extracted by 30%-60%,40%-100% and 30%-50% ammonium sulfate-fractionation, respectively. Then antibiotic substances were partially separated by means of DEAE-Sepharose Fast Flow column chromatography, sephadex G-75 column and second round DEAE-Sepharose Fast Flow column chromatography. One active peak of B. subtilis SL-13, SL-14 and SL-44 were yielded during step elution. The antifungal proteins recycling rate were 6.1%、8.7% and 4.2%, respectively. The concentration were 0.0052 mg/mL、0.0087 mg/mL and 0.0021 mg/mL, respectively. The molecular weights of the purified protein were confirmed as 33kD, 24kD and 17.8kD by SDS-PAGE, respectively.
Through examination of antifungal substances to B.subtilis SL-13, SL-14 and SL-44 and the plate experiments contain the chitin and the casein. We found that the B. subtilis strain SL-13 secretes proteins have high chitinase activity. The relations of B. subtilis SL-13 growth, chitinase activity and antifungal activity were studied in synthesized medium condition. With the extension of incubation the chitinase production had a positive correlation with the changing of antifungal activity. That is the chitinase was the main antifungal substances of strain SL-13. When detecting antifungal activity, the Rhizoctonia solani Kuhn treated by purified enzyme could not prolong, the cell wall broke, protoplasm leaked out, and the mycelia cracked. The microscopic analysis showed that the main effect of the purified enzyme was to degrade cell wall. The optimum temperature of the purified chitinase was 50?C. The enzyme was stable in the pH range of 5～9 for 1h at 50?C. It had an optimal pH of 7.0 when assayed with colloidal chitin as a substrate.
The growth promotion and disease control strains and salt-relieving and growth promotion strains were combined to screening multifunctional strains formulation. According to the results of antagonistic examination in vitro and greenhouse and plot experiment in vivo, The combination BCL-8+Rs-5 was found have remarkable growth promotion, salt-relieving and disease control effects. The control efficacy of the combination BCL-8+Rs-5 to seedling Rhizoctonia rot of cotton were 54.41% and 51.06% under greenhouse and plot conditions, respectively. The cotton seedling dry weight was increased by 40.85%. The study result indicated the control efficacy of BCL-8+Rs-5 to seedling Rhizoctonia root rot of cotton was 16.24% in field. The treatment of drip irrigation with BCL-8+Rs-5 could obvious promote growth of cotton seedling. The effect of increase production of cotton reached 10.51%. The control efficacy of treatment of drip irrigation with BCL-8+Rs-5 to seedling Rhizoctonia root rot of processing tomato was 26.34%. The number of blossoms was enhanced 30.31%, and the number of fruits was enhanced 29.07%. The effect of increase production of processing tomato reached 5.04%.
The activity of resistant enzyme (POD,PPO,PAL,SOD and CAT) increased after treated with BCL-8+Rs-5 in roots of cotton and processing tomato. It was obvious that the multifunctional strains formulation BCL-8+ Rs-5 have induction of systemic resistance. The influence of multifunctional strains formulation BCL-8+ Rs-5 is different to composition and quantity of plant rhizosphere microorganism. BCL-8+ Rs-5 can effectively raise the amount of the bacterial and actinomycete in soil and decrease the amount of the fungi. It conforms to the distributed tendency of healthy soil microecology. The result of 16S-rDNA DGGE fingerprint analysis showed consistent.
引文
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