冬枣采后病害拮抗菌的筛选、发酵及活性物质研究
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摘要
本文以冬枣病害研究中常见的病原菌细交链格孢、多隔镰孢霉和串珠霉为靶标,从森林和菜园土壤中筛选防治三种病原菌的拮抗菌。通过测定对不同病原菌的拮抗能力,确定拮抗菌的抑菌谱,从中筛选具有研究和开发价值的拮抗菌研究。对具有较好拮抗作用和较宽抑菌谱的拮抗菌B26确定其分类学地位；通过正交试验和单因子实验,研究B26产生拮抗物质的最优条件；并进一步研究拮抗菌粗蛋白对温度、酸碱度、紫外线照射、有机溶剂、蛋白酶的稳定性,结果如下：
从土壤中分离筛选出的11株对细交链格孢、多隔镰孢霉和串珠霉具有较好抑制作用的拮抗菌。平板对峙实验结果表明：B26的抑菌效果最显著,对细交链格孢的平均抑菌圈半径为15.8mm,抑制率达78.8%；对多隔镰孢霉和串珠霉的平均抑菌圈半径分别为14.3 mm和12.8 mm,抑制率分别为71.3%和63.8%,具有进一步研究的价值。通过观察在PDA培养基上生长的形态学特征、结合生理生化实验以及PCR产物测序结果,鉴定B26为枯草芽孢杆菌(Bacillus subtilis)。
通过正交试验设计对培养基成分及用量进行优化,单因子实验对B26产生活性物质的发酵条件进行选择。B26在种子培养基扩大培养后接入不同成分的发酵培养基,获得产生拮抗效果最好的培养基成分的用量是：蛋白胨25 g,淀粉40 g,氯化钠1.5 g,硫酸铵0.8 g,磷酸氢二钾1.5 g；产生活性物质的最优发酵条件为：种子菌龄24 h,接种量5%,初始pH 7,装液量20%,吐温80用量0.5%,发酵温度为30℃,转速150rpm,发酵时间为60 h,获得B26菌株发酵液对多隔镰孢霉的抑制直径为26mm,大于其他条件培养获得的拮抗菌直径。
枯草芽孢杆菌B26发酵液在4℃,经8000rpm,离心20 min后,过0.45μm微孔滤膜后,用70%的硫酸铵沉淀,透析后获得仍具有拮抗细交链格孢、多隔镰孢霉和串珠霉活性的无菌粗蛋白。进一步通过实验检测粗蛋白对温度、酸碱度、紫外线照射、有机溶剂和蛋白酶处理的稳定性,结果表明：B26的粗蛋白经100℃,20 min处理后,仍是常温处理下抑菌直径的78.2%,对高温具有一定的耐受性；但经121℃,20 min处理后,B26粗蛋白完全失去活性；粗蛋白在pH 3-pH 10范围内都具有抑菌活性,具有较宽的pH适应范围,但在pH为7时抑菌能力最高,所以应注意B26抑菌蛋白的纯化应在偏中性的条件下进行；粗蛋白对紫外线照射有部分敏感性,B26的抑菌粗蛋白经距离40 cm,20W紫外灯照射80 min后,抑菌活性是对照的78.1%;有机溶剂对枯草芽孢杆菌B26的抑菌粗蛋白的抑菌活性影响很小,经过乙醚,甲醇,氯仿,和丙酮有机溶剂处理过的粗蛋白的抑菌活性分别为对照的96.4%、85.7%、82.1%、81.5%；B26的抑菌粗蛋白对三种蛋白酶具有部分敏感性,B26的抑菌粗蛋白经过蛋白酶K处理后,拮抗直径活性为15.2 mm为对照的76.7%,经过胰蛋白酶处理的拮抗直径为16.2 mm,是未处理的81.8%,而经过胃蛋白酶处理过的,拮抗直径为16.3mm,抑制率是未处理的83.3%。
In this thesis, we set Alternaria alternate, Fusarium decemcellulare Brick and Monilia fructicola as target, which are common pathogens in the research of'Dongzao'Jujube, screened antagonists from the soil of the forest and garden aimed at controlling the three pathogens. By means of measuring antagonistic ability of the different we successfully determined its antagonistic spectrum, and then screened antagonists of important value for future research and development. We identified taxonomic status of antagonist B26 which is antagonistically more effective and with wider antimicrobial spectrum; By conducting the orthogonal experiments and single factor experiments, the optimal conditions for the fermentation of B26 was studied; also, we further studied the antagonistic extract's stability under the treatment of temperature, pH, ultraviolet radiation, organic solvents, proteases and the results were as follows:
Eleven strains of antagonists were isolated from the soil samples, which had better antagonistic effects on Alternaria alternate, Fusarium decemcellulare Brick and Monilia fructicola. The duel cultural tests showed B26 had the strongest antagonism against the three selected fungal pathogens, the average radius of inhibition zone against Alternaria alternate was 15.8 mm and inhibition rate against Alternaria alternate was 78.8%.The duel culture tests also showed B26 antagonistic had effect on Fusarium decemcellulare Brick and Monilia fructicola, the average radius of inhibition zone were 14.3 mm and 12.8 mm, inhibition rates were 71.3% and 63.8%, so B26 is valuable for further study. Based on the observation of B26's morphological features on PDA and combined usage of 16s rDNA sequence analysis with physiological and biochemical experiments, we initially identified B26 as Bacillus subtilis.
Through orthogonal experiments and single-factor experiments, the medium components and dosages were optimized and the optimum conditions of fermentation for producing antibacterial substances were selected. Microbial strains were inoculated into different fermentation mediums with different prescriptions after B26 was intermediate cultured in Seed culture medium, after that, the optimum medium components and dosages for producing antagonistic substances were obtained as follows: peptone 25 g, starch 40 g, sodium chloride 1.5 g, ammonium sulfate 0.8 g, potassium hydrogen phosphate 1.5 g. The optimum conditions of fermentation for producing antagonistic substances were:liquid seed age 24 h, inoculum sizes 5%, initial pH 7, aeration rates 20%, glycerol 0.5%, fermentation temperature 30℃, rotation speed 150 rpm, fermentation time 60 h.The average inhibition diameter of B26 fermented filtrates larger than other conditions against Fusarium decemcellulare Brick.
The fermented filtrates of B26 from optimum culture conditions were centrifuged for 20 min at 8000rpm,4℃and then filtered through 0.45μm filter membrane before analysis. After that, Fermentation supernatant was precipitated with (NH4)2SO4 solution at 70% saturation degree. Aseptic crude extractions were dialyzed from precipitation solution, which still have antagonistic ability against Alternaria alternate, Fusarium decemcellulare Brick and Monilia fructicola. The antagonistic substance stability with different treatments of temperature, pH, ultraviolet radiation, organic solvents, proteases were obtained by further study as follows:Inhibition diameter was 78.2% control after crude antagonistic protein of B26 was treated by 100℃,20min,it means crude antagonistic protein can be resistant to high temperature, but lost the antagonistic activity completely after the treatment with 121℃,20min. Purification of antagonistic protein of B26 should be at neutral pH for antagonistic ability was the highest, regardless that the antagonistic protein have antagonistic activity at pH 3-pH 10, a wide range of pH. Crude antagonist protein solution was partially sensitive to Ultraviolet. Inhibition diameter was 78.1% of control after exposed for 80 min under UV light with distance 40 cm, Power 20 w. Organic solvents have little effect on crude antagonist protein, Inhibition diameters were 96.4%、85.7%、82.1%、81.5% respectively after treatment with Ether, chloroform, methanol, and acetone for 30 min.Crude antagonist protein was partially sensitive to the three protease, when treated with Proteinase K, trypsin,pepsin, inhibition diameter was 15.2 mm,16.2 mm,16.3 mm, which was76.7%,81.8%,83.3% control respectively.
从土壤中分离筛选出的11株对细交链格孢、多隔镰孢霉和串珠霉具有较好抑制作用的拮抗菌。平板对峙实验结果表明：B26的抑菌效果最显著,对细交链格孢的平均抑菌圈半径为15.8mm,抑制率达78.8%；对多隔镰孢霉和串珠霉的平均抑菌圈半径分别为14.3 mm和12.8 mm,抑制率分别为71.3%和63.8%,具有进一步研究的价值。通过观察在PDA培养基上生长的形态学特征、结合生理生化实验以及PCR产物测序结果,鉴定B26为枯草芽孢杆菌(Bacillus subtilis)。
通过正交试验设计对培养基成分及用量进行优化,单因子实验对B26产生活性物质的发酵条件进行选择。B26在种子培养基扩大培养后接入不同成分的发酵培养基,获得产生拮抗效果最好的培养基成分的用量是：蛋白胨25 g,淀粉40 g,氯化钠1.5 g,硫酸铵0.8 g,磷酸氢二钾1.5 g；产生活性物质的最优发酵条件为：种子菌龄24 h,接种量5%,初始pH 7,装液量20%,吐温80用量0.5%,发酵温度为30℃,转速150rpm,发酵时间为60 h,获得B26菌株发酵液对多隔镰孢霉的抑制直径为26mm,大于其他条件培养获得的拮抗菌直径。
枯草芽孢杆菌B26发酵液在4℃,经8000rpm,离心20 min后,过0.45μm微孔滤膜后,用70%的硫酸铵沉淀,透析后获得仍具有拮抗细交链格孢、多隔镰孢霉和串珠霉活性的无菌粗蛋白。进一步通过实验检测粗蛋白对温度、酸碱度、紫外线照射、有机溶剂和蛋白酶处理的稳定性,结果表明：B26的粗蛋白经100℃,20 min处理后,仍是常温处理下抑菌直径的78.2%,对高温具有一定的耐受性；但经121℃,20 min处理后,B26粗蛋白完全失去活性；粗蛋白在pH 3-pH 10范围内都具有抑菌活性,具有较宽的pH适应范围,但在pH为7时抑菌能力最高,所以应注意B26抑菌蛋白的纯化应在偏中性的条件下进行；粗蛋白对紫外线照射有部分敏感性,B26的抑菌粗蛋白经距离40 cm,20W紫外灯照射80 min后,抑菌活性是对照的78.1%;有机溶剂对枯草芽孢杆菌B26的抑菌粗蛋白的抑菌活性影响很小,经过乙醚,甲醇,氯仿,和丙酮有机溶剂处理过的粗蛋白的抑菌活性分别为对照的96.4%、85.7%、82.1%、81.5%；B26的抑菌粗蛋白对三种蛋白酶具有部分敏感性,B26的抑菌粗蛋白经过蛋白酶K处理后,拮抗直径活性为15.2 mm为对照的76.7%,经过胰蛋白酶处理的拮抗直径为16.2 mm,是未处理的81.8%,而经过胃蛋白酶处理过的,拮抗直径为16.3mm,抑制率是未处理的83.3%。
In this thesis, we set Alternaria alternate, Fusarium decemcellulare Brick and Monilia fructicola as target, which are common pathogens in the research of'Dongzao'Jujube, screened antagonists from the soil of the forest and garden aimed at controlling the three pathogens. By means of measuring antagonistic ability of the different we successfully determined its antagonistic spectrum, and then screened antagonists of important value for future research and development. We identified taxonomic status of antagonist B26 which is antagonistically more effective and with wider antimicrobial spectrum; By conducting the orthogonal experiments and single factor experiments, the optimal conditions for the fermentation of B26 was studied; also, we further studied the antagonistic extract's stability under the treatment of temperature, pH, ultraviolet radiation, organic solvents, proteases and the results were as follows:
Eleven strains of antagonists were isolated from the soil samples, which had better antagonistic effects on Alternaria alternate, Fusarium decemcellulare Brick and Monilia fructicola. The duel cultural tests showed B26 had the strongest antagonism against the three selected fungal pathogens, the average radius of inhibition zone against Alternaria alternate was 15.8 mm and inhibition rate against Alternaria alternate was 78.8%.The duel culture tests also showed B26 antagonistic had effect on Fusarium decemcellulare Brick and Monilia fructicola, the average radius of inhibition zone were 14.3 mm and 12.8 mm, inhibition rates were 71.3% and 63.8%, so B26 is valuable for further study. Based on the observation of B26's morphological features on PDA and combined usage of 16s rDNA sequence analysis with physiological and biochemical experiments, we initially identified B26 as Bacillus subtilis.
Through orthogonal experiments and single-factor experiments, the medium components and dosages were optimized and the optimum conditions of fermentation for producing antibacterial substances were selected. Microbial strains were inoculated into different fermentation mediums with different prescriptions after B26 was intermediate cultured in Seed culture medium, after that, the optimum medium components and dosages for producing antagonistic substances were obtained as follows: peptone 25 g, starch 40 g, sodium chloride 1.5 g, ammonium sulfate 0.8 g, potassium hydrogen phosphate 1.5 g. The optimum conditions of fermentation for producing antagonistic substances were:liquid seed age 24 h, inoculum sizes 5%, initial pH 7, aeration rates 20%, glycerol 0.5%, fermentation temperature 30℃, rotation speed 150 rpm, fermentation time 60 h.The average inhibition diameter of B26 fermented filtrates larger than other conditions against Fusarium decemcellulare Brick.
The fermented filtrates of B26 from optimum culture conditions were centrifuged for 20 min at 8000rpm,4℃and then filtered through 0.45μm filter membrane before analysis. After that, Fermentation supernatant was precipitated with (NH4)2SO4 solution at 70% saturation degree. Aseptic crude extractions were dialyzed from precipitation solution, which still have antagonistic ability against Alternaria alternate, Fusarium decemcellulare Brick and Monilia fructicola. The antagonistic substance stability with different treatments of temperature, pH, ultraviolet radiation, organic solvents, proteases were obtained by further study as follows:Inhibition diameter was 78.2% control after crude antagonistic protein of B26 was treated by 100℃,20min,it means crude antagonistic protein can be resistant to high temperature, but lost the antagonistic activity completely after the treatment with 121℃,20min. Purification of antagonistic protein of B26 should be at neutral pH for antagonistic ability was the highest, regardless that the antagonistic protein have antagonistic activity at pH 3-pH 10, a wide range of pH. Crude antagonist protein solution was partially sensitive to Ultraviolet. Inhibition diameter was 78.1% of control after exposed for 80 min under UV light with distance 40 cm, Power 20 w. Organic solvents have little effect on crude antagonist protein, Inhibition diameters were 96.4%、85.7%、82.1%、81.5% respectively after treatment with Ether, chloroform, methanol, and acetone for 30 min.Crude antagonist protein was partially sensitive to the three protease, when treated with Proteinase K, trypsin,pepsin, inhibition diameter was 15.2 mm,16.2 mm,16.3 mm, which was76.7%,81.8%,83.3% control respectively.
引文
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