大丽轮枝菌拮抗细菌DM-54菌株的分离及抗菌蛋白纯化
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摘要
棉花黄萎病是由大丽轮枝菌(Verticillium dahliae)侵染引起的真菌性维管束系统病害,是棉花最严重的病害之一,给我国棉花生产造成严重损失,被称为“棉花的癌症”目前以菌治菌的生物防治被认为是一种最具有发展潜力的有效防治方法。
本研究从棉花根际土壤中分离到的891株细菌菌株中筛选出对大丽轮枝菌的生长有抑制作用的89株,其中10株拮抗菌株的无菌体发酵液表现拮抗活性,说明10株拮抗菌株主要是通过产生抗生类物质来抑制大丽轮枝菌的生长,其中DM-54菌株的活性最高。经进一步分析发现DM-54菌株所分泌的抗生类物质属于蛋白质类。DM-54菌株发酵液分别对12种病原菌进行抑菌实验,结果表明对其中大丽轮枝(Verticillium dahliae Kleb),胶胞炭疽菌(Colletotrichum gloeosporioides Penz),串珠镰孢菌(Fusarium moniliforme Sheld),剑兰炭疽(Glomerella cingulata)等8种病原菌有较高的拮抗活性,属较广抗菌谱的细菌,生防应用前景广阔。
通过结合形态特征、生理生化反应及16S rDNA基因序列分析确定DM-54菌株为解淀粉芽孢杆菌(Bacillus amyloliquefaciens)。
对解淀粉芽孢杆菌B. amyloliquefaciens DM-54菌株产抗菌物质的液态发酵条件进行优化,确定DM-54菌株发酵产抗菌蛋白最适发酵培养基为：糊精5%,大豆蛋白胨3%,MgSO4 0.02%, CaCl2 0.01%,初始pH 7.0；250 mL三角瓶装液量30 mL,种龄16h,接种量10%,摇床转速200r/min,发酵温度32℃,发酵时间48 h。优化前后蛋白抑菌活性差别明显,优化之前抑菌圈直径16.3 mm,优化后22.8 mm。
芽孢是微生物菌剂的最佳制剂形式,对恶劣的环境因素的具有抗性,可延长制剂保存期,稳定活性。对B. amyloliquefaciens DM-54菌株产芽孢条件进一步进行摸索,产芽孢最适发酵培养基为：乳糖0.5%、黄豆饼粉2.0%、MnSO4·H2O0.3%、培养液初始pH值为7.5；种龄18 h、装液量50 mL/250 mL、接种量10%、发酵时间36 h、温度为30℃、转速为220r/min。在此优化条件下,DM-54菌株发酵液中的菌体芽孢形成率大大提高,最终生物量达到2×109个/mL,芽孢形成率达到98.3%。
通过盆栽实验,考察棉花黄萎病拮抗细菌DM-54菌株在土壤中的定殖情况及在棉花根内的定殖情况。本实验分别对灭菌土和非灭菌土这两种土样进行拮抗菌的浸种、全部混土和部分混土三种不同处理进行盆栽实验。结果表明,30d后拮抗细菌DM-54菌株能够在灭菌土和非灭菌中定殖,定殖数量达106cfu/g土左右,经过30 d后并保持较高的抑菌活性,并且菌株在灭菌土中的定殖数量高于非灭菌土中的数量。施用方法中以其中全部混土情况下定殖能力最强。通过对棉苗根内细菌的回收,证实拮抗细菌DM-54能在棉花根内定殖,定殖数量达103cfu/g左右。
B. amyloliquefaciens DM-54菌株胞外分泌蛋白溶液经DEAE Sepharose Fast Flow阴离子交换层析后分离纯化到分子质量约为47 KDa的抗菌蛋白。DM-54抗菌蛋白最适作用温度为28℃,蛋白活性随着温度增加而下降。最适pH值为8.0。Cu2+则使蛋白完全失活,Mn2+对蛋白有明显的激活作用。
Cotton Verticillium wilt is one of the most dangerous fascicular disease caused by Verticillium dahliae Kleb. V. dahliae caused serious losses to cotton production in China, known as the "cancer of cotton". Biological control which people pay more attention now is a highly effective measure for management of cotton Verticillium wilt.
Eight hundred ninety one strains having antagonistic activities against V. dahliae Kleb were isolated and screened from the soil of cotton field from several different regions.89 strains had antagonistic activity.10 strains had antagonistic activity after secondary screening. A strain named DM-54 strain with a rather higher antagonistic activity was obtained. The experiment result showed that the antifungal substance of DM-54 is protein. The antifungal protein showed antibiosis activity to 9 pathogenic fungis, such as V. dahliae, Colletotrichum gloeosporioides Penz, Ceratosphaeria phyllostachydis, Colletotrichum lagenarium, Fusarium moniliforme Sheld and so on.
Thus the morphology characteristics, physiological and biochemical properties and 16S rDNA sequence of this strain were further studied. Through 16S rDNA sequence comparison analysis as well as the physiological and biochemical properties analysis referring to Bergey's Manual of Determinative Bacteriology and Manual of systematic and Determinative Bacteriology, the DM-54 strain was finally identified as a kind of Bacillus amyloliquefaciens.
The further studies on the antifungal protein-producing conditions of the strain was carried out. Through single factor experiment and orthogonal experiment, the optimal shaking flask fermentation conditions were determined as follow:media composed of 5% dextrin,3% soy peptone,0.02% MgSO4,0.3% CaCl2, initial pH 7.0 and 10% inoculum volume, media volume 30/250(mL/mL), fermentation temperature 32℃, rotating speed 200r/min, fermentation time 48h. A distinguished elevation of the antagonistic activity was observed with about 39.9%. This essay paves a bright way for the large scale production of the antagonistic protein.
Spore is the best preparation form of microbial agents. Strain DM-54(Bacillus amyloliquefaciens) which was isolated and screened by our lab had better antagonistic activity against V. dahliae. In order to improve the formation rate and spore quantity of antagonistic bacterium strain DM-54. We investigated the main factors influencing sporulation using shake-flask fermentation method. Through single factor experiment and orthogonal experiment, the optimal shaking flask fermentation conditions were determined as follow: media composed of 0.5% lactose,2% soybean meal,0.3% MnSO_5·H_2O, initial pH 7.5, incubation time 18 h and bottle filling capacity 50 mL/250mL,10% inoculum volume, fermentation time 36h, fermentation temperature 30℃, rotating speed 220r/min. Under this optimum condition, the spore formation rate achieved 98.3%. Biomass achieves 2×109 spore /mL.
Colonization of antagonistic bacteria DM-54 in root soil of cotton was studied through pot experiment of sterilized and non-sterilized soil, part and all mixed soil three kind of different methods. The results showed that antagonistic bacterium DM-54 could colonize in the sterilized and non-sterilized under laboratory conditions,the population in the sterilized and non-sterilized soil was 106cfu/g,and still kept the high bacteriostatic activity. The numbers in the non-sterilized soil were less than those in the sterilized soil. The colonization activity of all mixed soil is the highest. The results showed that antagonistic bacterium DM-54 could colonize in root of cotton on 30th day after treatment.
An antagonistic protein was purified from the fermented broth of strain DM-54 by ammonium sulphate fractional precipitation, exchange chromatography on DEAE-Sepharose Fast Flow and preparative PAGE. The protein was a single chain polypeptide with a predicted molecular weight of 47 kDa in SDS-PAGE. We have done a preliminary research of the antifungal protein nature. The results showed that the optimum temperature of the antifungal protein was 28℃and the activity of antifungal protein increased with the temperature dropped. The optimum pH was 5.0, and Cu2+ was completely inactivating the antifungal protein, Mn2+ could apparently activate the antifungal protein.
本研究从棉花根际土壤中分离到的891株细菌菌株中筛选出对大丽轮枝菌的生长有抑制作用的89株,其中10株拮抗菌株的无菌体发酵液表现拮抗活性,说明10株拮抗菌株主要是通过产生抗生类物质来抑制大丽轮枝菌的生长,其中DM-54菌株的活性最高。经进一步分析发现DM-54菌株所分泌的抗生类物质属于蛋白质类。DM-54菌株发酵液分别对12种病原菌进行抑菌实验,结果表明对其中大丽轮枝(Verticillium dahliae Kleb),胶胞炭疽菌(Colletotrichum gloeosporioides Penz),串珠镰孢菌(Fusarium moniliforme Sheld),剑兰炭疽(Glomerella cingulata)等8种病原菌有较高的拮抗活性,属较广抗菌谱的细菌,生防应用前景广阔。
通过结合形态特征、生理生化反应及16S rDNA基因序列分析确定DM-54菌株为解淀粉芽孢杆菌(Bacillus amyloliquefaciens)。
对解淀粉芽孢杆菌B. amyloliquefaciens DM-54菌株产抗菌物质的液态发酵条件进行优化,确定DM-54菌株发酵产抗菌蛋白最适发酵培养基为：糊精5%,大豆蛋白胨3%,MgSO4 0.02%, CaCl2 0.01%,初始pH 7.0；250 mL三角瓶装液量30 mL,种龄16h,接种量10%,摇床转速200r/min,发酵温度32℃,发酵时间48 h。优化前后蛋白抑菌活性差别明显,优化之前抑菌圈直径16.3 mm,优化后22.8 mm。
芽孢是微生物菌剂的最佳制剂形式,对恶劣的环境因素的具有抗性,可延长制剂保存期,稳定活性。对B. amyloliquefaciens DM-54菌株产芽孢条件进一步进行摸索,产芽孢最适发酵培养基为：乳糖0.5%、黄豆饼粉2.0%、MnSO4·H2O0.3%、培养液初始pH值为7.5；种龄18 h、装液量50 mL/250 mL、接种量10%、发酵时间36 h、温度为30℃、转速为220r/min。在此优化条件下,DM-54菌株发酵液中的菌体芽孢形成率大大提高,最终生物量达到2×109个/mL,芽孢形成率达到98.3%。
通过盆栽实验,考察棉花黄萎病拮抗细菌DM-54菌株在土壤中的定殖情况及在棉花根内的定殖情况。本实验分别对灭菌土和非灭菌土这两种土样进行拮抗菌的浸种、全部混土和部分混土三种不同处理进行盆栽实验。结果表明,30d后拮抗细菌DM-54菌株能够在灭菌土和非灭菌中定殖,定殖数量达106cfu/g土左右,经过30 d后并保持较高的抑菌活性,并且菌株在灭菌土中的定殖数量高于非灭菌土中的数量。施用方法中以其中全部混土情况下定殖能力最强。通过对棉苗根内细菌的回收,证实拮抗细菌DM-54能在棉花根内定殖,定殖数量达103cfu/g左右。
B. amyloliquefaciens DM-54菌株胞外分泌蛋白溶液经DEAE Sepharose Fast Flow阴离子交换层析后分离纯化到分子质量约为47 KDa的抗菌蛋白。DM-54抗菌蛋白最适作用温度为28℃,蛋白活性随着温度增加而下降。最适pH值为8.0。Cu2+则使蛋白完全失活,Mn2+对蛋白有明显的激活作用。
Cotton Verticillium wilt is one of the most dangerous fascicular disease caused by Verticillium dahliae Kleb. V. dahliae caused serious losses to cotton production in China, known as the "cancer of cotton". Biological control which people pay more attention now is a highly effective measure for management of cotton Verticillium wilt.
Eight hundred ninety one strains having antagonistic activities against V. dahliae Kleb were isolated and screened from the soil of cotton field from several different regions.89 strains had antagonistic activity.10 strains had antagonistic activity after secondary screening. A strain named DM-54 strain with a rather higher antagonistic activity was obtained. The experiment result showed that the antifungal substance of DM-54 is protein. The antifungal protein showed antibiosis activity to 9 pathogenic fungis, such as V. dahliae, Colletotrichum gloeosporioides Penz, Ceratosphaeria phyllostachydis, Colletotrichum lagenarium, Fusarium moniliforme Sheld and so on.
Thus the morphology characteristics, physiological and biochemical properties and 16S rDNA sequence of this strain were further studied. Through 16S rDNA sequence comparison analysis as well as the physiological and biochemical properties analysis referring to Bergey's Manual of Determinative Bacteriology and Manual of systematic and Determinative Bacteriology, the DM-54 strain was finally identified as a kind of Bacillus amyloliquefaciens.
The further studies on the antifungal protein-producing conditions of the strain was carried out. Through single factor experiment and orthogonal experiment, the optimal shaking flask fermentation conditions were determined as follow:media composed of 5% dextrin,3% soy peptone,0.02% MgSO4,0.3% CaCl2, initial pH 7.0 and 10% inoculum volume, media volume 30/250(mL/mL), fermentation temperature 32℃, rotating speed 200r/min, fermentation time 48h. A distinguished elevation of the antagonistic activity was observed with about 39.9%. This essay paves a bright way for the large scale production of the antagonistic protein.
Spore is the best preparation form of microbial agents. Strain DM-54(Bacillus amyloliquefaciens) which was isolated and screened by our lab had better antagonistic activity against V. dahliae. In order to improve the formation rate and spore quantity of antagonistic bacterium strain DM-54. We investigated the main factors influencing sporulation using shake-flask fermentation method. Through single factor experiment and orthogonal experiment, the optimal shaking flask fermentation conditions were determined as follow: media composed of 0.5% lactose,2% soybean meal,0.3% MnSO_5·H_2O, initial pH 7.5, incubation time 18 h and bottle filling capacity 50 mL/250mL,10% inoculum volume, fermentation time 36h, fermentation temperature 30℃, rotating speed 220r/min. Under this optimum condition, the spore formation rate achieved 98.3%. Biomass achieves 2×109 spore /mL.
Colonization of antagonistic bacteria DM-54 in root soil of cotton was studied through pot experiment of sterilized and non-sterilized soil, part and all mixed soil three kind of different methods. The results showed that antagonistic bacterium DM-54 could colonize in the sterilized and non-sterilized under laboratory conditions,the population in the sterilized and non-sterilized soil was 106cfu/g,and still kept the high bacteriostatic activity. The numbers in the non-sterilized soil were less than those in the sterilized soil. The colonization activity of all mixed soil is the highest. The results showed that antagonistic bacterium DM-54 could colonize in root of cotton on 30th day after treatment.
An antagonistic protein was purified from the fermented broth of strain DM-54 by ammonium sulphate fractional precipitation, exchange chromatography on DEAE-Sepharose Fast Flow and preparative PAGE. The protein was a single chain polypeptide with a predicted molecular weight of 47 kDa in SDS-PAGE. We have done a preliminary research of the antifungal protein nature. The results showed that the optimum temperature of the antifungal protein was 28℃and the activity of antifungal protein increased with the temperature dropped. The optimum pH was 5.0, and Cu2+ was completely inactivating the antifungal protein, Mn2+ could apparently activate the antifungal protein.
引文
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