西瓜枯萎病拮抗芽孢杆菌HD-5菌株的筛选与发酵工艺
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摘要
西瓜枯萎病又叫蔓割病,是一种世界性分布的土传病害,是由尖孢镰刀菌(Fusarium oxysporum)引起的。西瓜枯萎病的田间发病率一般为10%-30%,特别是重茬瓜田的发病率尤为严重,减产20%-50%,甚至绝收,造成了巨大的经济损失。目前,从土壤中筛选拮抗微生物来防治病害逐渐成为研究的热点课题。本研究旨在筛选到对西瓜枯萎病菌尖孢镰刀菌具有较好生物防治潜力的拮抗芽孢细菌,并对其进行相关研究,以便在西瓜种植业中用于西瓜枯萎病的防治。
对土样中分离的产芽孢细菌进行初筛和复筛,得到3株具有较强抑菌活性的拮抗芽孢细菌HD-5、CZ-10、BY-1菌株,并对该3菌株进行了实验室西瓜枯萎病盆栽防治效果试验,最终获得了对西瓜枯萎病具有较好防治效果的拮抗芽孢细菌HD-5菌株。
通过对HD-5菌株的形态特征、生理生化特征考察,以及16S rDNA全序列分析表明HD-5菌株与解淀粉芽孢杆菌(Bacillus amyloliquefaciens)的生理生化特征相符,与其16S rDNA序列同源性高达99.78%的。因此,判定HD-5菌株为解淀粉芽孢杆菌(Bacillus amyloliquefaciens)。
通过对B. amyloliquefaciens HD-5菌株抗菌物质的性质分析,初步确定其分泌的抗菌物质为蛋白质或多肽;为了提高HD-5菌株发酵产抑菌活性物质的量,采用单因素法对HD-5菌株发酵培养基所需的碳源、氮源、无机盐进行了筛选,并采用正交试验对HD-5菌株的发酵培养基组成及培养条件进行了优化,最终确定产抑菌活性物质的最适发酵培养基及培养条件为:葡萄糖5.0%,大豆蛋白胨5.0%,MnSO4 0.05%,MgSO4·7H2O 0.05%,培养基初始pH7.5,种龄14-16 h,接种量8%,于30℃、200 r/min摇床培养48 h。经过优化拮抗细菌HD-5菌株的发酵产抑菌活性物质能力大大提高了,抑菌圈直径增加了65.0%。
为了提高B. amyloliquefaciens HD-5菌株的芽孢形成率及芽孢数量,奠定芽孢菌剂的工业化生产基础,用摇瓶发酵对芽孢形成的主要影响因素进行了考察。通过单因素实验和正交实验对HD-5菌株产芽孢条件进行了分析,确定了摇瓶产芽孢最佳条件为:玉米粉0.5%,大豆蛋白胨0.5%,MgSO4·7H2O 0.05%,MnSO4 0.05%,初始pH 7.5,接种量5%,于37℃、180 r/min摇床培养48 h,最终可使HD-5菌株的芽孢形成率达到89.62%,生物量为1.2×10~9 cfu/mL。
为了进行B. amyloliquefaciens HD-5菌株芽孢菌剂的田间应用试验与最终推广应用,研究HD-5菌株的大型发酵工艺参数,在2 m3发酵罐进行了产芽孢试生产,确立了该菌株在2 m3发酵罐中的发酵工艺参数。经过发酵,发酵液菌浓达到1.0×109 cfu/mL,芽孢形成率在85.0%以上。最后经过添加锯末固定、干燥,制备出的固体菌剂为140 kg。经过测定,其活菌含量为7.0×10~9 cfu/g。
Watermelon fusarium wilt also known as Fusarium wilt, a worldwide distributed of soil-borne disease, is caused by Fusarium oxysporum. The incidence of watermelon fusarium wilt is generally 10%-30%, particularly serious in successive cropping melonfield. The disease leads to 20%-50% yield reduction, and in severe cases even to complete loss in harvest, which causes huge economic losses. At present, the screening of antagonistic microorganisms from soil to prevent and treat Watermelon fusarium wilt caused by Fusarium oxysporum, has gradually become one hot topic of research. Research was done to screen Bacillus bacteria with better biological control potential of Watermelon fusarium wilt caused by Fusarium oxysporum, in order to prevent and treat Watermelon fusarium wilt in watermelon farming.
In order to obtain antagonistic bacteria against Fusarium oxysporum, strains were isolated and screened with the improved agar plate diffusion method using the soil from different districts. The antagonistic activity of strain HD-5, strain CZ-10 and strain BY-1 against Fusarium oxysporum was tested via the pot test for preventing and treating of Watermelon fusarium wilt in the laboratory. A strain HD-5, exhibiting the strongest antagonistic activity against Fusarium oxysporum was obtained. The morphological characteristics, physiological and biochemical properties and 16S rDNA sequence of this strain were studied to determine the species. The similarity of the 16S rDNA sequences between strain HD-5 and Bacillus amyloliquefaciens was up to 99.78%. Finally, the strain HD-5 was identified as Bacillus amyloliquefaciens.
In order to increase the quantity of antifungal protein from Bacillus amyloliquefacien HD-5, the strain was cultured under different conditions. Through single factor experiments, the suitable carbon source, nitrogen source and inorganic salt were chosen. The optimal composition of media was determined by orthogonal experiments. The optimization of fermentation factors, such as initial pH, fermentation temperature and fermentation time, was made by single factor experiments. By the orthogonal design, the optimal proportions of fermentation medium were glucose 5.0%, soybean peptone 5.0%, MnSO4 0.05% , MgSO4·7H2O 0.05%. By single factor experiments, the optimal fermentation condition was initial pH 7.5 and inoculum age of 14~16 hours, shaking flask fermentation temperature is 30℃for 48 hours, under the listed conditions, the highest quantity of antifungal protein was obtained. After optimization, diameter of inhibition zone was increased by 65.0%
In order to improve the production rate and spore quantity of antagonistic strain HD-5 of Bacillus amyloliquefacien against Fusarium oxysporum. The main factors influencing sporulation were investigated using shake-flask fermentation method. Through the single factor experiment and orthogonal experiment, the optimal shaking flask fermentation condition was determined as follow: media composed of 0.5% corn meal, 0.5% soybean peptone, 0.05% MgSO4·7H2O, 0.05% MnSO4, initial pH at 7.5, 5% of inoculum size, fermentation for 48h at 37℃with rotating speed 180r/min. Under this optimum condition, the spore quantity was 1.2×10~9 spore /mL, and spore production rate achieved 89.62%.
For field application testing and extension application of B. amyloliquefaciens strain HD-5, fermentation parameters of strain HD-5 in large-scale production were investgated and obtained by trial production in 2 m3 fermenter. After fermentation, the fermentation broth of bacteria concentration was up to 1.0×109 cfu/mL and the sporulation rate up to 85.0%. Finally, 140 kg inocula solids was prepared by drying through adding sawdust. The viable cell number was determined to be 7.0×10~9 cfu/g.
对土样中分离的产芽孢细菌进行初筛和复筛,得到3株具有较强抑菌活性的拮抗芽孢细菌HD-5、CZ-10、BY-1菌株,并对该3菌株进行了实验室西瓜枯萎病盆栽防治效果试验,最终获得了对西瓜枯萎病具有较好防治效果的拮抗芽孢细菌HD-5菌株。
通过对HD-5菌株的形态特征、生理生化特征考察,以及16S rDNA全序列分析表明HD-5菌株与解淀粉芽孢杆菌(Bacillus amyloliquefaciens)的生理生化特征相符,与其16S rDNA序列同源性高达99.78%的。因此,判定HD-5菌株为解淀粉芽孢杆菌(Bacillus amyloliquefaciens)。
通过对B. amyloliquefaciens HD-5菌株抗菌物质的性质分析,初步确定其分泌的抗菌物质为蛋白质或多肽;为了提高HD-5菌株发酵产抑菌活性物质的量,采用单因素法对HD-5菌株发酵培养基所需的碳源、氮源、无机盐进行了筛选,并采用正交试验对HD-5菌株的发酵培养基组成及培养条件进行了优化,最终确定产抑菌活性物质的最适发酵培养基及培养条件为:葡萄糖5.0%,大豆蛋白胨5.0%,MnSO4 0.05%,MgSO4·7H2O 0.05%,培养基初始pH7.5,种龄14-16 h,接种量8%,于30℃、200 r/min摇床培养48 h。经过优化拮抗细菌HD-5菌株的发酵产抑菌活性物质能力大大提高了,抑菌圈直径增加了65.0%。
为了提高B. amyloliquefaciens HD-5菌株的芽孢形成率及芽孢数量,奠定芽孢菌剂的工业化生产基础,用摇瓶发酵对芽孢形成的主要影响因素进行了考察。通过单因素实验和正交实验对HD-5菌株产芽孢条件进行了分析,确定了摇瓶产芽孢最佳条件为:玉米粉0.5%,大豆蛋白胨0.5%,MgSO4·7H2O 0.05%,MnSO4 0.05%,初始pH 7.5,接种量5%,于37℃、180 r/min摇床培养48 h,最终可使HD-5菌株的芽孢形成率达到89.62%,生物量为1.2×10~9 cfu/mL。
为了进行B. amyloliquefaciens HD-5菌株芽孢菌剂的田间应用试验与最终推广应用,研究HD-5菌株的大型发酵工艺参数,在2 m3发酵罐进行了产芽孢试生产,确立了该菌株在2 m3发酵罐中的发酵工艺参数。经过发酵,发酵液菌浓达到1.0×109 cfu/mL,芽孢形成率在85.0%以上。最后经过添加锯末固定、干燥,制备出的固体菌剂为140 kg。经过测定,其活菌含量为7.0×10~9 cfu/g。
Watermelon fusarium wilt also known as Fusarium wilt, a worldwide distributed of soil-borne disease, is caused by Fusarium oxysporum. The incidence of watermelon fusarium wilt is generally 10%-30%, particularly serious in successive cropping melonfield. The disease leads to 20%-50% yield reduction, and in severe cases even to complete loss in harvest, which causes huge economic losses. At present, the screening of antagonistic microorganisms from soil to prevent and treat Watermelon fusarium wilt caused by Fusarium oxysporum, has gradually become one hot topic of research. Research was done to screen Bacillus bacteria with better biological control potential of Watermelon fusarium wilt caused by Fusarium oxysporum, in order to prevent and treat Watermelon fusarium wilt in watermelon farming.
In order to obtain antagonistic bacteria against Fusarium oxysporum, strains were isolated and screened with the improved agar plate diffusion method using the soil from different districts. The antagonistic activity of strain HD-5, strain CZ-10 and strain BY-1 against Fusarium oxysporum was tested via the pot test for preventing and treating of Watermelon fusarium wilt in the laboratory. A strain HD-5, exhibiting the strongest antagonistic activity against Fusarium oxysporum was obtained. The morphological characteristics, physiological and biochemical properties and 16S rDNA sequence of this strain were studied to determine the species. The similarity of the 16S rDNA sequences between strain HD-5 and Bacillus amyloliquefaciens was up to 99.78%. Finally, the strain HD-5 was identified as Bacillus amyloliquefaciens.
In order to increase the quantity of antifungal protein from Bacillus amyloliquefacien HD-5, the strain was cultured under different conditions. Through single factor experiments, the suitable carbon source, nitrogen source and inorganic salt were chosen. The optimal composition of media was determined by orthogonal experiments. The optimization of fermentation factors, such as initial pH, fermentation temperature and fermentation time, was made by single factor experiments. By the orthogonal design, the optimal proportions of fermentation medium were glucose 5.0%, soybean peptone 5.0%, MnSO4 0.05% , MgSO4·7H2O 0.05%. By single factor experiments, the optimal fermentation condition was initial pH 7.5 and inoculum age of 14~16 hours, shaking flask fermentation temperature is 30℃for 48 hours, under the listed conditions, the highest quantity of antifungal protein was obtained. After optimization, diameter of inhibition zone was increased by 65.0%
In order to improve the production rate and spore quantity of antagonistic strain HD-5 of Bacillus amyloliquefacien against Fusarium oxysporum. The main factors influencing sporulation were investigated using shake-flask fermentation method. Through the single factor experiment and orthogonal experiment, the optimal shaking flask fermentation condition was determined as follow: media composed of 0.5% corn meal, 0.5% soybean peptone, 0.05% MgSO4·7H2O, 0.05% MnSO4, initial pH at 7.5, 5% of inoculum size, fermentation for 48h at 37℃with rotating speed 180r/min. Under this optimum condition, the spore quantity was 1.2×10~9 spore /mL, and spore production rate achieved 89.62%.
For field application testing and extension application of B. amyloliquefaciens strain HD-5, fermentation parameters of strain HD-5 in large-scale production were investgated and obtained by trial production in 2 m3 fermenter. After fermentation, the fermentation broth of bacteria concentration was up to 1.0×109 cfu/mL and the sporulation rate up to 85.0%. Finally, 140 kg inocula solids was prepared by drying through adding sawdust. The viable cell number was determined to be 7.0×10~9 cfu/g.
引文
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