| 蟹源弗氏柠檬酸杆菌拮抗菌PNB3的分离鉴定与安全性分析 |
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| 引用本文： | 黄晓东,张健,朱元宏,安健,曹海鹏.蟹源弗氏柠檬酸杆菌拮抗菌PNB3的分离鉴定与安全性分析[J].上海海洋大学学报,2021,30(4):653-663. |
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| 作者姓名： | 黄晓东 张健 朱元宏 安健 曹海鹏 |
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| 作者单位： | 上海海洋大学,上海海洋大学 |
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| 基金项目： | 江苏省海洋与渔业科技创新与推广项目(Y2018-8)；上海市科学技术科研计划项目(19DZ1204800)；江苏省农业科技自主创新资金项目(CX(18)2010） |
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| 摘 要： | 摘 要：筛选和鉴定抗水产致病性弗氏柠檬酸杆菌的优良芽孢杆菌，并分析其安全性。以一株蟹源致病性弗氏柠檬酸杆C1作为筛选指示菌、抑菌圈直径和抑菌效果作为指标进行抗菌芽孢杆菌优良菌株的分离与筛选，综合生理生化鉴定与16S rRNA序列分析对抗菌芽孢杆菌优良菌株进行鉴定，并参照《HJ/T 415—2008 环保用微生物菌剂环境安全评价导则》、《NY/T1444-2007微生物饲料添加剂评价通则》等国家标准，从毒力基因、耐药性、对小球藻生长的影响以及对斑马鱼和中华绒螯蟹的致病性等方面分析抗菌芽孢杆菌优良菌株的安全性。结果显示，从养殖淤泥中分离并筛选出一株抗菌芽孢杆菌优良菌株PNB3，其在1.0×105 CFU/mL时与蟹源致病性弗氏柠檬酸杆菌C1共培养处理120 h的最高抑菌率达到了99.99%。结合生理生化鉴定以及16S rRNA序列分析，菌株PNB3被鉴定为地衣芽孢杆菌(Bacillus licheniformis)，不含溶血素BL基因、肠毒素Nhe基因、肠毒素T基因、肠毒素FM 基因、细胞毒素 K 基因、呕吐基因、多效调控因子等毒力基因，对复方新诺明、氨苄西林、诺氟沙星、卡那霉素、多粘菌素B、氟苯尼考、庆大霉素、依诺沙星、头孢拉定、青霉素等10种抗生素高度敏感，对多西环素、四环素等2种抗生素中度敏感，对链霉素耐药，在终浓度为2.0×104~2.0×108 CFU/mL时对小球藻的生长有促进作用，对斑马鱼的半数致死浓度（LC50）大于2.0×108 CFU/mL，对中华绒螯蟹的半数致死浓度（LC50）大于2.0×108 CFU/g。地衣芽孢杆菌PNB3是一株具有潜在安全性的弗氏柠檬酸杆菌拮抗菌，具备应用于中华绒螯蟹弗氏柠檬酸杆菌病生物防控的安全基础。
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| 关 键 词： | 弗氏柠檬酸杆菌 地衣芽孢杆菌 拮抗 鉴定 安全性 |
| 收稿时间： | 2020/4/8 0:00:00 |
| 修稿时间： | 2020/7/4 0:00:00 |
Isolation, identification and safety analysis of antagonistic strain PNB3 against crab-pathogenic Citrobacter freundii |
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| HUANG Xiaodong,ZHANG Jian,ZHU Yuanhong,AN Jian,CAO Haipeng.Isolation, identification and safety analysis of antagonistic strain PNB3 against crab-pathogenic Citrobacter freundii[J].Journal of Shanghai Ocean University,2021,30(4):653-663. |
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| Authors: | HUANG Xiaodong ZHANG Jian ZHU Yuanhong AN Jian CAO Haipeng |
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| Institution: | Shanghai Ocean University,Shanghai Ocean University |
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| Abstract: | The objective of the present study is to screen and identify potential antagonistic bacteria against aquatic pathogenic Citrobacter freundii, and to evaluate its safety. Using crab-pathogenic C. freundii C1 as the indicator and the diameter of inhibition zones as the evaluation index, the good Bacillus isolate with antagonistic activity was screened, and was identified by phenotypic identification and 16S rRNA gene analysis. The safety of the isolated antagonist was assessed by assaying virulence gene, antibiotic resistance, inhibitory effect on Chlorella sp, and pathogenicity for zebrafish and Chinese mitten crabs. Isolate PNB3 with good antagonistic activity was screened from the aquaculture sediment which reached 99.99% after co-culture with C. freundii C1 for 120 h at 1.0×105 CFU/mL, and was identified as Bacillus licheniformis based on phenotypic characterization and 16S rRNA gene analysis. It did not contain virulence genes including haemolysin BL gene, enterotoxin Nhe gene, enterotoxin T gene, enterotoxin FM gene, cytotoxin K gene, vomiting gene, multiple regulatory factors, and exhibited high sensitivity to compound sulfamethoxazole, ampicillin, norfloxacin, kanamycin, polymyxin B, florfenicol, gentamycin, enoxacin, cefradine, penicillin, and displayed a growth-promoting effect on Chlorella sp., and showed no pathogenicity for zebrafish and Chinese mitten crabs. In conclusion, B. licheniformis PNB3 is a potential safe antagonist against C. freundii, and has the safe potential in the biocontrol of C. freundii infection in Chinese mitten crabs. |
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| Keywords: | Citrobacter freundii Bacillus licheniformis Antagonism Identification Safety |
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