大黄鱼主要致病菌拮抗菌株的分离鉴定、抑菌谱及安全性分析

Isolation,Identification,Inhibition Spectrum and Safety Test of Antagonistic Bacteria to Major Pathogenic Bacteria in Larimichtys crocea

大黄鱼是我国重要的海水养殖鱼类,随着大黄鱼养殖规模的扩大,病害也日益增多,其中细菌性疾病是造成大黄鱼疾病暴发、死亡的重要病原。近年来,哈维弧菌(Vibrio harveyi)、溶藻弧菌(Vibrio alginolyticus)、杀香鱼假单胞菌(Pseudomonas plecoglossicida)、鰤鱼诺卡菌(Nocardia seriolea)等细菌造成了大黄鱼养殖重大经济损失。为寻找大黄鱼主要致病菌的拮抗菌,用于大黄鱼细菌病的生态控制,作者对大黄鱼及周边环境进行了拮抗菌的分离和鉴定。以哈维弧菌、杀香鱼假单胞菌作为拮抗活性筛选指示菌,用纸片法(KB)、菌落接种法从大黄鱼体内、养殖池周边土壤、藻类、芦苇等筛选出具抗菌活性物质菌株10株;分离菌株用16S rDNA通用引物序列分析鉴定为芽孢杆菌(Bacillus sp.)、产碱杆菌(Alcaligenes sp.)、类芽孢杆菌(Paenibacillus sp.)、赖氨酸芽孢杆菌(Lysinibacillus sp.)、弧菌(Vibrio sp.)和异常球菌(Daenococcus sp.)等。对具较强抑菌活性的碱性杆菌NBPa-7(Alcaligenes faecalis)和芽孢杆菌NBlm-36(Bacillus amyloliquefaciens)进行了抑菌活性测定,结果表明NBPa-7对溶藻弧菌、杀香鱼假单胞菌有良好拮抗作用,抑菌直径分别为23.20 mm、12.00 mm;NBlm-36对创伤弧菌(Vibrio vulnificus)、哈维弧菌、嗜水气单胞菌(Aeromonas hydrophila)、大肠杆菌(Escherichia coli)及葡萄球菌(Staphylococcus sp.)有拮抗作用,抑菌直径为6.00 mm-14.00 mm。胞外抑菌产物对热、酸、碱有较好耐受性。将拮抗菌以1×108CFU/ind腹腔注射成年鼠、口喂新生鼠,48 h内未出现毒性和死亡,将拮抗菌以3×108CFU/ind腹腔注射大黄鱼,2周内未出现发病死亡,大黄鱼内脏未发生病变,白细胞未出现明显升高现象,初步表明拮抗菌株不具有致病力。

Large yellow croaker Larimichtys crocea is an important marine cultivated fish in China.With the rapid expanding of large yellow croaker culture,the disease is increasing in recent years.Bacteria are the important agent causing diseases outbreak and the death of large yellow croaker.Vibrio harveyi,V.alginolyticus,Nocardia seriolea,Pseudomonas plecoglossicida and other bacterial agent were reported to cause the severe economic loss in the culture.Antagonistic bacteria were considered as a potential alternative measure for bacterial diseases instead of antibiotics,with its green and ecological friendship merits.Using V.harveyi and P.plecoglossicida as the indicator bacteria and via disk method(KB)and colony inoculation,10 antagonistic bacteria were screened from the large yellow croaker and soil,algae,and sea bass around fish culture ponds,and then identified as Bacillus sp.,Alcaligenes sp.,Paenibacillus sp.,Lysinibacillus sp.,Vibrio sp.,Daenococcus sp.by 16S rDNA sequence analysis.A.faecalis NBPa-7 and B.amyloliquefaciens NBlm-36,the antagonistic strains with strong inhibition to V.harveyi and P.plecoglossicida,the main bacterial pathogens of large yellow croaker,were used in this study.The results showed that NBPa-7 gave fine antagonism against V.alginolyticus and P.plecoglossicida,with inhibition zone of 23.20 mm,12.00 mm,respectively.The NBlm-36 showed antagonistic effects on V.vulnificus,A.hydrophila,Escherichia coli,and Staphylococcus sp.,with the inhibition zone of 6.00 mm-14.00 mm.Extracellular product presented favorable tolerance to heat,acid and alkali.The ICR mice were used for injection intraperitoneally with 1×10^8 CFU/ind of 2 antagonistic strains,and no toxicity and death occurred in 48 h after injection.There was no morbidity within 2 weeks,no lesion in the viscera of the large yellow croaker,and there was no obvious increase in white blood cells when the large yellow croakers were injected intraperitoneally with 3×10^8 CFU/ind of two antagonistic strains,indicating that antagonistic strains are non-pathogenic to mice and large yellow croaker.