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

doi:10.13560/j.cnki.biotech.bull.1985.2018-0565

Abstract

Abstract: 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 1×10⁸CFU/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 3×10⁸CFU/ind of two antagonistic strains,indicating that antagonistic strains are non-pathogenic to mice and large yellow croaker.

Key words: Larimichtys crocea, antagonistic bacteria, identification, antimicrobial spectrum

FU Chao-ying, WANG Jian-ping, SUN Chen, CHEN Lin, QIAN Dong. Isolation,Identification,Inhibition Spectrum and Safety Test of Antagonistic Bacteria to Major Pathogenic Bacteria in Larimichtys crocea[J]. Biotechnology Bulletin, 2019, 35(1): 67-75.

References

[1] 农业部渔业渔政管理局. 2014年中国渔业统计年鉴[M]. 北京:中国农业出版社, 2015.
[2] 李忠琴, 张坤, 林茂, 等. 大黄鱼(Pseudosciaena crocea)致病性维氏气单胞菌的分离鉴定与药敏特性研究[J]. 海洋与湖沼, 2017, 48(1):139-147.
[3] 黄志斌, 蔡其良. 大黄鱼几种暴发性疾病及防治方法新探[J]. 福建水产, 1999, 12(4):66-70.
[4] 汪长友, 郑尧兰. 大黄鱼网箱育种阶段的病害与诊治[J]. 齐鲁渔业, 2004(2):39-41.
[5] 王国良, 袁思平, 金珊. 大黄鱼结节病病原菌—诺卡氏菌的鉴定及其系统发育分析[J]. 中国水产科学, 2006(3):410-414.
[6] Zhang, JT, Zhou SM, An SW, et al. Visceral granulomas in farmed large yellow croaker, Larimichthys crocea(Richardson), caused by a bacterial pathogen, Pseudomonas plecoglossicida[J]. Journal of Fish Diseases, 2014, 37(2):113-121.
[7] 周瑞娟. 网箱养殖大黄鱼主要疾病的预报模型研究[D]. 宁波:宁波大学, 2011.
[8] Liu KF, Chiu CH, Shiu YL, et al.Effects of the probiotic, Bacillus subtilis E20, on the survival, development, stress tolerance, and immune status of white shrimp, Litopenaeus vannamei Larvae[J]. Fish Shellfish Immun, 2010, 28(5/6):837-844.
[9] Santos L, Ramos F.Analytical strategies for the detection and quantification of antibiotic residues in aquaculture fishes:A review[J]. Trends Food Sci Tech, 2016, 52(3):16-30.
[10] 夏汉钦. 活性和热灭活益生菌对斜带石斑鱼生长性能、肠道菌群和免疫基因表达的影响[D]. 厦门:集美大学, 2013.
[11] 瞿佳, 赵玲侠, 孙晓宇, 等. 一株抗铅细菌的分离鉴定与抗铅性能研究[J]. 西北农业学报, 2016, 25(8):1195-1200.
[12] 萨姆布鲁克J, 拉塞尔DW. 黄培堂, 等译. 分子克隆实验指南[M]. 第3版. 北京:科学出版社, 2008:26-32.
[13] Yamamoto S, Harayama S .PCR amplification and direct sequencing of gyrB genes with universal primers and their application to the detection and taxonomic analysis of Pseudomonas putida strains[J]. Applied Environmental Microbiology, 1995, 61:1104-1109.
[14] 喻国辉, 牛春艳, 陈远凤, 等. 利用16S rDNA 结合gyrA 和gyrB 基因对生防芽孢杆菌R31 的快速鉴定[J]. 中国生物防治, 2010, 26(2):160-166.
[15] Jandera P.Column selectivity for two dimensional liquid chromatography[J]. Sep Sci, 2006, 29:1763-1783.
[16] Wang LT, Lee FL, Tai CJ, et al.Comparison of gyrB gene sequences, 16S rRNA gene sequences and DNA-DNA hybridization in the Bacillus subtilis group[J]. International Journal of Systematic and Evolutionary Microbiology, 2007, 57:1846-1850.
[17] Chelo MI, Zé-Zé L, Tenreiro R.Congruence of evolutionary relationships inside the Leuconostoc-Oenococcus-Weissella clade assessed by phylogeneti c analysis of the 16S rRNA gene, dnaA, gyrB, rpoC and dnaK[J]. International Journal of Systemati c and Evolutionary Microbiology, 2007, 57:276-286.
[18] 鞠守勇. 秀丽隐杆线虫AMPK介导的抵御苏云金芽胞杆菌Cry5Ba毒素的研究[D]. 武汉:华中农业大学, 2015.
[19] 秦楠. 解淀粉芽抱杆菌HRH317抗菌蛋白鉴定及抑菌机理研究[D]. 太谷:山西农业大学, 2015.
[20] 彭研. 拮抗解淀粉芽孢杆菌的筛选鉴定及其产孢外抗菌物质的初步研究[D]. 曲阜:曲阜师范大学, 2013. 
[21] 宋博. 解淀粉芽孢杆菌anti-CA菌株抗污损生物的研究[D]. 青岛:中国海洋大学, 2014.
[22] Ongena M, Jacques P.Bacillus liopopetides:versatile weapons for plant disease biocontrol[J]. Trends in Microbiology, 2008, 6(3):115-125.
[23] Perpoux F, Bonmation JM, Wallach J.Recent trends in the biochemistry of surfactin[J]. Apply Microbio Biotechnol, 1999, 51:553-563.
[24] Yu GY, Sinclair JB, Hartman GL, et al.Production of iturin A by Bacillus amyloliquefaciens auppressing Rhizoctonia salani[J]. Soil Biology and Biochemistry, 2002, 34:955-963.
[25] Cazorla FM, Romero D, Perez-Garcia A, et al.Isolation and characterization of antagonistic Bacillus subtilis strains from the avocadorhzoplane displaying biocotrol activity[J]. Journal of Applied Microbiology, 2007, 103:1950-1959.