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
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摘要
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 largeyellow croaker culture,the disease is increasing in recent years. Bacteria are the important agent causing diseases outbreak and the death oflarge yellow croaker. Vibrio harveyi,V. alginolyticus,Nocardia seriolea,Pseudomonas plecoglossicida and other bacterial agent were reportedto cause the severe economic loss in the culture. Antagonistic bacteria were considered as a potential alternative measure for bacterial diseasesinstead of antibiotics,with its green and ecological friendship merits. Using V. harveyi and P. plecoglossicida as the indicator bacteria andvia disk method(KB)and colony inoculation,10 antagonistic bacteria were screened from the large yellow croaker and soil,algae,andsea bass around fish culture ponds,and then identified as Bacillus sp.,Alcaligenes sp.,Paenibacillus sp.,Lysinibacillus sp.,Vibrio sp.,Daenococcus sp. by 16 S rDNA sequence analysis. A. faecalis NBPa-7 and B. amyloliquefaciens NBlm-36,the antagonistic strains with stronginhibition to V. harveyi and P. plecoglossicida,the main bacterial pathogens of large yellow croaker,were used in this study. The resultsshowed 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 theinhibition zone of 6.00 mm-14.00 mm. Extracellular product presented favorable tolerance to heat,acid and alkali. The ICR mice were used forinjection intraperitoneally with 1×108 CFU/ind of 2 antagonistic strains,and no toxicity and death occurred in 48 h after injection. There wasno morbidity within 2 weeks,no lesion in the viscera of the large yellow croaker,and there was no obvious increase in white blood cells whenthe large yellow croakers were injected intraperitoneally with 3×108 CFU/ind of two antagonistic strains,indicating that antagonistic strains arenon-pathogenic to mice and large yellow croaker.
Large yellow croaker Larimichtys crocea is an important marine cultivated fish in China. With the rapid expanding of largeyellow croaker culture,the disease is increasing in recent years. Bacteria are the important agent causing diseases outbreak and the death oflarge yellow croaker. Vibrio harveyi,V. alginolyticus,Nocardia seriolea,Pseudomonas plecoglossicida and other bacterial agent were reportedto cause the severe economic loss in the culture. Antagonistic bacteria were considered as a potential alternative measure for bacterial diseasesinstead of antibiotics,with its green and ecological friendship merits. Using V. harveyi and P. plecoglossicida as the indicator bacteria andvia disk method(KB)and colony inoculation,10 antagonistic bacteria were screened from the large yellow croaker and soil,algae,andsea bass around fish culture ponds,and then identified as Bacillus sp.,Alcaligenes sp.,Paenibacillus sp.,Lysinibacillus sp.,Vibrio sp.,Daenococcus sp. by 16 S rDNA sequence analysis. A. faecalis NBPa-7 and B. amyloliquefaciens NBlm-36,the antagonistic strains with stronginhibition to V. harveyi and P. plecoglossicida,the main bacterial pathogens of large yellow croaker,were used in this study. The resultsshowed 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 theinhibition zone of 6.00 mm-14.00 mm. Extracellular product presented favorable tolerance to heat,acid and alkali. The ICR mice were used forinjection intraperitoneally with 1×108 CFU/ind of 2 antagonistic strains,and no toxicity and death occurred in 48 h after injection. There wasno morbidity within 2 weeks,no lesion in the viscera of the large yellow croaker,and there was no obvious increase in white blood cells whenthe large yellow croakers were injected intraperitoneally with 3×108 CFU/ind of two antagonistic strains,indicating that antagonistic strains arenon-pathogenic to mice and large yellow croaker.
引文
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[8]Liu KF, Chiu CH, Shiu YL, et al. Effects of the probiotic, Bacillus subtilis E20, on the survival, development, stress tolerance, andimmune status of white shrimp, Litopenaeus vannamei Larvae[J].Fish Shellfish Immun, 2010, 28(5/6):837-844.
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[15]Jandera P. Column selectivityfor two dimensionalliquidchromatography[J]. Sep Sci, 2006, 29:1763-1783.
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[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 forplant disease biocontrol[J]. Trends in Microbiology, 2008, 6(3):115-125.
[23]Perpoux F, Bonmation JM, Wallach J. Recent trends in thebiochemistry of surfactin[J]. Apply Microbio Biotechnol, 1999,51:553-563.
[24]Yu GY, Sinclair JB, Hartman GL, et al. Production of iturin A byBacillus amyloliquefaciens auppressing Rhizoctonia salani[J].Soil Biology and Biochemistry, 2002, 34:955-963.
[25]Cazorla FM, Romero D, Perez-Garcia A, et al. Isolation andcharacterization of antagonistic Bacillus subtilis strains from theavocadorhzoplane displaying biocotrol activity[J]. Journal ofApplied Microbiology, 2007, 103:1950-1959.
[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 farmedlarge yellow croaker, Larimichthys crocea(Richardson), caused bya bacterial pathogen, Pseudomonas plecoglossicida[J]. Journal ofFish 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, andimmune 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 andquantification of antibiotic residues in aquaculture fishes:Areview[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 directsequencing of gyrB genes with universal primers and theirapplication 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 selectivityfor two dimensionalliquidchromatography[J]. Sep Sci, 2006, 29:1763-1783.
[16]Wang LT, Lee FL, Tai CJ, et al. Comparison of gyrB genesequences, 16S r RNA gene sequences and DNA-DNA hybridizationin the Bacillus subtilis group[J]. International Journal ofSystematic and Evolutionary Microbiology, 2007, 57:1846-1850.
[17]Chelo MI, Zé-ZéL, Tenreiro R. Congruence of evolutionaryrelationships inside the Leuconostoc-Oenococcus-Weissella cladeassessed by phylogeneti c analysis of the 16S rRNA gene, dnaA,gyrB, rpoC and dnaK[J]. International Journal of Systemati cand 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 forplant disease biocontrol[J]. Trends in Microbiology, 2008, 6(3):115-125.
[23]Perpoux F, Bonmation JM, Wallach J. Recent trends in thebiochemistry of surfactin[J]. Apply Microbio Biotechnol, 1999,51:553-563.
[24]Yu GY, Sinclair JB, Hartman GL, et al. Production of iturin A byBacillus amyloliquefaciens auppressing Rhizoctonia salani[J].Soil Biology and Biochemistry, 2002, 34:955-963.
[25]Cazorla FM, Romero D, Perez-Garcia A, et al. Isolation andcharacterization of antagonistic Bacillus subtilis strains from theavocadorhzoplane displaying biocotrol activity[J]. Journal ofApplied Microbiology, 2007, 103:1950-1959.