[1]刘学实,蒋 葛,沈 辉,等.一株铜绿假单胞菌的分离鉴定及其对VPAHPND拮抗效果的评价[J].南京师大学报(自然科学版),2023,46(01):73-79,87.[doi:10.3969/j.issn.1001-4616.2023.01.011]
　Liu Xueshi,Jiang Ge,Shen Hui,et al.Isolation, Identification and uation of an Antagonistic Bacteria of VPAHPND Strain[J].Journal of Nanjing Normal University(Natural Science Edition),2023,46(01):73-79,87.[doi:10.3969/j.issn.1001-4616.2023.01.011]
点击复制

一株铜绿假单胞菌的分离鉴定及其对VPAHPND拮抗效果的评价()
分享到：

《南京师大学报（自然科学版）》[ISSN:1001-4616/CN:32-1239/N]

卷:
第46卷
期数:
2023年01期
页码:
73-79,87
栏目:
生物学
出版日期:
2023-03-15

文章信息/Info

Title:
Isolation, Identification and uation of an Antagonistic Bacteria of VPAHPND Strain
文章编号:
1001-4616(2023)01-0073-07
作者:
刘学实1; 蒋 葛2; 沈 辉2; 乔 毅2; 成 婕2; 曹晓慧2; 窦雅斌3; 温梦莹3; 万夕和2; 王李宝2; 黎 慧2; 史文军2
(1.南京师范大学海洋科学与工程学院，江苏 南京 210023)
(2.江苏省海洋水产研究所，江苏 南通 226007)
(3.上海海洋大学水产与生命学院，上海 201306)
Author(s):
Liu Xueshi1; Jiang Ge2; Shen Hui2; Qiao Yi2; Chen Jie2; Cao Xiaohui2; Dou Yabin3; Wen Mengying3; Wan Xihe2; Wang Libao2; Li Hui2; Shen Wenjun2
(1.College of Marine Science and Engineering, Nanjing Normal University, Nanjing 210023, China)
(2.Jiangsu Institute of Oceanology & Marine Fisheries, Nantong 226007, China)
(3.College of Fisheries and Life, Shanghai Ocean University, Shanghai 201306, China)
关键词:
VPAHPND; 凡纳滨对虾; 拮抗菌; 铜绿假单胞菌
Keywords:
Vibrio parahaemolyticus;  Litopenaeus vannamei;  antagonistic bacterium;  Pseudomonas aeruginosa
分类号:
Q939.9
DOI:
10.3969/j.issn.1001-4616.2023.01.011
文献标志码:
A
摘要:
近年来,急性肝胰腺坏死病(Acute hepatopancreatic necrosis disease,AHPND)造成了凡纳滨对虾(Litopenaeus vannamei)养殖业巨大的经济损失,其病原为一类含有致病基因PirAB的弧菌(Vibrio),主要为副溶血弧菌(Vibrio parahaemolyticus,VPAHPND). 本研究以VPAHPND为受试菌株,从江苏海域捕获的黄姑鱼(Nibea albiflora)体内分离筛选对VPAHPND具有抑制作用的拮抗菌株. 通过形态学、生理生化及分子生物学方法进行鉴定,研究了其拮抗效果、拮抗活性物质,并对生物安全性和被动保护效果进行评估. 结果表明,分离筛选出1株具有良好拮抗效果的菌株JSHY97,经鉴定该菌株为铜绿假单胞菌(Pseudomonas aeruginosa). 该菌株对3株具有不同分型的VPAHPND均具有良好的拮抗效果,其抑菌圈直径达 19.25 mm～21.80 mm,初步判断其活性物质来源于胞外产物. 口服JSHY97的凡纳滨对虾的7 d保护率也达到80%,拮抗组的凡纳滨对虾体内的毒力基因PirA拷贝数显著低于对照组(P<0.05). 安全性实验表明JSHY97对斑马鱼(Brachydanio reriovar)和凡纳滨对虾无毒力. 由此表明,本研究得到的铜绿假单胞菌菌株JSHY97对VPAHPND具有较好的抑制作用,为AHPND的防治提供了新的菌株资源.
Abstract:
Acute hepatopancreatic necrosis disease causes serious economic losses to the Litopenaeus Vannamei shrimp industry in recent years. Studies have shown that the pathogen responsible for this disease is a type of Vibrio containing PirAB virulence factors,specifically Vibrio parahaemolyticus(VPAHPND). In this paper,the antagonistic strain of VPAHPND was isolated from Nibea albiflora in the coastal waters of Jiangsu province,with identified by morphological characteristics,physiological and biochemical characteristics,and 16S rDNA sequence analysis. Its antagonistic effect and antagonistic active substances were preliminarily studied,and the assessment of bio-safety were preliminarily optimized. The results showed that the strain JSHY97 with powerful antagonistic effect was screened and identified as Pseudomonas aeruginosa. JSHY97 had an antagonistic effect against 3 strains of VPAHPND with a zone of inhibition diameter of 19.25 mm-21.80 mm,and its active substances were preliminarily judged to be extracellular products. After 7 days of diet,protection rates of 80% were also achieved by feeding JSHY97 to L. vannamei. The copy number of the virulence gene PirA in the JSHY97-fed L.vannamei was significantly lower than that in the control group(P<0.05). Safety experiments showed that JSHY97 was non-toxic to Brachydanio reriovar and L. vannamei. Pseudomonas aeruginosa strain JSHY97 has a good inhibitory effect on VPAHPND,which provides a new strain resource for the prevention and treatment of AHPND.

参考文献/References:

[1]唐小千,徐洪森,战文斌. 对虾急性肝胰腺坏死综合症研究进展[J]. 海洋湖沼通报,2016(2):90-93.
[2]TRAN L,NUNAN L,REDMAN R M,et al. Determination of the infectious nature of the agent of acute hepatopancreatic necrosis syndrome affecting penaeid shrimp[J]. Diseases of aquatic organisms,2013,105(1):45-55.
[3]LEE C,CHEN I,YANG Y,et al. The opportunistic marine pathogen Vibrio parahaemolyticus becomes virulent by acquiring a plasmid that expresses a deadly toxin[J]. Proceedings of the National Academy of Sciences of the United States of America,2015,112(34):10798-10803.
[4]李吉云,沈辉,孟庆国,等. 对虾急性肝胰腺坏死病(AHPND)流行病学、诊断方法及防控措施的研究进展[J]. 海洋科学,2021,45(3):163-172.
[5]李吉云. 急性肝胰腺坏死病致病菌VP_(AHPND)毒力差异的初步研究[D]. 南京:南京师范大学,2021.
[6]赵淑江,刘健,杨星星,等. 南麂岛海洋沉积物中抗大黄鱼(Pseudosciaena crocea)致病弧菌的放线菌分离和筛选研究[J]. 海洋与湖沼,2010,41(4):571-576.
[7]张欢欢,王秀华,李晨,等. 对虾养殖池中一株弧菌拮抗菌的分离鉴定[J]. 渔业科学进展,2016,37(3):85-92.
[8]肖国华. 一种复合微生态制剂对养殖水体中生物因子的影响[J]. 微生物学通报,2013,40(7):1154-1162.
[9]冯艳琴,沈辉,王兴强,等. VP(AHPND)拮抗菌筛选鉴定及其活性物质的初步研究[J]. 水产学杂志,2022,35(2):35-41.
[10]XU L L,YUAN J F,CHEN X X,et al. Screening of intestinal probiotics and the effects of feeding probiotics on the digestive enzyme activity,immune,intestinal flora and WSSV resistance of Procambarus clarkii[J]. Aquaculture,2021(540):736-748.
[11]HAN J E,TANG K F J,PANTOJA C R,et al. qPCR assay for detecting and quantifying a virulence plasmid in acute hepatopancreatic necrosis disease(AHPND)due to pathogenic Vibrio parahaemolyticus[J]. Aquaculture,2012,442:12-15.
[12]REYNOLDS D,KOLLEF M,REYNOLDS D,et al. The epidemiology and pathogenesis and treatment of Pseudomonas aeruginosa infections:an update. [J]. Drugs,2021,81(18):1-15.
[13]李连星,陈成勋,张扬,等. 铜绿假单胞菌YY24对凡纳滨对虾生长及水质的影响[J]. 水生态学杂志,2018,39(3):87-93.
[14]PRIYAJA P,JAYESH P,CORREYA N S,et al. Antagonistic effect of Pseudomonas aeruginosa isolates from various ecological niches on Vibrio species pathogenic to crustaceans[J]. Journal of coastal life medicine,2014,2(1):76-84.
[15]甄晓然,沈辉,万夕和,等. 对虾源副溶血弧菌拮抗菌的筛选与评价[J]. 水产科学,2019,38(6):767-773.
[16]冯艳琴. VPAHPND拮抗菌筛选鉴定及其活性物质的初步研究[D]. 连云港:江苏海洋大学,2021.
[17]TRUC L N T,NGOC A T,HONG T T T,et al. Selection of Lactic Acid Bacteria(LAB)Antagonizing Vibrio parahaemolyticus:the Pathogen of Acute Hepatopancreatic Necrosis Disease(AHPND)in Whiteleg Shrimp(Penaeus vannamei)[J]. Biology,2019,8(4):91.
[18]杨行,章翔,龙昊,等. 假交替单胞菌对哈维氏弧菌拮抗作用初探[J]. 水产科学,2019,38(6):833-838.
[19]WANG H,WANG C,TANG Y,et al. Pseudoalteromonas probiotics as potential biocontrol agents improve the survival of Penaeus vannamei challenged with acute hepatopancreatic necrosis disease(AHPND)—causing Vibrio parahaemolyticus[J]. Aquaculture,2018,494:30-36.
[20]WANG K R,LIU N,SHANG F,et al. Activation of secondary metabolism in red soil-derived streptomycetes via Co-Culture with mycolic acid-containing bacteria[J]. Microorganisms,2021,9(11):2187-2187.
[21]LUCIO G,GABRIEL E C,JOS?C,et al. Assessment of probiotic bacteria from marine coasts against Vibrio parahaemolyticus(AHPND strains)in Litopenaeus vannamei[J]. Aquaculture research,2021,52(12):6396-6409.
[22]MERY R,CRISTOBAL D,LIZETH S,et al. The probiotics Vibrio diabolicus(Ili),Vibrio hepatarius(P62),and Bacillus cereus sensu stricto(P64)colonize internal and external surfaces of Penaeus vannamei shrimp larvae and protect it against Vibrio parahaemolyticus[J]. Aquaculture,2022,549:737-826.

备注/Memo

备注/Memo:
收稿日期:2022-09-19.
基金项目:国家农业长期性基础性重大疫病监测项目(NAES-AH-011)、江苏省农业专项(2022-SJ-037-02)、中国盐城渔业高质量发展研究课题(YCSCYJ2021005)、南通市科技计划项目(JC2021089、JCZ20045、JC2020119、JC2020116).
通讯作者:沈辉,博士,正高级工程师,研究方向:水生动物病害防治. E-mail:darkhui@163.com
更新日期/Last Update: 2023-03-15