Biotechnology Bulletin ›› 2020, Vol. 36 ›› Issue (8): 120-128.doi: 10.13560/j.cnki.biotech.bull.1985.2019-1158

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Isolation,Identification and Antibiotic Sensitivity Analysis of Bacterial Pathogen from Litopenaeus vannamei with Black Gill Disease

LIN Jia-ming1, GE Hui1,2, LIN Ke-bing2, YANG Zhang-wu2, ZHOU Chen2, WU Jian-shao2, WANG Guo-dong1, ZHANG Zhe2, YANG Qiu-hua2, WANG Yi-lei1   

  1. 1. Key Laboratory of Healthy Mariculture for the East China Sea,Ministry of Agriculture,Fisheries College,Jimei University,Xiamen 361021;
    2. Key Laboratory of Cultivation and High-value Utilization of Marine Organisms in Fujian Province,Fisheries Research Institute of Fujian,Xiamen 361012
  • Received:2019-11-27 Online:2020-08-26 Published:2020-08-27

Abstract: In order to identify the pathogen of black gill disease in Litopenaeus vannamei overwintering parent shrimp and analyze its drug resistance,a dominant strain was isolated from the focus in the gill of a dying diseased shrimp via plate method,and then analyzed by combining the morphology of the strain and its ITS-rDNA gene sequence. The results showed that the dominant strain had 99% sequence homology with Fusarium solani,and the constructed phylogenetic tree clustered as one branch with F. solani;therefore the isolated dominant strain was determined as F. solani. The artificial infection was conducted based on Koch’s rule,and the results confirmed that the pathogen had strong pathogenicity and disease shrimps had black gill. The shrimps infected with concentrations of 5.0×105 CFU/mL(25 µL/shrimp)for 7 d died 53.33%. Agar dilution method and M38-A2 scheme of NCCLS were used for drug sensitivity test with selected 11 common drugs. After preliminary screening,it was found that the strain was sensitive to clotrimazole,econazole,terbinafine,nystatin,natamycin and ketoconazole. The MIC of econazole was 1 μg/mL,that of terbinafine(TBF)was 4 μg/mL,that of clotrimazole and natamycin was 8 μg/mL,and that of miconazole and itraconazole was 16 μg/mL. In conclusion,the pathogenicity,drug resistance and taxonomic status of the dominant strain were determined in this study,which may provide a reference for the prevention and control of black gill disease of overwintering parent shrimp.

Key words: Litopenaeus vannamei, isolation and identification of pathogen, Fusarium solani, drug sensitivity test