枯草芽孢杆菌LJ的分离鉴定及其对凡纳滨对虾幼体弧菌病防治效果

Isolation and Identification of Bacillus subtilis LJ and Its Control Effect Against Vibriosis in Litopenaeus vannamei Larvae

【目的】从对虾肠道分离鉴定拮抗病原弧菌的益生菌,对其生物安全性及对凡纳滨对虾(Litopenaeus vannamei)幼体弧菌病的控制效果进行评价,以促进益生菌对对虾弧菌病的防治效果。【方法】利用平板划线法从虾肠分离细菌,根据拮抗病原弧菌活性筛选潜在益生菌株,结合表型特征、16S rRNA和rpoB基因序列对目标菌株进行鉴定,利用纸片扩散法测试其对17种抗菌药物敏感性。通过水体添加目标菌评价其对凡纳滨对虾幼体感染副溶血弧菌(Vibrio parahaemolyticus)的生物防治效果。【结果】从对虾肠道分离筛选得到一株对副溶血弧菌、哈维氏弧菌(Vibrio harveyi)和坎氏弧菌(Vibrio campbellii)均有明显拮抗作用的菌株LJ;LJ不溶血且无多重耐药性,被鉴定为枯草芽孢杆菌(Bacillus subtilis)。水体添加1×10^(5)和1×10^(6)cfu/mL LJ均可显著提高感染副溶血弧菌的对虾幼体存活率和变态率(P<0.05),且高剂量处理组效果更明显;未添加病原菌条件下,高浓度(5×10^(7)cfu/mL)LJ对幼体无害,并能显著促进幼体存活和变态。【结论】对虾肠源枯草芽孢杆菌LJ生物安全性高,可有效拮抗病原弧菌和提高感染副溶血弧菌凡纳滨对虾幼体的存活率。

【Objective】To isolate and identify probiotics that antagonize pathogenic vibrios from shrimp intestines,and evaluate its biological safety and control effect against vibriosis in Litopenaeus vannamei larvae,so as to promote the prevention and bioconrol of shrimp vibriosis by probiotics.【Method】Shrimp intestinal bacteria were isolated by using streak plate method,and from which potential probiotic was screened based on antagonistic activity against pathogenic vibrios.The target strain was identified according to the phenotypical characteristics,16S rRNA and rpoB gene sequences,and its sensitivities to 17 kinds of antibacterial drugs were examined by disc diffusion method.The biocontrol effect of L.vannamei larvae infected with Vibrio parahaemolyticus was evaluated by adding target bacteria to water.【Result】A strain named LJ with obvious antagonism against V.parahaemolyticus,Vibrio harveyi,and Vibrio campbellii was isolated and selected from shrimp intestines.LJ has no multiple drug resistance and was identified as Bacillus subtilis.The addition of 1×10^(5) and 1×10^(6) cfu/mL LJ to water could significantly improve the survival and metamorphosis rates of shrimp larvae infected with V.parahaemolyticus(P<0.05),and the high-dose treatment group had a more significant effect.Under conditions without pathogen infection,high concentration of LJ(5×10^(7) cfu/mL)was harmless to larvae and could significantly promote their survival and metamorphosis.【Conclusion】The B.subtilis LJ derived from shrimp gut has high biosafety,can effectively antagonize pathogenic vibrios and improve the survival rate of L.vannamei larvae infected with V.parahaemolyticus.