两种培养基对对虾苗池海洋蛭弧菌的分离及其多样性分析

Comparison of two media for isolation and diversity analysis of marine Bdellovibrio-and-like organisms in shrimp hatchery system

摘要:

【目的】明确海水(Sw)和聚蛋白胨20 (Pp20)两种双层琼脂培养基对海洋蛭弧菌的分离计数效果，了解对虾苗池可培养海洋蛭弧菌多样性。【方法】采用双层平板法，比较Sw和Pp20培养基对2株海洋蛭弧菌和对虾苗池未知海洋蛭弧菌的计数效果。通过宿主范围测试和16S rRNA基因序列分析评估两种培养基分离苗池海洋蛭弧菌的多样性。【结果】宿主菌含量高时，Sw培养基对两株已知海洋蛭弧菌的计数值均显著高于(P<0.05) Pp20。Sw和Pp20培养基从同一苗池水样分别分离得到21和22株蛭弧菌。根据宿主裂解范围差异，43株分离物可分为15种裂解模式，其中Sw和Pp20培养基各分离到12和8种。16S rRNA基因序列分析表明，所有分离物都被鉴定为噬菌弧菌属(Bacteriovorax)菌株，并可分为6个类群，Sw和Pp20培养基分别分离到6和4个类群。【结论】Sw培养基在分离计数海洋蛭弧菌及其多样性检测上效果均优于Pp20；对虾苗池可培养海洋蛭弧菌具有较高多样性，并以类群XIII、X及一个潜在新类群为优势种群。

Abstract:

[Objective] To isolate and enumerate marine Bdellovibrio-and-like organisms (BALOs) by seawater (Sw) and polypeptone 20 (Pp20) double-layer agar media, and to reveal the diversity of cultivable marine BALOs in shrimp hatchery system. [Methods] We first compared the effects of the two media on enumeration of marine BALOs strains NA7 and NF1, as well as of BALOs in larval rearing water of Litopenaeus vannamei, then evaluated the diversities of BALOs isolated through prey range testing and 16S rRNA gene sequence analysis. [Results] Strains NA7 and NF1 both exhibited significantly higher (P<0.05) plaque numbers on Sw medium than on Pp20 medium if more prey bacteria were present. A total of 21 and 22 strains of BALOs were isolated from the same larviculture water by Sw and Pp20 media, respectively. According to prey range testing, 15 lytic patterns were differentiated from 43 isolates, of which 12 and 8 patterns corresponding to Sw and Pp20 were identified respectively. The 16S rRNA gene sequence analysis shows that all the BALOs isolates were affiliated to Bacteriovorax strains. Based on 97% sequence similarity, they could be divided into 6 phylogenetic clusters, and Sw and Pp20 media respectively recovered 6 and 4 clusters. [Conclusion] Medium Sw exhibited superior to medium Pp20 either for isolation and enumeration of marine BALOs or for diversity detection of their populations. The cultivable BALOs recovered from larviculture system of L. vannamei presented relatively high diversity. They all belong to the genus Bacteriovorax and are dominated by the clusters XIII, X and a potentially new cluster.