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

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.