Screening of antagonistic bacterium strain against Saprolegnia sp. and characterization of the antifungal stability of culture broth from the target strain

Abstract:

[Objective] Isolation of marine actinobacteria with antagonistic activity against pathogenic Saprolegnia sp., and the antifungal stability of culture broth from the target strain. [Methods] Serial dilution and plating method was employed to isolate actinobacteria from marine sediments. The antagonistic strains were screened by confrontation assay on PDA plates. The anti-Saprolegnia effects were characterized using cell-free culture broth as references. The stability of antagonistic activity was analyzed upon physical and chemical factors changes including temperature, pH, and proteinase treatments. Phylogenetic tree was constructed using the Neighbor-Joining method based on the 16S rRNA gene sequences. [Results] Dozens of actinobacteria strains were isolated, and five of them presented antagonistic activity against Saprolegnia sp. The strain of S26, which showed the strongest activity, was found closely related to Streptomyces violaceus NBRC 13103T (98.6% similarity) based on the 16S rRNA gene sequence analysis. This showed that S26 should be assigned to genus Streptomyces. The inhibition zones were 32.00 mm±0.81 mm for S26 fermented broth against germination of Saprolegnia spores and 39.75 mm±0.50 mm for five times concentrated cell-free culture against growth of Saprolegnia hypha. The minimum to inhibit Saprolegnia spores germination was 3.125% of the total activity of the five times concentrated cell-free culture. The substance responsible for antagonism showed strong resistance to high temperatures and a clear inhibition zone (25.50 mm±0.58 mm in diameter) was still obtained by the fermented broth even after intense heating treatment of 100 °C for 30 min. The antifungal activity of fermented broth was sensitive to extreme acid or alkaline condition (pH<5.0 or pH>9.0), but it was sustained throughout the pH range between 5.0 and 9.0. The active substance was partially sensitive to proteinase treatments. These suggested that it was highly possible for the antagonisitic substances to be both proteinaceous and non-proteinaceous molecules. [Conclusion] The marine actinobacterium strain of S26 isolated in this study showed strong inhibitory effects to the spores germination and hypha growth of Saprolegnia sp., and the compounds with the antagonistic activity were resistant to some extent against temperature, pH and proteinase treatments. These suggested that S26 was a promising microbial strain to control saprolegniasis occurred in aquatic animals. The study implies marine actinobacteria will play an important role in biocontrol of aquatic animal diseases.