水霉拮抗菌的筛选及其拮抗活性物质稳定性初步研究

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

摘要:

【目的】从海底沉积物中分离、筛选水霉拮抗放线菌菌株，鉴定目标菌株及其无菌发酵液对水霉生长的抑制效果，并初步分析拮抗活性物质的稳定性。【方法】用稀释涂布法从采集的海底沉积物中分离得到海洋放线菌，以水霉为靶菌，通过平板对峙法在PDA平板上筛选出对水霉有拮抗作用的菌株；利用其发酵液对水霉菌丝和孢子进行初步拮抗效果研究；通过16S rRNA基因序列分析对目标菌株的种属进行初步鉴定。【结果】从分离到的数十株海洋放线菌中筛选到5株水霉拮抗菌，其中拮抗效果最强的为S26菌株，16S rRNA基因序列分析结果显示其为链霉菌，并与紫色链霉菌具有较近的亲缘关系；S26马铃薯葡萄糖液体培养基发酵液在平板抑菌圈实验中，对水霉孢子萌发的抑菌圈直径达32.00 mm±0.81 mm，其5倍浓缩无菌发酵液对水霉菌丝的抑菌圈直径达39.75 mm±0.50 mm；5倍浓缩无菌发酵液抑菌活性的3.125%即能完全抑制水霉孢子的萌发；5倍浓缩液对温度具有较强耐受性，经100 °C高温30 min处理后平板抑菌圈直径为25.50 mm±0.58 mm；经不同pH值处理12 h后，pH 5.0–9.0之间仍保持较好的拮抗活性；在37 °C下蛋白酶处理2 h后实验组与对照组存在显著性差异，但平板抑菌圈直径仍可达33.25 mm以上，推测拮抗物质活性成分由多肽和非多肽类代谢物共同组成。【结论】海洋链霉菌株S26产生的活性物质对病原水霉真菌有较强的抑制作用，并对外界环境变化有较强的适应能力，因而在水霉病的生物防治中具有潜在的应用价值。研究结果同时也显示海洋链霉菌在水产病害生物防治应用领域有较好的发展前景和更广阔的挖掘空间。

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.