西沙海藻共附生放线菌的分离鉴定及其抗菌活性评价

Isolation and antimicrobial bioactivity of algae-associated actinomycetes from Xisha Islands

摘要:

【目的】为了探究南海海藻共附生放线菌资源的多样性及潜在的应用价值，对中国西沙群岛来源的海藻进行共附生放线菌的分离鉴定与抗菌活性筛选。【方法】利用稀释涂布平板法，采用2种不同分离培养基对不同采样位点的6种海藻进行放线菌分离；通过16S rRNA基因序列分析、构建系统发育树对分离的放线菌进行鉴定；用打孔法对无乳链球菌(Streptococcus agalactiae)等10种敏感细菌进行抗菌活性筛选；对筛选得到的目标活性菌株HZ014进行全基因组测序，通过AntiSMASH在线工具分析其次级代谢产物生物合成基因簇，预测其产生新型活性物质的潜力。【结果】从6种海藻中分离得到36株共附生放线菌，基于16S rRNA基因序列比对和系统发育分析，鉴定结果为链霉菌属(Streptomyces) 2株、红球菌属(Rhodococcus) 2株、诺卡氏菌属(Nocardia) 3株、小单孢菌属(Micromonospora) 5株和盐孢菌属(Salinispora) 24株；抗菌活性筛选结果表明，36株共附生放线菌发酵粗提物对至少1种敏感细菌表现出一定的抑制作用，不同菌株发酵粗提物的抗菌活性存在明显差异，盐孢菌发酵粗提物抑菌谱较另外4个属更广；AntiSMASH分析结果显示，菌株HZ014基因组中超过22.28%的基因序列与次级代谢产物合成相关，表明盐孢菌属存在巨大的生物合成潜能，值得深入发掘。【结论】西沙海藻中蕴藏着丰富的可培养稀有放线菌资源，首次从西沙海藻共附生环境中分离得到专性海洋稀有放线菌盐孢菌属，且分离得到的36株海藻共附生放线菌发酵粗提物对部分敏感细菌具有较好的抗菌活性，在渔业病害防治中具有潜在的开发应用价值，有望为海洋药物或生物菌剂的研发提供新资源。

Abstract:

[Objective] To investigate the diversity and potential value of the algae-associated symbiotic or epiphytic actinomycete resources from South China Sea, we isolated and identified actinomycetes from different algae samples collected from Xisha Islands and evaluated their antibacterial activities. [Methods] We used two types of media for the isolation and purification of actinomycetes and the isolates were identified according to their 16S rRNA gene sequences and the phylogenetic tree. The activities of their fermentation products against 10 pathogenic bacterial strains including Streptococcus agalactiae were tested with the punching method. The whole genome of the target active strain was sequenced and the biosynthetic gene clusters of secondary metabolites were predicted and analyzed by AntiSMASH. [Results] A total of 36 strains of actinomycetes were isolated from six kinds of algae. Based on 16S rRNA gene sequence and phylogeny analysis, they belonged to Streptomyces (2 strains), Rhodococcus (2 strains), Nocardia (3 strains), Micromonospora (5 strains), and Salinispora (24 strains), respectively. The antibacterial tests showed that the fermentation products of the 36 strains displayed inhibitory effect against at least one of ten sensitive bacteria. The antibacterial activities of Salinispora spp. were stronger than those of other actinomycetes. AntiSMASH analysis showed that more than 22.28% of the gene sequences in HZ014 genome were related to the biosynthesis of secondary metabolites, indicating their great potential in natural product biosynthesis. [Conclusion] The algae of Xisha Islands are home to abundant rare culturable actinomycetes. In this study, marine obligate rare actinomycetes associated with marine algae, Salinispora spp., were reported for the first time. The fermentation products of 36 strains of actinomycetes displayed antibacterial activity, indicating the potential value in the prevention of fish diseases. These rare actinomycetes are potential resources for the development of marine drugs or biocontrol agents.