涠洲岛2种造礁珊瑚共附生可培养潜在耐热细菌多样性

Diversity of potential heat-tolerant bacteria associated with two species of scleractinian corals in Weizhou Island

摘要:

【背景】珊瑚适应环境的能力与机体内共附生细菌有关，然而，这些细菌在珊瑚宿主适应环境变化过程中所起的作用尚不清楚。对珊瑚共附生细菌进行纯培养，探究其生物功能和生态作用，是解析珊瑚宿主环境适应机理的重要途径。【目的】研究热耐受性不同的2种造礁珊瑚共附生可培养潜在耐热细菌多样性和功能，为理解珊瑚适应环境的能力提供新的认识。【方法】从涠洲岛选取2种热耐受性差异显著的霜鹿角珊瑚(Acropora pruinosa)和丛生盔形珊瑚(Galaxea fascicularis)为研究对象，采用2216E、海水R₂A和海水GYP这3种琼脂培养基，于32 ℃ (珊瑚热耐受阈值)培养条件下对珊瑚共附生潜在耐热细菌进行分离培养，对分离菌株进行16S rRNA基因测序和序列相似性分析。选取代表菌株进行热耐受性检验，并利用平板对峙法进行抗菌活性检测。【结果】 2种造礁珊瑚共附生可培养潜在耐热细菌的多样性存在显著差异。从热敏型的霜鹿角珊瑚中获得44株细菌，隶属于4个门22个属，其中弧菌属(Vibrio)、假交替单胞菌属(Pseudoalteromonas)和Tenacibaculum为优势属；从热耐受性强的丛生盔形珊瑚中获得28株细菌，隶属于3个门11个属，其中弧菌属、假交替单胞菌属和鲁杰氏菌属(Rugeria)为优势属。此外，分离菌株中有17株菌与16S rRNA基因序列相似性低于98.65%，可能代表潜在的新分类单元。细菌热耐受性试验研究中，在26-37 ℃温度范围内，细菌生长速率均在34 ℃时最大，温度高于大多数海洋细菌的最适生长温度和珊瑚白化阈值，表明分离获得的细菌具有一定的耐热性。来源于丛生盔形珊瑚的2株鲁杰氏菌对珊瑚潜在病原弧菌具有抑制作用，而来自霜鹿角珊瑚Tenacibaculum的3株细菌对弧菌的抑制作用不明显。【结论】2种造礁珊瑚共附生可培养潜在耐热细菌具有丰富的多样性，而且蕴含着不少潜在新类群。另外，条件致病菌弧菌作为优势类群，但来源于热耐受性强的珊瑚共附生细菌对其有一定的拮抗作用。因此，本研究推测珊瑚的耐热特性与体内共生细菌对致病菌的抑制作用有关。

关键词:珊瑚礁;共附生细菌;纯培养;热胁迫;弧菌

Abstract:

[Background] The environmental resilience of scleractinian corals is related to the associated bacteria. However, it is unclear how these bacteria adapt to the environmental changes. Studying the biological and ecological roles of these bacteria isolated via pure culture method is a fundamental approach to decipher the environment adaptation mechanism of corals. [Objective] To study the diversity and function of the heat-tolerant bacteria associated with two species of corals and further provide new insights into the environmental resilience of scleractinian corals. [Methods] We used three media, 2216E, seawater GYP, and seawater R₂A agar, to isolate the heat-tolerant bacteria associated with two coral species (Acropora pruinosa and Galaxea fascicularis) with significant differences in heat tolerance from Weizhou Island, Beibu Gulf at 32 ℃ (heat tolerance threshold of corals). The 16S rRNA gene sequencing and sequence similarity analysis were then performed for the isolated bacteria. Predominant strains were selected and tested for the heat tolerance, and their antibacterial activity was analyzed by the plate confrontation method. [Results] The diversity of the potential heat-tolerant bacteria showed significant difference between the two coral species. A total of 44 strains of potential heat-tolerant bacteria belonging to 22 genera of 4 phyla were isolated from A. pruinosa, among which Vibrio, Pseudoalteromonas, and Tenacibaculum were predominant. A total of 28 heat-tolerant bacterial strains belonging to 11 genera of 3 phyla were isolated from G. fascicularis, among which Vibrio, Pseudoalteromonas, and Rugeria were predominant. Among the isolated bacteria, 17 strains shared the 16S rRNA gene sequence similarity below 98.65%, which might be new taxa. The growth conditions of bacteria were studied at 26-37 ℃, which showed the optimal growth temperature was 34 ℃, higher than the optimal growth temperature of most marine bacteria and coral bleaching threshold, indicating that the isolated bacteria had potential heat resilience. Two strains of Rugeria had an inhibitory effect on Vibrio, a potential pathogen associated with coral diseases, and 3 strains of Tenacibaculum did not exert significantly inhibitory activity. [Conclusion] The diversity of bacteria associated with A. pruinosa and G. fascicularis is high and needs to be further researched. Although the opportunistic pathogen Vibrio was the predominant genus, the bacteria associated with G. fascicularis had an inhibitory effect on it. Based on the above results, we hypothesized that the heat resilience of scleractinian corals was related to the inhibitory effect of the coral-associated bacteria on pathogenic bacteria.

Key words:coral reefs;associated bacteria;pure culture;heat stress;Vibrio