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微生物在黄河三角洲互花米草与日本鳗草生态竞争中的作用及机制分析
Alternative TitleAnalysis of the roles and mechanisms of microorganisms in the ecological competition between Spartina alterniflora and Zostera japonica in the Yellow River Delta
宋增磊
Subtype博士
Thesis Advisor胡晓珂
2023-05-28
Training institution中国科学院烟台海岸带研究所
Degree Grantor中国科学院大学
Place of Conferral中国科学院烟台海岸带研究所
Degree Name理学博士
Degree Discipline海洋生物学
Keyword互花米草 日本鳗草 沉积物微生物 叶际微生物 玫瑰杆菌
Abstract日本鳗草(Zostera japonica)作为海草床生态系统中的重要组成部分,具有重要的生态服务功能。但是由于近年来黄渤海区海草生境的恶化,已经很难再找到大面积连续分布的日本鳗草海草床。我们认为,互花米草(Spartina alterniflora)入侵是导致黄渤海区生境恶化、日本鳗草种群退化的重要原因。既有研究多从植物本身探究互花米草的入侵机制,且相关研究主要集中于微生物群体层面,从微生物个体层面研究互花米草入侵机制的研究相对较少。因此,本研究选择山东东营黄河三角洲互花米草入侵日本鳗草的典型生态区域,通过将传统的细菌分离纯培养法与高通量测序技术紧密结合,从微生物群体层面和个体层面系统开展如下研究:(1)群体层面调查互花米草与日本鳗草0-5厘米上层与15-20厘米下层沉积物中微生物的多样性、群落结构的差异,分析不同植物沉积物的优势微生物类群;(2)研究不同季节、不同潮间带位置的互花米草叶片的叶际微生物群落结构差异以及优势微生物类群的功能特性;(3)基于微生物群体层面的结果,本文以具有重要生态意义的优势菌群玫瑰杆菌类群(Roseobacter lineage)为例,从个体层面分离纯培养大量玫瑰杆菌,利用群体基因组学分析技术,一方面系统评估互花米草根际沉积物对特定微生物类群的影响,另一方面阐明特定微生物类群对互花米草入侵的响应机制。 主要研究结果如下: (1)无论是互花米草还是日本鳗草,其上层沉积物微生物的绝对丰度显著(P < 0.05)高于下层和退化区。沉积物的微生物群落组成在门水平上的变化与总有机碳(TOC)关系最密切,而重金属的含量会降低微生物的绝对丰度。与退化区和本地植物群落相比,互花米草入侵增加了上层沉积物样品中的微生物的绝对丰度。在大多数样品中,黄杆菌科(Flavobacteriaceae)的绝对丰度最高。脱硫棒菌科(Desulfobulbaceae)、脱硫杆菌科(Desulfobacteraceae)和除硫单胞菌科(Desulfuromonadaceae)等硫酸盐还原菌(Sulfate-reducing bacteria,SRB)与红杆菌科(Rhodobacteraceae)也是此生态系统的优势菌群。互花米草入侵后,拟杆菌(Bacteroidia)、酸微菌科(Acidimicrobiaceae)和Dehalococcoidaceae的丰度在互花米草沉积物中富集成为优势类群,这些优势类群可以促进互花米草根系生长,使其更好地适应环境,从而利于其完成入侵。 (2)对互花米草叶际微生物群落的调查研究发现,基于NMDS分析的β多样性显示,这些附生微生物群落的分布根据叶片位置和季节变化而聚集,不同环境样品类型和不同季节的微生物群落存在显著差异,并且不同组样品均存在适合环境的特征微生物群落。海水的温度、盐度、溶解氧和有机碳、氮含量与附生叶际微生物群落在两个季节都有密切的相关性。在夏季组中,叶片始终浸没在海水中的LDS组的微生物群落表现出较高的多样性,而在冬季组中,叶片一直不接触海水的LUW组的多样性最高。只有莫拉式菌科(Moraxellaceae)和周蝶菌科(Weeksellaceae)是夏季叶片一直不接触海水的LUS组的优势类群,这与其他组有显著差异。此外,除LUS组外,黄杆菌科和红杆菌科是所有组的优势科。与季节相比,潮汐作用对不同位置的叶际附生微生物群落结构的贡献更大。互花米草叶际微生物群落中功能相对丰度最高的是化能营养。夏季组和冬季组微生物群落的功能分布大致不同,冬季氮循环功能的相对丰度远高于夏季。从功能差异的比较分析结果发现,夏季组与冬季组在化能营养(P < 0.01)、有氧化能营养(P < 0.01)、芳香化合物降解(P < 0.01)、硝酸盐还原(P < 0.01)和硝酸盐呼吸(P < 0.01)等相关功能方面存在显著差异。其中,与人类病原体(P < 0.001)和动物寄生虫或共生体(P < 0.001)相关功能的相对丰度在夏季显著高于冬季。 (3)对沉积物细菌群落多样性分析结果表明,无论是基于所有的ASV,还是基于注释为红杆菌科的ASV,不同采样环境下的沉积物样品存在显著差异(P = 0.001, r = 0.36; P = 0.005, r = 0.27),尤其是互花米草根际环境与潮滩表层沉积物环境。通过优化选择培养基,本文共分离纯化了2,362株菌,其中玫瑰杆菌类群有1,117株,在不同的采样环境下,玫瑰杆菌类群占比都超过了1/3。进一步对在各个采样环境中占比均较高的3个菌属的229株(分别为Marivita属、Ruegeria属和Sulfitobacter属)细菌进行基因组测序。最终共获得了200个符合后续分析的基因组数据。随后,通过群体基因组学的方法系统评估了4个玫瑰杆菌种群趋异进化的贡献,结果表明,潮间带植物互花米草根际沉积物环境对玫瑰杆菌类群的趋异进化贡献是有限的。这是由于潮间带环境强烈的潮汐混合作用,使得根际沉积物环境未能完全形成“有效屏障”的生态位,从而使得玫瑰杆菌类群未能完成分化或处于分化阶段的早期。进一步地,本文通过玫瑰杆菌的基因组分析,发现玫瑰杆菌(尤其是Sulfitobacter属)的基因组中编码大量鞭毛生物合成基因以及IV型分泌系统基因,这些均可能促进玫瑰杆菌与互花米草的相互作用,使得互花米草入侵时大量定植的玫瑰杆菌类群可以作为致病菌对日本鳗草退化产生影响。再结合本文在前面研究得到的互花米草入侵增加了入侵地微生物的绝对丰度(这可能同时增加了病原菌的数量)的结果,可以推测玫瑰杆菌作为共生微生物参与互花米草入侵的可能性相对较小,因此,本文更倾向于认为玫瑰杆菌在互花米草入侵过程中符合“本地病原菌积累假说”的机制,即玫瑰杆菌可能作为一种病原微生物参与了互花米草的入侵过程。 综上,本研究从微生物群体层面和个体层面系统地探究了入侵物种互花米草对日本鳗草这一典型生境相关微生物的影响及机制。在微生物群体层面分析了两种植物沉积物上层与下层以及叶际微生物的多样性、丰度和功能在不同生态位、环境和季节间的分布规律,阐明了环境因素(如营养盐、重金属污染、季节变化和潮汐作用)与微生物的相关关系;个体层面上通过细菌纯培养分离得到了大量玫瑰杆菌优势类群,利用群体基因组学的方法分析了互花米草沉积物中特定微生物群对互花米草入侵的响应,并提出了玫瑰杆菌介导的“互花米草入侵-病原菌积累-日本鳗草退化-入侵成功”机制,有助于人们更好地理解微生物与植物之间的相互作用以及互花米草的入侵机制,并可为该生境日本鳗草海草床的恢复及湿地的生态保护提供重要的科学依据。
Other AbstractZostera japonica, as an important component of the seagrass bed ecosystem, plays an important role in the ecological service function. However, due to the deterioration of seagrass habitats in the Yellow Sea and Bohai Sea regions in recent years, it has been difficult to find large-scale and continuously distributed Z. japonica seagrass beds. The invasion of Spartina alterniflora is considered to be an important reason for the degradation of Z. japonica population. There are many studies on the invasion mechanism of S. alterniflora, but they usually focus on the plant itself. At present, research on microbial mechanisms in invasion ecology mainly focuses on the community level of microorganisms, and there is relatively little research on the invasion mechanism of S. alterniflora from the individual level of microorganisms. Therefore, this study selected the typical ecological area where S. alterniflora invaded Z. japonica in the Yellow River Delta of Dongying, Shandong Province. By combining the traditional bacteria isolation and pure culture method with high-throughput sequencing technology, the following research was carried out systematically from the community level and individual level of microorganisms: (1) At the microbial community level, the diversity and community structure differences of microorganisms in the upper (0-5 cm) and bottom (15-20 cm) sediment layers of S. alterniflora and Z. japonica were investigated, and the dominant microbial groups in different plant sediments were analyzed; (2) The differences of microbial community structure and functional characteristics of dominant microbial groups in the leaf of S. alterniflora in different seasons and different intertidal zone locations were studied; (3) Based on the results at the community level of microorganisms, the dominant microbial group Roseobacter lineage with significant ecological significance was selected as an example. At the individual level, a large number of Roseobacter strains was screened and isolated. Using population genomics analysis techniques, on the one hand, the impact of the rhizosphere sediments of S. alterniflora on specific microbial groups was systematically evaluated, and on the other hand, the response mechanism of specific microbial groups on the invasion of S. alterniflora was elucidated. The main results are listed as follows: (1) Whether it is S. alterniflora or Z. japonica, the absolute abundance of microorganisms in the upper layer sediments was significantly higher (P < 0.05) than that in the bottom layer sediments and degraded areas. The changes in microbial community composition in sediments at the phylum level were most closely related to Total Organic Carbon (TOC), and the content of heavy metals would reduce the absolute abundance of microorganisms. Compared with degraded areas and native plant communities, S. alterniflora invasion increased the absolute abundance of microbial communities in upper layer sediment samples. In most samples, Flavobacteriaceae has the highest absolute abundance, and Sulfate-reducing bacteria (SRB) such as Desulfobulbaceae, Desulfobacteriaceae and Desulfuromonadaceae, and Rhodobacteraceae were also the dominant flora in this ecosystem. With the invasion of S. alterniflora, the abundance of Bacteroidia, Acidimicrobiaceae, and Dehalococccoidaceae enriched in the sediments of S. alterniflora and became dominant groups, which could promote the growth of S. alterniflora root system and help them to adapt to the environment better, thus facilitating its invasion. (2) The investigation of the microbial community in S. alterniflora leaves showed that the distribution of these epiphytic microbial communities gathered according to leaf position and seasonal changes, and there were differences in microbial communities in different environmental samples and different seasons. There were characteristic microbial communities suitable for the environment in different groups of samples. The temperature, salinity, dissolved oxygen, organic carbon, and nitrogen contents of seawater are closely related to the epiphytic phyllosphere microbial community in both seasons. In the summer group, the diversity of microbial community in the LDS group was higher than that in the LUW group, while in the winter group, the diversity of microbial communities in the LUW group was the highest. Only Moraxellaceae and Weeksellaceae were the dominant groups in the LUS group, which was significantly different from other groups. In addition, Flavobacteriaceae and Rhodobacteraceae were the dominant families in all groups except for the LUS group. Compared with seasons, tidal action contributed more to the community structure of epiphytic phyllosphere microorganisms in different positions of the leaf. In the phyllosphere microbial community of S. alterniflora, chemotrophic nutrition has the highest relative functional abundance. The functional distribution of microbial communities in the summer group and winter group was roughly different. The relative abundance of nitrogen cycle function in winter was much higher than that in summer. The results showed that there were significant differences between the summer group and the winter group in chemoheterotrophy (P < 0.01), aerobic chemoheterotrophy (P < 0.01), aromatic compounds degradation (P < 0.01), nitrate reduction (P < 0.01) and nitrate respiration (P < 0.01). The relative abundance of functions associated with human pathogens (P < 0.001) and animal parasites or symbionts (P < 0.001) was significantly higher in summer than in winter. (3) The results of bacterial community diversity analysis in sediments showed that there were significant differences (P = 0.001, r = 0.36; P = 0.005, r = 0.27) in sediment samples under different sampling environments, whether based on all ASVs or ASVs annotated as Rhodobacteraceae, especially in S. alterniflora rhizosphere environment and tidal flat surface sediment environment. By optimizing the culture medium, we isolated and purified a total of 2,362 strains of bacteria, including 1,117 strains of the Roseobacter lineage. The proportion of the Roseobacter lineage in different sampling environments exceeded 1/3. Further genome sequencing was performed on 229 strains of bacteria belonging to three genera (Marivita, Ruegeria, and Sulfitobacter, respectively) with a high proportion in each sampling environment. In the end, we obtained a total of 200 genome data that met the subsequent analysis. Then, the contribution of four Roseobacter populations to divergent evolution was systematically evaluated by population genomics. The results showed that the contribution of rhizosphere sediment environment of intertidal plant S. alterniflora to divergent evolution of Roseobacter groups was limited, which was due to the strong tidal mixing in the intertidal zone environment, which made the rhizosphere sediment environment could not form an effective barrier niche completely, thus making Roseobacter groups fail to complect differentiation or being in the early stages of differentiation. Furthermore, according to the genome analysis of Roseobacter, we found that the genome of Roseobacter (especially the Sulfitobacter population) encodes a large number of flagellar biosynthetic genes and type IV secretion system genes, which may promote the interaction between Roseobacter and S. alterniflora, allowing the large number of colonized Roseobacter groups during S. alterniflora invasion to act as pathogenic bacteria and have an impact on the degradation of Z. japonica. Furthermore, based on our previous research findings that the invasion of S. alterniflora increased the absolute abundance of microorganisms in the invaded area (which may also increase the number of pathogenic bacteria), it is speculated that the possibility of Roseobacter participating in S. alterniflora invasion as a symbiotic microorganism is relatively small. We are more inclined to believe that Roseobacter conforms to the mechanism of "local pathogen accumulation hypothesis" during the invasion process of S. alterniflora, that is, Roseobacter may participate in the invasion process of Roseobacter as a pathogenic microorganism. In conclusion, this study systematically explored the influence and mechanism of the invasive species S. alterniflora on microorganisms related to Z. japonica, a typical habitat, from the microbial community and individual levels. At the microbial community level, the distribution patterns of the diversity, abundance and function of the microorganisms in the upper and bottom layers of the sediment and in the phyllosphere of the two plants in different ecological niche, environments and seasons were analyzed, and the correlation between environmental factors (such as nutrients, heavy metal pollution, seasonal changes and tidal effects) and microorganisms was clarified; At the individual level, a large number of dominant groups of Roseobacter were isolated through pure bacterial culture. The response of specific microbial communities in the sediment of S. alterniflora to S. alterniflora invasion was analyzed using population genomics methods, and a mechanism of S. alterniflora invasion - pathogen accumulation - Z. japonica degradation - invasion success" mediated by Roseobacter was proposed. This will help us better to understand the interaction between microorganisms and plants, as well as the invasion mechanism of S. alterniflora, and provide important scientific basis for the restoration of the Z. japonica seagrass bed in this habitat and the ecological protection of wetlands.
Pages121
Language中文
Document Type学位论文
Identifierhttp://ir.yic.ac.cn/handle/133337/32048
Collection中科院烟台海岸带研究所知识产出_学位论文
Recommended Citation
GB/T 7714
宋增磊. 微生物在黄河三角洲互花米草与日本鳗草生态竞争中的作用及机制分析[D]. 中国科学院烟台海岸带研究所. 中国科学院大学,2023.
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