|Other Abstract||Zostera 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.|