Nitrogen (N) is one of the essential primary macronutrients for plant growth and production (Li et al., 2018). Through the reduction of atmospheric N₂ to biologically available ammonium, about 70% of total N is fixed (Galloway et al., 2004; Lin et al., 2016). An efficient way to introduce N₂ into the biosphere is N fixation, since plants cannot directly utilize gaseous N₂. In the rhizosphere of plants, a wide variety of diazotrophs, including free living bacteria, sulfate reducing bacteria and symbiotic diazotrophs, usually dominated by Proteobacteria, Cyanobacteria and Firmicutes, inhabited (Wang et al., 2016; Zhang et al., 2021). These diazotrophs shared the same operon in which the nifH gene encoding the Fe protein subunit of the nitrogenase in the nitrogen fixation process has been widely used for phylogenetic analyses (Ininbergs et al., 2011; Ospina-Betancourth et al., 2020; Dong et al., 2022). Plant diversity could affect the composition and function of microbial community, which in return, increased soil N supply to plants (Zak et al., 2003). Due to the high genetic diversity, rhizospheric diazotrophs could play multiple roles in plant growth and development (Vejan et al., 2016; Oliveira et al., 2017). Generally, diazotrophs provided readily available N sources to host plants, and in return, host plants supplied carbon sources to diazotrophs through photosynthesis (Gupta et al., 2006; Liu et al., 2019).

The diversity and composition of microbial communities in the rhizosphere of plants were affected by soil properties (Garbeva et al., 2008; Tkacz et al., 2015; Jiang et al., 2017; Li et al., 2021). In some studies, a greater impact of host plant species on microbial assemblage composition, than of abiotic parameters, has been observed, since the selection of microbial communities in rhizospheric soils was dependent on the root morphology, root exudation and nutrient competition of plants (Rodrigo et al., 2006; Ladygina and Hedlund, 2010). In some studies, diazotrophic abundance and community composition were found to be mainly affected by soil characteristics, such pH, inorganic N and carbon/nitrogen (C/N) ratio, especially salinity and water content (Moisander et al., 2007; Collavino et al., 2014; Levy-Booth et al., 2014; Wang et al., 2017; Che et al., 2018; Lin et al., 2018). The effects of plants on microbial community in plant rhizosphere were species specific, and a synergistic relationship between plant species and environmental conditions existed (Morina et al., 2018). In addition, diazotroph assemblages were also strongly influenced by seasons, abiotic environmental parameters and host plants (Debra et al., 2011; Lin et al., 2017).

One of the major causes for soil salinization in the coastal region was the intrusion of seawater into coastal aquifers (Omuto et al., 2020). With the increase of distance from the vertical coastline, soil salinity and moisture of coastal wetlands changed significantly (Xian et al., 2019). In coastal sediments, higher rate of sediment N-fixation, which could be a significant N source, has been detected (Mortazavi et al., 2012; Bhavya et al., 2016). In coastal seagrass beds, microbial N-fixing provided 50% of the N requirements (Whiting et al., 1986). The biological N fixation (BNF) rates in coastal region can be affected by many biotic and abiotic factors, such as diazotroph diversity, soil C:N ratio and soil P level (Dang et al., 2013; Huang et al., 2016). Microbially-mediated N-dynamics were particular important to the biogeochemical functions in coastal marine wetlands (Affourtit et al., 2001; Moisander et al., 2007; Moseman-Valtierra et al., 2009). Nitrogen-fixing bacteria constituted a key functional group of microorganisms relevant to the functions of wetland ecosystems (Moseman et al., 2009). Meanwhile, human activity also generated a great amount of nitrogen, causing numerous ecological and environmental problems, such as eutrophication, in coastal region (Newell et al., 2016; Huang et al., 2021; Lin and Lin, 2022). Salinization could affect the transformation and uptake of N, and ultimately restrain the growth and decrease the production of plants (Midgley, 2012). To adapt to the salt stress condition, halophyte plants could shape specific rhizospheric microbiomes and increase the microbial diversities (Wang et al., 2010; Anburaj et al., 2012; Qiu et al., 2022). The growth of halophyte plants is usually limited by the availability of N, which is supplemented by the introduction of ‘new’ N in the ecosystems through plant associated diazotrophs (Debra et al., 2011; Rejmánková et al., 2018; Zhang et al., 2020; Qiu et al., 2022). The rate of microbial N fixation is correlated to plant photosynthetic activity, and the structure of diazotroph assemblage is significantly influenced by the variety of carbon sources associated with plant species. According to the mechanisms of salt tolerance, halophytes are divided into secretohalophyte, pseudohalophyte and euhalophyte. Dicotyledonous plants have more developed root system than monocotyledonous plants, and annual halophyte plants have developed a set of mechanisms safer than those of perennial halophyte plants, including seed dormancy when subjected to high salt stress, fast germination after rehydration, polymorphism in morphology and germination, and persistent seed bank and plastic resource allocation (Cao et al., 2021).

Since the 1980s, the coastal zone of Laizhou Bay-Yellow River Estuary, which cover an area of 2,870 km², has become one of the most serious seawater intruded regions in China (Guo and Gong, 2014). In this area, coastal saline soils show a gradient of salinity and water content from land to sea, accompanied with an apparent species succession of native plants. Although some studies on the characterization and evolution of vegetation in this area have been carried out for saline soil bioremediation, limited work has been done on the microbial community structure in the rhizosphere of plants grown in this area (Zhang et al., 2006; Ravit et al., 2007; Cao et al., 2015; Jing et al., 2018). In this study, we investigated the physicochemical factors, as well as the diversity and composition of rhizospheric N-fixing microbes in the coastal wetland. We demonstrate that the key driving factors affecting diazotrophic community structure in the coastal saline soil are physicochemical factors but not host plant traits.

The growth and health of plants are largely affected by soil physiochemical properties and microbial community structure (Marasco et al., 2018; Jing et al., 2018; Wang et al., 2020). And plant invasion and N enrichment can considerably affect BNF (Xu et al., 2012; Huang et al., 2016). To date, a number of studies based on the diversity and abundance of diazotrophic community in the soils of different habitats, such as paddy field, forest and desert, have been carried out (Wang et al., 2016; Meng et al., 2019; Wang et al., 2021).However, the knowledge about the diazotrophic community structures in the rhizosphere of plants grown in coastal saline soil is limited. In this work, we assess the diversity and community composition of diazotrophic microbes in the rhizosphere of plants grown at the coastal zone of Laizhou Bay-Yellow River estuary in China ( Figures 1A, B ). Similar to that of other coastal lands in the north part of China, the host plant community is mainly composed of various halophytes in this area, and the soil physiochemical properties are obviously varied at different sampling locations ( Figure 1C ; Table 1 ).

Previously it was found that the abundance of nifH gene was also significantly related to the BNF rates of in the temperate coastal region of the northwestern North Pacific (Shiozaki et al., 2015). Consistent with the soil physiochemical properties, we found that nifH gene copy numbers in the collected soil samples varied widely, with the highest nifH gene copy number observed in the rhizospheric soils of plants grown in high salinity and medium moister area ( Figure 2 ; Table 2 ). To further understand the correlation between the nifH gene copy numbers and soil physiochemical properties, we performed Spearman’s correlation analysis. The results demonstrated that, in addition to soil salinity and moister, the copy numbers of nifH gene were also closely related to the value of soil pH, and the levels of TN, TC, TS, NH₄ ⁺-N and NO₃ ^--N ( Table 3 ).

Due to its strong persistence across various soil environments, Bradyrhizobium has been shown to be the dominant diazotroph in soil system (Pereira et al., 2013; Piromyou et al., 2015). Bradyrhizobium was generally detected in the rhizosphere of plants forming root nodules with legumes for N fixation or plants participating in N fixation under non-symbiotic condition (Davis et al., 2011; Meng et al., 2019; Wang et al., 2020; Wang et al., 2021). Halorhodospira was also dominantly identified in the rhizospheric soils of coastal salt tolerant plants due to its strong salinity resistance (Imhoff and Truper, 1997). Azospirillum has been found to be associated with a variety of crops, and performed an important role in the non-symbiotic N fixation (Steenhoudt and Vanderleyden, 2000; Di Salvo et al., 2018). Our results expanded the distribution range of Bradyrhizobium and Azospirillum to the rhizosphere of coastal halophyte plants. In addition, Alpha- and Gammaproteobacteria have been identified as the dominant N fixers in coastal saline soil ecosystem (Yousuf et al., 2014). Similarly, a total number of 10 phyla, with Bradyrhizobium, Desulfovibrio, Halorhodospira and Azospirillum were the dominant diazotrophic groups at genus level, were identified in the collected rhizospheric soil samples ( Figures 3A, B ).

The growth and photosynthetic activity of plants driven by the energy from the sulfide oxidation in root tissues was stimulated by rhizodeposition (Mendelssohn and Morris, 2000; Rolando et al., 2022). In our study, some sulfate reducing bacteria, such as Desulfovibrio, Desulfomicrobium and Desulfobacter, were observed in the diazotrophic community ( Figure 3A ). This is consistent with previous reports that plant rhizodeposition stimulated N fixation by root-associated sulfate reducers in marsh and hypersaline soda lake environments, and Desulfovibrio, Desulfobulbus and Desulfatitalea coupled sulfate or sulfur respiration to N fixation (Gandy and Yoch, 1988; Tourova et al., 2014; Thajudeen et al., 2017; Rolando et al., 2022). A percentage of 17% energy produced in sulfate reduction was provided to the nitrogen fixation process (Nielsen et al., 2001). Thioalkalispira, the typical haloalkaliphilic sulfur-oxidizing bacteria (SOB), could remove the toxic sulphides from the root zone of plants, and couple S oxidation with C and N fixation (Barbieri et al., 2010; Petersen et al., 2016; Thajudeen et al., 2017). In all the collected rhizospheric soil samples, Thioalkalispira, were found to be the core diazotrophs ( Figure 3A ). Similar results were also observed in the coastal sediments of Bohai Bay, China (Wang et al., 2015). Furthermore, in the rhizosphere of each coastal host species grown under different salinity and moisture conditions, a high proportion of unclassified taxa (5.86%) was observed, suggesting that a substantially high and novel diversity of diazotrophic taxa existed in the coastal ecosystem ( Figures 3A, B ). This is possibly attributed to the special soil physicochemical and plant community property of the coastal ecosystem. Indeed, a total number of 495 shared OTUs among the soil samples with different salinity levels were identified, as indicated by the Venn diagram analysis ( Figures 4A-H ).

Under stress condition, specific root related microbial community could be formed in the rhizosphere of a certain plant. And in return, this microbiota may help to improve plant resistance to the corresponding stress (Bai et al., 2022). Ectothiorhodospira species were found in all kinds of aquatic environments, especially in the stagnant littoral, estuarine and highly saline area (Trüper & Imhoff, 1981). They were also found in the rhizospheric soils of plants grown on Ebinur Lake Wetlands, but not in the paddy fields (Davis et al., 2011; Wang et al., 2020; Zhang et al., 2021). In coastal alkaline soil, Ectothiorhodospira attached to anoxygenic purple sulfur bacteria and played a role in the process of N fixation (Tourova et al., 2014; Zhou et al., 2021). We observed that the community structure of diazotrophic microbes in the tested rhizospheric soil samples were significantly affected by the types of halophytes, and as indicated by the high throughput sequencing analysis, Ectothiorhodospira was significantly different in the diazotrophic microbial community of halophyte rhizosphere ( Table 2 ; Figures 5A-C ). No significant difference was found in the diazotrophic abundance and diversity between monocotyledon and dicotyledon, or perennial and annual plant rhizosphere ( Table 2 ; Figures 5D, E ).

When either NO₃ ^--N or NH₄ ⁺-N as N source was insufficient, N fixation occurred (Zhivotchenko et al., 1995). In the rhizosphere of annual halophytes, Synechococcales and Marichromatium might play an important role in N fixation, especiall in coastal saline soils (Rigonato et al., 2013). We observed that, for the community structure of diazotrophic microbes, more diazotrophic genus, such as Dechloromonas, Desmonostoc, Rhodobacter, Methylosinus, Erwiniaceae and Pantoea, were found in the rhizospheric soils of monocotyledonous plants, and the biomarkers were mostly clustered in Synechococcale and Marichromatium in the rhizospheric soils of annual plants ( Figures 5D, E ). These results may be largely explained by the adventitious root systems of monocotyledonous plants, the main organs for the absorption of nutrient and water, and for the recruiting of specific N-fixing microbes in the rhizosphere.

Previous study has showed that low salinity promoted, whereas high salinity inhibited, the growth of soil N-fixing bacteria (Hou et al., 2018). In addition to the salinity, soil electrical conductivity (EC) also functioned as the main factor driving the variation of microbial community composition (Wang et al., 2020). In our study, an influence of soil salinity and moisture on the abundance and community composition of diazotrophic microbes was observed ( Table 2 ; Figure 3B ). In coastal saline soils, the influence of soil salinity and moisture on diazotrophic community could be explained by several reasons (Herbert et al., 2015; Rolando et al., 2022). First, salinization increased NH₄ ⁺ release through ion substitution, which subsequently inhibited N fixation, leading to decreased nifH copies under severely saline environment. Second, seawater infiltration increased soil SO₄ ^2- content, which affected the coupling of chemolithotrophic S oxidation with N fixation, a sulfate reduction process by diazotrophic microorganisms. Third, salinity changed the stability of microbial extracellular enzymes, and affected the mineralization and decomposition of macromolecular organic matter. And fourth, the increase of ionic strength reduced the adsorption of organic matters, and affected the availability of organic substrates for the growth of heterotrophs including diazotrophs.

In conclusion, using high throughput sequencing and quantitative real-time PCR targeting nifH genes, we investigated the diazotrophic abundance and community composition in the coastal saline soils of Laizhou Bay-Yellow River estuary. A strong correlation between diazotrophic abundance and soil physicochemical factors was observed. The physicochemical factors were significantly co-varied with the OTU based community composition of diazotrophs. Our findings demonstrate that in coastal saline soils, the community structure of diazotrophic microbes in the rhizosphere of plants grown in coastal saline soil is mainly affected by soil physicochemical factors but not by the host plant traits.

The datasets presented in this study can be found at NCBI Sequence Read Archive, with accession number SRP411732.

YS, LM, HYZ, RF, XL, YS, ML and JuL conducted the experiments. YS, RF and JL analyzed data. YS, JC and HXZ wrote the manuscript. All authors contributed to the article and approved the submitted version.