Drivers on The Halophytes' Rhizosphere Bacteria Community and Functions in North China Salinized Areas

Soil salinity is a serious environmental issue in arid China. Soil bacteria play a fundamental role in soil systems and respond rapidly to environmental changes. However, the responses of soil bacterial community to the different halophytes remains poorly understood. We investigated rhizosphere soil bacterial community changes under different halophytes in north China using high-throughput sequencing. Three typical halophytes were Leymus chinensis (LC), Puccinellia tenuiora (PT), Suaeda glauca (SG). The dominant phyla were Proteobacteria, Actinobacteria, and Chloroexi across three halophytic vegetation. These bacteria have important assistance for halophytes adapt to saline soil. PICRUSt forecasts demonstrated that energy metabolism, amino acid metabolism and carbohydrate metabolism are main bacterial functions in halophyte vegetation soil, and the abundance of metabolism these bacterial functions in SG was signicantly higher than that in LC and PT. The pH value of different halophyte rhizosphere soils has a more signicant effect on bacterial diversity than EC and soil trophic status, and soil water content (SWC) was important effect factors leading to differences in bacterial functions. These results give us a deeper understanding of the diversity and functional differences of rhizosphere soil bacterial communities in the typical halophytic vegetation of northern China. a compound electrode (INESA Scientic PHSJ-3F) using a soil to water ratio of 1:2.5. Soil electrical conductivity (EC) were tested using a 1: 5 soil water suspension. The soil water content (SWC) was measured after drying in an oven at 105°C for 24 h. Soil organic matter (SOM) was determined by oxidizing organic C with potassium dichromate (K 2 Cr 2 O7), and alkali hydrolysable nitrogen (AN) was measured by alkaline hydrolysis. The available potassium (AK) was measured by ame photometry after 1 mol L −1 CH 3 CO 2 NH 4 neutral extraction 61 , and available phosphorus (AP) was extracted by NaHCO 3 and measured by molybdenum blue colorimetry.


Introduction
Soil salinity is one of the rising environmental issues causing considerable yield losses worldwide especially in arid and semiarid regions 1 . It damages the soil structure, reduces soil quality and limits the growing of crops 2 . Halophytes, such as Suaeda glauca (Bunge), Puccinellia tenui ora, Tamarix chinensis Lour and Leymus chinensis, are plant species that grow well in saline soil due to their saline-alkali tolerance features [3][4] . They contribute enormously to the developing countries supply of food, fuel, ber and fodder 5 . Saline soils are thought harsh environments for life, but such environments survive active and diverse microorganisms 6 . Soil microorganisms are crucial to the maintenance of ecosystem functions due to their contributions to soil structure formation and stability, organic matter decomposition, and nutrition cycling 7 . Soil microbial communities from salinity environment are more complex than soils under neutral pH or moderate salinity conditions 8 .
Although most studies on halophytes have concentrated solely on phytoremediation of saline land and heavy metal contaminated soils by planting halophytes, little known microbially mediated decomposition processes in the rhizosphere soil under different halophytes [9][10] .
The rhizosphere is a critical interface supporting the exchange of nutrients between plants and their associated soil environment 11 . It had long been recognized that bacteria are the most abundant and diverse group of microbes in the soil, and major drivers of biogeochemical cycles and participate in maintaining ecosystem functioning 12 . Many studies indicated that halotolerant rhizobacteria isolated from halophytes enhance salt tolerance in their host plants 13 . For example, Marasco et al. (2016) reported that rhizobacteria colonized in the Salicornia strobilacea rhizoplane is capable of improving plant growth 14 . Kearl et al. (2019) found that several bacteria communities, such as Halomonas, Bacillus, and Kushneria, have been observed to improve growth of alfalfa under saline conditions 15 . Likewise, several studies have been published on bene cial effects of bacterial application on wheat growth under salt conditions [16][17] . But, more information on the bacterial community present in the rhizospheric of various halophyte species is needed before these halotolerant rhizobacteria can be applied in salinity affected agriculture soil 18 .
Pyrosequencing is a novel technique to expand our understanding of the bacterial community composition, diversity and in relation to their environments 19 . Simultaneously, PICRUSt has been widely used to predict the bacterial functions based on the16S rRNA gene 20 . Here, we investigated the effect of various halophyte species on soil bacterial communities in rhizospheric soil using high-throughput sequencing in north China. The objectives of this study were to: (i) investigated the soil properties changes under different halophyte species, (ii) reveal the bacterial community composition, diversity and predicted functions in soils from different halophyte species, (iii) to determine the possible factors in shaping bacterial community changes in these rhizospheric soils.

Material And Methods
Site description, experimental design and sampling. Soil samples were collected from three typical salinized areas in the north of China (40°28'37"-44°34'19", 85°54'03"-123°17'45"). The three typical salinized areas are located in Tumochuan Plain in Inner Mongolia, Songnen Plain in Jilin Province, and Manasi River Basin in the Xinjiang. The dominant halophyte is Leymus chinensis (Trin.) Tzvel in Tumochuan Plain, Puccinellia tenui ora (Griseb.) Scribn. et Merr in Songnen Plain, and Suaeda glauca (Bunge) Bunge in Manasi River Basin (more than 85% of species present). Leymus chinensis (Trin.) Tzvel can tolerate cold, drought and alkali, has good nutritional value and high palatability. It has potential value as animal forage 57 . Puccinellia tenui ora (Griseb.) Scribn. et Merr is monocotyledonous halophyte and an alkali tolerant species that can survive in highly alkaline soil 58 . Suaeda glauca (Bunge) Bunge is a rigid, annual, 100 cm high grass occurring in alkali conditions such as coastal region, wastelands, canal banks, and elds and tender plants are delicious and edible 59 . A summary of each treatment is given in Table 1. Soil samples were collected in the middle of May 2019. Within an area of approximately 35 ha, we randomly selected nine plots (replicates) in each samples, each plot 3 m×3 m within monospeci c population of each halophyte species for sampling. With the use of soil corer (5 cm diameter), soil samples were collected from the roots of plants with the same canopies by using sterile brushes 60 . Soil samples of each replicate were put in individual plastic bags and transported to the laboratory. After visible stones and plant residues were removed, soil samples were sieved (2 mm) the separated into two subsamples, one portion was air-dried for the determination of chemical analysis, and the reminder was stored in a -20 °C refrigerator for molecular analysis.
Soil properties determination. Soil pH was measured with a compound electrode (INESA Scienti c PHSJ-3F) using a soil to water ratio of 1:2.5. Soil electrical conductivity (EC) were tested using a 1: 5 soil water suspension. The soil water content (SWC) was measured after drying in an oven at 105°C for 24 h. Soil organic matter (SOM) was determined by oxidizing organic C with potassium dichromate (K 2 Cr 2 O7), and alkali hydrolysable nitrogen (AN) was measured by alkaline hydrolysis. The available potassium (AK) was measured by ame photometry after 1 mol L −1 CH 3 CO 2 NH 4 neutral extraction 61 , and available phosphorus (AP) was extracted by NaHCO 3 and measured by molybdenum blue colorimetry.
DNA extraction and PCR ampli cation. Soil DNA was extracted from fresh soil sample (0.5 g) using Soil DNA Kit (Omega Biotek, Norcross, GA, U.S.), according to the manufacturer's protocols. The quality of extracted DNA was assessed by 1% agarose gel electrophoresis and substandard samples were extracted again until all the samples passed the quality control. All extracted DNA samples were stored at -20 °C for further analysis. The V4-V5 hypervariable regions of the soil bacterial 16S rRNA gene were subjected to high-throughput sequencing by Majorbio Pharmaceutical Technology Co., Ltd. (Shanghai, China) using PE300 sequencing platform (Illumina, Inc., CA, USA). The V4-V5 bacterial 16S rRNA gene were ampli ed by PCR using the primers pair 515 F/907R. The PCR program was as follows: denaturation at 95°C for 30 s; annealing at 5 °C for 30 s; extension at 72°C for 45 s; 27 cycles; holding at 72°C for 10 min and storing at 10°C. In order to guarantee the accuracy of the analysis results, Quantitative Insights into Microbial Ecology (QIIME software) (version1.9.0) was used for sequence ltering, and then the chimeric sequence was removed using Mothur software to obtain a high-quality sequence for subsequent analysis 62 . The Usearch program was used to cluster eligible sequences into operational classi cation units (OTUs) with a cutoff of 97% similarity. The representative sequence of each OTU is compared with the reference database.
Pyrosequencing data processing and statistical analysis. Venn diagrams were used to describe compare the similarities between soil bacterial communities at OTU level. According to the species abundance of each sample in the OTU list, Soil bacterial richness indices (Chao and Ace) and diversity index (Shannon) were calculated by using Mothur software 63 . We used nonmetric multidimensional scaling (NMDS) based on unweighted UniFrac distance to reveal changes in soil bacterial community structures 64 . Linear discriminant analysis (LDA) coupled with effect size measurements (LEfSe) analysis was conducted to search for statistically different biomarkers between groups 65 . The in uences of halophytic vegetations and soil properties on soil bacterial community structures were examined by Mantel test and distance-based redundancy analysis (db-RDA). Spearman correlation coe cients were calculated and tested for signi cance between the dominant OTUs and soil properties. PICRUSt (Phylogenetic Investigation of Communities by Reconstruction of Unobserved States) was performed to predict the relative abundance of main metabolic function genes 20 . The functional genes were predicted from the Kyoto Encyclopedia of Genes and Genomes (KEGG) catalogue 37 . We explored redundancy analysis (RDA) to understand the relationship between relative abundance of predicted functional genes and soil properties. SPSS version 20.0 (SPSS Inc., Chicago, USA) was utilized to data statistics and analysis of variance to determine the difference between soil properties, bacterial community and bacterial function spectrum. R was used to perform redundancy analysis. Venn diagram, cladogram, spearman correlation heatmap and abundance of KEGG heatmap were drawn using R 66 . The speci c functional differences were performed using Origin version 9.0 (Microcal Software, Inc., Northampton, MA, USA).

Results
Soil properties. The changes of rhizosphere soil properties under different halophytes were shown in Table 2. Signi cant differences of soil pH were found between soil samples from the different halophytes (P < 0.05). PT had the highest soil pH, whereas LC had the lowest pH. Soil EC signi cantly (P < 0.05) varied from 0.45 to 1.44 (mS·cm -1 ) across the soil samples where the LC treatment showed highest values. There was a signi cant variation in SOM under different halophytes rhizosphere soil (P < 0.05). The highest SOM content was found in samples from LC, which was 84.31% higher than that of SG.
SWC followed the order PT > LC > SG, and there was no signi cant difference between LC and PT (P > 0.05). There was a signi cant variation in available nutrients, which ranged from 7.28 to 17.05 mg kg −1 for available phosphorous (AP), from 115.2 to 240.73 mg kg −1 for available potassium (AK), and from 115.2 to 240.73 mg kg −1 for available nitrogen (AN), respectively (P < 0.05).
Soil bacterial diversity. Venn diagrams indicated that the sum of total observed OTUs in the soil samples from three halophytes rhizosphere was 10,154 (Fig. 1a), and 1,413 OTUs were shared by all three groups. The numbers of OTUs cooccurred in LC, PT and SG were 1 232, 522 and 931, respectively. To determine whether different halophytes are associated with alteration of soil bacterial community structure, we pro led the overall structural changes of bacterial community by using NMDS based on unweighted UniFrac dissimilarities (Fig. 1b). NMDS ordinations showed soil samples in LC are closely accumulated together, samples in PT and SG separated with LC along NMDS axis1 (P < 0.01). Coverage percentage of soil samples under three halophytes exceeded 98%, indicating that the sequencing results can be used for further analysis (Fig. 2).
LC had the highest Shannon index, but there was no signi cant difference in LC and SG (P > 0.05). The ACE index of samples under different halophytes had the same trends with Chao index, the highest values were recorded in LC, but there was no signi cant difference in SG and LC (P < 0.05) Soil bacterial community composition. A total of 38 bacterial phyla were procured from soil samples. Proteobacteria, Actinobacteria, Chloro exi, Gemmatimonadetes, Acidobacteria, Bacteroidetes, Planctomycetes, and Firmicutes were dominant phyla through entire soil samples (Fig. 3a). However, no signi cant difference was observed between the LC, PT and SG. To further compare the differences in bacterial community composition between rhizosphere soil under different halophytes, we conducted the relative abundance of bacteria (> 0.02%) at the class level (Fig. 3b). The dominant classes were Actinobacteria, Alphaproteobacteria, Gemmatimonadetes, Gammaproteobacteria and Anaerolineae in LC, PT and SG. Statistical analysis was performed from the phylum to the class level under the LEfSe tool. Groups were shown in cladograms, and LDA scores of 3 or greater were con rmed by LEfSe (Fig. 3c). Planctomycetes, Planctomycetacia and Rhodothermia were signi cantly enriched in LC. Rokubacteria, Deltaproteobacteria, Anaerolineae, NC10, Ignavibacteria and Thermodesulfovibrionia were signi cantly enriched in PT. Cyanobacteria, Chloro exia, Oxyphotobacteria, Nitrospira, Nitrospirae, Deinococci, Deinococcus_Thermus and Gitt_GS_136 were signi cantly enriched in SG.
Soil potential bacterial functions. We used PICRUSt to predict the soil bacterial community function in the rhizosphere soil under three halophytes (Fig. 5). The classi cation of KEGG functions at pathway Level 1 and pathway Level 2 was performed in Figure 4. The result showed that the predicted functions mainly the bacterial metabolism, and the relative abundance of three halophytes was above 60% at pathway Level 1. Carbohydrate metabolism, amino acid metabolism, energy metabolism, nucleotide metabolism and lipid metabolism were main metabolic functions at pathway Level 2. Interestingly, the carbohydrate metabolism, amino acid metabolism and energy metabolism were observed to account for more than 7% at pathway Level 2 throughout the three halophytes. Further statistical tests revealed the relative abundance of carbohydrate metabolism, Lipid metabolism and xenobiotics biodegradation and metabolism were highest in SG (Fig. 5). Nucleotide metabolism, Replication and repair, Folding, sorting and degradation and Transcription were no signi cant difference between LC and PT (P > 0.05), which was signi cantly higher than that in SG. Cell motility and Transport and catabolism in LC and SG, which was signi cantly higher than that in PT ( Fig. 5; P < 0.05).
Correlation between bacterial communities and soil properties. We explored the in uence of soil properties on bacterial community composition at OTU level through db-RDA analysis ( Fig. S1; Mantel test, R = 0.492, P = 0.001). It is apparent from this chart that the two axes of CAP axis explain 37% of total variation, and soil EC, pH, SOM, AK and AN were longer arrows.
The quantitative data between the soil properties and the bacterial communities analyzed by db-RDA at the OTU Leve l was shown in Table 3, Soil EC (P = 0.001), pH (P = 0.001), SOM (P = 0.002), AK (P = 0.001) and AN (P = 0.001) were closely correlated to the CAP axis. EC, pH, SOM and AN were good illustration that these soil properties were signi cantly correlated with the top 50 abundance of bacterial OTUs through further correlation analysis ( Fig. 6; Table S1), and this coincides with the results of db-RDA analysis. To determine which soil properties affected the metabolic functions, we performed RDA analysis of soil properties and pathway Level 2's KEGG function. We found that these metabolic functions are signi cantly correlated with soil SWC (P = 0.001) and AK (P = 0.001; Table 4). On the other hand, carbohydrate metabolism, amino acid metabolism, nucleotide metabolism, lipid metabolism, xenobiotics biodegradation and metabolism, glycan biosynthesis and metabolism, translation, replication and repair, folding, sorting and degradation, transport and catabolism and signaling molecules and interaction were signi cantly correlated with soil SWC (Table S3; P < 0.01).

Discussion
Soil properties. Our study found that soil properties, such as pH, EC, SOM, were changed signi cant in rhizosphere soil of three halophytes. Soil pH in PT was signi cantly higher than that of SG and LC, the changes in soil pH could be attributed to the higher concentration of Na 2 CO 3 in soil. Previous studies showed that saline-alkali soil increased soil pH by increasing soil Na + , CO 3 2− , and HCO 3 − concentration [21][22][23] . Excessive soluble salt content leads to the EC in LC being signi cantly higher than that in SG and PT. The current study found that soil SOM content is signi cant differences in rhizosphere soil of three halophytes, and the highest SOM content was found in samples from LC. It is consistent with prior studies that have noted the in uence of different halophytes type to soil organic matter 24 . This discrepancy could be attributed to different halophytes forms in uencing organic matter input by plant debris input and rooting depth 25 .
Soil bacterial community diversity of different halophytes. Soil bacterial diversity is critical to maintaining the soil ecosystems 26 . Yamamoto (2018) who reported the diversity of bacterial communities, depends on the halophytic plant species and the sampling site 13 . In our study, the soil samples from different regions with three halophytes are geographically distant, environment and climatic factors are different (Table 1). These factors can shape the diversity of rhizosphere soil bacterial community. The results of NMDS analysis con rm that soil bacterial community were signi cant difference in rhizosphere soil of three halophytes. Study has shown that the rhizosphere of plants has speci c selectivity for bacteria that colonize the rhizosphere, which change the species richness and homogeneity, leading to differences in alpha diversity 27  Proteobacteria accounting for over 40% in samples from Glaux maritima and Salicornia europaea 13 . We found that the relative abundance of Proteobacteria was more than 20% in three halophytes rhizosphere soils (Fig. 3a). This indicates that three halophytes may affected by the salt stress, and enriched Proteobacteria Actinobacteria and Chloro exi in the rhizosphere soil.
Plant species certainly affect the structure of bacterial communities, and select speci c microbial populations 34 . Speci c selection leads to differences in the rhizosphere bacterial communities of halophytes, and con rmed in our research (Fig. 3c).
These results indicate that bacteria have important assistance for halophytes adapt to saline soil, and the abundance bacteria is also affected by different halophytes vegetation types. Soil bacteria play a vital part in nutrient cycling, maintaining soil fertility, and carbon sequestration through amino acid and carbohydrate metabolism [35][36] . The relative abundance of bacterial metabolism was above 60% in soil from three halophytes rhizosphere. Carbohydrate metabolism, amino acid metabolism, rhizosphere soil bacterial communities. The microbial biogeography is controlled primarily by edaphic variables, especially by pH 50 . Our results con rm that soil EC (P = 0.001), pH (P = 0.001) and SOM (P = 0.002) are signi cantly related to the soil bacterial community and are consistent with previous research [51][52] . Correlation analysis showed that ACE, Chao, and Shannon indices negatively correlated with pH, while the effect of EC and SOM on bacterial diversity in this study was not signi cant (Table S2). It is consistent with prior studies who reported that the soil bacterial diversity effected by pH [53][54][55] . The pH not only affects the abundance but also the bacteria diversity, this suggesting that soil pH is important factor in shaping soil bacterial diversity in rhizosphere soil of halophytes. Simultaneously, this in uence of soil properties on bacterial functions is also re ected in the prediction function 34,49 . We found that the main metabolic functions signi cantly correlated with SWC (Table 4;