Metagenomic analysis of cucumber rhizospheric microbiome provides insights into NaHCO3 stress mitigation by Rheinheimera pacifica NYJ


 Background: NaHCO3 in soil leads to alkalinization and causes stress in plants, thus hindering agricultural sustainability. A strain NYJ was identified as Rheinheimera pacifica and applied into cucumber-planted soil to mitigate NaHCO3 stress in this study. We aimed to detect the taxonomy and functional potential of rhizospheric microbiome affected by NaHCO3 stress and NYJ application, obtaining NaHCO3-tolerant microbes and exploring one mechanism underlining NaHCO3 stress mitigation by NYJ application.Results: The strain NYJ grew well under 0.01-0.06 g L-1 NaHCO3 and had plant growth-promoting capacity. Application of NYJ into NaHCO3-contaminated and cucumber-planted soil improved plant growth, increased relative water contents and osmotic potential of leaves, and decreased malonaldehyde and hydrogen peroxide contents in seedlings, mitigating NaHCO3 stress in cucumber. In cucumber rhizospheric soil, Proteobacteria, Actinobacteria, and Bacteroidetes of microbes were dominant phyla with 69, 17, and 6% of total abundance. To respond to NaHCO3 stress, 669 species and 208 genera of microbes were enriched in rhizospheric soil, while 190 sodium ion-related genes, 1242 osmotic stress-related genes, and 121 other stress-related genes from microbial genera including Pseudomonas showed the increased abundances. Under NaHCO3 stress, 26 microbial genera were distributed in connectors, the interaction network of rhizospheric microbes was changed, and soil enzyme activities were decreased. When NYJ being applied into the NaHCO3-contaminated soil, 1327 species and 487 genera of microbes were enriched than those under NaHCO3 stress alone. Meantime, soil enzyme activities were increased, while 2238 symbiont- and immune-related genes and 2434 plant growth-promoting genes including 16 genes encoding chitinase had the elevated abundances in microbial genera. Moreover, NYJ negatively interacted with one bacterial genus and positively interacted with 3 microbial genera under NaHCO3 stress. As a result, 25 bacterial genera were distributed in connectors, and microbial interaction network in rhizospheric soil was different from the one caused by NaHCO3 stress alone.Conclusions: 669 NaHCO3-tolerant species and 208 NaHCO3-tolerant genera of microbes were identified. NYJ application affected microbial community structure and interaction network in rhizospheric soil, increased the abundances of genes related to symbiont, immune, and plant growth-promoting traits in microbes, and activated soil enzymes, thereby mitigating NaHCO3 stress in cucumber.1 These authors contributed equally to this work.

4 rhizospheric soil, increased the abundances of genes related to symbiont, immune, and plant growthpromoting traits in microbes, and activated soil enzymes, thereby mitigating NaHCO3 stress in cucumber. 1 These authors contributed equally to this work.

Background
Microbes in plant rhizosphere interact with each other and also with plants [1]. These interactions play important roles in many vital processes such as nutrient cycling [2] and have impacts on plant growth [3]. Therefore, rhizospheric microbiome has received an increasing attention in recent years [4]. In the rhizospheric microbiome, plant growth-promoting rhizobacteria (PGPR) are beneficial to plants [5]. When PGPR are isolated from plant rhizosphere and then applied into planted soil, they not only change the levels of phytohormones, antioxidants, and epoxypolysaccharide [6], but also influence the rhizospheric microbial communities [7] by having competitive skills over the existing rhizosphere microbes [8], thus improving plant resistance to environmental stresses. For example, inoculation with Bacillus amyloliquefaciens NBRISN13 stimulates the population of betaine-, sucrose-, trehalose-, and glutamine-utilizing bacteria in rhizosphere and induces salt tolerance in rice [9].
Application of Streptomyces canus GLY-P2 has positive impact on microbial community composition in rhizosphere and mitigates stresses of ferulic and p-hydroxybenzoic acids in cucumber [10].
Soil salinization and alkalinization hinder plant growth and lead to desertification [11]. Alkaline soils, which are mainly caused by NaHCO 3 or Na 2 CO 3 , have an area of 434 million hectares in the world [12]. So NaHCO 3 stress has become one of the major constraints to crop yield and agricultural sustainability [13]. Under the stress of NaHCO 3

, inoculation of Enterobacter aerogenes LJL-5 and
Pseudomonas aeruginosa LJL-13 increases alfalfa yield and quality [14]. Application of Trichoderma asperellum decreases the infection incidence of pathogenic fungi and alleviates NaHCO 3 stress in poplar [15]. To the best of our knowledge, it has not been reported that PGPR would mitigate the stress of NaHCO 3 in plants by changing the microbial community composition and structure in plant rhizosphere. Moreover, uncovering the functional information of microbiome might contribute to 5 further understanding the PGPR-mediated stress mitigation [6], however, the function potentials of genes have not been reported in the rhizospheric microbes when PGRR being applied under NaHCO 3 stress. In addition, salt affects soil microorganisms, and many salt tolerance bacteria have been isolated [16]. Up to now, effects of NaHCO 3 stress on rhizospheric microbiome have not been analyzed, and NaHCO 3 tolerance microbes have not been identified.
In this study, a strain NYJ was isolated from rhizospheric soil, identified as Rheinheimera pacifica, and then applied into cucumber-planted soil under NaHCO 3 stress. It was hypothesized that the strain NYJ might belong to PGPR and mitigate NaHCO 3 stress in cucumber by affecting rhizospheric microbiome.
We aimed to determine the changes in taxonomy and functional potential of rhizospheric microbiome caused by NaHCO 3 stress and NYJ application, thus obtaining NaHCO 3 tolerance microbes and exploring one mechanism underlining the NaHCO 3 stress mitigation by NYJ application.

Materials And Methods
Isolation, identification, and properties of the strain NYJ Using a spread plate technique [17], the strain NYJ was isolated from the rhizosphere of wheat seedlings that have been cultured in saline-alkaline soils for 6 months. Each dilution was added to 20 mL of beef extract-tryptone agar medium (containing 10 g L − 1 tryptone, 5 g L − 1 NaCl, 5 g L − 1 beef extract, and 1.5% agar), in which the pH value was adjusted to 10 with NaOH. To identify the strain NYJ, its 16S rRNA gene sequence was amplified with the primers 1492R (5'-GGTTACCTTGTTACGACTT-3') and 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and used to perform sequence alignment and phylogenetic analysis [18]. In order to analyze the ability of NYJ to tolerate NaHCO 3 , 1 mL of strain cultures grown exponentially in Luria-Bertani (LB) liquid medium were inoculated into 100 mL of beef extract-tryptone liquid medium supplemented with 0.01, 0.03, 0.04, 0.06, 0.08, or 0.1 g L − 1 NaHCO 3 . Subsequently, they were incubated at 37 °C and 180 rpm. The alteration of the optical density was observed at 600 nm [19]. To verify the NaHCO 3 tolerance ability of NYJ, 1 mL of strain cultures grown exponentially in LB medium were inoculated into 100 mL of the beef extract-tryptone liquid medium, in which the pH value was adjusted to 6, 6.5, 7, 8, 9, 10, 10.5, or 6 11. To determine the plant growth-promoting properties of NYJ and assess whether the strain would belong to PGPR, its ability of indole acetic acid (IAA) production [20], phosphate solubilization [21], and siderophore production [22] were detected, while the activity of 1-aminocyclopropane-1carboxylic acid (ACC) deaminase of the strain were analyzed [23].

Nahco Stress Mitigation In Cucumber By The Strain Nyj
According to the procedures of Zhang et al. [18], each cucumber seedling was cultured in one 10-cm plastic pot filled with 400 g of soil. The soil contained 1.37 g kg − 1 total N, 29.95 mg kg − 1 NO 3 -N, 13.87 mg kg − 1 NH 4 -N, 20.96 mg kg − 1 Olsen-P, 86.76 mg kg − 1 rapidly-available K, and 0.75% organic matter, and its pH value was 7.34. At the two-leaf stage, 32 cucumber seedlings were chosen and divided into four groups (8 plants per group) to impose the following treatments: I (control group), watered with 10 mL of autoclaved water; II (NaHCO 3 treatment group), watered with 10 mL of NaHCO 3 (0.12 g mL − 1 ) to make the concentration of NaHCO 3 reach 3 mg g − 1 dry soil, which induced stress in cucumber but did not cause plant wilting; III (NYJ treatment group), watered with 10 mL of NYJ (10 9 cfu mL − 1 ); IV (NYJ + NaHCO 3 treatment group), watered with 10 mL of solutions containing NYJ (10 9 cfu mL − 1 ) and NaHCO 3 (0.12 g mL − 1 ). Then, all of the seedlings were cultured according to Wu et al. [10]. After 12 days of treatment, the second leaves and rhizospheric soil of cucumber were collected.
In order to confirm the ability of NYJ to mitigate NaHCO 3 stress in cucumber seedlings, we measured the effects of the strain on cucumber seed germination under NaHCO 3 conditions. When being sterilized with 10% sodium hypochlorite, 60 cucumber seeds were put into the cultures of NYJ grown exponentially in LB liquid medium. Subsequently, the seeds were incubated at 28 °C and 180 rpm for 12 h. Meantime, another 60 sterilized seeds were incubated in LB liquid medium and were not treated with NYJ. After that, 30 NYJ-treated and 30 NYJ-untreated seeds were placed on sterile filter paper moistened with 3% NaHCO 3 in petri dishes. They were named as the "NYJ + NaHCO 3 treatment" and "NaHCO 3 treatment", respectively. Meantime, the remaining 30 NYJ-treated and 30 NYJ-untreated seeds were put on sterile filter paper moistened with sterilized distilled water, and they were named as the "NYJ treatment" and "control", respectively. Then, all of the filter paper was kept moistened with sterilized distilled water everyday. At 7 d, cucumber roots were collected.
To assess the effects of NYJ application on NaHCO 3 -stressed cucumber, the levels of plant growth parameters such as the fresh and dry weights of shoots and roots were determined [24]. Meantime, the activities of soil enzymes including catalase (CAT) [25], alkaline phosphatase [26], urease [27], and sucrase [28] were assayed in rhizospheric soil. The levels of relative water content [29], osmotic potential [30], and malonaldehyde (MDA) [31] in leaves were also measured. Since hydrogen peroxide (H 2 O 2 ) will accumulate in stressed plants and result in the over-production of MDA, the content of H 2 O 2 was assayed in leaves [32] to confirm the MDA level. In order to verify the H 2 O 2 content, the activities of H 2 O 2 -related antioxidant enzymes such as CAT [33], ascorbate peroxidase (APX) [34], guaiacol peroxidase (GPX) [35], dehydroascorbate reductase (DHAR) [36], monodehydroascorbate reductase (MDHAR) [37], glutathione reductase (GR) [38], and superoxide dismutase (SOD) [39] were determined in leaves. Since reduced glutathione (GSH) is regenerated by GR, its content were detected [40] to verify the activity of GR.
16s Rrna Amplicon Sequencing Of Microorganisms In Rhizospheric Soil Microorganism DNA in cucumber rhizospheric soil was extracted by using an EZNA® Soil DNA Kit (Omega Bio-tek, Norcross, GA, USA). Then, 16S rRNA amplicon sequencing and data processing were performed at Personal Biotechnology Co., Ltd. (Shanghai, China) [41].

Metagenome Sequencing Of Rhizospheric Microbes
When being extracted from rhizospheric soil, microbial genomic DNA was used to construct metagenome shotgun sequencing libraries by using the Illumina TruSeq Nano DNA LT Library Preparation Kit. Subsequently, each library was sequenced, and all data were processed at Personal Biotechnology Co., Ltd. (Shanghai, China) [42].

Results
NaHCO 3 tolerance ability and plant growth-promoting properties of the strain NYJ The 16S rRNA gene sequence of the strain NYJ showed 100% homology to Rheinheimera pacifica 8 (Additional file 1: Fig. S1), so the strain was identified as Rheinheimera pacifica NYJ. At 10 h, the cell numbers of NYJ under 0.01-0.06 g L − 1 NaHCO 3 were the same as the ones under 0 g L − 1 NaHCO 3 and were higher than those under 0.08-0.1 g L − 1 NaHCO 3 (Additional file 1: Fig. S2a). These were verified by the results that the strain NYJ had the same numbers of cells under pH 6.5-10.5 at 20 h and showed low numbers of cells under pH 6 and 11 (Additional file 1: Fig. S2b). Therefore, NYJ could grow under NaHCO 3 environment. On the other hand, NYJ had the capacity of IAA production, phosphate solubilization, and siderophore production and showed the activity of ACC deaminase, belonging to PGPR (Additional file 1: Fig. S3).

Nahco Stress Mitigation In Cucumber By Nyj Application
In comparison to the control, the shoot fresh and dry weights, plant height, areas of the third leaves, relative water contents and osmotic potential of the second leaves were significantly increased in cucumber seedlings of the NYJ treatment and were significantly decreased in the NaHCO 3 treatment, while the contents of MDA and H 2 O 2 in the second leaves were significantly decreased in the NYJ treatment and were significantly increased in the NaHCO 3 treatment ( Fig. 1a and Additional file 1: Fig.   S4). When compared the NYJ + NaHCO 3 treatment to the NaHCO 3 treatment, the shoot fresh and dry weights, plant height, areas of the third leaves, relative water contents and osmotic potential of the second leaves were significantly increased, and the contents of MDA and H 2 O 2 in the second leaves were significantly decreased. These were consistent with the results that the activities of APX, CAT, GPX, MDHAR, DHAR, GR, and SOD and the content of GSH in the second leaves were significantly increased in cucumber seedlings of the NYJ + NaHCO 3 treatment in comparison to the NaHCO 3 treatment (Additional file 1: Fig. S5). So inoculation with NYJ into soil mitigated NaHCO 3 stress in cucumber seedlings. Similarly, NYJ application increased root length, decreased the contents of MDA and H 2 O 2 , and elevated the activities of APX, CAT, DHAR, MDHAR, and SOD in roots, alleviating NaHCO 3 stress in germinating cucumber seeds (Additional file 1: Fig. S6).

Effects of NYJ application and NaHCO 3 stress on soil enzyme activity
Compared to the control, the activities of urease (Fig. 1b), phosphatase (Fig. 1c), and sucrase ( Fig. 1d) were significantly increased in the rhizospheric soil of the NYJ treatment and were significantly decreased in the NaHCO 3 treatment, while the activity of CAT (Fig. 1e) did not change in the NYJ treatment and were significantly reduced in the NaHCO 3 treatment. In the NYJ + NaHCO 3 treatment in comparison to the NaHCO 3 treatment, the activities of urease, phosphatase, sucrase, and CAT were significantly increased in the rhizospheric soil. Therefore, NYJ application increased soil enzyme activity under NaHCO 3 stress.
Effects of NYJ application and NaHCO 3 stress on microbial richness and diversity in rhizospheric soil Whether using the shotgun metagenomic sequencing or the 16S rRNA amplicon sequencing, the operational taxonomic units (OTUs) of observed species in the treatments of control, NYJ, NaHCO 3 , and NYJ + NaHCO 3 tended to be flat with the increasing sequence numbers (Additional file 1: Fig. S7).
Thus, the sequencing depth was reasonable to detect the majority of species in each treatment. The microbes in the rhizospheric soils of all treatments were identified ( Fig. 2 and Additional file 1: Fig. S8-S12). Based on the shotgun metagenomic sequencing, there were 7880 species shared in all treatments (Additional file 1: Fig. S13). Considerable overlap was observed in the NYJ-treated rhizospheric soils, with the NYJ and NYJ + NaHCO 3 treatments having 8404 shared species. Meantime, the rhizospheric soils treated with NaHCO 3 had considerable overlap, with the NaHCO 3 and NYJ + NaHCO 3 treatments having 8268 shared species. Moreover, in the control, NYJ, NaHCO 3 , and NYJ + NaHCO 3 treatments, the numbers of species were 8749, 8843, 8679, and 8772, respectively, and the distinct species were 64, 108, 97, and 74, respectively. Similarly, using the 16S rRNA amplicon sequencing, we not only observed the shared OTUs of microbial communities among four treatments, but also found the distinct ones in each treatment (Additional file 1: Fig. S14). According to the principal component analysis (PCA) based on the abundances of species (Additional file 1: Fig. S15) and genera (Additional file 1: Fig. S16), the microbial communities of the NaHCO 3 treatment separated from those of the control or NYJ + NaHCO 3 treatment. So soil microbial richness and diversity were changed by NaHCO 3 treatment and affected by NYJ application under NaHCO 3 stresses. 10 Effects of NYJ application and NaHCO 3 stress on microbial community composition and structure in rhizospheric soil In rhizospheric soil of the control, NYJ, NaHCO 3 , and NYJ + NaHCO 3 treatments, the majority of microbes were bacteria (Additional file 2: Table S1). Based on the shotgun metagenomic sequencing, Proteobacteria, Actinobacteria, and Bacteroidetes in rhizospheric soil were the dominant phyla with 69, 17, and 6% of total abundance, respectively ( Fig. 2a and Additional file 1: Fig. S17). The three phyla were also dominant in all treatments according to the 16S rRNA amplicon sequencing ( Fig. 2b and Additional file 1: Fig. S18).  (14), Acinetobacter (13), and Sphingomonas (13). Using the 16S rRNA amplicon sequencing, the genera Pseudomonas, Bacillus, Rhizobium, Microbacterium, and Acinetobacter were also found to respond to NYJ application (Additional file 1: Fig. S20 and Additional file 3: Table S3). Therefore, NYJ application affected microbial community composition and structure in rhizospheric soil.
To respond to NaHCO 3 stress, 834 OTUs were enriched and 1489 OTUs were depleted according to the shotgun metagenomic sequencing (Additional file 3: Table S4) Sphingomonas (14). Based on the 16S rRNA amplicon sequencing, the genera Pseudomonas, Rhizobium, Bradyrhizobium, Mycobacterium, and Sphingomonas were still in response to NaHCO 3 tress (Additional file 4: Table S5). Moreover, 208 genera were enriched and 421 genera were depleted in the NaHCO 3 treatment in comparison to the control by using the shotgun metagenomic sequencing (Additional file 4: Table S6), indicating that NaHCO 3 stress changed microbial community composition and structure.
Using the shotgun metagenomic sequencing, 1539 enriched and 612 depleted OTUs were found when compared the NYJ + NaHCO 3 treatment to the NaHCO 3 treatment, among which 1327 enriched and 489 depleted species were identified (Additional file 5: Table S7) Xanthomonas (12). Using the 16S rRNA amplicon sequencing, the genera Pseudomonas, Paenibacillus, Sphingomonas, Bradyrhizobium, Bacillus, and Legionella were also in response to NYJ application under NaHCO 3 stress (Additional file 6: Table S8). As shown in Additional file 6: Table S9, 487 genera were enriched and 137 genera were depleted in the NYJ + NaHCO 3 treatment when compared to the NaHCO 3 treatment. There were 257 depleted and 20 enriched genera in the NaHCO 3 treatment in comparison to the control, and these genera were enriched when compared the NYJ + NaHCO 3 treatment to the NaHCO 3 treatment (Additional file 7: Table S10). Meantime, 6 depleted and 78 enriched genera were found in the NaHCO 3 treatment when compared to the control, and they were depleted in the NYJ + NaHCO 3 treatment in comparison to the NaHCO 3 treatment. So NYJ application affected microbial community composition and structure under NaHCO 3 stress.

Effects of NYJ application and NaHCO 3 stress on the interactions of microbial communities
The interaction networks for rhizospheric microbes of the control, NYJ, NaHCO 3 , and NYJ + NaHCO 3 treatments were different from each other whether using the shotgun metagenomic sequencing or the 16S rRNA amplicon sequencing (Figs. 4 and 5, and Additional file 1: Fig. S21-S26). Based on the shotgun metagenomic sequencing, the strain NYJ negatively interacted with 10 species in the rhizospheric soil of the NYJ treatment (Additional file 1: Fig. S24), and it negatively interacted with 1 species and positively interacted with 11 species in the NYJ + NaHCO 3 treatment (Fig. 5). Meantime, using the 16S rRNA gene sequences, one OTU was identified as Rheinheimera pacifica, and its relative abundance was significantly increased in the NYJ treatment in comparison to the control or in the NYJ + NaHCO 3 treatment when compared to the NaHCO 3 treatment (Additional file 1: Fig. S27). The OTU of Rheinheimera negatively interacted with one genus in the NYJ treatment (Additional file 1: Fig.   S25), while it negatively interacted with one genus and positively interacted with 3 genera in the NYJ + NaHCO 3 treatment (Additional file 1: Fig. S26). So NYJ application and NaHCO 3 stress affected the interaction of microbes in rhizospheric soil.
The majority of the OTUs in the control, NYJ, NaHCO 3 , and NYJ + NaHCO 3 treatments were peripherals with most of their links being inside modules (Additional file 1: Fig. S28-S31). One network hub, which highly linked to many species in its own module and highly connected to several modules, was assigned to Acidovorax whether in the treatments of control, NYJ, NaHCO 3 , or NYJ + NaHCO 3 . There were three module hubs, which highly linked to many species in their own modules and respectively belonged to Pseudoxanthomonas, Flavobacterium, and Glutamicibacter in all treatments. In the connectors that highly connected to several modules, there were 18, 23, 26, and 25 identified genera in the control, NYJ, NaHCO 3 , and NYJ + NaHCO 3 treatments, respectively.

Effects of NYJ application and NaHCO 3 stress on the functional profiles of microbial communities
Based on the shotgun metagenomic sequencing, microbial communities in rhizospheric soil responded to abiotic and biotic stimulus and showed plant growth-promoting traits including nitrogen utilization (Additional file 1: Fig. S32). The genes related to osmotic stress were found in microbial communities (Fig. 6). When compared the NaHCO 3 treatment to the control, the relative abundances of osmotic stress-related genes were significantly decreased in GO:0006970, GO:0009651, GO:0071470, and GO:0030104 and did not change in GO:0061392, GO:0047484, GO:0006972, and GO:0071474 (Additional file 8: Table S11). However, 1242 osmotic stress-related genes showed significantly increased relative abundances in the NaHCO 3 treatment in comparison to the control. In 2 major functional genera for GO:0047484, 15 major functional genera for GO:0006970, 4 major functional genera for GO:0009651, 4 major functional genera for GO:0071470, 1 major functional genus for GO:0006972, 2 major genera for GO:0030104, and 1 major functional genus for GO:0071474, these abundance-increased genes were 22, 805, 165, 128, 35, 75, and 12, respectively. Among these, the osmotic stress-related genes of Pseudomonas had significantly enhanced relative abundances to respond to NaHCO 3 Table S12). There were 190 sodium ion-related genes, which showed significantly enhanced relative abundances in the NaHCO 3 treatment in comparison to the control (Additional file 8: Table S13). In 3 major functional genera (Pseudomonas, Bacillus, and Pseudoxanthomonas) for GO:0005343, 1 major functional genus (Pseudomonas) for GO:0005295, and 2 major functional genera (Pseudomonas and Pseudoxanthomonas) for GO:0005283, these abundance-increased genes were 96, 21, and 73, respectively.
In the microbial communities of rhizospheric soil, the genes implicated in other stresses except for the osmotic stress were found (Additional file 8: Table S14). When compared the NaHCO 3 treatment to the control, the relative abundances of other stress-related genes were significantly increased in GO:0043618, GO:0032101, GO:0032103, GO:0043619, GO:0034644, and GO:0009750. We observed 121 stress-related genes, which had significantly increased relative abundances in the NaHCO 3 treatment in comparison to the control (Additional file 8: Table S15). In 1 functional genus (Pseudomonas) for GO:0032101 and GO:0032103 and 2 major functional genera (Pseudomonas and Xanthomonas) for GO:0034644, these abundance-increased genes were 54, 54, and 13, respectively.

Discussion
Salt changes soil microbial community [16], and salt-tolerant microbes are screened by using a metagenomic library of saline soil [45]. In this study, 834 OTUs of microbes were enriched to respond to NaHCO 3 stress by using the shotgun metagenomic sequencing, among which 669 enriched species were identified. Meantime, there were 208 enriched genera by using the shotgun metagenomic sequencing when compared the NaHCO 3 treatment to the control in the current experiment. Although the 16S rRNA amplicon sequencing will show several artifacts [46], enriched genera were also found to respond to NaHCO 3 stress by using this approach. These results indicated that NaHCO 3 stress altered microbial community structure in rhizospheric soil, and 669 NaHCO 3 -tolerant species and 208 NaHCO 3 -tolerant genera were obtained for the first time in this paper. On the other hand, salt tolerance genes are mined from the metagenomic profiling of soil microbes [45]. In this study, 190 sodium ion-related genes, 1242 osmotic stress-related genes, and 121 other stress-related genes from the functional genera including Pseudomonas showed the increased relative abundances in the NaHCO 3 treatment in comparison to the control, suggesting that these sodium ion-, osmotic stress-, and other stress-related genes might play roles in tolerating NaHCO 3 stress. This could be verified by the result that the relative abundance of Pseudomonas was increased when compared the NaHCO 3 treatment to the control in this paper and would be confirmed by the report that Pseudomonas mendocina is tolerant to osmotic stress [47]. Moreover, elevated atmospheric concentrations of CO 2 change network of soil microbial communities [48]. In rhizospheric soil of this study, the interaction network of microbial communities and the OTUs of connectors had difference between the NaHCO 3 treatment and the control, indicating that NaHCO 3 stress altered the soil microbial interaction network and the connectors might contribute to the alteration.
Hou et al. [40] isolated a drought-tolerant strain of Bacillus, and they found that application of the strain into cucumber-planted soil improves plant growth, increases the relative water contents and osmotic potential of leaves, and decreases the MDA and H 2 O 2 contents of seedlings, thus mitigating drought stress in cucumber. In this study, inoculation with NaHCO 3 -tolerant NYJ into NaHCO 3supplemented and cucumber-planted soil had the same effects on plant growth, relative water contents, osmotic potential, MDA contents, and H 2 O 2 levels of leaves and thereby alleviated NaHCO 3 stress in cucumber seedlings. Application of Acinetobacter calcoaceticus CSY-P13 into soil mitigates the stresses of ferulic and p-hydroxybenzoic acids by altering soil bacterial community [7]. When compared the NYJ + NaHCO 3 treatment to the NaHCO 3 treatment in this paper, 1539 enriched OTUs of microbes were found, and the interaction networks were different. Moreover, NYJ negatively interacted with 1 species and positively interacted with 11 species in the NYJ + NaHCO 3 treatment of this study. Thus, application of NYJ affected microbial community structure and interaction network under NaHCO 3 stress. These results were consistent with the improved plant growth and the decreased contents of MDA and H 2 O 2 in the NYJ + NaHCO 3 treatment in comparison to the NaHCO 3 treatment in this paper. So NYJ application might mitigate NaHCO 3 stress by affecting microbial community in rhizospheric soil.
Symbiotic relationship between plant and microbes affects plant growth [49], and soil microbes modulate plant immune system [50]. Therefore, genes implicated in the symbiont and immune response were investigated in this study to understand the NaHCO 3 stress mitigation by NYJ application. In the NYJ + NaHCO 3 treatment in comparison to the NaHCO 3 treatment of this paper, the relative abundances of symbiont-or immune-related genes in GO:0002764, GO:0002757, GO:0002253, GO:0045087, GO:0045088, GO:0045089, GO:0002758, GO:0002218, GO:0044409, GO:0046718, and GO:0051828 were increased. Meantime, there were 2238 symbiont-and immunerelated genes, which were from major functional genera including Massilia and had the significantly increased relative abundances in this study. These results indicated that NYJ application might affect symbiotic relationship between plant and microbes and make some soil microbes to modulate plant immune system under NaHCO 3 stress.
Proteobacteria, Actinobacteria, and Bacteroidetes were the dominant phyla in rhizospheric soil of this paper whether using the shotgun metagenomic sequencing or the 16S rRNA amplicon sequencing.
Proteobacteria and Bacteroidetes are important contributors to N cycling in soil [51] and will induce phosphatase [52]. Actinobacteria plays roles in soil C mineralization [53]. Thus, N-related urease, phosphatase, and C-related sucrase were investigated in rhizospheric soil of this study to explore the NaHCO 3 stress mitigation by NYJ application. CAT in soil, an indicator of aerobic microbial activity [54], was also detected. Application of Pseudomonas putida CSY-P1 into soil increased soil enzyme activity under stress of p-hydroxybenzoic acid [17]. In this paper, inoculating NYJ into NaHCO 3supplemented and cucumber-planted soil elevated the activities of urease, phosphatase, sucrase, and CAT in rhizospheric soil, which were consistent with the improved plant growth and the decreased contents of MDA and H 2 O 2 in seedlings of the NYJ + NaHCO 3 treatment in comparison to the NaHCO 3 treatment. These results suggested that NYJ application might mitigate NaHCO 3 stress by inducing soil enzymes.
Plant growth-promoting traits of rhizospheric microbial communities contribute to root colonization of microbes and act beneficially on plant growth [55]. The strain NYJ in this study belonged to PGPR but did not produce chitinase. Compared the NYJ + NaHCO 3 treatment to the NaHCO 3 treatment, the relative abundances of chitinase-encoding genes were increased, and 16 chitinase-encoding genes of Massilia showed the enhanced relative abundances. These results indicated that NYJ application might induce microbes in rhizospheric soil to produce chitinase, which would play roles in NaHCO 3 stress mitigation by NYJ application. Similarly, the chitinase isolated from Pseudoalteromonas is salt-tolerant [56]. We also found some major functional genera, in which NYJ application induced genes implicated in nitrogen metabolism, IAA production, general plant growth promoting activity, phosphate solubilization, siderophore, polyamine, and extracellular polysaccharide under NaHCO 3 stress. These genera might contribute to plant growth [55] when NYJ being applied to mitigate NaHCO 3 stress in cucumber.

Conclusions
NaHCO 3 stress altered rhizospheric microbial community structure and interaction network, while 669 NaHCO 3 -tolerant species and 208 NaHCO 3 -tolerant genera were obtained from rhizospheric soil. NaHCO 3 -tolerant NYJ had plant growth-promoting capacity, and the strain negatively interacted with 1 species and positively interacted with 11 species under NaHCO 3 stress. As a result, application of NYJ into soil affected microbial community in rhizospheric soil, increased the activities of urease, phosphatase, sucrase, and CAT, changed the symbiotic relationship between plant and microbes, modulated plant immune system, and elevated the relative abundances of genes implicated in plant growth-promoting traits, thus mitigating NaHCO 3 stress in cucumber.

Declarations
Ethics approval and consent to participate Not applicable.

Consent for publication
Not applicable.

Availability of data and materials
The sequence of a 16S rRNA gene fragment in the strain NYJ and the raw data of the 16S rRNA amplicon sequencing and the shotgun metagenomic sequencing for rhizospheric microbes have been 19 submitted to the NCBI database under accession number MN197602, PRJNA615957, and PRJNA616167, respectively.

Competing interests
The authors declare that they have no competing interests.  Relative abundance of microbial phyla in rhizospheric soil based on the shotgun metagenomic sequencing (a) and 16S rRNA amplicon sequencing (b). Control, supplemented with water; NaHCO3, supplemented with NaHCO3; NYJ, supplemented with NYJ; NYJ+NaHCO3, supplemented with NYJ and NaHCO3.
28 Figure 3 Heatmap of the relative abundance of microbial genera in rhizospheric soil by using the shotgun metagenomic sequencing. Control, supplemented with water; NaHCO3, supplemented with NaHCO3; NYJ, supplemented with NYJ; NYJ+NaHCO3, supplemented with NYJ and NaHCO3. Heatmap of the relative abundance of genes, which are implicated in osmotic stress and annotated by mapping the metagenomic sequencing reads against the GO database.
Control, supplemented with water; NaHCO3, supplemented with NaHCO3; NYJ, supplemented with NYJ; NYJ+NaHCO3, supplemented with NYJ and NaHCO3. 32 Figure 7 Heatmap of the relative abundance of genes, which are implicated in the adaptation to sodium ion and annotated by mapping the metagenomic sequencing reads against the GO database. Control, supplemented with water; NaHCO3, supplemented with NaHCO3; NYJ, supplemented with NYJ; NYJ+NaHCO3, supplemented with NYJ and NaHCO3. Heatmap of the relative abundance of genes, which are implicated in plant growthpromoting traits and annotated by mapping the metagenomic sequencing reads against the GO database. Control, supplemented with water; NaHCO3, supplemented with NaHCO3; NYJ, supplemented with NYJ; NYJ+NaHCO3, supplemented with NYJ and NaHCO3.