Bacterial community and phosphorus species changes in pepper rhizosphere soils after Pseudomonas putida Rs-198 inoculation

Rhizosphere 11 (2019) 100164

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Bacterial community and phosphorus species changes in pepper rhizosphere soils after Pseudomonas putida Rs-198 inoculation

T

Yanhui Hea, Zhansheng Wua,b,*, Wenfei Wanga, Xiaochen Liub, Bang-Ce Yea a

School of Chemistry and Chemical Engineering, The Key Lab for Green Processing of Chemical Engineering of Xinjiang Bingtuan, Shihezi University, Shihezi, 832003, PR China b School of Environmental and Chemical Engineering, Xi'an Polytechnic University, Xi'an, 710048, PR China

A R T I C LE I N FO

A B S T R A C T

Keywords: Bacterial diversity Interaction Phosphorus Pseudomonas Salinity

The present study was designed to analyze the effect of Pseudomonas putida Rs-198 (Rs-198) on pepper plant nutrient accumulation and its root environment changes in slightly saline soil field condition at Xinjiang, China. Results showed that the application of Rs-198 increased the pepper biomass in shoot and root dry weight by 97% and 131%, respectively, and increased N, P and K uptake in pepper stem by 18.40%, 25.00% and 25.07%, respectively. Additionally, Rs-198 inoculated treatment significantly decreased Na content in pepper root by 59.71% likely for its salt stress alleviating ability. Differences in bacterial composition and predicted metagenome were observed between the Rs-198 inoculated treatment and the control in both bulk and rhizosphere soils. The gene abundance of Blastococcus, AKYG587, and Pseudomonas were significantly increased in bulk soil after Rs-198 inoculation, where as the abundance of Chloroflexi decreased at phyla level. Further analysis showed that Rs-198 inoculation increased the contents of available P, Ca2-P, Ca8-P, iron-P, aluminum-P and total-P by 34.28%, 32.68%, 18.04%, 9.90%, 14.51% and 9.01%. The bacterial community was changed with the variation of soil P form relative abundances and soil pH that associated with Rs-198 application. Overall, these findings indicate that the beneficial changes in soil environment condition, such as soil nutrition, bacterial community and functional bacterial diversity, could be attributed to rhizobacteria inoculation.

1. Introduction Phosphorus (P) is one of the most necessary elements for plant growth but it is also non-renewable resource (Hafeez et al., 2019; Zhu et al., 2018). Although the phosphorus reserve is in high storage capacity in many areas, crop yields are often limited by the unavailability of P in the soil (Rodríguez-Morgado et al., 2017). The soluble phosphates introduced into the soil can be immobilized easily by aluminum, iron and calcium ions present in the soil at pH value 5.55–5.91, which increases the immobilized P amount that plants are unable to access (Yang et al., 2010). Addition of organic amendments such as manure compost could alleviate this problem and provide a P source for the organic amendments making the soil pH closer to a neutral range, which P is more available (Meena et al., 2018; Wei et al., 2018; Yu et al., 2012). The increase of soil salinity has become a critical problem in arid and semiarid area of the world (Wu et al., 2014). Available P status in saline soils is highly variable and low availability due to its high

calcium phosphate sequestration (Meena et al., 2018). In order to make appropriate P management practices for improving plant productivity in saline soils, it is crucial to understand the transformation mechanism of availability P in rhizosphere soil. Rhizobacteria affect soluble phosphorous forms in slightly saline soil, and studies had reported that those Rhizobacteria (Pseudomonas spp., Agrobacterium spp., and Bacillus spp.) can be used as bio-fertilizers improving plant growth and P uptake (Alori et al., 2017; Khan et al., 2017). These phosphate-solubilizing bacteria (PSB) can transform insoluble P into soluble P forms by excretion of H+ during respiration or secretion of organic acids (Wei et al., 2018). It was reported that some PSB had the capacity to solubilize residual or fixed phosphorus and also affected plant growth through controlling plant pathogens in the soil (Alori et al., 2017). However, applications of these PSB are rarely effective in real agronomic practices due to the lack of knowledge of these microorganisms on ecology and functions under field conditions (Unno et al., 2015). Many factors, like pH, fertilizer, tillage, water, affect microbial community structure and functions in soil ecosystems at different

* Corresponding author. School of Chemistry and Chemical Engineering, The Key Lab for Green Processing of Chemical Engineering of Xinjiang Bingtuan, Shihezi University, Shihezi, 832003, PR China. E-mail address: [email protected] (Z. Wu).

https://doi.org/10.1016/j.rhisph.2019.100164 Received 20 May 2019; Received in revised form 3 July 2019; Accepted 5 July 2019 Available online 13 July 2019 2452-2198/ © 2019 Elsevier B.V. All rights reserved.

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inoculated at the plant base just below the soil level. In order to detect the maximum impacts that the bio-fertilizer had on the root associated bacteria, the period of late flowering stage (already fruit) for pepper plants was chosen as an ideal time to sample the soil (about 30 days after inoculation). Pepper plants were harvested and leaf number, fresh weight (g/plant) and dry weight (g/plant) of shoot and root were recorded. During those growth days, no fertilizer or pesticide were used and irrigated once a week.

scales, so it is difficult to determine the role of one individual factor (López-Carmona et al., 2019; Maul et al., 2014). In addition, the bacterial community composition in rhizosphere could be driven by P availability and nutrient acquisition of plants (Castrillo et al., 2017; Samaddar et al., 2019). Previous studies had showed that the bacterial community shifts associated with high abundance of Rhizobium spp. in potato roots under macronutrient-deficient conditions (Unno et al., 2015). In addition, the soil microbes can form a complex ecological community that affect plant growth and development through their metabolic activities and plant interactions (He et al., 2018; Hmaeid et al., 2019); including decomposition, nutrient cycling transformation, and virtually all soil processes (Hartmann et al., 2015; Hmaeid et al., 2019). The interaction between microbial community and nutrient cycling are still poorly understood. It is crucial to understand the mechanisms of P fractions and rhizosphere soil microbial community changes after biofertilizer application in saline soils. As well how these changes are observed in the soil microbial community, and feedback from microorganisms to plant nutrient uptake and stress tolerance to salinity. We hypothesized that salt stress to pepper will be alleviated and thus available P content can be improved in a semi-arid saline soil (Xinjiang, China) when inoculated with Pseudomonas putida Rs-198 strain. Thus, the specific aims of this study were (i) to study the effects of Rs-198 inoculation on microbial community of pepper soils; (ii) investigate P content in rhizosphere and bulk soils with Rs-198 inoculation; (iii) assess the effect of inoculated Rs-198 on indigenous bacteria and P fractions. The biometric observations recorded were dry matter accumulation of plants, N, P, K and Na uptake and chlorophyll content of pepper plants. This research will provide useful information about regulating the distribution of P fractions and bacterial communities in both rhizosphere and bulk soils when using Rs-198 as a bio-fertilizer.

2.4. Soil sampling and processing Samples of bulk and rhizosphere soil were both collected in triplicates from three replicates of each treatment at 36 days after transplanting. Bulk soil was collected away from the root using a little medicine spoon. In order to collect the rhizosphere fraction, the entire plant was removed from the soil and gently shaken free of loose soil, the roots were pulled and shaken to separate the rhizosphere soil particles attached. The five treatments collected soils were as follows: Pre-plant soil: CK; Bulk soil of non-inoculation treatment: CB; Rhizosphere soil of non-inoculation treatment: CR; Bulk soil of inoculation treatment: IB; Rhizosphere soil of inoculation treatment: IR. Each sample was separated into two parts. One part was air dried, passed through a 2 mm sieve and stored at room temperature for determining chemical properties. The other part was immediately stored at −20 °C for determining soil bacteria properties. Soil pH was measured at a soil to water ratio of 1:1 (w/v) with a pH meter (PHS-2F).

2.5. Analysis of soil P concentrations in soil Rhizosphere and bulk soils were sieved through 0.15 mm mesh for total P analysis and through 2 mm for other P analyses. Total soil P was determined by the H2SO4-HClO4 digestion method by putting 1 g of soil and 15 mL H2SO4-HClO4 into a 100 mL digestion tube and digesting about 3 h until dense white fumes appeared. Soil available P and various P pools were extracted using sequential P fractionation after Hedley (Hedley et al., 1982). All the resulting of PO43− were detected by molybdenum-blue colorimetric method at 700 nm (Wei et al., 2014).

2. Materials and methods 2.1. Experimental site and soil position The experimental site was carried out at the agricultural experimental station of Shihezi University (latitude 44°23' N, longitude 85°41' E), located in Xinjiang, China and the soil is classified as saline, desert grey soil. The soil properties of the cultivation layer were as follows: Field Moisture Capacity, 25%; pH 8.02, Electrical Conductivity, 0.304 ms cm−1; Organic Matter, 15.30 g kg−1; Alkali Hydrolysable Nitrogen, 75.00 mg kg−1; Available Phosphorus, 23.4 mg kg−1; Available Potassium, 251 mg kg−1.

2.6. Soil bacterial community diversity and composition analysis Total genomic DNA was extracted from 0.25 g of soil using the Soil DNA kit (MO Bio Laboratories, USA) according to the manufacturer's instruction. The quantity and quality of extracted genomic DNA were checked photometrically using a NanoDrop® ND-2000c UV-Vis spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). To identify the bacterial taxa, soil DNA samples were subjected to PCR amplification targeting the V3–V4 hypervariable region of 16S rRNA gene with primer: 338F/806R ACTCCTACGGGAGGCAGCA/GGACTACHVGGGTWTCTAAT. Each sample was quantified in a 25 μl reaction, including 5 μl reaction buffer (5 × ), 5 μl GC buffer (5 × ), 2 μl dNTP (2.5 mM) 1 μL Forward primer(10 μM), 1 μL Reverse primer (10 μM), 2 μL DNA Template, 8.75 μL ddH2O, 0.25 μL Q5 DNA Polymerase. The PCR thermal cycling conditions were as follows: an initial denaturation 98 °C for 2 min, 30 Cycles of 98 °C for 15 s, Annealing 55 °C 30 s, Extension 72 °C 30 s, Final extension 72 °C 5 min, 10 °C Hold. The soil bacterial diversity and community composition were examined by 16S rRNA amplicon sequencing. The V3-V4 region of the 16S rRNA gene was amplified using 338F/806R primer set and then sequenced on the Illumina MiSeq platform by the Next Generation Sequencing Service at Personal Bio Co., Ltd. (Shanghai, China). The remaining sequences were clustered using the open reference approach into OTUs at 97 percent similarity using UCLUST (Edgar, 2010). Taxonomy was assigned using the QIIME script with the Green Genes database (v.13_8) using UCLUST.

2.2. Bacterial strain and inoculums preparation The bacteria Rs-198 was isolated from the healthy cotton rhizosphere soil grown in a saline field in Xinjiang, China, and identified as Pseudomonas putida (He et al., 2016; Yao et al., 2010). And it had been reported to have good phosphate solubilization ability in our study (Fig. S1). The strain was incubated in fresh nutrient medium (beef extract 5 g, peptone 10 g, NaCl 5 g, H2O 1000 mL, pH = 7) and placed in a shaker at 30 °C for 24 h. 2.3. Experiment design Pepper seeds (Capsicum annuum L, open pollinated.), purchased from Xinjiang Changji City Xinke Seed Co., Ltd, was surface sterilized by immersion in 70% ethanol for 2 to 3 min for 3 times, and then washed with sterile distilled water several times. After sterilizing, seeds were sown in pre-sterilized tray in vermiculite for germination. 20 days after sowing, uniformed seedlings were transplanted to the field plot. Two treatments (control and Rs-198 inoculation) with 3 replicates were set in this study (each rectangle plot of 1.5 by 2 m). For the inoculation treatment, 1 mL Rs-198 liquid fermentation (1 × 109 cfu mL−1) was 2

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Table 1 Numbers of leaf and flower, fresh weight (FW) and dry weight (DW) of each pepper shoot and root after 30 days inoculation. Treatment

Leaf number

Flower number

Root FW (g)

Shoot FW (g)

Root DW (g)

Shoot DW (g)

Control Rs-198

16.00 ± 3.05 b 42.00 ± 14.78 a

5.40 ± 2.41 b 13.60 ± 4.03 a

1.534 ± 0.287 a 2.828 ± 1.039 a

8.152 ± 2.219 b 16.942 ± 4.318 a

0.220 ± 0.058 a 0.434 ± 0.194 a

1.192 ± 0.326 b 2.760 ± 0.522 a

FW: Fresh Weight; DW: Dry Weight; Values are means ± SD, n = 5, the different letters indicate significant differences among the treatments, P < 0.05.

coordinate analysis (PCoA) and unweighted pair group method with arithmetic mean (UPGMA) clustering were also calculated by this software. Data on assigned sequences at family level shared between samples were used to generate a Venn diagram. Statistical differences between groups of samples were tested using analysis of similarity (ANOSIM-available through QIIME).

2.7. Plant nutrient analysis Plant samples (root, stem, and leaves) were finely ground after drying at 65 °C for 2 days. Dried plant samples (aerial parts or roots) were milled, digested, and analyzed as described by Fan et al. (2016). The dried plant samples (0.2 g) were digested in 5 mL of concentrated H2SO4-H2O2 at 380 °C until solution clear, and then transferred to a volumetric flask fixing to a volume of 50 mL. The samples were then filtered, and total N was quantified as NH4+ using the DigiPREP TKN Systems (2300 Kjeltec Analyzer unit, Foss Tecator AB, Sweden), and total P concentration was quantified as PO43− using the phosphorus vanadium molybdate yellow colorimetric method at 400 nm. The K and Na concentration were estimated by flame photometry (FP6450, Shanghai Instrument Electric Analytical Co., LTD, China) (Yu et al., 2012).

3. Results 3.1. Biomass and nutrient uptake The current study on pepper plant planted in saline soil showed that the inoculation of Rs-198 to the soil has a significant positive effect on growth of pepper (Fig. S2a). Application of Rs-198 greatly enhanced the number of leaves and flowers by 162% and 152% than control, respectively, in just 5 weeks after the initial inoculation of Rs-198. Rs-198 treated plants yielded 84% and 108% more fresh biomass of root and shoot over control, respectively (Table 1). And application of Rs-198 also greatly enhanced the shoot and root dry weight by 97% and 131%, respectively, compared to non-inoculated treatment for its growth promoting property. The N concentration in the stem and leaf were significantly (p < 0.05) increased by 18.40% and 17.91% in Rs-198 inoculated treatment when compared to the control in saline soils (Fig. 1a). Similar, the P content in stem was observed significantly increase by 25.00% in the Rs-198 inoculated treatment, (one-way ANOVA, P < 0.05; Fig. 1b). Moreover, the application of Rs-198 significantly (p < 0.05) increased K uptake in the stem (25.07%) over control (Fig. 1c). However, Na concentrations in the root of pepper decreased (59.71%) considerably after Rs-198 inoculation and thus reduced the transportation of Na+ from root to stem and leaf. This decrease in Na uptake in the root of pepper due to the Rs-198 inducing the alteration of Na uptake in pepper by regulating the expression of Na+/H+ antiporter (Fig. 1d).

2.8. Chlorophyll contents measurement The chlorophyll contents were measured according to the Lichtenthaler and Wellbum's method (Lichtenthaler and Wellburn, 1983). Briefly, leaf tissue were cut and immersed in 96% ethanol over night and read the absorbance at wavelength 666, 649 and 470 nm. The concentrations (μg per ml of plant extract) of chlorophyll a (Chl a), chlorophyll b (Chl b), and carotenoids (Car) were calculated using the following equations: Chl a = 13.95 × OD666 - 6.88 × OD649 Chl b = 24.96 × OD649 - 7.32 × OD666 Car = 1000 × (OD470 - 2.05 Chl a −114.8 Chl b)/

245

2.9. Statistical analysis LEfSe (Linear Discriminant Analysis Effect Size) couples robust tests for measuring statistical significance (Kruskal-Wallis test) with quantitative tests for biological consistency (Wilcoxon-rank sum test). The differentially abundant and biologically relevant features are ranked by effect size after undergoing linear discriminant analysis. The functional analyses used in this study were PICRUSt 1.0.0. The OTUs were selected using the Greengenes release version 12 may 2012. The filtered OTU table was normalized by the 16S rRNA copy number abundance to identify true abundance followed by metagenome functional prediction for each sample. PICRUSt was used to value relative Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway abundance. Finally, the module ‘categorize by function’ was used to generate a table of the predicted metagenome functional content based on the Kyoto encyclopedia of genes and genomes (KEGG) classification at level 2. The data were analyzed statistically by using STAMP v 2.0.0. All physico-chemical data and P contents data were analyzed using Excel 2007 and Origin 8.5. Data were reported as the means of 9 values (3 duplicate samples from treatment, and every sample tested 3 times) and analyzed using one-way ANOVA. Differences among different treatments were analyzed using the least significant difference (LSD) test at the p < 0.05 probability level. Sequence data visualization was performed exclusively in R and statistical analyses were performed using a combination of QIIME scripts and R. Rarefaction curves were generated by using QIIME. And the weighted UniFrac for principal

3.2. Photosynthesis pigments The photosynthetic pigment contents of pepper leaves were significantly ranged between control and inoculation treatments (Fig. 2). The chlorophyll a and carotenoids were significantly (p ≤ 0.05) higher in the inoculation plant as compared to the untreated plant. 3.3. Bacterial community profiles A total of 1211541 high quality 16S rRNA gene sequence reads were obtained from the bulk and rhizosphere soil samples. The sequences from 15 samples possessed a 436-bp average length and will be submitted to the NCBI Sequence Read Archive according to MIMS standard. There were a total of 119,720 distinct OTUs (observations) across samples. The mean counts for CB, CR, IB, IR and CK were 13,647, 15,200.67, 13,881.67, 12,838.67 and 13,564, respectively (Fig. 3). 1,872 OTUs were common among the five groups. The unique OTUs in each group were also observed with 480, 421, 403, 253, and 285 unique OTUs in the CK, CB, CR, IB and IR groups, respectively, in the Venn diagram (Fig. 3). In our experimental conditions, the sampling curve rose rapidly in the beginning and then gradually levelled off suggesting that the 3

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Fig. 1. Changes in the nutrients contents in the pepper plants inoculated Rs-198 (a: Nitrogen content; b: Phosphorus content; b: Sodium content; d: Potassium content).

between the rhizosphere soil and bulk soil using the Simpson and Shannon richness estimators when comparing the set of samples (Fig. 4). While Chao1 and ACE indexes suggested that the compositions of soil bacterial communities were different among inoculation and non-inoculated soil samples. One-way test showed that α diversity of inoculation rhizosphere soil bacterial communities were significantly higher (p < 0.05) than those of non-inoculation rhizosphere soil bacterial community samples for Chao1 and ACE indexes. The diversity

number and quality of the sequences obtained are comparable in each rhizosphere soil sample (Fig. S3). The Shannon, Simpson profiles revealed that application of Rs-198 decreased bacterial diversity and abundance while improved bacterial community structure substantially. The diversity indices indicated the highest bacterial diversity and richness in soil samples of non-inoculated bulk soil (CB) with highest Shannon at 10.04, followed by non-inoculated rhizosphere soil (CR) at 9.89. It is interesting to note that no differences were observed

Fig. 2. Changes in the photosynthesis pigment contents in the pepper plants inoculated with Rs-198. 4

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investigate the possible correlations between communities' members, and the nutritional status of soil (Fig. 5). The soil microbial community structure refers to the abundance and relative proportions of the main bacteria in soil that were valued in this study. The most dominant phyla were classified as 5 phyla including Actinobacteria, Proteobacteria, Chloroflexi, Gemmatimonadetes, and Cyanobacteria (Fig. 5). All of those phyla significantly decreased in inoculated treatments soil compared to that non inoculated treatment. By contrast, Rs-198 application has been found to enhance the abundance of Cyanobacteria. In addition, the abundance of Cyanobacteria was significantly higher in rhizosphere soil than bulk soil. Rs-198 application can decrease the abundance of Latescibacteria at both bulk and rhizosphere soil. At the genus level (Fig. 5), the dominant genera accounting for more than 1% of the overall community were Sphingomonas, Solirubrobacter, Haliangium, Roseiflexus, Blastococcus in bulk and rhizosphere soil. Other genera, such as Iamia, Streptomyces, Nitrospira, Gemmatimonas, Nocardioides, Actinoplanes, g__H16, Bryobacter, Skermanella, AKYG587 and g_RB41 were detected in all soils and were dominant in several samples. The gene abundance of Blastococcus, AKYG587, and Pseudomonas were significantly increased in bulk soil after Rs-198 inoculation. While, the Solirubrobacter, Roseiflexus, Actinoplanes and Skermanella abundance were significantly decreased in bulk soil after Rs-198 inoculation. Across bulk and rhizosphere soil, Gossypium_arboreum were clearly more abundant in IR (11.09%) and CR (3.74%) soil than in IB (2.05%) and CB (0.11%) samples (ANOVA, P < 0.001), while rarely detected in CB soils. These significant differences were further tested by LEfSe and PLSDA analysis (Fig. 6). A taxonomy tree visualized by GraPhlAn was used to quickly find the dominant taxa from the complex bacteria data (Fig. 7). The colored nodes represented the 20 most abundant taxa signified by the letters in the tree, including Actinobacteria, Thermoleophilia, Solirubrobacterales, Cyanobacteria, Chloroplast, Gemmatimonadetes, Chloroflexi, Proteobacteria, Deltaproteobacteria, Alphaproteobacteria.

Fig. 3. Venn diagram showing the shared OTUs among the five samples (CK: non plants soils; CB: non inoculation bulk soils; CR: non inoculation rhizosphere soils; IB: inoculation bulk soils; IR: inoculation rhizosphere soils).

metrics were significantly difference (p < 0.05) between samples as per inoculation and non-inoculation treatments, since the inoculation of Rs-198 can influence the diversity metrics. The rarefaction curves showed the trend that CB samples have lower alpha diversity and CR have higher alpha diversity (IR < CK < CB < IB < CR, Fig. 4). 3.4. Rhizosphere bacterial community diversity The taxonomic distribution of OTUs (97% identity) were classified into phylum, class, order, family, and genus levels in order to

Fig. 4. Alpha diversity base of rhizosphere and bulk soil samples (CK: non plants soils; CB: non inoculation bulk soils; CR: non inoculation rhizosphere soils; IB: inoculation bulk soils; IR: inoculation rhizosphere soils). 5

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Fig. 5. Main bacteria phyla (4 a) and genera (4 b).

Fig. 6. Comparison of microbial variations at the genus level, using the LEfSe online tool (a: Histogram of the LDA scores for differentially abundant features among groups; b: Cladogram for taxonomic representation of significantly differences among groups. Differences are represented in the color of the most abundant taxa (red indicating CB, green indicating CR and blue indicating IR). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.) 6

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Fig. 7. Taxonomy visualization of the bacteria community (A taxonomy tree visualized by GraPhlAn. The background color of the letter is consistent with the color of corresponding node. The colored nodes from the inner to the outer circles represent the top 20 abundant taxa from the phylum to genus level, which are signified by the letters arranged from the outer to the inner circles). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

(Fig. 9). Two-sided Welch's t-test with multiple testing corrections in STAMP was performed to identify KEGG pathways at a significant different level of p < 0.05. At KEGG Level 2 metabolism, a total of 12 metabolic functions were predicted in all treatments with the most enrichment in xenobiotics biodegradation and metabolism, nucleotide metabolism, metabolism of terpenoids and polyketides, metabolism of other amino acids, metabolism of cofactors and vitamins, lipid metabolism, glycan metabolism and biosynthesis, enzyme families, energy metabolism, carbohydrate metabolism, biosynthesis of other secondary metabolites, and amino acid metabolism. As expected from the taxonomies of bacteria from the ISA, glycan biosynthesis and metabolism, enzyme families' energy metabolism were observed to be higher in the inoculated soil bacterial communities as compared to that of non-inoculated soil (Fig. 9). While, the xenobiotic biodegradation and metabolism, lipid metabolism and amino acids metabolism, all related to plant stress in soil ecosystem, was observed to be lower in the inoculation treatment. In addition, the carbohydrate metabolism and amino acid metabolism were observed higher in bulk soil than rhizosphere soil. The metabolism of cofactors and vitamins and energy metabolism were observed in higher relative abundance in rhizosphere

3.5. Beta diversity To determine the difference in community composition between the rhizosphere soil and the bulk soil samples, inoculated soil and non-inoculated soil, beta diversity metrics were calculated for the soil bacterial communities. We are surprised that we did not find very clear segregations in community structures among treatments. UPGMA hierarchical clustering analysis also showed that the treatments did not form well-separated clusters corresponding to the five treatments, suggesting the similarity in the bacterial community structures (Fig. 8, R = 0.1215, p = 0.165). Moreover, differences in Unifrac distances for pair wise comparison among groups were found (Fig. S4). Multivariate data analyses using adonis, ANOSIM, and MRPP on weighted Unifrac distances showed significant differences (p < 0.01) in the beta diversity of the bulk and rhizosphere soils. 3.6. Functional profiling of the bacterial communities For this reason, the 16S rRNA copy number normalized abundance was used to predict metagenome and collapse into KEGG pathways 7

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negatively correlated with Ca2-P (R = - 0.816), Fe-P (R = − 920) and Al-P (R = - 879). This indicted that application of PSB Rs-198 can dissolve insoluble O-P to available P (Ca2-P) or slow P (Fe-P, Al-P). 4. Discussion This study aims to compare different phosphorus chemical form in both bulk and rhizosphere soil, PSB Pseudomonas putida Rs-198 inoculated and non-inoculated soil. Our results showed that Rs-198 inoculation can increase plant biomass and change bulk and rhizosphere soil bacterial community. The fact that the strain has both indirect and direct effect on plant, which had been well established and documented (He et al., 2016; Yao et al., 2010). Salt stress is known to inhibit root systems, thus reducing plant ability to acquire nutrient resources in soil (He et al., 2015). In our study, pepper plants inoculated with Rs-198 strains showed stronger roots and more root hairs compared to noninoculated plants (Fig. S2b). Rs-198 also regulates photosynthetic pigment production leading to the acquisition of chemical energy, so changes in photosynthesis pigment content as a result of Rs-198 inoculation may reflect photosynthetic capacity and photosynthesis nutrient acquisition by pepper grown in saline soils (He et al., 2017). The redistributed of available phosphorus (A-P) and occupied phosphorus (O-P) contents in both bulk and rhizosphere soil was likely because that soil microorganisms play an important role in nutrient cycling and decomposition (Table S1, Fig. 10). It has also observed Ca-P is always as the main source of P in salinized soil, while, Fe-P, Al-P were less effective and O-P, Ca10-P was not effective for crop growth in acidic soil (Janegitz et al., 2017; Meena et al., 2018). The present results is similar to a reported in field experiments based on 31P nuclear magnetic resonance (NMR), A-P concentration and the quantity of PSM also could be increased by applying functional PSB inoculants (Wei et al., 2014). Pepper plants containing low P concentrations might be due to the increasing bacteria immobilization of P, thereby resulting in the decrease of A-P content. The increase of Al-P and Fe-P chemical form of P were due to the fact that Al and Fe bound P are chemically mobilized by carboxylic acids produced by phosphate solubilizing microorganism (PSMs) (Khan et al., 2015). It had been shown that aluminium and iron phosphate were observed after organic fertilizer (biosolids) application (Kar et al., 2012). And these changes would be an important reason for the increase of plant growth in Rs-198 inoculated treatment. It is important to point out that bacterial communities may affect plant production (López-Carmona et al., 2019; Meier et al., 2013). The changes of bacterial community composition resulted by Rs-198 inoculation in the rhizosphere soil probably influence soil P availability to plants (Marschner et al., 2006). Effects of microbial diversity on plant productivity probably also depends on the number of functional groups in the present (Van Der Heijden et al., 2008). Functional diversity is an aspect of the overall bacterial diversity in soil and encompasses a range of activities. There is currently much interest in the relationship between soil bacterial diversity and ecosystem functioning. The plant stress related metabolism in soil ecosystem, was observed to be lower in the inoculation treatment, since the decrease in lipid by inoculation of PGPR is well known to alleviate the salt stress. Previous studies also documented the negative correlation between A-P and calcium-P (Wei et al., 2014). We should pay more attention on that the chemical P applied into the field does not accumulate in plant uptake form but may turn over to other insoluble formulations. In addition, we know that soil pH is the best indicators of bacterial community changes. According to the data collected from the literatures, soil pH has positively correlated with some of genera abundant in soil, such as: Mycobacterium, Flavobacterium, Sphingomonas, Nitrososphaera, Steroidobacter, Nocardioides, Streptomyces, Micromonospora, Pseudomonas, Solirubrobacter and Methylibium, and pH negatively correlated with Solibacter, Clostridium and Actinomadura (Zhalnina et al., 2015). The Rs-198 addition may alter soil bacterial communities via direct P nutrient effects or indirectly by decreasing soil pH or by altering the

Fig. 8. UPGMA hierarchical clustering analysis based on weighted Unifrac distances.

soil. 3.7. P fractions change in soils associated with Rs-198 inoculation Among different phosphorous fractions in rhizosphere soils, contents of available P (A-P), Ca2-P, Ca8-P, iron-P (Fe-P), aluminum-P (AlP) and total-P (T-P) were increased by 34.28%, 32.68%, 18.04%, 9.90%, 14.51% and 9.01% after Rs-198 inoculation, while unavailable P including occluded-P (O-P) decreased 27.97%. As for the bulk soil, contents of available P (A-P), Ca2-P, Ca8-P, iron-P (Fe-P) and aluminumP (Al-P) were significantly increased by 61.81%, 34.31%, 32.97%, 9.52% and 13.13% after Rs-198 inoculation. In contrast, inoculation of Rs-198 significantly decreased bulk soil O-P content by 24.67% (Fig. 10). The A-P loss in non-inoculated bulk soil (CB) and non-inoculated rhizosphere soil (CR) appeared when compared to that in pre-plant soil (CK). There were significant differences between bulk soil and rhizosphere soil in the Fe-P and Al-P content. Fe-P contents of rhizosphere soil were found significantly increased by 20.21% and 20.62% over bulk soil in non-inoculated and inoculated treatments soil. Al-P contents also were significantly increased by 34.96% and 36.61% in rhizosphere soil when compared with bulk soil at non-inoculated and inoculated treatments soil. Rhizosphere soil Ca10-P contents were found significant increased by 13.11% and 24.61% over bulk soil in non-inoculated and inoculated treatments soil, respectively. While the rhizosphere soil O-P content were lower than that bulk soils (CB) at both non-inoculated (18.30%) inoculated treatments (21.88%) soil. The soil pH decreased from 7.86 to 7.67 after Rs-198 inoculation. Additionally, the soil pH negatively correlated with T-P content (R = 0.930, p < 0.05, Table S1). 93.33–95.52% of P species were unavailable P in most soil samples, (Fig. 11). Overall, the P form distribution indicated that Ca10-P was the major component of P species in this soil. Meanwhile, the composition of all the unavailable P species of both bulk and rhizosphere soils in inoculation treatment resulted in a much lower proportion than those in the non-inoculation treatment (Fig. 11). In the present study, we also found that the content of A-P had positive correlation with Ca2-P (R = 0.965) and Ca8-P (R = 0.840) (Table S1). Obviously, the Fe-P and Al-P also has a positive correlation at R = 0.9945 (Table S1). In addition, O-P contents also showed 8

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Fig. 9. Bacterial KEGG_L2_Metabolism function prediction by PICRUSt analysis.

Roseiflexus, Actinoplanes and Skermanella abundance were significantly decreased in bulk soil after Rs-198 inoculation. The chemotaxonomic analyses of different bacteria with Skermanella gene in previous work showed that the gene of Skermanella has the ability to tolerance 4% NaCl and has the potential to promote plant growth under salinity and drought conditions (Hezbri et al., 2016). Microbial activity probably increased with increasing C sources caused by root exudates (Hooper et al., 2000; Mommer et al., 2016). Gemmatimonadetes have been found in variety of arid soils, and a study conducted suggested that the distribution of the Gemmatimonadetes in soil tends to be determined by the water availability than aggregation, and confirm that Gemmatimonadetes prefer dryer soils (Chen et al., 2016). Given that the nutrients (e.g., organic matter, total N, total P) in the rhizosphere were more abundant than that in bulk soils, it was expected that Gemmatimonadetes in higher abundance in bulk soil (CB, 10.72%; IB, 12.07%) than rhizosphere soil (CR, 9.33%; IR, 9.69%) at phyla level (Fig. S7). The high relative abundance of Chloroflexi in bulk soil may be an adaptation of this phylum, which probably occurred due to N limitation and its flexibility for the limiting nutrient under salt soil condition (Das et al., 2017a). The quantity and quality of exudates released alter the soil chemistry and affect the bacterial community for the microorganisms colonizing in the rhizosphere (Mommer et al.,

supply and type of organic matter mentioned above (Castrillo et al., 2017; Hartmann et al., 2015). Microorganisms and plants carry out a chemical communication, and only the bacteria recognizing the signals produced by the plant are able to penetrate and colonize the roots (Venturi and Keel, 2016). Soil bacterial community played an important role in plant nutrients acquisition. Firstly, Cyanobacteria were found to enhance after Rs-198 application, significantly higher in rhizosphere soil than bulk soil. The present data showed that the abundance of Chloroflexi decreased after Rs-198 application at phyla level (Fig. S5). Garcia-Pichel reported that some Cyanobacteria can actively move in response to wetting by migrating to the soil surface (Garcia-Pichel and Pringault, 2001). Past studies had shown that the genera Chloroflexi has wide potential to fix carbon under poor nutrient soil, so the reduction of Chloroflexi may be due to an increase in nutrient availably after application of Rs-198 (Hug et al., 2013). The high relative abundance of phylum Actinobacteria in non-inoculation treatment probably for the formation of endospores, which could tolerance adverse environments (Das et al., 2017b). The gene abundance of Blastococcus AKYG587, and Pseudomonas were lower in inoculation treatment soil compared to non-inoculation treatment. The abundance of Blastococcus had been reported to decrease in drought-stressed plots (Bastida et al., 2017). While, the Solirubrobacter, 9

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Fig. 10. P contents in bulk and rhizosphere soils of pepper with different treatments (a: A-P, Ca2-P, Ca8-P; b: Fe-P and Al-P; c: occluded-P (O-P), Ca10-P, d: pH and TP, values are means of three replicates, and error bars represent standard deviation. Bars followed by different uppercase letters indicate differences (P < 0.05) among samples.

Fig. 11. Distribution of different chemical form P (Data represent averages of three replicates, and error bars are ± one standard deviation. CK: non plants soils; CB: non inoculation bulk soils; CR: non inoculation rhizosphere soils; IB: inoculation bulk soils; IR: inoculation rhizosphere soils). 10

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2016; Saleem et al., 2018). So, the high organic acid deposition may explain the specific rhizosphere community, signifying that secretion of phytochemicals by roots is crucial for these plant–microbe interactions and strongly influences the ratio of bacterial community and their functional in rhizosphere soil ecosystems. The ability of Arabidopsis thaliana produce strigolactones affects rhizosphere community composition of fungi but not bacteria (Carvalhais et al., 2019). In conclusion, the study aims to know the changed among P forms and bacterial diversity in a semi-arid salt stressed soil after inoculating Pseudomonas putida Rs-198 strain. And the results showed that the plant root, soil P content, bacterial diversity are a well regulating whole system. Application of additional microbe did reshape the bacterial community structure by strongly affecting soil pH and nutrition level, especially P chemical form. Lastly, the microorganisms that relate to all kinds of nutrient cycling will be considered to influence the nutrition chemical form and plant growth in return. Further studies would be necessary to evaluate the effects of P chemical form on mediating bacterial communities. These results provide a more detailed analysis of complex interactions between plant nutrient uptake and bacterial communities. The findings of our study imply that plant-microbe interactions may explain the growth promotion effect of Rs-198 on the pepper plant and improve our knowledge of the structure/function relationships and the effects of salt factors on soil bacterial communities. Further work should be done in soil N content or other nutrients cycle that associated microbial community after target PGPR application or reduction.

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Conflicts of interest The authors declare that they have no competing interests. Acknowledgement The work was financially supported by Natural Science Foundation of China [grant number 21566035 > , U1803332 > ], Postgraduate Education Reform and Innovation Program of Autonomous Regions [grant number XJGR12016031], Scientific Research Foundation for Changjiang Scholars of Shihezi University [grant number CJXZ201501], "Double First Class" Science and Technology Project of Shihezi University [grant number SHYL-ZD201805], and China Scholarship Council [grant number No. 201709505007]. We would like to thank Personalbio. Inc (Shanghai, China) for sequencing data analysis. And we would like to thank Mike DiLegge form Center of Rhizosphere Biology, Colorado State University for the review and language edit. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.rhisph.2019.100164. References Alori, E.T., Glick, B.R., Babalola, O.O., 2017. Microbial phosphorus solubilization and its potential for use in sustainable agriculture. Front. Microbiol. 8, 9. https://doi.org/10. 3389/fmicb.2017.00971. Bastida, F., Torres, I.F., Andrés-Abellán, M., Baldrian, P., López-Mondéjar, R., Větrovský, T., Richnow, H.H., Starke, R., Ondoño, S., García, C., López‐Serrano, F.R., Jehmlich, N., 2017. Differential sensitivity of total and active soil microbial communities to drought and forest management. Glob. Chang. Biol. 23, 4185–4203. https://doi.org/ 10.1111/gcb.13790. Carvalhais, L.C., Rincon-Florez, V.A., Brewer, P.B., Beveridge, C.A., Dennis, P.G., Schenk, P.M., 2019. The ability of plants to produce strigolactones affects rhizosphere community composition of fungi but not bacteria. Rhizosphere 9, 18–26. https://doi.org/ 10.1016/j.rhisph.2018.10.002. Castrillo, G., Teixeira, P.J.P.L., Paredes, S.H., Law, T.F., de Lorenzo, L., Feltcher, M.E., Finkel, O.M., Breakfield, N.W., Mieczkowski, P., Jones, C.D., Paz-Ares, J., Dangl, J.L., 2017. Root microbiota drive direct integration of phosphate stress and immunity. Nature 543, 513. https://doi.org/10.1038/nature21417.

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