ORIGINAL RESEARCH

published: 24 September 2021

doi: 10.3389/fmicb.2021.737116

Phosphate-Solubilizing Bacterium
Acinetobacter pittii gp-1 Affects
Rhizosphere Bacterial Community to
Alleviate Soil Phosphorus Limitation
for Growth of Soybean (Glycine max)
Donglan He 1 and Wenjie Wan 2,3,4*
1
College of Life Science, South-Central University for Nationalities, Wuhan, China, 2 Key Laboratory of Aquatic Botany
and Watershed Ecology Wuhan Botanical Garden, Chinese Academy of Sciences, Wuhan, China, 3 Center of the Plant
Ecology, Core Botanical Gardens, Chinese Academy of Sciences, Wuhan, China, 4 State Key Laboratory of Agricultural
Microbiology, Huazhong Agricultural University, Wuhan, China

Phosphorus (P) availability is a major restriction to crop production, and phosphate-

solubilizing bacteria (PSBs) in soils are responsible for P turnover. However, it remains
unknown whether the application of PSB can facilitate both inorganic and organic
P transformation and enhance function of plant rhizosphere bacteria. In this study,
Edited by: we applied Illumina MiSeq sequencing, plate-colony counting, quantitative PCR, and
Wei Zhang, multiple ecological analyses. We found that the inoculation of PSB Acinetobacter pittii
Michigan State University,
United States
gp-1 significantly promoted the growth of soybean represented by better vegetation
Reviewed by:
properties (e.g., plant height and root P) and increased activities of phosphatase
Anukool Vaishnav, (4.20–9.72 µg/g/h) and phytase (0.69–1.53 µmol/g/day) as well as content of indole
GLA University, India acetic acid (5.80–40.35 µg/g/h). Additionally, the application of strain A. pittii gp-
Anandham Rangasamy,
Tamil Nadu Agricultural University, 1 significantly increased abundances of both inorganic and organic P-cycling-related
India genes (i.e., phoD, bpp, gcd, and pstS). More importantly, the application of A. pittii
*Correspondence: gp-1 could increase the function represented by P-cycling-related enzymes (e.g.,
Wenjie Wan
wanwenjie@wbgcas.cn
phosphotransferase) of rhizosphere bacterial community based on functional profiling.
To our knowledge, this is the first report that the application of PSB A. pittii promotes
Specialty section: inorganic and organic P utilization and increases the function of rhizosphere bacterial
This article was submitted to
Terrestrial Microbiology,
community. Therefore, the PSB A. pittii gp-1 could be a good candidate for the
a section of the journal promotion of soybean growth.
Frontiers in Microbiology
Keywords: phosphorus-solubilizing bacteria, P-cycling-related gene, rhizosphere bacterial community, functional
Received: 06 July 2021 profiling, vegetation properties
Accepted: 27 August 2021
Published: 24 September 2021
Citation: INTRODUCTION
He D and Wan W (2021)
Phosphate-Solubilizing Bacterium Enhancing the yield of farmland is the most important agricultural issue (Mehrabi and Ramankutty,
Acinetobacter pittii gp-1 Affects
2019). P is an essential element for growth and development of plants and, thus, is of significance
Rhizosphere Bacterial Community
to Alleviate Soil Phosphorus Limitation
to the production of fiber and food crops (Hansen et al., 2020; Wan et al., 2020a). At present,
for Growth of Soybean (Glycine max). the major and wide input of P to farmland is non-renewable P fertilizer, which is often
Front. Microbiol. 12:737116. applied beyond the demand of crops due to soil P fixation to metal ions (Neal et al., 2017; Ye
doi: 10.3389/fmicb.2021.737116 et al., 2017). The accumulation of P in soil could lead to the waste of resources and potential

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He and Wan Plant Growth-Promoting Bacteria

environmental risks (e.g., soil compaction and water bacterial community to the inoculation of PSB. We hypothesized
eutrophication) (Hu et al., 2018). Rational fertilization and that the inoculation of PSB A. pittii gp-1 would increase P
improving utilization efficiency of P fertilizer are important availability and promote the growth of plant and might elevate
agricultural problems. the P-cycling-related gene abundance. To meet our purpose and
The transformation of plant-unavailable P (e.g., Ca3 (PO4 )2 , address our hypotheses, we conducted potted experiments and
phytate, phospholipid, and nucleic acid) to plant-available P Illumina MiSeq sequencing and evaluated soil properties.
(e.g., H2 PO4 − and HPO4 2− ions) needs the participation of
P-solubilizing microorganisms (Yu et al., 2011; Wan et al.,
2020b). PSBs are responsible for the solubilization of inorganic MATERIALS AND METHODS
P and mineralization of organic P (Oliveira et al., 2009; Liu
et al., 2014). Phospholipids and phytate are significant organic Potted Experiment Design
P pools in soils, which can be hydrolyzed by phosphatase and The previously isolated PSB A. pittii gp-1 (accession number:
phytase, respectively (Lim et al., 2007; Maougal et al., 2014; MK641660) with indole acetic acid production ability was
Wei et al., 2019). The inorganic P can be solubilized by small used in potted experiment. The strain gp-1 was inoculated to
molecular organic acids (e.g., gluconic acid and citric acid), 200 ml of the National Botanical Research Institute’s phosphate
and the formation of small molecular organic acids needs (NBRIP) medium and incubated at 28◦ C with shaking of
the participation of dehydrogenase (Hanif et al., 2015; Rasul 180 rpm for 5 days. NBRIP medium contained 10 g/L of
et al., 2019). Previous studies have reported that P-cycling- glucose, 5 g/L of Ca3 (PO4 )2 , 0.25 g/L of MgSO4 ·7H2 O, 5 g/L of
related genes of phoD, bpp, gcd, and pstS can encode alkaline MgCl2 ·7H2 O, 0.2 g/L of KCl, 0.1 g/L of (NH4 )2 SO4 , and 2 ml/L
phosphatase, phytase, glucose dehydrogenase, and phosphatase of trace element solution (EDTA, 10 g/L; MnSO4 ·H2 O, 2.2 g/L;
inorganic transporter system, respectively (Neal et al., 2017; Wan FeSO4 ·7H2 O, 1.0 g/L; CuSO4 ·5H2 O, 0.5 g/L; CoCl2 ·6H2 O,
et al., 2020a). Therefore, phoD, bpp, gcd, and pstS genes can be 0.3 g/L; Na2 MoO4 ·2H2 O, 0.2 g/L; and CaCl2 , 0.1 g/L) (Nautiyal,
good biomarkers to provide insight into soil P transformation. 1999). After incubation, bacteria were collected by centrifuging
Prior studies have reported that specific bacterial community and washed three times with sterile water.
including alkaline phosphomonoesterase-harboring bacterial The experimental potted soil was collected from an
community and phytase-producing bacterial community can uncultivated field in Wuhan, China (30◦ 280 N, 114◦ 210 E).
promote plant growth (Maougal et al., 2014; Hanif et al., 2015; The soil type is calcareous, with original pH, total carbon,
Ye et al., 2017; Wei et al., 2019). Additionally, many PSBs have total nitrogen, availability phosphorus, and total phosphorus of
been isolated from natural conditions and found to possess plant 6.9, 0.52, 0.68%, 0.22 mg/g, and 0.89 mg/g, respectively. These
growth-promoting capability, such as Acinetobacter (Collavino P-deficient soils were sieved through a 2-mm mesh to remove
et al., 2010; Liu et al., 2014), Pseudomonas (Yu et al., 2011), stones and plant residuals. TCP was applied as phosphorus
Burkholderia (Collavino et al., 2010), and Bacillus (Hanif et al., source in plant growth promotion experiment as described in
2015; Hansen et al., 2020). The application of PSB in agriculture previous literatures (Yu et al., 2011; Liu et al., 2014). Four potted
is a useful approach to enhance soil P availability and avoid treatments were designed: 200 g of sieved soil + 100 ml of sterile
excessive use of P fertilizer. Therefore, it is necessary to reveal water (CK treatment), 195 g of sieved soil + 5 g of TCP + 100 ml
plant growth-promoting mechanism of PSB. P solubilization and of sterile water (Tri treatment), 200 g of sieved soil + 10 ml of
mineralization of single PSB are gradually clarified; however, bacterial suspension (107 cfu/ml) + 90 ml of sterile water (Sup
effects of PSB on transformation of both inorganic and organic treatment), and 195 g of sieved soil + 5 g of TCP + 10 ml of
P and rhizosphere bacterial community are poorly understood. bacterial suspension (107 cfu/ml) + 90 ml of sterile water (Bac
To broaden candidates of P-solubilizing microorganisms, we treatment). Each treatment had five replications. Soybean seeds
isolated a PSB Acinetobacter pittii gp-1 from agricultural soils (Glycine max w82) were purchased from China National Seed
(Wan et al., 2020b). In a prior study, we found the strain Group, pre-cultivated in sterile nutritious soils, and allowed
A. pittii gp-1 showed good performances for utilizing tricalcium them grow to about 10-cm length of sprouts. Each sprout with
phosphate (TCP), aluminum phosphate, iron phosphate, and same growth potential was transplanted to each plastic pot as
phytate (Wan et al., 2020b). Soil-derived Acinetobacter bacteria described above, and the strain gp-1 was inoculated to soybean
present good P-solubilizing abilities and show great potentials rhizosphere in Sup and Bac treatments. Each plot was covered
in agroecosystems (Collavino et al., 2010; Yu et al., 2011; Marra with Nylon membrane. These pots were randomly placed in
et al., 2012; Rasul et al., 2019). However, responses of diversity, greenhouse and incubated at 25◦ C with the cycling treatment of
composition, and function of indigenous bacterial community 16-h light and 8-h dark for a total of 40 days.
to inoculation of PSB Acinetobacter remain unknown. Soybeans
are in great demand by human society, and P deficiency leads to
poor growth and low production of soybean (Bononi et al., 2020).
Determination of Phosphate-Solubilizing
This situation caught our interest to investigate the growth- Bacterium Abundance and Indole Acetic
promoting capacity of Acinetobacter bacteria for soybean. In Acid
the present study, we aimed to (i) investigate effects of PSB Every 10 days, we used alcohol-wiped shovels and tweezers to
inoculation on P transformation and plant growth-promoting collected about 5 g of bulk soils near soybean root from each
performance and (ii) explore responses of soybean rhizosphere pot. In the experiment of plate-colony counting for abundance

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He and Wan Plant Growth-Promoting Bacteria

of PSB, 1 g of freeze-dried soil was added to 10 ml of sterile water (Mori et al., 2013). A PCR of 20 µl was performed in triplicate
and shaken at 180 rpm for 30 min, and the mixture is allowed using a thermal cycler (ABI 9700, Thermo, United States) and
to stand for 10 min. Then 1 ml of soil suspension was diluted, conducted at the following conditions: an initial denaturation at
0.1 ml × 10−6 of diluent was evenly spread on NBRIP solid 95◦ C for 3 min, 30 cycles of 95◦ C for 40 s, 58◦ C for 40 s, and
medium containing 0.2 g/L of cycloheximide acting as fungicide 72◦ C for 50 s, and then a final extension at 72◦ C for 10 min.
and incubated at 28◦ C for 5 days. After incubation, the cfu in Sequencing was conducted on an Illumina MiSeq platform at
different plates were counted. We also estimated content of indole Majorbio Bio-Pharm Technology Co., Ltd., Shanghai, China.
acetic acid by using Van Urk Salkowski reagent, and the standard The raw reads were processed to gain purified sequences
approach has been described previously (Biswas et al., 2018). following the pathway of QIIME (Caporaso et al., 2010). We
eliminated (1) sequences that did not exactly match barcodes
Determination of Soil Physicochemical and primers; (2) sequences with an average quality score < 20;
Properties, Enzyme Activity, and (3) sequences with maximum homopolymers < 10 bp; and (4)
sequences that contained ambiguous bases call. The purified
Vegetation Properties
sequences were clustered into operational taxonomic units
After 40-day growth of soybean, we excluded pots with the
(OTUs) at 97% identity against the SILVA v128 reference set.
best and worst soybean growth in each treatment, and then 12
pots were left. We scraped rhizosphere soils by using a brush.
We measured soil physicochemical properties, including pH, Statistical Analysis
total carbon, total nitrogen, and available P, based on standard Significant differences were calculated by the one-way analysis
methods (Wan et al., 2021a). Microbial biomass P was evaluated of variance with means compared using Tukey’s test in R. Venn
by chloroform fumigation extraction and was calculated as the diagram and non-metric multidimensional scaling (NMDS) plot
difference between fumigated and non-fumigated subsamples were used to reflect bacterial community composition. Pairwise
and simultaneously revised for the incomplete recovery of a P analysis of similarity (ANOSIM) was applied to quantitatively
spike (Roberts et al., 2013; Ragot et al., 2016). evaluate difference in bacterial community composition by using
Soil alkaline phosphatase activity and phytase activity were the “anosim” function in the “vegan” package of R. Permutational
determined according to previous methods (Wan et al., 2020a). multivariate analysis of variance (PERMANOVA) was applied to
Phosphatase activity and phytase activity were expressed as µg evaluate pure effect of factors (e.g., physicochemical parameters
pNPP produced per gram of freeze-dried soil in 1 h and µmol P and enzyme activity) on vegetation properties by using the
produced per gram of freeze-dried soil in 1 day, respectively. “adonis” function in the “vegan” package of R. Linear
The pots in each group was kept to measure the plant height, discriminant analysis (LDA) effect size (LEfSe) statistical analysis
plant fresh weight, plant dry weight, leaf number, leaf fresh was conducted on the online interface Galaxy1 at a significant
weight, root length, and root fresh weight. Soybean shoots and level of p < 0.05 and an LDA score > 4. Functional
roots were separated from plants and dried at 60◦ C. The clean profiling of bacterial taxa was carried out by applying the
and dried root and shoot were separately cut into small pieces and “Tax4Fun2” package in R, and the functional redundancy
digested by concentrated H2 SO4 –H2 O2 . The digested solutions index for each sample was calculated based on 16S rRNA
were applied for measuring the content of root P and shoot P gene similarity (Wemheuer et al., 2020). Canonical analysis of
(Fraser et al., 2017). principal coordinates was applied to investigate influences of
components including soil physicochemical parameters, gene
DNA Extraction, Gene Quantification, abundance, cell exudates (include enzyme and indole acetic
acid), and relative abundances of phylum bacteria on the
Amplicon Sequencing, and Sequence vegetation properties. To identify core taxa, OTUs observed in
Processing more than 50% of all samples (> 6 samples, 875 OTUs) were
Three rhizosphere soils from each group were used to extract applied to build a co-occurrence network. The co-occurrence
total DNA using a DNA extraction kit (Mo Bio, Carlsbad, network was visualized using Gephi v. 0.9.22 at a significant
CA, United States) according to the manufacturer’s instruction. level of p < 0.01 and Spearman’s correlation coefficient higher
DNA concentrations were determined using a NanoDrop 2,000 than 0.67 (Wan et al., 2021b). Structural equation model
Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, was built to show relationships among vegetation properties,
United States). All extracted DNA samples were stored at –80◦ C. physicochemical properties, gene abundance, cell exudate, and
The absolute abundances of phosphorus-cycling-related genes bacterial community composition by using the packages of
in soil bacteria were measured using qPCR with SYBR green “sem” and “plspm” in R. The first principal component (PC1)
mix. Primer sequences for amplifying P-cycling-related genes value of soil physicochemical properties, P-cycling-related gene
(i.e., phoD, bpp, gcd, and pstS) and quantitation PCR condition abundance, bacterial community composition, cell exudate, and
are summarized in Supplementary Method 1. Additionally, we vegetation properties accounting for 96.19, 85.19, 41.56, 98.99,
used these primers to amplify bpp, phoD, gcd, and pstS from and 96.37% of the total variances, respectively, were used as a
A. pittii gp-1. proxy in structural equation model.
The V3–V4 region of bacterial 16S rRNA gene was amplified
using the primers 338F (50 -ACT CCT ACG GGA GGC AGC 1
http://huttenhower.sph.harvard.edu/lefse/
A-30 ) and 806R (50 -GGA CTA CHV GGG TWT CTA AT-30 ) 2
https://gephi.org/

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He and Wan Plant Growth-Promoting Bacteria

RESULTS Tri treatments (Figure 1B). Expectedly, the abundance of

gcd was significantly higher in Bac treatment (3.36 × 107
Shifts in Phosphate-Solubilizing copies/g soil) than that in CK treatment (1.86 × 106 copies/g
soil), Tri treatment (4.68 × 106 copies/g soil), and Sup
Bacterium Abundance and Indole Acetic
treatment (9.62 × 106 copies/g soil). Linear regressions reflected
Acid Content During Soybean Growth significantly positive correlations between phoD gene abundance
The PSB abundance represented by the number of cfu showed and alkaline phosphatase activity (R2 = 0.585, p < 0.01), between
significant difference in four treatments (CK, Tri, Sup, and Bac) bpp gene abundance and phytase activity (R2 = 0.892, p < 0.001),
during 40-day growth of soybean (Figure 1A). The abundance and between gcd gene abundance and indole acetic acid content
of PSB in Bac treatment dramatically increased from 3.57 × 107 (R2 = 0.854, p < 0.001) (Supplementary Figure 3). Additionally,
cfu/g soil at day 10 to 6.96 × 107 cfu/g soil at day 40 (p < 0.05). gcd and pstS could be amplified from strain A. pittii gp-1
The population of PSB fluctuated in CK, Tri, and Sup treatments using primers described above, while bpp and phoD did not.
during 40 days but did not significantly ascend at day 40 than These results might imply that the addition of A. pittii gp-
at day 10 (p > 0.05). The abundance of PSB in Bac treatment 1 could increase the abundances of organic P-cycling-related
was significantly higher than that in other groups; this difference bacterial abundance.
might be partially due to the input of A. pittii gp-1 and TCP.
In addition, we randomly picked 10 colonies from the plate and
found that 16S rRNA gene sequence of three bacterial colonies General Properties of Rhizosphere
presented 100% similarity with that of A. pittii gp-1. Bacterial Community
The indole acetic acid content was significantly higher A total of 2,829 OTUs were found across 12 soil samples.
in Bac treatment than in other treatments in each period The CK, Tri, Sup, and Bac treatments possessed 1,670, 1,556,
(p < 0.05; Figure 1B). Additionally, the indole acetic acid 1,413, and 906 OTUs, respectively; and they shared 181
content noticeably increased in Bac and Sup treatments during OTUs (Figure 2A). A total of 39 phyla were observed, and
40 days (p < 0.05), while in CK and Tri treatments, it did 11 phyla with relative abundance > 0.01% were found across
not (p > 0.05). Linear regression indicated that abundance of these 12 samples (Figure 2B). Proteobacteria, Chloroflexi,
PSB was significantly correlated with content of indole acetic Actinobacteria, and Firmicutes were the first level dominant
acid (Supplementary Figure 1). This suggests that PSB could bacteria, with corresponding relative abundance from 7.94
produce and release indole acetic acid, which in turn might to 48.22%, from 0.92 to 46.58%, from 8.86 to 44.47%, and
promote soybean growth. from 1.13 to 25.25%, respectively. Acidobacteria, Bacteroidetes,
Cyanobacteria, Deinococcus–Thermus, Gemmatimonadetes,
Nitrospirae, and Saccharibacteria were the secondary dominant
Vegetation Properties, Soil bacteria. The NMDS result showed that distinct difference
Physicochemical Properties, and in bacterial community composition among four treatments
P-Cycling-Related Gene Abundance (Figure 2C). ANOSIM confirmed further the significant
After 40 days’ growth, the soybean presented erect leaves that difference (R = 0.6451, p < 0.001). According to LEfSe
became dark green (Supplementary Figure 2). Differences in result, bacteria including Bacillus and Acinetobacter were
vegetation properties were found in four treatments, including dramatically abundant in Bac treatment, while bacteria
the plant height, plant fresh weight, plant dry weight, leaf number, including Acinetobacter, Nitrospira, and Rhodobacter were
leaf fresh weight, root length, root fresh weight, shoot P, and root significantly abundant in Sup treatment (Supplementary
P (Table 1). Plant dry weight, root length, shoot P, and root P Figure 4). According to PERMANOVA results, the application
were significantly higher in Bac group than in other three groups of TCP explained 23.40% of the total variation in community
(p < 0.05). More importantly, the plant length, plant fresh weight, composition (F = 5.27, p < 0.01), and the application of A. pittii
plant dry weight, leaf number, leaf fresh weight, root length, root gp-1 explained 29.11% of the total variation in community
fresh weight, root P, and shoot P were dramatically higher in Sup composition (F = 5.75, p < 0.001).
treatment than in CK treatment (p < 0.05). This suggests that the The bacterial community diversity represented by the
inoculation of PSB A. pittii gp-1 promotes soybean growth. Shannon–Wiener index (3.85–6.16) and community richness
The soil pH (6.1–7.0) was significantly lower in Bac and Sup represented by Chao1 index (687–1303) were significantly
treatments than that in CK and Tri treatments (Table 1). Total lower in Bac treatment than in other treatments (p < 0.05;
carbon (0.43–2.04%), total nitrogen (0.07–0.24%), available P Supplementary Figure 5). This suggests that the addition of
(0.19–0.99 mg/g), microbial biomass P (0.08–0.24 mg/g), alkaline A. pittii gp-1 and TCP decreased rhizosphere bacterial diversity.
phosphatase activity (3.79–10.25 µg/g/h), and phytase activity Based on functional profiling results, 3,113 functions at Kyoto
(0.66–1.63 µmol/g/day) were remarkably higher in Bac treatment Encyclopedia of Genes and Genomes (KEGG) pathway level 3,
than in other treatments (p < 0.05). These results indicate that including carbon-, nitrogen-, phosphorus-, and sulfate-cycling-
the inoculation of PSB A. pittii gp-1 increases P availability and related enzymes or proteins, displayed a higher functional
microbial activity. redundancy in CK + Tri (without A. pittii gp-1 addition),
Basically, the abundances of bpp, phoD, gcd, and psts genes whereas 3,772 functions had higher redundancies in Sup + Bac
were higher in Bac and Sup treatments than in CK and (with A. pittii gp-1 addition) (Figure 3A). It was worth noting

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He and Wan Plant Growth-Promoting Bacteria

FIGURE 1 | The colony-forming unit number of phosphate-solubilizing bacteria (A) and content of indole acetic acid (B) during 40 days. The results are the mean
value of five replicates; error bars represent standard error. Different letters above the column indicate significance (p < 0.05).

TABLE 1 | Vegetation properties, soil physicochemical properties, enzyme activity, and P-cycling-related gene abundance in four potted treatments.

Property CK treatment Tri treatment Sup treatment Bac treatment

Plant height/cm 26.67 ± 6.11 (c) 42.60 ± 5.72 (bc) 62.83 ± 7.42 (ab) 88.50 ± 17.76 (a)
Plant fresh weight/g 3.12 ± 0.29 (b) 8.85 ± 0.27 (b) 26.83 ± 3.07 (a) 35.18 ± 7.14 (a)
Plant dry weight/g 0.38 ± 0.04 (c) 0.73 ± 0.25 (c) 1.53 ± 0.09 (b) 2.02 ± 0.26 (a)
Leaf number 4.67 ± 0.58 (b) 13.00 ± 1.73 (a) 14.00 ± 0.00 (a) 17.33 ± 5.77 (a)
Leaf fresh weight/g 1.09 ± 0.19 (b) 3.77 ± 0.40 (b) 10.08 ± 0.38 (a) 12.45 ± 2.78 (a)
Root length/cm 2.60 ± 0.46 (c) 8.57 ± 0.40 (b) 10.33 ± 1.15 (b) 16.33 ± 2.08 (a)
Root fresh weight/g 0.12 ± 0.02 (b) 0.58 ± 0.14 (b) 8.85 ± 1.43 (a) 12.75 ± 3.88 (a)
Shoot P/(mg/g dw plant) 5.14 ± 0.21 (c) 6.04 ± 0.46 (bc) 7.21 ± 0.29 (b) 9.27 ± 0.80 (a)
Root P/(mg/g dw plant) 1.37 ± 0.22 (c) 2.07 ± 0.24 (c) 3.57 ± 0.38 (b) 4.95 ± 0.36 (a)
Microbial P/(mg/g soil) 0.09 ± 0.01 (c) 0.12 ± 0.01 (c) 0.16 ± 0.01 (b) 0.22 ± 0.02 (a)
Available P/(mg/g soil) 0.22 ± 0.03 (c) 0.34 ± 0.06 (c) 0.63 ± 0.08 (b) 0.89 ± 0.09 (a)
pH 6.91 ± 0.15 (a) 6.76 ± 0.07 (a) 6.43 ± 0.16 (b) 6.25 ± 0.12 (b)
Total carbon (%) 0.51 ± 0.07 (c) 0.53 ± 0.04 (c) 1.40 ± 0.22 (b) 1.93 ± 0.13 (a)
Total nitrogen (%) 0.07 ± 0.01 (c) 0.08 ± 0.01 (c) 0.13 ± 0.01 (b) 0.21 ± 0.03 (a)
Phytase (µmol/g/day) 0.71 ± 0.05 (c) 0.69 ± 0.01 (c) 1.13 ± 0.11 (b) 1.53 ± 0.11 (a)
Phosphatase (µg/g/h) 4.20 ± 0.40 (c) 4.13 ± 0.14 (c) 7.76 ± 0.44 (b) 9.72 ± 0.62 (a)
bpp (log10 copies/g soil) 6.43 ± 0.12 (b) 6.44 ± 0.10 (b) 6.93 ± 0.21 (a) 7.20 ± 0.06 (a)
phoD (log10 copies/g soil) 6.36 ± 0.21 (c) 7.13 ± 0.14 (b) 7.57 ± 0.13 (a) 7.64 ± 0.05 (a)
gcd (log10 copies/g soil) 6.26 ± 0.13 (c) 6.67 ± 0.11 (b) 6.98 ± 0.14 (b) 7.53 ± 0.13 (a)
pstS (log10 copies/g soil) 7.38 ± 0.15 (b) 7.61 ± 0.06 (ab) 7.68 ± 0.47 (ab) 8.09 ± 0.18 (a)

The results are the mean value of three replicates with standard errors. Different letters in the same row denote significance (p < 0.05).

that 206 functions representing P-cycling-related enzymes or vitamins, energy metabolism, and translation) were significantly
proteins were higher in Sup + Bac than in CK + Tri, higher in Sup + Bac than in CK + Tri (p < 0.05), but some
such as phosphoglycerate dehydrogenase (EC: 1.1.1.95) and functions were not (Figure 3B).
phosphoglycerate kinase (EC: 2.7.2.3). Additionally, 35 functions A co-occurrence network was constructed to reveal the
[(e.g., L-iduronidase (EC: 3.2.1.76), dCTP deaminase (EC: relationships among bacterial taxa (Figure 4A). We found 50,510
3.5.4.30), and phloroglucinol synthase (EC: 2.3.1.253)] were positive edges (represent significantly positive correlation) and
unique in CK + Tri, while 198 functions [e.g., phosphotransferase two negative edges (denote dramatically negative correlation),
(EC: 2.7.1.-), neamine phosphoribosyltransferase (EC: 2.4.2.49), suggesting that rhizosphere bacteria presented a less conflicting
5-phosphoribostamycin phosphatase (EC: 3.1.3.88), and uracil interaction. We also clarified the top 20 core nodes; i.e.,
phosphatase (EC: 3.1.3.104)] were exclusive in Sup + Bac. At those with the highest betweenness centrality were affiliated
KEGG level 2, some functions (e.g., metabolism of cofactors and with Acidobacteria (e.g., OTU522), Actinobacteria (e.g.,

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He and Wan Plant Growth-Promoting Bacteria

FIGURE 2 | Composition of rhizosphere bacterial community. (A) Venn diagram shows the shared core microbiomes among four groups. (B) Stacking diagram
reflects relative abundances of top 11 bacterial phyla (relative abundance > 1%) in 12 soil samples. (C) Non-metric multidimensional scaling plot exhibits difference
in bacterial community composition among four treatments. Asterisks denote significance (∗∗∗ p < 0.001).

OTU947), Chloroflexi (OTU67), Firmicutes (e.g., OTU601), significantly pure effects on vegetation properties based on
Gemmatimonadetes (OTU1967), and Proteobacteria (e.g., PERMANOVA (Figure 5).
OTU1813) (Figure 4B). Additionally, we also found that bacterial functions based on
functional profiling were responsible for vegetation properties
(Figure 3C). The function of metabolism of cofactors and
Effects of Abiotic and Biotic Factors on vitamins (R2 = 79.75%, F = 39.38; p < 0.01) showed greater
Vegetation Properties effect on vegetation properties than other functions according
According to PERMANOVA results, the application of TCP to PERMANOVA results. The core taxa identified from co-
could explain 13.69% of the total variation (F = 20.23, p < 0.01) occurrence network also have significant effects on vegetation
in vegetation properties, while the application of A. pittii gp- properties based on PERMANOVA (Figure 4B). The OTU1813
1 could explain 72.41% of the total variation (F = 107.05, regarded as Acinetobacter genus presented higher influence
p < 0.001). According to results of canonical analysis of principal (R2 = 52.08%, F = 10.87; p < 0.01) than other core taxa.
coordinates, soil physicochemical properties (Figure 5A), gene Ultimately, we used structural equation model to reveal
abundance (Figure 5B), cell exudates (Figure 5C), and relative interconnections among soil physicochemical properties,
abundances of bacterial phyla (Figure 5D) explained more P-cycling-related gene abundance, bacterial community
than 80% of the total variation in vegetation properties. composition, enzyme activity, and vegetation properties
Physicochemical parameter, gene abundance, enzyme activity (Figure 6). The model presented a good fit to our data, as
and IAA, and relative abundance of bacterial phylum showed indicated by the non-significant χ2 -test (N = 12, χ2 = 0.707,

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He and Wan Plant Growth-Promoting Bacteria

FIGURE 3 | Community functional differences and functional contributions to vegetation property. (A) Functional redundancy indices (FRIs) of bacterial community in
soils with inoculation of phosphate-solubilizing bacteria (PSBs) (Sup + Bac) and soils without addition of PSBs (CK + Tri) soils. A log ratio > 0 denotes that a function
is more redundant in soils without PSB addition. (B) Differences in bacterial functions between group with addition of PSBs (Sup + Bac) and group without addition
of phosphorus-solubilizing bacteria (CK + Tri) at Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway level 1 and level 2. (C) Effects of functions at KEGG
pathway level 2 on vegetation property determined by permutational multivariate analysis of variance (PERMANOVA). The abbreviations of F1–F21 represent
functions in panel B (from bottom to up, namely, from amino acid metabolism to replication and repair). Asterisks denote significance (∗ p < 0.05; ∗∗ p < 0.01;
∗∗∗ p < 0.001).

d.f. = 1, p = 0.400). On the one hand, bacterial community could Hanif et al., 2015; Wan et al., 2020b) and promise great
affect soil physicochemical properties and P-cycling-related application potentials in agriculture because PSB are responsible
gene abundance, which in turn affect vegetation properties; on for P availability and facilitate P uptake by crops (Richardson
the other hand, soil physicochemical properties and P-cycling- et al., 2011; Bononi et al., 2020; Pastore et al., 2020). However, the
related gene abundance could influence enzyme activity, which activity and abundance of PSB are subjected to the fertilization
in turn influences vegetation properties. These results indicated treatment and phosphorus fractions (Luo et al., 2017; Hu et al.,
that soil, plant, and bacteria presented close relationships. 2018; Wei et al., 2019; Wan et al., 2020a). Therefore, the isolation
and application of highly efficient PSB are meaningful in terms
of promoting soil P availability in agroecosystems.
DISCUSSION
Promoting efficient utilization of P is important in agriculture
Elucidating Soybean Growth-Promoting
due to rapidly increasing cost of fertilizers and big concerns by Phosphate-Solubilizing Bacteria
of environmental protection (Hu et al., 2018). The bacteria Acinetobacter Pittii gp-1
possessing P utilization capacity are widespread in the Applying PSB can increase soil available P content (Maougal
rhizosphere soils of different crops (Maougal et al., 2014; et al., 2014) and promote vegetation growth (Yu et al., 2011;

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He and Wan Plant Growth-Promoting Bacteria

FIGURE 4 | Co-occurrence network of rhizosphere bacteria (A) and contributions of core taxa to vegetation property based on permutational multivariate analysis of
variance (PERMANOVA) (B). Asterisks denote significance (∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001).

Biswas et al., 2018; Hansen et al., 2020). However, it should gcd, and pstS were higher in Sup and Bac treatments. These
be considered whether the PSB could maintain their activity, results and findings suggest that the inoculation of PSB A. pittii
function, and abundance after inoculation. In this study, the gp-1 might significantly increase both inorganic and organic
inoculation of PSB A. pittii gp-1 significantly promoted the P-cycling-related gene abundance of soil indigenous bacteria.
growth of soybean represented by better vegetation properties, This phenomenon might be due to the solubilization of inorganic
which is in accordance with prior findings describing that PSB P by added PSB A. pittii gp-1 via releasing small molecular
can enhance the growth of legume plant (Collavino et al., 2010; organic acid. Consequently, part of soluble P was assimilated by
Bononi et al., 2020; Cumpa-Velásquez et al., 2021) and other native bpp-harboring bacteria and phoD-harboring bacteria and
kinds of plants (Yu et al., 2011; Liu et al., 2014). In these studies, in turn enriched the abundances of bpp and phoD genes and
the increase in the content of available P or small molecular released more phosphatase and phytase. In addition, a part of
organic acid is closely correlated with the growth of plants. The inoculated A. pittii gp-1 might die; thus, the cell residues could
PSB Acinetobacter genus is reported to have the ability to release be treated as nutrient for indigenous microorganisms. Previous
small molecular organic acid (e.g., indole acetic acid, gluconic literatures have reported that gcd-harboring bacteria can produce
acid, oxalic acid, and citric acid) (Marra et al., 2012; Marwa et al., and release small organic acid to solubilize insoluble inorganic
2019; Rasul et al., 2019). Interestingly, we found the A. pittii gp-1 P, thus promoting the growth of plant (Wagh et al., 2014; Rasul
could produce indole acetic acid detected by using the Van Urk et al., 2019). The bpp-harboring bacteria and phoD-harboring
Salkowski reagent. Therefore, the inoculation of the A. pittii gp- bacteria are reported to be responsible for the turnover of soil
1 might increase the content of soil organic acid, which in turn organic P by releasing extracellular enzyme, which in return
increased the content of available P. Additionally, we detected promotes the growth of vegetation (Maougal et al., 2014; Ragot
Acinetobacter genus in Bac treatment by using simple 16S rRNA et al., 2016; Hu et al., 2018; Zhang et al., 2021). Therefore,
gene sequencing for single colony. Illumina MiSeq sequencing the application of PSB A. pittii gp-1 could enhance utilization
result also reflected that Acinetobacter dominated in Sup and Bac potentials of both inorganic and organic P.
groups. These results suggest that A. pittii gp-1 could survive after
inoculation and could promote the growth of soybean. Response of Rhizosphere Bacterial
In addition, we used four pairs of primers as described above Community to Inoculation of Strain gp-1
to amplify bpp, phoD, gcd, and pstS genes from A. pittii gp- Considering community diversity is closely correlated with
1. Unexpectedly, only gcd and pstS genes could be amplified. soil ecosystem functions (Wan et al., 2021c), it is important
Previous studies have reported that Acinetobacter genus harbors to decipher effects of the application of PSB on plant
gcd and pstS gene (Marra et al., 2012; Farrugia et al., 2015; rhizosphere bacterial community. We found significant decrease
Wan et al., 2020b), and almost no study has reported that in rhizosphere bacterial diversity and distinct change in bacterial
Acinetobacter genus possesses bpp and phoD genes. However, community composition, which is similar to findings in
the abundances of P-cycling-related genes including bpp, phoD, published literatures (Estrada-Bonilla et al., 2017; Wei et al., 2017;

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He and Wan Plant Growth-Promoting Bacteria

FIGURE 5 | Canonical analysis of principal coordinates showing effects of soil physicochemical properties (A), abundances of phosphorus-cycling-related genes (B),
cell exudates (C), and bacterial abundances at phylum level (D) on vegetation properties. The significance of factors was determined using permutational multivariate
analysis of variance (PERMANOVA) and is reflected by asterisks next to the variable names. Asterisks denote significance (∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001).

Widdig et al., 2019). In addition, earlier studies have reported that stronger interconnections among vegetation properties, soil
vegetation also affects the composition of bacterial community physicochemical properties, P-cycling-related gene abundance,
(Xue et al., 2017; Yang et al., 2018; Campos-Herrera et al., 2019). cell exudates, and bacterial community composition. This result
To the best of our knowledge, this is the first report that the is similar to our prior finding (Wan et al., 2021a). The co-
addition of PSB A. pittii could promote the community function occurrence network also showed that core taxa belonging
of rhizosphere bacteria especially phosphorus-cycling-related to Acidobacteria, Chloroflexi, Gemmatimonadetes, and
functions. This phenomenon might be due to elevated nutrient Proteobacteria presented significant effects on vegetation
caused by inoculation of PSB A. pittii, which in turn affected properties. Previous literature has reported that some specific
rhizosphere bacterial community composition and function. An phylum bacteria, such as Acidobacteria, Actinobacteria,
earlier study has reported that dead bacteria can be treated and Proteobacteria, are responsible for vegetation growth
as available nutrient to affect growth of other microorganisms under different P conditions (Bergkemper et al., 2016).
(Hanajima et al., 2019). Additionally, microbial biomass P Vegetation properties and microbes could also affect each
contributes to P solubility in riparian vegetated buffer strip soils other (Neal et al., 2017; Yang et al., 2018; Muñoz et al.,
(Roberts et al., 2013). 2021), suggesting that soil, plant, and bacteria have close
Based on these results and findings, we raised one question relationships. In the future, we will explore molecular
of whether there were close relationships among plant, soil, mechanisms to reveal close interconnections among soil,
and rhizosphere. The structural equation model reflected plant, and bacteria.

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He and Wan Plant Growth-Promoting Bacteria

FIGURE 6 | Structural equation model showing the hypothesized causal relationships among vegetation properties, soil physicochemical properties,
phosphorus-cycling-related gene abundance, cell exudates (include enzyme and indole acetic acid), and bacterial community composition. The width of the arrows
presents the strength of the standardized path coefficient. The blue lines indicate negative path coefficients, and yellow lines reflect positive path coefficients. Values
above the lines indicate path coefficients between two parameters. Asterisks denote significance (p < 0.05).

CONCLUSION AUTHOR CONTRIBUTIONS

The application of TCP and A. pittii gp-1 could significantly WW and DH designed the whole experiments. WW
increase soil available P, enrich both inorganic and organic conducted all the experiments, analyzed the data, and
P-cycling-related gene abundance, and promote the growth of wrote the manuscript. DH revised the manuscript. Both
soybean. Addition of TCP and A. pittii gp-1 significantly alters authors contributed to the article and approved the
the local bacterial community composition after 40-day soybean submitted version.
growth. To our knowledge, we firstly report that the addition
of Acinetobacter could promote both inorganic and organic
P utilization and could increase the function of rhizosphere FUNDING
bacterial community. Phosphate-solubilizing bacterium A. pittii
gp-1 could be a good candidate for the growth promotion of This work was supported by grants from the
soybean in agroecosystems, and experiments will be conducted National Natural Science Foundation of China
to estimate its growth-promoting performance for more different (Grant No. 31772399) and the Fundamental
plants in future studies. Research Funds for the Central Universities (Grant
No. 2662015PY116).

DATA AVAILABILITY STATEMENT

SUPPLEMENTARY MATERIAL
The datasets presented in this study can be found in online
repositories. The names of the repository/repositories and The Supplementary Material for this article can be found online
accession number(s) can be found below: https://www.ncbi.nlm. at: https://www.frontiersin.org/articles/10.3389/fmicb.2021.
nih.gov/, SRR8742689–SRR8742700. 737116/full#supplementary-material

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He and Wan Plant Growth-Promoting Bacteria

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T., et al. (2020). Tax4Fun2: prediction of habitat-specific functional profiles Publisher’s Note: All claims expressed in this article are solely those of the authors
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abundance of phosphorus-solubilizing bacteria and phosphorus activity
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