How can fertilization regimes and durations shape earthworm gut microbiota in a long-term field experiment?

The positive roles of earthworms on soil functionality has been extensively documented. The capacity of the earthworm gut microbiota on decomposition and nutrient cycling under long-term fertilization in field conditions has rarely been studied. Here, we report the structural, taxonomic, and functional responses of Eisenia foetida and Pheretima guillelmi gut microbiota to different fertilization regimes and durations using 16S rRNA gene-based Illumina sequencing and high-throughput quantitative PCR techniques. Our results revealed that the core gut microbiota, especially the fermentative bacteria were mainly sourced from the soil, but strongly stimulated with species-specificity, potential benefits for the host and soil health. The functional compositions of gut microbiota were altered by fertilization with fertilization duration being more influential than fertilization regimes. Moreover, the combination of organic and inorganic fertilization with the longer duration resulted in a higher richness and connectivity in the gut microbiota, and also their functional potential related to carbon (C), nitrogen, and phosphorus cycling, particularly the labile C decomposition, denitrification, and phosphate mobilization. We also found that long-term inorganic fertilization increased the abundance of pathogenic bacteria in the P. guillelmi gut. This study demonstrates that understanding earthworm gut microbiota can provide insights into how agricultural practices can potentially alter soil ecosystem functions through the interactions between soil and earthworm gut microbiotas.

Arsenic transformation mediated by gut microbiota affects the fecundity of Caenorhabditis elegans.

Arsenic biotransformation has been discovered in guts of soil invertebrates. Reproduction of invertebrates is sensitive to arsenic contamination in soils. However, little is known about the impact of gut microbe-mediated arsenic biotransformation on the fecundity of invertebrates. Here, Caenorhabditis elegans was firstly pre-fed with Escherichia coli BL21 possessing the capability of reducing arsenate [As(V)] or BL21M having the ability to reduce As(V) and methylate arsenite [As(III)], then inoculated worms were transferred to inactive E. coli AW3110 (harboring no arsenic transformation gene)-seeded plates treated with As(V) at different concentrations. Quantification of gut microbes showed that both E. coli BL21 and BL21M stably colonized in the guts after worms were cultured on inactive E. coli AW3110-seeded plates for 72 h. The analysis of arsenic species indicated that there was As(III) in C. elegans guts colonized with E. coli BL21, As(III) and dimethylarsinic acid [DMAs(V)] in C. elegans guts with E. coli BL21M exposed to As(V) for 6 h. After treatment of 100 µM As(V), decrease in brood sizes was observed for worms that were colonized with E. coli BL21 or BL21M compared to that with AW3110 in the guts. The levels of vitellogenin (VTG), glutathione S-transferases (GST) and superoxide dismutase (SOD), closely linked to reproduction and antioxidation-linked indicators, were the highest in worms whose guts colonized with E. coli BL21, followed by worms colonized with E. coli BL21M and worms colonized with inactive E. coli AW3110 exposed to As(V). Our results indicated the toxic impact of As(III) and DMAs(V) produced by gut microbes on reproduction of C. elegans. The work provides novel insight into the interplay between arsenic biotransformation mediated by gut microbes and the host fecundity in soils.

Partial replacement of inorganic phosphorus (P) by organic manure reshapes phosphate mobilizing bacterial community and promotes P bioavailability in a paddy soil.

The optimization of more sustainable fertilization practice to relieve phosphorus (P) resource scarcity and increase P fertilizer utilization, a better understanding of the regulatory roles of microbes in P mobilization is urgently required to reduce P input. The genes phoD and pqqC are responsible for regulating organic and inorganic P mobilization, respectively. Using high-throughput sequencing, the corresponding bacterial communities harbored by these genes were determined. We conducted a 4-year rice-rice-crop rotation to investigate the responses of phoD- and pqqC-harboring bacterial communities to the partial replacement of inorganic P fertilizer by organic manure with reduced P input. The results showed that a combination of organic and inorganic fertilization maintained high rice yield, and also produced a more complex and stable phosphate mobilizing bacterial community, which contributed to phosphatase activities more than their gene abundances in the model analysis. Compared with the conventional mineral fertilization, organic-inorganic fertilization with the reduced P input slightly increased pqqC gene abundance while significantly enhanced the abundance of phoD-harboring bacteria, especially the genera Bradyrhizobium and Methylobacterium known as potential organic P mineralizers which can maintain high rice production. Moreover, the increased pH was the most impactful factor for the phoD- and pqqC-harboring bacterial communities, by promoting microbial P turnover and greatly increasing bioavailable P pools (H2O-Pi and NaHCO3-Pi, NaOH-Pi) in this P-deficient paddy soil. Hence, our study demonstrated that the partial replacement of mineral P with organic manure could reshape the inorganic phosphate solubilizing and alkaline-phosphomonoesterase encoding bacterial communities towards more resilient and effective to the high P utilization and productivity over intense cultivation, providing insights into the potential of soil microbes in the efficient management of agricultural P fertilization.

Data on Quantitative Microbial Elemental Cycling (QMEC) primer design and validation.

This work included the primer design details of Quantitative Microbial Elemental Cycling (QMEC) method, a high-throughput qPCR method for microbial carbon (C), nitrogen (N), phosphorus (P), sulfur (S) and methane metabolism potential detection and assessment. We designed 36 novel primers based on their amino acid sequences. Via illumina sequencing technology, their phylogenetic taxonomy was identified and analyzed to validate the primer specificity.

Metabolic Inactivity and Re-awakening of a Nitrate Reduction Dependent Iron(II)-Oxidizing Bacterium Bacillus ferrooxidans.

Microorganisms capable of anaerobic nitrate-dependent Fe(II) (ferrous iron) oxidation (ANDFO) contribute significantly to iron and nitrogen cycling in various environments. However, lab efforts in continuous cultivation of ANDFO strains suffer from loss of activity when ferrous iron is used as sole electron donor. Here, we used a novel strain of nitrate-dependent Fe(II)-oxidizing bacterium Bacillus ferroxidians as a model and focused on the physiological activity of cells during ANDFO. It was shown that B. ferrooxidans entered a metabolically inactive state during ANDFO. B. ferrooxidans exhibited nitrate reduction coupled with Fe(II) oxidation, and the activity gradually declined and was hardly detected after 48-h incubation. Propidium monoazide (PMA) assisted 16S rRNA gene real-time PCR suggested that a large number of B. ferrooxidans cells were alive during incubation. However, 2H(D)-isotope based Raman analysis indicated that the cells were metabolically inactive after 120-h of ANDFO. These inactive cells re-awakened in R2A medium and were capable of growth and reproduction, which was consistent with results in Raman analysis. Scanning electron microscopy (SEM) observation and x-ray diffraction (XRD) revealed the formation of Fe minerals in close proximity of cells in the Fe(II)-oxidizing medium after Fe(II) oxidation. Overall, our results demonstrated that continued ANDFO can induce a metabolically inactive state in B. ferrooxidans, which was responsible for the loss of activity during ANDFO. This study provides an insight into the ANDFO process and its contribution to iron and nitrogen cycling in the environments.

The Draft Genome Sequence of Pseudomonas frederiksbergensis Strain 11-D3 Reveals Its Ability To Mobilize Phosphorus.

Pseudomonas frederiksbergensis strain 11-D3 is a Gram-negative, nonmotile, aerobic bacterium isolated from a managed maize field in North China. This strain displays high efficiency for solubilization of inorganic phosphorus (P). We present the draft genome sequence of strain 11-D3 with P cycling genes which indicate the presence of a probable mechanism for P mobilization.

Mobile Incubator for Iron(III) Reduction in the Gut of the Soil-Feeding Earthworm Pheretima guillelmi and Interaction with Denitrification.

Diets of soil-feeding earthworms contain abundant nitrate and iron(III) oxides, which are potential electron acceptors for mineralization of organic compounds. The earthworm gut provides an ideal habitat for ingested iron(III)-reducing microorganisms. However, little is known about iron(III) reduction and its interaction with other processes in the guts of earthworms. Here, we determined the dynamics of iron(III) and revealed its interaction with the turnover of organic acids and nitrate in the gut of the earthworm Pheretima guillelmi. Samples from gut contents combined with anoxic incubation were used for chemical analysis and 16S rRNA based Illumina sequencing. Chemical analysis showed that higher ratios of iron(II)/iron(III), nitrite/nitrate, and more abundant organic acids were contained in the in vivo gut of the earthworm P. guillelmi than those in the in situ soil. A higher rate of iron(III) reduction was detected in treatments of microcosmic incubation with gut contents (IG gut) than that with soil (IG soil), and nitrate reduction occurred earlier than iron(III) reduction in both treatments. Potential iron(III) reducers were dominated by fermentative genera Clostridium, Bacillus, and Desulfotomaculum in the treatment of IG gut, while they were dominated by dissimilatory iron(III)-reducing genera Geobacter in the treatment of IG soil. The iron(III)-reducing microbial community shared several genera with denitrifers in the treatment of IG gut, revealing a close link between iron(III) reduction and denitrification in the gut of earthworms. Collectively, our findings demonstrated that iron(III) reduction occurred along the gut and provided novel insights into the great contribution of earthworm gut microbiota on Fe and the associated C and N cycling in soil environments.

Responses to soil pH gradients of inorganic phosphate solubilizing bacteria community.

Soil pH is commonly considered a dominant factor affecting the function of microbiota. Few studies, however, have focused on communities of bacteria able to solubilize inorganic phosphate (iPSB), which are important for the mobilization of soil phosphorus (P), because finding an effective method to assess the abundance and diversity of iPSB communities is difficult. We used a newly reported method of database alignment and quantified the gene pqqC to analyze the compositions of iPSB communities from five soils with pH gradients ranging from 4 to 8. The iPSB community structure differed significantly between these soil types. Among iPSB community, Bacillus was the dominant genus, followed by Arthrobacter and Streptomyces. A redundancy analysis indicated that soil pH was the most important of 15 soil factors and their pairwise interactions, accounting for 5.12% of the variance. The abundance of the iPSB communities increased with pH within the gradients which was confirmed by experimental adjustment of pH, suggesting that the defect P status in high pH soil was speculated as the driving force of iPSB community population. Our study demonstrated the dominant role of soil pH on the iPSB community, which may contribute to the understanding the possible mechanism of microbial P mobilization for better improvement of P use-efficiency.

D2O-Isotope-Labeling Approach to Probing Phosphate-Solubilizing Bacteria in Complex Soil Communities by Single-Cell Raman Spectroscopy.

Increasing the bioavailability of immobilized phosphorus (P) in soil by phosphate-solubilizing bacteria (PSB) is an effective strategy for sustainable agronomic use of P and for mitigating the P crisis. Here, D2O isotope labeling combined with single-cell Raman spectroscopy (Raman-D2O) was developed as an efficient activity-based approach to characterizing the presence and activity of PSB in a culture-independent way. On the basis of the finding that PSB were significantly more active than non-PSB in the presence of insoluble P, a C-D Raman band from active assimilation of D2O-derived D was established as a biomarker for both inorganic-phosphate-solubilizing bacteria and organic-phosphate-solubilizing bacteria. C-D ratios (intensities of C-D bands as percentages of the intensities of both the C-D and C-H bands) were further established as semiquantitative indicators of P-releasing activities because of the consistency between the C-D ratio and the concentration of solubilized phosphate or acid phosphatase activity as measured by conventional bulk assays. By applying Raman imaging, single-cell Raman-D2O clearly discerned PSB in a mixed-soil bacterial culture and even in complex soil communities. Remarkable heterogeneity of microbial activity, ranging from 2 to 30% (close to that in medium without P and that in medium with sufficient soluble P, respectively), was revealed at the single-cell level and clearly illustrated the subpopulation of soil bacteria active in solubilizing P. This work not only enables probing PSB and their P-releasing activities but also opens a window to explore more diverse microbial resources when obtaining related isotope-labeled substrates is prohibitive.

Straw biochar increases the abundance of inorganic phosphate solubilizing bacterial community for better rape (Brassica napus) growth and phosphate uptake.

The direct application of inorganic-phosphate-solubilizing bacteria (iPSBs) for improving the efficiency of phosphorus (P) use leads to a low rate of bacterial survival. Biochar is a good inoculum carrier for microbial survival, and diverse feedstocks can have different effects. We generated an iPSB community using seven selected iPSB strains with various phylogenic taxonomies and P-solubilizing abilities. Biochar was then inoculated with the iPSB community and applied to soil in pots seeded with rape (Brassica napus). Growth of the rape for four weeks and the effects of biochars produced from six raw feedstocks, rice straw, rice husks, soybean straw, peanut shells, corn cobs and wood, were compared. The synthetic iPSB community had a larger capacity to solubilize inorganic P and exude organic anions than any of the individual strains. The structure of the iPSB community was analyzed by high-throughput sequencing four weeks after inoculation. All seven iPSB strains were detected, dominated by Arthrobacter defluvii 06-OD12. The abundance of the iPSB community was significantly correlated with rape biomass, P content and P uptake (P < 0.05). The biochar amendments conferred 6.86-24.24% survival of the iPSB community, with the straw biochars conferring the highest survival. The available-P content of the biochar rather than soil pH was the dominant factor for iPSB community structure, suggesting that the biochar material was critical for the survival and functioning of the iPSB community. Our study demonstrates the feasibility of biochar-assisted iPSB improvement of crop growth and P uptake.

Long-term organic fertilization increased antibiotic resistome in phyllosphere of maize.

Phyllosphere contains various microorganisms that may harbor diverse antibiotic resistance genes (ARGs). However, we know little about the composition of antibiotic resistome and the factors influencing the diversity and abundance of ARGs in the phyllosphere. In this study, 16S rRNA gene amplicon sequencing and high-throughput quantitative PCR approaches were employed to investigate the effects of long-term (over 10 years) organic fertilization on the phyllosphere bacterial communities and antibiotic resistome. Proteobacteria, Bacteroidetes, Actinobacteria and Firmicutes dominated in the phyllosphere bacterial communities. Long-term application of sewage sludge and chicken manure altered the phyllosphere bacterial community composition, with a remarkable decrease in bacterial alpha-diversity. A total of 124 unique ARGs were detected in the phyllosphere. The application of sewage sludge and chicken manure significantly increased the abundance of ARGs, with a maximum 2638-fold enrichment. Variation partitioning analysis (VPA) together with network analysis indicated that the profile of ARGs is strongly correlated with bacterial community compositions. These results improve the knowledge about the diversity of plant-associated antibiotic resistome and factors influencing the profile of ARGs in the phyllosphere.

Bacillus ferrooxidans sp. nov., an iron(II)-oxidizing bacterium isolated from paddy soil.

An endospore-forming bacterium, designated YT-3T, was isolated from a paddy soil in Yingtan, Jiangxi, China. Cells of strain YT-3T were Gram-positive, rod-shaped, facultative anaerobic, catalase, and oxidase positive. The optimum growth temperature and pH were 30°C (ranged from 15 to 50°C) and 6.5-7.0 (ranged from 3 to 11), respectively. Analysis of the 16S rRNA gene sequence showed that strain YT-3T was affiliated to the genus Bacillus and displayed the highest similarity to that of Bacillus drentensis JCM 21707T (98.3%), followed by B. ginsengisoli JCM 17335T (97.8%) and B. fumarioli JCM 21708T (97.0%). The similarity of rpoB gene sequence between strain YT-3T and B. drentensis JCM 21707T, B. ginsengisoli JCM 17335T and B. fumarioli JCM 21708T was 80.4%, 81.5%, and 82.1%, respectively. The genomic DNA G + C content was 44.9 mol%. The predominant respiratory quinone was Menaquinone-7, and meso-diaminopimelic acid was present in the peptidoglycan layer of cell wall. The major fatty acids were C15:0 anteiso (36.2%), C14:0 iso (19.6%), C15:0 iso (17.4%), and C16:0 iso (9.8%). The polar lipid profile consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phospholipids, and ammoniac phospholipids. The DNA-DNA hybridization values between isolate YT-3T and B. drentensis (JCM 21707T), B. ginsengisoli (JCM 17335T), and B. fumarioli (JCM 21708T) were 36.3%, 30.3%, and 25.3%, respectively. On the basis of physiological, genetic and biochemical data, strain YT-3T represented a novel species of the genus Bacillus, for which the name Bacillus ferrooxidans sp. nov was proposed. The type strain is YT-3T (= KCTC 33875T = CCTCC AB 2017049T).

Draft Genome Sequence of Rhodococcus opacus Strain 04-OD7, Which Can Mobilize Phosphate.

Rhodococcus opacus strain 04-OD7 (=CCTCC AB 2017148) is a Gram-positive bacterium showing inorganic phosphate solubilization capacity for the first time in the genus Rhodococcus We present here the draft genome description of R. opacus 04-OD7 along with multiple phosphorus (P) mobilization-related genes, supporting its inorganic phosphate solubilization.

Propionicimonas ferrireducens sp. nov., isolated from dissimilatory iron(III)-reducing microbial enrichment obtained from paddy soil.

A novel strain, designated Y1A-10 4-9-1T, with Gram-stain-positive and rod-shaped cells, was isolated from paddy soil in Yingtan, Jiangxi, China. Cells were 0.15-0.2 µm wide and 1.5-3.3 µm long. The optimal growth temperature was 30 °C and the optimal pH was 7.0. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the novel strain is closely related to Propionicimonas paludicola JCM 11933T (98.57 %). The genomic DNA G+C content was 63.9 mol%. The predominant menaquinone was MK-9(H4) and meso-diaminopimelic acid was present in the cell-wall peptidoglycan layer. The major polar lipids were diphosphatidylglycerol, one unidentified phospholipid and two unidentified lipids. The dominant cellular fatty acids detected were anteiso-C15 : 0 and iso-C16 : 0. The phylogenetic and phenotypic results supported that strain Y1A-10 4-9-1T is a novel species of the genus Propionicimonas, for which the name Propionicimonas ferrireducens sp. nov. is proposed. The type strain is Y1A-10 4-9-1T (=CCTCC AB 2016249T=KCTC 15566T=LMG 29810T).

Identification and characterization of inorganic-phosphate-solubilizing bacteria from agricultural fields with a rapid isolation method.

The ability to solubilize fixed inorganic phosphorus (P) for plant growth is important for increasing crop yield. More P can be released by inoculating soil with inorganic-phosphate-solubilizing bacteria (iPSBs). We used 96-well microplates instead of traditional 200-mm petri dishes to rapidly screen iPSB strains for their solubilizing ability. We simultaneously obtained 76 iPSB isolates from 576 wells containing two agricultural soils. This method conveniently identified positive iPSB strains and effectively prevented fungal cross-contamination. Maximum-likelihood phylogenetic trees of the isolated strains showed that Bacillus megaterium was the most dominant iPSB, and strains Y99, Y95, Y924 and Y1412 were selected as representatives for the analysis of P solubilization. Succinic acid was the main organic acid of B. megaterium for releasing P. It was strongly correlated with the increase in soluble P concentration during 168 h of incubation of these four strains. pH was negatively exponentially correlated with the amount of soluble P in the medium, and the amount of succinic acid was strongly linearly correlated with the amount of P released (P < 0.001), suggesting that organic acid may mobilize microbial P. Our study provides an efficient and effective method for identifying and analyzing the growth of iPSB strains able to solubilize inorganic P and gives a better understanding of the mechanism of P solubilization.

Effects of combined application of nitrogen fertilizer and biochar on the nitrification and ammonia oxidizers in an intensive vegetable soil.

Soil amended with single biochar or nitrogen (N) fertilizer has frequently been reported to alter soil nitrification process due to its impact on soil properties. However, little is known about the dynamic response of nitrification and ammonia-oxidizers to the combined application of biochar and N fertilizer in intensive vegetable soil. In this study, an incubation experiment was designed to evaluate the effects of biochar and N fertilizer application on soil nitrification, abundance and community shifts of ammonia-oxidizing bacteria (AOB) and ammonia oxidizing archaea (AOA) in Hangzhou greenhouse vegetable soil. Results showed that single application of biochar had no significant effect on soil net nitrification rates and ammonia-oxidizers. Conversely, the application of only N fertilizer and N fertilizer + biochar significantly increased net nitrification rate and the abundance of AOB rather than AOA, and only AOB abundance was significantly correlated with soil net nitrification rate. Moreover, the combined application of N fertilizer and biochar had greater effect on AOB communities than that of the only N fertilizers, and the relative abundance of 156 bp T-RF (Nitrosospira cluster 3c) decreased but 60 bp T-RF (Nitrosospira cluster 3a and cluster 0) increased to become a single predominant group. Phylogenetic analysis indicated that all the AOB sequences were grouped into Nitrosospira cluster, and most of AOA sequences were clustered within group 1.1b. We concluded that soil nitrification was stimulated by the combined application of N fertilizer and biochar via enhancing the abundance and shifting the community composition of AOB rather than AOA in intensive vegetable soil.

Massilia phosphatilytica sp. nov., a phosphate solubilizing bacteria isolated from a long-term fertilized soil.

A Gram-stain-negative and rod-shaped bacterial strain, 12-OD1T, with rock phosphate solubilizing ability was isolated from agricultural soil in Hailun, Heilongjiang, PR China. The isolate was affiliated to the genus Massilia, based on 16S rRNA gene sequence alignments, having the highest similarities with Massilia putida6 NM-7T (98.67 %), Massilia kyonggiensis TSA1T (98.28 %), and Massilia norwichensis NS9T (98.07 %), respectively. The DNA G+C content was 67.72 mol% and DNA-DNA hybridization showed low relatedness values (less than 47 %) between strain 12-OD1T and other phylogenetically related species of the genus Massilia. The predominant isoprenoid quinone was Q-8 and the polar lipid profile comprised diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine. The major fatty acids were C17 : 0 cyclo (25.4 %), C16 : 0 (23.4 %) and summed feature 3 (C16 : 1ω7c and/or C16 : 1 ω6c) (22.5 %), which differentiates it from close relatives within the genus Massilia. Combined genetic, physiological and biochemical properties indicate that strain 12-OD1T is a novel species of the genus Massilia, for which the name Massilia phosphatilytica sp. nov., is proposed, with the type strain 12-OD1T (=CCTCC AB 2016251T=LMG 29956T=KCTC 52513T).

Biochar Addition Increases the Rates of Dissimilatory Iron Reduction and Methanogenesis in Ferrihydrite Enrichments.

Biochar contains quinones and aromatic structures that facilitate extracellular electron transfer between microbial cells and insoluble minerals. In this study, granulated biochar (1.2-2 mm) and powdered biochar (<0.15 mm) were amended to two ferrihydrite (in situ ferrihydrite and ex situ ferrihydrite) enrichments to investigate the effect of biochar with different particle sizes on dissimilatory iron(III)-reducing bacteria (DIRB) and methanogens. Biochar addition significantly stimulated the reduction of both in situ ferrihydrite and ex situ ferrihydrite and the production of methane. Powdered biochar amendments increased iron reduction compared to granulated biochar amendment in both the in situ ferrihydrite and ex situ ferrihydrite enrichments. However, no significant difference was observed in methane production between the powdered biochar and granulated biochar amendments in the two ferrihydrite enrichments. Analysis of 16S rRNA gene sequences showed that both DIRB and methanogens were enriched after biochar amendments in the in situ ferrihydrite and ex situ ferrihydrite enrichments. Taxa belonging to the Geobacteraceae and methanogenic genus affiliated to Methanosarcina were detected with significantly higher relative abundances in powdered biochar amendments than those in granulated biochar amendments in both the ferrihydrite enrichments. X-ray diffraction analysis indicated green rust [Fe2(CO3) (OH)] and vivianite [Fe3(PO4)2 8(H2O)] formed in the ex situ ferrihydrite and in situ ferrihydrite enrichments without biochar addition, respectively. After granulated biochar amendment, the mineral phase changed from the green rust to vivianite in the ex situ ferrihydrite enrichment, while crystalline vivianite and iron oxide (γ-Fe2O3) were detected simultaneously in the in situ ferrihydrite enrichment. No crystalline iron compound was found in the powdered biochar amendments in both ferrihydrite enrichments. Overall, our study illustrated that the addition of biochar affected iron-reducing and methane-generating microbial communities to some extent.

Long-term nitrogen fertilization decreased the abundance of inorganic phosphate solubilizing bacteria in an alkaline soil.

Inorganic phosphate solubilizing bacteria (iPSB) are essential to facilitate phosphorus (P) mobilization in alkaline soil, however, the phylogenetic structure of iPSB communities remains poorly characterized. Thus, we use a reference iPSB database to analyze the distribution of iPSB communities based on 16S rRNA gene illumina sequencing. Additionally, a noval pqqC primer was developed to quantify iPSB abundance. In our study, an alkaline soil with 27-year fertilization treatment was selected. The percentage of iPSB was 1.10~2.87% per sample, and the dominant iPSB genera were closely related to Arthrobacter, Bacillus, Brevibacterium and Streptomyces. Long-term P fertilization had no significant effect on the abundance of iPSB communities. Rather than P and potassium (K) additions, long-term nitrogen (N) fertilization decreased the iPSB abundance, which was validated by reduced relative abundance of pqqC gene (pqqC/16S). The decreased iPSB abundance was strongly related to pH decline and total N increase, revealing that the long-term N additions may cause pH decline and subsequent P releases relatively decreasing the demands of the iPSB community. The methodology and understanding obtained here provides insights into the ecology of inorganic P solubilizers and how to manipulate for better P use efficiency.