Abstract
Phosphorus (P) availability remains a major constraint to crop productivity, and improving the efficiency of recycled P fertilizers requires biological strategies able to mobilize poorly soluble phosphate sources. Salmon-bone nanohydroxyapatites (SnHAs) represent a promising circular P fertilizer, but their agronomic effectiveness may depend on microbial mediation in the rhizosphere. The study tested, under controlled conditions, whether native phosphate-solubilizing bacterial (PSB) consortia isolated from long-term agroecosystems under contrasting climatic conditions improve early maize growth, P nutrition, and P-acquisition responses when applied alone or in combination with SnHAs. A total of 59 native PSB isolates were obtained from Mediterranean, Continental, and Arid soils and characterized for taxonomic identity, phosphate-solubilization ability, indole-3-acetic acid production, biological N2 fixation, and quantitative P release from SnHAs. Selected strains were assembled into site-specific consortia and applied to maize seeds grown in their corresponding soils with or without SnHA fertilization. Quantitative screening revealed significant strain-dependent differences in P solubilization from SnHAs, with efficiencies ranging from 4.8% to 11.4% and a maximum release of 34.5 mg L-1 soluble P. In vivo, PSB inoculation increased bacterial biomass in the substrate at all sites, with increases of 47%, 56%, and 155% in Mediterranean, Continental, and Arid soils, respectively. Maize responses were strongly soil-dependent: PSB inoculation and/or SnHA fertilization improved biomass accumulation mainly in Continental and Arid soils, while nutrient concentration responses varied among organs and elements. Root expression of ZmPHT1;1, ZmPHT1;2, and ZmPHT1;6 was significantly upregulated only in the Arid soil, where combined PSB inoculation and SnHA fertilization also increased P recovery efficiency. These findings show that native, site-specific PSB consortia can enhance the agronomic value of SnHAs, particularly under more limiting soil conditions, and highlight the potential of integrating locally adapted microbial resources with recycled phosphate nanofertilizers for sustainable P management in maize. However, field validation is required.
1 Introduction
Despite the widespread use of phosphate fertilizers, phosphorus (P) availability remains one of the major constraints to crop productivity worldwide. In agricultural soils, phosphate ions are readily immobilized through adsorption and precipitation reactions, while a substantial proportion of total soil P occurs in organic forms that require biological mineralization before becoming available for plant uptake (Schneider et al., 2019; Doydora et al., 2020). Consequently, crops typically recover only a limited fraction of the applied fertilizer P, resulting in low P-use efficiency and the progressive accumulation of poorly available “legacy P” in many agricultural systems.
The mechanisms controlling P availability vary among soil types. In acidic soils, phosphate is commonly adsorbed onto iron and aluminum oxides or occluded within clay minerals, whereas in alkaline and calcareous soils it tends to precipitate with calcium into sparingly soluble phosphate minerals (Jindo et al., 2023; McKenna et al., 2024). These processes substantially reduce the pool of plant-available P and represent a major challenge for sustainable crop production. Improving P-use efficiency has become a major challenge for sustainable agriculture. Phosphate fertilizers are derived from finite phosphate rock reserves that are geographically concentrated and increasingly subject to economic and geopolitical constraints (Blackwell et al., 2019). At the same time, excessive P fertilization contributes to nutrient losses and eutrophication of freshwater ecosystems (Pavinato et al., 2022). Therefore, innovative strategies capable of mobilizing poorly available soil P and improving fertilizer efficiency are urgently needed.
Phosphate-solubilizing bacteria (PSB) represent a promising biological approach to enhance P availability in the rhizosphere, the narrow soil zone where nutrient mobilization and root uptake are closely coupled. These microorganisms increase the accessibility of insoluble inorganic and organic P pools through the production of organic acids and phosphatase enzymes, thereby improving P acquisition by plants (Alori et al., 2017; Pan and Cai, 2023). In addition to P mobilization, many PSB exhibit plant growth-promoting traits (PGPR) such as phytohormone production, biological nitrogen (N) fixation, and stress-mitigation mechanisms, which can further stimulate root development and nutrient uptake (Backer et al., 2018; Bargaz et al., 2021; Timofeeva et al., 2023; Zholdasbek et al., 2026).
However, the agronomic performance of microbial inoculants, as direct inoculation or seed coating, is often inconsistent because introduced/exotic strains may fail to establish and persist under field conditions. Commercial microbial inoculants selected under laboratory conditions are not always adapted to local soils, climatic conditions, or resident microbial communities (Mawarda et al., 2022; Čaušević et al., 2024). In contrast, native microorganisms originating from local agroecosystems may be better adapted to site-specific environmental conditions and therefore exhibit greater persistence and functional stability (Janati et al., 2022, 2023; Wang et al., 2024). Long-term agricultural experiments (LTEs) provide particularly valuable reservoirs of microbial diversity because their communities have been shaped by decades of management practices, fertilization regimes, crop rotations, and tillage systems (Dai et al., 2020; Bolo et al., 2021; Pellegrino et al., 2022).
Alongside microbial inoculants, recycled and slow-release P fertilizers are attracting increasing attention as sustainable alternatives to conventional phosphate fertilizers. Nanohydroxyapatite (nHA), a calcium phosphate material that can be produced from different organic sources, including fish-processing residues, has emerged as a promising recycled P source because of its high surface reactivity and gradual nutrient release (Sajadinia et al., 2021; Jia et al., 2022; Kumar et al., 2024). The use of fish-derived nHA is also consistent with circular economy principles, as it valorizes food-industry by-products into agricultural inputs (Piccirillo et al., 2014; Pilotto et al., 2025). Nevertheless, the agronomic effectiveness of nHA may be constrained by its limited solubility, particularly in neutral and alkaline soils. Combining nHA with PSB may help overcome this limitation because bacterial metabolites can enhance calcium phosphate dissolution and increase P availability in the rhizosphere (Pilotto et al., 2025).
Recent studies have shown that the combined application of fish-derived nHA and PSB can improve maize growth, P uptake, and P-use efficiency, particularly when bacterial inoculants are delivered through seed coating (Quattrocelli et al., 2025). Seed coating represents a practical and cost-effective strategy for placing beneficial microorganisms near emerging roots, thereby promoting early plant-microbe interactions and nutrient acquisition (Ma, 2019; Cortés-Rojas et al., 2021; Raymond et al., 2021).
Despite the growing interest in both native microbial inoculants and recycled phosphate fertilizers, little is known about whether native PSB consortia adapted to contrasting agroclimatic conditions can enhance the agronomic effectiveness of fish-derived nHA. Furthermore, the interaction between native PSB consortia, recycled phosphate nanoparticles, and plant phosphate uptake mechanisms remain poorly understood.
Maize (Zea mays L.) is an ideal model crop to investigate these interactions because of its high P demand, global economic importance, and sensitivity to P availability during early developmental stages (Erenstein et al., 2022). In maize, phosphate uptake is mainly mediated by members of the PHT1 family of high-affinity phosphate transporters, whose expression is regulated by plant P status and interactions with beneficial soil microorganisms (Nagy et al., 2006; Nussaume et al., 2011; Liu et al., 2016). Among these transporters, ZmPHT1;1, ZmPHT1;2, and ZmPHT1;6 have been proposed as useful molecular markers for investigating plant responses to changes in P availability (Quattrocelli et al., 2025). Therefore, coupling nutrient uptake measurements with the analysis of root PHT1 gene expression may help clarify whether improvements in plant P nutrition result solely from enhanced P solubilization in the rhizosphere or also involve modulation of plant phosphate acquisition pathways.
The aim of this study was to determine whether native, phosphate-solubilizing bacterial (PSB) consortia adapted to contrasting agroclimatic conditions could improve maize growth and nutrient uptake, either alone or in combination with salmon-bone-derived nanohydroxyapatite (SnHA). Native PSB were isolated from long-term conventional tillage and no-tillage experiments located in Arid, Continental, and Mediterranean regions. After molecular and functional characterization, selected isolates were assembled into site-specific consortia and evaluated for their ability to enhance maize performance and phosphorus acquisition.
We hypothesized that: (i) native PSB isolated from long-term agroecosystems differ in their functional traits according to their agroclimatic origin; (ii) site-specific native PSB consortia enhance maize growth and nutrient uptake when applied as seed inoculants; (iii) the combined application of native PSB consortia and SnHA provides greater benefits than either component alone through complementary and potentially synergistic effects on P mobilization and availability; and (iv) native PSB consortia and SnHA influence the expression of root PHT1 phosphate transporter genes, thereby contributing to the regulation of maize phosphorus acquisition pathways. By integrating microbial ecology, recycled P fertilization, and plant molecular responses, this study advances our understanding of biologically mediated P acquisition and supports the development of locally adapted biofertilization strategies for sustainable crop production.
2 Materials and methods
2.1 Study sites and soil sampling
Soils were collected from three long-term field experiments located in contrasting agroclimatic regions: Córdoba, Spain (Mediterranean climate, Med), Hollabrunn, Austria (Continental climate, Cont), and Niamey, Niger (Arid climate, Arid), classified according to the Köppen-Geiger system (Kottek et al., 2006). Each site included long-term experiment (LTE) on conventional tillage (CT) and no-tillage (NT) systems (Med: Panattieri et al., 2013; Cont: Rosner et al., 2018; Arid: Bado et al., 2022). Detailed information on climate, main soil characteristics, year of establishment, cropping systems, and management practices of the long-term experimental (LTE) sites used for soil sampling are reported in Supplementary Materials and Methods and Supplementary Table 1.
Soil samples were collected from the topsoil layer (0–20 cm), which hosts the most active microbial communities involved in nutrient cycling. Sampling was conducted in March 2022 at the Mediterranean site during winter wheat cultivation and in June 2022 and July 2002 at the Continental and Arid sites, during soybean and pearl millet cultivation, respectively. For varieties, sowing density, sowing date and harvesting date see Supplementary Table 1.
2.2 Isolation of native PSB
Native phosphate-solubilizing bacteria were isolated using National Botanical Research Institute’s Phosphate (NBRIP) medium containing tricalcium phosphate (TCP) as the sole insoluble P source (Nautiyal, 1999). Soil suspensions were prepared by serial dilution in sterile 0.85% NaCl solution and plated on NBRIP agar. Colonies showing visible P-solubilization halos were purified and maintained for subsequent molecular and functional analyses. Details on the native PSB isolation is given in Supplementary Materials and Methods.
A total of 59 isolates were obtained and coded according to agroclimatic origin and tillage management. Isolates from the Arid site were coded as AT (CT) and HC (NT), those from the Continental site as T (CT) and NT (NT), and those from the Mediterranean region as CT (CT) and ZT (NT).
2.3 Qualitative assessment of phosphate solubilization
Phosphate-solubilizing activity of the 59 native isolates was evaluated using a qualitative spot-inoculation assay on NBRIP agar supplemented with tricalcium phosphate (TCP) as the insoluble P source. Bacterial cultures were grown to the exponential phase, standardized to OD600 = 0.1, and 50 µL aliquots were spotted in duplicate onto agar plates. Uninoculated plates were included as negative controls. After incubation at 28 °C in the dark for 7 days, phosphate solubilization was assessed by the formation of clear halos around colonies.
The phosphate solubilization index (PSI) was calculated according to Morales et al. (2011) as follows:
2.4 Molecular identification of native PSB isolates and evaluation of PGPR traits
Genomic DNA was extracted from bacterial cultures grown in LB broth for approximately 18 h using the DNeasy PowerLyzer Microbial Kit (Qiagen, The Netherlands). The 16S rRNA gene was amplified using primers 27F and 1492R, as described by Kuramae et al. (2010), generating a 1465-bp fragment. PCR reactions were performed in a final volume of 20 µL containing 4 µL of 5× Colorless Buffer, 1 µL of MgCl2 (25 mM), 0.4 µL of dNTPs (10 mM), 0.5 µL of each primer (0.5 µM), 0.125 µL of GoTaq DNA polymerase (5 U µL-1), 12.475 µL of deionized water, and 1 µL of DNA template. PCR cycling consisted of initial denaturation at 95 °C for 2 min, followed by 25 cycles of denaturation at 95 °C for 30 s, annealing at 51.9 °C for 1 min, and extension at 72 °C for 1 min, with a final extension at 75 °C for 5 min. Amplicons were checked by electrophoresis on a 1% agarose gel and purified using Agencourt AMPure® XP magnetic beads (Beckman Coulter, USA). DNA concentration was quantified with a Qubit™ 4 Fluorometer using the Qubit™ 1× dsDNA High Sensitivity Assay Kit (Invitrogen, Thermo Fisher Scientific, CA, USA), according to the manufacturer’s instructions. Purified amplicons were normalized to 1.33 ng µL-1 and Sanger sequenced on both forward and reverse strands by Eurofins Genomics (Germany) using the PCR primers.
Indole-3-acetic acid (IAA) production was quantified using the Salkowski colorimetric assay following Cumpa-Velásquez et al. (2021). IAA concentrations were determined spectrophotometrically at 530 nm using a standard calibration curve. Details are given in Supplementary Materials and Methods.
The potential ability of isolates to fix atmospheric N was assessed using N-free semi-solid New Fabio Pedrosa medium (Batista et al., 2020). Isolates were inoculated into the medium and incubated at 28 °C. Putative diazotrophic activity was inferred from bacterial growth under nitrogen-free conditions, indicated by pellicle formation and a color shift of the medium.
2.5 Synthesis and characterization of salmon-bone nanohydroxyapatite
Salmon-bone-derived nanohydroxyapatite was used as a recycled P source. Synthesis followed previously described protocols for fish-bone-derived apatite materials (Teixeira et al., 2017; Quattrocelli et al., 2025). Phase composition, functional groups, particle morphology, and surface charge were characterized by X-ray diffraction (XRD), Fourier-transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), and zeta potential analysis, respectively. Total P concentration was quantified using a Merck Spectroquant P reagent kit, following Piccirillo et al. (2014).
2.6 Quantitative evaluation of P solubilization from SnHAs
Twenty high-performing PSB isolates were selected for quantitative assessment of P solubilization from SnHAs. Standardized bacterial suspensions (OD600 = 0.1) were inoculated into liquid NBRIP medium containing SnHAs (5 g L-1) as the sole P source. Uninoculated controls were included to account for abiotic P release. After 7 days of incubation at 28 °C under 140 rpm shaking conditions, soluble P in culture supernatants was quantified spectrophotometrically using the Merck Spectroquant phosphorus reagent kit. Details are reported in Supplementary Materials and Methods. Results were expressed as the percentage of solubilized P relative to the total P content of SnHAs.
2.7 Assembly of native PSB consortia and in vivo experiment with maize
Based on molecular identification, phosphate-solubilizing ability, P mobilization from SnHAs, and PGPR traits, three site-specific bacterial consortia were assembled. The Med consortium consisted of Sinorhizobium meliloti CT1, Priestia aryabhattai CT4, and Arthrobacter oryzae ZT5. The Cont consortium included P. aryabhattai T4, Agromyces sp. T9, and Priestia megaterium T14. The Arid consortium included P. aryabhattai AT2, Bacillus subtilis HC3, and P. aryabhattai HC4.
Maize (Zea mays L. cv. Lolita, Pioneer Hi-Bred, USA) surface-sterilized seeds were coated with site-specific PSB consortia using carboxymethyl cellulose (CMC) as adhesive, following established microbial seed-coating procedures (Ma, 2019; Cortés-Rojas et al., 2021). Control seeds received a mock inoculum consisting of sterile bacterial growth medium and CMC. Details are given in Supplementary Materials and Methods. Seeds were pre-germinated for 5 days and transplanted into 1300 mL pots containing a mixture of native soil, steam-sterilized vermiculite, and quartz sand (1:1:1, v/v/v). SnHAs were applied at a rate of 0.5 g pot-1 in fertilized treatments. The physico-chemical parameters from the three climatic sites are reported in Supplementary Table 2.
The experiment followed a completely randomized factorial design with two factors: bacterial inoculation (non-inoculated, NB and inoculated, B), and fertilization (without SnHAs, NFert and with SnHAs, Fert). Each treatment was applied to the three soil types (Mediterranean, Continental, and Arid soils), with four replicates per treatment (48 pots in total). Plants were grown under controlled conditions (25/18 °C day/night, 16/8 h photoperiod) and irrigated regularly with deionized water. A modified Hoagland solution containing 25% of the standard P concentration was supplied every 10 days to meet the minimum plant P requirement (Gómez-Muñoz et al., 2018). This allowed a uniform low-P nutritional background across soils, thereby enabling the effects of SnHA and PSB inoculation to be evaluated under comparable P-limited conditions.
Plants were harvested at V3 growth stage (Hanway, 1963). Leaves, stems and roots were separated, dried at 65 °C to constant weight, and weighed. Total biomass was calculated as the sum of leaf, steam and root dry weights. Ground plant material was microwave-digested with nitric acid and analyzed for P, K, Ca, Fe, and Zn concentrations by ICP-OES.
Plant P uptake was calculated separately for each plant organ as the product of P concentration and dry weight:
P uptake = P concentration × dry weight
where P concentration was expressed on a dry weight basis and dry weight corresponded to the dry biomass of the same organ. Total plant P uptake was obtained by summing P content in roots, leaves, and stems. Phosphorus use efficiency (PUE) and phosphorus recovery efficiency (PRE) were then calculated as:
where P applied with SnHAs corresponded to the amount of P supplied through the nanohydroxyapatite fertilizer (i.e., 18% of the total of SnHA dose applied per pot).
2.8 Bacterial biomass in the substrate and expression analysis of phosphate transporter genes in roots
Bacterial biomass in the substrate was quantified as a proxy for the persistence of the native PSB inoculum. DNA was extracted from substrate and root samples using the DNeasy PowerSoil Kit (Qiagen, Venlo, Netherlands). Three biological replicates per treatment were analyzed, resulting in 12 substrate samples climatic sites. Bacterial abundance was estimated by qPCR targeting the bacterial 16S rRNA gene using the Eub338/Eub518 primer pair (Muyzer et al., 1993; Fierer et al., 2005). Standard curves were generated using triplicate plasmid dilution series, and each sample was analyzed in technical triplicate. The mean Ct value was used to calculate 16S rRNA gene copy numbers, which were expressed as 16S rRNA gene copies per gram of substrate dry weight. Detailed qPCR reaction conditions and thermal cycling parameters are provided in the Supplementary Material.
The expression of the maize phosphate transporter genes ZmPHT1;1, ZmPHT1;2, and ZmPHT1;6 was analyzed in root samples collected at harvest. Total RNA was extracted using the RNeasy Mini Kit (Qiagen, The Netherlands). During extraction, genomic DNA was removed by on-column digestion using the RNase-Free DNase Set (Qiagen, Venlo, The Netherlands), and further dissolved in nuclease free water. RNA integrity was checked by electrophoresis on 1% agarose gel, while RNA concentration and purity were assessed using a Qubit™ RNA HS Assay Kit (Life Technologies, Thermo Fisher Scientific, Waltham, MA, USA) and a NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Only high-quality RNA samples were used for downstream analysis, following general recommendations for quantitative real-time PCR experiments (Bustin et al., 2009). For each sample, ca. 650 ng of total RNA was reverse-transcribed into complementary DNA (cDNA) using the iScript™ Reverse Transcription Supermix (Bio-Rad, USA). Gene-specific primers, previously validated by Quattrocelli et al. (2025), were used for RT-qPCR analysis. Amplifications were performed using the SsoAdvanced™ Universal SYBR® Green Supermix (Bio-Rad, Hercules, CA, USA) on a CFX Connect Real-Time PCR System (Bio-Rad, USA). Details are given in Supplementary Materials and Methods. Relative expression levels were calculated using the 2−ΔΔCt method (Livak and Schmittgen, 2001), with poliub7 as the reference gene (Pii et al., 2016). For each soil type, the NB: NFert treatment was used as calibrator. Results were expressed as fold change relative to the corresponding control treatment.
2.9 Statistical analysis and bioinformatics
For the in vitro parameters, namely phosphate solubilization index (PSI), IAA production, biological N fixation, and P solubilization from SnHAs, each climatic site was analyzed separately using one-way ANOVA, with bacterial isolate (Isolate) as the fixed factor. When the effect of Isolate was significant, Tukey’s honestly significant difference (HSD) post hoc test was used for pairwise comparisons among isolates.
For the in vivo experiment, each climatic site was analyzed separately using two-way ANOVA to test the effects of bacterial inoculation (Inoc), salmon-bone nanohydroxyapatite fertilization (Fert), and their interaction on maize growth and nutritional parameters. Climatic sites were analyzed separately because they represented distinct soil-microbiome contexts, differing in soil origin, physicochemical properties, resident microbial communities, and site-specific native PSB consortia. Therefore, climatic site was not treated as a fully crossed experimental factor, but rather as a source of different biological contexts in which the effects of inoculation and SnHA fertilization were tested.
For PUE and PRE, the analysis was restricted to fertilized treatments only, comparing non-inoculated and inoculated plants receiving salmon-bone nanohydroxyapatite fertilization (-NB +Fert and +NB +Fert). Moreover, total P in plant was calculated for NB-NFert and B-NFert, and used to assess whether PSB mobilized native soil P in the absence of SnHAs. Differences between treatments were tested for each climatic site using a two-sample t-test.
For all ANOVA models, assumptions of normality and homogeneity of variances were assessed using the Shapiro–Wilk test on model residuals and Levene’s test, respectively. When model assumptions were not satisfied, data were transformed before analysis, using natural logarithm transformation (i.e., P % in the in vitro system; P % in leaves and roots and bacterial biomass as 16S gene copies g-1 substrate in the in vivo system of all sites). In the two-way ANOVAs, when a significant Inoc × Fert interaction was detected, Tukey’s HSD test was used for pairwise comparisons among the combined treatment levels. In the absence of a significant interaction, Tukey’s HSD test was applied to the significant main effects only. Multiple comparisons were controlled within each response variable using Tukey’s HSD test following ANOVA. Climatic sites and traits were analyzed separately because the objective was to evaluate treatment effects within distinct soil–microbiome contexts and on agronomically or physiologically distinct response variables, rather than to test a single global hypothesis across all measured traits. Therefore, no additional global correction was applied across different response variables; treatment effects were interpreted based on the consistency of responses across related traits.
For P-related gene expression, relative expression values were calculated using the 2−ΔΔCT method. Each treatment was compared against the reference group NB_Nfert, separately for each climatic site and gene. Statistical significance was tested using t-tests on relative expression values and was set at P ≤ 0.05.
Statistical significance was set at P ≤ 0.05. Compact letter displays were used to summarize post hoc comparisons. Statistical analyses were carried out in R (R Core Team, 2025) using the packages car, multcompView, dplyr, tidyr, and readxl. Bar plots showing means ± standard error and/or boxplots were generated in R using ggplot2.
Taxonomic identification was performed through BLAST searches against the NCBI database and confirmed by phylogenetic analysis. Bacterial sequences are available in the NCBI SUB16263280 from PZ558346 to PZ558402. The newly sequenced PSB isolates were included in the Neighbour-Joining (NJ) tree together with reference sequences retrieved from NCBI following BLAST searches. Multiple sequence alignment was performed using the online MAFFT server (Kuraku et al., 2013; Katoh et al., 2019). The phylogenetic tree was constructed using the Neighbour-Joining method (Saitou and Nei, 1987) and the Kimura 2-parameter model. Branch support was assessed using 1000 bootstrap replicates (Felsenstein, 1985). The tree was drawn to scale, with branch lengths expressed in the same units as the evolutionary distances used to infer the phylogeny (Tamura et al., 2004). Ambiguous positions were removed for each sequence pair using the pairwise deletion option. The final dataset included 114 nucleotide sequences and 1570 positions. Evolutionary analyses were conducted in MEGA11 (Stecher et al., 2020; Tamura et al., 2021).
3 Results
3.1 Isolation and molecular identification of native PSB
Based on halo formation on TCP-containing medium, 59 putative phosphate-solubilizing bacterial (PSB) strains were obtained from soils collected across the three agroclimatic regions (Supplementary Table 3). The isolates comprised 12 strains from the Mediterranean site (Med), 25 from the Continental site (Cont), and 22 from the Arid site.
At the Med site, five isolates were identified as Sinorhizobium meliloti (CT1, CT75, CT7, ZT3, and ZT4) (Supplementary Table 3). The remaining isolates were assigned to Priestia endophytica (CT2), Priestia aryabhattai (CT3, CT4), Pseudarthrobacter oxydans (CT6), Staphylococcus epidermidis (ZT1), Bacillus sp. (ZT2), and Arthrobacter sp. (ZT5). Sequence identity values ranged from 99.6% to 100%.
At the Cont site, P. aryabhattai was the best match for 13 isolates (T1, T2, T3, T4, T5, T6, T7, T8, T10, T13, T15, T17, and NT9), with sequence identity values ranging from 99.5% to 100% (Supplementary Table 3). Isolates NT5 was assigned to Streptomyces galilaeus, NT6 to Streptomyces graminilatus, NT7 and NT8 to Streptomyces spinicoumarensis, and NT3 and NT4 to Streptomyces sp., with sequence identity values ranging from 92% to 99%. Isolates T12, T18, and NT1 matched Bacillus sp., with identity values ranging from 99% to 100%. Isolates T9 and T14 were identified as Agromyces sp. and Priestia megaterium, respectively, both with 99% identity. For one isolate (NT10), no reliable taxonomic assignment could be obtained from the 16S rRNA gene sequence.
At the Arid site, P. aryabhattai was the best match for 13 isolates (AT1, AT2, AT3, AT4, AT5, AT6, AT7, HC1, HC4, HC6, HC7, HC12, and HC16), with sequence identity values ranging from 99.9% to 100% (Supplementary Table 3). Isolates HC2, HC5, HC8, H11 and HC17 showed the highest similarity to P. megaterium, with identity values ranging from 93.5% to 99.9%. Strains HC10 was assigned to Neobacillus niacini with 88.3% identity. The remaining strains, HC3, HC9, and HC18, were identified as Bacillus subtilis, Bacillus cereus, and Bacillus sp., with identity values of 99.9%, 100%, and 99.7%, respectively.
The NJ phylogenetic tree mostly confirmed the taxonomic assignments obtained by BLAST analysis, with bootstrap support values ≥70% for most clades (Figure 1; Supplementary Table 3). Overall, the isolated PSB strains belonged to the genera Sinorhizobium, Priestia, Pseudarthrobacter/Arthrobacter, Bacillus, Streptomyces, and Agromyces, with differences in taxonomic composition among climatic sites. Staphylococcus was not included in the NJ tree. The NJ tree did not show a clear separation between P. aryabhattai and P. megaterium. Some isolates classified as Bacillus sp. by BLAST clustered within the Priestia aryabhattai/P. megaterium group, including ZT2 from the Med site and T12, T18, and NT1 from the Cont site. In addition, HC18, assigned as Bacillus sp. by BLAST, clustered with Chungangia koreensis.
Figure 1
3.2 Qualitative screening of phosphate solubilization on TCP and PGPR traits
The qualitative screening on TCP plates showed that the P solubilization index (PSI) varied significantly among isolates within each climatic site (one-way ANOVA, P < 0.001). Overall, PSI values ranged from 1.0 to 2.2 across the 59 tested isolates (Figure 2). At the Med site, PSI values ranged from 1.0 to 1.4. Strains CT1 and ZT1 showed the highest PSI value, with a PSI of 1.4, followed by CT3, CT4, CT5, ZT2, and ZT3, which showed a PSI of 1.3 (Figure 2A). The remaining Med isolates displayed PSI values ranging from 1.0 to 1.2. At the Cont site, PSI values ranged from 1.2 to 2.2. The highest values were recorded for N6, T12, and N10, with PSI values of 2.2, 2.0, and 1.8, respectively (Figure 2B). The remaining Cont isolates showed PSI values ranging from 1.2 to 1.3. At the Arid site, PSI values ranged from 1.0 to 1.2 (Figure 2C). Twelve strains showed a PSI of 1.2, seven strains showed a PSI of 1.0, and two strains showed a PSI of 1.1.
Figure 2
Additional PGPR traits were assessed by measuring indole-3-acetic acid (IAA) production and biological N2 fixation. IAA production varied significantly among isolates within each climatic site (one-way ANOVA, P < 0.001; Figure 3). At the Med site, the highest IAA production were recorded for strains CT5, CT7, and ZT3, which produced 11.0, 10.9, and 10.1 μg mL-1 IAA, respectively (Figure 3A). At the Cont site, IAA production ranged from 0.2 μg mL-1 in strain T10 to 5.0 μg mL-1 in strain NT1 (Figure 3B). Strains T14, T18, NT8, and NT10 did not produce detectable amounts of IAA (Supplementary Table 4). At the Arid site, all strains produced detectable amounts of IAA except HC9, HC10, and HC18 (Figure 3C). The highest IAA production was observed in strains HC12, HC11, and HC1, with 13.4, 13.2, and 10.8 μg IAA mL-1, respectively.
Figure 3
The same strains were also tested for in vitro biological N2 fixation. Ten out of 12 strains from the Med site, seven out of 25 strains from the Cont site, and nine out of 22 strains from the Arid site tested positive for N2 fixation (Supplementary Table 4).
3.3 Quantitative solubilization of P from sHAs
Strains that tested positive for qualitative phosphate-solubilization activity were selected for quantitative evaluation of P solubilization from SnHAs. For each climatic site, strains were selected based on their phosphate-solubilization performance and phylogenetic diversity. The amount of P solubilized from SnHAs differed significantly among isolates within each climatic site (one-way ANOVA, P < 0.001) (Figure 4). In the Med site, P solubilization ranged from 4.8% to 11.4% of the total P (Figure 4D). Strain ZT3 showed the highest P solubilization value, with 11.4% of total P solubilized, corresponding to 34.5 mg P L-1, followed by CT1, which solubilized 8.4% of total P, corresponding to 18.5 mg P L-1 (Figure 4A; Supplementary Figure 1). Strains CT3 and ZT2 showed the lowest values, with 4.9% and 4.8% of total P solubilized, corresponding to 6.4 and 6.2 mg P L-1, respectively. In the Cont site, P solubilization ranged from 4.7% to 7.6% of the total P, corresponding to 5.8-15.5 mg P L-1 (Figure 4B; Supplementary Figure 1). The highest value was observed for strain T4, which solubilized 7.6% of total P, corresponding to 15.5 mg P L-1. In the Arid site, P solubilization ranged from 6.2% to 6.4% of the total P (Figure 4C). Strain H4 showed the highest value, with 6.4% of total P solubilized, corresponding to 10.9 mg P L-1 (Figure 4C; Supplementary Figure 1). Strains A2 and A6 solubilized 6.2% of total P, corresponding on average to 10.3 mg P L-1.
Figure 4
3.4 In vivo experiment on maize
The effects of native PSB inoculation and SnHA fertilization were evaluated in maize plants grown in soils from the three climatic sites. For each site, maize seeds were inoculated with the corresponding site-specific native PSB consortium and grown with or without SnHA fertilization. The analysis included bacterial biomass in the substrate, plant growth traits, P concentration, other nutrient concentrations, root phosphate transporter gene expression, and P-use indicators.
3.4.1 Effects of native PSB consortia and SnHAs on bacterial biomass in substrate, maize growth, P concentration, and other nutrient concentration
Bacterial biomass in the substrate, expressed as 16S rRNA gene copies per gram of substrate dry weight, was significantly affected by native PSB inoculation in all three climatic sites, whereas SnHA fertilization and the Inoc × Fert interaction were not significant (Figure 5; Supplementary Table 5). Within sites, the strongest increase due to Inoc was shown in the Arid site (47%, 56, and 155%, respectively) (Figures 5A–C).
Figure 5
The effects of Inoc and Fert on maize growth differed among the three climatic sites (Figure 6; Supplementary Table 6). At the Med site, neither PSB inoculation nor SnHA fertilization significantly affected root, leaf, and stem dry weight (RDW, LDW, and SDW, respectively) (Figures 6A–C). Conversely, a significant Inoc × Fert interaction was detected for plant dry weight (PDW) (Figure 6D). PSB inoculation and SnHA applied alone increased PDW by over 20%, whereas no further increase was observed when PSB inoculum was combined with SnHA fertilization. At the Cont site, PSB inoculation significantly increased LDW and SDW by 32% and 37%, respectively (Figures 6E, F; Supplementary Table 6). SnHA fertilization also significantly increased biomass across plant organs, with an average increase of approximately 30% (Figures 6H–J). SnHA and Inoc applied alone did not produce any significant effect on PDW, while their combination increased PDW by 109% compared with the control (Figure 6G). At the Arid site, PSB inoculation significantly increased LDW and SDW by 24% and 41%, respectively (Figures 6K, L). SnHA fertilization significantly increased LDW by 18%. Under PSB inoculation or SnHA fertilization, RDW and RDW increased by approximately 40%, and their combined application did not further increase values (Figures 6M, N).
Figure 6
The effects of native PSB inoculation and SnHA fertilization on P concentration varied according to climatic site and plant organ (Figure 7; Supplementary Table 7). At the Med site, neither PSB inoculation nor SnHA fertilization significantly affected P concentration in roots and leaves (Supplementary Table 7). In stems, SnHA fertilization increased P concentration by 23% (Figure 7C), whereas PSB inoculation reduced stem P concentration by 13% (Figure 7A). At the Cont site, SnHA fertilization increased leaf P concentration by 20% (Figure 7L), while PSB inoculation reduced root P concentration by 8% (Figure 7K). No other significant changes in P concentration were observed in the other organs. At the Arid site, SnHA fertilization increased P concentration in roots and leaves by 16% and 24%, respectively (Figures 7S, U). PSB inoculation increased root P concentration by 8% (Figure 7R), whereas leaf P concentration decreased by 20% under PSB inoculation (Figure 7T).
Figure 7
Similarly, the effects of native PSB inoculation and SnHA fertilization on K, Ca, Fe, and Zn concentrations varied according to climatic site, plant organ, and treatment combination (Figure 7; Supplementary Table 7). At the Med site, SnHA fertilization significantly decreased root K concentration by 15% (Figure 7D), while PSB inoculation decreased leaf K concentration by 19% (Figure 7B). Root Ca concentration was reduced under PSB inoculation both in the absence and presence of SnHA fertilization (Figure 7E). A similar response was observed for Fe concentration, with value decreasing by 44% under PSB inoculation in the absence of SnHA fertilization (Figure 7F). In leaves, Fe concentration decreased by 27% under PSB inoculation (Figure 7G). Root Zn concentration decreased by 24% under PSB inoculation (Figure 7H). In contrast, Zn concentration was the lowest with PSB inoculation in absence of SnHA, intermediate with no inoculation and SnHA application, and the highest with PSB inoculation and no fertilization and with both PSB and SnHa (Figure 7I). At the Cont site, K concentration in roots, leaves, or stems was not significantly affected by treatments (Supplementary Table 7). Root Ca concentration in untreated plants decreased by over 30% under PSB inoculation, SnHA fertilization and the combination of both treatments (Figure 7M). A similar pattern was observed for root Fe concentration, decreasing by over 40% under all treatments (Figure 7O). Leaf Ca concentration decreased by 26% following SnHA fertilization (Figure 7N), while leaf Fe concentration decreased by 20% under PSB inoculation (Figure 7P). Leaf Zn concentration decreased by 17% under PSB inoculation (Figure 7Q). At the Arid site, significant changes were mainly observed in leaves (Supplementary Table 6). Leaf K concentration decreased by 38% under PSB inoculation (Figure 7W). Fe concentration decreased by 25% in both roots and leaves under PSB inoculation (Figures 7Y, Z). Root Zn concentration decreased by 23% under PSB inoculation (Figure 7AA). Leaf Ca concentration decreased by over 50% under PSB inoculation, SnHA fertilization, and their combined application (Figure 7X). Similar leaf Ca values were observed in PSB-inoculated plants with and without SnHAs and in not inoculated plants receiving SnHA fertilization. Data for K, Ca, Fe, and Zn concentrations in roots, leaves, and stems are also summarized in Supplementary Table 8.
3.4.2 Expression of P-related genes in maize roots and P use and recovery efficiency
The expression of maize phosphate transporter genes varied according to climatic site and treatment (Figures 5D–N). At the Med site, the expression of ZmPHT1;1, ZmPHT1;2, and ZmPHT1;6 was not significantly affected by PSB inoculation, SnHA fertilization, or their combined application. Although higher mean expression values were observed in some fertilized treatments, the differences were not statistically significant for any of the three genes (Figures 5D, G, L). Similarly, at the Cont site, no significant changes were detected in the expression of the phosphate transporter genes across treatments (Figures 5E, H, M). At the Arid site, the expression of all three phosphate transporter genes was significantly increased by PSB inoculation, SnHA fertilization, and their combined application compared with the untreated control (Figures 5F, I, N) treatments. The pattern of response of the three genes was similar: the increase of expression was the highest with the combined application of Inoc and SnHA and progressively decreased with the application of Inoc and SnHA alone.
Phosphorus use efficiency of plants from the three areas was not affected by inoculation (Figures 4D–F; Supplementary Table 9). Similarly, phosphorus recovery efficiency of plants from Med and Cont sites was not modified by inoculation (Figures 4G, H), while it was significantly increased in plants grown in the substrate from the Arid site(Figure 4I). To assess whether the PSB mobilized native soil P in absence of SnHAs, plant P uptake was compared between the NB-NFert and B-NFert treatments for each soil. No significant effect of bacterial inoculation on plant P uptake was detected in any of the three soils (Arid: t = −0.857, P = 0.440; Continental: t = −0.838, P = 0.449; Mediterranean: t = 2.222, P = 0.090).
4 Discussion
4.1 Native PSB consortia as site-adapted tools for P mobilization
This study demonstrates that native PSB isolated from LTE soils can be assembled into site-specific consortia combining complementary plant-growth promoting traits. The selected isolates belonged to bacteria genera commonly associated with phosphate solubilization and plant growth promotion, including Sinorhizobium, Priestia, Bacillus, Streptomyces, Agromyces, Pseudarthrobacter, and Arthrobacter. Although taxonomic identity alone does not predict inoculant performance, integrating molecular identification with functional screening enabled the selection of strains differing in phosphate solubilization, P mobilization from SnHAs, IAA production, and biological N2 fixation. The resulting consortia therefore combined complementary functional traits that could contribute to improved plant P acquisition.
The in vitro screening further confirmed that the selected isolates were able to mobilize P from a SnHAs, a poorly soluble Ca phosphate source. Unlike, qualitative halo formation on TCP agar, this assay provided direct evidence that the selected strains could release P from the recycled fertilizers used in the subsequent plant experiemnt. Because the agronomic value of HA-based fertilizers depends largely on biological processes that promote their dissolution, this functional trait represents a key prerequisite for combining PSB with recycled phosphate nanofertilizers. Previous studies similarly demonstrated that PSB enhance P release from fish-derived HA through rhizosphere acidification and organic acid production, thereby increasing the availability of this otherwise sparingly soluble P source (Santana et al., 2019; Pilotto et al., 2025).
4.2 Soil context modulates the effectiveness of native PSB inoculation
The response of maize to native PSB inoculation and SnHA fertilization differed markedly among the three soils, indicating that the effectiveness of the biofertilization strategy was strongly influenced by soil context. Plant growth and P nutrition responded only modestly in the Mediterranean soil, whereas clearer improvements were observed in the Continental soil and, most notably, in theArid soil. These contrasting responses indicate that the same inoculation strategy does not generate equivalent outcomes across different soil environments.
The limited response observed in the Mediterranean soil may reflect a greater functional capacity of the resident microbial community to sustain P mobilization, thereby reducing the additional benefit provided by the introduced consortium. In contrast, the stronger responses recorded in the Continental and especially the Arid soil suggest that microbial inoculation becomes more effective where native microbial functions or P availability are more limiting. Similar patterns have been reported in previous studies showing that soil physicochemical properties and resident microbial communities are major determinants of microbial inoculant performance (Raymond et al., 2021; Mawarda et al., 2022; Čaušević et al., 2024; Wang et al., 2024).
Inoculation consistently increased bacterial biomass in all soils, although the magnitude of the response varied among sites. The largest increase occurred in the Arid soil where plant growth, P recovery, and the expression of phosphate transporter genes were also most strongly stimulates. While qPCR did not distinguish the introduced strains for the resident bacterial community, these results indicate that inoculation enhanced overall bacterial abundance and suggest a more pronounced biological response under the Arid soil conditions. Overall, these findings identify soil context as a key factor governing the establishment and functional expression of PSB inoculants.
4.3 Salmon-bone nanohydroxyapatite as a P source mobilized by native PSB
Salmon-bone nanohydroxyapatite represents a recycled Ca phosphate source whose agronomic effectiveness depends the biological and chemical processes regulating P dissolution in the rhizosphere (Alori et al., 2017; Raymond et al., 2021; Pan and Cai, 2023). In agreement with this concept the response to SnHA fertilization was not uniform across soils. While SnHAs alone improved some plant growth and P-related traits, the greatest benefits were generally observed when they were combined with native PSB inoculation, indicating that microbial activity plays a key role in enhancing the availability of this poorly soluble P source (Montalvo et al., 2015; Tian et al., 2019; Jia et al., 2022; Jindo et al., 2023; McKenna et al., 2024).
Previous studies demonstrated that PSB promoted the dissolution of fish-derived HA through rhizosphere acidification and organic acid production, thereby increasing P availability for plants (Santana et al., 2019). More recently, Pilotto et al. (2025) showed that PSB enhanced P release from biowaste-derived nHA under soil-column conditions, confirming that microbial activity is essential for mobilizing P source from recycled Ca phosphate materials. The present results are consistent with these findings, extending them by showing that the effectiveness of this biological interaction depends strongly on soil context.
A similar synergistic interaction between fish-derived nHA and PSB was recently reported by Quattrocelli et al. (2025), who observed improvements in maize biomass, P uptake, P-use efficiency, and P recovery following seed coating with a PSB consortium combined with fish-derived nHAs. In the present study, the strongest response was likewise obtained when SnHAs were combined with native PSB, but only in the Arid soil, where inoculation increased bacterial biomass, enhanced the expression of root phosphate transporters, and improved P recovery efficiency. Compared with the single-soil system investigated by Quattrocelli et al. (2025), our results demonstrate that the magnitude of the SnHA-PSB interaction is strongly influenced by soil characteristics. The particularly pronounced response in the Arid soil may reflect the lower biological fertility of this soil as suggested by its lower initial microbial biomass compared with the Mediterranean and Continental soils (data not shown).
4.4 Plant growth and nutrient responses reveal benefits and trade-offs of enhanced P acquisition
The improvement in maize growth promoted by PSB inoculation and/or SnHA fertilization was accompanied by changes in the concentration of several macro- and micronutrients, particularly in the Continental and Arid soils, where biomass accumulation was greatest. These responses indicate that enhanced P acquisition influenced the nutritional balance of the plant rather than simply increasing the concentration of all mineral elements (Jarrell and Beverly, 1981; White and Brown, 2010).
One likely explanation is a biomass dilution effect, whereby rapid plant growth reduces nutrient concentrations even when nutrient uptake is maintained or increased (Jarrell and Beverly, 1981). This mechanism may account for the lower concentrations of K, Ca, Fe, or Zn observed in treatments that stimulated biomass accumulation. However, dilution alone is unlikely to explain all the observed responses. Increased P availability can also modify nutrient acquisition through changes in rhizosphere chemistry and nutrient interactions, particularly for Ca, Zn, and Fe, whose availability and uptake are known to be influenced by soil P status (Grant et al., 2001; Hinsinger, 2001; Bouain et al., 2014; Xie et al., 2019).
The reduction in Fe and Zn concentrations observed across several treatments is therefore consistent with the well-documented interaction between P nutrition and micronutrient homeostasis. Similar responses have been reported in plants receiving enhanced P supply, where improved P acquisition is accompanied by lower tissue concentrations of Fe and Zn due to a combination of physiological regulation, altered nutrient partitioning, and growth dilution (Grant et al., 2001; Bouain et al., 2014; Xie et al., 2019). Likewise, the decrease in K concentration is more likely attributable to dilution and changes in nutrient allocation than to direct interactions with phosphate, since K thus not readily form insoluble phosphate compounds (White and Brown, 2010; Marschner, 2012). This interpretation is consistent with the findings of Quattrocelli et al. (2025), who reported increased K uptake despite lower tissue concentrations when fish-derived nHA and PSB stimulated maize biomass production.
These findings indicated that PSB-SnHA strategies improve early maize P acquisition and growth, but also alter plant nutrient balance during early development. Although these changes do not diminish the agronomic potential of the approach, they suggest that recycled P nanofertilizers combined with microbial inoculants should be evaluated within an integrated nutrient-management framework, particularly in soils susceptible to micronutrient limitations or P-induced nutrient imbalances.
4.5 Root PHT1 expression indicates site-specific activation of phosphate acquisition pathways
The analysis of maize root phosphate transporter genes revealed that the molecular response to PSB inoculation and SnHA fertilization was strongly dependent on soil context. While no significant changes in the expression of the selected ZmPHT1 genes were detected in the Mediterranean and Continental soils, the Arid soil showed a clear up-regulation of ZmPHT1;1, ZmPHT1;2, and ZmPHT1;6, particularly under combined PSB and SnHA application. This indicates that the strongest activation of root phosphate acquisition mechanisms occurred under the soil conditions where improvements in plant performance and P recovery were also most evident.
The PHT1 family plays a central role in phosphate uptake, and its expression is regulated by plant P status, external Pi availability, and interactions with beneficial microorganisms (Nussaume et al., 2011; Liu et al., 2016). Most studies have focused on the regulation of PHT1 transporters by arbuscular mycorrhizal fungi, which modulate both direct and symbiotic phosphate uptake pathways (Loth-Pereda et al., 2011; Tian et al., 2013; Sawers et al., 2017). Evidence for PSB-mediated regulation is more limited, although Alzate Zuluaga et al. (2021) demonstrated that bacterial inoculation can also alter the expression of maize PHT1 genes, suggesting that microbial effects extend beyond P solubilization in the rhizosphere to include modulation of plant P acquisition pathways.
Our results are consistent with the recent study of Quattrocelli et al. (2025), who reported a marked induction of maize PHT1 transporters following the application of fish-derived nHAs, either alone or in combination with a PSB consortium. However, whereas Quattrocelli et al. (2025) observed transporter induction in a single experimental soil, the present study demonstrates that this molecular response is not universal but is strongly influenced by soil conditions. The induction of ZmPHT1;1, ZmPHT1;2, and PHT1;6 exclusively in the Arid soil suggests that activation of high-affinity transport systems occurs primarily where microbial mobilization of recycled P provides the greatest benefit to the plant.
The coordinated increase in PHT1 expression, bacterial biomass and P recovery efficiency observed in the Arid soil further supports this interpretation. Although the present data do not establish a causal relationship between transporter induction and improved P recovery, they indicate that enhanced microbial access to SnHA-derived P was accompanied by activation of the plant phosphate uptake machinery. Together, these findings highlight that the molecular response of maize to recycled P fertilization is governed not only by fertilizer properties but also by the interaction between soil conditions and microbial activity.
4.6 Implications for sustainable phosphorus management and future application
The present findings suggest combining native PSB consortia with recycled SnHAs represents a promising strategy to improve P use efficiency while promoting the valorization of food-processing by-products. However, the contrasting responses observed among soils indicate that microbial biofertilizers should not be considered universal products, but rather site-adapted technologies whose performance depends on soil physicochemical characteristics, resident microbial communities, and plant-microbe interactions. This concept is consistent with growing evidence that the agronomic success of microbial inoculants relies not only on strain functionality but also on ecological compatibility and successful establishment within the target soil (Soumare et al., 2020; Raymond et al., 2021; Mawarda et al., 2022; Čaušević et al., 2024; Wang et al., 2024).
However, several limitations from this study should be considered before translating results to field management. The experiment was conducted under controlled conditions and focused on early maize growth; therefore, the persistence of the inoculated consortia, the stability of the observed responses throughout the crop cycle, and their effects on final yield remain to be validated under field conditions. Another limitation is the lack of a direct comparison with a commercial/exotic microbial PSB consortium which prevents to verify whether native PSB consortia outperform commercially available inoculants. Moreover, although qPCR demonstrated an increase in total bacterial biomass following inoculation, it did not distinguish the introduced strains from the resident bacterial community. Future studies should combine strain-specific tracking, microbial community profiling, and long-term field experiments to determine inoculant persistence, interactions with indigenous microbiota, and the long-term agronomic and environmental performance of recycled phosphate nanofertilizers (Soumare et al., 2020; Mawarda et al., 2022; Čaušević et al., 2024). Finally, because nanomaterials may exert variable effects on soil microbial processes depending on their physicochemical properties, application rate, and soil characteristics, long-term studies are also needed to evaluate their environmental safety alongside their agronomic effectiveness (Tian et al., 2019).
From a practical perspective, native PSB consortia could be manufactured using well-established industrial fermentation technologies and formulated as seed coatings or other conventional delivery systems (Soumare et al., 2020). The use of native strains does not substantially increase production complexity, although it requires robust procedures for strain selection, quality control, and formulation stability to ensure reproducible field performance and adequate shelf-life (Bashan et al., 2014). The economic feasibility of this approach may be further improved by coupling microbial inoculants with recycled SnHA, which valorizes fish-processing by-products as a secondary P resource. However, large-scale adoption by farmers will ultimately depend on the consistency of crop responses under field conditions, cost competitiveness with conventional fertilization strategies, ease of application, and compatibility with existing crop management practices (French et al., 2021). In addition, commercialization of microbial inoculants requires compliance with national and international regulatory frameworks governing microbial biostimulants and biofertilizers, including accurate taxonomic identification of microbial strains, demonstration of product safety and efficacy, manufacturing quality standards, and formulation stability before market authorization (Rakhmatova et al., 2026; Termorshuizen et al., 2026). These aspects highlight the importance of validating native PSB consortia under multi-site field conditions to support both regulatory approval and farmer confidence in their agronomic performance (Čaušević et al., 2024; Mawarda et al., 2022).
Beyond their agronomic potential, the combined use of native PSB and recycled SnHAs contributes to the transition toward more sustainable P management. Recovering P from fish-processing by-products supports circular nutrient use by returning secondary P resources to agricultural production rather than disposing them as waste, thereby reducing waste generation, improving resource efficiency, and closing P cycles in agri-food systems (Elser and Bennett, 2011; Withers et al., 2015). At the same time, improving the biological availability of recycled Ca phosphates through PSB-mediated P mobilization may reduce reliance on conventional phosphate fertilizers derived from phosphate rock, a finite and geographically concentrated resource whose extraction is associated with increasing economic, geopolitical, and environmental concerns (Cordell et al., 2009; Elser and Bennett, 2011; Withers et al., 2015). By enhancing the utilization of recycled P sources, the combined PSB-SnHA strategy has the potential to improve P-use efficiency while decreasing the demand for primary phosphate resources (Santana et al., 2019; Jindo et al., 2023; Pilotto et al., 2025).
5 Conclusions
Overall, these findings highlight three key principles. First, SnHA represents a versatile recycled P source capable of supporting maize growth across contrasting soil conditions. Second, native PSB constitute a valuable biological resource for improving P acquisition, particularly in soils with low indigenous microbial abundance. Third, effective biofertilization strategies must be tailored to local soil conditions, as the benefits of microbial inoculation depend on the interaction between soil properties, resident microbial communities, and plant nutrient demand. Our results support a site-specific approach to biofertilization and provide a scientific basis for the development of integrated microbial-recycled P fertilization strategies aimed at improving P-use efficiency while promoting the valorization of fish-processing by-products within a circular economy framework. From a practical perspective, the combination of native PSB consortia with recycled phosphate nanofertilizers represents a promising approach to support a more sustainable crop production. although further field validation is required, our approach has the potential to contribute to the development of site-adapted biofertilization strategies that combine higher P-use efficiency with more circular and resource-efficient nutrient management. Future research should focus on long-term field validation, rhizosphere process characterization, and microbial community dynamics to further optimize integrated nano-biofertilization strategies and improve the sustainability of P management in agricultural systems.
Statements
Data availability statement
The original contributions presented in the study are publicly available. This data can be found here: NCBI GenBank, accession PZ558346 to PZ558402.
Author contributions
PQ: Data curation, Software, Methodology, Formal analysis, Investigation, Validation, Conceptualization, Writing – review & editing. FP: Writing – review & editing, Investigation, Formal analysis, Software, Validation, Data curation. SP: Writing – review & editing. LE: Supervision, Writing – review & editing, Funding acquisition. EP: Data curation, Visualization, Conceptualization, Validation, Methodology, Supervision, Funding acquisition, Project administration, Writing – original draft, Formal analysis, Resources, Writing – review & editing.
Funding
The author(s) declared that financial support was received for this work and/or its publication. This work was funded by the European Agricultural Fund for Rural Development 2004–2020 for Tuscany (Italy) mis. 16.2 MICROforAGRI - MICRorganismi ed idrOssiapatatite da sOttoprodotti della lavorazione del pesce come Risorsa di fosforo in AGRIcoltura sostenibile (CUP ARTEA 1074061 - CUP CIPE J53C23002020002) and by AGRITECH National Research Centre for Agricultural Technologies (CUP J53C22001610007). Piera Quattrocelli was supported by a PhD scholarship in Agrobiosciences (XXXVII cycle) from the Scuola Superiore Sant’Anna (SSSA, Pisa, Italy).
Conflict of interest
The authors declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
The author EP declared that they were an editorial board member of Frontiers at the time of submission. This had no impact on the peer review process and the final decision.
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Supplementary material
The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fagro.2026.1916426/full#supplementary-material
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Summary
Keywords
microbial biomass, microbial inocula, nanofertilizers, nutrient uptake, P gene expression, phosphorus use efficiency, plant-growth promoting bacteria
Citation
Quattrocelli P, Pompilii F, Pecchia S, Ercoli L and Pellegrino E (2026) Soil-dependent effects of climate-adapted phosphate-solubilizing bacteria and salmon-bone nanohydroxyapatite on early maize phosphorus acquisition under controlled conditions. Front. Agron. 8:1916426. doi: 10.3389/fagro.2026.1916426
Received
22 June 2026
Revised
11 August 2026
Accepted
17 August 2026
Published
14 September 2026
Volume
8 - 2026
Edited by
Botagoz Mutaliyeva, M.Auezov South Kazakhstan State University, Kazakhstan
Reviewed by
Maria Carelli, Council for Agricultural Research and Agricultural Economy Analysis | CREA, Italy
Zhadyrassyn Nurbekova, L. N. Gumilyov Eurasian National University, Kazakhstan
Updates
Copyright
© 2026 Quattrocelli, Pompilii, Pecchia, Ercoli and Pellegrino.
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*Correspondence: Elisa Pellegrino, elisa.pellegrino@santannapisa.it
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