The study was conducted at the Dafeng experimental base (113°22′E, 23°09′N) of the Guangdong Academy of Agricultural Sciences, Guangdong Province, southern China. The area is characterized by a subtropical monsoon climate with an average annual air temperature of 21.8°C and an average annual precipitation of 1,694 mm. Roots of the Wufengyou615 variety were collected in October 2019 during the heading stage of the rice plant.

To ensure the reliability of the results, five healthy rice plants at the tillering stage were randomly sampled from different locations, placed on ice in a cooler box, and promptly transported to the laboratory. The samples were refrigerated at 4°C for later use and processed within 24 h of collection.

One gram soil tightly adhering to the roots of five rice plants was separately dissolved in 100 mL of sterile deionized water in 250 mL Erlenmeyer flasks. The flasks were incubated on a thermostatic shaker for 30 min at 28°C and 180 rpm. The supernatant was serially diluted to concentrations ranging from 10⁻¹ to 10⁻⁷ with sterile deionized water and plated on 10 cm petri dishes containing potato dextrose agar (PDA) medium: 200 g of potato was cut into small pieces, boiled for 30 min with 1,000 mL of water, and then filtered. The filtrate was mixed with 20 g of glucose and 20 g of agar, and deionized water was added to 1,000 mL. The medium was sterilized at 121°C for 20 min, cooled, and stored for later use. The fungal discs were incubated at 28 ± 2°C for 5–7 days in an incubator. Fungal colonies were purified by repeatedly transferring a single hyphal tip to PDA agar medium.

The fungal isolates were identified on the basis of their morphological characteristics and genetic analysis. The pure isolates of Pyrenochaetopsis tabarestanensis obtained from the above isolation and purification process were transferred to oatmeal medium for fungal morphology identification. The universal ITS rRNA sequence of the fungal isolates was used for genetic identification. The primers used for PCR amplification were ITS1F (5′-CTTGGTCATTTAGAGGAAGTAA-3′) and ITS2R (5′-GCTGCGTTCTTCATCGATGC-3′) (Adams et al., 2013). DNA extraction of the isolates was conducted following the procedure specified by the manufacturers of the Fungi kit (Omega Bio-Tek, Inc., US). DNA quality was determined with a NanoDrop 2000 spectrophotometer (Thermo Scientific, USA). PCR amplification of the target sequence was performed as previously described (Qarni et al., 2021). Amplified fragments were checked and purified by using QIA QuickPCR purification kit (QIAGEN) and then sequenced at BGI Genomics Co., Ltd. (Shenzhen, China).

The nucleotide sequences so generated were compared using National Centre of Biotechnology Information (NCBI) BLAST method (https://blast.ncbi.nlm.nih.gov/Blast.cgi), and the ITS sequence showed 99.77% homology to Pyrenochaetopsis tabarestanensis. The sequences of the novel isolates were deposited in the NCBI GenBank database under the accession numbers PP658459 and PP658460. The newly identified fungal strains were named Pyrenochaetopsis tabarestanensis WFY-1 (PtWFY-1) and WFY-2 (PtWFY-2), and they were preserved at the Guangdong Microbial Culture Collection Center (GDMCC), Guangzhou, China, accession numbers GDMCC No. 61861 and GDMCC No. 61862. The phylogenetic tree was constructed by the neighbor-joining (NJ) method using MEGA X software.

The isolates PtWFY-1 and PtWFY-2 were preliminarily screened for their ability to solubilize insoluble phosphate sources [Ca₃(PO₄)₂ and calcium phytate] on Pikovskaya's (PVK) agar. One liter (1.0 L) of PVK agar comprised the following (g/L): 10.0 g of glucose, 0.30 g of NaCl, 0.30 g of KCl, 0.5 g of (NH₄)₂SO₄, 0.30 g of MgSO₄·7H₂O, 0.03 g of FeSO₄·7H₂O, 0.03 g of MnSO₄·H₂O, 0.03 g of Ca3(PO₄)₂/Al₃(PO₄)₂/FePO₄/Mg₃(PO₄)₂/phosphate rock powder/calcium phytate, 5.0 g of agar, and 18.0 g of agar in 1,000 mL of distilled water (pH 7.0–7.2) (Pikovskaya, 1948). The medium was autoclaved at 121°C for 20 min. Fungal mycelium plugs (5 mm³), cut from the edges of actively growing colonies, were placed on PVK agar for 5 days at 28°C. Sterile PDA plugs served as controls. Three replicates were tested for each of the PtWFY-1 and PtWFY-2 isolates. The formation of visible halo zones around the microbial colonies on the plates indicated the phosphate solubilization capability of the strains. The diameter of the halo zones around the colonies and the diameter of the colonies were measured after 5 days of incubation. The phosphate solubilization index was calculated according to Premono et al. (1996).

Phosphate solubilization activity testing was conducted in 50 mL centrifuge tubes containing 40 mL of PVK broth in vitro, which had the same composition as the PVK agar but without agar. The insoluble phosphate sources included Ca₃(PO₄)₂, Al₃(PO₄)₂, FePO₄, Mg₃(PO₄)₂, phosphate rock powder, and calcium phytate. The initial pH of the medium was adjusted to 7.0 before sterilization. Spore suspensions of each Pyrenochaetopsis tabarestanensis isolate were prepared according to Elias et al. (2016). Two percentage spore suspensions (10⁷ spores/mL) were inoculated into sterilized PVK broth. The controls consisted of 2% sterile distilled water in sterilized PVK broth. Three replicates were maintained for each test. The cultures were incubated on a rotary shaker at 28°C and 180 rpm for 7 days. The culture supernatant was aseptically collected daily from days 1 to 7. The amount of available soluble phosphorus released from the insoluble sources by the fungal strains was estimated via the molybdenum blue method at 700 nm (Ryan et al., 2001). The pH of the culture supernatant in the PVK broth was measured daily using a digital pH meter with a glass electrode (Jingci, Shanghai, Co., Ltd.).

The in vitro antibacterial activity of the PtWFY-1 and PtWFY-2 strains was also evaluated through plate confrontation assays against the plant pathogen Magnaporthe oryzae Guy 11, as described by Singh et al. (2013, 2014). The mediums of PtWFY-1 and PtWFY-2 served as negative controls. Three replicates were tested for each isolate. Strains demonstrating more than 50% inhibition of mycelial growth were considered promising antagonists. A total of 1.0 L of Prune agar (PA) medium consisted of 5.0 g lactose, 1.0 g yeast extract powder, 40 mL prune juice, 20 g agar, and 1,000 mL distilled water (Pikovskaya, 1948). The medium was autoclaved at 121°C for 20 min. Growth inhibition rate was calculated from mean values as: Inhibition rate (%) = (Magnaporthe oryzae colony diameter in Control – Magnaporthe oryzae colony diameter in treatment)/Magnaporthe oryzae colony diameter in Control × 100% (Elsharkawy et al., 2014).

For organic acid measurement, PtWFY-1 and PtWFY-2 mycelia and spores were scraped into PVK liquid medium containing calcium phosphate to create a fungal spore suspension of 10⁶ spores/mL. This suspension was cultured on a shaking bed at 28°C and 180 rpm for 5 days and then centrifuged for 10 min at 12,000 r min⁻¹ at 4°C. The supernatant was collected and stored at −80°C for further measurement of organic acids. The external standard method was used for the determination of organic acids. A total of 74 different organic acids were screened. Nine kinds of organic acids, oxalic acid, citric acid, tartaric acid, formic acid, malonic acid, acetic acid, maleic acid, aconitate, and propionic acid, were measured on a Thermo U3000 HPLC platform. The remaining organic acids were detected via MetWare on the AB Sciex QTRAP 6500 LC-MS/MS platform. Standards for the nine acids were prepared in ddH₂O and diluted to concentrations of 0.1, 0.2, 0.5, 0.8, 1, 5, 10, 15, 20, 50, 80, 100, 150, and 200 μg/mL. The remaining standards were prepared in methanol to 1 mg/mL and diluted to concentrations of 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1, 2, 5, 10, 20, 50, 100, 200, 500, 1,000, 5,000, and 10,000 ng/mL. Standard curves were drawn with the concentration of the external standard as the abscissa and the peak area as the ordinate. The concentration of each acid in the samples was calculated by substituting the integral peak areas into the linear equation of the standard curve. The types and standard curves of the standards are listed in Supplementary Table S3.

For phytohormone measurement, PtWFY-1 and PtWFY-2 spores were scraped into PDB liquid medium to create a fungal spore suspension of 10⁶ spores/mL. The formula of the PDB liquid medium was the same as that of the PDA medium without agar. The suspension was incubated on a shaker at 28°C and 180 rpm for 15 days and then centrifuged for 10 min at 4°C, 12,000 r min⁻¹. The supernatant was stored at −80°C until further use. The external standard method and internal standard correction were applied for the quantification of target phytohormones. A total of 88 kinds of phytohormones were screened. Standards were prepared in methanol at a concentration of 1 mg/mL and then diluted to concentrations of 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 50, 100, 200, and 500 ng/mL. For the measurement of L-tryptophan and salicylic acid 2-O-β-glucoside, the standard curve concentration range was adjusted to 0.2–10,000 ng/mL. Standard curves were drawn with the concentration ratio of the external standard to the internal standard as the abscissa and the peak area ratio as the ordinate. The types and standard curves of the standards are listed in Supplementary Table S4. Phytohormones were detected by MetWare (http://www.metware.cn/) based on the AB Sciex QTRAP 6500 LC-MS/MS platform. The concentration of each phytohormone in the samples was calculated by substituting the integral peak areas into the linear equation of the standard curve.

The effects of inoculating soils with Pyrenochaetopsis tabarestanensis strains on P availability, P uptake, and rice growth were assessed through pot experiments, with four replicates per treatment. Paddy soil samples containing 9 mg kg⁻¹ available P and with a pH of 6.0 were air-dried, ground, sieved (< 2.0 mm), and sterilized before the experiments. Three rice seeds (Yangdao6 variety) were sown individually in pots containing 300 g of soil, and 1.0 mL of Pyrenochaetopsis tabarestanensis inoculant (10⁶ spores/mL) was applied. Uninoculated seeds served as controls. The plants were cultivated under greenhouse conditions with an average temperature of 28°C, a relative humidity of 50–60%, and a 12 h light and 12 h dark photoperiod with natural lighting. The soil moisture was maintained at 60% of its maximum water-holding capacity (WHC) to prevent nutrient leaching and root damage. After 30 days, three rice seedlings were harvested to measure their dry biomass and P concentration. The soil samples were analyzed for total P by the molybdenum blue method and for available P by the Olsen-P extraction method (Pansu and Gautheyrou, 2006).

The data were presented as the means with standard errors from three replicates. Differences between treatments were evaluated via one-way analysis of variance (ANOVA) followed by Duncan's multiple range tests, which were performed using the Statistical Package for the Social Sciences (SPSS) for Windows version 22 (SPSS, Inc., Chicago, IL, United States). Statistical significance was set at P ≤ 0.05. Additionally, the production of organic acids and phytohormones by the fungal strains was analyzed by cluster and heatmap analyses via the ggplots 3.0.3 package in R software version 3.2.3. UV (unit variance scaling) scaling was applied to the concentrations of organic acids and phytohormones before plotting.

Two novel strains of Pyrenochaetopsis tabarestanensis were isolated from the rice rhizosphere. Both fungi presented similar morphologies and growth rates. After 14 days of cultivation, both colonies were velvety and slightly flocculent, with a grayish-white center. However, the color of PtWFY-1 was grayish-brown, whereas that of PtWFY-2 was gray-brown (Figures 1A, B, D, E). The diameter of both colonies reached 50–60 mm within 14 days of culture. Thick-walled spores, characteristic of Pyrenochaetopsis tabarestanensis, were observed under a microscope in both colonies (Figures 1C, F). The length of the amplified ITS rDNA sequence was 442 bp for PtWFY-1 and 450 bp for PtWFY-2, with high homology (99.77%) to Pyrenochaetopsis tabarestanensis. The sequences of the novel isolates were deposited in the NCBI GenBank database under the accession numbers PP658459 and PP658460. On the basis of morphological characteristics and genetic analysis, both PtWFY-1 and PtWFY-2 were identified as Pyrenochaetopsis tabarestanensis. Additionally, the sequences of PtWFY-1 and PtWFY-2 with another eleven nucleotide sequences retrieved from the NCBI were processed for the phylogenetic tree, and their sequences revealed 61% similarity to the ITS gene sequences of Pyrenochaetopsis tabarestanensis 1 NV-2016 UTHSC: DI16-193 (Figure 1G).

PtWFY-1 and PtWFY-2 demonstrated their potential for phosphate solubilization by forming clear zones on PVK agar plates containing Ca₃(PO₄)_2and calcium phytate (Supplementary Figure S1). The solubilization index (SI) of PtWFY-1 was 3.49 and 3.67 for Ca₃(PO₄)_2and calcium phytate, respectively, after 5 days of incubation (Supplementary Figures S1A, C), whereas PtWFY-2 presented SI values of 3.00 and 3.40 for Ca₃(PO₄)_2and calcium phytate, respectively (Supplementary Figures S1B, D).

PtWFY-1 and PtWFY-2 were able to dissolve Ca₃(PO₄)₂, Mg₃(PO₄)₂, phosphate rock powder, and calcium phytate in PVK broth but not Al₃(PO₄)₂ or FePO₄ (Figure 2). When Ca₃(PO₄)₂ was utilized as the P source, the soluble P concentration increased by 21.8% (day 3) for PtWFY-1 and by 43.2% (day 5) for PtWFY-2 relative to the uninoculated control (Figure 2A). Similarly, for Mg₃(PO₄)₂, the soluble P concentration increased on day 2 for PtWFY-1 (19.6%) and day 3 for PtWFY-2 (25.4%) relative to the uninoculated control (Figure 2B). With phosphate rock powder as the P source, the soluble P concentrations in the culture medium of PtWFY-1 and PtWFY-2 increased by 16.4% and 25.0%, respectively, on day 5 compared with those in the uninoculated control (Figure 2C). In the case of calcium phytate, the soluble P concentration in the culture medium of PtWFY-1 and PtWFY-2 increased 21.6% and 24.5%, respectively, on days 6 and 7 relative to that of the uninoculated control (Figure 2D).

The PtWFY-1 and PtWFY-2 strains demonstrated antibacterial activity against the plant pathogen Magnaporthe oryzae Guy 11 on PA agar plates. Initially, the diameters of the PtWFY-1 and PtWFY-2 strains were 3.2 cm and 7.6 cm, respectively, and the diameter of Magnaporthe oryzae Guy 11 was 6.38 cm (Figures 3A, B, D, E). Upon placement of the PtWFY-1 and PtWFY-2 strains at opposite positions, the diameter decreased to 2.57 and 2.88 cm, respectively (Figures 3C, F). The inhibition rates of the PtWFY-1 and PtWFY-2 strains were 59.7% and 54.9%, respectively. Compared with the PtWFY-2 strain, the PtWFY-1 strain exhibited a relatively greater inhibitory effect on Magnaporthe oryzae. This characteristic makes the PtWFY-1 and PtWFY-2 strains desirable candidates for antibacterial purposes.

Inoculation with the PtWFY-1 and PtWFY-2 strains in the soil treatments resulted in a significant increase in the growth of the rice seedlings, plant P content, and total soil P (Figure 4). Compared with the uninoculated control, PtWFY-1 and PtWFY-2 inoculation resulted in 24.5% and 14.5% increases in fresh weight, respectively (Figure 4B). The total P concentration of the rice plants significantly increased by 69.8% and 60.3% in response to PtWFY-1 and PtWFY-2 inoculation, respectively (Figure 4C). Additionally, the available P content in the soil significantly increased by 5.80% and 7.70% following PtWFY-1 and PtWFY-2 inoculation, respectively (Figure 4D).

There was a significant decline in the pH of the PtWFY-1 and PtWFY-2 culture medium, indicating the secretion of organic acids by these strains to dissolve insoluble phosphate (Supplementary Figure S2). Compared with those in the control treatment, the pH levels of Ca₃(PO₄)₂, Al₃(PO₄)₂, FePO₄, Mg₃(PO₄)₂, and phosphate rock powder broth inoculated with both strains decreased by 5.0%, 36.5%, 25.5%, 7.4%, and 17.4%, respectively, around day 7 (Supplementary Figures S2A–E). Additionally, the pH of the calcium phytate broth decreased by 33.9% and 23.2% after PtWFY-1 and PtWFY-2 inoculation, respectively, around day 7 (Supplementary Figure S2F).

A total of 26 types of organic acids were produced by the PtWFY-1 isolate, including oxoglutaric acid (1,900.03 mg L⁻¹), acetic acid (1,478.47 mg L⁻¹), pyruvic acid (685.90 mg L⁻¹), citric acid (579.11 mg L⁻¹), malonic acid (306.97 mg L⁻¹), formic acid (250.13 mg L⁻¹), lactic acid (239.22 mg L⁻¹), succinic acid (205.17 mg L⁻¹), aconitate (175.60 mg L⁻¹), tartaric acid (166.49 mg L⁻¹), citraconic acid (132.73 mg L⁻¹), 5-hydroxymethyl-2-furoic acid (129.47 mg L⁻¹), oxalic acid (111.00 mg L⁻¹), L-malic acid (92.81 mg L⁻¹), and other organic acids (Supplementary Table S1). PtWFY-2 broth also contained 26 types of organic acids, with 23 types being the same as those observed in PtWFY-1 broth, including oxoglutaric acid, pyruvic acid, succinic acid, 5-hydroxymethyl-2-furoic acid, citraconic acid, L-malic acid, 4-aminobutyric acid, fumaric acid, pantothenic acid, kynurenic acid, aminobenzoic acid, 4-hydroxybenzoic acid, 3-phenyllactic acid, hydroxyphenyllactic acid, 3-hydroxymethylglutaric acid, oxalic acid, citric acid, tartaric acid, formic acid, malonic acid, acetic acid, aconitate, and propionic acid (Supplementary Table S1). The composition of the PtWFY-2 broth included oxoglutaric acid (2,441.67 mg L⁻¹), acetic acid (1,519.18 mg L⁻¹), pyruvic acid (888.30 mg L⁻¹), citric acid (867.65 mg L⁻¹), formic acid (328.86 mg L⁻¹), malonic acid (289.56 mg L⁻¹), citraconic acid (199.87 mg L⁻¹), aconitate (194.69 mg L⁻¹), L-malic acid (154.17 mg L⁻¹), tartaric acid (144.38 mg L⁻¹), oxalic acid (121.25 mg L⁻¹), 4-aminobutyric acid (79.10 mg L⁻¹), succinic acid (59.90 mg L⁻¹), and other organic acids (Supplementary Table S1). The PtWFY-1 strain uniquely secreted adipic acid, maleic acid, and lactic acid, whereas the PtWFY-2 strain specifically produced shikimic acid, taurine, and neochlorogenic acid (Figure 5). Furthermore, oxoglutaric acid, acetic acid, pyruvic acid, and citric acid were identified as the primary organic acids produced by both the PtWFY-1 and PtWFY-2 isolates compared with the control (Supplementary Table S1).

In the PDB broth of the PtWFY-1 isolate, 28 types of phytohormones were identified, whereas 30 types were observed in the PDB broth of the PtWFY-2 isolate (Supplementary Table S2). Among these, 23 types of phytohormones, including L-tryptophan, 1-aminocyclopropanecarboxylic acid, Indole-3-acetic acid, 2-oxindole-3-acetic acid, Indole-3-lactic acid, N6-isopentenyladenosine, Indole-3-carboxylic acid, 3-indole acetamide, cis-zeatin riboside, 2-methylthio-N6-isopentenyladenosine, Gibberellin A3, dihydrozeatin ribonucleoside, trans-zeatin-O-glucoside, 3-indoleacrylic acid, trans-zeatin, Indole-3-acetyl glutamic acid, kinetin riboside, 2-methylthio-cis-zeatin riboside, trans-zeatin riboside, methyl indole-3-acetate, N6-benzyladenine-9-glucoside, cis-zeatin, and Indole-3-acetyl-L-valine methyl ester (Supplementary Table S2), were common to both strains. The predominant composition of the PtWFY-1 broth was 84.68% L-tryptophan (3,541.57 mg/L), 13.38% 1-aminocyclopropanecarboxylic acid (559.54 mg/L), and 1.35% indole-3-acetic acid (56.60 mg/L). Similarly, the primary composition of the PtWFY-2 broth was 83.46% L-tryptophan (3,769.21 mg/L), 15.24% 1-aminocyclopropanecarboxylic acid (688.35 mg/L), and 0.82% indole-3-acetic acid (37.00 mg/L) (Supplementary Table S2). The unique phytohormone types produced by the PtWFY-1 strain included N-(3-indolylacetyl)-L-phenylalanine, meta-topolin-9-glucoside, dihydrozeatin, and jasmonic acid, whereas gibberellin A20, methyl jasmonate, 6-benzyladenosine, indole-3-acetyl glycine, N6-isopentenyl-adenine-7-glucoside, 3-indolepropionic acid, and gibberellin A9 were specific to the PtWFY-2 strain (Figure 6). L-tryptophan was the principal phytohormone produced by both the PtWFY-1 and PtWFY-2 isolates (Supplementary Table S2).

Pyrenochaetopsis species are ubiquitous in nature and have been isolated from various ecosystems, such as freshwater, rice paddies, and ornamental boxwood (Papizadeh et al., 2017; Wang et al., 2019; Magana-Duenas et al., 2021; Špetík et al., 2021). However, Pyrenochaetopsis tabarestanensis strains have not previously been reported to be isolated from the rice rhizosphere (Papizadeh et al., 2017; Chen et al., 2020). In this study, we successfully isolated two P. tabarestanensis strains, and for the first time, these strains were obtained from the rice rhizosphere. Pyrenochaetopsis species often exhibit overlapping features, making it challenging to differentiate between genera (Papizadeh et al., 2017). Therefore, the identities of the PtWFY-1 and PtWFY-2 strains in this study were determined by a polyphasic approach, including morphological observations, sequencing of ITS regions, and phylogenetic analysis. The presence of thick-walled spores serves as the basis for identifying these two strains as P. tabarestanensis (Valenzuela-Lopez et al., 2018). Additionally, ITS rRNA sequence analysis revealed 99.77% similarity with known P. tabarestanensis strains. Phylogenetic analysis further supported their taxonomic classification. The isolation and identification of these P. tabarestanensis strains from the rice rhizosphere provide a foundation for further exploration of the functions of Pyrenochaetopsis species.

The PtWFY-1 and PtWFY-2 strains exhibited solubilization of various phosphate sources, including Ca₃(PO₄)₂, Mg₃(PO₄)₂, phosphate rock powder, and calcium phytate, while showing no solubilization of Al₃(PO₄)_2and FePO₄ (Figure 2), which was consistent with previous findings (Satyaprakash et al., 2017). Previous studies have indicated that Ca₃(PO₄)₂ starts to dissolve when the pH drops below 4.0, whereas FePO₄ remains insoluble until the pH decreases to 2.5 or lower (Jiang et al., 2020). In this study, we observed that Ca₃(PO₄)₂ began to dissolve when the pH was below 6.0, thereby expanding the pH range for the dissolution of insoluble Ca₃(PO₄)₂. Furthermore, the pH of the PVK broth containing Al₃(PO₄)_2and FePO₄ remained above 3.0 on day 7 (Supplementary Figures S2A, B), resulting in the insolubilization of Al₃(PO₄)_2and FePO₄. Since calcium predominates were the primary phosphate fertilizer in paddy soils, the solubility of Ca₃(PO₄)₂ indicated that the PtWFY-1 and PtWFY-2 strains were suitable phosphate-solubilizing fungal fertilizer candidates for application in paddy fields.

In this investigation, the PtWFY-1 and PtWFY-2 strains were found to possess greater phosphate solubilizing abilities toward Ca₃(PO₄)₂ than Mg₃(PO₄)₂, phosphate rock powder, and calcium phytate. As a result, these strains are promising candidates for increasing P solubilization, particularly in paddy soils where calcium predominates as the primary phosphate fertilizer. However, compared with the phosphate solubilization efficiency of Aspergillus and Penicillium species isolated from soil samples in previous studies, the PtWFY-1 and PtWFY-2 strains exhibited lower efficacy (Saxena et al., 2013; Xie et al., 2019). This finding cannot deny their value in paddy fields because of their other ecological functions, such as rice growth-promoting effects and resistance against Magnaporthe oryzae. In the meantime, this suggests that factors such as application conditions (e.g., concentration, interactions with other microorganisms) and environmental variables (including soil element content, biotic, and abiotic stresses) should be carefully considered when employing these fungal strains in practical applications.

In this study, LC-MS/MS and HPLC analysis results (Supplementary Tables S1, S2) demonstrated that the PtWFY-1 and PtWFY-2 strains secreted primarily oxoglutaric acid, acetic acid, citric acid, and pyruvic acid, with most of these acids being involved in the Krebs cycle (Li et al., 2016). These findings partially align with previous research indicating that oxalic acid and citric acid are the main organic acids produced by PSF (Islam et al., 2019). Citric acid is an intermediate product of the Krebs cycle, and its secretion has been described in many species, such as Aspergillus, Penicillium, and Eupenicillium (Adhikari and Pandey, 2019; de Oliveira Mendes et al., 2014). According to earlier studies, acetic acid generated by the PtWFY-1 and PtWFY-2 strains can enter the Krebs cycle via conversion to acetyl-CoA, similar to its secretion by Penicillium oxalicum (Yang et al., 2022). Oxoglutaric acid, an essential intermediate in the microbial Krebs cycle, acts as a critical link between intracellular carbon and nitrogen metabolism and is positioned after isocitric acid and before succinyl coenzyme A (Miller and Smith-Magowan, 1990). The synthesis of oxoglutaric acid by phosphorus-soluble fungi has rarely been reported, suggesting that its secretion is an innovative mechanism for dissolving phosphate substrates (Zhang et al., 2022). Pyruvic acid, a key metabolite within cells, connects the central metabolic pathways of glycolysis and the Krebs cycle, linking to various branching metabolic pathways (Roosterman and Cottrell, 2021). Its involvement in phosphate solubilization has been observed in species of Aspergillus, Penicillium, and Talaromyces, highlighting its importance as another major component of phosphorus dissolution (Brazhnikova et al., 2022; Zúñiga-Silgado, 2020). Our findings also revealed no apparent correlation between the soluble phosphorus content and total acid production (data not shown). This observation is consistent with Jiang et al. (2018) research, suggesting variability among PSM strains in terms of organic acid types and concentrations, which may contribute to diverse solubilization mechanisms.

Liquid medium experiments are commonly employed to assess the ability of microorganisms to solubilize P. However, these experiments may not directly mirror the impact of these microbes on P availability in soil and subsequent plant uptake (Kanse et al., 2015; López et al., 2020). Consequently, this study sought confirmatory evidence regarding the efficacy of P. tabarestanensis as an inoculant in soil and its influence on P availability and nutrition in rice seedlings through greenhouse experiments (Figure 4). Similar capabilities have been documented for other fungi, such as Westerdykella, Trichoderma, Rhizopus, Lasiodiplodia (Srivastava et al., 2012), Trichoderma harzianum (Chagas et al., 2017), and Penicillium bilaii (Geethalakshmi et al., 2017). In this study, since the inoculated plants did not receive additional soluble P, it is plausible that the organic acids produced by the two strains played a role in desorbing P from the soil minerals. Organic acids can increase P accessibility in soil by excreting protons to reduce the soil pH, by forming complexes with cations on the surface of soil minerals, or by blocking P absorption sites on soil particles (Behera et al., 2017; Lobo et al., 2019; López et al., 2020).

P is an essential element for rice growth and is directly involved in the metabolism of sugars and proteins. An increase in soil available P is helpful for the rice growth process and development (Rose et al., 2013). The improvement in rice growth parameters, such as plant dry weight, induced by the PtWFY-1 and PtWFY-2 strains also led to a notable increase in rice phosphorus absorption. In addition to P solubilization, phytohormone production has been proposed as a significant factor in promoting rice growth (Fitriatin et al., 2022). In this study, the PtWFY-1 and PtWFY-2 strains secreted primarily L-tryptophan, which emerged as the major phytohormone (Figure 6; Supplementary Table S2). Auxin, a well-known phytohormone consisting mainly of indole acetic acid (IAA), is derived predominantly from L-tryptophan through the indole-3-pyruvic acid pathway (Ahmad et al., 2005). Auxin plays a pivotal role in regulating various agronomic traits in rice, such as root architecture, tillering, inflorescence architecture, seed quality, and stress responses (Wang et al., 2018). Therefore, the secretion of L-tryptophan represents another significant trait of the PtWFY-1 and PtWFY-2 strains that contributes to the increase in rice phosphorus uptake.

The strains PtWFY-1 and PtWFY-2 displayed plant growth-promoting characteristics, including their ability to combat Magnaporthe oryzae. Magnaporthe oryzae is notably damaging, as it causes rice blast, a highly destructive disease affecting rice plants throughout their growth stages. It leads to annual losses ranging from 10 to 30% in various regions where rice is cultivated (Law et al., 2017). Similar antagonistic effects against M. oryzae have been observed in other fungi, such as Cladosporium, Penicillium, Talaromyces, and Aspergillus (Chaibub et al., 2020; Landum et al., 2016). The findings from this study suggest that P. tabarestanensis holds promise as a biocontrol agent against M. oryzae. However, further research is warranted to elucidate the precise mechanisms by which P. tabarestanensis inhibits M. oryzae.

P is a pressing concern in paddy fields because of its limited availability and effectiveness in agriculture (Bünemann, 2015; Dash and Dangar, 2017). The exploration of PSMs has emerged as an innovative approach to address this issue, offering significant benefits for both environmental sustainability and agricultural productivity (Mahanty et al., 2017). The discovery of the PtWFY-1 and PtWFY-2 strains, which demonstrate phosphate solubilization and enhance plant P uptake in soils, highlights the potential of P. tabarestanensis for biofertilization strategies in paddy soils lacking available phosphorus. Future research directions should include (i) experimenting with various P sources in soil; (ii) examining the effects on soil-available P and rice P uptake across different growth stages; (iii) investigating the detailed mechanisms involved in rice P uptake; and (iv) determining the optimal application conditions (e.g., concentration, interaction with other microorganisms) and considering environmental factors (e.g., soil element content, biotic and abiotic stresses).