Wild rice (Oryza granulata) samples were collected from Xishuangbanna, Yunnan province, southwest of China, in November 2019. The isolation method of endophytic fungi referred to Yuan’s method (Yuan et al., 2010). Briefly, the healthy rice roots were gently rinsed with tap water, then immersed in 75% ethanol for 30 s and 1% sodium hypochlorite for 10 min. Subsequently, the roots were rinsed with sterile distilled water three times and cut into approximately 5 mm long segments. The segments were then transferred into a malt extract agar (MEA) medium (2% malt extract, 2% agar). The plates were incubated at 25°C in darkness. Fungal cultures were isolated and purified, saved on potato dextrose agar (PDA) slope (Yuan et al., 2010).

Fungal DNA was extracted by DNA extraction method (Chi et al., 2009). Six genes, internal transcribed spacer (ITS), large subunit (LSU) and small subunit (SSU) of ribosomal RNA genes, DNA replication licensing factor (MCM7), the largest subunit of RNA polymerase II (RPB1), and translation elongation factor 1-α (TEF1-α) genes, were amplified for identification (Zhang et al., 2011; Luo and Zhang, 2013). Primers are listed in Supplementary Table 1. PCR amplification refers to the method of Zhang et al. (2011). PCR products were sequenced by ABI3730 (Tsingke company, Beijing), and the sequencing results were compared with the BLAST sequence on the national center for biotechnology information (NCBI) website. All reference strain names used for phylogenetic analysis and isolate numbers, sources, hosts, and GenBank accession numbers were listed in Table 1 (Luo and Zhang, 2013; Luo et al., 2014). The partial sequences of strain P-B313 were submitted to the GenBank and obtained GenBank accession numbers (Table 1). Sequences of each gene were aligned with Clustal X 2.1 (Thompson et al., 1997) and manually corrected with Genedoc (Yuan et al., 2010). A six-gene dataset was generated by connecting the individual sequence alignments. JModel Test 2.1.7 (Posada, 2008) was used to calculate the best-fit nucleotide substitution models by computing likelihood scores and calculating AIC. Cryphonectria parasitica was chosen as the outgroup taxon. Bayesian inference (BI) trees were constructed in MrBayes v3.2.6 (Ronquist et al., 2012), using the optimal nucleotide substitution model. A total of 100,000 trees were produced. The latter 37,500 trees were selected to calculate the posterior probability values of each branch in the consensus tree. Maximum-likelihood (ML) analysis with the selected optimal model was executed in IQ-Tree (Nguyen et al., 2015). Branch support was evaluated by 1000 bootstraps replicates.

Strain P-B313 was cultured in 150 mL potato dextrose broth (PDB) at 25°C 150 rpm for 3 days. The mycelia and conidia were then collected and observed under a microscope (Carl Zeiss Inc., Germany).

P-B313 fungal plug (5 mm × 5 mm) was fixed into 2.5% glutaraldehyde solution at 4°C overnight. Then the samples were rinsed with 0.1 M phosphate buffer (pH = 7) three times (15 min each time), fixed in 1% OsO₄ for 2 h at 25°C, washed with phosphate buffer three times and dehydrated in a graded ethanol series. The samples were dried on HCP-2 critical point dryer (Hitachi, Japan) and coated. Finally, the samples were observed under SU-8010 scanning electron microscope (SEM) (Hitachi, Japan) (Liu X. H. et al., 2007).

The strain P-B313 was cultured in PDB for 3 days. And the conidia suspension with a concentration of 1 × 10⁶ spores/mL was collected. Agrobacterium tumefaciens strains containing PKD5-GFP vector with sulfonylureas resistance gene were mixed with P-B313 conidia suspension in equal volume (Lu et al., 2014). The transformants were screened on a defined complex medium (DCM) containing sulfonylurea (Dai et al., 2021). The fluorescence was detected by LSM880 confocal laser scanning microscope (Carl Zeiss Inc., Germany).

Rice seeds of blast-susceptible rice cultivar CO-39 (Oryza sativa) were surface-sterilized in 70% ethanol for 5 min, in 1.0% sodium hypochlorite solution for 20 min rinsed repeatedly using sterile water. Rice seeds were then planted in half-strength Murashige and Skoog medium (Murashige and Skoog, 1962) for 3 days, then transferred into tissue culture bottles (8 cm in width, 50 cm in height) containing half-strength Murashige and Skoog in which 10 seedlings were inoculated. We then inoculated three fresh mycelium plugs (diameter 8 mm, 7-day-old) in each tissue culture vessel. Blank agar blocks were used as control.

After 14 days of co-culture with GFP-tagged strain P-B313, the roots of the symbionts were collected and observed under an LSM880 confocal laser scanning microscope (Carl Zeiss Inc., Germany).

The fungus/plant DNA ratio (FPDR) was used to detect fungal infection in rice roots. The degree of fungal infection was determined by 2^{–Δ Ct} (Kenneth and Thomas, 2002), where ΔCt was the difference threshold value between strain P-B313 Tef-1α gene and rice Actin gene (Deshmukh et al., 2006; Deshmukh and Kogel, 2007). The specific primers were designed to be consistent with the tef-1α gene amplification primers. A total of 100 mg of root samples were collected at 5, 10, 15, and 20 days after inoculation (d.a.i.), respectively, according to Maciá-Vicente et al. (2009). The DNA was extracted using the nuclear plant genomic DNA kit (Tiangen, Beijing). The real-time PCR was performed in a total volume of 25 μL, including 10 ng of DNA, 12.5 μL of 2x SYBR Premix Ex Taq™ (Takara Bio Inc., Shiga, Japan), 1.25 μL of specific primer TEF1-F/R (or Actin-F/R for the rice Actin gene; Supplementary Table 1) and 10.25 μL of ddH₂O. Melting curve analysis was performed. Ct values were measured by using the Realplex software 2.2.10.84.

Strain P-B313 was cultured in 150 mL PDB at 25°C 150 rpm for 3 days. The mycelium suspension was then inoculated into sterilized barley grains (150 mL/200 g) and fermented at 25°C for 15 days. The germinated rice seeds were planted into pots containing fermented fungal fertilizer (75 g fertilizer, 30 seeds per pot). The controls were rice seeds inoculated with sterile barley grains. After 14 days of co-culture, the growth parameters, such as the chlorophyll content, shoot length, root length, shoot fresh weight, fresh root weight, and dry weight, were determined. A total of 30 rice plants were measured in the control and treatment groups, respectively. The length of the longest root was measured.

The pathogen Magnaporthe oryzae Guy11 was cultured in a complete medium (CM) 10 days. Then the spores were collected and prepared into suspension with a concentration of 5 × 10⁴ spores/mL. The rice leaves were sprayed with spore suspension and incubated in the dark at 22°C for 2 days, at 25°C for 4 days (light 16 h/darkness 8 h). The lesion area rate and disease index were calculated. The disease index was investigated according to the Standard Evaluation System for Rice (SES) of the International Rice Research Institute (IRRI 2002) (Supplementary Table 2). The disease equation is as follows: disease index = Σ(diseased level leaf number × representative value) / (total leaf number × heavy disease representative value) × 100% (Li et al., 2020).

The rice leaves and roots were collected separately and dried to constant weight under −80°C, then ground into dry powder. A total of 0.5 g of dry powder sample was placed in the digestion tank with 5 mL concentrated nitric acid and 1 mL hydrogen peroxide, shake well and let it stand for 1 min before digestion. After digestion, the acid was heated on an electric stove. And after cooling, use 2% nitric acid to make the volume 200 mL. Finally, phosphorus (P), potassium (K), magnesium (Mg), and iron (Fe) were determined by ICP-OES (IRIS Intrepid II XSP, Thermo, United States). The nitrogen (N) content is determined by Kjeldahl method (Stafilov et al., 2020).

After co-culture of strain P-B313 with rice for 14 days, rice plants were collected. Total rice RNA was extracted using TRIzol (Invitrogen, United States), followed by PrimeScript™ RT reagent Kit with gDNA Eraser (Perfect Real Time) (TaKaRa, Japan) kit for reverse transcription. The rice nutrition absorption-related genes OsPTR9, OsAMT3;2, OsMRS2-8, OsPT4, OsHAK16, OsIRO2, and OsYSL15 and rice disease resistance-related genes NAC, AOS, OsSAUR2, OsWRKY71, POX1, POX2, EL5, ERF4, PR1α, and PR1b were measured by quantitative analysis. The real-time PCR was performed in a total volume of 20 μL, including template cDNA (five times diluted) 1 μL, 10 μL of 2x SYBR Premix Ex Taq™ (Takara, Japan), 1 μL of specific primer (Supplementary Table 1) and 7 μL of ddH₂O. Reaction conditions: 95°C for 5 min, 40 cycles (95°C for 10 s, 60°C for 15 s), and the dissolution curve was set. The relative expression quantity of gene expression was calculated by 2^–ΔΔCt (Schmittgen and Livak, 2008).

Data were statistically analyzed by SPSS 16.0 version software (SPSS Inc., United States), expressed as mean ± standard deviation (SD). Graphs were created using GraphPad Prism 8.

The morphology of the colony, hyphae and conidia were observed. Strain P-B313 grew slowly on PDA medium, and the colony diameter reached 4 cm after growing at 25°C for 7 days. Aerial mycelia were white, prostrating on the medium surface. Mycelia were 0.5–4.0 μm in width, with a septum. Conidiophores were solitary, no branching. Conidia were elliptic or dumbbell-shaped, 11–15 × 3.5–6.5 μm (Figure 1).

We first blasted the similarity of the ITS sequence of the strain P-B313 on the NCBI website. The results showed that the identity between strain P-B313 and Pseudophialophora sp. (MK808146) was 99.4%. We conducted a phylogenetic analysis of strain P-B313 with the other related genus in Magnaporthaceae. It was found that there were 580 nucleotides in the ITS alignment, 869 in LSU, 1,032 in SSU, 926 in TEF1, 559 in MCM7, and 769 in RPB1. The 6-gene dataset involved 4,735 characters, including 925 parsimony informative, 722 variable and parsimony uninformative, and 3,088 constant. Calculated by jModel Test2.1.7, TN + F + R4 and TrN + I + G were selected as the optimal BI and ML analysis models. The two trees’ topological structures are similar using phylogenetic trees constructed by BI and ML methods. Only the BI tree is shown in Figure 2. Strain P-B313 belongs to the Pseudophialophora genus from the phylogenetic tree, but it exists in a separate clade independent of Pseudophialophora panicorum (Luo et al., 2014). In addition, the strain morphology and mycelium morphology of strain P-B313 and P. panicorum were quite different (Luo et al., 2014). Based on the molecular phylogeny and morphological, biological, and ecological characteristics, strain P-B313 was defined as a new species P. oryzae sp. nov (Collection Number: CCTCC M 2021504).

After five generations, intense green fluorescence was found to be uniformly distributed in the hyphae and conidiophores (Figure 3). The GFP-expressed transformant was selected as a candidate for further root inoculation.

The colonization pattern was monitored using GFP-labeled P. oryzae. Transversely, the fungus entered the root epidermis and then invaded the inner cortical layer, finally colonized in the inner cortical layer. No hyphae approached the central part of the roots. Concomitantly, abundant hyphae preferred to colonize in the epidermis and outer cortex (Figure 4A).

The FPDR was measured simultaneously to assess fungal growth and the respective plant response. It was shown that an early moderate increase in the FPDR from 1.20 ± 0.18 to 4.90 ± 2.43 occurred within 10 d.a.i., followed by a significant increase to 22.85 ± 9.51 at 20 d.a.i. (Figure 4B).

Pseudophialophora oryzae and rice were co-cultivated to investigate whether P. oryzae promotes rice growth. It was founded that the P. oryzae inoculated rice seedlings grew better and stronger than the control plants (Figures 5A,B), exhibiting higher chlorophyll content, shoot height, root length, fresh shoot weight, fresh root weight, and plant dry weight by 24.10, 35.32,19.35, 90.00, 33.3, and 79.17%, respectively (Figures 5C–H). These results indicated that P. oryzae possessed a positive capacity for plant growth.

We then investigated whether P. oryzae confers resistance to rice against rice blast under both plate and pot conditions. It was shown that the disease of the control rice plants grown in plates was serious, forming large circular or oval brown spots, disease spots densely covered (Figure 6A). The lesion area rate was 36.23%, and the disease index was 80.95% (Figures 6B,C). In contrast, the disease of rice plants inoculated with P. oryzae was relatively mild (Figure 6A), with a 9.91% lesion area rate (Figure 6B). The leaf area of the disease spot was small, accompanied by a few necrotic spots, and the disease index was only 25.92% (Figure 6C). The control effect of P. oryzae on rice blast reached 72.65%. Similarly, the disease resistance tests for potted plants were consistent with those for plates (Figure 6D). The lesion area rate of control and treatment was 53.13 and 12.95% (Figure 6E), respectively, and the disease index was 91.54and 28.04% (Figure 6F). The control effect of P. oryzae on rice blast reached 75.63% in pots. In conclusion, root colonization of P. oryzae can induce systemic disease resistance of hosts and has a positive control effect on rice blast.

Through the analysis of the nutrient element contents in the shoots and roots of rice plants, it was found that after inoculation with P. oryzae, the contents of N and K in the shoot tissues of rice plants increased significantly, which increased by 15.28 and 3.88% compared with the control group, respectively (Figure 7A). There was no significant change in P, Mg, and Fe content. Similarly, the contents of elements such as N, K, and Mg in the roots of the treatment group also increased significantly, increasing by 12.35, 3.29, and 0.36%, respectively (Figure 7B). There was no significant change in P and Fe content. Therefore, the root colonization of P. oryzae can effectively promote the absorption of nutrient elements in rice roots and increase the content of nutrient elements in the tissues.

We analyzed the expression levels of N, P, K, Fe, Mg, and other key genes for nutrient absorption and resistance-related genes. The results showed that the root colonization of P. oryzae significantly up-regulated the expression of peptide transporter OsPTR9 and potassium transporter OsHAK16, which were 7.28 ± 0.84 times and 2.57 ± 0.80 times higher than that of the control group, respectively. Genes such as OsAMT3;2 and OsMRS2-8 were significantly down-regulated. It can be seen that after P. oryzae infects and colonizes rice roots, it can significantly up-regulate the expression of genes related to N and K element absorption, thereby promoting nutrient element absorption (Table 2).

In addition, we found that the root colonization of P. oryzae significantly up-regulated the expression of NAC, OsSAUR2, OsWRKY71, EL5, and PR1α genes, which were 3.04 ± 0.72, 10.37 ± 0.34, 1.98 ± 0.13, 2.10 ± 0.35, and 1.46 ± 0.17 times of the control group, respectively. Compared with the control group, AOS, POX2, and PR1b were significantly down-regulated by 0.36 ± 0.05, 0.39 ± 0.24, and 0.38 ± 0.16 times. However, the expression levels of POX1 and ERF4 were not significantly changed. In conclusion, P. oryzae can induce up-regulated expression of some genes representing plant defense response and improve the host systemic disease resistance (Table 3).