A phialophora endophytic fungus pbx31 and application thereof

Abstract

This invention discloses an endophytic fungus of the genus *Pbx31* and its applications, belonging to the field of microbial technology. The endophytic fungus Pbx31 is classified and named... Phialocephala sp. Pbx31, deposited at the China Center for Type Culture Collection (CCTCC) under accession number M 20252563, was identified as a new species of the genus *Pyrtomyces* through morphological and molecular phylogenetic analysis. This endophytic fungus, when co-cultured with rice, can colonize the root tissues of rice. Its interaction with rice significantly promotes rice growth and enhances resistance to rice blast, as well as seedling resistance to leaf blast. Therefore, the endophytic fungus Pbx31 has significant potential for widespread application in promoting rice growth and improving rice blast control.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a type of bottle head mold ( Phialocephala The study focuses on the endophytic fungus Pbx31 and its application in promoting rice plant growth and enhancing rice resistance to rice blast fungus. Background Technology

[0002] Rice blast is caused by the pathogenic fungus *Pseudomonas oryzae* (rice blast fungus). Magnaporthe oryzae Rice blast, caused by pathogens, is one of the most destructive diseases limiting rice production. For a long time, the control of rice blast has mainly relied on the large-scale application of chemical fungicides or the breeding and promotion of resistant varieties. However, the long-term and extensive use of chemical pesticides has not only caused serious environmental pollution and agricultural product residues, but also accelerated the formation of drug-resistant pathogen populations. Furthermore, due to the high variability of physiological races of rice blast pathogens, resistant varieties often face the dilemma of "loss of resistance" after 3-5 years of widespread planting. Therefore, it is urgent to explore new, efficient, and environmentally friendly control strategies.

[0003] Plant endophytic fungi, as a natural biological resource, are considered one of the most promising technological pathways to replace chemical pesticides due to their green, environmentally friendly, and long-lasting effects. Plant endophytic fungi can colonize plant tissues for extended periods without causing obvious symptoms in the host, establishing a co-evolutionary symbiotic relationship with the host plant. They can directly promote host growth by secreting indoleacetic acid (IAA), siderophores, and engaging in phosphorus solubilization and nitrogen fixation. Furthermore, they can resist pathogen infection by producing antibacterial substances or inducing systemic resistance in the host.

[0004] In recent years, the research and application of endophytic fungi in the control of rice blast and the promotion of growth have been increasing. For example, patent document CN113215002A discloses an endophytic fungus of the family Megalocerosidae. Magnaporthaceae sp. M-B927, when co-cultured with germinating rice seeds, can colonize the roots of rice seedlings, achieving a 73.06% control effect against rice seedling leaf blast. Patent document CN121472051A discloses a strain of *Fusarium solani* (sp. M-B927). Falciphoriella LH002 sp., when co-cultured with rice and implanted at the roots of rice seedlings, can significantly increase the growth indicators of rice and reduce the leaf damage caused by rice blast fungus, achieving a control effect of 89.25% against rice blast.

[0005] Therefore, in-depth exploration of bacterial strains with dual functions of promoting growth and resisting disease, and their development into novel microbial agents, biopesticides, and plant growth promoters, can provide green solutions for disease control in various crops such as grains, vegetables, and fruit trees. This has profound theoretical value and practical significance for promoting agricultural development and ensuring food security.

[0006] Bottlehead mold ( ) Phialocephala As a typical dark-colored, septate endophytic fungus, *Botrytis cinerea* has been shown in studies to exhibit biocontrol activity against plant pathogenic fungi such as *Fusarium oxysporum* and *Rhizoctonia solani*. This activity can enhance host resistance and improve plant survival rates by increasing the activity of plant defense enzymes and accumulating osmotic regulators. Studies by Surono et al. have found that *Botrytis cinerea* strains... Phialocephala fortinii It can effectively inhibit crop diseases caused by *Fusarium oxysporum*, achieving significant disease mitigation effects (Surono, Narisawa, K. The inhibitory role of dark septateendophytic fungus). Phialocephala fortinii (Reference: against Fusarium disease on the growth of the Asparagus officinalis in organic source conditions. Biological Control, 2018, 120: 159-167.), but there are currently no research reports on the control efficacy of this genus of fungi against rice blast. Summary of the Invention

[0007] The purpose of this invention is to provide a new endophytic fungal strain with dual functions of promoting growth and resisting disease, and to develop it into bio-fertilizer and bio-pesticide, providing solutions for promoting rice growth and controlling rice blast disease.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: This invention isolated a strain belonging to the genus *Botrytis* from asparagus roots. Phialocephala The novel strain Pbx31 has the following main biological characteristics: after 7 days of growth on PDA plates at 25°C, the colony diameter is 5 cm; the central area of ​​the colony is dark gray to black with granular elevation, and the edge is light gray to white with a velvety appearance; under an optical microscope, the strain is observed to produce typical phialidiospores with dark septate hyphae. The ITS sequence of this endophytic fungus is shown in SEQ ID NO.1, the LSU sequence in SEQ ID NO.2, the RPB1 sequence in SEQ ID NO.3, the SSU sequence in SEQ ID NO.4, and the TEF sequence in SEQ ID NO.5.

[0009] Strain Pbx31 was identified as belonging to the genus *Bottlebill mold* through biological morphology and molecular tree analysis. Phialocephala A new species of () Phialocephala guttata sp. nov.), belonging to the kingdom Fungi ( Fungi Ascomycota ( Ascomycota ), Truffle class ( Leotiomycetes), Flexiomycetes ( Helotiales ), Floppy fungi family ( Mollisiaceae ), Bottlehead mold ( Phialocephala ).

[0010] Therefore, strain Pbx31 was classified and named Phialocephala sp. Pbx31 was deposited on November 17, 2025 at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: M20252563.

[0011] Furthermore, the culture conditions for the endophytic fungus strain Pbx31 were as follows: the endophytic fungus Pbx31 was inoculated into potato dextrose agar (PDA) medium and cultured in the dark at 22-25°C.

[0012] This invention provides a formulation of the endophytic fungus Pbx31, which is prepared into a liquid microbial fertilizer. The preparation method of the liquid microbial fertilizer includes: inoculating the endophytic fungus Pbx31 of the genus *Pyrtomyces* into PDB liquid culture medium, culturing until the culture medium is filled with mycelia, and obtaining the liquid microbial fertilizer.

[0013] The composition of the PDB liquid culture medium is: 5 g potato extract powder, 10 g peptone, and 15 g glucose per 1000 mL.

[0014] The liquid microbial fertilizer can be applied by mixing the liquid microbial fertilizer into the seedling substrate to obtain a mixed substrate, and then sowing the germinated crop seeds in the mixed substrate. During the seedling process, the endophytic fungus Pbx31 colonizes the roots of the seedlings.

[0015] This invention has found that colonizing the endophytic fungus Pbx31 into the roots of crops promotes crop growth and significantly improves the crop's resistance to rice blast fungus.

[0016] As a specific embodiment of the present invention, the present invention provides the application of the endophytic fungus Pbx31 of the genus *Pyrtomyces* in promoting rice growth, the application including: colonizing the endophytic fungus Pbx31 on the roots of rice.

[0017] This invention demonstrates that co-culturing the endophytic fungus Pbx31 with rice plants, allowing it to colonize the rice roots, significantly promotes rice plant growth. The interaction between the endophytic fungus Pbx31 and rice plants can significantly enhance the growth performance of rice plants.

[0018] Furthermore, the indicators for rice growth include plant height, stem width, and fresh weight.

[0019] Furthermore, the application includes: co-culturing rice seeds with the endophytic fungus Pbx31 after germination, allowing it to colonize the roots of rice seedlings to promote rice plant growth.

[0020] As another specific embodiment of the present invention, the present invention provides the application of the endophytic fungus Pbx31 of the genus *Pyrtomyces* in the prevention and control of rice blast disease, the application including: colonizing the endophytic fungus Pbx31 on the roots of rice.

[0021] This invention demonstrates that co-culturing the endophytic fungus Pbx31 with rice plants and allowing it to colonize the rice roots can significantly improve the rice plants' control over rice blast disease.

[0022] Furthermore, the rice blast is specifically leaf blast. Colonizing the endophytic fungus Pbx31 into the rice roots can reduce leaf damage caused by the rice blast fungus.

[0023] Furthermore, the application includes: co-culturing rice seeds with the endophytic fungus Pbx31 after germination, allowing it to colonize the roots of rice seedlings, thereby improving the resistance of rice seedlings to rice blast during the seedling stage.

[0024] Furthermore, this invention provides a tissue culture colonization method for the endophytic fungus Pbx31. Specifically, rice seeds are surface-sterilized and germinated at 22-25℃. After the seeds show white sprouts, they are transferred to 1 / 2 MS medium and simultaneously inoculated with Pbx31 mycelial cakes for co-culture.

[0025] Furthermore, the present invention provides another colonization form of the endophytic fungus Pbx31. Specifically, the endophytic fungus Pbx31 of the genus *Pyrtomyces* is inoculated into PDB liquid culture medium and cultured until the culture medium is covered with mycelia to obtain liquid microbial fertilizer; then the liquid microbial fertilizer is mixed with seedling substrate to obtain mixed substrate; then the germinated rice seeds are sown in the mixed substrate for co-cultivation.

[0026] Furthermore, the co-culture conditions are as follows: cultured at 22-25℃ until the three-leaf-one-heart stage, with 16 hours of light and 8 hours of dark culture per day.

[0027] The beneficial effects of this invention are as follows: This invention provides a novel endophytic fungus, Pbx31, belonging to the genus *Pyrtomyces*. Through biological morphology and molecular phylogenetic analysis, strain Pbx31 was identified as a new species within the genus *Pyrtomyces*. This endophytic fungus, when co-cultured with rice, can colonize the rice roots. Its interaction with rice significantly promotes rice growth and enhances rice resistance to rice blast, as well as strengthening seedling resistance to leaf blast. Therefore, the endophytic fungus Pbx31 has significant application value in promoting rice growth and improving rice blast control. Attached Figure Description

[0028] Figure 1 The colony morphology of strain Pbx31 is shown.

[0029] Figure 2 Microscopic images of hyphae of strain Pbx31.

[0030] Figure 3 Phylogenetic tree of strain Pbx31.

[0031] Figure 4 This image shows the colonization of strain Pbx31 in rice roots. A represents trypan blue staining results; B represents scanning electron microscopy results; the control represents rice not inoculated with strain Pbx31, and Pbx31 represents rice inoculated with strain Pbx31.

[0032] Figure 5 The effects of strain Pbx31 on rice growth are shown in Figure 1. A represents potted rice; B represents a single rice plant; C represents the effect of strain Pbx31 on rice plant height; D represents the effect of strain Pbx31 on rice stem width; and E represents the effect of strain Pbx31 on rice fresh weight. The bar chart represents the mean ± standard deviation (n=9). Significant differences were determined using Tukey's method with one-way ANOVA. * indicates a significant difference compared to the control group, * indicates p < 0.05, and ** indicates p < 0.01. In the figure, CK represents the uninoculated control group (strain Pbx31), and Pbx31 represents the inoculated strain Pbx31.

[0033] Figure 6 This study investigates the efficacy of strain Pbx31 in controlling rice blast leaf blast in potted rice. The left image shows rice leaves; the right image is a statistical chart of lesion incidence. The bar chart represents the mean ± standard deviation, n=9. Significant differences were determined using Tukey's method with one-way ANOVA for multiple comparisons. *** indicates p < 0.001. In the figure, CK represents the uninoculated control group (Pbx31), and Pbx31 represents the inoculated strain Pbx31.

[0034] Figure 7 This study investigates the efficacy of strain Pbx31 against leaf blast of tissue-cultured rice. The left image shows rice leaves; the right image is a statistical chart of lesion incidence. The bar chart represents the mean ± standard deviation, n=9. Significant differences were determined using Tukey's method with one-way ANOVA for multiple comparisons. ** indicates p < 0.01. In the figure, CK represents the uninoculated control strain Pbx31, and Pbx31 represents the inoculated strain Pbx31. Detailed Implementation

[0035] The present invention will be further described below with reference to specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the invention.

[0036] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0037] Example 1: Isolation, purification and identification of endophytic fungus strain Pbx31 I. Strains Isolation and Purification Strain Pbx31 was isolated from the roots of wild asparagus collected in Hangzhou. The specific method was as follows: the asparagus roots were continuously rinsed with tap water to carefully remove soil particles and appendages. Healthy root tissue was selected and surface disinfected. First, it was disinfected with 1% sodium hypochlorite for 20 min, then rinsed four times with sterile deionized water. The root tissue was then cut into 0.5 cm segments and placed in 2% malt extract agar (MEA, malt extract agar, OXOID; 50 mg / L chloramphenicol was added to inhibit the growth of endophytic bacteria) and incubated in the dark at 25°C. Hyphae were observed every 24 h. Endophytic fungal hyphae growing from the edges of the tissue cuts were carefully picked out with a toothpick, transferred to fresh PDA medium for purification, and the strain was recorded as Pbx31.

[0038] PDA medium: Each liter contains 20 g glucose, 200 g potato, and 15 g agar. Weigh the required amount of potato according to the volume of the medium to be prepared, boil it in water, mash and dissolve it, filter it, add glucose and agar, and autoclave at 121°C for 20 min.

[0039] II. Strain Identification 1. Morphological identification After isolation and purification, strain Pbx31 was inoculated onto PDA medium and cultured at 25°C for 7 days. A small amount of bacterial cells was picked up with a needle, prepared onto a glass slide, and observed and measured under a microscope.

[0040] The results are as follows Figure 1 and Figure 2 As shown, this strain forms circular colonies on PDA medium, with a dark gray to black, granular, raised central area and a light gray to white, fluffy edge; after 7 days of culture, the colony diameter is approximately 5 cm. Under an optical microscope, it was observed that this strain produces typical phialidiospores with dark, septate hyphae.

[0041] 2. Molecular identification (1) DNA extraction After culturing Pbx31 strain on PDA plates at 25°C for 7 days, mycelia were scraped from the plates with a toothpick and placed into a sterilized centrifuge tube containing 300 μL of extraction buffer (1 M KCl, 100 mM Tris HCl, 10 mM EDTA, pH 8.0). The mycelia were ground using an electric grinder and shaken vigorously for 2 min. The tube was then centrifuged at 10,000 rpm for 10 min. The supernatant was collected and transferred to a new centrifuge tube, and the precipitate was discarded. An equal volume of isopropanol (analytical grade) was added to the supernatant, and the mixture was gently inverted several times. The tube was then centrifuged at 12,000 rpm for 10 min to precipitate the nucleic acid. The supernatant was gently discarded, and the centrifuge tube containing the precipitate was inverted on absorbent paper to drain excess water. 300 μL of 70% ethanol was then added, and the mixture was gently inverted several times. The tube was then centrifuged at 12,000 rpm for 2 min. After 1 minute, gently pour off the supernatant and repeat the step once; invert the centrifuge tube onto absorbent paper to drain the water, and place it at 37°C for 15 minutes to allow the ethanol to evaporate completely; resuspend the precipitate with 50 μL ddH2O to obtain Pbx31 genomic DNA at a concentration of 30 ng / μL.

[0042] (2) PCR amplification of conserved fungal sequences PCR amplification was performed in a 20 μL reaction system containing: 1 μL each of forward and reverse primers, 10 μL of Green TaqMix enzyme, 7 μL of ddH2O, and 1 μL of template DNA.

[0043] The upstream primer ITS-F sequence is: 5'-TCCGTAGGTGAACCTGCGG-3'. The downstream primer ITS-R sequence is: 5'-TCCTCCGCTTATTGATATGC-3'.

[0044] The upstream primer LSU-F sequence is: 5'-GTACCCGCTGAACTTAAGC-3'. The downstream primer LSU-R sequence is: 5'-TCCTGAGGGAAACTTCG-3'.

[0045] The upstream primer RPB1-F sequence is: 5'-GARTGYCCDGGDCAYTTYGG-3'. The downstream primer RPB1-R sequence is: 5'-CCNGCDATNTCRTTRTCCATRTA-3'.

[0046] The upstream primer SSU-F sequence is: 5'-GTAGTCATATGCTTGTCTC-3'. The downstream primer SSU-R sequence is: 5'-CTTCCGTCAATTCCTTTAAG-3'.

[0047] The upstream primer TEF-F sequence is: 5'-GCYCCYGGHCAYCGTGAYTT-3'. The downstream primer TEF-R sequence is: 5'-ATGACACCRACRGCRACRGTYTGYAT-3'.

[0048] PCR amplification was performed on a Langqi MG96G PCR instrument. Reaction conditions: 94℃ pre-denaturation for 2 min; then 35 cycles including: 94℃ denaturation for 30 sec, 55℃ annealing for 40 sec, 72℃ extension for 1 min; and a final extension at 72℃ for 10 min.

[0049] (3) Gene sequencing and sequence analysis The purified and recovered target DNA fragments, after electrophoresis detection, were sent to the Hangzhou Sequencing Department of Youkang Biotechnology Co., Ltd. for sequencing. After rigorous verification, the sequencing results showed the following: ITS sequence (SEQ ID NO.1, 549 bp); LSU sequence (SEQ ID NO.2, 1125 bp); RPB1 sequence (SEQ ID NO.3, 782 bp); SSU sequence (SEQ ID NO.4, 1327 bp); and TEF sequence (SEQ ID NO.5, 981 bp).

[0050] On the NCBI website, the measured nucleotide sequences are used to search for and compare homologous or similar nucleotide sequences in the GenBank database using BLAST.

[0051] To determine the exact phylogenetic relationship of Pbx31, a search was conducted in NCBI for closely related genera (including...). Acephala , Phialocephala , Vibrissea , Chlorosplenium The relevant gene sequences of (etc.) were obtained, and a multi-gene phylogenetic tree (ML tree) was constructed, such as Figure 3 As shown. From the topological structure of the phylogenetic tree, strain Pbx31 and... Phialocephala bamuru , Phialocephala chinensis , Phialocephala hiberna The species clustered closely together (Bootstrap value > 90%), establishing its classification as belonging to the genus *Bootstrap*. Phialocephala ).

[0052] Based on morphological analysis, this strain forms circular colonies on PDA medium, with a dark gray to black, granular, raised central area and a light gray to white, fluffy periphery. Under a light microscope, the strain was observed to produce typical pyriform spores with dark, septate hyphae. Morphological characteristics and... Acephala The characteristics of genus Azotocinus differ significantly from those of genus Azotocinus. Phialocephala The characteristics of the genus are highly consistent.

[0053] Based on the topological structure of the multi-gene phylogenetic tree, BLAST sequence alignment, and conidial morphology and colony characteristics, strain Pbx31 was identified as belonging to the genus *Pyrtomyces*. Phialocephala A new species of () Phialocephala guttata sp. nov.), belonging to the kingdom Fungi ( Fungi Ascomycota ( Ascomycota ), (class Amygdaloides) Leotiomycetes ), Flexiomycetes ( Helotiales ), Flococcidae / Flococcidae ( Mollisiaceae, Vibrisseaceae ), Bottlehead mold ( Phialocephala Therefore, strain Pbx31 was classified and named... Phialocephala sp. Pbx31.

[0054] Strain Pbx31 was deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, on November 17, 2025, with accession number CCTCC NO: M 20252563. The collection was confirmed to be viable on November 24, 2025.

[0055] Example 2: Colonization of endophytic fungus Pbx31 in rice roots 1. Activation culture of bacterial strains The Pbx31 strain preserved on the slant was inoculated onto potato dextrose agar (PDA) solid medium for activation culture, and cultured in the dark at 25°C for 7 days for later use.

[0056] 2. Co-culture of Pbx31 strain with rice After removing the husks from rice seeds, shriveled, dry, and insect-infested grains were removed. Healthy rice seeds were disinfected with 75% alcohol for 5 minutes, followed by 1% NaClO for 20 minutes. The seeds were then rinsed five times with sterile water before use. The disinfected seeds were transferred to half MS medium using sterile forceps, sealed with sealing film, and incubated at 25°C for germination (16 h light / 8 h dark). After 3-4 days, the emerging seeds were inoculated into square dishes containing half MS + PDB medium, with 8-9 seeds per dish. For the treatment group, 6-7 Pbx31 mycelial discs (5 mm in diameter) were inoculated beneath the seeds; for the control group, sterile mycelial discs were inoculated into half MS + PDB medium. Each treatment was repeated in triplicate, incubated at 25°C under 16 h light / 8 h dark for 15-20 days until the three-leaf stage. In the subsequent results analysis, the control group consisted of rice seedlings that were not inoculated with the Pbx31 strain, while the treatment group consisted of rice seedlings that were inoculated with Pbx31 and co-cultured.

[0057] 1 / 2 MS + PDB medium: per 1000 mL contains 0.5 g MES, 2.2 g MS Salt, 5 g sucrose, 8 g agar powder, 17.5 g PDB, pH 5.7, autoclaved at 121℃ for 20 min.

[0058] 3. Observation of rice root colonization (1) Observation of rice root colonization using trypan blue staining method First, thoroughly rinse the rice roots, blot dry with absorbent paper, and cut them into 1cm lengths. Place the cuts in centrifuge tubes. Add 1.8 mL of 5% potassium hydroxide solution to the centrifuge tubes, mix well, and incubate at 90°C for 10 minutes. Discard the solution, rinse the roots with running water, and blot dry with absorbent paper. Add 1.8 mL of freshly prepared alkaline H₂O₂ solution, mix well, and incubate at room temperature for 15 minutes for bleaching. After 15 minutes, rinse the roots with running water and blot dry. Add 1.8 mL of 2% HCl solution, mix well, and incubate at room temperature for 5 minutes. Discard the acidification solution and blot dry with absorbent paper. Add 1.8 mL of 0.05% trypan blue staining solution, mix well, and incubate at 90°C for 60 minutes for staining. Discard the staining solution, rinse the roots with running water, and blot dry with absorbent paper. Add 1.8 mL of decolorizing solution, mix well, and let stand at room temperature for 3 h for decolorization; take a glass slide and place about 3 root segments on each slide; prepare 2 glass slides for each sample, add 50% glycerol mounting solution to mount the slides, observe under an inverted microscope, and take pictures to record the colonization of mycelium in the roots.

[0059] 0.05% trypan blue staining solution: 300 mg trypan blue, 200 mL single-distilled water, 200 mL lactic acid, 200 mL glycerol.

[0060] The control group consisted of rice that was not co-cultured with the strain, while the treatment group consisted of rice co-cultured with the strain. (2) Observation of fungal attachment on rice root surface using scanning electron microscopy Fresh rice roots were collected, and root segments approximately 3-5 mm in length were immersed in 2.5% glutaraldehyde fixative for 4-12 h. The root segments were then rinsed 3-4 times with 0.1 mol / L phosphate buffer, each rinse lasting 10-15 min. The ethanol was then replaced with isoamyl acetate. Subsequently, the samples were placed in a critical point desiccator and dried using liquid carbon dioxide. The dried rice root segments were then attached to the scanning electron microscope (SEM) stage using conductive double-sided tape. The samples were then placed in an ion sputtering apparatus, and platinum sputtering was performed on the sample surface under vacuum. Scanning observation was performed in the SEM sample chamber, and representative fields of view were photographed and recorded.

[0061] 4. Results Analysis Trypan blue staining results as follows Figure 4 As shown in Figure A, the root tissue of the control group was stained lighter, showing a uniform light blue color without obvious dark patches; the root tissue of the rice co-cultured with Pbx31 showed a large number of concentrated dark blue patches, indicating that there was a high density of fungal hyphae in the rice roots.

[0062] Scanning electron microscopy results as follows Figure 4 As shown in Figure B, the surface of the rice roots in the control group was relatively smooth and flat, with a dense and regular overall structure; the surface of the rice roots co-cultured with Pbx31 was covered with a large number of filamentous and net-like structures, with a robust and multi-branched hyphal network wrapping the roots, indicating that Pbx31 had attached to and infected the root surface.

[0063] Example 3: Endophytic fungus Pbx31 promotes rice growth 1. Activation culture of bacterial strains The Pbx31 strain preserved on the slant was inoculated onto potato dextrose agar (PDA) solid medium for activation culture, and cultured in the dark at 25°C for 7 days for later use.

[0064] 2. Preparation of Pbx31 liquid microbial fertilizer Endophytic fungal Pbx31 mycelial blocks (0.5 cm in diameter) cultured for 7 days were placed in PDB liquid fermentation medium (containing 5 g potato extract, 10 g peptone, and 15 g glucose per 1000 mL) for fermentation (25℃, 150 rpm, 7 days). Pbx31 liquid microbial fertilizer was obtained when the PDB liquid fermentation medium was fully colonized with mycelium.

[0065] 3. Co-culture of strain Pbx31 with rice Application of liquid microbial fertilizer: Mix the fermented liquid microbial fertilizer with the seedling substrate (liquid microbial fertilizer: substrate soil = 0.1 L : 3 L) and use it as the new substrate soil for rice pot cultivation. Soak the seeds of Zhejing 99 rice and then place them in a 37℃ dark constant temperature incubator to germinate for 2 days. Once the seeds show white sprouts, sow them evenly in the pots. Incubate at 22-25℃, 16 h light / 8 h dark for 14 days, observe and record the growth, and statistically analyze various growth indicators.

[0066] 4. Results Analysis like Figure 5 As shown, rice plants inoculated with strain Pbx31 under pot culture conditions exhibited significantly higher plant height, stem width, and fresh weight than the control group. Both the overall plant performance and individual plant performance indicated that the plants inoculated with strain Pbx31 showed better growth than the uninoculated group.

[0067] Example 4: Control efficacy of strain Pbx31 against rice blast fungus at the seedling stage. 1. Activation culture of bacterial strains The Pbx31 strain preserved on the slant was inoculated onto potato dextrose agar (PDA) solid medium for activation culture, and cultured in the dark at 25°C for 7 days for later use.

[0068] 2. Co-culture of Pbx31 strain with rice (1) Tissue culture After removing the husks from Co39 rice seeds, shriveled, dry, and insect-infested grains were removed. Healthy rice seeds were disinfected with 70% alcohol for 5 min, followed by 1% NaClO for 15 min, and rinsed 5 times with sterile water before use. The disinfected seeds were transferred to half MS medium using sterile forceps, sealed with sealing film, and germinated in a 25℃ incubator (16 h light / 8 h dark). After 3-4 days, the emerging seeds were inoculated into tissue culture flasks containing half MS medium, 10 seeds per flask, along with 3 Pbx31 mycelial cakes. The control group was inoculated with sterile PDA agar blocks. Each treatment had 3 replicates. The culturing was carried out at 25℃ for 15-20 days until the three-leaf stage, at which point the seeds were inoculated with rice blast fungus.

[0069] (2) Potted cultivation Preparation of Pbx31 liquid microbial fertilizer: Endophytic fungal Pbx31 mycelial blocks (0.5 cm in diameter) cultured for 7 days were placed in PDB liquid fermentation medium (containing 5 g potato starch, 10 g peptone, and 15 g glucose per 1000 mL) for fermentation (25℃, 150 rpm, 7 days). Pbx31 liquid microbial fertilizer was obtained when the PDB liquid fermentation medium was fully colonized with mycelium.

[0070] Application of liquid microbial fertilizer: Mix the fermented liquid microbial fertilizer with the seedling substrate (liquid microbial fertilizer: seedling substrate = 0.4 L: 40 L) and use it as new substrate soil for rice pot cultivation. Soak Co39 rice seeds and place them in a 37℃ dark constant temperature incubator to germinate for 2 days. When the seeds show white sprouts, sow them evenly in the pots. Incubate at 22-25℃, 16 h light / 8 h dark until the three-leaf stage, then inoculate with rice blast fungus.

[0071] 3. Spraying with conidia of rice blast fungus The rice blast fungus strain Guy11 was inoculated onto CM solid medium and cultured at 25°C for 12-14 days (12 h light / 12 h dark). Guy11 conidia were washed off with sterile water, filtered through three layers of filter paper, and the spore suspension was collected at a concentration of 2 × 10⁵ spores / mL. A 0.4% gelatin solution was prepared and mixed with an equal volume of the spore suspension.

[0072] CM medium (1 L): yeast extract (1 g), casein amino acids (1 g), glucose (10 g), potassium dihydrogen phosphate (1.52 g), sodium nitrate (6 g), peptone 140 (2 g), potassium chloride (0.52 g), magnesium sulfate heptahydrate (0.52 g), 0.1% (v / v) vitamin solution, 0.1% (v / v) trace element solution. Adjust the pH to 6.5 with sodium hydroxide, and add 15 g / L agar to the solid medium. Autoclave at 121°C for 15 min.

[0073] Spray the spore suspension evenly onto the leaves of tissue-cultured rice seedlings using a sprayer, using 1 mL per bottle. Place the tissue culture bottles in an incubator at 22℃ and incubate in the dark for 2 days. Then, incubate at 25℃ for 4-5 days with 16 h light / 8 h darkness, and count the lesion rate.

[0074] To further investigate the control efficacy of strain Pbx31 against rice blast fungus in the seedling stage, a spore suspension was evenly sprayed onto the leaves of potted rice seedlings using a sprayer and placed in an incubator at 22℃ in the dark for 2 days. Afterwards, the seedlings were incubated at 25℃ for 4-5 days under a 16-hour light / 8-hour dark cycle, and the lesion rate was recorded.

[0075] The formula for calculating the lesion rate is: Lesion area rate (%) = (lesion area / total leaf area) × 100%.

[0076] 4. Results Analysis like Figure 6 and Figure 7 As shown, regardless of whether it is under pot culture or tissue culture conditions, the control group had severe leaf blast disease, while the Pbx31 strain treatment group had milder disease and significant control effect.

[0077] The above description is merely a specific embodiment of the present invention, intended to enable those skilled in the art to understand the content of the present invention and implement it accordingly, and should not be construed as limiting the scope of protection of the present invention. All equivalent modifications or substitutions made based on the essence of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A species of bottlehead mold ( Phialocephala Endophytic fungus Pbx31, characterized in that, The endophytic fungus Pbx31 is classified as follows: Phialocephala sp. Pbx31, deposited at the China Center for Type Culture Collection, accession number CCTCC NO: M 20252563.

2. A liquid microbial fertilizer containing endophytic fungi Pbx31, characterized in that, The method for preparing the liquid microbial fertilizer includes: inoculating the endophytic fungus Pbx31 of the genus *Pyrtomyces* as described in claim 1 into PDB liquid culture medium, culturing until the culture medium is filled with mycelia, and obtaining the liquid microbial fertilizer.

3. The application of the endophytic fungus Pbx31 of the genus *Pyrtomyces* as described in claim 1 in promoting rice growth, characterized in that... The application includes colonizing the endophytic fungus Pbx31 into the roots of rice.

4. The application as described in claim 3, characterized in that, The indicators for rice growth include plant height, stem width, and fresh weight.

5. The application as described in claim 3, characterized in that, The application includes: co-culturing rice seeds with the endophytic fungus Pbx31 after germination, allowing it to colonize the roots of rice seedlings to promote rice plant growth.

6. The application of the endophytic fungus Pbx31 of the genus *Pyrtomyces* as described in claim 1 in the control of rice blast, characterized in that, The application includes colonizing the endophytic fungus Pbx31 into the roots of rice.

7. The application as described in claim 6, characterized in that, The rice blast disease mentioned is leaf blast.

8. The application as described in claim 6, characterized in that, The application includes: co-culturing rice seeds with the endophytic fungus Pbx31 after germination, allowing it to colonize the roots of rice seedlings, thereby improving the resistance of rice seedlings to rice blast during the seedling stage.

9. The application as described in claim 8, characterized in that, The co-culture conditions are as follows: cultured at 22-25℃ until the three-leaf-one-heart stage, with 16 hours of light and 8 hours of dark culture per day.

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