An endophytic fungus of the genus *M. spp.* Mskw1 and its applications

Abstract

This invention discloses an endophytic fungus of the genus *M. spp.*, ... and its applications, belonging to the field of microbial technology. The endophytic fungus *M. spp.* is classified and named... Mortierella sp. Mskw1, deposited at the China Center for Type Culture Collection (CCTCC) under accession number M20252566, demonstrates that co-culturing the endophytic fungus Mskw1 with rice allows it to colonize the rice roots and significantly promotes rice growth. It also enhances rice's resistance to rice blast and its tolerance to salt stress, with particularly pronounced effects during the seedling stage. Therefore, the endophytic fungus Mskw1 has significant potential for widespread application in ensuring plant health and promoting rice seedling growth.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a species of *Morchella* (…). Mortierella Endophytic fungus Mskw1 and its application in promoting rice growth, enhancing rice's resistance to rice blast fungus, and improving rice's tolerance to salt stress. Background Technology

[0002] Rice (Oryza sativa L.) is an annual aquatic herbaceous plant belonging to the Poaceae family and is one of the world's most important food crops. During its growth, it is subject to various abiotic stresses, such as drought, salinity, and extreme temperatures, which severely affect its growth and yield. For example, high salinity can negatively impact rice growth, leading to root damage and impaired nutrient absorption. These environmental stresses severely inhibit plant growth, affecting crop yield and quality.

[0003] In addition, pathogens in the environment also pose a serious threat to plants, among which rice blast fungus ( Magnaporthe oryzae This disease can cause rice blast, leading to lesions on leaves, nodes, and panicles. In severe cases, it can cause significant yield reduction or even total crop failure. The disease can occur at multiple growth stages of rice, from seedling to heading, infecting leaves, internodes, and panicle necks. Mild cases result in yield losses of 10%-30%, while severe cases lead to complete crop failure. Currently, production mainly relies on chemical fungicides and resistant varieties for control. However, the use of chemical pesticides not only increases production costs but also easily leads to pesticide resistance in pathogens with long-term use, and poses environmental residue risks. The physiological races of rice blast fungus mutate rapidly, causing resistant varieties to easily become ineffective.

[0004] Endophytic fungi, as a class of beneficial microorganisms to plants, are receiving increasing attention for their role in enhancing plant resistance to abiotic stresses (such as salinity stress) and biotic stresses (such as pathogen infection). These fungi are microorganisms that colonize healthy plant tissues during part or all of their life cycle without causing obvious symptoms in the host. Through long-term evolution, they have formed complex mutualistic symbiotic relationships with their host plants, promoting plant growth and enhancing their adaptability to adversity through various mechanisms.

[0005] For example, in terms of alleviating salt and alkali stress, studies have shown that Trichoderma ( Trichoderma koningii By releasing volatile organic compounds, it significantly reduces the accumulation of hydrogen peroxide and cell necrosis in plants under salt stress, thereby enhancing their antioxidant capacity.

[0006] In resisting the damage of rice blast fungus, endophytic fungi can enhance the plant's resistance to pathogen infection through various mechanisms, such as producing antimicrobial active metabolites, competing for ecological niches with pathogens, secreting cell wall degrading enzymes (such as chitinase and cellulase) to directly parasitize or destroy pathogen hyphae (i.e., hyperparasitism), and inducing host plants to develop systemic acquired resistance (Wang Qi et al. Resistance mechanisms of plant endophytic fungi to biological stress and their applications. Soil and Crops, 2026, 15(2): 282-296).

[0007] Given the broad application potential of endophytic fungi in promoting plant growth under multiple stresses, exploring a richer resource library of endophytic fungal strains and utilizing their interactions with crops to synergistically enhance crop resistance to complex stresses such as abiotic stresses like salinity and alkali soil, as well as biotic stresses like rice blast, is of great significance for ensuring food security and promoting green agriculture and sustainable land use.

[0008] genus *Morchella* Mortierella (This is a class of endophytic fungi that can exert growth-promoting, disease-resistant, and soil-improving functions through multiple pathways, including phosphorus solubilization, secretion of plant growth hormones, improvement of the rhizosphere microenvironment, and induction of plant defense responses. Among them, *Morchella alpina* (…) Mortierella alpina As a typical representative strain of the genus *Morchella*, previous studies have focused primarily on its lipid synthesis characteristics. Recent research has confirmed that some endophytic strains of this species can colonize plant roots, enhancing host resistance and inhibiting soil-borne pathogen infection, demonstrating good potential for biocontrol and growth promotion. However, currently reported alpine *Morchella* strains are mostly isolated from medicinal and economic crops, with functional verification concentrated on soil-borne disease control and conventional growth promotion effects in specialty crops. For rice, a major cultivated crop, alpine *Morchella* strains capable of simultaneously promoting plant growth, improving salt stress tolerance, and enhancing disease resistance are extremely scarce. Summary of the Invention

[0009] The purpose of this invention is to provide an endophytic fungal strain that can stably colonize rice roots and has significant growth-promoting, salt-tolerant, and disease-resistant activities, in order to solve the problems of limited rice growth and infection by rice blast fungus in saline-alkali soils.

[0010] To achieve the above objectives, the present invention adopts the following technical solution: This invention isolated a strain of the genus *Morchella* from *Pteris vittata* plants in Yinchuan. MortierellaThe endophytic fungal strain Mskw1 exhibits the following main biological characteristics: After 5 days of cultivation on PDA plates at 25°C, colonies grow rapidly, reaching a diameter of 7 cm. In the early stages of cultivation, aerial hyphae are weakly developed, with the hyphae mostly creeping and adhering to the culture medium surface. The colonies are white to milky white, with a raised, thick center and a clearly defined multi-layered concentric ring structure on the surface. As the cultivation time increases, the colony morphology shows significant differentiation, with the overall texture gradually changing from an adhering state to a fluffy to cottony texture. Numerous spores are observed in the central region of the colony. The hyphae are well-developed, with tubular hyphae interwoven into a wavy, irregularly branched network; the width of ordinary hyphae is 38 μm. Intercalary or lateral chlamydospores are produced, ranging from nearly spherical to elliptical, with some chlamydospores arranged in a beaded pattern. The spore diameter is 10-20 μm, with tuberculate protuberances on the surface. The hyphal surface is rough, and granular inclusions are visible inside.

[0011] The ITS sequence of the endophytic fungus Mskw1 is shown in SEQ ID NO.1. Phylogenetic tree construction confirmed that this strain belongs to the kingdom Fungi (…). Fungi ), Mucor phylum ( Mucoromycota ), Moniliformes ( Mortierellomycetes ), Moniliales ( Mortierellales ), Morphozoaceae ( Mortierellaceae ), genus *Morchella* ( Mortierella ), Alpine spores ( Mortierella alpina ).

[0012] Therefore, the endophytic fungus Mskw1 was classified and named Mortierella sp. Mskw1 was deposited on November 17, 2025 at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: M20252566.

[0013] Furthermore, the culture conditions for the endophytic fungus Mskw1 strain were as follows: the endophytic fungus Mskw1 was inoculated into potato dextrose agar (PDA) medium and cultured in the dark at 22-25°C.

[0014] As a specific embodiment of the present invention, the present invention provides a formulation of the endophytic fungus Mskw1, which is prepared into a liquid microbial fertilizer. Specifically, the preparation method of the liquid microbial fertilizer includes: inoculating the endophytic fungus Mskw1 of the genus *Morchella* into PDB medium, culturing until the medium is filled with mycelia, and obtaining the liquid microbial fertilizer.

[0015] As a specific embodiment of the present invention, the present invention provides another formulation of the endophytic fungus Mskw1, which is prepared into a solid microbial fertilizer. Specifically, the preparation method of the solid microbial fertilizer includes: inoculating the endophytic fungus Mskw1 of the genus *Morchella* into PDB medium to obtain a fermentation broth, then inoculating the fermentation broth onto sterile barley grains, and culturing in the dark until the mycelium grows and covers the barley grains, thereby obtaining Mskw1 solid microbial fertilizer.

[0016] The composition of the PDB liquid culture medium is: 5 g potato extract powder, 10 g peptone, and 15 g glucose per 1000 mL.

[0017] The application method of the liquid or solid microbial fertilizer is as follows: the liquid or solid microbial fertilizer is mixed into the seedling substrate to obtain a mixed substrate, and then the germinated crop seeds are sown in the mixed substrate. During the seedling process, the endophytic fungus Mskw1 colonizes the roots of the seedlings.

[0018] Furthermore, the mixing ratio of liquid microbial fertilizer to seedling substrate is 0.1 L: 3 L; the mixing ratio of solid microbial fertilizer to seedling substrate is 70 g: 800 g.

[0019] This invention has found that colonizing the endophytic fungus Mskw1 into the roots of rice significantly promotes rice growth and enhances the resistance of rice seedlings to rice pathogens. The study also found that colonizing the endophytic fungus Mskw1 into the roots of rice can significantly promote the growth of rice seedlings in salt-stressed soil and enhance their tolerance to salt stress.

[0020] Therefore, the present invention provides the application of the endophytic fungus Mskw1 of the genus *Morchella* in promoting rice growth and / or preventing rice blast and / or improving rice's tolerance to salt stress.

[0021] Furthermore, the application includes: co-culturing the endophytic fungus Mskw1 of the genus Monosporium with rice plants, so that it colonizes the roots of the rice plants.

[0022] Furthermore, the application includes: co-culturing rice seeds with the endophytic fungus Mskw1 of the genus *Morchella* after germination, allowing it to colonize the roots of rice seedlings, in order to promote rice growth and / or prevent rice blast and / or improve rice's tolerance to salt stress.

[0023] Specifically, the indicators for rice growth include: stem width, chlorophyll content, and fresh weight. This invention demonstrates that the interaction between the endophytic fungus Mskw1 and rice can significantly improve the growth performance of rice plants.

[0024] The rice blast disease mentioned is leaf blast. This invention demonstrates that colonizing the endophytic fungus Mskw1 into the roots of rice can reduce leaf damage caused by rice blast fungus.

[0025] Salt stress refers to the adverse effects of excessively high soluble salt content in the soil on the growth and development of rice. This invention demonstrates that the interaction between the endophytic fungus Mskw1 and rice can significantly promote the growth of rice seedlings in salt-stressed soil and enhance their tolerance to salt stress.

[0026] As a specific embodiment of the present invention, the present invention provides a colonization method of the endophytic fungus Mskw1. Specifically, rice seeds are surface disinfected and germinated at 22-25℃. After the seeds show white sprouts, they are transferred to 1 / 2 MS medium and simultaneously inoculated with a mycelial cake of the endophytic fungus Mskw1 for co-culture.

[0027] As a specific embodiment of the present invention, the present invention provides another colonization method for the endophytic fungus Mskw1. Specifically, the endophytic fungus Mskw1 of the genus Evodia is prepared into liquid or solid microbial fertilizer, and then mixed into a seedling substrate to obtain a mixed substrate. Then, the germinated rice seeds are sown in the mixed substrate for co-cultivation.

[0028] 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.

[0029] The beneficial effects of this invention are as follows: This invention provides a new strain of the genus *Morchella* ( Mortierella The endophytic fungus Mskw1, when co-cultured with rice, can colonize the rice roots and significantly promote rice growth. It also enhances rice's resistance to rice blast and tolerance to salt stress, with particularly noticeable effects during the seedling stage. Therefore, the endophytic fungus Mskw1 has significant potential for widespread application in ensuring plant health and promoting rice seedling growth. Attached Figure Description

[0030] Figure 1 The colony morphology of strain Mskw1 is shown.

[0031] Figure 2 The morphology of the Mskw1 strain under a microscope.

[0032] Figure 3 Phylogenetic tree of strain Mskw1.

[0033] Figure 4 This image shows the colonization of strain Mskw1 in rice roots. A represents trypan blue staining results; B represents scanning electron microscopy results; the control is uninoculated rice with strain Mskw1, and Mskw1 represents the inoculated strain Mskw1.

[0034] Figure 5Images show the growth-promoting effect of the Mskw1 strain in co-culture with rice. A represents the overall potted plant, B represents a single plant, CK represents the control without inoculation, and Mskw1 represents the inoculated strain Mskw1.

[0035] Figure 6 for Figure 5 Statistical results of stem width of medium-sized rice plants. The bar chart represents the mean ± standard deviation, n=9; significant differences were determined using Tukey's method of multiple comparisons in a one-way ANOVA: *** indicates a significant difference compared with the control group, P<0.001; where CK represents the uninoculated control and Mskw1 represents the inoculated strain Mskw1.

[0036] Figure 7 This study investigated the efficacy of strain Mskw1 against rice blast leaf blast in potted rice. Image A shows rice leaves, and image B shows the lesion rate. Bar charts represent mean ± standard deviation, n=9. Significant differences were determined using Tukey's method with one-way ANOVA: *** indicates significant difference (P < 0.001). CK represents the uninoculated control, and Mskw1 represents the inoculated strain Mskw1.

[0037] Figure 8 This study investigates the efficacy of strain Mskw1 against leaf blast in tissue-cultured rice. Image A shows rice leaves, and image B shows the lesion rate. Bar charts represent mean ± standard deviation, n=9. Significant differences were determined using Tukey's method with multiple comparisons and one-way ANOVA: ** indicates significant difference (P < 0.01). CK represents the uninoculated control, and Mskw1 represents the inoculated strain Mskw1.

[0038] Figure 9 Salt tolerance test of strain Mskw1 on plate.

[0039] Figure 10 Images show the effect of strain Mskw1 on salt tolerance in rice plants. A represents the overall potted plant, B represents a single plant; CK represents the uninoculated control, Mskw1 represents the inoculated strain Mskw1, CK+NaCl represents the control group under salt stress, and Mskw1+NaCl represents the experimental group inoculated with strain Mskw1 under salt stress.

[0040] Figure 11 for Figure 10Statistical results of chlorophyll content and fresh weight of rice seedlings. A represents chlorophyll content, B represents fresh weight, and the bar chart represents the mean ± standard deviation, n=9. Significant differences were determined using Tukey's method with multiple comparisons in a one-way ANOVA: *** represents P < 0.001, **** represents P < 0.0001; CK represents the uninoculated control, Mskw1 represents the inoculated strain Mskw1, CK+NaCl represents the control group under salt stress, and Mskw1+NaCl represents the experimental group inoculated with strain Mskw1 under salt stress. Detailed Implementation

[0041] 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.

[0042] 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.

[0043] The culture media involved in the following examples: MEA medium: 20 g malt extract and 15 g agar powder per liter, autoclaved at 121°C for 20 min.

[0044] Potato glucose agar (PDA) medium: 20 g glucose, 200 g potato, and 15 g agar per liter. Weigh the required amount of potato according to the volume of the medium to be prepared, boil in water, mash and dissolve, filter, add glucose and agar, and autoclave at 121°C for 20 min.

[0045] PDB liquid fermentation medium: 5 g potato extract powder, 10 g peptone, and 15 g glucose per liter, autoclaved at 121°C for 20 min.

[0046] Example 1: Isolation, purification and identification of endophytic fungus strain Mskw1 I. Strains Isolation and Purification (1) Gently rinse the root zone of the fern plant collected from Yinchuan with water, soak it in 75% ethanol for 10 min, and then rinse it twice with sterile water. (2) Soak in 1.5% sodium hypochlorite for 3-5 minutes, wash with sterile distilled water 3-5 times, and cut the plant roots into root segments about 5 mm long; (3) The root segments were transferred to MEA medium and cultured in the dark at 25°C; (4) The fungi that grow out are promptly transferred to PDA medium. After several generations of inoculation, purified fungi are obtained and numbered Mskw1. (5) The purified fungi were preserved on PDA slant and sealed with liquid paraffin.

[0047] II. Strain Identification 1. Morphological identification After isolation and purification, strain Mskw1 was inoculated onto PDA medium and cultured at 25°C for 5 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.

[0048] The results are as follows Figure 1 and Figure 2 As shown, its morphological characteristics are as follows: Strain Mskw1 grows rapidly on PDA plates, reaching a colony diameter of 6 cm after 5 days of growth at 25℃; aerial hyphae development is weak in the early stages of culture, with most hyphae growing in a creeping, adhering state close to the culture medium surface; the colonies are generally white to milky white, with a raised, thick center and a clearly defined multi-layered concentric ring structure on the surface. As the culture time extends to the later stages, the colony morphology shows significant differentiation, with the overall texture gradually changing from an adhering state to a fluffy to cottony state; a large number of spores can be observed in the central area of ​​the colony. The hyphae are well-developed, with tubular hyphae interwoven into a network, exhibiting a wavy twist, and numerous irregular branches; the width of ordinary hyphae is 38 μm; the hyphae produce intercalary or lateral, nearly spherical to elliptical chlamydospores, some arranged in a beaded pattern, with a spore diameter of 10-20 μm and tuberculate protrusions on the surface; the hyphal surface is rough, and granular inclusions are visible inside.

[0049] 2. Molecular identification (1) DNA extraction ① After culturing the Mskw1 strain on a PDA plate at 25°C for 5 days, scrape the mycelium from the plate with a toothpick and place it into a sterilized centrifuge tube containing 300 μL of extraction buffer (1 M KCl, 100 mM Tris HCl, 10 mM EDTA, pH=8.0). ② Grind with an electric grinder and shake vigorously for 2 minutes; ③ Centrifuge at 10,000 rpm for 10 min; ④ Aspirate the supernatant and transfer it to another new centrifuge tube, discarding the precipitate; ⑤ Add an equal volume of isopropanol (analytical grade) to the supernatant, gently invert and mix several times, then centrifuge at 12000 rpm for 10 min to precipitate nucleic acids; ⑥ Gently pour off the supernatant and invert the centrifuge tube containing the precipitate onto absorbent paper to drain the water; ⑦ Add 300 μL of 70% ethanol, gently invert and mix several times, then centrifuge at 12000 rpm for 2 min; ⑧ Gently pour off the supernatant and repeat step ⑦ once; ⑨ Invert the centrifuge tubes onto absorbent paper to drain excess water, and place them at 37°C for 15 minutes to allow the ethanol to evaporate completely; ⑩ The precipitate was resuspended in 50 μL of pure water to obtain Mskw1 genomic DNA at a concentration of 30 ng / μL.

[0050] (2) PCR amplification of fungal ITS rDNA gene 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 pure water, and 1 μL of template DNA.

[0051] The upstream primer ITS1 sequence is: 5'-TCCGTAGGTGAACCTGCGG-3'. The downstream primer ITS4 sequence is: 5'-TCCTCCGCTTATTGATATGC-3'.

[0052] PCR amplification was performed on a Langqi MG96G PCR instrument. Reaction conditions: 94℃ pre-denaturation for 3 min; then 35 cycles including: 94℃ denaturation for 30 sec, 55℃ annealing for 30 sec, 72℃ extension for 1 min; and finally 72℃ extension for 10 min, and storage at 4℃.

[0053] (3) Recovery and purification of PCR products After the PCR reaction was completed, the PCR products were detected by 1% agarose gel electrophoresis and then purified using the DNA gel purification kit from Aspirin Biotechnology Co., Ltd., following the instructions in the kit's manual.

[0054] (4) Gene sequencing and sequence analysis The purified and recovered target DNA fragment, after electrophoresis detection, was sent to the Hangzhou sequencing department of Youkang Biotechnology Co., Ltd. for sequencing. After rigorous verification, the sequencing results yielded a DNA fragment sequence of 675 bp, as shown in SEQ ID NO.1.

[0055] On the NCBI website, the determined nucleotide sequence was searched and compared with homologous or similar nucleotide sequences in the GenBank database using BLAST. After BLAST alignment, the sequence matched that of a *Morchella* strain with accession number PV834169.1. Mortierella alpina The ITS rDNA sequence coverage of strain MWH-E-2 was 100%, with a similarity of 99.85%.

[0056] To further clarify the phylogenetic position of this strain, related gene sequences from closely related genera were searched in the NCBI database, and a phylogenetic tree was constructed, such as... Figure 3 As shown, Mskw1 is associated with multiple strains of the genus *Morchella* (…). Mortierella The strain is closely related to *Morchella alpineensis*. Based on the combined results of nucleotide sequence analysis and phylogenetic tree, Mskw1 is considered to be a strain belonging to *Morchella alpineensis*. Mortierella alpina ) strains.

[0057] The above molecular and morphological identification results indicate that the identified strain Mskw1 belongs to the kingdom Fungi (…). Fungi ), Mucor phylum ( Mucoromycota ), Moniliformes ( Mortierellomycetes ), Moniliales ( Mortierellales ), Morphozoaceae ( Mortierellaceae ), genus *Morchella* ( Mortierella ), Alpine spores ( Mortierella alpina Therefore, the Mskw1 strain was named... Mortierella sp. Mskw1.

[0058] The Mskw1 strain was deposited at the China Center for Type Culture Collection (CCTCC) on November 17, 2025, at Wuhan University, Wuhan, China, with accession number CCTCC NO: M 20252566. The collection center confirmed its viability on November 24, 2025.

[0059] Example 2: Colonization of endophytic fungus Mskw1 in rice roots 1. Activation culture of bacterial strains The Mskw1 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.

[0060] 2. Co-culture of Mrkw1 strain with rice After removing the husks from Zhejing 99 rice seeds, shriveled, dry, and insect-infested grains were removed. Healthy rice seeds were disinfected with 75% alcohol for 5 min, followed by 1% NaClO for 20 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 incubated at 25℃ for germination (16h light / 8h dark). After 3-4 days, the emerging seeds were inoculated into square dishes containing half MS + PDB medium, with 8-9 seeds per dish. The treatment group had 6-7 5 mm diameter Mrkw1 mycelial cakes inoculated under the seeds, while the control group had sterile mycelial cakes in half MS + PDB medium. Each treatment was repeated in triplicate, incubated at 25℃ for 16h light / 8h 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 strain, while the treatment group consisted of rice seedlings that were co-cultured with Mskw1.

[0061] 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.

[0062] 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 1 cm lengths. Place the cuts in centrifuge tubes. Add 1.8 mL of 5% potassium hydroxide solution to the centrifuge tubes, mix well, and incubate in a 90°C water bath for 10 min. Discard the treatment solution, rinse the root sections 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 min to bleach. After 15 min, rinse the root sections with running water and blot dry. Add 1.8 mL of 2% HCl solution, mix well, and incubate at room temperature for 5 min. 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 min to stain. Discard the staining solution, rinse the root sections 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.

[0063] 0.05% trypan blue staining solution: 300 mg trypan blue, 200 mL single-distilled water, 200 mL lactic acid, 200 mL glycerol.

[0064] The control group consisted of rice that was not co-cultured with the Mrkw1 strain, while the treatment group consisted of rice that was co-cultured with the Mrkw1 strain.

[0065] (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 the surface was treated with platinum sputtering under vacuum. Scanning observation was performed in the SEM sample chamber, and representative fields of view were photographed and recorded.

[0066] 4. Results Analysis Trypan blue staining results as follows Figure 4 As shown in Figure A, the rice roots in the control group were stained lighter and had a more intact structure; while the Mskw1 treatment group showed scattered dark blue reticulated hyphae, indicating that Mskw1 attached to the rice roots in a relatively dispersed and slender form.

[0067] Scanning electron microscopy results as follows Figure 4 As shown in Figure B, the roots of the control group of rice showed smooth root surface texture and root hairs; while the root surface of the Mskw1 treatment group was tightly covered by an irregular, flat membrane-like substance.

[0068] The above microscopic morphological results fully demonstrate that, under the condition of co-culture with rice, Mskw1 mainly adheres tightly to the rice root surface with a fine and continuous flat membrane structure, thereby achieving and completing effective colonization on the rice root surface.

[0069] Example 3: Co-cultivation of Mrkw1 microbial fertilizer and rice in pots 1. Activation culture of bacterial strains The Mskw1 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 5 days for later use.

[0070] 2. Preparation of Mskw1 liquid fermentation broth Endophytic fungal Mskw1 mycelial blocks (0.5 cm in diameter) cultured for 5 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, 5 days). The Mskw1 liquid fermentation broth was obtained when the PDB liquid fermentation medium was fully covered with mycelia.

[0071] 3. Co-cultivation of Mskw1 microbial fertilizer and potted rice seedlings (1) Application of liquid fermentation liquid: Mix the fermented liquid fermentation liquid with the seedling substrate at a ratio of 100 mL / pot and use it as new substrate soil for rice pot cultivation. Soak rice seeds (Zhejiang Jing 99) in 70% alcohol for 15 min for surface disinfection, rinse three times with running water, and then place them in a 37℃ dark constant temperature incubator for 2 days to germinate. When the seeds show white sprouts, sow them evenly in the pots.

[0072] (2) Rice seedling pot cultivation: Rice was cultivated in pots at 25℃ for 16 h of light and 8 h of darkness for a total of 14 days. The growth was observed and recorded, and various growth indicators were statistically analyzed.

[0073] 4. Results Analysis like Figure 5 and Figure 6 As shown, the stem width of plants inoculated with the Mskw1 strain was significantly higher than that of the control group. The measured data showed that the stem width of the treatment group was 0.376±0.025 cm, which was 75.7% higher than that of the uninoculated group (0.214±0.022 cm). The difference reached a highly significant level (P<0.001), indicating that this strain has a significant promoting effect on rice stem growth.

[0074] Example 4: Control efficacy of strain Mskw1 against rice blast fungus 1. Activation culture of bacterial strains The Mskw1 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 5 days for later use.

[0075] 2. Co-cultivation of Mrkw1 microbial fertilizer and potted rice seedlings (1) Pot culture: The mycelial blocks (0.5 cm in diameter) of the endophytic fungus Mskw1, which had been cultured for 5 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 culture (25℃, 150 rpm, 5 days). The Mskw1 liquid fermentation broth was obtained when the PDB liquid fermentation medium was fully covered with mycelium.

[0076] Mix the fermented liquid fertilizer with the seedling substrate (liquid microbial fertilizer: seedling substrate = 0.1 L: 3 L) and use this mixture as new substrate for rice pot cultivation. Soak Co39 rice seeds and then place them in a 37℃ dark incubator to germinate for 2 days. Once the seeds show signs of germination, sow them evenly in the pots. Incubate at 22-25℃ with 16 hours of light / 8 hours of darkness for 14 days.

[0077] (2) 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, then with 1% NaClO for 15 min, and rinsed 5 times with sterile water before use. The disinfected seeds were transferred to 1 / 2 MS medium using sterile forceps, sealed with sealing film, and placed in a 25℃ incubator for germination (16 h light / 8 h dark). After 3-4 days, the emerging seeds were inoculated into tissue culture bottles containing 1 / 2 MS medium, with 10 seeds in each bottle and 3 Mskw1 mycelial cakes inoculated simultaneously. The control group was inoculated with sterile PDA agar blocks. Each treatment had 3 replicates. The culture was carried out at 25℃ for 16 h light / 8 h dark for 15-20 days until the three-leaf stage, and then inoculated with rice blast fungus.

[0078] 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 × 10⁻⁶. 5 spores / mL. Prepare a 0.4% gelatin solution and mix it with an equal volume of spore suspension.

[0079] CM medium (1 L): yeast extract (1 g), casein amino acids (1 g), D-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 pH to 6.5 with sodium hydroxide, and add 15 g / L agar to the solid medium. Autoclave at 121°C for 15 min.

[0080] 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.

[0081] To further investigate the control efficacy of strain Mskw1 against rice blast fungus, a spore suspension was evenly sprayed onto the leaves of potted rice seedlings using a sprayer and placed in a 22℃ incubator for 2 days in the dark. 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.

[0082] The formula for calculating the lesion rate is: Lesion area rate (%) = (lesion area / total leaf area) × 100%.

[0083] 4. Results Analysis like Figure 7 and Figure 8 As shown, Mskw1 induced a systemic defense response in rice under both sterilized tissue culture bottles and pot conditions. Pot experiment data showed that the lesion area rate in the control group was 21.6%, while it dropped to only 0.7% in the Mskw1-co-cultured plants, resulting in a relative control efficacy of 96.8%. In the tissue culture experiment, the lesion area rate plummeted from 17.6% in the control group to 1.3%, a reduction of 92.6%. Visually, the leaves of the Mskw1-treated rice in the pots showed only scattered, very small lesions, a stark contrast to the densely covered, typical dark brown blast disease lesions in the control group.

[0084] Example 5: Tolerance of endophytic fungus Mskw1 to salt stress 1. Activation culture of bacterial strains The Mskw1 strain preserved on the slant was inoculated onto PDA solid medium for activation culture and cultured in the dark at 25°C for 5 days for later use.

[0085] 2. Testing the tolerance of the Mskw1 strain to salt concentration gradients. The sodium chloride concentrations in the PDA medium were 0 mol / L, 0.2 mol / L, 0.4 mol / L, and 0.6 mol / L, respectively. The strain Mskw1 was inoculated onto PDA media with different salt concentration gradients and cultured at 25°C in the dark for 5 days.

[0086] 3. Results Analysis like Figure 9 As shown, different salt concentrations caused differences in the colony morphology of Mskw1. Under salt-free stress, the colonies were milky white overall, with underdeveloped aerial hyphae that crawled along the culture medium surface, forming distinct multi-layered concentric rings. As the salt concentration increased, the colonies gradually turned whiter and the colony diameter gradually decreased. When the salt concentration was 0.2 mol / L, the colony diameter slightly decreased, and the central area appeared white to milky white. The aerial hyphae in the center of the colony evolved to a fluffy to cottony texture, with obvious clusters of protrusions. When the salt concentrations were 0.4 mol / L and 0.6 mol / L, the colony diameter decreased significantly, the colonies remained milky white overall, and the number of aerial hyphae in the center of the colony decreased significantly.

[0087] Example 6: Co-cultivation of Mskw1 microbial fertilizer and potted rice seedlings under salt stress 1. Activation culture of bacterial strains The Mskw1 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 5 days for later use.

[0088] 2. Preparation of Mskw1 solid microbial fertilizer Endophytic fungal Mycelial Blocks (0.5 cm in diameter) of Mskw1, cultured for 5 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, 5 days). The fermentation liquid was then inoculated onto sterilized barley grains (150 mL / 200 g inoculation amount) and incubated in the dark for 15 days until the barley grains were fully covered with mycelium, yielding Mskw1 solid microbial fertilizer.

[0089] 3. Co-cultivation of Mskw1 microbial fertilizer and potted rice seedlings (1) Application of solid microbial fertilizer: Mix the fermented solid microbial fertilizer with the seedling substrate (solid microbial fertilizer: seedling substrate = 70g: 800g) and use it as new substrate soil for rice pot cultivation. Soak rice seeds (Zhejiang Jing 99) in 70% alcohol for 15 minutes to disinfect the surface, rinse them 3 times with running water, and then 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.

[0090] (2) Rice seedling pot culture: Rice was cultured in pots at 25℃ for 16 h of light and 8 h of darkness. After 5 days of culture, 100 mL of 0.2 mol / L NaCl solution was applied three times. The culture was carried out for a total of 14 days. The growth was observed and recorded, and various growth indicators were statistically analyzed.

[0091] 4. Results Analysis like Figure 10 and Figure 11 As shown, under pot cultivation conditions, and under both salt-free and salt-stressed conditions, plants inoculated with the Mskw1 strain exhibited significantly higher chlorophyll content and fresh weight than the control group. Both the overall plant performance and individual plant performance indicated that plants inoculated with the Mskw1 strain showed better growth than the uninoculated group.

[0092] 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 the genus *Morchella* ( Mortierella Endophytic fungus Mskw1, characterized by, The endophytic fungus Mskw1 is classified and named Mortierella sp. Mskw1, deposited at the China Center for Type Culture Collection, accession number CCTCC NO: M20252566.

2. The endophytic fungus Mskw1 of the genus *Morchella* as described in claim 1, characterized in that, The culture conditions for the strain were as follows: the endophytic fungus Mskw1 was inoculated into PDA medium and cultured in the dark at 22-25℃.

3. A liquid microbial fertilizer containing the endophytic fungus Mskw1 of the genus *Morchella*, characterized in that... The method for preparing the liquid microbial fertilizer includes: inoculating the endophytic fungus Mskw1 of the genus *Morchella* as described in claim 1 or 2 into PDB medium, culturing until the medium is filled with mycelia, and obtaining the liquid microbial fertilizer.

4. A solid microbial fertilizer containing the endophytic fungus Mskw1 of the genus *Morchella*, characterized in that, The method for preparing the solid microbial fertilizer includes: inoculating the endophytic fungus Mskw1 of the genus *Morchella* as described in claim 1 or 2 into a PDB medium to obtain a fermentation broth, then inoculating the fermentation broth onto sterile barley grains, and culturing in the dark until the mycelium grows and covers the barley grains to obtain Mskw1 solid microbial fertilizer.

5. The application of the endophytic fungus Mskw1 of the genus *Morchella* as described in claim 1 or 2 in promoting rice growth and / or preventing rice blast and / or improving rice's tolerance to salt stress.

6. The application as described in claim 5, characterized in that, The application includes: co-culturing the endophytic fungus Mskw1 of the genus Monsporium with rice plants, so that it colonizes the roots of the rice plants.

7. The application as described in claim 6, characterized in that, The application includes: rice seeds are surface-sterilized and then germinated at 22-25℃. After the seeds show white sprouts, they are transferred to 1 / 2 MS medium and simultaneously inoculated with a mycelial cake of the endophytic fungus Mskw1 of the genus Monsporium for co-culture.

8. The application as described in claim 6, characterized in that, The application includes: preparing liquid or solid microbial fertilizer from the endophytic fungus Mskw1 of the genus Monsporium, mixing it with a seedling substrate to obtain a mixed substrate, and then sowing the germinated rice seeds in the mixed substrate for co-cultivation.

9. The application as described in claim 7 or 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.

10. The application as described in claim 5, characterized in that, The indicators for rice growth include: stem width, chlorophyll content, and fresh weight; the rice blast disease is leaf blast.

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