Endophytic fungus LH002 and application thereof

Abstract

This invention discloses an endophytic fungus LH002 and its applications, belonging to the field of microbial technology. The endophytic fungus LH002 was isolated from rice roots and classified as *Fusarium solanum* LH002 (…). Falciphoriella sp. LH002), deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20252451. This invention provides a novel... Falciphoriella The endophytic fungus LH002 interacts with rice to promote rice growth and enhance its resistance to rice blast. Co-culturing the endophytic fungus LH002 with rice, allowing it to colonize the roots of rice seedlings, significantly increases rice growth indicators and reduces foliar damage caused by rice blast, thus enhancing seedling resistance to leaf blast. Therefore, the widespread application of endophytic fungus LH002 in agriculture has great value.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to endophytic fungi. Falciphoriella sp. LH002 and its application in promoting rice growth and preventing rice blast. Background Technology

[0002] Rice (Oryza sativa L.) is an annual aquatic herbaceous plant belonging to the Poaceae family and is an important grain widely cultivated in tropical Asia. During its growth, rice is frequently affected by many biotic and abiotic stresses, among which yield loss caused by pathogenic fungi and bacteria is one of the most significant threats to agricultural production.

[0003] Among fungal diseases, rice blast fungus is extremely destructive, causing crop yield reduction and threatening growth and development, and in severe cases, even resulting in total crop failure. Rice blast, also known as rice fever, can be classified into seedling blast, leaf blast, neck blast, branch blast, and grain blast, depending on the time and location of damage.

[0004] Currently, the main control measures for rice blast include breeding resistant varieties and using fungicides. Although these two methods are economical and effective, the numerous physiological races of rice blast pathogen can easily cause resistant varieties to lose their resistance; frequent use of fungicides can lead to drug resistance in the pathogen and also cause environmental pollution. Therefore, finding green and environmentally friendly control measures is of great significance for the safe production and increased yield of rice.

[0005] Endophytic fungi are a class of fungi that colonize at least a portion of their life cycle within healthy plant tissues without causing obvious symptoms to the plant. In recent years, an increasing number of endophytic fungi have been discovered that not only promote plant growth but also enhance the host plant's ability to resist biotic and abiotic stresses through various pathways. Endophytic fungi can promote plant growth by directly providing nutrients to the plant, increasing compounds such as iron or phosphates; promoting the absorption of nutrients; and producing plant hormones such as auxins and gibberellins. They also enhance the plant's resistance to biotic stresses through multiple mechanisms, including inducing systemic resistance, producing secondary metabolites, competing with pathogens for nutrients and space, and hyperparasitism. Therefore, endophytic fungi can be widely used as potential resource fungi in crops such as grains, vegetables, and fruit trees, serving as biocontrol agents and plant growth promoters.

[0006] In the development of rice endophytic fungal resources, patent documents CN113388526A and CN113355245A disclose a strain of the genus *Phyllostachys* (…). Falciphora oryzae Endophytic fungi, by colonizing the root tissues of rice, can significantly improve the quality of rice seedlings, increase rice yield, and enhance the rice's resistance to neck blast; patent document CN113249229A discloses a strain of the genus *Pseudophila* (…). PseudophialophoraEndophytic fungi (sp.) can enhance rice's resistance to seedling blight by colonizing the root tissue. Therefore, utilizing the symbiotic interaction between endophytic fungi and rice to improve rice's growth-promoting ability or its ability to resist external stress is an effective means of ensuring safe rice production and increasing yield.

[0007] In summary, identifying more endophytic fungi with biocontrol or growth-promoting effects is a problem that needs to be solved by those skilled in the art. Regarding *Fusarium solani* (… Falciphoriella solaniterrestris There are literature reports that this strain was isolated from the soil of Dutch potato fields and its morphological characteristics were described, but there are few studies on its related functions. Summary of the Invention

[0008] The purpose of this invention is to provide an endophytic fungus for rice that promotes rice growth and enhances rice resistance to rice blast through symbiotic interaction, thereby achieving the prevention and control of rice seedling and leaf blast.

[0009] To achieve the above objectives, the present invention adopts the following technical solution:

[0010] This invention isolates a new plant from the root system of rice. Falciphoriella This strain is an endophytic fungus. Its main biological characteristics are: after 10 days of growth on a PDA plate at 25°C, the colony diameter is 5.1 cm, the colony is pale yellow, and the aerial hyphae are white and cottony. The hyphae are septate, and no conidia are produced on the PDA plate. Its ITS sequence is shown in SEQ ID NO.1. Phylogenetic tree construction confirmed that this strain belongs to [the genus / family / organism / etc.]. Falciphoriella solaniterrestris It was classified and named Fusarium solani LH002 ( Falciphoriella sp. LH002 was deposited on November 4, 2025 at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: M 20252451.

[0011] The culture conditions for endophytic fungus LH002 are as follows: inoculate in PDA medium and culture in the dark at 22-25℃ for 7-12 days.

[0012] When endophytic fungus LH002 is co-cultured with rice, LH002 can colonize the epidermal and cortical cells of rice roots.

[0013] This invention has found that colonizing the endophytic fungus LH002 into the root tissue of rice plants has a growth-promoting effect, specifically manifested in a significant increase in leaf width, stem diameter, plant height, chlorophyll content, and fresh weight.

[0014] Therefore, the present invention provides the application of the endophytic fungus LH002 in promoting rice growth, the application comprising: co-culturing the endophytic fungus LH002 with rice to colonize the root tissue of rice.

[0015] Furthermore, the indicators for promoting rice growth include at least one of leaf width, stem diameter, plant height, chlorophyll content, and fresh weight.

[0016] This invention has found that colonizing the endophytic fungus LH002 into the root tissue of rice can achieve a control effect of 89.25% on rice seedling leaf blast, which is a significant effect.

[0017] Therefore, the present invention provides the application of the endophytic fungus LH002 in the prevention and control of rice blast, the application including: colonizing the endophytic fungus LH002 into the root tissue of rice.

[0018] Furthermore, the application includes: co-culturing rice seeds with the endophytic fungus LH002 after germination, allowing it to colonize the roots of rice seedlings, thereby improving the resistance of rice seedlings to leaf blight.

[0019] Furthermore, after surface disinfection, the rice seeds were germinated at 37°C. Once the seeds showed signs of germination, they were transferred to rice substrate soil and co-cultured with an endophytic fungal suspension of LH002.

[0020] The method for preparing the bacterial suspension includes: culturing strain LH002 in PDB medium at 25°C and 150 rpm for 5 days, collecting mycelia after filtration, and adding water to prepare the bacterial suspension.

[0021] Furthermore, the co-culture conditions are: 22-25℃, 16 hours of light and 8 hours of dark culture per day, for 14-20 days.

[0022] Specifically, after the bacterial suspension is evenly poured around rice seeds or rice roots and cultured for 14-20 days, the LH002 strain can colonize the root tissue of rice.

[0023] The beneficial effects of this invention are as follows:

[0024] This invention provides a new FalciphoriellaThe endophytic fungus LH002 interacts with rice to promote rice growth and enhance its resistance to rice blast. Co-culturing the endophytic fungus LH002 with rice, allowing it to colonize the roots of rice seedlings, significantly increases rice growth indicators and reduces foliar damage caused by rice blast, enhancing seedling resistance to leaf blast. Statistical analysis showed that, compared to the control group, rice plants co-cultured with the endophytic fungus LH002 exhibited increases in leaf width, stem diameter, plant height, chlorophyll content, and fresh weight of 28.69%, 38.29%, 21.68%, 17.08%, and 89.41%, respectively, demonstrating a significant growth-promoting effect. Furthermore, compared to the control group, rice plants co-cultured with the endophytic fungus LH002 showed an 89.25% control effect against rice blast. Therefore, the widespread application of the endophytic fungus LH002 in agriculture has significant value. Attached Figure Description

[0025] Figure 1 The images show the colony and hyphal morphology of strain LH002. A represents the colony morphology of LH002 on a PDA plate; B represents the hyphal morphology under an optical microscope, with a scale bar of 5 μm.

[0026] Figure 2 This is a phylogenetic tree constructed using fungal ITS sequences.

[0027] Figure 3 This image shows the colonization of strain LH002 in rice roots. A and B are longitudinal and transverse sections of rice roots inoculated with GFP-labeled LH002 strain, respectively, under a confocal microscope.

[0028] Figure 4 The growth of strain LH002 after co-culturing with rice.

[0029] Figure 5 This study investigated the growth-promoting effect of strain LH002 on rice. Specifically, it measured leaf width, stem diameter, plant height, chlorophyll content, and fresh weight after co-culturing with the control group and the LH002 group. Significance (t-test): *** indicates significance. P <0.001.

[0030] Figure 6 The control efficacy of strain LH002 against rice blast leaf blast is shown. A represents the disease incidence in the control group and LH002 group after foliar spraying with rice blast fungus; B represents the disease index in the control group and LH002 group; and C represents the lesion rate in the control group and LH002 group. Significance (t-test): * indicates significance. P <0.05; ** indicates P <0.01. Detailed Implementation

[0031] 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.

[0032] 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.

[0033] PDA solid medium is made from potato dextrose agar powder (containing 20 g glucose, 10 g potato extract powder, and 13 g agar per liter of distilled water) and autoclaved at 121°C for 15 min.

[0034] PDB medium was prepared using potato glucose broth powder (containing 5 g potato extract, 10 g peptone, 15 g glucose, and 5 g sodium chloride per liter of distilled water) and autoclaved at 121°C for 20 min.

[0035] 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 elements. Adjust the pH to 6.5 with NaOH, and add 15 g / L agar to the solid medium. Autoclave at 121°C for 15 min.

[0036] Example 1: Isolation and Identification of Strains LH002

[0037] I. Isolation and purification of strain LH002

[0038] Strain LH002 was isolated from rice roots. The specific method was as follows: Rice 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 medium (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. Mycelial growth was observed every 24 h. Endophytic fungal hyphae growing from the edges of the tissue cuts were carefully picked out with a toothpick and transferred to fresh potato dextrose agar (PDA) medium for purification and culture. The isolated and purified strains were numbered. The following section describes the identification of strain LH002.

[0039] II. Identification of LH002 strain

[0040] 1. Morphological identification

[0041] After isolation and purification, strain LH002 was inoculated onto PDA medium and cultured at 25°C for 10 days. Colony and hyphal morphology were observed.

[0042] Colony growth status as follows Figure 1 As shown in A, its morphological characteristics are as follows: on PDA plates, after growing at 25°C for 10 days, the colony diameter is 5.1 cm; the colony is pale yellow, and the aerial hyphae are white and cottony.

[0043] Hyphae morphology as Figure 1 As shown in B, the hyphae are septate and have a diameter of 2-5 μm. They do not produce conidia on PDA plates.

[0044] 2. Molecular identification

[0045] (1) DNA extraction

[0046] After culturing LH002 strain on PDA plates at 25°C for 7 days, mycelia were scraped from the plates with a toothpick and placed into a sterile 1.5 mL centrifuge tube containing 500 μL of extraction buffer (1 M KCl, 100 mM Tris-HCl, 10 mM EDTA, pH=8.0). The mycelia were ground with a grinder and shaken vigorously for 2 min. The tube was then centrifuged at 12000 rpm for 10 min. 300 μL of the supernatant was transferred to another new centrifuge tube, and the precipitate was discarded. An equal volume of isopropanol (analytical grade) was added to the supernatant, and the mixture was inverted several times. The tube was then centrifuged at 12000 rpm for 10 min to precipitate the nucleic acid. The supernatant was discarded, and 700 μL of 70% ethanol was added. The mixture was inverted and centrifuged at 12000 rpm for 2 min. The supernatant was discarded, and the centrifuge tube was dried in a clean bench to allow the ethanol to evaporate completely. The precipitate was dissolved in 50 μL of ddH2O to obtain LH002 genomic DNA.

[0047] (2) PCR amplification of fungal ITS rDNA gene

[0048] PCR amplification was performed in a 50 μL reaction system containing: 2 μM each of forward and reverse primers, 2 μL of template DNA, 25 μL of Green Taq Mix, and the remaining volume made up with ddH2O.

[0049] The upstream primer ITS1 sequence is: 5′-TCCGTAGGTGAACCTGCGG-3′;

[0050] The downstream primer ITS4 sequence is: 5′-TCCTCCGCTTATTGATATGC-3′.

[0051] The PCR amplification conditions were as follows: pre-denaturation at 94℃ for 3 min, followed by 35 cycles of denaturation at 94℃ for 30 sec, annealing at 58℃ for 30 sec, extension at 72℃ for 1 min, and a final extension at 72℃ for 10 min.

[0052] After electrophoresis, the PCR products were purified and recovered, and the recovered target DNA fragment was sent to Hangzhou Youkang Biotechnology Co., Ltd. for sequencing. After rigorous verification, the sequencing results yielded a DNA fragment sequence of 522 bp, as shown in SEQ ID NO.1.

[0053] Using the sequencing results described above, homologous or similar nucleotide sequences were searched and compared in the NCBI GenBank database to perform ITS rDNA gene alignment and construct a phylogenetic tree, such as... Figure 2 As shown, this strain is related to Falciphoriella solaniterrestris The homology of CBS117.83 is 100%.

[0054] Based on biological characteristics and ITS rDNA gene comparison, LH002 was identified as belonging to the kingdom Fungi ( ). Fungi Ascomycota ( Ascomycota ), class of fecal scabies ( Sordariomycetes ), Giant Crustacea ( Magnaporthales ), Giant Seashell Family ( Magnaporthaceae ), Falciphoriella Therefore, it was named Fusarium solanifolium LH002 ( ). Falciphoriella sp. LH002 was deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, on November 4, 2025, with accession number CCTCC NO: M 20252451. The collection was identified as viable by the collection center on November 11, 2025.

[0055] Example 2: Growth-promoting effect of strain LH002 on rice

[0056] Test plant: Rice (Oryza sativa L.) conventional variety CO39.

[0057] I. Activation and culture of LH002 strain

[0058] The LH002 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 10 days for later use.

[0059] II. Co-culture of LH002 strain with rice

[0060] 1. Observe colonization during tissue culture co-culture.

[0061] After removing the husks from rice seeds, shriveled, dry, and insect-infested grains were removed. Healthy rice seeds were disinfected with 70% alcohol for 5 minutes, followed by 1% NaClO for 20 minutes, and then rinsed 6 times with sterile water before use. The disinfected seeds were transferred to 1 / 2 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 tissue culture flasks containing 1 / 2 MS medium, 10 seeds per flask, along with 3 LH002 bacterial discs tagged with GFP. The control group was inoculated with sterile PDA agar blocks. Each treatment had 3 replicates. The culturing was carried out at 25°C for 16 h light / 8 h dark for 20 days until the three-leaf stage. The colonization of endophytic fungi in the rice roots was observed. The roots were cleaned, and transverse and longitudinal sections were prepared for observation under a fluorescence confocal microscope.

[0062] The GFP strain was constructed by scraping LH002 mycelia grown on a PDA plate with distilled water. An equal volume of Agrobacterium tumefaciens culture containing the PKD5-GFP vector (containing the sulfonylurea resistance gene) was mixed with the LH002 mycelial suspension. Transformants were then screened on defined complex medium (DCM) containing sulfonylureas, and those exhibiting green fluorescence were further screened under a Nikon DS-Qi2 microscope.

[0063] The colonization status of LH002 is as follows: Figure 3 As shown in Figures A and B, strain LH002 was found to colonize the epidermal and cortical cells of rice.

[0064] 2. Observe the growth promotion effect of potted plants in co-cultivation.

[0065] The LH002 strain was cultured in PDB medium at 25℃ and 150 rpm for 5 days, and the mycelium was collected after filtration. Rice seeds were surface-sterilized with 1% NaClO for 10 min, washed 5 times with sterile water, and then spread evenly in petri dishes and placed in a 37℃ incubator in the dark for 2 days to promote germination. Once the seeds showed signs of sprouting, they were transferred to rice substrate soil, 25 seeds per pot, and 30 g of mycelial suspension was evenly poured around the rice seeds. When the rice seedlings reached the three-leaf stage (14-20 days), the growth of the rice was observed, and growth indicators such as plant height, leaf width, stem diameter, chlorophyll content, and fresh weight were recorded. The chlorophyll content was measured using a plant nutrient analyzer (TYS-4N), with three replicates for each leaf and the average value taken.

[0066] The results are as follows Figure 4 and Figure 5As shown, leaf width, stem thickness, plant height, chlorophyll content, and fresh weight increased by 28.69%, 38.29%, 21.68%, 17.08%, and 89.41%, respectively, demonstrating a significant growth-promoting effect.

[0067] Example 3: Control efficacy of strain LH002 against rice blast fungus during seedling leaf blast.

[0068] Test plant: Rice (Oryza sativa L.) conventional variety CO39.

[0069] I. Activation and culture of LH002 strain

[0070] The LH002 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 10 days for later use.

[0071] II. Co-culture of LH002 strain with rice

[0072] The LH002 strain was cultured in PDB medium at 25°C and 150 rpm for 5 days, and the mycelium was collected after filtration. Rice seeds were surface-sterilized with 1% NaClO for 10 min, washed 5 times with sterile water, and then spread evenly in petri dishes and placed in a 37°C incubator in the dark for 2 days to promote germination. Once the seeds showed signs of sprouting, they were transferred to rice substrate soil, 25 seeds per pot, and 30g of mycelial suspension was evenly poured around the rice seeds. When the rice seedlings reached the three-leaf stage (14-20 days), rice blast fungus spores were sprayed.

[0073] III. Spraying with conidia of rice blast fungus

[0074] 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.

[0075] Spray the spore suspension evenly onto the leaves of rice seedlings using a sprayer, using 2 mL per pot. Place the pots in an incubator at 22℃ and incubate in the dark for 2 days. Then, incubate at 25℃ with 16 h light / 8 h darkness for 4-5 days, and calculate the disease index and lesion rate. Use Adobe Photoshop to calculate the lesion rate and the Rice Standard Evaluation System (SES) from the International Rice Research Institute to calculate the disease index.

[0076] The results are as follows Figure 6As shown, the control group suffered severe leaf blast disease, with a lesion rate of 50.79% and a disease index of 23.39. The LH002 strain treatment group showed milder disease, with a lesion rate of 5.46% and a disease index of 8.59. Compared with the control group, the LH002 strain treatment group showed a 45.33% decrease in lesion rate and a 14.8 decrease in disease index, achieving a control effect of 89.25%, significantly improving the rice's resistance to rice blast.

[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. An endophytic fungus, LH002, characterized in that, The endophytic fungus LH002 was isolated from rice roots and classified as *Fusarium solanum*. Falciphoriella sp.) LH002, deposited at the China Center for Type Culture Collection, accession number CCTCC NO: M 20252451.

2. The endophytic fungus LH002 of claim 1, wherein, The ITS sequence of the endophytic fungus LH002 is shown in SEQ ID NO.

1.

3. The endophytic fungus LH002 of claim 1, wherein, The endophytic fungus LH002 grew on PDA medium at 25°C for 10 days. The colonies were light yellow, the aerial hyphae were white and cottony, and the hyphae were septate. No conidia were produced on the plate.

4. The endophytic fungus LH002 of claim 1, wherein, The culture conditions for endophytic fungus LH002 are as follows: inoculate in PDA medium and culture in the dark at 22-25℃ for 7-12 days.

5. The use of the endophytic fungus LH002 of claim 1 in promoting the growth of rice, wherein the endophytic fungus LH002 is applied to the rice in an amount of 0.1-1000 ppm. The application includes: co-culturing endophytic fungus LH002 with rice to colonize the root tissue of rice.

6. Use according to claim 5, wherein The indicators for promoting rice growth include at least one of the following: leaf width, stem diameter, plant height, chlorophyll content, and fresh weight.

7. The application of the endophytic fungus LH002 as described in claim 1 in the control of rice blast, characterized in that, The application includes colonizing the endophytic fungus LH002 into the root tissue of rice.

8. Use according to claim 7, wherein the compound is ###0002### The application includes: co-culturing rice seeds with the endophytic fungus LH002 after germination, allowing it to colonize the roots of rice seedlings, thereby improving the resistance of rice seedlings to leaf blight.

9. Use according to claim 8, wherein the compound is ###0002### After surface disinfection, rice seeds were germinated at 37°C. Once the seeds showed signs of germination, they were transferred to rice substrate soil and co-cultured with an endophytic fungal suspension of LH002.

10. The use according to claim 8, wherein the compound is ###00002### The co-culture conditions are: 22-25℃, 16 hours of light and 8 hours of dark culture per day, for 14-20 days.

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