Endophytic fungus LH001 and application thereof

Abstract

The application discloses an endophytic fungus LH001 and application thereof, and belongs to the technical field of plant disease control. The endophytic fungus LH001 is separated from the root system of wild rice, is classified and named as Pseudophialophora sp. LH001, is preserved in the China Center for Type Culture Collection, and has a preservation number of CCTCC NO: M 20241751. The endophytic fungus LH001 strain is co-cultured with rice, is allowed to colonize the root of rice seedlings, can reduce the leaf damage caused by Magnaporthe oryzae, enhances the disease resistance of rice seedlings to leaf blight, and the control effect reaches 94.05%. Therefore, the biological control effect of the endophytic fungus LH001 on rice blast has great value in the popularization and application in the agricultural field.

Description

Technical Field

[0001] This invention relates to the field of plant disease control technology, specifically to the endophytic fungus Pseudophialophorasp.LH001 and its application in the control of rice blast. Background Technology

[0002] Rice blast is a disease of rice caused by the rice blast pathogen. It can occur throughout the entire growth period of rice, damaging seedlings, leaves, panicles, and nodes, and is respectively called seedling blast, leaf blast, panicle blast, and node blast. Among them, leaf blast and panicle blast are the most common and cause the most damage.

[0003] The main methods for controlling rice blast include selecting resistant varieties, cultivating high-quality seedlings, implementing fertilizer and water management techniques, strengthening field management, implementing control measures, and using chemical agents. Cultivating resistant varieties is one of the most economical and effective ways to control rice blast fungus, but the large number of physiological races of the fungus, its high mutagenicity, and rapid adaptability present significant challenges. Chemical control is also a common method for controlling rice blast, but this method pollutes the environment and easily leads to drug resistance in the fungus. Therefore, seeking efficient, environmentally friendly, green, and safe biological control measures is a research hotspot in plant disease control.

[0004] Biological control utilizes living organisms or metabolically active components produced by organisms to control plant pathogens. It has advantages such as being environmentally friendly, less prone to developing resistance, causing minimal harm to natural enemies of harmful organisms, and posing low risks to human and animal health. Compared to traditional chemical control methods, it is more in line with the development trend of modern agriculture. Common biological control measures include: 1. Applying antagonistic bacteria: Antagonistic bacteria are beneficial bacteria that can produce metabolic products such as antibiotics, enzymes, and hormones and can control pathogenic microorganisms through inhibition and competition. 2. Applying bioremediation agents: Bioremediation agents are microbial preparations that can decompose organic matter in the soil, build soil microbial communities, and improve soil fertility. 3. Applying biofertilizers: Biofertilizers are fertilizers rich in beneficial microorganisms that enhance rice immunity by leveraging the dominant role of the soil's microbial community. 4. Applying plant-derived biological agents: Plant-derived biological agents are organic compounds extracted from plants or extracted through fermentation. These agents can improve rice immunity through plant-plant interaction mechanisms and effectively control rice blast.

[0005] Natural ecosystems harbor a wealth of beneficial microorganisms, among which plant endophytic fungi are one. Plant endophytic fungi are fungi that can infect and colonize healthy plant tissues at least part of their life cycle, without causing obvious disease in the host. They are ubiquitous in ecosystems, forming a mutually beneficial symbiotic relationship with host plants; thus, they can promote plant growth and enhance the plant's resistance to biotic and abiotic stresses. Therefore, endophytic fungi can be widely used as potential resource bacteria as biocontrol agents, biological nitrogen fixatives, and plant growth promoters in crops such as grains, vegetables, and fruit trees.

[0006] Therefore, identifying beneficial microorganisms from plant endophytic fungi that are effective against rice blast is of great significance for the prevention and control of rice blast. Summary of the Invention

[0007] The purpose of this invention is to provide a wild rice endophytic fungus that enhances rice's resistance to rice blast through symbiotic interaction with rice, thereby achieving the prevention and control of rice seedling and leaf blast.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] This invention isolates a novel endophytic fungus of the genus *Pseudophialophora* from wild rice roots. Its main biological characteristics are: colony diameter reaches 5.4 cm after 12 days of growth on PDA plates at 25°C; colonies are yellowish-green with underdeveloped aerial hyphae that creep along the culture medium surface and have septa; conidia are elliptical or pear-shaped and without septa. Its ITS sequence is shown in SEQ ID NO. 1. Phylogenetic analysis identified this strain as belonging to *Pseudophialophora panicorum*. It is classified as *Pseudophialophora sp. LH001* and deposited on August 5, 2024, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: M 20241751.

[0010] The culture conditions for the endophytic fungus strain LH001 were as follows: the endophytic fungus LH001 was inoculated into PDA solid medium and cultured in the dark at 22-25℃ for 10-20 days.

[0011] This invention has found that colonizing the endophytic fungus LH001 into the root tissue of rice can achieve a 94.05% control effect on rice seedling leaf blast, which is a significant effect.

[0012] Therefore, the present invention provides the application of the endophytic fungus LH001 in the prevention and control of rice blast.

[0013] The application includes colonizing the endophytic fungus LH001 into the root tissue of rice.

[0014] Furthermore, the application includes: co-culturing rice seeds with the endophytic fungus LH001 after germination, allowing it to colonize the roots of rice seedlings, thereby improving the resistance of rice seedlings to leaf blight.

[0015] Furthermore, the method for seed germination includes: removing the husk from the rice seeds, surface disinfecting them with 2% NaClO for 15 minutes, and rinsing them 5 times with sterile water. The disinfected seeds are then placed in 1 / 2 MS medium and cultured at 25°C under 16h light / 8h darkness conditions until germination.

[0016] Furthermore, after surface disinfection, rice seeds were germinated at 22-25℃. Once the seeds showed white sprouts, they were transferred to 1 / 2 MS medium and simultaneously inoculated with endophytic fungal LH001 mycelial cakes for co-culture.

[0017] 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.

[0018] The beneficial effects of this invention are as follows:

[0019] This invention provides an endophytic fungus, LH001, which, when interacting with rice, enhances rice's resistance to rice blast. Co-culturing the LH001 strain with rice and allowing it to colonize the roots of rice seedlings reduces foliar damage caused by rice blast and enhances the seedlings' resistance to leaf blast. Statistical analysis showed that the lesion rate in the control group was 49.88%, while the lesion rate in the LH001 treatment group was 2.97%, achieving a control effect of 94.05%. Therefore, the biological control effect of the endophytic fungus LH001 against rice blast has significant value for widespread application in the agricultural field. Attached Figure Description

[0020] Figure 1 The images show the colony, hyphae, and spore morphology of strain LH001. A represents the colony morphology of LH001 on a PDA plate; B and C represent the hyphae and spore morphology under an optical microscope (scale bar: 10 μm); and D represents the hyphae and spore morphology of LH001 under a scanning electron microscope.

[0021] Figure 2 This is a phylogenetic tree constructed using fungal ITS sequences.

[0022] Figure 3 The colonization of strain LH001 on rice roots is shown. A and B are cross-sections and longitudinal sections of rice roots inoculated with GFP-labeled LH001 strain, respectively, under a confocal microscope; C shows the colonization trend of strain LH001 on rice roots; D and E show LH001 hyphae attached to the surface of rice roots under a scanning electron microscope.

[0023] Figure 4 The study aimed to assess the control efficacy of strain LH001 against rice blast leaf blast. In this study, A represents the disease incidence in the control group and the LH001 group after foliar spraying with rice blast fungus; B represents the lesion rate in the control group and the LH001 group; and C represents the disease index in the control group and the LH001 group. Significance (t-test): **, P < 0.01; ***, P < 0.001. Detailed Implementation

[0024] 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.

[0025] 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.

[0026] Example 1: Isolation and Identification of Strains LH001

[0027] I. Isolation and purification of strain LH001

[0028] Strain LH001 was isolated from the root system of wild rice in Yunnan. The specific method was as follows: the wild rice root system was 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 minutes, 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. Every 24 hours, the growth of hyphae was observed. Endophytic fungal hyphae growing from the edge of the tissue cut were carefully picked out with a toothpick, transferred to fresh PDA medium for purification, and the strain was recorded as LH001.

[0029] II. Identification of LH001 strain

[0030] 1. Morphological identification

[0031] After isolation and purification, strain LH001 was inoculated onto PDA medium and cultured at 25°C for 12 days. Colony, mycelial, and conidial morphology were observed. Colony growth status is as follows. Figure 1 As shown in A, the morphology of hyphae and conidia is as follows: Figure 1As shown in B and 1C. Its morphological characteristics are as follows: the colonies grow slowly on PDA plates, and the colony diameter reaches 5.4 cm after 12 days of growth at 25℃; the colonies are yellowish-green; the aerial hyphae are underdeveloped and creep along the surface of the culture medium; the hyphae have septa; the conidia are elliptical or pear-shaped and without septa.

[0032] 2. Molecular identification

[0033] (1) DNA extraction

[0034] ① After culturing strain LH001 on a PDA plate at 25℃ for 7 days, scrape the mycelium from the plate with a toothpick and put it into a sterilized 1.5mL centrifuge tube containing 500μL of extraction buffer (1M KCl, 100mM Tris-HCl, 10mM EDTA, pH=8.0).

[0035] ② Grind with a grinder and shake vigorously for 2 minutes;

[0036] ③ Centrifuge at 12000 rpm for 10 min;

[0037] ④ Aspirate the supernatant and transfer it to another new centrifuge tube, discarding the precipitate;

[0038] 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 acid;

[0039] ⑥ Discard the supernatant, add 700 μL of 70% ethanol, gently invert and mix several times, then centrifuge at 12000 rpm for 2 min;

[0040] ⑦ Discard the supernatant and dry the centrifuge tubes in a clean bench to allow the ethanol to evaporate completely;

[0041] ⑧ Dissolve the precipitate with 50 μL ddH2O to obtain LH001 genomic DNA.

[0042] (2) PCR amplification of fungal ITS rDNA gene

[0043] PCR amplification was performed in a 50 μL reaction system containing: 2 μM each of forward and reverse primers, 2 μL of template DNA, and 25 μL of Green Taq Mix.

[0044] The upstream primer ITS1 sequence is: 5'-TCCGTAGGTGAACCTGCGG-3';

[0045] The downstream primer ITS4 sequence is: 5'-TCCTCCGCTTATTGATATGC-3'.

[0046] The PCR amplification reaction 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.

[0047] 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 496 bp, as shown in SEQ ID NO.1.

[0048] Combining biological characteristics and ITS rDNA gene alignment, a phylogenetic tree was constructed. Figure 2 LH001 was identified as belonging to the kingdom Fungi, phylum Ascomycota, class Sordariomycetes, order Magnaporthales, family Magnaporthaceae, and genus Pseudophialophora. This strain showed 100% homology with Pseudophialophora panicorum strain CM9s6. Therefore, it was named Pseudophialophorasp.LH001 and deposited on August 5, 2024, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: M 20241751. The collection was confirmed as viable on August 12, 2024.

[0049] Example 2: Control efficacy of strain LH001 against rice blast fungus during seedling leaf blast.

[0050] Test plant: Rice (Oryza sativa L.) conventional variety CO39.

[0051] I. Activation and culture of LH001 strain

[0052] The LH001 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.

[0053] PDA solid medium: Each liter contains 20g glucose, 200g potato, and 15g 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℃ for 15 minutes.

[0054] II. Co-culture of LH001 strain with rice

[0055] After removing the husks from rice seeds, shriveled, dry, and insect-infested grains are removed. Healthy rice seeds are disinfected with 70% alcohol for 5 minutes, followed by 1% NaClO for 15 minutes. The seeds are then rinsed five times with sterile water before use. The disinfected seeds are transferred to 1 / 2 MS medium using sterile forceps, sealed with sealing film, and germinated in a 25°C incubator (16h light / 8h dark). After 3-4 days, the emerging seeds are inoculated into tissue culture flasks containing 1 / 2 MS medium, 10 seeds per flask, along with 3 LH001 mycelial cakes. The control group is inoculated with sterile PDA agar blocks. Each treatment has three replicates. The culturing is carried out at 25°C for 15-20 days until the three-leaf stage, followed by inoculation with rice blast fungus.

[0056] Meanwhile, to ensure the fungus could colonize the rice roots, the rice roots inoculated with the GFP-labeled LH001 strain were washed, cross-sectioned, and longitudinally sectioned, and observed under a fluorescence confocal microscope. The LH001 colonization status is shown below. Figure 3 As shown in A and 3B, strain LH001 was found to colonize the epidermis, cortex, and stele cells of rice.

[0057] III. Spraying with conidia of rice blast fungus

[0058] The rice blast fungus strain Guy11 was inoculated onto CM solid medium and cultured at 25°C for 12-14 days (12h light / 12h 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.

[0059] CM medium (1L): Yeast Extract (1g), Casamino acid (1g), D-glucose (10g), KH2PO4 (1.52g), NaNO3 (6g), Peptone 140 (2g), KCl (0.52g), MgSO4·7H2O (0.52g), 0.1% (v / v) Vitamin solution, 0.1% (v / v) Trace Element. Adjust pH to 6.5 with NaOH, and add 15g / L agar to the solid medium. Autoclave at 121℃ for 15min.

[0060] Spray the spore suspension evenly onto the leaves of rice seedlings using a sprayer, using 1 mL per bottle. Place the tissue culture bottles in an incubator at 25℃ and incubate in the dark for 2 days. Then, incubate for 4-5 days with 16 hours of light / 8 hours of darkness, and record the disease index and lesion rate.

[0061] The results are as follows Figure 4As shown, the control group suffered severe leaf blast disease, with a lesion rate of 49.88% and a disease index of 35.65. The LH001 strain treatment group had milder disease, with a lesion rate of 2.97% and a disease index of 11.99. Compared with the control group, the LH001 strain treatment group showed a 46.91% decrease in lesion rate and a 23.66 decrease in disease index, achieving a control effect of 94.05%, which was significant.

Claims

1. An endophytic fungus LH001, characterized in that, The endophytic fungus LH001 was isolated from the roots of wild rice and classified as follows: Pseudophialophora sp. It is deposited at the China Center for Type Culture Collection, with accession number CCTCC NO: M20241751.

2. The application of the endophytic fungus LH001 as described in claim 1 in the prevention and control of rice blast.

3. The application as described in claim 2, characterized in that, The application includes colonizing the endophytic fungus LH001 into the root tissue of rice.

4. The application as described in claim 2, characterized in that, The application includes: co-culturing rice seeds with the endophytic fungus LH001 after germination, allowing it to colonize the roots of rice seedlings, thereby improving the resistance of rice seedlings to leaf blight.

5. The application as described in claim 4, characterized in that, After surface disinfection, rice seeds were germinated at 22-25℃. Once the seeds showed signs of germination, they were transferred to 1 / 2 MS medium and simultaneously inoculated with endophytic fungal LH001 mycelial cakes for co-culture.

6. The application as described in claim 4, 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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