Endophytic fungus LH001 and application thereof

Abstract

The invention 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 (CCTCC), and has the preservation number of CCTCC NO: M 20241751. The endophytic fungus LH001 strain is co-cultured with rice and colonized at the root of a rice seedling, so that leaf harm caused by magnaporthe oryzae can be reduced, the disease resistance of the rice to leaf blast in the seedling stage is enhanced, and the prevention and treatment effect reaches 94.05%. Therefore, the endophytic fungus LH001 has great value in popularization and application of the biological control effect on the rice blast in the agricultural field.

Description

Technical Field

[0001] The invention relates to the technical field of plant disease prevention and control, and in particular to an endophytic fungus Pseudophialophorasp.LH001 and an application thereof in preventing and controlling rice blast. Background Art

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

[0003] The main methods for preventing and controlling rice blast include selecting disease-resistant varieties, cultivating high-quality seedlings, fertilizer and water management techniques, strengthening field management, implementing prevention and control measures, and chemical control. Cultivating disease-resistant varieties is one of the most economical and effective ways to prevent and control rice blast fungi, but due to the large number of physiological races of rice blast fungi, strong mutation, and fast adaptability, it faces great challenges in the cultivation of disease-resistant varieties. Chemical control is also a common method for preventing and controlling rice blast, but this method will cause environmental pollution and easily make rice blast fungi resistant to drugs. Therefore, seeking efficient, environmentally friendly, green, and safe biological control measures is a research hotspot for plant disease prevention and control.

[0004] Biological control is the use of living organisms or metabolically active ingredients produced by organisms to control plant pathogens. It has the advantages of being environmentally friendly, not easy to develop drug resistance, little harm to the natural enemies of harmful organisms, and low risk to human and animal health. Compared with traditional chemical control methods, it is more in line with the development trend of modern agriculture. The more common biological control measures are as follows: First, the application of antagonistic bacteria, which refers to beneficial bacteria that can produce metabolites such as antibiotics, enzymes, hormones, and can control pathogenic microorganisms through antibacterial and competition. Second, the application of bioremediation agents, which refers to microbial preparations that can decompose organic matter in the soil, build soil flora, and improve soil fertility. Third, the application of biological fertilizers, which refers to a fertilizer rich in beneficial microorganisms, achieves the effect of enhancing rice immunity by giving play to the dominant role of microbial communities in the soil environment. Fourth, the application of plant-derived biological preparations, which refers to some organic compounds extracted from plants or extracted by fermentation. These preparations can improve rice immunity through plant interaction mechanisms and effectively control rice blast.

[0005] There are a large number of beneficial microorganisms hidden in natural ecosystems, and endophytic fungi are one of them. Endophytic fungi refer to a type of fungi that can infect and colonize healthy plant tissues for at least part of their life cycle, and the host has no obvious symptoms. They are widely present in ecosystems and form a mutualistic symbiotic relationship with host plants, thereby promoting plant growth and improving the ability of plants to resist biotic and abiotic stresses. Therefore, endophytic fungi can be used as potential resource fungi such as biocontrol agents, biological nitrogen fixers, and plant growth promoters, and are widely used in crops such as grains, vegetables, and fruit trees.

[0006] Therefore, it is of great significance to discover beneficial microorganisms that have preventive effects on rice blast from plant endophytic fungi for the prevention and control of rice blast. Summary of the invention

[0007] The purpose of the present invention is to provide a wild rice endophytic fungus, which can improve the disease resistance of rice to rice blast through symbiotic interaction with rice, thereby achieving the prevention and treatment of rice seedling leaf blast.

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

[0009] The present invention isolates a new endophytic fungus of the genus Pseudophialophora from the root system of wild rice, and its main biological characteristics are: the colony diameter reaches 5.4 cm after growing at 25°C on a PDA plate for 12 days; the colony is yellow-green, the aerial hyphae are not well developed, creeping on the surface of the culture medium, and the hyphae have septa; the conidia are oval or pear-shaped and have no septa. Its ITS sequence is shown in SEQ ID NO.1, and the strain is identified as Pseudophialophora panicorum by constructing a phylogenetic tree. It is classified and named Pseudophialophora sp.LH001, and it was deposited in the China Center for Type Culture Collection on August 5, 2024, with a deposit address of Wuhan University, Wuhan, China, and a deposit number of CCTCC NO: M 20241751.

[0010] The culture conditions of the endophytic fungus LH001 strain are as follows: the endophytic fungus LH001 is inoculated into a PDA solid culture medium and cultured in the dark at 22-25° C. for 10-20 days.

[0011] The present invention has found that after the endophytic fungus LH001 is colonized in the root tissue of rice, the control effect on rice seedling leaf blight at the seedling stage reaches 94.05%, and the effect is significant.

[0012] Therefore, the present invention provides the use of the endophytic fungus LH001 in preventing and controlling rice blast.

[0013] The application comprises: colonizing the endophytic fungus LH001 in the root tissue of rice.

[0014] Furthermore, the application includes: co-culturing rice seeds with the endophytic fungus LH001 after germination, so that the fungus colonizes the roots of rice seedlings, so as to improve the resistance of rice seedlings to seedling leaf blight.

[0015] Furthermore, the seed germination method comprises: hulling the rice seeds, surface sterilizing them with 2% NaClO for 15 minutes, washing them with sterile water for 5 times, and then placing the sterilized seeds in 1 / 2MS culture medium, culturing them at 25°C, 16h light / 8h dark conditions, and waiting for them to germinate.

[0016] Furthermore, the rice seeds were surface sterilized and then germinated at 22-25°C. After the seeds turned white, they were transferred into 1 / 2MS culture medium and inoculated with endophytic fungus LH001 cake for co-cultivation.

[0017] Furthermore, the co-cultivation conditions are: culturing at 22-25° C. to the three-leaf one-heart stage, with 16 hours of light and 8 hours of dark cultivation per day.

[0018] The present invention has the following beneficial effects:

[0019] The present invention provides an endophytic fungus LH001, which can interact with rice to improve the resistance of rice to rice blast. The endophytic fungus LH001 strain is co-cultured with rice to colonize the roots of rice seedlings, which can reduce the leaf damage caused by rice blast pathogens and enhance the resistance of rice seedlings to leaf blast. According to statistics, the lesion rate of the control group was 49.88%, the lesion rate of the LH001 strain treatment group was 2.97%, and the control effect reached 94.05%. Therefore, the promotion and application of the biological control effect of the endophytic fungus LH001 on rice blast in the agricultural field has great value. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The colony, hyphae and spore morphology of LH001 strain. A is the colony morphology of LH001 on PDA plate; B and C are the hyphae and spore morphology under optical microscope, respectively, with a scale of 10 μm; D is the hyphae and spore morphology of LH001 under scanning electron microscope.

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

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

[0023] Figure 4 The control effect of LH001 strain on rice blast leaf blight. A is the disease incidence of the control group and LH001 group after spraying rice blast fungus on rice leaves; B is the lesion rate of the control group and LH001 group; C is the disease index of the control group and LH001 group. Significance (t-test): **, P<0.01; ***, P<0.001. DETAILED DESCRIPTION

[0024] The present invention will be further described below in conjunction with specific embodiments. The following embodiments are only used to illustrate the present invention and are not intended to limit the scope of application of the present invention. Without departing from the spirit and essence of the present invention, modifications or replacements made to the inventive method, steps or conditions all belong to the scope of the present invention.

[0025] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0026] Example 1: Isolation and identification of strain LH001

[0027] 1. Isolation and purification of LH001 strain

[0028] The LH001 strain was isolated from the roots of wild rice in Yunnan. The specific method is: rinse the wild rice roots continuously with tap water, and carefully remove soil particles and appendages. Select healthy root tissue and perform surface disinfection. First disinfect with 1% sodium hypochlorite for 20 minutes, and then wash 4 times with sterile deionized water. Then cut the root tissue into 0.5 cm small segments and put it into 2% malt extract agar medium (MEA, malt extract agar, OXOID; add 50 mg / L chloramphenicol to inhibit the growth of endophytic bacteria), and culture in the dark at 25°C. Observe whether hyphae grow every 24 hours. Carefully pick out the endophytic fungal hyphae growing from the edge of the tissue incision with a toothpick, transfer it to fresh PDA medium for purification and culture, and record the strain number as LH001.

[0029] 2. Identification of LH001 strain

[0030] 1. Morphological identification

[0031] After the LH001 strain was isolated and purified, it was inoculated on PDA medium and cultured at 25°C for 12 days. The morphology of colonies, hyphae and conidia was observed. Figure 1 As shown in A, the morphology of hyphae and conidia is as Figure 1B, 1C. Its morphological characteristics are: the colony grows slowly on the PDA plate, and the colony diameter reaches 5.4 cm after growing at 25°C for 12 days; the colony is yellow-green; the aerial hyphae are not well developed and creep on the surface of the culture medium; the hyphae have septa; the conidia are oval or pear-shaped and have no septa.

[0032] 2. Molecular identification

[0033] (1) DNA extraction

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

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

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

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

[0038] ⑤ Add an equal volume of isopropanol (analytical grade) to the supernatant, gently invert several times to mix, and centrifuge at 12000 rpm for 10 min to precipitate the nucleic acid;

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

[0040] ⑦Discard the supernatant and blow dry the centrifuge tube in a clean bench to allow the ethanol to fully evaporate;

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

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

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

[0044] The sequence of the upstream primer ITS1 was: 5′-TCCGTAGGTGAACCTGCGG-3′;

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

[0046] The PCR amplification reaction conditions were: 94°C pre-denaturation for 3 min, followed by 35 cycles including: 94°C denaturation for 30 sec, 58°C annealing for 30 sec, 72°C extension for 1 min, and finally 72°C extension for 10 min.

[0047] After electrophoresis detection, the PCR product was purified and recovered, and the recovered target DNA fragment was sent to Hangzhou Youkang Biotechnology Co., Ltd. for sequencing. After strict verification of the sequencing results, a DNA fragment sequence with a length of 496 bp as shown in SEQ ID NO.1 was obtained.

[0048] Combining biological characteristics and ITS rDNA gene comparison, an evolutionary tree was established ( Figure 2 ), LH001 was identified as belonging to Fungi, Ascomycota, Sordariomycetes, Magnaporthales, Magnaportaceae, Pseudophialophora, and the homology of the strain to Pseudophialophora panicorum strain CM9s6 reached 100%. Therefore, it was named Pseudophialophorasp.LH001, and was deposited in China Center for Type Culture Collection on August 5, 2024, with the deposit address: Wuhan, China, Wuhan University, and the deposit number is CCTCC NO: M 20241751. The collection center identified it as alive on August 12, 2024.

[0049] Example 2: Effect of strain LH001 on rice blast at seedling stage

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

[0051] 1. Activation culture of LH001 strain

[0052] The LH001 strain stored on the slant was inoculated on potato dextrose agar (PDA) solid medium for activation culture, cultured at 25°C in the dark for 10 days, and then used.

[0053] PDA solid culture medium: Each liter contains 20g glucose, 200g potato, and 15g agar. Weigh the required potatoes according to the volume of the culture medium to be prepared, boil them in water, mash them to dissolve, filter them, add glucose and agar, and sterilize them with high-pressure steam at 121℃ for 15 minutes.

[0054] 2. Co-cultivation of LH001 strain and rice

[0055] After the rice seeds are shelled, the shrunken grains, dry grains and insect grains are removed. The healthy rice seeds are disinfected with 70% alcohol for 5 minutes, and then disinfected with 1% NaClO for 15 minutes. They are washed with sterile water for 5 times and set aside. The disinfected seeds are transferred to 1 / 2MS culture medium with sterile tweezers, sealed with sealing film, and placed in a 25°C incubator for germination (16h light / 8h dark). After 3-4 days, the white seeds are inoculated into tissue culture bottles containing 1 / 2MS culture medium, with 10 seeds in each bottle and 3 LH001 bacterial cakes at the same time. The control group is inoculated with sterile PDA agar blocks. Each group is treated with 3 replicates. 25°C, 16h light / 8h dark, culture for 15-20 days to the three-leaf one-heart stage, and inoculate with rice blast fungus.

[0056] At the same time, to ensure that the fungus can colonize the rice roots, the rice roots inoculated with the GFP fluorescently labeled LH001 strain were cleaned, cut horizontally and vertically, and observed under a fluorescence confocal microscope. Figure 3 As shown in A and 3B, it was found that the LH001 strain could colonize the epidermis, cortex, and stele cells of rice.

[0057] 3. Spraying of rice blast conidia

[0058] The rice blast fungus strain Guy11 was inoculated on CM solid medium and cultured at 25°C for 12-14 days (12 h light / 12 h dark). The Guy11 conidia were washed with sterile water and filtered through three layers of filter paper to collect the spore suspension at a concentration of 2×10 5 Prepare 0.4% gelatin solution and mix it with the spore suspension in equal volumes.

[0059] CM medium (1L): Yeast Extract (1g), Casamino acid (1g), D-glucose (10g), KH2PO4 (1.52g), NaNO3 (6g), Peptone140 (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, add 15g / L agar to the solid medium. Autoclave at 121°C for 15min.

[0060] Use a sprayer to evenly spray the spore suspension onto the leaves of rice seedlings, with 1 mL per bottle. Place the tissue culture bottle in a 25°C incubator and culture in the dark for 2 days. Then, culture for 4-5 days with 16 hours of light / 8 hours of darkness, and calculate the disease index and lesion rate.

[0061] The results are as follows Figure 4As shown in the figure, the control group suffered from severe leaf blight, with a lesion rate of 49.88% and a disease index of 35.65. The LH001 strain-treated group suffered from milder disease, with a lesion rate of 2.97% and a disease index of 11.99. Compared with the control group, the lesion rate of the LH001 strain-treated group decreased by 46.91%, and the disease index decreased by 23.66, with a control effect of 94.05%, which was significant.

Claims

1. An endophytic fungus LH001, characterized in that The endophytic fungus LH001 is isolated from the root system of wild rice, classified and named Pseudophialophora sp. LH001, and preserved in the China Center for Type Culture Collection with a preservation number of CCTCC NO: M 20241751.

2. The endophytic fungus LH001 according to claim 1, characterized in that The ITS sequence of the strain is shown in SEQ ID NO.

1.

3. The endophytic fungus LH001 according to claim 1, characterized in that The colony diameter of the strain reached 5.4 cm after growing on a PDA plate at 25°C for 12 days; the colony was yellow-green, the aerial hyphae were not well developed, creeping on the surface of the culture medium, and the hyphae had septa; the conidia were oval or pear-shaped and had no septa.

4. The endophytic fungus LH001 according to claim 1, characterized in that The strain culture conditions are as follows: the endophytic fungus LH001 is inoculated into a PDA solid culture medium and cultured in the dark at 22-25° C. for 10-20 days.

5. Use of the endophytic fungus LH001 as claimed in claim 1 in preventing and controlling rice blast.

6. The use according to claim 5, characterized in that The application comprises: colonizing the endophytic fungus LH001 in the root tissue of rice.

7. The use according to claim 5, characterized in that The application comprises: co-culturing rice seeds with the endophytic fungus LH001 after germination, so that the endophytic fungus LH001 colonizes the roots of rice seedlings, so as to improve the resistance of rice seedlings to seedling leaf blight.

8. The use according to claim 7, characterized in that After surface disinfection, rice seeds were germinated at 22-25°C. After the seeds turned white, they were transferred into 1 / 2MS culture medium and inoculated with endophytic fungus LH001 cake for co-cultivation.

9. The use according to claim 7, characterized in that The co-cultivation conditions are: culturing at 22-25° C. until the three-leaf one-heart stage, with 16 hours of light and 8 hours of dark cultivation per day.

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