Fusarium endophytic fungus and application thereof
By inoculating rice with the endophytic fungus Fusarium annulatum, the problem of environmental stress in rice production was solved, the soil environment was improved and rice growth was promoted, the stress resistance and biomass of rice were increased, and a solution for sustainable agriculture was provided.
Patent Information
- Application Number
- CN202510803230.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-16
- Publication Date
- 2025-09-19
AI Technical Summary
Rice production faces multiple environmental stresses. The existing agricultural production model causes resource waste and ecological environment damage. Fusarium fungi often cause diseases that affect yield and commodity value. There is a lack of environmentally friendly and efficient disease prevention and control and promotion methods.
The endophytic fungus Fusarium annulatum is used to improve the soil environment, promote rice growth and enhance stress resistance. By inoculating Fusarium annulatum fungi, a symbiotic relationship is established in rice, enhancing its salt stress tolerance.
It significantly promotes the growth of rice seedlings, increases the biomass and antioxidant enzyme activity of rice, enhances salt tolerance, improves the root soil environment under salt stress, and enhances the stress resistance of rice.
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Figure CN120665723A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of rice and relates to an endophytic fungus of the genus Fusarium and an application thereof. Background Art
[0002] Endophytic fungi, as microorganisms that live in symbiosis with plants, complete some or all stages of their life cycle within the plant, typically without causing significant pathological damage to the host. In recent years, with the rapid development of molecular biology and omics technologies, the study of endophytic fungi has become a key research area in plant-microbe interactions. Research has demonstrated that the symbiotic relationship between endophytic fungi and plants is highly diverse and complex, playing an irreplaceable role in plant growth and development, enhancing stress tolerance, and maintaining ecosystem function. Endophytic fungi play a crucial protective role in plant stress tolerance. Studies have shown that endophytic fungi can enhance plant tolerance to biotic and abiotic stresses through a variety of mechanisms, including regulating hormone synthesis, signal transduction, and mineral absorption and utilization. Furthermore, endophytic fungi can enhance plant adaptation to adverse environments such as drought, salinity, and heavy metal pollution by producing antioxidants and stabilizing plant cell membrane structure, thereby enhancing plant resilience and protecting against environmental stresses. The symbiotic relationship between endophytic fungi and plants is not only of great ecological significance but also holds broad application prospects in agricultural production. By screening and utilizing beneficial endophytic fungal resources, new biofertilizers and biopesticides can be developed, reducing the use of chemical inputs and promoting the sustainable development of agriculture.
[0003] As the world's main food crop, rice faces severe challenges in its sustainable production due to multiple environmental stresses. The high-input model currently prevalent in agricultural production has ensured yields, but it has also resulted in resource waste and ecological damage. Studies have shown that the application of microbial fertilizers can effectively promote rice growth and development, ultimately increasing crop yields by increasing key yield components such as the number of grains per panicle and improving thousand-grain weight. With the continuous deepening of research, the potential of fertilizers and microorganisms in rice growth and development, improving stress resistance, and sustainable agricultural development has become increasingly prominent. Endophytic fungi can establish a unique mutually beneficial symbiotic relationship with host plants. When they colonize in the plant body, they not only do not affect the normal development of the host, but also produce a variety of growth-promoting effects. This special plant-microbe interaction model provides a new research direction for improving crop productivity.
[0004] Fusarium belongs to the Ascomycota, Hypodicotyledonous, Hypodicotales, and Hypodicotaceae families, and is widely distributed in nature. Fusarium fungi have strong adaptability to the environment and can survive in the soil for a long time. This pathogen can infect a variety of plant hosts, including food crops, cash crops, and ornamental plants, causing a variety of tissue diseases. The infected sites include the roots, stems, flowers, and ears of plants, mainly causing damage to the vascular system. At the same time, it secretes toxic metabolites, causing the plants to wilt or even die, seriously affecting the yield and commercial value of crops, and causing significant economic losses to agricultural production.
[0005] By deeply exploring the growth-promoting potential of Fusarium and developing new bio-fertilizers and bio-pesticides, it is expected to provide more environmentally friendly and efficient means of disease prevention and control and plant growth promotion for agricultural and forestry production. Summary of the Invention
[0006] The present invention provides an endophyte Fusarium annulatum Improve soil environment, promote rice growth and enhance stress resistance, and fully tap Fusarium annulatum The application value of this technology provides new solutions for sustainable agricultural development.
[0007] The present invention provides an endophytic fungus of the genus Fusarium, which is Fusarium annulatum 1#, deposited in the China General Microbiological Culture Collection Center, the deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, the deposit date is May 22, 2025, and the deposit number is CGMCC No. 41946.
[0008] The application of the endophytic fungus of the genus Fusarium of the present invention is characterized in that the endophytic fungus of the genus Fusarium is Fusarium annulatum , used to improve soil environment, promote rice growth and enhance rice resistance.
[0009] Furthermore, the promoting of rice growth is promoting the growth of rice under salt stress conditions.
[0010] Furthermore, the improvement of rice stress resistance is to improve rice salt tolerance.
[0011] Furthermore, the improvement of rice stress resistance is to increase the activity of antioxidant enzymes in rice leaves and roots, reduce the content of active oxygen, and enhance the stress resistance of rice.
[0012] Furthermore, the improving the soil environment is improving the soil environment of rice roots under salt stress conditions.
[0013] Furthermore, the improvement of the soil environment around the rice roots is to increase the pH value of the soil around the rice roots.
[0014] The present invention has the following beneficial effects: Endophytic fungi provided by the present invention Fusarium annulatum It has multiple effects in improving soil environment, promoting rice growth and improving stress resistance. Fusarium annulatum The fungus significantly promoted the growth and development of rice seedlings. Fusarium annulatum The strain has a significant promoting effect on the growth of rice seedlings and can increase physiological and biochemical indicators such as biomass, root length, and nutrient content of rice seedlings. Fusarium annulatum Fungi can improve the salt tolerance of rice. On the one hand, they can indirectly improve its salt tolerance by improving the physiological and biochemical indicators of rice. On the other hand, they can directly improve its salt tolerance by improving physiological indicators such as rice antioxidant enzymes. Fusarium annulatum In symbiosis with rice, the fungus not only promotes host growth and increases biomass, but also directly enhances the host's resistance to salt stress, fully demonstrating its excellent biological functions. This endophytic Fusarium has great potential application value and provides a new solution for the development of sustainable agriculture. BRIEF DESCRIPTION OF THE DRAWINGS
[0015] Figure 1 Figure 1 shows the isolation and purification of endophytic fungi from rice roots. Figure A shows the isolation and purification of endophytic fungi from rice roots. Figure a represents the control group, where 50 μL of sterile water used in the final rinse of the root segments was diluted and applied; Figure b represents the experimental group, where the washed and disinfected rice root segments were placed horizontally in the culture medium. Figure B shows the isolation and purification of endophytic fungi from rice roots. Figure 2 The chart below shows the salt tolerance of endophytic fungi in rice roots and the salt-alkali tolerance of fungus No. 1. A: salt tolerance analysis of endophytic fungi; B: salt-alkali tolerance analysis of fungus No. 1. Figure 3 The morphology of endophytic fungus No. 1 is shown; A: colony morphology of fungus No. 1 in culture medium, B: hyphae morphology of fungus No. 1, C: spore morphology of fungus No. 1; Figure 4 Identification and evolutionary analysis of fungus No. 1; A: ITS segment amplification, B: Evolutionary tree analysis based on ITS; Figure 5 for Fusarium annulatum Pathogenicity analysis diagram; Figure 6 Under salt stress Fusarium annulatum Analysis of the effects on rice growth and development; A: Phenotype of rice grown in culture medium for 7 days under 150 mM NaCl stress and the results of rice root length measurement; B: Phenotype of rice grown in pots under 150 mM NaCl stress for 4 weeks and the results of rice root length measurement; Figure 7 Rice under salt stress Fusarium annulatumColonization analysis and fungal structure observation; A: Trypan blue staining of endophytic fungi in rice roots, B: fungal hyphae and vesicle structure, C: microsclerotia, D: vesicle, E: endospore structure observation; Figure 8 for Fusarium annulatum Analysis of the impact on superoxide anion and H2O2 content in rice leaves; DAB and NBT staining Fusarium annulatum Effects of inoculation conditions on H2O2 and superoxide anion contents in rice leaves grown in salt-stressed pots, A: DAB staining, B: NBT staining; Figure 9 Under salt stress Fusarium annulatum Analysis of the effects on various enzyme activities of rice; among them, A: Fusarium annulatum Effects on enzyme activity in rice leaves, B: Fusarium annulatum Effects on enzyme activities in rice roots; Figure 10 Under salt stress Fusarium annulatum Analysis of the impact of soil pH on rice roots. DETAILED DESCRIPTION
[0016] In order to make the objectives, technical solutions and advantages of the embodiments of the present invention more clearly understood, the spirit of the contents disclosed in the present invention will be described in detail below. After understanding the embodiments of the contents of the present invention, any technician in the relevant technical field can change and modify the contents of the present invention based on the techniques taught by the contents of the present invention without departing from the spirit and scope of the contents of the present invention.
[0017] The exemplary embodiments of the present invention and the description thereof are used to explain the present invention but are not intended to limit the present invention.
[0018] Example 1 Isolation, Screening and Identification of Endophytic Fungi from Rice Roots 1 Experimental Materials 1.1 Plant materials Two-month-old Songjing No. 2 rice seedlings were dug out from pots and stored in a refrigerator at 4°C. Several sections of young rice fibrous roots were selected as experimental materials.
[0019] 1.2 Strains Rhizoctonia solaniAG1-IA (Rice sheath blight pathogen).
[0020] 2 Experimental methods 2.1 Preparation of saline-alkali stress culture medium (1) PDA solid culture medium with different NaCl concentrations Add 46 g of PDA powder to 1 L of water, and add 11.688 g, 23.376 g, and 35.064 g of NaCl, respectively, to prepare PDA culture media with NaCl concentrations of 200 mmol / L, 400 mmol / L, and 600 mmol / L. Sterilize at 115°C for 20 min.
[0021] (2) PDA solid culture medium with different NaHCO3 concentrations Add 46 g of PDA powder to 1 L of water, sterilize at 115°C for 20 min, cool to 45°C, and add 80 μL, 160 μL, and 240 μL of 1 M NaHCO3 stock solution, respectively, to prepare PDA culture medium with NaHCO3 concentrations of 200 mmol / L, 400 mmol / L, and 600 mmol / L.
[0022] 2.2 Rice root disinfection and isolation of endophytic fungi This example uses a direct root segment culture method to isolate endophytic fungi from plant roots. Take a small amount of tender, young, fibrous roots from rice plants and cut the roots into several 1-2 cm segments with a knife. Wrap the segments in gauze and rinse with running water for 1 hour. Sterilize the sections in a sterile operating table with 75% alcohol and 0.1% NaClO for 1 minute each, then rinse with sterile water for 1 minute, repeating three times. Use tweezers to pick up 6-9 root segments and place them flat on PDA culture medium (sterile-added streptomycin at a final concentration of 30 mg / L at a temperature below 60°C) as the experimental group. Use 50 μL of the sterile water diluted from the last rinse of the root segments to form a control group, then incubate in a dark incubator at 28°C for one week.
[0023] 2.3 Purification and preservation of strains When hyphae from the root segments on the culture medium form colonies, isolate the edge of the hyphae and transfer them to PDA medium. Purify the culture by incubating in the dark at 28°C for 7 days. Observe the consistency of the colony morphology and decide whether to repurify the strain until the colonies have consistent morphological characteristics. The resulting purified strain is plated on a slant medium and refrigerated at 4°C. It is then stored at -80°C in a 20% v / v glycerol suspension for further study.
[0024] 2.4 Colony gradient salt-alkali tolerance test In order to detect the effects of different concentrations of salt stress on the growth and other characteristics of endophytic fungi, the isolated and purified endophytic fungi were inoculated on PDA culture media with four NaCl concentrations (0mM, 200mM, 400mM, 600mM) and four NaHCO3 concentrations (0mM, 200mM, 400mM, 600mM), respectively. After cultured in a constant temperature incubator at 28°C in the dark for 5 days, the colony size was observed and photographed.
[0025] 2.5 Preliminary classification of endophytic fungi and extraction of fungal genomic DNA The strain was inoculated onto PDA medium and incubated in the dark at 25°C for one week. Preliminary identification was performed by observing colony morphology (including colony form, color, diameter, spore color, and growth rate) according to the Fungal Identification Manual. DNA was extracted using the Ezup Column-Based Fungal Genomic DNA Extraction Kit (B518255) according to the manufacturer's instructions.
[0026] 2.6 DNA electrophoresis detection DNA electrophoresis was performed (1.5% agarose, 1× TAE electrophoresis buffer, observation).
[0027] 2.7 PCR amplification 2.7.1 Universal primer sequences for fungi Table 1 Common primer sequences for fungi ; 2.7.2 PCR reaction system Table 2 PCR reaction system ; 2.7.3 PCR reaction conditions Table 3 PCR reaction conditions ; 2.7.4 PCR electrophoresis Electrophoresis was performed on 1.5% agarose gel, 1×TAE, 150 V, 100 mA, for 20 min.
[0028] 2.8 Identification of endophytic fungus species No. 1 in rice roots The strain was identified using 16S rRNA gene sequence analysis. Genomic DNA was first extracted, amplified by PCR, and sequenced. Sequence alignment and similarity analysis were then performed using the EzBioCloud platform. Multiple sequence alignment was performed using MEGA 7.0 software, based on the Kimura two-parameter model, and the ClustalW algorithm.
[0029] 2.9 Sequencing of PCR Products and Construction of ITS-Based Phylogenetic Trees The PCR products obtained by gel recovery were sent to the sequencing department of Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The sequencing results were compared on the NCBI website to identify and classify the fungi.
[0030] The predicted ITS sequences were aligned with the NCBI nt database ITS sub-library using the Blast tool, setting a similarity threshold of >95%. The 30 sequences with the highest similarity (or all if less than 95%) were selected for multiple sequence alignment and pruning using mafft software. A phylogenetic tree was then constructed using FastTree software.
[0031] 2.10 Pathogenicity analysis of endophytic fungus No. 1 in rice roots R. solani AG1-IA (rice sheath blight pathogen) and endophytic fungus No. 1, stored at 4°C, were inoculated onto PDA (containing 0.4% streptomycin) culture plates under a sterile workbench and incubated upside down at 28°C for approximately 48 hours to activate the strains. Sterile rice seedlings of uniform growth were selected and neatly arranged in a disposable sterile Petri dish. Two segments were secured with a thin glass strip. Small wounds were uniformly inflicted on the underside of the leaves using a sterile disposable syringe needle. Fungal masses (0.7 cm in diameter) of the same age were selected and inoculated into the wounds. The plates were sprayed with an appropriate amount of sterile water and incubated at 28°C (to facilitate infection by the sheath blight pathogen). Images were taken every 24 hours. After 7 days of co-cultivation, the effects of the fungus on the leaves were observed. Each experiment was replicated three times (all instruments used for the inoculation experiment were sterilized by high-temperature and high-pressure wet heat).
[0032] 3 Results 3.1 Isolation and purification of endophytic fungi from rice roots After culturing the sterilized rice roots in PDA medium for 7 days, 8 fungal colonies were isolated and purified ( Figure 1 Ab), and no colonies grew on the control plate coated with sterile water for cleaning the roots ( Figure 1 Aa). This indicates that the colonies growing on the culture medium are endophytic fungi from rice roots and have been successfully isolated. Further purification and morphological observation of the eight endophytic fungi revealed that 1#, 4#, 7#, and 8# endophytic fungi were pale yellow fuzzy colonies. 2#, 3#, 5#, and 6# fungi were milky white cotton-like colonies ( Figure 1 B).
[0033] 3.2 Analysis of salt-alkali tolerance characteristics of endophytic fungi in rice roots Analysis of the resistance of endophytic fungi to salt and alkali stress. Seven endophytic fungi (4# fungus contamination was absent) were cultured under salt conditions (0mM, 200mM, 400mM, and 600mM) for 5 days and then their colony sizes were observed. It was found that the colony sizes of endophytic fungi 1#, 3#, 7#, and 8# under high salt concentration of 600mM were close to those under salt-free conditions, while the colony growth of endophytic fungi 2#, 5#, and 6# was inhibited under 200mM salt conditions, and colony growth almost stopped under 600mM salt conditions ( Figure 2 A). Therefore, endophytic fungi of 1#, 3#, 7#, and 8# are salt-tolerant. In particular, the colony color, diameter, and density of endophytic fungi of 1# are close to those of colonies in salt-free conditions under various salt concentrations, indicating that endophytic fungi of 1# have halophilic biological characteristics and are significantly salt-tolerant ( Figure 2 B left).
[0034] At the same time, the growth status of endophytic fungus No. 1 after 5 days of cultivation under different concentrations (0mM, 200mM, 400mM, 600mM) of alkali showed that endophytic fungus No. 1 could still grow well under 200mM alkali stress conditions. Therefore, endophytic fungus No. 1 also has good alkali resistance ( Figure 2 B right).
[0035] 3.3 Morphological characteristics of endophytic fungus No. 1 Detailed observation of the morphology of fungus No. 1 after 7 days of growth on PDA medium showed that fungus No. 1 showed rapid growth characteristics. The colony was initially white aerial hyphae. As the colony expanded, the middle part gradually turned light yellow, and the edges remained white. Conidia were mainly born at the end of the short branches of hyphae, with a sickle shape, a pointed and slightly curved tip ( Figure 3 C).
[0036] 3.4 Identification and evolutionary analysis of endophytic fungus No. 1 In order to identify the species of endophytic fungus No. 1, the genomic DNA of fungus No. 1 was extracted and the ITS segment was amplified by PCR using universal primers ITS1 and ITS4. The target product fragment size was 533 bp ( Figure 4 A). The obtained ITS sequence was compared with the ITS sub-library in the NCBI database using the Blast method, with the parameter identify>95. The results showed that fungus No. 1 was the same as Fusarium circulans U61670 s Fusarium annulatum Has the greatest similarity, with a similarity rate of 99.8% ( Figure 4 A).
[0037] The top 30 ITS sequences with the highest identify were selected, sequence multiple alignment and shearing were performed using the mafft software, and the phylogenetic tree was constructed using the FastTree software ( Figure 4 B) Observing the bacterial community structure and the evolutionary relationship between different bacterial species Fusarium annulatum These results indicate that fungus No. 1 belongs to the genus Fusarium and the strain name is Fusarium annulatum .
[0038] 3.5 Fusariumannulatum Pathogenicity analysis Research has shown that Fusarium is often a pathogenic bacteria. R.solani AG1-IA (Rhizoctonia solani) as a control, the Fusariumannulatum The results showed that the pathogenicity of sterile inoculation and inoculation R.solani AG1-IA The rice leaves turned slightly darker and dried up after 3 days of cultivation, and the inoculated R.solani AG1-IA The blackening degree of rice leaves in the sterile inoculation group was more serious than that in the sterile inoculation group. Fusarium annulatumThe leaves are in good growth condition ( Figure 5 (middle picture). Continuing to observe after seven days, it was found that R.solani AG1-IA Most of the leaf tissues have died, but the inoculated Fusarium annulatum There is no obvious disease on the rice leaves. Fusarium annulatum The growth rate was extremely fast, but the damage to the leaves was minimal. These results indicate that Fusarium annulatum It is basically harmless to the growth of leaves and is a non-pathogenic symbiotic fungus ( Figure 5 ).
[0039] Example 2 Fusarium annulatum Effects of endophytic fungi on rice growth and development 1. Experimental Materials (1) Rice: The japonica rice variety Songjing No. 2, which is cultivated in northern China, was selected.
[0040] (2) Arabidopsis: wild-type Arabidopsis (Arabidopsis thaliana, Columbia) seeds.
[0041] 2. Experimental Methods 2.1 Preparation of saline-alkali stress culture medium (1) MS solid culture medium with different NaCl concentrations 8.766 g and 11.688 g of NaCl were added to 1 L of MS liquid culture medium to prepare MS culture medium with NaCl concentrations of 150 mmol / L and 200 mmol / L, respectively, and sterilized at 121°C for 20 min.
[0042] (2) MS solid culture medium with different NaHCO3 concentrations 1 L of MS liquid culture medium was sterilized at 121°C for 20 min, cooled to 45°C, and 2 μL and 4 μL of 1 M NaHCO3 stock solution were added to prepare MS culture medium with NaHCO3 concentrations of 5 mmol / L and 10 mmol / L, respectively.
[0043] 2.2 Fungal culture and preparation of fungal conidia The strain preserved in Example 1 was taken out from the -70°C freezer, and the bacterial block in the cryovial was inoculated into the PDA culture medium in a sterile operating table, and cultured in the dark at 25°C for 3-5 days until the mycelium germinated. The mycelium was then transferred to fresh PDA culture medium under sterile conditions and cultured in the dark at 25°C for 7-10 days until conidia were produced. In a sterile operating table, 5-8 mL of sterile saline (0.85%) was injected into the PDA plate, and the spores and mycelium were fully washed off with a coating rod. Filter through a double layer of sterile gauze to remove the mycelium and culture medium residue to obtain a spore suspension. Spores were counted under an optical microscope using a hemocytometer, and the spore concentration was adjusted to 1×105 pieces / mL.
[0044] 2.3 Rice seedling cultivation in culture medium and pots (1) Rice in culture medium: Songjing No. 2 rice seeds were disinfected with 75% alcohol and 3% NaClO and then cultured in 1 / 8 MS culture medium at 23°C. After the rice seeds germinated and the embryos appeared, rice seedlings with basically the same growth and size were selected and transferred to salt stress culture medium for use.
[0045] (2) Rice in pots: Use tweezers to transfer pre-selected rice seedlings of uniform size and consistent growth status from the rooting medium to a potting container (normal soil) for later use.
[0046] (3) Arabidopsis thaliana culture: Rinse Arabidopsis thaliana seeds with 75% alcohol and 0.1% NaClO (shaking and inverting continuously), rinse with sterile water four times, and evenly inoculate on the surface of 1 / 2 MS culture medium. Vernalize at 4°C in the dark for 3 days and transfer to a plant culture room with a constant temperature of 23°C and 16 h light and 8 h dark per day.
[0047] 2.4 Experimental design of fungal inoculation under salt stress conditions (1) Rice in culture medium Rice seedlings of the same growth and development status were transplanted using MS medium containing 150 mM NaCl. They were divided into one treatment group and one control group. The control group was sterile inoculated MS medium, and the treatment group was Fusarium annulatum Spore suspension (1×10 5 Rice roots were inoculated with 1 μL of spore solution (1 μL / mL) using sterile conditions throughout the inoculation process. Each sterile seedling in the treatment group was inoculated with 1 μL of spore solution using a pipette, while each sterile seedling in the control group was inoculated with 1 μL of sterile water using a pipette and evenly applied to the root system. The inoculated and transplanted seedlings were cultured in a plant incubator at 23°C with 16 hours of light and 8 hours of darkness daily for 7 days.
[0048] (2) Rice in pots The rice seedlings were divided into 1 treatment group and 1 control group, with 2 pots in each group and 4 seedlings planted in each pot. Fusarium annulatum (1×10 4 The rice plants were inoculated with a spore suspension (1 mL / mL) of spores per pot, mixed with 200 mL of sterile water, and watered. This was repeated three days later, for a total of two inoculations. For the control group, each bottle was inoculated with 200 mL of sterile water. After 10 days of growth, the rice plants were watered daily with 100 mL of a 200 mM NaCl solution. The plants were cultured in a constant temperature plant room at 23°C with 16 hours of light and 8 hours of darkness per day for a total of 30 days.
[0049] (3) Arabidopsis thaliana in culture medium Divide into 1 treatment group and 1 control group, each group has 3 plates, and each plate is planted with 6 Arabidopsis thaliana plants. Fusarium annulatum Spore suspension (1×10 5 Inoculation was performed using a microporous filter (MS / mL). Sterile conditions were maintained throughout the inoculation process. Each sterile Arabidopsis seedling in the treatment group was inoculated with 1 μL of spore solution using a pipette, while each sterile seedling in the control group was inoculated with 1 μL of sterile water using a pipette and evenly dripped onto the root system. The inoculated and transplanted seedlings were incubated in a plant culture room at 23°C with 16 hours of light and 8 hours of darkness daily for 7 days.
[0050] 2.5 Experimental design of fungal inoculation under alkaline stress conditions (1) Rice in culture medium: Divided into 1 treatment group and 1 control group, the alkaline stress culture medium is 5mM NaHCO3 and 10mM NaHCO3 MS medium. Each group has 3 plates, and 5 Songjing No. 2 rice seedlings are planted in each plate. Fusarium annulatum Spore suspension (1×10 5 Inoculate the seedlings with 1 μL of spore solution (100 μg / mL) using strict aseptic techniques. Each sterile seedling was inoculated with 1 μL of spore solution using a pipette and evenly dripped onto the roots. For the control group, each sterile seedling was inoculated with 1 μL of sterile water using a pipette and evenly dripped onto the roots. The inoculated and transplanted seedlings were cultured in a plant culture room at 23°C with 16 hours of light and 8 hours of darkness daily for 7 days.
[0051] (2) Rice in pots: Divide into 1 treatment group and 1 control group, with 2 pots in each group and 4 seedlings planted in each pot. After the rice seedlings grow for 2 weeks, the treatment group is used Fusarium annulatum (1×10 4 The rice plants were inoculated with a spore suspension (1 mL / mL) of spores per pot and mixed with 200 mL of sterile water. This was repeated three days later, for a total of two inoculations. For the control group, each pot was inoculated with 200 mL of sterile water. After 10 days of growth, the rice plants were watered daily with 100 mL of a 15 mM NaHCO solution. The plants were cultured in a constant temperature plant room at 23°C with 16 hours of light and 8 hours of darkness per day for a total of 30 days.
[0052] 2.6 Trypan blue staining observation A small amount of rice seedling roots from the experimental group were taken, and the agar or other appendages on the root surface were cleaned with sterile water. The roots were fixed in 50% ethanol for more than 24 hours; then washed with deionized water 3-4 times to remove the ethanol; then the roots were placed in a 5% KOH solution (85℃ water bath) for decolorization for 15-30 minutes (if the roots are young, the temperature and decolorization time can be reduced), and then washed again with sterile deionized water several times to remove the KOH solution, otherwise it will affect the subsequent staining; if the root color is dark, 15% hydrogen peroxide can be added for bleaching, followed by adding ammonia water to a final concentration of 0.05% for about 2-3 minutes; then washed with deionized water; then the roots were acidified with 2% lactic acid for 1 minute; finally, they were stained with 0.05%-0.1% trypan blue for 2-3 hours (adjusted according to the tenderness of the roots) and counterstained in 50% glycerol for 48 hours; the root tissue slides were placed in 50% glycerol for observation. The infection structural characteristics of endophytic fungi were observed under a fluorescence microscope.
[0053] 2.7 Rice root length measurement Rice in culture medium: After 7 days of co-cultivation, the experimental seedlings were harvested. After removal from the culture dish, the culture medium on the roots was washed away with sterile purified water. The sterile purified water on the surface of the seedlings was blotted dry with filter paper. The rice plants were photographed and the root length was measured using ImageJ software (www.imagej.nih.gov / ij / ). The average value was used for further analysis.
[0054] Rice in pots: After four weeks of co-cultivation, the experimental seedlings were harvested. After removing the rice from the pots, the soil surrounding the rhizosphere was washed with sterile purified water. The sterile purified water on the surface of the seedlings was blotted dry with filter paper. Images of the rice plants were taken, and the root lengths were measured using ImageJ software (www.imagej.nih.gov / ij / ). The average values were used for further analysis.
[0055] 2.8 Determination of antioxidant enzyme activities and ROS content in rice 2.8.1 Determination of antioxidant enzyme activities in leaves (1) Catalase (CAT) activity assay Weigh 0.1 g of the plant tissue to be tested into a pre-chilled mortar. Liquid nitrogen was added to the mortar in small amounts several times. After thorough grinding, 1 mL of pre-chilled PBS buffer (pH 7.8) was added for collection. Mix thoroughly by pipetting, centrifuge at 12,000 rpm for 15 minutes, and aspirate the supernatant for a second centrifugation. The second supernatant is the crude catalase extract and is stored at 4°C. Phosphate buffer, crude enzyme extract, and H2O were added to the experimental group, while the crude enzyme extract was not added to the control group. After preheating to 25°C, 0.03 mol / L H2O2 was added to each tube. Absorbance was measured at 240 nm for 30 seconds each, for 10 cycles.
[0056] (2) Superoxide dismutase (SOD) activity assay The samples were treated in the same manner as in (1) to obtain a crude superoxide dismutase extract. An experimental group and two control groups, one under light and one under darkness, were set up. The crude enzyme extract and reaction reagents (130 mM / L methionine, 750 μM / L nitroblue tetrazolium, 100 μM / LEDTA-Na2, and 20 μM / L riboflavin) were added to the experimental group, while the control group was treated with water instead of the crude enzyme extract. The absorbance was measured at 560 nm.
[0057] (3) Peroxidase (POD) activity assay The samples were treated in the same manner as in (1). 1 mL of pre-cooled phosphate buffer (pH = 7.0) was added, pipetting and mixing was performed, and the mixture was centrifuged at 12000 rpm for 15 min. The supernatant was aspirated and centrifuged again. The second supernatant was the crude peroxidase extract and stored at 4°C. The crude enzyme extract and reaction reagent (0.05 mol / L guaiacol, pH = 7.0 phosphate buffer, 40 mmol / L H2O2) were added to the experimental group, and H2O and reaction reagent were added to the control group. The absorbance was measured at 470 nm for 10 s each time for 6 cycles.
[0058] (4) Ascorbate peroxidase (APX) activity assay The samples were treated as in (1) to obtain a crude ascorbate peroxidase extract, which was then stored at 4°C. The crude enzyme extract and reaction reagents (5 mM / L ascorbic acid, 1 mM / L EDTA-Na₂, 1 mM / L H₂O₂) were added to the experimental group, while the crude enzyme extract was replaced with water in the control group. The absorbance was measured at 290 nm for 10 s each, for 6 cycles.
[0059] (5) Determination of soluble protein content The soluble protein content was determined using Coomassie Brilliant Blue. 30 μL of enzyme solution was added to 200 μL of Coomassie Brilliant Blue staining solution. After standing at room temperature for 5 min, the OD value was measured at a wavelength of 595 nm.
[0060] 2.8.2 Determination of leaf reactive oxygen species levels Determination of H2O2 content 0.2 g of rice leaves grown under various culture conditions for 60 days were weighed and ground into powder under liquid nitrogen. 1 mL of 0.1% trichloroformic acid (TCA) was added and centrifuged at 19,000 g for 20 min. The supernatant was collected.
[0061] (2) DAB staining Select rice leaves of well-grown, appropriately sized leaves in the tillering stage and place them in DAB staining solution (containing 0.1 mg DAB, 1 mL 0.05 M Tris-HAC, pH 5). Stain at 28°C in the dark for 6 h. After the reaction is complete, pour out the excess staining solution, add 80% ethanol, and boil at 100°C for more than 15 min. During this period, continuously change the ethanol until the leaves are completely decolorized. Then, add anhydrous ethanol again and take pictures under a microscope.
[0062] (3) NBT staining Select rice leaves at the tillering stage with good growth and appropriate size, place them in NBT staining solution (containing 0.5 mg NBT, 1 mL 25 mM HEPES, pH 7.6), and stain them in the dark at 28°C for 6 h. After the reaction is completed, pour out the excess staining solution, add 80% ethanol, and boil at 100°C for more than 15 min. During this period, continuously change the ethanol until the leaves are completely decolorized. Then add anhydrous ethanol again and take pictures under a microscope.
[0063] 2.8.3 Measurement of N, P, and K Content in Leaves and Roots After rinsing the roots of rice plants with sterile water, the surface moisture was blotted with filter paper. The plants were separated from the rhizome junction, and the roots and aerial parts were wrapped in red wax paper and dried in a 65°C oven for 3 days to constant weight. The roots and aerial parts were ground into powder using a ball mill and digested with sulfuric acid-hydrogen peroxide. Total nitrogen content was determined by the Kjeldahl method, total phosphorus content was determined by the molybdenum antimony colorimetric method, and total potassium content was analyzed by flame photometry.
[0064] 2.8.4 Soil pH measurement Soil samples were completely saturated with water at a ratio of 1:2.5 w / v and centrifuged at room temperature for 15 minutes to obtain a soil-water suspension. pH was measured using a pH meter by immersing the electrode in the supernatant and recording the pH value after the reading stabilized. This was repeated three times.
[0065] result 3.1 Under salt stress Fusarium annulatum Analysis of the impact on rice growth and development Under salt stress Fusarium annulatum Functional analysis, sterile culture conditions, inoculation Fusarium annulatum The plant height and root length of rice grown under 150mM NaCI for one week were higher than those of the control group, especially the root length increased significantly ( Figure 6 A). This shows that the inoculation under salt stress Fusarium annulatum It can promote the growth of rice seedlings.
[0066] Analyzed under potted conditions Fusarium annulatumThe results showed that under 200mM NaCI salt stress, the rice leaves of the control group after 4 weeks of growth showed yellowing and wilting, and the corresponding inoculated Fusarium annulatum The rice leaves of the group grew well, and the plant height and root length were significantly higher than those of the control group ( Figure 6 B). This shows that under the salt stress condition, Fusarium annulatum It can also significantly promote rice growth. These results prove Fusarium annulatum Endophytic fungi can improve the tolerance of rice to salt stress.
[0067] 3.2 Rice under salt stress Fusarium annulatum Colonization analysis Trypan blue staining was used to analyze the Fusarium annulatum Observation of the rice roots after 7 days of inoculation revealed that there were a large number of hyphae and spores on the rice roots, while there was no obvious fungal colonization on the sterile inoculated rice roots, and the fungal content in the roots was similar to that in the inoculated rice roots. Fusarium annulatum The difference between the groups is large ( Figure 7 A). This proves that rice roots are inoculated Fusarium annulatum The feasibility of the method. At the same time, in-depth observation revealed a large number of hyphae ( Figure 7 B) Microsclerotia ( Figure 7 C) Vesicles ( Figure 7 D) Endospores ( Figure 7 E) Equi-structures exist for inoculation Fusarium annulatum of rice roots.
[0068] 3.3 Salt stress Fusarium annulatum Analysis of the impact on the physical and chemical properties of rice 3.3.1 Fusarium annulatum Analysis of the effects on ROS content in rice leaves The DAB and NBT staining methods were used to investigate Fusarium annulatum Effects of inoculation on the contents of H2O2 and superoxide anions in potted rice leaves under salt stress. Fusarium annulatum The brown spots on rice leaves were lighter than those in the control group ( Figure 8 A). At the same time, NBT staining showed that the inoculation Fusariumannulatum The color and number of blue spots on rice leaves were significantly lower than those in the control group ( Figure 8 B). Therefore, Fusarium annulatum Inoculation plays an important role in preventing the accumulation of superoxide anions and H2O2 in rice leaves.
[0069] 3.3.2 Under salt stress Fusarium annulatum Analysis of the effects on rice enzyme activity In order to investigate the salt stress Fusarium annulatum Effects of inoculation on rice enzyme activity under 200mmolNaCl stress Fusarium annulatumThe activities of various antioxidant enzymes and H2O2 content in the leaves of inoculated potted rice were determined. Fusarium annulatum The activities of soluble protein, POD, SOD and CAT in rice leaves were higher than those without Fusarium annulatumu The APX activity of the control group was slightly lower than that of the control group. Fusarium annulatum The H2O2 content in the inoculated rice leaves was significantly lower than that in the control group, which was consistent with the results of DAB and NBT staining ( Figure 9 A). At the same time, under 200mmol NaCl stress treatment Fusarium annulatum The activities of various antioxidant enzymes and H2O2 content in the roots of inoculated potted rice were also measured. The results showed that Fusarium annulatum The soluble protein, POD and CAT activities in the roots of potted rice were higher than those in the control group, while the APX and SOD activities were slightly lower than those in the control group, and the H2O2 content was lower than that in the control group ( Figure 9 B). This indicates Fusarium annulatum The trends of physiological indicators of inoculated rice roots and rice leaves were basically consistent.
[0070] 3.3.3 Under salt stress Fusarium annulatum Analysis of the impact on N, P and K content of rice vaccination Fusarium annulatum Analysis of the physical and chemical properties of potted rice leaves showed that Fusarium annulatum The total nitrogen and total phosphorus contents in the inoculated rice leaves were significantly higher than those in the control group, and the total potassium content was slightly higher than that in the control group (Table 4). Fusarium annulatum Inoculation increased the N, P and K contents in potted rice leaves.
[0071] Table 4 Under salt stress Fusarium annulatum Analysis of the effects on N, P and K content in rice leaves ; The control group was rice that was not inoculated with endophytic fungi under salt stress conditions, and the experimental group was rice that was inoculated with endophytic fungi under the same salt stress conditions. Fusarium annulatum Rice. The same letters in the same column represent the mean value. p The differences were not significant at the 0.05 level; the data are the means ± standard errors of three replicates.
[0072] Further measurement of root N, P, and K content showed that the inoculation Fusarium annulatum The total nitrogen and total potassium contents of the rice roots of the treated rice were slightly higher than those of the control group, and the total phosphorus content was slightly lower than that of the control group (Table 5). Fusarium annulatum Inoculation had no significant effect on the N, P, and K contents in rice roots.
[0073] Table 5 Under salt stress Fusarium annulatum Analysis of the impact on N, P and K content in rice roots ; The control group was rice that was not inoculated with endophytic fungi under salt stress conditions, and the experimental group was rice that was inoculated with endophytic fungi under the same salt stress conditions. Fusarium annulatum Rice. The same letters in the same column represent the mean value. p The differences were not significant at the 0.05 level; the data are the mean ± standard error of three replicates.
[0074] 3.3.4 Under salt stress Fusarium annulatum Analysis of the impact of soil pH on rice roots Under salt stress conditions, inoculation Fusarium annulatum The rhizosphere soil of the potted rice was completely saturated with water at a ratio of 1:2.5 (w / v). After obtaining the soil-water suspension, the soil pH was measured. The results showed that the pH of the control group soil was 6.13. Fusarium annulatum The pH value of rice rhizosphere soil was 6.38. Fusarium annulatum The pH of rice rhizosphere soil was slightly higher than that of the control group, and had no significant effect on soil pH ( Figure 10 ).
[0075] It can be seen that the present invention has been confirmed under sterile culture conditions. Fusarium annulatum Inoculation can significantly promote the growth and development of rice in salt and alkali stress environments. The results of rice pot experiments confirmed that Fusarium annulatum It can effectively improve the tolerance of rice to saline-alkali stress. Microscopic observation of rice roots after 7 days of inoculation was conducted by trypan blue staining. Fusarium annulatum The successful colonization of rice roots was manifested by the presence of a large number of hyphae and spores, while no obvious fungal colonization was observed in the uninoculated control group, and the fungal content was significantly lower than that in the inoculated group, which fully confirmed that Fusarium annulatum Feasibility and effectiveness of vaccination methods.
[0076] At the physiological and biochemical level, studies have found Fusarium annulatum Inoculation can effectively inhibit the accumulation of superoxide anions and hydrogen peroxide in rice leaves, while significantly increasing the activity of antioxidant enzymes and the content of essential nutrients such as nitrogen, phosphorus, and potassium.
Claims
1. An endophytic fungus of the genus Fusarium, characterized in that: It is Fusarium annulatum 1#, deposited in the China General Microbiological Culture Collection Center, the deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, the deposit date is May 22, 2025, and the deposit number is CGMCC No. 41946.
2. The use of an endophytic fungus of the genus Fusarium according to claim 1, characterized in that: The endophytic fungus of the genus Fusarium is Fusarium annulatum , used to improve soil environment, promote rice growth and enhance rice resistance.
3. The use according to claim 2, characterized in that The promoting of rice growth is promoting the growth of rice under salt stress.
4. The use according to claim 2, characterized in that The method of improving the stress resistance of rice is to improve the salt tolerance of rice.
5. The use according to claim 2, characterized in that The method for improving the stress resistance of rice is to improve the activity of antioxidant enzymes in rice leaves and roots, reduce the content of active oxygen, and enhance the stress resistance of rice.
6. The use according to claim 2, characterized in that The improving of the soil environment is to improve the soil environment of rice roots under alkaline stress conditions.
7. The use according to claim 6, characterized in that The improving the soil environment around the rice roots is to increase the pH value of the soil around the rice roots.