Strain of salt-tolerant branch of 550w1 and its application to promoting nodulation and nitrogen fixation of leguminous plants

Abstract

This invention discloses a salt-tolerant Cladosporium 550W1 strain and its application in promoting nodulation and nitrogen fixation in leguminous plants, belonging to the field of microbial technology. The salt-tolerant Cladosporium (… Cladosporium halotolerans Cladosporium 550W1, deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 41943, provides a salt-tolerant Cladosporium 550W1 that can significantly promote nodulation and nitrogen fixation in legumes, increase plant biomass and nitrogen accumulation, and reduce dependence on chemical nitrogen fertilizers. It has broad application prospects and environmental and ecological benefits.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a salt-tolerant Cladosporium 550W1 strain and its application in promoting nodulation and nitrogen fixation in legumes. Background Technology

[0002] The symbiotic nitrogen fixation between legumes and rhizobia is the most common form of biological symbiotic nitrogen fixation. It converts atmospheric nitrogen into usable ammonium nitrogen, offering advantages unmatched by chemical nitrogen fixation, such as high efficiency, low cost, and no pollution. Legumes and rhizobia symbiotically produce new organs—root nodules—which are the primary site of the symbiotic nitrogen fixation reaction. Under nitrogen deficiency stress, common legumes secrete flavonoid signaling molecules, inducing rhizobia to secrete nodfactors (NFs). Plants use LysM receptors located on the cell surface to recognize these nodfactors. Through a series of signal transductions, rhizobia and plant recognize each other, invade each other, and enter the root nodule cells via infection threads (ITs) to form bacteroids. In mature root nodules, the bacteroids reduce nitrogen to ammonium nitrogen, providing the nitrogen source needed for plant growth. Simultaneously, nitrogen fixation is an energy-consuming process; the plant provides the carbon source fixed through photosynthesis to the rhizobia, providing the energy needed for their life activities and nitrogen fixation. To avoid unnecessary energy waste, plants have sophisticated mechanisms to control the growth of rhizobia and the number of root nodules, controlling and balancing the acquisition of nitrogen sources and the energy supply to rhizobia.

[0003] However, the symbiotic efficiency of rhizobia under natural conditions is often limited by environmental factors, making it of great significance to find microbial resources that can synergistically promote nodulation and nitrogen fixation. Summary of the Invention

[0004] The purpose of this invention is to provide a salt-tolerant Cladosporium 550W1 strain to solve the problems mentioned in the background art.

[0005] To achieve the above objectives, the present invention provides the following technical solution: a salt-tolerant Cladosporium strain ( Cladosporium halotolerans 550W1 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 41943, on May 20, 2025. The deposit address is: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.

[0006] Furthermore, the salt-tolerant Cladosporium 550W1 can grow normally in an environment with a pH of 4-11 and a temperature of 20-37℃.

[0007] Another object of the present invention is to provide an application of the above-mentioned salt-tolerant Cladosporium 550W1 or its bacterial solution in promoting nodulation and nitrogen fixation in legumes.

[0008] Furthermore, the legume in question is alfalfa.

[0009] Furthermore, a method to promote nitrogen fixation and nodulation in legumes is to inoculate legumes or their growing environment with salt-tolerant Cladosporium 550W1 or its bacterial solution.

[0010] Another object of the present invention is to provide a microbial preparation for promoting nodulation and nitrogen fixation in leguminous plants, comprising an agriculturally acceptable carrier and the aforementioned salt-tolerant Cladosporium 550W1 or its bacterial culture.

[0011] The salt-tolerant Cladosporium 550W1 strain provided by this invention can significantly promote nodulation and nitrogen fixation in legumes, increase the biomass and nitrogen accumulation of legumes, reduce dependence on chemical nitrogen fertilizers, and has broad application prospects and environmental and ecological benefits. Attached Figure Description

[0012] Figure 1 Phylogenetic tree of strain 550W1.

[0013] Figure 2 Phenotypes of plants inoculated with alfalfa tribulus in the control group (CK) and 550W1 were shown.

[0014] Figure 3 The root nodule phenotypes of plants inoculated with alfalfa tribulus terrestris (CK) and 550W1 were compared.

[0015] Figure 4 The figure shows the statistical results of plant and root nodule phenotypes after inoculation with alfalfa tribulus in the control group (CK) and 550W1. Detailed Implementation

[0016] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0017] In one embodiment of the present invention, a strain 550W1 is provided, which was isolated from Lushan hemp seed potato. No more than 50 mg of 550W1 mycelium was scraped under aseptic conditions, placed in a 2 mL sterile centrifuge tube, and rapidly ground into powder in a liquid nitrogen bath. Fungal DNA was extracted using a BioFlux fungal genomic DNA extraction kit according to the kit's instructions. The extracted total DNA from the strain was used as a template to amplify the ITS sequence. PCR amplification was performed using universal fungal primers ITS1 and ITS4.

[0018] The nucleotide sequence of primer ITS1 is 5'-TCCGYTAGGTGAACCTGCGG-3' (as shown in SEQ ID NO.1 in the sequence listing); the nucleotide sequence of primer ITS4 is 5'-TCCTCCGCTTATTGATATGC-3' (as shown in SEQ ID NO.2 in the sequence listing).

[0019] The PCR reaction system consisted of: 2 µL template DNA, 1 µL primer ITS1, 1 µL primer ITS4, 15 µL 2×SuperTaq PCRStarMix (Dye), and ddH2O to a final volume of 30 µL.

[0020] The PCR reaction conditions were: 95℃, 5 min; 95℃, 30 s, 55℃, 30 s, 72℃, 1 min, 30 cycles; 72℃, 5 min.

[0021] The PCR products obtained from PCR amplification were detected by electrophoresis on a 1.0% agarose gel, and the amplified products were sequenced. The sequencing results are shown in SEQ ID NO.3 of the sequence listing. The sequencing results were compared with the NCBI database, and phylogenetic analysis was performed with ITS sequences that showed high similarity, such as... Figure 1 As shown, strain 550W1 is related to salt-tolerant cladosporium ( Cladosporium halotolerans It is most similar to , so it is named Salt-tolerant Cladosporium ( Cladosporium halotolerans )550W1, and deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No.41943, deposited on May 20, 2025, at No.3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.

[0022] In practical applications, the culture conditions for the aforementioned salt-tolerant Cladosporium 550W1 are as follows: it can grow normally on MS and PDA media in an environment with a pH of 4-11 and a temperature of 20-37℃. The aforementioned salt-tolerant Cladosporium 550W1 or its bacterial suspension can significantly promote nodulation and nitrogen fixation in legumes; preferably, the legume is Alfalfa.

[0023] Specifically, the method to promote nodulation and nitrogen fixation in legumes is to inoculate legumes or their growing environment with salt-tolerant Cladosporium 550W1 or its bacterial solution.

[0024] In another embodiment of the invention, a microbial preparation for promoting nodulation and nitrogen fixation in legumes is also provided, comprising an agriculturally acceptable carrier and the aforementioned salt-tolerant Cladosporium 550W1 or its bacterial culture.

[0025] It should be noted that agriculturally acceptable carriers include, but are not limited to, water, buffer solutions, or culture media.

[0026] In the embodiments of the present invention, the above-mentioned salt-tolerant Cladosporium 550W1 can significantly promote nodulation and nitrogen fixation in legumes, increase the biomass and nitrogen accumulation of legumes, reduce dependence on chemical nitrogen fertilizers, and has broad application prospects and environmental and ecological benefits.

[0027] The following embodiments are implementation examples of the technical solution of the present invention in practical applications, but are not limited thereto. Unless otherwise specified, all materials and reagents involved are commercially available products; unless otherwise specified, all experimental methods used are conventional methods.

[0028] Example: This example provides an experiment to promote nitrogen fixation and nodulation in the legume alfalfa. The alfalfa had grown for approximately 10 days. The experiment used the aforementioned salt-tolerant Cladosporium 550W1 and a known alfalfa rhizobium (…). Sinorhizobium meliloti Plants were inoculated with ABS7, with a control group (CK) inoculated with *Alfalfa rhizobium* ABS7. Plant phenotype and root nodules were observed 21 days later. Plants inoculated with *Cladosporium halophilum* 550W1 showed significantly better growth than those inoculated only with *Alfalfa rhizobium* ABS7, and their root nodules were longer than those in the control group, as detailed below:

[0029] S1. Seed Germination: Peel the pericarp of Alfalfa A17, select plump seeds, and lightly sand the seed coat until it is scratched. Transfer the treated seeds to a petri dish, add appropriate amount of water, and treat on a horizontal shaker at room temperature in the dark for about 24 hours. The treatment time can be adjusted according to the seed coat breaking condition to avoid excessive treatment time, which may cause the hypocotyl to grow crooked and deformed, affecting root growth. Remove the seed coat, transfer the seeds with the emerging hypocotyl to a square petri dish, place 1-2 sheets of absorbent paper on the lid of the dish and moisten them with water, invert and place in a refrigerator at 4℃ for vernalization for 2-3 days. When the hypocotyl grows to 1-1.5cm, it can be transferred to a 1:1 mixture of vermiculite and nutrient soil and placed in a greenhouse for growth. After 7-10 days, transfer to sterilized green zeolite to prepare for inoculation with salt-tolerant Cladosporium 550W1 and Alfalfa rhizobium ABS7.

[0030] S2. Preparation of bacterial culture: 1. *Alfalfa rhizobia*: Dip a pipette tip into *Alfalfa rhizobia* ABS7 glycerol culture stored at -80℃ and streak it onto a TY solid culture plate containing TC resistance to obtain a single colony. Pick a single colony and transfer it to 5 mL of TY liquid medium containing TC resistance, and incubate at 28℃ and 180 rpm for 36-48 hours. Dilute the bacterial culture 1:100 to antibiotic-free TY medium and incubate at 28℃ on a shaker at 180 rpm until the OD600 reaches approximately 0.8, obtaining the ABS7 bacterial culture.

[0031] 2. Salt-tolerant Cladosporium 550W1: Dip the pipette tip into the glycerol culture of Salt-tolerant Cladosporium 550W1 stored at -80℃, spread it on PDA solid culture medium, and incubate at 28℃ for 7 days to obtain a single colony. Use a yellow pipette tip to print the colony block, add 100mL of PDB medium and shake. Incubate at 180rpm on a shaker at 28℃ for 3 days to obtain 550W1 bacterial solution.

[0032] S3. Inoculation: Aliquot the prepared bacterial cultures (ABS7 and 550W1) into 50mL centrifuge tubes, centrifuge at 5000rpm for 10 minutes, and discard the supernatant. Wash ABS7 and 550W1 with 1 / 2 BNM liquid medium and PBS, respectively, and add an appropriate amount of 1 / 2 BNM liquid medium or PBS buffer to the centrifuge tubes and vortex to mix.

[0033] After 7-10 days of growth in a 1:1 mixture of vermiculite and potting soil, alfalfa A17 seedlings were removed, their roots washed, and planted in sterilized zeolite. ABS7 seedlings were diluted with 1 / 2 BNM medium to an OD600 of 0.05 and inoculated near the roots at a rate of 5 mL per plant. 550W1 seedlings were diluted with PBS buffer to a volume of 30 mL and inoculated near the roots at a rate of 3 mL per plant. Results were observed and photographed after 21 days. The results are shown below. Figures 2-4 As shown.

[0034] As can be seen from the figure, compared with the control group (CK) inoculated with ABS7 rhizobium of alfalfa, the length of root nodules, fresh weight of plants, and fresh weight of aboveground parts of alfalfa were significantly increased after inoculation with 550W1. The number of root nodules and fresh weight of root nodules also increased, while the diameter of root nodules did not change significantly.

[0035] In summary, the salt-tolerant Cladosporium 550W1 provided in the embodiments of the present invention has the following beneficial technical effects:

[0036] 1. Significantly promotes nodulation and nitrogen fixation in legumes: Inoculation with this salt-tolerant Cladosporium 550W1 can effectively increase the number and length of root nodules in legumes (such as alfalfa), thereby enhancing their symbiotic nitrogen fixation ability with rhizobia and improving the plant's nitrogen acquisition.

[0037] 2. Promote plant growth and increase biomass: Experimental results show that the application of salt-tolerant Cladosporium 550W1 can significantly increase the fresh weight of plants and the fresh weight of aboveground parts, indicating that the salt-tolerant Cladosporium 550W1 can effectively promote plant vegetative growth and increase yield.

[0038] 3. Reduce dependence on chemical nitrogen fertilizers: By enhancing the plant's own biological nitrogen fixation efficiency, this invention provides a green alternative for agricultural production, which helps to reduce the amount of chemical nitrogen fertilizers applied, thereby reducing production costs and mitigating agricultural non-point source pollution.

[0039] 4. Environmentally friendly and highly ecologically safe: This salt-tolerant Cladosporium 550W1 is a natural fungal resource, a non-GMO microorganism, which avoids the biosafety controversy of GMO technology, has good environmental compatibility, and is in line with the direction of sustainable agricultural development.

[0040] 5. Strong adaptability and flexible cultivation conditions: This salt-tolerant Cladosporium 550W1 can grow normally in the temperature range of 20-37℃ and pH=4-11, making it easy to scale up cultivation and production, and has good application potential and commercial prospects.

[0041] Based on the above-described preferred embodiments of the present invention, and through the foregoing description, those skilled in the art can make various changes and modifications without departing from the inventive concept. The technical scope of this invention is not limited to the contents of the specification.

Claims

1. A salt-tolerant Cladosporium strain ( Cladosporium halotolerans The application of 550W1 or its bacterial solution in promoting nodulation and nitrogen fixation in leguminous plants is characterized by, The salt-tolerant Cladosporium 550W1 is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 41943; the legume is alfalfa.

2. The application according to claim 1, characterized in that, The salt-tolerant Cladosporium 550W1 can grow normally in an environment with a pH of 4-11 and a temperature of 20-37℃.

3. The application according to claim 1, characterized in that, The method to promote nodulation and nitrogen fixation in legumes is to inoculate legumes or their growing environment with salt-tolerant Cladosporium 550W1 or its bacterial solution.

4. A microbial preparation for promoting nodulation and nitrogen fixation in leguminous plants, comprising an agriculturally acceptable carrier, characterized in that, It also includes halophilic cladosporium 550W1 or its bacterial culture; the halophilic cladosporium 550W1 is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 41943; the legume is alfalfa.

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