Rossellomorea marisflavi YS17 and application thereof

Abstract

The application discloses a strain of Rossellomorea marisflavi YS17 and application thereof, which has been preserved in the China General Microbiological Culture Collection Center on August 2, 2024, and the preservation number is CGMCC No.31528. The strain of Rossellomorea marisflavi YS17 is a tomato rhizosphere growth promoting bacterium with the functions of dissolving inorganic phosphorus, degrading fat and nitrogen fixation. The strain has obvious growth promoting effect and salt resistance on tomatoes, and can significantly improve the physiological plant height, fresh weight and dry weight of tomatoes in a salt environment.

Description

Technical Field

[0001] This invention belongs to the field of salt-tolerant rhizosphere bacteria technology, specifically relating to a Rossellomoreamarisflavi YS17 strain and its applications. Background Technology

[0002] The statements herein provide only background information in relation to this invention and do not necessarily constitute prior art.

[0003] In recent years, various biotic and abiotic stresses have severely threatened crop production. Coupled with irrational agricultural practices, such as the use of chemical fertilizers, pesticides, herbicides, and untreated wastewater irrigation, these factors have posed a serious threat to the environment, leading to soil salinization and degradation. Plant growth-promoting rhizobacteria (PGPR) are a class of beneficial bacteria that live in the soil or attach to plant roots, promoting plant growth and the absorption and utilization of mineral nutrients. Microbial methods for improving saline-alkali land have shown great potential in the improvement of low-fertility saline-alkali land due to their advantages of high efficiency, energy saving, and stable effects.

[0004] Jagriti et al. isolated the salt-tolerant endophytic bacterium *Rosellomoreamarisflavi* from wheat shoots and roots. This bacterium significantly reduced the adverse effects of salt stress on wheat and improved various growth parameters of the salt-sensitive wheat variety "HD 2733," such as root length, shoot length, fresh weight, and dry weight. Yousef et al. found that *Rosellomoreamarisflavi* could be used for dark fermentation of wheat bran as a substrate to produce biogenic hydrogen. Paul et al. found that using the endophytic bacterium *Rosellomorea marisflavi* isolated from mushrooms improved the bioavailability (BE) and nutrient content of mushrooms. Previous studies have also found that the strain *Rosellomorea marisflavi* can significantly promote the growth of maize crops and increase maize's resistance to fall armyworm, making it a good biocontrol agent.

[0005] Current research has confirmed that the strain Rossellomorea marisflavi promotes the growth of wheat, mushrooms, and corn, but there is no research on its ability to promote the growth of tomato plants under saline-alkali conditions. Summary of the Invention

[0006] In view of the shortcomings of the existing technology, the purpose of this invention is to provide a Rossellomorea marisflavi YS17 and its applications.

[0007] To achieve the above objectives, the present invention is implemented through the following technical solution:

[0008] In a first aspect, the present invention provides a Rossellomorea marisflavi YS17 strain, which was deposited at the China General Microbiological Culture Collection Center on August 2, 2024, with accession number CGMCC No.31528.

[0009] In a second aspect, the present invention provides a mixed microbial agent, wherein the mixed microbial agent comprises at least the Rossellomoreamarisflavi YS17.

[0010] Thirdly, the present invention provides a product comprising the Rossellomorea marisflavi YS17 or the mixed microbial agent.

[0011] In some embodiments, the product is in powder form, solution form, suspension form, tablet form, or granule form.

[0012] Preferably, the product includes a carrier, which is a liquid or a solid.

[0013] Thirdly, the present invention provides the application of the Rossellomorea marisflavi YS17 or the mixed microbial agent in promoting the growth of tomato seedlings.

[0014] In some embodiments, promoting the growth of tomato seedlings means promoting the increase of physiological plant height, fresh weight, and dry weight of tomato plants.

[0015] Fourthly, the present invention provides the application of the Rossellomorea marisflavi YS17 or the mixed microbial agent in improving the salt stress resistance of tomato seedlings.

[0016] Fifthly, the present invention provides the use of the Rossellomorea marisflavi YS17 or the mixed microbial agent in the degradation of inorganic phosphorus.

[0017] In a sixth aspect, the present invention provides the use of the Rossellomorea marisflavi YS17 or the mixed microbial agent in the degradation of fats.

[0018] In a seventh aspect, the present invention provides the use of the Rossellomorea marisflavi YS17 or the mixed bacterial agent in nitrogen fixation.

[0019] The beneficial effects achieved by one or more embodiments of the present invention described above are as follows:

[0020] The strain Rossellomorea marisflavi YS17 is a tomato rhizosphere growth-promoting bacterium with the functions of dissolving inorganic phosphorus, degrading fat and fixing nitrogen. It has a significant growth-promoting effect and salt tolerance effect on tomatoes, and can significantly improve the physiological plant height, fresh weight and dry weight of tomatoes under high salt conditions. Attached Figure Description

[0021] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0022] Figure 1 This is a morphological diagram of strain YS17 on LB solid medium in an embodiment of the present invention;

[0023] Figure 2 This is the phylogenetic tree of strain YS17 in the embodiments of the present invention;

[0024] Figure 3 This is a colony growth diagram of strain YS17 in the Mongkina inorganic phosphorus bacteria culture medium in an embodiment of the present invention;

[0025] Figure 4 This is a colony growth diagram of strain YS17 in a lipid degradation medium in an embodiment of the present invention;

[0026] Figure 5 This is a colony growth diagram of strain YS17 in nitrogen-deficient Assumption medium in an embodiment of the present invention;

[0027] Figure 6 These are the tomato plant heights at 7d (A), 14d (B), and 21d (C) after adding YS17 bacterial solution in the embodiments of the present invention.

[0028] Figure 7 This is a comparative graph showing the effect of adding YS17 bacterial solution for 21 days on the fresh weight (A) and dry weight (B) of tomato plants in the embodiments of the present invention.

[0029] Figure 8 These are comparison photos of potted plants under different conditions in the embodiments of the present invention. Among them, A is the salt-free sterile control group; B is the salt-containing sterile control group; and C is the salt-containing experimental group with added YS17 bacterial solution. Detailed Implementation

[0030] It should be noted that the following detailed description is illustrative and intended to provide further explanation of the invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0031] The present invention will be further described below with reference to the embodiments.

[0032] Isolation and identification of test strains

[0033] 1. Isolation of strain YS17

[0034] The strains used in this experiment were screened from saline-alkali soil in Dongying City, Shandong Province. The specific isolation and screening methods are as follows: 10.0g of soil was weighed and placed in an Erlenmeyer flask containing sterile water, then placed on a constant temperature shaker at 37℃ and 180r / min for 30min to obtain a soil suspension; the soil suspension was then serially diluted using a gradient dilution method, and the strain at a dilution of 10-1 was collected. -4 10 -5 10 -6 Soil suspensions were spread onto LB + 8% NaCl solid medium and incubated at 37°C. After single colonies grew, they were picked out with toothpicks and purified on the medium using the three-zone streak method until single colonies were obtained.

[0035] The morphology of strain YS17 obtained from the above screening on LB solid medium is as follows: Figure 1 As shown, after streaking the strain in three zones on LB solid medium and culturing for 1 day, the resulting YS17 colonies were pale yellow, irregularly round in shape, with a rough and opaque surface and serrated edges.

[0036] The LB liquid culture medium formula is as follows: 1% peptone, 0.5% yeast extract, 1% sodium chloride, and 1000 mL deionized water.

[0037] The LB solid medium formula is: 1% peptone, 0.5% yeast extract, 1% sodium chloride, 15g agar, and 1000mL deionized water.

[0038] The formula for LB+8% NaCl solid culture medium is: 1% peptone, 0.5% yeast extract, 8% sodium chloride, 15g agar, and 1000mL deionized water.

[0039] 2. Identification of 16S rDNA from strain YS17

[0040] The 16S rDNA gene sequence of strain YS17 was sequenced by Sangon Biotech (Shanghai) Co., Ltd., and the sequencing results are shown in SEQ ID NO.1. The obtained 16S rDNA sequence was subjected to BLAST analysis with existing sequences in the NCBI database. Strains with similar homology were selected, and a phylogenetic tree was constructed using MEGA X software with a Neighbor-joining method. The results are as follows. Figure 2As shown; the results showed that the strain YS17 obtained by the above screening had 99% similarity to Rossellomoreamarisflavi TF-11 and was relatively close in evolutionary distance. Based on the physiological and biochemical characteristics of the strain, it was identified as Rossellomorea marisflavi.

[0041] The 16S rDNA sequence of strain YS17 is as follows:

[0042] YS17

[0043] GGCGTGGCGGCGTGCTATACATGCAGTCGAGCGGATCGATGGG

[0044] AGCTTGCTCCCTGAGATCAGCGGCGGACGGGTGAGTAACACGT

[0045] GGGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGG

[0046] GCTAATACCGGATAACACCTAACCCCCGCATGGGGGAAGGTTGA

[0047] AAGGTGGCTTCGGCTATCACTTACAGATGGACCCGCGGGCATT

[0048] AGCTAGTTGGTGAGGTAATGGCTCACCAAGGCGACGATGCGTA

[0049] GCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACAC

[0050] GGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCA

[0051] ATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGAAGAA

[0052] GGTTTTCGGATCGTAAAACTCTGTTGTTAGGGAAGAACAAGTG

[0053] CCGTTCGAATAGGGCGGCGCCTTGACGGTACCTAACCAGAAAG

[0054] CCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTG

[0055] GCAAGCGTTGTCCGGAATTATTGGGCGTAAAGCGCGCGCAGGT

[0056] GGTTTCTTAAGTCTGATGTGAAAGCCCACGGCTCAACCGTGGA

[0057] GGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGGAAAG

[0058] TGGAATTCCAAGTGTAGCGGTGAAATGCGTAGATATTTGGAGGA

[0059] ACACCAGTGGCGAAGGCGACTTTCTGGTCTGTAACTGACACTG

[0060] AGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGT

[0061] AGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGAGGGTTTC

[0062] CGCCCTTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGG

[0063] GAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGG

[0064] CCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGC

[0065] GAAGAACCTTACCAGGTCTTGACATCCTCTGACAACCCTAGAG

[0066] ATAGGGCTTTCCCCTTCGGGGACAGAGTGACAGGTGGTGCAT

[0067] GGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGC

[0068] AACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGC

[0069] ACTCTAAGATGACTGCCGGTGACAAACCGGAGGAAGGTGGGGA

[0070] TGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGT

[0071] GCTACAATGGACGGTACAAAGGGCTGCAAGACCGCGAGGTTTA

[0072] GCCAATCCCATAAAACCGTTCTCAGTTCGGATTGTAGGCTGCAA

[0073] CTCGCCTACATGAAGCTGGAATCGCTAGTAATCGCGGATCAGCA

[0074] TGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTC

[0075] ACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTTGGAGCCAGCCGCATAAGTGACCC.

[0076] Identification of growth-promoting function of strain YS17

[0077] 1. Identification of phosphorus solubility

[0078] The phosphorus-solubilizing ability of strain YS17 was determined using an inorganic phosphorus-containing bacterial culture medium. The specific method is as follows:

[0079] Straw strain YS17 was streaked onto the Mongkina inorganic phosphorus bacteria medium and incubated at 30°C for 5 days. Colony growth was then observed.

[0080] The composition of the *Montagna* inorganic phosphorus bacteria culture medium is as follows: 10g glucose, 0.5g (NH4)2SO4, 0.3g MgSO4·7H2O, 0.03g MnSO4·4H2O, 0.3g KCl, 0.03g FeSO4·7H2O, 0.3g NaCl, 10g Ca3(PO4)2, 15g agar, and 1000mL water.

[0081] Colony growth status as follows Figure 3As shown, strain YS17 exhibits a transparent degradation zone around its cells in the Mongkina inorganic phosphorus bacteria culture medium, indicating that strain YS17 has the ability to degrade inorganic phosphorus.

[0082] 2. Identification of fat-degrading function

[0083] The ability of strain YS17 to degrade lipids was determined using a lipid-degrading medium. The specific method is as follows:

[0084] Strain YS17 was inoculated onto a lipid degradation medium using a sterile toothpick and cultured at 30°C for 2 days. Colony growth was then observed.

[0085] The lipid degradation culture medium is composed of the following: 10g peptone, 5g yeast extract, 10g NaCl, 2mL glyceryl tartrate, 15g agar, and 1000mL water.

[0086] Colony growth status as follows Figure 4 As shown, YS17 exhibits a transparent degradation zone around its surface in the lipid degradation medium, indicating that strain YS17 has the ability to degrade lipids.

[0087] 3. Identification of nitrogen fixation function

[0088] The nitrogen-fixing ability of strain YS17 was determined using Assumption medium, and the specific method is as follows:

[0089] Strain YS17 was inoculated onto nitrogen-deficient Assumption medium with sterile toothpicks and cultured at 30°C for 7 days. Colony growth was then observed.

[0090] The nitrogen-deficient Ashube culture medium consists of the following components: 10g mannitol, 5g CaCO3, 0.2g KH2PO4, 0.2g MgSO4·7H2O, 0.2g NaCl, 0.1g CaSO4·2H2O, 15g agar, and 1000mL water.

[0091] Colony growth status as follows Figure 5 As shown, YS17 can grow in nitrogen-deficient Assumption medium with a transparent degradation zone around it, indicating that strain YS17 has the ability to fix nitrogen. Study on salt tolerance and growth promotion of tomato seedlings by strain YS17

[0092] (I) Preparation of potting soil and tomato seedlings

[0093] Soil was taken from the experimental field of Shandong Agricultural University's Panhe Campus, sieved, and mixed with vermiculite in a 2:1 ratio. The mixed soil was then filled into flowerpots, filling three-quarters of the pot. Zhongshu No. 4 tomato seeds were selected, frozen for 24 hours, then exposed to direct sunlight for 5 hours, and placed in a 37℃ incubator for germination in the dark. After germination, the seeds were planted in 72-cell trays. Once two leaves and a central bud had emerged, the seeds were transplanted into larger pots.

[0094] (II) Preparation of the Processing Team

[0095] Prepare 15 cultivation pots for cultivating tomato seedlings. Divide the 15 cultivation pots into three groups: salt-free and sterile group (salt-free blank), salt-containing and sterile group (salt-containing blank), and salt-containing and sterile group (YS17 group). Each group has 5 replicates.

[0096] The salt solution added to the cultivation pots of the salt-sterile group and the salt-infected group was NaCl with a concentration of 0.1 mol / L and an electrical conductivity of 0.76 S / cm.

[0097] (III) Preparation of strain YS17

[0098] (1) Transfer the YS17 strain to a test tube containing 5 mL of LB liquid medium and place it in a constant temperature shaker at 180 rpm and 37 ℃ for 12 h to obtain activated bacterial solution;

[0099] (2) The activated bacterial solution from step (1) was inoculated into 50 mL of LB liquid medium at an inoculation amount of 2% of the volume of LB liquid medium (1 mL), and cultured for 24 h to obtain bacterial solution of strain YS17.

[0100] The LB liquid culture medium consists of the following components: 5 g / L yeast extract, 10 g / L peptone, 10 g / L sodium chloride, 1 L distilled water, pH 7.0; sterilized at 121°C for 20 min.

[0101] (3) Dilute the bacterial solution of strain YS17 with water by 200 times, and apply 100 mL / Kg of bacterial solution to the pot.

[0102] (iv) Planting methods

[0103] Tomato seedlings were removed from 72-cell trays along with their soil and planted in cultivation pots, ensuring the root height was uniform in each pot. They were then cultured in an incubator for 2-3 days. Afterward, 50 mL of 0.1 mol / L NaCl solution was poured into the cultivation pots (salt group); 50 mL of water was poured into the cultivation pots (salt-free group); and the mixture was cultured in an incubator for 2-3 days. The YS17 bacterial suspension, after being treated in step (iii), was then poured into a portion of the salt group (salt-containing bacterial experimental group), with 50 mL poured into each pot.

[0104] (V) Result Measurement

[0105] After watering with YS17 bacterial solution, measure the plant height once a week. After 21 days, harvest the seedlings and measure their dry and fresh weights, then take photos and save the data. Figure 6-8 .

[0106] (1) Tomato plant height at 7, 14 and 21 days after inoculation with bacterial solution

[0107] Five treatments were set up in each group, with salt-free sterile and salt-treated sterile treatments serving as control groups and YS17 strain inoculated into the water as the experimental group. Plant height was measured weekly, and the weekly plant height results are shown below. Figure 6 As shown in the results, in the 14-day measurement, the YS17 treatment group showed a 24.8% improvement compared to the salt-treated sterile group (P<0.05); in the 21-day measurement, the YS17 treatment group showed a 35.7% improvement compared to the salt-treated sterile group (P<0.05).

[0108] (2) Dry and fresh weight

[0109] After harvesting the seedlings, clean the soil from the roots of the tomato plants, wipe them dry, and weigh them for fresh weight. Then, place them in an 80℃ oven to dry for 3-4 hours, and weigh them for dry weight. A bar chart of fresh weight and dry weight is shown below. Figure 7 As shown. Figure 7 As shown in Figure A, in terms of fresh weight, the YS17 treatment group increased by 108.7% compared to the salt-treated sterile group (P<0.01); Figure 7 As shown in Figure B, in terms of dry weight, the YS17 treatment group increased by 185.4% compared to the salt-treated sterile group (P<0.01).

[0110] (3) Photos of potted plants, such as Figure 8 As shown, YS17 can effectively improve the growth of tomato seedlings under saline conditions.

[0111] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A plant Rossellomorea marisflavi YS17 strain, characterized by: It is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 31528.

2. A mixed microbial agent, characterized in that: The mixed microbial agent comprises at least the components described in claim 1. Rossellomorea marisflavi YS17 strain.

3. A device comprising the features described in claim 1 Rossellomorea marisflavi The product of strain YS17 or the mixed bacterial agent of claim 2.

4. The product according to claim 3, characterized in that: The product may be in powder, solution, suspension, tablet, or granule form.

5. The claim 1 Rossellomorea marisflavi The application of strain YS17 or the mixed bacterial agent of claim 2 in promoting the growth of tomato seedlings under salt stress, wherein promoting the growth of tomato seedlings under salt stress means promoting the increase of physiological plant height, fresh weight and dry weight of tomato plants.

6. The claim 1 Rossellomorea marisflavi Application of YS17 strain or the mixed bacterial agent of claim 2 in improving the salt stress resistance of tomato seedlings.

7. The claim 1 Rossellomorea marisflavi Application of YS17 strain or the mixed bacterial agent of claim 2 in the degradation of inorganic phosphorus.

8. The claim 1 Rossellomorea marisflavi Application of YS17 strain or the mixed bacterial agent of claim 2 in the degradation of fats.

9. The claim 1 Rossellomorea marisflavi Application of YS17 strain or the mixed bacterial agent of claim 2 in nitrogen fixation.

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