Rosselomorpha marisflavi YS17 and application thereof

Abstract

The invention discloses a Rosselomorpha marisflavii YS17 and application thereof, the Rosselomorpha marisflavii YS17 is preserved in China General Microbiological Culture Collection Center (CGMCC) on August 2, 2024, and the preservation number is CGMCC No.31528. The Rosselomorpha marisflavii YS17 can be applied to the field of culture and application of the Rosselomorpha marisflavii YS17. The strain Rosselomorpha marisflavi YS17 is a tomato rhizosphere growth-promoting bacterium with the effects of dissolving inorganic phosphorus, degrading fat and fixing nitrogen, has an obvious growth-promoting effect and a salt-resistant effect on tomatoes, and can obviously improve the physiological plant height, the fresh weight and the dry weight of the tomatoes in a salt environment.

Description

Technical Field

[0001] The invention belongs to the technical field of salt-tolerant rhizobacteria, and particularly relates to a Rossellomoreamarisflavi YS17 strain and an application thereof. Background Art

[0002] The statements herein merely provide background information related to the present invention and do not necessarily constitute prior art.

[0003] In recent years, various biological and abiotic stresses have seriously threatened crop production. In addition, irrational agricultural production methods, such as the use of fertilizers, pesticides, herbicides and untreated wastewater irrigation, have posed a serious threat to the environment, leading to soil salinization or degradation. Plant Growth-Promoting Rhizobacteria (PGPR) are a class of beneficial bacteria in the soil or attached to plant roots that can promote plant growth and the absorption and utilization of mineral nutrients. The method of improving saline-alkali land with microorganisms has shown great potential in the process of improving low-fertility saline-alkali land due to its advantages of high efficiency, energy saving and stable effect.

[0004] Jagriti et al. isolated the salt-tolerant bacterial endophyte Rossellomoreamarisflavi from the shoots and roots of wheat, which 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 Rossellomoreamarisflavi can be used to produce biohydrogen by dark fermentation using wheat bran as a substrate. Paul et al. found that the use of endophyte Rossellomorea marisflavi isolated from mushrooms improved the biological efficiency (BE) and nutritional content of mushrooms. Previous studies have also found that the strain Rossellomorea marisflavi can significantly promote the growth of corn crops and increase corn resistance to fall armyworms, making it a good biocontrol agent.

[0005] In the current study, it was confirmed that the strain Rossellomorea marisflavi has a growth-promoting effect on wheat, mushrooms, and corn, but there has been no research on its ability to promote the growth of tomato plants in saline-alkali environments. Summary of the invention

[0006] In view of the shortcomings of the prior art, the object of the present invention is to provide a Rossellomorea marisflavi YS17 and applications thereof.

[0007] In order to achieve the above object, the present invention is implemented through the following technical solutions:

[0008] In the first aspect, the present invention provides a Rossellomorea marisflavi YS17, which was deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on August 2, 2024, with the deposit number CGMCC No.31528.

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

[0010] In a third aspect, the present invention provides a product comprising the Rossellomorea marisflavi YS17 or the mixed bacterial agent.

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

[0012] Preferably, the product comprises a carrier, and the carrier is liquid or solid.

[0013] In a third aspect, the present invention provides the use of the Rossellomorea marisflavi YS17 or the mixed bacterial agent in promoting the growth of tomato seedlings.

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

[0015] In a fourth aspect, the present invention provides the use of the Rossellomorea marisflavi YS17 or the mixed bacterial agent in improving the resistance of tomato seedlings to salt stress.

[0016] In a fifth aspect, the present invention provides the use of the Rossellomorea marisflavi YS17 or the mixed bacterial agent in degrading inorganic phosphorus.

[0017] In a sixth aspect, the present invention provides the use of the Rossellomorea marisflavi YS17 or the mixed bacterial agent in degrading fat.

[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 are as follows:

[0020] The strain Rossellomorea marisflavi YS17 is a tomato rhizosphere growth-promoting bacterium that can dissolve inorganic phosphorus, degrade fat and fix nitrogen. It has obvious growth-promoting and salt-resistant effects on tomatoes, and can significantly increase the physiological plant height, fresh weight and dry weight of tomatoes in a high-salt environment. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

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

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

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

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

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

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

[0028] Figure 7 This is a comparison chart of the effects of adding YS17 bacterial solution on the fresh weight (A) and dry weight (B) of tomato plants 21 days after the addition of YS17 bacterial solution in the embodiment of the present invention;

[0029] Figure 8 3 is a comparison of potted plant photos under different conditions in the embodiments of the present invention, wherein A is a salt-free sterile control group; B is a salt-containing sterile control group; and C is a salt-containing experimental group with YS17 bacterial solution added. DETAILED DESCRIPTION

[0030] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0031] The present invention will be further described below in conjunction with 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 separation and screening methods are as follows: weigh 10.0 g of soil and put it into a triangular flask filled with sterile water, then place it on a constant temperature shaker at 37°C and 180 r / min for 30 minutes to obtain a soil suspension; use the gradient dilution method to gradient dilute the soil suspension, and absorb the dilution degree of 10 -4 , 10 -5 , 10 -6 The soil suspension was spread on LB+8% NaCl solid culture medium and cultured in a constant temperature incubator at 37°C. After a single colony grew out, the single colony was picked out with a toothpick and purified on the culture medium using the three-zone line method until a single colony was obtained.

[0035] The morphology of strain YS17 obtained by the above screening on LB solid medium is as follows Figure 1 As shown, the strain was streaked in three zones on LB solid medium and cultured for 1 day. The grown YS17 colonies were light yellow in color, irregularly round in shape, rough and opaque on the surface, and serrated on the edges.

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

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

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

[0039] 2. 16S rDNA Identification of Strain YS17

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

[0077] 1. Identification of phosphate solubilization function

[0078] The phosphate solubilization ability of strain YS17 was determined by inorganic phosphorus bacterial culture medium. The specific method is as follows:

[0079] The strain YS17 was streaked onto the Montkina inorganic phosphorus bacterial medium and cultured at 30°C for 5 days to observe the growth of the colonies.

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

[0081] Colony growth Figure 3As shown, strain YS17 is surrounded by a transparent degradation zone in the Montgena inorganic phosphorus bacteria culture medium, which indicates that strain YS17 has the ability to degrade inorganic phosphorus.

[0082] 2. Identification of lipid degradation function

[0083] The ability of strain YS17 to degrade fat was determined using a fat degradation medium. The specific method is as follows:

[0084] The strain YS17 was inoculated onto the fat degradation medium with a sterilized toothpick and cultured at 30°C for 2 days to observe the growth of the colonies;

[0085] The fat degradation medium is composed of: 10 g peptone, 5 g yeast extract, 10 g NaCl, 2 mL tributyrin, 15 g agar, and 1000 mL water.

[0086] Colony growth Figure 4 As shown, YS17 is surrounded by a transparent degradation zone in the fat degradation medium, which indicates that strain YS17 has the ability to degrade fat.

[0087] 3. Identification of nitrogen fixation function

[0088] The nitrogen fixation ability of strain YS17 was determined by using Axubei medium. The specific method is as follows:

[0089] The strain YS17 was inoculated onto nitrogen-deficient Ashubei medium with a sterilized toothpick and cultured at 30°C for 7 days to observe the growth of the colonies.

[0090] The nitrogen-deficient Axubei culture medium components are as follows: mannitol 10g, CaCO3 5g, KH2PO4 0.2g, MgSO4·7H2O 0.2g, NaCl 0.2g, CaSO4·2H2O 0.1g, agar 15g, and water 1000mL.

[0091] Colony growth Figure 5 As shown, YS17 can grow in nitrogen-deficient Axubei medium with a transparent degradation zone around it, which indicates that strain YS17 has the ability to fix nitrogen. Study on the Salt Resistance and Growth Promotion of Tomato Seedlings by Strain YS17

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

[0093] Soil was collected from the experimental field of the Panhe Campus of Shandong Agricultural University, sieved and mixed with vermiculite in a ratio of 2:1, and then the mixed soil was put into the flower pot, occupying three quarters of the flower pot. Zhongshu No. 4 tomato seeds were selected, frozen for 24 hours, exposed to the sun for 5 hours, placed in a 37℃ incubator to germinate and culture in the dark, and planted in a 72-hole tray after germination. After growing two leaves and one heart, transplanted to a large pot.

[0094] (II) Preparation of treatment group

[0095] Prepare 15 cultivation pots for cultivating tomato seedlings, and divide the 15 cultivation pots into three groups, namely, salt-free sterile group (salt-free blank), salt-containing sterile group (salt blank), and salt-containing bacteria group (YS17 group), with 5 replicates in each group.

[0096] Among them, the salt solution added to the cultivation pots of the salt-free group and the salt-plus-bacteria group was NaCl with a concentration of 0.1 mol / L and a conductivity of 0.76 S / cm.

[0097] (III) Preparation of strain YS17

[0098] (1) The YS17 strain was transferred to a test tube containing 5 mL of LB liquid medium and cultured in a constant temperature shaker at 180 rpm and 37°C for 12 h to obtain an activated bacterial solution;

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

[0100] The components of the LB liquid culture medium are: 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 YS17 bacterial solution 200 times with water, and apply 100 mL / Kg of the solution to the potted plants.

[0102] (IV) Planting method

[0103] Take the tomato seedlings out of the 72-hole tray with the soil and plant them in the cultivation pots, making sure that the root height of each pot is consistent. Cultivate in the incubator for 2-3 days. Then pour 50mL of 0.1mol / L NaCl solution into the cultivation pots, which is the salt group; pour 50mL of water into the cultivation pots, which is the salt-free group; cultivate in the incubator for 2-3 days. After the strain YS17 bacterial solution is treated in step (iii), pour it into part of the salt group, which is the salt and bacteria experimental group, pour 50mL per pot.

[0104] 5. Outcome measurement

[0105] After watering with YS17 solution, the plant height was measured once a week. After 21 days, the seedlings were harvested, the dry weight and fresh weight were measured, and photos were taken for preservation. Figure 6-8 .

[0106] (1) Tomato plant height 7d, 14d, and 21d after inoculation

[0107] The control group was a control group with no salt and a control group with YS17 as the experimental group, and five treatments were set up for each group. The plant height was measured once a week, and the results of the plant height each week were as follows: Figure 6 In the 14-day measurement results, the YS17-treated group increased by 24.8% (P<0.05) compared with the salt-free group; in the 21-day measurement results, the YS17-treated group increased by 35.7% (P<0.05) compared with the salt-free group.

[0108] (2) Dry weight

[0109] After harvesting the seedlings, clean the soil around the roots of the tomato plants, wipe them dry and weigh their fresh weight; then put them in an oven at 80°C and dry them for 3-4 hours, then weigh their dry weight. Figure 7 As shown. Figure 7 As shown in Figure A, the fresh weight of the YS17-treated group increased by 108.7% compared with the salt-free and sterile group (P < 0.01). Figure 7 As shown in B, the dry weight of the YS17-treated group increased by 185.4% compared with the salt-free 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 salt environment.

[0111] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A Rossellomorea marisflavi YS17, characterized in that: It was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on August 2, 2024, with the deposit number CGMCC No.31528.

2. A mixed bacterial agent, characterized in that: The mixed bacterial agent at least includes Rossellomoreamarisflavi YS17 described in claim 1.

3. A product comprising Rossellomorea marisflavi YS17 according to claim 1 or the mixed bacterial agent according to claim 2.

4. The product according to claim 3, characterized in that: The product is in the form of powder, solution, suspension, tablet or granules.

5. Use of Rossellomorea marisflavi YS17 according to claim 1 or the mixed bacterial agent according to claim 2 in promoting the growth of tomato seedlings.

6. The use according to claim 5, characterized in that: The method of promoting the growth of tomato seedlings is to promote the growth of physiological plant height, fresh weight and dry weight of tomato plants.

7. Use of Rossellomorea marisflavi YS17 according to claim 1 or the mixed bacterial agent according to claim 2 in improving the resistance of tomato seedlings to salt stress.

8. Use of Rossellomorea marisflavi YS17 according to claim 1 or the mixed bacterial agent according to claim 2 in degrading inorganic phosphorus.

9. Use of Rossellomorea marisflavi YS17 according to claim 1 or the mixed bacterial agent according to claim 2 in degrading fat.

10. Use of Rossellomorea marisflavi YS17 according to claim 1 or the mixed bacterial agent according to claim 2 in nitrogen fixation.

Images