M. rossius and application thereof

Abstract

The application belongs to the field of agricultural microorganism technology, and particularly relates to a salt and alkali tolerant Moraxella rosenbachii and application thereof. The Moraxella rosenbachii strain is preserved in the China Center for Type Culture Collection, and is classified and named as Rossellomorea sp.DA9-6, with a preservation number of CCTCC No: M20242938, a preservation date of December 30, 2024, and a preservation address of Wuhan, China. Wuhan University. The Moraxella rosenbachii provided in the application has high salt and alkali tolerance and drought resistance, and can grow normally in an alkaline environment with a pH of 7-14, a salt environment with 1-16% NaCl, and a drought condition, and can improve the stress resistance performance of plants.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of agricultural microorganisms, and particularly relates to a salt and alkali tolerant Moraxella rossica and application thereof. BACKGROUND

[0002] Everything is born in the soil, and there is food with the soil. Soil is the material basis for human survival. There are about 500 million mu of salt and alkali land in China that can be developed as arable land. Through reasonable development and utilization, it is of great significance to break through the "ceiling" of increasing the area of grain planting, realize the expansion, upgrading and efficiency of arable land resources, and guarantee food security, ecological security and economic and social security.

[0003] The improved salt and alkali land usually adopts physical and chemical improvement methods. The physical improvement method such as deep ploughing, land leveling and salt washing by irrigation and drainage can reduce the salt content of the surface soil to a certain extent, but the engineering quantity is large, the cost is high, and the effect is easily affected by natural conditions and difficult to maintain. The chemical improvement method adjusts the soil pH value by applying gypsum, ferrous sulfate and other chemical improvers, but the large-scale use of chemical agents may cause soil structure damage, environmental pollution and other negative effects, and may also cause soil secondary salinization. With the continuous development of biotechnology, the use of microorganisms to improve the soil environment has become a research hotspot. Salt and alkali tolerant strains, as a special kind of microorganism, can survive in harsh salt and alkali environment and play a unique function. They can regulate soil physical and chemical properties, promote soil aggregate structure formation, improve soil fertility, and enhance soil water and fertilizer retention capacity through their own metabolic activities. At the same time, salt and alkali tolerant strains can form a symbiotic relationship with plant roots, help plants resist salt and alkali stress, and improve the survival rate and growth performance of plants in salt and alkali land. Compared with traditional improvement methods, microbial technology has the advantages of low cost, environmental friendliness and long-lasting effect, and opens up a new way for salt and alkali land improvement and agricultural production.

[0004] In-depth study on the application of salt and alkali tolerant strains in salt and alkali land has important theoretical and practical significance for developing efficient salt and alkali land improvement technology and promoting ecological restoration of salt and alkali land. SUMMARY

[0005] The purpose of the present application is to provide a Moraxella rossica strain and its application.

[0006] To achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows:

[0007] A Moraxella rossica strain, the Moraxella rossica strain is preserved in China Center for Type Culture Collection, and is classified and named as Rossellomorea sp.DA9-6, the preservation number is CCTCC No: M20242938, the preservation date is December 30, 2024, and the preservation address is China. Wuhan. Wuhan University.

[0008] The morphological characteristics of the Moraxella rossius are bacillary, gram-positive, and the size is 0.5-0.7 μm x 1.5-3.0 μm. The spore is oval mesospore or terminal spore, and the spore capsule is slightly swollen. On nutrient broth medium, the colony is round, raised, smooth, moist, light orange, and the edge is irregular.

[0009] The application of the Moraxella rossius in improving the stress resistance performance of crops.

[0010] The application of the Moraxella rossius in promoting the growth of crops under stress conditions.

[0011] The application of the Moraxella rossius as a preparation for improving the stress resistance and promoting the growth of crops in saline-alkali soil.

[0012] A preparation for improving the stress resistance and promoting the growth of crops, which contains the strain.

[0013] The preparation contains the culture, culture suspension or fermentation broth of the strain.

[0014] The concentration of the strain in the preparation is 3 x 10 9 cfu / mL to 7 x 10 9 cfu / mL.

[0015] The application of the preparation in promoting the growth of plants under stress conditions.

[0016] The crops include but are not limited to vegetables, fruits, food crops and pasture.

[0017] The beneficial effects of the present application are:

[0018] 1. The Moraxella rossius provided by the present application has high salt and alkali tolerance and drought resistance, and can grow normally in an alkaline environment with pH of 7-14, a salt environment with 1-16% NaCl and drought conditions, and can improve the stress resistance performance of plants.

[0019] 2. The Moraxella rossius provided by the present application can produce gibberellin 3 (GA3), cytokinin (CTK), ACC deaminase (ACCD) and extracellular polysaccharide (EPS), and has a growth-promoting effect.

[0020] 3、The Moraxella rossica bacterin provided by the application can be applied to vegetables, fruits, gramineae and the like, and is especially suitable for saline-alkali soil environment, and has the effects of improving the saline-alkali tolerance of crops, strengthening the plant root system and promoting the growth of crops. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 It is a colony morphology diagram of Moraxella rossica DA9-6;

[0022] Figure 2 It is a phylogenetic tree diagram of Moraxella rossica DA9-6;

[0023] Figure 3 It is the salt tolerance of Moraxella rossica DA9-6;

[0024] Figure 4 It is the alkali tolerance of Moraxella rossica DA9-6;

[0025] Figure 5 It is the salt-alkali tolerance of Moraxella rossica DA9-6;

[0026] Figure 6 It is the drought resistance of Moraxella rossica DA9-6;

[0027] Figure 7 It is the Moraxella rossica DA9-6 bacterin promoting the growth of corn;

[0028] Figure 8 It is the Moraxella rossica DA9-6 bacterin corn potting test data;

[0029] Figure 9 It is the Moraxella rossica DA9-6 bacterin promoting the growth of tomatoes;

[0030] Figure 10 It is the Moraxella rossica DA9-6 bacterin salt-alkali soil planting tomato potting test data;

[0031] Figure 11 It is the Moraxella rossica DA9-6 bacterin grass charcoal soil planting tomato potting test data. DETAILED DESCRIPTION

[0032] In order to make the objects, technical solutions and advantages of the present application clearer, further detailed description will be made to the present application in combination with the drawings and specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application, and are not used to limit the present application.

[0033] Example 1 Identification of strain DA9-6

[0034] (1) Screening: The strain was isolated and screened from the root soil of maize planted in the saline-alkali land of Da'an, Jilin Province. Under sterile conditions, 5g of crop root soil was taken and dissolved in 45mL of sterile distilled water. After shaking in a shaker at 220r / min for 30min, 1mL of soil suspension was taken and mixed with 9mL of sterile water to obtain strain 10. -1 Diluent, and so on, according to 10 -4 10 -5 10 -6 Three concentration gradients of soil dilution were each taken and spread onto LB agar medium containing pH 9 and 5% NaCl. The medium was incubated at 30°C for 24 hours. Single colonies were picked and streaked onto plates. This process was repeated multiple times until the strain DA9-6 was obtained after purification.

[0035] (2) Morphological identification and physiological and biochemical characteristics: such as Figure 1 As shown, strain DA9-6 forms round, raised, smooth, moist, pale orange colonies with irregular edges on LB agar. The bacterial cells are rod-shaped, measuring 0.5–0.7 μm × 1.5–3.0 μm. The spores are oval, centrally or apical, with slightly swollen sporangia.

[0036] The strain underwent the following physiological and biochemical characterization analysis. Morphological observation and physiological and biochemical characterization were performed according to the Common Bacterial System Identification Manual. Positive reactions included: Gram staining; spore staining; catalase; α-glucosidase; gelatin hydrolysis; starch hydrolysis; VP test; Tween 80 hydrolysis; utilization of glucose, mannitol, N-acetylglucosamine, and malic acid; and fermentation of ribose, fructose, maltose, and starch to produce acid. Negative reactions included: oxidase; arginine dihydrolase; urease; nitrate reduction; indole production; methyl red; utilization of mannose, decanoic acid, and citric acid; and fermentation of raffinose, D-arabinose, and galactose to produce acid. Hemolysis was observed as γ-hemolysis.

[0037] (3) Molecular identification: Genomic DNA of the strain was extracted using the bacterial genomic DNA extraction kit from Tiangen Biotech. Using the extracted DNA as a template, the 16S rRNA gene of the strain was amplified by PCR using the universal bacterial primer 27F / 1492R. The PCR product was sent to Beijing BGI Genomics Co., Ltd. for sequencing. The sequencing results were compared with NCBI BLAST. The comparison results showed that DA9-6 belonged to the genus *Rosellomorea*, and the 16S rRNA sequence of this strain showed more than 99% homology with sequences from multiple species within the genus *Rosellomorea*. Furthermore, relevant sequences of *Rosellomorea* were downloaded from NCBI and a phylogenetic tree of DA9-6 was constructed using BioEdit and PhyloSuite v1.2.1 software. Sequence similarity analysis was performed with *Rosellomorea* strains. The results are as follows: Figure 2As shown, DA9-6 is different from Rossellomorea sp. are in the same branch.

[0038] Further strain identification was carried out at the Ministry of Agriculture and Rural Affairs Microbial Fertilizer and Edible Fungus Strain Quality Inspection Test Center, and the results of whole genome ANI and dDDH analysis are shown in Table 1: the 16S rRNA sequence homology of the strain DA9-6 is more than 99%, and the strains are different species, combined with morphological analysis, the strain is determined to be Moraxella rossiae Rossellomorea sp . ).

[0039] Moraxella rossiae Rossellomorea sp. is preserved in China Center for Type Culture Collection, the preservation number is CCTCC No: M20242938, the preservation date is December 30, 2024, and the strain base sequence is shown in SEQ ID No. 1.

[0040] Table 1: Summary of whole genome ANI and dDDH analysis

[0041]

[0042] SEQ ID No. 1:

[0043]

[0044] Example 2 Determination of the growth-promoting ability of Moraxella rossii DA9-6

[0045] The gibberellin 3 (GA3), cytokinin (CTK), and ACC deaminase (ACCD) of Moraxella rossii DA9-6 were determined by enzyme-linked immunosorbent assay (ELISA) using a double antibody one-step sandwich method. The content of exopolysaccharide (EPS) secreted by Moraxella rossii DA9-6 strain was determined by the phenol-sulfuric acid method to determine whether the strain has growth-promoting ability.

[0046] The results are shown in Table 2. Moraxella rossii DA9-6 produced gibberellin GA3 100.83 pmol / L, cytokinin 30.41 ng / mL, ACC deaminase activity 0.148 U / mL, and exopolysaccharide 245.49 mg / L. Among them, GA3 promotes cell elongation and division, increases plant height and leaf area; CTK promotes cell division and differentiation, delays leaf senescence, promotes lateral bud germination, increases branch number, and enhances the absorption and utilization of nutrients by crops (such as promoting the absorption of nitrogen and phosphorus by roots); ACCD can degrade ethylene precursor ACC in plants, reduce ethylene levels under stress (such as drought and salt stress), alleviate the inhibitory effect of stress on crops, and promote root growth (such as increasing root length and root hair number), indirectly improving nutrient absorption capacity. EPS can improve soil structure, enhance water and fertilizer retention capacity, and create a favorable microenvironment for crops; protect the strain from harsh environments (such as high salt and extreme pH), and improve its survival rate in soil. Therefore, Moraxella rossii DA9-6 has growth-promoting effects on plants.

[0047] Table 2 Determination of the growth-promoting function of the strain

[0048]

[0049] Example 3 Salt and alkali stress tolerance of Moraxella rossii DA9-6

[0050] (1) Salt stress tolerance of the strain

[0051] LB solid medium containing different concentrations of NaCl (1%, 8%, 10%, 12%, 14%, 16%, 17%, and 18%) was set up to simulate different degrees of salt stress environment. In a clean bench, a single colony of strain DA9-6 was inoculated into LB liquid medium. The strain DA9-6 was cultured at 180 r / min for 18 h to obtain the bacterial solution. 100 μL of the bacterial solution was spread on the above LB solid medium and incubated at 30°C and 180 r / min for 48 h. The highest salt stress tolerance of the strain was observed. The results are shown in Table 3. Figure 3As shown, the strain can still grow normally under 16% NaCl salt concentration stress conditions.

[0052] (2) Alkali stress tolerance of the strain

[0053] Set up LB solid medium with different pH (7, 8, 9, 10, 11, 12, 13, 14) to simulate different degrees of alkali stress environment. In the clean bench, 100 μL of strain DA9-6 bacterial liquid was inoculated on the above LB solid medium by using a pipette gun, and incubated at 30°C, 180 r / min for 48 h. The highest alkali stress environment tolerated by the strain was observed. The results are shown in Figure 4 As shown, the strain can still grow normally under pH 14 conditions.

[0054] (3) Salt-alkali double stress tolerance of the strain

[0055] Set up LB liquid medium with different pH and different NaCl concentrations to simulate different degrees of salt-alkali stress environment. In the clean bench, 2% of DA9-6 bacterial liquid was inoculated into the above medium, and each treatment was repeated three times. The medium was incubated at 30°C, 180 r / min for 48 h. The highest salt-alkali double stress environment tolerated by the strain was evaluated by measuring the OD 600 value of different treatments. The greater the OD 600 value, the greater the biomass. The results are shown in Figure 5 As shown, the strain can still grow normally under pH 10, 10% NaCl conditions.

[0056] Example 4 Drought stress tolerance of Moraxella rossiae DA9-6

[0057] Set up LB liquid medium containing different concentrations of PEG6000 (0, 10%, 15%, 20%, 25%, 30%, 35%) to simulate different degrees of drought stress. In the clean bench, 2% of DA9-6 bacterial liquid was inoculated into the above medium, and each treatment was repeated three times. The medium was incubated at 30°C, 180 r / min for 72 h. The highest drought stress tolerance of the strain was evaluated by measuring the OD 600 value of different treatments. The results are shown in Figure 6 As shown, the strain can still grow normally under PEG6000 30% conditions.

[0058] Example 5 Potting growth promotion effect of microbial inoculant containing Moraxella rossiae DA9-6

[0059] A liquid microbial inoculant of DA9-6 strain with a viable bacterial count of ≥3×10 9 cfu / mL was prepared.

[0060] The preparation process of Roseburia hominis DA9-6 inoculant is as follows: the DA9-6 strain stored in a-80℃ refrigerator is inoculated on LB solid medium, and then cultured in a 30℃ constant temperature incubator for 24 hours; a single colony of the strain is picked and inoculated into LB liquid medium with pH 7.5, and then cultured at 30℃ and 180r / min for 24 hours. The concentration of the DA9-6 strain in the bacterial suspension is adjusted to ≥3×10 9 cfu / mL, and the obtained fermentation liquid is the inoculant.

[0061] (1) The corn potting test is carried out by using typical soda saline-alkali soil in Qian'an County, Songyuan City, Jilin Province. The physicochemical properties of the typical soda saline-alkali soil in Qian'an County, Songyuan City, Jilin Province are shown in Table 3. A non-inoculant group is set as a CK control group, and an inoculant group is set as a T1 treatment group. The T1 treatment group is irrigated with 100-fold diluted DA9-6 bacterial liquid, and the control group is irrigated with the same amount of water. The crops are grown under the conditions of 25℃-27℃ and natural light, and the inoculant is applied once every two weeks, and the compound water-soluble fertilizer (19-19-19) of Sinochem Fertilizer Co., Ltd. is applied, and the application amount is 5kg / acre. After 30 days, the morphology is observed, and the phenotypic indexes are measured.

[0062] The growth of corn is shown in Figure 7 The growth of corn is shown in Figure 8 The fresh weight and dry weight data of corn are shown in Table 4. The fresh weight of the aboveground part of the T1 treatment group is increased by 15%, the fresh weight of the underground part is increased by 28%, the dry weight of the aboveground part is increased by 21%, and the dry weight of the underground part is increased by 15%. As shown in

[0063] Table 3 Physicochemical properties of saline-alkali soil in Qian'an County

[0064]

[0065] Table 4 Average fresh weight and dry weight of corn

[0066]

[0067] (2) Tomato pot experiment adopts typical soda saline-alkali soil and grass charcoal soil (EC: 300 μs / cm, pH 5.0) in Qian'an County, Songyuan City, Jilin Province. Set up saline-alkali soil without adding microbial agent as control group CK1, and add DA9-6 microbial agent as treatment group T1. Set up grass charcoal soil without adding microbial agent as control group CK2, and add DA9-6 microbial agent as treatment group T2. Each treatment sets 24 pots, T1 and T2 use 100 times diluted DA9-6 microbial solution for irrigation, CK1 and CK2 use the same amount of water for irrigation. The crops grow under the condition of 25-27℃ natural light, and the microbial agent is applied once every 2 weeks, and the compound water-soluble fertilizer (19-19-19) of Sinochem Fertilizer Co., Ltd. is applied, and the application amount is 5 kg / acre. After 30 days, the morphology is observed and the phenotype index is measured.

[0068] The growth of tomatoes is shown in Figure 9 From Figure 9 It can be seen that, whether it is saline-alkali soil or grass charcoal soil, the crop growth of the microbial agent treatment group is better than that of the control group, and the root system is more developed. As can be seen from Table 5, the crop growth of the microbial agent treatment group T1 is obviously better than that of the control group CK1, the fresh weight of the aboveground part of the microbial agent treatment group T1 is increased by 58%, the fresh weight of the underground part is increased by 86%, the dry weight of the aboveground part is increased by 57%, and the dry weight of the underground part is increased by 92%. As can be seen from Table 5, Figure 10 It can be seen that, whether it is saline-alkali soil or grass charcoal soil, the crop growth of the microbial agent treatment group is better than that of the control group, and the root system is more developed. As can be seen from Table 5, the crop growth of the microbial agent treatment group T1 is obviously better than that of the control group CK1, the fresh weight of the aboveground part of the microbial agent treatment group T1 is increased by 58%, the fresh weight of the underground part is increased by 86%, the dry weight of the aboveground part is increased by 57%, and the dry weight of the underground part is increased by 92%. As can be seen from Table 5, Figure 11 It can be seen that, whether it is saline-alkali soil or grass charcoal soil, the crop growth of the microbial agent treatment group is better than that of the control group, and the root system is more developed. As can be seen from Table 5, the crop growth of the microbial agent treatment group T1 is obviously better than that of the control group CK1, the fresh weight of the aboveground part of the microbial agent treatment group T1 is increased by 58%, the fresh weight of the underground part is increased by 86%, the dry weight of the aboveground part is increased by 57%, and the dry weight of the underground part is increased by 92%. As can be seen from Table 5,

[0069] Table 5 Average fresh weight and dry weight data of tomatoes

[0070]

[0071] In summary, the Moraxella rosselloensis DA9-6 provided by the present application can tolerate salt, alkali and drought stress, can tolerate single alkali stress with pH of 14, single salt stress with NaCl of 16%, can tolerate salt-alkali double stress environment with 10% NaCl and pH of 10, and can tolerate 30% PEG6000 drought environment; can improve the stress resistance of plants, and can promote plant growth, and can increase the dry weight of corn seedlings in soda saline-alkali soil by 21%, and can increase the dry weight of tomato seedlings by 57%.

[0072] The above only describes the preferred embodiments of the present application, and is not intended to limit the present application. Any modification, equivalent replacement and improvement made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A strain of Moraxella rossii, characterized in that, The Moraxella rossica strain is preserved in the China Center for Type Culture Collection, classified and named as Rossellomorea sp. DA9-6, with a preservation number of CCTCC No: M20242938, a preservation date of December 30, 2024, and a preservation address of China. Wuhan. Wuhan University.

2. Use of Moraxella rossiae according to claim 1, characterized in that, The Rossellomorea in the application of improving the stress resistance of crops; the stress is one or several of salt stress, alkali stress, drought stress.

3. Use of the Moraxella rossius of claim 1, characterized in that, The Rossellomorea in the application of promoting the growth of crops under stress conditions; the stress conditions are one or several of salt stress, alkali stress, drought stress.

4. Use according to claim 3, characterized in that, The Rossellomorea as a preparation for improving the stress resistance of crops and promoting the growth of crops in saline-alkali soil.

5. A preparation for improving stress tolerance and promoting growth of crops, characterized in that: The preparation contains the strain of claim 1.

6. The preparation according to claim 5, characterized in that, The preparation contains the culture, culture suspension or fermentation broth of the strain of claim 1.

7. The formulation of claim 5, wherein, The concentration of the strain in the preparation is 3 x 10 9 cfu / mL ~ 7 x 10 9 cfu / mL.

8. Use of a preparation according to claim 5, characterized in that, The preparation in the application of promoting the growth of crops under stress conditions; the stress is one or several of salt stress, alkali stress, drought stress.

9. Use according to claim 8, characterized in that, The crops are vegetables, fruits, food crops and pasture.

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