A highly salt-alkali tolerant strain Rossellomorea aquimaris S-2 and its application

Abstract

The present invention relates to a highly salt-tolerant and alkali-tolerant strain Rossellomorea aquimaris S-2 and an application thereof. The present invention screens and obtains a strain of R.aquimaris S-2, which has the ability to dissolve potassium and can tolerate extreme saline-alkali conditions; applying the strain to root irrigation culture of corn and tomato seedlings can effectively improve the survival ability of crops in a high-salt-alkali environment, and provides an application of the strain in improving crop agronomic traits.

Description

Technical Field

[0001] The invention belongs to the technical field of agricultural microorganisms, and specifically relates to a Rossellomorea aquimaris S-2 strain, a bacterial agent containing the strain and application of the bacterial agent in improving agronomic traits of crops. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Salt is a very important factor affecting crop growth. Salt-tolerant microorganisms can promote plant growth and reduce damage caused by salt stress, which is a common method for crop cultivation. Existing studies have shown that Rossellomorea bacteria can promote the growth of crops: Zou Zhengwei proved that Rossellomorea has the function of producing ACC deaminase, which can reduce the ethylene content during crop growth and is one of the important indicators of strains promoting crop growth. Zhang Qi et al. found that Rossellomorea has the ability to deproteinize and produce lipase. Navarro-Torre et al. isolated an endophyte Rossellomorea sp.EAR8T from plants. The bacterium can grow under conditions of 15-45℃, pH 6.0-9.0 and tolerance to 0-25% NaCl (w / v), promoting plant growth while alleviating the stress caused by heavy metals in the soil to crops. Studies have shown that under salt stress conditions, the application of the bacterial agent Rossellomorea vietnamensis RL-WG62 can increase the survival rate, chlorophyll content, root length and dry and fresh weight of rice, and promote the growth of rice.

[0004] The inventors believe that there is a lack of research on Rossellomorea bacteria, and there is no clear report on the application of R. aquimaris in plant disease prevention and growth promotion. Only one article mentions that R. aquimaris 3.13 has the ability to degrade inulin and fructan, and can promote the fructan synthesis ability of plants. Research on R. aquimaris is expected to obtain microbial agents that improve the agronomic traits of crops. Summary of the invention

[0005] The present invention provides a Rossellomorea aquimaris S-2 strain, which can tolerate 16% NaCl, has potassium-dissolving ability and can grow under extreme alkaline conditions of pH=14. The present invention applies the strain to the cultivation of corn and tomato crops, and proves that the strain can promote the growth of corn and tomato crops, improve the surface agronomic traits of corn and tomatoes, and enhance the growth state of corn and tomato plants under high salinity and alkali conditions.

[0006] According to the above characteristics of the strain, the present invention provides the following technical solutions:

[0007] In the first aspect, a Rossellomorea aquimaris S-2 is provided. The strain was deposited in the General Microbiology Center of the China Microorganism Culture Collection Administration, referred to as CGMCC, on March 23, 2023. The address is: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, and its biological deposit number is: CGMCC No. 26878.

[0008] The present invention obtains the above-mentioned strain through screening. Through genome sequencing, the strain has a 16S rDNA sequence shown in SEQ ID NO: 1, has a high similarity with the known Rossellomorea aquimaris strain, and is named Rossellomoreaaquimaris S-2, hereinafter referred to as R.aquimaris S-2.

[0009] The morphological characteristics of the strain are as follows:

[0010] Bacterial characteristics: The bacteria are rod-shaped, spore-forming, and Gram-positive;

[0011] Colony characteristics: The colonies are yellow and round with dry surface and regular edges.

[0012] The physiological and biochemical characteristics of the strain R. aquimaris S-2 are as follows: it can utilize glucose, sorbitol, raffinose, mannitol, maltose and galactose, and can utilize a small amount of D-cellobiose.

[0013] The strain R.aquimaris S-2 is more preferably cultured in the following manner: inoculated in LB medium, and the culture conditions are as follows: cultured at 30-40°C and a rotation speed of 160-200rpm for 20-30h; further preferably, the culture temperature is 35-38°C and the rotation speed is 170-190rpm for 26-28h, and a specific example is cultured at 37°C and 180rpm for 24h.

[0014] The culture medium needs to be sterilized before inoculation, and conventional sterilization parameters in the art can be used, such as sterilization at 115-121° C. for 20-30 minutes.

[0015] Among them, the components of the LB medium are yeast powder, peptone, and NaCl, with a mass ratio of 1:1.5-3:1.5-3, and the pH is adjusted to 6-8; a further preferred mass ratio is 1:1.5-2.5:1.5-2.5, and the pH is adjusted to 6.5-7.5; in a specific example, the LB medium is prepared as follows: 5g yeast powder, 10g peptone, 10g NaCl, fixed to 1000mL, pH7.0.

[0016] In a second aspect, the present invention provides a bacterial agent, which comprises the R. aquimaris S-2 and / or a culture of the bacteria described in the first aspect.

[0017] Preferably, in the above-mentioned bacterial agent, the number of effective live bacteria is ≥ 10 8 cfu / mL; In a specific embodiment of this preferred embodiment, the bacterial agent is obtained by inoculating R.aquimaris S-2 into the above-mentioned LB medium.

[0018] Preferably, the bacterial agent dosage form of the second aspect includes liquid bacterial agents and solid preparations.

[0019] Furthermore, the liquid preparation is a water suspension, a dispersible oil suspension, a wettable powder or a water dispersible granule, and the liquid preparation further comprises an auxiliary material, and the auxiliary material is selected from one or more of a dispersant, a wetting agent, a disintegrant, a binder, a defoamer, an antifreeze agent, a thickener, a filler and a solvent. The present invention has no special restrictions on the source of the pesticide-acceptable auxiliary material, and generally commercially available products can be used.

[0020] In one embodiment of the solid preparation, a solid carrier is further included, which is a mineral material, a biological material or a polymer compound. The mineral material is at least one of clay, talc, kaolin, montmorillonite, white carbon, zeolite, silica, peat soil and diatomaceous earth; the biological material is at least one of various crop straws, pine husks, rice straw, peanut shells, corn flour, soybean flour, starch, peat and animal feces; and the polymer compound is polyvinyl alcohol and / or polyglycol.

[0021] The third aspect of the present invention provides the use of the strain described in the first aspect and the bacterial agent described in the second aspect in improving the agronomic traits of crop surfaces.

[0022] The above applications include improving the surface agronomic traits of crops, such as increasing the plant height, root length or fresh weight of crops; and also include improving the salt and alkali tolerance of crops.

[0023] In a specific embodiment provided by the present invention, the crop is corn or tomato.

[0024] A fourth aspect of the present invention provides a method for promoting the growth of corn or tomatoes, the method comprising applying the strain described in the first aspect and the bacterial agent described in the second aspect to corn or tomatoes.

[0025] The above-mentioned application method can adopt conventional methods in the field, such as seed soaking, root irrigation, spraying, etc.; the preferred method is seed soaking or root irrigation. During the root irrigation operation, the above-mentioned strain or microbial agent is applied to the soil at the rhizosphere of the plant after the corn or tomato germinates and the seedlings are established.

[0026] The beneficial effects of one or more of the above technical solutions are:

[0027] The present invention reports for the first time a R.aquimaris S-2 that has the effect of promoting the growth of corn and tomatoes. The bacterium has a certain potassium-dissolving ability, high salt resistance and extreme alkalinity resistance. Potted plant experiments have verified that R.aquimaris S-2 can increase the plant height of corn and tomatoes, promote the growth of corn and tomatoes, increase the fresh weight of plant stems and leaves, thereby increasing plant biomass, and at the same time enrich the strain resources of plant growth-promoting bacteria, and has good practical application value.

[0028] The microbial agent containing R.aquimaris S-2 of the present invention is pollution-free and has no toxic side effects. After repeated verification, the strain colonizes rapidly on corn and tomatoes, grows rapidly itself, and is easy to preserve; it can also alleviate the harm caused by saline-alkali stress to crops, thereby effectively meeting the nutritional needs of crop growth, improving the utilization rate of saline-alkali land in my country, and has good practical application value for the efficient development of saline-alkali land resources. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] 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.

[0030] Figure 1 This is the colony map of R. aquimaris S-2;

[0031] Figure 2 This is a microscopic examination of the cell morphology of R.aquimaris S-2;

[0032] Figure 3 This is the result of 16S rDNA phylogenetic tree analysis of R. aquimaris S-2 strain;

[0033] Figure 4 This is a graph showing the potassium solubilization effect of R. aquimaris S-2 strain;

[0034] Figure 5 This is a diagram of the salt tolerance effect of R. aquimaris S-2 strain;

[0035] Figure 6 This is a diagram showing the alkali resistance of the R. aquimaris S-2 strain;

[0036] Figure 7 This is a graph showing the growth-promoting effect of R. aquimaris S-2 strain on corn plants under nutrient solution hydroponic conditions;

[0037] Figure 8 This is a graph showing the growth-promoting effect of R.aquimaris S-2 strain on tomato plants under soil potting conditions. DETAILED DESCRIPTION

[0038] It should be noted that the following detailed descriptions are all illustrative and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0039] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0040] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0041] Example 1 Identification of strain S-2 and determination of its physicochemical properties

[0042] The present invention screened a strain that promotes corn growth, and determined the strain to be R. aquimaris S-2 through 16S rDNA sequence analysis and physiological and biochemical index identification. Figure 1 and Figure 2 As shown in the figure, the colony and bacterial characteristics of the R.aquimaris S-2 are: the colony is yellow and round, with a dry surface and regular edges. The bacterial body is rod-shaped, produces spores, and is Gram-positive. As shown in Table 1, the strain can utilize different sugar substances. The strain can utilize glucose, sorbitol, raffinose, mannitol, maltose and galactose, and can utilize a small amount of D-cellobiose. ("√" means it can be utilized, and "×" means it cannot be utilized.)

[0043] The 16S rDNA sequence of R. aquimaris S-2 is shown in SEQ ID NO:1.

[0044] Table 1 Results of determination of physiological and biochemical indexes of strains

[0045] feature S-2 Rhamnose × sucrose × glucose √ Sorbitol √ Raffinose √ D-Cellobiose weak Mannitol √ Trehalose × maltose √ lactose × Galactose √

[0046] Example 2 Determination of salt-alkali resistance and potassium-solubilizing ability of strain S-2

[0047] Determination of potassium-dissolving ability of strains: Inoculate the strain on silicate bacterial culture medium with a sterile toothpick and culture at 28°C for 3-4 days. Smooth and transparent oil-drop-shaped (capsule) colonies can grow on the plate. Figure 4 As shown, this strain has the function of potassium dissolving.

[0048] Determination of salt tolerance of strains: LB solid medium was used as the basic medium, and NaCl was added at a mass fraction of 0%, 1%, 10%, 12%, 14%, 15%, 16% and 17% to prepare medium with different salt concentrations. The strain was divided into three zones and inverted for overnight culture at 37°C. Single colonies were picked and the seed liquid was shaken in liquid LB medium to allow the bacterial liquid to grow to the middle of the logarithmic phase. 100 μL of the bacterial liquid was spread on solid medium with different NaCl concentrations, inverted for 24 hours at 37°C, and the growth of the colonies was observed. The results are as follows Figure 5 As shown, the strain can tolerate 0%-16% NaCl, but does not grow on 17% NaCl.

[0049] Determination of strain alkali resistance: Based on LB solid medium, adjust the pH to 7, 8, 9, 10, 11, 12, 13 and 14 with NaOH to prepare medium with different pH values. Incubate at 37℃ for 48h and observe the growth of colonies. Figure 6 As shown, the bacteria have the ability to grow under extremely alkaline conditions of pH = 14.

[0050] Example 3 Experiment on the growth promotion of corn plants by microbial agents under high-salt nutrient solution hydroponic conditions

[0051] Preparation of single microbial inoculant: transfer the R.aquimaris S-2 strain stored on the slant to a test tube containing 5 mL of LB liquid culture medium, and culture it in a constant temperature shaker for 12 h at 37 °C and 180 rpm; inoculate 1 mL of the activated bacterial solution into 50 mL of LB liquid culture medium, and culture it in a shaker for 24 h at 37 °C and 180 rpm until the late logarithmic growth phase, when the number of effective live bacteria is ≥10 8 cfu / mL.

[0052] The corn seeds were sterilized and placed in a tissue culture bottle for germination for 24 hours for later use. After the corn seedlings were settled, 2 mL of microbial agent was diluted (the dilution multiple was 100 times), and then 200 mL of the prepared agent was aspirated for root irrigation (CK group was treated by diluting 2 mL of sterilized LB liquid culture medium to 200 mL before root irrigation). After 12 days of liquid (Hoagland nutrient solution) culture, the growth-promoting effect of strain S-2 on the growth of corn plants was observed. Figure 7 ; At the same time, the growth characteristics of corn under salt (100mM NaCl) and salt-free conditions were detected, and the results are shown in Table 2: After the corn plants were irrigated with the above-mentioned single microbial agent, various indicators of the corn plants could be promoted. The plant height, root length and fresh weight of the corn plants were all promoted compared with the control group.

[0053] Table 2 Characteristics of hydroponic corn plants

[0054]

[0055] Note: "CK 0" is the control group without salt, "CK 100" is the control group with salt; "S-2 0" is the group without salt treated with bacterial agent S-2, and "S-2 100" is the group with salt treated with bacterial agent S-2. The salt used is 100mM NaCl.

[0056] The above embodiment studies the effect of promoting corn growth under the conditions of potted plants in a light incubator. The test method used is close to the actual application state and simulates the conditions of agricultural corn planting. It can be seen that adding the bacterial agent has a good effect under high-salt conditions. At the same time, the present invention detects the plant height, maximum root length, and fresh weight of potted corn plants, which strongly proves the growth-promoting effect of the strain on corn plants.

[0057] Example 4 Growth-promoting effect of microbial agents on corn plants under high saline-alkali soil potting conditions

[0058] The treated corn seeds were planted in a mixed salt-alkali (NaCl:Na 2 SO 4 :NaHCO 3 :Na 2 CO 3 The soil (pH = 8.5) was mixed with 1:9:9:1 molar ratio, and the microbial agent S-2 was applied 2 days later. The physiological plant height, stem diameter, aboveground fresh weight and underground fresh weight of corn were measured after 14 days of growth. The results are shown in Table 3. It was found that the application of microbial agent S-2 can promote the physiological plant height, stem diameter, aboveground fresh weight and underground fresh weight of corn plants.

[0059] Table 3 Characteristics of corn plants under high saline-alkali soil conditions

[0060] Physiological plant height (cm) Stem diameter (mm) Fresh weight above ground (g) Underground fresh weight (g) CK 0 37.42±1.13 3.45±0.11 1.34±0.11 0.44±0.01 S-2 0 41.94±2.39 4.00±0.30 1.85±0.18 0.54±0.10 CK+ 34.10±0.88 3.66±0.20 1.28±0.01 0.41±0.00 S-2+ 38.43±2.59 3.68±0.12 1.51±0.02 0.44±0.01

[0061] Note: Physiological plant height is net value added, net value added = final height - initial height. "CK 0" is the control group without salt, "CK+" is the control group with salt; "S-2 0" is the salt-free treatment group with the bacterial agent S-2, and "S-2+" is the salt-treated group with the bacterial agent S-2. Among them, "+" means adding mixed salt (NaCl:Na 2 SO 4 :NaHCO 3 :Na 2 CO 3 Mixed in a molar ratio of 1:9:9:1).

[0062] Example 5 Growth-promoting effect of microbial agents on tomato plants under high saline-alkali soil potting conditions

[0063] The tomato seeds were sterilized in the same manner as in Example 4. The tomato seeds were sown into ordinary soil, and the germination and seedling raising were carried out for about 14 days. After the seedlings were grown for 3 days, mixed saline-alkali water (NaCl:Na 2 SO 4 :NaHCO 3 :Na 2 CO 3 The mixture was mixed at a molar ratio of 1:9:9:1), soil pH = 8.5, and the tomato potted plants were treated with fungi after 10 days (the same as the fungicide treatment method for corn). The physiological plant height, plant height, stem diameter and fresh weight of tomatoes were measured after 18 days of growth. Figure 8 As shown, it is proved that the inoculant made from this strain has a growth-promoting effect on tomato plants.

[0064] Table 4 Tomato plant characteristics

[0065]

[0066] 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 strain of R.aquimaris S-2, which was deposited on March 23, 2023 in the General Microbiology Center of China Microorganism Culture Collection Administration, referred to as CGMCC, at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and its biological deposit number is: CGMCC NO.26878.

2. A bacterial agent, characterized in that Comprising the culture of R. aquimaris S-2 and / or bacteria as described in claim 1.

3. The bacterial agent according to claim 2, characterized in that In the bacterial agent, the number of effective live bacteria is ≥10 8 cfu / mL; the bacterial agent is obtained by inoculating R.aquimaris S-2 into the above LB medium.

4. The bacterial agent according to claim 2, characterized in that The bacterial agent dosage form includes liquid bacterial agent and solid preparation; the liquid preparation is a water suspension, a dispersible oil suspension, a wettable powder or a water dispersible granule, and the liquid preparation also includes an auxiliary material, and the auxiliary material is selected from one or more of a dispersant, a wetting agent, a disintegrant, a binder, a defoaming agent, an antifreeze agent, a thickener, a filler and a solvent; The solid preparation also includes a solid carrier, which is a mineral material, a biological material or a polymer compound. The mineral material is at least one of clay, talc, kaolin, montmorillonite, white carbon, zeolite, silica, peat soil and diatomaceous earth; the biological material is at least one of various crop straws, pine husks, straw, peanut shells, corn flour, soybean flour, starch, peat and animal feces; the polymer compound is polyvinyl alcohol and / or polyglycol.

5. Use of the strain according to claim 1 and / or the bacterial agent according to any one of claims 2 to 4 in improving agronomic traits of crops; The application includes improving the surface agronomic traits of crops and / or improving the salt and alkali tolerance of crops.

6. The use according to claim 5, characterized in that The surface agronomic traits of the crop are to increase the plant height, root length or fresh weight of the crop.

7. A method for promoting the growth of corn and tomatoes, characterized in that: The method comprises applying the strain according to claim 1 and the bacterial agent according to any one of claims 2 to 4 to corn and tomatoes; The application method is seed soaking, root irrigation or spraying.

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