Prosteria megatherium and application thereof

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to a Prlisteria megatherium strain and application thereof. The invention provides a strain of Pristeria megaterium A3, and the preservation number of the Pristeria megaterium A3 is CGMCC (China General Microbiological Culture Collection Center) No. 31931. The prelisteria megatherium A3 can generate indoleacetic acid (IAA), and can directly promote the elongation of a plant root system, so that the opportunity that a plant is in contact with nutrient substances in soil is increased, the content of endogenous IAA in a plant body is increased, the expression of a plant defense gene is induced, and the stress resistance such as disease resistance and drought resistance of the plant body is improved. Meanwhile, the prelisteria megatherium A3 disclosed by the invention has phosphorus solubilizing capacity, and can convert phosphorus which is difficult to utilize into available phosphorus, so that the content of available phosphorus in soil is increased. In addition, the prelisteria megatherium A3 also has potassium dissolving capacity, and can improve the utilization rate of the fertilizer and promote the growth and development of plants and the absorption of the fertilizer.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbiology, and particularly relates to a Priestia megaterium and its application. Background Art

[0002] Plant growth-promoting rhizobacteria (PGPR) refer to a class of beneficial bacteria that live freely in the soil or attach to the roots of plants, which can promote plant growth and the absorption and utilization of mineral nutrients, and inhibit harmful organisms. The direct promotion of plant growth by PGPR means that some plant growth-promoting rhizobacteria can synthesize substances that have a direct effect on plant growth and development to promote plant growth. It can also change the forms of some ineffective elements in the soil, such as promoting the dissolution of insoluble phosphorus and potassium in the soil, making them available for plant absorption. At present, the reports on multifunctional growth-promoting strains that can produce hormones and dissolve phosphorus and potassium are still limited. Summary of the Invention

[0003] The purpose of the present invention is to provide a Priestia megaterium and its application. The Priestia megaterium A3 of the present invention has a strong function of producing indoleacetic acid, can promote the dissolution of insoluble phosphorus and potassium in the soil, increase the nutrient content in the soil, promote plant growth, and improve plant quality.

[0004] The present invention provides a Priestia megaterium A3, and the preservation number is CGMCC No. 31931.

[0005] The present invention also provides a bacterial agent, and the active ingredient of the bacterial agent includes the Priestia megaterium A3 described in the above solution.

[0006] Preferably, the effective viable count of Priestia megaterium A3 in the bacterial agent is 10 8 ~10 11 CFU / g.

[0007] The present invention also provides the application of the Priestia megaterium A3 or the bacterial agent described in the above solution, and the application includes one or more of the following 1) to 5): 1) Producing indoleacetic acid; 2) Promoting plant growth; 3) Increasing the nutrient content in the soil; 4) Improving plant quality; 5) Controlling the pathogen of apple canker.

[0008] Preferably, the promoting plant growth includes at least one of promoting root growth, increasing the leaf area of plants, increasing plant height, and increasing plant weight.

[0009] Preferably, the plants include crops; the crops include cash crops, vegetable crops, or food crops.

[0010] Preferably, the increasing of nutrient content in the soil includes increasing the content of at least one of indoleacetic acid, available phosphorus and available potassium in the soil.

[0011] Preferably, the improving of plant quality includes increasing the content of at least one of total sugar, reducing sugar and potassium content in the plant.

[0012] Preferably, the application includes the following steps: applying the Priestia megaterium A3 described in claim 1 or the microbial agent described in claim 2 or 3 into the soil planted with plants.

[0013] Preferably, before applying the Priestia megaterium A3 into the soil planted with plants, it further includes: performing an enlarged culture on the Priestia megaterium A3 to obtain a bacterial suspension; the culture medium used for the enlarged culture is based on an inorganic salt culture medium and further includes components with the following mass percentage contents: 1% carbon source and 0.05% - 0.1% nitrogen source; the carbon source includes maltose or sucrose; the nitrogen source includes urea or peptone; the pH value of the culture medium used for the enlarged culture is 6, 7 or 8.

[0014] Beneficial effects:

[0015] The present invention provides a strain of Priestia megaterium A3, which is preserved in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms. The preservation date is September 10, 2024, and the preservation number is CGMCC No. 31931. The concentration of indoleacetic acid (IAA) produced by the Priestia megaterium A3 of the present invention can reach more than 55.47 mg / L, which can directly promote the elongation of plant roots, thereby increasing the chance of contact between plants and nutrients in the soil, increasing the content of endogenous IAA in plants, inducing the expression of plant defense genes, and improving the disease resistance, drought resistance and other stress resistance of plants. At the same time, the Priestia megaterium A3 of the present invention has the ability to dissolve phosphorus, and the concentration of dissolved insoluble inorganic phosphorus can reach more than 427.60 mg / L, which can convert the difficult-to-use phosphorus into available phosphorus and increase the content of available phosphorus in the soil. The Priestia megaterium A3 of the present invention also has the ability to release potassium, and the solubility of dissolved insoluble potassium reaches more than 172.29 mg / L, which can improve the utilization rate of fertilizers, promote the growth and development of plants and the absorption of fertilizers.

[0016] The Priestia megaterium A3 of the present invention can promote plant growth. The results of the examples show that applying the Priestia megaterium A3 to the planting of potted plants has a good improvement on the growth and development of economic crops such as tobacco, vegetable crops such as pakchoi and food crops such as wheat.

[0017] Applying Priestia megaterium A3 to field tobacco cultivation can effectively improve the yield and quality of tobacco. Experiments have proven that applying Priestia megaterium A3 to tobacco, pakchoi, and wheat potted plants can promote the increase of IAA content and nutrient content in the soil. At the same time, it promotes the roots of crops to be thicker, longer, and have a larger surface area, which is beneficial to the absorption of water and nutrients by crop roots, making the plant height, dry weight, and fresh weight of the above-ground parts of the crops significantly higher than those of the control treatment. In the tobacco field experiment, compared with the control, the treatment with Priestia megaterium A3 made the tobacco have a higher plant height and leaf area, and could increase the tobacco yield. The contents of total sugar and reducing sugar in the cured tobacco were increased by 12.03% and 12.37% respectively compared with the control treatment. At the same time, the potassium content in the cured tobacco leaves was significantly increased by 21.93% compared with the control treatment. It shows that Priestia megaterium A3 of the present invention can improve the yield and quality of tobacco. Brief Description of the Drawings

[0018] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments.

[0019] Figure 1 It is a colony map of Priestia megaterium A3 of the present invention;

[0020] Figure 2 It is a phylogenetic tree established by the neighbor-joining method based on the 16S rDNA sequences of Priestia megaterium A3 and related strains of the present invention;

[0021] Figure 3 It is the antagonistic effect diagram of Priestia megaterium A3 of the present invention against the pathogen of apple canker; the left figure is the effect diagram without inoculating Priestia megaterium A3; the right figure is the effect diagram after inoculating Priestia megaterium A3.

[0022] Biological Deposit Description

[0023] Priestia megaterium A3 was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on September 10, 2024. The unit abbreviation is CGMCC, and the address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing. The deposit number is CGMCC No. 31931. Detailed Embodiments

[0024] The present invention provides a strain of Priestia megaterium A3, characterized in that the deposit number is CGMCC No. 31931.

[0025] The Priestia megaterium A3 of the present invention was isolated from yellow brown soil. Through morphological and 16S rDNA molecular identification, the results showed that the Priestia megaterium A3 was Priestia megaterium. The cell morphology of the Priestia megaterium A3 is as follows: it has a capsule, no spore, a single polar flagellum, can move, and can form a 1.2 mm colony after culturing on a selective medium for 24 h. The colony morphology is that the colony is off-white, round, convex on the surface, smooth, moist, relatively viscous, easy to pick up, and the edge is neat. The physiological and biochemical characteristics of the Priestia megaterium A3 are: Gram-positive, facultative anaerobic, catalase-positive, M.R. test-positive, V.P. test-negative, starch hydrolysis-positive, gelatin liquefaction-positive, nitrate reduction-negative, citrate utilization-positive.

[0026] In the present invention, the Priestia megaterium A3 has the ability to secrete IAA and the ability to promote the dissolution of insoluble phosphorus and potassium in the soil; the strain can secrete more than 55.47 mg / L of IAA, which can directly promote the elongation of plant roots, thereby increasing the contact opportunity between plants and nutrients in the soil, increasing the content of endogenous IAA in plants, inducing the expression of plant defense genes, and improving the disease resistance, drought resistance and other stress resistances of plants. At the same time, the Priestia megaterium A3 of the present invention has the ability to dissolve phosphorus, and the concentration of dissolved insoluble inorganic phosphorus reaches more than 427.60 mg / L, which can convert the difficult-to-use phosphorus into available phosphorus and increase the content of available phosphorus in the soil. The Priestia megaterium A3 of the present invention also has the ability to release potassium, and the solubility of dissolved insoluble potassium reaches more than 172.29 mg / L, which can improve the utilization rate of fertilizers, promote the growth and development of plants and the absorption of fertilizers.

[0027] The present invention also provides a microbial agent, and the active ingredient of the microbial agent includes the Priestia megaterium A3 described in the above solution.

[0028] In the specific implementation process of the present invention, the effective viable count of Priestia megaterium A3 in the microbial agent is 10 8 ~10 11 CFU / g, and more preferably 5×10 11 CFU / g. In the specific implementation process of the present invention, the microbial agent includes Priestia megaterium A3 and a carrier; the present invention has no special restrictions on the type of the carrier, and the well-known carriers of microbial agents in the art can be used; in the embodiments of the present invention, the type of the carrier is bone meal. The present invention has no special restrictions on the preparation method of the microbial agent, and the well-known preparation methods of microbial agents in the art can be used.

[0029] The present invention also provides the application of the above-described Priestia megaterium A3 or the above-described microbial agent, and the application includes one or more of the following 1) to 5): 1) producing indole-3-acetic acid; 2) promoting plant growth; 3) increasing the nutrient content in the soil; 4) improving the quality of plants; 5) controlling the pathogen of apple canker.

[0030] The concentration of indole-3-acetic acid produced by the Priestia megaterium A3 of the present invention can reach more than 55.47 mg / L, which is significantly higher than 17.98 μg / mL secreted by the plant growth-promoting rhizobacterium YC5 disclosed in the prior art (Chinese Patent 201410761450.2). The Priestia megaterium A3 of the present invention can increase the content of endogenous IAA in plants, induce the expression of plant defense genes, and improve the disease resistance, drought resistance and other stress resistances of plants.

[0031] In the specific implementation process of the present invention, the promoting plant growth includes at least one of promoting root growth, increasing the leaf area of plants, increasing the plant height and increasing the plant weight.

[0032] In the specific implementation process of the present invention, the plants include crops; the crops include cash crops, vegetable crops or food crops; the cash crops include tobacco, cotton or fruit trees; the vegetable crops include pakchoi or tomatoes; the food crops include wheat, millet, corn or sorghum.

[0033] The indole-3-acetic acid produced by the Priestia megaterium A3 of the present invention can directly promote the growth of plant roots, make the roots thicker, longer and have a larger surface area, thereby increasing the contact opportunity between plants and nutrients in the soil, and increasing the plant height, leaf area and dry and fresh weights of the above-ground parts of plants.

[0034] In the specific implementation process of the present invention, the increasing the nutrient content in the soil includes increasing the content of at least one of indole-3-acetic acid, available phosphorus and available potassium in the soil.

[0035] The Priestia megaterium A3 of the present invention can promote the dissolution of insoluble phosphorus and / or potassium in the soil. The concentration of insoluble inorganic phosphorus dissolved by the Priestia megaterium A3 of the present invention can reach more than 427.60 mg / L, which can convert the difficult-to-use phosphorus into available phosphorus and increase the content of available phosphorus in the soil; and the Priestia megaterium A3 of the present invention also has the ability to decompose potassium, and the solubility of insoluble potassium reaches more than 172.29 mg / L, thereby improving the utilization rate of fertilizers, promoting the growth and development of plants and the absorption of fertilizers by plants.

[0036] In the specific implementation process of the present invention, the improving the quality of plants includes increasing the content of at least one of the total sugar, reducing sugar and potassium content of plants.

[0037] In the present invention, the Megasphaera elsdenii A3 can increase the contents of total sugar, reducing sugar and potassium in flue-cured tobacco, thereby increasing the yield and quality of tobacco.

[0038] In the specific implementation process of the present invention, the application includes the following steps: applying the Megasphaera elsdenii A3 or the bacterial agent described in the above solution into the soil planted with plants.

[0039] In the specific implementation process of the present invention, the soil water content of the soil is 50% - 60% of the maximum water holding capacity in the field; the application amount of the bacterial agent is 35 - 45 kg / hm 2 ; the inoculation amount of the Megasphaera elsdenii A3 or the bacterial agent is 1 - 9×10 8 CFU / g soil, further being 5×10 8 CFU / g soil. In the specific embodiment of the present invention, the application amount of the bacterial agent can be any value within 35 - 45 kg / hm 2 , such as 40 kg / hm 2 .

[0040] Before applying the Megasphaera elsdenii A3 to the soil planted with plants, it further includes: expanding and culturing the Megasphaera elsdenii A3 to obtain a bacterial suspension. In the specific implementation process of the present invention, the pH of the culture medium for the expansion culture is 6, 7 or 8; the expansion culture is carried out in a 250 mL Erlenmeyer flask; the culture medium used for the expansion culture is based on an inorganic salt medium, and further includes the following components in mass percentage: 1% carbon source and 0.05% - 0.1% nitrogen source; the inorganic salts include one or more of sodium chloride, potassium chloride, tricalcium phosphate, ammonium sulfate, magnesium sulfate heptahydrate, manganese sulfate and ferrous sulfate heptahydrate. As an implementation method, the formula of the inorganic salt medium is: ammonium sulfate 2.0 g, sodium dihydrogen phosphate 0.5 g, dipotassium hydrogen phosphate 0.5 g, magnesium sulfate heptahydrate 0.2 g, calcium chloride dihydrate 0.1 g and distilled water 1000 mL, pH 7.0, sterilized at 121 °C for 20 min. As an implementation method, the carbon source includes maltose or sucrose; the nitrogen source includes urea or peptone. The present invention has no special restrictions on the sources of the carbon source, nitrogen source and inorganic salts, and the above-mentioned sources of the carbon source, nitrogen source and inorganic salts well-known to those skilled in the art can be used. As an implementation method, the time for the expansion culture is 24 - 68 h; the temperature for the expansion culture is 30 °C. In the present invention, the expansion culture is an oscillating culture, and the rotation speed of the oscillating culture is 180 rpm; the volume of the culture medium filled during the oscillating culture is 30 - 50 mL / 250 mL conical flask. As an implementation method, when the carbon source is maltose, the nitrogen source is urea, the pH is 8, and the liquid loading volume is 30 mL / 250 mL, the effect of the Megasphaera elsdenii A3 producing IAA is the best; as another implementation method, when the carbon source is maltose, the nitrogen source is peptone, the pH is 6, and the liquid loading volume is 30 mL / 250 mL, the ability of the Megasphaera elsdenii A3 to dissolve insoluble inorganic phosphorus is the best; as another implementation method, when the carbon source is sucrose, the nitrogen source is peptone, the pH is 7, and the liquid loading volume is 50 mL / 250 mL, the ability of the Megasphaera elsdenii A3 to dissolve insoluble inorganic potassium is the best.

[0041] The Megasphaera elsdenii A3 of the present invention not only has a strong IAA-producing function, but also can promote the dissolution of insoluble phosphorus and potassium in the soil. In the cultivation of tobacco crops, it can not only promote plant growth but also greatly improve the tobacco yield. At the same time, this strain can also promote the growth and development of the vegetable crop pakchoi and the food crop wheat.

[0042] In order to further illustrate the present invention, the following describes in detail a strain of Megasphaera elsdenii and its application provided by the present invention with reference to the drawings and embodiments, but they cannot be understood as limiting the protection scope of the present invention.

[0043] Example 1

[0044] 1. Preparation of experimental culture media and isolation of bacteria

[0045] Formula of LB medium: 10 g of peptone, 5 g of yeast extract, 10 g of sodium chloride, 20 g of agar, and 1000 mL of distilled water, pH 7.0 - 7.2, sterilized at 121 °C for 20 min. On this basis, without adding agar, it is LB liquid medium.

[0046] Formula of inorganic phosphate bacteria medium (PKO medium): 5 g of tricalcium phosphate, 10 g of glucose, 0.5 g of ammonium sulfate, 0.3 g of sodium chloride, 0.3 g of magnesium sulfate heptahydrate, 0.3 g of potassium chloride, 0.03 g of manganese sulfate, 0.03 g of ferrous sulfate heptahydrate, and 1000 mL of distilled water, pH 7.0 - 7.2, sterilized at 121 °C for 20 min.

[0047] Formula of liquid potassium - releasing bacteria medium: 10.0 g of sucrose, 0.5 g of yeast extract, 1.0 g of ammonium sulfate, 2.0 g of sodium dihydrogen phosphate, 0.5 g of magnesium sulfate heptahydrate, CaCO 3 1.0 g, 1.0 g of potassium feldspar powder, and 1000 mL of distilled water, sterilized at 121 °C for 20 min.

[0048] Formula of inorganic salt medium: 2.0 g of ammonium sulfate, 0.5 g of sodium dihydrogen phosphate, 0.5 g of dipotassium hydrogen phosphate, 0.2 g of magnesium sulfate heptahydrate, 0.1 g of calcium chloride dihydrate, and 1000 mL of distilled water, pH 7.0, sterilized at 121 °C for 20 min.

[0049] A. Collect 10 g of tobacco - growing soil under natural conditions in Luoshan County, Xinyang City, Henan Province. The soil is yellow - brown soil, and its basic physical and chemical properties are shown in Table 1. B. Place the soil collected in step A into a 250 - mL Erlenmeyer flask containing 100 mL of sterilized water, shake it in a shaker at 30 °C and 150 rpm for 20 min, and then let it stand for 10 min to obtain a soil bacteria suspension. C. The soil bacteria suspension obtained in step B contains several plant - growth - promoting rhizobacteria. After dilution by the dilution method, it is spread on the LB medium. Invert the petri dish and culture it in an incubator at 30 °C for 24 h. Then, pick different types of typical single colonies, after purification on the plate, store them at 4 °C on the LB slant, and prepare an OD 600 as a seed solution for use, and screen out plant - growth - promoting bacteria that can secrete indole - 3 - acetic acid through qualitative determination and quantitative determination.

[0050] Table 1 Basic physical and chemical properties of the tested soil (yellow - brown soil)

[0051]

[0052] Next, screen out plant - growth - promoting bacteria that can secrete indole - 3 - acetic acid through qualitative determination and quantitative determination.

[0053] 2. Qualitative determination

[0054] A. The bacteria separated and purified from yellow brown soil were inoculated into LB liquid medium containing L-tryptophan (100 mg / L) at an inoculation amount of 1% with the seed liquid, and cultured on a shaker at 30 °C and 180 rpm for 1 day. B. After the culture was completed, 50 μL of the bacterial suspension in step A was dropped onto a white ceramic plate, and at the same time, 50 μL of Salkowski colorimetric solution (50 mL of 35% (w / v) HClO 4 + 1 mL of 0.5 M FeCl 3 ) was added. C. The positive control group was: 50 μL of 50 mg / L indoleacetic acid and 50 μL of Salkowski colorimetric solution were dropped onto a white ceramic plate. D. The white ceramic plate was placed in the dark at room temperature for 30 min and then observed. Those with a red color change indicated the ability to secrete indoleacetic acid.

[0055] 3. Quantitative determination

[0056] The bacteria secreting IAA obtained by the primary screening in step 2 were quantitatively determined, and the culture conditions were the same as those in A of step 2. Then, the cultured broth was centrifuged at 10000 rpm for 10 min, and the supernatant was taken and an equal volume of Salkowski colorimetric solution was added. It was allowed to stand in the dark for 30 min, and its OD 530 value was measured. A standard curve was drawn using an analytically pure indoleacetic acid gradient dilution solution. The content of IAA in each liter of the fermentation broth was detected, and the ability of the strain to produce IAA was determined. The results showed that the isolated strain D8 had the ability to secrete IAA, reaching 50.14 mg / L (Table 2).

[0057] 4. The bacteria with better IAA production were screened and their phosphorus solubilizing ability was determined.

[0058] The seed liquid of the isolated strains was inoculated into 250 mL Erlenmeyer flasks containing 50 mL of inorganic phosphorus medium at an inoculation amount of 1% respectively, placed in a shaker, and the shaker temperature was set at 30 °C and the rotation speed was 180 rpm. After culturing for 3 days, the culture broth was centrifuged at 10000 rpm for 5 min using a 2 mL centrifuge tube. Then, the centrifuge tube was taken out, the supernatant was transferred out, and the available phosphorus content was determined by the molybdenum blue colorimetric method. The phosphorus solubilizing ability of the strain was determined by the available phosphorus content in the supernatant. The results showed that the isolated Pantoea agglomerans A3 had the ability to dissolve inorganic phosphorus, reaching 457.67 mg / L (Table 2).

[0059] 5. The bacteria with better IAA production were screened and their potassium solubilizing ability was determined.

[0060] The seed liquid of the isolated strain was inoculated into 250 mL Erlenmeyer flasks containing 50 mL of liquid potassium-solubilizing bacteria medium at an inoculation amount of 1%, placed in a shaker, and the shaker temperature was set at 30 °C and the rotation speed was 180 rpm. After culturing for 3 days, the culture solution was centrifuged at 10000 rpm for 10 min using a 10 mL centrifuge tube. Then, the centrifuge tube was taken out, the supernatant was transferred out, and the potassium content was measured using a flame spectrophotometer. The potassium-solubilizing ability of the strain was determined by the potassium content in the supernatant. The results showed that the isolated Priestia megaterium A3 had the ability to dissolve inorganic potassium, reaching 172.29 mg / L (Table 2).

[0061] Table 2 Growth-promoting ability of Priestia megaterium A3

[0062]

[0063] Example 2

[0064] 1. Aerobic test

[0065] The sterilized LB medium was poured into 3 sterilized test tubes, about 2 / 3 full. On the sterile operating table, the Priestia megaterium A3 cultured on the slant was picked up with an inoculation needle and stabbed into the above medium (it must be stabbed to the bottom of the tube). Cultured at 30 °C, and the results were observed respectively from 3 days to 7 days. Those growing on the surface of the agar column were aerobic bacteria, and those growing along the puncture line were anaerobic or facultative anaerobic bacteria. The test results showed that the colonies of Priestia megaterium A3 grew along the surface of the agar column, and there were also colonies growing inside the puncture line, indicating facultative anaerobiosis.

[0066] 2. Determination of catalase

[0067] One drop of 3% H 2 O 2 was dropped on a clean glass slide. One loop of the Priestia megaterium A3 culture grown on the LB slant for 18 - 24 h was taken and smeared in the H 2 O 2 . If bubbles were produced, it was positive; otherwise, it was negative. The test results showed that Priestia megaterium A3 was catalase-positive (Table 3).

[0068] 3. Methyl red test (M.R. test)

[0069] a. Medium and reagents: 5 g of peptone, 5 g of glucose, 5 g of sodium chloride, 1000 mL of distilled water, adjust the pH to 7.0 - 7.2, dispense into test tubes, 4 - 5 mL per tube, sterilize at 121 °C for 20 min. Reagents: 0.1 g of methyl red, 300 mL of 95% alcohol, 200 mL of distilled water.

[0070] b. Bacterial strain culture and result observation: Inoculate Priestia megaterium A3 into the above culture medium and culture at 30 °C for 1 - 2 days. Add a few drops of methyl red reagent to the culture medium. If the culture medium turns red, it is methyl red positive; if it turns yellow, it is negative (the methyl red color change range is 4.4 red - 6.0 yellow).

[0071] The test results showed that Priestia megaterium A3 was methyl red positive (Table 3).

[0072] 4. Acetylmethylcarbinol test (VP test)

[0073] a. Culture medium: The culture medium is the same as that for the methyl red test.

[0074] b. Bacterial strain culture and result observation: The inoculation and culture are the same as those for the methyl red test. When performing the VP test, take the culture medium (about 2 mL) and mix it with an equal amount of 40% NaOH, add a small amount of creatine, and shake well for 2 - 5 min. If the culture medium turns red, it is VP positive. The test results showed that Priestia megaterium A3 was VP negative (Table 3).

[0075] 5. Starch hydrolysis test

[0076] a. Culture medium and reagent: Add 0.2% soluble starch to the broth peptone agar, dispense into Erlenmeyer flasks, and sterilize at 121 °C for 20 min for later use. Lugol's iodine solution: 1 g of iodine flakes and 2 g of potassium iodide. First, dissolve potassium iodide with a small amount (3 - 5 mL) of distilled water, then add iodine flakes. After the iodine is completely dissolved, dilute with water to 300 mL.

[0077] b. Bacterial strain culture and result observation: Inoculate strain A3 on the plate by spot inoculation and culture at 30 °C for 2 - 4 days. After colonies are formed, drop Lugol's iodine solution on the plate to cover the area around the colonies. If the plate turns blue and a colorless transparent circle appears around the colonies, it indicates that the starch has been hydrolyzed. The size of the transparent circle generally indicates the ability to hydrolyze starch. The test results showed that Priestia megaterium A3 was positive for starch hydrolysis (Table 3).

[0078] 6. Gelatin hydrolysis test

[0079] a. Culture medium and reagent: 5 g of peptone, 120 g of gelatin, and 1000 mL of distilled water. Adjust the pH to 7.2 - 7.4, dispense into test tubes, and the height of the culture medium is about 4 - 5 cm. Sterilize at 121 °C for 20 min.

[0080] b. Bacterial strain culture and result observation: Inoculate Priestia megaterium A3 in the center of the test tube by the stab inoculation method. Culture in an incubator at 30 °C for one month and observe whether the gelatin is liquefied. The test results showed that Priestia megaterium A3 was positive for gelatin hydrolysis (Table 3).

[0081] 7. Nitrate reduction test

[0082] a. Culture medium and reagents Nitrate liquid medium: 10 g of peptone, 1 g of potassium nitrate, 1000 mL of distilled water, pH 7.0 - 7.4. Gries reagent: Solution A: 0.5 g of sulfanilic acid, 150 mL of dilute acetic acid (about 10%); Solution B: 0.1 g of α-naphthylamine, 20 mL of distilled water, 150 mL of dilute acetic acid (about 10%). Diphenylamine reagent: 0.5 g of diphenylamine is dissolved in 100 mL of concentrated sulfuric acid and diluted with 20 mL of distilled water.

[0083] b. Strain culture and result observation Inoculate Priestia megaterium A3 into the nitrate liquid medium and culture at 30 °C for 1, 3, and 5 days. Pour a little culture solution into the small holes of a white porcelain plate, and then drop 1 drop of reagent A and solution B respectively. When the culture solution turns pink, rose red, orange, or brown, etc., it indicates the presence of nitrite, which is a positive reaction for nitrate reduction, otherwise it is negative. The test results show that strain A3 is negative for nitrate reduction (Table 3).

[0084] 8. Utilization of citrate

[0085] a. Culture medium and reagents 2 g of sodium citrate, 5 g of sodium chloride, 0.2 g of magnesium sulfate heptahydrate, 1 g of diammonium hydrogen phosphate, 10 mL of 1% aqueous bromothymol blue solution, 20 g of agar, 1000 mL of distilled water, pH 6.8 - 7.0, sterilize at 121 °C for 20 min.

[0086] b. Strain culture and result observation Inoculate fresh strain A3 on the slant and culture at 30 °C for 3 - 7 days. If the culture medium turns alkaline (blue), it is a positive reaction, otherwise it is negative. The test results show that Priestia megaterium A3 is positive (Table 3).

[0087] Table 3 Physiological and biochemical characteristics of Priestia megaterium A3

[0088] Item Result Item Result Gram staining + Starch hydrolysis + Aerobic test Facultative anaerobe Gelatin liquefaction + Catalase test + Nitrate reduction - Methyl red (M.R.) reaction + Citrate utilization + V-P test -

[0089] Note: +: Positive reaction; -: Negative reaction

[0090] The Priestia megaterium A3 screened and isolated by the above method was sequenced by Shanghai Yingjun Biotechnology Co., Ltd. According to the sequencing results of 16S rDNA, it was queried and analyzed online at http: / / www.ncbi.nlm.nih.gov. The Blast software was used to compare the homology with other 16S rDNA sequences in GenBank. The 16S rDNA phylogenetic tree of Priestia megaterium A3 was constructed using the MEGAversion3 software with the selected similar sequences and the sequence of Priestia megaterium A3, as Figure 2As shown. The sequence information of the 16S rDNA of the huge Priestia megaterium A3 is as shown in SEQ ID NO.1, specifically:

[0091]

[0092] Based on the physiological and biochemical characteristics of Priestia megaterium A3 of the present invention, it was identified as Priestia megaterium, and Priestia megaterium A3 of the present invention was deposited at the General Microbiology Center of the China Committee for Culture Collection of Microorganisms on September 10, 2024, with the deposit number CGMCC No. 31931.

[0093] The cell morphology of the said Priestia megaterium A3 is as follows: it has a capsule, no endospores, a single polar flagellum, can move, and can form a 1.2 mm colony after culturing on a selective medium for 24 h. The colony morphology is that the colony is off-white, round, convex on the surface, smooth, moist, relatively viscous, easy to pick up, and has a neat edge. The physiological and biochemical characteristics of the said Priestia megaterium A3 are: Gram-positive, facultative anaerobic, catalase-positive, M.R. test-positive, V.P. test-negative, starch hydrolysis-positive, gelatin liquefaction-positive, nitrate reduction-negative, citrate utilization-positive.

[0094] Example 3

[0095] In order to further verify the indole-3-acetic acid (IAA) production ability and the optimal conditions of the plant growth-promoting rhizobacterium A3 obtained in Example 1, the effects of different pH values, liquid loading amounts, different carbon sources, and different nitrogen sources on the IAA production were explored below.

[0096] An orthogonal experiment with three levels and four factors was set up to adjust the inorganic salt medium, as shown in Table 4 for details. The carbon and nitrogen sources were added in amounts of 1% and 0.1% (m / v) respectively. The screened strain was inoculated into a 250 mL Erlenmeyer flask and cultured at 30 °C and 180 rpm for 1 d. The amount of IAA produced was determined by the Salkowski colorimetric method; it was continuously cultured at 30 °C and 200 rpm for 3 d, and the supernatant was centrifuged and the available potassium content was determined by a flame spectrophotometer.

[0097] The results are shown in Table 5. The order of the influence of each factor on the IAA production ability of Priestia megaterium A3 is liquid loading amount > carbon source > nitrogen source > pH. By comprehensively comparing the means and intuitive analysis of the four factors, the best condition combination for A3 to produce IAA is that the carbon source is maltose, the nitrogen source is urea, the pH is 8, and the liquid loading amount is 30 mL / 250 mL.

[0098] Table 4 Orthogonal table for optimizing the plant growth-promoting function of Priestia megaterium A3

[0099]

[0100] Table 5 Results of the orthogonal experiment on the IAA production ability of Priestia megaterium A3

[0101]

[0102] Note: K1, K2, and K3 represent the average values of each factor, and R represents the maximum range among K1, K2, and K3. The same applies hereinafter.

[0103] Example 4

[0104] To further verify the inorganic phosphorus solubilizing ability and the optimal conditions of the plant growth promoting rhizobacteria A3 obtained in Example 1, the effects of different pH values, liquid volumes, different carbon sources, and different nitrogen sources on the inorganic phosphorus solubilizing ability were explored below.

[0105] An orthogonal experiment with three levels and four factors was set up to adjust the inorganic phosphorus bacteria medium respectively. See Table 4 for details. The carbon and nitrogen sources were added in amounts of 1% and 0.1% (M / V) respectively. The screened strain was inoculated into a 250 mL Erlenmeyer flask and cultured at 30 °C and 180 rpm for 1 d. The phosphorus content in the supernatant was determined by the molybdenum blue colorimetric method.

[0106] The results are shown in Table 6. The order of the influence of each factor on the inorganic phosphorus solubilizing ability of Priestia megaterium A3 is nitrogen source > liquid volume > pH > carbon source. Through comprehensive comparison of the means and intuitive analysis of the four factors, the optimal condition combination for the inorganic phosphorus solubilizing ability of A3 is carbon source: maltose, nitrogen source: peptone, pH: 6, and liquid volume: 30 mL / 250 mL.

[0107] Table 6 Results of the orthogonal experiment on the inorganic phosphorus solubilizing ability of Priestia megaterium A3

[0108]

[0109] Example 5

[0110] To further verify the optimal conditions for the potassium solubilizing ability of the plant growth promoting rhizobacteria A3 obtained in Example 1, the effects of different pH values, liquid volumes, different carbon sources, and different nitrogen sources on the potassium solubilizing ability were explored below.

[0111] An orthogonal experiment with three levels and four factors was set up to adjust the liquid potassium solubilizing bacteria medium respectively. See Table 4 for details. The carbon and nitrogen sources were added in amounts of 1% and 0.1% (M / V) respectively. The screened strain was inoculated into a 250 mL Erlenmeyer flask and cultured at 30 °C and 180 rpm for 1 d. The potassium content in the supernatant was determined by a flame spectrophotometer.

[0112] The results are shown in Table 7. The order of the influence of each factor on the potassium solubilizing ability of Priestia megaterium A3 is liquid volume > nitrogen source > pH > carbon source. Through comprehensive comparison of the means and intuitive analysis of the four factors, the optimal condition combination for the potassium solubilizing ability of A3 is carbon source: sucrose, nitrogen source: peptone, pH: 7, and liquid volume: 50 mL / 250 mL.

[0113] Table 7 Results of the orthogonal experiment on the potassium solubilizing ability of Priestia megaterium A3

[0114]

[0115] Example 6

[0116] To explore the biocontrol ability of *Priestia megaterium* A3, the apple rot pathogen, wheat root rot pathogen, bitter gourd wilt pathogen, and tobacco black shank pathogen stored in the laboratory were used as the test pathogens, and the antagonistic function of *Priestia megaterium* A3 against them was explored respectively. The plate confrontation method was used, with the pathogen as the target, and the antagonistic activity of the strain after isolation and purification was detected. The pathogen cake was placed at the center of the OA plate, and the strain was inoculated around the pathogen cake. Three replicates were set, and only the pathogen cake was used as the control. Incubate at 28 °C, and determine the antagonistic ability according to the presence or absence of an inhibition zone. As Figure 3 shown, A3 showed better antagonistic ability against the apple rot pathogen. The apple rot pathogen had filled the culture dish in the OA medium without inoculating *Priestia megaterium* A3, while there was an obvious inhibition zone around *Priestia megaterium* A3 in the medium inoculated with *Priestia megaterium* A3.

[0117] Example 7

[0118] *Priestia megaterium* A3 of the present invention has an obvious growth-promoting effect on tobacco, which is illustrated by the following pot experiment.

[0119] Fresh soil from the 0-20 cm soil layer of natural tobacco-growing soil in Luoshan County, Xinyang City, Henan Province was collected respectively, passed through a 5-mm sieve, and 700 g of soil was filled into each pot. Tobacco was planted, and the water content was adjusted to 60% of the maximum field water holding capacity. Tobacco seedlings with consistent growth vigor were planted. The test *Priestia megaterium* A3 was cultured and made into a bacterial water agent, and inoculated into the soil at an inoculation amount of 10 8 CFU / g. Without inoculating the strain as the control, there were 4 replicates for each treatment. The potted plants were placed in a light incubator. After 30 days, samples were taken. The content of soil IAA was determined by HPLC method, and the contents of soil pH, organic matter, available nitrogen, available phosphorus, available potassium, fresh weight of plants, plant height, and total nitrogen, total phosphorus, and total potassium contents were also determined.

[0120] Tobacco seeds: Tobacco seeds were surface-sterilized with 20% hydrogen peroxide for 20 min, rinsed with sterile water multiple times, germinated for 2 d, and seeds with consistent germination were selected for standby.

[0121] Inoculation treatment: A3 of the present invention was inoculated into LB liquid medium, cultured at 30 °C and 180 rpm on a shaker. When the bacteria grew to the logarithmic growth phase, the bacterial suspension was centrifuged at 3000 rpm for 10 min, then resuspended with sterile water, and centrifuged three times in the same way. The inoculation amount was 10 8 CFU·g -1 .

[0122] Control treatment: As a control, the soil was not sprayed with A3 bacterial solution, and an equal amount of sterile water was added.

[0123] The results are shown in Table 8. After inoculation with A3, compared with the CK treatment, the contents of soil IAA, available phosphorus, and available potassium increased significantly by 94.44%, 14.11%, and 8.99% respectively, indicating that this strain can effectively increase the nutrient content in the soil to promote the growth of tobacco. After inoculation with A3, compared with the CK treatment, the plant height, fresh weight, dry weight, total root length, root surface area, and root volume of tobacco seedlings increased significantly by 13.39%, 11.17%, 23.83%, 12.03%, 10.26%, and 40.93% respectively (Table 9). From the above results, it can be seen that the multifunctional plant growth-promoting rhizobacteria of the present invention can effectively increase the soil nutrient content, have an obvious effect on promoting the growth and development of tobacco roots, and can effectively promote the growth of tobacco.

[0124] Table 8 Effects of Priestia megaterium A3 on the physical and chemical properties of tobacco potting soil

[0125]

[0126] Note: "*" indicates a significant difference between treatments (p < 0.05), and "**" indicates a highly significant difference between treatments (p < 0.01). The same applies hereinafter.

[0127] Table 9 Effects of Priestia megaterium A3 on the biological traits of tobacco

[0128]

[0129] Example 8

[0130] The growth-promoting effect of Priestia megaterium A3 of the present invention is broad-spectrum, which is illustrated by the following pot experiment.

[0131] The tested soil used for collecting and screening the strains was passed through a 5-mm sieve. 700 g of soil was filled in each pot, and pakchoi was planted. The water content was adjusted to 60% of the maximum water-holding capacity in the field. Pakchoi seeds with consistent germination were planted. The tested Priestia megaterium A3 was cultured and made into a bacterial water agent, and inoculated into the soil at an inoculation amount of 10 8 CFU / g. Without inoculating the strain as a control, there were 4 replicates for each treatment. The pots were placed in a light incubator. After 30 days, samples were taken. The content of soil IAA was measured by HPLC method, and the contents of available phosphorus, available potassium in the soil, fresh weight of the plant, plant height, and total nitrogen, total phosphorus, and total potassium contents were also measured.

[0132] The pakchoi seeds were surface-sterilized with 20% hydrogen peroxide for 20 min, rinsed with sterile water for several times, and germinated for 2 d. The seeds with consistent germination were selected for standby.

[0133] Inoculation treatment: Inoculate A3 of the present invention into LB liquid medium, culture it in a shaker at 30 °C and 180 rpm until the bacteria grow to the logarithmic growth phase. Then centrifuge the bacterial suspension at 3000 rpm for 10 min, resuspend it with sterile water, and centrifuge three times in the same way. The inoculation amount is 10 8 CFU·g -1 .

[0134] Control treatment: As a control, the soil is not sprayed with A3 bacterial solution, and an equal amount of sterile water is added.

[0135] The results are shown in Table 10. After inoculation with A3, compared with the CK treatment, the contents of soil IAA, available phosphorus, and available potassium increased by 100.00%, 12.36%, and 8.56% respectively, indicating that this strain can effectively increase the nutrient content in the soil to promote the growth of pakchoi. After inoculation with A3, compared with the CK treatment, the plant height, fresh weight, dry weight, total root length, root surface area, and root volume of pakchoi seedlings increased by 14.16%, 36.53%, 30.00%, 12.75%, 14.48%, and 14.67% respectively (Table 11). From the above results, it can be seen that the multifunctional plant growth-promoting rhizobacteria of the present invention can effectively increase the soil nutrient content, have an obvious effect on promoting the growth and development of pakchoi roots, and can effectively promote the growth of pakchoi.

[0136] Table 10 Effects of Priestia megaterium A3 on the physical and chemical properties of pakchoi potting soil

[0137]

[0138] Table 11 Effects of Priestia megaterium A3 on the biological traits of pakchoi

[0139]

[0140] Example 9

[0141] The growth-promoting effect of Priestia megaterium A3 of the present invention is broad-spectrum, which is illustrated by the following pot experiment.

[0142] Collect the test soil used for screening the strain, pass it through a 5-mm sieve, fill 700 g of soil into each pot, plant wheat, and adjust the water content to 60% of the maximum water holding capacity of the field. Plant wheat seeds with consistent germination. After culturing the test Priestia megaterium A3, make it into a bacterial water agent, and inoculate it into the soil at an inoculation amount of 10 8 CFU / g, with no inoculation of the strain as the control. There are 4 replicates for each treatment. Place the pots in a light incubator, sample after 30 days, use the HPLC method to measure the soil IAA content, and measure the soil available phosphorus, available potassium contents, plant fresh weight, plant height, and total nitrogen, total phosphorus, and total potassium contents.

[0143] The wheat seeds were surface-sterilized with 20% hydrogen peroxide for 20 min, rinsed several times with sterile water, germinated for 2 d, and the seeds with consistent germination were selected for standby.

[0144] Inoculation treatment: A3 of the present invention was inoculated into LB liquid medium, cultured on a shaker at 30 °C and 180 rpm. When the cultured bacteria grew to the logarithmic growth phase, the bacterial suspension was centrifuged at 3000 rpm for 10 min, and then resuspended with sterile water. The same centrifugation was carried out three times, and the inoculation amount was 10 8 CFU·g -1 。

[0145] Control treatment: As a control, the soil was not sprayed with A3 bacterial solution, and an equal amount of sterile water was added.

[0146] As shown in Table 12. After inoculation with A3, compared with the CK treatment, the contents of IAA, available phosphorus, and available potassium in the soil increased significantly by 115.15%, 17.37%, and 8.47% respectively, indicating that this strain can effectively increase the nutrient content in the soil to promote the growth of wheat. After inoculation with A3, compared with the CK treatment, the plant height, fresh weight, dry weight, total root length, root surface area, and root volume of pakchoi seedlings increased significantly by 13.39%, 11.17%, 23.83%, 5.67%, 21.27%, and 53.52% respectively (Table 13). From the above results, it can be seen that the multifunctional rhizosphere growth-promoting bacteria of the present invention can effectively increase the soil nutrient content, have an obvious effect on promoting the growth and development of wheat roots, and can effectively promote the growth of wheat.

[0147] Table 12 Effects of Priestia megaterium A3 on the physical and chemical properties of wheat potting soil

[0148]

[0149] Table 13 Effects of Priestia megaterium A3 on the biological traits of wheat

[0150]

[0151] Example 10

[0152] Priestia megaterium A3 was made into a microbial agent with bone meal as the carrier, and its effective viable count was 10 11 CFU / g, and a field experiment was carried out to verify the efficacy of A3. Economic crop tobacco was planted at an appropriate planting time. The experiment was set with 3 replicates, arranged in a randomized block design, and the plot area was 6×3 m 2 。All treatments were applied with 15-15-15 compound fertilizer as the base fertilizer, and the application rate was 600 kg·hm -2 At the same time, 40 kg·hm of A3 microbial agent was applied -2 The control treatment was applied with an equal amount of bone meal, and the corresponding agronomic traits and quality indexes were measured at the harvest stage.

[0153] The results are shown in Table 14. Compared with the control, the treatment with Megasphaera elsdenii A3 significantly increased the plant height of tobacco by 6.99% and the maximum leaf area by 32.73%, indicating that this strain can increase the yield of tobacco in the field environment. At the same time, this strain can also improve the quality of tobacco. After inoculation with this strain, compared with the CK treatment, the total sugar, reducing sugar and potassium content of flue-cured tobacco leaves increased by 12.03%, 12.37% and 21.93% respectively (Table 15).

[0154] Table 14 Effects of Megasphaera elsdenii A3 on the biological traits of tobacco

[0155]

[0156] Table 15 Effects of Megasphaera elsdenii A3 on the chemical components of flue-cured tobacco

[0157]

[0158] As can be seen from the above embodiments, the application of Megasphaera elsdenii A3 provided by the present invention in plant cultivation can significantly improve the growth rate of plants, and increase the yield and quality of agricultural crops and products.

[0159] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, not all of them. People can also obtain other embodiments based on these embodiments without creative efforts, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A strain of Priestia megaterium A3, characterized in that The deposit number is CGMCC No.31931.

2. A bacterial agent, characterized in that The active ingredient of the bacterial agent includes the Priococcus megaterium A3 described in claim 1.

3. The bacterial agent according to claim 2, characterized in that The effective viable bacteria count of Priesterol A3 in the bacterial agent is 10 8 ~10 11 CFU / g.

4. Use of the Priococcus megaterium A3 according to claim 1 or the bacterial agent according to claim 2 or 3, wherein the use comprises one or more of the following 1) to 5): 1) Produce indoleacetic acid; 2) Promote plant growth; 3) Increase the nutrient content in the soil; 4) Improve plant quality; 5) Prevent and control apple rot pathogens.

5. The use according to claim 4, characterized in that: The promoting plant growth comprises at least one of promoting root growth, increasing plant leaf area, increasing plant height and increasing plant weight.

6. The use according to claim 4 or 5, characterized in that: The plants include crops; the crops include cash crops, vegetable crops or food crops.

7. The use according to claim 4, characterized in that: The method of increasing the nutrient content in the soil includes increasing the content of at least one of indoleacetic acid, available phosphorus and available potassium in the soil.

8. The use according to claim 4, characterized in that: The improving of plant quality comprises increasing the content of at least one of total sugar, reducing sugar and potassium in the plant.

9. The use according to claim 4, characterized in that: The application The method comprises the following steps: applying the Priococcus megaterium A3 described in claim 1 or the bacterial agent described in claim 2 or 3 to soil in which plants are planted.

10. The use according to claim 9, characterized in that: Before applying the huge Priesteria A3 to soil planted with plants, the method also includes: expanding the culture of the huge Priesteria A3 to obtain a bacterial suspension; the culture medium used for the expanded culture uses an inorganic salt culture medium as a basic culture medium and also includes the following components in percentage by mass: 1% carbon source and 0.05% to 0.1% nitrogen source; the carbon source includes maltose or sucrose; the nitrogen source includes urea or peptone; the pH value of the culture medium used for the expanded culture is 6, 7 or 8.

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