P.megatherium NZ-1, microbial inoculum and application of P.megatherium NZ-1

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to a P.megatherium NZ-1 strain, a microbial agent and application of the P.megatherium NZ-1 strain and the microbial agent. The classification name of the strain is Pristia megaterium, the strain is preserved in the China Center for Type Culture Collection on September 13, 2024, the preservation number is CCTCC NO: M 20241962, the preservation address is No. 299 on eight road, Wuchang District, Wuhan City, Hubei Province, and the postal code is 430000. The strain has the capabilities of decomposing zinc and secreting indoleacetic acid, and can convert zinc which is difficult to use into available zinc, increase the content of effective zinc in soil, improve the utilization rate of a fertilizer and promote plant development and fertilizer absorption, so that the biomass in the seedling stage and the yield in the whole growth period of wheat are improved, and the growth of wheat is promoted; strain resources are provided for agricultural production and soil improvement.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to Priesteria gigantea. Background Art

[0002] Loamy damp soil has strong permeability, good ventilation, and aerobic microbial activity is dominant, which can promote the decomposition of organic matter and accelerate the mineralization of organic matter. The soil has strong capillary action, fast water flow, strong drainage capacity, and "night tide" phenomenon. The suitable farming period is also long, and it is easy to establish seedlings; the nutrient content is high, the fertilizer retention performance is good, and it is easy to fertilize and cultivate. At present, most of this soil has been used as arable land, with two crops a year, and the annual grain yield per mu is generally about 800kg. Targeted improvement measures in the future: planting suitable crops, reasonable irrigation, reasonable application of chemical fertilizers, and formula fertilization; multi-channel application of organic fertilizers.

[0003] Plant Growth Promoting Bacteria (PGPB) are defined as bacteria that live freely in the soil, rhizosphere, root surface, and phyllosphere and are beneficial to plant growth under certain conditions. These bacteria can fix nitrogen, dissolve phosphorus, potassium, and iron, and produce plant hormones such as auxin, gibberellin, cytokinin, and ethylene. Patent 202410253561.6 discloses a strain of Priestia megaterium WYJ-1, which was isolated from the rhizosphere soil of green beans and named Priestia megaterium. It is preserved in the General Microbiological Center of the China Microbiological Culture Collection Administration Committee with a preservation number of CGMCC No. 26912; strain WYJ-1 has the ability to dissolve potassium, produce plant growth hormone IAA, and fix nitrogen, increase the biomass of green beans, soluble protein content, and significantly increase the vitamin C, crude protein, and potassium content of green beans. In addition, they can also improve the stress resistance of plants, including drought, high salt, heavy metal poisoning, and pesticides. There are also a large number of zinc-dissolving microorganisms in the soil, which play an important role in plant growth and development. Zinc-dissolving microorganisms can increase the soluble zinc content in the soil, thereby increasing crop yields. The use of zinc-dissolving microbial preparations can activate soil zinc in a shorter time and at a lower cost, improve the absorption and utilization of zinc by plants, and thus achieve the purpose of zinc biofortification. Zinc-dissolving bacteria can also increase the absorption of some trace nutrients (such as iron, copper, etc.) by plant roots and enhance the resistance of plants to diseases. Based on the ability to dissolve insoluble zinc in the soil, zinc-dissolving bacteria are widely used in inoculants to increase zinc absorption and crop yields.

[0004] At present, there are still certain technical difficulties and uncertainties in the screening and identification methods of zinc-degrading microorganisms, which limits their promotion and popularization in practical applications. In the future, we will continue to optimize the screening and identification methods to screen out more microorganisms with efficient zinc-degrading capabilities to better serve agricultural production and soil improvement, and provide more high-quality microbial resources and technical support for agricultural production. Summary of the invention

[0005] In view of the above problems existing in the prior art, the present invention provides a strain of Priesteria gigantea NZ-1, a bacterial agent and application thereof.

[0006] The technical solution of the present invention is achieved in this way:

[0007] A strain of Priestia megaterium NZ-1, whose classification name is Priestia megaterium, was deposited in the China Center for Type Culture Collection on September 13, 2024, with the deposit number: CCTCC NO: M 20241962, and the deposit address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, Postal Code 430000.

[0008] The Priesteria gigantea NZ-1 of the present invention is isolated and screened from wheat rhizosphere soil, and its colony characteristics are as follows: the colony is round, the surface is convex, the edge is complete, the surface is smooth, the colony is white at the beginning, and later turns yellow, rod-shaped, has spores, is relatively sticky, and is easy to pick up. The physiological and biochemical characteristics are catalase positive, methyl red (MR) negative, starch hydrolysis negative, gelatin hydrolysis positive, nitrate reduction positive, citrate utilization negative, urease reaction negative, sucrose fermentation reaction positive, glucose fermentation reaction positive, and indole reaction negative.

[0009] The 16S rDNA sequence of the strain NZ-1 of the present invention is clustered in the same branch as that of Pristia megaterium strain PB3. Combined with the physiological and biochemical characteristics of the strain, the strain is identified as Pristia megaterium.

[0010] This strain has the ability to secrete IAA and dissolve zinc, which can convert difficult-to-use zinc into usable zinc, improve the utilization rate of fertilizers, promote plant development and fertilizer absorption, promote plant growth, increase yields, and provide bacterial strain resources for growth-promoting agents that can be used in agricultural production.

[0011] In a second aspect, the method for culturing the Priesteria gigantea NZ-1 comprises the following steps: inoculating the Priesteria gigantea NZ-1 into a Tris liquid culture medium, and heating the culture medium at 36-38°C and 160-180 r·min -1 , culture for 12-16h under pH 5.0-7.5 conditions.

[0012] A third aspect is a bacterial agent containing the above-mentioned Priesteria gigantea NZ-1.

[0013] Preferably, the concentration of Priesteria gigantea NZ-1 in the above-mentioned bacterial agent is 10 6 -10 8 CFU·g -1 .

[0014] Preferably, the bacterial agent is obtained by inoculating Priesteria gigantea NZ-1 into Tris liquid culture medium and culturing the culture medium at 36-38° C. and pH 5.0-7.5.

[0015] Preferably, the Tris liquid culture medium uses any one of glucose, sucrose, fructose, xylose, mannitol, lactose and maltose as a carbon source; and uses any one of peptone, yeast powder, glutamic acid, potassium nitrate, ammonium chloride and ammonium sulfate as a nitrogen source.

[0016] In a fourth aspect, the above-mentioned Priesteria gigantea NZ-1 or the above-mentioned bacterial agent is used as a zinc-dissolving bacterial agent.

[0017] Preferably, the zinc-dissolving bacteria agent has any of the following applications:

[0018] ① Decompose the insoluble zinc in the soil and increase the effective zinc content in the soil;

[0019] ②Used to secrete indoleacetic acid;

[0020] ③ Promote plant growth;

[0021] ④Increase plant yield.

[0022] Preferably, the above-mentioned insoluble zinc is smithsonite.

[0023] Preferably, the above plant is wheat.

[0024] The present invention has the following beneficial effects:

[0025] 1. The present application discovered a Priesteria gigantea strain NZ-1, which has good stability and the ability to dissolve zinc and secrete indoleacetic acid (IAA), which can convert the zinc that is difficult to utilize in the soil into usable zinc, increase the content of effective zinc in the soil, improve the utilization rate of fertilizers, promote plant development and absorption of fertilizers, promote plant growth, and increase yields.

[0026] 2. The strain NZ-1 of the present application has the ability to secrete IAA, reaching 18.19 μg·mL -1 Soil culture experiments showed that the effective zinc content in the soil after inoculation with NZ-1 increased significantly by 6.86% and 4.16% at 7d and 14d, respectively.

[0027] Pot experiments at the seedling stage confirmed that strain NZ-1 has a significant growth-promoting effect on wheat. The aboveground dry weight of wheat plants inoculated with NZ-1 under CK and +N conditions increased significantly by 36.05% and 12.24%, respectively; the available zinc content in the soil decreased by 5.49%; the zinc content in wheat plants and the accumulated zinc in the aboveground parts increased significantly by 50.58% and 44.51%, respectively. The available zinc in the soil was absorbed by the plants. Strain NZ-1 can effectively promote the growth and development of wheat.

[0028] Pot experiments throughout the entire growth period confirmed that after inoculation with NZ-1, wheat yield increased significantly by 37.66%; the zinc content in roots, husks and grains increased by 36.52%, 32.94%, 44.95% and 35.31% respectively; the zinc content in stems and leaves increased by 15.95%; the zinc content in grains increased significantly by 24.32%; the available zinc content in the soil decreased by 8.70%, and the available zinc was absorbed by the plants.

[0029] Mineral verification tests confirmed that strain NZ-1 had a significant zinc-releasing effect on smithsonite. After strain NZ-1 was inoculated in liquid culture medium containing smithsonite, the zinc concentration increased significantly. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0031] Figure 1 This is the standard curve of IAA content.

[0032] Figure 2 This is a picture of the colony morphology of Priesteria gigantea strain NZ-1.

[0033] Figure 3 This is a picture of the hyphae morphology of Priesteria gigantea strain NZ-1.

[0034] Figure 4 This is a phylogenetic tree constructed based on 16S rDNA sequences.

[0035] Figure 5 The effect of different culture time on the zinc-solubilizing ability of strain NZ-1.

[0036] Figure 6 The effect of different pH on the zinc-solubilizing ability of strain NZ-1.

[0037] Figure 7The effect of different liquid volumes on the zinc-solubilizing ability of strain NZ-1.

[0038] Figure 8 The effect of different carbon sources on the zinc-degrading ability of strain NZ-1.

[0039] Fig. 9 The effect of different nitrogen sources on the zinc-solubilizing ability of strain NZ-1.

[0040] Fig.10 This is the effect of inoculation with strain NZ-1 on the available zinc content in soil.

[0041] Fig.11 This is the effect of inoculation strain NZ-1 on the zinc content in smithsonite. DETAILED DESCRIPTION

[0042] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0043] Unless otherwise specified, the experimental methods used in the following experimental examples are all conventional methods; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0044] Example 1

[0045] Screening of plant growth-promoting bacteria that secrete indoleacetic acid by qualitative and quantitative assays:

[0046] 1. Materials and methods:

[0047] Zinc-containing medium: zinc content 1g / L, Tris liquid culture medium purchased from Yuanye Biotechnology Co., Ltd., adjusted to pH 7.0, sterilized at 121℃ for 20min. Add 18g agar powder to the culture medium to obtain zinc-containing solid medium.

[0048] Weigh 10g of rhizosphere soil from the root box test wheat and place it in a conical flask containing 90mL of sterile water. Oscillate it at 180r / min for 1h in a shaker at 30℃, take it out and let it stand for 20min to obtain soil bacterial suspension. After gradient dilution with sterile water, apply the appropriate gradient on a zinc-containing medium plate and place it in a 30℃ incubator for culture. When transparent circle colonies appear, they are zinc-solubilizing bacteria. Purify the zinc-solubilizing bacteria and store them.

[0049] 2. Qualitative determination: The isolated and purified bacteria were inoculated into LB liquid medium containing L-tryptophan (100 mg / L) at 30°C and 180 r·min. -1Incubate on a shaker for 1 day. Take 50 μL of bacterial suspension and drop it on a 96-well plate, and add 50 μL of Salkowski colorimetric solution. Add 50 μL of 50 mg / L indoleacetic acid colorimetric solution as a positive control. Place the 96-well plate at room temperature and avoid light for 30 minutes, and observe. Those that turn red indicate that they can secrete indoleacetic acid.

[0050] 3. Quantitative determination: The IAA-secreting bacteria obtained in the initial screening were quantitatively determined under the same culture conditions as above. First, the OD600 value of the bacterial suspension was determined by spectrophotometry, and then the bacterial suspension was cultured at 10,000 r / min. -1 Centrifuge for 10 min, take the supernatant and add an equal volume of Salkowski colorimetric solution, keep it in the dark for 30 min, and measure its OD 530 Calculate the content of indoleacetic acid in unit volume of fermentation broth when the bacterial concentration OD600 value is 1. Standard curve ( Figure 1 ) was prepared by gradient dilution of analytically pure indoleacetic acid.

[0051] The IAA-producing bacteria were screened for zinc solubility. The test strains were inoculated into a 100 mL Erlenmeyer flask containing 50 mL of zinc liquid culture medium at 30 °C and 180 r / min. -1 After culturing for 14 days, the culture medium was centrifuged at 8000 r / min for 5 minutes in a centrifuge, and the supernatant was taken to determine the zinc content using an atomic absorption spectrophotometer.

[0052] The results showed that the screened strains had the ability to secrete IAA, reaching 18.19 μg mL -1 , which can convert difficult-to-use zinc into usable zinc and has the ability to decompose zinc.

[0053] Example 2

[0054] To explore the growth characteristics and physiological and biochemical characteristics of Priesteria gigantea strain NZ-1:

[0055] 1. Growth characteristics of Priesteria megaterium strain NZ-1

[0056] Materials and methods:

[0057] Zinc-containing medium: zinc content 1g / L, Tris liquid culture medium purchased from Yuanye Biotechnology Co., Ltd., adjusted to pH 7.0, sterilized at 121℃ for 20min. Add 18g agar powder to the culture medium to obtain zinc-containing solid medium.

[0058] Weigh 10g of rhizosphere soil from the root box test wheat and place it in a conical flask containing 90mL of sterile water. Oscillate it at 180r / min for 1h in a shaker at 30℃, take it out and let it stand for 20min to obtain soil bacterial suspension. After gradient dilution with sterile water, apply the appropriate gradient on a zinc-containing medium plate and place it in a 30℃ incubator for culture. When transparent circle colonies appear, they are zinc-solubilizing bacteria. Purify the zinc-solubilizing bacteria and store them.

[0059] The strains isolated by the above method were sequenced by Sangon Biotech (Shanghai) Co., Ltd., and the sequencing results of 16S rDNA were analyzed online at http: / / www.ncbi.nlm.nih.gov. The homology was compared with other 16S rDNA sequences in GenBank using Blast software, and the phylogenetic tree of 16S rDNA sequences of NZ-1 was constructed using MEGA software. Figure 4 As shown, strain NZ-1 and Pristia megaterium strain PB3 are clustered in the same branch. Combined with the physiological and biochemical characteristics of the strain, it was identified as Pristia megaterium. The strain was deposited in the China Center for Type Culture Collection on September 13, 2024, with the deposit number: CCTCC NO: M 20241962, and the deposit address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0060] The strain is Gram-positive, and the colony morphology of NZ-1 is as follows Figure 2 As shown in the figure, the mycelial morphology is as follows Figure 3 As shown, after culturing on the selective medium for 24 hours, 1.2 mm colonies can be formed. The colonies are round, convex, with complete edges and smooth surfaces. The colonies are white at first, yellow later, rod-shaped, with spores, relatively sticky and easy to pick up. The optimal growth temperature is 36°C.

[0061] 2. Physiological and biochemical characteristics of Priesteria megaterium strain NZ-1

[0062] 2.1 Determination of catalase

[0063] Take 100mL of 30% hydrogen peroxide solution, dissolve it in 900mL of distilled water, mix well, and dispense it into 5mL sterile test tubes for later use. Pick fresh bacteria and inoculate them into 3% hydrogen peroxide solution to observe whether bubbles are generated. If bubbles are generated, it is a positive reaction, and if not, it is a negative reaction.

[0064] 2.2 Methyl red test (MR test)

[0065] MR medium: potassium dihydrogen phosphate (K 2 HPO 4) 5.0g, glucose 5.0g, peptone 7.0g, distilled water 1000mL, pH 6.7-7.1, 121℃, autoclave sterilization for 20min. Dispense into 5mL sterile test tubes for later use.

[0066] Methyl red indicator: Dissolve 10 mg of methyl red in 30 mL of 95% ethanol, then add 20 mL of distilled water. Pick fresh bacteria, inoculate in a test tube, and culture at an appropriate temperature for 24 to 48 hours. After the culture is completed, add 2 to 3 drops of methyl red reagent, mix well and observe the results. If red appears, it is positive, and if yellow appears, it is a negative reaction.

[0067] 2.3 Starch hydrolysis test

[0068] Soluble starch medium: 10 g soluble starch, dipotassium hydrogen phosphate (K 2 HPO 4 )1g, magnesium sulfate (MgSO 4 )1g, calcium carbonate (CaCO 3 )2g, ammonium sulfate ((NH 4 ) 2 SO 4 )2g, sodium chloride (NaCl) 1g, zinc sulfate (ZnSO 4 )0.001g, manganese chloride (MnCl 2 )0.001g, ferrous sulfate (FeSO 4 )0.001g, distilled water 1000mL, pH 7.0-7.4, 121℃, autoclave sterilization for 20min. The sterilized liquid culture medium is divided into 5mL sterile test tubes for use. After 24h of culture, 50-100μL of the bacterial liquid is pipetted into a soluble starch test tube and cultured in an incubator at 36℃ for 48-96h. After the culture is completed, Lugol's iodine solution is added dropwise to observe the color change of the reagent. If it does not turn blue, it is positive, and if it turns blue, it is negative.

[0069] 2.4 Gelatin hydrolysis test

[0070] Culture medium: 10g peptone, 120g gelatin, 3g beef extract, 5g sodium chloride (NaCl), 1000mL distilled water, pH 7.2-7.4. Sterilize in a high pressure sterilizer at 121℃ for 20min. Dispense the sterilized gelatin culture medium into 5mL sterile test tubes and place them vertically until the gelatin solidifies for later use. Before use, liquefy the gelatin in a 30-40℃ incubator until it is frozen in a liquid state. Place it upright on a test tube rack, inoculate 50-100μL of the bacterial suspension that has been cultured for 24h into the gelatin test tube, wrap it with sealing film, set up 2 blank controls, and place it in a 36℃ incubator for culture. Since the time for bacteria to liquefy gelatin varies, observe after 1, 2, 3, 7, 10, 14, and 30 days. Before observation, place the gelatin test tube in a refrigerator at 2-8℃ for 10-30min, and observe whether the gelatin is liquefied after taking it out. If liquefaction occurs, record it as positive, otherwise it is negative.

[0071] 2.5 Nitrate reduction test

[0072] Nitrate medium (nitrate broth): peptone 5g, KNO 3 1.0 g, 1000 mL of distilled water, pH 7.0-7.5, 121°C, sterilize in an autoclave for 20 min, and dispense the nitrate medium into 5 mL sterile test tubes for later use.

[0073] Solution A (p-toluenesulfonic acid solution): 0.8g p-aminobenzenesulfonic acid is dissolved in 100mL 5mol / L acetic acid. Solution B: 5g α-naphthol is dissolved in 100mL 5mol / L acetic acid. 50-100μL of the bacterial solution after 24h of culture is taken into nitrate broth, and cultured in an incubator at about 36℃ for 1-5d. Take 2 test tubes of nitrate broth as blank control. After the culture is completed, add 2-3 drops of solution A and solution B respectively, mix well, and observe the color change immediately. If it turns red, it is a positive reaction; if it does not change color, continue to add a little zinc powder. If it still does not change color, it is also a positive reaction. If it turns red, it is a negative reaction.

[0074] 2.6 Utilization of Citrate

[0075] Simon's citrate medium: sodium chloride (NaCl) 5.0 g, potassium dihydrogen phosphate (K 2 HPO 4 )1.0g, magnesium sulfate (MgSO 4 )0.5g, potassium dihydrogen phosphate (KH 2 PO 4 )1.0g, sodium citrate (C 6 H 5 Na 3 O 7 ) 1.0g, agar 14.0g, bromothymol blue (C 27 H28 O 5 SBr 2 )0.08g, distilled water 1000mL, pH 6.7-6.9, 121℃, autoclave sterilization for 20min. Dispense into 5mL sterile test tubes, tilt to solidify into a slope, and set aside. Pick fresh colonies, streak inoculation on the slope, and culture in an incubator at an appropriate temperature for 24-96h. Observe the color of the culture medium, blue for positive, green for negative.

[0076] 2.7 Urease reaction

[0077] Urease agar medium: peptone 1.0g, sodium chloride (NaCl) 5.0g, glucose 1.0g, potassium dihydrogen phosphate (K 2 HPO 4 )2.0g, phenol red 0.012g, agar 15.0g, distilled water 1000mL, pH 6.9-7.1, 121℃, autoclave sterilization for 20min. Dispense into 5mL sterile test tubes, tilt and let stand to solidify into a slope for use. Pick fresh bacteria, streak inoculate on the slope, and pierce into the bottom of the culture medium. After inoculation, seal with sealing film. Set up 2 blank controls and place them in an incubator at 36℃ for 18-24h. After the culture is completed, observe the color of the culture medium. If it turns rose red, it is positive. If it does not change color or turns yellow, it is negative.

[0078] 2.8 Utilization of Sucrose

[0079] Sucrose fermentation medium: peptone 10.0g, sucrose 10.0g, sodium chloride (NaCl) 30.0g, beef extract powder 3.0g, bromocresol purple 0.04g, distilled water 1000mL, pH 6.8-7.2, 121℃, autoclave sterilization for 20min. Dispense into 5mL sterile test tubes for later use. Pick fresh strains, inoculate them into test tubes, culture at appropriate temperature for 18-24h, and observe the color of the test tube culture medium. Yellow is positive, blue or blue-green is negative.

[0080] 2.9 Glucose Utilization

[0081] Glucose fermentation medium: 30.0g sodium chloride (NaCl), 10.0g peptone, 3.0g beef extract powder, 0.04g bromocresol purple, 10.0g glucose, 1000mL distilled water, pH 6.8-7.2, 121℃, sterilize in an autoclave for 20min. Dispense into 5mL sterile test tubes for later use. Pick fresh strains, inoculate them into test tubes, culture at room temperature for 18-24h, and observe the color of the test tube culture medium. Yellow is positive, and blue or blue-green is negative.

[0082] 2.10 Peptone water reaction (indole reaction)

[0083] Tryptone water culture medium: 10.0g tryptone, 5.0g sodium chloride (NaCl), 1000mL distilled water, pH 7.2-7.4, 121℃, autoclave sterilization for 20min. The sterilized liquid culture medium is divided into 5mL test tubes for later use. Kovacs' indigo matrix reagent: 5.0g p-dimethylaminobenzaldehyde is dissolved in 75mL amyl alcohol, and then 25mL concentrated hydrochloric acid is slowly added. Inoculate an appropriate amount of bacterial liquid into the above culture medium and place it in a 36℃ incubator for 18-24h. After the culture is completed, add 2-3 drops of Kovacs' indigo matrix reagent to the test tube, shake well and observe the results within a few seconds. If a rose-red ring appears, it is a positive reaction, and if there is no color change (or yellow ring), it is a negative reaction.

[0084] The test results in Table 1 show that the physiological and biochemical characteristics of strain NZ-1 are catalase positive, methyl red (MR) negative, starch hydrolysis negative, gelatin hydrolysis positive, nitrate reduction positive, citrate utilization negative, urease reaction negative, sucrose fermentation reaction positive, glucose fermentation reaction positive, and indole reaction negative.

[0085] Table 1 Physiological and biochemical characteristics of strain NZ-1

[0086]

[0087] Note: “+” indicates positive; “-” indicates negative.

[0088] Example 3

[0089] In order to further verify the zinc-solubilizing ability and optimal conditions of rhizosphere zinc-solubilizing bacteria NZ-1, the following study explored the effects of different culture times, pH, liquid volume, different carbon sources, and different nitrogen sources on zinc-solubilizing ability:

[0090] Add 100 mL of Tris liquid culture medium to a 250 mL conical flask, use the culture medium without bacteria as a control, set up 4 replicates for each treatment, place it in a 36°C shaker for culture, and take samples at 10, 15, 20, 28, 36, 44, and 56 hours to test its effective zinc content. Figure 5 It can be seen that in the first 28 hours, with the increase of culture time, the zinc concentration in the culture medium showed a gradually increasing trend, and in 28-36 hours, the zinc concentration in the culture medium showed a gradually decreasing trend. At 44 hours, the zinc concentration in the culture medium reached the maximum value of 3.95 mg / L. From this analysis, it can be seen that 44 hours is the culture time with the best zinc decomposition effect.

[0091] The initial value of Tris medium was adjusted to pH 4, 5, 6, 7, 8, 9, 10, and the medium without bacteria was used as the control. Four replicates were set for each treatment. After 96 hours of incubation at 30°C in a shaking incubator, samples were taken to detect the effective zinc content. Figure 6 It can be seen that with the increase of pH gradient, the zinc concentration in the culture medium shows a gradually decreasing trend. When pH is 5, the zinc concentration in the culture medium is the highest, which is 113.43 mg / L. In the pH range of 6-7, the zinc concentration in the culture medium drops sharply, indicating that the zinc-degrading ability of the strain is reduced. From this analysis, it can be seen that pH 5 is the pH with the best zinc-degrading effect.

[0092] Different volumes of culture medium were added to 250 mL conical flasks. The volumes of culture medium were 25, 50, 75, 100, and 150 mL, respectively. Uninoculated culture medium was used as control. Four replicates were set for each treatment. After 96 hours of incubation at 30°C in a shaking incubator, samples were taken to detect the effective zinc content. Figure 7 It can be seen that when the volume of the culture medium is 25mL, the zinc content in the culture medium is the largest, which is 20.74mg / L. As the volume of the culture medium increases, the oxygen supply in the flask decreases, and the zinc decomposition of the strain NZ-1 decreases. The reduction in oxygen supply in the culture medium will affect the growth, reproduction and zinc decomposition ability of the strain NZ-1. It can be seen that the zinc decomposition effect of the bacteria is best when the volume of the culture medium is 25mL.

[0093] Different carbon sources were added to the Tris medium, including glucose, sucrose, mannose, lactose, xylose, and maltose. The medium without bacteria was used as the control. Four replicates were set for each treatment. After 96 hours of incubation at 30°C in a shaking incubator, samples were taken to detect the effective zinc content. Figure 8 It can be seen that under different carbon source conditions, the zinc solubilization effect of strain NZ-1 has significant differences. Among the 7 carbon source culture media given in this experiment, the zinc solubilization effect of strain NZ-1 is from large to small: xylose>glucose>fructose>mannitol>maltose>sucrose>lactose. When xylose is used as the carbon source of the culture medium, the zinc solubilization amount of the strain is significantly higher than that of the other 6 carbon sources (P<0.05), which is 55.83 mg / L; when lactose is used as the carbon source, the zinc solubilization amount of the strain is significantly lower than that of other carbon sources, which shows that xylose is the best carbon source.

[0094] Different nitrogen sources were added to the Tris medium, including peptone, glutamic acid, yeast powder, ammonium sulfate, potassium nitrate, and ammonium chloride. The medium without bacteria was used as a control. Four replicates were set for each treatment. After 96 hours of incubation at 30°C in a shaker, samples were taken to detect the effective zinc content. Fig. 9 It can be seen that the zinc solubilization ability of strain NZ-1 also has significant differences under different nitrogen source conditions. The zinc solubilization effect of strain NZ-1 is from large to small in the order of glutamate > ammonium sulfate > ammonium chloride > potassium nitrate > peptone > yeast powder. When glutamate is used as the nitrogen source, the zinc solubilization effect of NZ-1 is the strongest at 157.25 mg / L, but when yeast powder is used as the nitrogen source, the zinc solubilization effect is significantly lower than that of the other five nitrogen sources (P<0.05). Therefore, glutamate is the most suitable nitrogen source for the growth and zinc solubilization of strain NZ-1.

[0095] Application Example 1

[0096] 1. The strain NZ-1 of the present invention has the effect of increasing the effective zinc content in the soil, which is illustrated by a soil culture test as follows:

[0097] Take 250 g of the test soil shown in Table 2 and put it in a PVC cup. Cultivate the test strain NZ-1 to make a bacterial suspension. 8 CFU·g -1 The inoculum amount was inoculated into the soil, with the uninoculated strain as the control. Each treatment was replicated 4 times, and the moisture content was maintained at 60% of the maximum field water holding capacity. The potted plants were placed in a light culture room for dark culture, and samples were taken at 0, 7, 14, 21, and 28 days.

[0098] Table 2 Basic properties of the tested soil

[0099]

[0100] Inoculation treatment: The strain NZ-1 of the present invention was inoculated into Tris liquid medium at 30°C and 180 r·min -1 The culture was grown in a shaking incubator until the logarithmic growth phase, and then the bacterial suspension was shaken at 8000 r / min -1 Centrifuge for 10 minutes, resuspend in sterile water, repeat the operation three times, and the inoculum volume is 10 8 CFU·g -1 .

[0101] Control treatment: As a control, the soil was not sprayed with NZ-1 bacterial solution, and an equal amount of deionized water was added.

[0102] The results are as follows Fig.10 As shown, in the first 14 days, the available zinc content in the soil increased significantly after inoculation with NZ-1, and the increases were 6.86% and 4.16% at 7 and 14 days of incubation, respectively. After 14 days, the available zinc content in the soil decreased after inoculation with NZ-1, but not significantly.

[0103] 2. The strain NZ-1 of the present invention has a significant growth-promoting effect on wheat, which is illustrated below by pot tests at the seedling stage and pot tests during the entire growth period:

[0104] 2.1 Seedling pot test:

[0105] Take about 1.5 kg of test soil and put it in a pot. The wheat variety used in the test is "Bainong 307". The surface of the wheat is disinfected, and seeds with the same fullness are selected and planted in the pot with 5 grains per pot. The test strain NZ-1 is cultured and made into a bacterial suspension. 8 CFU·g -1The inoculum was inoculated into the soil, with the uninoculated strain as the control. Each treatment was repeated 4 times, and the water content was maintained at 60% of the maximum field water holding capacity. The potted plants were placed in a light culture room. Samples were taken after 45 days, and the basic physical and chemical properties of the soil and the zinc content of the plants were determined.

[0106] Wheat seeds: Disinfect the surface of wheat seeds with 20% hydrogen peroxide for 20 minutes, and select seeds with the same fullness for later use.

[0107] Inoculation treatment: The strain NZ-1 of the present invention was inoculated into Tris liquid medium at 30°C and 180 r·min -1 The culture was grown in a shaking incubator until the logarithmic growth phase, and then the bacterial suspension was shaken at 8000 r / min -1 Centrifuge for 10 minutes, resuspend in sterile water, repeat the operation three times, and the inoculum volume is 10 8 CFU·g -1 .

[0108] Control treatment: As a control, the soil was not sprayed with NZ-1 bacterial solution, and an equal amount of deionized water was added.

[0109] The results are shown in Table 3. It can be seen from Table 3 that the aboveground dry weight of wheat plants inoculated with NZ-1 under CK and +N conditions showed a significant growth trend, with increases of 36.05% and 12.24%, respectively; after inoculation with NZ-1 under +N conditions, the effective zinc content in the soil decreased, with a decrease of 5.49%, but it was not significant. The zinc content and aboveground zinc accumulation of wheat plants increased significantly, with increases of 50.58% and 44.51%, respectively, which may be because the effective zinc in the soil was absorbed by the plants. Combined with the above results, it can be seen that the rhizosphere zinc-solubilizing bacteria NZ-1 of the present invention has a significant effect on the growth and development of wheat, has a high IAA yield, and can effectively promote the growth and development of crops.

[0110] Table 3 Effects of strain NZ-1 on dry weight and zinc content of wheat plants at seedling stage

[0111]

[0112] 2.2 Potted plant test during the whole growth period:

[0113] About 5 kg of test soil was placed in a pot. The wheat variety used in the test was "Bainong 307". The surface of the wheat seeds was disinfected, and seeds with the same fullness were selected and planted in pots with 5 grains of wheat per pot. The test strain NZ-1 was cultured and made into a bacterial suspension. 8 CFU·g -1The inoculum was inoculated into the soil, with the uninoculated strain as the control. Each treatment was repeated 4 times, and the water content was maintained at 60% of the maximum field water holding capacity. The potted plants were placed in a greenhouse. After maturity, samples were collected and the basic physical and chemical properties of the soil and the zinc content of the plants were determined.

[0114] Wheat seeds: Disinfect the surface of wheat seeds with 20% hydrogen peroxide for 20 minutes, and select seeds with the same fullness for later use.

[0115] Inoculation treatment: The strain NZ-1 of the present invention was inoculated into Tris liquid medium at 30°C and 180 r·min -1 The culture was grown in a shaking incubator until the logarithmic growth phase, and then the bacterial suspension was shaken at 8000 r / min -1 Centrifuge for 10 minutes, resuspend in sterile water, repeat the operation three times, and the inoculum volume is 10 8 CFU·g -1 , vaccinated once every 2 months.

[0116] Control treatment: As a control, the soil was not sprayed with NZ-1 bacterial solution, and an equal amount of tap water was added.

[0117] The results are shown in Tables 4 and 5. As can be seen from Table 4, the yield increased significantly after inoculation of NZ-1 under +N conditions, with an increase of 37.66%. As can be seen from Table 5, the zinc content of roots and husks increased after inoculation of NZ-1 under CK and +N treatments, with increases of 36.52% and 32.94%, 44.95% and 35.31%, respectively; the zinc content of stems and leaves increased after inoculation of NZ-1 under CK conditions, with an increase of 15.95%; the zinc content of grains increased significantly after inoculation of NZ-1 under +N conditions, with an increase of 24.32%; the effective zinc content of soil decreased significantly after inoculation of NZ-1 under +N conditions, with a decrease of 8.70%, which is consistent with the results of the pot test at the seedling stage, which may be related to the absorption of effective zinc by plants.

[0118] Table 4 Effects of NZ-1 strain on wheat yield and its components during the whole growth period

[0119]

[0120] Table 5 Effects of strain NZ-1 on zinc content in various parts of wheat and available zinc content in soil during the whole growth period

[0121]

[0122] 3. The strain NZ-1 of the present invention has a significant zinc-releasing effect on smithsonite, which is illustrated by mineral verification tests below.

[0123] Add 1% zinc smithsonite to Tris liquid culture medium, use uninoculated culture medium as control, set 4 replicates for each treatment, place in 30℃ shaker culture for 7 days, and then take samples to detect effective zinc content. Fig.11 As shown, the zinc concentration increased significantly after inoculation with zinc-dissolving bacteria NZ-1, which indicates that strain NZ-1 has the ability to dissolve smithsonite.

[0124] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A strain of Priesteria gigantea NZ-1, whose taxonomic name is Priestia megaterium , deposited in the China Center for Type Culture Collection on September 13, 2024, with the deposit number: CCTCC NO: M 20241962, and the deposit address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, Postal Code 430000.

2. The method for culturing Priesteria gigantea NZ-1 according to claim 1, characterized in that: The method comprises the following steps: inoculating Priesteria gigantea NZ-1 into Tris liquid culture medium, and culturing the culture medium at 36-38° C. and pH 5.0-7.

5.

3. A bacterial agent containing the Priesteria gigantea NZ-1 according to claim 1.

4. The bacterial agent containing Priesteria gigantea NZ-1 according to claim 3, characterized in that: The concentration of Priesteria gigantea NZ-1 in the bacterial agent is 10 6 -10 8 CFU·g -1 .

5. The bacterial agent according to claim 3, characterized in that: The bacterial agent is obtained by inoculating Priesteria gigantea NZ-1 into Tris liquid culture medium and culturing the culture medium at 36-38°C and pH 5.0-7.

5.

6. The bacterial agent according to claim 5, characterized in that: The Tris liquid culture medium uses any one of glucose, sucrose, fructose, xylose, mannitol, lactose and maltose as a carbon source; and uses any one of peptone, yeast powder, glutamic acid, potassium nitrate, ammonium chloride and ammonium sulfate as a nitrogen source.

7. Use of the Priesteria gigantea NZ-1 described in claim 1 or the bacterial agent described in claim 3 or 4 as a zinc-solubilizing bacterial agent.

8. The use according to claim 7, characterized in that: The zinc dissolving bacteria agent has any of the following applications: ① Decompose the insoluble zinc in the soil and increase the effective zinc content in the soil; ②Used to secrete indoleacetic acid; ③ Promote plant growth; ④Increase plant yield.

9. The use according to claim 8, characterized in that: The insoluble zinc is smithsonite.

10. The use according to claim 8 or 9, characterized in that: The plant is wheat.

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