PHOSPHATE-SOLUBILIZING MICROBIAL AGENT FOR MAIZE, AND PREPARATION METHOD AND USE THEREOF

Abstract

A phosphate-solubilizing microbial agent for maize is provided. The phosphate-solubilizing microbial agent is obtained by fermentation of a strain CR50; and the strain CR50 is Bacillus stratosphericus (B. stratosphericus), which was deposited in the China Center for Type Culture Collection (CCTCC) on Nov. 10, 2020, with a deposit number of CCTCC M 2020721. The phosphate-solubilizing microbial agent has the ability to solubilize insoluble inorganic phosphorus and insoluble organic phosphorus such as calcium phosphate and calcium phytate, which can increase an available phosphorus content in soil, significantly promote the growth of maize, and increase a maize yield. The phosphate-solubilizing strain also has the ability to produce indoleacetic acid (IAA) and siderophore, and can undergo an agglutination reaction with maize agglutinin due to maize agglutinin affinity. The strain CR50 can colonize at maize roots for a long time under the mediation of agglutinin and stably exert the growth-promoting effect.

Claims

1. A phosphate-solubilizing microbial agent for maize, wherein the phosphate-solubilizing microbial agent is obtained by fermentation of a strain CR50; and the strain CR50 is Bacillus stratosphericus deposited in China Center for Type Culture Collection (CCTCC) on Nov. 10, 2020, with a deposit number of CCTCC M 2020721.

2. The phosphate-solubilizing microbial agent according to claim 1, wherein the phosphate-solubilizing microbial agent is a liquid microbial agent or a solid microbial agent.

3. The phosphate-solubilizing microbial agent according to claim 2, wherein the phosphate-solubilizing microbial agent is used in seed dressing, seed coating, and root watering for the maize.

4. The phosphate-solubilizing microbial agent according to claim 2, wherein the liquid microbial agent is applied at an amount of 5 L to 20 L per mu of a maize field, and the liquid microbial agent has a viable count of 1×10.sup.8 to 9×10.sup.8 CFU.mL.sup.−1.

5. The phosphate-solubilizing microbial agent according to claim 3, wherein the solid microbial agent is applied at an amount of 5 kg to 20 kg per mu of a maize field, and the solid microbial agent has a viable count of 2×10.sup.8 to 8×10.sup.8 CFU.mL.sup.−1.

6. A preparation method of the phosphate-solubilizing microbial agent according to claim 2, comprising: preparing the liquid microbial agent through the following steps: S61: strain activation: inoculating the strain CR50 to a Luria-Bertani (LB) slant, and culturing the strain CR50 at 25° C. to 30° C. for 18 h to 30 h; and S62: liquid medium culture: washing the strain CR50 on the LB slant with normal saline (NS), inoculating the strain CR50 in 80 mL to 120 mL of an LB liquid medium at a volume ratio of 2% to 5%, and culturing the strain CR50 on a shaker at 25° C. to 30° C. and 120 r.min.sup.−1 to 180 r.min.sup.−1 for 24 h to 28 h until a resulting CR50 bacterial solution has a concentration of OD.sub.680≥0.8 to obtain the liquid microbial agent.

7. A preparation method of the phosphate-solubilizing microbial agent according to claim 2, comprising: preparing the solid microbial agent through the following steps: S71: strain activation: inoculating the strain CR50 to an LB slant, and culturing the strain CR50 at 25° C. to 30° C. for 18 h to 30 h; S72: liquid medium culture: washing the strain CR50 on the LB slant with NS, inoculating the strain CR50 in 80 mL to 120 mL of an LB liquid medium at a volume ratio of 2% to 5%, and culturing the strain CR50 on a shaker at 25° C. to 30° C. and 120 r.min.sup.−1 to 180 r.min.sup.−1 for 24 h to 28 h until a resulting CR50 bacterial solution has a concentration of OD.sub.680≥0.8 to obtain the liquid microbial agent; and S73: preparation of the solid microbial agent: adding 0.5 g to 1 g of dry peat per ml of the liquid microbial agent into the liquid microbial agent obtained in S72, thoroughly mixing the dry peat and the liquid microbial agent to obtain a resulting mixture and fermenting the resulting mixture for 1 h to 3 h to obtain a product, and placing the product in a cool place to air-dry until a surface of the product has no moisture to obtain the solid microbial agent.

Description

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0028] The present disclosure will be further described below through examples.

Example 1

[0029] Strain screening

[0030] Maize plants with robust growth were selected, the whole root system with soil was dug out, and large clods and other useless residues were removed. 1 g of rhizosphere soil of each sample was collected and placed in nystatin-containing sterile NS, and cultured on a shaker at 160 r.min.sup.−1 for 0.5 h to obtain a rhizosphere soil bacterial suspension. 1 mL of the above bacterial suspension was taken and added to 9 mL of nystatin-containing sterile NS, and serially diluted to concentrations of 10.sup.−7. The bacterial suspension was coated on a nystatin-containing solid medium for phosphate-solubilizing bacteria and cultured at a constant temperature of 28° C. for more than 3 d until a transparent circle appeared, where 3 replicates were set for each concentration. The solid medium for phosphate-solubilizing bacteria had the following formula: glucose: 10 g, K.sub.2HPO.sub.4:2.0 g, ammonium sulfate: 0.5 g, NaCl: 0.3 g, KCl: 0.3 g, MgSO.sub.4.7H.sub.2: 0.3 g, FeSO.sub.4.7H.sub.2O: 0.03 g, MnSO.sub.4.4H.sub.2O: 0.03 g, Ca.sub.3(PO.sub.4).sub.2: 10 g, agar: 15 g, and distilled water: 1,000 mL. A transparent circle diameter H and a colony diameter C were observed and measured, and a ratio of the two (H/C) was calculated. The larger the H/C value, the stronger the phosphate-solubilizing ability of the strain.

[0031] The colonies with transparent circles were picked and inoculated in an LB medium with 50 mg.L.sup.−1 nystatin, and streaking purification was conducted multiple times to obtain a pure phosphate-solubilizing strain. A strain with phosphate-solubilizing ability screened out in the LB liquid medium was cultured at a constant temperature for 1 d under shaking until cells were at logarithmic growth phase; a resulting culture was transferred into centrifuge tubes and centrifuged at 3,000 r.min.sup.−1 for 10 min; bacteria were collected and added into sterile water, and a resulting mixture was repeatedly pipetted up and down with a pipette to allow uniform dispersion of the bacteria and then centrifuged; and the washing process was repeated 3 times to obtain a bacterial suspension. If the bacterial suspension was turbid, a small amount of sterile water could be added for dilution. 25 μL of the bacterial suspension was added dropwise to the center of a glass slide and mixed with an equal volume of maize agglutinin, the glass slide stood at room temperature for 0.5 h to air-dry, and a reaction was observed under a microscope after staining. The strain undergoing an agglutination reaction was picked out to obtain a maize-compatible phosphate-solubilizing strain. The CR50 used in this application is a maize-compatible phosphate-solubilizing strain obtained by this screening method.

[0032] The LB medium had the following formula: peptone: 10 g, yeast powder: 5 g, NaCl: 10 g, agar: 15 g, and distilled water: 1,000 mL.

Example 2

[0033] Determination of the phosphate-solubilizing ability of the strain CR50

[0034] A selective-medium and agglutinin dual screening method was used to screen out phosphate-solubilizing strains with specific affinity with maize from the rhizosphere of maize, and the phosphate-solubilizing ability was determined for these strains. The strains were prepared into bacterial suspensions, inoculated into different media for phosphate-solubilizing bacteria, and cultured at 28° C. and 160 r.min.sup.−1 for 5 d. The medium had the following formula: glucose: 10 g, K.sub.2HPO.sub.4: 2.0 g, ammonium sulfate: 0.5 g, NaCl: 0.3 g, KCl: 0.3 g, MgSO.sub.4.7H.sub.2O: 0.3 g, FeSO.sub.4.7H.sub.2O: 0.03 g, MnSO.sub.4.4H.sub.2O: 0.03 g, insoluble phosphorus: 10 g, and distilled water: 1,000 mL, pH: 7.2. The Mo—Sb anti-spectrophotometry method was used to determine a soluble phosphorus content in each culture.

[0035] The strains were compared for the phosphate-solubilizing ability, and the strain CR50 showed the highest phosphate-solubilizing ability. The ability of the strain CR50 to solubilize insoluble phosphorus was shown in Table 1. It can be seen from Table 1 that the strain CR50 exhibited a prominent solubilizing ability for both calcium phosphate (insoluble inorganic phosphorus) and calcium phytate (insoluble organic phosphorus).

TABLE-US-00001 TABLE 1 Ability of the strain CR50 to solubilize insoluble phosphorus Ability to solubilize Ability to solubilize calcium phosphate calcium phytate Strain (mg .Math. L.sup.−1) (mg .Math. L.sup.−1) CR50 125.28 ± 18.08 94.10 ± 12.72

Example 3

[0036] Identification of the strain

[0037] The morphological observation, the physiological and biochemical tests, and the 16SrRNA gene sequence alignment analysis were conducted to identify the strain CR50.

[0038] According to the Manual for Identification of Common Bacterial Systems and Berger's Bacterial Identification Manual, the physiological and biochemical characteristics of the strain CR50 were tested. The morphological test included Gram staining; and the physiological and biochemical tests included a starch hydrolysis test, a catalase test, a VP test, an M-R test, a gelatin liquefaction test, a glucose oxidative fermentation test, and an H.sub.2S production test. Results were shown in Table 2.

[0039] The 16SrRNA gene sequence of the strain CR50 was submitted to the NCBI database for BLAST alignment, and in combination with the results of the physiological and biochemical tests, it was determined that the strain CR50 was B. stratosphericus. The 16SrRNA gene sequence of the strain CR50 was shown in the appendix.

TABLE-US-00002 TABLE 2 Some physiological and biochemical characteristics of the strain CR50 Glucose Starch Gelatin oxidative H.sub.2S Gram Strain hydrolysis Catalase VP M-R liquefaction fermentation production staining CR50 + + + − − Oxidative − +

Example 4

[0040] Determination of the ability of the strain CR50 to produce IAA and siderophore

[0041] The strain CR50 was inoculated into a nitrogen liquid medium at an inoculation amount of 2% and cultured at 28° C. and 160 r.min.sup.−1 for 5 d; a resulting culture was centrifuged, a resulting precipitate was removed, and a resulting supernatant was collected and added with a corresponding Sackowski's chromogenic solution at a ratio of 1:2; a resulting mixture reacted for 30 min at 25° C. in the dark, and a nitrogen culture without bacteria was used as a blank control; and the absorbance at 530 nm was determined to calculate an IAA production level of the strain CR50. Preparation of the Sackowski's chromogenic solution: 1 mL of a 0.5 mol.L.sup.−1 FeCl.sub.3 solution was added to 50 mL of a 35% HClO.sub.4 solution, and a resulting mixture was well mixed. The nitrogen liquid medium had the following formula: sucrose: 10.0 g, K.sub.2HPO.sub.4: 2.0 g, MgSO.sub.4.7H.sub.2O: 0.5 g, NaCl: 0.1 g, yeast extract: 0.5 g, CaCO.sub.3: 0.5 g, agar: 20 g, and distilled water: 400 mL, pH: 7.0.

[0042] The strain CR50 was inoculated into an MKB liquid medium at an inoculation amount of 2% and cultured at 28° C. and 160 r.min.sup.−1 for 48 h; a resulting culture was centrifuged, and a resulting supernatant was collected and added with a CAS detection solution at a ratio of 1:1; a resulting mixture was thoroughly mixed and stood for 1 h, and distilled water was used as a control for zero adjustment; and the absorbance (A) at a wavelength of 630 nm was determined. In addition, the CAS detection solution was mixed with an MKB medium without bacteria at a ratio of 1:1, and the absorbance (Ar) at 630 nm was determined. The ability of the strain CR50 to produce siderophore was determined by ultraviolet spectrophotometry. The MKB medium had the following formula: casamino acid: 5.0 g, glycerol: 15 mL, K.sub.2HPO.sub.4: 2.5 g, MgSO.sub.4.7H.sub.2O: 2.5 g, and distilled water: 1,000 mL, pH: 7.2. Preparation of the CAS detection solution: 6 mL of a 10 mmol.L.sup.−1 cetyltrimethylammonium bromide (CTAB) solution was added to a 100 mL volumetric flask and slightly diluted with double distilled water (DDW); then 1.5 mL of a 1 mmol.L.sup.−1FeCl.sub.3 solution and 7.5 mL of a 2 mmol.L.sup.−1 chromeazurol (CAS) solution were mixed and slowly added into the volumetric flask along a glass rod; 4.307 g of anhydrous bisdimethylamine (anhydrous piperazine) was weighed and dissolved in about 30 mL of DDW, and 6.25 mL of 12 mol.L.sup.−1 HCl was added to obtain a buffer with a pH of 5.6; and the buffer was transferred to the volumetric flask, and a resulting solution was diluted with DDW to 100 mL for later use.

TABLE-US-00003 TABLE 3 Ability of the strain CR50 to produce IAA and siderophore Ability to produce IAA Ability to produce Strain (mg .Math. L.sup.−1) siderophore CR50 9.31 ± 0.46 ++

[0043] The ability of the strain CR50 to produce IAA and siderophore was shown in Table 3. It can be seen from Table 3 that the strain CR50 had a strong ability to produce IAA and siderophore, indicating that the strain has the potential to promote plant growth and resist pathogenic bacteria.

Example 5

[0044] Preparation of a microbial agent and a field test of the microbial agent for maize

[0045] 1. Preparation of the microbial agent

[0046] A CR50 strain on an LB slant was washed with 5 ml of NS, inoculated into an LB liquid medium at a volume ratio of 2%, and cultured at 28° C. and 160 r.min.sup.−1 for 24 h to obtain a CR50 liquid microbial agent. The microbial agent was diluted with water to OD.sub.680=0.8 before use.

[0047] 2. Field test of the microbial agent Two groups were set in the test, including an experimental group applied with the microbial agent and a control group applied with no microbial agent. 10 replicates were set for each group. Maize seeds were sown into the field, and after maize seedling emergence, 20 mL of a diluted microbial agent was applied to the roots. After the maize matured, a plant height, an ear diameter, an ear length, the number of filled grains per ear, an ear weight, a phosphorus content in leaves, and a phosphorus content in soil were determined for the experimental group and the control group. Results were shown in Tables 4 and 5.

TABLE-US-00004 TABLE 4 Influence of the CR50 liquid microbial agent on the agronomic traits and yield of maize Plant Ear Ear Number of filled Ear height diameter length grains per ear weight Treatment (cm) (cm) (cm) (grains) (g) Control 178.33 ± 12.90 ± 12.40 ± 364.00 ± 85.84 ± group 3.61 0.70 2.17 40.15 3.40 Experimental 218.53 ± 15.57 ± 18.63 ± 464.00 ± 116.13 ± group 16.29* 0.25** 0.42** 24.58* 8.57* Notes: *indicates a significant difference between treatments (p < 0.05), and **indicates an extremely-significant difference between treatments (p < 0.01).

[0048] It can be seen from Table 4 that the plant height, the ear diameter, ear length, the number of filled grains per ear, and the ear weight of the experimental group applied with the CR50 microbial agent increased by 22.54%, 20.70%, 50.24%, 27.47%, and 35.29% respectively compared with that of the control group applied with no microbial agent, where the increase in the plant height, the number of filled grains per ear, and the ear weight reached a significant level, and the increase in the ear diameter and the ear length reached an extremely-significant level.

TABLE-US-00005 TABLE 5 Influence of the CR50 liquid microbial agent on the phosphorus content in maize leaves and the phosphorus content in soil Phosphorus Available content in phosphorus leaves content in soil Treatment (g .Math. kg.sup.−1) (mg .Math. kg.sup.−1) Control group 2.74 ± 0.16 12.86 ± 0.69  Experimental   3.23 ± 0.025** 15.43 ± 0.50** group Note: **indicates an extremely-significant difference between treatments (p < 0.01).

[0049] It can be seen from Table 5 that the phosphorus content in leaves and the available phosphorus content in soil of the experimental group applied with the CR50 microbial agent increased by 17.88% and 19.98% respectively compared with that of the control group applied with no microbial agent, both reaching an extremely-significant level.

[0050] It can be seen from Tables 4 and 5 that the increase in the plant height, the ear diameter, the ear length, the number of filled grains per ear, the ear weight, the phosphorus content in leaves, and the available phosphorus content in soil of the experimental group applied with the CR50 microbial agent reached a significant level compared with that of the control group applied with no microbial agent, indicating that the CR50 liquid microbial agent can release insoluble phosphorus in soil, increase the available phosphorus content in soil, promote the phosphorus absorption of maize, and increase the phosphorus content in leaves, thereby promoting the maize growth and increasing the maize yield.

[0051] It should be emphasized that the phosphate-solubilizing microbial agent provided by the present disclosure can be used in combination with other microbial agents or products with growth-promoting effects. Although maize is used as an experimental object for the phosphate-solubilizing microbial agent of the present disclosure, the application of the phosphate-solubilizing microbial agent with strain CR50 on other plants shall also be regarded as an implementation of the present disclosure.

[0052] The above are only preferred examples of the present disclosure and are not intended to limit the present disclosure. Although the present disclosure is described in detail with reference to the foregoing examples, a person skilled in the art can still make modifications to the technical solutions described in the foregoing examples, or make equivalent replacement to some technical features. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and scope of the present disclosure should be included within the protection scope of the present disclosure.