Multifunctional bacillus velezensis as well as microbial agent and application thereof

Abstract

The invention relates to bacillus velezensis CGMCC (China General Microbiological Culture Collection Center) No.32593 as well as preparation and application of a microbial agent of the bacillus velezensis, and belongs to the technical field of microorganisms. The strain disclosed by the invention has the effects of dissolving phosphorus, dissolving potassium, fixing nitrogen and producing indoleacetic acid (IAA) at the same time. The method comprises the following steps: inoculating bacillus velezensis CGMCC No.32593 into an LB liquid culture medium, and fermenting by using a 5L fermentation tank to obtain the liquid microbial agent. The microbial agent provided by the invention is simple in preparation method and convenient for industrial production. By applying the liquid microbial agent, the plant height of tomatoes and the fresh weight of overground parts can be effectively increased, and the utilization rate of soil phosphorus in crop production is increased.

Description

Technical Field

[0001] The invention relates to the field of microbial technology, and more specifically to the preparation and application of a strain of Bacillus velezensis CGMCC NO.32593 and a microbial agent. Background Art

[0002] Phosphorus is one of the essential elements for plant growth and development, and plays an important role in plant resistance to stress and adaptation to adversity. About two-thirds of my country's cultivated land is seriously deficient in phosphorus. Most of the phosphorus in the soil is chelated in difficult-to-degrade minerals and organic compounds, and only a small amount of phosphorus is directly used by microorganisms and plants in the form of inorganic orthophosphates. Various insoluble ineffective phosphorus cannot be directly absorbed and utilized by plants, which greatly reduces the utilization rate of phosphorus in the soil and restricts crop growth.

[0003] Microorganisms play a vital role in decomposing insoluble phosphorus in the soil. Several plant rhizosphere growth-promoting bacteria have been reported to have good crop growth promotion and soil improvement effects. Bacillus velez is an emerging functional strain in the genus Bacillus. In the study of Yating Zhang et al., a strain of Bacillus velez D103 was isolated, and its gene sequence URL is www.ncbi.nlm.nih.gov / nuccore / CP095093. Studies have shown that strain D103 has the functions of nitrogen fixation, phosphorus solubilization, potassium solubilization, and production of auxin indoleacetic acid (IAA). Its ability to solubilize inorganic phosphorus is 68.67 mg / L. In hydroponic experiments, corn plants treated with D103 suspension showed a growth-promoting effect (Zhang Y, Microbiol Spectr, 2024, 12: e0114724).

[0004] Patent CN119020202A discloses a microbial agent with a phosphate-dissolving function, a preparation method and an application thereof, wherein the active ingredient of the microbial agent is Bacillus Velez CY30. The strain has an ability to dissolve calcium phytate of 23.80±1.08mg / L, and the number of viable bacteria of the microbial agent prepared by fermentation is 6 to 10 billion / g.

[0005] The existing phosphate-solubilizing strains have weak phosphate-solubilizing ability, single function, and low fermentation production level, which limits the application in the preparation of microbial agents. It is urgent to screen new high-quality phosphate-solubilizing bacteria and develop microbial agents with multiple functions. The Bacillus velezensis CGMCC NO.32593 and its microbial agent disclosed by the present invention have multiple functions. Only a single agent can be used to achieve the effects of phosphate, potassium, nitrogen fixation, and IAA production. The preparation method of the agent is simple, the production efficiency is high, the culture condition requirements are low, the cycle is short, and it is suitable for the needs of industrialized mass production and is convenient for promotion and use. The application of the agent can promote crop yield increase and reduce soil phosphate fertilizer dependence, which is of great significance for ensuring the sustainable utilization of phosphorus resources in the soil. Summary of the invention

[0006] In order to solve the above technical problems, the present invention provides a strain of Bacillus velezensis CGMCC NO.32593 and application thereof.

[0007] The present invention provides a strain of Bacillus velezensis CGMCC NO.32593 and its application, which adopts the following technical scheme:

[0008] A strain of Bacillus velezensis was deposited in the General Microbiology Center of China Culture Collection Administration, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with the deposit number CGMCC NO.32593.

[0009] The present invention provides a method for separating and culturing Bacillus velez CGMCC NO.32593, which adopts the following technical scheme:

[0010] A method for separating and screening Bacillus velez CGMCC NO.32593 comprises the following steps:

[0011] The soil was collected in Tianjin. 10 g of the collected soil sample was weighed and gradiently diluted. The obtained soil supernatant suspension was applied to Pikovskaya inorganic phosphorus and Pikovskaya organic phosphorus solid culture media for screening. The culture was carried out at 35-39° C. for 48-72 h. Single colonies that grew well on the culture media and had transparent circles were selected and continued to be cultured on the same solid culture media under the same culture conditions to purify the desired strains.

[0012] A method for culturing Bacillus Velez CGMCC NO.32593 comprises the following steps:

[0013] (1) Streak the culture medium on LB solid medium and culture it at 35-39°C for 12-14 h to obtain a single colony.

[0014] (2) Inoculate a single colony into 5 mL of LB liquid culture medium and culture at 35-39°C and 170-190 r / min for 12-14 h to obtain a primary seed solution.

[0015] Preferably, the pH of the LB medium is 7.0, the culture temperature is 37° C., and the culture time is 14 h.

[0016] The Bacillus Velez CGMCC NO.32593 of the present invention has multiple functions, including phosphorus solubilization, potassium solubilization, nitrogen fixation and IAA production.

[0017] The present invention also provides a microbial agent and a preparation method thereof, wherein the microbial agent comprises the above-mentioned Bacillus Velez CGMCC NO.32593, and the preparation method is as follows:

[0018] (1) Bacillus velezensis CGMCC No. 32593 was inoculated on LB solid medium and cultured at 37°C for 14 h to obtain a single colony.

[0019] (2) A single colony was inoculated into 5 mL of LB liquid culture medium and cultured at 37°C and 170-190 rpm for 14 h to obtain a primary seed solution.

[0020] (3) Inoculate the primary seed solution into 50 mL of LB liquid culture medium at a rate of 1-5%, and culture at 37° C. and 170-190 rpm for 14 h to obtain the secondary seed solution.

[0021] (4) The secondary seed liquid is inoculated into the fermentation medium at an inoculum rate of 1-5%, and fermentation is carried out in a 5L fermenter. The culture temperature is 35-39°C, and the dissolved oxygen (DO) is adjusted in series with the stirring speed to maintain the dissolved oxygen at 20-30%, and the fermentation culture is carried out for 24-36 hours.

[0022] Preferably, the culture rotation speed of the first and second seed solutions is 190 r / min, the first seed solution is inoculated into the second seed solution at an inoculation rate of 1% (V / V), and the second seed solution is inoculated into the fermentation medium at an inoculation rate of 1% (V / V).

[0023] Preferably, the fermentation medium formula (1 L) is: 10.0 g of tryptone, 5.0 g of yeast extract powder, 10.0 g of sodium chloride, 20.0 g of glucose, 1000 mL of distilled water, pH 7.0. The dissolved oxygen in the fermentation tank is maintained at 30%, and the culture time is 36 h.

[0024] Preferably, the microbial agent is a liquid agent, and the number of viable bacteria of Bacillus Velezii CGMCCNO.32593 in the microbial agent is 1 to 2×10 11 CFU / mL.

[0025] The present invention also provides the use of the microbial agent in promoting plant growth. Experiments have shown that the microbial agent can effectively increase the plant height and fresh weight of the aboveground part of tomatoes.

[0026] The microbial agent provided by the present invention has phosphorus-dissolving activity, can convert the insoluble phosphorus in the soil into soluble phosphorus that can be directly absorbed and utilized by plants, and can improve the utilization rate of soil phosphorus in crop production.

[0027] Beneficial effects:

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] 1. The Bacillus Velezii CGMCC NO.32593 provided by the present invention has multiple functions. A single strain can achieve the effects of solubilizing phosphorus, potassium, fixing nitrogen, and producing IAA.

[0030] 2. The preparation method of the microbial agent provided by the present invention is simple, has high production efficiency, low requirements on culture conditions, and a short cycle. It is suitable for the needs of industrial mass production and is easy to promote and use.

[0031] 3. Application of the microbial agent provided by the present invention can effectively increase the plant height and fresh weight of the aboveground part of tomatoes, promote plant growth, and at the same time improve the utilization rate of phosphorus in the soil by the plants. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 The morphological characteristics of Bacillus Velez CGMCC NO.32593 in the present invention on inorganic phosphorus and two organic phosphorus solid culture media: A is an inorganic phosphorus (calcium phosphate) solid culture medium, B is an organic phosphorus (lecithin) solid culture medium, and C is an organic phosphorus (calcium phytate) solid culture medium;

[0033] Figure 2 The colony morphology and microscopic morphology of Bacillus Velez CGMCC NO.32593 on LB solid medium in the present invention: A is the colony morphology, and B is the microscopic morphology;

[0034] Figure 3 This is a phylogenetic tree constructed based on the 16S rRNA gene sequence of Bacillus Velezii CGMCC NO.32593 in the present invention;

[0035] Figure 4The determination of the phosphorus solubilization ability of Bacillus Velez CGMCC NO.32593 in the present invention: A is a phosphorus standard curve, B is the determination of the ability to solubilize inorganic phosphorus (calcium phosphate), and C is the determination of the ability to solubilize organic phosphorus (calcium phytate);

[0036] Figure 5 It is the standard curve diagram of IAA in the present invention;

[0037] Figure 6 The growth of Bacillus Velez CGMCC NO.32593 in the present invention on Ashby nitrogen-free medium;

[0038] Figure 7 The growth of Bacillus Velez CGMCC NO.32593 in the present invention on Alexander Rove medium;

[0039] Figure 8 This is the growth curve of Bacillus Velez CGMCC NO.32593 in a 5L fermenter in the present invention;

[0040] Fig. 9 The liquid bacterial agent of the present invention has a growth-promoting effect on potted tomatoes. DETAILED DESCRIPTION

[0041] The present invention is further described in detail below in conjunction with the accompanying drawings and specific examples of the specification. The examples are only used to explain the present invention and are not used to limit the scope of the present invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0042] The culture medium and reagents involved in the embodiments of the present invention are as follows:

[0043] Pikovskaya inorganic phosphate medium (1L): glucose 10g, (NH4)2SO40.5g / L, yeast extract powder 0.5g, KCl0.3g, NaCl 0.3g, MgSO4·7H2O 0.3g, MnSO4·H2O 0.03g, K2SO40.3g, FeSO4·7H2O 0.03g, Ca3(PO4)2·2H2O 5.0g, distilled water 1000mL, pH 7.0-7.5 (solid culture medium adds 5g of agar powder on the basis of liquid culture medium).

[0044] Pikovskaya organophosphorus medium (1L): glucose 10g, (NH4)2SO40.5g / L, yeast extract powder 0.5g, KCl0.3g, NaCl 0.3g, MgSO4·7H2O 0.3g, MnSO4·H2O 0.03g, K2SO40.3g, FeSO4·7H2O 0.03g, calcium phytate 5g (or add lecithin 2g), distilled water 1000mL, pH 7.0-7.5 (solid culture medium adds 5g of agar powder on the basis of liquid culture medium).

[0045] LB medium (1 L): 10.0 g of tryptone, 5.0 g of yeast extract powder, 10.0 g of NaCl, 1000 mL of distilled water, pH 7.0 (5 g of agar powder is added to the liquid medium for solid culture medium).

[0046] IAA detection medium (1L): 10.0g tryptone, 5.0g yeast extract powder, 10.0g NaCl, 0.2g L-tryptophan, 1000mL distilled water, pH7.0.

[0047] Salkowski colorimetric reagent: Add 1 mL of 0.5 mol / LFeCl3·6H2O to 50 mL of 35% perchloric acid and prepare as needed.

[0048] Alexander Roth medium (1L): sucrose 5g, MgSO4·7H2O 0.5g, Na2HPO4·H2O 2.0g, CaCO3 0.1g, FeCl3·6H2O 0.05g, potassium feldspar powder 1.0g, distilled water 1000mL, pH 7.4 (solid culture medium adds 5g of agar powder on the basis of liquid culture medium).

[0049] Ashby nitrogen-free medium (1L): mannitol 10g, KH2PO4 0.2g, MgSO4·7H2O 0.5g, NaCl 0.2g, CaCO3 5g, CaSO4·H2O 0.5g, distilled water 1000mL, pH 7.0-7.5 (solid culture medium adds 5g of agar powder on the basis of liquid culture medium).

[0050] Example 1 Isolation and purification of phosphate-solubilizing strains.

[0051] The soil was collected in Tianjin, and the drilling depth was 20 cm. After removing the roots and stones and other impurities, it was fully mixed as the soil sample and stored in an ice box at low temperature. Weigh 10g of the collected soil sample and perform gradient dilution. The dilution factor was 10 -5 , 10 -6 , 10 -7The soil supernatant suspension was spread on Pikovskaya inorganic phosphorus and Pikovskaya organic phosphorus solid medium and cultured at 37℃ for 72h.

[0052] The organic acid produced by the strain on the phosphate-solubilizing medium forms a transparent circle around its colony through osmotic diffusion. The phosphate-solubilizing index (D / d) of the formed phosphate-solubilizing circle diameter (D) and the colony diameter (d) is used as an indicator to screen excellent phosphate-solubilizing strains, and finally obtain the phosphate-solubilizing bacteria BLS. The morphology of strain BLS on different phosphorus solid media is shown in Figure 1 , A is inorganic phosphorus (calcium phosphate) solid medium, B is organic phosphorus (lecithin) solid medium, and C is organic phosphorus (calcium phytate) solid medium. The solubility index is shown in Table 1.

[0053] Table 1 Phosphate solubilization index of strain BLS

[0054]

[0055] Example 2 Strain Classification and Identification

[0056] (1) Morphological identification

[0057] Transfer the purified strain BLS to LB solid medium and record the colony morphology of the strain on the culture dish. Figure 2 A.

[0058] The strain grows rapidly on LB solid medium. The colonies are round, light yellow, wrinkled, dry, and opaque. The edges of the colonies are neat in the early stages of growth, but not neat in the later stages. The bacteria are rod-shaped, have buds, and are about 2.5 μm long ( Figure 2 B).

[0059] (2) Physiological and biochemical identification

[0060] Physiological and biochemical identification indicators mainly include: Gram staining, VP test, MR test, catalase reaction, starch hydrolase activity, citrate test, gelatin liquefaction test, sugar fermentation test, etc. The specific determination method is carried out according to the method in the "Common Bacteria System Identification Manual" (Dong Xiuzhu, Cai Miaoying, Beijing: Science Press, 2001). Gram staining results are as follows Figure 2 B is purple, so the bacteria are Gram-positive bacteria. The rest of the experimental results are shown in Table 2. Combining the morphological and physiological and biochemical characteristics, the strain was identified as a Gram-positive spore-forming strain.

[0061] Table 2 Physiological and biochemical identification results

[0062]

[0063]

[0064] (3) 16S rRNA gene identification

[0065] The pure culture cultured to the logarithmic growth phase was collected by centrifugation, and the genomic DNA of the phosphate-solubilizing bacteria BLS was extracted using TaKaRa MiniBEST Bacteria Genomic DNA Extraction Kit Ver.3.0 produced by Baoriyi Biotechnology (Beijing) Co., Ltd. Then, the 16S rRNA gene was amplified by PCR using the universal bacterial identification primer (1492R) as a template.

[0066] The reaction conditions of PCR amplification include: ① pre-denaturation at 94°C for 5 min; ② denaturation at 94°C for 30 s, ③ annealing at 56°C for 30 s, ④ extension at 72°C for 90 s, ②-④ steps are cycled 30 times, and ⑤ final extension at 72°C for 10 min.

[0067] The obtained PCR product was sequenced, and the sequencing results were analyzed using the BLAST tool in the NCBI database (http: / / www.ncbi.nlm.nih.gov). Comparison analysis with the GenBank database showed that the 16S rRNA gene sequence homology between the strain of the present invention and Bacillus velezinoff was 99.79%. The 16S rRNA gene sequence with a high homology to the strain of the present invention was downloaded as a reference object, and the neighbor-joining (NJ) method in MEGA11.0 software was used to construct a phylogenetic tree, as shown in FIG. Figure 3 Phylogenetic tree analysis showed that the strain of the present invention was closely related to Bacillus velezensis KnL15 (www.ncbi.nlm.nih.gov / nuccore / NZ_JAJERE010000023.1?report=fasta). Therefore, it is believed that the strain of the present invention is Bacillus velezensis.

[0068] The 16S rRNA gene sequence of the strain is shown in SEQ ID No.1:

[0069] CTTCGGCGGCTGGCTCCTAAAGGTTACCTCACCGACTTCGGGTGTTACAAACTCTCGTG

[0070] GTGTGACGGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGC

[0071] GATTACTAGCGATTCCAGCTTCACGCAGTCGAGTTGCAGACTGCGATCCGAACTGAGAA

[0072] CAGATTTGTGGGATTGGCTTAACCTCGCGGTTTCGCTGCCCTTTGTTCTGTCCATTGTAG

[0073] CACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCC

[0074] GGTTTGTCACCGGCAGTCACCTTAGAGTGCCCAACTGAATGCTGGCAACTAAGATCAAG

[0075] GGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCAT

[0076] GCACCACCTGTCACTCTGCCCCCGAAGGGGACGTCCTATCTCTAGGATTGTCAGAGGAT

[0077] GTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTG

[0078] TGCGGGCCCCCGTCAATTCCTTTGAGTTTCAGTCTTGCGACCGTACTCCCCAGGCGGAG

[0079] TGCTTAATGCGTTAGCTGCAGCACTAAGGGGCGGAAACCCCCTAACACTTAGCACTCAT

[0080] CGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTCGCTCCCCACGCTTTCGCTCCT

[0081] CAGCGTCAGTTACAGACCAGAGAGTCGCCTTCGCCACTGGTGTTCCTCCACATCTCTAC

[0082] GCATTTCACCGCTACACGTGGAATTCCACTCTCCTCTTCTGCACTCAAGTTCCCCAGTTT

[0083] CCAATGACCCTCCCCGGTTGAGCCGGGGGCTTTCACATCAGACTTAAGAAACCGCCTGC

[0084] GAGCCCTTTACGCCCAATAATTCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGC

[0085] TGGCACGTAGTTAGCCGTGGCTTTCTGGTTAGGTACCGTCAAGGTGCCGCCCTATTTGAA

[0086] CGGCACTTGTTCTTCCCTAACAACAGAGCTTTACGATCCGAAAACCTTCATCACTCACGC

[0087] GGCGTTGCTCCGTCAGACTTTCGTCCATTGCGGAAGATTCCCTACTGCTGCCTCCCGTAG

[0088] GAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCCGATCACCCTCTCAGGTCGGCTACGCA

[0089] TCGTCGCCTTGGTGAGCCGTTACCTCACCAACTAGCTAATGCGCCGCGGGTCCATCTGTA

[0090] AGTGGTAGCCGAAGCCACCTTTTATGTCTGAACCATGCGGTTCAAACAACCATCCGGTAT

[0091] TAGCCCCGGTTTCCCGGAGTTATCCCAGTCTTACAGGCAGGTTACCCACGTGTTACTCAC

[0092] CCGTCCGCCGCTAACATCAGGGAGCAAGCTCCCATCTGTCCGCTCGACTTGCATGTATAG

[0093] Based on the morphological characteristics, physiological and biochemical properties and molecular biological identification results, the strain screened and identified was Bacillus velezensis.

[0094] The screened phosphate-solubilizing strain BLS is deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with a deposit number of CGMCC NO.32593 and a deposit date of November 13, 2024.

[0095] Example 3 Determination of various biological abilities of Bacillus velez CGMCC NO.32593

[0096] (1) Determination of phosphate solubilization capacity

[0097] Taking advantage of the fact that soluble phosphorus can combine with ammonium molybdate to form phosphomolybdic acid amide under acidic conditions, and that phosphomolybdic acid amide can be reduced by 2,6-dinitrophenol to form a blue compound, a visible light spectrophotometer was used to measure the absorbance of molybdenum antimony antibody at a wavelength of 700nm to quantitatively calculate the content of soluble phosphorus in the culture supernatant.

[0098] Bacillus Velezii CGMCC NO.32593 was activated in LB liquid medium and inoculated into a conical flask containing 50 mL Pikovskaya inorganic phosphorus (calcium phosphate) and organic phosphorus (calcium phytate) liquid medium at an inoculum size of 1%. Three replicates were set up, and the medium without bacteria was used as the control. The culture was carried out at 190 r / min and 37°C for 7 days.

[0099] Take 2 mL of fermentation broth every 24 hours, measure the pH and centrifuge at 4000 r / min for 20 min, take 1 mL of supernatant in a 50 mL volumetric flask, add 100 μL of 2,6-dinitrophenol as a reaction indicator, adjust the pH of the solution with sodium hydroxide or dilute sulfuric acid until the solution is slightly yellow, add 5 mL of molybdenum antimony anticolorimetric agent, make the volume up to 50 mL, and place it in an environment above 15°C to react for 30 min.

[0100] The absorbance of the reaction solution at 700 nm was measured using a UV-visible spectrophotometer, with the uninoculated culture medium as the zero point. The phosphorus standard curve y = 0.5146x + 0.0018 (R 2 =0.999) to calculate the soluble phosphorus content in the culture solution and record the data. Figure 4 As shown in A, Figure 4 B is the ability of Bacillus Velez CGMCC NO.32593 to dissolve inorganic phosphorus. Its maximum dissolution capacity of inorganic phosphorus (calcium phosphate) is 273.54 mg / L. Figure 4C is the determination of the ability of Bacillus velez CGMCC NO.32593 to dissolve organic phosphorus, and its maximum solubility of organic phosphorus (calcium phytate) is 177.03 mg / L. It proves that the strain Bacillus velez CGMCC NO.32593 has a good function of dissolving insoluble phosphorus. The phosphorus solubilization amount of the strain of the present invention and the phosphorus solubilization ability of other strains are compared in Table 3.

[0101] Table 3 Comparison of phosphate solubilization ability with other strains

[0102]

[0103] (2) Generate IAA capability

[0104] Bacillus Velez CGMCC NO.32593 was inoculated into a 5 mL LB liquid culture medium test tube and cultured at 37°C and 190 r / min for 14 h to serve as a seed culture medium. The seed culture medium was aspirated at a 1% inoculum amount and cultured in 50 mL IAA detection medium for 48 h to obtain a fermentation broth, which was repeated three times.

[0105] Take 50uL of fermentation liquid on a white ceramic plate, add the same volume of Salksowski colorimetric solution, and use 50μL of IAA (50mg / L) standard solution as a blank control. After placing it in a dark environment for 30 minutes, observe the color change. If the color turns red, it means that IAA can be produced.

[0106] IAA yield: The fermentation broth obtained above was centrifuged at 4000 r / min for 10 min, 4 mL of the supernatant was mixed with an equal volume of Salksowski colorimetric solution, shaken well, and placed at room temperature away from light for 30 min. The OD 530 The uninoculated liquid culture medium was mixed with the Salksowski colorimetric solution in equal volumes as the blank control group. According to the standard curve y = 0.0152x-0.0052 (R 2 =0.9923) to calculate the corresponding IAA content. IAA standard curve diagram is shown in Figure 5 .

[0107] The IAA production of Bacillus Velez CGMCC NO.32593 was calculated to be 20.19±1.27 mg / L. The strain can produce auxin, indicating that it has a certain ability to promote plant growth.

[0108] (3) Nitrogen fixation ability of strains

[0109] The preserved Bacillus Velez CGMCC NO.32593 was inoculated into 5 mL LB liquid culture medium. After activation culture for 14 hours, 2.5 μL of the bacterial solution was inoculated onto Ashby nitrogen-free solid culture medium. After the bacterial solution was absorbed by the culture medium, the plate was inverted and cultured in a 37°C constant temperature incubator. The results were observed after 72 hours.

[0110] After 72 hours of culture, the strain was able to grow well on Ashby solid medium, and the colonies were transparent and convex. Figure 6 shown.

[0111] Quantitative analysis: Bacillus Velez CGMCC NO.32593 was activated in a 5 mL LB liquid culture medium test tube and inoculated into a flask containing 50 mL Ashby nitrogen-free culture medium at a 1% inoculation amount. Three replicates were set up, and the uninoculated culture medium was used as the control. The culture was cultured at 190 r / min and 37°C for 48 h to obtain the fermentation broth.

[0112] The blank digestive tube was used as the blank group CK, the uninoculated Ashby liquid medium was used as the control group, and the fermentation broth was used as the experimental group. The nitrogen content of each group was measured by an automatic Kjeldahl nitrogen analyzer, and the data were recorded, as shown in Table 4.

[0113] Table 4 Nitrogen content of each group

[0114]

[0115] According to the data in the table, the nitrogen fixation capacity of Bacillus Velez CGMCC NO.32593 is 25.447±0.12 mg N / mL, which has good nitrogen fixation ability.

[0116] (4) Potassium-solubilizing ability of strains

[0117] The preserved Bacillus Velez CGMCC NO.32593 was inoculated into 5 mL LB liquid medium. After activation culture for 14 hours, 2.5 μL of bacterial solution was inoculated on the plate of Alexander Roth medium. After the bacterial solution was absorbed by the medium, the plate was inverted and placed in a 37°C constant temperature incubator for culture. The results were observed after 72 hours. Figure 7 shown.

[0118] The strain can grow well on Alexander Rolf medium solid culture medium and has potassium solubilizing ability.

[0119] Example 4 Preparation of liquid microbial agent

[0120] Fermentation tank culture medium formula (1L): 10.0g tryptone, 5.0g yeast extract powder, 10.0g sodium chloride, 20.0g glucose, pH 7.0.

[0121] Fermentation process:

[0122] (1) Streak Bacillus velezensis CGMCC No. 32593 on a LB solid plate and culture at 37°C for 14 h to obtain a single colony.

[0123] (2) A single colony was inoculated into 5 mL of LB liquid culture medium and cultured at 37°C and 190 rpm for 14 h to obtain a primary seed solution.

[0124] (3) The first-level seed solution was inoculated into 50 mL of liquid culture medium at an inoculum rate of 1% (V / V), and cultured at 37° C. and 190 rpm for 14 h to obtain the second-level seed solution.

[0125] (4) The secondary seed solution was inoculated into the fermentation medium at an inoculum rate of 1% (V / V), and fermentation was carried out in a 5L fermenter. The culture temperature was 37°C, and the dissolved oxygen (DO) and the stirring speed were adjusted in series to maintain them at 30%, and the fermentation was carried out for 36 hours.

[0126] During the fermentation process, samples were taken every 2 hours, diluted with fermentation medium, and the OD was measured by UV spectrophotometer. 600 The absorbance value was measured and the growth curve of the strain was drawn. Figure 8 .

[0127] like Figure 8 As shown in the figure, when the fermentation time is 12 to 16 hours, the OD 600 The maximum value is 16h~36h, and the fermentation liquid OD 600 The value tends to be stable and continues to decline slowly. As the nutrients in the tank are consumed, the bacteria gradually form spores. After 36 hours, the proportion of spores in the fermentation liquid remains basically stable, so the fermentation is ended at 36 hours to obtain a liquid bacterial agent. The effective viable bacteria count at the maximum absorbance value and at the end of fermentation is determined by the dilution coating method. The results are shown in Table 5 below.

[0128] Table 5 Effective viable bacteria count

[0129]

[0130] Example 5: Effect of liquid inoculant on tomato growth

[0131] (1) The following treatments were set for tomato potted plants:

[0132] Liquid bacterial agent: In order to eliminate the influence of other components in the culture medium, the liquid bacterial agent obtained in Example 4 was centrifuged, the supernatant was discarded, the bacterial cells were collected, and they were washed with distilled water, and the OD was adjusted with distilled water. 600 = 1.0. Take another 1 mL and dilute it 10 times to obtain the irrigation inoculant.

[0133] 1% diammonium phosphate: Prepare diammonium phosphate solution with distilled water at a mass-to-volume ratio.

[0134] CK: blank control group (irrigated with tap water only);

[0135] Microbial agent: experimental group (microbial agent treatment);

[0136] CK+diammonium phosphate: 1% diammonium phosphate was added to the CK group;

[0137] Bacterial agent + diammonium phosphate: Add 1% diammonium phosphate to the bacterial agent group.

[0138] (2) Potted plant treatment method:

[0139] Select high-quality tomato seeds and soak them in distilled water at room temperature for 6 to 10 hours. Prepare a plastic container covered with wet gauze, place the seeds on the gauze one by one, cover them with dry gauze, and place them in a constant temperature incubator at 28°C for germination. When more than 40% of the tomato seeds turn white, sow them in a seedling tray mixed with fine sand and vermiculite in a ratio of 1:2. After the seeds break through the soil and germinate, select seedlings with consistent growth conditions and transplant them into pots with the same soil conditions (12 cm high, 15.5 cm in diameter at the top).

[0140] (3) Watering treatment:

[0141] CK: No other exogenous substances are used, only tap water is used for irrigation;

[0142] Microbial agent: Apply liquid microbial agent to tomato pots, with a total of 10 mL applied to each pot;

[0143] CK+diammonium phosphate: Apply the aforementioned 1% diammonium phosphate to the roots of tomatoes on the basis of the CK group, with a total of 10 mL per pot;

[0144] Bacterial agent + diammonium phosphate: On the basis of the bacterial agent group, the aforementioned 1% diammonium phosphate was applied to the roots of tomatoes, with a total of 10 mL applied per pot.

[0145] Ensure that the time for applying the phosphate fertilizer suspension is consistent with the time for applying the liquid bacterial agent, and ensure that the amount of watering is the same each time.

[0146] (4) Cultivation conditions: Cultivate outdoors, maintain appropriate humidity, irrigate with tap water, and harvest after 30 days.

[0147] (5) Index determination: After the plants are harvested, the plant height is measured with a ruler and the stem diameter is measured with a vernier caliper. Then, the soil remaining on the surface of the plants is cleaned, and the plants are naturally dried after being rinsed. The fresh weight of each part is weighed, and the plants are placed in an oven at 105°C for 20 minutes. The oven temperature is adjusted to 60°C and dried until the weight no longer changes. The dry weight of each part of the plant is weighed.

[0148] The growth of tomatoes in the four treatment groups is as follows Fig. 9 Table 6 was obtained by statistical analysis of the tomato plant traits in the four treatment groups.

[0149] Table 6 Tomato plant traits under different treatment groups

[0150]

[0151] Note: Different lowercase letters in the above table indicate significant differences (P<0.05)

[0152] like Fig. 9 As shown in Table 6, compared with the blank control group CK, the height of tomato plants in the bacterial agent group increased by 15.1%, the stem diameter increased by 4.0%, the fresh weight of the aboveground part increased by 24.7%, the fresh weight of the underground part increased by 8%, the dry weight of the aboveground part increased by 21.4%, and the dry weight of the underground part increased by 25.0%. This is because the Bacillus Velez CGMCC NO.32593 contained in the bacterial agent has the function of solubilizing phosphorus and fixing nitrogen, which can provide the corresponding phosphorus and nitrogen elements for the growth of plants in the seedling stage. In addition, Bacillus Velez CGMCC NO.32593 has the ability to produce IAA, which is an important auxin that directly promotes plant growth. The results show that the application of bacterial agents can promote the growth of tomatoes.

[0153] like Fig. 9 As shown in Table 6, compared with the tomato plants treated with only diammonium phosphate, the treatment with simultaneous addition of the fungicide and diammonium phosphate increased the plant height, stem diameter, aboveground fresh weight, underground fresh weight, aboveground dry weight, and underground dry weight of tomatoes by 3.9%, 3.6%, 2.5%, 8.3%, 15%, and 20%, respectively, with a significant growth-promoting effect.

Claims

1. A strain of Bacillus velezensis, characterized in that: On November 13, 2024, it was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with the deposit number CGMCC NO.32593. The 16S rRNA gene sequence of the strain is shown in SEQID No.

1.

2. The Bacillus Velez CGMCC NO.32593 of claim 1, characterized in that: It has the ability to solubilize phosphorus, potassium, fix nitrogen and produce the growth hormone indoleacetic acid (IAA).

3. A microbial agent, characterized in that: The active ingredient is the Bacillus Velez CGMCC NO.32593 as claimed in claim 1.

4. The microbial agent according to claim 3, characterized in that The preparation method of the microbial agent is as follows: streak inoculation of Bacillus Velez CGMCC NO.32593 on an LB solid plate, culture at 37°C for 14 hours to obtain a single colony, inoculate the single colony in 5mL LB liquid culture medium, culture at 37°C, 190r / min for 14 hours to obtain a primary seed solution, inoculate the primary seed solution into 50mL LB liquid culture medium at an inoculation amount of 1%, culture at 37°C, 190r / min for 14 hours to obtain a secondary seed solution, inoculate the secondary seed solution into a fermentation medium at an inoculation amount of 1% (V / V), and ferment in a 5L fermenter. The culture temperature is 37°C, the dissolved oxygen (DO) is adjusted in series with the stirring speed to be maintained at 30%, and the fermentation culture is carried out for 36 hours.

5. The microbial agent according to claim 3, characterized in that: The number of viable bacteria of Bacillus Velezii CGMCC NO.32593 contained in the microbial agent is 1 to 2×10 11 CFU / mL.

6. Use of the Bacillus Velez CGMCC No. 325933 according to claim 1 or 2 or the microbial agent according to any one of claims 3 to 5 in promoting plant growth.

7. The use according to claim 6, characterized in that: The plant is tomato.

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