CN113980833B

CN113980833B - Bacillus megaterium and application thereof in soil phosphate solubilizing

Info

Publication number: CN113980833B
Application number: CN202111114279.2A
Authority: CN
Inventors: 孙敏; 张帅; 刘巍; 李洪顺; 鲍俊萍
Original assignee: Sinochem Agriculture Linyi Research and Development Center Co Ltd
Current assignee: Sinochem Agriculture Linyi Research and Development Center Co Ltd
Priority date: 2021-09-23
Filing date: 2021-09-23
Publication date: 2022-07-12
Anticipated expiration: 2041-09-23
Also published as: CN113980833A

Abstract

The invention relates to the field of microorganisms, and provides bacillus megaterium and application thereof in soil phosphate solubilizing. The Bacillus megaterium is preserved in the China general microbiological culture Collection center with the preservation number of CGMCC No. 21828. The provided bacillus megaterium can also be prepared into a microbial agent, a composite microbial agent or a microbial fertilizer. The strain shows good phosphate solubilizing effect, and shows excellent phosphate solubilizing capability on inorganic phosphorus and organic phosphorus. But also can promote the growth of crops and improve the yield of the crops.

Description

Bacillus megaterium and application thereof in soil phosphate solubilizing

Technical Field

The invention relates to the technical field of microorganisms, in particular to bacillus megaterium and application thereof in soil phosphate solubilizing.

Background

China is the first major country of world chemical fertilizer consumption, agricultural output depends on chemical fertilizer input, but the yield is not increased by increasing fertilizer, the fertility is excessive, soil hardening is caused by excessive chemical fertilizer application, the soil structure is damaged, and the like. Test data show that the microbial fertilizer can obtain high-yield and high-quality crops, change the physical and chemical properties of soil, improve the soil fertility and the like. Generally, the microbial fertilizer is an essential factor for the development of agriculture at present as a high-efficiency, high-yield, green, healthy and pollution-free plant fertilizer.

The bacillus megaterium is used as gram-positive aerobic bacteria, can decompose organic phosphorus substances and insoluble phosphorus compounds, is one of the most widely applied microorganisms in the prior agricultural microbial agents, and reportedly can improve the microbial community structure of soil, promote the growth of crops and improve the yield of the crops by stably colonizing in the soil. By screening excellent strains and preparing the microbial fertilizer by using phosphate solubilizing microorganisms, the utilization rate of phosphorus in soil can be improved, fertilizer is saved, yield is increased, soil organic matters can be improved, the development of microbial industry is promoted, and the microbial fertilizer has positive effects on crop yield and ecological environment.

However, Bacillus megaterium, which is excellent in performance, is in need of further improvement.

Disclosure of Invention

The present invention is directed to solving, at least to some extent, one of the technical problems in the related art. Therefore, the invention aims to provide a Bacillus megaterium, a microbial agent, a complex microbial agent, a microbial fertilizer and application thereof in soil phosphate solubilizing.

The first aspect of the invention provides a Bacillus megaterium which is preserved in China general microbiological culture Collection center with the preservation number of CGMCC No. 21828. The provided strain is screened, separated and identified as bacillus megaterium and is also named as LY 153-1. But also can change the microbial community structure and has the capability of efficiently dissolving phosphorus.

In a second aspect, the present invention provides a microbial inoculant comprising bacillus megaterium as described in the first aspect above.

The third aspect of the invention provides a complex microbial inoculant, which comprises a first microbial inoculant and a second microbial inoculant, wherein the first microbial inoculant is the microbial inoculant of the first aspect of the invention, and the second microbial inoculant comprises at least one of bacillus belgii and bacillus licheniformis.

In a fourth aspect, the invention provides a microbial fertilizer, which comprises bacillus megaterium of the first aspect, or a microbial agent of the second aspect, or a complex microbial agent of the third aspect.

The fifth aspect of the invention provides a use of bacillus megaterium in preparation of a microbial agent, a complex microbial agent or a microbial fertilizer, wherein the bacillus megaterium is the bacillus megaterium of the first aspect of the invention.

A sixth aspect of the present invention provides a method for solubilizing phosphorus in soil, comprising:

applying an effective amount of bacillus megaterium, a microbial agent, a composite microbial inoculum or a microbial fertilizer to soil, wherein the bacillus megaterium is the bacillus megaterium of the first aspect of the invention, the microbial agent is the microbial agent of the second aspect of the invention, the composite microbial inoculum is the composite microbial inoculum of the third aspect of the invention, and the microbial fertilizer is the microbial fertilizer of the fourth aspect of the invention. The phosphorus dissolving capacity to inorganic phosphorus in the soil is at least 200mg/L by dissolving phosphorus in the soil; the phosphate solubilizing capability for organic phosphorus in soil is at least 10 mg/L.

A seventh aspect of the present invention provides a method of fertilizing a crop, comprising: applying an effective amount of bacillus megaterium, a microbial agent, a compound microbial inoculum or a microbial fertilizer to the crops, wherein the bacillus megaterium is the bacillus megaterium of the first aspect of the invention, the microbial agent is the microbial agent of the second aspect of the invention, the compound microbial inoculum is the compound microbial agent of the third aspect of the invention, and the microbial fertilizer is the microbial fertilizer of the fourth aspect of the invention.

Information on strain preservation

Bacillus megaterium, with the preservation number of CGMCC No.21828, the preservation unit is China general microbiological culture Collection center, the preservation address is the institute of microbiology, China academy of sciences, 3, West Lu No. 1, North Cheng, south China, Beijing, and the preservation time is 2021 year, 02 month and 25 days.

Drawings

FIG. 1 is a graph showing the evaluation results of the indoor phosphate solubilizing ability of Bacillus megaterium according to the embodiment of the present invention.

FIG. 2 is a graph (species level) showing the change of microbial colonies in soil after administration of a Bacillus megaterium LY153-1 microbial inoculum, provided in accordance with an embodiment of the present invention.

FIG. 3 is a graph showing the results of culturing Bacillus megaterium LY153-1 in TSA medium at 30 ℃ for 18 hours, which was provided in accordance with the example of the present invention.

FIG. 4 is a graph showing the results of systematic analysis of 16S rDNA of Bacillus megaterium LY153-1, provided in accordance with an embodiment of the present invention.

FIG. 5 is the result of phylogenetic analysis of rpoB gene of Bacillus megaterium LY153-1 provided according to an embodiment of the present invention.

Detailed Description

The embodiments of the present invention will be described in detail below with reference to the accompanying drawings, which are intended to be illustrative and not to be construed as limiting the invention.

Herein, when the content of a certain substance is expressed, the mass of the substance is referred to as a percentage of the total substance weight, unless otherwise specified.

Herein, when referring to "prevention" of a disease, "prevention" includes not only prevention of occurrence of a certain disease but also treatment of a certain disease, thereby alleviating, reducing, alleviating symptoms of the disease.

The invention provides a Bacillus megaterium, which is preserved in the China general microbiological culture Collection center with the preservation number of CGMCC No. 21828. The provided strain is screened, separated and identified as bacillus megaterium. This Bacillus megaterium was also designated LY 153-1. The bacillus megaterium is obtained by LC-MS non-targeted metabonomics analysis, and organic acid compounds which can convert insoluble phosphorus into available phosphorus for plant decomposition and utilization exist in fermentation liquor of the bacillus megaterium, wherein the organic acid compounds comprise monoacid, succinic acid, acetic acid, formic acid, glutaric acid and the like. The provided bacillus megaterium can change the microbial community structure and has the capability of efficiently dissolving phosphorus.

The bacillus megaterium is identified to be positive in gram stain, and the bacillus megaterium is rod-shaped and has round ends. On TSA medium, the colonies were nearly round, smooth in surface, clean in edges, yellowish and opaque, and shiny. And the analysis result of the physiological and biochemical characteristics (whole cell fatty acid and API 50CH) of the Bacillus cell meets the biochemical metabolic characteristics of the Bacillus. Furthermore, rpoB conserved gene sequencing results of the strain show that the strain and Bacillus megaterium NBRC 15308T are polymerized into the same branch at 100% support rate. According to a specific embodiment of the present invention, there is provided Bacillus megaterium having a 16S rDNA sequence as shown in SEQ ID NO. 1. The 16S rDNA sequence mentioned is a gene encoding the 16S rRNA subunit. According to a specific embodiment of the present invention, Bacillus megaterium has the rpoB gene sequence shown in SEQ ID NO. 2.

The invention also provides a microbial agent, which comprises the bacillus megaterium. The result of a soil colonization test shows that the LY153-1 strain can exist for a long time in the soil applied with the Bacillus megaterium LY153-1 microbial inoculum, and the content of viable bacteria in the soil changes along with the growth period of plants and the change of environmental factors. After 180 days, the number of viable bacteria of the Bacillus megaterium LY153-1 in the soil is 260 ten thousand/g (the initial value is 500 ten thousand/g). And the change of the diversity of the soil microbial communities after the bacillus megaterium LY153-1 powder is applied is detected by a high-throughput sequencing technology, and the result shows that the ratio of the diversity of the soil microbial communities to the ratio of the diversity of the soil microbial communities without the addition of the CK: the relative abundance of the bacillus, the Germinatum and the Bacillus is increased more, and the relative abundance of the xanthogenic bacteria is reduced;

the phosphate solubilizing capability of the bacillus megatherium or the microbial agent provided by the invention for inorganic phosphorus is at least 200mg/L, which is higher than 191% of NY/T1847-2010 standard, and the phosphate solubilizing capability for organic phosphorus is 11mg/L, which is higher than 120% of NY/T1847-2010 standard.

The invention also provides a complex microbial inoculant which comprises a first microbial inoculant and a second microbial inoculant, wherein the first microbial inoculant is the microbial inoculant of the first aspect of the invention, and the second microbial inoculant comprises at least one of bacillus belgii and bacillus licheniformis.

In at least some embodiments, the complex microbial inoculant comprises 30-35 parts by weight of the first microbial inoculant, and at least one selected from the group consisting of: 30-35 parts by weight of Bacillus belgii and 30-35 parts by weight of Bacillus licheniformis; 30-35 parts by weight of bacillus subtilis. In at least some embodiments, the complex microbial inoculant comprises 30-35 parts by weight of Bacillus megaterium, 30-35 parts by weight of Bacillus belgii, and 30-35 parts by weight of Bacillus licheniformis.

In some embodiments, the effective viable count of the bacillus megaterium contained in the composite microbial inoculum is at least 1.0 multiplied by 10¹⁰CFU/g, and comprising at least one selected from the group consisting of: the effective viable count of Bacillus belezii is at least 1.0 × 10¹¹CFU/g; the effective viable count of Bacillus licheniformis is at least 1.0 × 10¹¹CFU/g. CFU/g is interpreted as meaning generally in the art and represents the number of colonies of microorganisms contained in 1g of test sample, and CFU represents a colony forming unit.

The provided strain, or microbial agent or composite microbial agent can be used alone or added into a fertilizer for use. Therefore, the invention also provides a microbial fertilizer, which comprises the bacillus megaterium, the microbial agent or the composite microbial agent.

The microbial fertilizer further comprises a base fertilizer. The basic fertilizer can be organic and inorganic fertilizer, compound fertilizer and the like. For example, the organic and inorganic fertilizers are 15-40/S (15-8-17), and the compound fertilizers are 15-15-15/S. The microbial fertilizer can be prepared by the following method: and uniformly mixing the coating working section of the basic fertilizer and the anti-caking agent, and then adding a microbial agent with the content of 2 per mill to obtain the microbial fertilizer. The microbial fertilizer contains at least 0.2 hundred million CFU/g of effective microbial viable count.

The invention also provides the application of the bacillus megaterium in preparing a microbial agent, a composite microbial agent or a microbial fertilizer.

The invention also provides a method for dissolving phosphorus in soil, which comprises the following steps: applying an effective amount of bacillus megaterium, a microbial agent, a composite microbial inoculum or a microbial fertilizer to soil, wherein the bacillus megaterium is the bacillus megaterium, the microbial agent is the microbial agent, the composite microbial inoculum is the composite microbial inoculum, and the microbial fertilizer is the microbial fertilizer. The phosphorus dissolving capacity to inorganic phosphorus in the soil is at least 200mg/L by dissolving phosphorus in the soil; the phosphate solubilizing ability for organic phosphorus in soil is at least 10 mg/L. The change of the diversity of the soil microbial community after the application of the bacillus megaterium LY153-1 microbial inoculum is detected by a high-throughput sequencing technology, and the result shows that compared with the ratio of the CK without the added bacteria: the relative abundance of the bacillus, the Germinatum and the Bacillus is increased more, and the relative abundance of the xanthogenic bacteria is reduced.

The invention also provides a method for fertilizing crops, which comprises the following steps: applying an effective amount of bacillus megaterium, a microbial agent, a composite microbial inoculum or a microbial fertilizer to the crops, wherein the bacillus megaterium is the bacillus megaterium, the microbial agent is the microbial agent, the composite microbial inoculum is the composite microbial inoculum, and the microbial fertilizer is the microbial fertilizer. The fertilizer has the effects of increasing both production and income by treating crops in soil, and particularly has the effects of increasing both production and income for potatoes and sweet potatoes. For example, the biological organic and inorganic fertilizers applied to the potatoes are increased by 15.6 percent and 5.7 percent respectively compared with the conventional fertilization and market products; compared with conventional fertilization and market products, the biological organic and inorganic fertilizer applied to sweet potatoes increases the yield by 6.93 percent and 4.2 percent respectively. The microbial fertilizer prepared by the strain is combined with biological organic and inorganic fertilizers, so that the microbial fertilizer has the functions of obviously promoting the growth of crops and improving the yield of the crops. The bacillus megaterium has obvious growth promoting effect on underground root crops such as potatoes, sweet potatoes and the like, and the yield is increased by 6.93-15.6%.

The scheme of the invention will be explained with reference to the following examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

Example 1 isolation and screening of Bacillus megaterium LY153-1

The method for separating and obtaining the bacillus megaterium LY153-1 from the surface of the potato root system by adopting a flat plate coating method and a flat plate marking method comprises the following steps:

healthy potato rhizosphere soil is selected from brown soil of the Xilinguoren union blue flag-positive potato planting base in the inner Mongolia autonomous region for screening. The method comprises the following specific steps: gently shaking off root soil, placing on weighing paper, scraping root system with a blade, collecting all materials, mixing, weighing 1g, placing in 100mL sterile water, oscillating at 150rpm and 30 deg.C for 30min, and performing gradient dilution to 10%⁴And (3) selecting 3 gradients for coating, wherein each gradient is 3 in parallel, after culturing for 2d in an incubator at 30 ℃, selecting strains with different colony morphologies, streaking on an LB (lysogeny broth) culture medium, and regularly observing the colony growth condition. Then, the strain was purified by plate streaking, and the strain was designated by LY153-1 and stored.

Example 2 measurement of phosphate solubilizing Effect of Bacillus megaterium LY153-1

(1) Evaluation method of phosphate solubilizing effect

Preparing a phosphate solubilizing liquid culture medium according to a detection method in general technology for quality evaluation of bacterial strains produced by the microbial fertilizer NY/T1847-2010, inoculating phosphate solubilizing bacteria, performing shake flask culture, sampling after a certain time to determine the content of soluble phosphorus, and comparing the increase amount of the soluble phosphorus in the liquid culture medium before inoculation and after inoculation.

Preparing an inorganic phosphorus dissolving culture medium: glucose 10g (NH4)₂SO₄ 0.5g，NaCl 0.3g，KCl 0.3g，MgSO₄.7H₂O 0.3g，FeSO₄.7 H₂O 0.03g，MnSO₄.4H₂O 0.03g，Ca₃(PO4)₂10g, pH 7.0, and constant volume to 1000 mL; preparing an organophosphorus phosphate-solubilizing culture medium: other components are the same as inorganic phosphorus medium, Ca₃(PO4)₂10g of lecithin needs to be replaced by 2g of lecithin, and the volume is up to 1000 mL.

Inoculating Bacillus megaterium LY153-1 into LB liquid culture medium, culturing for 3d, and measuring OD₆₀₀Value, adjusting the concentration of bacterial liquid 1 x 10⁸cfu/mL. The culture solution with the adjusted concentration is absorbed by 5mL and inoculated into a phosphate solubilizing culture medium, and meanwhile, the phosphate solubilizing effect control of the strains CK1 and CK2(CK1 and CK2 are all self-screening and preserving strains) with the same series of functions is set. Repeating the steps for 3 times, after culturing for 4d, centrifuging the sample at 4000rpm for 20min, and taking 10mL of supernatant for inspection.

(2) Evaluation results of phosphate solubilizing Effect

LY153-1 indoor evaluation results show, as shown in FIG. 1.

As can be seen from FIG. 1, compared with the control strains CK1 and CK2, the phosphate solubilizing effect of the strain LY153-1 is the best, the phosphate solubilizing capability for inorganic phosphorus is 204mg/L and is higher than that of standard 191 of NY/T1847-2010, the phosphate solubilizing capability for organic phosphorus is 11mg/L and is higher than that of standard 120 of NY/T1847-2010; compared with the control group CK1 and CK2, the phosphorus dissolving capacity of the phosphate fertilizer for inorganic phosphorus is improved by 603 percent and 98 percent, and the phosphorus dissolving capacity of the phosphate fertilizer for organic phosphorus is improved by 22 percent and 450 percent.

Example 3 Bacillus megaterium LC-MS non-targeted metabolomics assay

(1) LC-MS non-targeted metabonomics detection method

Accurately transferring 200 mu L of LY153-1 fermentation broth supernatant sample into a 1.5mL centrifuge tube; 800. mu.L of the extract (methanol: acetonitrile: 1(v: v)) and 0.02mg/mL of an internal standard (L-2-chlorophenylalanine) were added; mixing uniformly for 30s by vortex, and performing low-temperature ultrasonic extraction for 30min (5 deg.C, 40 KHz); standing the sample at-20 deg.C for 30 min; centrifuging for 15min (13000g, 4 ℃), transferring supernatant, and drying with nitrogen; adding 100 μ L of re-solution (acetonitrile: water 1:1) for re-dissolving, vortex mixing for 30s, ultrasonic extracting at low temperature for 5min (5 deg.C, 40KHz), centrifuging for 10min (13000g, 4 deg.C), and transferring supernatant to sample injection vial with cannula for analysis.

Chromatographic conditions are as follows: the column was ACQUITY UPLC HSS T3(100 mm. times.2.1 mm i.d.,1.8 μm; Waters, Milford, USA); mobile phase a was 95% water + 5% acetonitrile (containing 0.1% formic acid), mobile phase B was 47.5% acetonitrile + 47.5%, isopropanol + 5% water (containing 0.1% formic acid); the flow rate was 0.40mL/min, the amount of sample was 2. mu.L, and the column temperature was 40 ℃.

(2) LC-MS non-target metabonomics detection result

The results obtained by LC-MS non-targeted metabonomics analysis show that: the metabonomics in the fermentation liquor comprise 252 organic acids, 12 indoles, 133 amino acid polypeptides, 70 amines, 81 alcohols, 84 glucosides, 74 esters, 36 alkalis, 3 ethers, 68 ketones, 17 alkenes, 28 salts, 17 aldehydes, 13 antibiotics and 197 other groups. The analysis of organic acid substances in the fermentation liquor shows that the insoluble phosphorus can be converted into compounds which can be utilized by plants for decomposition, and the compounds comprise monoacid, succinic acid, acetic acid, formic acid, glutaric acid and the like.

Example 4 Bacillus megaterium soil colonization data determination

(1) Soil colonization test determination method

The soil and the bacillus megaterium LY153-1 powder are fully and uniformly mixed and potted (the initial concentration of soil in each pot is 500 ten thousand/g, and CK is not added with bacteria). Rape seeds were sown, 6 rape seeds were finally retained per pot, 20 pots were repeated per treatment. And (3) carrying out a long-term tracking test, taking out plant rhizosphere soil in pots from different periods of 10d, 15d, 20d, 30d, 40d, 60d, 70d, 90d, 120d, 150d and 180d after seedling emergence, diluting and coating rhizosphere microorganisms, and detecting the colonization condition of bacillus megatherium through colony morphology.

(2) Determination result of soil colonization test data

Sowing rape, harvesting and sowing again, wherein 4 batches of the seeds are continuously sowed for 180 days. And (3) detecting the viable count of the bacillus megaterium in the soil at 0 d-150 d, wherein the detection result shows a gradual descending trend, and is 90 ten thousand/g when the number reaches 150d, sampling and detecting the colonization condition of the bacillus megaterium LY153-1 in the growth period of the rape at 180d, detecting that the number of the LY153-1 colonization bacteria is 260 ten thousand/g, and greatly increasing compared with the number before 150d, wherein the analysis reason is likely to increase root secretions along with the growth of the rape plants, and the proliferation of the LY153-1 colonization strains in the soil is promoted. During the tracking, CK did not detect bacillus megaterium on the plate.

And (2) carrying out fluorescence labeling on the bacillus megaterium LY153-1 applied to the soil, sampling at regular intervals, sending the coated bacillus megaterium colony to an external detection mechanism for 16SrRNA sequencing, and comparing the sequence with a sequence in an NCBI database, wherein the comparison result shows that the homology between a suspected bacterial colony and the bacillus megaterium strain in the database is up to 99%. The bacterial colony obtained by diluting and coating the bacillus megaterium in the soil after the bacillus megaterium is applied is the bacillus megaterium, and the application of the bacillus megaterium to the soil optimizes a phosphorus circulation structure in the soil and improves the utilization rate of phosphorus.

Example 5 soil microbial community diversity assay for Bacillus megaterium

(1) Method for detecting diversity of soil microbial community

The experimental process for detecting the diversity of the microbial colonies mainly comprises the following steps: extracting environmental sample (soil) DNA, primer joint and DNA amplification, quantifying and homogenizing PCR products, constructing libraries, performing high-throughput sequencing and analyzing bioinformatics. The test is completed by an external detection company.

(2) Soil microbial community diversity detection result

On the basis of the structure of the soil community at a subordinate level, the relative abundance of genera in the soil applied with the powder of the Bacillus megaterium LY153-1 (500 ten thousand/g) is increased, and the relative abundance of the genera is reduced, in some cases, compared with the relative abundance of CK (no powder applied) and T1(170 ten thousand/g), and the specific change is shown in the following table 1 (the genera with the absolute change larger than 0.4% are shown). Wherein, the relative abundance of the bacillus, the Germinatum and the Bacillus is increased more, and the bacteria are all beneficial bacteria which can promote the growth of plants.

TABLE 1 Change in the soil microbial community following LY153-1 administration (genus)

Note: indicates that the DNA fragment was annotated, but no related genus was identified.

Removing unknown species (other), as shown in fig. 2, it was found that as the application amount of Bacillus megaterium LY153-1 microbial inoculum increases, the colonization in soil also increases, the occupation ratio in CK, T1, T2 is 0.27%, 0.73%, 1.83% respectively, and the red note Bacillus megaterium-NBRC-15308 ═ ATCC-14581 in the figure proves that the successful colonization in soil of Bacillus megaterium LY153-1 strain can be detected by the diversity detection method and the Bacillus _ unclassified, a bacterium of another Bacillus, is affected.

Example 6 identification of Bacillus megaterium LY153-1

Example 6 morphological characteristics, physiological and biochemical characteristics and molecular biological identification were carried out on Bacillus megaterium LY 153-1.

(1) Morphological characteristics:

gram staining is positive, and the thallus is rod-shaped and has round ends. On TSA medium, the colonies were nearly round, smooth in surface, clean in edges, yellowish and opaque, and shiny. As shown in fig. 3.

(2) Physiological and biochemical characteristics:

the fatty acid composition of the strain 153-1 is detected by a microorganism fatty acid rapid identification system (MIDI), and the main fatty acids of the strain to be detected are C15:0 anteiso, C15:0 iso, C14:0 iso and C16:1 w11C, wherein the contents of the main fatty acids are 42.73%, 27.26%, 7.90% and 4.46%, respectively. Conforms to the main cell fatty acid characteristics of Bacillus (Bacillus).

API 50CH assay results: the positive reactions comprise L-arabinose, L-xylose, glucose, fructose, mannitol, N-acetyl-glucosamine, esculetin, lactose, sucrose and trehalose; the weak positive reaction comprises glycerol, galactose, mannose, inositol, saligenin, maltose, starch, pangolin and D-turanose; negative reactions include control, erythrose, D-arabinose, ribose, D-xylose, adonitol, beta-methyl-D-xyloside, sorbose, rhamnose, dulcitol, sorbitol, alpha-methyl-D-mannoside, alpha-methyl-D-glucoside, amygdalin, arbutin, cellobiose, melibiose, inulin, sancose, raffinose, starch, glycogen, xylitol, D-lyxose, D-tagatose, D-fucose, L-fucose, D-arabitol, L-arabitol, gluconate, 2-keto-gluconate and 5-keto-gluconate. Meets the biochemical metabolism characteristics of Bacillus.

(3) Molecular biological characteristics:

1) 16S rDNA gene sequence (1454bp) of strain 153-1 and phylogenetic analysis

GCCTGGCGGCGTGCCTATACATGCAAGTCGAGCGAACTGATTAGAAGCTTGCTTCTATGACGTTAGCGGCGGACGGGTGAGTAACACGTGGGCAACCTGCCTGTAAGACTGGGATAACTTCGGGAAACCGAAGCTAATACCGGATAGGATCTTCTCCTTCATGGGAGATGATTGAAAGATGGTTTCGGCTATCACTTACAGATGGGCCCGCGGTGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCAACGATGCATAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGCTTTCGGGTCGTAAAACTCTGTTGTTAGGGAAGAACAAGTACGAGAGTAACTGCTCGTACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGAATTATTGGGCGTAAAGCGCGCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCACGGCTCAACCGTGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGAAAAGCGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGGCTTTTTGGTCTGTAACTGACGCTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGAGGGTTTCCGCCCTTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAACTCTAGAGATAGAGCGTTCCCCTTCGGGGGACAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTTAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGATGGTACAAAGGGCTGCAAGACCGCGAGGTCAAGCCAATCCCATAAAACCATTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGGAGTAACCGTAAGGAGCTAGCCGCATAAGGGGACAGA(SEQ ID NO:1)

The results of the systematic analysis of 16S rDNA are shown in FIG. 4.

2) rpoB gene sequence (385bp) of 153-1 strain and phylogenetic analysis

TGATCGAAACGGCTGAAGGTCCAAACATAGGTGAAGATGCGCTTCGCAACTTAGATGAGCGTGGAATCATCCGCATTGGTGCAGAAGTAAAAGACGGAGATCTTTTAGTTGGTAAAGTAACGCCAAAAGGTGTAACAGAACTAACAGCTGAAGAACGTCTTCTACACGCTATTTTCGGTGAAAAAGCGCGTGAAGTTCGTGATACTTCTCTTCGTGTACCGCACGGCGGCGGTGGAATCATTCTTGATGTTAAAGTCTTCAACCGTGAAGATGGGGACGAATTACCACCAGGTGTAAACCAATTAGTCCGTGTATATATTGTTCAGAAGCGTAAAATTTCTGAAGGTGACAAAATGGCCGGTCGTCACGGTAACAAGGGTGTAAA(SEQ ID NO:2)

The phylogenetic analysis results of rpoB gene are shown in FIG. 5.

The strain is identified as Bacillus megaterium through strain morphological characteristics, physiological and biochemical characteristics and molecular biological characteristic analysis. The strain is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.21828 and the preservation time of 2021, 02 and 25 days.

Example 7 Complex microbial Agents and methods of preparation thereof

Embodiment 7 provides a complex microbial inoculum, which is specifically prepared by the following method:

(1) bacillus megaterium LY153-1 stored in the plate was inoculated with an inoculating loop into a triangular flask (250mL) containing 100mL of LB medium, cultured at 37 ℃ and 210rpm in an incubator for 6 hours.

Meanwhile, purifying, inoculating and culturing the industrialized strains No. 5 (Bacillus subtilis), No. 8 (Bacillus subtilis), No. 31 (Bacillus licheniformis), No. 149-1 (Bacillus belgii) (5#, 8#, 31#, 149-1# are all functional strains which are independently screened by the Yiyi research and development center of chemical fertilizer Limited company, two strains are respectively selected to be compounded with the Bacillus megaterium LY153-1 in a compounding ratio of 1:1:1, and the cucumber is potted and the growth promoting effect of the field test is verified.

(2) Inoculating LY153-1 zymocyte liquid into an amplification fermentation tank (10L-50L-500L), culturing at 37 ℃, and fermenting step by step. Spray drying after fermentation, and subpackaging, wherein the viable count is 1000 hundred million/g.

Wherein the formula of the amplification fermentation medium comprises the following components in percentage by mass: 3% of soybean meal, 2% of corn flour, 0.5% of starch, 0.50% of glucose, 0.10% of sodium chloride, 0.3% of dipotassium hydrogen phosphate, 0.02% of manganese sulfate, 0.3% of defoaming agent and 7.0% of pH value.

The rest strains contained in the composite bacterial agent are prepared according to the preparation process of LY153-1 bacterial agent, and all the bacterial agents are mixed according to the proportion of 1:1:1, the theoretical addition amount of the live bacteria in the composite bacterial fertilizer is 1 hundred million/g, and the shelf life of the effective live bacteria number of 6 months is tracked.

The results of the compound microbial inoculum cucumber pot culture test data are shown in table 1, the obtained optimal compound combination is 31# +149-1+153-1 (referred to as compound bacterial fertilizer S2 for short), the compound proportion is 1:1:1, and the cucumber root length is increased by 35.4% compared with CK according to the cucumber root length data. The results of the cucumber field test data are shown in table 2, and the optimal compound fertilizer S2 is obtained, wherein the compound ratio is 1:1:1. According to the cucumber yield data, the cucumber yield per mu is 1.38 ten thousand kg, and is increased by 35.4 percent compared with CK. The shelf life tracking data of the effective viable count of the composite bacterial manure for 6 months shows that as shown in table 3, the retention rate of the effective viable count of the composite bacterial manure S26 months is the highest and is 83%, and compared with the retention rate of a control group, the retention rate is 48%, and is increased by 35%.

Table 1 root length data results of complex microbial inoculum cucumber pot culture test

Table 2 Compound fungicide cucumber field test data results

TABLE 3 shelf life tracking data of effective viable count of complex microbial inoculum

Treatment of	0d	15d	30d	60d	120d	180d
							Control group (Yi/g)	1	0.87	0.75	0.64	0.53	0.48
Test set S1 (Yi/g)	1	0.92	0.82	0.77	0.75	0.73
							Test set S2 (Yi/g)	1	0.96	0.92	0.87	0.86	0.83
Test set S3 (Yi/g)	1	0.94	0.87	0.82	0.76	0.75

Example 8 microbial Fertilizer and applications

Firstly, the microbial fertilizer is prepared according to the following method, which specifically comprises the following steps:

the compound microbial agents S1, S2 and S3 prepared in the example 7 are respectively added according to 2 per mill after being uniformly mixed with anti-caking powder in the coating working section of organic and inorganic fertilizers and compound fertilizers, and the corresponding fertilizers obtained after stable production are microbial fertilizers, wherein the effective viable count is not less than 0.2 hundred million/g. The basic fertilizers used for preparing the microbial fertilizer are respectively organic and inorganic fertilizers 15-40/S (15-8-17) and compound fertilizers 15-15-15/S.

The prepared microbial fertilizer is subjected to field effect verification, and experiments show that the planting of potatoes is carried out in the Xilinguo union positive blue flag potato planting base in the inner Mongolia autonomous region, and the planting of sweet potatoes is carried out in Kaifeng city Tong county in Henan province.

Carrying out potato development in the Xinling Guo Lui union positive blue flag potato planting base of the inner Mongolia autonomous region, and arranging a control group 1, a control group 2 and test groups S1-1, S1-2, S2-1, S2-2, S3-1, S3-2 and 6 treatment groups, wherein each treatment group comprises 3 cells, and each cell is 50m²The fertilizer application amount is 50 Kg/mu. The control group 1 is applied by conventional fertilization, the control group 2 is applied by a microbial fertilizer purchased in the market, and the test groups S1-1, S2-1 and S3-1 are applied by the microbial fertilizer. The specific effects are shown in Table 4.

TABLE 4 Potato test effects of the treatments

As can be seen from table 4, it is,

the test result shows that the test group has the best S2 treatment compared with the control group, the yield of the biological organic and inorganic fertilizers used by the S2-1 is respectively increased by 15.6 percent and 5.7 percent compared with the control group 1 and the control group 2, and the test result shows that the yield of the compound fertilizer used by the test group S2-2 is respectively increased by 14.7 percent and 4.9 percent compared with the control group 1 and the control group 2.

The sweet potato is carried out in Kangcity of Henan province, and comprises 5 treatment groups including control group 1, control group 2, test group S1, S2, and S3, wherein each treatment group comprises 3 cells, each cell has a size of 60m²The control group 1 is a conventional organic and inorganic fertilizer, the control group 2 is a conventional compound fertilizer, the test groups S1, S2 and S3 are organic and inorganic fertilizers 15-40/S (15-8-17), and the application amount of the fertilizer is 40 Kg/mu. The specific effects are shown in Table 5.

TABLE 5 sweet Potato test treatment Effect

As can be seen from the results shown in Table 5, the results of the tests showed that the biological organic and inorganic fertilizers 15-40/S (15-8-17) used in the test group S2 were the most effective than the control group in the test groups S1, S2 and S3, and the yields were increased by 6.93% and 4.2% respectively in comparison with the control group 1 and the control group 2.

In conclusion, the microbial fertilizer containing the bacillus megaterium 153-1 with the high phosphate solubilizing effect has the obvious effects of increasing both the yield and the income of potatoes and sweet potatoes, and the microbial fertilizer has the functions of promoting the growth of crops and improving the yield of the crops, and particularly has more remarkable effect of combining with organic and inorganic fertilizers.

In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "an implementation" or the like, mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.

Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.

SEQUENCE LISTING

<110> research and development center of Zhonghua agriculture (Linyi)

<120> bacillus megaterium and application thereof in soil phosphate solubilizing

<130> BI3211474

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 1454

<212> DNA

<213> Bacillus megaterium

<400> 1

gcctggcggc gtgcctatac atgcaagtcg agcgaactga ttagaagctt gcttctatga 60

cgttagcggc ggacgggtga gtaacacgtg ggcaacctgc ctgtaagact gggataactt 120

cgggaaaccg aagctaatac cggataggat cttctccttc atgggagatg attgaaagat 180

ggtttcggct atcacttaca gatgggcccg cggtgcatta gctagttggt gaggtaacgg 240

ctcaccaagg caacgatgca tagccgacct gagagggtga tcggccacac tgggactgag 300

acacggccca gactcctacg ggaggcagca gtagggaatc ttccgcaatg gacgaaagtc 360

tgacggagca acgccgcgtg agtgatgaag gctttcgggt cgtaaaactc tgttgttagg 420

gaagaacaag tacgagagta actgctcgta ccttgacggt acctaaccag aaagccacgg 480

ctaactacgt gccagcagcc gcggtaatac gtaggtggca agcgttatcc ggaattattg 540

ggcgtaaagc gcgcgcaggc ggtttcttaa gtctgatgtg aaagcccacg gctcaaccgt 600

ggagggtcat tggaaactgg ggaacttgag tgcagaagag aaaagcggaa ttccacgtgt 660

agcggtgaaa tgcgtagaga tgtggaggaa caccagtggc gaaggcggct ttttggtctg 720

taactgacgc tgaggcgcga aagcgtgggg agcaaacagg attagatacc ctggtagtcc 780

acgccgtaaa cgatgagtgc taagtgttag agggtttccg ccctttagtg ctgcagctaa 840

cgcattaagc actccgcctg gggagtacgg tcgcaagact gaaactcaaa ggaattgacg 900

ggggcccgca caagcggtgg agcatgtggt ttaattcgaa gcaacgcgaa gaaccttacc 960

aggtcttgac atcctctgac aactctagag atagagcgtt ccccttcggg ggacagagtg 1020

acaggtggtg catggttgtc gtcagctcgt gtcgtgagat gttgggttaa gtcccgcaac 1080

gagcgcaacc cttgatctta gttgccagca tttagttggg cactctaagg tgactgccgg 1140

tgacaaaccg gaggaaggtg gggatgacgt caaatcatca tgccccttat gacctgggct 1200

acacacgtgc tacaatggat ggtacaaagg gctgcaagac cgcgaggtca agccaatccc 1260

ataaaaccat tctcagttcg gattgtaggc tgcaactcgc ctacatgaag ctggaatcgc 1320

tagtaatcgc ggatcagcat gccgcggtga atacgttccc gggccttgta cacaccgccc 1380

gtcacaccac gagagtttgt aacacccgaa gtcggtggag taaccgtaag gagctagccg 1440

cataagggga caga 1454

<210> 2

<211> 385

<212> DNA

<213> Bacillus megaterium

<400> 2

tgatcgaaac ggctgaaggt ccaaacatag gtgaagatgc gcttcgcaac ttagatgagc 60

gtggaatcat ccgcattggt gcagaagtaa aagacggaga tcttttagtt ggtaaagtaa 120

cgccaaaagg tgtaacagaa ctaacagctg aagaacgtct tctacacgct attttcggtg 180

aaaaagcgcg tgaagttcgt gatacttctc ttcgtgtacc gcacggcggc ggtggaatca 240

ttcttgatgt taaagtcttc aaccgtgaag atggggacga attaccacca ggtgtaaacc 300

aattagtccg tgtatatatt gttcagaagc gtaaaatttc tgaaggtgac aaaatggccg 360

gtcgtcacgg taacaagggt gtaaa 385

Claims

1. A kind of giant bacillus (B), (B)Bacillus megaterium) LY153-1, which is preserved in China general microbiological culture Collection center with the preservation number of CGMCC No. 21828.

2. A microbial agent, comprising the bacillus megaterium of claim 1.

3. The microbial agent according to claim 2, wherein the microbial agent is in a dry powder form, and the effective viable count of the bacillus megaterium contained in the microbial agent is at least 1.0 x 10¹⁰CFU/g。

4. The method for preparing the microbial preparation according to claim 2 or 3, comprising:

carrying out fermentation culture on the bacillus megaterium so as to obtain a fermentation product;

and carrying out spray drying and crushing treatment on the basis of the fermentation product so as to obtain the microbial agent.

5. The method for producing a microbial agent according to claim 4, wherein the fermentation culture comprises:

carrying out activated fermentation culture on the bacillus megaterium so as to obtain a zymogen liquid;

and carrying out amplification fermentation culture on the zymophyte liquid so as to obtain a fermentation product.

6. The method according to claim 5, wherein the medium for the scale-up fermentation culture comprises soybean meal, corn meal, starch, glucose, sodium chloride, dipotassium hydrogen phosphate, manganese sulfate, and an antifoaming agent.

7. The method according to claim 6, wherein the medium for the amplified fermentation culture comprises, in parts by weight:

1 to 5 parts by weight of soybean meal,

1-5 parts by weight of corn flour,

0.1 to 1 part by weight of starch,

0.1 to 1 part by weight of glucose,

0.02 to 0.3 parts by weight of sodium chloride,

0.1 to 0.8 part by weight of dipotassium hydrogenphosphate,

0.01 to 0.2 parts by weight of manganese sulfate, and

0.1 to 1 part by weight of a defoaming agent.

8. A complex microbial inoculant, which comprises a first microbial inoculant and a second microbial inoculant, wherein the first microbial inoculant is the microbial inoculant in claim 2 or 3, and the second microbial inoculant comprises at least one of bacillus belgii, bacillus licheniformis and bacillus subtilis.

9. The complex microbial inoculant according to claim 8, wherein the complex microbial inoculant comprises 30-35 parts by weight of the first microbial inoculant, and at least one of the following components:

30-35 parts by weight of Bacillus belgii;

30-35 parts by weight of bacillus licheniformis;

30-35 parts by weight of bacillus subtilis.

10. The complex microbial inoculant according to claim 8, wherein effective live bacillus megaterium bacteria are contained in the complex microbial inoculantA number of at least 1.0 x 10¹⁰CFU/g, and comprising at least one selected from the group consisting of:

the effective viable count of Bacillus belgii is at least 1.0 × 10¹¹CFU/g；

The effective viable count of Bacillus licheniformis is at least 1.0 × 10¹¹CFU/g。

11. A microbial fertilizer, comprising the bacillus megaterium of claim 1, or the microbial agent of claim 2 or 3, or the complex microbial agent of any one of claims 8 to 10.

12. A microbial fertilizer according to claim 11, comprising effective viable microbial count of at least 0.2 hundred million CFU/g.

13. The microbial fertilizer according to claim 11, wherein said microbial fertilizer contains two per thousand of said bacillus megaterium, two per thousand of said microbial agent, or two per thousand of said complex microbial agent.

14. A microbial fertilizer according to claim 11, further comprising a base fertilizer selected from at least one of a compound fertilizer and an organic-inorganic fertilizer.

15. Use of the bacillus megaterium of claim 1 in the preparation of a microbial agent, a complex microbial agent or a microbial fertilizer.

16. A method of solubilizing phosphorus in soil, comprising:

applying an effective amount of bacillus megaterium, a microbial agent, a complex microbial agent or a microbial fertilizer to soil, wherein the bacillus megaterium is the bacillus megaterium of claim 1, the microbial agent is the microbial agent of claim 2 or 3, the complex microbial agent is the complex microbial agent of any one of claims 8 to 10, and the microbial fertilizer is the microbial fertilizer of any one of claims 11 to 14.

17. A method of fertilizing a crop, comprising:

applying an effective amount of bacillus megaterium, a microbial agent, a complex microbial agent or a microbial fertilizer to the crop, wherein the bacillus megaterium is the bacillus megaterium of claim 1, the microbial agent is the microbial agent of claim 2 or 3, the complex microbial agent is the complex microbial agent of any one of claims 8 to 10, and the microbial fertilizer is the microbial fertilizer of any one of claims 11 to 14.

18. The method of claim 17, wherein the crop is selected from at least one of potatoes and sweet potatoes.