CN113980835A

CN113980835A - Compound microbial agent and application thereof in fertilizer

Info

Publication number: CN113980835A
Application number: CN202111115788.7A
Authority: CN
Inventors: 战帅; 王子浩; 张帅; 李洪顺; 刘巍
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: 2022-01-28
Anticipated expiration: 2041-09-23
Also published as: CN113980835B

Abstract

The invention relates to the technical field of microorganisms, in particular to a compound microbial agent and application thereof in fertilizers. The compound microbial agent comprises at least two of Bacillus megaterium, Bacillus bleezensis and Bacillus subtilis subsp. The provided compound microbial agent can be used for preparing fertilizers, can promote the growth of crops, has the growth promoting effect on various crops such as peanuts, garlic, cucumbers, eggplants, hot peppers and oranges, and obviously improves the yield of the crops.

Description

Compound microbial agent and application thereof in fertilizer

Technical Field

The invention belongs to the technical field of agricultural microorganisms, and relates to a compound microbial agent and application thereof in fertilizers.

Background

In recent decades, in order to meet the demand of crop production in China, the application amount of fertilizers in China is continuously increased, the fertilizer industry is rapidly developed, the consumption amount (purity) of the fertilizers in China in 2019 reaches 5100 ten thousand tons, and the unit fertilizing amount reaches 21.9 kg/mu, which is 2.5 times of European Union and 2.6 times of that in the United states. The environmental problems caused by excessive fertilization are increasingly serious, inorganic fertilizers are emphasized, the phenomenon of biological organic fertilizers is slightly considered, the inorganic fertilizers are commonly generated all over the country, and the soil problems are increasingly serious. In order to respond to the two reduction policies of weight reduction and drug reduction of China, the biological ecological fertilizer is actively popularized and used, the application amount of chemical fertilizer is reduced, the purposes of weight reduction and yield increase are realized, and the sustainable development of agriculture is promoted.

The microbial inoculum has the advantages of environmental protection, no pollution, no residue in use, soil ecological environment improvement, soil-borne disease inhibition, soil nutrient activity improvement and the like, and is widely applied to agriculture in recent years. With the development of the technology, the application of the composite microbial inoculum gradually replaces the application of a single microbial inoculum, and people find that the complex formulation of microbial agents with different functions can compound the functions of different microbial agents, mutually promote and enhance the growth promotion effect, improve the adaptability of the microbial agents in different environments and improve the planting effect of microorganisms.

Meanwhile, the compound microbial agent is combined with the compound fertilizer to prepare the biological fertilizer, so that the application method of the microbial agent can be simplified, the effect of the fertilizer is improved, the yield of crops is improved, and the aims of fertilizer synergism and crop yield increase are finally fulfilled.

However, further improvements are still needed with respect to complex microbial agents.

Disclosure of Invention

The present invention is directed to solving, at least to some extent, one of the technical problems in the related art. The invention provides a compound microbial agent which can be applied to fertilizers to improve the fertilizer effect, promote the growth of crops and inhibit diseases. Also provides a preparation method of the compound microbial agent.

The first aspect of the present invention provides a complex microbial inoculant, comprising: at least two or three of bacillus megaterium, bacillus belgii and bacillus subtilis; the Bacillus megaterium is preserved in the China general microbiological culture Collection center with the preservation number of CGMCC No. 21828; the Bacillus velezensis is preserved in the China general microbiological culture Collection center with the preservation number of CGMCC No. 21827; the Bacillus subtilis subsp. subtilis is preserved in the China general microbiological culture Collection center of the culture Collection of microorganisms with the preservation number of CGMCC No. 21826.

In a second aspect of the present invention, there is provided a method for preparing a complex microbial inoculant, comprising: respectively carrying out fermentation treatment on the strains, and obtaining microbial agents based on products of the fermentation treatment; and compounding the microbial agent so as to obtain the compound microbial agent.

In a third aspect of the present invention there is provided a fertilizer comprising a complex microbial inoculant according to the first aspect of the present invention.

In a fourth aspect of the present invention there is provided the use of a complex microbial inoculant in the preparation of a fertilizer for use as a fertiliser for crops, said complex microbial inoculant being a complex microbial inoculant according to the first aspect of the present invention.

A fifth aspect of the present invention provides a method for fertilizing a crop, comprising: and (2) applying a compound microbial agent or a fertilizer to the crops, wherein the compound microbial agent is the compound microbial agent of the first aspect of the invention, and the fertilizer is the fertilizer of the third aspect of the invention.

The beneficial effects obtained by the invention are as follows: the microbial agent provided by the invention can be used as a fertilizer, can play a role in promoting growth, can obviously increase the yield of crops, and particularly can increase the yield of peanuts, garlic, cucumbers, eggplants, hot peppers, oranges and the like.

Information on strain preservation

The Bacillus megaterium has the preservation number of CGMCC No.21828, the preservation unit is China general microbiological culture Collection center, the preservation address is the microbiological research institute of China academy of sciences No. 3, West Lu No. 1 Hospital, North Kogyo, Beijing, and the preservation date is 2021 year, 02 month and 25 days.

The preservation number of the Bacillus velezensis is CGMCC No.21827, the preservation unit is the China general microbiological culture Collection center of China Committee for culture Collection, the preservation address is the microbiological research institute of China academy of sciences No. 3 of North Chen West Lu No. 1 of Yangxi of Beijing, and the preservation date is 2021 year, 02 month and 25 days.

Bacillus subtilis subsp. subtilis with the preservation number of CGMCC No.21826, the preservation unit is the common microorganism center of China Committee for culture Collection of microorganisms, the preservation address is the institute of microbiology of China academy of sciences, No. 3 of West Lu 1 of the North Chen West of the Korean area, Beijing, and the preservation date is 2021 year, 02 month and 25 days.

Drawings

FIG. 1 is a graph showing the results of evaluation of the indoor phosphate solubilizing ability of Bacillus megaterium provided in example 1 of the present invention.

FIG. 2 is a graph showing the results of culturing Bacillus megaterium in TSA medium according to an embodiment of the present invention.

FIG. 3 is a graph showing the results of systematic analysis of 16S rDNA of Bacillus megaterium provided in example 1 according to the present invention.

FIG. 4 is a graph showing the results of phylogenetic analysis of rpoB gene of Bacillus megaterium provided in example 1 according to the present invention.

FIG. 5 is a graph showing the results of the morphology of Bacillus belgii on TSA medium, provided in example 1 according to the present invention.

FIG. 6 is a graph showing the results of the evolutionary tree analysis of 16S rDNA of Bacillus belgii provided in example 1 according to the present invention.

FIG. 7 is a graph showing the results of a phylogenetic tree analysis of the gyrB gene of Bacillus belgii according to example 1 of the present invention.

FIG. 8 is a graph showing morphological results of Bacillus subtilis provided in example 1 according to the present invention.

FIG. 9 is a graph showing the results of a tree analysis of the 16S rDNA of Bacillus subtilis according to example 1 of the present invention.

FIG. 10 is a graph showing the results of a phylogenetic tree analysis of the gyrB gene of Bacillus subtilis, which is provided in example 1 according to 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 compound microbial agent, which comprises: two or three of bacillus megaterium, bacillus belgii and bacillus subtilis; the Bacillus megaterium is preserved in the China general microbiological culture Collection center with the preservation number of CGMCC No. 21828; the Bacillus velezensis is preserved in the China general microbiological culture Collection center with the preservation number of CGMCC No. 21827; the Bacillus subtilis subsp. subtilis is preserved in the China general microbiological culture Collection center of the culture Collection of microorganisms with the preservation number of CGMCC No. 21826. Each provided strain is isolated and stored in the company laboratory. The provided compound microbial agent can promote the growth of crops, such as various crops of peanuts, garlic, cucumbers, eggplants, hot peppers and oranges, has obvious effect, and can be produced and applied. The provided compound microbial agent can be combined with organic and inorganic fertilizers and compound fertilizers to prepare biological fertilizers, and compared with treatment without adding microbial agents, the yield increase effect is 4.27% -13.42%. The Bacillus subtilis referred to is identified as belonging to the Bacillus subtilis subsp.

In at least some embodiments, the complex microbial inoculant is in the form of a dry powder comprising between 500 and 800 million CFU of the Bacillus megaterium per gram of the complex microbial inoculant, between 1000 and 1500 million CFU of the Bacillus belgii per gram of the complex microbial inoculant, and between 1000 and 1500 million CFU of the Bacillus subtilis subspecies subtilis. Reference to a CFU is generally understood in the art as a colony forming unit.

In at least some embodiments, the complex microbial agent comprises: 30-35 parts of bacillus megaterium, 25-35 parts of bacillus belgii and 30-40 parts of bacillus subtilis subspecies subtilis.

The invention also provides a preparation method of the compound microbial agent, which comprises the following steps: respectively carrying out fermentation treatment on the strains, and obtaining microbial agents based on products of the fermentation treatment; and compounding the microbial agent so as to obtain the compound microbial agent.

In some embodiments, the fermentation process comprises: performing activated fermentation culture on the strain to obtain liquid zymogen liquid; and carrying out amplification fermentation culture on the liquid zymophyte liquid so as to obtain a product of the fermentation treatment.

In the case of activated fermentation culture, LB medium may be used. Activated cultureThe temperature of (1) is 37 ℃, the culture solution is cultured in a triangular flask under the conditions of 150-250rpm for 4-8 hours, for example, 6 hours. In a specific embodiment, Bacillus megaterium, Bacillus belgii and Bacillus subtilis stored in a plate can be respectively inoculated into a triangular flask (250mL) containing 100mL of LB culture solution by using inoculating loops, cultured for 6h at 37 ℃ and 250rpm in a constant temperature incubator until logarithmic growth phase and OD₆₀₀The value is 0.5-0.8, the concentration of the seed liquid is not lower than 1 hundred million/mL, and the bacillus megaterium seed liquid, the bacillus belgii seed liquid and the bacillus subtilis seed liquid which are inoculated into a fermentation culture medium are respectively obtained.

The culture medium for the bacillus megaterium amplification fermentation culture comprises soybean meal, corn flour, starch, glucose, sodium chloride, dipotassium hydrogen phosphate, manganese sulfate and a defoaming agent. In some embodiments, the medium for the amplified fermentation culture of bacillus megaterium comprises, in parts by weight: 1-5 parts of soybean meal, 1-5 parts of corn flour, 0.1-1 part of starch, 0.1-1 part of glucose, 0.02-0.3 part of sodium chloride, 0.1-0.8 part of dipotassium hydrogen phosphate, 0.01-0.2 part of manganese sulfate and 0.1-1 part of defoaming agent. In a specific embodiment, the culture medium for the bacillus megaterium amplified fermentation culture comprises the following components in percentage by weight: 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 the balance of water, and the pH value is adjusted to 7.0. The components can be sterilized under 0.1MPa and fermentation tank at 121 deg.C for 60min, and cooled under reduced pressure to normal pressure and temperature for use.

The culture medium for the amplification fermentation culture of the bacillus beleisi comprises corn starch, sucrose, soybean meal, yeast powder, peptone, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, calcium carbonate, sodium chloride and an antifoaming agent. In some embodiments, the medium for the amplified fermentation culture of bacillus belgii comprises, in parts by weight: 2.5-3 parts of corn starch, 0.5-1 part of cane sugar, 3-4.5 parts of soybean meal, 0.15-0.20 part of yeast powder, 0.15-0.20 part of peptone, 0.2-0.3 part of dipotassium hydrogen phosphate, 0.2-0.3 part of potassium dihydrogen phosphate, 0.1-0.15 part of calcium carbonate, 0.1-0.15 part of sodium chloride and 0.2-0.3 part of defoaming agent. In a specific embodiment, the medium for the amplified fermentation culture of bacillus belgii comprises, in weight percent: 2.5 to 3 percent of corn starch, 0.5 to 1 percent of cane sugar, 3 to 4.5 percent of soybean meal, 0.15 to 0.20 percent of yeast powder, 0.15 to 0.20 percent of peptone, 0.2 to 0.3 percent of dipotassium phosphate, 0.2 to 0.3 percent of monopotassium phosphate, 0.1 to 0.15 percent of calcium carbonate, 0.1 to 0.15 percent of sodium chloride, 0.2 to 0.3 percent of defoaming agent and the balance of water, and the pH is adjusted to 7.0. Mixing the above components, sterilizing at 0.1MPa and 121 deg.C for 60min, and cooling under reduced pressure to normal pressure and temperature.

The culture medium for the bacillus subtilis subspecies subtilis amplification fermentation culture comprises corn starch, cane sugar, bean pulp, yeast powder, peptone, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, calcium carbonate, sodium chloride and an antifoaming agent. In some embodiments, the medium for the bacillus subtilis subspecies-subtilis amplified fermentation culture comprises, in parts by weight: 3.0-3.5 parts by weight of corn starch, 0.8-1.0 part by weight of sucrose, 4.0-4.5 parts by weight of soybean meal, 0.20-0.50 part by weight of yeast powder, 0.20-0.50 part by weight of peptone, 0.2-0.3 part by weight of dipotassium hydrogen phosphate, 0.2-0.3 part by weight of potassium dihydrogen phosphate, 0.1-0.15 part by weight of calcium carbonate, 0.1-0.15 part by weight of sodium chloride, and 0.3-0.5 part by weight of antifoaming agent. In a specific embodiment, the culture medium for the bacillus subtilis subspecies subtilis amplified fermentation culture comprises the following components in percentage by weight: 3.0-3.5% of corn starch, 0.8-1.0% of sucrose, 4.0-4.5% of soybean meal, 0.20-0.50% of yeast powder, 0.20-0.50% of peptone, 0.2-0.3% of dipotassium hydrogen phosphate, 0.2-0.3% of potassium dihydrogen phosphate, 0.1-0.15% of calcium carbonate, 0.1-0.15% of sodium chloride, 0.3-0.5% of defoaming agent and the balance of water, and the pH is adjusted to 7.0. Mixing the above components, sterilizing at 121 deg.C under 0.1MPa for 60min, and cooling under reduced pressure to normal pressure and temperature.

In the case of the scale-up fermentation culture, the fermenter used may be a pilot plant fermenter, for example, a 10L, 50L or 500L pilot plant fermenter, and the stepwise fermentation may be carried out using these fermenters. The prepared bacillus megaterium seed liquid, the prepared bacillus belief-lsi seed liquid and the prepared bacillus subtilis seed liquid are respectively inoculated into a pilot-scale fermentation tank (10L-50L-500L), the initial concentration of thalli in each fermentation culture system is not lower than 100 ten thousand per ml, the tank pressure is kept at 0.05-0.10 KPa under the condition of 35-37 ℃, the dissolved oxygen content is kept at 20-25%, the rotating speed is 180r/min, the pH value is 7.0-8.0, and the culture is carried out for 36-48 h. After fermentation, fermentation liquor of bacillus megaterium, bacillus belgii and bacillus subtilis is obtained respectively, the sporulation rate reaches over 90 percent, and the viable count of the fermentation liquor reaches not less than 50 hundred million/mL, 300 hundred million/mL and 300 hundred million/mL respectively.

Adding 3-5% of diatomite into the obtained fermentation liquor in proportion, drying and subpackaging by using a spray drying device to obtain bacillus megaterium, bacillus belief and bacillus subtilis microbial agent preparations used for adding in the fertilizer, wherein the viable count of the microbial agent preparations respectively reaches the mass ratio of bacillus megaterium: 500-800 hundred million CFU/g, 1000-1500 hundred million CFU/g of Bacillus belgii and 1000-1500 hundred million CFU/g of Bacillus subtilis.

Then compounding the prepared different microbial agents according to different proportions to obtain the compound microbial agent. In some embodiments, 30-35 parts by weight of bacillus megaterium powder, 25-35 parts by weight of bacillus beiLeisi powder and 30-40 parts by weight of bacillus subtilis powder are fully mixed to prepare the compound microbial agent, and the number of viable bacteria reaches 800-1200 hundred million/g.

The invention also provides a fertilizer, which comprises the compound microbial agent. The fertilizer further comprises a base fertilizer, and the base fertilizer comprises at least one of compound fertilizer and organic and inorganic fertilizer. The content of the compound microbial agent in the fertilizer can be added according to needs, for example, the proportion of the compound microbial agent in the fertilizer can be one thousandth to five thousandth. The provided fertilizer can be obtained by the following method: the prepared compound microbial agent and the anti-caking agent are mixed uniformly, the mixed powder is added into a fertilizer roller in a coating working section of organic and inorganic fertilizer and compound fertilizer production through a chain dragon feeder and is coated on the surfaces of fertilizer particles, and the corresponding fertilizer is prepared after stable production, namely the bio-fertilizer, wherein the effective viable count is more than or equal to 0.2 hundred million/g.

For example, the fertilizer applied to peanut planting can play a role in promoting growth, and specifically, compared with a treatment group without adding the compound microbial agent, the fertilizer with the compound microbial agent increases the yield by more than 10%, for example, by 13% in a field test.

For another example, the provided fertilizer can play a role in promoting growth when applied to garlic planting, and specifically can be shown in that the fertilizer added with the compound microbial agent increases the yield by more than 5%, such as by about 7%, in a field test compared with a treatment group without the compound microbial agent. For another example, when the provided fertilizer is applied to cucumber planting, the growth promotion effect can be achieved, and specifically, the effect can be shown in that in a field test, compared with a treatment group without adding the compound microbial agent, the yield of the fertilizer with the compound microbial agent is increased by more than 10%, for example, by about 13%. For another example, when the provided fertilizer is applied to eggplant planting, the fertilizer can play a role in promoting growth, and specifically, the fertilizer added with the compound microbial agent can increase the yield by more than 10%, such as by about 11%, in a field test compared with a treatment group without the compound microbial agent. For another example, when the provided fertilizer is applied to pepper planting, the growth promoting effect can be achieved, and specifically, in a field test, compared with a fertilizer without the compound microbial agent, the fertilizer with the compound microbial agent is increased in yield by more than 3%, for example, by about 4%.

For another example, the provided fertilizer can play a role in promoting growth when applied to citrus (tribute citrus) planting, and specifically can be shown in that in a field test, compared with a fertilizer without the compound microbial agent, the fertilizer with the compound microbial agent increases the yield by more than 10%, for example, by about 12%. For another example, the provided fertilizer can play a role in promoting growth when applied to planting of citrus (crystal sugar orange), and specifically can be shown in a field test that the fertilizer added with the compound microbial agent increases the yield by more than 8%, for example, by about 9%, compared with a fertilizer without the compound microbial agent.

The invention also provides application of the compound microbial agent in preparation of fertilizer, wherein the fertilizer is used for crop fertilization, and the compound microbial agent is the compound microbial agent. The invention also provides a crop fertilizing method, which comprises the following steps: and (2) applying a compound microbial agent or a fertilizer to crops, wherein the compound microbial agent is the compound microbial agent, and the fertilizer is the fertilizer. The mentioned crops can be grain crops and also can be melon and fruit crops.

The scheme of the invention will be explained with reference to the 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

Example 1 Bacillus megaterium, Bacillus belgii and Bacillus subtilis subspecies, respectively, were obtained.

(I) Bacillus megaterium

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%⁴Selecting times ofAnd 3 gradients are coated, each gradient is 3 in parallel, after the culture is carried out in an incubator at 30 ℃ for 2d, strains with different colony morphologies are selected and streaked on an LB culture medium, and the colony growth condition is observed regularly. Then, the strain was purified by plate streaking, and the strain was designated by LY153-1 and stored.

The phosphate solubilizing effect of Bacillus megaterium LY153-1 was determined as follows:

(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 NY/T1847-2010 microbial fertilizer, inoculating phosphate solubilizing bacteria, culturing in a shake flask, sampling after a certain time to determine the content of soluble phosphorus, and comparing the increase of the soluble phosphorus in the liquid culture medium before 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，FeSO₄·7H₂O 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 153-1 into LB liquid culture medium, culturing for 3d, and measuring OD₆₀₀Value, adjusting the concentration of bacterial liquid 1 x 10⁸cfu/mL. Absorbing 5mL of the culture solution with the adjusted concentration, inoculating the culture solution into a phosphate solubilizing culture medium, and simultaneously setting the same series of functional strains CK1 and CK2(CK1 and CK2 are all laboratory self-screening and storage strains) for controlling the phosphate solubilizing effect. Repeating the steps for 3 times, after culturing for 4d, centrifuging the sample at 4000rpm for 20min, taking 10mL of supernatant, and inspecting.

(2) Evaluation results of phosphate solubilizing Effect

153-1 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 functional 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, and 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.

Then carrying out morphological characteristics, physiological and biochemical characteristics and molecular biological identification on the bacillus megaterium.

(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. 2.

(2) Physiological and biochemical characteristics:

the fatty acid composition of the strain LY153-1 is detected by a microorganism fatty acid rapid identification system (MIDI), and the main fatty acids of the strain to be detected are C15:0anteiso, C15:0iso, C14:0iso and C16:1w11C, 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 LY153-1 and phylogenetic analysis

GCCTGGCGGCGTGCCTATACATGCAAGTCGAGCGAACTGATTAGAAGCTTGCTTCTAT GACGTTAGCGGCGGACGGGTGAGTAACACGTGGGCAACCTGCCTGTAAGACTGGGAT AACTTCGGGAAACCGAAGCTAATACCGGATAGGATCTTCTCCTTCATGGGAGATGATT GAAAGATGGTTTCGGCTATCACTTACAGATGGGCCCGCGGTGCATTAGCTAGTTGGTG AGGTAACGGCTCACCAAGGCAACGATGCATAGCCGACCTGAGAGGGTGATCGGCCAC ACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCG CAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGCTTTCGGGTCG TAAAACTCTGTTGTTAGGGAAGAACAAGTACGAGAGTAACTGCTCGTACCTTGACGGT ACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTG GCAAGCGTTATCCGGAATTATTGGGCGTAAAGCGCGCGCAGGCGGTTTCTTAAGTCTG ATGTGAAAGCCCACGGCTCAACCGTGGAGGGTCATTGGAAACTGGGGAACTTGAGTG CAGAAGAGAAAAGCGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGG AACACCAGTGGCGAAGGCGGCTTTTTGGTCTGTAACTGACGCTGAGGCGCGAAAGCG TGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTA AGTGTTAGAGGGTTTCCGCCCTTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTG GGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCG GTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCC TCTGACAACTCTAGAGATAGAGCGTTCCCCTTCGGGGGACAGAGTGACAGGTGGTGC ATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAAC CCTTGATCTTAGTTGCCAGCATTTAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAA CCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACA CGTGCTACAATGGATGGTACAAAGGGCTGCAAGACCGCGAGGTCAAGCCAATCCCAT AAAACCATTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGCTGGAATCGC TAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGC CCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGGAGTAACCGTAAGGAGCT AGCCGCATAAGGGGACAGA(SEQ ID NO:1)

The results of the systematic analysis of 16S rDNA are shown in FIG. 3.

2) RpoB gene sequence (385bp) of LY153-1 strain and phylogenetic analysis

TGATCGAAACGGCTGAAGGTCCAAACATAGGTGAAGATGCGCTTCGCAACTTAGATG AGCGTGGAATCATCCGCATTGGTGCAGAAGTAAAAGACGGAGATCTTTTAGTTGGTAA AGTAACGCCAAAAGGTGTAACAGAACTAACAGCTGAAGAACGTCTTCTACACGCTAT TTTCGGTGAAAAAGCGCGTGAAGTTCGTGATACTTCTCTTCGTGTACCGCACGGCGGC GGTGGAATCATTCTTGATGTTAAAGTCTTCAACCGTGAAGATGGGGACGAATTACCAC CAGGTGTAAACCAATTAGTCCGTGTATATATTGTTCAGAAGCGTAAAATTTCTGAAGGT GACAAAATGGCCGGTCGTCACGGTAACAAGGGTGTAAA(SEQ ID NO:2)

The phylogenetic analysis results of rpoB gene are shown in FIG. 4.

The strain is identified as Bacillus megaterium through strain morphological characteristics, physiological and biochemical characteristics and molecular biological characteristic analysis.

Bacillus belgii (di)

The method for separating and obtaining the bacillus beiLeisi from the surface of the corn root system by adopting a flat plate coating method and a flat plate marking method comprises the following steps:

(1) isolation and screening of Bacillus beilis

Healthy corn rhizosphere soil is selected from yellow soil in Mei Tou street houzhou village in Hedong region of Shandong city and near-Yi city of Shandong province for screening. The method comprises the following specific steps: gently shaking off root soil, washing with clear water, placing on weighing paper, scraping root system with blade, collecting all materials, mixing, weighing 1g, placing in 100ml sterile water, oscillating at 150rpm at 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 strains are purified by adopting a plate marking method, and are respectively numbered and stored.

4 strains of Bacillus belgii with application potential are obtained by strain isolation, purification and identification, and are respectively numbered as 012-2, 025-2, 121-1 and LY 149-1.

(2) And (4) screening the strawberry root rot antagonistic bacteria.

Primary screening: preparing a PDA culture medium by adopting a plate confronting method, punching fungus cakes with the diameter of 5mm on the edges of plates of strawberry phytophthora, fusarium oxysporum, rhizoctonia solani and polyporus clavuligerus by using a puncher respectively, transplanting the fungus cakes into the center of a new PDA plate, inoculating the strains screened in the step 1 to the periphery of the plate by using toothpicks respectively, transferring the plates to a constant-temperature incubator at 25 ℃ for culture, and observing and recording the inhibition degree of the strains on pathogenic bacteria.

Re-screening: and comparing and screening again to obtain a microbial strain with the antagonistic effect on the strawberry root rot pathogenic bacteria and the best antagonistic effect, measuring the antagonistic rate, and finally obtaining a strain with the best antagonistic effect, namely the Bacillus belgii LY149-1, through the calculation of the antagonistic rate.

Wherein the antagonism rate is calculated by the following formula:

antagonism (%) (control colony radius-treated colony radius)/control colony radius 100

Wherein the control colony radius in the formula refers to the colony radius of corresponding pathogenic bacteria treated by the following PDA culture medium.

The antagonistic effect against fusarium pathogens of strain number LY149-1 is listed in table 1.

TABLE 1 antagonistic Effect of LY149-1 strains on Fusarium pathogenic bacteria

In addition, liquid chromatography-mass spectrometry (LC-MS) analysis of the strain metabolites revealed that Bacillus belgii LY149-1 was able to produce antagonistic active substances such as kanamycin, natamycin, biotin and the like.

Then, Bacillus belgii LY149-1 was identified:

(1) morphological characteristics:

the identification of Bacillus belgii with the code LY149-1 shows that: gram staining is positive, and the thallus is rod-shaped. On TSA medium, colonies were irregularly round, milky white, and thick in texture, as shown in FIG. 5.

(2) Physiological and biochemical characteristics:

the fatty acid composition of the strain LY149-1 is detected by a microorganism fatty acid rapid identification system (MIDI), and the main fatty acids of the strain to be detected are C15:0anteiso, C15:0iso, C17:0anteiso and C17:0iso, the contents of the main fatty acids are 42.09%, 12.85%, 18.71% and 10.36%, respectively, and the main fatty acid composition accords with the main cell fatty acid characteristics of Bacillus (Bacillus).

API 50CH assay results:

the positive reaction comprises L-arabinose, ribose, D-xylose, glucose, fructose, mannose, mannitol, alpha-methyl-D-glucoside, arbutin, esculetin, saligenin, cellobiose, sucrose, trehalose, glycogen, pangolin and D-turanose;

the weak positive reaction comprises glycerol, inositol, sorbitol, N-acetyl-glucosamine, amygdalin, maltose, inulin, starch and gluconate;

negative reactions include control, erythrose, D-arabinose, L-xylose, adonitol, beta-methyl-D-xyloside, galactose, sorbose, rhamnose, dulcitol, alpha-methyl-D-mannoside, lactose, melibiose, matsutake, raffinose, xylitol, D-lyxose, D-tagatose, D-fucose, L-fucose, D-arabitol, L-arabitol, 2-keto-gluconate and 5-keto-gluconate.

Meets the biochemical metabolism characteristics of Bacillus.

(3) Molecular biological characteristics:

1)16S rDNA gene sequence (1419bp) and phylogenetic analysis

The 16S rDNA gene sequence was identified as follows:

TTCGGCGGCTGGCTCCTAAAGGTTACCTCACCGACTTCGGGTGTTACAAACTCTCGTG GTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCC GCGATTACTAGCGATTCCAGCTTCACGCAGTCGAGTTGCAGACTGCGATCCGAACTGA GAACAGATTTGTGGGATTGGCTTAACCTCGCGGTTTCGCTGCCCTTTGTTCTGTCCATT GTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCT TCCTCCGGTTTGTCACCGGCAGTCACCTTAGAGTGCCCAACTGAATGCTGGCAACTAA GATCAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGAC GACAACCATGCACCACCTGTCACTCTGCCCCCGAAGGGGACGTCCTATCTCTAGGATT GTCAGAGGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATG CTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAGTCTTGCGACCGTAC TCCCCAGGCGGAGTGCTTAATGCGTTAGCTGCAGCACTAAGGGGCGGAAACCCCCTA ACACTTAGCACTCATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTCGCTCC CCACGCTTTCGCTCCTCAGCGTCAGTTACAGACCAGAGAGTCGCCTTCGCCACTGGTG TTCCTCCACATCTCTACGCATTTCACCGCTACACGTGGAATTCCACTCTCCTCTTCTGC ACTCAAGTTCCCCAGTTTCCAATGACCCTCCCCGGTTGAGCCGGGGGCTTTCACATCA GACTTAAGAAACCGCCTGCGAGCCCTTTACGCCCAATAATTCCGGACAACGCTTGCCA CCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTTAGGTACCG TCAAGGTGCCGCCCTATTTGAACGGCACTTGTTCTTCCCTAACAACAGAGCTTTACGA TCCGAAAACCTTCATCACTCACGCGGCGTTGCTCCGTCAGACTTTCGTCCATTGCGGA AGATTCCCTACTGCTGCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGC CGATCACCCTCTCAGGTCGGCTACGCATCGTCGCCTTGGTGAGCCGTTACCTCACCAA CTAGCTAATGCGCCGCGGGTCCATCTGTAAGTGGTAGCCGAAGCCACCTTTTATGTCTG AACCATGCGGTTCAGACAACCATCCGGTATTAGCCCCGGTTTCCCGGAGTTATCCCAG TCTTACAGGCAGGTTACCCACGTGTTACTCACCCGTCCGCCGCTAACATCAGGGAGCA AGCTCCCATCTGTCCGCTCGACTGC(SEQ ID NO:3)

the 16S rDNA tree analysis was performed on this strain, and the results are shown in FIG. 6.

The strain conforms to the characteristics of bacillus (sp) through analysis of a strain 16S rDNA evolutionary tree.

2) gyrB gene sequence (1162bp) and phylogenetic analysis

The gyrB gene sequence analysis was performed on the strain numbered LY149-1, and the results are shown below: GGATAACGCGCTTTTTCAAGATTAAAATCTTCTCCGATTCCTGTTCCGAGGGCCGTGAT CATTGATCTGACCTCATTGTTTGAGAGAATCTTATCAAGTCTGGCTTTCTCAACGTTCA GAATCTTACCGCGCAGCGGCAGAATGGCTTGGAAATGACGGTCCCGTCCCTGTTTCGC TGATCCGCCCGCAGAGTCACCCTCTACGATATACAGCTCGGAAATGCTCGGATCTTTAG AAGAACAGTCCGCCAGTTTGCCCGGCAGATTGGAAATCTCAAGCGCACTTTTGCGGC GGGTCAATTCCCGCGCTTTTTTCGCTGCCATCCGCGCTCTTGCGGCCATTAAACCTTTT TCAACGATTTTGCGGGCTGAGTCCGGATTTTCAAGAAGGAATGTTTCCAGCGCAGAA GAAAACAGCGTATCAGTGATCGTTCTCGCTTCGGAGTTGCCGAGCTTCGTTTTCGTCT GCCCTTCGAATTGCGGATCAGGGTGCTTAATTGAAATAATGGCAGTCAGCCCTTCCCT CACATCATCCCCGCTTAAATTCGGATCATTTTCTTTGAAAATCCCTTTTCTTCTTGCATA GTCGTTTATAACACGGGTCAGACCGGTTTTAAATCCGGCTTCGTGCGTGCCGCCTTCGT ATGTGTTGATATTATTTGTGAAAGAATAAATATTGCTTGTATAGCTGTCGTTGTATTGCA ATGCAACTTCAACCGTTATGCCGTCTTTCTCGCCTTCGATATAAATCGGCTCTTCATGA ACGACTTCTTTGGAACGGTTTAAGTACTCAACATAGCTTTTGATTCCGCCTTCGTAGTG GTACTCGTTTTTCCGTTCTTGTCCTTCACGTTTGTCTTCAATCGTGATGTTTACACCTTT TGTCAGGAAGGCCAATTCCCGGACACGGTTTGAAAGCAGGTCATAGTCGTATTCGGTT GTTTCTTTGAAAATTTCCGGATCCGGAACGAAGTGCGTAATCGTTCCGGTCTTATCAGT ATCACCGATCACTTCAAGATCGGCCACAGGTACACCGCGCTCGTACGCCTGATAGTGG ATTTTTCCGTCACGATGAACCGTAACGTCAAGAGTGGTCGACAAGGCGTTTACGACA GACGCCCCTACACCGTGAAGACCGCCGGATACTTATATCCGCCTCCCGT (SEQ ID NO:4)

Meanwhile, the gyrB gene was subjected to the phylogenetic tree analysis, as shown in FIG. 7.

Through the analysis of a gyrB gene evolutionary tree of the strain, the similarity of the strain and Bacillus velezensis strain A2 reaches 99.7 percent, and the genetic relationship is the most similar.

To summarize: through the analysis of the morphological characteristics, the physiological and biochemical characteristics and the molecular biological characteristics of the strains, the LY149-1 strain is identified as Bacillus velezensis.

(III) Bacillus subtilis subspecies subtilis

And separating the root soil of the spring sweet potatoes by adopting a flat plate coating method and a flat plate scribing method to obtain the bacillus subtilis subspecies. The method comprises the following steps:

(1) separating and screening bacillus subtilis subspecies: healthy spring sweet potato rhizosphere soil is selected from sandy soil in Zhang Ying county Zhuhuagang village in Kangcity of Kangnan province in Henan province for screening. The method comprises the following specific steps: gently shaking off soil at plant root, placing the root into a triangular flask filled with 200ml of sterile water, shaking for 10 minutes, collecting soil suspension, centrifuging to collect soil, weighing 1g, placing into 100ml of sterile water, shaking at 150rpm and 30 ℃ for 30 minutes, and performing gradient dilution to 10 DEG⁴And (3) multiplying, selecting 3 gradients, wherein each gradient is parallel to 3 gradients, selecting strains with different colony morphologies after culturing in an incubator at 30 ℃ for 2d, streaking on an LB (lysogeny broth) culture medium, and regularly observing the colony growth condition. Then, the strains were purified by plate-streaking, and the numbers of the strains were LY005, A21, A100 and A197, respectively, and stored.

(2) And (4) screening growth-promoting functional bacteria.

Screening by adopting a plate growth promotion test, preparing LB culture solution, shaking each screened strain, culturing for 3 days at 30 ℃, 180rpm, collecting fermentation liquor, diluting the fermentation liquor by 300 times, placing 20 wheat seeds in each culture dish, adding 5ml of diluted fermentation liquor, culturing for 3-5 days in an incubator with 25 ℃, detecting bud length, and taking pure water as a reference, wherein the test results are shown in Table 2.

TABLE 2 growth promoting effect of different strains on wheat seeds

Bacterial strains	A21	A197	A100	LY005	Control
						Length of bud cm	2.60	2.81	2.97	3.21	2.11
Is improved by comparison with the control group	23.22	33.18	40.76	52.13	/

(3) Metabolite identification

Culturing LY005 strain in LB culture solution for 3 days, collecting 10ml fermentation supernatant, extracting with 2 times volume of ethyl acetate, rotary evaporating, concentrating to 2ml, re-dissolving with small amount of acetonitrile, and transferring into liquid bottle. HPLC-MS is adopted to detect and analyze the fermentation supernatant, and the result shows that the metabolite contains growth promoting substances such as 3-indoleacetic acid and the like.

Then, morphological characteristics, physiological and biochemical characteristics, and molecular biological analysis were performed on the strain numbered LY005, respectively.

(1) Morphological characteristics:

gram staining is positive, and the thallus is rod-shaped. On TSA medium, the colonies were nearly circular, pale yellow, with rectangular teeth on the edges, as shown in FIG. 8.

(2) Physiological and biochemical characteristics:

the fatty acid composition of the strain with the serial number of LY005 is detected by a microorganism fatty acid rapid identification system (MIDI), and the main fatty acids of the strain are C15:0anteiso, C15:0iso, C17:0anteiso and C17:0iso, the contents of the main fatty acids are 42.09%, 12.85%, 18.71% and 10.36%, respectively, and the main fatty acid composition accords with the main cell fatty acid characteristics of Bacillus (Bacillus).

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

(3) Molecular biological characteristics:

1)16S rDNA gene sequence (1417bp) and phylogenetic analysis

GCGGCTGGCTCCTAAAAGGTTACCTCACCGACTTCGGGTGTTACAAACTCTCGTGGTG TGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGCG ATTACTAGCGATTCCAGCTTCACGCAGTCGAGTTGCAGACTGCGATCCGAACTGAGAA CAGATTTGTGGGATTGGCTTAACCTCGCGGTTTCGCTGCCCTTTGTTCTGTCCATTGTA GCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCC TCCGGTTTGTCACCGGCAGTCACCTTAGAGTGCCCAACTGAATGCTGGCAACTAAGAT CAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGAC AACCATGCACCACCTGTCACTCTGCCCCCGAAGGGGACGTCCTATCTCTAGGATTGTC AGAGGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTC CACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAGTCTTGCGACCGTACTCC CCAGGCGGAGTGCTTAATGCGTTAGCTGCAGCACTAAGGGGCGGAAACCCCCTAACA CTTAGCACTCATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTCGCTCCCCA CGCTTTCGCTCCTCAGCGTCAGTTACAGACCAGAGAGTCGCCTTCGCCACTGGTGTTC CTCCACATCTCTACGCATTTCACCGCTACACGTGGAATTCCACTCTCCTCTTCTGCACT CAAGTTCCCCAGTTTCCAATGACCCTCCCCGGTTGAGCCGGGGGCTTTCACATCAGAC TTAAGAAACCGCCTGCGAGCCCTTTACGCCCAATAATTCCGGACAACGCTTGCCACCT ACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTTAGGTACCGTC AAGGTACCGCCCTATTCGAACGGTACTTGTTCTTCCCTAACAACAGAGCTTTACGATCC GAAAACCTTCATCACTCACGCGGCGTTGCTCCGTCAGACTTTCGTCCATTGCGGAAGA TTCCCTACTGCTGCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCCGA TCACCCTCTCAGGTCGGCTACGCATCGTTGCCTTGGTGAGCCGTTACCTCACCAACTA GCTAATGCGCCGCGGGTCCATCTGTAAGTGGTAGCCGAAGCCACCTTTTATGTTTGAA CCATGCGGTTCAAACAACCATCCGGTATTAGCCCCGGTTTCCCGGAGTTATCCCAGTCT TACAGGCAGGTTACCCACGTGTTACTCACCCGTCCGCCGCTAACATCAGGGAGCAAG CTCCCATCTGTCCGCTCGACTTGC(SEQ ID NO:5)

The results of phylogenetic analysis of the 16S rDNA gene sequence are shown in FIG. 9.

2) gyrB gene sequence (1180bp) and phylogenetic analysis

CCGGGGAATGCGGCTATAAGTATCCGGAGGATTACACGGTGTAGGTGCGTCGGTCGTA AACGCACTATCAACAGAGCTTGATGTGACGGTTCACCGTGACGGTAAAATTCACCGCC AAACCTATAAACGCGGAGTTCCGGTTACAGACCTTGAAATCATTGGCGAAACGGATCA TACAGGAACGACGACACATTTTGTCCCGGACCCTGAAATTTTCTCAGAAACAACCGA GTATGATTACGATCTGCTTGCCAACCGCGTGCGTGAATTAGCCTTTTTAACAAAGGGC GTAAACATCACGATTGAAGATAAACGTGAAGGACAAGAGCGCAAAAATGAATACCAT TACGAAGGCGGAATTAAAAGTTATGTAGAGTATTTAAACCGCTCTAAAGAGGTTGTCC ATGAAGAGCCGATTTACATTGAAGGCGAAAAGGACGGCATTACGGTTGAAGTGGCTT TGCAATACAATGACAGCTACACAAGCAACATTTACTCGTTTACAAACAACATTAACAC GTACGAAGGCGGTACCCATGAAGCTGGCTTCAAAACGGGCCTGACTCGTGTTATCAA CGATTACGCCAGAAAAAAAGGGCTTATTAAAGAAAATGATCCAAACCTAAGCGGAGA TGACGTAAGGGAAGGGCTGACAGCGATTATTTCAATCAAACACCCTGATCCGCAGTTT GAGGGCCAAACAAAAACAAAGCTGGGCAACTCAGAAGCACGGACGATCACCGATAC GTTATTTTCTACGGCGATGGAAACATTTATGCTGGAAAATCCAGATGCAGCCAAAAAA ATTGTCGATAAAGGTTTAATGGCGGCAAGAGCAAGAATGGCTGCGAAAAAAGCGCGT GAACTAACACGCCGTAAGAGTGCTTTGGAAATTTCAAACCTGCCCGGTAAGTTAGCG GACTGCTCTTCAAAAGATCCGAGCATCTCCGAGTTATATATCGTAGAGGGTGACTCTGC CGGAGGATCTGCTAAACAAGGACGCGACAGACATTTCCAAGCCATTTTGCCGCTTAG AGGTAAAATCCTAAACGTTGAAAAGGCCAGACTGGATAAAATCCTTTCTAACAACGA AGTTCGCTCTATGATCACAGCGCTCGGCACAGGTATCGGAGAAGACTTCAACCTTGAG AAAGCCCGTACCACTAGAGAGATATCCAGC(SEQ ID NO:6)

The phylogenetic analysis results of the gyrB gene sequences are shown in FIG. 10.

The strain is determined to be Bacillus subtilis subsp. The strain is preserved in China general microbiological culture Collection center with the preservation number of CGMCC No. 21826.

Example 2 preparation of Complex microbial Agents

Example 2 provides a method of preparing a complex microbial inoculant comprising: 1) preparing a seed solution: respectively inoculating Bacillus megaterium, Bacillus belgii and Bacillus subtilis stored in a plate into a triangular flask (250mL) containing 100mL LB culture solution by using inoculating loops, culturing at 37 ℃ and 250rpm at 150-₆₀₀The value is 0.5-0.8, the concentration of the seed liquid is not less than 1 hundred million/mL, and the inoculated fermentation culture medium is respectively obtainedThe bacillus megaterium seed liquid, the bacillus belgii seed liquid and the bacillus subtilis seed liquid.

2) Respectively preparing a fermentation culture medium according to the following formula (in percentage by mass):

fermentation medium a: 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 the balance of water, carrying out high-pressure sterilization at 121 ℃ for 60min together with a fermentation tank under the conditions of pH adjustment to 7.0 and 0.1MPa, and cooling to normal pressure and normal temperature under reduced pressure for later use.

Fermentation medium B: 2.5 to 3 percent of corn starch, 0.5 to 1 percent of cane sugar, 3 to 4.5 percent of soybean meal, 0.15 to 0.20 percent of yeast powder, 0.15 to 0.20 percent of peptone, 0.2 to 0.3 percent of dipotassium phosphate, 0.2 to 0.3 percent of potassium dihydrogen phosphate, 0.1 to 0.15 percent of calcium carbonate, 0.1 to 0.15 percent of sodium chloride, 0.2 to 0.3 percent of defoaming agent and the balance of water, the pH is adjusted to 7.0, the mixture and a fermentation tank are sterilized under high pressure at the temperature of 121 ℃ for 60min under the condition of 0.1MPa, and the mixture is cooled under reduced pressure to normal pressure and normal temperature for standby.

Fermentation medium C: 3.0-3.5% of corn starch, 0.8-1.0% of sucrose, 4.0-4.5% of soybean meal, 0.20-0.50% of yeast powder, 0.20-0.50% of peptone, 0.2-0.3% of dipotassium phosphate, 0.2-0.3% of monopotassium phosphate, 0.1-0.15% of calcium carbonate, 0.1-0.15% of sodium chloride, 0.3-0.5% of defoaming agent and the balance of water, wherein the pH value is adjusted to 7.0 and 0.1MPa, the corn starch, the peptone, the sucrose, the dipotassium phosphate and the fermentation tank are sterilized at the high pressure of 121 ℃ for 60min together, and the corn starch, the sucrose, the soybean meal and the fermentation tank are cooled to normal pressure and normal temperature for later use under the conditions of reduced pressure and normal temperature.

3) Liquid fermentation:

respectively inoculating the bacillus megaterium seed liquid, the bacillus belgii seed liquid and the bacillus subtilis seed liquid prepared in the step 1) into a pilot fermentation tank (10L-50L-500L), wherein the bacillus megaterium adopts a fermentation culture medium A, the bacillus belgii adopts a fermentation culture medium B and the bacillus subtilis adopts a fermentation culture medium C, the initial concentration of thalli in each fermentation culture system is not lower than 100 ten thousand/mL, the tank pressure is kept at 0.05-0.10 KPa at the temperature of 35-37 ℃, the dissolved oxygen is ensured to be 20-25%, the rotating speed is 180r/min, the pH value is 7.0-8.0, and the culture is carried out for 36-48 h. After fermentation, fermentation liquor of bacillus megaterium, bacillus belgii and bacillus subtilis is obtained respectively, the sporulation rate reaches over 90 percent, and the viable count of the fermentation liquor reaches not less than 50 hundred million/mL, 300 hundred million/mL and 300 hundred million/mL respectively.

4) Adding 3% -5% of diatomite into the fermentation liquor prepared in the step 3) in proportion, drying and subpackaging by using a spray drying device to obtain bacillus megaterium, bacillus belief and bacillus subtilis microbial agent preparations used for adding in the fertilizer, wherein the viable count of the microbial agent preparations respectively reaches the bacillus megaterium: 500-800 hundred million/g, 1000-1500 hundred million/g Bacillus belgii and 1000-1500 hundred million/g Bacillus subtilis.

5) And (3) fully and uniformly mixing 30-35 parts of bacillus megaterium powder, 25-35 parts of bacillus belief strain powder and 30-40 parts of bacillus subtilis powder to prepare the compound microbial agent, wherein the viable count reaches 800-1200 hundred million/g. The specific ratio is shown in Table 3.

TABLE 3 Complex microbial inoculum compounding ratio and viable count

Example 3 application of Complex microbial Agents to potted peanuts

The potting test site is a greenhouse of a Linyi agricultural research and development center of Zhongyi chemical fertilizer Co Ltd in Linyi City, Shandong province, the crop to be tested is peanut, the variety is small white sand, the potting test is adopted in the test, soil for the test is taken from soil of 10-20cm soil layer of a perennial planting area of the peanut, various impurities, bricks and the like are removed, the soil sample is dried in the air, and the soil sample passes through a 2mm sieve. Then, potting is carried out, 8kg of soil is filled in each pot, and the specification of the test pot is that the diameter of the pot opening is 28 cm, the diameter of the pot bottom is 24 cm, and the height is 40 cm.

The test was conducted with 7 treatment groups, which were blank treatment, addition of Bacillus megaterium preparation, addition of Bacillus belgii preparation, addition of Bacillus subtilis preparation, and addition of 1-3 of the complex microbial preparation prepared in example 2, each treatment being performed with 2 g/pot of added bacterial powder. Planting 20 pots of peanuts in each treatment pot, planting 1 peanut in each pot, sowing according to a conventional method and managing conventionally.

The yield measurement standard is to take the peanut pods of each pot, dry the pods in an oven at 65 ℃ to constant weight, and measure.

TABLE 4 peanut pot culture test data

Treatment of	Yield (g/strain)
		Blank space	58.12
Compound microbial agent 1	65.11
		Compound microbial agent 2	63.22
Compound microbial agent 3	64.23
		Bacillus megaterium agent	62.12
Bacillus belgii agent	63.75
		Bacillus subtilis microbial inoculum	59.32

The test results show that the compound microbial agent 1 has the best treatment effect, and the yield is improved by 12.03 percent compared with the blank treatment; compared with Bacillus megaterium treatment, Bacillus belgii treatment and Bacillus subtilis treatment, the yield is respectively increased by 4.81%, 2.13% and 9.76%. Therefore, the peanut yield can be improved by applying the compound microbial agent, a statistical significant level is achieved, and the result shows that the effect of applying the compound microbial agent is better than that of applying a single microbial agent.

Example 4 application of composite microbial inoculant in peanut planting

Firstly, preparing a fertilizer containing a compound microbial agent, wherein the preparation process comprises the following steps: uniformly mixing the compound microbial agent prepared in the embodiment 2 with anti-caking powder, adding the uniformly mixed powder into a fertilizer roller through a flood chain feeder in a coating working section of organic and inorganic fertilizer production to coat the surface of fertilizer particles, and preparing a corresponding fertilizer after stable production to obtain the corresponding microbial fertilizer, wherein the organic matter in the used organic and inorganic fertilizer is more than or equal to 15 percent, the N is more than or equal to 16 percent, and the P is more than or equal to 16 percent₂O₅≥12％，K₂O is more than or equal to 12 percent, and the effective viable count contained in the prepared fertilizer is more than or equal to 0.2 hundred million/g.

The microbial fertilizer is subjected to field effect verification, a peanut field test is carried out in the village of Weizhu village of the panda village in Zhengyang county of Henan province, and the physicochemical properties of the soil in the test field are as follows: 21.14g/kg of organic matter, 5.7 of pH value, 132.59mg/kg of alkaline hydrolysis nitrogen, 24.39mg/kg of available phosphorus and 156.29mg/kg of quick-acting potassium.

The experiment set up 5 treatment groups, respectively:

15-15-15/S of conventional compound fertilizer,

16-12-12/S (containing 15% of organic matter) of organic and inorganic fertilizers,

16-12-12/S (containing 15% of organic matters and added with the compound microbial agent 1 in the example 2) of biological organic and inorganic fertilizers,

Biological organic and inorganic fertilizer 2 (added with the compound microbial agent 2 in the embodiment 2),

The number of living bacteria of the biological organic and inorganic fertilizers 3 (added with the compound microbial agent 3 in the example 2) is 0.2 hundred million/g.

All test treatments are applied to the soil in the form of base fertilizers, the application amount is 50 kg/mu, the application mode is machine ploughing after broadcasting, and the area of each plot is 30m²Repeat 3 times. Sowing and uniformly managing according to a conventional method.

Yield determination criteria 3 blocks of 1m per cell were selected²And (3) measuring the yield of the square plots with the sizes, picking all peanut pods, drying the peanut pods in an oven at 65 ℃ to constant weight, and measuring.

TABLE 5 peanut field test data

Treatment of	Yield (kg/mu)
		Conventional compound fertilizer	406.69
Organic and inorganic fertilizer	430.23
		Biological organic and inorganic fertilizer 1	456.38
Biological organic and inorganic fertilizer 2	432.16
		Biological organic and inorganic fertilizer 3	450.11

The test results show that the microbial fertilizer is superior to conventional compound fertilizers and organic and inorganic fertilizers, wherein the microbial fertilizer 1 has the best effect, and the yield is respectively increased by 12.21% and 6.08% compared with the conventional compound fertilizers and the organic and inorganic fertilizers. Therefore, the microbial fertilizer can effectively improve the peanut yield when being applied under the condition of the same fertilizing amount.

Example 5 application of Complex microbial Agents to potted Garlic

The potting test site is greenhouse of Linyi agricultural research and development center of Zhonghua chemical fertilizer Co Ltd in Linyi City, Shandong province, the crop to be tested is garlic, the test adopts potting test, soil for the test is soil of 10-20cm soil layer in a garlic perennial planting area, various impurities, bricks and the like are removed, the soil sample is dried in the air, and the soil sample is sieved by a 2mm sieve. Then, potting is carried out, 8kg of soil is filled in each pot, and the specification of the test pot is that the diameter of the pot opening is 28 cm, the diameter of the pot bottom is 24 cm, and the height is 40 cm.

The test was conducted with 7 treatment groups, which were blank treatment, addition of Bacillus megaterium preparation, addition of Bacillus belgii preparation, addition of Bacillus subtilis preparation, and addition of 1-3 of the complex microbial preparation prepared in example 2, each treatment being performed with 2 g/pot of added bacterial powder. Planting 20 pots of garlic in each treatment, planting 3 garlic in each pot, sowing according to a conventional method and managing conventionally. The height of the garlic plants is measured in the garlic sprout stage, the garlic sprout yield is obtained in the garlic sprout stage, and the garlic yield is obtained in the garlic mature stage. The test results are shown in Table 6.

The plant height determination standard is that the plant height of each pot of garlic is determined when the garlic is planted for 20 days, the young garlic shoot yield determination standard is that the young garlic shoot part of each pot is picked for determination, and the garlic yield determination standard is that the bulb part of each pot of garlic is picked for determination.

TABLE 6 Garlic potting test data

The test results show that the treatment effect of the compound microbial agent 1 is best, the plant height is improved by 11.98 percent compared with that of a blank treated plant, the yield of young garlic is improved by 15.15 percent, and the yield of garlic is improved by 18.98 percent; compared with Bacillus megaterium treatment, Bacillus belgii treatment and Bacillus subtilis treatment, the composite microbial agent 1 treatment increases the plant heights by 2.78%, 8.05% and 12.11%, increases the garlic sprout yields by 8.91%, 5.87% and 7.90%, and increases the garlic yields by 13.32%, 10.47% and 17.21%, respectively. Therefore, the compound microbial agent can promote the growth of garlic and improve the yield of garlic shoots and garlic, achieves the statistical significant level, and simultaneously the result shows that the effect of the compound microbial agent is better than that of the single microbial agent.

Example 6 application of composite microbial inoculant in garlic planting

Firstly, preparing a microbial fertilizer: uniformly mixing the compound microbial agent prepared in the embodiment 2 with the anti-caking agent, adding the uniformly mixed powder into a fertilizer roller through a chain flood dragon feeding machine in a coating working section of compound fertilizer production to coat the surface of fertilizer particles, and preparing a corresponding fertilizer after stable production, namely a microbial fertilizer, wherein N is more than or equal to 12 percent, and P is more than or equal to 12 percent₂O₅≥18％，K₂O is more than or equal to 15 percent, and the number of effective viable bacteria is more than or equal to 0.2 hundred million/g.

The field effect verification is carried out on the biological compound fertilizer, the field test of the garlic is carried out in Kaifeng city of Henan province, and the physicochemical properties of the soil in the test field are as follows: 65.21g/kg of organic matter, 5.5 of pH value, 144.19mg/kg of alkaline hydrolysis nitrogen, 84.55mg/kg of available phosphorus and 186.33mg/kg of quick-acting potassium.

The experiment set up 2 treatment groups, respectively:

12-18-15/S of compound fertilizer,

Biological compound fertilizer 1 (adding the compound microbial agent 1 in the embodiment 2),

Biological compound fertilizer 2 (adding the compound microbial agent 2 in the embodiment 2),

The number of viable bacteria of the biological compound fertilizer 3 (added with the compound microbial agent 3 in the example 2) is 0.2 hundred million/g.

All test treatments are applied to the soil in the form of base fertilizers, the application amount is 80 kg/mu, the application mode is machine ploughing after broadcasting, and the area of each plot is 50m²Repeat 3 times. Sowing and uniformly managing according to a conventional method.

Yield determination criteria 3 blocks of 1m per cell were selected²Measuring the yield of square plots, and picking all garlic bulbsAnd (4) carrying out measurement. The test data are shown in Table 7.

TABLE 7 Garlic field test data

Treatment of	Yield (kg/mu)
		Compound fertilizer	2569.77
Biological compound fertilizer 1	2749.52.
		Biological compound fertilizer 2	2678.12
Biological compound fertilizer 3	2635.66

The test results show that the effect of each biological compound fertilizer is better than that of compound fertilizer treatment, wherein the biological compound fertilizer 1 has the best effect, and the yield is increased by 6.99 percent compared with the compound fertilizer. Therefore, the application of the biological compound fertilizer under the condition of the same fertilizing amount can effectively improve the garlic yield.

Example 7 application of composite microbial inoculant in cucumber planting

Firstly, preparing a microbial fertilizer: uniformly mixing the compound microbial agent prepared in the example 1 with the anti-caking powder, adding the uniformly mixed powder into a fertilizer roller through a flood chain feeder in a coating working section of organic and inorganic fertilizer production to coat the surface of fertilizer particles, and preparing the corresponding fertilizer after stable production, namely the bio-organic and inorganic fertilizer, wherein the organic matter is more than or equal to 15 percent, and the N is more than or equal to 18 percent， P₂O₅≥10％，K₂O is more than or equal to 12 percent, and the effective viable count is more than or equal to 0.2 hundred million/g.

The biological organic and inorganic fertilizers are subjected to field effect verification, a cucumber field test is carried out in a test field of a specialized agriculture near-Yi research and development center in the near-Yi city in Shandong province, and the physicochemical properties of soil in the test field are as follows: 17.32g/kg of organic matter, 7.2 of pH value, 99.62mg/kg of alkaline hydrolysis nitrogen, 32.56mg/kg of available phosphorus and 142.36mg/kg of quick-acting potassium.

The test set up 5 treatments, respectively:

15-15-15/S of conventional compound fertilizer,

18-10-12/S (containing 15% of organic matter) of organic and inorganic fertilizers,

18-10-12/S of biological organic and inorganic fertilizers (containing 15 percent of organic matters, and added with the compound microbial agent 1 in the example 2),

biological organic and inorganic fertilizer 2 (added with compound microbial agent 2 in example 1),

the number of living bacteria of the biological organic and inorganic fertilizer 3 (added with the compound microbial agent 3 in the example 1) is 0.2 hundred million/g.

All test treatments are applied to the soil in the form of base fertilizers, the application amount is 40 kg/mu, the application mode is machine ploughing after broadcasting, and the area of each plot is 20m²Repeat 3 times. Transplanting seedlings and managing uniformly according to a conventional method.

Yield determination criteria 3 blocks of 1m per cell were selected²And (4) carrying out yield measurement on the land parcels with the sizes, and measuring by accumulating and calculating the yield after each picking. The test results are shown in Table 8.

Table 8 cucumber field test data

Treatment of	Yield (kg/mu)
		Conventional compound fertilizer	2356.2
Organic and inorganic fertilizer	2634.3
		Biological organic and inorganic fertilizer 1	2987.9
Biological organic and inorganic fertilizer 2	2879.3
		Biological organic and inorganic fertilizer 3	2744.5

The test results show that the effect of the biological organic and inorganic fertilizer is better than that of the conventional compound fertilizer and organic and inorganic fertilizer, wherein the effect of the biological organic and inorganic fertilizer 1 is the best, and the yield is respectively increased by 26.81% and 13.42% compared with the conventional compound fertilizer and organic and inorganic fertilizer. Therefore, the yield of the cucumbers can be effectively improved by applying the biological organic and inorganic fertilizers under the condition of the same fertilizing amount.

Example 8 application of composite microbial inoculant in eggplant planting

Firstly, preparing a microbial fertilizer: uniformly mixing the compound microbial agent prepared in the embodiment 2 with the anti-caking agent, adding the uniformly mixed powder into a fertilizer roller through a flood chain feeder in a coating working section of organic and inorganic fertilizer production to coat the surface of fertilizer particles, and preparing a corresponding fertilizer after stable production, namely the bio-organic and inorganic fertilizer, wherein the organic matter content is more than or equal to 15%, the N content is more than or equal to 18%, and the P content is more than or equal to 18%₂O₅≥10％，K₂O is more than or equal to 12 percent, and the effective viable count is more than or equal to 0.2 hundred million/g.

The field effect verification is carried out on the biological organic and inorganic fertilizers, the eggplant field test is carried out in a test field of a specialized agriculture near-Yi research and development center in the near-Yi city in Shandong province, and the soil physicochemical properties of the test field are as follows: 18.22g/kg of organic matter, 7.1 of pH value, 89.52mg/kg of alkaline hydrolysis nitrogen, 35.62mg/kg of available phosphorus and 151.23mg/kg of quick-acting potassium.

The experiment set up 5 treatment groups, respectively:

the conventional compound fertilizer is 15-15-15/S,

18-10-12/S of organic and inorganic fertilizers (containing 15 percent of organic matters),

biological organic and inorganic fertilizer 2 (added with compound microbial agent 2 in example 2),

the number of living bacteria of the biological organic and inorganic fertilizer 3 (added with the compound microbial agent 3 in the example 3) is 0.2 hundred million/g.

Yield determination criteria 3 blocks of 1m per cell were selected²And (4) carrying out yield measurement on the land parcels with the sizes, and measuring by accumulating and calculating the yield after each picking. The test results are shown in Table 9.

TABLE 9 eggplant field test data

Treatment of	Yield (kg/mu)
		Conventional compound fertilizer	3152.6
Organic and inorganic fertilizer	3501.0
		Biological organic and inorganic fertilizer 1	3898.5
Biological organic and inorganic fertilizer 2	3788.5
		Biological organic and inorganic fertilizer 3	3724.3

The test results show that the effect of the applied biological organic and inorganic fertilizers is better than that of the conventional compound fertilizer and organic and inorganic fertilizers, wherein the effect of the biological organic and inorganic fertilizer 1 is the best, and the yield is respectively increased by 23.66% and 11.35% compared with the yield of the conventional compound fertilizer and organic and inorganic fertilizers. Therefore, the yield of the eggplants can be effectively improved by applying the biological organic and inorganic fertilizers under the condition of the same fertilizing amount.

Example 9 application of Complex microbial Agents in Pepper planting

Firstly, preparing a microbial fertilizer: uniformly mixing the compound microbial agent prepared in the embodiment 2 with the anti-caking agent, adding the uniformly mixed powder into a fertilizer roller through a flood chain feeder in a coating working section of organic and inorganic fertilizer production to coat the surface of fertilizer particles, and preparing a corresponding fertilizer after stable production, namely the bio-organic and inorganic fertilizer, wherein the organic matter content is more than or equal to 15%, the N content is more than or equal to 16%, and the P content is more than or equal to 15%, the P content is more than or equal to 16%, and the corresponding fertilizer is obtained₂O₅≥8％，K₂O is more than or equal to 16 percent, and the number of effective viable bacteria is more than or equal to 0.2 hundred million/g.

The field effect verification is carried out on the biological organic and inorganic fertilizers, the field test of the pepper is carried out in a test field of a specialized agriculture near-Yi research and development center in the near-Yi city in Shandong province, and the physicochemical properties of soil in the test field are as follows: 15.22g/kg of organic matter, 7.2 of pH value, 102.35mg/kg of alkaline hydrolysis nitrogen, 41.23mg/kg of available phosphorus and 142.69mg/kg of quick-acting potassium.

The experiment set up 5 treatment groups, respectively:

the conventional compound fertilizer is 15-15-15/S,

16-8-16/S organic and inorganic fertilizers (containing 15 percent of organic matters),

16-8-16/S (containing 15% of organic matters, added with the compound microbial agent 1 in the example 2),

Yield determination criteria 3 blocks of 1m per cell were selected²And (4) carrying out yield measurement on the land parcels with the sizes, and measuring by accumulating and calculating the yield after each picking. The test results are shown in Table 10.

TABLE 10 Pepper field test data

Treatment of	Yield (kg/mu)
		Conventional compound fertilizer	1613.4
Organic and inorganic fertilizer	1710.6
		Biological organic and inorganic fertilizer 1	1783.7
Biological organic and inorganic fertilizer 2	1732.2
		Biological organic and inorganic fertilizer 3	1764.3

The test results show that the effect of the biological organic and inorganic fertilizer is better than that of the conventional compound fertilizer and organic and inorganic fertilizer, wherein the effect of the biological organic and inorganic fertilizer 1 is the best, and the yield is respectively increased by 10.56% and 4.27% compared with the conventional compound fertilizer and organic and inorganic fertilizer. Therefore, the yield of the pepper can be effectively improved by applying the biological organic and inorganic fertilizers under the condition of the same fertilizing amount.

Example 10 application of Complex microbial Agents in Citrus (Trigonggan) planting

The field effect verification is carried out on the biological organic and inorganic fertilizers, the field test of the citrus gonggan is carried out on the large roadside town of the Lizhou city of Qingyuan, Guangdong province, and the physicochemical property of the soil in the test field is as follows: 19.65g/kg of organic matter, 5.6 of pH value, 68.32mg/kg of alkaline hydrolysis nitrogen, 55.32mg/kg of available phosphorus and 188.65mg/kg of quick-acting potassium.

The experiment set up 5 treatment groups, respectively:

the conventional compound fertilizer is 15-15-15/S,

All test treatments were applied to the soil as a base fertilizer at an application rate of 1kg per plant to approximately 50kg per acre in a furrow application depth of 15-20cm with 20 trees per plot repeated 3 times.

The yield measurement standard is that 5 trees with uniform growth vigor are selected for each cell to carry out yield and fruit diameter measurement. The test results are shown in Table 11.

TABLE 11 Citrus gonggan field test data

Treatment of	Yield (kg/mu)	Fruit diameter (mm)
			Conventional compound fertilizer	800.2	52.3
Organic and inorganic fertilizer	814.0	54.6
			Biological organic and inorganic fertilizer 1	913.7	59.9
Biological organic and inorganic fertilizer 2	864.3	57.2
			Biological organic and inorganic fertilizer 3	884.3	58.6

The test results show that the effect of the biological organic and inorganic fertilizer is better than that of the conventional compound fertilizer and organic and inorganic fertilizer, wherein the effect of the biological organic and inorganic fertilizer 1 is the best, and the yield is respectively increased by 14.2% and 12.2% compared with the conventional compound fertilizer and organic and inorganic fertilizer; the fruit diameter is respectively increased by 14.6 percent and 9.7 percent. Therefore, the application of the biological organic and inorganic fertilizers under the condition of the same fertilizing amount can effectively improve the fruit diameter and the yield of the citrus gonggan.

Example 11 application of composite microbial inoculant in orange (crystal sugar orange) planting

Firstly, preparing a fertilizer: uniformly mixing the compound microbial agent prepared in the embodiment 2 with the anti-caking agent, adding the uniformly mixed powder into a fertilizer roller through a flood chain feeder in a coating working section of organic and inorganic fertilizer production to coat the surface of fertilizer particles, and preparing a corresponding fertilizer after stable production, namely the bio-organic and inorganic fertilizer, wherein the organic matter content is more than or equal to 15%, the N content is more than or equal to 16%, and the P content is more than or equal to 15%, the P content is more than or equal to 16%, and the corresponding fertilizer is obtained₂O₅≥8％，K₂O is more than or equal to 16 percent, and the number of effective viable bacteria is more than or equal to 0.2 hundred million/g.

The field effect of the biological organic and inorganic fertilizer is verified, the rock sugar orange field test is carried out in Chenzhou city Yongxing county in Hunan province, and the physicochemical property of the soil of the test field is as follows: 23.21g/kg of organic matter, 5.3 of pH value, 121.35mg/kg of alkaline hydrolysis nitrogen, 86.22mg/kg of available phosphorus and 176.59mg/kg of quick-acting potassium.

The experiment set up 5 treatment groups, respectively:

the conventional compound fertilizer is 15-15-15/S,

The yield measurement standard is that 5 trees with uniform growth vigor are selected for each cell to carry out yield and fruit diameter measurement. The test results are shown in Table 12.

TABLE 12 field test data for Crystal sugar oranges

Treatment of	Yield (kg/mu)	Fruit diameter (mm)
			Conventional compound fertilizer	1765.7	59.0
Organic and inorganic fertilizer	1839.1	61.5
			Biological organic and inorganic fertilizer 1	2010.9	65.7
Biological organic and inorganic fertilizer 2	1962.6	63.4
			Biological organic and inorganic fertilizer 3	1922.4	64.1

The test results show that the effect of the biological organic and inorganic fertilizer is better than that of the conventional compound fertilizer and organic and inorganic fertilizer, wherein the effect of the biological organic and inorganic fertilizer 1 is the best, and the yield is respectively increased by 13.9% and 9.3% compared with the conventional compound fertilizer and organic and inorganic fertilizer; the fruit diameter is respectively increased by 11.4 percent and 6.9 percent. Therefore, the application of the biological organic and inorganic fertilizers under the condition of the same fertilizing amount can effectively improve the fruit diameter and the yield of the crystal sugar oranges.

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> compound microbial agent and application thereof in fertilizer

<130> BI3211472

<160> 6

<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

<210> 3

<211> 1419

<212> DNA

<213> Bacillus velezensis

<400> 3

ttcggcggct ggctcctaaa ggttacctca ccgacttcgg gtgttacaaa ctctcgtggt 60

gtgacgggcg gtgtgtacaa ggcccgggaa cgtattcacc gcggcatgct gatccgcgat 120

tactagcgat tccagcttca cgcagtcgag ttgcagactg cgatccgaac tgagaacaga 180

tttgtgggat tggcttaacc tcgcggtttc gctgcccttt gttctgtcca ttgtagcacg 240

tgtgtagccc aggtcataag gggcatgatg atttgacgtc atccccacct tcctccggtt 300

tgtcaccggc agtcacctta gagtgcccaa ctgaatgctg gcaactaaga tcaagggttg 360

cgctcgttgc gggacttaac ccaacatctc acgacacgag ctgacgacaa ccatgcacca 420

cctgtcactc tgcccccgaa ggggacgtcc tatctctagg attgtcagag gatgtcaaga 480

cctggtaagg ttcttcgcgt tgcttcgaat taaaccacat gctccaccgc ttgtgcgggc 540

ccccgtcaat tcctttgagt ttcagtcttg cgaccgtact ccccaggcgg agtgcttaat 600

gcgttagctg cagcactaag gggcggaaac cccctaacac ttagcactca tcgtttacgg 660

cgtggactac cagggtatct aatcctgttc gctccccacg ctttcgctcc tcagcgtcag 720

ttacagacca gagagtcgcc ttcgccactg gtgttcctcc acatctctac gcatttcacc 780

gctacacgtg gaattccact ctcctcttct gcactcaagt tccccagttt ccaatgaccc 840

tccccggttg agccgggggc tttcacatca gacttaagaa accgcctgcg agccctttac 900

gcccaataat tccggacaac gcttgccacc tacgtattac cgcggctgct ggcacgtagt 960

tagccgtggc tttctggtta ggtaccgtca aggtgccgcc ctatttgaac ggcacttgtt 1020

cttccctaac aacagagctt tacgatccga aaaccttcat cactcacgcg gcgttgctcc 1080

gtcagacttt cgtccattgc ggaagattcc ctactgctgc ctcccgtagg agtctgggcc 1140

gtgtctcagt cccagtgtgg ccgatcaccc tctcaggtcg gctacgcatc gtcgccttgg 1200

tgagccgtta cctcaccaac tagctaatgc gccgcgggtc catctgtaag tggtagccga 1260

agccaccttt tatgtctgaa ccatgcggtt cagacaacca tccggtatta gccccggttt 1320

cccggagtta tcccagtctt acaggcaggt tacccacgtg ttactcaccc gtccgccgct 1380

aacatcaggg agcaagctcc catctgtccg ctcgactgc 1419

<210> 4

<211> 1162

<212> DNA

<213> Bacillus velezensis

<400> 4

ggataacgcg ctttttcaag attaaaatct tctccgattc ctgttccgag ggccgtgatc 60

attgatctga cctcattgtt tgagagaatc ttatcaagtc tggctttctc aacgttcaga 120

atcttaccgc gcagcggcag aatggcttgg aaatgacggt cccgtccctg tttcgctgat 180

ccgcccgcag agtcaccctc tacgatatac agctcggaaa tgctcggatc tttagaagaa 240

cagtccgcca gtttgcccgg cagattggaa atctcaagcg cacttttgcg gcgggtcaat 300

tcccgcgctt ttttcgctgc catccgcgct cttgcggcca ttaaaccttt ttcaacgatt 360

ttgcgggctg agtccggatt ttcaagaagg aatgtttcca gcgcagaaga aaacagcgta 420

tcagtgatcg ttctcgcttc ggagttgccg agcttcgttt tcgtctgccc ttcgaattgc 480

ggatcagggt gcttaattga aataatggca gtcagccctt ccctcacatc atccccgctt 540

aaattcggat cattttcttt gaaaatccct tttcttcttg catagtcgtt tataacacgg 600

gtcagaccgg ttttaaatcc ggcttcgtgc gtgccgcctt cgtatgtgtt gatattattt 660

gtgaaagaat aaatattgct tgtatagctg tcgttgtatt gcaatgcaac ttcaaccgtt 720

atgccgtctt tctcgccttc gatataaatc ggctcttcat gaacgacttc tttggaacgg 780

tttaagtact caacatagct tttgattccg ccttcgtagt ggtactcgtt tttccgttct 840

tgtccttcac gtttgtcttc aatcgtgatg tttacacctt ttgtcaggaa ggccaattcc 900

cggacacggt ttgaaagcag gtcatagtcg tattcggttg tttctttgaa aatttccgga 960

tccggaacga agtgcgtaat cgttccggtc ttatcagtat caccgatcac ttcaagatcg 1020

gccacaggta caccgcgctc gtacgcctga tagtggattt ttccgtcacg atgaaccgta 1080

acgtcaagag tggtcgacaa ggcgtttacg acagacgccc ctacaccgtg aagaccgccg 1140

gatacttata tccgcctccc gt 1162

<210> 5

<211> 1417

<212> DNA

<213> Bacillus subtilis

<400> 5

gcggctggct cctaaaaggt tacctcaccg acttcgggtg ttacaaactc tcgtggtgtg 60

acgggcggtg tgtacaaggc ccgggaacgt attcaccgcg gcatgctgat ccgcgattac 120

tagcgattcc agcttcacgc agtcgagttg cagactgcga tccgaactga gaacagattt 180

gtgggattgg cttaacctcg cggtttcgct gccctttgtt ctgtccattg tagcacgtgt 240

gtagcccagg tcataagggg catgatgatt tgacgtcatc cccaccttcc tccggtttgt 300

caccggcagt caccttagag tgcccaactg aatgctggca actaagatca agggttgcgc 360

tcgttgcggg acttaaccca acatctcacg acacgagctg acgacaacca tgcaccacct 420

gtcactctgc ccccgaaggg gacgtcctat ctctaggatt gtcagaggat gtcaagacct 480

ggtaaggttc ttcgcgttgc ttcgaattaa accacatgct ccaccgcttg tgcgggcccc 540

cgtcaattcc tttgagtttc agtcttgcga ccgtactccc caggcggagt gcttaatgcg 600

ttagctgcag cactaagggg cggaaacccc ctaacactta gcactcatcg tttacggcgt 660

ggactaccag ggtatctaat cctgttcgct ccccacgctt tcgctcctca gcgtcagtta 720

cagaccagag agtcgccttc gccactggtg ttcctccaca tctctacgca tttcaccgct 780

acacgtggaa ttccactctc ctcttctgca ctcaagttcc ccagtttcca atgaccctcc 840

ccggttgagc cgggggcttt cacatcagac ttaagaaacc gcctgcgagc cctttacgcc 900

caataattcc ggacaacgct tgccacctac gtattaccgc ggctgctggc acgtagttag 960

ccgtggcttt ctggttaggt accgtcaagg taccgcccta ttcgaacggt acttgttctt 1020

ccctaacaac agagctttac gatccgaaaa ccttcatcac tcacgcggcg ttgctccgtc 1080

agactttcgt ccattgcgga agattcccta ctgctgcctc ccgtaggagt ctgggccgtg 1140

tctcagtccc agtgtggccg atcaccctct caggtcggct acgcatcgtt gccttggtga 1200

gccgttacct caccaactag ctaatgcgcc gcgggtccat ctgtaagtgg tagccgaagc 1260

caccttttat gtttgaacca tgcggttcaa acaaccatcc ggtattagcc ccggtttccc 1320

ggagttatcc cagtcttaca ggcaggttac ccacgtgtta ctcacccgtc cgccgctaac 1380

atcagggagc aagctcccat ctgtccgctc gacttgc 1417

<210> 6

<211> 1180

<212> DNA

<213> Bacillus subtilis

<400> 6

ccggggaatg cggctataag tatccggagg attacacggt gtaggtgcgt cggtcgtaaa 60

cgcactatca acagagcttg atgtgacggt tcaccgtgac ggtaaaattc accgccaaac 120

ctataaacgc ggagttccgg ttacagacct tgaaatcatt ggcgaaacgg atcatacagg 180

aacgacgaca cattttgtcc cggaccctga aattttctca gaaacaaccg agtatgatta 240

cgatctgctt gccaaccgcg tgcgtgaatt agccttttta acaaagggcg taaacatcac 300

gattgaagat aaacgtgaag gacaagagcg caaaaatgaa taccattacg aaggcggaat 360

taaaagttat gtagagtatt taaaccgctc taaagaggtt gtccatgaag agccgattta 420

cattgaaggc gaaaaggacg gcattacggt tgaagtggct ttgcaataca atgacagcta 480

cacaagcaac atttactcgt ttacaaacaa cattaacacg tacgaaggcg gtacccatga 540

agctggcttc aaaacgggcc tgactcgtgt tatcaacgat tacgccagaa aaaaagggct 600

tattaaagaa aatgatccaa acctaagcgg agatgacgta agggaagggc tgacagcgat 660

tatttcaatc aaacaccctg atccgcagtt tgagggccaa acaaaaacaa agctgggcaa 720

ctcagaagca cggacgatca ccgatacgtt attttctacg gcgatggaaa catttatgct 780

ggaaaatcca gatgcagcca aaaaaattgt cgataaaggt ttaatggcgg caagagcaag 840

aatggctgcg aaaaaagcgc gtgaactaac acgccgtaag agtgctttgg aaatttcaaa 900

cctgcccggt aagttagcgg actgctcttc aaaagatccg agcatctccg agttatatat 960

cgtagagggt gactctgccg gaggatctgc taaacaagga cgcgacagac atttccaagc 1020

cattttgccg cttagaggta aaatcctaaa cgttgaaaag gccagactgg ataaaatcct 1080

ttctaacaac gaagttcgct ctatgatcac agcgctcggc acaggtatcg gagaagactt 1140

caaccttgag aaagcccgta ccactagaga gatatccagc 1180

Claims

1. A complex microbial inoculant, comprising:

at least two of bacillus megaterium, bacillus belgii and bacillus subtilis subspecies subtilis;

the Bacillus megaterium is preserved in the China general microbiological culture Collection center with the preservation number of CGMCC No. 21828;

the Bacillus velezensis is preserved in the China general microbiological culture Collection center with the preservation number of CGMCC No. 21827;

the Bacillus subtilis subsp. subtilis is preserved in the China general microbiological culture Collection center of the culture Collection of microorganisms with the preservation number of CGMCC No. 21826.

2. The complex microbial inoculant according to claim 1, wherein the complex microbial inoculant is in a dry powder form, each gram of the complex microbial inoculant contains 500-800 billion CFU of the bacillus megaterium, 1000-1500 billion CFU of the bacillus belgii and 1000-1500 billion CFU of the bacillus subtilis.

3. The complex microbial inoculant according to claim 1, comprising:

30 to 35 parts by weight of Bacillus megaterium,

25 to 35 parts by weight of Bacillus belgii,

30-40 parts of bacillus subtilis.

4. The method for preparing a complex microbial inoculant according to any one of claims 1 to 3, comprising:

respectively carrying out fermentation treatment on the strains, and obtaining microbial agents based on products of the fermentation treatment;

and compounding the microbial agent so as to obtain the compound microbial agent.

5. The method of claim 4, wherein the fermentation treatment comprises:

performing activated fermentation culture on the strain to obtain liquid zymogen liquid;

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

6. The production method according to claim 5, wherein the medium for the amplified fermentation culture of Bacillus megaterium comprises soybean meal, corn meal, starch, glucose, sodium chloride, dipotassium hydrogen phosphate, manganese sulfate and an antifoaming agent;

preferably, the culture medium for the bacillus megaterium amplified fermentation culture comprises the following components 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;

the culture medium for the amplification fermentation culture of the bacillus beleisi comprises corn starch, sucrose, soybean meal, yeast powder, peptone, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, calcium carbonate, sodium chloride and an antifoaming agent;

preferably, the culture medium for the amplification fermentation culture of the bacillus belgii comprises the following components in parts by weight:

2.5 to 3 parts of corn starch,

0.5 to 1 part of cane sugar,

3-4.5 parts of soybean meal,

0.15 to 0.20 portion of yeast powder,

0.15-0.20 parts of peptone,

0.2 to 0.3 portion of dipotassium hydrogen phosphate,

0.2 to 0.3 portion of monopotassium phosphate,

0.1 to 0.15 portion of calcium carbonate,

0.1 to 0.15 parts of sodium chloride, and

0.2-0.3 part of defoaming agent;

the culture medium for the bacillus subtilis amplification fermentation culture comprises corn starch, cane sugar, bean pulp, yeast powder, peptone, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, calcium carbonate, sodium chloride and an antifoaming agent;

preferably, the culture medium for the bacillus subtilis amplified fermentation culture comprises the following components in parts by weight:

3.0 to 3.5 parts by weight of corn starch,

0.8 to 1.0 part by weight of sucrose,

4.0 to 4.5 weight portions of soybean meal,

0.20-0.50 weight parts of yeast powder,

0.20-0.50 of peptone,

0.2 to 0.3 part by weight of dipotassium hydrogenphosphate,

0.2 to 0.3 part by weight of monopotassium phosphate,

0.1 to 0.15 parts by weight of calcium carbonate,

0.1 to 0.15 parts by weight of sodium chloride, and

0.3-0.5 parts by weight of a defoaming agent.

7. A fertilizer, characterized in that the fertilizer comprises the compound microbial agent as claimed in any one of claims 1 to 3;

optionally, the effective viable count of the compound microbial agent contained in each gram of fertilizer is more than 0.2 hundred million CFU.

8. The fertilizer according to claim 7, further comprising a base fertilizer selected from at least one of a compound fertilizer, an organic fertilizer and an inorganic fertilizer;

optionally, the proportion of the compound microbial agent in the fertilizer is one thousandth to five thousandth.

9. Use of a compound microbial inoculant in the preparation of a fertilizer for crop fertilization, wherein the compound microbial inoculant is as defined in any one of claims 1 to 3.

10. A method of fertilizing a crop, comprising:

applying a compound microbial agent or a fertilizer to crops, wherein the compound microbial agent is the compound microbial agent described in any one of claims 1-3, and the fertilizer is the fertilizer described in claim 7 or 8;

optionally, the crop is selected from at least one of peanut, garlic, cucumber, eggplant, pepper and citrus.