CN113980834A

CN113980834A - Bacillus subtilis and application thereof in fertilizer

Info

Publication number: CN113980834A
Application number: CN202111115767.5A
Authority: CN
Inventors: 刘巍; 邱鹏飞; 李洪顺; 王子浩; 孙鹰翔
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: CN113980834B

Abstract

The invention relates to the field of microorganisms, and provides bacillus subtilis and application thereof in a fertilizer. Bacillus subtilis is provided, which is preserved in China general microbiological culture Collection center with the preservation number of CGMCC No. 21826. The bacillus subtilis, the microbial agent or the composite microbial agent can be applied to fertilizers, can promote the growth of crops and improve the yield of the crops, and has remarkable effect.

Description

Bacillus subtilis and application thereof in fertilizer

Technical Field

The invention relates to the technical field of microorganisms, in particular to bacillus subtilis and application thereof, and particularly relates to bacillus subtilis and application thereof in a fertilizer.

Background

The production of chemical fertilizer brings about the yield increase of grains, but also brings about the environmental pollution and the land pollution. Especially in the current society, the environmental pollution and the land pollution are increasingly serious, which causes the sustainable development of the grains to appear crisis. Therefore, how to reduce the use of fertilizers is an important subject of research of scientists today. Practice proves that the microbial fertilizer has the advantages of high efficiency, high yield, green, healthy and pollution-free plant fertilizer. The use of microbial fertilizers is an essential factor in the development of agriculture today.

The application of the microbial fertilizer can promote the proliferation of beneficial microbes in soil, so that the beneficial microbes decompose more nutrients such as nitrogen, phosphorus, potassium and the like in the soil and promote the growth and development of plants. The bacillus subtilis is a microorganism which is most widely applied in the current agricultural microbial agents, and comprises subspecies such as natto subspecies (B.subtilis subsp.natto), bacillus subtilis subspecies (B.subtilis subsp.subtilis) and desert subspecies (B.subtilis subsp.inaquosum). In the microbial application process, the deep mechanism research has positive significance for the development of the microbial industry and the crop yield.

However, further improvements are needed for the use of bacillus subtilis in fertilizers.

Disclosure of Invention

The present invention is directed to solving, at least to some extent, one of the technical problems in the related art. Therefore, the invention aims to provide the bacillus subtilis, the microbial agent, the composite microbial agent, the microbial fertilizer and the application thereof in the treatment of crops.

The invention provides a Bacillus subtilis subsp, which is preserved in the China general microbiological culture Collection center with the preservation number of CGMCC No. 21826. The strain provided by the invention is identified as bacillus subtilis through 16S rRNA gene, gyrB conserved gene, cell gram stain, colony morphology and physiological and biochemical characteristic analysis. And the sequencing result of the Bacillus subtilis gyrB conserved gene shows that the Bacillus subtilis tetrasp.subtilis 6051-HGWT is polymerized into the same branch with 99% support rate. The provided bacillus subtilis has an obvious growth promoting effect on wheat seeds, the growth promoting effect of a dish germination test reaches 52.13%, and metabolites contain growth promoting substances such as indoleacetic acid and the like.

In a second aspect of the present invention, there is provided a microbial inoculant comprising the bacillus subtilis of the first aspect.

The third aspect of the present invention provides a method for preparing a microbial agent, comprising: performing activation culture on the strain so as to obtain zymogen liquid; and carrying out amplification fermentation culture on the zymophyte liquid so as to obtain a fermentation product.

The fourth aspect of the present invention provides a complex microbial inoculant, which comprises a first microbial inoculant and a second microbial inoculant, wherein the first microbial inoculant is the microbial inoculant of the first aspect of the present invention, and the second microbial inoculant comprises at least one selected from bacillus amyloliquefaciens, bacillus licheniformis, bacillus laterosporus and bacillus coagulans.

In a fifth aspect, the invention provides a microbial fertilizer, which comprises the bacillus subtilis in the first aspect, or the microbial fertilizer in the second aspect, or the composite microbial inoculum in the third aspect.

The sixth aspect of the invention provides an application of bacillus subtilis in preparation of a microbial agent, a composite microbial agent or a microbial fertilizer, wherein the bacillus subtilis is the bacillus subtilis in the first aspect.

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

Information on strain preservation

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 Inward area of Beijing, and the preservation time is 2021 year, 02 month and 25 days.

Drawings

FIG. 1 is a graph showing the results of culturing of strain No. LY005 on TSA medium at 30 ℃ for 18 hours, provided according to an example of the present invention.

FIG. 2 is a diagram showing the results of phylogenetic analysis of the 16S rDNA gene sequence of strain LY005 provided in accordance with an embodiment of the present invention.

Fig. 3 is a graph showing the results of phylogenetic analysis of the gyrB gene sequence of strain LY005 provided according to an embodiment of the present invention.

Detailed Description

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

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

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

The invention provides a Bacillus subtilis subsp. subtilis which is preserved in the China general microbiological culture Collection center with the preservation number of CGMCC No. 21826. The provided strain is screened from rhizosphere soil of the spring sweet potatoes, the soil depth is 0-20cm, and the strain is separated and identified to be bacillus subtilis. This Bacillus subtilis is also numbered LY 005. The bacillus subtilis is positive in gram staining, and the bacillus subtilis is rod-shaped. On TSA medium, the colonies were nearly circular, pale yellow, with rectangular teeth on the edges. And the analysis result of the physiological and biochemical characteristics (whole cell fatty acid and API 50CH) of the Bacillus strain conforms to the biochemical metabolic characteristics of the Bacillus strain.

And the sequencing result of the Bacillus subtilis gyrB conserved gene shows that the Bacillus subtilis tetrasp.subtilis 6051-HGWT is polymerized into the same branch with 99% support rate. The provided Bacillus subtilis can be further divided into Bacillus subtilis subsp. Thus, the Bacillus subtilis also provided may be referred to in the expression as Bacillus subtilis subspecies. According to a specific embodiment of the present invention, Bacillus subtilis is provided having a 16S rDNA sequence shown in SEQ ID NO. 1. The 16S rDNA sequence encodes the 16S rRNA subunit. According to a specific embodiment of the present invention, Bacillus subtilis is provided with the gyrB gene sequence shown in SEQ ID NO. 2.

The germination test result shows that the provided strain has an obvious growth promoting effect on wheat seeds, the growth promoting effect reaches 52.13%, and the metabolite of the bacillus subtilis subspecies subtilis contains growth promoting substances such as indoleacetic acid and the like through detection.

The invention also provides a microbial agent which comprises the bacillus subtilis subspecies.

The invention also provides a preparation method of the microbial agent, which comprises the following steps: performing activation culture on the strain so as to obtain zymogen liquid; and carrying out amplification fermentation culture on the zymophyte liquid so as to obtain a fermentation product.

The culture medium for amplified fermentation culture comprises corn starch, sucrose, soybean meal, yeast powder, peptone, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, calcium carbonate, sodium chloride and defoaming agent. In some embodiments, the medium for the scale-up 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.

The invention also provides a compound microbial inoculum, which comprises a first microbial agent and a second microbial agent, wherein the first microbial agent is the microbial agent, and the second microbial agent comprises at least one of bacillus amyloliquefaciens, bacillus licheniformis, bacillus laterosporus and bacillus coagulans.

In at least some embodiments, the complex microbial inoculant comprises 30-35 parts by weight of the first microbial inoculant, and at least one selected from the group consisting of: 15-20 parts of bacillus amyloliquefaciens, 20-25 parts of bacillus licheniformis, 15-20 parts of bacillus laterosporus and 10-15 parts of bacillus coagulans.

In at least some embodiments, the composite microbial inoculum comprises not less than 1000 hundred million CFU of effective viable bacteria of bacillus subtilis subspecies per gram, and at least one selected from the following: the effective viable count of the bacillus amyloliquefaciens is at least 1000 hundred million CFU; the effective viable count of the bacillus licheniformis is at least 1000 hundred million CFU; the effective viable count of the bacillus laterosporus is at least 100 hundred million CFU; the effective viable count of the bacillus coagulans is at least 100 hundred million CFU. As shown herein, CFU is generally understood in the art as a colony forming unit.

The provided strain, or microbial agent or composite microbial agent can be used alone or added into a fertilizer for use. Therefore, the invention also provides a microbial fertilizer, which comprises the bacillus subtilis subspecies, or the microbial agent, or the compound microbial agent. In some embodiments, the microbial fertilizer contains one to five per thousand of the bacillus subtilis subspecies, one to five per thousand of the microbial agent, or one to five per thousand of the complex microbial agent. For example, two to five per thousand bacillus subtilis, two to five per thousand microbial agents, or two to five per thousand composite microbial agents are included. In some embodiments, the microbial fertilizer contains microorganisms with an effective viable count of at least 0.2 billion CFU per gram of microbial fertilizer. The preparation process of the provided microbial fertilizer comprises the following steps: the prepared compound microbial agent is uniformly mixed with anti-caking agent in the coating working section of organic and inorganic fertilizers and compound fertilizers, and then added according to 1-5 per mill, and the corresponding fertilizer, namely the microbial fertilizer, is obtained after stable production.

The microbial fertilizer further comprises a base fertilizer. The basic fertilizer can be organic and inorganic fertilizer, compound fertilizer and the like. For example, the organic and inorganic fertilizers are 15-40/S (15-8-17), and the compound fertilizers are 15-8-20/S. The microbial fertilizer can be prepared by the following method: uniformly mixing the basic fertilizer coating workshop section and the anti-caking agent, and then adding 1-5 per mill of microbial agent to obtain the microbial fertilizer. The microbial fertilizer contains at least 0.2 hundred million CFU of effective microbial viable bacteria.

The provided microbial fertilizer can be used for crops. For example, field experiments show that the fertilizer has the effects of increasing both production and income for peanuts and sweet potatoes, and the specific effects are as follows: compared with conventional fertilization and market products, the biological organic and inorganic fertilizer applied to spring peanuts increases the yield by 13.3% and 5.5% respectively; compared with the yield of a control group, the biological organic and inorganic fertilizer applied to the sweet potatoes is increased by 6.93 percent, and compared with the yield of the control group, the microbial compound fertilizer is increased by 5.8 percent. The microbial fertilizer prepared by the strain has the functions of promoting the growth of crops and improving the yield of the crops.

The invention also provides application of the bacillus subtilis subspecies in preparation of microbial agents, complex microbial agents or microbial fertilizers.

The invention also provides a crop fertilizing method, which comprises the following steps: applying an effective amount of bacillus subtilis subspecies, a microbial agent, a compound microbial agent or a microbial fertilizer to the crops, wherein the bacillus subtilis subspecies is the bacillus subtilis subspecies, the microbial agent is the microbial agent, the compound microbial agent is the compound microbial agent, and the microbial fertilizer is the microbial fertilizer. By applying fertilizer to crops, the growth of the crops can be obviously promoted, the yield of the crops is improved, and the effect is very obvious. The mentioned crops include but are not limited to potato, peanut, sweet potato, wheat, etc. For example, the field experiment result shows that the production of spring flowers is improved by 13.3%, and the yield of sweet potatoes is improved by 6.93%; the sweet potato yield is improved by 5.8 percent when the sweet potato is added into the compound fertilizer.

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 isolation and screening of strains

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 1.

TABLE 1 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.

EXAMPLE 2 identification of the strains

Morphological characteristics, physiological and biochemical characteristics, and molecular biological analysis were respectively performed on the strain numbered LY005 in example 1.

(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. 1.

(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:1)

The results of phylogenetic analysis of the 16S rDNA gene sequence are shown in FIG. 2.

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:2)

The phylogenetic analysis results of the gyrB gene sequences are shown in FIG. 3.

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 3 preparation of Complex microbial inoculum

Example 3 a complex microbial inoculum was prepared, comprising the steps of:

(1) inoculating bacillus subtilis subspecies 05-1 stored in a plate into a triangular flask (250ml) containing 100ml of LB culture solution by using an inoculating loop, culturing for 6h in a constant-temperature incubator at 37 ℃ and 210rpm to obtain a zymogen solution;

(2) inoculating the zymophyte liquid into a pilot-scale fermentation tank (10L-50L-500L), culturing at 37 ℃, and fermenting step by step. Spray drying after fermentation, and subpackaging, wherein the viable count is 1700 hundred million CFU. Mixing with diatomite and other materials to obtain the bacillus subtilis subspecies 05-1 microbial inoculum for adding in fertilizers. Wherein the formula of the culture medium is as follows: 3.0 to 3.5 percent of corn starch, 0.8 to 1.0 percent of cane sugar, 4.0 to 4.5 percent of soybean meal, 0.20 to 0.50 percent of yeast powder, 0.20 to 0.50 percent of peptone, 0.2 to 0.3 percent of dipotassium hydrogen phosphate and potassium dihydrogen phosphate respectively, 0.1 to 0.15 percent of calcium carbonate and sodium chloride, 0.3 to 0.5 percent of defoaming agent and the pH value is adjusted to 7.0.

According to the method, the bacillus amyloliquefaciens microbial inoculum, the bacillus licheniformis microbial inoculum, the bacillus laterosporus microbial inoculum and the bacillus coagulans microbial inoculum are respectively prepared. Then, the microbial inoculum was mixed according to the example given in table 2 to obtain a complex microbial inoculum.

The compounding examples of the microbial inoculum are as follows:

TABLE 2 compounding examples of each fungicide

Example 4 preparation and application of microbial Fertilizer

Example 4 the microbial fertilizer is prepared, and in the process of preparing the microbial fertilizer, the microbial fertilizer can be added in a coating working section after organic and inorganic fertilizers and compound fertilizers are granulated. Specifically, the compound microbial inoculum prepared in example 3 is uniformly mixed with anti-caking powder in an organic and inorganic fertilizer and compound fertilizer coating working section, and then added according to 1-5 per mill, the corresponding fertilizer obtained after stable production is the microbial fertilizer, and the effective viable count per gram in the prepared microbial fertilizer is determined to be not less than 0.2 million CFU. Wherein the used organic and inorganic fertilizers are 15-40/S (15-8-17), and the used compound fertilizers are 15-8-20/S.

And then the prepared microbial fertilizer is subjected to field effect verification, experiments are carried out in Tong-permit counties in Kaifeng city of Henan province and Zhengyang counties in Juma city of Henan province, and crops comprise sweet potatoes and spring peanuts. The fertilizer is the microbial fertilizer (the addition amount is 2 per mill, and the effective viable count per gram is not less than 0.2 hundred million CFU): 15-40/S (15-8-17) of biological organic and inorganic fertilizer and 15-8-20/S of microbial compound fertilizer.

Spring peanut is developed in Zhengyang county of Chengma shop, Henan province, and is provided with three groups of treatment groups, namely a control group 1, a control group 2 and a test group, wherein each group is treated for 3 cells, and each cell is 60m²The fertilizer application amount is 50 kg/mu. The control group 1 is applied by a conventional method, the control group 2 is applied by microbial fertilizer purchased in the market 15-40 (the effective viable count per gram is not less than 0.2 hundred million CFU), the test group is applied by the prepared biological organic and inorganic fertilizer 15-40/S (15-8-17), and 10 test groups are correspondingly arranged according to the microbial agent compounding example. The specific effects are shown in Table 3.

TABLE 3 spring peanut Each treatment test Effect

The test results show that the effect of the biological organic and inorganic fertilizers used in the test group is better overall, the yield is increased by 11.97-14.40% compared with that of the control group 1, and the yield is increased by 3.81-6.06% compared with that of the control group 2.

The sweet potato is developed in Kangcity of Henan province, and comprises control group 1, control group 2, and test group 1-20, wherein each group is treated for 3 cells, each cell is 60m²The control group 1 is a conventional organic and inorganic fertilizer, the control group 2 is a conventional compound fertilizer, the test groups 1-10 are biological organic and inorganic fertilizers 15-40/S (15-8-17), and the test groups 11-20 are microbial compound fertilizers 15-8-20/S. The fertilizer application rate of the control group 1 and the test groups 1-10 is 40 kg/mu. The fertilizer application rate of the control group 2 and the test groups 11-20 is 50 kg/mu. The specific effects are shown in tables 4 and 5.

TABLE 4 biological organic and inorganic fertilizer sweet potato treatment effect

TABLE 5 microbial Compound fertilizer sweet potato test Each treatment effect

The test result shows that the yield of the biological organic and inorganic fertilizer used in the test group is 15-40/S (15-8-17), which is increased by 5.56-8.25% compared with the yield of the control group 1; compared with the control group 2, the yield of the used microbial compound fertilizer is increased by 2.29-6.84%.

In conclusion, the microbial fertilizer containing the bacillus subtilis subspecies LY005 has the effects of increasing both production and income of peanuts and sweet potatoes, and the microbial fertilizer has the functions of promoting the growth of crops and improving the yield of the crops, and particularly has a more prominent effect of combining organic and inorganic fertilizers.

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

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

SEQUENCE LISTING

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

<120> bacillus subtilis and application thereof in fertilizer

<130> BI3211475

<160> 2

<170> PatentIn version 3.5

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<211> 1417

<212> DNA

<213> Bacillus subtilis

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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> 2

<211> 1180

<212> DNA

<213> Bacillus subtilis

<400> 2

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

optionally, the bacillus subtilis has a 16S rDNA sequence shown in SEQ ID No. 1;

optionally, the Bacillus subtilis has a gyrB gene sequence shown in SEQ ID NO. 2.

2. The Bacillus subtilis of claim 1, wherein the Bacillus subtilis gyrB conserved gene sequencing result shows 99% support rate for branching to Bacillus subtilis subsp.subtilis 6051-HGWT;

optionally, the metabolite of bacillus subtilis contains indoleacetic acid.

3. A microbial inoculant comprising the bacillus subtilis of claim 1;

optionally, the microbial agent is in a dry powder form, and the effective viable count of the bacillus subtilis contained in each gram of the microbial agent is at least 1000 hundred million CFU.

4. The method for producing a microbial agent according to claim 3, comprising:

performing activation culture on the strain so as to obtain zymogen liquid;

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

5. The method for preparing a microbial agent according to claim 4, wherein a medium for the amplified fermentation culture comprises corn starch, sucrose, soybean meal, yeast powder, peptone, dipotassium hydrogenphosphate, potassium dihydrogenphosphate, calcium carbonate, sodium chloride, and an antifoaming agent;

optionally, the medium for the amplified fermentation culture comprises, 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.

6. A complex microbial inoculant, which comprises a first microbial inoculant and a second microbial inoculant, wherein the first microbial inoculant is the microbial inoculant of claim 3, and the second microbial inoculant comprises at least one of bacillus amyloliquefaciens, bacillus licheniformis, bacillus laterosporus and bacillus coagulans;

optionally, the complex microbial inoculant comprises 30-35 parts by weight of the first microbial inoculant and at least one of the following components:

15-20 parts by weight of bacillus amyloliquefaciens,

20 to 25 parts by weight of Bacillus licheniformis,

15 to 20 parts by weight of Bacillus laterosporus,

10-15 parts by weight of bacillus coagulans.

7. The complex microbial inoculum according to claim 6, which contains the effective viable count of bacillus subtilis not less than 1000 hundred million per gram, and at least one of the following substances:

the effective viable count of the bacillus amyloliquefaciens is at least 1000 hundred million CFU;

the effective viable count of the bacillus licheniformis is at least 1000 hundred million CFU;

the effective viable count of the bacillus laterosporus is at least 100 hundred million CFU;

the effective viable count of the bacillus coagulans is at least 100 hundred million CFU.

8. A microbial fertilizer, characterized in that the microbial fertilizer comprises the bacillus subtilis of claim 1 or 2, or the microbial agent of claim 3, or the complex microbial agent of claim 6 or 7;

optionally, the effective viable count of microorganisms contained in each gram of the microbial fertilizer is at least 0.2 hundred million CFU;

optionally, the microbial fertilizer contains one to five thousandth of the bacillus subtilis, one to five thousandth of the microbial agent, or one to five thousandth of the composite microbial agent;

optionally, the microbial fertilizer further comprises a base fertilizer, and the base fertilizer is selected from at least one of compound fertilizers and organic and inorganic fertilizers.

9. Use of the bacillus subtilis of claim 1 or 2 for preparing a microbial agent, a complex microbial agent or a microbial fertilizer.

10. A method of fertilizing a crop, comprising:

applying an effective amount of bacillus subtilis according to claim 1 or 2, a microbial agent according to claim 3, a complex microbial agent according to claim 6 or 7 or a microbial fertilizer according to claim 8 to the crop;

optionally, the crop is selected from at least one of peanut, potato and sweet potato.