CN112877258A - Bacillus subtilis JX-20 strain and application thereof - Google Patents

Abstract

The invention provides a bacillus subtilis JX-20 strain and application thereof, and relates to the technical field of microbial fertilizers. The JX-20 strain of the invention is deposited with a deposition number of GDMCCNo: 61546. The JX-20 strain has the beneficial effects of antagonizing pathogenic microorganisms, restoring and optimizing the ecology of a beneficial microorganism group in soil, decomposing phosphorus and potassium, increasing the availability and nutrition of crops, improving the immunity and stress resistance of the crops, promoting the healthy growth of the crops, increasing the yield and improving the quality, and can be used for preparing a plant antibacterial agent and an anti-nematode agent, promoting the growth of the plants and improving the resistance of the plants. The invention also provides a microbial fertilizer based on the JX-20 strain, which has the effects of antagonizing plant pathogens, restoring and improving the ecological environment of beneficial microbial groups, reasonably providing and balancing nutrient elements available for plants and promoting the healthy growth of the plants, and can be used for various types of soil, different crops and growth periods.

Description

Bacillus subtilis JX-20 strain and application thereof

Technical Field

The invention belongs to the technical field of microbial fertilizers, and particularly relates to a bacillus subtilis JX-20 strain and application thereof.

Background

China has more population and less cultivated land, and high yield per unit area has to be pursued in order to meet the food demand of people. In order to realize the aim, the planting in a protected area, the improvement of the multiple cropping index, the continuous cropping and the increase of the input of chemical fertilizers and pesticides become important measures for pursuing and ensuring the high yield of agricultural products. However, continuous cropping obstacles caused by increase of soil-dwelling pathogens, deterioration of soil nutrition and the like limit sustainable development of crop planting industry, so that the dosage of chemical fertilizers and pesticides continues to increase, and ecological environment safety and food quality safety are seriously threatened.

After the fertilizer is used in a large amount for a long time, soil hardening, accumulation of plant pathogenic microorganisms, change of beneficial microorganism ecological communities, loss of N, P, K large amount of nutrient elements, and difficulty in absorption of plant nutrient elements such as P and K fixed in soil by plants result in further increase of the fertilizer dosage and continuous deterioration of soil environment. With the knowledge of the plant nutrition demand rule and the nutrition absorption and utilization obstacle and mechanism, the microbial fertilizer is developed and used by modern biotechnology, so that the soil and the plant leaf periphery can repair and maintain good microbial clusters, the life activities and metabolites of microbes, such as polypeptide, amino acid, oligosaccharide or polysaccharide, fatty acid, biotin and the like, are utilized to provide direct biological nutritional ingredients for plants, the secretion products of microbes, such as protease and the like, are utilized to change the physical and chemical properties of the soil, the plant nutritional elements fixed by the soil are dissociated, the absorption and utilization of the nutrition of the plants are promoted, the antagonism of biological active substances, such as antibacterial peptide and the like, generated by the microbial fertilizer on plant pathogenic microorganisms and the induction of defense reaction on the plants are utilized to reduce the infection of pathogenic plants and increase the disease resistance of the crops, the microbial ecological environment is improved, the absorption and utilization of the nutrition of the plants are improved, promoting healthy growth and reducing the dosage of chemical fertilizers and pesticides.

Disclosure of Invention

In view of the above, the invention aims to provide a bacillus subtilis JX-20 strain and an application thereof, wherein the JX-20 strain has biological effects of antagonizing pathogenic microorganisms, restoring soil microorganism ecology, de-phosphorizing and de-potassium, increasing crop availability nutrition, improving crop immunity and stress resistance, promoting crop healthy growth, increasing yield and improving quality, and can be applied to preparation of safe and efficient biological fertilizers and biological pesticides.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a Bacillus subtilis JX-20 strain, wherein the preservation number of the JX-20 strain is GDMCC No. 61546.

Preferably, the nucleotide sequence of 16S rDNA of the JX-20 strain is shown in SEQ ID NO. 1.

The invention also provides application of the JX-20 strain or a fermentation product of the JX-20 strain in preparation of plant antibacterial agents and nematode resisting agents.

Preferably, the antibacterial spectrum of the plant antibacterial agent includes angular leaf spot of cucumber (Xanthomonas campestris), ralstonia solanacearum (Pseudomonas syringa), Chinese cabbage soft rot (Erwinia carotovora), Sclerotinia sclerotiorum (Sclerotinia sclerotiorum), Rhizoctonia solani (Rhizoctonia solani), rice seedling blight (Fusarium moniliforme), Magnaporthe oryzae (Magnaporthe grisea), Gibberella tritici (Fusarium spp.), Rhizoctonia cerealis (Rhizoctonia cerealis), early blight of tomato (Alternaria solani), Botrytis cinerea (Botrytis cinerea), Cucumis sativus (Fusarium oxysporum), potato late blight (Phytophora solani), yellow rot fungus (Rhizophora solani), melon blight (Rhizophora solani), and Cucumis cucumis (Cucuminum graminis) and Mucor melo graminis (Cucuminum graminearum);

the anti-nematode spectrum of the anti-nematode agent includes Meloidogyne incognita (melodogyne incognita).

Preferably, the fermentation product is prepared by fermenting the JX-20 strain in an NB culture medium for 48-72 hours, wherein the NB culture medium comprises the following components in concentration: 15g/L of sucrose, 5g/L of polypeptone, 1g/L of yeast powder and 3g/L of beef extract.

The invention also provides application of the JX-20 strain or a fermentation product of the JX-20 strain in promoting plant growth and/or improving plant resistance.

The invention also provides a microbial fertilizer based on the JX-20 strain, which comprises a zymogen liquid of the JX-20 strain of the bacillus subtilis, and also comprises major elements and trace elements;

the zymophyte liquid comprises 50 hundred million to 6000 hundred million spores per gram of microbial fertilizer and secondary metabolites accounting for 0.3 to 1.5 percent of the mass of the microbial fertilizer;

the total mass of the macroelements is 5-50% of the mass of the microbial fertilizer, and the mass of the microelements is 0.1-1% of the mass of the microbial fertilizer;

the macroelements include N, P and K;

the microelements comprise calcium, magnesium, zinc, copper, sulfur, iron, boron, manganese, chlorine and sodium.

Preferably, the secondary metabolite of the bacillus subtilis JX-20 strain comprises a mixture of one or more of oligosaccharides, fatty acids, amino acids, polypeptides and biotin.

Preferably, the microbial fertilizer also comprises a survival carrier and an auxiliary agent, wherein the mass of the survival carrier is 10-85% of the mass of the microbial fertilizer, and the mass of the auxiliary agent is 2-15% of the mass of the microbial fertilizer;

the survival carrier is selected from one or more of water, attapulgite, kaolin, light calcium carbonate, ammonium sulfate, borate, ethylene diamine tetraacetate, melamine, urea, ammonium sulfate, monopotassium phosphate, corn flour, soybean meal, humus, stable manure, human excrement, poultry manure, compost and talcum powder;

the auxiliary agent is selected from one or more of ethylene glycol, propylene glycol, NNO-1, NNO-7, xanthan gum, polyethylene glycol, glycerol, sodium dodecyl sulfate, sodium dodecyl benzene sulfonate, ammonium sulfate, urea, alkylphenol polyoxyethylene ether phosphate, fatty acid polyoxyethylene ester, benzoic acid, lauric acid, sodium lignin sulfonate, carboxymethyl cellulose, polyvinyl alcohol and potassium ferrocyanide.

The invention also provides a method for improving the crop yield and the disease and insect resistance by using the microbial fertilizer, which comprises the following steps: the microbial fertilizer is used as a base fertilizer, an additional fertilizer or a leaf fertilizer and is applied to the field according to the use amount of 15-450 kg/hectare;

or 10-3000 g of the microbial fertilizer is used for dressing or coating every 100kg of crop seeds.

The invention provides a bacillus subtilis JX-20 strain, wherein the JX-20 strain has the beneficial effects of antagonizing pathogenic microorganisms, repairing and optimizing the ecology of beneficial microorganism groups of soil, dissolving phosphorus and potassium, increasing the availability and nutrition of crops, improving the immunity and stress resistance of the crops, promoting the healthy growth of the crops and increasing the yield and quality, and oligosaccharides (extracellular polysaccharide, EPS), fatty acids, amino acids, polypeptides and biotin generated by metabolism not only have the biological fertilizer effect, but also have the biological effects of antagonizing the pathogenic microorganisms, inducing the disease resistance of the crops and promoting the healthy growth of the crops, so that the JX-20 strain can be used for preparing plant antibacterial agents and nematode inhibitors, promoting the growth of the plants and improving the plant resistance.

The invention also provides a microbial fertilizer based on the JX-20 strain, which breaks through the technical obstacles that microorganisms are easy to die in the transportation and storage processes and cause reduction or instability of biological effects, greatly improves the contents of spores, oligosaccharides, fatty acids, amino acids, polypeptides and biotin in fermentation products, ensures that the microbial fertilizer has the effects of antagonizing plant pathogens, restoring and improving the ecological environment of beneficial microbial groups, reasonably providing and balancing nutrient elements available for plants and promoting the healthy growth of the plants. The bacillus subtilis microbial fertilizer has wide application, can be used for various types of soil, different crops and growth periods, has various application methods, and can be applied before seeding and transplanting of the crops and in the growth period.

Biological preservation information

Bacillus subtilis JX-20 is preserved in Guangdong province microbial strain preservation center in 2021, 3, 4 days, with the preservation address of Guangzhou city Michelia Tokyo No. 59, 5, Guangdong province microbial research institute, the preservation number is GDMCC NO: 61546.

Detailed Description

The invention provides a Bacillus subtilis JX-20 strain, wherein the preservation number of the JX-20 strain is GDMCCNo: 61546.

The nucleotide sequence of 16S rDNA of the JX-20 strain is preferably shown in SEQ ID NO. 1. The JX-20 strain and the metabolite thereof have broad-spectrum antibiotic effect on pathogens, reduce the population quantity of pathogenic fungi, bacteria and nematodes in soil, leaf surfaces and seeds, repair and optimize the microbial ecology of soil environment, inhibit the growth development and infection hazards of plant pathogens, induce plant defense reaction, promote the healthy growth of plants, and particularly promote the healthy growth of cucumber angular leaf spot pathogen (Xanthomonas campestris), tomato ralstonia solanacearum (pseudomonis syringae), Chinese cabbage soft rot pathogen (Erwinia carotovora), Sclerotinia sclerotiorum (Sclerotinia sclerotiorum), Rhizoctonia solani (Rhizoctonia solani), rice bakanae (Fusarium moniliforme), rice blast fungus (Magnaporthe grisea), wheat scab (Fusarium graminearum), wheat scabies (Fusarium graminearum), tomato early blight fungus (Rhizoctonia solani), and strawberry blight (Rhizoctonia solani), and early blight (Botrytis) of cucumber, 15 pathogenic bacteria and fungi such as potato late blight bacteria (Phytophthora infestans), Verticillium solani (Verticillium dahliae) and Pythium cucumerinum (Pythium aphanidermatum) can generate a bacteriostatic circle with the diameter of 15.5-24.5 mm, wherein the antagonistic capacity to sclerotinia sclerotiorum and rhizoctonia cerealis is strongest, and the diameter of the bacteriostatic circle reaches 20.3-24.5 mm; the antagonism to the botrytis cinerea is the weakest, but the inhibition zone still reaches 15.5 mm. Meanwhile, the NB fermentation product of JX-20 also has the effects of preventing and treating Erysiphe graminis, and can also obviously inhibit the hatching of oocysts of tomato and cucumber root-knot nematodes (Meloidogyne incognita). The NB fermentation product is preferably prepared by fermenting the JX-20 strain in an NB culture medium for 48-72 h, wherein the NB culture medium comprises the following components in concentration: 15g/L of sucrose, 5g/L of polypeptone, 1g/L of yeast powder and 3g/L of beef extract. The temperature of the fermentation according to the invention is preferably 28 ℃ and the fermentation is preferably carried out on a shaker, the shaking frequency of which is preferably 150 rpm.

The invention also provides application of the JX-20 strain or a fermentation product of the JX-20 strain in preparation of plant antibacterial agents and nematode resisting agents.

The antibacterial spectrum of the plant antibacterial agent of the present invention preferably includes Xanthomonas campestris (Xanthomonas campestris), Ralstonia solani (Pseudomonas solanacearum), Brassica rapa (Erwinia carotovora), Sclerotia sclerotiorum (Sclerotinia sclerotiorum), Rhizoctonia solani (Rhizoctonia solani), Rhizoctonia oryzae (Fusarium moniliforme), Pyricularia oryzae (Magnaporthe grisea), Rhizoctonia cerealis (Fusarium graminearum, F.asiticum), Rhizoctonia cerealis (Rhizoctonia cerealis), Phytophthora solani (Alternaria solani), Botrytis cinerea (Botrytis cinerea), Fusarium oxysporum (Fusarium oxysporum), Solanum tuberosum (Phytophthora tenera), Phytophthora graminis (Blastomyces), and Cucumis sativum (Cucumis cinerea) bacteria (Cucuminum solani); the anti-nematode profile of the anti-nematode agent preferably comprises meloidogyne incognita. The preparation method of the fermentation liquid of the present invention is preferably the same as above, and will not be described herein again.

The invention also provides application of the JX-20 strain or a fermentation product of the JX-20 strain in promoting plant growth and/or improving plant resistance.

The method for preparing the fermentation product of the JX-20 strain of the present invention is preferably the same as described above, and thus, it is not described herein again. The JX-20 strain has the capabilities of dissolving phosphorus, potassium and nitrogen, and the leavening of the JX-20 strain has obvious promotion effect on the growth of wheat, rape and cucumber in seedling stage, and can improve the resistance to diseases and insect pests and the like.

The invention also provides a microbial fertilizer based on the JX-20 strain, which comprises a zymogen liquid of the JX-20 strain of the bacillus subtilis, and also comprises major elements and trace elements; the zymophyte liquid comprises 50 hundred million to 6000 hundred million spores per gram of microbial fertilizer and secondary metabolites accounting for 0.3 to 1.5 percent of the mass of the microbial fertilizer; the total mass of the macroelements is 5-50% of the mass of the microbial fertilizer, and the mass of the microelements is 0.1-1% of the mass of the microbial fertilizer; the macroelements include N, P and K; the microelements comprise calcium, magnesium, zinc, copper, sulfur, iron, boron, manganese, chlorine and sodium.

In the present invention, the method for producing spores is not particularly limited, and fermentation is preferably carried out in NB medium using the JX-20 strain. In the fermentation process, the invention also produces secondary metabolites, preferably including oligosaccharides, fatty acids, amino acids, polypeptides and biotin, preferably including xanthan gum, glucose, mannose, galactose and glucuronic acid. The biotin of the invention is also called vitamin H and coenzyme R, is water-soluble vitamin, also belongs to vitamin B group, and has the function of regulating and controlling the growth of plants. The total mass of N, P and K in the microbial fertilizer is 5% -50% of the microbial fertilizer, and the ratio of N, P to K is preferably (6-45): (6-12): 6. the secondary metabolite can fix N and dissociate P and K ions fixed by soil, and increase the absorption and utilization of P and K nutrient elements by plants.

The source of N, P and K in the microbial fertilizer of the invention preferably comprises bacillus subtilis JX-20 fermentation product and 1 or more inorganic fertilizers and/or organic fertilizers selected from ammonium nitrate, ammonium chloride, ammonium bicarbonate, urea, ammonium sulfate, melamine, calcium phosphate, triple superphosphate, rich superphosphate, ammonium phosphate, ground phosphate rock, calcium magnesium phosphate, calcium phosphate, potassium sulfate, potassium chloride, potassium nitrate, potassium ferrocyanide, monopotassium phosphate, potassium dihydrogen phosphate, potash spar fume, potassium magnesium salt, potassium ferrocyanide, calcium nitrate, copper sulfate, magnesium sulfate, zinc chloride, manganese nitrate, manganese chloride, ferric sulfate, borate, sodium chloride, edetate, corn flour, bean pulp, plant ash, human excrement, stable manure, compost, green manure, cake manure, biogas manure, humus. The trace elements are preferably selected from one or more of calcium phosphate, triple superphosphate, rich superphosphate, potassium magnesium salt, potassium ferrocyanide, calcium nitrate, copper sulfate, magnesium sulfate, zinc chloride, manganese nitrate, manganese chloride, ferric sulfate, borate and sodium chloride, and in the microbial fertilizer, the trace elements are mixed in equal mass.

The microbial fertilizer also preferably comprises a survival carrier and an auxiliary agent, wherein the mass of the survival carrier is preferably 10-85% of that of the microbial fertilizer, and the mass of the auxiliary agent is preferably 2-15% of that of the microbial fertilizer; the survival carrier is preferably selected from one or more of water, attapulgite, kaolin, light calcium carbonate, ammonium sulfate, borate, ethylene diamine tetraacetate, melamine, urea, ammonium sulfate, monopotassium phosphate, corn flour, soybean meal, humus, stable manure, human excrement, poultry manure, compost and talcum powder; the auxiliary agent is preferably selected from one or more of ethylene glycol, propylene glycol, NNO-1, NNO-7, xanthan gum, polyethylene glycol, glycerol, sodium dodecyl sulfate, sodium dodecyl benzene sulfonate, ammonium sulfate, urea, alkylphenol polyoxyethylene ether phosphate, polyoxyethylene fatty acid, benzoic acid, lauric acid, sodium lignin sulfonate, carboxymethyl cellulose, polyvinyl alcohol and potassium ferrocyanide.

The formulation of the microbial fertilizer is not particularly limited, and preferably comprises tablets, granules, water dispersible granules, powder, wettable powder, dry suspension, seed coating or aqueous solution.

The invention also provides a method for improving the crop yield and the disease and insect resistance by using the microbial fertilizer, which comprises the following steps: the microbial fertilizer is used as a base fertilizer, an additional fertilizer or a leaf fertilizer and is applied to the field according to the use amount of 15-450 kg/hectare;

or 10-3000 g of the microbial fertilizer is used for dressing or coating every 100kg of crop seeds.

The microbial fertilizer can be used for spreading, hole application and furrow application according to 15-450 kg per hectare before or during planting or in the growth period of plants, or diluted by water according to the ratio of 1: 200-1: 20000 and then irrigated, drip-irrigated or sprayed; or before sowing, 10-3000 g of the microbial fertilizer is used for mixing seeds or coating per 100kg of crop seeds.

The following examples are provided to illustrate the JX-20 strain of Bacillus subtilis and its application in detail, but they should not be construed as limiting the scope of the invention.

Example 1

Bacillus subtilis JX-20 strain source and identification

1. JX-20 Strain Source and screening

Separating from soil for planting tomato and cucumber in sunlight greenhouse by plate dilution method, culturing on solid culture medium plate in opposition to bacterial blight of rice and Sclerotinia sclerotiorum, and culturing in solid culture medium plate containing insoluble phosphate Ca₃(PO₄)₂The bacterial strain JX-20 which is the only phosphorus source and can be screened on a culture medium which can grow on both an Ashby culture medium and a potassium bacteria selective culture medium and has the functions of dissolving phosphorus, dissolving potassium, fixing N and resisting the plant pathogens.

2. JX-20 Strain identification

(1) And (5) physiological and biochemical identification. Referring to the conventional method of bacterial identification, 13 physiological and biochemical characteristics of JX-20 were determined in comparison with the physiological and biochemical characteristics of Bacillus subtilis in Berger's Manual of bacterial identification (eighth edition), and the results were identical to those of Bacillus subtilis (Table 1). Therefore, JX-20 can be identified as Bacillus subtilis.

TABLE 1 comparison of the physiological and biochemical Properties of JX-20 with Bacillus subtilis Properties

Note: "+" indicates that the reaction is positive or can be grown and utilized; "-" is negative or not able to grow and be used.

(2)16S rDNA sequence and its homology identification. Further adopting modern molecular biology identification technology, amplifying 16S rDNA fragments with the size of about 1500bp from the genome of the strain JX-20 by using the universal primers 27F and 1492R of bacteria, cloning the fragments onto a PMD19-T simple vector, and sequencing to obtain the full sequence of 1505bp of the 16S rDNA fragments. BLAST homology comparison is carried out on NCBI, and the sequence is found to achieve 100 percent of sequence homology with 16S rDNA of 20 strains of Bacillus subtilis strains, wherein the 16S rDNA sequence of the Bacillus subtilis strain YJ001 strain with the accession number of DQ444283.1 is 1511bp in size, the sequence homology is closest to the 16S rDNA of JX-20 and also achieves 100 percent of sequence comparison homology, and the JX-20 strain is further identified to be Bacillus subtilis (Bacillus subtilis) as a result.

Example 2

Biological characteristics of Bacillus subtilis JX-20 Strain

1. Antibiotic biological Activity of JX-20

The pathogens isolated and identified from different crops include Corynespora cucumerinum (Xanthomonas campestris), Ralstonia solani (Pseudomonas solanacearum), Botrytis brassicae (Erwinia carotovora), Sclerotia sclerotiorum (Sclerotinia sclerotiorum), Rhizoctonia solani (Rhizoctonia solani), Rhizoctonia oryzae (Fusarium moniliforme), Pyricularia oryzae (Magnaporthe grisea), Rhizoctonia cerealis (Fusarium graminearum, F. asiticum), Rhizoctonia cerealis (Rhizoctonia cerealis), Phytophthora solani (Alternaria solani), Botrytis cinerea (Botrytis cinerea), Rhizoctonia solani (Fusarium oxysporum), Rhizoctonia solani (Phytophora solani), Pseudoperonospora fragilis (Botrytis cinerea) and the species (Pythium cucurbitae) which are cultured in a medium and the central strain (Pythium cucurbitae) and the 4. solanacearum and the strain (Pythium cucurbitacearum) can be cultured in a species of the central strain and the 4-3-15-3-d-medium, and then 100 mu L of each fermentation filtrate is added into an Oxford cup, the Oxford cup is placed under the dark condition of 25 ℃ for culturing for 72 hours, and the width of the inhibition zone is measured), and the result shows that the JX-20 can generate the inhibition zone with the diameter of 15.5 mm-24.5 mm for the pathogenic bacteria and the fungi. Wherein the antagonism ability to Sclerotinia sclerotiorum and Rhizoctonia cerealis is strongest, and the diameter of the inhibition zone reaches 20.3 mm-24.5 mm; the antagonism to the botrytis cinerea is the weakest, but the inhibition zone still reaches 15.5 mm.

JX-20 NB broth (sucrose 15g, polypeptone 5g,1g of yeast powder, 3g of beef extract and ddH₂O to 1000ml) is sprayed and treated for 24 hours, then powdery mildew is inoculated, or sprayed and treated for 24 hours after powdery mildew is inoculated, and then cultivated in a greenhouse for 7 days to investigate the preventing and treating effects on the powdery mildew. The results (Table 2) show that the fermentation liquor of the strain has good prevention and treatment effects on wheat powdery mildew.

TABLE 2 protective and therapeutic Effect of JX-20 fermentation broth on wheat powdery mildew

Note: the capital letters in the disease index indicate that LSD is more heteropolar (P <0.01) and the lowercase letters indicate that LSD is more heteropolar (P <0.05), the same below.

Inoculating a JX-20 strain into an NB culture solution, shaking at 150rpm for 72h, treating tomato and cucumber root-knot nematode (Meloidogyne incognita) oocysts with filtrate of a bacterial filter, and incubating at 28 ℃ for 72h, wherein the incubation rate is reduced by 52.6-64.3% compared with that of tap water control treatment; when the second-instar larvae of the root-knot nematodes are treated, the death rate of the larvae in 48 hours reaches 85-96%.

The research result shows that the strain JX-20 has biological characteristics of broad-spectrum antibiotic activity and strong antagonistic capability on plant pathogenic bacteria, fungi and nematodes, and has certain biocontrol potential.

2. JX-20 ability to dissolve phosphorus, potassium and nitrogen

Adding 0.5 percent (volume: weight ratio) of phosphate rock powder (sieving with a 700-mesh sieve) into a phosphorus-free NB liquid culture medium, inoculating a JX-20 strain, shaking and culturing for 7 days at 28 ℃ and 180r/min, and measuring the content of soluble phosphorus (P capable of being absorbed and utilized by plants) in the culture solution by adopting a molybdenum-antimony colorimetric resistance method, wherein the content of the soluble phosphorus in the JX-20 culture solution is found to reach 5.43 mu g/mL, while the content of the soluble phosphorus in a control bacillus subtilis strain is only 2.25 mu g/mL, and the phosphorus-dissolving capacity is increased by 2.4 times.

In a potash feldspar-containing liquid culture medium, a JX-20 strain is inoculated, and the culture solution is shaken for 7 days at the temperature of 28 ℃ and at the speed of 180r/min, the content of soluble potassium in the culture solution is 53.4 mu g/mL measured by an atomic absorption method, while the content of soluble potassium in the control bacillus subtilis strain is only 19.8 mu g/mL, and the potassium-dissolving capacity is increased by 2.7 times.

JX-20 strains formed smooth, translucent brown to dark brown colonies on Ashby medium, indicating the ability to fix nitrogen spontaneously. The measured mass (micromole number) of the substance of the JX-20 strain fermentation liquor which reduces acetylene to generate ethylene in unit time is 0.25 mu mol/g.h, and the JX-20 strain fermentation liquor has certain nitrogen fixation capacity.

3. Metabolic nutrients of bacillus subtilis JX-20 strain

(1) Oligosaccharide, fatty acid

After shaking JX-20 in NB medium at 28 ℃ and 180rpm for 3 days, the supernatant was obtained by centrifugation. The relative viscosity of the culture broth measured at 25 ℃ with an NDJ-1 viscometer was 1.55 per mg of dry cell weight, which corresponds to about 15% to 18% glucose. Extraction by ethanol precipitation and redissolution with double distilled water and salt saturation of phenol and chloroform: strong shaking and centrifuging n-butanol (5:1), adding 95% ice-cold ethanol into the water phase for coagulation, centrifuging, washing with anhydrous ethanol to obtain white purified Exopolysaccharide (EPS), and performing hydrolysis with 0.5mol/L hydrochloric acid and high performance liquid chromatography to find that the exopolysaccharide of JX-20 mainly contains xanthan gum, glucose, mannose, galactose, glucuronic acid and fatty acid.

(2) Amino acids, proteins and polypeptides

Amino acid can be chemically reacted with a derivatization agent ninhydrin to generate a blue purple compound, and JX-20 can be metabolized in an NB culture medium to generate glutamic acid, tyrosine, phenylalanine and the like through detection of a high performance liquid chromatography, wherein the weight of the JX-20 accounts for 2.5-5.5% of the dry weight of thalli.

By adopting a Coomassie brilliant blue determination method, the extracellular protein content in the NB culture solution of JX-20 reaches 28-35 mu g/mg dry weight of the thallus, and the total content of the extracellular protein and the intracellular protein reaches 52.8-59.5 percent of the dry weight of the thallus. The main protease and amylase have the biological activities of degrading protein into amino acid and degrading starch into sugar, and are beneficial to absorption and utilization by plants.

(3) Biotin assay

The JX-20 can secrete biotin 0.05-0.08 mu g/g dry weight of thallus by adopting a Difco biotin test medium and a biotin Assay kit (Colorimetric).

(4) Induction and disease resistance of JX-20 culture solution to plant defense reaction

The JX-20 strain is inoculated into NB culture solution, shaken for 72H, and sprayed with 5-fold, 10-fold and 50-fold diluent to treat the Arabidopsis thaliana, and the disease resistance marker substances, namely active oxygen and callose in leaves are measured after 12H and 24H treatment, and the active oxygen and callose generated by spraying the 50-fold diluent are found to be the most, and the active oxygen H is₂O₂Respectively 9.3 μm/g and 9.8 μm/g fresh weight leaf, callose accounting for 0.64% and 2.4% of fresh weight of leaf, and disease resistant substance decreasing after 36 hr treatment.

After spraying 50 times of diluent of JX-20 strain fermentation liquor, spraying and inoculating pseudomonas syringae P.syringae for 12 hours, and after 5 days, the index of disease is reduced by 64 percent compared with that of clear water control treatment. Further research shows that the transcriptional expression level of PR1 gene in the leaf of Arabidopsis thaliana is up-regulated by 2.6 times compared with that of clear water treatment 24 hours after 50 times of dilution treatment of the culture solution of the JX-20 strain, which indicates that the culture solution of the JX-20 strain can induce the defense response of the salicylic acid pathway of plants.

(5) Colonization kinetics after JX-20 application

And after the obtained rifampicin-resistant marked JX-20 strain culture solution is used for soaking roots to treat rape seedlings for 90min, the rape seedlings are respectively transferred into pots filled with sterile soil and natural soil. Transplanting for 1, 4, 7, 10, 15, 20, 25, 30, 35 and 40 days, randomly pulling 10 strains from each treatment, washing, cutting the plantlets into three sections of roots, stems and leaves, weighing the weight of the three sections, grinding, coating the juice of the three sections onto an NA (20 g agar/L) plate containing 450 mu g/mL rifampicin in an NB culture medium, culturing for 24 hours at 28 ℃, checking the number of separated colonies, and determining the bacteriostatic activity of the separated bacteria on sclerotinia sclerotiorum.

The number of JX-20 colonies isolated from the rape plants (Table 3) showed that JX-20 was able to colonize not only the rape plants transplanted in natural and sterilized soil but also to distribute from the roots to the leaves. The JX-20 colonization quantity in the rape body cultivated in natural soil and sterilized soil is root > stem > leaf part, and the time for colonization in the leaf part is shorter than that in the root part. Except for the roots of the sterilized soil, the colonization number of the JX-20 in each tissue of the rape tends to increase firstly and then decrease, and the number of the JX-20 in each tissue of the plant after 10 days changes little and tends to be stable.

TABLE 3 Rifampicin-tagged JX-20 dynamics of colonization number in oilseed rape at different days post inoculation (× 10)³CFU/g fresh weight)

(6) Promoting effect of JX-20 on seedling growth of wheat, rape and cucumber

After the wheat in the seedling stage is subjected to root irrigation treatment for 15 days by using JX-20 fermentation stock solution and 100 times of diluent, the plant height, the fresh weight and the dry weight of wheat seedlings are obviously changed compared with those of a control, but the plant height is relatively small in growth promotion effect, and the JX-20 has the growth promotion effect of a microbial fertilizer on the growth of the wheat in the seedling stage (Table 4).

TABLE 4 influence of JX-20 on growth of wheat at seedling stage

Treatment of

Plant height/cm

Growth promoting rate (%)

Fresh weight (g/plant)

Growth promoting rate (%)

Dry weight (mg/strain)

Growth promoting rate (%)

Stock solution

16.5aA

6.5

0.35aA

25.0

32aA

18.5

Diluting by 100 times

15.91aA

2.6

0.30abA

7.1

29bA

7.4

CK

15.5aA

0.28bA

27bA

After the rape in the seedling stage is subjected to root irrigation treatment by using JX-20 fermentation stock solution and 100 times of diluent, the plant height, fresh weight and dry weight are all remarkably improved compared with those of a control (Table 5). Wherein the growth promoting rates of the rape plants are respectively 30.6 percent and 13.9 percent, the growth promoting rates of the fresh weight are respectively 41.9 percent and 6.5 percent, and the growth promoting rates of the dry weight of the rape are respectively 52.2 percent and 6.8 percent. The JX-20 has obvious biological fertilizer growth promotion effect on the rape.

TABLE 5 Effect of JX-20 on the growth of oilseed rape at seedling stage

Treatment of

Plant height/cm

Growth promoting rate (%)

Fresh weight (g/plant)

Growth promoting rate (%)

Dry weight (mg/strain)

Growth promoting rate (%)

Stock solution

9.4aA

30.6

0.44aA

41.9

24.5aA

52.2

Diluting by 100 times

8.2cC

13.9

0.33cB

6.5

17.2cC

6.8

CK

7.2dD

/

0.31cB

/

16.1cC

/

After the seedlings of cucumbers are subjected to root irrigation treatment by using JX-20 fermentation stock solution and 100 times of diluent, the plant height, the fresh weight and the dry weight of the seedlings of cucumbers are remarkably improved compared with a control, wherein the growth promoting rates of the plant height are respectively 17.9% and 7.5%, the growth promoting rates of the plant height are respectively 34.5% and 16.8% on the fresh weight, and the growth promoting rates of the plant height on the dry weight of the cucumbers are respectively 28.8% and 12.3% (Table 6). The JX-20 also has obvious growth promoting effect on the microbial fertilizer for the cucumber.

TABLE 6 influence of JX-20 on cucumber seedling growth

Treatment of

Plant height/cm

Growth promoting rate (%)

Fresh weight (g/plant)

Growth promoting rate (%)

Dry weight (mg/strain)

Growth promoting rate (%)

Stock solution

12.5aA

17.9

1.52aA

34.5

94aA

28.8

Diluting by 100 times

11.4AB

7.5

1.32bB

16.8

82cC

12.3

CK

10.6cB

/

1.13cC

/

73dD

/

Example 3

Application effect of bacillus subtilis JX-20 microbial fertilizer

1. 5500 hundred million spores/g bacillus subtilis microbial fertilizer granules have using effect

850g of 6500 hundred million bacillus subtilis JX-20 spores/g of dry powder is taken, and urea, ammonium sulfate, calcium phosphate and potassium phosphate which are crushed to 300 meshes are added to ensure that the final content of N, P, K (6:2:2) is 10 percent; adding calcium nitrate, zinc sulfate, manganese chloride, sodium tetraborate and organosilicon quaternary ammonium salt to make the final content of the mixed trace elements equal to 0.25%; 5 percent of 1:0.3:1 polyethylene glycol, ethylene diamine tetraacetic acid disodium/calcium and sodium lignosulphonate are added, kaolin is used as a carrier to supplement the total weight of 1000g, a small amount of water is added for wetting, and the bacillus subtilis microbial fertilizer tablet/granule with 5500 hundred million spores per gram is prepared.

750 g of 4000 hundred million bacillus subtilis JX-20 spores/g of dry powder is taken, and urea, ammonium sulfate, monopotassium phosphate, potassium nitrate, potassium chloride, calcium nitrate, magnesium sulfate, copper sulfate, zinc sulfate, manganese nitrate and ferrous sulfate which are crushed into 300 meshes are added to ensure that the final content of N, P, K (6:2.5:1.5) is 18 percent and the final content of equivalent mixed trace elements is 0.45 percent; adding 12% of the total weight of polyoxyethylene fatty acid, nekal, NNO-7, polyethylene glycol, urea, lauric acid and mixed auxiliary agent, and supplementing 300-mesh kaolin, dextrin, calcium bicarbonate, corn flour and soybean meal mixed carriers to the total weight of 1000g, so as to prepare the bacillus subtilis microbial fertilizer wettable powder of 3000 hundred million spores/g.

When tomatoes are transplanted in a sunlight greenhouse in autumn, 1-2 particles (1-2 g) of the 5500 hundred million spores per gram bacillus subtilis microbial fertilizer granules are applied to each hole, and 2.5kg of 0.5 percent abamectin granules are mixed with fine soil for broadcasting. Drip irrigation is carried out respectively 30 days, 90 days and 150 days after hole application of the granules and 15 kg/hectare of 3000 hundred million spores/g bacillus subtilis microbial fertilizer wettable powder. And when the tomato seedlings are transplanted for 30 days, the average fresh weight of the 30 tomato seedlings is measured to be 24.6-29.7% higher than that of the control without the biofertilizer, and the average plant height is increased by 2.7-3.6 cm. In 4 th month of the next year, the control effect of the root knot nematode disease reaches 81.5-90.4 percent (Table 7).

TABLE 7 influence of JX-20 on growth of tomato at seedling stage and control of root knot nematode disease

Treatment of	Plant height (cm)	Fresh weight (g)/plant	Finger for disease	Control effect (%)
					1 g/strain	40.6bB	315.3bB	6.8bB	81.5
2 g/strain	41.5aA	328.2aA	3.53aA	90.4
					0.5% of abamectin	38.1cC	251.1cC	14.2cC	61.3
CK	37.9cC	253.0cC	36.7dD	/

When cucumbers are sowed in spring, 1-2 prepared 5500 hundred million spores per gram of bacillus subtilis microbial fertilizer granules are applied to each hole, and 2.5 kg/mu of 0.5 percent abamectin granules are mixed with fine soil for broadcast application. 30 days after hole application of the granules, water is irrigated with 3000 hundred million spores per gram of bacillus subtilis microbial fertilizer wettable powder 60kg per hectare. The average plant height of 25 cucumber seedlings is increased by 20.5-24.6 percent and the fresh weight is increased by 28.9-34.1 percent when the cucumber seedlings are transplanted for 30 days. The control effect of root knot nematode disease detected by 60 days after transplanting reaches 68-83.5% (Table 8).

TABLE 8 influence of JX-20 on cucumber seedling growth and root knot nematode control

Treatment of	Plant height (cm)	Fresh weight (g)/plant	Finger for disease	Control effect (%)
					1 g/strain	69.75bB	455.60bB	5.29bB	68.0
2 g/strain	72.15aB	474.05aA	2.72aA	83.5
					0.5% of abamectin	58.13c	352.14cc	5.33cC	61.3
CK	57.90c	353.41cc	16.53dD	/

2. Application effect of 3000 hundred million spores per gram bacillus subtilis microbial fertilizer wettable powder

The preparation method of the bacillus subtilis microbial fertilizer wettable powder with 3000 hundred million spores per gram is the same as the preparation method.

And (3) pouring 100-200 mL of the prepared 3000-hundred million spores/g bacillus subtilis microbial fertilizer wettable powder in each hole by using 10000-20000 times of diluent of 1: 3000-20000 times of the wettable powder. After 30 days, the average fresh weight of 50 plants is 30.2-35.5% higher than that of a watering control, and more flower buds are 1.1-1.3 per plant than that of the control (Table 9).

And in the flowering and fruiting period of the strawberries, 1:3000-5000 times of the 3000 hundred million spores/g microbial fertilizer wettable powder is sprayed, and the microbial fertilizer wettable powder is continuously sprayed for 4 times every 7 days. The growth of strawberry plants is obviously stronger than that of the contrast (spraying water and applying a conventional fertilizer), the disease rate of gray mold is reduced by 78.1-82.3% compared with that of the contrast of spraying water, and the disease rate is slightly lower than the control effect of spraying the pyrimethanil and fludioxonil bactericide mixture; the leaf powdery mildew is reduced by 71.9-81.2% compared with the spraying water, and is reduced by 39-48% compared with the spraying of the mixed agent of pyrimethanil and fludioxonil bactericides (Table 9).

TABLE 9 influence of JX-20 on growth of strawberry at seedling stage and prevention and treatment of gray mold and powdery mildew

And when the cucumber is transplanted in autumn in a sunlight greenhouse, pouring 100-200 mL of the prepared 1: 10000-20000 times of bacillus subtilis microbial fertilizer wettable powder of 3000 hundred million spores/gram in each hole. After 30 days, the average height of 30 plants is higher than that of a watering control by 17.8-28.1 percent (table 10). Meanwhile, the cucumber seedling culture medium is sprayed with 1: 10000-20000 times of diluent (50-100 mL per plant) by drip irrigation at 30 days, 60 days, 90 days, 120 days and 150 days after transplantation and 1:5000 times of diluent at the middle interval of drip irrigation, the total yield of the cucumber seedling culture medium is increased by 34.6% compared with the conventional chemical fertilizer application, and the control effect of the root knot nematode disease reaches 82.3% -89.5%. The control effect of cucumber powdery mildew and target spot disease can reach 75.3 percent and 67.5 percent (Table 10).

TABLE 10 growth promoting effect of JX-20 as base fertilizer on cucumber seedlings and disease control effect of drip irrigation

100g, 300g, 1000g and 3000g of bacillus subtilis microbial fertilizer wettable powder are added with water to be mixed into paste for seed mixing according to the proportion of 3000 hundred million spores per gram of wheat seeds, and the seeds are sowed after being dried. After 30 days (before winter), the seedling condition was investigated, and the results are shown in Table 11. The fresh weight of the overground part is respectively increased by 8.4%, 17.3%, 21.3% and 31.6% compared with the control without the biofertilizer; the fresh weight of underground roots was increased by 11.4%, 14.0%, 18.9% and 26.1% respectively over the control. The root rot was investigated 4 months next year, with 14.2%, 25.7%, 56.3% and 86.3% lower incidence than the control, respectively. Meanwhile, the disease indexes of banded sclerotial blight are respectively reduced by 26.3%, 54.1%, 73.3% and 76.1% compared with the control. Along with the increase of the biological fertilizer for seed dressing, the tillering of wheat seedlings is increased, the leaf color is deepened, and the growth vigor is stronger.

TABLE 11 growth promoting Effect of JX-20 on wheat seedlings and controlling Effect on soil-borne diseases

100g, 300g, 500g and 1000g of bacillus subtilis microbial fertilizer wettable powder of 3000 hundred million spores per gram is added with water to be mixed into paste for seed mixing per 100kg of corn seeds, the mixture is sowed in a pot with the diameter of 15cm after being dried, the seedling condition of 30 plants is investigated after 20 days, and the average plant height is respectively increased by 5.4 percent, 12.2 percent, 19.5 percent and 28.8 percent compared with the control without the biological fertilizer; the fresh weight of underground roots was increased by 8.1%, 16.2%, 21.6% and 29.7% respectively over the control (table 12).

TABLE 12 growth promoting Effect of JX-20 seed treatment on maize seedlings

1g, 3g, 10g and 30g of 3000 hundred million spores per gram bacillus subtilis microbial fertilizer wettable powder are added with water to be mixed into paste for seed mixing according to 1kg of cucumber seeds, the mixture is sowed in a pot with the diameter of 15cm after being dried, and 30 seedlings are investigated after 35 days (Table 13). The average vine length is increased by 4.5 cm, 6.6 cm, 17.1 cm and 19.2cm respectively compared with the control; the number of melon buds treated by seed dressing is 0.45, 1.3 and 1.8 more in 3g, 10g and 30 g.

TABLE 13 growth promoting Effect of JX-20 seed treatment on cucumber seedlings

3. Use effect of 1000 hundred million spores/g bacillus subtilis microbial fertilizer wettable powder

200g of 5000 hundred million bacillus subtilis JX-20 spores/g of dry powder is added with urea, ammonium sulfate, ammonium nitrate, ammonium chloride, ammonium bicarbonate, calcium phosphate, monopotassium phosphate, potassium magnesium salt, copper sulfate, manganese nitrate, borate and silicate which are crushed to 300 meshes, so that the final content of N, P, K (7.5:1.5:1.0) is 32 percent, and the equivalent mixing final content of trace elements such as calcium (Ca), magnesium (Mg), zinc (Zn), copper (Cu), sulfur (S), iron (Fe), boron (B), manganese (Mn), chlorine (Cl), sodium (Na) and the like is 0.4 percent; adding 1: 0.5: 0.1: 0.5 percent of sodium lignosulfonate, nekal, lauric acid and carboxymethyl cellulose mixed auxiliary agent with the final content of 7.5 percent, and kaolin, bean pulp, corn meal, humus, stable manure, compost and talcum powder mixed carriers are supplemented until the total weight is 1000 grams, so that the bacillus subtilis microbial fertilizer wettable powder with 1000 hundred million spores per gram is prepared.

225kg of the prepared bacillus subtilis microbial fertilizer wettable powder with 1000 hundred million spores per gram is used for broadcasting as a base fertilizer per hectare and then ridging is carried out when tomatoes are transplanted. After 30 days, the average fresh weight of 50 plants is 107.1g, which is 29.7% higher than that of a watering control 82.6 g. Meanwhile, drip irrigation is carried out 30 days, 60 days, 90 days, 120 days and 150 days after transplantation by using 1:10000 times of diluent (50 mL of each plant) and 1:500-1000 times of liquid is sprayed in the middle period of drip irrigation interval, the average weight of single-plant tomato fruits is 222.0 g/plant, the average weight of the single-plant tomato fruits is increased by 23% compared with 180.4g of the average weight of fruits which are conventionally applied with the chemical compound fertilizer, the average weight of 50 abnormal tomato fruits (cracking and the like) treated by JX-20 is reduced by 18.75% compared with 0.32 single-plant tomato fruits, the control effect of root knot nematode disease reaches 88.4%, the control effect of early blight reaches 88.0%, the control effect of late blight reaches 83.9%, and the dosage of chemical bactericide is reduced by 36% (Table 14).

TABLE 14 control Effect of JX-20 drip irrigation on tomato diseases

When cucumber is sowed in spring, 1 spoon (5 g) of the prepared bacillus subtilis microbial fertilizer with 1000 hundred million spores per gram is broadcast in each hole. After 30 days, the average plant height of 25 plants is 52.9cm and the fresh weight is 293g, compared with the control plant height of 42.7cm, the fresh weight is 219g, the vine length is increased by 24 percent, and the fresh weight is increased by 34 percent. The self-flowering bearing cucumber is sprayed for 5 times by 1000 times of 1000 million spores/g bacillus subtilis microbial fertilizer 1: 500-.

TABLE 15 prevention and control of cucumber diseases by JX-20 hole application and spray treatment

In the flowering and fruiting period of the strawberries, the bacillus subtilis microbial fertilizer is sprayed by 1:800-1200 times of 1000 hundred million spores/g, and the spraying is continuously carried out for 4 times at intervals of 7 days. The disease rate of the gray mold of the strawberries is reduced by 74.5 percent compared with the control of spraying water, and is reduced by 42.4 percent compared with the mixed agent of spraying pyrimethanil and fludioxonil bactericides; leaf powdery mildew was reduced by 82.6% compared to water spray (results are shown in table 16).

TABLE 16 control of grey and powdery mildew of strawberry by JX-20 spray

150-450 kg of bacillus subtilis microbial fertilizer with 1000 hundred million spores per gram is used as base fertilizer for each hectare before wheat seeding, the fresh weight of wheat seedlings is 8.9-37.9% higher than that of the wheat seedlings without fertilizer after 30 days, the average increase of 100 tillers is 0.3-1.1, and the wheat seedlings have strong cold resistance and good growth vigor during overwintering. When the banded sclerotial blight is investigated at 4 months and 15 days, the banded sclerotial blight of the cell applied with the biological fertilizer is reduced by 46.6-71.5 percent compared with the control (Table 17).

TABLE 17 growth promotion and sheath blight control of wheat seedlings by JX-20 base fertilizer

4. Application effect of 1000 hundred million spores/mL bacillus subtilis microbial fertilizer suspending agent

Taking 200g of 5000 hundred million bacillus subtilis JX-20 spores/g dry powder, adding urea, ammonium sulfate, ammonium nitrate, ammonium chloride, ammonium bicarbonate, monopotassium phosphate, potassium magnesium salt, borate and trace elements to enable the final content of N, P, K (2:1:1) to be 20% (weight percentage, the same below), and the final content of the trace elements such as calcium (Ca), silicon (Si), magnesium (Mg), zinc (Zn), sulfur (S), iron (Fe), manganese (Mn), chlorine (Cl) and the like to be 0.5%; adding 1: 0.5: 0.3: 0.3: 0.5 xanthan gum, ethylene glycol, NNO-1, polyethylene glycol and glycerin with the final content of 10.4 percent, and supplementing water to the total volume of 1000mL to prepare the bacillus subtilis microbial fertilizer suspending agent/aqueous solution with the volume of 1000 hundred million spores/mL.

Irrigating with a diluent of which the ratio is 1: 1500-3000 times of a bacillus subtilis microbial fertilizer suspending agent is 1000 hundred million spores/g when the peppers are transplanted, investigating the height of 50 plants by 25.7cm after 21 days, and averagely increasing the height by 2.4cm compared with the height of 23.3cm of a control plant in which a compound fertilizer is applied; the fresh weight of the JX-20 treated plant is 128.8 g/plant, which is increased by 14.6 percent compared with the control plant of 112.4 g/plant.

Spraying a 1: 800-1500-fold diluent of the bacillus subtilis microbial fertilizer suspending agent every 7 days for 5 times in the growth period of the pepper, wherein the number of the diluent is 1000 hundred million spores/g, the early blight and the powdery mildew are respectively reduced by 56% and 71%, and the number of the diluent is reduced by 25% and the amount of the azoxystrobin is 1000 times is 2 times; fresh fruit yield increased 38% (table 18). The growth is close to that of the application of 300 kg/hectare of compound fertilizer.

TABLE 18 growth promotion and disease control effects of JX-20 on Capsicum annuum

1.5-7.5 kilograms of 1000 hundred million spores per gram of bacillus subtilis microbial fertilizer suspending agent is sprayed for 3 times by 1: 300-100 times of diluent in each hectare in the seedling stage, tillering stage and booting stage of rice, and the growth vigor and biomass of the bacillus subtilis microbial fertilizer suspending agent are close to 150-225 kilograms per hectare of urea sprayed for 1 time in the seedling stage.

In the later tillering stage of rice, 1500 times of 1: 1000-.

The method is characterized in that the method is implemented by spraying 1500-fold diluent of 1000 million spores/g bacillus subtilis microbial fertilizer 1: 1000-.

TABLE 19 field effect of JX-20 on false smut prevention

In the heading stage of wheat, the bacillus subtilis microbial fertilizer suspending agent is sprayed by a diluent of which the ratio is 1: 800-1500 times of that of the bacillus subtilis microbial fertilizer suspending agent per gram, so that the thousand-grain weight of wheat is increased by 6.8%, and the control effects on powdery mildew and scab are 56.3% and 47.8% (table 20).

TABLE 20 action of JX-20 spray increasing thousand-grain weight and disease control in wheat flowering phase

After 20 days, the fresh weights of 50 soybean seedlings and 50 corn seedlings are respectively increased by 8.5 percent and 14.2 percent and the root rot and damping off are respectively reduced by 46 to 63.4 percent and 63.2 to 76.9 percent (Table 21).

TABLE 21 growth promoting effects of JX-20 dressing on soybeans and corn and control effects on soil-borne diseases

5. Use effect of bacillus subtilis microbial fertilizer granules of 100 hundred million spores/g

Taking 40 g 2500 hundred million bacillus subtilis JX-20 spores/g dry powder, adding ammonium sulfate, ammonium nitrate, ammonium chloride, urea, ammonium phosphate, triple superphosphate, rich superphosphate, powdered rock phosphate, calcium magnesium phosphate fertilizer, potassium sulfate, potassium chloride, potassium nitrate, calcium nitrate, monopotassium phosphate, potassium magnesium salt, borate and trace elements which are crushed and pass through 300 meshes to ensure that the final content of N, P, K (7:2:1) is 45 percent, and the final content of mixed trace elements with the same quantity of magnesium (Mg), zinc (Zn), copper (Cu), sulfur (S), iron (Fe) and boron (B) and manganese (Mn) is 0.2 percent; adding 2:1: 0.5 alkylphenol polyoxyethylene ether, sodium lignin sulfonate and carboxymethyl cellulose with the final content of 7.5 percent, and 100 hundred million spores/gram of bacillus subtilis microbial fertilizer granules are prepared by supplementing kaolin, light calcium carbonate, soybean meal, corn meal, humus, stable manure and compost to the total weight of 1000 grams.

And (3) irrigating 200mL of the prepared bacillus subtilis microbial fertilizer granules with the ratio of 1:10000 times of the diluent in each hole when the tomatoes are transplanted. After 30 days, the average fresh weight of 40 plants is higher than that of a watering control by 26.3 percent. Meanwhile, the diluent (50 mL per plant) with the volume ratio of 1:10000 is dripped 30 days, 60 days, 90 days, 120 days and 150 days after transplanting, and the diluent is sprayed with the diluent with the volume ratio of 1: 800-1000 in the middle of the drip irrigation interval, so that the control effect of the root knot nematode disease reaches 66-83.1%, the control effect of the early blight reaches 62.3%, and the control effect of the late blight reaches 71.5% (Table 22).

When cucumber seeds are sown in spring, 1 spoon (about 5 g) of the prepared bacillus subtilis microbial fertilizer with 100 hundred million spores per gram is broadcast in each hole. After 30 days, the average plant height of 25 plants is 22.4 percent higher than that of the control, and the fresh weight is increased by 31 percent. The self-flowering melon seeds are sprayed for 5 times by using 1:1000-1500 times of 100 hundred million spores/g bacillus subtilis microbial fertilizer every 7-10 days, the total yield of the cucumber is increased by 25.8% compared with the conventional chemical fertilizer, the control effect of root knot nematode disease reaches 61.1-73.6%, and the control effects of cucumber powdery mildew and target spot disease reach 66.8% and 61.8% (Table 22).

TABLE 22 growth promoting and disease controlling effects of JX-20 base fertilizer + spray on tomato and cucumber

The bacillus subtilis microbial fertilizer granules with the concentration of 100 hundred million spores/mL are sprayed 1:500-1000 times in the growth period of the watermelons, 5 times are sprayed at intervals of 7 days, and the growth vigor of the watermelons is similar to that of 450kg of compound fertilizer and 300kg of magnesium sulfate.

Before transplanting rice, 180-450 kg of 100 hundred million spores per gram of bacillus subtilis microbial fertilizer is spread in each hectare, and the growth vigor and biomass of the bacillus subtilis microbial fertilizer are similar to 225-330 kg of urea spread in each hectare.

75-150 kg of 100 hundred million spores/g bacillus subtilis microbial fertilizer granules are sprayed for 1 time by 1:1000 times of liquid every 7 days in the growth period of rice and continuously sprayed for 5 times, and the growth vigor and the biomass of the bacillus subtilis microbial fertilizer granules are similar to those of 225-300 kg of urea in broadcasting application.

6. Use effect of 50 hundred million spores/g bacillus subtilis microbial fertilizer suspending agent

Taking 20g 2500 hundred million bacillus subtilis JX-20 spores/g dry powder, adding ammonium sulfate, urea, calcium superphosphate, potassium sulfate, potassium chloride, monopotassium phosphate, magnesium sulfate, manganese chloride, borax and trace elements to ensure that the final content of N, P, K (1:1:1) is 15 percent, and the final content of the trace elements such as calcium (Ca), magnesium (Mg), zinc (Zn), sulfur (S), boron (B), manganese (Mn) and the like is 0.25 percent; adding 2:1: the final content of 1 polyethylene glycol, glycerol and xanthan gum is 3.5%, water is supplemented until the total volume is 1000mL, and the bacillus subtilis microbial fertilizer suspending agent with 50 hundred million spores/mL is prepared.

In the flowering and fruiting period of the strawberries, the bacillus subtilis microbial fertilizer with 50 million spores/g in the ratio of 1:200 and 500 times is sprayed, the continuous spraying is carried out for 4 times at intervals of 7 days, and the pyrimethanil fludioxonil is sprayed for 1 time in the middle. The disease rate of the gray mold of the strawberries is reduced by 56.4 percent compared with the control of spraying water, and is reduced by 23.3 percent compared with the 4-time spraying of the mixture of pyrimethanil and fludioxonil bactericides; the leaf powdery mildew is reduced by 43.1 percent compared with spraying water and by 11.2 percent compared with spraying the mixture of pyrimethanil and fludioxonil bactericides (Table 23).

In the initial flowering period of rape, the 50 hundred million spores per gram bacillus subtilis microbial fertilizer is sprayed by 1: 200-fold, and is continuously sprayed for 3 times at intervals of 7 days, so that rape pod seeds are full, the yield is increased by 17.8% compared with the non-fertilization control, the sclerotiniose is reduced by 72.5% compared with the non-fertilization control, and no significant difference exists between the sclerotinia spraying and the control effect of 50 g/mu of sclerotinia (Table 23).

TABLE 23 growth promoting and disease control effects of JX-20 spray on strawberries and oilseed rape

225kg and 450kg of bacillus subtilis microbial fertilizer of 50 hundred million spores per gram are mixed with water and poured in seedling stage of winter wheat before winter, the growth vigor of wheat seedlings is better than that of the wheat seedlings of 450kg of wax fertilizer which is spread, the drought resistance and cold resistance of the overwintering wheat seedlings are improved, the green turning speed in spring is fast, the biomass of overground and underground wheat seedlings in the jointing stage is increased by 58 percent and 110.8 percent compared with the contrast without fertilization, and the tillering is increased (Table 24).

TABLE 24 growth promoting effect of JX-20 wax fertilizer on wheat seedlings

Treatment of	Biomass g/5 strain	Biomass increase%	Leaf color
				225kg/hm2	61.3B	58.0Cc	++++
450kg/hm2	81.8Aa	110.8Aa	+++++
				Composite fertilizer 450kg/hm2	69.9Bb	80.2Bb	++++
CK	38.8Cc	/	+++

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

<110> Nanjing GmbH Biotechnology development Co., Ltd

<120> bacillus subtilis JX-20 strain and application thereof

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1505

<212> DNA

<213> Bacillus subtilis

<400> 1

taccttgtta cgacttcacc ccaatcatct gtcccacctt cggcggctgg ctccataaag 60

gttacctcac cgacttcggg tgttacaaac tctcgtggtg tgacgggcgg tgtgtacaag 120

gcccgggaac gtattcaccg cggcatgctg atccgcgatt actagcgatt ccagcttcac 180

gcagtcgagt tgcagactgc gatccgaact gagaacagat ttgtgggatt ggcttaacct 240

cgcggtttcg ctgccctttg ttctgtccat tgtagcacgt gtgtagccca ggtcataagg 300

ggcatgatga tttgacgtca tccccacctt cctccggttt gtcaccggca gtcaccttag 360

agtgcccaac tgaatgctgg caactaagat caagggttgc gctcgttgcg ggacttaacc 420

caacatctca cgacacgagc tgacgacaac catgcaccac ctgtcactct gcccccgaag 480

gggacgtcct atctctagga ttgtcagagg atgtcaagac ctggtaaggt tcttcgcgtt 540

gcttcgaatt aaaccacatg ctccaccgct tgtgcgggcc cccgtcaatt cctttgagtt 600

tcagtcttgc gaccgtactc cccaggcgga gtgcttaatg cgttagctgc agcactaagg 660

ggcggaaacc ccctaacact tagcactcat cgtttacggc gtggactacc agggtatcta 720

atcctgttcg ctccccacgc tttcgctcct cagcgtcagt tacagaccag agagtcgcct 780

tcgccactgg tgttcctcca catctctacg catttcaccg ctacacgtgg aattccactc 840

tcctcttctg cactcaagtt ccccagtttc caatgaccct ccccggttga gccgggggct 900

ttcacatcag acttaagaaa ccgcctgcga gccctttacg cccaataatt ccggacaacg 960

cttgccacct acgtattacc gcggctgctg gcacgtagtt agccgtggct ttctggttag 1020

gtaccgtcaa ggtgccgccc tatttgaacg gcacttgttc ttccctaaca acagagcttt 1080

acgatccgaa aaccttcatc actcacgcgg cgttgctccg tcagactttc gtccattgcg 1140

gaagattccc tactgctgcc tcccgtagga gtctgggccg tgtctcagtc ccagtgtggc 1200

cgatcaccct ctcaggtcgg ctacgcatcg tcgccttggt gagccgttac ctcaccaact 1260

agctaatgcg ccgcgggtcc atctgtaagt ggtagccgaa gccacctttt atgtctgaac 1320

catgcggttc agacaaccat ccggtattag ccccggtttc ccggagttat cccagtctta 1380

caggcaggtt acccacgtgt tactcacccg tccgccgcta acatcaggga gcaagctccc 1440

atctgtccgc tcgacttgca tgtattaggc acgccgccag cgttcgtcct gagccaggat 1500

caaac 1505

Claims (10)

1. A Bacillus subtilis JX-20 strain is characterized in that the preservation number of the JX-20 strain is GDMCC No. 61546.

2. The JX-20 strain of claim 1, wherein the nucleotide sequence of the 16S rDNA of the JX-20 strain is as set forth in SEQ ID No. 1.

3. Use of the JX-20 strain of claim 1 or 2 or a fermentation product of the JX-20 strain for the preparation of a plant antibacterial and/or nematicidal agent.

4. The use according to claim 3, wherein the antibacterial spectrum of the plant antibacterial agent comprises the germs Xanthomonas campestris (Xanthomonas campestris), Ralstonia solani (Pseudomonas syringae), Brassica rapa (Erwinia carotovora), Sclerotinia sclerotiorum (Sclerotinia sclerotiorum), Rhizoctonia solani (Rhizoctonia solani), Rhizoctonia oryzae (Fusarium moniliforme), Pyricularia oryzae (Magnaporthe grisea), Rhizoctonia cerealis (Fusarium graminearum), Rhizoctonia cerealis (Rhizoctonia cerealis), Phytophthora solani (Alternaria solani), Botrytis neraria (Botrytis neria cinerea), Fusarium oxysporum (Fusarium oxysporum), Phytophthora nivale (Phytophthora nikohlia solani), Pyricularia solani (Rhizoctonia solani), and Rhizophora solani (Cucuminus graminis), Rhizoctonia solani (Rhizoctonia solani), Rhizoctonia solani (Rhizoctonii), Rhizoctonia solani) and Rhizoctonii (Rhizoctonii), Rhizoctonia solani (Rhizoctonia solani), Rhizoc;

the anti-nematode spectrum of the anti-nematode agent includes Meloidogyne incognita (melodogyne incognita).

5. The use according to claim 3, wherein the ferment is prepared by fermenting the JX-20 strain in NB medium for 48-72 h, wherein the NB medium comprises the following components in concentration: 15g/L of sucrose, 5g/L of polypeptone, 1g/L of yeast powder and 3g/L of beef extract.

6. Use of the JX-20 strain of claim 1 or 2 or a ferment of said JX-20 strain for promoting plant growth and/or increasing plant resistance.

7. A microbial fertilizer based on the JX-20 strain of claim 1 or 2, wherein the microbial fertilizer comprises a zymogen solution of the JX-20 strain of bacillus subtilis, and further comprises macro-elements and trace elements;

the zymophyte liquid comprises 50 hundred million to 6000 hundred million spores per gram of microbial fertilizer and secondary metabolites accounting for 0.3 to 1.5 percent of the mass of the microbial fertilizer;

the total mass of the macroelements is 5-50% of the mass of the microbial fertilizer, and the mass of the microelements is 0.1-1% of the mass of the microbial fertilizer;

the macroelements include N, P and K;

the microelements comprise calcium, magnesium, zinc, copper, sulfur, iron, boron, manganese, chlorine and sodium.

8. The microbial fertilizer according to claim 7, wherein the secondary metabolites of the JX-20 strain of Bacillus subtilis comprise a mixture of one or more of oligosaccharides, fatty acids, amino acids, polypeptides and biotin.

9. The microbial fertilizer as claimed in claim 7, further comprising a survival carrier and an auxiliary agent, wherein the mass of the survival carrier is 10-85% of the mass of the microbial fertilizer, and the mass of the auxiliary agent is 2-15% of the mass of the microbial fertilizer;

the survival carrier is selected from one or more of water, attapulgite, kaolin, light calcium carbonate, ammonium sulfate, borate, ethylene diamine tetraacetate, melamine, urea, ammonium sulfate, monopotassium phosphate, corn flour, soybean meal, humus, stable manure, human excrement, poultry manure, compost and talcum powder;

the auxiliary agent is selected from one or more of ethylene glycol, propylene glycol, NNO-1, NNO-7, xanthan gum, polyethylene glycol, glycerol, sodium dodecyl sulfate, sodium dodecyl benzene sulfonate, ammonium sulfate, urea, alkylphenol polyoxyethylene ether phosphate, lauric acid, polyoxyethylene fatty acid, benzoic acid, sodium lignin sulfonate, carboxymethyl cellulose, polyvinyl alcohol and potassium ferrocyanide.

10. A method for improving crop yield and disease and pest resistance by using the microbial fertilizer as claimed in any one of claims 7 to 9, which comprises the following steps: the microbial fertilizer is used as a base fertilizer, an additional fertilizer or a leaf fertilizer and is applied to the field according to the use amount of 15-450 kg/hectare;

or 10-3000 g of the microbial fertilizer is used for dressing or coating every 100kg of crop seeds.