A Bacillus zoogloea and its preparation method and application

Abstract

The present invention discloses a bacillus zoogloea, which comprises spores and extracellular polysaccharides produced by bacillus during fermentation. The extracellular polysaccharides form a composite colloid with fulvate and coat the outside of the spores, thereby forming the zoogloea. The present invention also discloses a preparation method of the zoogloea, a fermentation medium for preparing the zoogloea, and a microbial inoculant containing the zoogloea. By forming a composite colloid of extracellular polysaccharides produced during the fermentation of bacillus with fulvate, the zoogloea not only protects the bacterial cells and increases their vitality, but also prevents the loss of fulvate, thereby enabling the microbial inoculant to better promote the growth of crops.

Description

Field of the Invention

[0001] The present invention belongs to the field of microorganisms. In particular, the present disclosure provides a microbial composition and method useful for crop production. Specifically, the present invention provides a Bacillus zoogloea, and the present invention also relates to a preparation method and application of the zoogloea, as well as a fermentation medium for preparing the zoogloea. Background Art

[0002] Since Bacillus microorganisms have functions such as inhibiting pathogenic bacteria, promoting crop growth, and inducing crop stress resistance, they have become the most widely used types in microbial inoculants.

[0003] Bacillus mucilaginosus, Bacillus polymyxa, and Bacillus azotofixans are all Bacillus species that produce extracellular polysaccharides and can secrete water-soluble polysaccharides into the environment during growth and metabolism. Due to its hydrophilicity, spatial occupancy, and barrier effects, extracellular polysaccharides can effectively resist the phagocytosis of host phagocytes; it can also effectively protect the bacteria from damage by various lysozymes and antibacterial substances. Extracellular polysaccharides are highly hydrated molecules with a water content of more than 95%, which can also help beneficial bacteria resist the threat of drying to survival. However, the bacterial powder products produced by ordinary centrifugal spray drying processes do not contain extracellular polysaccharides.

[0004] Fulvate is a highly efficient and novel plant growth promoter. It can promote root development, improve root vitality and the activities of various enzymes, increase chlorophyll content and photosynthetic intensity, and improve yield and quality. It can inhibit the growth of certain plant pathogenic microorganisms and enhance the disease resistance of plants by increasing the activity of catalase in plants. Fulvate can be mixed with various fungicides and has a good slow-release and enhancing effect on fungicides. It can also reduce the toxicity of pesticides, reduce the number of pesticide applications and dosage, and is beneficial to environmental protection.

[0005] Based on the fact that Bacillus has functions such as inhibiting pathogenic bacteria and inducing crop stress resistance; fulvate can significantly promote the development of crop roots, etc. The combined use of the two can significantly promote crop growth and control soil-borne diseases. Therefore, there are many microbial inoculant products on the market that are a mixture of Bacillus and fulvate. However, such products simply mix the two substances in a certain weight ratio. After this product is applied to the soil, under hydraulic erosion and gravitational erosion, the spores and fulvate are easily separated, making the spores vulnerable to environmental factors and interference, resulting in problems such as an extended spore germination adaptation period and a low germination rate. Summary of the Invention

[0006] The first aspect of the present invention provides a Bacillus zoogloea, which comprises spores, extracellular polysaccharides and fulvate salts. Among them, the spores and extracellular polysaccharides are produced during the fermentation process of Bacillus, and the fulvate salts are fulvate salts added to the fermentation medium during the fermentation process. The extracellular polysaccharides and the fulvate salts form a composite colloid and coat the outside of the spores. The Bacillus is a Bacillus that produces extracellular polysaccharides.

[0007] Preferably, the Bacillus is selected from any one of Paenibacillus mucilaginosus, Paenibacillus polymyxa, Paenibacillus azotofixans.

[0008] Preferably, the fulvate salts account for 5-30% of the weight of the Bacillus zoogloea.

[0009] The second aspect of the present invention provides a fermentation medium for preparing the Bacillus zoogloea described in the first aspect of the present invention. The fermentation medium contains 0.01-0.05% of silicon dioxide and 1-5% of fulvate salts.

[0010] Preferably, the fermentation medium contains the following components in weight ratio: peanut meal 1-2%, glucose 2-3%, yeast extract 0.5-1%, corn steep liquor 1.5-2%, potassium dihydrogen phosphate 0.05-0.1%, silicon dioxide 0.01-0.05%, fulvate salts 1-5%, magnesium sulfate 0.05-0.1%, and the rest is made up to 100% with water. The pH of the medium is adjusted to 7.0-7.5.

[0011] The third aspect of the present invention provides a method for preparing the Bacillus zoogloea described in the first aspect of the present invention. The method comprises the following steps: inoculating Bacillus into a fermentation medium and culturing it, and drying and pulverizing the resulting fermentation broth to obtain the original powder of the Bacillus zoogloea.

[0012] Preferably, the medium consists of the following components in weight ratio: peanut meal 1-2%, glucose 2-3%, yeast extract 0.5-1%, corn steep liquor 1.5-2%, potassium dihydrogen phosphate 0.05-0.1%, silicon dioxide 0.01-0.05%, fulvate salts 1-5%, magnesium sulfate 0.05-0.1%, and the rest is made up to 100% with water. Adjust the pH to 7.0-7.5.

[0013] Preferably, the culture is liquid aeration culture, the culture temperature is 30-32 °C, the aeration rate is 0.8-1.2 VVM, the stirring speed of the fermentation tank is 375-525 rpm, and the tank pressure is 0.05-0.07 MP.

[0014] The fourth aspect of the present invention provides a microbial inoculant, which comprises the Bacillus zoogloea described in the first aspect of the present invention.

[0015] Preferably, the microbial inoculant further comprises other microorganisms and inorganic salts.

[0016] Preferably, the other microorganisms are selected from Bacillus subtilis.

[0017] Preferably, the inorganic salts may exist in the form of a complex with a carrier, and the complex is preferably a fulvic powder.

[0018] Preferably, the microbial inoculant comprises a bacillus zoogloea, a Bacillus subtilis original powder, a fulvic powder, and a fulvate. Among them, the externally added fulvate can be released as soon as possible to play the functions of activating the soil and regulating plant growth, while the fulvate bound inside the zoogloea can play a better role in promoting crop growth through slow release.

[0019] Compared with the prior art, the advantages of the present invention are as follows:

[0020] (1) During the fermentation process of bacillus, due to the addition of fulvate and silicon dioxide in the fermentation medium, the carboxyl groups in the extracellular polysaccharide first form a complex with SiO 2 and then strongly adsorb organic acids, thereby binding part of the carboxyl groups in the fulvate to form a composite colloid in the liquid environment. After the spores fall off at the end of fermentation, the composite colloid and the spores form a zoogloea. The fermentation broth containing the zoogloea is dried to obtain a bacillus zoogloea mother powder. After the microbial inoculant prepared from the mother powder is applied to the soil, since the zoogloea is wrapped with extracellular polysaccharide that binds fulvate, it can effectively protect the bacterial cells from being damaged by various lysozymes and antibacterial substances in the external environment, help the beneficial bacteria resist the threats of the environment. At the same time, the extracellular polysaccharide in the zoogloea has nutritional components, which can shorten the adjustment period of the beneficial bacteria, increase the number and vitality of the beneficial bacteria, enhance the fertilizer efficiency and inhibit the abundance of pathogenic bacteria.

[0021] (2) Since the zoogloea itself adsorbs a large amount of fulvate, under the protection of the composite colloid, it can prevent the flocculation of fulvic acid and its inactivation in saline-alkali soil or heavy metal soil, thereby better activating the soil, making it have higher vitality, and making the nutrients more sufficient and the supply more reasonable.

[0022] (3) Adding fulvate to the fermentation medium can also improve the fermentation level of bacillus. Description of the Drawings

[0023] Figure 1 It is a scanning electron micrograph of a zoogloea (Paenibacillus mucilaginosus).

[0024] Figure 2 It is a scanning electron micrograph of a zoogloea (Paenibacillus polymyxa).

[0025] Figure 3It is a scanning electron micrograph of zoogloea (Paenibacillus azotofixans).

[0026] Figure 4 It is a scanning electron micrograph of zoogloea (Bacillus megaterium).

[0027] Figure 5 It is a scanning electron micrograph of the microbial inoculant containing Bacillus zoogloea prepared by the present invention.

[0028] Figure 6 It is a scanning electron micrograph of a conventional product of Bacillus + fulvic acid on the market. Detailed implementation mode

[0029] The present invention will be further described in detail below in conjunction with embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the scope of the present invention.

[0030] The Paenibacillus mucilaginosus (product number BNCC193571), Paenibacillus polymyxa (BNCC1355575), Paenibacillus azotofixans (product number BNCC222299) and Bacillus megaterium (product number BNCC336464) used in the following examples were purchased from Beina Biology.

[0031] Example 1 Preparation of Zoogloea - Screening of Fulvic Acid Salts

[0032] (1) Pretreatment of fulvic acid salts

[0033] Potassium fulvate, sodium fulvate, calcium fulvate, iron fulvate, manganese fulvate, and zinc fulvate were filtered through a 200 - mesh sieve, and the fulvic acid salts with a fineness of more than 200 meshes were taken.

[0034] (2) Preparation of fermentation medium

[0035] Components of the 1# fermentation medium: peanut meal 2%, glucose 2%, yeast extract 1%, corn steep liquor 2%, potassium dihydrogen phosphate 0.1%, silicon dioxide 0.05%, magnesium sulfate 0.1%, and the rest was made up to 100% with water, and the pH was adjusted to 7.2; The 2 - 7# fermentation media were respectively added with 1% potassium fulvate, sodium fulvate, calcium fulvate, iron fulvate, manganese fulvate, and zinc fulvate in the 1# medium, and the addition amount was based on the weight of the fermentation medium.

[0036] (3) Inoculation and fermentation

[0037] The Paenibacillus mucilaginosus was respectively inoculated into the fermentation media numbered 1 - 7 described in step (2), and the inoculation amount was 2% of the volume of the fermentation medium.

[0038] (3) Fermentation

[0039] Rotary oscillation flask culture was adopted. The culture conditions were 30 °C, 180 rpm, and 50 h. After the fermentation was completed, the fermentation broth was collected.

[0040] (4) Preparation of bacterial powder

[0041] Drop the fermentation broth obtained in step (3) on a sterile petri dish, and then place it in a constant temperature forced air drying oven. Set the air temperature to 33 °C and dry it. After drying, scrape it off with a sterile inoculation shovel to obtain bacterial mass powder.

[0042] (5) Determination of the mass ratio of extracellular polysaccharide:

[0043] Take 2.0 g of the bacterial mass powder obtained in step (4) and place it in a 50 mL beaker. Add 15 mL of distilled water, sonicate for 10 min, stir for 10 min, repeat three times, and make up the volume to 25 mL to obtain the test solution for the determination of total polysaccharide.

[0044] The above test solution was centrifuged at 4 °C and 10000 r / min for 15 min to remove the precipitated bacteria, and the supernatant was retained; 3 times the volume of 95% ethanol was added to the supernatant, and it was left to stand at 4 °C for 24 h, then centrifuged for 15 min, and the supernatant was poured off. The precipitate was dissolved in an appropriate amount of distilled water and placed in a dialysis bag for dialysis at 4 °C for 24 h; 25% (v / v) of sevage reagent [V(chloroform):V(n-butanol)=4:1] was added to the liquid obtained by dialysis, and it was placed on a shaker and shaken at room temperature for 30 min to adsorb the protein in the organic phase, then centrifuged at 8000 r / min for 1 min, and the aqueous phase was retained. Repeat 4 - 5 times until the protein was completely removed.

[0045] The content of extracellular polysaccharide was determined by the phenol-sulfuric acid method. Using 0.2 mg / mL glucose as the standard solution, take 0, 0.1, 0.2, 0.4, 0.6, 0.8, 1.0 mL of the standard solution and make up the volume to 2.0 mL to measure the OD values of different concentrations of glucose solutions at 490 nm, and obtain the linear regression equation.

[0046] Transfer 200 μL of the dialyzed sample solution to a clean test tube, add 100 μL of 5% phenol solution, shake well, and then slowly add 500 μL of concentrated H 2 SO 4 . After standing in the dark for 30 min, measure the OD value at 490 nm and calculate its content by substituting it into the standard curve. Divide the mass of the obtained extracellular polysaccharide by the mass of the taken bacterial mass powder, which is the mass ratio of extracellular polysaccharide in the bacterial mass.

[0047] (6) Mass ratio of fulvate in the bacterial mass:

[0048] The content of fulvate was determined according to GB / T 34765-2017, and the ratio of the obtained result to the sample weight is the mass ratio of fulvate in the bacterial mass.

[0049] Results and analysis: As can be seen from Table 1, adding fulvate salts to the fermentation medium can not only increase the number of spores in the fermentation broth, but also increase the secretion of extracellular polysaccharide substances. The carboxyl groups in the extracellular polysaccharide form a complex with SiO in the medium, strongly adsorb organic acids, and form a composite colloid after binding to some carboxyl groups in the fulvate salts. Adding potassium fulvate to the fermentation medium results in higher spore counts and the mass ratio of extracellular polysaccharide in the mucilaginous aggregates in the fermentation broth compared to other fulvate salts. 2 in the fermentation medium, forming a complex that strongly adsorbs organic acids and forms a composite colloid after binding to some carboxyl groups in the fulvate salts. Adding potassium fulvate to the fermentation medium results in higher spore counts and the mass ratio of extracellular polysaccharide in the mucilaginous aggregates in the fermentation broth compared to other fulvate salts.

[0050] Table 1 Effects of fulvate salts on spore counts and extracellular polysaccharides

[0051]

[0052] Example 2 Preparation of mucilaginous aggregates - Screening of potassium fulvate dosage

[0053] (1) Pretreatment of potassium fulvate

[0054] Filter potassium fulvate through a 200-mesh sieve, and take potassium fulvate with a fineness of more than 200 meshes.

[0055] (2) Preparation of fermentation medium

[0056] Composition of Fermentation Medium 1#: Peanut meal 1%, glucose 2%, yeast extract 0.6%, corn steep liquor 1.5%, potassium dihydrogen phosphate 0.05%, silicon dioxide 0.05%, magnesium sulfate 0.05%, and the rest is made up to 100% with water, adjusting the pH to 7.2; Fermentation Media 2-6# are prepared by adding 1%, 2%, 5%, 7%, and 10% of potassium fulvate to Fermentation Medium 1# respectively, and the addition amount is based on the weight of the fermentation medium.

[0057] (3) Inoculation and fermentation

[0058] Inoculate Paenibacillus polymyxa into the fermentation media numbered 1-6 obtained in step (2) respectively, and the inoculation amount is 2% of the volume of the fermentation medium.

[0059] (3) Fermentation: The same as in Example 1.

[0060] (4) Preparation of bacterial powder: The same as in Example 1.

[0061] (5) Determination of the mass ratio of extracellular polysaccharide: The same as in Example 1.

[0062] (6) Mass ratio of fulvate salts in the mucilaginous aggregates: The same as in Example 1.

[0063] Results and analysis: As can be seen from Table 2, when the addition amount of potassium fulvate is in the range of 1%-5%, the number of spores in the fermentation broth is relatively high, and the extracellular polysaccharide can form a stable colloid with some carboxyl groups in the fulvate. When the addition amount of potassium fulvate continues to increase, not only the number of spores in the fermentation broth decreases, but also the viscosity of the colloid is too high, which is not conducive to the subsequent separation and drying of the fermentation broth.

[0064] Table 2 Screening results of the addition amount of potassium fulvate

[0065]

[0066] Example 3 Preparation of zoogloea - Screening of the dosage of silicon dioxide

[0067] (1) Pretreatment of potassium fulvate

[0068] Filter potassium fulvate through a 200-mesh sieve, and take potassium fulvate with a fineness of more than 200 meshes.

[0069] (2) Preparation of fermentation medium

[0070] Components of Fermentation Medium 1#: Peanut meal 1%, Glucose 2%, Yeast extract 0.5%, Corn steep liquor 1.5%, Potassium dihydrogen phosphate 0.05%, Potassium fulvate 1.5%, Magnesium sulfate 0.05%, and the rest is made up to 100% with water, and the pH is adjusted to 7.2; Fermentation Media 2 - 8# are respectively added with 0.01%, 0.012%, 0.015%, 0.018%, 0.02%, 0.05%, 0.1% of silicon dioxide in Fermentation Medium 1#, and the addition amount is based on the weight of the fermentation medium.

[0071] (3) Inoculation and fermentation

[0072] Inoculate Paenibacillus gelatinosus into the fermentation medium obtained in step (2), and the inoculation amount is 2% of the volume of the fermentation medium.

[0073] (3) Fermentation: The same as Example 1.

[0074] (4) Preparation of bacterial powder: The same as Example 1.

[0075] (5) Determination of the mass ratio of extracellular polysaccharide, the same as Example 1:

[0076] (6) Mass ratio of fulvate in zoogloea: The same as Example 1.

[0077] Results and analysis: As can be seen from Table 3, when the addition amount of silica is in the range of 0% - 0.1%, the content of extracellular polysaccharide in the zoogloea does not increase significantly. However, when the addition amount of silica reaches 0.02% - 0.05%, the mass ratio of potassium fulvate in the zoogloea increases significantly compared with that when the addition amount of silica is below 0.02%. When the silica content continues to increase to 0.1%, the mass ratio of potassium fulvate does not increase compared with that when the addition amount of silica is 0.05%. Excessive dosage does not adsorb more potassium fulvate, but will increase the fermentation cost.

[0078] Table 3 Screening results of silica addition amount

[0079]

[0080] Example 4 Preparation of zoogloea - Screening of applicable strains

[0081] Paenibacillus mucilaginosus, Paenibacillus polymyxa, Paenibacillus azotofixans, and Bacillus megaterium were respectively inoculated into the fermentation medium, and the inoculation amount was 2% of the volume of the medium. After fermentation and drying, electron microscopy imaging at 5.00 μm was performed. As Figure 1-4 can be seen, the bacterial powder prepared from Paenibacillus mucilaginosus, Paenibacillus polymyxa, and Paenibacillus azotofixans according to the method of the present invention contains a large amount of colloidal extracellular polysaccharide, while the bacterial powder inoculated with Bacillus megaterium hardly produces extracellular polysaccharide.

[0082] The components of the fermentation medium are: 2% peanut meal, 2% glucose, 1% yeast extract, 2% corn steep liquor, 0.1% potassium dihydrogen phosphate, 0.05% silica, 0.1% magnesium sulfate, 1% potassium fulvate, 0.02% silica, and the rest is made up to 100% with water, and the pH is adjusted to 7.2.

[0083] Therefore, according to the results, for the strains producing extracellular polysaccharide, adding silica and potassium fulvate during the fermentation process can form zoogloea. For the strains that do not produce extracellular polysaccharide, adding silica and potassium fulvate during the fermentation process cannot form zoogloea either.

[0084] Example 5 Proportion of fulvate in zoogloea

[0085] (1) Pretreatment of potassium fulvate

[0086] Potassium fulvate was filtered through a 200 - mesh sieve, and potassium fulvate with a fineness of more than 200 meshes was taken.

[0087] (2) Preparation of the fermentation medium

[0088] Prepare a fermentation medium by taking 1% peanut meal, 2% glucose, 0.5% yeast extract, 1.5% corn steep liquor, 0.05% potassium dihydrogen phosphate, 0.02% silicon dioxide, 2% potassium fulvate, 0.05% magnesium sulfate, and making up the rest to 100% with water, and adjusting the pH to 7.2.

[0089] (3) Inoculate and ferment

[0090] Add 8 L of the culture medium prepared in step (2) to the 15-L fermenters numbered 1, 2, and 3 respectively. After sterilization at 121 °C for 30 min, inoculate Paenibacillus mucilaginosus into fermenters 1, 2, and 3 respectively, and the inoculation amount is 2% of the volume of the fermentation medium. The fermentation process of the fermenter is liquid deep aeration culture. The culture conditions of fermenter No. 1 are 30 °C, aeration rate 0.8 VVM, rotation speed 375 rpm, and tank pressure 0.05 MP; the culture conditions of fermenter No. 2 are 32 °C, aeration rate 1.0 VVM, rotation speed 450 rpm, and tank pressure 0.06 MP; the culture conditions of fermenter No. 3 are 30 °C, aeration rate 1.2 VVM, rotation speed 525 rpm, and tank pressure 0.07 MP. All three fermenters are fermented for 50 h to obtain the fermentation broth.

[0091] After discharging the tank, the specific gravity of the fermentation broth in fermenter No. 1 is measured to be 1.008, the viscosity is 839 cps, and the spore count is 7.35×10 8 CFU / ml;

[0092] The specific gravity of the fermentation broth in fermenter No. 2 is 1.065, the viscosity is 1424 cps, and the spore count is 1.35×10 9 CFU / ml;

[0093] The specific gravity of the fermentation broth in fermenter No. 3 is 1.035, the viscosity is 2716 cps, and the spore count is 1.12×10 9 CFU / ml.

[0094] (4) Dry and pulverize

[0095] Drop the fermentation broth obtained in step (3) onto a cold roller shaft, scrape it off and then dry it. The air temperature is 33 °C. After drying, pulverize it and pass it through a 150-mesh sieve to obtain powdery bacterial gum masses. Fermenter No. 1 obtains bacterial gum mass powder No. 1, fermenter No. 2 obtains bacterial gum mass powder No. 2, and fermenter No. 3 obtains bacterial gum mass powder No. 3. The spore count in bacterial gum mass No. 1 is measured to be 590 million / g, and the potassium fulvate content measured according to GB / T 34765-2017 is 5.48%; the spore count in bacterial gum mass No. 2 is 1.07 billion / g, and the potassium fulvate content measured according to GB / T 34765-2017 is 30.58%. The spore count in bacterial gum mass No. 3 is 896 million / g, and the potassium fulvate content measured according to GB / T 34765-2017 is 23.79%.

[0096] The mass of potassium humate combined in the floc powder is 5%-30%.

[0097] Example 6 Microbial Agent Containing Floccules

[0098] Take 58 parts of the No. 2 bacterial floc powder obtained in step (4) of Example 5, 5 parts of 10 billion CFU / g Bacillus subtilis original powder, 2 parts of fulvic powder, and 35 parts of potassium humate and mix them to obtain the finished product of the Bacillus floc composition, that is, the microbial agent of the present invention, which contains 1.1 billion CFU / g of viable spores.

[0099] We conducted electron microscopic observation on the product prepared in this example and similar products on the market that were compounded with potassium fulvic acid and Bacillus. Figure 5 It can be seen that the product of the present invention has obvious complex adsorption of Bacillus and fulvic acid salt particles to form a bacterial floc complex, and the complex colloid is coated on the outside of the spores. Similar products on the market do not have bacterial flocs of Bacillus and fulvic acid salts, and the fulvic acid salts and spores are dispersed ( Figure 6 ).

[0100] Example 7

[0101] The sample of Example 6 was taken for field test in Zhongzhan Village, Hepu County, Guangxi. The test set up a conventional treatment area where conventional potassium humate and Bacillus mixed bacterial fertilizer (purchased in Yantai, Shandong) was applied, a sample treatment area of ​​Example 6, and a blank treatment area. The specific use method is to flush and apply once in the cucumber seedling and pre-flowering stage, respectively, with an application amount of 1kg / mu·time, and investigate the transplant survival rate, plant stem diameter and total number of cucumbers during the melon setting period; investigate the number of melons per plant when the first batch of cucumbers are harvested. The results are shown in Table 4.

[0102] Table 4 Survey of cucumber characteristics in treatment and control areas

[0103]

[0104] The test results show that the microbial agent containing Bacillus flocculus of the present invention (sample of Example 6) was used at the cucumber seedling stage. In the first investigation at the fruit-setting stage, it was found that the transplant survival rate of the seedlings was high in the treatment area using the sample of Example 6, the cucumber seedlings had thick stems and developed root systems, and the total number of fruit-setting per 100 cucumber plants was high, which was significantly better than the effect of conventional products on the market. When the first batch of cucumbers was harvested, the number of fruit-setting per plant was investigated, and the number of fruit-setting per plant in the treatment area using the sample of Example 6 was also significantly better than conventional products on the market.

[0105] Example 8

[0106] The samples of Example 6 were taken for field test in Qingdao, Shandong. The test set up a conventional treatment area where conventional potassium humate and Bacillus mixed fertilizer (purchased in Yantai, Shandong) were applied, a sample treatment area of ​​Example 6, and a blank treatment area. The specific method of use was to apply once during the fruiting period of blueberries, with an application amount of 1 kg / mu·time, and investigate the color change of blueberries and the sugar content of the fruit 30 days after the application. The results are shown in Table 5.

[0107] Sugar content determination method: Use a handheld sugar refractometer.

[0108] Table 5 Survey on color change period and sugar content of blueberries in treatment area and control area

[0109] Processing Picking date of the first batch of fruits Sugar content % Average weight of single fruit g Sample of Example 6 May 20, 2020 12.3 2.46 Market conventional Bacillus + potassium fulvate product June 8, 2020 10.05 2.04 Blank control June 8, 2020 9.11 1.84

[0110] From the above experiments, it can be seen that the blueberries in the treated area using the microbial agent containing Bacillus flocculus (sample in Example 6) of the present application have early color change, and the picking date of the first batch of fruits can be more than 15 days earlier than the conventional products on the market. The fruit has a good shape, an average weight of 2.46g per fruit, and a sugar content of 12.3%. This shows that the use of the microbial agent of the present invention can significantly improve the yield and quality of blueberries, and early listing can increase farmers' income.

[0111] Example 9

[0112] The sample of Example 6 was taken for field test in Changyang, Hubei. The test set up a conventional treatment area where conventional potassium humate and Bacillus mixed bacterial fertilizer (purchased in Yantai, Shandong) were applied, a sample treatment area of ​​Example 6, and a blank treatment area. The specific method of use was to flush and apply once in the tomato seedling and pre-flowering stage, respectively, with an application amount of 1kg / mu·time. The transplant survival rate, stem diameter and root length were investigated 30 days after transplanting, and the flowering period of tomatoes was recorded during the test. The results are shown in Table 6.

[0113] Table 6 Survey table of tomato traits in treatment and control areas

[0114]

[0115] From the above experiments, it can be seen that in the area treated with the microbial agent containing Bacillus flocculus (sample in Example 6) of the present application, the survival rate of tomatoes is high, the plants are sturdy, the leaves are dark green, the root system is well-developed, and the fibrous roots are well-developed. During the experiment, it was observed that the flowering period of tomatoes can be advanced by more than 10 days compared with conventional products on the market. This shows that the use of the microbial agent of the present invention can significantly improve the transplant survival rate of tomatoes, promote the growth of crops, and make tomatoes bloom early.

[0116] It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention. In addition, those skilled in the art can make various modifications or equivalent replacements to the present invention within the essence and protection scope of the present invention, and such modifications or equivalent replacements should also be regarded as falling within the protection scope of the present invention.

Claims

1. A method for preparing a microbial inoculant, characterized in that: the microbial inoculant contains a Bacillus mycelium aggregate, the mycelium aggregate contains spores, extracellular polysaccharides and fulvate salts, wherein the spores and extracellular polysaccharides are the spores and extracellular polysaccharides produced during fermentation, the extracellular polysaccharides form a composite colloid with the fulvate salts and coat the outside of the spores, and the Bacillus is a Bacillus that produces extracellular polysaccharides. The method comprises the following steps: inoculating Bacillus into a fermentation medium and culturing, drying and pulverizing the obtained fermentation broth, the culturing is liquid aerated culturing, the culturing temperature is 30-32 °C, the aeration rate is 0.8-1.2 VVM, the stirring speed of the fermentation tank is 375-525 revolutions per minute, and the tank pressure is 0.05-0.07 MP; the fermentation medium contains the following components in weight ratio: peanut meal 1-2%, glucose 2-3%, yeast extract 0.5-1%, corn steep liquor 1.5-2%, potassium dihydrogen phosphate 0.05-0.1%, silicon dioxide 0.01-0.05%, fulvate salts 1-5%, magnesium sulfate 0.05-0.1%, and the pH of the medium is 7.0-7.5; the Bacillus is selected from any one or more of Paenibacillus mucilaginosus, Paenibacillus polymyxa, and Paenibacillus azotofixans.

2. The method for preparing a microbial inoculant according to claim 1, wherein the fulvate salts account for 5-30% of the weight of the Bacillus mycelium aggregate.

3. The method for preparing a microbial inoculant according to claim 1, wherein the fulvate salts are selected from any one or more of potassium fulvate, sodium fulvate, calcium fulvate, iron fulvate, manganese fulvate, or zinc fulvate.

4. The method for preparing a microbial inoculant according to claim 3, wherein the fulvate salts are potassium fulvate.

5. A microbial inoculant, characterized in that: it contains a Bacillus mycelium aggregate prepared by the method according to any one of claims 1-4.

6. Use of the microbial inoculant according to claim 5 in improving the survival rate of plant seedlings.

7. Use of the microbial inoculant according to claim 5 in promoting plant growth.

8. Use of the microbial inoculant according to claim 5 in increasing plant yield.

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