阅读说明：本技术 一种适用于盐碱土壤的微生物肥料及其制备方法 (Microbial fertilizer suitable for saline-alkali soil and preparation method thereof ) 是由 刘丽英 邵光杰 古今朝 孙中涛 侯汉学 崔树君 徐燕 徐风英 于 2021-07-08 设计创作，主要内容包括：本发明属于农业生物技术领域,公开了一种适用于盐碱土壤的微生物肥料及其制备方法,所述适用于盐碱土壤的微生物肥料按照质量份数计,由贝莱斯芽孢杆菌BOB-2菌粉100份,巨大芽孢杆菌菌粉20份,矿源腐植酸粉50份,七水硫酸锌10份,七水硫酸镁10份,以及七水硫酸亚铁10份组成。本发明以贝莱斯芽孢杆菌(Bacillus velezensis)BOB-2菌株和巨大芽孢杆菌(Bacillus megaterium)ACCC04314为有效菌,贝莱斯芽孢杆菌BOB-2菌株分离自盐碱地土壤,具有耐盐碱、促进作物生长、提高土壤酶活、拮抗作物病原菌等优良特性或有益作用,可用于生产微生物肥料,获得更好应用效果。(The invention belongs to the technical field of agricultural biology, and discloses a microbial fertilizer suitable for saline-alkali soil and a preparation method thereof, wherein the microbial fertilizer suitable for the saline-alkali soil comprises, by mass, 100 parts of Bacillus belgii BOB-2 bacterial powder, 20 parts of Bacillus megaterium bacterial powder, 50 parts of mineral humic acid powder, 10 parts of zinc sulfate heptahydrate, 10 parts of magnesium sulfate heptahydrate, and 10 parts of ferrous sulfate heptahydrate. The Bacillus velezensis BOB-2 strain and the Bacillus megaterium ACCC04314 are effective bacteria, and the Bacillus velezensis BOB-2 strain is separated from the saline-alkali soil, has excellent characteristics or beneficial effects of salt and alkali resistance, crop growth promotion, soil enzyme activity improvement, crop pathogenic bacteria antagonism and the like, can be used for producing microbial fertilizers, and obtains better application effect.)

1. The microbial fertilizer suitable for the saline-alkali soil is characterized by comprising, by mass, 100 parts of Bacillus belgii BOB-2 bacterial powder, 20 parts of Bacillus megaterium bacterial powder, 50 parts of mineral humic acid powder, 10 parts of zinc sulfate heptahydrate, 10 parts of magnesium sulfate heptahydrate and 10 parts of ferrous sulfate heptahydrate.

2. The microbial fertilizer suitable for saline-alkali soil according to claim 1, wherein the Bacillus belgii BOB-2 has a preservation number of CGMCC No. 22606; the stock strain of the bacillus megaterium is purchased from China agricultural microbial strain preservation management center, and the preservation number is ACCC 04314.

3. The microbial fertilizer applicable to saline-alkali soil of claim 1, wherein the effective viable bacteria content of the bacillus belgii BOB-2 is (0.5-1.0) x 10¹⁰cfu/g, the effective viable bacteria content of the bacillus megaterium is (1.0-2.0) multiplied by 10⁹cfu/g。

4. The method for preparing the microbial fertilizer applicable to the saline-alkali soil according to any one of claims 1 to 3, wherein the method for preparing the microbial fertilizer applicable to the saline-alkali soil comprises the following steps:

activating Bacillus belgii strains;

step two, preparing a Bacillus beilesiensis triangular flask seed solution;

step three, preparing seeds of the Bacillus belgii liquid fermentation tank;

step four, carrying out solid state fermentation culture on the Bacillus belgii;

step five, preparing bacillus megaterium powder;

step six, preparing the microbial fertilizer.

5. The method for preparing a microbial fertilizer suitable for saline-alkali soil according to claim 4, wherein in the first step, the activation of Bacillus belgii strain comprises:

the Bacillus belgii BOB-2 strain is transferred to an LB culture medium test tube slant, and cultured for 24h at 37 ℃ for activation.

6. The method for preparing microbial fertilizer suitable for saline-alkali soil as claimed in claim 4, wherein in the second step, the preparation of Bacillus beiLeisi seed solution comprises:

scraping the activated Bacillus beilesiensis BOB-2 lawn by using an inoculating loop, inoculating the Bacillus beilesiensis BOB-2 lawn in the LB liquid culture medium, and performing shake culture at 37 ℃ at 180r/min for 24 h; wherein the LB liquid culture medium comprises the following components in percentage by weight: 10g of peptone, 5g of yeast powder, 10g of sodium chloride, 1000mL of water and pH 7.5.

7. The method for preparing microbial fertilizer suitable for saline alkali soil as claimed in claim 4, wherein the Bacillus belgii liquid fermenter seed preparation in step three comprises:

inoculating 2-5% of the seed in a triangular flask into a liquid fermentation tank containing LB liquid culture medium, and culturing at 37 ℃ for 24 h; wherein the volume of the seeding tank is 10L, the liquid loading amount is 7L, the stirring speed is 200r/min, and the ventilation amount is 7L/min.

8. The method for preparing a microbial fertilizer suitable for saline-alkali soil according to claim 4, wherein in the fourth step, the solid-state fermentation culture of Bacillus belgii comprises:

culturing with 60cm × 120cm stainless steel tray;

sterilizing bran culture medium at 121 deg.C for 30min, cooling, spreading in a pre-sterilized tray, and making the material layer 4-7cm thick; wherein the formula of the bran culture medium is as follows: bran 80%, rice straw powder 19%, Ca (OH)₂1％，MnSO₄0.1 percent and the initial water content is 50 to 60 percent;

inoculating seed liquid of Bacillus belgii BOB-2 into culture medium in an inoculation amount of 2-5%, and fermenting and culturing for 36-48 h; controlling the temperature of the product to be 33-40 ℃ and the water content to be 45-60% in the fermentation process, and turning over once every 2-5h to adjust the temperature and supply oxygen; after fermentation, drying the culture at 40-45 deg.C, and pulverizing to 80-100 mesh to obtain Bacillus beiLeisi BOB-2 powder with viable bacteria content of preferably (1.0-2.0) x 10¹⁰cfu/g, used for preparing microbial fertilizer.

9. The method for preparing microbial fertilizer suitable for saline-alkali soil as claimed in claim 4, wherein in the fifth step, the preparation of the bacillus megaterium powder comprises the following steps:

the method for activating the strain of the bacillus megaterium, preparing the seed solution in a triangular flask and preparing the seeds in a liquid fermentation tank is the same as that of the bacillus belief BOB-2 strain; the solid state fermentation culture method of the bacillus megaterium is the same as the Bacillus beleisi BOB-2 strain, but the formula of the fermentation culture medium is as follows: 99% of bran, Ca (OH)₂ 1％，MnSO₄0.1 percent and the initial water content is 50 to 60 percent; after fermentation, drying the culture at 40-45 deg.C, and pulverizing to 80-100 mesh to obtain Bacillus megaterium powder with viable bacteria content of preferably (1.0-2.0) x 10¹⁰cfu/g, used for preparing microbial fertilizer.

10. The method for preparing microbial fertilizer suitable for saline-alkali soil according to claim 4, wherein in the sixth step, the preparation of microbial fertilizer comprises:

100 parts of Bacillus beilesiensis BOB-2 bacterial powder, 20 parts of Bacillus megaterium bacterial powder, 50 parts of mineral humic acid powder, 10 parts of zinc sulfate heptahydrate, 10 parts of magnesium sulfate heptahydrate and 10 parts of ferrous sulfate heptahydrate are uniformly mixed to obtain the microbial fertilizer product, wherein the total humic acid content of the mineral humic acid powder is more than or equal to 50% (w/w).

Technical Field

The invention belongs to the technical field of agricultural biology, and particularly relates to a microbial fertilizer suitable for saline-alkali soil and a preparation method thereof.

Background

At present, China has a large amount of saline-alkali soil which is mainly distributed in northwest, northChina, northeast and coastal areas, and the total area is about 4-5 hundred million acres. When the salt content of the soil is higher than 0.2% -0.5%, the salt content has obvious adverse effect on the growth of plants. Too high salinity can cause soil water salt movement unbalance, soil structure to change, leads to soil unit weight to increase, and soil particle clearance reduces, and the permeability of soil weakens, and the permeability of soil increases to cause soil to harden, the fertility descends, and the plant roots absorbs water the difficulty, can not grow even, finally causes the crop to subtract the output and abandons the cultivation even. Therefore, how to improve saline-alkali soil, enlarge the cultivated land area and improve the ecological environment is a problem which needs to be solved urgently at present.

At present, the saline-alkali soil is mainly treated by physical and chemical methods in agriculture. However, most of these methods have high treatment cost, great damage to soil texture and difficulty in solving the problems of saline-alkali soil for a long time and effectively. With the increasing concern of the country on the ecological environment, the biological improvement of saline-alkali soil becomes a great hot spot in agricultural production and research.

The biological improvement means that saline-alkali soil is improved by using organisms in the soil and planting saline-alkali tolerant plants. The soil contains a large amount of microorganisms, and has remarkable promotion effects on soil nutrients, organic matter content, soil physicochemical properties and crop growth. The secretion discharged in the soil during the growth of the microorganism and saline-alkali components have complex chemical reaction, so that the structural characteristics of the soil can be improved, and the continuous utilization of the saline-alkali soil is promoted. Meanwhile, the microorganism has the functions of promoting the growth of crops, antagonizing pathogenic bacteria, inducing the resistance of crops and the like, and can also improve the tolerance of the crops to saline-alkali stress, thereby improving the growth condition of the crops and improving the yield and the quality.

The microbial fertilizer is a green input product for saline-alkali soil improvement and fertilization. However, the application effect of the common microbial fertilizer in saline-alkali soil is not ideal, and the main reason is that the saline-alkali environment is not beneficial to the field planting and growth of microorganisms, so that the application effect of the microbial fertilizer is reduced. Therefore, functional strains which have strong saline-alkali tolerance, can be planted in saline-alkali soil and have better disease resistance and growth promotion effects need to be selected from the habitat of the saline-alkali soil for producing the microbial fertilizer suitable for the saline-alkali soil.

In recent years, the separation of saline-alkali tolerant strains and the application of the strains in the development of microbial fertilizers suitable for saline-alkali soil become research hotspots, and a better application effect is achieved. The Bacillus belgii is a bacillus with disease-resistant and growth-promoting effects, but has few saline-alkali resistant strains separated from saline-alkali soil, and is not suitable for producing microbial fertilizers suitable for saline-alkali soil. The reported saline-alkali tolerant Bacillus belgii is few, and Liuhuan and the like are separated from the nature to promote a saline-alkali tolerant strain Bacillus velezensisYM6 of corn, the strain can remarkably promote the growth of corn seedlings under the saline-alkali condition, but the strain does not have the capability of antagonizing crop pathogenic bacteria such as Rhizoctonia solani. Generally speaking, compared with a single microbial inoculum, the composite microbial fertilizer prepared by preferably combining multiple strains and combining the composite microbial fertilizer with raw materials such as humic acid and the like has better application effect.

Through the above analysis, the problems and defects of the prior art are as follows:

(1) most of the methods for treating the saline-alkali soil by adopting physical and chemical methods have higher treatment cost and are difficult to solve the problem of the saline-alkali soil durably and effectively.

(2) The effective microorganisms in the common microbial fertilizer have poor field planting capability in saline-alkali soil, and the saline-alkali environment is not beneficial to the growth and the propagation of the microorganisms, so that the application effect of the microbial fertilizer is reduced, and the effects of promoting the growth and resisting diseases of crops are not ideal when the microbial fertilizer is applied to the saline-alkali soil.

(3) The existing Bacillus belgii has fewer saline-alkali tolerant strains separated from saline-alkali soil, and is not suitable for producing microbial fertilizers suitable for saline-alkali soil.

The difficulty in solving the above problems and defects is: (1) separating and screening out functional strains with strong saline-alkali tolerance and good growth promoting and disease resisting effects on crops; (2) a fermentation process of saline-alkali tolerant strains is established, and economic and clean production is realized; (3) the biological fertilizer can be reasonably mixed with other beneficial bacteria and chemical components to develop a biological fertilizer product which can be produced in a large scale and has better disease-resistant and growth-promoting effects.

The significance of solving the problems and the defects is as follows: provides green investment for the development and utilization of saline-alkali soil, promotes the growth of saline-alkali soil crops, reduces the occurrence of diseases, increases the yield, improves the quality and increases the income of farmers.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides a microbial fertilizer suitable for saline-alkali soil and a preparation method thereof.

The microbial fertilizer is prepared from 100 parts by mass of Bacillus belgii BOB-2 powder, 20 parts by mass of Bacillus megaterium powder, 50 parts by mass of mineral humic acid powder, 10 parts by mass of zinc sulfate heptahydrate, 10 parts by mass of magnesium sulfate heptahydrate and 10 parts by mass of ferrous sulfate heptahydrate.

Further, the preservation number of the Bacillus belgii BOB-2 in the common microorganism center of China Committee for culture Collection of microorganisms is CGMCC No. 22606; the stock strain of the bacillus megaterium is purchased from China agricultural microbial strain preservation management center, and the preservation number is ACCC 04314.

Further, the content of effective viable bacteria of the Bacillus belgii BOB-2 is (0.5-1.0) multiplied by 10¹⁰cfu/g, the effective viable bacteria content of the bacillus megaterium is (1.0-2.0) multiplied by 10⁹cfu/g。

The invention also aims to provide a preparation method of the microbial fertilizer applicable to saline-alkali soil, which comprises the following steps:

activating Bacillus belgii strains;

step two, preparing a Bacillus beilesiensis triangular flask seed solution;

step three, preparing seeds of the Bacillus belgii liquid fermentation tank;

step four, carrying out solid state fermentation culture on the Bacillus belgii;

step five, preparing bacillus megaterium powder;

step six, preparing the microbial fertilizer.

Further, in the first step, the activation of the bacillus beilesensis strain comprises:

the Bacillus belgii BOB-2 strain is transferred to an LB culture medium test tube slant, and cultured for 24h at 37 ℃ for activation.

Further, in the second step, the preparation of the bacillus beilesensis triangular flask seed solution comprises the following steps:

scraping the activated Bacillus beilesiensis BOB-2 lawn by using an inoculating loop, inoculating the Bacillus beilesiensis BOB-2 lawn in the LB liquid culture medium, and performing shake culture at 37 ℃ at 180r/min for 24 h; wherein the LB liquid culture medium comprises the following components in percentage by weight: 10g of peptone, 5g of yeast powder, 10g of sodium chloride, 1000mL of water and pH 7.5.

Further, in step three, the preparation of the seeds for the liquid fermentation tank of bacillus beijerinckii comprises the following steps:

inoculating 2-5% of the seed in a triangular flask into a liquid fermentation tank containing LB liquid culture medium, and culturing at 37 ℃ for 24 h; wherein the volume of the seeding tank is 10L, the liquid loading amount is 7L, the stirring speed is 200r/min, and the ventilation amount is 7L/min.

Further, in the fourth step, the solid state fermentation culture of bacillus beleisi comprises:

culturing with 60cm × 120cm stainless steel tray;

sterilizing bran culture medium at 121 deg.C for 30min, cooling, spreading in a pre-sterilized tray with a material layer thickness of 4-7 cm; wherein the formula of the bran culture medium is as follows: bran 80%, rice straw powder 19%, Ca (OH)₂1％，MnSO₄0.1 percent and the initial water content is 50 to 60 percent; inoculating seed liquid of Bacillus belgii BOB-2 into culture medium in an inoculation amount of 2-5%, and fermenting and culturing for 36-48 h; controlling the temperature of the product to be 33-40 ℃ and the water content to be 45-60% in the fermentation process, and turning over once every 2-5h to adjust the temperature and supply oxygen; after fermentation, drying the culture at 40-45 deg.C, and pulverizing to 80-100 mesh to obtain Bacillus beiLeisi BOB-2 powder with viable bacteria content of preferably (1.0-2.0) x 10¹⁰cfu/g, used for preparing microbial fertilizer.

Further, in the fifth step, the preparation of the bacillus megaterium powder comprises the following steps:

the method for activating the strain of the bacillus megaterium, preparing the seed solution in a triangular flask and preparing the seeds in a liquid fermentation tank is the same as that of the bacillus belief BOB-2 strain; the solid state fermentation culture method of the bacillus megaterium is the same as the Bacillus beleisi BOB-2 strain, but the formula of the fermentation culture medium is as follows: 99% of bran, Ca (OH)₂ 1％，MnSO₄0.1 percent and the initial water content is 50 to 60 percent; after fermentation, drying the culture at 40-45 deg.C, and pulverizing to 80-100 mesh to obtain Bacillus megaterium powder with viable bacteria content of preferably (1.0-2.0) x 10¹⁰cfu/g, used for preparing microbial fertilizer.

Further, in the sixth step, the preparation of the microbial fertilizer comprises:

100 parts of Bacillus beilesiensis BOB-2 bacterial powder, 20 parts of Bacillus megaterium bacterial powder, 50 parts of mineral humic acid powder, 10 parts of zinc sulfate heptahydrate, 10 parts of magnesium sulfate heptahydrate and 10 parts of ferrous sulfate heptahydrate are uniformly mixed to obtain the microbial fertilizer product, wherein the total humic acid content of the mineral humic acid powder is more than or equal to 50% (w/w).

By combining all the technical schemes, the invention has the advantages and positive effects that: the microbial fertilizer suitable for the saline-alkali soil provided by the invention is a compound microbial fertilizer suitable for the saline-alkali soil, which is developed on the basis of the screened saline-alkali resistant disease-resistant growth-promoting strain Bacillus velezensis BOB-2 strain and preferably by strain combination.

The Bacillus subtilis BOB-2 strain and the Bacillus megaterium ACCC04314 are effective bacteria, the adopted Bacillus subtilis BOB-2 strain is separated from saline-alkali soil, has excellent characteristics or beneficial effects of salt and alkali resistance, crop growth promotion, soil enzyme activity improvement, crop pathogenic bacteria antagonism and the like, can be used for producing microbial fertilizers, is particularly suitable for microbial fertilizers of the salt and alkali soil, and has antagonism on pathogenic bacteria Rhizoctonia solani (Rhizoctonia solani) of rice sheath blight; the Bacillus beilesiensis BOB-2 strain, the Bacillus megaterium ACCC04314 strain, mineral humic acid and the like are formulated together to prepare the composite microbial fertilizer, so that a better application effect can be obtained.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments of the present invention will be briefly described below, and it is obvious that the drawings described below are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to the drawings without creative efforts.

FIG. 1 is a flow chart of a preparation method of a microbial fertilizer suitable for saline-alkali soil according to an embodiment of the invention.

FIG. 2 is a schematic diagram of the colony morphology of Bacillus belgii BOB-2 strain provided in the examples of the present invention.

FIG. 3 is a cell morphology of Bacillus belgii BOB-2 strain according to an embodiment of the present invention, wherein FIG. 3(b) is a partially enlarged view of FIG. 3 (a).

FIG. 4 is a schematic diagram of a phylogenetic tree constructed based on a 16S rDNA partial sequence of the Bacillus belgii BOB-2 strain provided by the embodiment of the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

Aiming at the problems in the prior art, the invention provides a microbial fertilizer suitable for saline-alkali soil and a preparation method thereof, and the invention is described in detail below with reference to the accompanying drawings.

The microbial fertilizer suitable for the saline-alkali soil provided by the embodiment of the invention comprises, by mass, 100 parts of Bacillus beijerinckii BOB-2 bacterial powder, 20 parts of Bacillus megaterium bacterial powder, 50 parts of mineral humic acid powder, 10 parts of zinc sulfate heptahydrate, 10 parts of magnesium sulfate heptahydrate and 10 parts of ferrous sulfate heptahydrate.

As shown in fig. 1, the preparation method of the microbial fertilizer suitable for saline-alkali soil provided by the embodiment of the invention comprises the following steps:

s101, activating the Bacillus belgii strain;

s102, preparing a Bacillus beilesiensis triangular flask seed solution;

s103, preparing seeds of the Bacillus belgii liquid fermentation tank;

s104, carrying out solid state fermentation culture on the Bacillus belgii;

s105, preparing bacillus megaterium powder;

and S106, preparing the microbial fertilizer.

The technical solution of the present invention will be further described with reference to the following examples.

Example 1

The invention discloses a microbial fertilizer suitable for saline-alkali soil and a preparation method thereof. The microbial fertilizer suitable for the saline-alkali soil is prepared from a saline-alkali-resistant disease-resistant growth-promoting bacterium Bacillus velezensis BOB-2, a Bacillus megaterium ACCC04314, mineral humic acid and other raw materials. The Bacillus belgii BOB-2 strain is preserved in the China general microbiological culture Collection center with the preservation number of CGMCC No.22606, has the excellent characteristics or beneficial effects of salt and alkali resistance, crop growth promotion, soil enzyme activity improvement, crop pathogenic bacteria antagonism and the like, can be used for producing microbial fertilizers, and is particularly suitable for microbial fertilizers of salt and alkali soil. The stock of the bacillus megaterium ACCC04314 is purchased from China agricultural microbial strain preservation management center, and the preservation number is ACCC 04314.

The microbial fertilizer comprises 2 strains: bacillus velezensis (Bacillus velezensis) BOB-2 strain and Bacillus megaterium (Bacillus megaterium) ACCC 04314.

The strain is Bacillus velezensis (Bacillus velezensis) BOB-2. The preservation number of the strain in China general microbiological culture Collection center (CGMCC) is CGMCC No. 22606; the preservation address is microbial research institute of China academy of sciences No. 3 of Xilu No.1 of Beijing, Chaoyang district, and the preservation date is 2021 year, 5 month and 25 days; the referenced biological materials: BOB-2; suggested classification nomenclature: bacillus velezensis; the depository is received at 25/5/2021 and registered into a book, and is detected to be alive; storage for thirty years, as requested, from 5 months and 25 days 2021, continues for five years after receiving a request to provide a sample of biological material before expiration. The Bacillus belgii BOB-2 has excellent characteristics or beneficial effects of salt and alkali resistance, crop growth promotion, soil enzyme activity improvement, pathogenic bacteria antagonism, rice sheath blight prevention and control and the like.

The stock of the bacillus megaterium ACCC04314 is purchased from China agricultural microbial strain preservation management center, and the preservation number is ACCC 04314.

The microbial fertilizer also comprises humic acid and trace elements. The microbial fertilizer comprises the following components in percentage by weight: 100 parts of Bacillus belgii BOB-2 powder, 20 parts of Bacillus megaterium powder, 50 parts of mineral humic acid powder, 10 parts of zinc sulfate heptahydrate, 10 parts of magnesium sulfate heptahydrate and 10 parts of ferrous sulfate heptahydrate.

After amplification culture, the Bacillus beilesensis BOB-2 is inoculated in a solid fermentation culture medium, cultured for 36-48h at 33-40 ℃, dried, crushed and uniformly mixed with raw materials such as Bacillus megaterium powder, humic acid and the like to obtain the microbial fertilizer; the formula of the solid fermentation medium is as follows: bran 80%, rice straw powder 19%, Ca (OH)₂ 1％，MnSO₄0.1 percent and 50 to 60 percent of initial water content.

The viable bacteria content of the microbial fertilizer is preferably as follows: bacillus belgii BOB-2 (0.5-1.0) x 10¹⁰cfu/g, Bacillus megaterium (1.0-2.0). times.10⁹cfu/g。

Example 2: screening of Bacillus beilis BOB-2

(1) Isolation of salt and alkali tolerant strains

Collecting saline-alkali soil, weighing 10g of soil sample, placing the soil sample in a sterilized 250mL conical flask filled with 90mL deionized water and 10-20 glass beads, and placing the conical flask in a shaking table at 180r/min to shake for 30 min. Diluting the soil suspension to 10 deg.C by gradient dilution method^-2，10^-3，10^-4，10^-5，10^-6，10^-7. 0.1mL of each of the soil suspensions of different dilutions was pipetted into an LB medium plate using a pipette gun and spread uniformly. Placing into an incubator at 37 deg.C, and culturing for 48-72 h. And selecting strains with different colony morphological characteristics, streaking and culturing the strains on a separation plate, and repeatedly streaking, separating and purifying until a single colony is obtained. The LB culture medium comprises the following components: tryptone 10 g/L; 5g/L of yeast extract; 10g/L of sodium chloride (NaCl); agar 20g/L, pH 7.5.

Inoculating the separated bacterial strain into LB saline-alkali culture medium, culturing at 37 ℃ for 48-72h, observing and recording the growth condition of the bacterial strain, and selecting the bacterial strain with relatively good growth condition for subsequent tests. The LB saline-alkali culture medium comprises the following components in percentage by weight: tryptone 10 g/L; 5g/L of yeast extract; 50g/L of sodium chloride (NaCl); agar 20g/L, pH 9.0.

(2) Screening of saline-alkali tolerant growth-promoting strains

And carrying out germination test on the screened saline-alkali tolerant strains under the saline-alkali stress condition, and further screening the strains with better growth promoting effect. The test was performed in a petri dish with filter paper, using cream pakchoi seeds as the test material. The test uses a saline-alkali culture solution with the following composition: NaCl 25mmol/L, Na₂SO₄ 50mmol/L，NaHCO₃25mmol/L, pH 8.0. Adding 7mL of saline-alkali culture solution into blank control group, and adding 7mL of saline-alkali culture solution containing different bacteria to be screened (bacterial suspension with bacterial content of 1 × 10) into test group⁷cfu/mL). Each treatment setting is 3 levelsAnd (3) putting 30 seeds in each parallel, and culturing for 3-7 days in a light incubator at 28 ℃. And (4) screening out strains with relatively good growth promoting effect according to the growth condition of the young seedlings of the pakchoi.

(3) Screening of antagonistic strains

Screening antagonistic strains by taking rhizoctonia solani, a pathogenic bacterium of rice sheath blight, as a tested pathogenic bacterium. And (3) culturing rhizoctonia solani on a PDA culture medium plate to obtain a bacterial colony. Taking a fungus block with the diameter of 5mm by using a puncher, inoculating the fungus block to the center of a PDA culture medium flat plate, inoculating strains to be screened at equal distances at a position 2.0cm away from the center of the flat plate, culturing for 10 days at 28 ℃ by taking the flat plate without the strains to be screened as a contrast, selecting the strains with larger diameter of a bacteriostatic zone, and storing for later use. Each treatment was repeated 3 times. The PDA culture medium formula comprises: 200g of potato, 20g of glucose, 20g of agar and 1000mL of distilled water, and the pH value is natural.

Through the multiple screening, the saline-alkali resistance, the growth promoting capability and the pathogenic bacterium antagonistic capability of each strain are comprehensively considered, and a relatively good strain is screened out and numbered as BOB-2.

Example 3: identification of Bacillus beleisi BOB-2 strains

(1) Morphological and physiological biochemical identification

Through observation and microscopic examination, the morphological characteristics of the BOB-2 strain are as follows: culturing in LB medium at 37 deg.C for 48 hr to obtain colony diameter of 0.5-0.8mm, white semitransparent, round, smooth surface, swelling, moistening, viscous, glossy, uniform texture, and tidy edge (see FIG. 2); the bacterial body is in the shape of a short rod (see FIG. 3).

The physiological and biochemical characteristics of the BOB-2 strain were determined with reference to Bergey's Manual of bacteriological identification (eighth edition). The physiological and biochemical characteristics of the BOB-2 strain are as follows: the test results show that the test results show spore formation, gram staining positive, catalase test positive, methyl red reaction negative, V-P test negative, malonate utilization test positive, nitrate reduction test positive, starch hydrolysis test positive, motility negative, maltose utilization test positive, mannitol utilization test positive, glucose utilization test positive and lactose utilization test positive.

(2)16S rDNA sequence analysis

The BOB-2 strain is inoculated into an LB culture medium and cultured for 24 hours at 37 ℃ and 180r/min in a shaking way. Collecting thalli, extracting total DNA, and then taking the total DNA as a template, wherein the total DNA is expressed in the expression vector of a universal primer F27 of a prokaryotic 16S rRNA gene: 5'-AGA GTT TGA TCA TGG CTC AG-3' and F27: 5'-AGA GTT TGA TCA TGG CTC AG-3', the 16S rDNA gene was amplified by PCR. The amplification product is separated by 1% agarose gel electrophoresis, recovered by gel recovery kit, and sequenced by Celastri Borcico Inc., and the obtained sequence is shown in sequence table SEQ No. 1. The 16S rDNA sequences tested were aligned to sequences in the GenBank database, multiple sequence homology analysis was performed using MEGA5.5 software, and a phylogenetic tree was constructed, as shown in figure 4.

Through morphological, physiological and biochemical characteristics and 16S rDNA sequence analysis, the strain is Bacillus belgii and is named as Bacillus belgii (Bacillus velezensis) BOB-2.

Example 4: preparation of microbial fertilizer

(1) Activation of Bacillus belgii strains

The Bacillus belgii BOB-2 strain is transferred to an LB culture medium test tube slant, and cultured for 24h at 37 ℃ for activation.

(2) Preparation of seed solution of Bacillus beleisi in triangular flask

Scraping the activated Bacillus beilesiensis BOB-2 lawn by using an inoculating loop, inoculating the lawn in the LB liquid culture medium, and performing shake culture at 37 ℃ at 180r/min for 24 h. The LB liquid culture medium comprises the following components in percentage by weight: 10g of peptone, 5g of yeast powder, 10g of sodium chloride, 1000mL of water and pH 7.5.

(3) Bacillus belgii liquid fermentation tank seed preparation

Inoculating 2-5% of seed in triangular flask into liquid fermenter containing LB liquid culture medium, and culturing at 37 deg.C for 24 hr. The volume of the seeding tank is 10L, the liquid loading amount is 7L, the stirring speed is 200r/min, and the ventilation amount is 7L/min.

(4) Solid state fermentation culture of Bacillus belgii

The culture was carried out using a 60cm X120 cm stainless steel tray. Sterilizing bran culture medium at 121 deg.C for 30min, cooling, spreading in a pre-sterilized tray, and making the material layer 4-7cm thick. Mixing the sprouts with a solventInoculating seed liquid of bacillus BOB-2 into culture medium at an inoculation amount of 2-5%, and fermenting and culturing for 36-48 h. During the fermentation process, the temperature of the product is controlled to be 33-40 ℃, the water content is controlled to be 45-60%, and the product is turned over once every 2-5h to adjust the temperature and supply oxygen. The formula of the bran culture medium is as follows: bran 80%, rice straw powder 19%, Ca (OH)₂ 1％，MnSO₄0.1 percent and 50 to 60 percent of initial water content. After fermentation, drying the culture at 40-45 deg.C, and pulverizing to 80-100 mesh to obtain Bacillus beiLeisi BOB-2 powder with viable bacteria content of preferably (1.0-2.0) x 10¹⁰cfu/g, used for preparing microbial fertilizer.

(5) Preparation of Bacillus megaterium powder

The method for activating the strain of the bacillus megaterium, preparing the seed solution in the triangular flask and preparing the seeds in the liquid fermentation tank is the same as the method for preparing the strain of the bacillus beilesiensis BOB-2. The solid state fermentation culture method of the bacillus megaterium is the same as the Bacillus beleisi BOB-2 strain, but the formula of the fermentation culture medium is as follows: 99% of bran, Ca (OH)₂ 1％，MnSO₄0.1 percent and 50 to 60 percent of initial water content. After fermentation, drying the culture at 40-45 deg.C, and pulverizing to 80-100 mesh to obtain Bacillus megaterium powder with viable bacteria content of preferably (1.0-2.0) x 10¹⁰cfu/g, used for preparing microbial fertilizer.

(6) Preparation of microbial fertilizer

The formula of the microbial fertilizer comprises: 100 parts of Bacillus belgii BOB-2 powder, 20 parts of Bacillus megaterium powder, 50 parts of mineral humic acid powder, 10 parts of zinc sulfate heptahydrate, 10 parts of magnesium sulfate heptahydrate and 10 parts of ferrous sulfate heptahydrate. The preparation method of the microbial fertilizer comprises the following steps: taking the raw materials according to the formula amount, and uniformly mixing to obtain the microbial fertilizer product, wherein the viable bacteria content of the microbial fertilizer product is preferably Bacillus beiLeisi BOB-2 strain (0.5-1.0) multiplied by 10¹⁰cfu/g, Bacillus megaterium (1.0-2.0). times.10⁹cfu/g. The mineral humic acid powder is purchased from Shandong Innovative humic acid science and technology Limited, and the total humic acid content is more than or equal to 50% (w/w).

The technical solution of the present invention is further described below with reference to application examples.

Application example 1: growth promoting effect of microbial fertilizer on tomatoes

The pot experiment was performed in 1 treatment group (T) and 1 control group (CK) in total, each treatment group consisting of 25 pots. The treatment group T was applied with 0.1% (dry fertilizer/dry soil) per pot of the microbial fertilizer according to the invention, and the control group CK was applied with 0.1% (dry fertilizer/dry soil) per pot of the pre-sterilized microbial fertilizer. The soil for test is taken from reclamation areas in Dongying cities and is slight saline-alkali soil. The microbial fertilizer is applied as a base fertilizer, namely the microbial fertilizer and soil are uniformly mixed when the pot is filled. Transplanting the tomato seedlings with basically consistent growth vigor and good growth conditions into pots (the specifications of the pots are that the upper inner diameter is 20cm, the lower inner diameter is 14cm, the height is 16cm, and 3kg of soil is filled in each pot), and only 1 tomato seedling is planted. At day 60 of the potting test, the correlation index measurements were performed. The plant height and the stem thickness are respectively measured by a tape measure and a vernier caliper; both the fresh weight and the dry weight were measured by a weighing method. Determination of rhizosphere soil enzyme activity: the urease determination adopts a sodium phenolate colorimetric method, the catalase determination adopts a potassium permanganate titration method, the phosphatase determination adopts a disodium phenyl phosphate colorimetric method, and the sucrase determination adopts a DNS colorimetric method.

The test results are shown in tables 1 and 2. Compared with the CK of the control group, the plant height, the stem thickness, the fresh weight and the dry weight of the T of the treatment group are improved to different degrees, and are respectively improved by 35.41 percent, 26.70 percent, 33.73 percent and 30.18 percent. This shows that the application of the microbial fertilizer according to the invention has a significant effect of promoting the growth of tomatoes. Compared with a control group CK, the activity of urease, catalase and sucrase in the treatment group T is respectively improved by 36.14%, 25.00% and 19.76%, which shows that the microbial fertilizer can improve the activity of tomato rhizosphere soil enzyme.

TABLE 1 Effect of microbial fertilizers on tomato growth

TABLE 2 influence of microbial fertilizers on the enzyme activity of tomato rhizosphere soil

Application example 2: prevention and treatment effect of microbial fertilizer on rice sheath blight disease

The pot experiment was performed in 1 treatment group (T) and 1 control group (CK) in total, each treatment group consisting of 25 pots. The treatment group T was applied with 0.1% (dry fertilizer/dry soil) per pot of the microbial fertilizer according to the invention, and the control group CK was applied with 0.1% (dry fertilizer/dry soil) per pot of the pre-sterilized microbial fertilizer. The soil for test is taken from reclamation areas in Dongying cities and is slight saline-alkali soil. The microbial fertilizer is applied as a base fertilizer, namely the microbial fertilizer and soil are uniformly mixed when the pot is filled. Two rice plants were planted in each pot, and after 5 days of seedling revival, 30mL of Rhizoctonia solani spore suspension (1X 10) was poured into each pot⁶spores/mL). And (5) after the root of the pathogenic bacteria is irrigated for 10 days, 20 days and 30 days, observing symptoms and counting the severity and the control effect of the disease.

Sheath blight grading standard: level 0: the whole plant is disease-free; level 1: the 4 th leaf and the leaf sheaths and leaves below the 4 th leaf (the 1 st leaf is sword leaf); and 3, level: the 3 rd leaf and the leaf sheaths and leaves below the 3 rd leaf are attacked; and 5, stage: the 2 nd leaf and the leaf sheaths and leaves below the 2 nd leaf are attacked; and 7, stage: attack of Jianye leaf and the leaf sheaths and leaves below the Jianye leaf; and 9, stage: the whole plant is attacked and withered in advance. Disease index ∑[ (number of diseased leaves per stage × representative value per stage)/(9 × total number of plants) ] × 100. Control effect ═ [ (control zone disease index-treatment zone disease index)/control zone disease index ] × 100%.

The pot test results are shown in table 3, and the control effect of the treatment group T is 83.33% 10 days after inoculation of pathogenic bacteria; after 20 days, the control effect is 59.78%; after 30 days, the control effect is 44.78 percent. The microbial fertilizer has a better prevention and treatment effect on rice sheath blight disease.

TABLE 3 prevention and treatment of rice sheath blight with microbial fertilizer

Application example 3: growth promoting effect on paddy field rice

The land for growing field crops is located in the reclamation and development area of Dongying city in Shandong province, the demonstration area is 60 mu, and the land for growing field crops is a slight saline-alkali land. Two treatments were set for the experiment, each treatment 30 acres. Control treatment CK: habitually applying 30kg of urea, 15kg of potassium chloride and 30kg of calcium magnesium phosphate fertilizer per mu. Experimental treatment T: 40kg of biological fertilizer, 27kg of urea, 13.5kg of potassium chloride and 27kg of calcium magnesium phosphate fertilizer are applied to each mu. The calcium magnesium phosphate fertilizer is used as a base fertilizer at one time, 60 percent of the biological fertilizer is used as the base fertilizer, 40 percent of the biological fertilizer is used as a tillering fertilizer, 40 percent of urea and potassium chloride are used as the base fertilizer, and 60 percent of the biological fertilizer is used as the tillering fertilizer. The control of diseases and pests and the water management during the test are carried out according to the conventional method. The test variety is rice Yanfeng 47, uniformly sown 5-month and 25-day 2020, and harvested 5-month and 5-day 11 to measure yield. The experimental results are as follows: the average yield per mu of CK treated by contrast is 820Kg, and the average yield per mu of T treated by experiment is 918Kg, so that the yield is increased by 11.95%; the organic matter content of CK soil is 1.52g/g soil (dry basis) through contrast treatment, the organic matter content of T soil is 1.68g/g soil (dry basis) through experiment treatment, and the improvement is 10.53%. This demonstrates that application of the bio-fertilizer product of the present invention has the effect of improving soil and increasing rice yield.

The above description is only for the purpose of illustrating the present invention and the appended claims are not to be construed as limiting the scope of the invention, which is intended to cover all modifications, equivalents and improvements that are within the spirit and scope of the invention as defined by the appended claims.

Sequence listing

<110> Wanlong agriculture and forestry economic Co., Ltd in reclamation area of Dongying City

<120> microbial fertilizer suitable for saline-alkali soil and preparation method thereof

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1458

<212> DNA

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