CN111909863B

CN111909863B - Bacillus amyloliquefaciens and application thereof

Info

Publication number: CN111909863B
Application number: CN201910379871.1A
Authority: CN
Inventors: 王敬敬; 黄志勇; 徐松; 赵维; 杨榕; 李晴晴; 赵思崎
Original assignee: Tianjin Institute of Industrial Biotechnology of CAS
Current assignee: Tianjin Institute of Industrial Biotechnology of CAS
Priority date: 2019-05-08
Filing date: 2019-05-08
Publication date: 2021-10-22
Anticipated expiration: 2039-05-08
Also published as: CN111909863A

Abstract

The invention discloses a Bacillus amyloliquefaciens and application thereof, wherein the Bacillus amyloliquefaciens is Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) FH1, and the preservation number of the Bacillus amyloliquefaciens in the common microorganism center of the China Committee for culture Collection of microorganisms is CGMCC No. 17050. Experiments prove that Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) FH1 has the functions of potassium dissolving, phosphorus dissolving and nitrogen fixing and can promote plants to absorb potassium elements, phosphorus elements and nitrogen elements; can produce iron carrier to promote the absorption of plant to iron element; the ACC deaminase can be produced, and the stress resistance of the plant can be enhanced; the yield and the quality of crops can be improved; can inhibit fusarium oxysporum, fusarium graminearum and rhizoctonia solani, thereby preventing and treating various plant fungal diseases. The invention has important application value.

Description

Bacillus amyloliquefaciens and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to bacillus amyloliquefaciens and application thereof.

Background

The soil is a material foundation on which people rely to live, is an extremely precious natural resource, and plays an extremely important role in the sustainable development of agriculture and animal husbandry. China has more people and less land, land resources are extremely limited, and the per-capita cultivated land area is far lower than the average level in the world. In order to increase the yield of crops per unit area, fertilizers and pesticides are used in large quantities. The use of chemical fertilizers and pesticides in large quantities not only causes serious ecological environmental problems (such as soil deterioration, water pollution, air pollution and the like), but also directly harms human health. Therefore, there is a high necessity to develop novel fertilizers and pesticides that are environmentally friendly to realize sustainable development of agriculture and society.

The microbial fertilizer is a product containing specific microorganism living bodies, is applied to agricultural production, and can increase the supply of plant nutrients or promote the growth of plants, prevent and control crop diseases, improve the yield, and improve the quality of agricultural products and the agricultural ecological environment through the life activities of the microorganisms contained in the microbial fertilizer. However, in the microbial fertilizer industry, the problems of single function of strains, unstable application effect and the like still exist. Therefore, it is highly desirable to screen strains with high activity, multiple functions and stable effect to promote the vigorous development of the microbial industry. At present, strains of microbial fertilizers are more heavily weighted than rhizobia, bacillus, pseudomonas and the like with the capabilities of fixing nitrogen, dissolving phosphorus, dissolving potassium, secreting hormones and the like, and strains of biological pesticides are more heavily weighted than bacillus, pseudomonas and the like which antagonize phytopathogens. Meanwhile, the strain with growth promoting and disease resisting capabilities can enhance the function of the microbial fertilizer, improve the effect and stability of the microbial fertilizer and enlarge the application range.

Fusarium oxysporum (Fusarium oxysporum) is a worldwide-distributed soil-borne pathogenic fungus, has a wide host range, and can cause blight of more than 100 plants such as melons, solanaceae, bananas, cotton, leguminous and flowers. Fusarium graminearum (Fusarium graminearum) belongs to fungi of Deuteromycotina, and can cause diseases of gramineous crops in fields (such as wheat scab caused by the nature); fusarium graminearum can cause heat and mildew of grain crops such as wheat, corn and the like in the disease of stored grains. Rhizoctonia solani (Rhizoctonia) belongs to fungi of the subdivision Deuteromycotina, and is a soil inhabitant which does not produce any asexual spores, widely exists in nature and is distributed worldwide. As an important bacterial species causing root rot and stem rot symptoms, rhizoctonia solani is considered to be one of the most destructive soil-borne plant pathogens. At present, the rhizoctonia solani has quite common distribution and extremely wide host range, can infect over 200 plants of 50 families, such as wheat, rice, sesame, peanut, cotton, corn and the like, and is extremely easy to cause serious harm.

Disclosure of Invention

The invention aims to provide a multifunctional plant rhizosphere growth-promoting bacterium.

The invention firstly protects Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) FH1, the strain is preserved in China general microbiological culture Collection center (CGMCC for short, with the address of No. 3 Siro 1 of Beijing city Kogyo-Yang district) in 29 months in 2018, and the preservation number is CGMCC No. 17050. Bacillus amyloliquefaciens FH1 CGMCC No.17050 is called Bacillus amyloliquefaciens FH1 for short.

The invention also protects a microbial inoculum which can contain bacillus amyloliquefaciens FH 1.

The microbial inoculum can be used as at least one of the following a1) -a 26): a1) potassium is dissolved; a2) preparing a product with potassium-dissolving capacity; a3) dissolving phosphorus; a4) preparing a product with phosphate solubilizing capability; a5) fixing nitrogen; a6) preparing a product with nitrogen fixation capacity; a7) generating a siderophore; a8) preparing a siderophore production product; a9) regulating and controlling the absorption of nutrient elements; a10) preparing a product for regulating and controlling the absorption of nutrient elements; a11) regulating and controlling the yield of the plant; a12) preparing a product for regulating and controlling the yield of the plant; a13) regulating and controlling the quality of the plant; a14) preparing a product for regulating and controlling the quality of the plant; a15) regulating and controlling the growth of plants; a16) preparing a product for regulating and controlling the growth of plants; a17) producing ACC deaminase; a18) preparing a product that produces ACC deaminase; a19) regulating and controlling the stress resistance of the plants; a20) preparing a product for regulating and controlling the stress resistance of the plants; a21) regulating and controlling the disease resistance of plants; a22) preparing a product for regulating and controlling the disease resistance of the plants; a23) inhibiting plant pathogenic fungi; a24) preparing a product for inhibiting plant pathogenic fungi; a25) preventing and controlling plant fungal diseases; a26) preparing the product for preventing and treating plant fungal diseases.

The invention also provides a preparation method of the microbial inoculum, which comprises the following steps: inoculating the bacillus amyloliquefaciens FH1 to a bacterial culture medium and culturing to obtain a bacterial liquid, namely the microbial inoculum.

The bacterial culture medium can be LB liquid culture medium.

In the preparation method of the microbial inoculum, the specific culture conditions can be as follows: culturing at 30 + -2 deg.C and 150-.

The microbial inoculum may include a carrier in addition to the active ingredient. The carrier may be a solid carrier or a liquid carrier. The solid carrier may be a mineral material, a plant material or a polymeric compound. The mineral material may be at least one of clay, talc, kaolin, montmorillonite, white carbon, zeolite, silica, and diatomaceous earth. The plant material may be at least one of bran, soybean meal, corn flour, bean flour and starch. The high molecular compound may be polyvinyl alcohol and/or polyglycol. The liquid carrier can be an organic solvent, vegetable oil, mineral oil, or water. The organic solvent may be decane and/or dodecane. In the microbial inoculum, the active ingredient may be present in the form of cultured living cells, a fermentation broth of living cells, a filtrate of a cell culture, or a mixture of cells and a filtrate. The composition can be prepared into various dosage forms, such as liquid, emulsion, suspending agent, powder, granules, wettable powder or water dispersible granules.

According to the requirement, the microbial inoculum can also be added with a surfactant (such as Tween 20, Tween 80 and the like), a binder, a stabilizer (such as an antioxidant), a pH regulator and the like.

The invention also protects the application of the bacillus amyloliquefaciens or the microbial inoculum containing the bacillus amyloliquefaciens, which can be at least one of the following a1) -a 28):

a1) potassium is dissolved;

a2) preparing a product with potassium-dissolving capacity;

a3) dissolving phosphorus;

a4) preparing a product with phosphate solubilizing capability;

a5) fixing nitrogen;

a6) preparing a product with nitrogen fixation capacity;

a7) generating a siderophore;

a8) preparing a siderophore production product;

a9) regulating and controlling the absorption of nutrient elements;

a10) preparing a product for regulating and controlling the absorption of nutrient elements;

a11) regulating and controlling the yield of the plant;

a12) preparing a product for regulating and controlling the yield of the plant;

a13) regulating and controlling the quality of the plant;

a14) preparing a product for regulating and controlling the quality of the plant;

a15) regulating and controlling the growth of plants;

a16) preparing a product for regulating and controlling the growth of plants;

a17) producing ACC deaminase;

a18) preparing a product that produces ACC deaminase;

a19) regulating and controlling the stress resistance of the plants;

a20) preparing a product for regulating and controlling the stress resistance of the plants;

a21) regulating and controlling the disease resistance of plants;

a22) preparing a product for regulating and controlling the disease resistance of the plants;

a23) inhibiting plant pathogenic fungi;

a24) preparing a product for inhibiting plant pathogenic fungi;

a25) preventing and controlling plant fungal diseases;

a26) preparing a product for preventing and treating plant fungal diseases;

a27) improving the soil;

a28) preparing a product for improving soil.

In the application, in a2), a4), a6), a8), a10), a12), a14), a16), a18), a20) or a22), the product can be a fertilizer or a seed dressing agent. In the a24) or the a26), the product can be a pesticide, a bacteriostatic agent or a seed dressing agent. In the a28), the product can be a soil conditioner.

In any of the above applications, the bacillus amyloliquefaciens may be bacillus amyloliquefaciens FH 1. The microbial inoculum containing the bacillus amyloliquefaciens FH1 can be any one of the microbial inoculants.

The invention also protects a product containing the bacillus amyloliquefaciens or a microbial inoculum containing the bacillus amyloliquefaciens; the function of the product may be a1) or a3) or a5) or a7) or a9) or a11) or a13) or a15) or a17) or a19) or a21) or a23) or a25) or a 27):

a1) potassium is dissolved;

a3) dissolving phosphorus;

a5) fixing nitrogen;

a7) generating a siderophore;

a9) regulating and controlling the absorption of nutrient elements;

a11) regulating and controlling the yield of the plant;

a13) regulating and controlling the quality of the plant;

a15) regulating and controlling the growth of plants;

a17) producing ACC deaminase;

a19) regulating and controlling the stress resistance of the plants;

a21) regulating and controlling the disease resistance of plants;

a23) inhibiting plant pathogenic fungi;

a25) preventing and controlling plant fungal diseases;

a27) improving the soil.

The product of a1), a3), a5), a7), a9), a11), a13), a15), a17), a19) or a21) can be a fertilizer or a seed dressing. The product of a23) or a25) can be a pesticide, a bacteriostatic agent or a seed dressing agent. The product of a27) can be a soil conditioner.

In any of the above products, the bacillus amyloliquefaciens may be bacillus amyloliquefaciens FH 1. The microbial inoculum containing the bacillus amyloliquefaciens FH1 can be any one of the microbial inoculants.

The invention also provides any one of the following methods.

K1) A method for improving the yield and/or quality of plants is to treat the plants with Bacillus amyloliquefaciens so as to improve the yield and/or quality of the plants.

K2) A method for improving the stress resistance of plants adopts bacillus amyloliquefaciens to treat the plants so as to improve the stress resistance of the plants.

K3) A method for improving disease resistance of plants comprises treating plants with Bacillus amyloliquefaciens to improve disease resistance of plants.

K4) A method for promoting plant growth comprises treating plant with Bacillus amyloliquefaciens to promote plant growth.

K5) A method for promoting absorption of plant nutrient elements comprises treating plants with Bacillus amyloliquefaciens to promote absorption of plant nutrient elements.

K6) A method for improving soil comprises treating soil with Bacillus amyloliquefaciens.

Any of the above "treating a plant with Bacillus amyloliquefaciens" can be achieved by applying Bacillus amyloliquefaciens to the plant roots.

Any of the above "treating a plant with Bacillus amyloliquefaciens" can be achieved by adding Bacillus amyloliquefaciens to the plant seeds, i.e., Bacillus amyloliquefaciens is one of the components of the seed dressing agent.

Any of the above "treating a plant with Bacillus amyloliquefaciens" can also be achieved by spraying Bacillus amyloliquefaciens to aerial parts (e.g., leaves) of the plant.

Any one of the above-mentioned "treating a plant with Bacillus amyloliquefaciens" may specifically be treating a plant with any one of the above-mentioned microbial agents.

Any one of the above-mentioned "treating a plant with any one of the above-mentioned microbial agents" can be achieved by applying any one of the above-mentioned microbial agents to a plant root system.

The step of treating the plant with any of the microbial inoculum can be realized by adding any of the microbial inoculum into a plant seed, namely, any of the microbial inoculum is used as one of the components of the seed dressing agent.

The step of treating the plant with any of the above-mentioned microbial agents can be realized by spraying any of the above-mentioned microbial agents on the overground part (such as leaves) of the plant.

Any of the above "treating a plant with the bacillus amyloliquefaciens FH1 or any of the above inoculants" can be applying the fertilizer to the root system of a plant, adding the fertilizer to the seeds of a plant (i.e., the fertilizer is one of the components of the seed dressing agent), or spraying the fertilizer to the aerial parts (e.g., leaves) of a plant.

The "treating soil with Bacillus amyloliquefaciens" can be achieved by applying Bacillus amyloliquefaciens to the soil.

The step of treating the soil by using the bacillus amyloliquefaciens can be realized by applying any of the microbial inoculum to the soil.

In any of the above methods, the bacillus amyloliquefaciens can be bacillus amyloliquefaciens FH 1.

Any of the above-described methods of increasing plant yield may be characterized by at least one increase in ear length, ear per plant, grain per ear, and thousand kernel weight.

Any of the above described improvements in plant quality may be manifested by an increase in amylose content.

Any of the above stress resistance may be salt resistance.

Any of the above promoting plant growth is manifested by at least one of an increase in plant height, an increase in dry weight of the plant, an increase in fresh weight of the stem, an increase in dry weight of the stem, an increase in length of the stem, an increase in fresh weight of the root, an increase in dry weight of the root, an increase in length of the root, and an increase in root length.

Any of the above regulating nutrient absorption may be promoting nutrient absorption.

Any of the above methods of modulating plant yield may comprise increasing plant yield.

Any of the above described methods of modulating plant quality may be plant quality enhancement.

Any of the above methods of regulating plant growth may be promoting plant growth.

Any one of the above methods for regulating plant stress resistance may be for improving plant stress resistance.

Any of the above methods for modulating disease resistance in a plant can be used to increase disease resistance in a plant.

Any one of the above plant pathogenic fungi can be at least one of fusarium oxysporum, fusarium graminearum and rhizoctonia solani.

Any one of the above plant fungal diseases may be at least one of blight, head blight, root rot and stem rot.

The plant fungal disease can be a disease (such as fusarium wilt) caused by fusarium oxysporum.

The plant fungal disease can be specifically a disease (such as gibberellic disease) caused by fusarium graminearum.

The plant fungal diseases can be diseases (such as root rot and stem rot) caused by rhizoctonia solani.

Any one of the above nutrient elements may be at least one of potassium, iron, nitrogen and phosphorus.

Any of the plants described above may be any of the following c1) to c 13): c1) a dicotyledonous plant; c2) a monocot plant; c3) a gramineous plant; c4) a plant of the Cucurbitaceae family; c5) cucumber; c6) number 8 cucumber cultivar zhongnong; c7) rice; c8) rice variety Yongyou 1540; c9) corn; c10) sesame seeds; c11) peanut; c12) cotton; c13) wheat (Triticum aestivum L.).

Any of the above fertilizers can be a microbial fertilizer. The microbial fertilizer can be a compound microbial fertilizer and/or a biological organic fertilizer. The compound microbial fertilizer can be a fertilizer compounded by microbial inoculum, nutrient substances and organic matters. The compound microbial fertilizer has the functions of both microbes and chemical fertilizers. The biological organic fertilizer can be a fertilizer formed by compounding a microbial inoculum and a decomposed organic fertilizer. The dosage form of the compound microbial fertilizer and/or the biological organic fertilizer can be granules.

Experiments prove that the bacillus amyloliquefaciens FH1 can dissolve insoluble phosphorus in soil into effective phosphorus and insoluble potassium in soil into effective potassium, and can promote plants to absorb phosphorus and potassium elements; the fertilizer has nitrogen fixation capacity, can fix nitrogen in the air into effective nitrogen, and is beneficial to the absorption of nitrogen elements by plants; ACC deaminase can be generated, and the stress resistance of crops is enhanced; the yield and the quality of crops can be improved; can also produce pig carriers, and has antagonistic effect on pathogenic fungi (such as Fusarium graminearum, Rhizoctonia solani, and Fusarium oxysporum), thereby antagonizing pathogenic fungi and preventing and treating plant fungal diseases. Therefore, the bacillus amyloliquefaciens FH1 can be used as plant rhizosphere growth-promoting bacteria and can promote plant growth, prevent and control plant fungal diseases and enhance plant stress resistance. The invention has important application value.

Drawings

FIG. 1 shows the single colony morphology of bacterium FH1 cultured on LB solid medium (A), LB liquid medium (B), Allium cepa solid medium (D), potassium-solubilizing solid medium (E) and ADF solid medium (F), and the amylolytic ability identification (C) of bacterium FH 1.

FIG. 2 shows the determination of the phosphate solubilizing ability of Bacillus amyloliquefaciens FH1 (A) and the determination of the siderophore producing ability (B).

FIG. 3 shows the antagonistic effect of Bacillus amyloliquefaciens FH1 on Fusarium graminearum (A), Rhizoctonia solani (B) and Fusarium oxysporum (C).

FIG. 4 shows that Bacillus amyloliquefaciens FH1 can improve rice yield and quality.

FIG. 5 shows that Bacillus amyloliquefaciens FH1 can improve stress tolerance of rice.

Deposit description

The strain name is as follows: bacillus amyloliquefaciens

Latin name: bacillus amyloliquefaciens

The strain number is as follows: FH1

The preservation organization: china general microbiological culture Collection center

The preservation organization is abbreviated as: CGMCC (China general microbiological culture Collection center)

Address: xilu No.1 Hospital No. 3 of Beijing market facing Yang district

The preservation date is as follows: 29/12/2018

Registration number of the preservation center: CGMCC No.17050

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention.

The experimental procedures in the following examples are conventional unless otherwise specified.

The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified.

In the experiments in the following examples, three or four replicates were set up and the results averaged.

LB liquid medium: adding yeast extract 5g, tryptone 10g and sodium chloride 10g into appropriate amount of distilled water, adjusting pH to 7.0-7.5, adding distilled water to constant volume of 1.0L, and sterilizing at 121 deg.C for 15 min.

LB solid medium: adding yeast extract 5g, tryptone 10g, sodium chloride 10g and agar 20g into appropriate amount of distilled water, adjusting pH to 7.0-7.5, diluting to 1.0L with distilled water, and sterilizing at 121 deg.C for 15 min.

Example 1 isolation, identification and preservation of Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) FH1 CGMCC No.17050

First, separate

1. Adding 10g of soil sample (rhizosphere soil collected from tea trees in south town of xi, mountain, Huangshan, Anhui, province) into 90mL of sterile water, oscillating at high speed for 10min, and standing for 30s to obtain supernatant; heating the supernatant in 100 deg.C boiling water for 10min, and naturally cooling to obtain soil suspension (the dilution is 10)^-1)。

2. Sucking 1mL of soil suspension, adding into a test tube containing 9mL of sterile water, and mixing well (the dilution is 10 at this time)^-2) Then, 1mL of the solution was aspirated from the test tube, and the solution was added to another test tube containing 9mL of sterile water and mixed well to prepare a diluted solution (dilution 10)^-3)。

3. The diluted solution was spread on LB solid medium uniformly and cultured at 30 ℃ for 3 days. Bacterial colonies that could grow on LB solid medium were strain purified.

The screened bacterium is named as bacterium FH 1.

II, identification

1. Morphological identification

The bacteria FH1 were inoculated to LB solid medium, cultured at 30 ℃ and observed for the morphology of single colonies after 3 days.

The results are shown in FIG. 1, panel A. The results show that bacterial colony of bacteria FH1 is milky white, protruding in the center, opaque, irregular in shape, and wrinkled on the surface.

The bacteria FH1 were inoculated into LB liquid medium, cultured at 30 ℃ and observed for 2 days by means of a scanning electron microscope (Hitachi SU 8010).

The results are shown in FIG. 1B. As a result, the bacterial cell of FH1 was a bacillus having a length of about 2.25 μm and a width of about 688nm, and it was blunt-rounded at both ends and crosshatched.

2. 16S rRNA sequence homology analysis

(1) Genomic DNA of bacterium FH1 was extracted and used as template with primer 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' and 1492R: 5'-TACGGCTACCTTGTTACGACTT-3' to obtain PCR amplification product.

The reaction system was 25. mu.L composed of 12.5. mu.L of 2 XMaster Mix (product of TaKaRa Co.), 1. mu.L of an aqueous solution of the primer 27F (concentration: 2.5 pmol/. mu.L), 1. mu.L of an aqueous solution of 1492R (concentration: 2.5 pmol/. mu.L), 1. mu.L of the template, and 9.5. mu.L of ddH₂And (C) O.

The reaction conditions are as follows: 3min at 94 ℃; 30 cycles of 94 ℃ for 30s, 55 ℃ for 30s, and 72 ℃ for 90 s; 10min at 72 ℃.

(2) Sequencing the PCR amplification product obtained in the step (1).

And (3) sequencing results show that the PCR amplification product obtained in the step (1) contains DNA molecules shown in a sequence 1 in a sequence table.

And (3) comparing the nucleotide sequences shown in the sequence 1 in the sequence table on NCBI. The alignment result shows that the bacterial FH1 has the highest homology with Bacillus amyloliquefaciens (Bacillus amyloliquefaciens). Therefore, the bacterium FH1 was identified as Bacillus amyloliquefaciens (Bacillus amyloliquefaciens).

3. Identification of starch-dissolving ability

Bacterium FH1 was inoculated to LB solid medium containing 0.2% soluble starch, cultured at 30 ℃ for 3 days, stained with Lugol iodine solution (obtained by dissolving 5g of iodine and 10g of potassium iodide in 85mL of distilled water) added dropwise to the medium, and then the plate was washed with 70% (v/v) aqueous ethanol solution, followed by the following judgment: if transparent circles are generated around the bacterial colonies, the bacillus amyloliquefaciens FH1 has the amylolytic capacity; otherwise bacillus amyloliquefaciens FH1 did not have amylolytic capacity.

The results are shown in FIG. 1C. The result shows that transparent halo is generated around the colony of the bacillus amyloliquefaciens FH1, which indicates that the bacillus amyloliquefaciens has the amylolytic capacity.

III, preservation

The separated bacteria FH1 in the first step are preserved in China general microbiological culture Collection center (CGMCC, address: No. 3 Xilu 1 Beijing Kogyo-Kchen) in 2018, 12 and 29, and the preservation number is CGMCC No. 17050. The bacteria FH1 are all called Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) FH1 CGMCC No.17050, abbreviated as Bacillus amyloliquefaciens FH 1.

Example 2 determination of Nitrogen-fixing ability of Bacillus amyloliquefaciens FH1

An abb solid culture medium: adding mannitol 10.0g and KH into appropriate amount of distilled water₂PO₄0.2g、MgSO₄·7H₂O 0.2g、NaCl 0.2g、CaCO₃5.0g、CaSO₄·2H₂0.1g of O and 20g of agar powder, adjusting the pH value to 7.0-7.5, then using distilled water to fix the volume to 1.0L, and sterilizing for 15min at 121 ℃.

1. Inoculating the single clone of the bacillus amyloliquefaciens FH1 to 10mL of LB liquid culture medium, and carrying out shaking culture at 30 ℃ and 180rpm for 48h to obtain a culture solution.

2. After the step 1 is finished, taking the culture bacterial liquid, centrifuging for 5min at 4 ℃ and 10000rpm, and collecting thalli.

3. After completion of step 2, the cells were taken and diluted with sterile water to a concentration of 1X 10⁸The strain suspension of bacillus amyloliquefaciens FH1 of individual/mL.

4. After completing step 3, 5 μ L of the bacterial suspension of bacillus amyloliquefaciens FH1 was spotted on the solid culture medium of the alunite avenae, cultured at 30 ℃ for 3d, and then judged as follows: if the bacillus amyloliquefaciens FH1 can grow on the Artocarpus solid culture medium, the bacillus amyloliquefaciens FH1 has the nitrogen fixation capacity; if bacillus amyloliquefaciens FH1 could not grow on the aphrodisiac solid medium, bacillus amyloliquefaciens FH1 did not have nitrogen fixation ability.

The results are shown in FIG. 1, panel D. The results show that bacillus amyloliquefaciens FH1 can grow on the solid culture medium of the alunite, which indicates that the bacillus amyloliquefaciens has the nitrogen fixation capacity. Therefore, the bacillus amyloliquefaciens FH1 has the capacity of fixing nitrogen in the air into effective nitrogen, and is helpful for the absorption of nitrogen elements by plants, so that the growth of the plants is promoted.

Example 3 determination of Potassium-solubilizing ability of Bacillus amyloliquefaciens FH1

Potassium-dissolving solid culture medium: adding 2.0g of disodium hydrogen phosphate, 0.5g of magnesium sulfate heptahydrate, 0.005g of ferric chloride, 0.1g of calcium carbonate, 5.0g of sucrose, 1.0g of potassium feldspar powder and 20g of agar powder into a proper amount of distilled water, adjusting the pH value to 7.0-7.5, then using distilled water to fix the volume to 1.0L, and sterilizing at 121 ℃ for 15 min.

mu.L of the bacterial suspension of Bacillus amyloliquefaciens FH1 prepared in step 3 of example 2 was spotted on potassium-solubilizing solid medium and cultured at 30 ℃ for 3 days, and then judged as follows: if the bacillus amyloliquefaciens FH1 can grow on a potassium-solubilizing solid medium, the bacillus amyloliquefaciens FH1 has the potassium-solubilizing capability; if Bacillus amyloliquefaciens FH1 could not grow on the potassium-solubilizing solid medium, then Bacillus amyloliquefaciens FH1 did not have potassium-solubilizing ability.

The results are shown in FIG. 1, panel E. The result shows that the bacillus amyloliquefaciens FH1 can grow on the potassium-dissolving solid culture medium, which indicates that the bacillus amyloliquefaciens FH1 has the potassium-dissolving capacity. Therefore, the bacillus amyloliquefaciens FH1 has the capacity of converting insoluble potassium in soil into effective potassium, and is helpful for plants to absorb the potassium in the soil, so that the plant growth is promoted.

Example 4 determination of the Capacity of Bacillus amyloliquefaciens FH1 to produce ACC deaminase

Component one solution: adding H into appropriate amount of distilled water₃BO₃10mg、MnSO₄·H₂O 11.19mg、ZnSO₄·7H₂O 124.6mg、CuSO₄·5H₂O78.22 mg and MoO₃10mg, then made up to 100mL with distilled water, filtered and sterilized.

Solution of component two: FeSO (ferric oxide) is added₄·7H₂O100 mg was dissolved in 10mL of distilled water, and filtered and sterilized.

DF liquid medium: adding KH into appropriate amount of distilled water₂PO₄4.0g、Na₂HPO₄6.0g、MgSO₄·7H₂0.2g of O, 2.0g of glucose, 2.0g of sodium gluconate, 2.0g of citric acid, 0.1mL of the first component solution and 0.1mL of the second component solution, adjusting the pH value to 7.2, then using distilled water to fix the volume to 1.0L, and sterilizing for 15min at 121 ℃.

ADF liquid medium: after the DF broth was cooled to about 55 ℃, ACC (1-aminocyclopropane-1-carboxyl) aqueous solution (which had been filter-sterilized) was added to obtain the ADF broth. ACC concentration in ADF liquid medium was 3.0 mmol/L.

ADF solid medium: adding 20g/L agar into DF liquid culture medium, sterilizing at 121 deg.C for 15min, cooling to about 55 deg.C, adding ACC (1-aminocyclopropane-1-carboxyl) water solution (filter-sterilized) to obtain ADF solid culture medium. ACC concentration in ADF solid medium was 3.0 mmol/L.

Coomassie brilliant blue G250 solution: 100mg of Coomassie brilliant blue G250 is accurately weighed, dissolved by 50mL of 95% (v/v) ethanol water solution, then 100mL of 85% (v/v) phosphoric acid water solution is added, and the volume is adjusted to 1000mL by using distilled water. Filtered off with suction and stored in a brown bottle for later use.

The solute and the concentration of the 2, 4-dinitrophenylhydrazine solution are 0.2 percent (M/v) of 2, 4-dinitrophenylhydrazine, and the solvent is HCl solution with the concentration of 2M. 2, 4-dinitrophenylhydrazine is a product of Sigma-Aldrich company.

ACC deaminase is one of indexes for determining the stress resistance of rhizosphere growth-promoting bacteria.

First, qualitative determination of ACC deaminase producing ability of Bacillus amyloliquefaciens FH1

The bacterial suspension of bacillus amyloliquefaciens FH1 prepared in step 3 of example 2 was inoculated into ADF solid medium, cultured at 30 ℃ for 3 days, and then judged as follows: if bacillus amyloliquefaciens FH1 can grow on the ADF solid medium, bacillus amyloliquefaciens FH1 has the ability to produce ACC deaminase; if Bacillus amyloliquefaciens FH1 could not grow on ADF solid medium, then Bacillus amyloliquefaciens FH1 did not have the ability to produce ACC deaminase.

The results are shown in FIG. 1, panel F. The results show that bacillus amyloliquefaciens FH1 can grow on ADF solid medium, indicating its ability to produce ACC deaminase.

Second, quantitative determination of ACC deaminase producing ability of Bacillus amyloliquefaciens FH1

1. A single clone of bacillus amyloliquefaciens FH1 was inoculated into 10mL of LB liquid medium and subjected to shaking culture at 30 ℃ and 180rpm for 48 hours to obtain a culture solution 1.

2. Centrifuging the culture solution 1 at 4 deg.C and 8000rpm for 10min, and collecting thallus 1.

3. The thallus 1 is taken, firstly centrifuged and washed for 2 times by 15mL of DF liquid culture medium, and then resuspended by 24mL of DF liquid culture medium to obtain a resuspension 1.

4. Taking the resuspension 1, carrying out shaking culture at 30 ℃ and 180rpm for 24h to obtain a culture bacterial liquid 2.

5. Taking the culture solution 2, firstly using Tris-HCl buffer solution with pH7.6 and 0.1mol/L for centrifugal washing for 2 times, and then using 1mL Tris-HCl buffer solution with pH7.6 and 0.1mol/L for heavy suspension to obtain a heavy suspension 2.

6. The suspension was centrifuged at 12000rpm for 5min at 2, 4 ℃ to collect the cells 2.

7. The cell 2 was resuspended in 600. mu.L of Tris-HCl buffer (0.1 mol/L) at pH8.5 to obtain a resuspension 3.

8. Taking the heavy suspension 3, adding 30 mu L of toluene, and rapidly shaking for 30s to break the cells to obtain a crude enzyme solution.

9. Taking the crude enzyme solution, and determining the specific activity of ACC (ACC deaminase) (repeating the experiment for three times and taking an average value), the method comprises the following specific steps:

(1) determination of Total protein content

(1-1) establishing a bovine serum albumin standard curve

Taking 0.0mL, 0.1mL, 0.2mL, 0.4mL, 0.6mL, 0.8mL or 1.0mL of bovine serum albumin solution (the concentration is 100 mu G/mL), adding water to a constant volume of 1mL, uniformly mixing, adding 5mL of Coomassie brilliant blue G250 solution, and shaking up; after standing for 3min, the absorbance value was measured at 595 nm. And drawing a bovine serum albumin standard curve by taking the bovine serum albumin content as an abscissa and the corresponding absorbance value as an ordinate.

(1-2) determination of Total protein content

Taking 100 mu L of crude enzyme solution, adding water to a constant volume of 1mL, uniformly mixing, adding 5mL of Coomassie brilliant blue G250 solution, and shaking uniformly; after standing for 3min, the absorbance value was measured at 595 nm.

And obtaining the total protein content in the crude enzyme solution according to a bovine serum albumin standard curve.

(2) Determination of ACC deaminase Activity

(2-1) establishing a tetronic acid standard curve

Diluting ketobutyric acid to 0.1-1 μmol in gradient; respectively adding 300 mu L of 2, 4-dinitrophenylhydrazine solution into the solution of tetronic acid after gradient dilution, and preserving heat for 30min at 30 ℃; then, 2mL of an aqueous NaOH solution (concentration: 2mol/L) was added thereto, mixed well, and the absorbance value was measured at 540 nm. And drawing a tetronic acid standard curve by taking the amount (mu mol) of the tetronic acid substance as an abscissa and the corresponding absorbance value as an ordinate.

(2-2) measurement of ACC deaminase Activity

(2-2-1) taking 200 mu L of crude enzyme solution, adding 20 mu L of ACC aqueous solution (the concentration is 0.5mol/L), mixing uniformly, and reacting for 15min at 30 ℃.

(2-2-2) after completion of the step (2-2-1), 1mL of an aqueous HCl solution (concentration: 0.56mol/L) was added to terminate the reaction, and then the reaction mixture was centrifuged at 12000rpm for 5min at 4 ℃ to collect the supernatant.

(2-2-3) after the step (2-2-2) is finished, taking the supernatant, adding 800 mu L of HCl aqueous solution (the concentration is 0.56mol/L) and 300 mu L of 2, 4-dinitrophenylhydrazine solution, and preserving the temperature for 30min at 30 ℃; finally, 2mL of NaOH aqueous solution (the concentration is 2mol/L) is added and mixed evenly, and the absorbance value is detected at 540 nm.

And obtaining the amount of the ketobutyric acid substance generated in the crude enzyme solution according to a ketobutyric acid standard curve.

(3) ACC deaminase specific activity was calculated according to the following formula:

the result shows that the ACC deaminase specific activity of the bacillus amyloliquefaciens FH1 is 0.42U/mg.

Example 5 determination of phosphate solubilizing ability of Bacillus amyloliquefaciens FH1

Inorganic phosphorus solid medium: adding 0.30g of sodium chloride, 0.30g of sulfuric acid, 0.30g of potassium chloride, 0.50g of ammonium sulfate, 0.03g of ferrous sulfate, 0.03g of manganese sulfate, 5.00g of calcium phosphate, 10.00g of grapes and 20.00g of agar into a proper amount of distilled water, adjusting the pH value to 7.0-7.5, then using distilled water to fix the volume to 1.0L, and sterilizing at 121 ℃ for 15 min.

mu.L of the bacterial suspension of Bacillus amyloliquefaciens FH1 prepared in step 3 of example 2 was spotted on an inorganic phosphorus solid medium, cultured at 30 ℃ for 3 days, observed for color change around colonies, and then judged as follows: if the colony is surrounded by transparent rings, the bacillus amyloliquefaciens FH1 has the phosphate solubilizing capability; otherwise bacillus amyloliquefaciens FH1 did not have phosphate solubilizing ability.

The results are shown in FIG. 2A. The results show that transparent halo is generated around the colony of the bacillus amyloliquefaciens FH1, which indicates that the bacillus amyloliquefaciens has the phosphate solubilizing capability.

5. The diameter of the resulting transparent circle and the colony was measured.

The results showed that the diameter ratio of the generated clearing zones to colonies was 2.00.

The results show that the bacillus amyloliquefaciens FH1 has the phosphate solubilizing capability.

Example 6 determination of the ability of Bacillus amyloliquefaciens FH1 to produce siderophores

CAS dyeing liquid: 50mL of a solution (0.079g of chrom azurol S, CAS) dissolved in 50mL of deionized water) and 10mLb solution (0.00162g of FeCl6H)₂Dissolving O in 10mL of HCl aqueous solution (concentration is 12mM) and mixing to obtain 60mL of solution c; 60mL of solution c was added slowly along the wall of the beaker to 40mL of solution d (0.069g HDTMA (cetyltrimethyl ammonium bromide) dissolved in 40mL deionized water) and mixed to give 100mL of CAS stain which was sterilized at 121 ℃ for 15 min.

0.1mol/L phosphate buffer (pH 6.8): na is contained in each 100mL of deionized water₂HPO₄·12H₂O 2.427g、NaH₂PO₄·2H₂O 0.5905g、KH₂PO₄0.075g、NH₄0.250g of Cl and 0.125g of NaCl, sterilized at 121 ℃ for 15min, and diluted 10 times when used.

CAS solid medium: adding 1mL of 20% sucrose solution, 3mL of 10% acid hydrolyzed casein and 1mmol/L CaCl into proper deionized water ₂100 μ L and 1mmol/L MgSO₄2mL, adding deionized water to 100mL, adding 2g of agar, and sterilizing at 121 ℃ for 15 min. When the culture medium is cooled to 60 ℃, 5mL of 0.1mol/L phosphate buffer solution (pH6.8) and 10mL of CAS dye solution are slowly added, and after uniform mixing, the mixture is poured into a plate to obtain the CAS solid culture medium.

1. The suspension of bacillus amyloliquefaciens FH1 prepared in step 3 of example 2 was inoculated on a CAS solid medium, cultured at 30 ℃ for 5 days, observed for color change around colonies, and then judged as follows: if an orange transparent halo is produced around the colony, bacillus amyloliquefaciens FH1 has the capacity to produce siderophores; otherwise bacillus amyloliquefaciens FH1 did not have the ability to produce iron carriers.

The results are shown in fig. 2B. The results showed that an orange-yellow transparent halo was produced around the colony of bacillus amyloliquefaciens FH1, indicating its ability to produce siderophores.

2. The diameter of the resulting orange-yellow transparent halo was measured.

The results showed that the ratio of the diameters of the resulting orange-yellow transparent halo to the colonies was 2.3.

The above results indicate that bacillus amyloliquefaciens FH1 has the ability to produce siderophores.

Example 7 antagonistic action of Bacillus amyloliquefaciens FH1 on pathogenic bacteria

PDA solid medium: cutting peeled potato 200g into small pieces, adding 1.0L distilled water, and boiling for 30 min; filtering with gauze, collecting filtrate, adding glucose 20.0g and KH₂PO₄3.0g、MgSO₄.7H₂O1.5 g, vitamin B110 μ g and agar 20.0g, adjusting pH to 6.0 and adding distilled water to volume of 1.0L, and sterilizing at 115 deg.C for 15 min.

PDA solid plate: and (3) pouring 20mL of potato solid culture medium with the temperature of about 55 ℃ into a culture dish, and cooling to obtain the PDA solid flat plate.

Fusarium graminearum, rhizoctonia solani and fusarium oxysporum are all described in the following documents: jingjing Wang, Huiwen Zhang, Xiaooli Zhang, Shenghong Qin, Huangbo Tan, Xinyu Li. effects of Long-term chloride-ethyl application on the diversity and anti-reflection activity of solvent was used in a solvent field in the north phase of microorganisms of Microbiology 2013, Volume 63, Issue 1, 335 pp 341.

The antagonistic action of the bacillus amyloliquefaciens FH1 on fusarium graminearum, rhizoctonia solani and fusarium oxysporum is researched by adopting a plate opposing method. The method comprises the following specific steps:

1. the bacillus amyloliquefaciens FH1 bacterial suspension was prepared according to the method of step 3 in example 2.

2. Inserting agar blocks with the diameter of 5mm into the edges of colonies of pathogenic bacteria (fusarium graminearum, rhizoctonia solani or fusarium oxysporum) to be detected by using a sterile gun head with the specification of 1mL, and placing the agar blocks in the center of a PDA solid flat plate.

3. And (3) after the step 2 is completed, uniformly dividing the PDA solid plate into four equal parts, respectively carrying out point connection on 2 mu L of bacillus amyloliquefaciens FH1 bacterial suspension at the center of each equal part, carrying out a plate confrontation experiment, carrying out static culture at 30 ℃ for 5d, and observing the bacteriostatic effect.

The bacteriostatic effect is shown in figure 3(A is fusarium graminearum, B is rhizoctonia solani, and C is fusarium oxysporum). The results show that the bacillus amyloliquefaciens FH1 produced obvious inhibition zones on plates where fusarium graminearum, fusarium solani or fusarium oxysporum grew, and the inhibition rates (inhibition rate: antagonistic fungus colony radius/non-antagonistic fungus colony radius) of the bacillus amyloliquefaciens FH1 on the fusarium oxysporum, fusarium oxysporum and fusarium graminearum were 47%, 41% and 43%, respectively.

Therefore, the bacillus amyloliquefaciens FH1 have a certain inhibiting effect on fusarium oxysporum, fusarium oxysporum and fusarium graminearum, namely the bacillus amyloliquefaciens FH1 has a strong capability of antagonizing fusarium oxysporum, fusarium graminearum and fusarium solani, and can be used for preparing biological pesticides or biocontrol agents and the like for preventing and treating fungal diseases (such as diseases caused by fusarium oxysporum, diseases caused by fusarium oxysporum and diseases caused by fusarium graminearum).

Example 8 growth promoting Effect of Bacillus amyloliquefaciens FH1 on cucumber seedlings

The soil to be tested was taken from the airport economic area of Tianjin.

The cucumber to be tested is cucumber variety Zhongnong No. 8.

A pot experiment is adopted to carry out the growth promotion experiment of cucumber seedlings, the experiment is repeated three times to obtain an average value, and the specific steps of each experiment are as follows:

1. the soil to be tested was dried in the shade, sieved (aperture 1mm), and 200g of the soil was placed in a flowerpot (diameter 8 cm). The total number of 6 pots is filled.

2. After step 1, the cucumber plants to be tested (of substantially uniform size) grown up to 7 days were transplanted in pots, 2 cucumber plants to be tested being transplanted per pot.

3. After completion of step 2, 3 arbitrary pots were randomly selected and 20mL of the bacterial suspension of bacillus amyloliquefaciens FH1 (prepared in step 3 of example 2) was uniformly added, and the remaining 3 pots were uniformly added with 20mL of sterile physiological saline (as a control).

4. After the step 3 is finished, the flowerpot with the tested cucumber plant is placed in an artificial climate incubator and cultured (28 ℃, 14h illumination culture; 12 ℃, 16h dark culture) for 50 days, and 30mL of water is added every 48h during the culture period.

5. After step 4, measuring the plant height of the cucumber plant to be tested, the fresh weight of the cucumber plant to be tested, the dry weight of the cucumber plant to be tested, the fresh weight of the stem, the dry weight of the stem, the length of the stem, the fresh weight of the root, the dry weight of the root and the length of the root respectively.

The statistical results are shown in Table 1. The results show that the fresh weight of the cucumber plant tested, the dry weight of the cucumber plant tested, the plant height of the cucumber plant tested, the fresh weight of the stem, the dry weight of the stem, the length of the stem, the fresh weight of the root, the dry weight of the root and the length of the root were all significantly increased by applying the bacterial suspension of bacillus amyloliquefaciens FH1 compared to applying sterile physiological saline (i.e., control).

TABLE 1

Example 9 improvement Effect of Bacillus amyloliquefaciens FH1 on Rice yield and quality

The bacterial liquid (with the concentration of 10) of the bacillus amyloliquefaciens FH1 with the temperature of-80℃ is added¹⁰CFU/mL) is inoculated into 100mL LB liquid culture medium according to the proportion of 1% (v/v), and shaking culture is carried out at 30 ℃ and 180rpm for 48 h; then carrying out amplification culture in an LB liquid culture medium according to the proportion of 5% (v/v); finally, the culture was carried out in a fermenter containing 50L of the industrial LB liquid medium at a ratio of 5% (v/v) (the culture conditions were: the temperature is 30 ℃, the rotating speed is 150r/min, and the ventilation volume is 3m³And/h, the pot pressure is 0.05Mpa) for 24h, and obtaining FH-1 bacterial liquid. The industrial LB liquid culture medium is prepared from industrial yeast powder and peptone which are low in price and simple in formula.

2. Influence of Bacillus amyloliquefaciens FH1 on rice yield and quality

The experimental place is Huzhou city of Zhejiang province, and the experimental place is a rice planting area and is 2-3 seasons per year. The climate in the region is humid, belongs to subtropical monsoon, and has an average annual temperature of 16 ℃ and an average annual rainfall of 1200 mm.

The experiment set up two treatments:

blank Control (CK): 5 kg/mu of urea, 7-8 kg/mu of potassium chloride and 80 kg/mu of organic fertilizer are applied in the rice transplanting period; applying 80 kg/mu of organic fertilizer in the middle stage of rice; spreading 80 kg/mu in the heading stage of the rice;

FH-1 treatment (FH): 5 kg/mu of urea, 7-8 kg/mu of potassium chloride and 80 kg/mu of organic fertilizer are applied in a rice transplanting period, and 2L/mu of FH-1 bacterial liquid is applied in a flushing manner; applying 80 kg/mu of organic fertilizer in the middle period of rice, and spraying 2L/mu of FH-1 bacterial liquid; and (3) applying 80 kg/mu of organic fertilizer in the heading stage of the rice, and spraying 2L/mu of FH-1 bacterial liquid.

Each treatment is repeated for 5 times, the total number of the treatment cells is 10, each treatment cell is 130 square meters, and the experiment blocks are arranged by adopting a random design. Management measures such as fertilization and watering are carried out according to local farmer habits, the cultivated crops are rice, and the variety is Yongyou 1540.

In the rice maturation period, 5 Yongyou 1540 rice plants are collected in each cell according to a rice maturation sampling method, the rice is removed from the soil by using a shovel, the plant height, the root length and the ear length of the rice are measured by using a ruler, and the ear number, the single ear number, the thousand kernel weight and the yield of the rice are counted.

The chalkiness rate of rice was determined. The rice grains with opaque parts of the endosperm which are white (including the abdominal, cardiac and dorsal) are chalky grains. The chalky percentage of the number of grains in the sample is the chalky rate.

The method for measuring the protein content of the rice flour comprises the following specific steps:

(1) preparation of Standard Curve

The test tubes with 6 plugs were numbered, and protein standard solution (product of Bijing Ding Guosheng Biotech, LLC), distilled water and Coomassie brilliant blue G250 solution were added according to Table 2, shaken well, left for 3min, and the absorbance at 595nm was measured.

And drawing a standard curve by taking the protein content as an abscissa and the corresponding light absorption value at the wavelength of 595nm as an ordinate.

TABLE 2

(2) Determination of protein content in a sample

Accurately weighing 0.5g of rice flour, putting the rice flour into a mortar, adding 2mL of 0.1mol/L NaOH aqueous solution, grinding for 10min to obtain homogenate, transferring the homogenate into a10 mL centrifuge tube, washing the mortar by using 6mL of 0.1mol/L NaOH aqueous solution for three times, transferring the washing liquid into a 50mL centrifuge tube, stirring by using a glass rod, standing for 30min, and intermittently stirring for several times during the standing process. Centrifuging at 4000r/min for 15min, transferring the supernatant into a 50mL volumetric flask, and then repeating the above process with 8mL of 0.1mol/L NaOH aqueous solution to wash the sample once more. And (5) adding distilled water to a constant volume to reach a scale, and shaking up to obtain the alkali-soluble protein extracting solution. Sucking 0.1mL of alkali soluble protein extract, placing into 10mL test tube with plug scale, adding 5mL of Coomassie brilliant blue G250 solution, mixing, standing for 2min, performing colorimetric at 595nm with 10mm cuvette (0.1 mL of distilled water is used as colorimetric blank to replace extract and 5mL of Coomassie brilliant blue G250 solution is mixed), and recording OD_595nmThe value is obtained. Then according to the measured OD_595nmValues the corresponding protein concentration was found on the standard curve.

The method for determining the content of the amylose in the rice comprises the following specific steps:

(1) pretreatment of standard and sample

(1-1) degreasing

Respectively weighing 150mg of an amylose standard product, a amylopectin standard product and a sample which is crushed and then passes through a 80-mesh sieve, wrapping the samples with a filter paper cylinder, respectively placing the wrapped samples in a Soxhlet extractor, degreasing the samples with 85% (v/v) methanol water solution, and performing reflux extraction for 4 to 6 hours at the speed of 5 to 6 drops/s to sequentially obtain a degreased amylose standard product, a degreased amylopectin standard product and a degreased sample.

(1-2) moisture calibration

Spreading the degreased amylose standard substance, the degreased amylopectin standard substance and the degreased sample in a culture dish, standing for 3-4h, drying in a constant-temperature drying oven at the temperature of 130 ℃ for 3h after methanol volatilizes, then cooling in a dryer for 1h, and sequentially obtaining the amylose standard substance after moisture calibration, the amylopectin standard substance after moisture calibration and the sample after moisture calibration.

(1-3) preparation of solution

Accurately weighing amylose standard substance after water calibration or amylopectin standard substance (100 +/-0.5) mg after water calibration, placing the amylose standard substance or the amylopectin standard substance after water calibration into a 100mL conical flask, carefully adding 1.0mL 95% (v/v) ethanol water solution, and flushing the standard substance stuck on the wall of the conical flask; adding 9.0mL of 1.0mol/L sodium hydroxide aqueous solution, and shaking up gently to obtain a mixture; the mixture was heated in a boiling water bath for 10min to completely disperse the starch standard. Taking out, cooling to room temperature, transferring to a 100mL volumetric flask, adding distilled water to a constant volume, and violently shaking and mixing uniformly to obtain a standard solution. The concentration of the amylose standard solution was 1 mg/mL. The concentration of the amylopectin standard solution is 1 mg/mL.

Accurately weighing mg of a sample (100 +/-0.5) with calibrated moisture, placing the sample in a 100mL conical flask, carefully adding 1.0mL of 95% (v/v) ethanol aqueous solution, and washing down a standard substance stuck on the wall of the flask; adding 9.0mL of 1.0mol/L sodium hydroxide aqueous solution, and shaking up gently to obtain a mixture; and heating the mixture in a boiling water bath for 10min, then cooling to room temperature, transferring to a 100mL volumetric flask, adding distilled water to a constant volume, and violently shaking and mixing uniformly to obtain a sample solution.

Taking 5.0mL of 0.09mol/L sodium hydroxide aqueous solution, adding 9.0mL of 1.0mol/L sodium hydroxide aqueous solution, and shaking up gently to obtain a mixture; heating the mixture in a boiling water bath for 10min, then cooling to room temperature, transferring to a 100mL volumetric flask, adding distilled water to a constant volume, and violently shaking and mixing uniformly to obtain a blank solution.

(2) Drawing of calibration curve and sample determination

Preparing a series of standard solutions: accurately sucking 0.0mL, 2.00mL, 4.00 mL, 5.00mL, 6.00 mL and 7.00mL of the amylose standard solution into 6 volumetric flasks with 100mL, accurately sucking 18.0 mL, 16.0 mL, 14.0 mL, 13.0 mL, 12.0 mL and 11.0mL of the amylopectin standard solution into the 6 volumetric flasks respectively, adding 2.00mL of a sodium hydroxide aqueous solution with the concentration of 0.09mol/L respectively, and uniformly mixing to obtain a standard solution series with the amylose contents of rice of 0, 10%, 20%, 25%, 30% and 35% respectively.

And (3) drawing a calibration curve: accurately sucking 5.00mL of standard solution series into 6 100mL volumetric flasks previously added with about 50mL of distilled water, respectively adding 1.0mL of acetic acid aqueous solution with the concentration of 1.0mol/L, and shaking up; then respectively adding 2.0mL of iodine reagent, adding water to scale, shaking up, and standing for 10 min. The blank solution was zeroed and the absorbance of the series of standards was measured at 720 nm. And drawing a calibration curve by taking the absorbance as an ordinate and the amylose content as an abscissa.

And (3) sample determination: accurately sucking 5.00mL of sample solution into a 100mL volumetric flask which is pre-added with about 50mL of distilled water, then adding 1.0mL of 1.0mol/L acetic acid aqueous solution, and shaking up; then adding 2.0mL iodine reagent, adding water to scale, shaking up, and standing for 10 min. The absorbance of the sample solution was measured at 720nm with a blank solution for zeroing. And calculating the content of amylose in the tested sample according to the calibration curve.

The results are shown in Table 3 and FIG. 4. Results show that compared with a blank control, the FH-1 treated rice plants have the advantages of obviously increased root length, plant height, spike length, spike number per plant, spike number per spike, thousand kernel weight, yield and amylose content, and obviously reduced chalkiness rate.

TABLE 3

	Control	Bacillus amyloliquefaciens FH1 microbial inoculum	Lifting Rate (%)
				Root length (cm)	10.48±0.72b	16.38±2.22a	56.30
Plant height (cm)	113.16±3.96b	130.02±2.51a	14.90
				Ear length (cm)	17.16±0.26b	23.32±1.64a	35.90
Single plant ear number	12.20±1.79b	23.00±5.24a	88.52
				Single ear grain number	199.40±41.25b	309.00±23.23a	54.96
Thousand Kernel weight (g)	19.72±1.52b	22.22±1.01a	12.68
				Yield (Kg/mu)	550±20.00a	600±26.46a	9.09
Chalkiness ratio (%)	7.60±2.88a	5.60±1.51a	-26.32
				Protein content (g)	6.38±0.29a	4.80±0.23b	-24.76
Amylose content (%)	15.82±0.47a	16.82±1.74a	6.32

Note: the different lower case letters represent that the data differ significantly (P <0.05) for each property of rice in the different treatment groups.

Example 10 Bacillus amyloliquefaciens FH1 was able to improve stress tolerance in rice

The experiment was repeated three times to obtain an average, and the procedure for each repetition was as follows:

1. inoculating a monoclonal of bacillus amyloliquefaciens FH1 into 100mL of LB liquid culture medium, and carrying out shaking culture at 30 ℃ and 180rpm for 48h to obtain a culture solution.

2. The method comprises the steps of soaking plump seeds of the rice variety Yongyou 1540 with the same size in a 2% (v/v) sodium hypochlorite aqueous solution for 15min, and then cleaning the seeds with deionized water to obtain the cleaned seeds.

3. Placing 40 cleaned seeds in 100mL LB liquid culture medium, placing 40 cleaned seeds in 100mL culture medium, culturing at 30 deg.C and 180rpm for 8h under shaking, and treating as follows:

CK: taking 20 cleaned seeds from an LB liquid culture medium, placing the seeds in a glass culture dish paved with a layer of sterile filter paper, and pouring 20mL of distilled water;

CK 100: taking 20 cleaned seeds from an LB liquid culture medium, placing the seeds in a glass culture dish paved with a layer of sterile filter paper, and pouring 20mL of NaCl aqueous solution with the concentration of 100 mmol;

FH: taking 20 cleaned seeds from the culture solution, placing the seeds in a glass culture dish paved with a layer of sterile filter paper, and pouring 20mL of distilled water;

FH 100: 20 washed seeds were taken from the culture broth, placed in a glass petri dish with a layer of sterile filter paper, and 20mL of 100mmol NaCl aqueous solution was poured in.

4. After completion of step 3, the plate was dark-treated at 30 ℃ (during which time water was added to maintain humidity in the glass plate).

Germination rates were counted and averaged by group on day 3 and 8 of the dark treatment, respectively.

On day 8 of dark treatment, the weight of rice seedlings was measured and averaged by group.

The experimental results are shown in FIG. 5(A is the growth state of the seeds on the 3 rd day of dark treatment, B is the statistical result of germination percentage on the 3 rd day of dark treatment, C is the statistical result of germination percentage on the 8 th day of dark treatment, and D is the statistical result of rice seedling weight on the 8 th day of dark treatment). The results show that the germination rate and the weight of the applied culture bacteria liquid are obviously increased compared with the distilled water under the salt stress.

The results show that the bacillus amyloliquefaciens FH1 can dissolve insoluble phosphorus in soil into effective phosphorus and insoluble potassium in soil into effective potassium, and can promote the absorption of phosphorus and potassium elements by plants; the fertilizer has nitrogen fixation capacity, can fix nitrogen in the air into effective nitrogen, and is beneficial to the absorption of nitrogen elements by plants; ACC deaminase can be generated, and the stress resistance of crops is enhanced; the yield and the quality of crops can be improved; can also produce pig carriers, and has antagonistic effect on pathogenic fungi (such as Fusarium graminearum, Rhizoctonia solani, and Fusarium oxysporum), thereby antagonizing pathogenic fungi and preventing and treating plant fungal diseases. Therefore, the bacillus amyloliquefaciens FH1 can be used as plant rhizosphere growth-promoting bacteria and can promote plant growth, prevent and control plant fungal diseases and enhance plant stress resistance.

<110> institute of biotechnology for Tianjin industry of Chinese academy of sciences

<120> bacillus amyloliquefaciens and application thereof

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 1393

<212> DNA

<213> Artificial sequence

<400> 1

gtcgagcgga cagatgggag cttgctccct gatgttagcg gcggacgggt gagtaacacg 60

tgggtaacct gcctgtaaga ctgggataac tccgggaaac cggggctaat accggatggt 120

tgtttgaacc gcatggttca gacataaaag gtggcttcgg ctaccactta cagatggacc 180

cgcggcgcat tagctagttg gtgaggtaac ggctcaccaa ggcgacgatg cgtagccgac 240

ctgagagggt gatcggccac actgggactg agacacggcc cagactccta cgggaggcag 300

cagtagggaa tcttccgcaa tggacgaaag tctgacggag caacgccgcg tgagtgatga 360

aggttttcgg atcgtaaagc tctgttgtta gggaagaaca agtgccgttc aaatagggcg 420

gcaccttgac ggtacctaac cagaaagcca cggctaacta cgtgccagca gccgcggtaa 480

tacgtaggtg gcaagcgttg tccggaatta ttgggcgtaa agggctcgca ggcggtttct 540

taagtctgat gtgaaagccc ccggctcaac cggggagggt cattggaaac tggggaactt 600

gagtgcagaa gaggagagtg gaattccacg tgtagcggtg aaatgcgtag agatgtggag 660

gaacaccagt ggcgaaggcg actctctggt ctgtaactga cgctgaggag cgaaagcgtg 720

gggagcgaac aggattagat accctggtag tccacgccgt aaacgatgag tgctaagtgt 780

tagggggttt ccgcccctta gtgctgcagc taacgcatta agcactccgc ctggggagta 840

cggtcgcaag actgaaactc aaaggaattg acgggggccc gcacaagcgg tggagcatgt 900

ggtttaattc gaagcaacgc gaagaacctt accaggtctt gacatcctct gacaatccta 960

gagataggac gtccccttcg ggggcagagt gacaggtggt gcatggttgt cgtcagctcg 1020

tgtcgtgaga tgttgggtta agtcccgcaa cgagcgcaac ccttgatctt agttgccagc 1080

attcagttgg gcactctaag gtgactgccg gtgacaaacc ggaggaaggt ggggatgacg 1140

tcaaatcatc atgcccctta tgacctgggc tacacacgtg ctacaatgga cagaacaaag 1200

ggcagcgaaa ccgcgaggtt aagccaatcc cacaaatctg ttctcagttc ggatcgcagt 1260

ctgcaactcg actgcgtgaa gctggaatcg ctagtaatcg cggatcagca tgccgcggtg 1320

aatacgttcc cgggccttgt acacaccgcc cgtcacacca cgagagtttg taacacccga 1380

agtcggtgag gta 1393

Claims

1. Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) FH1, the preservation number of which in China general microbiological culture Collection center is CGMCC No. 17050.

2. A bacterial agent comprising Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) FH1 CGMCC No.17050 according to claim 1.

3. The method for preparing the microbial inoculum according to claim 2, which comprises the following steps: inoculating the Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) FH1 CGMCC No.17050 of the claim 1 to a bacterial culture medium and culturing to obtain a bacterial liquid, namely a microbial inoculum.

4. The application of the Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) FH1 CGMCC No.17050 or the microbial inoculum of claim 2 in at least one of the following a1) -a28) is as follows:

a1) potassium is dissolved;

a2) preparing a product with potassium-dissolving capacity;

a3) dissolving phosphorus;

a4) preparing a product with phosphate solubilizing capability;

a5) fixing nitrogen;

a6) preparing a product with nitrogen fixation capacity;

a7) generating a siderophore;

a8) preparing a siderophore production product;

a9) promoting the absorption of nutrient elements;

a10) preparing a product for promoting the absorption of nutrient elements;

a11) the rice yield is improved;

a12) preparing a product for improving the rice yield;

a13) the quality of the rice is improved;

a14) preparing a product for improving the quality of rice;

a15) promoting the growth of cucumbers;

a16) preparing a product for promoting the growth of cucumbers;

a17) producing ACC deaminase;

a18) preparing a product that produces ACC deaminase;

a19) the stress resistance of the rice is improved;

a20) preparing a product for improving the stress resistance of the rice;

a21) the disease resistance of the rice is improved;

a22) preparing a product for improving the disease resistance of the rice;

a23) inhibiting plant pathogenic fungi;

a24) preparing a product for inhibiting plant pathogenic fungi;

a25) preventing and controlling plant fungal diseases;

a26) preparing a product for preventing and treating plant fungal diseases;

a27) improving the soil;

a28) preparing a product for improving soil;

the plant pathogenic fungi is at least one of fusarium oxysporum, fusarium graminearum and rhizoctonia solani;

the plant fungal disease is at least one of fusarium wilt, gibberellic disease, root rot and stem rot.

5. A product containing the Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) FH1 CGMCC No.17050 of claim 1 or the bacterial agent of claim 2; the function of the product is a1) or a3) or a5) or a7) or a9) or a11) or a13) or a15) or a17) or a19) or a21) or a23) or a25) or a 27):

a1) potassium is dissolved;

a3) dissolving phosphorus;

a5) fixing nitrogen;

a7) generating a siderophore;

a9) promoting the absorption of nutrient elements;

a11) the rice yield is improved;

a13) the quality of the rice is improved;

a15) promoting the growth of cucumbers;

a17) producing ACC deaminase;

a19) the stress resistance of the rice is improved;

a21) the disease resistance of the rice is improved;

a23) inhibiting plant pathogenic fungi;

a25) preventing and controlling plant fungal diseases;

a27) improving the soil;

6. A method for improving the yield and/or quality of rice, which comprises the step of treating rice with Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) FH1 CGMCC No.17050 according to claim 1, so as to improve the yield and/or quality of rice.

7. A method for improving stress resistance of rice, which is to adopt Bacillus amyloliquefaciens FH1 CGMCC No.17050 to treat the rice so as to improve the stress resistance of the rice.

8. A method for improving disease resistance of rice, which is to adopt Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) FH1 CGMCC No.17050 to treat rice so as to improve the disease resistance of the rice.

9. A method for promoting cucumber growth, which is to adopt Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) FH1 CGMCC No.17050 to treat cucumber so as to promote the cucumber growth.

10. A method for improving soil, which is to treat soil by using Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) FH1 CGMCC No.17050 as described in claim 1.