CN110734872B

CN110734872B - Bacillus methylotrophicus and application thereof in agricultural production

Info

Publication number: CN110734872B
Application number: CN201910812784.0A
Authority: CN
Inventors: 周英俊; 苑伟伟; 凌红丽; 吕宾; 孙文丽; 梁莉
Original assignee: Shandong Vland Biotech Co ltd; SHANDONG KDN BIOTECH CO Ltd
Current assignee: Shandong Vland Biotech Co ltd; SHANDONG KDN BIOTECH CO Ltd
Priority date: 2019-08-30
Filing date: 2019-08-30
Publication date: 2021-08-20
Anticipated expiration: 2039-08-30
Also published as: CN110734872A

Abstract

The invention relates to the technical field of functional microorganism screening and application, and particularly provides a novel bacillus methylotrophicus (bacillus methylotrophicus)Bacillus methylotrophicus) And provides its application in agricultural production. The bacillus methylotrophicus is screened from soil in Minskus white areas, has a preservation number of CCTCC NO: M2019439, has a remarkable inhibiting effect on various pathogenic bacteria, can effectively prevent and treat common crop diseases and improve crop yield, and has a wide application prospect.

Description

Bacillus methylotrophicus and application thereof in agricultural production

Technical Field

The invention relates to the technical field of functional microorganism screening and application, in particular to bacillus methylotrophicus and application thereof in agricultural production.

Background

The microorganisms have a close relationship with agriculture, and in soil which is called 'microbial capital nutrition', the microorganisms play a main role in the qualitative circulation and have irreplaceable functions. They decompose the animal and plant residual wastes to convert them into humus, promoting the formation of a good structure of the soil. Many soil microorganisms can fix nitrogen in the air and convert various organic matters, and continuously provide various nutrient elements such as carbon, nitrogen, phosphorus, potassium and the like which can be effectively utilized for plants. Antagonistic microorganisms of pathogenic microorganisms of insects and plant germs are also widely present in nature and can be used for the control of plant diseases and insect pests to partially replace chemical pesticides. In addition, various products such as organic acid, amino acid, growth hormone, antibiotics, various enzyme preparations and the like can be produced through microbial propagation and fermentation, can be respectively used as feed additives, food additives, pesticides and the like, and are increasingly widely applied.

1. Microbial fertilizer

Microbial fertilizers, also known as bacterial fertilizers or biofertilizers, are a class of microbial preparations produced by artificially culturing microorganisms beneficial to plants. The microbial fertilizer has comprehensive effect, does not directly provide nutrient elements for crops generally, has indirect nutrition effect, and can improve soil fertility, improve soil structure, stimulate crop growth, improve crop quality, and enhance disease resistance (pest) and stress resistance of plants. After the microbial fertilizer is used, the application amount of the fertilizer can be effectively reduced, and the utilization rate of the fertilizer is improved.

There are five major groups according to the microbial species: bacterial fertilizers (such as rhizobium fertilizer, azotobacter fertilizer, phosphate solubilizing bacterial fertilizer, potassium solubilizing bacterial fertilizer and photosynthetic bacterial fertilizer); ② actinomycete fertilizer (such as antibiotic fertilizer); thirdly, fungus fertilizer (mycorrhizal fungus fertilizer, including ectomycorrhizal fungus agent and endophytic fungus agent); algae fertilizer (such as nitrogen-fixing blue algae bacterial fertilizer); compound microbial fertilizer, i.e. the fertilizer is formed by combining more than two microbes according to a certain proportion.

2. Microbial pesticide

The microbial pesticide is a non-chemically synthesized microbial preparation with the functions of killing insects and preventing diseases, such as a microbial insecticide, a bactericide, an agricultural antibiotic and the like, and the microorganisms comprise bacteria, viruses and fungi for killing insects and preventing diseases.

The most studied and used amount of the insecticidal microorganisms is bacillus thuringiensis, and one or more protein crystals called endotoxin with strong bactericidal action can be formed in the bacillus during spore generation, so that the bacillus thuringiensis can be widely used for moisture prevention of grains, economic crops, vegetables, forestry and certain sanitary pests. The bacillus thuringiensis has the other outstanding advantage of strong selectivity, is very safe to people, livestock, natural enemies and plants, and can be called as 'pollution-free pesticide'. However, the bacterium also has some disadvantages and shortcomings, such as the toxin protein crystal is easy to be decomposed by the effect of environmental factors; the insecticidal action is not lasting, and the field control effect can only be maintained for 3 to 4 days; the insecticidal spectrum is narrow, the insecticidal composition is only effective to partial lepidoptera pests, and the pests may generate drug resistance after being used for a long time.

In addition, many microorganisms in nature have inhibitory and antagonistic effects on plant pathogens. For example, many bacteria, actinomycetes and fungi prevent the occurrence and spread of diseases by producing antibiotics or competing for nutrition and living spaces, but the effect of controlling diseases is also affected by complicated environmental conditions and cannot be stabilized or sustained.

3. Microbial preparation for feed

The research of novel feed microbial preparation becomes a main means for feed industry technical innovation and product updating, and plays an important role in eliminating anti-nutritional factors, improving resource utilization rate, opening up new feed sources and solving the environmental pollution of animal husbandry.

4. Environmental decontamination

Microorganisms can play a unique role in the decomposition of various agricultural environmental pollutants, and microbial strains capable of effectively decomposing various pollutants such as plant straws, livestock and poultry manure, residual pesticides, industrial wastewater, household garbage and the like are continuously developed and applied. The biodegradation and biological recovery technology has the obvious advantages of high efficiency, high speed, low cost, mild reaction condition, no secondary pollution and the like, and becomes an important development direction for treating agricultural environmental pollution internationally. Due to the complexity of pollutant components and environmental pollution factors, microorganisms used for environmental pollution purification should have not only strong degradability and environmental tolerance, but also diversity of degradability.

The wide application of the microorganism in agricultural production is beneficial to improving the agricultural ecological environment, increasing the grain yield, reducing the environmental pollution, realizing the sustainable development of agriculture, and has obvious practical significance and profound historical influence on promoting the revolution of agricultural production. Digging, screening and expanding new functional microbial strains, breeding microbial products with excellent crop affinity, regionality, pertinence, adaptability and productivity, and improving the stability of application effect, and is the research focus in the field at present.

Disclosure of Invention

The invention aims to provide a novel Bacillus methylotrophicus (Bacillus methylotrophicus) and application thereof in agricultural production. The bacillus methylotrophicus is screened from soil in Russian Minskysk areas, has obvious inhibition effect on various pathogenic bacteria, can effectively prevent and treat common crop diseases and improve crop yield, and has wide application prospect.

The invention provides a Bacillus methylotrophicus (Bacillus methylotrophicus) named as Bacillus methylotrophicus VB1263(Bacillus methylotrophicus VB1263), which is preserved in China center for type culture collection at Wuhan university in Wuhan Han, China in 6 months and 6 days in 2019, wherein the preservation number is CCTCC NO: and M2019439.

On the one hand, the invention provides the application of the bacillus methylotrophicus in plant disease control.

The plant diseases comprise any one of neck rot, anthracnose, banded sclerotial blight, curvularia, root rot, southern blight, powdery mildew, scab, gray mold, fusarium wilt, brown spot and fruit rot.

The invention provides an application of the bacillus methylotrophicus in a biological fertilizer.

The invention also provides a microbial preparation which contains the bacillus methylotrophicus VB 1263.

The microbial preparation also comprises any one or the combination of two or more of bacillus, pseudomonas, agrobacterium, azotobacter, rhizobium, penicillium, aspergillus, rhizopus and streptomyces.

The viable bacteria amount of the Bacillus methylotrophicus VB1263 in the microbial preparation is at least 10⁸CFU/g。

The invention also provides application of the microbial preparation in plant disease control.

The invention also provides application of the microbial preparation in a biological fertilizer.

The bacillus methylotrophicus VB1263 screened by the method has a strong inhibition effect on common pathogenic bacteria such as fusarium oxysporum, alternaria alternate, pyelomyces graminis, botrytis cinerea, rhizoctonia solani and the like, particularly has a strongest inhibition effect on the pyelomyces graminis, rhizoctonia solani, agrobacterium tumefaciens and carrot soft rot pectin bacillus, and the width of an inhibition zone exceeds 24 mm. The bacillus methylotrophicus can be widely applied to prevention and control of common plant diseases, wherein the prevention and control efficiency on the ginger neck rot disease and the corn curvularia disease is respectively as high as 75.2% and 82.7%. The strain can also obviously improve the yield of the melons and improve the quality of the melons. Compared with a control group, the yield of the melons in the treatment group applied with the VB1263 powder of the bacillus methylotrophicus is generally improved by 5.1-25.4%, and the yield increase effect is obvious; the content of soluble solids in the processed muskmelon is improved by 10.3-24.1%, the content of vitamin C is improved by 10.7-33.5%, and unexpected technical effects are achieved.

The bacillus methylotrophicus VB1263 has stronger nitrogen fixation capacity, and the nitrogen fixation enzyme activity is as high as 40.9 nmol/(mg.h). Effective nitrogen, phosphorus and potassium nutrients in soil of a treatment group which applies the Bacillus methylotrophicus VB1263 powder in the seedling stage of the muskmelon are obviously improved, and the contents of alkaline hydrolysis nitrogen, effective phosphorus and quick-acting potassium in the soil are respectively improved by 53.9%, 44.2% and 46.9% compared with those of a control group, so that unexpected technical effects are achieved. Therefore, the bacillus methylotrophicus VB1263 provided by the invention has obvious effects of fixing nitrogen, dissolving phosphorus and dissolving potassium, can effectively improve soil fertility and soil environment, and is beneficial to improving crop yield and increasing economic benefits.

In conclusion, the bacillus methylotrophicus VB1263 provided by the invention can be used as a bio-control microbial inoculum, a bio-fertilizer and the like, is widely applied to the field of agricultural production, and has a remarkable effect and a wide application prospect.

Drawings

FIG. 1 shows matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) of VB1263 strain.

Detailed Description

The equipment and reagents used in the examples of the present invention may be selected from any commercially available ones. For the specific methods or materials used in the embodiments, those skilled in the art can make routine alternatives based on the existing technologies based on the technical idea of the present invention, and not limited to the specific descriptions of the embodiments of the present invention.

The invention is further illustrated by the following specific examples.

Example 1 Strain screening and identification

1. Strain screening:

(1) soil sample: collected from the soil in the area of Minskysk, Russia.

(2) Preparing a soil diluent by adopting a gradient dilution method, then carrying out flat plate coating and streak culture, and separating to obtain a plurality of strains of soil microorganisms; and further screening three strains with the most obvious bacteriostatic effects on pathogenic bacteria such as rhizoctonia solani, fusarium graminearum and strawberry anthrax by adopting a transparent ring method, wherein the three strains are respectively named as VB1263, VB1264 and VB 1266.

2. And (3) strain identification:

the VB1263 strain morphological characteristics: the bacterial colony is circular, the diameter is 3-6mm, the edges of the bacterial colony are neat, and the surface is smooth and moist; after long-time culture, the surface of the bacterial colony is rough and flat; gram-positive bacteria, which produce spores; the spore is oval, the local part is round or approximately round, and the sporangium is not expanded; the bacterial cells are present singly or in pairs, or in short chains.

The VB1263 strain is subjected to molecular biological identification, and a 16S rDNA sequence and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) are measured (shown in figure 1).

Combining the above biological characteristics and molecular biological identification results, the applicant confirmed that the strain VB1263 is Bacillus methylotrophicus, named as Bacillus methylotrophicus VB1263(Bacillus methylotrophicus VB 1263).

The applicant has deposited the Bacillus methylotrophicus VB1263(Bacillus methylotrophicus VB1263) in the China center for type culture collection at Wuhan university in Wuhan, China at 6.6.2019 with the preservation number of CCTCC NO: M2019439.

Example 2 evaluation of bacteriostatic ability of Bacillus methylotrophicus VB1263

1. Preparation of bacterial liquid

Firstly activating Bacillus methylotrophicus VB1263, then selecting the activated Bacillus methylotrophicus VB1263 and inoculating the activated Bacillus methylotrophicus VB1263 in an LB liquid culture medium, and carrying out inoculation at 37 ℃ and 2 DEG CCulturing at 20r/min for 14h to obtain viable bacteria amount of 10⁸-10⁹CFU/ml bacterial fluid.

2. Preparation of pathogenic bacteria

Respectively inoculating 10 kinds of pathogenic bacteria (provided by plant protection institute of agricultural academy in Shandong province) including Fusarium oxysporum, Alternaria alternata, Cytospora graminicola, Botrytis cinerea, Rhizoctonia solani, Agrobacterium tumefaciens, Xanthomonas campestris, Gum mucilaginosus, Michelia mikimura and Myxobacter paraguai on a PDA culture medium, and culturing at 30 deg.C for 5 days.

3. Plate bacteriostasis test

Inoculating a pathogenic bacteria cake with the diameter of 7mm in the center of a nutrient agar culture medium, placing a sterilized filter paper sheet at a position 25mm away from the center of a culture dish on two sides of the bacteria cake, and sucking 20ul of a liquid of bacillus methylotrophicus VB1263 to infiltrate the filter paper sheet; the petri dish was then placed in a 30 ℃ incubator for 5 days and then removed, and the width of the zone was measured. Triplicates were made for each pathogen and the average was taken. The filter paper method can prevent bacteria liquid from diffusing to the outside to form irregular colonies, and can quickly and accurately count the antibacterial effect.

The bacteriostatic effect of the bacillus methylotrophicus VB1263 on the 10 pathogenic bacteria is detailed in table 1.

TABLE 1 bacteriostatic effect of Bacillus methylotrophicus VB1263 on different pathogenic bacteria

Pathogens	The width of the bacteriostatic strip is mm
		Fusarium oxysporum	22.0±1.0
Alternaria alternata	23.0±1.5
		Leptospira interrogans (Fr.) Kuntze	24.0±1.0
Botrytis cinerea (Botrytis cinerea)	23.0±1.5
		Rhizoctonia solani	24.0±1.0
Agrobacterium tumefaciens	24.0±1.5
		Xanthomonas campestris	22.0±0.5
Bacillus mucilaginosus for carrot soft rot	24.0±1.5
		Leptomonas miehei	23.0±1.0
Myxobacterium baterii	23.0±1.5

The results in table 1 show that the bacillus methylotrophicus VB1263 provided by the invention has obvious inhibition effect on the 10 pathogenic bacteria, wherein the inhibition effect on the coenospora graminicola, rhizoctonia solani, agrobacterium tumefaciens and carrot soft rot disease pectin bacillus is strongest, and the width of the inhibition zone exceeds 24 mm.

Example 3 evaluation of Nitrogen fixation Effect of Bacillus methylotrophicus VB1263

1. The method for measuring the activity of the azotase comprises the following steps:

(1) ethylene content determination

Adding 4mL of nitrogen-fixing culture medium into a screw test tube with the volume of 21mL to prepare a slant, inoculating a strain to be tested, culturing in an incubator at 28 ℃, and respectively setting 7 times of operations by taking an uninoculated empty slant as a negative control and taking azotobacter chroococcum with known enzyme activity as a positive control. After 72h, the rubber stopper is replaced, acetylene gas is injected until the final concentration is 10%, the culture is sealed by a sealing film, and the culture is continued for 72 h. The amount of ethylene produced was measured by a gas chromatograph using 100. mu.L of the reaction gas.

Standard curve of ethylene content:

injecting 4, 8, 10, 15, 20, 40, 60 and 80 mu L of pure ethylene into a screw test tube with a certain volume, culturing for 72h in an incubator at 28 ℃, and measuring the peak height value. The volume of ethylene (μ L) was taken as the abscissa and the corresponding peak height (mv) was taken as the ordinate to make a standard curve.

(2) Determination of mycoprotein- -Bradford method (Coomassie Brilliant blue method)

Washing the strain to be detected on the inclined plane into a centrifugal tube by using 5mL of physiological saline, and centrifugally collecting thalli at 8000 r/min; adding 3mL of 0.5mol/L NaOH into the precipitate, and boiling for 5 min; adding 3mL of 0.5mol/L HCl, mixing uniformly, centrifuging at 5000r/min, taking 1.0mL of supernatant, adding 5mL of Coomassie brilliant blue, immediately mixing on a vortex mixer, and measuring the light absorption value OD595 of each sample at 595nm on a spectrophotometer after 2-5 min.

Protein standard solution: 10mg of bovine serum albumin was weighed and dissolved in 100mL of distilled water to prepare a 100. mu.g/mL stock solution.

Protein standard curve: accurately sucking 0.1mL, 0.2mL, 0.4mL, 0.6mL and 0.8mL of bovine serum-containing 100 mu G/mL stock solution, adding the solution into a test tube, respectively adding water to 1mL, adding 10, 20, 40, 60 and 80 mu G of bovine serum to each tube, adding 5.0mL of Coomassie brilliant blue G-250 protein reagent, and carrying out the same operation as above. The light absorption value is used as the ordinate, the protein content is used as the abscissa, and a standard curve is drawn.

And calculating the mycoprotein amount of the to-be-detected strain on the inclined plane according to a bovine serum albumin standard curve.

(3) Conversion to nitrogenase activity according to the following formula:

azotoxin activityProperty [ nmol/(mg. h)]＝C₂H₄Number of moles (nmol)/[ amount of bacterial protein (mg) × reaction time (h)]，C₂H₄Mole number (nmol) ═ C₂H₄Volume (. mu.L). times.273 XP/[ 22.4 × (273+ t ℃ C.) times.760]。

2. The result of the azotobacter activity measurement:

the azotobacter activity of the Bacillus methylotrophicus VB1263 is measured to be 40.9 nmol/(mg.h) according to the method, so that the strain has strong azotobacter capacity and unexpected technical effect.

Example 3 application of Bacillus methylotrophicus VB1263 in prevention and treatment of ginger neck rot disease

1. Preparation of Bacillus methylotrophicus VB1263 powder

Carrying out liquid fermentation on Bacillus methylotrophicus VB1263 in a 5-ton fermentation tank, and stopping fermentation when the microscopic spore rate reaches more than 90%; centrifuging at 5000rpm for 10min, removing supernatant, and spray drying to obtain powder with viable bacteria amount of 10 hundred million/g.

2. Experiment for preventing and treating ginger neck rot

(1) The experimental site: in the Changyi city foxtail ginger planting field, the land is planted with ginger in successive years and has serious continuous cropping disease, namely neck rot disease.

(2) Experiment design:

10m multiplied by 8m areas are selected as an experimental area, 10 ridges of gingers are planted in each experimental area, and about 400 +/-20 gingers are planted in each experimental area. 15 experimental areas are arranged in total, and a protection row is arranged between each experimental area. 3 experimental zones were randomly selected for each treatment group.

(1) Blank control group: directly planting ginger in a ginger ditch without spreading any fungus powder;

(2) VB1263 bacterial powder treatment group: firstly, VB1263 fungus powder (the living fungus amount is 10 hundred million/g) is uniformly scattered in a ginger ditch according to the use amount of 2-5 kg/mu, and then ginger is planted in the ginger ditch. Wherein:

treatment group 1: the consumption of VB1263 bacterial powder is 2 kg/mu;

treatment group 2: the consumption of VB1263 bacterial powder is 3 kg/mu;

treatment group 3: the consumption of VB1263 bacterial powder is 4 kg/mu;

treatment group 4: the consumption of VB1263 bacterial powder is 5 kg/mu.

In the growth process of the ginger, the same field management method is adopted for each test area. When the ginger is harvested, the occurrence condition of the ginger neck rot disease is counted, the prevention and treatment efficiency of the bacillus methylotrophicus VB1263 on the ginger neck rot disease is calculated, and specific results are shown in table 2.

Disease grading standard: level 0, healthy ginger plants are disease-free; level 1, the mother ginger plants are locally attacked, and the offspring ginger plants are healthy and disease-free; grade 2, the offspring ginger has disease spots but no withering; grade 3, local withering of the rhizomes (30-50%); 4 grade, the ginger cluster is basically withered or completely withered, and the ginger flesh is discolored and rotted by less than 60%; grade 5, the ginger cluster is completely withered, and the ginger pulp is rotted by more than 60%.

The disease index and the relative prevention and treatment effect are respectively calculated by the following formulas:

the disease index is (Σ (number of diseased plants at each stage × corresponding number of disease stages)/(number of total investigated plants × highest number of disease stages)) × 100;

relative control effect (%) (disease index of control group-disease index of treatment group)/disease index of control group x 100%.

TABLE 2 prevention and treatment effect of Bacillus methylotrophicus VB1263 on ginger neck rot disease

As can be seen from the data in Table 2, compared with the control group, the indexes of the diseased state of the ginger of each treatment group, which is obtained by scattering the Bacillus methylotrophicus VB1263 powder before the ginger is planted, are greatly reduced, so that the Bacillus methylotrophicus VB1263 provided by the invention has a remarkable prevention and treatment effect on the ginger diseased state, the prevention and treatment efficiency reaches 75.2% at most, and an unexpected technical effect is obtained.

Example 4 application of Bacillus methylotrophicus VB1263 in prevention and treatment of Curvularia zeae

1. The experimental site:

the planting time of the corns in the planting area is the same, and the growth vigor of the corns is similar.

2. Curvularia zeae prevention and treatment experiment:

a square area of 10m multiplied by 10m is selected as an experimental area, 15 experimental areas are arranged in total, and an interval of 5 meters is kept between every two experimental areas. 3 experimental zones were randomly selected for each treatment group.

(1) Blank control group: irrigating the corn roots with clear water;

(2) VB1263 bacterial powder treatment group: VB1263 bacterial powder (the living bacterial quantity is 10 hundred million/g) is applied to the root of the seedling corn along with water according to the dosage of 1-4 kg/mu, and the bacterial powder is used once every 7 days and is continuously used for three times. Wherein:

treatment group 1: the consumption of VB1263 bacterial powder is 1 kg/mu;

treatment group 2: the consumption of VB1263 bacterial powder is 2 kg/mu;

treatment group 3: the consumption of VB1263 bacterial powder is 3 kg/mu;

treatment group 4: the consumption of VB1263 bacterial powder is 4 kg/mu.

In the growth period of the corn, the incidence condition of the curvularia zeae is counted, the prevention and treatment efficiency of the bacillus methylotrophicus VB1263 on the curvularia zeae is calculated, and specific results are shown in table 3.

Disease grading standard: no disease spot at grade 0; level 1: the area of the lesion spots accounts for less than 1/4 of the area of the leaves; and 2, stage: 1/4-2/4 of the area of the lesion spots in the leaf area; and 3, level: 2/4-3/4 of the area of the lesion spots in the leaf area; 4, level: the lesion area is above 3/4 of the leaf area.

The disease index is [ Σ (disease level value × number of diseased leaves at this level)/(total number of investigated leaves × highest disease level value) ] × 100.

The calculation formula of the prevention and treatment efficiency is [1- (disease index of treatment group/disease index of control group) ] × 100%.

TABLE 3 preventive and therapeutic effects of Bacillus methylotrophicus VB1263 on Curvularia zeae

As can be seen from the data in Table 3, the curvularia pathogenicity index of the corn of each treatment group which is applied with the powder of the VB1263 strain of Bacillus methylotrophicus is only 12.1-28.6, which is far lower than that of the control group. Therefore, the bacillus methylotrophicus VB1263 provided by the invention has an obvious prevention and treatment effect on corn curvularia, the prevention and treatment efficiency reaches 82.7% at most, and an unexpected technical effect is achieved.

Example 5 application of Bacillus methylotrophicus VB1263 in melon planting

1. The experimental site:

big-arch shelter is planted to chinese countryside melon in lai xi city of Qingdao.

2. Melon planting experiment:

a10 m multiplied by 8m area is selected as an experimental area, and 10 ridges of melons are arranged in each experimental area, and about 500 +/-10 plants are obtained. 15 experimental areas are arranged in total, and a protection row is arranged between each experimental area. 3 experimental zones were randomly selected for each treatment group.

(1) Blank control group: watering the roots of the sweet melon seedlings with clear water;

(2) VB1263 bacterial powder treatment group: VB1263 strain powder (the amount of viable bacteria is 10 hundred million/g) is applied with water after the melon seedlings are transplanted according to the dosage of 1-8 kg/mu, and the application is carried out once after 7 days. Wherein:

treatment group 1: the consumption of VB1263 bacterial powder is 1 kg/mu;

treatment group 2: the consumption of VB1263 bacterial powder is 3 kg/mu;

treatment group 3: the consumption of VB1263 bacterial powder is 5 kg/mu;

treatment group 4: the consumption of VB1263 bacterial powder is 8 kg/mu.

After the muskmelon is ripe, centrally picking the muskmelons with the maturity of more than 8 days at 6:00-8:00 a day, weighing, and recording the daily output of the muskmelons in each experimental area; 20 melons were randomly picked from the daily melons picked in each experimental area, and the soluble solid content (mass%) and the vitamin C content (mg/100g) were measured.

After all melons are harvested, the total yield of the melons in each experimental area is counted, the average total yield and the yield increase rate of the melons in the control group and the melons in the treatment group are calculated respectively, and specific results are shown in table 4; the average content of soluble solids and vitamin C in the melons of the control group and the treated group were calculated, and the specific results are shown in table 5.

Yield increase is (melon yield in treatment group-melon yield in blank control group)/melon yield in blank control group x 100%.

TABLE 4 influence of Bacillus methylotrophicus VB1263 on melon yield

Experiment grouping	Average yield (kg/mu)	Increase of yield
			Blank control group	2461	-
VB1263 treatment group 1	2672.54	5.1％
			VB1263 treatment group 2	2768.31	12.5％
VB1263 treatment group 3	2908.79	18.2％
			VB1263 treatment group 4	3086.36	25.4％

TABLE 5 influence of Bacillus methylotrophicus VB1263 on melon quality

Experiment grouping	Soluble sugar content	VC content (mg/100g)
			Blank control group	8.7％	5.4
VB1263 treatment group 1	9.6％	5.98
			VB1263 treatment group 2	9.8	6.31
VB1263 treatment group 3	10.5	7.19
			VB1263 treatment group 4	10.8	7.21

From the results in tables 4 and 5, it is understood that the yield of melons can be significantly increased and the quality of melons can be improved by applying the powder of bacillus methylotrophicus VB1263 in the seedling stage of melons. Compared with a control group, the yield of the melons in the treatment group is generally improved by 5.1-25.4%, and the yield increasing effect is obvious; the content of soluble solids in the processed muskmelon is improved by 10.3-24.1%, the content of vitamin C is improved by 10.7-33.5%, and unexpected technical effects are achieved.

3. And (3) soil fertilizer efficiency determination:

after the melons were harvested, the overground sweet melon seedlings were removed, and 5 sampling points were selected for each experimental area using the diagonal method. At each sampling point, 1kg of soil layers 10cm and 20cm below the surface of the earth were collected.

And (3) air-drying, removing impurities, grinding, sieving and uniformly mixing the collected soil samples, then respectively detecting the contents of the alkaline hydrolysis nitrogen, the available phosphorus and the available potassium in each soil sample, and calculating the average contents of the alkaline hydrolysis nitrogen, the available phosphorus and the available potassium in the soil of the blank control group and the soil of the treatment group, wherein the specific results are shown in table 6.

TABLE 6 influence of Bacillus methylotrophicus VB1263 on nitrogen, phosphorus and potassium nutrients in soil

As can be seen from the data in table 6, compared with the control group, the effective nitrogen, phosphorus and potassium nutrients in the soil of the treatment group to which the powder of the bacillus methylotrophicus VB1263 is applied in the seedling stage of the melon are all significantly improved, and particularly, the contents of alkaline-hydrolyzable nitrogen, effective phosphorus and quick-acting potassium in the soil of the treatment group 4 are the highest, and are respectively improved by 53.9%, 44.2% and 46.9%, so that unexpected technical effects are achieved. Therefore, the bacillus methylotrophicus VB1263 provided by the invention has obvious effects of fixing nitrogen, dissolving phosphorus and dissolving potassium, can effectively improve soil fertility and soil environment, and is beneficial to improving crop yield and increasing economic benefits.

In conclusion, the bacillus methylotrophicus VB1263 provided by the invention can be independently used as a bio-control microbial inoculum, a bio-fertilizer and the like to be widely applied to the field of agricultural production, can be combined with any one or more of other bacillus, pseudomonas, agrobacterium tumefaciens, azotobacter, rhizobium, penicillium, aspergillus, rhizopus and streptomyces to be used for preventing and treating plant diseases, improving soil fertility and improving crop quality, and has wide application prospect.

Claims

1. Bacillus methylotrophicus (A)Bacillus methylotrophicus) VB1263, wherein the preservation number of the bacillus methylotrophicus is CCTCC NO: and M2019439.

2. The use of Bacillus methylotrophicus according to claim 1 for controlling a plant disease, wherein the plant disease is ginger neck rot or corn curvularia.

3. Use of bacillus methylotrophicus according to claim 1 in the preparation of a biological fertilizer.

4. A microbial preparation comprising the bacillus methylotrophicus of claim 1.

5. The microbial preparation of claim 4, wherein the viable count of Bacillus methylotrophicus in the microbial preparation is at least 10⁸CFU/g。

6. The use of the microbial preparation according to claim 4 or 5 for the control of plant diseases, wherein the plant diseases are ginger neck rot or corn curvularia.

7. Use of a microbial preparation according to claim 4 or 5 in the preparation of a biological fertilizer.