CN113186119A

CN113186119A - Bacillus methylotrophicus and application thereof in plant disease control

Info

Publication number: CN113186119A
Application number: CN202110332031.7A
Authority: CN
Inventors: 吕宾; 苑伟伟; 凌红丽; 周英俊; 赵凯; 张春玉
Original assignee: Shandong Vland Biotech Co ltd; SHANDONG KDN BIOTECH CO Ltd
Current assignee: QINGDAO VLAND BIOTECH Inc; SHANDONG KDN BIOTECH CO Ltd; Shandong Vland Biotech Co ltd; Qingdao Vland Biotech Group Co Ltd
Priority date: 2021-03-29
Filing date: 2021-03-29
Publication date: 2021-07-30
Anticipated expiration: 2041-03-29
Also published as: CN113186119B

Abstract

The invention relates to the technical field of functional microorganism screening and application, and particularly provides a novel bacillus methylotrophicus ZB2073(Bacillus methylotrophicusZB2073), accession number CCCTCC NO: m2020739. The bacillus methylotrophicus ZB2073 has the characteristics of wide antibacterial spectrum and remarkable antagonistic effect, has a good effect mainly aiming at soil-borne fungal diseases of plants, can promote the growth of crops and improve the yield of the crops, and has a wide application prospect.

Description

Bacillus methylotrophicus and application thereof in plant disease control

Technical Field

The invention relates to the technical field of functional microorganism screening and application, in particular to a bacillus methylotrophicus and application thereof in plant disease control.

Background

The chemical pesticide is used for preventing and treating plant diseases and insect pests, has the advantages of high speed, high efficiency, low cost, simple method and the like, is a main mode for preventing and treating the plant diseases and insect pests all the time, but is easy to degrade soil, pollute ecological environment, influence human health and the like due to long-term use of the chemical pesticide, and the diseases and insect pests are easy to generate drug resistance. The prevention and control of plant diseases and insect pests by microbial pesticides prepared from microorganisms and metabolites thereof are important points of biological control research in recent years. The microbial pesticide has the characteristics of specificity, high insecticidal rate, no pollution to the environment, no toxicity to people and animals and the like, can enhance the resistance of plants and promote the plants to grow better, and has various varieties, and fungi, bacteria, actinomycetes and the like are widely applied to production.

Trichoderma as a biological fungus widely used for preventing and controlling plant diseases and insect pests mainly exists in soil, air, plant surface and other ecological environments, and can effectively prevent and control various plant diseases and insect pests. A large number of studies have shown that most trichoderma can produce biologically active substances and have antagonistic effects against phytopathogenic fungi, phytopathogenic nematodes, etc. Researches on Jingfang and the like find that the trichoderma longibrachiatum T6 biocontrol microbial inoculum has a good control effect on pepper damping off, can effectively control the spread of pepper diseases, has a control effect as high as 54.8 percent, and is 12.5 percent higher than the control effect of a chemical pesticide carbendazim. The yayanpo research shows that the trichoderma harzianum has good control effect on the blight of hot pepper and potato, and can not only inhibit the growth of blight in soil and reduce the population quantity of pathogenic bacteria, but also effectively reduce the plant death rate and disease index.

Beauveria bassiana is a broad-spectrum entomogenous fungus, and can parasitize more than 700 kinds of insects and part of acarids; the insecticidal composition has strong selectivity on hosts and weak attack capability on infection of ladybug, lacewing, eclosia, horsefly and the like, and the characteristic is beneficial to protecting natural enemy insects and reducing damage to ecological communities of the insects. At present, beauveria bassiana is widely applied to prevention and control of agricultural and forestry pests such as tetranychus urticae, bemisia tabaci and thrips. The data show that the beauveria bassiana has good inhibition rate on the tobacco alternaria alternata, the beauveria bassiana is gradually enhanced on the pathogenic fungi tobacco alternaria alternata along with the time extension, and the beauveria bassiana spore suspension with the concentration of 1.0 multiplied by 107 has the most obvious inhibition effect on the tobacco alternaria alternata.

The mycorrhizal fungi also has better effect in the aspect of plant disease and insect pest control, and is a green and environment-friendly biocontrol factor. The mycorrhizal fungi which are most researched are Arbuscular Mycorrhizal (AM) fungi, and the AM fungi improve the insect resistance and the pest tolerance of plants and promote the plants to grow better by promoting symbiotic plants to generate secondary metabolites and improving the growth, the plant nutrition, the root activity and the like of the plants. Zhujian discloses AM mycorrhizal fungi MW21, and a biocontrol microbial inoculum and application thereof. The arbuscular mycorrhizal fungi MW21 powder mainly comprises giant micro-bustle (Gigaspora albica) MW21, the prevention effect of the microbial inoculum containing the mycorrhizal fungi spores on woody plant diseases reaches more than 52.29%, the growth promoting effect reaches more than 30.60%, the transplanting survival rate of disease-free woody plants can be improved by more than 26.66%, the stress resistance of the woody plants is improved, and the growth promoting effect reaches more than 10%.

The bacillus is mainly used for preventing and controlling plant diseases and insect pests, and has at least one or more of nitrogen fixation, phosphorus dissolution, potassium release, phytohormone production and antibiotic secretion. It can not only promote plant growth, but also inhibit harmful organisms. Plum crystal and the like disclose bacillus subtilis PB12 for preventing and treating potato scab, and the occurrence of the potato scab can be reduced by dressing seeds with PB12 bacteria and applying PB12 granular fertilizer, and the prevention and treatment rate reaches 54.42%.

Actinomycetes are a special branch of bacteria, streptomycete and variants thereof are mostly applied, and a plurality of biocontrol agents are widely applied to the fields of industry, agriculture, medicine and the like. Zhang Sina screens out 1 Streptomyces microflavus actinomycetes G33 from a tomato planting field, the experiment of preparing the actinomycetes into an organic fertilizer pot plant shows that the biocontrol effect on tomato bacterial wilt reaches 51.72 percent, and the field test result shows that the biocontrol effect reaches 48.72 percent.

The biocontrol bacteria can inhibit pathogenic bacteria of plants by utilizing the characteristics of the biocontrol bacteria, and more beneficial nonpathogenic microorganisms exist in the nature, so that scientific researchers need to continuously screen and optimize the biocontrol bacteria, reduce a plurality of hazards such as environmental pollution and the like by utilizing the biocontrol bacteria, and promote the vigorous development of pollution-free and green modern agriculture in China.

Disclosure of Invention

The invention aims to provide a Bacillus methylotrophicus (Bacillus methylotrophicus) and application thereof in agricultural production. The bacillus methylotrophicus can effectively prevent and treat common crop diseases and has a wide application prospect.

The invention provides a methylotrophic Bacillus named as Bacillus methylotrophicus ZB2073 (ZB 2073), which is preserved in China center for type culture Collection of Wuhan university in Wuhan, China at 11 and 16 months in 2020 with the preservation number of CCTCC NO: M2020739.

The MALDI-TOF-MS protein mass spectrum of the Bacillus methylotrophicus ZB2073 strain is shown in figure 2, and the Riboprinter fingerprint spectrum is shown in figure 3.

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

The plant disease is leaf spot, stem base rot or root rot.

In one aspect of the present invention, there is provided a microbial preparation comprising the above-mentioned Bacillus methylotrophicus ZB 2073.

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 bacillus is preferably bacillus subtilis, bacillus licheniformis, bacillus pumilus, bacillus megaterium, bacillus coagulans, bacillus laterosporus or bacillus siamensis.

The viable bacteria amount of Bacillus methylotrophicus ZB2073 in the microbial preparation is at least 10⁹CFU/g。

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

The invention also provides a fertilizer containing the microbial preparation.

The methylotrophic bacillus ZB2073 provided by the invention has strong bacteriostatic ability and wide bacteriostatic spectrum, and has strong inhibitory action on various crop frequently-occurring pathogenic bacteria such as fusarium oxysporum, rhizoctonia solani, sclerotinia sclerotiorum, pyricularia oryzae, rhizoctonia solani, fusarium moniliforme, botrytis cinerea, ralstonia solani, pythium aphanidermatum and the like. Wherein the inhibition effect on the pythium is best, and the width of the inhibition zone reaches 32.3 mm.

The bacillus methylotrophicus ZB2073 can produce Surfactin and pangamin Fengycin. The Surfactin has inhibitory activity on various plant pathogenic fungi, and the pansin Fengycin has excellent inhibitory effect on filamentous fungi as lipopeptide antifungal.

The bacillus methylotrophicus ZB2073 can be widely applied to the control of common plant diseases such as leaf spot, stem basal rot and root rot, and has very obvious effect. Compared with a control group, the growth vigor of the corn in the treatment group applied with the bacillus methylotrophicus ZB2073 microbial inoculum is obviously improved, the plant height and the number of developed leaves of the corn in the treatment group are gradually increased along with the increase of the using amount of the microbial inoculum, the increase rates are respectively as high as 61.4% and 45.5%, and the growth promotion effect is obvious. In addition, the disease index of the stem basal rot of the corn in the treatment group is obviously reduced, and the relative prevention effect can reach as high as 90.94%. The bacillus methylotrophicus ZB2073 can obviously promote the growth of peanuts, increase the yield of the peanuts and effectively prevent and control the root rot of the peanuts. The main stem of the peanuts in the treatment group is high, the pod number, the plumpness and the weight average of all the peanuts are improved to different degrees, the yield is increased by up to 25.8 percent, and the growth-promoting and yield-increasing effects are obvious. Moreover, the disease index of the root rot of the peanuts in the treatment group is obviously lower than that of the peanuts in the control group, and the control effect of the bacillus methylotrophicus ZB2073 fungus powder on the root rot can reach 79.1 percent at most.

The bacillus methylotrophicus ZB2073 provided by the invention can be used as a biocontrol microbial inoculum to be applied to agricultural planting, has the characteristics of wide antibacterial spectrum and remarkable antagonistic effect, mainly has a good effect on soil-borne fungal diseases of plants, can promote the growth of crops and improve the yield of the crops, and has a wide application prospect.

Drawings

FIG. 1 is a colony diagram of ZB2073 strain;

FIG. 2 is a protein peak of ZB2073 strain;

FIG. 3 is a gene fingerprint of ZB2073 strain;

FIG. 4 shows the inhibition of Pythium species by ZB2073 strain;

FIG. 5 is an LC-MS spectrum of a crude extract of ZB2073 strain;

FIG. 6 is a graph showing the control effect of ZB2073 strain on leaf spot of excised leaf.

Detailed Description

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 isolation and screening of strains

1. Sample source: rhizosphere soil of a Laiyang pear tree planting base in Yantai city of Shandong province.

2. Strain separation:

a10 g sample of the soil was weighed into a 250mL Erlenmeyer flask containing sterile glass beads and 90mL sterile water and shaken well. Standing, and performing gradient dilution on an ultraclean workbench to obtain 10^-4、10^-5、10^-6Three dilutions, 100 μ L each, were spread on nutrient agar plates and repeated 3 times. Culturing in 37 deg.C incubator for 24 hr, observing growth condition, classifying according to colony size, shape and color, and selecting single colony for purification culture. The applicant finally selected 6 strains of the strain, designated as L1-L6, and performed liquid glycerol conservation.

3. Strain screening:

6 strains are preliminarily screened through a flat plate bacteriostasis experiment, and strains with better bacteriostasis effect on pathogenic bacteria of three plant diseases including rhizoctonia solani, fusarium and gibberella bardawil are screened. Inoculating a pathogenic bacteria cake with the diameter of 5mm in the center of a PDA culture medium, sucking 10 mu L of a bacterial liquid point to be detected at a position 3cm away from the bacteria cake by using a liquid transfer gun, culturing in a constant-temperature incubator at 26 ℃, and observing and measuring the bacteriostatic effect at regular time. Specific results are shown in table 1.

TABLE 1 bacteriostatic effect of different strains on pathogenic bacteria

From the results shown in table 1, it is clear that the L2 strain among the 6 strains screened by the present invention has a strong inhibitory effect on three pathogenic bacteria, namely rhizoctonia solani, fusarium and gibberella bardawil, and has an unexpected technical effect.

The applicant named the L2 strain ZB2073 and evaluated it further.

Example 2 identification of the Strain ZB2073

2.1 colony morphology identification

As shown in figure 1, the colony of the ZB2073 strain on the nutrient agar medium is round, has neat edges, flat and non-wrinkled surface, is opaque and has white color.

2.216S rRNA molecular identification

The genome of the strain ZB2073 was extracted using the kit. Then, the 16S rRNA was amplified using the genome as a template and a specific primer. Sequencing results of the PCR amplification products are compared with BLAST in NCBI database, and the similarity of the sequencing results with Bacillus methylotrophicus (Bacillus methylotrophicus) is the highest. Therefore, the ZB2073 strain was preliminarily determined to be Bacillus methylotrophicus (Bacillus methylotrophicus).

2.3 MALDI-TOF-MS protein mass spectrometry identification

Coating a small amount of ZB2073 single colony on a target plate in a thin film form; adding 1 mu L of lysate in the mass spectrum sample pretreatment kit, and naturally airing at room temperature; adding 1 mu L of matrix solution in the mass spectrum sample pretreatment kit to cover the sample, and naturally airing at room temperature; and putting the sample target into a mass spectrometer for identification. The identification result shows that the ZB2073 strain is Bacillus methylotrophicus (Bacillus methylotrophicus), and the protein spectrum peak diagram is shown in figure 2.

2.4 RiboPrinter full-automatic microbial Gene fingerprint identification

The strain ZB2073 was identified on the computer according to the full-automatic microbial gene fingerprint identification system operating instructions to obtain its rRNA gene fingerprint map, as shown in FIG. 3. The similarity of the strain ZB2073 and the Bacillus methylotrophicus is more than 90 percent through comparison with a known standard strain library fingerprint map, so that the strain is identified as the Bacillus methylotrophicus.

In conclusion, the applicant utilizes three molecular biological methods of 16S rRNA identification, MALDI-TOF-MS protein mass spectrum identification and RiboPrinter full-automatic microorganism gene fingerprint identification to identify the strain ZB2073, and the identification results are consistent. In combination with the colony morphology of the strain ZB2073, the applicant determined that the strain was Bacillus methylotrophicus (Bacillus methylotrophicus) named Bacillus methylotrophicus ZB2073(Bacillus methylotrophicus ZB 2073).

The applicant has deposited the above-mentioned Bacillus methylotrophicus ZB2073(Bacillus methylotrophicus ZB2073) in China center for type culture Collection, CCTCC NO: M2020739, at 16/11/2020.

Example 3 evaluation of bacteriostatic ability of Bacillus methylotrophicus ZB2073

1. Preparation of Bacillus methylotrophicus ZB2073 bacterial liquid

Selecting Bacillus methylotrophicus ZB2073, inoculating into nutrient broth culture medium, culturing at 37 deg.C and 220r/min for 18h to obtain viable bacteria amount of 10⁸-10⁹CFU/ml bacterial liquid for use.

2. Preparation of pathogenic bacteria

Various crop frequently-occurring pathogenic bacteria (provided by plant protection institute of agricultural academy of Shandong province) such as fusarium oxysporum, rhizoctonia solani, sclerotium solani, rice blast, rhizoctonia solani, fusarium moniliforme, botrytis cinerea, ralstonia solani, pythium is respectively inoculated on a PDA culture medium for purification culture, and the culture is carried out for 5 days at 25 ℃ for later use.

3. Plate bacteriostasis test

In the plate confrontation experiment, 10 mu L of bacillus methylotrophicus ZB2073 bacterial liquid is firstly taken and spotted in the middle of a PDA plate, and the liquid is cultured for 12h in an incubator at 25 ℃. Making pathogenic fungi into hypha suspension, uniformly spraying onto a flat plate with biocontrol bacteria liquid by using a small spray can, comparing with the hypha suspension only sprayed with pathogenic fungi, culturing in an incubator at 25 ℃ for 24h, and measuring and recording the size of a bacteriostatic zone. Each pathogenic bacterium was replicated three times, and the average value was taken, and the bacteriostatic effect is detailed in Table 2.

TABLE 2 bacteriostatic effect of Bacillus methylotrophicus ZB2073 on different pathogenic bacteria

Pathogenic bacteria	Average width of antibacterial belt (mm)
		Pythium species	32.3±0.5
Rhizoctonia solani	30.0±1.0
		Leaf spot pathogen	28.0±0.8
Magnaporthe grisea	28.0±1.2
		Rhizoctonia solani	25.0±1.0
Fusarium moniliforme	30.5±1.5
		Botrytis cinerea (Fr.) Kuntze	28.0±1.0
Ralstonia solanacearum	26.0±1.0
		Fusarium oxysporum	28.0±1.2
Gibberella fujikuroi	31.0±1.0

As can be seen from Table 2, in the plate confrontation experiment, Bacillus methylotrophicus ZB2073 has obvious inhibition effect on the 10 pathogenic bacteria and obvious inhibition zone, wherein the inhibition effect on Pythium is the best, and the inhibition zone width reaches 32.3mm (figure 4). Therefore, the bacillus methylotrophicus ZB2073 provided by the invention has strong bacteriostatic ability and a wider bacteriostatic spectrum.

Example 4 Mass Spectrometry of Bacillus methylotrophicus ZB2073 lipopeptide Compounds

1. Bacillus methylotrophicus ZB2073 culture and preparation of extracellular substance

The Bacillus methylotrophicus ZB2073 is cultured in Landy culture medium specially for producing antibacterial active substance at 35 deg.C and 200rpm for 48 h. After the completion of the reaction, the precipitated cells were removed by centrifugation, and the supernatant was added with 6mol/L HCI to adjust the pH to 2.0, gently stirred for 2 hours, and stored overnight. Centrifuging to collect precipitate, adding 4ml methanol, adjusting pH to 7.0 with 1mol/L NaOH, extracting with 2ml methanol for 2 times, and mixing the extractive solutions to obtain crude extracellular extract of ZB2073 strain.

2. Mass spectrometric detection of lipopeptide compounds

The type of the lipopeptide compound generated by the ZB2073 strain is analyzed by a liquid chromatograph-mass spectrometer, and the experimental method, the type of the lipopeptide compound and the corresponding ion mass-to-charge ratio refer to NY/T3264-2018 detection standards.

By analyzing LC-MS (FIG. 5) and the types of lipopeptide compounds, it was found that Bacillus methylotrophicus ZB2073 can produce Surfactin and pangamin Fengycin. The Surfactin has inhibitory activity on various plant pathogenic fungi, and the pansin Fengycin has excellent inhibitory effect on filamentous fungi as lipopeptide antifungal.

In conclusion, the bacillus methylotrophicus ZB2073 provided by the invention can generate lipopeptide compounds capable of inhibiting plant pathogenic bacteria, so that a good antibacterial effect is shown.

Example 5 disease prevention Effect of Bacillus methylotrophicus ZB2073 on Ex vivo leaves

1. Preparation of Bacillus methylotrophicus ZB2073 bacterial liquid

The bacteria amount is 10⁸-10⁹CFU/ml ZB2073 bacterial liquid for later use.

2. Preparation of a suspension of pathogenic hyphae

Pouring a small amount of sterile water into the purified and cultured leaf spot germ plate, scraping bacterial colony hyphae with an operating knife, soaking the hyphae in water for 30min, shaking in a test tube, or grinding with water, and filtering to obtain hyphae suspension.

3. In vitro leaf pathogenic bacteria inoculation protection experiment

Taking healthy peanut compound leaf blades with consistent growth vigor, soaking sterilized cotton wrapped petioles in 0.1% benzimidazole cotton under aseptic condition, wrapping with tin foil paper to play a role in keeping fresh, putting into an aseptic culture dish, and spraying sterile water on filter paper to keep moisture. The treatment group was sprayed with ZB2073 bacterial liquid and the suspension of leaf spot pathogen, and the control group was sprayed with the same amount of sterile water and the suspension of leaf spot pathogen. Culturing in a constant temperature box with light and dark alternation at 25 deg.C, and observing and recording the disease condition and disease index of leaves.

The investigation result after 3 days shows that the control group has leaf spot disease, while the treatment group sprayed with ZB2073 bacterial liquid has no disease spot and the leaves are healthy and dark green (figure 6); the prevention effect of the bacillus methylotrophicus ZB2073 on the leaf spot can be up to 95.6 percent through counting the disease index and the prevention effect. Therefore, the bacillus methylotrophicus ZB2073 can effectively prevent and treat the leaf spot, has very obvious effect and obtains unexpected technical effect.

Example 6 control Effect of Bacillus methylotrophicus ZB2073 on Stem rot of potted corn

1. Preparation of Bacillus methylotrophicus ZB2073 bacterial liquid

The bacteria amount is 10⁸-10⁹CFU/ml ZB2073 bacterial liquid for later use.

2. Preparation of a suspension of pathogenic hyphae

Pouring a small amount of sterile water on a purified and cultured corn stalk basal rot plate, scraping bacterial colony hyphae with an operating knife, soaking the hyphae in water for 30min, shaking in a test tube, or grinding with water, and filtering to obtain a hyphae suspension.

3. Planting of potted corn and pathogenic bacteria inoculation protection

The soil used for potting is sterilized, corn seeds are sowed in flowerpots after pregermination treatment, 3 corn seeds are planted in each flowerpot, the operation is repeated for 10 times, and inoculation of stem base rot pathogenic bacteria is carried out when four unfolded leaves grow out of the corn, and the specific grouping and treatment are as follows:

control group: pouring 1L of clear water in the pot;

treatment group 1: pouring 100ml of ZB2073 bacterial liquid and 900ml of clear water in a pot;

treatment group 2: pouring 500ml of ZB2073 bacterial liquid and 500ml of clear water in a pot;

treatment group 3: and pouring 1L of ZB2073 bacterial liquid in the pot.

Periodically investigating the growth condition of the potted corn plants, investigating the plant height and the number of the unfolded leaves of the corn plants to evaluate the growth condition of the plants; and (5) investigating the disease index of stem basal rot of the potted corn and calculating the control effect.

Disease grading standard: grade 0, no scab; grade 1, the disease leaf rate is below 5%; grade 3, the disease lobe rate is 6-10%; and 5, stage: the disease leaf rate is 11% -30%; and 7, stage: the disease leaf rate is 31-40%; and 9, stage: the disease rate of the leaves is more than 41 percent.

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.

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

TABLE 3 effect of Bacillus methylotrophicus ZB2073 on corn pot culture

Experiment grouping	Plant height (cm)	Number of leaves developed	Index of disease condition	Relative prevention and cure effect
					Control group	35.5	6.6	98.13	-
ZB2073-1	47.5	8.3	43.33	55.84％
					ZB2073-2	53.8	8.9	37.67	61.61％
ZB2073-3	57.3	9.6	8.89	90.94％

As can be seen from the data in Table 3, compared with the control group, the growth vigor of the corn treated by the Bacillus methylotrophicus ZB2073 microbial inoculum is obviously improved, and the plant height and the number of developed leaves of the corn treated by the Bacillus methylotrophicus ZB2073 microbial inoculum are gradually increased along with the increase of the consumption of the microbial inoculum, the increase is respectively as high as 61.4 percent and 45.5 percent, and the growth promotion effect is obvious. In addition, the disease index of the stem basal rot of the corn in the treatment group is obviously reduced, and the relative prevention effect can reach as high as 90.94%. Therefore, the bacillus methylotrophicus ZB2073 provided by the invention can obviously promote the growth of corn and effectively prevent and control the basal rot of the corn.

Example 7 controlling Effect of Bacillus methylotrophicus ZB2073 on rooting and rot of flowers

1. Preparation of bacillus methylotrophicus ZB2073 powder

Carrying out liquid fermentation on bacillus methylotrophicus ZB2073 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. General description of the Experimental site

Experiments are carried out in peanut demonstration gardens in Lyocell of Qingdao, the experimental land is a multi-year continuous cropping land, root rot occurs all the year round, and the soil type is sandy loam. The peanut variety is No. 22 flower cultivation, ridge forming and film covering cultivation are carried out, the ridge height is 8cm, the ridge width is 65cm, double-row sowing is carried out, the hole distance is 12cm, and 2 seeds are planted in each hole.

3. Experimental methods

The experiment is provided with 4 treatments in total, the treatments are grouped according to different dosages of microbial inoculum, clear water is used as blank control, each treatment is repeated for 3 times, the treatments are arranged in random blocks, and the area of each cell is 100m². Each treating agent is used for 3 times, the first time is applied in the soil-mixing ditch before sowing, and the second time is appliedThe watering is carried out once every 10 days. Other planting methods and field management are the same as those of the production field. The specific grouping and processing are as follows:

control group: watering with clear water without using a microbial inoculum;

treatment group 1: 100g of 10 hundred million/g ZB2073 bacterial powder is used;

treatment group 2: 200g of 10 hundred million/g ZB2073 bacterial powder is used;

treatment group 3: 500g of 10 hundred million/g ZB2073 bacterial powder was used.

4. Agronomic traits and yield

When the peanuts are harvested, each district actually harvests 100m²And (4) counting yield, randomly taking 10 typical plants in each cell to investigate the main stem height, the pod number of a single plant and the plumpness rate, weighing and counting yield after the pods are dried in the sun, and measuring the weight of the hundreds of fruits.

5. Investigation of prevention and treatment effects

In the growth process of peanuts, a five-point sampling method is adopted to investigate the occurrence condition of peanut root rot, the occurrence condition is recorded according to the damage classification standard, and the disease index and the prevention effect are calculated. The classification criteria and formula are as follows:

grading the disease condition of the root rot of the peanut in the field according to the wilting degree of the peanut leaves: level 0, no wilting of the plant; grade 1, a few leaves show recoverable wilting; grade 3, leaf wilting by 10.1-30% and leaves are still green; grade 5, 30.1 to 50 percent of leaves are obviously wilted; 7 grade, plant wilting, 50.1-80% of leaves becoming yellow and wilting, and rootstock becoming brown; grade 9, the whole plant died, and the roots rotten.

Disease index [ ∑ (disease grade value × number of diseased leaves at this grade)/(investigation total number of leaves × highest disease grade value) ] × 100

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

TABLE 4 effect of Bacillus methylotrophicus ZB2073 on peanut field

As can be seen from the field plot experimental data in Table 4, compared with the control group, the height of the main stem, the number of pods, the plumpness and the weight average of all the peanuts of the treatment group applied with the Bacillus methylotrophicus ZB2073 powder are improved to different degrees, the yield is increased by up to 25.8%, and the growth promoting and yield increasing effects are obvious. Moreover, the disease index of the root rot of the peanuts in the treatment group is obviously lower than that of the peanuts in the control group, and the control effect of the bacillus methylotrophicus ZB2073 fungus powder on the root rot can reach 79.1 percent at most. Therefore, the bacillus methylotrophicus ZB2073 can obviously promote the growth of the peanuts, increase the yield of the peanuts and effectively prevent and control the root rot of the peanuts.

In conclusion, the bacillus methylotrophicus ZB2073 provided by the invention can be independently used as a biocontrol microbial inoculum to be applied to agricultural planting, can be used in combination with any one or two or more of bacillus, pseudomonas, agrobacterium tumefaciens, azotobacter, rhizobium, penicillium, aspergillus, rhizopus and streptomyces, has the characteristics of wide antibacterial spectrum and remarkable antagonistic effect, mainly has a good effect on plant soil-borne fungal diseases, can promote the growth of crops and improve the yield of the crops, and has a wide application prospect.

Claims

1. The bacillus methylotrophicus is characterized in that the preservation number of the bacillus methylotrophicus is CCTCC NO: m2020739.

2. Use of the Bacillus methylotrophicus of claim 1 for controlling a plant disease.

3. The use according to claim 2, wherein the plant disease is leaf spot, stem rot or root rot.

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

5. The microbial preparation of claim 4, further comprising any one or a combination of two or more of Bacillus, Pseudomonas, Agrobacterium, Azotobacter, Rhizobium, Penicillium, Aspergillus, Rhizopus, and Streptomyces.

6. The microbial formulation of claim 5 wherein the bacillus is any one or a combination of two or more of bacillus subtilis, bacillus licheniformis, bacillus pumilus, bacillus megaterium, bacillus coagulans, bacillus laterosporus or bacillus siamensis.

7. A microbial preparation according to any one of claims 4 to 6 wherein the viable count of Bacillus methylotrophicus in the microbial preparation is at least 10⁹CFU/g。

8. Use of the microbial preparation according to any one of claims 4 to 7 for the control of plant diseases.

9. A fertilizer, characterized in that it comprises a microbial preparation according to any one of claims 4 to 7.