CN111690557B

CN111690557B - Bacillus amyloliquefaciens LXJDF-1 and application thereof

Info

Publication number: CN111690557B
Application number: CN202010460147.4A
Authority: CN
Inventors: 许曼琳; 迟玉成; 张霞; 于静; 郭志青
Original assignee: Shandong Peanut Research Institute
Current assignee: Shandong Peanut Research Institute
Priority date: 2020-05-27
Filing date: 2020-05-27
Publication date: 2022-02-15
Anticipated expiration: 2040-05-27
Also published as: CN111690557A

Abstract

The invention belongs to the technical field of bacillus amyloliquefaciens, and particularly relates to bacillus amyloliquefaciens (Bacillus amyloliquefaciens)Bacillus amyloliquefaciens) LXJDF-1 and applications thereof. Discloses a bacillus amyloliquefaciens (Bacillus amyloliquefaciens) The bacillus amyloliquefaciens LXJDF-1 is preserved in the China general microbiological culture Collection center of China Committee for culture Collection of microorganisms with the preservation number of: CGMCC No.18009, and the preservation date is 6 months and 21 days in 2019. The strain has the function of preventing and treating bacterial wilt.

Description

Bacillus amyloliquefaciens LXJDF-1 and application thereof

Technical Field

The invention belongs to the technical field of Bacillus amyloliquefaciens, and particularly relates to Bacillus amyloliquefaciens (LXJDF-1) and application thereof.

Background

Peanuts are important economic and oil crops in China, the largest peanut planting country and export big country in the world, and the peanuts occupy an extremely important position in world peanut production. The planting area of Chinese peanuts is about 20 percent of the area of peanuts in the world, and the total production and the single production are listed as the first in the world just after Indian.

Peanut bacterial wilt (Pseudomonas solanacearum) was first reported in Indonesia in 1950, and then occurrence and prevalence of peanut bacterial wilt in southeast Asia countries, south Africa countries and multiple countries in the United states are reported. China reports about peanut bacterial wilt are reported in the later stage of 60 years, the peanut bacterial wilt is widely distributed in peanut producing areas, the actual disease area of the whole country is more than 40 ten thousand hm2 every year, the disease is particularly serious in southern provinces, the disease is also generated in mountains, Liaoning, Hebei, Henan and other places, and the harm of partial regions is gradually increased. The diseased plant can die quickly in a short period. The field with diseases after peanut pod bearing generally reduces yield, and the field with diseases before pod bearing can be harvested absolutely. It can be attacked from seedling stage to harvest stage, and is most serious in full-bloom stage. Typical symptoms are acute plant wilting and vascular bundles browning. At the initial stage of disease occurrence, the first leaves and the second leaves at the top tips of the main lotuses of the diseased plants are withered due to water loss in the daytime and can be recovered in the morning and evening, then the leaves of the diseased plants are not recovered along with the development of disease conditions, the leaves of the diseased plants are gradually withered from top to bottom, the color of the leaves is dark and greenish, and finally the diseased plants die. Generally, the period from onset to plant death lasts 7-15 days, and the number is about 20 days. The tip of the main root of the diseased plant turns brown and is in a wet rot shape, and the root nodule is in dark green. The stem of the plant with the longitudinal section is seen to turn light brown to black brown, until the top and the root of the stem. The vascular bundles arranged in a ring shape are seen to be brown and small spots in the root and stem parts of the plants. The incision was squeezed with a hand and the pus of the white bacteria was found to overflow. Early diseased plants, stalks and pods turned into a dark brown wet rot, and the nuts were not practical. The symptoms of the plants with diseases in the pod bearing stage are not as obvious as those of the plants with diseases in the prophase.

Because the bacillus has wide development and application prospects, the development of newly screened bacillus of indefinite species and the identification of the bacterial wilt prevention and control effect of a new strain are of great significance.

Disclosure of Invention

Aiming at the requirements in the prior art, the invention aims to provide a Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) LXJDF-1 which is a brand-new screened strain and a method for preventing and treating bacterial wilt by using the Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) LXJDF-1, and the method has obvious effect of preventing and treating the bacterial wilt and has good application prospect.

In order to achieve the purpose, the invention adopts the following technical scheme:

the invention provides a Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) LXJDF-1, which is preserved by China general microbiological culture Collection center (CGMCC) (China institute of microbiology, institute of sciences, Japan, No.1, No. 3, China academy of sciences, North Jing City, Chaoyang district, North Cheng, Xilu, No. 1) in 6-21 days in 2019, with the preservation number of CGMCC No. 18009.

The invention provides application of Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) LXXJDF-1 in inhibition of ralstonia solanacearum.

The invention provides application of Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) LXJDF-1 in preventing and treating bacterial wilt of crops.

The invention provides a ralstonia solanacearum inhibitor which contains Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) LXXJDF-1.

The invention provides a solid composition for controlling bacterial wilt, which contains Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) LXXJDF-1.

Compared with the prior art, the invention has the beneficial effects that: the bacillus amyloliquefaciens LXJDF-1 has good control effect on fungi and bacteria, has the advantages of high growth speed, simple nutritional requirement, strong heat resistance, strong stress resistance and the like, is safe to people and livestock, is non-toxic and environment-friendly, and has no pollution.

Drawings

FIG. 1: a colony morphology laboratory effect chart of the bacillus amyloliquefaciens on an LB culture medium.

FIG. 2: and (3) an evolutionary tree diagram constructed by the bacillus amyloliquefaciens LXJDF-1 and other bacteria 16S rDNA.

FIG. 3: a diagram of the inhibition effect of group I bacillus amyloliquefaciens LXXJDF-1 on Ralstonia solanacearum.

FIG. 4: and the inhibition effect of the group II bacillus amyloliquefaciens LXXJDF-1 on the ralstonia solanacearum.

FIG. 5: and the experiment result chart of the group III filter paper sheets for resisting the ralstonia solanacearum by using clear water instead of LXXJDF-1 as a control.

The bacillus amyloliquefaciens LXXJDF-1 is preserved in 6.8.21.2019 by the China general microbiological culture Collection center (CGMCC) (the microbiological research institute of China academy of sciences, No. 3, Taihe Xilu No.1, Beijing, the rising area, the Beijing) with the preservation number of CGMCC No. 18009.

Detailed Description

The present invention discloses a bacillus amyloliquefaciens and uses thereof, and features and aspects of the present invention are described in detail below with reference to specific embodiments. Unless otherwise indicated, the test methods and test reagents used in the present invention are those well known to those skilled in the art. In addition, the embodiments are to be construed as illustrative and not limitative of the scope of the present invention, and it is apparent to those skilled in the art that various changes or modifications to the components, contents, culture conditions, separation and processing conditions of the culture medium in the embodiments are possible without departing from the spirit and scope of the present invention.

The present invention will be described in further detail below with reference to specific embodiments in order to enable those skilled in the art to better understand the present invention.

Example one

The Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) LXXJDF-1 is obtained by separating rhizosphere soil of healthy peanuts in the south of Juglans, and is already stored in China general microbiological culture Collection center (CGMCC) (China academy of sciences institute of microbiology, 3, North Cheng West Lu 1 institute of China, Tokyo ward Yangtze, Beijing city) at 21 days 6 and 2019, and the preservation number is CGMCC No. 18009. The bacillus amyloliquefaciens is obtained by diluting soil on an LB (Luzhou-juba) flat plate, sterilizing, coating and culturing at constant temperature, and can be selected from rhizosphere soil of the sciaeus hierarchia. 1. Isolation of Bacillus amyloliquefaciens LXJDF-1

Weighing 5g of rhizosphere soil of the Acorus sieboldii healthy peanuts, putting the soil into a triangular flask with 45ml of sterile water, uniformly mixing, and sequentially diluting the soil in a gradient manner by 10^-1-10^-6And (3) sucking 50 mu L of soil suspension with different dilution times respectively by using a micropipette to the LB plate, uniformly coating the soil suspension by using a sterilization coater, sealing the plate, and then inversely placing the plate in a constant-temperature incubator at 28 ℃ for culturing for 48 hours. And (3) selecting a batch of single colonies on the plate, drawing lines on an LB solid culture medium plate, and inversely placing the plate in a constant temperature incubator at 28 ℃ for culturing for 1-2 d. Screening different bacteria by a confronting culture method to inhibit peanut ralstonia solanacearum, and carrying out classification and identification on the selected inoculated bacteria.

2. Morphological observation of Bacillus amyloliquefaciens LXJDF-1

Referring to fig. 1, the colonies were circular, in which the colonies were milky translucent and the surface protrusions were semi-wet. The diameter is 1.2-2.1m, and the shape of the thallus is gram-positive bacteria and rod-shaped when observed by a microscope.

Bacillus amyloliquefaciens LXXJDF-1 is gram-positive and aerobic, grows weakly under anaerobic condition, has positive contact enzyme activity and positive V-P experiment, can degrade starch and xylan, and can utilize saccharides such as glucose, arabinose, mannose, xylose and the like.

3. Physiological and biochemical characteristics

TABLE 1 physio-biochemical characteristics of the active Strain LXJDF-1

Note: "+" indicates that the reaction result was positive; "-" indicates that the reaction result was negative.

4. 16S rRNA molecular identification of Bacillus amyloliquefaciens LXXJDF-1

Selecting bacterial strains with antagonistic activity on fungi, selecting 10 identical colonies, and extracting genome DNA by mainly referring to the instruction of a TIANGEN TIANAmp BACTERIA DNA Kit, wherein the steps are as follows:

(1) centrifuging 1mL of the bacterial culture solution at 10000rpm for 1min, and completely sucking the supernatant as much as possible;

(2) adding 200 mu L of buffer solution GA into the thallus precipitate, and shaking until the thallus is completely suspended;

(3) adding 4 μ LRNAase (100mg/mL) solution, shaking for 15s, standing at room temperature for 5 min;

(4) adding 20 mu L of proteinase K solution into the tube, and uniformly mixing;

(5) adding 220 μ L buffer solution GB, shaking for 15s, standing at 70 deg.C for 10min, centrifuging to remove water droplets on the inner wall of the tube cover;

(6) adding 220 μ L of anhydrous ethanol, shaking thoroughly, mixing for 15s to obtain flocculent precipitate, and centrifuging briefly to remove water drop on the inner wall of the tube cover;

(7) adding the solution and flocculent precipitate obtained in the previous step into an adsorption column GB3 (the adsorption column is put into a collecting pipe), centrifuging at 12000rpm for 30s, pouring off waste liquid, and putting the adsorption column CB3 into the collecting pipe;

(8) adding 500 μ L buffer GD (checking whether absolute ethanol is added before use) into adsorption column CB3, centrifuging at 12000rpm for 30s, pouring off waste liquid, and placing adsorption column CB3 into a collection tube;

(9) adding 700 μ L of rinsing solution PW (added with anhydrous ethanol before use) into adsorption column CB3, centrifuging at 12000rpm for 30s, pouring off waste liquid, and placing adsorption column CB3 into a collecting tube;

(10) putting 500 microliter rinsing liquid PW into an adsorption column CB3, centrifuging at 12000rpm for 30s, pouring off waste liquid, and putting the adsorption column CB3 into a collecting pipe;

(11) putting the adsorption column CB3 back into the collecting pipe, centrifuging at 12000rpm for 2min, pouring off waste liquid, and placing the adsorption column CB3 at room temperature for a plurality of minutes to thoroughly dry the residual rinsing liquid in the adsorption material;

(12) transferring the adsorption column CB3 into a clean centrifuge tube, suspending and dripping 50-200 mu L of elution buffer TE into the middle part of the adsorption film, standing at room temperature for 2-5min, centrifuging at 12000rpm for 2min, collecting the solution into the centrifuge tube, and storing at-20 ℃.

PCR and sequencing

(1) The extracted DNA was subjected to PCR amplification with reference to Kit instructions of 16s rDNA Bacterial Identification PCR Kit of TaKaRa, wherein the forward primers were: 5'-AGAGTTTGATCATGGCTCAG-3' (27F), the reverse primer is: 3 '-CGCTTACCTTGTTACGACTT-5' (1492R). The PCR amplification conditions were as follows: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 deg.C for 1 min; annealing at 53 deg.C for 1 min; extension at 72 ℃ for 90 s; extension was carried out for 5min at 72 ℃ for a total of 30 cycles. The PCR products were separated by electrophoresis on a 1% agarose gel, stained with EB, and observed under a 3 UVTMTransillementor (UVP, USA). Electrophoresis detection shows that the size of the 16s rDNA fragment of the LXXJDF-1 strain is about 1500bp, the size of the fragment accords with the expected design of a kit, and the amplification result is shown in SEQ ID NO. 1.

(2) According to the size of a pre-designed primer and an expected amplified fragment, combining a Marker, cutting a target fragment under an ultraviolet lamp, and recovering DNA by using a recovery Kit Silica Bead DNA GelExtraction Kit. 16s rDNA sequencing was performed by Qingdao Okagaku Biotechnology Co. The sequence determination result shows that the 16s rDNA fragment of the LXJDF-1 strain has 1511nt base pair composition, and the sequence table information is shown in SEQ ID NO. 1. The determined sequence was compared with the sequences in GenBank (NCBI, Website http:// BLAST. NCBI. nlm. nih. gov/BLAST. cgi) by BLAST program, and then the 16s rDNA sequence of the species and genus similar to the test strain sequence was obtained from GenBank for determination. The alignment result is shown in FIG. 2, and the result shows that the 16s rDNA sequence of Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) of Paenibacillus in the GenBank gene library of the active strain is highly homologous, and the homology rate reaches 100%. The results of the development tree construction and homology analysis show that as shown in FIG. 2, the strain LXXJDF-1 and Bacillus amyloliquefaciens (Accession No: MN795904) of Bacillus form a branch independently, the evolutionary distance is nearest, the genetic relationship between the strain and Bacillus amyloliquefaciens is reflected to be nearest, and the homology between the strain and the Bacillus amyloliquefaciens is 99% by analysis of DANMAN software. And determining the strain LXJDF-1 as the bacillus amyloliquefaciens by combining the results of the traditional physiological and biochemical characteristic identification and 16S rDNA sequence analysis.

Example two

Preparation of liquid microbial inoculum containing Bacillus amyloliquefaciens (LXJDF-1)

1. Activation of amylobacillus amyloliquefaciens (Bacillus amyloliquefaciens) LXXJDF-1 and preparation of seed liquid

Activation of Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) LXXJDF-1: a part of bacterial powder is picked up to an LB shaking flask (250mL shaking flask with the liquid filling amount of LB liquid culture medium being 50mL) from a freeze-drying tube of amyloliquefaciens (Bacillus amyloliquefaciens) LXXJDF-1 by using a sterile inoculating needle, and the LB culture medium becomes turbid after being cultured for 24 hours at 30 ℃ and 150 r/min. Taking a bacterial liquid to an LB solid culture medium for streak culture for 24-36 h, wherein the amylolytic Bacillus (Bacillus amyloliquefaciens) LXXJDF-1 grows well in the LB solid culture medium soil, and bacterial colonies are white or grey white, irregular in surface and opaque.

Preparing a first-level seed solution: and selecting a single colony on an LB solid culture medium to be inoculated into a first-stage seed shake flask, wherein the volume of the shake flask is 250mL, and the liquid loading capacity of the LB liquid culture medium is 50mla, and the first-stage seed shake flask is cultured for 24 hours at 30 ℃ at 150r/min to obtain a first-stage seed liquid.

Preparing a secondary seed solution: the formula (g/L) of the culture medium in the seeding tank is as follows: 10g of tryptone, 5g of yeast extract and 10g of NaCl, and the pH value is adjusted to 7.0 by using 5mol/L of NaOH. Inoculating the first-stage seed liquid into a seed tank, wherein the inoculation amount is 1-5% (V/V), and the culture conditions are as follows: the aeration ratio is 1:1, the stirring speed is 200r/min, the culture temperature is 30 ℃, the tank pressure is 0.0lMPa, and the culture time is 20 h. Obtaining secondary seed liquid with viable bacteria content of 1-3 × 10⁸cfu/mL, at which the cell viability was high.

2. Fermentation preparation of liquid microbial inoculum containing Bacillus amyloliquefaciens (LXJDF-1)

The formula (g/L) of the fermentation medium is as follows: 10g of tryptone, 5g of yeast extract and 10g of NaCl, and the pH value is adjusted to 7.0 by using 5mol/L of NaOH.

Placing the fermentation medium in a fermentation tank, and sterilizing.

Inoculating the second-stage seed liquid of Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) LXXJDF-1 into a fermentation tank according to the inoculation amount of 1-5% (V/V), wherein the culture conditions are as follows: the aeration ratio is 1:1, the stirring speed is 150r/min, the tank pressure is 0.0lMPa, the culture temperature is 30 ℃, and the culture time is 48h, so that the liquid microbial inoculum 1 is obtained. The content of viable bacteria in the liquid microbial inoculum 1 is 30 multiplied by 10⁸cfu/mL。

When the fermentation tank culture is finished, the viable bacteria content in the liquid microbial inoculum is ensured to be 5-100 multiplied by 10⁸cfuThe fermentation medium and the fermentation conditions may be adjusted on the premise of/mL.

The fermentation medium is LB medium.

LB culture medium formula (g/L): 10 parts of tryptic orange, 5 parts of yeast extract, 10 parts of NaCl10g and water as a solvent.

EXAMPLE III

Preparation of solid microbial inoculum containing Bacillus amyloliquefaciens (LXJDF-1)

The liquid microbial inoculum 1 (viable cell content 30X 10) prepared in example 1 was added⁸cfu/mL), and the weight ratio of the liquid microbial inoculum to the kaolin is 100: 25. And stirring the liquid-solid mixture for 30 minutes, standing for 60 minutes, and conveying the feed liquid into a centrifugal spray tower by using a conveying pump for drying to obtain the solid microbial inoculum 1. The drying conditions in the centrifugal spray tower were: the temperature of the air inlet is 200 ℃, and the temperature of the air outlet is 85 ℃. The water content in the solid microbial inoculum 1 is 4 percent (W/W), and the content of t bacteria is 60 multiplied by 10⁸cfu/g。

The drying conditions are to satisfy: the air inlet temperature is not higher than 250 ℃, and the air outlet temperature is not higher than 100 ℃.

The solid microbial inoculum needs to meet the following requirements: water content not higher than 5% (W/W), viable bacteria content of 1 × 10^6-1×10¹²cfu/g。

The liquid microbial inoculum can also be concentrated and filtered by a plate frame and then dried in vacuum to prepare various national microbial inocula.

Adding an ammonium sulfate aqueous solution accounting for 5 percent of the weight of the liquid microbial inoculum 1 into the liquid microbial inoculum 1, then adding calcium carbonate accounting for 3 percent of the weight of the liquid microbial inoculum and perlite accounting for 1 percent of the weight of the liquid microbial inoculum for assisting filtration, stirring for 30 minutes, standing for 60 minutes, and then pressing into a plate and frame filter for filtration. Wherein, the ammonium sulfate, the calcium carbonate and the perlite are filter aids.

The filtration conditions were: the filtration temperature was 40 ℃ and the filtration pressure was 4kg/cm²And the water content of the material filtered by the plate frame is kept at 30% (W/W).

Vacuum drying filter cake obtained by plate-frame filtration at 80 deg.C to obtain water content of 4% (W/W) and viable bacteria content of 120 × 10⁸cfu/g solids.

Adding the solid matter to the solid matterKaolin accounting for 4 times of the weight of the solid is added, sodium dodecyl benzene sulfonate accounting for 25 percent of the weight of the solid is added, and the solid microbial inoculum 2 can be obtained after the even mixing and the crushing. In the solid microbial inoculum 2, the content of viable bacteria is 22 multiplied by 10⁸cfu/g。

Example four

Determination of capability of bacillus amyloliquefaciens LXXJDF-1 strain in inhibiting ralstonia solanacearum

Selecting LXJDF-1 and peanut bacterial wilt pathogen (Pseudomonas solanacearum) single colonies, respectively inoculating in LB culture medium at 28 deg.C for 12 hr, respectively inoculating 100uL culture solution in LB culture medium at 20ml, respectively culturing LXJDF-1 and peanut bacterial wilt pathogen at 28 deg.C and 180r/min in shaker at concentration of 10⁶cfu/mL。

The experiment was divided into I, II and III groups.

Three LB plates were evenly spread with 50uL of peanut wilt disease, three groups I, II and III, on a sterile operating table. LXJDF-1 medium was diluted to 10^-5Respectively taking 3 circular filter papers with the diameter of 0.5cm by using tweezers to fully absorb the diluted LXXJDF-1 culture solution, placing the LXXJDF-1 culture solution in group I LB culture medium containing ralstonia solanacearum, and carrying out inverted culture in a greenhouse at 28 ℃ for 2-3 days; LXJDF-1 medium was diluted to 10^-5Respectively taking 3 circular filter papers with the diameter of 0.5cm by using tweezers to fully absorb the diluted LXXJDF-1 culture solution, placing the LXXJDF-1 culture solution in a group II LB culture medium containing ralstonia solanacearum, and carrying out inverted culture in a greenhouse at 28 ℃ for 2-3 days; taking 3 circular filter papers with diameter of 0.5cm with tweezers to fully absorb clear water, placing in group III LB culture medium containing Ralstonia solanacearum, and performing inverted culture in a greenhouse at 28 deg.C for 2-3 days.

Obvious inhibition zones appear around the filter paper dipped with the LXXJDF-1 culture solution, namely the group I and the group II, the diameter of the inhibition zone is 3cm, and as shown in figures 3 and 4, no inhibition zone appears around the filter paper dipped with clear water.

EXAMPLE five

Experiment for pot culture control of LXXJDF-1 strain on peanut ralstonia solanacearum

The experiments were divided into groups A, B and C.

And (3) treating group A bacteria: taking peanut seeds of No. 20, accelerating germination in culture dish, and planting to a container with sterilized water4 seeds are planted in each flowerpot, 20 peanuts are inoculated, the seedlings grow to 5-6 leaves, the roots of the seedlings are irrigated with bacillus amyloliquefaciens LXJDF-1, and the concentration of the bacterial suspension reaches 10⁶cfu/mL, 10mL per strain.

Group A clear water control: another 20 peanuts are taken to be germinated in a culture dish and then planted in flowerpots filled with sterilized soil, 4 seeds are planted in each flowerpot, 20 peanuts are inoculated in total, and 15mL of clear water is added after the seedlings grow to 5-6 leaves.

After 48h of group A fungus treatment and clear water control, the fungus is inoculated with the fungus liquid of peanut, 5d later, the growth and disease condition of peanut plants are recorded, and the observation is continued for 15 days.

Control effect%

Table 2: potted plant control effect of LXJDF-1 strain on peanut ralstonia solanacearum

Treatment of	Incidence (%)	Control effect (%)
			Group A: LXJDF-1	50.0	42.3
Group A: clear water control	85.0	0
			Group B: LXJDF-1	40.0	53.8
Group B: clear water control	90.0	0
			Group C: LXJDF-1	35.0	59.6
Group C: control	85.0	0

From the experimental results it can be seen that: the three groups of pot experiment results show that the LXJDF-1 strain has good prevention and treatment effects on ralstonia solanacearum, the prevention and treatment effects are 42.3% of group A, 53.8% of group B and 59.6% of group C respectively, the average prevention and treatment effect is 51.9%, and the LXJDF-1 strain has good application prospects for prevention and treatment of peanut bacterial wilt.

While specific embodiments of the invention have been described above, it will be appreciated by those skilled in the art that these are by way of example only, and that the examples will assist those skilled in the art in further understanding the invention, but are not intended to limit the invention in any way. It will be apparent to those skilled in the art that various changes and modifications can be made without departing from the spirit and scope of the invention. The present invention is not limited to the above-mentioned preferred embodiments, and any other products in various forms can be obtained by anyone in the light of the present invention, but any changes in the shape or structure thereof, which have the same or similar technical solutions as those of the present application, fall within the protection scope of the present invention.

Sequence listing

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agagtttgat cctggctcag gacgaacgct ggcggcgtgc ctaatacatg caagtcgagc 60

ggacagatgg gagcttgctc cctgatgtta gcggcggacg ggtgagtaac acgtgggtaa 120

cctgcctgta agactgggat aactccggga aaccggggct aataccggat ggttgtctgg 180

accgcatggt tcagacataa aaggtggctt cggctaccac ttacagatgg acccgcggcg 240

cattagctag ttggtgaggt aacggctcac caaggcgacg atgcgtagcc gacctgagag 300

ggtgatcggc cacactggga ctgagacacg gcccagactc ctacgggagg cagcagtagg 360

gaatcttccg caatggacga aagtctgacg gagcaacgcc gcgtgagtga tgaaggtttt 420

cggatcgtaa agctctgttg ttagggaaga acaagtgccg ttcaaatagg gcggcacctt 480

gacggtacct aaccagaaag ccacggctaa ctacgtgcca gcagccgcgg taatacgtag 540

gtggcaagcg ttgtccggaa ttattgggcg taaagggctc gcaggcggtt tcttaagtct 600

gatgtgaaag cccccggctc aaccggggag ggtcattgga aactggggaa cttgagtgca 660

gaagaggaga gtggaattcc acgtgtagcg gtgaaatgcg tagagatgtg gaggaacacc 720

agtggcgaag gcgactctct ggtctgtaac tgacgctgag gagcgaaagc gtggggagcg 780

aacaggatta gataccctgg tagtccacgc cgtaaacgat gagtgctaag tgttaggggg 840

tttccgcccc ttagtgctgc agctaacgca ttaagcactc cgcctgggga gtacggtcgc 900

aagactgaaa ctcaaaggaa ttgacggggg cccgcacaag cggtggagca tgtggtttaa 960

ttcgaagcaa cgcgaagaac cttaccaggt cttgacatcc tctgacaatc ctagagatag 1020

gacgtcccct tcgggggcag agtgacaggt ggtgcatggt tgtcgtcagc tcgtgtcgtg 1080

agatgttggg ttaagtcccg caacgagcgc aacccttgat cttagttgcc agcattcagt 1140

tgggcactct aaggtgactg ccggtgacaa accggaggaa ggtggggatg acgtcaaatc 1200

atcatgcccc ttatgacctg ggctacacac gtgctacaat ggacagaaca aagggcagcg 1260

aaaccgcgag gttaagccaa tcccacaaat ctgttctcag ttcggatcgc agtctgcaac 1320

tcgactgcgt gaagctggaa tcgctagtaa tcgcggatca gcatgccgcg gtgaatacgt 1380

tcccgggcct tgtacacacc gcccgtcaca ccacgagagt ttgtaacacc cgaagtcggt 1440

gaggtaacct tttaggagcc agccgccgaa ggtgggacag atgattgggg tgaagtcgta 1500

acaaggtagc c 1511

Claims

1. Bacillus amyloliquefaciens (A)Bacillus amyloliquefaciens) The application of LXJDF-1 in inhibiting ralstonia solanacearum of peanuts and/or preventing and treating bacterial wilt of peanuts is characterized in that: bacillus amyloliquefaciens (A)Bacillus amyloliquefaciens) The preservation number of the LXJDF-1 is CGMCC No. 18009.

2. Bacillus amyloliquefaciens (A)Bacillus amyloliquefaciens) The application of LXJDF-1 in preparing bacterial agents for inhibiting peanut bacterial wilt and/or preventing peanut from producing bacterial wilt is characterized in that: using Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) LXJDF-1 is an active ingredient, the bacillus amyloliquefaciens (A)Bacillus amyloliquefaciens) The preservation number of the LXJDF-1 is CGMCC No. 18009.

3. The Bacillus amyloliquefaciens of claim 2Bacillus amyloliquefaciens） LXJDF-1 application in preparing bacterial agent for inhibiting peanut bacterial wilt and/or preventing peanut from producing bacterial wilt, which is characterized in that: bacillus amyloliquefaciens in liquid microbial inoculumBacillus amyloliquefaciens) The content of LXJDF-1 is 1 x 10⁷-1×10¹¹cfu/ml。

4. The Bacillus amyloliquefaciens of claim 2Bacillus amyloliquefaciens) The application of LXJDF-1 in preparing bacterial agents for inhibiting peanut bacterial wilt and/or preventing peanut from producing bacterial wilt is characterized in that: bacillus amyloliquefaciens in solid microbial inoculumBacillus amyloliquefaciens) The content of LXJDF-1 is 1 x 10⁶-1×10¹²cfu/ml。

5. The Bacillus amyloliquefaciens of claim 4 (C)Bacillus amyloliquefaciens) The application of LXJDF-1 in preparing bacterial agents for inhibiting peanut bacterial wilt and/or preventing peanut from producing bacterial wilt is characterized in that: the solid microbial inoculum also contains an adsorption matrix, wherein the adsorption matrix is a carrier or a mixture of the carrier and an auxiliary agent, the carrier is one or a mixture of two or more of kaolin, light calcium carbonate, diatomite and white carbon black, and the auxiliary agent is one or a mixture of two or more of sodium dodecyl benzene sulfonate, sodium lignin sulfonate and sodium alkyl naphthalene sulfonate polycondensate.

6. The Bacillus amyloliquefaciens of claim 3 (C)Bacillus amyloliquefaciens) The application of LXJDF-1 in preparing bacterial agents for inhibiting peanut bacterial wilt and/or preventing peanut from producing bacterial wilt is characterized in that: mixing Bacillus amyloliquefaciens (A), (B) and (C)Bacillus amyloliquefaciens) LXJDF-1 is inoculated in a culture medium and cultured for 24-72 hours at the temperature of 28-30 ℃ to obtain a liquid microbial inoculum.

7. The Bacillus amyloliquefaciens of claim 6 (C.), (C. and (C.), (C. and (C.), (C. in accordance with claim 6)Bacillus amyloliquefaciens) The application of LXJDF-1 in preparing bacterial agents for inhibiting peanut bacterial wilt and/or preventing peanut from producing bacterial wilt is characterized in that:the culture medium is 10g of tryptone, 5g of yeast extract and 10g of NaCl, and the pH value is adjusted to 7.0 by NaOH.

8. The Bacillus amyloliquefaciens of claim 4 or 5Bacillus amyloliquefaciens) The application of LXJDF-1 in preparing bacterial agents for inhibiting peanut bacterial wilt and/or preventing peanut from producing bacterial wilt is characterized in that: bacillus amyloliquefaciens (A), (B) and (C)Bacillus amyloliquefaciens) LXJDF-1 is inoculated in a culture medium and cultured for 24h-72h at the temperature of 28-30 ℃ to obtain a liquid microbial inoculum; adding an adsorption matrix into the liquid microbial inoculum, and drying to obtain the solid microbial inoculum, or filtering and drying the liquid microbial inoculum, adding the adsorption matrix, mixing and crushing to obtain the solid microbial inoculum.