CN112746043B

CN112746043B - Bacillus belgii K01 and application thereof

Info

Publication number: CN112746043B
Application number: CN202110114861.2A
Authority: CN
Inventors: 郭岩彬; 邢明振; 张云鸽; 王秀艳; 柴同海; 吕硕; 李伟欣
Original assignee: Genliduo Bio Tech Corp ltd
Current assignee: Genliduo Bio Tech Corp ltd
Priority date: 2021-01-26
Filing date: 2021-01-26
Publication date: 2021-09-17
Anticipated expiration: 2041-01-26
Also published as: CN112746043A

Abstract

The invention provides a Bacillus belezii K01 strain and application thereof, wherein the strain K01 not only has the growth promoting functions of phosphate solubilizing, indoleacetic acid production, cellulase production, protease production and the like, but also can produce antibacterial active substances and volatile antibacterial substances, can inhibit the growth of various pathogenic bacteria, has a good biocontrol function, can well colonize in and at the rhizosphere of plants, can be applied to plant growth promotion and biological control of plant diseases, and has a good application prospect.

Description

Bacillus belgii K01 and application thereof

Technical Field

The invention relates to the technical fields of microbiology, biotechnology and biological control, and particularly relates to a bacillus beiLeisi K01 and application thereof.

Background

The bacillus is a gram-positive bacterium which is aerobic or facultative anaerobic and can produce spores, and the thallus is in a straight rod shape and is arranged in pairs or chains. Compared with other microorganisms, the bacillus has the most outstanding characteristics that spores with strong stress resistance can be generated in a severe environment to overcome poor environmental conditions, the bacillus is ubiquitous in nature, harmless to people and livestock and non-pathogenic to plants, and most of the bacillus can also generate various antibacterial active substances. Therefore, bacillus is widely researched and applied as a model research species, and becomes one of the most deeply researched and widely applied plant growth promoting and biocontrol bacteria in the world at present.

Bacillus can affect the growth and development of plants in two ways, directly or indirectly: direct effects include stimulating plant growth, improving nutrient uptake, seed germination, etc. by producing plant regulators; indirect effects include indirect enhancement of plant growth by helping plants fight abiotic stress, competing for nutrient-inhibiting pathogens, producing diffusible antibacterial compounds and releasing bacterial Volatile compounds (VOCs) (Qu et al 2020).

In the research of preventing and treating plant diseases, the bacillus has the advantages of high safety evaluation, good prevention and treatment effect and easy preparation. Currently, the types of bacillus suitable for pathogen control are mainly: bacillus subtilis, Bacillus amyloliquefaciens, Bacillus methylotrophicus, Bacillus licheniformis, Bacillus pumilus, Bacillus megaterium, and Bacillus firmus (Fan, Blum et al.2017). Meanwhile, with the progress of research on Bacillus, more and more new Bacillus species with antibacterial activity are found, such as Bacillus belief (Bacillus velezensis) and the like.

In recent years, researches report that Bacillus belgii can prevent and treat plant diseases, but most of the bacterial inhibition spectrums are narrow; for example, the Cao Weiping et al (2020) reports that Bacillus belezii Hsg1949 has good application to prevention and treatment of peanut fruit rot, the Yi Cheng Mi et al (2020) reports that Bacillus belezii XC1 has good application to apple continuous cropping obstacle, the research reports that Bacillus belezii can generate volatile substances to inhibit pathogenic bacteria, for example, Bacillus belezii JS25R can generate volatile substances to inhibit the thallus growth (the inhibition rate is 27.7%) and spore germination (the inhibition rate is 26.7%) (Yinzong et al, 2017) of Fusarium graminearum, and the research on other aspects of phosphorus dissolving, growth promotion, enzyme production and the like of the Bacillus belezii is less, for example, the Zhang Kantong et al (2020) reports that the Bacillus belezii KY01 has application to degradation of kitchen garbage. However, no bacterial strain with broad-spectrum antibacterial activity, multiple disease prevention and control and plant growth promotion is reported at present, and particularly the bacterial strain can play a role in promoting growth under the adverse environments such as high salinity and the like.

Disclosure of Invention

The invention aims to provide a broad-spectrum disease-resistant growth-promoting Bacillus belgii K01 and application thereof, and particularly relates to application of the strain in plant growth promotion and biological control of plant diseases.

In order to achieve the purpose of the invention, in a first aspect, the invention provides a strain of Bacillus belief that has a biological control effect and an application thereof, wherein the Bacillus belief (Bacillus velezensis) K01 is separated from the company chenchentai city, north river in 2012 and is currently deposited in the center of typical culture collection of china, addresses: wuhan, Wuhan university, zip code 430072, preservation number CCTCC NO: m2020871, preservation date 2020, 12 months and 8 days.

In a second aspect, the present invention provides a bacterial agent comprising Bacillus belgii K01.

In a third aspect, the present invention provides a growth promoting substance secreted by bacillus belgii K01, the growth promoting substance comprising IAA (indoleacetic acid) and a bacteriostatic active substance (volatile bacteriostatic substance). The antibacterial active substance can resist 100 deg.C high temperature, ultraviolet irradiation, and pH of 1.0-11.0.

In a fourth aspect, the invention provides enzymes secreted by bacillus belgii K01, including but not limited to cellulases, proteases.

In a fifth aspect, the present invention provides a biopesticide, biofertilizer, phosphorus activator or plant growth promoter prepared from bacillus belgii K01 or a microbial agent thereof.

In a sixth aspect, the present invention provides a biocontrol agent, an antibacterial agent, a preservative, a disinfectant or a food preservative prepared from Bacillus belgii K01.

In a seventh aspect, the present invention provides any one of the following uses of bacillus belgii K01 or its microbial inoculum:

1) for promoting plant growth and development and increasing crop yield;

2) the plant stress resistance is improved;

3) dissolving phosphorus;

4) for the preparation of biofertilizers;

5) used for preparing a phosphorus activator;

6) for the preparation of plant growth promoters;

7) is used for preparing biological pesticide.

Preferably, the plant includes, but is not limited to, tomato, corn, pepper, sunflower, sugar beet.

In an eighth aspect, the invention provides any one of the following uses of bacillus beijerinckii K01 and/or a bacteriostatic active substance produced therefrom:

a) used against pathogenic bacteria and related diseases or plant diseases caused by pathogenic bacteria;

b) for inducing plants to increase resistance to diseases or plant diseases caused by pathogenic bacteria;

c) can be used for preparing biocontrol agents, antibacterial agents, antiseptics, plant immunity inducing agents, disinfectants or food preservatives.

The pathogenic bacteria may be selected from Botrytis cinerea, Fusarium oxysporum (Fusarium oxysporum), Alternaria solani (Alternaria solani), Ceratophyllum microphyllum (Ceratophyllum oxysporum), Fusarium moniliforme (Fusarium moniliforme), Rhizoctonia lilacinus (Fusarium oxysporum.sp.Lili), Fusarium oxysporum (Fusarium oxysporum.sp.niveum), Monilinia fructicola (Monilinia laxa), Rhizoctonia cerealis (Rhizoctonia cerealis), Rhizoctonia solani (Rhizoctonia solani), Xanthomonas gossypii (Verticillium dahliae), Rhizoctonia cerealis (Septoria nigra), anthrax (Colotrichum), Helianthus annuus (Leptospira), and Rhizoctonia solani (Rhizoctonia solani), and the like.

In a ninth aspect, the invention provides the use of bacillus beilaisi K01 or its microbial inoculum for dissolving insoluble phosphorus under high salt conditions. The high-salt condition means that the concentration of NaCl is 13-15%.

In a tenth aspect, the invention provides a preparation process of a bacillus beiLeisi K01 microbial inoculum, which comprises the following steps:

1) preparing a seed solution: inoculating the activated Bacillus belgii K01 into a seed culture medium for culture; the inoculation amount is 1.2 percent, the culture temperature is 28 ℃, the rotating speed is 200rpm, and the culture time is 12 hours;

wherein the seed culture medium comprises the following components: peptone 12g/L, yeast extract 5g/L, glucose 10g/L, NaCl 10g/L, pH7.2;

2) fermentation culture: inoculating the cultured seed solution into a 10-ton fermentation tank according to the proportion of 5% v/v, wherein the liquid loading amount is 70%, the fermentation temperature is 37 ℃, the stirring speed is 150rpm, the ventilation quantity is adjusted to ensure that the dissolved oxygen is more than 50%, the tank pressure is 0.05MPa, the fermentation time is 24h, the fermentation is stopped when the spore amount reaches more than 95%, and the temperature is reduced to 20 ℃;

wherein the fermentation medium comprises the following components: soybean powder 20g/L, potato starch 15g/L, yeast extract powder 2.5g/L, peptone 3.0g/L, sucrose 5g/L, KH₂PO₄ 0.6g/L，MgSO₄·7H₂O 0.08g/L，MnSO₄·7H₂O 0.02g/L，(NH₄)₂SO₄5g/L, 1.2g/L of polyether defoamer, and pH7.2;

3) preparation of microbial inoculum

Liquid preparation: diluting the fermentation liquor obtained in the step 2) with sterile water according to a certain proportion, and then carrying out sterile filling to prepare a liquid microbial inoculum; alternatively, the first and second electrodes may be,

powder preparation: concentrating the fermentation liquid obtained in the step 2) by 3 times by using a centrifugal machine, adding 5% of protective agent, stirring for 30min, drying by using a centrifugal spray drying tower, setting the air inlet temperature to be 160 ℃ and the air outlet temperature to be 75 ℃, collecting dry powder, and diluting the dry powder to the required concentration by using zeolite powder;

wherein the protective agent is: dextrin, soluble starch and diatomite in the weight ratio of 70 to 25 to 5.

The Bacillus belgii K01 genome contains multiple antibacterial substance gene clusters, can generate multiple antibacterial substances, has a wide antibacterial spectrum, has an inhibition effect on sixteen pathogenic bacteria, can play a role in biocontrol in the plant growth process, can inhibit the pathogenic bacteria by spraying after fruits are picked, and can reduce fruit rot, so that the quality guarantee period of the fruits is prolonged; the K01 strain can stably colonize plant root systems, can mutualize with plants, and can also produce volatile antibacterial substances, so that the field growth promotion and biocontrol effects of the K01 strain are very obvious. The strain K01 has good application value in the aspects of plant growth promotion, disease biocontrol and the like.

Strain K01 is resistant to Botrytis cinerea (Botrytis cinerea), Fusarium oxysporum (Fusarium oxysporum), Alternaria solani (Alternaria solani), Ceratophyllus microphyllus (Ceratophylla micrantis), Fusarium moniliforme (Fusarium moniliforme), Rhizopus lilacinus (Fusarium oxysporum.sp.Lili), Fusarium oxysporum (Fusarium oxysporum.sp.niveum), Monilinia persica (Monilinia laxa), Rhizoctonia cerealis (Rhizoctonia cerealis), Rhizoctonia solani (Rhizoctonia solani), Rhizoctonia gossypii (Verticillium dahliae), Rhizoctonia solani (Sepiella apiacea), Bacillus anthracis (Colostreatus), Helianthus annuus (Leptosporium nigra solani), and Rhizoctonia solani (Rhizoctonia solani) with a solani (Rhizoctoniensis) rates of 50% -97%. In vitro leaf, fruit and field experiments prove that the K01 fermentation liquor has 78 to 100 percent of control effect on pepper gray mold and tomato gray mold caused by Botrytis cinerea; the control effect of Rhizoctonia solani (Rhizoctonia cerealis) on wheat sharp eyespot reaches 62.01%, the control effect of sunflower Sclerotinia sclerotiorum (sclerotiniorum) on sunflower detached leaves reaches 96%, and the control effect of peach brown rot (Monilinia laxa) on peach and cherry fruits reaches 89% -87%; the volatile bacteriostatic substance pair produced by K01 has a wide bacteriostatic spectrum, the inhibitory rate of the volatile bacteriostatic substance pair on twelve pathogenic fungi reaches 14.11-46.46%, and the K01 strain on pepper leaves has 70% of effect of preventing botrytis cinerea of pepper through fumigation. Besides, K01 can dissolve insoluble phosphorus, produce auxin (IAA), cellulase, protease and the like, and can well colonize tomato roots and corn roots, the dry stem weight and the dry root weight of corn seedlings soaked in K01 are respectively increased by 33.33% and 36.33% compared with those of a control group, and the control effects on sunflower sclerotiniose and beet root rot in a field demonstration test are respectively 23.29% + -1.50% and 54.76% + -14.90%. The Bacillus belgii K01 has a very good application prospect in the aspects of biological fertilizers and biological pesticides.

Drawings

FIG. 1 shows the colony morphology of K01 in the preferred embodiment of the present invention.

FIG. 2 is a transmission electron micrograph of K01 according to the preferred embodiment of the present invention.

FIG. 3 is a phylogenetic tree of K01 strain based on 9 housekeeping genes such as 16S rRNA, atpD, gyrA, topA, gyrB, recN, infB, uvrC, ftsZ, etc. in the preferred embodiment of the present invention.

FIG. 4 is a diagram showing the detection and analysis of the functional gene of strain K01 in the preferred embodiment of the present invention; wherein, M: DNA molecular weight standard; 1: negative control (ddH)₂O); 2: fend gene; 3: the ituD gene; 4: the srfA gene; 5: the bacC1 gene; 6: the bmyB gene; 7: the spaS gene.

FIG. 5 shows the gene cluster of K01 metabolite synthase in accordance with a preferred embodiment of the present invention.

FIG. 6 shows the growth of K01 in different salt concentrations in the preferred embodiment of the present invention.

FIG. 7 shows the growth of K01 in LB medium with 13% NaCl in a preferred embodiment of the present invention.

FIG. 8 shows the phosphorus-dissolving cycle of K01 and the phosphorus-dissolving cycle under the condition of high salt content in the preferred embodiment of the present invention.

FIG. 9 shows the ability of K01 to produce protease in a preferred embodiment of the invention.

FIG. 10 shows the cellulase production ability of K01 in the preferred embodiment of the present invention.

FIG. 11 shows the growth promoting effect of K01 on young corn plants in the preferred embodiment of the present invention.

FIG. 12 shows the bacteriostatic spectrum of K01 in the preferred embodiment of the invention.

FIG. 13 shows the control effect of K01 on Botrytis cinerea on Piper kaki fruits according to the preferred embodiment of the present invention; wherein, from top to bottom are: k01 bacterial liquid, medicament control and positive control.

FIG. 14 shows the control effect of K01 on Botrytis cinerea on pepper fruits according to the preferred embodiment of the present invention; wherein, from left to right in sequence: k01 bacterial liquid, medicament control and positive control.

FIG. 15 shows the control effect of K01 on Botrytis cinerea on the Zanthoxylum piperitum leaves in the preferred embodiment of the present invention; wherein, from top to bottom are: k01 bacterial liquid, medicament control and positive control; the left side was taken with normal light and the right image was taken with black light.

FIG. 16 shows the control effect of K01 on Botrytis cinerea on tomato leaves in the preferred embodiment of the present invention.

FIG. 17 shows the control effect of K01 on tomato gray mold in the preferred embodiment of the present invention.

FIG. 18 shows the control effect of K01 on peach brown rot in the wild peach fruit according to the preferred embodiment of the present invention.

FIG. 19 is a graph showing the control effect of K01 cherry fruit on peach brown rot in the preferred embodiment of the present invention.

FIG. 20 shows the control effect of K01 on Rhizoctonia cerealis on wheat seedlings in the preferred embodiment of the present invention.

FIG. 21 shows the control effect of K01 on sunflower sclerotia in sunflower leaves according to the preferred embodiment of the present invention.

FIG. 22 shows the inhibition spectrum of K01 (the left of each pathogen is control, and the right is the inhibition result of K01) as a volatile bacteriostatic substance in the preferred embodiment of the present invention.

FIG. 23 is a graph showing the effect of K01 bacteriostatic agents on inhibiting Botrytis cinerea infection of pepper leaves in a preferred embodiment of the present invention.

FIG. 24 shows the biofilm formed by K01 in the preferred embodiment of the invention.

FIG. 25 is a graph showing the colonization of K01 on corn roots in a preferred embodiment of the present invention.

FIG. 26 is the colonization of tomato rhizosphere and soil by K01 in the preferred embodiment of the present invention; wherein, 1-soil and 2-tomato rhizosphere.

FIG. 27 shows the sensitivity of K01 active substance to different temperatures in a preferred embodiment of the present invention.

FIG. 28 shows the sensitivity of K01 active substance to UV light in a preferred embodiment of the present invention.

FIG. 29 is a graph showing the pH sensitivity of K01 actives in accordance with a preferred embodiment of the present invention.

FIG. 30 shows the inhibition of bacteria by K01 in a preferred embodiment of the invention.

FIG. 31 is a graph of K01 enhancing resistance of tomato seedlings to Botrytis cinerea in a preferred embodiment of the invention.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are commercially available products.

The strains and sources referred to in the following examples are as follows:

EXAMPLE 1 isolation screening and identification of Strain K01

1. Isolation and screening of Strain K01

Collecting soil from the Shichen city of Hebei in 2012, adding 10g of the soil sample into a triangular flask filled with 100ml of sterile physiological saline, standing for 20min, shaking with a shaking table at 28 deg.C and 200rpm for 30min, adding 1ml of the soil sample into 9ml of sterile physiological saline, and sequentially diluting by 10²，10³，10⁴Taking 100 mul of the soil suspension, respectively, uniformly coating the soil suspension on an LB (LB) culture medium (5 g/L of yeast powder, 10g/L of peptone, 5g/L of NaCl and 15g/L of agar) plate, culturing for 48h in an incubator at 28 ℃, picking out single bacterial colonies with different shapes by using a sterile toothpick, streaking and purifying, and storing for later use. A total of 892 strains of bacteria were isolated.

Adopting a confrontation culture method to detect the biocontrol effect of the separated strains, firstly activating Botrytis cinerea (Botrytis cinerea) on a PDA plate, making a pathogenic bacteria cake by using a 5mm puncher, inoculating the pathogenic bacteria cake to the center of the PDA plate, respectively inoculating 892 strains separated from the PDA plate at a position 2.5cm away from the bacteria cake, culturing for 4-7 days at 25 ℃, respectively measuring a control diameter and an inhibition diameter, and calculating the inhibition rate according to a formula I):

158 bacillus with inhibiting effect on gray mold are primarily screened out from 892 strains, and 5 strains with inhibiting effect more than 40% are secondarily screened out, wherein the bacteriostasis rate is shown in Table 1. The K01 has the best bacteriostatic effect, and K01 is selected for subsequent experiments.

TABLE 1 bacteriostatic ratio of the strains

Strain name	K01	K36	K128	K69	K77
						Inhibition rate/%)	53.76±1.23	47.12±1.72	46.22±1.65	42.87±0.86	40.94±2.49

2. Identification of strains

The strain K01 is identified as Bacillus velezensis (Bacillus velezensis) by integrating the physiological and biochemical characteristics and the molecular biological detection result. The method comprises the following specific steps:

(1) morphological characteristics of bacterial colony and bacterial cell

After the strain K01 is cultured on an LB solid medium at 28 ℃ for 24h, a milky colony is formed, the edge is regular, the shape is nearly circular, the shape is slightly convex, and the surface is wet (figure 1). K01 was inoculated on LB medium, and the medium was shaken for 12 hours, and a drop of the resulting suspension was pipetted onto a copper mesh with a clean pipette, and the resulting suspension was air-dried and observed by a Transmission Electron Microscope (TEM) (H7500, Hitachi, Japan). The transmission electron micrograph is shown in FIG. 1. K01 has long-rod shape, length of about 2-3 μm, width of 0.5-1 μm, and circumferential flagellum.

(2) Physiological and biochemical characteristic identification

The physiological and biochemical characteristics were tested using a merriella VITEK2 Compact fully automated bacterial identification and susceptibility analysis system. The biochemical characteristics of 46 carbon source utilization, inhibition and drug resistance tests and biochemical tests of enzyme activity are measured by using a BCL biochemical identification card, and the result is shown in Table 2, and the identification result is Bacillus (Bacillus).

TABLE 2 physiological and biochemical test results of Strain K01

Note: "+" and "-" indicate positive and negative reactions, respectively.

(3) K01 Whole genome sequencing

The biocontrol strain B.velezensis K01 is cultured in LB culture medium at 37 ℃ for 12h at 200r/min, then the strain is centrifugally collected at 1000g, the total DNA of the genome is extracted, and after the DNA purity is detected to be qualified, sequencing analysis is entrusted to Beijing and kang biotechnology Limited. Sequencing was accomplished using a combination of traditional Sanger sequencing and IlluminaSolexa high throughput sequencing. 16S rRNA, atpD, gyrA, topA, gyrB, recN, infB, uvrC and ftsZ9 housekeeping genes are selected from a K01 genome to carry out phylogenetic evolution analysis, and an evolutionary tree is constructed.

The K01 genome size was 3927799bp, the G + C content was 46.5%, there were 3657 genes in total, the coding region accounted for 87.2% of the genome, there were 9 ribosomal RNA operons, and 86 transfer RNAs (tRNAs) (Table 3).

TABLE 3 genomic information of Strain K01

The 16S rRNA, atpD, gyrA, topA, gyrB, recN, infB, uvrC, ftsZ9 housekeeping genes are shown in Table 4, and the nucleotide sequences are shown in SEQ ID NO: 1-9. According to the whole genome sequencing result and the combination of the evolutionary tree and the physiological and biochemical analysis result, the strain K01 is identified as Bacillus velezensis (Bacillus velezensis), and the result is shown in figure 3.

TABLE 4 16S rRNA, atpD, gyrA, topA, gyrB, recN, infB, uvrC, ftsZ Gene information of Strain K01

3. Genomic analysis of Strain K01

(1) PCR detection and sequence analysis of K01 antibiotic substance synthesis related gene

6 pairs of primers are designed according to the methods of Joshi (Joshi, et al, 2006) and Isabel Mora (Isabel Mora et a1., 2011) and the like, the genomic DNA of a strain is obtained according to the operation method of the bacterial genomic DNA extraction kit, the genomic DNA of the strain K01 is used as a template for PCR amplification, and whether the genomic DNA contains related genes for antibacterial substance synthesis is screened, wherein the related genes mainly comprise srfAA (surfactin), fend (fengycin), bacC1 (lysobactin), ituD (itu-in), bmyA (bacillomycin D Bacilomycin D) and spa (subtilin). A total of 5 DNA bands were obtained by electrophoretic detection analysis, the size of the target fragment was correct and was as expected (FIG. 4), and the amplification product was purified and sequenced, which revealed that the strain K01 contained genes related to the synthesis of antibacterial substances such as toyocin, surfactin, iturin, lysobactin, and baccatin D.

(2) Metabolite synthetase gene cluster analysis

Through PCR sequencing results and genome sequence analysis, the main genes of each secondary metabolism antibacterial substance synthetase are shown in Table 5; mapping K01 secondary metabolite genes by DNAMAN software, 8 antibacterial substance synthetase gene cluster maps are drawn and shown in figure 5.

The similarity rate of the fenC gene in the fengycin synthetase gene of K01 and the fenC gene of international strain FZB42 is 97%; the similarity rate of the bmYC gene in the Bacillus D synthetase gene of K01 and the bmYC gene of the international strain FZB42 is 75%, 82% and 97% respectively. It can be seen that the K01 strain produced an antibacterial substance different from the reported Bellis strain. The nucleotide sequences of the bmYB and bmYC genes of K01 are respectively shown in SEQ ID NO: 10-11.

TABLE 5K 01 Synthesis of Key genes for antibacterial substances

EXAMPLE 2 determination of salt tolerance of Strain K01

Firstly, the strain K01 is activated on LB liquid culture medium, and the activated strain is inoculated to the strain containing 5%, 9%, 13%, 15%, 17% NaCl (w/v) according to the inoculation amount of 1%In LB liquid medium, shaking at 28 deg.C and 150rpm for 48h, and determining OD₆₀₀The value is obtained. Meanwhile, LB medium was not inoculated as a Control (CK), and each treatment was repeated 3 times. The results show that the growth of the strain K01 is not obviously inhibited in the culture medium with the salt concentration below 13%, and the significant inhibition is only observed in the 15% NaCl solution (figure 6), which shows that the strain K01 can well grow in the environment with the salt content of 13% (figure 7).

Example 3 detection of phosphorus solubilizing function of Strain K01

The strain K01 was first activated on LB medium. The activated strain was inoculated into phosphorus-solubilizing medium (glucose 10.00g, (NH) shown in appendix A of NY/T1847-2010₄)₂SO₄ 0.50g，MnSO₄·7H₂O 0.3g，NaCl 0.3g，KCl 0.30g，FeSO₄·4H₂O 0.036g，MnSO₄.4H₂0.03g of O, 1000mL of distilled water and pH 7.0; the phosphorus source is calcium phytate, and the addition amount is 2 g; solid medium: add 1.5% agar powder in proportion). And detecting whether the phosphorus-dissolving function is achieved, wherein K01 has the capacity of dissolving phosphorus, and the diameter (D) of a dissolving ring of the phosphorus-dissolving function reaches 16.07 +/-0.48 mm (figure 8).

After the phosphorus dissolving function of the strain is determined, the phosphorus dissolving capacity of the strain is quantitatively determined, and the content of the effective phosphorus in the fermentation supernatant is determined by a molybdenum-antimony colorimetric resistance method. After activating the strain K01 on an LB liquid culture medium, inoculating the strain onto a liquid phosphorus dissolving culture medium, shaking at 28 ℃ and 150rpm for 7 days, centrifuging the strain liquid at 12000rpm and 4 ℃ for 5min, and leaving a supernatant. The content of soluble phosphorus in the supernatant was determined by molybdenum-antimony colorimetry (auspicious spot, 2008). And (3) preparing a standard curve: weighing KH dried at 105 deg.C for 2 hr₂PO₄0.2195g are dissolved in 400ml water, 5m 1g of concentrated H are added₂SO₄Transferring (analytically pure) into a 1L volumetric flask, adding water to a constant volume, and storing the stock solution for a long time. 25.0ml of phosphorus standard stock solution with rho (P) of 50mg/L is accurately sucked, and is accurately diluted by 10 times with water to obtain standard working solution with rho (P) of 5mg/L, which is prepared for use. Accurately sucking 0, 0.50, 1.00, 2.00, 4.00, 6.00 and 8.00ml of phosphorus standard working solution (the concentration of the corresponding phosphorus in the standard series solution is 0, 0.05, 0.10, 0.20, 0.40, 0.60 and 0.80mg/LP in sequence),putting into 50ml volumetric flask, adding water to about 30m1, adjusting pH and color development of the solution (same method as above), measuring absorbance of the serial solutions, drawing calibration curve, and obtaining phosphorus concentration value of each solution according to the drawn standard curve.

The results show that the standard curve equation is y-2.8109 x-0.1526 (R)²0.9996) was well linear over the range of concentrations tested. The calculated phosphorus dissolving capacity of the strain K01 reaches 75.25 +/-1.42 mg/L.

After K01 is confirmed to have the phosphorus dissolving function, the phosphorus dissolving function of K01 in a high-salt solution is further verified, NaCl concentration is added to 13% on the basis of the original culture medium, and the phosphorus dissolving function of K01 is detected according to the method. The results show that the phosphorus dissolving capacity of the K01 in high-salt solution does not change significantly, and the diameter (D) of a dissolving ring reaches 15.69 +/-0.89 mm (figure 8).

EXAMPLE 4 determination of protease-producing ability of Strain K01

Firstly, activating a strain K01 on an LB culture medium, inoculating the activated strain on a culture medium for detecting protease (5.00 g of tryptone, 3.00g of yeast extract, 1.00g of glucose, 15.00g of agar, 1000mL of distilled water, pH7.0 and 121 ℃ for 30min under high pressure, cooling the sterilized detection culture medium to about 50 ℃, adding skimmed milk into the culture medium according to the proportion of 10%, uniformly mixing, pouring into a culture dish, cooling for later use, culturing at 28 ℃ for 48h, repeating for 3 times, and observing whether a dissolving ring appears, wherein the result shows that K01 has good capability of dissolving protein, high-yield protease, the diameter (D) of the dissolving ring reaches 24.15 +/-0.91 mm, the diameter (D) of a colony is 11.57 +/-0.46 mm, and the enzyme yield (D/D) is 2.09 +/-0.20 (figure 9).

And (3) detecting the activity of the protease: the K01 strain was inoculated at 5% inoculum size to a liquid assay medium (tryptone 5.00g, yeast extract 3.00g, glucose 1.00g, agar 15.00g, distilled water 1000mL, pH7.0, autoclaved 30min at 121 ℃ C.), shake-cultured at 28 ℃ and 200rpm for 48 h. Centrifuging the cultured fermentation liquid at 10000rpm for 5min, taking the supernatant, and determining the activity of the neutral protease by adopting a Folin-phenol method according to the standard of GB/T23527-. The protease activity was 2683.17. + -. 13.62U/ml.

EXAMPLE 5 determination of cellulase-producing ability of Strain K01

Firstly, the strain K01 was activated on LB medium, and the activated strain was inoculated on a medium for detecting cellulose (MgSO)₄·7H₂O 0.25g，K₂HPO₄ 0.50g，(NH₄)₂SO₄0.5g, sodium carboxymethylcellulose 1.88g, agar 15.0g, distilled water 1000mL, pH7.0, autoclaving at 121 ℃ for 30 min). Culturing at 28 ℃ for 7 days, adding 5mL of 0.2mg/mL Congo red dye solution into each plate after 7 days, dyeing for 1h, removing the Congo red dye solution, adding 1MNaCl for washing for 1h, removing the washing solution, observing the generation of a hydrolysis ring around a bacterial colony, wherein the generation of the cellulase is indicated by the occurrence of the hydrolysis ring, and repeating for 3 times. The results showed that K01 has a very good ability to dissolve cellulose, and its lysis circle diameter (D) reached 28.35. + -. 0.75mm, colony diameter (D) was 13.86. + -. 1.08mm, and enzyme yield (D/D) was 2.05. + -. 0.19 (FIG. 10).

And (3) detecting the activity of the cellulase: drawing a glucose standard curve according to a method disclosed in the Jiang national loyalty paper screening, application and enzyme production condition research on high-yield cellulase bacillus subtilis, determining the activity of the cellulase, inoculating the K01 strain to a liquid detection culture medium (5.00 g of yeast extract, 10.00g of tryptone, 10.00g of NaCl and MgSO 5%) with an inoculation amount of 5%₄·7H₂O 0.25g，K₂HPO₄ 0.50g，(NH₄)₂SO₄0.5g, 1000mL of distilled water, pH7.0, autoclaving at 121 ℃ for 30min), shake culturing at 28 ℃ and 200rpm for 48h, repeating for 3 times. Centrifuging the cultured fermentation broth at 4000rpm for 15min, collecting 0.5mL of centrifuged supernatant as crude enzyme solution, adding 2mL of sodium carboxymethylcellulose substrate solution (1% sodium carboxymethylcellulose solution dissolved in phosphate buffer solution containing 11.876g/L Na₂HPO₄·2H₂O solution and 9.078g/L KH₂PO₄Mixing the solutions in equal volume), performing enzymolysis in a 50 deg.C water bath for 0.5h, adding 2mL dinitrosalicylic acid (DNS) color development solution, heating in boiling water bath for 10min, cooling, and measuring absorbance at 540 nm. The calculation formula of the cellulase activity is as follows:

cellulase activity (U/ml) × reduced sugar amount (mg) × 1/enzyme solution volume (ml) × enzyme sample dilution factor

Drawing a glucose standard curve, wherein the standard curve is that y is 0.3983x +0.0091, and r is²0.9987, indicating OD_540nmThe cellulase activity of the strain K01 can be obtained by the equation and the enzyme activity calculation formula, and the cellulase activity reaches 7.59 +/-0.32U/mL.

Example 6 detection of IAA-producing ability of Strain K01

Firstly, the strain K01 was activated on LB liquid medium, and the activated strain was inoculated in 1% amount into DF + medium (peptone 5.00g, yeast extract 1.50g, beef extract 1.50g, NaCl 5.00g, tryptophan 0.50g, distilled water 1000mL, pH7.0, autoclaving at 121 ℃ for 30 min). Shaking at 150rpm for 7 days at 28 deg.C, taking out the fermentation broth after 7 days, centrifuging at 12000rpm for 5min, measuring IAA content in the fermentation broth by Salkowkin colorimetry, and repeating for 3 times. The result shows that K01 produces IAA in an amount of 2.51 + -0.19 mg/L. Further analysis by HPLC confirmed that the strain synthesized IAA, the strain was cultured for 7 days, centrifuged at 12000rpm for 5min, 30mL of the supernatant was taken, extracted with twice the volume of ethyl acetate in a constant temperature shaker for 3 times, the combined extracts were distilled with ethyl acetate under reduced pressure, then dissolved in 5mL of methanol, made to volume, and filtered through a 0.22um filter. Sample detection was performed with reference to methods of delphinium (2006) and high cassia twig (2007). A detection instrument: waters2998 high performance liquid chromatography, column: AgilertZorbaxSB-C₁₈250mm × 4.6mm, 5 um. Mobile phase, methanol: acetonitrile: 0.6% aqueous glacial acetic acid (50: 5: 45, v/v/v). Sample introduction amount: 20 μ l. The flow rate was 0.8 mL/min. Column temperature: and (4) room temperature. Detection wavelength: 255 nm. The detection result shows that the IAA content is 2.75 +/-0.11 mg/L.

Example 7 detection of growth promoting ability of Strain K01

Selecting single colony from a flat plate of a test strain, activating the single colony in an LB liquid culture medium, inoculating the activated bacterial liquid into a DF culture medium according to the bacterial inoculation amount of 1%, shaking at 28 ℃, 150rpm for 72 hours, and diluting the bacterial liquid to 10⁵cfu/mL. According to the method for detecting the growth promoting effect of plant rhizosphere growth promoting bacteria disclosed in CN101984067A, the growth promoting capability of K01 fermentation liquor on corn seedlings is detected, and the growth promoting capability is measuredThe stem height, the root length, the fresh weight of the stem, the fresh weight of the root, the dry weight of the stem and the dry weight of the root of the corn seedling.

The results show that strain K01 has a good growth promoting effect (FIG. 11). Can increase the stem height of corn seedling by 14.23 +/-0.27%, increase the root length by 6.92 +/-0.13%, increase the fresh weight of stem by 22.41 +/-0.11%, increase the fresh weight of root by 22.14 +/-0.13%, increase the stem weight by 36.33 +/-0.09% and increase the dry weight of root by 33.33 +/-0.15% (Table 6).

TABLE 6 growth promoting effect of Strain K01 on maize

Treatment of

Stem height/cm

Root length/cm

Fresh weight of stem/g

Fresh weight of root/g

Dry weight of stem/g

Root Dry weight/g

Control (CK)

12.44±0.45b

17.11±1.33a

0.522±0.063b

0.514±0.097b

0.044±0.0051b

0.048±0.0085b

K01

14.21±1.01a

18.54±2.47a

0.639±0.061a

0.628±0.034a

0.059±0.0058a

0.064±0.0047a

Increase ratio (%)

14.23±0.27

6.92±0.13

22.41±0.11

22.14±0.13

36.33±0.09

33.33±0.15

Note: different letters indicate significant differences at the level of P < 0.05.

Example 8 inhibition of plant pathogenic fungi by Strain K01

The fungal inhibition spectrum of K01 was examined by the counter culture method, and the effect of inhibiting Botrytis cinerea (Botrytis cinerea), Fusarium oxysporum (Fusarium oxysporum), Alternaria solani (Alternaria solani), Ceratophyllus microphyllus (Ceratophylla fimbriata), Fusarium moniliforme (Fusarium moniliforme), Lilium radicle (Fusarium oxysporum.Lili), Fusarium oxysporum (Fusarium oxysporum.sp.niveum), Monilinia persica (Monilinia laxa), Rhizoctonia cerealis (Rhizoctonia cerealis), Rhizoctonia oryzae (Rhizoctonia solani), Rhizoctonia gossypii (Verticillum verticillii), Bacillus cereus (Bacillus subtilis), Bacillus cerealis (Rhizoctonia solani), Bacillus subtilis (Rhizoctonia solani), Rhizoctonia solani (Rhizoctonia solani) and Rhizoctonia solani (Rhizoctonia solani) were inoculated on a solani, Sphingomonas a roseum, Sphingcan be, Sphingomonas a, Sphingulata, Sphingensis (Rhizoctonia sp.5, Sphingaria, Klebsiella pneumoniae (Klebsiella pneumoniae, Klebsiella sp.5. origin, Klebsiella pneumoniae, Klebsiella sp.5, Klebsiella pneumoniae, botrytis cinerea (Botrytis cinerea), Fusarium oxysporum (Fusarium oxysporum) and Helianthus annuus (Sclerotinia sclerotiorum) were cultured at 25 ℃ for 4 days, and the rest were cultured for 7 days, and the control diameter and the inhibition diameter were measured, respectively, and the inhibition ratio was calculated according to formula I).

The results show that the strain K01 has a wide antibacterial spectrum, has an inhibitory effect on many pathogenic fungi, and shows that the strain K01 has a great application potential in the prevention and treatment of fungal diseases, the results show that the K01 has a wide antibacterial spectrum (figure 12), the antibacterial rate on Botrytis cinerea (Botrytis cinerea) is 53.76 +/-1.23%, the antibacterial rate on Fusarium oxysporum (Fusarium oxysporum) is 64.30 +/-1.86%, the antibacterial rate on Alternaria solani (Alternaria solani) is 52.86 +/-0.79%, the antibacterial rate on Peucenophora microphylla (Ceratophyceae) is 86.62 +/-1.83%, the antibacterial rate on Fusarium moniliforme (Fusarium moniliforme) is 68.47 +/-5.55%, the antibacterial rate on Liriophyria solani (Fusarium oxysporum) is 51.62 +/-2.46%, the antibacterial rate on Fusarium oxysporum (Fusarium oxysporum) is 0.84%, the antibacterial rate on Rhizoctonia solani is 0.97% and the antibacterial rate on Rhizoctonia solani is 25.97.97.80%, and the antibacterial rate on Rhizoctonia solani.97.97.80% The bacteriostatic ratio of Verticillium dahliae Kleb of cotton is 87.62 + -2.43%, the bacteriostatic ratio of celery spot blight (Septoria apiicola Speg) is 55.08 + -1.96%, the bacteriostatic ratio of Colletotrichum (Colletotrichum) is 69.23 + -1.87%, the bacteriostatic ratio of sunflower black stem germ (Leptosphaeria Lindsii) is 97.45 + -1.62%, the bacteriostatic ratio of sunflower sclerotium (Sclerotinia sclerotiorum) is 50.01 + -2.47%, and the bacteriostatic ratio of rice bakanae disease (Fusarium moniliforme Shell) is 66.56 + -0.92% (Table 7).

TABLE 7 bacteriostasis spectra of K01

Pathogenic bacteria	Inhibition rate/%)
		Botrytis cinerea (Botrytis cinerea)	53.76±1.23
Rhizoctonia solani (Rhizoctonia solani)	57.82±1.49
		Peach brown rot pathogen (Monilinia laxa)	59.05±2.04
Rhizoctonia cerealis (Rhizoctonia cerealis)	62.82±1.85
		Tomato early blight (Alternaria solani)	52.86±0.79
Lily root rot (Fusarium oxygen sporum f.sp.Lili)	51.62±2.46
		Anthrax bacteria (Colletotrichum)	69.23±1.87
Helianthus annuus L.var.nigra (Leptosphaeria lindquisii)	97.45±1.62
		Fusarium oxysporum (Fusarium oxysporum)	64.30±1.86
Watermelon wilt bacterium (Fusarium oxysporum f.sp.niveum)	60.84±0.97
		Fusarium moniliforme (Fusariuummoniliforme)	68.47±5.55
Pediobolus paragua (Ceratocysis fimbriata)	86.62±1.83
		Verticillium dahliae Kleb)	87.62±2.43
Pythium celebratum (Septoria apiicola Speg)	55.08±1.96
		Sunflower sclerotium (sclerotinia sclerotiorum)	50.01±2.47
Bakanae disease of rice (Fusarium moniliforme Shell)	66.56±0.92

EXAMPLE 9 prevention and treatment Effect of Strain K01 on Gray mold on Capsicum fruits

Selecting pepper fruits with uniform size and no mechanical wound, wiping the pepper fruits with 75% ethanol for disinfection, and drying in the air. Cutting 3mm × 3mm wound with sterile scalpel, picking pericarp, air drying, inoculating K01 bacterial solution (10)⁸cfu/ml) of 10 mu L, carrying out moisturizing culture at room temperature (18-23 ℃) to ensure that the medicament is fully absorbed, inoculating a botrytis cinerea cake with the thickness of 3mm after 24 hours, carrying out moisturizing culture at 25 ℃, counting the disease area of each treated pepper fruit after 5 days, and calculating the preventing and treating effect according to a formula II). The 500-fold liquid of 50 percent carbendazim is used as a positive medicament control, sterile water is used as a negative control, and the treatment method and the using amount are the same as those of a K01 microbial inoculum. Each treatment was 5 fruits, 3 replicates.

Area of spread of lesion: (mm²) Total lesion area-wound area

The result shows that the K01 has good control effect on the gray mold of the pepper (figure 13 and figure 14), the control effect on the gray mold of the bell pepper fruits reaches 78.36 percent (Table 8), and the control effect is not obviously different from that of a carbendazim treatment group; the control effect on gray mold on pepper fruits reaches 80.70 percent, which is 89.7 percent of the control effect of a carbendazim treatment group (Table 9).

TABLE 8K 01 preventive effect on Botrytis cinerea on Piper kaki fruits

Treatment of	Extended area of lesion (mm)²)	Control effect (%)
			Control	297.21±28.21a	-
Carbendazim	54.59±5.94b	81.63±5.29a
			K01	64.31±7.16b	78.36±4.48a

TABLE 9K 01 control Effect on Botrytis cinerea on Pepper fruits

Treatment of	Extended area of lesion (mm)²)	Control effect (%)
			Control	81.41±8.21a	-
Carbendazim	8.15±0.94c	90.00±8.21a
			K01	15.71±1.52b	80.70±7.29b

EXAMPLE 10 control Effect of K01 on Gray mold on Pepper leaves

Selecting healthy pepper leaves with the same leaf age and basically consistent leaf size, and cleaning the pepper leaves with sterile water for later use. Firstly, K01 strain fermentation liquor (10)⁸cfu/m1) is uniformly sprayed on the blades, and the condition that the blade surfaces just overflow is taken as the standard; after air drying, 1 botrytis cinerea bacterial cake with the diameter of 3mm is inoculated in the middle of each leaf, and the leaf is subjected to moisture-preserving culture at 25 ℃ for 3 days. The 500-fold liquid of 50 percent carbendazim is used as a positive medicament control, sterile water is used as a negative control, and the treatment method and the using amount are the same as those of a K01 microbial inoculum. Each treatment contained 9 leaves and was repeated 3 times.

Counting the disease area of each pepper leaf, and calculating the control effect of K01 on the gray mold according to the formula II). The result shows that K01 has good control effect on the gray mold of hot pepper on the isolated leaves (figure 15), and the control effect reaches 87.84% (Table 10).

Extended area of lesion (mm)²) Area of the disease plaque-area of the fungus cake

TABLE 10K 01 control Effect on Botrytis cinerea on Pepper leaves

Treatment of	Extended area of lesion (mm)²)	Control effect (%)
			Control	143.17±7.25a	-
Carbendazim	12.89±2.98b	90.99±4.24a
			K01	17.41±3.13c	87.84±4.32a

Example 11 control Effect of K01 on Botrytis cinerea on tomato leaves

Selecting healthy tomato leaves with the same leaf age and basically consistent leaf size, and cleaning the tomato leaves with sterile water for later use. Firstly, K01 strain fermentation liquor (10)⁸cfu/ml) is uniformly sprayed onOn the blades, the condition that the blade surfaces just overflow is taken as the standard; after air drying, 1 Botrytis cinerea fungus cake with the diameter of 3mm is inoculated in the middle of each leaf, and the leaf is subjected to moisture-preserving culture at 25 ℃ for 3 days. The 500-fold liquid of 50 percent carbendazim is used as a positive medicament control, sterile water is used as a negative control, and the treatment method and the using amount are the same as those of a K01 microbial inoculum.

Counting the disease area of each tomato leaf, and calculating the control effect of K01 on gray mold according to the formula II). The result shows that K01 has good control effect on tomato gray mold on the isolated leaves (figure 16), and the control effect reaches 81.86% (Table 11).

TABLE 11K 01 control Effect on Botrytis cinerea on Pepper leaves

Treatment of	Extended area of lesion (mm)²)	Control effect (%)
			Control	103.26±8.19a	-
Carbendazim	13.07±1.46c	87.34±7.24
			K01	18.73±2.33b	81.86±7.32

EXAMPLE 12K 01 controlling Effect on tomato Botrytis cinerea on tomato fruit

Selecting tomato fruits with uniform size and without mechanical wounds, wiping the tomato fruits with 75% ethanol for disinfection, and drying in the air. Pricking a round wound with a diameter of 2mm with a self-made needle bundle, picking pericarp, airing the wound, and inoculating K01 bacterial liquid (10)⁸cfu/m1)10 μ L, and is preserved at room temperature (15-18 deg.C) to make the medicine be absorbed sufficiently. After 24h, 3mm Botrytis cinerea (Botrytis cinerea) cakes were inoculated and incubated for 3 days at 25 ℃ with moisture retention. The 500-fold liquid of 50 percent carbendazim is used as a positive medicament control, sterile water is used as a negative control, and the treatment method and the using amount are the same as those of a K01 microbial inoculum.

Counting the disease area of each treated tomato fruit, and calculating the control effect according to the formula II). Each treatment was 5 fruits, 3 replicates.

Extended area of lesion (mm)²) Total lesion area-wound area

The experimental result is shown in FIG. 17, the spread area of the control treatment lesion is 283.45 + -14.52 mm²The K01 treatment and the carbendazim treatment have no morbidity at all, and the control effect reaches 100 percent.

EXAMPLE 13 field control Effect of K01 on Botrytis cinerea

Tomato and pepper field biocontrol tests were performed in 2019 in greenhouse growing areas of agricultural scientific and technological gardens of large gezhai, Wei county, chentai, province, Hebei. Respectively divided into 3 treatment areas, and field management is carried out according to conventional cultivation management technology, wherein the area of each treatment area is 15m²Length 5m, width 3m, 4 repeats. Fermenting K01 (with bacterial load of 10)⁹cfu/ml) is diluted by 100 times, and the whole plant is uniformly sprayed until the surface of the leaf is completely wetted and dropped. The 500-fold liquid of 50 percent carbendazim is used as a positive medicament control, sterile water is used as a negative control, and the treatment method and the using amount are the same as those of a K01 microbial inoculum. Spraying the tomato and pepper after 20 days of field planting, investigating a cell after 15 days, sampling at five points, investigating 2-3 plants at each point, investigating all leaves of each plant, and recording according to the following grading method. Calculating disease index and prevention effect.

Leaf disease grading method (leaf as unit):

level 0: no scab.

Level 1: 1-3 scabs exist on a single leaf.

And 3, level: 4-6 scabs exist on a single leaf.

And 5, stage: 7-10 scabs exist on a single leaf.

And 7, stage: the single leaf has 11-20 scabs.

And 9, stage: the single leaf has dense lesion spots occupying more than one fourth of the leaf area.

The experimental results are shown in table 12, and compared with the control, K01 significantly reduces the disease indexes of tomato gray mold and pepper gray mold; the K01 strain has the control effect of 74.99 percent on the botrytis cinerea and 70.83 percent on the botrytis cinerea.

TABLE 12 field control of Botrytis cinerea and Capsici fructus by K01

Example 14K 01 control of peach Brown rot (Monilinia laxa) in the wild peach fruit

Selecting peach fruits with uniform size and no mechanical wound, wiping the peach fruits with 75% ethanol, sterilizing, and air drying. Pricking a round wound with a diameter of 2mm with a self-made needle bundle, picking pericarp, airing the wound, and inoculating K01 bacterial liquid (10)⁸cfu/ml) 10. mu.L, and the medicament is fully absorbed by moisturizing culture at room temperature (15-18 ℃). After 24h, a 5mm cake of Monilinia fructicola (Monilinia laxa) was inoculated and incubated at 25 ℃ for 3d with moisture retention. Using 50% carbendazim 500 times liquid as positive drug controlThe treatment method and the dosage are the same as those of K01 microbial inoculum by taking sterile water as a negative control.

Counting the disease area of each treated wild peach fruit, and calculating the control effect according to the formula II). Each treatment was 5 fruits, 3 replicates.

Extended area of lesion (mm)²) Total lesion area-wound area

The results show that K01 significantly reduced the incidence area of brown rot of peach compared with the control, and the control effect reached 89.82%, which is 91.6% of that of the carbendazim treatment group (table 13). The K01 shows that the pesticide composition has good control effect on the peach brown rot (figure 18).

TABLE 13 prevention and control of peach brown rot by K01 in wild peach fruit

Treatment of	Extended area of lesion (mm)²)	Control effect (%)
			Control	345.03±29.49c	-
Carbendazim	6.86±0.71a	98.01±4.28a
			K01	35.11±3.96b	89.82±3.12b

Example 15K 01 control Effect of cherry fruit on peach Brown rot (Monilinia laxa)

Selecting cherry fruits with uniform size and no mechanical wound, wiping the cherry fruits with 75% ethanol for disinfection, and drying in the air. Pricking a round wound with a diameter of 2mm with a self-made needle bundle, picking pericarp, airing the wound, and inoculating K01 bacterial liquid (10)⁸cfu/ml) 10. mu.L, and the medicament is fully absorbed by moisturizing culture at room temperature (15-18 ℃). After 24h, the Monilinia fructicola (Monilinia laxa) cake of 3mm is inoculated, and the cake is subjected to moisture-preserving culture at 25 ℃ for 2 d. The 500-fold liquid of 50 percent carbendazim is used as a positive medicament control, sterile water is used as a negative control, and the treatment method and the using amount are the same as those of a K01 microbial inoculum.

Counting the disease area of each processed cherry fruit, and calculating the control effect according to the formula II). Each treatment was 5 fruits, 3 replicates.

Extended area of lesion (mm)²) Total lesion area-wound area

The results show that K01 has good control effect on the peach brown rot on cherry fruits (figure 19), and compared with a control, K01 obviously reduces the morbidity area of the peach brown rot on cherries, the control effect reaches 87.02 percent, and the control effect is 89.3 percent of that of a carbendazim treatment group (Table 14).

TABLE 14K 01 prevention and treatment of peach brown rot by cherry fruit

Treatment of	Extended area of lesion (mm)²)	Control effect (%)
			Control	149.96.01±16.48a	-
Carbendazim	3.81±0.95c	97.46±7.81a
			K01	19.47±4.71b	87.02±8.62a

Example 16K 01 Effect on wheat seedling control of Rhizoctonia cerealis

Inoculating Rhizoctonia cerealis into corn grit culture medium, culturing at room temperature until mycelia overgrow (about 10 days), and taking out. Mixing germs bred by corn sand with sterile soil (grass carbon, vermiculite, sandy soil and humic acid soil in a volume ratio of 1: 1) uniformly according to a volume ratio of 1: 30 to prepare bacterial soil; suspending K01 bacteria (10)⁸cfu/m1) and sowing 10 grains of the wheat seeds in flowerpots filled with bacterial soil; soaking the seed inoculation bacterial soil in sterile normal saline as a negative control, diluting the seed inoculation bacterial soil by 500 times of 50% carbendazim wettable powder as a positive control, and repeating three times of treatment; and investigating the disease condition, investigating the disease index and the disease incidence and calculating the control effect fifteen days after the emergence of seedlings.

Grading standard of wheat seedling stage disease condition:

level 0: no symptoms;

level 1: browning leaf sheath;

and 2, stage: obvious striae withering disease spots are formed outside the leaf sheath, and the diameter of the disease spots is less than 0.5 cm;

and 3, level: 1 to a plurality of disease spots are arranged outside the leaf sheath, and the diameter of the disease spots is more than 0.5 cm;

4, level: scabs are in the leaf sheath;

and 5, stage: the stalk has disease spots or the wheat plant dies.

The result shows that the K01 has good control effect on the wheat sharp eyespot (figure 20), and compared with the control, the K01 obviously reduces the disease index of the wheat sharp eyespot, and the control effect reaches 62.01 percent (Table 15).

TABLE 15 control of Rhizoctonia cerealis in wheat seedlings by K01

Treatment of	Incidence (%)	Disease index (%)	Control effect (%)
				Control	90.72±3.17	66.67±3.05a	-
Carbendazim	26.67±1.22	10.67±1.39c	83.33±8.02a
				K01	33.33±2.05	25.33±2.31b	62.01±5.18b

Example 17K 01 preventive and therapeutic effects on sunflower Sclerotinia sclerotiorum (sclerotiorum) on sunflower leaves

Selecting healthy sunflower leaves with the same leaf age and basically consistent leaf size, and cleaning the leaves with sterile water for later use. Fermentation broth of K01 strain for test (10)⁸cfu/ml), and is preserved and cultured under room temperature (15-18 ℃) to ensure that the medicament is fully absorbed. Each treatment contained 9 leaves and was repeated 3 times. Uniformly spraying the treatments on the blades, wherein the water overflow of the blade surfaces is the standard; after air-drying, 1 sclerotinia sclerotiorum cake with the diameter of 3mm is inoculated in the middle of each leaf, and the sclerotinia sclerotiorum cake is kept moisture and cultured for 3 days at 25 ℃. The 500-fold liquid of 50 percent carbendazim is used as a positive medicament control, sterile water is used as a negative control, and the treatment method and the using amount are the same as those of a K01 microbial inoculum.

Counting the incidence area of each sunflower leaf, and calculating the control effect of K01 on sunflower sclerotium according to formula II).

The result shows that K01 has a good control effect on sunflower sclerotinia sclerotiorum (figure 21), and compared with a control, K01 obviously reduces the lesion area of sunflower leaf sclerotinia sclerotiorum, the control effect is 96.64%, and the control effect is not obviously different from that of validamycin on sunflower sclerotinia sclerotiorum (Table 16).

TABLE 16 control of sunflower sclerotia in sunflower leaves by K01

Treatment of	Incidence of disease	Extended area of lesion (mm)²)	Control effect (%)
				CK	100.00％	57.88±19.36a	-
Validamycin	80.00％	2.35±1.09b	95.95±5.63％a
				K01	66.67％	1.95±1.27b	96.64±6.56％a

EXAMPLE 18 bacteriostatic Spectrum of volatile bacteriostatic substances of K01

The plate-buckled method is used for detecting the inhibiting effect of K01 volatile substances on Botrytis cinerea (Botrytis cinerea), Fusarium oxysporum (Fusarium oxysporum) of the root rot of beet, tomato early blight (Alternaria solani), sunflower Sclerotinia sclerotiorum (Sclerotinia sclerotiorum), Fusarium moniliforme (Fusarium moniliforme), anthracnose (Colletotrichum), watermelon Fusarium oxysporum (Fusarium oxysporum.sp.niveum), peach blossom (Monilinia laxa), wheat sharp leaf blight (Rhizoctonia cerealis), rice sharp leaf blight (Rhizoctonia solani), cotton Verticillium dahliae (Kleb), and celery spot blight (Septoria apiaria speciosa) respectively. First, a Botrytis cinerea cake of 5mm was inoculated in the middle of a PDA plate, and 100. mu.L of 10 cells were inoculated on an LB medium⁸cfu K01 bacterial solution, which was applied uniformly, and treated with the same amount of sterile physiological salineAs a comparison, the two plates are buckled and sealed by a preservative film, and are placed in a constant temperature incubator at 25 ℃ for culturing for 5d, each treatment is repeated for 3 times, the colony diameter of the botrytis cinerea capsici in the plates of the treatment group and the control group is determined by a cross method, and the inhibition rate of the volatile bacteriostatic substance on the botrytis cinerea is calculated according to the formula I). The results show that the strain K01 volatile antibacterial substance also has a very wide antibacterial spectrum, has an inhibitory effect on a plurality of pathogenic fungi (figure 22), has an antibacterial rate of 41.91 +/-2.26% on Botrytis cinerea, 14.11 +/-1.40% on Fusarium oxysporum (Fusarium oxysporum), 41.96 +/-1.71% on Alternaria solani (Alternaria solani), 24.92 +/-2.55% on Sclerotinia helianthi (Sclerotinia sclerotiorum), 17.87 +/-1.34% on Fusarium moniliforme, 19.94 +/-1.63% on Colletotrichum, 33.03 +/-1.58% on Colletotrichum, 19.94 +/-1.63% on Fusarium oxysporum, 56.56.94% on Xanthomonas oryzae and 14.5.94 +/-1.94% on Rhizoctonia solani (Klebsiella solani) and 14.5929.5% on Rhizoctonia solani (Rhizoctonia solani) on, The inhibition rate of bacterial blight of celery (Septoria apiicola Speg) was 35.04. + -. 1.53% (Table 17).

TABLE 17 bacteriostatic effect of K01 volatile bacteriostatic substance

Pathogenic bacteria	Inhibition rate/%)
		Fusarium moniliforme (Fusarium moniliforme)	17.87±1.34
Tomato early blight (Alternaria solani)	41.96±1.71
		Rhizoctonia cerealis (Rhizoctonia cerealis)	37.43±2.37
Anthrax bacteria (Colletotrichum)	33.03±1.58
		Botrytis cinerea (Botrytis cinerea)	41.91±2.26
Fusarium oxysporum (Fusarium oxysporum)	14.11±1.40
		Sunflower sclerotium (Sclerotinia sclerotiorum)	24.92±2.55
Peach brown rot pathogen (Monilinia laxa)	36.57±1.94
		Verticillium dahliae Kleb)	46.46±3.24
Pythium celebratum (Septoria apiicola Speg)	35.04±1.53
		Rhizoctonia solani (Rhizoctonia solani)	14.57±4.17
Watermelon wilt bacterium (Fusarium oxysporum f.sp.niveum)	19.94±1.63

Example 19 inhibitory Effect of K01 volatile bacteriostatic substances on infection of Pepper leaves by Botrytis cinerea

A plate buckling method is adopted to detect the inhibition effect of K01 volatile substances on botrytis cinerea infected pepper leaves. Inoculating Botrytis cinerea cake of 3mm in the middle of Capsici fructus leaf, placing on the lower plate, inoculating 10 μ L of 100 μ L on LB culture medium⁸Evenly coating the cfu/ml K01 bacterial liquid, contrasting with the treatment of the same amount of sterile normal saline, buckling the two plates, sealing the plates by using a preservative film, placing the plates in a constant-temperature incubator at 25 ℃ for culturing for 3 days, repeating each treatment for 3 times, counting the disease area of each pepper leaf, and calculating the disease incidence and prevention effect of the volatile antibacterial substances on the botrytis cinerea according to the formula II).

The control effect of K01 on Botrytis cinerea is shown in FIG. 23, and the control effect reaches 70.97% (Table 18).

TABLE 18 inhibition of Botrytis cinerea infection of Capsicum frutescens leaves by volatile bacteriostatic substances with K01

Treatment of	Extended area of lesion (mm)²)	Control effect (%)
			Control	322.01±16.47	-
K01	93.47±8.25*	70.97±6.48

Note: indicates significant differences at the level of P < 0.05.

Example 20K 01 improving resistance of tomato to Botrytis cinerea infection

Selecting tomato seedlings with seedling stage of about 45 days, and using 10⁷10mL of cfu/mL bacterial solution is treated by dipping roots and is used as a blank control at the same time, the bacterial solution is transplanted into a flowerpot, after 3 days of growth, the leaves are inoculated with the activated 3mm botrytis cinerea, the leaves are cultured for 48 hours at a constant temperature and under a constant humidity of 25 ℃, the morbidity area of each inoculated pathogenic bacterium leaf is counted, and the control effect is calculated. The results show that. K01 increased the overall resistance of tomato seedlings and increased the disease resistance of leaves after root use (fig. 31).

Table 19K 01 enhances resistance of tomato to Botrytis infection

	Extended area of lesion (mm2)	Control effect (%)
			CK	151.29±7.19a
K01	63.07±14.42b	58.31±5.21

Example 21K 01 detection of biofilm Forming ability

The biofilm-forming ability of strain K01 was tested by reference to the crystal violet staining method of O 'Toole and Kolter (O' Toole and Kolter, 1998), and the following procedure was carried out: k01 was activated in LB medium, shaken overnight at 28 ℃ and OD adjusted₆₀₀0.6, EPS medium (K) containing 2% glucose was inoculated at an inoculum size of 1%₂HPO₄ 7.0g/L，KH₂PO₄ 3.0g/L，MgSO₄*7H₂O 0.1g/L，CaCl₂ 0.01g/L，FeSO₄0.001g/L, NaCl 0.1g/L, glucose 1.0g/L, yeast extract 0.125g/L, pH7.0), standing at 28 deg.C for 48h, discarding the supernatant, washing the thallus with sterile water once, air drying completely, adding 1.5 times of 0.1% crystal violet dye solution, dyeing at room temperature for 30min, removing the dye solution, washing with sterile water twice, air drying at room temperature, adding equal volume of 95% ethanol to wash the purple ring, and measuring OD with a spectrophotometer₅₇₀. The results show that the strain K01 has strong capability of generating biological membrane, OD₅₇₀0.98 ± 0.23, as shown in fig. 24, biofilm formation is important for plant rhizosphere colonization and interaction with plants.

Example 22K 01 colonization of tomato and corn roots

1. Colonization of K01 on maize

The green fluorescent protein plasmid is transferred into a K01 strain by adopting an electric shock transformation method, so that the green fluorescent protein plasmid can be stably expressed, and the strain K01-GFP with a green fluorescent marker is obtained. According to the method for detecting the growth promoting effect of plant rhizosphere growth promoting bacteria disclosed in CN101984067A, the colonization ability of K01 strain on corn seedlings is detected, and 10 is used⁶After the cfu/mL seed soaking growth is carried out for 5 days, whether the root has fluorescence or not is observed under a Leica laser confocal scanning microscope, and the experimental result shows that the K01 strain has good colonization capacity and can be well colonized on the corn root after the seed soaking growth is carried out for 5 days (figure 25).

2. Colonization of K01 on tomato

The K01 strain was continuously subcultured on rifampicin-resistant plates to gradually increase rifampicin concentrations of 2. mu.g/mL, 5. mu.g/mL, 10. mu.g/mL, 20. mu.g/mL, 50. mu.g/mL, 100. mu.g/mL, 150. mu.g/mL, and 200. mu.g/mLAnd (3) the rifampicin resistance concentration induces the strain to generate rifampicin resistance, and finally reaches 200 mu g/mL to obtain the rifampicin resistance strain K01-RFP capable of being stably inherited. The strain is shaken in a common LB culture medium to be made into 10⁶And (3) immersing the tomato seedlings growing in the seedling tray for 40 days in cfu/mL bacterial solution for 30min, transplanting the tomato seedlings into a flowerpot, adding the bacterial solution with the same concentration into flowerpot soil, uniformly mixing, culturing in a constant temperature room at 21 ℃, and illuminating for 12h every day and culturing in darkness for 8 h. Respectively measuring the viable count of the soil and the plant rhizosphere soil in the initial period, 2 days, 5 days, 10 days, 15 days, 20 days and 30 days, simultaneously cutting off the tomato leaves in 30 days, weighing, sterilizing in 75% alcohol for 1min, cleaning 4 times by using sterile water, crushing in a mortar, adding 3 times of sterile water for dilution and plate coating, and detecting the bacterial content of the top leaves of the tomatoes. The results show that the bacteria in the soil are rapidly attenuated, and the bacteria are detected from the initial 10 days after 15 days⁷Attenuation of cfu/mL to about 10³The strain at tomato rhizosphere can be kept at 10 days⁶cfu/mL, which indicates that the K01 strain can be planted in tomato rhizosphere, the stem is cut at a position 2cm away from the ground surface on the 6 th day, the leachate is collected, and a graph tests K01, so that the concentration of K01 in the vascular bundle leachate reaches 9.25 x 10³cfu/mL; the bacterial content in the tomato leaves reaches 10 days³cfu/g, indicating that strain K01 can not only colonize tomato roots and enter into the tomato body for colonizing growth, but also be transported from tomato roots to the overground part through vascular bundles to reach leaf colonization (FIG. 26).

Example 23 stability test of K01 antibacterial active substance

Activating K01 on an LB plate, inoculating the activated K01 into an LB culture medium, shaking at 28 ℃, 150rpm for 48h, centrifuging at 8000 rpm for 3min, collecting supernatant, and respectively processing the supernatant as follows: (1) treating at 40 deg.C, 60 deg.C, 80 deg.C, and 100 deg.C for 30min respectively; (2) adjusting the pH of the supernatant to 1, 3, 5, 7, 9, 11, and 13 with 6M hydrochloric acid and 6M sodium hydroxide, respectively, standing at room temperature for 30min, and adjusting to original pH; (3) and (5) placing the supernatant under an ultraviolet lamp for irradiating for 30 min.

Filtering the above treated supernatant with 0.22 μm sterile filter membrane, diluting the treated filtrate by 25 times, adding into PDA culture medium, inoculating activated Fusarium oxysporum cake of 5mm, and simultaneously making positive control (untreated supernatant) and negative control (sterile water), each treatment is repeated for 3 times. After 5 days of culture at 25 ℃ the colony diameter was measured using the cross method. The results show that the K01 antimicrobial active substance is resistant to high temperatures (Table 20, FIG. 27), stable under UV light (Table 21, FIG. 28), stable between pH 1 and pH 11, and has no effect on activity (Table 22, FIG. 29).

TABLE 20K 01 sensitivity of antibacterial substances to temperature

Treatment of	Diameter of bacterial colony/mm
		Negative control	69.94±1.69a
Positive control	61.84±0.08b
		Treatment at 40 deg.C	60.33±0.26b
Treatment at 60 deg.C	60.77±1.18b
		Treatment at 80 deg.C	61.85±0.96b
Treatment at 100 deg.C	62.08±0.64b

TABLE 21K 01 sensitivity of antibacterial substances to UV

Treatment of	Diameter of bacterial colony/mm
		Negative control	64.46±0.78a
Positive control	54.36±0.94b
		Ultraviolet treatment	53.33±1.29b

TABLE 22K 01 sensitivity of antibacterial substances to pH

Treatment of	Diameter of bacterial colony/mm
		Negative control	64.46±0.78a
Positive control	54.36±0.94bc
		pH 1.0	55.00±0.63b
pH 3.0	53.26±0.48c
		pH 5.0	53.40±1.07c
pH 7.0	54.35±1.43bc
		pH 9.0	53.42±1.31c
pH 11.0	54.05±1.15bc
		pH 13.0	64.92±1.10a

Example 24 inhibition of pathogenic bacteria by Strain K01

Regulating pathogenic bacteria concentration to 10 with sterile physiological saline⁸cfu/mL, 1mL was added to 50mL of LB medium sterilized and cooled to 50 ℃ and the plate was inverted. Inoculating 10 after the culture medium is solidified⁸2 μ L of cfu/mL K01 strain, 3 replicates of each treatment. The inhibitory effect on cucumber angular leaf spot (Pseudomonas syringae pv. lachrymans), Pectobacterium carotovorum (Pectonia carotovorum) and citrus canker (Xanthomonas citri) was determined separately.

The results are shown in fig. 30 and table 23, the strain K01 has a wider antibacterial spectrum and a better inhibitory effect on the pathogenic bacteria, and the strain K01 has great application potential in prevention and treatment of bacterial diseases.

TABLE 23 inhibition of bacteria by K01

Example 25K 01 field control of Sclerotinia sclerotiorum

In 2019, a sunflower sclerotiniose field biocontrol test is carried out in a test base of Furui species industry Limited company in Heqing river county, Heilongjiang province, of Tongliu city, an autonomous region of inner Mongolia. Respectively divided into 3 treatment areas, and field management is carried out according to conventional cultivation management technology, wherein the area (length 5.5m × width 5.4m) of each plot is 30m²4 repeats. Fermenting K01 (with bacterial load of 10)⁹cfu/ml) is diluted by 100 times, and sunflower discs are uniformly sprayed in the initial flowering phase and the full flowering phase of the sunflower respectively. 2000 times of solution of 75% trifloxystrobin and tebuconazole water dispersible granules is used as a positive medicament control, sterile water is used as a negative control, and the treatment method and the using amount are the same as those of a K01 microbial inoculum. Sunflower harvest time was surveyed in plots, using five-point sampling, 3 plants per plot, and the floral disc of each plant was surveyed and recorded according to the following grading method. Calculating disease index and prevention effect.

The disease grading standard of the disc rot type sclerotinia sclerotiorum is determined according to the proportion of the lesion area on the back of the flower disc to the area of the flower disc: level 0: no symptoms; level 1: less than 25%; and 2, stage: 26 to 50 percent; and 3, level: 51 to 75 percent; 4, level: more than 76 percent.

The experimental results are shown in table 24, K01 significantly reduced the disease index of sunflower sclerotinia sclerotiorum compared to the control; the K01 strain has 23.29 percent of effect on preventing sunflower sclerotinia rot.

TABLE 24K 01 field control of Helianthus annuus sclerotinia

Treatment of	Disease index (%)	Control effect (%)
			Control	23.6±0.73a	-
Medicament	17.21±0.14b	27.04±2.21
			K01	18.11±0.92b	23.29±1.50

Example 26 field control of K01 against beet root rot (Fusarium oxysporum)

In 2020, field biocontrol tests of beet root rot are carried out in a Huohjia camp county, a Hehajia camp county and a Yang home camp army. The beet variety BTS356 is divided into 2 processing areas, field management is carried out according to the conventional cultivation management technology, and the area of each cell is 24.7m multiplied by 1.8m which is 44.47m²And 3 repetitions. 50Kg of biological protein controlled-release fertilizer with the K01 content of 0.5 hundred million/g is applied to each mu of ground, the negative control is the application of fertilizer without K01, and the processing method and the using amount are the same as those of K01 microbial inoculum. And (4) surveying the incidence of the beet and calculating the control effect by adopting five-point sampling in the harvest period and surveying 4 beet plants at each point.

The experimental results are shown in table 25, K01 significantly reduced the incidence of beet root rot compared to the control; the K01 strain has the prevention effect of 54.76 percent on the root rot of the beetroot.

TABLE 25K 01 field control of beet root rot

Note: indicates significant differences at the level of P < 0.05.

Example 27K 01 preparation of microbial inoculum

1. Strain activation

Firstly, activating the strain K01 on an LB culture medium, inoculating the activated strain into an LB liquid culture medium, and culturing for 12h at 28 ℃.

2. Cultivation of seed liquid

The seed culture medium comprises the following components: peptone 12g/L, yeast extract 5g/L, glucose 10g/L, NaCl 10g/L, pH7.2. Inoculating the activated strain into a seed liquid culture medium according to the inoculation amount of 1.2%, and performing shake culture at 28 ℃ on a shaking table at the rotating speed of 200rpm for 12 hours.

3. Fermenting in a fermentation tank

The fermentation medium comprises the following components: soybean powder 20g/L, potato starch 15g/L, yeast extract powder 2.5g/L, peptone 3.0g/L, sucrose 5g/L, KH₂PO₄ 0.6g/L，MgSO₄·7H₂O 0.08g/L，MnSO₄·7H₂O 0.02g/L，(NH₄)₂SO₄5g/L, 1.2g/L of polyether defoamer, and pH 7.2. The cultured seed liquid OD₆₀₀When the inoculation amount reaches 5.00 percent, the mixture is inoculated into a fermentation tank (10 ton fermentation tank, the liquid loading amount is 70 percent), the stirring speed is 150rpm at 37 ℃, and the ventilation quantity is adjusted to ensure that the dissolved oxygen is more than 50 percent and the tank pressure is 0.05 MPa. The bacterial quantity can reach 10 after 24 hours of fermentation¹⁰cfu/ml, stopping when the spore amount reaches more than 95 percentStopping fermentation, cooling, placing in a tank, and optimizing the formula of the culture medium to obtain 10 of Bacillus beiLeisi K01¹⁰cfu/mL bacterial liquid.

4. Preparation of the formulations

Liquid preparation: diluting the fermented microbial inoculum by using sterile water according to a certain proportion, and then carrying out sterile filling to prepare the liquid microbial inoculum.

Powder preparation: concentrating the fermented microbial inoculum by 3 times by using a disc centrifuge, and adding 5% of protective agent, wherein the protective agent comprises the following components in percentage by mass: dextrin, soluble starch and diatomite are mixed at a ratio of 70:25:5, a protective agent is added and stirred for 30min, a centrifugal spray drying tower is used for drying, air inlet temperatures of 140 ℃, 160 ℃ and 180 ℃ and an air outlet temperature of 75 ℃ are respectively set, dry powder is collected, the yield and the treatment capacity are measured, the result is shown in table 26, experiments show that the yield is lower when the temperature is higher, but the treatment capacity is lower when the temperature is lower, the air inlet temperature is 160 ℃ is comprehensively considered, and the spray dried powder is diluted to the required concentration by using zeolite powder.

TABLE 26 spray drying yield and throughput at different temperatures of K01

Air intake temperature/. degree.C	Yield (%)	Throughput (L/h)
			140	93.02±2.65	98.23±7.23
160	87.23±3.01	120.54±5.45
			180	75.98±2.05	142.00±7.69

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Sequence listing

<110> Gen Lian Multi-Biotechnology Ltd

<120> Bacillus belgii K01 and application thereof

<160> 11

<170> SIPOSequenceListing 1.0

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cggacagatg ggagcttgct ccctgatgtt agcggcggac gggtgagtaa cacgtgggta 120

acctgcctgt aagactggga taactccggg aaaccggggc taataccgga tggttgtctg 180

aaccgcatgg ttcagacata aaaggtggct tcggctacca cttacagatg gacccgcggc 240

gcattagcta gttggtgagg taacggctca ccaaggcgac gatgcgtagc cgacctgaga 300

gggtgatcgg ccacactggg actgagacac ggcccagact cctacgggag gcagcagtag 360

ggaatcttcc gcaatggacg aaagtctgac ggagcaacgc cgcgtgagtg atgaaggttt 420

tcggatcgta aagctctgtt gttagggaag aacaagtgcc gttcaaatag ggcggcgcct 480

tgacggtacc taaccagaaa gccacggcta actacgtgcc agcagccgcg gtaatacgta 540

ggtggcaagc gttgtccgga attattgggc gtaaagggct cgcaggcggt ttcttaagtc 600

tgatgtgaaa gcccccggct caaccgggga gggtcattgg aaactgggga acttgagtgc 660

agaagaggag agtggaattc cacgtgtagc ggtgaaatgc gtagagatgt ggaggaacac 720

cagtggcgaa ggcgactctc tggtctgtaa ctgacgctga ggagcgaaag cgtggggagc 780

gaacaggatt agataccctg gtagtccacg ccgtaaacga tgagtgctaa gtgttagggg 840

gtttccgccc cttagtgctg cagctaacgc attaagcact ccgcctgggg agtacggtcg 900

caagactgaa actcaaagga attgacgggg gcccgcacaa gcggtggagc atgtggttta 960

attcgaagca acgcgaagaa ccttaccagg tcttgacatc ctctgacaat cctagagata 1020

ggatgtcccc ttcgggggca gagtgacagg tggtgcatgg ttgtcgtcag ctcgtgtcgt 1080

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ttgggcactc taaggtgact gccggtgaca aaccggagga aggtggggat gacgtcaaat 1200

catcatgccc cttatgacct gggctacaca cgtgctacaa tggacagaac aaagggcagc 1260

gaaaccgcga ggttaagcca atcccacaaa tctgttctca gttcggatcg cagtctgcaa 1320

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ttcccgggcc ttgtacacac cgcccgtcac accacgagag tttgtaacac ccgaagtcgg 1440

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gtacaattgc catggcatct acggacggaa ttcagcgcgg aatggaagca gttgatacag 240

gtgctccgat ttccgttcct gtcggtgatg tgactctcgg acgcgtattt aacgtactgg 300

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gatcagctcc ttcattcgat gagctttcaa ctgaggttga aattcttgaa acgggaatta 420

aggttgttga cctgctcgct ccttacatca agggcggtaa aatcggattg ttcggcggcg 480

ccggcgtagg taaaaccgta ttaatccagg aattaatcaa caatatcgcg caagagcacg 540

gcggtatttc cgtattcgcc ggtgtaggag agcgtactcg tgaagggaac gaccttttct 600

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cgggttcaga ggtttccgcg cttctcggcc gtatgccttc tgcggttggt tatcagccga 840

cgcttgcgac agaaatgggt cagcttcaag agcgtattac gtctacaaac gttggttcag 900

ttacatctat ccaggcgatc tacgtgcctg ccgatgacta cactgacccg gctccggcga 960

caacgttcgc tcacttggat gcgacgacta accttgagcg taaattaact gaaatgggta 1020

tctaccctgc ggttgatccg ctggctggct tccacttccc gcgcgcttgc tccggaaatt 1080

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cagtttacgg gacaaaaggg ttcttacgtg cctgttaaag aaacggtacg aggctttaaa 1320

gaaatccttt ccggtaaata tgaccatctt ccggaagatg cattccgtct tgtgggacgc 1380

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tgacaaacca tataaaaaat ctgcccgtat cgtcggtgaa gttatcggta agtaccaccc 240

gcacggtgac tcagcggttt acgaatcaat ggtcagaatg gcgcaggatt ttaactaccg 300

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gaatcctgag attacaaacc aggagctgat ggaatacatc ccgggcccgg attttccgac 660

tgcaggtcag attttgggcc ggagcggcat ccgcaaggca tatgaatccg gacggggatc 720

cattacgatc cgggctaagg ctgaaatcga agagacatca tcgggaaaaa agaaagaatt 780

attgtcacag aacttcctta tcaggtgaac aaagcgagat taattgaaaa aatcgcagag 840

atcttgtccg ggacaaaaaa atcgaaggaa ttaccgatct gcgtgacgaa tccgaccgta 900

acggaatgag aatcgtcatt gagatccgcc gtgacgccaa tgctcacgtc attttgaata 960

acctgtacaa acaaacggcc ctgcagacgt ctttcggaat caacctgctg gcgctcgttg 1020

acggacagcc gaaggtacta agcctgaaac aatgtctgga gcattatctt gatcaccaaa 1080

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ttctggaagg tttaagaatt gctctcgatc atttagatgc tgttatttct cttatccgca 1200

gttcccagac ggctgaaatt gcgagaacag gtttgatcga acaattctca ctgactgaaa 1260

aacaagcgca agccattctg gatatgcgcc ttcagcgatt aaccggtctg gagcgcgaaa 1320

agattgaaga ggaatatcaa tcacttgttg cgcttatcgc cgagctgaaa gatattttag 1380

ccaatgaaga aagagttctt gagatcatcc gtgaagaact taatgaaatc aaagagcgtt 1440

tcaatgatga gagacgcact gaaatcgtga cgtccggtct tgaaacaatt gaagatgaag 1500

atcttatcga gagagaaaac atcgtcatca cactgaccca taacggctat gttaaacgtc 1560

tcccggcgtc cacataccgc agccaaaaac ggggcggaaa aggcgtacag ggaatgggaa 1620

cgaatgaaga tgactttgtg gaacacttaa tttccacatc aacgcacgat acgatcctgt 1680

tcttctctaa taaagggaag gtctaccgtt ctaaaggtta tgaaatccct gaatacggcc 1740

gtacggcaaa aggcattccg attattaacc ttctggaagt tgaaaaaggc gaatggatta 1800

atgccattat tccggtcagc acgtttgatg aagagcaata cctcttcttt acgaccaaac 1860

aaggggtgtc gaaacgtacg gcactgtccc agtttgcgaa tatacggaat aacgggctga 1920

tcgccctagg tctccgggaa gacgatgaac tgatggccgt ccgcctgact gacggcaaga 1980

aacaaatcat catcggaacg aagaacggtt tgctgatccg tttccctgaa gaagatgtaa 2040

gacaaatggg acgcacagct gcgggtgtta aaggcatcac gctgacagat gatgatgtcg 2100

ttgtaggcat ggagattctt gaagaagact cccacgtcct catagtgaca gaaaacggct 2160

acggcaaacg cacaccggct tcagaataca gagttcaaag ccggggcgga aaaggcctta 2220

aaacatgtaa aatcactgac agcaatggtc cgttagtaac cgtgaaagca acaaaaggcg 2280

aagaggactt aatgattatt acggcaagcg gcgtgttaat cagaatggat attaatgata 2340

tgatatttcc actacgggac gcgtaacaca aggcgtccgt ctgatccgca tgtctgatca 2400

agagcatgta gccactgtgg cgcttgtgga aaagaatgaa gaagagccgg aagaaacgga 2460

agaagtataa 2470

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<213> Bacillus belgii (Bacillus velezensis)

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atgtctgatt atctagtcat cgtggaatcg ccggcaaagg cgaaaacgat tgaacgttat 60

ttagggaaga aatataaagt caaagcttca atgggacatg tccgggatct tccgaaaagt 120

caaatgggag ttgacatcga acagaacttt gaaccgaaat atattacgat ccgcggaaaa 180

ggccctgttt taaaagaatt aaaaacagcg gcaaagaaag caaaaaaagt ctatctcgcg 240

gccgaccccg acagagaagg ggaagcgatt gcatggcatc tcgcacacag tcttgatctg 300

gatctcagct cggactgccg ggtcgtcttt aatgaaataa cgaaagacgc cattaaagaa 360

tcatttaaac atccgcgcat gattaatatg gatcttgtcg atgcgcagca ggcgcgccgc 420

atactagaca gactcgtcgg atacaaaatc agcccgattc tttggaaaaa agtaaaaaaa 480

ggcctcagtg cgggccgtgt gcaatccgtt gcgctccggc tgatcatcga ccgggaaaag 540

gaaatcaacg actttaaacc tgaagagtac tggacgatta ccggctcatt tttaaaggga 600

aaagagacgt ttgaagccgg atttttcgga aaaaacggca aaaagcttcc tttaaaaaac 660

gaagaagacg taaaagccat ccttgcccag ctgaaaggca ataagtatac agttgacaag 720

gttacgaaaa aagagcgcaa acgaaatccc gctctgccgt ttacaacgtc cactctgcag 780

caggaggctg caagaaagct gaatttcaga gcgaagaaaa cgatgatgat cgctcagcaa 840

ttatatgaag gaattgatct cggcaaagaa gggacagtcg ggctcatcac gtatatgaga 900

acggactcaa cgcggatttc caacacagcc gttgaagaag cgtcagcttt tattgatcag 960

gcgtacggaa aagagtactt agcgggaaaa cggaaaccgg caaagaaaaa tgaaaacgct 1020

caagatgccc acgaagcgat ccgcccgact tcggtgctca gaaaacctgc cgacttaaaa 1080

gcggtgcttg gaagagacca gctcagatta tacaagctga tttgggagcg ctttgttgca 1140

agccagatgg cgcctgccgt ccttgatacg atgagcgtcg atctcagcaa taacggattg 1200

acattccggg caaacggaag taaagtcaaa ttcgccggct tcatgaaagt ctatgtagag 1260

ggtaaagacg atcagctgga agaaaaagac cgcatgcttc cggaccttaa agagggagac 1320

acggttttat cgaaagatat tgagcctgag agcagcactt tactcagccg cctccgcgtt 1380

atacagaggc gcgcctggtt aaaacgcttg aagaactggg gatcggccgt ccttcgacat 1440

atgcgccgac tcttgacacc atccagcgcc gcggttacgt gggattagac aataaacggt 1500

ttgtgccgac tgagcttggg cagatcgtgc tggatcttat catggagttt ttcccggaaa 1560

tcattaacgt tgaattcacg gctaaaatgg aaagagacct tgaccatgtt gaagatggcc 1620

agacagaatg ggttcaaatc attgacagct tctatactga tttcgaaaaa cgcgtgaaaa 1680

aagcggaagc cgaaatgaaa gaagtggaaa ttgaacctga atacgccgat gaagattgtg 1740

agttatgcgg ctcccggatg gtttataaaa tgggccggta cggcaaattt atggcgtgct 1800

ccaacttccc tgactgccgg aatacaaaac cgattgtcaa acaaatcggc gtaaaatgcc 1860

ctaagtgcca tgaaggaaac attgtcgaac gaaaatcaaa aaagaaacgc atcttttacg 1920

gctgtgaccg ttatcctgaa tgcgacttcg tatcttggga caaaccgatt gaacgaaaat 1980

gcccgaaatg tgaaaatatg ctcgtagaga aaaagcttaa aaaaggcata caagtacaat 2040

gcgtcgaatg cgattataag gaagaaccac agaagtag 2078

<210> 5

<211> 1923

<212> DNA

<213> Bacillus belgii (Bacillus velezensis)

<400> 5

atggaacagc agcaaaatag ttatgatgag aatcagatac aggtattaga aggtttggaa 60

gctgttcgaa agagaccggg gatgtacatc ggatcaacta acagcaaagg ccttcaccac 120

ttggtgtggg aaatcgtcga caacagtatt gacgaagccc tggccggtta ttgtacagat 180

attaacatcg agattgaaaa agataacagc attaccgtta aggacaacgg gcgcggaatt 240

ccggtcggta tccaggagaa gatgggccgc cctgcggttg aagtcatcat gaccgttctc 300

cacgccggcg gtaaatttga cggaagcgga tataaagtat ccggcggtct tcacggtgta 360

ggggcgtctg tcgtaaacgc cttgtcgacc actcttgacg ttacggttca tcgtgacgga 420

aaaatccact atcaggcgta cgagcgcggt gtacctgtgg ccgatcttga agtgatcggt 480

gatactgata agaccggaac gattacgcac ttcgttccgg atccggaaat tttcaaagaa 540

acaaccgaat acgactatga cctgctttca aaccgtgtcc gggaattggc cttcctgaca 600

aaaggtgtaa acatcacgat tgaagacaaa cgtgaaggac aagaacggaa aaacgagtac 660

cactacgaag gcggaatcaa aagctatgtt gagtacttaa accgttccaa agaagtcgtt 720

catgaagagc cgatttatat cgaaggcgag aaagacggca taacggttga agttgcattg 780

caatacaacg acagctatac aagcaatatt tattctttca caaataatat caacacatac 840

gaaggcggca cgcacgaagc cggatttaaa accggtctga cccgtgttat aaacgactat 900

gcaagaagaa aagggatttt caaagaaaat gatccgaatt taagcgggga tgatgtgagg 960

gaagggctgg ctgactgcca ttatttcaat taagcaccct gatccgcaat tcgaagggca 1020

gacgaaaacg aagctcggca actccgaagc gagaacgatc actgatacgc tgttttcttc 1080

tgcgctggaa acattccttc ttgaaaatcc ggactcagcc cgcaaaatcg ttgaaaaagg 1140

tttaatggcc gcaagagcgc ggatggcagc gaaaaaagcg cgggaattga cccgccgcaa 1200

aagtgcgctt gagatttcca atctgccggg caaactggcg gactgttctt ctaaagatcc 1260

gagcatttcc gagctgtata tcgtagaggg tgactctgcg ggcggatcag cgaaacaggg 1320

acgggaccgt catttccaag ccattctgcc gctgcgcggt aagattctga acgttgagaa 1380

agccagactt gataagattc tctcaaacaa tgaggtcaga tcaatgatca cggccctcgg 1440

aacaggaatc ggagaagatt ttaatcttga aaaagcgcgc gttatcataa agtggtcatc 1500

atgacggatg ccgatgttga cggcgcccac atcagaacgc ttttattaac gttcttctac 1560

agatacatgc gggaaatcat cgaaaacggc tatgtctaca ttgcccagcc gccgctttat 1620

aaagtgcagc agggaaaacg ggtggaatac gcttataatg ataagcagct tgatgagctg 1680

ttaaaagaac ttccgcaatc acctaagccc ggcctccagc gttataaagg tcttggagaa 1740

atgaacgcga ctcagctttg ggaaacgaca atggaccctg cgaccagaac gcttctgcaa 1800

gtcaatcttg aagatgcaat ggacgctgac gagacttttg aaatgctgat gggtgacaaa 1860

gtagaaccgc ggagaaactt catagaagca aacgccagat acgtgaaaaa tcttgatatt 1920

taa 1923

<210> 6

<211> 1737

<212> DNA

<213> Bacillus belgii (Bacillus velezensis)

<400> 6

ttgttagctg aactatcaat aaaaaaactt tgccattata gaggaactga cgatttcgtt 60

tgaacggggg ctcaccgtcc tgacgggaga aacaggggcc ggcaagtcaa ttattataga 120

cgccgtttcc ctattagtcg gcggccgcgg ctcgtctgaa tttgtccgtt acggtgaaac 180

aaaggccgag cttgaaggac tgtttctttt ggacagcggt catcccgttt ttgatgtatg 240

ccgtgagcag ggaatagacg catctgatga catgattgtc atgagaaggg acatcaatgc 300

cggaggaaaa agcgtctgcc gtgtcaacgg aaaattagtg acgattgcgg ctttaaggga 360

aatcggcaga cttttattag atattcacgg acagcatgat aaccagctgt taatggagga 420

cgataaacac ttggagcttt tggacaggtt tgcgggcgct gaggccgaat ccgcgcttca 480

ggcttaccat gaaggctatg agcgttatat gaagctgctc aaaaaagtga agaagctgtc 540

agaaagcgag caggaaatgg cgcatcggtt agacctgatt caatttcagc ttgaagaaat 600

cgagtcggcg aagcttgaat tgaatgaaga cgaacttctt caggaagagc ggaagcagat 660

atccaactga aaaaaaaatt tatgaatcat tgcaaaacgc ctataatgcg ctccgcagcg 720

agcagggggg gcttgactgg gtcggaatgg cctctgccca gcttgaagat atttcggata 780

tcaaatgaac cgctgcaaaa aaagctgtca gaaagtgtgt caagttccta ttatctgctg 840

gaggatgcaa cctttcagat gcgcaatctg ttagatgatc tggaatatga cccggaacgg 900

ctgaatttta ttgaaacccg tttaaatgag atcaaacagc tgaagcgcaa atacggagcg 960

acggtagaag acattttgga atacggctcc aaaatagaag aggaaatcga tcaaatcgag 1020

aaccgcgaca gccaccttga agcgctgaag aaggaattgg agtcggtcgg caaagatgtg 1080

gccgtagaag ccgccaattt atcaaaaatc agaaaagctt gggctaaaaa actggcggaa 1140

gcgattcatc aggaattaaa aagtctgtat atgggcaaat caacgtttga tacggaattt 1200

ctcgtgaaaa ccgatccgtc agccagtgaa gcaccggttg ttaacgggca gcctgtgcag 1260

cttacccaaa aggggattga ccttgtgaag tttttaattt caacaaacac cggtgagcct 1320

ttaaaaccgc tgtcaaaggt cgcttccgga ggcgagcttt caagggtgat gctggcgatg 1380

aagagtattt tttcttccca gcaggatgtc acctccatca tctttgacga agtggacacc 1440

ggtgtaagcg ggcgggttgc ccaagccatc gcggaaaaaa tccataaagt atcgaccggt 1500

tcacaggtgc tctgcattac acacctgccg caggttgccg ccatggcgga tacacacctt 1560

ctgatcgcaa aagaattcaa agacggccgg acgacaacgc atgtcacgcc gctttccaaa 1620

caggacaagg tagcggaaat cggccgcatg attgcgggcg ttgaagtgac cgatctgaca 1680

aaacggcatg cacaagagct tctaaaacag gccggacagg taaaaacgac aggataa 1737

<210> 7

<211> 1857

<212> DNA

<213> Bacillus belgii (Bacillus velezensis)

<400> 7

aaaaacaaga agaacaacaa caacaacaaa aacaaaaaca aaacgcaatc ataacaacaa 60

aaatcagtat cagcaaaaac cgctgaagcc gaaaaaagag cttcctgaga aaattacgtt 120

ttccggctct ttaacagtcg gagctttagc cgaggaactg ggcaaagagc cgtctgaact 180

cattaaaaaa ctgatgcttc tgggcgtaat ggctacgatt aaccaggagc ttgacaaaga 240

cacaatcgaa ctgatcgcat ctgaatacgg tgtggaaaca gaagaagtga ttgtgcttga 300

agaaactgag cttgaaaaat atgaagaggc tgacaaagaa gaagatcttc aaatccgtcc 360

gcctgtcgtg acgatcatgg gccacgttga ccacgggaaa acgaccctcc ttgacagcat 420

cagaaaaaca aaagttgttg aaggtgaagc cggcggaatc acccagcata tcggggctta 480

ccagatcgaa gaaaacggca aaaaaatcac gttcctagat actccaggac acgcggcgtt 540

cacaacaatg cgtgcgcgcg gtgcggaagt aactgatatt accattttag tcgtagcggc 600

tgatgacggc gttatgccgc aaacagtcga agccatcaac catgcgaaag cggctgaggt 660

tccgattatc gttgccgtga ataaagtgga taaagaaagc gcgaaccctg accgtgtcat 720

gcaggagctg actgaatacg gactcgttcc tgaagcatgg ggaggagaaa cgattttcgt 780

tcctctgtcc gctcttacag gcaagggaat tgacgagctt gtcgaaatga ttctgcttgt 840

cagtgaagtg gaagagctga aagccaatcc gaaccgtcag gcaaaaggaa cggttattga 900

agctgagctc gataaaggaa gaggatctgt cgcgacactt ctcgttcaaa cgggaacatt 960

gaatgtcgga gatccgatcg ttgtcgaaat acattcggcc gcgtgcgggc catggtcaat 1020

gatctcggcc gccgcgtgaa aacagccgga ccgtccacac ctgtagaaat tacgggctta 1080

aacgacgtac cgcaggctgg cgatcaattc ctcgtcttta aagacgaaaa aaaaacagcc 1140

cgttctgtcg gtgaagcacg cgcttccaag caactggaag aacagcgcag cgacaaagcg 1200

aagctgagcc tcgatgatct gtttgaacaa attaaacaag gcgatgtcaa agacatcaat 1260

ttaatcgtaa aagctgacgt tcaaggttca gccgaagcgt taacagccgc tcttcaaaaa 1320

attgaagtag aaggcgtaaa agtgaaaatc attcacacag gcgtcggtgc gattacggaa 1380

tcagacatca ttctcgcatc tgcttcaaat gcaatcgtta tcgggtttaa tgtgcgccca 1440

gacggaaatg ctaagagcac ggctgaagct gaaaatgtag atatccgtct tcaccgtatc 1500

atttacaaag tcatcgaaga aattgaagcg gcgatgaaag gaatgcttga tcctgaatac 1560

gaagaaaaag tcatcggtca ggttgaagtg cgccaaacat tcaaagtgtc gaaaatcggc 1620

acgattgccg gcggatatgt aacagacggc cacattacac gtgacagcgg actccgcctg 1680

atccgtgacg gcgtggtcat cttcgaaggg gaagtagacg ttctgaaacg cttcaaagat 1740

gatgtgaaag aagtttcaca aggatatgaa tgtggtatta caattaagaa atacaatgac 1800

attcgcgaag gcgacatcat tgaagcgtat gtcatgcagg aaatcgaaag aaagtga 1857

<210> 8

<211> 1773

<212> DNA

<213> Bacillus belgii (Bacillus velezensis)

<400> 8

atgaataaac aactgaaaga aaagctggcc ctccttcccg atcagccggg ctgttattta 60

atgaaagacc gtcaaaaaac agtcatctac gtggggaaag cgaaagtctt gaaaaacagg 120

gtgcggtctt atttcaccgg ttctcatgac gcaaaaaccc aacggcttgt gacggagatt 180

gaggactttg aatatatcgt gacttcctcc aatcttgaag cgcttatttt ggaaatgaat 240

ctgattaaaa aatacgatcc gaaatacaat gtcatgctga aagatgataa aacctatccc 300

tttattaaaa tcacccatga acgccatccg cggctgattg tcactcggaa tgtcaaaaaa 360

gacaaaggac gctatttcgg cccgtatccg aatgtgcagg cggcgcggga aacgaaaaaa 420

ctgctggacc gcctttaccc gctcaggaaa tgctcaaagc ttccggaccg cgtctgtctc 480

tattatcatc tcggacaatg cctcgcccca tgtgtaaagg atatatctga ggagacaaac 540

cgcgagattg ttgaagacat cacccgtttt ttaaagggag ggcataacga aatcaaaaaa 600

gagctcaaag cgaaaatggc agaggcggcg gaaaagcttg aatttgaacg ggctaaagaa 660

ttccgcgacc agcttgctca catcgaatcg acgatggaga agcaaaaaat gaccatgaat 720

gaccttctgg accgcgatgt gtttgcatat gcctacgata aaggctggat gtgcgtccaa 780

gtctttttca ttcgccaagg aaaactgatc gaacgcgatg tcagcatgtt tccgatgtat 840

caggaagcgg acgaagagtt tctgaccttc atcggccagt tctattccaa aaacaatcac 900

tttctgccta aggaaattct ggtgcctgac agcgttgaca gggacatgat cagcgagctg 960

ttggaaaccg ccgtgcatca gccgaaaaaa ggcccgaaaa aagagctgct tttgcttgcg 1020

cacaaaaatg cgaaaatcgc tctgcgggaa aaattctcgt taatcgagcg ggatgaggaa 1080

cggtcaatcg gtgcagccga acggctcggc gaagcgctga atatttacac gccgcatcgg 1140

attgaagcgt ttgacaactc caatattcaa ggcacaaacc ccgtatcggc catgattgtc 1200

tttattgacg gaaaaccgaa taaaaaggaa taccgcaaat ataaaattaa aaccgtaaca 1260

gggccggatg attacggttc aatgagggaa gtcgtcagaa gaaggtacac gagggtgctc 1320

cgcgaaaacc ttccgctgcc ggatttaatc atcattgacg gcggaaaagg gcagattaac 1380

gcggcgcgcg acgtgctcga gaatgagctc ggccttgatg tcccggtagc gggattggcg 1440

aaagatgata aacaccgaac atccaacctt ttgatcggcg acccgcttga accggtgttt 1500

ttggagcgga acagccaaga gttttacctg cttcagcgta ttcaggacga agttcaccgt 1560

tttgcgatca gctttcacag acagatccga ggaaaaagcg tctttcaatc cgttctggat 1620

gatatccccg gaatcggcga aaaacgcaaa aaaatgctct taaaacattt cggatctgtc 1680

aaaaagatga aggaagcaag ccttgacgat atcaaaaaag cgggtgtgcc ggccgcggcg 1740

gcccagctgc tgtttgaaaa attaaaaaaa tag 1773

<210> 9

<211> 1155

<212> DNA

<213> Bacillus belgii (Bacillus velezensis)

<400> 9

atgttggatt tcgaaacaaa catagacggc ttagcatcaa ttaaagtaat cggagtagga 60

ggcggcggta ataacgcggt caaccgaatg attgaaaatg aagtgcaggg tgtagagtag 120

agtacattgc ggtaaacacg gacgcgcaag ctcttaacct gtcaaaagcg gaagtgaaaa 180

tgcagatcgg ggaaaagctg acccgcggtc tcggagcggg agcgaatccg gaagtcggca 240

aaaaagccgc tgaagaaagt aaggaacaaa tcgaagaagc gctaaaaggc gctgatatgg 300

tgttcgttac agccggaatg ggaggcggta ccggaacggg agccgcaccg gttatcgcgc 360

aaatcgcaaa ggacttaggc gctttgactg tcggtgtcgt aacaagaccg tttacatttg 420

aaggaagaaa acgccagctg caggctgcag gcggtatcac ggcaatgaaa gaagccgttg 480

acacactgat cgttattccg aatgaccgca ttcttgaaat tgtcgataaa aacacaccga 540

tgcttgaagc attccgtgaa gctgataacg tgcttcgcca aggggtgcaa ggtatctctg 600

acctcatcgc gacgcctggt ttaatcaact tggactttgc cgatgtcaaa accatcatgt 660

ccaacaaagg ttcagcattg atgggtatcg ggatcgctac gggcgaaagc cgtgctgctg 720

aagccgccaa aaaggcgatc tccagtccgt tactggaagc ggcaatcgac ggagcgcaag 780

gtgttttgat gaacattacg ggaggtacaa acctgagcct ctacgaagtg caggaagcag 840

ccgacatcgt cgcttctgca tctgaccctg atgtcaacat gattttcggt tcagttatca 900

atgaaaatct gaaagatgaa atcgtcgtca ccgttattgc gaccggtttt atcgaacagg 960

aaaaagatga ctcaaaaccg cagcgaccga ctttttaacc aaggattgaa gtctcaaagc 1020

caaaacgctg cgaaacggga gccgaaacga gaagaaacac agcatcaaaa cacggtgaac 1080

cgccatacat cacagccggc ggatgatgcc ctcgacattc ctacattttt aagaaaccgc 1140

aacaaacgcg gctga 1155

<210> 10

<211> 16086

<212> DNA

<213> Bacillus belgii (Bacillus velezensis)

<400> 10

atgtcggtat ttaaaactca agaaacgtac tgggaaaacc tgtttgatga agaagacggc 60

ctaagcgcat tcccttactt taaagcagcg gataaagcgt cgttggcccg taccggctac 120

caggaaaaat gcatctgccg ttccttatct ccggaagtat ctcaaagaat catgacaatg 180

gcgaatcatt ccgatatggc ggcctatctg attttattgg cgggtattga atgcttgttg 240

tataaatata cggatcgaac gagcctgatt cttggcattc caacggtatc gaagcaaaaa 300

gccggtcagt cagccgttaa caacattgtt ctcttaaaaa atacgctttc aaacgagagc 360

acatttaaaa ccgtattcgg gcagctgaaa gaagccgtta atgattctct gaaaaatcaa 420

aacctgccgt ttagaaaaat ggtccggcat ttaagcgtgc aatacaatga cgagcatatg 480

cctttgattc ataccgtcgt ttcgcttaac gaaattcatt ccttgcaatt taaagaagat 540

actgcaaccg atacgctgtt tcactttgac ttggagaaca acgggattca tttgaagctt 600

ttttataacg gcaatctgta cgatgagcgc tatattaatc aaattgtatc ccatttggat 660

cagctgctgt ctgtgattct gtttcagcct caagccgcga tccatacggc agagatctta 720

ccggaagcgg aaaaacagaa gctattgttt gattttaacg acaccggcac agatttctcc 780

gggagcagaa cagtctatca attatttgaa gagcaagcag aaagaacgcc tgagcatacg 840

gccgtcaagt ttaaaaacga ccatttgacg tacagggaac tgaatgagaa ggcgaaccgt 900

ttggcaagaa cattgcgtaa ttgcggggtt caacccgata cgttagtcgc cattctcgct 960

gatcgttcat tagaaatgat cgtatccatt atagccgttt ggaaggcagg gggcgcttat 1020

gtgcccctgg atcctgaata tccaaaagag cggcttcaat atttgcttca tgatgcggat 1080

gcagacgttc tccttgttca gcaacattta aaaaacagcc tcgcctttga cggccgggtg 1140

attgatttga atgatgaaac atcataccat gccgattgct cattgttatc accaatagcg 1200

gaacacagcc atttagccta tgtcatttat acgtcgggta caacaggaaa gccgaaaggg 1260

gtcatggttg aacacggcgg cattgtgaat tcgctccaat ggaagaaggc gtttttcaag 1320

cattctccgg cggatcgcgt gcttgtcttg tacccgtatg tttttgatgc gttcatttta 1380

aacttcttcg gcccgctcat ctcaggagca actttgcacc ttttgcctaa tgaagagaat 1440

aaagaaactt tcgccattca aaatgccata aaacaagaga gaattactca tttttcaact 1500

tctccccgtc ttctgaaaac gatgattgaa cagatgaacg cagaggattt catccatgtt 1560

cagcatgttg ttgtaggcgg tgagcagctg gaaacagata cagttgagaa gctgcattct 1620

ctgcagcctc gcattcggat caataatgaa tacggaccga cagaaaacag cgtggtatct 1680

acatttcacc ctgttcagtc cgctgatgaa cagatcacaa tcggcagccc ggtggccaac 1740

catcaagctt atattttagg agcgcaccat caaatccagc cgatcggcgt accgggagag 1800

ctgtatgtcg gaggagcggg agtggcccgc ggctacctca atcgggctga attgacggag 1860

gaaaaatttg tggagcatct gcatgtccct ggtcaaaaga tgtataaaac gggggatctc 1920

gccagatggc tgccggatgg aagaatcgaa tatttagggc ggattgatca tcaagtgaaa 1980

attcgcggct accggattga gatcggcgag gtagaggcag ccatgttcaa tctggaaaac 2040

gttcgtgaag ccgcagtcgt cgcgcgggaa gacgcagacg gggccaaaca attgtatgcc 2100

tattatgtcg gcgagccttc tttgaccgct gcgcagttca gagaagactt atcccgagaa 2160

ctgccgaatt atatgattcc ctcccgcttt atccctttgg aacgcattcc gctcacttca 2220

aatgggaaga ttgatctgaa agctttgcct gctgctgacg aaaatacgcg ggcggaatac 2280

gaatacatag ccccgcggaa cacaatagaa gaactgctgg tgtccatctg gcaagaagta 2340

ttaggggcag aacgtatagg aatttcggat aatttctttg attttggcgg agattcgatt 2400

aaatcaattc aggtgtcatc cagattgtat caagccggat acaaggttga tatgaagcat 2460

ttgtttaaac acccctcgat tgctgaactt agtcagtttg tagcgccggt cagcagggtg 2520

gctgatcaag gagaagtgaa tggcggaacc aagctgaccc cgatacagca ttggtttttt 2580

gagcaaaaaa tgccgcatgc acaccattac aatcaggccg tcatgttata ttcggcggaa 2640

ggctttaaag aggctccgct tcgccggacg atggaacgga tcgcgtcgca tcacgatgcc 2700

ttgaggatga tttttgaaaa aacgccggat ggatatgctc cgcgaattac aggaacagat 2760

gaaagtaaac tataccatct ggaagtgatg aattataaag gggaaaccga tccggctcaa 2820

gcgattgccg ataaagcgaa tgagattcaa agcagcatgg tattagacaa agggccattg 2880

atgaagctcg gtttgttcca atgtccggac ggggatcatt tgctgattgc aattcatcat 2940

ttgctgattg acggagtatc ctggcgtatt ttactcgaag attttgcaag cggatatgag 3000

caggcagaac gcggacaaac aatccagctc ccgcaaaaaa cagactcgtt tccattctgg 3060

gccgatcaat tgtccaaata tgcggcggaa accgatatgg aagaggagat tgcgtattgg 3120

actgagcttt caagtataaa gcctcagcca ttgcctaagg acactatcag cgaaggatcg 3180

ttattaagag acagcgaaga agtgacgatt caatggacaa aagaggaaac ggagcaatta 3240

ttaaaacatg cgaaccgcgc ctacaacacg gatatcaacg atctgctgct gacttcgtta 3300

gggctggctg ttcacaagtg gacaggcacg gaagacattg tcgtaaattt ggaaggccat 3360

gggagagaac cgattattcc ggatgctgac atttcccgca cgataggatg gttcacaagc 3420

cagtaccctg tcgtcctgag gatggaagcc ggaaaaaacc tgtcgcagcg gataaagatc 3480

gtaaaagaag ggctgcgccg catccctgac aaaggaatga attacagcat catcaaatat 3540

atatccgggc atccggaagc cgacagcctg cagctgaatc cggagatatc gtttaactat 3600

ttgggacaat ttgaccaaga cctgaagcac caggctttac gcatctcgcc tttttccaca 3660

gggttatcaa tgaacgaaaa ccaagagaga acggccgtgc ttgatttgaa cggcatgatt 3720

gccgaaggga cattatcact gacactgagc tacagcagca aacaatacga aagatctact 3780

atggcgcaat ttgcccgggg gctgaaagaa agtctgcaag aagtcattgc gcattgcgtg 3840

agccggcagc agacttctct cacaccaagt gacatcctgt taaaagacat ctcaatcgat 3900

gaactggaac agcttttgga gcagacgcgt gaattaggcg aagcggaaaa tatttatcca 3960

ttaacgccga tgcagaaggg catgctgttt cacagcttat ttgatccgaa ttcaggtgct 4020

tattttcagc aaacgatgtt tgatctgcat ggagatctgg agattgattc attttcaaaa 4080

agcttggacg gcttatcgca aaaatatgac attttccgca cgaattttta cagaggctgg 4140

aaagatcagc cgctccaaat catattcaaa acgaaaaaaa tcggttttca attcaatgac 4200

ttacgagaga tgaaggaatc acaaaaagaa gcgatgattc aggaatatgc cagagaagat 4260

aagatgcggg ggttcgactt agaaaaaggc gcgttaatgc ggttattcat ccttcgtacg 4320

gatgaaaaga cataccgttt tatctggagt ttccaccata ttttgatgga cggctggtgc 4380

ctgccgctga ttacaaaaga aatattcgag aattactttg ccttgcttca gcaaaagcag 4440

cctgaacaat catcaattac tccgtacagt caatatattg aatggctcgg ccggcaagat 4500

gcgaaagagg ccgcggctta ttgggatcag tacttggagg gatatgagga gcaaaccggc 4560

cttcctaagg atcaccatgc agcggaagac ggacgatacg ttcctgaaaa agtgacgtgt 4620

gacataagct ccgatctcac ttcgaaaatg aagcggacgg caggcaagca tcatgtcacg 4680

ctgaacacgc tgctgcagac ggcatgggcg gttttgctgc agaaatataa ccgtagccgg 4740

gacgttgtgt ttggaagtgt tgtatccgga agacctgctg gaattccaaa cgtggaaacc 4800

atgatcggct tattcattaa caccattccc gtccggttcc gatgtgaagc cgggacaacg 4860

tttgctgagc ttatgaaaga ggcgcaggaa agggctgtag cgtctcaaca atttgagaca 4920

cacccgctgt acgatattca agcgcgtaca acgcaaaagc aagatttaat tacacattta 4980

atgatattcg aaaattatcc ggttgatcag tatatggaaa gtataggcag ccagaacgaa 5040

acatccatca aaatttccaa cgtacaaatg gaagaacaga ccaactacga ttttaatttg 5100

accgttatac cgggcgacga gatgaacatc tcttttgaat acaatgcaaa tgtgtatgat 5160

caggcaagca tagagcgtgt tcgggaacat ttcatgcaaa tcctgcatca agtcgtgaca 5220

gatgcggata tccgggtgga tcaagcggaa ttgttaacgg aaggcgaaag aagaaccctt 5280

ctgcagacgc tgaacgacac ggccgcgccg ttcccgcaga cgccggttca tcaactattt 5340

gaagagcaat cgcagcgtac gcccgatcaa gcagccgtca ttgataagga caggcagctc 5400

acgtacggag agctcaataa gcgggccaac cgactggcgc gaacgttaag agcgaaagga 5460

gtgcagacgg atcagcctgt agccatcatc acaagaaaca gcatcgaatc ggtggtcgga 5520

atccttgccg ttttaaaatc aggcggagcc tatgtcccta tagatccgga atatccgcaa 5580

gaccgcatcc ggtatatgct ggatgactcc caagccggaa tcgttttaat gcagcgggat 5640

gtcaggaaac agcttgccta tgaaggcgcc acggttttac ttgatgatga aagttcttat 5700

caccaagatg actctgacct tgctccgata aatgatgtga gccatttggc atatgtcatt 5760

tatacgtcag gttccacagg ccggccaaaa ggcgtactaa tcgagcacgg aggcttgacc 5820

aattatattt ggtgggcgaa agaggtttat gtaaaaggcg agaaagccaa cttcccattg 5880

tattcgtcca tttcttttga tctgacggtg acttctattt tcacgccgct tgtcacaggc 5940

aatgcaatca tcgtctatga cggagaggat aaaacagctc tgcttgaatc gattgtgcgg 6000

gacccgaggg tggatatcat taaattgacc ccggcccatt tgcaggtttt gaaagaaatg 6060

aacattgccg atcaaacggc tgtcagaaga atgattgtgg gcggggaaaa cttaagcacc 6120

cggttagccc gcagcattca tgaacagttc gaaggccgga ttgaaatatg caatgagtac 6180

ggaccgacgg aaaccgttgt cggctgcatg atttaccgtt acgatgccgc aaaggacaga 6240

cgggaatcgg taccgatcgg aaccgctgcg gcaaacacga gcatttacgt tcttgatgag 6300

aatatgaaac ccgcaccgat cggagtgccg ggagagatct atatcagcgg cgccggtgtg 6360

gctagaggat atttaaaccg tcccgaatta acagcagaga aatttgtcgt tgacccgttt 6420

gaacccggag cgaagatgta taagaccgga gatttggcga agtggctggc ggacggaaac 6480

attgaatatg cgggcagaat agatgaacaa gtgaaaatca gaggctatcg tatcgaatta 6540

ggcgagatcg aggcggcact gcttcaagaa gaagcgatca aggaagctgt cgttacagcc 6600

agagaggacg tccacgggtt taaacaattg tgtgcatatt acgtgagcgg cgggcaaaca 6660

acggcagccc ggctccggaa acaattgtct caaacccttg caagttatat ggttccggct 6720

tattttatcg agcttgatga aatgccttta acttctaacg gaaaaataaa cagaaaaggg 6780

ctgccggctc cggatttcga actgcaggac agagcagagt ataaagctcc ccgaacaaaa 6840

gctgaagaaa tattagtttc tgcctgggaa tcggtattag gcgctgaaaa cgtcagtatt 6900

cttgataatt tctttgacct tggcggagat tccattaaat ccattcaagt atcctccaga 6960

ttaaatcagc aaggatacaa aatggaaatc aaggacttgt ttcaatatgc aacaattgct 7020

gaattgagtc cgcatatcaa gcagaatctg cgtatagccg atcaagatga ggtcaaagga 7080

aaagtcagcc tgactcctat tcagcattgg tttttcgaac aaacaacaac agatcagcat 7140

tattataatc aagccgtcat gctgcatgcg ccggaaggtt ttcaggaaac gcagcttcgt 7200

caaaccctgc aaaagcttgc tgaacatcac gatgcgctcc gcatgacgtt ccggactacg 7260

gaaaatggct gcgaggcgca gaatgaagaa atcgcccaaa gcggactgta ccgcttagaa 7320

gtcctgaatc tgaaagaaga ttccgatccg ggacggacaa ttgaagccaa ggctgatgag 7380

atacaaagca gtatgcgttt aagtgatggg ccgttaatga aagccgtgtt gtttcaatgc 7440

gcaaatggag atcatctgct cattgccatc catcatttga ttatagacgg gatttcatgg 7500

cgcatcttac tggaagatat cgttagcggc tacaagcaag ccgagaacgg gcgagtgatt 7560

caattgccgc aaaaaacaga ctctttccaa ttatgggcta agagactttc agaatacgcg 7620

caaagtgaaa ccataaaaca agaacaggaa tattggacaa agatcgaaca aaccgatgtc 7680

aaaccgctgc ctaaagattt tcatgaaacc catacaactg caaaagacag tgaaacagca 7740

actgtggaat ggacgaaaga ggaaacggat ctgctattaa aacaagcgaa tcgcgcttat 7800

aacacggaaa taaacgattt gcttttaact tctctgggtc tttccatatc gcattggtca 7860

ggactggaac aaattccgat tcatttagag ggacacggca gagaacagat tatacaagat 7920

atagatatct cccgcacggt cgggtggttt acaagtctgt atccggttgt gctgcacgcg 7980

cagccgggca aagaaatctc tgattatatc aagacgacca aagaaggatt gcgccaaatt 8040

ccgcataaag ggatcggata tggaatagca aggtacttaa gcggcggaat gccatcgaaa 8100

ctcaatcctg agatcagctt taactacctg ggacagtttg accaagattt acagcagcat 8160

ggggttcaat tatcttctta ttcttgcggt tcagactcaa gcgaaaatca agaaagaccg 8220

tatgtattga atataaacgg aatgatcacc gagggccggc tgaggcttac gatcagctac 8280

agcagcaaac agtacgcaaa agaaacgatc atgcggttat cggaaacgat tcaaagccgc 8340

ctgcggtcca tcattacgca ttgcgttcat aaagaacaat cggaactgac gccaagcgat 8400

attttgttaa agggcatgtc cattgatgaa ttggatcagc tcctcattca actgccgcat 8460

gcaggtgagg ttgaaaacgt gtatccgctc actccgatgc agaaaggaat gctcttccac 8520

agcctgctgg acgaggcctc aagttcttat tttgaacaag catcgttcga tttgcaggga 8580

gagttaaaga ttgattggtt taaagcaagc cttgaacgat tatttgaaac atacgccgtg 8640

ctcagaaccc gtttttacag cggctggaac gatactcctt tacaaattgt ctataaaacg 8700

caaacaccgc aaattcattt tacagatttg cgtgacatag aggagcacct gcgagaggat 8760

gcaatcgcag cctatcaaag agaagacaag gcaaaaggat ttgacttggc tcgggatccg 8820

ttgatgcgca ttgcgatttt ccgtatggaa gaccgcaaat atcatttgat ttggagcttc 8880

catcatattg tgatggatgg ctggtgcctg tctcttatca cgaaggaagt gtttgatcat 8940

tatagcgcct tacaggaagg cagggaacca gaaccgccat cggcagctcc ttacagcgac 9000

tatatcgagt ggcttggccg ccaagatcag ggagcggcaa agcggtactg gagcaggtat 9060

cttgaaggat ataaaggtga aaccacgctt cttcataaaa tagcgcagca tgaacaaaaa 9120

gaatacgcct atgccaatgt gatctgccga tttgatcacg aacagacaaa acaattacag 9180

cagatcgcaa atcagcatca ggttacatta aatacgttga tccaaacgtt atggggaatt 9240

ttattacaga aatacagcgg gtccgcagat gttgtcttcg gcagtgtcgt gtcgggaagg 9300

ccggcggaaa ttcctgatgt ggaacagatg atcggcctgt tcatcaatac cattccggtt 9360

cgcatccgct gcgaggagga cagcactttt gctgatacga tgcaaatggt tcaacaaaac 9420

gcgctggctt cccaatcgta cgatacctat cctttatacg aaattcaggc ccaaacggaa 9480

caaaagcaga acctgattga ccatatcatg atttttgaaa actatccgat cgggcagcag 9540

gtcgaagaga cgggacatca tggtacggag ctgaacatta cgaattttca tatgcaggaa 9600

cactcccact atgatttgaa tgtggttgtt attcctggcg aacagctggc cgttcatttc 9660

ggttttaatg aaaatgaata tgaaaaatca gaagcggaac ggctctgcgg acattttgaa 9720

cagctcatgc agcaagtatt acagcagccg tctgtcaaaa tagaagaatt ggagcttctc 9780

tctcaacaag aaaaagaaca tctgctcagc cgctttcagt ctaatgacat gcaatacccg 9840

cgtgaaaaaa cgattcatga gttgttcgag gaacaggcgc aacgtactcc ggacaataca 9900

gcggttgtat tcgaggataa acaactcacc tacggagaac tgaatcggcg tgccaatcag 9960

cttgcgcgaa ccttgcaggc aaaaggtgtg cggaacgatc agcttgtcgg tattatgact 10020

gaacgctcgc ttgagatgat tgtaggcatc ttgggagttt taaaagcggg cggcgcatat 10080

ttgccgatag atcctgattc tccgtctgaa cgcatccgtt atattttgag tgattcaagt 10140

ctcagcgtat tgctttaccg cgggaagctg caggatgaca tcggcttctc aggaacatgt 10200

atcgaactca tggaggaaca tttctatcac gaaaaagaca gcagtcttgc actctcttat 10260

cagtccagtc aactggccta tgccatttat acttccggta cgacaggcaa gcctaaaggc 10320

acgttaattg aacatcgcca ggtcattcac ctgattgaag gacttcgacg ccaaatttac 10380

tctgcctatg aggcggagct gaatgtagcc atgctcgctc catattattt cgatgcttct 10440

gttcagcaaa tgtatgcttc cttgctgtca ggacatacct tgtttattgt tccgaaagag 10500

attgtgtcag acggtgctgc cttatgccgt tactaccggc agcattcgat tgacatcacc 10560

gacggaacac ctgcccactt aaagcttttg atagctgcgg gtgatttgca gggagtcacg 10620

cttcggcatc ttttaatcgg aggagaggct ctttccaaaa cgacggtaaa taagctcatg 10680

caattattcg gcgatcatgg cgctgcgcct gggattacca atgtatatgg gcctacggag 10740

acttgcgtag acgcgtctct gttcaacatt gaatgttctt ccgatgcgtg ggcacgcagc 10800

caaaactatg tccctatagg caagccgtta ggcagaaacc gcatgtacat tcttgattca 10860

aaaaaacggc tgcagccggc gggcgtccaa ggcgagctgt atattgcagg ggacggtgtc 10920

gggcggggtt acttgaacct gccggaactg acggacgaaa aatttgttgc cgatcagttt 10980

gtaccggagg accgaatgta cagaaccggt gatctggcct gccttctgcc ggatggaaat 11040

attgaatata tcggccggat tgatcatcag gtgaaaattc aaggttttcg gattgaactc 11100

ggtgaaatcg aatcggtgat gctgaacgtt cctgatattc aagaagccgc cgcagctgcc 11160

cttaaagatg cagatgatga atattacttg tgcggatact ttgctgcgga taagacaata 11220

cagatcagtg aacttcgcga aagaatggcc cggcatttac cgggatatat gatcccggct 11280

cattttgttc agctggataa gatgccgctg actccaaacg ggaagttgaa tcgccagtta 11340

ttgccggctc cggtcaaaaa gcgtgacagc ggcatagagt acgtcccgcc gcaaacttcg 11400

gcagaaatcc agctgacagc aatttgggag gatgtcctcg gattagagca ggtggggatc 11460

agagatcact tttttgagat cggcggacac tccctgcggg caacggcgct gattgcaaaa 11520

atacaaaagc aattgcatgt ccaaattcct ttgcgggacg tcttccgttt cccaaccatt 11580

gaacagctcg cacagacgat cacgaagacg gagctgaccg gatatgctgc gattcctgca 11640

attgaaaaaa gaccttatta tccggtatcc tcagcacaaa aacggttgta catcctgaat 11700

cacttggaag gcggagaact cagttacaac atgctgggac tgatggctgt cgaaggcaag 11760

cttgaccgtg acaaactgca gcaagccttc cgcacgctga ttttgcgcca tgaatctttg 11820

cgcaccggtt ttaaaatggc tgacggagaa cccgttcagt atgtcttgga tcatgcagcg 11880

tttgaaacag agtggtacca aggggaagaa gatggtgctc acctttatat ccgccaattt 11940

attcgtcctt ttcacctgga tgagccgccg ctgcttcgcg tggggctgat tgaacttcaa 12000

cctgaccgcg gaattctgat gtttgatatg caccacatta tttctgacgg aacgtccatg 12060

agcgtattga tcaaagaatt tatccgcatt tatgaaggag agacattacc gcccctgcgc 12120

atccagtaca aggactacgc ggtatggcag accggcgaag cgagattaca acagattcaa 12180

aaacaagaag cctattggtt agagctgtac agcggcgacg ttcctgtcct tcatctgccg 12240

accgattata tccggccgtc ggcacgggat tttgcgggag ccacaatgca gtttacgctt 12300

gataagcaaa aaagcgatgg gttaaagcag ctggcatccc aaactgaatc gactttatat 12360

atggtgctcc ttgcgtctta tacattattg ctttcaaaat acagcggcca ggaagacatt 12420

attgtcggca gtcctatagc gggaagaccg cacgccgatt tggaaccgat tatcggaatg 12480

tttgtcaata cattggccat gagaaattac cctgaaaaag ggaagacgtt ctctcagtat 12540

ctgtccgagg tgaaggagaa tgcattaaaa gcttatgaac atcaagatta tccgttcgaa 12600

gcgctgatcg atcaactgaa tatcgccaga gacttgagcc gtaatccgtt gtttgacacc 12660

atgtttgtgc ttcaaaacac ggagcaggaa caattagaga taagtgacgt cactttcaaa 12720

ccatatccaa acggacatac gatggcgaaa tttgatctaa ctttaactgc cgtggaagaa 12780

gaggcgggta ttcagttcac gatggaatac ttgacggccc tgtttaaacc ggaaacgatc 12840

gaacgtatga tggggcattt tgtgaagctg attgattcga ttattaaaca gcctgatgcc 12900

gagctggctc gtctgaatat aatgacccag gaagaggaaa gagacattca tcagctcttt 12960

aacgatactg cagcagaaac acgtattccg actacggttc atcagctttt tgagcagcag 13020

gctgtgctta atccggacca tgaagccatc atgtttggaa atcagacgct gacttatcgt 13080

cagctgaatg agcgttctaa tcagctggcg cgcgtccttc aagataaagg tgttggcact 13140

gatcaggtgg ttgccgttct cacggatcgc tcagcaaata tgatcatcgg cattttagcc 13200

atattaaagg ccggcgcagc atttctccct atcgatccgg agcttcccga aaaacggcgg 13260

gcttttatgc tgaaagacag cggtgccgac gtgcttttga cttgtgcagg tcccgctatt 13320

ccgccgctct ttgaaggaga agtgcttctg cttgatgatc cgatgatata tcaaggcaga 13380

gcagacaatc ttaatctttc ctgctcggaa aatgatttaa tgtatgttat ttacacgtca 13440

ggcacaacgg ggcagccgaa aggcgttcag ctggagcaca agaccatgac gaatctgctc 13500

gcttatgagc aggatcatac gcagctgcag tttgacagag tgctgcaatt tgccgcaatg 13560

agctttgatg tctgctatca agaaatgttc tccgcgcttt caagcggagg aacactcttt 13620

atcatcggca atgaggcaaa acgtgacata cgtcagctca atgattttgt cagaacgcac 13680

gggatacaaa cagcatttct tccgacagcg ttccttaagc ttcttgcctc ggagaaacac 13740

tatttcgaac catttgccga gtgcgtagat cacatcatcg ctgcggggga acagctcatc 13800

gtaacaagta tgctgcgtga catgctggcg cgccagcaag tcacattgca taatcattac 13860

ggtccgtcgg agacgcatgt cgtgaccatg tataccgttg atccgaatac agatcaggag 13920

cttcagccga tcggcaaacc gatttcaaat acggagatgt tcattttgaa tgaagccgga 13980

acgcttcagc ctgtcggagt cgtcggtgaa ctttgtattt caggagtgag cctggccaga 14040

ggctatcata acagggagtc tttgacgctc gaaacttttg ttccgcaccc atatgactcg 14100

aatcagcgga tgtataaaac aggcgatctg gcccgttacc ttcctgacgg aaacattgaa 14160

tatgcgggaa ggagggacca tcaggtcaaa atccgcggtt accgggttga actcggtgag 14220

gtggaggccg ctcttctcaa gcacgtacag gaagcagttg tcctagcaaa agaaaatacc 14280

gatggccaaa gtgatttata tgcctatttt acagcggagc aatcactctc gatcagtcag 14340

ctgaaagaaa aacttgctgg tcaaataccg ggatacatga tcccttcata cttcatccag 14400

ctggagaaac tgccgctaac cggcaatgga aaagtaaatc gaagagcatt accgatgcct 14460

gaagctggtt tgcaaacggg aaccgactac gttgctccgc ggacgaatat ggaagagcag 14520

ctgatatgca tctggcaaga cgttttaaag gtaaaagaaa taggtgtaaa agataacttt 14580

ttcgatttgg gcggccactc gttgcggggg atgacgctca tcgccaaaat tcataagcaa 14640

ttcagcaaaa acatttcatt aagagaagtc tttcaatgtc cgacagttga ggaaatggca 14700

aaggtaattg caggagctga aaccaacggg ccggattaca ttccgaaagc caaagcaaaa 14760

gatgtctatc ctgtatcttc cgtgcagaag atggtttacc tgtcaacaca aatagaaggc 14820

ggagaactta gctacaatat gccgggaatc cttacattag aaggaaaact cgacatgaat 14880

cgcctgcaaa ccgctttcca aaaactgatc cagcgtcatg aatcgctgcg taccggtttt 14940

gaaatggtcc gcggagaacc ggtgcaggtg atcaaaccgc aagtggagtt ttcgatggaa 15000

agatacaagg caacagctga cgaggttgag gaatgtttcc gcaccttcgt gcggccgttc 15060

gatctcagcc aggctccatt acttagggcc ggactcatcg agttagaaca agacctgaat 15120

atttttatgt ttgacatgca ccacatcatc acggacggcg cctccatgaa tatttttgtc 15180

gaagaactga ttcaaatgta tgacgaaaaa gagttagctc cgctccgtat tcagtacaaa 15240

gactttacgg aatggaaaca tcaaaaagag caaagagaac gaatcaagaa gcaggaagag 15300

tattggcttg gtgtatttca tgaagagctc ccgtcattcg agcttccgaa agattttgcg 15360

agaccgccgg ttagaagctt tgatgggaaa aggcacaatt ttacgctcga taagactgtc 15420

acccaaggga taaagcagct ggaagaattg actggaacga ccgcttacat gattttgttc 15480

tcggcctatt ccatactgct ggcaaaatac agcggtcagg atgatatcgt cgtcggcacc 15540

cctattgcgg ggagaccgca tgcggatttg gaacccatta tcggaatgtt tgtcaataca 15600

ttagccatcc gcaccgctcc gatggcagaa aggacatttt tggattatat aacggaaacg 15660

aaagaaacca tgctgaaggc ttttgaacat caagagtatc cttttgaaga attagttgaa 15720

aaactgggtg tgaaacggga cttaagccgc aatccattgt ttgatacgat gtttgtcctg 15780

caaaataccg aacaaacgga tattgaagtg gactctctcg cagtcagacc ttatgaacaa 15840

acggagacgg cagccaagtt tgatttacag ctgaacttct taatagatca agatgagatt 15900

cagggcagtt ttgattactg tacaaaacta tttaaaaaga aaaccattgc tgtattggac 15960

aaggattacg tcatgatcct ttcggcgatt atgagaaacc catccattcc tttaaaagac 16020

attcaattaa gcgaaaaagt aaacaaaagt aaacatctcg caagcacaat caaattggat 16080

ttctaa 16086

<210> 11

<211> 7860

<212> DNA

<213> Bacillus belgii (Bacillus velezensis)

<400> 11

atgtcagaat ttaagcaaca ggagttgttc tggagcagca tgttcgatgc ggaagatcgt 60

cccagtgcaa tcccttcgtt tcaaatgtcg gactcaacaa tagagcacga tgcgtcgagt 120

gcacccaacc gtattcacag ttcattgagt tccgatgtat ccctgcgcat catgaagatg 180

acgaataaat cgccaatggc cgtttatatg gttctcctgg tcggaatcga gtgtcttttg 240

tacaaatata cgggggaaga gggcgttgtt gtaggtgttc cgacctttga agacgaaacc 300

gatgaagacc ttcgtttaga tcaagtcatg ttaatcaaac aaaacataaa tgcggacagc 360

acatttaaat ccatatttaa tgaattcaaa cacaagctga acggagccat cttacatcag 420

cacgttcctt tcgataaaat ggccggtcca ttaaatctga actacgattc aaatcatttg 480

ccaatgatcc atacgatcgt atcactagat cagctgcacc ccattcgttt tatagaaacg 540

gctgctgctg atacattatt tcagttttcc atagaaaatg attcgattca tttaaagtta 600

acttataatg aacaagctta tgatcgtcag tatatgatgc aagtgatcga acatgtaaac 660

cggatttttt cgattttgtt attccaaccc gaccttatga tccgccagct taatattctg 720

tcagactcag agacaaatca actcatcgcc tataatcaaa cggctgcaga gtatccaaga 780

gaaaaaacga ttcatcaatt tttggaggaa caagcggaac gtacccctga tcaaacggct 840

gttgtctacg aggatagccg tctgacgtac cgggaattga atgaaagggc gaatcaactc 900

gctcggacac tgcaatctga aggcgtgcag ccggatcagc cggtcgggat tatggccgaa 960

cgctccctgg atatgattgt agggatcttc gggatcttga aagccggcgg ggcgtatgta 1020

ccgattgacc cgggctatcc cgaggaacgg gttcgataca tattggagga ttcggatacg 1080

aagttgttgc tcgtgcagaa tcaatcgcaa gagagagtac cttttacggg aaaagtgctg 1140

gatatgaaag atccgcaaaa cttttgtgaa gacgggtcga atgtagagcc ggctgctggc 1200

ccggatcatc tggcctatgt tatttataca tcgggttcaa cgggcaagcc gaaaggggtt 1260

atggttgagc accggtcggt catcaatcgg ctggtgtgga tgcaggaaaa ataccctctc 1320

gatgagcggg atgcgattct gcaaaaaact gcgatcacct ttgatgtatc agtgtgggaa 1380

ctgttctggt ggacgatatc aggttccaga ttggttctgt tgccaaacgg aggagaaaag 1440

aatcccgagc tgattttaga tacaattgca caaaaaggtg tcagcacaat gcattttgta 1500

ccggcgatgc tgcatgcctt cctggaatcc atggatcaga agccaagcgg gatgttgaaa 1560

caaaaattag cgtcattaag acatgtgttt gcaagcggtg aggctttgaa gcctgtacat 1620

gtcgcaggct tcaaacgcat cataacgtcg gtgagccagg ctcaaatcat caacttgtac 1680

ggaccgactg aggcaacaat tgatgtatca tactttgatt gccagacgga agaaacgtat 1740

gcatcgattc cgataggcaa accgatttcc aacattcaat tgtatatatt acatgctgat 1800

ttggaacata tgcagccgat cggtgtggcg ggagaactgt gcatagcggg cgatgggctt 1860

gcaaggggct acttgaatcg tcccgaactg acggccgaga agtttgtaaa ccatccgatt 1920

gcatcaggag aacggatata ccggacggga gacttggcaa gatggctgcc ggacggcaat 1980

atcgagtatt tgggccggat tgatcaccag gtgaaaatcc gcggttaccg cattgaaatc 2040

ggcgaagtcg aaggcgcttt ttttcagctg ccggcgatca aagaagccat cattatcgcc 2100

cgggaaatcg atggggaaac atccttgtgc gcctattata ccgcacaaca tgctttaact 2160

gcaggcgagc tccgtgaaga gctgtcccgc cagcttccat cctatatgat acctgcgtat 2220

ttcgttcagc tggaagagat gccgcttact ttcaacggga aaatagaccg taaatccctt 2280

ccttctccaa gagagaatct tacaggcatg aactatgaag ctcctcgaac agaactggaa 2340

aaaatactgg ccgcggtgtg ggaggcggtc ttaggcttag aacgggtcgg aatctcagat 2400

catttcttcg aattgggggg cgattccatt aagtcgattc aagtgtcctc aagattatac 2460

caagccgggt ataagtttga aatcaaacac ttgtttaaat acccaacgat ctccgagctt 2520

gttccatacg tagagccggt aacccgaatc gcagaacaag gagaaatcaa aggccgggcg 2580

ctgctgaccc cgatacagca ttggtttttt gaccagaaat acccggaatt acaccattac 2640

aatcaagcgg ttatgcttta ttggaaagag gaactggatg aatcaaagct tcgggacgtc 2700

atgaagaaga tcacagagca tcacgatgct ttgcgcatgg tctatgttcc gacagaagat 2760

ggatatgaag cccgaaatcg cgggatcgat gaaggtgatc tgttcagcct tgaagtgatc 2820

tcattacgcg aagaaaaaaa tgtgtcccag acgatagaaa ccatttctaa tgagattcag 2880

caatccattc atttacccga aggaccgctc atgaaactgg gattgttccg gtgtcaagag 2940

ggagatcatt tgctgattgc ggttcatcat ctggtcatcg acggcgtatc gtggaggatt 3000

ttacttgagg atatagcggc agcttatgag cagcttcaaa acggcgaagc gatccggctt 3060

ccgaagaaaa cggactctta cctgttatgg gctgaacagt tgaatcgtta tgcagaaagt 3120

caggaattcg aagcggaaaa tcaatactgg ttcagacaga aacataatcc gcaacttaca 3180

ttgccaaagg acaatgaaca ggagaccggt ctggccaaag accgggaaac ggtcatcgtt 3240

caatggaccg ttgaggaaac cgaacgttta ttaaaaaacg ctcaccgggc ttattcgacc 3300

gatatgaacg atttattact gaccggtctg ggaactgcta ttcatcgttg gaccggatat 3360

gaagacattc tcattgatct tgaaggacac ggcagagagt ctatcattcc ggatctcgat 3420

atttcgcgta cggtgggatg gttcacaagc ctatatccgg tttcgcttca aatcaaggct 3480

gatcaggata tcccgcagcg gattaaaaca gtgaaagaaa atttgcgtca gatcccccaa 3540

aaaggaatag gttacggcct gatcaaatat ttgtccgatc acccgaaggc acatgaatgg 3600

accggacatc cggaaatacg tttcaattat ttgggtcaat ttgatcaaga tgtccggaac 3660

ggcaagatgg aggtatcccc ttactcaagc gggaaaacag ccagtgacaa tcgtcccttg 3720

acctatacac ttgatatcaa cggcatgatt tcagacggcc gattgtcgct ggccatcagc 3780

tattgcggta aacaatatca aagagaaaca atggaagcct gtgccgatct cttaaaaagc 3840

agcctgcagc aagtcatcgc acattgtgat gctcaagatc aaattcacct gacgccaagc 3900

gatatttcgt taaaaggtat aacgatcggt gaactggatc aatttgtgca gcaaacgagt 3960

catctcggtg acattgaaaa tatataccca ttaaccccga tgcagaaagg aatgttgttc 4020

cacagcttga tcgattcggc ctccgaagct tactttgaac aagccgcttt tgatctgaaa 4080

ggtttcttgg atatcgatgc attcaagatg agtttggcgc atctggccga aaaatatgac 4140

attcttcgga ctctttttta tacagaatgg aaagatcagc ctttgcagat tgtattccgg 4200

caaaagccga ttgaaacggc cgttgaagat attcgcagca tgaacagtca tcagcgcagc 4260

gagtttattg ccgattttgc aagaagagat aaggcgcggg gattcaatct cacccgggat 4320

gctttaatgc gcgtatcgat cctgcggaca gaagaagacc aagcgcgatt gatttggagc 4380

tttcatcata ttttaatgga tggctggtgt ttgcctcttg ttacgaaaga agtgtttgaa 4440

acctattatg cgatacttga gcaaagacag ccgaagcggg gagccgtcac cccatacagc 4500

cgatatatcg aatggctgga tcaacaagat cacaagcagg cgtcagccta ttggcgtaac 4560

tatttagaag gttatgaagg gcagactgtt ttgttacaag aacaatcttc cgaccgggcg 4620

aaaggatacg aaaaaggtga gcatgagttt cgtttaggaa agcggctcac agacgaaata 4680

aaacgggccg caagccgcca gcaagtcacc gtcaatacat ggatacaaac cgcatggggc 4740

ttattgctgc aaagatacaa cgggagtcag gatgtcgtct tcggcactgt cgtatccggc 4800

agaccggcag aaattccggg aattgaaacc atggtcggtt tatttatcaa taccattccc 4860

gtacgtattc atacgcagcc tgaaatgaca gctgcacaag tattgaaaat gaaccaggag 4920

cgggcactgg cttctcaaaa atatgataca tttccgttgt atgacattca ggctcaaact 4980

gagcaaaagc agcagttgat caatcatatt atggtatttg agaattaccc ggttgaaaaa 5040

caaatagaac atatgaagca ggatgacaac gcattagaca ttctcgattt tcatatggaa 5100

gaacatactc attacgattt tacttttata gtgatgccgg atggagaaat tgacattcgt 5160

tttgtataca atcgagatgt ttatgaccaa gcaagtgtcg aacggatgca gacgcatttc 5220

atgcaaatta tgaagcaaat ggcggacgat caggaaattc gcgttcaaga cctcgatatc 5280

gtaacagcgg atgagcgatc gctcctgata gataagttca acgatacggc ggctgagtat 5340

ccgaaggaaa agacgattca tcaattgttt gaagaacaag cggagcgaac gccggagcaa 5400

gcagcgattg tttttgaaga taagaagatg acgtatcgga tagtaaatga acgcgccaat 5460

cagctggccc gaaccttggt tgccaaagga cttcaggccg aggagcttgt cggcattatg 5520

gcggaacgtt caccggagat ggtgataggc atccttgcta ttttgaaagc tgggggcgtt 5580

tacgtaccga tcgatccgga ttacccgaaa gaacgcatcc attatatgct tgaggattct 5640

aacgtctcta tcttattatt gcagcatcat cttctggaag gtaccgatta tcaaagtcat 5700

actgtcttcc ttgacgatcc gtcatcatac ggcgccgaag catctaatct caaactgaac 5760

gtgatgccaa atcaattagc ctacgttatt tatacatcag gaacaacggg gaatccgaaa 5820

ggcacgctca ttgaacataa aaacgtggtt cgtctgttat ttaacaacaa aaatgttttt 5880

gattttaatg catcggacac atggactttg tttcattcct tctgctttga tttttcagta 5940

tgggaaatgt acggagcgtt gctgtatggg ggcaaattag tgattattcc gaaacaaata 6000

gcaaaaaacc cggaaaggta tttgcaattg ttaaaatcgg aagctgtgac tattttaaat 6060

caaacgccga gttatttcta tcaactgatg caggaggaga gagcagatcc cgaatccaat 6120

ttaaacatcc gaaagatcat atttggcgga gaggcgttaa atccatcctt cttgaaagac 6180

tggaagctta aatatccttt aacccaatta atcaatatgt acggcatcac cgaaaccacc 6240

gttcatgtca catataaaga aatcacggaa cgggaaatcg acgaaggcag aagcaacatc 6300

ggacaaccta ttcctactct gcaagcgtat attttggacg aatatcagcg cattcaggta 6360

atgggcatac cgggcgagct ctatgtggca ggagagggac tggccagagg atatttgaac 6420

cgacctgaat taacgggcga gaaatttgtt gaacacccgt ttgccgcagg cgaaaaaatg 6480

tacaaaacgg gagatgtcgc ccggtggctg ccggacggca atatcgagta tttgggaaga 6540

attgatcatc aggtgaaaat acggggctac cggattgaaa ttggcgaggt cgaagcggct 6600

ttattgcaac tggaatctgt caaagaagcg gtcgtcatcg caattgaaga agagggctca 6660

aagcagctgt gcgcctactt gtcaggagac gattcattaa atacggcgca gctgaagcat 6720

catttactga acaaactgcc cgcttatatg ataccggctt actttgtaca aatggagaag 6780

atgccgataa ccgcaaatgg aaaaatcgat cgaaaagctt tgccggcccc tgagggaaat 6840

aggctcaccg gaaccgaata cgaagcgccc ggaacgttaa ttgaaaagca gctggccgag 6900

atatggaaga acattttagc tctcagcgac ccgggaatta aagataactt ctttgacgtt 6960

ggcggccatt cgctcaaagt attgcaagtt attcatcaaa tcaacgaccg tatgggaatc 7020

aaaatgcatt atcaagccgt atacgatttt cctaccatcg aaacaatggc acgcgccatt 7080

caagcggcag ttttcgagtc caagacggat aacgtattcg tcaagatgaa tcagaacggt 7140

tcaatccctg tgttctgttt cccgccttta atcggatacg ggctggtcta taatgaaatg 7200

gcgaaaagac ttgacggccg ctgcaccgtc tatgccgctg atttcctaga agagccgtct 7260

tacgaaaaag agatcgttga ccggtatgca gaaagcatga taggcattca ggaacaaggg 7320

ccctttgttt tacttggtta ctcctcgggg tcgaatttgg cttttgaggt tgccaaagcc 7380

ttggagaagc gcggacgcat cgtatcggac attatgatgc ttgattctaa aagagccgtt 7440

tcggtgaatt atttttcgga agaggaaacc gaagagatca ttcatcggaa tctggatatc 7500

attccggatt attatagaga attattaacc attccttcca ttaaggataa aatcagaagc 7560

tatctcacat atcacaataa actgatcaat tccggcgcgg tgaacgccaa cattcatcat 7620

tttctatgcg gcgagttgac cgatagagga tggaagcaat caaccgcaca gcattacctt 7680

gagtacaaat taaaaggaga ccatgtgacg atctttgacc ctcacaatat tgaagaaaat 7740

acggatacaa ttcgatctat tatcaaaagg attgaagaac ggcatcatca cggacttgtt 7800

cttgaagaac aactgtcaat gggatcgttt gcgggggacg caaagtttga caaaatgtaa 7860

Claims

1. Bacillus velezensis K01 with the preservation number of CCTCC NO: m2020871.

2. A bacterial agent comprising Bacillus belgii K01 according to claim 1.

3. A biopesticide, biofertilizer, phosphorus activator or plant growth promoter prepared from Bacillus belgii K01 according to claim 1 or the microbial agent according to claim 2.

4. A biocontrol agent, an antibacterial agent, a preservative, a disinfectant or a food preservative prepared from the bacillus belgii K01 of claim 1.

5. The use of the Bacillus belgii K01 of claim 1 or the microbial inoculum of claim 2 in any one of the following applications:

1) for promoting plant growth and development and increasing crop yield;

2) dissolving phosphorus;

3) for the preparation of biofertilizers;

4) used for preparing a phosphorus activator;

5) for the preparation of plant growth promoters;

6) for the preparation of biopesticides;

wherein the plant is maize.

6. The use of Bacillus belgii K01 according to claim 1 for any one of:

a) for combating pathogenic bacteria and plant diseases caused by pathogenic bacteria;

b) for inducing plants to increase resistance to plant diseases caused by pathogenic bacteria;

c) used for preparing biocontrol agents, antibacterial agents, preservatives, plant immunity inducing agents, disinfectants or food preservatives;

the pathogenic bacteria are selected from the group consisting of Botrytis cinerea, Fusarium oxysporum (Fusarium oxysporum), Alternaria solani (Alternaria solani), Ceratophyllum microphyllum (Ceratophyllum fischeriana), Fusarium moniliforme (Fusarium moniliforme), Rhizopus liliius (Fusarium oxysporum.sp.Lii), Fusarium oxysporum (Fusarium oxysporum.sp.niveum), Fusarium moniliforme (Monilinia laxa), Rhizoctonia cerealis (Rhizoctonia cerealis), Rhizoctonia oryzae (Rhizoctonia solani), Xanthomonas gossypii (Verticillium dahliae), Rhizoctonia cerealis (Septoria nigra spe), anthrax (Colotrichum), Helianthus annuus (Leptospira), and Rhizoctonia solani (Rhizoctonia solani).

7. Use of the Bacillus belgii K01 of claim 1 or the microbial agent of claim 2 for dissolving poorly soluble phosphorus under high salt conditions; the high-salt condition means that the concentration of NaCl is 13-15%.

8. The process for preparing the microbial inoculum according to claim 2, which comprises the following steps:

1) preparing a seed solution: inoculating the activated Bacillus belgii K01 of claim 1 into a seed culture medium for culture; the inoculation amount is 1.2 percent, the culture temperature is 28 ℃, the rotating speed is 200rpm, and the culture time is 12 hours;

3) preparation of microbial inoculum

wherein the protective agent is: dextrin, soluble starch and diatomite in a mass ratio of 70:25: 5.