CN113717908B - Bacillus licheniformis and application thereof - Google Patents

Abstract

The invention discloses bacillus licheniformis and application thereof. The bacillus licheniformis, named FB8, is preserved in the China general microbiological culture Collection center with the preservation number of CGMCC No. 20021. The bacillus licheniformis CGMCC No.20021 has bacteriostatic action on plant pathogenic fungi, plant pathogenic bacteria, bee pathogenic bacteria and conditional pathogenic bacteria.

Description

Bacillus licheniformis and application thereof

Technical Field

The invention belongs to the field of microorganisms, and relates to bacillus licheniformis (Bacillus licheniformis)Bacillus licheniformis) And applications thereof.

Background

Bacillus (A), (B), (C) and (C)Bacillus) Bacteria are a type of aerobicOr facultative anaerobes, rod-shaped bacteria capable of producing resistant endospores. The bacillus can secrete and produce various active proteases and bacteriostatic active substances, so that the bacillus can be widely applied to the fields of medicine and agriculture. In the agricultural field in particular, bacteria of the genus bacillus can provide a wide range of benefits to animals and plants, and they can secrete large amounts of bacteriostatic active substances to kill some common pathogenic bacteria in animals and plants, including protection of animals and plants against the hazards of pathogenic microorganisms, insects and nematodes. In addition, bacillus can improve rhizosphere microbial flora, induce plant self-resistance and promote plant growth, does not damage the environment, and is increasingly paid more attention to the prevention and control of animal and plant diseases.

The commonly used Bacillus bacteria for controlling animal and plant diseases are mainly Bacillus subtilis (B.)Bacillus subtilis) Bacillus licheniformis (B), (B)Bacillus licheniformis) Bacillus amyloliquefaciens (A) and (B)Bacillus amyloliquefaciens) And the like, the antibacterial polypeptide secreted by the bacillus subtilis BS-2 has strong inhibition effect on various plant pathogenic fungi and bacteria such as plant anthracnose pathogen, tomato ralstonia solanacearum and the like, and has 69.79 percent of disease prevention effect on pepper fruit anthracnose. Meanwhile, with the continuous and intensive research, more and more other kinds of bacillus bacteria are applied to biological control, such as the commercial product "Yield Shield" produced by Bayer crops science inc, USA from bacillus pumilus GB34 (b) (b.pumilus), which is a product of Bayer crop science incBacillus pumilus) Compositions, which are used to induce systemic resistance and promote plant growth, are registered by the U.S. environmental protection agency and are used to control soybean sheath blight and fusarium. Recent studies have also shown that Bacillus belgii (B.), (B.velezensis) Has multiple application potentials, can produce the iturin, the pheninterleukin and the surfactant, can inhibit pathogenic bacteria and fungi (including Listeria monocytogenes and a plurality of fungi of aspergillus and fusarium), and when being applied to corn, the Bacillus beijerinckii can inhibit the growth of the pathogenic fungi and reduce the production of the aflatoxin and the ochratoxin. Therefore, the bacterium belonging to the genus Bacillus is considered to be useful for biological control of diseases of animals and plantsThe microbial resource library is increasingly studied at home and abroad. However, due to the limitations of the bacteriostatic range and effect, there is still a need to develop strains for agricultural biocontrol that are excellent in efficacy.

Disclosure of Invention

In order to solve the problems, the invention aims to provide bacillus licheniformis and application thereof.

The bacillus licheniformis provided by the invention is classified and named as bacillus licheniformisBacillus licheniformisEntitled Bacillus licheniformis (Bacillus licheniformis) Strain FB8, which was deposited in the common microbiology center of the china committee for culture collection on 04 th month 06 in 2020 (address: west road No. 1 hospital No. 3, north kyo, chaoyang district, beijing), the collection number is CGMCC number 20021.

The bacillus licheniformis FB8 CGMCC number 20021 has bacteriostatic action on plant pathogenic fungi, plant pathogenic bacteria, bee pathogenic bacteria and conditional pathogenic bacteria.

Another object of the present invention is to provide the use of said Bacillus licheniformis in inhibiting phytopathogenic fungi, phytopathogenic bacteria, bee pathogenic bacteria and/or conditionally pathogenic bacteria.

It is still another object of the present invention to provide a bacteriostatic agent for inhibiting plant pathogenic fungi, plant pathogenic bacteria, bee pathogenic bacteria and conditionally pathogenic bacteria.

Wherein the plant pathogenic fungus is Sclerotinia sclerotiorum (A)Sclerotinia sclerotiorum) Alternaria brassicae (S.brassicae) ((S.brassicae))Alternaria brassicicola) Fusarium oxysporum (F.), (Fusarium Oxysporum) Colletotrichum bacteria (A), (B), (C), (B), (C), (B), (C)Colletotrichum orbiculare (Berk.&Mont.) Arx) Sclerotinia virescens (A), (B), (C), (B), (C), (B), (C)Ustilaginoidea oryzae (Patou.) Bref=U.virens (Cooke) Tak.) Rhizoctonia solani (A), (B), (C), (B), (C), (B), (C)Rhizoctonia cerealis Vander Hoeven) And Humicola lanuginosa (L.) TokayataValsa mali Miyabe et Yamada.) One or more of (a).

The plant pathogenic bacteria is pseudomonas syringae (Pseudomonas syringae)Pseudomonas syringae) Xanthomonas gossypii (A. gossypii:)Xanthomonas campestris pv. malvacearum (Smith) Dye) Pseudomonas solanacearum (B) ((B))Pseudomonas solanacearum) One or more of; the bee pathogenic bacteria are Bacillus larvae (B), (C)Paenibacillus larvae) (ii) a The conditionally pathogenic bacterium is Micrococcus luteus (A)Micrococcus luteus）。

The bacteriostatic agent provided by the invention comprises the bacillus licheniformis FB8 as an active ingredient.

The bacillus licheniformis strain FB8 CGMCC No.20021 of the invention can obviously inhibit sclerotinia sclerotiorum (B)Sclerotinia sclerotiorum) Alternaria brassicae (S.brassicae) ((S.brassicae))Alternaria brassicicola) Fusarium oxysporum (F.), (Fusarium Oxysporum) Colletotrichum bacteria (A), (B), (C), (B), (C), (B), (C)Colletotrichum orbiculare (Berk.&Mont.) Arx) Sclerotinia virescens (A), (B), (C), (B), (C), (B), (C)Ustilaginoidea oryzae (Patou.) Bref=U.virens (Cooke) Tak.) Rhizoctonia solani (A), (B), (C), (B), (C), (B), (C)Rhizoctonia cerealis Vander Hoeven) And Humicola lanuginosa (L.) TokayataValsa mali Miyabe et Yamada.) The 7 kinds of pathogenic fungi have strong bacteriostatic activity and can kill pathogenic bacteria Pseudomonas syringae ((A))Pseudomonas syringae) Xanthomonas gossypii (A. gossypii:)Xanthomonas campestris pv. malvacearum (Smith) Dye) Pseudomonas solanacearum (B) ((B))Pseudomonas solanacearum) Bacillus larvae (b), (c)Paenibacillus larvae) And Micrococcus luteus: (Micrococcus luteus) And the like show strong bacteriostatic activity.

Drawings

FIG. 1 shows the colony morphology of strains FB8, FB17 and FB 23.

Fig. 2 shows the results of starch hydrolysis tests of strains FB8, FB17 and FB 23.

Fig. 3 shows the results of the grease hydrolysis test for strains FB8, FB17, and FB 23.

Fig. 4 shows the results of the gelatin hydrolysis test of strains FB8, FB17 and FB 23.

Fig. 5 shows the results of the casein hydrolysis assays of strains FB8, FB17 and FB 23.

FIG. 6 shows the results of D-glucose fermentation experiments for strains FB8, FB17 and FB 23.

FIG. 7 shows the results of D-fructose fermentation experiments for strains FB8, FB17 and FB 23.

FIG. 8 shows the results of sucrose fermentation experiments for strains FB8, FB17 and FB 23.

FIG. 9 shows the results of D-mannitol fermentation experiments for strains FB8, FB17 and FB 23.

Fig. 10 shows the results of hydrogen sulfide tests of strains FB8, FB17 and FB 23.

Fig. 11 shows the results of indole tests of strains FB8, FB17 and FB 23.

Fig. 12 shows the results of methyl red tests of strains FB8, FB17 and FB 23.

FIG. 13 shows the results of V-P tests of strains FB8, FB17 and FB 23.

Fig. 14 shows the results of gram staining of strains FB8, FB17 and FB 23.

Fig. 15 shows the results of spore staining of strains FB8, FB17 and FB 23.

FIG. 16 shows the result of agarose gel electrophoresis of the PCR amplification product of 16S rDNA.

FIG. 17 showsgyrBAgarose gel electrophoresis results of PCR amplification products of the genes.

FIG. 18 shows a schematic diagram of 3 antibacterial peptide gene clusters synthesized by Bacillus licheniformis FB8 CGMCC number 20021.

Detailed Description

The methods in the following examples are conventional methods unless otherwise specified.

The present invention will be described in detail with reference to specific examples. The following examples will assist those skilled in the art in further understanding the invention, but are not intended to limit the invention in any way. It should be noted that it would be obvious to those skilled in the art that various changes and modifications can be made without departing from the spirit of the invention. All falling within the scope of the present invention.

Examples

First, separation and identification of bacterial strains FB8, FB17 and FB23

Isolation of the Strain

Isolation of strains FB8 and FB23From the date honey sample (table 1) in elm city, shanxi province, the sample was collected and stored at room temperature. Taking 50 mL of date honey, heating in water bath at 45 ℃ for 30 min to increase the fluidity of the honey. 10 mL of jujube honey is uniformly mixed with 10 mL of sterile normal saline (preheated). Diluting the diluted date honey to 10 times of gradient again^-2For each dilution, 100. mu.L of each of samples diluted 2-, 20-and 200-fold was plated on LB medium plate and repeated 3 times for each dilution. The plates were incubated at 37 ℃ for 24-48 h. Selecting single colonies of different forms, plating on LB medium plate, purifying to obtain pure culture of bacteria, numbering different strains, and storing at-80 deg.C in 20% glycerol.

Strain FB17 was isolated from a bee sample (Table 1) in Xiaogan City of Hubei province and collected and stored in a refrigerator at 4 ℃. Placing 2-3 bees into a sterile 1.5 mL centrifuge tube, adding 600 μ L sterile normal saline, grinding with a grinding rod to homogenate, and vortex mixing. Slightly standing, sucking turbid solution, and diluting with sterile physiological saline 10 times in gradient to 10^-5. Respectively take 10^-1To 10^-5The diluted samples were 100 μ L spread on LB medium plates, and each dilution was repeated 3 times. The plates were incubated at 37 ℃ for 24-48 h. Selecting single colonies of different forms, plating on LB medium plate, purifying to obtain pure culture of bacteria, numbering different strains, and storing at-80 deg.C in 20% glycerol.

TABLE 1 sample Collection information

The colony morphology of the 3 strains is shown in figure 1, and after the strain FB8 is cultured on an LB solid medium at 37 ℃ for 48 hours, the colonies are irregular, the edges are notched, hair-shaped grows, and the strains are milky white, opaque, dense in texture and loose in edges. After the bacterial strain FB17 is cultured on an LB solid culture medium at 37 ℃ for 48 hours, the bacterial colony is round and slightly regular, the edge is notched, the middle is provided with an annular bulge, the inside of the annular bulge is provided with a fold, the bacterial colony is milky white and opaque, the inside of the bacterial colony is in a mucus shape, and the surface texture of the bacterial colony is compact. After the bacterial strain FB23 is cultured for 48 hours at 37 ℃ on an LB solid culture medium, the bacterial colony is regular in circle and neat in edge, has irregular folds, is light yellow and opaque and is dense in texture.

(II) identification of the Strain

1. Physiological and biochemical identification of strains

1.1 starch hydrolysis test:

streaking a fresh solid culture of bacillus into a starch culture medium, carrying out inversion culture at a constant temperature of 37 ℃ for 48 h to form obvious bacterial colonies, and dripping iodine solution on a flat plate, wherein the flat plate is blue black, and transparent circles are formed around bacterial colonies to show that the starch is hydrolyzed positively; the colony is blue-black, and the starch is hydrolyzed negatively.

Starch medium (LB medium +1.0% soluble starch) (100 mL): tryptone 1.0 g, yeast extract 0.5 g, NaCl 1.0 g, soluble starch 1.0 g, agar 1.5 g, distilled water 100 mL. Dissolving in water bath, autoclaving at 121 deg.C for 20 min, and pouring into plate.

After 3 strains were cultured in a starch medium for 48 hours, iodine solution was added dropwise around the colonies for observation. The results are shown in fig. 2, a transparent circle is formed around the strains FB8 and FB17, but no transparent circle is formed around the strain FB23, which indicates that the strains FB8 and FB17 can secrete amylase hydrolyzed starch, and the strain FB23 cannot secrete amylase hydrolyzed starch, so that the strains FB8 and FB17 are starch hydrolyzed positively, and the strain FB23 is starch hydrolyzed negatively.

1.2 grease hydrolysis test:

cooling the melted solid oil culture medium to about 50 ℃, fully shaking to uniformly distribute the oil, and pouring the oil on a flat plate. The bacillus was streaked into a lipid medium. The culture was carried out at 37 ℃ for 24 hours by inversion. Observing the color of the lawn. If a red spot appears, fat hydrolysis is indicated, and the reaction is positive.

Fat medium (100 ml): 1.0 g of peptone, 0.5 g of beef extract, 0.5 g of NaCl, 1.0 g of peanut oil or sesame oil, 0.1 mL of 1.6% neutral red water solution, 1.5 g of agar, 100 mL of distilled water, pH7.2, autoclaving at 121 ℃ for 20 min, and pouring the mixture into a flat plate for later use.

1.6% neutral red aqueous solution: neutral red 1.6 g, 95% ethanol 28 mL, distilled water 72 mL.

The results are shown in FIG. 3, the bacterial colony of the strain FB23 shows red spots, the fatty acid is hydrolyzed, which shows that the bacterial colony can produce lipase, and the lipase is hydrolyzed positively. No red spots appeared around the colonies of strains FB8 and FB17, indicating that they were not lipase producing and lipase hydrolysis negative.

1.3 gelatin hydrolysis test:

taking a gelatin culture medium test tube, and respectively puncturing and inoculating the bacillus by using an inoculating needle. The inoculated tubes were incubated at 20 ℃ for 2-5 days. And observing the liquefaction condition of the gelatin.

Gelatin medium (100 mL): LB culture medium, gelatin 12-18 g, pH 7.2-7.4. Dissolving the above components in water bath, adjusting pH to 7.2-7.4, packaging into test tubes, and autoclaving at 121 deg.C for 20 min.

After 3 strains of Bacillus were inoculated by puncture on a gelatin medium, the cells were cultured at 20 ℃ for 3 to 4 days for observation. As shown in FIG. 4, it was observed that gelatin medium was liquefied, and therefore, the strains FB8, FB17, and FB23 produced gelatinase-hydrolyzed gelatin, indicating that the strains FB8, FB17, and FB23 were positive in the gelatin hydrolysis test.

1.4 Casein hydrolysis assay (litmus milk test):

inoculating Bacillus into milk litmus culture medium test tube, culturing at 37 deg.C for 24-48 hr, and determining the milk is positive.

Milk litmus medium: 100 mL of skimmed milk and 4 mL of 2.5% litmus aqueous solution (used after overnight filtration) are mixed to obtain clove or purple, and the mixture is packaged into test tubes, the height of the milk is 4 cm, and the milk is autoclaved at 121 ℃ for 15 min.

The strains FB8, FB17 and FB23 were inoculated with milk litmus medium, respectively, and the blank control was milk litmus medium without inoculated strain, and cultured at 37 ℃ for 48 h. The results are shown in FIG. 5, in which the media inoculated with FB17, FB23 were milk-cleared, whereas the media inoculated with FB8 were not significantly changed from the control (milk-not-cleared). The results show that the casein hydrolysis experiment of the strain FB8 is negative and can not hydrolyze casein, while the casein hydrolysis experiment of the strains FB17 and FB23 is positive and can hydrolyze casein.

1.5 sugar alcohol fermentation experiments

Inoculating bacillus cultured for 18 h into sugar fermentation medium, setting negative control, culturing at 37 deg.C for 24-48 h, and checking the result. The color of each tube was observed to change and the presence or absence of air bubbles in the Duchen tubes was observed. The indicator turns yellow from purple to indicate that the fermentation of the saccharides produces acid, and the indicator is positive.

Sugar fermentation medium (100 mL): 1 g tryptone, 0.5 g NaCl, 0.05 mL of 1.6% bromocresol purple ethanol solution, pH 7.6, constant volume to 100 mL, subpackaging test tubes (10 mL/tube), aseptically adding 0.5 mL of 20% sugar solution into each tube, placing one Duchen small tube into each tube to fill the culture solution, and autoclaving at 121 ℃ for 20 min.

20% sugar solution (D-glucose, D-fructose, sucrose, D-mannitol) was autoclaved at 113 deg.C for 30 min.

The results of the D-glucose fermentation experiments are shown in FIG. 6, the color of the glucose fermentation culture medium inoculated with the strains FB8, FB17 and FB23 is changed from purple to yellow, and no bubble is generated in the Duchen tubes, which indicates that the strains FB8, FB17 and FB23 can decompose D-glucose, produce acid and produce no gas. The results of the D-fructose fermentation experiments are shown in FIG. 7, the color of the fructose fermentation culture medium inoculated with the strains FB8, FB17 and FB23 is changed from purple to yellow, and no bubble is generated in Duchen tubes, which indicates that the strains FB8, FB17 and FB23 can decompose D-fructose and produce no acid or gas. The results of the sucrose fermentation experiments are shown in FIG. 8, the color of the sucrose fermentation culture medium inoculated with the strains FB8, FB17 and FB23 changes from purple to yellow, and no bubble is generated in Du's tubes, which indicates that the strains FB8, FB17 and FB23 can decompose fructose, produce acid and produce no gas. The D-mannitol fermentation experiment result is shown in FIG. 9, the color of the D-mannitol fermentation medium inoculated with the strain FB8 is changed from purple to yellow, no bubbles are generated in the Duchen tube, while the color of the D-mannitol fermentation medium inoculated with the strains FB17 and FB23 is unchanged, the D-mannitol fermentation medium is still purple, and no bubbles are generated in the Duchen tube, which indicates that the strain FB8 can decompose D-mannitol, produce no acid and produce no gas, while the strains FB17 and FB23 can not decompose D-mannitol, produce no acid and produce no gas.

1.6 Hydrogen sulfide test:

inoculating Bacillus into lead acetate culture medium, culturing at 37 deg.C for 48 hr, and culturing for 6 days.

Lead acetate medium (100 mL): 0.3 g of beef extract, 1.0 g of peptone, 0.5 g of NaCl and 1.5 g of agar, metering the volume to 100 mL, heating for dissolving, cooling to 60 ℃, adding 0.25 g of sodium thiosulfate, adjusting the pH value to 7.2, subpackaging in a triangular flask, and sterilizing at 115 ℃ for 15 min; taking out, cooling to 55-60 ℃, adding 1mL of 10% lead acetate aqueous solution (sterile), mixing uniformly, and pouring into a sterile test tube.

As shown in FIG. 10, after inoculating strains FB8, FB17 and FB23 by puncturing on a lead acetate medium respectively, and culturing at 37 ℃ for 48 h, no blackening occurs in 3 tubes, and when the culture is continued to the 5 th day, the strain FB8 is not blackened near the puncturing line, and the strains FB17 and FB23 are blackened near the puncturing line, which indicates that the strain FB8 does not produce hydrogen sulfide, the hydrogen sulfide test is negative, and the strains FB17 and FB23 can produce hydrogen sulfide, and the hydrogen sulfide test is positive.

1.7 indole test:

inoculating Bacillus into indole culture medium, and culturing at 37 deg.C for 48 hr. And (3) slowly adding 2 drops of indole reagent on the surface of the tube wall, wherein the interface of the liquid layer is red, the reaction is positive, if the reaction is not obvious, adding 4-5 drops of ether into the culture solution, standing for 1 min, adding the indole reagent after the ether rises, and observing the color reaction. The production of a red circle between ether and culture was positive.

Indole medium (100 mL): tryptone 1.0 g, NaCl 0.5 g and L-tryptophan 0.05 g, pH 7.6, autoclaved at 121 ℃ for 15 minutes for use.

Indole reagent: 2 g of p-dimethylaminobenzaldehyde, 190 mL of 95% ethanol and 40 mL of concentrated HCl.

After inoculating strains FB8, FB17 and FB23 to an indole culture medium, culturing for 48 h at 37 ℃, and slowly adding 2 drops of an indole reagent along the tube wall. As a result, as shown in FIG. 11, no red color was formed on the interface between the liquid layers of the 3 strains, indicating that the indole reaction of the 3 strains was negative.

1.8. Methyl red test:

selecting bacillus, inoculating the bacillus to a glucose peptone water culture medium, culturing at 37 ℃, taking 1mL of culture solution every day from the next day, adding 1-2 drops of methyl red indicator, and enabling the positive to be bright red, the weak positive to be light red and the negative to be yellow. If the result is positive or negative by day 5, the result can be determined.

Glucose peptone water medium (100 mL): 0.5 g peptone, 0.5 g glucose, 0.2 g K₂HPO₄Dissolving the above components in 100 mL of water, adjusting pH to 7.0-7.2, subpackaging with 10 mL of test tube, and sterilizing at 113 deg.C for 30 min.

Methyl red reagent: 0.04 g methyl red, 60 mL 95% ethanol, 40 mL distilled water. Dissolving in ethanol, and adding distilled water.

The strains FB8, FB17 and FB23 were inoculated with a peptone water medium and cultured at 37 ℃. As shown in FIG. 12, after 24 h of culture, 1mL of the culture solution was added with 1-2 drops of methyl red indicator after the bacterial strains FB8 and FB23 were cultured, and the bacterial solution was bright red, and sampled until day 5 after the bacterial strain FB17 was cultured, and the bacterial solution was yellow in color similar to that of the blank control (non-inoculated culture medium). The above results indicate that the FB8 and FB23 methyl red reactions are positive, while the FB17 methyl red reaction is negative.

1.9V-P test

Inoculating bacillus into a glucose peptone water culture medium (V-P determination culture medium), culturing at 37 ℃ for 48 h, adding 1mL of 40% NaOH and 1mL of 5% alpha-naphthol into a test tube, shaking with force, and then placing at 37 ℃ for heat preservation for 15-30 min to accelerate the reaction speed, wherein if the culture is red, the V-P reaction is positive.

V-P assay Medium (100 mL): dissolving the above components in 100 mL water, adjusting pH to 7.0-7.2, packaging into 4-5 mL test tube, and sterilizing at 113 deg.C for 30 min. 5% alpha-naphthol: 5 g of alpha-naphthol and 100 mL of absolute ethanol.

The strains FB8, FB17 and FB23 were inoculated with a glucose peptone water medium, respectively, and after culturing at 37 ℃ for 48 h, 1mL of 40% NaOH and 1mL of 5% alpha-naphthol were added, respectively. As shown in FIG. 13, the fermentation liquid of 3 strains turned red, but the color of the blank medium without inoculated strain did not change, indicating that the V-P reactions of 3 strains were all positive.

1.10 determination of Catalase

A small amount of Bacillus culture was picked from the LB slant, spread on a clean slide, and 10% hydrogen peroxide was added dropwise, and the positive was found when bubbles were produced.

A small amount of culture was picked from each of the plates streaked and purified from the strains FB8, FB17 and FB23, spread on a clean glass slide and 10% hydrogen peroxide was added dropwise. All 3 strains produced bubbles, and therefore all of the FB8, FB17, and FB23 catalase reactions (catalase reactions) were positive.

2. Gram stain (gram stain kit)

Gram staining Using the Solebao gram staining kit (cat # G1060), the specific procedures were as follows:

(1) the strains FB8, FB17 and FB23 cultured on the plate are picked to inoculate an LB test tube and shake-cultured for 24 h at 37 ℃. (2) Fixing a smear: 10 mul of bacterial liquid is absorbed on a glass slide and fixed by flame for 1-2 times. (3) Dyeing: adding crystal violet, dyeing for 1 min, and washing with water. Adding iodine solution, dyeing for 1 min, and washing with water. Adding decolorizing solution, shaking slide, decolorizing for 20-60 s according to smear thickness, washing with water, and absorbing water. Adding safranin, dyeing for 1 min, and washing with water. After being sucked dry or dried in the air, the test is performed by oil microscope. Gram-negative bacteria are red, and gram-positive bacteria are purple.

The gram stain results are shown in FIG. 14, in which the bacterial cells of the strain FB8 are long rod-shaped, while the bacterial cells of the strains FB17 and FB23 are short rod-shaped and have blunt ends. In addition, after gram staining of 3 strains of bacteria, cells are purple, which indicates that 3 strains of bacteria are all gram-positive bacteria.

3. Spore dyeing (spore dyeing kit)

Spore staining A Scharffer-Fulton method, namely a malachite green staining method, is adopted by using a spore staining kit (product number R23109) of the original leaf biotechnology, Inc., and the specific operation steps are as follows: (1) selecting strains FB8, FB17 and FB23 cultured on a plate to inoculate an LB test tube, and performing shake culture at 37 ℃ for 48 hours; (2) sucking 10 mu L of bacterial liquid on a clean grease-free glass slide to prepare a smear, and naturally drying; (3) dripping malachite green staining solution at the coating solution to ensure that bacteria are uniformly distributed; (4) intermittently heating and dyeing for 10 min with weak fire; (5) lightly washing with water; (6) dyeing with spore dye liquor for 1 min, and washing with water; (7) microscopic examination: after drying, the spore is stained green, and the thallus is stained red.

The spore staining results are shown in fig. 15, and the spores in the 3 bacillus FB8, FB17 and FB23 cells were stained green, which indicates that all the 3 bacillus can produce spores, wherein the spores of the strain FB8 are mesogenic and enlarged, the spores of the strain FB17 are telogenic, and the spores of the strain FB23 are subtogenic and enlarged.

4. Movement property

Inoculating by puncturing with an inoculating needle, inoculating the bacillus on LB culture medium, culturing at 37 ℃ for 48 h, and observing the growth condition visually every 24 h after inoculation. If the growth spreads in a cloud around the puncture line, it indicates that the test strain is motile and positive.

LB medium (100 mL): tryptone 1.0 g, yeast extract 0.5 g, NaCl 1.0 g, agar 1.5 g, distilled water 100 mL, pH =7.2, subpackaged with tubes, and autoclaved at 121 ℃ for 20 min.

After 3 strains of bacillus are punctured and inoculated into an LB culture medium for 48 hours of culture, the three strains grow along the puncture line to the periphery, and the 3 strains have motility. The strain FB8 grows in a remarkable cloud form, the motility is strongest, the motility of the strain FB23 is the second to be strongest, and the motility of the strain FB17 is weaker.

In summary, the basic biological characteristics of the 3 strains are shown in table 2 below:

TABLE 2.3 basic biological characteristics of the strains

5. Molecular characterization of strains

5.1 extraction of genomic DNA

Selecting single colony from the lineation activated LB plate to inoculate fresh LB liquid culture medium, shaking culturing at 37 ℃ and 200 rpm until bacterial liquid OD₆₀₀When the bacterial strain grows to 0.8-1.0, 5 mL of bacterial liquid is taken, and the bacterial strain is collected by centrifugation. Using a bacterial genomic DNA extraction kit (CW 0552S, kang is a century,china) to extract genomic DNA.

5.216 PCR amplification of S rDNA

Using the extracted genomic DNA as a template, primers (bmede biotechnology limited, china) were designed and synthesized to amplify 16S rDNA:

27F: 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO: 3 in the sequence Listing)

1492R: 5'-GGTTACCTTGTTACGACTT-3' (SEQ ID NO: 4 in the sequence Listing)

Then, the 16S rDNA fragment was amplified by Q5 high fidelity polymerase (BDTP 1180, NEB, USA), and the amplified fragment was detected as a single band by 1% agarose gel electrophoresis (the fragment length was about 1500 bp, and the result is shown in FIG. 16), and then sent to the sequencing company for sequencing (Bomaide Biotechnology Co., Ltd., China). After obtaining the 16S rDNA fragment sequence by sequencing, the sequence is compared and analyzed by BLAST (http:// www.ncbi.nlm.nih.gov) in GenBank to obtain the identified strain name similar to the 3 strains, and the strain is identified. And (3) PCR system: genomic DNA 50-100 ng, 27F 0.5 mM, 1492R 0.5 mM, 2 XQ 5 mix 25. mu.L, H₂O was supplemented to 50. mu.L. DNA amplification procedure: first 98 ℃ for 30 seconds, then 98 ℃ for 10 seconds, 50 ℃ for 15 seconds, 72 ℃ for 45 seconds, for 30 cycles, and finally 72 ℃ for 10 minutes.

5.3 gyrBPCR amplification of genes

Using genome DNA as template, designing degenerate primer and synthesizing primer (Bomaide Biotech limited, China), amplifyinggyrBThe gene (DNA gyrase subunit B gene, partial sequence, length about 1200 bp), the underlined parts are sequencing primers UP-1S and UP-2 Sr:

gyrB-F: (SEQ ID NO: 5 in the sequence Listing)

5'-GAAGTCATCATGACCGTTCTGCAYGCNGGNGGNAARTTYGA-3'

gyrB-R: (SEQ ID NO: 6 in the sequence Listing)

5'-AGCACGGTACGGATGTGCGAGCCRTCNACRTCNGCRTCNGCRTCNGTCAT-3'

Amplification was then performed using Q5 high fidelity polymerase (BDTP 1180, NEB, USA)gyrBGene fragment amplified toThe fragment was detected as a single band by 1% agarose gel electrophoresis (the fragment length was about 1200 bp, and the result is shown in FIG. 17), and the sequencing primers UP-1S and UP-2Sr were used to sequence the fragment by the sequencer (Bomader Biotech, Inc., China). And (3) PCR system: 50-100 ng of genome DNA is obtained,

gyrB-F 0.5 mM ，gyrB-R 0.5 mM，2×Q5 mix 25 μL，H₂o make up to 50. mu.L. DNA amplification procedure: first 98 ℃ for 30 seconds, then 98 ℃ for 10 seconds, 55 ℃ for 15 seconds, 72 ℃ for 36 seconds, for 30 cycles, and finally 72 ℃ for 10 minutes.

5.416S rDNA andgyrBanalysis of Gene sequences

16S rDNA and the DNA of 3 strains (FB 17, FB8, FB 23)gyrBThe sequences obtained after gene sequencing were BLAST aligned in the NCBI database, and the alignment results are shown in the table below.

As shown in Table 3, the 16S rDNA sequence of the strain FB8 (sequence 1 in the sequence listing) and Bacillus licheniformis (B.licheniformis)B. licheniformis) Bacillus solinola desert (B.sp.) (B. sonorensis) Bacillus licheniformis (A), (B)B. paralicheniformis) The sequence similarity of (A) is highest and is 100%, of the strain FB8gyrBGene sequence (sequence 2 in sequence table) and bacillus licheniformis(B. licheniformis)The sequence similarity of the strain is the highest and is 99.42 percent, and the strain FB8 is determined to be the bacillus licheniformis: (Bacillus licheniformis）。

TABLE 3.3 molecular characterization of Bacillus bacteria

Bacillus licheniformis (Bacillus licheniformis) Strain FB8, classified and named as Bacillus licheniformisBacillus licheniformisAnd the strain has been preserved in China general microbiological culture Collection center (address: No. 3 of Xilu No. 1 on the North Chen of the Chaoyang district, Beijing) in 04.06.04.2020 with the preservation number of CGMCC number 20021.

Bacillus belgii (B.), (Bacillus velezensis) BacteriaStrain FB17, designated Bacillus belgiiBacillus velezensisAnd the strain has been preserved in China general microbiological culture Collection center (address: No. 3 of Xilu No. 1 on the North Chen of the Chaoyang district, Beijing) in 04.06.04.2020 with the preservation number of CGMCC number 20022.

Bacillus pumilus (Bacillus pumilus) strain FB23, which is classified and named as Bacillus pumilus, has been preserved in China general microbiological culture Collection center (address: No. 3 Xilu-Shih 1 Beijing, Chaoyang, area) in 04.2020 by CGMCC, and has a preservation number of CGMCC 20023.

Secondly, determining the bacteriostatic activity of the bacillus licheniformis strain FB8 CGMCC number 20021, the Bacillus belgii FB17 and the bacillus pumilus FB23

The bacteriostatic activity of the bacillus on pathogenic fungi and bacteria is detected in vitro by a double-culture method, the bacteriostatic activity of the bacillus on the pathogenic bacteria is detected in vitro by a hole digging diffusion method, 17 plant pathogenic fungi, 4 plant pathogenic bacteria, 1 bee pathogenic fungus, 1 bee pathogenic bacterium and 1 conditional pathogenic bacterium (24 pathogenic bacteria are shown in table 4 and provided by plant disease and pest laboratories of the institute of plant protection and research of Chinese academy of agricultural sciences and honeybee disease and pest laboratories of the institute of honeybee research) are taken as target bacteria, and the bacteriostatic activity of the FB8, the FB17 and the FB23 strains is respectively determined.

TABLE 4.24 plant (or bee) pathogenic fungi and bacteria

1. Pathogenic bacteria activation

And (3) fungus activation: the pathogenic fungi strain preserved at 4 deg.C is inoculated with sterilized inoculating loop, and cultured in new PDA culture medium plate at 28 deg.C for 4-5 days.

And (3) activating bacteria: taking out bacterial strains, carrying out streak inoculation on the bacterial strains to a fresh LB solid culture medium by using a sterile inoculating loop, and putting the bacterial strains into a constant-temperature incubator at 37 ℃ for culture for 24-48 h for later use. In liquid culture, single colony is picked up and put into 3 mL liquid LB culture medium, at 37 ℃, 200 r/min, and shake culture is carried out for 12 h.

2. Double culture method for determining bacteriostasis of antagonistic bacteria to pathogenic fungi

The activated pathogenic fungi were punched out into 6 mm-diameter blocks by a sterilization punch, bacteria were inoculated at equal distances around the blocks (distance about 3 cm), the culture without any bacteria was repeated 3 times for each treatment, and after 5-7 days of incubation at 28 ℃, the width of the zone of inhibition (distance of antagonistic bacteria from the edge of the pathogenic fungi) was measured.

3. Method for measuring bacteriostatic action of antagonistic bacteria on pathogenic bacteria by hole digging diffusion method

Preparing pathogenic bacteria liquid, when bacteria grow to logarithmic phase, coating LB solid culture medium with 100 μ L of the liquid, perforating uniformly (diameter 6 mm) around the culture medium, then adding 50 μ L of antagonistic bacteria liquid (FB 8 CGMCC No.20021, FB17, FB 23) with filtration sterilization into the holes, repeating for 3 times in each culture dish, culturing at 37 ℃, and measuring the diameter of the inhibition zone after 1-2 days.

4. Results and analysis

4.1 the results of the bacterial strains FB8 CGMCC No.20021, FB17 and FB23 for the determination of the bacteriostatic activity of the plant pathogenic fungi are shown in Table 5.

TABLE 5 determination of the bacteriostatic Activity of plant (or bee) pathogenic fungi

Note: weak indicates that there is a zone of inhibition, the hypha of the fungus grows and collapses, and the fungus continues to grow.

The bacteriostatic activity of the strains FB8 CGMCC No.20021, FB17 and FB23 on 18 pathogenic fungi is respectively measured by a double culture method, and the measurement results are shown in Table 5. According to experimental results, the bacterial strains FB8 CGMCC No.20021, FB17 and FB23 have different degrees of bacteriostatic activity, and the maximum bacteriostatic band width can reach 14.3 mm.

The bacterial strain FB8 can obviously inhibit 7 pathogenic fungi such as sclerotinia sclerotiorum, alternaria brasiliensis, rhizoctonia solani and the like, particularly has strong bacteriostatic activity on rhizoctonia solani, has the bacteriostatic zone width of 11.8 mm, has certain bacteriostatic action on other pathogenic fungi, but has weak inhibitory action.

The bacteriostatic activity of the strains FB8 CGMCC No.20021, FB17 and FB23 on 6 pathogenic bacteria was determined by a hole digging diffusion method, and the results are shown in Table 6.

TABLE 6 measurement results of bacteriostatic activity against pathogenic bacteria

The result shows that 3 strains basically have stronger bacteriostatic activity on 6 pathogenic bacteria (including bee pathogenic bacteria), the bacteriostatic diameter of FB8 CGMCC No.20021 on the larval bacillus of the bee pathogenic bacteria can reach 37.0 mm, and the bacteriostatic diameter on the xanthomonas gossypii can reach 21.8 mm.

And thirdly, identifying antibacterial peptide genes produced by the bacillus licheniformis FB8 CGMCC number 20021, the bacillus belgii FB17 and the bacillus pumilus FB 23.

The 3 strains of bacillus were mainly subjected to third generation Nanopore sequencing of the whole genome, and the results of genome information assembly, analysis and comparison by the third generation sequencing sequence are shown in table 7.

TABLE 7.3 statistics of Bacillus subtilis genome information

The biological information analysis of the whole genome is carried out on 3 strains of bacillus by using anti SMASH (antimicrobial peptide prediction software), and the antimicrobial peptide gene cluster possibly contained in each strain is predicted, and the result shows that bacillus licheniformis FB8 CGMCC number 20021 at least has the gene cluster for synthesizing 3 antimicrobial peptides (figure 18), bacillus belgii FB17 at least has the gene cluster for synthesizing 7 antimicrobial peptides, and bacillus pumilus FB23 at least has the gene cluster for synthesizing 3 antimicrobial peptides. The types of the antibacterial peptide synthesis gene clusters contained in the genome of the FB8 CGMCC number 20021 strain are shown in the following table 8.

Table 8, antibacterial peptide synthesis gene cluster in Bacillus licheniformis FB8 CGMCC number 20021 genome

The results show that 3 strains of bacillus have the potential of synthesizing various antibacterial peptides.

Sequence listing

<110> bee institute of Chinese academy of agricultural sciences

<120> Bacillus licheniformis and application thereof

<130> WHOI210060

<160> 6

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1432

<212> DNA

<213> Bacillus licheniformis

<400> 1

ttcggcggct ggctccaaag gtgtacctca ccgacttcgg gtgttacaaa ctctcgtggt 60

gtgacgggcg gtgtgtacaa ggcccgggaa cgtattcacc gcggcatgct gatccgcgat 120

tactagcgat tccagcttca cgcagtcgag ttgcagactg cgatccgaac tgagaacaga 180

tttgtgggat tggcttagcc tcgcggcttc gctgcccttt gttctgccca ttgtagcacg 240

tgtgtagccc aggtcataag gggcatgatg atttgacgtc atccccacct tcctccggtt 300

tgtcaccggc agtcacctta gagtgcccaa ctgaatgctg gcaactaaga tcaagggttg 360

cgctcgttgc gggacttaac ccaacatctc acgacacgag ctgacgacaa ccatgcacca 420

cctgtcactc tgcccccgaa ggggaagccc tatctctagg gttgtcagag gatgtcaaga 480

cctggtaagg ttcttcgcgt tgcttcgaat taaaccacat gctccaccgc ttgtgcgggc 540

ccccgtcaat tcctttgagt ttcagtcttg cgaccgtact ccccaggcgg agtgcttaat 600

gcgtttgctg cagcactaaa gggcggaaac cctctaacac ttagcactca tcgtttacgg 660

cgtggactac cagggtatct aatcctgttc gctccccacg ctttcgcgcc tcagcgtcag 720

ttacagacca gagagtcgcc ttcgccactg gtgttcctcc acatctctac gcatttcacc 780

gctacacgtg gaattccact ctcctcttct gcactcaagt tccccagttt ccaatgaccc 840

tccccggttg agccgggggc tttcacatca gacttaagaa accgcctgcg cgcgctttac 900

gcccaataat tccggacaac gcttgccacc tacgtattac cgcggctgct ggcacgtagt 960

tagccgtggc tttctggtta ggtaccgtca aggtaccgcc ctattcgaac ggtacttgtt 1020

cttccctaac aacagagttt tacgatccga aaaccttcat cactcacgcg gcgttgctcc 1080

gtcagacttt cgtccattgc ggaagattcc ctactgctgc ctcccgtagg agtctgggcc 1140

gtgtctcagt cccagtgtgg ccgatcaccc tctcaggtcg gctacgcatc gtcgccttgg 1200

tgagccgtta cctcaccaac tagctaatgc gccgcgggtc catctgtaag tggtagctaa 1260

aagccacctt ttataattga accatgcggt tcaatcaagc atccggtatt agccccggtt 1320

tcccggagtt atcccagtct tacaggcagg ttacccacgt gttactcacc cgtccgccgc 1380

taacatcagg gagcaagctc ccatctgtcc gctcgacttg catgtattag ca 1432

<210> 2

<211> 1213

<212> DNA

<213> Bacillus licheniformis

<400> 2

cgctggtggg aaatttgacg gaagcggata taaagtttca ggcggtttgc acggcgtcgg 60

tgcatctgtt gttaacgccc tttcaaccga gctcgatgta acggtttaca gagatggaaa 120

agtccattac caggaatttg aacggggcgt tccgaaagct gatttgaaag tcatcggaga 180

tacggaagtg acgggaacga ccactcactt caagcctgat ccggaaatat tcacggaaac 240

gacggaatac gactatgata cgcttgccac tcgtgtccgg gagctcgctt tcttgacaaa 300

aggcgtcaaa atcacgattg aagacaagcg agaaggaaaa gaacgcaaga atgattactg 360

ctatgaaggc ggtattaaaa gctatgttga acacttgaac cgttcacggg aagtggttca 420

tgaagagcca gtctatattg aaggatccaa agacggcatt acggtcgagg tggctcttca 480

atacaacgac agctatacca gcaacattta ttcgtttgcc aataacattc atacgtatga 540

aggcggaacg catgaagccg gctttaagac cggtttgacg agagtcatca atgattacgc 600

gagaaggaac ggtgtcttca aagaaagcga tccgaactta agcggggaag acgtccgtga 660

aggtttgaca gcgatcattt caatcaagca tccggatcct caatttgaag ggcagacgaa 720

aacaaagctt ggcaactcag aagcgcggac gataacagat gcgctatttt cagaagcgct 780

cgaaaagttt ctgcttgaaa acccggattc ggcgaaaaaa atcgttgaaa aaggggttat 840

ggccgccaga gcacgaatgg ctgcaaagaa agcacgtgaa ctgacgcgca gaaaaagcgc 900

ccttgaagtg tcgaatctgc cggggaaact ggctgactgt tcttctaaag acccgacgat 960

ttccgaactt tacatcgttg agggtgactc tgcgggcgga tcggcaaaac aaggccgcga 1020

tcgtcatttc caagccattt tgcctttgag agggaaaatt ttgaacgtcg aaaaagcacg 1080

cctggacaaa attttgtcca acaatgaggt tcgttctatg atcaccgcgc ttggcaccgg 1140

tatcggggaa gatttcaatc ttgaaaaagc ccgctaccac aaagtcgtga ttatgaccga 1200

tgccgacgtc gac 1213

<210> 3

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

agagtttgat cctggctcag 20

<210> 4

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

ggttaccttg ttacgactt 19

<210> 5

<211> 41

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

gaagtcatca tgaccgttct gcaygcnggn ggnaarttyg a 41

<210> 6

<211> 50

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

agcacggtac ggatgtgcga gccrtcnacr tcngcrtcng crtcngtcat 50

Claims (3)

1. The bacillus licheniformis is named as bacillus licheniformis FB8, and the bacillus licheniformis FB8 is preserved in the China general microbiological culture Collection center of the Committee for culture Collection of microorganisms with the preservation number of CGMCC No. 20021.

2. Use of a bacillus licheniformis according to claim 1 for the preparation of a biological agent inhibiting phytopathogenic fungi, phytopathogenic bacteria, honey bee pathogenic bacteria and/or conditionally pathogenic bacteria; the plant pathogenic fungus is sclerotinia sclerotiorum (A)Sclerotinia sclerotiorum) Alternaria brassicae (S.brassicae) ((S.brassicae))Alternaria brassicicola) Fusarium oxysporum (F.), (Fusarium Oxysporum) Colletotrichum cucurbitacearum (A)Colletotrichum orbiculare (Berk.&Mont.) Arx) Sclerotinia virescens (A), (B), (C), (B), (C), (B), (C)Ustilaginoidea oryzae (Patou.) Bref=U.virens (Cooke) Tak.) Rhizoctonia cerealis (a)Rhizoctonia cerealis Vander Hoeven) And apple black beancurd sheetsShell bacterium (A), (B)Valsa mali Miyabe et Yamada.) One or more of; the plant pathogenic bacteria is pseudomonas syringae (Pseudomonas syringae)Pseudomonas syringae) Xanthomonas gossypii (A. gossypii:)Xanthomonas campestris pv. malvacearum (Smith) Dye) Pseudomonas solanacearum (B) ((B))Pseudomonas solanacearum) One or more of; the bee pathogenic bacteria are Bacillus larvae (B), (C)Paenibacillus larvae) (ii) a The conditionally pathogenic bacterium is Micrococcus luteus (A)Micrococcus luteus）。

3. A bacteriostatic agent for inhibiting phytopathogenic fungi, phytopathogenic bacteria, bee pathogenic bacteria and conditionally pathogenic bacteria, which comprises the Bacillus licheniformis of claim 1 as an active ingredient; the plant pathogenic fungus is sclerotinia sclerotiorum (A)Sclerotinia sclerotiorum) Alternaria brassicae (S.brassicae) ((S.brassicae))Alternaria brassicicola) Fusarium oxysporum (F.), (Fusarium Oxysporum) Colletotrichum cucurbitacearum (A)Colletotrichum orbiculare (Berk.& Mont.) Arx) Sclerotinia virescens (A), (B), (C), (B), (C), (B), (C)Ustilaginoidea oryzae (Patou.) Bref=U.virens (Cooke) Tak.) Rhizoctonia cerealis (a)Rhizoctonia cerealis Vander Hoeven) And Humicola lanuginosa (L.) TokayataValsa mali Miyabe et Yamada.) One or more of; the plant pathogenic bacteria is pseudomonas syringae (Pseudomonas syringae)Pseudomonas syringae) Xanthomonas gossypii (A. gossypii:)Xanthomonas campestris pv. malvacearum (Smith) Dye) Pseudomonas solanacearum (B) ((B))Pseudomonas solanacearum) One or more of; the bee pathogenic bacteria are Bacillus larvae (B), (C)Paenibacillus larvae) (ii) a The conditionally pathogenic bacterium is Micrococcus luteus (A)Micrococcus luteus）。

Images