CN116731928B - Bacillus bailii YC5 and application thereof in preventing and treating black skin disease of Chinese yam - Google Patents

Bacillus bailii YC5 and application thereof in preventing and treating black skin disease of Chinese yam Download PDF

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CN116731928B
CN116731928B CN202310801773.9A CN202310801773A CN116731928B CN 116731928 B CN116731928 B CN 116731928B CN 202310801773 A CN202310801773 A CN 202310801773A CN 116731928 B CN116731928 B CN 116731928B
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bacillus
skin disease
yam
black skin
strain
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CN116731928A (en
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陈晓蓉
曾粮斌
龙文平
熊灵芝
孙佳莉
许文丽
栾明宝
李柏花
欧阳昌东
颜雪梅
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Yichun Academy Of Sciences Jiangxi Selenium Industry Research Institute
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Abstract

The invention discloses bacillus belicus YC5 and application thereof in prevention and treatment of yam black skin disease, and belongs to the technical field of biology. The bacillus beleiensis YC5 is preserved in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) for 2023, 6 and 8 days, and the preservation number is CGMCC NO.27583. The bacillus beliae YC5 provided by the invention can inhibit the growth and the morbidity of Fusarium solani, has an undetermined antibacterial effect, and has obvious control effect on the black skin disease data of the Chinese yam through an indoor in-vitro test and a field control test, thereby providing a new direction for the control of the black skin disease of the Chinese yam.

Description

Bacillus bailii YC5 and application thereof in preventing and treating black skin disease of Chinese yam
Technical Field
The invention relates to the technical field of biology, in particular to bacillus bailii YC5 and application thereof in preventing and treating yam black skin disease.
Background
Soil-borne diseases refer to diseases caused by the invasion of pathogens such as fungi, bacteria, nematodes and viruses from the roots or stems of crops when the conditions are appropriate, by living in the soil with the disease residues. These diseases usually cause significant economic losses by infecting the roots or stems of the plants, thereby causing the onset of the roots or stems of the crops, and even the whole plant. The black skin disease of the Chinese yam is the most common soil-borne disease in the Chinese yam cultivated species, at present, the prevention and control are mainly carried out by adopting chemical agents, and the long-term use of the chemical agents can cause a series of serious consequences such as water source soil pollution, ecological balance being destroyed, pesticide residues, secondary diseases being rampant, pathogenic microorganisms generating drug resistance and the like, so that the prevention and control of the black skin disease of the Chinese yam encounters bottlenecks.
With the enhancement of people's environmental awareness, attach importance to food safety problem and the requirement of ecological environment construction, protection biodiversity and agricultural sustainable development for biological prevention and control soil-borne disease becomes the hot spot of current research and development. Bacillus sp is a kind of gram positive bacteria producing spores, and has the advantages of being capable of producing endospores with heat resistance, drought resistance, ultraviolet resistance and organic solvent resistance in aerobic or facultative anaerobic life, being an important biological control resource, and the control mechanism is mainly as follows: the bacillus colonizes the root, body surface or body of the plant, competes with pathogenic bacteria for nutrition around the plant, secretes antibacterial substances to inhibit the growth of the pathogenic bacteria, and induces a plant defense system to resist the invasion of the pathogenic bacteria, so that the aim of biological control is achieved. At present, part of excellent bacillus is separated out and successfully applied to biological control of plant diseases, such as bacillus subtilis BLG010 and bactericide thereof have good control effect on banana wilt; the Paenibacillus terrae NK3-4 has good control effect on soybean root rot; the bacillus subtilis BS-KA2 and the biological agent thereof play a good role in preventing and controlling peanut rot.
Although the biocontrol microbial inoculum of bacillus exists at present, the biocontrol microbial inoculum has the defects of fewer biocontrol microbial strains, single antibacterial effect and instability, and in order to enrich the types of biocontrol microbial inoculum and improve the comprehensive control effect of the yam black skin disease, the biocontrol microbial inoculum for controlling the yam black skin disease needs to be further researched.
Disclosure of Invention
The invention aims to provide bacillus bailii YC5 and application thereof in preventing and treating yam black skin diseases, so as to solve the problems in the prior art. The bacillus belgium YC5 provided by the invention can inhibit the growth and the morbidity of Fusarium solani, has high and stable antibacterial effect, and can effectively prevent and treat the black skin disease of Chinese yam caused by Fusarium solani.
In order to achieve the above object, the present invention provides the following solutions:
the invention provides bacillus bailii (Bacillus velezensis) YC5 which is preserved in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) for 2023, 6 and 8 days, and has a preservation number of CGMCC NO.27583.
The invention also provides application of the bacillus belgium YC5 in preparation of a biocontrol agent, wherein the biocontrol agent is used for controlling plant diseases caused by Fusarium solani.
Further, the plant disease includes yam black skin disease.
The invention also provides a biocontrol microbial inoculum for preventing and treating plant soil-borne fungal diseases, which comprises bacillus bailii YC5 and/or fermentation products thereof.
The invention also provides a method for preventing and controlling soil-borne fungus diseases of plants, which comprises the step of applying the biocontrol microbial inoculum of bacillus belicus YC5 or more households to plants so as to prevent the plants from being infected by pathogenic bacteria.
Further, the pathogenic bacteria are Fusarium solani.
Further, the soil-borne fungus disease is yam black skin disease.
The invention also provides application of the bacillus belicus YC5 or the biocontrol microbial inoculum in preventing and controlling plant soil-borne fungus diseases.
Further, the plant soil-borne fungus is Fusarium solani.
Further, the soil-borne fungus disease is yam black skin disease.
The invention discloses the following technical effects:
the bacillus bailii YC5 is screened out from the rhizosphere soil of the Chinese yam, is a gram positive bacterium, can generate spores, and can generate IAA and ammonia, secrete amylase and contact enzyme. According to the invention, the bacillus belicus YC5 is discovered for the first time, the growth and the morbidity of Fusarium solani can be inhibited, the antibacterial effect is not definite, and the bacillus belicus YC5 has obvious control effect on the black skin disease data of the Chinese yam through an indoor in-vitro test and a field control test, so that a new direction is provided for the control of the black skin disease of the Chinese yam.
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In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a colony morphology of Bacillus belicus YC 5;
FIG. 2 is a gram (a) and a spore stain (b) of Bacillus bailii YC 5;
FIG. 3 is a phylogenetic tree of 16S rDNA sequence construction of Bacillus bailii YC 5;
FIG. 4 shows the results of the contact enzyme test (a), amylolytic ability test (b), IAA production test (c), phosphate production ability test (d) and ammonia production ability (e) of Bacillus belicus YC 5;
FIG. 5 is a plate inhibition zone experiment of Bacillus belicus YC5 on Fusarium solani;
FIG. 6 is an in vitro indoor test of Bacillus belicus YC5 against yam black skin disease;
fig. 7 is a field control test of bacillus belicus YC5 against yam black skin disease.
Detailed Description
Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the invention described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present invention. The specification and examples of the present invention are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.
Example 1 isolation screening and identification of Bacillus bailii YC5
1. Separation and screening of Bacillus bailii YC5
S01: collecting yam rhizosphere soil by a multipoint sampling method, and collecting Yu Yi yam rhizosphere soil in the embodiment as a yam test base in the academy of sciences of spring and market;
s02: placing the obtained rhizoma Dioscoreae rhizosphere soil sample into a centrifuge tube with sterile water, and shaking thoroughly to obtain rhizoma Dioscoreae rhizosphere soil sample with concentration of 10 -1 CFU/mL of suspension;
s03: gradually and gradiently diluting the suspension to obtain suspensions with different concentrations;
s04: selecting concentration of 10 -3 、10 -4 And 10 -5 Three gradient soil suspensions are added to an LB culture medium flat plate for coating treatment, and the three gradient soil suspensions are placed into a 30 ℃ incubator for culturing for 48 hours, so that bacterial colonies are obtained;
s05: selecting bacillus single bacteria with different morphologies after morphological observation by a microscope, performing streak preservation on an inclined plane, culturing for 24 hours, and placing in a refrigerator at 4 ℃ for later use;
s06: the separated bacteria are evenly inoculated on a KB solid culture medium plate coated with 100 mu L of kiwi fruit canker bacteria liquid, cultured for 48 hours at the temperature of 25 ℃, repeated experiments are carried out for 3 times each time, and the bacteria with the largest inhibition zone are selected and marked as YC5.
The LB culture medium is a nutrient agar culture medium, and the nutrient agar culture medium comprises 15g/L of agar, 10g/L of tryptone, 5g/L of yeast extract and 10g/L of NaCl according to the concentration. The LB medium was prepared at pH 7.4 and subjected to sterilization treatment for 20min at 121 ℃. The KB culture medium consists of 15g/L of agar powder, 20g/L of peptone and K 2 HPO 4 1.5g/L, glycerin 10mL/L, mgSO 4 ·7H 2 O1.5 g/L, pH remains natural.
2. Identification of bacillus beleiensis YC 5:
1. morphological identification
The YC5 was streaked on a LB medium plate, then the plate was inverted, cultured for 24 hours at a temperature of 30℃and the growth of colonies on the plate was observed and recorded, and the colony morphology on the plate was as shown in FIG. 1, as can be seen from the graph, the colony growth morphology of the strain prepared in this example exhibited an irregular circular shape, the surface was convex, and the color was milky and opaque.
YC5 was gram stained and spore stained with the kit, and the strain was observed under an oil microscope and photographed. The gram staining and spore staining of the strain are shown in (a) of fig. 2 and (b) of fig. 2 respectively, and as can be seen from (a) of fig. 2, the strain YC5 is in a rod shape and in a blue-purple color after gram staining, and is a gram positive bacterium; as can be seen from FIG. 2 (b), after spore staining, the YC5 cells of the strain showed blue color and sporulation, thereby demonstrating that the strain provided in this example was capable of producing spores.
2. Physiological and biochemical identification
(1) Contact enzyme assay
3% hydrogen peroxide was directly added dropwise to the liquid culture medium of strain YC5, and immediately observed. If a large number of bubbles are generated, the result is positive; if no bubble is generated, the result is negative. The strain YC5 provided by the present invention immediately produced a large amount of bubbles, and the experimental result was positive (FIG. 4 (a)).
(2) Oxidase test
A small amount of bacterial strain colonies prepared in the embodiment is dipped in one corner of white clean filter paper, one drop of 1% dimethyl p-phenylenediamine hydrochloride aqueous solution is added, and the positive person immediately turns pink and the color gradually deepens. In this experiment, the colonies did not change color, and the experimental result was negative.
(3) Starch hydrolysis experiments
The strain YC5 was inoculated onto a starch medium, cultured at 37℃for 24 hours, and a small amount of iodine solution was added dropwise onto a starch medium plate, and the mixture was gently swirled to uniformly distribute the iodine solution on the starch medium plate, whereby the condition around the colony was observed. If a colorless transparent ring appears around the colony, this indicates the ability to hydrolyze starch, otherwise, it is not. The colony of the strain YC5 was surrounded by a transparent ring, whereby it could be explained that the strain YC5 had the ability to hydrolyze starch (FIG. 4 (b)).
(4) Methyl red MR experiment
Selecting a small amount of strain YC5, inoculating on a general culture medium, culturing for 3-5d at 30 ℃, taking 1mL of culture solution after the culture is finished, adding 1-2 drops of methyl red indicator, wherein the positive is bright red, the weak positive is light red, and the negative is yellow. In this experiment, the bacterial liquid turned yellow, and was therefore negative.
(5) VP experiment
Selecting a small amount of strain YC5, inoculating to a general culture medium, culturing for 4d at 30 ℃, taking 2.5mL of culture solution after the culturing is finished, adding 0.6mL of alpha-naphthol pure alcohol solution, adding 0.2mL of 40% potassium hydroxide aqueous solution, shaking for 2-5min, enabling positive bacteria to normally immediately appear red, standing in an incubator at 30 ℃ if no red appears, and judging negative if no red appears within 2 h. In this experiment, the bacterial liquid became red immediately, so it was positive.
(6) Gelatin liquefaction experiment
Strain YC5 was taken and inoculated by puncture into gelatin and placed at 2/3 of the gelatin depth. Culturing at 20deg.C for 7d. Observing whether the strain is liquefied by bacteria every day, and if so, determining that the strain is positive in test; if not liquefied, it is negative. In this experiment, gelatin was not liquefied, so the reaction characteristic was negative.
(7) Nitrate reduction experiment
The strain YC5 is inoculated in a nitrate culture medium, shake culture is carried out for 3d at the temperature of 30 ℃, then 5mL of culture solution is taken, a color-developing agent is added according to the instruction of a kit (a nitrate reduction kit of the Haibo biotechnology Co., ltd.) and turns yellow to be positive, otherwise, no color change is negative. In this experiment, the bacterial liquid turned yellow, indicating that the reaction was positive in character.
(8) Experiment for producing hydrogen sulfide
The strain YC5 was inoculated by puncture into a lead acetate medium, cultured at a temperature of 35℃for 48 hours, and the result was observed. If the culture medium turns black, the result is positive; and if the color is not black, the color is negative. In this experiment, the medium turned black, indicating that the reaction was positive.
(9) Citrate utilization experiments
The selected strain YC5 was streaked onto a slope of a citrate medium of Western Meng Sishi, and cultured at 37℃for 7 days. If the culture medium is alkaline, the indicator is blue or pink positive; if the medium does not change color, it is negative. In this experiment, the medium was discoloured, indicating that the reaction was characterized as positive.
(10) Lecithin enzyme Activity assay
Sterilizing fresh egg surface with 75% ethanol, perforating egg with sterilized forceps, removing egg white, sucking yolk with sterile suction tube, adding into NA culture medium cooled to about 50deg.C after thawing, mixing, placing flat plate, inoculating strain YC5, culturing at 30deg.C for 24 hr, and observing. If the colony edge appears cloudy circles, the colony edge is enzyme positive. In this experiment, a clear cloudy ring appeared at the edge of the colony, indicating a positive reaction profile.
(11) Malonate utilization experiments
The strain YC5 cultured for 12 hours is selected and inoculated in malonate culture medium, and is cultured for 48 hours at the temperature of 35 ℃, the culture medium is positive when the color of the culture medium changes from green to blue, and the culture medium is negative when the color of the culture medium does not change. In this experiment, the color of the medium was unchanged, indicating that the reaction characteristic was negative.
(12) Glucose fermentation experiments
A small amount of strain YC5 is selected and inoculated on a glucose oxidation fermentation culture medium in a penetrating way, and is cultured for 3d under the condition of 30 ℃ to observe the color change of the culture medium. If there is no color change, the observation is continued for 7 days, and the medium turns yellow to be fermented. In this experiment, the medium turned yellow, indicating that the reaction was positive.
(13) D-galactose utilization test positivity
Strain YC5 was inoculated into D-galactose medium and cultured at 30 ℃ for 2D to observe colony growth, and if colony formation was observed, D-galactose could be used, whereas galactose could not be used. In this experiment, the cell growth indicated that the strain YC5 could utilize D-galactose.
(14) D-arabinose utilization experiment
Strain YC5 was inoculated into an arabinose medium, and cultured at 30 ℃ for 2D to observe colony growth, and if colony formation was observed, D-arabinose could be used, whereas otherwise, it could not be used. In this experiment, the cell was unable to grow, indicating that the strain YC5 could not utilize D-arabinose.
(15) D-mannose utilization experiment
Strain YC5 was inoculated into D-mannose medium, cultured at 30 ℃ for 2D, and colony growth was observed. In this experiment, the strain YC5 was grown using D-mannose.
(16) D-fructose utilization experiment
Strain YC5 was inoculated into D-fructose medium, cultured at 30 ℃ for 2D, and colony growth was observed, and if colony formation was observed, D-fructose could be used, whereas otherwise, it could not be used. In this experiment, the strain YC5 was grown using D-fructose.
(17) D-xylose utilization experiments
The strain YC5 was inoculated into a D-xylose medium, and cultured at 30℃for 2 days, and the colony growth was observed, and if colonies were formed, D-xylose could be used, whereas otherwise, it could not be used. In this experiment, the strain YC5 was grown using D-xylose.
Part of biocontrol bacteria can promote plant growth and development by secreting growth promoting substances such as indoleacetic acid (IAA), ferrite and the like, and seedlings can be strengthened by using the biocontrol bacteria. Therefore, the strain YC5 is subjected to qualitative detection of functional substances, and the IAA production, phosphate dissolution and ammonia production capacities are measured.
(18) IAA production ability assay: the strain YC5 was inoculated into a TSA medium (tryptone 10.0g, yeast extract 5.0g, naCl 5.0g, distilled water to 1000mL, pH was adjusted to 7.5) containing 5mmol/L L-tryptophan, cultured for 36 hours on a shaker at a constant temperature of 30℃at 160r/min, the supernatant was retained by low-speed centrifugation, and after removing the cells by filtration through a 0.22 μm sterile filter, 1mL of the sterile filtrate was added to 2mL of Salkawski developer, and the mixture was allowed to stand at room temperature for 30 minutes. Using sterile TSA medium as a control, if the sterile filtrate turned pink, IAA could be produced, and conversely, could not be produced. The change of sterile filtrate to pink indicates IAA production (fig. 4 (c)).
(19) Determination of phosphate-solubilizing ability: the strain prepared in this example was inoculated into NBRIP solid medium (agar powder 15g, glucose 10.0g, bromophenol blue (0.4%, pH 6.7) 6mL, alPO 4 2 g，(NH 4 ) 2 SO 4 0.5g, yeast extract 0.5g,KCl 0.2g,NaCl 0.2g,MgSO 4 0.1 g，FeSO 4 0.002 g，MnSO 4 0.002 g, after each component is fully dissolved, distilled water is used for fixing the volume to 1000mL, the pH is kept natural), the center of the flat plate is inversely cultured for 4 days at the temperature of 30 ℃ in a biochemical incubator, whether a culture medium around a colony generates a transparent ring or not is observed, if the transparent ring exists, the bacterial strain has the phosphate dissolving capability, otherwise, the bacterial strain does not have the phosphate dissolving capability. The presence or absence of a permeable ring in the medium surrounding the colony resulted in the strain having phosphate-solubilizing ability (FIG. 4 (d)).
(20) Determination of Ammonia production Capacity: the strain prepared in this example was inoculated into peptone ammoniation medium (peptone 5g, mgSO 4 0.5 g，KH 2 PO 4 0.5 g，K 2 HPO 4 0.5g, adding water to 1000mL and keeping the volume constant and the pH value to 7.0), and culturing for 24h at the constant temperature of 30 ℃ and 160r/min by taking the mixture as an experimental group. With untreated medium as control group CK, 5 drops of Nessler reagent were added to each of the test group and CK, and the test group produced yellow or reddish brown precipitate, and CK did not appear, indicating that strain YC5 had ammonia-producing ability (FIG. 4 (e)).
In summary, the physiological and biochemical identification results of strain YC5 are shown in Table 1:
TABLE 1
Note that: +: a positive reaction; -: negative reaction.
3. 16S rDNA sequence analysis
The method adopts a bacterial liquid PCR method to carry out PCR amplification on ITS sequences of the bacterial strain YC5, and comprises the following specific operation steps: 25. Mu.L of a PCR reaction system comprising 12.5. Mu.L of a2 XMaster Mix; 1. Mu.L of upstream primer 27F (5'-AGAGTTTGATCCTGGCTCAG-3', SEQ ID NO. 2); 1. Mu.L of the downstream primer 1492R (5'-GGTTACCTTGTTACGACTT-3', SEQ ID NO. 3); 9 mu L ddH 2 O; 1.5. Mu.L of template DNA.
The PCR reaction conditions were: pre-denaturing at 94 ℃ for 2min; denaturation at 94℃for 30s; annealing at 55 ℃ for 30s; extending at 72 ℃ for 30s;35 cycles, 2min extension at 72℃and storage at 4 ℃. The obtained PCR product was sequenced by Hunan qing Kogyo Co., ltd. And the nucleotide sequence of strain YC5 was shown as SEQ ID NO. 1.
SEQ ID NO.1:
AGGTTGCGCTGCTATACTGCAGTCGAGCGGACAGATGGGAGCTTGCTCCCTGATGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATGGTTGTCTGAACCGCATGGTTCAGACATAAAAGGTGGCTTCGGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTGCCGTTCAAATAGGGCGGCACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAATCCTAGAGATAGGACGTCCCCTTCGGGGGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACAGAACAAAGGGCAGCGAAACCGCGAGGTTAAGCCAATCCCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTTAGGAGCCAGCCGCCGAAGGGACAGGGAAGT。
The nucleotide sequence obtained is subjected to homologous sequence comparison analysis through NCBI-BLAST to obtain a sequence with higher similarity. The 16SrDNA sequence of the strain prepared in this example has homology of 99.79% with Bacillus belicus (Bacillus velezensis) by constructing phylogenetic tree (shown in FIG. 3) using MEGA6.0 software.
From the above morphological observation, physiological biochemical identification and 16S rDNA sequence analysis results, it was confirmed that the strain prepared in this example was Bacillus bailii (Bacillus velezensis), which was designated Bacillus bailii strain YC5.
The bacillus belay strain prepared by the embodiment of the invention is bacillus belay strain YC5 which is preserved in China general microbiological culture Collection center (CGMCC for short, the preservation unit address: north Chen West Lu No.1, 3 of the Korean region of Beijing city, and the institute of microorganisms of the China academy of sciences, postal code: 100101) in the 6 th month 8 day 2023, and the preservation number is CGMCC No.27583.
Example 2 Petri dish inhibition zone test of Bacillus bailii YC5 against yam black skin disease
Test pathogenic bacteria: fusarium solani (strain number CGMCC 3.2889, available from China general microbiological culture Collection center).
The method comprises the following steps: the preparation method comprises the steps of taking fusarium solani as a target bacteria, inoculating a fusarium solani block with the diameter of 5mm to the center of a culture dish, inoculating bacillus beijerinus strain YC5 to the position 2.5cm away from the center of a flat plate, and repeating each treatment for 3 times by taking a flat plate without inoculating YC5 bacteria as a control. The flat plate is placed in a 28 ℃ incubator in an inverted mode, when pathogenic fungi of a control group grow up on the flat plate, the diameters of pathogenic fungi of the control group, pathogenic fungi of a treatment group, the diameters of biocontrol bacteria and the diameters of bacteriostasis circles of bacteria on the pathogenic fungi are measured, and the bacteriostasis rate and antagonism index of the bacteria are calculated:
antibacterial ratio (%) = (control pathogen diameter-counter pathogen diameter)/control pathogen diameter×100
Antagonistic index (%) = (inhibition zone diameter-antagonistic diameter)/antagonistic diameter×100.
The antibacterial effect of the strain YC5 on Fusarium solani is shown in figure 5, and as can be seen from figure 5, fusarium solani can obviously inhibit the growth of Fusarium solani, the diameter of pathogenic bacteria of a control group is 83.45 +/-0.74 mm, the diameter of pathogenic bacteria of a YC5 treatment group is 23.71+/-0.38 mm, and the antibacterial rate is 71.59 +/-0.45%; the diameter of the YC5 pathogenic bacteria inhibition zone for the yam black skin disease is 27.45 plus or minus 0.31mm, the diameter of the YC5 is 14.57 plus or minus 0.34mm, and the antagonism index is 88.40 plus or minus 2.15%.
EXAMPLE 3 control Effect of Bacillus bailii YC5 on black skin disease of Dioscorea opposita
1. Preparation of microbial inoculum
Inoculating strain YC5 of example 1 into sterilized LB liquid medium (10 g/L tryptone, 5g/L yeast extract, 10g/L NaCl; pH 7.4), culturing at 30deg.C, shaking for 4d at 180r/min, centrifuging for 5min 12000r/min, and diluting the precipitate with sterile water to a concentration of 10 -5 The CFU/mL suspension is used for obtaining the biocontrol microbial inoculum containing bacillus bailii YC5.
2. Indoor ex-vivo test
Cutting rhizoma Dioscoreae into slices with thickness of 3mm, and preparing into tablet with concentration of 10 -5 CFU/mL biocontrol microbial inoculum containing bacillus bailii YC5 is soaked for 5min, a control group is soaked for 5min by clean water, fusarium solani with the diameter of 5mm is inoculated in the center of a Chinese yam slice, the Chinese yam slice is moisturized and placed in a biochemical incubator at 25 ℃, the diameter of a disease spot is measured after 5 days, and the control effect is calculated. The results are shown in FIG. 6 and Table 2.
TABLE 2
Treatment of Diameter of disease spot (cm) Preventing effect (%)
Clear water control 27.10±0.46 -
YC5(10 -5 CFU/mL) 10.56±0.32 61.03±1.18
As can be seen from fig. 6 and table 2, the biocontrol agent containing bacillus belicus YC5 has a control effect of 61.18% on yam black skin disease in an indoor in vitro test.
3. Field test
Will be formulated at a concentration of 10 -5 CFU/mL biocontrol microbial inoculum containing bacillus bailii YC5 is sprayed with stump 100mL after the emergence of the Chinese yam seedlings, stump is sprayed with stump once every stump at intervals of 20 days, the Chinese yam is dug out to investigate the disease index of the Chinese yam black skin disease when the Chinese yam is harvested, and the control effect is calculated. The results are shown in FIG. 7 and Table 3.
TABLE 3 Table 3
Treatment of Index of disease condition Preventing effect (%)
Clear water control 31.56±0.75 -
YC5(10 -5 CFU/mL) 11.68±0.52 62.99±1.65
As can be seen from fig. 7 and table 3, the biocontrol agent containing bacillus belicus YC5 has a control effect of 62.99% on yam black skin disease in field experiments.
The above embodiments are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solutions of the present invention should fall within the protection scope defined by the claims of the present invention without departing from the design spirit of the present invention.

Claims (7)

1. Bacillus bailii strainBacillus velezensis) YC5, which is preserved in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) for 2023, 6 and 8 days, and has a preservation number of CGMCC No.27583.
2. The application of bacillus belgium YC5 according to claim 1, in preparing a biocontrol agent, wherein the biocontrol agent is used for preventing and controlling fusarium solani from fusarium solaniFusarium solani) Caused plant diseases.
3. The use according to claim 2, wherein the plant disease comprises yam black skin disease.
4. A biocontrol microbial agent for controlling soil-borne microbial diseases of plants, which is characterized by comprising bacillus belicus YC5 and/or fermentation products thereof according to claim 1.
5. A method for controlling black skin disease of yam, comprising the step of applying the bacillus belicus YC5 of claim 1 or the biocontrol agent of claim 4 to yam to prevent the yam from being infected by pathogenic bacteria.
6. The method of claim 5, wherein the pathogenic bacteria is fusarium solani.
7. Use of bacillus belgium YC5 according to claim 1 or a biocontrol agent according to claim 4 for controlling black skin disease of yam.
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