CN114250171A

CN114250171A - Bacillus amyloliquefaciens Yb-2 and separation method and application thereof

Info

Publication number: CN114250171A
Application number: CN202111493745.2A
Authority: CN
Inventors: 严婉荣; 赵志祥; 王宝; 肖彤斌; 吉训聪; 陈圆
Original assignee: Institute Of Plant Protection Hainan Academy Of Agricultural Sciences Research Center For Quality Safety And Standards Of Agricultural Products Hainan Academy Of Agricultural Sciences
Current assignee: Institute Of Plant Protection Hainan Academy Of Agricultural Sciences Research Center For Quality Safety And Standards Of Agricultural Products Hainan Academy Of Agricultural Sciences
Priority date: 2021-12-08
Filing date: 2021-12-08
Publication date: 2022-03-29
Anticipated expiration: 2041-12-08
Also published as: CN114250171B

Abstract

The invention discloses a bacillus amyloliquefaciens Yb-2 and a separation method and application thereof, relating to the technical field of biology, wherein the strain is preserved in the China general microbiological culture Collection center (CGMCC) at 6, 7 and 2021 with the preservation number of CGMCC No. 22660. The invention separates a bacillus amyloliquefaciens Yb-2 which can generate antagonistic action on pepper colletotrichum, eggplant verticillium wilt, tomato gray mold and tomato bacterial scab from vegetable crops for the first time; according to the germination growth promotion test of the bacillus amyloliquefaciens Yb-2 on vegetable seeds, the bacillus amyloliquefaciens Yb-2 has a growth promotion effect on seed bud growth and root growth, and the germination rate and the rooting rate of the seeds can be improved; according to the invention, the safety of bacillus on pepper and tomato is evaluated by a root irrigation method, a needle punching method and a leaf cutting method, and the result shows that the bacillus amyloliquefaciens Yb-2 is safe to pepper and tomato after being inoculated.

Description

Bacillus amyloliquefaciens Yb-2 and separation method and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to bacillus amyloliquefaciens Yb-2 and a separation method and application thereof.

Background

Plant diseases are important factors threatening agricultural production, and long-term large-scale use of chemical pesticides always causes drug resistance of pathogenic bacteria and pesticide residues to be harmful to human, storage and environment. With the enhancement of awareness of human beings on environmental protection and food safety, a biological control strategy with good control effect draws high attention, the biological control is a method for inhibiting or eliminating harmful organisms by using beneficial organisms, the biological control is harmless, non-toxic, pollution-free and difficult to generate drug resistance, and chemical pesticides can be gradually replaced to become an environment-friendly and safe choice in the future. The plant endophytic bacteria exist in the plant body, have high reproduction capacity, multiple varieties and short life cycle, are easy to culture, are important microbial resources for preventing and treating plant diseases, can promote healthy growth of plant tissues and control generation and development of the plant diseases, and are widely applied to the research of preventing and treating the plant diseases. Bacillus like Bacillus (Bacillus), Pseudomonas (Pseudomonas), Serratia (Serratia) and Arthrobacter (Arthrobacter) all have outstanding effects in controlling fungal diseases of plants. In an agricultural ecosystem, the biological potential of a biocontrol strain is fully utilized, thereby being conductive to reducing the input of chemical fertilizers and pesticides, promoting the growth of plants, reducing the environmental pollution and realizing the sustainable development of agriculture.

The plant endophyte can not only promote the growth of plants and increase the crop yield, but also enhance the disease resistance of the plants by inducing the disease resistance potential of the plants, is not a foreign species for the plants per se, and has most disease prevention potential and application value. The bacillus is a gram-positive bacterium which is aerobic or facultative anaerobic and can produce stress-resistant endospore, is rod-shaped or spherical, and has the advantages of rapid growth, strong stress resistance, wide application range and the like. The bacillus can generate a plurality of antibacterial substances in the growth and metabolism process, including low molecular weight antibacterial peptide, antibacterial protein and volatile antibacterial substances, which play an important role in preventing and treating plant diseases, so that the bacillus has a good application prospect and a great development potential.

Despite the considerable research on biological control of plant diseases, the actual application of bacillus to production is still limited by a number of factors. The bacillus does not pollute the environment, is safe to people, livestock and plants, has a long-term control effect on plant diseases, is easily interfered by chemical bactericides and has slow effect. Sometimes, the bioassay results of the bacillus in vitro and in vivo are often inconsistent, and in the application process, the behavior and the action mode of the bacillus in a field large ecological system are greatly different due to the influence on the colonization and the propagation of the bacillus caused by the continuous change of field environmental factors such as temperature, humidity, illumination and the like. Therefore, further research on the biological control of plant diseases by taking the bacillus in plants as a main object has great significance.

Disclosure of Invention

In order to solve the defects in the prior art, the invention aims to provide the bacillus amyloliquefaciens Yb-2 and the separation method and the application thereof, provide technical support for the production safety and the quality safety of crops, reduce the use of field chemical pesticides, reduce solanaceae vegetables and soil pesticide residues, and create remarkable economic, social and ecological benefits.

The technical purpose of the invention is realized by the following technical scheme:

the invention provides a Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) Yb-2 which is characterized in that the strain is preserved in the common microorganism center of China Committee for culture Collection of microorganisms (CGMCC for short, the address: No. 3 of West Lu 1 of Beijing Inward Yangxi district, the institute of microbiology of China academy of sciences, zip code 100101) in 6 months and 7 days in 2021, and the preservation number is CGMCC No. 22660.

The Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) Yb-2 provided by the invention is a gram-positive bacterium, has an oval spore, and has no expanded spore sac and two blunt ends. The bacteria are rod-shaped, 0.3-2.2X 1.2-7.0 μm, and flagellum is lateral. The colony surface is dry and rough, irregular in edge, white or milk white. Facultative anaerobic. Catalase positive, oxidase negative. A variety of sugars are available.

The invention provides a method for separating Bacillus amyloliquefaciens Yb-2, which comprises the following steps:

obtaining a vegetable sample, separating bacillus by adopting grinding, diluting and scribing methods, and primarily separating bacillus strains after carrying out purification culture on the separated bacillus for multiple times;

performing physiological and biochemical characteristic identification analysis on the preliminarily separated bacillus strains, and screening and identifying the bacillus strains according to obvious differences of basic characteristics to obtain the bacillus amyloliquefaciens Yb-2 as claimed in claim 1.

Preferably, the preliminary isolation of the bacillus strain is specifically:

selecting a vegetable sample, and cutting the vegetable sample into squares of about 4 mm;

placing the square vegetable sample into a culture dish filled with 75% absolute ethyl alcohol for disinfection, wherein the disinfection time is 30s-2min according to different materials, and cleaning for 2 times by using sterile water;

placing the disinfected and cleaned vegetable sample into a drip dish, adding sterile water for grinding, sucking about 50 mu L of the vegetable sample by using a gun head, placing the vegetable sample on an LB (Langmuir-Blodgett) plate for scribing, and culturing for 2-3d at 30 ℃;

and (3) observing the growth condition of the bacillus, picking suspected colonies, performing purification culture, and primarily separating bacillus strains.

The microbial agent provided by the invention comprises one or more of the bacillus amyloliquefaciens Yb-2, a fermentation culture and a fermentation product thereof; or the microbial inoculum consists of one or more of the bacillus amyloliquefaciens Yb-2, the fermentation culture and the fermentation product thereof.

The two microbial agents provided by the invention are applied to being used as antagonists of colletotrichum capsici.

The two microbial agents provided by the invention are applied to being used as an antagonist of verticillium wilt of eggplants.

The two microbial agents provided by the invention are applied to being used as antagonists of botrytis cinerea.

The two microbial agents provided by the invention are applied to being used as antagonists of tomato bacterial scab bacteria.

The invention provides an application of bacillus amyloliquefaciens Yb-2 in biological control of plant diseases.

Specifically, the method comprises the following steps. The plant diseases are caused by one or more of colletotrichum capsici, verticillium wilt of eggplants, botrytis cinerea and bacterial scab of tomatoes.

The invention provides application of bacillus amyloliquefaciens Yb-2 in promoting vegetable growth and simultaneously improving seed germination rate and rooting rate.

Placing pepper colletotrichum gloeosporioides, eggplant verticillium wilt, tomato botrytis cinerea and tomato bacterial scab on a PDA culture medium for culturing for 6 days at 30 ℃, and preparing a PDA culture medium plate; drying the PDA culture medium flat plate, punching holes with the diameter of 4mm in triangular distribution on the flat plate by using a sterilized puncher, and drying the moisture in the holes; and adding 30 mu l of bacillus liquid cultured to logarithmic phase into the hole, placing a strain to be detected in the center of the plate, using sterile water as a reference, inverting the plate, culturing at constant temperature of 30 ℃ for 7d, and measuring the diameter of the inhibition zone by a ten-finger cross method.

Compared with the prior art, the invention has the following beneficial effects:

1. the invention separates a bacillus amyloliquefaciens Yb-2 which can generate antagonistic action on pepper colletotrichum, eggplant verticillium wilt, tomato gray mold and tomato bacterial scab from vegetable crops for the first time;

2. according to the germination growth promotion test of the bacillus amyloliquefaciens Yb-2 on vegetable seeds, the bacillus amyloliquefaciens Yb-2 has a growth promotion effect on seed bud growth and root growth, and the germination rate and the rooting rate of the seeds can be improved;

3. according to the invention, the safety of bacillus to pepper and tomato is evaluated by a root irrigation method, a needle punching method and a leaf cutting method, and the result shows that the bacillus amyloliquefaciens Yb-2 is safe to pepper and tomato after being inoculated;

4. the invention provides technical support for the production safety and quality safety of crops, reduces the use of chemical pesticides in fields, reduces the pesticide residues in solanaceae vegetables and soil, and creates remarkable economic, social and ecological benefits.

Drawings

The accompanying drawings, which are included to provide a further understanding of the embodiments of the invention and are incorporated in and constitute a part of this application, illustrate embodiment(s) of the invention and together with the description serve to explain the principles of the invention. In the drawings:

FIG. 1 is a schematic diagram of a process for isolating Bacillus bacteria in an embodiment of the present invention;

FIG. 2 is a graph showing the effect of initial separation of a part of Bacillus bacteria in the examples of the present invention;

FIG. 3 is a diagram showing the effect of the colony of Bacillus bacteria after purification and culture in the example of the present invention;

FIG. 4 is a diagram showing the bacteriostatic effect of a part of Bacillus bacteria in the example of the present invention;

FIG. 5 shows the growth promoting effect of Bacillus amyloliquefaciens Yb-2 on vegetable seeds in the example of the present invention

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to examples and accompanying drawings, and the exemplary embodiments and descriptions thereof are only used for explaining the present invention and are not meant to limit the present invention.

Example 1: rapid separation and identification method of vegetable endogenous bacillus amyloliquefaciens Yb-2

The method comprises the following steps: as shown in the figure 1-3, a vegetable sample is obtained, bacillus is separated by adopting grinding, diluting and streaking methods, and bacillus strains are preliminarily separated after the separated bacillus is subjected to purification culture for multiple times.

The primary separation of the bacillus strain comprises the following steps: selecting a vegetable sample, and cutting the vegetable sample into squares of about 4 mm; placing the square vegetable sample into a culture dish filled with 75% absolute ethyl alcohol for disinfection, wherein the disinfection time is 30s-2min according to different materials, and cleaning for 2 times by using sterile water; placing the disinfected and cleaned vegetable sample into a drip dish, adding sterile water for grinding, sucking about 50 mu L of the vegetable sample by using a gun head, placing the vegetable sample on an LB (Langmuir-Blodgett) plate for scribing, and culturing for 2-3d at 30 ℃; and observing the growth condition of the bacillus, and selecting suspected colonies for purification culture to obtain the target strain Yb-2.

Step two: and (3) carrying out identification and analysis on physiological and biochemical characteristics of the primarily separated bacillus strains, and screening and identifying the bacillus strains according to the obvious difference of the basic characteristics.

The physiological and biochemical characteristic determination analysis specifically comprises the following steps: the bacillus strain Yb-2 is subjected to physiological and biochemical measurements such as colony morphology description, gram staining, optimum temperature, pH measurement, glucose utilization, sodium citrate utilization, starch hydrolysis, gelatin liquefaction, catalase test, oxidase test and the like.

(1) Gram stain

a. The slide was wiped dry with gauze and a small circle of marker strokes (to approximately locate the drop of bacteria) was made on one side of the slide. The part coated with the bacteria is baked on flame to remove oil.

b. Smearing: liquid culture medium: holding the bacteria liquid test tube with the left hand, and opening a tube cover about 5cm near the flame of the alcohol lamp; burning and sterilizing the inoculating loop in flame, dipping a bacterial liquid loop from the test tube after cooling, coating a film with the diameter of about 2mm on a clean grease-free glass slide, and finally burning and sterilizing the inoculating loop on the flame. Solid medium: a drop of sterile water is first dropped onto the slide, and a small amount of bacteria is then inoculated and spread onto the slide to make it thin and uniform.

c. Drying: the smear was allowed to dry naturally in air.

d. Fixing: the mycoderm is allowed to face upwards and fixed by flame for 2-3 times (preferably without scalding hands).

e. Dyeing: and (3) placing the fixed smear on a newspaper, dropwise adding ammonium oxalate crystal violet solution, and dyeing for 1 min.

f. Washing with water: the staining solution on the smear was slowly rinsed with water and blotted dry with absorbent paper. The cell morphology can be observed after simple staining.

g. Mordant dyeing: dripping 1 drop of iodine solution, dyeing for 1min, and washing with water.

h. And (3) decoloring: absorbing residual water, continuously dropwise adding 95% ethanol for decolorizing for 20-30s until effluent liquid is purple, and immediately washing with water.

i. Counterdyeing: and (5) dripping lycopene for redyeing for 3-5min and washing with water. At this point, gram staining is complete. Gram-positive bacteria stain purple to blue-black, and gram-negative bacteria stain red.

(2) Optimum temperature and pH measurement

Setting different temperatures of 26 ℃, 28 ℃, 30 ℃, 32 ℃, 34 ℃ and 36 ℃ for culturing bacillus, setting different pH values of 5-9 for culturing bacillus, and measuring the concentration of bacterial liquid after culturing and shaking at different temperatures and pH values, wherein the temperature and pH value when the bacteria grow most vigorously are the optimal temperature and pH value of the bacteria.

(3) Utilization of glucose

Glucose culture medium: (NH4)2HPO4 is 1g, MgSO4 is 0.2g, yeast extract is 0.2g, glucose is 1%, water-washed agar is 5-6g, distilled water is 1000mL, bromcresol purple is 0.4% ethanol solution is 2mL (dissolved by 95% ethanol and then added with water to prepare 0.4% solution), and pH is 6.8-7.0. The indicator is added after the pH is adjusted. Subpackaging the above culture medium with a height of about 4-5cm, and sterilizing at 115 deg.C for 20 min. Inoculating 18-24h of young strain into culture medium with puncture needle, culturing at room temperature for 1d, 3d, and 5d, observing, wherein the color turns yellow to positive and does not turn to negative, and gas is generated due to bubbles.

(4) Utilization of sodium citrate

Sodium citrate medium: 2g of sodium citrate, 1g of K2HPO4, 1g of NH4H2PO4, 5g of NaCL, 0.2g of MgSO4, 15-20g of agar, 10ml of 1% bromothymol blue (alcoholic solution) or 0.04% phenol red and 1000ml of water. Bacteria can decompose citrate to produce carbonate, which makes the culture medium alkaline. The bromothymol blue indicator in the medium changed from green to dark blue at this time. Bacteria that cannot utilize citrate as a carbon source do not grow on the medium and the medium does not change color.

(5) Starch hydrolysis

Bacteria were inoculated onto starch agar plates for 2d and then the plates were soaked with Lugol iodine. A clear, colorless area indicates starch hydrolysis. Note that: some bacillus species produce limited regions, so colonies should be scraped off for observation. Lugol iodine: 5g of iodine and 10.0g of potassium iodide in each 100ml of the wine, dissolving iodine and potassium iodide in 10ml of water, and fixing the volume by using distilled water. When in use, the extract is diluted by 5 times by using distilled water. Starch agar (8 g of nutrient agar per liter of distilled water, 10.0g of soluble potato starch).

(6) Catalase and oxidase test

And (3) contact enzyme test: the bacteria with catalase can catalyze hydrogen peroxide to generate water and nascent oxygen, then molecular oxygen is formed to generate bubbles, a colony loop on a solid culture medium is picked, the colony loop is placed in a clean test tube, 2mL (temporary configuration) of 3% hydrogen peroxide solution is dripped, and the result is observed. The positive one was found to be positive when bubbles were generated within half a minute, and the negative one was found to be negative when bubbles were not generated.

Oxidase test: and taking white clean filter paper to dip colonies. Adding one drop of 1% dimethyl p-phenylenediamine hydrochloride solution, wherein the positive part is pink and gradually deepens; a drop of 1% alpha-naphthol solution was added, and the positive one appeared bright blue within half a minute. Negative did not change color within two minutes. The reagent is absorbed by a capillary pipette and directly dripped on the bacterial colony, and the color reaction is the same as above. (1% dimethyl-p-phenylenediamine hydrochloride solution: little fresh, dry refrigerator dark preservation 1% alpha-naphthol-ethanol solution.)

After primary separation and physiological and biochemical determination of the bacillus Yb-2, the Yb-1 is gram-positive bacteria, and the colony surface is dry and rough, irregular in edge and milky white. The results are shown in table 1:

TABLE 1 physiological and biochemical basic characteristics of Bacillus Yb-2

Step three: molecular identification of Bacillus Yb-2. The bacillus is cultured to logarithmic phase, genome is extracted, and identification is carried out by adopting 16SrRNA universal primer 27F/1492R and a method for combining multiple genes such as gyrA, gyrB, rpoA, yyaR R, yyaO and the like. And recovering and purifying the amplified product, performing a parallel reaction, and connecting the amplified product to a vector containing a lethal gene of pLBVecter for connection transformation without blue-white spot screening. Positive clones were picked directly on LB plates containing 100. mu.g/mL ampicillin, sequenced, and the sequencing results were logged in the NCBI website for Blast. The results are shown in table 2:

TABLE 2 basic information and identification results of Bacillus

Example 2: bacillus subtilis Yb-2 bacteriostatic activity identification

Four pathogenic bacteria harmful to vegetables are selected for carrying out bacteriostatic activity identification on the bacillus subtilis Yb-2, and the bacteriostatic effect of the bacillus subtilis Yb-2 is determined.

In this example, the pathogens are four species of colletotrichum capsici, verticillium wilt, botrytis cinerea and tomato bacterial scab.

The identification of antagonism of colletotrichum capsici, verticillium wilt of eggplant and botrytis cinerea specifically comprises the following steps: placing pepper colletotrichum gloeosporioides, eggplant verticillium wilt and tomato botrytis cinerea on a PDA culture medium, and culturing at 30 ℃ for 6 days to prepare a PDA culture medium plate; drying the PDA culture medium flat plate, punching holes with the diameter of 4mm in triangular distribution on the flat plate by using a sterilized puncher, and drying the moisture in the holes; and adding 30 mu l of bacillus liquid cultured to logarithmic phase into the hole, placing a strain to be detected in the center of the plate, using sterile water as a reference, inverting the plate, culturing at constant temperature of 30 ℃ for 7d, and measuring the diameter of the inhibition zone by a ten-finger cross method.

The calculation of the bacteriostatic rate of the pepper colletotrichum, the eggplant verticillium wilt and the tomato botrytis cinerea is as follows: taking a PDA (personal digital Assistant) plate without a microbial inoculum as a control, culturing for 8 days in a constant-temperature incubator at 25 ℃, measuring the diameter of a colony by a cross method, and calculating the average value of the diameter of the colony and the hypha growth inhibition rate, wherein the hypha growth inhibition rate (%) is [ (the diameter of the control colony-the diameter of a treated colony)/(the diameter of the control colony-0.4) ] × 100%.

The identification of the antagonistic action of the tomato bacterial scab bacteria specifically comprises the following steps: placing tomato bacterial scab bacteria on YDC culture medium, culturing at 28 deg.C for 2d, adding 5ml of Xanthomonas cultured to logarithmic phase into 200ml of LB solid culture medium liquefied at 45 deg.C under aseptic operation table, mixing, and pouring into flat plate; after the flat plate is dried, punching triangular holes with the diameter of 4mm on the flat plate by using a sterilized puncher, and drying the moisture in the holes; adding the bacillus to be detected after the logarithmic phase culture into LB culture medium holes containing xanthomonas, adding 30 mu l of bacillus to be detected into each hole, repeating each concentration for 3 times, using sterile water as a control, inverting the plate, culturing at the constant temperature of 28 ℃ for 24h, and then measuring the diameter of the inhibition zone by a ten-finger cross method.

The calculation of the bacteriostasis rate of the tomato bacterial scab germs is as follows: and calculating the average value of the inhibition zones, and calculating the relative inhibition rate according to the diameter of the inhibition zones, wherein the relative inhibition rate is (the diameter of the treated inhibition zone-the diameter of the control inhibition zone)/(the diameter of the treated inhibition zone) multiplied by 100%.

FIG. 4 is a partial bacillus bacteriostatic effect diagram, wherein a is the bacteriostatic effect of Yb-2 on tomato bacterial scab pathogen, and b is the bacteriostatic effect of Yb-2 on tomato botrytis cinerea.

The preliminarily separated bacillus amyloliquefaciens Yb-2 is selected for bacteriostasis determination, and the results are shown in Table 3:

TABLE 3 antagonistic action of Bacillus on 4 pathogens (unit: cm)

The inhibition rate (%) of hyphal growth according to the calculation formula [ (control colony diameter-treated colony diameter)/(control colony diameter-0.4) ] × 100%, the inhibition rate of Yb-2 against 4 pathogens is shown in table 4:

TABLE 4 bacteriostatic ratio (%) of Bacillus amyloliquefaciens Yb-2 against 4 pathogens

Example 3: germination growth promotion test of bacillus amyloliquefaciens Yb-2 on vegetable seeds

The LB plate activates the Yb-2 strain, picks up a single colony, cultures to logarithmic phase at 28 ℃ with shaking at 180 rpm. According to initial preliminary experiments, different concentration gradients were set for the strains. Taking cruciferous vegetables such as Chinese cabbages and water spinach, solanaceous vegetables such as hot peppers and tomatoes, cucurbitaceous vegetables such as cucumbers and leguminous vegetables such as cowpea seeds as objects, respectively taking 20-40 seeds according to the sizes of the seeds, putting water-absorbing filter paper at the bottom of a culture dish, soaking the seeds in bacterial solutions with different concentrations, accelerating germination at room temperature, and periodically wetting. Each concentration was 3 replicates and sterile water was used as a control. Because the germination time of each seed is different, the water spinach and the Chinese cabbage germinate earlier, and the pepper and the tomato germinate later, the growth conditions of the seed buds and the roots need to be observed regularly. And measuring the bud length and the root length for statistical analysis, calculating the germination rate and the rooting rate, researching the growth promoting or inhibiting effect of each concentration on the seeds, and selecting the bacillus with better growth promoting effect for carrying out pot culture test.

TABLE 5 growth promoting units of Yb-2 on vegetable seeds: mm is

The growth promoting effect of Yb-2 on seeds is shown in FIG. 5 and Table 5. In fig. 5, a is pepper, b is tomato, c is cabbage, d is water spinach, and e is cowpea seed. In the growth promotion experiment of Yb-2 on vegetable seeds, 3 concentration gradients are set according to the early test result, one control is carried out, and the steps are repeated for 3 times. The average value for each concentration was taken at the time of statistics. In the growth promoting effect of Yb-2 on pepper seeds, when Yb-2 is diluted by 10X and 100X, the Yb-2 can promote the bud length and the root length of pepper, the germination rate is 26.67% higher than that of a control at 10X, the rooting rate is 38.34% higher than that of the control at 100X, the germination rate is 20.84% higher than that of the control at 100X, and the rooting rate is 30.84% higher than that of the control. In tomato seeds, when the Yb-2 concentration is diluted to 10X, the germination and rooting rates are weaker than those of the control, and when the Yb-2 concentration is 100X, the germination rate is equivalent to that of the control, but the germination rate is 3.33 percent higher than that of the control, and the rooting rate is 5.00 percent higher than that of the control. In Chinese cabbage seeds, when the Yb-2 concentration is diluted to 100X, the growth promotion effect is achieved on both bud length and root length, the germination rate is 15.00% higher than that of a control, and the rooting rate is 12.50% higher than that of the control. In the water spinach seeds, the stock solution can ensure that the seeds germinate, the average bud length is 11.29, the germination rate is only 11.67 percent, when the Yb-2 concentration is diluted to 100X, the seeds have growth promoting effect on both the bud length and the root length, and the germination rate and the rooting rate are 6.67 percent higher than those of a control. In cucumber seeds, when the Yb-2 concentration is diluted to 100X, the cucumber seeds have growth promoting effect on both bud length and root length, and the germination rate and the rooting rate are 11.67 percent higher than those of a control. In cowpea seeds, when the Yb-2 concentration is diluted to 100X, the cowpea seeds have growth promoting effect on both bud growth and root growth, but the germination rate is 3.33 percent lower than that of a control, and the rooting rate is 6.67 percent higher than that of the control. In conclusion, when the Yb-2 concentration is diluted to 100X, the germination and rooting of pepper, Chinese cabbage, water spinach and cucumber seeds are promoted, the germination rate and rooting rate of the seeds can be improved, and the tomato and cowpea growth promoting effect is not obvious.

The above-mentioned embodiments are intended to illustrate the objects, technical solutions and advantages of the present invention in further detail, and it should be understood that the above-mentioned embodiments are merely exemplary embodiments of the present invention, and are not intended to limit the scope of the present invention, and any modifications, equivalent substitutions, improvements and the like made within the spirit and principle of the present invention should be included in the scope of the present invention.

Claims

1. The Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) Yb-2 is characterized in that the strain is preserved in the China general microbiological culture Collection center (CGMCC) at 6-7 th month in 2021, and the preservation number is CGMCC No. 22660.

2. A method for separating Bacillus amyloliquefaciens Yb-2 is characterized by comprising the following steps:

3. The method for separating the bacillus amyloliquefaciens Yb-2 as claimed in claim 2, wherein the primary separation of the bacillus strain comprises:

4. A microbial agent, characterized in that the agent comprises one or more of bacillus amyloliquefaciens Yb-2, a fermentation culture thereof and a fermentation product thereof according to claim 1; or, the bacterial agent consists of one or more of the bacillus amyloliquefaciens Yb-2, the fermentation culture and the fermentation product thereof of the claim 1.

5. Use of a microbial inoculant according to claim 4 as an antagonist of Colletotrichum capsici (Colletotrichum capsicii).

6. Use of a microbial inoculant according to claim 4 as an antagonist of Verticillium dahliae (Verticillium dahliae).

7. Use of a microbial inoculant according to claim 4 as an antagonist against Botrytis cinerea (Botrytis cinerea).

8. Use of a microbial inoculant according to claim 4 as an antagonist against bacterial species of tomato scab (Xanthomonas campestris pv.

9. The use of a bacillus amyloliquefaciens Yb-2 according to claim 1 for biological control of a plant disease.

10. The use of the bacillus amyloliquefaciens Yb-2 of claim 1 for promoting vegetable growth while increasing seed germination and rooting.