CN114250171B - Bacillus amyloliquefaciens Yb-2 and separation method and application thereof - Google Patents

Abstract

The invention discloses bacillus amyloliquefaciens Yb-2, a separation method and application thereof, and relates to the technical field of biology, wherein the strain is preserved in China general microbiological culture collection center (CGMCC) No.22660 in the year 6 and the day 7 of 2021. The bacillus amyloliquefaciens Yb-2 which can antagonize pepper anthracnose, eggplant verticillium, tomato gray mold bacteria and tomato bacterial scab bacteria is separated 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 growth promotion effect on the bud length and root length of the seeds, and the germination rate and rooting rate of the seeds can be improved; according to the invention, the safety of bacillus to peppers and tomatoes is evaluated by a root irrigation method, a needling method and a leaf shearing method, and the result shows that bacillus amyloliquefaciens Yb-2 is safe to peppers and tomatoes 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 and large-scale use of chemical pesticides has long been used to cause drug resistance of pathogenic bacteria, and pesticide residues are harmful to people, storage and environment. With the enhancement of environmental protection and food safety consciousness of human beings, biological control strategies with good control effects have attracted great attention, biological control is a method for inhibiting or eliminating harmful organisms by utilizing beneficial organisms, and is harmless, nontoxic, pollution-free and not easy to generate drug resistance, and chemical pesticides can be gradually replaced in the future to become an environment-friendly and safe choice. The plant endophytic bacteria exist in the plant body, have high fertility, multiple species, short life cycle and easy culture, are important microbial resources for preventing and treating plant diseases, can promote healthy growth of plant tissues and simultaneously control occurrence and development of the plant diseases, and are widely applied to research on preventing and treating the plant diseases. Like Bacillus, pseudomonas, serratia and Arthrobacter have a prominent effect on controlling fungal diseases of plants. In the agricultural ecological system, the biological potential of the biocontrol strain is fully utilized to help reduce the investment of chemical fertilizers and pesticides, promote the plant growth, reduce the environmental pollution and realize the sustainable development of agriculture.

The endophyte not only can promote plant growth and increase crop yield, but also can enhance disease resistance of plants by inducing disease resistance potential of the plants, is not a foreign species for the plants, and has disease resistance potential and application value. Bacillus is a kind of aerobic or facultative anaerobic gram-positive bacteria producing stress-resistant endospores, and has the advantages of fast growth, strong stress resistance, wide application range and the like. The bacillus can produce various antibacterial substances including low molecular weight antibacterial peptide, antibacterial protein and volatile antibacterial substances in the growth and metabolism process, and the substances play an important role in preventing and controlling plant diseases, so that the bacillus has good application prospect and great development potential.

Although biological control of plant diseases has been studied in a large amount, the actual application of bacillus to production is still limited by many factors. The bacillus does not pollute the environment, is safe to people, livestock and plants, has longer-term control effect on plant diseases, is easily interfered by chemical bactericides, and has slow effect. Sometimes, the bioassay results of bacillus in vitro and in vivo often show inconsistency, and during the application process, the colonization and reproduction of bacillus are affected due to continuous changes of field environmental factors such as temperature, humidity, illumination and the like, so that great differences of behaviors and action modes of the bacillus in a field large ecological system can occur. Therefore, the further research of taking bacillus in plants as a main object has great significance for biological control of plant diseases.

Disclosure of Invention

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

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

the bacillus amyloliquefaciens (Bacillus amyloliquefa ciens) Yb-2 provided by the invention is characterized in that the strain is preserved in China general microbiological culture Collection center (CGMCC) in 6-7 days of 2021, and the preservation number is CGMCC No.22660.

The bacillus amyloliquefaciens (Bacillus amyloliquefa ciens) Yb-2 provided by the invention is gram positive bacteria, the spore is oval, the spore bag is not expanded, and the two ends of the spore bag are rounded. The thallus is in the shape of rod and 0.3-2.2X1.2-7.0 μm, and the flagellum is laterally grown. The colony surface is dry and rough, the edge is irregular, white or milky. Facultative anaerobism. Contact enzyme positive and oxidase negative. A variety of sugars can be utilized.

The invention provides a separation method of bacillus amyloliquefaciens Yb-2, which comprises the following steps:

obtaining a vegetable sample, separating bacillus by adopting grinding, diluting and streaking methods, purifying and culturing the separated bacillus for multiple times, and primarily separating bacillus strains;

the bacillus strain which is primarily separated is subjected to physiological and biochemical characteristic identification analysis, and is screened and identified according to the obvious difference of basic characteristics, so that the bacillus amyloliquefaciens Yb-2 is obtained.

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

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

placing square vegetable samples into a culture dish filled with 75% absolute ethyl alcohol for sterilization, sterilizing for 30s-2min according to different materials, and cleaning with sterile water for 2 times;

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

observing the growth condition of bacillus, picking suspected bacterial colony for purifying culture, and primarily separating bacillus strain.

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 is composed of one or more of bacillus amyloliquefaciens Yb-2, a fermentation culture and a fermentation product thereof.

The application of the two microbial agents provided by the invention as antagonists of pepper anthracnose bacteria.

The application of the two microbial agents provided by the invention as the antagonists of eggplant verticillium wilt bacteria.

The application of the two microbial agents provided by the invention as antagonists of botrytis cinerea.

The invention provides application of the two microbial agents in serving as antagonists of tomato bacterial scab germs.

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

In particular. The plant disease is caused by one or more of pepper anthracnose, eggplant verticillium, tomato gray mold, tomato bacterial scab.

The bacillus amyloliquefaciens Yb-2 provided by the invention is applied to promoting the growth of vegetables and simultaneously improving the germination rate and rooting rate of seeds.

According to the invention, pepper anthracnose, eggplant verticillium, tomato gray mold, tomato bacterial scab bacteria are placed on a PDA culture medium for 6d at 30 ℃ 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 sterilizing puncher, and drying the water in the holes; and (3) adding 30 mu l of bacillus liquid cultured to the logarithmic phase into the hole, placing a strain to be detected in the center of the flat plate, taking sterile water as a reference, inverting the flat plate, culturing at the constant temperature of 30 ℃ for 7d, and measuring the diameter of a bacteriostasis ring by a ten-finger crossing method.

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

1. the bacillus amyloliquefaciens Yb-2 which can antagonize pepper anthracnose, eggplant verticillium, tomato gray mold bacteria and tomato bacterial scab bacteria is separated 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 growth promotion effect on the bud length and root length of the seeds, and the germination rate and rooting rate of the seeds can be improved;

3. according to the invention, the safety of bacillus to peppers and tomatoes is evaluated by a root irrigation method, a needling method and a leaf shearing method, and the result shows that bacillus amyloliquefaciens Yb-2 is safe to peppers and tomatoes after inoculation;

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

Drawings

The accompanying drawings, which are included to provide a further understanding of embodiments of the invention and are incorporated in and constitute a part of this application, illustrate embodiments of the invention. In the drawings:

FIG. 1 is a schematic diagram of a bacillus separation process in an embodiment of the present invention;

FIG. 2 is a graph showing the primary separation effect of a part of bacillus in the embodiment of the present invention;

FIG. 3 is a graph showing the effect of purifying cultured bacillus colonies in the examples of the present invention;

FIG. 4 is a graph showing the bacteriostatic effect of a portion of Bacillus bacteria in an embodiment 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

For the purpose of making apparent the objects, technical solutions and advantages of the present invention, the present invention will be further described in detail with reference to the following examples and the accompanying drawings, wherein the exemplary embodiments of the present invention and the descriptions thereof are for illustrating the present invention only and are not to be construed as limiting the present invention.

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

Step one: as shown in fig. 1-3, a vegetable sample is obtained, bacillus is separated by grinding, diluting and streaking, and bacillus strains are primarily separated after the separated bacillus is subjected to purification culture for a plurality of times.

The bacillus strain primary separation is specifically as follows: selecting a vegetable sample, and cutting the vegetable sample into squares with the length of about 4 mm; placing square vegetable samples into a culture dish filled with 75% absolute ethyl alcohol for sterilization, sterilizing for 30s-2min according to different materials, and cleaning with sterile water for 2 times; placing the sterilized and cleaned vegetable sample into a drip dish, adding sterile water, grinding, sucking about 50 mu L by using a gun head, placing the vegetable sample on an LB plate for scribing, and culturing for 2-3d at 30 ℃; observing the growth condition of bacillus, and selecting suspected bacterial colony to purify and culture to obtain target strain Yb-2.

Step two: and (3) carrying out physiological and biochemical characteristic identification analysis on 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 measurement and analysis concretely comprises: the bacillus strain Yb-2 is subjected to physiological and biochemical measurement such as colony morphology description, gram staining, optimal temperature, pH measurement, glucose utilization, sodium citrate utilization, starch hydrolysis, gelatin liquefaction, contact enzyme test, oxidase test and the like.

(1) Gram staining

a. The slide was wiped dry with gauze and a small circle of strokes was made on one side of the slide with a marker (to roughly determine the location of the bacterial droplets). The coated part is baked on flame to remove grease.

b. Smearing: liquid medium: the bacterial liquid test tube is held on the left, and a tube cover is opened about 5cm near the flame of the alcohol lamp; the right hand holding inoculating loop burns and sterilizes in flame, after cooling, the inoculating loop is dipped with bacterial liquid one loop from test tube, the clean non-fat slide glass is coated with coating film with diameter of about 2mm, finally the inoculating loop burns and sterilizes in flame. Solid medium: firstly, a drop of sterile water is dropped on a glass slide, then a small amount of thalli is taken out by an inoculating loop, and the thalli is coated on the glass slide, so that the thalli are thin and uniform.

c. And (3) airing: allowing the smear to dry naturally in air.

d. Fixing: the fungus film is kept upwards, and the fungus film is fixed for 2-3 times by flame (preferably without scalding hands).

e. Dyeing: placing the fixed smear on newspaper, dripping ammonium oxalate crystal violet solution, and dyeing for 1min.

f. Washing: the staining solution on the smear was slowly rinsed with water and blotted dry with absorbent paper. Cell morphology can be observed at the end of simple staining.

g. Mordant dyeing: 1 drop of iodine solution is added dropwise for dyeing for 1min, and the mixture is washed with water.

h. Decoloring: residual water is sucked off, 95% ethanol is continuously added dropwise for decolorization for 20-30s until the effluent is purple, and the effluent is immediately washed.

i. Counterstaining: dripping lycopene for counterstaining for 3-5min, and washing with water. The gram staining ends up. Gram positive bacteria appear purple to bluish black after staining and gram negative bacteria appear red after staining.

(2) Determination of optimum temperature, pH

Different temperatures of 26 ℃,28 ℃,30 ℃, 32 ℃, 34 ℃ and 36 ℃ are set, different pH values of pH5-pH9 are set for culturing bacillus, the concentration of bacterial liquid is measured after culturing and shaking at different temperatures and pH values, and the temperature and the pH value when the bacterial growth is most vigorous are the optimal temperature and the pH value of the bacterial body.

(3) Glucose utilization

Glucose medium: (NH 4) 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, bromocresol purple is 0.4% ethanol solution is 2mL (firstly, 95% ethanol is used for dissolution, then water is added for preparing 0.4% solution), and pH is 6.8-7.0. The pH is adjusted first and then the indicator is added. Subpackaging the above culture medium, sterilizing at 115 deg.C for 20min at a height of about 4-5 cm. Inoculating young strain of 18-24 hr into culture medium with puncture needle, culturing at room temperature for 1d,3d, and 5d, observing to get yellow positive, and generating gas with bubbles.

(4) Utilization of sodium citrate

Sodium citrate medium: sodium citrate 2g, K2HPO4 1g, NH4H2PO4 1g, naCL 5g, mgSO4 0.2g, agar 15-20g,1% bromothymol blue (alcoholic solution) or 0.04% phenol red 10ml, water 1000ml. Bacteria can break down citrate to produce carbonate, which makes the medium alkaline. At this point the bromothymol blue indicator in the medium changed from green to dark blue. Bacteria which cannot use 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 immersed with Lugol iodine. Clear, non-colored areas indicate starch hydrolysis. Note that: some bacillus species produce limited areas, so colonies should be scraped off for observation. Lugol iodine: iodine 5g and potassium iodide 10.0g in 100ml, and iodine and potassium iodide were dissolved in 10ml water, and distilled water was used to determine the volume. When in use, the solution is diluted by 5 times by distilled water. Starch agar (8 g of nutrient agar per liter of distilled water, 10.0g of soluble potato starch).

(6) Contact enzyme and oxidase test

Contact enzyme test: bacteria with catalase can catalyze hydrogen peroxide to generate water and nascent oxygen, then molecular oxygen is formed to generate bubbles, a colony on a solid culture medium is picked up to form an inoculating loop, the inoculating loop is placed in a clean test tube, 2mL of 3% hydrogen peroxide solution is dripped (temporary configuration), and the result is observed. The cells were positive when they were in half a minute, and the cells were negative when they were not in half a minute.

Oxidase test: white clean filter paper is taken to be dipped into bacterial colonies. Adding 1% dimethyl p-phenylenediamine hydrochloride solution into the mixture to make the positive person appear pink and deepen gradually; then adding 1% alpha-naphthol solution into the mixture for one drop, and making positive person appear bright blue within half a minute. Negative, no discoloration was observed within two minutes. The reagent was sucked by a capillary pipette and directly added dropwise onto the colony, and the chromogenic reaction was the same as above. ( 1% solution of dimethyl p-phenylenediamine hydrochloride: and (5) preparing a small amount of fresh dry ice, and storing in a dry ice box in a dark place. 1% alpha-naphthol-ethanol solution. )

After preliminary separation and physiological and biochemical determination of bacillus Yb-2, yb-1 is gram positive bacteria, and the colony surface is dry and rough, and the edge is irregular and milky. 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. Culturing bacillus to logarithmic phase, extracting genome, and identifying by adopting a method of combining multiple genes such as 16SrRNA universal primer 27F/1492R, gyrA, gyrB, rpoA, yyaR, yyaO and the like. The amplified product is recovered and purified, subjected to flattening reaction, and connected to a vector containing a lethal gene of pLBVecter for connection transformation without blue and white spot screening. Positive clones were picked directly on LB plates containing ampicillin at 100. Mu.g/mL, sequenced, and the sequencing results were logged on NCBI website for Blast. The results are shown in Table 2:

TABLE 2 basic information of Bacillus and identification results

Example 2: identification of bacteriostatic activity of bacillus subtilis Yb-2

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

In the embodiment, the pathogens are four species of pepper anthracnose, eggplant verticillium, botrytis cinerea and tomato bacterial scab.

The antagonism of the pepper anthracnose, the eggplant verticillium wilt and the tomato botrytis is specifically identified as follows: placing pepper anthracnose, eggplant verticillium wilt and tomato gray mold bacteria on a PDA culture medium, and culturing for 6d at 30 ℃ 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 sterilizing puncher, and drying the water in the holes; and (3) adding 30 mu l of bacillus liquid cultured to the logarithmic phase into the hole, placing a strain to be detected in the center of the flat plate, taking sterile water as a reference, inverting the flat plate, culturing at the constant temperature of 30 ℃ for 7d, and measuring the diameter of a bacteriostasis ring by a ten-finger crossing method.

The bacteriostasis rate of the pepper anthracnose, the eggplant verticillium wilt and the tomato gray mold bacteria is calculated as follows: PDA plates without microbial inoculum are used as a control, colony diameters are measured by a crisscross method after culturing for 8 days in a constant temperature incubator at 25 ℃, and the average value of the colony diameters and the hypha growth inhibition rate are calculated, wherein the hypha growth inhibition rate (%) = [ (control colony diameter-treated colony diameter)/(control colony diameter-0.4) ]multipliedby 100%.

The antagonism of tomato bacterial scab bacteria is identified as follows: culturing tomato bacterial scab bacteria on YDC culture medium at 28deg.C for 2d, taking 5ml of Xanthomonas cultured to logarithmic phase under aseptic operation table, adding into 200ml of LB solid culture medium liquefied at 45deg.C, mixing, and pouring into plate; after the flat plate is dried, a hole with the diameter of 4mm, which is triangular, is punched on the flat plate by a sterilizing puncher, and moisture in the hole is dried; and (3) adding the bacillus to be detected which is cultivated to the logarithmic phase into LB culture medium holes containing xanthomonas, adding 30 mu l of each hole, repeating each concentration for 3 times, taking sterile water as a reference, inverting a flat plate, and carrying out constant temperature cultivation at 28 ℃ for 24 hours, and then measuring the diameter of a bacteriostasis ring by a ten-finger crossing method.

The calculation of the bacteriostasis rate of the tomato bacterial scab bacteria is specifically as follows: and calculating an average value of the bacteriostasis zones, and calculating a relative bacteriostasis rate according to the diameter of the bacteriostasis zones, wherein the relative bacteriostasis rate is = (the diameter of the treated bacteriostasis zone-the diameter of the control bacteriostasis zone)/(the diameter of the treated bacteriostasis zone) ×100%.

FIG. 4 is a graph showing the antibacterial effect of part of bacillus, a is the antibacterial effect of Yb-2 on tomato bacterial scab bacteria, and b is the antibacterial effect of Yb-2 on tomato gray mold bacteria.

The bacteriostasis condition of the primarily separated bacillus amyloliquefaciens Yb-2 is determined, and the results are shown in Table 3:

TABLE 3 antagonism of Bacillus against 4 pathogens (units: cm)

Hypha growth inhibition (%) = [ (control colony diameter-treated colony diameter)/(control colony diameter-0.4) ]100% according to the calculation formula, and the inhibition ratio of Yb-2 to 4 pathogens is shown in table 4:

TABLE 4 antibacterial Rate of Bacillus amyloliquefaciens Yb-2 against 4 pathogens (%)

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

The LB plate activates Yb-2 strain, single colony is picked up at 28 ℃ and shake-cultivated at 180rpm to logarithmic phase. According to the initial pre-experiments, different concentration gradients were set for the strains. Taking cabbage and water spinach of cruciferous vegetables, capsicum and tomato of Solanaceae vegetables, cucumber of cucurbitaceae vegetables and cowpea seeds of leguminous vegetables as objects, respectively taking 20-40 grains according to the sizes of the seeds in a culture dish, filling water-absorbing filter paper into the bottom of the culture dish, soaking the seeds with bacterial liquid with different concentrations, accelerating germination at room temperature, and wetting regularly. Each concentration was replicated 3 times and sterile water was used as a control. Because the germination time of each seed is inconsistent, the germination of the water spinach and the cabbage is early, the germination of the pepper and the tomato is late, and the growth condition of the seed buds and the roots needs to be observed regularly. And measuring the bud length and root length, performing statistical analysis, calculating the germination rate and rooting rate, researching the growth promotion or inhibition effect of each concentration on seeds, and selecting bacillus with better growth promotion effect for potting test.

Table 5 growth promoting action units of Yb-2 on vegetable seeds: mm (mm)

The growth promoting effect of Yb-2 on seeds is shown in FIG. 5 and Table 5. In fig. 5, a capsicum, 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, one control and 3 repetitions are set according to the early-stage test result. The average value of each concentration was taken at the time of statistics. In the growth promoting effect of Yb-2 on pepper seeds, when the concentration of Yb-2 is diluted by 10X and 100X, the growth promoting effect can be generated on the bud length and root length of the peppers, 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 rate is weaker than that of the control, and the germination rate is 3.33% higher than that of the control and the rooting rate is 5.00% higher than that of the control at 100X. In the cabbage seeds, when the Yb-2 concentration is diluted to 100X, the growth of the cabbage seeds is promoted, 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 seed, the stock solution can enable the seed to sprout, the average sprout length is 11.29, the germination rate is only 11.67%, when the Yb-2 concentration is diluted to 100X, the growth promotion effect is achieved on the sprout length and the root length, and the germination rate and the rooting rate are 6.67% higher than those of a control. In cucumber seeds, when the Yb-2 concentration is diluted to 100X, the cucumber seeds have growth promotion effects on bud length and root length, and the germination rate and rooting rate are 11.67% higher than those of a control. In cowpea seeds, when the Yb-2 concentration is diluted to 100X, the cowpea seeds have growth promotion effects on bud length and root length, but the germination rate is 3.33% lower than that of a control, and the rooting rate is 6.67% higher than that of the control. In summary, when the Yb-2 concentration is diluted to 100X, the seed germination and rooting agent has growth promotion effect on pepper, cabbage, water spinach and cucumber seeds, can improve the germination rate and rooting rate of the seeds, and has no obvious growth promotion effect on tomatoes and cowpeas.

The foregoing description of the embodiments has been provided for the purpose of illustrating the general principles of the invention, and is not meant to limit the scope of the invention, but to limit the invention to the particular embodiments, and any modifications, equivalents, improvements, etc. that fall within the spirit and principles of the invention are intended to be included within the scope of the invention.

Claims (8)

1. Bacillus amyloliquefaciens @Bacillus amyloliquefaciens) Yb-2, wherein the strain is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.22660 in 2021, 6 and 7.

2. A microbial agent comprising a bacillus amyloliquefaciens Yb-2 according to claim 1.

3. A microbial agent as claimed in claim 2 for use as a pepper anthracnose pathogenColletotrichum capsiciThe use of the antagonists of (a).

4. A microorganism according to claim 2The microbial inoculum is used as verticillium wilt of eggplantVerticillium dahliaeThe use of the antagonists of (a).

5. A microbial agent as claimed in claim 2 for use as botrytis cinereaBotrytis cinereaThe use of the antagonists of (a).

6. A microbial agent as claimed in claim 2 for use as tomato bacterial scab pathogenXanthomonas campestris pv.vesicatoriaThe use of the antagonists of (a).

7. The use of Bacillus amyloliquefaciens Yb-2 according to claim 1 for biological control of plant diseases, wherein the pathogen of the plant diseases is pepper anthracnose pathogenColletotrichum capsici、Eggplant verticillium wilt bacteriaVerticillium dahliae、Botrytis cinereaBotrytis cinereaOr tomato bacterial scab pathogenXanthomonas campestris pv.vesicatoria。

8. The use of bacillus amyloliquefaciens Yb-2 according to claim 1 for increasing germination rate and rooting rate of seeds, wherein the seeds are cabbage seeds, cucumber seeds, water spinach seeds or capsicum seeds.