Bacillus, fungicide and application of bacillus in disease control and plant growth promotion

Abstract

The invention discloses bacillus, a fungicide and application of the fungicide in disease control and plant growth promotion, and relates to the technical field of biocontrol bacteria. The preservation number of the bacillus is CGMCC (China General Microbiological Culture Collection Center) No.3265. The bacillus RD22S2 strain of bacillus is separated and screened from rhizosphere soil of eucommia ulmoides, and experiments prove that the bacillus RD22S2 strain can effectively prevent and control gray mold of eucommia ulmoides leaves and has broad-spectrum fungal disease resistance and growth promoting functions. Therefore, the bacillus provided by the invention not only can be used for preventing and treating plant diseases caused by fungi, but also can be used for promoting the growth of various plants and improving the yield and quality of the plants. Good application prospects of preventing and treating plant diseases and promoting plant growth are realized.

Description

Technical Field

[0001] The present invention relates to the technical field of biocontrol bacteria, and in particular, to a Bacillus, a bacterial agent and their applications in disease control and plant growth promotion. Background Art

[0002] Eucommia ulmoides is a traditional Chinese medicinal material in China and is a forest-source medicinal tree species and a forage tree species in the field of traditional Chinese medicine in China. Eucommia ulmoides is full of treasures, and its dried bark and leaves are used as raw materials for medicinal ingredients. Eucommia ulmoides leaves are the dried leaves of the medicinal plant Eucommia ulmoides of the Eucommiaceae family. As a traditional Chinese medicine, they have been included in the Chinese Pharmacopoeia in 2005, 2015 and 2020 editions, and have the effects of lowering blood pressure, strengthening bones and muscles, treating liver and kidney deficiency, dizziness and vertigo, and lumbar pain. Its leaves were first recorded and eaten in ancient times. The Illustrated Classic of Materia Medica in the Song Dynasty once recorded that Eucommia ulmoides "the newly grown tender leaves can be eaten". And in 2023, Eucommia ulmoides leaves were officially included in the list of homologous medicines and foods, and their raw materials were processed and developed into health products such as Eucommia ulmoides tea, Eucommia ulmoides wine, Eucommia ulmoides vinegar, and Eucommia ulmoides fine powder, as well as used for green breeding. Therefore, the chemical components, pharmacological activities, nutrients, etc. of Eucommia ulmoides leaves are widely used in medicines, foods, health products, and feeds, and have high development and comprehensive utilization value.

[0003] With the increasing market demand for Eucommia ulmoides raw materials, its artificial cultivation area is also constantly expanding. Due to the over-concentration of planting plots, the fungal diseases of Eucommia ulmoides leaves have become increasingly serious, such as leaf spot disease, leaf blight, gray mold disease, etc., which seriously restricts the development of the Eucommia ulmoides industry. After the occurrence of Eucommia ulmoides leaf blight, many leaves initially show some lesions, the area continues to expand, and the disease spreads rapidly, resulting in the withering of the leaves of the whole tree. Especially the 1-2-year-old Eucommia ulmoides seedlings are severely affected, resulting in a reduction in the yield and quality of Eucommia ulmoides leaves, which causes huge economic losses to planting farmers and planting parks.

[0004] At present, for the prevention and control of pathogenic bacteria of traditional Chinese medicines, chemical agents are mainly used. Chemical agents will cause the gray mold pathogen to develop drug resistance to a variety of chemical agents, and the chemical disease prevention method will also cause the problem of residues in traditional Chinese medicines. In addition, there are few targeted bacterial agent prevention and applications for the pathogen of Eucommia ulmoides gray mold, and there are limitations.

[0005] In view of this, the present invention is specifically proposed. Summary of the Invention

[0006] The purpose of the present invention is to provide a Bacillus, a bacterial agent and their applications in disease control and plant growth promotion to solve the above technical problems.

[0007] The present invention is implemented as follows:

[0008] In a first aspect, the present invention provides a Bacillus sp. which is deposited with the China General Microbiological Culture Collection Center, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, deposit date: November 15, 2024, deposit number: CGMCC No. 32656.

[0009] In a second aspect, the present invention provides a microbial agent which comprises the above-mentioned Bacillus sp.

[0010] In a third aspect, the present invention provides the application of the Bacillus sp. or the above-mentioned microbial agent in preventing and controlling plant diseases caused by fungi.

[0011] In a fourth aspect, the present invention provides the application of the Bacillus sp. or the above-mentioned microbial agent in promoting plant growth, and the plants are selected from tomato, pepper, wheat, rice, barley, oats, corn, sorghum, millet, buckwheat, broomcorn millet, sweet potato, potato, cotton, sesame, peanut, sunflower, radish, carrot, eggplant, Chinese chives, green onion, onion, leek, spinach, celery, amaranth, lettuce, crown daisy, daylily, grape, strawberry, sugarcane, tobacco, cruciferous vegetables, cucurbitaceae plants, leguminous plants, tea, cassava and Chinese herbal medicine plants.

[0012] In a fifth aspect, the present invention provides a method for promoting plant growth, which comprises the following step: applying the above-mentioned Bacillus sp. or the above-mentioned microbial agent to plants.

[0013] The present invention has the following beneficial effects:

[0014] By isolating and identifying the pathogenic bacteria of the diseased samples, the present invention finds that the pathogenic bacteria causing the wilt disease of Eucommia ulmoides leaves is Botrytis cinerea. The pathogenic bacteria of gray mold have characteristics such as diverse hosts, high genetic variation, and resistance to a variety of chemical agents, and the currently widely used chemical disease prevention methods will also cause the problem of chemical agent residues in Chinese medicinal materials. In view of this, the present invention provides a new biocontrol bacterium for effectively controlling pathogenic bacteria.

[0015] The present invention isolates and screens a Bacillus sp. strain RD22S2 from the rhizosphere soil of Eucommia ulmoides. Through experiments, it is proved that this bacterium can effectively control the gray mold of Eucommia ulmoides leaves and has both broad-spectrum antifungal disease prevention and plant growth promotion functions. Therefore, the Bacillus sp. provided by the present invention can be used both for preventing and controlling plant diseases caused by fungi and for promoting the growth of various plants, improving the yield and quality of plants. It has good application prospects for preventing and controlling plant diseases and promoting plant growth. Description of the Drawings

[0016] To more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the attached drawings required for the embodiments. It should be understood that the following attached drawings only show some embodiments of the present invention, and therefore should not be regarded as a limitation of the scope. For those of ordinary skill in the art, without creative efforts, other related attached drawings can also be obtained based on these attached drawings.

[0017] Figure 1 For Bacillus sp. strain RD22S2 (A, colony morphology on PDA medium; B, phylogenetic tree of the 16S rDNA gene sequence).

[0018] Figure 2 For the antagonistic effect of Bacillus sp. strain RD22S2 against Botrytis cinerea in a plate confrontation assay (A, front view of the antagonistic effect on 1 / 2 PDA medium; B, back view of the PDA plate with the antagonistic effect).

[0019] Figure 3 For the antagonistic effect of Bacillus sp. strain RD22S2 against Fusarium oxysporum causing Codonopsis pilosula root rot in a plate confrontation assay (A, antagonistic effect diagram on 1 / 2 PDA medium; B, control diagram of the Fusarium oxysporum colony).

[0020] Figure 4 For the antagonistic effect of Bacillus sp. strain RD22S2 against Plectosphaerella cucumerina causing cucumber fusarium wilt in a plate confrontation assay (A, front view of the antagonistic effect on 1 / 2 PDA medium; B, control diagram of the Plectosphaerella cucumerina colony).

[0021] Figure 5 For the antagonistic effect of Bacillus sp. strain RD22S2 against Fusarium tricinctum in a plate confrontation assay (A, front view of the antagonistic effect on 1 / 2 PDA medium; B, control diagram of the Fusarium tricinctum colony).

[0022] Figure 6 For the detection result diagram of the IAA secretion ability of Bacillus sp. strain RD22S2.

[0023] Figure 7 For the detection result diagram of the siderophore secretion ability of Bacillus sp. strain RD22S2.

[0024] Figure 8 For the detection result diagram of the protease secretion ability of Bacillus sp. strain RD22S2.

[0025] Figure 9 For the detection result diagram of the ability of Bacillus sp. strain RD22S2 to dissolve inorganic phosphorus.

[0026] Figure 10 Effect diagram of Bacillus sp. RD22S2 for controlling Botrytis cinerea disease on pepper fruits;

[0027] Figure 11 Effect diagram of Bacillus sp. RD22S2 for controlling Botrytis cinerea disease on pepper leaves;

[0028] Figure 12 Effect diagram of Bacillus sp. RD22S2 for controlling Botrytis cinerea disease on tomato seedlings;

[0029] Figure 13 Effect diagram of Bacillus sp. RD22S2 for promoting the growth of pepper seedlings;

[0030] Figure 14 Effect diagram of Bacillus sp. RD22S2 for promoting the growth of tomato seedlings. Detailed implementation manners

[0031] Reference to the embodiments of the present invention will now be provided in detail, one or more examples of which are described below. Each example is provided by way of explanation and not limitation of the present invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the scope or spirit of the present invention. For example, features described or illustrated as part of one embodiment can be used in another embodiment to yield a still further embodiment.

[0032] Unless otherwise indicated, the practice of the present invention will employ conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry, and immunology, which are within the capabilities of those skilled in the art. Such techniques are well explained in the literature, such as "Molecular Cloning: A Laboratory Manual" (Second Edition, Sambrook et al., 1989); "Oligonucleotide Synthesis" (edited by M.J. Gait, 1984); "Animal Cell Culture" (edited by R.I. Freshney, 1987); "Methods in Enzymology" (Academic Press, Inc.); "Handbook of Experimental Immunology" (edited by D.M. Weir and C.C. Blackwell); "Gene Transfer Vectors for Mammalian Cells" (edited by J.M. Miller and M.P. Calos, 1987); "Current Protocols in Molecular Biology" (edited by F.M. Ausubel et al., 1987); "PCR: The Polymerase Chain Reaction" (edited by Mullis et al., 1994); and "Current Protocols in Immunology" (edited by J.E. Coligan et al., 1991), each of which is hereby expressly incorporated by reference.

[0033] By isolating and identifying the pathogenic bacteria in the diseased samples, it was found that the pathogenic bacteria causing the wilt disease of Eucommia ulmoides leaves was Botrytis cinerea. The pathogenic bacteria of gray mold have characteristics such as diverse hosts, high genetic variation, and resistance to a variety of chemical agents, and the currently widely used chemical disease prevention methods are likely to cause problems of residues in traditional Chinese medicines. Therefore, there are limitations in the targeted bactericide prevention and application against the pathogen of gray mold on Eucommia ulmoides leaves at present.

[0034] In a first aspect, the present invention provides a Bacillus sp. which is deposited in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, deposition date: November 15, 2024, deposition number: CGMCC No. 32656. The name of the biological material submitted for deposition is RD22S2, and the proposed taxonomic name is Bacillus sp. The identification result is viable.

[0035] The biocontrol bacterium Bacillus RD22S2 against Botrytis cinerea on Eucommia ulmoides leaves, which is screened from the rhizosphere soil of Eucommia ulmoides, is identified as the genus Bacillus through morphological and molecular biological species identification. On the one hand, this strain has strong abilities of secreting IAA, protease, producing siderophores, and dissolving inorganic phosphorus. On the other hand, this strain has an inhibitory effect on the pathogen Botrytis cinerea of Eucommia ulmoides leaf blight, and has a broad-spectrum antibacterial effect on pathogenic fungi of plant rhizosphere diseases such as Fusarium oxysporum f. sp. codonopsis, Fusarium oxysporum f. sp. cucumerinum, and Fusarium tricinctum. The RD22S2 strain promotes plant growth by secreting IAA, and provides essential nutrients for plant growth by dissolving inorganic phosphorus. By secreting siderophores, it competes with plant pathogenic fungi for iron resources, disrupts the iron homeostasis of the pathogen, thereby reducing the damage of the pathogen to plants, and by secreting siderophores, it is a strategy for its survival in an iron-deficient environment, and the bacteria can obtain iron elements to maintain their growth and survival. Therefore, the RD22S2 strain has both biocontrol and growth-promoting functions of promoting plant growth and controlling plant diseases.

[0036] Studies have shown that plant growth-promoting rhizobacteria (PGPR) are beneficial bacteria that can stably survive in the rhizosphere or on the root surface and can secrete various secondary metabolites to directly or indirectly promote plant growth and prevent and control diseases. The promotion of plant growth by PGPR is mainly reflected in promoting plant growth by secreting secondary metabolites such as the plant growth hormone 3-indoleacetic acid (IAA) and siderophores. Therefore, the RD22S2 strain also belongs to plant growth-promoting rhizobacteria and has both biocontrol and growth-promoting functions of promoting plant growth and controlling plant diseases.

[0037] In a second aspect, the present invention provides a bacterial agent which includes the above-mentioned Bacillus sp. The bacterial agent is prepared, for example, by diluting the fermentation product.

[0038] It includes, but is not limited to, being obtained by solid fermentation or liquid fermentation of the above-mentioned Bacillus sp. The bacterial agent includes, but is not limited to, at least one of a concentrate, a paste, a dry product, a liquid product, a diluent, and a crushed product of Bacillus sp. The dry product includes, but is not limited to, a spray-dried product, a freeze-dried product, a vacuum-dried product, a drum-dried product, etc.

[0039] In a third aspect, the present invention provides the use of Bacillus or the above-mentioned bacterial agent in preventing and controlling plant diseases caused by fungi.

[0040] Bacillus or the above-mentioned bacterial agent secretes siderophores, competes with plant pathogenic fungi for iron resources, disrupts the iron homeostasis of the pathogen, thereby reducing the invasion of the pathogen to the plant, and by secreting siderophores, it is a strategy for its survival in an iron-deficient environment. Bacteria can obtain iron elements to maintain their growth and survival. Therefore, the Bacillus or bacterial agent provided by the present invention has good application prospects in preventing and controlling plant diseases caused by fungi.

[0041] In a preferred embodiment of the application of the present invention, the fungal disease is a plant disease caused by at least one selected from Botrytis cinerea, Fusarium oxysporum, Fusarium oxysporum f. sp. cucumerinum, and Fusarium tricinctum.

[0042] The results of the antibacterial experiment show that the Bacillus provided by the present invention has a high antibacterial effect on Botrytis cinerea, Fusarium oxysporum, Fusarium oxysporum f. sp. cucumerinum, and Fusarium tricinctum. Therefore, the Bacillus and its bacterial agent provided by the present invention can prevent and control plant diseases caused by at least one of Botrytis cinerea, Fusarium oxysporum, Fusarium oxysporum f. sp. cucumerinum, and Fusarium tricinctum.

[0043] In a preferred embodiment of the application of the present invention, the plants are selected from tomato, pepper, wheat, rice, barley, oats, corn, sorghum, millet, buckwheat, proso millet, sweet potato, potato, cotton, sesame, peanut, sunflower, radish, carrot, eggplant, leek, green onion, onion, leek, spinach, celery, amaranth, lettuce, crown daisy, daylily, grape, strawberry, sugarcane, tobacco, Brassica vegetables, cucurbitaceae plants, leguminous plants, tea, cassava, and Chinese herbal medicine plants.

[0044] In a preferred embodiment of the application of the present invention, the Chinese herbal medicine plants are selected from Codonopsis pilosula, Eucommia ulmoides, Scutellaria baicalensis, Cannabis sativa, Isatis indigotica, Astragalus membranaceus, Lonicera japonica, Forsythia suspensa, Phellodendron amurense, Coptis chinensis, Polygonum cuspidatum, Gardenia jasminoides, Paris polyphylla, Bupleurum chinense, Isatis indigotica, Taraxacum mongolicum, Lycium chinense, Anemarrhena asphodeloides, Rehmannia glutinosa, Scrophularia ningpoensis, Sterculia lychnophora, Sargentodoxa cuneata, Paeonia veitchii, Pulsatilla chinensis, and Paeonia suffruticosa.

[0045] In a preferred embodiment of the application of the present invention, the application includes the following application methods: Bacillus secretes siderophores. By secreting siderophores, it competes with plant pathogenic fungi for iron resources, disrupts the iron homeostasis of the pathogen, thereby reducing the invasion of the pathogen to the plant.

[0046] Fourthly, the present invention provides the application of Bacillus or the above-mentioned microbial agent in promoting plant growth, and the plants are selected from tomato, pepper, wheat, rice, barley, oats, corn, sorghum, millet, buckwheat, proso millet, sweet potato, potato, cotton, sesame, peanut, sunflower, radish, carrot, eggplant, leek, green onion, onion, leek, spinach, celery, amaranth, lettuce, crown daisy, daylily, grape, strawberry, sugarcane, tobacco, Brassica vegetables, cucurbit plants, leguminous plants, tea, cassava and Chinese herbal medicine plants.

[0047] In a preferred embodiment of the application of the present invention, the Chinese herbal medicine plants are selected from Codonopsis pilosula, Eucommia ulmoides, Scutellaria baicalensis, Cannabis sativa, Isatis tinctoria, Astragalus membranaceus, Lonicera japonica, Forsythia suspensa, Phellodendron amurense, Coptis chinensis, Polygonum cuspidatum, Gardenia jasminoides, Paris polyphylla, Bupleurum chinense, Isatis tinctoria, Taraxacum mongolicum, Lycium chinense, Anemarrhena asphodeloides, Rehmannia glutinosa, Scrophularia ningpoensis, Sterculia lychnophora, Sargentodoxa cuneata, Paeonia veitchii, Pulsatilla chinensis and Paeonia suffruticosa.

[0048] In a preferred embodiment of the application of the present invention, the application includes the following application methods:

[0049] (1) Bacillus secretes IAA;

[0050] (2) Bacillus secretes siderophores;

[0051] (3) Bacillus dissolves inorganic phosphorus;

[0052] (4) Bacillus secretes protease.

[0053] In a preferred embodiment of the application of the present invention, the application includes at least one of the following application methods:

[0054] (1) Used as a root irrigation agent;

[0055] (2) Used as a seed soaking agent;

[0056] (3) Used as a foliar spray agent;

[0057] In a preferred embodiment of the application of the present invention, promoting plant growth includes at least one of the following applications:

[0058] (1) Increasing the fresh weight of plants;

[0059] (2) Increasing the dry weight of plants;

[0060] (3) Increasing the plant height.

[0061] Fresh Weight refers to the weight of an organism or cell in its natural state, including all the water and other components inside the cell. Dry Weight refers to the weight of an organism or cell after removing all free water.

[0062] In a preferred embodiment of the application of the present invention, the use concentration of Bacillus is 1×10 5 -1×10 7 CFU / mL.

[0063] In a preferred embodiment of the application of the present invention, the use concentration of Bacillus is 1×10 5 -1×10 7 CFU / mL. The actual use concentration can be adjusted as needed. For example, the use concentration can be 1×10 5 CFU / mL, 2×10 5 CFU / mL, 3×10 5 CFU / mL, 4×10 5 CFU / mL, 5×10 5 CFU / mL, 6×10 5 CFU / mL, 7×10 5 CFU / mL, 8×10 5 CFU / mL, 9×10 5 CFU / mL, 1×10 6 CFU / mL, 2×10 6 CFU / mL, 3×10 6 CFU / mL, 4×10 6 CFU / mL, 5×10 6 CFU / mL, 8×10 6 CFU / mL, 9×10 6 CFU / mL, 1×10 7 CFU / mL.

[0064] In a preferred embodiment of the application of the present invention, at least one of a surfactant, a binder, a stabilizer, a pH regulator, a protective agent, an excipient, a disintegrant, a lubricant, a fragrance, a preservative, a suspending agent, a dispersing agent, and a diluent is further added to the root irrigation agent, the seed soaking agent, or the foliar spraying agent.

[0065] Examples include: surfactants such as Tween 20, Tween 80, etc.; pH regulators such as disodium hydrogen phosphate, dipotassium hydrogen phosphate, etc.; excipients such as sucrose, starch, mannitol, sorbitol, lactose, glucose, cellulose, talc, calcium phosphate, calcium carbonate; binders such as cellulose, methylcellulose, hydroxypropylcellulose, polypropylpyrrolidone, gelatin, gum arabic, polyethylene glycol, sucrose, starch; disintegrants such as starch, hydrolyzed starch, carboxymethylcellulose, calcium carboxymethylcellulose, hydroxypropyl starch, sodium glycol starch, sodium bicarbonate, calcium phosphate, calcium citrate; lubricants such as magnesium stearate, talc, sodium lauryl sulfate; flavors such as citric acid, menthol, glycine, orange powder; preservatives such as sodium benzoate, sodium bisulfite, parabens (such as methyl paraben, ethyl paraben, propyl paraben, butyl paraben); stabilizers such as citric acid, sodium citrate, acetic acid and polycarboxylic acids from the titriplex series such as diethylenetriaminepentaacetic acid (DTPA); suspending agents such as methylcellulose, polyvinylpyrrolidone, aluminum stearate; dispersants; diluents such as water, organic solvents; waxes, fats and oils such as beeswax, cocoa butter; polyethylene glycol; white petrolatum, etc.

[0066] When used as a root drench, the timing of root drenching includes but is not limited to the seedling stage, maturity stage, etc. When used as a seed soaking agent, it includes but is not limited to soaking the seeds of plants and then sowing them. In an alternative embodiment, Bacillus can be attached to the surface of the seeds as a component of the seed coating, for example, as a filler in the seed coating. When used as a foliar spray bacterium, a foliar spray agent containing Bacillus can be sprayed onto the leaf surface of the target plant.

[0067] In a fifth aspect, the present invention provides a method for promoting plant growth, which includes the following steps: applying the above-mentioned Bacillus or the above-mentioned bacterial agent to plants;

[0068] In a preferred embodiment of the application of the present invention, Bacillus is used for root drenching, seed soaking, or foliar spraying of plants.

[0069] The Bacillus or the above-mentioned bacterial agent provided by the present invention can increase the plant height, fresh weight and dry weight of plant seedlings, and can effectively promote the growth of plant seedlings. Especially for peppers and tomatoes.

[0070] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Those not specified in the embodiments are carried out according to conventional conditions or conditions recommended by the manufacturer. Those reagents or instruments not specified by the manufacturer can be obtained as conventional products through commercial purchase.

[0071] The features and performance of the present invention will be further described in detail below in conjunction with embodiments.

[0072] Example 1

[0073] In this example, the isolation and identification of Bacillus were carried out.

[0074] 1. Sample source

[0075] In 2021, disease investigations and samplings were carried out in Jinjiahe, Lueyang County, Hanzhong, the main Eucommia ulmoides production area in China (33°20'01.6"N, 105°59'10.9"E). Five diseased plants and their leaves, five healthy seedlings and five rhizosphere soil samples were collected in the Eucommia ulmoides planting area. The areas where the diseased plants were located were all infected, and the healthy samples were taken from adjacent areas. The rhizosphere soil was collected by shaking off the soil on the root surface and gently brushing with a sterilized toothbrush. It was transported back to the laboratory on the same day in a sampling box equipped with ice packs and immediately stored in a 4°C refrigerator, and then the fungal pathogens and antagonistic bacteria were isolated immediately.

[0076] 2. Source of the pathogen Botrytis cinerea of Eucommia ulmoides leaves

[0077] The pathogen Botrytis cinerea of Eucommia ulmoides leaf blight was isolated from the above-mentioned diseased leaves of Eucommia ulmoides with leaf blight, from three wilted leaves of Eucommia ulmoides saplings showing typical leaf blight symptoms. At the junction of the diseased area and the healthy area, the leaf samples were surface-sterilized, that is, rinsed twice with sterile water, soaked in 75% absolute ethanol for 30 s, rinsed twice with sterile water, soaked in 3% sodium hypochlorite for 5 minutes, and rinsed three times with sterile water. The surface-sterilized sample materials were cut into small pieces (each about 1×0.5 inches in size) with a sterile scalpel. Every three small pieces were placed on Rose Bengal agar medium, which was prepared by mixing 5 g of peptone, 10 g of glucose, 1 g of potassium dihydrogen phosphate, 0.5 g of magnesium sulfate, 0.033 g of Rose Bengal, 0.1 g of chloramphenicol and 15 g of agar per liter. After culturing at 25°C for 5 - 7 days, the mycelium was taken from the edge of the primary colony and placed on a PDA plate for further purification. After obtaining the purified fungus, it was identified by morphology, molecular biology, and pathogenicity re-inoculation experiments, and it was verified that the pathogen causing Eucommia ulmoides leaf blight was Botrytis cinerea.

[0078] 3. Isolation and culture of antagonistic bacteria in the rhizosphere of Eucommia ulmoides

[0079] To isolate antagonistic bacteria against Botrytis cinerea of Eucommia ulmoides leaf blight, rhizosphere soil samples were collected from healthy 2-year-old trees in adjacent planting areas. Four gradient dilutions (10 2 、10 3 、10 4) Mix 5 parts of rhizosphere soil samples, and culture them in R2A medium (0.5 g peptone, 0.3 g potassium dihydrogen phosphate, 0.5 g casein, 0.5 g soluble starch, 0.5 g yeast extract, 0.5 g glucose, 0.5 g magnesium sulfate heptahydrate, 0.3 g sodium pyruvate, add 15 g agar to every liter of ultrapure water, make up to 1 L with distilled water, and adjust the pH to 7.2) at 30 °C for 2 - 5 days. After three purification processes, remove duplicates according to the morphology and characteristics of the newly grown colonies. All 29 isolated strains were stored in 40% glycerol at -80 °C, except for the strains that could not be further subcultured.

[0080] Screen out the antagonistic bacteria of Botrytis cinerea from the numerous bacteria isolated from Eucommia ulmoides rhizosphere. Inoculate the antagonistic bacterial strain into liquid LB medium and shake culture for 48 hours. Centrifuge the precipitated cells at 5000 rpm for 5 minutes and resuspend with an equal volume of sterile water. After Botrytis cinerea pathogenic fungi grow on the 1 / 2 PDA plate for 10 days, inoculate a small amount of mycelia in the center of a new 1 / 2 PDA plate. Then, add 5 μL of bacterial liquid to each of the four symmetric points at the cross. Use sterile water as a control. Use ImageJ v.1.53a (NIH) software to measure the colony radius of Botrytis cinerea on these antagonistic assay plates after culturing at 25 °C for 5 days. Calculate the fungal growth inhibition rate (PFGI) using the formula: PFGI = (R1 - R2) / R1 * 100%. R1 represents the average diameter of the mycelia in the blank test, and R2 is the diameter of the inhibited cells.

[0081] The antagonistic effect of Bacillus sp. RD22S2 against Botrytis cinerea in the plate confrontation assay is shown in Figure 2 as follows, Figure 2 Figure A in

[0082] Figure 2 is the front view of the antagonistic effect on the 1 / 2 PDA medium;

[0083] Figure B in

[0084] The present invention also provides a method for culturing the bacterial agent of the above-mentioned Bacillus sp. RD22S2, which includes the following steps: After preparing a single colony of Bacillus sp. RD22S2 into a seed suspension, inoculate it into LB liquid medium, and shake culture at 30 °C and 200 r / min on a shaker for 24 - 48 hours, and the inoculation concentration is 10 6 -10 8 CFU / mL.

[0085] 3. Observation of the colony morphological characteristics of RD22S2

[0086] Streak a single colony of RD22S2 on 1 / 2 PDA, seal it with parafilm, and place it in an incubator at 30 °C for 48 h, then take it out and observe the characteristics of the single colony such as its morphology, color, and texture. The observation results show that the single colony of RD22S2 on the 1 / 2 PDA medium is white with a blue center, rough surface, and uneven edges, as specifically shown in Figure 1 Figure A

[0087] 4. 16S rDNA gene sequence and phylogenetic identification of the RD22S2 bacterium

[0088] Pick a single colony of the purified RD22S2 bacterium strain, culture it overnight at 30 °C with a rotation speed of 180 rpm in LB liquid medium, and collect the bacterial cells after centrifugation at 10000 rpm for 2 minutes. Use the EZ-10 Column Bacterial Genomic DNA Extraction Kit (Shanghai Sangon Biotech Co., Ltd.) to extract the genomic DNA of the bacterial cells of this strain according to the steps in the instruction manual. Using the obtained DNA as a template, perform PCR amplification with the universal primers 27F (5’-AGAGTTTGATCCTGGCTCAG-3’) and 1492R (5’-GGTTACCTTGTTACGACTT-3’) of the bacterial 16S rDNA gene. The amplification conditions are 93 °C for 4 minutes; 30 cycles: 94 °C for 30 seconds, 48 °C for 15 seconds, 72 °C for 1 minute; 72 °C for 10 minutes. Detect the amplification quality of the PCR product by 1% agarose gel electrophoresis. Subsequently, send the product to Shanghai Sangon Biotech Co., Ltd. for Sanger bidirectional sequencing

[0089] Use Seqman software to splice the gene sequences obtained from the bidirectional sequencing. The 16S rDNA nucleotide sequence of the RD22S2 strain obtained is shown in SEQ ID NO.1, with a length of 1354 bp. Upload the spliced sequence to the NCBI database and perform Blastn alignment similarity to obtain related sequences. Align these sequences using Align and use Mega 11 software, and then construct a phylogenetic tree using the Neighbor-joining method. The results are as shown in Figure 1As shown by B in []. Although the 16S rDNA related sequence of the RD22S2 strain is Bacillus, the phylogenetic classification analysis result does not cluster with other strains. Therefore, the RD22S2 strain belongs to the genus Bacillus and was deposited in the China General Microbiological Culture Collection Center (CGMCC) on January 15, 2024. The deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit name is Bacillus RD22S2, and the deposit number is: CGMCC No. 32656.

[0090] The following is the sequencing sequence splicing result (as shown in SEQ ID NO.1):

[0091] GCTTGCTCTTATGAAGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCCATAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATAACATTTTGAACTGCATGGTTCGAAATTGAAAGGCGGCTTCGGCTGTCACTTATGGATGGACCCGCGTCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCAACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGCTTTCGGGTCGTAAAACTCTGTTGTTAGGGAAGAACAAGTGCTAGTTGAATAAGCTGGCACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGAATTATTGGGCGTAAAGCGCGCGCAGGTGGTTTCTTAAGTCTGATGTGAAAGCCCACGGCTCAACCGTGGAGGGTCATTGGAAACTGGGAGACTTGAGTGCAGAAGAGGAAAGTGGAATTCCATGTGTAGCGGTGAAATGCGTAGAGATATGGAGGAACACCAGTGGCGAAGGCGACTTTCTGGTCTGTAACTGACACTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGAGGGTTTCCGCCCTTTAGTGCTGAAGTTAACGCATTAAGCACTCCGCCTGGGGAGTACGGCCGCAAGGCTGAAACTCAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGAAAACCCTAGAGATAGGGCTTCTCCTTCGGGAGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCATCATTAAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACGGTACAAAGAGCTGCAAGACCGCGAGGTGGAGCTAATCTCATAAAACCGTTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAA。

[0092] Example 2

[0093] This example provides an inhibitory experiment on the mycelial growth of Bacillus sp. RD22S2 against Fusarium oxysporum, Plectosphaerella cucumerina, and Fusarium tricinctum, which cause root rot in Codonopsis pilosula. Using the bacterial agent or bacterial liquid of Bacillus sp. RD22S2 screened in Example 1 above as the active ingredient, a co-culture confrontation test was carried out with the above three pathogenic bacteria on a PDA plate.

[0094] The specific steps are as follows:

[0095] 1. Source of pathogenic bacteria

[0096] Fusarium oxysporum was isolated from the roots of Codonopsis pilosula with black root rot (published in the literature [First report of root rot caused by the Fusarium oxysporum species complex on Codonopsis pilosula in China, Zhao Xia et al., Plant Disease. 105.11: 3742. 2021]), Plectosphaerella cucumerina was isolated from the diseased roots of Codonopsis pilosula (published in the literature [Continuous monoculture alters the fungal community and accumulates potential pathogenic strains in the rhizosphere of Codonopsis pilosula. Zhao Xia et al., Phytobiomes Journal: PBIOMES - 11, 2024]), and Fusarium tricinctum preserved in this laboratory.

[0097] 2. Test on the inhibitory activity of antagonistic bacterium Bacillus sp. RD22S2 against multiple pathogenic fungi of Codonopsis pilosula.

[0098] The antagonistic bacterium Bacillus sp. RD22S2 strain was inoculated into liquid LB medium and cultured with shaking for 48 hours. The precipitated cells were centrifuged at 5000 rpm for 5 minutes and resuspended with an equal volume of sterile water. After the five pathogenic fungi of Fusarium oxysporum causing root rot of Codonopsis pilosula, Plectosphaerella cucumerina causing root rot of Codonopsis pilosula, and Fusarium tricinctum had grown on 1 / 2 PDA plates for 3 days and 7 days respectively, a small amount of mycelia was inoculated in the center of a new 1 / 2 PDA plate. Then, 5 μL of the RD22S2 bacterial liquid was added dropwise at each of the four symmetric points of the cross. Sterile water was used as a control. These antagonistic assay plates were cultured at 25 °C for 6 days, 9 days, and 7 days respectively and observed. The results showed the inhibitory effects of Bacillus sp. RD22S2 against Fusarium oxysporum causing root rot of Codonopsis pilosula ( Figure 3 ), Plectosphaerella cucumerina causing root rot of Codonopsis pilosula ( Figure 4 ), and Fusarium tricinctum ( Figure 5 ).

[0099] Example 3

[0100] In this example, a growth promotion experiment was conducted on Bacillus sp. RD22S2. A plant growth-promoting bactericide was provided, and it was found that the bactericide or bacterial liquid containing the above-mentioned Bacillus sp. RD22S2 as the active ingredient had the potential to promote plant growth by secreting IAA, siderophores, protease, and dissolving inorganic phosphorus.

[0101] 1. Determination of the ability to secrete IAA.

[0102] A single colony of Bacillus sp. RD22S2 was picked and transferred to 1 mL of LB liquid medium, and cultured at 180 rpm in a shaker at 30 °C for 24 hours for activation. 10 μL of the activated RD22S2 bacterial liquid was pipetted and added to the LB liquid medium containing 200 mg / L of L-tryptophan, and incubated at 180 rpm in a shaker at 30 °C for 4 days. Then, 50 μL of the bacterial liquid was dropped into the depression of a white ceramic plate, and then 50 μL of Salkowski colorimetric solution (Salkowski colorimetric solution preparation: 50 mL of 30% HClO4, 1 mL of 0.5 mol / L FeCl3) was added for color reaction. 50 μL of the colorimetric solution containing 50 mg / L IAA was used as the positive control. 50 μL of the un-inoculated liquid LB medium was used as the negative control. The ceramic plate was placed in the dark for 30 minutes for color development. If the color turned red, it indicated that the strain had the ability to produce IAA.

[0103] The test results were as Figure 6 shown. The reaction solution inoculated with Bacillus sp. RD22S2 showed an obvious red color, indicating that Bacillus sp. RD22S2 had the ability to produce IAA.

[0104] 2. Determination of the ability to produce siderophores.

[0105] 10 μL of the above-activated RD22S2 bacterial liquid was pipetted and dropped onto the surface of the CAS solid medium (CAS medium preparation: 0.59 g of disodium hydrogen phosphate dihydrate, 2.43 g of dodecahydrate disodium hydrogen phosphate, 0.25 g of ammonium chloride, 0.075 g of potassium dihydrogen phosphate, 0.125 g of sodium chloride were used to prepare 100 mL of phosphate solution; 6.04 g of PIPES was mixed with 100 mL of phosphate solution, and the pH was adjusted to 6.8 with 50% NaOH solution, 3.2 g of agar powder, sterilized at 121 °C for 15 min; 1 mmol / L CaCl2 solution, 1 mmol / L magnesium sulfate tetrahydrate, 10% acid-hydrolyzed casein, 20% glucose solution, sterilized at 121 °C for 15 min; at 60 °C, 0.2 mL of calcium chloride, 4 mL of magnesium sulfate heptahydrate, 2 mL of glucose, and 6 mL of acid-hydrolyzed casein solution were added) plate surface. After the plate inoculated with Bacillus sp. RD22S2 was statically cultured in an incubator at 37 °C for 48 hours, it was observed whether an obvious orange-yellow transparent circle was produced around the colony. The presence or absence of the transparent circle could reflect whether the strain had the ability to produce siderophores.

[0106] The detection results are as follows Figure 7 shown, indicating that Bacillus sp. RD22S2 has the ability to produce siderophores.

[0107] 3. Determination of protease secretion ability.

[0108] Take 10 μL of the activated RD22S2 bacterial solution in the above-mentioned example and drop it on the surface of a 90-mm diameter plate containing skim milk powder medium (Skim milk powder medium preparation: 3.0 g skim milk powder, 15 g agar powder, 1 L distilled water, pH = 7.0 - 7.2, sterilized at 120 °C for 20 min). After placing the plate inoculated with RD22S2 in an incubator at 30 °C and statically culturing for 48 hours, observe whether a clear zone appears around the colony. The presence or absence of the clear zone reflects whether the strain has the ability to produce protease.

[0109] The detection results are as shown in Figure 8 shown, and the results show that Bacillus sp. RD22S2 has good protease production ability.

[0110] 4. Determination of inorganic phosphorus solubilization ability.

[0111] A single colony of RD22S2 was picked and cultured in LB liquid at 180 rpm with shaking at 30 °C overnight for activation. Take 10 μL of the above-activated RD22S2 bacterial solution and drop it on the surface of a solid inorganic phosphorus medium (Inorganic phosphorus medium preparation: 10 g glucose, 0.5 g ammonium sulfate, 0.3 g magnesium sulfate heptahydrate, 0.03 g manganese sulfate tetrahydrate, 0.3 g potassium chloride, 0.03 g ferrous sulfate heptahydrate, 0.3 g sodium chloride, 10 g calcium phosphate, 15 g agar powder, 1 L distilled water, pH = 7.0) plate. After statically culturing at 30 °C for 2 to 4 days, observe and record whether a clear zone exists around the colony to determine whether the strain can dissolve inorganic phosphorus.

[0112] The detection results are as follows Figure 9 shown. There is an obvious clear zone around the RD22S2 colony, so it indicates that Bacillus sp. RD22S2 has the ability to dissolve inorganic phosphorus.

[0113] Example 4

[0114] In this example, an experiment on controlling Botrytis cinerea disease of pepper fruits was carried out with Bacillus sp. RD22S2. Spraying the above-mentioned broad-spectrum antibacterial agent RD22S2 and inoculating the fungal plug of Botrytis cinerea on the wounds of pepper fruits can effectively reduce the incidence of Botrytis cinerea disease of pepper fruits.

[0115] 1. Preparation of RD22S2 bacterial agent.

[0116] The activated RD22S2 bacterial solution in the above-mentioned example was transferred to 50 mL of liquid LB medium for activation and cultured in a shaker at 37 °C at 180 rpm for 48 hours. The cultured bacterial solution was centrifuged at 8000 g for 5 min, the supernatant was discarded, and the precipitated bacteria were collected. Then, they were resuspended with 50 mL of sterile water to prepare an RD22S2 bacterial agent, and the viable bacteria count was detected under a microscope to be 1×10 6 -1×10 7 CFU / mL.

[0117] 2. Application in the control of Botrytis cinerea on pepper fruits.

[0118] The pepper fruits were soaked in 1% (v / v) sodium hypochlorite solution for 2 minutes and then rinsed 3 times in sterile distilled water. Then, the above-mentioned RD22S2 biocontrol bacterial solution was sprayed with a sterilized sterile sprayer, and 0.3 mL was sprayed on each pepper fruit in the experimental group. 0.3 mL of sterile water was sprayed in the control group. After air-drying, wounds were made vertically at the tip, center, and bottom of each pepper fruit with a sterile pipette tip. Discs with a diameter of about 3 mm were cut from the mycelia of Botrytis cinerea cultured on PDA medium for 14 days, and the mycelia were attached to the wound sites with the mycelia facing downwards. The treated fruits were placed in a sterilized plastic box and placed in an incubator at about 24 °C with a relative humidity of 100% for 5 days, and the results were observed and recorded. Also, the number of diseased sites on the inoculated parts of the pepper fruit surface was counted, and the control effect of Bacillus sp. RD22S2 bacterial agent on Botrytis cinerea of pepper fruits was quantitatively analyzed using the calculation formula of the number of diseased sites / the total number of inoculated sites × 100% = incidence rate (%). The control group also had to be treated in the same way, inoculating Botrytis cinerea mycelia.

[0119] According to Figure 10 The results showed that the RD22S2 biocontrol bacterial agent could inhibit Botrytis cinerea on pepper fruits. The statistical results of the incidence rate in Table 1 showed that the incidence rate of Botrytis cinerea on pepper fruits was significantly reduced after inoculation with the RD22S2 biocontrol bacterial agent compared with the control. Compared with 74.11% of the control group for the incidence rate of pepper fruits, the incidence rate decreased to 22.67% after spraying the RD22S2 biocontrol bacterial agent, and the relative control effect reached 69.41%. The above results indicated that the Bacillus sp. RD22S2 biocontrol bacterial agent had a good inhibitory effect on Botrytis cinerea of pepper fruits and could effectively control Botrytis cinerea on pepper fruits.

[0120] Table 1. Control effect of Bacillus sp. RD22S2 bacterial agent on Botrytis cinerea of pepper fruits

[0121]

[0122] Example 5

[0123] This embodiment provides a control method for preventing and treating Botrytis cinerea diseases on the leaves of pepper and tomato seedlings with Bacillus sp. RD22S2. It includes spraying the above-mentioned RD22S2 biocontrol agent on the leaves of pepper and tomato, as well as inoculating Botrytis cinerea plugs on pepper seedlings and spraying spore suspension on tomato seedlings, all of which can effectively reduce the incidence of Botrytis cinerea diseases on the leaves of pepper and tomato.

[0124] The specific steps are as follows:

[0125] 1. Preparation of the pathogen spore suspension.

[0126] Add 20 mL of sterile water to the PDA plate of Botrytis cinerea cultured for 30 days, gently brush it with a sterile toothbrush, filter the mixture of spores and hyphae through a sterile four-layer cotton gauze to obtain a spore suspension, and detect it with a microscope and adjust the concentration to 1×10 5 -1×10 6 spores / mL to make the pathogen spore suspension.

[0127] 2. Application of the control of Botrytis cinerea diseases on pepper and tomato seedlings.

[0128] Spray the above-mentioned RD22S2 biocontrol liquid on pepper and tomato seedlings respectively. Each seedling in the experimental group is sprayed with 0.5 mL (containing 0.2% Tween 20). Each seedling in the control group is sprayed with 0.3 mL of sterile water (containing 0.2% Tween 20). After the water stains on the pepper leaves dry, punch a disc with a diameter of about 3 mm from the Botrytis cinerea hyphae cultured on the PDA medium for 14 days and stick it on the surface of the pepper leaves with the hyphae facing down. After the water stains on the tomato leaves dry, spray the above-mentioned pathogen spore suspension on the tomato leaves, and each seedling is sprayed with 0.5 mL of spore suspension, and let it stand until the water stains on the leaf surface dry. Place the treated pepper and tomato seedlings in a greenhouse at about 24°C with a relative humidity of more than 70%, record the wilting situation of the seedling leaves, and conduct statistics on the leaf incidence rate.

[0129] According to Figure 11 and Figure 12 The results show that the RD22S2 biocontrol agent can inhibit Botrytis cinerea diseases on the leaves of pepper and tomato. According to the statistical results of the incidence rate in Table 2, compared with the control, the incidence rate of Botrytis cinerea on pepper and tomato after inoculation with the RD22S2 biocontrol agent is significantly reduced. Compared with 67.01% of the control group for the incidence rate of Botrytis cinerea on pepper leaves, the incidence rate after spraying the RD22S2 biocontrol agent decreased to 33.00, and the relative control effect reached 50.75%; compared with 88.83% of the control group for the incidence rate of Botrytis cinerea on tomato leaves, the incidence rate after spraying the RD22S2 biocontrol agent decreased to 62.67%, and the relative control effect reached 29.45%. The above results indicate that the RD22S2 biocontrol agent of Bacillus sp. has a good inhibitory effect on Botrytis cinerea on the leaves of pepper and tomato and can effectively prevent and treat Botrytis cinerea diseases on pepper and tomato.

[0130] Table 2. Control effect of Bacillus sp. RD22S2 agent on gray mold of pepper and tomato leaves

[0131]

[0132] Example 6

[0133] This example provides a method for promoting the growth of pepper and tomato seedlings. By inoculating the agent RD22S2 into the soil at the roots of potted pepper and tomato seedlings, the plant height, fresh weight and dry weight of pepper and tomato seedlings are increased, which can effectively promote the growth of pepper and tomato seedlings.

[0134] Use seed sowing, thin out or fill in seedlings after emergence to ensure that there are 3 seedlings in each hole (9 cm × 9 cm) of the seedling tray. The above-mentioned RD22S2 agent inoculation treatment is carried out on 30-day-old pepper and tomato seedlings, and the inoculation method mainly adopts root irrigation. In the experimental group, 1 mL of RD22S2 agent is inoculated at the rhizosphere of each seedling, and the second inoculation of the agent is carried out after one week, and tap water inoculation is used as the control group. The growth conditions of pepper and tomato are observed and recorded 30 days after inoculation, and the plant height and fresh weight of each seedling are counted. After the harvested seedlings are dried in a ventilated manner in an envelope bag at room temperature for 14 days, the dry weight is measured. The plant height is measured by ImageJ software, and the data statistical analysis is carried out by spss 18 software. The significance of the differences in various parameters (fresh weight, dry weight, plant height) between the inoculated RD22S2 and the control group is tested by one-way ANOVA (Duncan method, P < 0.05).

[0135] According to Figure 13 (pepper) and Figure 14 (tomato) results show that the growth-promoting agent RD22S2 can significantly increase the growth of pepper and tomato. According to the statistics of the plant height, fresh weight and dry weight of pepper and tomato in Table 3, compared with the average plant height of 13.64 cm and 24.70 cm in the control group, the average plant height increased to 15.76 cm and 31.02 cm after inoculating the growth-promoting agent RD22S2; compared with the average fresh weight of 1.02 g and 3.14 g in the control group, the average fresh weight increased to 1.13 g and 4.27 g after inoculating the growth-promoting agent RD22S2; compared with the average dry weight of 0.10 g and 0.28 g in the control group, the average dry weight increased to 0.14 g and 0.41 g after inoculating the growth-promoting agent RD22S2. The above results show that the growth-promoting agent of Bacillus sp. RD22S2 has a good application effect on promoting the growth and development of pepper and tomato.

[0136] Table 3. The application of Bacillus sp. RD22S2 agent increases the plant height, fresh weight and dry weight of tomato seedlings.

[0137]

[0138] Note: The data in the table are the mean ± standard error per seedling. Different letters indicate significant differences at the P<0.05 level by Duncan's test.

[0139] In summary, a biocontrol bacterium Bacillus RD22S2 against Botrytis cinerea on Eucommia ulmoides leaves, screened from the rhizosphere soil of Eucommia ulmoides, was identified as Bacillus by morphological and molecular biological species identification. On the one hand, this strain has strong abilities of secreting IAA, protease, siderophore production, and dissolving inorganic phosphorus. On the other hand, this strain has an effective inhibitory effect on Botrytis cinerea, the pathogen of Eucommia ulmoides leaf blight, and has a broad-spectrum antibacterial effect on pathogenic fungi of plant rhizosphere diseases such as Fusarium oxysporum f. sp. codonopsis, Fusarium oxysporum f. sp. cucumerinum, and Fusarium tricinctum. Strain RD22S2 promotes plant growth by secreting IAA, provides essential nutrients for plant growth by dissolving inorganic phosphorus. By secreting siderophores, it competes with plant pathogenic fungi for iron resources, disrupts the iron homeostasis of the pathogen, thereby reducing the damage of the pathogen to plants, and by secreting siderophores, it is a strategy for its survival in an iron-deficient environment, enabling the bacteria to obtain iron elements and maintain their growth and survival. Therefore, strain RD22S2 has both biocontrol and growth-promoting functions of promoting plant growth and preventing plant diseases.

[0140] The Bacillus RD22S2 bacterium, bactericide, and application provided by the present invention can effectively control Botrytis cinerea on pepper fruits, pepper leaves, and tomato leaves, and have good application prospects for preventing and controlling plant diseases.

[0141] In addition, the Bacillus RD22S2 bacterium, bactericide, and application provided by the present invention can effectively promote the growth and development of peppers and tomatoes, and can improve the yield and quality of various plants.

[0142] The above are only the preferred embodiments of the present invention and are not used to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A Bacillus sp., characterized in that, It is preserved in the General Microbiological Center of the China National Center for Culture Collection of Microorganisms, Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Preservation Date: November 15, 2024, Preservation Number: CGMCC No. 32656.

2. A bacterial agent, characterized in that, It includes the Bacillus described in claim 1.

3. Use of the Bacillus described in claim 1 or the microbial agent described in claim 2 in controlling plant diseases caused by fungi.

4. The application according to claim 3, wherein The fungal diseases are selected from plant diseases caused by at least one of Botrytis cinerea, Fusarium oxysporum, Fusarium oxysporum f. sp. cucumerinum, and Fusarium tricinctum; Preferably, the plants are selected from tomato, pepper, wheat, rice, barley, oats, corn, sorghum, millet, buckwheat, proso millet, sweet potato, potato, cotton, sesame, peanut, sunflower, radish, carrot, eggplant, leek, green onion, onion, leek, spinach, celery, amaranth, lettuce, crown daisy, daylily, grape, strawberry, sugarcane, tobacco, Brassica vegetables, cucurbit plants, leguminous plants, tea, cassava, and Chinese herbal medicine plants; Preferably, the Chinese herbal medicine plants are selected from Codonopsis pilosula, Eucommia ulmoides, Scutellaria baicalensis, Cannabis sativa, Isatis indigotica, Astragalus membranaceus, Lonicera japonica, Forsythia suspensa, Phellodendron amurense, Coptis chinensis, Polygonum cuspidatum, Gardenia jasminoides, Paris polyphylla, Bupleurum chinense, Isatis indigotica, Taraxacum mongolicum, Lycium chinense, Anemarrhena asphodeloides, Rehmannia glutinosa, Scrophularia ningpoensis, Sterculia lychnophora, Sargentodoxa cuneata, Paeonia lactiflora, Pulsatilla chinensis, and Paeonia suffruticosa.

5. The application according to claim 3, wherein The use includes the following application method: the Bacillus secretes siderophores.

6. Use of the Bacillus according to claim 1 or the microbial agent according to claim 2 in promoting plant growth, characterized in that, The plants are selected from tomato, pepper, wheat, rice, barley, oats, corn, sorghum, millet, buckwheat, proso millet, sweet potato, potato, cotton, sesame, peanut, sunflower, radish, carrot, eggplant, leek, green onion, onion, leek, spinach, celery, amaranth, lettuce, crown daisy, daylily, grape, strawberry, sugarcane, tobacco, Brassica vegetables, cucurbit plants, leguminous plants, tea, cassava, and Chinese herbal medicine plants; Preferably, the Chinese herbal medicine plants are selected from Codonopsis pilosula, Eucommia ulmoides, Scutellaria baicalensis, Cannabis sativa, Isatis indigotica, Astragalus membranaceus, Lonicera japonica, Forsythia suspensa, Phellodendron amurense, Coptis chinensis, Polygonum cuspidatum, Gardenia jasminoides, Paris polyphylla, Bupleurum chinense, Isatis indigotica, Taraxacum mongolicum, Lycium chinense, Anemarrhena asphodeloides, Rehmannia glutinosa, Scrophularia ningpoensis, Sterculia lychnophora, Sargentodoxa cuneata, Paeonia lactiflora, Pulsatilla chinensis, and Paeonia suffruticosa.

7. The application according to claim 6, characterized in that The use includes the following application method: (1) The Bacillus secretes IAA; (2) The Bacillus secretes siderophores; (3) The Bacillus dissolves inorganic phosphorus; (4) The Bacillus secretes protease.

8. The application according to claim 7, wherein The use includes at least one of the following application methods: (1) Used as a root drench; (2) Used as a seed soaking agent; (3) Used as a foliar spray. Preferably, the promoting plant growth includes at least one of the following applications: (1) Increasing the fresh weight of the plant; (2) Increasing the dry weight of the plant; (3) Increasing the plant height. Preferably, the use concentration of the Bacillus is 1×10 5 -1×10 7 CFU / mL.

9. The application according to claim 8, characterized in that, At least one of surfactant, binder, stabilizer, pH regulator, protective agent, excipient, disintegrant, lubricant, fragrance, preservative, suspending agent, dispersing agent, and diluent is further added to the root drench, seed soaking agent, or foliar spray.

10. A method for promoting plant growth, characterized in that, It includes the following steps: applying the Bacillus described in claim 1 or the microbial agent described in claim 2 to plants; Preferably, the Bacillus is used for root irrigation, seed soaking of the plant, or spraying on the leaf surface of the plant.

Images