Synergistic bactericide containing bacillus turicatus and application thereof

Abstract

The present application relates to fungicides, in particular to synergistic fungicide containing bacillus takil and application thereof. The active ingredient of the fungicide composition comprises: bacillus takil 2_2a and fungicide; the fungicide is selected from any one of fluazinam, triflumizole, triforine, guazatine acetate or catechin. The bacillus takil 2_2a is compounded with fluazinam, triflumizole, triforine, guazatine acetate and catechin, has synergistic fungicidal effect, and can be used for plant disease control.

Description

Technical Field

[0001] This invention relates to bactericides, specifically to synergistic bactericides containing Bacillus tekirae and their applications. Background Technology

[0002] *Corynespora cassiicola* (Berk. & Curt.) Wei., also known as *Corynespora cassiicola*, causes leaf spot disease in vegetables, soybeans, trees, and ornamental crops. This pathogen primarily infects the leaves of host plants, but can also infect stems, flowers, and fruits, causing severe leaf and fruit drop. The earliest reports of *Corynespora cassiicola* leaf spot disease were found in vegetables such as cucumbers and cowpeas. *Corynespora cassiicola* has diverse and widely distributed host species, varied transmission methods, and the pathogen is prone to mutation. After infecting plants, it exhibits strong destructive power, and the lack of effective control measures has led to *Corynespora cassiicola* leaf spot disease rising from a minor disease to a globally recognized major disease, becoming a significant factor restricting the development of industries in its main host plants.

[0003] Chemical control of *Cladosporium* leaf spot mainly involves periodically spraying chemical agents during the incubation period or early stages of disease to inhibit pathogen infection and the spread of the disease. Chemical agents reported as effective against *Cladosporium* leaf spot both domestically and internationally include mancozeb, thiram, thiophanate-methyl, azoxystrobin, prochloraz, metalaxyl, iprodione, carbendazim, and chlorothalonil. However, due to the pathogen's strong infectivity and rapid spread, the control effectiveness of chemical agents decreases significantly once more than 3% of the plant's leaves are infected. In recent years, despite the application of various fungicides during host plant cultivation, *Cladosporium* leaf spot has not been effectively controlled. The difficulty in controlling the disease is due to two main reasons: firstly, the widely adopted continuous cropping system promotes the accumulation of pathogens year after year; secondly, *Cladosporium* strains are highly prone to mutation and easily develop resistance to multiple fungicides. Therefore, in the control of *Cladosporium* leaf spot, it is essential to reduce the frequency and dosage of fungicide use to inhibit the emergence of resistant strains. Recent screening tests for fungicides to control *Cyclocarya paliurus* have demonstrated that benzimidazole and QoI fungicides have almost lost their effectiveness against *Cyclocarya paliurus* leaf spot. Therefore, screening for scientifically effective fungicides for controlling *Cyclocarya paliurus* leaf spot will provide crucial evidence and technical support for the effective control of this disease.

[0004] In recent years, cucumber leaf spot disease caused by *Cyclocarya spp.*, also known as brown spot or target spot, has become severe, and in many areas, it is more difficult to control than cucumber downy mildew. Protected greenhouses are severely affected, with disease incidence exceeding 70% in severely affected areas. Studies have found that in cucumber greenhouses where the same chemical agent has been sprayed more than three times consecutively, the probability of resistance to the pathogen *Cyclocarya spp.* increases significantly. Therefore, in the control of *Cyclocarya spp.* leaf spot, it is essential to reduce the frequency and dosage of fungicides used, and to rotate or mix fungicides with different mechanisms of action to inhibit the emergence of resistant strains.

[0005] Soybean target spot, also known as *Cyclocarya spp.* leaf spot, is caused by *Cyclocarya spp.* and primarily affects leaves, stems, and pods. It can occur in both seedlings and mature plants. This disease is common in soybean production. Affected plants experience premature leaf drop and reduced pod quality; susceptible varieties can suffer yield losses of 18-32%. Cowpea gray spot is also caused by *Cyclocarya spp.* It is widely distributed and a common disease in cowpeas. It primarily affects cowpea leaves, mainly during the flowering and podding stages. Eggplant black rot, also known as *Cyclocarya spp.* leaf spot, is caused by *Cyclocarya spp.* It mainly affects leaves, stems, and fruits. In recent years, field incidence rates have reached 30%-50%, severely reducing eggplant yield and quality, causing significant economic losses.

[0006] Fluopyram is a novel pyrazole amide fungicide. It works by inhibiting succinate dehydrogenase in the mitochondrial respiration of pathogens. In 2019, fluopyram received its first global registration in Paraguay, with the registered product being a 100g / L fluopyram + 100g / L tetraflufenicol emulsifiable concentrate formulation for controlling Asian soybean rust. In 2021, fluopyram was approved in the United States for use in field crops such as lawns to control various diseases. In early 2021, a mixture of fluopyram and difenoconazole was launched in Argentina, exhibiting advantages such as strong systemic activity and rapid onset of action. Recently, FMC launched a ternary mixture containing fluopyram (fluopyram + fenpyroxetine + azoxystrobin) for use in barley, wheat, corn, sorghum, etc., to control anthracnose leaf blight, gray leaf spot, common rust, northern corn leaf blight, and southern rust.

[0007] Azoxystrobin is a methoxyacrylate fungicide developed by Bayer. It has a broad fungicidal spectrum, high fungicidal activity, and is resistant to rain washout. It is used on many crops such as cereals, soybeans, corn, rice, rapeseed, cotton, and sugar beets to control diseases caused by ascomycetes, deuteromycetes, basidiomycetes, and oomycetes, and is particularly effective against Asian rust on soybeans. Azoxystrobin is a mitochondrial respiration inhibitor, suppressing mitochondrial respiration by blocking electron transfer at the cytochrome bc1 Qo site. Azoxystrobin has systemic and penetrating properties, enabling rapid distribution within the plant, good resistance to rain washout, and a long residual effect. Zhou Quankang compared the activity and fungicidal spectrum of several major methoxyacrylate fungicides. The results showed that azoxystrobin, pyraclostrobin, and azoxystrobin had comparable activity, but the activity was lower than that of pyraclostrobin. Similar to other methoxyacrylate fungicides, pyrostrobin has a single site of action, making it prone to resistance development; currently, several pathogens have developed resistance to it. In practical production applications, the development and progression of resistance to azoxystrobin can be delayed by limiting the frequency of use, compounding, and alternating with fungicides of different mechanisms of action. Among these measures, compounding is the main way to avoid the risk of resistance.

[0008] Biguanide trioctylbenzene sulfonate is a biguanide fungicide that primarily inhibits spore germination, germ tube elongation, appressorium formation, and mycelial formation. Introduced in the 1990s, biguanide trioctylbenzene sulfonate fungicide was mainly used to control diseases in citrus fruits during storage; its application in vegetable disease control has been less reported. While there are currently no reports on the development of resistance to biguanide trioctylbenzene sulfonate in pathogens, it has been suggested that when using biguanide trioctylbenzene sulfonate in the integrated management of cucumber scab leaf spot, further careful screening of compatible fungicides is necessary.

[0009] Catechins are a new generation of plant-derived fungicides. Early studies found that catechins can inhibit the mycelial growth, sporulation, and spore germination of *Fusarium wilt*, the causal agent of cotton wilt. Provisional registrations have been made for 20% catechin technical material and 1.1% wettable powder for the control of fungal diseases such as gray mold in tomatoes and black spot in cucumbers. Catechins have also been reported to inhibit the growth of *Fusarium wilt*. Furthermore, catechins can effectively inhibit downy mildew, leaf mold, black spot, anthracnose, gray mold, rice blast, onion soft rot, wheat powdery mildew, and various other crop diseases caused by bacteria, fungi, and viruses.

[0010] As people's living standards continue to improve, they are paying more attention to ecological methods when controlling plant diseases. Biological control is a non-toxic, harmless, environmentally friendly, and highly efficient method of disease treatment. Biological control does not aim to completely eliminate diseases, but rather to minimize their harm through economic means, thereby achieving the goal of profitability. Chemical control has always been controversial, but its rapid effectiveness and ease of control are undeniable. Appropriate amounts of chemical agents can enhance the vitality and vigor of biological strains. If biological and chemical control can be effectively combined, the best way to control diseases can be found while meeting environmental carrying capacity requirements. Summary of the Invention

[0011] This invention reveals that *Bacillus tequilensis* 2_2a possesses natural resistance to fluopyram, azoxystrobin, biguanide trioctylbenzenesulfonate, and catechins. This strain can grow on LB medium containing 1000 μg / mL of these fungicides. Therefore, *Bacillus tequilensis* 2_2a can be formulated with fluopyram, azoxystrobin, biguanide trioctylbenzenesulfonate, or catechins to create a fungicide, allowing for long-term coexistence. Further research has shown that *Bacillus tequilensis* 2_2a, when combined with fluopyram, azoxystrobin, biguanide trioctylbenzenesulfonate, and catechins, exhibits synergistic fungicidal activity and can be used for plant disease control.

[0012] The *Bacillus tequilensis* 2_2a described in this invention was obtained through screening by the Institute of Plant Protection, Hebei Academy of Agricultural and Forestry Sciences. This *Bacillus tequilensis* 2_2a was deposited on March 3, 2020, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 19446; its Latin name is *Bacillus tequilensis* 2_2a. This strain has been disclosed in CN111172084A. This strain is characterized by high efficiency, broad antibacterial spectrum, ease of use, low cost, and no environmental pollution.

[0013] A bactericidal composition comprising, as its active ingredients: Bacillus tequilensis 2_2a and a bactericide; wherein the preservation number of Bacillus tequilensis 2_2a is CGMCC No. 19446; wherein the bactericide is selected from any one of fluopyram, azoxystrobin, biguanide trioctylbenzene sulfonate, or catechin.

[0014] In some specific embodiments, in the bactericidal composition, the weight ratio of the bactericide to the *Bacillus tekirae* 2_2a is (1-99):(99-1); the total viable count of the *Bacillus tekirae* 2_2a is (7×10⁻⁶). 9 -2.4×10 10 )CFU / g.

[0015] In some specific embodiments, the Bacillus tekirae is its fermentation broth or lyophilized powder.

[0016] In some specific embodiments, the *Bacillus tekiria* 2_2a is used as its fermentation broth, and the total viable count of the fermentation broth is (7 × 10⁻⁶). 9 -1×10 10 CFU / g, for example 8×10 9 CFU / g.

[0017] In some specific embodiments, the *Bacillus tekiria* 2_2a is its freeze-dried bacterial powder, and the total viable count of the freeze-dried bacterial powder is (1.8 × 10⁻⁶). 10 -2.4×10 10 CFU / g, for example, a total viable count of 2.0 × 10⁻⁶ CFU / g. 10 CFU / g.

[0018] Furthermore, the weight ratio of the bactericide to the Bacillus tekirae 2_2a is (1-50):(50-1).

[0019] Furthermore, the weight ratio of the bactericide to the Bacillus tekirae 2_2a is 1:(1-50).

[0020] In some preferred embodiments, the weight ratio of the bactericide to the Bacillus tekirae 2_2a is 1:1, 1:2, 1:4, 1:10, 1:25, or 1:50.

[0021] Furthermore, the active ingredient of the bactericidal composition consists of Bacillus tekirae 2_2a and a bactericide.

[0022] In this article, fluopyram, oxadiazon, biguanide trioctylbenzene sulfonate, and catechins are all commercially available.

[0023] In some specific embodiments, the mass concentration of fluopyram is 95%.

[0024] In some specific embodiments, the mass concentration of oxime ester is 97%.

[0025] In some specific embodiments, the mass concentration of biguanide trioctylbenzene sulfonate is 90%.

[0026] In some specific embodiments, the mass concentration of catechins is 98%.

[0027] In some embodiments, the *Bacillus tekiria* 2_2a is its cells and / or its metabolites.

[0028] The fermentation broth of Bacillus tekiria 2_2a described in this invention can be prepared by existing technology such as the method described in Chinese Patent CN202010171544.X; or by the following method.

[0029] A method for preparing Bacillus tekirae 2_2a fermentation broth includes the following steps: First, activate Bacillus tekirae 2_2a in LB agar plates, pick a single colony and streak it onto a Kesselrins flask. After 3 days, wash the bacterial growth with sterile water and inoculate it into a seed tank (e.g., a 1-ton tank) as seed liquid. After culturing at 31°C for 12 hours, inoculate the seed liquid into a fermenter (e.g., a 5-ton tank) for fermentation. After 48 hours, remove the fermenter from the tank to obtain Bacillus tekirae 2_2a fermentation broth.

[0030] The freeze-dried Bacillus tekirae 2_2a bacterial powder of the present invention can be obtained from the Bacillus tekirae 2_2a fermentation broth using conventional methods in the prior art. For example, after the Bacillus tekirae 2_2a fermentation broth is placed in a tank, it is centrifuged and freeze-dried to obtain the freeze-dried Bacillus tekirae 2_2a bacterial powder.

[0031] The present invention also provides a synergistic bactericide containing Bacillus tekirae, comprising the above-mentioned bactericidal composition.

[0032] In some embodiments, the synergistic bactericide containing Bacillus tekirae uses the above-described bactericidal composition as the sole active ingredient.

[0033] Furthermore, the synergistic fungicide containing Bacillus tekiria also contains adjuvants or excipients that are applicable to pesticides.

[0034] The synergistic bactericide containing Bacillus tekirae described in this invention is a suspension concentrate, wettable powder, or aqueous solution, and can be prepared according to existing technical methods.

[0035] Furthermore, the synergistic bactericide containing Bacillus tekirae also contains one or more of the following: stabilizer, wetting and dispersing agent, ultraviolet protectant, antifreeze agent, dispersant, wetting agent, thickener, defoamer, spore germination agent, desiccant, solubilizer, preservative, pH adjuster, and filler.

[0036] In some embodiments, the synergistic bactericide containing Bacillus tekirae comprises, by weight: 20-50 parts of the above-mentioned bactericidal composition; and further comprises any one or more of the following adjuvants: 5-20 parts of stabilizer, 5-10 parts of wetting and dispersing agent, 0.1-5 parts of UV protectant, 4-5 parts of antifreeze agent, 5-6 parts of dispersant, 1-6 parts of wetting agent, 0.5-2 parts of thickener, 0.5-2 parts of defoamer, 1-2 parts of spore germination agent, 3-7 parts of desiccant, 3-5 parts of cosolvent, 0.2-0.5 parts of preservative, 0.01-0.02 parts of pH adjuster, and 30-50 parts of filler.

[0037] For suspending agents, the following additives can be used:

[0038] The stabilizer is selected from ethyl oxalate, butyl oxalate, methyl vanillate or ethyl vanillate.

[0039] UV protectants include one or more of the following: dextrin, carboxymethyl cellulose, urea, starch, Congo red, ascorbic acid (VC), xanthan gum, and pure milk.

[0040] The wetting and dispersing agent is selected from one or more of the following: EO-PO block copolymers, phosphate ester anionic compounds, sulfate anionic compounds, aryl polyoxyalkyl sulfates, polycarboxylates, succinates, alkyl naphthalene sulfonates, alkylphenol polyoxyethylene ethers, lignin sulfonates, fatty alcohol polyoxyethylene ethers, sorbitan fatty acid ester polyoxyethylene ethers, isooctyl alcohol polyoxyethylene polyoxypropylene ethers, polyvinyl alcohol monooleate, and methacrylic acid grafted comb copolymers.

[0041] The thickener is selected from one or more of xanthan gum, bentonite, gelatin, gum arabic, polyvinyl acetate, polyvinylpyrrolidone, magnesium aluminum silicate, polyvinyl alcohol, polyethylene glycol, phenolic resin, shellac, and hydroxymethyl cellulose.

[0042] The defoamer is selected from one or more of SAG-1522, SAG-1572, magnesium aluminum silicate, silicones, C8-10 fatty alcohols, phosphate esters, C10-20 saturated fatty acids (such as decanoic acid) and amides;

[0043] The antifreeze is selected from one or more of ethanol, ethylene glycol, propylene glycol, glycerol or urea.

[0044] For wettable powders, the following adjuvants can be used:

[0045] UV protectant: one or a mixture of two of ascorbic acid (VC) and lecithin.

[0046] Spore germination agent: one or more of alanine, inosine, fructose, and serine.

[0047] Desiccant: one or a mixture of maltose, methylcellulose, sucrose, and mannitol.

[0048] The dispersant is one or a mixture of sodium lignosulfonate, sodium carboxymethyl cellulose, desaccharified sodium lignosulfonate, sulfite pulp waste liquor, alkylphenol polyoxyethylene ether, ethylene glycol monobutyl ether, polyoxyethyl alkyl ether, nonylphenol polyoxyethylene ether, sodium dodecylbenzene sulfonate, and sodium alkylnaphthalene sulfonic acid condensate.

[0049] The wetting agent is one or a mixture of Morwet EFW, tea saponin, sodium dibutylnaphthalene sulfonate (spreading powder BX), wetting agent wgwin D30, fatty alcohol polyoxyethylene ether, sodium dodecyl sulfonate, sodium dodecylbenzene sulfonate, and alkylphenol polyoxyethylene formaldehyde condensate sulfonate.

[0050] The filler is one or a mixture of silica, kaolin, light calcium carbonate, diatomaceous earth, light calcium carbonate, and bentonite.

[0051] For aqueous solutions, the following additives may be used:

[0052] The wetting agent is one or more of the following: alkylbenzene sulfonates, alkyl sulfonates, alkyl succinate sulfonates, alkyl naphthalene sulfonates, fatty amide taurines, phenethylphenol ether sulfonates, alkoxy polyoxyethylene ether sulfonates, alkyl phosphate esters, fatty acid polyoxyethylene ester phosphates, alkyl polyoxyethylene ether phosphates, fatty alcohol polyoxyethylene ethers, fatty acid polyoxyethylene ethers, castor oil ethylene oxide and its derivatives, sorbitan fatty acids and their ethylene oxide, alkylphenol polyoxyethylene ethers and their formaldehyde condensates, and alkyl naphthalene polyoxyethylene ethers and their formaldehyde condensates.

[0053] The co-solvent is one or more of methanol, acetonitrile, acetone, ethylene glycol, glycerol, N,N-dimethylformamide, N,N-dimethylacetamide, N-methylpyrrolidone, and dimethyl sulfoxide;

[0054] The preservative is one or more of formaldehyde, isothiazolinone, nifedipine methyl ester, potassium sorbate, and sodium benzoate;

[0055] The antifreeze is one or more of ethylene glycol, propylene glycol, glycerin, and urea;

[0056] The pH adjuster is one or more of sodium hydroxide, ammonium hydroxide, acetic acid, hydrochloric acid, and phosphoric acid.

[0057] Specifically, the pH value of the synergistic bactericide containing Bacillus tekirulatus is 6.5-7.5.

[0058] The present invention also provides the application of the above-mentioned bactericidal composition or the above-mentioned synergistic bactericide containing Bacillus tekirius in the prevention and control of plant diseases caused by Bacillus multifungus.

[0059] The plants include, but are not limited to, fruits, vegetables, soybeans, and flowers, such as tomatoes, strawberries, cucumbers, zucchini, soybeans, grapes, lettuce, romaine lettuce, cabbage, eggplants, peppers, green beans, bottle gourds, chrysanthemums, apples, and more than 200 other plants; cucumbers, soybeans, and eggplants are preferred.

[0060] The above-mentioned bactericidal composition or the above-mentioned synergistic bactericide containing Bacillus tekiria of the present invention has significant effects on the prevention and control of Cucumber Corynebacterium leaf spot, Soybean Corynebacterium leaf spot, Cowpea gray spot and Eggplant Corynebacterium leaf spot, with the control effect reaching more than 90%.

[0061] The method of using the synergistic fungicide containing Bacillus tekirae of the present invention includes: diluting the synergistic fungicide containing Bacillus tekirae and spraying it on the stems and leaves of the plant (e.g., cucumber, soybean, eggplant) to prevent and control plant diseases caused by Bacillus multiflora; preferably, the control is carried out during the growth period of the plant (e.g., cucumber, soybean, eggplant).

[0062] Generally, the synergistic bactericide containing Bacillus tekirae is used with an active ingredient content of at least 300 μg / mL; preferably 400 μg / mL-600 μg / mL.

[0063] Generally, the synergistic bactericide containing Bacillus tekiria is diluted with water before use.

[0064] The weight parts mentioned in this invention can be weight units known in the art such as μg, mg, g, kg, or multiples thereof, such as 1 / 10, 1 / 100, 10 times, 100 times, etc.

[0065] The advantages of this invention are as follows: By utilizing Bacillus tergentii 2_2a, which is safe for humans and animals and has an inhibitory effect on Cladosporium multiflorum, and combining it with low-toxicity chemical agents such as fluopyram, azoxystrobin, biguanide trioctylbenzene sulfonate, or catechin in a certain proportion, complementary advantages are achieved, the amount of chemical fungicides used is reduced, the occurrence and development of drug resistance are delayed, and the control effect is improved while improving the ecological environment and reducing the residue of chemical agents. Detailed Implementation

[0066] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention.

[0067] Experimental Example - Screening of Synergistic Combinations

[0068] Unless otherwise specified, the following concentrations of fluopyram, oxadiazon, biguanide trioctylbenzene sulfonate, and catechin are 95%, 97%, 90%, and 98%, respectively, provided by Hailier Pharmaceutical Group, Hebei Weiyuan Biochemical Co., Ltd., Nippon Soda Co., Ltd., and Shanghai Shidande Biotechnology Co., Ltd.

[0069] The accession number for Bacillus tequilensis 2_2a is CGMCC No. 19446.

[0070] Bacillus tequilensis 2_2a fermentation broth (total viable count 8×10⁻⁶) 9 The CFU / g was provided by the Institute of Plant Protection, Hebei Academy of Agricultural and Forestry Sciences.

[0071] The tested pathogenic bacteria strains were *Corynebacterium cucumeris* (cucumber leaf spot pathogen), *Corynebacterium spp.* (soybean leaf spot pathogen), *Corynebacterium spp.* (cowpea leaf spot pathogen), and *Corynebacterium spp.* (eggplant leaf spot pathogen), purchased from Ningbo Mingzhou Biotechnology Co., Ltd., with corresponding numbers BMZ075695, B237717, BMZ096647, and BMZ075660. They were pre-cultured for 5 days at 27℃±1℃ using PDA medium (200g potato, 20g glucose, 10g agar, 1000mL distilled water) for later use.

[0072] The fermentation broth of *Bacillus tekiria* 2_2a was diluted with sterile water and mixed with PDA medium at a volume ratio of 1:9 to prepare *Bacillus tekiria*-containing plates with concentrations of 5 μg / mL, 1 μg / mL, 0.5 μg / mL, 0.1 μg / mL, 0.05 μg / mL, and 0.025 μg / mL. 95% fluopyram, 97% azoxystrobin, 90% biguanide trioctylbenzene sulfonate, and 98% catechin technical grade were dissolved in an appropriate amount of acetone to prepare stock solutions with a concentration of 1000 μg / mL. These stock solutions were then serially diluted with sterile water and mixed with PDA medium at a volume ratio of 1:9 to prepare *Bacillus tekiria*-containing plates with concentrations of 5 μg / mL, 1 μg / mL, 0.5 μg / mL, 0.1 μg / mL, 0.05 μg / mL, and 0.025 μg / mL. Simultaneously, different ratios of 1:99, 1:50, 1:25, 1:10, 1:4, 1:2, 1:1, 2:1, 4:1, 10:1, 25:1, 50:1, and 99:1 were combined to prepare drug-containing plates with concentrations of 5 μg / mL, 1 μg / mL, 0.5 μg / mL, 0.1 μg / mL, 0.05 μg / mL, and 0.025 μg / mL. Mycelial cakes (5 mm in diameter) of *Corynebacterium cucumeris* (cucumber leaf spot pathogen), *Corynebacterium spp.* (soybean leaf spot pathogen), *Corynebacterium glutamicum* (cowpea leaf spot pathogen), and *Corynebacterium spp.* (eggplant leaf spot pathogen), which had been pre-cultured for 5 days, were inoculated onto the drug-containing plates with the mycelial side down. An equal volume of sterile water was added as a blank control (CK). Each treatment was replicated four times, and the plates were incubated at 27±1℃. When the control colonies had grown to 1 / 2-2 / 3 of the culture dish, the colony diameter of each treatment was measured using the cross-sectional method, and the effective inhibitory concentration (EC) was calculated. 50 .

[0073] The inhibition rate of each treatment was calculated using Excel. Based on the concentration and inhibition rate of each treatment, the EC was determined using DPSv6.55 data processing software. 50 (The treatment concentration at which pathogen growth is effectively inhibited by 50%, in μg / mL). The synergistic effect coefficients of different compound agents were calculated using the Wadley (1967) formula:

[0074]

[0075] SR=EC 50 (th) / EC 50 (ob)

[0076] In the above formula, a and b represent the percentages of two fungicides, fluopyram / oximetronidazole / biguanidine trioctylbenzene sulfonate / catechin (A) and Bacillus terrestris (B), respectively, in the mixture; EC 50 (A) and EC 50 (B) represents the actual observations of EC for A and B, respectively.50 Value, EC 50 (th) represents the theoretical EC value of a mixture of fungicides A and B in a:b ratio. 50 Value, EC 50 (ob) represents the actual EC value after mixing fungicides A and B in a:b ratio. 50 Values. SR>1.5 indicates synergistic effect; SR<0.5 indicates antagonistic effect; SR between 0.5 and 1.5 indicates additive effect.

[0077] The experimental results are as follows.

[0078] Table 1. Virulence of Bacillus tekiria and four fungicides against different pathogens.

[0079]

[0080] Table 2. Virulence of Bacillus tergentii and fluopyram compound against pathogens of different diseases.

[0081]

[0082]

[0083] As shown in Table 2, the synergistic effect of fluopyram combined with Bacillus tekirae 2_2a on Cucumber Corynebacterium leaf spot, Soybean Corynebacterium leaf spot, Cowpea Gray Spot, and Eggplant Corynebacterium leaf spot is greater than 1.5. The optimal weight ratios of fluopyram to Bacillus tekirae 2_2a are 1:10 and 1:4, respectively.

[0084] Table 3. Virulence of Bacillus terrestris and azoxystrobin compound against pathogens of different diseases.

[0085]

[0086]

[0087] As shown in Table 3, the synergistic effect of azoxystrobin combined with Bacillus tekirae 2_2a on *Corynebacterium cucumeris* leaf spot pathogen of cucumber, *Corynebacterium tekirae* leaf spot pathogen of soybean, *Corynebacterium citrinum* leaf spot pathogen of cowpea, and *Corynebacterium citrinum* leaf spot pathogen of eggplant is greater than 1.5; the optimal weight ratio of azoxystrobin to Bacillus tekirae 2_2a is 1:10 and 1:4, respectively.

[0088] Table 4. Virulence of Bacillus tergentii and biguanide trioctylbenzene sulfonate compound against pathogens of different diseases.

[0089]

[0090]

[0091] As shown in Table 4, the synergistic effect of biguanide trioctylbenzene sulfonate combined with Bacillus tekirae 2_2a on Cucumber Corynebacterium leaf spot, Soybean Corynebacterium leaf spot, Cowpea gray spot, and Eggplant Corynebacterium leaf spot is greater than 1.5; the optimal ratio of biguanide trioctylbenzene sulfonate to Bacillus tekirae 2_2a is 1:2 and 1:1, respectively.

[0092] Table 5. Virulence of catechin-Bacillus tekirica compound against pathogens of different diseases.

[0093]

[0094]

[0095] As shown in Table 5, the synergistic effect of catechin combined with Bacillus tekirae 2_2a on Cucumber Corynebacterium leaf spot, Soybean Corynebacterium leaf spot, Cowpea Gray Spot, and Eggplant Corynebacterium leaf spot is greater than 1.5; the optimal weight ratio of catechin to Bacillus tekirae 2_2a is 1:50 and 1:25, respectively.

[0096] Example: Preparation of the combination formulation and its field application and control effect

[0097] The optimal combination of formulations was used to prepare compound formulations for field control efficacy against Cucumber Corynebacterium leaf spot, Soybean Corynebacterium leaf spot, Cowpea gray spot, and Eggplant Corynebacterium leaf spot.

[0098] Example 1

[0099] This embodiment provides a compound fungicide containing Bacillus tekirae 2_2a and indoxazole, comprising the following components: 4 kg of 95% fluopyram, and lyophilized Bacillus tekirae 2_2a bacterial powder (total viable count 2.0 × 10⁻⁶). 10 The following ingredients were added to a mixing tank: 40 kg CFU / g (CFU / g), 10 kg ethyl oxalate (stabilizer), 0.1 kg β-cyclodextrin (UV protectant), 2 kg TERGITOL W-600 (wetting and dispersing agent), 5 kg DOWFAX D-865 (wetting and dispersing agent), 4 kg propylene glycol (antifreeze agent), 0.12 kg xanthan gum (thickener), 1.5 kg bentonite (thickener), 0.8 kg SAG-1522 (defoamer), and water to a final volume of 100 kg. The mixture was stirred and mixed using a high-shear disperser, and then milled using a sand mill to obtain the suspension.

[0100] Example 2

[0101] This embodiment provides a compound fungicide containing Bacillus tekirae 2_2a and indoxazole, comprising the following components: 6 kg of 95% fluopyram, and lyophilized Bacillus tekirae 2_2a bacterial powder (total viable count 2.0 × 10⁻⁶).10 The following ingredients were added to a mixing tank: 24 kg CFU / g (CFU / g), 15 kg butyl oxalate (stabilizer), 4 kg urea (UV protectant), 6 kg Morwet D-425 (wetting and dispersing agent), 2 kg Efhylan-324 (wetting and dispersing agent), 0.12 kg xanthan gum (thickening agent), 1.5 kg bentonite (thickening agent), and water to a final volume of 100 kg. The mixture was stirred and mixed using a high-shear disperser, and then milled using a sand mill to obtain the suspension.

[0102] Example 3

[0103] This embodiment provides a compound bactericide containing Bacillus tekirae 2_2a and azoxystrobin, comprising the following components: 4 kg of 97% azoxystrobin, and lyophilized Bacillus tekirae 2_2a bacterial powder (total viable count 2.0 × 10⁻⁶). 10 The following ingredients are added to a mixing tank: 40 kg CFU / g (CFU / g), 5 kg ethyl oxalate (stabilizer), 0.1 kg VC (UV protectant), 2 kg TERGITOL W-600 (wetting and dispersing agent), 4 kg Tensiofix 96DB08 (wetting and dispersing agent), 4 kg ethylene glycol (antifreeze agent), 0.08 kg xanthan gum (thickener), 1.5 kg magnesium aluminum silicate (defoamer), 0.4 kg BS-12 (thickener), and water to a final volume of 100 kg. The mixture is stirred and mixed using a high-shear disperser, and then milled using a sand mill to obtain the suspension.

[0104] Example 4

[0105] This embodiment provides a compound bactericide containing Bacillus tekirae 2_2a and azoxystrobin, comprising the following components: 6 kg of 97% azoxystrobin, and lyophilized Bacillus tekirae 2_2a bacterial powder (total viable count 2.0 × 10⁻⁶). 10 The following ingredients were added to a mixing tank: 24 kg CFU / g (CFU / g), 12 kg butyl oxalate (stabilizer), 4 kg Soprophor FD (wetting and dispersing agent), 2 kg YUS-D3020 (wetting and dispersing agent), 4 kg ethylene glycol (antifreeze agent), 0.12 kg xanthan gum (thickener), 1.5 kg magnesium aluminum silicate (defoamer), 0.2 kg SAG-1572 (defoamer), and water to a final volume of 100 kg. The mixture was stirred and mixed using a high-shear disperser, and then milled using a sand mill to obtain the suspension.

[0106] Example 5

[0107] This embodiment provides a compound bactericide containing Bacillus tekirae 2_2a and biguanide trioctylbenzene sulfonate, comprising the following components: 10 kg of 90% biguanide trioctylbenzene sulfonate, and lyophilized Bacillus tekirae 2_2a bacterial powder (total viable count 2.0 × 10⁻⁶).10 The following ingredients were added: 20 kg CFU / g, 1 kg ascorbic acid (UV protectant), 0.5 kg lecithin (UV protectant), 5 kg sodium lignosulfonate (dispersant), 1 kg ethylene glycol monobutyl ether (dispersant), 6 kg Morwet EFW (wetting agent), 0.5 kg L-alanine (spore germination agent), 0.1 kg inosine (spore germination agent), 0.5 kg fructose (spore germination agent), 2 kg maltose (desiccant), 1 kg methylcellulose (filler), 10 kg silica (filler), and light calcium carbonate to a final volume of 100 kg. The mixture was stirred according to the specified proportions and then subjected to a first-stage air jet milling process to achieve a particle size of 80 mesh. After stirring, the mixture was further subjected to a second-stage air jet milling process to achieve a particle size of 325 mesh, thus obtaining a wettable powder.

[0108] Example 6

[0109] This embodiment provides a compound bactericide containing Bacillus tekirae 2_2a and biguanide trioctylbenzene sulfonate, comprising the following components: 15 kg of 90% biguanide trioctylbenzene sulfonate, and lyophilized Bacillus tekirae 2_2a bacterial powder (total viable count 2.0 × 10⁻⁶). 10 The following ingredients were used: 15 kg CFU / g UV protectant (ascorbic acid 1 kg), 0.8 kg sodium carboxymethyl cellulose dispersant, 3 kg tea saponin wetting agent, 2 kg Morwet D-425 wetting agent, 0.3 kg inosine spore germination agent, 0.8 kg serine spore germination agent, 6 kg sucrose desiccant, 0.8 kg mannitol desiccant, 10 kg diatomaceous earth filler, and light calcium carbonate filler to a total of 100 kg. The various components were mixed and stirred according to the formula, and then subjected to a first-stage air jet milling process to achieve a particle size of 80 mesh. After mixing and stirring, the mixture was further subjected to a second-stage air jet milling process to achieve a particle size of 325 mesh, thus obtaining a wettable powder.

[0110] Example 7

[0111] This embodiment provides a compound bactericide containing Bacillus tekirae 2_2a and catechins, comprising the following components: 0.5 kg of 98% catechins, and lyophilized Bacillus tekirae 2_2a bacterial powder (total viable count 2.0 × 10⁻⁶). 10 The following ingredients were added: 25 kg CFU / g (CFU / g), wetting agent: 1 kg fatty alcohol polyoxyethylene ether, cosolvent: 3 kg N-methylpyrrolidone, antifreeze: 5 kg ethylene glycol, preservative: 0.5 kg isothiazolinone, pH adjuster: 0.01 kg hydrochloric acid, and deionized water to a final volume of 100 kg. The mixture was thoroughly stirred and then filtered to obtain the aqueous solution.

[0112] Example 8

[0113] This embodiment provides a compound bactericide containing Bacillus tekirae 2_2a and catechins, comprising the following components: 1 kg of 98% catechins, and lyophilized Bacillus tekirae 2_2a bacterial powder (total viable count 2.0 × 10⁻⁶). 10 25 kg of CFU / g, wetting agent: fatty amide taurine 2 kg, solubilizer: glycerol 5 kg, preservative: potassium sorbate 0.5 kg, pH adjuster: phosphoric acid 0.02 kg, and deionized water to 100 kg. The components are thoroughly mixed and filtered to obtain the aqueous solution.

[0114] Field verification

[0115] Comparative agents sourced from: 42% fluopyram suspension concentrate, FMC Corporation, USA; 25% azoxystrobin suspension concentrate, Shandong Binhai Hansheng Biotechnology Co., Ltd.; 40% biguanide trioctylbenzene sulfonate wettable powder, Nippon Soda Co., Ltd.; and 1.1% catechin wettable powder, Pingshan Forestry Pharmaceutical Factory, Heilongjiang Province.

[0116] Cucumber leaf spot disease

[0117] Two replicate trials were conducted in December 2022 and April 2023 in a cucumber greenhouse in Dingxing County, Hebei Province, using the cucumber variety Qiande No. 2, to verify the field efficacy of different fungicide combinations against cucumber scab leaf spot. Large-scale field trials were also conducted when cucumber scab leaf spot occurred sporadically and naturally in the field. Each plot was 3m². 2 (Plant spacing 25cm*60cm), approximately 20 cucumber plants were planted, with 4 replicates. The pesticide or water was sprayed at a uniform rate using a MATABI-16 backpack manual sprayer (pressure: 0.3MPa, nozzle: conical), with a water flow rate of 900L / ha. Each treatment consisted of 4 plots, all of which were completely randomized. The pesticide was applied three times during the experiment, with a 7-day interval between applications. Seven days after the last application, the disease index of all plants was assessed, and the disease index and control efficacy were calculated. Disease severity is assessed by classifying the disease into grades 0 to 10 based on the percentage of lesion area: grade 0 represents no disease, and grades 1-10 represent the percentage of lesion area to leaf area as follows: <10%, 10%-20%, 20%-30%, 30%-40%, 40%-50%, 50%-60%, 60%-70%, 70%-80%, 80%-90%, and >90% (Ishii et al., 2007). Disease index = ∑[(number of diseased leaves per disease grade × disease grade) / (total number of leaves × 10)] × 100, control efficacy (%) = [(disease index of clean water control group - disease index of treatment group) / disease index of clean water control group] × 100.

[0118]

[0119]

[0120] The results showed that the compound fungicide in the embodiments of the present invention had a control effect of more than 90% on cucumber leaf spot disease caused by Corynebacterium tumefaciens, which was significantly better than the control agent.

[0121] Soybean leaf spot disease

[0122] Two replicate trials were conducted in July 2022 and July 2023 in soybean fields in Gaoyang County, Hebei Province, using the soybean variety Jidou No. 1, to verify the field efficacy of different fungicide combinations against soybean causal leaf spot. Large-scale field trials were conducted when soybean causal leaf spot occurred spontaneously and sporadically in the field. Each plot was 2.4 m². 2 (Plant spacing 30cm*40cm), approximately 20 soybean plants were planted. A MATABI-16 backpack manual sprayer (pressure: 0.3MPa, nozzle: conical) was used to spray the pesticide or water at a uniform rate, with a water volume of 900L / ha. Four plots were divided into each treatment, and all plots were completely randomized. Two applications were made during the experiment, with a 7-day interval between applications. Seven days after the last application, the disease index of all plants was assessed, and the disease index and control efficacy were calculated. Disease grading criteria: Grade 0: No disease; Grade 1: Lesions covering less than 5% of the total leaf area; Grade 3: Lesions covering 6%-25% of the total leaf area; Grade 5: Lesions covering 26%-50% of the total leaf area; Grade 7: Lesions covering 51%-75% of the total leaf area; Grade 9: Lesions covering more than 76% of the total leaf area. Disease index = ∑[(number of diseased leaves per disease level × disease level) / (total number of leaves × 9)] × 100, control efficacy (%) = [(disease index of the clean water control group - disease index of the treatment group) / disease index of the clean water control group] × 100.

[0123]

[0124]

[0125] The results showed that the compound fungicide in the embodiments of the present invention had a control effect of more than 90% on soybean leaf spot disease caused by Corynebacterium tumefaciens, which was significantly better than the control agent.

[0126] Cowpea gray spot disease

[0127] Two replicate trials were conducted in June 2022 and June 2023 in cowpea fields in Dingxing County, Hebei Province, using the white cowpea variety, to verify the field efficacy of different fungicide combinations against cowpea gray leaf spot. Large-scale field trials were also conducted when cowpea gray leaf spot occurred spontaneously and sporadically in the field. Each plot was 3.9 m². 2(Plant spacing 30cm*65cm), approximately 20 cowpea plants were planted. A MATABI-16 backpack manual sprayer (pressure: 0.3MPa, nozzle: conical) was used to spray the pesticide or water at a uniform rate, with a water volume of 900L / ha. Four plots were divided into each treatment, and all plots were completely randomized. The pesticide was applied three times during the experiment, with a 7-day interval between applications. Seven days after the last application, the disease index of all plants was assessed, and the disease index and control efficacy were calculated. Disease severity is assessed by classifying the disease into grades 0 to 10 based on the percentage of lesion area: grade 0 represents no disease, and grades 1-10 represent the percentage of lesion area to leaf area as follows: <10%, 10%-20%, 20%-30%, 30%-40%, 40%-50%, 50%-60%, 60%-70%, 70%-80%, 80%-90%, and >90% (Ishii et al., 2007). Disease index = ∑[(number of diseased leaves per disease grade × disease grade) / (total number of leaves × 10)] × 100, control efficacy (%) = [(disease index of the clean water control group - disease index of the treatment group) / disease index of the clean water control group] × 100.

[0128]

[0129]

[0130] The results showed that the compound fungicide in the embodiments of the present invention had a control effect of more than 90% on cowpea gray spot disease, which was significantly better than the control agent.

[0131] Control efficacy against eggplant leaf spot disease caused by Cladosporium wilt:

[0132] The experiment was repeated twice, in February and August 2022. Field trials were conducted in an eggplant greenhouse in Dingxing County, Hebei Province, using the variety Qiezha 2. Eggplant seeds were germinated before sowing, and efficacy was tested when the eggplants reached the three-leaf stage. The experiment included three treatments, each with three replicates, with 20 seedlings per replicate and a plot area of ​​4.2 m². 2 (Plant spacing 35cm*60cm). The eggplant leaf spot pathogen *Cladosporium* was cultured on PDA plates at 25℃ in the dark for 10 days. Mycelia were scraped off with a sterile scalpel, and the mixture was cultured under a BLB (black light blue) lamp at 25℃ for 3 days to produce conidia. Spores were collected by brushing and dissolved in sterile water, diluting the spore concentration to 1×10⁻⁶. 4 pcs·mL -1Prepare for later use. Spray eggplant plants evenly with a suspension of pathogenic fungal spores. 72 hours later, spray the leaves with different fungicide combinations and single agents according to the experimental concentrations in the table; use sterile water as a control treatment. Apply the fungicide or water at a uniform rate using a MATABI-16 backpack manual sprayer (pressure: 0.3 MPa, nozzle: conical), with a water volume of 900 L / ha. After the control group has fully developed disease, count the disease index of all plants in each treatment and calculate the control efficacy. Disease severity is assessed by classifying the disease into grades 0 to 10 based on the percentage of lesion area: grade 0 represents no disease, and grades 1-10 represent the percentage of lesion area to leaf area as follows: <10%, 10%-20%, 20%-30%, 30%-40%, 40%-50%, 50%-60%, 60%-70%, 70%-80%, 80%-90%, and >90% (Ishii et al., 2007). Disease index = ∑[(number of diseased leaves per disease grade × disease grade) / (total number of leaves × 10)] × 100, control efficacy (%) = [(disease index of the clean water control group - disease index of the treatment group) / disease index of the clean water control group] × 100.

[0133]

[0134]

[0135] The results showed that the compound fungicide in the embodiments of the present invention had a control effect of more than 90% on eggplant leaf spot disease caused by Corynebacterium tumefaciens, which was significantly better than the control agent.

[0136] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A bactericidal composition, characterized in that, Its active ingredients include: Bacillus equilensis 2_2a and a fungicide; the preservation number of Bacillus equilensis 2_2a is CGMCC No. 19446; the fungicide is selected from any one of fluopyram, azoxystrobin, biguanide trioctylbenzene sulfonate or catechin; In the bactericidal composition, the weight ratio of the bactericide to the *Bacillus tekirae* 2_2a is (1-99):(99-1); the total viable count of the *Bacillus tekirae* 2_2a is (7×10⁻⁶). 9 -2.4×10 10 ) CFU / g.

2. The bactericidal composition according to claim 1, characterized in that, The *Bacillus tekirii* was used as its fermentation broth; the total viable count of the fermentation broth was (7 × 10⁻⁶). 9 -1×10 10 ) CFU / g.

3. The bactericidal composition according to claim 2, characterized in that, The total number of viable bacteria in the fermentation broth was 8 × 10⁻⁶. 9 CFU / g.

4. The bactericidal composition according to claim 1, characterized in that, The *Bacillus tekirae* is its lyophilized powder; the total viable count of the lyophilized powder is 2.0 × 10⁻⁶. 10 CFU / g.

5. The bactericidal composition according to any one of claims 1-4, characterized in that, The weight ratio of the bactericide to the Bacillus tekiria 2_2a is (1-50):(50-1).

6. The bactericidal composition according to claim 5, characterized in that, The weight ratio of the bactericide to the Bacillus tekirae 2_2a is 1:(1-50).

7. The bactericidal composition according to claim 6, characterized in that, The weight ratio of the bactericide to the Bacillus tekirae 2_2a is 1:2, 1:4, 1:10, 1:25 or 1:

50.

8. A synergistic bactericide containing Bacillus tektii, characterized in that, The bactericidal composition includes any one of claims 1-7.

9. The synergistic bactericide containing Bacillus tekiria according to claim 8, characterized in that, The synergistic fungicide containing Bacillus tekiria also contains adjuvants or excipients that are applicable to pesticides.

10. The synergistic bactericide containing Bacillus tekiria according to claim 9, characterized in that, The synergistic bactericide containing Bacillus tekiria is a suspension concentrate, wettable powder, or aqueous solution.

11. The synergistic bactericide containing Bacillus tekiria according to claim 8, characterized in that, The synergistic bactericide containing Bacillus tekirae comprises, by weight: 20-50 parts of the bactericidal composition; and further comprises any one or more of the following adjuvants: 5-20 parts of stabilizer, 5-10 parts of wetting and dispersing agent, 0.1-5 parts of UV protectant, 4-5 parts of antifreeze agent, 5-6 parts of dispersant, 1-6 parts of wetting agent, 0.5-2 parts of thickener, 0.5-2 parts of defoamer, 1-2 parts of spore germination agent, 3-7 parts of desiccant, 3-5 parts of cosolvent, 0.2-0.5 parts of preservative, 0.01-0.02 parts of pH adjuster, and 30-50 parts of filler.

12. The use of the bactericidal composition according to any one of claims 1-7 or the synergistic bactericide containing Bacillus tekirii according to any one of claims 8-11 in the prevention and control of plant diseases caused by Bacillus multifungus.

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