Microbial source fungicidal composition for preventing and treating fire blight of pear and application thereof

Abstract

The application discloses a microbial source bactericidal composition containing Leuconostoc mesenteroides WZ-44 and kasugamycin and application thereof. The bactericidal composition takes Leuconostoc mesenteroides WZ-44 fermentation liquor (or fermentation sterile filtrate) and kasugamycin as main effective components, wherein the mass ratio of the Leuconostoc mesenteroides WZ-44 fermentation liquor (or fermentation sterile filtrate) to the kasugamycin is 4999:1-99:1, and is further preferably 1499:1-499:1. The bactericidal composition shows a synergistic effect within the above-mentioned ratio range, overcomes and delays drug resistance of pathogenic bacteria, has a good prevention and control effect on pear fire blight, a long effective period and low application cost, is obviously superior to a single agent in the prevention and control effect, and greatly reduces the use amount of the kasugamycin.

Description

Technical Field

[0001] This invention belongs to the field of biological pesticide technology, specifically relating to a bactericidal composition with Leuconostoc mesenteroides WZ-44 fermentation broth and kasugamycin as the main active ingredients and its application. Background Technology

[0002] Pear fire blight (Erwinia amylovora) is a quarantine bacterial disease affecting Rosaceae fruit trees such as pear, apple, crabapple, and hawthorn. This disease primarily damages flowers, fruits, leaves, young shoots, and branches. It has occurred in pear and apple producing areas in Xinjiang and in some pear orchards in the Hexi Corridor of Gansu, and there is a risk of it spreading to major pear and apple producing areas in my country. Pear fire blight not only significantly reduces fruit yield and quality but also causes tree decline, tree death, and even orchard destruction. Control of pear fire blight mainly relies on chemical agents and agricultural antibiotics, such as kasugamycin, thiamethoxam, thiamethoxam, and copper hydroxide. However, the long-term and excessive use of these pesticides has led to increased resistance in pathogens, high costs, and the risk of phytotoxicity when applied during flowering. For already infected branches, spraying is ineffective; control is mainly achieved through timely pruning and scraping with pesticides, which is labor-intensive, time-consuming, and ineffective, posing a serious threat to fruit safety and the orchard's ecological environment.

[0003] In recent years, microbial pesticides, with their advantages of safety, no residue, and environmental friendliness, have developed rapidly and have become a key focus of international green pesticide development. Developing pesticides that can improve antagonistic activity against target pathogens and disease control efficacy, reduce the use of chemical pesticides, improve fruit quality and economic benefits, and improve the orchard ecological environment is of great significance. Summary of the Invention

[0004] In view of the above-mentioned technical problems, one of the objectives of the present invention is to provide a microbial bactericidal composition and its application in the prevention and control of pear fire blight.

[0005] The objective of this invention is achieved through the following technical measures:

[0006] A bactericidal composition comprising Leuconostoc mesenteroides WZ-44 fermentation broth and kasugamycin, wherein the mass ratio of Leuconostoc mesenteroides WZ-44 fermentation broth to kasugamycin is 4999:1 to 99:1.

[0007] The bactericidal composition of the present invention preferably has a mass ratio of Leuconostoc mesenteroides WZ-44 to kasugamycin within any range of 1500:1 to 450:1, including but not limited to any range of values ​​such as 1499, 1399, 1299, 1199, 1100, 1099, 999, 900, 899, 799, 699, 599, 550, 499, and 450, including but not limited to 1499:1 to 499:1, 1399:1 to 499:1, 1299:1 to 499:1, 1199:1 to 499:1, 1099:1 to 499:1, 999:1 to 499:1, 900:1 to 1100:1, or 450:1 to 550:1. The inventors discovered that within this range, the bactericide composition not only has a better synergistic effect, but also requires a lower dosage and has a longer duration of action; further preferred ratios are 999:1 and 499:1.

[0008] The *Leuconostoc mesenteroides* WZ-44 fermentation broth of this invention can be the bacterial broth after fermentation of *Leuconostoc mesenteroides* WZ-44 or the sterile filtrate after filtration of the fermented bacterial broth. The pH of the *Leuconostoc mesenteroides* WZ-44 fermentation broth is 3.8–4.8. The inventors have found that when the pH of the fermentation broth meets this range, a synergistic effect can be guaranteed when it is combined with kasugamycin in the proportions described in this invention. For example, the pH can be any value from 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, or any range of these values, including but not limited to 3.9–4.5 and 3.9–4.2. In one specific example, the pH is 4.1.

[0009] The bacterial cell concentration in the fermentation solution of Leuconostoc mesenteroides WZ-44 described in this invention is (1×10⁻⁶). 9 ~10×10 9 CFU / mL, preferably 2.5 × 10⁻⁶. 9 ~4.0×10 9 When the bacterial concentration is within this range (CFU / mL), the pH of the fermentation broth formed by the bacterial solution or the sterile filtrate after filtration is 3.8 to 4.8. At this point, when combined with kasugamycin in the proportion described in this invention, a synergistic effect can be guaranteed.

[0010] The fermented Leuconostoc mesenteroides WZ-44 culture medium of this invention can be prepared according to conventional methods in the art. In a specific example, Leuconostoc mesenteroides WZ-44 is inoculated into MRS liquid culture medium at a volume ratio of 1% to 10%, with the liquid volume being 20% ​​to 80% of the container volume. Fermentation is carried out statically at 20 to 35°C, with shaking every 4 to 12 hours, for a fermentation time of 16 to 48 hours. The pH of the fermentation broth is measured using a pH meter to be 3.8 to 4.8, more preferably 3.9 to 4.2, thus obtaining the fermented Leuconostoc mesenteroides WZ-44 culture medium. Preferably, the bacterial concentration in the fermented Leuconostoc mesenteroides WZ-44 culture medium is (1×10⁻⁶)⁻¹. 9 ~10×10 9 CFU / mL, preferably 2.5 × 10⁻⁶. 9 ~4.0×10 9 CFU / mL.

[0011] In a more specific example, the preparation method of the fermented broth of Leuconostoc mesenteroides WZ-44 is as follows: Leuconostoc mesenteroides WZ-44 seed culture is inoculated into MRS liquid medium at a volume ratio of 1%, filling the container to 50% of its volume. The medium is allowed to ferment statically at 28°C, shaken once every 6–12 hours, for a fermentation time of 35–37 hours. The fermentation broth is stopped when the pH reaches 4.1 using a pH meter, thus obtaining the fermented broth of Leuconostoc mesenteroides WZ-44. The fermented broth of Leuconostoc mesenteroides WZ-44 is centrifuged at 6000 rpm for 10 min, and the supernatant is collected and filtered through a 0.22 μm bacterial filter to obtain a sterile filtrate.

[0012] The preparation of the *Leuconostoc mesenteroides* WZ-44 seed culture of this invention can be carried out according to conventional methods in the art, and the preparation process does not affect the results. For example, it may include the following steps:

[0013] A. Microbial culture

[0014] (1) Activation culture: Take the glycerol-preserved strain of Leuconostoc mesenteroides WZ-44, streak it onto a fresh MRS solid plate, and incubate it at 25-32℃ for 24-36h.

[0015] (2) Seed culture preparation: Use a sterile toothpick to pick a single colony and inoculate it into MRS liquid culture, and incubate it at 25-32℃ for 16-24h.

[0016] A second objective of this invention is to provide a compound fungicide formulation, which includes the fungicidal composition described in this invention and excipients acceptable in the field of pesticide formulations. The formulation can be prepared in any commonly used agricultural formulation, such as, but not limited to, soluble concentrates, suspension concentrates, water-in-oil emulsions, or suspension concentrates. The excipients can be commonly used agricultural excipients, such as, but not limited to, at least one of wetting agents, dispersants, spreading agents, stabilizers, penetrants, thickeners, antifreeze agents, defoamers, fillers, or solvents.

[0017] The compound bactericide formulation of the present invention can be prepared according to conventional methods in the art. In some specific examples, the mass content of Leuconostoc mesenteroides WZ-44 and kasugamycin in the compound bactericide formulation of the present invention is preferably 5% to 99%, more preferably 80% to 98.5%, and most preferably 90% to 98.5%.

[0018] The excipients described in this invention can be commonly used adjuvants in the art. In some embodiments, the total mass of the adjuvants is 1.0% to 5.0% (m / m). The adjuvants can be one or two to three of the following: glycerin, agricultural organosilicon, 98B, etc. This is only an example of one embodiment, but it is not limited to these adjuvant addition methods. Other schemes that can form a fungicide composition are also possible, and can be formulated according to conventional methods in the art.

[0019] The MRS liquid or solid culture medium used in this invention can be prepared according to conventional methods in the art, and minor differences in the culture medium formulation have no significant impact on the effect.

[0020] In some embodiments of the present invention, the MRS liquid culture medium consists of: 10g peptone, 10g beef extract, 5g yeast extract, 2g dipotassium hydrogen phosphate, 2g diammonium citrate, 5g sodium acetate, 0.58g MgSO4·7H2O, 0.25g MnSO4·4H2O, 1mL Tween 80, diluted with water to a final volume of 900mL, pH 6.8–7.0, sterilized at 121°C for 15min, and then 100mL of filtered sterilized 20% glucose solution is added to the sterilized culture medium and shaken to mix.

[0021] In some embodiments of the present invention, the MRS solid culture medium consists of: 10g peptone, 10g beef extract, 5g yeast extract, 2g dipotassium hydrogen phosphate, 2g diammonium citrate, 5g sodium acetate, 0.58g MgSO4·7H2O, 0.25g MnSO4·4H2O, 1mL Tween 80, and 16g agar powder. Water is added to bring the volume to 900mL, pH 6.8–7.0, and the medium is sterilized at 121°C for 15 minutes. Then, 100mL of filtered and sterilized 20% glucose solution is added to the sterilized liquid culture medium, and the mixture is shaken to mix.

[0022] The third objective of this invention is to provide the application of the above-mentioned microbial bactericidal composition or compound bactericidal formulation in the prevention and control of pear fire blight. The application can be carried out according to conventional methods in the art, for example, diluting the bactericidal composition 10 to 100 times, preferably 10 to 50 times, and spraying it evenly before or at the early stage of pear fire blight. The number of applications can be increased according to the fruit tree's growth stage and the development of the disease, and 2 to 3 consecutive applications can be made.

[0023] The microbial bactericidal composition provided by this invention is a novel bactericidal composition. The *Leuconostoc mesenteroides* WZ-44 and kasugamycin used have different mechanisms of action and good compatibility, exhibiting high efficacy in controlling pear fire blight and demonstrating a significant synergistic effect. This bactericidal composition has a simple preparation process, low cost, and convenient application, and is characterized by safety, high efficiency, and no pesticide residue. Attached Figure Description

[0024] Figure 1 The results of the tolerance test of Leuconostoc mesenteroides WZ-44 and Phytophthora pearis KL20-28 to kasugamycin are presented. Detailed Implementation

[0025] The present invention will be further described in detail below with reference to specific embodiments.

[0026] Unless otherwise specified, the Leuconostocmesenteroides WZ-44 involved in the following examples is the antagonistic microorganism disclosed in patent ZL202310135903.X, classified and named Leuconostocmesenteroides, and deposited on August 19, 2021, at the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, postcode 100101), with the culture accession number CGMCC No. 23157.

[0027] The pear fire blight pathogen KL20-28 strain used in this invention was isolated and identified by our laboratory from diseased samples of fragrant pears from Korla, Xinjiang, and is preserved in our laboratory, which promises to make it publicly available.

[0028] Unless otherwise specified, the techniques and methods used in the following embodiments are all conventional methods; unless otherwise specified, the experimental materials used in the following embodiments are all conventional reagents; unless otherwise specified, all percentages in the following embodiments are mass percentages.

[0029] The embodiments described below are merely preferred embodiments of the present invention and are used only to describe the present invention. They should not be construed as limiting the scope of the present invention. It should be noted that any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

[0030] Unless otherwise specified, the culture medium preparations used in the following examples are as follows:

[0031] The MRS liquid culture medium consists of: 10g peptone, 10g beef extract, 5g yeast extract, 2g dipotassium hydrogen phosphate, 2g diammonium citrate, 5g sodium acetate, 0.58g MgSO4·7H2O, 0.25g MnSO4·4H2O, and 1mL Tween 80. Water is added to bring the volume to 900mL, pH 6.8–7.0, and the medium is sterilized at 121℃ for 15 minutes. Then, 100mL of filtered and sterilized 20% glucose solution is added to the sterilized medium and the mixture is shaken to mix.

[0032] The MRS solid culture medium consists of: 10g peptone, 10g beef extract, 5g yeast extract, 2g dipotassium hydrogen phosphate, 2g diammonium citrate, 5g sodium acetate, 0.58g MgSO4·7H2O, 0.25g MnSO4·4H2O, 1mL Tween 80, and 16g agar powder. Water is added to bring the volume to 900mL, pH 6.8–7.0, and the medium is sterilized at 121℃ for 15 minutes. Then, 100mL of filtered and sterilized 20% glucose solution is added to the sterilized liquid culture medium and the mixture is shaken to mix.

[0033] The NA solid culture medium consists of: 5g peptone, 10g sucrose, 1g yeast extract, 3g beef extract, and 15g agar powder. Water is added to bring the volume to 1000mL, pH 6.8–7.2, and the medium is sterilized at 121℃ for 15min.

[0034] Example 1: Test of tolerance of Leuconostoc mesenteroides WZ-44 to kasugamycin

[0035] Kasugamycin is one of the main fungicides used in production to control pear fire blight. This experiment determined the tolerance of *Leuconostoc mesenteroides* WZ-44 to this fungicide in vitro, using *Leuconostoc mesenteroides* KL20-28 as a control. The specific method is as follows: First, kasugamycin was dissolved in water to prepare NA solid plates with final concentrations of 10 mg / L, 20 mg / L, 40 mg / L, 80 mg / L, 160 mg / L, and 320 mg / L. *Leuconostoc mesenteroides* WZ-44 culture medium (OD...) was then... 600 ≈1.0) and pear fire blight pathogen KL20-28 culture medium (OD 600≈1.0) was diluted 5-, 25-, and 125-fold, respectively. 2 μL of the stock solution of WZ-44 and KL20-28, as well as each serially diluted solution, was spotted onto NA solid plates containing kasugamycin bactericide and incubated at 28°C for 36 h. The results showed that compared to *Pyracantha fortuneana* KL20-28, *Leuconostoc mesenteriae* WZ-44 exhibited stronger tolerance to kasugamycin, and could still grow normally on NA solid plates at a concentration of 320 mg / L. Figure 1 As shown in the figure. This result indicates that Leuconostoc mesenteroides WZ-44 can be used in combination with kasugamycin, showing good compatibility.

[0036] Example 2: Preparation of Leuconostoc mesenteroides WZ-44 fermentation broth and sterile fermentation filtrate

[0037] (1) Activation culture: Take the glycerol-preserved strain of Leuconostoc mesenteroides WZ-44, streak it onto a fresh MRS solid plate, and incubate at 28℃ for 24-36 h;

[0038] (2) Seed culture preparation: A single colony was picked up with a sterile toothpick and inoculated into MRS liquid culture, and then incubated at 28°C for 16 hours.

[0039] (3) Preparation of biocontrol bacterial solution: The seed solution obtained in step (2) was inoculated into MRS liquid culture medium at a ratio of 1% (v / v), with the liquid volume being 50% of the container volume. The mixture was allowed to ferment statically at 28℃, shaken once every 6–12 hours, for a total fermentation time of 36 hours. The pH of the fermentation broth was measured to be 4.1 using a pH meter, and the bacterial cell concentration in the fermentation solution was (2.5 × 10⁻⁶) / 2.5 × 10⁻⁶. 9 ~4.0×10 9 CFU / mL.

[0040] (4) Preparation of sterile fermentation filtrate: Centrifuge the WZ-44 fermentation filtrate prepared in step (3) above at 6,000 rpm for 10 min, take the supernatant, and filter it using a 0.22 μm bacterial filter to obtain sterile fermentation filtrate.

[0041] Unless otherwise specified, the bacterial solutions or sterile filtrates used in the following examples are all prepared using the bacterial solutions or sterile filtrates of this example.

[0042] Example 3: Indoor bioassay of two microbial bactericidal components and their different combinations against *Pyrus pyriformis* causal agent.

[0043] OD used indoors 600 The absorbance method was used to determine the EC50 of WZ-44 fermentation supernatant, kasugamycin, and its different combinations in order to inhibit the growth of pear fire blight pathogen. 50 The co-toxicity coefficient (CTC) of the mixture was calculated using the co-toxicity coefficient calculation method to determine its synergistic effect. The specific calculation method is as follows:

[0044] Using a single agent from the mixture as the standard reagent (usually EC) 50 The lower-ranking agent), and other single-agent agents were used as test reagents for calculation:

[0045] Single-dose toxicity index = EC of standard drug 50 / Test reagent EC 50 ×100

[0046] Theoretical Toxicity Index (TTI) = Toxicity Index of Single Agent A × Proportion of Single Agent A in the Mixture + Toxicity Index of Single Agent B × Proportion of Single Agent B in the Mixture

[0047] Actual Toxicity Index (ATI) = EC of Standard Agent 50 EC value / mixture 50 value × 100

[0048] Cotoxicity coefficient (CTC) = Measured toxicity index / Theoretical toxicity index × 100

[0049] Co-toxicity coefficient classification: When CTC is greater than 120, the mixture has synergistic effect; when CTC is less than 80, it is antagonistic; when CTC is between 80 and 120, it is additive.

[0050] The *Leuconostoc mesenteroides* WZ-44 fermentation sterile filtrate prepared in Example 2 above was used for experiments, and the effective ingredient *Leuconostoc mesenteroides* WZ-44 fermentation sterile filtrate (A):kasugamycin (B) was tested at mass ratios of 1499:1, 999:1, 749:1, and 499:1. The dilution gradients for the 1499:1 ratio were set at 20x, 40x, 80x, 160x, 320x, 640x, and 1280x; the dilution gradients for the 999:1 and 749:1 ratios were set at 20x, 40x, 80x, 160x, 320x, 640x, 1280x, and 2560x; the dilution gradients for the 499:1 ratio were set at 20x, 40x, 80x, 160x, 320x, 640x, 1280x, and 2560x. The dilution gradients for WZ-44 fermentation sterile filtrate were set at 0x, 160x, 320x, 640x, 1280x, 2560x, and 5120x; the effective concentration gradients for kasugamycin were set at 1mg / L, 2mg / L, 4mg / L, 8mg / L, 16mg / L, 32mg / L, and 64mg / L. The EC50 of single agents and various combinations against *Pyrus pyrifolia* KL20-28 was determined. 50 The co-virulence coefficients of each formulation combination against pear fire blight pathogen were calculated. Serial dilutions were performed using sterile water.

[0051] Table 1. Indoor bioassay results of different ratios of Leuconostoc mesentery WZ-44 fermentation sterile filtrate and kasugamycin against Pear blight pathogens.

[0052]

[0053]

[0054] The above-mentioned indoor bioassay results show that the co-toxicity coefficients of the four combinations of WZ-44 fermentation sterile filtrate and kasugamycin set in this embodiment ranged from 230.2 to 452.2, significantly higher than 120, indicating a significant synergistic effect on the inhibitory effect against pear fire blight pathogen. The co-toxicity coefficient (CTC) of the WZ-44 fermentation sterile filtrate and kasugamycin in a 999:1 ratio was as high as 452.2, and the EC50 of the inhibitory effect on the growth of pear fire blight pathogen was also significant. 50 The concentration was only 327.5 mg / L, with the WZ-44 fermentation aseptic filtrate component concentration being 327.17 mg / L and the kasugamycin component effective concentration being 0.33 mg / L, which are the single-agent EC values ​​of WZ-44 fermentation aseptic filtrate. 50 (3061.8 mg / L) 10.7%, Kasugamycin single dose EC 50 11.5% of (2.8639 mg / L).

[0055] Example 4: Preparation method of sterile fermentation filtrate of Leuconostoc mesenteroides WZ-44 and bactericidal composition with kasugamycin

[0056] The sterile filtrate of Leuconostoc mesenteroides WZ-44 prepared in Example 2 above after fermentation for 36 hours was mixed with kasugamycin at a mass ratio of 999:1. 1.0% (m / m) of glycerol and 0.75% (m / m) of agricultural organosilicon were added to the bactericidal composition, and the mixture was stirred thoroughly to obtain the microbial bactericidal composition.

[0057] Example 5: Preparation method of the fermentation broth of Leuconostoc mesenteroides WZ-44 and the bactericidal composition of Kasugamycin

[0058] The fermentation broth of Leuconostoc mesenteroides WZ-44 prepared in Example 2 above, fermented for 36 hours, was mixed with kasugamycin at a mass ratio of 999:1. 1.0% (m / m) of glycerol and 0.75% (m / m) of agricultural organosilicon were added to the bactericidal composition, and the mixture was stirred thoroughly to obtain the microbial bactericidal composition.

[0059] Example 6: Preparation method of the fermentation broth of Leuconostoc mesenteroides WZ-44 and the bactericidal composition of Kasugamycin

[0060] The fermentation broth of Leuconostoc mesenteroides WZ-44 prepared in Example 2 above, fermented for 36 hours, was mixed with kasugamycin at a mass ratio of 499:1. Then, 5% (m / m) of 98B agricultural adjuvant was added, and the mixture was stirred thoroughly to obtain the microbial bactericidal composition.

[0061] Example 7: Field plot trial of the microbial bactericidal composition prepared in Example 4 above for the control of pear fire blight.

[0062] Test reagent: The microbial bactericidal composition prepared in Example 4 above.

[0063] Control reagent: Fermentation broth of Leuconostoc mesenteroides WZ-44 fermented for 36 h (prepared in Example 2) + 1.0% (m / m) glycerol + 0.75% (m / m) agricultural organosilicon

[0064] Sterile filtrate of Leuconostoc mesenteroides WZ-44 fermented for 36 h (prepared in Example 2) + 1.0% (m / m) glycerol + 0.75% (m / m) agricultural organosilicon

[0065] 2% Kasugamycin soluble solution (commercially available)

[0066] Blank control: Spray with clean water.

[0067] Experimental setup: The experiment consisted of 5 treatments, with 3 replicates per treatment and 10 mature pear trees per replicate. The specific treatment setup is as follows:

[0068] Treatment 1: A 20-fold dilution of the microbial bactericidal composition prepared in Example 4

[0069] Treatment 2: Fermentation broth of Leuconostoc mesenteroides WZ-44 prepared in Example 2, fermented for 36 hours, + 1.0% (m / m) glycerol + 0.75% (m / m) agricultural organosilicon biological agent diluted 20 times.

[0070] Treatment 3: Aseptic filtrate of Leuconostoc mesenteroides WZ-44 prepared in Example 2 after fermentation for 36 h + 1.0% (m / m) glycerol + 0.75% (m / m) agricultural organosilicon biological agent diluted 20 times.

[0071] Treatment 4: 2% Kasugamycin soluble solution diluted 400 times

[0072] Process 5: Blank control

[0073] Basic information about the pear orchard: Korla fragrant pears, 7 years old, with a spacing of 1.5m × 5m between trees.

[0074] Application method: Spray for the first time at the early flowering stage (about 5% flowering), spray for the second time when 80% of the flowers have fallen (after flowering), and spray for the third time about 20 days after flowering, for a total of 3 applications. Each treatment was set up with 3 replicates, with 10 trees per plot, 1.25 kg of pesticide solution sprayed per tree, and 12.5 kg of pesticide solution sprayed per plot.

[0075] Investigation methods: Investigate the number of diseased plants and the total number of plants in each plot, as well as the number of diseased branches and the severity of disease per plant. Investigate the baseline disease incidence before the first application (if there are no diseases or only sporadic cases, this investigation is unnecessary). Investigate the efficacy at the time of the last application and 15-20 days after the last application. The disease grading standards are as follows:

[0076] Grade 0: No disease occurs, and there are no ulcer spots on the entire trunk and branches;

[0077] Level 1: The number of branches affected by flower rot, fruit rot, and branch dieback accounts for less than 10% of the total number of branches surveyed, and there is no sterile purulent exudate on the branches and trunk;

[0078] Level 3: The number of branches affected by flower rot, fruit rot, and branch dieback accounts for 11% to 20% of the total number of branches surveyed; there are individual bacterial oozes on branches and trunks;

[0079] Level 5: The number of branches affected by flower rot, fruit rot, and branch dieback accounts for 21% to 35% of the total number of branches surveyed; a small amount of bacterial ooze is present on the branches and trunk.

[0080] Level 7: The number of branches affected by flower rot, fruit rot, and branch dieback accounts for 36% to 50% of the total number of branches surveyed; there is a lot of bacterial ooze on the branches and trunks;

[0081] Level 9: More than 51% of the branches affected by flower rot, fruit rot, and branch dieback are affected; a large amount of bacterial ooze is exuded from the branches and trunk.

[0082] The method for calculating the control efficacy is as follows: the incidence rate is calculated according to Formula 1, the disease index is calculated according to Formula 2, and the control effect is calculated according to Formula 3, as detailed below:

[0083] Formula 1: Disease incidence rate (%) = (Number of diseased plants / Total number of plants surveyed) × 100

[0084] Formula 2: Disease index = ∑(Number of diseased plants at each level × Relative level value) / (Total number of plants surveyed × 9) × 100

[0085] Formula 3: Control efficacy (%) = (Disease index in blank control area - Disease index in drug-treated area) / Disease index in blank control area × 100

[0086] The experimental data were statistically analyzed using Duncan's New Multiple Range (DMRT) method. Specific experimental results are shown in Table 2.

[0087] Table 2 Results of field plot trials of microbial bactericidal compositions for the control of pear fire blight.

[0088]

[0089] Note: abc indicates a significant difference (P<0.05), and AB indicates a highly significant difference (P<0.01).

[0090] The above field trial results showed that the treatment with a 20-fold dilution of the aseptic fermentation filtrate of Leuconostoc mesenteroides WZ-44 and a bactericidal composition of kasugamycin (999:1) had the highest control efficacy against pear fire blight. The control efficacy reached 97.2% after the third application and 91.7% 20 days after the third application, which was significantly better than the control efficacy of single treatment (P<0.05). It has a significant synergistic effect, a long-lasting effect, and high safety for pear flowers, young fruits, and trees, and has good application and development prospects.

[0091] Example 8: Field trial of the microbial bactericidal composition prepared in Examples 5 and 6 above for the control of pear fire blight.

[0092] Test reagent: The microbial bactericidal composition prepared in Example 5 above.

[0093] The microbial bactericidal composition prepared in Example 6 above

[0094] Control reagent: Fermentation broth of Leuconostoc mesenteroides WZ-44 fermented for 36 h (prepared in Example 2) + 5% (m / m) 98B adjuvant.

[0095] Sterile filtrate of Leuconostoc mesenteroides WZ-44 fermented for 36 h (prepared in Example 2) + 5% (m / m) 98B adjuvant.

[0096] 2% Kasugamycin soluble solution (commercially available)

[0097] Blank control: Spray with clean water.

[0098] Experimental treatment settings: The experiment consists of 6 treatments, and the specific treatment settings are as follows:

[0099] Treatment 1: A 30-fold dilution of the microbial bactericidal composition prepared in Example 5

[0100] Treatment 2: A 50-fold dilution of the microbial bactericidal composition prepared in Example 6

[0101] Treatment 3: Fermentation broth of Leuconostoc mesenteroides WZ-44 prepared in Example 2, fermented for 36 hours, plus 5% (m / m) 98B adjuvant biological agent diluted 20 times.

[0102] Treatment 4: Aseptic filtrate of Leuconostoc mesenteroides WZ-44 prepared in Example 2 after fermentation for 36 h + 5% (m / m) 98B adjuvant biological agent diluted 20 times.

[0103] Treatment 5: 400-fold dilution of 2% kasugamycin soluble solution

[0104] Process 6: Blank Control

[0105] Basic information about the pear orchard: Early-maturing pears, 8-year-old trees, with a spacing of 1.3m × 5m.

[0106] Application method: Spray for the first time at the early flowering stage (about 5% flowering), spray for the second time when about 85% of the flowers have fallen (after flowering), and spray for the third time about 20 days after the flowers have fallen, for a total of 3 applications. Each agent is used to treat 100 pear trees, with 20 pear trees as a blank control. No replicates are set. The amount of pesticide solution sprayed per tree is 1.25 kg.

[0107] Investigation methods: Investigate the number of diseased plants and the total number of plants in the region, the number of diseased branches per plant, and the severity of the disease. Investigate the baseline disease incidence before the first application (if there are no diseases or only sporadic cases, this investigation is unnecessary). Investigate the efficacy at the time of the last application and 15-20 days after the last application. The disease grading standards are as follows:

[0108] Grade 0: No disease occurs, and there are no ulcer spots on the entire trunk and branches;

[0109] Level 1: The number of branches affected by flower rot, fruit rot, and branch dieback accounts for less than 10% of the total number of branches surveyed, and there is no sterile purulent exudate on the branches and trunk;

[0110] Level 3: The number of branches affected by flower rot, fruit rot, and branch dieback accounts for 11% to 20% of the total number of branches surveyed; there are individual bacterial oozes on branches and trunks;

[0111] Level 5: The number of branches affected by flower rot, fruit rot, and branch dieback accounts for 21% to 35% of the total number of branches surveyed; a small amount of bacterial ooze is present on the branches and trunk.

[0112] Level 7: The number of branches affected by flower rot, fruit rot, and branch dieback accounts for 36% to 50% of the total number of branches surveyed; there is a lot of bacterial ooze on the branches and trunks;

[0113] Level 9: More than 51% of the branches affected by flower rot, fruit rot, and branch dieback are affected; a large amount of bacterial ooze is exuded from the branches and trunk.

[0114] The method for calculating the control efficacy is as follows: the incidence rate is calculated according to Formula 1, the disease index is calculated according to Formula 2, and the control effect is calculated according to Formula 3, as detailed below:

[0115] Formula 1: Disease incidence rate (%) = (Number of diseased plants / Total number of plants surveyed) × 100

[0116] Formula 2: Disease index = ∑(Number of diseased plants at each level × Relative level value) / (Total number of plants surveyed × 9) × 100

[0117] Formula 3: Control efficacy (%) = (Disease index in blank control area - Disease index in drug-treated area) / Disease index in blank control area × 100

[0118] The experimental data were statistically analyzed using Excel software. The specific experimental results are shown in Table 3.

[0119] Table 3. Results of field trials of microbial bactericidal compositions for the control of pear fire blight.

[0120]

[0121] The above-mentioned field efficacy trials showed that the combination of *Leuconostoc mesenteroides* WZ-44 fermentation broth and kasugamycin (treatment 1: 999:1, 30-fold dilution; treatment 2: 499:1, 50-fold dilution) achieved a control efficacy of 85.5%–89.7% against pear fire blight. This was superior to a 20-fold dilution of *Leuconostoc mesenteroides* WZ-44 fermentation broth (78.7%–81.8%), a 20-fold dilution of *Leuconostoc mesenteroides* WZ-44 fermentation sterile filtrate (69.0%–70.9%), and a control efficacy of 400-fold dilution of 2% kasugamycin soluble concentrate 18 days after the third application (79.4%). The treatment demonstrated a significant synergistic effect, a long-lasting effect, and high safety for pear blossoms, young fruit, and the tree itself, indicating promising application prospects.

[0122] The results of the above-mentioned indoor bioassay and field efficacy tests show that the combination of *Leuconostoc mesenteroides* WZ-44 fermentation sterile filtrate (or fermentation broth) and kasugamycin has a significant synergistic effect on the antagonistic activity against pear fire blight pathogens and the control efficacy against pear fire blight. The control effect is better than that of single-agent components, while significantly reducing the dosage of kasugamycin. It also has a long-lasting effect and good safety. It is a brand-new microbial bactericidal composition with great application value in pesticides.

Claims

1. A bactericidal composition containing *Leuconostoc mesenteroides* WZ-44 fermentation broth and kasugamycin, characterized in that, In this bactericidal composition, the mass ratio of *Leuconostoc mesenteroides* WZ-44 fermentation broth to kasugamycin is 1499:1 to 499:1; the *Leuconostoc mesenteroides* WZ-44 is classified as *Leuconostoc mesenteroides* (…). Leuconostoc mesenteroides The strain is preserved under the CGMCC No. 23157. The fermentation broth of Leuconostoc mesenteroides WZ-44 is the sterile filtrate obtained after filtration of the fermented bacterial broth. The pH of the fermentation broth of Leuconostoc mesenteroides WZ-44 is 3.8~4.

8. Specifically, the sterile filtrate is obtained by inoculating Leuconostoc mesenteroides WZ-44 seed culture into MRS liquid medium at a volume ratio of 1%~10%, filling the container to 20%~80% of its volume, allowing it to ferment statically at 20~35℃, shaking it once every 4~12 hours, and fermenting for 16~48 hours. The fermentation broth is measured with a pH meter until the target pH is reached to obtain the fermented bacterial broth of Leuconostoc mesenteroides WZ-44. After centrifugation and filtration, the sterile filtrate is obtained.

2. The bactericidal composition according to claim 1, characterized in that, The mass ratio of the fermentation broth of Leuconostoc mesenteroides WZ-44 to the bactericidal composition of kasugamycin is 999:1 or 499:

1.

3. The bactericidal composition according to claim 1 or 2, characterized in that, The pH of the fermentation broth of Leuconostoc mesenteroides WZ-44 was 3.9-4.

2.

4. The bactericidal composition according to claim 3, characterized in that, The pH of the fermentation broth of Leuconostoc mesenteroides WZ-44 was 4.

1.

5. The bactericidal composition according to claim 1, characterized in that, The method for preparing the sterile filtrate is as follows: Leuconostoc mesenteroides WZ-44 seed culture is inoculated into MRS liquid medium at a volume ratio of 1%, and the liquid volume is 50% of the container volume. It is allowed to ferment statically at 28 ℃, and shaken once every 6~12 h. The fermentation time is 35~37 h. The pH of the fermentation broth is measured with a pH meter until the target pH is reached to obtain the bacterial broth of Leuconostoc mesenteroides WZ-44 after fermentation. The broth is centrifuged at 6000 rpm for 10 min, the supernatant is collected, and the sterile filtrate is obtained by filtering it through a 0.22 µm bacterial filter.

6. A compound bactericide formulation, characterized in that, It contains the bactericidal composition according to any one of claims 1 to 5 and excipients acceptable in the field of pesticide formulations.

7. The compound bactericide formulation according to claim 6, characterized in that, The dosage form of the preparation is a soluble concentrate, an emulsion, or a suspension.

8. The compound bactericide formulation according to claim 6, characterized in that, The excipients are at least one of the following: wetting agent, dispersant, spreading agent, stabilizer, penetrant, thickener, antifreeze, defoamer, filler, or solvent.

9. The compound bactericide formulation according to any one of claims 6 to 8, characterized in that, The total mass content of Leuconostoc mesenteroides WZ-44 fermentation broth and kasugamycin in the compound bactericide formulation is 5%~99%.

10. The compound bactericide formulation according to claim 9, characterized in that, The total mass content of Leuconostoc mesenteroides WZ-44 fermentation broth and kasugamycin in the compound bactericide formulation is 80%~98.5%.

11. The compound bactericide formulation according to claim 10, characterized in that, The total mass content of Leuconostoc mesenteroides WZ-44 fermentation broth and kasugamycin in the compound bactericide formulation is 90%~98.5%.

12. The application of the bactericidal composition according to any one of claims 1 to 5 or the compound bactericidal formulation according to any one of claims 6 to 11 in the prevention and control of fire blight in pear trees.

13. The application according to claim 12, characterized in that, The specific application involves diluting the bactericidal composition 10 to 100 times and spraying it evenly before or at the early stage of pear blight. The number of applications can be increased according to the fruit tree's growth stage and the development of the disease, with 2 to 3 consecutive applications.

14. The application according to claim 13, characterized in that, The application specifically involves diluting the bactericidal composition by 10 to 50 times.

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