Compound agent for preventing and treating fire blight of pear, and preparation method and application thereof

Abstract

This invention relates to the field of biological control, and in particular to a compound agent for controlling pear fire blight, its preparation method, and its application. This invention provides a compound agent with Bacillus vesicularis FX1 fermentation broth and kasugamycin aqueous solution as active ingredients, which can effectively control pear fire blight. The compound agent, by combining Bacillus vesicularis FX1 fermentation broth and kasugamycin aqueous solution, exhibits a synergistic effect. It leverages the high efficiency and rapid bactericidal advantage of kasugamycin while increasing the colonization of Bacillus vesicularis FX1 on the tree surface, making it the dominant foliar flora and constructing a stable and balanced micro-ecosystem. This can reduce the use of chemical pesticides by 60%, leveraging the synergistic and complementary effects of bacteria and pesticides to improve disease control, achieving the goal of safe, efficient, and sustainable control of the occurrence, spread, and diffusion of the disease.

Description

Technical Field

[0001] This invention relates to the field of biological control, and in particular to a compound agent for controlling pear blight, its preparation method, and its application. Background Technology

[0002] Pear fire blight is a newly emerging and sudden major quarantine bacterial disease of fruit trees in my country, listed in the "List of Class I Crop Diseases and Pests" and the "List of Key Managed Invasive Alien Species." The pathogen of pear fire blight is *Erwinia amylovora*, one of the world's top ten plant pathogenic bacteria, capable of infecting more than 220 species of plants in over 40 genera of the Rosaceae family, seriously damaging pome fruit trees such as pears, apples, hawthorns, and crabapples. Pear fire blight was first discovered in apple trees in Ili Prefecture, Xinjiang Uygur Autonomous Region in 2016. In 2017, an outbreak occurred in Korla fragrant pears in Bayingolin Mongol Autonomous Prefecture, Xinjiang, and rapidly spread throughout the region. Currently, pear fire blight poses a significant threat and risk to the healthy development of the apple and pear industries nationwide.

[0003] Chemical control is an important means of preventing and controlling pear fire blight. my country has screened out several agents for the sudden outbreak of pear fire blight. Currently registered agents for the control of pear fire blight include: organic copper (thiabendazole copper), inorganic copper (copper hydroxide), antibiotics (kasugamycin, kasugamycin), and thiazoles (thiamethoxam zinc, thiamethoxam), etc. Indoor or field trials have confirmed that these registered agents have a certain degree of control effect against pear fire blight. However, overall, the types and varieties of pesticides specifically for the control of pear fire blight in production are still very limited. Long-term and frequent use of relatively single agents not only leads to accelerated drug resistance in pathogens, but also causes a series of problems such as increased pesticide residues, decreased fruit quality, and aggravated environmental pollution.

[0004] In-depth research into the microecology and infection biology of *Pseudomonas fluorescens*, the causal agent of pear fire blight, has spurred progress in the biocontrol of pear fire blight using beneficial microorganisms. Commercially available microbial agents developed abroad, such as *Pseudomonas fluorescens* A506, *Pantoea agglomerans* D325 P10c, *E. herbicola* C9-1, *Bacillus subtilis* QST713, and BD170, have all seen significant application, with some products exhibiting control effects comparable to antibiotics. Domestically, a number of antagonistic strains with good activity against *Pseudomonas fluorescens* have recently been screened, demonstrating promising potential for biocontrol. Research and practice in the biocontrol of plant diseases show that using antagonistic microorganisms to control plant diseases offers significant advantages, including high selectivity, low risk of resistance development, safety, high efficiency, and environmental friendliness, making it an inevitable trend in modern agricultural green development and pesticide research. However, the stability of the disease prevention effect of live microbial pesticides is easily affected by environmental factors, and the effect is relatively slow. These factors have become key factors restricting the promotion and application of microbial pesticides.

[0005] In recent years, the synergistic control of some plant diseases using bio-chemical methods has achieved good results. It has gained increasing attention as a practical and environmentally friendly control strategy. Combining microbial inoculants with chemical fungicides to control plant diseases can rapidly reduce the pathogen population, solving the problem of slow efficacy of biocontrol bacteria; it can also significantly alleviate the development of resistance to chemical pesticides by pathogens, reducing the amount of chemical pesticides used, ultimately achieving the goal of green, safe, and efficient disease control. However, research has also found that the synergistic effect of the two is not a simple additive; chemical pesticides and their adjuvants may have beneficial or detrimental effects on biocontrol microorganisms. Only biocontrol bacteria and fungicides with good compatibility can produce a synergistic effect in disease control; otherwise, negative effects will occur.

[0006] Currently, no research has been reported on the use of biocontrol bacteria and fungicides in the combined control of pear fire blight. Therefore, this invention aims to find a highly efficient compound formulation for the combined control of biocontrol bacteria and fungicides against pear fire blight, thereby enriching the product range of control agents for pear fire blight and improving disease prevention efficacy. Summary of the Invention

[0007] The purpose of this invention is to provide a compound agent for the prevention and control of pear fire blight, its preparation method, and its application, thereby solving the problems existing in the prior art. This invention provides a compound agent with Bacillus vesiculosus FX1 fermentation broth and kasugamycin aqueous solution as active ingredients. This compound agent can effectively prevent and control pear fire blight, and the Bacillus vesiculosus FX1 fermentation broth and kasugamycin aqueous solution have a synergistic effect.

[0008] To achieve the above objectives, the present invention provides the following solution:

[0009] This invention provides a compound preparation for the prevention and control of pear fire blight, comprising Bacillus velezensis FX1 fermentation broth and kasugamycin aqueous solution (AS);

[0010] The volume ratio of the Bacillus Fischer-Flavor FX1 fermentation broth to the Kasugamycin aqueous solution is (9-4):(1-6).

[0011] Preferably, the effective viable count of Bacillus FX1 in the Bacillus Fischerpyrifos FX1 fermentation broth is 1.0 × 10⁻⁶. 6 cfu·mL -1 The kasugamycin aqueous solution is a 2% kasugamycin aqueous solution, that is, the mass percentage of kasugamycin in the kasugamycin aqueous solution is 2%.

[0012] Preferably, the volume ratio of the Bacillus Fibrosum FX1 fermentation broth to the Kasugamycin aqueous solution is (6-4):(4-6).

[0013] Preferably, the compounding agent further includes Tween-80 and gelatin.

[0014] More preferably, the volume ratio of the Bacillus Fibrosum FX1 fermentation broth to the Kasugamycin aqueous solution is 6:4.

[0015] This invention provides a method for preparing the above-mentioned compound agent, comprising the following steps:

[0016] The compound agent is obtained by mixing the Bacillus FX1 fermentation broth and the Kasugamycin aqueous solution.

[0017] Preferably, the effective viable count of Bacillus FX1 in the Bacillus Fischerpyrifos FX1 fermentation broth is 1.0 × 10⁻⁶. 6 cfu·mL -1 The kasugamycin aqueous solution is a 600-fold dilution of 2% kasugamycin aqueous solution.

[0018] Preferably, the volume ratio of the Bacillus Fibrosum FX1 fermentation broth to the Kasugamycin aqueous solution is (6-4):(4-6).

[0019] Preferably, after mixing the Bacillus Fischer-Flavor FX1 fermentation broth and the Kasugamycin aqueous solution, the mixture further includes the steps of adding Tween-80 and gelatin.

[0020] More preferably, the volume ratio of the Bacillus Fibrosum FX1 fermentation broth to the Kasugamycin aqueous solution is 6:4.

[0021] This invention provides the application of the above-mentioned compound agent in the prevention and control of pear fire blight.

[0022] This invention provides a method for preventing and controlling pear blight, comprising the following steps:

[0023] Spray the above-mentioned compound agent onto the plant surface.

[0024] The present invention discloses the following technical effects:

[0025] This invention, based on preliminary laboratory work, screened out the biocontrol strain *Bacillus velezensis* FX1, which exhibits high antagonistic activity and good disease control efficacy against pear fire blight pathogen. Building upon this, the compatibility of this strain with fungicides and the synergistic control effect of its combination with fungicides against pear fire blight were studied. Optimal mixing ratios with strong synergistic effects were screened to improve disease control efficacy and stability. This invention used plate count and turbidimetric methods to determine the biocompatibility of the fermentation broth of the biocontrol bacterium *Bacillus velezensis* FX1 with seven fungicides. Among the tested fungicides, Bacillus Fischer-Belley's FX1 strain showed poor compatibility with 5% Kasugamycin WP, 20% Thiamethoxam SC, 20% Thiazole Zinc SC, 3% Thiamethoxam WDG, and 46% Copper Hydroxide WDG; it showed some compatibility with 40% Kasugamycin·Thiamethoxam SC; and it showed the best compatibility with 2% Kasugamycin AS. Moreover, at low concentrations and field application concentrations, the growth inhibition rate of FX1 strain was less than 14%, and it had no significant effect on its spore germination. After 30 days of mixing, the germination rate remained above 83%. Subsequently, the in vitro toxicity against *Bacillus vesiculosus* strain FX1 fermentation broth was determined by co-culturing it with 2% kasugamycin AS. The Horsfall method was used to determine the optimal ratio of the mixture for synergistic effects. The results showed that when the ratio of *Bacillus vesiculosus* strain FX1 fermentation broth to kasugamycin was 6:4, 5:5, and 4:6, the inhibition rate against *Bacillus vesiculosus* was above 70%, with an synergistic effect ratio (IR) greater than 1.7, indicating a significant synergistic effect. To improve the stability of the FX1 / kasugamycin mixture, 0.1% TW-80 and 0.1% gelatin, which are compatible with FX1, were screened as wetting and dispersing agents for the mixture. The colonization dynamics of Bacillus vesiculosus FX1 on Korla pear were detected in combinations of Bacillus vesiculosus FX1 and kasugamycin at ratios of 6:4 and 5:5. The results showed that 21 days after spraying the compound agent, the colonization rate of Bacillus vesiculosus FX1 on the leaves and branches of Korla pear remained at 10. 3The concentration of cfu / g in fresh samples was more than 10 times higher than that of the Bacillus vesicularis FX1 single-agent treatment, indicating that the combination treatment facilitated the colonization of biocontrol bacteria on Korla pear plants. High-throughput sequencing was used to detect the effect of the combined application of Bacillus vesicularis FX1 and kasugamycin on the phyllosphere bacterial community structure of Korla pear. The results showed that on day 7 after spraying, the abundance of Bacillus vesicularis in the leaf bacteria of the combined treatment (31.38%) was significantly higher than that of the FX1 single-agent treatment (13.13%); while the abundance of pear fire blight pathogens (0.0079%) was significantly lower than that of the FX1 single-agent treatment (4.89%). On day 14 after spraying, the abundance of Bacillus vesicularis in the phyllosphere bacteria of Korla pear leaves treated with the combined treatment remained at 18.48%, while it was only 5.67% in the FX1 single-agent treatment. The results indicate that the combined application of the compound foliar spray helps Bacillus vesicles FX1 establish a significant colonization advantage among the phyllosphere bacteria of Pear leaves, thereby significantly reducing the population of Pear Fire Blight Pathogen. Therefore, spraying the compound foliar spray of Bacillus vesicles FX1 and kasugamycin helps Bacillus vesicles FX1 occupy the leaf niche and become the dominant species in the leaf epiphytic flora, significantly reducing the types and numbers of other bacteria, greatly reducing the population of Pear Fire Blight Pathogen, and increasing the protection and disease prevention effect on the leaves. The disease control effect was evaluated through experiments on detached inflorescences of fragrant pear, potted wild pear seedlings, and orchards. The results showed that the combination of Bacillus vesicularis FX1 and kasugamycin in a ratio of 6:4 and 5:5 had an average protective effect against fragrant pear flower rot for 3-5 days, and a protective and curative effect against wild pear seedlings for 7-14 days, both of which were above 73%. The combination of Bacillus vesicularis FX1 and kasugamycin aqueous solution in a ratio of 6:4 had the best control effect, with protective and curative effects reaching 79.18% and 78.82%, respectively, which were significantly higher than other treatments. When the compound of Bacillus vesicularis FX1 and kasugamycin aqueous solution in a ratio of 6:4 was sprayed in diseased orchards, the disease control effect reached more than 87% 60 days after spraying from the flowering period, and the control effect remained stable.

[0026] Based on the results of virulence determination of Bacillus vesiculosus FX1 and 2% kasugamycin AS combination against pear fire blight pathogen, effect on FX1 colonization ability, and evaluation of control efficacy against pear fire blight, the optimal combination ratio of Bacillus vesiculosus FX1 fermentation broth and 2% kasugamycin AS was determined to be 6:4, considering the excellent and stable disease control effect and the environmental principle of minimizing fungicide usage.

[0027] The advantage of this invention lies in the combined use of Bacillus frenulum FX1 fermentation broth and 2% kasugamycin AS, which not only reduces the amount of chemical agents used by 60%, but also creates a synergistic effect. While exerting the highly efficient and rapid antibacterial effect of kasugamycin, it also helps Bacillus frenulum FX1 to stably colonize on pears, allowing it to occupy its ecological niche and become the dominant species, thus constructing a stable and balanced micro-ecosystem. This further reduces the number of pear fire blight pathogens and their infection sites, fully leveraging its antibacterial and disease-preventing functions. Simultaneously, this compound agent also exhibits a synergistic effect between the bacteria and the drug, further improving the disease prevention effect, ultimately achieving the goal of safely, efficiently, and sustainably controlling the occurrence, spread, and diffusion of diseases. Attached Figure Description

[0028] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0029] Figure 1 Bar chart showing the distribution of bacterial species in the phyllosphere of Pear after 1 day of different high-throughput sequencing treatments;

[0030] Figure 2 Bar chart showing the distribution of bacterial species in the phyllosphere of Pear after 7 days of different high-throughput sequencing treatments;

[0031] Figure 3 Bar chart showing the distribution of bacterial species in the phyllosphere of Pear after 14 days of different high-throughput sequencing treatments;

[0032] Among them, FP1, FP2, and FP3 represent treatment 3 (3 replicates); FX11, FX12, and FX13 represent treatment 4 (3 replicates); CL1, CL2, and CL3 represent treatment 5 (3 replicates); D1, D2, and D3 represent treatment 2 (3 replicates); CK1, CK2, and CK3 represent treatment 1 (3 replicates); FPb1-1 and FPb2-1 represent treatment 3 treated with the compound agent and sprayed for 7 days, followed by a second spray; FPb2-2 represents treatment 3 treated with the compound agent and sprayed for 14 days, followed by a second spray; FX1b1-1 and FX1b2-1 represent Bacillus belysin FX1 single agent sprayed for 7 days, followed by a second spray; FX1b2-2 represents Bacillus belysin FX1 single agent sprayed for 14 days, followed by a second spray. Detailed Implementation

[0033] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0034] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0035] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0036] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0037] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0038] Bacillus Fibrosum FX1 has been published in the literature "Optimization of Culture Medium and Shake Flask Fermentation Conditions for FX1, an Antagonistic Bacterium of Pear Fireblight".

[0039] Example 1: Screening of biocontrol agents compatible with Bacillus frenulum FX1 strain

[0040] (1) Effects of fungicides on the growth of biocontrol bacterium Bacillus Fischerpie FX1 strain

[0041] Seven fungicides registered and already widely used in production for controlling pear fire blight were selected from the China Pesticide Information Network. Specific information is shown in Table 1. Three experimental concentrations were set for the tested fungicides: the field application concentration recommended by the registered agent as the intermediate concentration, twice the intermediate concentration as the high concentration, and half the intermediate concentration as the low concentration. A co-culture method combining fungicide and biocontrol bacteria was used. The tested fungicides were diluted and added to 50mL Erlenmeyer flasks containing sterilized NB medium to prepare the set final experimental concentrations, resulting in the dosing medium. Bacillus Fibrosum FX1 bacterial suspension was added to the dosing medium at a volume ratio of 1%. After 24 hours of incubation, the inhibition rate of bacterial growth was determined using turbidimetric and plate count methods. The results are shown in Table 2.

[0042] Table 1. Information on the tested fungicides

[0043]

[0044]

[0045] Note: WP is wettable powder, SC is suspending agent, WDG is water dispersant, and AS is aqueous solution.

[0046] Table 2. Effects of the tested agents on the growth of biocontrol bacterium Bacillus belye FX1

[0047]

[0048] Note: Data are mean ± standard error. Different lowercase letters in the same column indicate significant differences between treatments (p < 0.05).

[0049] The experimental results showed that all tested fungicides inhibited the growth of *Bacillus belyssiensis* strain FX1 to varying degrees. The inhibitory effects of the seven fungicides on *Bacillus belyssiensis* strain FX1 were as follows: 5% kasugamycin WP > 20% thiabendazole copper SC > 20% thiabendazole zinc SC > 46% copper hydroxide WDG > 3% thiamethoxam WDG > 40% kasugamycin·thiabendazole zinc SC > 2% kasugamycin AS. Plate colony counting showed that 5% kasugamycin WP, 20% thiabendazole copper SC, 20% thiabendazole zinc SC, 3% thiamethoxam WDG, and 46% copper hydroxide WDG strongly inhibited the growth of *Bacillus belyssiensis* strain FX1. At medium concentrations (field application concentration) and high concentrations (twice the field application concentration), the inhibition rate was over 88%, and at low concentrations (half the field application concentration), the inhibition rate was over 77%. 40% kasugamycin·thiazolium zinc SC showed a 67.3% inhibition rate against the growth of *Bacillus belyssiensis* FX1 at medium concentrations and a 55.4% inhibition rate at low concentrations, indicating a relatively weak inhibitory effect. 2% kasugamycin AS showed inhibition rates of 13.5% and 7.6% against *Bacillus belyssiensis* FX1 at medium and low concentrations, respectively, with a 21.1% inhibition rate at high concentrations, indicating the least inhibitory effect. Turbidimetric assays of the growth of *Bacillus belyssiensis* FX1 cells co-cultured with the tested bactericides showed that 5% kasugamycin WP, 20% copper thiabendazole SC, 20% thiazolium zinc SC, 3% thiamethoxam WDG, and 46% copper hydroxide WDG significantly inhibited the growth of *Bacillus belyssiensis* FX1 at high, medium, and low concentrations, with inhibition rates exceeding 73%. 40% kasugamycin·thiazolium zinc SC showed some inhibitory effect on *Bacillus belyssin* FXI at medium and low concentrations, with an inhibition rate of less than 31%. 2% kasugamycin AS showed inhibition rates of 8.5% and 0.67% on *Bacillus belyssin* FX1 at medium and low concentrations, respectively, while the inhibition rate at high concentrations was 19.3%, indicating weak inhibitory effect on *Bacillus belyssin* FX1. These results are basically consistent with the results obtained by the plate colony counting method (Table 2). Therefore, the bactericide with the least inhibitory effect on the growth of *Bacillus belyssin* FX1 was selected as 2% kasugamycin aqueous solution.

[0050] (2) Effect of fungicide on the survival of Bacillus belyss FX1 spores: Same as experiment (1), the tested fungicide was added to test tubes to prepare the set concentration, with sterilized NB medium without fungicide as the control. 1×10 7A CFU / mL suspension of *Bacillus belyceae* FX1 spores was incubated at room temperature and periodically shaken. *Bacillus belyceae* FX1 spores were collected after 3, 15, and 30 days and washed with sterile water to remove bactericides. The bacterial suspension was serially diluted and plated on NA agar plates. The spore germination rate was calculated as: spore germination rate (%) = (number of colonies in treatment groups) / number of colonies in control groups × 100. The effect of 2% kasugamycin AS on the spore germination of *Bacillus belyceae* FX1 strain is shown in Table 3.

[0051] Table 3. Effects of 2% Kasugamycin AS on the germination of Bacillus belysin FX1 spores.

[0052]

[0053] The results showed that treatment of Bacillus belyss FX1 spore suspension with 2% kasugamycin AS at different dilutions maintained a spore germination rate above 83% for 3-30 days, with little impact on spore survival. However, at high concentrations (300×), spore survival was somewhat affected, with the germination rate decreasing to 69.3% after 30 days. In summary, 2% kasugamycin AS had minimal impact on the growth and spore germination of Bacillus belyss FX1 strain, and the two showed good compatibility, making it suitable as a fungicide for constructing bacterial-drug compound formulations (Table 3).

[0054] Example 2: Combination of biocontrol strain Bacillus freundii FX1 with a compatible fungicide

[0055] (1) Indoor toxicity determination of Bacillus vesiculosus FX1 fermentation broth and 2% kasugamycin fungicide against pear fire blight: 2% kasugamycin was screened in Example 1 to show good compatibility with the biocontrol bacterium Bacillus vesiculosus FX1. The inhibitory effects of the two on pear fire blight were further determined. Pear fire blight bacterial suspension was prepared using E. a001-GFP strain (green fluorescent protein labeled strain, with kanamycin resistance of 50 μg / mL). This strain was constructed in our laboratory and published in the literature "Study on infection, colonization and spread characteristics of pear fire blight in branches of Korla fragrant pear" [J]. (Lv Tianyu, Xu Linyun, Xi Haishen, et al. Journal of Fruit Science, 2023, 40(08): 1692-1702.). The bacterial concentration was 1×10⁻⁶. 9cfu / mL. The fermentation broth of *Bacillus belyssus* FX1 was prepared. The fermentation medium formula is disclosed in "Optimization of Culture Medium and Shake Flask Fermentation Conditions for *Bacillus thuringiensis* Antagonistic Bacterium FX1" (Lv Tianyu, He Xu, Luo Ming, et al. Optimization of Culture Medium and Shake Flask Fermentation Conditions for *Bacillus thuringiensis* Antagonistic Bacterium FX1 [J]. Chinese Journal of Biological Control, 2022, 38(06): 1553-1565.). *Bacillus belyssus* FX1 fermentation broth at different concentrations and 2% kasugamycin AS were co-cultured with *Bacillus thuringiensis*, and the inhibition rate against *Bacillus thuringiensis* was determined. Data processing was performed using Origin2021 software. A virulence regression equation was established with the logarithm of the 2% kasugamycin AS concentration (or the concentration of *Bacillus belyssus* FX1 fermentation broth) as the independent variable (x) and the inhibition rate as the dependent variable (y), and the coefficient of determination R was calculated. 2 and inhibition of medium concentration EC 50 The survey results are shown in Tables 4 and 5.

[0056] Table 4. Results of virulence assay of Bacillus vesiculosus FX1 strain fermentation broth against Pear blight pathogen.

[0057] <![CDATA[Concentration of Bacillus velezensis FX1 fermentation broth / (cfu·mL -1 )]]> <![CDATA[E.a001 - viable count of GFP bacteria / (cfu·mL -1 )]]> Antibacterial rate / % <![CDATA[1.0×10 5 ]]> <![CDATA[1.14×10 9 ]]> 15.34 <![CDATA[1.0×10 6 ]]> <![CDATA[4.28×10 8 ]]> 68.52 <![CDATA[1.0×10 7 ]]> <![CDATA[2.86×10 8 ]]> 78.81 <![CDATA[1.0×10 8 ]]> <![CDATA[1.39×10 8 ]]> 89.69 <![CDATA[1.0×10 9 ]]> <![CDATA[1.35×10 5 ]]> 99.90 CK <![CDATA[1.35×10 9 ]]> -

[0058] Table 52 shows the virulence test results of kasugamycin AS against pear blight pathogen.

[0059] <![CDATA[春雷霉素浓度2% / (μg·mL -1 )]]> <![CDATA[E.a001 - viable count of GFP bacteria / (cfu·mL -1 )]]> Antibacterial rate / % 500 <![CDATA[1.17×10 9 ]]> 22.51 800 <![CDATA[9.45×10 8 ]]> 37.41 1000 <![CDATA[8.18×10 8 ]]> 45.82 1300 <![CDATA[6.69×10 8 ]]> 55.70 1600 <![CDATA[5.53×10 8 ]]> 63.40 1800 <![CDATA[3.96×10 8 ]]> 73.77 CK <![CDATA[1.51×10 9 ]]> -

[0060] The inhibition rate of different concentrations of Bacillus vesiculosus FX1 fermentation broth against Pyrus pyriformis (E. a001-GFP strain) is shown in Table 4. The concentrations of Bacillus vesiculosus FX1 fermentation broth at concentrations of 1.0 × 10⁻⁶ were as follows: 5 -1.0×10 9 cfu·mL -1 Between these values, the inhibition rate against pear fire blight pathogens ranged from 15.34% to 99.9%. Based on this, a virulence regression equation was established as y = 3.5759x - 15.9732, with a correlation coefficient R0. 2 =0.966, inhibiting medium concentration EC 50 The value is 7.33 × 10 5 The inhibition rates of different concentrations of 2% kasugamycin AS against pear fire blight pathogens are shown in Table 5. The inhibition rates of different concentrations of kasugamycin against pear fire blight pathogens ranged from 22.51% to 73.77%. An independent regression equation was established: y = 2.2031x - 1.7174, with a correlation coefficient R0. 2 =0.972, inhibiting medium concentration EC 50 The value was 1119.51 μg / mL.

[0061] (2) Virulence determination of Bacillus belyceta FX1 and 2% kasugamycin compound against Pyrus pyrifolia (E. a001-GFP strain): The Horsfall method was used for experimental design. Based on the results of single-agent virulence determination, the median inhibitory concentration (EC50) of Bacillus belyceta FX1 and 2% kasugamycin AS single agents was used as the criterion. 50 Based on the values, effective medium-concentration solutions of two single agents were prepared and mixed at different volume ratios: V1 (Bacillus belyssus FX1 fermentation broth): V2 (2% kasugamycin AS) = 10:0, 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9, 0:10. The synergistic ratio (IR) was calculated. The formula for calculating the IR is: IR = E(ob) / E(th); where: E(ob) is the actual inhibition rate of the mixture, and E(th) is the theoretical inhibition rate of the mixture; E(th) = (a+b) - ab / 100; where a and b represent the antibacterial effects of the biocontrol bacteria and the chemical agent used alone at their respective mass concentrations in the mixture. IR ≥ 1 indicates an additive effect; IR < 1 indicates an antagonistic effect. The survey results are shown in Table 6.

[0062] Table 6. Virulence test results of Bacillus vesiculosus FX1 fermentation broth combined with 2% kasugamycin aqueous solution against Pear fire blight pathogen.

[0063]

[0064] Will suppress medium concentrations of EC 50 The fermentation broth of *Bacillus belyssinus* FX1 was combined with 2% kasugamycin AS at different volume ratios, and the results of the combined toxicity assay are shown in Table 6. The results showed that when V1 (*Bacillus belyssinus* FX1 fermentation broth):V2 (2% kasugamycin AS) = 9:1, 8:2, 7:3, 6:4, 5:5, and 4:6, an IR ≥ 1 indicated a synergistic effect. The synergistic ratios of 6:4, 5:5, and 4:6 were 1.811, 1.801, and 1.703, respectively, with actual inhibition rates of 73.35%, 74.47%, and 70.25%, demonstrating a significant synergistic effect.

[0065] Example 3: Screening of wetting agents and dispersants when Bacillus vesiculosus FX1 is combined with kasugamycin

[0066] Five wetting agents (Tween-80, glycerol, sodium dodecyl sulfate (SDS), calcium dodecylbenzenesulfonate, and Tween-20)) and four dispersants (alkylphenol polyoxyethylene ether (OP-10), sodium lignosulfonate, gelatin, and polycarboxylate) were added to the Bacillus freundii FX1 fermentation medium at mass fractions of 0.1%, 1%, 2%, 3%, and 5%, respectively. The medium was cultured at 28°C and 200 rpm for 48 h with shaking. Each treatment was repeated three times. The Bacillus freundii FX1 fermentation medium without wetting and dispersants was used as a control (CK). The number of viable FX1 cells in the fermentation broth was measured, and the effects of wetting agents and dispersants on cell growth were compared. The results are shown in Table 7.

[0067] Table 7. Effects of different wetting and dispersing agents on the viable count of Bacillus belyss FX1 fermentation broth

[0068]

[0069]

[0070] The experimental results showed that among the five wetting agents, adding 0.1 wt.% TW-80 to the fermentation medium of *Bacillus belyssiensis* FX1 had no significant effect on the growth of *Bacillus belyssiensis* FX1, and the viable cell count was not significantly different from the control (CK). Among the four dispersants, adding 0.1 wt.% gelatin had a relatively low effect on the viable cell count of *Bacillus belyssiensis* FX1 fermentation broth (Table 7). Therefore, 0.1 wt.% TW-80 and 0.1 wt.% gelatin were selected as the wetting agent and dispersant, respectively, for the *Bacillus belyssiensis* FX1 fermentation broth and the kasugamycin compound.

[0071] Example 4: Colonization dynamics of Bacillus vesicularis FX1 combined with kasugamycin in the leaf margin of Korla pear and its effect on the leaf margin bacterial community structure.

[0072] (1) Isolation culture method to detect the colonization performance of Bacillus vesicularis FX1 on pear trees: Bacillus vesicularis FX1 antibiotic resistance marker strain (resistant to streptomycin 1000μg / mL) was screened by multi-gradient concentration antibiotic acclimatization culture.

[0073] Three treatment groups were set up: Treatment group 1: Bacillus flocculation strain FX1 fermentation broth (concentration 1.0 × 10⁻⁶) 6 cfu·mL -1 The mixture was prepared with 2% kasugamycin AS (600x dilution recommended for field use) at a volume ratio of 6:4; two treatment groups were treated: Bacillus thuringiensis FX1 fermentation broth (concentration 1.0×10⁻⁶). 6 cfu·mL -1Mixed with 2% kasugamycin AS (600-fold dilution) at a volume ratio of 5:5; 3 treatment groups: Bacillus thuringiensis FX1 fermentation broth (concentration 1.0 × 10⁻⁶). 6 cfu·mL -1 In the preparation of Bacillus flocculation FX1 fermentation broth, each treatment added 0.1% TW-80 and 0.1% gelatin to the fermentation medium as wetting and dispersing agents.

[0074] Afterwards, each treatment was sprayed onto the surface of pear branches and leaves until completely moistened. 24 hours later, a bacterial suspension of pear fire blight pathogen (E. a001-GFP strain) (effective viable count 1.0 × 10⁻⁶) was sprayed. 8 cfu·mL -1 Samples were taken on days 0, 1, 3, 5, 7, 10, 14, and 21 post-inoculation. After serial dilution, 100 μL of each sample was plated on antibiotic agar plates. Each treatment was repeated three times, and colony counts were recorded. The number of viable FX1 colonies per gram of fresh tissue (CFU·g fresh tissue) was calculated. -1 The results are shown in Table 8.

[0075] Table 8. Colonization dynamics of Bacillus vesiculosus FX1 fermentation broth combined with 2% kasugamycin aqueous solution on pear leaves and branches (unit: cfu / g fresh tissue)

[0076]

[0077]

[0078] The results showed that the colonization of *Bacillus berleis* FX1 on pear branches and leaves gradually decreased over time after spraying. However, the treatment with a mixture of *Bacillus berleis* FX1 fermentation broth and 2% kasugamycin AS at volume ratios of 6:4 and 5:5 resulted in a significantly smaller decrease in the colonization of *Bacillus berleis* FX1 on pear branches and leaves compared to the treatment with *Bacillus berleis* FX1 alone. By 21 days after spraying, viable *Bacillus berleis* FX1 was undetectable on branches treated with the single agent, while the viable *FCI* in the mixed agent treatment remained at 10. 3 CFU·g Fresh Tissue -1 In the compound formulation for leaf application, the colonization rate of Bacillus vesiculosus FX1 cells on Korla pear leaves was maintained at 10. 3 CFU·g Fresh Tissue -1 It is 10 times that of a single treatment with Bacillus vesiculosus FX1 (Table 8).

[0079] (2) High-throughput sequencing was used to monitor the effect of the combined application of Bacillus FX1 and kasugamycin on the phyllosphere bacterial community structure.

[0080] Five treatment groups were set up: Treatment 1: Sterile water treatment (CK); Treatment 2: Pear fire blight fungus suspension (E. a001-GFP strain suspension, concentration 1×10⁻⁶). 8 cfu·mL -1 Treatment 3: Bacillus belyss FX1 fermentation broth (concentration 1×10⁻⁶) 6 cfu·mL -1 Treatment 4: A mixture of 2% kasugamycin AS (600x dilution) and 2% kasugamycin AS at a volume ratio of 6:4; Treatment 4: Bacillus belye FX1 fermentation broth (1×10⁻⁶) 6 cfu·mL -1 Single-agent treatment; Treatment 5: 2% Kasugamycin AS (600 times dilution) single-agent treatment; In the preparation of Bacillus flocculation FX1 fermentation broth, each treatment added 0.1 wt.% TW-80 and 0.1 wt.% gelatin as wetting and dispersing agents to the fermentation medium.

[0081] Spray the leaves of the fragrant pear tree, ensuring complete wetting of the branches and leaf surfaces. Collect leaves from each treatment on days 1, 7, and 14 after spraying. For each treatment, add 10g of leaves to 100mL of PBS buffer, vortex at 200rpm for 120min, and transfer the supernatant to a 50mL centrifuge tube. Centrifuge at 8000g to collect the precipitate in a sterile centrifuge tube, freeze in liquid nitrogen, and transport on dry ice. Total DNA extraction, PCR amplification, library construction, and sequencing were performed by Beijing Biomarker Biotechnology Co., Ltd. Results are as follows: Figures 1-3 As shown. Figures 1-3 This is a bar chart of species distribution, where one color represents one species and the length of the color block indicates the relative abundance of the species. Only the top ten species by abundance level are shown in the chart, and other species are grouped into Others. Unclassified represents species that have not received taxonomic annotations. Figure 1 , Figure 2 and Figure 3 The values ​​represent the results of different treatments on day 1, day 7, and day 14, respectively. FP1, FP2, and FP3 represent treatment 3 (3 replicates); FX11, FX12, and FX13 represent treatment 4 (3 replicates); CL1, CL2, and CL3 represent treatment 5 (3 replicates); D1, D2, and D3 represent treatment 2 (3 replicates); CK1, CK2, and CK3 represent treatment 1 (3 replicates); FPb1-1 and FPb2-1 represent a second spray after 7 days of treatment with the compound agent of treatment 3; FPb2-2 represents a second spray after 14 days of treatment with the compound agent of treatment 3; FX1b1-1 and FX1b2-1 represent a second spray after 7 days of single-agent spray of Bacillus belyssus FX1; and FX1b2-2 represents a second spray after 14 days of single-agent spray of Bacillus belyssus FX1.

[0082] The results show that at day 1 ( Figure 1 The proportion of Bacillus velezensis in the compound treatment FP1 was 3.24%, while the proportion of Bacillus velezensis in the single treatment of Bacillus velezensis FX11 fermentation broth reached 15.45%. This result is related to the dilution of the Bacillus velezensis FX1 concentration after compounding. On the 7th day after spraying ( Figure 2 The proportion of *Bacillus belyssus* in the compound treatment (FP2) reached 31.38%, significantly higher than the 13.13% in the single-agent treatment (FX12). Furthermore, the abundance of *Erwinia amylovora* (yellow) as the pear blight pathogen on the leaves of the pear trees treated with the compound treatment was less than 1% (0.0079%), while the proportion of *Erwinia amylovora* as the pear blight pathogen on the leaves of the pear trees treated with the single-agent treatment was 4.89%. On day 14 after spraying... Figure 3 The abundance of *Bacillus belyssus* in the phyllosphere bacteria of *Pyracantha fortuneana* leaves treated with the compound combination spray remained at 18.48%, while it was only 5.67% in the single-agent treatment with *Bacillus belyssus* FX1. This indicates that compared to the single-agent treatment, the compound combination spray helped *Bacillus belyssus* FX1 establish a significant colonization advantage in the phyllosphere bacteria of *Pyracantha fortuneana* leaves, thus significantly reducing the population of pear fire blight pathogens.

[0083] The species distribution bar chart also shows that on day 1 of the compound treatment ( Figure 1 The top ten most abundant species were *Streptococcus macedonicus* (43.10%), *Blautia* (19.63%), other species (19.58%), *Lachnospira* (9.07%), *Bacillus velezensis* (3.24%), *Oscillospira* (2.83%), *Clostridium saudiense* (1.66%), *Cyanophyceae* (0.80%), and *Erwinia amylovora* (0.008%). At day 7 of spraying ( Figure 2 The types and quantities of bacteria in the phyllosphere decreased significantly, with cyanobacteria (65.52%), Bacillus belyssus (31.38%), Citrobacter murliniae (0.04%), and other species (3.04%). On day 14 after spraying ( Figure 3The bacterial composition included cyanobacteria (74.11%), *Bacillus belye* (18.48%), *Kocuria rosea* (1.35%), *Roseburia* (0.03%), *Oscillatoria* (0.02%), and other species (2.41%). Compared to single treatments with FX1 and kasugamycin, the combined treatment resulted in *Bacillus belye* achieving population dominance among chloroplasts, significantly reducing the diversity and abundance of other bacterial species.

[0084] Example 5: Evaluation of the efficacy of Bacillus vesiculosus FX1 fermentation broth combined with 2% Kasugamycin AS in controlling pear fire blight.

[0085] (1) Disease control effect on detached inflorescences of fragrant pear: Fragrant pear flower branches at the initial flowering stage were collected from the orchard and hydroponically cultured in a 3% sucrose solution. After the flower buds opened, Bacillus vesiculosus FX1 fermentation liquid (live bacteria concentration 1×10⁻⁶) was sprayed separately. 6 cfu·mL -1 The preparation method is the same as in Example 4), 2% kasugamycin AS (600 times dilution) and different ratios of Bacillus belyssus FX1 (live bacteria concentration 1×10⁻⁶). 6 cfu·mL -1 A combination solution of 2% kasugamycin AS (600-fold dilution) was prepared, with 0.1% TW-80 and 0.1% gelatin added as wetting and dispersing agents to each treatment. Each treatment was sprayed on at least 50 flowers, and repeated three times. A blank control was prepared by spraying an equal volume of sterile water, and a 3% thiamethoxam WDG solution (800-fold dilution) was used as a control.

[0086] After culturing in an artificial climate chamber for 24 hours, the bacteria were spray-inoculated with a bacterial suspension of *E. a001-GFP* strain (1×10⁻⁶ viable cells). 8 cfu·mL -1 The plants were then cultured in an artificial climate chamber. Disease incidence was observed and recorded, the rate of flower rot was calculated, and the control efficacy against flower rot was determined. Flower rot rate (%) = (number of diseased flowers / total number of flowers) × 100; Flower rot control efficacy (%) = (control flower rot rate - treatment flower rot rate) / control flower rot rate × 100. The results are shown in Table 9.

[0087] Table 9. Protective efficacy of Bacillus vesiculosus FX1 fermentation broth combined with 2% kasugamycin AS against fire blight in detached inflorescences of Prunus camphora.

[0088]

[0089] The results of the control efficacy test on the inflorescence of fragrant pear showed that when the fermentation broth of Bacillus belye FX1 was mixed with 2% kasugamycin AS at volume ratios of 9:1, 7:3, 6:4, and 5:5, the control efficacy against flower rot of fragrant pear inflorescence could reach more than 74% within 3-5 days. Among them, the 9:1 ratio had the highest control efficacy of 77.21%, which was significantly higher than the control efficacy of other ratios, single agents, and the control agent 3% thiamethoxam WDG treatment (Table 9).

[0090] (2) Test of the efficacy of potted pear seedlings:

[0091] 1) Protective efficacy: The experiment was conducted in a greenhouse using tender branches of 2-year-old pear seedlings as inoculation material. Bacillus belye FX1 fermentation broth (live bacteria concentration 1×10⁻⁶) was used. 6 cfu·mL -1 The preparation method is the same as in Example 4), 2% kasugamycin AS (600 times dilution) and different ratios of Bacillus belyssus FX1 (live bacteria concentration 1×10⁻⁶). 6 cfu·mL -1 A combination solution of 2% kasugamycin AS (600-fold dilution) was prepared, with 0.1% TW-80 and 0.1% gelatin added as wetting and dispersing agents to each treatment. A control was prepared by spraying thiamethoxam (3% water-dispersible granules, Shaanxi Xida Huate Technology Industry Co., Ltd.) at 800-fold dilution, with sterile water spray as a blank control. Each treatment was sprayed on 5 pots (approximately 25 branches), repeated 3 times. The pots were placed in a plastic film greenhouse for 48 hours, and then sprayed with a bacterial suspension of *Pyrus pyrifolia* (E. a001-GFP strain, viable count 1×10⁻⁶). 8 cfu·mL -1 Observe the disease situation daily, record the number of affected branches and the disease severity level. Calculate the incidence rate and disease index, and statistically analyze the control effect.

[0092] 2) Therapeutic efficacy: In the therapeutic efficacy test, the inoculation sequence of Bacillus vesicularis FX1 fermentation broth, 2% kasugamycin, and different ratios of Bacillus vesicularis FX1-kasugamycin compound solutions and Pear fire blight pathogens was reversed compared to the protective test. Other experimental materials, culture conditions, and efficacy investigation methods were consistent with the protective test. The disease severity grading standards for Pear fire blight in Pear seedlings were as follows: Grade 0, no lesions on branches; Grade I, lesion length accounting for 1 / 3 of the inoculated branch length; Grade III, lesion length accounting for 1 / 3-2 / 3 of the inoculated branch length; Grade V, lesion length accounting for 2 / 3 of the inoculated branch length. The efficacy of each treatment was calculated based on the disease index. Disease index = ∑(number of diseased branches at each level × representative value of disease grade) / (total number of inoculated branches × highest grade value) × 100; Efficacy calculation formula: Efficacy (%) = (Control disease index - Treatment disease index) / Control disease index × 100.

[0093] 3) Experimental results:

[0094] The results of the efficacy test on fire blight prevention of potted pear seedlings are shown in Tables 10 and 11.

[0095] Table 10. Results of protective efficacy of Bacillus vesicularis FX1 fermentation broth combined with 2% kasugamycin AS against pear fire blight in potted pear seedlings.

[0096]

[0097] Table 11 Results of therapeutic efficacy determination of Bacillus vesiculosus FX1 fermentation broth combined with 2% Kasugamycin AS on potted pear seedlings

[0098]

[0099]

[0100] The results showed that treatments with a volume ratio of Bacillus belyceta FX1 fermentation broth to 2% kasugamycin AS of 6:4 and 5:5 resulted in protective and therapeutic efficacy exceeding 73% after 7-14 days. The 6:4 volume ratio of Bacillus belyceta FX1 fermentation broth to 2% kasugamycin AS yielded the best efficacy, with protective and therapeutic efficacy reaching 79.18% and 78.82%, respectively, significantly higher than other treatments, and the efficacy remained stable (Tables 10 and 11).

[0101] (3) Evaluation of the field efficacy of the compound combination against fire blight in early-ripening pear: The field trial was conducted at the Zhangye Water-Saving Agriculture Experimental Station of the Gansu Academy of Agricultural Sciences. Prior to the trial, the incidence of fire blight in pears was investigated in both locations, and the trial was conducted in areas with similar disease prevalence. Two treatments were set up: Treatment 1 consisted of 200 pear trees treated with conventional chemical control (CK); Treatment 2 consisted of Bacillus belyss FX1 fermentation broth (live bacteria concentration 1×10⁻⁶). 6 cfu·mL -1 The preparation method was the same as in Example 4) and 2% kasugamycin AS (600 times diluted solution) were mixed at a volume ratio of 6:4 to treat 230 pear trees. Each pear tree was sprayed with 5L of the compound solution. Disease index and control effect were regularly investigated after spraying. The disease index was calculated as follows: Disease Index (DI) = ∑(Number of diseased trees at each level × Representative value of disease level) × 100 / Total number of trees × Highest level value. The control effect was calculated as follows: Control effect (%) = (Control disease index - Treatment disease index) × 100% / Control disease index. The field disease grading standards for pear fire blight were: Level 0: No symptoms; Level I: Only twigs are affected, with no more than 10 twig diebacks, flower rot, and fruit rot in total; Level III: More than 10 twig diebacks, flower rot, and fruit rot in total, or large branches are affected; Level V: Bacterial ooze from the main trunk or infection spreading to the main trunk from new shoots. The results of the field trial on fire blight in early crisp pear are shown in Table 12.

[0102] Table 12 Results of field control efficacy of Bacillus vesiculosus FX1 fermentation broth combined with 2% kasugamycin AS against fire blight in early crisp pear.

[0103]

[0104] The results showed that the combination of Bacillus vesiculosus FX1 fermentation broth and 2% kasugamycin in a 6:4 volume ratio exhibited good control efficacy against pear fire blight. The average control efficacy reached 87.66% from the flowering period to 60 days after spraying, and remained stable (Table 12).

[0105] Based on the results of virulence determination of Bacillus vesiculosus FX1 fermentation broth and 2% kasugamycin AS combination against pear fire blight, effect determination on colonization ability of Bacillus vesiculosus FX1 combination, and evaluation of control efficacy against pear fire blight, the optimal combination ratio of Bacillus vesiculosus FX1 fermentation broth and 2% kasugamycin AS was determined to be 6:4, based on the principles of excellent and stable disease control effect and environmental protection that minimizes pesticide use.

[0106] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A compound preparation for preventing and controlling pear blight, characterized in that, Including Bacillus belesiensis ( Bacillus velezensis FX1 fermentation broth and kasugamycin aqueous solution; The volume ratio of the Bacillus Fischer-Flavor FX1 fermentation broth to the Kasugamycin aqueous solution is (6-4):(4-6). The effective viable count of the Bacillus FX1 fermentation broth was 1.0 × 10⁻⁶. 6 cfu·mL -1 The kasugamycin aqueous solution is a 2% kasugamycin aqueous solution.

2. The compounding agent according to claim 1, characterized in that, The compounding agents also include Tween-80 and gelatin.

3. The method for preparing the compounding agent according to claim 1, characterized in that, Includes the following steps: The Bacillus Fischer-Belase FX1 fermentation broth and the kasugamycin aqueous solution are mixed to obtain the compound agent; the volume ratio of the Bacillus Fischer-Belase FX1 fermentation broth to the kasugamycin aqueous solution is (6-4):(4-6). The effective viable count of Bacillus FX1 in the Bacillus Fibre FX1 fermentation broth was 1.0 × 10⁻⁶. 6 cfu·mL -1 The kasugamycin aqueous solution is a 600-fold dilution of 2% kasugamycin aqueous solution.

4. The preparation method according to claim 3, characterized in that, The mixture of the Bacillus FX1 fermentation broth and the Kasugamycin aqueous solution further includes the steps of adding Tween-80 and gelatin.

5. The application of the compound agent according to claim 1 or 2 in the prevention and control of pear blight.

6. A method for preventing and controlling pear blight, characterized in that, Includes the following steps: The compound agent according to claim 1 or 2 is sprayed onto the plant surface.

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