Compound agent for preventing and treating pear fire blight as well as preparation method and application of compound agent

Abstract

The invention relates to the field of biological prevention and treatment, in particular to a compound agent for preventing and treating pear fire blight as well as a preparation method and application of the compound agent. The invention provides a compound agent taking bacillus velezensis FX1 fermentation liquor and kasugamycin aqueous solution as effective components, and the compound agent can be used for effectively preventing and treating pear fire blight of fruit trees. According to the compound agent, the bacillus velezensis FX1 fermentation liquor and the kasugamycin water aqua are combined for use, so that a synergistic interaction effect is achieved, the advantages of high efficiency and rapid sterilization of the kasugamycin can be exerted, the colonization quantity of the bacillus velezensis FX1 on the surface of a tree body is increased, a phyllospheric dominant flora is formed, a stable and balanced micro-ecological system is constructed, and the growth of the tree body is promoted. The application amount of chemical pesticides can be reduced by 60%, the advantages of bacterium-pesticide synergistic complementation and disease prevention effect improvement are exerted, and the purpose of safely, efficiently and sustainably preventing and controlling occurrence, prevalence and spreading of diseases is achieved.

Description

Technical Field

[0001] The invention relates to the field of biological control, and in particular to a compound agent for preventing and controlling fire blight of amylovora, and a preparation method and application thereof. Background Art

[0002] Pear fire blight is a major quarantine bacterial disease of fruit trees that has recently emerged and has been listed in my country's "List of Class I Crop Pests and Diseases" and "List of Key Alien Invasive Species". The pathogen of apricot fire blight is Erwinia amylovora, which is one of the world's top ten plant pathogens. It can infect more than 220 species of plants in more than 40 genera of the Rosaceae family, and seriously harm pear, apple, hawthorn, crabapple and other pome fruit trees. In 2016, my country first discovered pear fire blight on apple trees in Ili Prefecture, Xinjiang Uygur Autonomous Region. In 2017, an outbreak occurred on Korla fragrant pears in Bayingolin Mongol Autonomous Prefecture, Xinjiang, and quickly spread throughout the region. At present, the apricot fire blight pathogen poses a huge threat and risk to the healthy development of the apple and pear industries in the country.

[0003] Chemical control is an important means of preventing and controlling apricot fire blight. my country has screened out some agents for sudden outbreaks of apricot fire blight. Currently, the registered products for the prevention and control of apricot fire blight include: organic copper (thiophanate-methyl), inorganic copper (copper hydroxide), antibiotics (kasugamycin, sinomenine) and thiazoles (zinc thiazole, thiophanate-methyl), etc. Indoor or field tests have shown that the registered agents have a certain disease prevention effect on apricot fire blight. But in general, the types and varieties of special agents for the prevention and control of apricot fire blight in production are still very limited. The long-term and frequent use of a relatively single agent will not only accelerate the resistance of pathogens to drugs, but also cause a series of problems such as increased pesticide residues, reduced fruit quality and increased environmental pollution.

[0004] With the in-depth study of the microecology and infection biology of the amylopectin disease, the progress of biological control of amylopectin disease with beneficial microorganisms has been promoted. Commercial bacterial agents developed abroad, such as Pseudomonasfluorescens A506, Pantoea agglomerans D325 P10c, E.herbicola C9-1, Bacillus subtilis QST713 and BD170, have been used on a certain scale, and the control effect of some products is equivalent to that of antibiotics. Recently, a number of antagonistic strains with good activity against amylopectin disease have been screened in China, showing good biological control potential. Research and practice on biological control of plant diseases have shown that the use of antagonistic microorganisms to control plant diseases has outstanding advantages such as strong selectivity, low resistance to drug resistance, safety, high efficiency and environmental friendliness, and is an inevitable trend in the green development of modern agriculture and the research and development of pesticides. However, the stability of the disease prevention effect of living microbial pesticides is easily affected by environmental factors, and the efficacy is relatively slow, which have become key factors restricting the promotion and application of microbial pesticides.

[0005] In recent years, biological-chemical synergistic control of some plant diseases has achieved good results. As a practical and environmentally friendly control strategy, it has received increasing attention. The combination of microbial agents and chemical fungicides to control plant diseases can not only quickly reduce the pathogen base and solve the problem of slow control effect of biocontrol bacteria, but also greatly alleviate the resistance of pathogens to chemical pesticides, reduce the application of chemical pesticides, and ultimately achieve the goal of green, safe and efficient disease control. However, the study also found that the synergistic effect of the two is not a simple superposition, and chemical pesticides and their adjuvants may have favorable or unfavorable effects on biocontrol microorganisms. Only when the biocontrol bacteria and fungicides have good compatibility can the bacteria-drug combination produce a synergistic effect on disease control, otherwise it will produce a negative effect.

[0006] At present, there is no report on the research of using biocontrol bacteria in combination with fungicides to control apricot fire blight. Therefore, the present invention intends to find an efficient compound agent for controlling apricot fire blight by combining biocontrol bacteria with fungicides, enrich the control preparation products of apricot fire blight, and improve the disease prevention effect. Summary of the invention

[0007] The purpose of the present invention is to provide a compound agent for preventing and treating amylopectin fire blight, and a preparation method and application thereof, so as to solve the problems existing in the above-mentioned prior art. The present invention provides a compound agent with Bacillus velez FX1 fermentation broth and kasugamycin aqueous solution as active ingredients, the compound agent can effectively prevent and treat amylopectin fire blight, and at the same time, the Bacillus velez FX1 fermentation broth and kasugamycin aqueous solution have a synergistic effect.

[0008] To achieve the above object, the present invention provides the following solutions:

[0009] The invention provides a compound agent for preventing and treating fire blight of apricot, comprising Bacillus velezensis FX1 fermentation liquid and kasugamycin aqueous solution (AS);

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

[0011] Preferably, the effective viable count of Bacillus velez FX1 in the fermentation broth of Bacillus velez FX1 is 1.0×10 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 Velez subtilis FX1 fermentation broth to the kasugamycin aqueous solution is (6-4): (4-6).

[0013] Preferably, the compound further comprises Tween-80 and gelatin.

[0014] Further preferably, the volume ratio of the Bacillus Velez subtilis FX1 fermentation broth to the kasugamycin aqueous solution is 6:4.

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

[0016] The Bacillus Velez subtilis FX1 fermentation liquid and the kasugamycin aqueous solution are mixed to obtain the compound agent.

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

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

[0019] Preferably, after the Bacillus Velez subtilis FX1 fermentation broth and the kasugamycin aqueous solution are mixed, the method further comprises the step of adding Tween-80 and gelatin.

[0020] Further preferably, the volume ratio of the Bacillus Velez subtilis FX1 fermentation broth to the kasugamycin aqueous solution is 6:4.

[0021] The invention provides application of the compound agent in preventing and treating fire blight of apricot.

[0022] The present invention provides a method for preventing and treating fire blight of apricot, comprising the following steps:

[0023] The compound agent is sprayed on the plant surface.

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

[0025] In the preliminary laboratory work, the present invention screened out the biocontrol strain Bacillus velezensis FX1 strain with high antagonistic activity and good disease prevention effect on amylovora blight. On this basis, the compatibility of the strain with fungicides and the synergistic prevention and control effect of the strain with the fungicides on amylovora blight were studied, and the optimized mixing ratio with strong synergistic effect was screened out to improve the disease prevention effect and stability. The present invention uses the plate colony counting method and the turbidimetry method to determine the biocompatibility of the fermentation liquid of the biocontrol bacteria Bacillus velezensis FX1 strain of amylovora blight with 7 fungicides. Among the tested fungicides, the compatibility of Bacillus velezensis FX1 with 5% cypermethrin WP, 20% thiophanate-copper SC, 20% thiophanate-zinc SC, 3% thiophanate-methyl WDG, and 46% copper hydroxide WDG was poor; it had a certain compatibility with 40% pyraclostrobin·thiophanate-zinc SC; and it had the best compatibility with 2% pyraclostrobin AS. At low concentrations and field use concentrations, the growth inhibition rate of FX1 strain was less than 14%, and there was no obvious effect on its spore germination. After 30 days of mixing, the germination rate remained above 83%. Afterwards, the fermentation liquid of Bacillus velezensis FX1 was co-cultured with 2% kasugamycin AS to determine the indoor toxicity to E. amylovora. The Horsfall method was used to determine the compound ratio with synergistic effect. The test results showed that when the compound ratio of Bacillus velezensis FX1 fermentation liquid and kasugamycin was 6:4, 5:5, and 4:6, the inhibition rate of E. amylovora was above 70%, and the synergistic ratio (IR) was greater than 1.7, which had a significant synergistic effect. In order to improve the stability of the compound of FX1 and kasugamycin, 0.1% TW-80 and 0.1% gelatin with good compatibility with FX1 were selected as the wetting agent and dispersant of the compound. The colonization dynamics of Bacillus Velez FX1 on the leaves and branches of the fragrant pear tree were detected in the combination of Bacillus Velez FX1 and kasugamycin with a ratio of 6:4 and 5:5. The results showed that 21 days after spraying the combination, the colonization amount of Bacillus Velez FX1 on the leaves and branches of the fragrant pear tree remained at 10 3cfu / g fresh sample, which is more than 10 times higher than that of the single-agent treatment of Bacillus Velez FX1, indicating that the combination is helpful for the colonization of biocontrol bacteria on fragrant pear plants. The effect of the combined application of Bacillus Velez FX1 and kasugamycin on the bacterial community structure of the pear leaves was detected by high-throughput sequencing. The results showed that on the 7th day after spraying, the abundance ratio of Bacillus Velez in leaf bacteria in the combined combination (31.38%) was significantly higher than that of the single-agent treatment of FX1 (13.13%); while the abundance ratio of the amylovora pathogen (0.0079%) was significantly lower than that of the single-agent treatment of FX1 (4.89%). On the 14th day after spraying, the abundance ratio of Bacillus Velez in the leaf bacteria on the leaves of the fragrant pear trees treated with the combined combination was maintained at 18.48%, while it was only 5.67% in the single-agent treatment of FX1. This indicates that spraying the compound combination can help Bacillus Velez FX1 establish a significant colonization advantage in the leaf phyllosphere bacteria of the fragrant pear tree, and significantly reduce the population of the amylopectin pathogen. It can be seen that spraying the compound agent of Bacillus Velez FX1 and kasugamycin can help Bacillus Velez FX1 occupy the leaf ecological niche and become the dominant population in the leaf epiphytic flora, significantly reducing the species and number of other bacteria, greatly reducing the number of amylopectin pathogens, and increasing the protection and disease prevention effect on the leaves. The disease prevention effect was evaluated through experiments on in vitro inflorescences of fragrant pear, potted pear seedlings and orchards. The results showed that the combination of Bacillus Velezii FX1 and kasugamycin in a ratio of 6:4 and 5:5 had an average protective effect on 3-5d fragrant pear blossom end rot, and both the protective and therapeutic effects on 7-14d pear seedlings were above 73%. The best prevention effect was achieved when Bacillus Velezii FX1 and kasugamycin aqueous solution were mixed in a ratio of 6:4, with the protective and therapeutic effects reaching 79.18% and 78.82%, respectively, which were significantly higher than other treatments. When the diseased orchard was sprayed with a combination of Bacillus Velezii FX1 and kasugamycin aqueous solution in a ratio of 6:4, the disease prevention effect could reach above 87% 60d after spraying during the flowering period, and the prevention effect was sustained and stable.

[0026] Based on the test results of the virulence of the combination of Bacillus Velez subtilis FX1 and 2% kasugamycin AS against Erwinia amylovora, the effect on the colonization ability of FX1, and the evaluation of the prevention effect on Erwinia amylovora, the optimal combination ratio of Bacillus Velez subtilis FX1 fermentation broth and 2% kasugamycin AS was determined to be 6:4, combining the excellent and stable disease prevention effect with the environmental protection principle of minimizing the use of fungicides.

[0027] The invention has the advantage that after the fermentation liquid of Bacillus velez FX1 and 2% kasugamycin AS are used in combination, not only the amount of chemical agents used is reduced by 60%, but also a synergistic effect of complementary advantages can be formed. While exerting the efficient and rapid antibacterial effect of kasugamycin, it is also helpful for Bacillus velez FX1 to be stably colonized on the fragrant pear, so that it occupies an ecological niche and becomes a dominant population, constructs a stable and balanced micro-ecological system, further reduces the number of bacteria and infection sites of the amylopectin blight pathogen, and gives full play to its antibacterial and disease prevention function; at the same time, the compound can also exert the synergistic and complementary effect of bacteria and drugs, further improve the disease prevention effect, and finally achieve the purpose of safely, efficiently and sustainably preventing and controlling the occurrence, prevalence and spread of diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0029] Figure 1 This is a bar graph showing the distribution of bacterial species in the pear phyllosphere after high-throughput sequencing with different treatments for 1 day;

[0030] Figure 2 This is a bar graph showing the distribution of bacterial species in the pear phyllosphere after 7 days of high-throughput sequencing with different treatments;

[0031] Figure 3 This is a bar graph showing the distribution of bacterial species in the pear phyllosphere after high-throughput sequencing with different treatments for 14 days;

[0032] Among them, FP1, FP2, and FP3 represent treatment 3 (3 repetitions), FX11, FX12, and FX13 represent treatment 4 (3 repetitions); CL1, CL2, and CL3 represent treatment 5 (3 repetitions); D1, D2, and D3 represent treatment 2 (3 repetitions); CK1, CK2, and CK3 represent treatment 1 (3 repetitions); FPb1-1 and FPb2-1 represent treatment with the compound agent of treatment 3 and additional spraying once 7 days after spraying; FPb2-2 represents treatment with the compound agent of treatment 3 and additional spraying for the second time 14 days after spraying; FX1b1-1 and FX1b2-1 represent additional spraying once 7 days after single-dose spraying of Bacillus velezensis FX1; FX1b2-2 represents additional spraying for the second time 14 days after single-dose spraying of Bacillus velezensis FX1. DETAILED DESCRIPTION

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

[0034] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0035] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0036] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

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

[0038] Bacillus velez FX1 has been disclosed in the document "Optimization of culture medium and shake flask fermentation conditions of antagonistic bacteria FX1 against Erwinia amylovora".

[0039] Example 1 Screening of compatible fungicides for biocontrol of Bacillus Velez FX1 strain

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

[0041] The China Pesticide Information Network was consulted, and 7 fungicides that have been registered and promoted in production to prevent and treat fire blight were selected. The specific information is shown in Table 1. Three test concentrations were set for the test fungicides, namely, the recommended field use concentration of the registered agent as the intermediate concentration, 2 times the intermediate concentration as the high concentration, and 1 / 2 times the intermediate concentration as the low concentration. The fungicide + biocontrol bacteria co-cultivation method was adopted, and the test fungicides were diluted and added to 50mL triangular flasks containing sterilized NB culture medium to prepare the set test final concentration to obtain the dosing culture medium. Bacillus Velez FX1 bacterial solution was added to the dosing culture medium at a volume ratio of 1%. After culturing for 24 hours, the inhibition rate of bacterial growth was determined by turbidimetry and plate colony counting methods. The survey results are shown in Table 2.

[0042] Table 1 Related information of the tested fungicides

[0043]

[0044]

[0045] Note: WP is wettable powder, SC is suspension concentrate, WDG is water dispersible concentrate, and AS is aqueous solution.

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

[0047]

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

[0049] The test results showed that the tested fungicides had different degrees of inhibition on the growth of Bacillus velez FX1. The inhibitory effects of the seven fungicides on the growth of Bacillus velez FX1 were as follows: 5% zhongshengmycin WP> 20% thiophanate-copper SC> 20% zinc thiophanate SC> 46% copper hydroxide WDG> 3% thiophanate-methyl WDG> 40% kasugamycin·zinc thiophanate SC> 2% kasugamycin AS. The results of plate colony counting showed that 5% zhongshengmycin WP, 20% thiophanate-copper SC, 20% zinc thiophanate SC, 3% thiophanate-methyl WDG and 46% copper hydroxide WDG had strong inhibitory effects on the growth of Bacillus velez FX1. At medium concentration (field concentration) and high concentration (2 times field concentration), the inhibition rate was more than 88%, and at low concentration (1 / 2 field concentration), the inhibition rate was more than 77%. The inhibition rate of 40% kasugamycin·thiazole zinc SC on the growth of Bacillus velez FX1 was 67.3% at medium concentration and 55.4% at low concentration, which was relatively weak. The inhibition rates of 2% kasugamycin AS on the growth of Bacillus velez FX1 were 13.5% and 7.6% at medium and low concentrations, respectively, and the inhibition rate was 21.1% at high concentration, which had the least inhibitory effect on the growth of Bacillus velez FX1. The results of turbidimetric determination of the effect of co-culture of Bacillus velez FX1 with the tested fungicides on the growth of Bacillus velez FX1 showed that 5% zhongshengmycin WP, 20% thiazole copper SC, 20% thiazole zinc SC, 3% thiazole WDG and 46% copper hydroxide WDG highly inhibited the growth of Bacillus velez FX1 at high, medium and low concentrations, with an inhibition rate of more than 73%. 40% kasugamycin·thiazole zinc SC has a certain inhibitory effect on Bacillus Velez FXI at medium and low concentrations, and the inhibition rate is less than 31%; 2% kasugamycin AS has an inhibition rate of 8.5% and 0.67% on the growth of Bacillus Velez FX1 at medium and low concentrations, respectively, and an inhibition rate of 19.3% at high concentration, which has a weak inhibitory effect on Bacillus Velez FX1. This result is basically consistent with the results of the plate colony count method (Table 2). Therefore, the fungicide with the least inhibitory effect on the growth of Bacillus Velez FX1 was selected as 2% kasugamycin aqueous solution.

[0050] (2) Effect of fungicides on the survival of spores of Bacillus velez FX1 strain: As in experiment (1), add the test fungicide to the test tube to prepare the set concentration, and use the sterilized NB medium without fungicide as the control. Add 1×10 7cfu / mL Velez Bacillus FX1 spore suspension was placed at room temperature and shaken regularly. After 3d, 15d and 30d, Velez Bacillus FX1 spores were collected and washed with sterile water to remove the bactericide. The bacterial solution was gradient diluted and spread on NA medium plates to calculate the spore germination rate, spore germination rate (%) = (number of colonies in the treatment group) / number of colonies in the control group × 100. The effect of 2% kasugamycin AS on spore germination of Velez Bacillus FX1 strain is shown in Table 3.

[0051] Table 3 Effect of 2% Kasugamycin AS on spore germination of Bacillus velez FX1

[0052]

[0053] The results showed that the spore suspension of Bacillus Velezii FX1 was treated with 2% kasugamycin AS at different dilution multiples, and the spore germination rate was kept above 83% from 3 to 30 days, which had little effect on its survival. However, at high concentration (300×), it had a certain effect on spore survival, and the spore germination rate was reduced to 69.3% at 30 days. The comprehensive measurement results showed that 2% kasugamycin AS had little effect on the growth and spore germination of Bacillus Velezii FX1 strain, and the two had good compatibility and could be used as a fungicide for constructing a bacterium-drug combination (Table 3).

[0054] Example 2 Compounding of the biocontrol Bacillus Velez FX1 strain and compatible fungicides

[0055] (1) Indoor toxicity determination of fermentation broth of Bacillus Velez subtilis FX1 strain and 2% kasugamycin fungicide against amylovora: The compatibility of 2% kasugamycin and the biocontrol bacterium Bacillus Velez subtilis FX1 strain was screened out through Example 1, and the antibacterial effect of the two on amylovora was further determined. The amylovora blight bacterial solution was prepared using the E.a001-GFP strain (green fluorescent protein-labeled strain with kanamycin resistance 50 μg / mL). This strain was constructed in this laboratory and published in the document "Study on the infection, colonization and expansion characteristics of amylovora blight in Korla fragrant pear branches" [J]. (Lv Tianyu, Xu Linyun, Xi Haishen, et al. Journal of Fruit Science, 2023, 40(08):1692-1702.), and the bacterial concentration was 1×10 9cfu / mL. Prepare Bacillus velezensis FX1 fermentation broth. The fermentation medium formula is disclosed in "Optimization of culture medium and shake flask fermentation conditions for antagonistic bacteria FX1 against amylovora amylovora" (Lv Tianyu, He Xu, Luo Ming, et al. Optimization of culture medium and shake flask fermentation conditions for antagonistic bacteria FX1 against amylovora amylovora [J]. Chinese Journal of Biological Control, 2022, 38(06): 1553-1565.). Different concentrations of Bacillus velezensis FX1 fermentation broth and 2% kasugamycin AS were co-cultured with amylovora amylovora, and the inhibition rates of amylovora amylovora were determined respectively. Data processing was performed using Origin2021 software. Taking the logarithm of 2% kasugamycin AS concentration (or Bacillus velezensis FX1 fermentation broth concentration) as the independent variable (x), and the inhibition rate as the dependent variable (y), a toxicity regression equation was established to calculate the determination coefficient R 2 and inhibited by EC 50 The survey results are shown in Tables 4 and 5.

[0056] Table 4 Results of toxicity test of Bacillus Velezii FX1 fermentation liquid against Erwinia amylovora

[0057] <![CDATA[Concentration of Bacillus velezensis FX1 fermentation broth / (cfu·mL -1 )]]> <![CDATA[E.a001 - viable count of GFP / (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 5 Results of toxicity test of 2% Kasugamycin AS against Erwinia amylovora

[0059] <![CDATA[2% kasugamycin concentration / (μg·mL -1 )]]> <![CDATA[E.a001 - viable count of GFP / (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 results of the determination of the inhibition rate of different concentrations of Bacillus Velezii FX1 fermentation broth against E. a001-GFP strain are shown in Table 4. 5 -1.0×10 9 cfu·mL -1 The inhibition rate of amylopectin is between 15.34% and 99.9%. The toxicity regression equation is y = 3.5759x-15.9732, and the correlation coefficient is R 2 =0.966, inhibition of medium concentration EC 50 The value is 7.33×10 5 cfu / mL. The results of the determination of the inhibition rate of 2% kasugamycin AS at different concentrations against Erwinia amylovora are shown in Table 5. The inhibition rate of kasugamycin at different concentrations against Erwinia amylovora was between 22.51% and 73.77%, and an independent regression equation y=2.2031x-1.7174 was established, with a correlation coefficient R 2 =0.972, inhibition of medium concentration EC 50 The value is 1119.51μg / mL.

[0061] (2) Toxicity test of the combination of Bacillus velezensis FX1 and 2% kasugamycin against Erwinia amylovora (E.a001-GFP strain): The experiment was designed using the Horsfall method. Based on the results of the single-dose toxicity test, the inhibitory concentration EC of Bacillus velezensis FX1 and 2% kasugamycin AS was 50 The value was used as the basis to prepare two single doses of effective medium mass concentration drug solutions, and the mixtures were mixed according to different volume ratios such as V1 (Bacillus Velez 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, etc., and the synergistic ratio IR was calculated. The calculation formula of the synergistic ratio 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 biocontrol bacteria and chemical agents when used alone at the mass concentration in the mixture. Among them, IR≥1 is an additive effect; IR<1 is an antagonistic effect. The survey results are shown in Table 6.

[0062] Table 6 The results of toxicity test of the complex of Bacillus Velezii FX1 fermentation broth and 2% kasugamycin aqueous solution against Erwinia amylovora

[0063]

[0064] Will inhibit the concentration of EC 50 The fermentation broth of Bacillus Velez subtilis FX1 and 2% kasugamycin AS were compounded in different volume ratios, and the results of the combined toxicity test of the two are shown in Table 6. The results showed that when V1 (fermentation broth of Bacillus Velez subtilis FX1): V2 (2% kasugamycin AS) = 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, IR≥1 had a synergistic effect, among which the synergistic ratios of the compounding ratios of 6:4, 5:5, and 4:6 were 1.811, 1.801, and 1.703, respectively, and the actual inhibition rates were 73.35%, 74.47, and 70.25%, respectively, which had a significant synergistic effect.

[0065] Example 3 Screening of wetting agents and dispersants when compounding Bacillus Velezii FX1 with Kasugamycin

[0066] Five wetting agents (Tween-80 (TW-80), glycerol, sodium dodecyl sulfate (SDS), calcium dodecylbenzene sulfonate, Tween-20 (TW-20)) and four dispersants (alkylphenol polyoxyethylene ether (OP-10), sodium lignin sulfonate, gelatin, polycarboxylates) were added to the fermentation medium of Bacillus Velez FX1 at a mass fraction of 0.1%, 1%, 2%, 3%, and 5%, respectively. The culture was shaken at 28°C and 200 r / min for 48 h. Each treatment was repeated 3 times. The fermentation medium of Bacillus Velez FX1 without wetting and dispersant was used as the control (CK). The number of viable FX1 cells in the fermentation broth was determined, and the effects of wetting agents and dispersants on bacterial growth were compared. The results are shown in Table 7.

[0067] Table 7 Effects of different wetting and dispersing agents on the number of viable cells in fermentation broth of Bacillus velez FX1

[0068]

[0069]

[0070] The test results show that among the five wetting agents, when preparing the fermentation broth of Bacillus Velez FX1, adding 0.1wt.% TW-80 to its fermentation medium has no obvious effect on the growth of Bacillus Velez FX1, and there is no significant difference in the number of viable bacteria compared with CK. Among the four dispersants, adding 0.1wt.% gelatin has a relatively low effect on the number of viable bacteria in the fermentation broth of Bacillus Velez FX1 (Table 7). Therefore, 0.1wt.% TW-80 and 0.1wt.% gelatin were selected as wetting agents and dispersants for the compound of Bacillus Velez FX1 fermentation broth and kasugamycin.

[0071] Example 4 Colonization dynamics of the combination of Bacillus velez FX1 and kasugamycin in the pear phyllosphere and its effect on the bacterial community structure in the phyllosphere

[0072] (1) The isolation and culture method was used to detect the colonization performance of Bacillus Velez subtilis FX1 on pear trees: the drug-resistant marker strain of Bacillus Velez subtilis FX1 (resistant to streptomycin 1000 μg / mL) was screened out through acclimation culture with multiple gradient concentrations of antibiotics.

[0073] Three treatment groups were set up: Treatment 1: Velezac spore Bacillus FX1 fermentation broth (concentration 1.0×10 6 cfu·mL -1 ) and 2% kasugamycin AS (recommended field concentration 600-fold dilution) at a volume ratio of 6:4; Treatment 2: Velezacacillus FX1 fermentation broth (concentration 1.0×10 6 cfu·mL -1) and 2% kasugamycin AS ((600-fold dilution) were mixed at a volume ratio of 5:5; three groups were treated with Bacillus Velezii FX1 fermentation broth (concentration 1.0×10 6 cfu·mL -1 ) treatment, in each treatment, 0.1% TW-80 and 0.1% gelatin were added to the fermentation medium as wetting agent and dispersant when preparing the fermentation broth of Bacillus Velezii FX1.

[0074] Afterwards, each treatment was sprayed on the branches and leaves of the fragrant pear until they were completely wet. After 24 hours, the bacteria solution of E.a001-GFP strain (the effective number of viable bacteria was 1.0×10 8 cfu·mL -1 ). Samples were taken at 0, 1, 3, 5, 7, 10, 14, and 21 days after inoculation, and 100 μL was applied to antibiotic plates after gradient dilution. Each treatment was repeated 3 times, and the number of colonies was recorded. The number of viable bacteria colonized by FX1 per gram of fresh tissue (CFU g fresh tissue) was calculated. -1 ), and the results are shown in Table 8.

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

[0076]

[0077]

[0078] The results showed that the colonization of Bacillus Velez FX1 on the branches and leaves of fragrant pear gradually decreased with the extension of time after spraying. However, the reduction in the colonization of Bacillus Velez FX1 on the branches and leaves of fragrant pear in the treatment of the compound treatment of Bacillus Velez FX1 fermentation liquid and 2% kasugamycin AS at a volume ratio of 6:4 and 5:5 was significantly lower than that in the treatment of Bacillus Velez FX1 alone. By 21 days after spraying, no live Bacillus Velez FX1 bacteria could be detected on the branches in the treatment of Bacillus Velez FX1 alone, while the live bacteria of FXI in the treatment of the compound agent could be maintained at 10 3 CFU·g fresh tissue -1 ; The colonization of Bacillus Velezii FX1 on the leaves of the fragrant pear was maintained at 10 3 CFU·g fresh tissue -1 , which was 10 times that of the single-dose treatment with Bacillus Velezii FX1 (Table 8).

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

[0080] Five treatment groups were set up: treatment 1: sterile water treatment (CK); treatment 2: amylovora blight bacterial solution (E.a001-GFP strain bacterial solution, concentration 1×10 8 cfu·mL -1 ) treatment; Treatment 3: Bacillus Velez FX1 fermentation broth (concentration 1×10 6 cfu·mL -1 ) and 2% kasugamycin AS (600-fold dilution) were mixed at a volume ratio of 6:4; Treatment 4: Bacillus velezensis FX1 fermentation broth (1×10 6 cfu·mL -1 ) single dose treatment; treatment 5: 2% kasugamycin AS (600-fold dilution) single dose treatment; in each treatment, 0.1wt.% TW-80 and 0.1wt.% gelatin were added to the fermentation medium as a wetting agent and dispersant when preparing the Velezac spore Bacillus FX1 fermentation broth.

[0081] Spray the leaves of the fragrant pear tree until the branches and leaves are completely wet. Collect the leaves from each treatment on the 1st, 7th and 14th day after spraying. Take 10g of leaves from each treatment and add them to 100mL PBS buffer. Oscillate at 200r / min for 120min. Take the supernatant and add it to a 50mL centrifuge tube. Centrifuge at 8000g to collect the precipitate in a sterile centrifuge tube. Quick-freeze with liquid nitrogen and transport on dry ice. The total DNA extraction, PCR amplification, library construction and sequencing of the samples were commissioned to Beijing Biomarker Biotechnology Co., Ltd. The results are as follows Figure 1-Figure 3 shown. Figure 1-Figure 3 It is a species distribution bar chart, in which one color represents one species, and the length of the color block indicates the relative abundance ratio of the species. Only the top ten species in abundance level are shown in the figure, and other species are combined into Others. Unclassified represents species that have not been taxonomically annotated. Figure 1 , Figure 2 and Figure 3 Represent different treatment results on the 1st day, 7th day and 14th day respectively, among which FP1, FP2 and FP3 represent treatment 3 (repeated 3 times), FX11, FX12 and FX13 represent treatment 4 (repeated 3 times); CL1, CL2 and CL3 represent treatment 5 (repeated 3 times); D1, D2 and D3 represent treatment 2 (repeated 3 times); CK1, CK2 and CK3 represent treatment 1 (repeated 3 times); FPb1-1 and FPb2-1 represent treatment with the compound agent of treatment 3 and then sprayed once after 7 days of spraying; FPb2-2 represents treatment with the compound agent of treatment 3 and then sprayed for the second time after 14 days of spraying; FX1b1-1 and FX1b2-1 represent treatment with Bacillus velezensis FX1 and then sprayed once after 7 days of spraying; FX1b2-2 represents treatment with Bacillus velezensis FX1 and then sprayed for the second time after 14 days of spraying.

[0082] The results showed that: on the first day ( Figure 1 ), the proportion of Bacillus velezensis in the compound treatment FP1 was 3.24%, and the proportion of Bacillus velezensis in the single-dose treatment of Bacillus velezensis FX11 fermentation liquid reached 15.45%. This result is related to the dilution of the concentration of Bacillus velezensis FX1 after compounding. Figure 2 ), the proportion of Bacillus Velez in the compound combination (FP2) reached 31.38%, which was significantly higher than that of the single agent treatment of Bacillus Velez FX1 (FX12) by 13.13%; and the abundance of Erwinia amylovora (yellow) in the leaf bacteria of the fragrant pear tree leaves sprayed with the compound treatment was less than 1% (0.0079%), while the abundance of Erwinia amylovora (yellow) in the leaf bacteria of the fragrant pear tree leaves sprayed with the single agent of Bacillus Velez FX1 was 4.89%. On the 14th day after spraying ( Figure 3 ), the abundance of Bacillus Velez in the phyllosphere bacteria on the leaves of the fragrant pear tree treated with the compound combination remained at 18.48%, while it was only 5.67% in the single-agent treatment of Bacillus Velez FX1. It can be seen that compared with the single-agent treatment, spraying the compound combination can help Bacillus Velez FX1 establish a significant colonization advantage in the phyllosphere bacteria of the fragrant pear tree leaves, and significantly reduce the population of the amylopectin pathogen.

[0083] It can also be seen from the species distribution bar graph that on the first day of the compound combination treatment ( Figure 1 ), the top ten species in terms of abundance 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%). Figure 2 ), the number and species of leaf bacteria were greatly reduced, including Cyanobacteria (65.52%), Bacillus Velezii (31.38%), Citrobacter murliniae (0.04%), and other species (3.04%). Figure 3), Cyanobacteria (74.11%), Bacillus Velezii (18.48%), Kocuria rosea (1.35%), Roseburia (0.03%), Oscillatory Spirochete (0.02%), and other species (2.41%). Compared with the single-agent FX1 and single-agent kasugamycin treatments, the combined treatment can make Bacillus Velezii occupy the population dominance in the leaf-sphere bacteria and significantly reduce the species and number of other bacterial populations.

[0084] Example 5 Evaluation of the efficacy of the fermentation broth of Bacillus velez FX1 combined with 2% kasugamycin AS against fire blight

[0085] (1) Disease prevention effect on in vitro inflorescence of pear: Pear branches in the early flowering period were collected from the orchard and inserted into a 3% sucrose solution for hydroponics. After the buds opened, the fermentation liquid of Bacillus Velezii FX1 (live bacteria concentration 1×10 6 cfu·mL -1 , the preparation method is the same as that of Example 4), 2% kasugamycin AS (600-fold dilution) and different proportions of Bacillus Velezii FX1 (live bacteria concentration 1×10 6 cfu·mL -1 ) and 2% kasugamycin AS compound solution (600 times dilution), and 0.1% TW-80 and 0.1% gelatin were added to each treatment as wetting agent and dispersant. More than 50 flowers were sprayed for each treatment, and repeated 3 times. The blank control was sprayed with the same amount of sterile water, and the 3% thiamethoxam WDG 800 times solution was used as the drug control.

[0086] After culturing in an artificial climate chamber for 24 h, the samples were sprayed with a solution of E. a001-GFP strain, with a viable count of 1 × 10 8 cfu·mL -1 ), continue to cultivate in the artificial climate box, observe and record the disease situation, count the blossom end rot rate and calculate the blossom end rot prevention effect. Blossom end rot rate (%) = (number of diseased flowers / total number of flowers) × 100; blossom end rot prevention effect (%) = (blossom end rot rate of control - blossom end rot rate of treatment) / blossom end rot rate of control × 100. The results are shown in Table 9.

[0087] Table 9 The protective effect of the fermentation liquid of Bacillus Velezii FX1 combined with 2% kasugamycin AS on the fire blight of fragrant pear in vitro inflorescence

[0088]

[0089] The results of the test on the efficacy of pear inflorescence showed that when the volume ratio of Bacillus Velez FX1 fermentation broth and 2% kasugamycin AS was 9:1, 7:3, 6:4, and 5:5, the efficacy of preventing pear inflorescence blossom end rot could reach more than 74% within 3-5 days, among which the 9:1 compound ratio had the highest efficacy of 77.21%, which was significantly higher than the efficacy of other compound ratios, single agent and the control agent 3% thiamethoxam WDG treatment (Table 9).

[0090] (2) Determination of the protective effect of potted pear seedlings:

[0091] 1) Protective effect: The test was conducted in a solar greenhouse with young branches of two-year-old Pyrus betel nut seedlings as inoculation materials. The fermentation liquid of Bacillus Velezii FX1 (live bacteria concentration 1×10 6 cfu·mL -1 , the preparation method is the same as that of Example 4), 2% kasugamycin AS (600-fold dilution) and different proportions of Bacillus Velezii FX1 (live bacteria concentration 1×10 6 cfu·mL -1 ) and 2% kasugamycin AS (600-fold dilution) were mixed into a combination liquid, and 0.1% TW-80 and 0.1% gelatin were added to each treatment as a wetting agent and dispersant. At the same time, a 800-fold solution of thiophanate-methyl (3% water-dispersible granules, Shaanxi Xida Huate Technology Industry Co., Ltd.) was sprayed as a control, and sterile water spray was used as a blank control. Five pots (about 25 branches) were sprayed for each treatment, and repeated 3 times. Put it in a plastic film moisturizing shed, and spray it with a solution of amylovora amylovora (E.a001-GFP strain solution, the number of viable bacteria is 1×10 8 cfu·mL -1 ). Observe the disease situation every day, record the number of diseased branches and disease grade. Calculate the incidence rate and disease index, and calculate the prevention effect.

[0092] 2) Curative control effect: The order of spraying the fermentation liquid of Bacillus Velez subtilis FX1, 2% kasugamycin and different ratios of Bacillus Velez subtilis FX1-kasugamycin compound liquid and the inoculation of fire blight pathogen in the curative control test is opposite to that in the protective test. Other test materials, culture conditions and control effect investigation methods are consistent with those in the protective test. Among them, the disease grading standard of fire blight of Pyrus betulaceae seedlings is: grade 0, no lesions on the branches; grade I, the length of the lesions on the branches accounts for 1 / 3 of the length of the inoculated branches; grade III, the length of the lesions on the branches accounts for 1 / 3-2 / 3 of the length of the inoculated branches; grade V, the length of the lesions on the branches accounts for 2 / 3 of the length of the inoculated branches. The control effect of each treatment in the test is calculated based on the disease index. Disease index = ∑ (number of diseased branches at each level × representative value of disease level) / (total number of inoculated branches × highest level value) × 100; the formula for calculating the control effect is: control effect (%) = (control disease index-treatment disease index) / control disease index × 100.

[0093] 3) Experimental results:

[0094] The results of the test on the prevention effect of potted Pyrus betulae var. betulae seedlings against fire blight are shown in Tables 10 and 11.

[0095] Table 10 The protective efficacy test results of the combination of Bacillus Velezii FX1 fermentation liquid and 2% kasugamycin AS on potted Pyrus beleue seedlings against fire blight

[0096]

[0097] Table 11 Results of the therapeutic efficacy test of the combination of Bacillus Velezii FX1 fermentation liquid and 2% Kasugamycin AS on potted Pyrus betulaceae seedlings

[0098]

[0099]

[0100] The results showed that the protective and therapeutic efficacy of the treatments with a volume ratio of 6:4 and 5:5 of Bacillus Velez FX1 fermentation broth to 2% kasugamycin AS was above 73% for 7-14 days. Among them, the protective efficacy was best when the volume ratio of Bacillus Velez FX1 fermentation broth to 2% kasugamycin AS was 6:4, with the protective and therapeutic efficacy reaching 79.18% and 78.82% respectively, which was significantly higher than other treatments, and the protective efficacy was sustained and stable (Table 10 and Table 11).

[0101] (3) Field evaluation of the efficacy of the compound combination on fire blight of Zaosu pear: The field experiment was conducted at the Zhangye Water-Saving Agricultural Experiment Station of Gansu Academy of Agricultural Sciences. The incidence of fire blight in the two areas was investigated before the experiment, and the experiment was conducted in areas with similar incidence. The experiment was set up with two treatments: treatment group 1: 200 pear trees, conventional chemical control (CK); treatment group 2: fermentation liquid of Bacillus Velezii FX1 (live bacteria concentration 1×10 6 cfu·mL -1 , the preparation method is the same as that in Example 4) and 2% kasugamycin AS (600 times diluted solution) were compounded at a volume ratio of 6:4 to obtain a compound agent to treat 230 pear trees, and each pear tree was sprayed with 5L of the compound combination liquid. After spraying, the disease index and control effect were regularly investigated; the disease index calculation formula is: disease index (DI) = ∑ (number of diseased plants at each level × disease level representative value) × 100 / total number of plants × highest level value; the control effect calculation formula is: control effect (%) = (control disease index-treatment disease index) × 100% / control disease index; the field survey disease grading standard for pear fire blight is, level 0: no symptoms; level I: only branches and tips are affected, and the total number of dead branches, flower rot, and fruit rot does not exceed 10; level III: dead branches, flower rot, and fruit rot exceed 10 in total or large branches are affected; level V: bacterial pus flows from the main trunk or new branches on the main trunk are affected to the main trunk. The field test results of pear fire blight on Zaosu pear are shown in Table 12.

[0102] Table 12 Field efficacy test results of the combination of Bacillus Velezii FX1 fermentation liquid and 2% Kasugamycin AS against early crisp pear fire blight

[0103]

[0104] The results showed that the 6:4 volume mixture of Bacillus Velezii FX1 fermentation broth and 2% kasugamycin showed good efficacy against apricot fire blight, with an average efficacy of 87.66% from flowering stage to 60 days after spraying, and remained stable (Table 12).

[0105] Based on the test results of the toxicity of the complex of Bacillus Velez FX1 fermentation broth and 2% kasugamycin AS against Erwinia amylovora, the effect on the colonization ability of Bacillus Velez FX1 and the evaluation of the prevention effect against Erwinia amylovora, the optimal combination ratio of Bacillus Velez FX1 fermentation broth and 2% kasugamycin AS was determined to be 6:4, based on the environmental protection principle of excellent and stable disease prevention effect and the ability to minimize the use of pesticides.

[0106] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A compound agent for preventing and treating fire blight of apricot, characterized in that: It includes Bacillus velezensis FX1 fermentation liquid and kasugamycin aqueous solution; The volume ratio of the Bacillus Velez subtilis FX1 fermentation broth to the kasugamycin aqueous solution is (9-4): (1-6).

2. The compound according to claim 1, characterized in that The effective viable bacteria count in the fermentation broth of Bacillus Velez FX1 is 1.0×10 6 cfu·mL -1 : The kasugamycin aqueous solution is 2% kasugamycin aqueous solution.

3. The compound according to claim 1, characterized in that: The volume ratio of the Bacillus Velez subtilis FX1 fermentation broth to the kasugamycin aqueous solution is (6-4): (4-6).

4. The compound according to claim 3, characterized in that The compound further comprises Tween-80 and gelatin.

5. The method for preparing the compound according to claim 1, characterized in that: The following steps are involved: The Bacillus Velez subtilis FX1 fermentation liquid and the kasugamycin aqueous solution are mixed to obtain the compound agent.

6. The preparation method according to claim 5, characterized in that: The effective viable bacteria count of Bacillus velez FX1 in the fermentation liquid of Bacillus velez FX1 is 1.0×10 6 cfu·mL -1 The kasugamycin aqueous solution is a 600-fold dilution of a 2% kasugamycin aqueous solution.

7. The preparation method according to claim 5, characterized in that: The volume ratio of the Bacillus Velez subtilis FX1 fermentation broth to the kasugamycin aqueous solution is (6-4): (4-6).

8. The preparation method according to claim 7, characterized in that: After the Velez subtilis FX1 fermentation liquid and the kasugamycin aqueous solution are mixed, the method further comprises the step of adding Tween-80 and gelatin.

9. Use of the compound according to any one of claims 1 to 4 in preventing and treating fire blight of apricot.

10. A method for preventing and treating fire blight of amylopectin, characterized in that: The following steps are involved: The compound according to any one of claims 1 to 4 is sprayed on the plant surface.

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