A wettable powder and its preparation method and application

Abstract

The present application provides a kind of wettable powder and its preparation method and application, belong to forest disease biological control technical field.The wettable powder of the present application is made of the following mass fraction components: Bacillus subtilis WB1 fermentation liquor 90-110 parts, filler 42-48 parts, auxiliary 28-32 parts.The wettable powder of Bacillus subtilis WB1 prepared by the present application has high viable count, good suspensibility, dispersibility and wettability, can be stored, transported and sprayed in large area in forest at room temperature for a long time, has broad-spectrum antibacterial property, and has good control effect on walnut bacterial black spot.The preparation method of the wettable powder of the present application is simple, low in cost, and suitable for large-scale popularization and application.

Description

Technical Field

[0001] This invention relates to the field of biological control technology for forest tree diseases, and in particular to a wettable powder, its preparation method, and its application. Background Technology

[0002] Bacillus subtilis is a thermophilic, aerobic, spore-forming rod-shaped bacterium with diverse physiological characteristics, wide distribution, and easy isolation and culture. This bacterium is widely distributed in nature, non-toxic and harmless to humans and animals, does not pollute the environment, and can produce various antibiotics and enzymes, exhibiting broad-spectrum antibacterial activity. It is currently one of the most studied microorganisms in biopesticides and microbial fertilizers. Patent application number "201310628960.8" discloses a Bacillus subtilis strain WB1, whose fermentation broth is used to prepare biocontrol agents for walnut canker and horse chestnut canker. However, the content of active ingredients in the fermentation broth is low, and the fermentation broth is difficult to store, transport, and use, easily contaminated, and has unstable efficacy.

[0003] Wettable powder (WP) is a formulation made by thoroughly mixing and pulverizing pesticide technicals and inert fillers (such as bentonite and kaolin) in a certain proportion to achieve a specific particle size. Visually, it is indistinguishable from regular powder, but due to the addition of wetting agents, dispersants, and other adjuvants, it can be wetted and dispersed in water to form a suspension for spraying. While this formulation is convenient for storage and transportation, it faces many limitations in production and application. For example, most wettable powders rely solely on spores as the sole criterion for evaluating the active ingredient; if the number of viable bacteria is low, the application effect will be poor. Existing technologies have made significant improvements in spore inducers and the industrial processing of wettable powders, as exemplified by patents with application numbers "20151095109.2, 20171117130.1, and 201910440874.1," but these patents suffer from drawbacks such as complex processes, complex compositions, and high costs.

[0004] In addition, due to the different compatibility of Bacillus species with wetting agents, dispersants and other adjuvants, the adsorption capacity of carriers for various Bacillus species varies. The adsorption amount, survival rate, control effect on harmful bacteria and dosage stability of wettable powders vary greatly. Therefore, the existing formulations or preparation processes of Bacillus wettable powders are not very effective in preparing wettable powders of Bacillus subtilis strain WB1. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a wettable powder for Bacillus subtilis strain WB1, its preparation method and application, which can maintain a high number of viable bacteria and spores, and the preparation process is simple and reduces production costs.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides a wettable powder, which is made from the following components in parts by weight: 90-110 parts of Bacillus subtilis WB1 fermentation broth, 42-48 parts of filler, and 28-32 parts of additives; wherein the filler includes diatomaceous earth, and the additives include Tween-80, PEG6000, and dextrin.

[0008] The Bacillus subtilis WB1 has the accession number CGMCC No. 8439.

[0009] Preferably, the viable cell count in the Bacillus subtilis WB1 fermentation broth is 1×10⁻⁶. 9 ~9×10 9 CFU / mL.

[0010] Preferably, the mass ratio of Tween-80, PEG6000 and dextrin is 9-11:14-16:14-16.

[0011] This invention provides a method for preparing the wettable powder, comprising the following steps:

[0012] (1) Bacillus subtilis WB1 was inoculated into the culture medium for fermentation to obtain fermentation broth;

[0013] (2) Mix the fermentation broth, filler and additives, and dry them to obtain the wettable powder.

[0014] Preferably, the culture medium is supplemented with NH4NO3, and the final concentration of NH4NO3 is 4.5 to 5.5 / L.

[0015] Preferably, the fermentation occurs at a pH of 6.8–7.2, a temperature of 28–33°C, and a rotation speed of 170–220 r·min. -1 .

[0016] Preferably, the drying temperature is 28–32°C and the drying time is 3.5–4.5 h.

[0017] The present invention also provides the application of the wettable powder in the prevention and control of walnut diseases.

[0018] Preferably, the walnut disease includes walnut black spot disease.

[0019] By adopting the above technical solution, the present invention has the following beneficial effects: the Bacillus subtilis WB1 wettable powder prepared by the present invention has a high viable count, with a viable count of 2.5 × 10⁻⁶. 9 CFU / g, spore count 1.7×10 9With a CF / g content, good suspension, dispersibility, and wettability, the powder suspension rate is 87%, and the wetting time is 25 seconds. This Bacillus subtilis WB1 wettable powder exhibits high stability, allowing for long-term storage, transportation, and large-scale spraying in forests at room temperature. It also possesses broad-spectrum antibacterial properties and demonstrates good control efficacy against bacterial black spot disease of walnuts. The preparation method of this wettable powder is simple and low-cost, making it suitable for large-scale application. Attached Figure Description

[0020] Figure 1 The results show the antagonistic activity of Bacillus subtilis WB1 fermentation broth against pathogens, where A is Fusarium solani, B is Dothidea viticola, C is Colletotrichium gloeosporioides, D is Boeremiaexigua, E is Phopsis sp., F is Botryosphaeria dothidea, G is Cytospora juglandis, and H is Xanthomonas arboricola var. Juglandis.

[0021] Figure 2 The results show the performance test results of the secreted protein of Bacillus subtilis WB1. In the figure, A and B are the front and back sides of the colloidal chitin medium, respectively; C and D are the front and back sides of the skim milk medium, respectively.

[0022] Figure 3 The results of lipopeptide gene detection for Bacillus subtilis WB1 secreted protein.

[0023] Figure 4 OD of fermentation broth prepared in Examples 2 and Comparative Examples 1-5 600 Values ​​and protein content graph.

[0024] Figure 5 OD of fermentation broth prepared for Examples 2 and Comparative Examples 6-9 600 Values ​​and protein content graph.

[0025] Figure 6 OD of fermentation broth prepared for Examples 2 and Comparative Examples 10-13 600 Values ​​and protein content graph.

[0026] Figure 7 OD of fermentation broth prepared for Examples 2 and Comparative Examples 14-17 600 Values ​​and protein content graph.

[0027] Figure 8 The interaction between fermentation temperature and pH on the OD of fermentation broth 600 Response surface plot of the value.

[0028] Figure 9 The interaction between fermentation temperature and pH on the OD of fermentation broth 600 Contour plot of values.

[0029] Figure 10 The interaction between rotation speed and temperature on the OD of fermentation broth 600 Response surface plot of the value.

[0030] Figure 11 The interaction between rotation speed and temperature on the OD of fermentation broth 600 Contour plot of values.

[0031] Figure 12 The interaction between rotation speed and pH value on the OD of fermentation broth 600 Response surface plot of the value.

[0032] Figure 13 The interaction between rotation speed and pH value on the OD of fermentation broth 600 Contour plot of values.

[0033] Figure 14 The interaction between fermentation temperature and pH on the OD of fermentation broth 600 Response surface plot of the value.

[0034] Figure 15 The interaction between fermentation temperature and pH on the OD of fermentation broth 600 Contour plot of values.

[0035] Figure 16 The interaction between rotation speed and temperature on the OD of fermentation broth 600 Response surface plot of the value.

[0036] Figure 17 The interaction between rotation speed and temperature on the OD of fermentation broth 600 Contour plot of values.

[0037] Figure 18 The interaction between rotation speed and pH value on the OD of fermentation broth 600 Response surface plot of the value.

[0038] Figure 19 The interaction between rotation speed and pH value on the OD of fermentation broth 600 Contour plot of values.

[0039] Figure 20 The results are for the screening of carriers and additives.

[0040] Figure 21 The graph shows the effect of different amounts of PEG6000 added on the viable bacteria count in wettable powder.

[0041] Figure 22The graph shows the effect of different amounts of Tween-80 added on the viable bacteria count in wettable powder.

[0042] Figure 23 The graph shows the effect of different amounts of dextrin added on the viable bacteria count in wettable powder.

[0043] Figure 24 3D response surface plot of the interaction between the amount of PEG6000 and Tween-80 added and the number of viable bacteria.

[0044] Figure 25 Contour plot of the interaction between PEG6000 and Tween-80 dosage on viable cell count.

[0045] Figure 26 3D response surface plot of the interaction between dextrin and PEG6000 addition amounts on viable cell count.

[0046] Figure 27 Contour plot showing the effect of the interaction between dextrin and PEG6000 additions on viable cell count.

[0047] Figure 28 3D response surface plot of the interaction between dextrin and Tween-80 addition amounts on viable cell count.

[0048] Figure 29 Contour plot showing the effect of the interaction between dextrin and Tween-80 additions on viable cell count.

[0049] Figure 30 This refers to the wettable powder prepared in Example 2 of the present invention.

[0050] Figure 31 The inhibitory effects of wettable powders on pathogens are shown, with the following order of AG: Colletotrichium gloeosporioides, Botryosphaeria dothidea, Phopsis sp., Dothidea viticola, Fusarium solani, Boeremia exigua, and Cytosporajuglandis.

[0051] Figure 32 The results of the field trial of the wettable powder prepared in Example 2 are shown.

[0052] Biological Preservation Instructions

[0053] The Bacillus subtilis WB1 strain provided by this invention is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 8439, deposited on November 6, 2013, at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. Detailed Implementation

[0054] This invention provides a wettable powder, which is made from the following components in parts by weight: 90-110 parts of Bacillus subtilis WB1 fermentation broth, 20-30 parts of filler, and 15-20 parts of additives; wherein the filler includes diatomaceous earth, and the additives include Tween-80, PEG6000, and dextrin.

[0055] The Bacillus subtilis WB1 has the accession number CGMCC No. 8439.

[0056] The Bacillus subtilis WB1 fermentation broth is 90-110 parts by weight, more preferably 95-105 parts, and even more preferably 100 parts.

[0057] The filler is 42-48 parts by weight, more preferably 44-46 parts, and even more preferably 45 parts; the filler includes diatomaceous earth.

[0058] The additive is present in 28 to 32 parts by weight, more preferably 29 to 31 parts, and even more preferably 30 parts;

[0059] The adjuvants include Tween-80, PEG6000 and dextrin, wherein the mass ratio of Tween-80, PEG6000 and dextrin is 9-11:14-16:14-16, more preferably 9.5-10.5:14.5-15.5:14.5-15.5, and even more preferably 10:15:15.

[0060] The Bacillus subtilis WB1 strain has the preservation number CGMCC No. 8439 and is the strain in the invention patent with patent number "201310628960.8" and invention title "A Walnut Endophytic Bacterial Strain Resisting Walnut Canker Disease and Its Application".

[0061] The present invention also provides a method for preparing the wettable powder, comprising the following steps:

[0062] (1) Bacillus subtilis WB1 was inoculated into the culture medium for fermentation to obtain fermentation broth;

[0063] (2) Mix the fermentation broth, filler and additives, and dry them to obtain the wettable powder.

[0064] In this invention, the Bacillus subtilis WB1 culture tube is first removed, and the culture is revived and expanded. This invention does not have specific limitations on the revival and expansion culture of Bacillus subtilis WB1, as long as the revival and expansion culture of the culture can be achieved. When the strain is in the exponential growth phase, it is transferred to a fermenter for further cultivation. The culture medium in the fermenter is based on LB medium, with additional carbon and nitrogen sources added. The carbon source is glucose, sucrose, or starch, and the amount of carbon source added to the culture medium is 4.5–5.5 g / L, more preferably 4.7–5.2 g / L, and more preferably 5.0 g / L. The nitrogen source is NH4NO3, and the final concentration of NH4NO3 in the culture medium is 4.5–5.5 g / L, more preferably 4.8–5.3 g / L, and more preferably 5.0 g / L. During fermentation, the pH of the culture medium is 6.8–7.2, more preferably 6.9–7.1, and even more preferably 7.0; the fermentation temperature is 28–33°C, more preferably 29–32°C, and even more preferably 30°C; the fermentation is accompanied by stirring at a speed of 170–220 r / min. -1 More preferably, it is 180–210 r·min -1 More preferably 200 r·min -1 OD 600 Maximum (2.03), with a spore content exceeding 1×10⁻⁶. 9 Fermentation can be stopped when CFU / mL is reached, and the resulting product is the Bacillus subtilis WB1 fermentation broth.

[0065] In this invention, Bacillus subtilis WB1 fermentation broth is mixed with filler and additives in the above proportions, stirred evenly, and then dried. The drying temperature is 28-32°C, more preferably 29-31°C, and even more preferably 30°C. The drying time is 3.5-4.5 hours, more preferably 4 hours. The drying is preferably carried out in a forced-air drying oven. After drying, the powder is allowed to cool to room temperature to obtain the wettable powder of this invention.

[0066] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0067] In this embodiment of the invention, the NA culture medium consists of 10g peptone, 3g beef extract, 5g sodium chloride, and 1000mL of 2% agar water, adjusted to pH 7.0.

[0068] The Bacillus subtilis WB1 liquid culture medium consisted of 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, and water, adjusted to pH 7.0.

[0069] The fermentation medium consisted of 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, 5 g / L glucose, 5 g / L NH4NO3, and water, with the pH adjusted to 7.0.

[0070] The sterilization conditions for each culture medium were: 0.10 MPa, 121℃ for 30 min.

[0071] Example 1

[0072] Remove the bacterial culture from the preservation tube and reactivate it using NA solid medium streaks, incubating at 28°C for 48 hours. Pick a single colony from the plate and inoculate it onto NA solid medium in an eggplant bottle, spreading it evenly across the entire bottle. Incubate at 28°C for 48 hours. Wash the bacterial growth from the eggplant bottle with 1L of sterile water. Inoculate the washed Bacillus subtilis into a 200L seed fermentation tank containing 120L of Bacillus subtilis liquid medium. The initial pH is 6.8, the temperature is 28°C, and the fermentation speed is 170 rpm. -1 After 24 hours of cultivation, the bacterial cells were in the exponential growth phase. The culture medium was then transferred to a 2000L fermenter at 70% capacity, with a pH of 6.8, a temperature of 28℃, and an operating rate of 170 rpm. -1 Fermentation was carried out. After 24 hours of cultivation, the spore content was found to be greater than 1×10⁻⁶. 9 Fermentation can be stopped when CFU / mL is reached, and the resulting product is the Bacillus subtilis WB1 fermentation broth.

[0073] 90 kg of Bacillus subtilis WB1 fermentation broth, 42 kg of diatomaceous earth, 6.82 kg of Tween-80, 10.59 kg of PEG6000 and 10.59 kg of dextrin were mixed, stirred evenly and placed in a forced-air drying oven and dried at 28℃ for 3.5 h to obtain a wettable powder.

[0074] Example 2

[0075] Remove the bacterial culture from the preservation tube and reactivate it using NA solid medium, incubating at 30°C for 48 hours. Pick a single colony from the plate and inoculate it onto NA solid medium in an eggplant bottle, covering the entire bottle. Incubate at 30°C for 48 hours. Wash the bacterial growth from the eggplant bottle with 1L of sterile water. Inoculate the washed Bacillus subtilis into a 200L seed fermentation tank containing 120L of Bacillus subtilis liquid medium. The initial pH is 7.0, the temperature is 30°C, and the fermentation speed is 200 rpm. -1 After 24 hours of cultivation, the bacterial cells were in the exponential growth phase. The culture medium was then transferred to a 2000L fermenter at 70% capacity, with a pH of 7.0, a temperature of 30℃, and an incubation time of 200 rpm. -1 Fermentation was carried out. After 24 hours of cultivation, the spore content was found to be greater than 1×10⁻⁶. 9Fermentation can be stopped when CFU / mL is reached, and the resulting product is the Bacillus subtilis WB1 fermentation broth.

[0076] 100 kg of Bacillus subtilis WB1 fermentation broth, 44.5 kg of diatomaceous earth, 7.5 kg of Tween-80, 11.25 kg of PEG6000 and 11.25 kg of dextrin were mixed, stirred evenly and placed in a forced-air drying oven and dried at 30°C for 4 hours to obtain a wettable powder.

[0077] Example 3

[0078] Remove the bacterial culture from the preservation tube and reactivate it using NA solid medium, incubating at 32°C for 48 hours. Pick a single colony from the plate and inoculate it onto NA solid medium in an eggplant bottle, spreading it evenly across the entire bottle. Incubate at 32°C for 48 hours. Wash the bacterial growth from the eggplant bottle with 1L of sterile water. Inoculate the washed Bacillus subtilis into a 200L seed fermentation tank containing 120L of Bacillus subtilis liquid medium. The initial pH is 7.2, the temperature is 32°C, and the fermentation speed is 220 rpm. -1 After 24 hours of cultivation, the bacterial cells were in the exponential growth phase. The culture medium was then transferred to a 2000L fermenter at 70% capacity, with a pH of 7.2, a temperature of 32℃, and an operating rate of 220 rpm. -1 Fermentation was carried out. After 24 hours of cultivation, the spore content was found to be greater than 1×10⁻⁶. 9 Fermentation can be stopped when CFU / mL is reached, and the resulting product is the Bacillus subtilis WB1 fermentation broth.

[0079] 110 kg of Bacillus subtilis WB1 fermentation broth, 48 kg of diatomaceous earth, 8.2 kg of Tween-80, 11.9 kg of PEG6000 and 11.9 kg of dextrin were mixed, stirred evenly and placed in a forced-air drying oven and dried at 32℃ for 4.5 h to obtain a wettable powder.

[0080] Experiment Example 1: Antibacterial Spectrum and Secretory Protein Performance Test

[0081] Under aseptic conditions, using a sterile punch with a diameter of 3 mm, mycelial discs of eight strains—Fusarium solani, Dothidea viticola, Colletotrichium gloeosporioides, Boeremia exigua, Phopsis sp., Botryosphaeria dothidea, Cytospora juglandis, and Xanthomonas arboricola var. Juglandis—were cut from the edge of the colony. The mycelial discs were inoculated into the center of a PDA agar plate using an inoculation needle. Fresh WB1 fermentation broth prepared in Example 2 was then drawn up with 3 cm cotton thread and placed 2.5 cm away from both sides of the central mycelial disc. The plate was inverted and incubated statically at 25°C. LB plates were used as a blank control. Results are as follows: Figure 1 As shown.

[0082] Five additional petri dishes were prepared, each containing one of the following media: ABP (Poria cocos powder) medium, colloidal chitin medium, cellulose-decomposing bacteria medium, skim milk medium, and lipase detection medium, respectively. 20 μL of fresh fermentation broth was dropped into the center of each of the five petri dishes. The dishes were inverted and incubated at 28°C. The presence of hydrolysis zones around the colonies was observed. The results are as follows: Figure 2 As shown in Table 1. After culturing for 24 hours, DNA was extracted using the CTAB method, and the lipopeptide synthesis gene was detected by homologous PCR amplification. The primers used are shown in Table 1. The PCR reaction conditions were 95℃ for 10 min, 94℃ for 45 s; 50℃ for 45 s (annealing temperature depends on different primers); 72℃ for 2 min, 35 cycles; 72℃ for 10 min; 4℃ forever.

[0083] Table 1 Primer sequences

[0084]

[0085] Depend on Figure 1 It can be seen that the fermentation broth of Bacillus subtilis strain WB1 showed significant inhibitory effects on all tested forest tree pathogens; Figure 2 As can be seen, hydrolysis zones are generated around the colonies in colloidal chitin medium and skim milk medium, indicating that Bacillus subtilis strain WB1 can secrete chitinase and protease. Figure 3 The results of lipopeptide gene detection for Bacillus subtilis strain WB1 show that the genome of Bacillus subtilis strain WB1 contains genes for the synthesis of surfactant, fengycin, bacillomycin, and bacylisin, and can secrete related antibiotics.

[0086] Experiment Example 2: Optimization of Fermentation Conditions

[0087] Based on LB medium, the optimal carbon and nitrogen sources and their concentrations, as well as the optimal fermentation conditions, were determined through single-factor screening of pH, temperature, and rotation speed ranges. After fermentation, the OD value and protein content of the Bacillus subtilis WB1 fermentation broth were measured at 600 nm. The results are as follows: Figures 4-7 As shown. By Figure 4 It can be seen that when ammonium sulfate is used as an additional nitrogen source, the protein content is significantly reduced; when urea is used as an additional nitrogen source, both the viable bacteria concentration and the protein content are significantly reduced. Figure 5 It can be seen that when the fermentation pH is 5 or 6, the concentration of viable bacteria and the protein content are slightly lower, while when the fermentation pH is 8 or 9, the concentration of viable bacteria and the protein content decrease sharply. Figure 7 It can be seen that when the rotational speed is 200 r·min -1 At that time, it has the highest concentration of live bacteria and protein content.

[0088] Box-Behnken experiments were designed using Design-Expert 13 software, and analysis of variance was performed on the results. The interaction of fermentation temperature, pH, and rotation speed on OD6 was investigated. 00 The 3D response surface plot and contour plot of the impact are as follows: Figures 8-19 As shown in the figure, when the pH is 6, the temperature is 33℃, and the rotation speed is 200 r·min -1 At that time, OD in the fermentation broth 600 Reaching its maximum value, OD 600 The value is 2.008; the 3D response surface plot and contour plot of the interaction of various factors on protein content are as follows: Figures 18-23 As shown in the figure. It can be seen from the graph that when the pH is 7, the temperature is 33℃, and the rotation speed is 200 r·min... -1 At that time, the protein content in the fermentation broth was 8.947 mg / mL. This invention was carried out at a pH of 7, a temperature of 30°C, and a rotation speed of 200 r / min. -1 Under the fermentation conditions specified, the maximum bacterial yield and maximum secretion antagonistic protein of WB1 can be obtained. Verification experiments were conducted to obtain the OD values. 600 =2.03, protein content is 8.79 mg / mL.

[0089] Experimental Example 3: Determination of the types and proportions of fillers and additives for wettable powders

[0090] Diatomaceous earth, calcium carbonate, and kaolin were added to LB solid medium at a concentration of 5 g / L, sterilized, and then poured into plates. 1 mL of fresh fermentation broth was then used for serial dilution, with each dilution derived from the previous step. Each step involved a 1:10 dilution, for a total of eight dilutions. (Dilution 10...)8 After dilution, 100 μL of the diluted solution was evenly spread onto LB agar plates. Using a plate without the carrier as a control, the experiment was repeated three times, inverted, and incubated at 28°C for 2 days. Colonies on the plates were counted to study the effect of each carrier on strain WB1. By comparing the number of colonies, the optimal carrier for the wettable powder was determined.

[0091] Carrier adsorption capacity: Based on the optimal carrier selected through screening, under aseptic conditions, accurately weigh 10.0 g of diatomaceous earth carrier into a beaker, denoted as M (unit: kg). Slowly pour the fresh fermentation broth of strain WB124 h into the beaker while stirring with a glass rod until fully mixed. Stop when the mixture is moist, uniform, free of lumps, and no water flows out of the beaker. Record the volume of fermentation broth poured in, denoted as V (unit: L), and calculate the maximum adsorption capacity H (H = V·M). -1 ).

[0092] Sodium dodecylbenzenesulfonate (5 g / L), Tween-80 (5 mL / L), PEG6000 (5 g / L), sodium lignosulfonate (5 g / L), and dextrin (1 g / L) were added to LB solid medium and sterilized to prepare plates. 1 mL of fresh fermentation broth was then used for serial dilution, with each dilution derived from the previous step, and each solution was diluted 1:10, for a total of eight dilutions. 100 μL of each diluted solution was then evenly spread onto plates. The effects of different carriers on strain WB1 were investigated, and the optimal adjuvant for the wettable powder was determined by comparing the number of colonies.

[0093] Results: Diatomaceous earth was the optimal carrier, and Tween-80, PEG6000, and dextrin were the optimal adjuvants. The adsorption capacity of diatomaceous earth carrier was 2.24 L / kg. When the addition amount of Tween-80 was in the range of 5–10 mL, the viable bacterial count of the wettable powder peaked; when the addition amount of PEG6000 was in the range of 8–12 g, the viable bacterial count of the wettable powder was the highest; when the addition amount of dextrin was in the range of 10–14 g, the viable bacterial count of the wettable powder peaked. Figures 20-23 As shown.

[0094] Box-Behnken experiments were designed using Design-Expert 13 software, and analysis of variance was performed on the experimental results. The coefficient of determination R0 of the Box-Behnken model was calculated. 2 =0.9066, indicating good model correlation. The order of influence of each factor on the viable count of wettable powder is: PEG6000, dextrin, Tween-80, with dextrin having the greatest interaction effect. According to the model prediction, when the bacterial solution volume is 100mL, the maximum viable count of the powder is reached when the addition amount of Tween-80 is 7.5mL, PEG6000 is 11g, and dextrin is 11.5g, with a viable count of 4.2×10⁻⁶.9 CFU / g, such as Figures 24-29 As shown. A model validation experiment was conducted on the response surface methodology predictions, yielding a viable bacterial count of 4.18 × 10⁻⁶. 9 CFU / g. The experimental value was basically the same as the predicted value, verifying that the regression equation and response surface model could accurately reflect the influence of various factors on the viable count of wettable powder. The optimal mass ratio of excipients for wettable powder was finally determined to be 60% diatomaceous earth, 10% Tween-80, 15% PEG6000, and 15% dextrin.

[0095] Experimental Example 4: Quality Testing and Antagonistic Effect Testing of Wettable Powder

[0096] The wettable powder prepared in Example 2 was miscible with water at a ratio of 1g:500mL. The suspension rate, wettability, and pH of the powder were determined according to national standards [suspension rate determination (GB / T14825-2006), wettability determination (GB / T5451-2001), pH determination (GB / T1601-1993)]. The antibacterial effect of the wettable powder on pathogens was determined by plate confrontation test.

[0097] Plate confrontation test: The wettable powder was diluted according to the commonly used dilution ratio of commercial powders (500 times dissolved in water), and after being fully soaked in sterile cotton, it was placed in 1 / 3 of the plate, with pathogenic bacterial cake in the middle. The plate was then placed in the dark at 25℃ for 5 days to determine the antibacterial effect of the wettable powder on the pathogenic bacteria.

[0098] Results: According to the national standard test results, the number of viable bacteria in the wettable powder was 2.5 × 10⁻⁶. 9 CFU·g -1 The number of spores was 1.7 × 10⁻⁶. 9 CFU·g -1 The powder suspension rate was 87%, the wetting time was 25 seconds, and the pH was 6.18, all within the national standard range. The powder prepared by this invention is qualified and can be used to prevent and control plant diseases.

[0099] The antibacterial effect of wettable powder on pathogens is as follows: Figure 31 As shown. By Figure 31It is known that the wettable fungicide of the present invention has a good inhibitory effect on Fusarium solani, Dothidea viticola, Colletotrichium gloeosporioides, Boeremia exigua, Phoopsis sp., Botryosphaeria dothidea, and Cytosporajuglandis.

[0100] Experimental Example 5: Field control effect of Bacillus subtilis WB1 wettable powder

[0101] The wettable powder prepared in Example 2 was dissolved in water at a ratio of 1g:500mL. In May, when bacterial black spot disease of walnuts first appeared, the powder was sprayed onto the upper and lower surfaces of walnut leaves using an agricultural sprayer. The control effect was observed one week later. Simultaneously, walnut trees from the same orchard that were sprayed with tap water served as a control, with 10 trees per treatment. The control efficacy was statistically analyzed based on the incidence rate of the upper, middle, and lower leaves of the canopy. One hundred leaves were randomly selected from each location for statistical analysis, and the incidence rate and control efficacy were recorded.

[0102] Incidence rate = (Number of diseased leaves / Total number of leaves surveyed) × 100%

[0103] Prevention efficacy = (incidence rate in control group - incidence rate in treatment group) / incidence rate in control group × 100%;

[0104] The results are as follows Figure 32 As shown, the incidence of disease in the upper, middle and lower leaves of trees sprayed with Bacillus subtilis WB1 wettable powder was significantly lower than that in the control group, and the control efficacy of the treatment group was between 85% and 97.6% after one week.

[0105] Experimental Example 6

[0106] The wettable powder prepared in Example 2 of this invention, after being stored at room temperature for 6 months and then at low temperature for 2 years, showed no significant weakening of its antibacterial effect, with viable cell counts of 1.3 × 10⁻⁶. 5 CFU / g, 3.7×10 7 CFU / g; After 1 hour of ultraviolet irradiation in the clean bench, the viable bacterial count was 3.7 × 10⁻⁶. 5 The CFU / g indicates that the wettable powder prepared in this application has storage stability and UV stability.

[0107] As can be seen from the above embodiments, the present invention provides a wettable powder, its preparation method and application. The wettable powder of the present invention has the characteristics of high viable bacteria count, low cost and easy storage.

[0108] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A wettable powder characterized by, It is made from the following components in parts by weight: Bacillus subtilis ( Bacillus subtilis The fermentation broth of Bacillus subtilis WB1 consists of 90-110 parts, packing material of 42-48 parts, and auxiliary agent of 28-32 parts; the packing material is diatomaceous earth, and the auxiliary agent is composed of Tween-80, PEG6000 and dextrin; the biological preservation number of Bacillus subtilis WB1 is CGMCC No.8439. The viable cell count in the fermentation broth of the Bacillus subtilis WB1 is 1 x 10 9 9 x 10 9 CFU / mL; The mass ratio of Tween-80, PEG6000 and dextrin is 9-11:14-16:14-16.

2. A process for the preparation of the wettable powder of claim 1, characterized in that, The method comprises the following steps: (1) inoculating Bacillus subtilis WB1 into a culture medium to perform fermentation, and obtaining a fermentation liquor; (2) mixing the fermentation liquor, a filler and an additive, and drying to obtain the wettable powder.

3. The preparation method according to claim 2, characterized in that, The nitrogen source in the culture medium comprises NH4NO3, and the final concentration of NH4NO3 in the culture medium is 4.5-5.5 g / L.

4. The production method according to claim 2 or 3, characterized by, The fermentation has a pH value of 6.8-7.2, a temperature of 28-33℃, and a rotating speed of 170-220 r·min -1 .

5. The preparation method according to claim 4, characterized in that, The drying temperature is 28-32 DEG C, and the drying time is 3.5-4.5 h.

6. The wettable powder of claim 1 is used for preventing and treating walnut diseases.

7. Use according to claim 6, characterized in that, The walnut diseases comprise walnut black spot.

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