Composition for controlling grape gray mold and use thereof

Abstract

The application discloses a composition for preventing and treating grape gray mold and application thereof. The composition for preventing and treating grape gray mold disclosed by the application is composed of iprodione and Bacillus velezensis GSBZ09. Experiments prove that the composition of iprodione and Bacillus velezensis GSBZ09 can prevent and treat grape gray mold, and the effect is best when the ratio of iprodione and Bacillus velezensis GSBZ09 is 300 mu:10 8 CFU. It is illustrated that the composition of the application can be used for preventing and treating grape gray mold, and has a good application prospect.

Description

Technical Field

[0001] This invention relates to the field of plant protection, specifically to compositions for controlling grape gray mold and their applications. Background Technology

[0002] Currently, with the rapid development of the grape industry and the continuous expansion of grape planting area, the occurrence of grape diseases is also increasing year by year. Grape gray mold is an important fungal disease of grapes, occurring in grape-producing regions worldwide. Its pathogen is *Botrytis cinerea*. Under natural conditions, *Botrytis cinerea* can infect grape plants, and under suitable environmental conditions, it can cause a large-scale outbreak, resulting in economic losses of approximately 20%, and in severe cases, more than 80%, causing huge economic losses to grape cultivation. Currently, the control of grape gray mold in my country mainly relies on chemical agents. However, chemical pesticides not only pollute the environment but also cause pathogens to develop resistance, leading to reduced control effectiveness and seriously affecting the sustainable development of agriculture in my country. Developing environmentally friendly biological pesticides is crucial for the efficient and green production of agriculture in my country.

[0003] As an economic crop that can be eaten fresh or used for winemaking, grapes have high requirements for their intrinsic quality. However, pesticide residues, pathogen resistance, and toxin content seriously affect the safe production of grapes and limit the large-scale use of pesticides.

[0004] Bacillus belye possesses excellent growth-promoting and disease-preventing functions, a broad antibacterial spectrum, and great development potential. It has been found to have a strong inhibitory effect on the pathogens of grape white rot, grape canker, grape anthracnose, and grape crown gall.

[0005] As the grape industry gradually develops towards ecological, safe, and high-quality practices, and as people increasingly value their own health and living environment, they are gradually realizing the importance of safe, pollution-free, and environmentally friendly prevention and control of grape diseases. Therefore, in controlling grape gray mold, how to reduce the use of chemical pesticides to minimize environmental pollution and improve safety has become a pressing technical problem for those skilled in the art. Summary of the Invention

[0006] The technical problem to be solved by this invention is how to prevent and control grape gray mold.

[0007] To solve the above-mentioned technical problems, the present invention first provides a composition composed of iprodione and Bacillus velezensis GSBZ09.

[0008] In the above composition, the ratio of iprodione to Bacillus velezensis GSBZ09 can be (100 / 3μg-300μg): 10 8 CFU.

[0009] Specifically, the ratio of iprodione to Bacillus velezensis GSBZ09 in the composition can be 300 μg: 10 μg. 8 CFU.

[0010] The composition can be used to inhibit the growth of grape gray mold and to control grape gray mold.

[0011] The application of the composition in inhibiting the growth of grape botrytis cinerea is also within the scope of protection of this invention.

[0012] The application of the composition in the preparation of products that inhibit the growth of grape botrytis cinerea is also within the scope of protection of this invention.

[0013] In the above applications, the grape gray mold fungus can be Botrytis cinerea.

[0014] The application of the composition in the prevention and control of grape gray mold is also within the scope of protection of this invention.

[0015] The application of the composition in the preparation of products for the prevention and control of grape gray mold is also within the scope of protection of this invention.

[0016] The present invention also provides a method for controlling grape gray mold, the method comprising: applying the composition to grape plants infected with grape gray mold to achieve control of grape gray mold.

[0017] The present invention also provides a product for controlling grape gray mold, wherein the active ingredient is the aforementioned composition.

[0018] Experiments have shown that the combination of iprodione and Bacillus velezensis GSBZ09 can control grape gray mold, with the optimal ratio of iprodione to Bacillus velezensis GSBZ09 being 300 μg: 10 μg. 8 The effect is best with CFU. This indicates that the composition of the present invention can be used for the control of grape gray mold and has excellent application prospects.

[0019] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way. Attached Figure Description

[0020] Figure 1 The effects of Bacillus belyssus GSBZ09 and Bacillus belyssus GS09 on the growth and hyphae of Botrytis cinerea.

[0021] Figure 2 This is a graph showing the inhibitory effects of different concentrations of iprodione on Bacillus belysin GSBZ09.

[0022] Figure 3 This is a diagram illustrating the indoor in vitro control effect of iprodione combined with Bacillus vesicles GSBZ09 on grape gray mold. A: Iprodione solution; B: GSBZ09 fermentation broth; C: Iprodione solution: GSBZ09 fermentation broth = 1:1; D: GS09 fermentation broth; E: Iprodione solution: GS09 fermentation broth = 1:1; F: Water.

[0023] Figure 4 This is a diagram showing the indoor in vitro control effect of this composition against grape gray mold. A: Iprodione solution: GSBZ09 fermentation broth = 3:1; B: Iprodione solution: GSBZ09 fermentation broth = 1:1; C: Iprodione solution: GSBZ09 fermentation broth = 1:3; D: Water; E: Iprodione solution: GS09 fermentation broth = 3:1; F: Iprodione solution: GS09 fermentation broth = 1:1; G: Iprodione solution: GS09 fermentation broth = 1:3; H: Water.

[0024] Figure 5 The results show the potting control effect of iprodione combined with Bacillus vesicae GSBZ09 on botrytis cinerea. A: Botrytis cinerea; B: CK; C: GSBZ09; D: iprodione; E: iprodione + GSBZ09; F: fluazinam. Detailed Implementation

[0025] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials, reagents, instruments, etc., used in the following embodiments are commercially available. All quantitative experiments in the following embodiments were performed in at least three replicates, and the results were averaged.

[0026] The grape gray mold fungus, Botrytis cinerea, described in the following examples is described in the literature “Yin Xiangtian, Yang Liying, Wang Chaoping, Chen Yingchun, Wu Xinying. Indoor toxicity determination of different types of fungicides against 6 grape rot pathogens. Jiangsu Agricultural Sciences, 2021, 49(03):99-104.”, which is available to the public from the Shandong Grape Research Institute.

[0027] 99% Iprodione technical grade: Qingdao Fengfei Biotechnology Co., Ltd., 36734-19-7.

[0028] 99% imazalil technical grade: Anhui Haozhou Baier Pharmaceutical Co., Ltd., 35554-44-0.

[0029] 99% azoxystrobin technical grade: Hubei Bolan Chemical Co., Ltd., 131860-33-8.

[0030] 99% cyazofamid technical grade: Shandong Lutong Biotechnology Co., Ltd., 188425-85-6.

[0031] 99% iprodione technical grade: Shaanxi Yinong Gaoke Pharmaceutical Co., Ltd., 32809-16-8.

[0032] 99% pyrimethanil technical grade: Hebei Mojin Biotechnology Co., Ltd., 53112-28-0.

[0033] 99% Fluazinam technical grade: Hubei Hanwei Chemical Co., Ltd., 79622-59-6.

[0034] The test culture media in the following examples are as follows:

[0035] Potato glucose medium (PDA): 200g potatoes (cooked until soft), 20g glucose, 15g agar, and dilute to 1L with deionized water.

[0036] Tryptone liquid medium (LB): 5g yeast extract, 10g tryptone, 10g sodium chloride, distilled water to a final volume of 1L.

[0037] Tryptone solid medium (LB): 5g yeast extract, 10g tryptone, 10g sodium chloride, 15g agar powder, 1L distilled water.

[0038] Example 1: Effects of different chemical pesticides on indoor toxicity of grape gray mold

[0039] 1. Preparation of Grape Botrytis cinerea mycelium

[0040] Take sterilized PDA medium, prepare PDA plates, pick out mycelia of Grape Botrytis cinerea and place them in the center of the PDA plate, incubate at 20℃ for 5 days, use a 0.4cm diameter punch to make holes in the PDA plate covered with mycelia, and use an inoculation needle to pick out the mycelial cake for later use.

[0041] 2. Preparation of the medicine solution

[0042] Accurately weigh 0.101 g of each of the following pesticides (99% iprodione, 99% imazalil, 99% pyraclostrobin, 99% boscalid, 99% procymidone, 99% pyrimethanil, and 99% fluazinam) into a 10 mL volumetric flask. Dissolve the pesticides thoroughly in 2 mL of sterile water, then dilute to the mark with sterile purified water to prepare a 10 mg / mL stock solution of each pesticide. Further dilute the 10 mg / mL stock solution to 100 μg / mL, 50 μg / mL, 10 μg / mL, 5 μg / mL, and 1 μg / mL concentrations for use.

[0043] 3. Effects of pesticides on Grape Botrytis cinerea

[0044] Sterilized PDA medium was used to prepare PDA plates containing different mass concentrations of the pesticide, with final mass concentrations of 1, 5, 10, 50, and 100 μg / mL. After cooling, 5.0 mm diameter mycelial discs were inoculated into the center of the pesticide-containing plates, and the plates were incubated upside down at 24℃ for 3 days. The colony diameter of each treatment plate was measured using the cross-cross method, and the inhibition rate was calculated. Water was used as a control, and each treatment was replicated three times. The inhibition rate of a single chemical pesticide against the mycelial growth of *Botrytis cinerea* was calculated. The inhibition rate was converted into an inhibition probability value (y). Based on the logarithm of the chemical pesticide mass concentration and the logarithm of the biocontrol fungus concentration (x) and the inhibition probability value (y), the regression equation y = bx + a was obtained, and the median mass concentration (EC) of the chemical pesticide for inhibition was calculated. 50 Values. The results are shown in Table 1.

[0045] The formula for calculating the inhibition rate is: Inhibition rate (%) = (Control colony growth diameter - Treatment colony net growth diameter) / Control colony net growth diameter × 100%.

[0046] Table 1. Results of toxicity assay of the test agents against grape gray mold

[0047] Iprodione 1，5，10，50，100 y = 0.8424x + 4.3172 0.9525 6.46 Azoxystrobin 1，5，10，50，100 y = 0.6905x + 4.2570 0.9200 12.02 pyrimethanil 1，5，10，50，100 y = 2.6792x + 1.3419 0.9590 23.44 Fluazinam 1，5，10，50，100 y = 1.0475x + 3.2049 0.9157 51.29 iprodione 1，5，10，50，100 y = 0.7601x + 3.6602 0.9238 57.22 Imazalil 1，5，10，50，100 y = 1.2380x + 2.7963 0.9586 60.15 Cyclomethasone 1，5，10，50，100 y = 0.4636x + 3.688 0.9499 670.50

[0048] Based on the above results, iprodione EC 50 The value was 6.46, indicating the best inhibitory effect.

[0049] Example 2: Effect of Bacillus belye GSBZ09 on the growth of Botrytis cinerea.

[0050] Bacillus velezensis GSBZ09 is a biocontrol strain isolated by the applicant. Its accession number at the China General Microbiological Culture Collection Center is CGMCC No. 23947, and this strain is described in Chinese patent application 202210132776.3.

[0051] Bacillus amyloliquefaciens GS09 is described in the literature “Yin Xiangtian, Ji Shaolan, Yang Yang. Growth-promoting effects of three Bacillus species on chili peppers. Anhui Agricultural Sciences, 2017, 45(1):55-57.” and can be obtained from the Shandong Grape Research Institute.

[0052] 1. The effect of Bacillus vesiclei on Botrytis cinerea, the causal agent of grape gray mold.

[0053] The strain GSBZ09 was inoculated into liquid LB medium and cultured with shaking at 28°C for 16 hours to obtain the biocontrol solution.

[0054] Grape gray mold was inoculated into the center of PDA medium. Four holes were made at four opposite points 10 mm from the edge of the petri dish using a pipette tip, and 5 μl of biocontrol bacterial suspension was added to each hole. A blank control was included, inoculated only with LB liquid medium. The inoculation was repeated three times. The plates were incubated at 28°C. After the control group mycelia had fully colonized the plates, the width of the inhibition zone was measured, and the colony diameter of grape gray mold was also measured. The colony growth inhibition rate was calculated using the following formula:

[0055] Colony growth inhibition rate (%) = (control colony growth diameter - treated colony net growth diameter) / control colony net growth diameter × 100%.

[0056] Experimental results are as follows Figure 1 As shown, *Bacillus belyceae* GSBZ09 exhibited a 68.15% inhibition rate against *Botrytis cinerea* colony growth, while *Bacillus amyloliquefaciens* GS09 showed a 32.02% inhibition rate. Therefore, strain GSBZ09 has a strong inhibitory effect on *Botrytis cinerea*, while *Bacillus amyloliquefaciens* GS09 has a relatively low inhibitory effect.

[0057] 2. Determination of safe concentrations of different concentrations of iprodione against Bacillus vesiculosus GSBZ09

[0058] 99% iprodione was diluted with sterile water and mixed with PDA medium to prepare iprodione-containing plates with concentrations of 50 μg / mL, 100 μg / mL, 200 μg / mL, and 500 μg / mL. The plates were then poured and air-dried. Bacillus belye GSBZ09 was streaked onto each concentration plate, with a medium without iprodione serving as a control. The growth of the strain was observed.

[0059] like Figure 2 As shown, compared with the control, 50-500 μg / mL of iprodione did not significantly inhibit the growth of Bacillus belyssus GSBZ09.

[0060] Example 3: The combination of iprodione and Bacillus vesalivarius GSBZ09 can be used for the control of gray mold in grapes.

[0061] Preparation of Botrytis cinerea spore suspension: After activation of Botrytis cinerea, the spores were incubated in a constant temperature incubator at 23℃. After spore production, the spores were washed off with sterile water, the mycelium was removed by filtration, the spores were collected by centrifugation at 4000 r / min for 10 min, the supernatant was discarded, and the spores were resuspended in sterile water to adjust the spore concentration to 1×106 spores / mL.

[0062] Bacillus belye GSBZ09 fermentation broth: Single colonies of strain GSBZ09 were picked and incubated in 50 mL LB medium at 28°C and 180 rpm. -1 Incubate for 24 h; then transfer to LB medium at a volume ratio of 0.1% and incubate at 28 °C and 180 rpm. -1 After culturing for 48 hours, fermentation broth of strain GSBZ09 was obtained, in which the concentration of Bacillus belyssus GSBZ09 was 10. 8 CFU / mL.

[0063] Bacillus amyloliquefaciens GS09 fermentation broth: Pick a single colony of strain GS09 and incubate it in 50 mL LB medium at 28°C and 180 rpm. -1 Incubate for 24 h; then transfer to LB medium at a volume ratio of 0.1% and incubate at 28 °C and 180 rpm. -1 After culturing for 48 hours, the fermentation broth of strain GS09 was obtained, in which the concentration of Bacillus amyloliquefaciens GS09 was 10. 8 CFU / mL.

[0064] Iprodione solution: 99% of the original iprodione drug is dissolved in sterile water, with a concentration of 100 μg / mL.

[0065] Combination solution 1: Iprodione solution and Bacillus vesiculosus GSBZ09 fermentation broth are mixed at a volume ratio of 1:1.

[0066] Combination solution 2: Iprodione solution and Bacillus amyloliquefaciens GS09 fermentation broth are mixed at a volume ratio of 1:1.

[0067] Botrytis cinerea spores at a concentration of 1×10⁶ spores / mL were inoculated onto Red Globe grape berries in an inoculation volume of 10 μL. 24 h after inoculation, control was achieved by spraying with iprodione solution, Bacillus vesiculosus GSBZ09 fermentation broth, and combination solution 1-2, respectively, in a spray volume of 20 μL. Water was used as a control to replace the pesticide / bacterial solution.

[0068] Results after 7 days of spraying: Figure 3As shown, the control effect of iprodione combined with Bacillus vesiculosus GSBZ09 in a 1:1 ratio is significantly better than that of iprodione alone or Bacillus vesiculosus alone, and also better than the mixture of iprodione and Bacillus amyloliquefaciens GS09.

[0069] Example 4: Screening for the optimal combination ratio of Bacillus belycetamol GSBZ09 and iprodione

[0070] The iprodione solution and Bacillus vesicle GSBZ09 fermentation broth from Example 3 were selected and sprayed at volume ratios of 1:1, 1:3, and 3:1 to control grape gray mold.

[0071] The *Botrytis cinerea* spore suspension at a concentration of 1 × 10⁶ spores / mL, as described in Example 3, was inoculated onto grape berries at a volume of 10 μL. 24 h after inoculation, control was achieved by spraying with different volume ratios of iprodione-*Bacillus belycetamol* combination solutions at a volume of 20 μL. Water was used as a control (CK) to replace the pesticide / fermentation broth.

[0072] Following the above method, the fermentation broth of *Bacillus belye* GSBZ09 was replaced with fermentation broth of *Bacillus amyloliquefaciens* GS09 as a control experiment. Specifically, for the *Bacillus amyloliquefaciens* GS09 fermentation broth: a single colony of strain GS09 was picked and incubated in 50 mL of LB medium at 28°C and 180 rpm. -1 Incubate for 24 hours; then transfer to LB medium at a ratio of 0.1% and incubate at 28°C and 180 rpm. -1 After culturing for 48 hours, the fermentation broth of strain GS09 was obtained, in which the concentration of Bacillus amyloliquefaciens GS09 was 10. 8 CFU / mL.

[0073] Results after 5 days of spraying: Figure 4 As shown in Table 2, when the volume ratio of iprodione solution to Bacillus vesalis GSBZ09 fermentation broth is 3:1, the average diameter of the diseased wounds is minimized, resulting in good control of grape gray mold and reducing the amount of chemical agents used. At this ratio, the combined solution of iprodione and Bacillus vesalis GSBZ09 is 300 μg: 10 μg. 8 The control efficacy of CFU. GS09 combined with iprodione was lower than that of GSBZ09.

[0074] Table 2. Average diameter of the affected wound

[0075]

[0076] Note: Different lowercase letters indicate significant differences in results (p<0.05).

[0077] Example 5: Determination of the potted plant efficacy of the combination of iprodione and Bacillus vesalivarius GSBZ09 in controlling grape gray mold.

[0078] 1. Inoculation with pathogens

[0079] 20 mL of the Botrytis cinerea spore suspension with a concentration of 1×10⁶ spores / mL from Example 3 was sprayed onto grape seedlings.

[0080] 2. Control of Grape Gray Mold

[0081] Experimental spraying treatment: Spraying was used to control the disease by moistening the leaves. Four treatments were set up in the experiment:

[0082] (1) Spray with 100 μg / mL iprodione solution as described in Example 3;

[0083] (2) Spray the combined solution obtained by spraying iprodione solution and Bacillus vesiculosus GSBZ09 fermentation broth in Example 3 at a volume ratio of 3:1;

[0084] (3) Spray the fermentation broth of Bacillus vesiculosus GSBZ09 from Example 3;

[0085] (4) Spray iprodione solution, wherein the iprodione solution is obtained by fully dissolving 99% fluazinam technical in sterile water, wherein the concentration of fluazinam is 100 μg / mL;

[0086] (5) Spray with clean water;

[0087] (6) Healthy controls without any treatment, i.e., those not inoculated with botrytis cinerea spore suspension (CK).

[0088] Each treatment was repeated three times. Control measures were initiated 24 hours after inoculation with Grape Botrytis cinerea, with a spray volume of 20 mL. Disease surveys were conducted 5 days later.

[0089] Investigation method: 10 grapevines were investigated for each treatment, and all leaves of each vine were investigated. Statistics were collected by vine and disease index was calculated.

[0090] Grading standards:

[0091] Grade 0: Fruit is disease-free;

[0092] Grade 1: Diseased fruit (lesions) cover 5% or less of the total fruit (leaf) area;

[0093] Grade 3: Diseased fruit (lesions) account for 5% to 15% of the total fruit (leaf) area (excluding 5%, including 15%);

[0094] Level 5: Diseased fruit (lesions) account for 15% to 25% of the total fruit (leaf) area (excluding 15%, including 25%);

[0095] Grade 7: Diseased fruit (lesions) account for 25% to 50% of the total fruit (leaf) area (excluding 25% and 50%);

[0096] Level 9: Diseased fruit (lesions) cover 50% or more of the total fruit (leaf) area.

[0097] Disease index = ∑(number of diseased leaves at each level × level) / (total number of leaves surveyed × highest disease level) × 100;

[0098] Prevention and control effect (%) = (Control disease index - Treatment disease index) / Control disease index × 100%.

[0099] The results are shown in Table 3. The combination of iprodione and GSBZ09 showed the strongest control effect against gray mold in potted grapevines, with a disease index of 4.25 and a control efficacy of 80.59%. Iprodione alone was the second most effective, with a disease index of 5.25 and a control efficacy of 76.04%. GSBZ09 alone showed slightly lower control efficacy than iprodione alone, with a disease index of 7.65 and a control efficacy of 65.02%. Fluazinam showed the lowest control effect against gray mold.

[0100] Table 3. Control efficacy of Bacillus GSBZ09 combined with iprodione against potted grape plants.

[0101]

[0102] Note: Different lowercase letters indicate significant differences in results (p<0.05).

[0103] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A composition for controlling grape gray mold, comprising iprodione and Bacillus vesicularis (… Bacillus velezensis Composed of GSBZ09; In the composition, iprodione and Bacillus belye ( Bacillus velezensis The ratio of GSBZ09 is (100 / 3 μg-300 μg): 10 8 CFU.

2. The composition according to claim 1, characterized in that: In the composition, iprodione and Bacillus belye ( Bacillus velezensis The ratio of GSBZ09 is 300μg: 10 8 CFU.

3. The use of the composition according to claim 1 or 2 in inhibiting the growth of Botrytis cinerea; The grape gray mold fungus is Botrytis cinerea (Botrytis cinerea). Botrytis cinerea ).

4. The use of the composition according to claim 1 or 2 in the preparation of a product that inhibits the growth of Botrytis cinerea; The grape gray mold fungus is Botrytis cinerea (Botrytis cinerea). Botrytis cinerea ).

5. The composition according to claim 1 or 2 in controlling Botrytis cinerea (… Botrytis cinerea Application in the treatment of gray mold disease in grapes caused by ).

6. The composition according to claim 1 or 2 in the preparation of a treatment for controlling Botrytis cinerea (… Botrytis cinerea Application of products containing grape gray mold caused by ) 7. Methods for controlling grape gray mold include: Applying the composition of claim 1 or 2 to grapes infected with gray mold achieves the control of gray mold. The grape gray mold disease is caused by Botrytis cinerea (Glaucus spp.) Botrytis cinerea )cause.

8. A product for the prevention and control of grape gray mold, wherein the active ingredient is the composition described in claim 1 or 2; The grape gray mold disease is caused by Botrytis cinerea (Glaucus spp.) Botrytis cinerea )cause.

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