Application of bacillus velezensis TCS001 to improvement of grape quality and prevention and treatment of grape downy mildew

Abstract

The invention relates to application of bacillus velezensis TCS001 as a medicine for preventing and treating grape downy mildew and application of bacillus velezensis TCS001 as a medicine for improving grape quality. The method comprises the following steps: irrigating roots once by using a 400-fold solution of the bacillus velezensis TCS001 before germination of grapes; spraying 300-fold solution of bacillus velezensis TCS001 once a week from grape germination to a fruit swelling period; the method comprises the following steps: spraying 300-fold solution of bacillus velezensis TCS001 once every two weeks from a grape fruit swelling period to harvesting; it is found that the control effect of the bacillus velezensis TCS001 on the grape downy mildew is 68.30%. The grape total polyphenol content is increased by 14.30%, the single fruit weight is increased by 15.40%, the coloring consistency of the whole string of grapes is improved, the soluble solid content and the total sugar content are slightly reduced, the acidity is slightly increased, and the grape flavor is obviously improved. The bacillus velezensis TCS001 is found to be capable of promoting the coloring consistency of grapes for the first time.

Description

Technical Field

[0001] The invention relates to the field of plant protection and plant growth regulation, and in particular to application of Bacillus Velez TCS001 in grapes. Background Art

[0002] Bacillus velezensis is a new type of biocontrol bacteria. In 2005, Spanish scholars Ruiz-García et al. isolated two strains, CR-14b and CR-502T, which can synthesize a large amount of lipopeptides and have strong antibacterial activity. Bacillus velezensis is widely distributed and can survive in adverse conditions such as drought and salinity. There are few related products, and the development prospects are broad.

[0003] Bacillus velezensis TCS001 was isolated from Bohai silt. The original strain was marine Bacillus CT2628. After mutagenesis and stabilization, it was identified and named Bacillus velezensis TCS001. It has been deposited in the General Microbiological Center of China Microbiological Culture Collection Administration (CGMCC) with the deposit number CGMCC No.8921, and was first published in patent ZL201410168402.2. After growing on NA medium, the single colony morphology of TCS001 is nearly round, light yellow, and opaque; the surface of the colony is smooth and the edges are neat in the early stage of culture, and the surface is wrinkled in the later stage, the edges are slightly irregular, there is a bulge in the middle, and it spreads in a cloud-like manner around. Gram staining showed that the Velez Bacillus TCS001 strain was Gram-positive and rod-shaped; it had a certain inhibitory effect on cucumber gray mold, cucumber vine blight, cucumber sclerotinia, cucumber brown spot, cotton wilt and banana leaf spot, among which the inhibition rate against cucumber gray mold was the highest, reaching 87.66%.

[0004] At present, it is registered that using 5000 times of 10% S-inducible agent when irrigating grape roots can enhance plant photosynthesis, promote the growth of grape buds and new shoots, promote the horizontal and vertical diameters of buds, increase grain weight and bunch weight, and have a good promoting effect on grape growth. Spraying 300 times of 2.5% amino oligosaccharide on the leaves of grapes from the young fruit stage to the mature stage can increase VC content and yield.

[0005] In the prior art, there is no report on the application of Bacillus Velez TCS001 to grapes. Summary of the invention

[0006] The main purpose of the present invention is to provide the use of Bacillus Velez TCS001 as a drug for preventing and treating grape downy mildew.

[0007] The main purpose of the present invention is to provide the use of Bacillus Velez TCS001 as a drug for improving the quality of grapes.

[0008] Preferably, the improvement of grape quality includes one or more of increasing the total polyphenol content of grapes, increasing the weight of grape berries, increasing the content of vitamin C in grapes, increasing the content of total acid in grapes, improving the color consistency of the whole bunch of grapes, reducing the content of soluble solids and total sugar, etc.

[0009] Preferably, the Bacillus Velez subtilis TCS001 is a fermentation broth of Bacillus Velez subtilis TCS001.

[0010] Preferably, the Bacillus Velez TCS001 fermentation broth is prepared as a suspension.

[0011] Preferably, the formula of the suspension concentrate includes 40% of Bacillus Velez TCS001 fermentation broth, 3% of dispersant SXC, 3% of dispersant UNA, 0.15% of thickener xanthan gum, 0.2% of preservative kason, 0.2% of preservative sodium benzoate, and 53.45% of water.

[0012] Preferably, the application concentration of the suspension is 200-600 times diluted.

[0013] Preferably, the application concentration of the suspension is 300 times diluted.

[0014] Preferably, the Bacillus Velez subtilis TCS001 is applied by root irrigation and / or spraying.

[0015] Preferably, the application time of the Bacillus Velez TCS001 is before germination, after germination to fruit swelling period, and from fruit swelling period to harvest period of grape.

[0016] Beneficial technical effects of the present invention

[0017] Before the grapes sprouted, the roots were irrigated with 400 times concentration of Bacillus Velez TCS001; from the budding period to the fruit swelling period, the grapes were sprayed with 300 times concentration of Bacillus Velez TCS001 once a week; from the fruit swelling period to before harvest, the grapes were sprayed with 300 times concentration of Bacillus Velez TCS001 once every two weeks; the test found that the control effect of Bacillus Velez TCS001 on grape downy mildew was 68.30%. The total polyphenol content of grapes increased by 14.30%, the weight of single fruit increased by 15.40%, the color consistency of the whole bunch of grapes was improved, the soluble solids and total sugar content were slightly reduced, the acidity was slightly increased, and the grape flavor was significantly improved.

[0018] The present invention discovers for the first time that Bacillus Velez TCS001 can promote the coloring consistency of grapes. Specific embodiments

[0019] Materials and Methods

[0020] Test Materials:

[0021] The test bacterial agent of the present invention is the suspension concentrate of Bacillus Velezii TCS001 (3.9×10 9 CFU / mL), the specific formula is: 40% of Bacillus Velez TCS001 fermentation broth, 3% of dispersant SXC, 3% of dispersant UNA, 0.15% of thickener xanthan gum, 0.2% of preservative Kathon, 0.2% of preservative sodium benzoate, and 53.45% of water.

[0022] Test plants:

[0023] Experimental variety: Grape (Kyoho).

[0024] Main instrument: ME104E electronic balance (Sartorius Scientific Instruments Co., Ltd.)

[0025] Test method: See Table 1 for treatment methods

[0026] Table 1 Treatment methods of suspension concentrate of Bacillus Velez TCS001 in grape test

[0027]

[0028]

[0029] According to GB / T 17980.122-2004 "Guidelines for Field Efficacy Tests of Pesticides (II) Part 122: Fungicides for Control of Grape Downy Mildew", 10 new vines grown that year were randomly surveyed in each plot, and all leaves were surveyed from top to bottom. The number of diseased leaves at each level and the total number of leaves were recorded according to the following classification method.

[0030] Leaf grading method:

[0031] Level 0: no lesions;

[0032] Level 1: The lesion area accounts for 5% or less of the entire leaf area;

[0033] Level 3: The lesion area accounts for 6%-25% of the entire leaf area;

[0034] Level 5: The lesion area accounts for 26-50% of the entire leaf area;

[0035] Level 7: The lesion area accounts for 51%-75% of the entire leaf area;

[0036] Level 9: The lesion area accounts for more than 76% of the total leaf area.

[0037] The survey calculation results are retained to two decimal places, and the disease index is calculated according to formula (1):

[0038]

[0039] The control effect is calculated according to formula (2):

[0040]

[0041] Where:

[0042] CK1-----Disease index of blank control area after application of pesticide;

[0043] PT1-----Disease index after application of pesticide in the treatment area.

[0044] Grape quality index determination

[0045] Soluble sugar determination method: NY / T 2742-2015

[0046] 1. Prepare the raw materials: Wash and dry the fruit, take the edible part, chop and mix, sample by quartering, and make a homogenate with a tissue masher (juicy fruits are directly homogenized, and fruits with low water content are added with water in a 1+1 ratio before homogenization). Weigh 10.00g (m) of the sample, wash it with water into a volumetric flask, add 3mL each of potassium ferrocyanide solution (4.5) and zinc acetate solution (4.6), shake well, make up to 250mL (V), let it stand for a while, filter, and use the filtrate for later use.

[0047] 2. Drawing of standard curve: Use a pipette to accurately draw 0.0mL, 0.2mL, 0.4mL, 0.8mL, 1.0mL, and 1.2mL of glucose standard solution into 6 10mL stoppered test tubes, add water to make the solution volume 2.0mL, add 400mL 3,5-dinitrosalicylic acid reagent, and heat in a boiling water bath for 5min. Take it out, immediately put it in cold water, cool to room temperature, make up to volume, and shake well. The concentrations of the obtained series of glucose standard solutions are 0mg / mL, 0.02mg / mL, 0.04mg / mL, 0.08mg / mL, 0.10mg / mL, and 0.12mg / mL, respectively. Use a spectrophotometer to measure the absorbance value at 540nm. Use the glucose concentration (mg / mL) as the ordinate (y) and the absorbance value as the abscissa (x) to draw a standard curve.

[0048] 3. Determination of reducing sugar: According to the sugar content in different samples, use a pipette to draw 5mL-20mL (V2) of the filtrate into a volumetric flask, and dilute to 100mL (V) with water. Draw 1.00mL (V) of sample solution from the volumetric flask into a 10mL (V) volumetric flask or a stoppered graduated test tube, and add water to 2.0mL. Follow the steps for drawing the standard curve above. Record the absorbance value measured, and obtain the concentration of reducing sugar in the test solution from the standard curve.

[0049] 4. Determination of soluble sugar: According to the sugar content of the sample, use a pipette to draw 5mL-10mL (V) of the sample solution into a volumetric flask, add 1mL of 6mol / L hydrochloric acid solution, place in a constant temperature water bath (80±2)℃ and heat for 10min, take out, place in a cold water tank and cool to room temperature, add 3 drops of methyl red indicator and neutralize with 6mol / L sodium hydroxide solution until light orange, dilute to 100mL (V) with water and mix well. The following steps are followed for the determination of reducing sugar. The soluble sugar content of the sample is calculated by mass fraction according to the following formula:

[0050]

[0051] Where:

[0052] X: Soluble sugar content in the sample, in %;

[0053] P: the concentration of reducing sugar in the test solution, in mg / mL;

[0054] V1: sample solution volume, in mL;

[0055] V2: sample volume, in mL;

[0056] V3: fixed volume of sample solution, in mL;

[0057] V4: the volume of the test liquid absorbed, in mL;

[0058] V5: volume of sample solution, in mL;

[0059] A: Dilution multiple, 1 for juicy fruits and 2 for fruits with less water content;

[0060] m: sample mass, in g;

[0061] 10: Conversion factor for converting the measurement result into mass percentage.

[0062] Detection of polyphenols: refer to GB / T 8313-2018

[0063] 1. Principle

[0064] The total polyphenols in the ground grape sample were extracted with 70% methanol aqueous solution in a 70°C water bath. The -OH groups in the tea polyphenols were oxidized by the Folin phenol reagent and showed blue color with a maximum absorption wavelength of 765nm. Gallic acid was used as a calibration standard to quantify the polyphenols.

[0065] 2. Instruments

[0066] Analytical balance: accuracy 0.001g

[0067] Water bath: 70℃±1℃

[0068] Centrifuge: speed 3500r / min.

[0069] Spectrophotometer

[0070] 3. Reagents

[0071] 10% Folin-phenol reagent (freshly prepared): Transfer 20 mL of Folin-phenol reagent to a 200 mL volumetric flask, make up to volume with water and shake well.

[0072] 7.5% Sodium carbonate (NaCO 3 ) Solution: Weigh 37.50g of sodium carbonate and dissolve it in water, transfer it to a 500mL volumetric flask and dilute to the mark.

[0073] Gallic acid standard stock solution (1000g / mL): Weigh 0.110g of gallic acid (GA, relative molecular mass 188.14), dissolve it in a 100mL volumetric flask and dilute to the scale; Gallic acid working solution: Use a pipette to transfer 1.0mL, 2.0mL, 3.0mL, 4.0mL, and 5.0mL of gallic acid standard stock solution into a 100umL volumetric flask, dilute to the scale with water, shake well, and the concentrations are 10μg / mL, 20μg / mL, 30μg / mL, 40ug / mL, and 50μg / mL, respectively.

[0074] 4. Operation method

[0075] Use a pipette to transfer 1.0 mL of gallic acid working solution, water (for blank control) and test solution into graduated test tubes, add 5.0 mL of Folin phenol reagent to each test tube and shake well. React within 3-8 minutes, add 4.0 mL of 7.5% sodium carbonate solution and dilute to the scale with water, shake well. Leave at room temperature for 60 minutes. Use a 10 mm cuvette to measure the absorbance with a spectrophotometer at a wavelength of 765 nm. Prepare a standard curve based on the absorbance (A) of the gallic acid working solution (4.3.8) and the gallic acid concentration of each working solution.

[0076] The results are calculated using the following formula:

[0077] Compare the absorbance of the sample and the standard working solution, and calculate according to formula (4):

[0078]

[0079] Where:

[0080] C TP ——Tea polyphenols content, %;

[0081] A——absorbance of sample test solution;

[0082] A 0 ——Absorbance of reagent blank solution;

[0083] SLOPE Std ——The slope of the gallic acid standard curve;

[0084] m——sample mass, in grams (g);

[0085] V——volume of sample extract, in milliliters (mL);

[0086] d——dilution factor (usually 1 mL is diluted into 100 mL, then the dilution factor is 100);

[0087] ω——dry matter content of sample (mass fraction), %.

[0088] Total acid determination method: GB12456-2021

[0089] Determination of titratable acid:

[0090] 1. Prepare raw materials: remove the inedible part of the sample (frozen products should be thawed in a covered container in advance), use the quartering method to divide the edible part, chop and mix, weigh 250g, accurate to 0.1g, put it into a high-speed tissue pounder, add an equal amount of water, and pound for 1 to 2min. Every 2g of homogenate is converted into 1g of sample, weigh 50g of homogenate, accurate to 0.1g, wash it into a 250mL container bottle with 100mL of water, and heat it in a water bath at 75℃ to 80℃ for 30min. Shake it several times during the process, take it out to cool, add water to the scale, shake well and filter.

[0091] 2. Indicator titration

[0092] Use a pipette to draw 50 mL of sample solution, add 5 drops of phenolphthalein indicator, and titrate with standard sodium hydroxide solution until a slightly reddish color appears that does not fade within 30 seconds. Record the consumed volume.

[0093] The measurement results are calculated according to the following formula:

[0094]

[0095] Where:

[0096] V1: Volume of sodium hydroxide standard solution consumed during titration, mL;

[0097] C: molar concentration of sodium hydroxide standard solution, mol / L;

[0098] m: sample mass, g;

[0099] 250: Fixed volume of the sample after extraction, mL.

[0100] Soluble solids determination method: NY / T2637-2014

[0101] Determination of soluble solids:

[0102] 1. Prepare the raw materials: Take the edible part of the sample, chop it and mix it evenly (frozen products must be thawed in advance), weigh 250g, accurate to 0.1g, put it into a high-speed tissue grinder and grind it, and use two layers of lens paper or gauze to squeeze out the homogenized juice for determination.

[0103] 2. Determination:

[0104] 1) Adjust the temperature of the circulating water in the constant temperature water bath to 20±0.5℃, and let the water flow through the thermostat of the refractometer. The circulating water can also be adjusted within the range of 15-25℃, and the temperature should not exceed ±0.5℃.

[0105] 2) Calibrate the refractometer reading with distilled water and adjust the soluble solids to 0% at 20°C; when the temperature is not 20°C, calibrate according to the correction values ​​in the table below.

[0106] 3) After wiping the surface of the prism dry, add 2 to 3 drops of the sample solution to be tested to the center of the prism, immediately close the upper and lower prisms, aim at the light source, turn the achromatization adjustment knob to divide the field of view into light and dark parts, then turn the prism knob to make the light and dark dividing line fit at the cross point of the objective lens, read the percentage shown on the scale, and record the temperature during the measurement.

[0107] Vitamin C (ascorbic acid) determination method: GB5009.86-2016

[0108] Determination of Vitamin C (Ascorbic Acid)

[0109] 1. Sample solution preparation: weigh 10g of slurry sample, transfer the sample into a 100mL volumetric flask with an extractant, dilute to scale, shake well and filter. If the filtrate is colored, add 0.4g of white clay per gram of sample to decolorize it before filtering.

[0110] 2. Titration: Pipette an appropriate amount of filtrate into a 50mL conical flask and titrate with a calibrated 2,6-dichloroindophenol solution until the solution turns pink and does not fade for 15 seconds. Perform a blank test at the same time. Vitamin C is calculated as follows:

[0111]

[0112] Where:

[0113] V 1 : The volume of dye solution consumed when titrating the sample (mL);

[0114] V 0 : The volume of dye solution consumed during the blank titration (mL);

[0115] T: 2,6-dichloroindophenol dye titer (mg / mL);

[0116] F: dilution factor;

[0117] M: sample weight (g).

[0118] Test results:

[0119] As shown in Table 2, continuous spraying of 300 times diluted Bacillus subtilis TCS001 once every two weeks from the fruit swelling stage to before harvesting has a control effect of 68.30% on grape downy mildew, which is equivalent to the control effect of 1800 times diluted 40% dimethomorph suspension of the chemical control.

[0120] Table 2 Grape downy mildew test results (spray treatment from fruit swelling stage to before harvest)

[0121]

[0122]

[0123] As shown in Table 3, before grape budding, Bacillus Velez TCS001400 was used for root irrigation once; after grape budding, before the fruit swelling period, Bacillus Velez TCS001 300 times concentration was used for continuous spraying once a week; from the fruit swelling period to before harvest, Bacillus Velez TCS001 300 times concentration was used for continuous spraying once every two weeks. The total polyphenol content of grapes increased by 14.30%, and the single fruit weight increased by 15.40%; the soluble sugar decreased, and the vitamin C and total acid increased slightly, the sour and sweet flavor was better, and the hardness and gloss were better than CK. It was observed that Bacillus Velez TCS001 can promote the consistency of grape coloring.

[0124] Table 3 Grape quality test results

[0125]

[0126] The specific implementation methods described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above description is only a specific implementation method of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. Use of Bacillus Velez TCS001 as a drug for preventing and treating grape downy mildew.

2. Use of Bacillus Velez TCS001 as a drug for improving grape quality.

3. The use according to claim 2, characterized in that: The improvement of grape quality includes one or more of increasing the total polyphenol content of grapes, increasing the weight of grape berries, increasing the content of vitamin C in grapes, increasing the content of total acid in grapes, improving the color consistency of the whole bunch of grapes, and reducing the content of soluble solids and total sugar.

4. The use according to any one of claims 1 to 3, characterized in that: The Bacillus Velez TCS001 is the fermentation liquid of Bacillus Velez TCS001.

5. The use according to claim 4, characterized in that The Bacillus Velez TCS001 fermentation liquid is prepared into a suspension.

6. The use according to claim 5, characterized in that The suspending agent comprises 40% of Bacillus Velez TCS001 fermentation liquid, 3% of dispersant SXC, 3% of dispersant UNA, 0.15% of thickener xanthan gum, 0.2% of preservative kason, 0.2% of preservative sodium benzoate and 53.45% of water.

7. The use according to claim 6, characterized in that The application concentration of the suspension is 200-600 times diluted, preferably, the application concentration is 300 times diluted.

8. The use according to any one of claims 1 to 3, characterized in that: The application time of the Bacillus Velezii TCS001 can be before germination, after germination, during the fruit expansion period, or during one or more of the fruit expansion period and the harvest period.

9. The use according to claim 8, characterized in that The application time of Bacillus Velez TCS001 is to irrigate the roots of the grape seedlings once before budding; and / or spray once a week from budding to the fruit swelling stage; and / or spray once every 2 weeks from the fruit swelling stage to harvest.

10. The use according to any one of claims 1 to 3, wherein the application method of Bacillus Velez subtilis TCS001 is spraying and / or root irrigation.

Images