Preparation containing bacillus velezensis TCS001 and use thereof

Abstract

Provided in the present invention is a formulation for a Bacillus velezensis TCS001 suspension. The suspension has good dispersibility and a high suspension rate, adheres well to plant body surfaces, is resistant to flushing by rainwater, and has more significant and longer-lasting efficacy in comparison to wettable powder. Applying the suspension improves the quality of cherries and blueberries, and promotes growth in strawberries. The suspension can further be used as a drug in the prevention and control of diseases throughout the growth of cucumbers and tomatoes and / or in the improvement of the quality thereof; application of the microbial agent throughout crop growth effectively controls diseases in facility crops, having a prevention and control effect superior to that of conventional chemical agents; furthermore, both the quality and the yield of the crops are improved in comparison to chemical agent-treated groups.

Description

A preparation containing Bacillus velezensis TCS001 and its application Technical Field

[0001] The present invention relates to the fields of biological strain preparation formulas, crop disease prevention and control, and plant growth regulation, and particularly relates to a suspension concentrate and wettable powder formula containing Bacillus Velez TCS001, and the use of the suspension concentrate of Bacillus Velez TCS001 in regulating the growth quality of cherries and blueberries, promoting early ripening of blueberries, improving the growth characteristics of strawberries, and preventing and controlling diseases and improving the quality of cucumbers and tomatoes. Background Art

[0002] Bacillus velezensis is a novel biocontrol bacterium. Spanish researchers Ruiz-García et al. first isolated two strains, CR-14b and CR-502T, in 2005. These strains are capable of producing large quantities of lipopeptides and exhibit strong antibacterial activity. Bacillus velezensis is widely distributed and can survive adverse conditions such as drought and salinity. However, few related products exist, suggesting promising development prospects.

[0003] Bacillus velezensis TCS001 was isolated from Bohai Sea mud. The original strain was Bacillus marineus CT2628. After mutagenesis and stabilization, it was identified and named Bacillus velezensis TCS001. It has been deposited with the General Microbiology Center of the China General Culture Collection (CGMCC) under the accession number CGMCC No. 8921 and was first published in Patent ZL201410168402.2. When grown on NA medium, TCS001 colonies are nearly round, light yellow, and opaque. Initially, the surface is smooth with neat edges, but later, the surface becomes wrinkled, with slightly irregular edges and a central bulge, which diffuses in a cloud-like pattern. Gram staining showed that the Bacillus Velezii TCS001 strain is Gram-positive and rod-shaped; it has 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 is the highest, reaching 87.66%.

[0004] Currently, the registered microbial pesticide formulations on the market include wettable powders (WP), suspension concentrates (SC), flowable concentrates for seed coating (FSC), water dispersible granules (WG), dust power (DP), granules (GR), pesticide water agents (AS), emulsions in water (EW), and encapsulated granules (CG). WP accounts for 66.7%, SC for 8.5%, WG for 5.9%, AS for 1.9%, FSC for 1.3%, GR for 5.2%, DP for 1.3%, CG for 1.3%, and EW for 0.65% (data source: http: / / www.icama.org.cn / hysj / index.jhtml).

[0005] Judging from the registration situation up to now, the dosage form of microbial pesticide fungicides is still mainly wettable powders, and the number of other dosage forms is relatively small.

[0006] Wettable powders are a formulation in which the original drug, inert fillers, and adjuvants are fully mixed in a certain proportion and crushed to achieve a certain powder fineness. The laboratory where the inventor of this application is located has previously developed a wettable powder containing Bacillus velezensis TCS001. However, due to the serious loss of secondary metabolites produced by the strain during the development of wettable powders, the large amount of sediment produced during use, the low suspension rate, and the easy clogging of the sprayer nozzle. The low suspension rate can lead to uneven concentration of the drug solution, reducing the prevention and control effect and increasing the probability of disease occurrence. Therefore, wettable powders have not been used as a commercial formulation.

[0007] The inventors of this application believe that microbial pesticide suspension concentrates (SC) account for an increasing proportion, and green and environmentally friendly formulations are a new trend in the future development of pesticides. The developed Bacillus Velez TCS001 suspension concentrate has not yet been applied to cherries and blueberries. In addition, in previous studies on the prevention and control of strawberry gray mold, it was unexpectedly discovered that Bacillus Velez TCS001 can not only prevent and control diseases, but also significantly improve the growth traits of strawberries. Based on this discovery, the present invention proposes to use Bacillus Velez TCS001 as an active ingredient and apply it to cherries, blueberries, strawberries, tomatoes and cucumbers, in order to reduce the use of chemical pesticides and fertilizers while preventing and controlling diseases, improving quality and increasing yields. This provides a new direction for future research and application.

[0008] Summary of the Invention

[0009] The main purpose of the present invention is to provide a suspension concentrate formula containing Bacillus Velez TCS001, which can meet the requirements of pesticide registration and commercial application.

[0010] Another object of the present invention is to find the use of SXC and UNA in preparing a preparation containing Bacillus Velez sp. TCS001.

[0011] Preferably, the mass ratio of SXC to UNA is 1:1.

[0012] Another object of the present invention is to find the use of kasone and sodium benzoate in preparing a preparation containing Bacillus Velez TCS001.

[0013] Preferably, the mass ratio of the kasonon to sodium benzoate is 1:3-3:1.

[0014] Another object of the present invention is to find the use of xanthan gum in preparing a preparation containing Bacillus Velez TCS001.

[0015] Preferably, the above-mentioned preparation containing Bacillus Velez subtilis TCS001 is a suspension concentrate containing Bacillus Velez subtilis TCS001.

[0016] Another object of the present invention is to provide a suspension concentrate containing Bacillus Velez TCS001, comprising 10-60% of Bacillus Velez TCS001 fermentation broth, 2-10% of a wetting and dispersing agent, 0.1-1% of a thickener, 0.1-0.5% of a preservative, and water to 100%, wherein the wetting and dispersing agent is SXC and UNA, and the preservative is kason and sodium benzoate.

[0017] Preferably, the mass ratio of the kasonon to sodium benzoate is 1:1.

[0018] Preferably, the mass ratio of SXC to UNA is 1:1.

[0019] Preferably, the thickener is xanthan gum.

[0020] Another object of the present invention is to provide a suspension concentrate containing Bacillus Velez TCS001, comprising 40% of Bacillus Velez TCS001 fermentation broth, 3% of a wetting and dispersing agent SXC, 3% of a wetting and dispersing agent UNA, 0.15% of a thickener xanthan gum, 0.2% of a preservative kason, 0.2% of a preservative sodium benzoate, and 100% water.

[0021] Another object of the present invention is to provide a wettable powder containing Bacillus Velez TCS001, which comprises 70% of Bacillus Velez TCS001 fermentation broth, 10% of carrier diatomaceous earth, 5% of dispersant polyvinyl alcohol, 10% of wetting agent sodium dodecylbenzenesulfonate, 2.5% of stabilizer potassium phosphate, and 2.5% of protective agent dextrin.

[0022] Another object of the present invention is to provide a method for improving cherry quality using the Bacillus Velez TCS001 suspension concentrate.

[0023] Preferably, the improving of cherry quality includes one or more of increasing cherry soluble sugar, increasing cherry soluble solids, reducing cherry vitamin C, increasing cherry anthocyanin, increasing cherry β-carotene, reducing cherry acidity, etc.

[0024] Preferably, the application concentration of the suspension is 300-700 times diluted.

[0025] Preferably, the application concentration of the suspension is 400 times diluted.

[0026] Preferably, the Bacillus Velez subtilis TCS001 suspension concentrate is applied by spraying.

[0027] Preferably, the application time of the Bacillus Velez TCS001 suspension concentrate is one or more of the following stages: flower bud differentiation stage, early flowering stage, full flowering stage, young fruit stage, and harvest stage of cherry;

[0028] Preferably, the Bacillus Velez TCS001 suspension concentrate is applied once a week from the young fruit stage to the harvest stage until the end of harvest.

[0029] Preferably, the Bacillus Velez TCS001 suspension concentrate is applied once within 5 days after harvesting.

[0030] Another object of the present invention is to provide a method for improving the quality of blueberries using the Bacillus Velez TCS001 suspension concentrate.

[0031] Preferably, the improving the quality of blueberries includes one or more of increasing the sugar content and anthocyanin of blueberries, reducing the acidity of blueberries, increasing the vitamin C content, color, size, glossiness, etc.

[0032] Another main object of the present invention is to provide a method for promoting early ripening of blueberries, comprising treating blueberries with a suspension concentrate of Bacillus Velez TCS001.

[0033] Optimization promotes the ripening of blueberries 5-7 days earlier.

[0034] Preferably, the application concentration of the suspension is 300-700 times diluted.

[0035] Preferably, the application concentration of the suspension is 500 times diluted.

[0036] Preferably, the Bacillus Velez subtilis TCS001 suspension concentrate is applied by root irrigation and / or spraying.

[0037] Preferably, the Bacillus Velez TCS001 suspension concentrate is applied during one or more of the following periods: flower bud differentiation, early flowering, full flowering, young fruit, and harvest.

[0038] Preferably, the Bacillus Velez TCS001 suspension concentrate is applied once a week from the young fruit stage to the harvest stage until the end of harvest.

[0039] Preferably, the Bacillus Velez TCS001 suspension concentrate is applied once within 5 days after harvesting.

[0040] Another object of the present invention is to provide a method for improving the growth characteristics of strawberries using the Bacillus Velez TCS001 suspension concentrate.

[0041] The strawberry growth traits include leaf chlorophyll concentration, single plant leaves, stem and leaf fresh weight, root fresh weight, leaf area, plant height, leaf IAA content, ABA content, SOD activity, leaf GA content, CAT activity, POD activity, etc.

[0042] Preferably, the application concentration of the suspension is 200-500 times diluted.

[0043] Preferably, the application concentration of the suspension is 300 times diluted.

[0044] Preferably, the Bacillus Velez subtilis TCS001 suspension concentrate is applied during the seedling stage.

[0045] Preferably, the Bacillus Velez subtilis TCS001 suspension concentrate is applied by root irrigation and / or spraying.

[0046] The main purpose of the present invention is to provide an application of the Bacillus Velez subtilis TCS001 suspension concentrate for the prevention and control of diseases and improvement of quality in greenhouse crops, wherein the greenhouse crops are tomatoes and cucumbers;

[0047] The diseases include one or more of tomato damping-off, bacterial wilt, early blight, gray mold, and wilt; cucumber damping-off, bacterial angular spot, powdery mildew, downy mildew, and wilt;

[0048] The quality aspects include one or more of the following: plant height, stem diameter, average single fruit weight, average single plant yield, soluble solids, vitamin C, soluble sugar, and titratable acid for tomatoes; and the circumference of the main stem base, relative chlorophyll content, number of roots, average fruit weight, soluble solids, vitamin C, and water content for cucumbers;

[0049] Another object of the present invention is to provide a method for improving tomato quality, comprising applying the Bacillus Velez TCS001 suspension concentrate during one or more of the tomato seedling stage, transplanting day, seedling stage, flowering stage, and fruiting stage.

[0050] Another object of the present invention is to provide a method for improving the quality of cucumbers, wherein the suspension concentrate of Bacillus Velez TCS001 is applied during one or more of the cucumber seedling stage, transplanting day, planting stage, vine sprouting stage, and flowering and fruiting stage.

[0051] The method of the present invention further comprises applying amino oligosaccharides during the tomato seedling stage, and applying amino oligosaccharides during the cucumber planting stage and the vine-growing stage.

[0052] Beneficial technical effects of the present invention

[0053] In order to retain the effective ingredients such as the secondary metabolites of the Velez subtilis TCS001 strain to the greatest extent, the present invention selects the fermentation broth of the Velez subtilis TCS001 strain as the main ingredient to prepare a suspension concentrate, which has better efficacy than a wettable powder, does not require an organic solvent, has low toxicity, low volatility, is safe for the environment, and has a low cost.

[0054] Since the survival period of Bacillus Velez TCS001 in the suspension concentrate is short, it cannot meet the needs of practical applications. In addition, it is inconvenient to package and transport, easily produces precipitation during storage, and has poor fluidity. In order to solve the high mortality rate of live bacteria in the suspension concentrate due to environmental problems during storage, the present invention adds an appropriate amount of preservative to solve this key problem.

[0055] The present invention screens out for the first time the use of SXC and UNA in preparing a preparation containing Bacillus Velez TCS001; the use of casone and sodium benzoate in preparing a preparation containing Bacillus Velez TCS001; and the use of xanthan gum in preparing a preparation containing Bacillus Velez TCS001.

[0056] Compared with the previous wettable powder, the suspension concentrate of the present invention has no dust hazard and is relatively safe for operators and the environment. In addition, the suspension concentrate diffuses well in water and can be directly prepared into a spray liquid for use. It has good dispersibility, a high suspension rate, a strong ability to adhere to the surface of plants, and is resistant to rain erosion. Therefore, the efficacy is more significant and more lasting than that of the wettable powder. In addition, it has the advantages of the wettable powder, can be wetted by water, and has better suspension properties after being diluted with water.

[0057] Treatment with the suspension concentrate of Bacillus Velez TCS001 significantly improved cherry fruit quality. Spraying the cherry fruit with a 400-fold concentration of Bacillus Velez TCS001 suspension concentrate during the flower bud differentiation, initial flowering, peak flowering, young fruit, and harvest stages increased beta-carotene by 13.6%, anthocyanin by 9.8%, and soluble sugar by 7.3% compared to the control (CK). Vitamin C decreased by 19.4%, and total acidity decreased by 7%. Observations during the trial revealed that Bacillus Velez TCS001 significantly improved cherry size and gloss, as well as other traits, compared to the blank control. The fruit also tasted better when ate.

[0058] Treatment with Bacillus Velez TCS001 suspension concentrate significantly improved blueberry fruit quality, marking the first discovery that Bacillus Velez TCS001 can promote earlier maturity and marketability of blueberries. Root irrigation with a 500-fold concentration of Bacillus Velez TCS001 suspension concentrate (SC) advanced ripening by 5-7 days compared to the control (CK). Root irrigation with SC at a 500-fold concentration during the flower bud differentiation, initial flowering, peak flowering, young fruit, and harvest stages increased anthocyanin content by 10%, soluble sugar by 4%, and vitamin C by 5%, while reducing total acidity by 20%. Observations during the trial revealed that Bacillus Velez TCS001 significantly improved other blueberry traits, including color, size, and gloss, compared to the blank control, and the taste was positive.

[0059] Bacillus Velez TCS001 was first shown to improve strawberry growth traits. After spraying with a 300-fold suspension concentrate of Bacillus Velez TCS001, chlorophyll concentrations in strawberry leaves peaked at 14 and 21 days old, increasing by 11% and 14.89%, respectively, compared to the control. Fresh weights of leaves, stems, and roots per plant increased by 26.5%, 79.4%, and 120%, respectively, compared to the control. Root irrigation with a 300-fold suspension concentrate of Bacillus Velez TCS001 increased leaf area and plant height by 68.41% and 29.68%, respectively, compared to the control. Root irrigation with a 300-fold suspension concentrate of Bacillus Velez TCS001 increased IAA content, ABA content, and SOD activity in strawberry leaves by 38.47%, 36.04%, and 21.41%, respectively, compared to the control. After treatment with a 300-fold spray concentration of Bacillus Velez TCS001, GA content, CAT activity, and POD activity in strawberry leaves increased by 34.04%, 21.23%, and 23.21% respectively, compared to the control group, on day 28. Treatment with Bacillus Velez TCS001 significantly promoted strawberry plant growth and induced a faster and stronger protective enzyme defense response in strawberry leaves.

[0060] Applying Bacillus Velez TCS001 suspension concentrate throughout the entire growth period of greenhouse crops effectively controlled tomato and cucumber diseases, outperforming conventional chemical pesticides. Furthermore, crop quality and yield improved compared to those treated with conventional chemical pesticides. By applying Bacillus Velez TCS001 suspension concentrate throughout the entire growth period, the company achieved both disease control and prevention while simultaneously reducing the use of chemical pesticides and fertilizers and increasing their efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Figure 1 shows the biocompatibility test results of different types of wetting and dispersing agents with Bacillus velezensis TCS001;

[0062] Figure 2 shows the biocompatibility test data of different wetting and dispersing agents with Bacillus velezensis TCS001;

[0063] FIG3 shows the biocompatibility test results of different types of thickeners with Bacillus velezensis TCS001;

[0064] FIG4 shows the biocompatibility test results of different types of preservatives with Bacillus velezensis TCS001;

[0065] FIG5 is the test data of the low-temperature storage stability of the Bacillus Velez TCS001 suspension concentrate of the present invention at 0°C;

[0066] FIG6 is the test data of the low-temperature storage stability of the Bacillus Velez TCS001 suspension concentrate of the present invention at 4° C.;

[0067] FIG7 is the storage stability test data of the Bacillus Velez TCS001 suspension concentrate of the present invention at room temperature of 28° C.;

[0068] FIG8 is the test data of the high temperature storage stability of the Bacillus Velez TCS001 suspension concentrate of the present invention at 35° C.;

[0069] FIG9 is the test data of the storage stability of the Bacillus Velez TCS001 suspension concentrate of the present invention at 45° C.;

[0070] FIG10 is a test data of the high temperature storage stability of the Bacillus Velez TCS001 suspension concentrate of the present invention at 54° C.;

[0071] Figure 11 shows the cherry fruits after the control and Bacillus Velez TCS001 suspension concentrate shed treatment;

[0072] Figure 12 shows the cherry fruits after the control and Bacillus Velez TCS001 suspension concentrate shed treatment;

[0073] Figure 13 shows fruits on cherry branches after treatment with the suspension concentrate of Bacillus Velez TCS001 in a shed;

[0074] Figure 14 shows fruits on control cherry branches;

[0075] Figure 15 shows fruits on cherry branches after treatment with the suspension concentrate of Bacillus Velez TCS001 in a shed;

[0076] Figure 16 shows fruits on control cherry branches;

[0077] FIG17 shows strawberry plants treated with various methods according to the present invention;

[0078] FIG18 shows the changes in chlorophyll content (A), number of leaves per plant (B), plant height (C), stem and leaf fresh weight (D), root fresh weight (E), and leaf area (F) of strawberries in each treatment group of the present invention at different time periods;

[0079] FIG19 shows the changes in ABA (A), IAA (B), and GA (C) content and CAT (D), SOD (E), and POD (F) activity in strawberry leaves of each group treated with different days of the present invention.

[0080] Figure 20 shows the blueberry fruits of the control group and the blueberry fruits after root irrigation treatment with the Bacillus Velez TCS001 suspension concentrate. Specific embodiments

[0081] Materials and Methods

[0082] Test materials

[0083] Wetting and dispersing agents: SXC, UNA, NN9401, 9160, J401, SP-3060, NS-500LQ, 4913, 33SC, 92FS1, 20TX, SP-2833, MNS / 90;

[0084] Preservatives: Kasone, sodium benzoate, S30N;

[0085] Thickeners: xanthan gum, magnesium aluminum silicate;

[0086] The test bacterial agent of the present invention is the suspension concentrate of Bacillus velezensis TCS001 (3.9×10 9CFU / mL), the specific formula is: Bacillus velezensis TCS001 fermentation broth 40%, dispersant SXC 3%, dispersant UNA 3%, thickener xanthan gum 0.15%, preservative kason 0.2%, preservative sodium benzoate 0.2%, water 53.45% (National and Local Joint Engineering Laboratory of High-efficiency Preparation Technology of Biopesticides, Zhejiang Agricultural and Forestry University), 2% amino oligosaccharide aqueous solution (Shandong Heyi Biotechnology Co., Ltd.), 60% sulfur and sodium sulfonamide wettable powder (Dandong Pesticide Factory, Liaoning Province), 3% zhongshengmycin wettable powder (Shandong Zhaofengnian Biotechnology Co., Ltd.), 75% chlorothalonil water dispersible granules (American Shike) Company), 40% pyrimethanil wettable powder (Jiangsu Fengdeng Crop Protection Co., Ltd.), 66.5% propamocarb hydrochloride aqueous solution (Qingdao Huizhi Biotechnology Co., Ltd.), 6% kasugamycin wettable powder (Shaanxi Thompson Biotechnology Co., Ltd.), 5% zhongshengmycin wettable powder (Fujian Kaili Biological Products Co., Ltd.), 430 g / L tebuconazole suspension concentrate (Zhejiang Qianjiang Biochemical Co., Ltd.), 40% dimethomorph suspension concentrate (Jiangsu Jianpai Agrochemical Co., Ltd.).

[0087] Test plants:

[0088] Experimental varieties: cherry, four-leaf red strawberry, cucumber, tomato, blueberry (AFP).

[0089] Main instruments:

[0090] ME104E electronic balance (Sartorius Scientific Instruments Co., Ltd.), chlorophyll tester (Zhejiang Top Yunnong Technology Co., Ltd.), electric constant temperature blast drying oven (DHG-9023A)

[0091] method

[0092] The types and concentrations of the above three types of additives were screened respectively to obtain the optimal suspending agent additive ratio, and finally the performance index was tested. The wetting and dispersing agent was evaluated by measuring the suspension rate.

[0093] Additive compatibility

[0094] The biocompatibility of wetting dispersants, preservatives and thickeners (4 concentrations 0, 0.1%, 0.3%, 0.5%) with Bacillus Velez TCS001 was determined by the toxicity plate method (4 concentrations 0, 1%, 3%, 5%). For wetting dispersants that were incompatible with LB solid culture medium, their biocompatibility with Bacillus Velez TCS001 was determined by the growth method.

[0095] Screening of wetting and dispersing agents

[0096] Determine the appropriate wetting and dispersing agent according to the highest standard of suspension rate and excellent dispersion. After screening the appropriate wetting and dispersing agent by the above method, add the test wetting and dispersing agent to 100 mL of fermentation broth according to different proportions (0, 1%, 3%, 6%). After standing at room temperature for 24 hours, the CFU method is used to determine the content of the active ingredient in the suspension. The grade is determined according to the dispersion state according to Table 1, and the optimal dosage of the wetting and dispersing agent is determined.

[0097] Table 1 Dispersion performance classification standard

[0098] Thickener dosage screening

[0099] Thickeners of different concentrations (0, 0.05%, 0.1%, 0.15%, 0.2%, and 0.25%) were added to the fermentation broth containing the wetting and dispersing agent. The water separation rate and viscosity at room temperature for one month were observed. 8 mL of the fermentation broth was added to a 10 mL centrifuge tube and centrifuged at low speed for half an hour at room temperature. The precipitation was then closely observed, and 1 mL of the upper suspension after centrifugation was taken to detect its CFU content to determine the appropriate dosage of the thickener.

[0100] Screening of preservatives

[0101] During storage, suspension concentrates must be protected against bacterial contamination, so a small amount of preservatives must be added. The type and concentration of preservatives are determined through biocompatibility testing. Using the adjuvants selected in the tests, different concentrations of preservatives (0, 0.1%, 0.2%, and 0.3%) are added to prepare different samples. The samples are then stored at room temperature for 14 days and their suspension rate and degree of mildew are measured.

[0102] Determination method of Bacillus velez content

[0103] Dilution solution preparation

[0104] Heat 802 mL of Tween, 18 g of NaCl, and 2000 mL of distilled water until fully dissolved, then dispense into conical flasks and sterilize at 121°C for 30 min.

[0105] Dilution of sample (determination of viable bacterial count)

[0106] Under aseptic conditions, use a pipette to accurately pipette 1.0 mL of sample (accurate to 0.01 mL) and place it in 9.0 mL of diluent solution, mixing thoroughly to obtain a 10-fold diluted sample solution, labeled as 0. Then, perform a serial dilution according to Table 2. (To minimize sample dilution errors, during serial dilutions, thoroughly shake each dilution for uniformity. At each dilution, use a new pipette and pipette the bacterial solution along the tube wall from the previous gradient to the next. Shake manually for 30 seconds to ensure uniform dilution.)

[0107] Table 2 Serial dilution

[0108] Dilution of sample (spore count determination)

[0109] Ultrasonicate the sample to be tested in an ultrasonic water bath for 10 minutes. Pipette 5.0 mL of the sample into a conical flask containing 45 mL of diluent. Then fully shake on a shaker at 200 r / min for 30 minutes to obtain a 1:10 bacterial suspension. Place the 1:10 bacterial suspension in an 80℃±1℃ water bath for 10 minutes. After cooling, dilute it according to the gradient in Table 2.

[0110] Plate count (viable bacteria and spore count)

[0111] Under sterile conditions, pour sterilized LB medium into a culture dish and cool to room temperature.

[0112] Pipette 100 μL of each of the three gradient dilutions No. 6, 7, and 8 onto an LB plate and evenly spread it over the entire surface of the plate using a curved glass rod. Repeat three times for each dilution, and then culture in a constant temperature incubator at 28°C in the dark for 48 h.

[0113] pH determination

[0114] Weigh 1.0g of the sample into a 100mL beaker, add 100mL of water, stir vigorously for 1 minute, and let it sit for 1 minute. Insert a rinsed glass electrode and a saturated calomel electrode into the sample solution and measure its pH. Perform at least three replicate measurements. The absolute difference in the results should be less than 0.1. The arithmetic mean of these results is the pH value of the sample.

[0115] Suspension rate determination

[0116] The specific operation process is as follows: take 1 mL of the evenly mixed product into a measuring cylinder containing 249 mL of standard hard water, and continuously turn it upside down with the middle of the measuring cylinder as the axis. Then, treat it in a constant temperature water bath at 30°C for 30 minutes. Then, use a pipette to suck out 9 / 10 of the top of the measuring cylinder and determine the CFU of the remaining suspension.

[0117] The suspension rate is calculated according to the following formula:

[0118] W1(%)=[(M1-M2) / M1]×10 / 9×100

[0119] Where:

[0120] M1——the total number of Bacillus in the sample taken to prepare the suspension, in pieces;

[0121] M2——the total number of bacteria in the corresponding bottom suspension, in units;

[0122] 10 / 9——Conversion factor.

[0123] Determination of persistent foaming

[0124] Add standard hard water (15°C to 25°C) to the 180mL mark in the measuring cylinder. Place the measuring cylinder on a balance and weigh 1.0g of sample. Add hard water to the mark 9cm±0.1cm from the bottom of the measuring cylinder stopper. Cover the cylinder with the stopper. With the center of the measuring cylinder as the center, invert it 180° up and down 30 times (2 seconds each time). Place it vertically on the test bench and let it rest. Record the foam volume at 1min±10s (accurate to 2mL). Repeat the measurement three times and take the arithmetic mean as the persistent foaming test result for the sample.

[0125] Determination of the pourability of suspension concentrates

[0126] Determination of residue after dumping

[0127] Weigh the stoppered graduated cylinder (accurate to 0.1 g); add the sample to the 80% mark on the cylinder's total volume, replace the stopper, and weigh again (accurate to 0.1 g). After standing for 24 hours at room temperature (the specific temperature can be determined according to actual needs), rotate the cylinder 135° from its upright position, invert for 60 seconds, and then invert for 60 seconds. Reweigh the stoppered graduated cylinder (accurate to 0.1 g). Express the sample residue after inversion as a mass fraction, calculated according to formula (1):

[0128] Where:

[0129] W1 - residue after pouring, expressed in %;

[0130] M2 - the sum of the mass of the residue after pouring and the mass of the stoppered measuring cylinder, in grams (g);

[0131] M0——mass of the stoppered graduated cylinder, in grams (g);

[0132] M1 is the sum of the mass of the sample and the mass of the measuring cylinder with stopper, in grams (g);

[0133] Determination of residue after washing

[0134] Add 20°C distilled water to the 80% mark on the measuring cylinder's total volume, cover with a stopper, and invert the cylinder 10 times (when inverting the cylinder, ensure that each time the center of the cylinder is used as the center, the cylinder should be turned 180° from the upright position and then returned to the original position in about 2 seconds. The operation should be completed smoothly and evenly). Then, pour out the water using the same method as when pouring, cover with a lid, and weigh (accurate to 0.1g). Express the sample's post-wash residue as a mass fraction and calculate according to formula (2):

[0135] W1 - residue after washing, expressed in %;

[0136] M2 - the sum of the mass of the residue after washing and the mass of the stoppered measuring cylinder, in grams (g);

[0137] M0——mass of the stoppered graduated cylinder, in grams (g);

[0138] M1 is the sum of the mass of the sample and the mass of the measuring cylinder with stopper, in grams (g);

[0139] Wet sieve test

[0140] The finished product of Bacillus Velez suspension concentrate should pass through a 75μm standard sieve. Repeat the test three times and take the average value.

[0141] Determination of miscellaneous bacteria rate

[0142] The number of bacterial colonies growing on the PDA plate is the total bacterial count in the sample. Typical Bacillus velezensis can be identified based on colony morphology and microscopic observation. The ratio of contaminants to the total bacterial count is the contaminant bacterial rate of the product.

[0143] The bacteria that are inconsistent with the typical colony morphology are foreign bacteria, and the suspicious colonies are morphologically observed and verified. The number of foreign bacteria colonies B (unit: CFU / mL) of the sample is calculated according to formula (2): The foreign bacteria rate X (%) is calculated according to formula (3):

[0144] Where:

[0145] c——the average number of non-Velez Bacillus TCS001 colonies on the plate, in CFU;

[0146] 0.1——the amount of sample taken from the plate, in mL;

[0147] n——sample dilution multiple.

[0148] Where:

[0149] B-----The number of bacteria in each mL of sample, the unit is CFU / mL;

[0150] A-----The number of typical Bacillus Velezii TCS001 colonies per mL of sample, in CFU / mL.

[0151] Storage stability determination

[0152] Low temperature (0±2℃) storage stability test

[0153] The Bacillus Velez TCS001 suspension concentrate needs to be tested for low-temperature stability for 2 weeks. The TCS001 suspension concentrate sample is stored in an environment of 0±2℃. After 2 weeks of storage, its active ingredient content, i.e. the number of viable bacteria and spores, is determined using the dilution spread plate method.

[0154] Low temperature (4±2℃) storage stability test

[0155] The Bacillus Velez TCS001 suspension concentrate is subject to a two-year low-temperature stability test. The TCS001 suspension concentrate sample is stored at 4±2°C and its active ingredient content, i.e., the number of viable bacteria and spores, is measured every three months using the dilution spread plate method.

[0156] Storage stability test at room temperature (28±2℃)

[0157] The Bacillus Velez TCS001 suspension concentrate needs to be tested for stability at room temperature for one year. The TCS001 suspension concentrate sample is stored in an environment of 28±2℃, and its active ingredient content, i.e. the number of viable bacteria and spores, is measured every one month using the dilution spread plate method.

[0158] High temperature (54±2℃) storage stability test

[0159] The TCS001 suspension concentrate of Bacillus velezensis needs to be tested for high temperature stability for 2 weeks. The TCS001 suspension concentrate sample is stored in an environment of 54±2℃. After 2 weeks, its active ingredient content, i.e. the number of viable bacteria and spores, is measured using the dilution spread plate method.

[0160] High temperature (45±2℃) storage stability test

[0161] The Bacillus Velez TCS001 suspension concentrate needs to be tested for high temperature stability for 6 weeks. The TCS001 suspension concentrate sample is stored in an environment of 45±2℃, and its active ingredient content, i.e. the number of viable bacteria and spores, is measured every 3 weeks using the dilution spread plate method.

[0162] High temperature (35±2℃) storage stability test

[0163] The Bacillus Velez TCS001 suspension concentrate needs to be tested for high temperature stability for 12 weeks. The TCS001 suspension concentrate sample is stored in an environment of 35±2℃, and its active ingredient content, i.e. the number of viable bacteria and spores, is measured every 3 weeks using the dilution spread plate method.

[0164] Experimental method for treating cherries and blueberries with suspension concentrate of Bacillus Velez TCS001

[0165] Table 3 Treatment methods of Bacillus Velez TCS001 suspension concentrate cherry test

[0166] The suspension concentrate of Bacillus Velez TCS001 was diluted with water to the corresponding concentration and then sprayed.

[0167] Spray treatment: Spray once during the cherry flower bud differentiation period, early flowering period, and peak flowering period, and spray continuously once a week from the young fruit period to the harvest period.

[0168] Table 4 Treatment methods for the blueberry test of Bacillus velezensis TCS001 suspension concentrate

[0169] Bacillus Velez TCS001 suspension concentrate was diluted with water to the appropriate concentration and then irrigated the roots of the blueberry trees with 500 ml of suspension concentrate per tree.

[0170] Root irrigation: Root irrigation should be carried out once during the blueberry flower bud differentiation period, early flowering period, and peak flowering period, and root irrigation should be carried out once a week from the young fruit period to the harvest period.

[0171] Determination of quality indicators of cherries and blueberries

[0172] Soluble sugar determination method: NY / T2742-2015

[0173] 1. Prepare the ingredients: Wash and dry the fruit, remove the edible portion, chop and mix thoroughly, quarter the sample, and prepare a homogenate using a tissue masher (juicy fruits can be homogenized directly; fruits with low water content can be homogenized by adding water in a 1:1 ratio before homogenization). Weigh 10.00 g (m2) of the sample and rinse it with water into a volumetric flask. Add 3 mL each of potassium ferrocyanide solution (4.5) and zinc acetate solution (4.6), shake well, and dilute to 250 mL (V). Let stand for a while, filter, and set aside the filtrate.

[0174] 2. Plotting the Standard Curve: Use a pipette to accurately pipette 0 mL, 0.2 mL, 0.4 mL, 0.8 mL, 1.0 mL, and 1.2 mL of glucose standard solution into six 10 mL stoppered test tubes. Add water to bring the volume to 2.0 mL. Add 400 mL of 3,5-dinitrosalicylic acid reagent and heat in a boiling water bath for 5 minutes. Remove the solution and immediately place it in cold water. Cool to room temperature, bring to volume, and shake well. The resulting series of glucose standard solutions have concentrations of 0 mg / mL, 0.02 mg / mL, 0.04 mg / mL, 0.08 mg / mL, 0.10 mg / mL, and 0.12 mg / mL, respectively. Measure the absorbance at 540 nm using a spectrophotometer. Plot a standard curve with glucose concentration (mg / mL) as the y-axis and absorbance as the x-axis.

[0175] 3. Determination of Reducing Sugars: Depending on the sugar content of the sample, pipette 5 mL to 20 mL (V2) of the filtrate into a volumetric flask and dilute to 100 mL (V) with water. Pipette 1.0 mL (V) of the sample solution from the volumetric flask into a 10 mL (V) volumetric flask or a graduated test tube with a stopper and add water to 2.0 mL in each case. Proceed as described above for the standard curve. Record the absorbance readings and calculate the reducing sugar concentration in the test solution from the standard curve.

[0176] 4. Determination of soluble sugar: Depending on 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 10 minutes, remove, 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 it turns 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 as mass fraction according to the following formula:

[0177] Where:

[0178] X: soluble sugar content in the sample, in %;

[0179] P: the concentration of reducing sugar in the test solution, in mg / mL;

[0180] V1: volume of sample solution, in mL;

[0181] V2: sample volume, in mL;

[0182] V3: fixed volume of sample solution, in mL;

[0183] V4: the volume of the test solution absorbed, in mL;

[0184] V5: volume of sample solution, in mL;

[0185] A: Dilution factor, 1 for juicy fruits and 2 for fruits with less water content;

[0186] m: sample mass, in g;

[0187] 10: Conversion factor for converting the measurement result into mass percentage.

[0188] Total acid determination method: GB12456-2021

[0189] The steps for determining titratable acidity are as follows:

[0190] 1. Prepare the ingredients: Quarter the edible portion, mince and mix thoroughly. Weigh 250 g, accurate to 0.1 g, and place in a high-speed tissue pounder. Add an equal amount of water and pound for 1 to 2 minutes. Every 2 g of homogenate is converted into 1 g of sample. Weigh 50 g of the homogenate, accurate to 0.1 g, and wash it with 100 mL of water into a 250 mL container. Heat in a water bath at 75°C to 80°C for 30 minutes, shaking several times. Remove and cool. Add water to the mark, shake well, and filter.

[0191] 2. Indicator titration method: According to the predicted acidity, use a pipette to draw 50 mL of sample solution, add 5 drops of phenolphthalein indicator, and titrate with sodium hydroxide standard solution until a slightly reddish color appears that does not fade within 30 seconds. Record the consumed volume.

[0192] The measurement results are calculated according to the following formula:

[0193] Where:

[0194] V1: The volume of sodium hydroxide standard solution consumed during titration

[0195] Volume, mL

[0196] C: molar concentration of sodium hydroxide standard solution, mol / L;

[0197] m: sample mass, g;

[0198] 250: The fixed volume of the sample after extraction, mL.

[0199] Determination of soluble solids: NY / T 2637-2014

[0200] The refractometer method is as follows:

[0201] 1. Sample preparation: Chop blueberries and cherries and mix them evenly. Weigh an appropriate amount of sample (generally 250 g for samples with high water content and 125 g for samples with low water content). Add appropriate amount of water and grind in a high-speed tissue grinder. Use two layers of mirror paper or four layers of gauze to squeeze out the homogenized juice for measurement.

[0202] 2. Instrument calibration: Calibrate the refractometer with distilled water at 20°C and adjust the soluble solids content reading to 0.

[0203] 3. Sample Liquid Measurement: Maintain a stable measurement temperature, with fluctuations not exceeding ±0.5°C. Clean the prism surface with a soft flannel. Add 2-3 drops of the sample solution to be tested, distributing it evenly across the entire prism surface. Aim at the light source (for non-digital refractometers, turn the achromatic adjustment knob to divide the field of view into light and dark areas, then turn the prism knob so that the light-dark dividing line is aligned with the cross-section of the objective lens). Record the refractometer reading. For refractometers without automatic temperature compensation, record the measured temperature. Clean the prism surface with distilled water and a soft flannel.

[0204] 4. Calculation: For undiluted samples, the refractometer reading is the soluble solids content of the sample. For samples diluted with distilled water, the soluble solids content is calculated according to the formula.

[0205] The measurement results are calculated according to the following formula:

[0206] Where:

[0207] X: Soluble solids content of the sample, in percentage

[0208] (%)

[0209] P: Soluble solid content of the sample solution, expressed as a percentage

[0210] (%)

[0211] m0: sample mass, in grams (g);

[0212] m1: The mass of distilled water added to the sample, in grams

[0213] (g).

[0214] Vitamin C (ascorbic acid) determination method: GB5009.86-2016

[0215] Determination of Vitamin C (Ascorbic Acid)

[0216] 1. Sample Preparation: Weigh 10g of slurry sample, transfer the sample to a 100mL volumetric flask with the extractant, dilute to the mark, shake well, and filter. If the filtrate is colored, add 0.4g of white clay per gram of sample to decolorize it before filtering.

[0217] 2. Titration: Pipette an appropriate amount of filtrate into a 50mL conical flask and titrate with a standardized 2,6-dichloroindophenol solution until the solution turns pink and does not fade for 15 seconds. Perform a blank test at the same time. Calculate vitamin C using the following formula:

[0218] Where:

[0219] V1: Volume of dye solution consumed when titrating the sample (mL);

[0220] V0: volume of dye solution consumed during blank titration (mL);

[0221] T: 2,6-dichloroindophenol dye titer (mg / mL);

[0222] F: dilution factor;

[0223] M: sample weight (g).

[0224] Anthocyanin determination method: T / QAS075-2022

[0225] Determination of anthocyanins:

[0226] 1. Prepare the raw materials: Take an appropriate amount or all of the raw materials by quartering, remove the branches, leaves, broken fruits and other impurities visible to the naked eye except the fruit and stalk, and use a homogenizer to homogenize and disperse at 15000r / min for 20 seconds to make a homogenate. Use it immediately for testing. The entire testing process should be carried out under light-proof conditions.

[0227] 2. Weigh 5.0 g of fresh fruit homogenate, accurate to ±1 mg, and 0.1 g of extract, accurate to ±0.1 mg, into a 150 mL ground-mouth Erlenmeyer flask with a stopper. Add 50 mL of extract solution (concentrated HCl: 80% ethanol solution = 3:97, v / v), stopper tightly, and weigh thoroughly. Ultrasonicate and extract at 50°C for 30 minutes in an ultrasonic bath, shaking every 10 minutes to ensure complete dispersion of the solid phase. After extraction, cool to room temperature, weigh again, and make up the lost weight with the extract solution. Mix thoroughly. Centrifuge a portion of the extract at 8000 rpm for 3 minutes, and reserve the supernatant.

[0228] 3. The prepared test solution is first diluted 2-10 times (containing 5mg-60mg anthocyanins) by adding the extract (concentrated hydrochloric acid: 80% ethanol solution = 3:97, v / v), and then diluted 5 times with the buffer solution to prepare two test solutions. One is diluted with potassium chloride buffer solution (0.025mol / L, pH 1.0) (1mL test solution + 4mL potassium chloride buffer solution), and the other is diluted with sodium acetate buffer solution (0.4mol / L, pH 4.5) (1mL test solution + 4mL sodium acetate buffer solution).

[0229] 4. After the test solution has been allowed to stand for 10 minutes, scan a portion of the visible band between 400 nm and 600 nm to determine the wavelength of maximum absorption. Measure the absorbance of the test solution diluted with pH 1.0 buffer solution and the test solution diluted with pH 4.5 buffer solution at the wavelength of maximum absorption and at 700 nm, respectively.

[0230] The content of anthocyanidins (anthocyanidins) X (calculated as petunidin components) is calculated according to the following formula:

[0231] Where:

[0232] X: the content of anthocyanins in cherries calculated as petunidin components, in grams per 100 grams (g / 100g);

[0233] A: The absorbance difference between the test solution at pH 1.0 and pH 4.5, A = (Amax nm - A700 nm) pH1.0 - (Amax nm - A700 nm) pH4.5;

[0234] MW: 912.7, the average molar mass of petunidin-like components, in grams per mole (g / mol);

[0235] DF: dilution factor;

[0236] V: total volume of the extract, in milliliters (mL);

[0237] ε: 29591, the average molar extinction coefficient of petunidin components, in liters per mole centimeter (L / (mol·cm));

[0238] l: cuvette thickness, in centimeters (cm);

[0239] m: the mass of the sample, in grams (g);

[0240] 10: Conversion factor from g / kg to g / 100g.

[0241] Determination of β-carotene in cherry: GB 5009.83-2016

[0242] Reverse phase chromatography determination, the steps are as follows:

[0243] 1. Prepare the raw materials: Accurately weigh 1-5 g (accurate to 0.001 g) of the mixed sample, transfer it to a 250 mL conical flask, add 1 g of ascorbic acid and 75 mL of anhydrous ethanol, and shake in a water bath at 60°C ± 1°C for 30 minutes.

[0244] 2. Saponification: Add 25 mL of potassium hydroxide solution and cap the bottle. Place in a preheated, vibrating water bath at 53°C ± 2°C and saponify for 30 minutes. Remove, allow to stand, and cool to room temperature.

[0245] 3. Sample Extraction: Transfer the saponified solution to a 500mL separatory funnel, add 100mL of petroleum ether, gently shake to degas, cap the bottle, and shake at room temperature for 10 minutes. After stratification, transfer the aqueous phase to another separatory funnel and perform a second extraction as described above. Combine the organic phases and wash with water until nearly neutral. Discard the aqueous phase and filter the organic phase through anhydrous sodium sulfate to dehydrate it. Collect the filtrate into a 500mL evaporating flask and concentrate to near dryness on a rotary evaporator at 40°C ± 2°C under reduced pressure. Blow dry with nitrogen. Accurately add 5.0mL of dichloromethane using a pipette, cap the bottle, and dissolve the extract thoroughly. Filter through a 0.45μm membrane, discard the initial approximately 1mL of filtrate, and collect it in a sample injection vial for later use.

[0246] 4. Sample determination: Under the same chromatographic conditions, the test sample solution was injected into the liquid chromatograph for HPLC analysis. The retention time was used for qualitative analysis and the peak area was used for quantitative analysis by the external standard method. The concentration of β-carotene in the test solution was calculated according to the regression equation of the standard curve.

[0247] The measurement results are calculated according to the following formula:

[0248] Where:

[0249] ρβ: β-carotene concentration in the test solution obtained from the standard curve2, in μg / mL;

[0250] V: fixed volume of sample solution, in mL;

[0251] 100: factor to express the result as μg / 10g2;

[0252] m: sample mass, in g.

[0253] Experimental method for promoting strawberry growth by suspension concentrate of Bacillus Velez TCS001

[0254] Table 5 Treatment methods of strawberry pot test with Bacillus velezensis TCS001 suspension concentrate

[0255] The suspension concentrate of Bacillus Velez TCS001 was diluted with sterile water to the corresponding concentration and then used for root irrigation and spraying treatments respectively.

[0256] The potting soil was sterilized by dry heat sterilization at 160°C for 2 h in an oven, and each pot contained 960 g of sterile soil.

[0257] Root irrigation: After the strawberry seedlings were transplanted, each plant was irrigated with 100 mL of 300-fold diluted Bacillus Velez subtilis TCS001 suspension concentrate, and each treatment was repeated in 5 pots.

[0258] Spraying: After transplanting, the strawberry seedlings were sprayed for the first time. Every 7 days, 5 mL of spray per plant was applied (1, 2, and 3 times). Each treatment was repeated in 5 pots at a spray pressure of 0.1 MPa. Growth index measurements were performed.

[0259] Determination of strawberry morphological indicators

[0260] The basic morphological indexes (plant height) of strawberries after treatment with Bacillus Velez TCS001 suspension concentrate (14d, 21d, 28d) were measured using a ruler (graduation value 1mm). Five plants with consistent growth were selected for measurement in each treatment.

[0261] Determination of chlorophyll content in strawberry leaves

[0262] The relative chlorophyll content (SPAD) of the second expanded leaf outside the heart leaf of strawberry after treatment with TCS001 suspension concentrate (14d, 21d, and 28d) was determined using a chlorophyll meter (Zhejiang Top Yunnong Technology Co., Ltd.). Five plants with consistent growth were selected for measurement in each treatment.

[0263] Determination of fresh weight of strawberry roots and leaves

[0264] After treatment with TCS001 suspension concentrate (14d, 21d, 28d), the strawberry plants were dried with paper towels and weighed with an electronic balance to determine the fresh weight of their roots and leaves. The fresh weight of the roots and leaves of a single strawberry plant was considered one replicate, and each treatment had 5 replicates.

[0265] Determination of leaf number per strawberry plant

[0266] The number of leaves per strawberry plant was determined after treatment with TCS001 suspension concentrate (14d, 21d, and 28d). The number of leaves per strawberry plant was considered one replicate, and each treatment had 5 replicates.

[0267] Strawberry leaf area measurement

[0268] The leaf area of ​​strawberries was measured 28 days after treatment with TCS001 suspension concentrate using a leaf area meter (Zhejiang Top Yunnong Technology Co., Ltd.). The third expanded leaf outside the heart leaf of the strawberry was taken, and each treatment was replicated five times.

[0269] Enzyme activity and endogenous hormones

[0270] After 7 days of incubation following spray treatment (1, 2, or 3 times), root length, plant height, stem and leaf fresh and dry weight, and chlorophyll were measured. Leaf peroxidase (POD), superoxide dismutase (SOD), and catalase (CAT) activities were determined using corresponding enzyme-linked immunosorbent assay kits. The contents of abscisic acid (ABA), indoleacetic acid (IAA), and gibberellin (GA) in strawberry leaves were also measured using corresponding enzyme-linked immunosorbent assay kits.

[0271] Application of Bacillus Velez TCS001 Suspension Concentrate in Facility Tomatoes

[0272] Experimental design

[0273] This experiment set up three treatment groups:

[0274] Group A was the group treated with Bacillus lyssum TCS001;

[0275] Group B was the chemical agent treatment group;

[0276] Group C was the blank control group (not treated with any drug).

[0277] Each treatment area is 200 square meters. The treatment plans for Groups A and B are shown in the table below.

[0278] Table 6 Application scheme of tomato A treatment group

[0279] Table 7 Treatment and application plan of tomato B

[0280] Disease prevention effectiveness survey

[0281] Investigation on the effectiveness of prevention of tomato damping-off disease

[0282] After tomato seedlings emerged, when the blank control group showed obvious symptoms of disease, the seedling mortality rate was checked and the control effect was calculated. Three seedling trays were selected for each treatment for investigation.

[0283] Investigation on the effectiveness of control of tomato bacterial wilt

[0284] When obvious bacterial wilt symptoms appeared in the blank control group, the control efficacy of tomato bacterial wilt was investigated. According to the method of "NY / T 1464.32-2010 Pesticide Field Efficacy Test Guidelines Part 32 - Fungicide Control of Tomato Bacterial Wilt", 4 random sampling points were taken for each treatment, and each point was investigated for 8 m 2 , investigate the total number of tomato plants and the number of diseased plants respectively. Calculate the diseased plant rate and prevention effect.

[0285] Investigation on the effectiveness of prevention of tomato early blight

[0286] When obvious early blight symptoms appeared in the blank control group, the control efficacy against tomato bacterial wilt was assessed. Following the method outlined in "GB / T 17980.31-2000 Guidelines for Field Efficacy of Pesticides (I) - Control of Tomato Early and Late Blight by Fungicides," five random sampling points were selected for each treatment, with two plants surveyed at each point. Ten leaves from each plant were surveyed, divided into the upper, middle, and lower sections. The percentage of lesion area on each leaf to the total leaf area was used for grading.

[0287] Leaf grading method:

[0288] Level 0: no lesions;

[0289] Level 1: The lesion area accounts for less than 5% of the entire leaf area;

[0290] Level 3: The lesion area accounts for 6%-10% of the entire leaf area;

[0291] Level 5: The lesion area accounts for 11%-20% of the entire leaf area;

[0292] Level 7: The lesion area accounts for 21%-50% of the entire leaf area;

[0293] Level 9: The area of ​​lesions accounts for more than 50% of the total leaf area.

[0294] According to the survey results, the disease index and prevention effect are calculated according to the following formula.

[0295] Investigation on the Control Effect of Tomato Gray Mold

[0296] When obvious symptoms of gray mold appear in the blank control group, investigate the gray mold control efficacy. Refer to the method in "GB / T 17980.28-2000 Guidelines for Field Efficacy Tests of Pesticides (I) - Control of Gray Mold of Vegetables with Fungicides." Samples were collected at five locations per treatment, and two plants were surveyed at each location. All fruits from each plant were surveyed, and the diseased fruit rate was calculated and recorded according to the following grading method.

[0297] Fruit grading method:

[0298] Level 0: no lesions;

[0299] Level 1: Remaining petals are diseased or stigma is diseased;

[0300] Level 3: Sepal rot or stigma disease spreads to the navel;

[0301] Level 5: There are infiltration spots on the navel of the fruit without mold layer;

[0302] Grade 7: There is a mold layer at the navel but it does not spread to other parts of the fruit;

[0303] Level 9: The mold layer extends to other parts of the fruit.

[0304] According to the survey results, the disease index and prevention effect are calculated according to the following formula.

[0305] Investigation on the effectiveness of control of tomato wilt

[0306] When the blank control group showed obvious symptoms of wilt, the wilt control efficacy was investigated. According to the method of "GB / T 17980.113-2004 Guidelines for Field Efficacy Tests of Pesticides (II) - Control of Fusarium Wilt by Fungicides for Cucurbits", 4 random sampling points were taken for each treatment, and each point was investigated for 8 m 2 , investigate the total number of tomato plants and the number of diseased plants respectively. Calculate the diseased plant rate and prevention effect.

[0307] Tomato biomass determination

[0308] Four points were randomly selected for each treatment, and 15 consecutive plants were selected at each point, totaling 60 plants. They were marked with red lines. The plant height and stem diameter of the selected tomato plants were measured at the initial flowering stage of tomatoes.

[0309] Tomato yield determination

[0310] Starting from the red ripe stage, ripe fruits of marked tomatoes were picked every 7 days until the end of the vine pulling stage. The yield per plant was the sum of the number of times and weight of red fruits picked. The number of fruits was recorded at each picking.

[0311] Tomato quality measurement

[0312] The vitamin C content was determined with reference to GB 5009.86-2016 National Food Safety Standard - Determination of Ascorbic Acid in Foods; the total acid content was determined with reference to GB / T 12456-2008 Determination of Total Acid in Foods; the soluble sugar content was determined with reference to GB 5009.8-2016 National Food Safety Standard - Determination of Fructose, Glucose, Sucrose, Maltose and Lactose in Foods; and the protein content was determined with reference to GB 5009.9-2016 National Food Safety Standard - Determination of Starch in Foods.

[0313] Application of Bacillus Velez TCS001 on greenhouse cucumbers

[0314] Experimental design

[0315] This experiment involved three treatment groups: Group A was treated with Bacillus subtilis TCS001; Group B was treated with a chemical agent; and Group C served as a blank control (no chemical treatment). Each treatment area was 200 square meters. The treatment plans for Groups A and B are shown in the table below.

[0316] Table 8 Spraying scheme for cucumber A treatment group

[0317] Table 9 Cucumber B treatment application plan

[0318] Investigation on the effectiveness of prevention of cucumber damping-off disease

[0319] After cucumber seedlings emerged, when obvious disease symptoms appeared in the blank control group, the seedling mortality rate was checked and the control effect was calculated. Three seedling trays were selected for each treatment for investigation.

[0320] Investigation on the control effect of bacterial angular leaf spot of cucumber

[0321] When obvious angular leaf spot symptoms appeared in the blank control group, the angular leaf spot control efficacy was investigated. This was conducted in accordance with the provisions of "GB / T17980.110-2004 Guidelines for Field Efficacy of Pesticides (II): Control of Cucumber Bacterial Angular Leaf Spot with Fungicides." Samples were collected at three locations per treatment, with five plants surveyed at each location and all leaves surveyed for each plant. Grades were determined based on the percentage of lesion area on each leaf to the total leaf area.

[0322] Grading method:

[0323] Level 0: no lesions;

[0324] Level 1: lesions occupy less than 5% of the entire leaf area;

[0325] Level 3: lesions occupy 6%-10% of the entire leaf area;

[0326] Level 5: lesions occupy less than 11%-20% of the entire leaf area;

[0327] Level 7: lesions occupy less than 21%-50% of the entire leaf area;

[0328] Level 9: Lesions cover more than 51% of the entire leaf area.

[0329] According to the survey results, the disease index and prevention effect are calculated according to the following formula.

[0330] Investigation on the effectiveness of controlling cucumber powdery mildew

[0331] When obvious powdery mildew symptoms appeared in the blank control group, the powdery mildew control efficacy was investigated. This was conducted in accordance with the provisions of "GB-T 17980.30-2000 Guidelines for Field Efficacy of Pesticides (I) Part 30: Control of Cucumber Powdery Mildew with Fungicides." Five random surveys were conducted per treatment, with two plants surveyed at each survey point and all leaves surveyed for each plant. The percentage of lesion area on each leaf to the total leaf area was used to grade the disease, and the disease index and control efficacy were calculated.

[0332] Grading method (leaf unit):

[0333] Level 0: no lesions;

[0334] Level 1: lesions occupy less than 5% of the entire leaf area;

[0335] Level 3: lesions occupy 6%-10% of the entire leaf area;

[0336] Level 5: lesions occupy less than 11%-20% of the entire leaf area;

[0337] Level 7: lesions occupy less than 21%-40% of the entire leaf area;

[0338] Level 9: Lesions cover more than 40% of the entire leaf area.

[0339] According to the survey results, the disease index and prevention effect are calculated according to the following formula.

[0340] Investigation on the effectiveness of prevention against cucumber downy mildew

[0341] When obvious downy mildew symptoms appeared in the blank control group, the control efficacy was assessed. This was conducted in accordance with the requirements of "GB-T 17980.26-2000 Guidelines for Field Efficacy of Pesticides (I) Part 26: Control of Cucumber Downy Mildew with Fungicides." Five random sites were surveyed in each plot, with three plants at each site and ten leaves from each plant. The percentage of lesion area on each leaf to the total leaf area was used for grading.

[0342] Grading method (leaf unit):

[0343] Level 0: no lesions;

[0344] Level 1: The lesion area accounts for less than 5% of the entire leaf area;

[0345] Level 3: The lesion area accounts for 6%-10% of the entire leaf area;

[0346] Level 5: The lesion area accounts for 11%-20% of the entire leaf area;

[0347] Level 7: The lesion area accounts for 21%-50% of the entire leaf area;

[0348] Level 9: The lesion area accounts for more than 50% of the entire leaf area.

[0349] According to the survey results, the disease index and prevention effect are calculated according to the following formula.

[0350] Investigation on the effectiveness of prevention of cucumber wilt

[0351] When the blank control group showed obvious symptoms of wilt, the wilt control efficacy was investigated. According to the method of "GB / T 17980.113-2004 Guidelines for Field Efficacy Tests of Pesticides (II) - Control of Fusarium Wilt by Fungicides for Cucurbits", 4 random sampling points were taken for each treatment, and each point was investigated for 8 m 2 , investigate the total number of cucumber plants and the number of diseased plants respectively. Calculate the diseased plant rate and prevention effect.

[0352] Cucumber biomass determination

[0353] Four points were randomly selected for each treatment, and 15 consecutive plants were selected at each point, totaling 60 plants. They were marked with red lines. At the initial flowering stage of cucumber, the stem diameter of the cucumber plants was measured, and the relative chlorophyll content of the fourth to last leaf was determined using a handheld chlorophyll meter TYS-A.

[0354] Cucumber yield determination

[0355] After the fruits matured, cucumber fruits with a diameter of 3 to 4 cm were collected regularly for 8 consecutive times to calculate the cumulative yield of cucumbers.

[0356] Cucumber quality determination

[0357] The vitamin C content was determined in accordance with GB 5009.86-2016 National Food Safety Standard - Determination of Ascorbic Acid in Foods; the moisture content was determined in accordance with GB 5009.3-2016 National Food Safety Standard - Determination of Water in Foods; and the soluble solids content was determined in accordance with NY-T 2637-2014 Fruits and Vegetables - Determination of Soluble Solids Content.

[0358] Test results

[0359] It can be seen from the biocompatibility test results in Figures 1 and 2 that Bacillus velez TCS001 grows better in SXC, 4913, UNA, NN9401, and NS-500LQ, while the other wetting and dispersing agents inhibit the growth of Bacillus velez TCS001 as the concentration increases. Therefore, the five wetting and dispersing agents with better compatibility were re-screened in the next step.

[0360] Table 10 Wetting and dispersing agent rescreening results

[0361] Table 10 shows that the addition of SXC, UNA, and NN9401 resulted in higher suspension efficiency than 4913 and NS-500LQ, but all were below 90%. Further experiments were conducted using two-by-two combinations to improve suspension efficiency. Table 11 shows that the optimal ratio of wetting and dispersing agents is 3% SXC + 3% UNA, which achieves the highest suspension efficiency.

[0362] Table 11 Wetting and dispersing agent combination screening results

[0363] As shown in Figure 3, while magnesium aluminum silicate did not inhibit the growth of Bacillus Velez TCS001 as its concentration increased, it was difficult to dissolve in the culture medium and formed small clumps. Xanthan gum, on the other hand, did not exhibit this phenomenon and did not inhibit the growth of Bacillus Velez TCS001. Therefore, xanthan gum was selected as the thickener. Table 12 shows that the optimal xanthan gum concentration was 0.15%.

[0364] Table 12 Screening results of xanthan gum addition amount of thickener

[0365] As shown in Figure 4, S30N inhibited the growth of Bacillus velezensis TCS001, so kasone and sodium benzoate were selected as preservatives for the next test.

[0366] As shown in Table 13, the optimal ratio of kasonon to sodium benzoate is 0.2% + 0.2%, which results in the highest suspension rate and the lowest mildew rate.

[0367] Table 13 Anticorrosive effect of preservatives

[0368] According to the optimization results of the adjuvants of wetting and dispersing agent, thickener and preservative, Table 14 shows the optimal formula of the suspension concentrate of Bacillus velezensis TCS001.

[0369] Table 143.9 billion CFU / mL Velezella TCS001 suspension formulation

[0370] Table 15 shows the performance index test results of 3.9 billion CFU / mL Bacillus velezensis TCS001 suspension concentrate, and all performance indicators meet the qualified standards.

[0371] Table 1 Performance test results of 539 billion CFU / mL Velezella TCS001 suspension concentrate

[0372] According to FIG5 , the viable bacteria of the 3.9 billion CFU / mL Velez subtilis TCS001 suspension concentrate of the present invention decreased from 3.9×10 9 The CFU / mL dropped to 3.86×10 9 CFU / mL, the active ingredients after storage are almost not reduced compared with those before storage, that is, it is stable when stored at 0℃.

[0373] According to FIG6 , the viable bacteria of the 3.9 billion CFU / mL Velez subtilis TCS001 suspension concentrate of the present invention decreased from 3.9×10 9 The CFU / mL dropped to 3.73×10 9 CFU / mL, the effective ingredient after storage was only reduced by 4.3% compared with that before storage, that is, it was stable when stored at 4°C.

[0374] According to FIG7 , the viable bacteria of the 3.9 billion CFU / mL Velez subtilis TCS001 suspension concentrate of the present invention decreased from 3.9×10 9 The CFU / mL dropped to 2.36×10 9 CFU / mL, the active ingredient after storage decreased by 39.4% compared with that before storage, that is, it is unstable when stored at 28℃.

[0375] As shown in FIG8 , the viable bacteria of the 3.9 billion CFU / mL Velez subtilis TCS001 suspension concentrate of the present invention decreased from 3.73×10 9 The CFU / mL dropped to 3.56×10 9 CFU / mL, the effective ingredient after storage decreased by 4.5% compared with that before storage, that is, it is stable when stored at 35℃.

[0376] As shown in FIG9 , the viable bacteria of the 3.9 billion CFU / mL Velez subtilis TCS001 suspension concentrate of the present invention decreased from 3.73×10 9 The CFU / mL decreased to 1.73×10 9 CFU / mL, the effective ingredients after storage decreased by 53.6% compared with those before storage, that is, it is unstable when stored at 45℃.

[0377] According to FIG10 , the viable bacteria of the 3.9 billion CFU / mL Velez subtilis TCS001 suspension concentrate of the present invention decreased from 3.76×10 9 The CFU / mL dropped to 0.15×10 9 CFU / mL, the effective ingredients after storage decreased by 96% compared with those before storage, that is, it is unstable when stored at 54℃.

[0378] As shown in Table 16, the spraying treatment with the suspension concentrate of Bacillus Velez TCS001 diluted 400 times, once each during the flower bud differentiation period, early flowering period and full flowering period of cherry, and once a week from the young fruit period to the harvest period, can increase the β-carotene content of cherry by 13.6%, anthocyanin by 9.8%, soluble sugar by 7.3%, reduce vitamin C by 19.4%, and reduce the total acid of cherry by 7%.

[0379] Table 16 Cherry quality measurement results

[0380] As shown in Table 17, the root irrigation treatment with the suspension concentrate of Bacillus Velez TCS001 diluted 500 times, once during the flower bud differentiation stage, early flowering stage and full flowering stage of blueberries, and once a week from the young fruit stage to the harvest stage, can increase the anthocyanin content of blueberries by 10%, the soluble sugar content by 4%, the vitamin C content by 5%, and reduce the total acid content of blueberries by 20%.

[0381] Table 17 Blueberry quality measurement results

[0382] During the experiment, it was observed that Bacillus Velez TCS001 had a positive effect on other characteristics of blueberries, such as color, size, and glossiness. The five groups of people who tasted the blueberries found the flavor to be favorable, as shown in Table 18 below.

[0383] Table 18 Blueberry tasting results

[0384] The plants after the spraying and root irrigation treatments of Bacillus Velez TCS001 suspension concentrate at 300 times concentration are shown in FIG17 .

[0385] The growth index measurement results are shown in Figures 18 and 19.

[0386] As shown in Figure 18, after treatment with 300 times the suspension concentrate of Bacillus Velez TCS001, the chlorophyll concentration of strawberry leaves was the highest at 14d and 21d, respectively, compared with CK, which increased by 11% and 14.89%. The fresh weight of leaves, stems and leaves, and roots of individual strawberries at 28d increased by 26.5%, 79.4%, and 120%, respectively, compared with CK.

[0387] After root irrigation with 300 times the suspension concentrate of Bacillus Velez TCS001, the leaf area and plant height of strawberry increased by 68.41% and 29.68% respectively compared with CK on day 28.

[0388] Endogenous hormone and enzyme activity assay results

[0389] As shown in Figure 19, after root irrigation with 300-fold suspension concentrate of Bacillus Velez TCS001, the IAA content, ABA content, and SOD activity of strawberry leaves increased by 38.47%, 36.04%, and 21.41% on day 28 compared with CK.

[0390] After treatment with 300 times the concentration of Bacillus Velez TCS001 suspension concentrate, the GA content, CAT activity and POD activity of strawberry leaves increased by 34.04%, 21.23% and 23.21% respectively compared with CK on day 28.

[0391] Effect of application of suspension concentrate of Bacillus Velez TCS001 on greenhouse tomatoes

[0392] Table 19 Effect of disease prevention and control on tomatoes throughout the whole process

[0393] As can be seen from Table 19, the full application of Bacillus Velez TCS001 suspension concentrate has a good preventive effect on important diseases such as damping-off, bacterial wilt, early blight, gray mold and wilt in the growth process of greenhouse tomatoes, which is better than conventional chemical agents.

[0394] Table 20 Results of the tomato quality improvement test throughout the entire process

[0395] It can be seen from Table 20 that after applying the TCS001 bacterial agent composition, the quality and yield of greenhouse tomatoes were significantly improved. The biomass of tomatoes increased, the yield was significantly improved, the soluble solid content, vitamin C, and soluble sugar content were all increased, and the titratable acid content was reduced.

[0396] Effect of application of Bacillus Velez TCS001 suspension concentrate on greenhouse cucumbers

[0397] Table 21 Effect of cucumber disease prevention and control throughout the whole process

[0398] As can be seen from Table 21, the full application of Bacillus Velez TCS001 suspension concentrate has a good preventive effect on important diseases of cucumber growth in the greenhouse, including damping-off, bacterial angular leaf spot, powdery mildew, downy mildew and wilt. Except for bacterial angular leaf spot, which is equivalent to conventional chemical agents, the other diseases are better than chemical agents.

[0399] Table 22 Results of the cucumber quality improvement test throughout the entire process

[0400] As can be seen from Table 22, after applying the TCS001 microbial agent composition, the yield of cucumber was significantly increased; the vitamin C and moisture content were both increased; it can be seen that the application of the Bacillus Velez TCS001 microbial agent composition has a significant effect on improving the quality and increasing the yield of greenhouse cucumbers.

[0401] The laboratory of the inventor of this application has previously developed a wettable powder containing Bacillus Velez TCS001 with the following formula: 70% fermentation broth of Bacillus Velez TCS001, 10% carrier diatomaceous earth, 5% dispersant polyvinyl alcohol, 10% wetting agent sodium dodecylbenzenesulfonate, 2.5% stabilizer potassium phosphate, and 2.5% protective agent dextrin.

[0402] After testing, the wettable powder containing Bacillus Velez TCS001 of the present invention has a suspension rate of 80.2% and a wetting time of 20 seconds, which can meet the requirements for pesticide registration.

[0403] However, due to the shortcomings of wettable powders such as the serious loss of secondary metabolites produced by the strains during the development process, the large amount of sediment produced during use, the low suspension rate, and the easy clogging of the sprayer nozzle, the low suspension rate will lead to uneven concentration of the liquid medicine, reducing the prevention and control effect and increasing the probability of disease occurrence, the inventors did not consider wettable powders as a commercial dosage form.

[0404] Compared with the previous powder, the suspension concentrate of the present invention has no dust hazard and is relatively safe for operators and the environment. In addition, the suspension concentrate diffuses well in water and can be directly prepared into a spray liquid for use. It has good dispersibility, a high suspension rate, a strong ability to adhere to the surface of plants, and is resistant to rain erosion. Therefore, the efficacy is more significant and more lasting than that of wettable powders. It also has the advantages of wettable powders, can be wetted by water, and has better suspension properties after dilution with water.

[0405] Experimental results show that treatment with the Bacillus Velez-like TCS001 suspension concentrate significantly improved the quality of cherries and blueberries, accelerating blueberry ripening and improving taste. It also significantly promoted strawberry plant growth and induced faster and stronger protective enzyme responses in strawberry leaves. Furthermore, application of the suspension concentrate to cucumbers and tomatoes effectively controlled diseases, outperforming conventional chemical agents and resulting in increased yield and quality.

[0406] 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 SXC and UNA in the preparation of a preparation containing Bacillus velezensis TCS001, preferably, the mass ratio of SXC to UNA is 1:

1.

2. Use of kason and sodium benzoate in the preparation of a preparation containing Bacillus Velez TCS001, preferably, the mass ratio of kason and sodium benzoate is 1:3-3:

1.

3. Use of xanthan gum in the preparation of a preparation containing Bacillus Velez TCS001.

4. The use according to any one of claims 1 to 3, characterized in that: The preparation containing Bacillus Velez subtilis TCS001 is a suspension containing Bacillus Velez subtilis TCS001.

5. A suspension concentrate containing Bacillus Velez TCS001, characterized in that: The invention comprises 10-60% of Bacillus Velez TCS001 fermentation liquid, 2-10% of a wetting and dispersing agent, 0.1-1% of a thickener, 0.1-0.5% of a preservative, and 100% of water. The wetting and dispersing agent is SXC and UNA, and the preservative is kason and sodium benzoate.

6. The suspension concentrate containing Bacillus Velez subtilis TCS001 according to claim 5, characterized in that: The mass ratio of the kasonite to sodium benzoate is 1:

1.

7. The suspension concentrate containing Bacillus Velez subtilis TCS001 according to claim 5, characterized in that: The mass ratio of SXC to UNA is 1:

1.

8. The suspension concentrate containing Bacillus Velez subtilis TCS001 according to claim 5, characterized in that: The thickener is xanthan gum.

9. A suspension concentrate containing Bacillus Velez TCS001, characterized in that: The invention comprises 40% of fermentation broth of Bacillus Velez TCS001, 3% of wetting and dispersing agent SXC, 3% of wetting and dispersing agent UNA, 0.15% of thickener xanthan gum, 0.2% of preservative kason, 0.2% of preservative sodium benzoate, and 100% of water.

10. A wettable powder containing Bacillus Velez TCS001, characterized in that: The invention comprises 70% of fermentation liquid of Bacillus Velez TCS001, 10% of carrier diatomaceous earth, 5% of dispersant polyvinyl alcohol, 10% of wetting agent sodium dodecylbenzene sulfonate, 2.5% of stabilizer potassium phosphate and 2.5% of protective agent dextrin.

11. Use of the Bacillus Velez TCS001 suspension according to any one of claims 5 to 9 as a drug for improving the quality of cherries and blueberries.

12. The use according to claim 11, characterized in that: The method of improving cherry quality includes increasing cherry soluble sugar; increasing cherry soluble solids, reducing cherry vitamin C, increasing cherry anthocyanins, increasing cherry β-carotene, reducing cherry acidity, etc.; the method of improving blueberry quality includes increasing blueberry sugar content and anthocyanins, reducing blueberry acidity, increasing vitamin C content, color, size, glossiness, etc. ...

13. A method for improving the quality of cherries and blueberries using the Bacillus Velez TCS001 suspension concentrate according to any one of claims 5 to 9, characterized in that: The method of improving cherry quality includes one or more of increasing cherry soluble sugar, increasing cherry anthocyanin, increasing cherry soluble solids, reducing cherry vitamin C, increasing cherry anthocyanin, increasing cherry beta-carotene, reducing cherry acidity, etc.; the method of improving blueberry quality includes one or more of increasing blueberry sugar content and anthocyanin, reducing blueberry acidity, and increasing vitamin C content, color, size, glossiness, etc.

14. A method for promoting early ripening of blueberries, characterized in that: The method comprises treating blueberries with the Bacillus Velez TCS001 suspension concentrate according to any one of claims 5 to 9; preferably, the method promotes the blueberries to mature 5 to 7 days earlier.

15. The use according to any one of claims 11-12 or the method according to any one of claims 13-14, characterized in that: The concentration of the suspension when applied to cherries is 300-700 times diluted; preferably, 400 times diluted; the concentration when applied to blueberries is 300-700 times diluted; preferably, 500 times diluted.

16. The use or method according to any one of claims 11 to 14, characterized in that: The Bacillus Velez TCS001 suspension concentrate can be applied to cherries at one or more of the following stages: flower bud differentiation stage, early flowering stage, full flowering stage, young fruit stage, and harvesting stage.

17. The use or method according to any one of claims 11 to 14, characterized in that: The application method of the Bacillus Velez TCS001 on cherries is spraying treatment; the application method of the Bacillus Velez TCS001 on blueberries is root irrigation and / or spraying treatment.

18. A method for increasing the size and / or glossiness and / or taste of cherries, characterized in that: The method comprises treating cherries with the Bacillus Velez TCS001 described in any one of claims 5 to 9.

19. Use of the Bacillus Velez TCS001 suspension concentrate according to any one of claims 5 to 9 as a drug for improving the growth traits of strawberries.

20. The use according to claim 19, characterized in that The strawberry growth traits include one or more of leaf chlorophyll concentration, single plant leaves, stem and leaf fresh weight, root fresh weight, leaf area, plant height, leaf IAA content, ABA content, SOD activity, leaf GA content, CAT activity, POD activity, etc.

21. A method for improving the growth characteristics of strawberries using the Bacillus Velez TCS001 suspension concentrate according to any one of claims 5 to 9, characterized in that: The strawberry growth traits include one or more of leaf chlorophyll concentration, single plant leaves, stem and leaf fresh weight, root fresh weight, leaf area, plant height, leaf IAA content, ABA content, SOD activity, leaf GA content, CAT activity, POD activity, etc.

22. The use according to claims 19-20 or the method according to claim 21, characterized in that: The application concentration of the suspension is 200-500 times diluted; preferably, 300 times diluted.

23. The use according to any one of claims 19-20 or the method according to claim 21, characterized in that: The application time of the Bacillus Velez TCS001 is the seedling stage.

24. The use according to claim 23 or the method according to claim 23, characterized in that The application method of the Bacillus Velez TCS001 is root irrigation and / or spraying treatment.

25. Use of the Bacillus Velez TCS001 suspension concentrate according to any one of claims 5 to 9 as a drug for full-process disease prevention and control and / or quality improvement on greenhouse crops, wherein the greenhouse crops are tomatoes and cucumbers.

26. The use according to claim 25, characterized in that The diseases include one or more of tomato damping-off, bacterial wilt, early blight, gray mold, and wilt; cucumber damping-off, bacterial angular spot, powdery mildew, downy mildew, and wilt.

27. The use according to claim 25, characterized in that The quality aspects include one or more of the plant height, stem thickness, average single fruit weight, average single plant yield, soluble solids, vitamin C, soluble sugars, and titratable acid of tomatoes; and the circumference of the base of the main stem of cucumbers, relative chlorophyll content, number of roots, average fruit weight, soluble solids, vitamin C, and water content.

28. A method for improving tomato quality, characterized in that: The Bacillus Velez TCS001 suspension concentrate according to any one of claims 5 to 9 is applied during one or more of the tomato seedling stage, transplanting day, seedling stage, flowering stage, and fruiting stage.

29. A method for improving the quality of cucumbers, characterized in that: The Bacillus Velez TCS001 suspension concentrate according to any one of claims 5 to 9 is applied during one or more of the cucumber seedling stage, transplanting day, planting stage, vine growth stage, and flowering and fruiting stage.

30. The method according to any one of claims 28-29, characterized in that: It also includes applying amino-oligosaccharides during the tomato seedling stage and applying amino-oligosaccharides during the cucumber planting and vine-forming stages.

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