Application of bacillus velezensis TCS001 in facility crops

Abstract

The invention relates to application of bacillus velezensis TCS001 as a drug for whole-course disease prevention and control and / or quality improvement on facility crops, wherein the facility crops are tomatoes and cucumbers. After the bacillus velezensis TCS001 is applied in the whole growth process of the facility crops, the diseases of the facility crops are effectively controlled, the prevention and control effect of the bacillus velezensis TCS001 is better than that of a conventional chemical agent, and the quality and yield of the crops are improved compared with those of a chemical agent treatment group.

Description

Technical Field

[0001] The present invention relates to the field of crop disease prevention and control and crop growth regulation, and in particular to the application of Bacillus Velezii TCS001 in the whole-process disease prevention and control and quality improvement of facility crops. 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] Protected cultivation can provide people with off-season agricultural products and increase yields, but the increasing popularity of vegetable cultivation technology has caused diseases to occur all year round, especially with continuous high temperature and humidity and continuous cultivation, which has increased the number of diseases year by year, seriously harming vegetable crops such as tomatoes and cucumbers, and directly affecting vegetable yield and quality. At present, the prevention and control of crop diseases in facilities is mainly chemical, but the frequent use of chemical pesticides poses a hidden danger to the environment and food safety on the table. In order to effectively control the use of chemical pesticides and promote the sustainable development of agriculture, we emphasize the research and development of biological pesticides, effectively promote the green prevention and control of crop diseases and insects, and reduce the use of pesticides and fertilizers.

[0005] This invention uses Bacillus subtilis TCS001 as the active ingredient for the first time and applies it throughout the growth process of greenhouse tomatoes and cucumbers, aiming to prevent and control diseases, improve quality and increase yield, while reducing the use of chemical pesticides and fertilizers. Summary of the invention

[0006] The main purpose of the present invention is to provide an application of Bacillus Velez TCS001 in the whole process of disease prevention and control and quality improvement on facility crops, wherein the facility crops are tomatoes and cucumbers;

[0007] The diseases include one or more of tomato damping-off, bacterial wilt, early blight, gray mold, wilt, etc.;

[0008] The diseases include one or more of cucumber damping-off, bacterial angular spot, powdery mildew, downy mildew, wilt, etc.;

[0009] The quality aspects include one or more of tomato plant height, stem thickness, average single fruit weight, average single plant yield, soluble solids, vitamin C, soluble sugar, and titratable acid;

[0010] The quality aspects include one or more of the circumference of the base of the cucumber main stem, relative chlorophyll content, number of roots, average fruit weight, soluble solids, vitamin C, and water content;

[0011] The Bacillus Velez TCS001 is the fermentation liquid of Bacillus Velez TCS001.

[0012] The Bacillus Velez TCS001 fermentation broth is prepared into a suspension;

[0013] The formula of the suspension is 40% of Bacillus Velez TCS001 fermentation liquid, 3% of dispersant SXC, 3% of dispersant UNA, 0.15% of thickener, 0.2% of preservative Kathon, 0.2% of preservative sodium benzoate, and 53.45% of water.

[0014] Another object of the present invention is to provide a method for improving tomato quality, wherein Bacillus Velez TCS001 is applied during one or more of the seedling stage, transplanting day, seedling stage, flowering stage, and fruiting stage of the tomato.

[0015] Another object of the present invention is to provide a method for improving the quality of cucumbers, wherein Bacillus Velez TCS001 is applied during one or more of the cucumber seedling stage, transplanting day, planting stage, vine growth stage, and flowering and fruiting stage.

[0016] The Bacillus Velez TCS001 is the fermentation liquid of Bacillus Velez TCS001.

[0017] 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.

[0018] Beneficial technical effects of the present invention

[0019] After applying Bacillus Velez TCS001 throughout the growth process of facility crops, tomato and cucumber diseases are effectively controlled, and the prevention and control effect is better than that of conventional chemical agents; at the same time, the crop quality and yield are improved compared with the conventional chemical agent treatment group.

[0020] By applying Bacillus Velez TCS001 throughout the entire process, we can not only prevent and control major diseases, but also reduce the amount of chemical pesticides and fertilizers and increase their efficiency. Specific embodiments

[0021] Example 1

[0022] Application of Bacillus Velez TCS001 in greenhouse tomatoes

[0023] Test agents and sources

[0024] 3.9 billion CFU / mL Bacillus Velez TCS001 suspension, Zhejiang Agriculture and Forestry University, the specific formula is as follows:

[0025] 3.9 billion CFU / mL Bacillus Velez TCS001 suspension formula

[0026]

[0027] 2% amino oligosaccharide aqueous solution, Shandong Heyi Biotechnology Co., Ltd.;

[0028] 60% sulfur·sodium sulfamethoxam wettable powder, Dandong Pesticide Factory, Liaoning Province;

[0029] 3% Zhongshengmycin wettable powder, Shandong Zhaofengnian Biotechnology Co., Ltd.;

[0030] 75% chlorothalonil water dispersible granules, Sipcam, USA;

[0031] 40% pyrimethanil wettable powder, Jiangsu Fengdeng Crop Protection Co., Ltd.; Experimental design

[0032] This experiment set up 3 treatment groups:

[0033] Group A was the group treated with Bacillus velezensis TCS001;

[0034] Group B was the chemical agent treatment group;

[0035] Group C was the blank control group (without any drug treatment).

[0036] Each treatment area is 200 square meters.

[0037] The treatment plans for Groups A and B are shown in the table below.

[0038] Table 1 Application scheme of tomato A treatment group

[0039]

[0040] Table 2 Tomato B treatment application plan

[0041]

[0042]

[0043] Disease prevention effect survey

[0044] Investigation on the efficacy of preventing tomato damping-off disease

[0045] After tomato seedlings emerged, when the blank control group showed obvious symptoms of disease, the seedling death rate was checked and the control effect was calculated. Three seedling trays were selected for each treatment for investigation.

[0046]

[0047] Investigation on the efficacy of controlling tomato bacterial wilt

[0048] When the blank control group showed obvious symptoms of bacterial wilt, the control efficacy of tomato bacterial wilt was investigated. According to the method of "NY / T1464.32---2010 Pesticide Field Efficacy Test Guidelines Part 32-Fungicide Control of Tomato Bacterial Wilt", 4 random sampling points were selected for each treatment, and each point was investigated for 8m 2 , investigate the total number of tomato plants and the number of diseased plants respectively. Calculate the diseased plant rate and prevention effect.

[0049]

[0050] Investigation on the efficacy of early blight control in tomatoes

[0051] When the blank control group showed obvious early blight symptoms, the control efficacy of tomato bacterial wilt was investigated. According to the method of "GB / T17980.31---2000 Pesticide Field Efficacy Test Guidelines (I)-Fungicide Control of Tomato Early Blight and Late Blight", 5 random sampling points were selected for each treatment, 2 plants were investigated at each point, and 10 leaves of each plant were investigated at the upper, middle and lower parts. The grades were based on the percentage of the lesion area on each leaf to the entire leaf area.

[0052] Leaf grading method:

[0053] Level 0: no lesions;

[0054] Level 1: The lesion area accounts for less than 5% of the entire leaf area;

[0055] Level 3: The lesion area accounts for 6%-10% of the entire leaf area;

[0056] Level 5: The lesion area accounts for 11%-20% of the entire leaf area;

[0057] Level 7: The lesion area accounts for 21%-50% of the entire leaf area;

[0058] Level 9: The lesion area accounts for more than 50% of the total leaf area.

[0059] According to the survey results, the disease index and prevention effect are calculated according to the following formula.

[0060]

[0061]

[0062] Investigation on the efficacy of controlling tomato gray mold

[0063] When the blank control group showed obvious symptoms of gray mold, the gray mold control efficacy was investigated. According to the method of "GB / T17980.28---2000 Pesticide Field Efficacy Test Guidelines (I)-Fungicide Control of Vegetable Gray Mold", 5 points were sampled for each treatment, 2 plants were investigated at each point, all fruits of each plant were investigated, and the diseased fruit rate was calculated and recorded according to the following grading method.

[0064] Fruit grading method:

[0065] Level 0: no lesions;

[0066] Level 1: The remaining petals are diseased or the stigma is diseased;

[0067] Level 3: Sepal rot or column hair disease spreads to the navel;

[0068] Level 5: There are infiltration spots on the navel of the fruit without mold layer;

[0069] Grade 7: There is a mold layer at the navel but it does not spread to other parts;

[0070] Level 9: The mold layer extends to other parts of the fruit.

[0071] According to the survey results, the disease index and prevention effect are calculated according to the following formula.

[0072]

[0073]

[0074] Investigation on the efficacy of preventing tomato wilt

[0075] When the blank control group showed obvious symptoms of wilt, the wilt control efficacy was investigated. According to the method of "GB / T17980.113---2004 Pesticide Field Efficacy Test Guidelines (II)-Fungicide Control of Melon Wilt", 4 random sampling points were selected for each treatment, and each point was investigated for 8m 2 , investigate the total number of tomato plants and the number of diseased plants respectively. Calculate the diseased plant rate and prevention effect.

[0076]

[0077] Tomato biomass determination

[0078] 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 thickness of the selected tomato plants were measured at the initial flowering stage of tomatoes.

[0079] Tomato yield determination

[0080] Starting from the red ripe stage, the 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 quality of the red fruits picked. The number of fruits was recorded each time they were picked.

[0081] Tomato quality measurement

[0082] The vitamin C content was determined by referring to GB 5009.86-2016 National Food Safety Standard Determination of Ascorbic Acid in Foods; the total acid content was determined by referring to GB / T 12456-2008 Determination of Total Acid in Foods; the soluble sugar content was determined by referring to GB 5009.8-2016 National Food Safety Standard Determination of Fructose, Glucose, Sucrose, Maltose and Lactose in Foods; the protein content was determined by referring to GB 5009.9-2016 National Food Safety Standard Determination of Starch in Foods.

[0083] Example 2

[0084] Application of Bacillus Velez TCS001 on greenhouse cucumber

[0085] Test agents and sources

[0086] 3.9 billion CFU / mL Bacillus Velez TCS001 suspension, Zhejiang Agriculture and Forestry University;

[0087] 2% amino oligosaccharide aqueous solution, Shandong Heyi Biotechnology Co., Ltd.;

[0088] 66.5% propamocarb hydrochloride aqueous solution, Qingdao Huizhi Biotechnology Co., Ltd.;

[0089] 6% kasugamycin wettable powder, Shaanxi Thompson Biotechnology Co., Ltd.;

[0090] 5% Zhongshengmycin wettable powder, Fujian Kaili Biological Products Co., Ltd.;

[0091] 430 g / L tebuconazole suspension, Zhejiang Qianjiang Biochemical Co., Ltd.;

[0092] 40% oxadimethoxine suspension concentrate, Jiangsu Jianpai Agrochemical Co., Ltd.

[0093] Experimental Design

[0094] This experiment set up three treatment groups: Group A was treated with Bacillus lyssum TCS001; Group B was treated with chemical agents; Group C was a blank control group (no treatment with any agent). Each treatment area was 200 square meters.

[0095] The treatment plans for Groups A and B are shown in the table below.

[0096] Table 3 Application scheme of cucumber A treatment group

[0097]

[0098]

[0099] Table 4. Spraying scheme for cucumber B treatment

[0100]

[0101]

[0102] Investigation on the efficacy of preventing cucumber damping-off disease

[0103] After cucumber seedlings emerged, when the blank control group showed obvious symptoms of disease, the seedling death rate was checked and the control effect was calculated. Three seedling trays were selected for each treatment for investigation.

[0104]

[0105] Investigation on the efficacy of preventing bacterial angular leaf spot of cucumber

[0106] When the blank control group showed obvious symptoms of angular leaf spot, the angular leaf spot control efficacy was investigated. The method was carried out in accordance with the provisions of "GB / T17980.110-2004 Pesticide Field Efficacy Test Guidelines (II): Fungicide Control of Cucumber Bacterial Angular Leaf Spot", sampling was carried out at 3 points for each treatment, 5 plants were investigated at each point, and all leaves of each plant were investigated. The grading was based on the percentage of the lesion area on each leaf to the entire leaf area.

[0107] Grading method:

[0108] Level 0: no lesions;

[0109] Level 1: The lesions occupy less than 5% of the entire leaf area;

[0110] Level 3: lesions occupy 6%-10% of the entire leaf area;

[0111] Level 5: The lesions occupy less than 11%-20% of the entire leaf area;

[0112] Level 7: The lesions occupy less than 21%-50% of the entire leaf area;

[0113] Level 9: Lesions occupy more than 51% of the entire leaf area.

[0114] According to the survey results, the disease index and prevention effect are calculated according to the following formula.

[0115]

[0116]

[0117] Investigation on the efficacy of preventing cucumber powdery mildew

[0118] When the blank control group showed obvious symptoms of powdery mildew, the powdery mildew control efficacy was investigated. The investigation was carried out in accordance with the provisions of "GB-T17980.30-2000 Pesticide Field Efficacy Test Guidelines (I) Part 30: Fungicide Control of Cucumber Powdery Mildew". Five random points were investigated for each treatment, two plants were investigated at each point, and all leaves of each plant were investigated. The grades were based on the percentage of the lesion area on each leaf to the entire leaf area.

[0119] Grading method (based on leaves):

[0120] Level 0: no lesions;

[0121] Level 1: The lesions occupy less than 5% of the entire leaf area;

[0122] Level 3: lesions occupy 6%-10% of the entire leaf area;

[0123] Level 5: The lesions occupy less than 11%-20% of the entire leaf area;

[0124] Level 7: The lesions occupy less than 21%-40% of the entire leaf area;

[0125] Level 9: The lesions occupy more than 40% of the entire leaf area.

[0126] According to the survey results, the disease index and prevention effect are calculated according to the following formula.

[0127]

[0128]

[0129] Investigation on the efficacy of cucumber downy mildew control

[0130] When the blank control group showed obvious symptoms of downy mildew, the downy mildew control efficacy was investigated. According to the provisions of GB-T17980.26-2000 Pesticide Field Efficacy Test Guidelines (I) Part 26: Fungicide Control of Cucumber Downy Mildew, 5 points were randomly selected for investigation in each plot, 3 plants were investigated at each point, and 10 leaves were investigated for each plant. The lesion area on each leaf was graded as a percentage of the total leaf area.

[0131] Grading method (based on leaves):

[0132] Level 0: no lesions;

[0133] Level 1: The lesion area accounts for less than 5% of the entire leaf area;

[0134] Level 3: The lesion area accounts for 6%-10% of the entire leaf area;

[0135] Level 5: The lesion area accounts for 11%-20% of the entire leaf area;

[0136] Level 7: The lesion area accounts for 21%-50% of the entire leaf area;

[0137] Level 9: The lesion area accounts for more than 50% of the total leaf area.

[0138] According to the survey results, the disease index and prevention effect are calculated according to the following formula.

[0139]

[0140]

[0141] Investigation on the efficacy of preventing cucumber wilt

[0142] When the blank control group showed obvious symptoms of wilt, the wilt control efficacy was investigated. According to the method of "GB / T17980.113---2004 Pesticide Field Efficacy Test Guidelines (II)-Fungicide Control of Melon Wilt", 4 random sampling points were selected for each treatment, and each point was investigated for 8m 2 , investigate the total number of cucumber plants and the number of diseased plants, and calculate the diseased plant rate and prevention effect.

[0143]

[0144] Cucumber biomass determination

[0145] 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 measured using a handheld chlorophyll meter TYS-A.

[0146] Cucumber yield determination

[0147] 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 cucumber yield.

[0148] Cucumber quality test

[0149] The vitamin C content was determined by referring to "GB 5009.86-2016 National Food Safety Standard Determination of Ascorbic Acid in Foods"; the moisture content was determined by referring to "GB 5009.3-2016 National Food Safety Standard Determination of Water in Foods"; the soluble solids content was determined by referring to "NY-T 2637-2014 Determination of Soluble Solids Content of Fruits and Vegetables".

[0150] Test results

[0151] Effect of application of Bacillus Velez TCS001 on greenhouse tomatoes

[0152] Table 5 Effect of disease prevention and control on tomato throughout the whole process

[0153]

[0154] It can be seen from Table 5 that 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.

[0155] Table 6 Results of the test on improving the quality of tomatoes throughout the process

[0156]

[0157] It can be seen from Table 6 that the application of the TCS001 bacterial agent composition has a significant effect on improving the quality and increasing the yield of greenhouse tomatoes. The biomass of tomatoes has increased, the yield has increased significantly, the soluble solid content, vitamin C, and soluble sugar content have all increased, and the titratable acid content has decreased.

[0158] Application Effect of Bacillus Velez TCS001 on Cucumber in Greenhouse

[0159] Table 7 Effect of cucumber disease prevention and control throughout the process

[0160]

[0161] It can be seen from Table 7 that the full application of Bacillus Velez TCS001 suspension concentrate has a good preventive effect on important diseases of cucumber growth in the greenhouse, such as 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.

[0162] Table 8 Results of the test on improving the quality of cucumber throughout the whole process

[0163]

[0164] It can be seen from Table 8 that after applying the TCS001 bacterial agent composition, the yield of cucumber was significantly improved; the vitamin C and water content were both increased; it can be seen that the application of the Bacillus Velez TCS001 bacterial agent composition has a significant effect on improving the quality and increasing the yield of greenhouse cucumbers.

[0165] 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. Application of Bacillus Velez TCS001 as a drug for full-process disease prevention and control and / or quality improvement on facility crops, wherein the facility crops are tomatoes, cucumbers, etc.

2. The use according to claim 1, characterized in that: The diseases include one or more of tomato damping-off, bacterial wilt, early blight, gray mold, wilt, etc.; and one or more of cucumber damping-off, bacterial angular spot, powdery mildew, downy mildew, wilt, etc.

3. The use according to claim 1, characterized in that: The quality aspects include one or more of the tomato plant height, stem thickness, average single fruit weight, average single plant yield, soluble solids, vitamin C, soluble sugar, titratable acid, etc.; one or more of the cucumber main stem base circumference, relative chlorophyll content, root number, average fruit weight, soluble solids, vitamin C, water content, etc.

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 formula of the suspension is 40% of Bacillus Velez TCS001 fermentation liquid, 3% of dispersant SXC, 3% of dispersant UNA, 0.15% of thickener, 0.2% of preservative Kathon, 0.2% of preservative sodium benzoate, and 53.45% of water.

7. A method for improving tomato quality, characterized in that: Bacillus Velez TCS001 is applied during one or several of the tomato seedling stage, transplanting day, seedling stage, flowering stage, and fruiting stage.

8. A method for improving the quality of cucumbers, characterized in that: Bacillus Velez TCS001 is applied during one or several stages of the cucumber seedling stage, transplanting day, planting stage, vine growth stage, and flowering and fruiting stage.

9. The method according to any one of claims 7-8, characterized in that: The Bacillus Velez TCS001 is the fermentation liquid of Bacillus Velez TCS001.

10. The method according to any one of claims 7-8, characterized in that: The method further comprises applying amino-oligosaccharides during the tomato seedling stage; and applying amino-oligosaccharides during the cucumber planting stage and the cucumber vine-raising stage.

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