Broad-spectrum biocontrol preparation for preventing and treating tomato soil-borne diseases as well as preparation method and application of broad-spectrum biocontrol preparation

Abstract

The invention provides a broad-spectrum biocontrol preparation for preventing and treating tomato soil-borne diseases and a preparation method and application thereof.The preparation method comprises the steps that 1, according to the thallus number ratio of 1: (1-2), bacterial powder of bacillus amyloliquefaciens and bacterial powder of bacillus atrophaeus are mixed to obtain a thallus mixture, then the thallus mixture is evenly mixed with culture freeze-dried powder of pseudomonas stutzeri, and a mixture is obtained; the weight ratio of the thallus mixture to the culture freeze-dried powder of the pseudomonas stutzeri is 1: (2-3); and (2) adding the mixture obtained in the step (1) into sterile water, so that the weight ratio of the mixture is 2-3%. The biocontrol agent disclosed by the invention has an excellent prevention and treatment effect on various tomato diseases, and the morbidity of crops is generally low after the biocontrol agent is applied; meanwhile, the preparation process of the biocontrol agent is simple, large-scale production is easy, and the biocontrol agent has good popularization value.

Description

Technical Field

[0001] The invention belongs to the technical field of vegetable soil-borne disease prevention and control, and specifically relates to a broad-spectrum biocontrol agent for preventing and controlling tomato soil-borne diseases, and a preparation method and application thereof. Background Art

[0002] Solanaceae crops include a variety of important vegetables, cash crops and ornamental plants. Among them, Solanaceae crops represented by potatoes, tomatoes and peppers, as important food and vegetable crops, occupy an important position in the global daily diet and have important economic value. However, Solanaceae crops are susceptible to a variety of diseases during their growth. If they are not controlled in time, their yield and quality will be significantly reduced. The main diseases of Solanaceae vegetable crops include tomato virus disease, tomato bacterial wilt, tomato late blight, eggplant cotton blight, root-knot nematode disease, pepper virus disease and pepper anthracnose.

[0003] At present, in terms of the prevention and control of Solanaceae crop diseases, it is mainly divided into agricultural prevention, physical prevention, chemical prevention, biological prevention or the comprehensive application of several prevention methods. Among them, agricultural prevention is mainly achieved through the comprehensive application of agricultural technical measures, including the selection of excellent Solanaceae vegetable varieties resistant to diseases and insects, the improvement of Solanaceae vegetable cultivation methods, the strengthening of vegetable field management, the scientific use of water and fertilizer, etc., so as to achieve the occurrence of diseases. Physical prevention refers to the use of the reactions of pests and diseases to temperature, humidity, spectrum, color, sound, etc. to control the occurrence of pests and diseases, kill, repel or isolate pests. Chemical prevention is the use of the toxicity of chemical agents to prevent and control pests and diseases. It has the advantages of good prevention and control effect, quick results, easy use, less seasonal influence, and suitable for large-scale use. However, if used improperly, it can cause poisoning of humans and animals, pollute the environment, kill natural enemies and cause pesticide damage; long-term use of chemical pesticides can cause certain pests and diseases to develop different degrees of resistance. Biological control refers to the method of using biological natural enemies of pests and diseases or beneficial biological products to prevent and control pests and diseases of Solanaceae vegetables. The main methods include using insects to control insects, using bacteria to control insects, using viruses to control insects, and using other beneficial biological agents to control pests and diseases. Biological control does not pollute the environment and is safe for humans and animals. Once successful, it can play a lasting role and achieve the purpose of controlling pests and diseases of Solanaceae vegetables. The goals of biological control are often very specific or highly specific, but the effect takes a long time to take effect.

[0004] Generally speaking, agricultural control and physical control are limited by the specific planting environment and are not suitable for large-scale promotion and application. Although chemical agents are easy to mass produce and have strong universality in various environments, they are prone to environmental pollution and pose safety hazards. Therefore, in recent years, people have focused their research on biological control technology and have achieved some encouraging results. For example, CN108293480B provides a method for controlling tomato gray mold using biocontrol agents, which is achieved by mixing a mixed fermentation broth of Trichoderma koningii and Trichosporon spp. and a culture broth of Pseudomonas stutzeri in a volume ratio of 3:2, and then vacuum freeze-drying to obtain a bacterial powder, and adding the bacterial powder to water to spray tomatoes, thereby achieving effective control of tomato gray mold, with a control effect of more than 80%; For example, CN112493256B provides a green muscardine preparation for promoting tomato growth and preventing and controlling tomato anthracnose. It was found that green muscardine CQMa421 has a good prevention and control effect on tomato anthracnose, with an incidence rate of only 6% in field trials and a prevention effect of more than 70%. For another example, CN112640916B provides the application of microbial preparations in the prevention and control of tomato bacterial wilt. By combining Bacillus jamillaris W51 with an adsorbent to form a microbial preparation, a prevention effect of 79.35% was achieved on tomato bacterial wilt.

[0005] However, biological control technologies, including the above-mentioned invention patents, usually can only have good prevention effects on one disease and lack broad spectrum. Crops are usually inevitably affected by multiple diseases during the cultivation process. Therefore, these technologies lack good promotion and application value. Summary of the invention

[0006] In view of the shortcomings of the prior art, the purpose of the present invention is to provide a biocontrol technology that can have excellent control effects on various soil-borne diseases of tomatoes, specifically to provide a broad-spectrum biocontrol preparation for various soil-borne diseases of tomatoes, and its preparation method and application.

[0007] In order to achieve the above object, the present invention provides the following technical solutions:

[0008] A method for preparing a broad-spectrum biocontrol agent for preventing and controlling soil-borne diseases of tomatoes, the method comprising the following steps:

[0009] (1) mixing bacterial powder of Bacillus amyloliquefaciens and bacterial powder of Bacillus atrophaeus at a bacterial cell number ratio of 1:1-2 to obtain a bacterial cell mixture, and then mixing the mixture evenly with freeze-dried culture powder of Pseudomonas stutzeri, wherein the weight ratio of the bacterial cell mixture to the freeze-dried culture powder of Pseudomonas stutzeri is 1:2-3;

[0010] (2) adding the mixture obtained in step (1) to water so that the weight ratio of the mixture is 2 to 3%;

[0011] The accession number of the Bacillus amyloliquefaciens is CGMCC No.3789; the accession number of the Bacillus atrophaeus is CCTCC NO: M2010316; the accession number of the Pseudomonas stutzeri is ATCC 17588; the bacterial powder of the Bacillus amyloliquefaciens is obtained by centrifuging the culture solution of Bacillus amyloliquefaciens to obtain a bacterial precipitate and freeze-drying it; the bacterial powder of the Bacillus atrophaeus is obtained by centrifuging the culture solution of Bacillus atrophaeus to obtain a bacterial precipitate and freeze-drying it; the culture freeze-dried powder of Pseudomonas stutzeri is obtained by centrifuging the culture solution of Pseudomonas stutzeri to obtain a supernatant and freeze-drying it.

[0012] Preferably, the soil-borne diseases include at least one of tomato bacterial wilt, gray mold or late blight.

[0013] Preferably, when preparing the Bacillus amyloliquefaciens culture solution, the culture medium used is prepared by weighing 5 g of tryptone, 5 g of yeast extract, and 5 g of glucose, putting them into a 1000 ml conical flask, mixing them thoroughly and adjusting the pH to 6.8-7.2, filling the 1000 ml conical flask with 300 ml of culture solution, plugging the conical flask mouth with a stopper, sterilizing with wet heat at 121° C. for 30 minutes, and cooling for use.

[0014] Preferably, when preparing the Bacillus atrophaeus culture solution, the culture medium formula used is: 1.5% w / w glucose, 1.5% w / w soybean powder, 1% w / w sodium chloride, and the rest is water.

[0015] Preferably, when preparing the Pseudomonas stutzeri culture solution, the culture medium formula used is: 12 g / L corn starch, 10 g / L yeast extract, 5 g / L sodium chloride, 2 g / L urea, 0.1 g / L potassium dihydrogen phosphate, 0.1 g / L dipotassium hydrogen phosphate, 0.01 g / L ferrous sulfate, 0.01 g / L manganese sulfate, and the rest is water.

[0016] As a preferred technical solution, a method for preparing a broad-spectrum biocontrol agent for controlling soil-borne diseases of tomatoes comprises the following steps:

[0017] (1) mixing bacterial powder of Bacillus amyloliquefaciens and bacterial powder of Bacillus atrophaeus at a bacterial cell number ratio of 1:1 to obtain a bacterial cell mixture, and then mixing the mixture evenly with freeze-dried culture powder of Pseudomonas stutzeri, wherein the weight ratio of the bacterial cell mixture to the freeze-dried culture powder of Pseudomonas stutzeri is 1:3;

[0018] (2) Add the mixture obtained in step (1) to water so that the weight ratio of the mixture is 2%.

[0019] Another object of the present invention is to provide a broad-spectrum biocontrol agent for controlling soil-borne diseases of tomatoes prepared by the above method.

[0020] Another object of the present invention is to provide the use of the above-mentioned broad-spectrum biocontrol agent for controlling soil-borne diseases of tomatoes in controlling tomato crop diseases.

[0021] Optionally, when applying the biocontrol agent, the application methods include foliar spraying and root irrigation.

[0022] As shown in the embodiments of the present invention, the present invention obtains a preparation having excellent control effects on various diseases of tomatoes by mixing the powder of Bacillus amyloliquefaciens, the powder of Bacillus atrophaeus and the freeze-dried powder of the culture of Pseudomonas stutzeri. Before the present invention, the Pseudomonas stutzeri described in the present invention was used in CN 108293480 B, which mainly produced the control effect on tomato gray mold by mixing with Trichoderma koningii and Trichosporon fermentation broth, but when Pseudomonas stutzeri fermentation broth (containing thalli) was used alone, the control effect on tomato gray mold was limited. The present invention found through exploration that when the culture of Pseudomonas stutzeri (without thalli) was mixed with the powder of Bacillus amyloliquefaciens and the powder of Bacillus atrophaeus, an excellent control effect could be produced, but after the culture of Pseudomonas stutzeri was replaced with the thalli of Pseudomonas stutzeri, the corresponding control effect was significantly reduced. This indicates that the culture of Pseudomonas stutzeri has a synergistic effect with the powder of Bacillus amyloliquefaciens and the powder of Bacillus atrophaeus.

[0023] The Bacillus amyloliquefaciens described in the present invention was first reported in CN 101985608 B and was used to control rice bacterial leaf streak. When the inventor used it for the control of soil-borne diseases of tomatoes, the effect was not ideal, and the control efficiency did not exceed 50%, see Comparative Example 3 (see experimental group 6 of Table 1 and Table 2 respectively). The Bacillus atrophaeus described in the present invention was first reported in CN102559533B and was used to antagonize sea cucumber pathogens. As far as the inventor knows, there is no report on the use of this bacterium for the control of soil-borne diseases of crops. As shown in Comparative Examples 2 and 4, whether the Bacillus atrophaeus described in the present invention is used alone or in combination with the Bacillus amyloliquefaciens described in the present invention, satisfactory results were not achieved in the control of soil-borne diseases of tomatoes, and even a mild antagonistic effect occurred.

[0024] Beneficial effects of the present invention:

[0025] The biocontrol agent of the present invention can have excellent control effects on a variety of soil-borne diseases of tomatoes, and the incidence of crops is generally low; at the same time, the preparation process of the biocontrol agent of the present invention is simple, and it is easy to mass produce, and has good promotion value. DETAILED DESCRIPTION

[0026] The present invention is described in detail below through examples. It is necessary to point out that the following examples are only used to further illustrate the present invention and cannot be understood as limiting the scope of protection of the present invention. Some non-essential improvements and adjustments made by technical personnel in this field based on the above invention content still fall within the scope of protection of the present invention.

[0027] Example 1

[0028] 1. Bacterial strains and culture medium

[0029] Bacillus amyloliquefaciens: accession number CGMCC No.3789, commercially available through the General Microbiological Center of China Microbiological Culture Collection Administration;

[0030] Bacillus atrophaeus: CCTCC NO: M2010316, commercially available through China Center for Type Culture Collection;

[0031] Pseudomonas stutzeri: deposit number ATCC 17588, purchased from Shanghai Huzheng Biotechnology Co., Ltd.

[0032] YPG culture medium: Weigh 5g of tryptone, 5g of yeast extract and 5g of glucose, put them into a 1000ml Erlenmeyer flask, mix thoroughly and adjust the pH to 6.8-7.2. Fill the 1000ml Erlenmeyer flask with 300ml of culture medium, plug the mouth of the Erlenmeyer flask with a stopper, sterilize with wet heat at 121℃ for 30 minutes, and set aside after cooling.

[0033] Bacillus atrophaeus fermentation medium: glucose 1.5% w / w, soybean powder 1.5% w / w, sodium chloride 1% w / w, and the rest is water.

[0034] Pseudomonas stutzeri culture medium: corn starch 12g / L, yeast extract 10g / L, sodium chloride 5g / L, urea 2g / L, potassium dihydrogen phosphate 0.1g / L, dipotassium hydrogen phosphate 0.1g / L, ferrous sulfate 0.01g / L, manganese sulfate 0.01g / L, and the rest is water.

[0035] 2. Raw Materials Preparation

[0036] 2.1 Cultivation of Bacillus amyloliquefaciens:

[0037] After the bacteria were activated, 2% of the inoculum was added to 300 ml of YPG culture medium in a 1000 ml triangular flask. After culturing at 120 rpm and 28°C for 48 hours, the bacterial content in the culture medium was adjusted to 10 9 cfu / ml.

[0038] 2.2 Preparation of Bacillus amyloliquefaciens powder:

[0039] The fermentation broth of Bacillus amyloliquefaciens obtained by the above-mentioned culture was centrifuged (conditions: 20000rpm, 5min, 4°C), the bacteria were collected, resuspended with PBS and centrifuged again (conditions: 20000rpm, 5min, 4°C) for washing, the washing was repeated 3 times, and the obtained centrifugal precipitate was freeze-dried to obtain bacterial powder of Bacillus amyloliquefaciens.

[0040] 2.3 Cultivation of Bacillus atrophaeus:

[0041] After the strain was activated, it was inoculated into the fermentation medium of Bacillus atrophaeus at a rate of 2%, and cultured at 28°C and 170 rpm for 96 hours. The bacterial content in the culture medium was adjusted to 10 9 cfu / ml.

[0042] 2.4 Preparation of Bacillus atrophaeus powder:

[0043] The fermentation broth of Bacillus atrophaeus obtained by the above-mentioned culture was centrifuged (conditions: 20000rpm, 5min, 4°C), the bacteria were collected, resuspended with PBS and centrifuged again (conditions: 20000rpm, 5min, 4°C) for washing, the washing was repeated 3 times, and the obtained centrifugal precipitate was freeze-dried to obtain the bacterial powder of Bacillus atrophaeus.

[0044] 2.5 Cultivation of Pseudomonas stutzeri:

[0045] After the strain was activated, it was inoculated into a Pseudomonas stutzeri culture medium at a 10% inoculum volume and cultured at 28°C and 140 rpm for 24 h. The bacterial content in the culture medium was adjusted to 10 9 cfu / ml.

[0046] 2.6 Preparation of Pseudomonas stutzeri bacterial powder:

[0047] The Pseudomonas stutzeri fermentation broth obtained by the above-mentioned culture was centrifuged (conditions: 20000rpm, 5min, 4°C), the bacteria were collected, resuspended with PBS and centrifuged again (conditions: 20000rpm, 5min, 4°C) for washing, and the washing was repeated 3 times. The obtained centrifugal precipitate was freeze-dried to obtain Pseudomonas stutzeri bacterial powder.

[0048] 2.7 Lyophilized powder of Pseudomonas stutzeri culture

[0049] The fermentation liquid of Pseudomonas stutzeri obtained by the above culture was centrifuged (conditions: 20000 rpm, 5 min, 4°C), the supernatant was collected, and the obtained supernatant was freeze-dried to obtain freeze-dried powder of the culture of Pseudomonas stutzeri.

[0050] A method for preparing a broad-spectrum biocontrol agent for preventing and controlling soil-borne diseases of tomatoes comprises the following steps:

[0051] (1) mixing bacterial powder of Bacillus amyloliquefaciens and bacterial powder of Bacillus atrophaeus at a bacterial cell number ratio of 1:1 to obtain a bacterial cell mixture, and then mixing the mixture evenly with freeze-dried culture powder of Pseudomonas stutzeri, wherein the weight ratio of the bacterial cell mixture to the freeze-dried culture powder of Pseudomonas stutzeri is 1:3;

[0052] (2) Add the mixture obtained in step (1) to water so that the weight ratio of the mixture is 2%.

[0053] Example 2

[0054] The strains, culture medium and raw materials were the same as those in Example 1.

[0055] A method for preparing a broad-spectrum biocontrol agent for preventing and controlling soil-borne diseases of tomatoes comprises the following steps:

[0056] (1) mixing bacterial powder of Bacillus amyloliquefaciens and bacterial powder of Bacillus atrophaeus at a bacterial cell number ratio of 1:1.5 to obtain a bacterial cell mixture, and then mixing the mixture evenly with freeze-dried culture powder of Pseudomonas stutzeri, wherein the weight ratio of the bacterial cell mixture to the freeze-dried culture powder of Pseudomonas stutzeri is 1:2.5;

[0057] (2) The mixture obtained in step (1) was added to water so that the weight ratio of the mixture was 1.5%.

[0058] Example 3

[0059] The strains, culture medium and raw materials were the same as those in Example 1.

[0060] A method for preparing a broad-spectrum biocontrol agent for preventing and controlling soil-borne diseases of tomatoes comprises the following steps:

[0061] (1) mixing bacterial powder of Bacillus amyloliquefaciens and bacterial powder of Bacillus atrophaeus at a bacterial cell number ratio of 1:2 to obtain a bacterial cell mixture, and then mixing the mixture evenly with freeze-dried culture powder of Pseudomonas stutzeri, wherein the weight ratio of the bacterial cell mixture to the freeze-dried culture powder of Pseudomonas stutzeri is 1:2;

[0062] (2) Add the mixture obtained in step (1) to water so that the weight ratio of the mixture is 1%.

[0063] Comparative Example 1

[0064] The relevant strains, culture medium and raw materials are the same as those in Example 1. Compared with the example, this comparative example uses only one kind of bacterial powder, namely, Bacillus amyloliquefaciens powder, and does not add freeze-dried powder of Pseudomonas stutzeri culture.

[0065] A method for preparing a broad-spectrum biocontrol agent for preventing and controlling soil-borne diseases of tomatoes comprises the following steps:

[0066] The powder of Bacillus amyloliquefaciens was added to water so that the weight content of the bacteria was 2%.

[0067] Comparative Example 2

[0068] The relevant strains, culture medium and raw materials are the same as those in Example 1. Compared with the example, the comparative example does not add the lyophilized culture powder of Pseudomonas stutzeri.

[0069] A method for preparing a broad-spectrum biocontrol agent for preventing and controlling soil-borne diseases of tomatoes comprises the following steps:

[0070] (1) mixing bacterial powder of Bacillus amyloliquefaciens and bacterial powder of Bacillus atrophaeus at a bacterial cell number ratio of 1:2 to obtain a bacterial cell mixture;

[0071] (2) Add the bacterial cell mixture obtained in step (1) to water so that the weight content of the mixture is 1%.

[0072] Comparative Example 3

[0073] The relevant strains, culture medium and raw materials are the same as those in Example 1. Compared with the example, this comparative example uses only one kind of bacterial powder, namely, Bacillus amyloliquefaciens powder.

[0074] A method for preparing a broad-spectrum biocontrol agent for preventing and controlling soil-borne diseases of tomatoes comprises the following steps:

[0075] (1) taking powder of Bacillus amyloliquefaciens and freeze-dried powder of Pseudomonas stutzeri and mixing them evenly, the weight ratio of the powder of Bacillus amyloliquefaciens to the freeze-dried powder of Pseudomonas stutzeri is 1:3;

[0076] (2) Add the mixture obtained in step (1) to water so that the weight content of the mixture is 2%.

[0077] Comparative Example 4

[0078] The relevant strains, culture medium and raw materials are the same as those in Example 1. Compared with the example, this comparative example uses only one kind of bacterial powder, namely, Bacillus atrophaeus powder.

[0079] A method for preparing a broad-spectrum biocontrol agent for preventing and controlling soil-borne diseases of tomatoes comprises the following steps:

[0080] (1) Mix the powder of Bacillus atrophaeus and the freeze-dried powder of Pseudomonas stutzeri culture evenly, with the weight ratio of the powder of Bacillus atrophaeus to the freeze-dried powder of Pseudomonas stutzeri culture being 1:3;

[0081] (2) Add the mixture obtained in step (1) to water so that the weight content of the mixture is 2%.

[0082] Comparative Example 5

[0083] The relevant strains, culture medium and raw materials are the same as those in Example 1. Compared with the example, the culture freeze-dried powder of Pseudomonas stutzeri is replaced with bacterial powder of Pseudomonas stutzeri in this comparative example.

[0084] A method for preparing a broad-spectrum biocontrol agent for preventing and controlling soil-borne diseases of tomatoes comprises the following steps:

[0085] (1) mixing bacterial powder of Bacillus amyloliquefaciens, bacterial powder of Bacillus atrophaeus, and bacterial powder of Pseudomonas stutzeri at a bacterial cell number ratio of 1:1:1 to obtain a bacterial cell mixture;

[0086] (2) Add the bacterial cell mixture obtained in step (1) to water so that the weight ratio of the mixture is 2%.

[0087] Experimental Example 1: Tomato bacterial wilt control experiment

[0088] Transplant tomato seedlings at the 4-leaf stage into 12cm×10.5cm pots, 4 plants in each pot, and cultivate them in sterile soil. When the tomatoes grow to the 12-leaf stage, cut off two tomatoes from each pot, and keep two tomatoes with similar growth in each pot, so that all the test tomato plants grow in the same way, and irrigate the roots with activated bacterial wilt culture solution for pathogen inoculation.

[0089] The preparations obtained in Examples 1-3 and Comparative Examples 1-5 were used as experimental groups, and the groups were set in order as experimental groups 1-8, and each group was irrigated with 100 mL of root. A control group was set up, and the roots were irrigated with 100 mL of sterile water per plant. Three replicates were set up for each group, with 20 pots of tomatoes in each replicate, and the temperature was controlled at 28°C, the relative humidity was 60% to 80%, and the photoperiod was 12 hours. The organic fertilizers used in the treatment of each group were consistent, and no other fungicides were applied. After irrigating the roots for 3 weeks, the incidence rate and disease index of bacterial wilt in each group were counted.

[0090] The investigation on the incidence of tomato bacterial wilt was based on the "Technical Procedures for Identification of Tomato Resistance to Bacterial Wilt" (NY / T1858.4-2010) and combined with the actual situation of this experiment, the test data were recorded and analyzed.

[0091] Grade 0, asymptomatic;

[0092] Level 1, 1 leaf withers;

[0093] Level 2, 2-3 leaves withered;

[0094] Level 3: All leaves except the bottom 2-3 are withered;

[0095] Level 4, the entire plant leaves wither;

[0096] Disease severity (disease index) = ∑ (disease level × number of plants at that disease level) / (highest disease level × total number of plants) × 100%;

[0097] Biological control effect = (disease severity of the control group - disease severity of the treatment group) / disease severity of the control group × 100%.

[0098] After 3 weeks of treatment, the disease index and biocontrol effect of each experimental group and control group are shown in Table 1.

[0099] Table 1

[0100]

[0101]

[0102] Experimental Example 2: Tomato gray mold control experiment

[0103] When the tomato seedlings grew to 4 leaves and 1 heart, the spores of Botrytis cinerea cultured on PDA solid medium for 5-7 days were eluted from the medium with sterile water and filtered, centrifuged at 6000r / min for 5min, and the supernatant was discarded to collect the Botrytis cinerea spores. The spores were resuspended in 2% sucrose solution to a concentration of 2×10 5 cfu / mL, spray the tomato gray mold spore suspension evenly on the leaves and stems of tomato plants until the leaves are moistened. After inoculation, keep the relative humidity of the air in the light incubator above 90%. The preparations obtained in Examples 1-3 and Comparative Examples 1 to 5 were used as experimental groups, and the groups were set up in sequence as experimental groups 1-8. After 24 hours of spraying the gray mold spore suspension, the biocontrol preparation was sprayed on the leaves and stems of each group until the leaves were completely moistened. Set up a control group and spray with the same volume of sterile water. Set up 3 replicates for each group, and each replicate has 20 pots of tomatoes.

[0104] After the plants are infected, the disease situation of each group is investigated and graded. The classification standard for tomato gray mold leaves is:

[0105] Grade 0, no lesions;

[0106] Level 1: the lesion area accounts for less than 5% of the entire leaf area;

[0107] Level 3: the lesion area accounts for 6% to 10% of the entire leaf area;

[0108] Level 5: the lesion area accounts for 11% to 20% of the entire leaf area;

[0109] Level 7: the lesion area accounts for 21% to 50% of the entire leaf area;

[0110] Level 9: The lesion area accounts for more than 50% of the total leaf area.

[0111] Disease severity (disease index) = ∑ (disease level × number of plants at that disease level) / (highest disease level × total number of plants) × 100%;

[0112] Biological control effect = (disease severity of the control group - disease severity of the treatment group) / disease severity of the control group × 100%.

[0113] Table 2

[0114]

[0115] Experimental Example 3: Tomato late blight control experiment

[0116] After disinfection, tomato seeds were sown in seedling pots. After the plants grew for 35 days, they were inoculated with a suspension of tomato late blight pathogenic bacteria (2×10 4 cfu / ml), spray until the leaves are wet. After inoculation, keep the relative humidity of the air in the light incubator above 90%. The preparations obtained in Examples 1-3 and Comparative Examples 1-5 were used as experimental groups, and the groups were set up in sequence as experimental groups 1-8. Each group sprayed the biocontrol preparation on the leaves and stems 24 hours after spraying the suspension of tomato late blight pathogen spores until all the leaves were wet. Set up a control group and spray with the same volume of sterile water. Set up 3 replicates for each group, and each replicate has 20 pots of tomatoes.

[0117] After the plants become diseased, the disease conditions of each group are graded and investigated. The grading standards for tomato late blight leaves are:

[0118] Grade 0, no lesions;

[0119] Level 1: the lesion area accounts for less than 5% of the entire leaf area;

[0120] Level 3: the lesion area accounts for 6% to 10% of the entire leaf area;

[0121] Level 5: the lesion area accounts for 11% to 20% of the entire leaf area;

[0122] Level 7: the lesion area accounts for 21% to 50% of the entire leaf area;

[0123] Level 9: The lesion area accounts for more than 50% of the total leaf area.

[0124] Disease severity (disease index) = ∑ (disease level × number of plants at that disease level) / (highest disease level × total number of plants) × 100%;

[0125] Biological control effect = (disease severity of the control group - disease severity of the treatment group) / disease severity of the control group × 100%.

[0126] Table 3

[0127]

[0128]

Claims

1. A method for preparing a broad-spectrum biocontrol agent for preventing and controlling soil-borne diseases of tomatoes, characterized in that: The preparation method comprises the following steps: (1) mixing bacterial powder of Bacillus amyloliquefaciens and bacterial powder of Bacillus atrophaeus at a bacterial cell number ratio of 1:1-2 to obtain a bacterial cell mixture, and then mixing the mixture evenly with freeze-dried culture powder of Pseudomonas stutzeri, wherein the weight ratio of the bacterial cell mixture to the freeze-dried culture powder of Pseudomonas stutzeri is 1:2-3; (2) adding the mixture obtained in step (1) to water so that the weight proportion of the mixture is 2 to 3%; The accession number of the Bacillus amyloliquefaciens is CGMCC No.3789; the accession number of the Bacillus atrophaeus is CCTCCNO:M2010316; the accession number of the Pseudomonas stutzeri is ATCC 17588; The bacterial powder of Bacillus amyloliquefaciens is obtained by centrifuging a culture solution of Bacillus amyloliquefaciens to obtain a bacterial precipitate and then freeze-drying it; the bacterial powder of Bacillus atrophaeus is obtained by centrifuging a culture solution of Bacillus atrophaeus to obtain a bacterial precipitate and then freeze-drying it; the culture freeze-dried powder of Pseudomonas stutzeri is obtained by centrifuging a culture solution of Pseudomonas stutzeri to obtain a supernatant and then freeze-drying it.

2. The preparation method according to claim 1, characterized in that: The soil-borne diseases include at least one of tomato bacterial wilt, gray mold or late blight.

3. The preparation method according to claim 1, characterized in that: When preparing the amyloliquefaciens culture solution, the culture medium used is prepared as follows: 5 g of tryptone, 5 g of yeast extract, and 5 g of glucose are weighed and put into a 1000 ml conical flask, and the pH is adjusted to 6.8-7.2 after being fully mixed. The 1000 ml conical flask is filled with 300 ml of culture solution, the conical flask mouth is plugged with a stopper, and the flask is sterilized by wet heat at 121° C. for 30 minutes, and then cooled for use.

4. The preparation method according to claim 1, characterized in that: When preparing the culture fluid of Bacillus atrophaeus, the culture medium formula used is: 1.5% w / w glucose, 1.5% w / w soybean powder, 1% w / w sodium chloride, and the rest is water.

5. The preparation method according to claim 1, characterized in that: When preparing the Pseudomonas stutzeri culture solution, the culture medium formula used is: 12 g / L corn starch, 10 g / L yeast extract, 5 g / L sodium chloride, 2 g / L urea, 0.1 g / L potassium dihydrogen phosphate, 0.1 g / L dipotassium hydrogen phosphate, 0.01 g / L ferrous sulfate, 0.01 g / L manganese sulfate, and the rest is water.

6. The preparation method according to claim 1, characterized in that: The preparation method comprises the following steps: (1) mixing bacterial powder of Bacillus amyloliquefaciens and bacterial powder of Bacillus atrophaeus at a bacterial cell number ratio of 1:1 to obtain a bacterial cell mixture, and then mixing the mixture evenly with freeze-dried culture powder of Pseudomonas stutzeri, wherein the weight ratio of the bacterial cell mixture to the freeze-dried culture powder of Pseudomonas stutzeri is 1:3; (2) Add the mixture obtained in step (1) to water so that the weight ratio of the mixture is 2%.

7. A broad-spectrum biocontrol agent for controlling soil-borne diseases of tomatoes, characterized in that: The biocontrol agent is prepared by the preparation method according to any one of claims 1 to 6.

8. Use of the broad-spectrum biocontrol agent for controlling soil-borne diseases of tomatoes as claimed in claim 7 in controlling soil-borne diseases of tomatoes.

9. The use according to claim 8, characterized in that: When the biocontrol agent is used, the application methods include foliar spraying and root irrigation.

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