Microbial agent for preventing and treating typical diseases of tomatoes as well as preparation method and application of microbial agent

Abstract

The invention discloses a microbial agent for preventing and treating typical diseases of tomatoes, and belongs to the technical field of microorganisms. The microbial agent comprises the following components: trichoderma evergreen, trichoderma hamatum, bacillus licheniformis and azotobacter chroococcum in a volume ratio of 1: 1: 1: 1. Fungi and bacteria with disease-resistant and growth-promoting effects are reasonably combined and compounded for use, various typical tomato diseases are effectively improved, through combined and compounded use of the fungi and bacteria of the microbial agent, the practical problems that prevention and control of a single biocontrol strain are unstable and the persistence is weak are solved, the biocontrol effect is greatly improved, and the method is suitable for large-scale popularization and application. Particularly, the composition has a remarkable prevention and treatment effect on typical tomato diseases such as root rot, gray mold, fusarium wilt and early blight; in addition, the microbial agent can be harmonious and compatible with soil ecology, the advantages of strains can be brought into full play, and the tomato yield and quality are effectively guaranteed and improved.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to a microbial agent for preventing and treating typical tomato diseases and a preparation method thereof. Background Art

[0002] Tomato (Lycopersicon esculentum Mill.), Solanaceae. Originated from South America, it contains a variety of vitamins, minerals and cellulose that are beneficial to the human body. It has the effects of anti-aging and reducing the occurrence of cancer. It has great development prospects in the fields of medicine, food, health care, etc.

[0003] At present, tomatoes are widely planted in various places, and the cultivated area is expanding year by year. There are many kinds of diseases in the planting process, and they are serious. The occurrence of diseases reduces the yield and quality of tomatoes, causing great economic losses to farmers. Common diseases of tomatoes mainly include gray mold, early blight, leaf mold, virus disease, bacterial wilt, late blight, nematode disease, ulcer disease, etc. With the continuous expansion of the planting area and continuous cropping for many years, the occurrence of diseases has gradually increased, new diseases have appeared one after another, and often multiple diseases occur at the same time, which increases the difficulty of prevention and control.

[0004] Microbial agents are products that utilize microorganisms and their life activities. They have the functions of directly or indirectly improving soil, restoring soil fertility, preventing various plant diseases, maintaining the balance of rhizosphere microbial flora, and degrading toxic substances. The use of microbial agents can reduce the drug resistance of pathogenic bacteria, and is safe and healthy for fruits and pollution-free to the environment. At present, most of the microbial agents studied for tomato diseases have good control effects on a certain common disease of tomatoes, and cannot achieve efficient control effects on multiple diseases and insect pests at the same time. For example, Chinese patent CN104087541A discloses a biocontrol strain for preventing and controlling tomato early blight and its application. Chinese patent CN103952352A discloses a circulative Bacillus and a microbial agent and their application, which mainly have good control effects on tomato gray mold. Although such microbial strains have obvious control effects on a certain type of tomato disease, in actual production, various diseases of tomatoes are not produced alone, and may be accompanied by multiple diseases at the same time during the growth process. Therefore, this type of microbial agent has a single function and is difficult to be effectively utilized and promoted in actual agricultural production. Therefore, developing a new microbial agent for preventing and controlling various tomato diseases is a technical problem that needs to be solved urgently. Summary of the invention

[0005] The invention provides a microbial agent for preventing and controlling typical tomato diseases and a preparation method thereof. The microbial agent is rationally compounded and used so that fungi and bacteria act synergistically to effectively prevent and control a variety of typical tomato diseases.

[0006] In order to achieve the above technical objectives, the technical solution adopted by the present invention is:

[0007] A microbial agent for preventing and controlling typical diseases of tomatoes, comprising the following components: Trichoderma viridescens, Trichoderma hamatum, Bacillus licheniformis, and Azotobacter chroococcum, in a volume ratio of 1:1:1:1;

[0008] The Trichoderma chlororaphis was purchased from the General Microbiological Center of China Microorganism Culture Collection Administration (CGMCC), with the strain number of CGMCC No.5.1040; the original preservation time was August 8, 2005;

[0009] The hooked Trichoderma was purchased from the General Microbiological Center of China Microorganism Culture Collection Administration (CGMCC), with the strain number of CGMCC No.3.7249; the original preservation time was July 8, 2005;

[0010] The Bacillus licheniformis was purchased from the General Microbiology Center of China Microorganism Culture Collection Administration (CGMCC), and the strain collection number is CGMCC No.1.7677; the original collection time is June 24, 2008;

[0011] The brown ball nitrogen-fixing bacteria were purchased from the General Microbiological Center of China Microorganism Culture Collection Administration (CGMCC), with the strain number of CGMCC No.1.5803; the original preservation time was June 27, 2005.

[0012] The four microbial strains used in the present invention can be purchased through the strain catalog of the General Microbiological Center of the China Microbiological Culture Collection Administration, without the need for repeated biological preservation.

[0013] Preferably, the microbial agent is a liquid agent.

[0014] Preferably, the spore count of Trichoderma chlororaphis is 2×10 8 cfu / mL; the spore count of the hooked Trichoderma is 2×10 8 cfu / mL; the amount of live Bacillus licheniformis in the microbial agent is 2×10 8 cfu / mL, the viable bacterial count of brown spherical nitrogen-fixing bacteria is 2×10 8 cfu / mL.

[0015] A method for preparing a microbial agent for preventing and controlling typical tomato diseases comprises the following preparation steps:

[0016] (1) Activate the slant strains of Trichoderma chlororaphis and Trichoderma hooked, pick the strains and put them into 100 ml of potato glucose liquid culture medium, culture them at 170 r / min, 25-28° C. for 48 hours to obtain seed liquid; inoculate the seed liquid into the fermentation culture medium at an inoculum rate of 10%, culture them at 170 r / min, 25-28° C. for 96 hours to obtain culture liquid, at which time the culture liquid is full of hyphae and spores of Trichoderma chlororaphis and Trichoderma hooked, wash the spores with sterile water, filter them with four layers of gauze, and dilute them again to obtain Trichoderma chlororaphis liquid and Trichoderma hooked liquid, respectively, to ensure that the spore concentrations of the two Trichoderma liquids obtained after dilution are both 2×10 8 cfu / mL;

[0017] (2) Thawing the brown ball nitrogen-fixing bacteria, inoculating them into PDA medium, and activating them at 28°C; after activation, taking 1% of the inoculum, adding it to the fermentation medium, and fermenting it at 25-28°C and 150 r / min until the viable bacteria amount is 2×10 8 cfu / mL of fermentation broth;

[0018] (3) Thawing the Bacillus licheniformis, inoculating it into LB solid medium, culturing it at 28-30°C for 1 day to activate the strain; picking the activated Bacillus licheniformis, inoculating it into LB liquid medium, fermenting it at 28-30°C, 220r / min, until the viable bacteria amount is 2×10 8 cfu / mL of fermentation broth;

[0019] (4) The four fermentation broths were mixed in a volume ratio of 1:1:1:1 to obtain the final product microbial agent.

[0020] LB solid medium uses conventional culture medium.

[0021] Preparation method of PDA culture medium: 200 grams of potatoes are boiled in water, filtered and the filtrate is added with 20 grams of glucose and 15-20 grams of agar, and water is added to 1000 ml, with natural pH.

[0022] The composition of the fermentation medium, namely potato glucose liquid medium (PDB), is as follows: 300 g / L potato, 20 g / L glucose, distilled water added to 1000 mL, and pH 5.6-5.8.

[0023] The microbial agent of the invention has good prevention and control effects on tomato root rot, gray mold, wilt and early blight.

[0024] The microbial agent of the present application can be used in the seed stage, or in the plant growth stage or seedling stage for root irrigation. The prepared microbial agent is diluted 100 times, and 25 ml is used for each plant. It is used twice in a row, with an interval of 10 days between each use.

[0025] The present invention has the following beneficial effects:

[0026] First, the present invention combines Trichoderma chlororaphis and Trichoderma hamata, which have antagonistic effects on a variety of tomato pathogens and can prevent and treat a variety of typical tomato diseases. The Trichoderma chlororaphis and Trichoderma hamata selected by the present invention can secrete a variety of extracellular enzymes, such as cellulase, chitinase, etc., which can degrade the cell walls of pathogens, thereby inhibiting the growth of pathogens; at the same time, they can produce a variety of volatile and non-volatile metabolites, which have obvious antibacterial effects on pathogens;

[0027] Secondly, Bacillus licheniformis and brown spherical nitrogen-fixing bacteria were introduced. Bacillus licheniformis can produce a variety of antibacterial substances, directly inhibit the growth and reproduction of pathogens. At the same time, Bacillus licheniformis can induce plants to produce systemic resistance, which can significantly reduce the incidence of plant diseases. At the same time, the selected Bacillus licheniformis and brown spherical nitrogen-fixing bacteria have good nitrogen-fixing ability, improve the soil microecological environment, and increase the soil nutrient content, thereby providing a better growth environment for other plant growth-promoting bacteria. They can also generate indoleacetic acid, phosphorus-dissolving activity and iron carrier production to promote plant growth, help alleviate the harm of diseases to tomato yield, and promote the improvement of tomato yield and quality. At the same time, the nitrogen-fixing bacteria have a certain inhibitory effect on the pathogens of gray mold and wilt of fruits and vegetables.

[0028] The combined use of Trichoderma chlororaphis and Trichoderma hooked, Bacillus licheniformis and Nitrogen-fixing Bacteria brown promotes each other and shows good synergistic inhibition against pathogens of tomato root rot, gray mold, wilt and early blight. The synergy of fungi and bacteria not only solves the practical problems of unstable and weak sustainability of single biocontrol strains, but also greatly improves the biocontrol effect and effectively prevents diseases such as root rot, gray mold, wilt and early blight; in addition, this microbial agent is harmoniously compatible with the soil ecology, which is conducive to giving full play to the advantages of strains and effectively ensuring and increasing tomato yields. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 It is the growth status diagram of different strains in LB medium;

[0030] Figure 2 The figure is a graph showing the inhibitory effect of the bacterial solution of Example 1 of the present invention on different pathogens, wherein A0: clean water + Pythium, A1: Example 1 + Pythium; B0: clean water + Botrytis cinerea, B1: Example 1 + Botrytis cinerea; C0: clean water + Fusarium oxysporum, C1: Example 1 + Fusarium oxysporum; D0: clean water + Alternaria solani, D1: Example 1 + Alternaria solani;

[0031] Figure 3 The figure is a graph showing the inhibitory effect of Example 1 and Comparative Examples 1-4 on Fusarium oxysporum. DETAILED DESCRIPTION

[0032] The technical solution of the present invention is further described below in conjunction with specific embodiments, but is not limited thereto.

[0033] Example 1

[0034] A microbial agent for preventing and controlling typical diseases of tomatoes, comprising the following components: Trichoderma viridescens, Trichoderma hamatum, Bacillus licheniformis, and Azotobacter chroococcum, in a volume ratio of 1:1:1:1;

[0035] The Trichoderma chlororaphis was purchased from the General Microbiological Center of China Microorganism Culture Collection Administration (CGMCC), with the strain number of CGMCC No.5.1040; the original preservation time was August 8, 2005;

[0036] The hooked Trichoderma was purchased from the General Microbiological Center of China Microorganism Culture Collection Administration (CGMCC), with the strain number of CGMCC No.3.7249; the original preservation time was July 8, 2005;

[0037] The Bacillus licheniformis was purchased from the General Microbiology Center of China Microorganism Culture Collection Administration (CGMCC), and the strain collection number is CGMCC No.1.7677; the original collection time is June 24, 2008;

[0038] The brown ball nitrogen-fixing bacteria were purchased from the General Microbiological Center of China Microorganism Culture Collection Administration (CGMCC), with the strain number of CGMCC No.1.5803; the original preservation time was June 27, 2005.

[0039] The four microbial strains used in this example can all be purchased through the strain catalog of the General Microbiological Center of the China Culture Collection Administration, without the need for repeated biological deposits.

[0040] The microbial agent is a liquid agent.

[0041] The spore count of the Trichoderma chlororaphis was 2×10 8 cfu / mL; the spore count of the hooked Trichoderma is 2×10 8 cfu / mL; the amount of live Bacillus licheniformis in the microbial agent is 2×10 8 cfu / mL, the viable bacterial count of brown spherical nitrogen-fixing bacteria is 2×10 8 cfu / mL.

[0042] A method for preparing a microbial agent for preventing and controlling typical tomato diseases comprises the following preparation steps:

[0043] (1) Activate the slant strains of Trichoderma chlororaphis and Trichoderma hooked, pick the strains and put them into 100 ml of potato glucose liquid culture medium, culture them at 170 r / min, 25-28° C. for 48 hours to obtain seed liquid; inoculate the seed liquid into the fermentation culture medium at an inoculum rate of 10%, culture them at 170 r / min, 25-28° C. for 96 hours to obtain culture liquid, at which time the culture liquid is full of hyphae and spores of Trichoderma chlororaphis and Trichoderma hooked, wash the spores with sterile water, filter them with four layers of gauze, and dilute them again to obtain Trichoderma chlororaphis liquid and Trichoderma hooked liquid, respectively, to ensure that the spore concentrations of the two Trichoderma liquids obtained after dilution are both 2×10 8 cfu / mL;

[0044] (2) Thawing the brown ball nitrogen-fixing bacteria, inoculating them into PDA medium, and activating them at 28°C; after activation, taking 1% of the inoculum, adding it to the fermentation medium, and fermenting it at 25-28°C and 150 r / min until the viable bacteria amount is 2×10 8 cfu / mL of fermentation broth;

[0045] (3) Thawing the Bacillus licheniformis, inoculating it into LB solid medium, culturing it at 28-30°C for 1 day to activate the strain; picking the activated Bacillus licheniformis, inoculating it into LB liquid medium, fermenting it at 28-30°C, 220r / min, until the viable bacteria amount is 2×10 8 cfu / mL of fermentation broth;

[0046] (4) The four bacterial solutions were mixed in a volume ratio of 1:1:1:1 to obtain the final product microbial agent.

[0047] LB solid medium uses conventional culture medium.

[0048] Preparation method of PDA culture medium: 200 grams of potatoes are boiled in water, filtered and the filtrate is added with 20 grams of glucose and 15-20 grams of agar, and water is added to 1000 ml, with natural pH.

[0049] The composition of the fermentation medium, namely potato glucose liquid medium (PDB), is as follows: 300 g / L potato, 20 g / L glucose, distilled water added to 1000 mL, and pH 5.6-5.8.

[0050] The microbial agent of this embodiment has good control effects on tomato root rot, gray mold, wilt and early blight.

[0051] Verification of strain antagonism:

[0052] 1. Activate the frozen Trichoderma chlororaphis, Trichoderma hooked, Bacillus licheniformis, and Azotobacter fusca, streak them on LB medium plates, and culture them at 25-28℃ for 3-5 days.

[0053] Select three bacteria and streak them on one culture medium to fully prove that there is no mutual inhibition between the strains:

[0054] Horizontal line Vertical Line Slash LB1 Trichoderma chlororaphis Trichoderma hook Bacillus licheniformis LB2 Trichoderma hook Bacillus licheniformis Azotobacter nitrofurantoides LB3 Trichoderma chlororaphis Bacillus licheniformis Azotobacter nitrofurantoides LB4 Trichoderma chlororaphis Trichoderma hook Azotobacter nitrofurantoides

[0055] Observe the growth of the four strains tested on LB medium and whether there is any antagonism between them. Figure 1 As shown, there is no obvious inhibition zone between the four bacteria, indicating that there is no obvious inhibitory effect between them, and composite fermentation or compounding of the bacterial liquid can be carried out.

[0056] Comparative Example 1

[0057] A method for preparing a microbial agent for preventing and controlling tomato diseases comprises the following preparation steps:

[0058] (1) Activate the slant strains of Trichoderma chlororaphis and Trichoderma hooked, pick the strains and put them into 100 ml of potato glucose liquid culture medium, culture them at 170 r / min, 25-28° C. for 48 hours to obtain seed liquid; inoculate the seed liquid into the fermentation culture medium at an inoculum rate of 10%, culture them at 170 r / min, 25-28° C. for 96 hours to obtain culture liquid, at which time the culture liquid is full of hyphae and spores of Trichoderma chlororaphis and Trichoderma hooked, wash the spores with sterile water, filter them with four layers of gauze, and dilute them again to obtain Trichoderma chlororaphis liquid and Trichoderma hooked liquid, respectively, to ensure that the spore concentrations of the two Trichoderma liquids obtained after dilution are both 2×10 8 cfu / mL;

[0059] (2) The two spore liquids are mixed in a volume ratio of 1:1 to obtain the final product microbial agent.

[0060] The difference in this comparative example is that only the Trichoderma chlororaphis solution and the Trichoderma hooked solution obtained in step (1) of the preparation method of Example 1 are mixed in equal volumes to obtain a microbial agent. The remaining raw materials and preparation method are the same as those of Example 1.

[0061] Comparative Example 2

[0062] A method for preparing a microbial agent for preventing and controlling typical tomato diseases comprises the following preparation steps:

[0063] (1) Thaw the brown ball nitrogen-fixing bacteria and inoculate them in PDA medium for activation culture at 28°C; after activation culture, pick 1% of the inoculum and add it to the fermentation culture medium, and ferment at 25-28°C and 150r / min until the viable bacteria amount is 2×10 8cfu / mL of fermentation broth;

[0064] (2) Thaw the Bacillus licheniformis and inoculate it into LB solid medium, culture it at 28-30°C for 1 day to activate the strain; pick the activated Bacillus licheniformis and inoculate it into LB liquid medium, ferment it at 28-30°C and 220r / min until the viable bacteria amount is 2×10 8 cfu / mL of fermentation broth;

[0065] (3) The two fermentation broths were mixed in a volume ratio of 1:1 to obtain the final product microbial agent.

[0066] The difference in this comparative example is that only the brown spherical nitrogen-fixing bacteria solution and the Bacillus licheniformis solution obtained in the preparation method of Example 1 are mixed in equal volumes to obtain a microbial agent. The remaining raw materials and preparation method are the same as those in Example 1.

[0067] Comparative Example 3

[0068] A method for preparing a microbial agent for preventing and controlling typical tomato diseases comprises the following preparation steps:

[0069] (1) Activate the slant strains of Trichoderma chlororaphis and Trichoderma hooked, pick the strains and put them into 100 ml of potato glucose liquid culture medium, culture them at 170 r / min, 25-28° C. for 48 hours to obtain seed liquid; inoculate the seed liquid into the fermentation culture medium at an inoculum rate of 10%, culture them at 170 r / min, 25-28° C. for 96 hours to obtain culture liquid, at which time the culture liquid is full of hyphae and spores of Trichoderma chlororaphis and Trichoderma hooked, wash the spores with sterile water, filter them with four layers of gauze, and dilute them again to obtain Trichoderma chlororaphis liquid and Trichoderma hooked liquid, respectively, to ensure that the spore concentrations of the two Trichoderma liquids obtained after dilution are both 2×10 8 cfu / mL;

[0070] (2) Thawing the brown ball nitrogen-fixing bacteria, inoculating them into PDA medium, and activating them at 28°C; after activation, taking 1% of the inoculum, adding it to the fermentation medium, and fermenting it at 25-28°C and 150 r / min until the viable bacteria amount is 2×10 8 cfu / mL of fermentation broth;

[0071] (3) Thawing the Bacillus licheniformis, inoculating it into LB solid medium, culturing it at 28-30°C for 1 day to activate the strain; picking the activated Bacillus licheniformis, inoculating it into LB liquid medium, fermenting it at 28-30°C, 220r / min, until the viable bacteria amount is 2×10 8 cfu / mL of fermentation broth;

[0072] (4) The four bacterial solutions were mixed in a volume ratio of 2:2:1:1 to obtain the final product microbial agent.

[0073] The difference of this comparative example is that the volume ratio of the four bacterial liquids is changed. The other raw materials and preparation methods are the same as those in Example 1.

[0074] Comparative Example 4

[0075] A method for preparing a microbial agent for preventing and controlling typical tomato diseases comprises the following preparation steps:

[0076] (1) Activate the slant strains of Trichoderma chlororaphis and Trichoderma hooked, pick the strains and put them into 100 ml of potato glucose liquid culture medium, culture them at 170 r / min, 25-28° C. for 48 hours to obtain seed liquid; inoculate the seed liquid into the fermentation culture medium at an inoculum rate of 10%, culture them at 170 r / min, 25-28° C. for 96 hours to obtain culture liquid, at which time the culture liquid is full of hyphae and spores of Trichoderma chlororaphis and Trichoderma hooked, wash the spores with sterile water, filter them with four layers of gauze, and dilute them again to obtain Trichoderma chlororaphis liquid and Trichoderma hooked liquid, respectively, to ensure that the spore concentrations of the two Trichoderma liquids obtained after dilution are both 2×10 8 cfu / mL;

[0077] (2) Thawing the brown ball nitrogen-fixing bacteria, inoculating them into PDA medium, and activating them at 28°C; after activation, taking 1% of the inoculum, adding it to the fermentation medium, and fermenting it at 25-28°C and 150 r / min until the viable bacteria amount is 2×10 8 cfu / mL of fermentation broth;

[0078] (3) Thawing the Bacillus licheniformis, inoculating it into LB solid medium, culturing it at 28-30°C for 1 day to activate the strain; picking the activated Bacillus licheniformis, inoculating it into LB liquid medium, fermenting it at 28-30°C, 220r / min, until the viable bacteria amount is 2×10 8 cfu / mL of fermentation broth;

[0079] (4) The four bacterial solutions were mixed in a volume ratio of 1:1:2:2 to obtain the final product microbial agent.

[0080] The difference of this comparative example is that the volume ratio of the four bacterial liquids is changed. The other raw materials and preparation methods are the same as those in Example 1.

[0081] Antibacterial effect of bacterial solution:

[0082] The microbial cultures prepared in the examples and comparative examples were tested.

[0083] The pathogens of tomato root rot (Pythium), gray mold (Botrytis cinerea), wilt (Fusarium oxysporum), and early blight (Alternaria solani) were selected, and the growth inhibition experiment was carried out by plate confrontation method. The growth inhibition activity of the target bacterial solution on plant pathogens was detected by observing the size of the antagonistic circle. The method is as follows: select newly activated pathogens with good growth, punch out a bacterial cake with a diameter of 5mm, place the bacterial cake of the pathogen in the center of the PDA plate, add 10μL of the test bacterial solution at about 22mm around both sides, and use an equal amount of sterile water as a control. Each treatment was repeated 3 times and cultured at a constant temperature of 28-30℃ for 3d. Observe the antibacterial effect. Figure 2 The inhibitory effect of the bacterial solution in Example 1 on four pathogens is shown in Figure 2 We can see that the bacterial solution of the present invention has a good inhibitory effect on the four pathogens.

[0084] Taking Fusarium oxysporum, a major pathogen of tomatoes, as an example, referring to the above test method, a bacterial cake of Fusarium oxysporum was placed in the center of a PDA plate, and circular filter papers were placed about 22 mm on both sides thereof, and 10 μL of bacterial solution of Example 1 and Comparative Examples 1-4 were added dropwise, and cultured at a constant temperature of 28-30° C. for 3 days. The antibacterial effect was observed. Figure 3 The graph is a graph showing the inhibitory effect of Example 1 and Comparative Examples 1-4 on Fusarium oxysporum. From the graph, we can see that compared with the embodiments, the antibacterial effect of Comparative Examples 1-2, which only uses fungi or bacteria, is significantly reduced, while the synergistic balance between the two is broken in Comparative Examples 3-4, which changes the dosage relationship of fungi and bacteria, and thus the antibacterial effect is weakened.

[0085] Planting test

[0086] Disease prevention efficacy test:

[0087] The surface of crop seeds is disinfected, germinated in a culture dish with multiple layers of wet filter paper, and moisturized with sterile water for several days. After the seeds have lost their seed coat, they are transplanted into sterile soil according to the conventional planting density. When the tomato seedlings grow to three leaves and one heart, the corresponding pathogen suspension is poured to the roots of the seedlings.

[0088] After inoculation of the pathogen, the tomato seedlings were cultured for 2 days, and then the Example bacterial agent of the present invention and the microbial agent prepared in the comparative example diluted 100 times were respectively irrigated to the roots of the seedlings, and the dosage was calculated based on 25 ml per plant, and the control group was treated with clean water. Each treatment was repeated 3 times with 20 tomato seedlings, and the results were averaged. The control effect of tomatoes was observed after 25 days, and the control experiment results are shown in Table 1.

[0089] The disease classification standards for tomato root rot are as follows:

[0090] Level 0: no symptoms, no lesions at the stem base;

[0091] Level 1: Yellowing of cotyledons or slight browning of stem base;

[0092] Level 2: Cotyledons wilt or less than 50% of the stem base turns brown;

[0093] Level 3: Plants wilt or 50%-75% of the stem base turns brown;

[0094] Level 4: The plant's vascular bundles turn brown and the roots become necrotic.

[0095] Disease index = Σ(disease level × number of plants at that disease level) / (highest disease level × total number of plants) × 100% Prevention effect = (disease index of control group – disease index of treatment group) / disease index of control × 100%

[0096] The classification standards for tomato gray mold disease are as follows:

[0097] Level 0: no lesions;

[0098] Level 1: The lesion area accounts for less than 5% of the entire leaf area;

[0099] Level 2: The lesion area accounts for 5%-25% of the entire leaf area;

[0100] Level 3: The lesion area accounts for 25%-50% of the entire leaf area;

[0101] Level 4: The lesion area accounts for more than 50% of the total leaf area.

[0102] Disease index = Σ(number of diseased leaves × disease level) / (total number of leaves surveyed × highest disease level) × 100%

[0103] Prevention effect = (control disease index - treatment disease index) / control disease index × 100%

[0104] The classification standards for tomato wilt disease are as follows:

[0105] Grade 0: no symptoms;

[0106] Level 1: 1-2 true leaves turn yellow or wilt and droop;

[0107] Level 2: 3-4 true leaves turn yellow or wilt and droop;

[0108] Level 3: 5-6 true leaves turn yellow or wilt and droop;

[0109] Level 4: The whole plant is severely wilted.

[0110] Disease index = Σ(number of diseased plants at each level × relative level value) / (total number of plants surveyed × highest disease level) × 100%; prevention effect = (1-disease index of test area / disease index of control area) × 100%.

[0111] The disease classification of tomato early blight is as follows:

[0112] Level 0: No lesions

[0113] Level 1: The lesion area accounts for less than 25% of the entire leaf area;

[0114] Level 2: The lesion area accounts for 25% to 50% of the entire leaf area;

[0115] Level 3: The lesion area accounts for 51% to 75% of the entire leaf area;

[0116] Level 4: The lesion area accounts for more than 75% of the entire leaf area.

[0117] Disease index = Σ(number of diseased leaves × disease level) / (total number of leaves surveyed × highest disease level) × 100%

[0118] Preventive effect = (control group disease index - treatment group disease index) / control disease index × 100%

[0119] Table 1. Control effect on typical tomato diseases (%)

[0120] Pathogens Tomato root rot Tomato gray mold Tomato wilt Tomato Early Blight Example 1 88.3 81.3 89.2 86.5 Comparative Example 1 48.7 52.8 51.4 46.2 Comparative Example 2 50.9 49.5 47.0 53.4 Comparative Example 3 64.2 64.2 68.5 57.8 Comparative Example 4 67.5 62.1 56.3 69.7 Control group -- -- -- --

[0121] It can be seen from the data in Table 1 that the microbial agent of the present invention has a significant control effect on typical tomato diseases, and the control effect on multiple diseases is higher than 81%. The control effect of the microbial agent compounded with four different fungal and bacterial species is significantly higher than that of the microbial agent compounded with only fungi or only bacteria. Example 1 of the present invention, in which the ratio of the four species is 1:1:1:1, has the best control effect on typical tomato diseases.

[0122] Tomato growth status and quality detection:

[0123] 10 fields with the same terrain and similar soil conditions were selected and planted with tomato seedlings of the same quality, and divided into ten groups of experiments, with 20 seeds planted in each group. After the tomato seedlings grew slowly for one week, they were irrigated once every 10 days with 25 ml of the bacterial agent prepared in the embodiment and the comparative example, for 2 consecutive times; the control group was treated with sterile water, and the net weight of a single tomato fruit and the number of fruits per plant were measured after harvest, as shown in Table 2 below, and the quality of the tomatoes was measured, as shown in Table 3 below.

[0124] Table 2. Net weight of tomato fruit and number of fruits per plant

[0125] Single fruit net weight (g) Number of fruits per plant Example 1 0.302 8.6 Comparative Example 1 0.219 7.4 Comparative Example 2 0.206 7.2 Comparative Example 3 0.253 7.9 Comparative Example 4 0.248 7.7 Control group 0.197 7.1

[0126] Table 3 Tomato quality

[0127]

[0128]

[0129] The experimental results show that the microbial agent of the present invention can not only increase the net weight of a single tomato fruit, but also increase the number of fruits set in tomato plants. By detecting the VC content and soluble protein content of tomatoes, it can be seen that compared with the control group, the microbial agent of the present invention can significantly increase the VC content and soluble protein content of tomatoes, thereby improving the quality of tomatoes.

[0130] Soil physical and chemical properties testing:

[0131] Experimental group: Sterile soil was placed in a 20 cm diameter flower pot, and tomato seedlings of uniform size and good growth were selected for transplanting, with one plant per pot and 6 pots per group. The prepared microbial agent was diluted 100 times with sterile water according to Example 1, and 25 ml of the microbial agent was taken for root irrigation after planting, for 2 consecutive times, with an interval of 10 days each time.

[0132] Control group: The control group was irrigated with sterile water.

[0133] After 90 days, soil from the tomato root zone was collected and tested for soil physical and chemical properties. The test results are shown in Table 4.

[0134] Table 4 Physical and chemical properties of soil

[0135] Control group Example 1 Microbial Agent Available phosphorus (mg / kg) 189.47 285.69 Organic matter (g / kg) 65.43 85.79 Ammonium nitrogen (mg / kg) 76.96 101.28 Nitrate nitrogen (mg / kg) 8.77 29.14

[0136] As can be seen from the data in Table 4, the physical and chemical properties of the soil treated with the composite microbial agent of the present invention are significantly better than those of the control group. The N content and organic matter content of the soil treated with the microbial agent are significantly higher than those of the control group, and the activity of microorganisms is also conducive to the increase of soil organic matter. This shows that the microbial agent of the present invention can increase the nitrogen and phosphorus content in the soil and the decomposition of organic matter, thereby helping to improve soil fertility and promote crop growth.

[0137] It should be noted that the above embodiments are only partial embodiments of the preferred methods of implementing the present invention, rather than all embodiments. Obviously, based on the above embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

Claims

1. A microbial agent for preventing and treating typical tomato diseases, characterized in that: The invention comprises the following ingredients: Trichoderma viridescens, Trichoderma hamatum, Bacillus licheniformis and Azotobacter chroococcum, in a volume ratio of 1:1:1:1; The Trichoderma chlororaphis was purchased from the General Microbiological Center of China Microorganism Culture Collection Administration (CGMCC), with the strain number of CGMCC No.5.1040; the original preservation time was August 8, 2005; The hooked Trichoderma was purchased from the General Microbiological Center of China Microorganism Culture Collection Administration (CGMCC), with the strain number of CGMCC No.3.7249; the original preservation time was July 8, 2005; The Bacillus licheniformis was purchased from the General Microbiology Center of China Microorganism Culture Collection Administration (CGMCC), and the strain collection number is CGMCC No.1.7677; the original collection time is June 24, 2008; The brown ball nitrogen-fixing bacteria were purchased from the General Microbiological Center of China Microorganism Culture Collection Administration (CGMCC), with the strain number of CGMCC No.1.5803; the original preservation time was June 27, 2005.

2. A microbial agent for controlling typical tomato diseases according to claim 1, characterized in that: The microbial agent is a liquid agent.

3. The microbial agent for controlling typical tomato diseases according to claim 1, characterized in that: The spore count of the Trichoderma chlororaphis was 2×10 8 cfu / mL; the spore count of the hooked Trichoderma is 2×10 8 cfu / mL; the amount of live Bacillus licheniformis in the microbial agent is 2×10 8 cfu / mL, the viable bacterial count of brown spherical nitrogen-fixing bacteria is 2×10 8 cfu / mL.

4. A method for preparing a microbial agent for preventing and treating typical diseases according to any one of claims 1 to 3, characterized in that: The method comprises the following preparation steps: (1) Activating the slant strains of Trichoderma chlororaphis and Trichoderma hooked, picking the strains and placing them in 100 ml of potato glucose liquid culture medium, culturing at 170 r / min, 25-28° C. for 48 h to obtain seed solution; The seed liquid was inoculated into the fermentation culture medium at an inoculum rate of 10%, and cultured at 170 r / min and 25-28°C for 96 hours to obtain a culture medium. At this time, the culture medium was full of hyphae and spores of Trichoderma chlororaphis and Trichoderma hamata. The spores were washed with sterile water, filtered through four layers of gauze, and diluted to obtain Trichoderma chlororaphis liquid and Trichoderma hamata liquid, respectively, to ensure that the spore concentrations of the two Trichoderma liquids obtained after dilution were both 2×10 8 cfu / mL; (2) Thawing the brown ball nitrogen-fixing bacteria, inoculating them into PDA medium, and activating them at 28°C; after activation, taking 1% of the inoculum, adding it to the fermentation medium, and fermenting it at 25-28°C and 150 r / min until the viable bacteria amount is 2×10 8 cfu / mL of fermentation broth; (3) Thawing the Bacillus licheniformis, inoculating it into LB solid medium, culturing it at 28-30°C for 1 day to activate the strain; picking the activated Bacillus licheniformis, inoculating it into LB liquid medium, fermenting it at 28-30°C, 220r / min, until the viable bacteria amount is 2×10 8 cfu / mL of fermentation broth; (4) The four bacterial solutions were mixed in a volume ratio of 1:1:1:1 to obtain the final product microbial agent.

5. Use of the microbial agent according to any one of claims 1 to 3 in preventing and controlling typical tomato diseases.

6. The use of the microbial agent according to claim 5 in preventing and controlling typical tomato diseases, characterized in that: The typical diseases are root rot, gray mold, wilt and early blight.

Images