Microbial agent for preventing and treating tomato root rot

Abstract

The invention discloses a microbial agent for preventing and treating tomato root rot, and belongs to the technical field of microbial materials. According to the invention, three biocontrol strains, namely bacillus licheniformis, bacillus subtilis and monascus curvularia, are obtained through screening, and the three biocontrol strains have an inhibition effect on a plurality of pathogenic bacteria, and particularly have a relatively strong inhibition effect on fusarium oxysporum. A small amount of glucose, soybean cake powder and the like are added to form a composite nutrient solution, and sufficient nutrition is provided for microbial activities. A microbial activity accelerant composed of alginate oligosaccharide and 5-aminolevulinic acid is added, so that the microbial colonization efficiency is improved. In actual tomato planting, the tomato root rot can be effectively prevented and treated, meanwhile, tomato plant growth is promoted, tomato quality is improved, the method is green, safe and pollution-free, environmental pollution is avoided, the dosage of other corresponding chemical pesticides can be reduced, expenditure is saved, cost is reduced, potential economic benefits and environmental benefits are achieved, and the method is worthy of application and popularization.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial materials, and in particular relates to a microbial agent for preventing and treating tomato root rot. Background Art

[0002] Tomato is one of the most widely planted economic crops in the world. It is favored by growers and consumers for its variety, wide adaptability, high yield, rich nutrition, good edible quality and wide range of uses. It is an important vegetable crop widely distributed in different parts of the world. Tomato is rich in vitamins, carbohydrates, minerals, organic acids and a small amount of protein. It can not only be eaten as fruits and vegetables, but also as an important processing raw material. It has a wide range of uses in the consumer field. However, various diseases of tomatoes have always been a major limiting factor in tomato yield and economic benefits. How to improve the disease resistance of tomatoes has become an important issue in tomato breeding.

[0003] Tomato root rot is a soil-borne disease that seriously affects tomato growth and yield, mainly caused by fungi. Symptoms of tomato root rot include rotting of the base of the plant stem and roots, causing the leaves to turn yellow and wilt, and may eventually lead to plant death. This disease not only affects the growth of tomatoes, but also leads to a significant decrease in yield and quality.

[0004] The importance of controlling tomato root rot lies in its impact on agricultural production. Due to the widespread spread and serious harmfulness of root rot, if it is not controlled, it may lead to complete crop loss. Therefore, taking effective control measures is the key to ensure tomato yield and quality. Control methods include chemical control, biological control, agronomic control and physical control. Among them, biological control is considered to be an environmentally friendly and efficient method because it can reduce the use of chemical pesticides, thereby reducing potential harm to the environment and human health.

[0005] For example, Chinese patent application CN202010257789.4 discloses a composite microbial agent and its use in preventing and controlling various plant diseases, wherein the active ingredients are Bacillus thuringiensis, Bacillus amyloliquefaciens, Bacillus megaterium, Burkholderia pyrrocinia, Acinetobacter oleivorans and Pseudomonas fluorescens.

[0006] Chinese patent application CN202010306775.7 discloses a composite microbial agent for preventing and controlling soil-borne diseases, its preparation method and application, and discloses a composite microbial agent, its preparation method and application, which belongs to the field of microbial technology. The composite microbial agent is composed of the following components in parts by weight: 1-2 parts of Paecilomyces lilacinus, 0.5-1.5 parts of Paecilomyces polymyxa, 3-10 parts of calcium lignin sulfonate, 2-8 parts of sodium carboxymethyl cellulose, 7-17 parts of smoke foam, 8-15 parts of Fumei powder, 3-7 parts of starch, 8-16 parts of composite synergist, 5-15 parts of diatomaceous earth, 7-14 parts of attapulgite and 23.5-35.5 parts of water. The present invention combines Paecilomyces lilacinus with Paecilomyces polymyxa to effectively prevent and control soil-borne diseases and pests such as root-knot nematodes, Chinese cabbage soft rot, pepper root rot, tomato damping-off and pepper blight.

[0007] Most of the current existing technologies combine and compound multiple commercial strains with biocontrol effects. They have poor targeting, large usage, high cost, and unsatisfactory effects, making them difficult to be effectively used in actual agricultural production. Summary of the invention

[0008] In view of the problems existing in the prior art, the present invention provides a microbial agent for efficiently preventing and treating tomato root rot. The three strains screened are combined with microbial active agents and plant extracts to effectively inhibit the pathogens of tomato root rot, while promoting tomato growth and improving tomato quality.

[0009] In order to achieve the above technical objectives, the technical solution adopted by the present invention is:

[0010] A microbial agent for preventing and treating tomato root rot comprises disease-resistant microbial solution, compound nutrient solution and microbial promoter, wherein the mass volume ratio of the three is 10 mL:0.5 mL:0.1 g.

[0011] Furthermore, the anti-disease microbial liquid comprises Bacillus licheniformis, Bacillus subtilis and Monascus lunisporas, and the volume ratio of the three is 1:1:1.

[0012] Furthermore, the Bacillus licheniformis is deposited in the General Microbiology Center of the China Culture Collection Administration, the preservation date is September 12, 2023, the preservation number is CGMCC No. 28445, and the preservation address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; the Bacillus subtilis is deposited in the General Microbiology Center of the China Culture Collection Administration, the preservation date is June 16, 2023, the preservation number is CGMCC No. 27647, and the preservation address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0013] Furthermore, the Bacillus licheniformis was donated by Linyi Mingying Industry and Trade Co., Ltd. and deposited in the General Microbiological Center of China National Committee for the Preservation and Administration of Microbiological Cultures; the Bacillus subtilis is disclosed in the invention patent CN116836896B authorized by the applicant.

[0014] Furthermore, the strain number of the Monascus lunulatus is CGMCC No.3.15356, which was purchased from the General Microbiological Center of the China Microbiological Culture Collection Administration. The strain can be purchased through the strain catalog of the collection center without the need for repeated biological preservation.

[0015] Furthermore, the preparation method of the anti-disease microbial liquid is:

[0016] a. After activating Bacillus licheniformis, Bacillus subtilis and Monascus lunulatus, they were cultured in LB liquid medium at 25-28°C and 150-200rpm with shaking for 12-24h to obtain seed solutions of the three bacteria, and the seed solutions of the three bacteria were mixed in a volume ratio of 1:1:1 to obtain a mixed seed solution;

[0017] b. Inoculate the mixed seed solution at a 1% inoculation rate into a fermentation culture tank and culture at 25-28°C and 150-200rpm. When the total concentration of viable bacteria is 1×10 9 -1×10 10 CFU / mL, stop the culture and obtain the anti-disease microbial liquid.

[0018] Furthermore, the method for strain activation in step a. is: inoculate the frozen target strain on an LB solid culture medium plate and culture it at 25-28°C for 36 hours; pick a single colony and streak it again on an LB solid culture medium plate, and culture it at 25-28°C for 24 hours to complete the strain activation.

[0019] The composition of the LB liquid culture medium is: 10 g / L peptone, 5 g / L yeast powder, 5 g / L sodium chloride, and the pH value is 7.0.

[0020] Furthermore, the composition of the fermentation medium in the fermentation culture tank is: 4wt% corn flour, 2wt% bean cake powder, 4wt% bran powder, 3wt% K2HPO4, 1wt% KH2PO4, 0.065wt% MgSO4, 0.3wt% peanut oil and balance water, and the pH value is 7.0.

[0021] Furthermore, the composite nutrient solution is composed of: 1g glucose, 0.5g bean cake powder, 3g yeast powder, 3g KH2PO4, 2g NaHCO3, 0.5g MgCl2·6H2O, 0.01g FeCl2·7H2O, 1000ml water, pH value 7.0-7.2, and sterilized at 121°C for 20min.

[0022] Furthermore, the microbial promoter is composed of brown algae oligosaccharide and 5-aminolevulinic acid mixed in a mass ratio of 1:0.1-0.3. All raw materials used in the present invention are commercially available.

[0023] Furthermore, the method for using the microbial agent of the present invention is: when using, the disease-resistant microbial solution, the compound nutrient solution and the microbial promoter are evenly dispersed, diluted 100 times with water, and then irrigated the roots after transplanting the seedlings, 10-30 mL per plant.

[0024] Fusarium oxysporum is one of the pathogens that cause tomato root rot, which can cause browning and rot at the stem base and roots of tomato plants, greatly affecting tomato yield.

[0025] Beneficial effects:

[0026] (1) The present invention screened and obtained three biocontrol strains, namely, Bacillus licheniformis, Bacillus subtilis and Monascus lunulatus, which have inhibitory effects on a variety of pathogenic bacteria, especially have a strong inhibitory effect on Fusarium oxysporum. Among them, Bacillus licheniformis can secrete lipopeptide bioactive substances to inhibit the growth of Fusarium oxysporum. These lipopeptide substances can not only interfere with the normal growth of Fusarium oxysporum, but also affect the development of its mycelium, thereby achieving an inhibitory effect. Bacillus subtilis can produce a variety of secondary metabolites, such as proteases, cellulases, amylases, etc. These substances have antibacterial activity and effectively inhibit the growth of pathogens. The added Monascus lunulatus has a weaker antagonistic and inhibitory effect on Fusarium oxysporum than Bacillus licheniformis and Bacillus subtilis, but when it is mixed with Bacillus licheniformis and Bacillus subtilis in equal proportions and used in combination, it can enhance the natural competitiveness and survival rate of Bacillus, improve the efficiency of rhizosphere colonization, improve the effect and stability of disease control, and play a synergistic and antibacterial effect.

[0027] (2) The present invention simultaneously adds a small amount of composite nutrient solution composed of glucose, bean cake powder, etc. to provide sufficient nutrition supply for microbial activities. Adding a microbial activity promoter composed of brown algae oligosaccharide and 5-aminolevulinic acid, brown algae oligosaccharide can enhance the stress resistance of plants by regulating the hormone level in plants. On the other hand, when brown algae oligosaccharide is used in combination with Bacillus, it can significantly promote the motility of Bacillus, so that it can quickly and effectively colonize in the rhizosphere of crops, thereby promoting plant growth. The added 5-aminolevulinic acid can promote the growth and metabolism of beneficial microorganisms, improve the activity of beneficial microorganisms, and induce microorganisms to produce a series of stress resistance substances, such as antioxidant enzymes, osmotic regulating substances, etc., to help microorganisms resist the invasion of adversity and maintain their normal growth and metabolic functions. The combination of brown algae oligosaccharide and 5-aminolevulinic acid can effectively improve the colonization efficiency of functional microorganisms while improving microbial activity, efficiently inhibit pathogens and improve crop resistance.

[0028] (3) The microbial agent of the present invention can effectively prevent and treat tomato root rot in actual tomato cultivation, while promoting the growth of tomato plants and improving the quality of tomatoes. It is green, safe, and harmless, and does not cause environmental pollution. It can eliminate or reduce the amount of other corresponding chemical pesticides, save farmers' expenses, and reduce planting costs. It has potential economic and environmental benefits and is worthy of application and promotion. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The figure is a result of the antagonistic experiment of Bacillus licheniformis, Bacillus subtilis and Monascus lunulatus of the present invention;

[0030] Figure 2 The diagram is a diagram of the antibacterial effect of Bacillus licheniformis, Bacillus subtilis and Monascus lunulatus of the present invention, wherein a is Monascus lunulatus, b is Bacillus licheniformis, c is Bacillus subtilis, and d is a blank control;

[0031] Figure 3 The antibacterial effect diagram of the bacterial solution obtained in Example 1 of the present invention and Comparative Examples 1-3;

[0032] Figure 4 This is the result of the microbial colonization experiment on tomato roots using different experimental groups of bacterial solutions. DETAILED DESCRIPTION

[0033] The technical solution of the present invention is further described below in conjunction with specific embodiments, but is not limited thereto.

[0034] Example 1

[0035] A microbial agent for preventing and treating tomato root rot comprises disease-resistant microbial solution, compound nutrient solution and microbial promoter, wherein the mass volume ratio of the three is 10 mL:0.5 mL:0.1 g.

[0036] The anti-disease microbial liquid comprises Bacillus licheniformis, Bacillus subtilis and Monascus lunisporas, and the volume ratio of the three is 1:1:1.

[0037] The Bacillus licheniformis is deposited in the General Microbiology Center of the China Culture Collection Administration, the preservation date is September 12, 2023, the preservation number is CGMCC No. 28445, and the preservation address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; the Bacillus subtilis is deposited in the General Microbiology Center of the China Culture Collection Administration, the preservation date is June 16, 2023, the preservation number is CGMCC No. 27647, and the preservation address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0038] The Bacillus licheniformis was donated by Linyi Mingying Industry and Trade Co., Ltd.; the Bacillus subtilis is disclosed in the invention patent CN116836896B authorized by the applicant.

[0039] The strain number of Monascus lunulatus is CGMCC No.3.15356, which was purchased from the General Microbiology Center of China National Microbiological Culture Collection Administration.

[0040] The preparation method of the anti-disease microbial liquid is as follows:

[0041] a. After activating Bacillus licheniformis, Bacillus subtilis and Monascus lunulatus, they were cultured in LB liquid medium at 25-28°C and 150-200rpm with shaking for 12h to obtain seed solutions of the three bacteria, and the seed solutions of the three bacteria were mixed in a volume ratio of 1:1:1 to obtain a mixed seed solution;

[0042] b. Inoculate the mixed seed solution at a 1% inoculation rate into a fermentation culture tank and culture at 25-28°C and 150-200rpm. When the total concentration of viable bacteria is 1×10 9 -1×10 10 CFU / mL, stop the culture and obtain the anti-disease microbial liquid.

[0043] The method for strain activation in step a. is as follows: the frozen target strain is inoculated on an LB solid culture medium plate and cultured at 25-28°C for 36 hours; a single colony is picked and streaked again on an LB solid culture medium plate and cultured at 25-28°C for 24 hours to complete strain activation.

[0044] The composition of the LB liquid culture medium is: 10 g / L peptone, 5 g / L yeast powder, 5 g / L sodium chloride, and the pH value is 7.0.

[0045] The fermentation medium in the fermentation culture tank is composed of: 4wt% corn flour, 2wt% bean cake flour, 4wt% bran flour, 3wt% K2HPO4, 1wt% KH2PO4, 0.065wt% MgSO4, 0.3wt% peanut oil and the balance water, and the pH value is 7.0.

[0046] The composite nutrient solution comprises: 1 g of glucose, 0.5 g of bean cake powder, 3 g of yeast powder, 3 g of KH2PO4, 2 g of NaHCO3, 0.5 g of MgCl2·6H2O, 0.01 g of FeCl2·7H2O, 1000 ml of water, a pH value of 7.0-7.2, and sterilization at 121°C for 20 min.

[0047] The microbial promoter is composed of brown algae oligosaccharide and 5-aminolevulinic acid mixed in a mass ratio of 1:0.1.

[0048] Example 2

[0049] A microbial agent for preventing and treating tomato root rot comprises disease-resistant microbial solution, compound nutrient solution and microbial promoter, wherein the mass volume ratio of the three is 10 mL:0.5 mL:0.1 g.

[0050] The anti-disease microbial liquid comprises Bacillus licheniformis, Bacillus subtilis and Monascus lunisporas, and the volume ratio of the three is 1:1:1.

[0051] The acquisition method of Bacillus licheniformis, Bacillus subtilis and Monascus lunulatus is the same as that of Example 1.

[0052] The preparation method of the anti-disease microbial liquid is as follows:

[0053] a. After activating Bacillus licheniformis, Bacillus subtilis and Monascus lunulatus, they were cultured in LB liquid medium at 25-28°C and 150-200rpm with shaking for 24h to obtain seed solutions of the three bacteria, and the seed solutions of the three bacteria were mixed in a volume ratio of 1:1:1 to obtain a mixed seed solution;

[0054] b. Inoculate the mixed seed solution at a 1% inoculation rate into a fermentation culture tank and culture at 25-28°C and 150-200rpm. When the total concentration of viable bacteria is 1×10 9 -1×10 10CFU / mL, stop the culture and obtain the anti-disease microbial liquid.

[0055] The strain activation method and related culture medium composition of this example are the same as those of Example 1.

[0056] The microbial promoter is composed of brown algae oligosaccharide and 5-aminolevulinic acid mixed in a mass ratio of 1:0.2.

[0057] Example 3

[0058] A microbial agent for preventing and treating tomato root rot comprises disease-resistant microbial solution, compound nutrient solution and microbial promoter, wherein the mass volume ratio of the three is 10 mL:0.5 mL:0.1 g.

[0059] The anti-disease microbial liquid comprises Bacillus licheniformis, Bacillus subtilis and Monascus lunisporas, and the volume ratio of the three is 1:1:1.

[0060] The acquisition method of Bacillus licheniformis, Bacillus subtilis and Monascus lunulatus is the same as that of Example 1.

[0061] The preparation method of the anti-disease microbial liquid is as follows:

[0062] a. After activating Bacillus licheniformis, Bacillus subtilis and Monascus lunulatus, they were cultured in LB liquid medium at 25-28°C and 150-200rpm with shaking for 20h to obtain seed solutions of the three bacteria, and the seed solutions of the three bacteria were mixed in a volume ratio of 1:1:1 to obtain a mixed seed solution;

[0063] b. Inoculate the mixed seed solution at a 1% inoculation rate into a fermentation culture tank and culture at 25-28°C and 150-200rpm. When the total concentration of viable bacteria is 1×10 9 -1×10 10 CFU / mL, stop the culture and obtain the anti-disease microbial liquid.

[0064] The strain activation method and related culture medium composition of this example are the same as those of Example 1.

[0065] The microbial promoter is composed of brown algae oligosaccharide and 5-aminolevulinic acid mixed in a mass ratio of 1:0.3.

[0066] Comparative Example 1

[0067] A microbial agent for preventing and treating tomato root rot comprises disease-resistant microbial solution, compound nutrient solution and microbial promoter, wherein the mass volume ratio of the three is 10 mL:0.5 mL:0.1 g.

[0068] The anti-disease microbial liquid contains Bacillus subtilis and Monascus lunisporas, and the volume ratio of the two is 1:1.

[0069] The acquisition method of Bacillus subtilis and Monascus lunulatus is the same as that of Example 1.

[0070] The preparation method of the anti-disease microbial liquid is as follows:

[0071] a. After activating Bacillus subtilis and Monascus lunulatus, they were cultured in LB liquid medium at 25-28°C and 150-200rpm with shaking for 12h to obtain seed solutions of the two bacteria, and the seed solutions of the two bacteria were mixed in a volume ratio of 1:1 to obtain a mixed seed solution;

[0072] b. Inoculate the mixed seed solution at a 1% inoculation rate into a fermentation culture tank and culture at 25-28°C and 150-200rpm. When the total concentration of viable bacteria is 1×10 9 -1×10 10 CFU / mL, stop the culture and obtain the anti-disease microbial liquid.

[0073] The strain activation method and related culture medium composition are the same as in Example 1.

[0074] The microbial promoter is composed of brown algae oligosaccharide and 5-aminolevulinic acid mixed in a mass ratio of 1:0.1.

[0075] In this comparative example, except that only Bacillus subtilis and Monascus lunulatus were used in the anti-pathogenic microbial liquid, the other raw materials and preparation process were the same as those in Example 1.

[0076] Comparative Example 2

[0077] A microbial agent for preventing and treating tomato root rot comprises disease-resistant microbial solution, compound nutrient solution and microbial promoter, wherein the mass volume ratio of the three is 10 mL:0.5 mL:0.1 g.

[0078] The anti-disease microbial liquid contains Bacillus licheniformis and Monascus lunisporas, and the volume ratio of the two is 1:1.

[0079] The acquisition method of Bacillus licheniformis and Monascus lunulatus is the same as that of Example 1.

[0080] The preparation method of the anti-disease microbial liquid is as follows:

[0081] a. After activating Bacillus licheniformis and Monascus lunulatus, they were cultured in LB liquid medium at 25-28°C and 150-200rpm with shaking for 12h to obtain seed solutions of the two bacteria, and the seed solutions of the two bacteria were mixed in a volume ratio of 1:1 to obtain a mixed seed solution;

[0082] b. Inoculate the mixed seed solution at a 1% inoculation rate into a fermentation culture tank and culture at 25-28°C and 150-200rpm. When the total concentration of viable bacteria is 1×10 9 -1×10 10 CFU / mL, stop the culture and obtain the anti-disease microbial liquid.

[0083] The strain activation method and related culture medium composition are the same as in Example 1.

[0084] The microbial promoter is composed of brown algae oligosaccharide and 5-aminolevulinic acid mixed in a mass ratio of 1:0.1.

[0085] In this comparative example, except that only Bacillus licheniformis and Monascus lunulatus were used in the anti-pathogenic microbial liquid, the other raw materials and preparation process were the same as those in Example 1.

[0086] Comparative Example 3

[0087] A microbial agent for preventing and treating tomato root rot comprises disease-resistant microbial solution, compound nutrient solution and microbial promoter, wherein the mass volume ratio of the three is 10 mL:0.5 mL:0.1 g.

[0088] The anti-disease microbial liquid contains Bacillus licheniformis and Bacillus subtilis, and the volume ratio of the two is 1:1.

[0089] The method for obtaining Bacillus licheniformis and Bacillus subtilis is the same as that in Example 1.

[0090] The preparation method of the anti-disease microbial liquid is as follows:

[0091] a. After activating Bacillus licheniformis and Bacillus subtilis, they were cultured in LB liquid medium at 25-28°C and 150-200rpm with shaking for 12h to obtain seed solutions of the two bacteria, and the seed solutions of the two bacteria were mixed in a volume ratio of 1:1 to obtain a mixed seed solution;

[0092] b. Inoculate the mixed seed solution at a 1% inoculation rate into a fermentation culture tank and culture at 25-28°C and 150-200rpm. When the total concentration of viable bacteria is 1×10 9 -1×10 10 CFU / mL, stop the culture and obtain the anti-disease microbial liquid.

[0093] The strain activation method and related culture medium composition are the same as in Example 1.

[0094] The microbial promoter is composed of brown algae oligosaccharide and 5-aminolevulinic acid mixed in a mass ratio of 1:0.1.

[0095] In this comparative example, except that only Bacillus licheniformis and Bacillus subtilis were used in the anti-pathogenic microbial liquid, the other raw materials and preparation process were the same as those in Example 1.

[0096] Comparative Example 4

[0097] A microbial agent for preventing and treating tomato root rot comprises disease-resistant microbial solution, compound nutrient solution and microbial promoter, wherein the mass volume ratio of the three is 10 mL:0.5 mL:0.1 g.

[0098] The microbial promoter is brown algae oligosaccharide.

[0099] In this comparative example, except that only brown algae oligosaccharide was used as the microbial promoter, the other raw materials and preparation process were the same as those in Example 1.

[0100] Comparative Example 5

[0101] A microbial agent for preventing and treating tomato root rot comprises disease-resistant microbial solution, compound nutrient solution and microbial promoter, wherein the mass volume ratio of the three is 10 mL:0.5 mL:0.1 g.

[0102] The microbial promoter is 5-aminolevulinic acid.

[0103] Comparative Example 6

[0104] A microbial agent for preventing and treating tomato root rot comprises a disease-resistant microbial solution and a compound nutrient solution, wherein the volume ratio of the three is 1 mL:0.5 mL.

[0105] In this comparative example, except that no microbial promoter is added, the other raw materials and preparation method are the same as those of Example 1.

[0106] Performance Testing

[0107] The antagonistic effects of three biocontrol bacteria: Bacillus licheniformis, Bacillus subtilis and Monascus lunulatus were determined by the following test methods:

[0108] The frozen Bacillus licheniformis, Bacillus subtilis and Monascus lunulatus were activated by the following method: the frozen target strains were inoculated on LB solid culture medium plates and cultured at 25-28℃ for 36h; a single colony was picked and streaked again and transferred to LB solid culture medium plates, and cultured at 25-28℃ for 24h to complete the strain activation. The three strains to be tested were inoculated on LB solid culture medium respectively, and incubated inverted in a constant temperature incubator at 25-30℃ for 48h. The antagonism between the biocontrol bacteria was judged based on the degree of growth. Figure 1 We can see that the three strains of bacteria grow well on the LB solid culture medium, and there is no antagonism between them, so they can be used for mixing in composite bacterial agents.

[0109] Determination of biocontrol ability of strains:

[0110] (1) Antibacterial effect test: The target strains Bacillus licheniformis, Bacillus subtilis and Monascus lunulatus were activated respectively, inoculated into LB liquid culture medium after activation, and cultured in a shaking incubator at 25°C and 160 r / min for 24 h to obtain bacterial suspensions of the three bacteria.

[0111] (2) Use a sterile puncher to punch a bacterial cake with a diameter of 8 mm on the OA plate full of tomato Fusarium oxysporum, use a pick needle to inoculate it to the center of a new OA plate, punch two holes with a diameter of 8 mm symmetrically 2.5 cm away from the center of the plate, and pick out the culture medium; add the bacterial suspension into the holes, 50 μL per well, seal the film and transfer the plate to a 25-28℃ constant temperature box, culture for 4 days, measure the colony diameter of each treatment (the line connecting the two holes), and calculate the inhibition rate. A total of 3 treatments were set up, with 3 replicates for each treatment, and the results were averaged. The inhibition rate calculation formula is as follows: Inhibition rate (%) = (control colony diameter - treatment colony diameter) / control colony diameter × 100%.

[0112] OA is oat culture medium, and its specific composition is: 30g / L oat, 15g / L agar. 30g oat was placed in 1L deionized water and boiled for 20min, and the leaching liquid was filtered through 4 layers of gauze, and 15g agar was added, and the volume was fixed to 1L with deionized water, and the pH was adjusted to 7.2±0.2, and sterilized at 121℃ for 20min. The experimental results are shown in Table 1:

[0113] Table 1 Verification of antibacterial effect of strains

[0114] deal with Colony diameter cm Antibacterial rate% Bacillus licheniformis 1.12 86.68 Bacillus subtilis 1.05 87.51 Monascus lunulatus 1.52 81.93 Blank control 8.41 -

[0115] From the data in Table 1, it can be seen that the three strains have good inhibitory effects on Fusarium oxysporum, and the inhibition rates are all above 80%.

[0116] Determination of biocontrol ability of compound strains:

[0117] Taking Example 1 as an example, the antibacterial effect of the antimicrobial liquid obtained in Example 1 and Comparative Examples 1-3 was further determined, and the test method was as follows:

[0118] Select newly activated pathogens with good growth, use a hole punch to make a 5mm diameter bacterial cake, place the bacterial cake of Fusarium oxysporum pathogen in the center of the PDA plate, add 10μL of the test bacterial solution at about 22mm on both sides, use an equal amount of sterile water as a control, and culture at a constant temperature of 28-30℃ for 10d.

[0119] Test results such as Figure 3 As shown. Figure 3 It can be seen that the antibacterial microbial liquid obtained by simultaneously using three strains of bacteria in Example 1 of the present invention has a much higher antibacterial effect than that of Comparative Examples 1-3, and the lack of any one of the bacteria will lead to a decrease in the antibacterial effect.

[0120] Planting experiment:

[0121] Test crops: Tomato variety was Jinpeng No. 1, purchased from Xi'an Jinpeng Seedlings Co., Ltd.

[0122] Test substrates: peat, perlite, and vermiculite, all provided by Ningxia Kerui Hengsheng Agricultural Technology Co., Ltd., peat: perlite: vermiculite = 3:1:1.

[0123] Application method: Start transplanting when tomatoes have 6-7 true leaves. During transplanting, use the spore suspension root irrigation method to inoculate tomato root rot pathogens. Dilute the test agent 100 times with water and irrigate the roots 10 days after transplanting the seedlings. 10mL per plant. Use clean water as a control. After 20 days of treatment, investigate the field incidence and control effect. Incidence = number of investigated diseased plants / total number of investigated plants × 100%; control effect = [(number of diseased plants in the control area - number of diseased plants in the treatment area) / number of diseased plants in the control area] × 100%.

[0124] Treatment 1: 10 mL of the bacterial agent from Example 1 was used for root irrigation per plant;

[0125] Treatment 2: Example 2 bacterial agent, 10 mL per plant root irrigation treatment;

[0126] Treatment 3: Example 3 bacterial agent, 10 mL per plant root irrigation treatment;

[0127] Treatment 4: Comparative Example 1 bacterial agent, 10 mL per plant root irrigation treatment;

[0128] Treatment 5: Comparative Example 2 bacterial agent, 10 mL per plant for root irrigation;

[0129] Treatment 6: Comparative Example 3 bacterial agent, 10 mL per plant root irrigation treatment;

[0130] Treatment 7: Comparative Example 4 bacterial agent, 10 mL per plant for root irrigation treatment;

[0131] Treatment 8: Comparative Example 5 bacterial agent, 10 mL per plant for root irrigation;

[0132] Treatment 9: Comparative Example 6 bacterial agent, 10 mL per plant for root irrigation treatment;

[0133] Treatment 10: 10 mL of clean water was applied to the roots of each plant.

[0134] Each treatment group was repeated five times, and all test results were averaged.

[0135] Table 2 Results of the experimental study on the prevention of tomato root rot

[0136] Incidence % Control effect% Example 1 5.2 91.90 Example 2 4.7 92.68 Example 3 4.5 92.99 Comparative Example 1 22.3 65.26 Comparative Example 2 23.1 64.02 Comparative Example 3 21.0 67.29 Comparative Example 4 22.8 64.49 Comparative Example 5 20.1 68.69 Comparative Example 6 25.6 60.12 Comparison 64.2 -

[0137] In the peak fruiting period, 30 fruits of the same maturity were randomly harvested from each treatment group to determine the tomato quality. The vitamin C content was determined by 2,6-dichlorophenol indophenol titration method; the soluble sugar content was determined by direct titration method; and the titratable acid content was determined by acid-base titration method. The test results are shown in Table 3.

[0138] Table 3 Effects of different treatments on tomato quality

[0139]

[0140] From the experimental results in Table 2-3, it can be seen that the experimental group using the bacterial agent of the embodiment of the present invention has good prevention effect on tomato root rot and good tomato quality, while the prevention effect and various indicators of Comparative Examples 1-3 with changed microbial composition and Comparative Examples 4-6 with changed microbial promoters are significantly reduced. This is because the balance of the three strains is broken in Comparative Examples 1-2, which lack microbial strains, resulting in a decrease in the antibacterial and growth-promoting effects. Comparative Examples 4-5, which lack brown algae oligosaccharides or 5-aminolevulinic acid, have reduced colonization and promotion effects on microorganisms, resulting in reduced prevention effect and quality.

[0141] To further verify the colonization effect of the microorganisms in the embodiment and the comparative example on the roots of tomatoes, an experimental verification is conducted, and the method is as follows: select full tomato seeds, disinfect the tomato seeds with 70% alcohol solution, and rinse with sterile water. The sterilized seeds are placed in a glass culture dish covered with two layers of filter paper moistened with sterile water, keep the filter paper moist, and place at 28°C until the seeds germinate. The above-mentioned test soil is sieved to remove large particles, and then the obtained natural soil is sterilized at 121°C for 1h, taken out overnight, and then repeated at 121°C for 1h, taken out overnight to obtain sterilized soil. The experiment adopts a potted plant experiment, and seeds with consistent growth after germination are selected and sown in a seedling pot. Each seedling pot is pre-filled with 150g of sterile soil, and 3 seeds are sown in each pot. The tomato plant is irrigated by root irrigation. After 3 cotyledons grow, the test bacterial solution is added to the root of the tomato by root irrigation, 10mL per plant. Samples were taken on the 28th day after adding the bacterial solution, and tomato roots were taken for plating. Three replicates were set for each treatment, and the results were averaged.

[0142] The sampling method is as follows: Gently pull out the tomato roots, shake off the soil, wash with sterile water three times, and use filter paper to absorb the residual moisture on the roots; weigh 0.02g of tomato roots, cut into small pieces with sterile scissors, and grind in a sterilized mortar; after grinding into a homogenate, transfer to a 2mL centrifuge tube, add 1mL of sterile water, mix well, and select LB plates for plate coating. After the coated plates are sealed, they are placed in a constant temperature incubator at 28℃ and inverted for 48h. After the colonies grow, count them and calculate the colonization of the strains. The results are as follows Figure 4 As shown, the embodiment of the present invention is in 10 5 -10 6 The colonization level of CFU / g showed that the applied functional microorganisms had good colonization ability in the tomato rhizosphere. The colonization level of comparative examples 4-6, which changed the composition of the microbial promoter, decreased significantly. The synergistic cooperation of brown algae oligosaccharides and 5-aminolevulinic acid can effectively increase the colonization level of functional microorganisms in the rhizosphere so that the microorganisms can continue to play a role.

[0143] 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 tomato root rot, characterized in that: It includes disease-resistant microbial liquid, compound nutrient solution and microbial promoter, and the mass volume ratio of the three is 10mL:0.5mL:0.1g.

2. The microbial agent for preventing and controlling tomato root rot according to claim 1, characterized in that: The anti-disease microbial liquid comprises Bacillus licheniformis, Bacillus subtilis and Monascus lunisporas, and the volume ratio of the three is 1:1:

1.

3. The microbial agent for preventing and controlling tomato root rot according to claim 2, characterized in that: The Bacillus licheniformis was deposited in the General Microbiology Center of the China National Microorganism Culture Collection Administration on September 12, 2023, and the deposit number was CGMCC No. 28445; the Bacillus subtilis was deposited in the General Microbiology Center of the China National Microorganism Culture Collection Administration on June 16, 2023, and the deposit number was CGMCC No. 27647; the strain number of the Monascus lunulatus was CGMCC No. 3.15356, which was purchased from the General Microbiology Center of the China National Microorganism Culture Collection Administration.

4. The microbial agent for preventing and controlling tomato root rot according to any one of claims 1 to 3, characterized in that: The preparation method of the anti-disease microbial liquid is as follows: a. After activating Bacillus licheniformis, Bacillus subtilis and Monascus lunulatus, they were cultured in LB liquid medium at 25-28°C and 150-200rpm with shaking for 12-24h to obtain seed solutions of the three bacteria, and the seed solutions of the three bacteria were mixed in a volume ratio of 1:1:1 to obtain a mixed seed solution; b. Inoculate the mixed seed solution at a 1% inoculation rate into a fermentation culture tank and culture at 25-28°C and 150-200rpm. When the total concentration of viable bacteria is 1×10 9 -1×10 10 CFU / mL, stop the culture and obtain the anti-disease microbial liquid.

5. The microbial agent for preventing and controlling tomato root rot according to claim 1, characterized in that: The composite nutrient solution is composed of: 1g glucose, 0.5g bean cake powder, 3g yeast powder, 3g KH2PO4, 2g NaHCO3, 0.5g MgCl2·6H2O, FeCl2·7H2O 0.01g, water 1000ml, pH 7.0-7.2, sterilize at 121℃ for 20min.

6. The microbial agent for preventing and controlling tomato root rot according to claim 1, characterized in that: The microbial promoter is composed of brown algae oligosaccharide and 5-aminolevulinic acid mixed in a mass ratio of 1:0.1-0.3.

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