A microbial agent for preventing and treating tomato root rot

Abstract

The present invention discloses a microbial agent for preventing and treating tomato root rot, and belongs to the technical field of microbial materials. The present invention screens and obtains three biocontrol strains, Bacillus licheniformis, Bacillus subtilis and Monascus lunulatus, which have an inhibitory effect on a variety of pathogenic bacteria, especially a strong inhibitory effect on Fusarium oxysporum. The present invention also adds a small amount of composite nutrient solution composed of glucose, bean cake powder, etc. to provide sufficient nutrition supply for microbial activity. A microbial activity promoter composed of brown algae oligosaccharide and 5-aminolevulinic acid is added to improve the efficiency of microbial colonization. In actual tomato planting, it can effectively prevent and treat tomato root rot, promote the growth of tomato plants, improve tomato quality, and is green, safe and pollution-free, does not cause environmental pollution, can reduce the amount of other corresponding chemical pesticides, save expenses, reduce costs, has potential economic and environmental benefits, and is worthy of application and promotion.

Description

Technical Field

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

[0002] Tomatoes are one of the most widely cultivated economic crops worldwide. Favored by growers and consumers for their diverse varieties, wide adaptability, high yield, nutritious nutrition, excellent edible quality, and versatility, they are an important vegetable crop distributed across diverse regions of the world. Rich in vitamins, carbohydrates, minerals, organic acids, and a small amount of protein, tomatoes are not only edible as fruits and vegetables but also serve as an important processing ingredient, enjoying a wide range of applications in the consumer sector. However, various tomato diseases have been a major limiting factor in tomato yield and economic profitability. Improving tomato disease resistance has become a key issue in tomato breeding.

[0003] Tomato root rot is a soil-borne disease that severely impacts tomato growth and yield, primarily caused by a fungus. Symptoms include rot at the base of the stem and roots, leading to yellowing and wilting of leaves, and ultimately, plant death. This disease not only impacts tomato growth but also significantly reduces yield and quality.

[0004] The importance of controlling tomato root rot lies in its impact on agricultural production. Due to its widespread spread and severe detrimental effects, uncontrolled root rot can lead to complete crop loss. Therefore, effective control measures are crucial for ensuring tomato yield and quality. Control methods include chemical, biological, agronomic, and physical control. Biological control is considered an environmentally friendly and highly effective method, as it reduces 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. 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 and Paecilomyces polymyxa to effectively prevent and control soil-borne pests and diseases such as root-knot nematodes, soft rot of Chinese cabbage, root rot of pepper, damping-off of tomato and blight of pepper.

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

[0008] In response to 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 a disease-resistant microbial solution, a compound nutrient solution and a microbial promoter, wherein the mass-to-volume ratio of the three is 10 mL:0.5 mL:0.1 g.

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

[0012] Furthermore, the Bacillus licheniformis was deposited in the General Microbiology Center of the China Culture Collection Administration Committee, the preservation date was September 12, 2023, the preservation number was CGMCC No. 28445, and the preservation address was No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; the Bacillus subtilis was deposited in the General Microbiology Center of the China Culture Collection Administration Committee, the preservation date was June 16, 2023, the preservation number was CGMCC No. 27647, and the preservation address was 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 Microbiology Center of China Culture Collection Administration; the Bacillus subtilis is disclosed in the invention patent CN116836896B authorized by the applicant.

[0014] Furthermore, the strain number of Monascus lunulatus is CGMCC No. 3.15356, purchased from the General Microbiology Center of China Culture Collection Administration. This strain can be purchased through the collection center's strain catalog without the need for repeated biological deposits.

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

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

[0017] b. The mixed seed solution was inoculated into a fermentation culture tank at a 1% inoculum volume and cultured at 25-28°C and 150-200 rpm. When the total concentration of viable bacteria reached 1×10 9 -1×10 10 CFU / mL, stop the culture and obtain the anti-pathogenic microbial liquid.

[0018] Furthermore, 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 the strain activation.

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

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

[0021] Furthermore, the composite nutrient solution is composed of: 1g glucose, 0.5g soybean 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 a mixture of brown algae oligosaccharide and 5-aminolevulinic acid 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 as follows: when using, the disease-resistant microbial liquid, the compound nutrient solution and the microbial promoter are mixed and dispersed evenly, diluted 100 times with water, and then irrigated the roots of the seedlings after transplanting, with 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 base of the stem 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 on Fusarium oxysporum. Among them, Bacillus licheniformis can secrete lipopeptide bioactive substances to inhibit the growth of Fusarium oxysporum. These lipopeptides 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. However, 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 role.

[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 activity. Adding a microbial activity promoter composed of brown algae oligosaccharide and 5-aminolevulinic acid, brown algae oligosaccharide can, on the one hand, enhance the stress resistance of plants by regulating the hormone level in the plant body. On the other hand, when brown algae oligosaccharide is used in combination with Bacillus, it can significantly promote the motility of Bacillus, allowing it to 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 at the same time 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, effectively inhibit pathogens and improve crop resistance.

[0028] (3) The microbial agent of the present invention can effectively prevent and control 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 1. It is a graph showing the results of the antagonistic experiment of Bacillus licheniformis, Bacillus subtilis and Monascus lunata of the present invention;

[0030] Figure 2 Figure 1 is a diagram showing the antibacterial effects 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 after applying different experimental groups of bacterial solutions. DETAILED DESCRIPTION

[0033] The technical solution of the present invention will be further described below with reference to specific embodiments, but is not limited thereto.

[0034] Example 1

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

[0036] The anti-pathogenic microbial liquid contains 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, and it was purchased from the General Microbiology Center of China Culture Collection Administration of Microorganisms.

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

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

[0042] b. The mixed seed solution was inoculated into a fermentation culture tank at a 1% inoculum volume and cultured at 25-28°C and 150-200 rpm. When the total concentration of viable bacteria reached 1×10 9 -1×10 10 CFU / mL, stop the culture and obtain the anti-pathogenic 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 the strain activation.

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

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

[0046] The composite nutrient solution comprises: 1 g of glucose, 0.5 g of soybean 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 is sterilized 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 a disease-resistant microbial solution, a compound nutrient solution and a microbial promoter, wherein the mass-to-volume ratio of the three is 10 mL:0.5 mL:0.1 g.

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

[0051] The acquisition methods of Bacillus licheniformis, Bacillus subtilis and Monascus lunata were the same as those in Example 1.

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

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

[0054] b. The mixed seed solution was inoculated into a fermentation culture tank at a 1% inoculum volume and cultured at 25-28°C and 150-200 rpm. When the total concentration of viable bacteria reached 1×10 9 -1×10 10CFU / mL, stop the culture and obtain the anti-pathogenic microbial liquid.

[0055] The strain activation method and related culture medium composition of this example are the same as those in 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 a disease-resistant microbial solution, a compound nutrient solution and a microbial promoter, wherein the mass-to-volume ratio of the three is 10 mL:0.5 mL:0.1 g.

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

[0060] The acquisition methods of Bacillus licheniformis, Bacillus subtilis and Monascus lunata were the same as those in Example 1.

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

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

[0063] b. The mixed seed solution was inoculated into a fermentation culture tank at a 1% inoculum volume and cultured at 25-28°C and 150-200 rpm. When the total concentration of viable bacteria reached 1×10 9 -1×10 10 CFU / mL, stop the culture and obtain the anti-pathogenic microbial liquid.

[0064] The strain activation method and related culture medium composition of this example are the same as those in 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 a disease-resistant microbial solution, a compound nutrient solution and a microbial promoter, wherein the mass-to-volume ratio of the three is 10 mL:0.5 mL:0.1 g.

[0068] The anti-pathogenic 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 lunata was the same as that in Example 1.

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

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

[0072] b. The mixed seed solution was inoculated into a fermentation culture tank at a 1% inoculum volume and cultured at 25-28°C and 150-200 rpm. When the total concentration of viable bacteria reached 1×10 9 -1×10 10 CFU / mL, stop the culture and obtain the anti-pathogenic 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 remaining 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 a disease-resistant microbial solution, a compound nutrient solution and a microbial promoter, wherein the mass-to-volume ratio of the three is 10 mL:0.5 mL:0.1 g.

[0078] The anti-pathogenic 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 lunata was the same as that in Example 1.

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

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

[0082] b. The mixed seed solution was inoculated into a fermentation culture tank at a 1% inoculum volume and cultured at 25-28°C and 150-200 rpm. When the total concentration of viable bacteria reached 1×10 9 -1×10 10 CFU / mL, stop the culture and obtain the anti-pathogenic 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 remaining 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 a disease-resistant microbial solution, a compound nutrient solution and a microbial promoter, wherein the mass-to-volume ratio of the three is 10 mL:0.5 mL:0.1 g.

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

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

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

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

[0092] b. The mixed seed solution was inoculated into a fermentation culture tank at a 1% inoculum volume and cultured at 25-28°C and 150-200 rpm. When the total concentration of viable bacteria reached 1×10 9 -1×10 10 CFU / mL, stop the culture and obtain the anti-pathogenic 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 remaining 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 a disease-resistant microbial solution, a compound nutrient solution and a microbial promoter, wherein the mass-to-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 remaining 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 a disease-resistant microbial solution, a compound nutrient solution and a microbial promoter, wherein the mass-to-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 was added, the rest of the raw materials and preparation method were the same as those in Example 1.

[0106] Performance Testing

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

[0108] The frozen Bacillus licheniformis, Bacillus subtilis and Monascus lunulatus were activated. The activation method is as follows: the frozen target strains were inoculated on LB solid culture medium plates and cultured at 25-28℃ for 36 hours; a single colony was picked and streaked again and transferred to LB solid culture medium plates, and cultured at 25-28℃ for 24 hours to complete the strain activation. The three strains to be tested were inoculated on LB solid culture medium respectively, and cultured in an inverted incubator at 25-30℃ for 48 hours. The antagonism between the biocontrol bacteria was judged based on the growth degree. Figure 1 We can see that the three strains grew well on the LB solid culture medium and had no antagonistic effect on each other, so they can be used for mixing with each other in compound bacterial agents.

[0109] Determination of biocontrol ability of strains:

[0110] (1) Antibacterial effect test: The target strains Bacillus licheniformis, Bacillus subtilis and Monascus lunata were activated separately, 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 out a bacterial cake with a diameter of 8 mm on an OA plate covered with tomato Fusarium oxysporum. Use a pick needle to inoculate the center of a new OA plate. Punch two symmetrical holes with a diameter of 8 mm 2.5 cm from the center of the plate and pick out the culture medium. Add the bacterial suspension to the wells, 50 μL per well. After sealing the film, transfer the plate to a 25-28°C incubator and incubate for 4 days. Measure the diameter of the colony in each treatment (at the line connecting the two holes) and calculate the inhibition rate. A total of three treatments were set up, with three replicates per treatment, and the results were averaged. The inhibition rate was calculated as follows: Inhibition rate (%) = (control colony diameter - treatment colony diameter) / control colony diameter × 100%.

[0112] OA is oatmeal culture medium, specifically composed of 30g / L oats and 15g / L agar. 30g of oats were placed in 1L of deionized water and boiled for 20 minutes. The extract was filtered through four layers of gauze, and 15g of agar was added. The volume was then adjusted to 1L with deionized water. The pH was adjusted to 7.2±0.2, and the culture was sterilized at 121°C and autoclaved for 20 minutes. 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 all reach over 80%.

[0116] Determination of biocontrol ability of composite 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 tested. The test method is as follows:

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

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

[0120] Planting experiment:

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

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

[0123] Application Method: Transplant tomatoes when they have 6-7 true leaves. During transplanting, inoculate the tomato root rot pathogen with a spore suspension using root irrigation. Dilute the test agent 100-fold with water and apply 10 mL per plant to the roots 10 days after transplanting. A water treatment served as a control. Field disease incidence and control efficacy were assessed 20 days after treatment. Incidence = number of diseased plants in survey / total number of plants surveyed × 100%; control efficacy = [(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: 10 mL of the bacterial agent from Example 2 was used for root irrigation per plant;

[0126] Treatment 3: 10 mL of the bacterial agent from Example 3 was used for root irrigation per plant;

[0127] Treatment 4: 10 mL of the fungicide from Comparative Example 1 was used for root irrigation of each plant;

[0128] Treatment 5: 10 mL of the fungicide from comparative example 2 was used for root irrigation per plant;

[0129] Treatment 6: 10 mL of the fungicide from Comparative Example 3 was used for root irrigation per plant;

[0130] Treatment 7: 10 mL of the bacterial agent from Comparative Example 4 was used for root irrigation per plant;

[0131] Treatment 8: 10 mL of the fungicide from comparative example 5 was used for root irrigation per plant;

[0132] Treatment 9: 10 mL of the bacterial agent from Comparative Example 6 was used for root irrigation per plant;

[0133] Treatment 10: 10 mL of clean water was applied to each plant for root irrigation.

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

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

[0139]

[0140] The experimental results in Tables 2-3 show that the experimental groups using the inoculants of the present invention showed good efficacy against tomato root rot and produced high-quality tomatoes. However, the control efficacy and various indicators of Comparative Examples 1-3, which varied the microbial composition, and Comparative Examples 4-6, which varied the microbial promoter, were significantly reduced. This is because Comparative Examples 1-2 lacked the microbial strains, disrupting the balance between the three strains and leading to a decrease in antibacterial and growth-promoting effects. Furthermore, Comparative Examples 4-5, which lacked either fucoidan or 5-aminolevulinic acid, showed reduced effects on microbial colonization and growth promotion, resulting in decreased control efficacy and quality.

[0141] To further verify the colonization effect of microorganisms in the examples and comparative examples on tomato roots, further experimental verification was conducted. The method was as follows: plump tomato seeds were selected, surface-sterilized with a 70% alcohol solution, and rinsed with sterile water. The sterilized seeds were placed in a glass petri dish lined with two layers of filter paper moistened with sterile water, the filter paper kept moist, and the dish was incubated at 28°C until the seeds germinated. The test soil was sieved to remove large particles. The resulting natural soil was then sterilized at 121°C for 1 hour, removed overnight, and then sterilized at 121°C for 1 hour, removed overnight, to obtain sterilized soil. Potted seeds with consistent growth after germination were selected and sown in seedling pots. Each pot was pre-filled with 150g of sterile soil, and three seeds were sown per pot. After the tomato plants grew three cotyledons, 10mL of the test solution was added to the roots of the tomatoes using root irrigation. 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 them three times with sterile water, and then use filter paper to absorb the residual moisture on the roots; weigh 0.02g of tomato roots, cut them into small pieces with sterile scissors, and grind them in a sterilized mortar; after grinding into a homogenous slurry, transfer them to a 2mL centrifuge tube, add 1mL of sterile water, mix well, and spread them on LB plates. Seal the coated plates and place them in a constant temperature incubator at 28℃, invert and culture for 48 hours. 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 was 2.5, indicating that the applied functional microorganisms have a good ability to colonize in the tomato rhizosphere. In contrast, in Comparative Examples 4-6, where the composition of the microbial promoter was changed, the colonization level decreased significantly. The synergistic effect of fucoidan and 5-aminolevulinic acid effectively increased the colonization level of functional microorganisms in the rhizosphere, allowing the microorganisms to continue to function.

[0143] It should be noted that the above embodiments are only some of the preferred embodiments of the present invention, and not all of them. Obviously, based on the above embodiments of the present invention, all other embodiments obtained by those skilled in the art 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: The invention comprises an anti-pathogenic microbial liquid, a compound nutrient solution and a microbial promoter, wherein the mass volume ratio of the three is 10 mL: 0.5 mL: 0.1 g; the anti-pathogenic microbial liquid contains Bacillus licheniformis ( Bacillus licheniformis) , Bacillus subtilis ( Bacillus subtilis ) and Monascus lunulatus ( Monascus lunisporas ), the volume ratio of the three is 1:1:1; the Bacillus licheniformis is deposited in the General Microorganism Center of China Culture Collection of Microorganisms, the preservation time is September 12, 2023, and the preservation number is CGMCC No. 28445; the Bacillus subtilis is deposited in the General Microorganism Center of China Culture Collection of Microorganisms, the preservation time is June 16, 2023, and the preservation number is CGMCC No. 27647; the strain number of Monascus lunulatus is CGMCC No. 3.15356, which was purchased from the General Microorganism Center of China Culture Collection of Microorganisms.

2. The microbial agent for preventing and treating tomato root rot according to claim 1, characterized in that: The preparation method of the anti-pathogenic microbial liquid is as follows: a. After activation, Bacillus licheniformis, Bacillus subtilis and Monascus lunulatus were cultured in LB liquid medium at 25-28 ° C and 150-200 rpm with shaking for 12-24h to obtain three kinds of bacterial seed solution. The seed solution of the three bacteria was mixed in a volume ratio of 1:1:1 to obtain a mixed seed solution. b. The mixed seed solution was inoculated into a fermentation culture tank at a 1% inoculum volume and cultured at 25-28°C and 150-200 rpm. When the total concentration of viable bacteria reached 1×10 9 -1×10 10 CFU / mL, stop the culture and obtain the anti-pathogenic microbial liquid.

3. The microbial agent for preventing and treating tomato root rot according to claim 1, characterized in that: The composite nutrient solution comprises: 1 g of glucose, 0.5 g of soybean 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 is sterilized at 121°C for 20 min.

4. The microbial agent for preventing and treating 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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