Microbial preparation for improving cold resistance and preventing and treating pepper fusarium wilt and application thereof

Abstract

The present application relates to a kind of microbial preparation for improving the cold resistance of pepper and preventing and treating pepper wilt and application thereof, and the active component in the microbial preparation includes probiotic bacterial inoculant and functional adjuvant;The probiotic bacteria is FangSG106 strain, and the preservation number is CGMCC No.31655;The functional adjuvant is brassinolide, methyl jasmonate and proline.The present application develops a kind of brand-new microbial preparation, which is with FangSG106 strain as functional strain, and is assisted by three kinds of adjuvants for combination, and research finds that FangSG106 strain has the efficacy of improving the cold resistance of pepper and preventing and treating pepper wilt, and three kinds of adjuvants cooperate with each other, synergistic effect, can further improve the effect of strain in improving the cold resistance of pepper and preventing and treating pepper wilt.

Description

Technical Field

[0001] This invention belongs to the field of agricultural microbial agents technology, and relates to a microbial preparation for improving the cold resistance of chili peppers and preventing chili pepper wilt disease, and its application. Background Technology

[0002] Chili peppers are an important economic crop in my country and are widely cultivated throughout the country. However, they face two major constraints during cultivation, which seriously affect yield and quality: First, the damage caused by soil-borne diseases. Among them, Fusarium wilt is a devastating disease caused by Fusarium oxysporum sp. This fungus can survive in the soil for a long time, infecting plants through the root system, leading to wilting, yellowing of leaves, browning of vascular bundles, and ultimately death of the entire plant, resulting in total crop failure in severe cases. Currently, chemical control easily causes soil pollution and pesticide residues, and the fungus is prone to developing resistance. Second, the impact of low temperature stress. Chili peppers are a warm-loving crop with an optimal growth temperature of 20-30℃. When the temperature is below 10℃, plant growth is inhibited, resulting in leaf curling, wilting, and slow growth. Prolonged low temperatures can lead to cell damage, increased electrolyte leakage, inhibited photosynthesis, and even plant death. Low temperature stress occurs frequently, especially in winter and spring in greenhouse cultivation and in early spring and late autumn in open-field cultivation, causing huge economic losses.

[0003] Existing technologies for the control of Fusarium wilt in peppers mainly include chemical control, agricultural control, and biological control. Chemical control commonly uses fungicides such as hymexazol and carbendazim, which can be effective in the short term, but long-term use can damage the soil microecology, pollute the environment, and easily lead to drug resistance in pathogens. Agricultural control (such as crop rotation and soil disinfection) is cumbersome, time-consuming, and labor-intensive, with limited control effects. Biological control, due to its green, environmentally friendly, and sustainable nature, has become a research hotspot in recent years.

[0004] Current technologies for improving the cold resistance of chili peppers mainly rely on breeding cold-resistant varieties and physical insulation (such as mulching and greenhouse heating). However, breeding cold-resistant varieties is time-consuming and costly; physical insulation is energy-intensive and inconvenient to operate, failing to fundamentally improve the plant's inherent cold resistance. Utilizing microorganisms to induce plant cold resistance is a green, low-cost, and efficient stress-resistance measure, and has become a research hotspot. However, its core lies in screening strains with significant cold resistance induction effects and stable colonization capabilities. Currently, the screening and application of such strains are still lacking, especially multifunctional strains that can simultaneously control diseases and induce cold resistance.

[0005] Therefore, screening out a probiotic strain and its agent that can simultaneously and effectively prevent and control wilt disease in peppers, induce cold resistance in peppers, and is environmentally friendly and easy to apply is of great significance in production practice. Summary of the Invention

[0006] In view of the shortcomings of the prior art, the purpose of this invention is to provide a microbial preparation for improving the cold resistance of chili peppers and preventing wilt disease in chili peppers, and its application.

[0007] To achieve this objective, the present invention adopts the following technical solution:

[0008] In a first aspect, the present invention provides a microbial preparation for enhancing the cold resistance of chili peppers and preventing wilt disease in chili peppers, wherein the active components of the microbial preparation include probiotic agents and functional adjuvants;

[0009] The probiotic is strain FangSG106, classified as Paenibacillus polymyxa, with accession number CGMCC No.31655;

[0010] The functional additives are brassinolide, methyl jasmonate, and proline.

[0011] This invention develops a novel microbial preparation using FangSG106 strain as the functional strain, supplemented with three adjuvants: brassinolide, methyl jasmonate, and proline. Studies have shown that FangSG106 strain enhances both cold resistance and wilt disease control in chili peppers. Furthermore, the three adjuvants work synergistically to further improve the effectiveness of FangSG106 strain in enhancing cold resistance and controlling wilt disease in chili peppers. This microbial preparation simultaneously addresses disease control and cold resistance induction in chili peppers, overcoming two key constraints in chili pepper cultivation and improving both yield and quality.

[0012] Preferably, the probiotic agent is in the form of a solution or a powder.

[0013] Preferably, the probiotic agent is a solution, which is prepared by a method comprising the following steps:

[0014] FangSG106 strain was inoculated into a culture medium and activated and fermented sequentially to obtain fermentation broth.

[0015] Preferably, the probiotic agent is a powder, which is prepared by a method comprising the following steps:

[0016] FangSG106 strain was inoculated into a culture medium and activated and fermented sequentially to obtain fermentation broth; the fermentation broth was centrifuged, mixed with a protectant, and then dried to obtain bacterial powder.

[0017] Preferably, the live bacteria content in the probiotic agent is not less than 1×10⁻⁶. 8 CFU / g or 1×10 8 CFU / mL, for example 1×10 8CFU / mL (CFU / g), 1×10 9 CFU / mL (CFU / g), 1×10 10 CFU / mL (CFU / g), 5×10 10 CFU / mL (CFU / g), 1×10 11 CFU / mL (CFU / g), 5×10 11 CFU / mL (CFU / g), 1×10 12 CFU / mL (CFU / g), etc.; other specific point values ​​within this range can be selected, which will not be elaborated here.

[0018] Preferably, the active components in the microbial preparation include, by weight, 20-30 parts of probiotic inoculum (e.g., 20, 22, 24, 25, 26, 28, 30, etc.) and 5-20 parts of functional adjuvants (e.g., 5, 8, 10, 12, 14, 16, 18, 20, etc.). Other specific values ​​within the above range are optional and will not be elaborated further here.

[0019] Preferably, the mass ratio of brassinolide, methyl jasmonate and proline is (3-6):(3-6):1, wherein the specific values ​​in (3-6) can be 3, 3.5, 4, 4.5, 5, 5.5, 6, etc. Other specific values ​​within this range can be selected, and will not be elaborated here.

[0020] Based on the synergistic relationship between brassinolide, methyl jasmonate, and proline in further enhancing the cold resistance and controlling wilt of peppers by strain FangSG106, the effect is better when they meet the above-mentioned specific quality ratio.

[0021] Preferably, the microbial preparation further contains a nutritional adjuvant selected from any one or a combination of at least two of sucrose, glucose, yeast extract, peptone, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium sulfate, ammonium sulfate, manganese sulfate, ferrous sulfate, or B vitamins.

[0022] In a second aspect, the present invention provides the application of the microbial preparations according to the first aspect in enhancing the cold resistance of chili peppers and / or preventing and controlling chili pepper wilt disease.

[0023] Preferably, the application methods of the microbial preparation include: seed dressing, hole application, drip irrigation, or root irrigation.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] This invention develops a novel microbial preparation using FangSG106 strain as the functional strain, supplemented with three adjuvants: brassinolide, methyl jasmonate, and proline. Studies have shown that FangSG106 strain enhances both cold resistance and wilt disease control in chili peppers. Furthermore, the three adjuvants work synergistically to further improve the effectiveness of FangSG106 strain in enhancing cold resistance and controlling wilt disease in chili peppers. This microbial preparation simultaneously addresses disease control and cold resistance induction in chili peppers, overcoming two key constraints in chili pepper cultivation and improving both yield and quality.

[0026] The strain FangSG106 involved in this invention is classified as Paenibacillus polymyxa, and its depositary is the China General Microbiological Culture Collection Center (CGMCC), with a deposit date of August 12, 2024, accession number CGMCC No. 31655, and address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing. Detailed Implementation

[0027] To further illustrate the technical means and effects of the present invention, the following describes the technical solution of the present invention in conjunction with preferred embodiments of the present invention. However, the present invention is not limited to the scope of the embodiments.

[0028] The strain FangSG106 involved in the following experiments is classified as Paenibacillus polymyxa, with accession number CGMCC No. 31655.

[0029] The pathogenic fungus *Fusarium oxysporum* used in the following experiments was kindly provided by the Beijing Academy of Agricultural and Forestry Sciences.

[0030] Preparation Example 1

[0031] A microbial preparation is provided, and its preparation method is as follows:

[0032] (1) Take Bacillus polymyxa FangSG106 strain and inoculate it into a 500 mL Erlenmeyer flask containing 100 mL LB broth at a ratio of 2% (v / v). Incubate the flask at 30℃ and 220 r / min for 36 h on a shaker. Adjust the viable count of the fermentation broth to 1×10⁻⁶. 8 CFU / mL available for use.

[0033] (2) Mix 25 parts of FangSG106 fermentation broth and 10 parts of functional additives evenly, wherein the functional additives are brassinolide, methyl jasmonate and proline in a mass ratio of 5:4:1 to obtain a microbial preparation.

[0034] Preparation Example 2

[0035] A microbial preparation is provided, and its preparation method is as follows:

[0036] (1) Take Bacillus polymyxa FangSG106 strain and inoculate it into a 500 mL Erlenmeyer flask containing 100 mL LB broth at a ratio of 2% (v / v). Incubate the flask at 30℃ and 220 r / min for 36 h on a shaker. Adjust the viable count of the fermentation broth to 1×10⁻⁶. 8 CFU / mL available for use.

[0037] (2) Mix 30 parts of FangSG106 fermentation broth and 13 parts of functional additives evenly, wherein the functional additives are brassinolide, methyl jasmonate and proline in a mass ratio of 6:6:1 to obtain a microbial preparation.

[0038] Preparation Example 3

[0039] A microbial preparation is provided, and its preparation method is as follows:

[0040] (1) Take Bacillus polymyxa FangSG106 strain and inoculate it into a 500 mL Erlenmeyer flask containing 100 mL LB broth at a ratio of 2% (v / v). Incubate the flask at 30℃ and 220 r / min for 36 h on a shaker. Adjust the viable count of the fermentation broth to 1×10⁻⁶. 8 CFU / mL available for use.

[0041] (2) Mix 20 parts of FangSG106 fermentation broth and 10 parts of functional additives evenly, wherein the functional additives are brassinolide, methyl jasmonate and proline in a mass ratio of 4:5:1 to obtain a microbial preparation.

[0042] Preparation Example 4

[0043] A formulation is provided, the preparation method of which is as follows:

[0044] (1) Take Bacillus polymyxa FangSG106 strain and inoculate it into a 500 mL Erlenmeyer flask containing 100 mL LB broth at a ratio of 2% (v / v). Incubate the flask at 30℃ and 220 r / min for 36 h on a shaker. Adjust the viable count of the fermentation broth to 1×10⁻⁶. 8 CFU / mL available for use.

[0045] (2) Mix 25 parts of FangSG106 fermentation broth and 10 parts of functional additives evenly, wherein the functional additives are methyl jasmonate and proline in a mass ratio of 4:1.

[0046] Preparation Example 5

[0047] A formulation is provided, the preparation method of which is as follows:

[0048] (1) Take Bacillus polymyxa FangSG106 strain and inoculate it into a 500 mL Erlenmeyer flask containing 100 mL LB broth at a ratio of 2% (v / v). Incubate the flask at 30℃ and 220 r / min for 36 h on a shaker. Adjust the viable count of the fermentation broth to 1×10⁻⁶. 8 CFU / mL available for use.

[0049] (2) Mix 25 parts of FangSG106 fermentation broth and 10 parts of functional additives evenly, wherein the functional additives are brassinolide and proline in a mass ratio of 5:1.

[0050] Preparation Example 6

[0051] A formulation is provided, the preparation method of which is as follows:

[0052] (1) Take Bacillus polymyxa FangSG106 strain and inoculate it into a 500 mL Erlenmeyer flask containing 100 mL LB broth at a ratio of 2% (v / v). Incubate the flask at 30℃ and 220 r / min for 36 h on a shaker. Adjust the viable count of the fermentation broth to 1×10⁻⁶. 8 CFU / mL available for use.

[0053] (2) Mix 25 parts of FangSG106 fermentation broth and 10 parts of functional additives evenly, wherein the functional additives are brassinolide and methyl jasmonate in a mass ratio of 5:4.

[0054] Preparation Example 7

[0055] A formulation is provided, the preparation method of which is as follows:

[0056] Bacillus polymyxa strain FangSG106 was inoculated at a ratio of 2% (v / v) into a 500 mL Erlenmeyer flask containing 100 mL of LB broth, and incubated at 30 °C and 220 r / min on a shaker for 36 h. The viable cell count of the fermentation broth was then adjusted to 1 × 10⁻⁶. 8 CFU / mL available for use.

[0057] Preparation Example 8

[0058] A formulation is provided, the preparation method of which is as follows:

[0059] Mix 25 parts water and 10 parts functional additives evenly, wherein the functional additives are brassinolide, methyl jasmonate and proline in a mass ratio of 5:4:1.

[0060] Example 1

[0061] Verification of the control effect of pepper wilt disease:

[0062] (1) Preparation of pathogen: Fusarium oxysporum was cultured on a PDA at 28°C for 5 days. Then, 10 5 mm diameter bacterial blocks were taken using a punch and transferred to a 500 mL Erlenmeyer flask containing 100 mL of PDB medium. The flask was incubated in a shaker at 28°C and 180 r / min for 3 days. The Fusarium oxysporum culture was filtered through four layers of sterile gauze, and the concentration of the filtered spore suspension was adjusted to 10 using a hemocytometer. 6 spores / mL.

[0063] (2) Disinfection: Refer to GB / T3543.4-1995 Crop Seed Inspection Procedure (Germination Test), select plump pepper seeds, soak them in 75% alcohol for 30 seconds, then rinse them with sterile water until there is no alcohol residue; then soak them in 1% sodium hypochlorite solution for 20 minutes, and then rinse them several times with sterile water to complete the seed disinfection.

[0064] (3) Germination: Place sterile filter paper in a petri dish (9cm in diameter), and evenly sprinkle the sterilized chili seeds (25 seeds / dish) on the filter paper in a clean bench. Place the dish in a 30℃ constant temperature incubator to moisturize and germinate.

[0065] (4) Sowing: Select chili seeds that have germinated and grown evenly, and transfer them to sterilized nutrient soil pots (11 cm in diameter, 9 cm in height, and 7.5 cm in bottom diameter). Sow one chili seed in each pot, and then cover the surface with 2 cm of sterilized nutrient soil. When the chili seedlings have grown two true leaves, irrigate the roots.

[0066] (5) Root irrigation: Groups 1-8 were irrigated with 30 mg of the preparation prepared in Examples 1-8 near the roots of pepper seedlings. The blank control group was irrigated with sterile water of the same mass, and the positive control group was irrigated with 2000 times diluted solution of 98% oxamyl soluble powder of the same mass. The total number of plants investigated in each group was 15.

[0067] (6) Pathogen inoculation: 24 h after inoculation of experimental groups 1-8 and the control group, the roots of each seedling were punctured with a sterile needle and inoculated with 30 mL of 1×10⁻⁶ solution. 6 A spore suspension of *Fusarium oxysporum* was prepared at spore density of 1 / mL. The suspension was then cultured in a greenhouse at 25°C, 75% relative humidity, and a photoperiod of 14 hours light / 10 hours darkness to induce disease.

[0068] (7) Disease assessment: After 21 days, the soil around the roots of the pepper seedlings was cleaned, and the disease incidence of plants in different treatment groups was counted. The severity of the disease was assessed, and the incidence rate, disease index and relative control efficacy were calculated using the following formula (Table 1 shows the disease grading standards for pepper wilt).

[0069] Incidence rate = (Number of infected plants / Total number of plants) × 100%

[0070] Disease index = 100% × ∑(number of diseased plants at each level × representative value at each level) / (total number of plants surveyed × 4)

[0071] Relative prevention and control effect = (Control disease index - Treatment disease index) / Control disease index × 100%

[0072] Table 1

[0073]

[0074] The statistical results of the prevention and control effects of each group are shown in Table 2.

[0075] Table 2

[0076]

[0077] As shown in Table 2, compared with the blank control group (sterile water), the FangSG106 fermentation broth of Preparation Example 7, which is the subject of this invention, has excellent efficacy in preventing and controlling wilt of pepper. Furthermore, Preparation Example 1 is more effective in preventing and controlling wilt of pepper than Preparation Example 7 and Preparation Examples 4-6. This indicates that adding three functional adjuvants to the microbial preparation can further enhance the efficacy in preventing and controlling wilt of pepper, and the three functional adjuvants have a synergistic effect in achieving the above-mentioned efficacy.

[0078] Example 2

[0079] Verification of the cold resistance induction effect of chili peppers:

[0080] (1) Disinfection: Refer to GB / T3543.4-1995 Crop Seed Inspection Procedure (Germination Test), select plump pepper seeds, soak them in 75% alcohol for 30 seconds, then rinse them with sterile water until there is no alcohol residue; then soak them in 1% sodium hypochlorite solution for 20 minutes, and then rinse them several times with sterile water to complete the seed disinfection.

[0081] (2) Germination: Place sterile filter paper in a petri dish (9cm in diameter), and evenly sprinkle the sterilized chili seeds (25 seeds / dish) on the filter paper in a clean bench. Place the dish in a 30℃ constant temperature incubator to moisturize and germinate.

[0082] (3) Sowing: Select chili seeds that have germinated and grown evenly, and transfer them to sterilized nutrient soil pots (11 cm in diameter, 9 cm in height, and 7.5 cm in bottom diameter). Sow one chili seed in each pot, and then cover the surface with 2 cm of sterilized nutrient soil. When the chili seedlings have grown two true leaves, irrigate the roots.

[0083] (4) Root irrigation: Groups 1-8 were irrigated with 30 mg of the preparation prepared in Examples 1-8 near the roots of the pepper seedlings. Sterile water of the same quality was used as a blank control group. The total number of plants in each group was 15.

[0084] (5) Chilling stress treatment: Chilling stress was carried out in a 4℃ low-temperature incubation room for 5 days. Light cycle: 40% light intensity light treatment for 10 hours, 0% light intensity darkness treatment for 14 hours.

[0085] (6) Recovery treatment: Seedlings that have been subjected to cold stress in a 5℃ incubator for 5 days are transferred to a room temperature incubator for recovery growth for 7 days. Light cycle: 40% light intensity light treatment for 10 hours, 0% light intensity darkness treatment for 14 hours.

[0086] (7) Assessment of seedling cold resistance: The survival rate of chili seedlings was calculated by statistically analyzing the survival status of each pot of chili seedlings. Survival rate = number of surviving chili seedlings per pot ÷ total number of chili seedlings per pot × 100%.

[0087] (8) Electrolyte permeability assessment: Chili leaf slices treated with different samples were selected, soaked in deionized water for 1 hour, and the initial conductivity value R1 was measured. After being treated with boiling water for 10 minutes, the total conductivity value R2 was measured. Calculation formula: Relative conductivity REC (%) = R1 / R2 × ​​100. The ratio of the two reflects the change in cell membrane permeability. The lower the relative conductivity, the more stable the cell membrane structure and the more effective the osmotic pressure control.

[0088] (9) Evaluation of SOD and CAT activities: Pepper leaves treated with different samples were selected, and the activities of different defense enzymes in the plant leaves were measured using an enzyme activity assay kit. Catalase (CAT) and superoxide dismutase (SOD) were measured using a test kit purchased from Beijing Solarbio Technology Co., Ltd., and the detection methods were in accordance with the kit instructions.

[0089] The statistical results for each group are shown in Table 3.

[0090] Table 3

[0091]

[0092] As shown in Table 3, compared with the blank control group (sterile water), the FangSG106 fermentation broth prepared in Example 7, which is the subject of this invention, has excellent effects in improving the cold resistance of chili peppers. Specifically, it improves the survival rate of chili pepper seedlings under low temperature stress, and the electrolyte permeability is lower than that of the blank control group (sterile water), indicating that the degree of cell membrane damage is significantly reduced. The SOD activity and CAT activity are significantly higher than those of the blank control group (sterile water), indicating that the antioxidant capacity of the plants is significantly improved.

[0093] Furthermore, Preparation Example 1 was superior to Preparation Example 7 and Preparation Examples 4-6 in enhancing the cold resistance of chili peppers, indicating that adding three functional adjuvants to the microbial preparation can further enhance the induction of cold resistance in chili peppers, and the three functional adjuvants have a synergistic effect in exerting the above-mentioned effects.

[0094] The applicant declares that the technical solution of this invention is illustrated by the above embodiments, but this invention is not limited to the above embodiments, that is, it does not mean that this invention must rely on the above embodiments to be implemented. Those skilled in the art should understand that any improvements to this invention, equivalent substitutions of raw materials for the products of this invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of this invention.

[0095] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0096] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

Claims

1. A microbial preparation for enhancing the cold resistance of chili peppers and preventing wilt disease in chili peppers, characterized in that, The active components in the microbial preparation consist of 20-30 parts by mass of probiotic agent and 5-20 parts by mass of functional adjuvant. The probiotic is strain FangSG106, classified as Paenibacillus polymyxa, with accession number CGMCC No.31655; The probiotic agent contains no less than 1×10⁻⁶ live bacteria. 8 CFU / g or 1×10 8 CFU / mL; The functional additives are brassinolide, methyl jasmonate and proline in a mass ratio of (3-6):(3-6):

1.

2. The microbial preparation according to claim 1, characterized in that, The probiotic preparation can be in the form of a solution or a powder.

3. The microbial preparation according to claim 2, characterized in that, The probiotic agent is a solution, which is prepared by a method including the following steps: FangSG106 strain was inoculated into a culture medium and activated and fermented sequentially to obtain fermentation broth.

4. The microbial preparation according to claim 2, characterized in that, The probiotic agent is a powder, which is prepared by a method including the following steps: FangSG106 strain was inoculated into a culture medium and activated and fermented sequentially to obtain fermentation broth; the fermentation broth was centrifuged, mixed with a protectant, and then dried to obtain bacterial powder.

5. The microbial preparation according to claim 1, characterized in that, The microbial preparation also contains a nutritional adjuvant selected from any one or a combination of at least two of the following: sucrose, glucose, yeast extract, peptone, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium sulfate, ammonium sulfate, manganese sulfate, ferrous sulfate, or B vitamins.

6. The application of the microbial preparation according to any one of claims 1-5 in enhancing the cold resistance of chili peppers and / or preventing and controlling chili pepper wilt disease.

7. The application according to claim 6, characterized in that, The application methods of the microbial agents include: seed dressing, hole application, drip irrigation, or root irrigation.

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