Compound microbial agent and application thereof in prevention and control of tomato fusarium wilt and promotion of tomato growth

Abstract

The invention discloses a compound microbial agent and application of the compound microbial agent in prevention and control of tomato fusarium wilt and promotion of tomato growth. The compound microbial agent is prepared from bacillus siamensis, bacillus velezensis and bacillus solidus, and the compound microbial agent is prepared from bacillus siamensis, bacillus velezensis and bacillus solidus. Wherein the ratio of the number of the bacterial strains of the bacillus siamensis to the number of the bacterial strains of the bacillus velezensis to the number of the bacterial strains of the bacillus subtilis to the number of the bacterial strains of the bacillus subtilis to the number of the bacterial strains of the bacillus velezensis to the number of the bacterial strains of the bacillus subtilis is (1.5-2.5) to (0.5-1.5) to (0.5-1.5). The compound microbial agent can prevent and control tomato fusarium wilt, inhibit fusarium oxysporum and promote tomato plant growth.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and specifically relates to a composite microbial agent and application thereof in preventing and controlling tomato wilt and promoting tomato growth. Background Art

[0002] The basic mechanisms of microbial control of crop diseases mainly include competitive interactions, antagonistic activities, quantitative induction inhibition, induction of plant resistance, and promotion of plant growth. A large number of studies have shown that the survival and successful colonization of microorganisms in the plant rhizosphere are crucial for their effective control of crop diseases. Traditional biological control techniques usually involve the formulation of biological control agents into liquid or powder formulations for application, which has been proven to effectively control the reproduction and invasion of pathogenic microorganisms. However, there are some challenges in inoculating biological control agents into plant roots, such as malnutrition, which makes it difficult for them to establish and survive, resulting in unstable and unsatisfactory long-term effects. Therefore, it is necessary to develop new procedures to directly use biological control agents for crop disease management.

[0003] Tomato wilt is a soil-borne fungal disease caused by Fusarium oxysporum f. sp. lycopersici FOL, which poses a serious threat to tomato crops. It is a fungus widely distributed in the soil and mainly harms the vascular bundles of tomato stems. Globally, a variety of economic crops and vegetables may be affected by wilt, affecting the roots of plants. Fusarium oxysporum is spread through the soil and can spread from bottom to top in the vascular bundles of plants. The pathogen can overwinter in the soil and survive for several years. Once infected, it will cause annual disease and is difficult to cure. Recent research has focused on the cultivation of resistant varieties to reduce the occurrence of the disease. Bacillus siamensis can inhibit Fusarium oxysporum by competition and enhance the systemic resistance of plants. Studies have reported transcriptome reprogramming after Fusarium oxysporum infection in tomatoes to reveal the plant's response mechanism to the disease and to find effective prevention and control strategies. These research advances provide new perspectives and methods for the prevention and control of tomato Fusarium wilt, which will help develop more effective management strategies and reduce the impact of the disease on agricultural production. Summary of the invention

[0004] The purpose of this section is to summarize some aspects of embodiments of the present invention and to briefly introduce some preferred embodiments.

[0005] The invention provides a composite microbial agent for preventing and controlling tomato wilt disease, which consists of Bacillus siamensis Bs-9 (Bacillus siamensis), Bacillus velezensis Bv (Bacillus velezensis) and Bacillus firmus Bf (Bacillus firmus).

[0006] Among them, Bacillus siamensis Bs-9 was deposited in the General Microbiology Center of China Culture Collection Administration on June 15, 2022. The deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing. The deposit number is CGMCC No. 25088. It is a publicly available strain.

[0007] Bacillus ve / ezensis was deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on May 16, 2018. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. The deposit number is CGMCC No. 15766. It is a publicly available strain.

[0008] Bacillus firmus Bf (Bacil / us firmus) was purchased, and the strain deposit number is CCTCC AB94028, which is a published strain.

[0009] Preferably, the strain quantity ratio of Bacillus siamensis, Bacillus velezensis and Bacillus firmus is 1.5-2.5:0.5-1.5:0.5-1.5.

[0010] Preferably, the strain quantity ratio of Bacillus siamensis, Bacillus velezensis and Bacillus firmus is 1.9-2.2:1-1.1:0.9-1.

[0011] The composite microbial agent of the present invention has an antagonistic effect on tomato wilt, and the pathogenic bacteria include: Fusarium oxysporum f.sp.lycopersici FOL.

[0012] The beneficial effects of the present invention are as follows: when the composite bacterial agent of the present invention is subjected to plate confrontation, the antibacterial radius of the composite bacterial agent after confronting the wilt pathogen is higher than that of the single bacterial agent. The application of the composite bacterial agent can effectively improve the ability to confront the wilt pathogen and can induce plants to produce resistance.

[0013] When conducting plate confrontation, under the action of the same bacterial solution concentration, the composite bacterial solution composed of Bacillus siamensis Bs-9, Bacillus velezensis Bv and Bacillus firmus Bf in a certain proportion (Bs-9: Bv: Bf = (1.9-2.1): (1.1): (0.9)) can significantly improve the inhibitory effect on Fusarium oxysporum f.sp.lycopersici FOL, and the inhibition radius reaches 11.4 mm, indicating that the composite bacterial agent has a synergistic effect to inhibit the infection of Fusarium oxysporum f.sp.lycopersici FOL.

[0014] The content of IAA (indole-3-acetic acid) and IAA-ASP (indole-3-acetic acid aspartate, IAA-aspartate) per gram of tomato plants treated with single strains and compound strains was higher than that of the control (CK). The IAA and IAA-ASP plant hormones in the seeds of the compound strains were higher than those of the single strains, indicating that the combination of strains can promote the elongation growth of the vegetative organs of the plants, promote cell division and organ construction, inhibit the formation of abscission layers, and promote the formation of xylem and phloem cells. And then confront the negative effects of tomato wilt. IAA-ASP is a derivative of the IAA hormone. Its content is increased at the same time. It may act as an active regulator to regulate plant growth by affecting the release and degradation of IAA; improve the chemical stability of IAA to extend the half-life of IAA and help plants regulate the distribution of IAA at different growth stages. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the technical solution of the embodiment of the present invention, the following briefly introduces the drawings required for describing the embodiment, wherein:

[0016] Figure 1 This is a picture showing the effects of different bacterial solutions against tomato wilt disease.

[0017] Figure 2 is the standard curve of IAA and its derivatives.

[0018] Figure 3 It is the extraction peak of IAA and its derivatives. DETAILED DESCRIPTION

[0019] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with specific embodiments.

[0020] Embodiment 1:

[0021] The plate confrontation of compound fungicide against tomato wilt:

[0022] Colonies of Bacillus siamensis Bs-9, Bacillus velezensis and Bacillus firmus were picked respectively, and the colonies were streaked for purification. After the single colony was purified, it was picked into 200 ml LB medium and cultured at 25°C for 48 hours. The subsequent confrontation experiment was carried out.

[0023] Four groups of bacterial agents were prepared respectively, and the total concentration of strains in each group of bacterial agents was controlled to be 3.0×10 7 CFU / g: (1) Bacillus velezensis Bv group; (2) Bacillus siamensis Bs-9 group; (3) Bacillus firmus Bf group; (4) Composite bacterial agent group: based on the strain quantity ratio, Bacillus siamensis Bs-9: Bacillus velezensis Bv: Bacillus firmus Bf = 2:1.1:0.9.

[0024] The Oxford cup confrontation method was used to inoculate Fusarium oxysporum stored at 20°C onto the PDA plate for activation. After the fungus grew all over the plate, it was evenly beaten into round fungus blocks with a diameter of 5 mm using a sterilized blue pipette tip.

[0025] Prepare a PDA plate with a diameter of 90 mm and uniform thickness in advance, and take two inoculation points on the culture dish at a distance of 3.5 cm from the center of the circle using the cross method. Inoculate the mycelium block at the inoculation point of the PDA culture medium, place an Oxford cup at another inoculation point, and inject 80 μm (1) to (4) group of bacterial liquid respectively, and repeat four times. Culture at 25℃ for 5 days. After the control mycelium has grown all over the plate, use a vernier caliper to measure the radius of the inhibition zone (mm), that is, the distance from the center of the target bacterial colony to the edge of the target bacterial colony.

[0026] Table 1 Plate confrontation results of different bacterial suspensions against tomato wilt

[0027] Group Bacterial liquid ratio Antibacterial radius (mm) (1) Bv 5.7 (2) Bs-9 3.4 (3) Bf 6.2 (4) Compound group 11.4

[0028] like Figure 1 As shown, Figure 1The results of different bacterial solutions against tomato wilt disease show that on the 5th day of culturing Fusarium oxysporum, the target bacterial colony showed a relatively obvious and stable inhibition zone, so the data was measured on the 5th day. Among the three groups of treatments, the lowest (3.4mm) and the largest (11.4mm) inhibition radius were the Bacillus siamensis Bs-9 single bacteria confrontation group and the fourth group of compound bacteria confrontation group Bs-9: Bv: Bf = 2: (1.1): (0.9) group. The antibacterial radius of the mixed bacterial liquid group was significantly higher than that of the single bacteria confrontation group of Bv, Bf and Bs-9. The antibacterial effect of the mixed bacterial agent group of Bacillus Velez, Bacillus siamense and Bacillus firmus was significantly improved, indicating that the mixed bacterial agent of Bacillus Velez, Bacillus siamense and Bacillus firmus played a synergistic role in inhibiting Fusarium oxysporumf.sp.lycopersiciFOL. Figure 1 Among them, (a) Bv: single bacteria confrontation of Bacillus velezensis (group (1)); (b) Bs-9: single bacteria confrontation of Bacillus siamensis (group (2)); (c) single bacteria confrontation of Bacillus firmus (group (3)); (d) mixed bacteria group of Bacillus velezensis, Bacillus siamensis and Bacillus firmus (group (4)).

[0029] Embodiment 2:

[0030] Determination of endogenous hormone content in tomato plants with compound fungi:

[0031] The experimental groups were: (1) clean water (control group); (2) Bacillus siamensis Bs-9 single bacterial solution, with a concentration of 3.0×10 7 CFU / g; (3) Compound bacterial agent group: based on the strain number ratio, Bacillus siamensis Bs-9: Bacillus velezensis Bv: Bacillus firmus Bf were mixed at a ratio of 2:1.1:0.9, with a total bacterial solution concentration of 3.0×10 7 CFU / g.

[0032] Tomato plants with similar plant height and the same growth status were divided into three groups. Each group of plants was sprayed with the bacterial agent of each experimental group once every three days, with a total amount of 100 mL of bacterial solution each time. After two sprayings, samples were taken for testing at intervals of three days.

[0033] Determination of endogenous hormone content: Grind all samples in liquid nitrogen until they are crushed, accurately weigh the samples in a test tube and add 10 mL of acetonitrile solution, and add 8 μL of internal standard stock solution; extract at 4°C overnight, centrifuge at 4°C, 12000g for 5 min, and take the supernatant.

[0034] The precipitate was added with 5 mL of acetonitrile solution again, extracted twice, the supernatant obtained was combined, and appropriate amounts of C18 and GCB were added to purify the impurities, centrifuged at 4°C and 12000g for 5 min, and the supernatant was taken. It was blown dry with nitrogen, re-dissolved with 400 μl of methanol, filtered through a 0.22 μm organic phase filter, and placed in a -20°C refrigerator for detection.

[0035] A high performance liquid chromatography tandem mass spectrometry (HPLC-MS / MS) scheme was used, wherein the liquid phase conditions were: chromatographic column: poroshell 120SB-C18 reverse phase chromatographic column (2.1×150, 2.7 μm); column temperature: 30° C.; mobile phase: A:B=(water / 0.02% formic acid):(chromatographic methanol); injection volume: 2 μl; elution gradient was as follows:

[0036] Table 2H PLC elution gradient parameters

[0037] Time (min) Flow rate (mL / min) A％ 0-1 0.3 95 1-9 0.3 Decrease from 95 to 40 9-11 0.3 Decrease from 40 to 5 11-13 0.3 5 13-13.2 0.3 Increase from 5 to 95 13.2-15 0.3 95

[0038] Mass spectrometry parameters: ionization mode: ES1 positive and negative ion mode monitoring respectively; scan type: M RM; curtain gas: 15psi; spray voltage: +5500v, -5000V; nebulizing gas pressure: 65psi; auxiliary gas pressure: 70psi; nebulizing temperature: 300℃.

[0039] Table 3 Selected reaction monitoring conditions for protonation or deprotonation of plant hormones ([M+H]+ or [MH]-)

[0040]

[0041] Table 4 Plant hormone detection concentrations under different treatments

[0042]

[0043] The content of plant hormones per gram of sample can be obtained by multiplying the fixed volume by the detection concentration. The content of plant hormones per gram of sample is calculated to obtain the following table:

[0044] Table 5 Plant hormone content per gram sample under different treatments

[0045]

[0046] The results showed that the content of IAA per gram of sample in tomato plants treated with bacterial solution was higher than that in the control group. The content of IAA and IAA-ASP per gram of sample in tomato plants treated with compound bacterial solution was higher than that in the experimental group treated with single bacterial solution Bs-9.

[0047] Based on the above results, it can be further inferred that the combination treatment of strains will promote the elongation growth of the plant's nutritional organs, promote cell division and organ construction, inhibit the formation of abscission layer, promote the formation of xylem and phloem cells, etc., thereby counteracting the negative effects of tomato wilt disease and improving the plant's root growth ability.

[0048] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. A composite microbial agent, characterized in that: The composite microbial agent is composed of Bacillus siamensis, Bacillus velezensis and Bacillus firmus; The ratio of the number of strains of Bacillus siamensis, Bacillus velezensis and Bacillus firmus is 1.5-2.5:0.5-1.5:0.5-1.

5.

2. The composite microbial agent according to claim 1, characterized in that: The strain quantity ratio of Bacillus siamensis, Bacillus velezensis and Bacillus firmus is 1.9-2.2:1-1.1:0.9-1.

3. The composite microbial agent according to claim 1 or 2, characterized in that: The Bacillus siamensis includes Bacillus siamensis Bs-9.

4. The composite microbial agent according to claim 1 or 2, characterized in that: The Bacillus velezensis includes Bacillus velezensis Bv.

5. The composite microbial agent according to claim 1 or 2, characterized in that: The Bacillus firmus includes Bacillus firmus Bf.

6. Use of the composite microbial agent according to claim 1 in preventing and controlling tomato wilt disease.

7. The use according to claim 6, characterized in that: The composite microbial agent can inhibit Fusarium oxysporumf.sp.lycopersici FOL.

8. Use of the composite microbial agent according to claim 1 in promoting tomato growth.

9. The use according to claim 8, characterized in that: The composite microbial agent can increase the indole-3-acetic acid and indole-3-acetic acid aspartic acid contents of tomato plants.

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