Synthetic bacterial flora co-culture fermentation broth, bacterial agent and application thereof

Abstract

This invention relates to the field of agricultural biological control technology, specifically to a synthetic microbial co-culture fermentation broth, its inoculum, and its application. The synthetic microbial co-culture fermentation broth is prepared by sequential co-inoculation fermentation of *Trichoderma harzianum* TW21990, *Burkholderia viminalis* P418, and *Bacillus atrophicus* BsR05; wherein *Trichoderma harzianum* TW21990 has the CGMCC No. 12864, *Burkholderia viminalis* P418 has the CGMCC No. 1212, and *Bacillus atrophicus* BsR05 has the CGMCC No. 11665. Compared to single biocontrol strains, the antibacterial and growth-promoting secondary metabolites produced by the above-mentioned synthetic microbial co-culture fermentation are broad-spectrum, making it less likely to induce drug resistance in the control of gray mold in leeks. The control effect is stable, there is no antagonism within the synthetic microbial community, and the efficacy of the strains is synergistically enhanced, with a significant growth-promoting effect on leeks.

Description

Technical Field

[0001] This invention relates to the field of agricultural biological control technology, specifically to a synthetic microbial co-culture fermentation broth, its inoculant, and its application. Background Technology

[0002] Gray mold has become one of the major diseases restricting the development of the leek industry. Gray mold in leeks is caused by the genus *Botrytis* (…). Botrytis Gray mold is caused by pathogens (Botrytis cinerea, Botrytis sinensis, and Botrytis cinerea), which can cause rotting of chive leaves. Currently, pathogens found to cause gray mold in chives include Botrytis cinerea, Botrytis chinensis, and Botrytis cinerea. Control of gray mold is primarily based on chemical methods, using fungicides such as iprodione, pyrimethanil, prochloraz, boscalid, and fludioxonil, applied through fumigation or spraying. However, chemical control presents environmental problems such as pesticide residues and soil pollution. Biological control, on the other hand, has become a research hotspot due to its green and environmentally friendly nature, broad application prospects, and low cost.

[0003] Syncoms are a promising tool for fully utilizing the beneficial functions of the entire bacterial community. They are an effective means of understanding microbe-plant interactions, combining multiple microorganisms with biocontrol potential to create synergistic effects and enhance biocontrol efficacy. Syncoms can also more comprehensively regulate soil microbial composition, increasing the stability of soil ecosystems and plant health. However, the complexity of reconstructed synthetic microbial communities often limits their application in sustainable agriculture. Furthermore, since pathogens may develop multidrug resistance, constructing synthetic communities can reduce the risk of target pathogens developing resistance to single biocontrol agents, improving the persistence and stability of biocontrol.

[0004] Therefore, it is necessary to utilize synthetic microbial community technology to develop highly effective microbial agents for the prevention and control of gray mold in chives, breaking through the limitations of single biocontrol agents and providing microbial agents that can provide chives with a variety of plant hormones and antifungal compounds. Summary of the Invention

[0005] To address the technical problems of single biocontrol agents for controlling gray mold in chives easily developing resistance and having insufficient long-lasting biological control effects, this invention provides a synthetic microbial co-culture fermentation broth composed of Trichoderma, Burkholderia, and Bacillus, along with its inoculum and applications. Compared to single biocontrol strains, the synthetic microbial co-culture fermentation produces a wide range of antibacterial and growth-promoting secondary metabolites, making it less likely to induce drug resistance in controlling gray mold in chives and exhibiting significant efficacy. Compared to traditional "compound microbial inoculum" products obtained by mixing single-strain cultures, the synthetic microbial co-culture fermentation broth / inoculum of this invention does not exhibit antagonism among the strains; rather, the effects of the strains are synergistically enhanced. The preparation process is also simpler, making it highly valuable for application.

[0006] The technical solution of this invention is as follows: In a first aspect, the present invention provides a synthetic microbial co-culture fermentation broth, composed of Trichoderma harzianum (… Trichoderma harzianum TW21990, Burkholderia viminalis ( Burkholderia vietnamiensis P418 and Bacillus atrophus ( Bacillus atrophaeus It was prepared by co-culturing and fermenting BsR05; Among them, *Trichoderma harzianum* TW21990 was deposited on September 1, 2016, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 12864, and classified as *Trichoderma harzianum*. Trichoderma harzianum ; Burkholderia viviparus P418 was deposited on August 30, 2004, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 1212, and classified as Burkholderia viviparus. Burkholderia vietnamiensis ; Bacillus atrophicus BsR05 was deposited on November 16, 2015, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 11665. The classification name is... Bacillus atrophaeus .

[0007] Furthermore, Trichoderma harzianum TW21990, Burkholderia viminalis P418, and Bacillus atrophus BsR05 were inoculated into MKB fermentation medium at a viable count ratio of 1~2:1~2:1~2 and co-cultured for fermentation.

[0008] Furthermore, the co-culture fermentation conditions are: shaking at 180-200 rpm at 28-30℃ for 5-7 days.

[0009] Furthermore, the concentration was 1.0 × 10 8 CFU / mL Trichoderma harzianum TW21990 seed culture and a concentration of 1.0 × 10⁻⁶ CFU / mL 8 CFU / mL Burkholderia viminalis P418 seed culture was inoculated onto MKB medium at a volume percentage of 10% and cultured for 24–48 h, followed by inoculation at a concentration of 1.0 × 10⁻⁶. 8 Co-culture with Bacillus atrophus BsR05 at cfu / mL.

[0010] Furthermore, the co-culture fermentation broth of the synthetic microbial community includes the fermentation cells, fermentation supernatant, and the resulting metabolites. The metabolites include the following major secondary metabolites: cyclic (L-pro-L-va) dipeptide, cyclic (L-pro-L-tyrosine) dipeptide, indoleacetic acid (IAA), DL-indole-3-lactic acid (ILA), butyric acid, 4-aminobutyric acid (GABA), 2-hydroxycinnamic acid, stachydrine, betaine, bafloxacin B1, 1-deoxynojirimycin, and styraxin.

[0011] Secondly, the present invention provides a synthetic microbial agent composed of Trichoderma, Burkholderia, and Bacillus, prepared by the following method: The above-mentioned synthetic microbial co-culture fermentation broth is adsorbed using a carrier to form a solid microbial agent. The solid microbial agent is then mixed with a binder to obtain the synthetic microbial agent.

[0012] Furthermore, the carrier is selected from diatomaceous earth, maifanite, or peat moss, and the mass ratio of fermentation broth to carrier is 4~5:1.

[0013] Furthermore, the binder is selected from sodium carboxymethyl cellulose and / or sodium alginate, and the mass ratio of solid bacterial agent to binder is 50:3~5.

[0014] Thirdly, the present invention also provides an application of the above-mentioned synthetic microbial co-culture fermentation broth and / or synthetic microbial agent, for the prevention and control of gray mold in leeks and the promotion of leek growth and the increase of leek yield.

[0015] The beneficial effects of this invention are as follows: 1. This invention provides a synthetic microbial co-culture fermentation broth and a synthetic microbial agent containing *Trichoderma harzianum* TW21990, *Burkholderia viminalis* P418, and *Bacillus atrophicus* BsR05, which are sequentially inoculated and co-cultured. Compared with single-strain culture, this method increases the production of secondary metabolites that inhibit pathogenic fungi and promote plant growth. Among these, cyclic (L-pro-L-va) dipeptide, cyclic (L-pro-L-tyrosine) dipeptide, stachydrine, betaine, bafloxacin B1, 1-deoxynojirimycin, and plasminogen sulfadiazine exhibit strong inhibitory effects against *Botrytis cinerea*, thereby achieving a stable control of gray mold in leeks.

[0016] 2. The Trichoderma harzianum TW21990, Burkholderia viminalis P418, and Bacillus atrophicus BsR05 provided by this invention all have rhizosphere growth-promoting effects. They can colonize and occupy locations in the roots, inside the plant, or in the soil ecosystem, and direct the secretion of secondary metabolites such as indoleacetic acid, DL-indole-3-lactic acid, and 4-aminobutyric acid in the plant rhizosphere. These can induce systemic defense responses and disease resistance in leeks, and have a synergistic nutritional effect, thereby improving the microecological environment of the soil for leek cultivation, improving soil structure and physicochemical properties, and increasing the biological yield and quality of leeks.

[0017] 3. Compared to most "compound microbial agents" obtained by single-strain culture and subsequent mixing, the *Trichoderma harzianum* TW21990, *Burkholderia viminalis* P418, and *Bacillus atrophicus* BsR05 in this synthetic microbial agent can coexist symbiotically, with synergistic effects. There is no antagonism or inhibition between the strains, nor any impact on their individual growth. Furthermore, the preparation process of this synthetic microbial agent is cost-effective, the fermentation method is stable, and the application is simple, making it an environmentally friendly product. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, for those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 These are the results of single and co-culture of Trichoderma harzianum TW21990, Burkholderia viminalis P418, and Bacillus atrophicus BsR05 on PDA plates; where A is the single culture result of TW21990, B is the single culture result of P418, C is the single culture result of BsR05, D is the co-culture result of TW21990 and P418, E is the co-culture result of TW21990 and BsR05, F is the co-culture result of P418 and BsR05, and G is the co-culture result of TW21990, P418, and BsR05.

[0020] Figure 2 The results show the plate inhibition of *Botrytis cinerea* on single-cell fermentation filtrates and co-culture fermentation filtrates of *Trichoderma harzianum* TW21990, *Burkholderia viminalis* P418, and *Bacillus atrophicus* BsR05. A represents the inhibition of *Botrytis cinerea* on single-cell fermentation filtrate of the synthetic microorganisms; B represents the inhibition of *Botrytis cinerea* on single-cell fermentation filtrate of TW21990; C represents the inhibition of *Botrytis cinerea* on single-cell fermentation filtrate of P418; D represents the inhibition of *Botrytis cinerea* on single-cell fermentation filtrate of BsR05; and E represents the blank plate control for *Botrytis cinerea*. Detailed Implementation

[0021] To enable those skilled in the art to better understand the technical solutions of this invention, the technical solutions of the embodiments of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of this invention.

[0022] The *Trichoderma harzianum* TW21990 used in this invention was deposited on July 19, 2013, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 12864, and classified as *Trichoderma harzianum*. Trichoderma harzianum This strain has been disclosed in Chinese invention patent ZL202010256670.5.

[0023] Burkholderia viviparus P418 was deposited on August 30, 2004, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 1212, and classified as Burkholderia viviparus. Burkholderia vietnamiensis This strain has been disclosed in Chinese invention patent ZL202411570586.5.

[0024] Bacillus atrophicus BsR05 was deposited on November 16, 2015, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 11665. The classification name is... Bacillus atrophaeus This strain has been disclosed in Chinese invention patent ZL201610056373.X.

[0025] All culture media used in this invention can be obtained using in-house methods or by purchasing commercially available culture media products. PDA culture medium consists of 20% peeled potato, 2% glucose, 1.5% agar powder, and 1L distilled water, with a natural pH. TY culture medium consists of 1% tryptone, 0.5% yeast extract, 0.02% calcium chloride, 1.5% agar powder, and 1L distilled water, with a pH of 7.2-7.4. TY liquid culture medium consists of 1% tryptone, 0.5% yeast extract, 0.02% calcium chloride, and 1L distilled water, with a pH of 7.2-7.4. LB culture medium consists of 1% tryptone, 0.5% yeast extract, 1% sodium chloride, 1.5% agar powder, and 1L distilled water, with a pH of 7.0±0.1. LB liquid culture medium consists of 1% tryptone, 0.5% yeast extract, 1% sodium chloride, and 1L distilled water, with a pH of 7.0±0.1. The MKB fermentation medium consists of 2% casein hydrolysate, 1% glycerol, 0.15% dipotassium hydrogen phosphate, 0.15% magnesium sulfate, 1L distilled water, and pH 7.2±0.2.

[0026] Example 1: Pre-evaluation of strain co-culture To assess the interactions among different biocontrol microorganisms, *Trichoderma harzianum* TW21990, *Burkholderia viminalis* P418, and *Bacillus atrophicus* BsR05 were cultured as single organisms, in pairs, and in three groups on PDA plates. The results are shown in the table below. Figure 1 By observing colony morphology, growth rate, and mutual inhibition or coexistence among colonies, the antagonistic or synergistic effects between strains can be analyzed.

[0027] like Figure 1 As shown in Figure A, when TW21990 single bacteria were cultured alone on a PDA plate, the colonies grew rapidly, with dense hyphae and a yellowish-green color; as... Figure 1 As shown in B, single-cell cultures of P418 form smooth, semi-transparent colonies; as... Figure 1 As shown in C, BsR05 single-cell culture shows smooth, dry, milky white and opaque colonies.

[0028] Under co-culture conditions, TW21990 and P418 ( Figure 1 D) No obvious inhibition zone was observed during co-culture. TW21990 showed a strong growth advantage, while the edge morphology of the P418 streaked colony remained unchanged. TW21990 mycelia covered the P418 colony and continued to grow, indicating good affinity between them. TW21990 and BsR05 ( Figure 1 E) During co-cultivation, the colony morphology of both remained unchanged. TW21990 hyphae could cover the BsR05 colony, but failed to continue spreading outwards, indicating that their affinity was slightly weaker than that of TW21990 and P418; P418 and BsR05 were co-cultured ( Figure 1 When F), both can fuse and grow simultaneously when lines are drawn on the plate, and no inhibition phenomenon occurs, indicating that they have good affinity.

[0029] In a three-strain co-culture system, such as Figure 1 As shown in G, the colony morphology of the three colonies remained unchanged. The hyphae of TW21990 covered the P418 and BsR05 colonies, which grew to cover the entire plate. The upper half of the P418 colony was almost completely covered, with only the lower part of the plate visible. The BsR05 colony grew normally in the horizontal direction, indicating that there was no antagonistic effect among the three and that they had good affinity.

[0030] The above results indicate that when TW21990 is co-cultured with P418 and BsR05, the three bacteria grow in a balanced manner, providing experimental basis for the subsequent construction of composite biocontrol bacteria and their combined application.

[0031] Example 2: Preparation of co-culture fermentation broth and fermentation filtrate of synthetic microbial communities (1) Activation of strain: Trichoderma harzianum TW21990 was inoculated onto PDA medium and placed in a constant temperature incubator at 28℃ for 5 days for activation.

[0032] Select Burkholderia viminalis P418 colonies and streak them on TY medium. Incubate at 30°C in the dark until single colonies grow.

[0033] Select Bacillus atrophus BsR05 colonies and streak them on LB medium. Incubate at 30°C in the dark until single colonies grow.

[0034] (2) Seed liquid preparation: Spores of Trichoderma harzianum TW21990 were scraped with sterile water and diluted to 1.0 × 10⁻⁶. 8 cfu / mL, as seed solution A.

[0035] Single colonies of Burkholderia viminalis P418 were transferred to TY liquid medium and cultured in a shaker at 30°C and 180 rpm until the logarithmic growth phase. OD 660 The value is approximately 0.6~0.8, and the concentration is 1.0×10. 8 cfu / mL, as seed solution B.

[0036] Single colonies of Bacillus atrophus BsR05 were transferred to LB liquid medium and cultured in a shaker at 30°C and 180 rpm until the logarithmic growth phase. OD 660 The value is approximately 0.6~0.8, and the concentration is 1.0×10. 8 cfu / mL, as seed solution C.

[0037] (3) Co-culture fermentation: Seed liquid A and seed liquid B obtained in step (2) were inoculated into MKB fermentation medium at an inoculation amount of 10% (volume percentage), and co-cultured at 28~30℃ and 180~200rpm for 2 days. Then, seed liquid C was inoculated into each of them and co-cultured for 6 days to obtain the co-culture fermentation broth of synthetic microorganisms. The co-culture fermentation broth of synthetic microorganisms contains fermentation cells of Trichoderma harzianum TW21990, Burkholderia viminalis P418 and Bacillus atrophicus BsR05, fermentation supernatant and their metabolites.

[0038] (4) Filtration and sterilization: The co-culture fermentation broth of synthetic microorganisms is filtered and sterilized using a 0.22μm microporous membrane to obtain the co-culture fermentation filtrate of synthetic microorganisms; the co-culture fermentation filtrate of synthetic microorganisms contains the fermentation supernatant of co-culture and the generated metabolites and other components.

[0039] Simultaneously, seed solutions A, B, and C were inoculated into three MKB fermentation media at an inoculation rate of 10% (volume percentage) for single-cell culture. The fermentation conditions were the same as those in step (3) for co-culture fermentation: 28-30℃, 180-200 rpm shaking, and co-cultured for 7 days to obtain TW21990, P418, and BsR05 single-cell fermentation broths. The TW21990, P418, and BsR05 single-cell fermentation broths were filtered through a 0.22 μm microporous membrane to remove bacteria, yielding TW21990, P418, and BsR05 single-cell fermentation filtrates, respectively.

[0040] (5) Analysis and identification of metabolites from co-culture of synthetic microorganisms: The co-culture fermentation filtrate of synthetic microorganisms, the fermentation filtrate of TW21990 single strain, the fermentation filtrate of P418 single strain and the fermentation filtrate of BsR05 single strain were freeze-dried in a freeze dryer, 100 μL of 80% methanol aqueous solution was added, the mixture was vortexed for 30 s, and then allowed to stand in an ice bath for 5 min. After that, it was centrifuged at 4℃ and 12000 rpm for 15 min. The supernatant was taken and diluted with mass spectrometry grade water to a methanol content of 53%. It was centrifuged at 4℃ and 12000 rpm for 15 min. The supernatant sample was taken for LC-MS analysis and identification.

[0041] The differential metabolites produced by co-culture fermentation of synthetic colonies are shown in Table 1 below. Compared with the fermentation broth of TW21990 single strain, co-culture fermentation produced substances not found in TW21990 single strain fermentation, such as cyclic (L-pro-L-va) dipeptide, cyclic (L-pro-L-tyrosine) dipeptide, 1-deoxynojirimycin, and plasmin, and significantly increased the content of substances such as indoleacetic acid, DL-indole-3-lactic acid, and bafloxacin B1. Compared with the fermentation broth of P418 single strain, co-culture fermentation produced substances not found in P418 single strain fermentation, such as indoleacetic acid, DL-indole-3-lactic acid, stachydrine, betaine, 1-deoxynojirimycin, and plasmin, and significantly increased the content of substances such as cyclic (L-pro-L-va) dipeptide, cyclic (L-pro-L-tyrosine) dipeptide, 4-aminobutyric acid, and 2-hydroxycinnamic acid. Compared with the BsR05 single-strain fermentation broth, co-culture fermentation produced substances not found in BsR05 single-strain fermentation, such as cyclic (L-pro-L-va) dipeptide, cyclic (L-pro-L-tyrosine) dipeptide, indoleacetic acid, DL-indole-3-lactic acid (ILA), and bafloxacin B1, and significantly increased the content of butyric acid, stachydrine, betaine, 1-deoxynojirimycin, and acetophenone. Among these, indoleacetic acid, DL-indole-3-lactic acid, and 4-aminobutyric acid have plant growth-promoting effects, while 2-hydroxycinnamic acid, bafloxacin B1, 1-deoxynojirimycin, and acetophenone have antifungal effects, laying the foundation for the development and application of synthetic microbial agents.

[0042] Table 1. Differential metabolites in the co-culture fermentation broth of synthetic microorganisms

[0043] (6) Determination of the inhibitory and killing effects of the metabolites from the co-culture of synthetic microorganisms on Botrytis cinerea: The fermentation filtrates of the synthetic microbial co-culture, TW21990 single-cell fermentation filtrate, P418 single-cell fermentation filtrate, and BsR05 single-cell fermentation filtrate were mixed with PDA medium at 10% by volume and poured into plates as three treatment groups. PDA medium supplemented with 10% sterile water served as a blank control. Botrytis cinerea cakes were inoculated in the center of each plate, and the plates were incubated at 28°C for 5 days. Results are as follows: Figure 1 As shown.

[0044] The distance between the center and edge hyphae of *Botrytis cinerea* colonies was measured using the cross-cross method, and the inhibition rate against *Botrytis cinerea* was calculated using the following formula:

[0045] Among them, L 对照组 The hyphal length of the blank plate control; L 处理组 The mycelial length of the treatment group is shown.

[0046] Calculations showed that the co-culture fermentation filtrate of the synthetic microorganisms exhibited a 99.50% inhibition rate against Botrytis cinerea, significantly higher than the 55.59% of the TW21990 single-strain fermentation filtrate, the 25.05% of the P418 single-strain fermentation filtrate, and the 66.20% of the BsR05 single-strain fermentation filtrate. This indicates that under the co-culture fermentation mode, Trichoderma harzianum TW21990, Burkholderia viminalis P418, and Bacillus atrophicus BsR05 in the synthetic microorganisms can produce more secondary metabolites with antibacterial effects, thereby enhancing their antagonistic activity against Botrytis cinerea.

[0047] Example 3 Preparation of Synthetic Microbial Inoculants Using diatomaceous earth as a carrier, the co-culture fermentation broth of the synthetic microbial community from Example 2 was adsorbed. The mass ratio of the co-culture fermentation broth to diatomaceous earth was 5:1, forming a solid microbial agent. 4 wt% sodium carboxymethyl cellulose and 4 wt% sodium alginate were added to the solid microbial agent, and the mixture was stirred to obtain synthetic microbial community agent A. Synthetic microbial community agent A contains fermentation cells of co-cultured Trichoderma harzianum TW21990, Burkholderia viminalis P418, and Bacillus atrophicus BsR05, fermentation supernatant, and their metabolites.

[0048] Using peat moss as a carrier, the co-culture fermentation broth of the synthetic microbial community from Example 2 was adsorbed. The mass ratio of the co-culture fermentation filtrate to peat moss was 4:1, forming a solid microbial agent. 6 wt% sodium carboxymethyl cellulose was added to the solid microbial agent, and the mixture was stirred to obtain synthetic microbial community agent B. Synthetic microbial community agent B contains fermentation cells of *Trichoderma harzianum* TW21990, *Burkholderia viminalis* P418, and *Bacillus atrophicus* BsR05, fermentation supernatant, and their metabolites.

[0049] Using maifanite as a carrier, the co-culture fermentation broth of the synthetic microbial community from Example 2 was adsorbed. The mass ratio of the co-culture fermentation filtrate to maifanite was 4.5:1, forming a solid microbial agent. 10 wt% sodium alginate was added to the solid microbial agent, and the mixture was stirred to obtain synthetic microbial community agent C. Synthetic microbial community agent C contains fermentation cells of *Trichoderma harzianum* TW21990, *Burkholderia viminalis* P418, and *Bacillus atrophicus* BsR05, fermentation supernatant, and their metabolites.

[0050] Example 4: Application of synthetic microbial inoculants in the control of gray mold in leeks Experimental location: Shagou Town, Yishui County, Linyi City, Shandong Province, a leek-growing area where gray mold of leeks has occurred in previous years, with an experimental area of ​​90 square meters. The row and hole spacing of leeks was 25cm×20cm, with 25-30 plants per hole. The cultivation conditions (soil, fertilizer, tillage, and plant spacing) of all experimental plots were uniform.

[0051] The crop tested was Shagou leeks.

[0052] The experiment consisted of six treatment groups: a blank control group, a pyrimethanil treatment group (positive control), a TW21990 treatment group, a P418 treatment group, a BsR05 treatment group, and a synthetic microbial agent treatment group. The blank control group received no intervention. The other groups received the following treatments: Pyrimethanil treatment group: 40% pyrimethanil suspension was applied according to the field efficacy test dose of 100g / mu.

[0053] TW21990 treatment group: Trichoderma harzianum TW21990 single-strain agent was applied at a dosage of 100g / mu.

[0054] P418 treatment group: Apply Burkholderia vivax P418 single-strain agent at a dosage of 100g / acre.

[0055] BsR05 treatment group: Apply Bacillus atrophicus BsR05 single-strain agent at a dosage of 100g / mu.

[0056] Synthetic microbial agent treatment group: Synthetic microbial agent A from Example 2 was applied at a dosage of 100g / acre.

[0057] The preparation methods for Trichoderma harzianum TW21990 single-strain agent, Burkholderia viminalis P418 single-strain agent, and Bacillus atrophicus BsR05 single-strain agent are as follows: The single-strain fermentation broth is mixed with diatomaceous earth at a mass ratio of 5:1 to form a solid inoculum. 4 wt% sodium carboxymethyl cellulose and 4 wt% sodium alginate are added to the solid inoculum and then mixed with a mixer to obtain the single-strain agent of the corresponding strain.

[0058] The first application of pesticide was carried out when the average height of the chive plants was about 10cm, followed by a second application 10 days later. A survey was conducted 10 days after the second spraying. A 5-point sampling method was used, with 5 points surveyed per plot, and 20-25 leaves examined at each point. The severity of gray mold on the leaves was graded. Grading standards: Grade 0, no lesions; Grade 1, lesions covering less than 5% of the total leaf area; Grade 3, lesions covering 6%-10% of the total leaf area; Grade 5, lesions covering 11%-20% of the total leaf area; Grade 7, lesions covering 21%-50% of the total leaf area; Grade 9, lesions covering more than 50% of the total leaf area.

[0059] Calculate the disease index and prevention and control effectiveness using the following formula:

[0060]

[0061] Table 2 Statistical Table of Chive Field Plot Experiment

[0062] The field control results of gray mold in leeks and the growth of leeks are shown in Table 2 above. It can be seen that the synthetic microbial agent of this invention can utilize Trichoderma harzianum TW21990, Burkholderia viminalis P418 and Bacillus atrophicus BsR05 to construct a micro-ecological balance in the soil. The antibacterial substances produced by the symbiosis of the three can also antagonize the growth of pathogens and effectively reduce the incidence of gray mold in leeks. Its control effect on gray mold in leeks reaches 93.65%, which is higher than the 90.32% of the single treatment of Trichoderma harzianum TW21990 and the 88.72% of the single treatment of Bacillus atrophicus BsR05, and significantly higher than the 76.64% of the treatment of the chemical pesticide pyrimethanil. Furthermore, the application of synthetic microbial agents significantly improved various growth indicators of chives. Plant height and yield per plant showed significant differences compared to the control group (p<0.05). The promoting effect on plant height and yield per plant was 18.45% and 25.78% higher than the control group, respectively, and higher than the single-strain treatment of *Trichoderma harzianum* TW21990 (13.61% and 16.44%, respectively) and the single-strain treatment of *Bacillus atrophus* BsR05 (9.39% and 12.03%, respectively), and significantly higher than the treatment with pyrimethanil (7.59% and 10.22%, respectively). This indicates that the application of synthetic microbial agents can not only effectively control gray mold in chives, but also significantly promote yield, thereby reducing the use of pesticides and fertilizers, demonstrating significant application value and economic and social benefits.

[0063] Although the present invention has been described in detail with reference to the accompanying drawings and preferred embodiments, the present invention is not limited thereto. Various equivalent modifications or substitutions can be made to the embodiments of the present invention by those skilled in the art without departing from the spirit and essence of the invention, and such modifications or substitutions should all be within the scope of the present invention. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should also be covered within the protection scope of the present invention.

Claims

1. A synthetic microbial co-culture fermentation broth, characterized in that, Trichoderma harzianum ( Trichoderma harzianum TW21990, Burkholderia viminalis ( Burkholderia vietnamiensis P418 and Bacillus atrophus ( Bacillus atrophaeus It was obtained through co-cultivation and fermentation; Among them, Trichoderma harzianum TW21990 was deposited at the China General Microbiological Culture Collection Center on September 1, 2016, at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 12864. Burkholderia viviparus P418 was deposited on August 30, 2004, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 1212. Bacillus atrophus BsR05 was deposited on November 16, 2015, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 11665.

2. The synthetic microbial co-culture fermentation broth as described in claim 1, characterized in that, Trichoderma harzianum TW21990, Burkholderia viminalis P418 and Bacillus atrophus BsR05 were inoculated into MKB fermentation medium in a live count ratio of 1~2:1~2:1~2.

3. The synthetic microbial co-culture fermentation broth as described in claim 1, characterized in that, The culture conditions for co-culture fermentation are: shaking at 180-200 rpm at 28-30℃ for 5-7 days.

4. The synthetic microbial co-culture fermentation broth as described in claim 2, characterized in that, The concentration is 1.0 × 10 8 CFU / mL Trichoderma harzianum TW21990 seed culture and a concentration of 1.0 × 10⁻⁶ CFU / mL 8 CFU / mL Burkholderia viminalis P418 seed culture was inoculated onto MKB medium at a volume percentage of 10% and cultured for 24–48 h, followed by inoculation at a concentration of 1.0 × 10⁻⁶. 8 Co-culture with Bacillus atrophus BsR05 at cfu / mL.

5. The synthetic microbial co-culture fermentation broth as described in claim 1, characterized in that, It includes fermentation cells obtained through co-culture, fermentation supernatant, and the resulting metabolites. The metabolites include the following major secondary metabolites: cyclic (L-pro-L-va) dipeptide, cyclic (L-pro-L-tyrosine) dipeptide, indoleacetic acid, DL-indole-3-lactic acid, butyric acid, 4-aminobutyric acid, 2-hydroxycinnamic acid, stachydrine, betaine, bafloxacin B1, 1-deoxynojirimycin, and styraxin.

6. A synthetic microbial agent, characterized in that, The preparation method is as follows: the synthetic microbial co-culture fermentation broth as described in any one of claims 1 to 5 is adsorbed using a carrier to form a solid microbial agent, and the solid microbial agent is mixed with a binder to obtain the synthetic microbial agent.

7. The synthetic microbial agent as described in claim 6, characterized in that, The carrier is selected from diatomaceous earth, maifanite or peat moss, and the mass ratio of fermentation broth to carrier is 4~5:

1.

8. The synthetic microbial agent as described in claim 6, characterized in that, The binder is selected from sodium hydroxymethyl cellulose and / or sodium alginate, and the mass ratio of solid bacterial agent to binder is 50:3~5.

9. The application of the synthetic microbial co-culture fermentation broth as described in any one of claims 1 to 5 and / or the synthetic microbial inoculant as described in any one of claims 6 to 8, characterized in that, It is used to prevent gray mold in chives, promote chive growth, and increase chive yield.

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