A compound bacterial agent containing Pseudomonas and its application

Abstract

This invention discloses a compound microbial agent containing Pseudomonas and its application, belonging to the field of agricultural microbiology technology. The compound microbial agent of this invention includes a Pseudomonas synthetic flora and N-acetyl-L-tryptophan. N-acetyl-L-tryptophan can effectively inhibit the growth of pathogens and effectively improve the colonization ability of Pseudomonas in the soil. The combined use of Pseudomonas and N-acetyl-L-tryptophan can increase the relative abundance of beneficial bacteria and decrease the relative abundance of harmful bacteria in continuously cropped soils; increase the positive correlation ratio of microbial interactions in continuously cropped soils; reshape the rhizosphere microenvironment of apples; and promote the growth of apple seedlings in continuously cropped soils, thereby significantly improving the control effect on continuous cropping obstacles in apples.

Description

Technical Field

[0001] This invention relates to the field of agricultural microbial technology, specifically to a compound microbial agent containing Pseudomonas and its application. Background Technology

[0002] Apple replanting disease (ARD, also known as apple continuous cropping obstacle) is a major soil-borne disease problem commonly faced during the renewal of aging apple orchards worldwide, and has become a key bottleneck restricting the sustainable development of the apple industry. Replanting disease is mainly characterized by weakened tree vigor, increased pests and diseases, yield reduction of 20%-50%, and even total crop failure (Somera & Mazzola, 2022).

[0003] Traditional control measures are becoming increasingly unsustainable due to the cumulative effects of pathogens persisting in the soil and the deterioration of the soil microecology. In recent years, environmentally friendly biological control technologies have shown great application potential because they can balance maintaining crop productivity with protecting ecological balance.

[0004] Pseudomonas ( Pseudomonas Pseudomonas bacteria are widely distributed and abundant in soil, and their simple nutrient requirements, rapid reproduction, and strong competitive colonization capabilities make them a key species and core resource for resisting pathogen infection. Applying Pseudomonas is one of the relatively green and sustainable measures for controlling plant diseases. However, after Pseudomonas is applied to the soil, its growth rate may slow down due to changes in the soil environment, the influence of plant root metabolites, and effective competition with existing soil microorganisms. This may affect its colonization efficiency in the soil, preventing it from reaching the minimum quantity required for beneficial bacteria to exert biocontrol functions, ultimately reducing its control efficiency. Summary of the Invention

[0005] In view of the above-mentioned prior art, the purpose of this invention is to provide a compound microbial agent containing Pseudomonas and its application. This invention combines Pseudomonas synthetic flora with N-acetyl-L-tryptophan to form a compound microbial agent, which effectively promotes the colonization of Pseudomonas in the soil, optimizes the rhizosphere microbial community structure of apple trees, and thus significantly improves the control effect on continuous cropping obstacles in apples.

[0006] Specifically, the present invention relates to the following technical solutions:

[0007] In a first aspect, the present invention provides a compound bacterial agent containing Pseudomonas, comprising: a Pseudomonas synthetic flora and N-acetyl-L-tryptophan;

[0008] The Pseudomonas synthetic flora consists of alkylphenol Pseudomonas (… Pseudomonas alkylphenolica S4, Pseudomonas oleifera ( Pseudomonas oleovorans S38 and Pseudomonas putida ( Pseudomonas putida It is constructed from S42.

[0009] Preferably, the alkylphenol Pseudomonas ( Pseudomonas alkylphenolica The preservation number of S4 is CGMCC No. 36078; the *Pseudomonas oleifera* ( Pseudomonas oleovorans The preservation number of S38 is CGMCC No. 36079; the *Pseudomonas putida* ( Pseudomonas putida The accession number for S42 is CGMCC No. 36080. All of the aforementioned Pseudomonas species are described in patent CN 121472069A.

[0010] Preferably, the Pseudomonas synthetic microbiota is constructed by the following method:

[0011] Alkylphenol Pseudomonas ( Pseudomonas alkylphenolica S4, Pseudomonas oleifera ( Pseudomonas oleovorans S38 and Pseudomonas putida ( Pseudomonas putida The activated bacterial solution of S42 was mixed with an equal number of live bacteria to obtain a mixed seed solution;

[0012] The mixed seed culture was inoculated into LB liquid medium and fermented for 24-48 hours to prepare a synthetic Pseudomonas colony.

[0013] Furthermore, the fermentation conditions were: 30℃, 180rpm shaker culture.

[0014] Furthermore, the viable count in the Pseudomonas synthetic flora is greater than or equal to 10. 8 cfu / ml.

[0015] Preferably, in the compound microbial agent, the mass ratio of Pseudomonas synthetic microbiota to N-acetyl-L-tryptophan is 1:(0.1-0.2); more preferably, the mass ratio of Pseudomonas synthetic microbiota to N-acetyl-L-tryptophan is 1:0.125.

[0016] The compound microbial agent can be in the form of a liquid or a solid. The liquid agent uses water as the dispersion medium and is formulated with effective active bacterial strains and suitable adjuvants, protectants, and stabilizers, featuring uniform dispersion, ease of use, and easy absorption and colonization. The solid agent can be prepared using processes such as carrier adsorption and drying, and includes forms such as powders and granules. It offers advantages such as good stability, convenient storage and transportation, and a long shelf life, allowing for flexible selection based on actual application scenarios, application methods, and field conditions.

[0017] In a second aspect, the present invention provides the application of the above-mentioned compound microbial agent containing Pseudomonas in promoting soil colonization of Pseudomonas.

[0018] The present invention contains a compound microbial agent containing Pseudomonas, wherein N-acetyl-L-tryptophan can effectively inhibit the growth of pathogens Fusarium solani and Fusarium oxysporum; it has a strong chemotactic effect on Pseudomonas S4, S38 and S42, and significantly promotes the colonization of Pseudomonas in the soil by reshaping the soil microenvironment.

[0019] A third aspect of the present invention provides the application of the above-mentioned compound bacterial agent containing Pseudomonas in alleviating apple replanting obstacles.

[0020] In the above applications, the compound bacterial agent containing Pseudomonas aeruginosa alleviates apple replanting obstacles through at least one of the following pathways (1)-(3):

[0021] (1) Promotes the growth of apple seedlings in continuously cropped soil;

[0022] (2) Increase the relative abundance of beneficial bacteria in continuously cropped soil and decrease the relative abundance of harmful bacteria in continuously cropped soil;

[0023] (3) Optimize the structure of apple rhizosphere microbial community.

[0024] The beneficial effects of this invention are:

[0025] This invention is the first to discover a key metabolite, N-acetyl-L-tryptophan, in the rhizosphere metabolites of healthy kiwifruit trees. N-acetyl-L-tryptophan exhibits strong chemotaxis towards three beneficial rhizosphere bacteria, Pseudomonas, S4, S38, and S42, promoting their colonization in the soil. Combining these two metabolites into a Pseudomonas synbiotic for controlling apple continuous cropping obstacles significantly promotes apple plant growth, optimizes the rhizosphere microbial community structure, increases the positive correlation ratio of microbial interactions, and reshapes the microbial interaction network. It demonstrates excellent efficacy in mitigating apple continuous cropping obstacles and can be used for the prevention and control of these obstacles. Attached Figure Description

[0026] Figure 1 Volcano diagram of differential metabolites.

[0027] Figure 2 : Relative abundance of differential metabolites; In the figure, A represents the root exudate components of apple (A); D represents the root exudate components of kiwifruit.

[0028] Figure 3 Mantel Test Network and Heatmap Analysis.

[0029] Figure 4 The results of the antibacterial test of N-acetyl-L-tryptophan; In the figure, A is the plate antagonism test diagram of N-acetyl-L-tryptophan and pathogens; B is the hyphal diameter heatmap, where the numbers 3, 4, 5...9 represent the hyphal diameter of the pathogens, in cm.

[0030] Figure 5 Results of chemotactic assays for N-acetyl-L-tryptophan; In the figure, A represents the qualitative detection of the chemotaxis of Pseudomonas S4, S38, and S42 towards N-acetyl-L-tryptophan; B represents the OD concentration of the strains measured in the chemotactic assay. 600 Value heatmap, 0 represents OD 600 =0, 0.8 indicates OD 600 =0.8.

[0031] Figure 6 qPCR standard curves for Pseudomonas S4, S38, and S42; In the figure, A is the qPCR standard curve for Pseudomonas S4; B is the qPCR standard curve for Pseudomonas S38; and C is the qPCR standard curve for Pseudomonas S42.

[0032] Figure 7 The results of the colonization experiments of N-acetyl-L-tryptophan on Pseudomonas S4, S38, and S42 are shown in the figure. In the figure, A represents the colonization ability of Pseudomonas S4, where S4 is the dynamic colonization curve without N-acetyl-L-tryptophan and S4+ is the dynamic colonization curve with N-acetyl-L-tryptophan. B represents the colonization ability of Pseudomonas S38, where S38 is the dynamic colonization curve without N-acetyl-L-tryptophan and S38+ is the dynamic colonization curve with N-acetyl-L-tryptophan. C represents the colonization ability of Pseudomonas S42, where S42 is the dynamic colonization curve without N-acetyl-L-tryptophan and S42+ is the dynamic colonization curve with N-acetyl-L-tryptophan.

[0033] Figure 8 Results of colonization of Pseudomonas S4, S38 and S42 in pot experiments.

[0034] Figure 9 Kruskal-Wallis analysis determined the differences in relative abundance at the bacterial and fungal genera levels under different treatments; in the figure, (a) shows the difference in relative abundance at the bacterial genera level; (b) shows the difference in relative abundance at the fungal genera level.

[0035] Figure 10 Analysis of bacterial and fungal community structure in pot experiments; In the figure, (a) is the number of edges; (b) is the number of nodes; (c) is the mean degree; and (d) is the mean clustering coefficient. Detailed Implementation

[0036] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0037] In their preliminary research, the inventors isolated three strains of Pseudomonas aeruginosa from the rhizosphere soil of healthy kiwifruit trees for the first time. These strains were identified as *Pseudomonas alkylphenol*. (Pseudomonas alkylphenolica) S4, *Pseudomonas oleifera* (Pseudomonas oleovorans) S38, *Pseudomonas putida* (Pseudomonas putida) S42, each possesses potential antagonistic growth-promoting abilities; both exhibit inhibition rates exceeding 70% against Fusarium oxysporum and Fusarium solani, which cause continuous cropping obstacles in apples, and can be applied to the control of continuous cropping obstacles in apples. However, as mentioned earlier, due to limited soil nutrients, the application of Pseudomonas bacteria to control continuous cropping obstacles cannot achieve its maximum control effect because they are difficult to proliferate in large quantities in the soil.

[0038] To further enhance the application effect of Pseudomonas synthetic flora and improve its colonization ability in soil, this invention discovered a key metabolite—N-acetyl-L-tryptophan—from the rhizosphere metabolites of healthy kiwifruit trees. N-acetyl-L-tryptophan effectively inhibits the growth of pathogens *Fusarium solani* and *Fusarium oxysporum*. Furthermore, N-acetyl-L-tryptophan exhibits strong chemotaxis towards three beneficial rhizosphere bacteria, *Pseudomonas* S4, S38, and S42, promoting their colonization in the soil. When combined with the *Pseudomonas* synthetic flora constructed from *Pseudomonas* S4, S38, and S42 to form a compound microbial agent, and applied to continuously cropped soil, it can reshape the soil microenvironment, increase the relative abundance of beneficial bacteria, decrease the relative abundance of harmful bacteria, and optimize the soil microbial interaction network structure, exhibiting a synergistic effect in controlling continuous cropping obstacles in apples.

[0039] To enable those skilled in the art to more clearly understand the technical solution of this application, the technical solution of this application will be described in detail below with reference to specific embodiments. If specific experimental conditions are not specified in the embodiments, they are generally based on conventional conditions or conditions recommended by the reagent company; the reagents, consumables, etc. used in the following embodiments, unless otherwise specified, can be obtained commercially. Wherein:

[0040] LB liquid medium: 10.0 g tryptone, 5.0 g yeast extract, 10.0 g sodium chloride, 1000 mL distilled water.

[0041] LB solid medium: 10.0 g tryptone, 5.0 g yeast extract, 10.0 g sodium chloride, 15 g agar, 1000 mL distilled water.

[0042] PDA medium: 200.0 g peeled potato, 20.0 g glucose, 20.0 g agar, 1000 mL distilled water.

[0043] Alkylphenol Pseudomonas ( Pseudomonas alkylphenolicaThe preservation number for S4 is CGMCC No. 36078; *Pseudomonas oleifera* ( Pseudomonas oleovorans The accession number for S38 is CGMCC No. 36079; *Pseudomonas putida* ( Pseudomonas putida The accession number for strain S42 is CGMCC No. 36080; the above strain was deposited on September 26, 2025, at the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing). All three Pseudomonas strains are described in patent CN 121472069A.

[0044] N-acetyl-L-tryptophan (CAS: 1218-34-4), purchased from Maclean's Reagent Company.

[0045] The soil used for the pot experiment was taken from a 39-year-old orchard in Manzhuang, Tai'an City, Shandong Province. The soil's physicochemical properties were ammonium nitrogen 4.38 mg·kg⁻¹. -1 Nitrate nitrogen 5.29 mg·kg -1 Available phosphorus 43.49 mg·kg -1 Available potassium 16.58 mg·kg -1 Organic matter 8.94 mg·kg -1 .

[0046] Example 1: Extraction of root metabolites and metabolomics sequencing analysis

[0047] 1. Extraction of root metabolites

[0048] When the above-ground parts of the kiwifruit and apple plants reach about 30cm in height, remove the plants completely from the soil. First, rinse off the attached soil with clean water, then rinse with deionized water until there is no soil on the root surface. Place 50 cleaned plants in a container and fix them in place. Add 500mL of deionized water and aerate for 24 hours. Filter the mixture twice with paper and bring the volume to 500mL with deionized water to achieve a final concentration of 10ml / plant for the root exudates. Finally, filter the mixture through a 0.45μm filter membrane, seal it, and store it in a refrigerator at 4℃ for later use.

[0049] 2. Metabolomics sequencing and analysis

[0050] Extensive targeted metabolomics analysis was performed using UHPLC-Q Exactive LC-MS / MS to determine the components of root exudates from Actinidia cuspidatum (D) and apple (A). Differential metabolites were screened using principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA), and pathway annotation was performed using the KEGG database.

[0051] The results are as follows Figure 1 - Figure 2As shown, differential analysis of root metabolites revealed that 944 substances were significantly upregulated in the rhizosphere of *Actinidia cuspidatum*, with six substances, including N-acetyl-L-tryptophan and N-acetyl-D-tryptophan, showing the most significant differences. Spearman correlation analysis showed that N-acetyl-L-tryptophan, α-aminobutyric acid, and N-acetyl-D-tryptophan were significantly positively correlated with the rhizosphere microbial community of *Actinidia cuspidatum*. Figure 3 Therefore, we selected N-acetyl-L-tryptophan, which showed the most significant difference, as the specific metabolite for subsequent experiments.

[0052] Example 2: N-acetyl-L-tryptophan antibacterial test, chemotaxis test, and beneficial bacteria colonization promotion test

[0053] 1. Antibacterial test

[0054] Weigh out 0.25g and 0.5g of N-acetyl-L-tryptophan, respectively, and add them to 200ml of PDA medium. Pour the medium into plates to make the N-acetyl-L-tryptophan concentrations 0.125% and 0.25%, respectively. The control is PDA medium without any added substances. After the medium solidifies, inoculate the center of the medium with pathogenic bacterial cakes. Seal the plates and incubate them in a 28℃ incubator for 4-5 days. Measure the diameter of the pathogenic bacteria.

[0055] The results are as follows Figure 4 As shown, N-acetyl-L-tryptophan can effectively inhibit the growth of pathogenic fungal hyphae. When the N-acetyl-L-tryptophan concentration is 0.125%, the hyphae of Fusarium solani and Fusarium oxysporum are reduced by 49.44% and 52.81% respectively compared with the culture alone. When the N-acetyl-L-tryptophan concentration is 0.25%, the hyphae of Fusarium solani and Fusarium oxysporum are reduced by 77.53% and 65.17% respectively compared with the culture alone.

[0056] 2. Chemotaxis experiment

[0057] Pseudomonas S4, S38, and S42 were inoculated into LB liquid medium and cultured at 37°C and 170 rpm until OD200. 600 =0.8, centrifuge to collect bacterial cells and resuspend in 12 mL chemotactic buffer (100 mM potassium phosphate, pH 7.0, containing 20 μM EDTA); add 4 mL of 1% hydroxypropyl methylcellulose solution, and add 10 μL of N-acetyl-L-tryptophan (0.1 M) to the center of the culture dish. Incubate at 37℃ for 3 h, then observe the turbidity and measure the OD. 600 Value; with an equal amount of sterile water added as a control.

[0058] The results are as follows Figure 5 As shown, N-acetyl-L-tryptophan exhibited strong positive chemotaxis towards all three Pseudomonas strains.

[0059] 3. Colonization experiment

[0060] Use 10 respectively 8 Apple seedlings were treated with bacterial suspensions of Pseudomonas S4, S38, and S42 (CFU / mL). Twelve seedlings of each strain were treated at a rate of 10 mL / kg soil. Six seedlings were treated with 0.125% N-acetyl-L-tryptophan (by soil mass) in aqueous solution (applied every 7 days), while the other six seedlings served as controls, treated with an equal volume of sterile water. Rhizosphere soil samples were collected periodically, flash-frozen in liquid nitrogen, and stored at -80℃. After all samples were collected, soil DNA was extracted and qPCR was performed to determine the colonization rate of the strains. The colonization rate was calculated using a standard curve.

[0061] The standard curve is constructed as follows:

[0062] Specific primers were designed using Primer Premier 5.0 software (Table 1). Primer specificity was verified by PCR and agarose gel electrophoresis. Target fragment cloning was then performed using the following program: 98℃, 3 min; 95℃, 30 s; 60℃, 15 s; 72℃, 15 s; 40 cycles; 72℃, 5 min. The target band was purified and recovered, ligated into a recombinant vector, and transformed into *E. coli*. High-purity recombinant plasmids were extracted from correctly identified positive clones verified by sequencing. The recombinant plasmids were serially diluted tenfold to six orders of magnitude, and qPCR was performed using the following program: 95℃, 3 min; 95℃, 10 s; 60℃, 30 s; 40 cycles; 72℃, 3 min. The Ct values ​​for each gradient were recorded. A linear regression equation was plotted with log(initial template copy number) on the x-axis and the average Ct value on the y-axis.

[0063] Table 1: qPCR-specific primers for strains

[0064]

[0065] To analyze the colonization patterns of each strain and the regulation of the colonization process by N-acetyl-L-tryptophan, a qPCR standard curve for each strain was constructed. Figure 6 Following this, a soil colonization experiment was conducted. The results showed that all three tested Pseudomonas strains were able to effectively colonize within a 90-day experimental period, with their soil copy number consistently maintained at 10. 3 -10 5 CFU・g -1 Levels. Adding N-acetyl-L-tryptophan to the soil significantly increased the colonization of the strains, and the colonization dynamics became more stable. At 90 days, the colonization of Pseudomonas S4, S38, and S42 were 5.36, 2.66, and 7.74 times that of the control, respectively. Figure 7 The results showed that N-acetyl-L-tryptophan significantly improved the colonization of the three Pseudomonas strains in the soil.

[0066] Example 3: Preparation of a compound bacterial agent containing Pseudomonas aeruginosa

[0067] Alkylphenol Pseudomonas ( Pseudomonas alkylphenolica S4, Pseudomonas oleifera ( Pseudomonas oleovorans S38, *Pseudomonas putida* ( Pseudomonas putida S42 cells were streaked onto LB solid medium and incubated at 30°C for 24 hours. Single colonies were then picked and inoculated onto LB liquid medium and incubated at 30°C and 180 rpm. -1 After 24 hours of shaker fermentation, activated bacterial solutions of S4, S38, and S42 were obtained. The bacterial concentration of the activated solutions was determined by counting the bacteria under a microscope. The activated bacterial solutions of S4, S38, and S42 were then mixed at a viable cell ratio of 1:1:1 to obtain a mixed seed culture.

[0068] The mixed seed culture was inoculated into LB liquid medium and cultured at 30°C and 180 rpm for 48 hours to prepare a Pseudomonas synthetic microbiota. The viable count in the Pseudomonas synthetic microbiota was adjusted to 1 × 10⁻⁶ cells / year. 8 cfu / ml.

[0069] A compound bacterial agent containing Pseudomonas was prepared by mixing Pseudomonas synthetic bacterial groups and N-acetyl-L-tryptophan at a mass ratio of 1:0.125.

[0070] Example 4: Pot experiment on the reduction of continuous cropping obstacles in apples by compound inoculants containing Pseudomonas aeruginosa

[0071] 1. Test Methods

[0072] The experiment was conducted from April to October 2024 at the National Apple Engineering Experimental Center, using seedlings of Malushupehensis Rehd. as the test plants. Malushupehensis seeds were stratified at 4 ℃ for approximately 30 days until they sprouted, then sown in culture pots containing seedling substrate. When the seedlings had 5-6 true leaves, plants with uniform growth and free from pests and diseases were selected and transplanted in early May into clay pots (25 cm top diameter, 17 cm bottom diameter, and 18 cm height) containing 7.5 kg of soil from different treatments. The continuously cropped soil used in the experiment was randomly sampled from multiple points in a 39-year-old apple orchard in Manzhuang, Daiyue District, Tai'an City, Shandong Province, China, at a distance of 80 cm from the trunk and a depth of 10-40 cm, and mixed thoroughly.

[0073] The experiment was set up with the following 5 treatments:

[0074] Continuous cropping control (CK1): Only soil from continuous cropping was used;

[0075] LB liquid medium control (CK2): LB liquid medium was added to the continuously cropped soil at a concentration of 1% of the soil mass.

[0076] N-acetyl-L-tryptophan treatment (CK3): N-acetyl-L-tryptophan was added to the continuously cropped soil at a concentration of 0.125% of the soil mass.

[0077] SynCom treatment with Pseudomonas synthetic microbial community (SynCom_3): The SynCom prepared in Example 3 was added to the continuously cropped soil. The viable count of the SynCom was 1 × 10⁻⁶. 8 The concentration of cfu / ml was increased to 1% of the mass of the continuously cropped soil.

[0078] SynCom 3+ treatment with a compound microbial agent containing Pseudomonas: A compound microbial agent consisting of Pseudomonas synthetic microbial community and N-acetyl-L-tryptophan was added to continuously cropped soil. The viable count of the Pseudomonas synthetic microbial community was 1 × 10⁻⁶. 8 The concentration of cfu / ml was 1% of the mass of the continuously cropped soil, and the concentration of N-acetyl-L-tryptophan was 0.125% of the mass of the continuously cropped soil.

[0079] Each treatment consisted of 20 pots, with one seedling per pot; all treatments were subsequently managed with the same water and fertilizer. Samples were taken 90 days after each treatment to measure the following indicators:

[0080] Biomass determination: Plant height and diameter at ground level were measured using a meter stick and a vernier caliper, respectively. When measuring fresh weight, the above-ground stems and leaves and underground roots were first washed with tap water, dried, and then measured using an electronic balance. After drying at 80 ℃ to constant weight, the weight was measured.

[0081] Colony capacity testing: Fresh soil from the rhizosphere of Pingyi sweet tea seedlings was collected, and DNA was extracted using a soil DNA extraction kit. qPCR was then performed to determine the colonization rate of the strains. Primers and standard curves for the strains are shown in Table 1 and [Table data missing]. Figure 6 .

[0082] Pathogen quantification and high-throughput sequencing: Fresh rhizosphere soil from Pingyi sweet tea seedlings was collected, and DNA was extracted using the Tiangen Soil DNA Extraction Kit. The gene copy numbers of *Fusarium oxysporum*, *Fusarium solani*, *Fusarium flocculation*, and *Fusarium moniliforme* were determined according to the method of Duan et al. (2021). Primer sequences are shown in Table 2. High-throughput sequencing of soil bacteria and fungi was commissioned to Shanghai Meiji Biomedical Technology Co., Ltd. Three treatments were performed: continuous cropping control (CK1), *Pseudomonas* inoculum treatment (SynCom_3), and synbiotic treatment (synthetic flora 3+N-acetyl-L-tryptophan) (SynCom_3+).

[0083] Table 2: Primers for PCR amplification of four Fusarium species in soil

[0084]

[0085] Data analysis: One-way ANOVA was performed using IBM SPSS 27.0 (IBM SPSS Statistics 27, IBM Corporation, USA), with multiple comparisons conducted using the Least Significant Difference (LSD) method. Charts were created using Microsoft Excel 2013 and GraphPad Prism 8.0.2. Correlation analysis and visualization were performed using R based on OTU abundance. Data are expressed as mean ± standard error, and p < 0.05 was considered statistically significant.

[0086] 2. Test Results

[0087] (1) Effects of different treatments on the biomass of Pingyi sweet tea seedlings

[0088] Table 3 shows that, under pot cultivation conditions, the application of a compound microbial agent (SynCom_3+) composed of Pseudomonas synthetic flora and N-acetyl-L-tryptophan to the continuously cropped soil significantly promoted the increase of biomass in Pingyi sweet tea seedlings. Compared with CK1, the height, stem diameter, dry weight, and fresh weight of apple seedlings increased by 47.98%, 65.80%, 63.06%, and 109.40%, respectively. Notably, the addition of N-acetyl-L-tryptophan alone had no significant promoting effect on plant growth, but its growth-promoting effect was significantly enhanced in the presence of Pseudomonas, confirming that its effect depends on the presence of Pseudomonas strains. Compared with the application of Pseudomonas synthetic flora (SynCom_3) alone, the seedlings treated with the compound microbial agent (SynCom_3+) showed significantly enhanced growth, with increases in seedling height, ground diameter, fresh weight, and dry weight of 11.53%, 14.28%, 43.72%, and 19.87%, respectively.

[0089] The results showed that the Pseudomonas synthetic flora and N-acetyl-L-tryptophan could synergistically increase the biomass of apple seedlings in continuously cropped soil.

[0090] Table 3: Effects of different treatments on the biomass of Pingyi sweet tea seedlings

[0091]

[0092] (2) Effects of different treatments on the colonization of Pseudomonas aeruginosa in the root soil of Pingyi sweet tea

[0093] like Figure 8As shown, the addition of N-acetyl-L-tryptophan in the compound microbial agent treatment (SynCom_3+) effectively promoted the colonization of Pseudomonas aeruginosa in the rhizosphere soil of Pingyi sweet tea seedlings, increasing the colonization of Pseudomonas aeruginosa S4, S37 and S42 by 5.3%, 24.3% and 36.8% respectively compared with the treatment of only the synthetic microbial community of Pseudomonas aeruginosa (SynCom_3).

[0094] (3) Effects of different treatments on the root microbial community structure of Pingyi sweet tea

[0095] High-throughput sequencing results from soil showed that treatment with the compound microbial agent (SynCom_3+) significantly improved the rhizosphere microbial community structure of apple trees. At the bacterial genus level, treatment with the compound microbial agent (SynCom_3+) reduced the concentration of Pseudomonas spp. (…). Pseudomonas The relative abundance of Bacillus was increased by 302.04% compared to the control, and it was also enriched with Bacillus spp. Bacillus ) and Sphingosomalidone spp. Sphingomonas The number of bacteria in the soil increased by 90.20% and 84.42% respectively compared to the control group; Xanthomonas spp. in the soil... Xanthomonas The relative abundance of *Trichoderma* was significantly reduced, decreasing by 38.30% compared to the control group. At the fungal genus level, the compound inoculant treatment (SynCom_3+) significantly increased the abundance of *Trichoderma* (…). Trichoderma )and( Condenascus The relative abundance of the genus *Fusarium* increased by 432.33% and 1225% respectively compared to the control group; while the abundance of the genus *Fusarium*... Fusarium ) and Fusarium spp. ( Bisifusarium The relative abundance of bacteria and fungi decreased by 57.76% and 58.93% respectively compared to the control group. Furthermore, the compound microbial agent treatment (SynCom_3+) significantly increased the number of soil bacterial and fungal network interactions, network density, and mean density compared to the control. Figure 10 ).

[0096] In summary, treatment with a composite microbial agent consisting of Pseudomonas synthetic microbial communities and N-acetyl-L-tryptophan can effectively optimize the structure of soil microbial interaction networks and enhance the complexity of microbial associations within the network.

[0097] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A compound bacterial agent containing Pseudomonas, characterized in that, include: Pseudomonas synthetic flora and N-acetyl-L-tryptophan; The Pseudomonas synthetic flora consists of alkylphenol Pseudomonas (… Pseudomonas alkylphenolica S4, Pseudomonas oleifera ( Pseudomonas oleovorans S38 and Pseudomonas putida ( Pseudomonas putida It is constructed from S42; The alkylphenol Pseudomonas ( Pseudomonas alkylphenolica The preservation number of S4 is CGMCC No. 36078; the *Pseudomonas oleifera* ( Pseudomonas oleovorans The preservation number of S38 is CGMCC No. 36079; the *Pseudomonas putida* ( Pseudomonas putida The accession number for S42 is CGMCC No. 36080; The Pseudomonas synthetic microbiota was constructed using the following method: Alkylphenol Pseudomonas ( Pseudomonas alkylphenolica S4, Pseudomonas oleifera ( Pseudomonas oleovorans S38 and Pseudomonas putida ( Pseudomonas putida The activated bacterial solution of S42 was mixed with an equal number of live bacteria to obtain a mixed seed solution; The mixed seed culture was inoculated into LB medium and fermented for 24-48 h to prepare a synthetic Pseudomonas bacterial community. The viable count of the Pseudomonas synthetic flora is greater than or equal to 10. 8 cfu / ml; In the compound bacterial agent, the mass ratio of Pseudomonas synthetic flora to N-acetyl-L-tryptophan is 1:(0.1-0.2).

2. The compound bacterial agent containing Pseudomonas as described in claim 1, characterized in that, The fermentation conditions were: 30℃, 180rpm shaker culture.

3. The compound bacterial agent containing Pseudomonas as described in claim 1, characterized in that, The compound microbial agent is available in liquid or solid form.

4. The application of the compound microbial agent containing Pseudomonas as described in any one of claims 1-3 in promoting the colonization of Pseudomonas in soil.

5. The application of the compound microbial agent containing Pseudomonas as described in any one of claims 1-3 in alleviating apple continuous cropping obstacles.

6. The application according to claim 5, characterized in that, The compound inoculant containing Pseudomonas bacteria alleviates apple replanting obstacles through at least one of the following pathways (1)-(3): (1) Promotes the growth of apple seedlings in continuously cropped soil; (2) Increase the relative abundance of beneficial bacteria in continuously cropped soil and decrease the relative abundance of harmful bacteria in continuously cropped soil; (3) Optimize the structure of apple rhizosphere microbial community.

Images