Pseudomonas synthetic bacterial flora and application thereof in prevention and treatment of apple continuous cropping obstacles

Abstract

This invention discloses a synthetic microbial community of Pseudomonas and its application in controlling continuous cropping obstacles in apples, belonging to the field of agricultural microbiology technology. The synthetic microbial community is constructed from three strains of Pseudomonas: *Pseudomonas alkylphenol*, *Pseudomonas oleophila*, and *Pseudomonas putida*, with bioaccession numbers of *Pseudomonas alkylphenol*, *Pseudomonas oleophila*, and *Pseudomonas putida*. (Pseudomonas alkylphenolica) S4, CGMCC No. 36078; *Pseudomonas oleifera* (Pseudomonas) oleovorans) S38, CGMCC No. 36079; *Pseudomonas putida* (Pseudomonas putida) S42, CGMCC No. 36080. This synthetic microbial community exhibits strong antagonistic effects against the main pathogens causing apple continuous cropping obstacles (Fusarium oxysporum and Fusarium solani), and its inhibitory ability is significantly better than that of single strains. Field and pot experiments show that both liquid and solid microbial fertilizers from the synthetic community can promote the growth of the aboveground and underground parts of continuously cropped apple trees; significantly reduce the number of harmful fungi in the continuously cropped soil; increase the activity of root protective enzymes and reduce MDA content; optimize the rhizosphere microecology of continuously cropped apples; and significantly alleviate apple continuous cropping obstacles.

Description

Technical Field

[0001] This invention relates to the field of agricultural microbiology, specifically to a Pseudomonas synthetic flora and its application in controlling continuous cropping obstacles in apples. Background Technology

[0002] my country accounts for over 50% of the global apple cultivation area and output, making it a crucial pillar industry for increasing farmers' income and rural revitalization (Liu et al., 2024). However, due to aging trees, over 50% of apple orchards will require renewal and reconstruction in the next 10 years, and over 90% will require continuous cropping, further exacerbating the problem of continuous cropping obstacles. Continuous cropping obstacles are mainly manifested as weakened tree vigor, increased pests and diseases, yield reduction of 20%–50%, or even crop failure, and have become a major bottleneck restricting the sustainable development of the apple industry (Duan et al., 2022a). Studies have shown that continuous cropping obstacles in apples are closely related to soil microbial community imbalance, with harmful fungi such as Pythium, Fusarium, Cyclospora, and Phytophthora being the main pathogenic factors (Mannai et al., 2023; Ajeethan et al., 2023; Manici et al., 2021; Somera et al., 2021).

[0003] To address the causes of apple cropping disorder, scholars both domestically and internationally have proposed agronomical measures such as rational crop rotation, intercropping, and application of organic matter (Gao et al., 2020; He et al., 2023; Zydlik et al., 2023); soil disinfection measures such as chemical fumigation and physical disinfection (Wieczorek et al., 2023); biological control measures such as antagonistic bacteria and antagonistic fungi (Li et al., 2024; Wang et al., 2024); and resistance breeding measures such as the selection of resistant rootstocks to control apple cropping disorder (Fazio et al., 2023; Siefen et al., 2024). However, these measures suffer from drawbacks such as time-consuming and labor-intensive agronomical measures, high implementation difficulty, high investment costs and environmental pollution associated with soil disinfection measures, and lengthy breeding processes for resistant rootstocks.

[0004] Biological control is currently an important means of controlling soil-borne diseases and an effective strategy for alleviating continuous cropping obstacles in apple orchards. At present, Trichoderma, Bacillus, Pseudomonas, and Streptomyces are widely used as biological control agents. However, the control effect of single-strain bio-fertilizers is not ideal, with a narrow spectrum of inhibition, poor colonization effect, and poor application stability, making it difficult to completely eliminate continuous cropping obstacles.

[0005] Recent studies have shown that soil microbiomes are closely related to various plant diseases, and are even key drivers of some soil-borne diseases. Academician Shen Qirong's team successfully cultivated disease-suppressing soil by applying bio-organic fertilizers and regulating rhizosphere bacterial communities (Deng et al., 2022). Rhizosphere microbiomes can predict plant health status at an early stage (Gu et al., 2022). Professor Wei Gehong's team successfully suppressed Astragalus root rot by constructing simplified synthetic microbial communities (Li et al., 2021). Synthetic microbial communities inhibit fungal diseases through chitin lysis and secondary metabolite production (Carrión, 2019). Compared to single-strain systems, synthetic microbial communities have the advantage of division of labor and cooperation, enabling them to perform complex functions and enhance environmental adaptability (Mehlferber et al., 2024).

[0006] Pseudomonas (Pseudomonas) As a core group of rhizosphere growth-promoting bacteria (PGPR), they have shown significant potential in the field of agricultural biological control (Madigan M & Martinko J 2005). Pseudomonas microorganisms are easily cultured under indoor conditions, and their high nutrient utilization rate and rapid community growth make them excellent model strains. Treatment of potato seed tubers with *Pseudomonas fluorescens* F113 can induce scab disease. (Streptomyces scabies) The incidence rate decreased by 72%, and the tuber yield increased by 19% (Chen et al., 2016). *Pseudomonas aeruginosa* (Pseudomonas aeruginosa) Four applications of WKT-26 fermentation broth as a root drench resulted in a 62.63% control efficacy against soybean Phytophthora root rot in potted plants, and an increase in soybean biomass of 57.90%. However, current research on Pseudomonas mainly focuses on the use of single strains to control plant diseases, and there are no reports on the use of Pseudomonas compound biocontrol agents in controlling apple continuous cropping obstacles. Summary of the Invention

[0007] In view of the above-mentioned prior art, the purpose of this invention is to provide a Pseudomonas synthetic microbial community and its application in preventing and controlling apple continuous cropping obstacles.

[0008] Specifically, the present invention relates to the following technical solutions:

[0009] In a first aspect, the present invention provides a Pseudomonas synthetic flora composed of alkylphenol Pseudomonas (… Pseudomonas alkylphenolica S4, Pseudomonas oleifera ( Pseudomonas oleovorans S38 and Pseudomonas putida ( Pseudomonas putida It is constructed from S42.

[0010] in:

[0011] Alkylphenol Pseudomonas ( Pseudomonas alkylphenolica S4, this strain was deposited on September 26, 2025 at the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing), with the biological accession number: CGMCC No. 36078.

[0012] Oil-producing Pseudomonas ( Pseudomonas oleovorans S38, this strain was deposited on September 26, 2025 at the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing), with the biological accession number: CGMCC No. 36079.

[0013] Pseudomonas putida ( Pseudomonas putida S42, this strain was deposited on September 26, 2025 at the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing), with the biological accession number: CGMCC No. 36080.

[0014] The above-mentioned alkylphenol Pseudomonas ( Pseudomonas alkylphenolica S4, Pseudomonas oleifera ( Pseudomonas oleovorans S38 and Pseudomonas putida ( Pseudomonas putida S42 samples were all isolated from the rhizosphere soil of healthy Actinidia chinensis trees and have the following characteristics:

[0015] S4: When grown on LB solid medium, single colonies are round, white, translucent, with a smooth, moist, mucous-like surface, neat edges, and flattened sides. Under an optical electron microscope (100× / 1.30 oil immersion), they stain red with Gram staining, indicating they are Gram-negative bacteria. The cells are straight, arranged in a single row, and are short rod-shaped.

[0016] S38: When grown on LB solid medium, single colonies are round, slightly yellow, opaque, with a smooth and moist surface, neat edges, and slightly convex sides. Under an optical electron microscope (100× / 1.30 oil immersion), they stain red after Gram staining, indicating they are Gram-negative bacteria. The cells are straight, arranged in a single row, and are short rod-shaped.

[0017] S42: When grown on LB solid medium, single colonies are round, white, translucent, with a smooth and moist surface, neat edges, and flat sides. Under an optical electron microscope (100× / 1.30 oil immersion), they stain red after Gram staining, indicating they are Gram-negative bacteria. The cells are straight, arranged in a single row, and are short rod-shaped.

[0018] Preferably, among the Pseudomonas synthetic flora, alkylphenol Pseudomonas (… Pseudomonas alkylphenolica S4, Pseudomonas oleifera ( Pseudomonas oleovorans S38 and Pseudomonas putida ( Pseudomonas putida The ratio of live bacteria in S42 is 1:1:1.

[0019] In a second aspect, the present invention provides a microbial agent containing a synthetic flora of Pseudomonas.

[0020] Preferably, in the bacterial agent, the Pseudomonas synthetic flora exists in one or more forms among the cultured live bacteria, bacterial suspension, and fermentation broth.

[0021] Furthermore, the fermentation broth refers to the liquid produced after inoculating the microbial strain into a culture medium and culturing it for a period of time.

[0022] The bacterial suspension refers to the bacterial precipitate obtained by resuspending the precipitate after centrifugation of the fermentation broth.

[0023] In a preferred embodiment of the present invention, the fermentation broth is prepared by the following method:

[0024] 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;

[0025] The mixed seed culture was inoculated into LB medium and cultured at 30°C and 180 rpm for 24-48 hours to obtain the fermentation broth.

[0026] Preferably, the bacterial agent is in the form of a liquid bacterial agent or a solid bacterial agent.

[0027] A third aspect of the present invention provides the application of the above-mentioned Pseudomonas synthetic flora or inoculants in inhibiting the growth of plant pathogens.

[0028] In the above application, the plant pathogen is Fusarium oxysporum (Fusarium oxysporum). Fusarium oxysporum ) or Fusarium solani ( Fusarium solani) .

[0029] In a fourth aspect, the present invention provides the use of the above-mentioned Pseudomonas synthetic flora or bacterial agent in any of the following (1)-(5):

[0030] (1) Produces gluten;

[0031] (2) Secretion of siderophores;

[0032] (3) Phosphorus dissolution;

[0033] (4) Potassium dissolution;

[0034] (5) Ammonia production.

[0035] In a fifth aspect, the present invention provides the use of the above-mentioned Pseudomonas synthetic flora or bacterial agents in the following (1) or (2):

[0036] (1) Reduce the obstacles of continuous cropping of apples;

[0037] (2) Prepare biocontrol agents to reduce the obstacle of continuous cropping of apples.

[0038] In a sixth aspect, the present invention provides a biocontrol agent for mitigating continuous cropping obstacles in apples, wherein the biocontrol agent uses fermentation broth of a Pseudomonas synthetic flora as its active ingredient.

[0039] Preferably, the biocontrol agent is prepared by the following method:

[0040] The fermentation broth of the Pseudomonas synthetic microbial community and the sterilized carrier were mixed at a weight ratio of 1:10 and fermented at 30°C for 7-10 days.

[0041] The carrier is made of cow dung and corn stalks mixed in a weight ratio of 3:1.

[0042] A seventh aspect of the present invention provides the use of the above-described biocontrol agent in at least one of the following (1)-(3):

[0043] (1) Promotes the growth of apple seedlings planted in continuously cropped soil;

[0044] (2) Improve the root vigor and root defense enzyme activity of apple seedlings planted in continuously cropped soil;

[0045] (3) Increase the number of bacteria / fungi in the apple root soil under continuous cropping conditions.

[0046] An eighth aspect of the present invention provides a method for mitigating apple continuous cropping obstacles, comprising the following steps: applying the above-mentioned Pseudomonas synthetic microbial community, microbial agent or the above-mentioned biocontrol agent to apple continuous cropping soil.

[0047] The beneficial effects of this invention are:

[0048] This invention is the first to isolate three Pseudomonas strains from the rhizosphere soil of healthy Actinopterygium 'Cephalotaxus' trees, namely, *Pseudomonas alkylphenolus*. (Pseudomonas alkylphenolica) S4, *Pseudomonas oleifera* (Pseudomonas oleovorans) S38, *Pseudomonas putida* (Pseudomonas putida) S42, each possesses potential antagonistic growth-promoting abilities. Under pot and field trial conditions, applying solid biocontrol agents or liquid microbial fertilizers to the soil of continuously cropped apple orchards showed that the microbial fertilizer could promote the growth of both the above-ground and below-ground parts of continuously cropped apple trees; to a certain extent increase the content of culturable bacteria in the soil and reduce the number of culturable fungi in the soil; increase the activity of root protective enzymes; reduce oxidative damage to plant roots; increase the activity of soil enzymes and root protective enzymes in continuously cropped apple soil; and reduce oxidative damage to plants. It has excellent effects in alleviating continuous cropping obstacles in apples and can be used for the prevention and control of continuous cropping obstacles in apples. Attached Figure Description

[0049] Figure 1 : Strain Alkylphenol Pseudomonas (Pseudomonas alkylphenolica) S4, *Pseudomonas oleifera* (Pseudomonas oleovorans) S38, *Pseudomonas putida* (Pseudomonas putida) A single colony image of S42 on a plate, and Gram staining images of strains S4, S38, and S42.

[0050] Figure 2 : Strain Alkylphenol Pseudomonas (Pseudomonas alkylphenolica) S4, *Pseudomonas oleifera* (Pseudomonas oleovorans) S38, *Pseudomonas putida* (Pseudomonas putida) Phylogenetic tree of the 16S rDNA sequence of S42.

[0051] Figure 3 : Strain Alkylphenol Pseudomonas (Pseudomonas alkylphenolica) S4, *Pseudomonas oleifera* (Pseudomonas oleovorans) S38, *Pseudomonas putida* ( Pseudomonas putida )S42 and synthetic bacteria and pathogenic bacteria plate antagonism test diagram.

[0052] Figure 4 : Strain Alkylphenol Pseudomonas (Pseudomonas alkylphenolica) S4, *Pseudomonas oleifera* (Pseudomonas oleovorans) S38, *Pseudomonas putida* (Pseudomonas putida) Compatibility test diagram between S42

[0053] Figure 5 : Strain Alkylphenol Pseudomonas (Pseudomonas alkylphenolica) S4, *Pseudomonas oleifera* [[ID= S38, *Pseudomonas putida* ( ​ S42 effectively colonizes in the rhizosphere soil of apple trees.

[0054] ​: Pot experiment diagram of synthetic microbial community treatment. Continuous cropping control (CK1), methyl bromide fumigation control (CK2), blank carrier treatment (CK3), S4 solid microbial fertilizer treatment (S4), S38 solid microbial fertilizer treatment (S38), S42 solid microbial fertilizer treatment (S42), synthetic microbial community solid inoculant treatment (Syncom_3).

[0055] ​ : Field experiment diagram of synthetic microbial community treatment. Continuous cropping control (CK1), blank vector treatment (CK2), synthetic microbial community solid inoculum treatment (Syncom_3). Detailed Implementation

[0056] 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.

[0057] As described in the background section, continuous cropping obstacles in apple orchards weaken the tree, exacerbate pests and diseases, reduce fruit yield and quality, and even lead to tree death, causing severe economic losses to fruit growers. Current research on continuous cropping obstacles largely focuses on the plant-microbe interaction system. However, long-term continuous cropping leads to a highly specific co-evolutionary relationship between pathogens and the host, making it easy for pathogens to adapt and overcome homologous resistance resources (pathogens easily adapt to the host's defense mechanisms). Currently, there is no research on microbial transplantation from heterologous plants, and there are no reports on its application in controlling continuous cropping obstacles in apple orchards.

[0058] In light of this, this invention innovatively proposes the concept of a "broad-spectrum resistance resource bank"—by screening heterologous resistant rootstocks unrelated to apples, whose root microorganisms have developed more universal disease-suppressing mechanisms due to long-term resistance to non-apple-specific pathogens. Actinopterygium is a long-domesticated resistant variety, and its root system has formed a unique "disease-resistant microbiome." Therefore, transplanting the "disease-resistant microbiome" of continuously cropped plants for the control of apple continuous cropping obstacles has great development potential.

[0059] This invention, for the first time, isolated three bacterial strains (S4, S38, and S42) from the rhizosphere soil of healthy *Actinidia cuspidatum* trees. Based on morphological, physiological, and biochemical characteristics, and phylogenetic analysis using 16S rDNA, all three strains were identified as *Pseudomonas* species: *Pseudomonas alkylphenolae*, *Pseudomonas oleifera*, and *Pseudomonas putida*. The three isolated *Pseudomonas* strains exhibit good application potential, combining multiple functions such as antibacterial activity, auxin production, secretion of growth-promoting substances, promotion of apple seedling growth, and alleviation of continuous cropping obstacles in apples. This invention used the plate confrontation method to study their antagonistic effects against *Fusarium oxysporum* and *Fusarium solani*, which cause continuous cropping obstacles in apples. The inhibition rates of the three *Pseudomonas* strains against both pathogens were all above 70%, and the synthetic flora composed of the three *Pseudomonas* strains showed better antagonistic effects compared to individual strains. Under potted conditions, the synthetic microbial inoculant applied to continuously cropped soil effectively colonized the plant, significantly promoted the growth of both above-ground and below-ground parts of seedlings, reduced oxidative damage to apple seedlings, improved root vitality, promoted the activity of root protective enzymes and reduced MDA content, increased the bacterial / fungal ratio in the soil, reduced the gene copy number of four Fusarium species in continuously cropped soil, and promoted plant growth. This indicates that the synthetic microbial inoculant has a good effect on preventing and controlling continuous cropping obstacles in apples.

[0060] 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:

[0061] LB medium: 10.0 g tryptone, 5.0 g yeast extract, 10.0 g sodium chloride, 1000 mL distilled water.

[0062] PDA medium: 200.0 g peeled potato, 20.0 g glucose, 20.0 g agar, 1000 mL distilled water.

[0063] Phosphate-soluble medium: 10 g glucose, 5 g ammonium sulfate, 3 g sodium hypochlorite, 0.3 g magnesium sulfate heptahydrate, 0.3 g potassium chloride, 0.036 g ferrous sulfate, 0.03 g manganese sulfate, 2.0 g calcium phytate, 20.0 g agar, 1000 mL distilled water.

[0064] Potassium-solubilizing medium: sucrose 5.0g, ammonium sulfate 0.5g, yeast powder 0.5g, magnesium sulfate 0.3g, disodium hydrogen phosphate 2.0g, ferrous sulfate 0.03g, manganese sulfate 0.03g, potassium feldspar 2.0g, agar 20.0g, distilled water 1000mL.

[0065] Ferrocarrier medium (CAS medium): Chromium azurite S (CAS) 60.5 mg, cetyltrimethylammonium bromide 72.9 mg, piperazine-1,4-diethanesulfonic acid 2.4 g, 1 mM FeCl3·6H2O added to 10 mM hydrochloric acid and agarose (0.9% w / v), 20.0 g agar, and 1000 mL distilled water.

[0066] Example 1: Isolation and Identification of Strains

[0067] 1. Isolation and purification of the strain:

[0068] In July 2023, rhizosphere soil from healthy *Actinidia chinensis* trees was collected from Zibo and brought back to the laboratory. The dilution plate method was used for separation. The collected soil sample was filtered to remove impurities. 5 g of rhizosphere soil was weighed and added to 45 mL of sterile deionized water. The mixture was shaken at 180 rpm for 30 min, allowed to stand for 5 min, and then diluted with sterile water to a final concentration of 10. -3 —10 -6 Take 100 μL and spread it evenly on LB medium. Incubate at 37°C for 24 h. Pick colonies with different morphologies and transfer them to LB plates for streaking isolation and purification. Store at 4°C. A total of 295 soil bacteria strains were isolated.

[0069] 2. Screening of strains:

[0070] The antagonistic effects of isolated strains against *Fusarium solani* and *Fusarium oxysporum* were determined using the plate confrontation method. 295 isolated and purified strains were confronted with activated *Fusarium solani* and *Fusarium oxysporum* on PDA plates. Each treatment was repeated three times, incubated at 28°C, and strains exhibiting inhibition zones were selected after 5 days. The inhibition rate was calculated. After repeated screening, three strains with the best antagonistic effects against the tested pathogens were obtained and labeled S4, S38, and S42, respectively.

[0071] Inhibition rate = [(control colony diameter - treated colony diameter) / control colony diameter] × 100%.

[0072] 3. Identification of the strain:

[0073] (1) Morphological identification:

[0074] The isolated strains S4, S38, and S42 were streaked onto LB medium and incubated at 37°C for 24 h to grow single colonies. The morphological characteristics of the colonies were then observed.

[0075] Gram staining procedure: First, stain with ammonium oxalate crystal violet, then treat with Lugol's iodine solution as a mordant, and then treat with 95% ethanol as a decolorizing agent. Observe under an oil immersion microscope. Gram staining that appears red indicates Gram-negative bacteria, and Gram staining that appears blue-purple indicates Gram-positive bacteria.

[0076] Colony morphology and Gram staining results are as follows: ​ As shown:

[0077] S4 strain single colonies are round, white, translucent, with a smooth, moist, mucous-like surface, neat edges, and flat sides. ​ A); Under a fluorescence microscope at 100× / 1.30 oil immersion, the strain appears red after Gram staining, indicating it is a Gram-negative bacterium. The cells are straight, arranged in a single row, and are short rod-shaped. ​ B).

[0078] Strain S38 forms single colonies that are round, slightly yellow, opaque, with a smooth, moist surface, neat edges, and slightly convex sides. ​ C), after Gram staining, the strain appears red, indicating it is a Gram-negative bacterium. The cells are straight, arranged in a single row, and are short and rod-shaped. ​ D).

[0079] S42 strain single colonies are round, white, translucent, with a smooth, moist surface, neat edges, and flat sides. ​ E), after Gram staining, the strain appears red, indicating it is a Gram-negative bacterium. The cells are straight, arranged in a single row, and are short and rod-shaped. ​ F).

[0080] (2) 16S rDNA sequence analysis and phylogenetic analysis:

[0081] Bacterial genomic DNA was extracted using a bacterial genomic DNA kit (Beijing Kangwei Century Technology Co., Ltd.). Universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3') were used for 16S rDNA gene amplification. Thermal cycling was performed with the following parameters: initial denaturation at 94°C for 1 min; denaturation at 94°C for 1.5 min; primer annealing at 55°C for 1 min; extension at 74°C for 90 s, for 30 cycles; and final extension at 72°C for 10 min. Primer synthesis and sequencing of the amplified fragments were performed by Riboscience. Sequencing results were BLAST-aligned using the NCBI nucleotide database. A phylogenetic tree was constructed using the neighbor-joining method in MEGA 11 software, with bootstrap values ​​tested with 1000 replicates.

[0082] The 16S rDNA sequence of strain S4 is 1401 bp in length. This sequence was BLAST-aligned with the NBCI GenBank database, and the sequence matched that of *Pseudomonas alkylphenol* strains already registered in GenBank. ​ ​ The MBH (PP334175.1) sequence showed 100% homology. Phylogenetic tree analysis revealed that strain S4 was related to *Pseudomonas alkylphenol* (…). ​ The highest homology was found in the group that clustered into a single branch ( ). ​ The results of morphological analysis indicate that strain S4 is *Pseudomonas alkylphenol* (…). ​ ).

[0083] The 16S rDNA sequence of strain S38 is 1398 bp in length. A BLAST comparison of this sequence with the NBCI GenBank database showed that the sequence matched that of *Pseudomonas oleifera* already registered in the GenBank database. ​ ​ The Rhs-L7 (OQ605543.1) sequence showed 100% homology. Phylogenetic tree analysis revealed that strain S38 was related to *Pseudomonas oleifera* (…). ​ The highest homology was found in the group that clustered into a single branch ( ). Figure 2 The results of morphological analysis indicate that strain S38 is *Pseudomonas oleifera* (…). 食油假单胞菌 ).

[0084] The 16S rDNA sequence of strain S42 is 1399 bp in length. A BLAST comparison of this sequence with the NBCI GenBank database showed that the sequence matched that of *Pseudomonas putida* already registered in the GenBank database. 恶臭假单胞菌 The sequence 31920-1 (FJ932760.1) showed 100% homology. Phylogenetic tree analysis revealed that strain S42 was related to *Pseudomonas putida* (…). 恶臭假单胞菌 The highest homology was found in the group that clustered into a single branch ( Figure 2 The results of morphological analysis indicate that strain S42 is *Pseudomonas putida* (…). 恶臭假单胞菌 ).

[0085] Based on the above morphological and molecular biological identification and analysis results of the strain, the isolated S4 strain was identified as *Pseudomonas alkylphenol*. 烷基酚假单胞菌 Strain S38 was identified as *Pseudomonas oleifera* (…). 食油假单胞菌 Strain S42 was identified as *Pseudomonas putida* (…). 恶臭假单胞菌 The three strains were biopreserved.

[0086] Example 2: Alkylphenol Pseudomonas ( 烷基酚假单胞菌 S4, Pseudomonas oleifera ( 食油假单胞菌S38 and Pseudomonas putida ( 恶臭假单胞菌 Functional identification of S42

[0087] 1. Antibacterial performance evaluation:

[0088] Strains S4, S38, and S42, individually and collectively (SynCom_3), were subjected to plate confrontation experiments against *Fusarium oxysporum* and *Fusarium solani*, respectively. A bacterial disc was inoculated at the center of the culture medium. Strains S4, S38, and S42 were then inoculated onto the culture medium plates using the streak method. For the collective strain (SynCom_3), strain S4 was inoculated on the left side of the plate, strain S38 on the top, and strain S42 on the right. The plates were incubated at 28 °C for 5 days, and the inhibition levels were observed and the inhibition rates were calculated.

[0089] Inhibition rate = [(control colony diameter - treated colony diameter) / control colony diameter] × 100%.

[0090] The results are as follows Figure 3 As shown, strains S4, S38, and S42, individually and collectively, all exhibited significant inhibition zones against both *Fusarium oxysporum* and *Fusarium solani*. The inhibition rates of strains S4, S38, S42, and the synthetic flora SynCom_3 against *Fusarium solani* were 80.8%, 79.7%, 76.1%, and 82.5%, respectively; and against *Fusarium oxysporum*, the rates were 40.7%, 44.4%, 77.8%, and 82.1%, respectively. Therefore, the synthetic flora composed of S4, S38, and S42 showed a more significant inhibitory effect on the pathogens.

[0091] 2. Evaluation of growth-promoting properties:

[0092] (1) Phosphorus solubility performance evaluation:

[0093] Strains S4, S38, and S42 were inoculated into phosphate-solubilizing medium and cultured for 3 days. The presence of a clear zone around the strain was observed. If a clear zone was present, it indicated that the strain possessed the ability to solubilize organic phosphorus.

[0094] (2) Potassium solubilization performance evaluation:

[0095] Strains S4, S38, and S42 were inoculated into potassium-solubilizing medium and cultured for 3 days. The presence of a clear zone around the strain was observed. If a clear zone was present, it indicated that the strain possessed potassium-solubilizing properties.

[0096] (3) Investigation of iron-producing carriers:

[0097] Strains S4, S38, and S42 were inoculated into CAS medium and cultured for 3 days. Observe whether a clear zone is formed around the strain. If so, it indicates that the strain has the ability to produce hydroxyl-type siderophores.

[0098] (4) Ammonia production detection:

[0099] Strains S4, S38, and S42 were inoculated into test tubes containing 10 mL of 5% peptone broth and cultured at 28°C for 7 days. After adding 1 mL of Nesler's reagent, the color change was observed. If the color turned yellowish-brown, it indicated that ammonia was produced during the growth of the strain.

[0100] (4) Detection of endothelin (IAA):

[0101] Strains S4, S38, and S42 were inoculated into LB liquid medium containing 1 g / L tryptophan and cultured at 28°C for 7 days. The filtrate was filtered through Whatman filter paper, and 1 mL of the filtrate was mixed with 2 mL of Salkowski reagent in a test tube and incubated at room temperature for 20 min. The color change was observed; if the color turned pink, it indicated the production of IAA.

[0102] The results of the growth-promoting performance tests of strains S4, S38, and S42 are shown in Table 1.

[0103] Table 1: Results of growth-promoting performance test of strains

[0104]

[0105] Note: In the table, "+" indicates that the relevant performance is present; "-" indicates that the relevant performance is not present.

[0106] 3. Bacterial compatibility test:

[0107] Strawberries S4, S38, and S42 were streaked in pairs on LB agar. The plates were then sealed and incubated at 37°C for 24 hours. The presence of inhibition zones was observed. If no inhibition zones were observed, the tested strains were compatible and could be co-cultured. If inhibition zones were observed, the strains were incompatible and antagonistic effects would occur during co-culture.

[0108] The results are as follows Figure 4 As shown, no obvious inhibition zone was formed between the cross lines where the three strains intersected, indicating that the three strains are compatible and do not have antagonistic effects, and can be co-cultured.

[0109] 4. Colonization capacity test:

[0110] Strains S4, S38, and S42 were inoculated onto LB liquid medium and cultured to obtain an initial concentration of 10. 8CFU / mL bacterial suspensions were prepared. Apple seedlings were treated with bacterial suspensions of strains S4, S38, and S42 at a rate of 100 mL / kg soil. Soil samples were collected on days 0, 3, 6, 12, 18, 24, 30, 40, 50, 60, and 90. The samples were rapidly frozen in liquid nitrogen and stored. Soil DNA was extracted and analyzed by qPCR after all samples were collected. The colonization rate in the soil was calculated according to the standard curve of each strain.

[0111] The results are as follows Figure 5 As shown, after a brief adaptation period following application to the soil, strains S4, S38, and S42 exhibited rapid growth in bacterial populations, peaking around the third day. They then entered a slow decline phase, maintaining a population density of around 10 from day 3 to day 30. 5 After 30 days, the population of the inoculated strain with CFU / g or higher slowly and continuously declined, eventually stabilizing at a sustainable level after 60 days, and remaining at 10 at 90 days. 3 The CFU / g viable count indicates that strains S4, S38, and S42 have the ability to survive in the soil for a long time.

[0112] Example 3: Pot Experiment

[0113] 1. Experimental Design

[0114] The pot experiment was conducted from March to October 2021 at the National Apple Engineering Experimental Center on the South Campus of Shandong Agricultural University. The test plant was Pingyi Sweet Tea (Pingyi Sweet Tea). 湖北海棠 Seedling cultivation. Pingyi sweet tea seeds were stratified at 4 ℃ for approximately 30 days. Once the seeds showed signs of sprouting, they were 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 with different treatments.

[0115] The soil samples were taken from a 38-year-old apple orchard in Manzhuang, Daiyue District, Tai'an City, Shandong Province, China. Multiple random samples were taken from an area 80cm from the tree trunk and 10-40cm deep, and the soil was thoroughly mixed. The soil texture was sandy loam.

[0116] The S4, S38, S42, and SynCom_3 inoculants used in potted plants are solid inoculants. Solid inoculants are prepared using the following method:

[0117] Alkylphenol Pseudomonas ( 烷基酚假单胞菌 S4, Pseudomonas oleifera ( 食油假单胞菌 S38, *Pseudomonas putida* ( 恶臭假单胞菌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 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 S4, S38, and S42 were then mixed at a viable cell ratio of 1:1:1 to obtain the SynCom_3 activated bacterial solution.

[0118] Activated bacterial cultures of S4, S38, S42, and SynCom_3 were inoculated into LB medium and cultured at 30°C and 180 rpm for 48 hours to obtain S4, S38, S42, and SynCom_3 fermentation broths, respectively. The viable cell count in each fermentation broth was adjusted to ensure a viable cell count of 10-1. 8 cfu / ml.

[0119] The S4, S38, S42, and SynCom_3 fermentation broths, after adjusting the viable cell count, were mixed with sterilized carriers at a weight ratio of 1:10 and fermented at 30°C for 8 days to obtain S4 solid inoculant, S38 solid inoculant, S42 solid inoculant, and SynCom_3 solid inoculant, respectively. The carrier was a mixture of cow dung and corn stalks at a weight ratio of 3:1.

[0120] This experiment included seven treatments: continuous cropping control (CK1), methyl bromide fumigation control (CK2), blank carrier treatment (CK3), S4 solid inoculant treatment (S4), S38 solid inoculant treatment (S38), S42 solid inoculant treatment (S42), and SynCom_3 treatment (SynCom_3).

[0121] Twenty pots were used per treatment, with one seedling planted in each pot, and uniform fertilizer and water management was implemented. CK1 used only continuously cropped soil; CK2 used continuously cropped soil treated with methyl bromide fumigation; CK3 used continuously cropped soil with a carrier added; S4, S38, S42, and SynCom_3 used continuously cropped soil with the corresponding solid inoculant added. The amount of carrier and solid inoculant used was 1% of the soil mass. Before planting, the continuously cropped soil was thoroughly mixed with the carrier or solid inoculant. Samples were taken 90 days after the treatment to measure the following indicators.

[0122] 2. Measurement Indicators

[0123] 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.

[0124] Soil microorganism determination: The number of culturable bacteria and fungi in the soil was determined using LB and PDA mediums respectively by plate dilution spread method (Shen Ping and Chen Xiangdong, 2007). Subsequently, 0.5g of sieved fresh soil was taken, and DNA was extracted using a soil DNA extraction kit. The gene copy number of four Fusarium species in the soil was determined using a CFX96™ Thermal Cycler (Bio-Rad).

[0125] Soil enzyme determination: Fresh soil from the rhizosphere of Pingyi sweet tea seedlings was collected and dried according to the method of Guan Songyin et al. (1986), and then passed through a 2 mm sieve. Urease activity was determined by the indophenol blue colorimetric method, neutral phosphatase activity by the disodium phenyl phosphate colorimetric method, sucrase activity by the 3,5-dinitrosalicylic acid colorimetric method, and catalase activity by potassium permanganate titration.

[0126] Root vigor assay: Remove the root tip and upper old roots, take 0.4-0.5g of roots, cut them into 2cm long segments, and place them in test tubes. For the control, add 2mL of 1mol / L sulfuric acid. For the other test tubes, add equal volumes of 0.4% TTC and phosphate buffer (1 / 15mol / L, pH=7.0) and mix 10mL. Seal the tubes and place them in a 37℃ incubator for 4h. Remove the tubes and, except for the control, add 2mL of 1mol / L sulfuric acid to the other tubes to stop the reaction. After 15min, remove the roots, blot dry, and return them to the original test tubes. Add 10mL of 95% ethanol to each test tube, seal the tubes, and extract for 24h until the roots turn white. Dilute the roots 3-5 times according to the color and compare the color at 485nm.

[0127] Data Analysis: All statistical analyses were performed using IBM SPSS 26.0 (IBM SPSS Statistics, IBM Corporation, Armonk, NY, United States). Different lowercase letters indicate significant differences between treatments (one-way ANOVA, p < 0.05), based on Duncan's multiple range test. Graphs were plotted using Microsoft Excel 2013 and GraphPad Prism 7.0.

[0128] 3. Test Results

[0129] (1) Effect of applying synthetic microbial agents on the biomass of Pingyi sweet tea seedlings

[0130] As shown in Table 2, under potted conditions, the application of SynCom_3, a synthetic microbial solid inoculant, to the soil after continuous cropping significantly promoted the increase of biomass in Pingyi sweet tea seedlings. The plant height, diameter at root, fresh weight, and dry weight increased by 31.80%, 38.54%, 113.96%, and 78.20%, respectively. Although the seedling growth after the synthetic inoculant treatment (SynCom_3) did not exceed that after the methyl bromide fumigation treatment (CK2), the effect was significantly better than that after continuous cropping control (CK1), microbial fertilizer carrier treatment (CK2), and single microbial fertilizer treatment (S4, S38, S42).

[0131] Table 2: Effects of SynCom_3 treatment on biomass of Pingyi sweet tea seedlings

[0132]

[0133] (2) Effects of applying synthetic inoculant SynCom_3 on culturable microorganisms in continuously cropped soil

[0134] As shown in Table 3, after treatment with synthetic microbial agent (SynCom_3), the content of culturable bacteria in the continuously cropped soil increased by 74.3% compared with the control (CK1), while the content of culturable fungi decreased by 55.80% compared with the control. This significantly improved the bacteria / fungus ratio in the continuously cropped soil, second only to the methyl bromide treatment (CK2) and significantly more effective than the single-strain microbial fertilizer treatment (S4, S38, S42).

[0135] Table 3: Effects of SynCom_3 treatment on culturable microorganisms in continuously cropped soil

[0136]

[0137] The copy numbers of four Fusarium species (Alternaria alternata, Cassia spp., Cassia spp., and Cassia spp.) in continuously cropped soil were determined by real-time fluorescence quantitative PCR. As shown in Table 4, the synthetic inoculant treatment (SynCom_3) can significantly reduce the number of the four Fusarium species in the soil, and its overall effect is significantly better than that of the single-strain inoculant treatment (S4, S38, S42). This indicates that the application of synthetic inoculants in continuously cropped soil can effectively inhibit the reproduction of pathogens.

[0138] Table 4: Effects of SynCom_3 treatment on pathogen copy number in continuously cropped soil

[0139]

[0140] (3) Effects of applying synthetic inoculant SynCom_3 on soil enzyme activity in continuously cropped soil

[0141] As shown in Table 5, the treatment with the synthetic microbial agent SynCom_3 significantly increased the enzyme activity in the soil. Compared with the control soil (CK1), the levels of sucrase, urease, neutral phosphatase, and catalase were 2.85, 1.88, 2.86, and 1.78 times higher than those of the control, respectively. Furthermore, the synthetic microbial agent treatment (SynCom_3) showed a more significant promoting effect than the single-strain microbial fertilizer treatments (S4, S38, and S42).

[0142] Table 5: Effects of SynCom_3 treatment on soil enzyme activity in continuously cropped soil

[0143]

[0144] (4) Effect of application of synthetic inoculant SynCom_3 on root vigor of Pingyi sweet tea seedlings

[0145] As shown in Table 6, treatment with the synthetic inoculant SynCom_3 can increase the root activity of Pingyi sweet tea seedlings by 1.88 times that of the continuous cropping soil control (CK1) and slightly higher than that of the methyl bromide fumigation treatment (CK2).

[0146] Table 6: Effects of SynCom_3 treatment on root vigor of Pingyi sweet tea seedlings

[0147]

[0148] Example 4: Field Experiment

[0149] 1. Experimental Design

[0150] The field experiments were conducted in mid-April 2025 in three locations: Xicheng Town, Qixia City, Yantai City, Shandong Province; Kunyu Town and Wanggezhuang Town, Muping District, Yantai City, Shandong Province. One-year-old grafted seedlings were used as the rootstock (M9T337) and the scion (Yanfu 9). Seedlings with consistent growth were selected from each experimental site (due to the distance between the three sites, the one-year-old grafted seedlings were purchased by the farmers themselves; the initial growth of seedlings varied across different sites, but the growth of seedlings treated at the same site remained consistent). The solid inoculant SynCom_3 used in the experiments was the same as in the pot experiments.

[0151] The field experiment consisted of three treatments: continuous cropping soil (CK1), blank microbial fertilizer carrier substrate treatment (CK2), and synthetic microbial agent SynCom_3 treatment (SynCom_3). Each treatment had 10 trees. The amount of microbial fertilizer and carrier used was 1% of the mass of the soil excavated from the tree pit. Before planting, the excavated soil and microbial fertilizer were thoroughly mixed and backfilled.

[0152] 2. Measurement Indicators and Methods

[0153] Biomass indicators such as plant height, stem length, number of branches, and branch length of one-year-old grafted seedlings in the field were measured 120 days after treatment with solid inoculant. Plant height and branch length were measured with a meter stick, and stem diameter was measured with a vernier caliper.

[0154] 3. The growth-promoting effect of the synthetic inoculant SynCom_3 on one-year-old grafted seedlings in continuously cropped apple orchards under field conditions.

[0155] As shown in Table 7, under field conditions, compared with the continuous cropping control (CK1), treatment with the synthetic inoculant SynCom_3 (SynCom_3) promoted the increase of biomass in one-year-old grafted apple seedlings. Specifically, the plant height increased by 23.1%, 43.4%, and 20.0% in the Xicheng Town, Wanggezhuang Town, and Kunyu Town experimental sites, respectively, while the rootstock length increased by 15.8%, 46.4%, and 21.9%, respectively. Furthermore, the number and length of branches also increased to varying degrees in the three experimental sites.

[0156] Table 7: Effects of SynCom_3 treatment on one-year-old grafted seedlings in continuously cropped apple orchards under field conditions.

[0157]

[0158] The results showed that the growth-promoting effect of the solid inoculant SynCom_3 in the field environment proved that this strain of bacteria performed excellently in reducing apple replanting obstacles in actual field production and can be used for the prevention and control of apple replanting obstacles.

[0159] 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 Pseudomonas complex, characterized in that, Pseudomonas alkylphenolica (CGMCC No. 36078) Pseudomonas alkylphenolica ) S4, Pseudomonas oleovorans (CGMCC No. 36079) Pseudomonas oleovorans ) S38 and Pseudomonas putida (CGMCC No. 36080) Pseudomonas putida ) S42.

2. The Pseudomonas complex according to claim 1, characterized in that, The Pseudomonas complex includes alkylphenol Pseudomonas (Pseudomonas). Pseudomonas alkylphenolica S4, Pseudomonas oleifera ( Pseudomonas oleovorans S38 and Pseudomonas putida ( Pseudomonas putida The ratio of live bacteria in S42 is 1:1:

1.

3. A microbial agent, characterized in that, The bacterial agent contains the Pseudomonas complex as described in claim 1 or 2.

4. The microbial agent according to claim 3, characterized in that, In the bacterial agent, the Pseudomonas complex exists in one or more forms, including cultured live bacteria, bacterial suspension, and fermentation broth.

5. The microbial agent according to claim 4, characterized in that, The fermentation broth is prepared by 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 cultured at 30°C and 180 rpm for 24-48 hours to obtain the fermentation broth.

6. The microbial agent according to claim 3, characterized in that, The bacterial agent is in the form of a liquid bacterial agent or a solid bacterial agent.

7. The application of the Pseudomonas complex of claim 1 or the inoculant of claim 3 in inhibiting the growth of plant pathogens, characterized in that, The plant pathogen is Fusarium oxysporum ( ). Fusarium oxysporum ) or Fusarium solani ( Fusarium solani) .

8. The use of the Pseudomonas complex of claim 1 or the microbial agent of claim 3 in any one of the following (1)-(5): (1) Produces gluten; (2) Secretion of siderophores; (3) Phosphorus dissolution; (4) Potassium dissolution; (5) Ammonia production.

9. The use of the Pseudomonas complex of claim 1 or the microbial agent of claim 3 in the following (1) or (2): (1) Reduce the obstacles of continuous cropping of apples; (2) Prepare biocontrol agents to reduce the obstacle of continuous cropping of apples.

10. A biocontrol agent for mitigating continuous cropping obstacles in apples, characterized in that, The biocontrol agent uses the fermentation broth of the Pseudomonas complex as described in claim 1 as its active ingredient; The biocontrol agent is prepared by the following method: The fermentation broth of the Pseudomonas complex was mixed with the sterilized carrier at a weight ratio of 1:10 and fermented at 30°C for 7-10 days. The fermentation broth of the Pseudomonas complex was prepared by 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 solution was inoculated into LB medium and cultured in a shaker at 30℃ and 180rpm for 24-48 hours to obtain the fermentation broth; The carrier is made of cow dung and corn stalks mixed in a weight ratio of 3:

1.

11. The use of the biocontrol agent according to claim 10 in either (1) or (2) below: (1) Promotes the growth of apple seedlings planted in continuously cropped soil; (2) Increase the number of bacteria / fungi in the apple root soil under continuous cropping conditions.

12. A method for mitigating continuous cropping obstacles in apple orchards, characterized in that, Includes the following steps: Apply the microbial agent of claim 3 or the biocontrol agent of claim 10 to the soil in which apples are continuously cropped.

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