Pseudomonas chlororaphis subsp. aurantiaca, complex microbial agent and application thereof in relieving apple continuous cropping obstacles

Abstract

The application discloses a Pseudomonas chlororahis subsp. aurantiaca strain, a compound microbial inoculum and application of the compound microbial inoculum in relieving apple continuous cropping obstacles, and belongs to the technical field of agricultural microorganisms. Pseudomonas chlororaphis subsp.aurantiaca The application obtains a Pseudomonas chlororahis subsp. aurantiaca strain (S74) from a rhizosphere, the strain has antagonistic effects on various pathogenic bacteria causing apple continuous cropping obstacles, and has the abilities of producing protease, secreting iron carrier, dissolving inorganic phosphorus, fixing nitrogen and producing ammonia. The strain S74 is applied to apple continuous cropping soil in cooperation with AMF, so that the number of bacteria in the soil is increased, the number of pathogenic bacteria is reduced, plant growth is promoted, the chlorophyll content of leaves of an aboveground part of a plant is improved, AMF colonization in continuous cropping soil and a plant root system is promoted, and the strain S74 can relieve apple continuous cropping obstacles in cooperation with AMF. The application method of the strain S74 belongs to the category of biological control, and provides technical support for comprehensive green control of apple continuous cropping obstacles.

Description

Technical Field

[0001] This invention relates to the field of agricultural microbial technology, specifically to a strain of Pseudomonas aeruginosa orange subspecies, a compound inoculant, and its application in alleviating continuous cropping obstacles in apples. Background Technology

[0002] Many aging orchards repeatedly plant the same variety of apple, leading to stunted trees, root necrosis, reduced yield and quality, and consequently, apple replanting obstacle (ARD). It is generally believed that soil microbial community imbalance is the main cause of ARD (Mazzola and Manici, 2012; Manici et al., 2018). Wang et al. (2018) showed a significant positive correlation between Fusarium fungi and the severity of ARD in the Bohai Bay region of China, and identified Fusarium as the main pathogen causing ARD in this region.

[0003] Chemical fumigation is the most effective measure to control continuous cropping obstacles in apple orchards. For example, fumigating continuously cropped soil with methyl bromide is the most effective way to overcome these obstacles. However, methyl bromide fumigation easily pollutes the environment, harms the atmospheric ozone layer, and is detrimental to sustainable ecological development. Therefore, chemical control has gradually been replaced by biological control. Biological control, as a green and environmentally friendly method, utilizes the antagonistic effect between beneficial and harmful microorganisms to reduce the impact of pathogens and protect plants from disease infection (Gerbore et al., 2014). Currently, Bacillus, Pseudomonas, and Streptomyces are widely used as biological control agents.

[0004] Arbuscular mycorrhizal fungi (AMFs) are rich in germplasm and have wide ecological adaptability. They can promote nutrient absorption by expanding the root system's absorption area (Bhantana et al., 2021), while also enhancing the activity of protective enzymes and inducing the synthesis of secondary metabolites, thereby improving the soil environment in continuous cropping and enhancing plant tolerance to abiotic or biotic stresses and resistance to soil-borne diseases (Wen et al., 2025). However, existing AMFs suffer from insufficient infection rates during use, limiting their application. Mycorrhizal bacteria are specific bacteria that colonize the rhizosphere microdomain after AMFs form mycorrhizae with plant roots (Song Xiaoshuang et al., 2020). They participate in the "plant-AMF-bacteria" symbiotic network and possess core functions such as antagonizing pathogens, activating nutrients, and synergistically strengthening plant stress resistance with AMFs.

[0005] Pseudomonas aeruginosa orange subspecies ( Pseudomonas chlororaphis subsp. aurantiaca As an important functional group of the genus Pseudomonas, current systematic research on this group is still relatively limited, and mainly focuses on broad-spectrum antibacterial activity, plant growth promotion, and pesticide compatibility. However, there are no reports on the role of Pseudomonas aeruginosa orange subspecies in synergistic control of apple continuous cropping obstacles with AMF. Summary of the Invention

[0006] In view of the above-mentioned prior art, the purpose of this invention is to provide a strain of Pseudomonas aeruginosa orange subspecies, a compound inoculum, and its application in alleviating apple continuous cropping obstacles.

[0007] Specifically, the present invention relates to the following technical solutions:

[0008] In a first aspect, the present invention provides a strain of *Pseudomonas aeruginosa* subsp. orange (… Pseudomonas chlororaphis subsp. aurantiaca S74, this strain was deposited on November 21, 2025, at the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing), with accession number CGMCC No. 36725; its classification name is: *Pseudomonas aeruginosa* subsp. orange. Pseudomonas chlororaphis subsp. aurantiaca .

[0009] The present invention, *Pseudomonas aeruginosa* subspecies S74, has the following characteristics:

[0010] (1) It can secrete siderophores, fix nitrogen, produce proteases, dissolve inorganic phosphorus and produce ammonia;

[0011] (2) It has broad-spectrum antibacterial activity against Fusarium oxysporum ( ). Fusarium oxysporum Fusarium solani ( ), Fusarium solani Fusarium moniliforme ( Fusarium moniliforme ) and apple-specific Fusarium moniliforme ( Fusarium proliferatum MR5 has good antibacterial effects.

[0012] (3) It can promote AMF infection, increase the amount of AMF in the soil and the mycorrhizal infection rate of apple roots.

[0013] (4) It can alleviate the problem of continuous cropping of apples.

[0014] In a second aspect, the present invention provides a microbial agent containing the above-mentioned Pseudomonas aeruginosa orange subspecies S74.

[0015] Preferably, in the bacterial agent, *Pseudomonas aeruginosa* subspecies S74 exists in the form of cultured live bacteria, fermentation broth, or bacterial suspension.

[0016] In some preferred embodiments of the present invention, the fermentation broth is prepared by the following method:

[0017] Inoculate Pseudomonas aeruginosa orange subspecies S74 into the fermentation medium and ferment at 25-30℃ for 30-40 hours.

[0018] In some preferred embodiments of the present invention, the bacterial suspension is prepared by the following method:

[0019] Centrifuge the fermentation broth of Pseudomonas aeruginosa orange subspecies S74 and collect the bacterial precipitate; resuspend the bacterial precipitate in physiological saline to obtain a bacterial suspension.

[0020] A third aspect of the present invention provides the use of the above-mentioned *Pseudomonas aeruginosa* subspecies S74 or the inoculum in at least one of the following (1)-(4):

[0021] (1) Inhibit the growth of plant pathogens;

[0022] (2) Prepare products for inhibiting plant pathogens;

[0023] (3) Prevention and control of diseases caused by plant pathogens;

[0024] (4) Prepare products for the prevention and control of diseases caused by plant pathogens.

[0025] In the above application, the plant pathogen is Fusarium oxysporum (Fusarium oxysporum). Fusarium oxysporum Fusarium solani ( ), Fusarium solani Fusarium moniliforme ( Fusarium moniliforme ) and apple-specific Fusarium moniliforme ( Fusarium proliferatum One or more of MR5.

[0026] In a fourth aspect, the present invention provides the use of the above-mentioned *Pseudomonas aeruginosa* subspecies S74 or the inoculum in the following (1) or (2):

[0027] (1) Alleviating apple cropping obstacles;

[0028] (2) Prepare biocontrol agents to alleviate the continuous cropping obstacles of apple trees.

[0029] In a fifth aspect, the present invention provides the application of the above-mentioned *Pseudomonas aeruginosa* subspecies S74 or fungal agent in promoting the colonization of arbuscular mycorrhizal fungi in continuously cropped soils or plant roots.

[0030] In a sixth aspect, the present invention provides a compound microbial agent, which is composed of the above-mentioned microbial agent of Pseudomonas aeruginosa orange subspecies S74 and AMF microbial agent;

[0031] The AMF is a genus of mycorrhizal fungi with accession number CGMCC NO.20744. (Paraglomus sp.) .

[0032] In some preferred embodiments of the present invention, the AMF inoculant is prepared by the following method:

[0033] First, the AMF strain was inoculated into the roots of clover that had been growing for one week, and single-spore culture was carried out in a greenhouse for one month to further propagate the bacteria. The clover roots were then crushed to obtain powder containing AMF. The sterilized substrate and the powder containing AMF were mixed at a mass ratio of 3:1, and sterilized clover seeds were sown to expand the propagation under greenhouse conditions. After three months, the infection rate was tested. When the infection rate was greater than 50%, the above-ground parts were removed, and the clover roots and substrate were crushed together to obtain the AMF inoculum.

[0034] Preferably, in the compound bacterial agent, the bacterial agent of *Pseudomonas aeruginosa* subsp. orange S74 exists in the form of a bacterial suspension, and the effective viable bacteria concentration in the bacterial suspension is 1×10⁻⁶. 8 CFU / mL; the equivalent ratio of Pseudomonas aeruginosa orange subspecies S74 inoculum to AMF solid inoculum is 1:1.

[0035] Preferably, the total application amount of the compound microbial agent is 2% of the soil weight. The application method of the compound microbial agent is as follows: taking 1 kg of soil as an example, first mix in 10 g of the AMF microbial agent, and after the plant has been growing stably for two weeks, then irrigate the root zone of the plant with 10 mL of the S74 microbial suspension.

[0036] A seventh aspect of the present invention provides the use of the above-mentioned compound microbial agent in at least one of the following (1)-(6):

[0037] (1) Increase the ratio of bacteria to fungi in soil where apples are continuously cropped;

[0038] (2) Improve the root vigor of apple seedlings;

[0039] (3) Increase the chlorophyll content of the aboveground leaves of apple seedlings;

[0040] (4) Increase the amount of AMF in soils continuously cropped with apples;

[0041] (5) Increase the AMF mycorrhizal infection rate of the underground root system of apple seedlings;

[0042] (6) Alleviate the problem of continuous cropping of apples.

[0043] Under potted conditions, applying strain S74 in conjunction with AMF to apple continuous cropping soil can increase the number of bacteria in the soil, reduce the number of pathogens, promote plant growth, increase the root respiration rate, and increase the chlorophyll content of the above-ground leaves. At the same time, it can promote the colonization of AMF in continuous cropping soil and plant roots. Strain S74 can work with AMF to alleviate apple continuous cropping obstacles.

[0044] The beneficial effects of this invention are:

[0045] This invention is the first to isolate an orange subspecies of *Pseudomonas aeruginosa* from the rhizosphere of *Malus*. Pseudomonas chlororaphis subsp. aurantiaca S74 strain significantly inhibits four major pathogens causing continuous cropping obstacles in apples: *Fusarium oxysporum*, *Fusarium solani*, *Fusarium moniliforme*, and *Fusarium leucosus* MR5, a specialized type of *Fusarium*. Under potted conditions, applying strain S74 in conjunction with AMF to continuously cropped apple soil increases the number of bacteria in the soil, reduces the number of pathogens, promotes plant growth, increases root respiration rate, and enhances chlorophyll content in the above-ground leaves. Simultaneously, it promotes the colonization of AMF in the continuously cropped soil and plant roots. Strain S74, in synergy with AMF, can alleviate continuous cropping obstacles in apples. Attached Figure Description

[0046] Figure 1 Morphological image of strain S74 on a TSA plate.

[0047] Figure 2 Gram staining image of strain S74.

[0048] Figure 3 Phylogenetic tree of the 16S rDNA sequence of strain S74.

[0049] Figure 4 Results of extracellular enzyme production capacity of strain S74: a) Siderophore secretion capacity of strain S74; b) Nitrogenase production capacity of strain S74; c) Protease production capacity of strain S74; d) Inorganic phosphorus decomposition capacity of strain S74; e) Ammonia production capacity of strain S74.

[0050] Figure 5 : antagonistic experiment diagram of strain S74; where a is the confrontation experiment between strain S74 and Fusarium moniliforme; b is the confrontation experiment between strain S74 and Fusarium solani; c is the confrontation experiment between strain S74 and Fusarium oxysporum; d is the confrontation experiment between strain S74 and MR5.

[0051] Figure 6 Effects of different treatments on the biomass of Pingyi sweet tea seedlings.

[0052] Figure 7 The effect of different treatments on the ratio of culturable bacteria to fungi in soil.

[0053] Figure 8 The effects of different treatments on the gene copy number of four Fusarium species in apple-continuously cropped soil; where a represents the copy number detection results of Fusarium rotundum; b represents the copy number detection results of Fusarium oxysporum; and c represents the copy number detection results of Fusarium moniliforme.

[0054] Figure 9 Effects of different treatments on root vigor of Pingyi sweet tea seedlings.

[0055] Figure 10Effects of different treatments on chlorophyll content in leaves of Pingyi sweet tea seedlings. 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 mentioned earlier, continuous cropping obstacles in apple orchards can lead to stunted growth, root necrosis, and reduced apple yield and quality. Arbuscular mycorrhizal fungi (AMFs) are rich in germplasm and have wide ecological adaptability. They can promote nutrient absorption by expanding the root system's absorption area, while also enhancing the activity of protective enzymes and inducing the synthesis of secondary metabolites. This, in turn, can improve the soil environment after continuous cropping, enhance the plant's tolerance to abiotic and biotic stresses, and increase its resistance to soil-borne diseases. However, AMFs suffer from insufficient infection rates during use, which limits their application.

[0058] In view of this, the present invention isolated a bacterium from the rhizosphere of *Pseudomonas aeruginosa*, named S74. Based on morphological, physiological and biochemical identification and 16S rDNA phylogenetic analysis, this bacterium was identified as *Pseudomonas aeruginosa* subsp. *orange*. Pseudomonas chlororaphis subsp. aurantiaca This invention employs the plate confrontation method to study the antagonistic effects of *Fusarium oxysporum*, *Fusarium moniliforme*, *Fusarium solani*, and *Fusarium leptospirum* MR5, all of which cause continuous cropping obstacles in apples. The strains of this invention produce proteases, secrete siderophores, can dissolve inorganic phosphorus, have nitrogen-fixing capabilities, and can produce ammonia. Under pot conditions, strain S74 of this invention can synergistically apply AMF to continuously cropped apple soil, increasing the number of bacteria in the soil, reducing the number of pathogens, promoting plant growth, increasing root respiration rate, and increasing chlorophyll content in the above-ground leaves. Simultaneously, it promotes the colonization of AMF in continuously cropped soil and plant roots. Strain S74 can synergistically alleviate continuous cropping obstacles in apples.

[0059] 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:

[0060] The culture medium used in this invention has the following composition:

[0061] PDA medium: 200.0 g peeled potato, 20.0 g glucose, 20.0 g agar, 1000 mL distilled water.

[0062] TSA medium: 15.0 g tryptone, 5.0 g soybean papain hydrolysate, 5.0 g sodium chloride, 15.0 g agar, pH 7.3±0.2, 1000 mL distilled water.

[0063] Fermentation medium: 15.0 g tryptone, 5.0 g soybean papain hydrolysate, 5.0 g sodium chloride, pH 7.3±0.2, 1000 mL distilled water.

[0064] Gao's No. 1 culture medium: 20g soluble starch, 0.5g sodium chloride, 1.0g potassium nitrate, 0.5g dipotassium hydrogen phosphate trihydrate, 0.5g magnesium sulfate heptahydrate, 0.01g ferrous sulfate heptahydrate, 15-20g agar, pH 7.4-7.6, 1000mL distilled water.

[0065] LB medium: 10.0 g peptone, 5.0 g yeast extract, 10.0 g sodium chloride, 1000 mL distilled water.

[0066] Protease culture medium: 10.0 g skim milk powder, 20.0 g agar, 1000 mL distilled water.

[0067] CAS detection medium: acid-hydrolyzed casein 3.0 g, chromaine S 0.0605 g, hexadecyltrimethylammonium bromide (HDTMA) 0.0729 g, ferric chloride hexahydrate 0.002645 g, disodium hydrogen phosphate 3.58 g, ammonium chloride 1.0 g, potassium dihydrogen phosphate 3.0 g, sodium chloride 0.5 g, magnesium sulfate 0.05 g, calcium chloride 0.01 g, agar 15.0 g, pH 6.8±0.2, distilled water 1000 mL.

[0068] Ammonia-producing medium: liquid medium of 5% peptone broth.

[0069] Inorganic phosphorus bacteria culture medium: glucose 10.0 g, ammonium sulfate 0.5 g, sodium chloride 0.3 g, potassium chloride 0.3 g, potassium sulfate heptahydrate 0.3 g, ferrous sulfate heptahydrate 0.03 g, manganese sulfate tetrahydrate 0.03 g, calcium phosphate 5.0 g, agar 15.0 g, pH 7.0-7.5, distilled water 1000 mL.

[0070] Assab's medium for nitrogen-fixing bacteria: mannitol 10 g, potassium dihydrogen phosphate 0.2 g, magnesium sulfate heptahydrate 0.2 g, sodium chloride 0.2 g, calcium sulfate dihydrate 0.1 g, calcium phosphate 5 g, agar powder 18 g, pH 6.9±0.1, distilled water 1000 mL.

[0071] The pathogen used in this invention is *Fusarium moniliforme*, a species of apple-specific fungus. Fusarium proliferatumMR5 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 22426, on May 17, 2021, and is described in patent CN113881573B. The *Fusarium moniliforme* used is... F. moniliforme AMCC300086), Fusarium solani ( F. solani AMCC300028), Fusarium oxysporum ( F. oxysporum AMCC300029 was obtained from the Shandong Agricultural Microbial Culture Collection Center of Shandong Agricultural University, and all of these are pathogens already existing in the prior art. The AMF strain used in this invention is a mycorrhizal fungus of the genus *Gloydiomycota* (…). Paraglomus sp. SW1, this strain was deposited on October 16, 2020 at the China General Microbiological Culture Collection Center, with the biological accession number: CGMCC NO.20744, and is recorded in patent CN113337405B.

[0072] The sterilization substrate used in the preparation of AMF inoculant is a common commercially available seedling substrate, which can be obtained through commercial channels.

[0073] Example 1: Isolation and Identification of Strains

[0074] 1. Isolation and purification of strains:

[0075] In October 2024, rhizosphere soil and root systems from healthy apple trees inoculated with AMF in old orchards were collected from Zibo and Yantai and brought back to the laboratory. The dilution plate method was used for separation. Large impurities were filtered out from the collected soil samples using a sieve. 10 g of the mycotoxin rhizosphere soil was weighed and added to 90 mL of sterile deionized water. The mixture was shaken at 180 rpm for 30 min. The soil sample was then diluted to 10 g with sterile water. -3 -10 -6 150 μL of the sample was spread onto four different media: LB medium, TSA medium, nutrient agar (NA), and beef extract peptone medium. The mixture was spread evenly with a spreader and incubated at 37°C. Larger soil clumps were removed from the roots. The roots were repeatedly rinsed with sterile water and then disinfected with 75% ethanol solution for 2 minutes. After disinfection, the roots were rinsed four times with sterile water, placed in a sterilized mortar, and ground with MgCl2 until a homogenate was formed. The homogenate was then diluted with sterile water to a final concentration of 1:10. -2 -10 -4 150 μL of each culture medium was spread evenly on LB, TSA, NA, and beef extract peptone media, and incubated at 37°C. After 36 h of incubation, colonies with different morphologies were picked and streaked onto TSA plates for purification. A total of 125 bacterial strains were isolated and stored at 4°C.

[0076] 2. Screening of strains:

[0077] The antagonistic effect of isolated strains against MR5 was determined using the plate confrontation method. The preserved strains were inoculated onto fresh TSA medium for activation at 37°C for 24 h. The MR5 pathogen strain was then inoculated onto PDA medium for activation at 28°C for 5 days. From the activated pathogen, a bacterial cake was collected at the edge of the pathogen using a 0.25 mm blue pipette tip and placed in the center of a PDA plate. Activated strains were streaked around the bacterial cake at equal intervals. This process was repeated three times. PDA plates inoculated only with the pathogen served as a control. After 5 days of incubation at 28°C, the colony radius of the pathogen was measured using the cross-cross method. Bacterial strains with higher inhibition rates were selected for further screening.

[0078] The results showed that 26 strains had an inhibitory effect on MR5, among which strain No. 74, isolated from rhizosphere soil, had the highest inhibition rate of 66.67%, and was selected as the target strain and named S74.

[0079] The calculation formula is: Inhibition rate = (Control colony radius - Treated colony radius) / Control colony radius × 100%

[0080] 3. Identification of the strain:

[0081] (1) Morphological and physiological / biochemical identification:

[0082] The isolated strain S74 was streaked onto TSA medium and incubated at 37°C for 36 h. Single colonies grew, and the morphological characteristics of the colonies were observed.

[0083] The results are as follows Figure 1 As shown, a single colony is round, small in diameter, yellowish-white, produces orange-yellow pigment, has a bright and uniform color, a smooth and moist surface, neat edges, is opaque, and the colony is uniform in size with no light-transmitting areas.

[0084] Gram staining results of strain S74 are as follows Figure 2 As shown, bacteria appear red after Gram staining under a 40x optical microscope, indicating they are Gram-negative bacteria.

[0085] The physiological and biochemical identification of the strain was performed according to the methods described in Bergey's Manual of Bacteriological Identification (Second Edition) and the Manual of Systematic Identification of Common Bacteria. Physiological and biochemical tests were conducted on strain S74, and the results are shown in Table 1.

[0086] Table 1: Physiological and biochemical characteristics of strain S74

[0087]

[0088] (2) Molecular biological identification:

[0089] The 16S rDNA gene of strain S74 was amplified using universal primers 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' (sequence 1); 1492R: 5'-GGTTACCTTGTTACGACTT-3' (sequence 2). The PCR reaction mixture (50 μL) consisted of: 3 μL total DNA template, 1 μL each primer, 1 μL dNTP, 5 μL 10x PCR buffer, 0.6 μL Taq DNA polymerase, and 38.4 μL ddH2O (Weisburg et al., 1991). The PCR amplification program was: 94 °C pre-denaturation for 4 min; 94 °C denaturation for 1.5 min; 55 °C annealing for 1 min; 72 °C extension for 1.5 min; for a total of 30 cycles, with a final extension at 72 °C for 10 min, followed by incubation at 4 °C.

[0090] Primer synthesis and amplified fragment sequencing were performed by Riboscience. Sequencing results were BLAST-aligned using the NCBI nucleotide database, and a phylogenetic tree was constructed using MEGA 7, with bootstrap values ​​tested 1000 times.

[0091] The 16S rDNA sequence of strain S74 was determined to be 1406 bp in length, as shown in sequence 3.

[0092] The sequence was analyzed in the NBCI GenBank database and BLAST aligned. Strain S74 was found to be similar to *Pseudomonas aeruginosa* subsp. orange. Pseudomonas chlororaphis subsp. aurantiaca The similarity was 99%. A phylogenetic tree was constructed using MEGA 7.0 software based on the 16S rDNA of strain S74. Figure 3 ).

[0093] Based on morphological, physiological, biochemical, and molecular biological identification, strain S74 was determined to be *Pseudomonas aeruginosa* subsp. orange. Pseudomonas chlororaphis subsp. aurantiaca The strain was then preserved, and the preservation information is as follows:

[0094] Reference biological material (strain): S74

[0095] Classification and nomenclature: Pseudomonas aeruginosa orange subspecies Pseudomonas chlororaphis subsp. aurantiaca

[0096] Preservation Institution: China General Microbiological Culture Collection Center, China Microbiological Culture Collection Committee

[0097] Collection institution abbreviation: CGMCC

[0098] Address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing

[0099] Deposit date: November 21, 2025

[0100] Registration number at the Preservation Center: CGMCC No. 36725.

[0101] Example 2: Functional identification of Pseudomonas aeruginosa orange subspecies S74

[0102] 1. Detection of extracellular enzyme production capacity:

[0103] (1) Determination of siderophore production capacity: The activated strain S74 was inoculated onto CAS detection medium and cultured at 37℃ for 48h. The orange-yellow transparent ring was observed around the strain S74.

[0104] (2) Determination of nitrogen fixation ability: The activated strain S74 was inoculated onto Assabbey medium for nitrogen fixation bacteria and cultured at 37°C for 48 h. The strain was observed to see if it could grow normally on the medium. If it grew, it could fix nitrogen; otherwise, it could not fix nitrogen.

[0105] (3) Determination of protease production capacity: The activated strain S74 was inoculated onto the protease medium and cultured at 37°C for 48 h. The presence of a clear zone around the strain S74 was observed.

[0106] (4) Determination of phosphorus solubility: The activated strain S74 was inoculated onto an inorganic phosphorus bacteria culture medium and cultured at 37°C for 48 h. The presence of a transparent zone around the strain S74 was observed.

[0107] (5) Ammonia production capacity determination: The activated strain S74 was inoculated into an Erlenmeyer flask containing 10 mL of 5% peptone broth and cultured at 37°C for 48 h. After adding 1 mL of Nesler's reagent, the color turned orange, indicating that the S74 strain produced ammonia during its growth.

[0108] The results are as follows Figure 4 As shown, strain S74 produces a clear zone on protease medium and inorganic phosphorus bacteria medium, can grow normally on Assumption medium for nitrogen-fixing bacteria, and produces an orange-yellow clear zone on CAS detection medium; the results indicate that strain S74 has the ability to produce siderophores, protease, inorganic phosphorus, nitrogen fixation, and ammonia.

[0109] 2. Determination of broad-spectrum antibacterial effect:

[0110] The antagonistic activity of strain S74 against *Fusarium moniliforme*, *Fusarium solani*, *Fusarium oxysporum*, and *Fusarium leucosus* MR5 (a specific type of *Fusarium*) was evaluated using a plate confrontation assay. The cultures were incubated at 28°C for 5 days.

[0111] The results are as follows Figure 5 As shown, the results indicate that strain S74 has a significant inhibitory effect on all four pathogens. The inhibition rates of this strain against Fusarium moniliforme, Fusarium solani, Fusarium oxysporum, and MR5 were 62.35%, 62.96%, 60.76%, and 66.67%, respectively.

[0112] Inhibition rate = [(Control colony diameter - Treated colony diameter) / Control colony diameter] × 100%

[0113] The results indicate that strain S74 has broad-spectrum antibacterial activity against plant pathogens and is expected to become a promising biocontrol agent.

[0114] Example 3: Pot Experiment

[0115] 1. Experimental Design:

[0116] The pot experiment was conducted from March to October 2025 at the National Apple Engineering Experimental Center and the State Key Laboratory of Crop Biology, Shandong Agricultural University. Soil samples were taken from multiple random locations in a 38-year-old apple orchard in Manzhuang, Daiyue District, Tai'an City, Shandong Province, China. Samples were taken from 80 cm away from the tree trunk and at depths of 10–40 cm, and mixed thoroughly. The soil texture was sandy loam.

[0117] The S74 inoculant used for potted plants has an effective viable bacteria concentration of approximately 1×10⁻⁶. 8 S74 bacterial suspension at CFU / mL. The S74 bacterial suspension was prepared as follows: Activated *Pseudomonas chlororaphis* subsp. *aurantiaca* S74 was inoculated into the fermentation medium at a rate of 3% (v / v) and incubated at 30°C and 200 r·min. -1 After culturing for 36 hours, the fermentation broth was obtained. The broth was centrifuged at 4°C and 8000 rpm for 20 minutes, the supernatant was removed, and the cells were resuspended in physiological saline to OD0.05. 600 =1.

[0118] The AMF inoculant for potted plants was prepared by the following method: AMF strains were inoculated into the roots of clover plants that had grown for one week. Single-spore culture was carried out in a greenhouse for one month for further propagation. The clover roots were then pulverized to obtain AMF-containing powder. Sterilized substrate (common seedling substrate, commercially available) was mixed with the AMF-containing powder at a mass ratio of 3:1. Sterilized clover seeds were sown, and propagation was expanded under greenhouse and potted conditions. After three months, the infection rate was tested. If the infection rate was greater than 50%, the above-ground parts were removed, and the clover roots and substrate were pulverized together to obtain the AMF inoculant for subsequent experiments. The AMF was then air-dried in a cool place and stored at 4℃.

[0119] In March, seedlings of *Malus hupehensis* Rehd. were planted. The seeds were stratified at approximately 4℃ for 40 days until they sprouted, and then sown in March. A layer of sterilized substrate (common seedling substrate, commercially available) was first laid out and thoroughly watered. Some of the stratified seeds were sown in seedling pots containing sterilized substrate, while others were sown in seedling pots containing a mixture of sterilized substrate and AMF (Amino Acid) inoculant at a mass ratio of 3:1. At the end of April, healthy seedlings of similar size were transplanted into clay pots with a top diameter of 25 cm, a bottom diameter of 17 cm, and a height of 18 cm, each containing 7 kg of soil for the experiment.

[0120] Potted plant experimental treatment:

[0121] Five treatments were set up: continuous cropping soil control (CK1), continuous cropping soil treated with methyl bromide fumigation (Br), continuous cropping soil treated with AMF inoculant (AMF), continuous cropping soil treated with S74 inoculant (S74), and continuous cropping soil co-inoculated with AMF and S74 inoculants (S74+AMF).

[0122] Continuous cropping soil (CK1): As a blank control, no treatment was given to the continuous cropping soil. Seedlings of Pingyi sweet tea were transplanted from ordinary seedling pots.

[0123] Methyl bromide fumigation of continuously cropped soil (Br): Place the continuously cropped soil in a double-layered plastic film, put in a methyl bromide gas canister, and place a board of nails underneath the gas canister. After sealing the opening of the plastic film, tread through the methyl bromide gas canister. After fumigating for about three weeks, tear open the plastic film, ventilate for one week, and it can be used directly. Transplant Pingyi sweet tea seedlings from ordinary seedling pots.

[0124] AMF-treated continuous cropping soil (AMF): Add 70g of AMF to each pot of continuous cropping soil, mix well, and transplant Pingyi sweet tea seedlings grown in seedling pots containing AMF substrate.

[0125] S74 microbial agent treatment for continuous cropping soil (S74): Two weeks after transplanting Pingyi sweet tea seedlings into ordinary seedling pots, 70 mL of S74 microbial agent was added to each pot of continuous cropping soil.

[0126] Co-inoculation of continuous cropping soil with AMF and S74 microbial agents (S74+AMF): 70g of AMF microbial agent was added to each pot of continuous cropping soil, mixed evenly, and Pingyi sweet tea seedlings grown in seedling pots with AMF microbial agent substrate were transplanted. Two weeks after the seedlings grew, 70 mL of S74 microbial agent was added to each pot of continuous cropping soil.

[0127] Each replicate consisted of 12 pots, with uniform fertilizer and water management. Samples were collected in August 2025.

[0128] When sampling soil, remove the topsoil and take soil from 10 cm below the root zone. After collecting the fresh soil, pass it through a 20-mesh sieve as soon as possible and divide it into portions. Place one portion of the soil sample in a 4°C refrigerator for soil microbial spread counting; place another portion in a -80°C refrigerator for soil DNA extraction; and place a portion in a ventilated area to dry for determining other soil parameters.

[0129] When sampling the plants, three healthy Pingyi sweet tea seedlings of uniform size and growth were taken from each treatment. After breaking the pot, the surface soil and the soil around the pot were removed. The root zone soil, seedling leaves and roots were brought back to the laboratory. After cleaning the roots, white roots were immediately taken for root vigor testing and fresh weight measurement.

[0130] 2. Measurement Indicators

[0131] Biomass determination: Plant height and diameter at ground level were measured using a meter stick and a vernier caliper, respectively. The above-ground and underground roots of the plants were rinsed with tap water and dried. Fresh weight was measured on an electronic balance. Then, the plants were blanched at 102℃ for 30 min, dried at 60℃ to constant weight, and the dry weight was measured again.

[0132] Soil microorganisms were determined using the plate count method. Soil samples were diluted and spread onto LB medium, PDA medium, and Gao's No. 1 medium to calculate the number of bacteria, fungi, and actinomycetes in the soil.

[0133] Take a fresh rhizosphere soil sample and add it to 90 mL of sterile deionized water. Shake at 180 rpm for 30 min, then dilute with sterile water to a final concentration of 10. -2 10 -3 10 -4 The soil dilution was diluted, and 150 μL was added to the pre-prepared culture medium, spread evenly, and the number of microorganisms was recorded. Fungi were cultured on PDA medium with the addition of streptomycin and penicillin antibiotics to inhibit bacterial growth, and counted after 2-3 days of growth in an incubator at 28°C; actinomycetes were cultured on Gao's No. 1 medium with the addition of 3% potassium dichromate solution, and counted after about 7 days of growth in an incubator at 28°C; bacteria were cultured on LB medium and counted after 1-2 days of growth in an incubator at 37°C.

[0134] Quantitative Analysis of Arbuscular Mycorrhizal Fungi and Fusarium in Soil: Total DNA was extracted from soil using the EZNATM Soil DNA Extraction Kit (OMEGA). The copy numbers of genes from arbuscular mycorrhizal fungi, *Fusarium oxysporum*, *Fusarium solani*, and *Fusarium moniliforme* in the soil were quantitatively analyzed using a CFX96TM Thermal (Cycler Bio-Rad). First, standard curves for the ITS gene fragments of these four fungi were established. Then, using the primer pairs shown in Table 2, real-time fluorescence quantitative analysis of the gene copy numbers of the four fungi in the soil was performed. The PCR reaction system consisted of 25 μL: DNA template 1.5 μL; SYBR Premix Ex Taq II 12.5 μL; primers 1 μL each; ddH2O 9 μL. The reaction program is shown in Table 1, with a total of 40 cycles. The PCR products were verified by melting curve analysis and agarose gel electrophoresis to confirm their specificity and effectiveness. Based on the linear equation of gene copy number and Cycle threshold (Ct), the gene copy numbers of arbuscular mycorrhizal fungi, Fusarium oxysporum, Fusarium solani, and Fusarium moniliforme were calculated using the extrapolation method based on the Ct value of environmental samples.

[0135] Table 2: Primer sequences and amplification reaction procedures for fungi

[0136]

[0137] Soil AMF spore density: Spore density was calculated using the wet soil sieving method. A 10 g soil sample was taken from the mixed soil and placed in a 1 L beaker. 500 mL of sterile water was added, and the sample was stirred thoroughly. The sample was then passed through a 20-mesh sieve and a 400-mesh sieve. All residue from the 400-mesh sieve was transferred to a 50 mL centrifuge tube, centrifuged at 4000 rpm for 3 min, and the supernatant was removed. 20 mL of a 60% sucrose solution was added, and the mixture was stirred thoroughly. The sample was then centrifuged at 3000 rpm for 2 min to collect spores. All supernatant was transferred to a new petri dish, and the soil spore density was calculated under a stereomicroscope.

[0138] Mycorrhizal infection rate: Fresh root segments approximately 1 cm in length were selected and decolorized by boiling in 10% potassium hydroxide solution at high temperature for 20 min, acidified with 2% hydrochloric acid for 5 min, and stained at high temperature in 0.05% tribenzene blue lactic acid glycerol solution (lactic acid: glycerol = 1:1) for 30 min. Finally, they were placed in lactic acid glycerol solution (lactic acid: glycerol: water = 1: 1:1) for decolorization overnight. At least 100 root segments were observed under a microscope. The colonization rate of each root segment was assessed by the number of mycorrhizal fungal structures in each segment and expressed as 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100%. The mycorrhizal infection rate (%) was calculated as follows:

[0139] Mycorrhizal infection rate = ∑ (0 × number of root segments + 10% × number of root segments + 20% × number of root segments + ... + 100% × number of root segments) / total number of root segments.

[0140] Root activity assay: Take 0.5 g of white root, cut it into 2 cm long segments, place them in a test tube, add an equal volume of 0.4% TTC and phosphate buffer (1 / 15 mol / L, pH=7.0) and mix 10 mL. For the control, first add 2 mL of 1 mol / L sulfuric acid to terminate the reaction, then add an equal volume of 0.4% TTC and phosphate buffer (1 / 15 mol / L, pH=7.0) and mix 10 mL. Seal the tube and incubate at 37℃ for 4 h. Remove the tube after this step. For all other samples, add 2 mL of 1 mol / L sulfuric acid to terminate the reaction, let stand for 15 min, remove the roots, aspirate the solution, and return them to the test tube. Add 10 mL of 95% ethanol to each test tube, seal the tube, and extract for 24 h until the roots turn white. Dilute 3-5 times according to the color and measure the color at 485 nm.

[0141] Chlorophyll content determination: Wash fresh leaves, dry them with filter paper, remove the midrib, and cut them into small pieces. Weigh 0.05 g of the chopped fresh leaves and place them in a 25 ml colorimetric tube. Add 5 ml of 95% ethanol, stopper the tube, shake, and store in the dark for 48 hours, shaking 2-3 times during this period. The supernatant is the chlorophyll extract. Take 1 ml of the chlorophyll extract, dilute it with 3 ml of 95% ethanol, and measure the absorbance at wavelengths of 665 nm, 649 nm, and 470 nm. Use 95% ethanol as a blank control.

[0142] Data Processing: 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. GraphPad Prism 9.0 was used to create the images.

[0143] 3. Experimental Results:

[0144] (1) Effects of different treatments on the biomass of Pingyi sweet tea seedlings

[0145] From Table 3 and Figure 6 It can be seen that, compared with the continuous cropping soil control (CK), both AMF and S74 inoculants, whether inoculated alone or in combination (S74+AMF), can promote the increase of biomass in Pingyi sweet tea seedlings. The combination of AMF and S74 has a better promoting effect than inoculation of either alone.

[0146] In August, the plant height, ground diameter, fresh weight, and dry weight of the treatments co-inoculated with AMF and S74 inoculants (S74+AMF) were 1.53 times, 1.47 times, 3.33 times, and 3.43 times that of the continuous cropping soil control (CK), respectively. Moreover, all indicators of the treatments inoculated with AMF (AMF) and S74 (S74) alone were lower than those of the treatments co-inoculated with AMF and S74 inoculants (S74+AMF).

[0147] Although the growth of Pingyi sweet tea seedlings co-inoculated with AMF and S74 inoculant treatment (S74+AMF) did not exceed that of the methyl bromide fumigation treatment (Br), it showed significant differences from the continuous cropping control treatment (CK), AMF inoculant treatment (AMF), and S74 inoculant treatment (S74).

[0148] Table 3: Effects of different treatments on the biomass of Pingyi sweet tea seedlings grown in pots under continuous apple cropping

[0149]

[0150] (2) Effects of different treatments on soil microorganisms

[0151] As shown in Table 4, in August, compared with the continuous cropping soil control (CK), both AMF and S74 inoculants, whether inoculated alone or in combination (S74+AMF), could increase the number of soil bacteria and decrease the number of soil fungi. The combination of AMF and S74 inoculation had a more significant effect than inoculation alone. The combination of AMF and S74 inoculation (S74+AMF) increased the number of bacteria by 5.24 times and decreased the number of fungi by 1.15 times compared with the continuous cropping soil control (CK).

[0152] Table 4: Effects of different treatments on soil microorganisms

[0153]

[0154] Figure 7 The results showed that in August, the bacterial to fungal ratio in the co-inoculation with AMF and S74 inoculant treatment (S74+AMF) was 6.03 times higher than that in the continuous cropping soil control (CK), and was further improved by... Figure 8 The results showed that, compared with the continuous cropping soil control (CK), the number of three Fusarium species (Alternaria alternata, Leymus chinensis, and Caryophyllaria spp.) in the soil was significantly reduced after co-inoculation with AMF and S74 inoculant (S74+AMF), and the effect was better than that of single inoculation.

[0155] (3) Effects of different treatments on soil AMF levels

[0156] As shown in Table 5, in August, compared with the continuous cropping soil control (CK), both AMF and S74 inoculants alone and co-inoculation with AMF and S74 (S74+AMF) could increase the spore density and gene copy number of AMF in the soil. Moreover, the co-inoculation with AMF and S74 (S74+AMF) had a significantly greater effect than the two treatments alone.

[0157] In August, the AMF spore density in the soil treated with both AMF and S74 (S74+AMF) was 2.32 times, 1.18 times, and 1.93 times that of the continuous cropping control (CK), AMF treatment (AMF), and S74 treatment (S74), respectively. The AMF gene copy number in the soil was 3.26 times, 1.37 times, and 3.02 times that of the continuous cropping control (CK), AMF treatment (AMF), and S74 treatment (S74), respectively. This indicates that the use of S74 can promote the abundance of AMF in the soil, providing data support for S74 as a helper bacterium promoting AMF colonization.

[0158] Table 5: Effects of different treatments on soil AMF spore density and gene copy number

[0159]

[0160] (4) Effects of different treatments on the mycorrhizal infection rate of roots of Pingyi sweet tea seedlings

[0161] As shown in Table 6, compared with the continuous cropping soil control (CK), both AMF and S74 inoculants alone and co-inoculation with AMF and S74 (S74+AMF) can increase the mycorrhizal infection rate of Pingyi sweet tea seedlings. Moreover, the co-inoculation with AMF and S74 (S74+AMF) has a significantly greater effect on promoting mycorrhizal fungal infection than the two treatments alone.

[0162] In August, the inoculation rates of AMF and S74 fungal agents (S74+AMF) were 1.94 times, 1.20 times, and 1.18 times higher than those of the continuous cropping control (CK), AMF treatment (AMF), and S74 treatment (S74), respectively. This indicates that the addition of S74 fungal agent can promote the colonization of AMF in the root system of Pingyi sweet tea seedlings and increase the mycorrhizal infection rate of AMF, providing data support for S74 as an auxiliary bacterium to promote AMF colonization.

[0163] Table 6: Effects of different treatments on apple root mycorrhizal infection rate

[0164]

[0165] (5) Effects of different treatments on root vigor of Pingyi sweet tea seedlings

[0166] Figure 9 The results showed that in August, co-inoculation with AMF and S74 inoculants (S74+AMF) significantly improved the root vigor of apple seedlings, which was 1.43 times, 1.26 times, and 1.12 times that of the continuous cropping control (CK), AMF treatment (AMF), and S74 treatment (S74), respectively. Moreover, co-inoculation with AMF and S74 inoculants (S74+AMF) was more effective in improving the root vigor of the plants than inoculation with either treatment alone.

[0167] (6) Effects of different treatments on chlorophyll content in leaves of Pingyi sweet tea seedlings

[0168] like Figure 10 As shown, in August, co-inoculation with AMF and S74 inoculant treatment (S74+AMF) increased the chlorophyll a content, chlorophyll b content, and total chlorophyll content of plant leaves. The total chlorophyll content was 1.24 times, 1.12 times, and 1.06 times that of the continuous cropping control (CK), AMF inoculant treatment (AMF), and S74 inoculant treatment (S74), respectively. Co-inoculation with AMF and S74 inoculant treatment (S74+AMF) was more effective in increasing the chlorophyll content of the aboveground leaves of plants than inoculation with either treatment alone.

[0169] 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 strain of *Pseudomonas aeruginosa* subsp. orange ( Pseudomonas chlororaphis subsp. aurantiaca S74, characterized in that, The accession number is: CGMCC No.36725.

2. A microbial agent, characterized in that, The bacterial agent contains Pseudomonas aeruginosa orange subspecies S74 as described in claim 1.

3. The microbial agent according to claim 2, characterized in that, In the bacterial agent, *Pseudomonas aeruginosa* subspecies S74 exists in the form of cultured live bacteria, fermentation broth, or bacterial suspension.

4. The use of the *Pseudomonas aeruginosa* subspecies S74 of claim 1 or the inoculum of claim 2 or 3 in at least one of the following (1)-(4): (1) Inhibit the growth of plant pathogens; (2) Prepare products for inhibiting plant pathogens; (3) Prevention and control of diseases caused by plant pathogens; (4) Prepare products for the prevention and control of diseases caused by plant pathogens; The plant pathogen is one or more of the following: Fusarium oxysporum, Fusarium solani, Fusarium moniliforme, and Fusarium MR5, a specialized type of Fusarium moniliforme.

5. The use of the *Pseudomonas aeruginosa* subspecies S74 as described in claim 1 or the inoculum as described in claim 2 or 3 in the following (1) or (2): (1) Alleviating apple cropping obstacles; (2) Prepare biocontrol agents to alleviate the continuous cropping obstacles of apple trees.

6. The application of the *Pseudomonas aeruginosa* subspecies S74 as described in claim 1 or the fungal agent as described in claim 2 or 3 in promoting the colonization of arbuscular mycorrhizal fungi in continuously cropped soils or plant roots.

7. A compound microbial agent, characterized in that, It is a compound of the bacterial agent described in claim 2 or 3 and the AMF bacterial agent; The AMF is a mycorrhizal fungus of the genus Gymnomycorrhiza, with accession number CGMCC No. 20744.

8. The compound microbial agent according to claim 7, characterized in that, The AMF bacterial agent is prepared by the following method: First, the AMF strain was inoculated into the roots of clover that had been growing for one week. Single spores were cultured for one month for further propagation. The clover roots were then crushed to obtain powder containing AMF. The sterilized substrate and the AMF-containing powder were mixed at a mass ratio of 3:

1. Sterilized clover seeds were then sown and propagated under greenhouse conditions. After three months, the infection rate was tested. Once the infection rate was greater than 50%, the above-ground parts were removed, and the clover roots and substrate were crushed together to obtain the AMF inoculum.

9. The use of the compound microbial agent according to claim 7 or 8 in at least one of the following (1)-(6): (1) Increase the ratio of bacteria to fungi in soil where apples are continuously cropped; (2) Improve the root vigor of apple seedlings; (3) Increase the chlorophyll content of the aboveground leaves of apple seedlings; (4) Increase the amount of AMF in soils continuously cropped with apples; (5) Increase the AMF mycorrhizal infection rate of the underground root system of apple seedlings; (6) Alleviate the problem of continuous cropping of apples.

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