Pseudomonas aeruginosa strain Pa605 and application thereof

Abstract

The invention provides pseudomonas aeruginosa Pa605 and application thereof, and belongs to the technical field of microorganisms. The invention provides a Pseudomonas aeruginosa strain Pa605, and the preservation number of the Pseudomonas aeruginosa strain Pa605 is CGMCC (China General Microbiological Culture Collection Center) NO.28510. The Pseudomonas aeruginosa strain Pa605 has the advantages that the preservation number is CGMCC NO.28510; a radicle inoculation test and a potting test show that the pseudomonas aeruginosa strain Pa605 has a relatively good inhibition effect on fusarium oxysporum, fusarium laminatum, fusarium solani and fusarium moniliforme, can effectively prevent and treat plant diseases caused by various fusarium, also has an effect of promoting plant growth, and in addition, the pseudomonas aeruginosa strain Pa605 can be used for preventing and treating plant diseases caused by fusarium oxysporum, fusarium laminatum, fusarium solani and fusarium moniliforme. Field colonization experiments show that the pseudomonas aeruginosa strain Pa605 can be colonized in soil for a long time, the effects of preventing and treating plant diseases and promoting plant growth can be achieved for a long time, and the research result provides a scientific basis for biological prevention and treatment of apple replanting diseases.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a strain of Pseudomonas aeruginosa, Pa605, and its applications. Background Technology

[0002] Apple replanting disease (ARD), also known as continuous cropping obstacle, is a serious problem in apple orchards and nurseries, caused by the accumulation of complex soil biota during the replanting cycle. Mild cases affect crop yield and quality, while severe cases can lead to complete crop failure, causing significant losses for farmers. Many pathogenic fungi cause apple replanting disease, and their pathogens vary across different regions. Liu Zhi's research found that the main pathogenic fungi causing apple continuous cropping obstacle in the Bohai Bay region of China are *Fusarium* species *F. proliferatum*, *F. moniliforme*, *F. solani*, and *F. oxysporum*. Wang Gongshuai's research showed that these four pathogenic *Fusarium* species are highly pathogenic to Pingyi sweet tea, reducing seedling dry weight by 35.60%, 51.92%, 40.96%, and 40.96% respectively one month after inoculation compared to the control. Two months later, the mortality rate of the four Fusarium species all exceeded 50%. Zou Qingjia found that Fusarium species have a strong pathogenicity to crabapple seedlings, which can not only cause browning of the radicle, but also affect the elongation of the radicle, making it shorter. Therefore, it is of great significance to find effective methods to reduce the infection of plants by pathogens.

[0003] Microbial inhibitors and microbial fertilizers play a role in controlling plant diseases and promoting plant growth. However, currently reported biocontrol bacteria cannot simultaneously inhibit *F. proliferatum*, *F. moniliforme*, *F. solani*, and *F. oxysporum*, and these biocontrol bacteria generally fail to successfully colonize in the soil, leading to inconsistent effectiveness in controlling plant diseases and promoting plant growth. Commonly used methods for colonization detection include antibiotic labeling, exogenous gene labeling, and immunological techniques. Screening for biocontrol bacteria that simultaneously inhibit multiple pathogens and can colonize the soil long-term is of great significance. Summary of the Invention

[0004] The purpose of this invention is to provide a strain of Pseudomonas aeruginosa and its applications, in order to solve the problems existing in the prior art.

[0005] To achieve the above objectives, the present invention provides the following solution:

[0006] One of the technical solutions of the present invention is a strain of Pseudomonas aeruginosa, Pa605, with the accession number CGMCC NO.28510.

[0007] The second technical solution of the present invention: an antibacterial agent comprising the aforementioned Pseudomonas aeruginosa strain Pa605 and excipients.

[0008] The third technical solution of the present invention: a microbial fertilizer, comprising the aforementioned Pseudomonas aeruginosa strain Pa605 and auxiliary materials.

[0009] The fourth technical solution of the present invention: the application of the Pseudomonas aeruginosa strain Pa605, the microbial agent, or the microbial fertilizer in plant cultivation.

[0010] Preferably, the plant cultivation includes promoting plant growth and / or preventing plant diseases.

[0011] Preferably, the promotion of plant growth includes promoting seed germination and / or promoting plant growth.

[0012] Preferably, the plant diseases include those caused by the pathogen Fusarium.

[0013] Preferably, the plant pathogens include one or more of Fusarium oxysporum, Fusarium laminarum, Fusarium solani, and Fusarium moniliforme.

[0014] Preferably, the working concentration of the *Pseudomonas aeruginosa* strain Pa605 is 1 × 10⁻⁶. 6 ~2×10 8 CFU / mL.

[0015] Preferably, the plant includes any one of the following: mung bean, crabapple, apple, or Pingyi sweet tea.

[0016] The present invention discloses the following technical effects:

[0017] This invention obtained a Pseudomonas aeruginosa strain Pa605 from 156 strains previously preserved in the laboratory. This strain exhibits good antagonistic effects against Fusarium oxysporum, Fusarium solani, and Fusarium moniliforme, and can promote plant growth.

[0018] This invention investigated the growth-promoting effects of Pa605 on plants through radicle inoculation and pot experiments. The radicle inoculation experiment showed that, in terms of dry weight, mung bean sprouts soaked in the fermentation broth of strain Pa605 increased by approximately 32.85% compared to the control (CK); and in terms of the number and length of fibrous roots, the Pa605-treated mung bean sprouts increased by more than 65% compared to the CK. The pot experiment showed that Pa605 treatment significantly increased the plant height, fresh weight, dry weight, root length, root weight, and number of fibrous roots in mung beans, and at a rate of 5 × 10⁻⁶... 7 The best results were achieved at a concentration of CFU / mL. When crabapple seedlings were treated with this concentration, the results obtained by Pa605 on mung beans were basically consistent with the results obtained on crabapple.

[0019] This invention investigated the disease resistance effect of Pa605 on plants through radicle inoculation and pot experiments. The radicle inoculation experiment showed that mung bean seeds soaked in the fermentation broth of strain Pa605 had a significantly reduced mortality rate, indicating that the fermentation broth of strain Pa605 can enhance the disease resistance of mung bean radicles. In the prevention experiment in the pot experiment, crabapple seedlings inoculated with four pathogens of apple replanting diseases, Fusarium, showed severe disease, exhibiting yellowing and wilting of leaves. However, crabapple seedlings treated with Fusarium spore suspension 24 hours after inoculation with strain Pa605 only showed mild symptoms in a few plants, indicating that the antagonistic strain has a significant preventive effect against apple replanting diseases. In the treatment experiment, although many crabapple seedlings were infected with Fusarium spore suspension 24 hours after inoculation with biocontrol bacteria suspension, the disease was milder, showing a significant difference compared to plants only inoculated with the pathogen, indicating that Pa605 reduces the infectivity of the pathogen to a certain extent.

[0020] Using the natural resistance of biocontrol bacteria to antibiotics, antibiotic labeling is a traditional method for determining colonization in application environments. This method is simple, rapid, low-cost, and allows for statistical analysis of the results. This invention labeled strain Pa605 with rifampicin resistance and investigated its colonization in the roots of crabapple seedlings. The results showed that in both continuously cropped and newly cropped soils, the labeled strain successfully colonized in the root zone of crabapple seedlings, and the Pa605 inoculum remained at 10⁻⁶ cells / day until 90 days later. 5 At CFU / g levels, strain Pa605 not only exhibits strong environmental adaptability and field stability, but also, after 90 days of root colonization at the roots of crabapple seedlings in the field, its effects on seedling height, stem diameter, chlorophyll content, and survival rate were further determined. The results indicate that strain Pa605 can promote the growth and control diseases of crabapple seedlings over a long period. Attached Figure Description

[0021] Figure 1The image shows the results of a plate confrontation experiment between Pseudomonas aeruginosa strain Pa605 and four Fusarium species, which are pathogens causing apple replanting.

[0022] Figure 2 The effects of treatment with six different concentrations of spore suspensions from four Fusarium pathogens on mung bean growth were investigated (Fo: Fusarium oxysporum; Fp: Fusarium flocculationii; Fs: Fusarium solani; Fm: Fusarium moniliforme).

[0023] Figure 3 The protective effect of Pseudomonas aeruginosa strain Pa605 against replanting diseases caused by Fusarium wilt;

[0024] Figure 4 The colonization amount of Pseudomonas aeruginosa marker strain Pa605 at different depths in soils of plots with positive cropping and negative cropping;

[0025] Figure 5 The effect of Pseudomonas aeruginosa marker strain Pa605 on the growth of crabapple seedlings in fields with positive and negative cropping conditions;

[0026] Figure 6 Colony morphology and cell microstructure of Pseudomonas aeruginosa strain Pa605;

[0027] Figure 7 Agarose gel electrophoresis image of the PCR product of the rDNA gene of Pseudomonas aeruginosa strain Pa60516S.

[0028] Figure 8 Phylogenetic analysis of the 16S rDNA gene of Pseudomonas aeruginosa strain Pa605.

[0029] Information on the preservation of biological materials

[0030] The strain of Pseudomonas aeruginosa was deposited on September 22, 2023, at the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing), with the accession number CGMCC No. 28510 and the strain number Pa605. Detailed Implementation

[0031] This invention provides a strain of *Pseudomonas aeruginosa*, Pa605, with accession number CGMCC NO.28510. The 16S rDNA sequence of the *Pseudomonas aeruginosa* strain Pa605 is shown in SEQ ID NO: 1.

[0032] SEQ ID NO: 1:

[0033]

[0034] This invention provides an antibacterial agent comprising the aforementioned *Pseudomonas aeruginosa* strain Pa605 and excipients. The *Pseudomonas aeruginosa* strain Pa605 can inhibit any one or more of *F. oxysporum*, *F. proliferatum*, *F. solani*, and *F. moniliforme*. The antibacterial agent is available in powder or aqueous solution form, wherein the effective viable count of *Pseudomonas aeruginosa* strain Pa605 in the aqueous solution is preferably not less than 1 × 10⁻⁶. 6 CFU / ml, more preferably 1×10 7 ~2×10 8 CFU / ml, the optimal value is 5×10⁻⁶. 7 CFU / ml, preferably not less than 1×10 in powder form. 6 CFU / g.

[0035] This invention provides a microbial fertilizer, comprising the aforementioned *Pseudomonas aeruginosa* strain Pa605 and excipients. The microbial fertilizer may be in powder or aqueous form, and the effective viable count of *Pseudomonas aeruginosa* strain Pa605 is preferably the same as that of the antibacterial agent.

[0036] This invention provides the application of the aforementioned Pseudomonas aeruginosa strain Pa605, the aforementioned microbial inoculant, or the aforementioned microbial fertilizer in plant cultivation.

[0037] In this invention, the plant cultivation preferably includes promoting plant growth and / or preventing plant diseases.

[0038] In this invention, the promotion of plant growth preferably includes promoting seed germination and / or promoting plant growth.

[0039] This invention, through radicle inoculation experiments, shows that in terms of dry weight, mung bean sprouts soaked in the fermentation broth of strain Pa605 increased by approximately 32.85% compared to the control (CK); and in terms of the number and length of fibrous roots, the mung bean sprouts treated with Pa605 increased by more than 65% compared to the CK; pot cultivation growth promotion experiments show that Pa605 treatment significantly increased the plant height, fresh weight, dry weight, root length, root weight, and number of fibrous roots of mung beans, and at 5×10 7 The best results were achieved at a concentration of CFU / mL. When crabapple seedlings were treated with this concentration, the results obtained by Pa605 on mung beans were basically consistent with the results obtained on crabapple.

[0040] In this invention, the plant diseases preferably include those caused by pathogenic Fusarium fungi. The plant pathogens include one or more of Fusarium oxysporum, Fusarium proliferatum, Fusarium solani, and Fusarium moniliforme.

[0041] This invention demonstrates through radicle inoculation experiments that mung bean seeds soaked in the fermentation broth of strain Pa605 showed a significant reduction in disease mortality, indicating that the fermentation broth of strain Pa605 can enhance the disease resistance of mung bean radicles. In the prevention experiment in pot cultivation, crabapple seedlings inoculated with four pathogens of apple replanting diseases, Fusarium, showed severe disease, exhibiting yellowing and wilting of leaves. However, crabapple seedlings treated with Fusarium spore suspension 24 hours after inoculation with strain Pa605 only showed mild disease in a few plants, indicating that the antagonistic strain has a significant preventive effect against apple replanting diseases. In the treatment experiment, although many crabapple seedlings were infected with Fusarium spore suspension 24 hours after inoculation with biocontrol bacteria suspension, the disease was milder, showing a significant difference compared to plants inoculated only with the pathogen, indicating that Pa605 reduces the infectivity of the pathogen to a certain extent.

[0042] Using the natural resistance of biocontrol bacteria to antibiotics, antibiotic labeling is a traditional method for determining colonization in application environments. This method is simple, rapid, low-cost, and allows for statistical analysis of the results. This invention labeled strain Pa605 with rifampicin resistance and investigated its colonization in the roots of crabapple seedlings. The results showed that in both continuously cropped and newly cropped soils, the labeled strain successfully colonized in the root zone of crabapple seedlings, and the Pa605 inoculum remained at 10⁻⁶ cells / day until 90 days later. 5 At CFU / g levels, strain Pa605 not only exhibits strong environmental adaptability and field stability, but also, after 90 days of root colonization at the roots of crabapple seedlings in the field, its effects on seedling height, stem diameter, chlorophyll content, and survival rate were further determined. The results indicate that strain Pa605 can promote the growth and control diseases of crabapple seedlings over a long period.

[0043] In this invention, the plant preferably includes any one of the following: mung bean, crabapple, apple, or Pingyi sweet tea.

[0044] In this invention, the method of application preferably includes spraying or irrigation.

[0045] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0046] The experimental locations and materials used in the following embodiments of the present invention are as follows:

[0047] 1. Test orchard, seeds, and strains

[0048] The test orchard was the apple experimental garden of Hebei Agricultural University, located in Lianchi District, Hebei Province.

[0049] Fusarium oxysporum, Fusarium proliferatum, Fusarium solani, and Fusarium moniliforme were provided by the Plant Disease Epidemiology and Integrated Management Laboratory of the College of Plant Protection, Hebei Agricultural University; 156 biocontrol strains, including Pa605, were isolated and identified by the Plant Disease Epidemiology and Integrated Management Laboratory of Hebei Agricultural University; Malus robusta Rehd. seeds were purchased from Haoyun Malus robusta Nursery Base in Huailai County, Hebei Province; and mung bean seeds were purchased from the Agricultural Materials Market in Baoding City, Hebei Province.

[0050] 2. Data Analysis

[0051] Data from radicle inoculation experiments, growth indicators of Malus baccata seedlings, and antibacterial activity of biocontrol bacteria were analyzed using SPSS 22.0 software with one-way ANOVA. The significance of differences was tested using Duncan's new multiple range test (DMRT) (p = 0.05).

[0052] Example 1

[0053] Antagonistic effect test of biocontrol bacteria

[0054] 1. Preparation of the test biocontrol bacterial solution

[0055] Preparation of biocontrol bacterial fermentation broth: After culturing the biocontrol strain in LB solid plates for 12 h, a single colony was picked and inoculated into LB liquid medium and cultured on a shaker for 24 h (30℃, 180 r·min⁻¹). The culture was then transferred to a 50 mL centrifuge tube, centrifuged at 10000 r·min⁻¹ for 20 min, and the supernatant was filtered through a 0.22 μm bacterial filter and stored at 4℃ for later use.

[0056] Preparation of biocontrol bacterial suspension: In a clean bench, prepare the fermentation broth according to the above method. Transfer the fermentation broth of the biocontrol strain to a 50mL sterile centrifuge tube, centrifuge at 10000r·min-1, 4℃ for 20min, discard the supernatant, add sterile water to the centrifuge tube, mix thoroughly, and make the OD value of the bacterial suspension reach 1 to obtain the biocontrol bacterial suspension for later use.

[0057] 2. Antagonistic effect test of biocontrol bacteria

[0058] Using the plate confrontation culture method, 156 biocontrol strains isolated in our laboratory were confronted with four apple pathogenic Fusarium species (Fusarium oxysporum, Fusarium moniliforme, Fusarium moniliforme, and Fusarium rotundifolium) to screen for strains with antagonistic effects.

[0059] Initial screening: Four Fusarium pathogenic fungal discs (Φ6mm) were inoculated into the center of PDA plates. Purified biocontrol strains were inoculated in four directions (top, bottom, left, and right) 2cm from the edge of the disc. A control group treated only with pathogenic fungi was used. The plates were incubated at 28℃. After one week, the mycelial diameter was measured, and the inhibition rate was calculated using Formula I.

[0060]

[0061] The results showed that 41 out of 156 biocontrol strains were able to inhibit the four pathogenic Fusarium species.

[0062] Secondary screening: Forty-one biocontrol bacteria that showed antagonistic activity against all four pathogens in the initial screening were selected for secondary screening using the same method as above. The screening results are shown in Table 1.

[0063] Table 1. Inhibition rates of biocontrol strains against four pathogens (secondary screening)

[0064]

[0065]

[0066] According to the data in Table 1, the inhibition diameter ranges of strain Pa605 against *Fusarium oxysporum*, *Fusarium solani*, *Fusarium moniliforme*, and *Fusarium solani* were 2.87±0.24 cm, 3.30±0.12 cm, 3.33±0.12 cm, and 3.20±0.26 cm, respectively, all of which showed significant differences compared to the control. Furthermore, the inhibition rate against all four pathogenic *Fusarium* species was above 55%, effectively inhibiting the mycelial growth of all four pathogenic *Fusarium* species. Figure 1 ).

[0067] Example 2

[0068] Growth promotion experiment of strain Pa605

[0069] 1. Detection of the growth-promoting effect of biocontrol strains on mung bean radicles using the radicle inoculation method.

[0070] Mung bean seeds with a radicle length of 2 mm and soaked in Pa605 fermentation broth were selected and cultured in water agar medium. Simultaneously, seed treatments with strains PGL7 and PGL22, which showed similar antagonistic effects, were compared. LB seed treatment served as a blank control. After one week, the radicle length of the mung beans was measured, and the incidence rate was calculated using Formula II. The study was repeated three times, with five mung bean seeds per replicate. Statistical results are shown in Table 2.

[0071]

[0072] Table 2. Growth-promoting effect of strain Pa605 on mung beans.

[0073]

[0074] Note: "-" indicates no growth rate.

[0075] As shown in Table 2, compared with the control (CK), there were no significant differences in the length of the radicle and the fresh weight of mung bean sprouts soaked in Pa605 fermentation broth. In terms of dry weight, the Pa605-treated mung bean sprouts increased by approximately 32.85% compared to the CK. Furthermore, the number and length of fibrous roots increased by more than 65% compared to the CK. Compared with the PGL7 and PGL22 fermentation broth treatment groups, the Pa605 strain showed a better growth-promoting effect, with a significantly higher growth rate in both the number and length of fibrous roots.

[0076] 2. Pot experiment to detect the growth-promoting effect of strain Pa605 on mung beans.

[0077] Using mung beans as the experimental material, mung bean seeds were cleaned, sterilized with 3% NaClO for 10 minutes, rinsed 3-5 times with sterile water, and then placed on damp gauze to germinate at 28℃ for 1 day. Once the seeds showed signs of germination, they were sown in seedling trays (bottom × height: 3cm × 6cm), one seed per cell, with 20g of sterilized soil. When the mung beans reached the two-leaf stage in the trays, a root drench treatment with a biocontrol bacteria suspension was initiated, with a drench volume of 10mL / 20g soil. Three concentrations of biocontrol bacteria were prepared for each plant: 5 × 10⁻⁶. 7 CFU / mL, 1×10 8 CFU / mL, 2×10 8 CFU / mL. Three replicates were performed for each concentration, with three seedlings per replicate. Water was used as a control. The seedlings were cultured in a greenhouse (relative humidity of 60%, with 16h / 8h light and dark alternation every day, the same below). They were watered every 2 days. After 20 days, the plant height, fresh weight, dry weight, root length, root weight and number of fibrous roots of mung bean seedlings were investigated. The results are shown in Table 3.

[0078] Table 3 shows the effects of different concentrations of strain Pa605 on the growth of potted mung beans.

[0079]

[0080]

[0081] As shown in Table 3, at 1×10 8 CFU / mL and 2×10 8At both CFU / mL concentrations, the height of mung bean seedlings was significantly increased compared to the control (CK); in terms of fresh weight, mung bean seedlings showed a significant increase only at 5 × 10⁻⁶ CFU / mL for strain Pa605. 7 At a CFU / mL concentration, the fresh weight was significantly different from the control (CK); measurements of the dry weight of mung beans showed that at a concentration of 5 × 10⁻⁶ CFU / mL, the fresh weight was significantly different from the control (CK). 7 Treatment with the Pa605 strain at a CFU / mL concentration increased the dry weight of mung beans, and by a significant margin, exceeding 1×10⁻⁶. 8 CFU / mL and 2×10 8 Two treatments were performed using CFU / mL; measurements of the number of rootlets in mung beans showed that at 5 × 10... 7 Treatment with CFU / mL of strain Pa605 increased the number of fibrous roots in mung beans, with a better effect than treatment with 1×10⁶ CFU / mL. 8 CFU / mL and 2×10 8 Two concentrations of CFU / mL; regarding root weight, 5 × 10⁻⁶ 7 Treatment with CFU / mL significantly increased root weight; measurements of mung bean root length revealed that strain Pa605 at a concentration of 5 × 10⁻⁶ CFU / mL significantly increased root weight. 7 CFU / mL and 1×10 8 Both CFU / mL treatments had a certain promoting effect on the root length of mung beans, with 5×10⁶ CFU / mL being the highest concentration. 7 Treatment with CFU / mL concentration resulted in a more significant growth-promoting effect. In conclusion, it is speculated that treatment at 5×10⁻⁶ CFU / mL... 7 The biocontrol strain at a concentration of CFU / mL produced the best growth-promoting effect on mung beans, and subsequent experiments were conducted based on this concentration.

[0082] 3. Pot experiment to detect the growth-promoting effect of strain Pa605 on crabapple seedlings

[0083] Malus halliana was selected as the experimental material. First, the seeds were stratified in sand. Washed seeds were placed in a 37℃ water bath overnight. Sterilized sand and seeds were mixed evenly at a ratio of 4:1 (50%-60% water holding capacity), placed in a breathable woven bag, and stored at 4℃ for 60 days. After stratification, the seeds were cleaned, sterilized with 3% NaClO for 10 minutes, and rinsed 3-5 times with sterile water. Germination was then carried out at 28℃ for 2 days. Germinated seeds were sown in flowerpots (bottom × height: 9cm × 12cm), with one seed and 60g of sterilized soil per pot. Nutrient soil was mixed with vermiculite in a 2:1 ratio. The seedlings were cultivated in a greenhouse. When the seedlings reached the 4-leaf stage, they were treated with Pa605 bacterial suspension (5×10⁻⁶). 7The seedlings were treated with root irrigation (CFU / mL) at a rate of 30 mL / 60 g soil. A control group was irrigated only with plain water. Nine seedlings were used as one treatment, and three seedlings were used as one replicate, for a total of three replicates. The seedlings were watered every two days. After 30 days, the seedling height, fresh weight, root fresh weight, root dry weight, and taproot length were measured. The results are shown in Table 4.

[0084] Table 4 shows the effects of strain Pa605 on the growth of crabapple seedlings.

[0085]

[0086]

[0087] As shown in Table 4, the plant height, fresh weight and root dry weight of crabapple treated with strain Pa605 were increased compared with those of the control group.

[0088] Example 3

[0089] Biocontrol experiment of strain Pa605

[0090] 1. Determination of the anti-infection efficacy of Pa605 against four pathogenic Fusarium species using the radicle inoculation method.

[0091] Radicle inoculation can quickly detect the pathogenicity of pathogens on mung bean radicles. The radicle length of mung bean seedlings inoculated with pathogens will be significantly limited, and some will develop browning, mold, rot, and eventually die.

[0092] First, mung bean seeds with a radicle length of 2 mm were soaked in Pa605 bacterial suspension. Then, the soaked mung bean seeds were placed on water agar medium containing four pathogenic Fusarium species (when the pathogenic mycelia reached half the length of the culture dish), with the radicle facing inwards. CK1 was LB soaking, and CK2 was LB soaking plus pathogen inoculation. Simultaneously, the treatments were compared with those of strains PGL7 and PGL22, which showed similar antagonistic effects. After one week of culture, the length of the mung bean radicle was measured, and the incidence rate was calculated using Formula II. Each treatment was repeated three times, with five mung bean seeds per replicate. The statistical results of the inhibitory effects of different strains on Fusarium spores, a pathogen causing replanting diseases, are shown in Table 5.

[0093] Table 5 shows the inhibitory effects of different strains on Fusarium oxysporum, a pathogen causing replanting diseases.

[0094]

[0095] Note: CK1 represents LB graft soaking with pathogen inoculation; CK2 represents LB graft soaking without pathogen inoculation.

[0096] As shown in Table 5, in the seedling inoculation experiment, the radicle length of mung bean seeds soaked with strain Pa605 was significantly different from that of CK1, indicating that strain Pa605 has a certain promoting effect on the elongation of mung bean radicles and can weaken the pathogenicity of pathogens to a certain extent. Furthermore, the mortality rate of mung bean seeds was significantly reduced after treatment with biocontrol bacteria, indicating that the fermentation broth of strain Pa605 can enhance the disease resistance of mung bean radicles. Moreover, compared with other strains PGL7 and PGL22, Pa605 showed more prominent anti-infection effects against the four pathogenic Fusarium species.

[0097] 2. Pot experiment to determine the anti-infection effect of Pa605 against four pathogenic Fusarium species.

[0098] (1) Suspensions of four pathogenic Fusarium spores and inoculation methods

[0099] Preparation of spore suspension: First, the prepared wheat inoculum was placed in a clean bench. Then, four strains of Fusarium pathogens were used to create mycelial cakes using a punch (Φ6mm) and inoculated onto the wheat. The mixture was thoroughly mixed and placed in a 28℃ incubator for constant temperature cultivation until a large number of mycelia and spores were produced. After 7 days, 100mL of sterile water was added to an Erlenmeyer flask for dilution. The mixture was stirred thoroughly with a glass rod and filtered through sterile gauze to obtain a spore suspension. 100μL of the spore suspension was then placed on a hemocytometer for observation and counting.

[0100] Using mung bean seeds as experimental material, the mung beans were cleaned, disinfected with 3% NaClO, rinsed 3-5 times with sterile water, and germinated at 28℃ for 1 day. After the seeds showed signs of sprouting, they were sown in seedling trays (bottom × height: 3cm × 6cm), with one mung bean and 20g of sterilized soil per cell. When the mung beans reached the two-leaf stage, the roots were drenched with a Fusarium spore suspension at a rate of 10mL / 20g soil. Six concentrations of the spore suspension irrigation treatment were set up: 4 × 10⁻⁶. 6 cells / mL, 5×10 6 cells / mL, 7.5×10 6 cells / mL, 1×10 7 cells / mL, 1.25×10 7 cells / mL, 1.5×10 7 The concentration of pathogenicity was measured per mL. A water treatment was used as a control, with the same water irrigation volume as the treatment. Each group consisted of 15 seedlings, for a total of three replicates. Seedlings were cultivated in a greenhouse, watered every two days. After 20 days, the plant height, fresh weight, dry weight, root length, and number of fibrous roots of the mung bean seedlings were assessed. The optimal pathogenic concentration was determined as the basis for subsequent experiments.

[0101] The results show that ( Figure 2 Measurements of mung bean plant height revealed that after inoculation with 4×10⁶ plants... 6 cells / mL and 5×106 After inoculating with a spore suspension at a concentration of 7.5 × 10⁶ spores / mL, there was no significant difference compared to the control (CK). 6 After inoculating with a spore suspension of *Fusarium moniliforme* and *Fusarium oxysporum* at a concentration of 1 × 10⁶ spores / mL, the height of mung bean plants began to drop sharply. 7 After inoculating mung bean seedlings with spore suspensions of *Fusarium solani* and *Fusarium moniliforme* at a concentration of 4 × 10⁶ spores / mL, the plant height of mung bean seedlings began to decrease significantly; measurements of fresh weight showed that mung bean seedlings inoculated with *Fusarium moniliforme* and *Fusarium solani* spore suspensions showed a significant decrease in plant height at 4 × 10⁶ spores / mL. 6 At a concentration of [number] cells / mL, the fresh weight of mung beans began to decrease significantly, reaching 7.5 × 10 [units / mL]. 6 At a concentration of [number] spores / mL, the fresh weight of mung bean seedlings inoculated with *Fusarium oxysporum* and *Fusarium chrysogenum* spore suspensions was significantly lower than that of the control (CK). Regarding dry weight, the overall dry weight of mung beans inoculated with spore suspensions of the four pathogens showed a decreasing trend, with the dry weight of mung beans treated with *Fusarium chrysogenum*, *Fusarium moniliforme*, and *Fusarium solani* reaching 4 × 10⁻⁶. 6 The concentration of spores / mL decreased significantly, while the dry weight of mung beans inoculated with Fusarium oxysporum spore suspension was 7.5 × 10⁻⁶. 6 The number of rootlets dropped sharply at a concentration of [number] spores / mL; measurements of the number of fibrous roots revealed that, compared to the control (CK), the number of mung bean rootlets inoculated with suspensions of four Fusarium spores decreased by 4 × 10 [units / mL]. 6 At a concentration of [number] cells / mL, the effect was significant; regarding root length, inoculation at 7.5 × 10 [units] showed a significant decrease. 6 After inoculating mung bean seedlings with a spore suspension of *Fusarium moniliforme* and *Fusarium oxysporum* at a concentration of 4 × 10⁶ spores / mL, the root length began to decrease. 6 Fusarium moniliformes at a concentration of 1 × 10⁶ cells / mL and 1 × 10 7 After inoculation with a *Fusarium solani* spore suspension at a concentration of 4 × 10⁶ spores / mL, root length was significantly reduced compared to the control (CK), showing a sharp decreasing trend. Overall, inoculation with 4 × 10⁶ spores / mL... 6 Suspensions of *Fusarium moniliforme* and *Fusarium solani* at a concentration of 7.5 × 10⁻⁶ spores / mL and 7.5 × 10⁻⁶ spores / mL. 6 After applying a spore suspension of *Fusarium solani* and *Fusarium oxysporum* at a concentration of 4 × 10⁶ spores / mL, mung bean seedlings began to show signs of infection, with physiological indicators gradually decreasing. Therefore, subsequent experiments will use a concentration of 4 × 10⁶ spores / mL. 6 7.5 × 10⁶ cells / mL (Fusarium moniliforme and Fusarium solani spore suspension) 6 Two concentrations of spores / mL (Fusarium oxysporum and Fusarium solani suspension) were used for inoculation treatment.

[0102] (2) Control test of Pa605 against replanting disease caused by Fusarium oxysporum

[0103] Protective effect: Uniformly growing crabapple seedlings (4-leaf stage) were selected, and Pa605 bacterial suspension was applied to the roots of the seedlings via root drenching. 24 hours later, four different Fusarium spore suspensions were added to the seedlings treated with the biocontrol bacterial suspension via root drenching, with treatments of Fusarium spore suspension alone and water serving as controls. Each treatment consisted of 9 crabapple seedlings, with 3 seedlings per replicate, for a total of 3 replicates. The seedlings were cultivated in a greenhouse, watered every 2 days. One month after inoculation, the disease severity was assessed, and the disease index and control effect were calculated. The disease grading criteria were based on the method of Zhang Yanjie, Mohd-Yusuf et al., with slight modifications. The assessment criteria are as follows: 0 = no symptoms; 1 = yellowing leaves, number of affected leaves < 1 / 8; 2 = yellowing leaves, stunted plant, number of affected leaves between 1 / 8 and 1 / 4; 3 = yellowing leaves, stunted plant and browning roots, number of affected leaves between 1 / 4 and 1 / 2; 4 = wilted, dried-out plant, necrotic roots, number of affected leaves > 1 / 2.

[0104]

[0105]

[0106] Therapeutic effect: Selected crabapple seedlings of uniform growth (4-leaf stage) and applied spore suspensions of four pathogens, Fusarium spores, to the roots of the seedlings by root irrigation (the inoculation concentration was based on the experimental results obtained in 2.2.5(1)). 24 hours later, Pa605 bacterial suspension was added to the crabapple seedlings treated with Fusarium spore suspension by root irrigation. Water treatment was used as a negative control, and treatments with Fusarium spore suspension and water alone were used as controls. Each treatment had 9 crabapple seedlings, with 3 crabapple seedlings as one replicate, for a total of 3 replicates. The seedlings were cultivated in a greenhouse (relative humidity of 60%, 16h / 8h light and dark alternation per day) and watered every 2 days. One month after inoculation, the severity of disease in the plants was assessed. The protective effect of Pa605 against replanting diseases caused by the four Fusarium spores is shown in Table 6, and the therapeutic effect of Pa605 against replanting diseases caused by the four Fusarium spores is shown in Table 7.

[0107] Table 6. Protective effect of Pa605 against four Fusarium-induced replanting diseases.

[0108]

[0109] As shown in Table 6, most crabapple seedlings treated with Fusarium oxysporum exhibited wilting and death. However, the protective and control effect of Fusarium oxysporum spore suspension on crabapple seedlings treated with Pa605 bacterial suspension 24 hours after inoculation was 88.38%; the protective and control effect of Fusarium oxysporum spore suspension on crabapple seedlings treated with Pa605 bacterial suspension 24 hours after inoculation was 91.27%. Figure 3); successively with 5×10 7 Pa605 bacterial suspension at CFU / mL and 4×10 6 Inoculating crabapple seedlings with a *Fusarium moniliforme* spore suspension at a concentration of 1 / mL significantly reduced the severity of disease compared to seedlings inoculated only with *Fusarium moniliforme* spore suspension, achieving a protective control effect of 93.80%. Furthermore, crabapple seedlings inoculated with biocontrol strain Pa605 suspension for 24 hours followed by treatment with *Fusarium solani* achieved a protective control effect of 96.69%. In conclusion, Pa605 can effectively protect crabapple seedlings from pathogens, promoting healthy seedling growth, and can serve as a potential antagonist for controlling replanting diseases in apples.

[0110] Table 7. The therapeutic effects of Pa605 on replanting diseases caused by four Fusarium species.

[0111]

[0112] As shown in Table 7, applying Fusarium oxysporum spore suspension followed by Pa605 bacterial suspension 24 hours after inoculation of crabapple seedlings resulted in a 71.91% therapeutic effect. Crabapple seedlings inoculated only with Fusarium oxysporum spore suspension showed the most severe disease, while seedlings treated with Pa605 bacterial suspension 24 hours after inoculation with Fusarium oxysporum spore suspension showed an 84.78% therapeutic effect (Table 9). Crabapple seedlings inoculated with Fusarium moniliforme spore suspension 24 hours after root drenching with Pa605 bacterial suspension showed a 75.62% therapeutic effect compared to seedlings inoculated only with Fusarium moniliforme spore suspension. Crabapple seedlings inoculated with Fusarium solani spore suspension 24 hours after inoculation with biocontrol strain Pa605 bacterial suspension showed a 92.89% therapeutic effect. In summary, strain Pa605 showed good therapeutic effects against replanting diseases caused by Fusarium oxysporum.

[0113] Example 4

[0114] Colonization ability of labeled strains in soil

[0115] 1. Rifampicin was used as an antibiotic marker to induce resistance in Pa605, PGL7, and PGL22 strains. The rifampicin resistance induction concentration gradients were 1, 5, 10, 20, 40, 60, 80, 100, 150, and 200 μg / mL. The genetic stability of the resistant strains was assessed by repeatedly subculturing them on NA plates without rifampicin using the streak plating method. After observing the fifth generation of strains cultured on NA plates containing 200 μg / mL rifampicin, the morphology of Pa605 strain remained unchanged compared to the original strain, indicating no significant variation in genetic material. In contrast, the morphology of PGL7 and PGL22 strains changed significantly, and their growth was slow. To further verify the genetic stability of the Pa605 marker strain, a plate confrontation experiment was conducted. The results showed that the antagonistic activity of the marker strain was not significantly different from that of the original strain. These results indicate that the Pa605 marker strain has good genetic stability and maintains its original antagonistic activity, making it suitable for application in production practice.

[0116] 2. Field planting:

[0117] (1) In mid-February 2022, the germinated crabapple seeds were transplanted to a greenhouse for cultivation. When they grew to the 6-leaf stage, healthy and uniformly growing crabapple seedlings were selected and planted in the apple experimental garden of Hebei Agricultural University. Before planting, the plots in the experimental garden were tilled. One month after planting, the crabapple seedlings were treated with root irrigation.

[0118] (2) Five biocontrol bacteria solutions (concentration of 2×10⁹ CFU / mL) cultured for 24 h in LB liquid medium containing 200 μg / mL rifampicin were applied to the soil of the orchard where crabapple seedlings were planted. 300 mL of the solution was applied to the roots of each seedling. Six seedlings were treated with each biocontrol bacteria. A control group without the biocontrol solution was used. The planting sites for the crabapple seedlings were divided into two types: one was a replanted plot where the previous crabapple trees had been cut down, and the other was a plot where the previous crop was vegetables, and this was the first time crabapple seedlings were being planted.

[0119] (3) Sampling was carried out immediately after treatment. The sampling point was the root irrigation site, and a sampling point was taken every 3 cm for a total of 3 points. After the soil suspension was prepared, it was serially diluted and plated on NA plates (containing 200 μg / mL rifampicin) for 24 h. The number of labeled strains was recorded as the initial bacterial load.

[0120] (4) Subsequently, at 1, 2, 3, 5, 10, 15, 20, 30, 60 and 90 days after root irrigation inoculation, 3g of soil was taken from the sampling point at the root of each crabapple seedling (soil sampling depth: 0-10cm and 10-20cm). Soil was randomly taken from plots that had not been irrigated with biocontrol solution as a control. 1g of the soil was taken from the control and then mixed with sterile water for gradient dilution to detect the number of labeled strains per gram of soil. The dried soil was weighed to determine the amount of bacteria per gram of dry soil.

[0121] (5) After the labeled strain colonized the roots of crabapple seedlings in the field for 90 days, the plant height, stem diameter and chlorophyll content of the crabapple seedlings were measured to detect whether it had a certain growth-promoting effect on the crabapple seedlings.

[0122] Antibiotic labeling was used to label strains Pa605, PGL7, and PGL22. All three labeled strains grew normally at a concentration of 200 μg / mL rifampicin. After inoculation with the labeled strains in both continuously cropped and alternating cropped soils, only the Pa605 labeled strain showed significant re-isolation of labeled strains from the root soil of crabapple seedlings within 0 h–90 days post-inoculation, indicating that the Pa605 labeled strain could successfully colonize the root soil of crabapple seedlings in both continuously cropped and continuously cropped soils. Furthermore, from… Figure 4 The results show that the two plots did not differ significantly in their impact on the colonization ability of the strain. Two soil sampling depths were used for each plot: one was within the range of 0-10cm, and the other was within the range of 10-20cm.

[0123] After inoculating the Pa605 labeled strain into the newly planted plot, soil samples were taken at depths of 0-10 cm and 10-20 cm on the day of inoculation and 24 hours after inoculation, respectively. The number of labeled strains per gram of soil was measured to be 5.10 × 10⁻⁶. 7 CFU / g and 2.27×10 7 CFU / g (wet soil, the same below) was used as the initial bacterial load for this treatment. The number of Pa605 labeled strains initially decreased rapidly after treatment, then stabilized after reaching a certain range. On the 3rd day after inoculation, the bacterial load in samples at soil depths of 0-10 cm and 10-20 cm decreased to 1.88 × 10⁻⁶. 7 CFU / g and 1.05×10 7 CFU / g. Thereafter, until day 90, the bacterial load of Pa605 at both depths in the field remained at 10. 5 The CFU / g level was around [value missing]. Simultaneously, soil samples were taken from untreated, un-irrigated soil samples and diluted on antibiotic-free medium for plating; the bacterial count was approximately 10 [value missing]. 6When the CFU / g level was around [value missing], and when diluted and plated on rifampicin-containing medium, the microorganisms in the blank soil showed almost no growth. In summary, this indicates that the Pa605-labeled strain not only successfully colonized in the field but also maintained a high level of [value missing] CFU / g at 90 days. 5 CFU / g or above bacterial load ( Figure 4 ).

[0124] In the case of land with continuous cropping, the initial bacterial count of soil samples at depths of 0-10 cm and 10-20 cm was 1.71 × 10⁻⁶. 7 CFU / g and 1.04×10 7 CFU / g. Similar to the soil in the original stubble field, the number of Pa605-labeled strains initially decreased rapidly after treatment, then stabilized after reaching a certain range. On day 3 post-inoculation, the bacterial count in samples at soil depths of 0-10 cm and 10-20 cm decreased to 9.90 × 10⁻⁶ CFU / g. 6 CFU / g and 1.18×10 6 CFU / g. Thereafter, until day 90, the bacterial load of Pa605 at both depths in the field remained at 10. 5 The CFU / g level was around [value missing]. However, the microorganisms in the blank soil samples did not grow on the rifampicin antibiotic medium. In summary, this indicates that the Pa605-labeled strain not only successfully colonized in the field but also maintained a high level of [value missing] CFU / g at 90 days. 4 ~10 5 Approximately CFU / g ( Figure 4 After 90 days of colonization of crabapple seedlings with Pa605-labeled strains, PGL7, and PGL22, the plant height, stem diameter, chlorophyll content, and seedling mortality rate of the crabapple seedlings were measured. The results are shown in Table 8.

[0125] Table 8. Effects of three biocontrol fungi, Pa605, PGL7, and PGL22, on the growth of crabapple seedlings.

[0126]

[0127]

[0128] As shown in Table 8, in both continuous and continuous cropping plots, the Pa605 treatment promoted the growth of crabapple seedlings, with higher plant height and chlorophyll content than the CK group. However, the treatments with marker strains PGL7 and PGL22 showed no difference in plant height or chlorophyll content compared to the CK group. Furthermore, no disease or mortality was observed in the Pa605-treated crabapple seedlings in either continuous or continuous cropping plots. Figure 5 In conclusion, strain Pa605 can protect crabapple seedlings from pathogen infection by establishing stable colonization in the field, reduce seedling mortality, and promote crabapple growth.

[0129] Example 5

[0130] Species identification of strain Pa605

[0131] 1. Morphological characteristics identification

[0132] The morphology of antagonistic colonies on the culture medium and the individual morphology of antagonistic bacteria after Gram staining were observed, and identification was performed according to the methods of Dong Xiuzhu and Cai Miaoying. After cultivation on LB medium, strain Pa605 colonies were round, with smooth, moist edges, translucent, grayish-green, and Gram-negative. Figure 6 ).

[0133] 2. Measurement of physiological and biochemical characteristics

[0134] The methods of Dong Xiuzhu and Cai Miaoying were followed for determination, and the measured indicators included glucose, fructose, galactose, mannitol, maltose, sucrose, gelatin liquefaction, and methyl red test. The physiological and biochemical characteristics of the biocontrol bacterium Pa605 are shown in Table 9. The results indicate that the physiological and biochemical characteristics of strain Pa605 are similar to those of Pseudomonas spp.

[0135] Table 9. Physiological and biochemical characteristics of strain Pa605

[0136]

[0137] 3. Molecular identification and phylogenetic tree construction

[0138] Identification of the 16S rDNA gene sequence. DNA was extracted from the strain using a rapid DNA extraction kit, and PCR amplification was performed using specific primers. The amplified products were observed as specific target bands by 1% agarose gel electrophoresis. Figure 7 The PCR product containing the target band was sent to Beijing Liuhe BGI Genomics Co., Ltd. for sequencing. The sequence results were compared for similarity in the GenBank database, and a phylogenetic tree was constructed using the neighbor-joining (NJ) method with MEGA 7 software. The final identification results showed that strain Pa605 is *Pseudomonas aeruginosa*. Figure 8 ).

[0139] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A strain of Pseudomonas aeruginosa ( Pseudomona aeruginosa strain Pa605, characterized in that, Its accession number is CGMCC NO.28510.

2. An antibacterial agent, characterized in that, It includes the Pseudomonas aeruginosa strain Pa605 as described in claim 1 and excipients.

3. A microbial fertilizer, characterized in that, It includes the Pseudomonas aeruginosa strain Pa605 as described in claim 1 and excipients.

4. The application of the Pseudomonas aeruginosa strain Pa605 according to claim 1, the microbial agent according to claim 2, or the microbial fertilizer according to claim 3 in plant cultivation.

5. The application according to claim 4, characterized in that, The plant cultivation includes promoting plant growth and / or preventing plant diseases.

6. The application according to claim 5, characterized in that, The promotion of plant growth includes promoting seed germination and / or promoting plant growth.

7. The application according to claim 5, characterized in that, The plant diseases mentioned include those caused by the pathogen Fusarium.

8. The application according to claim 6, characterized in that, The plant pathogens include one or more of Fusarium oxysporum, Fusarium laminarum, Fusarium solani, and Fusarium moniliforme.

9. The application according to claim 4, characterized in that, The working concentration of the *Pseudomonas aeruginosa* strain Pa605 was 1×10⁻⁶. 6 ~2×10 8 CFU / mL.

10. The application according to any one of claims 4 to 9, characterized in that, The plant includes any one of the following: mung bean, crabapple, apple, or Pingyi sweet tea.

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