Pseudomonas chlororaphis subsp. Aurantiaca EG-228 and application thereof

Abstract

The invention discloses a pseudomonas chlororaphis subsp. Aurantii EG-228 and application thereof, and belongs to the technical field of microorganisms. The invention provides a pseudomonas chlororaphis subsp. Aurantiaca EG-228 with broad-spectrum antibacterial activity, and the preservation number of the pseudomonas chlororaphis subsp. Aurantiaca EG-228 is CCTCC (China Center for Type Culture Collection) NO: M 20251124. The pseudomonas chlororaphis subsp. Aurantiaca EG-228 has the broad-spectrum antibacterial activity, and the preservation number of the pseudomonas chlororaphis subsp. Aurantiaca EG-228 is CCTCC NO: M 20251124. Through verification, EG-228 has a remarkable effect of preventing and treating apple rot, and the prevention and treatment effect reaches 90% or above; the antibacterial agent has an inhibiting effect on various plant pathogenic bacteria and is wide in antibacterial spectrum; in addition, EG-228 has an obvious growth promoting effect on seed germination; and the EG-228 can be applied in a natural environment with proper high temperature and strong ultraviolet rays. In conclusion, the EG-228 can be used for preparing a biocontrol microbial inoculum or a seed germination accelerator, and is beneficial to safely and effectively controlling plant diseases or promoting seed germination.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, in particular to a strain of Pseudomonas chlororaphis subspecies orange EG-228 and an application thereof. Background Art

[0002] The Xinjiang wild apple, Malus sieversii (Ledeb.) Roem., belongs to the Rosaceae Juss. family and the genus Malus Mill. It is a Tertiary relict species native to the Tianshan Mountains of Central Asia and the progenitor of modern cultivated apples. With rich intraspecific variation, it plays an irreplaceable role in germplasm conservation and genetic development research, and is now listed as a priority protected species in China. Large-scale outbreaks of apple rot are one of the main causes of widespread mortality in wild apple orchards. Apple rot is primarily a fungal disease caused by the black rot fungus Cytospora mali (also known as Valsa mali). Currently, the rot is more severe in production areas due to poor management, poor soil quality, and insufficient fertilizer and water. Trees weakened by frost and salt damage are susceptible to the disease.

[0003] Xinjiang's wild apples are primarily concentrated in the Tianshan Mountains, found in Gongliu, Xinyuan, Huocheng, and Yumin counties in the Ili region of the Xinjiang Uyghur Autonomous Region. However, due to differences in management practices, the incidence of wild apples varies across regions. An outbreak of wild apple rot has caused devastating ecological damage to Xinjiang's wild apple orchards, leading to widespread decline and even death, posing a serious threat to the conservation and healthy development of wild apple germplasm resources.

[0004] Zhang Qin et al. (2024) collected diseased skin samples of wild apple rot from a wild fruit forest in Xinyuan County, Xinjiang Uygur Autonomous Region. Through tissue separation and purification, they found that the pathogenic bacteria belonged to two categories, Cytospora mali and Cytospora parasitica, and the pathogenicity of the pathogen Cytospora mali was stronger than that of Cytospora parasitica.

[0005] At present, there have been many studies on the screening of antagonistic bacteria against fungi that cause apple rot, but there are relatively few studies on the biological control of wild apples in Xinjiang. Dou Yanan et al. (2018) isolated and screened a strain of Bacillus, and found through indoor confrontation tests that it had a good inhibitory effect on apple rot. Ma Rong et al. (2020) found that Bacillus atrophaeus showed a good protective effect on detached apple branches. Among them, Bacillus is the dominant species. However, the above research has not yet been transformed and applied to the prevention and control of apple rot in production.

[0006] CN112961784B discloses an endophytic Alternaria strain Aa-Lcht and its use in preventing and treating apple tree rot. The endophytic Alternaria strain Aa-Lcht can inhibit the mycelial growth of apple tree rot pathogens by up to 93.33%, and can effectively prevent and treat apple tree rot. CN106906172B discloses a strain of Streptomyces albiflorus and its application in the prevention and treatment of apple tree rot. The strain can grow and reproduce by using the mycelium of the pathogen of apple tree rot as nutrition, which is beneficial for the long-term colonization of the biocontrol bacteria in the rot spots of apple trees, playing a long-term biological control role. At the same time, the strain induces itself to produce a variety of extracellular cell wall hydrolases, which disintegrate the pathogen cells through synergistic enzymolysis, and can produce strong antibacterial active substances. The antibacterial rate against the pathogen of apple tree rot is 89.82%, and it has broad-spectrum antibacterial properties. The strain is used as the main biocontrol bacteria to prevent and treat fruit and vegetable pathogens such as apple tree rot, and has multiple advantages such as good prevention and treatment effect (100%), high efficiency, low recurrence rate (0), strong adaptability to the environment, strong stability, and not easy to develop drug resistance. It is of great significance to improve the prevention and treatment effect of fruit and vegetable pathogens such as apple tree rot, prevent the recurrence of pathogens, and protect the environment.

[0007] The above studies indicate that Bacillus, Alternaria endophytica, and Streptomyces albiflora all have varying degrees of efficacy against apple rot, further inhibiting the pathogen through the production of their metabolites. However, their effectiveness in plant disease control is less than ideal, with drawbacks such as a narrow inhibition spectrum, unstable effects, and a limited scope of application. Further research is needed to investigate the degradation of metabolites during application with changes in temperature and UV light intensity, as well as the effects of volatile metabolites on seed germination. Summary of the Invention

[0008] The purpose of the present invention is to provide a strain of Pseudomonas chlororaphis subspecies orange EG-228 and its application to solve the problems existing in the above-mentioned prior art. EG-228 has a significant effect on preventing and controlling apple rot, with a prevention and control effect of more than 90%.

[0009] To achieve the above object, the present invention provides the following solutions:

[0010] The present invention provides a strain of Pseudomonas Chlororaphis subsp. Aurantiaca EG-228, which was deposited in the China Center for Type Culture Collection on May 20, 2025, with a deposit number of CCTCC NO: M 20251124, and a deposit address of Wuhan University, Wuhan, China.

[0011] The present invention also provides a microbial agent containing the Pseudomonas chlororaphis subspecies aurantiacus EG-228 or its fermentation filtrate.

[0012] The present invention also provides use of the Pseudomonas chlororaphis subspecies orange EG-228 or the microbial agent in inhibiting the growth of pathogens, wherein the pathogens include apple rot pathogen (Valsa mali), pear rot pathogen (Cytospora ambiens), sharp knife fusarium (Fusarium oxysporum), dahliae verticillium (Verticillium dahliae), sclerotinia (Sclerotinia sclerotiorum), botrytis cinerea, botryosphaeria sp., Fusarium acuminatum, watermelon wilt pathogen (Fusarium oxysporum f.sp. Niveum) and Alternaria alternat.

[0013] The present invention also provides use of the Pseudomonas chlororaphis subspecies orange EG-228 or the microbial agent in preventing and controlling plant diseases, wherein the plant diseases include apple rot.

[0014] The present invention also provides use of the Pseudomonas chlororaphis subspecies aurantiacus EG-228 or the microbial agent in promoting plant seed germination.

[0015] The present invention also provides a method for promoting plant seed germination, comprising the step of fumigating the plant seeds with the fermentation filtrate of the Pseudomonas chlororaphis subspecies aurantiacus EG-228.

[0016] The present invention also provides a method for inhibiting the growth of pathogenic bacteria, comprising the following steps:

[0017] The Pseudomonas chlororaphis subspecies orange EG-228 or its fermentation filtrate is used to treat pathogens, thereby inhibiting the growth of pathogens;

[0018] The pathogens include apple rot pathogen (Valsa mali), pear rot pathogen (Cytospora.ambiens), sharp knife fusarium (Fusarium oxysporum), dahliae verticillium (Verticillium dahliae), sclerotinia (Sclerotinia sclerotiorum), cinerea botrytis (Botrytis cinerea), grape seat cavity fungus (Botryosphaeria sp), sharp top fusarium (Fusarium acuminatum), watermelon wilt pathogen (Fusariumoxysporum f.sp.Niveum) and Alternaria alternat.

[0019] The present invention also provides a biocontrol preparation for preventing and controlling apple rot, which comprises the Pseudomonas chlororaphis subspecies aurantiacus EG-228 or its fermentation filtrate.

[0020] The present invention also provides a seed germination promoter, which comprises the Pseudomonas chlororaphis subspecies aurantiacus EG-228 or its fermentation filtrate.

[0021] The present invention discloses the following technical effects:

[0022] The present invention provides a strain of Pseudomonas Chlororaphis subsp. Aurantiaca EG-228 with broad-spectrum antibacterial activity, with a deposit number of CCTCC NO: M 20251124. It has been verified that EG-228 has a significant effect on the prevention and treatment of apple rot, with a prevention and treatment effect of more than 90%; it also has an inhibitory effect on a variety of plant pathogens, with a broad antibacterial spectrum; in addition, EG-228 has a significant growth-promoting effect on seeds; and EG-228 can be used in a suitable natural environment of high temperature and strong ultraviolet rays. In summary, the Pseudomonas Chlororaphis subsp. Aurantiaca EG-228 provided by the present invention can be used to prepare biocontrol agents or seed germination promoters, and has the advantages of simplicity, low cost, non-toxicity, harmlessness, and no environmental pollution, which is conducive to the safe and effective control of plant diseases or the promotion of seed germination. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0024] Figure 1 Figure 4 is the morphological characteristics of strain EG-228. A: morphology of strain EG-228 cultured on PDA plate at 28°C for 24 h; B: morphology of strain EG-228 cultured on LB plate at 28°C for 24 h.

[0025] Figure 2 This is the phylogenetic tree of strain EG-228 constructed based on the 16S rDNA gene sequence;

[0026] Figure 3 The results of the antibacterial test of EG-228 against V. mali, the pathogen of apple rot; A: blank control; B: EG-228 streak inoculation; C: EG-228 equidistant inoculation with bacterial cakes;

[0027] Figure 4 The experimental results of the effect of EG-228 on the mycelial growth of V. mali, the pathogen of apple rot; A: blank control, B: the opposite treatment inoculated with EG-228;

[0028] Figure 5 The experimental results of the control effect of EG-228 on apple rot pathogen V. mali; A: blank control, B: control treatment with EG-228;

[0029] Figure 6 The results of the antifungal test of EG-228 against 10 fungal pathogens are shown below: A: Botrytis cinerea; B: Botryosphaeria sp.; C: Valsa mali; D: Cytospora ambiens; E: Fusarium oxysporum; F: Verticillium dahliae; G: Fusarium acuminatum; H: Alternaria alternata; I: Sclerotinia sclerotiorum; J: Fusarium oxysporum f.sp. niveum.

[0030] Figure 7 The results of the thermal stability test of EG-228; A: Colony diameter of V. mali treated with EG-228 at different temperatures; B: Inhibition rate of V. mali treated with EG-228 at different temperatures;

[0031] Figure 8 The UV light stability test results of EG-228; A is the colony diameter of EG-228 on V. mali treated with different UV light irradiation times; B is the inhibition rate of EG-228 on V. mali treated with different UV light irradiation times;

[0032] Figure 9 Effects of EG-228 on the germination of wheat and cotton seeds; a: germination rate test device; b: wheat seed germination test group, where A is the control group and B is the treatment group; c: cotton seed germination test group, where A is the control group and B is the treatment group. DETAILED DESCRIPTION

[0033] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0034] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0035] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0036] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0037] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0038] The pathogens used in the present invention are apple rot pathogen (Valsa mali), pear rot pathogen (Cytospora.ambiens), sharp knife fusarium (Fusarium oxysporum), dahliae verticillium (Verticillium dahliae), sclerotinia (Sclerotinia sclerotiorum), cinerea botrytis (Botryosphaeria sp), acuminatum fusarium (Fusarium acuminatum), watermelon wilt pathogen (Fusarium oxysporum f.sp.Niveum) and Alternaria alternat. Among them, the apple rot pathogen is from the Xinjiang Institute of Ecology and Geography of the Chinese Academy of Sciences, and the other pathogenic fungi are donated by Chen Xiaofei, a teacher of the Agricultural College of Tarim University.

[0039] Example 1 Isolation and Identification of Pseudomonas Chlororaphis subsp. Aurantiaca EG-228

[0040] In this example, the apple tree rot pathogen was used as the target bacteria, and antagonistic bacteria were isolated and screened from the rhizosphere of a natural wild fruit forest in Xinyuan County, Xinjiang, and the strains were identified. The specific steps are as follows.

[0041] Ten healthy, well-growing wild apple trees were selected using a checkerboard sampling method. Five samples were collected from each tree. Before collection, the blades, pruning shears, and the sampling area were disinfected with 75% ethanol. Roots approximately 1 cm thick and 10 cm long were cut from within the 30-50 cm soil layer. Samples were placed in sealed, numbered, and stored at 4°C.

[0042] PDA medium: 200 g potato, 20 g glucose, 20 g agar powder, 1000 mL distilled water; PDB medium: 200 g potato, 20 g D-glucose, 1000 mL distilled water; LB solid medium: 5 g yeast extract, 10 g peptone, 10 g sodium chloride, 20 g agar powder, 1000 mL distilled water; LB liquid medium: 5 g yeast extract, 10 g peptone, 10 g sodium chloride, 1000 mL distilled water.

[0043] 1. Isolation of antagonistic bacteria

[0044] Sample surface disinfection and testing. Rinse the collected tissue blocks with distilled water and air dry. Cut the roots into 1.5 cm long segments using sterilized scissors under a clean bench. Place the roots in a beaker, rinse three times with sterile water, soak in 75% ethanol for 2 minutes, rinse again with sterile water three times, disinfect with 0.6% sodium hypochlorite for 2 minutes, rinse three times with sterile water, soak in 75% ethanol for 30 seconds, and finally rinse three times with sterile water. Take 100 μL of the sterile water from the last rinse and inoculate it onto an LB plate. Incubate at 28°C. If no bacteria grow on the LB plate, the surface disinfection is complete and the next step can be performed.

[0045] Isolation and purification: Using a sterile blade, cut the root tissue into 20 5 mm × 5 mm blocks on a clean bench. These blocks were inoculated into LB medium, with 5–8 blocks per dish. The culture was incubated at 28°C. After colonies emerged from the cuts, the cells were transferred to new LB plates for purification. After purification, the strains were stored on slants at 4°C.

[0046] 2. Screening of antagonistic bacteria

[0047] The plate confrontation method was used to screen antagonistic bacteria with potential antibacterial effects on apple tree rot pathogen V. mali. The specific steps are as follows.

[0048] After activation and culture, the preserved single colonies were streaked onto PDA plates inoculated with the apple tree rot pathogen at equal distances (about 2.5 cm from the pathogen). A blank control was used without inoculation of the candidate antagonistic bacteria, and three replicates were performed for each treatment. The plates were cultured at a constant temperature of 26°C. When the control mycelium was about to grow full, the diameters of the pathogen colonies in each treatment were measured, and the inhibition rate was calculated according to the following formula.

[0049] Inhibition rate (%) = (colony diameter on control plate - colony diameter on treated plate) / colony diameter on control plate × 100;

[0050] An antagonistic strain with an inhibition rate of more than 90% was screened out and numbered EG-228.

[0051] 3. Identification of antagonistic bacteria

[0052] (1) Refer to the Manual of Identification of Common Bacteria Systems to observe the morphology of antagonistic bacteria and colony culture morphology, and determine the physiological and biochemical indicators of antagonistic bacteria such as aerobic characteristics, organic phosphorus, inorganic phosphorus, and cellulose decomposition.

[0053] The results of morphological observation showed that the strain EG-228 was cultured on LB agar plates at 28℃ for 24h, and the colonies were orange, round, with convex, smooth, sticky, easy to pick up, and neat edges. The strain EG-228 was cultured on PDA agar plates at 28℃ for 24h, and the colonies were small, round, convex, and secreted yellow pigment ( Figure 1 ).

[0054] The results of physiological and biochemical indexes are shown in Table 1.

[0055] Table 1 Physiological and biochemical analysis of strain F-04

[0056]

[0057] (2) 16S rDNA sequencing was performed on strain EG-228, and the sequencing result (SEQ ID NO.1) was compared with the NCBI database for homology and phylogenetic tree analysis was constructed ( Figure 2 ), and combined with morphological characteristics ( Figure 1 ), physiological and biochemical characteristics (Table 1), the strain was finally identified as Pseudomonas Chlororaphis subsp. Aurantiaca and named Pseudomonas Chlororaphis subsp. Aurantiaca EG-228.

[0058] This strain was deposited in the China Center for Type Culture Collection (CCTCC, address: Wuhan University, Wuhan, China) on May 20, 2025, with the deposit number CCTCC NO: M 20251124.

[0059] SEQ ID NO.1 (16S rRNA sequence of EG-228):

[0060]

[0061] Example 2 Preparation of EG-228 bacterial suspension and sterile fermentation filtrate

[0062] 1. Preparation of EG-228 bacterial suspension

[0063] A single colony of the EG-228 strain was picked and inoculated into liquid LB medium, and cultured at 28°C and 200 rpm for 24 h to obtain a bacterial suspension.

[0064] 2. Preparation of EG-228 sterile fermentation filtrate

[0065] Pipette 1 mL of the above EG-228 bacterial suspension into liquid LB medium, culture at 28°C, 200 rpm, and shaking for 24 h, then centrifuge at 12,000 rpm and 4°C for 20 min. Take the supernatant and filter it through a 0.22 μm microporous membrane. The filtrate is the sterile fermentation filtrate.

[0066] Example 3: Verification of the antibacterial effect of EG-228 on apple rot pathogens

[0067] 1. Observation of the antibacterial effect of EG-228 on the apple rot pathogen Valsa mali using the plate confrontation method

[0068] A V. mali cake was inoculated in the center of the PDA plate. A blank control was used without EG-228 inoculation. Two treatment groups were set up, one with equidistantly streaked EG-228 lines and the other with EG-228 cake inoculation. Each test plate was incubated at 26°C. When the control group mycelium was about to be fully grown, the width of the inhibition zone between the EG-228 strain and the V. mali cake in each treatment group was observed. The results are shown in the figure below. Figure 3 shown.

[0069] from Figure 3 It can be seen that when the mycelium of the pathogen in the control group was fully grown, the mycelium of the pathogen in the two treatment groups stopped growing outward, indicating that EG-228 had a significant antibacterial effect on V. mali, with an inhibition rate of up to 91.17%.

[0070] 2. Effect of EG-228 sterile fermentation filtrate on V. mali mycelial morphology

[0071] Take 20mL of PDA culture medium cooled to 50℃ and mix it evenly with 5mL of EG-228 sterile fermentation filtrate, pour a layer of the mixture on a glass slide, wait for it to solidify, and use a blade to cut off a width of about 1cm of culture medium in the center of the glass slide with the mixture; use an inoculation needle to pick up V.mali hyphae that have been cultured for 3 days, place it on the cross section, and cover it with a coverslip; after culturing at a constant temperature of 26℃ for 3 days in a culture dish covered with two layers of moist filter paper, observe the morphology of the pathogenic hyphae under a microscope. At the same time, use PDA culture medium without mixing with EG-228 sterile fermentation filtrate as a blank control to observe the hyphae morphology. The results are as follows Figure 4 shown.

[0072] from Figure 4 It can be seen that the V. mali hyphae in the blank control plate are complete in morphology, slender, uniform and smooth in surface, with sharp tips and complete structure ( Figure 4 A); however, the mycelial morphology of V. mali in the EG-228-treated plate was severely distorted, with mycelium breaking and even melting ( Figure 4 Middle B) shows that EG-228 can destroy the hyphal morphology of V. mali.

[0073] 3. Effects of different concentrations of EG-228 sterile fermentation filtrate on the mycelial growth of V. mali

[0074] Sterile fermentation filtrate and melted PDA medium were mixed to prepare medium plates with sterile fermentation filtrate contents of 0%, 2%, 4%, 6%, 8%, 10%, and 12%, respectively, with 0% serving as a blank control. A 3-day-old V. mali bacterial cake (5 mm in diameter) was inoculated in the center of the plate, and the experiment was repeated three times. After incubation at 26°C for 3 days on each plate, the pathogen colony diameter was measured, and the inhibition rate was calculated according to the following formula. The results are shown in Table 2.

[0075] Inhibition rate (%) = [(control colony diameter - treated colony diameter) / control colony diameter] × 100.

[0076] The results in Table 2 show that different concentrations of EG-228 sterile fermentation filtrate have significant inhibitory effects on the growth of V. mali mycelium, and as the concentration increases, the inhibitory effect becomes more obvious, with the highest inhibition rate reaching 90.18%.

[0077] Table 2 Effects of different concentrations of EG-228 sterile fermentation filtrate on the mycelial growth of V. mali

[0078]

[0079]

[0080] Note: The data in the table are mean ± standard deviation. Different lowercase letters indicate significant differences at the P < 0.05 level as tested by Duncan's new multiple range test. Same below.

[0081] Example 4: Verification of the Control Effect of EG-228 on Apple Rot

[0082] 1. Detection of damage to apple leaves by EG-228 sterile fermentation filtrate

[0083] ① Leaf treatment: Collect leaves of uniform health and size, rinse with tap water, disinfect with 0.6% (w / w) sodium hypochlorite solution for 30 minutes, and then rinse with sterile water five times until the odor of sodium hypochlorite solution disappears. Allow to dry naturally at room temperature, seal the petiole with melted paraffin to retain moisture, and allow to dry for later use.

[0084] ② Wound inoculation treatment

[0085] Treated leaves were perforated with a sterile needle (5 mm diameter) to create wounds. Two treatments were set up: a control group, where 50 μL of LB liquid culture medium was dripped onto the wounds; a treatment group, where 50 μL of EG-228 sterile fermentation filtrate was dripped onto the wounds. The experiment was replicated three times.

[0086] After each branch was cultured under natural conditions for 5 days with moisturizing, the wound expansion of the apple leaves was observed, and the lesion area was calculated according to the following formula. The results are shown in Table 3.

[0087] Lesion area (cm 2 )=1 / 4×π×long diameter of lesion×short diameter of lesion.

[0088] As can be seen from Table 3, the average lesion area of ​​the treatment group was 0.023 cm 2 , while the average lesion area of ​​the control group was 1.878 cm 2 The results showed that EG-228 did not cause serious damage to apple leaves.

[0089] Table 3 Effects of EG-228 on detached leaf damage

[0090]

[0091] 2. In vitro efficacy determination of EG-228 sterile fermentation filtrate against apple tree rot

[0092] ① Leaf treatment: Healthy and uniform apple leaves were collected, rinsed with tap water, disinfected with a 0.6% (w / w) sodium hypochlorite solution for 30 minutes, and then rinsed five times with sterile water until the odor of the sodium hypochlorite solution disappeared. The leaves were then air-dried at room temperature, and the petioles were sealed with melted paraffin wax to retain moisture. The leaves were then left to dry for later use.

[0093] ② Wound inoculation treatment

[0094] The treated leaves were wounded with a sterilized needle (aperture 5 mm), and two treatments were set up: control group: a brush was dipped in an appropriate amount of LB liquid culture medium and the leaves were brushed 3 times (each time it was naturally dried before the next brushing); treatment group: a brush was dipped in an appropriate amount of EG-228 sterile fermentation filtrate and the leaves were brushed 3 times (each time it was naturally dried before the next brushing). The experiment was repeated 6 times.

[0095] After the leaves were dried, V. mali cakes were inoculated on the wounded areas of the leaves, with two inoculation points on each leaf. After 7 days of moisturizing and incubation at 26°C, the occurrence of apple rot was observed. The lesion area and control effect were calculated according to the following formula. The results are shown in Table 4 and Figure 5 shown.

[0096] Lesion area (cm 2 )=1 / 4×π×long diameter of lesion×short diameter of lesion.

[0097] Control effect (%) = [(control lesion area - treated lesion area) / control lesion area] × 100.

[0098] From Table 4 and Figure 5 It can be seen that the lesions in the treatment group expanded less, with an average lesion area of ​​0.48 cm 2 Compared with the control group, the control effect of the treatment group on V. mali was 89.35%. The test results show that EG-228 has a good control effect on apple rot.

[0099] Table 4 The control effect of EG-228 on detached leaves of apple tree rot

[0100]

[0101] Example 5 Determination of the antagonistic activity of EG-228 against 10 pathogens

[0102] The 10 target pathogens are: apple rot pathogen Valsa mali, fragrant pear rot pathogen Cytospora.ambiens, cotton wilt pathogen Fusarium oxysporum, cotton Verticillium dahliae, rapeseed sclerotinia Sclerotinia sclerotiorum, Botrytis cinerea, Botryosphaeria sp, wheat gibberel Fusarium acuminatum, watermelon wilt pathogen Fusarium oxysporumf.sp.niveum, and walnut brown spot pathogen Alternaria alternata.

[0103] EG-228 was drawn on the plate at equal intervals, 2.5 cm from the center, to prepare an antibacterial test; a PDA plate without EG-228 was used as a blank control.

[0104] The target pathogen cakes with a diameter of 5 mm were inoculated into the center of each plate. Three replicates were used for each treatment. Each plate was incubated at a constant temperature of 26°C. When the mycelium of the control plate was about to be fully grown, the colony diameter of the target pathogen in each plate was measured and the inhibition rate was calculated. The results are shown in Table 5 and Figure 6 shown.

[0105] As can be seen from Table 5, strain EG-228 has different degrees of inhibitory effect on the hyphae of 10 pathogens, and the inhibition rate is above 70%. Among them, the inhibition rate of Fusarium fungal diseases can reach 80%, and the inhibition rate of apple rot and cotton Verticillium wilt is 91.17% and 90.98% respectively. Figure 6 It can be seen that the hyphae growth of each pathogen close to EG-228 is severely restricted, and the colony edge gradually becomes sparse. This shows that EG-228 has a broad-spectrum inhibitory effect and can be used for the biological control of various plant diseases.

[0106] Table 5 Inhibition rate of EG-228 against 10 target pathogens

[0107]

[0108]

[0109] Example 6 Stability Test of EG-228

[0110] 1. Thermal stability test

[0111] Five 50 mL centrifuge tubes containing EG-228 sterile fermentation filtrate were placed in a water bath at 45°C, 55°C, 65°C, 75°C, 85°C, and 95°C for 30 min, respectively. After the sterile fermentation filtrate was cooled to room temperature, it was mixed with melted PDA culture medium at a volume ratio of 1:10 and poured into a plate. The sterile fermentation filtrate at room temperature was used as the positive control, and the PDA blank culture medium without the addition of sterile fermentation filtrate was used as the blank control.

[0112] A fresh V. mali cake with a diameter of 5 mm was inoculated in the center of each treatment group and control plate; each treatment was repeated 5 times and cultured at a constant temperature of 26°C. When the mycelium of the blank control plate was about to be fully grown, the colony diameter of V. mali on each plate was measured and the inhibition rate was calculated. The results are as follows: Figure 7 shown.

[0113] from Figure 7 It can be seen that after the EG-228 sterile fermentation filtrate was treated at five high-temperature temperatures, the inhibition rate decreased with increasing temperature. However, after treatment at 45-65°C, it had a good control effect on V. mali. This shows that EG-228 has good thermal stability and the temperature can be set in the range with the most significant inhibition effect in subsequent production.

[0114] 2. UV light stability test

[0115] Place the small triangular bottles containing the EG-228 sterile fermentation filtrate under ultraviolet light for 20 minutes, 30 minutes, 40 minutes, 50 minutes and 60 minutes respectively, then mix with the melted PDA culture medium at a volume ratio of 1:10 and pour into the plates. The EG-228 sterile fermentation filtrate that has not been irradiated with ultraviolet light is used as the positive control, and the PDA blank culture medium without the addition of the EG-228 sterile fermentation filtrate is used as the blank control. A fresh V.mali cake with a diameter of 5 mm is inoculated in the center of each treatment group and control group plate; each treatment is repeated 5 times and cultured at a constant temperature of 26°C. When the blank control plate is about to be filled with mycelium, the colony diameter of V.mali in each plate is measured and the inhibition rate is calculated. The results are as follows: Figure 8 shown.

[0116] from Figure 8 It can be seen that after the EG-228 sterile fermentation filtrate was treated with UV light for five different durations, the inhibition rate decreased with increasing treatment time, but within an appropriate range, it showed good inhibition. The influence of temperature can be considered in subsequent biocontrol agent preparation.

[0117] Example 7 Effect of EG-228 Volatile Substances on Seed Germination

[0118] Prepare the test plant materials: take wheat seeds and cotton seeds, disinfect them with 75% ethanol for 30 seconds, rinse them with sterile water three times, and then treat them as a control group: soak them in LB and 150 mg / mL water for 30 minutes and then culture them at 28°C; treat the experimental group: soak them in EG-228 in LB and 150 mg / mL water for 30 minutes and then place them in the designed device (such as Figure 9 As shown in Figure a, the bottom of the dish contains sterile fermentation filtrate of EG-228. A perforated dish is placed on top of the dish, and the seeds are cultured on top of this dish. Finally, another dish is placed upside down on top to fully expose the seeds to the volatile substances of EG-228. The seeds are incubated at 28°C. Root length is measured after the seeds have rooted and whitened (Table 6). Photos are taken on the third day.

[0119] from Figure 9 It can be seen from the graph that the germination rates of wheat and cotton seeds treated with EG-228 volatile gas were significantly promoted compared with the control group, and the root lengths of wheat and cotton increased by 76.87% and 47.20% respectively compared with the control group.

[0120] Table 6 Root length of wheat and cotton

[0121]

[0122] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A strain of Pseudomonas Chlororaphis subsp. Aurantiaca EG-228, characterized in that: It was deposited in the China Center for Type Culture Collection on May 20, 2025, with the deposit number being CCTCCNO: M 20251124.

2. A microbial agent, characterized in that: Contains the Pseudomonas chlororaphis subspecies aurantiacus EG-228 or its fermentation filtrate according to claim 1.

3. Use of the Pseudomonas chlororaphis subspecies aurantiacus EG-228 according to claim 1 or the microbial agent according to claim 2 in inhibiting the growth of pathogenic bacteria, characterized in that: The pathogens include apple rot pathogen (Valsa mali), pear rot pathogen (Cytospora.ambiens), sharp knife fusarium (Fusarium oxysporum), dahliae verticillium (Verticillium dahliae), sclerotinia (Sclerotinia sclerotiorum), cinerea botrytis (Botrytis cinerea), grape seat cavity fungus (Botryosphaeria sp), sharp top fusarium (Fusarium acuminatum), watermelon wilt pathogen (Fusarium oxysporum f.sp.Niveum) and Alternaria alternat.

4. Use of the Pseudomonas chlororaphis subsp. aurantiacus EG-228 according to claim 1 or the microbial agent according to claim 2 in preventing and controlling plant diseases, characterized in that: The plant diseases include apple rot.

5. Use of the Pseudomonas chlororaphis subsp. aurantiacus EG-228 according to claim 1 or the microbial agent according to claim 2 in promoting plant seed germination.

6. A method for promoting plant seed germination, characterized in that: The method comprises the step of fumigating plant seeds with the fermentation filtrate of Pseudomonas chlororaphis subspecies aurantiacus EG-228 according to claim 1.

7. A method for inhibiting the growth of pathogenic bacteria, characterized in that: The following steps are involved: The pathogen is treated with the Pseudomonas chlororaphis subspecies aurantiacus EG-228 or its fermentation filtrate according to claim 1, thereby inhibiting the growth of the pathogen; The pathogens include apple rot pathogen (Valsa mali), pear rot pathogen (Cytospora.ambiens), sharp knife fusarium (Fusarium oxysporum), dahliae verticillium (Verticillium dahliae), sclerotinia (Sclerotinia sclerotiorum), cinerea botrytis (Botryosphaeria sp), acuminatum fusarium (Fusarium acuminatum), watermelon wilt pathogen (Fusariumoxysporum f.sp.Niveum) and Alternaria alternat.

8. A biocontrol agent for preventing and controlling apple rot, characterized in that: The biocontrol agent comprises the Pseudomonas chlororaphis subspecies aurantiacus EG-228 or its fermentation filtrate according to claim 1.

9. A seed germination promoter, characterized in that The seed germination promoter comprises the Pseudomonas chlororaphis subspecies aurantiacus EG-228 or its fermentation filtrate according to claim 1.

Images