Enterobacter ludwigii SLGJ4, microbial agent and application of enterobacter ludwigii SLGJ4

Abstract

The invention belongs to the technical field of biological control, and particularly relates to an enterobacter ludwigii SLGJ4, a fungicide and application of the enterobacter ludwigii SLGJ4, the preservation number of the enterobacter ludwigii SLGJ4 is CCTCC No: M 20251540, the bacterial strain has broad-spectrum antibacterial activity, and the antibacterial rates of the bacterial strain on Colletotrichum gloeosporioides of pomegranate, Colletotrichum truncatum of pepper, rhizoctonia cerealis of wheat and Botryis cinerea of tomato are 55.48%, 51.03%, 48.67% and 47.15% respectively; the control effect on pomegranate fruit anthracnose is 60.99%, the enzyme activity of defensive enzyme POD of pomegranate fruits is remarkably improved, the activity of PPO is inhibited, and enzymatic browning of the pomegranate fruits is delayed; after colletotrichum gloeosporioides hyphae are treated by the strain SLGJ4, the growth of the hyphae is obviously inhibited In addition, the strain can also cause accumulation of intracellular active oxygen of colletotrichum gloeosporioides, destroy plasma membrane integrity, has a good control effect on pomegranate fruit anthracnose, and provides a theoretical basis for development of pomegranate biocontrol inoculants and preservatives.

Description

Technical Field

[0001] This invention belongs to the field of biological control technology, and specifically relates to a strain of Enterobacter ludwig's bacterium, its bacterial agent, and its application. Background Technology

[0002] Pomegranate( Punica granatum Pomegranate (L.) is a common fruit crop in China, originating from the Balkans and extending to Iran and neighboring regions. It is cultivated in temperate and tropical zones worldwide. The history of pomegranate cultivation in China can be traced back to the Han Dynasty. The pomegranate is a berry, rich in nutrients, with a vitamin C content 1-2 times higher than apples and pears. After ripening, the entire pomegranate plant is usable; the peel can be used medicinally, and the fruit can be eaten or juiced. However, anthracnose (… Colletotrichum gloeosporioides Anthracnose has become one of the core diseases restricting the development of the pomegranate industry. This disease is prone to outbreaks in the hot and humid summer. Initially, it forms small, slightly sunken, yellowish-brown round spots on the fruit surface with water-soaked edges. When moist, small orange-red, sticky dots (conidia) appear on the surface of the spots. Later, the spots enlarge and merge, forming large, irregular, dark brown spots that penetrate deep into the flesh beneath the peel. Diseased fruits rot and fall off or dry up, becoming shriveled and hanging from the branches. Statistics show that in rainy years, anthracnose can cause pomegranate yield losses of over 30% and a 50% decrease in marketable fruit rate, severely weakening the economic benefits for fruit farmers.

[0003] Currently, chemical control remains the primary method for controlling pomegranate anthracnose. Spraying with prochloraz, difenoconazole, or pyraclostrobin after flowering and during fruit enlargement is effective against anthracnose. However, chemical control has drawbacks such as pesticide residues, environmental damage, and increased pesticide resistance in pests and diseases. Biological control is an inevitable trend in plant disease management. Patent 201610890367.4 discloses a strain of Bacillus stolonifera Bf-01 and its application in controlling anthracnose in figs and pomegranates. This strain shows good control effects against pomegranate anthracnose and can reduce the use of chemical pesticides. Therefore, exploring different antagonistic microorganisms for green control of pomegranate diseases provides more theoretical basis for the development of pomegranate biocontrol agents and preservatives, which is of great significance for the healthy development of pomegranate ecological agriculture. Summary of the Invention

[0004] To address the above problems, this invention proposes a strain of Enterobacter ludwig's SLGJ4, a bacterial agent, and its applications.

[0005] The technical solution of this invention is implemented as follows: On the one hand, this invention applies for protection of a strain of Ludwig's Enterobacter SLGJ4, which is classified and named as follows: Enterobacter ludwigii It was deposited on July 7, 2025 at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC No: M20251540, and the deposit address is Wuhan University, Wuhan, China.

[0006] The morphological characteristics of Ludwig's Enterobacter SLGJ4 are: pale yellow, round colonies with smooth edges and a moist, slightly sticky surface.

[0007] Secondly, this invention also seeks to protect a bacterial agent comprising the aforementioned Ludwig's Enterobacter SLGJ4.

[0008] Thirdly, the application of the aforementioned Ludwig's Enterobacter SLGJ4 or the aforementioned bacterial agents in antagonizing plant pathogens.

[0009] Preferably, the above-mentioned antagonism is achieved by disrupting the hyphal morphology of plant pathogens, promoting the accumulation of intracellular reactive oxygen species, and disrupting the integrity of the plasma membrane.

[0010] After treating Colletotrichum gloeosporioides JBTJ1 with Ludwig's Enterobacter SLGJ4, the mycelial growth morphology of the pathogen was abnormal, with mycelial swelling and significant enlargement of the mycelial tips, indicating that the biocontrol bacterium SLGJ4 has a significant inhibitory effect on Colletotrichum gloeosporioides.

[0011] Preferably, the aforementioned plant pathogen is any one of *Colletotrichum gloeosporioides*, *Colletotrichum gloeosporioides*, *Rhizoctonia solani*, and *Botrytis cinerea*.

[0012] Fourthly, the application of the aforementioned Ludwig's Enterobacter SLGJ4 or the aforementioned inoculants in the prevention and control of plant diseases.

[0013] Preferably, the above-mentioned prevention and control is achieved by regulating the activity of plant defense enzymes, significantly increasing the activity of POD enzyme in pomegranate fruit, inhibiting PPO activity, and delaying the enzymatic browning of pomegranate fruit.

[0014] Preferably, the plant disease mentioned above is any one of pomegranate anthracnose, pepper anthracnose, wheat sheath blight, and tomato gray mold.

[0015] In the pomegranate diseased group, the lesion area expanded, black juice oozed out, and the flesh softened, with an incidence rate of 93.29% and a disease index of 62.09. In contrast, the disease incidence in the biological control group was greatly reduced, with very little browning of the peel, an incidence rate of 62.83%, and a disease index of 24.22. Both the incidence rate and disease index of the biological control group decreased significantly, and the control effect was 60.99%, indicating that Ludwig's Enterobacter SLGJ4 has a good control effect.

[0016] Fifthly, this invention claims a method for preventing and controlling pomegranate anthracnose, which involves inoculating the aforementioned fungal agent onto pomegranate fruits.

[0017] Preferably, the concentration of Ludwig's Enterobacter SLGJ4 in the above-mentioned bacterial agent is 1×10⁻⁶. 6 -1×10 7 per mL.

[0018] The present invention has the following beneficial effects: This invention isolated, screened, and identified an excellent biocontrol strain, SLGJ4, from the rhizosphere soil of healthy pomegranate trees. Its classification name is... Enterobacter ludwigii The strain SLGJ4, with accession number CCTCC No: M 20251540, exhibits pale yellow, round colonies with smooth edges and a moist, slightly sticky surface. It possesses broad-spectrum antibacterial activity, showing inhibition rates of 55.48% against *Colletotrichum gloeosporioides* of pomegranate, *Colletotrichum platyphyllum* of pepper, *Rhizoctonia graminearum* of wheat, and *Botrytis cinerea* of tomato, respectively. SLGJ4 also demonstrates a 60.99% control effect against anthracnose in pomegranate fruit, significantly increasing the activity of the defensive enzyme POD and inhibiting PPO activity, thus delaying the progression of anthracnose in pomegranate fruit. Enzymatic browning of pomegranate fruit; after treatment of *Colletotrichum gloeosporioides* mycelium with strain SLGJ4, the mycelial growth morphology became abnormal, the mycelium swelled, and the mycelial tip swelled significantly, significantly inhibiting the growth of *Colletotrichum gloeosporioides*; microscopic observation revealed that strain SLGJ4 also caused the accumulation of reactive oxygen species (ROS) in the cells of *Colletotrichum gloeosporioides*, disrupting the integrity of the *Colletotrichum gloeosporioides* plasma membrane; all the above studies indicate that the biocontrol agent SLGJ4 has a good control effect on anthracnose in pomegranate fruit. This application provides a theoretical basis for the development of pomegranate biocontrol agents and preservatives, and is of great significance to the healthy development of pomegranate ecological agriculture. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 Morphological observation of the pathogen causing pomegranate anthrax; where A is the frontal morphology of the pathogen and B is the reverse morphology of the pathogen.

[0021] Figure 2 Microscopic morphological observation of the pathogen causing pomegranate anthracnose; where A represents hyphae morphology and B represents spore morphology.

[0022] Figure 3 The pathogenicity of *Pycnopyrum spp.* was determined; where A represents inoculation with the pathogen for 2 days, B represents inoculation with the pathogen for 4 days, and C represents inoculation with the pathogen for 9 days.

[0023] Figure 4 The study investigated the antagonistic effect of the biocontrol strain SLGJ4 against Colloidal anthracnose; where A represents the control group and B represents the confrontation between SLGJ4 and Colloidal anthracnose.

[0024] Figure 5Morphological observation of biocontrol strain SLGJ4; where A is the frontal morphology of the strain and B is the reverse morphology of the strain.

[0025] Figure 6 Phylogenetic tree of the biocontrol strain SLGJ4 based on the 16S rDNA sequence.

[0026] Figure 7 The control effect of different treatment groups on day 7 is shown; where A is the control group, B is the pathogenic group, C is the biocontrol group, and D is the preservation group.

[0027] Figure 8 The changes in the activity of defense enzymes in pomegranate fruits under different treatment groups are shown in Figure A, where A represents the change in POD activity, B represents the change in SOD activity, and C represents the change in PPO activity.

[0028] Figure 9 The study aimed to observe the microscopic inhibitory effect of biocontrol strain SLGJ4 on *Bacillus anthracis* foetida; where A was the control group and B was the treatment group.

[0029] Figure 10 The results of PI staining observation of Ludwig's Enterobacter SLGJ4 inhibiting Colloidal Anthrax are shown; where A is the treatment group, B is the dark field of the treatment group, C is the control group, and D is the dark field of the control group.

[0030] Figure 11 The results of DAPI staining observation of Ludwig's Enterobacter SLGJ4 inhibiting Colloidal Anthrax are shown; where A is the treatment group, B is the dark field of the treatment group, C is the control group, and D is the dark field of the control group. Detailed Implementation

[0031] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0032] Unless otherwise specified, the experimental methods used in the following experimental examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0033] The biocontrol bacterium Ludwig's Enterobacter SLGJ4 provided by this invention was isolated in October 2023 from the rhizosphere soil of healthy pomegranates in Shiliu Town, Jialou Village, Shangqiu City, Henan Province. The pathogen was isolated from diseased pomegranate fruits in the same area.

[0034] The present invention provides a strain of Ludwig's Enterobacter SLGJ4, which is classified and named as follows: Enterobacter ludwigiiIt was deposited on July 7, 2025 at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC No: M20251540, and the deposit address is Wuhan University, Wuhan, China.

[0035] Example 1: Isolation, purification, and molecular biological identification of JBTJ1, the pathogen of pomegranate anthracnose. 1. Isolation and purification of pathogens Pomegranate fruits exhibiting consistent disease symptoms were selected, and the pathogen was isolated and purified using tissue isolation. A 1 cm × 1 cm tissue sample was taken from the diseased fruit at the junction of the diseased tissue and the infected tissue using sterile scissors. The sample was then soaked in 2% sodium hypochlorite for 4 minutes, disinfected with 75% alcohol for 30 seconds, and rinsed three times with sterile water. After air-drying, the tissue was inoculated onto PDA medium plates and incubated at 28°C.

[0036] After the mycelium has grown, select edge mycelia and inoculate them onto PDA medium to purify the pathogen. Incubate at a constant temperature for 3 days, observing colony growth until a pure strain is obtained. Store the purified strain on PDA medium at 4°C for later use.

[0037] 2. Microscopic morphological observation of pathogens Preparation of pathogenic mycelial observation slides using the slide insertion method: A sterilized, dried coverslip is obliquely inserted next to a pathogenic colony cultured for 2 days, with the coverslip at a 45°C angle to the surface of the culture medium. After 3 days of culture, the coverslip is removed and observed under a microscope.

[0038] A pathogenic fungal strain, designated JBTJ1, was isolated from pomegranate fruits infected with anthracnose. The pathogen's morphological characteristics include cottony hyphae, a yellowish-white color, regular edges, dense hyphae, and concentric rings on the surface. Figure 1 ).

[0039] Under a microscope, the hyphae of strain JBTJ1 were slender, septate, and clearly defined, with fine branching. The conidia were straight, single, colorless, oblong, with a blunt apex, measuring (7.7-16.8) × (2.8-6.1) µm, and contained oil globules. Figure 2 ).

[0040] 3. Molecular biological identification of pathogens The preserved pathogen strain was inoculated onto PDA medium. A small amount of mycelium, about the size of a bean, was scraped from the cultured colony with a toothpick, and then DNA was extracted using the CTAB method.

[0041] The extracted DNA was amplified by PCR using the universal ITS primers ITS1 and ITS4.

[0042] 50 μL reaction system: 2 μL template DNA, 1 μL each of ITS4 and ITS1 primers, 25 μL 2×Taq DNA polymerase, and 23 μL ddH2O.

[0043] PCR amplification program: 94℃ pre-denaturation for 3 min; 94℃ denaturation for 30 s, 55℃ annealing for 45 s, 72℃ extension for 1 min, 30 cycles; after the cycles, 72℃ extension for 10 min. The amplified products were detected by electrophoresis and then sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing.

[0044] Using DNA from strain JBTJ1 as a template, PCR amplification was performed using the universal fungal primers ITS1 / ITS4, and the PCR products were sequenced. The obtained ITS sequences were then compared with the NCBI gene database using BLAST homology analysis, revealing that strain JBTJ1 is similar to... Colletotrichum gloeosporioides The homology reached 99.45%. Based on the ITS sequence homology analysis of the pathogen, the pathogen causing pomegranate anthracnose was preliminarily identified as *Colletotrichum gloeosporioides*. Colletotrichum gloeosporioides ).

[0045] Example 2: Pathogenicity identification of JBTJ1 strain, the pathogen of pomegranate anthracnose. After wiping healthy, disease-free pomegranate fruits with alcohol, a 0.5 cm deep wound was made with a sterile toothpick, and a fungal cake of pathogenic strain JBTJ1 was inoculated into the wound. The control group was inoculated with PDA culture medium blocks, and the disease incidence in the pomegranates was observed.

[0046] Brown spots appeared on pomegranate fruits 2 days after inoculation with strain JBTJ1. Figure 3 A), and gradually spread outwards from this center point (A). Figure 3 B), ultimately leading to a further expansion of the lesion area and rotting of the fruit pulp. Figure 3 C). In the early stage, reddish-brown juice will flow out, and in the later stage, yellowish-brown juice will flow out. This symptom is consistent with the natural disease state of pomegranate anthracnose, both of which will show brown lesions that gradually spread, causing the peel and pulp to rot. The control group did not develop the disease.

[0047] Example 3: Isolation, purification and screening of biocontrol strain SLGJ4 Take 1 g of rhizosphere soil and add it to 10 mL of sterile water. After shaking, collect the supernatant in a centrifuge tube and label it as 10. -1 Then dilute this solution 10 times to obtain 10 -2 10 -3 10 -4 10 -5Soil dilutions. 200 μL of soil dilutions of different concentrations were spread onto LB agar plates, with three replicates for each dilution. The plates were incubated at 28°C inverted positions. Growth was observed, and once clear colonies formed, colonies with distinct characteristics were selected for purification.

[0048] Initial screening: The 63 isolated bacteria were screened using the plate confrontation method. A 7 mm diameter anthracnose pathogen mycelium was placed in the center of an agar plate, and 5 mm diameter filter paper discs were placed at three points 2.5 cm from the center of the mycelium. 1.5 μL of different bacterial suspensions were added to the filter paper discs, and the plates were sealed after standing for 1 min. The plates were then incubated upside down at 28℃. Observations were made every 24 hours, and colonies exhibiting inhibition zones were counted after 4 days, resulting in 4 bacterial species.

[0049] Secondary screening: Four types of bacteria were screened using the plate confrontation method. A 7 mm diameter *Colletotrichum pungens* mycelial cake was placed in the center of an agar plate, and a 5 mm diameter filter paper disc was placed 2.5 cm from the center of the cake. 1.5 μL of the same bacterial suspension was added to the filter paper disc, and after standing for 1 min, the plate was sealed with film and incubated upside down at 28°C. Observations were made every 24 hours, and the inhibition rate was calculated after 5 days. A control group was formed by placing only the mycelial cake without adding bacterial suspension.

[0050] Inhibition rate = (Coronavirus diameter of control group - Coronavirus diameter of antagonistic group) / (Coronavirus diameter of control group - Diameter of mycelial cake) × 100%.

[0051] Broad-spectrum antibacterial screening: Bacteria with high inhibition rates were selected for broad-spectrum antibacterial screening. Seven-mm diameter mycelial cakes of *Anthracis capsulatum*, *Rhizoctonia solani*, and *Botrytis cinerea* were placed in the center of agar plates. A five-mm diameter filter paper disc was placed 2.5cm from the center of each mycelial cake. 1.5 μL of the same bacterial suspension was added to the filter paper disc. After standing for 1 min, the plates were sealed with film and incubated upside down at 28℃. Observations were made every 24 hours, and the inhibition rate was calculated after 5 days. A control group consisted of only mycelial cakes without bacterial suspension.

[0052] Table 1. Broad-spectrum antibacterial rate of biocontrol bacteria SLGJ4 After initial and secondary screening, the size of the inhibition zone was observed, and the diameter of the anthracnose pathogen was measured under different biocontrol treatment groups to calculate the inhibition rate. From the 63 isolated strains, one biocontrol strain with the best antagonistic effect was selected and designated SLGJ4. Measurements and calculations showed that this strain had an inhibition rate of 55.48% against the anthracnose pathogen.

[0053] Broad-spectrum antibacterial tests on the SLGJ4 strain showed that it was effective against *Anthracis chinensis*, a type of causal agent of anthracnose in chili peppers. Figure 4The fungicides showed antagonistic effects against Rhizoctonia granatum of wheat and Botrytis cinerea of ​​tomato, with inhibition rates of 51.03%, 48.67%, and 47.15%, respectively (Table 1).

[0054] Morphological observation of biocontrol strain SLGJ4 revealed that its colonies were pale yellow, round, with smooth edges, and a moist, slightly sticky surface. Figure 5 ).

[0055] Example 4: Identification and physiological and biochemical index determination of biocontrol strain SLGJ4 DNA was extracted from strain SLGJ4 using the CTAB method. Using this DNA as a template, PCR amplification was performed using the universal bacterial primers 27F and 1429R, and the PCR amplification products were sequenced.

[0056] Reaction system (20 µL): template 2 µL, upstream primer (27F) 1 µL, downstream primer (1492R) 1 µL, dNTP 1.6 µL, 10×PCR buffer 2 µL, ddH2O 11.9 µL, Taq enzyme 0.5 µL.

[0057] Reaction procedure: 94℃ pre-denaturation for 3 min; 94℃ denaturation for 30 s, 55℃ annealing for 45 s, 72℃ extension for 1 min, 30 cycles; 72℃ extension for 10 min. The PCR product obtained from amplifying the bacterial 16S rDNA gene was sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The bacterial strain was identified based on physiological and biochemical characteristics.

[0058] Table 2 Physiological and biochemical indicators of Enterobacter ludwig's bacillus Using SLGJ4 strain DNA as a template, PCR amplification was performed using universal bacterial primers, and the PCR products were sequenced. The obtained sequences were then compared with the NCBI gene database using BLAST homology. Enterobacter ludwigii The homology reached 99.64%. A phylogenetic tree was constructed by comparing the 16S rRNA sequences of strain SLGJ4 with those of the reference strain. Figure 6 ), found that strain SLGJ4 and E. ludwigii (JTLO01000001) clustered together with a confidence level of 96%, preliminarily identifying the bacterium as Ludwig's Enterobacter (JTLO01000001). E. ludwigii The results of the physiological and biochemical index determination of this strain are shown in Table 2.

[0059] Application Example 1: Control of anthracnose in pomegranate fruits by biocontrol strain SLGJ4 The preserved antagonistic bacterium SLGJ4 was cultured on LB liquid medium at 37℃ and 200 r / min for 15 h with shaking. Preserved anthrax bacteria were inoculated into liquid PDA medium and cultured at 37℃ and 200 r / min for 3 days with shaking to obtain the pathogenic bacterial solution. Fresh, healthy pomegranates of uniform size were selected, rinsed thoroughly with running water, and disinfected with alcohol. Approximately 1 cm deep wounds were made on both sides of the pomegranate peel, and liquid PDA, pathogenic bacterial solution, and antagonistic bacterium solution were added to each wound, respectively. The control group was inoculated only with PDA medium; the pathogenic group was inoculated with pathogenic bacterial solution; the preservation group was inoculated with antagonistic bacterial solution; and the biocontrol group was inoculated with both antagonistic and pathogenic bacterial solutions. Each treatment was replicated six times, with five pomegranates treated per group. After inoculation, the pomegranates were cultured at 25℃ and 65%-80% relative humidity. Disease incidence was observed every 3 days, and after 10 days, the number of infected pomegranates and the severity of disease in each pomegranate were recorded. Based on the disease severity classification, the incidence rate and control effect were calculated. Enzyme activity was measured every two days after the appearance of symptoms, and the data were recorded.

[0060] The disease severity grading criteria for anthracnose in pomegranate fruits are as follows: Disease severity: Grade 0, all fruit is free of disease spots; Grade 1: Only light brown spots appear on the fruit surface, with the affected area less than 5%. Grade 2, with lesions covering 6% to 15% of the entire fruit surface; Grade 3, with lesions covering 16% to 30% of the entire fruit surface; Grade 4, with lesions covering 31% to 50% of the entire fruit surface; Grade 5, with lesions covering more than 51% of the entire fruit surface.

[0061] Disease index = [∑(representative value of each level × number of results at each level) / (total number of results in the survey × 5)] × 100%.

[0062] Prevention and control effect = [(disease index of control group - disease index of treatment group) / disease index of control group] × 100%.

[0063] Table 3. Control efficacy of strain SLGJ4 against anthracnose in pomegranate fruit. Different treatment groups of pomegranates exhibited different disease symptoms in the later stages of the disease. In the diseased group, the lesion area expanded, black juice oozed, and the flesh softened. In contrast, the disease incidence in the biocontrol group was significantly reduced, with minimal browning of the peel. Statistical analysis showed that no pomegranates in the control group showed disease. The disease incidence rate in the diseased group was 93.29%, with a disease index of 62.09. In the biocontrol group, the disease incidence rate was 62.83%, and the disease index was 24.22. The biocontrol group showed a significant decrease in both disease incidence and disease index, with a control effect of 60.99%, indicating a good control effect against *Enterobacter ludwigschneideri*. Figure 7 (Table 3).

[0064] Application Example 2: Determination of the activity of defense enzymes in pomegranate fruit POD activity assay: Weigh 3.0 g of pomegranate tissue sample and place it in a pre-cooled mortar. Add 5 mL of 0.1 mol / L phosphate buffer and grind into a homogenate under ice bath conditions. Centrifuge at 12000 r / min for 30 min at 4℃. Collect the supernatant as the enzyme extract and store it at low temperature for later use. Using distilled water as a reference solution, place the sample chamber of a UV spectrophotometer and zero it at 470 nm. Take another cuvette, add 3 mL of 25 mmol / L guaiacol and 0.5 mL of the enzyme extract, then add 0.2 mL of 0.5 mol / L H2O2 solution and mix rapidly to start the reaction. Start timing immediately and measure the absorbance after 15 s of reaction, recording it every 1 min. OD value per minute is recorded as the absorbance value. 470 The change is 0.1 to 1 U.

[0065] POD activity = (reaction solution absorbance termination value - reaction solution absorbance initial value) × total volume of extract / [volume taken during measurement × sample mass × (reaction termination time - reaction start time) × 0.1].

[0066] SOD activity assay: The SOD activity in pomegranate was determined using the nitroblue tetrazolium photoreduction method. 3 g of pomegranate was weighed, and 2 mL of pre-cooled SOD extraction medium was added. The mixture was ground in an ice bath and brought to a final volume of 5 mL. The mixture was centrifuged at 10,000 r / min for 15 min at 4℃ to obtain 5 mL of crude enzyme solution. Then, 1.3 mL of 50 mmol / L phosphate buffer (pH 7.8), 0.3 mL of 130 mmol / L methionine, 0.3 mL of 750 mol / L nitroblue tetrazolium, 0.3 mL of 100 mol / L disodium ethylenediaminetetraacetate, and 0.3 mL of 20 mol / L riboflavin solution were added sequentially. Next, 0.5 mL of 50 mmol / L pH 7.8 phosphate buffer and the enzyme extract from each treatment were added. The mixture was irradiated at 4000 1x for 10 min, and the absorbance was measured at 560 nm to calculate the SOD activity.

[0067] SOD activity = [(OD under light)] 560 -Sample OD 560 [(OD under illumination) × total volume] / [(OD under illumination)] 560 (×0.5×sample mass×volume of enzyme solution)).

[0068] PPO activity assay: Weigh 3.0 g of pomegranate tissue sample and place it in a pre-cooled mortar. Add 5 mL of extraction buffer (containing 1 mmol PEG, 4% PVPP, and 1% Triyon X-100), and grind into a homogenate under ice bath conditions. Centrifuge at 12000 r / min for 30 min at 4℃, and collect the supernatant as the enzyme extract. Store at low temperature for later use. Add 3 mL of 50 mmol / L acetate buffer and 0.75 mL of catechol solution, and finally add 0.075 mL of the enzyme extract. Start timing immediately. Using distilled water as a reference, record the absorbance of the reaction system at 420 nm after 15 s of reaction, and record every 1 min. The amount of enzyme required for a 0.1 change in absorbance at 420 nm per minute is defined as one unit of activity.

[0069] PPO activity = (absorbance termination value of reaction solution - absorbance initial value of reaction solution) × total volume of extract / [volume taken during measurement × sample mass × (reaction termination time - reaction start time) × 0.1].

[0070] POD enzyme activity generally showed an initial increase followed by a decrease with prolonged treatment time. All treatment groups reached their peak activity on day 3. The enzyme activity in the biocontrol group was higher than that in the pathogenic group for the first 1-5 days, indicating that the biocontrol bacterium SLGJ4 significantly increased the POD enzyme activity in pomegranate fruits. Figure 8 A).

[0071] The overall trend of SOD enzyme activity in pomegranates was an initial increase followed by a decrease. The control and preserved groups reached their peak activity on day 3, while the pathogenic and biocontrol groups reached their peak activity on day 5. At day 3 of treatment, the enzyme activity in both the pathogenic and biocontrol groups significantly increased, with the biocontrol group showing lower activity than the pathogenic group. Figure 8 B).

[0072] Significant differences in PPO enzyme activity were observed among the groups. The overall trend of enzyme activity changes was not significant between the biocontrol and preservation groups. In the pathogenic group, enzyme activity increased significantly on day 3, followed by a downward trend. On days 3 and 5, the PPO activity in the biocontrol group was significantly lower than that in the pathogenic group, indicating that the surface biocontrol bacteria effectively inhibited PPO activity and delayed enzymatic browning of pomegranate fruits. Figure 8 C).

[0073] Application Example 3: Effect of biocontrol strain SLGJ4 on the microstructure of *Bacillus anthracis*. Antagonistic bacterial strains preserved at 4℃ were inoculated into LB liquid medium and cultured at 37℃ and 120 r / min for 24 h on a shaker. The bacterial culture was then centrifuged at 10000 r / min for 10 min at 4℃, and the supernatant was collected. The supernatant was filtered through a 0.22 μm microporous membrane for sterilization, and the sterile filtrate was collected and stored at 4℃ for later use. Pathogenic bacterial strains preserved at 4℃ were inoculated onto PDA plates and cultured at 28℃ for 4 days for activation. Subsequently, mycelial discs were inoculated into the center of PDA plates containing sterile filtrate, and mycelial discs were inoculated onto PDA plates without sterile filtrate as a control. All plates were incubated at 28℃ for 4 days, and bacterial growth was observed. Hyphae morphology was observed under an optical microscope.

[0074] The pathogenic fungal hyphae in the control group grew normally. Figure 9 A), while the mycelial growth morphology of the treatment group was abnormal, with mycelial swelling and obvious swelling of the mycelial tips (A). Figure 9 B). This indicates that the biocontrol bacterium SLGJ4 has a significant inhibitory effect on Colletotrichum gloeosporioides.

[0075] Application Example 4: Effects of biocontrol strain SLGJ4 on intracellular reactive oxygen species (ROS) and plasma membrane integrity in *Bacillus anthracis*. 20 mL of biocontrol bacterial solution cultured for 2 days was filtered through a 0.22 μm filter and then added to 50 mL of pathogenic bacterial solution cultured for 5 days prior, for a total culture period of 2 days. A control group containing 20 mL of LB medium was used. Two groups of cultured pathogenic mycelia were divided into three portions each and added to 2 mL centrifuge tubes. The pathogenic mycelia were washed three times with phosphate-buffered saline (0.1 M, pH 7.0), and then stained with 1 mL of 10 µM DCFH-DA solution for 30 min and 1 mL of 10 µg / mL DAPI solution for 15 min in the dark at 28°C, respectively, and thoroughly mixed. The effect of the biocontrol bacterial solution on intracellular reactive oxygen species (ROS) and plasma membrane integrity of the pathogenic bacteria was studied. After staining in the dark, an appropriate amount of solution containing mycelia was pipetted onto a clean glass slide, excess water was blotted off with filter paper, and the solution was observed under a fluorescence microscope.

[0076] Results of DCFH-DA staining observation on the inhibition of *Enterobacter ludwigschneidera* strain SLGJ4 by *Colletotrichum gloeosporioides*. Dark field control group ( Figure 10 D) No green fluorescence was observed, while the treatment group emitted obvious green fluorescence. Figure 10 B). The results showed that strain SLGJ4 caused the accumulation of reactive oxygen species (ROS) in the cells of *Colletotrichum gloeosporioides*.

[0077] Dark field control group ( Figure 11 D) No red fluorescence was observed; the treated experimental group emitted weaker red fluorescence. Figure 11B). The results showed that *Enterobacter ludwigii* disrupted the integrity of the cell membrane of *Colletotrichum gloeosporioides*.

[0078] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A strain of Enterobacter ludwigii SLGJ4, which is classified and named as Enterobacter ludwigii , and preserved in the China Center for Type Culture Collection on July 7, 2025, with a preservation number of CCTCC No: M 20251540 and a preservation address of Wuhan, China. Wuhan University.

2. An inoculant characterized in that: Enterobacter ludwigii SLGJ4 of claim 1.

3. Use of Enterobacter ludwigii SLGJ4 of claim 1 or the bacterial agent of claim 2 in antagonizing plant pathogenic fungi.

4. Use according to claim 3, characterized in that: The antagonism is achieved by disrupting hyphal morphology of the plant pathogenic fungi, promoting accumulation of intracellular reactive oxygen species, and disrupting plasma membrane integrity.

5. Use according to claim 4, characterized in that: The plant pathogenic fungi is any one of Colletotrichum gloeosporioides, C. truncatum, Rhizoctonia cerealis, and Botrytis cinerea.

6. Use of Enterobacter ludwigii SLGJ4 of claim 1 or the bacterial agent of claim 2 in controlling plant diseases.

7. Use according to claim 6, characterized in that: The control is achieved by modulating defense enzyme activities of the plants.

8. Use according to claim 7, characterized in that: The plant diseases are any one of anthracnose of pomegranate, anthracnose of pepper, wheat sharp eyespot, and tomato gray mold.

9. A method of controlling anthracnose of pomegranate, characterized by: The bacterial agent of claim 2 is inoculated on pomegranate fruits.

10. A method of controlling grape anthracnose according to claim 9, characterized in that: The concentration of Enterobacter ludwigii SLGJ4 used in the bacterial agent is 1 x 10 6 -1 x 10 7 / mL.

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