Burkholderia gladioli and uses thereof

Abstract

The application discloses a strain of Burkholderia gladioli CT-5 and application thereof, and relates to the technical field of microorganisms. The strain is preserved in the China Center for Type Culture Collection, and the preservation number is CCTCC NO: M20242732, and the preservation date is December 5, 2024. The Burkholderia gladioli CT-5 provided by the application has a significant prevention and treatment effect (P<0.05) on root rot of Idesia polycarpa caused by Fusarium solani, and has a significant inhibitory effect on other various plant pathogenic fungi such as Schizophyllum commune, Diaporthe eres and Colletotrichum acutatum. In a pot experiment, the biomass of Idesia polycarpa seedlings, soil nutrients, SOD, POD and CAT enzyme activities of the CT-5 treatment group are significantly improved (P<0.05) compared with the pathogenic fungus control group (FP), indicating that the strain can promote plant growth, activate the activity of defense enzymes in the body, and thus effectively control the root rot of Idesia polycarpa, which provides strong theoretical basis and technical support for the sustainable development of Idesia polycarpa.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a strain of Burkholderia gladioli and its applications. Background Technology

[0002] Guizhou Province is one of the main distribution areas of *Vernicia fordii*. Since its introduction and cultivation in 2018, the planting area of ​​*Vernicia fordii* in Guizhou Province has gradually increased. With the large-scale planting of *Vernicia fordii*, pests and diseases have become one of the main factors restricting the development of the industry, and therefore have attracted much attention. Root rot of *Vernicia fordii* is one of the main diseases of this plant and is a common one. With the increase in the planting area of ​​*Vernicia fordii*, root rot has appeared in various regions. This disease mainly affects the roots of *Vernicia fordii*. After infection, the leaves of the entire plant turn yellow and wilt, the roots rot and turn black, and a large number of leaves fall off. In severe cases, the entire plant dies. From August to September 2023, investigations in Renhuai City, Dushan County, and Sandu County found that the damage caused by root rot of *Vernicia fordii* was serious, with an incidence rate of 28-35%, and there is a trend of continuous expansion and spread.

[0003] Currently, the main method for controlling root rot is to use chemical pesticides to drench the roots. However, this method is not only ineffective but also causes problems such as pesticide residues, pathogen resistance, and environmental pollution.

[0004] Therefore, providing a green prevention and control method that can effectively control the occurrence of root rot in *Vernicia fordii*, providing necessary technical support for the scientific prevention and control of root rot in *Vernicia fordii*, and ultimately ensuring the healthy and sustainable development of the *Vernicia fordii* industry is the problem that this invention urgently needs to solve. Summary of the Invention

[0005] The present invention provides a strain of Burkholderia gladioli and its application, aiming to solve the problems existing in the above-mentioned background art.

[0006] To achieve the above-mentioned technical objectives, the present invention mainly adopts the following technical solutions:

[0007] In a first aspect, the present invention discloses a strain of Burkholderia gladioli CT-5, which is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCCNO: M20242732 and deposit date of December 5, 2024.

[0008] In a preferred embodiment of the present invention, the Burkholderia gladioli CT-5 has a 16S rDNA sequence as shown in SEQ ID No:1.

[0009] In a second aspect, the present invention discloses a bacterial agent containing Burkholderia gladioli CT-5 as described in the first aspect.

[0010] In a preferred embodiment of the present invention, the Burkholderia gladioli CT-5 is present in the bacterial agent in the form of cultured live bacteria, fermentation broth, or bacterial suspension.

[0011] Thirdly, the present invention discloses the application of Burkholderia gladioli CT-5 as described in the first aspect in inhibiting root rot of *Vernicia fordii* or in the preparation of *Vernicia fordii* growth promoters.

[0012] Fourthly, this invention discloses the application of Burkholderia gladioli CT-5 as described in the first aspect in inhibiting Fusarium solani, Schizophyllum commune, Ulmus parvifolium, Anthracnose spp., Alternaria alternata, Anthracnose siamensis, and Anthracnose spp.

[0013] In a sixth aspect, the present invention discloses the application of Burkholderia gladioli CT-5 as described in the first aspect in enhancing the activity of SOD, POD, and CAT enzymes.

[0014] In a seventh aspect, the present invention discloses the application of Burkholderia gladioli CT-5 as described in the first aspect in increasing the content of organic matter, total phosphorus, total nitrogen, total potassium, alkaline nitrogen, available phosphorus, and available potassium in soil.

[0015] Eighthly, this invention discloses the application of non-volatile metabolites produced by Burkholderia gladioli CT-5 as described in the first aspect in inhibiting root rot of *Vernicia fordii*, preparing *Vernicia fordii* growth promoters, increasing the activity of SOD, POD, and CAT enzymes, or increasing the content of organic matter, total phosphorus, total nitrogen, total potassium, alkaline nitrogen, available phosphorus, and readily available potassium in soil.

[0016] Furthermore, the non-volatile metabolites include betaine, phenazine, phenylalanine, pyrocyanin, piperidine, erucamide, pyrrolidine, L-tyrosine, glutamic acid, cephalexin, L-lysine, chlorogenic acid, dipropyl phthalate, dioctyl phthalate, and benzophenone.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The *Burkholderia gladioli* CT-5 strain provided by this invention has a significant control effect on root rot of *Vernicia fordii* (P<0.05). *Bacillus subtilis* (wettable powder) showed moderate control, with a relative efficacy of 50.76%; 50% carbendazim (wettable powder) showed poor control, with a relative efficacy of 30.96%. In pot experiments, the bacterial treatment group showed significantly higher biomass, soil nutrients, SOD, POD, and CAT enzyme activities in *Vernicia fordii* seedlings compared to the pathogen control group (FP) (P<0.05). This indicates that inoculation with *Burkholderia gladioli* CT-5 can promote plant growth, activate the activity of internal defense enzymes, and thus control root rot of *Vernicia fordii*. This provides a strong theoretical basis and technical support for the prevention and control of root rot of *Vernicia fordii*, the scientific control of pests and diseases in *Vernicia fordii*, and the promotion of the green and sustainable development of the *Vernicia fordii* industry. Attached Figure Description

[0019] Figure 1 Morphological image of Burkholderia gladioli CT-5 provided by the present invention;

[0020] Figure 2 The CT-5 electron microscope scan image of Burkholderia gladioli provided by this invention;

[0021] Figure 3 This is a Gram staining image of Burkholderia gladioli CT-5 provided by the present invention;

[0022] Figure 4 The image shows the effect of Burkholderia gladioli CT-5 on the hyphae of the pathogen, provided by this invention; the left image shows hyphal breakage and entanglement, and the right image shows hyphal swelling.

[0023] Figure 5 The image shows the inhibitory effect of Burkholderia gladioli CT-5 on pathogens provided by this invention.

[0024] Figure 6 The image shows the secretory characteristics of Burkholderia gladioli CT-5 provided by this invention, where, from left to right, they are protease, hepatophile, and organophosphates. Detailed Implementation

[0025] To better illustrate the purpose, technical solution, and advantages of the present invention, the present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0026] In the following embodiments, unless otherwise specified, the experimental methods used are conventional methods, and the materials and reagents used are commercially available unless otherwise specified.

[0027] Example 1: Isolation and identification of Burkholderia gladioli CT-5

[0028] 1. Separation location

[0029] In Chishui City (28°28′4″N, 105°59′28″E, H: 823), healthy *Vernicia fordii* (tree) plants were selected. The root system was dug up 30 cm from the trunk, excess soil was removed from the roots, and the rhizosphere soil was obtained using a shaking method. The soil was placed in sealed plastic bags, stored in an icebox, and brought back to the laboratory at 5°C.

[0030] 2. Separation Method

[0031] Bacteria were isolated from collected rhizosphere soil samples using the dilution plating method. 10g of soil sample was weighed and placed in 90mL of sterile water (containing 10 sterile glass beads). The solution was shaken on a shaker at 25°C for 10 minutes. Sterile water was then added to dilute the soil solution to 10:1. -1 10 -2 10 -3 10 -4 Dilute the solution by 200 μL and spread it onto LB medium, with three replicates for each concentration. Incubate at 28°C and observe regularly. After a period of time, different bacterial colonies will appear. Pick a single colony and transfer it to a new LB medium. Purify repeatedly until a pure culture with uniform and single colonies is obtained. Store the obtained pure culture at 4°C.

[0032] Biocontrol bacteria exhibiting significant antagonistic activity against pathogens were screened using the plate confrontation method. On a clean bench, mycelial cakes of the pathogen causing root rot of *Vernicia fordii* were punched using a sterile punch (5 mm diameter) and inoculated into the center of PDA plates. Biocontrol bacteria were then inoculated using a streak loop at 3.0 cm from both ends of the mycelial cake. The plates were incubated at 28°C. Plates inoculated only with the pathogen served as the control group, and the experiment was conducted in triplicate. After 7 days of incubation, the colony diameters of *Vernicia fordii* root rot pathogens in both the control and confrontation groups were measured using a ruler, and the inhibition rate was calculated using the following formula.

[0033] Inhibition rate = (Coronavirus diameter of control group - Coronavirus diameter of treatment group / Coronavirus diameter of control group) × 100%.

[0034] 3. Identification of strains

[0035] Morphological identification: The selected biocontrol bacteria (i.e., Burkholderia gladioli CT-5) were streaked on LB solid medium. After 5 days, the colony shape, size, color, transparency, and moisture content were observed. The results are as follows: Figure 1 As shown. The morphological characteristics of biocontrol bacteria were observed using a Hitachi SUB900 scanning electron microscope, and the results are as follows. Figure 2 As shown.

[0036] After being cultured on LB medium for 48 hours, Burkholderia gladioli was pale yellow, opaque, moist, and raised, with regular colony edges and a distinctive odor. It was Gram-negative. Electron microscopy showed that the bacteria were short cylindrical, uniform and plump, with wrinkled surfaces, clear outlines, and no obvious flagella.

[0037] Physiological and biochemical identification: The physiological and biochemical characteristics of the screened biocontrol bacteria were determined with reference to books such as *Bergey's Manual of Bacterial Identification* and *Manual of Systematic Identification of Common Bacteria*. The indicators measured included Gram staining, glucose oxidation-fermentation, nitrate reduction, catalase, methyl red (MR), VP assay, starch hydrolysis, indole test, oxidase, gelatin liquefaction, and citrate utilization. The results showed that the bacteria exhibited Gram staining (e.g., Gram staining...). Figure 3 The results (shown) were negative for nitrate reduction and methyl red test, and positive for starch hydrolysis reaction. The results for catalase, VP assay, starch hydrolysis, indole test, gelatin liquefaction, and citrate utilization were all negative.

[0038] Molecular biological identification: DNA was extracted using the BioTeke bacterial genomic DNA extraction kit. PCR amplification was performed using the universal 16S rDNA primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') / 1492R (5'-GGTTACCTTGTTACGACTT-3'). After confirming the PCR products were correct, the samples were sent to Shanghai Bioengineering Co., Ltd. (Chengdu) for sequencing. Sequence alignment was performed using NCBI, and the sequences were downloaded. A phylogenetic tree was constructed using MEGA-11 software and the neighbor-joining method. The strain was identified as *Burkholderia gladiol i*, named *Burkholderia gladiol i* CT-5, and deposited at the China Center for Type Culture Collection (CCT CCNO: M20242732) on December 5, 2024.

[0039] The primer sequence of the 16S rDNA molecule of this bacterium: GGTCAGCGTACTGCTCGGCAGCACGGGTCGCTTG CACCTGGTGGCGAGTGGCGAACGGGTGAGTAATACATCGGAACATGTCCTGTAGTGGGGGATAGCCCGGCGAAAGCCGGATTAATACCGCATACGATCTACGGATGAAAGCGGGGGACCTTCGGGCCTCGCGCTATAGGGTTGGCCGATGGCTGATTAGCTAGTTGGTGGGGTAAAGGCCCACCAAGGCGACGATCAGTAGCTGGTCTGAGAGGACGACCAGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATTTTGGACAATGGGCGAAAGCCTGATCCAGCAATGCCGCGTGTGTGAAGAAGGCCTTCGGGTTGTAAAGCACTTTTGTCCGGAAAGAAATCCTGAGGGCTAATATCCTTCGGGGATGACGGTACCGGAAGAATAAGCACCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCGAGCGTTAATCGGAATTACTGGGCGTAAAGCGTGCGCAGGCGGTTTGTTAAGACCGATGTGAAATCCCCGGGCTCAACCTGGGAACTGCATTGGTGACTGGCAAGCTAGAGTATGGCAGAGGGGGGTAGAATTCCACGTGTAGCAGTGAAATGCGTAGAGATGTGGAGGAATACCGATGGCGAAGGCAGCCCCCTGGGCCAATACTGACGCTCATGCACGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCCTAAACGATGTCAACTAGTTGTTGGGGATTCATTTCCTTAGTAACGTAGCTAACGCGTGAAGTTGACCGCCTGGGGAGTACGGTCGCAAGATTAAAACTCAAAGGAATTGACGGGGACCCGCACAAGCGGTGGATGATGTGGATTAATTCGATGCAACGCGAAAAACCTTACCTACCCTTGACATGGTCGGAACCTTGGAGAGATCCGAGGGTGCTCGAAAGAGAACCGATACACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTCCTTAGTTGCTACGCAAGAGCACTCTAGGGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAGTCCTCATGGCCCTTATGGGTAGGGCTTCACACGTCATACAATGGTCGGAACAGAGGGTCGCCAACCCGCGAGGGGGAGCTAATCCCAGAAAACCGATCGTAGTCCGGATTGCACTCTGCAACTCGAGTGCATGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTCCTACACGAGAATTATCTAGTCTCACCACGTGGTGGCAAGGTAACCATAGAGTAACAA。

[0040] Example 2. Test on the inhibition of the growth of the pathogen causing Idesia polycarpa root rot by Burkholderia gladioli CT-5

[0041] (1) Effect of Burkholderia gladioli CT-5 on the hyphal morphology of the pathogen causing Idesia polycarpa root rot

[0042] Place a glass slide in a 9cm sterile petri dish, and pour sterile PDA medium into the culture medium until it completely covers the slide. Let it cool. Inoculate the root rot pathogen of *Vernicia fordii* at one end of the slide, 2cm from the edge, and inoculate the other end, 2cm from the edge, with biocontrol bacteria activated for 48 hours. Incubate at 28℃. When the hyphae reach the center of the slide, remove the slide, discard any excess culture medium, and observe it under an optical microscope. Use the culture medium without biocontrol bacteria as a control to observe whether the pathogen exhibits abnormal changes such as hyphal swelling or deformity.

[0043] (2) Effects of Burkholderia gladioli CT-5 fermentation broth on pathogen spores under liquid culture conditions

[0044] Two μL of Burkholderia gladioli CT-5 strain was inoculated into a 250 mL Erlenmeyer flask containing 100 mL of LB liquid medium. The flask was incubated at 28°C with shaking at 180 rpm for 48 h to obtain the biocontrol bacterial fermentation broth. Sterile water was added to adjust the biocontrol bacterial fermentation broth to 1×10⁸ CFU / mL. Two pieces of activated root rot pathogen mycelium (5 mm) were added to PDB medium, followed by 1 mL of the 1×10⁸ CFU / mL biocontrol bacterial fermentation broth. The flask was incubated at 28°C with 280 rpm for 5 days, with PDB medium containing only pathogen mycelium serving as a control. After 5 days, 1 mL of the culture was examined under an electron microscope to observe the morphology of root rot pathogen spores, and the number of spores in the culture was counted using a hemocytometer.

[0045] The result is Figure 4 It is known that Burkholderia gladioli CT-5 can cause the pathogen's hyphae to swell, bend, entangle, and dissolve, thus destroying the pathogen's hyphae. In a liquid culture environment, it can significantly inhibit the growth of pathogen spores, with a sporulation rate of 0.

[0046] (3) Inhibitory effect of volatile metabolites of Burkholderia gladioli CT-5 on pathogens

[0047] The inhibitory effect of volatile metabolites of *Burkholderia gladioli* CT-5 on the root rot pathogen of *Vernicia fordii* was detected using the plate-on-plate culture method. In a clean bench, pathogenic mycelial cakes were cut using a sterile punch (5 mm) and inoculated into the center of PDA medium. 2 μL of biocontrol bacteria was then inoculated into the center of fresh PDA medium using an inoculation loop. The two plates were inverted and incubated at 28℃ for 7 days. A control group was used, with the pathogen-inoculated PDA medium inverted onto a blank PDA medium. Mycelial growth was observed periodically. Each treatment was repeated three times. After 7 days of incubation, the mycelial diameter of the pathogen in the PDA medium of the control and treatment groups was measured. The inhibition rate was calculated using the following formula:

[0048] Inhibition rate = (Coronavirus colony diameter in control group - Coronavirus colony diameter in treatment group / Coronavirus colony diameter in control group) × 100%.

[0049] The results are as follows Figure 5 As shown in Figure 1 and Table 1. From Figure 5 As shown in Table 1, by comparing the control group (pathogen group) and the bacterial group, it can be seen that the volatile metabolites of this bacterium have no inhibitory effect on the pathogen.

[0050] Table 1. Inhibitory rate of volatile metabolites against pathogens

[0051]

[0052] (4) Inhibitory effect of non-volatile metabolites of Burkholderia gladioli CT-5 on pathogens

[0053] Two μL of Burkholderia gladioli CT-5 strain was inoculated into a 250 mL Erlenmeyer flask containing 100 mL of LB liquid medium and cultured at 28°C with shaking at 180 rpm for 48 h to obtain the biocontrol strain fermentation broth. The fermentation broth was centrifuged at 4°C and 12000 rpm for 10 min, and the supernatant was collected and filtered through a 0.22 μm microporous membrane to obtain a sterile supernatant. The sterile supernatant was mixed with PDA solid medium cooled to 45°C at a ratio of 1:2 and poured into plates. After the plates cooled and solidified, mycelial cakes of the pathogen causing root rot of *Vernicia fordii* were punched out using a 5 mm sterile punch and inoculated into the center of the plates. The plates were incubated at 28°C with three replicates for each treatment. The colony diameter of the pathogen was measured and recorded using a graduated ruler on days 5, 10, and 15 after inoculation, and the inhibition rate was calculated using the following formula.

[0054] Antibacterial rate = (diameter of pathogenic bacteria colonies in PDA plate - diameter of pathogenic bacteria colonies in fermentation broth antibacterial plate) / diameter of pathogenic bacteria colonies in PDA plate × 100%.

[0055] The results are as follows Figure 6 As shown in Figure 1 and Table 2. From Figure 6As shown in Table 2, the strain showed the best inhibitory effect on the pathogenic mycelium on the fifth day, at 22.21%.

[0056] Table 2. Inhibitory rates of non-volatile metabolites against pathogens.

[0057]

[0058] (5) Determination of non-volatile metabolites of Burkholderia gladioli CT-5

[0059] The activated biocontrol bacteria were inoculated into LB liquid medium and cultured at 28°C and 180 rpm for 48 h with shaking. After centrifugation at 12000 rpm and 4°C, the supernatant was collected and stored at -80°C. Three tubes of supernatant were collected from each strain. These tubes were then mailed on dry ice to Zhongke New Life Biotechnology Co., Ltd. in Jinhua City, Zhejiang Province for the determination of non-volatile metabolites.

[0060] The results showed that the strain produced 15 non-volatile metabolites, i.e., antibacterial substances: betaine, phenazine, phenylalanine, pyocyanin, piperidine, erucamide, pyrrolidine, L-tyrosine, glutamic acid, cephalexin, L-lysine, neochlorogenic acid, dipropyl phthalate, dioctyl phthalate, and benzophenone.

[0061] (6) Detection of secretory substances of Burkholderia gladioli CT-5

[0062] The activated bacteria to be tested were inoculated onto protease detection plates, chitin detection plates, organophosphate detection plates, and ferrophilic detection plates, respectively, and then incubated at a constant temperature of 28°C for 48–96 hours. The presence of a transparent zone at the edge of the colonies on each detection plate was observed; for the ferrophilic detection plate, the presence of a yellow halo around the colonies was observed.

[0063] The bacteria to be tested were inoculated onto cellulase detection medium and incubated at 28°C for 72–96 h. The detection medium plates were washed with 0.9% NaCl solution, and the NaCl solution was rinsed off every 2 h, followed by the addition of unused 0.9% NaCl solution. Then, the inside of the plates was stained with 0.2% Congo red stain. Finally, the color of the Congo red stain was washed off with 1 mol / L NaCl solution. The clear zone around the colonies was observed after 6 h.

[0064] The results are as follows Figure 6 As shown in Table 3. From Figure 6 As shown in Table 3, the strain can produce proteases, organophosphates, and hematophiles, but does not secrete chitin.

[0065] Table 3. Detection results of characteristic substances secreted by Burkholderia gladioli CT-5

[0066]

[0067] Example 3: Determination of the broad-spectrum antibacterial activity of Burkholderia gladioli CT-5

[0068] (1) Test pathogens: Schizophyllum commune, Alternaria alternata, Colletotrichum siamense, Diaporthe eres, Colletotrichum fructicola, and Colletotrichum acuminata.

[0069] (2) Experimental Design: The plate confrontation method was used to determine the inhibitory effect of Burkholderia gladioli CT-5 on seven common plant pathogens and to clarify its broad-spectrum antibacterial activity. In a clean bench, pathogens that had been cultured at 28℃ for 7 days were inoculated into mycelial cakes using a sterile punch (d=5mm) and placed in the center of PDA medium. Four symmetrical points were selected at a distance of 2.5cm from the pathogen mycelial cakes to inoculate Burkholderia gladioli CT-5. PDA medium without biocontrol bacteria was used as a control. Each treatment was repeated three times and cultured at 28℃ for 7 days. After 7 days, the diameter of pathogen colonies was measured using the cross-cross method, and the inhibition rate was calculated.

[0070] Inhibition rate = (Coronavirus colony diameter in control group - Coronavirus colony diameter in treatment group) / Coronavirus colony diameter in control group × 100%

[0071] (3) Experimental results: The biocontrol strain CT-5 showed the strongest inhibitory effect on Schizophyllum commune, with an inhibition rate of 66.74%; the weakest inhibitory effect on Alternaria alternata, with an inhibition rate of 41.1%; and the inhibition rates on Colletotrichum siamense, Diaporthe eres, Colletotrichum fructicola, and Colletotrichumacutatum were 51.1%, 64.51%, 41.37%, and 58.44%, respectively.

[0072] Table 4. Inhibition rate of Burkholderia gladioli CT-5 against several plant pathogens.

[0073]

[0074] Example 4: Effect of Gladiolus Burkholderia CT-5 inoculation on growth-promoting and disease-resistant effects of potted *Vernicia fordii* seedlings.

[0075] (1) Effect test of Burkholderia purpurea CT-5 on root rot of potted Michelia alba.

[0076] ① Preparation of biocontrol bacterial culture: Burkholderia gladioli CT-5 bacteria, stored at low temperature, were taken out and inoculated onto LB solid medium for activation for 2 days. The activated biocontrol bacteria were then inoculated into a 250mL Erlenmeyer flask containing 100mL of LB liquid medium and cultured at 28℃ with shaking at 180r / min for 48h. Sterile water was then added to prepare a solution with a concentration of 1×10⁻⁶. 8 CFU / mL bacterial suspension, for later use.

[0077] ② Experimental Design: Burkholderia CT-5 of Gladiolus, 50% carbendazim wettable powder (a chemical agent), and Bacillus subtilis wettable powder (a biological agent) were co-inoculated onto potted seedlings of *Vernicia fordii* (the root rot pathogen of *Vernicia fordii*). (*Vernicia fordii* seedlings: one-year-old healthy seedlings from a *Vernicia fordii* planting base in Anshun City, Guizhou Province, with an average plant height of 32cm). Specific inoculation treatments are as follows:

[0078] a. Control group: Inoculated with 50 mL of sterile LB medium (LB).

[0079] b. Control group: Inoculated with 50 mL of root rot pathogen suspension (FP).

[0080] c. Chemical agents: Inoculate with 50 mL (0.01 g / mL) of 50% carbendazim wettable powder (FP+DJL).

[0081] d. Biological agents: Inoculate with 50 mL (0.01 g / mL) of Bacillus subtilis wettable powder (FP+YBG).

[0082] e. Inoculate with 50 mL of Burkholderia gladioli CT-5 bacterial suspension.

[0083] For the treatment where pathogens and biocontrol bacteria were inoculated onto the same plant, seedlings were first inoculated with 50 mL of biocontrol bacteria suspension, followed by 50 mL of pathogen suspension 7 days later. Each treatment was set up in triplicate, with 5 seedlings per replicate, and 1 seedling of *Vernicia fordii* per pot. The potted seedlings were cultivated at (25±2)℃ under a light-dark ratio of 12:12h. The growth and disease incidence of *Vernicia fordii* seedlings were observed, and the disease incidence and disease index were investigated. The disease index and relative control efficacy were calculated.

[0084] Incidence rate (%) = [Number of infected trees / Total number of trees surveyed] × 100

[0085] Disease severity index grading standard, divided into 5 levels according to the degree of disease occurrence:

[0086] Grade 0: No symptoms;

[0087] Grade 1: The roots of the seedlings of *Vernicia fordii* have lesions, which are less than 1.0 cm in size, but the plants are healthy and do not wilt.

[0088] Grade 2: Root lesions of *Vernicia fordii* seedlings reach 1.0-2.0 cm in size; seedling leaves show slight wilting; and lower leaves are slightly lost.

[0089] Level 3: The lesions on the roots of the *Vernicia fordii* seedlings are 2.0 cm or more in size, the seedling leaves are obviously wilted, the leaves turn yellow, and the lower leaves fall off;

[0090] Level 4: The roots of the seedlings of *Vernicia fordii* turn brown and rot, or the entire plant wilts;

[0091] Level 5: The entire seedling of *Vernicia fordii* withers and dies.

[0092] Disease index = 100 × ∑ number of cases at each level × representative value at each level / total number of plants surveyed × highest representative value

[0093] ③ Experimental Results. Inoculation with Burkholderia calendula CT-5 in gladiolus effectively prevented root rot of *Vernicia fordii*, achieving a relative control efficacy of 74.20%. This efficacy was higher than that of the Bacillus subtilis treatment group (50.76%) and the 50% carbendazim treatment group (30.96%), indicating that this bacterial inoculation has a good control effect on root rot of *Vernicia fordii*, and its efficacy is higher than that of the biological agent Bacillus subtilis and the chemical agent 50% carbendazim, as shown in Table 5.

[0094] Table 5. Effects of Burkholderia gladioli CT-5 inoculation on root rot control in potted plants of *Vernicia fordii*.

[0095]

[0096] (2) Effects of Burkholderia gladioli CT-5 inoculation on height and biomass of potted *Vernicia fordii* seedlings

[0097] Three *Vernicia fordii* seedlings were randomly selected from each treatment group in the pot experiment. Seedling height and diameter at ground level were measured, and the fresh and dry weights of the samples were weighed using a balance. The results are shown in Table 5 below. Comparison with the control group FP shows that inoculation with this bacterium increased seedling height, diameter at ground level, fresh weight, and dry weight, demonstrating its growth-promoting effect, as shown in Table 6.

[0098] Table 6. Effects of Burkholderia gladioli CT-5 inoculation on seedling height and biomass of *Vernicia fordii*.

[0099]

[0100] (3) Effects of Burkholderia gladioli CT-5 inoculation on POD, SOD, and CAT enzyme activities in potted *Vernicia fordii* seedlings

[0101] Three seedlings of *Vernicia fordii* were randomly selected from each treatment group in the pot experiment. The leaves and roots of the seedlings were taken and the activities of POD, SOD, and CAT enzymes were detected using the following methods.

[0102] The POD enzyme activity assay was performed using a POD kit.

[0103] (1) Sample preparation: Weigh about 0.1g of tissue, add 1mL of extraction solution, and homogenize in an ice bath. Centrifuge at 4℃×12000rpm for 10min, collect the supernatant, and place on ice for testing.

[0104] (2) Operating steps: Follow the instructions in the kit.

[0105] Calculation formula: POD (U / mL) = ΔA ÷ V1 ÷ 1 ÷ T

[0106] ΔA = A blank tube (absorbance value) - A test tube (absorbance value);

[0107] V1 --- Add sample volume, 0.01 mL;

[0108] T---Reaction time, 1 min.

[0109] SOD enzyme activity was measured using an SOD kit.

[0110] (1) Sample preparation: Take about 0.1g of tissue, add 1mL of extraction solution, and homogenize at 4℃ or in an ice bath. Centrifuge at 4℃×12000rpm for 10min, and take the supernatant as the test solution.

[0111] (2) Operating steps: Follow the instructions in the kit.

[0112] Calculation formula: SOD activity (U / mL) = [Inhibition percentage ÷ (1 - Inhibition percentage) × V2] ÷ V1 × D

[0113] V1 --- The volume of sample added to the reaction system, 0.02 mL;

[0114] V2 --- Total volume of the reaction system, 0.2 mL;

[0115] D --- Sample dilution factor, 1 for undiluted sample;

[0116] CAT enzyme activity was measured using a CAT kit.

[0117] (1) Sample preparation: Weigh about 0.1g of tissue, add 1mL of extraction solution, and homogenize in an ice bath. Centrifuge at 4℃×12000rpm for 10min, collect the supernatant, and place on ice for testing.

[0118] (2) Operating steps: Follow the instructions in the kit.

[0119] Calculation formula: CAT (U / mL)=[(ΔA+0.0137)÷0.1412]÷V1÷T

[0120] ΔA = A blank tube (absorbance value) - A test tube (absorbance value);

[0121] V1 --- Add sample volume, 0.01 mL;

[0122] T---Reaction time, 5 min.

[0123] All of the above processes were repeated 3 times.

[0124] The results are shown in Table 7 below. As can be seen from Table 7, this bacterial inoculation can increase the activity of SOD, POD and CAT enzymes in the roots and leaves of *Vernicia fordii* seedlings, and can effectively improve the plant's resistance to pathogen invasion.

[0125] Table 7 Effects of Burkholderia gladioli CT-5 inoculation on POD, SOD, and CAT enzyme activities in potted *Vernicia fordii* seedlings

[0126]

[0127]

[0128] (4) Effects of Burkholderia gladiolus CT-5 inoculation on the physicochemical properties of rhizosphere soil of potted *Vernicia fordii* seedlings

[0129] For each treatment group in the pot experiment, root soil samples from the root systems of 5 *Vernicia fordii* plants were collected, mixed thoroughly, and placed in sterile plastic-sealed bags. These samples were then brought back to the laboratory. The soil samples were allowed to air dry in a cool, well-ventilated place, ground, and sieved. Equal masses of soil samples from the same treatment group were mixed thoroughly and divided into three equal portions, each placed in a sterile plastic-sealed bag for soil physicochemical property testing. Soil physicochemical properties were determined as follows: total nitrogen was determined using the Kjeldahl method; total phosphorus and available potassium were determined using the molybdenum-antimony colorimetric method; total potassium and available potassium were determined using flame photometry; alkaline nitrogen was determined using diffusion absorption; and soil organic matter was determined using chromate-oxygenation titration. All experiments were performed in triplicate.

[0130] The results are shown in Table 8 below. As can be seen from Table 8, comparing the FP treatment group and the FP+CT-5 treatment group, it can be seen that the inoculation of this bacterium can increase the content of organic matter, total phosphorus, total nitrogen, total potassium, alkaline nitrogen, available phosphorus, and available potassium in the rhizosphere soil of *Vernicia fordii* seedlings, thereby improving soil nutrition and promoting plant growth.

[0131] Table 8 Effects of Burkholderia gladioli CT-5 inoculation on the physicochemical properties of rhizosphere soil in *Vernicia fordii* seedlings

[0132]

[0133] In summary, the *Burkholderia gladioli* CT-5 strain provided by this invention can cause hyphal breakage, deformation, and swelling of *Fusarium solani*, and can inhibit the production of pathogenic spores. Furthermore, detection of characteristic substances produced by *Burkholderia gladioli* CT-5 reveals that the strain produces proteases, organophosphates, and heptaphiles, but does not secrete chitin. Antibacterial experiments using biocontrol bacterial metabolites show that the volatile metabolites of the biocontrol bacteria have no inhibitory effect on the pathogen; however, the non-volatile metabolites of the strain have a better inhibitory effect on the pathogen, and analysis of the non-volatile metabolites yielded 15 antibacterial active substances.

[0134] A pot experiment on the efficacy of Burkholderia CT-5 in Gladiolus spp. showed that the biocontrol bacteria had a significant control effect on root rot of *Vernicia fordii* (P<0.05), while Bacillus subtilis (wettable powder) had a moderate control effect with a relative efficacy of 50.76%, and 50% carbendazim (wettable powder) had a poor control effect with a relative efficacy of 30.96%. In the pot experiment, the biomass, soil nutrients, SOD, POD, and CAT enzyme activities of *Vernicia fordii* seedlings in the bacterial treatment group were significantly higher than those in the pathogen control group (FP) (P<0.05), indicating that inoculation with biocontrol bacteria can promote plant growth, activate the activity of internal defense enzymes, and thus control root rot of *Vernicia fordii*.

[0135] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A strain of Burkholderia gladioli CT-5, characterized in that, This strain is deposited at the China Center for Type Culture Collection (CCTCCNO: M20242732) on December 5, 2024.

2. An agent containing Burkholderia gladioli CT-5 as described in claim 1.

3. The microbial agent according to claim 2, characterized in that, In the bacterial agent, the Burkholderia gladioli CT-5 exists in the form of cultured live bacteria, fermentation broth, or bacterial suspension.

4. The application of Burkholderia gladioli CT-5 as described in claim 1 in inhibiting root rot of *Vernicia fordii* or in the preparation of *Vernicia fordii* growth promoters.

5. The application of Burkholderia gladioli CT-5 as described in claim 1 in inhibiting Fusarium solani, Schizophyllum commune, Ulmus parvifolia, Anthrax oxysporum, and Anthrax siamensis.

6. The application of Burkholderia gladioli CT-5 as described in claim 1 in improving the activity of SOD, POD, and CAT enzymes in the roots and leaves of *Vernicia fordii* seedlings.

7. The application of Burkholderia gladioli CT-5 as described in claim 1 in increasing the content of organic matter, total phosphorus, total nitrogen, total potassium, alkaline nitrogen, available phosphorus, and available potassium in soil.

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