Burkholderia gladioli and application thereof

Abstract

The invention discloses a Burkholderia gladioli (Burkholderia gladioli) CT-5 strain and an application of the Burkholderia gladioli CT-5 strain, and relates to the technical field of microorganisms. The strain is preserved in the China Center for Type Culture Collection, the preservation number is CCTCC NO: M20242732, and the preservation date is December 05, 2024. The Burkholderia gladioli (Burkholderia gladioli) CT-5 provided by the invention has a remarkable prevention and treatment effect (Plt, Plt) on idesia root rot caused by Fusarium solani (Fusarium solani), and the Burkholderia gladioli (Burkholderia gladioli) CT-5 has a remarkable prevention and treatment effect on idesia root rot caused by Fusarium solani (Busarium solani). The compound has the advantages that the compound can be used for inhibiting pathogenic bacteria of various plants, and the compound can be used for inhibiting pathogenic bacteria of various plants, such as Schizophyllum commune, Diaporthe erens, Colletotrichum acutatum and the like, and has a remarkable inhibiting effect on other various plant pathogenic bacteria such as the Schizophyllum commune, the Diaporthe erens, the Colletotrichum acutatum and the like. In a pot experiment, the biomass, soil nutrients, SOD, POD and CAT enzyme activities of idesia polycarpa seedlings in a CT-5 treatment group are remarkably improved compared with those of a pathogenic bacteria control group (FP) (Plt; 0.05), which indicates that the strain can promote plant growth and activate the activity of defensive enzyme in the body, so that effective control of idesia root rot is realized, and powerful theoretical basis and technical support are provided for sustainable development of idesia.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and in particular to a gladiolus Burkholderia and application thereof. Background Art

[0002] Guizhou Province is one of the main distribution areas of Castanopsis chinensis. Since the introduction and planting of Castanopsis chinensis in 2018, the planting area of Castanopsis chinensis has gradually increased in Guizhou Province. With the continuous planting of Castanopsis chinensis, pests and diseases have become one of the main factors restricting the development of the Castanopsis chinensis industry, and therefore have attracted much attention. Castanopsis chinensis root rot is one of the main diseases of Castanopsis chinensis and a common disease among Castanopsis chinensis diseases. With the increase in the planting area of Castanopsis chinensis, root rot has appeared in various regions. The disease mainly harms the roots of Castanopsis chinensis. 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, surveys in Renhuai City, Dushan County, Sandu County and other places found that the damage caused by Castanopsis chinensis root rot was serious, with an incidence rate of 28-35%, and there was a trend of continuous expansion and spread.

[0003] At present, the prevention and control of root rot mainly adopts root irrigation with chemical pesticides. Not only is the prevention effect not obvious, but there are also 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 Castanopsis chinensis root rot, providing necessary technical support for the scientific prevention and control of Castanopsis chinensis root rot, and ultimately ensuring the healthy and sustainable development of the Castanopsis chinensis industry are the problems that the present invention urgently needs to solve. Summary of the Invention

[0005] The present invention provides a Burkholderia gladiolus strain and application thereof, aiming to solve the problems existing in the above-mentioned background technology.

[0006] In order to achieve the above technical objectives, the present invention mainly adopts the following technical solutions:

[0007] In a first aspect, the present invention discloses a Burkholderia gladioli CT-5 strain, which is deposited in the China Center for Type Culture Collection with a deposit number of CCTCCNO: M20242732 and a 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 the Burkholderia gladioli CT-5 described in the first aspect.

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

[0011] In a third aspect, the present invention discloses a use of the Burkholderia gladioli CT-5 as described in the first aspect in inhibiting the root rot of Castanopsis truncatula or preparing a Castanopsis truncatula growth promoter.

[0012] In a fourth aspect, the present invention discloses a use of Burkholderia gladioli CT-5 as described in the first aspect in inhibiting Fusarium solani, Schizophyllum, Aspergillus niger, Colletotrichum oxysporum, Alternaria alternifolia, Colletotrichum siamensis, and Colletotrichum citrinum.

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

[0014] In a seventh aspect, the present invention discloses an 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-hydrolyzable nitrogen, available phosphorus, and available potassium in soil.

[0015] In an eighth aspect, the present invention discloses an application of a non-volatile metabolite produced by Burkholderia gladioli CT-5 as described in the first aspect in inhibiting tung oil tree root rot, preparing a tung oil tree growth promoter, increasing the activity of SOD, POD, and CAT enzymes, or increasing the content of organic matter, total phosphorus, total nitrogen, total potassium, alkaline-hydrolyzable nitrogen, available phosphorus, and fast-acting potassium in the soil.

[0016] Furthermore, the non-volatile metabolites include Betaine, Phenazine, Phenylalanine, Pyocyanin, Piperidine, Erucamide, Pyrrolidine, L-Tyrosine, Glutamic acid, Cephalexin, L-Lysine, Neochlorogenic 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 provided by the present invention has a significant control effect on tung oil tree root rot (P<0.05), while Bacillus subtilis (wettable powder) has a general control effect, with a relative control effect of 50.76%; and 50% carbendazim (wettable powder) has a poor control effect, with a relative control effect of 30.96%. In a potted plant experiment, the biomass, soil nutrients, and SOD, POD, and CAT enzyme activities of tung oil tree seedlings in the bacteria-treated group were significantly improved compared with the pathogen control group (FP) (P<0.05), indicating that inoculation with Burkholderia gladioli CT-5 can promote plant growth, activate the activity of defense enzymes in the body, and thus control tung oil tree root rot. This provides a strong theoretical basis and technical support for the control of tung oil tree root rot, the scientific prevention and control of tung oil tree diseases and pests, and the promotion of the green and sustainable development of the tung oil tree industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The morphological diagram of Burkholderia gladiolus CT-5 provided by the present invention;

[0020] Figure 2 This is an electron microscope scan of the gladiolus Burkholderia CT-5 provided by the present invention;

[0021] Figure 3 The Gram staining pattern of Burkholderia gladiolus CT-5 provided by the present invention;

[0022] Figure 4 The diagram of the effect of Burkholderia gladioli CT-5 on the mycelium of pathogenic bacteria provided by the present invention; wherein the left diagram shows the mycelium breakage and entanglement, and the right diagram shows the mycelium expansion;

[0023] Figure 5 This is a diagram showing the inhibitory effect of the gladiolus Burkholderia CT-5 on pathogens provided by the present invention;

[0024] Figure 6 This is a diagram of the characteristic substances secreted by Burkholderia gladioli CT-5 provided by the present invention, wherein from left to right are protease, siderophore and organophosphate. DETAILED DESCRIPTION

[0025] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below with reference to the accompanying drawings and specific embodiments.

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

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

[0028] 1. Separation location

[0029] A sample plot of healthy Castanopsis truncatula plants was selected in Chishui City (28°28′4″N, 105°59′28″E, H:823). Plant roots were excavated 30 cm from the trunk, and excess soil was removed from the roots. The rhizosphere soil was obtained by shaking out the soil. The soil was placed in a plastic bag, placed in an ice box, and brought back to the laboratory for storage at 5°C.

[0030] 2. Separation method

[0031] The bacteria in the rhizosphere soil samples were isolated by the dilution plate method. 10 g of soil sample was weighed and placed in 90 mL of sterile water (containing 10 sterile glass beads). The sample was shaken on a shaker at 25°C for 10 min. Sterile water was added to dilute the soil solution to 10 -1 , 10 -2 , 10 -3 , 10 -4 Spread 200 μL of each dilution 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 form. Pick a single colony and transfer it to new LB medium. Purify it multiple times until a single, uniform pure culture is obtained. Store the obtained pure culture at 4°C.

[0032] Biocontrol bacteria with significant antagonistic activity against pathogens were screened using the plate standoff method. On a clean bench, a sterile hole punch (5 mm diameter) was used to punch a cake of the pathogen of Castanopsis chinensis root rot and inoculate it in the center of a PDA plate. Using an inoculation loop, the biocontrol bacteria were inoculated 3.0 cm from each end of the cake and incubated in a 28°C incubator. The plate inoculated with the pathogen alone served as the control group, and the experiment was replicated three times. After 7 days of incubation, the colony diameters of the Castanopsis chinensis root rot pathogen in the control and standoff groups were measured with a ruler, and the inhibition rate was calculated according to the following formula.

[0033] Inhibition rate = colony diameter of control group - colony diameter of treated group / colony diameter of control group × 100%.

[0034] 3. Identification of strains

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

[0036] After being cultured on LB medium for 48 hours, the gladiolus Burkholderia was light yellow, opaque, moist, and raised, with regular colony edges, a special odor, and Gram-negative staining; the electron microscopy scanning results of this strain showed that the bacteria were short cylinders, the individuals were uniformly plump, with wrinkles on the surface, clear outlines, and no obvious flagella were seen.

[0037] Physiological and biochemical identification: The physiological and biochemical characteristics of the biocontrol bacteria screened were determined with reference to books such as "Berger's Manual of Bacterial Identification" and "Manual of Systematic Identification of Common Bacteria". The indicators measured included Gram staining, glucose oxidation and fermentation, nitrate reduction, catalase, methyl red (MR), VP determination, starch hydrolysis, indole test, oxidase, gelatin liquefaction, and citrate utilization. The results showed that the Gram staining of the bacteria (such as Figure 3 As shown), nitrate reduction and methyl red tests were all negative, and starch hydrolysis reactions were all positive. 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 and PCR amplified using the 16S rDNA universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') / 1492R (5'-GGTTACCTTGTTACGACTT-3'). After the PCR product was confirmed, it was sent to Shanghai Bioengineering Co., Ltd. (Chengdu) for sequencing. After sequencing, the sequences were aligned and downloaded from NCBI. A phylogenetic tree was constructed for related sequences using the neighbor joining method using MEGA-11 software. The strain was identified as Burkholderia gladiol i and named Burkholderia gladiol i CT-5. It has been deposited in the China Center for Type Culture Collection with the accession number CCT CCNO: M20242732 and the deposit date is December 5, 2024.

[0039] 16S rDNA molecular primer sequence of the bacterium: GGTCAGCGTACTGCTCGGCAGCACGGGTCGCTTG CACCTGGTGGCGAGTGGCGAACGGGTGAGTAATACATCGGAACATGTCCTGTAGTGGGGGATAGCCCGGCGAAAGCCGGATTAATACCGCATACGATCTACGGATGAAAGCGGGGGACCTTCGGGCCTCGCGCTATAGGGTTGGCCGATGGCTGATTAGCTAGTTGGTGGGGTAAAGGCCCACCAAGGCGACGATCAGTAGCTGGTCTGAGAGGACGACCAGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATTTTGGACAATGGGCGAAAGCCTGATCCAGCAATGCCGCGTGTGTGAAGAAGGCCTTCGGGTTGTAAAGCACTTTTGTCCGGAAAGAAATCCTGAGGGCTAATATCCTTCGGGGATGACGGTACCGGAAGAATAAGCACCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCGAGCGTTAATCGGAATTACTGGGCGTAAAGCGTGCGCAGGCGGTTTGTTAAGACCGATGTGAAATCCCCGGGCTCAACCTGGGAACTGCATTGGTGACTGGCAAGCTAGAGTATGGCAGAGGGGGGTAGAATTCCACGTGTAGCAGTGAAATGCGTAGAGATGTGGAGGAATACCGATGGCGAAGGCAGCCCCCTGGGCCAATACTGACGCTCATGCACGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCCTAAACGATGTCAACTAGTTGTTGGGGATTCATTTCCTTAGTAACGTAGCTAACGCGTGAAGTTGACCGCCTGGGGAGTACGGTCGCAAGATTAAAACTCAAAGGAATTGACGGGGACCCGCACAAGCGGTGGATGATGTGGATTAATTCGATGCAACGCGAAAAACCTTACCTACCCTTGACATGGTCGGAACCTTGGAGAGATCCGAGGGTGCTCGAAAGAGAACCGATACACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTCCTTAGTTGCTACGCAAGAGCACTCTAGGGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAGTCCTCATGGCCCTTATGGGTAGGGCTTCACACGTCATACAATGGTCGGAACAGAGGGTCGCCAACCCGCGAGGGGGAGCTAATCCCAGAAAACCGATCGTAGTCCGGATTGCACTCTGCAACTCGAGTGCATGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTCCTACACGAGAATTATCTAGTCTCACCACGTGGTGGCAAGGTAACCATAGAGTAACAA。

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

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

[0042] Place a glass slide in a 9cm sterile Petri dish and pour sterile PDA medium into the culture medium until the medium covers the slide. Allow to cool. Inoculate the Castanopsis chinensis root rot pathogen 2cm from one end of the slide and the biocontrol bacteria that have been activated for 48 hours 2cm from the other end. Incubate at 28°C. When hyphae grow to the center of the slide, remove the slide, remove any excess medium, and observe under an optical microscope. Use a culture medium uninoculated with biocontrol bacteria as a control to observe any abnormal changes in the pathogen, such as enlarged or deformed hyphae.

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

[0044] 2 μL of Burkholderia gladioli CT-5 strain was inoculated into a 250 mL Erlenmeyer flask containing 100 mL of LB liquid medium. The culture was shaken at 28°C and 180 rpm for 48 hours to obtain a 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 cake (5 mm) were added to PDB medium. Subsequently, 1 mL of the 1 × 108 CFU / mL biocontrol bacterial fermentation broth was added and incubated at 28°C and 280 rpm for 5 days. PDB medium supplemented with the pathogen cake alone served as a control. After 5 days, 1 mL of the culture medium was collected and examined for root rot pathogen spore morphology under an electron microscope. The number of spores in the culture broth was counted using a hemocytometer.

[0045] The results are Figure 4 It can be seen that Burkholderia gladioli CT-5 can cause the pathogen's hyphae to swell, bend, entangle, dissolve and lyse, etc., which has a destructive effect on the pathogen's hyphae. In liquid culture, it can significantly inhibit the growth of pathogen spores, with spore production reaching zero.

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

[0047] The inhibitory effect of volatile metabolites of Burkholderia gladioli CT-5 on the pathogen of Castanopsis chinensis root rot was tested using a plate-to-plate culture method. In a clean bench, a 5mm sterile punch was used to cut a cake of the pathogen and inoculated it in the center of a PDA culture medium. Using an inoculation loop, 2 μL of the biocontrol bacteria was inoculated in the center of a fresh PDA culture medium. The two plates were then placed side by side and incubated at 28°C for 7 days. PDA culture medium inoculated with the pathogen was placed side by side with a blank PDA culture medium as a control. Mycelial growth was regularly observed. Each treatment was replicated three times. After 7 days of incubation, the mycelial diameters of the pathogen in the PDA culture medium of the control and treatment groups were measured. The inhibition rate was calculated according to the following formula:

[0048] Inhibition rate = colony diameter of root rot pathogen in the control group - colony diameter of root rot pathogen in the treatment group / colony diameter of root rot pathogen in the control group × 100%.

[0049] The results are as follows Figure 5 As shown in Table 1. Figure 5 As shown in Table 1, by comparing the control group (pathogen group) and the bacteria group, it was found that the volatile metabolites of the bacteria had no inhibitory effect on pathogens.

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

[0051]

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

[0053] 2 μL of Burkholderia gladioli CT-5 strain was inoculated into 100 mL of LB liquid medium in a 250 mL Erlenmeyer flask. The culture was shaken at 28°C and 180 rpm for 48 hours to obtain a fermentation broth of the biocontrol bacteria. The fermentation broth was then centrifuged at 4°C and 12,000 rpm for 10 minutes. The supernatant was collected and filtered through a 0.22 μm microporous filter 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 onto a plate. After the plate cooled and solidified, a 5 mm sterile punch was used to punch a cake of the Tung tung root rot pathogen into the center of the plate. The plate was then incubated at 28°C, with three replicates for each treatment. The colony diameters of the pathogen were measured and recorded using a graduated ruler 5, 10, and 15 days after inoculation. The inhibition rate was calculated according to the following formula.

[0054] Inhibition rate = (pathogenic bacteria colony diameter on PDA plate - pathogenic bacteria colony diameter on fermentation broth inhibition plate) / pathogenic bacteria colony diameter on PDA plate × 100%.

[0055] The results are as follows Figure 6 As shown in Table 2. Figure 6As shown in Table 2, the strain had the best inhibitory effect on pathogenic fungi hyphae on the fifth day, which was 22.21%.

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

[0057]

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

[0059] The activated biocontrol bacteria were inoculated into LB liquid medium and cultured at 28°C, 180 rpm, and shaken for 48 hours. The culture was then centrifuged at 12,000 rpm and 4°C. The supernatant was collected and stored at -80°C. Three tubes of supernatant were collected for each strain and then mailed on dry ice to Zhongkexin Life Biotechnology Co., Ltd. in Jinhua, Zhejiang Province, for analysis of non-volatile metabolites.

[0060] Results The strain produced 15 non-volatile metabolites, i.e., antibacterial substances, including 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 characteristic substances secreted by Burkholderia gladiolus CT-5

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

[0063] Inoculate the test bacteria onto a cellulase assay medium and incubate at 28°C for 72–96 hours. Wash the assay medium plate with 0.9% NaCl solution, rinsing it off every 2 hours and then replacing it with fresh 0.9% NaCl. Stain the interior of the plate with 0.2% Congo red stain. Finally, remove the Congo red stain with 1 mol / L NaCl solution. After 6 hours, observe the clearing zone surrounding the colonies.

[0064] The results are as follows Figure 6 As shown in Table 3. Figure 6 As shown in Table 3, the strain can produce protease, organophosphate, and siderophore but does not secrete chitin.

[0065] Table 3 Detection results of characteristic substances secreted by Burkholderia gladiolus 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, Colletotrichum oxysporum.

[0069] (2) Experimental design: The plate confrontation method was used to determine the inhibitory effect of gladiolus Burkholderia CT-5 on seven common plant pathogens to clarify its broad-spectrum antibacterial activity. In a clean bench, a sterile punch (d = 5 mm) was used to punch out a bacterial cake from the pathogen cultured at 28°C for 7 days and inoculated into the center of the PDA culture medium. Four symmetrical points were selected 2.5 cm away from the pathogen cake and inoculated with gladiolus Burkholderia CT-5. PDA culture medium without biocontrol bacteria was used as a control. Each treatment was repeated three times and cultured at 28°C for 7 days. After 7 days, the diameter of the pathogen colony was measured using the cross-cross method to calculate the inhibition rate.

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

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

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

[0073]

[0074] Example 4 Experiment on the Growth-Promoting and Disease-Resistant Effect of Inoculation of Burkholderia gladiolus CT-5 on Potted Castanopsis chinensis Seedlings

[0075] (1) Experiment on the efficacy of Burkholderia gladioli CT-5 against root rot of potted Castanopsis chinensis

[0076] ① Preparation of biocontrol bacterial solution: Take out the Burkholderia gladioli CT-5 bacteria stored at low temperature and inoculate them into LB solid medium for activation for 2 days. Inoculate the activated biocontrol bacteria into a 250mL conical flask containing 100mL LB liquid medium and shake culture at 28℃ and 180r / min for 48h. Add sterile water to make the concentration of 1×10 8 cfu / mL bacterial suspension for later use.

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

[0078] a. Blank group: inoculate 50 mL of sterile LB medium (LB)

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

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

[0081] d. Biological agent: inoculate 50mL (0.01g / mL) Bacillus subtilis wettable powder (FP+YBG)

[0082] e. Inoculate 50 mL of Burkholderia gladiolus CT-5 suspension

[0083] For treatments where the pathogen and biocontrol bacteria were inoculated on the same plants, the seedlings were first inoculated with 50 mL of the biocontrol bacterial suspension, followed by another 50 mL of the pathogen suspension 7 days later. Each treatment was replicated three times, with five seedlings per replicate and one Castanopsis chinensis seedling per pot. The potted seedlings were incubated at (25±2)°C with a light-dark ratio of 12:12 hours. The growth and disease progression of the Castanopsis chinensis seedlings were observed, and the incidence and disease index were measured. The disease index and relative control efficacy were calculated.

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

[0085] The disease index grading standard is divided into 5 levels according to the degree of disease occurrence:

[0086] Level 0: no disease;

[0087] Level 1: There are lesions on the roots of the Castanopsis chinensis seedlings, the size of the lesions is less than 1.0 cm, but the plants are healthy and not wilting;

[0088] Level 2: The root spots of the Castanopsis chinensis seedlings are 1.0-2.0 cm in diameter, the leaves of the seedlings are slightly wilted, and the lower leaves rarely fall off.

[0089] Level 3: The root lesions of Castanopsis chinensis seedlings are larger than 2.0 cm, the leaves of the seedlings are obviously wilted, the leaves turn yellow, and the lower leaves fall off;

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

[0091] Level 5: The entire Castanopsis chinensis seedling 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 Test Results. Inoculation with Burkholderia gladiolus CT-5 effectively prevented the occurrence of tung oil tree root rot, achieving a relative efficacy of 74.20%. This efficacy was higher than the 50.76% of the Bacillus subtilis-treated group and the 30.96% of the 50% carbendazim-treated group. This indicates that this bacterial inoculation has a good preventive effect on tung oil tree root rot, 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 Control effect of gladiolus Burkholderia CT-5 inoculation on Tung oil tree root rot in potted plants

[0095]

[0096] (2) Effects of inoculation with Burkholderia gladioli CT-5 on the height and biomass of potted Castanopsis chinensis seedlings

[0097] Three Castanopsis truncatula seedlings were randomly selected from each treatment group in the potted experiment. Their seedling height and ground diameter were measured, and the fresh and dry weights of the samples were weighed using a balance. The results are shown in Table 5 below. Compared with the control group (FP), inoculation with the bacterium increased the seedling height, ground diameter, fresh weight, and dry weight of the plants, demonstrating its growth-promoting effect, as shown in Table 6.

[0098] Table 6 Effects of inoculation with Burkholderia gladioli CT-5 on the height and biomass of Castanopsis chinensis seedlings

[0099]

[0100] (3) Effects of inoculation with Burkholderia gladioli CT-5 on the activities of POD, SOD, and CAT enzymes in potted Castanopsis chinensis seedlings

[0101] Three Castanopsis chinensis seedlings were randomly selected from each treatment group in the pot experiment, and the leaves and roots of the seedlings were taken to detect the activities of POD, SOD, and CAT enzymes according to the following method.

[0102] Among them, POD enzyme activity was determined using a POD kit:

[0103] (1) Sample preparation: Weigh approximately 0.1 g of tissue, add 1 mL of extraction solution, and homogenize on ice. Centrifuge at 4°C × 12,000 rpm for 10 min, remove the supernatant, and place on ice for testing.

[0104] (2) Operation steps: Follow the instructions of the kit.

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

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

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

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

[0109] SOD enzyme activity was determined using a SOD kit:

[0110] (1) Sample preparation: Take approximately 0.1 g of tissue, add 1 mL of extraction solution, and homogenize at 4°C or on ice. Centrifuge at 12,000 rpm for 10 min at 4°C, and use the supernatant as the test solution.

[0111] (2) Operation steps: Follow the instructions of 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 reaction system, 0.2mL;

[0115] D---sample dilution multiple, undiluted is 1;

[0116] CAT enzyme activity was determined using a CAT kit:

[0117] (1) Sample preparation: Weigh approximately 0.1 g of tissue, add 1 mL of extraction solution, and homogenize on ice. Centrifuge at 4°C × 12,000 rpm for 10 min, remove the supernatant, and place on ice for testing.

[0118] (2) Operation steps: Follow the instructions of the kit.

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

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

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

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

[0123] The above treatments were repeated 3 times.

[0124] The results are shown in Table 7 below. As shown in Table 7, the bacterial inoculation can increase the activities of SOD, POD, and CAT enzymes in the roots and leaves of Castanopsis chinensis seedlings, and can effectively improve the plant's ability to resist the invasion of pathogens.

[0125] Table 7 Effects of inoculation with Burkholderia gladioli CT-5 on the activities of POD, SOD and CAT enzymes in potted Castanopsis chinensis seedlings

[0126]

[0127]

[0128] (4) Effects of inoculation with Burkholderia gladioli CT-5 on the physical and chemical properties of the rhizosphere soil of potted Castanopsis chinensis seedlings

[0129] In each potted experiment, soil from the root systems of five Castanopsis chinensis plants was collected from each treatment group, mixed thoroughly, placed in sterile plastic bags, and brought back to the laboratory. The soil samples were placed in a cool, ventilated area to air dry, ground, and sieved. Soil samples of equal mass from the same treatment groups were mixed thoroughly, divided into three equal portions, and placed in sterile plastic bags for testing of soil physical and chemical properties. Soil physical and chemical properties were determined as follows: total nitrogen in the soil 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 the diffusion absorption method; and soil organic matter was determined using the chromate redox titration method. All experiments were replicated three times.

[0130] The results are shown in Table 8 below. As can be seen from Table 8, by 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 fast-acting potassium in the rhizosphere soil of Castanopsis chinensis seedlings, which can improve soil nutrition and promote plant growth.

[0131] Table 8 Effects of inoculation with Burkholderia gladioli CT-5 on the physical and chemical properties of rhizosphere soil of Castanopsis chinensis seedlings

[0132]

[0133] In summary, the Burkholderia gladioli CT-5 strain provided by the present invention can cause mycelial breakage, deformation, and swelling of Fusarium solani, while also inhibiting the production of pathogenic spores. Furthermore, detection of characteristic substances produced by Burkholderia gladioli CT-5 revealed that the strain produces proteases, organophosphates, and siderophores, and does not secrete chitin. Antibacterial tests on biocontrol bacterial metabolites showed that volatile metabolites of the biocontrol bacteria had no inhibitory effect on pathogens, while non-volatile metabolites of the strain showed a more potent inhibitory effect. Analysis of these non-volatile metabolites revealed 15 antibacterial active substances.

[0134] Potted plant trials using Burkholderia gladiolus CT-5 showed that the biocontrol bacteria had a significant control effect on Alpinia truncatula root rot (P < 0.05). Bacillus subtilis wettable powder (WP) showed moderate control efficacy, with a relative efficacy of 50.76%. A 50% WP of carbendazim was less effective, with a relative efficacy of 30.96%. In the potted plant trials, Alpinia truncatula seedling biomass, soil nutrients, and SOD, POD, and CAT enzyme activities in the bacterial-treated group were significantly higher than those in the pathogen-treated group (FP) (P < 0.05). This suggests that inoculation with the biocontrol bacteria can promote plant growth and activate defense enzymes, thereby controlling Alpinia truncatula root rot.

[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 the technical solutions of the present invention may be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A Burkholderia gladioli CT-5 strain, characterized in that: The strain is deposited in the China Center for Type Culture Collection with the deposit number CCTCCNO: M20242732 and the deposit date is December 5, 2024.

2. The Burkholderia gladioli CT-5 according to claim 1, wherein The gene has a 16S rDNA sequence as shown in SEQ ID No:

1.

3. A bacterial agent containing the Burkholderia gladioli CT-5 according to claim 1.

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

5. Use of the Burkholderia gladioli CT-5 according to claim 1 in inhibiting the root rot of Castanopsis chinensis or preparing a Castanopsis chinensis growth promoter.

6. Use of the Burkholderia gladioli CT-5 according to claim 1 in inhibiting Fusarium solani, Schizophyllum sp., Aspergillus niger, Colletotrichum oxysporum, Alternaria alternifolia, Colletotrichum siamensis, and Colletotrichum citrinum.

7. Use of the Burkholderia gladioli CT-5 according to claim 1 in improving the activities of SOD, POD and CAT enzymes.

8. Use of the Burkholderia gladioli CT-5 according to claim 1 in increasing the contents of organic matter, total phosphorus, total nitrogen, total potassium, alkaline-hydrolyzable nitrogen, available phosphorus and available potassium in soil.

9. Use of the non-volatile metabolites produced by Burkholderia gladioli CT-5 as claimed in claim 1 in inhibiting root rot of Castanopsis chinensis, preparing a Castanopsis chinensis growth promoter, increasing the activities of SOD, POD, and CAT enzymes, or increasing the contents of organic matter, total phosphorus, total nitrogen, total potassium, alkaline-hydrolyzable nitrogen, available phosphorus, and available potassium in soil.

10. The use according to claim 9, characterized in that The non-volatile metabolites include Betaine, Phenazine, Phenylalanine, Pyocyanin, Piperidine, Erucamide, Pyrrolidine, L-Tyrosine, Glutamic acid, Cephalexin, L-Lysine, Neochlorogenic acid, Dipropyl phthalate, Dioctyl phthalate, and Benzophenone.

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