Burkholderia gladioli and its applications

Abstract

This invention discloses a species of *Burkholderia gladioli* and its applications. This invention provides *Burkholderia gladioli* (… Burkholderia gladioli The strain HZX-8, with accession number CGMCC No. 33009, is also described. The application of this strain, its culture medium, its bacterial suspension, or its fermentation broth in the prevention or treatment of anthracnose and / or twig blight in walnut plants is also provided. The *Burkholderia gladiolus* obtained in this invention... Burkholderia gladioli HZX-8 strain, in the prevention and control of anthrax caused by Guangyuan anthrax bacteria. Colletotrichum guangyuanense Walnut anthracnose caused by *Metacarpa spp.* Diaporthe chaotianensis It shows significant potential for biological control of walnut branch blight.

Description

Technical Field

[0001] This invention belongs to the field of microbial biological control technology, and relates to Burkholderia gladioli and its applications. Background Technology

[0002] Walnut( Juglans regia Walnuts (L.) are an important economic crop, widely cultivated in many regions around the world. However, walnut production faces threats from various diseases, among which walnut anthracnose and walnut twig blight are two major diseases that seriously affect walnut yield and quality. Walnut anthracnose is caused by a fungus, manifesting as lesions on leaves, fruits, and branches, leading to premature leaf drop and fruit rot; while walnut twig blight is caused by multiple pathogens, causing branches to die, affecting tree growth and yield.

[0003] Traditional disease control methods mainly rely on the use of chemical pesticides. While effective in the short term, long-term use can lead to environmental pollution, ecological imbalance, and the development of pesticide resistance in diseases. Therefore, developing safe and effective biological control technologies has become an important direction in current agricultural research.

[0004] In recent years, microbial biocontrol technology has gradually gained attention. Specific microbial strains can inhibit the growth of plant pathogens and enhance the disease resistance of plants through various mechanisms. Therefore, in-depth exploration of the application potential of microbial strains in walnut disease management is of great significance for promoting the sustainable development of walnut cultivation. Summary of the Invention

[0005] The technical problem solved by this invention is how to effectively manage and control two plant diseases that cause serious damage to walnut crops: walnut anthracnose and / or walnut twig blight.

[0006] To solve the above-mentioned technical problems, the first aspect of the present invention provides *Burkholderia gladioli* (… Burkholderia gladioli The strain HZX-8, with accession number CGMCC No. 33009.

[0007] Secondly, the present invention provides the use of the strain or its culture medium or its bacterial suspension or its fermentation culture medium described in the first aspect in any of the following:

[0008] A1) Prevention or treatment of anthracnose in walnut plants;

[0009] A2) Prevention or treatment of walnut branch blight in plants;

[0010] A3) Inhibits the pathogens causing walnut anthracnose;

[0011] A4) Inhibits the pathogens of walnut twig blight;

[0012] A5) Prepare products for the prevention or treatment of anthracnose in walnut plants;

[0013] A6) Prepare products for the prevention or treatment of walnut twig blight;

[0014] A7) Prepare products that inhibit the pathogens causing walnut anthracnose;

[0015] A8) Prepare products that inhibit the pathogens causing walnut twig blight.

[0016] In the above application, the pathogen of walnut anthracnose is *Anthracnose guangyuanensis* (…). Colletotrichum guangyuanense);

[0017] Alternatively, the pathogen causing the walnut branch blight is *Pterocarya stenoptera* (also known as *Pterocarya stenoptera*). Diaporthe chaotianensis).

[0018] In the applications described above, the product is a microbial agent, a microecological preparation, or a biological pesticide.

[0019] In the above-described applications, the microbial agent is a biocontrol agent.

[0020] Thirdly, the present invention provides a product in which the active ingredient is the strain or its culture medium or its bacterial culture or its fermentation culture medium described in the first aspect.

[0021] The product described above has any of the following functions:

[0022] A1) Prevention or treatment of anthracnose in walnut plants;

[0023] A2) Prevention or treatment of walnut branch blight in plants;

[0024] A3) Inhibits the pathogens causing walnut anthracnose;

[0025] A4) Inhibits the pathogens of walnut twig blight.

[0026] In the product described above, the pathogen causing walnut anthracnose is *Anthracnose guangyuanensis* (…). Colletotrichum guangyuanense);

[0027] Alternatively, the pathogen causing the walnut branch blight is *Pterocarya stenoptera* (also known as *Pterocarya stenoptera*). Diaporthe chaotianensis).

[0028] The products mentioned above are microbial agents, microecological preparations, or biological pesticides.

[0029] The product mentioned above is a microbial agent; specifically, a biocontrol microbial agent.

[0030] The inhibition of various pathogens mentioned above can be described as inhibiting various pathogens under a high-salt environment.

[0031] The plants mentioned above are either dicotyledonous or monocotyledonous plants;

[0032] The dicotyledonous plants mentioned above are plants of the Juglandaceae family.

[0033] The walnut tree mentioned above is a member of the Juglandaceae family.

[0034] The plant described above can grow in high-salt soil or saline-alkali land.

[0035] The Gladiolus Burkholderia obtained in this invention ( Burkholderia gladioli HZX-8 strain, in the prevention and control of anthrax caused by Guangyuan anthrax ( Colletotrichum guangyuanense Walnut anthracnose caused by *Hemiberleinii* and *Hemiberleinii var. chinensis* (… Diaporthe chaotianensis This strain demonstrates significant potential for biological control of walnut twig blight caused by [unspecified organism]. As a biocontrol agent, this strain shows broad application prospects in both the development of novel bio-inoculants and the preparation of biopesticides. This invention provides a new and environmentally friendly method for the biological control of walnut anthracnose, reducing the use of chemical pesticides, lowering environmental pollution, protecting ecological balance, improving walnut crop yield and quality, and contributing to the sustainable development of agriculture.

[0036] Preservation Instructions

[0037] Strain name: HZX-8

[0038] Latin name: Burkholderia gladioli .

[0039] Preservation Institution: China General Microbiological Culture Collection Center, China Microbiological Culture Collection Committee

[0040] Collection institution abbreviation: CGMCC

[0041] Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing

[0042] Deposit date: December 10, 2024

[0043] CGMCC Registration Number: 33009

[0044] Classified and named as: Burkholderia gladioli Burkholderia gladioli Attached Figure Description

[0045] Figure 1 Burkholderia gladioli ( Burkholderia gladioli Macroscopic morphology of HZX-8 colonies and individual morphology. The left image shows the macroscopic morphology of HZX-8 colonies; the right image shows the individual morphology of HZX-8.

[0046] Figure 2Burkholderia gladioli ( Burkholderia gladioli Phylogenetic tree diagram of HZX-8.

[0047] Figure 3 Burkholderia gladioli ( Burkholderia gladioli The antagonistic effect of HZX-8 on walnut anthracnose and walnut twig blight. The left image in the first row from top to bottom shows a blank control of *Anthracnose guangyuanensis*, the pathogen of walnut anthracnose, while the right image shows the antagonistic effect of HZX-8 against *Anthracnose guangyuanensis*. The left image in the second row from top to bottom shows a blank control of *Eclipta prostrata*, the pathogen of walnut twig blight, while the right image shows the antagonistic effect of HZX-8 against *Eclipta prostrata*.

[0048] Figure 4 Burkholderia gladioli ( Burkholderia gladioli The antagonistic effect of HZX-8 fermentation broth on walnut anthracnose and walnut twig blight. The left image in the first row from top to bottom shows the blank control of *Anthracnose guangyuanensis*, the pathogen of walnut anthracnose, while the right image shows the antagonistic effect of HZX-8 fermentation broth on *Anthracnose guangyuanensis*. The left image in the second row from top to bottom shows the blank control of *Echinochloa crus-galli*, the pathogen of walnut twig blight, while the right image shows the antagonistic effect of HZX-8 fermentation broth on *Echinochloa crus-galli*, the pathogen of walnut twig blight.

[0049] Figure 5 Burkholderia gladioli cultured at different salt concentrations Burkholderia gladioli The effect of HZX-8 on the antibacterial activity of walnut anthracnose. The top image in the first column from left to right shows the blank control of *Anthracnose guangyuanensis*, the pathogen of walnut anthracnose, at a 2% NaCl concentration; the bottom image in the first column from left to right shows the antagonistic effect of HZX-8 cultured at a 2% NaCl concentration on *Anthracnose guangyuanensis*, the pathogen of walnut anthracnose. The top image in the second column from left to right shows the blank control of *Anthracnose guangyuanensis*, the pathogen of walnut anthracnose, at a 5% NaCl concentration; the bottom image in the first column from left to right shows the antagonistic effect of HZX-8 cultured at a 5% NaCl concentration on *Anthracnose guangyuanensis*, the pathogen of walnut anthracnose. The top image in the third column from left to right shows the blank control of *Anthracnose guangyuanensis*, the pathogen of walnut anthracnose, at a 7% NaCl concentration; the bottom image in the third column from left to right shows the antagonistic effect of HZX-8 cultured at a 7% NaCl concentration on *Anthracnose guangyuanensis*, the pathogen of walnut anthracnose.

[0050] Figure 6 The image shows Burkholderia gladioli cultured at different salt concentrations. Burkholderia gladioliEffect of HZX-8 on the antibacterial activity of walnut twig blight. The top image in the first column from left to right shows the blank control of *Ichthyophthirius multifiliis*, the pathogen of walnut twig blight, at a 2% NaCl concentration; the bottom image in the first column from left to right shows the antagonistic effect of HZX-8 cultured at a 2% NaCl concentration on *Ichthyophthirius multifiliis*. The top image in the second column from left to right shows the blank control of *Ichthyophthirius multifiliis*, the pathogen of walnut twig blight, at a 5% NaCl concentration; the bottom image in the second column from left to right shows the antagonistic effect of HZX-8 cultured at a 5% NaCl concentration on *Ichthyophthirius multifiliis*. The top image in the third column from left to right shows the blank control of *Ichthyophthirius multifiliis*, the pathogen of walnut twig blight, at a 7% NaCl concentration; the bottom image in the second column from left to right shows the antagonistic effect of HZX-8 cultured at a 7% NaCl concentration on *Ichthyophthirius multifiliis*.

[0051] Figure 7 A bar chart showing the significant effects of different salt concentrations on the Guangyuan anthracnose fungus.

[0052] Figure 8 A bar chart showing the significant effects of different salt concentrations on the shell formation of walnut twig blight at the top of the stem. Detailed Implementation

[0053] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0054] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0055] Unless otherwise specified, the quantitative experiments in the following examples are all repeated three times, and the results are averaged.

[0056] The composition of 1L PDA medium is: 200g peeled potato, 20g glucose, 20g agar, and 1000mL distilled water.

[0057] Composition of 1L LB nutrient agar medium: 10g tryptone, 5g yeast extract, 10g NaCl, 15g agar, 1000mL distilled water.

[0058] Composition of 1L LB liquid culture medium: 10g tryptone, 5g yeast extract, 10g sodium chloride, 1000mL distilled water.

[0059] Composition of 1L NA medium: 10g peptone, 5g sodium chloride, 3g beef extract powder, 15g agar, 1000mL distilled water.

[0060] The composition of 1L TSA medium is: 15g tryptone, 5g soybean peptone, 5g sodium chloride, 15g agar, and 1000mL distilled water.

[0061] The pathogen of walnut anthracnose is *Anthracnose guangyuanensis* (… Colletotrichum guangyuanense The following non-patent literature describes it: "Li Y, Lin L, Cao J, Gan M, Fan X. Three new species of Colletotrichum (Glomerellales, Glomerellaceae) associated with walnut (Juglansregia) anthracnose from China. MycoKeys. 2024 Sep 3;108:147-167." It is available to the public from Beijing Forestry University. This biological material is only for repeating the relevant experiments of this invention and cannot be used for other purposes.

[0062] The pathogen of walnut twig blight is *Pterocarya stenoptera* ( Diaporthe chaotianensis The biomaterial is described in the following non-patent literature: "Jia, A.; Lin, L.; Li, Y.; Fan, X. Diversity and Pathogenicity of SixDiaporthe Species from Juglans regia in China. J. Fungi 2024, 10, 583." It is available to the public from Beijing Forestry University. This biomaterial is only for repeating the relevant experiments of this invention and may not be used for other purposes.

[0063] Example 1: Burkholderia gladioli ( Burkholderia gladioli Isolation, screening and identification of HZX-8

[0064] I. Isolation of strain HZX-8

[0065] Samples of walnut fruits infected with anthracnose were collected from the Hezhang Yelang National Forest Park in Shuitangbao Township, Hezhang County, Bijie City, Guizhou Province. The diseased and healthy fruit samples were mixed at the interface between the diseased and healthy tissues and placed in an Erlenmeyer flask containing 45 mL of sterile water and sterile steel balls. The mixture was shaken at 30℃ and 180 rpm for 60 min to prepare a bacterial suspension. The suspension was filtered through sterile gauze, and the volume was adjusted to 100 mL to form the original diseased fruit solution. Further 10-fold serial dilutions were prepared. -2 and 10-3 Dilute the diseased fruit solution to a multiple of 100 μL. Take 100 μL from each of these dilutions, spread them evenly on LB agar, and incubate upside down at 28°C. Transfer single colonies with different morphological characteristics to fresh LB agar using the streak plate method, purify them, and store them at 4°C for later use to obtain the test strains.

[0066] 2. Preliminary screening

[0067] Preliminary screening was conducted using the plate-holding method, targeting the pathogen *Anthracnose guangyuanensis*, the pathogen causing walnut anthracnose. Colletotrichum guangyuanense This bacterium can cause walnut anthracnose. The target pathogen, *Anthracnose guangyuanensis*, is used to treat walnut anthracnose. Colletotrichum guangyuanense The bacteria were inoculated onto PDA medium, with four test strains spotted onto each petri dish to form a cross pattern. Simultaneously, a setting was created where only the target pathogen, *Bacillus guangyuanensis*, was inoculated. Colletotrichum guangyuanense A culture dish containing the control group was used as a blank control. The dishes were incubated at 28°C until the control group completely covered the culture dish. Strains exhibiting antibacterial effects were then selected for further screening.

[0068] 3. Secondary screening

[0069] A 4mm sample of *Anthracnose guangyuanensis*, the pathogen causing walnut anthracnose, was inoculated into the center of each PDA plate. Colletotrichum guangyuanense ) and the walnut twig blight pathogen, *Heterophyllum sp.* Diaporthe chaotianensis The bacterial blocks were inoculated, and the strains with antibacterial effects selected in the initial screening were inoculated 2 cm from both sides of the pathogen blocks using the plate confrontation method. Simultaneously, only *Anthracnose guangyuanensis*, the pathogen causing walnut anthracnose, was inoculated. Colletotrichum guangyuanense ) and the walnut twig blight pathogen, *Heterophyllum sp.* Diaporthe chaotianensis As a blank control, each treatment was repeated three times. The cultures were incubated at 28°C until the control group completely covered the culture dish. The antagonistic strain with the strongest antibacterial effect was then screened. The strain with the largest inhibition zone was the antagonistic strain with the strongest antibacterial effect and was named HZX-8.

[0070] II. Burkholderia gladioli ( Burkholderia gladioli Morphological and physiological-biochemical identification of HZX-8

[0071] When HZX-8 is inoculated onto LB agar plates, the colonies are pale yellow, round, moist, slightly raised, opaque, with smooth edges, and the culture medium is yellow (as shown in the image). Figure 1 (As shown). Strain HZX-8 can also grow well on NA and TSA plates. The Gram reaction of strain HZX-8 is negative, and the cells are short rod-shaped.

[0072] Strain HZX-8 is a facultative anaerobic bacterium; growth temperature: 25-37℃, not heat-resistant.

[0073] The physiological and biochemical characteristics of HZX-8 are shown in Table 1. It grows well in LB medium with a concentration of 2%-7% NaCl; it can liquefy gelatin and produce siderophores; it cannot hydrolyze starch or produce H2S.

[0074]

[0075] Note: + indicates a positive reaction or that growth is possible; - indicates a negative reaction or that growth is not possible.

[0076] III. Burkholderia gladioli ( Burkholderia gladioli Molecular classification of HZX-8

[0077] Single colonies of HZX-8 were selected from LB agar plates and transferred to 2 mL sterile centrifuge tubes containing LB liquid medium. These tubes were then incubated in a shaker at 30°C with shaking at 180 rpm for 48 h. After incubation, 200 μL of culture was aspirated from each tube and transferred to 1.5 mL sterile centrifuge tubes. These tubes were then heated in a 100°C water bath for 10 min to lyse the cells and release DNA. The supernatant, containing the strain's DNA, was collected by centrifugation and stored at 4°C for subsequent analysis.

[0078] For molecular identification of the bacterial strain, the PCR reaction system consisted of 1 μL of forward and reverse primers, 12.5 μL of 2x PCR mixture, 2 μL of DNA template, and an appropriate amount of sterile water, for a total volume of 25 μL. The primers used were 27F (5'-AGAGTTTGATCCTGGCTCAG-3', SEQ ID NO:2) and 1492R (5'-GGTTACCTTGTTACGACTT-3', SEQ ID NO:3), which specifically amplify the bacterial 16S rRNA gene. The PCR program included: 94°C pre-denaturation for 5 minutes, 30 cycles, each cycle consisting of 94°C denaturation for 30 seconds, 55°C annealing for 30 seconds, and 72°C extension for 45 seconds, followed by a final extension at 72°C for 10 minutes, and storage at 4°C to obtain the PCR product.

[0079] The PCR products were sent to Beijing Nuosai Genome Research Center Co., Ltd. for 16S rDNA sequencing. The nucleotide sequence is SEQ ID NO:1. The sequencing results were aligned and spliced ​​using MEGA 6.0 software. Phylogenetic analysis was performed using maximum likelihood and Bayesian methods to determine the taxonomic position of the strain.

[0080] The results are as follows Figure 2 As shown, HZX-8 and Burkholderia gladioliIt showed the highest phylogenetic relationship, therefore it was identified as Burkholderia gladioli (…). Burkholderia gladioli ).

[0081] Based on the above morphological, physiological and biochemical characteristics and 16S rDNA sequence homology analysis results, the strain HZX-8 isolated and purified in step one was identified as Burkholderia gladioli (…). Burkholderia gladioli ).

[0082] Gladiolus Burkholderia ( Burkholderia gladioli HZX-8 was deposited on December 10, 2024, at the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing), with accession number CGMCC No. 33009. It will be referred to as strain HZX-8 or HZX-8.

[0083] Example 2, Burkholderia gladioli ( Burkholderia gladioli Antagonistic effect of HZX-8 on walnut anthracnose and walnut twig blight

[0084] The bacterial blocks for each pathogen described below are the bacterial blocks taken when each pathogen has been cultured in PDA medium until it has completely filled the plate.

[0085] (1) Preparation of bacterial culture

[0086] Burkholderia gladioli, identified in Example 1 ( Burkholderia gladioli HZX-8 was inoculated into a 2 mL sterile centrifuge tube containing LB liquid medium, and a sterile filter paper disc (4 mm in diameter) was placed in the tube. The centrifuge tube was placed in a constant temperature shaker and cultured at 30 °C and 180 r / min for 48 h to obtain a filter paper disc containing HZX-8 bacterial culture.

[0087] Sterile filter paper discs were placed in centrifuge tubes containing sterile water, and filter paper discs containing sterile water were obtained as a control, following the preparation method for filter paper discs containing HZX-8 bacterial solution.

[0088] (2) Pathogen inoculation and resistance experiment

[0089] The experiment was repeated three times, with each repetition as follows:

[0090] Experimental group of *H. guangyuanensis*, the pathogen of walnut anthracnose (HZX-8): Place 4 mm *H. guangyuanensis* bacterial blocks of walnut anthracnose on PDA medium plates, and place filter paper discs containing HZX-8 bacterial solution on both sides of the pathogen blocks using the confrontation inoculation method, 2 cm away from the bacterial blocks.

[0091] Control group (CK) of *Anthracnose guangyuanensis*, the pathogen of walnut anthracnose: Filter paper containing sterile water was used instead of filter paper containing HZX-8 bacterial solution, and the rest of the operation was the same as that of the experimental group of *Anthracnose guangyuanensis*, the pathogen of walnut anthracnose.

[0092] Walnut twig blight pathogen *Cyclocarya paliurus* experimental group (HZX-8): *Cyclocarya paliurus* mycelium blocks were used instead of *Anthracnose guangyuanensis* mycelium blocks for walnut anthracnose, and the remaining operations were the same as those for the *Anthracnose guangyuanensis* experimental group for walnut anthracnose.

[0093] Control group (CK) of *Anthracnose bacillus guangyuanensis*, the pathogen of walnut anthracnose: filter paper containing sterile water was used instead of filter paper containing HZX-8 bacterial solution, and the rest of the operation was the same as that of the experimental group of *Cyclocarya paliurus*, the pathogen of walnut branch blight.

[0094] Each of the above groups was incubated at 28°C until the pathogens in each control group covered the entire culture dish.

[0095] (3) Inhibition rate calculation

[0096] The inhibition rate of HZX-8 bacterial suspension against pathogen growth was calculated using the following formula: Relative inhibition rate = (Coronary diameter of control group - Colony diameter of experimental group) / Colony diameter of control group * 100%. The colony diameter was measured using the cross-multiplication method (colonial diameter = hyphae diameter minus bacterial block diameter).

[0097] The results are shown in Table 2 and Figure 3 As shown, Burkholderia gladioli ( Burkholderia gladioli HZX-8 has a significant antagonistic effect on the pathogens of walnut anthracnose and walnut twig blight, providing a new strategy for the biological control of these two diseases.

[0098]

[0099] Example 3, Burkholderia gladioli ( Burkholderia gladioli HZX-8 fermentation broth showed high inhibition rate against walnut anthracnose and walnut twig blight.

[0100] (1) Preparation of fermentation broth

[0101] Burkholderia gladioli, identified in Example 1 ( Burkholderia gladioli A single colony of HZX-8 was inoculated into a 2 mL sterile centrifuge tube containing LB liquid medium. The centrifuge tube was placed in a constant temperature shaker and incubated at 30°C and 180 rpm for 48 h until OD was reached. 600nm With a value ≈ 0.5, the fermentation broth of HZX-8 was obtained.

[0102] (2) Culture medium preparation and inoculation

[0103] The experiment was repeated three times, with each repetition as follows:

[0104] Experimental group of *Anthracnose guangyuanensis*, the pathogen of walnut anthracnose (HZX-8): The fermentation broth of HZX-8 was mixed with PDA medium at a volume ratio of 1:10 and evenly spread in a petri dish. After the medium solidified, a 4 mm diameter *Anthracnose guangyuanensis* mycelial block was placed in the center of the medium.

[0105] Control group (CK) of *Anthracnose guangyuanensis*, the pathogen of walnut anthracnose: Sterile water was used instead of HZX-8 fermentation medium, and the rest of the operation was the same as that of the experimental group of *Anthracnose guangyuanensis*, the pathogen of walnut anthracnose.

[0106] Walnut twig blight pathogen *Cyclocarya paliurus* experimental group (HZX-8): *Cyclocarya paliurus* mycelium blocks were used instead of *Anthracnose guangyuanensis* mycelium blocks for walnut anthracnose, and the remaining operations were the same as those for the *Anthracnose guangyuanensis* experimental group for walnut anthracnose.

[0107] Control group (CK) of *Anthracnose bacillus guangyuanensis*, the pathogen of walnut anthracnose: Sterile water was used instead of HZX-8 fermentation medium, and the rest of the operation was the same as that of the experimental group of *Heterophyllum chapensis*, the pathogen of walnut branch blight.

[0108] Each of the above groups was incubated at 28°C until the pathogens in each control group covered the entire culture dish.

[0109] (3) Inhibition rate calculation

[0110] Relative inhibition rate = (colon diameter of control group - colony diameter of experimental group) / colony diameter of control group × 100%. The colony diameter was measured using the cross-multiplication method (colon diameter = hyphae diameter minus block diameter).

[0111] The results are shown in Table 3 and Figure 4 As shown, Burkholderia gladioli ( Burkholderia gladioli HZX-8 fermentation broth showed significant inhibitory effects on the pathogens of walnut anthracnose and walnut twig blight.

[0112]

[0113] Example 4: Burkholderia gladioli under different salt concentrations ( Burkholderia gladioli HZX-8's inhibition rate against walnut anthracnose and walnut twig blight

[0114] (1) Culture medium preparation

[0115] Prepare LB liquid culture media containing different salt concentrations (2%, 5%, 7%) to simulate different environmental conditions. The specific composition is as follows:

[0116] Composition of LB liquid medium with 2% NaCl: NaCl is added to LB liquid medium to obtain the medium, wherein the mass-volume percentage (g:ml) of NaCl is 2%.

[0117] Composition of LB liquid medium with 5% NaCl: NaCl is added to LB liquid medium to obtain the medium, wherein the mass-volume percentage (g:ml) of NaCl is 5%.

[0118] Composition of LB liquid medium with 7% NaCl: NaCl is added to LB liquid medium to obtain the medium, wherein the mass-volume percentage (g:ml) of NaCl is 7%.

[0119] (2) Culture of strains

[0120] Burkholderia gladioli (identified in Example 1) was selected. Burkholderia gladioli Single colonies of strain HZX-8 were inoculated into LB liquid medium with different salt concentrations, and sterile filter paper was added. 2 mL sterile centrifuge tubes were placed in a constant temperature shaker and cultured at 30℃ and 180 rpm for 48 h to obtain HZX-8 bacterial suspension filter paper at different salt concentrations. The filter paper prepared by culturing in LB liquid medium containing 2% NaCl, 5% NaCl, and 7% NaCl was named 2% NaCl + HZX-8 bacterial suspension filter paper, 5% NaCl + HZX-8 bacterial suspension filter paper, and 7% NaCl + HZX-8 bacterial suspension filter paper, respectively.

[0121] Sterile filter paper discs were placed in centrifuge tubes containing sterile water, and filter paper discs containing sterile water were obtained according to the HZX-8 bacterial solution filter paper disc preparation method, which served as a control.

[0122] (3) Pathogen inoculation and resistance experiment

[0123] The experiment was repeated three times, with each repetition as follows:

[0124] ①The pathogen of walnut anthracnose, the Guangyuan anthracnose fungus group:

[0125] Experimental group of *Anthracnose guangyuanensis*, the pathogen of walnut anthracnose, with 2% NaCl: A 4 mm block of *Anthracnose guangyuanensis*, the pathogen of walnut anthracnose, was placed on a PDA medium plate. Filter paper discs of 2% NaCl + HZX-8 bacterial solution were placed 2 cm on both sides of the pathogen block using the confrontation inoculation method.

[0126] 2% NaCl + *Anthracnose guangyuanensis* control group (CK): Filter paper containing sterile water was used instead of filter paper containing 2% NaCl + HZX-8 bacterial solution. All other procedures were the same as in the 2% NaCl + *Anthracnose guangyuanensis* experimental group.

[0127] Experimental group of 5% NaCl + Guangyuan anthracnose pathogen of walnut: The filter paper of 5% NaCl + HZX-8 bacterial solution was replaced with the filter paper of 2% NaCl + HZX-8 bacterial solution, and the rest of the operation was the same as the experimental group of 2% NaCl + Guangyuan anthracnose pathogen of walnut.

[0128] 5% NaCl + *Bacillus guangyuanensis* control group (CK): Filter paper containing sterile water was used instead of filter paper containing 5% NaCl + HZX-8 bacterial solution. All other procedures were the same as in the 5% NaCl + *Bacillus guangyuanensis* experimental group.

[0129] Experimental group of 7% NaCl + Guangyuan anthracnose pathogen of walnut: The filter paper of 2% NaCl + HZX-8 bacterial solution was replaced with filter paper of 7% NaCl + HZX-8 bacterial solution, and the rest of the operation was the same as the experimental group of 7% NaCl + Guangyuan anthracnose pathogen of walnut.

[0130] 7% NaCl + *Anthracnose guangyuanensis* control group (CK): Filter paper containing sterile water was used instead of filter paper containing 7% NaCl + HZX-8 bacterial solution. All other procedures were the same as in the 7% NaCl + *Anthracnose guangyuanensis* experimental group.

[0131] Each of the above groups was incubated at 28°C until the pathogens in each control group covered the entire culture dish.

[0132] ②The pathogen of walnut branch blight is *Schizophyllum commune*.

[0133] Experimental group of 2% NaCl + walnut twig blight pathogen *Ceratophyllum demersum*: Place 4 mm *Ceratophyllum demersum* mycelial blocks on PDA medium plates, and place 2 cm on both sides of the pathogen blocks using the confrontation inoculation method with 2% NaCl + HZX-8 bacterial solution filter paper discs.

[0134] 2% NaCl + Walnut twig blight pathogen *Ceratophyllum demersum* control group (CK): Filter paper containing sterile water was used instead of the filter paper containing 2% NaCl + HZX-8 bacterial solution. All other procedures were the same as in the 2% NaCl + Walnut twig blight pathogen *Ceratophyllum demersum* experimental group.

[0135] Experimental group of 5% NaCl + walnut twig blight pathogen *Tetranychus chapensis*: The filter paper of 5% NaCl + HZX-8 bacterial solution was used instead of the filter paper of 2% NaCl + HZX-8 bacterial solution. The rest of the operation was the same as the experimental group of 2% NaCl + walnut twig blight pathogen *Tetranychus chapensis*.

[0136] 5% NaCl + Walnut twig blight pathogen *Tetranychus chapensis* control group (CK): Filter paper containing sterile water was used instead of the filter paper containing 5% NaCl + HZX-8 bacterial solution. All other procedures were the same as in the 5% NaCl + Walnut twig blight pathogen *Tetranychus chapensis* experimental group.

[0137] Experimental group of 7% NaCl + walnut twig blight pathogen *Tetranychus chapensis*: The filter paper of 7% NaCl + HZX-8 bacterial solution was used instead of the filter paper of 2% NaCl + HZX-8 bacterial solution. The rest of the operation was the same as the experimental group of 7% NaCl + walnut twig blight pathogen *Tetranychus chapensis*.

[0138] 7% NaCl + Walnut twig blight pathogen *Tetranychus chapensis* control group (CK): Filter paper containing sterile water was used instead of the filter paper containing 7% NaCl + HZX-8 bacterial solution. All other procedures were the same as in the 7% NaCl + Walnut twig blight pathogen *Tetranychus chapensis* experimental group.

[0139] Each of the above groups was incubated at 28°C until the pathogens in each control group covered the entire culture dish.

[0140] (4) Inhibition rate calculation:

[0141] The relative inhibition rate was calculated using the formula: Relative inhibition rate = (Control group colony diameter − Experimental group colony diameter) / Control group colony diameter × 100%. The colony diameter was measured using the cross-multiplication method (colony diameter = hyphae diameter minus block diameter).

[0142] (5) Results Analysis

[0143] The experimental results are shown in Table 4. Figure 5 , Figure 6 , Figure 7 and Figure 8 As shown, the antagonistic effect of strain HZX-8 gradually decreased with increasing salt concentration. However, even at higher salt concentrations, strain HZX-8 maintained a significant inhibitory effect on the pathogens causing walnut anthracnose and walnut twig blight.

[0144]

[0145] Note: P < 0.05 indicates a significant difference, and P < 0.01 indicates a highly significant difference.

[0146] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. Burkholderia gladioli ( Burkholderia gladioli The HZX-8 strain, with accession number CGMCCNo.33009.

2. The use of the strain or its bacterial culture according to claim 1 in any of the following: A1) Prevention or treatment of anthracnose in walnut plants; A2) Prevention or treatment of walnut branch blight in plants; A3) Inhibits the pathogens causing walnut anthracnose; A4) Inhibits the pathogens of walnut twig blight; A5) Prepare products for the prevention or treatment of anthracnose in walnut plants; A6) Prepare products for the prevention or treatment of walnut twig blight; A7) Prepare products that inhibit the pathogens causing walnut anthracnose; A8) Prepare products that inhibit the pathogens causing walnut twig blight; The pathogen causing walnut anthracnose is *Anthracnose guangyuanensis* (…). Colletotrichumguangyuanense ) ； Alternatively, the pathogen causing the walnut branch blight is *Pterocarya stenoptera* (also known as *Pterocarya stenoptera*). Diaporthechaotianensis.

3. The application according to claim 2, characterized in that: The product is a microecological preparation or a biological pesticide.

4. The application according to claim 2, characterized in that: The product is a microbial agent.

5. The application according to claim 4, characterized in that: The bacterial agent is a biocontrol agent.

6. A product, wherein the active ingredient is the bacterial strain or its broth as described in claim 1. 。 7. The product according to claim 6, characterized in that: The product is a microecological preparation or a biological pesticide.

8. The product according to claim 6, characterized in that: The product is a microbial agent.

9. The product according to claim 8, characterized in that: The bacterial agent is a biocontrol agent.

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