Burkholderia gladioli and application thereof

Abstract

The invention discloses burkholderia gladioli and an application of the burkholderia gladioli. The invention provides a Burkholderia gladioli HZX-8 strain, and the preservation number of the Burkholderia gladioli HZX-8 strain is CGMCC (China General Microbiological Culture Collection Center) No.33009. The Burkholderia gladioli HZX-8 strain has the advantages that the The invention also provides an application of the strain or a culture solution or a bacterial solution or a fermentation culture solution of the strain in preventing or treating plant walnut anthracnose and / or walnut branch blight. The Burkholderia gladioli HZX-8 strain disclosed by the invention has the advantages that the Burkholderia gladioli HZX-8 strain obtained by the invention shows a remarkable biological control potential in the aspects of preventing and treating the walnut anthracnose caused by the Colletotrichum guangyuanense and the walnut branch blight caused by the Diapotheca chaotianensis, and the Burkholderia gladioli HZX-8 strain can be used for preventing and treating the walnut branch blight caused by the Colletotrichum guangyuanense and the Burkholderia gladioli HZX-8 strain can be used for preventing and treating the walnut branch blight caused by the Diapotheca chaotianense.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial biological control and relates to gladiolus Burkholderia and application thereof. Background Art

[0002] Walnut( Juglans regia L.) is an important economic crop, widely planted in many regions around the world. However, walnut production is threatened by a variety of diseases, of which walnut anthracnose and walnut branch dieback are two major diseases, which seriously affect the yield and quality of walnuts. Walnut anthracnose is caused by fungi and manifests as lesions on leaves, fruits and branches, leading to early leaf fall and fruit rot; while walnut branch dieback is caused by a variety of pathogens, causing branch death, affecting the growth and yield of trees.

[0003] Traditional disease control methods mainly rely on the use of chemical pesticides, which are effective in the short term, but long-term use may lead to environmental pollution, ecological imbalance and the emergence of disease resistance. Therefore, the development of safe and effective biological control technology has become an important direction of current agricultural research.

[0004] In recent years, microbial biocontrol technology has gradually received attention. Specific microbial strains can inhibit the growth of plant pathogens through various mechanisms and enhance the disease resistance of plants. 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 planting. Summary of the invention

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

[0006] In order to solve the above technical problems, the present invention provides, in a first aspect, Burkholderia gladiolus ( Burkholderia gladioli ) HZX-8 strain, whose deposit number is CGMCC No.33009.

[0007] In a second aspect, the present invention provides the use of the strain or its culture solution or its bacterial solution or its fermentation culture solution according to the first aspect in any of the following: A1) Prevent or treat walnut anthracnose in plants; A2) Prevent or treat walnut blight in plants; A3) Inhibit the pathogen of walnut anthracnose; A4) Inhibit the pathogen of walnut blight; A5) Preparation of products for preventing or treating walnut anthracnose; A6) Preparation of products for preventing or treating walnut blight in plants; A7) Preparation of products for inhibiting the pathogen of walnut anthracnose; A8) Preparation of products for inhibiting the pathogen of walnut blight.

[0008] In the above application, the pathogen of walnut anthracnose is Guangyuan anthracnose ( Colletotrichum guangyuanense); Or, the pathogen of walnut branch blight is Aspergillus asafoetida ( Diaporthe chaotianensis).

[0009] In the applications described above, the product is a bacterial agent, a microecological preparation, or a biological pesticide.

[0010] In the application described above, the bacterial agent is a biocontrol agent.

[0011] In a third aspect, the present invention provides a product, the active ingredient of which is the strain or its culture solution or its bacterial solution or its fermentation culture solution as described in the first aspect.

[0012] The products mentioned above have any of the following functions: A1) Prevent or treat walnut anthracnose in plants; A2) Prevent or treat walnut blight in plants; A3) Inhibit the pathogen of walnut anthracnose; A4) Inhibit the pathogen of walnut blight.

[0013] In the above-mentioned product, the pathogen of walnut anthracnose is Guangyuan anthracnose ( Colletotrichum guangyuanense); Or, the pathogen of walnut branch blight is Aspergillus asafoetida ( Diaporthe chaotianensis).

[0014] The products mentioned above are bacterial agents, microecological preparations, or biological pesticides.

[0015] The product mentioned above is a bacterial agent; specifically a biocontrol agent.

[0016] In the above, the inhibiting of each pathogen may be inhibiting each pathogen in a high-salt environment.

[0017] In the above, the plant is a dicotyledon or a monocotyledon; In the above, the dicotyledonous plant is a plant of the Juglandaceae family.

[0018] In the above text, the Juglandaceae plant is walnut tree.

[0019] In the above, the plant can grow in high-salt soil or saline-alkali land.

[0020] The gladiolus Burkholderia obtained by the present invention Burkholderia gladioli) HZX-8 strain, in the prevention and treatment of Guangyuan anthrax ( Colletotrichum guangyuanense ) and walnut anthracnose caused by Aspergillus asafoetida ( Diaporthe chaotianensis ) showed significant biological control potential in the treatment of walnut branch blight caused by walnut twig blight. As a biological control agent, the strain has broad application prospects in the development of new biological agents and the preparation of biological pesticides. The present invention provides a new and environmentally friendly method for biological control of walnut anthracnose, which reduces the use of chemical pesticides through biological control, reduces environmental pollution, protects ecological balance, improves the yield and quality of walnut crops, and helps promote the sustainable development of agriculture.

[0021] Collection Instructions Bacteria name: HZX-8 Latin name: Burkholderia gladioli . Depository: China National Microbiological Culture Collection Administration General Microbiology Center Abbreviation of depository institution: CGMCC Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing Deposit date: December 10, 2024 CGMCC Registration Number: CGMCC No. 33009 Classification name: Burkholderia gladiolus Burkholderia gladioli BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Burkholderia gladiolus ( Burkholderia gladioli ) The colony macromorphology and individual morphology of HZX-8. The left picture shows the colony macromorphology of HZX-8; the right picture shows the individual morphology of HZX-8.

[0023] Figure 2 Burkholderia gladiolus ( Burkholderia gladioli ) Phylogenetic tree of HZX-8.

[0024] Figure 3 Burkholderia gladiolus ( Burkholderia gladioli ) The antagonistic effect of HZX-8 on walnut anthracnose and walnut branch blight. The left picture of the first row from top to bottom is the blank control of Colletotrichum guangyuanensis, the pathogen of walnut anthracnose, and the right picture is the antagonistic effect of HZX-8 on Colletotrichum guangyuanensis, the pathogen of walnut anthracnose; the left picture of the second row from top to bottom is the blank control of Aspergillus asafoetida, the pathogen of walnut branch blight; the right picture is the antagonistic effect of HZX-8 on Aspergillus asafoetida, the pathogen of walnut branch blight.

[0025] Figure 4 Burkholderia gladiolus ( Burkholderia gladioli) The antagonistic effect of HZX-8 fermentation culture fluid on walnut anthracnose and walnut branch blight. The left picture of the first row from top to bottom is the blank control of Guangyuan Anthracnose, the pathogen of walnut anthracnose, and the right picture is the antagonistic effect of HZX-8 fermentation culture fluid on Guangyuan Anthracnose, the pathogen of walnut anthracnose; the left picture of the second row from top to bottom is the blank control of Aspergillus asafoetida, the pathogen of walnut branch blight, and the right picture is the antagonistic effect of HZX-8 fermentation culture fluid on Aspergillus asafoetida, the pathogen of walnut branch blight.

[0026] Figure 5 The Burkholderia gladiolus cultured under different salt concentrations ( Burkholderia gladioli ) Effect of HZX-8 on the antibacterial activity of walnut anthracnose. The upper picture in the first column from left to right is the blank control of Guangyuan anthracnose pathogen of walnut anthracnose at 2% NaCl concentration, and the lower picture in the first column from left to right is the antagonistic effect of HZX-8 cultured at 2% NaCl concentration on Guangyuan anthracnose pathogen of walnut anthracnose; the upper picture in the second column from left to right is the blank control of Guangyuan anthracnose pathogen of walnut anthracnose at 5% NaCl concentration, and the lower picture in the first column from left to right is the antagonistic effect of HZX-8 cultured at 5% NaCl concentration on Guangyuan anthracnose pathogen of walnut anthracnose; the upper picture in the third column from left to right is the blank control of Guangyuan anthracnose pathogen of walnut anthracnose at 7% NaCl concentration, and the lower picture in the third column from left to right is the antagonistic effect of HZX-8 cultured at 7% NaCl concentration on Guangyuan anthracnose pathogen of walnut anthracnose.

[0027] Figure 6 Shown are the results of the culture of Burkholderia gladiolus ( Burkholderia gladioli ) Effect of HZX-8 on the antibacterial activity of walnut branch blight. The upper picture in the first column from left to right is the blank control of the pathogen of walnut branch blight, Aspergillus asafoetida at 2% NaCl concentration, and the lower picture in the first column from left to right is the antagonistic effect of HZX-8 cultured at 2% NaCl concentration on the pathogen of walnut branch blight; the upper picture in the second column from left to right is the blank control of the pathogen of walnut branch blight, Aspergillus asafoetida at 5% NaCl concentration, and the lower picture in the second column from left to right is the antagonistic effect of HZX-8 cultured at 5% NaCl concentration on the pathogen of walnut branch blight; the upper picture in the third column from left to right is the blank control of the pathogen of walnut branch blight, Aspergillus asafoetida at 7% NaCl concentration; the lower picture in the second column from left to right is the antagonistic effect of HZX-8 cultured at 7% NaCl concentration on the pathogen of walnut branch blight.

[0028] Figure 7 This is a bar graph showing the significant effects of different salt concentrations on walnut anthracnose.

[0029] Figure 8 This is a bar graph showing the significant effects of different salt concentrations on the walnut branch blight disease. DETAILED DESCRIPTION

[0030] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0031] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0032] Unless otherwise specified, the quantitative tests in the following examples were performed three times and the results were averaged.

[0033] 1L PDA culture medium composition: 200g peeled potatoes, 20g glucose, 20g agar, 1000mL distilled water.

[0034] 1L LB nutrient agar medium composition: 10g tryptone, 5g yeast extract, 10g NaCl, 15g agar, 1000mL distilled water.

[0035] 1L LB liquid culture medium composition: 10g tryptone, 5g yeast extract powder, 10g sodium chloride, and 1000mL distilled water.

[0036] 1L NA medium composition: peptone 10g, sodium chloride 5g, beef extract powder 3g, agar 15g, distilled water 1000mL.

[0037] 1L TSA medium composition: 15g tryptone, 5g soy peptone, 5g sodium chloride, 15g agar, and 1000mL distilled water.

[0038] The pathogen of walnut anthracnose is Colletotrichum guangyuanensis ( Colletotrichum guangyuanense ) is recorded in the following non-patent literature: "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.", which is available to the public from Beijing Forestry University. The biological material is only used to repeat the relevant experiments of the present invention and cannot be used for other purposes.

[0039] The pathogen of walnut branch blight is Aspergillus asafoetida ( Diaporthe chaotianensis ) is recorded 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.", which is available to the public from Beijing Forestry University. The biological material is only used to repeat the relevant experiments of the present invention and cannot be used for other purposes.

[0040] Example 1, Burkholderia gladiolus ( Burkholderia gladioli )Isolation, screening and identification of HZX-8 1. Isolation of strain HZX-8 Samples of walnut anthracnose-infected fruits were collected in Hezhang Yelang National Forest Park in Shuitangbao Township, Hezhang County, Bijie City, Guizhou Province. The diseased and healthy fruit samples were mixed and placed in a conical flask containing 45 mL of sterile water and sterile steel balls, and shaken at 30°C and 180 r / min for 60 min to prepare a bacterial suspension. The obtained suspension was filtered through sterile gauze and its volume was adjusted to 100 ml to form a diseased fruit stock solution. A 10-fold gradient dilution method was used to further prepare 10 -2 and 10 -3 100 μL of each of these dilutions was evenly spread on LB medium and inverted at 28°C for culture. Single colonies with different morphological characteristics were transferred to new LB medium by the plate streak method, purified and cultured, and stored at 4°C for future use to obtain the strain to be tested.

[0041] 2. Preliminary screening The plate pair method was used for preliminary screening, and the target pathogen was Colletotrichum guangyuanensis, the pathogen of walnut anthracnose ( Colletotrichum guangyuanense ), which can cause walnut anthracnose. The target pathogen, walnut anthracnose pathogen Guangyuan anthracnose bacteria ( Colletotrichum guangyuanense ) were inoculated on PDA medium, and 4 strains to be tested were inoculated on each culture dish to form a cross layout. At the same time, only the target pathogen Guangyuan anthrax ( Colletotrichum guangyuanense ) as a blank control. Culture at 28°C. After the control group covers the entire culture dish, select the strain with antibacterial effect for further screening.

[0042] 3. Rescreening Inoculate 4 mm-sized walnut anthracnose pathogen Guangyuan anthracnose bacteria in the center of the PDA plate. Colletotrichumguangyuanense ) and the walnut branch blight pathogen Aspergillus asafoetida ( Diaporthe chaotianensis ) bacteria block, and the strains with antibacterial effect selected in the initial screening were inoculated 2 cm on both sides of the pathogenic bacteria block using the plate confrontation method. At the same time, only the Guangyuan anthracnose pathogen of walnut anthracnose ( Colletotrichum guangyuanense ) and the walnut branch blight pathogen Aspergillus asafoetida ( Diaporthe chaotianensis ) as a blank control, and each treatment was repeated three times. Cultured at a constant temperature of 28°C, after the control group covered the entire culture dish, the antagonistic strain with the strongest antibacterial effect was screened out. The strain with the largest inhibition zone was the antagonistic strain with the strongest antibacterial effect, and it was named HZX-8.

[0043] 2. Burkholderia gladiolus ( Burkholderia gladioli )Morphological and physiological and biochemical identification of HZX-8 When HZX-8 was inoculated on LB plates, the colonies were light yellow, round, moist, slightly convex, opaque, with smooth edges, and the medium was yellow (e.g. Figure 1 Strain HZX-8 can also grow well on NA and TSA plates. The Gram reaction of strain HZX-8 is negative and the bacteria are short rod-shaped.

[0044] Strain HZX-8 is a facultative anaerobic bacterium; growth temperature: 25-37℃, not heat-resistant.

[0045] The physiological and biochemical characteristics of HZX-8 are shown in Table 1. It grows well in LB medium with a NaCl concentration of 2%-7%; it can liquefy gelatin and produce iron carriers; it cannot hydrolyze starch and produce H2S.

[0046]

[0047] Note: +, positive reaction or can be utilized, growth; -, negative reaction or cannot grow, utilization.

[0048] 3. Burkholderia gladiolus ( Burkholderia gladioli ) Molecular classification and identification of HZX-8 Single colonies of HZX-8 were picked from the LB agar plates and transferred to 2 mL sterile centrifuge tubes containing LB liquid medium. These centrifuge tubes were placed in a constant temperature shaker and cultured at 30°C at a speed of 180r / min for 48 h. After the culture was completed, 200 μL of the culture was taken from each centrifuge tube and transferred to a 1.5 mL sterile centrifuge tube. Next, these centrifuge tubes were placed in a 100°C water bath and heated for 10 minutes to lyse the cells and release the DNA. The supernatant containing the DNA of the strain was collected by centrifugation and stored at 4°C for subsequent analysis.

[0049] For molecular identification of the strain, the PCR reaction system included 1 μL of forward and reverse primers, 12.5 μL of 2xPCR mixture, 2 μL of DNA template, and an appropriate amount of sterile water, with 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 can specifically amplify the 16S rRNA gene of bacteria. The PCR program included: 94℃ pre-denaturation for 5 minutes, 30 cycles, each cycle including 94℃ denaturation for 30 seconds, 55℃ annealing for 30 seconds, and 72℃ extension for 45 seconds, and finally extension at 72℃ for 10 minutes, and stored at 4℃ to obtain PCR products.

[0050] The PCR product was sent to Beijing Nosai Genome Research Center Co., Ltd. for 16S rDNA sequencing, and the nucleotide sequence is SEQ ID NO: 1. The sequencing results were aligned and sheared using MEGA 6.0 software, and phylogenetic analysis was performed using the maximum likelihood method and Bayesian method to determine the taxonomic status of the strain.

[0051] The results are as follows Figure 2 As shown, HZX-8 and Burkholderia gladioli , so it was identified as Burkholderia gladiolus ( Burkholderia gladioli ).

[0052] 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 1 was identified as Burkholderia gladiolus ( Burkholderia gladioli ).

[0053] Burkholderia gladiolus ( Burkholderia gladioli )HZX-8 was deposited in the General Microbiology Center of China Microorganism Culture Collection Committee (referred to as CGMCC, address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing) on ​​December 10, 2024, and the deposit number is CGMCC No. 33009. Hereinafter referred to as strain HZX-8 or HZX-8.

[0054] Example 2, Burkholderia gladiolus ( Burkholderia gladioli ) Antagonistic effect of HZX-8 on walnut anthracnose and walnut branch blight The bacterial masses of each pathogen described below are obtained when each pathogen is cultured in PDA culture medium until it fills the entire dish.

[0055] (1) Preparation of bacterial culture The Burkholderia gladiolus 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 (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 containing HZX-8 bacterial liquid.

[0056] A sterile filter paper piece was placed in a centrifuge tube containing sterile water, and a filter paper piece containing sterile water was obtained according to the preparation method of the filter paper piece containing HZX-8 bacterial solution as a control.

[0057] (2) Pathogen inoculation and confrontation experiments The experiment was repeated three times, and each repetition was as follows: Experimental group of Guangyuan anthracnose pathogen of walnut anthracnose (HZX-8): Place a 4 mm block of Guangyuan anthracnose pathogen of walnut anthracnose on a PDA culture medium plate, and place the filter paper containing HZX-8 bacterial solution on both sides of the pathogen block using the confrontation inoculation method, 2 cm away from the block.

[0058] Walnut anthracnose pathogen Guangyuan anthracnose control group (CK): the filter paper containing sterile water was used to replace the filter paper containing HZX-8 bacterial solution, and the rest of the operations were the same as those of the walnut anthracnose pathogen Guangyuan anthracnose pathogen experimental group.

[0059] Walnut branch blight pathogen Aspergillus chinensis experimental group (HZX-8): The fungus blocks of Aspergillus chinensis, the pathogen of walnut anthracnose, were replaced by the fungus blocks of Aspergillus chinensis, the pathogen of walnut anthracnose, and the rest of the operations were the same as the experimental group of Aspergillus chinensis, the pathogen of walnut anthracnose, Guangyuan.

[0060] Control group (CK) of Colletotrichum guangyuanensis, the pathogen of walnut anthracnose: the filter paper containing HZX-8 bacterial solution was replaced by the filter paper containing sterile water, and the rest of the operations were the same as those of the experimental group of Aspergillus chondriasis, the pathogen of walnut branch blight.

[0061] The above groups were cultured at 28°C until the pathogens in each control group covered the entire culture dish.

[0062] (3) Calculation of inhibition rate The inhibition rate of HZX-8 bacterial solution on pathogen growth was calculated using the following formula: relative inhibition rate = (colony diameter of control group - colony diameter of experimental group) / colony diameter of control group * 100%. The cross method was used to measure the colony diameter (colony diameter = mycelium diameter minus bacterial block diameter).

[0063] The results are shown in Table 2 and Figure 3 As shown, Burkholderia gladiolus ( Burkholderia gladioli ) HZX-8 has a significant antagonistic effect on the pathogens of walnut anthracnose and walnut branch blight, providing a new strategy for the biological control of these two diseases.

[0064]

[0065] Example 3, Burkholderia gladiolus ( Burkholderia gladioli )Inhibition rate of HZX-8 fermentation culture fluid against walnut anthracnose and walnut branch blight (1) Preparation of fermentation broth The Burkholderia gladiolus 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 cultured at 30°C and 180 r / min for 48 h until the OD 600nm value ≈ 0.5, and the fermentation broth of HZX-8 was obtained.

[0066] (2) Culture medium preparation and inoculation The experiment was repeated three times, and each repetition was as follows: Walnut anthracnose pathogen Guangyuan anthracnose bacteria experimental group (HZX-8): HZX-8 fermentation culture medium and PDA medium were mixed at a volume ratio of 1:10 and evenly spread on the culture dish. After the culture medium solidified, a walnut anthracnose pathogen Guangyuan anthracnose bacteria block with a diameter of 4 mm was placed in the center of the culture medium.

[0067] Walnut anthracnose pathogen Guangyuan Colletotrichum control group (CK): Sterile water was used instead of HZX-8 fermentation culture medium, and other operations were the same as those of the walnut anthracnose pathogen Guangyuan Colletotrichum experimental group.

[0068] Walnut branch blight pathogen Aspergillus chinensis experimental group (HZX-8): The fungus blocks of Aspergillus chinensis, the pathogen of walnut anthracnose, were replaced by the fungus blocks of Aspergillus chinensis, the pathogen of walnut anthracnose, and the rest of the operations were the same as the experimental group of Aspergillus chinensis, the pathogen of walnut anthracnose, Guangyuan.

[0069] The control group (CK) of Colletotrichum guangyuanensis, the pathogen of walnut anthracnose, was treated with sterile water instead of HZX-8 fermentation medium. Other operations were the same as those of the experimental group of Aspergillus chondriasis, the pathogen of walnut branch dieback.

[0070] The above groups were cultured at 28°C until the pathogens in each control group covered the entire culture dish.

[0071] (3) Calculation of inhibition rate Relative inhibition rate = (colony diameter of control group − colony diameter of experimental group) / colony diameter of control group × 100%. The cross method was used to measure the colony diameter (colony diameter = mycelium diameter minus bacterial block diameter).

[0072] The results are shown in Table 3 and Figure 4 As shown, Burkholderia gladiolus ( Burkholderia gladioli)HZX-8 fermentation culture fluid showed significant inhibitory effect on walnut anthracnose and walnut branch blight pathogens

[0073] Example 4, Burkholderia gladiolus at different salt concentrations ( Burkholderia gladioli )Inhibition rate of HZX-8 against walnut anthracnose and walnut branch blight (1) Culture medium preparation Prepare LB liquid culture medium containing different salt concentrations (2%, 5%, 7%) to simulate different environmental conditions. The specific composition is as follows: The composition of 2% NaCl LB liquid culture medium is as follows: NaCl is added to LB liquid culture medium to obtain a culture medium in which the mass volume percentage content (g:ml) of NaCl is 2%.

[0074] The composition of LB liquid culture medium with 5% NaCl is as follows: NaCl is added to LB liquid culture medium to obtain a culture medium in which the mass volume percentage content (g:ml) of NaCl is 5%.

[0075] The composition of 7% NaCl LB liquid culture medium is as follows: NaCl is added to the LB liquid culture medium to obtain a culture medium in which the mass volume percentage content (g:ml) of NaCl is 7%.

[0076] (2) Strain culture The Burkholderia gladiolus identified in Example 1 was selected ( Burkholderia gladioli ) Single colonies of the HZX-8 strain were inoculated in LB liquid culture media with different salt concentrations, and sterile filter paper was added. The inoculated 2 mL sterile centrifuge tube was placed in a constant temperature shaker and cultured at 30°C and 180 r / min for 48 h to obtain HZX-8 bacterial liquid filter paper discs at different salt concentrations. The filter paper discs prepared by culturing in LB liquid culture media containing 2% NaCl, LB liquid culture media containing 5% NaCl, and LB liquid culture media containing 7% NaCl were specifically named 2% NaCl + HZX-8 bacterial liquid filter paper discs, 5% NaCl + HZX-8 bacterial liquid filter paper discs, and 7% NaCl + HZX-8 bacterial liquid filter paper discs.

[0077] Place a sterile filter paper piece into a centrifuge tube containing sterile water, and obtain a filter paper piece containing sterile water according to the HZX-8 bacterial liquid filter paper preparation method as a control.

[0078] (3) Pathogen inoculation and confrontation experiments The experiment was repeated three times, and each repetition was as follows: ①Walnut anthracnose pathogen Guangyuan anthracnose group: 2% NaCl + Guangyuan anthracnose pathogen of walnut anthracnose experimental group: 4mm-sized Guangyuan anthracnose pathogen block of walnut anthracnose pathogen was placed on the PDA culture medium plate, and 2% NaCl + HZX-8 bacterial liquid filter paper was placed 2cm on both sides of the pathogen block using the confrontation inoculation method.

[0079] 2% NaCl + Colletotrichum guangyuanensis, the pathogen of walnut anthracnose, control group (CK): the filter paper containing sterile water was used to replace the 2% NaCl + HZX-8 bacterial solution filter paper, and the other operations were the same as the 2% NaCl + Colletotrichum guangyuanensis, the pathogen of walnut anthracnose, experimental group.

[0080] 5% NaCl + Guangyuan anthracnose pathogen of walnut anthracnose experimental group: 5% NaCl + HZX-8 bacterial solution filter paper was used to replace 2% NaCl + HZX-8 bacterial solution filter paper, and the rest of the operations were the same as the 2% NaCl + Guangyuan anthracnose pathogen of walnut anthracnose experimental group.

[0081] 5% NaCl + Colletotrichum guangyuanensis, the pathogen of walnut anthracnose, control group (CK): the filter paper containing sterile water was used to replace the 5% NaCl + HZX-8 bacterial solution filter paper, and the other operations were the same as the 5% NaCl + Colletotrichum guangyuanensis, the pathogen of walnut anthracnose, experimental group.

[0082] 7% NaCl + Guangyuan anthracnose pathogen of walnut anthracnose experimental group: 7% NaCl + HZX-8 bacterial solution filter paper was used to replace 2% NaCl + HZX-8 bacterial solution filter paper, and the rest of the operations were the same as the 7% NaCl + Guangyuan anthracnose pathogen of walnut anthracnose experimental group.

[0083] 7% NaCl + Colletotrichum guangyuanensis, the pathogen of walnut anthracnose, control group (CK): the filter paper containing sterile water was used to replace the 7% NaCl + HZX-8 bacterial solution filter paper, and the other operations were the same as those of the 7% NaCl + Colletotrichum guangyuanensis, the pathogen of walnut anthracnose, experimental group.

[0084] The above groups were cultured at 28°C until the pathogens in each control group covered the entire culture dish.

[0085] ②Walnut branch blight pathogen Aspergillus group: 2% NaCl + Aspergillus niger experimental group: 4 mm sized Aspergillus niger bacteria blocks, the pathogen of walnut branch blight, were placed on the PDA culture medium plate, and 2% NaCl + HZX-8 bacterial solution filter paper sheets were placed 2 cm on both sides of the pathogen blocks using the confrontation inoculation method.

[0086] 2% NaCl + Aspergillus chinensis control group (CK): The filter paper containing sterile water was used to replace the 2% NaCl + HZX-8 bacterial solution filter paper, and the other operations were the same as the 2% NaCl + Aspergillus chinensis experimental group.

[0087] 5% NaCl + Aspergillus chinensis experimental group, the pathogen of walnut branch blight: 5% NaCl + HZX-8 bacterial solution filter paper was used to replace the 2% NaCl + HZX-8 bacterial solution filter paper, and the rest of the operations were the same as the 2% NaCl + Aspergillus chinensis experimental group, the pathogen of walnut branch blight.

[0088] 5% NaCl + Aspergillus niger control group (CK): The filter paper containing sterile water was used to replace the 5% NaCl + HZX-8 bacterial solution filter paper, and the other operations were the same as the 5% NaCl + Aspergillus niger experimental group.

[0089] 7% NaCl + Aspergillus chinensis experimental group, the pathogen of walnut branch blight: 7% NaCl + HZX-8 bacterial solution filter paper was used to replace 2% NaCl + HZX-8 bacterial solution filter paper, and the rest of the operations were the same as the 7% NaCl + Aspergillus chinensis experimental group, the pathogen of walnut branch blight.

[0090] 7% NaCl + Aspergillus chinensis control group (CK): The filter paper containing sterile water was used to replace the 7% NaCl + HZX-8 bacterial solution filter paper, and the other operations were the same as the 7% NaCl + Aspergillus chinensis experimental group.

[0091] The above groups were cultured at 28°C until the pathogens in each control group covered the entire culture dish.

[0092] (4) Calculation of inhibition rate: The relative inhibition rate was calculated using the formula: relative inhibition rate = (colony diameter of control group − colony diameter of experimental group) / colony diameter of control group × 100%. The colony diameter was measured using the cross method (colony diameter = mycelium diameter minus bacterial block diameter).

[0093] (5) Results analysis The experimental results are shown in Table 4. Figure 5 , Figure 6 , Figure 7 and Figure 8 As shown in the figure, with the increase of salt concentration, the antagonistic effect of HZX-8 strain gradually decreased. However, even at higher salt concentrations, HZX-8 strain still maintained a significant inhibitory effect on walnut anthracnose and walnut branch blight pathogens.

[0094]

[0095] Note: P < 0.05 indicates a significant difference, and P < 0.01 indicates an extremely significant difference.

[0096] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. Burkholderia gladiolus ( Burkholderia gladioli ) HZX-8 strain, whose deposit number is CGMCC No.33009.

2. Use of the strain according to claim 1 or its culture solution or its bacterial solution or its fermentation culture solution in any of the following: A1) Prevent or treat walnut anthracnose in plants; A2) Prevent or treat walnut blight in plants; A3) Inhibit the pathogen of walnut anthracnose; A4) Inhibit the pathogen of walnut blight; A5) Preparation of products for preventing or treating walnut anthracnose; A6) Preparation of products for preventing or treating walnut blight in plants; A7) Preparation of products for inhibiting the pathogen of walnut anthracnose; A8) Preparation of products for inhibiting the pathogen of walnut blight.

3. The use according to claim 2, characterized in that: The pathogen of walnut anthracnose is Guangyuan anthracnose ( Colletotrichum guangyuanense ) ； Or, the pathogen of walnut branch blight is Aspergillus asafoetida ( Diaporthe chaotianensis).

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

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

6. A product, the active ingredient of which is the strain according to claim 1 or its culture solution or its bacterial liquid or its fermentation culture solution.

7. The product according to claim 6, characterized in that: The product has any of the following functions: A1) Prevent or treat walnut anthracnose in plants; A2) Prevent or treat walnut blight in plants; A3) Inhibit the pathogen of walnut anthracnose; A4) Inhibit the pathogen of walnut blight.

8. The product according to claim 7, characterized in that: The pathogen of walnut anthracnose is Guangyuan anthracnose ( Colletotrichum guangyuanense); Or, the pathogen of walnut branch blight is Aspergillus asafoetida ( Diaporthechaotianensis).

9. The product according to any one of claims 6 to 8, characterized in that: The product is a bacterial agent, a microecological preparation, or a biological pesticide.

10. The product according to any one of claims 6 to 8, characterized in that: The product is a bacterial agent; The bacterial agent is a biocontrol agent.

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