Burkholderia gladioli NDG19 and its application in disease resistance

Abstract

The application relates to a Burkholderia gladioli NDG19 and an application of the Burkholderia gladioli NDG19 in disease resistance, and belongs to the technical field of microorganisms. The Burkholderia gladioli NDG19 is preserved in the Guangdong Provincial Microbial Culture Collection Center on July 31, 2023, the strain preservation number is 63701, and the preservation address is No. 59 Building, 5th Floor, Guangzhou, Guangzhou, China. The Burkholderia gladioli NDG19 provided in the application can effectively inhibit the growth and reproduction of Fusarium oxysporum and Alternaria tenuis, and can effectively prevent and treat plant fusarium wilt such as sharp banana fusarium wilt and melon root rot of cucurbitaceae crops, solves the problems of environmental pollution and food safety caused by chemical fungicides, the strain has the functions of iron carrier production, phosphorus dissolution and potassium dissolution, can effectively improve the utilization rate of phosphorus and potassium in soil as a biological bacterial fertilizer, promotes the growth of plants, is safe and environment-friendly, and has a wide application prospect.

Description

Technical Field

[0001] This application relates to the field of microbial technology, and in particular to Burkholderia gladioli NDG19 and its application in disease resistance. Background Technology

[0002] With the rapid development of my country's economy and the acceleration of urbanization and industrialization, the area of ​​arable land in my country is gradually decreasing. This inevitably leads to the need for continuous cultivation on the same piece of land. However, continuous cultivation can easily lead to problems such as soil degradation and the aggravation of diseases. Diseases (such as wilt and root rot) are difficult to prevent and treat effectively, which has a significant impact on agricultural production.

[0003] Currently, controlling soil-borne crop diseases in agricultural production mainly relies on spraying or irrigating the soil with chemical fungicides, in addition to selecting improved varieties and improving crop cultivation practices. However, most chemical fungicides have varying degrees of toxicity to humans and animals, and their pesticide residues are often excessive, leading to a severe decline in crop yield and quality, serious damage to the farmland ecosystem, and hindering sustainable social and economic development. Furthermore, these chemical fungicides applied to the soil are absorbed by crop roots and translocated to the edible parts of plants, causing agricultural product safety issues and threatening human health.

[0004] In related technologies, to address the aforementioned problems, biological control techniques, especially those utilizing microorganisms to control crop diseases, are receiving increasing attention. Although there are sporadic reports of strains that can control certain crop diseases, their effectiveness is unsatisfactory. Microorganisms require a certain biological cycle for reproduction and growth, and they must colonize in the soil to be effective. Compared to chemical methods such as fungicides, microorganisms do not show immediate effects. Furthermore, living microbial communities are easily affected by various environmental conditions during reproduction, leading to poor application results, and complex extraction processes of active substances are also required. Currently, research on strains that can simultaneously and efficiently control multiple crop diseases faces the problem of insufficient quantity and poor efficacy of microorganisms for crop disease control, limiting the application of biological control technologies.

[0005] Therefore, a new strain is needed that can effectively inhibit the growth and reproduction of pathogens such as Fusarium oxysporum and Alternaria alterniflora, and can be used to control root rot of cucurbit crops such as watermelon, melon, and cucumber, as well as wilt diseases of bananas. It can also effectively dissolve insoluble phosphorus and potassium in the rhizosphere soil of plants, promote plant growth, and provide a solid foundation for subsequent plant protection, biofertilizers and other related biological protection fields. Summary of the Invention

[0006] To overcome the problems existing in related technologies, this application provides a *Burkholderia gladioli* NDG19 strain and its disease-resistant applications. This *Burkholderia gladioli* NDG19 strain, which solubilizes phosphorus and potassium and has disease-resistant applications, can effectively inhibit the growth and reproduction of *Fusarium oxysporum* and *Alternaria alternata*, thereby effectively preventing and controlling plant wilt diseases such as banana wilt and root rot diseases of cucurbitaceous crops. It solves the environmental pollution and food safety problems caused by the use of chemical fungicides. Moreover, this strain has the functions of producing iron carriers, solubilizing phosphorus and potassium, and as a microbial fertilizer, it can effectively promote plant growth, is safe and environmentally friendly, and has broad application prospects.

[0007] One of the purposes of this application is to provide a Burkholderia gladioli NDG19 strain and its application in disease resistance, characterized in that it was deposited on July 31, 2023, at the Guangdong Provincial Center for Microbial Culture Collection, with the strain accession number 63701, and the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0008] In a preferred embodiment, the *Burkholderia gladioli* NDG19 has the following morphology: the colonies are pale yellow, round, with neat edges, smooth and moist, and have a certain degree of adhesiveness; it is obligate aerobic, the bacteria are rod-shaped, and it does not produce spores or buds, and it is Gram-negative.

[0009] In a preferred embodiment, the conditions for subculturing *Burkholderia gladioli* NDG19 on LB solid medium include:

[0010] The temperature is 20-45℃, the pH value is 6.0-10.0, the rotation speed is 160-200 r / min, and the time is 24-48h.

[0011] In a preferred embodiment, the LB solid culture medium comprises 10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, and 20 g / L agar powder.

[0012] In a preferred embodiment, the *Burkholderia gladioli* NDG19 possesses the following physiological and biochemical characteristics:

[0013] D-glucose+, sucrose-, amylase-, gelatin liquefaction+, methyl red test+, VP test-, indoleacetic acid-, citric acid+, oxidase+, catalase+, nitrate reduction-, urease+, ammonia production+.

[0014] The second objective of this application is to provide the application of the aforementioned Burkholderia gladioli NDG19 in inhibiting Fusarium oxysporum.

[0015] The third objective of this application is to provide the application of the aforementioned Burkholderia gladioli NDG19 in inhibiting Alternaria spp.

[0016] The fourth objective of this application is to provide the application of the aforementioned Burkholderia gladioli NDG19 in the control of plant wilt disease.

[0017] The fifth objective of this application is to provide the application of the aforementioned Burkholderia gladioli NDG19 in the control of banana wilt disease.

[0018] The sixth objective of this application is to provide the application of the aforementioned Burkholderia gladioli NDG19 in the prevention and control of plant root rot.

[0019] The seventh objective of this application is to provide the application of the siderophore-producing, phosphorus-solubilizing, and potassium-solubilizing functions of the aforementioned Burkholderia gladioli NDG19 in promoting plant growth.

[0020] The technical solution provided in this application may include the following beneficial effects:

[0021] (1) This invention isolated Burkholderia gladioli NDG19 from the roots of Reed chinensis and identified it morphologically, physiologically and biochemically, and genetically. 16S rRNA identification confirmed its identity as Burkholderia gladioli. Antagonistic effects experiments against pathogens such as Fusarium oxysporum and Alternaria alternata showed that this strain effectively inhibited the growth and reproduction of Fusarium oxysporum and Alternaria alternata, thus effectively preventing plant wilt diseases such as banana wilt and root rot in cucurbitaceous crops, solving the environmental pollution and food safety problems caused by the use of chemical fungicides.

[0022] (2) The Gladiolus Burkholderia provided in this application can produce iron carriers and has functions such as phosphorus solubilization and potassium solubilization. It can effectively dissolve insoluble phosphorus and potassium in the rhizosphere soil of plants and can significantly increase the content of available phosphorus and potassium in the soil. As a biological fertilizer, it can effectively improve the utilization rate of phosphorus and potassium in the soil by plants, thereby promoting plant growth, reducing the amount of chemical fertilizer used, increasing crop yield, and is safe and environmentally friendly, with broad application prospects.

[0023] It should be understood that the above general description and the following detailed description are exemplary and explanatory only, and do not limit this application. Attached Figure Description

[0024] The above and other objects, features and advantages of this application will become more apparent from the more detailed description of exemplary embodiments thereof in conjunction with the accompanying drawings, wherein the same reference numerals generally represent the same components in the exemplary embodiments thereof.

[0025] Figure 1 This is a schematic diagram of the colony morphology of Burkholderia gladioli NDG19 strain after 24 hours of culture on LB solid medium, as shown in the embodiments of this application.

[0026] Figure 2 This is a schematic diagram of the colony of Burkholderia gladioli NDG19 after seven days of culture on PKO solid medium, as shown in the embodiments of this application.

[0027] Figure 3 This is a schematic diagram of the colony of Burkholderia gladioli NDG19 after seven days of culture on potassium feldspar solid medium, as shown in the embodiments of this application.

[0028] Figure 4 This is a schematic diagram of the control group in the antagonistic experiment of Burkholderia gladioli NDG19 against Fusarium oxysporum, as shown in the embodiments of this application.

[0029] Figure 5 This is a schematic diagram of the treatment group in the antagonistic experiment of Burkholderia gladioli NDG19 against Fusarium oxysporum, as shown in the embodiments of this application.

[0030] Figure 6 This is a schematic diagram of the control group in the antagonistic experiment of Burkholderia gladioli NDG19 against Fusarium oxysporum, as shown in the embodiments of this application.

[0031] Figure 7 This is a schematic diagram of the treatment group in the antagonistic experiment of Burkholderia gladioli NDG19 against Fusarium oxysporum, as shown in the embodiments of this application.

[0032] Figure 8 This is a schematic diagram of the control group in the antagonistic experiment of Burkholderia gladioli NDG19 against Alternaria spp., as shown in the embodiments of this application.

[0033] Figure 9 This is a schematic diagram of the treatment group in the antagonistic experiment of Burkholderia gladioli NDG19, as shown in the embodiments of this application.

[0034] Figure 10 This invention relates to the determination of the siderophore production capacity of the strain Burkholderia gladioli NDG19 shown in the embodiments of this application. Detailed Implementation

[0035] Preferred embodiments of the present application will now be described in more detail with reference to the accompanying drawings. While preferred embodiments of the present application are shown in the drawings, it should be understood that the present application may be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided to make the present application more thorough and complete, and to fully convey the scope of the present application to those skilled in the art.

[0036] The terminology used in this application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. The singular forms “a,” “the,” and “the” used in this application and the appended claims are also intended to include the plural forms unless the context clearly indicates otherwise. It should also be understood that the term “and / or” as used herein refers to and includes any or all possible combinations of one or more of the associated listed items.

[0037] Currently, biological control technologies, especially those utilizing microorganisms to control crop diseases, are receiving increasing attention due to their safety, environmental friendliness, and harmlessness. However, the limited number and relatively low effectiveness of microorganisms currently available for crop disease control restricts the application of biological control technologies.

[0038] To address the aforementioned issues, this application provides a strain of Burkholderia gladioli that can effectively inhibit the growth and reproduction of Fusarium oxysporum and Alternaria alternata, thereby effectively preventing and controlling plant wilt diseases such as banana wilt and root rot. This solves the environmental pollution and food safety problems caused by the use of chemical fungicides. Furthermore, this strain can produce iron carriers and has functions such as phosphorus solubilization and potassium solubilization. As a microbial fertilizer, it can effectively promote plant growth, is safe and environmentally friendly, and has broad application prospects.

[0039] The technical solutions of the embodiments of this application are described in detail below with reference to the accompanying drawings.

[0040] Example 1: Screening of strain NDG19

[0041] 1. Strains are isolated, purified, and preserved.

[0042] Healthy *Dioscorea nipponica* plants were selected from Anqing County, Anhui Province and brought back to the laboratory. Strains NDG19 were isolated and purified from the *Dioscorea nipponica* plants. The specific isolation and purification steps are as follows:

[0043] Pretreatment of *Reedia spp.* involved the following steps: First, the roots were cut into 1cm sections using sterilized scissors. Then, the surface dust was washed away with sterile water to obtain clean roots. The cleaned roots were then immersed in a 2% sodium hypochlorite solution for 2 minutes, followed by rinsing with sterile water once, then soaking in 70% alcohol for 5 minutes. This process was repeated 8 times with sterile water, each time for 5 minutes. 200μL of the final rinse solution was spread onto LB agar plates for incubation at 37℃ for 24-48 hours. After 24 hours of incubation, bacterial growth on the LB agar plates was observed. Figure 1 The diagram shows the colony morphology of strain NDG19 after 24 hours of culture on LB solid medium. If no bacteria grow on LB solid medium after 24 hours, it indicates that the roots of *Dioscorea opposita* have been thoroughly sterilized, and sterilized *Dioscorea opposita* roots are obtained.

[0044] Using sterile tweezers, place the naturally dried roots and stalks of *Imperata cylindrica* into a mortar, add 5 mL of sterile water, and grind thoroughly with a grinding stick to obtain a grinding liquid. It is important to note that the grinding operation must be performed in a sterile environment; that is, both the mortar and grinding stick must be sterilized beforehand.

[0045] Take 1 mL of the above-mentioned homogenate and add it to 9 mL of sterile water. Mix thoroughly, and then continue to dilute with sterile water in a 10-fold serial dilution series to obtain concentrations of 10... －4 10 －5 and 10 －6 Dilute the grinding slurry, take 200 μL of each with a concentration of 10 -4 10 -5 and 10 -6The diluted grinding solution was evenly spread onto DN solid culture medium that had been sterilized at 121℃ for 20 min under high pressure, and incubated at 30℃ for 3-5 days to generate single bacteria; wherein, the DN solid culture medium (1.0L) includes 10.0g sucrose, 0.2g magnesium sulfate (MgSO4), 0.1g sodium chloride (NaCl), 0.2g dipotassium hydrogen phosphate (K2HPO4), 0.4g potassium dihydrogen phosphate (KH2PO4), 0.02g calcium chloride (CaCl2), 0.01g ferric chloride (FeCl3), 0.002g sodium molybdate (Na2MoO4), 5g malic acid, 2.0g agar powder, and the pH value is 7.0.

[0046] The newly generated single bacteria were isolated and purified using the streak plating method, specifically including: picking newly generated single bacteria using the streak plating method and purifying them on LB solid medium, and repeatedly subculturing them until the colonies had the same color, shape, size, texture, and transparency. It should be noted that the LB solid medium components include 10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, and 20 g / L agar powder; the subculturing conditions on LB solid medium are a temperature of 20-45℃ and a pH of 6.0-10.0. The subculturing conditions on LB liquid medium are a rotation speed of 160-200 rpm for 24-48 h, with a preferred temperature of 37℃ and a pH of 7.0.

[0047] Finally, its morphology was further observed by simple staining (carbohydrate fuchsin staining) and microscopic examination (oil immersion), with uniform length, consistent width, and uniform staining as the purification criteria for the strain. Three to four loops of the purified strain were picked up using a sterilized inoculation loop and preserved in a preservation solution (20% glycerol), and stored at -20℃ and -80℃ to obtain Burkholderia gladioli NDG19.

[0048] To verify the culture effect of LB solid medium, two control groups were added in this example:

[0049] Control group 1: The operation steps and culture conditions were the same as above. The only two variables were that the LB solid medium was replaced with PKO solid medium and the culture time was 7 days. A clear zone was observed around the colony, indicating that the strain has the ability to solubilize phosphorus.

[0050] Control group 2: The operation steps and culture conditions were the same as above. The only two variables were that the LB solid medium was replaced with potassium feldspar solid medium and the culture time was 7 days. A clear zone was observed around the colony, indicating that the strain has the ability to solubilize phosphorus.

[0051] Note: The culture time for control groups 1 and 2 was set to 7 days, instead of 1 day in this example, because strain NDG19 in control groups 1 and 2 did not show significant changes after 1 day of culture.

[0052] Example 2: Determination of the siderophore production capacity of the strain

[0053] Siderophores are low-molecular-weight substances that can bind ferric ions and supply them to microbial cells. Bacteria secrete siderophores to compete with plant pathogens for iron ions, thereby inhibiting the growth and reproduction of pathogens. The chromate azuril (CAS) assay can be used to detect whether bacteria are capable of producing siderophores.

[0054] The culture medium used in the siderophore production capacity determination experiment was:

[0055] Add 50 mL of CAS staining solution to 1.0 L of MSA medium, then add 50 mL of phosphate buffer solution (0.1 M) and mix well to obtain MSA-CAS medium.

[0056] MSA medium (1.0L): Peptone 5.0g, Glucose 4.0g, KCl 0.5g, MgSO4·7H2O 0.5g, pH 7.0. CAS staining solution (100mL): Cetyltrimethylammonium bromide 0.15g, FeCl3 0.003g, Chromium azurite 0.0653g, add ddH2O to bring the volume to 100mL. Phosphate buffer solution (0.1M): NaH2PO3 0.5g, Na2HPO3 2.0g, NH4Cl 0.25g, KH2PO4 0.075g, NaCl 0.2g, sterile water 100mL. It should be noted that the MSA medium, CAS staining solution, and phosphate buffer solution should be dispensed separately into Erlenmeyer flasks and sterilized at 121℃ for 20min before use.

[0057] Control group: NDG19 strain was inoculated into LB liquid medium and cultured at 30°C with shaking at 150 rpm for 72 h to obtain bacterial suspension. The bacterial suspension was centrifuged and then diluted with sterile water to prepare OD (diethyltoluene) concentration. 600 =1 bacterial suspension, 1.0 mL of bacterial suspension was transferred to a glass test tube containing 5.0 mL of MSA-CAS liquid culture medium, and cultured in a shaker at 130 rpm and 37 ℃ for 72 h.

[0058] Treatment group: NDG19 strain was inoculated into LB liquid medium and cultured at 30°C with shaking at 150 rpm for 72 h to obtain bacterial suspension. The bacterial suspension was centrifuged and then diluted with sterile water to prepare OD (diethyltoluene) concentration. 600=1 bacterial suspension, 1.0 mL of bacterial suspension was transferred to a glass test tube containing 5.0 mL of MSA-CAS liquid culture medium, and cultured in a shaker at 130 rpm and 37 ℃ for 72 h.

[0059] Observe the color changes in the control and treatment groups. If the culture medium changes from blue to pink, purplish-red, or orange-yellow, the strain can produce siderophores; the darker the color, the higher the siderophore content. Figure 10 As shown, the color of the treatment group was more orange-yellow than that of the control group, indicating that the siderophore content was higher and that NDG19 could secrete siderophores.

[0060] Example 3: Physiological and biochemical characteristics and taxonomic identification of the strain

[0061] The strain NDG19 isolated in Example 1 has the following morphological and physiological and biochemical characteristics:

[0062] a. Cell morphology: Colonies are pale yellow, round, with neat, smooth, and moist edges, exhibiting a certain degree of adhesiveness; they are obligate aerobic, rod-shaped, and do not produce spores or endospores; they are Gram-negative. The growth temperature range is 20-45℃, and the growth pH is 6.0-10.0.

[0063] b. Physiological and biochemical characteristics: D-glucose+, sucrose-, amylase-, gelatin liquefaction+, methyl red test+, VP test-, indoleacetic acid-, citric acid+, oxidase+, catalase+, nitrate reduction-, urease+, ammonia production+.

[0064] c. Biological characteristics: It has functions such as iron production carrier, phosphorus solubilization and potassium solubilization.

[0065] When this strain NDG19 is applied to bio-fertilizer, it can effectively dissolve insoluble phosphorus and potassium in the rhizosphere soil of plants, significantly increase the content of available phosphorus and potassium in the soil, improve the utilization rate of phosphorus and potassium in the soil by plants, thereby promoting plant growth, reducing the amount of chemical fertilizer used, and increasing crop yield.

[0066] d. Determination of molecular taxonomic status:

[0067] DNA was extracted from strain NDG19 isolated in Example 1, the 16S rRNA gene was amplified, and the results were detected using agarose gel electrophoresis. The PCR amplification products were sent to a testing company for sequencing to obtain the strain's 16S rRNA sequence, which is as follows:

[0068] GGTGGGGCGATGCTTACCATGCAGTCGAACGGCAGCACGGGTGCTTGCACCTGGTGGCGAGTGGCGAACGGGTGAGTAATACATCGGAACATGTCCTGTAGTGGGGGATAGCCCGGCGAAAGCCGGATTAATACCGCATACGATCTACGGATGAAAGCGGGGGACCTTCGGGCCTCGCGCTATAGGGTTGGCCGATGGCTGATTAGCTAGTTGGTGGGGTAAAGGCCCACCAAGGCGACGATCAGTAGCTGGTCTGAGAGGACGACCAGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATTTTGGACAATGGGCGAAAGCCTGATCCAGCAATGCCGCGTGTGTGAAGAAGGCCTTCGGGTTGTAAAGCACTTTTGTCCGGAAAGAAATCCTGAGGGCTAATATCCTTCGGGGATGACGGTACCGGAAGAATAAGCACCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCGAGCGTTAATCGGAATTACTGGGCGTAAAGCGTGCGCAGGCGGTTTGTTAAGACCGATGTGAAATCCCCGGGCTCAACCTGGGAACTGCATTGGTGACTGGCAAGCTAGAGTATGGCAGAGGGGGGTAGAATTCCACGTGTAGCAGTGAAATGCGTAGAGATGTGGAGGAATACCGATGGCGAAGGCAGCCCCCTGGGCCAATACTGACGCTCATGCACGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCCTAAACGATGTCAACTAGTTGTTGGGGATTCATTTCCTTAGTAACGTAGCTAACGCGTGAAGTTGACCGCCTGGGGAGTACGGTCGCAAGATTAAAACTC

[0069] The 16S rRNA sequence was entered into GenBank for BLAST alignment, and it was found that the strain NDG19 of this invention has 99.49% similarity to the type strain of Burkholderia gladioli, NBRC 13700. Therefore, the strain NDG19 of this invention is classified as Burkholderia gladioli.

[0070] e. Biological Preservation Information

[0071] The *Burkholderia gladioli* strain provided by this invention was deposited on July 31, 2023, at the Guangdong Provincial Center for Microbial Culture Collection, with accession number 63701, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0072] Example 4: Antagonistic Experiment of Strains Against Plant Wilt Pathogen

[0073] Fusarium oxysporum is a globally distributed soil-borne pathogenic fungus with a wide host range, causing wilt diseases in more than 100 plant species, including cucurbits, solanaceous plants, bananas, cotton, legumes, and flowers. The causative agent of plant wilt used in this experiment is Fusarium oxysporum race 4 (Cuba Physiological).

[0074] The culture medium used for pathogen culture and antagonistic experiments is:

[0075] PDA liquid culture medium (1.0L): Wash and peel the potatoes, weigh 200g of potatoes and cut them into small pieces. Add water and boil for 20-30 minutes until the potatoes are cooked through. Filter with gauze and add 20g of glucose. Add water to make up to 1000mL, stir well, heat thoroughly to dissolve, and then dispense into Erlenmeyer flasks. Sterilize at 121℃ for 20 minutes before use.

[0076] PDA solid medium (1.0L): Wash and peel the potatoes, weigh 200g of potatoes and cut them into small pieces. Add water and boil for 20-30 minutes until the potatoes are cooked through. Filter with gauze and add 20g of glucose and 15-20g of agar. Add water to a final volume of 1000mL, stir well, and sterilize at 121℃ for 20 minutes before use.

[0077] LB solid medium (1.0L): 10g peptone, 5g yeast extract, 10g sodium chloride (NaCl), 20g agar powder.

[0078] The antagonistic effect of the strains was observed using the plate confrontation method. Specifically, Fusarium oxysporum was inoculated into PDA liquid medium and cultured at 30°C and 150 r / min for 24 h to obtain the pathogenic bacterial suspension.

[0079] The strain NDG19 isolated in Example 1 was streaked into LB solid medium and incubated upside down at 37°C for 24 hours to obtain a single colony of NDG19.

[0080] Control group: 200 μL of pathogenic bacterial suspension was spread on PDA solid medium. After it grew to full size, a circular pathogenic bacterial cake was taken out by punching a hole with a sterilized Oxford punching ring and placed in the center of the blank PDA solid medium.

[0081] Treatment group: 200 μL of pathogenic bacterial suspension was spread on PDA solid medium. After it was fully grown, a circular pathogenic bacterial disc was punched with a sterilized Oxford punching ring and placed in the center of a blank PDA solid medium. Then, a single NDG19 colony was picked up with a sterilized bamboo stick and spotted 2 cm away from the center of the pathogenic bacterial disc. Three replicates were set up.

[0082] The petri dishes of the control group and the treatment group were incubated in a 30℃ incubator for one week, and the results were as follows: Figure 4-5 As shown: From Figure 4 As can be seen, without the addition of NDG19 single colonies, the pathogens grew well, almost completely covering the entire PDA solid medium. Figure 5 As can be seen, when NDG19 single colonies were added 2 cm away from the pathogen, obvious transparent halos appeared around the NDG19 single colonies, indicating that NDG19 has an antagonistic effect on the pathogen (i.e., Fusarium oxysporum). This further shows that NDG19 single colonies have a significant inhibitory effect on the growth of the pathogen (i.e., Fusarium oxysporum), thus effectively preventing and controlling more than 100 kinds of plant wilt diseases such as banana wilt.

[0083] Example 5: Antagonistic Experiment of Strains Against Plant Wilt Pathogen

[0084] Fusarium wilt of banana is a soil-borne disease caused by a fungus that infects the vascular bundles of banana plants. It is a devastating disease and is subject to international plant quarantine. The pathogen used in this experiment is Fusarium oxysporum f.sp.cubense.

[0085] The culture medium used for pathogen culture and antagonistic experiments is:

[0086] PDA liquid culture medium (1.0L): Wash and peel the potatoes, weigh 200g of potatoes and cut them into small pieces. Add water and boil for 20-30 minutes until the potatoes are cooked through. Filter with gauze and add 20g of glucose. Add water to make up to 1000mL, stir well, heat thoroughly to dissolve, and then dispense into Erlenmeyer flasks. Sterilize at 121℃ for 20 minutes before use.

[0087] PDA solid medium (1.0L): Wash and peel the potatoes, weigh 200g of potatoes and cut them into small pieces. Add water and boil for 20-30 minutes until the potatoes are cooked through. Filter with gauze and add 20g of glucose and 15-20g of agar. Add water to a final volume of 1000mL, stir well, and sterilize at 121℃ for 20 minutes before use.

[0088] LB solid medium (1.0L): 10g peptone, 5g yeast extract, 10g sodium chloride (NaCl), 20g agar powder.

[0089] The antagonistic effect of the strains was observed using the plate confrontation method. Specifically, Fusarium oxysporum was inoculated into PDA liquid medium and cultured at 30°C and 150 r / min for 24 h to obtain the pathogenic bacterial suspension.

[0090] The strain NDG19 isolated in Example 1 was streaked into LB solid medium and incubated upside down at 37°C for 24 hours to obtain a single colony of NDG19.

[0091] Control group: 200 μL of pathogenic bacterial suspension was spread on PDA solid medium. After it grew to full size, a circular pathogenic bacterial cake was taken out by punching a hole with a sterilized Oxford punching ring and placed in the center of the blank PDA solid medium.

[0092] Treatment group: 200 μL of pathogenic bacterial suspension was spread on PDA solid medium. After it was fully grown, a circular pathogenic bacterial disc was punched with a sterilized Oxford punching ring and placed in the center of a blank PDA solid medium. Then, a single NDG19 colony was picked up with a sterilized bamboo stick and spotted 2 cm away from the center of the pathogenic bacterial disc. Three replicates were set up.

[0093] The petri dishes of the control group and the treatment group were incubated in a 30℃ incubator for 2 weeks, and the results were as follows: Figure 6-7 As shown: From Figure 6 As can be seen, without the addition of NDG19 single colonies, the pathogens grew well, almost completely covering the entire PDA solid medium. Figure 7 As can be seen, when NDG19 single colonies were added 2 cm away from the pathogen, obvious transparent halos appeared around the NDG19 single colonies, indicating that NDG19 has an antagonistic effect on the pathogen (i.e., Fusarium oxysporum). This further shows that NDG19 single colonies have a significant inhibitory effect on the growth of the pathogen (i.e., Fusarium oxysporum), thus effectively preventing and controlling banana wilt disease.

[0094] Example 6: Antagonistic Experiment of Strains Against Plant Root Rot Pathogens

[0095] Root rot is one of the most serious diseases affecting cucurbitaceous crops, commonly occurring in watermelons, melons, cucumbers, and other crops. It is another devastating soil-borne disease following cucurbit wilt. Panax notoginseng, belonging to the Araliaceae family and the Panax genus, is a precious and unique traditional Chinese medicine. Root rot in Panax notoginseng is also a significant factor hindering its industrial development. The pathogen used in this experiment is *Alternaria tenuissima*.

[0096] The culture medium used for pathogen culture and antagonistic experiments is:

[0097] PDA liquid culture medium (1.0L): Wash and peel the potatoes, weigh 200g of potatoes and cut them into small pieces. Add water and boil for 20-30 minutes until the potatoes are cooked through. Filter with gauze and add 20g of glucose. Add water to make up to 1000mL, stir well, heat thoroughly to dissolve, and then dispense into Erlenmeyer flasks. Sterilize at 121℃ for 20 minutes before use.

[0098] PDA solid medium (1.0L): Wash and peel the potatoes, weigh 200g of potatoes and cut them into small pieces. Add water and boil for 20-30 minutes until the potatoes are cooked through. Filter with gauze and add 20g of glucose and 15-20g of agar. Add water to a final volume of 1000mL, stir well, and sterilize at 121℃ for 20 minutes before use.

[0099] LB solid medium (1.0L): 10g peptone, 5g yeast extract, 10g sodium chloride (NaCl), 20g agar powder.

[0100] The antagonistic effect of the strains was observed using the plate confrontation method. Specifically, Alternaria spp. was inoculated into PDA liquid medium and cultured at 30°C with shaking at 150 r / min for 24 h to obtain the pathogenic bacterial suspension.

[0101] The strain NDG19 isolated in Example 1 was streaked into LB solid medium and incubated upside down at 37°C for 24 hours to obtain a single colony of NDG19.

[0102] Control group: 200 μL of pathogenic bacterial suspension was spread on PDA solid medium. After it grew to full size, a circular pathogenic bacterial cake was taken out by punching a hole with a sterilized Oxford punching ring and placed in the center of the blank PDA solid medium.

[0103] Treatment group: 200 μL of pathogenic bacterial suspension was spread on PDA solid medium. After it was fully grown, a circular pathogenic bacterial disc was punched with a sterilized Oxford punching ring and placed in the center of a blank PDA solid medium. Then, a single NDG19 colony was picked up with a sterilized bamboo stick and spotted 2 cm away from the center of the pathogenic bacterial disc. Three replicates were set up.

[0104] The petri dishes of the control group and the treatment group were incubated in a 30℃ incubator for one week, and the results were as follows: Figure 8-9 As shown: From Figure 8 As can be seen, without the addition of NDG19 single colonies, the pathogens grew well, almost completely covering the entire PDA solid medium. Figure 9 The results show that when NDG19 single colonies were added 2 cm away from the pathogen, a clear halo appeared around each NDG19 single colony, indicating that NDG19 single colonies have an antagonistic effect on the pathogen (i.e., Alternaria spp.). This further indicates that NDG19 single colonies have a significant inhibitory effect on the growth of the pathogen (i.e., Alternaria spp.), thus effectively preventing and controlling root rot of Panax notoginseng and other cucurbit crops. This provides strain resources for the biological control of root rot of Panax notoginseng and other cucurbit crops, as well as for effectively managing and solving continuous cropping obstacles in production.

[0105] This invention isolated Burkholderia gladioli NDG19 from the roots of Reed ferns and identified it morphologically, physiologically, biochemically, and genetically. 16S rRNA identification confirmed its identity as Burkholderia gladioli. Antagonistic experiments against pathogens such as Fusarium oxysporum and Alternaria alternata showed that this strain effectively inhibited the growth and reproduction of these pathogens, thus effectively controlling plant wilt diseases such as banana wilt and root rot in cucurbitaceous crops. This solves the environmental pollution and food safety problems caused by the use of chemical fungicides. Furthermore, this strain has functions such as iron production, phosphorus solubilization, and potassium solubilization. As a bio-fertilizer, it can effectively improve the utilization rate of phosphorus and potassium in the soil by plants, promote plant growth, reduce the amount of chemical fertilizer used, increase crop yield, and is safe and environmentally friendly, with broad application prospects.

[0106] The solution of this application has been described in detail above with reference to the accompanying drawings. In the above embodiments, the descriptions of each embodiment have different emphases; parts not described in detail in a certain embodiment can be referred to in the relevant descriptions of other embodiments. Those skilled in the art should also understand that the actions and modules involved in the specification are not necessarily essential to this application. Furthermore, it is understood that the steps in the method of this application embodiment can be adjusted, combined, and deleted according to actual needs, and the modules in the device of this application embodiment can be combined, divided, and deleted according to actual needs.

[0107] The various embodiments of this application have been described above. These descriptions are exemplary and not exhaustive, nor are they limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terminology used herein is chosen to best explain the principles, practical application, or improvement of the technology in the market, or to enable others skilled in the art to understand the embodiments disclosed herein.

Claims

1. A species of Burkholderia gladioli ( Burkholderia gladioli NDG19, characterized in that: It was deposited on July 31, 2023 at the Guangdong Provincial Center for Microbial Culture Collection, with the strain accession number GDMCC NO: 63701, and the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

2. The Gladiolus Burkholderia as described in claim 1 ( Burkholderia gladioli Application of NDG19 in inhibiting Fusarium oxysporum.

3. The Gladiolus Burkholderia as described in claim 1 ( Burkholderia gladioli Application of NDG19 in inhibiting Alternaria spp.

4. The Gladiolus Burkholderia as described in claim 1 ( Burkholderia gladioli Application of NDG19 in the control of plant wilt disease.

5. The Gladiolus Burkholderia as described in claim 1 ( Burkholderia gladioli Application of NDG19 in the control of banana wilt disease.

6. The Gladiolus Burkholderia as described in claim 1 ( Burkholderia gladioli Application of NDG19 in the prevention and control of plant root rot.

7. The Gladiolus Burkholderia as described in claim 1 ( Burkholderia gladioli Application of NDG19's iron-carrier, phosphorus-solubilizing, and potassium-solubilizing functions in promoting plant growth.

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