Burkholderia parakholderia D15 and application thereof

Abstract

The invention belongs to the technical field of biological prevention and control, and particularly relates to a Burkholderia paragallinarum D15 and application of the Burkholderia paragallinarum D15. The Burkholderia parakholderia sp. D15 is preserved in China General Microbiological Culture Collection Center (CGMCC) on November 25, 2024, the address is No.3, No.1 yard, Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC NO. 1.61962. The Burkholderia parakholderia sp. D15 has the advantages that the Burkholderia parakholderia sp. According to the invention, a multifunctional strain Burkholderia paragallinarum D15 is separated from the rock wall of Danxia red rock, and the Burkholderia paragallinarum D15 has a relatively good antagonistic effect on most plant pathogens and has a relatively strong bacteriostatic effect on xanthomonas oryzae Xoo causing rice bacterial blight; the compound also has a relatively strong bacteriostatic effect on other pathogenic bacteria causing crop diseases, such as rice sheath blight disease, rice blast, fusarium graminearum, sclerotium rolfsii and ralstonia solanacearum, and lays a foundation for prevention and treatment of rice bacterial blight and development of related biological preparations.

Description

Technical Field

[0001] The invention belongs to the field of biological control technology, and specifically relates to a strain of Burkholderia parakreuteria D15 and application thereof. Background Art

[0002] Xanthomonas is a plant-associated gram-negative bacterium belonging to the family Xanthomonadaceae. γ -Proteobacteria, most strains in this genus are pathogenic to plants. And Xanthomonas genus infects a wide range of plants, being pathogenic to about 400 species of plants, including economic crops such as rice, sugarcane, beans, cassava, cabbage, banana, citrus and tomatoes. Xanthomonas oryzae pv. oryzae , Xoo ) is a pathogen that grows in the xylem of plants and causes yellow or white lesions on the veins of leaves. It is the pathogen that causes rice bacterial blight. Bacterial blight is one of the main diseases of rice in Asian countries. High-yielding rice varieties in these countries are often susceptible to the disease, resulting in crop failures. The yield of severely affected rice can drop by 50%-60%. Studies have found that the pathogenicity of Xanthomonas is associated with several virulence factors, such as allergic reactions and pathogenicity ( hrp ) genes, products dependent on type III and type IV secretion systems, extracellular polysaccharides, diffusible signaling factors and extracellular enzymes, etc.

[0003] Although the pathogenicity of Xanthomonas has been well studied, the genus remains the culprit for many economic crop diseases, resulting in annual yield losses of important economic crops worldwide. Current methods for controlling Xanthomonas-related diseases include physical control, such as debudding, uprooting, burying and burning infected plant tissues, and disinfecting gardening tools. Chemical fungicides include the use of copper-based fungicides or antibiotics such as streptomycin. However, physical control is costly, and chemical fungicides and antibiotics not only cause bacterial resistance problems, but also have a certain impact on the ecological environment.

[0004] In this context, biological control is a promising alternative that offers advantages over chemical pesticides in terms of sustainability, mode of action, and toxicity. Microbial-based products are used in plantations and greenhouses to reduce diseases caused by foliar or post-harvest pathogens in a variety of cereals, vegetables, fruits, flowers, and ornamental plants. Microbial-based products decompose faster in the environment and are generally less toxic to non-target species. In addition, due to their different mode of action from traditional fungicides, they may help suppress resistant pathogens and promote plant growth and yield improvement during the crop growth stage. Summary of the invention

[0005] The purpose of the present invention is to solve the deficiencies of the prior art and provide a strain of Burkholderia parabacterium D15 and its application, which specifically adopts the following technical solutions: In a first aspect of the present invention, a strain of Burkholderia parakholderia D15 is provided, wherein the Burkholderia parakholderia ( Paraburkholderia sp. ) D15 was deposited in the General Microbiological Center of China Microbiological Culture Collection Committee on November 25, 2024, with the address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC NO.1.61962. The gene sequence of 16S rDNA of the paraburkholderia D15 is shown in SEQ ID No: 1.

[0006] SEQ ID No: 1:

[0007] The second aspect of the present invention provides the use of the above-mentioned Burkholderia parakholderia D15 in preventing and controlling bacterial diseases and / or fungal diseases of crops.

[0008] As a further preferred embodiment, the bacterial diseases include bacterial diseases caused by Xanthomonas oryzae, Psoralea corylifolia, citrus canker or sugarcane white streak disease. Xoo .

[0009] As a further preferred embodiment, the fungal diseases include fungal diseases caused by rice sheath blight, rice blast, Fusarium graminearum, Fusarium oxysporum or Sclerotium uniformis.

[0010] The third aspect of the present invention provides a biological agent for preventing and controlling bacterial diseases and / or fungal diseases of crops, wherein the biological agent comprises the above-mentioned Burkholderia parakholderia D15.

[0011] As a further preferred embodiment, the biological preparation is prepared from the bacterial liquid and / or fermentation liquid of Burkholderia parakholderia D15.

[0012] The fourth aspect of the present invention also provides a method for preventing and controlling bacterial diseases and / or fungal diseases of crops, wherein the above-mentioned biological agent is used to treat plant materials.

[0013] As a further preferred embodiment, the above treatment is spraying treatment, and the plant material is rice.

[0014] The beneficial effects of the present invention are: The present invention isolated a multifunctional strain of Burkholderia parasitica D15 from the Danxia red rock wall, which has a good antagonistic effect on most plant pathogens and is effective against Xanthomonas oryzae that causes rice bacterial blight. Xoo It has a strong antibacterial effect, and it also has a strong antibacterial effect on other pathogens that cause crop diseases, such as rice sheath blight, rice blast, Fusarium graminearum, Sclerotium uniformis, and bacterial wilt, laying the foundation for the prevention and control of rice bacterial leaf blight and the development of related biological agents. The strain grows fast, and the antibacterial substance reaches the best effect within 24 hours, greatly shortening the fermentation time. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Shown is a growth curve graph of Burkholderia parabacterium D15; Figure 2Shown is the phylogenetic tree constructed from the 16S rDNA sequences of Burkholderia paraburkholderia D15 and other strains of the same genus. Node values ​​represent bootstrap values, and values ​​below 50% are not shown, with Burkholderia stagnalis LMG28156 as the outgroup; Figure 3 Shown is the phylogenetic tree constructed from the genome sequences of Paraburkholderia D15 and other strains of the same genus; Figure 4 Shown are the effects of Burkholderia parabacterium D15 and some antagonistic strains on Xcc and Xoo Antibacterial effect; Figure 5 Shown is the effect of different culture media on the antibacterial activity of D15 sterile metabolic fluid; Figure 6 Shown is the effect of different fermentation times on the antibacterial activity of D15 sterile metabolic fluid; Figure 7 Shown is the effect of different stress conditions on the stability of D15 sterile metabolic fluid; Figure 8 The figure shows the control effect of Burkholderia parakholderiae D15 and its sterile metabolite on rice bacterial blight; Fig. 9 Shown is the antibacterial effect of Burkholderia parasitica D15 on various plant pathogens. DETAILED DESCRIPTION

[0016] The following will be combined with the embodiments and drawings to clearly and completely describe the concept, specific structure and technical effects of the present invention, so as to fully understand the purpose, scheme and effect of the present invention. It should be noted that the embodiments and features in the embodiments of this application can be combined with each other without conflict.

[0017] Example 1 Isolation and Screening of Burkholderia parabacterium D15 1. Isolation of Burkholderia parasitica D15 Soil was collected from a Danxia red rock wall in Xinzhou District, Shangrao City, and sterile saline was used to -1 ~10 -7 Gradient dilution, then spread on NYG medium, culture at 28℃ for 24h, pick single colonies with an inoculation loop for streaking separation according to the different characteristics of colony color, morphology, size, edge neatness and transparency, until pure culture is obtained, a total of 12 strains were isolated, numbered D1, D3, D4, D5, D6, D9, D10, D11, D12, D15, D19, D21. Pick single colonies for liquid culture, add glycerol at a final concentration of 15%, and store at -80℃ for later use.

[0018] 2. Screening of Burkholderia parasitica D15 This experiment uses the filter paper inhibition zone method. Xoo In NA liquid medium, shake at 28°C, 200 rpm for 24 hours. Collect the cells by centrifugation at 5000 rpm for 5 minutes, then resuspend the cells in sterile NA liquid medium and adjust the OD 600 =0.2, as seed solution. Add the seed solution to the NA medium cooled to 45-50 ℃ at a 1% inoculation rate, mix well and pour into a plate. Place a 6 mm diameter sterile filter paper in a culture dish, then drop 5 μL of the test bacterial solution on the filter paper, and observe and measure the size of the inhibition zone after culturing at 28 ℃ for 24 h. Rescreen the strains with inhibition zones again, and select the strains with larger and more stable inhibition zones for subsequent experiments.

[0019] 3. Cultivation and growth curve of Burkholderia parabacterium D15 Use an inoculation loop to scrape some bacteria from the bacteria stored at -80 ℃, and activate them by streaking in NYG solid culture medium. Place it in a constant temperature incubator at 28 ℃ and culture it upside down for 2-3 days. Strain D15 forms bright yellow colonies in NYG culture medium. The colonies are round, with regular edges, smooth surfaces, and raised colonies. Use an inoculation loop to pick a single colony and inoculate it into NYG liquid culture medium. Shake it at 28 ℃ and 200 rpm for 24 hours. Transfer the bacterial suspension to new NYG liquid culture medium at a 1% inoculation rate, and culture it at 28 ℃ and 200 rpm with shaking. Measure the OD600 value of the bacterial solution every 2-3 hours, and draw a growth curve with time as the horizontal axis and OD600 value as the vertical axis. The results are shown below. Figure 1 shown.

[0020] 4. Identification of Burkholderia parasitica D15 (hereinafter referred to as D15) (1) Morphological and physiological and biochemical identification D15 was isolated from the Danxia Red Rock Wall in Shangrao and is a short rod-shaped, Gram-negative bacterium. Based on molecular biology, physiological and biochemical characteristics, D15 was classified as Paraburkholderia , and named Paraburkholderia sp. D15 ( Figure 2 ).

[0021] The results of physiological and biochemical studies showed that D15 formed bright yellow colonies in R2A medium and could grow and reproduce in a culture environment with a pH of 3-11, with the most suitable pH being 7. The results of the growth temperature range showed that D15 could grow at 4-37°C, with the most suitable growth temperature being 28°C. This low temperature adaptability may be because strain D15 was isolated from an exposed rock wall, where the temperature was lower in winter. At the same time, D15 had a tolerance of 0%-4% to NaCl, with the most suitable NaCl concentration being 1%. Enzyme activity assays showed that D15 had enzyme activity characteristics such as oxidase, catalase and β-galactosidase, as well as characteristics such as starch and fat degradation (degradation of Tween20 / 80).

[0022] (2) Molecular identification In order to clarify the taxonomic status of Burkholderia parakholderia D15, 16S rDNA sequence was used for phylogenetic analysis and combined with physiological and biochemical assays to identify the Burkholderia parakholderia D15 strain.

[0023] Phylogenetic tree analysis of 16S rRNA sequences showed that D15 was not clustered in the same branch as other strains in the genus, but in a separate branch (Figure 2). Further phylogenetic tree construction using the strain's whole genome revealed that the strain was clustered in the same branch as Paraburkholderia bryophila LMG23644 ( Figure 3 ). By comparing the average nucleotide identity (ANI) and DNA hybridization homology analysis (dDDH) of D15 and P. bryophila LMG23644, it was found that the ANI and dDDH between the two were 85.97% and 31.5%, respectively, which were lower than the strain identification threshold (ANI lower than 94%, dDDH lower than 70%), indicating that strain D15 is a new species.

[0024] Example 2 To explore the inhibitory effect of strain D15 and other antagonistic strains on the pathogens of rice bacterial blight and cabbage black rot Xanthomonas is a genus that infects a wide range of plants and is pathogenic to approximately 400 plant species, including economic crops such as rice, sugarcane, beans, cassava, cabbage, bananas, citrus, and tomatoes. Rice bacterial blight is caused by Xanthomonas oryzae pv. Xanthomonas oryzae pv. oryzae , Xoo), white leaf blight is one of the major rice diseases in Asian countries. High-yielding rice varieties in these countries are often susceptible to the disease, resulting in crop failures. Severely affected rice production can reduce yields by 50%-60%. Cabbage black rot is caused by Xanthomonas campestris ( Xanthomonas campestris pv.campestris. Xcc). Xoo and Xcc The antibacterial effect Xoo PXO99A and Xcc Activate in NA or NYG solid medium, then pick a single colony and inoculate it into NA or NYG liquid medium at 28°C, 200 rpm, and shake for 24 hours. Centrifuge at 5000 rpm for 5 minutes to collect the bacteria, then resuspend the bacteria in sterile NA or NYG liquid medium and adjust the OD 600 =0.2, as seed solution. Add the seed solution to NA or NYG medium cooled to 45-50℃ at a 1% inoculum, mix well and pour into a plate. Add 5μL OD 600 The D15 suspension with a value of 1 was used as the control group, and the same amount of sterile NYG liquid culture medium was used instead. The culture dishes were then sealed and placed in a 28°C constant temperature incubator for 2-3 days to observe the effect of D15 on the Xoo and Xcc The results are as follows Figure 4 As shown, it is Figure 4 It can be seen that D15 can effectively inhibit Xoo Compared with ZR2 (Pseudomonas cohnii), ZR62 (Pyrroholdenia pyrrolizumab), ZR86 (Bacillus polymyxa), ZR93 (Bacillus orientalis) and ZR118 (Streptomyces yunnanensis), D15 had the best antibacterial effect and Xcc The antibacterial effect of D15 was also stronger than that of other strains, indicating that D15 has great development potential.

[0025] Example 3 This example explores the effects of different culture conditions on strain D15 (1) Investigate the effects of different culture media on the antibacterial activity of D15 sterile metabolic fluid Pick a single colony of strain D15 in NYG liquid medium, culture at 28 °C and 200 rpm for 24 h, centrifuge at 5000 rpm for 10 min, remove the supernatant, and then resuspend the cells in sterile water to adjust the OD 600=1. The bacterial suspension was inoculated into LB, NYG, NA, GYM and YDP liquid culture media at 1% inoculation volume and cultured at 28 °C and 200 r / min for 24 hours. Then, the supernatant was collected by centrifugation at 12000 r / min for 5 min and filtered with a 0.22 μm sterile microporous filter to obtain a sterile metabolic solution. Xoo NA medium was placed in a sterile Oxford cup, and 200 μL of sterile metabolic fluid of each medium was added to it. The culture was placed in a 28°C constant temperature incubator and cultured upright for 24-36 hours. The diameter of the inhibition zone was observed. The results showed that the sterile metabolic fluid of D15 cultured in GYM medium had the best antibacterial effect ( Figure 5 ), the antibacterial effect was significantly higher than that of NYG and YPD medium. Therefore, GYM medium was used as the fermentation medium in subsequent experiments.

[0026] (2) Investigate the effect of different culture times on the antibacterial activity of D15 metabolites The production of bacterial metabolites is related to the growth cycle, and the types and yields of metabolites vary greatly at different culture stages. Pick a single colony of strain D15 and place it in GYM liquid medium, culture it at 28 °C and 200 r / min for 24 hours, centrifuge it at 5000 rpm for 10 minutes, remove the supernatant, and then resuspend the bacteria in sterile water to adjust the OD 600 =1. The bacterial suspension was inoculated into GYM liquid medium at 1% inoculation volume and cultured at 28 ℃ and 200 r / min. The bacterial suspension was collected at 24, 36, 48 and 60 hours, respectively, and the bacterial suspension was centrifuged at 12000 r / min for 5 min. The supernatant was collected and sterilized by filtering with a 0.22 μm sterile microporous filter membrane to obtain a sterile metabolic solution, which was stored at -20 ℃ for later use. Prepare the solution containing Xoo NA medium was added, and a sterile Oxford cup was placed in the medium. 200 μL of sterile metabolic solution of different time was added to it. The culture was placed in a 28 ℃ constant temperature incubator and cultured for 24-60 hours. The diameter of the inhibition zone was observed. Xoo The antibacterial effects are almost the same, with no difference ( Figure 6 ). This indicates that strain D15 can produce antibacterial active substances in a short time, which is of great significance for the production of such substances. In order to save costs, fermentation can be selected for 24 hours, and metabolites can be collected for extraction and preparation of antibacterial active substances.

[0027] (3) Investigate the effects of different stress conditions on the stability of D15 metabolites The stability of the substance is the basis for ensuring the shelf life and activity of the product during use. The external environment may contain adverse factors such as strong acid, strong alkali, ultraviolet light, high temperature and protease. In order to further determine the stability of D15 antibacterial active substances, the sterile metabolic fluid was collected and cultured for 24 hours according to the previous method, and the sterile metabolic fluid was divided into 2 ml centrifuge tubes, 1 ml per tube, and the pH of the sterile metabolic fluid was adjusted to 3-12 with 6 mol / L hydrochloric acid and 10 mol / L sodium hydroxide, respectively. After standing at room temperature for 24 hours, the pH was adjusted to the original pH value with hydrochloric acid and sodium hydroxide. Ultraviolet treatment: The sterile metabolic fluid was placed under an ultraviolet lamp for 30, 60 and 120 minutes respectively. High temperature treatment: The centrifuge tubes containing the sterile metabolic fluid were placed in a water bath at 60, 80 and 100 °C for high temperature treatment for 1 hour, and 121 °C was treated in a high pressure steam sterilizer at 121 °C for 1 hour, and then cooled at room temperature. Protease treatment: Add 20 mg / L proteinase K to the sterile metabolic solution to make the final concentration 200 μg / L, incubate at 37°C for 1 hour, and then cool to room temperature. Prepare the culture medium containing Xoo according to the previous experimental method, add 200 μL of the sterile metabolic solution treated under each stress into the Oxford cup, place it in a 28°C constant temperature incubator and culture it upright for 24-36 hours, and observe the diameter of the inhibition zone.

[0028] The results are as follows Figure 7 shown by Figure 7 It can be seen that the metabolites of strain D15 have strong stability, and strong acid, strong base, proteinase K and ultraviolet light do not affect the antibacterial activity. High temperatures of 60, 80 and 100°C have no effect on the stability of the sterile metabolic solution. The antibacterial activity only decreases at 121°C, but still retains more than 80% of the antibacterial activity. This shows that the antibacterial metabolites of D15 have strong stability, laying a foundation for the development of biological pesticides of D15.

[0029] Example 4 Study on the control effect of Burkholderia parasitica D15 on rice bacterial blight Twenty seeds of rice cultivar Yuanfengzao were sown in plastic pots (16 × 12 cm) and kept in a greenhouse for 14 10 h light (30 °C) / 10 h dark (28 °C) and 70% humidity. After 21 days of cultivation (3-6 leaf stage), rice seedlings were used for biological control experiments. Xoo PXO99A (OD 600 =0.6)、 D15 (OD 600 =0.5) bacterial suspension and D15 sterile metabolic solution. Sterile NYG medium was used as control. A biological control test was conducted on rice seedlings at the three-leaf stage, and inoculation was performed by syringe injection. The experiment was divided into 5 treatment groups, treatment group (1): inoculated with a sterile syringe Xoo Inject sterile NYG medium (CK) 3 hours later; Treatment group (2): inoculated with a sterile syringe Xoo 3 hours later, inoculate with D15 bacterial suspension (X-DJ); treatment group (3): inoculate with a sterile syringe Xoo Inoculate with D15 sterile metabolic liquid (X-DL) 3 hours later; Treatment group (4): Inoculate with D15 bacterial suspension using a sterile syringe 3 hours later Xoo Bacterial suspension (DJ-X); Treatment group (5): sterile syringe inoculated with D15 sterile metabolic fluid 3 hours later Xoo The bacterial suspension (DL-X) was cultured in the greenhouse under the same culture conditions, and the length of the disease was measured after 7 days. The above inoculation experiment was repeated three times independently.

[0030] The results are as follows Figure 8 shown by Figure 8 It can be seen that the four different treatments can significantly reduce the lesion length of rice bacterial leaf blight, and the incidence rate was reduced by 81%, 69%, 83% and 83% respectively compared with the control group; this shows that strain D15 and its metabolites can effectively prevent and control rice bacterial leaf blight, providing a theoretical basis for the development of biological pesticides.

[0031] Example 5 In order to better study the biocontrol potential of the strain Burkholderia parasitica D15 obtained in Example 1, the antibacterial spectrum of the bacterium was determined. The specific operation is as follows: Antagonistic effect on pathogenic bacteria: Pathogenic bacteria such as bacterial wilt, sugarcane white streak disease and citrus canker were activated in NYG medium, and then single colonies were picked and inoculated into each liquid culture medium and shaken to the logarithmic phase. The OD600 was adjusted to 0.2, and the bacterial suspension was added to the NYG medium cooled to about 45-50 ℃ at a 1% inoculation amount. After mixing, pour the plate, and after cooling and solidification, 5 μL of the prepared D15 bacterial suspension was added to the center of the medium. The same amount of GYM medium was added as a control and cultured at 28 ℃. Antagonistic effect on pathogenic fungi: 5 mm rice sheath blight, rice blast, Fusarium graminearum, Fusarium oxysporum and Sclerotium uniformis were inoculated into PDA medium, and 5 μL of the prepared D15 bacterial suspension was added 2 cm away from the bacterial cake. The culture was cultured at 28 ℃ to observe the antibacterial effect of D15 on different pathogens.

[0032] The results are as follows Fig. 9 shown by Fig. 9 It can be seen that D15 has a relatively good antibacterial effect on plant diseases caused by various bacteria and fungi, such as fungal diseases such as rice sheath blight, rice blast, Fusarium graminearum, Fusarium oxysporum, and Sclerotium uniformis, and bacterial diseases such as Ralstonia solanacearum, citrus canker, and sugarcane white streak disease. This shows that strain D15 is a broad-spectrum antagonistic bacterium with high development potential.

[0033] Although the description of the present invention has been quite detailed and specifically describes several described embodiments, it is not intended to be limited to any of these details or embodiments or any particular embodiment, but should be regarded as providing a broad possible interpretation of these claims in view of the prior art by reference to the appended claims, thereby effectively covering the intended scope of the present invention. In addition, the above description of the present invention is based on the embodiments foreseeable by the inventor, and its purpose is to provide a useful description, and those non-substantial changes to the present invention that have not yet been foreseen may still represent equivalent changes to the present invention.

Claims

1. A strain of Burkholderia parasitica D15, characterized in that The Burkholderia parasitica ( Paraburkholderia sp. )D15 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on November 25, 2024, with the address at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC NO.1.61962.

2. The Burkholderia parakholderiae D15 according to claim 1, characterized in that The gene sequence of 16S rDNA of the paraburkholderia D15 is shown in SEQ ID No:

1.

3. Use of the Burkholderia parakholderia D15 according to any one of claims 1 to 2 in preventing and controlling bacterial diseases and / or fungal diseases of crops.

4. The use according to claim 3, characterized in that: The bacterial diseases include bacterial diseases caused by rice bacterial blight, bacterial wilt, citrus canker or sugarcane white streak disease.

5. The use according to claim 4, characterized in that: The rice bacterial blight pathogen is Xanthomonas oryzae Xoo .

6. The use according to claim 3, characterized in that: The fungal diseases include fungal diseases caused by rice sheath blight, rice blast, Fusarium graminearum, Fusarium oxysporum or Sclerotium uniformis.

7. A biological agent for preventing and controlling bacterial diseases and / or fungal diseases of crops, characterized in that: The method comprises the Burkholderia parakholderia D15 according to any one of claims 1 to 2.

8. The biological agent according to claim 7, characterized in that The biological preparation is prepared from the bacterial liquid and / or fermentation liquid of Burkholderia parakholderia D15.

9. A method for preventing and controlling bacterial diseases and / or fungal diseases of crops, comprising treating plant materials with the biological agent according to any one of claims 7 to 8.

10. The method according to claim 9, characterized in that The treatment is a spraying treatment and the plant material is rice.

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