Burkholderia gladioli BG-GF5 as well as composition and application thereof

Abstract

The invention discloses burkholderia gladioli BG-GF5 as well as a composition and application thereof, the burkholderia gladioli BG-GF5 is preserved in the China Center for Type Culture Collection with the preservation number of CCTCC NO: M20242573, and the preservation date is November 25, 2024. The application of the BG-GF5 is mainly reflected in the inhibition effect of the BG-GF5 on pathogenic bacteria and the potential of the BG-GF5 in crop disease control. The strain or the fermentation product and the metabolite thereof can be prepared into a composition for inhibiting pathogenic bacteria and crop diseases caused by the pathogenic bacteria. Particularly, the application to crops such as bee sugar plums, chilies and kiwi fruits shows a wide application prospect in the field of agricultural biological control.

Description

Technical Field

[0001] The invention belongs to the technical field related to biological agriculture, and in particular relates to a new strain and a composition thereof, and uses thereof in biological control of plant diseases. Background Art

[0002] In the agricultural field, diseases caused by plant pathogenic fungi have always been an important factor affecting crop yield and quality. Although traditional chemical control methods are effective in the short term, long-term use will lead to environmental problems and increased resistance of pathogens. Therefore, the development of new, environmentally friendly and sustainable biological control methods is an important direction of current agricultural scientific research.

[0003] Biological control is a method of controlling pests and pathogens using biological resources, among which microbial control is valued for its environmental friendliness and sustainability. Among microbial control, bacteria, as an effective biological control agent, have been widely studied because of their ability to produce substances that inhibit or kill pathogenic fungi.

[0004] Burkholderia is a genus of Gram-negative bacteria that is widely found in soil and plant rhizosphere, and some species have been shown to have anti-plant pathogenic fungal activity. However, some species in the genus Burkholderia are also associated with human diseases, so it is of great significance to develop new safe and harmless Burkholderia strains for biological control.

[0005] The gladiolus Burkholderia BG-GF5 strain involved in the present invention is a new Burkholderia strain isolated from the root nodules of white clover. It has broad-spectrum antibacterial activity, can effectively inhibit the growth of various plant pathogenic fungi, and has been proven to be safe and harmless through safety evaluation, providing a new option for agricultural biological control. Summary of the invention

[0006] The present invention aims to provide Burkholderia gladiolus BG-GF5, its composition and use, especially

[0007] According to the first main aspect of the present invention, a new strain of Burkholderia gladioli BG-GF5 is provided, which is deposited in the China Center for Type Culture Collection (address: Wuhan University, Wuhan, China, postal code: 430072, telephone: 027-68754052) with the deposit number CCTCC NO: M20242573, the deposit date is: November 18, 2024, and the classification name is: Burkholderia gladioli BG-GF5.

[0008] The 16S rDNA sequence of the gladiolus Burkholderia BG-GF5 is shown in Seq ID No. 1. The 16S rDNA sequence is the gold standard for bacterial classification and identification because it is highly conserved in bacterial genomes and has sufficient variability between different species to distinguish different species.

[0009] Furthermore, the gladiolus Burkholderia BG-GF5 was isolated from the root nodules of white clover. The strain was isolated from the natural environment, has adapted to a specific ecological niche, and has biological activity that has not been fully studied, providing a potential resource for the development of new biological control agents. In addition, the natural source of the strain also means that it is more suitable and safer in the environment because it has coexisted with a specific plant species for a long time.

[0010] In some embodiments, as a further preferred embodiment, the culture method of gladiolus Burkholderia BG-GF5 comprises the following steps: using a culture medium combination for culture; the culture condition is culturing at 30° C. for 2 to 3 days; and obtaining a pure culture by gradient dilution method and spreading method.

[0011] Through standardized culture processes, the consistency and repeatability of strains can be guaranteed, which is crucial for subsequent experimental research and commercial production. In addition, gradient dilution and coating methods are classic methods for obtaining pure cultures, which can effectively isolate single colonies and lay the foundation for further identification and application.

[0012] Wherein, the culture medium combination comprises at least one of the following culture medium:

[0013] a. NA medium, which consists of 5 g / L beef extract, 10 g / L peptone, 5 g / L NaCl, 20 g / L agar powder, and 1000 mL sterile water;

[0014] b. PDA medium, which consists of 200 g potatoes, 20 g glucose, 17 g agar powder, and 1000 mL sterile water;

[0015] c. LB medium, which consists of 10 g of tryptone, 5 g of yeast extract, and 10 g of sodium chloride;

[0016] d.NB medium: peptone 10g, beef extract 3g, glucose 10g, yeast extract 1g.

[0017] By providing a variety of culture media options, the culture conditions can be flexibly adjusted according to the experimental purpose and strain characteristics to optimize the growth and activity expression of the strain, which is of great significance for improving the application effect and production efficiency of the strain.

[0018] In some embodiments, as a further preferred embodiment, the gladiolus Burkholderia BG-GF5 is amplified by PCR using a primer pair of 16S rRNA gene, wherein the primer pair includes a forward primer 16SF, the sequence of which is: 5′-AGA GTT TGA TCC TGG CTC AG-3′;

[0019] The primer pair includes a reverse primer 16SR, the sequence of which is: 5′-AAG GAG GTG ATC CAG CCGCA-3′.

[0020] By using a specific primer pair, the 16S rRNA gene of BG-GF5 can be accurately amplified, providing accurate molecular evidence for the identification and classification of the strain. The high specificity and sensitivity of this method allows even trace amounts of DNA samples to be successfully amplified and identified, which is very useful for identifying specific strains from complex environmental samples. In addition, the method has a high degree of standardization and automation, which allows a large number of samples to be processed quickly and accurately, improving work efficiency.

[0021] According to the second main aspect of the present invention, a composition is provided, which comprises the aforementioned gladiolus Burkholderia BG-GF5 or its fermentation product or metabolite. Based on the ability of microorganisms to produce biologically active substances, that is, BG-GF5 can produce fermentation products or metabolites with biological activity, these substances can be used to prevent and control crop diseases. Therefore, this composition can directly utilize the biological activity of BG-GF5 without the need to directly use live bacteria, which may be more convenient or safe in certain application scenarios. In addition, the stability and easy storage characteristics of the fermentation products and metabolites make this composition more suitable for commercial and large-scale applications.

[0022] According to the third main aspect of the present invention, there is provided the use of Burkholderia gladiolus BG-GF5, which includes use in inhibiting pathogens, use in inhibiting crop diseases caused by pathogens, use in preparing pathogen inhibitors and use in preparing crop disease inhibitors caused by pathogens.

[0023] In the present invention, based on the biological control potential of BG-GF5, that is, its ability to inhibit the growth of pathogens by competition, antagonism or production of antibiotics, the advantage is that BG-GF5 provides an environmentally friendly alternative to control crop diseases, reducing dependence on chemical pesticides and contributing to sustainable agricultural development. In addition, the use of BG-GF5 as a biological control agent can reduce the development of pathogen resistance because its mechanism of action is different from that of chemical pesticides.

[0024] Among them, the crops include honey plum, pepper and kiwi. By targeting the disease control of specific crops, the application of BG-GF5 is more targeted and effective. This not only helps to improve the yield and quality of these crops, but also reduces the economic losses caused by diseases, especially in the main planting areas of these crops.

[0025] In some embodiments, as a further preferred embodiment, the use includes preparing a bacterial suspension of the gladiolus Burkholderia or a composition thereof, and applying the bacterial suspension to target crops, and the application method includes soil application, seed treatment, foliar spraying or irrigation system application.

[0026] The above flexible application methods can be adjusted according to the growth stage of the crop, the occurrence of the disease and the field conditions to achieve the best control effect. In addition, the diversity of this method also allows BG-GF5 to adapt to different agricultural operations and management systems, improving its applicability in actual agricultural production.

[0027] Advantages and beneficial effects of the present invention:

[0028] The gladiolus Burkholderia BG-GF5 provided by the present invention is a microorganism separated from the root nodules of white clover. The safety of the gladiolus Burkholderia BG-GF5 has been verified through pathogenicity and hemolysis tests, which proves that it is safe and harmless to human body and environment, and provides a basis for its application in biological control.

[0029] In the present invention, the culture method of gladiolus Burkholderia BG-GF5 uses a specific culture medium combination, including NA culture medium, PDA culture medium and LB culture medium, which provides a suitable nutritional environment for the growth and reproduction of the strain. In addition, the present invention can obtain a pure culture of BG-GF5 through a gradient dilution method and a coating method, and a specific 16S rRNA gene primer pair is used to identify BG-GF5 through PCR amplification, ensuring accurate identification and identification of the strain.

[0030] The application of the gladiolus Burkholderia BG-GF5 of the present invention is mainly reflected in its inhibitory effect on pathogens and its potential in crop disease prevention and control. The strain or its fermented product and metabolite can be made into a composition for inhibiting pathogens and crop diseases caused by pathogens. In particular, in the application on crops such as honey plum, pepper and kiwi fruit, BG-GF5 can be made into a bacterial suspension and applied to crops, and can effectively prevent and control diseases through soil application, seed treatment, foliar spraying or irrigation system application, etc., showing its wide application prospects in the field of agricultural biological control. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, without paying creative labor, other drawings obtained based on these drawings still belong to the scope of the present invention.

[0032] Figure 1 The colony morphology of Burkholderia gladiolus BG-GF5 in one embodiment of the present invention is shown;

[0033] Figure 2 An electron microscope scanning image of Burkholderia gladiolus BG-GF5 in one embodiment of the present invention is shown;

[0034] Figure 3 A schematic diagram of the 16s rRNA phylogenetic tree of Burkholderia gladiolus BG-GF5 in one embodiment of the present invention is shown;

[0035] Figure 4 The results of evaluating the safety of the isolate BG-GF5 using a pathogenicity test on honey plums in one embodiment of the present invention are shown;

[0036] Figure 5 The results of evaluating the safety of isolate BG-GF5 using a hemolytic test in one embodiment of the present invention are shown;

[0037] Figure 6 The results of the broad-spectrum antibacterial activity test of the BG-GF5 bacterial suspension against Phytophthora capsici in one embodiment of the present invention are shown;

[0038] Figure 7 The results of the broad-spectrum antibacterial activity test of the BG-GF5 bacterial suspension against Fusarium proliferatum in one embodiment of the present invention are shown;

[0039] Figure 8 The results of the broad-spectrum antibacterial activity test of BG-GF5 bacterial suspension against Robillarda africana in one embodiment of the present invention are shown;

[0040] Fig. 9 The results of the broad-spectrum antibacterial activity test of the BG-GF5 bacterial suspension against the (Plum) American Australian type drupe chain sclerotinia Monilinia fructicola in one embodiment of the present invention are shown;

[0041] Fig.10The results of the broad-spectrum antibacterial activity test of the BG-GF5 bacterial suspension against Phomopsis (kiwifruit) in one embodiment of the present invention are shown;

[0042] Fig.11 The results of the broad-spectrum antibacterial activity test of the BG-GF5 bacterial suspension against Alternaria alternata (kiwifruit) in one embodiment of the present invention are shown;

[0043] Fig.12 The results of the broad-spectrum antibacterial activity test of the BG-GF5 bacterial suspension against Botryosphaeria dothidea of ​​(kiwifruit) are shown in one embodiment of the present invention;

[0044] Fig.13 The results of the broad-spectrum antibacterial activity test of the sterile fermentation liquid of BG-GF5 against Phytophthora capsici in one embodiment of the present invention are shown;

[0045] Fig.14 The results of the broad-spectrum antibacterial activity test of the BG-GF5 sterile fermentation liquid against Fusarium proliferatum in one embodiment of the present invention are shown;

[0046] Fig.15 The results of the broad-spectrum antibacterial activity test of BG-GF5 sterile fermentation broth against Robillarda africana in one embodiment of the present invention are shown;

[0047] Fig.16 The results of the broad-spectrum antibacterial activity test of the aseptic fermentation liquid of BG-GF5 against the American-Australian type Monilinia fructicola in one embodiment of the present invention are shown;

[0048] Fig.17 The results of the broad-spectrum antibacterial activity test of the aseptic fermentation liquid of BG-GF5 against Phomopsis (kiwifruit) in one embodiment of the present invention are shown;

[0049] Fig.18 The results of the broad-spectrum antibacterial activity test of the BG-GF5 sterile fermentation liquid against Alternaria alternata (kiwifruit) in one embodiment of the present invention are shown;

[0050] Fig.19 The results of the broad-spectrum antibacterial activity test of the BG-GF5 sterile fermentation broth against Botryosphaeria dothidea of ​​(kiwifruit) are shown in one embodiment of the present invention. DETAILED DESCRIPTION

[0051] The preferred embodiments of the present invention will be described in detail below in conjunction with the accompanying drawings so that the purpose, features and advantages of the present invention can be more clearly understood. It should be understood that the embodiments shown in the accompanying drawings are not intended to limit the scope of the present invention, but are only intended to illustrate the essential spirit of the technical solution of the present invention.

[0052] In the following description, certain specific details are set forth for the purpose of illustrating the various disclosed embodiments to provide a thorough understanding of the various disclosed embodiments. However, those skilled in the relevant art will recognize that the embodiments may be practiced without one or more of these specific details. In other cases, well-known devices, structures, and techniques associated with the present application may not be shown or described in detail to avoid unnecessarily obscuring the description of the embodiments.

[0053] References throughout the specification to "one embodiment" or "an embodiment" indicate that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of "in one embodiment" or "in an embodiment" in various places throughout the specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any manner in one or more embodiments.

[0054] The present invention provides a gladiolus Burkholderia BG-GF5, which is deposited in China Center for Type Culture Collection (address: Wuhan University, Wuhan, China, zip code: 430072, telephone: 027-68754052) with a deposit number of CCTCCNO: M20242573, and the deposit date is November 18, 2024.

[0055] Example 1: Isolation and identification of strains

[0056] 1. Strain Isolation

[0057] The root nodules of Trifolium repens collected from Xibei Community, Huaxi District, Guiyang City, Guizhou Province and the West Campus of Guizhou University (26.45°N, 106.66°E,) were rinsed with running water in the laboratory, and then rinsed with 75% alcohol for 15 to 20 seconds in a sterile operating table, and then rinsed with sterile water three times.

[0058] Add the nodules to a 1.5 mL centrifuge tube, add 1 mL of sterile water, and grind thoroughly with two grinding beads. Take 1 mL of the ground mother liquor and add 9 mL of sterile water, and dilute to 10% by gradient dilution. -3 , 10 -4 , 10 -5 , take 10 respectively -3 , 10 -4 , 10 -5Spread 100 μL of the dilution on NA medium and invert it in a 30°C incubator for 2 to 3 days.

[0059] Single colonies with different morphological characteristics were picked and purified by line drawing on new NA medium to obtain pure culture. The pure culture was sent for sequencing to obtain strain BG-GF5, which was added to 50% glycerol and stored in a -80°C refrigerator.

[0060] 2. Strain Identification

[0061] (1) Identification of physiological and biochemical characteristics

[0062] Morphological observation and physiological and biochemical identification were determined according to the "Common Bacteria System Identification Manual". The colonies are non-transparent, non-fluorescent, with neat and smooth edges, milky white or yellowish, slightly raised, and jelly-like and sticky. Electron microscope scanning images show that the bacterial cells are rod-shaped. The colony morphology is as follows Figure 1 As shown in the scanning electron microscope image Figure 2 shown.

[0063] (2) Molecular identification

[0064] The total DNA of BG-GF5 was extracted using a bacterial DNA extraction kit, and the 16S sequence was amplified using primers 16SF (5′-AGA GTT TGA TCCTGG CTC AG-3′) and 16SR (5′-AAG GAG GTG ATC CAG CCG CA-3′).

[0065] The amplified PCR products were detected by agarose gel electrophoresis, and the PCR products with a single band were sent to a sequencing company for sequencing.

[0066] The sequencing results were compared using BLAST in the NCBI database, and the strain sequences with higher homology were selected. The phylogenetic tree of the 16S rRNA sequence of BG-GF5 was constructed using the maximum likelihood method (ML) of MEGA 7.0 software, as shown in the following figure: Figure 3 shown.

[0067] Example 2: Safety Test

[0068] The safety of isolated strains is a necessary prerequisite for the application of biological control microorganisms. The present invention uses pathogenicity and hemolysis tests to evaluate the safety of isolated strain BG-GF5.

[0069] The test results showed that the honey plum fruits inoculated with BG-GF5 did not show any disease symptoms or necrosis (such as Figure 4 (shown in A / B).

[0070] Considering human safety, a hemolysis test was performed, and the Columbia blood plate results (such as Figure 5 Part C) shows that no hemolytic ring was found around the BG-GF5 colony, indicating the absence of α-hemolysin. In summary, strain BG-GF5 is safe and harmless.

[0071] Example 3: Determination of the broad-spectrum antibacterial activity of BG-GF5 against several plant pathogenic fungi

[0072] Activate the relevant pathogenic fungi on PDA culture medium, use a 6mm puncher to punch out the fungal cakes, and inoculate them in the center of a new PDA plate.

[0073] A 6 mm round filter paper soaked in BG-GF5 bacterial suspension was evenly placed 25 mm away from each pathogenic fungus cake. The treatment was repeated 3 times for each pathogenic fungus, with no filter paper and in the center.

[0074] The plate with pathogenic fungi was used as a control. The culture dish was inverted and cultured in a 28°C incubator. When the plate without filter paper and inoculated with pathogenic fungi in the center grew to two-thirds of the culture dish, the growth of pathogenic fungi was observed. The inhibition rate was calculated according to the following formula:

[0075] Inhibition rate (%) = (pathogen diameter of control group - pathogen diameter of treatment group) / pathogen diameter of control group × 100%

[0076]

[0077] Figure 6-Figure 12 The results of the broad-spectrum antibacterial activity test of BG-GF5 bacterial suspension against the above 7 pathogens were presented respectively. The results showed that BG-GF5 had the best inhibitory effect on the American and Australian type drupe chain fungus Monilinia fructicola.

[0078] Example 4: Determination of broad-spectrum antibacterial activity of BG-GF5 sterile fermentation broth against several plant pathogenic fungi

[0079] The sterile fermentation broth of BG-GF5 after 2 days of fermentation was evenly mixed with PDA medium at a ratio of 1:10 (v:v). 10 mL of the mixed medium was measured with a measuring cup and poured into the culture dish to make the concentration of the BG-GF5 sterile fermentation broth in the culture dish reach 100 mL / L. The fungal cakes of 7 pathogenic fungi were punched out with a 6 mm puncher and inoculated in the center of the PDA plate containing the sterile fermentation broth.

[0080] Add the same volume of sterile water as a control and repeat three times. Place the culture dish upside down in an incubator at 28°C for a suitable number of days to observe the growth of pathogens.

[0081] The inhibition rate was calculated according to the following formula:

[0082] Inhibition rate (%) = (pathogen diameter of control group - pathogen diameter of treatment group) / pathogen diameter of control group × 100%

[0083]

[0084] Figure 13-Figure 19 The results of the broad-spectrum antibacterial activity test of BG-GF5 sterile fermentation broth against the above 7 pathogens were displayed respectively. The results showed that BG-GF5 had the best inhibitory effect on the American and Australian type drupe chain fungus Monilinia fructicola.

[0085] Example 5: Method of applying strains

[0086] Prepare a culture of Burkholderia gladioli BG-GF5, obtained by the above-mentioned culture method.

[0087] The BG-GF5 culture is made into a bacterial suspension, and the concentration and formula of the bacterial suspension are adjusted according to the target crops and disease characteristics.

[0088] The bacterial suspension is applied to the target crops by soil application, seed treatment, foliar spraying or irrigation system application.

[0089] Adjust the application frequency and amount regularly during the crop growth cycle or according to the occurrence of diseases to effectively inhibit or reduce the occurrence of crop diseases.

[0090] The matters not described in detail in the present invention are all known technologies to those skilled in the art.

[0091] The above shows and describes the basic principles and main features of the present invention and the advantages of the present invention. It should be understood by those skilled in the art that the present invention is not limited to the above embodiments. The above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention to be protected. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. Burkholderia gladiolus BG-GF5, which is deposited in the China Center for Type Culture Collection with the deposit number CCTCC NO:M20242573, and the deposit date is November 18, 2024.

2. The Burkholderia gladiolus BG-GF5 according to claim 1, characterized in that Its 16SrDNA sequence is shown in Seq ID No.

1.

3. The Burkholderia gladiolus BG-GF5 according to claim 1, characterized in that The gladiolus Burkholderia BG-GF5 is isolated from the root nodules of white clover.

4. The Burkholderia gladiolus BG-GF5 according to claim 1, characterized in that The method for culturing gladiolus Burkholderia BG-GF5 comprises the following steps: Growing using a media combination; and The culture condition is culturing at 30°C for 2 to 3 days; and Pure cultures were obtained by serial dilution and plating methods.

5. The Burkholderia gladiolus BG-GF5 according to claim 4, characterized in that The culture medium combination comprises at least one of the following culture media: a. NA medium, which consists of 5 g / L beef extract, 10 g / L peptone, 5 g / L NaCl, 20 g / L agar powder, and 1000 mL sterile water; b. PDA medium, which consists of 200 g potatoes, 20 g glucose, 17 g agar powder, and 1000 mL sterile water; c. LB medium, which consists of 10 g of tryptone, 5 g of yeast extract, and 10 g of sodium chloride; d.NB medium: peptone 10g, beef extract 3g, glucose 10g, yeast extract 1g.

6. The Burkholderia gladiolus BG-GF5 according to claim 1, characterized in that The gladiolus Burkholderia BG-GF5 is amplified by PCR using a primer pair of 16S rRNA gene, wherein the primer pair includes a forward primer 16SF, the sequence of which is: 5′-AGA GTT TGA TCC TGG CTC AG-3′; The primer pair includes a reverse primer 16SR, the sequence of which is: 5′-AAG GAG GTG ATC CAG CCG CA-3′. 7 . A composition comprising the Burkholderia gladiolus BG-GF5 or a fermentation product or metabolite thereof according to claim 1 .

8. A use of the gladiolus Burkholderia BG-GF5 as claimed in claim 1, characterized in that: The uses include application in inhibiting pathogens, application in inhibiting crop diseases caused by pathogens, application in preparing pathogen inhibitors and application in preparing crop disease inhibitors caused by pathogens.

9. The use of Burkholderia gladiolus BG-GF5 according to claim 8, characterized in that: The crops include honey plums, peppers and kiwifruit.

10. The use of Burkholderia gladiolus BG-GF5 according to claim 8, characterized in that: The use comprises preparing a bacterial suspension from the gladiolus Burkholderia BG-GF5 or a composition thereof, and applying the bacterial suspension to target crops, wherein the application method comprises soil application, seed treatment, foliar spraying or irrigation system application.

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