Bordetella trematum, compositions and uses thereof

Abstract

The application discloses a Burkholderia gladioli BG-GF5, a composition and application thereof. The strain is preserved in the China Center for Type Culture Collection with a preservation number of CCTCC NO: M20242573, and the preservation date is November 18, 2024. The application of the BG-GF5 mainly reflects the inhibition effect on pathogenic bacteria and the potential in crop disease control. The strain or the fermentation product or the metabolic product thereof can be made into a composition for inhibiting pathogenic bacteria and crop diseases caused by pathogenic bacteria. Especially in the application of crops such as honey plum, pepper and kiwi, the application shows a wide application prospect in the field of agricultural biological control.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field related to bio-agriculture, and particularly relates to a new bacterial strain and a composition thereof, and use thereof in biological control of plant diseases. BACKGROUND

[0002] In the field of agriculture, 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 can lead to environmental problems and increased resistance of pathogenic fungi. Therefore, developing new, environmentally friendly, and sustainable biological control methods is an important direction of current agricultural scientific research.

[0003] Biological control is a method of using biological resources to control pests and pathogens, and microbial control is valued for its environmental friendliness and sustainability. In microbial control, bacteria are an effective biological control agent because they can produce substances that inhibit or kill pathogenic fungi.

[0004] Burkholderia is a group of gram-negative bacteria widely present in soil and plant rhizosphere, and some species have been proven to have activity against plant pathogenic fungi. However, some species of Burkholderia are also associated with human diseases, so it is important to develop new safe and harmless Burkholderia strains for biological control.

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

[0006] The purpose of the present application is to provide Burkholderia gladioli BG-GF5, a composition thereof, and use thereof, especially

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

[0008] The 16S rDNA sequence of the Gladiolus B Burkholderia BG-GF5 is shown in Seq ID No. 1. The 16S rDNA sequence is the gold standard for bacterial classification and identification, as it is highly conserved in the bacterial genome, while having sufficient variability between different species to distinguish different species.

[0009] Further, the Gladiolus B Burkholderia BG-GF5 is isolated from white clover root nodules. This strain is isolated from the natural environment, has adapted to a specific ecological niche, and has biological activities that have not been fully studied, providing potential resources for the development of new biological control agents. In addition, the natural source of the strain also means that it has higher applicability and safety in the environment, as it has coexisted with specific plant species for a long time.

[0010] In some embodiments, as a further preferred solution, the culture method of the Gladiolus B Burkholderia BG-GF5 includes the following steps: using a culture medium combination for culture; the culture condition is 30°C for 2-3 days; obtaining pure culture by gradient dilution method and coating method.

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

[0012] The culture medium combination includes at least one of the following culture media:

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

[0014] b. PDA culture medium, consisting of potato 200 g, glucose 20 g, agar powder 17 g, sterile water 1000 mL;

[0015] c. LB culture medium, consisting of tryptone 10 g, yeast extract 5 g, sodium chloride 10 g;

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

[0017] By providing a variety of culture medium options, the culture conditions can be flexibly adjusted according to the experimental purpose and strain characteristics, optimizing 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 aspect, the B. glumae BG-GF5 is subjected to PCR amplification using a primer pair for 16S rRNA gene, which includes a forward primer 16SF with the sequence of 5'-AGA GTT TGA TCC TGG CTC AG-3';

[0019] which includes a reverse primer 16SR with the sequence of 5'-AAG GAG GTG ATC CAG CCG CA-3'.

[0020] By using the specific primer pair, the 16S rRNA gene of BG-GF5 can be accurately amplified, providing accurate molecular evidence for strain identification and classification. The high specificity and sensitivity of this method enable 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, this method has high standardization and automation, which can quickly and accurately process a large number of samples, improving work efficiency.

[0021] According to a second main aspect of the present application, a composition comprising the aforementioned B. glumae BG-GF5 or its fermentation or metabolite is provided. Based on the bioactivity of microorganisms, BG-GF5 can produce fermentation or metabolites with biological activity, which can be used to control crop diseases. Therefore, this composition can directly utilize the biological activity of BG-GF5 without the need to use live bacteria directly, which may be more convenient or safe in certain application scenarios. In addition, the stability and easy-to-store characteristics of fermentation and metabolites make this composition more suitable for commercialization and large-scale application.

[0022] According to a third main aspect of the present application, the use of B. glumae BG-GF5 is provided, which includes the use in inhibiting pathogenic bacteria, the use in inhibiting crop diseases caused by pathogenic bacteria, the use in preparing pathogenic bacteria inhibitors, and the use in preparing crop disease inhibitors caused by pathogenic bacteria.

[0023] In the present application, based on the biological control potential of BG-GF5, it can inhibit the growth of pathogenic bacteria through competition, antagonism, or production of antibiotics, etc. The advantage is that BG-GF5 provides an environmentally friendly alternative to control crop diseases, reducing the dependence on chemical pesticides, and contributing to sustainable agricultural development. In addition, using BG-GF5 as a biological control agent can reduce the development of pathogenic bacteria resistance, as its mechanism of action is different from chemical pesticides.

[0024] Among them, the crops include honey plums, peppers and kiwifruits. Through 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 solution, the use comprises preparing a bacterial suspension of the B. gladioli or its composition, and applying the bacterial suspension to the target crops, including soil application, seed treatment, foliar spraying or irrigation system application.

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

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

[0028] The B. gladioli BG-GF5 provided by the present application is a microorganism isolated from white clover root nodules. The safety of the B. gladioli BG-GF5 has been verified by pathogenicity and hemolysis tests, proving that it is safe to human body and environment, which provides a basis for its use in biological control.

[0029] In the present application, the culture method of B. gladioli BG-GF5 uses specific medium combinations, including NA medium, PDA medium and LB medium, which provides a suitable nutrient environment for the growth and reproduction of the strain. Moreover, the present application can obtain pure culture of BG-GF5 through gradient dilution method and coating method, and specific 16S rRNA gene primers are used for PCR amplification to identify BG-GF5, ensuring accurate identification and identification of the strain.

[0030] The application of B. gladioli BG-GF5 of the present application mainly reflects its inhibition effect on pathogenic bacteria and its potential in crop disease control. The strain or its fermentation products, metabolites can be made into compositions for inhibiting pathogenic bacteria and crop diseases caused by pathogenic bacteria. Especially in the application of honey plums, peppers and kiwifruits, BG-GF5 can effectively prevent diseases through soil application, seed treatment, foliar spraying or irrigation system application, etc. by making bacterial suspension and applying it to crops, which shows its wide application prospect in the field of agricultural biological control. BRIEF DESCRIPTION OF DRAWINGS

[0031] In order to make the technical solutions in the embodiments of the present application or the prior art clearer, the accompanying drawings needed in the embodiments or prior art description will be briefly introduced. Obviously, the accompanying drawings in the following description only need to be some embodiments of the present application, and for those skilled in the art, other drawings obtained according to these drawings without creative labor still belong to the scope of the present application.

[0032] Figure 1 Colony morphology of B. gladioli BG-GF5 in one embodiment of the present application is shown;

[0033] Figure 2 Scanning electron microscope (SEM) image of B. gladioli BG-GF5 in one embodiment of the present application is shown;

[0034] Figure 3 16s rRNA phylogenetic tree of B. gladioli BG-GF5 in one embodiment of the present application is shown;

[0035] Figure 4 Safety evaluation results of isolate BG-GF5 using pathogenicity test on Prunus avium in one embodiment of the present application are shown;

[0036] Figure 5 Safety evaluation results of isolate BG-GF5 using hemolytic test in one embodiment of the present application are shown;

[0037] Figure 6 Broad-spectrum antibacterial activity of BG-GF5 bacterial suspension against Phytophthora capsici in one embodiment of the present application is shown;

[0038] Figure 7 Broad-spectrum antibacterial activity of BG-GF5 bacterial suspension against Fusarium proliferatum in one embodiment of the present application is shown;

[0039] Figure 8 Broad-spectrum antibacterial activity of BG-GF5 bacterial suspension against Robillarda africana in one embodiment of the present application is shown;

[0040] Figure 9 Broad-spectrum antibacterial activity of BG-GF5 bacterial suspension against Monilinia fructicola in one embodiment of the present application is shown;

[0041] Figure 10The results of the determination of the broad-spectrum bacteriostatic activity of the BG-GF5 bacterial suspension on Phomopsis (kiwi) in one embodiment of the application are shown;

[0042] Figure 11 The results of the determination of the broad-spectrum bacteriostatic activity of the BG-GF5 bacterial suspension on Alternaria alternata (kiwi) in one embodiment of the application are shown;

[0043] Figure 12 The results of the determination of the broad-spectrum bacteriostatic activity of the BG-GF5 bacterial suspension on Botryosphaeria dothidea (kiwi) in one embodiment of the application are shown;

[0044] Figure 13 The results of the determination of the broad-spectrum bacteriostatic activity of the BG-GF5 sterile fermentation broth on Phytophthora capsici (pepper) in one embodiment of the application are shown;

[0045] Figure 14 The results of the determination of the broad-spectrum bacteriostatic activity of the BG-GF5 sterile fermentation broth on Fusarium proliferatum (plum) in one embodiment of the application are shown;

[0046] Figure 15 The results of the determination of the broad-spectrum bacteriostatic activity of the BG-GF5 sterile fermentation broth on Robillarda africana (plum) in one embodiment of the application are shown;

[0047] Figure 16 The results of the determination of the broad-spectrum bacteriostatic activity of the BG-GF5 sterile fermentation broth on Monilinia fructicola (plum) in one embodiment of the application are shown;

[0048] Figure 17 The results of the determination of the broad-spectrum bacteriostatic activity of the BG-GF5 sterile fermentation broth on Phomopsis (kiwi) in one embodiment of the application are shown;

[0049] Figure 18 The results of the determination of the broad-spectrum bacteriostatic activity of the BG-GF5 sterile fermentation broth on Alternaria alternata (kiwi) in one embodiment of the application are shown;

[0050] Figure 19 The results of the determination of the broad-spectrum bacteriostatic activity of the BG-GF5 sterile fermentation broth on Botryosphaeria dothidea (kiwi) in one embodiment of the application are shown. DETAILED DESCRIPTION

[0051] The preferred embodiments of the present application will be described in detail below with reference to the drawings. It should be understood that the embodiments shown in the drawings are not intended to limit the scope of the present application, but merely to describe the essential spirit of the technical solutions of the present application.

[0052] In the following description, for the purpose of explaining various disclosed embodiments, certain specific details are set forth in order to provide a thorough understanding of various disclosed embodiments. However, one skilled in the relevant arts will recognize that embodiments can be practiced without one or more of the specific details, or with other methods, components, materials, and so forth. In other instances, well-known structures, materials, or operations are not shown or described in detail in order to avoid obscuring the description of embodiments.

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

[0054] The present application provides a strain of Burkholderia gladioli BG-GF5, which is preserved in the China Center for Type Culture Collection (address: Wuhan University, Wuhan, China, postcode: 430072, telephone: 027-68754052) with the accession number CCTCC NO: M20242573, and the preservation date is November 18, 2024.

[0055] Example 1: Isolation and identification of the strain

[0056] 1. Isolation of the strain

[0057] The root nodules of white clover collected from Xibei community of Huaxi district, Guiyang city, Guizhou province (26.45 °N, 106.66 °E, ) were washed with running water in the laboratory, then the root nodules were passed through 75% alcohol for 15 to 20 seconds, and then passed through sterile water three times in a sterile operation table.

[0058] The root nodules were added to a 1.5 mL centrifuge tube, 1 mL of sterile water was added, and two grinding beads were added for sufficient grinding. 1 mL of the ground mother liquor was taken and added to 9 mL of sterile water, and gradient dilution was performed in sequence to 10 -3 , 10 -4 , 10 -5 , 10 -3 , 10 -4 , 10 -5The diluted solution 100 μL was coated on NA medium and cultured in an inverted incubator at 30℃ for 2-3 days.

[0059] The single colony with different morphological characteristics was picked and drawn on a new NA medium to obtain a pure culture. The pure culture was sequenced to obtain strain BG-GF5, which was added to 50% glycerol and stored in a refrigerator at -80℃.

[0060] 2. Strain identification

[0061] (1) Physiological and biochemical characteristic identification

[0062] Morphological observation and physiological and biochemical identification were determined according to the Common Bacteria System Identification Manual. The colony was non-transparent, non-fluorescent, with a neat and smooth edge, a milky white or yellowish color, slightly raised, and a sticky gel. The scanning electron microscope showed that the bacterial cells were rod-shaped. The colony morphology is shown in Figure 1 , and the scanning electron microscope image is shown in Figure 2 .

[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 TCC TGG CTC AG-3') and 16SR (5'-AAG GAG GTG ATC CAG CCG CA-3').

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

[0066] The sequencing results were compared using BLAST in the NCBI database, and the sequences of strains with higher homology were obtained. The maximum likelihood method (Maximum Likelihood, ML) of MEGA 7.0 software was used to construct the phylogenetic tree of the 16S rRNA sequence of BG-GF5, as shown in Figure 3 .

[0067] Example 2: Safety test

[0068] The safety of the isolated strain is a necessary prerequisite for the application of biological control microorganisms. The pathogenicity and hemolysis test was used to evaluate the safety of the isolated strain BG-GF5.

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

[0070] Considering human safety, a hemolysis test was performed, and the Columbia blood platelet results (such as...) Figure 5 As shown in section C, no hemolytic zone was found around the BG-GF5 colony, indicating the absence of α-hemolysin. In conclusion, strain BG-GF5 is safe and harmless.

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

[0072] The relevant pathogenic fungi were activated on PDA medium, and fungal discs were punched out using a 6 mm punch and inoculated into the center of a new PDA plate.

[0073] 6 mm circular filter paper pieces soaked in BG-GF5 bacterial suspension were evenly placed 25 mm away from each pathogenic fungal cake. The treatment was repeated 3 times for each pathogenic fungus, with the filter paper pieces not being used and the center being the site of the infection.

[0074] Using a plate containing the pathogenic fungus as a control, the petri dish was inverted and incubated in a 28°C incubator. When the plate, without filter paper and inoculated with the pathogenic fungus in the center, grew to two-thirds full, the growth of the pathogen was observed. The inhibition rate was calculated using the following formula:

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

[0076] No. Species name CK diameter (cm) Colony diameter (cm) Inhibition rate (%) 1 Phytophthora capsici (pepper) 6.05 2.11 65.12 2 Robillarda africana (peach) 6.5 2.62 59.69 3 Fusarium proliferatum (peach) 5.72 3.03 49.58 4 Monilinia fructicola (peach) 6.48 0.84 87.03 5 Phomopsis (kiwi) 5.68 1.58 72.18 6 Alternaria alternata (kiwi) 6.23 3.12 49.92 7 Botryosphaeria dothidea (kiwi) 6.77 2.43 64.11

[0077] Figure 6 - Figure 12 The results of the broad-spectrum antibacterial activity assay of BG-GF5 bacterial suspension against the above 7 pathogens were presented. The results showed that BG-GF5 had the best inhibitory effect on Monilinia fructicola (Australia-type).

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

[0079] After 2 days of fermentation, the sterile fermentation broth of BG-GF5 was mixed with PDA medium at a ratio of 1:10 (v:v). 10 mL of the mixed medium was measured and poured into a petri dish to make the concentration of BG-GF5 sterile fermentation broth in the petri dish 100 mL / L. Seven kinds of pathogenic fungi were punched out with a 6 mm punch and inoculated into the center of the PDA plate containing sterile fermentation broth.

[0080] Using the same volume of sterile water as a control, the experiment was repeated three times. The petri dishes were inverted and incubated in a 28°C incubator for the appropriate number of days, and the growth of the pathogens was observed.

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

[0082] Inhibition rate (%) = (diameter of pathogenic bacteria in the control group - diameter of pathogenic bacteria in the treatment group) / diameter of pathogenic bacteria in the control group x 100%

[0083] No. Species name CK diameter (cm) Colony diameter (cm) Inhibition rate (%) 1 Phytophthora capsici (pepper) 6.15 3.25 47.15 2 Robillarda africana (peach) 5.8 3.1 46.55 3 Fusarium proliferatum (peach) 6.3 2.62 58.42 4 Monilinia fructicola (peach) 6.35 1.1 82.67 5 Phomopsis (kiwi) 6.2 2 67.74 6 Alternaria alternata (kiwi) 6.5 2.1 67.69 7 Botryosphaeria dothidea (kiwi) 9 5.85 35

[0084] Figure 13 - Figure 19 The results of the broad-spectrum antibacterial activity determination of the BG-GF5 sterile fermentation broth on the above seven pathogenic bacteria are shown respectively, and the results show that the BG-GF5 has the best inhibition effect on Monilinia fructicola (Lee).

[0085] Example 5: Method for applying the strain

[0086] Prepare the culture of Burkholderia gladioli BG-GF5 by the culture method described above.

[0087] Prepare a bacterial suspension from the BG-GF5 culture, and adjust the concentration and formula of the bacterial suspension according to the target crop and disease characteristics.

[0088] Apply the bacterial suspension to the target crop, and the application method can be soil application, seed treatment, foliar spraying, or irrigation system application.

[0089] Adjust the application frequency and dosage periodically during the growth cycle of the crop or according to the occurrence of the disease, so as to effectively inhibit or reduce the occurrence of crop diseases.

[0090] The parts not described in the present application are well known to those skilled in the art.

[0091] The above shows and describes the basic principles and main features of the present application and the advantages of the present application. It should be understood by those skilled in the art that the present application is not limited by the above examples, and the above examples and descriptions in the specification are only to illustrate the principles of the present application. Without departing from the spirit and scope of the present application, various changes and improvements can be made to the present application, and these changes and improvements all fall within the scope of the claimed present application. The scope of protection of the present application is defined by the appended claims and their equivalents.

Claims

1. Burkholderia gladioli ( Burkholderia gladioli BG-GF5, with accession number CCTCC NO:M20242573, is deposited at the China Center for Type Culture Collection on November 18, 2024.

2. The Burkholderia caryophila (BG-GF5) according to claim 1, characterized in that Burkholderia gladioli BG-GF5, characterized in that The 16S rDNA sequence thereof is shown as Seq ID No.

1.

3. The B. taraxacum of claim 1, which is characterized by, Burkholderia gladioli ) BG-GF5, characterized by, The Glauber's salt Burkholderia ( Burkholderia gladioli BG-GF5 was isolated from the root nodules of white clover.

4. The B. taraxacum of claim 1, which is characterized by, Burkholderia gladioli ) BG-GF5, characterized by, The Glauber's salt Burkholderia ( Burkholderia gladioli The cultivation method for BG-GF5 includes the following steps: The culture is carried out using a medium combination; and The culture condition is culturing at 30℃ for 2-3 days; and The pure culture is obtained by gradient dilution method and coating method.

5. The B. taraxacum of claim 4, which is characterized by, Burkholderia gladioli ) BG-GF5, characterized in that, The medium combination comprises at least one of the following media: a. NA medium, the composition of which is beef extract 5 g / L, peptone 10 g / L, NaCl 5 g / L, agar powder 20 g / L, sterile water 1000 mL; b. PDA medium, the composition of which is potato 200 g, glucose 20 g, agar powder 17 g, sterile water 1000 mL; c. LB medium, the composition of which is tryptone 10 g, yeast extract 5 g, sodium chloride 10 g.

6. The B. taraxacum of claim 1, which is characterized by, Burkholderia gladioli ) BG-GF5, characterized in that, The following primer pairs were used to target the *Burkholderia gladioli* strain (…). Burkholderia gladioli The 16S rRNA gene of BG-GF5 was amplified by PCR. The primer pair included a forward primer 16SF with the sequence: 5′-AGAGTT TGA TCC TGG CTC AG-3′. The primer pair comprises a reverse primer 16SR, the sequence of which is: 5'-AAG GAG GTG ATC CAG CCG CA-3'.

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