Burkholderia cepacia MZ-8 and application thereof

Abstract

The invention discloses a Burkholderia cepacia MZ-8 which is preserved in Guangdong Microbial Culture Collection Center (GDMCC) on April 8, 2025, and the preservation number of the Burkholderia cepacia MZ-8 is GDMCC No: 66106. The research finds that the burkholderia cepacia MZ-8 can efficiently inhibit the growth of pathogenic bacteria of citrus canker, the inhibition rate of the burkholderia cepacia MZ-8 to the pathogenic bacteria of citrus canker reaches 98.27% in a pot culture test, and meanwhile, the burkholderia cepacia MZ-8 can dissolve organic phosphorus, generate iron carriers and fix nitrogen and promote plant growth. Therefore, the burkholderia cepacia MZ-8 disclosed by the invention has good application potential in the aspects of preventing and treating citrus canker, promoting absorption and growth of phosphorus, iron and nitrogen of plants and the like. The discovery of the bacterial strain enriches beneficial microbial resources for biocontrol and growth promotion, and provides a bacterial strain which is stable in antibacterial effect, efficient and environment-friendly for green biological control.

Description

Technical Field

[0001] This invention relates to the field of agricultural microbiology, specifically to a strain of Burkholderia cepacia MZ-8 and its applications. Background Technology

[0002] Citrus canker is a major bacterial disease affecting the global citrus industry. It is widespread in citrus-growing regions worldwide, causing significant economic losses. Citrus canker primarily damages leaves, fruit, shoots, and stems. Under natural conditions, the pathogen mainly enters through stomata and wounds. Severe infections lead to leaf drop, branch wilting, and unnatural fruit drop, resulting in reduced yield and compromised fruit quality. If this pathogen spreads extensively within citrus-producing areas, it will directly or indirectly cause substantial negative impacts on the entire citrus industry in those regions, resulting in incalculable losses for fruit growers.

[0003] Currently, the main control method for citrus canker is chemical control, which typically involves applying pesticides including copper-based agents, agricultural antibiotics, thiazoles, and microbial agents. While spraying plants with copper-based agents is very helpful in controlling citrus canker, it leads to the accumulation of copper ions in the soil, which is detrimental to citrus root growth and nutrient absorption. Furthermore, frequent use of pesticides causes environmental pollution, such as damage to soil and water bodies. Therefore, green and efficient biological control will be the future trend. Biological control of citrus canker utilizes biological metabolites or heterologous gene expression to enhance plant resistance, limit the living space of the canker pathogen, and thus inhibit the pathogen or prevent infection—an environmentally friendly technology.

[0004] Numerous studies have shown that endophytic bacteria play a variety of beneficial biological roles in the defense against plant diseases, such as producing antimicrobial proteins, chitinases, glucanases, and other antibiotics to induce plant resistance, and even altering the host's genotype. The production of antibiotics and the induction of systemic resistance are among the most common. Biocontrol bacteria for citrus canker are currently the most studied, finely classified, and widely used group, mainly including Bacillus, Pseudomonas, and Actinomycetes, with the first two being the most frequently reported. Currently reported Bacillus species with antagonistic effects against citrus canker mainly include Bacillus subtilis, Bacillus amyloliquefaciens, and Bacillus belyssus, while there are relatively few reports on the use of Burkholderia sp. for the control of citrus canker. The article "Study on Endophytic Antagonistic Bacterium Bc51 in Citrus Canker Disease" points out that the formation of citrus canker lesions is inhibited by Burkholderia cepacia Bc51. The inhibition rate is highest (60.3%) when lesions are inoculated with both the canker pathogen and strain Bc51 simultaneously. The inhibition rate is lowest (24.91%) when inoculated with the canker pathogen for 2 days followed by strain Bc51. The inhibition rate is moderate (48.63%) when inoculated with strain Bc51 for 2 days followed by the canker pathogen. Research on other Burkholderia species mainly focuses on their ability to dissolve insoluble phosphorus and potassium, fix nitrogen, and secrete ferophiles. Examples include patents CN112625970A (Burkholderia cepacia JT79 and its application), CN119242527A (a compound microbial agent for decomposing insoluble phosphorus in soil and its application), and CN114134084A (Burkholderia cepacia Bckms21 and its application). Therefore, it is of great significance to provide Burkholderia cepacia, which has the functions of dissolving organophosphates, producing iron carriers, fixing nitrogen, and has a better inhibitory effect on the pathogen of citrus canker, so as to promote plant growth while defending against plant diseases. Summary of the Invention

[0005] The purpose of this invention is to overcome the above-mentioned defects and deficiencies in the prior art and to provide a strain of Burkholderia cepacia MZ-8.

[0006] A second objective of this invention is to provide a microbial agent / biological preparation.

[0007] The third objective of this invention is to provide a microbial fertilizer / additive / pesticide.

[0008] The fourth objective of this invention is to provide the application of the above-mentioned Burkholderia cepacia MZ-8, bacterial agent / biological agent, microbial fertilizer / additive / pesticide in promoting plant growth, decomposing organic phosphorus, producing iron carriers, fixing nitrogen, and controlling citrus canker and citrus canker pathogens.

[0009] The above-mentioned objective of this invention is achieved through the following technical solution:

[0010] This invention provides a strain of Burkholderia cepacia MZ-8, which was deposited on April 8, 2025, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), with accession number GDMCC No: 66106. The address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province, 510070, China.

[0011] This invention isolated a round, medium-sized, pale yellow, smooth, opaque, Gram-negative strain MZ-8 from a diseased citrus tree in the orchard of Cuitian Agricultural Technology Co., Ltd., Huangtian Town, Sihui City, Zhaoqing City, Guangdong Province. After sequencing and sequence comparison, strain MZ-8 was identified as Burkholderia cepacia. The strain was deposited on April 8, 2025, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), with accession number GDMCC No: 66106, classified as Burkholderia cepacia MZ-8, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, Guangdong Province, 510070, China.

[0012] The present invention also provides a bacterial agent / biological preparation, wherein the bacterial agent contains one or more of the above-mentioned Burkholderia cepacia MZ-8 and its culture medium, bacterial suspension and bacterial cells.

[0013] The present invention also provides a microbial fertilizer / additive / pesticide, wherein the microbial fertilizer / microbial additive contains one or more of the above-mentioned Burkholderia cepacia MZ-8 and its culture medium, bacterial suspension and bacterial cells.

[0014] Furthermore, the preparation method of the suspension, culture medium and bacterial cells is as follows: inoculate and culture Burkholderia cepacia MZ-8 to obtain a culture medium; centrifuge the culture medium and resuspend it to obtain a bacterial suspension; centrifuge the culture medium to obtain bacterial cells.

[0015] This invention has revealed that *Burkholderia cepacia* MZ-8 can effectively inhibit the growth of citrus canker pathogens, achieving an inhibition rate of 98.27% in pot tests. Simultaneously, it can dissolve organic phosphorus, produce iron carriers, and fix nitrogen, thus promoting plant growth. Therefore, *Burkholderia cepacia* MZ-8 of this invention has great application potential in the prevention and control of citrus canker and in promoting the absorption of phosphorus, iron, and nitrogen in plants and their growth.

[0016] Therefore, the present invention also provides the application of the above-mentioned Burkholderia cepacia MZ-8 or the above-mentioned microbial agent / biological agent or the above-mentioned bio-fertilizer / additive / pesticide in promoting plant growth.

[0017] The present invention also provides the application of the above-mentioned Burkholderia cepacia MZ-8 or the above-mentioned bacterial agent / biological agent or the above-mentioned bio-fertilizer / additive / pesticide in the decomposition of organophosphates, iron carrier production and nitrogen fixation.

[0018] Furthermore, the application involves applying the aforementioned Burkholderia cepacia MZ-8, or the aforementioned microbial agent / biological preparation, or the aforementioned bio-fertilizer / additive / pesticide to the soil.

[0019] The present invention also provides the application of the above-mentioned Burkholderia cepacia MZ-8 or the above-mentioned bacterial agent / biological agent or the above-mentioned bio-fertilizer / additive / pesticide in the prevention and control of citrus canker and citrus canker pathogen.

[0020] Furthermore, the application involves directly applying or spraying the aforementioned Burkholderia cepacia MZ-8, or the aforementioned microbial agent / biological preparation, or the aforementioned bio-fertilizer / additive / pesticide, onto the plant control area.

[0021] Furthermore, the inoculation concentration of Burkholderia cepacia MZ-8 is in the ratio of 1:0.8 to 3 to the concentration of the citrus canker pathogen.

[0022] Preferably, the inoculation concentration of Burkholderia cepacia MZ-8 is in a ratio of 1:2 to the concentration of the citrus canker pathogen.

[0023] Furthermore, the plant in question is a member of the Rutaceae family.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] This invention discloses a strain of *Burkholderia cepacia* MZ-8, which was deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC) on April 8, 2025, with accession number GDMCC No: 66106. This invention found that *Burkholderia cepacia* MZ-8 can effectively inhibit the growth of the pathogen causing citrus canker, achieving an inhibition rate of 98.27% in pot tests. Simultaneously, it can dissolve organic phosphorus, produce iron carriers, and fix nitrogen, promoting plant growth. Therefore, *Burkholderia cepacia* MZ-8 of this invention has great application potential in the control of citrus canker and in promoting the absorption of phosphorus, iron, and nitrogen in plants and their growth. The discovery of this strain enriches my country's resources of beneficial microorganisms used for biocontrol and growth promotion, providing a stable, efficient, and environmentally friendly strain for green biological control. Attached Figure Description

[0026] Figure 1 The colony morphology of Burkholderia cepacia MZ-8 on LB medium.

[0027] Figure 2Phylogenetic tree of Burkholderia cepacia MZ-8 based on 16S rRNA gene.

[0028] Figure 3 To test the inhibitory effect of Burkholderia cepacia MZ-8 on Xcc, the pathogen of citrus canker.

[0029] Figure 4 Tests on the dissolution of organophosphates by Burkholderia cepacia MZ-8.

[0030] Figure 5 Tests for siderophore production by Burkholderia cepacia MZ-8.

[0031] Figure 6 Tests on the nitrogen-fixing capacity of Burkholderia cepacia MZ-8.

[0032] Figure 7 Potted plant efficacy test for Burkholderia cepacia MZ-8. Detailed Implementation

[0033] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0034] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0035] Example 1: Isolation and Identification of Burkholderia cepacia MZ-8

[0036] Diseased citrus trees were selected from the orchard of Cuitian Agricultural Technology Co., Ltd. (N 23°26'13.315", E112°31'40.894") in Huangtian Town, Sihui City, Zhaoqing City, Guangdong Province. Fresh branches and leaves were cut using sterile scissors and placed in sterile sampling bags. The samples were then stored in a 4°C sampling box and brought back to the laboratory. 50 mg of leaf samples were cut from the leaves in a clean bench, washed with sterile water, and added to a 50 mL centrifuge tube containing 30 mL of sterile PBS buffer (0.1 mol / L, pH = 7.0). The mixture was shaken well and placed in a 28°C constant temperature shaker at 180 rpm for 15 min. This process was repeated three times. The leaves were then removed, and residual PBS was blotted dry with sterile filter paper. The leaves were placed in a 1.5 mL centrifuge tube, and 200 μL of 10 mM sterile MgCl2 was added. The mixture was then homogenized using a sterile pestle. The homogenate was then transferred to 50 mL centrifuge tubes, and 25 mL of 10 mM sterile MgCl2 was added. The mixture was thoroughly mixed and incubated at room temperature for 20 min to release bacteria. A suitable amount of homogenate was added to 10% TSB liquid medium and diluted 222, 666, 2000, 6000, and 18000 times. After mixing, 160 μL of the medium was added to a 96-well cell culture plate using a pipette, with each dilution gradient repeated three times. The 96-well plates were sealed with sealing film and incubated in a 28°C incubator in the dark for 2 weeks. Bacterial growth was observed in the 96-well plates; dilutions showing visible bacterial growth in approximately 30% of the wells were considered the ODC (optimal dilution). A 96-well plate with the optimal dilution gradient was used to streak all wells showing visible bacterial growth, one well per plate. The plates were then incubated at room temperature for 1–5 days. By observing characteristics such as colony size, color, dryness / wetness, smoothness, and the presence or absence of halos, different single colonies were picked from the culture medium and transferred to fresh TSB medium. The culture was purified through repeated streak filtration until a pure culture was obtained. The purified strains were numbered, mixed with 25% glycerol (v / v), and stored in an ultra-low temperature freezer, thus obtaining strain MZ-8.

[0037] Strain MZ-8 was Gram-negative. The colony morphology of this strain after 4 days of culture on LB medium was as follows: Figure 1 As shown, single colonies are round, medium-sized, pale yellow, smooth, opaque, and have neat edges.

[0038] Example 2: 16S rRNA gene sequence analysis of Burkholderia cepacia MZ-8

[0039] Genomic DNA was extracted and purified from strain MZ-8 using the TIANamp bacterial DNA extraction kit (Beijing Tiangen Biotech, catalog number DP302-02). Using the purified genomic DNA as a template, PCR amplification was performed using the universal bacterial 16S rDNA primers 27F / 1492R, i.e., 27F (5'-AGAGTTTGATCCTGGCTCAG-3', SEQ ID No. 2) and 1492R (5'-GGTTACCTTGTTACGACTT-3', SEQ ID No. 3). After amplification, an appropriate amount of PCR product was analyzed by 1.2% agarose gel electrophoresis. Once the target band was confirmed, the remaining PCR product was sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing.

[0040]

[0041] BLAST sequence alignment was performed in the NCBI (National Center for Biotechnology Information) database to download the gene base sequences of related strains with high homology. A phylogenetic tree was constructed using MEGA software with a neighbor-joining method. The results are as follows: Figure 2 As shown, this strain has a similarity of 99.73% to *Burkholderia cepacia*, and is therefore identified as *Burkholderia cepacia* strain MZ-8. Strain MZ-8 is a potential new species in the genus *Burkholderia*, hence its name *Burkholderia cepacia* MZ-8. This strain is deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou, Guangdong Province, 510070, China, with accession number GDMCC No. 66106 and deposit date of April 8, 2025.

[0042] Example 3: Identification of the inhibitory effect of Burkholderia cepacia MZ-8 on the pathogen of citrus canker.

[0043] The inhibitory effect of Burkholderia cepacia MZ-8 on the pathogen of citrus canker was determined using the plate agar diffusion method. Single colonies of both Burkholderia cepacia Xcc and Burkholderia cepacia MZ-8 were inoculated into LB liquid medium for activation, and the fermentation broth was collected after 24 h of incubation. LB solid medium (2% agar content) was prepared, sterilized, and cooled to 50°C. The Xcc fermentation broth was added at a ratio of 1:100, mixed thoroughly, and poured onto plates of appropriate thickness. 10 μL of Burkholderia cepacia MZ-8 bacterial suspension was spotted onto the solid medium, with three replicates, and incubated at 28°C for 48 h. The diameter of the inhibition zone was observed and measured.

[0044] like Figure 3 As shown, after the addition of Burkholderia cepacia MZ-8, the citrus canker pathogen Xcc formed a clear transparent ring around the well, with an average diameter of 25.0 mm. The results indicate that Burkholderia cepacia MZ-8 has a significant inhibitory effect on the growth of citrus canker pathogen Xcc.

[0045] Example 4: Determination of the organophosphate solubility of Burkholderia cepacia MZ-8

[0046] The ability of Burkholderia cepacia MZ-8 to dissolve organophosphates was determined using phosphate agar medium. Phosphate agar medium was prepared, sterilized, and poured onto plates of moderate thickness for later use. Single colonies of Burkholderia cepacia MZ-8 were transferred to liquid LB medium and incubated for 48 hours. 10 μL of the Burkholderia cepacia MZ-8 bacterial suspension was then spotted onto phosphate agar medium, with three replicates. The plates were incubated at 28°C, and the formation of a clear zone was observed. The diameter of the clear zone (D) and the colony diameter (d) were recorded after 48 hours of incubation.

[0047] like Figure 4 As shown, in phosphate agar medium, Burkholderia cepacia MZ-8 formed a clearly visible clear zone. After 48 hours of incubation, the average diameter (D) of the clear zone was 20.83 mm, the average colony diameter (d) was 6.27 mm, and the D / d ratio was 3.32. These results indicate that Burkholderia cepacia MZ-8 has a strong ability to dissolve insoluble phosphorus.

[0048] Example 5: Determination of the siderophore-producing capacity of Burkholderia cepacia MZ-8

[0049] The ability of Burkholderia morganii MZ-8 to produce siderophores was determined using CAS agar medium. CAS agar medium was prepared, sterilized, and poured onto plates of appropriate thickness for later use. Single colonies of activated MZ-8 were picked and transferred to LB liquid medium, incubated at 28°C for 48 h to obtain the seed culture. The MZ-8 seed culture and LB liquid medium were then mixed at a 1:10 ratio and incubated at 28°C in a shaker for 48 h to obtain the fermentation broth (MZ-8 fermentation broth concentration: 1.0 × 10⁻⁶). 8 CFU / mL). Take 10 μL of Burkholderia MZ-8 fermentation broth and inoculate it into CAS agar medium. Incubate at 28℃ for 72 h. Perform 3 replicates. Observe the orange halo around the colony and record the halo diameter D and the colony diameter d.

[0050] like Figure 5 As shown, in CAS agar medium, Burkholderia cepacia MZ-8 formed a clearly visible orange halo, with an average halo diameter D of 35.2 mm; the average colony diameter d was 6.83 mm; and the D / d ratio was 5.15. These test results indicate that Burkholderia cepacia MZ-8 has a good ability to produce siderophores. The siderophores it secretes can efficiently bind to iron in the surrounding environment, forming stable chelates, thereby effectively absorbing iron with very low solubility in the environment to promote plant growth.

[0051] Example 6: Determination of nitrogen fixation capacity of Burkholderia cepacia MZ-8

[0052] A single colony of Burkholderia cepacia MZ-8 strain was collected and placed in liquid LB medium and shaken for 48 hours. 10 μL of the bacterial suspension was then inoculated into nitrogen-fixing bacteria selection medium and incubated at 28°C for 48 hours. The growth of the strain on the medium was observed. If the strain could grow, it was identified as a nitrogen-fixing bacterium.

[0053] like Figure 6 As shown, Burkholderia cepacia MZ-8 can grow on nitrogen-fixing bacteria selection medium, indicating that this bacterium MZ-8 has nitrogen-fixing ability.

[0054] Example 7: Potted plant control efficacy test of Burkholderia cepacia MZ-8

[0055] The experimental cultivar "Orah mandarin" was purchased from Tianhe Nursery in Tianhe District, Guangdong Province, and potted in the greenhouse of South China Agricultural University. Five healthy potted seedlings with 15-20 healthy leaves were selected for the experiment. Single colonies of the citrus canker pathogen were incubated in 5 mL of NA liquid medium at 220 rpm and 28℃ for 24 h. Subsequently, 10% of the bacterial culture was transferred to 40 mL of NA liquid medium and incubated at 220 rpm and 28℃ for 24 h. The bacterial concentration was then adjusted to OD = 0.6 (2*10) with sterile water. 9 The inoculum solution was prepared as follows: CFU / mL. The inoculum solution of Burkholderia cepacia MZ-8 was prepared in the same manner. Subsequently, the inoculum solutions of *Citrus canker* and *Burkholderia cepacia* MZ-8 were mixed at a ratio of 1:2, and 0.1% Silwet L-77 was added. After thorough mixing, the inoculum solution was evenly sprayed onto both the upper and lower surfaces of the leaves using a sprayer. The mixture was incubated at 28℃ for 2–3 weeks. The number of needle pricks in the control and experimental groups was counted, and the number of diseased needle pricks was recorded. The results were then analyzed based on the formula... Calculate the incidence rate of inoculated leaves.

[0056] like Figure 7 As shown, leaves inoculated with Xcc exhibited obvious disease symptoms, including a yellow halo at the wound site and slight corking on the underside of the leaves. In contrast, potted plants inoculated with Burkholderia cepacia MZ-8 maintained good growth throughout, with an inhibition rate of 98.27%. This indicates that Burkholderia cepacia MZ-8 has a good inhibitory effect on Xcc.

[0057] The above results indicate that *Burkholderia cepacia* MZ-8, isolated in this invention, is a potential new species of the *Burkholderia* genus, possessing a significant ability to inhibit the growth of *Xcc*, the pathogen of citrus canker. It is also a highly efficient biocontrol strain, capable of being used directly as a spray for the control of citrus canker, or for the production of microbial fertilizers and inoculants, thus applicable to the control of the aforementioned plant diseases. *Burkholderia cepacia* MZ-8 can dissolve organic phosphorus, produce siderophores, and fix nitrogen, therefore it has great application potential in promoting the absorption of phosphorus, iron, and nitrogen in crops and in promoting crop growth.

[0058] The above are merely preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be considered as limitations on the present invention, and the scope of protection of the present invention should be determined by the scope defined in the claims. For those skilled in the art, several improvements and modifications can be made without departing from the spirit and scope of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A Burkholderia cepacia MZ-8 strain, characterized in that: The strain was deposited in Guangdong Provincial Microbiological Culture Collection Center (GDMCC) on April 8, 2025, with the deposit number GDMCC No: 66106.

2. A bacterial agent / biological agent, characterized in that: The bacterial agent / biological preparation contains the Burkholderia cepacia MZ-8 according to claim 1 and one or more of its culture solution, bacterial suspension and bacterial cells.

3. A microbial fertilizer / additive / pesticide, characterized in that: The microbial fertilizer / additive / pesticide contains one or more of the Burkholderia cepacia MZ-8 and its culture solution, bacterial suspension and bacterial cells as described in claim 1.

4. The microbial agent / biological agent according to claim 2 or the microbial fertilizer / additive / pesticide according to claim 3, characterized in that: The preparation method of the suspension, culture solution and bacterial cells comprises the following steps: inoculating and culturing Burkholderia cepacia MZ-8 to obtain a culture solution; centrifuging the culture solution and then resuspending the culture solution to obtain a bacterial suspension; and centrifuging the culture solution to obtain bacterial cells.

5. Use of the Burkholderia cepacia MZ-8 according to claim 1, the bacterial agent / biological agent according to claim 2, or the biological fertilizer / additive / pesticide according to claim 3 in promoting plant growth.

6. Use of the Burkholderia cepacia MZ-8 of claim 1, the bacterial agent / biological agent of claim 2, or the biological fertilizer / additive / pesticide of claim 3 in decomposing organic phosphorus, producing siderophores, and fixing nitrogen.

7. The use according to any one of claims 5 or 6, characterized in that: The application is to apply the Burkholderia cepacia MZ-8 described in claim 1 or the bacterial agent / biological agent described in claim 2 or the biological fertilizer / additive / pesticide described in claim 3 into the soil.

8. Use of the Burkholderia cepacia MZ-8 of claim 1, the bacterial agent / biological agent of claim 2, or the biological fertilizer / additive / pesticide of claim 3 in preventing and controlling citrus canker and the pathogens of citrus canker.

9. The application according to claim 8, characterized in that: The application is to directly apply or spray the Burkholderia cepacia MZ-8 described in claim 1 or the bacterial agent / biological agent described in claim 2 or the biological fertilizer / additive / pesticide described in claim 3 on the plant control area.

10. The use according to any one of claims 5 or 9, characterized in that: The plant is a Rutaceae plant.

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