Burkholderia metalloids BW17 with weight loss, disease resistance, and growth-promoting functions, and its inoculum and applications.

Abstract

This invention relates to the field of microbial technology, specifically to Burkholderia metalloids BW17, which possesses functions of reducing fertilizer use, resisting disease, and promoting growth, along with its inoculants and applications. This strain exhibits excellent abilities in IAA production, nitrogen fixation, phosphorus solubilization, and broad-spectrum antagonism against pathogens. Pot experiments have demonstrated that this strain significantly promotes the growth of pear and tomato seedlings, improves root morphology, increases biomass, and significantly enhances the appearance and internal quality of tomato fruits. Furthermore, BW17 exhibits significant inhibitory effects against various plant pathogens. This strain can be widely used as a bio-organic fertilizer or microbial inoculant in the cultivation of fruit trees, vegetables, and other crops, partially replacing chemical fertilizers, reducing environmental pollution, and improving the quality and yield of agricultural products, thus possessing significant potential for widespread application.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to Burkholderia metalloids BW17, which has functions of weight loss, disease resistance, and growth promotion, as well as its inoculants and applications. Background Technology

[0002] In agricultural planting, there are various methods to promote plant growth and improve fruit quality, but traditional methods have some limitations. For example, while the use of chemical fertilizers can provide the nutrients needed for plant growth to a certain extent, long-term excessive use can lead to soil compaction, disrupt the soil's micro-ecological balance, reduce the sustainability of soil fertility, and pose risks such as environmental pollution. Moreover, their effect on improving fruit quality at a deeper level is limited, making it difficult to meet the current demand for high-quality agricultural products.

[0003] Currently, common biological agents mainly include single-strain agents, such as Trichoderma ( Trichoderma spp. This includes various plant growth-promoting bacteria (PGPRs). Among them, plant rhizosphere growth-promoting bacteria (PGPBs) play an important role in improving soil nutrient supply and promoting plant growth. Certain strains in the Burkholderiaceae family have shown great potential in agriculture. These microorganisms can promote plant growth through multiple pathways, such as secreting plant hormones (IAA, etc.), producing ACC deaminase to reduce ethylene concentration in plants, and increasing plant nutrient supply through nitrogen fixation, phosphorus solubilization, and potassium solubilization, thereby significantly promoting the growth of plant roots and aboveground parts.

[0004] Furthermore, compared to Bacillus, Burkholderia has a stronger colonization ability, effectively establishing itself in the rhizosphere and endogenous tissues of plants, forming a close symbiotic relationship with them, and providing long-term growth-promoting and biocontrol effects. It also promotes root development, enhances crop resistance to adverse conditions, and inhibits pathogen growth through antagonistic effects. Combining these functions with traditional fertilization can not only improve fertilizer utilization but also achieve the goals of green and sustainable agriculture.

[0005] Burkholderia metalloids is a genus within the family Burkholderiaceae. Currently, research on the mechanisms by which Burkholderia metalloids promote plant growth and improve fruit quality is relatively scarce. Further in-depth research is urgently needed to reveal its potential value and provide more theoretical support and technical means for the development of green agriculture. Summary of the Invention

[0006] In view of the soil and environmental problems caused by the use of chemical fertilizers in existing technologies, and the limitations of current biological agents in promoting growth and disease resistance, the applicant has screened and isolated a strain of Burkholderia metalloids from the rhizosphere of a peach orchard in Wuxi. This strain has multiple growth-promoting functions, excellent colonization ability, and can significantly improve fruit quality. Burkholderia metallica BW17 is a strain that can promote plant growth through mechanisms such as IAA production, nitrogen fixation, and phosphorus solubilization, and enhance plant disease resistance by antagonizing pathogens. It provides a highly efficient and environmentally friendly microbial agent for green agriculture, achieving the goals of reducing fertilizer use, increasing efficiency, improving quality, and increasing yield.

[0007] To achieve the above objectives, this invention screened out a strain of Burkholderia metalloids. Burkholderia metallica Strain BW17 is classified as follows: Burkholderia metallica BW17, with accession number CCTCCNO: M20251607, is deposited at the China Center for Type Culture Collection, Wuhan University, Wuhan, China, on July 15, 2025.

[0008] The present invention also provides Burkholderia metalloids described above. Burkholderia metallica Inoculum of strain BW17.

[0009] The present invention also provides the aforementioned Burkholderia metalloids. Burkholderia metallica Application of strain BW17 or the aforementioned microbial agent in the preparation of bio-organic fertilizer.

[0010] The present invention also provides the aforementioned Burkholderia metalloids. Burkholderia metallica Application of strain BW17 or the aforementioned microbial agent in promoting plant growth.

[0011] To further clarify, the plant in question is a tomato and / or a wild pear.

[0012] The present invention also provides the aforementioned Burkholderia metalloids. Burkholderia metallica Application of strain BW17 or the aforementioned inoculant in improving the quality of plant fruits.

[0013] The present invention also provides the aforementioned Burkholderia metalloids. Burkholderia metallica The application of strain BW17 or the aforementioned bacterial agent in the preparation of biocontrol agents for the control of plant pathogens.

[0014] To further clarify, the plant pathogen is *Oryza sativa* (the causal agent of rice blast). Magnaporthe oryzae Alternaria ( Alternaria alternata ) and / or Fusarium oxysporum ( Fusarium oxysporum ).

[0015] The present invention also provides the aforementioned Burkholderia metalloids. Burkholderia metallicaApplication of strain BW17 or the aforementioned bacterial agent in the production of IAA, nitrogen fixation, and the solubilization of organic and / or inorganic phosphorus.

[0016] This invention also provides the application of the aforementioned Burkholderia metalloids. Burkholderia metallica A method for promoting plant growth using strain BW17 or the aforementioned bacterial agent, the method comprising: administering Burkholderia metalloids... Burkholderia metallica The bacterial suspension of strain BW17 was applied to the root zone of the plant seedlings.

[0017] To further clarify, the final concentration of the bacterial suspension is 10. 7 CFU / g soil.

[0018] In summary, due to the adoption of the above technical solution, the beneficial effects of the present invention are:

[0019] The *Burkholderia metalloids* BW17 strain screened in this invention possesses multiple growth-promoting and disease-resistant functions. Strain BW17 can secrete IAA, fix nitrogen, and dissolve organic and inorganic phosphorus, significantly promoting the growth of pear and tomato seedlings, increasing biomass, improving root morphology, and enhancing plant resistance. Furthermore, this strain exhibits significant antagonistic effects against various plant pathogens, reducing disease occurrence. Inoculation with BW17 can also significantly improve the appearance and internal quality of tomato fruits, resulting in larger fruits, increased sugar content, and decreased acidity, demonstrating high application value and market potential. This strain can be used to develop bio-fertilizers, partially replacing chemical fertilizers, meeting the development needs of green and sustainable agriculture. Attached Figure Description

[0020] Figure 1 This is a colony morphology diagram of strain BW17.

[0021] Figure 2 This is a phylogenetic tree diagram of strain BW17.

[0022] Figure 3 Phenotypic diagram of the effect of inoculation with strain BW17 on the growth of pear seedlings.

[0023] Figure 4 Figure showing the effect of inoculation with strain BW17 on the root morphology of pear seedlings.

[0024] Figure 5 Phenotypic diagram showing the effect of inoculation with strain BW17 on the growth of tomato seedlings.

[0025] Figure 6 Figure showing the effect of inoculation with strain BW17 on the root morphology of tomato seedlings.

[0026] Figure 7 The figure shows the effect of inoculation with strain BW17 on the appearance quality of tomato fruits.

[0027] Figure 8 The figure shows the effect of inoculation with strain BW17 on the internal quality of tomato fruits.

[0028] Figure 9 This is a graph showing the inhibition results of strain BW17 against different pathogens. Detailed Implementation

[0029] All features disclosed in this specification, or steps in all methods or processes disclosed herein, may be combined in any way, except for mutually exclusive features and / or steps.

[0030] Unless otherwise stated, each feature disclosed in this specification (including any appended claims and abstract) is merely one example of a series of equivalent or similar features.

[0031] The culture media, reagents, and instruments involved in this experiment are all commercially available. Example 1

[0032] This example describes the screening, identification, and biological characteristics of Burkholderia metalloid BW17 strain.

[0033] 1. Isolation and purification of strain BW17: 1g of rhizosphere soil was collected from the rhizosphere of peach trees in Yangshan Peach Garden, Wuxi. It was added to a 50ml Erlenmeyer flask containing glass beads and 9ml of SM buffer. The soil suspension was obtained by shaking at 170°C for 30min at 30°C. The suspension was then gradually diluted with sterile water to a concentration of 10⁻⁵ to 10⁻⁷. The suspension was then spread on TSA medium and incubated at 30°C for 48h. Single bacteria were picked using the streak plate method to purify the strain.

[0034] 2. Identification of strain BW17: The specific procedure for morphological identification is as follows: BW17 strain was inoculated onto TSA medium and incubated in a constant temperature incubator. After 24 hours of incubation, round colonies were observed to appear on the culture dish. These colonies were non-spore-forming, moist, and pale yellow (e.g., ...). Figure 1 ). Identified by molecular methods (e.g.) Figure 2 This strain is classified and named Burkholderia metallica Its Chinese classification name is Burkholderia metalloids.

[0035] The applicant has deposited Burkholderia metalloids strain BW17 at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M20251607; deposit address: Wuhan University, Wuhan, China; deposit date: July 15, 2025. Example 2

[0036] This example illustrates the growth-promoting function of Burkholderia metalloids strain BW17.

[0037] 1. Qualitative determination of IAA production function of the strain: Burkholderia metalloids strain BW17 was added to TSB medium containing L-tryptophan at an inoculum of 1%, and incubated at 30℃ and 170 r.min. -1 After shaking for 48 hours, the supernatant was obtained by centrifugation at 10,000 rpm for 5 minutes. 100 μl of the supernatant was mixed with an equal volume of Salkowski colorimetric solution. 100 μl of uninoculated culture medium was mixed with an equal volume of Salkowski colorimetric solution as a blank control. The mixture was reacted for 30 minutes in the dark. The solution turning red indicates that the strain has the ability to produce IAA.

[0038] 2. Determination of nitrogen fixation capacity of strains: Strains with nitrogen fixation capacity were qualitatively screened using Assumption nitrogen fixation medium.

[0039] 3. Determination of the ability of strains to dissolve inorganic phosphorus: Strains with inorganic phosphorus dissolving ability were qualitatively screened using the PKO inorganic phosphorus plate method.

[0040] 4. Determination of the ability of strains to dissolve organophosphates: The Monkina organophosphate plate method was used to screen strains with organophosphate dissolving ability.

[0041] The above growth-promoting characteristics were all measured using Burkholderia metalloid BW17 strain. The results are shown in Table 1.

[0042]

[0043] As shown in Table 1, strain BW17 has the ability to produce IAA, fix nitrogen, and solubilize organic and inorganic phosphorus. Example 3

[0044] This example studies the growth-promoting effect of Burkholderia metalloid BW17 inoculum on pear seedlings.

[0045] Pear seedling cultivation: Soak pear seeds in sterile water for 12 hours, then disinfect with 1% hydrogen peroxide for 20 minutes, and finally rinse thoroughly with sterile water. Spread the disinfected seeds evenly on a culture medium lined with sterile water-moistened filter paper, and place them in a 28℃ constant temperature incubator to germinate for 3-5 days. Select pear seedlings with sprouting white leaves and sow them in sterilized seedling substrate. When the pear seedlings have 2-3 true leaves, select healthy and uniform seedlings and transplant them into sterilized cultivation substrate pots.

[0046] Inoculation: Inoculate the prepared BW17 bacterial suspension (final concentration 10). 7 CFU / g soil was inoculated into the rhizosphere of *Pyrus pyrifolia* seedlings, with the uninoculated solution serving as a control (CK). Each treatment was replicated 6 times, and samples were collected after 30 days of culture to determine the phenotypic traits of *Pyrus pyrifolia*.

[0047] Experimental results:

[0048] 1. For example Figure 3 As shown, BW17 inoculant significantly promoted the growth of pear seedlings. Compared with the control (CK), the aboveground dry weight, root dry weight, and total dry weight of pear seedlings treated with BW17 were significantly increased by 46.5%, 34.2%, and 43.0%, respectively. The plant height, stem diameter, and SPAD of pear seedlings treated with BW17 were significantly increased by 75.1%, 32.7%, and 32.1%, respectively, compared with the CK.

[0049] 2. For example Figure 4 As shown, BW17 inoculant can improve the root morphology of pear seedlings, increasing root surface area, root length and root volume by 38.7%, 13.0% and 14.8%, respectively. Example 4

[0050] This example studies the growth-promoting effect of Burkholderia metalloid BW17 inoculum on pear seedlings.

[0051] (1) Tomato seedling cultivation: Seed treatment: Take healthy tomato seeds and soak them in sterile water for 24 hours; then soak them in 75% alcohol for 30 seconds, and then rinse them with sterile water 3-5 times; then soak them in 2% sodium hypochlorite solution for 20 minutes, and finally rinse them thoroughly with plenty of sterile water.

[0052] Germination and sowing: Spread the sterilized seeds evenly on a culture medium lined with sterile, water-moistened filter paper, and place them in a 28℃ constant temperature incubator for 3-5 days to germinate. Select seeds with good germination and sow them in sterile culture medium or sterile substrate for seedling cultivation.

[0053] Transplanting: Once the tomato seedlings have grown to 2-3 true leaves and are growing evenly, select healthy and uniform seedlings and transplant them into sterilized substrate pots for pot cultivation.

[0054] (2) Inoculation treatment: Preparation of bacterial suspension: According to the above method, a bacterial suspension of Burkholderia metallica BW17 was prepared and its final concentration was adjusted to 10. 7 CFU / g soil.

[0055] CK group (control group): Tomato seedlings were cultivated alone without any bacterial inoculation.

[0056] Group BW17: Inoculated only with Burkholderia metallica BW17 bacterial suspension at a concentration of 10. 7 CFU / g soil.

[0057] Inoculation procedure: The prepared bacterial suspension is evenly inoculated into the root zone of tomato seedlings.

[0058] Experimental design: Six replicates were set up for each treatment group and cultured under the same environmental conditions.

[0059] Results collection: After 30 days of cultivation, the growth phenotypes of tomato seedlings in each group were measured and recorded, including indicators such as plant height, root development, and biomass.

[0060] Experimental results:

[0061] 1. For example Figure 5 As shown, inoculation with BW17 bacterial agent significantly promoted the growth of tomato seedlings. Compared with the control CK, the aboveground dry weight, root dry weight and total dry weight of tomato seedlings treated with BW17 bacterial suspension increased significantly by 43.9%, 167.6% and 48.0%, respectively, while the stem diameter and plant height increased by 27.3% and 46.1%, respectively, compared with the CK.

[0062] 2. For example Figure 6 As shown, inoculation with BW17 bacterial agent can significantly improve the root morphology of tomatoes. Compared with the control CK, the root length, root surface area, number of root tips and average root diameter of tomatoes treated with BW17 bacterial suspension increased by 196.7%, 210.5%, 34.7% and 37.17%, respectively. Example 5

[0063] This example illustrates the effect of Burkholderia metalloid BW17 inoculum on the quality of tomato fruits.

[0064] (1) Tomato seedling cultivation: Seed treatment: Take healthy tomato seeds and soak them in sterile water for 24 hours; then soak them in 75% alcohol for 30 seconds, and then rinse them with sterile water 3-5 times; then soak them in 2% sodium hypochlorite solution for 20 minutes, and finally rinse them thoroughly with plenty of sterile water.

[0065] Germination and sowing: Spread the sterilized seeds evenly on a culture medium lined with sterile, water-moistened filter paper, and place them in a 28℃ constant temperature incubator for 3-5 days to germinate. Select seeds with good germination and sow them in sterile culture medium or sterile substrate for seedling cultivation.

[0066] Transplanting: Once the tomato seedlings have grown to 2-3 true leaves and are growing evenly, select healthy and uniform seedlings and transplant them into sterilized substrate pots for pot cultivation.

[0067] (2) Inoculation treatment: Preparation of bacterial suspension: According to the above method, a bacterial suspension of Burkholderia metallica BW17 was prepared and its final concentration was adjusted to 10. 7 CFU / g soil.

[0068] CK group (control group): Tomato seedlings were cultivated alone without any bacterial inoculation.

[0069] Group BW17: Inoculated only with Burkholderia metallica BW17 bacterial suspension at a concentration of 10. 7 CFU / g soil.

[0070] Inoculation procedure: The prepared bacterial suspension is evenly inoculated into the root zone of tomato seedlings.

[0071] Experimental design: Each treatment group was set up with 6 replicates and cultured under the same environmental conditions. Fruit quality and other relevant indicators were then measured.

[0072] Experimental results:

[0073] 1. For example Figure 7 As shown, inoculation with BW17 bacterial suspension significantly improved the appearance quality of tomato fruits. Compared with the control (CK), the longitudinal diameter, transverse diameter, and single fruit weight of tomatoes treated with BW17 bacterial suspension increased significantly by 33.1%, 34.7%, and 139.3%, respectively.

[0074] 2. For example Figure 8 As shown, inoculation with BW17 bacterial suspension significantly improved the internal quality of tomato fruits. Compared with the control (CK), the water content, total sugar content, and soluble solids content of tomato fruits treated with BW17 bacterial suspension increased significantly by 15.8%, 13.8%, and 9.4%, respectively; the titratable acid content of tomato fruits treated with BW17 bacterial suspension decreased significantly by 18.6% compared with the CK treatment. Example 6

[0075] This example is a plate confrontation test between Burkholderia metalloid BW17 strain and various pathogens.

[0076] Multiple pathogens were cultured on PDA medium. Then, the activated fungal strains were prepared into 5mm mycelial discs and transferred to the center of a new PDA plate. 1μl of OD200 was then spotted around the fungal discs. 600 The bacterial suspension was prepared at 0.80. After the suspension dried, the plates were placed in an incubator at 28°C for incubation. Sterile water was spotted around the perimeter as a control group. Each treatment was performed in triplicate. The plate phenomena were observed and photographed after 5 days of incubation.

[0077] Result: As Figure 9 As shown, strain BW17 can significantly antagonize rice blast fungus (… Magnaporthe oryzae Alternaria ( Alternaria alternata ) and Fusarium oxysporum tomato-specific ( Fusarium oxysporum ), while specifically targeting the cucumber type of Fusarium oxysporum ( Fusarium oxysporum It played a certain inhibitory role.

[0078] In summary, the above experiments demonstrate that the *Burkholderia metalloids* strain BW17, selected by the applicant, possesses excellent abilities in IAA production, nitrogen fixation, organic phosphorus solubilization, and inorganic phosphorus solubilization. It also significantly promotes the growth of pear and tomato seedlings, improves root morphology, and increases plant biomass. Furthermore, strain BW17 exhibits significant antagonistic effects against pathogens such as *Bacillus oryzae*, *Alternaria alternata*, and *Fusarium oxysporum* tomato-specific strain. Pot experiments showed that inoculation with BW17 bacterial suspension significantly increased aboveground dry weight, root dry weight, plant height, and stem diameter in pear and tomato, and improved the appearance and internal quality of tomato fruits, with significant increases in longitudinal and transverse diameter, single fruit weight, total sugar content, and soluble solids content, while reducing acidity. This strain can be used to prepare bio-inoculants or bio-organic fertilizers, reducing the use of chemical fertilizers and promoting green agriculture.

[0079] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. Burkholderia metalloids ( Burkholderia metallica Strain BW17, its classification name is: Burkholderia metallica BW17, with accession number CCTCC NO: M20251607, is deposited at the China Center for Type Culture Collection, Wuhan University, Wuhan, China, on July 15, 2025.

2. An inoculant comprising the metal Burkholderia strain BW17 of claim 1.

3. Use of the metal Burkholderia strain BW17 of claim 1 or the inoculant of claim 2 in preparing bio-organic fertilizer.

4. Use of the metal Burkholderia strain BW17 of claim 1 or the inoculant of claim 2 in promoting plant growth; the plant is tomato or pear.

5. Use of the metal Burkholderia strain BW17 of claim 1 or the inoculant of claim 2 in improving fruit quality of the plant; the plant is tomato; the improving fruit quality of the plant comprises improving appearance quality and internal quality of tomato fruit; the improving appearance quality of tomato fruit comprises increasing longitudinal diameter, transverse diameter and single fruit weight of tomato fruit; the improving internal quality of tomato fruit comprises increasing water content, total sugar content and soluble solid content and decreasing titratable acid content of tomato fruit.

6. Use of the Burkholderia metallica strain BW17 of claim 1 or the microbial agent of claim 2 in the preparation of a biocontrol agent for preventing and controlling plant pathogenic fungi; the plant pathogenic fungi being Magnaporthe grisea (Pyricularia grisea) Magnaporthe oryzae , Alternaria alternata (Alternaria alternata) Alternaria alternata and / or Fusarium oxysporum (Fusarium oxysporum) Fusarium oxysporum .​​​​​​ 7. Use of the metal Burkholderia strain BW17 of claim 1 or the inoculant of claim 2 in producing IAA, fixing nitrogen, degrading organic phosphorus and / or degrading inorganic phosphorus.

8. The method for promoting plant growth by applying the Burkholderia metallica strain BW17 according to claim 1 or the microbial agent according to claim 2, characterized in that, The method is: applying the metal Burkholderia strain BW17 to the rhizosphere of the plant seedling; the plant is tomato or pear. Burkholderia metallica Burkholderia metallica strain BW17 to the rhizosphere of the plant seedling; the plant is tomato or pear.

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