Burkholderia metallica BW17 with weight-losing, disease-resisting and growth-promoting functions as well as fungicide and application of burkholderia metallica BW17

Abstract

The invention relates to the technical field of microorganisms, in particular to burkholderia metallica BW17 with weight-losing, disease-resisting and growth-promoting functions as well as a fungicide and application thereof, and the strain has good capabilities of producing IAA, fixing nitrogen, dissolving phosphorus and broad-spectrum antagonizing pathogenic bacteria. Pot experiments prove that the strain can significantly promote growth of pyrus betulaefolia and tomato seedlings, improve root morphology, increase biomass, and significantly improve appearance and internal quality of tomato fruits. In addition, the BW17 has an obvious inhibition effect on various phytopathogens. The strain can be widely applied to planting of crops such as fruit trees and vegetables as a bio-organic fertilizer or a microbial agent, partially replaces chemical fertilizers, reduces environmental pollution, improves the quality and yield of agricultural products, and has good popularization and application values.

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 the prior art, and the limitations of current biological agents in promoting growth and disease resistance, the present applicant has screened and isolated a strain of Burkholderia metallica from the rhizosphere of a peach orchard, which has multiple growth-promoting functions, excellent colonization ability and can significantly improve fruit quality Burkholderia metallica The strain BW17 can promote plant growth through mechanisms such as IAA production, nitrogen fixation and phosphorus solubilization, and enhance plant disease resistance by antagonizing pathogenic bacteria, thereby providing an efficient and environmentally friendly microbial agent for green agriculture, achieving the goal of reducing fertilizer and increasing efficiency, improving quality and increasing yield.

[0007] To achieve the above-mentioned purposes, the present application screens a strain of Burkholderia metallica Burkholderia metallica The strain BW17 is classified and named as: Burkholderia metallica BW17, which has a preservation number of CCTCC NO: M20251607; the strain is preserved in the China Center for Type Culture Collection, located in Wuhan, Wuhan University, China, and was preserved on July 15, 2025.

[0008] The present application also provides a microbial agent containing the Burkholderia metallica Burkholderia metallica strain BW17.

[0009] The present application also provides the Burkholderia metallica Burkholderia metallica strain BW17 or the microbial agent in the preparation of a bio-organic fertilizer.

[0010] The present application also provides the Burkholderia metallica Burkholderia metallica strain BW17 or the microbial agent in the preparation of a bio-organic fertilizer.

[0011] Further, the plant is tomato and / or pear.

[0012] The present application also provides the Burkholderia metallica Burkholderia metallica strain BW17 or the microbial agent in the preparation of a bio-organic fertilizer.

[0013] The present application also provides the Burkholderia metallica Burkholderia metallica strain BW17 or the microbial agent in the preparation of a bio-organic fertilizer.

[0014] Further, the plant pathogenic fungus is Magnaporthe oryzae ( Magnaporthe oryzae ), Alternaria alternata ( Alternaria alternata ) and / or Fusarium oxysporum ( Fusarium oxysporum ).

[0015] The present application also provides the Burkholderia metallica Burkholderia metallicaThe application of the strain BW17 or the bacterial agent in IAA production, nitrogen fixation, organic phosphorus and / or inorganic phosphorus decomposition.

[0016] The application also provides the application of the metal Burkholderia Burkholderia metallica The method for promoting plant growth by the strain BW17 or the bacterial agent, which comprises inoculating the metal Burkholderia Burkholderia metallica The strain BW17 bacterial suspension is irrigated to the rhizosphere of the plant seedlings.

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

[0018] As described above, the beneficial effects of the application are as follows:

[0019] The metal Burkholderia BW17 screened by the application has multiple growth-promoting and disease-resistant functions. The strain BW17 can secrete IAA, fix nitrogen, decompose organic phosphorus and inorganic phosphorus, significantly promote the growth of pear and tomato seedlings, improve the biomass, improve the root morphology, and enhance the stress resistance of the plants. In addition, the strain has significant antagonistic effect on multiple plant pathogens, and can reduce the occurrence of diseases. Inoculation of BW17 can also significantly improve the appearance and internal quality of tomato fruits, increase the fruit size, increase the sugar content, and reduce the acidity, which has high application value and market prospect. The strain can be used for developing biological bacterial fertilizer, partially replacing chemical fertilizer, and meeting the development needs of green and sustainable agriculture. BRIEF DESCRIPTION OF DRAWINGS

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

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

[0022] Figure 3 It is a phenotype diagram of the effect of inoculation of the strain BW17 on the growth of pear seedlings.

[0023] Figure 4 It is a result diagram of the effect of inoculation of the strain BW17 on the root morphology of pear seedlings.

[0024] Figure 5 It is a phenotype diagram of the effect of inoculation of the strain BW17 on the growth of tomato seedlings.

[0025] Figure 6 It is a result diagram of the effect of inoculation of the strain BW17 on the root morphology of tomato seedlings.

[0026] Figure 7 It is a result diagram of the effect of inoculation of the strain BW17 on the appearance quality of tomato fruits.

[0027] Figure 8 The results of the influence of inoculating strain BW17 on the internal quality of tomato fruits are shown in the figure.

[0028] Figure 9 The results of the inhibition of strain BW17 on different pathogens are shown in the figure. DETAILED DESCRIPTION

[0029] All features disclosed in this specification, and / or all steps of any methods or processes disclosed, may be combined in any combination, except combinations where at least some of such features and / or steps are mutually exclusive.

[0030] Any of the features disclosed in this specification, unless explicitly stated otherwise, are examples of a generic series of equivalent or similar features.

[0031] The culture medium, reagents and instruments involved in the experiment can be purchased on the market. Example 1

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

[0033] 1. Isolation and purification of strain BW17: 1 g of rhizosphere soil was collected from the rhizosphere of peach trees in Yangshan Peach Orchard in Wuxi, added to a 50 ml flask containing glass beads and 9 ml of SM buffer, and 30°C, 170 shaking for 30 min to obtain a soil suspension. Sterile water was gradually diluted to a concentration of 10-5~10-7, and then plated on TSA medium and cultured at 30°C for 48 h. Single bacteria were picked by plate streaking method to purify the strain.

[0034] 2. Identification of strain BW17: The morphological identification was performed as follows: BW17 strain was inoculated on TSA medium and cultured in a constant temperature incubator. After 24 h of culture, round colonies appeared on the culture dish, no spores, moist colonies, and light yellow color (as shown in Figure 1 ). According to the molecular identification (as shown in Figure 2 ), the strain was classified and named as Burkholderia metallica , and the Chinese classification name was Burkholderia metallica.

[0035] The applicant has preserved the Burkholderia metallica strain BW17 in the China Center for Type Culture Collection, with the preservation number CCTCC NO: M20251607, the preservation address being Wuhan, Wuhan University, China, and the preservation date being July 15, 2025. Example 2

[0036] This example is the study on the growth-promoting function of Burkholderia metallica strain BW17.

[0037] 1. Qualitative determination of IAA production function of the strain: the Burkholderia metallica BW17 strain was added to the TSB medium containing L-tryptophan at an inoculation amount of 1%, and 170 r.min-1 shaking cultivation was carried out at 30°C for 48 h. -1 After 48 h of shaking, the supernatant was obtained by centrifugation at 10,000 rpm for 5 min, 100 μl of the supernatant was mixed with an equal volume of Salkowski colorimetric solution, 100 μl of uninfected medium was mixed with an equal volume of Salkowski colorimetric solution as a blank control, and the solution was reacted for 30 min under dark conditions. The color of the solution turned red, indicating that the strain had the ability to produce IAA.

[0038] 2. Determination of the nitrogen fixation ability of the strain: the Asubie nitrogen fixation medium was used to qualitatively screen strains with nitrogen fixation ability.

[0039] 3. Determination of the ability of the strain to dissolve inorganic phosphorus: the PKO inorganic phosphorus plate method was used to qualitatively screen strains with inorganic phosphorus dissolution ability.

[0040] 4. Determination of the ability of the strain to dissolve organic phosphorus: the Mengjina organic phosphorus plate method was used to screen strains with organic phosphorus dissolution ability.

[0041] The above-mentioned determination of growth-promoting properties was carried out using the Burkholderia metallica BW17 strain, and the results are shown in Table 1.

[0042] As shown in Table 1, the BW17 strain has the abilities of IAA production, nitrogen fixation, organic phosphorus dissolution, and inorganic phosphorus dissolution. Example 3

[0043] This example is a study on the growth-promoting effect of the Burkholderia metallica BW17 bacterial agent on pear seedlings.

[0044] Pear seedling cultivation: the Du pear seeds were soaked in sterile water for 12 h, then disinfected with 1% hydrogen peroxide for 20 min, and finally washed clean with sterile water. The disinfected seeds were spread on the culture medium with sterile water-soaked filter paper, and germinated in a 28°C constant temperature incubator for 3-5 days. The white pear seedlings were selected and sown in sterilized seedling substrate. When the pear seedlings grew to 2-3 true leaves, healthy and uniform seedlings were selected and transplanted into sterilized cultivation substrate pots.

[0045] Inoculation: the prepared BW17 bacterial agent suspension (final concentration 10 7 CFU / g soil) was inoculated into the rhizosphere of Du pear seedlings, and the non-inoculated agent liquid was used as a control treatment (CK). Each treatment had 6 replicates, and the samples were collected after 30 d of cultivation for determination of the phenotypic traits of Du pear.

[0046] Test results:

[0047] 1. AsFigure 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.

[0048] 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

[0049] This example studies the growth-promoting effect of Burkholderia metalloid BW17 inoculum on pear seedlings.

[0050] (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.

[0051] 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.

[0052] 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.

[0053] (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.

[0054] CK group (control group): Tomato seedlings were cultivated alone without any bacterial inoculation.

[0055] Group BW17: Inoculated only with Burkholderia metallica BW17 bacterial suspension at a concentration of 10. 7 CFU / g soil.

[0056] Inoculation procedure: The prepared bacterial suspension is evenly inoculated into the root zone of tomato seedlings.

[0057] Experimental design: Six replicates were set up for each treatment group and cultured under the same environmental conditions.

[0058] 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.

[0059] Experimental results:

[0060] 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.

[0061] 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

[0062] This example illustrates the effect of Burkholderia metalloid BW17 inoculum on the quality of tomato fruits.

[0063] (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.

[0064] 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.

[0065] 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.

[0066] (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.

[0067] CK group (control group): Tomato seedlings were cultivated alone without any bacterial inoculation.

[0068] Group BW17: Inoculated only with Burkholderia metallica BW17 bacterial suspension at a concentration of 10. 7 CFU / g soil.

[0069] Inoculation procedure: The prepared bacterial suspension is evenly inoculated into the root zone of tomato seedlings.

[0070] 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.

[0071] Experimental results:

[0072] 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.

[0073] 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

[0074] This example is a plate confrontation test between Burkholderia metalloid BW17 strain and various pathogens.

[0075] 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.

[0076] 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.

[0077] In summary, the test proves that the metal Burkholderia BW17 strain screened by the applicant has good abilities of IAA production, nitrogen fixation, organic phosphorus degradation and inorganic phosphorus degradation, and can significantly promote the growth of pear and tomato seedlings, improve the root morphology and increase the plant biomass. In addition, the strain BW17 has obvious antagonistic effect on pathogenic fungi such as Magnaporthe oryzae, Alternaria alternata and Fusarium oxysporum f. sp. lycopersici. The pot experiment shows that the inoculation of BW17 bacterial suspension can significantly improve the growth indexes of pear and tomato such as the dry weight of aboveground part, the dry weight of root system, the plant height and the stem diameter, and can improve the appearance and internal quality of tomato fruits, significantly improve the longitudinal diameter, transverse diameter, single fruit weight, total sugar content and soluble solid content of tomato fruits, and reduce the acidity. The strain can be used for preparing biological bacterial agent or biological organic fertilizer, reducing the use of chemical fertilizer and promoting the development of green agriculture.

[0078] Although the present application has been described in detail with general description and specific embodiments above, some modifications or improvements can be made on the basis of the present application, which is obvious to those skilled in the art. Therefore, these modifications or improvements made on the basis of not deviating from the spirit of the present application, all belong to the scope of the present application claimed.

Claims

1. Metallo-bacillus Burkholderia metallica Strain BW17, which is classified and named as: Burkholderia metallica BW17, with the accession number CCTCC NO: M20251607; the strain is preserved in the China Center for Type Culture Collection, Wuhan University, Wuhan, China, and the preservation date is July 15, 2025.

2. A composition comprising the metallo-Burkholderia of claim 1 Burkholderia metallica Inoculum of strain BW17.

3. The metallo-Burkholderia of claim 1 Burkholderia metallica Use of the bacterial agent of strain BW17 or of claim 2 in the preparation of bio-organic fertilizers.

4. The metallo-Burkholderia of claim 1 Burkholderia metallica Use of the bacterial inoculant of strain BW17 or of claim 2 in promoting plant growth.

5. The use according to claim 4, wherein the compound is ###0002### The plant is tomato and / or pear.

6. The metallo-Burkholderia of claim 1 Burkholderia metallica Use of the bacterial inoculant of strain BW17 or claim 2 to improve fruit quality in plants.

7. The metallo-Burkholderia of claim 1 Burkholderia metallica The use of the bacterial strain BW17 or the bacterial agent of claim 2 in the preparation of a biocontrol agent for preventing and controlling plant pathogenic bacteria.

8. Use according to claim 7, wherein the compound is ###0002### The plant pathogen is *Oryza sativa* (*Oryza sativa*). Magnaporthe oryzae Alternaria ( Alternaria alternata ) and / or Fusarium oxysporum ( Fusarium oxysporum ).

9. The metallo-Burkholderia of claim 1 Burkholderia metallica Use of the bacterial agent of strain BW17 or of claim 2 for the production of IAA, nitrogen fixation, organic and / or inorganic phosphorus solubilization.

10. Use of the metallo-Burkholderia as claimed in claim 1 Burkholderia metallica The method for promoting plant growth by the bacterial agent of strain BW17 or as claimed in claim 2, characterized in that, The method is to apply a suspension of the Burkholderia metallica Burkholderia metallica Strain BW17 bacterial suspension is applied to the rhizosphere of the plant seedlings.

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