Paenibacillus polymyxa strain ynk-fb0060 and application thereof

Abstract

The application belongs to the technical field of microorganisms, and relates to a Paenibacillus polymyxa YNK-FB0060 and application thereof. Paenibacillus polymyxa The Paenibacillus polymyxa YNK-FB0060 is an advantageous functional strain with high cellulase yield, and has a preservation number of CCTCC NO: M2026411. In a fermentation process, the strain has strong cellulase secretion capacity, wherein CMCase activity reaches 17.41+ / -1.30 U / mL at the third day of culture, and FPase activity reaches 5.42+ / -1.93 U / mL at the fifth day. Meanwhile, the strain has the characteristics of decomposing organic phosphorus, dissolving zinc, oxidizing sulfur, producing siderophore, producing protease and producing IAA, and has a significant inhibiting effect on a plurality of plant pathogenic fungi such as tomato early blight, rice leaf spot disease and banana wilt. The strain provided in the application has the advantages of efficient cellulose degradation, plant growth promotion and broad-spectrum disease resistance, and has a good application prospect in agricultural waste resource utilization, soil nutrient transformation and green plant disease control.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a polymyxa bacillus strain YNK-FB0060 and its applications. Background Technology

[0002] Cellulose agricultural waste (such as straw, bamboo processing waste, and forestry residues) is generated in large quantities during agricultural production and processing. Direct incineration or landfill not only wastes renewable biomass resources but also causes air pollution, increased greenhouse gas emissions, and ecological degradation. Therefore, utilizing highly efficient cellulose-degrading microorganisms to achieve the biotransformation and resource utilization of such waste has significant ecological and economic implications.

[0003] In the process of cellulose biodegradation, the cellulase system mainly consists of various enzymes such as endocarboxymethyl cellulase (CMCase), exocarboxymethyl cellulase, and β-glucosidase. Among them, CMCase mainly acts on the amorphous structure of cellulose, while the activity of filter paper enzyme (FPase) can comprehensively reflect the overall degradation ability of the cellulase system on natural cellulose substrates, and is an important indicator for evaluating the potential of microbial cellulose degradation. Existing studies have systematically evaluated the cellulose degradation ability of cellulose-degrading bacteria by isolating and screening them, and combining cellulase activity determination and straw degradation rate as indicators. "Si Haili, Ji Lidong, Li Xiaohui, et al. Screening, identification and straw degradation effect of cellulose-degrading bacteria [J]. Jiangsu Agricultural Sciences, 2024, 52(18):277-283.DOI:10.15889 / j.issn.1002-1302.2024.18.034" records Bacillus polymyxa ( Paenibacillus polymyxa Within 60 hours of cultivation, the filter paper strips showed significant disintegration, and within 21 days, the degradation rate of rice straw reached 48.26%, demonstrating significant potential for the degradation of natural cellulose.

[0004] In addition to its cellulose degradation function, Polymyxin Bacillus (… Paenibacillus polymyxa It is also an important plant growth-promoting rhizosphere microorganism, with multiple functions in promoting plant growth and improving crop stress resistance. The article “Bu Fan, Han Sining, Zhu Rengui, et al. Isolation, identification and evaluation of salt-tolerant growth-promoting effect of a salt-tolerant polymyxa strain TaRb44 [J]. Acta Microbiologica Sinica, 2025, 65(04):1498-1511. DOI:10.13343 / j.cnki.wsxb.20240726” records the isolation and identification of a salt-tolerant polymyxa strain TaRb44 as… Paenibacillus polymyxaThis strain not only has good salt and alkali tolerance, but also produces a variety of plant growth-promoting active substances, including siderophores (side carriers), amylases and water-organic phosphorus enzymes, and also has biological nitrogen fixation activity. In greenhouse pot experiments, this strain can significantly promote the growth of wheat seedlings and increase root biomass, indicating that it has potential application value in enhancing plant nutrient absorption and promoting plant growth. In addition, this strain can also synthesize plant growth regulators such as indoleacetic acid (IAA), and has shown significant growth-promoting effects in a variety of crop systems (Wang Fei, Li Xuemeng, Yang Jin, et al. Screening, identification and effect evaluation of three rhizosphere growth-promoting bacteria of Salvia miltiorrhiza [J]. Henan Agricultural Sciences, 2022, 51(12):81-89.DOI:10.15933 / j.cnki.1004-3268.2022.12.010). In terms of biological control, Bacillus polymyxa has significant inhibitory activity against a variety of plant pathogenic fungi. For example, it shows significant inhibitory activity against Botrytiscinerea, the pathogen of tomato gray mold, with an inhibition rate of up to 41.04% (Zhang Jianqiang, Wei Tao, Lai Xin, et al. Preliminary study on the biocontrol mechanism of Bacillus polymyxa YF against tomato gray mold [J]. China Vegetables, 2025, 38(10):61-67.DOI:10.16861 / j.cnki.zggc.2025.0020). At the same time, it also has a high inhibitory effect on the anthracnose pathogen of watermelon, with an inhibition rate of 81.00%, showing good potential for biocontrol application (Zhang Yan, Xu Yanping, Qiu Jiajia, et al. Effect of Bacillus polymyxa NX6 on antioxidant enzymes in watermelon seedlings infected with anthracnose [J]. Jiangsu Agricultural Sciences, 2024, 52(18):147-154.DOI:10.15889 / j.issn.1002-1302.2024.18.019).

[0005] However, existing microbial resources have significant limitations: some strains have low enzyme production capacity, incomplete cellulase systems, or insufficient enzyme activity stability; while some strains possess cellulose degradation capabilities, they lack complex functions such as promoting plant growth or inhibiting plant pathogenic fungi, exhibiting overall single-function characteristics. As agriculture develops towards high efficiency, greenness, and sustainability, single-function strains are insufficient to meet the integrated application needs of agricultural waste biological treatment, crop yield enhancement, and green disease control. Therefore, there is an urgent need to develop novel microbial resources that combine high-efficiency cellulose degradation capacity, significant plant growth-promoting effects, and broad-spectrum stable biocontrol activity. This would enable the synergistic application of multiple functions within the same biological agent, improving the efficiency of waste resource utilization, reducing fertilizer and pesticide inputs, and promoting healthy crop growth. This would overcome the technical bottlenecks of existing strains in terms of enzyme production levels, functional diversity, and application stability, possessing significant theoretical value and broad application prospects. Summary of the Invention

[0006] To address the above shortcomings, this invention provides a strain of *Bacillus polymyxa* YNK-FB0060 and its applications. The *Bacillus polymyxa* strain provided by this invention (… Paenibacillus polymyxa This strain, isolated from soil from tobacco-growing farmland in Lijiang City, Yunnan Province, exhibits strong cellulase production capabilities, as well as the ability to produce indoleacetic acid (IAA) and oxidize thiosulfate to sulfate. Furthermore, this strain can inhibit early blight of tomato. Alternaria solani Rice leaf spot disease ( Bipolaris oryzae Anthracnose of avocado ( Colletotrichum aeschynomenes ); Blueberry Alternaria fruit rot ( Alternaria sp . Tobacco red star disease ( Alternaria alternata ); banana wilt disease ( Fusarium oxysporum f. sp . cubense It has an inhibitory effect on a variety of plant pathogenic fungi.

[0007] To achieve the above objectives, the first aspect of the present invention provides a strain of *Bacillus polymyxa* (…). Paenibacillus polymyxa The strain is numbered YNK-FB0060 and its preservation number is CCTCC NO: M2026411.

[0008] A second aspect of the present invention provides a bacterial agent, the active ingredient of which is Bacillus polymyxa of the first aspect.

[0009] The third aspect of this invention provides the application of the *Bacillus polymyxa* described in the first aspect or the bacterial agent described in the second aspect in degrading cellulose and / or promoting plant growth, and / or controlling early blight of tomato, leaf spot of rice sesame, anthracnose of avocado, *Alternaria alternata* fruit rot of blueberry, red spot of tobacco, and wilt of banana.

[0010] Furthermore, the pathogen of early blight in tomatoes is Alternaria alternata (Solanum lyratum). Alternaria solani The pathogen of rice leaf spot disease is *Helicobacter pylori* (rice leaf spot). Bipolaris oryzae The pathogen causing avocado anthracnose is *Colletotrichum spp.* Colletotrichum aeschynomenes The pathogen of blueberry Alternaria fruit rot is a fungus of the genus Alternaria. Alternaria The pathogen of tobacco red spot disease is Alternaria sp. Alternaria alternata The pathogen causing banana wilt is Fusarium oxysporum, specifically the Cuban variant. Fusarium oxysporum f. sp. cubense ).

[0011] Through the above technical solution, the present invention can achieve at least the following beneficial effects: (1) The polymyxa bacillus provided by the present invention has a significant cellulase production capacity, wherein the carboxymethyl cellulase (CMCase) activity is 17.41±1.30U / mL and the filter paper enzyme (FPase) activity is 5.42±1.93U / mL, which can effectively promote the degradation of cellulose and cellulose-based agricultural waste.

[0012] (2) The polymyxa bacillus provided by the present invention can produce indoleacetic acid (IAA) with a yield of 8.039 μg / mL, which can promote plant root growth, cell division and differentiation, and improve crop growth and development.

[0013] (3) The polymyxa bacillus provided by the present invention has sulfur oxidation function, which can convert sulfur into a form that can be absorbed by plants, improve the availability of sulfur in the soil, and promote crop nutrient absorption.

[0014] (4) The polymyxa bacillus provided by the present invention can decompose organic phosphorus, enhance the effective supply of phosphorus in the soil, and benefit crop absorption and growth.

[0015] (5) The polymyxa bacillus provided by the present invention can synthesize siderophores, improve the solubility and availability of inefficient iron, and promote the absorption of iron by plants.

[0016] (6) The polymyxa bacillus provided by the present invention can produce proteases, which help decompose organic nitrogen in the soil and improve nitrogen use efficiency.

[0017] (7) The polymyxa bacillus provided by the present invention can dissolve zinc, improve the availability of zinc in the soil, and promote crop nutrient absorption.

[0018] (8) The polymyxa bacillus provided by this invention can inhibit the growth of various plant pathogenic fungi, including Alternaria alternata, the pathogen of early blight of tomato. Alternaria solani The pathogen of rice leaf spot disease is *Helicobacter oryzae* (rice leaf spot). Bipolaris oryzae The pathogen of avocado anthracnose is *Colletotrichum spp.* Colletotrichum aeschynomenes The pathogen of blueberry Alternaria fruit rot is a fungus of the genus Alternaria. Alternaria sp.), Alternaria, the pathogen of tobacco acorn disease ( Alternaria alternata The pathogen of banana wilt is *Fusarium oxysporum* var. *cubicans* (specific strain). Fusarium oxysporum f. sp. cubense This reduces the risk of disease occurrence and provides new resources for green prevention and control. Attached Figure Description

[0019] Figure 1 This is a colony morphology diagram of strain YNK-FB0060 cultured on a plate in Example 1; Figure 2 This is the phylogenetic tree of strain YNK-FB0060 constructed based on the 16S sequence in Example 1; Figure 3 This is the growth curve of strain YNK-FB0060 in LB medium for 2-24 hours in Example 2; Figure 4 This is a plate image showing the effect of the YNK-FB0060 strain producing Congo red cellulose in Example 2; Figure 5 This is a graph showing the degradation effect of cellulose filter paper produced by strain YNK-FB0060 in Example 2; Figure 6 This is the glucose standard curve from Example 2; Figure 7 This is the IAA standard curve diagram from Example 3; Figure 8 This is a diagram illustrating the inhibitory effect of strain YNK-FB006 in Example 4 on pathogens. In the diagram: (A) Inhibition of early blight pathogen Alternaria alternata (Alternaria alternata). Alternaria solani (A) Effect diagram; (B) Inhibition of rice leaf spot pathogen *Helicobacter pylori* ( ) Bipolaris oryzae (C) Effect diagram; Inhibition of the anthracnose pathogen *Colletotrichum spp.* (a type of fungus) Colletotrichum aeschynomenes (D) Effect diagram; Inhibition of blueberry Alternaria fruit rot ( Alternaria sp . (E) Effect diagram; Alternaria alternata, the pathogen of tobacco red spot disease ( Alternaria alternata (F) Effect diagram; Fusarium oxysporum, the pathogen of banana wilt, is a Cuban-specific strain. Fusarium oxysporum f. sp . cubense ).

[0020] Figure 9 This is a graph showing the growth-promoting effects of strain YNK-FB0060 in Example 5. In the graph: (A) production of siderophores; (B) production of organophosphates; (C) zinc dissolution; (D) production of proteases; (E) sulfur oxidation. Biological Preservation The polymyxin Bacillus provided by this invention ( Paenibacillus polymyxa ), categorized and named as: Paenibacillus polymyxa YNK-FB0060 was deposited on March 10, 2026, at the China Center for Type Culture Collection, No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province, China (Wuhan University), with accession number CCTCC NO: M2026411. Detailed Implementation

[0021] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0022] In this invention, unless otherwise specified, the term "side carrier" refers to a small-molecule organic compound synthesized and secreted by microorganisms that can form stable chelates with ferric ions, promoting the dissolution and absorption of low-available iron. "Antibacterial activity" refers to the activity of inhibiting or hindering the growth of plant pathogens. "Cellulase activity" refers to the catalytic ability of cellulose-degrading enzymes (including carboxymethyl cellulase CMCase, filter paper enzyme FPase, etc.) produced by microorganisms to hydrolyze cellulose or its derivative substrates to generate soluble sugars; its magnitude is usually expressed as unit enzyme activity. "Life-promoting activity" refers to the comprehensive biological effects of microbial strains in promoting plant seed germination, root growth, nutrient absorption, and plant growth and development through the production of plant hormones, improvement of nutrient supply, or regulation of rhizosphere microecology. "Indoleacetic acid (IAA)" is an important plant auxin that can be synthesized and secreted by certain microorganisms, possessing physiological regulatory functions such as promoting root elongation, cell division, and differentiation, playing a key role in plant growth promotion. "Sulfur oxidation capacity" refers to the ability of microorganisms to oxidize low-valent sulfides or thiosulfates (such as S²⁻, S₂O₃²⁻) into high-valent sulfides or sulfates (SO₄²⁻) through metabolism. This capacity promotes the transformation and efficient utilization of sulfur in the soil, while regulating soil pH and microbial community structure, thereby improving soil physicochemical properties and providing a favorable nutrient environment for plant growth.

[0023] During their research, the inventors of this invention isolated a strain of *Bacillus polymyxa* from soil in tobacco-growing farmland in Lijiang City, Yunnan Province. Test results showed that this strain is a dominant microbial strain with multiple functions. This strain not only has a strong cellulase production capacity but also produces plant growth-promoting substances such as indoleacetic acid (IAA), and possesses various plant growth-promoting functions including sulfur oxidation, decomposition of organic phosphorus, siderophore synthesis, protease production, and zinc dissolution. This strain exhibits significant inhibitory activity against various plant pathogenic fungi, including *Alternaria alternata*, the pathogen of early blight of tomato. Alternaria solani The pathogen of rice leaf spot disease is *Helicobacter oryzae* (rice leaf spot). Bipolaris oryzae The pathogen of avocado anthracnose is *Colletotrichum spp.* Colletotrichum aeschynomenes The pathogen of blueberry Alternaria fruit rot is a fungus of the genus Alternaria. Alternariasp.), Alternaria, the pathogen of tobacco acorn disease ( Alternaria alternata The pathogen of banana wilt is *Fusarium oxysporum* var. *cubicans* (specific strain). Fusarium oxysporum f. sp. cubense In summary, this strain exhibits a synergistic effect in promoting efficient nutrient absorption by plants and inhibiting various plant pathogens. Further research results indicate that this strain can significantly promote plant growth and development, increase crop yield and stress resistance, and has significant application potential and promotional value in the resource utilization of agricultural waste and green agricultural production systems.

[0024] Based on the above findings, the first aspect of the present invention provides a strain of *Bacillus polymyxa* (… Paenibacillus polymyxa The strain has the accession number CCTCC NO: M2026411.

[0025] A second aspect of the present invention provides a bacterial agent, the active ingredient of which is Bacillus polymyxa of the first aspect.

[0026] The third aspect of this invention provides the use of the *Bacillus polymyxa* described in the first aspect or the bacterial agent described in the second aspect in the decomposition of organophosphates, the dissolution of zinc, the oxidation of thiosulfate to sulfate, and the production of iron carriers, proteases, and indoleacetic acid.

[0027] This invention further provides a quantitative analytical method for verifying the cellulase production activity of a bacterial strain. The method includes: selecting single colonies of YNK-FB0060 strain that have grown well on a solid culture medium and inoculating them into LB liquid medium, then shaking and culturing them at 28℃ and 180 rpm; inoculating the culture medium into an enzyme-producing fermentation medium at an appropriate ratio and culturing under the same conditions; taking samples at predetermined times during fermentation, centrifuging to remove the bacterial cells, and obtaining the supernatant as the crude enzyme solution; determining the activities of carboxymethyl cellulase (CMCase) and filter paper enzyme (FPase) in the crude enzyme solution using the DNS colorimetric method, and converting the enzyme activity values ​​according to a standard curve; analyzing the enzyme production dynamics of the strain and determining the optimal enzyme production time point based on the enzyme activity data measured at different fermentation times. The culture medium is LB liquid medium: peptone 10.0 g / L, yeast extract 3.0 g / L, NaCl 5.0 g / L. Fermentation enzyme production medium: CMC-Na 20.0 g / L, peptone 10.0 g / L, NaCl 3.0 g / L.

[0028] The fourth aspect of the present invention provides the use of the polymyxa bacteria described above in the production of indoleacetic acid (IAA) and sulfur oxidation.

[0029] This invention further provides a quantitative analysis method for indoleacetic acid (IAA) production by a bacterial strain. The method includes: culturing the strain in KB medium containing tryptophan (20 g / L peptone, 15 mL / L glycerol, 1.5 g / L K₂HPO₄, 1.5 g / L MgSO₄·7H₂O, 0.1 g / L tryptophan, pH 7.2±0.2) at 30℃ and shaking at 180 rpm for 24 h to induce IAA secretion; qualitative detection using Sackowcki's chromogenic reagent (prepared with concentrated sulfuric acid, deionized water, and FeCl₃·6H₂O); a pink color after the fermentation broth reacts with the reagent indicates that the strain can produce IAA; quantitative analysis is performed by plotting an IAA standard curve (0–24 μg / mL) and analyzing the results at OD₂O₅. 535 The absorbance of the fermentation supernatant reacting with the colorimetric reagent was measured for 5 consecutive days. The IAA yield at different fermentation times was calculated by combining the standard curve, thereby determining the optimal fermentation time for IAA production by the strain.

[0030] The fifth aspect of this invention provides the application of the polymyxa bacillus described in the first aspect in degrading cellulose and / or promoting plant growth, and / or controlling early blight of tomato, leaf spot of rice sesame, anthracnose of avocado, Alternaria alternata fruit rot of blueberry, red spot of tobacco, and wilt of banana.

[0031] According to a preferred embodiment of the present invention, the bacterial agent is a liquid bacterial agent.

[0032] The present invention will be described in detail below through embodiments. It should be understood that the following embodiments are only used to further explain and illustrate the content of the present invention by way of example, and are not intended to limit the present invention.

[0033] Unless otherwise specified, the reagents and materials used in the following examples are all commercially available products purchased from regular chemical / biological reagent or material suppliers, and all reagents are of analytical grade.

[0034] Unless otherwise specified, the operating temperature in the following embodiments is room temperature (25±5℃).

[0035] Example 1 This embodiment illustrates the acquisition, identification, and preservation of Bacillus polymyxa CCTCC NO: M2025645.

[0036] (I) Strain Isolation and Purification In August 2025, a strain of bacteria was isolated from tobacco-growing farmland soil (100°42′E, 26°42′N) in Yongsheng County, Lijiang City, Yunnan Province, China, and deposited at the China General Microbiological Culture Collection Center, with accession number CCTCC NO: M2026411.

[0037] Nutrient agar (NA) medium was used for the isolation and purification of the strain. The medium was prepared as follows: peptone 10.0 g / L, beef extract 3.0 g / L, sodium chloride 5.0 g / L, and agar 15.0 g / L. The medium was autoclaved at 121℃ for 25 min.

[0038] Weigh 2g of soil sample and add it to an Erlenmeyer flask containing 198mL of sterile distilled water. Add an appropriate amount of sterile glass beads and shake on a shaker at 28℃ and 180r / min for 2 hours to fully disperse soil aggregates and release attached microorganisms. After shaking, take the supernatant and mix it according to 10... -7 10 -8 10 -9 The culture was diluted in three gradients, and an appropriate amount of each dilution was inoculated onto NA solid medium using the spread plate method. The culture was then incubated at 28 °C for 2 days. After incubation, single colonies were picked based on colony morphology and repeatedly purified using the streak plate method on NA plates until a pure culture with stable morphology and single colonies was obtained.

[0039] (II) Strain Identification 1. 16S Characterization and Molecular Genetic Classification Colonies: After inoculating Bacillus polymyxa YNK-FB0060 onto NA medium and culturing at 28°C for 48 hours, opaque colonies with a diameter of 2–4 mm were observed. The colonies were milky white to pale yellow with slightly wavy edges; the colony surface was relatively rough and did not exhibit obvious mucilage; the entire colony appeared... Figure 1 As shown.

[0040] Bacillus polymyxa ( Paenibacillus polymyxa The strain was inoculated into NA solid medium and cultured at 28°C for 48 hours. The strain was then sent to Beijing Qingke Biotechnology Co., Ltd. for 16S sequencing. The sequencing results (SEQ ID NO. 1) were imported into NCBI for comparison. NCBI BLAST homology comparison revealed that the strain obtained in this study is similar to the model strain *Bacillus polymyxa*. Paenibacillus polymyxa The sequence similarity of SQR-21 was as high as 99.94%. To further verify this result, a phylogenetic tree was constructed using standard strains with high homology. Figure 2 In the phylogenetic tree, this strain (YNK-FB0060) is associated with Bacillus polymyxa (…). Paenibacillus polymyxaThe DSM 36 clustered in the same branch, and the branch had a self-expansion support value of 99%, indicating that the strain has a very high phylogenetic relationship with Bacillus polymyxa, which is completely consistent with the sequence similarity results.

[0041] SEQ ID NO.1:

[0042] 2. Appraisal Results Based on the molecular detection results and morphological characteristics of strain YNK-FB0060, this strain was identified as *Bacillus polymyxa*. Paenibacillus polymyxa ) (III) Strain Preservation The categories obtained above are named as follows: Paenibacillus polymyxa YNK-FB0060, a polymyxin Bacillus, was deposited on March 31, 2025, at the China Center for Type Culture Collection (CCTCC), No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province, China (Wuhan University), with accession number CCTCC NO: M2026411. Example 2 This embodiment is used to illustrate the cellulase-producing ability of the strain involved in this invention. The cellulase production capacity was initially determined using sodium carboxymethyl cellulose medium and Congo red staining, as follows: Prepare sodium carboxymethyl cellulose medium (g / L): CMC-Na 10.0 g / L, (NH4)2SO4 4 g / L, KH2PO4 2.0 g / L, MgSO4 0.5 g / L, peptone 2.0 g / L, and agar 15.0 g / L. Inoculate strain YNK-FB0060 onto the sodium carboxymethyl cellulose medium and incubate upside down at 28℃ for 2 days. After colonies have grown, stain with 1 mg / mL Congo red dye, ensuring the dye completely covers the colony surface. After standing for 15 min, discard the Congo red dye and decolorize with 1 mol / L NaCl solution for 15 min. Measure the colony diameter and the diameter of the surrounding hydrolysis zone, denoted as D and d, respectively.

[0043] Table 1: Clear zone and cell diameter of strain YNK-FB0060 The filter paper disintegration test was used for secondary screening, and the method is as follows: LB liquid medium formula: peptone 10.0 g / L, yeast extract 3.0 g / L, sodium oxide 5.0 g / L.

[0044] Hutchison's medium formula: KH2PO4 1g / L, NaCl 0.1g / L, MgSO4·7H2O 0.3g / L, NaNO3 2.5g / L, FeCl3 0.01g / L, CaCl2 0.1g / L.

[0045] Under aseptic conditions, a single isolated and purified strain was picked up with an inoculation loop and inoculated into LB liquid medium. The culture was then incubated at 28°C and 180 rpm for 24 h to prepare a seed culture. One 6 cm × 1 cm filter paper strip was added to 100 mL of Hutchinson's medium and sterilized at 121°C, then cooled to room temperature. Five mL of the seed culture was then inoculated and incubated at 28°C and 120 rpm for 7 days. The degree of breakage of the filter paper strip was observed after incubation to assess the degradation ability of the strain. By the seventh day, the filter paper had been completely degraded.

[0046] Plotting the growth curve of the strain: Single colonies of the target strain growing well on solid medium were selected and inoculated into Erlenmeyer flasks containing LB broth and incubated with shaking at 28 ℃ and 180 rpm. Starting from the inoculation time (0 h), samples were taken every 2 h, and the OD of the bacterial suspension was measured at 600 nm using a spectrophotometer. 600 Absorbance values ​​were measured continuously for 24 hours. The values ​​were plotted with incubation time as the x-axis and OD value as the y-axis. 600 The growth curve is plotted with the vertical axis as the ordinate, thereby analyzing the characteristics of different growth stages of the strain (e.g., ...). Figure 3 ).

[0047] The quantitative determination of the cellulase CMCase and filter paper enzyme FPase activities of this strain was performed using the following method: The fermentation for cellulase (CMCase) and filter paper enzyme (FPase) production was carried out as follows: Single colonies of the target strain that grew well on solid culture medium were selected and inoculated into LB seed medium, and cultured with shaking at 28 ℃ and 180 r / min. The highest OD value of the strain was determined based on its growth curve. 600 The optimal inoculation time was determined by taking the seed culture after 20 h of culture as the inoculum suspension. The seed culture was inoculated into the enzyme-producing fermentation medium at an inoculation rate of 5% (v / v). The enzyme-producing fermentation medium consisted of: CMC-Na 20.0 g / L, peptone 10.0 g / L, and NaCl 3.0 g / L. The culture system was incubated with shaking at 28 ℃ and 180 r / min to induce the strain to secrete cellulose degradation-related enzymes. Samples were taken at 24, 72, 120, and 168 h of fermentation. Each time, 1.0 mL of fermentation broth was placed in a 1.5 mL centrifuge tube and centrifuged at 4 ℃ and 10000 r / min for 10 min. The supernatant was used as the crude enzyme solution for subsequent enzyme activity analysis.

[0048] (1) Glucose curve plotting: Take seven 15mL graduated test tubes and number them 0 to 6. Add distilled water and 1mg / mL glucose standard solution to the test tubes respectively to prepare glucose solutions of different concentrations (see Table 2). Add 2mL of DNS reagent to each test tube, shake well, and then boil in a water bath for 5 min. After cooling to room temperature, add distilled water to each test tube to make up to 10mL. Shake well and place in an ELISA reader. Under a wavelength of 540nm, the blank control group is the solution in test tube 0. Measure and record the optical density of each test tube solution. Plot the glucose content (mg) on ​​the x-axis and measure the OD of each group. 540 The values ​​were used as the ordinate to plot the glucose standard curve. Figure 6 The final standard curve is obtained as y = 0.7363x - 0.0265, with R² = 0.9939.

[0049] Table 2: Glucose solution concentration (2) Determination of carboxymethyl cellulase activity (CMCase): Take four clean test tubes of the same specifications, one as a blank control, and the other three as test tubes. Accurately weigh 1 mL of 1% CMC-Na standard solution (containing 1% CMC-Na in citrate buffer) and add it to each of the four test tubes. Add 1 mL of diluted crude enzyme solution to each of the three test tubes. Add 1 mL of boil-inactivated diluted crude enzyme solution to the blank control. Shake well and place the four test tubes in a 50℃ water bath for 30 min. Remove and cool rapidly to room temperature. Immediately add 2 mL of DNS reagent to each test tube, boil in a water bath for 5 min, remove and cool rapidly to room temperature, and add distilled water to each test tube to make up to 10 mL. Shake well and let stand. Measure the absorbance of the test solution at a wavelength of 540 nm using an ELISA reader. Determine the reducing sugar content according to the glucose standard curve, and then calculate the carboxymethyl cellulase activity.

[0050] (2) Filter paper enzyme activity (FPase) determination: Take 4 clean test tubes of the same specifications, one test tube as a blank control, and the other 3 test tubes. Weigh 0.05 g of filter paper strips cut into 1.0 cm x 1.0 cm pieces and put them into each test tube. Then add 1.0 mL of diluted crude enzyme solution and 1.0 mL of citrate buffer solution to submerge the filter paper strips. Add 1 mL of boil-inactivated diluted crude enzyme solution to the blank control. Shake well and place the 4 test tubes in a 50℃ water bath for 30 min. Remove and cool rapidly to room temperature. Immediately add 2 mL of DNS reagent to each test tube, boil in a water bath for 5 min, remove and cool rapidly to room temperature, and add distilled water to each test tube to make up to 10 mL. Shake well and let stand. Measure the absorbance of the test solution at a wavelength of 540 nm using an ELISA reader. Determine the reducing sugar content according to the glucose standard curve, and then calculate the filter paper enzyme activity.

[0051] In the formula: C is the mass of glucose (mg); N is the dilution factor; V is the volume of crude enzyme solution (mL); t is the reaction time (min).

[0052] The final results (Table 3) show that the CMCase enzyme activity reached its highest value of 17.41 ± 1.30 U / mL at 72 h of fermentation, and the FPase enzyme activity reached its highest value of 5.42 ± 1.93 U / mL at 120 h of fermentation.

[0053] Table 3: CMCase and FPAase enzyme activities Example 3 Secretion of indoleacetic acid (IAA): KB medium: 20g peptone, 15ml glycerol, 1.5g K2HPO4, 1.5g MgSO4·7H2O, 0.1g tryptophan, 1L distilled water, pH 7.2±0.2.

[0054] Sackowcki's colorimetric reagent: Slowly add 150 mL of concentrated sulfuric acid to 250 mL of deionized water while stirring. After the solution cools, add 7.5 mL of 0.5 mol / L FeCl3·6H2O solution.

[0055] Qualitative methods: Pick a single colony and inoculate it into KB medium. Incubate at 30℃ with shaking at 180 rpm / min for 24 h. Under aseptic conditions, transfer mL of fermentation broth into a centrifuge tube and quickly mix with 4 mL of Sackowcki's chromogenic reagent. Incubate at room temperature in the dark for 40 min to develop color. Observe and record the color change. If pink appears, it is positive, indicating that the strain can secrete IAA.

[0056] Quantitative: Plotting the standard curve: Weigh 10 mg of IAA standard and dissolve it in a small amount of ethanol. Dilute to 100 mL with distilled water (concentration 100 μg / mL). Perform serial dilutions to 0, 4, 8, 12, 16, 20, and 24 μg / mL. Take 1 mL of each dilution, add 4 mL of colorimetric reagent, and incubate at 40°C in the dark for 40 min. Measure the color at OD. 535 The OD value was measured and a standard curve was plotted. Figure 7 The standard curve y = 0.0068x + 0.0549 was obtained, R0. 2 =0.997.

[0057] Quantitative detection: Prepare 100ml of KB medium and add 1% OD of NB medium. 600 After culturing the bacterial suspension for one day, the bacterial suspension and blank control were centrifuged at 12500 r / min for 10 min. 4 mL of the supernatant was collected, and 4 mL of colorimetric solution was added. The mixture was then incubated in the dark for 40 min, and its OD value was measured using a spectrophotometer. 535 The value was repeated 3 times, and the blank colorimetric solution was used to zero the test. The standard curve was compared with the value. The test was conducted continuously for 5 days, and the color change at different fermentation times was recorded. The IAA yield of the strain at each time period was calculated. As shown in Table 4, the optimal fermentation time for the strain to produce IAA was on the second day, with a yield of 8.039 μg / mL.

[0058] Table 4: Dynamic changes in IAA yield of strain YNK-FB0060 Note: The unit is μg / mL.

[0059] Example 4 This embodiment is used to illustrate Polymyxin Bacillus (… Paenibacillus polymyxa ) and early blight of tomato ( Alternaria solani Rice leaf spot disease ( Bipolaris oryzae Anthracnose of avocado ( Colletotrichum aeschynomenes ); Blueberry Alternaria fruit rot ( Alternaria sp. Tobacco red star disease ( Alternaria alternata ); banana wilt disease ( Fusarium oxysporum f. sp. cubense The inhibitory effect of ).

[0060] Test pathogen: Tomato early blight ( Alternaria solani Rice leaf spot disease ( Bipolaris oryzae Anthracnose of avocado ( Colletotrichum aeschynomenes ); Blueberry Alternaria fruit rot ( Alternaria sp. Tobacco red star disease ( Alternaria alternata ); banana wilt disease ( Fusarium oxysporum f. sp. cubense (Provided by the Laboratory of Agricultural Resources and Environment Research Institute, Yunnan Academy of Agricultural Sciences)

[0061] Plate confrontation experiment: using tomato early blight ( Alternaria solani Rice leaf spot disease ( Bipolaris oryzae Anthracnose of avocado ( Colletotrichum aeschynomenes ); Blueberry Alternaria fruit rot ( Alternaria sp. Tobacco red star disease ( Alternaria alternata ); banana wilt disease ( Fusarium oxysporum f. sp. cubenseAs an indicator pathogen, a 5 mm pathogenic bacterial disc was inoculated in the center of the PDA medium, and the strain YNK-FB0060 obtained in Example 1 was inoculated in a cross shape at a distance of 25 mm from the disc. Uninoculated plates served as controls. Each strain was replicated in triplicate and incubated in a constant temperature incubator at 25-30℃ in the dark for 5-7 days. The inhibition rate was then calculated.

[0062] After the culture is completed, the average value and inhibition rate are calculated. Inhibition rate (%) = (colon diameter of control group - colony diameter of treatment group) / (colon diameter of control group - 5) × 100.

[0063] Figure 8 Images (A)-(F) demonstrate the effectiveness of strain YNK-FB0060 against tomato early blight (… Alternaria solani Rice leaf spot disease ( Bipolaris oryzae ), and avocado anthracnose ( Colletotrichum aeschynomenes ); Blueberry Alternaria fruit rot ( Alternaria sp. Tobacco red star disease ( Alternaria alternata ); banana wilt disease ( Fusarium oxysporum f. sp. cubense The inhibitory effect of strain YNK-FG0001 was determined by the plate confrontation method. The results showed that strain YNK-FG0001 could inhibit the above six pathogens, with inhibition rates of 79.08%, 71.71%, 79.48%, 64.64%, 72.84%, and 75.07% for each pathogen.

[0064] Example 5 This embodiment utilizes a plate qualitative detection method to evaluate the functional characteristics of the strain, such as siderophoresis, decomposition of organophosphates, zinc solubility, protease production, and sulfur oxidation (e.g., ...). Figure 9 The specific method is as follows.

[0065] (a) Testing of iron production capacity: Iron-free Czapek's medium: 30.0g glucose, 3.0g sodium nitrate, 1.0g dipotassium hydrogen phosphate, 0.5g potassium chloride, 0.5g magnesium sulfate, 15-20g agar, 1000 mL distilled water.

[0066] Detection method: The test strain was inoculated on a plate using a four-point inoculation method, and incubated upside down in a 28 ℃ constant temperature incubator for 5 days. The appearance of a yellow halo around the colony indicates that the strain has the ability to produce siderophores.

[0067] (II) Test for decomposition of organophosphorus compounds: Organic phosphorus decomposition medium: ammonium sulfate 0.5g, yeast extract 0.5g, sodium chloride 0.3g, potassium chloride 0.3g, magnesium sulfate 0.3g, ferrous sulfate 0.03g, manganese sulfate 0.03g, lecithin 0.2g, calcium carbonate 1.0g, agar 15-20g, distilled water 1000mL, sterilize at 121℃ for 15 min.

[0068] Detection method: The strain was inoculated into the above-mentioned solid culture medium using a four-point inoculation method and incubated at 28 ℃ for 5 days. The appearance of a transparent or yellow halo around the colony indicates that the strain has the ability to decompose organophosphates.

[0069] (III) Zinc dissolving capacity test Zinc-soluble bacteria screening medium: ZnO 1.0g, glucose 10.0g, KCl 0.2g, (NH4)2SO4 1.0g, MgSO4 0.2g, K2HPO4 0.1g, agar 15-20, distilled water 1000mL. Sterilize at 121 ℃ for 20 min.

[0070] Detection method: The strain was inoculated into the above-mentioned solid culture medium using a four-point inoculation method and incubated at 28°C for 5 days. The appearance of a clear zone around the colony indicates that the strain has zinc-dissolving ability.

[0071] (iv) Protease production capacity test Protease screening medium: glucose 10.0 g / L, yeast extract 4.0 g / L, casein 10.0 g / L, dipotassium hydrogen phosphate 1.0 g / L, magnesium sulfate heptahydrate 0.2 g / L, agar 20.0 g / L, pH 7.0.

[0072] Detection method: The same strain was inoculated into the protease selection medium at four points, and incubated upside down in a 28℃ constant temperature incubator for 5 days. The presence of a clear zone was then observed.

[0073] (v) Sulfur oxidation capacity test Modified thiosulfate medium (MST): Weigh out 5.0 g Na₂S₂O₃, 0.1 g K₂HPO₄, 0.2 g NaHCO₃, 0.1 g NH₄Cl, and C₆H₂O. 12 Dissolve 65.0g of O2 and 5.0g of yeast extract in 1000mL of water, adjust the pH to 8±0.1, and add 20g of agar. Autoclave the culture medium at 121℃ for 20min, and then cool it to 50-60℃. Then filter the solution of Na2S2O3 (5.0g) and bromocresol purple (0.008g, colorimetric reagent) through a 0.22μm filter membrane and add it to the culture medium, mix well and set aside.

[0074] Detection method: The same strain was inoculated into modified thiosulfate medium at four points, inverted and incubated in a constant temperature incubator at 28℃ for 5 days, and the appearance of a transparent zone was observed.

[0075] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. A polymyxa bacillus strain used to dissolve zinc, oxidize thiosulfate to sulfate, and produce proteases ( Paenibacillus polymyxa YNK-FB0060, with accession number CCTCC NO: M2026411.

2. A microbial agent, characterized in that, The active ingredient of the bacterial agent is the polymyxa bacillus described in claim 1.

3. The microbial agent according to claim 2, characterized in that: The bacterial agent is a liquid bacterial agent.

4. The application of the *Bacillus polymyxa* as described in claim 1 or the inoculant as described in claim 2 in the degradation of cellulose and / or the prevention and control of early blight in tomatoes, leaf spot in rice sesame, anthracnose in avocados, *Alternaria alternata* fruit rot in blueberries, and red spot disease in tobacco; wherein the pathogen of early blight in tomatoes is *Alternaria alternata* (…). Alternaria solani The pathogen of rice leaf spot disease is *Helicobacter pylori* (rice leaf spot). Bipolaris oryzae The pathogen causing avocado anthracnose is *Colletotrichum spp.* Colletotrichum aeschynomenes The pathogen of blueberry Alternaria fruit rot is a fungus of the genus Alternaria. Alternaria The pathogen of tobacco red spot disease is Alternaria sp. Alternaria alternata ).

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