Mycobacterium YNK-FB0058 and application thereof
By screening and identifying the genus YNK-FB0058, soil degradation problems have been solved, agricultural productivity has been improved, and green and sustainable development of ecological agriculture has been achieved.
Patent Information
- Application Number
- CN202511371970.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-24
- Publication Date
- 2025-12-09
AI Technical Summary
Existing technologies have failed to effectively address soil degradation, particularly soil compaction, continuous cropping obstacles, salinization, and heavy metal pollution caused by excessive application of chemical fertilizers and pesticides, which affect crop yields and food security.
A strain of the genus *Bacillus*, YNK-FB0058, was screened and identified. It possesses the ability to fix nitrogen, dissolve zinc, decompose organic and inorganic phosphorus, produce iron carriers, and secrete indoleacetic acid. It can be used in liquid fermentation broth preparations to promote plant growth.
It significantly improves agricultural productivity, promotes the green and sustainable development of ecological agriculture, reduces adverse environmental impacts, and has a stable growth-promoting effect.
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Figure CN121086940A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of microbial technology, specifically to a Bacillus spp. strain YNK-FB0058 and its applications. Background Technology
[0002] Soil health is crucial for the green and sustainable development of agriculture. However, due to the excessive use of chemical fertilizers and pesticides and improper planting practices, my country faces a series of soil degradation problems, including soil compaction, continuous cropping obstacles, salinization, and heavy metal pollution, which seriously affect crop yields and food security. Therefore, finding a green and environmentally friendly measure that can effectively improve soil conditions is particularly urgent.
[0003] The rhizosphere is a dynamic region surrounding the roots that provides an ideal environment for various microbial communities that greatly influence plant growth and development. Within the rhizosphere, all processes and functions are primarily influenced by the plant roots, the microorganisms present in the rhizosphere, and the interactions between roots and microorganisms.
[0004] Rhizosphere microorganisms participate in various biochemical processes in the soil. Plant growth-promoting rhizobacteria (PGPRs) are beneficial rhizosphere microorganisms that promote crop nutrient absorption, inhibit pathogenic microorganisms, improve abiotic stress environments, and secrete plant hormones, playing a crucial role in promoting plant growth and maintaining soil ecological health. The diverse microbial communities in the soil assist in fulfilling a series of important soil ecological functions by regulating soil nutrient cycling, decomposing soil organic matter, inhibiting soil-borne diseases, improving soil structure, and supporting plant production. They are a vital link in the material cycle and energy flow of the ecosystem, maintaining its sustainable development. Utilizing soil microorganisms for soil improvement, removal, and remediation of toxic pollutants is an economical, efficient, and environmentally friendly method.
[0005] Currently, the remediation effects of PGPR on obstacle soils and its growth-promoting effects on crops are receiving increasing attention. Microbial fertilizers, biopesticides, and seedling substrates developed with rhizosphere microorganisms as their core components have been widely used. Therefore, further exploration and screening of more PGPR strains with efficient colonization capabilities and broad functions will not only help develop new biofertilizer products and improve agricultural production efficiency, but also further promote the development of agriculture towards a greener, more environmentally friendly, and sustainable direction. Summary of the Invention
[0006] This invention addresses the current lack of a novel Phyllobacterium sp. YNK-FB0058 strain, obtained through screening numerous batches of strains isolated from soil samples obtained from flue-cured tobacco and safflower rotation in Yongsheng County, Lijiang City, Yunnan Province. The invention provides a Phyllobacterium sp. strain YNK-FB0058 and its applications. This strain, obtained through screening numerous batches of strains isolated from soil samples obtained from flue-cured tobacco and safflower rotation in Yongsheng County, Lijiang City, Yunnan Province, possesses the abilities to fix nitrogen, dissolve zinc, solubilize organic and inorganic phosphorus, produce iron carriers, and produce indoleacetic acid. Scientific identification through strain classification confirms it as a novel Phyllobacterium sp. strain. The liquid fermentation broth preparation of this strain can be applied to promote plant growth with significant and stable technical effects, improve agricultural productivity, and promote the green and sustainable development of ecological agriculture. It has broad application value in the field of microbial strain application technology.
[0007] To achieve the above objectives, the present invention provides the following technical solution: In its first aspect, the present invention provides a Phyllobacterium sp. strain YNK-FB0058 CCTCC M 20251973. A new Phyllobacterium sp. strain, designated YNK-FB0058, was isolated from flue-cured tobacco and safflower rotation soil in Yongsheng County, Lijiang City, Yunnan Province. Phyllobacterium sp. YNK-FB0058 was identified as a new strain using common bacterial identification methods. The isolated strain exhibits nitrogen fixation, zinc solubilization, inorganic phosphorus solubilization, organic phosphorus solubilization, iron carrier production, and indoleacetic acid secretion. The liquid fermentation broth preparation of this strain can be used to promote plant growth, demonstrating significant and stable technical effects, reflecting the clear application value of this strain.
[0008] Specifically, this invention involves culturing and isolating microbial strains from soil samples obtained from a tobacco and safflower rotation crop in Yongsheng County, Lijiang City, Yunnan Province. A batch of superior strains were screened, and from these, a strain named YNK-FB0058, *Phyllobacterium* sp., was isolated and identified as belonging to the genus *Phyllobacterium* through microbiological classification and identification. Referring to common microbial identification manuals such as Bergey's Manual of Systematic Bacteriology (9th Edition), the morphological characteristics and molecular identification of strain YNK-FB0058 were performed, confirming that strain YNK-FB0058 is a member of the genus *Phyllobacterium*, but with characteristics different from common *Phyllobacterium* species, exhibiting distinct features of a new species.
[0009] Furthermore, this invention performed gene sequencing on the strain YNK-FB0058, the sequence of which is shown in SEQ ID NO:1 provided below. The obtained sequence was compared and analyzed on a common website. The results showed that the 16S rRNA gene sequence of strain YNK-FB0058 had the highest homology with Phyllobacterium myrsinacearum IAM 13584D12789, at only 98.22%. Strain YNK-FB0058 is most closely related to Phyllobacterium myrsinacearum IAM13584D12789. A phylogenetic tree was constructed using MEGA 7.0 software, a common tool in this field, via the Neighbor-Joining method. Comparative analysis revealed that strain YNK-FB0058 and strain Phyllobacterium pellucidum BT25 M658537 share a 97% confidence level in originating from the same branch, indicating a very high support rate for this new species and excellent stability within the phylogenetic tree. Further identification confirmed that strain YNK-FB0058 is a new species of Phyllobacterium sp., exhibiting typical characteristics of a new species. From a taxonomic perspective, it is tentatively named Phyllobacterium sp. YNK-FB0058.
[0010] In addition, this strain YNK-FB0058 was deposited on September 8, 2025, at the China Center for Type Culture Collection (CCTCC), located at 299 Bayi Road, Wuchang District, Wuhan, Hubei Province, China (Wuhan University), with accession number CCTCC M 20251973. It has been microbiologically identified and tentatively named Phyllobecacterium sp. YNK-FB0058.
[0011] The new strain Phyllobacterium sp. YNK-FB0058 grew well on NA medium. After culturing at 28°C for 48 hours, visible colonies were formed. The colonies were round, raised, shiny, pale yellow, with a smooth and viscous surface. The Gram staining result was G-, indicating that it is a facultative anaerobe, consistent with the colony characteristics of Phyllobacterium.
[0012] A second aspect of the present invention provides a microbial agent, the active ingredient of which is a *Bacillus* species as described in the first aspect.
[0013] The third aspect of this invention provides the use of the strains of the first aspect or the bacterial agents of the second aspect in nitrogen fixation, zinc dissolution, organic phosphorus solubilization, inorganic phosphorus solubilization, iron carrier production, and indoleacetic acid production.
[0014] Compared with the prior art, the beneficial effects of the present invention are:
[0015] (1) The Phyllobacterium sp. YNK-FB0058 CCTCCM20251973 strain provided by this invention is a typical new strain. The isolated strain has the characteristics of nitrogen fixation, zinc solubilization, inorganic phosphorus solubilization, organic phosphorus solubilization, iron carrier production and indoleacetic acid secretion. The liquid fermentation broth preparation of this strain can be used to promote plant growth and has significant and stable technical effects, which reflects the clear application value orientation of screening this strain.
[0016] (2) The *Bacillus* strain provided by this invention is a strain isolated from soil in a rotation of flue-cured tobacco and safflower. Compared with strains screened by extreme environments and laboratory mutagenesis, it is more adaptable to the planting environment and can stably colonize in the plant rhizosphere and soil, thus playing a long-term and stable role in promoting growth. Moreover, since it is a strain isolated from farmland soil, the risk of adverse effects on the local environment and soil microbial ecology caused by the application of this strain in farmland is reduced. Attached Figure Description
[0017] Figure 1 This is a colony morphology diagram of strain YNK-FB0058 cultured on a plate in Example 1;
[0018] Figure 2 This is the phylogenetic tree of strain YNK-FB0058 constructed based on the ITS gene sequence in Example 1;
[0019] Figure 3 The following are the results of the YNK-FB0058 strain in Example 2: (A) nitrogen fixation effect; (B) zinc dissolution effect; (C) siderophore production effect; qualitative and quantitative analysis of IAA production capacity; (D) quantitative analysis of IAA production capacity;
[0020] Figure 4 The qualitative analysis of the inorganic phosphorus solubilization ability (A), the qualitative analysis of the organic phosphorus solubilization ability (B), the phosphorus solubilization standard curve (C), and the quantitative analysis of the inorganic and organic phosphorus solubilization ability (D) of strain YNK-FB0058 in Example 2 are as follows:
[0021] Figure 5 This is the growth curve of strain YNK-FB0058 in Example 3. Detailed Implementation
[0022] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0023] Biological Preservation
[0024] The Phyllobacterium species provided by this invention, classified and named Phyllobacterium sp. YNK-FB0058, was deposited on September 8, 2025, at the China Center for Type Culture Collection, located at No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, Wuhan University, with accession number CCTCC M20251973.
[0025] Please see Figures 1-5 This invention provides a technical solution: a strain of Phyllobacterium sp. isolated from the soil of flue-cured tobacco and safflower rotation in Yongsheng County, Lijiang City, Yunnan Province. Testing revealed that this strain exhibits excellent nitrogen fixation, inorganic phosphorus solubilization, organic phosphorus solubilization, siderophore production, and indoleacetic acid production. Further research has also shown that it can effectively promote plant growth, improve agricultural productivity, and promote the green and sustainable development of ecological agriculture.
[0026] Based on the above findings, the first aspect of the present invention provides a Phyllobacterium sp. YNK-FB0058 strain. After a series of bacterial identifications, it was determined that strain YNK-FB0058 is a new species of Phyllobacterium sp. The preservation number of this strain is CCTCC M 20251973.
[0027] A second aspect of the present invention provides a microbial inoculant that promotes plant growth, the microbial inoculant comprising Phyllobacterium sp. YNK-FB0058 as described in the first aspect and an optional carrier.
[0028] According to a preferred embodiment of the present invention, the bacterial agent is a liquid bacterial agent.
[0029] Preferably, the effective viable count of *Bacillus* spp. YNK-FB0058 in the liquid bacterial agent is 1×10⁶ to 1×10¹¹ CFU / mL. More preferably, it is 1×10⁷×10¹⁰ CFU / mL.
[0030] The third aspect of this invention provides the application of Phyllobacterium sp. YNK-FB0058, as described in the first aspect, in nitrogen fixation, inorganic phosphorus hydrolysis, organic phosphorus hydrolysis, iron carrier production, and indoleacetic acid production.
[0031] 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.
[0032] 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.
[0033] Unless otherwise specified, the operating temperature in the following embodiments is room temperature (25±5℃).
[0034] Example 1
[0035] This embodiment illustrates the acquisition, identification, and preservation of Phyllobacterium sp. YNK-FB0058 CCTCCM20251973.
[0036] (I) Strain Isolation and Purification
[0037] In April 2025, a bacterium was isolated from the soil of flue-cured tobacco and safflower rotation in Yongsheng County, Lijiang City, Yunnan Province, China (100°61'E, 26°28'N). It was deposited at the China General Microbiological Culture Collection Center with the accession number CCTCC M 20251973.
[0038] NA medium was used for the isolation and purification of the strain. The preparation method is as follows: Weigh 10g of peptone, 3g of beef powder and 5g of sodium chloride, add them to 1000mL of water, adjust the pH to 7.3±0.1, then add 15g of agar, and autoclave at 121℃ for 25min.
[0039] A bacterium, named YNK-FB0058, was isolated and purified from soil samples of flue-cured tobacco and safflower rotation using the dilution plating method and streak plating method.
[0040] (II) Strain Identification
[0041] 1. Identification of bacterial morphological characteristics and molecular genetic classification
[0042] Colony cell morphology characteristics: Strain YNK-FB0058 was inoculated on NA medium and cultured at 28°C. Figure 1 The colony morphology of strain YNK-FB0058 on NA medium is shown. As can be seen from the figure, the colonies of this strain are nearly round, with smooth surface and regular edges, translucent, pale yellow, moist and glossy. They are Gram-negative, facultative anaerobic bacteria, and observed under an optical microscope, this strain is a cocci.
[0043] Molecular identification
[0044] Total DNA from strain YNK-FB0058 was extracted using the Chelex extraction method and used as a template. 27F (5'-AGAGTTTGATCCTGGCTCAG-3') was used as the upstream primer and 1492R (5'-TACGGCTACCTTGTTACGACTT-3') was used as the downstream primer. 16S rRNA amplification was performed using the reaction system and conditions shown in Table 1.
[0045] Table 1. PCR System and Conditions
[0046]
[0047] The amplified products were purified by 1% agarose gel electrophoresis (using a gel extraction and purification kit manufactured by Guangzhou Meiji Biotechnology Co., Ltd.) and then sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. The sequencing results are shown in SEQ ID NO.1. The sequencing results were compared and analyzed in the EZBiocloud database (https: / / www.ezbiocloud.net / ). Phylogenetic analysis was performed, using the 16S rRNA sequence of a highly homologous type strain as a reference. Multiple sequence alignment was performed using Clustal X 1.8 software to calculate the sequence similarity between the tested strain and the reference strain. Base deletion sites were excluded during phylogenetic analysis, and a phylogenetic tree between the tested strain and the reference strain was constructed using MEGA 7.0 using the neighbor-joining method. The Bootstrap value was set to 1000, and the rest were default values. The 16S rRNA (SEQ ID NO.1) sequence of strain YNK-FB0058 is provided in the appendix.
[0048] The 16S rRNA gene sequence of strain YNK-FB0058, when compared and analyzed in the EZBiocloud database (https: / / www.ezbiocloud.net / ), showed the highest homology (98.22%) with Phyllobacterium myrsinacearum IAM 13584D12789. Strain YNK-FB0058 is most closely related to Phyllobacterium myrsinacearum IAM13584D12789.
[0049] Phylogenetic trees of strains were constructed using MEGA7. Figure 2The phylogenetic tree of strain YNK-FB0058 is shown. It can be seen that YNK-FB0058 is most closely related to the genus Phyllobacterium and is from the same branch as strain Phyllobacterium pellucidum BT25 M658537 with a confidence level of 97%.
[0050] 3. Appraisal Results
[0051] Based on the molecular detection results and bacterial morphological characteristics of strain YNK-FB0058, strain YNK-FB0058 was identified as a new species of Phyllobacterium sp. The preservation number of this strain is CCTCC M20251973.
[0052] (III) Strain Preservation
[0053] The obtained (Phyllobacterium sp.) YNK-FB0058 was deposited on September 8, 2025, at the China Center for Type Culture Collection, No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province, China (Wuhan University), accession number CCTCC M20251973.
[0054] Example 2
[0055] This example illustrates the effects of Phyllobacterium sp. YNK-FB0058 on nitrogen fixation, organic phosphorus solubilization, inorganic phosphorus solubilization, iron carrier production, zinc solubilization, and indoleacetic acid production.
[0056] (I) Nitrogen Fixation Effect Test
[0057] Preparation method of Asbhy nitrogen-free medium: Weigh 10g glucose, 0.2g dipotassium hydrogen phosphate, 0.2g sodium chloride, 0.2g magnesium sulfate monohydrate, 0.2g potassium sulfate, and 5g calcium carbonate, dissolve them in 1000mL of water, and add 18±2g agar. Autoclave at 121℃ for 20min.
[0058] The four-point inoculation method was used to inoculate strain YNK-FB0054 obtained in Example 1 onto Asbey nitrogen-free culture plates, with three replicates. After inoculation, the plates were placed in a 30°C incubator for 7 days, during which the bacterial growth and the formation of clear zones around colonies were observed daily.
[0059] Figure 3Figure (A) shows the nitrogen fixation effect of strain YNK-FB0058. As can be seen from the figure, strain YNK-FB0058 produces a clear zone when cultured on Asbey nitrogen-free medium, indicating that this strain has nitrogen fixation ability. The size of the clear zone was measured using the cross-multiplication method, and the ratio of the clear zone diameter (D) to the colony diameter (d) was calculated to be D / d = 6.19 ± 0.55, indicating that strain YNK-FB0058 has a strong nitrogen fixation ability.
[0060] (II) Testing of the effectiveness of inorganic and organic phosphorus desiccation
[0061] 1. Qualitative detection of inorganic phosphorus
[0062] Preparation method of inorganic phosphorus solid culture medium: Weigh 10g glucose, 0.5g ammonium sulfate, 0.3g sodium chloride, 0.3g potassium chloride, 0.3g magnesium sulfate heptahydrate, 0.03g ferrous sulfate heptahydrate, 0.03g manganese sulfate tetrahydrate, and 5g tricalcium phosphate. Dissolve in 1000mL of water, adjust the pH to 7.3±0.2, and add 18±2g agar. Autoclave at 121℃ for 20min.
[0063] The four-point inoculation method was used to inoculate strain YNK-FB0058 obtained in Example 1 onto inorganic phosphorus culture plates, with three plates repeated. After inoculation, the plates were placed in a 30°C incubator for 7 days, during which the bacterial growth and the formation of clear zones around the colonies were observed daily.
[0064] Figure 4 Figure (A) shows the inorganic phosphate solubilizing effect of strain YNK-FB0058. As can be seen from the figure, strain YNK-FB0058 produces a clear zone when cultured on an inorganic phosphate plate, indicating that this strain has the ability to solubilize inorganic phosphate. The size of the clear zone was measured using the cross-multiplication method, and the ratio of the clear zone diameter (D) to the colony diameter (d) was calculated to be D / d = 1.80 ± 0.21.
[0065] 2. Qualitative detection of organophosphorus compounds
[0066] Preparation method of organophosphorus solid culture medium: Weigh 10g glucose, 0.5g ammonium sulfate, 0.5g yeast extract, 0.3g sodium chloride, 0.3g potassium chloride, 0.3g magnesium sulfate, 0.03g ferrous sulfate, 0.03g manganese sulfate, 0.2g lecithin, and 1g calcium carbonate. Dissolve in 1000mL of water, adjust the pH to 7.3±0.2, and add 18±2g agar. Autoclave at 121℃ for 20min.
[0067] The four-point inoculation method was used to inoculate the strain YNK-FB0058 obtained in Example 1 onto organophosphate culture plates, with three repeat inoculations. After inoculation, the plates were placed in a 30°C incubator for 7 days, during which the bacterial growth and the formation of clear zones around the colonies were observed daily.
[0068] Figure 4 (B) shows the organophosphate solubilizing effect of strain YNK-FB0058. As can be seen from the figure, strain YNK-FB0058 produces a clear zone when cultured on an organophosphate plate. The size of the clear zone was measured using the cross-crossing method, and the ratio of the clear zone diameter (D) to the colony diameter (d) was calculated to be D / d = 2.10 ± 0.18. This indicates that the strain has a good organophosphate solubilizing ability.
[0069] 3. Quantitative detection of dissolved inorganic phosphorus and dissolved organic phosphorus
[0070] 50 mL each of organic-phosphorus and inorganic-phosphorus liquid culture medium were added to 150 mL Erlenmeyer flasks and autoclaved (120 °C, 30 min). Subsequently, bacteria grown on LB liquid medium for 24 hours were prepared into a bacterial suspension (approximately 10⁸ CFU / mL), and 500 μL were inoculated into both organic-phosphorus and inorganic-phosphorus liquid media. This process was repeated three times daily for seven days, with a control group (without inoculation) included. The cultures were incubated on a shaker at 28 °C and 150 rpm, with samples taken every 24 hours and stored in a refrigerator for later use.
[0071] Take 0, 2, 4, 6, 8, and 10 mL of 5 mg / L phosphorus standard solution and add them to 50 mL volumetric flasks. Dilute with water to approximately 20 mL, add 5 mL of molybdenum antimony colorimetric reagent, shake well, and make up to volume to obtain a series of phosphorus standard solutions with concentrations of 0, 0.2, 0.4, 0.6, 0.8, and 1.0 mg / L. After standing at room temperature (20–25 °C) for 4 hours, using the 0 mg / L phosphorus standard solution as a reference solution, scan each phosphorus standard solution within a wavelength range of 720 nm to find the highest absorbance peak and record its absorbance value. The color stabilization time is 8 hours. Plot the working curve with the absorbance at the highest peak as the ordinate and the phosphorus concentration (mg / L) as the abscissa, as shown below. Figure 4 (C).
[0072] Take an appropriate amount of supernatant and centrifuge at 4℃ (10000 r / min, 30 minutes) for subsequent determination of phosphorus content, organic acid content, and alkaline, neutral, and acidic phosphatase activities. Dilute the supernatant appropriately with distilled water and add a few milliliters to a 50 mL volumetric flask. Add 2 drops of 2,4-dinitrophenol indicator solution, adjust the pH to a slightly yellow color with 1 mol / L sodium hydroxide or sulfuric acid solution, then add 5 mL of molybdenum antimony colorimetric reagent, shake well, and dilute to the mark. Perform a blank test. After standing at room temperature (20–25℃) for 4 hours, measure the absorbance at 720 nm using a spectrophotometer. Use the blank test as a reference solution to zero the sample, read the absorbance value, and find the concentration of the phosphorus standard solution on the working curve.
[0073] Available phosphorus content (μg / mL) = (ρ×V×Ts) / Vo.
[0074] In the formula: ρ represents the mass concentration of available phosphorus (μg / mL); V represents the fixed volume (mL), which is 50mL here; Ts is the dispensing factor, which is 17 times here; Vo represents the volume of fermentation broth added (mL), which is 5mL here.
[0075] The inorganic and organic phosphorus soluble abilities of the 5-day-old strain were quantitatively determined using the molybdenum-antimony colorimetric method. The results are as follows: Figure 4 As shown in (D), the soluble inorganic phosphorus content of the fermentation broth of strain YNK-FB0058 reached a maximum of 474.46 mg / L on day 3, and the soluble organic phosphorus content reached a maximum of 566.3 mg / L on day 2.
[0076] (III) Iron-generating carrier effect test
[0077] Preparation method of CAS solid detection culture medium: Weigh 60.5 mg of Chromium Azurite S (CAS), 72.9 mg of hexadecyltrimethylammonium bromide (HDTMA), 2.645 mg of ferric chloride hexahydrate, 295.25 mg of sodium dihydrogen phosphate dihydrate, 1213.5 mg of disodium hydrogen phosphate dodecahydrate, 125 mg of ammonium chloride, 37.5 mg of potassium dihydrogen phosphate, and 62.5 mg of sodium chloride. Dissolve in 1000 mL of water, adjust the pH to 6.8 ± 0.1, and add 18 ± 2 g of agar. Autoclave at 121℃ for 20 min.
[0078] The four-point inoculation method was used to inoculate the strain YNK-FB0058 obtained in Example 1 onto CAS solid culture plates, with three plates repeated. After inoculation, the plates were placed in a 30°C incubator for 7 days. During the incubation period, the bacterial growth and the formation of orange-yellow halos around the colonies were observed daily.
[0079] Figure 3(C) illustrates the siderophore-producing effect of strain YNK-FB0058. As shown in the figure, strain YNK-FB0058 produces an orange-yellow halo when cultured on CAS solid culture plates, indicating that this strain possesses siderophore-producing ability. The size of the clear zone was measured using the cross-crossing method, and the ratio of the clear zone diameter (D) to the colony diameter (d) was calculated to be D / d = 2.57 ± 0.36.
[0080] (iv) Zinc dissolving effect test
[0081] Preparation method of zinc-soluble solid culture medium: Weigh 1g of zinc oxide, 10g of glucose, 0.2g of potassium chloride, 1g of ammonium sulfate, 0.2g of magnesium sulfate, and 0.1g of dipotassium hydrogen phosphate, add them to 1000mL of water to dissolve, adjust the pH to 7.3±0.1, add 20g of agar, and autoclave at 121℃ for 20min.
[0082] The four-point inoculation method was used to inoculate strain YNK-FB0058 obtained in Example 1 onto zinc dissolution culture plates, with three plates repeated. After inoculation, the plates were placed in a 30°C incubator for 5 days, during which the bacterial growth and the formation of clear zones around the colonies were observed daily.
[0083] Figure 3 (B) shows the zinc-dissolving effect of strain YNK-FB0058. As can be seen from the figure, strain YNK-FB0058 produces a clear zone when cultured on a zinc-dissolving plate, indicating that this strain has the ability to dissolve zinc. The size of the clear zone was measured using the cross-multiplication method, and the ratio of the clear zone diameter (D) to the colony diameter (d) was calculated to be D / d = 3.58 ± 0.64.
[0084] (V) Test on the effect of producing indoleacetic acid
[0085] 1. Qualitative testing
[0086] KB medium: Dissolve 20g peptone, 15ml glycerol, 1.5g K₂HPO₄, 1.5g MgSO₄·7H₂O, and 0.1g tryptophan in 1000mL of water and adjust the pH to 7.2±0.2. Sackowcki's colorimetric reagent: Slowly add 150mL of concentrated sulfuric acid to 250mL of deionized water while stirring. After the solution cools, add 7.5mL of 0.5mol / L FeCl₃·6H₂O solution.
[0087] A single colony of strain YNK-FB0058 obtained in Example 1 was inoculated into KB medium and cultured at 30°C and 180 rpm / min for 24 h with shaking. Under aseptic conditions, 1 mL of fermentation broth was taken into a centrifuge tube and quickly mixed with 4 mL of Sackowcki's chromogenic reagent. The mixture was then incubated at room temperature in the dark for 40 min for color development. The color change was observed and recorded. If a pink color appeared, it was considered positive, indicating that the strain could secrete IAA. Figure 3 (D) The left test tube is the control, and the right test tube shows the color development of strain YNK-FB0058. The pink color indicates that IAA is produced.
[0088] 2. Quantitative detection
[0089] After culturing the strain in NB medium for 1 day, a seed culture was prepared and inoculated at a 1% inoculum into NB liquid medium containing L-tryptophan. The culture was then placed in a 30°C shaker at 180 rpm. Each day, 4 ml of the supernatant was mixed with 4 ml of Sackowcki's chromogenic reagent and incubated in the dark for 40 minutes. The OD value was then measured at 535 nm. The absorbance values were substituted into a standard curve to calculate the indoleacetic acid content produced by the strain. Figure 3 (E) The strain YNK-FB0058 was found to produce the highest indoleacetic acid content of 19.319 μg / mL on day 4.
[0090] Example 3
[0091] This example illustrates the plotting of the growth curve of Phyllobacterium sp. YNK-FB0058 CCTCCM20251973.
[0092] Strain YNK-FB0058 was inoculated into LB liquid medium, with the inoculation amount controlled to achieve an OD600 of 0.01. The medium was then incubated at 28°C and 180 rpm for 36 hours. Samples were taken every 2 hours from 0 to 4 hours, and every 4 hours from 4 to 36 hours. 1 mL of each sample was used to determine the bacterial concentration (expressed as an OD600 value, measured using a UV spectrophotometer). A growth curve for strain YNK-FB0058 was then plotted. Three replicates were performed, and the average value was used.
[0093] The results are as follows Figure 5 As shown, the OD600 value of strain YNK-FB0058 exhibits an "S"-shaped change over time. The lag phase in LB liquid medium is approximately 2 hours, during which the strain grows relatively slowly. The logarithmic growth phase is from 2 to 16 hours, during which the strain grows rapidly, and the OD600 value reaches approximately 1 at 16 hours.
[0094] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.
Claims
1. A species of Phyllostachys sp., classified as Phyllostachys sp. YNK-FB0058, characterized in that, The *Bacillus* strain YNK-FB0058 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on September 2025, with accession number CCTCC M 20251973.
2. A microbial agent, characterized in that, The active ingredient of the bacterial agent includes the *Bacillus* genus YNK-FB0058 as described in claim 1.
3. The bacterial agent according to claim 2, wherein the bacterial agent is a liquid bacterial agent; the content of Bacillus spp. YNK-FB0058 in the bacterial agent is 1×106-1×108 cells / mL.
4. The application of the *Bacillus* genus YNK-FB0058 as described in claim 1 or the bacterial agent as described in claim 2 in nitrogen fixation, zinc dissolution, organic phosphorus decomposition, inorganic phosphorus decomposition, iron carrier production, and indoleacetic acid production.