A highly efficient straw-degrading Bacillus strain Z161 and its screening method and applications

Abstract

This invention belongs to the field of microbiology, specifically relating to a highly efficient straw-degrading Bacillus Z161 strain, its screening method, and its applications. The Bacillus Z161 is *Bacillus mexicanus* (…). Bacillus mexicanus The Bacillus Z161 strain obtained in this invention, with accession number CGMCC NO.36393, was deposited at the China General Microbiological Culture Collection Center on October 29, 2025. It exhibits strong laccase activity and high-temperature resistance, enabling efficient degradation of lignin at 50°C. This avoids the problem of existing technologies, which often employ ambient-temperature fermentation bacteria and require stringent fermentation conditions, thus facilitating rapid composting. The Bacillus Z161 strain provided by this invention possesses stable genetic and functional traits, exhibits good high-temperature tolerance, rapidly depolymerizes lignin, and improves composting efficiency.

Description

Technical Field

[0001] This invention belongs to the field of microbiology, specifically relating to a highly efficient straw-degrading Bacillus Z161 strain and its screening method and application. Background Technology

[0002] Straw is a widely available, carbon-neutral, inedible biological resource that can be recycled globally in large quantities every year. It is primarily composed of cellulose, hemicellulose, and lignin, forming a natural biological complex that makes it difficult to utilize. Widely used composting methods generally face technical bottlenecks such as long decomposition periods and low efficiency, and also increase the risk of releasing greenhouse gases (such as CO2 and CH4).

[0003] Recently, lignocellulosic wastes (such as rice straw and corn stalks) have attracted particular attention because they are well-suited for energy applications and can be obtained on a large scale and at low cost. Currently, straw is mainly used as animal feed, livestock bedding, or as natural fertilizer. However, large quantities of straw are left idle or burned, often in open environments: from an environmental perspective, this practice is unsustainable. Lignin suffers from difficult depolymerization and low degradation efficiency due to the complexity of its spatial structure, the diversity of its monomers, the complexity of its bonds, and the lack of lignin-degrading microorganisms. Previous research has shown that straw waste is rich in nutrients, and aerobic composting can gradually degrade unstable organic matter in compost materials into stable, crop-beneficial, and soil-improving compost products, playing an important role in meeting the growing energy demand. Wastes such as wheat, rice, and corn stalks are sustainable, low-cost raw materials. The discovery and application of lignin-degrading microorganisms offer an opportunity to address these challenges. Among the most discovered and studied fungal degradation of lignin are white-rot fungi and brown-rot fungi. Studies have found that they produce reducing free radicals and lignin depolymerases. These substances can depolymerize lignin into smaller molecules, which can then be further decomposed by other microbial communities, ultimately achieving efficient degradation of lignin.

[0004] Previous studies have found that in aerobic composting, the microorganisms involved in cellulose degradation are mainly thermotolerant bacteria, but most of these strains have not yet been clearly identified. Furthermore, many lignin-degrading fungi, such as white-rot fungi, have weak tolerance to high temperatures, with their optimal growth temperature not exceeding 40℃. However, to meet the requirements for harmlessness during aerobic composting, a relatively high temperature environment exceeding 45℃ must be maintained. This mismatch in temperature conditions makes it difficult for composting fermentation to proceed completely. Summary of the Invention

[0005] To address the problems of poor composting efficiency and slow straw degradation efficiency in existing technologies, this invention provides a Bacillus Z161 strain that is resistant to high-temperature straw degradation.

[0006] Another object of the present invention is to provide a screening method for the above-mentioned Bacillus Z161.

[0007] Another objective of this invention is to provide the application of the aforementioned Bacillus in straw composting and other applications.

[0008] The technical solution adopted by the present invention to achieve the above objectives is as follows:

[0009] This invention provides a highly efficient straw-degrading Bacillus strain Z161, wherein Bacillus Z161 is Bacillus mexicanus (… Bacillus mexicanus (), its accession number is CGMCC NO.36393, and it was deposited at the China General Microbiological Culture Collection Center of the China Committee on Microbial Culture Collection on October 29, 2025, and classified and named as follows: Bacillus mexicanus .

[0010] Preferably, the 16S rDNA sequence of the Bacillus Z161 is shown in SEQ ID NO.1.

[0011] The present invention also provides a screening method for the above-mentioned Bacillus Z161, comprising the following steps:

[0012] (1) The collected fermentation samples were placed in straw-MSM medium and shaken on a shaker to obtain a culture medium; then the culture medium was diluted to 10. -1 -10 -6 times;

[0013] (2) The diluted solution was spread on LBA plates, inverted and cultured, and purified to obtain Bacillus Z161 with high temperature resistance to straw degradation.

[0014] Preferably, the straw-MSM culture medium consists of 3.5 g of dry straw, 0.645 g of MSM inorganic salt culture medium, and distilled water to a final volume of 0.15 L.

[0015] The composition and concentration of the above-mentioned MSM inorganic salt medium include: Na₂HPO₄, 2.8 g / L, KH₂PO₄, 1.0 g / L, (NH₄)₂SO₄, 0.5 g / L, MgCl₂, 0.053 g / L, Ca(NO₃)₂·4H₂O, 0.05 g / L, and Na₂EDTA, 0.5 × 10⁻⁶ g / L. -3 g / L, FeSO4·7H2O, 0.2×10 -3 g / L, ZnSO4·7H2O, 0.1×10 -4 g / L, MnCl2·4H2O, 0.3×10 -5 g / L, H3BO3, 0.3×10 -4 g / L, CoCl2·6H2O, 0.2×10 -3g / L, CuCl2·2H2O, 0.1×10 -5 g / L, NiCl2·6H2O, 0.2×10 -5 g / L, Na₂MoO₄·2H₂O, 0.3×10 -5 g / L.

[0016] Preferably, the LBA plates are composed of: glucose, 10.0 g / L, MgCl2·6H2O, 5.0 g / L, MgSO4·7H2O, 0.25 g / L, (NH4)2SO4, 0.1 g / L, KCl, 0.2 g / L, Ca3(PO4)2, 5.0 g / L, agar, 15 g / L, pH 7.5~8.0, and are used after autoclaving at 115°C for 30 minutes.

[0017] Another objective of this invention is to provide the application of the aforementioned Bacillus Z161 in degrading straw, preventing crop diseases, and composting straw.

[0018] The beneficial effects of this invention are as follows:

[0019] 1. This invention presents, for the first time, a novel Bacillus strain capable of efficiently degrading straw under high-temperature conditions. This strain overcomes the limitations of traditional straw-degrading microorganisms in terms of temperature adaptability, enabling it to rapidly and efficiently decompose complex components in straw, such as cellulose, hemicellulose, and lignin, in high-temperature environments. Its straw degradation performance under high-temperature conditions solves the industry problem of low efficiency caused by temperature limitations in straw degradation and provides a new microbial resource for the resource utilization of agricultural straw.

[0020] 2. The Bacillus Z161 provided by this invention, when cultured with straw as the sole carbon source, achieves a straw degradation rate of 48.63% after 15 days of culture at 28℃ and 39.46% after 15 days of culture at 50℃.

[0021] 3. The enzyme activities of the strain provided by this invention were measured after culture. At 28℃, the cellulase activity was 23.03 ug / min / mL, hemicellulase 360.2 nmol / min / mL, laccase 481.52 nmol / min / L, manganese peroxidase 2.478 nmol / min / mL, and peroxidase 4.5 U / mL; at 50℃, the cellulase activity was 7.13 ug / min / mL, hemicellulase 40.32 nmol / min / mL, laccase 296.32 nmol / min / L, manganese peroxidase 0.826 nmol / min / mL, and peroxidase 11.75 U / mL.

[0022] 4. The strain provided by this invention has the ability to produce IAA, which can promote the development of plant roots and stem elongation, enhance the plant's ability to absorb nutrients and water, thereby enhancing the photosynthetic efficiency of the plant, promoting the overall growth and development of the plant, and increasing crop yield; moreover, the strain has the ability to antagonize pathogenic bacteria, is non-pathogenic to plants, animals and humans, and can be used to develop green agricultural products such as bio-organic fertilizers and bio-pesticides.

[0023] Preservation Information

[0024] Preservation period: October 29, 2025;

[0025] Preservation institution: China General Microbiological Culture Collection Center, China Committee on the Preservation and Management of Microbial Culture Collections;

[0026] Accession number: CGMCC No. 36393;

[0027] Address of the depositary: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing;

[0028] Postal code: 100101;

[0029] Category Naming: Bacillus mexicanus . Attached Figure Description

[0030] Figure 1 The streak colony morphology of Bacillus Z161 (LBA plate).

[0031] Figure 2 Phylogenetic tree of Bacillus Z161;

[0032] Figure 3 The cellulase activity of Bacillus Z161 at 28℃ and 50℃ was measured.

[0033] Figure 4 The hemicellulase activity of Bacillus Z161 at 28℃ and 50℃ was measured.

[0034] Figure 5 The peroxidase activity of Bacillus Z161 at 28℃ and 50℃ was measured.

[0035] Figure 6 The manganese peroxidase activity of Bacillus Z161 at 28℃ and 50℃ was measured.

[0036] Figure 7 The laccase activity of Bacillus Z161 at 28℃ and 50℃ was measured.

[0037] Figure 8 This is a colorimetric control diagram of the IAA test for Bacillus Z161 (left side is CK without bacteria, right side is with Z161).

[0038] Figure 9 The images show the plate confrontation between Bacillus Z161 and Fusarium oxysporum 2CM2W-4; where A is the growth of Fusarium oxysporum 2CM2W-4 in PDA; and B is the plate confrontation image. Detailed Implementation

[0039] The present invention will now be described in detail with reference to specific accompanying drawings and embodiments. The examples described below are merely preferred embodiments of the present invention. It should be noted that the following description is only for explaining the present invention and is not intended to limit the present invention in any way. Any simple modifications, equivalent changes, and alterations made to the embodiments based on the technical essence of the present invention shall fall within the scope of the technical solution of the present invention.

[0040] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0041] Example 1: Screening of Bacillus

[0042] The bacterial strain was isolated from samples collected during the high-temperature fermentation phase of lignocellulosic compost. 3g of the sample was weighed and added to 150 mL of pre-sterilized straw-MSM medium (3.5g dry straw, 0.645g MSM inorganic salt medium, and distilled water to a final volume of 0.15 L; the MSM inorganic salt medium consisted of: Na₂HPO₄, 2.8 g / L, KH₂PO₄, 1.0 g / L, (NH₄)₂SO₄, 0.5 g / L, MgCl₂, 0.053 g / L, Ca(NO₃)₂·4H₂O, 0.05 g / L, and Na₂EDTA, 0.5 × 10⁻⁶ g / L). -3 g / L, FeSO4·7H2O, 0.2×10 -3 g / L, ZnSO4·7H2O, 0.1×10 -4 g / L, MnCl2·4H2O, 0.3×10 -5 g / L, H3BO3, 0.3×10 -4 g / L, CoCl2·6H2O, 0.2×10 -3 g / L, CuCl2·2H2O, 0.1×10 -5 g / L, NiCl2·6H2O, 0.2×10 -5 g / L, Na₂MoO₄·2H₂O, 0.3×10 -5 g / L).

[0043] Shake in a shaker at 50°C for 7 days, then the well-shaken culture medium is prepared as 10 -1 Diluent. Add 1 mL of 10 to a test tube containing 9 mL of sterile water. -1The diluted solution is mixed by vortexing for 30 seconds to prepare 10. -2 Diluent. Then dilute sequentially in 10-fold increments to 10. -6 Take 100 µL of 10 -3 ~10 -6 The diluted solution was spread onto LBA plates (glucose, 10.0 g / L; MgCl2·6H2O, 5.0 g / L; MgSO4·7H2O, 0.25 g / L; (NH4)2SO4, 0.1 g / L; KCl, 0.2 g / L; Ca3(PO4)2, 5.0 g / L; agar, 15 g / L; pH 7.5–8.0; autoclaved at 115°C for 30 minutes) using a spreader. The plates were incubated upside down at 28°C for 2 days. Single colonies were observed and selected periodically. The culture was then purified at least five times on new LBA plates to obtain strain Z161 (Bacillus mexicans), which exhibits high-temperature tolerance to straw degradation.

[0044] Example 2: Morphological observation and physiological and chemical identification of strain Z161

[0045] This invention screened a strain of Bacillus ( ) from samples of lignocellulosic compost at high temperatures. Bacillus mexicanus Z161 was amplified by PCR using primer pairs; the primer pair sequences are as follows:

[0046] 27F (shown as SEQ ID NO.2): AGAGTTTGATCMTGGCTCAG;

[0047] 1492R (shown as SEQ ID NO.3): TACGGYTACCTTGTTACGACTT.

[0048] The PCR amplification system consists of the following components and composition: 25 μL of 2×HiFi PCR Master Mix, 2 μL of 27F primers, 2 μL of 1492R primers, 1 μL of gDNA, and ddH2O to a final volume of 50 μL.

[0049] The PCR cycling parameters were as follows: pre-denaturation: 95℃, 10 min; denaturation: 95℃, 15 s; annealing: 55℃, 30 s; extension: 72℃, 1 min (35 cycles in total); extension: 72℃, 10 min; storage: 4℃. A 3.0% agarose gel was then prepared, and the electrophoresis voltage was set to 18 V / cm for 20 min. Agarose gel electrophoresis was performed using nucleic acid dyes, and images were taken using a UV gel imaging system. The target fragment was recovered from the agarose gel using a standard agarose gel DNA recovery kit, and the recovered product was sequenced. The 16S rDNA amplification and sequencing sequence of strain Z161 is shown in SEQ ID NO.1, and it was identified as [missing information - likely a specific DNA sequence] in both Genbank and EzBioCloud Databases. Bacillus mexicanus .

[0050] The Z161 strain was successfully sequenced using forward primer 27F and reverse primer 1492R. The assembled result is shown below (SEQ ID NO.1, 1452bp):

[0051]

[0052] Morphologically, strain Z161 is a Gram-positive bacterium with typically rod-shaped cells, blunt at both ends, and approximately 1.0–1.5 μm in length. Colonies grown on LBA medium are round or nearly round, milky white or pale yellowish-white, with a glossy surface, a slightly convex center, and no or only slight wrinkles. Specifically, as shown... Figure 1 As shown.

[0053] Phylogenetic tree of strain Z161 as follows Figure 2 As shown: strain Z161 and Bacillus mexicanus The branching support rate of strain M5604 is 80%, indicating that the two are closely related.

[0054] Example 3: Determination of High-Temperature Straw Degradation Capacity

[0055] (1) Add 3.5 g of dried straw fibers with a length of 1-2 cm to 150 mL of MSM medium and sterilize in a pressure steam sterilizer at 115℃ for 30 minutes. After sterilization, add the pre-shaken bacterial solution to the conical flask to make the bacterial concentration in the experimental group conical flask 10. 6 The concentration was cfu / mL, with 6 biological replicates. The prepared conical flasks were then placed in a shaker at 50°C and 180 r / min for 15 days.

[0056] After the experiment, the degradation rate of strain Z161 under the condition of straw as the sole carbon source was determined by gravimetric method. The results showed that the straw degradation rate could reach 48.63% after 15 days of cultivation at 28 ℃ and 39.46% after 15 days of cultivation at 50 ℃. The degradation rates of other selected strains were lower than those of strain Z161, as shown in Table 1.

[0057] The components and concentrations of MSM medium include: Na₂HPO₄, 2.8 g / L, KH₂PO₄, 1.0 g / L, (NH₄)₂SO₄, 0.5 g / L, MgCl₂, 0.053 g / L, Ca(NO₃)₂·4H₂O, 0.05 g / L, and Na₂EDTA, 0.5 × 10⁻⁶ g / L. -3 g / L, FeSO4·7H2O, 0.2×10 -3 g / L, ZnSO4·7H2O, 0.1×10 -4 g / L, MnCl2·4H2O, 0.3×10 -5 g / L, H3BO3, 0.3×10 -4 g / L, CoCl2·6H2O, 0.2×10 -3 g / L, CuCl2·2H2O, 0.1×10 -5g / L, NiCl2·6H2O, 0.2×10 -5 g / L, Na₂MoO₄·2H₂O, 0.3×10 -5 g / L.

[0058] Table 1. High-temperature straw degradation rate of screened strains

[0059] .

[0060] Example 4: Determination of enzyme activity of degradative enzyme system of strain Z161

[0061] Preparation of crude enzyme solution: After thoroughly mixing the bacterial culture of strain Z161 in straw-MSM medium, 1 mL was transferred to a 1.5 mL centrifuge tube and centrifuged at 10000 r / min for 3 min. Then, 500 μL of the supernatant was transferred to a 10K ultrafiltration tube and centrifuged at 14000 g for 10 min to separate the enzyme protein from the liquid. After separation, the inner ultrafiltration tube containing the enzyme protein was inverted into a new outer tube and centrifuged at 10000 g in reverse for 5 min to recover the enzyme protein. 500 μL of distilled water was then used to thoroughly dissolve and mix the recovered enzyme protein to prepare the crude enzyme solution to be tested.

[0062] The kits for detecting laccase, manganese peroxidase, and lignin peroxidase were purchased from Isehisa (Lianyungang, Jiangsu) Biotechnology Co., Ltd.; the kits for detecting cellulase and neutral xylan (hemicellulase) activity were purchased from Shanghai Enzyme-Linked Biotechnology Co., Ltd.

[0063] (1) Determination of laccase:

[0064] Lac activity was detected using the ABTS method. The ABTS radicals generated by laccase lysis of the substrate ABTS have an absorbance coefficient at 420 nm that is much greater than that of the substrate ABTS. The laccase activity can be calculated by measuring the rate of increase of ABTS radicals.

[0065] Two reaction systems were set up using 1.5 mL centrifuge tubes: one control tube and one assay tube. 45 μL of crude enzyme solution was added to each reaction tube. Then, 255 μL of prepared citrate-disodium hydrogen phosphate buffer was added to the control tube, and 255 μL of prepared ABTS solution was added to the assay tube. Both control and assay tubes were placed in a dry, temperature-controlled metal bath at 60°C for 20 min. After cooling to room temperature, 200 μL of the reaction solution was pipetted into a 96-well plate. After preheating the microplate reader for 1 h, the absorbance of the reaction system was measured at a wavelength of 420 nm. The Lac activity unit is defined as the amount of enzyme required to oxidize 1 nmol of ABTS substrate per minute per liter of culture medium.

[0066] .

[0067] Note: A 测定 : The absorbance of the measuring tube at 420 nm; A 对照 : Absorbance of the control tube at 420 nm; ε: ABTS millimolectic extinction coefficient, 36 L / mmol / cm; d: Cuvette path length, 0.5 cm; V 反总 Total reaction volume, 0.3 mL; V 样 : Sample volume during reaction, 0.045 mL; T: Reaction time, 20 min.

[0068] (2) Determination of manganese peroxidase:

[0069] The activity of manganese peroxidase (MnP) was detected using the MnSO4 method. Manganese peroxidase in Mn... 2+ Under certain conditions, guaiacol will be oxidized to tetra-o-methoxyl, which has a characteristic absorption peak at 465 nm. By measuring the absorbance of tetra-o-methoxyl at 465 nm at different reaction times, the manganese peroxidase activity can be calculated.

[0070] Add 20 μL of crude enzyme solution to a 96-well plate, then add 100 μL of a prepared succinic acid and NaOH mixed aqueous solution, 20 μL of MnSO4 solution, 40 μL of a prepared guaiacol and ethanol mixed aqueous solution, and 20 μL of H2O2 solution sequentially. Mix thoroughly, zero the plate with distilled water, preheat the microplate reader for 1 h, adjust the wavelength to 465 nm, and measure the absorbance A1 after 30 s and the absorbance A2 after 150 s. MnP activity is defined as the amount of enzyme required to oxidize 1 nmol of guaiacol per minute per milliliter of culture medium.

[0071] .

[0072] Note: ε: Molar extinction coefficient of guaiacol: 1.21 × 10⁻⁶ 4 L / mol / cm; d: optical path length of 96-well plate, 0.5 cm; V 反总 Total volume, 2×10 -4 L;V 样 : Sample volume during reaction, 0.02 mL; T: Reaction time, 120 s.

[0073] (3) Determination of lignin peroxidase:

[0074] The activity of lignin peroxidase (LiP) was determined using the azurite B method. Linin peroxidase catalyzes the demethylation of azurite B solution, resulting in a decrease in absorbance at 651 nm. The lignin peroxidase activity can be calculated by measuring the absorbance at 651 nm of the reaction system at different time points.

[0075] First, prepare the working solution by mixing tartaric acid-sodium tartrate solution, azurite B solution, and H2O2 solution in a 2:1:1 ratio. Add 40 μL of the crude enzyme solution to a pre-prepared 96-well plate, then add 160 μL of the prepared working solution and mix thoroughly. Zero the plate with distilled water, preheat the microplate reader for 1 h, adjust the wavelength to 651 nm, and measure the absorbance A1 after 10 s and the absorbance A2 after 130 s. LiP activity is defined as a change of 0.01 A651 per minute per mL of culture medium in the reaction system, which is considered one unit of enzyme activity.

[0076] .

[0077] Note: V 反总 Total reaction volume, 0.2 mL; V 样 : Reaction sample volume, 0.04 mL; T: Reaction time, 2 min.

[0078] (4) Determination of cellulase activity

[0079] The content of reducing sugars produced by the degradation of sodium carboxymethyl cellulose catalyzed by CL was determined using the DNS colorimetric method. The content was calculated based on the volume of serum (plasma) or other liquids.

[0080] Definition of unit: One unit of enzyme activity is defined as the amount of glucose produced per minute by the catalytic activity of 1 μg of serum (plasma).

[0081] CL activity (μg / min / mL) = [(ΔA + 0.2885) ÷ 2.754 × 10 3 ] ×(V 反总 ÷V 样 )÷T×D=24.2072×(ΔA+0.2885) ×D.

[0082] ΔA=A 测定 -A 对照；

[0083] V: Volume of extraction solution added, 1 mL;

[0084] V 反总 Total volume of the reaction system: 0.2 mL;

[0085] V 样 Add 0.05 mL of sample volume;

[0086] T: Reaction time, 60 min;

[0087] 10 3 Conversion factor from mg to μg, 1000;

[0088] D: Additional dilution factor, which is 1 for undiluted.

[0089] (5) Determination of hemicellulase activity:

[0090] Xylanase catalyzes the degradation of xylan into reducing oligosaccharides and monosaccharides in a neutral environment. Under boiling water bath conditions, it further undergoes a colorimetric reaction with 3,5-dinitrosalicylates, exhibiting a characteristic absorption peak at 540 nm. The intensity of the color of the reaction solution is directly proportional to the amount of reducing sugars produced by enzymatic hydrolysis. The NEX activity can be calculated by measuring the rate of increase in absorbance at 540 nm of the reaction solution.

[0091] Calculated based on liquid volume:

[0092] Enzyme activity is defined as the amount of enzyme required to break down xylan into 1 nmol of reducing sugar per minute per milliliter of liquid sample at 50°C and pH 6.0.

[0093] NEX activity (nmol / min / mL) = [(ΔA + 0.0436) ÷ 0.6476 - 150.13] × 10 6 ÷T×D=342.8502×(ΔA+0.0436)×D.

[0094] For detailed test results, please see [link / details]. Figures 3-7 As shown: The enzyme activity of the strain provided by this invention was measured after culturing. The Z161 strain was inoculated into LB liquid medium at an inoculation concentration of 10. 6 The enzymes were tested at CFU / mL at 28℃ and 50℃, respectively. The activities of the five enzymes were measured on days 0, 1, 2, and 3. The enzyme activities were calculated using the enzyme method described above. (The vertical axis represents the different enzymes and their activities, and the horizontal axis represents the number of days of measurement.)

[0095] At 28℃, the highest Z161-related enzyme activities reached the following values: cellulase 23.03 μg / min / mL, hemicellulase 360.2 nmol / min / mL, laccase 481.52 nmol / min / L, manganese peroxidase 2.478 nmol / min / mL, and peroxidase 4.5 U / mL. At 50℃, the highest Z161-related enzyme activities reached the following values: cellulase 7.13 μg / min / mL, hemicellulase 40.32 nmol / min / mL, laccase 296.32 nmol / min / L, manganese peroxidase 0.826 nmol / min / mL, and peroxidase 11.75 U / mL.

[0096] Example 5: IAA Performance Testing

[0097] 1. Experimental reagents:

[0098] (1) Salkowski colorimetric reagent: Prepare 50 mL of 35% perchloric acid solution and add 1 mL of 0.5 mol / L FeCl3;

[0099] (2) LB liquid culture medium: Weigh 10g of Tryptone, 5g of Yeast Extract, 10g of Sodium Chloride and 15g of Agar and add them to 1L of water;

[0100] (3) L-Tryptophan solution (200 mg / L): Weigh 0.02 g of L-Tryptophan and add it to 100 ml of water.

[0101] 2. Quantitative determination method for IAA:

[0102] (1) The test strain (Bacillus Z161 strain) was inoculated into LB medium and cultured in a shaker at 28 ℃ and 180 r / min for 24 hours. 200 μL of bacterial culture was inoculated into 4 mL of LB medium (with 40 μL of 200 mg / L L-tryptophan added) as the experimental group, and the control group was LB medium without bacterial inoculation (with 40 μL of 200 mg / L L-tryptophan added). Each treatment was repeated three times and cultured in a shaker at 28 ℃ and 180 rpm for 24 hours.

[0103] (2) Centrifuge the cultured experimental group and control group at 12000 r / min for 10 min, take 500 μL of supernatant from each group, add them to a 2 mL centrifuge tube that has been sterilized in advance, add 500 μL of Salkowski colorimetric reagent and let stand in the dark for 30 min, then observe the color change.

[0104] As shown in Figure 8, the two Bacillus strains labeled Z161 on the right are from the Z161 group. The color of the liquid inside the tubes reflects the ability to produce indoleacetic acid (IAA), with a redder color indicating a stronger IAA production capacity. As can be seen from the figure, the Bacillus Z161 group's bacterial solution appears reddish compared to the control, indicating that the Bacillus Z161 strain screened in this invention has the ability to produce IAA.

[0105] Example 6: Antagonistic effect of the strain on the pathogen.

[0106] The selected Bacillus Z161 strain was initially screened using the plate confrontation method. For the test bacteria, strains stored at -80℃ were activated and then inoculated into LB liquid medium and cultured for 1-2 days. A 5 mm mycelial cake was punched at the edge of the pathogen using a punch, and the cake was placed on PDA medium. Bacterial suspension was added dropwise to both sides of the cake 3 cm from the edge of the medium. The control group was treated with LB liquid medium. Each treatment was repeated in triplicate. After 7 days of incubation at 28℃, the diameter of the pathogen was measured, and the growth inhibition rate was calculated.

[0107] Plate confrontation experiments revealed that the Bacillus Z161 screened in this invention is effective against 2CM2W-4 Fusarium oxysporum ( ). Fusarium oxysporum It has an inhibitory effect, with an inhibition rate of 55.29%. Figure 9 ).

Claims

1. A highly efficient Bacillus strain that degrades straw ( Bacillus mexicanus Z161, characterized in that, The Bacillus Z161 mentioned is Bacillus mexicanus, with accession number CGMCC NO.36393, deposited on October 29, 2025, at the China General Microbiological Culture Collection Center, and classified as follows: Bacillus mexicanus The 16S rDNA sequence of the Bacillus Z161 is shown in SEQ ID NO.

1.

2. The application of Bacillus Z161 as described in claim 1 in the degradation of straw.

3. The application according to claim 2, characterized in that, The application of Bacillus Z161 in straw composting.

4. The application according to claim 2, characterized in that, The Bacillus Z161 possesses a complex enzyme activity system consisting of laccase, manganese peroxidase, lignin peroxidase, cellulase, and hemicellulase at 50°C.

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