A Bacillus velezensis strain and its application in the preparation of a multifunctional straw composting agent

Abstract

The present invention relates to a strain of Bacillus velezensis and its application in the preparation of a multifunctional straw decomposer, belonging to the field of microbial technology. The Bacillus velezensis has the functions of producing cellulase, producing xylanase, degrading avermectin, decomposing inorganic phosphorus, and degrading straw. When returning straw to the field, using the multifunctional straw decomposer provided by the present invention can not only accelerate the decomposition rate of straw, decompose the avermectin residue in the soil, improve the soil ecology, but also promote the growth of pepper plants and enhance the stress resistance of pepper plants, showing good comprehensive application effects.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, in particular to a strain of Bacillus Velezii and application thereof in preparing a multifunctional straw composting agent. Background Art

[0002] China is a major agricultural producer. The straw of crops such as corn, wheat, and rice is a very valuable biomass resource. The comprehensive utilization of straw is of great significance to farmers' income increase, environmental protection, resource conservation, and sustainable agricultural development. In recent years, the comprehensive utilization rate of straw of major food crops such as corn, wheat, and rice in my country has increased year by year. Among them, fertilizer utilization is the most important way to comprehensively utilize straw, which includes direct return of straw to the field and return to the field after composting. Direct return of straw to the field is economical, convenient, labor-saving, and is the most widely used way to utilize straw as fertilizer; however, the straw has a slow decomposition speed and a long decomposition cycle after returning to the field, which may bring many negative effects, such as affecting the growth of crop roots or aggravating pests and diseases. Therefore, developing straw decomposition agents to accelerate the decomposition speed of straw after returning to the field is of great significance to preventing and controlling negative effects and promoting the return of straw to the field.

[0003] Straw composting agent is a biological agent that can promote the composting of crop straw. The use of straw composting agent can accelerate the biodegradation of straw after returning it to the field, shorten the composting cycle, improve the composting quality, and reduce the negative effects of directly returning straw to the field. There are many types of straw composting agents currently on the market, and they have achieved good application results in agricultural production. However, the functions of the straw composting agents currently on the market are relatively simple, and they often only consider their ability to degrade straw, without paying attention to other beneficial functions, such as promoting crop growth, preventing and controlling crop diseases, improving soil, and degrading pesticide residues.

[0004] Abamectin is a commonly used pesticide with high toxicity. Abamectin residues in the soil will not only affect the growth and metabolism of soil microorganisms and destroy the ecological balance of the soil, but may also be absorbed into plants, which will have an adverse effect on the growth of crops. Therefore, the use of microorganisms with avermectin degradation function to develop straw composting agents can not only promote straw composting, but also promote the biodegradation of avermectin residues in straw and soil, eliminating the adverse effects of avermectin on soil ecology and crops.

[0005] Therefore, developing a multifunctional straw composting agent that can degrade pesticide residues, improve soil ecology and promote plant growth while degrading straw will be of great significance to the development of my country's agriculture. Summary of the invention

[0006] In view of the deficiencies of the prior art, the present invention provides a strain of Bacillus velezensis and its application in the preparation of a multifunctional straw decomposing agent. When returning straw to the field, using the multifunctional straw decomposing agent provided by the present invention can not only accelerate the decomposition rate of straw, decompose the abamectin pesticide residues in the soil, improve the soil ecology, but also promote the growth of pepper plants and enhance the stress resistance of pepper plants.

[0007] The technical solution of the present invention is as follows:

[0008] A strain of Bacillus velezensis PAPM-LX25 was deposited at the General Microbiology Center of the China Committee for Culture Collection of Microorganisms on November 06, 2024. The address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 32499.

[0009] Among them, the 16S rDNA sequence of the Bacillus velezensis PAPM-LX25 is shown in SEQ ID NO.1.

[0010] The cultivation method of the Bacillus velezensis PAPM-LX25 includes the following steps:

[0011] (1) Inoculate Bacillus velezensis PAPM-LX25 into LB solid medium and activate it at 35-38 °C to obtain single colonies;

[0012] (2) Inoculate the single colonies in step (1) into LB liquid medium and shake culture at 180-220 r / min and 35-38 °C to obtain a seed solution;

[0013] (3) Inoculate the seed solution in step (2) into LB liquid medium at an inoculation amount of 3-5% by volume and shake culture at 180-220 r / min and 35-38 °C to obtain the Bacillus velezensis PAPM-LX25 bacterial solution.

[0014] The application of the Bacillus velezensis PAPM-LX25 in producing cellulase, producing xylanase, degrading abamectin, decomposing inorganic phosphorus, and degrading straw.

[0015] A multifunctional straw decomposing agent includes the Bacillus velezensis PAPM-LX25.

[0016] Preferably, the multifunctional straw decomposing agent further includes Trichoderma viride and Bacillus subtilis.

[0017] Further preferably, the multifunctional straw decomposing agent comprises the following components: 560-80 parts by weight of Bacillus velezensis PAPM-LX25, 15-25 parts by weight of Trichoderma viride, and 5-15 parts by weight of Bacillus subtilis.

[0018] For the preparation method of the multifunctional straw decomposing agent, inoculate Bacillus velezensis PAPM-LX25 into a solid fermentation medium, ferment at 33-42 °C for 24-48 h to obtain a fermentation product; dry the fermentation product at 42-45 °C and crush it to 80-100 mesh to obtain Bacillus velezensis PAPM-LX25 bacterial powder; mix the Bacillus velezensis PAPM-LX25 bacterial powder with Trichoderma viride powder and Bacillus subtilis powder to obtain the multifunctional straw decomposing agent.

[0019] Preferably, the solid fermentation medium comprises the following components according to the mass ratio: 40% wheat bran, 10% soybean meal powder, 49% corn straw powder, and 1% Ca(OH)2.

[0020] Further preferably, the initial water content of the solid fermentation medium is 50%-60%; during the fermentation process, the water content of the solid fermentation medium is maintained at 45%-55%.

[0021] Further preferably, in the multifunctional straw decomposing agent, the viable bacteria content in the Bacillus velezensis PAPM-LX25 bacterial powder is (2.0-3.0)×10 10 cfu / g, the viable bacteria content in the Trichoderma viride powder is (3.0-5.0)×10 9 cfu / g, and the viable bacteria content in the Bacillus subtilis powder is (2.0-3.0)×10 10 cfu / g.

[0022] Application of the multifunctional straw decomposing agent in producing cellulase, producing xylanase, degrading avermectin, decomposing inorganic phosphorus, degrading straw, promoting the growth of pepper plants, and enhancing the stress resistance of pepper plants.

[0023] Preferably, the application method of the multifunctional straw decomposing agent in degrading straw is: mix the multifunctional straw decomposing agent and water in a weight ratio of 1-2:50-100, and evenly sprinkle it on the straw.

[0024] Preferably, the application method of the multifunctional straw decomposing agent in promoting the growth of pepper plants or enhancing the stress resistance of pepper plants is: mix the multifunctional straw decomposing agent and straw evenly and apply them to the soil.

[0025] Advantages of the present invention:

[0026] The present invention provides a strain of Bacillus velezensis and a multifunctional straw decomposing agent prepared therefrom. When returning straw to the field, the multifunctional straw decomposing agent provided by the present invention can not only accelerate the decomposition rate of straw, decompose the abamectin residues in the soil, improve the soil ecology, but also promote the growth of pepper plants and enhance the stress resistance of pepper plants, showing good comprehensive application effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 is the colony morphology of strain PAPM-LX25.

[0028] Figure 2 is the cell morphology of strain PAPM-LX25.

[0029] Figure 3 is the phylogenetic tree of strain PAPM-LX25.

[0030] Figure 4 is the temperature change during the composting process in Application Example 2. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0031] The following is described in conjunction with specific embodiments:

[0032] Description of the source of experimental materials:

[0033] Trichoderma viride: Purchased from the China Center for Type Culture Collection of Agricultural Microorganisms, with the preservation number ACCC 30794.

[0034] Bacillus subtilis: Purchased from the China Center for Type Culture Collection of Agricultural Microorganisms, with the preservation number ACCC 04396.

[0035] Example 1: Isolation and screening of Bacillus velezensis PAPM-LX25

[0036] Samples were collected from the abamectin fermentation waste residue compost in Ancheng Town, Pingyin County, Jinan City, Shandong Province, China. The specific isolation and screening methods are as follows: Weigh 10.0 g of the compost sample, add 100 mL of sterile water, place it on a constant temperature shaker at 35 °C and 180 r / min, and oscillate and extract for 30 min to obtain the extract.

[0037] (1) Screening of abamectin-degrading strains: Inoculate the above-mentioned leaching solution into an LB liquid medium containing 200 mg / L abamectin, and culture it at 35 °C with shaking at 180 r / min for 3 days; then transfer the culture solution successively to LB liquid media containing 400 mg / L, 600 mg / L, and 800 mg / L abamectin, and culture for 3 days respectively to obtain enriched cultures; appropriately dilute the enriched cultures, and then spread them on a 1 / 10 concentration LB solid medium containing 800 mg / L abamectin, and culture at 35 °C until single colonies grow; isolate the single colonies to obtain strains with different colony morphologies, and then inoculate them into a medium with abamectin as the sole carbon source respectively, and culture at 35 °C with shaking at 180 r / min for 24 - 72 h. Pick out the strains that can make the medium turbid, that is, obtain the strains that can degrade abamectin, and store them for later use.

[0038] Among them, the components of the LB liquid medium are: peptone 10 g, yeast extract 5 g, NaCl 10 g, distilled water 1000 mL; pH 7.0, sterilized at 121 °C for 20 min.

[0039] Among them, the 1 / 10 concentration LB solid medium containing 800 mg / L abamectin is a medium with the concentration of each nutrient being 1 / 10 of the conventional LB solid medium and containing 800 mg / L abamectin; the specific components are: peptone 1 g, yeast extract 0.5 g, NaCl 1 g, abamectin 800 mg, agar 20 g, distilled water 1000 mL.

[0040] Among them, the components of the medium with abamectin as the sole carbon source are: (NH4)2SO4 2 g, MgSO4 0.2 g, NaH2PO4 0.5 g, CaCl2 0.1 g, K2HPO4 0.5 g, abamectin 800 mg, distilled water 1000 mL.

[0041] (2) Screening of straw-degrading strains: The strains capable of degrading avermectin screened above were inoculated onto a sodium carboxymethyl cellulose medium and cultured at 35 °C for 2 days. Strains capable of producing a hydrolysis zone were screened out, and the diameter of the clear zone (D) and the diameter of the colony (d) were recorded. Strains with a larger D / d value were preferred; the screened strains were inoculated into a filter paper disintegration medium and cultured with shaking at 35 °C and 180 r / min for 3 days. Strains with stronger filter paper disintegration ability were screened out; the screened strains were further screened by the straw weight loss method. The specific method was as follows: Wheat straw was cut into small sections of 3 - 5 cm, dried at 85 °C, and 50.00 g was weighed and placed into a nylon mesh bag; 1 mL of the culture solution of the strain to be screened was taken, diluted 50 times with water, 0.1 g of urea was added, stirred and dissolved, and then sprinkled on the wheat straw; the straw was buried in the field to a depth of 15 - 20 cm, taken out after 15 days, washed with clean water, dried and weighed. Strains with a higher straw weight loss rate were preferred for subsequent screening.

[0042] Among them, the components of the filter paper disintegration medium were: 1 g of (NH4)2SO4, 1 g of KH2PO4, 0.7 g of MgSO4·7H2O, 0.5 g of NaCl, 1000 mL of distilled water, and 1 strip of filter paper with a size of 6 cm × 1 cm.

[0043] (3) Screening of phosphorus-solubilizing strains: The strains with both strong avermectin degradation ability and straw degradation ability screened above were inoculated onto a Meng Jinna inorganic phosphorus bacteria medium and cultured at 35 °C for 5 days. Strains capable of producing a larger clear zone were screened out.

[0044] Among them, the components of the Meng Jinna inorganic phosphorus bacteria medium were: 10.0 g of glucose, 0.5 g of (NH4)2SO4, 0.3 g of MgSO4·7H2O, 0.03 g of MnSO4·4H2O, 0.3 g of KCl, 0.03 g of FeSO4·7H2O, 0.3 g of NaCl, 10.0 g of Ca3(PO4)2, 16 g of agar, 1000 mL of distilled water; pH 7.0 - 7.5.

[0045] Through the above separation and screening work, we finally screened out a strain with the characteristics of degrading avermectin, degrading straw, and solubilizing inorganic phosphorus, and named it "PAPM-LX25".

[0046] Example 2: Identification of Bacillus velezensis PAPM-LX25

[0047] The colony morphology of the strain PAPM-LX25 screened above on the LB solid medium was as Figure 1 shown. It can be seen that the colonies were round, milky white, opaque, and the surface was dry and wrinkled; the cell morphology of the strain PAPM-LX25 was as Figure 2As shown, the bacteria are rod-shaped and have endospores.

[0048] In addition, the physiological and biochemical characteristics of strain PAPM-LX25 were identified, and the identification results are as follows: catalase test +; methyl red test +; starch hydrolysis test +; denitrification test +; ammonia production test +; lactose fermentation test -; sucrose fermentation test +; xylose fermentation test +; arabinose test +; salicin test -; melibiose fermentation test +; rhamnose fermentation test -; mannitol fermentation test +; glucose fermentation test + (+, positive; -, negative).

[0049] The genomic DNA of strain PAPM-LX25 was extracted and PCR amplification was carried out using 16S rDNA universal primers. The amplification conditions were: pre-denaturation at 95°C for 3 min; denaturation at 94°C for 1 min, annealing at 55°C for 1 min, extension at 72°C for 1.5 min, for a total of 30 cycles; extension at 72°C for 10 min. The amplified 16S rDNA gene sequence was sequenced, and the sequencing results are shown in SEQ ID NO.1; the obtained 16S rDNA sequence was subjected to BLAST analysis with the existing sequences in the NCBI database, and strains with similar homology were selected. A phylogenetic tree was constructed using MEGA7.0 software, and the constructed phylogenetic tree is as Figure 3 shown; it was found that the above-selected strain PAPM-LX25 was relatively close to Bacillus velezensis strain C13 in terms of evolutionary distance. Combining the physiological and biochemical characteristics of the strain, it was identified as "Bacillus velezensis".

[0050] Bacillus velezensis PAPM-LX25 was deposited on November 06, 2024 at the General Microbiological Center of the China National Center for Biotechnology Development, address: No. 3, Building 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC No. 32499.

[0051] Example 3: Cultivation of Bacillus velezensis PAPM-LX25

[0052] (1) Bacillus velezensis PAPM-LX25 was inoculated onto LB solid medium and cultured at 35°C for 20 h for activation to obtain single colonies;

[0053] (2) The single colonies from step (1) were inoculated into LB liquid medium and cultured with shaking at 180 r / min and 35°C for 20 h to obtain a seed solution;

[0054] (3) Inoculate the seed liquid from step (2) into LB liquid medium at an inoculation amount of 5% by volume, and culture it with shaking at 180 r / min and 35 °C for 20 h to obtain Bacillus velezensis PAPM-LX25 bacterial liquid.

[0055] Example 4: Determination of beneficial characteristics of Bacillus velezensis PAPM-LX25

[0056] (1) Enzyme-producing ability of Bacillus velezensis PAPM-LX25

[0057] Take a mixture of wheat bran and corn straw powder (4:6, w / w), a total of 15 g, put it into a 250 mL Erlenmeyer flask, add 15 mL of distilled water, mix well, sterilize it by moist heat at 121 °C for 30 min, and cool it naturally; inoculate the Bacillus velezensis PAPM-LX25 bacterial liquid prepared in Example 3 into the above Erlenmeyer flask at an inoculation amount of 5% (w / w), and culture it by fermentation at 35 °C for 48 h, shaking the flask every 6 - 8 h during this period; after the fermentation is completed, dry the fermentation product at 50 °C and grind it; weigh 1.0 g of the fermentation product, add 100 mL of distilled water, extract it by shaking at 180 rpm for 10 min, and filter to obtain the crude enzyme solution.

[0058] The DNS method is used to determine the enzyme activities of cellulase and xylanase in the crude enzyme solution. The specific steps are as follows: Place 1.5 mL of 1.00% CMC solution or 1.5 mL of 1.00% xylan solution and the appropriately diluted crude enzyme solution in a water bath at 50 °C and preheat for 2 min; Pipette 0.5 mL of the crude enzyme solution into the CMC solution or xylan solution, accurately time, react for 10 min, quickly add 3 mL of DNS reagent to terminate the reaction, and then develop color in a boiling water bath for 15 min; After taking it out, make up the volume to 25 mL with distilled water, shake well, cool, and measure the absorbance at a wavelength of 540 nm. Blank control: First add DNS reagent to the CMC solution or xylan solution, place it in a boiling water bath, and then add the appropriately diluted crude enzyme solution, with other conditions the same as the normal measurement method.

[0059] Definition of cellulase enzyme activity unit: Under the conditions of 50 °C and pH 5.0, reacting for 10 min, the amount of enzyme required to hydrolyze sodium carboxymethyl cellulose to produce 1 g of reducing sugar per minute is defined as one enzyme activity unit, denoted by U.

[0060] Definition of xylanase enzyme activity unit: Under the conditions of 50 °C and pH 5.0, reacting for 10 min, the amount of enzyme required to hydrolyze xylan to produce 1 mol of reducing sugar per minute is defined as one enzyme activity unit, denoted by U.

[0061] The experimental results show that in the crude enzyme solution prepared from the fermentation products of Bacillus velezensis PAPM-LX25, the enzyme activity of cellulase is 956 U / g, and the enzyme activity of xylanase is 268 U / g. This indicates that Bacillus velezensis PAPM-LX25 has a strong ability to produce cellulase and xylanase, which will be beneficial to the degradation of crop straw by this strain.

[0062] (2) Degradation ability of Bacillus velezensis PAPM-LX25 to avermectin

[0063] Bacillus velezensis PAPM-LX25 was inoculated into the avermectin fermentation residue medium and cultured at 35 °C and 200 r / min with constant shaking for 72 h to obtain the culture solution. The avermectin fermentation residue medium without inoculating any strain was used as the blank control. Among them, the preparation method of the avermectin fermentation residue medium is as follows: after the avermectin fermentation residue is dried, it is crushed to 300 meshes, 10 g (the avermectin residue is 1.638 mg / g) is weighed, 100 mL of distilled water is added, the pH value is adjusted to 6.5, and it is sterilized at 121 °C for 15 min.

[0064] The liquid chromatography method was used to determine the residue amount of avermectin in the culture solution and calculate the degradation rate.

[0065] The experimental results show that the residue amount of avermectin in the culture solution inoculated with Bacillus velezensis PAPM-LX25 is 0.0093 mg / mL; the residue amount of avermectin in the blank control is 0.1513 mg / mL. This indicates that Bacillus velezensis PAPM-LX25 has a strong degradation ability to avermectin.

[0066] (3) Inorganic phosphorus solubilizing ability of Bacillus velezensis PAPM-LX25

[0067] Bacillus velezensis PAPM-LX25 was inoculated onto the Meng Jinna inorganic phosphorus bacteria medium and cultured at 35 °C for 3 d. The diameter of the clear zone (D) and the diameter of the colony (d) were measured, and the HC value (D / d) was calculated.

[0068] The experimental results show that the diameter of the clear zone (D) of Bacillus velezensis PAPM-LX25 is 13.8 mm, the diameter of the colony (d) is 5.2 mm, and the calculated HC value is 2.65. This indicates that Bacillus velezensis PAPM-LX25 has the ability to degrade inorganic phosphorus.

[0069] Example 5: Preparation of a multifunctional straw decomposer

[0070] The preparation of the multifunctional straw decomposing agent mainly includes the following steps: (1) Preparation of Bacillus velezensis PAPM-LX25 bacterial powder; (2) Preparation of Trichoderma viride powder; (3) Preparation of Bacillus subtilis bacterial powder; (4) Preparation of the multifunctional straw decomposing agent. The specific operation methods are as follows:

[0071] (1) Preparation of Bacillus velezensis PAPM-LX25 bacterial powder:

[0072] Strain activation: Inoculate Bacillus velezensis PAPM-LX25 onto the slant of a test tube containing LB solid medium and culture at 35°C for 20 h to obtain the activated strain.

[0073] Seed liquid preparation: Inoculate the activated strain into LB liquid medium and culture with shaking at 180 r / min and 35°C for 20 h to obtain the Bacillus velezensis PAPM-LX25 seed liquid.

[0074] Bacterial liquid culture: Inoculate the Bacillus velezensis PAPM-LX25 seed liquid into a seed tank containing LB liquid medium at an inoculation amount of 5% (v / v), and culture at 35°C for 20 h to obtain the Bacillus velezensis PAPM-LX25 bacterial liquid. Among them, the volume of the seed tank is 10 L, the liquid filling amount is 7 - 8 L, the stirring speed is 220 - 250 r / min, and the ventilation volume is 7 - 8 L / min.

[0075] Fermentation culture (using a stainless steel shallow tray of 55 cm × 100 cm for culture): Sterilize the solid fermentation medium at 121°C for 20 min, cool it and spread it evenly in the shallow tray with a spreading thickness of 5 cm; inoculate the Bacillus velezensis PAPM-LX25 bacterial liquid into the solid fermentation medium at an inoculation amount of 5% (v / v), and carry out fermentation culture for 40 h. During the fermentation process, control the product temperature at 33 - 42°C and keep the water content of the solid fermentation medium at 45 - 55% to obtain the fermented product; after the fermentation is completed, dry the fermented product at 45°C and crush it to 90 meshes to obtain the Bacillus velezensis PAPM-LX25 bacterial powder, and its viable bacteria content is 2.5×10 10 cfu / g. Among them, the formula of the solid fermentation medium by mass ratio is: wheat bran 40%, soybean meal powder 10%, corn straw powder 49%, Ca(OH)2 1%, and the initial water content is 55%.

[0076] (2) Preparation of Trichoderma viride powder:

[0077] Strain activation: Inoculate Trichoderma viride onto the slant of a test tube containing PDA solid medium and culture at 25°C for 48 h to obtain the activated strain. Among them, the components of the PDA solid medium are: 200 g of potato, 20 g of glucose, 20 g of agar powder, 1000 mL of distilled water; natural pH value.

[0078] Preparation of seed liquid: Scrape the activated strain with an inoculation loop and inoculate it into the PDB liquid medium. Incubate it at 180 r / min and 25 °C with shaking for 48 h to obtain the Trichoderma viride seed liquid. Among them, the composition of the PDB liquid medium is: 200 g of potato, 20 g of glucose, 1000 mL of distilled water; natural pH value.

[0079] Cultivation of bacterial liquid: Inoculate the Trichoderma viride seed liquid into a seed tank containing the PDB liquid medium at an inoculation amount of 5% (v / v), and culture it at 25 °C for 48 h to obtain the Trichoderma viride bacterial liquid. Among them, the volume of the seed tank is 10 L, the liquid loading is 7 - 8 L, the stirring speed is 200 - 220 r / min, and the ventilation volume is 7 - 8 L / min.

[0080] Fermentation culture (cultured in a 55 cm × 100 cm stainless steel shallow tray): Sterilize the Trichoderma viride - solid fermentation medium at 121 °C for 20 min, cool it and spread it flat in the shallow tray with a spreading thickness of 5 cm; inoculate the Trichoderma viride bacterial liquid into the Trichoderma viride - solid fermentation medium at an inoculation amount of 5% (v / v), and ferment for 72 h. During the fermentation process, control the product temperature at 28 - 37 °C, keep the water content of the Trichoderma viride - solid fermentation medium at 45 - 50%, and obtain the fermented product; after the fermentation is completed, dry the fermented product at 42 °C and crush it to 90 mesh to obtain the Trichoderma viride powder, and its viable bacteria content is 4.0×10 9 cfu / g. Among them, the formula of the Trichoderma viride - solid fermentation medium by mass ratio is: 40% wheat bran, 10% corn straw powder, 0.1% MnSO4, 0.1% ZnSO4, and the initial water content is 45%.

[0081] (3) Preparation of Bacillus subtilis powder:

[0082] Activation of the strain: Inoculate Bacillus subtilis onto the test tube slant containing LB solid medium and culture it at 33 °C for 20 h to obtain the activated strain.

[0083] Preparation of seed liquid: Inoculate the activated strain into the LB liquid medium and incubate it at 180 r / min and 33 °C with shaking for 20 h to obtain the Bacillus subtilis seed liquid.

[0084] Cultivation of bacterial liquid: Inoculate the Bacillus subtilis seed liquid into a seed tank containing the LB liquid medium at an inoculation amount of 5% (v / v), and culture it at 33 °C for 20 h to obtain the Bacillus subtilis bacterial liquid. Among them, the volume of the seed tank is 10 L, the liquid loading is 7 - 8 L, the stirring speed is 200 - 250 r / min, and the ventilation volume is 7 - 8 L / min.

[0085] Fermentation culture (cultured in a 55 cm × 100 cm stainless steel shallow tray): Sterilize the Bacillus subtilis - solid fermentation medium at 121 °C for 20 min, and after cooling, spread it flat in the shallow tray with a spreading thickness of 5 cm; inoculate the Bacillus subtilis bacterial liquid into the Bacillus subtilis - solid fermentation medium at an inoculation amount of 5% (v / v), and ferment for 40 h. During the fermentation process, control the product temperature at 33 - 42 °C, and keep the water content of the Bacillus subtilis - solid fermentation medium at 45 - 55% to obtain the fermented product; after the fermentation is completed, dry the fermented product at 43 °C and crush it to 90 mesh to obtain the Bacillus subtilis bacterial powder, and its viable bacteria content is 2.5×10 10 cfu / g. Among them, the components of the Bacillus subtilis - solid fermentation medium by mass ratio are: wheat bran 50%, soybean meal powder 10%, corn straw powder 38.4%, urea 0.5%, Ca(OH)2 1%, MnSO4 0.1%, and the initial water content is 55%.

[0086] (4) Preparation of the multifunctional straw decomposer:

[0087] Mix 70 parts by weight of Bacillus velezensis PAPM - LX25, 20 parts by weight of Trichoderma viride, and 10 parts by weight of Bacillus subtilis evenly to obtain the multifunctional straw decomposer.

[0088] Application Example 1: Decomposition effect of the multifunctional straw decomposer on wheat straw

[0089] To investigate the decomposition effect of the multifunctional straw decomposer of the present invention on straw, a decomposition test was carried out in Ancheng Town, Pingyin County, Shandong Province from July to September 2021. The test method is as follows:

[0090] Randomly select wheat straws with similar thickness and intactness, remove the leaves, cut them into straw segments of 3 - 5 cm, place them in a blast drying oven at 85 °C for 12 h, and take them out; weigh the straws (accurate to 0.01 g), put them into nylon mesh bags, and the mass of each straw sample in the bag is 50 g. Weigh 45 bags of straw samples according to this requirement, and the mass range of the straws between each bag does not exceed 0.5 g.

[0091] The straw samples were respectively subjected to the following 3 treatments:

[0092] T1 treatment group: Weigh urea (as a nitrogen source) and the multifunctional straw decomposer prepared in Example 5 respectively. The amount of urea used is 0.5% of the straw weight, and the amount of the multifunctional straw decomposer used is 0.2% of the straw weight; mix urea and the multifunctional straw decomposer, and then mix them with water according to a weight ratio of 1:50 to obtain a suspension; use the suspension to soak the straw, and after the straw is fully soaked, evenly spread the remaining suspension on the surface of the test area.

[0093] T2 treatment group: The Bacillus subtilis powder in equal amount was used to replace the Bacillus velezensis PAPM-LX25 powder, and the straw decomposer was prepared according to the same method as in Example 5; the remaining treatment methods were the same as those in the T1 treatment group.

[0094] T3 treatment group: Different from the T1 treatment group, no straw decomposer was added.

[0095] An experimental plot with flat terrain, uniform soil fertility, and good irrigation and drainage conditions was selected. 15 bags of straw samples treated in the same way were divided into 5 groups, with 3 bags in each group tied together with a thin string; the 5 groups of samples were evenly buried in the experimental area in a five-point plum blossom shape, and the straw in the bags was evenly spread out, with the burial depth of 5 - 15 cm, so that the soil humidity was maintained at 60% - 80% of the field water holding capacity.

[0096] Samples were taken on the 10th day, 20th day, and 30th day of the experiment respectively. Each time samples were taken, 1 bag of straw sample was randomly taken from each group of the same treatment, with a total of 5 bags for each treatment; the obtained samples were washed clean with tap water, dried at 85 °C in a blast drying oven, weighed, and its mass was recorded as N x . Calculate the straw weight loss rate (ω x ) of each bag of sample according to the following formula:

[0097] ω x =(N0 - N x ) / N0×100%.

[0098] In the formula, N0 is the dry weight of the straw before decomposition (g); N x is the dry weight of the straw at a certain decomposition time (g).

[0099] The calculation results of the wheat straw weight loss rate under different treatment conditions are shown in Table 1 below:

[0100] Table 1. Wheat straw weight loss rate (average value) under different treatment conditions

[0101]

[0102] As can be seen from Table 1, compared with the application of the straw decomposer without Bacillus velezensis PAPM-LX25 (Treatment Group T2) and the non-application of the decomposer (Treatment Group T3), the application of the multifunctional straw decomposer of the present invention (Treatment Group T1) can increase the weight loss rate of wheat straw on the 10th day, 20th day and 30th day of treatment. Specifically, on the 10th day of treatment, the weight loss rate of wheat straw in Treatment Group T1 increased by about 6.00% compared with Treatment Group T2 and by about 45.02% compared with Treatment Group T3; on the 20th day of treatment, the weight loss rate of wheat straw in Treatment Group T1 increased by about 13.36% compared with Treatment Group T2 and by about 27.00% compared with Treatment Group T3; on the 30th day of treatment, the weight loss rate of wheat straw in Treatment Group T1 increased by about 7.48% compared with Treatment Group T2 and by about 21.16% compared with Treatment Group T3. The above results indicate that the multifunctional straw decomposer provided by the present invention has a good decomposition effect on wheat straw.

[0103] Application Example 2: Decomposition effect of the multifunctional straw decomposer on corn straw

[0104] To verify the application effect of the multifunctional straw decomposer of the present invention in straw composting, a composting experiment was carried out in Ancheng Town, Pingyin County, Shandong Province in 2021 with corn straw and livestock manure as raw materials. The experimental method is as follows:

[0105] The compost reactor is an EPP foam box with an inner length of 60 cm, an inner width of 45 cm, an inner height of 40 cm and a wall thickness of 3 cm. The experiment was set up with 1 treatment group T and 2 control groups CK1 and CK2, and 3 compost boxes were set in each group, where:

[0106] Treatment Group T: Mix corn straw and livestock manure in a weight ratio of 1:1, inoculate the multifunctional straw decomposer prepared in Example 5, with an inoculation amount of 0.1% (w / w), and adjust the initial water content to 55%.

[0107] Control Group CK1: Inoculate 0.1% (w / w) of a commercially available decomposer purchased from the local agricultural materials market, and its effective microorganisms are Bacillus subtilis and Aspergillus niger, and the effective viable count ≥ 2.0×10 9 cfu / g; the remaining treatment methods are the same as those in Treatment Group T.

[0108] Control Group CK2: Different from Treatment Group T, no straw decomposer is added.

[0109] During the composting process, turn it once every day from 14:00 to 16:00 to supply oxygen and mix the materials; measure the temperature of the compost pile every day from 9:00 to 10:00, and take the average value as the daily temperature of the compost pile. The influence of each treatment group on the composting temperature is as Figure 4 shown; by Figure 4It can be seen that, compared with the control groups CK1 and CK2, the temperature of the treatment group T applying the multifunctional straw composting agent of the present invention rises faster and the high-temperature period is longer, which is beneficial to accelerating straw composting and shortening the composting cycle.

[0110] After the composting is completed, samples are taken, dried in a drying oven at 40 °C, and crushed to 60 meshes for nutrient content analysis; the results are shown in Table 2 below:

[0111] Table 2. Nutrient content (average value) under different treatment conditions

[0112] Group CK1 CK2 T Organic matter (%) 67.06 67.58 66.72 N(％) 2.28 2.04 2.78 <![CDATA[P2O5(%)]]> 1.24 1.07 1.47 <![CDATA[K2O (%)]]> 1.29 1.25 1.38 Humic acid (%) 36.87 35.36 38.64 Seed germination index (%) 98 95 107

[0113] It can be seen from Table 2 that, compared with the control groups CK1 and CK2, the total nutrients (N + P2O5 + K2O), humic acid content and seed germination index of the straw compost after applying the multifunctional straw composting agent of the present invention (treatment group T) are higher. Specifically, the total nutrients (N + P2O5 + K2O) in the treatment group T are increased by about 17.05% compared with the control group CK1 and by about 29.13% compared with the control group CK2; the humic acid in the treatment group T is increased by about 4.8% compared with the control group CK1 and by about 9.28% compared with the control group CK2; the seed germination index in the treatment group T is increased by about 9.18% compared with the control group CK1 and by about 12.63% compared with the control group CK2. The above results indicate that the composting quality of the straw compost after applying the multifunctional straw composting agent of the present invention is better, with higher fertilizer efficiency and biological safety.

[0114] Application Example 3: Promoting effect of the multifunctional straw composting agent on the growth of peppers

[0115] A pot experiment was used to evaluate the effect of the multifunctional straw composting agent of the present invention combined with wheat straw returning to the field on the growth of peppers. The experimental method is as follows:

[0116] The specifications of the flower pots used in the experiment are 20 cm in upper inner diameter, 14 cm in lower inner diameter, and 16 cm in height, filled with 3.5 Kg of soil. The experiment was set with 1 treatment group T and 1 control group CK, with 20 pots in each group, and 2 pepper seedlings were transplanted into each pot, where:

[0117] Treatment group T: 20 g of wheat straw powder and 0.2 g of the multifunctional straw composting agent prepared in Example 5 were mixed evenly, then mixed with the soil, and then filled into the flower pots.

[0118] Control group CK: The multifunctional straw composting agent prepared in Example 5 was inactivated in a high-pressure sterilizer in advance; the remaining treatment methods were the same as those in the treatment group T.

[0119] When the chili peppers grow to the initial fruit stage, take the chili pepper plants out of the pots, rinse the roots with clear water, and then measure the plant height, stem diameter, fresh weight, dry weight, total chlorophyll content of the leaves, as well as the activities of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT) in the roots, and the content of malondialdehyde (MDA) in the roots. Among them, the plant height and stem diameter are measured using a meter stick and a vernier caliper respectively; the fresh weight and dry weight of the plants are measured by the weighing method; the total chlorophyll content of the leaves is measured by the ultraviolet spectrophotometry; the activity of SOD is measured by the nitroblue tetrazolium (NBT) reduction method; the activity of POD is measured by the guaiacol method; the activity of CAT is measured by the ultraviolet spectrophotometry; the content of MDA is measured by the thiobarbituric acid (TBA) colorimetric method.

[0120] The measurement results are shown in Table 3 as follows:

[0121] Table 3. Growth conditions of chili peppers under different treatment conditions

[0122]

[0123]

[0124] It can be seen from Table 3 that compared with the control group CK, after applying the multifunctional straw decomposer of the present invention (treatment group T), the plant height, stem diameter, fresh weight and dry weight of the chili peppers have all increased significantly. Specifically, the plant height in the treatment group T has increased by about 11.54% compared with the control group CK, the stem diameter has increased by about 14.69% compared with the control group CK, the fresh weight has increased by about 17.09% compared with the control group CK, the dry weight has increased by about 18.94% compared with the control group CK, and the total chlorophyll content of the leaves has increased by about 8.86% compared with the control group CK. The above results indicate that the multifunctional straw decomposer of the present invention can significantly promote the growth of chili pepper plants.

[0125] In addition, compared with the control group CK, the activities of antioxidant enzymes such as SOD, POD and CAT in the chili pepper roots of the treatment group T have increased significantly, and the content of MDA has decreased significantly. Specifically, the activity of SOD in the treatment group T has increased by about 109.12% compared with the control group CK, the activity of POD has increased by about 17.40% compared with the control group CK, the activity of CAT has increased by about 22.39% compared with the control group CK, and the content of MDA has decreased by about 20.66% compared with the control group CK. The above results indicate that the multifunctional straw decomposer of the present invention can significantly enhance the stress resistance of chili peppers.

Claims

1. A strain of Bacillus velezensis ( Bacillus velezensis ), named PAPM-LX25, was deposited at the China General Microbiological Culture Collection Center on November 06, 2024. The address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 32499.

2. Use of the Bacillus velezensis PAPM-LX25 as claimed in claim 1 in producing cellulase, producing xylanase, degrading avermectin, decomposing inorganic phosphorus, and degrading straw.

3. A multifunctional straw decomposer, characterized in that, Comprising the Bacillus velezensis PAPM-LX25 as claimed in claim 1.

4. The multifunctional straw composting agent according to claim 3, characterized in that, The multifunctional straw decomposing agent further comprises Trichoderma viride ( Trichoderma viride ), and Bacillus subtilis ( Bacillus subtilis ).

5. The multifunctional straw composting agent according to claim 4, characterized in that, The multifunctional straw decomposing agent comprises the following components: 60-80 parts by weight of Bacillus velezensis PAPM-LX25, 15-25 parts by weight of Trichoderma viride, and 5-15 parts by weight of Bacillus subtilis.

6. The preparation method of the multifunctional straw composting agent according to claim 3, characterized in that, Inoculate Bacillus velezensis PAPM-LX25 into a solid fermentation medium, ferment at 33-42 °C for 24-48 h to obtain a fermented product; dry the fermented product at 42-45 °C and crush it to 80-100 meshes to obtain Bacillus velezensis PAPM-LX25 bacterial powder; mix the Bacillus velezensis PAPM-LX25 bacterial powder with Trichoderma viride powder and Bacillus subtilis powder to obtain a multifunctional straw decomposing agent.

7. The preparation method according to claim 6, characterized in that, The solid fermentation medium comprises the following components according to a mass ratio: 40% wheat bran, 10% soybean meal powder, 49% corn straw powder, and 1% Ca(OH)2.

8. The preparation method according to claim 6, characterized in that, The initial water content of the solid fermentation medium is 50%-60%, and the water content of the solid fermentation medium is maintained at 45%-55% during the fermentation process.

9. The preparation method according to claim 6, characterized in that, The viable count in the Bacillus velezensis PAPM-LX25 bacterial powder is (2.0 - 3.0) × 10 10 cfu / g, the viable count in the Trichoderma viride bacterial powder is (3.0 - 5.0) × 10 9 cfu / g, and the viable count in the Bacillus subtilis bacterial powder is (2.0 - 3.0) × 10 10 cfu / g.

10. Use of the multifunctional straw decomposing agent as claimed in claim 3 in producing cellulase, producing xylanase, degrading avermectin, decomposing inorganic phosphorus, degrading straw, promoting the growth of pepper plants, and enhancing the stress resistance of pepper plants.

11. The application according to claim 10, wherein, The application method of the multifunctional straw decomposing agent in degrading straw is: mix the multifunctional straw decomposing agent and water in a weight ratio of 1-2:50-100, and evenly sprinkle it on the straw.

12. The application according to claim 10, wherein The application method of the multifunctional straw decomposing agent in promoting the growth of pepper plants or enhancing the stress resistance of pepper plants is: mix the multifunctional straw decomposing agent and straw evenly and apply them to the soil.

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