Bacillus velezensis and application thereof in preparation of multifunctional straw-decomposing inoculant

Abstract

The invention relates to bacillus velezensis and application thereof in preparation of a multifunctional straw decomposition agent, and belongs to the technical field of microorganisms. The bacillus velezensis has the functions of producing cellulase, producing xylanase, degrading abamectin, decomposing inorganic phosphorus and degrading straws; when straw is returned to the field, the multifunctional straw decomposition agent provided by the invention not only can accelerate the decomposition speed of straw, decompose abamectin residues in soil and improve the soil ecology, but also can promote the growth of pepper plants and enhance the stress resistance of the pepper plants, and has a good comprehensive application effect.

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 in the prior art, the present invention provides a strain of Bacillus Velezii and an application thereof in the preparation of a multifunctional straw composting agent. When returning straw to the field, the multifunctional straw composting agent provided by the present invention is used, which can not only accelerate the composting speed of the straw, decompose the avermectin pesticide residues in the soil, and 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 on November 6, 2024 at the General Microbiology Center of China Culture Collection Administration, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC No.32499.

[0009] The 16S rDNA sequence of the Bacillus Velezii PAPM-LX25 is shown in SEQ ID NO.1.

[0010] The culture method of the Bacillus Velez PAPM-LX25 comprises the following steps:

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

[0012] (2) inoculating the single bacterial colony obtained in step (1) into LB liquid culture medium, shaking and culturing at 180-220 r / min and 35-38° C. to obtain seed solution;

[0013] (3) The seed solution of step (2) is inoculated into LB liquid culture medium at an inoculum amount of 3-5% by volume, and cultured with shaking at 180-220 r / min and 35-38° C. to obtain a bacterial solution of Bacillus Velez PAPM-LX25.

[0014] The Velez Bacillus PAPM-LX25 is used in producing cellulase, producing xylanase, degrading avermectin, degrading inorganic phosphorus and degrading straw.

[0015] A multifunctional straw composting agent comprises the Velez Bacillus PAPM-LX25.

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

[0017] Further preferably, the multifunctional straw composting agent comprises the following components: 60-80 parts by weight of Bacillus Velez PAPM-LX25, 15-25 parts by weight of Trichoderma viride, and 5-15 parts by weight of Bacillus subtilis.

[0018] The preparation method of the multifunctional straw composting agent comprises the following steps: inoculating Bacillus Velez PAPM-LX25 into a solid fermentation medium, fermenting at 33-42° C. for 24-48 hours to obtain a fermented product; drying the fermented product at 42-45° C. and crushing the product to 80-100 meshes to obtain Bacillus Velez PAPM-LX25 bacterial powder; and mixing the Bacillus Velez PAPM-LX25 bacterial powder with Trichoderma viride powder and Bacillus subtilis bacterial powder to obtain a multifunctional straw composting agent.

[0019] Preferably, the solid-state fermentation medium comprises the following components in terms of mass ratio: 40% bran, 10% soybean meal powder, 49% corn stalk powder, and 1% Ca(OH)2.

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

[0021] Further preferably, in the multifunctional straw composting agent, the content of live bacteria in the powder of Bacillus Velez PAPM-LX25 is (2.0-3.0)×10 10 cfu / g, the viable bacteria content in green Trichoderma powder is (3.0~5.0)×10 9 cfu / g, the content of live bacteria in Bacillus subtilis powder is (2.0~3.0)×10 10 cfu / g.

[0022] The multifunctional straw composting agent is used in producing cellulase, producing xylanase, degrading avermectin, degrading 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 composting agent in degrading straw is: mixing the multifunctional straw composting agent with water in a weight ratio of 1 to 2: 50 to 100, and evenly sprinkling the mixture on the straw.

[0024] Preferably, the application method of the multifunctional straw composting agent in promoting the growth of pepper plants or enhancing the stress resistance of pepper plants is: after mixing the multifunctional straw composting agent and straw, applying the mixture to the soil.

[0025] Beneficial effects of the present invention:

[0026] The present invention provides a strain of Bacillus Velezii and a multifunctional straw decomposing agent prepared therefrom. When returning straw to the field, the multifunctional straw decomposing agent provided by the present invention is adopted, which can not only accelerate the decomposition speed of the straw, decompose the avermectin residue in the soil, and improve the soil ecology, but also promote the growth of pepper plants and enhance the stress resistance of the pepper plants, and has a good comprehensive application effect. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

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

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

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

[0032] Source of experimental materials:

[0033] Trichoderma viride: purchased from China Agricultural Microbiological Culture Collection Center, with the collection number ACCC 30794.

[0034] Bacillus subtilis: purchased from China Agricultural Microbiological Culture Collection Center, with the collection number of ACCC 04396.

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

[0036] The samples were collected from the compost of avermectin fermentation waste residue in Ancheng Town, Pingyin County, Jinan City, Shandong Province, China. The specific separation and screening methods were as follows: 10.0 g of the compost sample was weighed, 100 mL of sterile water was added, and the sample was placed on a constant temperature shaker at 35°C and 180 r / min for 30 min of oscillation to obtain an extract.

[0037] (1) Screening of avermectin-degrading strains: The above-mentioned extract was inoculated into LB liquid culture medium containing 200 mg / L avermectin, and cultured at 35° C. and 180 r / min for 3 days; the culture solution was then transferred to LB liquid culture medium containing 400 mg / L, 600 mg / L, and 800 mg / L avermectin in turn, and cultured for 3 days respectively to obtain enriched culture; the enriched culture was appropriately diluted, and then spread on LB solid culture medium with a concentration of 1 / 10 containing 800 mg / L avermectin, and cultured at 35° C. until a single colony grew; the single colony was separated to obtain strains with different colony morphologies, and then inoculated into culture medium with avermectin as the sole carbon source, and cultured at 35° C. and 180 r / min for 24 to 72 h, and the strain that could make the culture medium turbid was selected, i.e., the strain that could degrade avermectin was obtained, and stored for later use.

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

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

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

[0041] (2) Screening of Straw Degrading Bacteria: The strains that can degrade avermectin selected above were inoculated on sodium carboxymethyl cellulose medium and cultured at 35°C for 2 days to screen out strains that can produce hydrolysis circles, and the diameter of the transparent circle (D) and the diameter of the colony (d) were recorded. The strains with a larger D / d value were preferred; the selected strains were inoculated into filter paper disintegration medium and cultured at 35°C and 180 r / min for 3 days to screen out strains with stronger filter paper disintegration ability; the selected strains were further screened using the straw weight loss method, specifically: wheat straw was cut into small pieces of 3 to 5 cm, dried at 85°C, and 50.00 g was weighed and put 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 to dissolve, and then sprinkled on the wheat straw; the straw was buried in the field at a depth of 15 to 20 cm, taken out after 15 days, washed with clean water, dried and weighed, and strains with higher straw weight loss rate were preferred for subsequent screening.

[0042] The components of the filter paper disintegration medium are: (NH4)2SO4 1g, KH2PO4 1g, MgSO4.7H2O 0.7g, NaCl 0.5g, distilled water 1000mL, and 1 piece of 6cm×1cm filter paper strip.

[0043] (3) Screening of phosphate-solubilizing strains: The strains screened above that have both strong avermectin-degrading and straw-degrading abilities were inoculated onto the Montgena inorganic phosphorus bacteria culture medium and cultured at 35°C for 5 days to screen out strains that can produce larger transparent zones.

[0044] The components of the Montgina inorganic phosphorus bacterial culture medium are: 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, and 1000 mL of distilled water; pH 7.0-7.5.

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

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

[0047] The colony morphology of the strain PAPM-LX25 obtained by the above screening on LB solid medium is as follows: Figure 1 As shown in the figure, the colonies are round, milky white, opaque, and have a dry, wrinkled surface. The bacterial morphology of strain PAPM-LX25 is as follows Figure 2As shown, the bacteria are rod-shaped and have spores.

[0048] In addition, the physiological and biochemical characteristics of strain PAPM-LX25 were identified, and the identification results were 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 amplified using 16S rDNA universal primers. The amplification conditions were: 95℃ pre-denaturation for 3min; 94℃ denaturation for 1min, 55℃ annealing for 1min, 72℃ extension for 1.5min, a total of 30 cycles; 72℃ extension for 10min. 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. The phylogenetic tree was constructed using MEGA7.0 software. The constructed phylogenetic tree is shown in the figure. Figure 3 As shown; the results showed that the strain PAPM-LX25 obtained by the above screening was relatively close to Bacillus velezensis strain C13 in evolutionary distance. Combined with the physiological and biochemical characteristics of the strain, it was identified as "Bacillus velezensis".

[0050] Bacillus velezensis PAPM-LX25 was deposited on November 6, 2024 at the General Microbiology Center of China Culture Collection Administration, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC No.32499.

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

[0052] (1) Inoculate Bacillus velezensis PAPM-LX25 into LB solid medium and activate and culture at 35°C for 20 h to obtain a single colony;

[0053] (2) inoculating the single colony obtained in step (1) into LB liquid culture medium, shaking and culturing at 180 rpm and 35° C. for 20 h to obtain seed solution;

[0054] (3) The seed solution of step (2) was inoculated into LB liquid culture medium at an inoculum volume percentage of 5%, and cultured with shaking at 180 r / min and 35° C. for 20 h to obtain a bacterial solution of Bacillus Velez PAPM-LX25.

[0055] Example 4: Determination of beneficial properties of Bacillus Velez PAPM-LX25

[0056] (1) Enzyme production ability of Bacillus velez PAPM-LX25

[0057] A total of 15 g of a mixture of bran and corn stalk powder (4:6, w / w) was taken, loaded into a 250 mL conical flask, 15 mL of distilled water was added, mixed, sterilized at 121° C. for 30 min, and cooled naturally; the Bacillus Velez PAPM-LX25 bacterial solution prepared in Example 3 was inoculated into the above-mentioned conical flask at an inoculum amount of 5% (w / w), fermented and cultured at 35° C. for 48 h, and the flask was shaken once every 6 to 8 h; after the fermentation was completed, the fermentation product was dried at 50° C. and ground; 1.0 g of the fermentation product was weighed, 100 mL of distilled water was added, and the product was shaken and extracted at 180 rpm for 10 min, and filtered to obtain a crude enzyme solution.

[0058] The DNS method was used to determine the enzyme activity of cellulase and xylanase in the crude enzyme solution. The specific steps are as follows: 1.5 mL of 1.00% CMC solution or 1.5 mL of 1.00% xylan solution, and the crude enzyme solution diluted appropriately were placed in a 50°C water bath to preheat for 2 minutes; 0.5 mL of crude enzyme solution was added to the CMC solution or xylan solution, and the reaction was carried out for 10 minutes with precise timing, and 3 mL of DNS reagent was quickly added to terminate the reaction, and then color was developed in a boiling water bath for 15 minutes; after taking out, the volume was made up to 25 mL with distilled water, shaken, cooled, and the absorbance was measured at a wavelength of 540 nm. Blank control: DNS reagent was first added to the CMC solution or xylan solution, placed in a boiling water bath, and then the crude enzyme solution diluted appropriately was added. Other conditions were consistent with the normal determination method.

[0059] Definition of cellulase activity unit: Under the conditions of 50℃ and pH 5.0, the reaction time is 10min, and the amount of enzyme required to hydrolyze sodium carboxymethyl cellulose to produce 1g reducing sugar per minute is one enzyme activity unit, expressed as U.

[0060] Definition of xylanase activity unit: Under the conditions of 50℃ and pH 5.0, the reaction time is 10min. The amount of enzyme required to hydrolyze xylan to produce 1 mol of reducing sugar per minute is one enzyme activity unit, expressed as U.

[0061] The experimental results showed that the crude enzyme solution prepared from the fermentation product of Bacillus Velez PAPM-LX25 had an enzyme activity of 956U / g for cellulase and 268U / g for xylanase. This indicates that Bacillus Velez 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 on avermectin

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

[0064] The residual amount of avermectin in the culture medium was determined by liquid chromatography and the degradation rate was calculated.

[0065] The experimental results showed that the residual amount of avermectin in the culture medium inoculated with Bacillus Velez PAPM-LX25 was 0.0093 mg / mL; the residual amount of avermectin in the blank control was 0.1513 mg / mL. This shows that Bacillus Velez PAPM-LX25 has a strong ability to degrade avermectin.

[0066] (3) Inorganic phosphorus degrading ability of Bacillus velez PAPM-LX25

[0067] Bacillus Velez PAPM-LX25 was inoculated into Montkina inorganic phosphorus bacterial medium and cultured at 35°C for 3 days. The diameter of the transparent zone (D) and the diameter of the colony (d) were measured, and the HC value (D / d) was calculated.

[0068] The experimental results showed that the transparent zone diameter (D) of Bacillus Velez PAPM-LX25 was 13.8 mm, the colony diameter (d) was 5.2 mm, and the calculated HC value was 2.65, which showed that Bacillus Velez PAPM-LX25 had the ability to degrade inorganic phosphorus.

[0069] Example 5: Preparation of a multifunctional straw composting agent

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

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

[0072] Activation of bacterial strains: Inoculate Bacillus Velez PAPM-LX25 into the slant of a test tube containing LB solid medium and culture at 35°C for 20 hours to obtain activated bacterial strains.

[0073] Preparation of seed solution: The activated strain was inoculated into LB liquid culture medium, and cultured at 180 r / min and 35° C. for 20 h to obtain the seed solution of Bacillus Velez PAPM-LX25.

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

[0075] Fermentation culture (using a 55 cm × 100 cm stainless steel shallow dish for culture): the solid-state fermentation culture medium was sterilized at 121° C. for 20 min, and after cooling, it was spread in a shallow dish with a thickness of 5 cm; the Velez subtilis PAPM-LX25 bacterial solution was inoculated into the solid-state fermentation medium at an inoculum amount of 5% (v / v), and the fermentation culture was carried out for 40 h. During the fermentation process, the product temperature was controlled at 33-42° C., and the water content of the solid-state fermentation medium was maintained at 45-55%, thereby obtaining a fermentation product; after the fermentation was completed, the fermentation product was dried at 45° C. and crushed to 90 meshes to obtain Velez subtilis PAPM-LX25 bacterial powder, and the live bacteria content thereof was 2.5×10 10 cfu / g. The solid fermentation medium has a mass ratio of: 40% bran, 10% soybean meal powder, 49% corn stalk powder, 1% Ca(OH)2, and an initial water content of 55%.

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

[0077] Activation of strains: Inoculate the green Trichoderma into the slant of a test tube containing a PDA solid culture medium, and culture at 25°C for 48 hours to obtain an activated strain. The components of the PDA solid culture medium are: 200g potato, 20g glucose, 20g agar powder, 1000mL distilled water; natural pH value.

[0078] Preparation of seed liquid: scrape the activated bacteria with an inoculation loop and inoculate into PDB liquid culture medium, shake and culture at 180r / min and 25°C for 48h to obtain green Trichoderma seed liquid. The components of the PDB liquid culture medium are: 200g potato, 20g glucose, 1000mL distilled water; natural pH value.

[0079] Bacterial liquid culture: Inoculate the green Trichoderma seed liquid into a seed tank containing PDB liquid culture medium at an inoculum rate of 5% (v / v), and culture at 25°C for 48 hours to obtain green Trichoderma liquid. The volume of the seed tank is 10L, the liquid volume is 7-8L, the stirring speed is 200-220r / min, and the ventilation volume is 7-8L / min.

[0080] Fermentation culture (using a 55 cm × 100 cm stainless steel shallow dish for culture): sterilize the green Trichoderma-solid fermentation culture medium at 121 ° C for 20 min, and spread it in a shallow dish after cooling, with a paving thickness of 5 cm; inoculate the green Trichoderma liquid into the green Trichoderma-solid fermentation medium at an inoculum amount of 5% (v / v), and ferment and culture for 72 hours. During the fermentation process, the product temperature is controlled at 28-37 ° C, and the water content of the green Trichoderma-solid fermentation medium is maintained at 45-50%, to obtain a fermentation product; after the fermentation is completed, the fermentation product is dried at 42 ° C and crushed to 90 meshes to obtain green Trichoderma powder, and the viable bacteria content is 4.0×10 9 cfu / g. The formula of the green Trichoderma-solid fermentation medium is as follows according to the mass ratio: bran 40%, corn stalk powder 10%, MnSO4 0.1%, ZnSO4 0.1%, and the initial water content is 45%.

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

[0082] Activation of bacterial strains: Inoculate Bacillus subtilis into the slant of a test tube containing LB solid culture medium and culture at 33°C for 20 hours to obtain activated bacterial strains.

[0083] Preparation of seed solution: The activated strain was inoculated into LB liquid culture medium, and cultured at 180 r / min and 33° C. for 20 h to obtain Bacillus subtilis seed solution.

[0084] Bacterial liquid culture: Inoculate the Bacillus subtilis seed liquid into a seed tank containing LB liquid culture medium at an inoculum rate of 5% (v / v), and culture at 33°C for 20 hours to obtain the Bacillus subtilis bacterial liquid. The volume of the seed tank is 10L, the liquid volume is 7-8L, the stirring speed is 200-250r / min, and the ventilation volume is 7-8L / min.

[0085] Fermentation culture (using a 55 cm × 100 cm stainless steel shallow dish for culture): sterilize the Bacillus subtilis-solid fermentation culture medium at 121° C. for 20 min, and spread it in a shallow dish after cooling, with a paving thickness of 5 cm; inoculate the Bacillus subtilis bacterial liquid into the Bacillus subtilis-solid fermentation medium at an inoculum amount of 5% (v / v), and ferment and culture for 40 h. During the fermentation process, the product temperature is controlled at 33-42° C., and the water content of the Bacillus subtilis-solid fermentation medium is maintained at 45-55%, thereby obtaining a fermented product; after the fermentation is completed, the fermented product is dried at 43° C. and crushed to 90 meshes to obtain a Bacillus subtilis powder, wherein the viable bacteria content is 2.5×10 10 cfu / g. The components of the Bacillus subtilis solid fermentation medium according to mass ratio are: bran 50%, soybean meal powder 10%, corn stalk powder 38.4%, urea 0.5%, Ca(OH)2 1%, MnSO4 0.1%, and the initial water content is 55%.

[0086] (4) Preparation of multifunctional straw composting agent:

[0087] 70 parts by weight of Bacillus Velez PAPM-LX25, 20 parts by weight of Trichoderma viride and 10 parts by weight of Bacillus subtilis are uniformly mixed to obtain a multifunctional straw composting agent.

[0088] Application example 1: The effect of multifunctional straw composting agent on the composting of wheat straw

[0089] In order to investigate the composting effect of the multifunctional straw composting agent of the present invention on straw, a composting 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 intact wheat straws of similar thickness, remove the leaves, cut into 3-5 cm straw segments, place in an 85℃ forced air drying oven to dry for 12 hours, and take out; weigh the straw (accurate to 0.01g), put it into a nylon mesh bag, the mass of the straw sample in each bag is 50g, and weigh 45 bags of straw samples according to this requirement. The extreme difference in straw mass between bags does not exceed 0.5g.

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

[0092] T1 treatment group: urea (as a nitrogen source) and the multifunctional straw composting agent prepared in Example 5 were weighed separately, wherein the amount of urea was 0.5% of the weight of the straw, and the amount of the multifunctional straw composting agent was 0.2% of the weight of the straw; the urea and the multifunctional straw composting agent were mixed, and then mixed with water in a weight ratio of 1:50 to obtain a suspension; the straw was soaked with the suspension, and after the straw was fully soaked, the remaining suspension was evenly spread on the surface of the test area.

[0093] T2 treatment group: an equal amount of Bacillus subtilis powder was used to replace Bacillus Velez PAPM-LX25 powder, and the straw composting agent was prepared in the same manner as in Example 5; the rest of the treatment methods were the same as those of the T1 treatment group.

[0094] T3 treatment group: Unlike T1 treatment group, no straw composting agent was added.

[0095] A test site with flat terrain, uniform soil fertility and good irrigation and drainage conditions was selected. Fifteen bags of straw samples using the same treatment were divided into five groups, with three bags in each group tied together with a thin rope. The five groups of samples were evenly buried in the test area in a five-point plum blossom shape, and the straw in the bags was evenly spread out. The burial depth was 5 to 15 cm, so that the soil moisture was maintained at 60% to 80% of the field water holding capacity.

[0096] Sampling was performed on the 10th, 20th, and 30th days of the experiment. Each time a sample was taken, one bag of straw sample was randomly taken from each group of the same treatment, with a total of 5 bags per treatment. The obtained samples were rinsed with tap water, dried in a forced air drying oven at 85°C, and weighed. The mass was recorded as N. x The straw weight loss rate (ω) of each bag of sample is calculated as follows: x ):

[0097] ω x =(N0-N x ) / N0×100%.

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

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

[0100] Table 1. Weight loss rate of wheat straw under different treatment conditions (average value)

[0101]

[0102] As shown in Table 1, compared with the application of the straw decomposing agent without Bacillus Velez PAPM-LX25 (T2 treatment group) and the application of no decomposing agent (T3 treatment group), the application of the multifunctional straw decomposing agent of the present invention (T1 treatment group) can increase the weight loss rate of wheat straw on the 10th, 20th and 30th day of treatment. Specifically, on the 10th day of treatment, the weight loss rate of wheat straw in the T1 treatment group increased by about 6.00% compared with the T2 treatment group, and increased by about 45.02% compared with the T3 treatment group; on the 20th day of treatment, the weight loss rate of wheat straw in the T1 treatment group increased by about 13.36% compared with the T2 treatment group, and increased by about 27.00% compared with the T3 treatment group; on the 30th day of treatment, the weight loss rate of wheat straw in the T1 treatment group increased by about 7.48% compared with the T2 treatment group, and increased by about 21.16% compared with the T3 treatment group. The above results show that the multifunctional straw composting agent provided by the present invention has a good composting effect on wheat straw.

[0103] Application Example 2: The effect of multifunctional straw composting agent on the composting of corn straw

[0104] In order to verify the application effect of the multifunctional straw composting agent of the present invention in straw composting, a composting test was carried out in Ancheng Town, Pingyin County, Shandong Province in 2021 using corn straw and livestock and poultry manure as raw materials. The test method is as follows:

[0105] The composting 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 has a total of 1 treatment group T and 2 control groups CK1 and CK2, each of which has 3 composting boxes, including:

[0106] Treatment group T: corn straw and livestock and poultry manure were mixed at a weight ratio of 1:1, and the multifunctional straw composting agent prepared in Example 5 was added at an inoculation amount of 0.1% (w / w), and the initial moisture content was adjusted to 55%.

[0107] Control group CK1: inoculated with 0.1% (w / w) commercially available composting agent, which was purchased from the local agricultural supply market. Its effective microorganisms were Bacillus subtilis and Aspergillus niger, and the effective viable count was ≥2.0×10 9 cfu / g; the rest of the treatments were the same as those in treatment group T.

[0108] Control group CK2: Unlike treatment group T, no straw composting agent was added.

[0109] During the composting process, the compost was turned once between 14:00 and 16:00 every day to supply oxygen and mix the materials; the temperature of the compost was measured between 9:00 and 10:00 every day, and the average value was taken as the temperature of the compost for the day. Figure 4 shown by Figure 4It can be seen that compared with the control group CK1 and the control group CK2, the application of the multifunctional straw composting agent of the present invention (treatment group T) causes a faster temperature rise and a longer high temperature period, which is beneficial to accelerate the composting of the straw and shorten the composting cycle.

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

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

[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] As shown in Table 2, compared with the control group CK1 and the control group CK2, the total nutrients (N+P2O5+K2O) and humic acid content and seed germination index of the straw compost (treatment group T) after the application of the multifunctional straw decomposing agent of the present invention 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 show that the compost has better decomposition quality after the application of the multifunctional straw decomposing agent of the present invention, and has higher fertilizer efficiency and biosafety.

[0114] Application example 3: The effect of 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 returning wheat straw to the field on the growth of peppers. The test method is as follows:

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

[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, and then mixed evenly with soil, and then placed in a flower pot.

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

[0119] When the peppers grew to the initial fruit stage, the pepper plants were taken out of the pots, and the roots were washed with clean water. The plant height, stem diameter, fresh weight, dry weight, total chlorophyll content of the leaves, and the root superoxide dismutase (SOD) enzyme activity, peroxidase (POD) enzyme activity, catalase (CAT) enzyme activity, and root malondialdehyde (MDA) content of the pepper plants were measured. Among them, the plant height and stem diameter were measured using a ruler and a vernier caliper, respectively; the plant fresh weight and dry weight were measured by weighing; the total chlorophyll content of the leaves was measured by ultraviolet spectrophotometry; the SOD enzyme activity was measured by the nitroblue tetrazolium (NBT) reduction method; the POD enzyme activity was measured by the guaiacol method; the CAT enzyme activity was measured by ultraviolet spectrophotometry; and the MDA content was measured by the thiobarbituric acid (TBA) colorimetric method.

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

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

[0122]

[0123]

[0124] As shown in Table 3, compared with the control group CK, after applying the multifunctional straw decomposing agent of the present invention (treatment group T), the plant height, stem thickness, fresh weight and dry weight of pepper are significantly increased. Specifically, the plant height in the treatment group T is increased by about 11.54% compared with the control group CK, the stem thickness is increased by about 14.69% compared with the control group CK, the fresh weight is increased by about 17.09% compared with the control group CK, the dry weight is increased by about 18.94% compared with the control group CK, and the total chlorophyll content of the leaves is increased by about 8.86% compared with the control group CK. The above results show that the multifunctional straw decomposing agent of the present invention can significantly promote the growth of pepper plants.

[0125] In addition, compared with the control group CK, the enzyme activities of antioxidant enzymes such as SOD, POD and CAT in the pepper root system of the treatment group T were significantly increased, and the MDA content was significantly reduced. Specifically, the SOD enzyme activity in the treatment group T was increased by about 109.12% compared with the control group CK, the POD enzyme activity was increased by about 17.40% compared with the control group CK, the CAT enzyme activity was increased by about 22.39% compared with the control group CK, and the MDA content was reduced by about 20.66% compared with the control group CK. The above results show that the multifunctional straw composting agent of the present invention can significantly enhance the stress resistance of peppers.

Claims

1. A strain of Bacillus velezensis PAPM-LX25, deposited on November 6, 2024 at the General Microbiology Center of China Culture Collection Administration, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC No. 32499.

2. Use of the Bacillus Velez PAPM-LX25 according to claim 1 in producing cellulase, producing xylanase, degrading avermectin, degrading inorganic phosphorus, and degrading straw.

3. A multifunctional straw composting agent, characterized in that: The invention comprises the Bacillus Velez PAPM-LX25 as described in claim 1.

4. The multifunctional straw composting agent according to claim 3, characterized in that: The multifunctional straw decomposing agent also includes Trichoderma viride and Bacillus subtilis; Preferably, the multifunctional straw composting agent comprises the following components: 60 to 80 parts by weight of Bacillus Velez PAPM-LX25, 15 to 25 parts by weight of Trichoderma viride, and 5 to 15 parts by weight of Bacillus subtilis.

5. The method for preparing the multifunctional straw composting agent according to claim 3, characterized in that: The Bacillus Velez PAPM-LX25 is inoculated into a solid fermentation medium, and fermented at 33-42° C. for 24-48 hours to obtain a fermentation product; the fermentation product is dried at 42-45° C. and crushed to 80-100 meshes to obtain Bacillus Velez PAPM-LX25 powder; the Bacillus Velez PAPM-LX25 powder is mixed with Trichoderma viride powder and Bacillus subtilis powder to obtain a multifunctional straw composting agent.

6. The preparation method according to claim 5, characterized in that: The solid-state fermentation medium comprises the following components in terms of mass ratio: 40% bran, 10% soybean meal powder, 49% corn stalk powder, and 1% Ca(OH)2; Preferably, the initial water content of the solid-state fermentation medium is 50% to 60%; during the fermentation process, the water content of the solid-state fermentation medium is maintained at 45% to 55%.

7. The preparation method according to claim 5, characterized in that: The live bacteria content of the Velez subtilis PAPM-LX25 powder is (2.0-3.0)×10 10 cfu / g, the viable bacteria content in green Trichoderma powder is (3.0~5.0)×10 9 cfu / g, the content of live bacteria in Bacillus subtilis powder is (2.0~3.0)×10 10 cfu / g.

8. Use of the multifunctional straw composting agent according to claim 3 in producing cellulase, producing xylanase, degrading avermectin, degrading inorganic phosphorus, degrading straw, promoting the growth of pepper plants, and enhancing the stress resistance of pepper plants.

9. The use according to claim 8, characterized in that The application method of the multifunctional straw decomposing agent in degrading straw is as follows: the multifunctional straw decomposing agent is mixed with water in a weight ratio of 1 to 2: 50 to 100, and then the mixture is evenly sprinkled on the straw.

10. The use according to claim 8, characterized in that 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: after mixing the multifunctional straw decomposing agent and straw, applying the mixture to the soil.

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