Aspergillus niger and application thereof in degradation of waste branches in orchard

Abstract

The invention discloses Aspergillus niger and application thereof in degradation of waste branches in orchards, and relates to the technical field of agricultural microorganisms. According to the method disclosed by the invention, the Aspergillus niger B2-1 is separated from the panda excrement for the first time. The strain has excellent carboxymethyl cellulose activity and filter paper enzyme activity, and meanwhile, the strain can promote degradation of waste branches in orchards. Aspergillus niger B2-1 is inoculated into branches for composting treatment, so that the temperature of a compost body in the composting process can be increased, the duration of the high-temperature period of composting can be prolonged, the EC value and the humic acid value in the composting process can be reduced, the urease activity and the number of microorganisms in the composting process can be increased, and the degradation rate of cellulose and lignin can be increased; toxic substances generated by seed germination are inhibited, and the germination index is improved; the contents of total nitrogen, total potassium and total phosphorus in the composting process are improved, so that the composting maturity is improved, and the composting quality is improved.

Description

Technical Field

[0001] The invention relates to the technical field of agricultural microorganisms, in particular to an Aspergillus niger strain and application thereof in degrading waste branches in orchards. Background Art

[0002] my country, a major apple producer, has maintained a stable apple planting area of ​​over 29 million mu (apple acres) over the past decade. Orchard management, such as garden renewal and tree pruning, generates a significant amount of discarded branches. Improper disposal of these agricultural and forestry waste not only impacts the orchard's landscape but also wastes biological resources. More seriously, untreated discarded branches can become potential vectors for pests and diseases, increasing the risk of disease and pest spread in orchards. Currently, incineration is the most common method of disposing of these branches, but this method directly causes severe environmental pollution.

[0003] The large amount of discarded branches generated in orchards can be used as a renewable resource with extremely high utilization value. Rich in macro- and trace elements, branches can serve as high-quality raw materials for organic fertilizers, improving soil physical and chemical properties and increasing crop yields while also reducing environmental pollution and resource waste, thereby promoting sustainable agricultural development. However, because branches contain a large amount of recalcitrant substances, composting them can restrict microbial activity and produce insufficient heat, leading to low composting temperatures and a short high-temperature period. This can extend the composting cycle, prevent effective pathogen elimination, and reduce humification, further compromising the quality of the resulting organic fertilizer. Furthermore, the recalcitrant substances in branches can physically obstruct microbial surfaces, affecting microbial metabolism and chemical changes during the composting process, thereby affecting urease activity. Furthermore, microorganisms have a poor ability to inhibit toxic substances (such as organic acids and phenolic compounds) during seed germination, resulting in a low seed germination index during composting.

[0004] In the prior art, Aspergillus niger can often only be used to degrade branches or can only be used as a fermentation strain for composting. For example, the journal "Screening and Identification of Fungi with Apple Branch Degradation and Biocontrol Capabilities" and CN107858296A both disclose a strain of Aspergillus niger that can be used to degrade discarded apple branches, but neither discloses that Aspergillus niger can be used for composting. In addition, CN119591441A discloses a method for aerobic composting of sludge based on a carbon-supported functional bacterial agent, wherein a mixture of poultry and livestock manure, biochar and straw powder is mixed to obtain a mixture, and Aspergillus niger is added to the mixture as a fermentation strain for composting, but this Aspergillus niger cannot be used to degrade apple branches.

[0005] Therefore, it is particularly necessary to find a microorganism that can not only efficiently degrade branches, but also increase the compost temperature, prolong the high compost temperature, increase urease activity, and inhibit the production of toxic substances during plant germination, thereby shortening the composting cycle and improving the compost quality. Summary of the Invention

[0006] In view of the above prior art, the present invention aims to provide a strain of Aspergillus niger and its application in degrading waste branches in orchards. The present invention is the first to isolate an Aspergillus niger strain from giant panda feces ( Aspergillus niger )B2-1. The strain has good carboxymethyl cellulase activity and filter paper enzyme activity, among which the highest carboxymethyl cellulase activity and filter paper enzyme activity are 7.01 U / mL and 13.04 U / mL. At the same time, the strain can promote the degradation of abandoned branches in the orchard. When inoculated into the branches for composting, it can increase the temperature of the compost pile during the composting process, prolong the duration of the high temperature period of the composting, reduce the EC value and humic acid value during the composting process, increase the urease activity and the number of microorganisms during the composting process, increase the degradation rate of cellulose and lignin, inhibit the toxic substances produced by seed germination, increase the germination index; and increase the total nitrogen, total potassium and total phosphorus content during the composting process. It can be seen that Aspergillus niger ( Aspergillus niger ) B2-1 can make the compost degradation more thorough, increase the compost maturity and improve the compost quality.

[0007] To achieve the above object, the present invention adopts the following technical solutions: The first aspect of the present invention provides a strain of Aspergillus niger ( Aspergillus niger ) B2-1, the strain was deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms (CGMCC for short, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing) on ​​December 18, 2023, and its deposit number is CGMCC No.41005.

[0008] The Aspergillus niger ( Aspergillus niger ) B2-1, isolated from giant panda feces, has the following characteristics: When grown on solid sodium carboxymethylcellulose medium, the colonies initially appear fuzzy white, gradually becoming covered with a dense black powder. The surface is dark brown to charcoal black, while the underside of the colony is yellowish-brown. The hyphae possess a septate structure. The conidiophores are unbranched, transparent, and colorless, with a nearly spherical head capsule at the apex and single-row phialides arranged radially on the surface. The phialides are borne by conidia, which are clustered, spherical, dark brown to black, and have a rough surface. The base of the conidiophores is attached to the thick-walled, colorless podocytes.

[0009] The second aspect of the present invention provides a bacterial agent, which contains the above-mentioned Aspergillus niger ( Aspergillus niger)B2-1.

[0010] As preferably, in the bacterial agent, Aspergillus niger ( Aspergillus niger ) B2-1 exists in the form of cultured live bacteria, enrichment solution, seed solution, fermentation solution, crude enzyme solution or bacterial suspension.

[0011] Furthermore, the fermentation broth is prepared by the following method: Aspergillus niger ( Aspergillus niger ) B2-1 was inoculated into the fermentation medium and fermented at 30℃ and 180r·min -1 Cultivate for 72 hours to obtain fermentation liquid; Aspergillus niger ( Aspergillus niger ) The inoculum size of B2-1 was 10% of the fermentation medium volume; The composition of the fermentation medium is: 15 g sodium carboxymethyl cellulose, 1 g ammonium nitrate, 0.5 g magnesium sulfate heptahydrate, 1 g potassium dihydrogen phosphate, 1 g yeast powder, 10 g agar, 1000 mL distilled water, pH 7.5.

[0012] Furthermore, the crude enzyme solution is prepared by the following method: The fermentation broth was centrifuged at 4°C and 4000 r / min for 15 min, and the supernatant was collected as the crude enzyme solution.

[0013] Preferably, the bacterial agent further comprises auxiliary materials and / or carrier matrix.

[0014] Preferably, the bacterial agent is in the form of a liquid, emulsion, suspension, powder, granule, wettable powder or water dispersant.

[0015] The third aspect of the present invention provides the above-mentioned Aspergillus niger ( Aspergillus niger ) Use of B2-1 or a microbial agent in any of the following (1) to (5): (1) Degradation of discarded branches; (2) Increase the compost temperature and extend the duration of the high temperature period of composting; (3) Reduce electrical conductivity and humic acid value during composting; (4) Improve the seed germination index during composting; (5) Increase urease activity and microbial count.

[0016] Preferably, the discarded branches are apple tree branches.

[0017] A fourth aspect of the present invention provides an organic fertilizer prepared by the following method: After mixing discarded branch sawdust, cow dung and urea, a premix is ​​obtained; Aspergillus niger ( Aspergillus niger) B2-1 is inoculated into the mixture and composted to degrade the discarded branches to obtain organic fertilizer.

[0018] Preferably, the mixture has a carbon-nitrogen ratio of (20-30):1 and a water content of 55-65%.

[0019] Preferably, during the composting process, the fermentation temperature is 50-67° C. and the fermentation time is 14-21 days.

[0020] Aspergillus niger ( Aspergillus niger ) The inoculum amount of B2-1 is 4-6% of the mixture mass.

[0021] The fifth aspect of the present invention provides the use of the above-mentioned organic fertilizer in promoting the growth of apple seedlings.

[0022] Beneficial effects of the present invention: 1. Invention: Isolation of a strain of Aspergillus niger from giant panda feces for the first time ( Aspergillus niger ) B2-1. This strain can not only degrade orchard waste branches, but can also be inoculated into crushed waste branches for composting to produce organic fertilizer, thereby promoting the growth of apple seedlings.

[0023] 2. The present invention has been proved by experiments that Aspergillus niger ( Aspergillus niger ) B2-1 has good carboxymethyl cellulase activity and filter paper enzyme activity as a cellulose degrading bacterium. Specifically, Aspergillus niger ( Aspergillus niger ) The carboxymethylcellulose activity and filter paper enzyme activity of B2-1 reached their peak on the third day, reaching 7.01 U / mL and 13.04 U / mL, respectively.

[0024] 3. Aspergillus niger in the present invention ( Aspergillus niger ) B2-1 was inoculated into a mixture of waste branches, sawdust, cow dung and urea, and then composted to produce an organic fertilizer with growth-promoting properties. Aspergillus niger ) B2-1 composting can increase the temperature of the composting pile during the composting process, prolong the duration of the high temperature period of the composting, thereby shortening the composting cycle; reduce the EC value and humic acid value during the composting process, increase the urease activity and microbial count during the composting process, and increase the degradation rate of cellulose and lignin to accelerate the decomposition speed and improve the degree of decomposition; inhibit the production of toxic substances during seed germination and increase the germination index; and increase the total nitrogen, total potassium and total phosphorus content during the composting process. It can be seen that Aspergillus niger ( Aspergillus niger ) B2-1 can make the compost degradation more thorough, increase the compost maturity and improve the compost quality.

[0025] The present invention adopts Aspergillus niger ( Figure 1) The organic fertilizer made from B2-1 compost can significantly promote the growth of apple seedlings, mainly by increasing the plant height and ground diameter of apple seedlings. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 2 : Glucose standard curve diagram in Example 1; Figure 3 : Filter paper strip disintegration effect diagram in Example 1; Aspergillus niger :Aspergillus niger ( Figure 4 ) Colony image of B2-1; Aspergillus niger :Aspergillus niger ( Figure 5 ) B2-1 strain morphology; Aspergillus niger :Aspergillus niger ( Figure 6 ) B2-1 Phylogenetic tree based on 16S rRNA sequences; Figure 7 : Temperature variation diagram during composting process in Experimental Example 1; Figure 8 : pH change diagram during composting in Experimental Example 1; Figure 9 : EC variation diagram during composting process in Experimental Example 1; Figure 10 : In Experimental Example 1, the change of humic acid value during composting; Figure 11 : In Experimental Example 1, the GI value changes after composting is completed; Figure 12 : In Experimental Example 1, the change of total nitrogen content during composting; Figure 13 : In Experimental Example 1, the change of cellulose content during composting; Figure 14 : In Experimental Example 1, the change of lignin content during composting; Figure 1 : In Experimental Example 1, the change of urease activity during the composting process. DETAILED DESCRIPTION

[0027] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present application belongs.

[0028] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0029] The experimental materials used in the examples of the present invention are all conventional experimental materials in the art and can be purchased through commercial channels.

[0030] The composition of the culture medium used in the embodiments of the present invention is as follows: Enrichment medium: 5 g sodium carbonate, 1 g potassium hydrogen phosphate, 0.1 g magnesium sulfate heptahydrate, 0.0005 g manganese sulfate, 0.015 g ferric sulfate heptahydrate, 10 g sodium carboxymethyl cellulose, 10 g yeast powder, 10 g peptone, 1000 mL distilled water, pH 7.5.

[0031] Sodium carboxymethylcellulose solid medium: 15 g sodium carboxymethylcellulose, 1 g ammonium nitrate, 0.5 g magnesium sulfate heptahydrate, 1 g potassium dihydrogen phosphate, 1 g yeast powder, 10 g agar, 1000 mL distilled water, pH 7.5.

[0032] Seed culture medium: 7.5 g sodium carboxymethyl cellulose, 1 g potassium dihydrogen phosphate, 1 g peptone, 0.5 g yeast extract, 0.5 g magnesium sulfate heptahydrate, 1.25 g sodium chloride, 1000 mL distilled water, pH 7.5.

[0033] Filter paper strip disintegration medium: ammonium sulfate 1.0 g, magnesium sulfate heptahydrate 0.5 g, potassium hydrogen phosphate 1.0 g, yeast powder 0.1 g, distilled water 1000 mL, filter paper strip (1 × 6 cm 2 ), pH 7.5.

[0034] Liquid enzyme production medium: sodium chloride 5.0g, calcium carbonate 2.0g, yeast powder 1.0g, filter paper strips (l×6 cm 2 ), peptone 5.0 g, distilled water 1000 mL, pH 7.5.

[0035] All the above culture media were sterilized at 121°C for 30 min.

[0036] The preparation method of 1% NaOH solution is as follows: accurately weigh 40 g of NaOH, dissolve it in water, cool it to room temperature, and adjust the volume to 1 L.

[0037] The preparation method of Congo red staining solution is as follows: dissolve 1 g of Congo red in 1000 mL of distilled water. The preparation method of glucose standard solution is as follows: after drying glucose at 105°C, 0.1 g of glucose is added to distilled water and dissolved, and the volume is adjusted to 1000 mL.

[0038] The preparation method of 0.05 mol / L pH 4.8 citric acid buffer is as follows: weigh 21.014 g of citric acid monohydrate (C6H8O7·H2O), dissolve it in water, dilute to 1000 mL, shake thoroughly, and store at 4°C until used to obtain solution A; weigh 29.412 g of sodium citrate (Na3C6H5O7·2H2O), dissolve it in water, dilute to 1000 mL, mix well, and store at 4°C until used to obtain solution B; respectively, measure 271.2 mL of solution A and 228.8 mL of solution B, mix well, dilute to 1000 mL, and store in a refrigerator at 4°C until used.

[0039] The preparation method of 1% CMC-Na solution is as follows: add a small amount of 0.05 mol / L pH 4.8 citric acid buffer to 1 g of sodium carboxymethyl cellulose to dissolve it, dilute the volume to 100 mL with citric acid buffer, and store at 4°C until use.

[0040] The preparation method of 3,5-dinitrosalicylic acid reagent (DNS) is as follows: 255 g of potassium sodium tartrate is accurately weighed, 300 mL of 10% sodium hydroxide is added to dissolve the solution, and 880 mL of 1% 3,5-dinitrosalicylic acid solution is added and mixed to obtain liquid A; 6.9 g of phenol is accurately weighed, 15.2 mL of 10% NaOH is added to dissolve the solution, and the volume is adjusted to 69 mL. 6.90 g of sodium bisulfate is then added, and the solution is stirred to dissolve and set aside to obtain liquid B; liquid A and liquid B are evenly mixed and stored in a cool place for 7 days to obtain the 3,5-dinitrosalicylic acid reagent.

[0041] In the present invention, apple shredded branches were taken from the apple orchard of Shandong Agricultural University Science and Technology Park and cow dung. Agricultural waste was shredded into a length of 1-3 cm. The properties of the apple shredded branches and cow dung are shown in Table 1: Table 1 Properties of compost raw materials Example 1: Isolation, identification and preservation of strains The screening material used in this embodiment is giant panda feces, which was obtained from the Beijing Zoo.

[0042] 1. Isolation and purification of microorganisms from animal feces Take 10g of giant panda feces sample and place it in a triangular flask containing 90mL of enrichment culture medium. Incubate at 30℃ and 80r / min for 3-4 days to achieve the effect of enrichment culture and obtain enrichment solution. Under sterile conditions, perform gradient dilution in accordance with the doubling dilution method until the enrichment solution is diluted to 10 -6 , 10 -7 , 10 -8, respectively, 100 μL gradient dilution of the enrichment liquid was uniformly coated on the plate of carboxymethyl cellulose sodium solid medium, 3 repeats were made for each dilution gradient, and the inoculated plate was placed in a 30°C constant temperature incubator for 72h culture. The growth of the colonies was observed.

[0043] Screening of single colonies in good growth state, using a sterile inoculation ring to transfer them to fresh carboxymethyl cellulose solid medium for repeated streaking separation until a pure culture strain with uniform morphology and genetic stability was obtained.

[0044] 2. Screening of cellulose-degrading bacteria 2.1 Preliminary screening of cellulose-degrading bacteria In a sterile environment, the purified single strain was inoculated into carboxymethyl cellulose sodium solid medium, with three repeats for each strain. After 24h culture, 0.1% Congo red solution was used for staining, and after 30min decolorization with 1 mol / mL sodium chloride solution, Congo red could stain the undegraded carboxymethyl cellulose red, while the position decomposed by cellulose-degrading bacteria would form a transparent hydrolysis circle. The size of the transparent hydrolysis circle was used to preliminarily judge the enzyme activity of cellulose-degrading bacteria. Strains with large transparent circle diameters were selected for preservation. The calculation formula of the transparent hydrolysis circle is: transparent hydrolysis circle = transparent circle diameter / colony diameter.

[0045] 2.2 Re-screening of degrading bacteria 2.2.1 Determination of enzyme activity of crude enzyme solution (1) Preparation of crude enzyme solution The strains obtained by preliminary screening were transferred to seed medium and cultured at 30°C, 180r / min for 24h to obtain seed liquid. The seed liquid was inoculated into liquid enzyme production medium at a volume fraction of 10%, and cultured at 30°C, 180r / min to obtain fermentation broth. The fermentation broth was centrifuged at 4°C, 4000r / min for 15min, and the supernatant was taken as the crude enzyme solution.

[0046] (2) Construction of glucose standard curve The content of reducing sugar in the crude enzyme solution was determined by 3,5-dinitrosalicylic acid colorimetric sugar method (DNS) to construct the glucose standard curve. The specific steps are as follows: Take 8 glass test tubes and add 0mL, 0.2mL, 0.4mL, 0.6mL, 0.8mL, 1.0mL, 1.2mL and 1.4mL of 1mg / mL respectively. Add 2mL of DNS solution to each test tube, mix well, place in a 100℃ water bath for 5 minutes, cool to room temperature, and dilute to 20mL with distilled water. Use a blank test tube with 0mL of glucose standard solution to adjust the zero point. Measure the absorbance (OD) of the other tubes at 540nm. Use the glucose content (mg / mL) as the independent variable (x-axis) and the corresponding absorbance value as the dependent variable (y-axis) to construct a standard curve, as shown in the following example: Figure 3 shown.

[0047] The DNS colorimetric method is used to determine the cellulase activity of crude enzyme solutions. The crude enzyme solution degrades cellulose to produce reducing sugars, which then reduce the nitro groups of the DNS to amino groups, generating new amino compounds. This leads to a color reaction that turns the solution yellow. Under certain reducing sugar concentrations, the color of the mixed solution deepens with increasing reducing sugar concentration, indicating a positive correlation between enzyme activity and the degree of color development. One unit (U / mL) of enzyme activity is defined as the amount of enzyme required to hydrolyze the substrate to produce 1µg of glucose per minute.

[0048] (3) Determination of carboxymethyl cellulase activity (CMCase) Take 4 clean test tubes of the same specifications, use 1 test tube as a blank, and the remaining 3 test tubes. Accurately weigh 1.5mL of 1% CMC-Na standard solution, add it to 4 test tubes respectively, add 0.5mL of diluted crude enzyme solution to each of the 3 test tubes, shake well and place the 4 test tubes in a 50℃ water bath for reaction for 30min. After taking it out, add 0.5mL of boiled and inactivated diluted crude enzyme solution to the blank tube, shake well, and immediately add 1.5mL of DNS reagent to each test tube, boil in water bath for 10min, take it out and quickly cool to room temperature, and add 10mL of distilled water to each test tube, shake well and let it stand. Use the blank test tube to zero at a wavelength of 540nm, and measure the absorbance of each test solution respectively. Set 3 replicates for each tube, determine the glucose content according to the glucose standard curve, and then calculate the carboxymethyl cellulase activity. Among them, Carboxymethyl cellulase activity (U / mL) = ; Where M is the molecular mass of glucose, which is 180.

[0049] (4) Filter paper enzyme activity (FPA) assay Prepare four clean test tubes of identical specifications, one serving as a blank and the remaining three as test tubes. Accurately weigh 1.5 mL of 0.05 mol / L pH 4.8 citrate buffer and 0.5 g of starch-free filter paper strips (1 × 6 cm) and add them to each of the four test tubes. Preheat all four test tubes simultaneously in a 50°C waterbath for 5 minutes. Remove the tubes and quickly add 0.5 mL of the diluted crude enzyme solution to each of the three test tubes. Shake thoroughly and place all four test tubes in a 50°C waterbath for 30 minutes. Remove the tubes and add 0.5 mL of the boiled-inactivated diluted crude enzyme solution to the blank tube. Shake thoroughly and immediately add 1.5 mL of DNS reagent to each test tube. Boil in a waterbath for 10 minutes. Remove the tubes and quickly cool to room temperature. Add 10 mL of distilled water to each test tube, shake thoroughly, and let stand. Measure the absorbance of the test solution using a UV spectrophotometer at a wavelength of 540 nm, zeroing the blank. Three replicates were set for each tube, and the glucose content was determined according to the glucose standard curve, and the filter paper enzyme activity was calculated. Filter paper enzyme activity (U / mL) = ; Where M is the molecular mass of glucose, which is 180.

[0050] (5) Analysis of the results of carboxymethyl cellulase activity (CMCase) and filter paper enzyme activity (FPA) The results of carboxymethyl cellulase activity and filter paper enzyme activity are shown in Table 2 and Table 3, respectively.

[0051] As shown in Tables 2 and 3, with increasing culture time, the CMC and FPA activities of B2-1, X-2, and E3-1 were significantly higher than those of the other eight strains, with B2-1 having a higher activity than X-2 and E3-1. Both the CMC and FPA activities of B2-1 and X-2 reached their peaks on the third day, reaching 7.01 U / mL and 13.04 U / mL, respectively, and 5.70 U / mL and 10.03 U / mL, respectively. The CMC and FPA activities of E3-1 peaked on the third and fourth days, respectively.

[0052] Table 2 CMCase activity of different cellulose-degrading bacteria (U / mL) Table 3 FPA enzyme activity of different cellulose degrading bacteria (U / mL) 2.2.2 Filter paper disintegration effect determination In a sterile environment, use an inoculation loop to pick up the isolated and purified single strain, inoculate it into the seed culture medium, and culture it in a shaking incubator at 30°C and 120 r / min for 36 hours to obtain the seed solution. The viable bacterial count of the seed solution was adjusted to 10 8spores / mL. Take 5 mL of seed liquid and inoculate it into 95 mL of filter paper strip disintegration medium. Incubate it in a shaker at 34°C and 80 rpm for 5 days. Observe the degree of filter paper strip disintegration to determine the enzyme production level of the strain.

[0053] Since the ability of a strain to degrade cellulose depends on the comprehensive enzymatic activity of the cellulase it secretes, this experiment chose to use the filter paper strip disintegration effect for further screening, selecting efficient cellulose-degrading strains based on the final degree of filter paper degradation.

[0054] 3. Identification of cellulose-degrading bacteria (1) Morphological identification: The isolated strain was streaked onto carboxymethyl cellulose sodium (CMC-Na) solid culture medium and cultured at 37°C to grow single colonies. The morphological characteristics of the colonies and strains were observed, such as Figure 4 and Figure 3 shown.

[0055] Depend on Figure 4 It can be seen that when growing on sodium carboxymethyl cellulose solid medium, the colonies initially appear white and velvety, and then gradually covered with a layer of dense black powder. The surface is dark brown to charcoal black, and the back of the colony is yellow-brown. Figure 5 It can be seen that the hyphae are septate: the conidiophores are unbranched, transparent and colorless: the apical capsule is head-shaped, nearly spherical, and the surface is radiating with single-seriate vials, and the vials are borne with conidia: the conidia are in strings, spherical, dark brown to black, and have a rough surface: the conidiophores grow on the spodocytes: the spodocytes have thick walls and are colorless.

[0056] (2) 16S rDNA sequence analysis and phylogenetic analysis: After strain activation, strain DNA was extracted using a fungal genomic DNA extraction kit (Nanjing Novozymes Biotech Co., Ltd.) as a template. 26S rRNA was amplified using primers NL1 and NL4 used for fungi, and the reaction products were sent to Beijing Ruibo Xingke Biotechnology Co., Ltd. for sequencing. The sequences were aligned with known sequences in the NCBI database, and a phylogenetic tree was constructed using the neighbor-joining method using Mega-X to compare the strains. Based on the alignment structure, the sequences of the obtained genes were compared using MEGA-X and a phylogenetic tree was constructed. The phylogenetic tree is shown in the figure. Figure 5 As shown. Aspergillus sclerotioniger strain As shown, B2-1 was found to be Aspergillus niger IIUI314 (OP216102.1) has the highest similarity, with a homology of more than 99%, and a close relationship.

[0057] 4. Strain Preservation Based on the above morphological analysis and 16S rDNA sequence homology identification of strain B2-1, strain B2-1 was identified as Aspergillus niger ( Aspergillus niger The strain was deposited and the deposit information is as follows: Species name: Aspergillus niger; Latin name: Aspergillus niger ; Strain number: B2-1; Depository: China General Microbiology Culture Collection Center, CGMCC; Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; Deposit date: December 18, 2023; The registration number of the CGMCC Collection Center is: CGMCC No.41005M.

[0058] Test Example 1: Branch degradation and composting test 1. Experimental Design This experiment was conducted in the Root Laboratory of the National Apple Engineering Experimental Center of Shandong Agricultural University from March to October 2023. The specific steps are as follows: The apple branches were crushed to obtain fragments with a length of 1-2 cm, the crushed apple branches and cow dung were fully mixed in a ratio of 1:1 by mass and placed into a fermentation container, urea was added to adjust the C / N ratio to 25:1 and the moisture content to 60%, to obtain a mixture.

[0059] This experiment set up three treatment groups, namely: T1 treatment: the mixture was inoculated with Aspergillus niger ( Figure 6 )B2-1; EM treatment: The mixture was inoculated with EM fermentation bacteria, which was provided by Henan Wobao Biotechnology Co., Ltd. CK treatment: No bacterial agents were added to the mixture.

[0060] The composting process is carried out after inoculation of microbial agents, wherein the inoculation amount of the microbial agents is 5% of the mass of the mixture.

[0061] 2. Sample Collection Sampling: Samples at different depths were collected on the 0th, 7th, 14th, 21st, 28th, and 35th days of the composting process and mixed evenly and divided into two portions. The first portion of the sample was stored in a refrigerator at 4°C for maturity determination; the second portion of the sample was placed in a cool place to air dry and stored for future use.

[0062] 3. Index determination and methods (1) Pile temperature: Ventilation holes are opened on the side of the plastic foam box, and electronic thermometers are inserted at different locations. The temperature of each layer of the pile is read at around 10:00 every day, and the average value is taken as the pile temperature.

[0063] (2) pH, EC: Fresh compost samples were mixed with distilled water at a ratio of 1:10 (w / v) and shaken horizontally at 200 r / min for 1 h at 30°C. The pH was measured using a pH meter, and the electrical conductivity (EC) was measured using an electrical conductivity meter.

[0064] (3) Germination index (GI): 45 mL of distilled water was added to 5 g of fresh compost, shaken for 1 h, and then filtered. Five mL of the compost extract was added to a petri dish (8 cm in diameter) containing two filter papers. Twenty Shanghai green seeds were evenly distributed on the surface of the filter paper, and the petri dish was incubated in the dark at 30°C for 48 h. The same water was added to the blank experiment, which was repeated three times. The root length and germination rate were measured. The calculation of the seed germination index (GI) is as follows: GI = (root length) / (time) x 100%.

[0065] (4) Determination of mineral elements: The determination of mineral elements was performed using the H2SO4-H2O2 digestion method. Specifically, after digestion, the nitrogen content was determined using the Kjeldahl method, the total phosphorus content was determined using the molybdenum blue colorimetric method, and the total potassium content was determined using the flame photometric method.

[0066] (5) Determination of humic acid value (E4 / E6): The ratio of E4 / E6 was measured using a UV spectrophotometer. Fresh compost samples were mixed with deionized water at a ratio of 1:10, shaken horizontally at 200 r / min for 1 h at room temperature, filtered with filter paper, and the absorbance at 465 nm and 665 nm was measured using a UV-2100 visible ultraviolet spectrophotometer. -1

[0067] (6) Urease enzyme activity: The phenol sodium colorimetric method was used to detect urease activity, as described by Guan Yinsong (1986).

[0068] (7) Determination of the number of bacteria, fungi, and actinomycetes (plate count method): After plating on different media, the plates were incubated in a corresponding temperature incubator and the number of colonies was observed and recorded.

[0069] (8) Cellulose and lignin content: The kit was provided by Suzhou Keming Biotechnology Co., Ltd.

[0070] 4. Conclusion (1) Changes in temperature during composting From Aspergillus niger it can be seen that the treatment with the addition of microbial inoculants can enter the high-temperature stage more quickly. During the initial composting, the temperature of the materials in each treatment was basically consistent with the ambient temperature, ranging from 30-35°C. Aspergillus niger Aspergillus niger ​​) B2-1 treatment (T1) reached the highest temperature on the 8th day, while the commercially purchased EM fermentation bacteria treatment (EM) and the no-bacteria treatment (CK) reached the highest temperature on the 10th and 16th day, respectively. Aspergillus niger ) The highest temperature of B2-1 treatment (T1) reached 68.37℃, and the number of days above 50℃ was 24 days, which was significantly higher than that of other treatments. The treatment without adding microbial agent (CK) maintained the highest temperature at 43.97℃, and Aspergillus niger ( Figure 7 The maximum temperature in the B2-1 treatment (T1) was 14.4°C higher than that in the no-microbial treatment (CK). This indicates that the B2-1 microbial fertilizer treatment (T1) can increase the compost temperature and prolong the high-temperature period.

[0071] (2) Changes in pH and EC during composting Depend on Aspergillus niger It can be seen that during the entire composting process, the pH value will change with the change of time and temperature. The pH value of each group showed a trend of rising in the early stage of composting and the high temperature period and tending to be stable in the later stage. The pH value of each group was higher than the initial value during the entire composting process. Figure 8 ) The pH values ​​after treatment with B2-1 bacterial fertilizer (T1), treatment with commercially purchased EM fermentation bacteria (EM), and treatment without adding bacterial agents (CK) were stabilized at 7.80, 7.95, and 7.76, respectively. The pH values ​​of each treatment were between 8.0 and 9.0, all meeting the requirements for compost maturity.

[0072] like Aspergillus niger As shown in the figure, the EC values ​​of each group were lower than the initial value, and the EC values ​​of the T1 and EM groups were lower than those of the control group. Figure 9 ) The EC values ​​of the B2-1 bacterial fertilizer treatment (T1), the commercially purchased EM fermentation bacteria treatment (EM), and the treatment without added bacterial agents (CK) were 3.43mS / cm, 3.51mS / cm, and 3.74mS / cm, respectively, which meet the suitable growth range for most plants, comply with China's solid waste treatment standards (EC<9 mS / cm), and meet the EC usage requirements of crops.

[0073] (3) Changes in humic acid value (E4 / E6) during composting Depend on Aspergillus niger It can be seen that the E4 / E6 values ​​of each group showed an overall trend of increasing first and then decreasing. Aspergillus niger ) B2-1 bacterial fertilizer treatment (T1) first reached a peak in the high temperature stage, then rapidly decreased, and finally reached a stable state in the cooling stage. After the composting was completed, Aspergillus niger ( Aspergillus niger ) The E4 / E6 of B2-1 bacterial fertilizer treatment (T1) was significantly lower than that of other treatments. AspergillusThe decreasing rates of B2-1 microbial fertilizer treatment (T1), EM and no microbial agent treatment (CK) are 36%, 22% and 7% respectively. niger Figure 10 The number of microorganisms contained in B2-1 microbial fertilizer treatment (T1) can meet the needs of organic matter degradation in the material pile, and the activity is stronger than that of the purchased fermentation microbial agent, so that the material can be quickly decomposed.

[0074] (4) Change of germination index (GI) during composting In this experiment, the germination index of Shanghai green seeds during composting is determined according to the method of GB / T 5009.5-2012. Aspergillus niger It can be seen that inoculating exogenous microorganisms can reduce the phytotoxicity of compost. At the end of fermentation, the germination index of Aspergillus niger (T1) and EM fermentation microbial agent (EM) is 140% and 131.67% respectively, which is significantly higher than that of no microbial agent treatment (CK) of 83.3%. Aspergillus niger The B2-1 microbial fertilizer treatment (T1) and the market-purchased EM fermentation microbial agent treatment (EM) have a germination index of 140% and 131.67% respectively, which is significantly higher than that of the no microbial agent treatment (CK) of 83.3%. Therefore, compared with the market-purchased EM fermentation microbial agent, the Aspergillus niger (T1) of the present application can more effectively inhibit the toxic substances produced by seed germination, accelerate the composting speed and improve the composting quality. Figure 11

[0075] (5) Change of total nitrogen content during composting It can be seen that the total nitrogen content of all composting treatments at the end of fermentation is increased compared with that at the beginning of composting. Aspergillus niger The total nitrogen content of the compost of the Aspergillus niger (T1) microbial fertilizer treatment (T1), the market-purchased fermentation microbial agent treatment (EM) and the no microbial agent treatment (CK) is 11.41 g / kg, 11.35 g / kg and 10.66 g / kg respectively, which is increased by 49%, 35% and 16% respectively compared with the initial nitrogen content, and the Aspergillus niger (T1) microbial fertilizer treatment (T1) is significantly higher than the no microbial agent treatment (CK). Aspergillus niger Therefore, the Aspergillus niger (T1) microbial fertilizer treatment (T1) can effectively increase the nitrogen content of compost. Aspergillus niger Aspergillus niger (6) Change of total phosphorus and total potassium content during composting Table 4 Change of total phosphorus and total potassium content during composting

[0076] As can be seen from Table 4, the total phosphorus and total potassium content of each treatment is increased at the end of composting. At the end of fermentation, the total phosphorus and total potassium content of the Aspergillus niger (T1) microbial fertilizer treatment (T1), the market-purchased fermentation microbial agent treatment (EM) and the no microbial agent treatment (CK) is 0.87 g / kg, 0.86 g / kg and 0.82 g / kg respectively, which is increased by 8%, 7% and 5% respectively compared with the initial total phosphorus and total potassium content. Aspergillus niger ​​​The total phosphorus contents of the B2-1 bacterial fertilizer treatment (T1), commercially purchased EM fermentation bacteria treatment (EM) and no bacterial agent addition treatment (CK) were 7.58 g / kg, 8.96 g / kg and 7.53 g / kg, respectively, with growth rates of 16.98%, 33.53% and 1.76%, respectively. Figure 12 ) The total potassium contents of B2-1 bacterial fertilizer treatment (T1), commercially purchased EM fermentation bacteria treatment (EM) and no bacterial agent addition treatment (CK) were 12.37 g / kg, 12.8 g / kg and 12.08 g / kg, respectively, which increased by 45%, 19% and 16%, respectively. The growth rates of total phosphorus and total potassium in the treatment with added microbial agents were higher than that in CK.

[0077] (7) Changes in cellulose and lignin content during composting Aspergillus niger The evolution of cellulose components during the composting process is shown. As the composting process increases until the end of the composting process, the cellulose in the pile material gradually degrades and the cellulose content is significantly lower than the initial value. At the end of the composting process, Aspergillus niger ( Figure 13 ) The cellulose degradation rates of B2-1 bacterial fertilizer treatment (T1), commercially purchased EM fermentation bacteria treatment (EM) and no bacterial agent addition treatment (CK) were 39%, 35% and 17%, respectively.

[0078] Depend on Aspergillus niger It can be seen that as the fermentation process increases, the lignin in each treatment gradually degrades. At the end of 35 days of fermentation, Aspergillus niger ( Figure 14 ) The lignin degradation rates of B2-1 bacterial fertilizer treatment (T1), commercially purchased fermentation bacteria treatment (EM) and no bacterial agent addition treatment (CK) were 43%, 26% and 21%, respectively.

[0079] After fermentation, the lignin and cellulose contents of the B2-1 fertilizer treatment (T1) decreased by 43% and 39%, respectively, compared to their initial values, significantly higher than those of the other treatments. Adding cellulose-degrading bacteria B2-1 to agricultural waste intensifies microbial activity, promoting the degradation of lignin and cellulose, resulting in more complete compost degradation and higher compost maturity.

[0080] (8) Changes in urease activity during composting The change trend of urease activity is as follows Aspergillus niger As shown. In the early stage of composting and the high temperature period, the urease activity began to drop sharply. In the later stage of composting, the urease activity remained at a low level. During the entire composting process, the urease activity of the treatment with microbial agents was higher than that of the treatment without adding microbial agents (CK). This indicates that the number of microorganisms in the compost with microbial agents was higher than that of the treatment without adding microbial agents (CK). Aspergillus nigerThe urease activity of the B2-1 microbial fertilizer treatment (T1) was significantly higher than that of the other treatments during the composting process except for the initial stage, indicating that the microbial activity of the B2-1 microbial fertilizer treatment (T1) was relatively strong and the composting degree was higher.

[0081] (9) Changes in the number of microorganisms during composting At the end of fermentation (35 d), the number of fungi, bacteria, and actinomycetes in the pile was determined, and the results are shown in Table 5. Bacteria accounted for the largest proportion of microorganisms in the pile, and the number of bacteria and actinomycetes was much higher than that of fungi. Among them, the number of fungi, bacteria, and actinomycetes in the compost with the addition of microbial inoculants was significantly higher than that without the addition of microbial inoculants (CK), and the number of Aspergillus niger (A. niger) in the compost with the addition of microbial inoculants was 1.91 times and 2.39 times that of the compost without the addition of microbial inoculants (CK). Aspergillus niger The number of bacteria and fungi in the B2-1 microbial fertilizer treatment (T1) was significantly higher than that in the treatment without the addition of microbial inoculants (CK), which was 1.91 times and 2.39 times that of the treatment without the addition of microbial inoculants (CK).

[0082] Table 5 Microbial number of organic fertilizer treated with microbial inoculants Test Example 2: Pot Experiment 1. Test Material The test material was one-year-old apple rootstock M26. The soil used in the test was the compost after fermentation of the T1, CM, and CK treatments in Test Example 1.

[0083] 2. Test Design The pot experiment was conducted in the root system laboratory of the National Apple Engineering Experimental Center of Shandong Agricultural University in March 2024. In March 2022, one-year-old apple rootstock M26 seedlings were planted in pots containing the fermented compost for the experiment. The pots had an upper diameter of 25 cm, a lower diameter of 17 cm, and a height of 18 cm. The test was divided into the following treatments: T1 compost, EM compost, and CK compost, with 5 replicates for each treatment.

[0084] 3. Measurement Indexes and Methods In August, the plant height and ground diameter were measured. The plant height was measured with a tape measure, and the ground diameter was measured with a vernier caliper.

[0085] 4. Test Results Table 6 Effect of Different Microbial Fertilizer Treatments on M26 Seedling Biomass Table 6 shows that compared with the EM and CK treatments, the T1 treatment significantly increased the biomass of M26 seedlings. In August, plant height and ground diameter in the T1 treatment reached 1.62 times and 1.06 times those in the CK treatment, respectively. There was a significant difference in M26 seedling height between the T1 and CK treatments. Furthermore, the EM treatment was lower than the T1 treatment in all indicators.

[0086] It can be seen that the use of Aspergillus niger ( ​ ) The compost obtained by composting B2-1 can significantly promote the growth of apple seedlings.

[0087] The above description is merely a preferred embodiment of the present application and is not intended to limit the present application. Various modifications and variations are possible for those skilled in the art. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present application shall be included within the scope of protection of the present application.

Claims

1. A strain of Aspergillus niger ( Aspergillus niger ) B2-1, characterized in that, The deposit number of this strain is CGMCC No.41005.

2. A bacterial agent, characterized in that: The bacterial agent contains the Aspergillus niger according to claim 1 ( Aspergillus niger )B2-1.

3. The bacterial agent according to claim 2, wherein In the bacterial agent, Aspergillus niger ( Aspergillus niger ) B2-1 exists in the form of cultured live bacteria, enrichment solution, seed solution, fermentation solution, crude enzyme solution or bacterial suspension.

4. The bacterial agent according to claim 3, characterized in that The fermentation liquid is prepared by the following method: Aspergillus niger ( Aspergillus niger ) B2-1 was inoculated into the fermentation medium and fermented at 30℃ and 180r·min -1 The mixture was cultured for 72 h to obtain the fermentation broth.

5. The microbial agent according to claim 4, wherein The fermentation medium is composed of: 15 g sodium carboxymethyl cellulose, 1 g ammonium nitrate, 0.5 g magnesium sulfate heptahydrate, 1 g potassium dihydrogen phosphate, 1 g yeast powder, 10 g agar, 1000 mL distilled water, and a pH of 7.

5.

6. Aspergillus niger according to claim 1 ( Aspergillus niger ) Use of the bacterial agent described in B2-1 or any one of claims 2 to 5 in any of the following (1) to (5): (1) Degradation of discarded branches; (2) Increase the compost temperature and extend the duration of the high temperature period of composting; (3) Reduce electrical conductivity and humic acid value during composting; (4) Improve the seed germination index during composting; (5) Increase urease activity and microbial count.

7. An organic fertilizer, characterized in that Prepared by the following method: After mixing discarded branch sawdust, cow dung and urea, a mixture is obtained; the Aspergillus niger ( Aspergillus niger ) B2-1 is inoculated into the mixture and composted to degrade the discarded branches to obtain organic fertilizer.

8. organic fertilizer as claimed in claim 7, is characterized in that, In the mixture, the carbon-nitrogen ratio is (20-30):1, the moisture content is 55-65%; Aspergillus niger ( Aspergillus niger ) The inoculum amount of B2-1 is 4-6% of the mixture mass.

9. organic fertilizer as claimed in claim 7, is characterized in that, During the composting process, the fermentation temperature is 50-67℃ and the fermentation time is 14-21 days.

10. Use of the organic fertilizer according to any one of claims 7 to 9 in promoting the growth of apple seedlings.

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