A strain of Aspergillus DLF18 and its application

Abstract

This invention discloses a strain of Aspergillus sp., DLF18, and its applications, belonging to the field of microbial technology, with accession number CGMCC NO.41737. This strain can effectively utilize rice straw and / or sugarcane bagasse to produce humic acid, with a humic acid content of 16.5% per gram of fermentation supernatant after rice straw fermentation. Applying the fermentation product of DLF18 to crops can significantly improve crop growth and increase the humic content in the soil. Simultaneously, metagenomic analysis results show that, compared with the control group, the application of DLF18 to the soil significantly altered species β-diversity, functional gene abundance, and inter-group differences. These findings indicate that DLF18 has broad application prospects in agricultural straw utilization and soil remediation.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and more specifically to an Aspergillus sp. DLF18 strain and its applications. Background Technology

[0002] Humic acid is a type of natural organic matter, mainly found in soil, sediment, and water bodies. It is a complex mixture formed after organic matter has undergone long-term decomposition and transformation. Humic acid is one of the main components of soil humus, possessing strong adsorption, water retention, and fertility-regulating properties. It is the final product of organic matter decomposition and usually exists in acidic form in soil and water bodies. Based on its solubility in solvents and color, humic acid is classified into three categories: fulvic acid, humic acid glycosides, and humic acid glycosides. As a core component of soil humus, humic acid plays a crucial role in maintaining soil fertility, improving soil structure, and promoting plant growth. Furthermore, humic acid is also significant for environmental remediation, helping to reduce the bioavailability of heavy metal pollution and promoting the degradation of pollutants.

[0003] The specific role of microorganisms in humic acid formation is currently debated, with four main hypotheses: plant transformation hypothesis, biochemical hypothesis, cell autolysis, and microbial synthesis. It is difficult to determine which hypothesis is closer to reality; perhaps multiple processes need to work synergistically to form humic acid. The microbial formation hypothesis emphasizes the role of microorganisms in humic acid formation. Microorganisms play a multifaceted role, not only as decomposers of large polymer molecules but also as producers that recombine smaller molecules. Therefore, lignocellulose decomposition products serve as the framework and substrate for humic acid formation. Adding protein-rich organic waste at the appropriate time can increase the activity of lignocellulase, providing a necessary option for the conversion of lignocellulose waste into humic acid by controlling key factors. Lignocellulose, as the largest renewable biological resource in the biological world, has a global annual production of 150 billion tons, of which straw production reaches 6 billion tons, with China's annual straw production at 1.113 billion tons.

[0004] However, the available microbial resources that can efficiently utilize agricultural straw to produce humic acid through fermentation are currently limited, which restricts the development and application of agricultural straw. Therefore, providing more microbial resources that can efficiently degrade agricultural straw to produce humic acid has become an urgent problem to be solved by those skilled in the art. Summary of the Invention

[0005] In view of this, the present invention provides an Aspergillus strain DLF18 and its applications.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A strain of Aspergillus sp. DLF18, with accession number CGMCCNO.41737.

[0008] Another object of the present invention is to provide a microbial inoculant, including the above-mentioned Aspergillus.

[0009] Another object of the present invention is to provide the application of the above-mentioned Aspergillus or the above-mentioned microbial agent in the production of humic acid.

[0010] Preferably, the humic acid includes soluble humic acid and fulvic acid, as well as insoluble humin.

[0011] Preferably, the Aspergillus or the microbial agent is made from rice straw and / or sugarcane bagasse.

[0012] Another object of the present invention is to provide the application of the above-mentioned Aspergillus or the above-mentioned microbial agents in the analysis of intergroup differences in soil microbial abundance, species composition, functional level, or functional abundance.

[0013] Preferably, the soil microbial abundance includes the abundance of microbial species and quantity, the abundance of carbohydrate active enzymes, and the abundance of genes for carbon metabolism pathways, nitrogen metabolism pathways, phosphorus metabolism pathways, and sulfur metabolism pathways.

[0014] Preferably, the application involves fermenting rice straw and / or sugarcane bagasse using the Aspergillus or the microbial agent, and applying the resulting fermentation product as fertilizer to the soil.

[0015] Another object of the present invention is to provide the application of the above-mentioned Aspergillus or the above-mentioned microbial inoculants in promoting plant growth.

[0016] Preferably, the application involves fermenting rice straw and / or sugarcane bagasse using the Aspergillus or the microbial agent, and applying the resulting fermentation product as fertilizer to the soil.

[0017] Beneficial Effects: This invention isolated a strain of Aspergillus DLF18 from the soil of Canglang Peak in Cangshan Mountain, Dali. This strain can effectively utilize rice straw and / or sugarcane bagasse to produce humic acid, with a humic acid content of 16.5% in the fermentation broth after rice straw fermentation. Applying the F18 fermentation product to crops significantly increased wheat plant height, leaf length, and leaf width, and increased soil humus content by 5.2 g / kg; for Chinese cabbage, the average increases in plant height, root length, leaf width, leaf number, above-ground fresh weight, and above-ground dry weight were 6.4 cm, 3.34 cm, 3.98 cm, 3 leaves, 3.49 g, and 0.225 g, respectively. Metagenomic analysis showed that compared with the control group, the application of F18 to the soil significantly altered species β-diversity, functional gene abundance, and inter-group differences. These findings indicate that F18 has broad application prospects in agricultural straw utilization and soil remediation. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0019] Figure 1 The image shows the morphological identification of strain DLF18, where a is the sporulation structure, b is the conidia, and c is the pure culture result.

[0020] Figure 2 This is the phylogenetic tree of strain DLF18.

[0021] Figure 3 The growth status of strain DLF18 at different temperatures is shown. "+" indicates the growth status of the strain, with more "+" indicating better growth, " / " indicates no growth of the strain, "1" indicates ck (uninoculated control), and "2" indicates the experimental group inoculated with F18.

[0022] Figure 4 The yield of humic acid produced by strain DLF18 under different carbon sources.

[0023] Figure 5 To accurately determine the humic acid content of strain DLF18 in the fermentation supernatant.

[0024] Figure 6 The effect of strain DLF18 on wheat growth phenotype.

[0025] Figure 7 This study aimed to quantitatively determine the effects of strain DLF18 on wheat growth traits.

[0026] Figure 8This study compares the content of humic components in soil under different treatments.

[0027] Figure 9 The microbial culture status in soil under different treatments.

[0028] Figure 10 The image shows a 1% gel electrophoresis result of soil metagenomic DNA, where M is a 15000bp DNA marker, 1-3 represent CKT1, CKT2, and CKT3 respectively, and 4-6 represent F181, F182, and F183 respectively.

[0029] Figure 11 Species abundance at the phylum level under different treatments.

[0030] Figure 12 Species abundance at different treatment levels.

[0031] Figure 13 Species abundance at the seed level under different treatments.

[0032] Figure 14 PCoA analysis at the phylum, genus, and species levels under different treatments.

[0033] Figure 15 Heatmap for analyzing differences in species abundance under different treatments.

[0034] Figure 16 Heatmaps for functional abundance analysis under different treatments.

[0035] Figure 17Heatmaps showing the abundance of functional genes in the carbon cycle under different treatments; where 1 represents: 4-aminobutyrate aminotransferase and related aminotransferases; 2 represents: acetaldehyde => ethanol; 3 represents: acetate => acetaldehyde; 4 represents: acyl-CoA dehydrogenase; 5 represents: alpha-amylase; 6 represents: aminotransferase class I and II; 7 represents: arabinosidase; 8 represents: aspB; 9 represents: bcrA; 10 represents: bcrB; 11 represents: bcrC; 12 represents: bcrD; 13 represents: beta-galactosidase; 14 represents: beta-glucosidase; 15 represents: beta-glucuronidase; 16 represents: beta-mannosidase; 17 represents: beta-xylosidase; 18 represents: branched-chain amino acid aminotransferase / 4-amino-4-deoxychorismate lyase; 19 represents: bsdC; 20 represents: catA; 21 represents: cellobiosidase; 22 represents: cellulase; 23 represents: chitiniase; 24 represents: fae; 25 represents: fdhA; 26 represents: fdhB; 27 represents: fdoG; 28 represents: fdoH; 29 represents: fghA; 30 represents: frmA; 31 represents: glucoamylase; 32 represents: hexosaminidase; 33 represents: histidinol-phosphate / aromatic aminotransferase; 34 represents: isoamylase; 35 represents: mannan endo-1,4-beta-mannosidase; 36 represents: mauA; 37 represents: mauB; 38 represents: mxaF; 39 represents: ornithine / acetylornithine aminotransferase; 40 represents: phosphoserine aminotransferase; 41 represents: pullulanase; 42 represents: serine-pyruvate aminotransferase / archaeal aspartate aminotransferase; 43 represents: ubiX; 44 represents: Form II;45 represents: aclA; 46 represents: aclB; 47 represents: cdhE; 48 represents: cooS; 49 represents: acdA; 50 represents: ack; 51 represents: acs; 52 represents: adh; 53 represents: ldh; 54 represents: pflD; 55 represents: porA; 56 represents: pta; 57 represents: pmoA; 58 represents: pmoB; 59 represents: pmoC.

[0036] Figure 18 Heatmaps showing the abundance of nitrogen cycle functional genes under different treatments.

[0037] Figure 19 Heatmaps showing the abundance of functional genes in the phosphorus cycle under different treatments.

[0038] Figure 20 Heatmaps showing the abundance of functional genes in the sulfur cycle under different treatments.

[0039] Figure 21 Anosim analysis based on functional gene abundance for different treatments.

[0040] Figure 22 The effect of strain DLF18 on the growth phenotype of Chinese cabbage.

[0041] Figure 23 This study aimed to quantitatively determine the effects of strain DLF18 on the growth traits of Chinese cabbage.

[0042] Note: In this invention, different lowercase letters indicate significant differences between groups (p<0.05), and different uppercase letters indicate extremely significant differences between groups (p<0.01). Detailed Implementation

[0043] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0044] The culture medium and main reagents used in this invention are as follows:

[0045] PDA medium: 200 g / L potato, 20 g / L glucose, natural pH; solid medium supplemented with 20 g / L agar.

[0046] R2A medium: peptone 0.5 g / L, sodium chloride 0.5 g / L, glucose 0.5 g / L, sodium citrate 0.5 g / L, K2HPO4 0.3 g / L, pH natural; solid medium supplemented with 20 g / L agar.

[0047] LB medium: trypsin 10g / L, yeast extract 5g / L, NaCl 10g / L, pH natural; solid medium supplemented with 20g / L agar.

[0048] Sodium carboxymethyl cellulose basal medium: sodium carboxymethyl cellulose 5 g / L, (NH4)2SO4 g / L, MgSO4·(7H2O) 1.2 g / L, CaCl2 0.3 g / L, K2HPO4 1 g / L, KH2PO4 1 g / L, NaNO3 1 g / L, pH natural.

[0049] Rice straw carbon source basal culture medium: rice straw powder 20 g / L, (NH4)2SO4 4 g / L, MgSO4·(7H2O) 1.2 g / L, CaCl2 0.3 g / L, K2HPO4 1 g / L, KH2PO4 1 g / L, NaNO3 1 g / L, pH natural.

[0050] Corn stalk carbon source basal culture medium: 20 g / L corn stalk, 4 g / L (NH4)2SO4, 1.2 g / L MgSO4·(7H2O)2, 0.3 g / L CaCl2, 1 g / L K2HPO4, 1 g / L KH2PO4, 1 g / L NaNO3, pH natural.

[0051] Basic culture medium with wheat straw as carbon source: 20 g / L wheat straw, 4 g / L (NH4)2SO4, 1.2 g / L MgSO4·(7H2O)2, 0.3 g / L CaCl2, 1 g / L K2HPO4, 1 g / L KH2PO4, 1 g / L NaNO3, pH natural.

[0052] Sugarcane bagasse carbon source basal culture medium: sugarcane bagasse 20 g / L, (NH4)2SO4 4 g / L, MgSO4·(7H2O) 1.2 g / L, CaCl2 0.3 g / L, K2HPO4 1 g / L, KH2PO4 1 g / L, NaNO3 1 g / L, pH natural.

[0053] Pine wood residue carbon source basal culture medium: pine wood residue 20 g / L, (NH4)2SO4 4 g / L, MgSO4·(7H2O) 1.2 g / L, CaCl2 0.3 g / L, K2HPO4 1 g / L, KH2PO4 1 g / L, NaNO3 1 g / L, pH natural.

[0054] Poplar wood residue carbon source basal culture medium: poplar wood residue 20 g / L, (NH4)2SO4 4 g / L, MgSO4·(7H2O) 1.2 g / L, CaCl2 0.3 g / L, K2HPO4 1 g / L, KH2PO4 1 g / L, NaNO3 1 g / L, pH natural.

[0055] 0.2 mol / L sodium pyrophosphate alkaline extract: Weigh 0.53 g of sodium pyrophosphate and dissolve it in 10 mL of pure water to obtain the extract.

[0056] 0.5 mol / L hydrochloric acid solution, 6X DNA Loading Buffer, 0.1 mol / L sodium pyrophosphate-sodium hydroxide, potassium dichromate.

[0057] Example 1

[0058] 1. Isolation of strains

[0059] Weigh 10g of soil sample (Canglang Peak, Cangshan Mountain, Dali: 25°50'28.90"N, 100°03'7.60"E) into 10mL of sterile water, and dilute to 10... -3 10 -4 10 -5 The culture medium was spread on a basal medium containing lignocellulose (such as rice straw, corn straw, wheat straw, sugarcane bagasse, pine wood chips, and poplar wood chips) as the sole carbon source, and incubated at 20°C. After bacterial growth, single colonies were picked from the plates for transfer and purification.

[0060] 2. Screening of strains

[0061] Initial screening: The selected and purified strains were inoculated into 10 mL of LB liquid medium and cultured at 20℃ and 180 rpm for 2 days to prepare seed culture. The seed culture was then inoculated at 2% of 100 mL of rice straw carbon source basal medium and cultured at 20℃ and 180 rpm for 7 days. Then, 0.2 mol / L sodium pyrophosphate extract was added to the fermentation broth to bring the pH to 12, and the mixture was allowed to stand at room temperature for 24 hours. The supernatant was scanned between 190 nm and 320 nm. Three biological replicates were set up for each group, and strains with absorbance values ​​greater than zero were selected.

[0062] Secondary screening: While ultraviolet spectroscopy can determine whether a strain produces humic acid, it cannot accurately measure the yield. Therefore, secondary screening is necessary to determine the humic acid yield of each strain. The strains obtained from the initial screening were inoculated onto a basal medium with lignocellulose (such as rice straw, corn stalks, wheat straw, sugarcane bagasse, pine wood, and poplar wood residue) as the sole carbon source. Fermentation was carried out at 20℃ and 180 rpm for 7 days, and the humic acid yield in the fermentation broth was preliminarily determined. Then, 0.2 mol / L sodium pyrophosphate alkaline extract was added to the fermentation broth to bring the pH to 12. The mixture was allowed to stand at room temperature for 24 hours, and then the pH was adjusted to 1-2 with hydrochloric acid and allowed to stand overnight. Centrifugation was performed, the supernatant was discarded, and the precipitate was dried. The precipitate obtained was crude humic acid. Based on the yield of crude humic acid, strains with high humic acid production capacity were screened and named DLF18 (hereinafter referred to as F18).

[0063] In the initial screening of F18 strain, OD 200 The presence of a maximum absorption peak at nm and an absorbance of 0.207 indicates that strain F18 has the ability to produce humic acid.

[0064] In the secondary screening, strain F18 was able to produce humic acid in both rice straw and sugarcane bagasse as the sole carbon source, with the highest yield in rice straw (0.047 g / 10 mL). Furthermore, strain F18 could also grow in corn stalks and wheat stalks, but the humic acid yield was almost zero (see appendix). Figure 4 ).

[0065] 3. Identification of strains

[0066] (1) The selected F18 strain was inoculated on PDA medium and cultured at 25°C for 5 days. The size and color of the colonies were then observed and recorded. Microscopic images of the strain were taken using a Nikon ECLIPSENi-U biological microscope, and the morphological characteristics of the strain were recorded.

[0067] The results showed that strain F18 was a typical Aspergillus fungus, with... Figure 1 Image a shows the sporulation structure of Aspergillus fungi. Aspergillus conidiophores are relatively long, with small spore-forming areas visible at the upper part of the conidiophore. Spores are scattered at the end of the conidiophore, demonstrating a typical Aspergillus spore arrangement. (Appendix) Figure 1 Image b shows conidia. The images show round or oval spores, ranging in color from brown to dark brown. They are neatly distributed, similar in size, and their surface exhibits minute textures or spots. (Attached) Figure 1 Photo c shows a pure culture (left side is the front of the culture medium, right side is the back of the culture medium), where the mycelium appears black.

[0068] (2) DNA was extracted from strain F18 and amplified by PCR using Taq polymerase with universal primers for fungal ribosomal rDNA intergenic spacer region (ITS) sequences (ITS4: 5'-TCCTCCGCTTATTGATATGC-3', ITS5: 5'-GGAAGTAA AAGTCGTAACAAGG-3'). Amplification parameters: pre-denaturation 94℃, 4 min; denaturation 94℃, 30 s; annealing 55℃, 35 s; extension 72℃, 90 s; 32 cycles; extension 72℃, 5 min. The PCR products were gel electrophoresed and then sent to Sangon Biotech for sequencing. The obtained sequences were submitted to GenBank (http: / / www.ncbi.nlm.nih.gov). Similarity comparisons were performed between the BLAST database and the ITS sequences. A phylogenetic tree was constructed using the Neighbor-Joining Method in MEGA 7.0 to determine the species position of the strain.

[0069] The phylogenetic tree shows that strain F18 clusters with Aspergillus niger in one clade, with 81% support (see appendix). Figure 2 ).

[0070] Based on colony morphology, microscopic photographs, and a phylogenetic tree constructed based on ITS rRNA sequence alignment, strain F18 was identified as a strain of the genus Aspergillus and named Aspergillus sp.F18.

[0071] The strain DLF18 was deposited on December 30, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.41737 and classified as Aspergillum sp. The deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0072] Example 2

[0073] 1. Inoculate the F18 strain onto R2A medium and observe the growth of the strain at different temperatures (4℃, 20℃, 25℃, 30℃, 37℃, 45℃, 50℃), with three biological replicates for each group.

[0074] The results showed that strain F18 ceased growth at 4℃ and 50℃, but grew well within the range of 20℃-45℃. The mycelia were dense and slender, changing color from white to yellow upon maturity. (See appendix) Figure 3 The above demonstrates that this strain has a wide range of temperature adaptability.

[0075] 2. The F18 strain was inoculated into LB liquid medium and cultured with shaking at 20℃ and 180 rpm for 2 days to prepare a seed culture. The seed culture was then inoculated at a rate of 2% into 100 mL of rice straw carbon source basal medium. The control was an uninoculated medium, and three biological replicates were performed. After 7 days of shaking culture at 20℃ and 180 rpm, 10 mL of the fermentation supernatant was collected and sent to Wuhan Punais Testing Technology Co., Ltd. for analysis (NY / T1971-2010 Determination of Humic Acid Content in Water-Soluble Fertilizers) to determine the humic acid content in the fermentation supernatant.

[0076] Appendix Figure 5 This study compared the humic acid content in the fermentation supernatant of strain F18 with that in the control group (CK, uninoculated rice straw basal salt liquid medium). The results showed that the humic acid content in each gram of F18 fermentation supernatant was 16.5%, while that in the control group (CK) was 9.59%. There was a highly significant difference (p<0.01) between F18 and the control group (CK), with F18 exhibiting a 72% higher humic acid content. This further demonstrates the strong humic acid production capacity of strain F18.

[0077] Example 3

[0078] 1. This experiment used barren soil in the suburbs of Chenggong District, Kunming City, and the gramineous wheat variety "Shannong 42" as research subjects to conduct a pot experiment to further explore whether humic acid-producing strains have a growth-promoting effect on wheat growth.

[0079] The growth of wheat in the control group (pure soil) and the experimental group (pure soil + F18 microbial fertilizer) was observed, and their agronomic traits were measured and analyzed.

[0080] Preparation method of F18 microbial fertilizer: Mix rice straw powder and wheat bran in a 7:3 ratio, add water and stir until moist but not waterlogged. Dispense into mushroom bags and seal them completely with rings. Sterilize each bag (1.5 kg) and store for later use (121℃, 120 min). Inoculate the strain into 50 mL of LB liquid medium (containing 50 μL of kanamycin) and culture at 20℃ and 180 rpm for 2 days to prepare seed culture. Inoculate the seed culture into sterilized mushroom bags and culture in a 25℃ incubator until the mycelium covers the entire bag.

[0081] After mixing the fermented inoculum with soil at a ratio of 1:8, the mixture was divided into equal portions and placed into flowerpots, with 6 replicates per group. After 15 days of soil repair by the inoculum, pre-prepared germinated wheat was inoculated into the flowerpots, with 3 pots in each group. The growth of the wheat was monitored, and agronomic traits such as plant height, leaf length, leaf width, and number of leaves were measured using SPASS software.

[0082] Appendix Figure 6The images show wheat growth at 12 and 56 days after sowing. It can be seen that the wheat growth after the addition of F18 microbial fertilizer was significantly better than that of the control group in pure soil. Data analysis of wheat growth traits such as plant height (a), leaf length (b), leaf width (c), and number of leaves (d) using SPASS software showed that the addition of F18 microbial fertilizer significantly increased plant height, leaf length, leaf width, and number of leaves compared to the control group (p<0.05). (See Appendix) Figure 7 This further demonstrates that the humic acid fertilizer produced by strain F18 from agricultural straw can promote wheat growth.

[0083] 2. Determination of soil humus content: Weigh 10g of soil from the wheat-grown soil in step 1, 10g per pot. Send the soil to Wuhan Punes Testing Company using dry ice to determine the content and changes in humus in the soil.

[0084] Soil samples were dried to constant weight at (105±5)℃. The dry matter and moisture content were calculated based on the difference in soil sample mass before and after drying, expressed as mass fractions. Soil humus was classified into soluble humus (humic acid and fulvic acid) and insoluble humus (humin) according to its solubility. Soluble humus was extracted with a 0.1 mol / L sodium pyrophosphate-sodium hydroxide mixture. The total amount of humic acid and fulvic acid was determined using the potassium dichromate oxidation capacity method. Humic acid was separated by acidification precipitation of the extract, and its content was determined. The fulvic acid content was then calculated. The total carbon content of the soil sample was determined, and the humin content was obtained by subtracting the humic acid and fulvic acid contents. The results are shown in Table 1 and Appendix. Figure 8 .

[0085] Table 1. Humus content in soils under different treatments

[0086]

[0087] Note: "g / kg" indicates the humic content per kg of dry soil.

[0088] The results showed that the humic content (including fulvic acid and humin) in soils treated with F18 was significantly increased. Figure 8 As shown in Figure a, the humic content in the experimental group was significantly different from that in the control group (p<0.01), with the humic content in the experimental group increasing by 5.19 g / kg compared to the control. (See attached figure.) Figure 8 c in the middle, attached Figure 8 As can be seen from the data, the fulvic acid and humin content in the experimental group were significantly different from those in the control group (p<0.05), with the fulvic acid and humin content increasing by 1.081 g / kg and 3.73 g / kg, respectively, compared to the control group.

[0089] 3. Changes in Soil Microbial Quantities: To investigate whether there were differences in the microbial community in soils treated with F18 microbial fertilizer, a dilution-spreading experiment was conducted on the soil previously planted with wheat in step 1. 10g of the wheat-planted soil was weighed and diluted to 10mL of sterile water. -5 The samples were spread on PDA medium, with three replicates per pot. They were incubated at 25°C for 3 days, and the changes in colony counts on the plates were observed.

[0090] Appendix Figure 9 To dilute to 10 -5 The growth period of soil sample solutions on PDA medium was observed. Group a represents the control group, and group b represents the experimental group treated with F18 microbial fertilizer, with three biological replicates per group. It can be seen that group b plates were covered with microbial colonies (>1000 colonies / plate), while group a plates had fewer microbial colonies (<10 colonies / plate). The colony count in the experimental groups was significantly higher than that in the control group, indicating that the addition of F18 microbial fertilizer can, to some extent, increase the number and species richness of culturable microbial communities in the soil.

[0091] 4. Soil metagenomic analysis: Soil metagenomic samples were extracted using the MP (MP Biomedicals Soil DNA Isolation Kit), with three biological replicates per group. 0.3 g of soil sample was weighed from each replicate. The extracted metagenomic DNA was mixed with 6X DNA Loading Buffer (5:1) and then subjected to agarose gel electrophoresis (see Table 2 and Appendix). Figure 10 After passing gel electrophoresis, samples were stored on dry ice and sent to Wuhan Punes Testing Co., Ltd. for metagenomic sequencing. Following sample testing, sequence libraries were constructed and high-throughput sequencing was performed. High-quality sequences were assembled using assembly software, and gene prediction was performed. A bar chart is used to illustrate the species composition of each sample and the proportion of different species in each sample (see appendix). Figure 11 PCoA analysis was performed using the unconstrained ordination (Classical Multidimensional Scaling, cMDScale) method to assess differences in microbial community composition among samples and to analyze species β-diversity (see appendix). Figure 14 Based on metagenomeSeq heatmap analysis, the differences in species abundance among samples were statistically analyzed (see appendix). Figure 15 Using the CAZy database, the predicted genes are annotated and classified according to species and function, and their functions, classifications, and metabolic pathways are predicted (see appendix). Figure 16 The intergroup differences in biogeochemical cycle functional abundance were investigated using metagenomeSeq heatmaps and Anosim analysis (see Appendix). Figure 17 -Appendix Figure 21 ).

[0092] Appendix Figure 10 The image shows a 1% gel electrophoresis result of the submitted samples after electrophoresis at 120V for 30 min, with a sample loading volume of 5 μl. Differences in the brightness and total amount of metagenomic DNA can be observed in different treatments. Compared to the control group CKT, the concentration and total amount of F18 in the experimental group were significantly increased, indicating that the addition of F18 to the soil increased the types and numbers of microorganisms to some extent.

[0093] Table 2. Sample DNA Detection Results

[0094]

[0095] Appendix Figure 11 -Appendix Figure 13 This section presents bar charts showing the species composition of CKT and F18 at the phylum, genus, and species levels. The charts visually illustrate the species composition of each sample and the proportion of different species within each sample. Using R software, bar charts were created for the dominant species (the top 30 species in overall abundance) at each taxonomic level in each sample. The horizontal axis (x-axis) shows the different sample groups, with three replicates per group. The vertical axis (y-axis) represents the relative abundance of each microbial community, ranging from 0 to 1. The height of each bar represents the relative abundance of microorganisms in that sample group. (Appendix) Figure 11 The abundance at the phylum level is shown: the pink area (Proteobacteria) and the green area (Actinobacteria) have the highest relative abundance, exceeding 0.5%. Significant differences exist between F18 and CKT in certain phyla, such as Actinobacteria and Bacteroidetes, showing marked abundance variations. (Appendix) Figure 12 The abundance at the genus level is shown: Excluding unclassifiable or few species, *Sphingomonas* (pink area) and *Nocardioides* (orange area) accounted for a large proportion of abundance in CKT. In F18, the relative abundance of unclassified groups within *Acidobacteria* (green area) and *Steroidobacter* (blue area) was higher. There were significant abundance variations in *Sphingomonas* and *Nocardioides* between F18 and CKT, with opposite trends in species abundance between the two groups. (Appendix) Figure 13The abundance at the species level is shown: the proportion of unclassifiable microorganisms exceeds 0.5. Furthermore, in the CKT, *Acidobacteria bacterium* (pink area) and *Chloroflexi bacterium* (green area) account for a large proportion of abundance. In the F18, the relative abundance of *Acidobacteria bacterium* (pink area) and *Proteobacteria bacterium* (orange area) is relatively high. Significant differences exist between F18 and CKT in some species, such as marked changes in the species abundance of *Acidobacteria bacterium* and *Chloroflexi bacterium*. (Appendix) Figure 11 -Appendix Figure 13 Stacked bar charts were used to display the relative abundance of different microbial phyla, genera, and species in the experimental and control groups, showing the differences in microbial community composition and reflecting the impact of the F18 treatment on the soil microbial community. This invention utilizes the unconstrained ordination (Classical Multidimensional Scaling, cMDScale) analysis method for PCoA analysis. Figure 14 The study illustrates the differences in species structure between CKT and F18 at the three taxonomic levels: phylum, genus, and species. PCoA1 represents the first principal component and its contribution to sample differences; this axis shows the major differences in most samples. PCoA2 represents the second principal component and its contribution to sample differences, showing the second largest major difference. The CKT group (blue) and the F18 group (dark blue) are clearly separated in the principal coordinate plot; the microbial community structure of the F18 group clusters together and is farther away from the structure of the CKT group. This indicates a significant structural difference between the microorganisms in these two groups. (See attached...) Figure 14 In Figure a, the first two axes of the PCoA analysis explained 74.34% and 21.07% of the total variance at the gate level, respectively. (See Appendix...) Figure 14 In section b, the first two axes of the PCoA analysis explained 59.65% and 28.46% of the total variance at the genus level, respectively. (See appendix...) Figure 11 In Figure c, the first two axes of the PCoA analysis explained 56.1% and 30.17% of the total variance at the species level, respectively. (From Appendix...) Figure 14 As can be seen, at the phylum level, the CKT group and the F18 group already showed significant differences in overall community composition; at the genus and species level, these differences were further amplified, specifically manifested in the distribution differences of individual genera or species. Therefore, with the addition of F18, the soil microbial community structure underwent significant changes.

[0096] Appendix Figure 15This study used metagenome-Seq analysis based on a zero-inflation model to assess abundance differences between groups under different treatments (with or without F18). Heatmaps were used to visualize species with significant differences at the phylum level. Blue represents low abundance (negative values) for that taxa, while red indicates high abundance (positive values). Yellow represents abundance close to the median. The color contrast between samples provides a visual understanding of the differences in microbial community composition. *Candidatus Pacebacteria*, *Candidate division*, *Chrysiogenetes*, and *Candidatus Kryptonia* showed significantly high abundance in F18 but low abundance in CKT. Conversely, *Armatimonadetes* and *Candidatus Cryosericota* showed significantly high abundance in CKT but low abundance in F18. (Appendix) Figure 15 The study demonstrated the differences in microbial community composition between CKT and F18. Through row and column cluster analysis, significant abundance differences were identified between groups at the phylum level, indicating that the addition of F18 significantly altered the richness and composition of the soil microbial community.

[0097] The CAZy (Carbohydrate-Active enZYmes) database is a database specifically designed for classifying and annotating enzymes related to carbohydrate metabolism. This study utilized the CAZy database to functionally annotate and classify predicted genes, assessing the impact of adding F18 on soil microbial functional abundance. (Appendix) Figure 16 To create heatmaps based on the functional annotations and abundance information of all samples in the CAZy database, clustering was performed at both the functional and sample levels. Rows in the heatmap represent different glycoenzyme families, and colors indicate the relative abundance of each glycoenzyme family in different samples: red indicates high abundance (>0); blue indicates low abundance (<0); and yellow indicates near-neutral abundance (0), meaning the abundance of the glycoenzyme in the sample is moderate. (Appendix) Figure 16The abundance of carbohydrate-active enzymes (CAZymes) in different samples (CKT group and F18 group) is shown. The heatmap visually illustrates the expression levels of different CAZymes in each sample through color changes. For example, in the CKT group (columns labeled CKT1, CKT2, CKT3), the abundance of CAZymes showed higher levels in the GT87 and GT39 families, while lower abundance in the GT5 and GH95 families. In contrast, the F18 group (F181, F182, F183) showed a clear opposite trend, with higher abundance in the GT5 and GH95 families, while lower abundance in the GT87 and GT39 families. We can clearly see the difference in abundance of CAZymes between the CKT and F18 groups. Specifically, the different expression levels of CAZymes in different sample groups reflect the differences in the carbohydrate metabolism potential of the microbial community after the addition of F18 to the soil.

[0098] Microorganisms are key participants in biogeochemical cycles, promoting the transformation and cycling of elements such as carbon, nitrogen, phosphorus, and sulfur through metabolic activities. This study assessed the impact of F18 on the biogeochemical cycling function of soil microorganisms by analyzing the relative abundance and inter-group differences of functional genes for carbon, nitrogen, phosphorus, and sulfur cycling. MetagenomeSeq heatmaps and Anosim analyses were used to study the inter-group differences in biogeochemical cycling function (see appendix). Figure 17 -Appendix Figure 21 Appendix Figure 17 The dendrograms on the left and top of the metagenomeSeq heatmap (a, b, c, and d represent the carbon, nitrogen, phosphorus, and sulfur cycles, respectively) show the clustering relationships between different samples and genes. The clustering results demonstrate the similarity of metabolic pathways between samples and genes. The vertical axis shows different genes associated with the carbon cycle. The horizontal axis shows gene expression data for different samples (CKT1, CKT2, CKT3, F181, F182, and F183). Red (high expression) to blue (low expression) represents gene expression levels. (See appendix...) Figure 17 In the study, the CKT group showed higher expression of genes such as ldh and acs, while the F18 group showed lower expression of these genes. (See attached...) Figure 18 In the F18 group, higher expression levels were observed in some nitrogen cycle genes such as nrfH, nrfA, and norC, while relatively lower expression levels were observed in these genes in the CKT group. (See attached...) Figure 19 In the F18 group, high expression was observed in multiple genes, particularly phoB and spoT. (The text abruptly ends here.) Figure 20The CKT group showed high expression of several sulfur metabolism genes, such as soxY and soxZ, while the F18 group showed relatively low expression. Heatmaps revealed differences in gene expression across carbon, nitrogen, phosphorus, and sulfur metabolic pathways between the CKT and F18 groups. The high expression of certain genes in specific metabolic pathways may indicate that microorganisms involved in these pathways dominate in the corresponding samples, thus influencing elemental transformation pathways and rates.

[0099] Appendix Figure 21 Anosim analysis (where a, b, c, and d represent the carbon, nitrogen, phosphorus, and sulfur cycles, respectively) was used to statistically analyze the differences in microbial communities across different elemental cycles. R represents the degree of difference between sample groups. A higher R value indicates a greater difference between groups; R greater than 0 indicates a significant difference between groups. (See attached...) Figure 21 In the carbon cycle of group a, R = 0.556 and P = 0.1. The relatively high R value (close to 1) indicates a significant difference between the CKT and F18 groups. (Appendix) Figure 21 From b, we can see that R = 0.556 and P = 0.2. Compared to the carbon cycle, the nitrogen cycle has a lower R value, but differences still exist between groups. (See attached...) Figure 21 From c, we know that R = 0.63 and P = 0.1. Similar to the carbon cycle, there are significant differences between different sample groups in the phosphorus cycle. (See appendix...) Figure 21 Significant differences were also observed between the two groups in the sulfur cycle of medium d, with R = 0.333 and P = 0.1. This indicates that the microbial communities differ between different samples in different biogeochemical cycles, especially in the carbon, phosphorus, and sulfur cycles.

[0100] Overall, the biogeochemical cycle function analysis revealed how microbial communities play a role in these processes and the changes in the activity of related microbial communities in each cycle, further suggesting that these differences may be related to the effects of soil addition of F18.

[0101] Example 4

[0102] Application of F18 microbial fertilizer in promoting the growth of Chinese cabbage

[0103] This experiment used barren soil in Yinjie Town, Midu County, Dali Prefecture (25°15'26"N, 100°31'52"E) and the "Hantian No. 1" Chinese cabbage (Caulis Haworthia) of the Brassicaceae family as the research subjects to conduct a pot experiment in a greenhouse to investigate whether the F18 strain had a growth-promoting effect on Chinese cabbage. The growth of Chinese cabbage in the control group (pure soil) and the experimental group (pure soil + F18 bacterial fertilizer) was observed, and their agronomic traits were measured and analyzed.

[0104] The preparation method of F18 microbial fertilizer is the same as in Example 3. The fermented microbial bags are mixed evenly with the soil, and 5% microbial fertilizer is added to each pot (i.e., 200g of microbial fertilizer is added to 4kg of soil). The mixture is then divided into flowerpots, with 5 replicates per group. The control group consists of 4kg of pure soil. After the microorganisms have repaired the soil for 15 days, 10 cabbages are inoculated into each flowerpot. The growth of the cabbages is monitored, and during this period, seedlings are thinned to 5 plants to measure agronomic traits. SPASS software is used to analyze the agronomic traits of the cabbages, including plant height, root length, leaf width, number of leaves, above-ground fresh weight, and above-ground dry weight.

[0105] Appendix Figure 22 These are growth images of Chinese cabbage taken 19 days and 40 days after sowing. It can be seen that at 19 days, there is almost no difference in growth between CKT and F18 Chinese cabbage. At 40 days, the Chinese cabbage treated with F18 microbial fertilizer shows significantly better growth than the CKT variety.

[0106] Appendix Figure 23 Data analysis of Chinese cabbage biomass measurements (a) plant height, b) root length, c) leaf width, d) number of leaves, e) aboveground fresh weight, and f) aboveground dry weight was performed using SPASS software. Results showed that the biomass of Chinese cabbage (plant height, root length, leaf width, number of leaves, aboveground fresh weight, and aboveground dry weight) was significantly increased (p<0.01) in soil treated with F18. Compared to CKT, plant height, root length, leaf width, number of leaves, aboveground fresh weight, and aboveground dry weight increased by an average of 6.4 cm, 3.34 cm, 3.98 cm, 3 leaves, 3.49 g, and 0.225 g, respectively. In conclusion, the addition of F18 microbial fertilizer to the soil promotes the growth of Chinese cabbage to a certain extent.

[0107] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0108] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A strain of Aspergillus ( Aspergillus sp.) DLF18, characterized in that, The accession number of the DLF18 is CGMCCNO.41737.

2. A microbial inoculant, characterized in that, Includes the Aspergillus described in claim 1.

3. The application of the Aspergillus of claim 1 or the microbial agent of claim 2 in the production of humic acid, characterized in that, The Aspergillus or the microbial agent is made from rice straw and / or sugarcane bagasse.

4. The application according to claim 3, characterized in that, The humic acid includes soluble humic acid and fulvic acid, as well as insoluble humin.

5. The application of the Aspergillus of claim 1 or the microbial agent of claim 2 in improving soil microbial abundance, characterized in that, The fermentation products obtained by fermenting rice straw and / or sugarcane bagasse using the Aspergillus or the microbial agent are applied to the soil as fertilizer.

6. The application according to claim 5, characterized in that, The soil microbial abundance includes the abundance of microbial species and quantity, the abundance of carbohydrate active enzymes, and the abundance of genes for carbon metabolism pathways, nitrogen metabolism pathways, phosphorus metabolism pathways, and sulfur metabolism pathways.

7. The application of the Aspergillus of claim 1 or the microbial agent of claim 2 in promoting plant growth, characterized in that, Rice straw and / or sugarcane bagasse are fermented using the Aspergillus or the microbial agent, and the resulting fermentation product is applied to the soil as fertilizer. The plants are wheat and cabbage.

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