Aspergillus DLF18 and application thereof

Abstract

The invention discloses Aspergillus sp. DLF18 and application thereof, and relates to the technical field of microorganisms, and the preservation number of the Aspergillus sp. DLF18 is CGMCC (China General Microbiological Culture Collection Center) NO.41737. The strain can effectively utilize rice straws and / or bagasse to produce humic acid, and the content of humic acid in each gram of fermentation supernatant after the rice straws are fermented is 16.5%. When the DLF18 fermentation product is applied to crops, the growth condition of the crops can be remarkably improved, and the humus content in soil is increased. Meanwhile, a metagenomics analysis result shows that compared with a control group, after the DLF18 is applied into the soil, the species beta diversity is remarkably changed, and the functional gene abundance and the inter-group difference are remarkably changed. Therefore, the DLF18 has a wide application prospect in the aspects of agricultural straw utilization and soil remediation.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and more particularly to an Aspergillus sp. DLF18 and applications thereof. Background Art

[0002] Humic acid is a type of natural organic matter found primarily in soil, sediment, and water. It is a complex mixture formed by the long-term decomposition and transformation of organic matter. Humic acid is one of the main components of soil humus and has strong adsorption, water-retention, and fertility-regulating properties. It is the end product of organic matter decomposition and typically exists in an acidic form in soil and water. Humic acid is divided into three categories based on its solubility in solvents and color: fulvic acid, humin, and humic acid. As a core component of soil humus, humic acid plays a vital role in maintaining soil fertility, improving soil structure, and promoting plant growth. Furthermore, humic acid is of great significance for environmental remediation, helping to reduce the bioavailability of heavy metal pollution and promoting the degradation of pollutants.

[0003] There is ongoing debate about the specific role of microorganisms in humic acid formation. Four main hypotheses exist: the plant transformation hypothesis, the biochemical hypothesis, cell autolysis, and microbial synthesis. It is currently difficult to determine which hypothesis is more accurate, as humic acid formation may require the coordinated action of multiple processes. The microbial formation hypothesis emphasizes the role of microorganisms in humic acid formation. Microorganisms play a multifaceted role in humic acid formation, acting not only as decomposers, breaking down macromolecular polymers, but also as producers, recondensing decomposed small molecules. Therefore, the decomposition products of lignocellulose serve as the backbone and substrate for humic acid formation. Adding protein-rich organic waste at the appropriate time to increase lignocellulosic enzyme activity offers a crucial option for converting lignocellulosic waste into humic acid by controlling key factors. Lignocellulose, the most abundant renewable bioresource in the biological world, produces 150 billion tons annually worldwide, of which straw accounts for 6 billion tons, and China produces 1.113 billion tons annually.

[0004] However, the microbial resources that can efficiently utilize agricultural straw to ferment and produce humic acid are 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 that needs to be solved by technicians in this field. Summary of the Invention

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

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] A strain of Aspergillus sp. DLF18, the deposit number of DLF18 is CGMCC NO.41737.

[0008] Another object of the present invention is to provide a microbial agent comprising the above-mentioned Aspergillus.

[0009] Another object of the present invention is to provide the use 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, and insoluble humin.

[0011] Preferably, the Aspergillus or the microbial agent uses rice straw and / or sugarcane bagasse as raw materials.

[0012] Another object of the present invention is to provide the use of the above-mentioned Aspergillus or the above-mentioned microbial agent in improving the abundance of soil microorganisms, or the inter-group difference analysis of species composition, or the functional level analysis, or the inter-group difference analysis of 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 in the carbon metabolism pathway, nitrogen metabolism pathway, phosphorus metabolism pathway, and sulfur metabolism pathway.

[0014] Preferably, the application is to ferment rice straw and / or sugarcane bagasse using the Aspergillus or the microbial agent, and the obtained fermentation product is applied to the soil as a fertilizer.

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

[0016] Preferably, the application is to ferment rice straw and / or sugarcane bagasse using the Aspergillus or the microbial agent, and the obtained fermentation product is applied to the soil as a fertilizer.

[0017] Beneficial effects: The present invention isolated a strain of Aspergillus DLF18 from the soil of Canglang Peak, Cangshan Mountain, Dali. This strain can effectively utilize rice straw and / or sugarcane bagasse to produce humic acid, and the humic acid content in the fermentation liquid after the rice straw fermentation is 16.5%. When the F18 fermentation product is applied to crops, the plant height, leaf length, leaf width, etc. of wheat are significantly improved, and the soil humus content increases by 5.2g / kg; the plant height, root length, leaf width, number of leaves, aboveground fresh weight and aboveground dry weight of cabbage are increased by an average of 6.4cm, 3.34cm, 3.98cm, 3 leaves, 3.49g and 0.225g, respectively. The results of metagenomic analysis showed that compared with the control group, after the application of F18 in the soil, the species β diversity changed significantly, and the functional gene abundance and inter-group differences changed significantly. The above shows that F18 has broad application prospects in agricultural straw utilization and soil remediation. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are merely embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying any creative work.

[0019] Figure 1 The morphological identification diagram of strain DLF18, where a is the spore-forming 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 conditions of strain DLF18 at different temperatures, where "+" indicates the growth of the strain, the more the better, and " / " indicates that the strain does not grow; "1" indicates the ck control group without inoculation, 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 This is the effect of strain DLF18 on wheat growth phenotype.

[0025] Figure 7 This is a quantitative determination of the effect of strain DLF18 on wheat growth traits.

[0026] Figure 8Comparison of humus group content in soil under different treatments.

[0027] Figure 9 Microbial culture conditions in soil under different treatments.

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

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

[0030] Figure 12 Species abundance at the genus level under different treatments.

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

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

[0033] Figure 15 Heat map of species abundance difference analysis under different treatments.

[0034] Figure 16 Heat map of functional abundance analysis under different treatments.

[0035] Figure 17This is a heat map of the abundance analysis of carbon cycle functional genes under different treatments; among them, 1 represents: 4-aminobutyrateaminotransferase 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: bsdC; 20: catA; 21: cellobiosidase; 22: cellulase; 23: chitiniase; 24: fae; 25: fdhA; 26: fdhB; 27: fdoG; 28: fdoH; 29: fghA; 30: frmA; 31: glucoamylase; 32: hexosaminidase; 33: histidinol-phosphate / aromatic aminotransferase; 34: isoamylase; 35: mannan endo-1,4-beta-mannosidase; 36: mauA; 37: mauB; 38: mxaF; 39: ornithine / acetylornithine aminotransferase; 40 represents: phosphoserineaminotransferase; 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 Heat map of nitrogen cycle functional gene abundance analysis under different treatments.

[0037] Figure 19 Heat map of abundance analysis of phosphorus cycle functional genes under different treatments.

[0038] Figure 20 Heat map showing the abundance analysis of sulfur cycle functional genes under different treatments.

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

[0040] Figure 22 This is the effect of strain DLF18 on the growth phenotype of pakchoy.

[0041] Figure 23 This is a quantitative determination of the effect of strain DLF18 on the growth traits of pakchoy.

[0042] Note: The data marked with different lowercase letters indicate significant differences among the groups (p<0.05), and the data marked with different uppercase letters indicate extremely significant differences among the groups (p<0.01). DETAILED DESCRIPTION

[0043] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0044] The culture medium and main reagents used in the present 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, natural pH; solid medium supplemented with 20 g / L agar.

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

[0048] Sodium carboxymethylcellulose basal medium: sodium carboxymethylcellulose 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, natural pH.

[0049] Rice straw carbon source basal 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, natural pH.

[0050] Corn stalk carbon source basal medium: corn stalk 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, natural pH.

[0051] Wheat straw carbon source basal medium: wheat straw 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, natural pH.

[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, natural pH.

[0053] Pinewood residue carbon source basic culture medium: pinewood 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, natural pH.

[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, natural pH.

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

[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 10 g of soil sample (Canglang Peak, Cangshan Mountain, Dali: 25°50'28.90"N, 100°03'7.60"E) in 10 mL of sterile water and dilute to 10 -3 , 10 -4 , 10 -5 The bacteria are then spread on a basal medium containing lignocellulose such as rice straw, corn straw, wheat straw, sugarcane bagasse, pine wood residue, or poplar wood residue as the sole carbon source and cultured in a constant temperature incubator at 20°C. After the bacteria grow, single colonies are picked from the plate for transfer and purification.

[0060] 2. Screening of strains

[0061] Initial screening: Inoculate the selected, purified strains into 10 mL of LB liquid medium and shake culture at 20°C, 180 rpm for 2 days to prepare seed culture. Inoculate 2% of the seed culture into 100 mL of rice straw-based carbon source medium and shake culture at 20°C, 180 rpm for 7 days. Add 0.2 mol / L sodium pyrophosphate alkaline extract to the fermentation broth to raise its pH to 12. Allow the supernatant to stand at room temperature for 24 hours, and scan the supernatant between 190 nm and 320 nm. Set up three biological replicates for each group, and select strains with absorbance values ​​greater than zero.

[0062] Rescreening: While UV spectroscopy can confirm whether a strain produces humic acid, it cannot accurately measure humic acid production. Therefore, rescreening is required to determine the humic acid production of each strain. The strains obtained from the initial screening were inoculated onto a basal medium containing lignocellulose, such as rice straw, corn stalks, wheat straw, sugarcane bagasse, pine wood, or poplar wood residue, as the sole carbon source. Fermentation was carried out at 20°C and 180 rpm for 7 days, and the humic acid production in the fermentation broth was initially determined. The fermentation broth was then treated with 0.2 mol / L sodium pyrophosphate alkaline extract to raise the pH to 12. The broth was allowed to stand at room temperature for 24 hours, followed by adjusting the pH to 1-2 with hydrochloric acid and allowing to stand overnight. The broth was centrifuged, the supernatant discarded, and the precipitate dried to obtain crude humic acid. Based on the crude humic acid production, a strain with high humic acid production was identified and designated DLF18 (hereinafter referred to as F18).

[0063] The F18 strain had a maximum absorption peak at OD 200 nm and the absorbance value was 0.207, indicating that the F18 strain had the ability to produce humic acid.

[0064] The F18 strain could produce humic acid under the condition of rice straw and sugarcane residue as the sole carbon source, with the highest yield in rice straw (0.047 g / 10 mL). In addition, the F18 strain could also grow in corn stalks and wheat stalks, but the humic acid yield was almost zero (see Figure 2). Figure 4 )。

[0065] 3. Identification of the strain

[0066] (1) The F18 strain was inoculated on PDA medium and incubated at 25°C for 5 days, then the size and color of the colony were observed and recorded. Nikon ECLIPSE Ni-U biological microscope was used to take micrographs and record the morphological characteristics of the strain.

[0067] The results showed that the F18 strain was a typical Aspergillus fungus, as shown in Figure 3. Figure 1 Figure 3a shows the sporulation structure of Aspergillus fungus. The conidial phialide of Aspergillus fungus is relatively long, and some small spore-forming areas can be seen on the upper part of the conidial phialide. The spores are scattered at the end of the phialide, showing a typical arrangement of Aspergillus spores. Figure 1 Figure 3b shows the conidial phialide. In the figure, round or oval spores can be seen, with brown to brown color. They are evenly distributed and similar in size, with a small texture or spot on the surface of the spores. Figure 1 Figure 3c is a photo of pure culture (the left side is the front of the culture medium, and the right side is the back of the culture medium). The mycelium is black.

[0068] (2) DNA of the F18 strain was extracted and PCR amplified using universal primers for the fungal ribosomal rDNA intergenic region (ITS) sequence (ITS4: 5'-TCCTCCGCTTATTGATATGC-3', ITS5: 5'-GGAAGTAA AAGTCGTAACAAGG-3') using Taq enzyme. Amplification parameters were as follows: pre-denaturation at 94°C for 4 min, denaturation at 94°C for 30 s, annealing at 55°C for 35 s, extension at 72°C for 90 s, 32 cycles; extension at 72°C for 5 min. The PCR product was subjected to gel electrophoresis and sent to Sangon Biotech Co., Ltd. for sequencing. The obtained sequence was submitted to GenBank (http: / / www.ncbi.nlm.nih.gov). The strain was compared with the ITS sequence in the BLAST database and a phylogenetic tree was constructed using the neighbor-joining method using MEGA7.0 to determine the species status of the strain.

[0069] The phylogenetic tree showed that strain F18 and Aspergillus niger were clustered in one branch with a support rate of 81% (see Appendix Figure 2 ).

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

[0071] The strain DLF18 was deposited in the General Microbiology Center of China Culture Collection Administration on December 30, 2024, with the deposit number CGMCC NO.41737, and the classification name was Aspergillus sp. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0072] Example 2

[0073] 1. The F18 strain was spotted onto R2A medium and the growth of the strain was observed at different temperatures (4°C, 20°C, 25°C, 30°C, 37°C, 45°C, 50°C), with three biological replicates per group.

[0074] The results showed that the F18 strain stopped growing at 4℃ and 50℃, but grew well at 20℃-45℃. The hyphae were dense and slender, and the color changed from white to yellow when mature. (See Appendix Figure 3 ). The above shows that the strain has a wide range of temperature adaptability.

[0075] 2. Inoculate the F18 strain into LB liquid medium and shake culture at 20°C and 180 rpm for 2 days to prepare seed liquid. Inoculate the seed liquid into 100 mL of rice straw carbon source basal medium at a 2% inoculum size, with uninoculated medium as the control, for three biological replicates. Shake culture at 20°C and 180 rpm for 7 days. Collect 10 mL of the fermentation supernatant and send it to Wuhan Pu Neisi Testing Technology Co., Ltd. for testing (NY / T1971-2010 Determination of Humic Acid Content of Water-soluble Fertilizers) to determine the humic acid content in the fermentation supernatant.

[0076] Attachment Figure 5 This study compared the humic acid content of the supernatant of F18 fermentation with that of CK (a basal salt liquid medium containing uninoculated rice straw). The results showed that the humic acid content per gram of F18 fermentation supernatant was 16.5%, while the humic acid content per gram of the control (CK) was 9.59%. There was a highly significant difference (p<0.01) between the F18 and CK cultures, with the humic acid content increasing by 72% compared to the control. This further demonstrates the F18 strain's strong humic acid production capacity.

[0077] Example 3

[0078] 1. This experiment used the poor soil in the suburbs of Chenggong, Kunming City and the "Shannong 42" wheat of the Poaceae family as research objects for a pot experiment to further explore whether humic acid 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 bacterial fertilizer) was observed, and their agronomic traits were measured and analyzed.

[0080] Preparation of F18 biofertilizer: Mix rice straw powder and wheat bran in a 7:3 ratio, then add water and stir until moist without water accumulation. Dispense into bags, sealing them tightly with a collar. Each bag should contain 1.5 kg of the bacteria. Sterilize (121°C, 120 minutes) and set aside. Inoculate the strain into 50 mL of LB liquid medium (containing 50 μL of kanamycin) and incubate at 20°C, 180 rpm, and shake for 2 days to prepare the seed solution. Inoculate the seed solution into sterilized bags and incubate in a 25°C incubator until the bags are completely covered with mycelium.

[0081] After mixing the fermented fungus bags with soil at a ratio of 1:8, the mixture was distributed by equal weight into flower pots, with six replicates per group. After the fungus had remediated the soil for 15 days, the germinated wheat prepared in advance was inoculated into the flower pots, with three per group. The wheat growth was monitored, and agronomic traits such as plant height, leaf length, leaf width, and leaf number were measured using SPASS software.

[0082] Attachment Figure 6The growth chart of wheat taken at 12d and 56d after sowing shows that the growth of wheat after adding F18 bacterial fertilizer is obviously better than that of the control group of pure soil. The data analysis of the growth traits of wheat such as plant height (a), leaf length (b), leaf width (c), and leaf number (d) measured by SPASS software shows that the plant height, leaf length, leaf width, and leaf number are significantly (p<0.05) higher than those of the control group (see Figs. 1-4). Figure 7 It further illustrates that the humic acid fertilizer produced by F18 strain using agricultural straw can promote the growth of wheat.

[0083] 2. Soil humus group content determination: 10g of the soil planted with wheat in step 1 was weighed, 10g per pot. The dry ice was sent to Wuhan Pnaes Detection Company to determine the content and change of humus in the soil.

[0084] The soil sample was dried at (105±5)℃ until constant weight, and the content of dry matter and water was calculated by the difference in mass before and after drying, expressed as mass fraction. Soil humus is divided into soluble humus (humic acid and fulvic acid) and insoluble humus (humin) according to its solubility. The soluble humus was extracted with 0.1mol / L sodium pyrophosphate-sodium hydroxide mixed solution, and the total amount of humic acid and fulvic acid was determined by potassium dichromate oxidation volumetric method. The extraction liquid was acidified and precipitated to separate humic acid, and its content was determined to calculate the content of fulvic acid. The total carbon content of the soil sample was determined, and the humin content was obtained by subtracting the content of humic acid and fulvic acid, as shown in Table 1 and Fig. 5. Figure 8

[0085] Table 1 Humus group content in soil of different treatments

[0086]

[0087] Note: "g / kg" means the content of humus group per kg of dry soil.

[0088] The results show that the content of humus (including fulvic acid and humin) in the soil with F18 added is significantly improved. Among them, Fig. 1a shows that there is a very significant difference (p<0.01) in the content of humus between the experimental group and the control group, and the content of humus in the experimental group is increased by 5.19g / kg compared with the control. Figure 8 Fig. 1c and Fig. 1d show that there is a significant difference (p<0.05) in the content of fulvic acid and humin between the experimental group and the control group, and the content of fulvic acid and humin in the experimental group is increased by 1.081g / kg and 3.73g / kg respectively compared with the control. Figure 8 Figure 8

[0089] ​​​3. Changes in the number of soil microorganisms: In order to explore whether there are differences in the microbial community in the soil with F18 bacterial fertilizer, a dilution plating experiment was conducted on the soil planted with wheat in step 1. 10 g of soil planted with wheat was weighed into 10 mL of sterile water, diluted to 10 -5 , and plated on PDA medium, with 3 replicates per flowerpot. Incubate in a 25°C constant temperature incubator. After 3 days, observe the number of colonies on the plates.

[0090] Figure 2 Figure 9 is a growth period of the soil sample solution diluted to 10 -5 on PDA medium. a is the control group, and b is the experimental group with F18 bacterial fertilizer, with 3 biological replicates in each group. It can be seen that the b group is covered with microbial colonies (>1000 colonies / plate), while the a group has fewer microbial colonies (<10 colonies / plate). The number of colonies in the experimental group is significantly higher than that in the control group, indicating that the addition of F18 bacterial fertilizer can increase the number and species richness of cultivable soil microorganisms to a certain extent.

[0091] 4. Soil metagenome analysis: The MP (MP Biomedicals Soil DNA Isolation Kit) kit was used to extract the soil metagenome sample, with 3 biological replicates in each group, and 0.3 g of soil sample was weighed for each replicate. The extracted metagenome DNA was mixed with 6X DNA Loading Buffer (5:1) and subjected to agarose gel electrophoresis (see Table 2 and Figure 3 Figure 10 ). After gel electrophoresis detection, store in dry ice and send to Wuhan Pnaes Detection Company for metagenome sequencing. After the sample is detected, construct a sequence library and perform high-throughput sequencing. Use splicing software to splice and assemble high-quality sequences and perform gene prediction. Display the species composition of each sample and the proportion of different species in each sample through a column chart (see Figure 4 Figure 11 ). Perform PCoA analysis based on the non-restricted sorting (Classical Multidimensional Scaling, cMDScale) analysis method to evaluate the differences in microbial community composition between samples and analyze species beta diversity (see Figure 5 Figure 14 ). Based on metagenomeSeq heat map analysis, statistically analyze the species abundance differences between samples (see Figure 6 Figure 15 ). Use the CAZy database to annotate and classify the predicted genes in terms of species and function, and predict the function, classification, and metabolic pathways of the genes (see Figure 7 Figure 16 ). Use metagenomeSeq heat map and Anosim analysis to study the differences in biogeochemical cycle function abundance between groups (see Figure 8 Figure 17 - Figure 8 Figure 21 ).

[0092] Attachment Figure 10 This is a 1% gel electrophoresis image of the samples tested at 120V for 30 minutes. The sample volume was 5μl. The brightness and total amount of metagenomic DNA differed between treatments. Compared to the CKT control, the concentration and total amount of DNA in the F18 treatment group increased significantly, indicating that the addition of F18 to the soil increased the number and diversity of microorganisms.

[0093] Table 2 Sample DNA test results

[0094]

[0095] Attachment Figure 11 -Attached Figure 13 The species bar graphs of CKT and F18 at three taxonomic levels, namely, phylum (Phylum), genus (Genus), and species (Species), are displayed. The species composition of each sample and the proportion of different species in each sample can be intuitively seen from the figure. Using R software, a bar graph was drawn for the composition of the dominant species in each sample (here the top 30 species in terms of overall abundance) at each taxonomic level. The horizontal axis (x-axis): shows different sample groups, with 3 replicates in each group. The vertical axis (y-axis) represents the relative abundance of each microbial group, ranging from 0 to 1. The height of each bar represents the relative abundance of microorganisms in that group of samples. Attached Figure 11 The abundance at the phylum level is shown: the pink area of ​​Proteobacteria and the green area of ​​Actinobacteria have the highest relative abundance, accounting for more than 0.5. There are obvious differences between F18 and CKT in some microbial phyla, such as Actinobacteria and Bacteroidetes, which have obvious abundance changes. Figure 12 The abundance at the genus level is shown: in addition to the microorganisms that cannot be classified or have a small number of species, the abundance of Sphingomonas (Sphingomonas genus) in the pink area and Nocardioides (Nocardioides genus) in the orange area in CKT accounts for a large proportion. The relative abundance of the Acidobacteria that are not classified into clear categories in the green area of ​​F18 and the Steroidobacter (steroid bacteria genus) in the blue area is high. There are obvious changes in the abundance of Sphingomonas and Nocardioides between F18 and CKT, and the species abundance between the two groups shows opposite trends. Attachment Figure 13The abundance at the species level is shown: the unclassified microorganisms account for more than 0.5, and the abundance of Acidobacteria bacterium in the pink area and Chloroflexi bacterium in the green area in CKT accounts for a large proportion. The relative abundance of Acidobacteria bacterium in the pink area and Proteobacteria bacterium in the orange area in F18 is relatively high. The difference between F18 and CKT in some species is obvious, such as the significant change in the species abundance of Acidobacteria bacterium and Chloroflexi bacterium. Figure 11 -Appendix Figure 13 The relative abundance of different microbial phyla, genera and species in the experimental group and the control group is shown by stacking the column chart, which shows the difference in the composition of the microbial community and reflects the effect of F18 treatment on the microbial population in the soil. The present application performs PCoA analysis based on the non-restricted sorting (Classical Multidimensional Scaling, cMDScale) analysis method, and the results are shown in the following figures. Figure 14 The species structure difference between CKT and F18 at the phylum, genus and species levels is shown. PCoA1 represents the first principal component and its contribution to the difference between samples, and this axis shows the main difference between most samples. PCoA2 represents the second principal component and its contribution to the difference between samples, showing the second major difference. The CKT group (blue) and the F18 group (dark blue) are clearly separated in the principal coordinate diagram, and the microbial community structure of the F18 group is clustered together and far away from the structure of the CKT group. It shows that there is a large structural difference between the microorganisms of the two groups of samples. In the following figures, Figure 14 In a of the following figures, the first two axes of the PCoA analysis explain 74.34% and 21.07% of the total variation in the data at the phylum level, respectively. Figure 14 In b of the following figures, the first two axes of the PCoA analysis explain 59.65% and 28.46% of the total variation in the data at the genus level, respectively. Figure 11 In c of the following figures, the first two axes of the PCoA analysis explain 56.1% and 30.17% of the total variation in the data at the species level, respectively. From the following figures, Figure 14 It can be seen from the following figures that at the phylum level, there is already a significant difference in the overall community composition between the CKT group and the F18 group; at the genus and species levels, these differences will be further amplified, which is specifically manifested as the distribution difference of individual genera or species. Therefore, with the addition of F18, the structure of the soil microbial community has changed significantly.

[0096] Appendix Figure 15For this study, metagenomeSeq analysis was used based on the zero-inflated model to evaluate the abundance differences between groups under different treatments (whether F18 was added), and a heatmap visualization method was used to display species with significant differences at the phylum level. Blue represents a low abundance of the taxonomic group (negative value), while red represents a high abundance (positive value). Yellow represents an abundance close to the median value. By contrasting the colors between samples, the differences in the microbial community composition of these samples can be intuitively understood. Candidatus Pacebacteria, Candidate division, Chrysiogenetes, and Candidatus Kryptonia showed significantly high abundance in F18, but lower abundance in CKT. On the contrary, Armatimonadetes and Candidatus Cryosericota showed significantly high abundance in CKT, but lower abundance in F18. Appendix Figure 15 The differences in microbial community composition between CKT and F18 were demonstrated. Through row and column cluster analysis, it was confirmed that there were significant abundance differences between groups at the phylum level, indicating that the addition of F18 significantly changed the richness and composition of soil microbial communities.

[0097] The CAZy (Carbohydrate-Active enZYmes) database is a database specifically used to classify and annotate enzymes related to carbohydrate metabolism. This study used the CAZy database to functionally annotate and classify the predicted genes and evaluate the effect of adding F18 on the functional abundance of soil microorganisms. Figure 16 A heat map was drawn based on the functional annotation and abundance information of all samples in the CAZy database, and clustering was performed at both the functional and sample levels. The rows in the heat map represent different carbohydrate-active enzyme families, and the colors represent the relative abundance of each carbohydrate enzyme family in different samples: red indicates that the carbohydrate enzyme family has a high abundance in the sample (>0); blue indicates a low abundance (<0); and yellow indicates that the abundance is close to neutral (0), that is, the carbohydrate enzyme has a moderate abundance in the sample. Figure 16The abundance of CAZymes in different samples (CKT group and F18 group) is shown. The heatmap visually shows the expression levels of different CAZymes in each sample through color changes. The abundance of carbohydrate enzymes in the CKT group (column labels CKT1, CKT2, CKT3) shows higher levels in the GT87 and GT39 families. While in the GT5 and GH95 carbohydrate enzyme families, it shows lower abundance. In contrast, the F18 group (F181, F182, F183) shows higher abundance in the GT5 and GH95 families. While in GT87 and GT39, it is lower. We can clearly see the difference in the abundance of CAZymes families between the CKT group and the F18 group. Specifically, the carbohydrate enzyme families in different sample groups have different expression levels, which reflects the difference in the potential of microbial community carbohydrate metabolism in soil after adding F18.

[0098] Microorganisms are key players in biogeochemical cycles, and they promote the transformation and cycling of elements such as carbon, nitrogen, phosphorus, and sulfur through metabolic activities. In this study, we analyzed the relative abundance and inter-group differences of functional genes related to carbon, nitrogen, phosphorus, and sulfur cycles to evaluate the impact of adding F18 on the biogeochemical cycling functions of soil microorganisms. Based on metagenomeSeq heat maps and Anosim analysis, we studied the inter-group differences in biogeochemical cycling functions (see Figure 17 -Appendix Figure 21 ). In the Appendix Figure 17 , the left and top tree diagrams of the metagenomeSeq heat map (a, b, c, d represent carbon cycle, nitrogen cycle, phosphorus cycle, and sulfur cycle, respectively) show the clustering relationship of different samples and genes. The clustering results show the similarity between samples and genes in metabolic pathways. The vertical axis shows different genes related to the carbon cycle. The horizontal axis shows the gene expression data of different samples (CKT1, CKT2, CKT3, F181, F182, F183). From red (high expression) to blue (low expression) represents the expression amount of the gene. In the Appendix Figure 17 , the CKT group shows higher expression in genes such as ldh and acs, while the F18 group shows lower expression in these genes. In the Appendix Figure 18 , the F18 group shows higher expression in some nitrogen cycle genes such as nrfH, nrfA, and norC, while the CKT group shows relatively lower expression in these genes. In the Appendix Figure 19 , the F18 group shows higher expression in multiple genes, especially in genes such as phoB and spoT. In the Appendix Figure 20The CKT group showed higher expression in multiple sulfur metabolism genes such as soxY and soxZ, while the F18 group showed relatively lower expression. The gene expression differences between the CKT group and the F18 group in carbon, nitrogen, phosphorus, and sulfur metabolism pathways can be seen through the heat map. The higher expression of some genes in specific metabolic pathways may indicate that the microorganisms involved in the relevant metabolic pathways dominate in the corresponding samples, thereby affecting the transformation path and rate of elements.

[0099] Appendix Figure 21 Anosim analysis (a, b, c, d represent carbon cycle, nitrogen cycle, phosphorus cycle, and sulfur cycle, respectively) was performed to statistically analyze the differences in microbial communities in different element cycles. R represents the degree of difference between sample groups. The higher the R value, the greater the difference between groups. An R value greater than 0 indicates that the difference between groups is significant. In the Appendix Figure 21 In the carbon cycle of a, R = 0.556, P = 0.1. The R value is relatively high (close to 1), indicating that there is a large difference between the CKT and F18 groups. In the Appendix Figure 21 In b, R = 0.556, P = 0.2. Compared to the carbon cycle, the R value of the nitrogen cycle is lower, but there is still a difference between the groups. In the Appendix Figure 21 In c, R = 0.63, P = 0.1. Similar to the carbon cycle, there is a large difference between different sample groups in the phosphorus cycle. In the Appendix Figure 21 In the sulfur cycle of d, there is also a large difference between the two groups, R = 0.333, P = 0.1. This indicates that there are differences in microbial communities between different biogeochemical cycles in different samples, especially in carbon, phosphorus, and sulfur cycles.

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

[0101] Example 4

[0102] Application of F18 bacterial fertilizer to promote the growth of Chinese cabbage

[0103] In this experiment, the poor soil in Yinjie Town, Midu County, Dali Prefecture (25°15'26"N, 100°31'52"E) and the Brassica oleracea "Cold Ice No. 1" were used as research objects for a pot experiment in a greenhouse to explore whether F18 strains have a growth-promoting effect on Chinese cabbage. The growth of the control group (pure soil) and the experimental group (pure soil + F18 bacterial fertilizer) was observed, and the agronomic traits were measured and analyzed.

[0104] The preparation method of F18 bacterial fertilizer is the same as that in Example 3. The fermented bacterial bag is mixed with the soil, and 5% bacterial fertilizer is added to each pot, that is, 200g of bacterial fertilizer is added to 4kg of soil. The fertilizer is divided into flower pots, and 5 replicates are repeated in each group. The control group is 4kg of pure soil. After the bacteria have repaired the soil for 15 days, the cabbage is inoculated into the flower pot, with 10 cabbages per pot. The growth of the cabbage is monitored, and the agronomic traits are measured by thinning the seedlings to 5 during the period. The agronomic traits of the cabbage, such as plant height, root length, leaf width, number of leaves, fresh weight above ground, and dry weight above ground, are analyzed using SPASS software.

[0105] Attachment Figure 22 These photos show the growth of Chinese cabbage taken 19 and 40 days after sowing. It can be seen that at 19 days, there was little difference in growth between the CKT and F18 varieties. At 40 days, the cabbage grown with the F18 fertilizer showed significantly better growth than the CKT variety.

[0106] Attachment Figure 23 This data analysis uses SPASS software to analyze cabbage biomass measurements (a plant height, b root length, c leaf width, d number of leaves, e aboveground fresh weight, f aboveground dry weight). The results show that cabbage biomass (plant height, root length, leaf width, number of leaves, aboveground fresh weight, and aboveground dry weight) significantly increased (p < 0.01) in soils containing 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. This suggests that the addition of F18 fertilizer to soil promotes cabbage growth to a certain extent.

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

[0108] The above description of the disclosed embodiments is intended to enable one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to one 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 present invention. Therefore, the present invention is not limited to the embodiments shown herein but is intended to conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A strain of Aspergillus sp. DLF18, characterized in that: The deposit number of DLF18 is CGMCC NO.41737.

2. A microbial agent, characterized in that: The invention comprises the Aspergillus according to claim 1.

3. Use of the Aspergillus according to claim 1 or the microbial agent according to claim 2 in the production of humic acid.

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

5. The use according to claim 3, characterized in that The Aspergillus or the microbial agent uses rice straw and / or sugarcane bagasse as raw materials.

6. Use of the Aspergillus according to claim 1 or the microbial agent according to claim 2 in improving soil microbial abundance, or inter-group difference analysis of species composition, or functional level analysis, or inter-group difference analysis of functional abundance.

7. The use according to claim 6, 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 in the carbon metabolism pathway, nitrogen metabolism pathway, phosphorus metabolism pathway, and sulfur metabolism pathway.

8. The use according to claim 6, characterized in that The rice straw and / or sugarcane bagasse is fermented by using the Aspergillus or the microbial agent, and the obtained fermentation product is applied to the soil as a fertilizer.

9. Use of the Aspergillus according to claim 1 or the microbial agent according to claim 2 in promoting plant growth.

10. The use according to claim 9, characterized in that The Aspergillus or the microbial agent is used to ferment rice straw and / or sugarcane bagasse, and the obtained fermentation product is applied to the soil as a fertilizer.

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