Mucor racemosus strain dls157 and use thereof

Abstract

The application discloses a strain of total branch Mucor DLS157 and an application thereof, and belongs to the technical field of microorganisms. The preservation number of the strain is CGMCC No.41738. The strain has a strong ability of producing humic acid by fermenting lignocellulose. The DLS157 bacterial fertilizer is prepared by fermenting rice straw powder and wheat bran with the strain. After the DLS157 bacterial fertilizer is added to soil and wheat and Chinese cabbage are sowed, the agronomic characters such as plant height, leaf length, leaf width and leaf number of the wheat are significantly promoted, and the biomass such as plant height, leaf width, leaf number, aboveground fresh weight and aboveground dry weight of the Chinese cabbage is significantly improved. Through the research on the soil after the sowing of the wheat is completed, it is found that the addition of the DLS157 bacterial fertilizer significantly changes the soil microbial community, increases the diversity and abundance of the soil microbial community, and improves the content of humus in the soil. It is further indicated that the DLS157 bacterial fertilizer is beneficial to the regulation of the structure of the soil microbial community, and can be used for soil improvement. The application has important significance for realizing the resource utilization of agricultural straw.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and more specifically to a strain of Mucor racemose DLS157 and its application in soil improvement and crop growth promotion. Background Technology

[0002] Humus is a complex, amorphous, high-molecular-weight colloidal compound formed by the long-term decomposition and transformation of dead plant and animal remains by microorganisms and by geophysical and chemical processes in the presence of moisture and air. In my country, humic acids are classified into three categories based on their solubility in solvents and their color: fulvic acid, humin, and humic acid. Fulvic acid is soluble in acidic, alkaline, and neutral media, promoting the decomposition of minerals and the release of nutrients in the soil, and plays an important role in the accumulation and regeneration of humic acid. Humin carries a negative charge and is a hydrophilic colloid, capable of absorbing or displacing potassium, sodium, and ammonium ions in the soil, making soil particles act as small reservoirs for water and fertilizer retention, increasing soil porosity, and improving soil fertility. Humic acid is the most active component of humic substances, with a high cation exchange capacity, which can improve the soil's water and fertilizer retention capacity and adsorption capacity, contributing to the formation of a well-structured soil structure. Therefore, humic acid can improve soil, activate insoluble mineral elements, promote the absorption of micronutrients by plants, enhance the slow release of nutrients, and stimulate plant development and enhance plant resistance.

[0003] While the theory that humic acid originates from plants is widely accepted, the exact components of plants that make up humic acid remain a point of contention. There are four main theories regarding the origin of humic acid: the lignin-protein theory, polyphenol self-condensation, the polyphenol-protein pathway, and the Maillard reaction. The specific role of microorganisms in humic acid formation is also debated, with four main hypotheses: plant transformation hypothesis, biochemical hypothesis, cell autolysis, and microbial synthesis. It is currently 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 in humic acid formation, not only decomposing large polymer molecules but also acting as producers, re-condensing 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 lignocellulosic waste into humic acid by controlling key factors.

[0004] Lignocellulose is a relatively abundant component in agricultural waste, mainly composed of a three-dimensional network structure of cellulose, hemicellulose, and lignin. As the most abundant renewable biological resource in the biological world, lignocellulose is produced globally at a rate of 150 billion tons annually, of which straw accounts for 6 billion tons. As a major agricultural country, China produces 1.113 billion tons of straw annually, accounting for approximately one-fifth of the global straw resource.

[0005] Therefore, screening strains that efficiently produce humic acid from agricultural straw is crucial for the resource utilization of agricultural straw. This is of great significance for agricultural treatment, soil remediation, and the microbial fermentation production of humic acid. Summary of the Invention

[0006] To address the aforementioned problems, this invention provides a strain of Mucor racemosa DLS157 and its application in soil improvement and crop growth promotion.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] A strain of *Mucor racemosus* DLS157, classified as *Mucor racemosus*, was deposited on December 30, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 41738, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0009] Another object of the present invention is to provide the application of Mucor racemosa DLS157 in the preparation of high-value humic acid products.

[0010] Another object of the present invention is to provide a high-value humic acid product comprising a culture or processed product of the aforementioned Mucor racemose DLS157.

[0011] Another object of the present invention is to provide a microbial fertilizer comprising the above-mentioned Mucor racemose DLS157 culture or a processed product thereof.

[0012] Preferably, the preparation method of the microbial fertilizer is as follows: rice straw powder and wheat bran are mixed at a mass ratio of 7:3, water is added and stirred evenly to achieve a moist but not waterlogged state, and the mixture is divided into microbial bags, sealed and sterilized to obtain a fermentation substrate. The seed liquid of strain DLS157 is inoculated into the above fermentation substrate at a ratio of 1mL:30g, and cultured at a constant temperature of 25℃ until the mycelium covers the microbial bag.

[0013] Preferably, the bacterial concentration of the DLS157 seed culture is 8.3 × 10⁻⁶. 5 CFU / mL.

[0014] Another object of the present invention is to provide the application of Mucor racemosa DLS157 in crop growth promotion.

[0015] Preferably, the crops are wheat and cabbage.

[0016] Preferably, when the crop is wheat, it has a promoting effect on plant height, leaf length, leaf width and number of leaves.

[0017] Preferably, when the crop is Chinese cabbage, it has a promoting effect on plant height, leaf width, number of leaves, above-ground fresh weight and above-ground dry weight.

[0018] Another object of the present invention is to provide the application of Mucor racemosa DLS157 in soil improvement.

[0019] Specifically, the improvements include the following: (1) increasing the content of humus in the soil; (2) increasing the diversity and abundance of microbial colonies in the soil; (3) regulating the structure of microbial colonies in the soil; and (4) promoting the transformation and cycling of carbon, phosphorus, and sulfur elements in the soil.

[0020] As can be seen from the above technical solution, compared with the prior art, this invention discloses a new strain of *Mucor* DLS157 that produces high-value humic acid from straw. This strain has a strong ability to ferment lignocellulose to produce humic acid. DLS157 microbial fertilizer is prepared by fermenting rice straw powder and wheat bran with this strain. Adding DLS157 microbial fertilizer to the soil before sowing wheat and cabbage not only significantly promotes agronomic traits such as plant height, leaf length, leaf width, and number of leaves in wheat, but also significantly increases biomass such as plant height, leaf width, number of leaves, above-ground fresh weight, and above-ground dry weight in cabbage.

[0021] Studies of soil after wheat sowing revealed that the addition of DLS157 microbial fertilizer significantly altered the soil microbial community, increasing its diversity and abundance, and raising the humus content. Significant functional changes were also observed, primarily in the abundance differences within the carbohydrate-active enzyme family (CAZymes) and the relative abundance and intergroup differences of genes involved in biogeochemical cycling. This indicates alterations in the structure and abundance of the microbial community involved in related metabolic pathways, thereby affecting metabolic transformation pathways and rates. This further demonstrates that DLS157 microbial fertilizer is beneficial for regulating soil microbial community structure and can be used for soil improvement.

[0022] In summary, this invention utilizes the ability of strain DLS157 to efficiently produce humic acid from agricultural straw, and prepares it into high-value humic acid products (such as microbial fertilizers). This not only promotes crop growth but also improves soil, which is of great significance for realizing the resource utilization of agricultural straw. Attached Figure Description

[0023] Figure 1Comparison of humic acid production by strain DLS157 under different carbon sources;

[0024] Figure 2 Morphological identification of strain DLS157: a: Colony morphology characteristics; b: Conidial morphology characteristics.

[0025] Figure 3 Phylogenetic tree of strain DLS157 constructed based on ITS rRNA sequence;

[0026] Figure 4 : Growth of strain DLS157 on rice straw carbon source liquid medium at different temperatures, where a: cultured at 4℃, b: cultured at 25℃, c: cultured at 30℃, d: cultured at 37℃, e: cultured at 45℃; +: indicates the growth of the strain, the more the better the growth, / : the strain does not grow, 1: control group without inoculation, 2: experimental group inoculated with strain DLS157;

[0027] Figure 5 Comparison of humic acid content in fermentation broth after different treatments: CK: uninoculated rice straw carbon source liquid basal medium, S157: rice straw basal salt medium inoculated with strain DLS157. Different capital letters indicate extremely significant differences between groups (p<0.01).

[0028] Figure 6 Comparison of wheat growth on different soils 12 and 56 days after sowing. CKT: pure soil, S157: pure soil + DLS157 microbial fertilizer.

[0029] Figure 7 The results of growth traits of wheat on different soils 75 days after sowing were measured. Among them, I: plant height, II: leaf length, III: leaf width, IV: number of leaves, CKT: pure soil, S157: pure soil + DLS157 microbial fertilizer. Different lowercase letters indicate significant differences between groups (p<0.05).

[0030] Figure 8 Comparison of Chinese cabbage growth in different soils 19 and 40 days after sowing. CKT: pure soil, S157: pure soil + DLS157 microbial fertilizer.

[0031] Figure 9 Biomass of Chinese cabbage in different soils 43 days after sowing: I: plant height, II: root length, III: leaf width, IV: number of leaves, V: aboveground fresh weight, VI: aboveground dry weight, CKT: pure soil, S157: pure soil + DLS157 microbial fertilizer. Different lowercase letters indicate significant differences between groups (p<0.05), and different uppercase letters indicate extremely significant differences between groups (p<0.01).

[0032] Figure 10The growth period of soil samples diluted to 10⁻⁵ after wheat sowing on PDA medium: a: ​​soil without DLS157 microbial fertilizer, b: soil with DLS157 microbial fertilizer.

[0033] Figure 11 Comparison of humic component content in different soils after wheat sowing, where I: humic content, II: humic acid content, III: fulvic acid content, IV: humin content, CKT: soil without DLS157 microbial fertilizer, S157: soil with DLS157 microbial fertilizer. Different lowercase letters indicate significant differences between groups (p<0.05), and different uppercase letters indicate extremely significant differences between groups (p<0.01).

[0034] Figure 12 : 1% gel electrophoresis images of soil samples from different groups after wheat sowing. M: 15000bp DNA Marker. 1-3 are soil samples without DLS157 microbial fertilizer, specifically, 1: CKT-1 soil sample, 2: CKT-2 soil sample, 3: CKT-3 soil sample; 4-6 are soil samples with DLS157 microbial fertilizer, specifically, 4: S157-1 soil sample, 5: S157-2 soil sample, 6: S157-3 soil sample.

[0035] Figure 13: Composition analysis of different microorganisms by phylum, genus, and species in different groups of soil samples after wheat sowing. Figure 13A Bar chart showing species distribution at the phylum level for each group of soil samples. Figure 13B Bar chart showing species distribution at the genus level for each group of soil samples. Figure 13C : Bar chart of species distribution at the species level for each group of soil samples;

[0036] Figure 14 PCoA analysis diagrams of different microorganisms at the phylum, genus, and species levels in different groups of soil samples after wheat sowing, where a: species structure differences at the phylum level, b: species structure differences at the genus level, and c: species structure differences at the species level.

[0037] Figure 15 Heatmap of species with significant differences at the phylum level in soil samples from different groups after wheat sowing;

[0038] Figure 16 Abundance heatmap of carbohydrate-active enzymes (CAZymes) in different groups of soil samples after wheat sowing;

[0039] Figure 17: Metagenome-Seq heatmaps of carbon, nitrogen, phosphorus, and sulfur cycles in different groups of soil samples after wheat sowing.

[0040] in, Figure 17A Carbon cycle. Captions: 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;

[0041] 33 represents hisidinol-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 FormII; 45 represents aclA; 46 represents... Representative: aclB; 47: cdhE; 48: cooS; 49: acdA; 50: ack; 51: acs; 52: adh; 53: ldh; 54: pflD; 55: porA; 56: pta; 57: pmoA; 58: pmoB; 59: pmoC; Figure 17B Nitrogen cycle Figure 17C Phosphorus cycle Figure 17D Sulfur cycle;

[0042] In Figures 13-17, CKT-1, CKT-2, and CKT-3 are soils without DLS157 microbial fertilizer, while S157-1, S157-2, and S157-3 are soils with DLS157 microbial fertilizer.

[0043] Figure 18 Anosim analysis of functional gene abundance in different groups of soil samples after wheat sowing, where A: carbon cycle, B: nitrogen cycle, C: phosphorus cycle, D: sulfur cycle; CKT: soil without DLS157 microbial fertilizer, and S157: soil with DLS157 microbial fertilizer. Detailed Implementation

[0044] The technical solutions in the embodiments of the present invention will be clearly and completely described below. 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.

[0045] All reagents used in the embodiments of this invention were purchased from commercial channels. Methods not mentioned are conventional experimental methods and will not be described in detail here.

[0046] The following reagents are provided as examples:

[0047] LB liquid medium: trypsin 10g / L, yeast extract 5g / L, NaCl 10g / L, pH natural.

[0048] LB liquid medium containing 0.05 mg / mL kanamycin: kanamycin 0.05 mg / mL, trypsin 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, pH natural.

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

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

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

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

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

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

[0055] Solid culture medium is the corresponding liquid culture medium with 20 g / L agar added.

[0056] PDA medium: 200 g / L potato, 20 g / L glucose, 20 g / L agar, natural pH.

[0057] Mix (Product No.: TSE004, Specification: 5×1mL, Beijing Qingke Biotechnology Co., Ltd.).

[0058] Example 1: Isolation, Screening and Identification of Strains

[0059] 1. Isolation and screening of strains

[0060] (1) Isolation of strains

[0061] This strain was derived from soil from Canglang Peak, Cangshan Mountain, Dali (25°50'28.90"N, 100°03'7.60"E). The specific culture method was as follows: 10g of soil sample was added to 10mL of sterile water as the stock solution. 1mL of each stock solution was then added to sterile water to dilute the concentration to 10 times the stock solution concentration. 3 10 4 10 5 The bacteria were spread onto basal culture media containing lignocellulose (such as rice straw, corn straw, wheat straw, sugarcane bagasse, pine wood, and poplar wood) as the sole carbon source and incubated at 20°C. After bacterial growth, single colonies exhibiting fluffy or flocculent appearance were picked from the plates for transfer and purification.

[0062] (2) Screening of strains

[0063] The selected and purified strains were selected, and single colonies were inoculated into 10 mL of LB liquid medium containing 0.05 mg / mL kanamycin. The cultures were shaken at 20℃ and 180 rpm for 2 days to obtain seed culture. The seed culture was then inoculated into 100 mL of rice straw carbon source liquid basal medium at a 2% inoculation rate. After shaking culture at 20℃ and 180 rpm for 7 days, 0.2 mol / L sodium pyrophosphate solution was added to the fermentation broth to bring the pH to 12. After standing at room temperature for 24 hours, the supernatant was collected and scanned in the range of 200 nm-320 nm using a UV-Vis spectrophotometer. Each group was repeated 3 times. Strains with absorbance values ​​>0 were selected to have the ability to produce humic acid. Based on the absorbance values, 8 humic acid-producing strains were initially screened.

[0064] The eight strains obtained from the initial screening were selected, and single colonies were inoculated into 10 mL of LB liquid medium containing 0.05 mg / mL kanamycin. The cultures were incubated at 20℃ and 180 rpm for 2 days with shaking to obtain seed culture. The seed culture of each strain was then inoculated at a 2% inoculation rate into equal volumes of liquid culture medium with different lignocellulose as the sole carbon source (rice straw, corn stalk, wheat straw, sugarcane bagasse, pine wood, and poplar wood). Fermentation was carried out at 20℃ and 180 rpm for 7 days, and the yield of humic acid in the fermentation broth was preliminarily determined. Then, 0.2 mol / L sodium pyrophosphate alkaline extract was added to the fermentation broth to adjust the pH to 12, and the mixture was allowed to stand at room temperature for 24 hours. The pH was then adjusted to 1-2 with hydrochloric acid and allowed to stand overnight. After centrifugation, the supernatant was discarded, and the precipitate was dried. The precipitate obtained was crude humic acid. The strain with the strongest humic acid production capacity was selected and named DLS157. Figure 1 The comparison of humic acid production of strain DLS157 under different carbon sources shows that strain DLS157 can produce humic acid when rice straw and sugarcane bagasse are the only carbon sources, with rice straw producing the highest yield. In addition, strain DLS157 can also grow in corn stalks and wheat stalks, but the humic acid production is almost zero.

[0065] 2. Identification of strains

[0066] (1) Morphological characteristics identification

[0067] Strain strain DLS157 was inoculated onto PDA medium and cultured at 25°C for 5 days. Colony size, color, and other characteristics were then observed and recorded. Microscopic images of the strain were taken using a Nikon ECLIPSENi-U biological microscope to record its morphological characteristics. Results are shown below. Figure 2 The hyphae are multinucleate and septate, with long, slender hyphae, smooth surfaces, and highly branched structures. They are white and form a dense layer of hyphae on the substrate. Figure 2 (as shown in a); conidia are spherical or elliptical, varying in size ( Figure 2(As shown in b). It exhibits typical morphological characteristics of Mucor fungi.

[0068] (2) Molecular biological identification

[0069] DNA was extracted from strain DLS157. Using universal primers ITS4 and ITS5 (both purchased from Sangon Biotech (Shanghai) Co., Ltd.) for fungal ITS identification, PCR amplification was performed using Taq polymerase. The PCR amplification reaction system (40 μL) consisted of: 1 μL each of forward and reverse primers, 1 μL of DNA template, and 37 μL of Mix. The PCR amplification program was as follows: 94℃ pre-denaturation for 4 min; 94℃ denaturation for 30 s, 55℃ annealing for 35 s, 72℃ extension for 90 s, for 32 cycles; and 72℃ extension for 5 min.

[0070] After agarose gel electrophoresis, the amplified products were sent to Sangon Biotech Co., Ltd. for sequencing. The obtained sequences were submitted to GenBank (http: / / www.ncbi.nlm.nih.gov). Similarity comparisons were performed between the BLAST database and ITS sequences. A phylogenetic tree of the ITS rRNA sequences was constructed using the Neighbor-Joining Method in MEGA 7.0 (e.g., [example missing]). Figure 3 Phylogenetic analysis showed that strain DLS157 clustered with *Mucor racemosus* in one clade, with a support rate of 85%. Based on colony morphology, microscopic photographs, and a phylogenetic tree constructed using ITS rRNA sequence alignment, strain DLS157 was identified as *Mucor racemosus*.

[0071] Example 2: Determination of humic acid production performance of the strain

[0072] 1. Effects of different temperatures on the growth of bacterial strains

[0073] Take the seed culture of strain DLS157 after expansion (bacterial concentration of 8.3 × 10⁻⁶). 5 CFU / mL was inoculated at a rate of 2% into rice straw carbon source liquid basal medium and cultured at different temperatures (4℃, 25℃, 30℃, 37℃, 45℃) for 7 days, designated as the experimental group. Uninoculated rice straw carbon source liquid basal medium served as the control group. Three replicates were set up for each temperature to detect the effect of different temperatures on the growth of the strain. Results are shown below. Figure 4 .

[0074] Depend on Figure 4It can be seen that strain DLS157 grows best at 25℃ (b), with dense and slender hyphae. Growth declines progressively at 30℃ (c) and 37℃ (d). Furthermore, the strain ceases growth at 4℃ and 45℃. DLS157 can grow within the temperature range of 25℃-37℃, indicating that this fungus is a low-temperature fungus.

[0075] 2. Assessment of the strain's ability to produce humic acid

[0076] Take the seed culture of strain DLS157 after expansion (bacterial concentration of 8.3 × 10⁻⁶). 5 The bacterial strain (CFU / mL) was inoculated at a rate of 2% into 100 mL of rice straw carbon source liquid basal medium. The control was uninoculated medium, and three biological replicates were performed. After culturing at 20℃ and 180 rpm with shaking for 7 days, 10 mL of the fermentation supernatant and liquid medium were collected and sent to Wuhan Punais Testing Technology Co., Ltd. for humic acid content determination (NY / T 1971-2010 Determination of Humic Acid Content in Water-Soluble Fertilizers). The results are shown below. Figure 5 .

[0077] Depend on Figure 5 The humic acid content of the fermentation supernatant of strain DLS157 was compared with that of the control group CK (uninoculated rice straw carbon source liquid basal medium). The results showed that the humic acid content per gram of fermentation broth of strain DLS157 was 14.03%, while that in the control group (CK) was 9.59%. There was a highly significant difference (p<0.01) between strain DLS157 and the control group (CK), with the humic acid content in the fermentation supernatant of strain DLS157 increasing by 46.30%. This further demonstrates that strain DLS157 has a strong humic acid production capacity.

[0078] Example 3: Determination of the growth-promoting properties of the strain on crops.

[0079] Rice straw powder and wheat bran were mixed at a mass ratio of 7:3. Water was added and stirred until the mixture was moist but not waterlogged. The mixture was then divided into 1.5 kg bags, sealed tightly with rings, and sterilized at 121°C for 120 min to obtain the fermentation substrate for later use. Strain DLS157 was inoculated into 50 mL of LB liquid medium containing 0.05 mg / mL kanamycin and cultured at 20°C and 180 rpm for 2 days with shaking to obtain the seed culture. The bacterial concentration was measured to be 8.3 × 10⁻⁶. 5 CFU / mL. Each sterilized culture bag was inoculated with 50 mL of DLS157 seed culture (8.3 × 10⁵ CFU / mL), and cultured in a 25℃ incubator until the mycelium completely covered the bag, yielding DLS157 microbial fertilizer. This fertilizer was used for subsequent growth-promoting experiments.

[0080] 1. Promotes the growth of grain crops

[0081] A pot experiment was conducted using barren soil in the suburbs of Chenggong District, Kunming City, and the gramineous wheat variety "Shannong 42" (approval number: 20210097) as the research object to further explore whether strain DLS157 has a growth-promoting effect on wheat.

[0082] The DLS157 microbial fertilizer was mixed with infertile soil from the suburbs of Chenggong District, Kunming City, at a ratio of 125g of microbial bag per kilogram of soil. The mixture was then divided into equal weight portions and placed into flowerpots (400g / pot), with 6 replicates per group. The experimental group was defined as the group where the microbial cells repaired the soil for 15 days. An equal amount of pure soil was used as the control group (CKT). Germinated wheat was inoculated into flowerpots of different treatments, with 3 pots in each group and 10 plants per pot. The growth of wheat in the control group (pure soil) and the experimental group (pure soil + DLS157 microbial fertilizer) was monitored. The growth comparison of wheat at 12 days and 56 days after sowing is shown in the figure. Figure 6 .Depend on Figure 6 It can be seen that the wheat growth after the addition of DLS157 microbial fertilizer was significantly better than that of the control group in pure soil.

[0083] The agronomic traits of wheat, including plant height, leaf length, leaf width, and leaf number, were measured 75 days after sowing using SPASS software. The results are shown below. Figure 7 .Depend on Figure 7 The results showed that, compared to the control group (CKT), the addition of DLS157 significantly improved plant height, leaf length, leaf width, and number of leaves. This further demonstrates that the humic acid fertilizer produced by strain DLS157 from agricultural straw can promote wheat growth.

[0084] 2. Promotes the growth of vegetable crops

[0085] A pot experiment was conducted in a greenhouse using barren soil in Yinjie Town, Midu County, Dali Prefecture (25°15 '26"N, 100°31 '52"E) and the "Hantian No. 1" Chinese cabbage of the Brassicaceae family as the research object to investigate whether the strain DLS157 has a growth-promoting effect on the growth of Chinese cabbage.

[0086] 4 kg of barren soil from Yinjie Town, Midu County, Dali Prefecture was mixed with 200 g of DLS157 microbial fertilizer at a ratio of 5%. The mixture was then divided into 5 equal-weight portions and stored in 5 flowerpots. The experimental group was established 15 days after the microbial cells repaired the soil. The control group consisted of 4 kg of pure soil. Ten cabbages were inoculated into each pot. The growth of the cabbages in the control group (pure soil, denoted as CKT) and the experimental group (pure soil + DLS157 microbial fertilizer, denoted as S157) was monitored. During this period, seedlings were thinned to 5 plants each to measure agronomic traits. The growth comparison of cabbages at 19 and 40 days after sowing is shown in the figure. Figure 8 .Depend on Figure 8It can be seen that at 19 days, there was almost no difference in the growth of cabbage between the control group (pure soil) and the experimental group (pure soil + DLS157 microbial fertilizer). At 40 days, the growth of cabbage with added DLS157 microbial fertilizer was significantly better than that of the control group without added DLS157 microbial fertilizer.

[0087] The agronomic traits of Chinese cabbage, including plant height, root length, leaf width, leaf number, above-ground fresh weight, and above-ground dry weight, were measured and analyzed using SPASS software 43 days after sowing. The results are shown below. Figure 9 .Depend on Figure 9 The results showed that in soil treated with DLS157 microbial fertilizer, the biomass of Chinese cabbage, including plant height, leaf width, number of leaves, above-ground fresh weight, and above-ground dry weight, was significantly increased. Figure 9-I As can be seen from 9-Ⅲ, 9-Ⅴ, and 9-Ⅵ, there were extremely significant differences in plant height, leaf width, above-ground fresh weight, and above-ground dry weight between the experimental group and the control group (p<0.01). Compared with the control group, plant height, leaf width, above-ground fresh weight, and above-ground dry weight increased by an average of 1.62cm, 1.64cm, 0.746g, and 0.0512g, respectively. Figure 9-Ⅳ It can be seen that there was a significant difference in the number of leaves between the experimental group and the control group (p<0.05), with the experimental group having an average increase of 1.2 leaves compared to the control group. However, there was no significant difference in the root length of the cabbage between the two groups.

[0088] In summary, this demonstrates that adding DLS157 microbial fertilizer to the soil can promote the growth of cabbage to a certain extent.

[0089] Example 4: Determination of the effects of the strain on soil

[0090] 1. Dilution Coating Experiment

[0091] To investigate whether there are differences in the microbial community in soil with added humic acid, a dilution-spreading experiment was conducted on the soil from Example 3 that had been planted with wheat. Specifically, 10g of soil from two groups of potted plants from Example 3 that had been planted with wheat was weighed and dissolved in 10mL of sterile water as a stock solution. 1mL of the stock solution was then diluted with sterile water to a concentration of 10⁻⁵ and spread onto PDA medium. Each group was repeated three times. After incubation at 25℃ for 3 days, the changes in the number of colonies on the plates were observed. The results are shown below. Figure 10 .

[0092] Depend on Figure 10It can be seen that the plates in group b (soil treated with DLS157 microbial fertilizer) were covered with microbial colonies (>500 colonies / plate), while the plates in group a (soil not treated with DLS157 microbial fertilizer) had fewer microbial colonies (<10 colonies / plate). The colony count in the soil treated with DLS157 microbial fertilizer was significantly higher than that in the soil not treated with DLS157 microbial fertilizer, indicating that the addition of DLS157 microbial fertilizer can, to some extent, increase the quantity and species richness of culturable microbial communities in the soil.

[0093] 2. Determination of soil humus content

[0094] Soil from two groups of flowerpots where wheat had been planted in Example 3 was collected. 10g of soil was taken from each pot (control group: CKT-1, CKT-2, CKT-3; experimental group: S157-1, S157-2, S157-3). The soil samples were sent to Wuhan Punes Testing Company using dry ice to determine the content and changes in humic substances. The testing principle is as follows: 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 humic substances are classified into soluble humic substances (humic acid and fulvic acid) and insoluble humic substances (humin) according to their solubility. Soluble humic substances were extracted using a 0.1mol / L sodium pyrophosphate-sodium hydroxide mixture. The total amount of humic acid and fulvic acid was determined using the potassium dichromate oxidation capacity method. The extract was acidified to precipitate and separate humic acid, and its content was determined. The fulvic acid content was then calculated. The total carbon content of the soil sample was determined, and the humic acid and fulvic acid contents were subtracted to obtain the humin content. Specific test results are shown in Table 1 and... Figure 11 .

[0095]

[0096] From Table 1 and Figure 11 The results showed that the humic content (including humic acid, fulvic acid, and humin) was significantly increased in soils treated with DLS157. Figure 11-I As shown in Figure 11-Ⅳ, the humic and humin contents in the experimental group were significantly different from those in the control group (p<0.01). Compared with the control group in soil without DLS157, the humic and humin contents increased by 3.99 g / kg and 2.75 g / kg, respectively. Figure 11-II As can be seen from 11-III, the humic acid and fulvic acid contents in the experimental group were significantly different from those in the control group (p<0.05), and the humic acid and fulvic acid contents in the experimental group increased by 0.395 g / kg and 0.843 g / kg, respectively, compared with the control group.

[0097] 3. Soil metagenomic sequencing analysis

[0098] (1) Agarose gel electrophoresis analysis

[0099] Soil samples from two groups of flowerpots where wheat had been grown, as described in Example 3, were extracted using the MP (MP Biomedicals Soil DNA Isolation Kit). Three biological replicates were used for each group, with 0.3 μL of soil sample taken from each replicate (control group: CKT-1, CKT-2, CKT-3; experimental group: S157-1, S157-2, S157-3). The extracted metagenomic DNA was mixed with 6X DNA Loading Buffer (5:1), and 5 μL was subjected to 1% agarose gel electrophoresis at 120V for 30 min. The results are shown in the figure. Figure 12 And Table 2.

[0100]

[0101] Figure 12 The differences in metagenomic DNA brightness and total amount can also be seen in Table 2. Compared with the control group without DLS157 microbial fertilizer, the concentration and total amount of DLS157 microbial fertilizer in the experimental group were significantly increased, indicating that the types and numbers of microorganisms in the soil increased to some extent after adding DLS157 microbial fertilizer.

[0102] (2) Species composition analysis

[0103] After passing the above gel electrophoresis test, samples from each group were stored on dry ice and sent to Wuhan Punes Testing Co., Ltd. for metagenomic sequencing. Specifically, after the samples passed the test, sequence libraries were constructed and high-throughput sequencing was performed. High-quality sequences were assembled using assembly software, and gene prediction was performed. Using the CAZy database, the predicted genes were annotated and classified according to species and function, predicting gene function, classification, and metabolic pathways. Using R software, bar charts were created to depict the composition of 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 group of samples. The bar charts illustrate the species composition of each sample and the proportion of different species in each sample.

[0104] The species bar charts at the phylum, genus, and species levels for the control group (CKT-1, CKT-2, CKT-3) of soil without DLS157 microbial fertilizer and the experimental group (S157-1, S157-2, S157-3) of soil with DLS157 microbial fertilizer are shown below. Figures 13A-13CAs shown.

[0105] At the door level (see) Figure 13A As can be seen, the pink area contains the highest relative abundance of Proteobacteria, and the green area contains the highest abundance of Actinobacteria, exceeding 0.5%. Significant differences were observed between the experimental and control groups in certain microbial phyla, such as obvious changes in the abundance of Actinobacteria and Bacteroidetes.

[0106] At the genus level (see) Figure 13B It can be seen that, apart from unclassifiable or few species of microorganisms, *Sphingomonas* (pink region) and *Nocardioides* (green region) accounted for a large proportion of abundance in the CKT. In the experimental group, the relative abundance of *Sphingomonas* (pink region) and *Saccharothrix* (orange region) was higher. There were significant differences in the abundance of *Sphingomonas* and *Nocardioides* between the experimental and control groups.

[0107] At this level (see Figure 13C It can be seen that the proportion of unclassifiable microorganisms exceeded 0.5%. Furthermore, the abundance of *Acidobacteria bacterium* in the pink region and *Chloroflexi bacterium* in the green region was relatively high in the CKT. In the experimental group, the relative abundance of *Acidobacteria bacterium* in the pink region and *Saccharothrix sp.* in the green region was higher. Significant differences were observed between the experimental and control groups in certain species, such as obvious abundance changes in *Sphingomonas edaphi* and *Saccharothrix sp.*.

[0108] In summary, Figure 13, through a stacked bar chart, shows the relative abundance of different microbial phyla, genera, and species in the experimental groups (S157-1, S157-2, S157-3) of soil treated with DLS157 microbial fertilizer and the control groups (CKT-1, CKT-2, CKT-3) of soil not treated with DLS157 microbial fertilizer. This demonstrates the differences in the composition of the microbial community and reflects the impact of DLS157 microbial fertilizer on the microbial community in the soil.

[0109] (3) Dimensionality reduction analysis based on species abundance

[0110] PCoA analysis was performed on the sequenced samples using the unconstrained ordination (Classical Multidimensional Scaling, cMDScale) method to assess differences in microbial community composition and analyze species β-diversity. 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. Differences in species structure at the phylum, genus, and species levels were shown in the control group (CKT-1, CKT-2, CKT-3) of soil without DLS157 fertilizer and the experimental group (S157-1, S157-2, S157-3) of soil with DLS157 fertilizer. Figure 14 As shown in (a, b, c).

[0111] As can be seen, the control group (CKT, blue) and the experimental group (S157, dark blue) are clearly separated on the principal coordinate plot. The microbial community structure of the experimental group clusters together and is far from the structure of the control group. This indicates a significant structural difference between the microorganisms of the two groups. At the phylum level, the first two axes of the PCoA analysis explained 78.45% and 17.57% of the total variance at the phylum level, respectively (see [link to PCoA analysis]). Figure 14 a). At the genus level, the first two axes of the PCoA analysis explained 68.86% and 20.4% of the total variance in the data at the genus level, respectively (see [link to analysis]). Figure 14 (b) At the species level, the first two axes of the PCoA analysis explained 67.17% and 20.24% of the total variance in the data at the species level, respectively (see [link to analysis]). Figure 14 c). At the phylum level, the control group (CKT, blue) and the experimental group (S157, dark blue) already showed significant differences in overall community composition; at the genus and species level, these differences were further amplified, specifically manifested as differences in the distribution of individual genera or species. Therefore, with the addition of DLS157 microbial fertilizer, the soil microbial community structure underwent significant changes.

[0112] (4) Species abundance difference analysis

[0113] MetagenomeSeq analysis was used on the sequenced samples to assess species abundance differences between the control group (CKT-1, CKT-2, CKT-3) of soil without DLS157 fertilizer and the experimental group (S157-1, S157-2, S157-3) of soil with DLS157 fertilizer, based on a zero-expansion model. Heatmaps were used to visualize species with significant differences at the phylum level. Blue indicates low abundance (negative values), while red indicates high abundance (positive values). Yellow indicates abundance close to the median. The color contrast between samples provides a visual understanding of the differences in microbial community composition.

[0114] The results are as follows Figure 15 As shown, *Candidatus Vogelbacteria*, *Candidatus Woesearchaeota*, and *Candidatus Saccharibacteri* exhibited significantly high abundance in the experimental groups (S157-1, S157-2, S157-3), while their abundance was lower in the control groups (CKT-1, CKT-2, CKT-3). Conversely, *Armatimonadetes*, *Candidatus Odinarchaeota*, and *Acidobacteria* showed significantly high abundance in the control groups (CKT-1, CKT-2, CKT-3), while their abundance was lower in the experimental groups (S157-1, S157-2, S157-3). Figure 15 The study demonstrated the differences in microbial community composition between the control and experimental groups. Through row and column cluster analysis, significant abundance differences were identified between the groups at the phylum level, indicating that the addition of DLS157 microbial fertilizer significantly altered the richness and composition of the soil microbial community.

[0115] (5) Functional relative abundance analysis

[0116] Using the CAZy database, predicted genes were functionally annotated and categorized to evaluate the impact of DLS157 microbial fertilizer application on soil microbial functional abundance. Specifically, heatmaps were constructed based on the functional annotations and abundance information of the control group (CKT-1, CKT-2, CKT-3) of soil without DLS157 fertilizer and the experimental group (S157-1, S157-2, S157-3) of soil with DLS157 fertilizer, using data from the CAZy database. Clustering was performed at both the functional and sample levels. Rows in the heatmap represent different saccharide-active enzyme families, and colors indicate the different samples of each saccharide-active enzyme family.

[0117] The relative abundance in the data is as follows: red indicates a high abundance (>0) of the glycozyme family in the sample; blue indicates a low abundance (<0); and yellow indicates a near-neutral abundance (0), meaning the abundance of the glycozyme in the sample is moderate. The abundance of glycozymes (CAZymes) in different samples is shown below. Figure 16 As shown.

[0118] Figure 16 The heatmap shown visually illustrates the expression levels of different saccharide-active enzymes in each sample through color changes. For example, the control groups (CKT-1, CKT-2, CKT-3) exhibited higher abundance of saccharide enzymes in the GH77 and GT39 families, while showing lower abundance in the GH5 and GH37 saccharide enzyme families. In contrast, the experimental groups (S157-1, S157-2, S157-3) showed the opposite trend, with higher abundance in the GH5 and GH37 families, but lower abundance in the GH77 and GT39 families. The difference in abundance of saccharide-active enzyme families between the control and experimental groups is clearly visible in the figure. Specifically, the different expression levels of saccharide enzyme families in different sample groups reflect the differences in the saccharide metabolism potential of the microbial community after the addition of DLS157 microbial fertilizer to the soil.

[0119] (6) Biogeochemical cycle function analysis

[0120] The effects of adding DLS157 microbial fertilizer on soil microbial geochemical cycling function were evaluated by analyzing the relative abundance and inter-group differences of functional genes for carbon, nitrogen, phosphorus, and sulfur cycles. Specifically:

[0121] ① Based on the metagenomeSeq heatmap, the clustering relationships of samples and genes in the control group (CKT-1, CKT-2, CKT-3) of soil without DLS157 microbial fertilizer and the experimental group (S157-1, S157-2, S157-3) of soil with DLS157 microbial fertilizer were analyzed. The dendrograms on the left and top of the metagenomeSeq heatmap show the clustering relationships of different samples and genes. The clustering results demonstrate the similarity of metabolic pathways between samples and genes. The vertical axis shows different genes related to carbon cycling, and the horizontal axis shows gene expression data for different samples, with red (high expression) to blue (low expression) representing gene expression levels. The metagenomeSeq heatmaps of different groups of soil samples in terms of carbon, nitrogen, phosphorus, and sulfur cycling are shown below. Figures 17A-17D As shown.

[0122] exist Figure 17AIn the carbon cycle metagenomeSeq heatmap shown, the control group (numbered CKT-1, CKT-2, CKT-3) of soil without DLS157 microbial fertilizer showed high expression of genes such as bcrA and fdhA, while the experimental group (numbered S157-1, S157-2, S157-3) of soil with DLS157 microbial fertilizer showed low expression of these genes.

[0123] exist Figure 17B In the nitrogen cycle metagenomeSeq heatmap shown, the experimental groups (S157-1, S157-2, S157-3) of soil treated with DLS157 microbial fertilizer showed higher expression levels of nitrogen cycle genes such as napA, while the control groups (CKT-1, CKT-2, CKT-3) of soil not treated with DLS157 microbial fertilizer showed relatively lower expression of this gene.

[0124] exist Figure 17C In the metagenomeSeq heatmap of phosphorus cycling shown, the experimental groups (numbered S157-1, S157-2, and S157-3) of soils treated with DLS157 microbial fertilizer showed high expression of multiple genes, especially olpA and gcd.

[0125] exist Figure 17D In the sulfur cycle metagenomeSeq heatmap shown, the control group (numbered CKT-1, CKT-2, CKT-3) of soil without DLS157 microbial fertilizer showed high expression of multiple sulfur metabolism genes such as sat and sqr, while the experimental group (numbered S157-1, S157-2, S157-3) of soil with DLS157 microbial fertilizer showed relatively low expression.

[0126] In summary, the heatmap reveals differences in gene expression along carbon, nitrogen, phosphorus, and sulfur metabolic pathways between the control and experimental groups. The higher expression of certain genes in specific metabolic pathways may indicate that microorganisms involved in these pathways dominate the corresponding samples, thereby influencing the transformation pathways and rates of elements.

[0127] ② Based on Anosim analysis, further statistical analysis was performed on the differences in microbial communities in carbon, nitrogen, phosphorus, and sulfur cycles between the control group (CKT) of soil without DLS157 microbial fertilizer and the experimental group (S157) of soil with DLS157 microbial fertilizer. In the figure, 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 [link to specific results] Figure 18 A-18D.

[0128] exist Figure 18 In the carbon cycle of group A, R = 0.556, P = 0.1. The relatively high R value (close to 1) indicates a significant difference between the control and experimental groups. Figure 18 In the nitrogen cycle of group B, R = 0.222 and P = 0.2. Compared to the carbon cycle, the R value of the nitrogen cycle is lower, but differences still exist between groups. Figure 18 In the phosphorus cycle of C, R = 0.556, P = 0.1. Similar to the carbon cycle, there are significant differences between different sample groups in the phosphorus cycle. Figure 18 In the sulfur cycle of D, significant differences were also observed between the two groups (R = 0.556, P = 0.1), indicating variations in microbial communities across different biogeochemical cycles, particularly in the carbon, phosphorus, and sulfur cycles. Overall, the biogeochemical cycle functional analysis revealed how microbial communities function in these processes and the changes in the activity of related microbial communities during each cycle, further suggesting that these differences may be related to the effects of DLS157 microbial fertilizer application to the soil.

[0129] 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.

[0130] 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 single strain of *Mucor* (racemose mold) Mucor racemosus DLS157, characterized in that, It was deposited on December 30, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 41738, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

2. The application of *Mucor racemosa* DLS157 according to claim 1 in the preparation of humic acid products using rice straw or sugarcane bagasse as a carbon source.

3. A humic acid product, characterized in that, It was obtained by fermentation using *Mucor racemosa* DLS157 as described in claim 1, with rice straw or sugarcane bagasse as the carbon source for fermentation.

4. A microbial fertilizer, characterized in that, It was prepared by fermenting straw powder and wheat bran using the *Mucor racemosa* DLS157 as described in claim 1.

5. The application of the microbial fertilizer according to claim 4 in promoting crop growth.

6. The application according to claim 5, characterized in that, The crops mentioned are wheat and cabbage.

7. The application according to claim 6, characterized in that, When the crop is wheat, it has a promoting effect on plant height, leaf length, leaf width and number of leaves; When the crop is Chinese cabbage, it has a promoting effect on plant height, leaf width, number of leaves, above-ground fresh weight and above-ground dry weight.

8. The application of the microbial fertilizer according to claim 4 in soil improvement.

9. The application according to claim 8, characterized in that, Including the following improvements: (1) Increase the content of humus in the soil; (2) Increase the diversity and abundance of microbial colonies in the soil; (3) Regulate the structure of microbial colonies in the soil; (4) Promote the transformation and cycling of carbon, phosphorus and sulfur elements in the soil.

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