Cladomucor racemosus DLS157 and application thereof

Abstract

The invention discloses a Cladomucor racemosus DLS157 strain and application thereof, and belongs to the technical field of microorganisms. The preservation number of the strain is CGMCC (China General Microbiological Culture Collection Center) No.41738. The strain is high in humic acid production capacity when being used for fermenting lignocellulose, and DLS157 bacterial fertilizer is prepared from rice straw powder and wheat bran which are fermented by the strain. After the DLS157 bacterial fertilizer is added into soil, wheat and Chinese cabbage are sown, so that agronomic characters such as plant height, leaf length, leaf width and leaf number of wheat are remarkably promoted, and biomass such as plant height, leaf width, leaf number, overground fresh weight and overground dry weight of Chinese cabbage are remarkably improved. Research on the soil after wheat sowing is completed shows that the addition of the DLS157 bacterial fertilizer significantly changes the microbial community of the soil, increases the diversity and abundance of the microbial community, and increases the content of humus in the soil. It is further shown that the DLS157 bacterial fertilizer is beneficial to adjustment of the soil microbial community structure and can be used for soil improvement. And the method has important significance for realizing resource utilization of agricultural straws.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and more particularly to a strain of Mucor racemosus DLS157 and its application in soil improvement and crop growth promotion. Background Art

[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 in the presence of water and air, as well as the accumulation of geophysical and chemical processes. Humic acid is classified in my country into three categories based on its solubility in solvents and 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, playing a vital role in the accumulation and regeneration of humic acid. Huminin, a negatively charged hydrophilic colloid, absorbs or replaces potassium, sodium, and ammonium ions in the soil, turning soil particles into small reservoirs for water and fertilizer retention, increasing soil porosity and improving soil fertility. Humic acid is the most active component of humus, with a high cation exchange capacity, which improves soil water and fertilizer retention and adsorption capacity, contributing to the formation of a well-developed soil structure. Therefore, humic acid can improve the soil, activate insoluble mineral elements, promote the absorption of trace elements 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 generally accepted, the exact components of plants that humic acid is composed of remain a point of debate. 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. There is also debate regarding the specific role of microorganisms in humic acid formation, with four main hypotheses proposed: the plant transformation hypothesis, the biochemical hypothesis, cell autolysis, and microbial synthesis. It is currently difficult to determine which hypothesis is more accurate, as the formation of humic acid may require the coordinated action of multiple processes. The microbial formation hypothesis primarily emphasizes the role of microorganisms in humic acid formation. Microorganisms play a multifaceted role in humic acid formation, acting not only as decomposers to break down macromolecular polymers but also as producers to recondense decomposed small molecules. Therefore, the products of lignocellulose decomposition serve as the skeleton and substrate for humic acid formation. Adding protein-rich organic waste at the appropriate time to increase the activity of lignocellulosic enzymes offers a crucial option for converting lignocellulosic waste into humic acid by controlling key factors.

[0004] Lignocellulosic components are the most abundant in agricultural waste, primarily composed of a three-dimensional network of cellulose, hemicellulose, and lignin. As the most abundant renewable bioresource in the biological world, lignocellulosic production reaches 150 billion tons annually worldwide, 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 out strains that can efficiently produce humic acid from agricultural straw and produce high-value-added humic acid, thus making agricultural straw a resource, is of great significance for agricultural treatment, soil remediation, and microbial fermentation to produce humic acid. Summary of the Invention

[0006] In response to the above problems, the present invention provides a strain of Mucor racemosus DLS157 and its application in soil improvement and crop growth promotion.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] A strain of Mucor racemosus DLS157, classified and named Mucor racemosus, was deposited in the General Microbiology Center of the China Culture Collection Administration on December 30, 2024, with the deposit number CGMCC No. 41738, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0009] Another object of the present invention is to provide the use of Mucor racemosus 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 the culture or processed product of the above-mentioned Mucor racemosus DLS157.

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

[0012] Preferably, the preparation method of the bacterial fertilizer is as follows: rice straw powder and wheat bran are mixed in a mass ratio of 7:3, water is added and stirred evenly to reach a moist state without water accumulation, the mixture is divided into bags, sealed and sterilized to obtain a fermentation substrate, the strain DLS157 seed liquid is inoculated into the above-mentioned fermentation substrate at a ratio of 1mL:30g, and cultured at a constant temperature of 25°C until the mycelium covers the bag.

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

[0014] Another object of the present invention is to provide the use of Mucor racemosus DLS157 in promoting crop growth.

[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 leaf number.

[0017] Preferably, when the crop is cabbage, it has a promoting effect on plant height, leaf width, leaf number, aboveground fresh weight and aboveground dry weight.

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

[0019] Specifically, the improvements include: (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 circulation of carbon, phosphorus, and sulfur elements in the soil.

[0020] Through the above technical solution, it can be seen that compared with the existing technology, the present invention discloses a new strain of Mucor racemosus DLS157 that produces high-value humic acid from straw. This strain has a strong ability to ferment lignocellulose to produce humic acid. The bacteria are used to ferment rice straw powder and wheat bran to prepare DLS157 bacterial fertilizer. After adding DLS157 bacterial fertilizer to the soil and sowing wheat and cabbage, it not only significantly promotes the agronomic traits of wheat such as plant height, leaf length, leaf width and leaf number, but also significantly increases the biomass of cabbage such as plant height, leaf width, leaf number, aboveground fresh weight and aboveground dry weight.

[0021] Studies on soil after wheat sowing revealed that the addition of DLS157 fertilizer significantly altered the soil microbial community, increasing its diversity and abundance and boosting the humus content. Significant changes also occurred at the functional level, primarily manifested in differences in the abundance of carbohydrate-active enzymes (CAZymes) and the relative abundance and intergroup differences of genes involved in biogeochemical cycling functions. This indicates changes in the structure and abundance of microbial communities involved in related metabolic pathways, thereby influencing the pathways and rates of metabolic conversion. This further demonstrates that DLS157 fertilizer is beneficial for regulating soil microbial community structure and can be used for soil improvement.

[0022] In summary, the present invention utilizes the property of strain DLS157 to efficiently produce humic acid from agricultural straw, and prepares it into high-value humic acid products (such as bacterial fertilizer), which not only promotes crop growth but also improves soil, and is of great significance for realizing the resource utilization of agricultural straw. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0024] Figure 2 :Morphological identification of strain DLS157, a: colony morphology, b: conidia morphology;

[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 culture medium at different temperatures, where a: cultured at 4°C, b: cultured at 25°C, c: cultured at 30°C, d: cultured at 37°C, and e: cultured at 45°C; +: indicates the growth of the strain, the more the better, / : no growth of the strain, 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: liquid basal medium with rice straw carbon source without inoculation, S157: basal salt medium with rice straw inoculated with strain DLS157, different capital letters indicate extremely significant differences among groups (p < 0.01);

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

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

[0030] Figure 8 : Comparison of the growth of cabbage on different soils 19 days and 40 days after sowing, CKT: pure soil, S157: pure soil + DLS157 bacterial fertilizer;

[0031] Figure 9 : Biomass determination results of cabbage grown on different soils 43 days after sowing, where 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 among groups (p<0.05), different uppercase letters indicate extremely significant differences among groups (p<0.01);

[0032] Figure 10:Different soils after wheat sowing were diluted to 10 -5 The growth period of soil sample solution on PDA medium, a: soil without DLS157 bacterial fertilizer, b: soil with DLS157 bacterial fertilizer;

[0033] Figure 11 : Comparison of humus content in different soils after wheat sowing, where Ⅰ: humus content, Ⅱ: humic acid content, Ⅲ: fulvic acid content, Ⅳ: humin content, CKT: soil without DLS157 bacterial fertilizer, S157: soil with DLS157 bacterial fertilizer, different lowercase letters indicate significant differences among groups (p<0.05), different uppercase letters indicate extremely significant differences among 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 application of DLS157 bacterial fertilizer, specifically, 1: CKT-1 soil sample, 2: CKT-2 soil sample, 3: CKT-3 soil sample; 4-6 are soil samples with application of DLS157 bacterial 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 in soil samples from different groups after wheat sowing at the level of phylum, genus, and species, among which: Figure 13A : Species distribution histogram of each group of soil samples at the phylum level, Figure 13B : Species distribution histogram of each group of soil samples at the genus level, Figure 13C : Species distribution histogram of each group of soil samples at the species level;

[0036] Figure 14 : PCoA analysis of different microorganisms in soil samples from different groups after wheat sowing at the phylum, genus, and species level, where a: species structure differences at the phylum (Phylum) taxonomy level, b: species structure differences at the genus (Genus) taxonomy level, c: species structure differences at the species (Species) taxonomy level;

[0037] Figure 15 : Heat map of significantly different species at the phylum level in soil samples of different groups after wheat sowing;

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

[0039] Figure 17: MetagenomeSeq heat map of carbon cycle, nitrogen cycle, phosphorus cycle and sulfur cycle in different groups of soil samples after wheat sowing.

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

[0041] 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 all soils without the application of DLS157 fertilizer, and S157-1, S157-2, and S157-3 are all soils with the application of DLS157 fertilizer;

[0043] Figure 18 : Anosim analysis of functional gene abundance based on different groups of soil samples after wheat sowing, where A: carbon cycle, B: nitrogen cycle, C: phosphorus cycle, and D: sulfur cycle; CKT refers to soil without DLS157 fertilizer, and S157 refers to soil with DLS157 fertilizer. DETAILED DESCRIPTION

[0044] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. 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 any creative efforts are within the scope of protection of the present invention.

[0045] The reagents involved in the embodiments of the present invention were all purchased from commercial channels, and the methods not mentioned are conventional experimental methods and will not be described in detail here.

[0046] The following reagents are illustrative:

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

[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, natural pH.

[0049] Rice straw carbon source liquid basal medium: rice 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.

[0050] Corn stalk carbon source liquid basal culture 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 liquid basal culture 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] Pine wood carbon source liquid basic culture medium: pine wood powder 20g / L, (NH4)2SO44g / L, MgSO4·7H2O1.2g / L, CaCl20.3g / L, K2HPO41g / L, KH2PO41g / L, NaNO31g / L, natural pH.

[0053] Sugarcane bagasse carbon source liquid basic culture medium: sugarcane bagasse 20g / L, (NH4)2SO44g / L, MgSO4·7H2O1.2g / L, CaCl20.3g / L, K2HPO41g / L, KH2PO41g / L, NaNO31g / L, pH natural.

[0054] Poplar carbon source liquid basic culture medium: poplar powder 20g / L, (NH4)2SO44g / L, MgSO4·7H2O1.2g / L, CaCl20.3g / L, K2HPO41g / L, KH2PO41g / L, NaNO31g / L, natural pH.

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

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

[0057] Mix (catalog number: TSE004, specification: 5×1 mL, 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] The strain was derived from the soil of Canglang Peak, Cangshan Mountain, Dali (25°50'28.90"N, 100°03'7.60"E). The specific culture method was as follows: 10 g of soil sample was added to 10 mL of sterile water as the mother solution, 1 mL of the mother solution was added to sterile water to dilute the concentration to 10% of the mother solution concentration. -3 , 10 -4 , 10 -5 , respectively, on a basal culture medium containing rice straw, corn stalks, wheat straw, sugarcane bagasse, pine wood, poplar wood, and other lignocelluloses as the sole carbon source, and cultured in a constant temperature incubator at 20°C. After the bacteria grow, single, fuzzy, or flocculent colonies are picked from the plates for transfer and purification.

[0062] (2) Screening of strains

[0063] The strains purified by selective culture were picked and single colonies were inoculated into 10 mL of LB liquid culture medium containing 0.05 mg / mL kanamycin, and cultured at 20°C and 180 rpm for 2 days to obtain seed liquid. The seed liquids obtained above were inoculated into 100 mL of rice straw carbon source liquid basal culture medium at an inoculum size of 2%. After shaking culture at 20°C and 180 rpm for 7 days, 0.2 mol / L sodium pyrophosphate solution was added to the obtained fermentation liquid to pH = 12. After standing at room temperature for 24 hours, the supernatant was taken and scanned in the range of 200 nm-320 nm using a UV-visible spectrophotometer. Each group was repeated 3 times. Strains with absorbance values ​​> 0 were selected as having the ability to produce humic acid. Eight humic acid-producing strains were preliminarily screened based on the absorbance values.

[0064] The 8 strains obtained from the initial screening were picked up and single colonies were inoculated into 10 mL of LB liquid culture medium containing 0.05 mg / mL kanamycin. They were shaken and cultured at 20°C and 180 rpm for 2 days to obtain seed liquid. The seed liquid of each strain was inoculated at a 2% inoculation rate into an equal amount of liquid culture medium with different lignocellulose as carbon source (rice straw, corn straw, wheat straw, sugarcane bagasse, pine wood, and poplar wood as the only carbon source). They were fermented at 20°C and 180 rpm for 7 days, and the yield of humic acid in the fermentation liquid was preliminarily determined. Then 0.2 mol / L sodium pyrophosphate alkaline extract was added to the fermentation liquid to pH = 12, and the solution 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. Centrifuge, discard the supernatant, and dry the precipitate at the same time. The precipitate obtained was crude humic acid. The strain with stronger humic acid production ability was selected and named DLS157. Figure 1The 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, among which rice straw has the highest yield. In addition, strain DLS157 can also grow in corn straw and wheat straw, but the humic acid production is almost zero.

[0065] 2. Identification of strains

[0066] (1) Morphological characteristics identification

[0067] The strain DLS157 was inoculated on PDA medium and cultured at 25℃ for 5 days, and then the size and color of the colonies were observed and recorded. Microscopic images were taken using a Nikon ECLIPSENi-U biological microscope to record the morphological characteristics of the strain. Figure 1 The mycelium is multinucleate and has no septate shape. The mycelium is slender, smooth in surface, highly branched, and white in color. It forms a dense mycelium layer on the substrate ( Figure 2 a); conidia are spherical or ellipsoidal, with different sizes ( Figure 2 b). This is a typical morphological feature of the genus Mucor.

[0068] (2) Molecular biological identification

[0069] DNA of strain DLS157 was extracted, and PCR amplification was performed using Taq enzyme using the universal primers ITS4 and ITS5 for fungal ITS identification (both ITS4 and ITS5 were purchased from Sangon Biotech (Shanghai) Co., Ltd.). The PCR amplification reaction system (40 μL) was as follows: 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: pre-denaturation at 94°C for 4 min; denaturation at 94°C for 30 s, annealing at 55°C for 35 s, and extension at 72°C for 90 s, for 32 cycles; and extension at 72°C for 5 min.

[0070] After agarose gel electrophoresis, the amplified products were sent to Sangon Biotech Co., Ltd. for sequencing, and the obtained sequences were submitted to GenBank (http: / / www.ncbi.nlm.nih.gov). Based on the similarity comparison with the ITS sequence in the BLAST database, the ITS rRNA sequence phylogenetic tree was constructed using MEGA7.0 using the neighbor-joining method (e.g. Figure 3 Phylogenetic analysis showed that strain DLS157 clustered with Mucor racemosus in a clade with 85% support. Based on colony morphology, microscopic photographs, and a phylogenetic tree constructed based on ITS rRNA sequence alignment, strain DLS157 was identified as Mucor racemosus.

[0071] Example 2 Determination of humic acid production performance of strains

[0072] 1. Effects of different temperatures on strain growth

[0073] Take the seed liquid of strain DLS157 after expansion (bacterial concentration is 8.3×10 5 CFU / mL) was inoculated into a rice straw carbon source liquid basal medium at an inoculum size of 2%, and cultured at different temperatures (4°C, 25°C, 30°C, 37°C, 45°C) for 7 days as the experimental group, and the rice straw carbon source liquid basal medium without inoculation was used as the control group. Three parallels were set for each temperature to detect the effect of different temperatures on the growth of the strain. The results are shown in the figure. Figure 4 .

[0074] Depend on Figure 4 It can be seen that strain DLS157 grows best at 25°C (b), with dense and elongated hyphae. Growth deteriorates at 30°C (c) and 37°C (d). Furthermore, growth ceases at 4°C and 45°C, while DLS157 can grow within the 25°C-37°C range, demonstrating that it is a low-temperature fungus.

[0075] 2. Evaluation of the Humic Acid Production Capacity of the Strain

[0076] Take the seed liquid of strain DLS157 after expansion (bacterial concentration is 8.3×10 5 CFU / mL) was inoculated into 100mL of rice straw carbon source liquid basal medium at an inoculum size of 2%, and the control was a medium without bacteria, with three biological replicates. After shaking culture at 20°C and 180rpm for 7 days, 10mL of the strain fermentation supernatant and liquid culture medium were collected and sent to Wuhan Pu Neisi Testing Technology Co., Ltd. for testing of humic acid content (NY / T 1971-2010 Determination of humic acid content in water-soluble fertilizers). The test results are shown in 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 (uninoculated rice straw carbon source liquid basal medium) culture medium. The results showed that the humic acid content per gram of DLS157 fermentation broth was 14.03%, while the humic acid content per gram of the control (CK) was 9.59%. There was a highly significant difference (p < 0.01) between strain DLS157 and the control (CK), with the humic acid content in the fermentation supernatant of strain DLS157 increasing by 46.30%. This further demonstrates the strong humic acid production capacity of strain DLS157.

[0078] Example 3 Determination of the growth-promoting performance of strains on crops

[0079] After mixing rice straw powder and wheat bran in a mass ratio of 7:3, add water and stir evenly until it is moist without water accumulation. Then, divide it into bacteria bags (1.5 kg / bag), seal it with a ring, and sterilize it at 121℃ for 120 minutes to obtain the fermentation substrate for use. Strain DLS157 was inoculated into 50 mL of LB liquid medium containing 0.05 mg / mL kanamycin and cultured at 20℃ and 180 rpm for 2 days to obtain seed liquid. The bacterial concentration was measured to be 8.3×10 5 CFU / mL. 50 mL of strain DLS157 seed solution (bacterial concentration of 8.3×10 5 CFU / mL) and cultured in a 25°C constant temperature incubator until the mycelium covered the bag, obtaining DLS157 bacterial fertilizer, which was used in subsequent growth promotion experiments.

[0080] 1. Promote the growth of food crops

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

[0082] The above-mentioned DLS157 bacterial fertilizer was mixed with the barren soil in the suburbs of Chenggong, Kunming at a ratio of 125g of bacterial bag per kilogram of soil, and then divided into flower pots of equal weight (400g / pot), with 6 replicates per group. After the bacteria repaired the soil for 15 days, it was recorded as the experimental group, and the same amount of pure soil was used as the control group CKT. The germinated wheat prepared in advance was inoculated into the flower pots with different treatments, with 3 pots inoculated in each group and 10 plants in each pot. The growth of wheat in the control group (pure soil) and the experimental group (pure soil + DLS157 bacterial fertilizer) was monitored. The growth comparison of wheat at 12d and 56d after sowing is shown in Figure 6 .Depend on Figure 6 It can be seen that the growth of wheat after adding DLS157 bacterial fertilizer is significantly better than that of the pure soil in the control group.

[0083] The agronomic traits of wheat such as plant height, leaf length, leaf width, and leaf number were measured using SPASS software 75 days after sowing. Figure 7 .Depend on Figure 7 The results showed that the plant height, leaf length, leaf width, and leaf number of DLS157 strains were significantly higher than those of the control group (CKT), further demonstrating that the humic acid fertilizer produced by the strain DLS157 using agricultural straw can promote wheat growth.

[0084] 2. Promote the growth of vegetable crops

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

[0086] Take 4kg of barren soil from Yinjie Town, Midu County, Dali Prefecture, add 200g of the above-mentioned DLS157 fertilizer at a ratio of 5%, mix well and divide into 5 flower pots by equal weight. After the bacteria repair the soil for 15 days, it is recorded as the experimental group, and the control group is 4kg of pure soil. The cabbage is inoculated into flower pots with different treatments, with 10 cabbages in each pot. The growth of cabbage in the control group (pure soil, recorded as CKT) and the experimental group (pure soil + DLS157 fertilizer, recorded as S157) was monitored, and the seedlings were thinned to 5 during the period to measure agronomic traits. The growth comparison of cabbage at 19d and 40d after sowing is shown in the figure. Figure 8 .Depend on Figure 8 It 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 fertilizer). At 40 days, the growth of cabbage with the addition of DLS157 fertilizer was significantly better than that of the control group without DLS157 fertilizer.

[0087] The agronomic traits of cabbage, such as plant height, root length, leaf width, leaf number, aboveground fresh weight and aboveground dry weight, were measured and analyzed using SPASS software 43 days after sowing. Figure 9 .Depend on Figure 9 The results showed that the plant height, leaf width, leaf number, aboveground fresh weight and aboveground dry weight of cabbage in the soil with DLS157 bacterial fertilizer were significantly improved. Figure 9-Ⅰ , 9-Ⅲ, 9-Ⅴ, 9-ⅥIt can be seen that the plant height, leaf width, aboveground fresh weight and aboveground dry weight in the experimental group were significantly different from those in the control group (p<0.01). Compared with the control group, the plant height, leaf width, aboveground fresh weight and aboveground dry weight increased by an average of 1.62 cm, 1.64 cm, 0.746 g and 0.0512 g, respectively. Figure 9-IV As can be seen, the number of leaves in the experimental group was significantly different from that in the control group (p < 0.05), with 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. In summary, the addition of DLS157 microbial fertilizer to the soil can promote cabbage growth to a certain extent.

[0088] Example 4 Determination of the Effect of Bacteria on Soil

[0089] 1. Dilution coating experiment

[0090] In order to explore whether there are differences in the microbial communities in the soil with humic acid added, a dilution coating experiment was conducted on the soil planted with wheat in Example 3. Specifically, 10 g of the soil from the two groups of flower pots planted with wheat in Example 3 was weighed and dissolved in 10 mL of sterile water as the mother solution. 1 mL of the mother solution was diluted with sterile water to a concentration of 10% of the mother solution. -5 , spread on PDA culture medium, repeat 3 times for each group. After culturing in a 25℃ constant temperature incubator for 3 days, observe the changes in the number of colonies on the plate. The results are shown in Figure 10 .

[0091] Depend on Figure 10 The plates in group b (soil treated with DLS157 fertilizer) were densely populated with microbial colonies (>500 colonies per plate), while the plates in group a (soil not treated with DLS157 fertilizer) were relatively few (<10 colonies per plate). The colony counts in the soil treated with DLS157 fertilizer were significantly higher than those in the soil not treated with DLS157 fertilizer, indicating that the addition of DLS157 fertilizer can, to a certain extent, increase the number and species richness of culturable microbial communities in the soil.

[0092] 2. Determination of soil humus content

[0093] Take the two groups of flowerpot soils planted with wheat in Example 3, take 10g of soil from each pot (control group number CKT-1, CKT-2, CKT-3, experimental group number S157-1, S157-2, S157-3), and send them to Wuhan Pu Naisi Testing Company with dry ice to measure the content and change of humus in the soil. The detection principle is as follows: the soil sample is dried to constant weight at (105±5)°C, and the dry matter and moisture content are calculated by the difference in soil sample 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. Soluble humus is extracted with 0.1mol / L sodium pyrophosphate-sodium hydroxide mixed solution, and the total amount of humic acid and fulvic acid is determined by potassium dichromate oxidation capacity method. The extract is separated by acidification precipitation and its content is determined, and the fulvic acid content can be calculated. The total carbon content of the soil sample was measured, and the humin content was obtained by subtracting the humic acid and fulvic acid content. The specific test results are shown in Table 1 and Figure 11 .

[0094]

[0095]

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

[0097] 3. Soil Metagenome Sequencing Analysis

[0098] (1) Agarose gel electrophoresis analysis

[0099] The soil from the two sets of flowerpots planted with wheat in Example 3 was taken and the soil metagenomic samples of each group were extracted using the MP Biomedicals Soil DNA Isolation Kit. Each group had three biological replicates, and 0.3 soil samples were weighed for each replicate (control group numbers CKT-1, CKT-2, CKT-3, experimental group numbers 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 minutes. The results are shown in Table 1. Figure 12 and Table 2.

[0100]

[0101] Figure 12 The differences in the brightness and total amount of metagenomic DNA can also be seen in Table 2. Compared with the control group without DLS157 fertilizer, the concentration and total amount in the experimental group with DLS157 fertilizer increased significantly, indicating that the addition of DLS157 fertilizer to the soil increased the number and species of microorganisms to a certain extent.

[0102] (2) Species composition analysis

[0103] After passing the above-mentioned gel electrophoresis test, each group of samples was stored on dry ice and sent to Wuhan Punaisi Testing Company for metagenomic sequencing. Specifically, after the samples were qualified, a sequence library was constructed and high-throughput sequencing was performed. Splicing software was used to assemble high-quality sequences and perform gene prediction. Using the CAZy database, the predicted genes were annotated and classified by species and function to predict the function, classification and metabolic pathway of the genes. Using R software, a bar graph was drawn for the composition of the dominant species in each sample (here the top 30 species in overall abundance) at each classification level. The horizontal axis (x-axis): shows different sample groups, with 3 replicates per 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. The bar graph shows the species composition of each sample and the proportion of different species in each sample.

[0104] The species histograms at the three taxonomic levels of Phylum, Genus, and Species for the control group (number: CKT-1, CKT-2, CKT-3) of the soil without DLS157 fertilizer and the experimental group (number: S157-1, S157-2, S157-3) of the soil with DLS157 fertilizer are shown as follows: Figures 13A-13C shown.

[0105] At door level (see Figure 13A ) As can be seen, the pink region (Proteobacteria) and the green region (Actinobacteria) have the highest relative abundance, exceeding 0.5. Differences were evident between the experimental and control groups in certain microbial phyla, such as Actinobacteria and Bacteroidetes, which showed significant abundance changes.

[0106] At the genus level (see Figure 13B ) shows that, in addition to unclassifiable or rare microorganisms, the abundance of Sphingomonas (pink) and Nocardioides (green) in CKT accounts for a large proportion. In the experimental group, the relative abundance of Sphingomonas (pink) and Saccharothrix (orange) was higher. Significant changes in the abundance of Sphingomonas and Nocardioides were observed between the experimental and control groups.

[0107] At the species level (see Figure 13C) As can be seen, the largest proportion of unclassified microorganisms exceeded 0.5. Furthermore, the abundance of Acidobacteria bacteria in the pink area and Chloroflexi bacteria in the green area of ​​the CKT cells was relatively high. The relative abundance of Acidobacteria bacteria in the pink area and Saccharothrix sp. in the green area was higher in the experimental group. Certain species showed significant differences between the experimental and control groups, such as Sphingomonase daphi and Saccharothrix sp., which showed significant changes in abundance.

[0108] In summary, Figure 13 shows the relative abundance of different microbial phyla, genera, and species in the experimental group of soil applied with DLS157 bacterial fertilizer (number: S157-1, S157-2, S157-3) and the control group of soil not applied with DLS157 bacterial fertilizer (number: CKT-1, CKT-2, CKT-3) through a stacked bar chart, showing the differences in the composition of the microbial community and reflecting the effect of DLS157 bacterial fertilizer on the microbial population in the soil.

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

[0110] The PCoA analysis of each group of samples after sequencing was performed based on the unconstrained sorting (Classical Multidimensional Scaling, cMDScale) analysis method to evaluate the differences in microbial community composition between samples and analyze the species β diversity. PCoA1 represents the first principal component and its contribution to sample differences. This axis shows the main differences in most samples. PCoA2 represents the second principal component and its contribution to sample differences, showing the second largest major difference. The differences in species structure at the three taxonomic levels of phylum (Phylum), genus (Genus), and species (Species) between the control group of soil without DLS157 fertilizer (No.: CKT-1, CKT-2, CKT-3) and the experimental group of soil with DLS157 fertilizer (No.: S157-1, S157-2, S157-3) are as follows 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 in the principal coordinates. The microbial community structure of the experimental group is clustered together, which is far away from the structure of the control group. This indicates that there are large structural differences between the microorganisms of the two groups of samples. At the phylum level, the first two axes of the PCoA analysis explain 78.45% and 17.57% of the total variation of the data at the phylum level, respectively (see Figure 14a). At the genus level, the first two axes of the PCoA analysis explained 68.86% and 20.4% of the total variation of the data at the genus level, respectively (see Figure 14 b). At the species level, the first two axes of the PCoA analysis explained 67.17% and 20.24% of the total variation in the data at the species level, respectively (see Figure 14 c). Significant differences in overall community composition were already observed between the control (CKT, blue) and experimental (S157, dark blue) groups at the phylum level. These differences were further amplified at the genus and species levels, manifesting as differences in the distribution of individual genera or species. Therefore, the addition of DLS157 fertilizer significantly altered the soil microbial community structure.

[0112] (4) Analysis of species abundance differences

[0113] MetagenomeSeq analysis was performed on each of the sequenced samples using a zero-inflated model to evaluate the abundance differences between the control group (numbers: CKT-1, CKT-2, CKT-3) of the soil without DLS157 fertilizer and the experimental group (numbers: S157-1, S157-2, S157-3) of the soil with DLS157 fertilizer. A heatmap visualization method was used to display species with significant differences at the phylum level. Blue represents a low abundance (negative value) of the taxonomic group, while red represents a high abundance (positive value). Yellow represents an abundance close to the median value. By comparing the colors between the samples, the differences in microbial community composition of these samples can be intuitively understood.

[0114] The results are as follows Figure 15 As shown, Candidatus Vogelbacteria, Candidatus Woesearchaeota, and Candidatus Saccharibacteri showed significantly higher abundance in the experimental groups (numbers: S157-1, S157-2, S157-3), but lower abundance in the control groups (numbers: CKT-1, CKT-2, CKT-3). Conversely, Armatimonadetes, Candidatus Odinarchaeota, and Acidobacteria showed significantly higher abundance in the control groups (numbers: CKT-1, CKT-2, CKT-3), but lower abundance in the experimental groups (numbers: S157-1, S157-2, S157-3). Figure 15The differences in microbial community composition between the control group and the experimental group were shown. Through row and column cluster analysis, it was confirmed that there were significant abundance differences between the groups at the phylum level, indicating that the addition of DLS157 fertilizer significantly changed the richness and composition of the soil microbial community.

[0115] (5) Functional relative abundance analysis

[0116] The predicted genes were functionally annotated and classified using the CAZy database to evaluate the effect of adding DLS157 fertilizer on the functional abundance of soil microorganisms. Specifically, a heat map was drawn based on the functional annotation and abundance information of the control group (number: CKT-1, CKT-2, CKT-3) of the soil without DLS157 fertilizer and the experimental group (number: S157-1, S157-2, S157-3) of the soil with DLS157 fertilizer applied in the CAZy database, and clustered at 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 an abundance close to neutral (0), that is, the carbohydrate enzyme has a moderate abundance in the sample. The abundance of carbohydrate-active enzymes (CAZymes) in different samples is shown in Figure 2. Figure 16 shown.

[0117] Figure 16 The heat map shown here visually displays the expression levels of different carbohydrate-active enzymes in each sample through color changes. For example, the carbohydrate enzyme abundance of the control group (numbers: CKT-1, CKT-2, CKT-3) showed higher levels in the GH77 and GT39 families, but lower abundance in the GH5 and GH37 carbohydrate enzyme families. In contrast, the experimental group (numbers: S157-1, S157-2, S157-3) showed a clear opposite abundance in the GH5 and GH37 families, with higher abundance in GH77 and GT39. The figure clearly shows the difference in the abundance of carbohydrate-active enzyme families between the control and experimental groups. Specifically, the carbohydrate enzyme families in different sample groups have different expression levels, which reflects the differences in the carbohydrate metabolism potential of the microbial community after the addition of DLS157 fertilizer to the soil.

[0118] (6) Analysis of biogeochemical cycle functions

[0119] The relative abundance and inter-group differences of carbon cycle, nitrogen cycle, phosphorus cycle and sulfur cycle functional genes were analyzed to evaluate the effect of adding DLS157 fertilizer on the geochemical cycle function of soil microorganisms. Specifically:

[0120] ①Based on the metagenomeSeq heat map, the clustering relationship of the samples and genes of the control group (numbered: CKT-1, CKT-2, CKT-3) of the soil without applying DLS157 microbial fertilizer and the experimental group (numbered: S157-1, S157-2, S157-3) of the soil applying DLS157 microbial fertilizer was analyzed. The tree diagram on the left and top of the metagenomeSeq heat map shows the clustering relationship of different samples and genes. The clustering results show the similarity of the metabolic pathways between samples and genes. The vertical axis shows different genes related to carbon cycle, and the horizontal axis shows the gene expression data of different samples. The expression amount of the gene is represented from red (high expression) to blue (low expression). The metagenomeSeq heat maps of the soil samples of different groups in carbon cycle, nitrogen cycle, phosphorus cycle and sulfur cycle are shown in Figures 17A-17D .

[0121] In the carbon cycle metagenomeSeq heat map shown in Figure 17A , the control group (numbered: CKT-1, CKT-2, CKT-3) of the soil without applying DLS157 microbial fertilizer shows higher expression on genes such as bcrA and fdhA, while the experimental group (numbered: S157-1, S157-2, S157-3) of the soil applying DLS157 microbial fertilizer shows lower expression on these genes.

[0122] In the nitrogen cycle metagenomeSeq heat map shown in Figure 17B , the experimental group (numbered: S157-1, S157-2, S157-3) of the soil applying DLS157 microbial fertilizer shows higher expression level on nitrogen cycle genes such as napA, while the control group (numbered: CKT-1, CKT-2, CKT-3) of the soil without applying DLS157 microbial fertilizer shows relatively lower expression on this gene.

[0123] In the phosphorus cycle metagenomeSeq heat map shown in Figure 17C , the experimental group (numbered: S157-1, S157-2, S157-3) of the soil applying DLS157 microbial fertilizer shows higher expression on multiple genes, especially on genes such as olpA and gcd.

[0124] In the sulfur cycle metagenomeSeq heat map shown in Figure 17D , the control group (numbered: CKT-1, CKT-2, CKT-3) of the soil without applying DLS157 microbial fertilizer shows higher expression on multiple sulfur metabolism genes such as sat and sqr, while the experimental group (numbered: S157-1, S157-2, S157-3) of the soil applying DLS157 microbial fertilizer shows relatively lower expression.

[0125] In summary, the heat map shows differences in gene expression between the control and experimental groups in the carbon, nitrogen, phosphorus, and sulfur metabolic pathways. The higher expression of some genes in specific metabolic pathways may indicate that the microorganisms involved in these metabolic pathways dominate in the corresponding samples, thereby affecting the conversion pathways and rates of these elements.

[0126] ②Based on Anosim analysis, we further conducted statistical analysis on the differences in microbial communities in the carbon cycle, nitrogen cycle, phosphorus cycle, and sulfur cycle between the control group (CKT) of the soil without DLS157 fertilizer and the experimental group (S157) of the soil with DLS157 fertilizer. The R value in the figure represents the degree of difference between the sample groups. The higher the R value, the greater the difference between the groups. R greater than 0 indicates that the difference between the groups is significant. Figure 18 A-18D.

[0127] exist Figure 18 In the carbon cycle of A, R=0.556, P=0.1, and the R value is high (close to 1), indicating that there is a large difference between the control group and the experimental group. Figure 18 In the nitrogen cycle of B, R=0.222, P=0.2, the R value of nitrogen cycle is lower than that of carbon cycle, but there are still differences between the groups. Figure 18 In the phosphorus cycle of C, R = 0.556, P = 0.1. Similar to the carbon cycle, there are large differences between different sample groups in the phosphorus cycle. Figure 18 The sulfur cycle of D also showed significant differences between the two groups, with R = 0.556 and P = 0.1, indicating that microbial communities within different biogeochemical cycles differed between samples, particularly within the carbon, phosphorus, and sulfur cycles. Overall, the functional analysis of biogeochemical cycles revealed how microbial communities function within these processes and the changes in activity of related microbial communities during each cycle, further suggesting that these differences may be related to the effects of the addition of DLS157 fertilizer to the soil.

[0128] 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.

[0129] 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 Mucor racemosus DLS157, characterized in that It was classified and named Mucor racemosus, and was deposited in the General Microbiology Center of China Culture Collection Administration on December 30, 2024, with the deposit number CGMCC No.41738, and the deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

2. Use of Mucor racemosus DLS157 according to claim 1 in the preparation of high-value humic acid products.

3. A high-value humic acid product, comprising the culture or processed product of Mucor racemosus DLS157 according to claim 1.

4. A bacterial fertilizer, characterized in that The bacterial fertilizer comprises the culture of Mucor racemosus DLS157 or a processed product thereof according to claim 3.

5. Use of Mucor racemosus DLS157 according to claim 1 in promoting crop growth.

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

7. The use 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 leaf number; When the crop is cabbage, it has a promoting effect on plant height, leaf width, leaf number, aboveground fresh weight and aboveground dry weight.

8. Use of Mucor racemosus DLS157 according to claim 1 in soil improvement.

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

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