Streptomyces DLS188 and application thereof

Abstract

The invention discloses streptomyces DLS188 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.33250. The strain is high in humic acid production capacity when being used for fermenting lignocellulose, and the strain is used for fermenting rice straw powder and wheat bran to prepare DLS188 bacterial fertilizer. After the DLS188 bacterial fertilizer is added into soil and wheat is sown, agronomic characters such as the plant height, the leaf length and the leaf number of the wheat can be remarkably promoted. Research on the soil after wheat sowing is completed shows that the addition of the DLS188 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 DLS188 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 Streptomyces DLS188 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 humic acid formation 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 of macromolecular polymers but also as producers, recondensing decomposed small molecules into new forms. Therefore, the products of lignocellulose decomposition serve as the backbone and substrate for humic acid formation. Adding protein-rich organic waste at the appropriate time to increase lignocellulase activity 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 Streptomyces DLS188 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 Streptomyces DLS188, classified and named Streptomyces sp., was deposited in the General Microbiology Center of the China Culture Collection Administration on December 30, 2024, with the deposit number CGMCC No. 33250, 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 Streptomyces DLS188 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 Streptomyces DLS188.

[0011] Another object of the present invention is to provide a bacterial fertilizer comprising the above-mentioned Streptomyces DLS188 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 bacteria bags, sealed and sterilized to obtain a fermentation substrate, the strain DLS188 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 bacteria bag.

[0013] Preferably, the OD of the strain DLS188 seed solution is 600 It is 0.313.

[0014] Another object of the present invention is to provide application of Streptomyces DLS188 in promoting crop growth.

[0015] Preferably, the crop is wheat.

[0016] Preferably, it has a promoting effect on plant height, leaf length and leaf number.

[0017] Another object of the present invention is to provide application of Streptomyces DLS188 in soil improvement.

[0018] 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.

[0019] As can be seen from the above technical solution, compared with the existing technology, the present invention provides a new strain of Streptomyces DLS188 for producing high-value humic acid from straw. This strain has a strong ability to ferment cellulose to produce humic acid. DLS188 bacterial fertilizer was prepared by fermenting rice straw powder and wheat bran with this strain. When DLS188 bacterial fertilizer is added to soil and then sown with wheat, it significantly improves agronomic traits such as plant height, leaf length, and leaf number.

[0020] Studies on soil after wheat sowing revealed that the addition of DLS188 fertilizer significantly altered soil microbial communities, increasing their diversity and abundance and boosting soil humus content. Significant changes also occurred at the functional level, primarily manifested in differences in the abundance of carbohydrate-active enzymes (CAZymes) and in the relative abundance and intergroup differences of genes involved in biogeochemical cycling functions. These findings indicate 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 DLS188 fertilizer is beneficial for regulating soil microbial community structure and can be used for soil improvement.

[0021] In summary, the present invention utilizes the property of strain DLS188 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

[0022] Figure 1 :Comparison of humic acid production by strain DLS188 under different carbon sources;

[0023] Figure 2 :Morphological identification of strain DLS188, a: microscopic structure of the strain, b: morphological characteristics of hyphae after pure culture;

[0024] Figure 3 : Phylogenetic tree of strain DLS188 constructed based on 16S rRNA sequence;

[0025] Figure 4 Comparison of humic acid content in fermentation broth after different treatments, CK: rice straw liquid basal medium without inoculation, S188: rice straw basal salt medium inoculated with strain DLS188, different capital letters represent extremely significant difference (p<0.01) between groups;

[0026] Figure 5 Comparison of growth of wheat on different soils at 12d and 56d after sowing, CKT: pure soil, S188: pure soil + DLS188 microbial fertilizer;

[0027] Figure 6 Determination results of growth traits of wheat on different soils at 75 days after sowing, wherein, I: plant height determination, II: leaf length determination, III: leaf width determination, IV: leaf number determination, CKT: pure soil, S188: pure soil + DLS188 microbial fertilizer, different lowercase letters represent significant difference (p<0.05) between groups, and different capital letters represent extremely significant difference (p<0.01) between groups;

[0028] Figure 7 Growth period of soil sample solution diluted to 10 -5 in different soils after sowing of wheat on PDA medium, a: soil without DLS188 microbial fertilizer, b: soil with DLS188 microbial fertilizer;

[0029] Figure 8 Comparison of humus component content in different soils after sowing of wheat, wherein, I: humus content, II: humic acid content, III: fulvic acid content, IV: humin content, CKT: soil without DLS188 microbial fertilizer, S188: soil with DLS188 microbial fertilizer, different capital letters represent extremely significant difference (p<0.01) between groups;

[0030] Figure 9 1% gel electrophoresis map of different soil samples after sowing of wheat, M: 15000bp DNA Marker, 1-3 are soil samples without DLS188 microbial fertilizer, specifically, 1: CKT-1 soil sample, 2: CKT-2 soil sample, 3: CKT-3 soil sample; 4-6 are soil samples with DLS188 microbial fertilizer, specifically, 4: S188-1 soil sample, 5: S188-2 soil sample, 6: S188-3 soil sample;

[0031] Figure 10: Composition analysis diagram of different microorganisms at the level of door, genus and species in different soil samples after sowing of wheat, wherein, Figure 10A : species distribution column chart of each group of soil samples at the level of door (Phylum), Figure 10B: Species distribution histogram of each group of soil samples at the genus level, Figure 10C : Species distribution histogram of each group of soil samples at the species level;

[0032] Figure 11 : 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;

[0033] Figure 12 : Heat map of significantly different species at the phylum level in soil samples of different groups after wheat sowing;

[0034] Figure 13 : Abundance heat map of carbohydrate-active enzymes (CAZymes) in different groups of soil samples after wheat sowing;

[0035] Figure 14: MetagenomeSeq heat map of carbon cycle, nitrogen cycle, phosphorus cycle and sulfur cycle in different groups of soil samples after wheat sowing.

[0036] in, Figure 14A: 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;

[0037] Figure 14B : Nitrogen cycle, Figure 14C : Phosphorus cycle, Figure 14D : Sulfur cycle;

[0038] In Figures 10-14, CKT-1, CKT-2, and CKT-3 are all soils without DLS188 fertilizer, and S188-1, S188-2, and S188-3 are all soils with DLS188 fertilizer applied.

[0039] Figure 15 : 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 DLS188 bacterial fertilizer, and S188 refers to soil with DLS188 bacterial fertilizer. DETAILED DESCRIPTION

[0040] 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.

[0041] 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.

[0042] The following reagents are illustrative:

[0043] LB liquid medium: 10 g / L trypsin, 5 g / L yeast extract, 10 g / L NaCl, natural pH.

[0044] 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.

[0045] 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.

[0046] 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.

[0047] 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.

[0048] 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.

[0049] 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.

[0050] Solid culture medium is the corresponding liquid culture medium with 20 g / L agar added.

[0051] PDA medium: potato 200 g / L, glucose 20 g / L, agar 20 g / L, natural pH.

[0052] Mix (catalog number: TSE004, specification: 5×1 mL, Beijing Qingke Biotechnology Co., Ltd.).

[0053] Example 1 Isolation, screening and identification of strains

[0054] 1. Isolation and screening of strains

[0055] (1) Isolation of strains

[0056] The strain is derived from the soil of Dali Cangshan Canglang Peak (25°50'28.90"N, 100°03'7.60"E), and the specific culture is as follows: 10 g of soil sample is taken in 10 mL of sterile water as a mother liquor, 1 mL of the mother liquor is respectively added into sterile water to dilute the concentration to 10 -3 , 10 -4 , 10 -5 , respectively, and is coated on the basic medium with rice straw, corn stalk, wheat stalk, sugarcane residue, pine wood, poplar wood and other lignocellulose as the only carbon source, and is cultured in a 20°C constant temperature incubator. After the bacteria grow, round smooth or wrinkled single colonies are picked from the plate for transfer and purification.

[0057] (2) Strain screening

[0058] The strain selected and purified above is picked for single colony inoculation in 10 mL of LB liquid medium, and is cultured at 20°C, 180 rpm for 2d to prepare a seed liquid. The seed liquid obtained above is inoculated in 100 mL of rice straw liquid medium at a 2% inoculation amount, and is cultured at 20°C, 180 rpm for 7d. Then, 0.2 mol / L sodium pyrophosphate solution is added to the obtained fermentation liquor to pH=12, and the supernatant is taken after the room temperature standing for 24h. The scanning is performed in the range of 200nm-320nm by using a UV-visible spectrophotometer, and each group is repeated for 3 times. The strain with absorbance value >0 has the ability to produce humic acid, and 8 strains producing humic acid are preliminarily screened according to the absorbance value.

[0059] The 8 strains obtained by the preliminary screening above are picked for single colony inoculation in 10 mL of LB liquid medium, and are cultured at 20°C, 180 rpm for 2d to prepare a seed liquid. The seed liquid of each strain is inoculated in equal amount of liquid medium with different lignocelluloses (rice straw, corn stalk, wheat stalk, sugarcane residue, pine wood, poplar wood as the only carbon source) as the carbon source at a 2% inoculation amount, and is fermented at 20°C, 180 rpm for 7d to preliminarily determine the yield of humic acid in the fermentation liquor. Then, 0.2 mol / L sodium pyrophosphate alkali extraction solution is added to the fermentation liquor to pH=12, and the supernatant is taken after the room temperature standing for 24h. The pH is adjusted to 1-2 by using hydrochloric acid, and is statically placed overnight. After centrifugation, the supernatant is discarded, and the precipitate is dried to obtain crude humic acid. The strain with stronger humic acid production capacity is selected and named as DLS188. It can be known from the humic acid yield comparison of the strain DLS188 under different carbon sources shown in Table 1 that the strain DLS188 can produce humic acid under the conditions that rice straw, corn stalk, wheat stalk and sugarcane residue are the only carbon source, and the yield of humic acid is the highest under the condition that rice straw is the only carbon source. In addition, the strain DLS188 hardly grows in pine wood and poplar wood, and the yield of humic acid is zero. Figure 1

[0060] 2. Strain identification ​

[0061] (1) Morphological characteristics identification

[0062] The strain DLS188 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 strain cells present a chain-like branched hyphae structure, like a net-like or filamentous substance, which is different from the single cell morphology of conventional bacteria ( Figure 2 a), pure culture mycelium appears white ( Figure 2 b). This is a typical morphological feature of Streptomyces.

[0063] (2) Molecular biological identification

[0064] The DNA of strain DLS188 was extracted, and PCR amplification was performed using the universal primers 27F and 1492R for bacterial 16S identification (27F and 1492R were purchased from Sangon Biotech (Shanghai) Co., Ltd.) as primers using Taq enzyme. The PCR amplification reaction system (40 μL) was: 1 μL each of forward and reverse primers, 1 μL of DNA template, and 37 μL of Mix; the PCR amplification program was: 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.

[0065] 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). The 16S rRNA sequence phylogenetic tree was constructed using MEGA7.0 using the neighbor-joining method (e.g., Figure 3 Phylogenetic analysis showed that strain DLS188 clustered with Streptomyces cyaneofuscatus in a clade with 88% support. Based on colony morphology, microscopic photographs, and a phylogenetic tree constructed based on 16S rRNA sequence alignment, strain DLS188 was identified as a Streptomyces strain.

[0066] Example 2 Determination of humic acid production performance of strains

[0067] Take the seed liquid (OD 600=0.313) was inoculated with 2% inoculum into 100 mL of rice straw carbon source liquid basal medium, with the control being a medium without bacteria, and three biological replicates. After shaking culture at 20°C and 180 rpm for 7 days, 10 mL of the 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 4 .

[0068] Depend on Figure 4 Comparative analysis of the humic acid content of the fermentation supernatant of strain DLS188 and that of CK (uninoculated rice straw-based salt liquid medium) showed that the humic acid content per gram of DLS188 fermentation broth was 14.47%, while the humic acid content per gram of the control (CK) was 9.59%. A highly significant difference (p < 0.01) was observed between the DLS188 and CK cultures, with the humic acid content in the DLS188 fermentation supernatant increasing by 50.89%. This further demonstrates the strong humic acid production capacity of strain DLS188.

[0069] Example 3 Determination of the growth-promoting performance of strains on crops

[0070] 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 pack 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 DLS188 was inoculated into 50 mL LB liquid medium and cultured at 20℃ and 180 rpm for 2 days to obtain seed liquid, and the OD was measured. 600 =0.313. 50 mL of strain DLS188 seed solution (OD 600 =0.313), and cultured in a 25°C constant temperature incubator until the mycelium covered the bag, obtaining DLS188 bacterial fertilizer, which was used for subsequent growth promotion experiments.

[0071] 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 DLS188 has a growth-promoting effect on wheat growth.

[0072] The above-mentioned DLS188 bacterial fertilizer was mixed with the barren soil in the suburbs of Chenggong, Kunming at a ratio of 125g of bacterial fertilizer per kilogram of soil, and then divided into flower pots (400g / pot) by equal weight, 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 + DLS188 bacterial fertilizer) was monitored. The growth comparison of wheat at 12d and 56d after sowing is shown in Figure 5 .Depend on Figure 5 It can be seen that the growth of wheat after adding DLS188 bacterial fertilizer is significantly better than that of the pure soil in the control group.

[0073] 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 6 .Depend on Figure 6 The results showed that there was no significant difference in leaf width between the two groups, while the experimental group with DLS188 showed significantly higher plant height, leaf length, and leaf number than the control group (CKT), with extremely significant differences (p < 0.01). This further demonstrates that the humic acid fertilizer produced by the strain DLS188 using agricultural straw can promote wheat growth.

[0074] Example 4 Determination of the Effect of Bacteria on Soil

[0075] 1. Dilution coating experiment

[0076] 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 7 .

[0077] Depend on Figure 7 The plates in group b (soil treated with DLS188 fertilizer) are densely populated with microbial colonies (>50 colonies / plate), while the plates in group a (soil not treated with DLS188 fertilizer) are relatively few (<10 colonies / plate). The colony counts in the soil treated with DLS188 fertilizer were significantly higher than those in the soil not treated with DLS188 fertilizer, indicating that the addition of DLS188 fertilizer can, to a certain extent, increase the number and species richness of culturable microbial communities in the soil.

[0078] 2. Determination of soil humus content

[0079] Take the two groups of flower pot soil in which wheat is planted in Example 3, take 10 g of soil from each pot (control group numbers CKT-1, CKT-2, CKT-3, experimental group numbers S188-1, S188-2, S188-3), and send to Wuhan Pnaes Detection Company with dry ice to determine the content and change of humus in the soil. The detection principle is as follows: the soil sample is baked at (105±5)℃ to constant weight, and the content of dry matter and water is calculated by the difference in mass before and after baking, expressed by mass fraction. Soil humus is divided into soluble humus (humic acid and fulvic acid) and insoluble humus (humin) according to its solubility. The soluble humus is extracted with 0.1 mol / L sodium pyrophosphate-sodium hydroxide mixed solution, and the total amount of humic acid and fulvic acid is determined by potassium dichromate oxidation volumetric method. The extraction liquid is acidified and precipitated to separate humic acid, and its content is determined to calculate the content of fulvic acid. The total carbon content of the soil sample is determined, and the humin content is obtained by subtracting the content of humic acid and fulvic acid. The specific test results are shown in Table 1 and Figure 8 .

[0080]

[0081] The results in Table 1 and Figure 8 show that the humus content (including: humic acid, fulvic acid, humin) in the soil added with DLS188 is significantly improved. Among them Figure 8-I It can be seen that the humus content in the experimental group has a very significant difference (p<0.01) with the control group, and the humus content in the experimental group is increased by 4.93 g / kg compared with the control. Figure 8-II , 8-Ⅲ, 8-Ⅳ can be seen that the humic acid, fulvic acid and humin content in the experimental group has a very significant difference (p<0.01) with the control group, and the humic acid, fulvic acid and humin content in the experimental group is increased by 0.62 g / kg, 1.42 g / kg and 2.88 g / kg respectively compared with the control.

[0082] 3. Soil metagenome sequencing analysis

[0083] (1) Agarose gel electrophoresis analysis

[0084] 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 S188-1, S188-2, S188-3). 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 9 and Table 2.

[0085]

[0086]

[0087] Figure 9 The differences in the brightness and total amount of metagenomic DNA can also be seen in Table 2. Compared with the control group without DLS188 fertilizer, the concentration and total amount in the experimental group with DLS188 fertilizer increased significantly, indicating that the addition of DLS188 fertilizer to the soil increased the number and species of microorganisms to a certain extent.

[0088] (2) Species composition analysis

[0089] 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.

[0090] 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 DLS188 fertilizer and the experimental group (number: S188-1, S188-2, S188-3) of the soil with DLS188 fertilizer are as follows: Figure 10A-10Cshown.

[0091] At the phylum level (see Figure 10A ), it can be seen that the relative abundance of Proteobacteria (pink area) and Actinobacteria (green area) is the highest, accounting for more than 0.5. The difference between the experimental group and the control group is obvious in some microbial phyla, such as Proteobacteria and Bacteroidetes (obvious abundance changes).

[0092] At the genus level (see Figure 10B ), it can be seen that, except for uncategorized or less species of microorganisms, the abundance of Sphingomonas (pink area) in CKT and Nocardioides (orange area) accounts for a large proportion. The relative abundance of Streptomyces (green area) in the experimental group is relatively high. The experimental group and the control group have obvious abundance changes in Sphingomonas, Streptomyces and Nocardioides, and the species abundance between the two groups shows opposite trends.

[0093] At the species level (see Figure 10C ), it can be seen that uncategorized microorganisms account for the largest proportion, more than 0.5, in addition to the abundance of Acidobacteria bacterium (pink area) in CKT and Chloroflexi bacterium (green area) accounts for a large proportion. The relative abundance of Streptomyces phaeochromogenes (purple area) and Ohtaekwangia koreensis (cyan area) in the experimental group is relatively high. The difference between the experimental group and the control group is obvious in some species, such as Acidobacteria bacterium, Sphingomonas edaphi and Chloroflexi bacterium, and the species abundance has changed significantly.

[0094] In summary, Figure 10 shows the relative abundance of different microbial phyla, genera and species in the experimental group (numbered: S188-1, S188-2, S188-3) of soil applied with DLS188 microbial fertilizer and the control group (numbered: CKT-1, CKT-2, CKT-3) of soil without DLS188 microbial fertilizer, which shows the composition difference of microbial community and reflects the influence of DLS188 microbial fertilizer on the microbial population in the soil.

[0095] (3) Dimensionality reduction analysis based on species abundance

[0096] 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 DLS188 bacterial fertilizer (No.: CKT-1, CKT-2, CKT-3) and the experimental group of soil with DLS188 bacterial fertilizer (No.: S188-1, S188-2, S188-3) are as follows Figure 11 As shown in (a, b, c).

[0097] As can be seen, the control group (CKT, blue) and the experimental group (S188, 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 56.34% and 40.23% of the total variation of the data at the phylum level, respectively (see Figure 11 a). At the genus level, the first two axes of the PCoA analysis explained 62.8% and 23.8% of the total variation of the data at the genus level, respectively (see Figure 11 b). At the species level, the first two axes of the PCoA analysis explained 59.78% and 25.12% of the total variation in the data at the species level, respectively (see Figure 11 c). Significant differences in overall community composition were already observed between the control (CKT, blue) and experimental (S188, 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 DLS188 fertilizer significantly altered the soil microbial community structure.

[0098] (4) Analysis of species abundance differences

[0099] The abundance difference between species in the control group (numbered: CKT-1, CKT-2, CKT-3) of soil without DLS188 microbial fertilizer and the experimental group (numbered: S188-1, S188-2, S188-3) of soil with DLS188 microbial fertilizer was evaluated based on the zero-inflated model using metagenomeSeq analysis after sequencing of each group of samples. The significantly different species at the Phylum level were visualized using a heatmap visualization method. Blue represents a lower abundance of the taxon (negative value), while red represents a higher abundance (positive value). Yellow represents an intermediate value of abundance. By comparing the colors between samples, the differences in microbial community composition can be intuitively understood.

[0100] As shown in the results Figure 12 Candidatus Levybacteria, Candidatus Yonathbacteria, Zoopagomycota, Candidatus Portnoybacteria, and Candidatus UhrbacteRia showed significantly high abundance in the experimental group (numbered: S188-1, S188-2, S188-3), while in the control group (numbered: CKT-1, CKT-2, CKT-3) the abundance was lower. Conversely, Nitrospirae, CRenarchaeota, Thaumarchaeota, and Candidatus Spechtbacteria showed significantly high abundance in the control group (numbered: CKT-1, CKT-2, CKT-3), while in the experimental group (numbered: S188-1, S188-2, S188-3) the abundance was lower. Figure 12 The differences in microbial community composition between the control group and the experimental group were demonstrated. Through cluster analysis of rows and columns, it was clear that there were significant abundance differences between groups at the Phylum level, indicating that the addition of DLS188 microbial fertilizer significantly changed the richness and composition of soil microbial communities.

[0101] (5) Functional relative abundance analysis

[0102] The CAZy database was used to annotate and classify the predicted genes in terms of function, and to evaluate the impact of adding DLS188 microbial fertilizer on the functional abundance of soil microorganisms. Specifically, the functional annotation and abundance information of the samples in the CAZy database were plotted into a heatmap for the control group (numbered: CKT-1, CKT-2, CKT-3) and the experimental group (numbered: S188-1, S188-2, S188-3) of soil samples with DLS188 microbial fertilizer, and clustering was performed from the function and sample levels. The rows in the heatmap represent different carbohydrate-active enzyme families, and the colors represent the relative abundance of each carbohydrate enzyme family in different samples: red indicates a higher abundance (>0) of the carbohydrate enzyme family in the sample; blue indicates a lower abundance (<0); yellow represents a near-neutral abundance (0), i.e., the carbohydrate enzyme has a moderate abundance in the sample. The abundance of carbohydrate-active enzymes (CAZymes) in different samples is shown in the heatmap as follows. Figure 13

[0103] Figure 13 The heatmap visually displays the expression levels of different carbohydrate-active enzymes in each sample, as shown in the following table. For the control group (numbered: CKT-1, CKT-2, CKT-3), the carbohydrate enzyme abundance in the GT35 and GH102 families shows a higher level, while the GH26 and GH30 carbohydrate enzyme families show a lower abundance. In contrast, the experimental group (numbered: S188-1, S188-2, S188-3) shows the opposite, with higher abundance in the GH26 and GH30 families and lower abundance in the GT35 and GH102 families. The differences in abundance of carbohydrate-active enzyme families between the control group and the experimental group can be clearly seen in the figure. Specifically, the carbohydrate enzyme families in different sample groups have different expression levels, which reflects the differences in the carbohydrate metabolic potential of microbial communities in soil after the addition of DLS188 microbial fertilizer.

[0104] (6) Biogeochemical cycle function analysis

[0105] By analyzing the relative abundance and inter-group differences of carbon cycle, nitrogen cycle, phosphorus cycle, and sulfur cycle functional genes, the impact of adding DLS188 microbial fertilizer on the biogeochemical cycle function of soil microorganisms was evaluated. Specifically:

[0106] ​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 DLS188 microbial fertilizer and the experimental group (numbered: S188-1, S188-2, S188-3) of the soil applying DLS188 microbial fertilizer was analyzed. The tree diagram on the left and top of the metagenomeSeq heat map showed the clustering relationship of different samples and genes. The clustering results showed the similarity of the metabolic pathways between the samples and genes. The vertical axis showed different genes related to carbon cycle, and the horizontal axis showed the gene expression data of different samples. The expression amount of the genes was 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 Figure 14A-14D .

[0107] In the carbon cycle metagenomeSeq heat map shown in Figure 14A , the experimental group (numbered: S188-1, S188-2, S188-3) of the soil applying DLS188 microbial fertilizer showed higher expression on the genes such as beta-glucuronidase, alpha-amylase and mauA, while the control group (numbered: CKT-1, CKT-2, CKT-3) of the soil without applying DLS188 microbial fertilizer showed lower expression on these genes.

[0108] In the nitrogen cycle metagenomeSeq heat map shown in Figure 14B , the control group (numbered: CKT-1, CKT-2, CKT-3) of the soil without applying DLS188 microbial fertilizer showed higher expression on some nitrogen cycle genes such as niRS, niRK and noRB, while the experimental group (numbered: S188-1, S188-2, S188-3) of the soil applying DLS188 microbial fertilizer showed relatively lower expression on these genes.

[0109] In the phosphorus cycle metagenomeSeq heat map shown in Figure 14C , the experimental group (numbered: S188-1, S188-2, S188-3) of the soil applying DLS188 microbial fertilizer showed higher expression on multiple genes, especially on the genes such as phoP, phoR and olpA.

[0110] In the sulfur cycle metagenomeSeq heat map shown in Figure 14DIn the sulfur cycle metagenomeSeq heat map shown, the control group (number: CKT-1, CKT-2, CKT-3) of the soil without DLS188 bacterial fertilizer application had higher expression in multiple sulfur metabolism genes such as soxZ, sat and sqR, while the experimental group (number: S188-1, S188-2, S188-3) of the soil with DLS188 bacterial fertilizer application showed relatively low expression.

[0111] 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.

[0112] 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 DLS188 fertilizer and the experimental group (S188) of the soil with DLS188 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 15 A-15D.

[0113] exist Figure 15 In the carbon cycle of A, R = 0.741, 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 15 In the nitrogen cycle of B, R=0.519, P=0.1. Compared with the carbon cycle, the R value of the nitrogen cycle is lower, but there are still differences between the groups. Figure 15 In the phosphorus cycle of C, R = 0.851, P = 0.1. Similar to the carbon cycle, there are large differences between different sample groups in the phosphorus cycle. Figure 15 The sulfur cycle of D also showed significant differences between the two groups (R = 0.593, P = 0.1). This suggests 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 DLS188 fertilizer added to the soil.

[0114] 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.

[0115] The foregoing description of the disclosed embodiments enables a person skilled in the art to make or use the application. Modifications of these embodiments will occur to persons of skill in the art, and that the appended claims are intended to cover all such modifications that do not depart from the true spirit and scope of the application. Therefore, the application is not limited to the embodiments shown but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A strain of Streptomyces DLS188, characterized in that It is classified and named Streptomyces sp. and deposited in the General Microbiology Center of China Culture Collection Administration on December 30, 2024. The deposit number is CGMCC No. 33250, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

2. Use of Streptomyces DLS188 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 Streptomyces DLS188 according to claim 1.

4. A bacterial fertilizer, characterized in that The bacterial fertilizer comprises the Streptomyces DLS188 culture or a processed product thereof according to claim 3.

5. Use of Streptomyces DLS188 according to claim 1 in promoting crop growth.

6. The use according to claim 5, characterized in that The crop is wheat.

7. The use according to claim 5, characterized in that It has a promoting effect on plant height, leaf length and leaf number.

8. Use of Streptomyces DLS188 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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