Penicillium chrysogenum strain dls189 and its use

Abstract

The application discloses a Penicillium chrysogenum DLS189 and application thereof, relates to the technical field of microorganisms, and has a preservation number of CGMCC NO.41739. The strain can effectively utilize rice straw and / or sugarcane residue to produce humic acid, and the humic acid content in the fermentation supernatant of the rice straw is 16.84% per gram. The fermentation product of the DLS189 is applied to crops, so that the growth condition of the crops can be significantly improved, and the humic substance content in the soil is improved. Meanwhile, the result of metagenomics analysis shows that, compared with a control group, after the DLS189 is applied to the soil, the species beta diversity is significantly changed, and the functional gene abundance and the difference between groups are significantly changed. The above shows that the DLS189 has a wide application prospect in the utilization of agricultural straw and soil remediation.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and more specifically to a strain of Penicillium chrysogenum DLS189 and its applications. Background Technology

[0002] Humic acid is a type of natural organic matter, mainly found in soil, sediments, and water bodies. It is a complex mixture formed after the long-term decomposition and transformation of organic matter. Humic acid is one of the main components of soil humus and has strong adsorption, water retention, and fertility regulating effects. It is the final product of organic matter decomposition and usually exists in acidic form in soil and water bodies. In my country, humic acid is classified into three categories based on its solubility in solvents and its color: fulvic acid, humic acid glycosides, and humic acid glycosides. As a natural organic polymer, humic acid has multiple functions, including improving soil, enhancing soil fertility, and promoting plant growth. Humic acid can improve the physical and chemical properties of soil, increase soil organic matter content, enhance soil water and fertilizer retention capacity, and promote plant growth and development. In recent years, the production of humic acid using microbial fermentation technology has become a research hotspot.

[0003] Lignocellulose, the most abundant renewable biological resource in the biosphere, is produced globally at a rate of 150 billion tons annually, of which straw accounts for 6 billion tons. China's annual straw production is approximately 1.1 billion tons, accounting for about one-fifth of the global straw resource. Agricultural straw, as a rich biomass resource, has high nutritional value. However, traditional methods of straw disposal, such as returning straw to the field and burning it, are not only inefficient but may also lead to environmental pollution and resource waste.

[0004] Therefore, how to efficiently utilize agricultural straw resources and improve the utilization rate of agricultural waste has become an urgent problem to be solved by those skilled in the art. Summary of the Invention

[0005] In view of this, the present invention provides a strain of Penicillium chrysogenum DLS189 and its applications.

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

[0007] A strain of Penicillium chrysogenum DLS189, with accession number CGMCC NO.41739.

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

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

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

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

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

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

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

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

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

[0017] Beneficial Effects: This invention isolated a Penicillium chrysogenum strain, DLS189, from the soil of Canglang Peak in Cangshan Mountain, Dali. This strain can effectively utilize rice straw and / or sugarcane bagasse to produce humic acid, with a humic acid content of 16.84% per gram of fermentation supernatant after rice straw fermentation. Applying the fermentation product of S189 to crops significantly increased wheat plant height, leaf length, and leaf width, and increased soil humus content by 4.04 g / kg; for Chinese cabbage, the average increases in plant height, root length, leaf width, leaf number, above-ground fresh weight, and above-ground dry weight were 3.4 cm, 2.74 cm, 2.54 cm, 1.2 leaves, 1.49 g, and 0.09 g, respectively. Metagenomic analysis results showed that, compared with the control group, the application of DLS189 to the soil significantly altered species β-diversity, functional gene abundance, and inter-group differences. These findings indicate that DLS189 has broad application prospects in agricultural straw utilization and soil remediation. Attached Figure Description

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

[0019] Figure 1 The images show the morphological identification of strain DLS189, where a represents the colony morphology and b represents the microscopic morphology of the strain.

[0020] Figure 2 This is the phylogenetic tree of strain DLS189.

[0021] Figure 3 The growth status of strain DLS189 at different temperatures is shown. "+" indicates the growth status of the strain, with more "+" indicating better growth, " / " indicates no growth of the strain, "1" indicates ck (control group without inoculation), and "2" indicates experimental group inoculated with DLS189.

[0022] Figure 4 The yield of humic acid produced by strain DLS189 under different carbon sources.

[0023] Figure 5 To accurately determine the humic acid content in the fermentation supernatant of DLS189 strain.

[0024] Figure 6 The effect of strain DLS189 on wheat growth phenotype.

[0025] Figure 7 This study aimed to quantitatively determine the effects of strain DLS189 on wheat growth traits.

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

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

[0028] Figure 10 The image shows a 1% gel electrophoresis result of soil metagenomic DNA, where M is a 15000bp DNA marker, 1-3 represent CKT1, CKT2, and CKT3 respectively, and 4-6 represent S1891, S1892, and S1893 respectively.

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

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

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

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

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

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

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

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

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

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

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

[0040] Figure 22 The effect of strain DLS189 on the growth phenotype of Chinese cabbage.

[0041] Figure 23 This study aimed to quantitatively determine the effects of strain DLS189 on the growth traits of Chinese cabbage.

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

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

[0044] The culture medium used in this invention is as follows:

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

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

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

[0048] Sodium carboxymethyl cellulose basal medium: sodium carboxymethyl cellulose 5 g / L, (NH4)2SO4 4 g / L, MgSO4·(7H2O) 1.2 g / L, CaCl2 0.3 g / L, K2HPO4 1 g / L, KH2PO4 1 g / L, NaNO3 1 g / L, pH natural.

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

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

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

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

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

[0054] Poplar carbon source basal culture medium: poplar wood residue 20g / L, (NH4)2SO4 4g / L, Mg / SO4MgSO4·(7H2O) 1.2g / L, CaCl2 0.3g / L, K2HPO4 1g / L, KH2PO4 1g / L, NaNO3 1g / L, pH natural.

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

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

[0057] Example 1

[0058] 1. Isolation of strains

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

[0060] 2. Screening of strains

[0061] Preliminary screening: The selected and purified strains were inoculated into 10 mL of LB liquid medium (containing 10 μL of kanamycin) and cultured at 20℃ and 180 rpm for 2 days to prepare seed culture. The seed culture was then inoculated at 2% of 100 mL of rice straw carbon source basal medium and cultured at 20℃ and 180 rpm for 7 days. Then, 0.2 mol / L sodium pyrophosphate extract was added to the fermentation broth to bring the pH to 12, and the mixture was allowed to stand at room temperature for 24 h. The supernatant was scanned between 200 nm and 320 nm. Three biological replicates were set up for each group, and strains with absorbance values ​​greater than zero were selected. Eight humic acid-producing strains were preliminarily screened.

[0062] Secondary screening: While ultraviolet spectroscopy can determine whether a strain produces humic acid, it cannot accurately measure the yield. Therefore, secondary screening is necessary to further determine the humic acid yield of each strain. The strains obtained from the initial screening were inoculated onto a basal medium with lignocellulose (such as rice straw, corn stalks, wheat straw, sugarcane bagasse, pine wood, and poplar wood residue) as the sole carbon source. Fermentation was carried out at 20℃ and 180 rpm for 7 days, and the humic acid yield in the fermentation broth was preliminarily determined. Then, 0.2 mol / L sodium pyrophosphate alkaline extract was added to the fermentation broth to bring the pH to 12. The mixture was allowed to stand at room temperature for 24 hours, and then the pH was adjusted to 1-2 with hydrochloric acid and allowed to stand overnight. Centrifugation was performed, the supernatant was discarded, and the precipitate was dried. The precipitate obtained was crude humic acid. Based on the yield of crude humic acid, strains with high humic acid production capacity were screened and named DLS189 (hereinafter referred to as S189).

[0063] In the initial screening, strain S189 showed higher absorbance differences than the control at OD200nm-OD240nm. A maximum absorption peak was observed at OD200nm, with an absorbance difference of 0.176 compared to the control; the largest absorbance difference at OD210nm was 1.344 compared to the control. This indicates that strain S189 has the ability to produce humic acid. In the secondary screening, strain S189 was able to produce humic acid using rice straw, corn stalks, wheat straw, and sugarcane bagasse as the sole carbon source, with rice straw yielding the highest amount (0.025 g / 10 mL). Furthermore, S189 could not grow in pine and poplar sawdust, with almost zero humic acid production (see Appendix). Figure 4 ).

[0064] 3. Identification of strains

[0065] (1) The selected S189 strain was inoculated on PDA medium and cultured at 25°C for 5 days. The size and color of the colonies were then observed and recorded. Microscopic images of the strain were taken using a Nikon ECLIPSENi-U biological microscope, and the morphological characteristics of the strain were recorded.

[0066] The results showed that strain S189 is a typical Penicillium fungus, with bluish-green colonies that are dry, loose, and have a fuzzy appearance. The mycelium penetrates the surface of the culture medium and invades deeper layers, producing yellow metabolites that cause discoloration of the medium (see Appendix). Figure 1 (a) Microscopic morphology of the strain shows that its conidia are spherical and bluish-green in color (see Appendix). Figure 1 (b)

[0067] (2) DNA was extracted from strain S189 and amplified by PCR using Taq polymerase with universal primers for the intergenic spacer region (ITS) sequence of fungal ribosomal rDNA (ITS4: 5'-TCCTCCGCTTATTGATATGC-3', ITS5: 5'-GGAAGTA AAAGTCGTAACAAGG-3'). Amplification parameters: pre-denaturation 94℃, 4 min; denaturation 94℃, 30 s; annealing 55℃, 35 s; extension 72℃, 90 s, 32 cycles; extension 72℃, 5 min. After gel electrophoresis, the PCR products were sent to Sangon Biotech 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, a phylogenetic tree was constructed using the maximum likelihood estimation method in MEGA 7.0 to determine the species position of the strain.

[0068] The phylogenetic tree shows that strain S189 clusters with Penicillium chrysogenum in one clade, with a support rate of 92% (see appendix). Figure 2 ).

[0069] Based on colony morphology, microscopic photographs, and a phylogenetic tree constructed based on ITS rRNA sequence alignment, strain S189 was identified as Penicillium chrysogenum and named Penicillium chrysogenum S189.

[0070] The strain DLS189 was deposited on December 30, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.41739 and classified as *Penicillium chrysogenum*. The deposit address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0071] Example 2

[0072] 1. Inoculate the S189 strain into 10 mL of LB medium (containing 10 μL of kanamycin) and culture at 20 °C for 2 days to prepare seed culture.

[0073] Seed culture was inoculated at a rate of 2% into rice straw carbon source basal medium, with three biological replicates. The effects of different temperatures (4℃, 25℃, 30℃, 37℃, 45℃) on the growth of the strain were examined under natural pH conditions.

[0074] The results showed that strain S189 grew best at 30℃, producing bluish-green colonies with dense hyphae. Growth decreased progressively at 25℃ and 37℃. Furthermore, the strain ceased growth at 4℃ and 45℃. S189 could grow within the 25℃-37℃ range, indicating that this is a low-temperature fungus (see appendix). Figure 3 ).

[0075] 2. The S189 strain was inoculated into 10 mL of LB liquid culture medium and cultured with shaking at 20℃ and 180 rpm for 2 days to prepare a seed culture. This seed culture was then inoculated at a rate of 2% into 100 mL of rice straw carbon source basal medium. The control was an uninoculated medium, and three biological replicates were performed. After 7 days of shaking culture at 20℃ and 180 rpm, 10 mL of the fermentation supernatant and liquid culture medium were collected and analyzed using Wuhan Punes Testing Technology Co., Ltd. (NY / T 1971-2010 Determination of Humic Acid Content in Water-Soluble Fertilizers) to determine the humic acid content in the fermentation broth.

[0076] Appendix Figure 5 The results show a comparative analysis of the humic acid content in the fermentation supernatant of strain S189 and the humic acid content in the control group (CK, uninoculated rice straw basal salt liquid medium). The results indicated that the humic acid content per gram of S189 fermentation broth was 16.84%, while the humic acid content in the control group (CK) was 9.59%. There was a highly significant difference (p<0.01) between S189 and the control group (CK), with S189 exhibiting a 76% higher humic acid content than the control group. This further demonstrates the strong humic acid production capacity of strain S189.

[0077] Example 3

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

[0079] The growth of wheat in the control group (pure soil) and the experimental group (pure soil + S189 microbial fertilizer) was observed, and their agronomic traits were measured and analyzed.

[0080] Preparation method of S189 microbial fertilizer: Mix rice straw powder and wheat bran in a 7:3 ratio, add water and stir until moist but not waterlogged. Dispense into mushroom bags and seal them completely with rings. Sterilize each bag (1.5 kg) and store for later use (121℃, 120 min). Inoculate the strain into 50 mL of LB liquid medium (containing 50 μL of kanamycin) and culture at 20℃ and 180 rpm for 2 days to prepare seed culture. Inoculate the seed culture into sterilized mushroom bags and culture in a 25℃ incubator until the mycelium covers the entire bag.

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

[0082] Appendix Figure 6 The images show wheat growth at 12 and 56 days after sowing. It can be seen that the wheat growth after the addition of S189 microbial fertilizer was significantly better than that of the control group in pure soil. Data analysis of wheat growth traits such as plant height (a), leaf length (b), leaf width (c), and number of leaves (d) using SPASS software showed that, compared to the control group (CKT), the leaf width after the addition of S189 microbial fertilizer was significantly higher (p<0.05), and the plant height and leaf length were extremely significantly higher (p<0.01) than the control group (see Appendix). Figure 7 This further demonstrates that the humic acid fertilizer produced by strain S189 from agricultural straw can promote wheat growth.

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

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

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

[0086]

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

[0088] The results showed that the humic content (including humic acid, fulvic acid, and humin) in soil treated with S189 microbial fertilizer was significantly increased. Specifically, the humic content in the experimental group was significantly different from that in the control group (p<0.01), increasing by 4.04 g / kg compared to the control. The humic acid and humin contents in the experimental group were also significantly different from those in the control group (p<0.01), increasing by 0.74 g / kg and 2.21 g / kg, respectively. The fulvic acid content in the experimental group was significantly different from that in the control group (p<0.05), increasing by 1.1 g / kg compared to the control.

[0089] 3. Changes in Soil Microbial Quantities: To investigate whether there were differences in the microbial community in soils treated with S189 microbial fertilizer, a dilution-spreading experiment was conducted on the soil previously planted with wheat in step 1. 10g of the wheat-planted soil was weighed and diluted to 10mL of sterile water. -5 The samples were spread on PDA medium, with three replicates per pot. They were incubated at 25°C for 3 days, and the changes in colony counts on the plates were observed.

[0090] Appendix Figure 9 To dilute to 10 -5 The growth period of soil sample solutions on PDA medium was observed. Group a represents the control group, and group b represents the experimental group treated with S189 microbial fertilizer, with three biological replicates per group. It can be seen that group b plates were teeming with microorganisms (>1000 colonies / plate), while group a plates had fewer microorganisms (<10 colonies / plate). The colony count in the experimental groups was significantly higher than that in the control group, indicating that the addition of S189 microbial fertilizer can, to some extent, increase the number and species richness of culturable microbial communities in the soil.

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

[0092] Appendix Figure 10 The image shows a 1% gel electrophoresis result of the submitted samples after electrophoresis at 120V for 30 min, with a sample loading volume of 5 μl. Differences in the brightness and total amount of metagenomic DNA can be observed in different treatments. Compared to the control group CKT, the concentration and total amount of S189 in the experimental group were significantly increased, indicating that the addition of S189 to the soil increased the types and numbers of microorganisms to some extent.

[0093] Table 2. Sample DNA Detection Results

[0094]

[0095] Appendix Figure 11 - Appendix Figure 13 This section presents bar charts showing the species composition of CKT and S189 at the phylum, genus, and species levels. The charts clearly illustrate the species composition of each sample and the proportion of different species within each sample. Using R software, bar charts were created for the dominant species (the top 30 species in overall abundance) at each taxonomic level in each sample. The horizontal axis (x-axis) shows the different sample groups, with three replicates per group. The vertical axis (y-axis) represents the relative abundance of each microbial community, ranging from 0 to 1. The height of each bar represents the relative abundance of microorganisms in that sample group. (Appendix) Figure 11 The abundance at the phylum level is shown: the pink area (Proteobacteria) and the green area (Actinobacteria) have the highest relative abundance, exceeding 0.5%. Significant differences exist between S189 and CKT in certain microbial phyla, such as obvious abundance variations in Actinobacteria and Proteobacteria. (Appendix) Figure 12 The abundance at the genus level is shown: Excluding unclassifiable or few species, *Sphingomonas* (pink area) and *Nocardioides* (orange area) accounted for a large proportion of abundance in CKT. *Steroidobacter* had a relatively high abundance in the blue area of ​​S189. There were significant abundance variations in *Sphingomonas*, *Steroidobacter*, and *Nocardioides* between S189 and CKT, with opposite trends in species abundance between the two groups. (Appendix) Figure 13 Abundance at the species level: the proportion of unclassifiable microorganisms exceeded 0.5. Furthermore, in CKT, *Acidobacteria bacterium* (pink area) and *Chloroflexibacterium* (orange area) accounted for a large proportion of abundance. In S189, *Acidobacteria bacterium* (pink area) and *Myxococcales bacterium* (green area) had higher relative abundances. Significant differences were observed between S189 and CKT in certain species, such as significant changes in the abundance of *Myxococcales bacterium* and *Proteobacteria bacterium*. These results, presented as stacked bar charts, illustrate the relative abundance of different microbial phyla, genera, and species in the experimental and control groups, demonstrating the differences in microbial community composition and reflecting the impact of the S189 treatment on the soil microbial community.

[0096] This invention performs PCoA analysis based on the unconstrained ordination (Classical Multidimensional Scaling, cMDScale) analysis method, with appended... Figure 14 The study illustrates the species structure differences between CKT and S189 at the three taxonomic levels: phylum, genus, and species. PCoA1 represents the first principal component and its contribution to sample differences; this axis shows the major differences among most samples. PCoA2 represents the second principal component and its contribution to sample differences, showing the second largest major difference. The CKT group (blue) and the S189 group (dark blue) are clearly separated on the principal coordinate plot. The microbial community structure of the S189 group clusters together, while its structure is more distant from that of the CKT group. This indicates a significant structural difference between the microorganisms in these two groups. (Appendix) Figure 14 In the middle section (a), the first two axes of the PCoA analysis explained 78.51% and 14.48% of the total variance at the gate level, respectively. (Appendix) Figure 14In section b, the first two axes of the PCoA analysis explained 59.9% and 27.86% of the total variance at the genus level, respectively. (Appendix) Figure 14 In the middle, the first two axes of the PCoA analysis explained 57.49% and 28.61% of the total variance at the species level, respectively. (From the appendix...) Figure 14 It can be seen that at the phylum level, the CKT group and the S189 group already showed significant differences in overall community composition; at the genus and species level, these differences are further amplified, specifically manifested in the distribution differences of individual genera or species. Therefore, with the addition of S189, the soil microbial community structure underwent significant changes.

[0097] Appendix Figure 15 This study used metagenome-Seq analysis based on a zero-inflation model to assess abundance differences between groups under different treatments (with and without S189). Heatmaps were used to visualize species with significant differences at the phylum level. Blue indicates low abundance (negative values) for that taxa, while red indicates high abundance (positive values). Yellow indicates abundance close to the median. The color contrast between samples provides a visual understanding of the differences in microbial community composition. *Calditrichaeota*, *Spirochaetes*, *Candidatus Buchananbacteria*, and *Proteobacteria* showed significantly high abundance in S189 but low abundance in CKT. Conversely, *Candidatus Liptonbacteria* and *Actinobacteria* showed significantly high abundance in CKT but low abundance in S189. (Appendix) Figure 15 The study demonstrated the differences in microbial community composition between CKT and S189. Through row and column cluster analysis, significant abundance differences were identified between groups at the phylum level, indicating that the addition of S189 significantly altered the richness and composition of the soil microbial community.

[0098] The CAZy (Carbohydrate-Active enZYmes) database is a database specifically designed for classifying and annotating enzymes related to carbohydrate metabolism. This study utilized the CAZy database to functionally annotate and classify predicted genes, and to assess the impact of adding S189 on soil microbial functional abundance. (Appendix) Figure 16To create heatmaps based on the functional annotations and abundance information of all samples in the CAZy database, clustering was performed at both the functional and sample levels. Rows in the heatmap represent different glycoenzyme families, and colors indicate the relative abundance of each glycoenzyme family in different samples: red indicates high abundance (>0); blue indicates low abundance (<0); and yellow indicates near-neutral abundance (0), meaning the abundance of the glycoenzyme family in the sample is moderate. (Appendix) Figure 16 The abundance of carbohydrate-active enzymes (CAZymes) in different samples (CKT group and S189 group) is shown. The heatmap visually illustrates the expression levels of different CAZymes in each sample through color changes. For example, the CKT group (columns labeled CKT1, CKT2, CKT3) showed higher abundance of CAZymes in the GT4, GT81, and GH32 families, while exhibiting lower abundance in the GH8 and GH23 families. In contrast, the S189 group (S1891, S1892, S1893) showed a clear opposite trend, with higher abundance in the GH8 and GH23 families, while lower abundance in the GT4, GT81, and GH32 families. We can clearly see the difference in abundance of CAZymes between the CKT and S189 groups. Specifically, the different expression levels of CAZymes in different sample groups reflect the differences in the carbohydrate metabolism potential of the microbial community after the addition of S189 to the soil.

[0099] Microorganisms are key participants in biogeochemical cycles, promoting the transformation and cycling of elements such as carbon, nitrogen, phosphorus, and sulfur through metabolic activities. This study assessed the impact of S189 addition on the biogeochemical cycling function of soil microorganisms by analyzing the relative abundance and inter-group differences of functional genes for carbon, nitrogen, phosphorus, and sulfur cycles. MetagenomeSeq heatmaps and Anosim analyses were used to study the inter-group differences in biogeochemical cycling function (see appendix). Figure 17 - Appendix Figure 21 Appendix Figure 17 The dendrograms on the left and top of the metagenomeSeq heatmap (a, b, c, and d represent the carbon, nitrogen, phosphorus, and sulfur cycles, respectively) show the clustering relationships between different samples and genes. The clustering results demonstrate the similarity of metabolic pathways between samples and genes. The vertical axis shows different genes associated with the carbon cycle. The horizontal axis shows gene expression data for different samples (CKT1, CKT2, CKT3, S1891, S1892, and S1893). Red (high expression) to blue (low expression) represents gene expression levels. (See appendix...) Figure 17 In the study, the CKT group showed higher expression of genes such as fdhA, catA, and acs, while the S189 group showed lower expression of these genes. (See attached...) Figure 18In the study, the S189 group showed higher expression levels of some nitrogen cycle genes such as norB, napB, and amoA, while the CKT group showed relatively lower expression levels of these genes. (See attached...) Figure 19 In the S189 group, high expression was observed in multiple genes, particularly phnA, surE, and spoT. (The remaining text appears to be incomplete and requires further context.) Figure 20 The CKT group showed high expression of several sulfur metabolism genes, such as sqr and soxY, while the S189 group showed relatively low expression. Heatmaps revealed differences in gene expression across carbon, nitrogen, phosphorus, and sulfur metabolic pathways between the CKT and S189 groups. The high expression of certain genes in specific metabolic pathways may indicate that microorganisms involved in these pathways dominate in the corresponding samples, thus influencing elemental transformation pathways and rates.

[0100] Appendix Figure 21 Anosim analysis (where a, b, c, and d represent the carbon, nitrogen, phosphorus, and sulfur cycles, respectively) was used to statistically analyze the differences in microbial communities across different elemental cycles. R represents the degree of difference between sample groups. A higher R value indicates a greater difference between groups; R greater than 0 indicates a significant difference between groups. (See Appendix...) Figure 21 In the carbon cycle of group a, R = 0.593 and P = 0.1. The relatively high R value indicates a significant difference between the CKT and S189 groups. (Appendix) Figure 21 From b, we can see that R = 0.333 and P = 0.1. Compared to the carbon cycle, the nitrogen cycle has a lower R value, but differences still exist between groups. (See attached...) Figure 21 From c, we know that R = 0.63 and P = 0.1. Similar to the carbon cycle, there are significant differences between different sample groups in the phosphorus cycle. (See appendix...) Figure 21 Significant differences were also observed between the two groups in the sulfur cycle of medium d, with R = 0.296 and P = 0.1. This indicates that the microbial communities differ between different samples in different biogeochemical cycles, especially in the carbon, phosphorus, and sulfur cycles.

[0101] Overall, the biogeochemical cycle function analysis revealed how microbial communities play a role in these processes and the changes in the activity of related microbial communities in each cycle, further suggesting that these differences may be related to the effects of soil addition of S189.

[0102] Example 4

[0103] Application of S189 microbial fertilizer in promoting the growth of Chinese cabbage

[0104] This experiment used barren soil in Yinjie Town, Midu County, Dali Prefecture (25°15'26"N, 100°31'52"E) and the "Hantian No. 1" Chinese cabbage (Caulis Haworthia) of the Brassicaceae family as the research subjects to conduct a pot experiment in a greenhouse to investigate whether the S189 strain has a growth-promoting effect on Chinese cabbage. The growth of Chinese cabbage in the control group (pure soil) and the experimental group (pure soil + S189 microbial fertilizer) was observed, and their agronomic traits were measured and analyzed.

[0105] The preparation method of S189 microbial fertilizer is the same as in Example 3. The fermented microbial bags are mixed evenly with the soil, and 5% microbial fertilizer is added to each pot (i.e., 200g of microbial fertilizer is added to 4kg of soil). The mixture is then divided into flowerpots, with 5 replicates per group. The control group consists of 4kg of pure soil. After the microorganisms have repaired the soil for 15 days, 10 cabbages are inoculated into each flowerpot. The growth of the cabbages is monitored, and during this period, seedlings are thinned to 5 plants to measure agronomic traits. SPASS software is used to analyze the agronomic traits of the cabbages, including plant height, root length, leaf width, number of leaves, above-ground fresh weight, and above-ground dry weight.

[0106] Appendix Figure 22 These are growth images of Chinese cabbage taken 19 and 40 days after sowing. It can be seen that at 19 days, there is almost no difference in the growth of Chinese cabbage grown with CKT and S189. At 40 days, the Chinese cabbage grown with the addition of S189 microbial fertilizer grows significantly better than that grown with CKT.

[0107] Appendix Figure 23 Data analysis of Chinese cabbage biomass measurements (a) plant height, b) root length, c) leaf width, d) number of leaves, e) aboveground fresh weight, and f) aboveground dry weight was performed using SPASS software. Results showed that the biomass of Chinese cabbage (plant height, root length, leaf width, number of leaves, aboveground fresh weight, and aboveground dry weight) was significantly increased (p<0.01) in soil treated with S189. Compared to CKT, plant height, root length, leaf width, number of leaves, aboveground fresh weight, and aboveground dry weight increased by an average of 3.4 cm, 2.74 cm, 2.54 cm, 1.2 leaves, 1.49 g, and 0.09 g, respectively. In conclusion, the addition of S189 microbial fertilizer to the soil significantly promotes the growth of Chinese cabbage.

[0108] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0109] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A strain of Penicillium chrysogenum ( Penicillium chrysogenum DLS189, characterized in that, The accession number of DLS189 is CGMCC NO.41739.

2. A microbial inoculant, characterized in that, Includes the Penicillium chrysogenum as described in claim 1.

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

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

5. The application of the Penicillium chrysogenum according to claim 1 or the microbial agent according to claim 2 in improving soil microbial abundance, characterized in that, The Penicillium chrysogenum or the microbial agent uses rice straw and / or sugarcane bagasse as raw materials, and the resulting fermentation product is applied to the soil as fertilizer.

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

7. The application of the Penicillium chrysogenum according to claim 1 or the microbial agent according to claim 2 in promoting plant growth, characterized in that, The fermentation products obtained by fermenting rice straw and / or sugarcane bagasse using the Penicillium chrysogenum or the microbial agent are applied to the soil as fertilizer.

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