Penicillium chrysogenum DLS189 and application thereof

Abstract

The invention discloses Penicillium chrysogenum DLS189 and application thereof, and relates to the technical field of microorganisms, and the preservation number of the Penicillium chrysogenum DLS189 is CGMCC (China General Microbiological Culture Collection Center) NO.41739. The invention further discloses a preparation method of the Penicillium chrysogenum DLS189. The strain can effectively utilize rice straws and / or bagasse to produce humic acid, and the content of humic acid in each gram of fermentation supernatant after the rice straws are fermented is 16.84%. When the DLS189 fermentation product is applied to crops, the growth condition of the crops can be remarkably improved, and the humus content in soil is increased. Meanwhile, a metagenomics analysis result shows that compared with a control group, after the DLS189 is applied into the soil, the species beta diversity is remarkably changed, and the functional gene abundance and the inter-group difference are remarkably changed. Therefore, the DLS189 has a wide application prospect in the aspects of agricultural straw utilization and soil remediation.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and more particularly to a strain of Penicillium chrysogenum DLS189 and an application thereof. Background Art

[0002] Humic acid is a type of natural organic matter found primarily in soil, sediment, and water. It is a complex mixture formed by the long-term decomposition and transformation of organic matter. As one of the main components of soil humus, humic acid possesses strong adsorption, water-retention, and fertility-regulating properties. It is the end product of organic matter decomposition and typically exists in an acidic form in soil and water. In my country, humic acid is classified into three categories based on its solubility in solvents and color: fulvic acid, humin, and humic acid. As a natural organic polymer, humic acid has multiple functions, including improving soil quality, 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, and promote plant growth and development. In recent years, the production of humic acid using microbial fermentation technology has become a hot topic of research.

[0003] Lignocellulose, the most abundant renewable bioresource in the biosphere, produces 150 billion tons annually worldwide, of which straw accounts for 6 billion tons. China produces approximately 1.1 billion tons of straw annually, accounting for approximately one-fifth of the global straw resource. Agricultural straw, as a rich biomass resource, possesses high nutritional value. However, traditional straw disposal methods, such as returning straw to the fields and burning it, are not only inefficient but can 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 that needs 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 application.

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

[0007] The invention relates to a Penicillium chrysogenum DLS189, whose deposit number is CGMCC NO.41739.

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

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

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

[0012] Another object of the present invention is to provide the use of the above-mentioned Penicillium chrysogenum or the above-mentioned microbial agent in improving soil microbial abundance, or inter-group difference analysis of species composition, or functional level analysis, or inter-group difference analysis of functional abundance.

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

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

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

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

[0017] Beneficial effects: The present invention isolated a strain of Penicillium chrysogenum DLS189 from the soil of Canglang Peak, Cangshan Mountain, Dali. This strain can effectively utilize rice straw and / or sugarcane bagasse to produce humic acid. The humic acid content in each gram of fermentation supernatant after fermentation of rice straw is 16.84%. When the fermentation product of S189 was applied to crops, the plant height, leaf length, leaf width, etc. of wheat were significantly improved, and the soil humus content increased by 4.04g / kg; the plant height, root length, leaf width, number of leaves, aboveground fresh weight and aboveground dry weight of cabbage increased by an average of 3.4cm, 2.74cm, 2.54cm, 1.2 leaves, 1.49g and 0.09g, respectively. The results of metagenomic analysis showed that compared with the control group, after DLS189 was applied to the soil, the species β diversity changed significantly, and the functional gene abundance and inter-group differences changed significantly. The above shows that DLS189 has broad application prospects in agricultural straw utilization and soil remediation. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to make the technical solutions in the embodiments of the present application or the prior art clearer, the accompanying drawings needed in the embodiments or prior art description will be briefly introduced. Obviously, the accompanying drawings in the following description only need to be drawn for the embodiments of the present application, and other drawings can be obtained by those skilled in the art without any creative effort on the basis of the provided drawings.

[0019] Figure 1 Fig. 1 is a morphological identification chart of strain DLS189, wherein a is the colony morphology, and b is the microscopic morphology of the strain.

[0020] Figure 2 Fig. 2 is a phylogenetic tree of strain DLS189.

[0021] Figure 3 Fig. 3 is the growth condition of strain DLS189 at different temperatures, wherein "+" represents the growth condition of the strain, the more "+" is, the better the growth is, " / " represents that the strain does not grow; "1" represents the ck, i.e. the control without inoculation, and "2" represents the experimental group inoculated with DLS189.

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

[0023] Figure 5 Fig. 5 is the accurate determination of the humic acid content in the fermentation supernatant of DLS189 strain.

[0024] Figure 6 Fig. 6 is the effect of strain DLS189 on the growth phenotype of wheat.

[0025] Figure 7 Fig. 7 is the quantitative determination of the effect of strain DLS189 on the growth traits of wheat.

[0026] Figure 8 Fig. 8 is the comparison of the humus group content in the soil under different treatments.

[0027] Figure 9 Fig. 9 is the microbial culture condition in the soil under different treatments.

[0028] Figure 10 Fig. 10 is a 1% gel electrophoresis diagram of soil metagenomic DNA, wherein 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 Fig. 11 is the species abundance at the door level under different treatments.

[0030] Figure 12 Fig. 12 is the species abundance at the sub-level under different treatments.

[0031] Figure 13 Species richness for different treatments at different levels.

[0032] Figure 14 PCoA analysis for different treatments at different levels of genera and species.

[0033] Figure 15 Heatmap of species richness difference analysis for different treatments.

[0034] Figure 16 Heatmap of functional richness analysis for different treatments.

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

[0036] Figure 18 Heat map of nitrogen cycle functional gene abundance under different treatments.

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

[0038] Figure 20 Heat map of sulfur cycle functional gene abundance under different treatments.

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

[0040] Figure 22 Effect of strain DLS189 on the growth phenotype of pakchoi.

[0041] Figure 23 Quantitative determination of the effect of strain DLS189 on the growth traits of pakchoi.

[0042] Note: The present application is marked with different lowercase letters to represent significant differences (p<0.05) between groups, and marked with different capital letters to represent extremely significant differences (p<0.01) between groups. DETAILED DESCRIPTION

[0043] The technical solutions in the embodiments of the present application will be described clearly and completely below in combination with the drawings in the embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0044] The culture medium used in the present application is as follows:

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

[0046] R2A culture 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 is added with 20 g / L agar.

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

[0048] Carboxymethylcellulose sodium base medium: carboxymethylcellulose sodium 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 base 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, pH natural.

[0050] Corn stalk carbon source base 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, pH natural.

[0051] Wheat stalk carbon source base medium: wheat 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, pH natural.

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

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

[0054] Poplar wood carbon source base medium: poplar wood residue 20 g / L, (NH4)2SO4 4 g / L, MgSO4-(7H2O) 1.2 g / L, CaCl2 0.3 g / L, K2HPO4 1 g / L, KH2PO4 1 g / L, NaNO3 1 g / L, pH natural.

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

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

[0057] Example 1

[0058] 1. Isolation of strains

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

[0060] 2. Screening of strains

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

[0062] Rescreening: While UV spectroscopy can confirm whether a strain produces humic acid, it cannot accurately measure humic acid production. Therefore, rescreening is required to further determine the humic acid production of each strain. The strains identified in the initial screening were inoculated onto a basal medium containing lignocellulosic materials 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°C and 180 rpm for 7 days, and the humic acid production in the fermentation broth was initially determined. The fermentation broth was then treated with 0.2 mol / L sodium pyrophosphate alkaline extract to raise the pH to 12. The broth was allowed to stand at room temperature for 24 hours, followed by adjusting the pH to 1-2 with hydrochloric acid and allowing to stand overnight. The broth was centrifuged, the supernatant discarded, and the precipitate dried to obtain crude humic acid. Based on the crude humic acid production, a strain with high humic acid production was identified and designated DLS189 (hereinafter referred to as S189).

[0063] In the initial screening of the S189 strain, the absorbance difference at OD200nm-OD240nm was higher than that of the control. There was a maximum absorption peak at OD200nm, and the absorbance difference with the control was 0.176; the maximum absorbance difference compared with the control at OD210nm was 1.344. This shows that the S189 strain has the ability to produce humic acid. In the re-screening of the S189 strain, humic acid was produced when rice straw, corn straw, wheat straw and sugarcane bagasse were the only carbon sources, among which rice straw had the highest yield (0.025g / 10mL). In addition, S189 could not grow in pine sawdust and poplar sawdust, and the humic acid production was almost zero (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 were taken using a Nikon ECLIPSENi-U biological microscope to record the morphological characteristics of the strain.

[0066] The results showed that the S189 strain was a typical Penicillium fungus. Its colonies were green in color, and the surface of the colonies was dry, loose, and hairy. The mycelium penetrated the surface of the culture medium and invaded deep layers, producing yellow metabolites that caused the culture medium to change color (see Appendix Figure 1 The microscopic morphology of the strain showed that its conidia were spherical and green in color (see Appendix Figure 1 (b)

[0067] (2) DNA of the S189 strain was extracted and PCR amplified using universal primers for the fungal ribosomal rDNA intergenic region (ITS) sequence (ITS4: 5'-TCCTCCGCTTATTGATATGC-3', ITS5: 5'-GGAAGTA AAAGTCGTAACAAGG-3') using Taq enzyme. Amplification parameters were as follows: pre-denaturation at 94°C for 4 min, denaturation at 94°C for 30 s, annealing at 55°C for 35 s, extension at 72°C for 90 s, 32 cycles; extension at 72°C for 5 min. The PCR product was subjected to gel electrophoresis and sent to Sangon Biotech Co., Ltd. for sequencing. The obtained sequence was submitted to GenBank (http: / / www.ncbi.nlm.nih.gov). The strain was compared with the ITS sequence in the BLAST database and a phylogenetic tree was constructed using the maximum likelihood estimation method using MEGA7.0 to determine the species status of the strain.

[0068] The phylogenetic tree showed that strain S189 and Penicillium chrysogenum were clustered in one branch 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 in the General Microbiology Center of China Culture Collection Administration on December 30, 2024, with the deposit number CGMCC NO.41739, and the classification name was Penicillium chrysogenum. The deposit address is No. 3, Yard 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 solution.

[0073] The seed liquid was inoculated into rice straw carbon source basal medium at an inoculum size of 2%, with three biological replicates. The effects of different temperatures (4°C, 25°C, 30°C, 37°C, 45°C) on the growth of the strain were tested under natural pH conditions.

[0074] The results showed that the S189 strain grew best at 30°C, with green colonies and dense hyphae. The growth rate deteriorated at 25°C and 37°C. The strain stopped growing at 4°C and 45°C. S189 could grow in the range of 25°C-37°C, indicating that the strain was a low-temperature fungus (see Appendix). Figure 3 ).

[0075] 2. The S189 strain was inoculated into 10 mL of LB liquid medium and cultured at 20°C, 180 rpm for 2 days to prepare a seed solution. The seed solution was inoculated into 100 mL of rice straw carbon source basal medium at a 2% inoculum size, and the control was a non-inoculated medium, with three biological replicates. After 7 days of shaking culture at 20°C, 180 rpm, 10 mL of the fermentation supernatant and liquid culture were collected and tested based on the Wuhan Pu Neisi 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 liquid.

[0076] Attachment Figure 5 Shown is a comparative analysis of the humic acid content in the fermentation supernatant of the S189 strain and in a CK (uninoculated rice straw-based salt liquid medium). The results showed that the humic acid content per gram of S189 fermentation broth was 16.84%, while the humic acid content in the control (CK) was 9.59%. There was a highly significant difference (p < 0.01) between the S189 and CK cultures, with the humic acid content increasing by 76% compared to the control. This further demonstrates the S189 strain's strong ability to produce humic acid.

[0077] Example 3

[0078] 1. This experiment used the poor soil in the suburbs of Chenggong, Kunming City and the "Shannong 42" wheat of the Poaceae family as research objects for a pot experiment to further explore whether humic acid strains have a growth-promoting effect on wheat growth.

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

[0080] Preparation of S189 biofertilizer: Mix rice straw powder and wheat bran in a 7:3 ratio, then add water and stir until moist without water accumulation. Dispense into bags, sealing tightly with a collar. Each bag should contain 1.5 kg of the bacteria. Sterilize (121°C, 120 minutes) and set aside. Inoculate the strain into 50 mL of LB liquid medium (containing 50 μL of kanamycin) and incubate at 20°C, 180 rpm, and shake for 2 days to prepare the seed solution. Inoculate the seed solution into sterilized bags and incubate in a 25°C incubator until the mycelium covers the entire bag.

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

[0082] Attachment Figure 6 The growth graphs of wheat taken 12 days and 56 days after sowing show that the growth of wheat after adding S189 bacterial fertilizer was significantly better than that of the pure soil control group. The data of wheat growth traits such as plant height (a), leaf length (b), leaf width (c), and leaf number (d) were analyzed using SPASS software. The results showed that compared with the control group (CKT), the leaf width after adding S189 bacterial fertilizer was significantly higher (p<0.05) than the control group, 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 the S189 strain using agricultural straw can promote the growth of wheat.

[0083] 2. Determination of soil humus content: Weigh 10g of soil from the wheat plantation in step 1, 10g per pot. Ship on dry ice to Wuhan Purness Testing Co., Ltd. to determine the soil humus content and changes.

[0084] Soil samples were dried at (105±5)℃ to constant weight, and the dry matter and water content were calculated based on the difference in mass between the soil samples before and after drying, and expressed as mass fraction. Soil humus is divided into soluble humus (humic acid and fulvic acid) and insoluble humus (humin) according to its solubility. Soluble humus was extracted with a 0.1mol / L sodium pyrophosphate-sodium hydroxide mixture, and the total amount of humic acid and fulvic acid was determined by the potassium dichromate oxidation capacity method. The extract was acidified and precipitated to separate humic acid, and its content was determined to calculate the fulvic acid content. 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 Humic substance content in soils with different treatments

[0086]

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

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

[0089] 3. Changes in soil microbial populations: To explore whether there are differences in the microbial communities in the soils that were treated with S189 fertilizer, a dilution and coating experiment was conducted on the soil that had been planted with wheat in step 1. Weigh 10 g of soil that had been planted with wheat in 10 mL of sterile water and dilute to 10 -5 , spread on PDA medium, with 3 replicates per pot. Culture in a 25℃ constant temperature incubator. After 3 days, observe the changes in the number of colonies on the plate.

[0090] Attachment Figure 9 To dilute to 10 -5 The growth period of soil sample solutions on PDA medium is shown in Figure 1. Group a represents the control group, while group b represents the experimental group treated with S189 fertilizer. Each group had three biological replicates. It can be seen that the plates in group b were densely populated with microorganisms (>1000 colonies / plate), while the plates in group a were relatively few (<10 colonies / plate). The colony counts in the experimental group were significantly higher than those in the control group, indicating that the addition of S189 fertilizer can, to a certain extent, increase the number and species richness of culturable soil microbial communities.

[0091] 4. Soil metagenomic analysis: Soil metagenomic samples were extracted using the MP Biomedicals Soil DNA Isolation Kit. Three biological replicates were performed in each group, and 0.3 soil samples were weighed for 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 the gel electrophoresis test, the samples were stored in dry ice and sent to Wuhan Puneisi Testing Company for metagenomic sequencing. After the samples passed the test, a sequence library was constructed and high-throughput sequencing was performed. The high-quality sequences were assembled using splicing software and gene prediction was performed. The species composition of each sample and the proportion of different species in each sample were displayed using a bar graph (see Appendix Figure 11PCoA analysis was performed based on the unconstrained ordination (Classical Multidimensional Scaling, cMDScale) analysis method to evaluate the differences in microbial community composition between samples and analyze species β diversity (see Appendix Figure 14 Based on metagenomeSeq heat map analysis, the species abundance differences between samples were statistically analyzed (see Appendix Figure 15 Using the CAZy database, the predicted genes were annotated and classified into species and functions, and the functions, classifications and metabolic pathways of the genes were predicted (see Appendix Figure 16 The differences in the abundance of biogeochemical cycle functions among groups were investigated using metagenomeSeq heatmap and Anosim analysis (see Appendix). Figure 17 -Attached Figure 21 ).

[0092] Attachment Figure 10 This is a 1% gel electrophoresis image of the samples run at 120V for 30 minutes. The sample volume was 5μl. The brightness and total amount of metagenomic DNA differed between treatments. Compared to the CKT control, the concentration and total amount of S189 in the soil increased significantly, indicating that the addition of S189 to the soil increased the number and diversity of microorganisms.

[0093] Table 2 Sample DNA test results

[0094]

[0095] Attachment Figure 11 -Attached Figure 13 The species bar graphs of CKT and S189 at three taxonomic levels, namely, phylum (Phylum), genus (Genus), and species (Species), are displayed. The species composition of each sample and the proportion of different species in each sample can be intuitively seen from the figure. Using R software, a bar graph was drawn for the composition of the dominant species in each sample (here the top 30 species in terms of overall abundance) at each taxonomic level. The horizontal axis (x-axis): shows different sample groups, with 3 replicates in each group. The vertical axis (y-axis) represents the relative abundance of each microbial group, ranging from 0 to 1. The height of each bar represents the relative abundance of microorganisms in that group of samples. Attached Figure 11 The abundance at the phylum level is shown: the pink area of ​​Proteobacteria and the green area of ​​Actinobacteria have the highest relative abundance, accounting for more than 0.5. There are obvious differences between S189 and CKT in some microbial phyla, such as Actinobacteria and Proteobacteria, which have obvious abundance changes. Figure 12 The abundance at the genus level is shown: In addition to the microorganisms that cannot be classified or have a small number of species, the abundance of Sphingomonas (Sphingomonas genus) in the pink area and Nocardioides (Nocardioides genus) in the orange area in CKT accounts for a large proportion. The relative abundance of Steroidobacter in the blue area in S189 is higher. There are obvious changes in the abundance of Sphingomonas, Steroidobacter and Nocardioides between S189 and CKT, and the species abundance between the two groups shows opposite trends. Figure 13 Abundance displayed at the species level: the largest proportion of unclassifiable microorganisms exceeded 0.5. In addition, the abundance of Acidobacteria bacterium in the pink area and Chloroflexibacterium in the orange area in CKT accounted for a large proportion. In S189, the relative abundance of Acidobacteria bacterium in the pink area and Myxococcales bacterium in the green area was higher. The differences between S189 and CKT in some species were quite obvious, such as the species abundance of Myxococcales bacterium and Proteobacteria bacterium changed significantly. The above results show the relative abundance of different microbial phyla, genera, and species in the experimental and control groups through stacked bar charts, showing the differences in the composition of the microbial community and reflecting the impact of S189 treatment on the microbial population in the soil.

[0096] The present invention conducts PCoA analysis based on the unconstrained sorting (Classical Multidimensional Scaling, cMDScale) analysis method. Figure 14 The differences in species structure between CKT and S189 at the three taxonomic levels of Phylum, Genus, and Species are shown. 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 main difference. The CKT group (blue) and the S189 group (dark blue) are clearly separated in the principal coordinate diagram. The microbial community structure of the S189 group is clustered together, which is far away from the structure of the CKT group. This shows that there are large structural differences between the microorganisms of the two groups of samples. Attached Figure 14 In (a), the first two axes of the PCoA analysis explained 78.51% and 14.48% of the total variation in the data at the phylum level, respectively. Figure 14In the middle b, the first two axes of PCoA analysis explained 59.9% and 27.86% of the total variation of data at the genus level, respectively. Appendix Figure 14 In the middle c, the first two axes of PCoA analysis explained 57.49% and 28.61% of the total variation of data at the species level, respectively. From the Appendix Figure 14 It can be seen that at the door level, CKT group and S189 group have significant differences in the overall community composition; at the genus and species levels, these differences will be further amplified, which is embodied in the distribution difference of individual genus or species. Therefore, with the addition of S189, the soil microbial community structure has changed significantly.

[0097] Appendix Figure 15 To evaluate the abundance difference between groups under different treatments (whether to add S189) based on zero-inflated model in this study, metagenomeSeq analysis was used, and heatmap visualization method was used to show the significantly different species at the door (Phylum) level. Blue represents lower abundance (negative value) of the classification group, while red represents higher abundance (positive value). Yellow represents the abundance close to the intermediate value. Through the color contrast between samples, the difference in microbial community composition of these samples can be intuitively understood. Calditrichaeota, Spirochaetes, Candidatus Buchananbacteria and Proteobacteria showed obvious high abundance in S189, while the abundance was lower in CKT. On the contrary, Candidatus Liptonbacteria and Actinobacteria showed obvious high abundance in CKT, while the abundance was lower in S189. Appendix Figure 15 The difference in microbial community composition between CKT and S189 is shown, and through the clustering analysis of rows and columns, it is clear that there are significant abundance differences between groups at the door (Phylum) level, which shows that the addition of S189 has a significant change on the richness and composition of soil microbial community.

[0098] CAZy (Carbohydrate-Active enZYmes) database is a database specially used for classifying and annotating enzymes related to carbohydrate metabolism. In this study, CAZy database was used to annotate and classify the predicted genes in terms of function, to evaluate the influence of adding S189 on the functional abundance of soil microorganisms. Appendix Figure 16A heat map was drawn based on the functional annotation and abundance information of all samples in the CAZy database, and clustering was performed at both the functional and sample levels. The rows in the heat map represent different carbohydrate-active enzyme families, and the colors represent the relative abundance of each carbohydrate enzyme family in different samples: red indicates that the carbohydrate enzyme family has a high abundance in the sample (>0); blue indicates a low abundance (<0); and yellow indicates that the abundance is close to neutral (0), that is, the carbohydrate enzyme has a moderate abundance in the sample. Figure 16 The abundance of carbohydrate-active enzymes (CAZymes) in different samples (CKT group and S189 group) is shown. The heatmap visually displays the expression levels of different carbohydrate-active enzymes in each sample through color changes. For example, the abundance of carbohydrate enzymes in the CKT group (columns labeled CKT1, CKT2, and CKT3) is high in the GT4, GT81, and GH32 families, while the abundance is low in the GH8 and GH23 carbohydrate enzyme families. In contrast, the abundance of the S189 group (S1891, S1892, and S1893) is significantly different, with higher abundance in the GH8 and GH23 families, while the abundance is lower in GT4, GT81, and GH32. The differences in the abundance of carbohydrate-active enzyme families between the CKT and S189 groups are clearly visible. Specifically, the carbohydrate enzyme families in different sample groups have different expression levels, reflecting the differences in the carbohydrate metabolism potential of the microbial community after the addition of S189 to the soil.

[0099] Microorganisms are key players in biogeochemical cycles. They promote the transformation and circulation of elements such as carbon, nitrogen, phosphorus, and sulfur through metabolic activities. This study evaluated the effect of adding S189 on the biogeochemical cycle function of soil microorganisms by analyzing the relative abundance and inter-group differences of carbon cycle, nitrogen cycle, phosphorus cycle, and sulfur cycle functional genes. Figure 17 -Attached Figure 21 ). Figure 17 The dendrogram on the left and above the metagenomeSeq heat map (a, b, c, d represent the carbon cycle, nitrogen cycle, phosphorus cycle, and sulfur cycle, respectively) shows the clustering relationship between different samples and genes. The clustering results show the similarity of metabolic pathways between samples and genes. The vertical axis shows different genes related to the carbon cycle. The horizontal axis shows the gene expression data of different samples (CKT1, CKT2, CKT3, S1891, S1892, S1893). The expression level of the gene is represented from red (high expression) to blue (low expression). In the attached Figure 17 In the CKT group, the expression of fdhA, catA and acs genes was higher, while the expression of these genes in the S189 group was lower. Figure 18In the CKT group, the S189 group showed higher expression levels of some nitrogen cycle genes such as norB, napB and amoA, while the CKT group had relatively lower expression levels of these genes. Figure 19 Among them, the S189 group showed higher expression in multiple genes, especially in genes such as phnA, surE and spoT. Figure 20 The CKT group showed higher expression of several sulfur metabolism genes, such as sqr and soxY, while the S189 group exhibited relatively lower expression. Heat maps reveal differences in gene expression between the CKT and S189 groups in carbon, nitrogen, phosphorus, and sulfur metabolic pathways. The higher expression of some genes in specific metabolic pathways may indicate that microorganisms involved in these pathways dominate in the corresponding samples, thereby influencing the conversion pathways and rates of these elements.

[0100] Attachment Figure 21 Anosim analysis (a, b, c, d represent carbon cycle, nitrogen cycle, phosphorus cycle, and sulfur cycle, respectively) was used to statistically analyze the differences in microbial communities in different element cycles. R represents the degree of difference between sample groups. A higher R value indicates a greater difference between groups, and an R value greater than 0 indicates a significant difference between groups. Figure 21 The carbon cycle of a in R = 0.593, P = 0.1. The high R value indicates that there is a large difference between the CKT and S189 groups. Figure 21 In Figure b, we can see that R=0.333, 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 21 From c, we can see that R=0.63, P=0.1. Similar to the carbon cycle, there are large differences between different sample groups in the phosphorus cycle. Figure 21 The sulfur cycle in the middle d also showed significant differences between the two groups, with R = 0.296, P = 0.1, indicating that the microbial communities of different biogeochemical cycles differed between samples, especially in the carbon, phosphorus, and sulfur cycles.

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

[0102] Example 4

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

[0104] This experiment, conducted in a greenhouse potted plant experiment, investigated the growth-promoting effect of the S189 bacterial strain on the poor soil of Yinjie Town, Midu County, Dali Prefecture (25°15'26"N, 100°31'52"E) and the Brassica rapa variety "Hantian No. 1." The results were analyzed using the S189 bacterial strain in a control (pure soil) and an experimental (pure soil + S189 bacterial fertilizer) group. Agronomic traits were also measured and analyzed.

[0105] The preparation method of S189 bacterial fertilizer is the same as that in Example 3. The fermented bacterial bag is mixed with the soil, and 5% bacterial fertilizer is added to each pot, that is, 200g of bacterial fertilizer is added to 4kg of soil. The fertilizer is divided into flower pots, and 5 repetitions are repeated in each group. The control group is 4kg of pure soil. After the bacteria have repaired the soil for 15 days, the cabbage is inoculated into the flower pot, and 10 cabbages are inoculated per pot. The growth of the cabbage is monitored, and the agronomic traits are measured by thinning the seedlings to 5 during the period. The agronomic traits of the cabbage, such as plant height, root length, leaf width, number of leaves, fresh weight above ground, and dry weight above ground, are analyzed using SPASS software.

[0106] Attachment Figure 22 These photos show the growth of Chinese cabbage taken 19 and 40 days after sowing. It can be seen that at 19 days, there was little difference in the growth of cabbage grown with CKT and S189. At 40 days, the cabbage grown with S189 fertilizer significantly outperformed the CKT.

[0107] Attachment Figure 23 Data from cabbage biomass measurements (a plant height, b root length, c leaf width, d number of leaves, e aboveground fresh weight, f aboveground dry weight) were analyzed using SPASS software. The results showed that cabbage biomass (plant height, root length, leaf width, number of leaves, aboveground fresh weight, and aboveground dry weight) significantly increased (p < 0.01) in 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. This suggests that the addition of S189 to soil significantly promotes cabbage growth.

[0108] 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.

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

Claims

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

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

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

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

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

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

7. The use according to claim 6, characterized in that The soil microbial abundance includes the abundance of microbial species and quantity, the abundance of carbohydrate-active enzymes, and the abundance of genes in the carbon metabolism pathway, nitrogen metabolism pathway, phosphorus metabolism pathway, and sulfur metabolism pathway.

8. The use according to claim 6, characterized in that The Penicillium chrysogenum or the microbial agent is used to ferment rice straw and / or sugarcane bagasse, and the obtained fermentation product is applied to the soil as a fertilizer.

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

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

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