Application of Leucopaxillus mongolicus LY9 in the remediation of heavy metal contaminated soil

Abstract

The application discloses a kind of mongolian white lily mushroom (Lepista sordida) Leucocalocybe mongolica ) LY9 strain in the application of heavy metal contaminated soil treatment, the strain is preserved in Guangdong Microbial Culture Collection Center, and the preservation number is GDMCC No.63678.The application method is that the LY9 strain containing bacterial agent fermentation culture medium is prepared into conversion matrix, and then it is applied to heavy metal contaminated soil and mixed evenly.The mycelium of the strain can effectively adsorb and transform heavy metals, and its unique broad-spectrum heavy metal response mechanism can activate the synergistic gene and metabolic detoxification network, thereby simultaneously reducing the effective state content of iron, cadmium, lead, manganese and other heavy metals in the soil, and maintaining or increasing the concentration of plant essential nutrients, realizing selective repair and considering soil fertility.The application belongs to the field of environmental friendly microbial remediation technology, without secondary pollution, simple process, low cost, easy to popularize and apply.

Description

Technical Field

[0001] This invention belongs to the field of microbial application technology, and relates to the application of a strain of *Mammillaria mongholica* LY9 in the remediation of heavy metal contaminated soil. Background Technology

[0002] Soil heavy metal pollution is a current global environmental challenge, posing a serious threat to agricultural production and ecological security. Current soil heavy metal remediation technologies are mainly divided into three categories: physical remediation, chemical remediation, and bioremediation. Physical remediation methods (such as soil replacement) are costly and damage soil structure, leading to a significant increase in soil bulk density, making them unsuitable for large-scale agricultural soils. Chemical remediation methods (such as adding chelating agents) are relatively low-cost, but they are prone to secondary pollution, pose a risk of heavy metal leaching, and can only provide short-term relief, not eradication.

[0003] Microbial remediation has attracted much attention due to its environmental friendliness and low cost. However, existing fungal remediation strains (such as Aspergillus niger and yeast) have significant limitations: First, their remediation efficiency is low and their broad-spectrum activity is poor, with removal rates for soluble iron generally below 15%, and insufficient capacity for simultaneous remediation of multiple heavy metal contaminations; second, they exhibit weak tolerance to high concentrations of heavy metals, particularly in Fe... 2+ When the concentration exceeds 20 mg / L or under acid stress, the biomass and activity of the strain are severely inhibited; thirdly, the remediation mechanism is simple, mainly relying on simple effects such as biosorption, lacking systematic regulation of soil microenvironment, gene transcription and metabolic pathways, resulting in unstable remediation efficiency, long cycle (usually 6-12 months), and risk of heavy metal rebound after remediation.

[0004] Mongolian white mushroom ( Leucocalocybe mongolica LY9 is a rare edible fungus with potential for agricultural and ecological applications. Existing research indicates that this strain and its fermentation products exhibit unique advantages in promoting crop growth. However, whether this strain possesses heavy metal tolerance and whether it can remediate heavy metal contaminated soil remains completely undiscovered in current technologies. Applying LY9 to the remediation of heavy metal-contaminated soil faces a complete technological gap, from functional verification to mechanism exploration, and its remediation efficacy and tolerance are highly unpredictable.

[0005] Therefore, there is an urgent need in this field for a new microbial remediation technology that combines high remediation capacity, strong environmental tolerance, and applicability to agricultural soils to overcome the shortcomings of existing remediation methods in terms of efficiency, stability, and practicality. Developing and utilizing the novel functions of the *Mammillaria mongholica* strain LY9 in heavy metal pollution remediation offers a completely new possibility for overcoming the aforementioned technical bottlenecks. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a Mongolian white mushroom ( Leucocalocybe mongolica Application of LY9 strain in the remediation of heavy metal contaminated soil; The above-mentioned Mongolian white mushroom ( Leucocalocybe mongolica The LY9 strain is deposited at the Guangdong Microbial Culture Collection Center, with accession number GDMCC No. 63678 and deposit date of August 22, 2023.

[0007] Preferably, the heavy metal is iron, cadmium, lead, or manganese.

[0008] The above application method involves using mushrooms containing Mongolian white mushrooms ( Leucocalocybe mongolica The inoculum of strain LY9 is used to ferment the culture medium to obtain the transformation substrate. The transformation substrate is then added to the heavy metal contaminated soil, mixed evenly, and the treatment of the heavy metal contaminated soil is completed.

[0009] The beneficial effects of this invention are: This invention is the first to discover and utilize the Mongolian white mushroom ( Leucocalocybe mongolica The function of LY9 strain in the remediation of heavy metal pollution in soil.

[0010] The mycelium of this strain can effectively adsorb and transform heavy metals. Molecular biological studies have shown that when faced with different heavy metal stresses, the LY9 strain can activate a universal and synergistic gene regulation and metabolic detoxification network (such as the glutathione pathway), forming a stable broad-spectrum heavy metal response mechanism. This overcomes the limitations of traditional strains that are single-function and only adaptable to specific heavy metals. By applying the LY9 transformation substrate, the bioavailable content of various heavy metals (such as iron, cadmium, lead, and manganese) in the soil can be effectively reduced, while maintaining or increasing the concentration of essential plant nutrients in the soil, achieving selective remediation and taking into account soil fertility.

[0011] This invention pertains to microbial remediation technology, posing no risk of secondary contamination. The preparation process of the microbial agent and transformation substrate is simple, easy to apply, low in cost, and readily applicable. Attached Figure Description

[0012] Figure 1 The effects of LY9 conversion matrix on soil ionosphere remodeling and iron biosorption; A: Hierarchical cluster analysis (Z-standardization) of elemental distribution in LY9-treated soils (10%, 30%, 50%) and control group (CK). Red indicates high abundance, and green indicates low abundance. B: Principal component analysis plot between CK and LY9 groups. Boxes highlight significant differences between groups. C: Quantitative analysis results of iron content.

[0013] Figure 2 Spatial distribution characteristics of LY9 in soil matrix; A: Control group (CK) sterilized soil bag, showing the homogeneous state before inoculation. B: Typical soil bag transformed with LY9 strain, showing three distinct sampling areas: the transformation zone (T) densely covered with white mycelium, the adjacent transition zone (B), and the untransformed zone (U) without mycelium. C: Horizontal profile of transformed soil covered with LY9 mycelium.

[0014] Figure 3 This is a hierarchical cluster analysis diagram of soil ionomics in different regions (transformation zone T, boundary zone B, and untransformed zone U) of soils from the control group (CK) and LY9-transformed soils. exist Figure 3 Medium red indicates high abundance, and green indicates low abundance; Figure 4 The results show the iron tolerance and biosorption capacity of strain LY9.

[0015] Figure 5 Under iron coercion, Mongolian white mushroom ( Leucocalocybe mongolica Transcriptome analysis results of strain LY9; A-D is a transcriptome heatmap of the upregulation of key functional gene clusters in LY9 under iron stress (Fe15 vs CK), containing genes with significant differential expression (Z-normalized FPKM values) (|log2FC| ≥ 1 and FDR < 0.05). Rows represent genes, and columns represent biological repeats. A: Iron homeostasis and binding; B: Antioxidant and glutathione metabolism; C: Energy metabolism, including key subunits of the mitochondrial electron transport chain and enzymes in the tricarboxylic acid (TCA) cycle to meet energy requirements; D: Fungal cell wall remodeling, including hydrophobic proteins, glucan remodeling enzymes, and a series of carbohydrate-active enzymes (CAZymes), reflecting adaptive enhancement and reorganization.

[0016] E: KEGG pathway enrichment analysis of the top 20 pathways with significant alterations in DEGs identified in D; Radar plot of the 14-fold change (FC) gene in the F:Fe15 vs CK comparison.

[0017] Figure 6 A diagram illustrating the dynamic metabolic reprogramming caused by iron supplementation in LY9; exist Figure 6 In the diagram, C is a Venn diagram of differentially expressed metabolites (DEMs) in different comparisons; F is a heatmap of HCA of metabolites in the "cofactor biosynthesis" pathway (ko01240); G is a radar diagram of the top 10-fold changes in metabolites in Fe15 and CK. Detailed Implementation

[0018] The following embodiments further illustrate the content of the present invention, but should not be construed as limiting the present invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the present invention.

[0019] Example 1: Mongolian white mushroom ( Leucocalocybe mongolica Preparation of LY9 liquid culture and transformation substrate 1. Mongolian white mushroom ( Leucocalocybe mongolica ) LY9 Propagation Prepare PDA solid culture medium and place Mongolian white mushroom ( Leucocalocybe mongolica The LY9 strain was inoculated into PDA solid medium and the inoculated medium was placed in a constant temperature incubator at 25°C for 20-30 days.

[0020] 2. Mongolian white mushroom ( Leucocalocybe mongolica Preparation of LY9 liquid culture and transformation substrate To prepare PDA liquid culture medium, mycelia from PDA solid culture medium were transferred into the PDA liquid culture medium and cultured in a shaker (25℃, 120 rpm) in the dark for 20 days to obtain LY9 liquid culture. Peat moss was diluted with distilled water to a moisture content of over 80%, and then placed into mushroom inoculation bags. The bags were autoclaved at 121℃ for 2 hours. After sterilization and cooling, 10 ml of LY9 liquid culture was inoculated into each bag, and the bags were cultured in a 25℃ incubator in the dark for 30-60 days until the mycelia completely colonized the bags, yielding the LY9 transformation substrate.

[0021] Example 2: Mongolian white mushroom ( Leucocalocybe mongolica Pot experiment of rice cultivation with LY9 strain 1. Rice cultivation in pots Rice was grown in pots, with LY9 conversion substrate application groups (applied with 10%, 30%, and 50% of the soil mass of decomposed LY9 conversion substrate) and control groups (applied with an equal amount of sterile decomposed peat soil), each replicated 3 times, and cultured for 30 days in a simulated paddy field waterlogged environment (soil moisture content maintained at 120% of field capacity).

[0022] 2. Soil sample collection After cultivation, soil from the topsoil layer (0-20 cm) of each group was collected, air-dried naturally, and then passed through a 0.15 mm sieve for homogenization.

[0023] 3. Heavy metal content detection After cultivation, the Fe content in the two soil groups was determined by ICP-OES. 2+ Cd 2+ Pb 2+ Mn 2+ The total amount and soluble content of heavy metals were compared and analyzed to determine the changes in heavy metal content.

[0024] 4. Experimental Results The test results are as follows Figure 1 As shown in A, Figure 1 A shows the hierarchical cluster analysis (Z-score normalization) of elemental distribution in soils treated with LY9 conversion matrix (10%, 30%, 50%) and the control group (CK), with red indicating high abundance and green indicating low abundance. From Figure 1 A shows that the soluble Fe in the soil of the LY9 conversion substrate application group was higher than that of the LY9 conversion substrate application group. 2+ Cd 2+ Pb 2+ Mn 2+ The content of heavy metals in paddy field soil decreased by 22.3%, 18.7%, 15.2%, and 16.5% respectively compared with the control group, confirming that the LY9 conversion matrix can reduce the heavy metal content in paddy field soil. On the contrary, the concentration of major plant nutrients (K, Mg, Ca) remained stable or increased, indicating that LY9 preferentially adsorbs harmful heavy metals while possibly retaining essential macro-elements.

[0025] Example 3: Mongolian white mushroom ( Leucocalocybe mongolica LY9 mycelium culture and mycelium weight metal enrichment verification experiment 1. Preparation of mushroom bags Dilute peat moss with distilled water to a moisture content of over 80%, pack it into mushroom inoculation bags, and autoclave at 121℃ for 2 hours. After sterilization and cooling, inoculate each bag with 10ml of LY9 liquid culture and incubate in a light-protected incubator at 25℃. Observe the mycelial growth status regularly. 2. Zoned sampling The mycelium in the substrate bag forms a distinct structure of "conversion zone (T zone, dense mycelium zone), transition zone (B zone, mycelial growth stage), and unconverted zone (U zone, no mycelium zone)" (e.g.) Figure 2 After that (as shown), matrix and mycelial samples were collected from three regions respectively; 3. Content determination The total amount and soluble content of heavy metals in the matrix of each region, as well as the amount of heavy metal accumulation in the mycelium, were determined by ICP-MS. 4. Experimental Results Experimental results are as follows Figure 3 As shown, Figure 3 This study presents hierarchical cluster analysis (Z-score normalization) of soil ionic omics in different regions (transformation zone T, boundary zone B, and untransformed zone U) of the control group (CK) and LY9-transformed soil. Red indicates high abundance, and green indicates low abundance. Figure 3 It can be seen that Fe in the mycelium of region B (mycelial growth stage) 2+ Cd 2+ Pb 2+ Mn 2+The accumulation amounts reached 38.6 mg / kg, 32.4 mg / kg, 45.7 mg / kg, and 30.2 mg / kg (based on mycelial dry weight), respectively, which were significantly higher than those in zones T and U. At the same time, the content of soluble heavy metals in the matrix of zone B was 25%-30% lower than that in zone U, directly demonstrating that LY9 mycelium can adsorb and dissolve heavy metals during its growth period.

[0026] Example 4: Soil Improvement Experiment and Ionic Composition Analysis 1. Soil sample processing Soil was collected from rice planting experimental fields, air-dried naturally, ground in an agate mortar, and homogenized by passing through a 0.15mm sieve. LY9 transformation substrate addition groups (10%, 30%, 50% w / w) and a control group (CK, no strain) were set up and cultured at constant temperature and humidity (temperature 25℃, air humidity 50%) for 30 days, with each group replicated 3 times.

[0027] 2. Soil digestion Weigh 0.5g of homogeneous soil sample into a PTFE container, add 5mL concentrated nitric acid, 2mL hydrofluoric acid, and 1mL 30% hydrogen peroxide; seal and digest using a drying oven with a constant temperature heating plate (80℃ 1-2h → 120℃ 1-2h → 160℃ 4h); after cooling, transfer to a 25mL volumetric flask, dilute to volume with 1% nitric acid, and filter. 3. Elemental Analysis Elemental quantitative analysis was performed using a Thermo Fisher iCAP 7200 HS Duo ICP-OES to determine major elements such as magnesium, iron, and calcium. For trace elements requiring higher sensitivity, an iCAP RQ ICP-MS (with helium / hydrogen mixed gas for interference suppression) was used. Calibration curves were established for all instruments using multi-element mixed standard solutions, and the concentrations of oxides (such as Fe2O3) were calculated from the corresponding elemental results using stoichiometric coefficients.

[0028] 4. Experimental Results Analysis results as follows Figure 1 B and Figure 1 As shown in C. Figure 1 B is the principal component analysis plot between the CK and LY9 groups. The boxes highlight the significant differences between the groups. It can be seen that the control group (CK) and the LY9 treatment group are clearly separated by the first principal component (PC1). PC1 explains 55.08% of the total variation. The horizontal box plot shows the significant differences along PC1, which significantly distinguishes the CK and LY9-modified soils. This indicates that the introduction of LY9 is the main factor driving the changes in the multi-element spectrum. Figure 1C represents the quantitative analysis results of iron content. It can be seen that the 50% LY9 treatment achieved the highest iron chelation rate, reducing soil iron content by 29.5% compared to the control (CK). The 10% and 30% LY9 treatments also showed significant reductions of 16.7% and 17.5%, respectively. This dose-response relationship provides clear evidence for the effectiveness of LY9 in iron bioremediation.

[0029] Example 5: Mongolian white mushroom ( Leucocalocybe mongolica Iron tolerance and biosorption experiment of LY9 strain 1. Strains culture and culture medium preparation At 25℃, the LY9 strain of *Mammillaria mongholica* was first inoculated onto PDA agar plates for activation.

[0030] Iron tolerance and adsorption tests were performed using potato-sucrose medium (potato 200 g / L, sucrose 20 g / L): FeSO4•7H2O was added to the treatment groups to allow Fe to adsorb. 2+ The final concentration reached 25 mg / L; no additional iron salt was added to the control group (CK).

[0031] 2. Inoculation and Culture Take 500 mL Erlenmeyer flasks, fill each flask with 250 mL of potato-sucrose medium, and autoclave at 121℃ for 20 min. After cooling, inoculate with 1 g of LY9 mycelial blocks approximately 2 mm × 2 mm in size.

[0032] The experiment included a Fe treatment group and a blank control (CK) group, with 12 replicates for each group; three bottles of uninoculated blank culture medium were also prepared to correct for abiotic adsorption. All bottles were placed in a shaker at 25°C in the dark and incubated at 120 r / min for 20 days.

[0033] 3. Sampling and Processing The Fe treatment group was sampled three times on days 5, 10, 15, and 20, while the CK group was sampled on day 20. The bacterial cells were isolated by filtration, frozen in liquid nitrogen, and stored at -80°C (for multi-omics analysis). 4. Determination of iron content in bacterial cells Take 10 mL of fermentation broth sample, add 5 mL of concentrated nitric acid, and heat on a hot plate to 160°C until it is nearly dry. Dissolve the residue in 1% nitric acid and dilute to 25 mL. Determine the iron concentration according to the ICP-OES method. 5. Experimental Results Experimental results are as follows Figure 4 As shown, Figure 4 A in the figure shows the performance of LY9 colonies after 20 days of culture in the control group (CK) and the iron-containing (Fe, 25 mg / L Fe) group. 2+The morphology of LY9 in potato-sucrose medium showed that it exhibited dense and fully developed growth in CK medium and maintained robust and adaptive growth in Fe-containing medium, demonstrating significant iron tolerance. Figure 4 Figure B shows the quantitative time-varying curves (n=3) of the iron content (mg / kg) adsorbed by LY9 biomass in Fe and CK media. The data show that LY9 rapidly and significantly accumulates iron, reaching a peak at 15 days, highlighting its active biosorption capacity.

[0034] Example 6: Iron stress on *White Mulberry* (Mongolian white mushroom) Leucocalocybe mongolica Transcriptome analysis of LY9 strain 1. Strains culture and culture medium preparation At 25℃, the LY9 strain of *Mammillaria mongholica* was first inoculated onto PDA agar plates for activation.

[0035] Preparation of potato-sucrose medium (potato 200 g / L, sucrose 20 g / L): FeSO4•7H2O was added to the treatment group to increase the Fe content. 2+ The final concentration reached 25 mg / L; no additional iron salt was added to the control group (CK).

[0036] 2. Inoculation and Culture Take 500 mL Erlenmeyer flasks, fill each flask with 250 mL of potato-sucrose medium, and autoclave at 121℃ for 20 min. After cooling, inoculate with 1 g of LY9 mycelial blocks approximately 2 mm × 2 mm in size.

[0037] The experiment included an iron-treated group and a control group. The iron-treated group had 12 replicates, and three uninoculated blank controls (iron medium) were included to exclude interference from non-biosorbent assays. The Fe treatment groups were Fe5, Fe10, Fe15, and Fe20, representing days 5, 10, 15, and 20 of culture, respectively, with three replicates per group. All bottles were placed in a 25°C, light-protected shaker at 120 r / min for 20 days. Three replicates were collected from the iron-treated group on days 5, 10, 15, and 20, while samples were collected from the control group on day 20. Fungal biomass was separated from the medium by filtration, rapidly frozen in liquid nitrogen, and stored at -80°C for subsequent multi-omics analysis.

[0038] 3. RNA extraction Total RNA was extracted from 100 mg of ground bacterial cells using TRIzol reagent. The integrity was verified using an Agilent 2100 bioanalyzer (RIN > 8.0), and the concentration was measured using a Qubit 4.0. 4. Library Construction Oligo (dT) magnetic beads were used to enrich eukaryotic mRNA, which was then fragmented and synthesized into double-stranded cDNA. After end repair, adapter ligation, and fragment selection, the cDNA was amplified by PCR. The quality of the library and the length of the inserted fragment were verified, and the cDNA was sequenced using the Illumina NovaSeq 6000 platform (150 bp paired-end reads). 5. Data quality control fastp (v0.23.2) processes raw data, removing connectors and low-quality bases to obtain Clean Data; 6. Comparison and quantification: HISAT2 (v2.2.1) aligns Clean Data to the reference genome of *Dendrobium Mongolianum*; featureCounts (v2.0.3) quantifies gene expression levels and normalizes them to FPKM.

[0039] 7. Difference Analysis The R language DESeq2 package (v1.22.1) is used to analyze the differences between the Fe group (Fe5 / 10 / 15 / 20) and the CK group. Genes with |log2FC|≥1 and FDR-corrected p-value <0.05 are differentially expressed genes (DEGs).

[0040] 8. Functional enrichment analysis Protein sequences were annotated with the KEGG database using the Diamond tool; the clusterProfiler software package was used for hypergeometric testing to enrich the KEGG pathways of DEGs, with FDR < 0.05 considered significant.

[0041] 9. Experimental Results The experimental results are shown in Table 1. Most of the 120-150 key functional genes activated by LY9 under iron stress are conserved genes responding to heavy metals, and can broadly regulate the repair processes of cadmium, lead, and manganese. Table 1. Results of transcriptome analysis of eukaryotic fungi, showing the upregulation and downregulation of gene expression in each control group (significance threshold |log2FoldChange|≥1).

[0042] As shown in Table 1, the Fe10_vs_CK group contained as many as 5947 differentially regulated genes, with the upregulation rate remaining stable at 45%-63% at each time point, indicating that the strain maintained high-intensity gene regulation under iron stress. Similarly, these conserved genes will also be activated when exposed to cadmium and lead, forming a broad-spectrum heavy metal response, overcoming the limitation of traditional strains with single gene regulation, adapting only to a certain type of heavy metal.

[0043] Figure 5 In the diagram, A~D represent the transcriptome heatmap of upregulated key functional gene clusters in LY9 under iron stress (Fe15 vs CK); for Figure 5 KEGG pathway enrichment analysis of the top 20 pathways with significant DEG alterations identified in D is shown in E; F is a radar chart of genes with a 14-fold change (FC) in the Fe15 vs CK comparison. A and F show that the expression levels of iron-sulfur cluster biosynthesis genes (iscU, iscS) increased 3-5 times under iron stress, and the proteins encoded by these genes are involved in Cd... 2+ Mn 2+ Intracellular complexation of iron-sulfur clusters stabilizes heavy metal ions and reduces their toxicity. The expression level of the heme-binding protein gene (hbpA) increases 4-6 times under iron stress, binding to Pb. 2+ Formation of stable complexes, reducing Pb 2+ Damage to the cell membrane; as shown in D and F, the expression level of the hydrophobic protein gene (hyd1) increased 5-8 times under iron stress, enhancing the hydrophobicity of the hyphal surface and promoting Cd... 2+ Pb 2+ The adsorption of [something] enhances the colonization ability of the strain under heavy metal stress; Figures B and E show that the expression level of the glutathione S-transferase gene (gst) increases 6-10 times under iron stress, catalyzing the reaction of GSH with Cd. 2+ Pb 2+ The binding reaction of this gene indicates that it is a core gene for the detoxification of multiple heavy metals. From C and E, it can be seen that the expression levels of mitochondrial electron transport chain-related genes increase 2-3 times under iron stress, and the expression levels of TCA-related genes increase 3-4 times under iron stress. These genes encode the protein Cd. 2+ Mn 2+ It provides energy for transport and metabolism, ensuring the efficient execution of the heavy metal detoxification process.

[0044] Example 7 Mongolian white mushroom ( Leucocalocybe mongolica Comprehensive metabolomics analysis of LY9 strain: 1. LC-MS / MS analysis of samples Take 30 mg of lyophilized and pulverized bacterial cells, add 1500 μL of 70% methanol aqueous solution (containing internal standard) pre-cooled at -20℃; vortex (5 minutes 30 seconds each time, 6 times in total), centrifuge at 12000 rpm for 3 minutes, and filter the supernatant through a 0.22 μm filter membrane.

[0045] 2. GC-MS analysis of samples Take 0.5 g of powder and extract with 1.7 mL of methanol:isopropanol:water (3:3:2 v / v); after centrifugation, add internal standard to the supernatant and dry under nitrogen; first add 0.1 mL of 15 mg / mL methoxyamine pyridine hydrochloride solution (37℃ for 2 h) for methoxyamination, then add 0.1 mL of BSTFA containing 1% TMCS (37℃ for 30 min) for silanization; dilute and filter before analysis.

[0046] 3. LC-MS / MS Separation was performed using an ExionLC AD UPLC system (Agilent SB-C18 column, 1.8 μm 2.1 mm × 100 mm), with a linear gradient of mobile phase A (0.1% formic acid water) - B (0.1% formic acid acetonitrile); detection was performed using a 6500 Q TRAP mass spectrometer (ESI source, positive and negative ion modes, MRM data acquisition).

[0047] 4. GC-MS 1 μL of derivatized sample was split and injected (5:1) into an Agilent 8890 GC-5977B MSD; separation was performed on a DB-5MS column (30 m × 0.25 mm × 0.25 μm) with helium carrier gas (1.2 mL / min) and programmed temperature rise (40 °C for 1 min → 300 °C); data were acquired in EI mode (70 eV) with full scan (m / z 50-600).

[0048] Data processing and metabolite identification MultiQuant (v3.0.3) handles LC-MS / MS peak integration and correction, while MassHunter handles GC-MS data. By comparing MS / MS spectra, retention times, retention indices with the MWDB database and internal standard library, metabolites are confirmed when MF > 70.

[0049] 6. Metabolomics Statistical and Bioinformatics Analysis First, the data was scaled using the `prcomp` function in R to unit variance, followed by PCA to identify the natural clustering trends of the samples. Then, an OPLS-DA model was constructed using the MetaboAnalyst R package, and its reliability was ensured through 200 permutation tests. Differential metabolites were screened based on VIP > 1 and |log2FC| ≥ 1. Finally, hierarchical clustering of the differential metabolites was performed using the ComplexHeatmap package, and the significantly enriched KEGG pathway was identified through hypergeometric testing, thus relating metabolic changes to biological functions.

[0050] 7. Experimental Results The LC-MS / MS detection results are shown in Tables 2 and 3: Table 2. Comparison of differentially expressed metabolites (DEMs) between the LY9 group and the control group (CK) at different time points after iron supplementation.

[0051] Table 3. Metabolites of strain LY9 that were significantly altered compared to the control group (CK) under iron supplementation (Fe15) conditions (fold change (FC) > 1 indicates upregulation in the Fe15 group, while FC < 1 indicates downregulation).

[0052] Table 2 shows that there were 1518-1696 differentially expressed metabolites (DEMs) at each time point, with upregulated DEMs accounting for 50%-56%. Table 3 shows that glutathione (GSH) and its precursors were significantly upregulated metabolites, indicating that the metabolic pathways were fully activated under iron stress. By analogy, these metabolic pathways will be simultaneously enhanced when facing cadmium and lead stress, forming a broad-spectrum metabolic detoxification system to ensure the stable repair of multiple heavy metals by the strain.

[0053] Figure 6 This is a diagram illustrating the dynamic metabolic reprogramming caused by iron supplementation in LY9. Figure 6 In the diagram, C is a Venn diagram of differentially expressed metabolites (DEMs) in different comparisons; F is a heatmap of HCA of metabolites in the "cofactor biosynthesis" pathway (ko01240); G is a radar diagram of the top 10-fold changes in metabolites in Fe15 and CK.

[0054] From Table 3 and Figure 6 As can be seen from Table 3, the intracellular accumulation of γ-glutamylcysteine ​​(GSH precursor) in the glutathione (GSH) synthesis pathway reached 120-150 nmol / g (an increase of 165-fold), as shown in Table 3. Figure 6 As can be seen from G, GSH reaches 80-100 nmol / g (a 124-fold increase); GSH can not only chelate intracellular Fe... 2+ It can also be used with Cd 2+ Pb 2+ Stable GSH-heavy metal complexes are formed (with binding equilibrium constants reaching 10). 9·3 10 10·1 L / mol), is the core substance for detoxifying multiple heavy metals; From Table 3 and Figure 6 As can be seen from F, the NAD+ content in energy and redox carrier metabolism reached 50-60 nmol / g (an increase of 50.74 times), as shown in Table 3 and Figure 6 As can be seen from C, NADP+ reaches 30-40 nmol / g (a 40.96-fold increase). By analogy, if LY9 affects Cd... 2+ Pb 2+ Mn 2+ The detoxification mechanism is consistent with that of iron stress (both rely on energy-driven transport and chelation), so the continuous activation of energy metabolism genes may maintain a stable respiratory rate.

Claims

1. A type of Mongolian white mushroom ( Leucocalocybe mongolica Application of LY9 strain in the remediation of heavy metal contaminated soil; the Mongolian white mushroom ( Leucocalocybe mongolica The LY9 strain is deposited at the Guangdong Microbial Culture Collection Center, with accession number GDMCC No. 63678 and deposit date of August 22, 2023. in, The heavy metal is at least one of iron, cadmium, lead, and manganese.

2. The application according to claim 1, characterized in that: The method of application is to include Mongolian white mushrooms ( Leucocalocybe mongolica The inoculum of strain LY9 is used to ferment the culture medium to obtain the transformation substrate. The transformation substrate is then added to the heavy metal contaminated soil, mixed evenly, and the treatment of the heavy metal contaminated soil is completed.

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