To elucidate the patterns of metabolite accumulation in the fruiting bodies and cultured mycelia of Collybia nuda and evaluate the potential of the cultured mycelia as a substitute for the fruiting bodies in terms of active components and antioxidant activity, the mature fruiting bodies and the mycelia cultured for 10, 20, and 30 days were examined for contents of major active components, and the in vitro antioxidant activities of the aqueous and 70% ethanol extracts of the fruiting bodies and mycelia were assessed. Additionally, metabolite profiles were analyzed using non-targeted metabolomics. The results indicated that the contents of total triterpenoids in the mycelia significantly surpassed that in the fruiting bodies. The contents of total flavonoids and total phenols increased with culture time, reaching levels that did not significantly differ from those of the fruiting bodies on the 30th day. The contents of total polysaccharides in the 30-day-old mycelium was significantly higher than in the fruiting bodies. The free radical scavenging capacity of the mycelia were ranked in the decreasing order of hydroxyl radicals > superoxide anion radicals > 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals. The superoxide anion radical scavenging rate of the mycelium aqueous extract on the 10th day of fermentation was (64.73 ± 1.68)%, while the hydroxyl radical scavenging rate of the ethanol extract was (76.71 ± 1.19)%, demonstrating strong antioxidant activity. Non-targeted metabolomics analysis identified 517 and 450 metabolites as significantly different between the fruiting bodies and the mycelia of different ages and between the mycelia of different ages, respectively. These were primarily classified as lipids and lipid-like molecules, organic heterocyclic compounds, and organic acids and derivatives. K-means clustering revealed time-dependent dynamic changes in differential metabolites from the mycelia during the culture period. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis indicated that some differential metabolites were significantly enriched in the basic metabolic pathways and the ATP-binding cassette (ABC) transporter pathway, which were key pathways related to the metabolic differences between the fruiting bodies and the mycelia. In the early stage of culture, the differential metabolites in the mycelia were mainly enriched in the biosynthesis of amino acids, carbon metabolism, and various antibiotic biosynthesis pathways. In the mid-to-late stages, they were significantly enriched in propionate metabolism, biosynthesis of various secondary metabolites, fatty acid biosynthesis, and the pentose phosphate pathway. Additionally, bioactive metabolites such as ergothioneine, dehydroevodiamine and panaxacol were significantly up-regulated in the late culture stage, reflecting a metabolic shift from basal metabolism toward bioactive compound accumulation and metabolic homeostasis. This study provides a theoretical foundation for understanding the metabolic mechanisms of the fruiting bodies and mycelium of C. nuda and for exploiting their high-value bioactive constituents.
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To explore the regulatory effect of Lactarius akahatsu on soil enzyme activities and ecological stoichiometry characteristics in three different forest stands, and to clarify the effect of ectomycorrhizal fungi on forest soil nutrient cycling.
The Lactarius akahatsu shiroes soil and adjacent control soil were collected from Pinus massoniana pure forest, Cyclobalanopsis glauca pure forest and Pinus massoniana -Cyclobalanopsis glauca mixed forest. Soil physicochemical properties, including organic carbon (SOC), total nitrogen (TN), total phosphorus (TP), acid available phosphorus (AAP), ammonium nitrogen (NH4+-N), nitrate nitrogen (NO3--N), and pH value were analyzed. Enzyme activities related to carbon, nitrogen, and phosphorus cycling, including β-glucosidase (BG), N-acetyl-glucosaminidase (NAG), leucine aminopeptidase (LAP), urease (URE), acid phosphatase (AP), peroxidase (PER) were measured. The enzyme ecological stoichiometric ratio (EC∶N、EC∶P、EN∶P) and vector analysis (VA、VL) were calculated. Principal component analysis and redundancy analysis were employed to identify key drivers of enzymatic variation.
Compared to control soils, the contents of SOC, TN, TP, NH4+-N and NO3--N in Lactarius akahatsu shiroes soil in the three stands were significantly higher than control soil. Lactarius akahatsu shiroes soil in all three stands demonstrated increased activities of BG, NAG, LAP, and URE, while significantly reduced PER activity, though AP activity remained statistically invariant, with the most pronounced variation observed in Pinus massoniana -Cyclobalanopsis glauca mixed forest. The results of soil enzyme stoichiometry revealed that the EC∶P and EN∶P ratios in Lactarius akahatsu shiroes soil in the three stands significantly increased, with the highest values in Cyclobalanopsis glauca pure forest. In the Pinus massoniana-Cyclobalanopsis glauca mixed forest, the EC∶N ratio in shiroes soil decreased significantly, while the EC∶P and EN∶P ratios increased significantly. All three stands exhibited carbon and phosphorus limitations, with the Cyclobalanopsis glauca pure forest experiencing the most severe phosphorus limitation. Lactarius akahatsu was more effective at relieving soil phosphorus limitation in the Pinus massoniana-Cyclobalanopsis glauca mixed forest. Redundancy analysis and vector analysis identified soil C∶N ratio and NH4+-N as critical regulatory factors affecting soil enzyme activities, with URE serving as a key indicator for distinguishing soil properties among the three forest stands.
Lactarius akahatsu altered soil physicochemical properties, enzyme activities and ecological stoichiometry characteristics in three forest stands, it significantly enhanced soil nutrient accumulation and enzyme activities in the Pinus massoniana -Cyclobalanopsis glauca mixed forest, and effectively alleviated soil phosphorus limitation, which could provide a reference for mycorrhizal management of plantations.
This study aims to analyze and compare the differences in root metabolites before and after mycorrhizal synthesis between Lactarius akahatsu and Pinus massoniana, in order to provide reference for exploring the potential substances regulating the symbiosis between L. akahatsu and P. massoniana.
The mycorrhizal of L. akahatsu and P. massoniana were synthesized on pure media. Liquid chromatography-mass spectrometry were used to determine the metabolic components of root system of P. massoniana and mycorrhizal of symbiotic system between L. akahatsu and P. massoniana. The metabolites were quantified and annotated using KEGG and HMDB databases. Principal component analysis, orthogonal partial least-squares discriminant analysis and other multivariate statistical methods were used to compare the metabolome data and screen the different metabolic components.
627 metabolites were detected from mycorrhizal between L. akahatsu and P. massoniana, of them 95 ones were differential metabolites, 39 ones were up-regulated and 56 were down-regulated. The main metabolites were flavonoids, terpenoids, amino acid and derivatives, phenols, alkaloids, organic acids and derivatives, nucleotide and its derivates, phenylpropanoids and lignan, phytohormone, etc. KEEG metabolic pathway analyses showed that different metabolites were significantly enriched in 9 metabolic pathways. They were biosynthesis of secondary metabolites, biosynthesis of phenylpropanoids, flavonoid biosynthesis, neuroactive ligand-receptor interaction, biosynthesis of siderophore group nonribosomal peptides, benzoate degradation, biosynthesis of plant hormones, biosynthesis of alkaloids derived from shikimate pathway and aminobenzoate degradation, which might be an important regulatory role in the formation of mycorrhizal synthesis between L. akahatsu and P. massoniana.
Using UPLC-MS/MS metabolomics technique, the metabolomics differences of mycorrhizal synthesis between L. akahatsu and P. massoniana can be revealed on an overall level. The results can provide a scientific basis for clarifying the symbiotic mechanism and the mycorrhizal synthesis of L. akahatsu and P. massoniana.
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