Search

Soil microbial communities play a vital role in accelerating nutrient cycling and stabilizing ecosystem functions in forests. However, the diversity of soil microbiome and the mechanisms driving their distribution patterns along elevational gradients in montane areas remain largely unknown. In this study, we investigated the soil microbial diversity along an elevational gradient from 650 m to 3,800 m above sea level in southeast Tibet, China, through DNA metabarcode sequencing of both the bacterial and fungal communities. Our results showed that the dominant bacterial phyla across elevations were Proteobacteria, Acidobacteriota and Actinobacteriota, and the dominant fungal phyla were Ascomycota and Basidiomycota. The Simpson indices of both soil bacteria and fungi demonstrated a hollow trend along the elevational gradient, with an abrupt decrease in bacterial and fungal diversity at 2,600 m a.s.l. in coniferous and broad-leaved mixed forests (CBM). Soil bacterial chemoheterotrophy was the dominant lifestyle and was predicted to decrease with increasing elevation. In terms of fungal lifestyles, saprophytic and symbiotic fungi were the dominant functional communities but their relative abundance was negatively correlated with increasing elevation. Environmental factors including vegetation type (VEG), altitude (ALT), soil pH, total phosphorus (TP), nitrate nitrogen (NO3−-N), and polyphenol oxidase (ppo) all exhibited significant influence on the bacterial community structure, whereas VEG, ALT, and the carbon to nitrogen ratio (C/N) were significantly associated with the fungal community structure. The VPA results indicated that edaphic factors explained 37% of the bacterial community variations, while C/N, ALT, and VEG explained 49% of the total fungal community variations. Our study contributes significantly to our understanding of forest ecosystems in mountainous regions with large elevation changes, highlighting the crucial role of soil environmental factors in shaping soil microbial communities and their variations in specific forest ecosystems.

【Objective】Cordyceps guangdongensis, a rare edible-medicinal fungus in China, has been approved as a new resource food, with a broad prospect of industrial application. The fruitingbodies of this fungus are rich in nutrients and have significant active effect. The protoplast preparation and genetic transformation methods of Cordyceps guangdongensis were established in order to provide references for new variety breeding and gene function research of this fungus.【Method】Protoplasts were separated from the mycelia of C. guangdongensis by various enzymes. During the process of protoplasts preparation, the enzyme combinations, enzyme digestion time and temperature, regeneration medium of protoplasts and resistance marker were selected by using single-factor analyses. Plasmid pCAMBIA1300 was used as transformation fragment to establish a genetic transformation system, and the concentration of spermidine, PEG addition interval and resistance marker addition time during PEG-mediated protoplast transformation were also screened by single-factor analyses. The knockout fragment of Zn2Cys6-type transcription factor CgPRo1 in C. guangdongensis was used as transformation fragment to verify the transformation system.【Result】The optimal condition for protoplast preparation was mycelium treated with lytic enzyme and driselase (1:1) for 5.5 h at 28 ℃. The highest protoplast regeneration rate of C. guangdongensis was observed on TB3 medium, followed by 0.6 mol/L KCl-PDA. During the transformation process, adding 5 mmol/L spermidine, PEG at an interval of 10 min and screening medium with 250 μg/mL hygromycin after protoplast regeneration for 3 days showed the optimal transformation efficiency. In addition, three CgPro1 knockout mutants and three heterozygotes were obtained through the established transformation system. Phenotypic analysis results showed that CgPro1 was involved in the fruit body development of C. guangdongensis.【Conclusion】In the study, the optimized protoplast preparation method and PEG-mediated protoplast transformation system for C. guangdongensis were established. The techniques and procedures described will lay a solid foundation for future researches on gene function and protein localization, and also provide important application value in genetic breeding of in C. guangdongensis.

Obesity has caused serious health and economic problems in the world. Cordyceps guangdongensis is a high-value macrofungus with broad application potential in the food and bio-medicine industry. This current study aimed to estimate the role of C. guangdongensis lipid-lowering compound formula (CGLC) in regulating fat and lipid accumulation, gut microbiota balance, short-chain fatty acid (SCFA) contents, and expression levels of genes involved in fat and lipid metabolism in high-fat diet (HFD) mice. The results showed that CGLC intervention markedly reduced body weights and fat accumulation in HFD mice, improved glucose tolerance and blood lipid levels, and decreased lipid droplet accumulation and fat vacuole levels in the liver. CGLC decreased the ratio of Firmicutes and Bacteroidetes and increased the relative abundances of Bacteroides (B. acidifaciens) and Bifidobacterium (B. pseudolongum). In addition, CGLC treatment significantly promoted the production of SCFAs and regulated the relative expression levels of genes involved in fat and lipid metabolism in liver. Association analysis showed that several species of Bacteroides and most of SCFAs were significantly associated with serum lipid indicators. These results suggested that CGLC is a novel candidate formulation for treating obesity and non-alcohol fatty liver by regulating gut microbiota, SCFAs, and genes involved in fat and lipid metabolism.

Melatonin, a bioactive compound and an important signaling molecule produced in plants and animals, is involved in many biological processes. However, its function and synthetic pathways in fungi are poorly understood. Here, the samples from Tolypocladium guangdongense, a highly valued edible fungus with functional food properties, were collected under different experimental conditions to quantify the levels of melatonin and its intermediates. The results showed that the intracellular melatonin content was markedly improved by Congo red (CR), cold, and heat stresses; the levels of intracellular melatonin and its intermediates increased at the primordial (P) and fruiting body (FB) stages. However, the levels of most intermediates exhibited a notable decrease under CR stress. Several genes related to melatonin synthesis, excluding AADC (aromatic-L-amino-acid decarboxylase), were markedly upregulated at an early stage of CR stress but downregulated later. Compared to the mycelial stage, those genes were significantly upregulated at the P and FB stages. Additionally, exogenous melatonin promoted resistance to several abiotic stressors and P formation in T. guangdongense. This study is the first to report melatonin biosynthesis pathway in macro-fungi. Our results should help in studying the diversity of melatonin function and melatonin-synthesis pathways and provide a new viewpoint for melatonin applications in the edible-medicinal fungus.

Tolypocladium guangdongense has a similar metabolite profile to Ophiocordyceps sinensis, a highly regarded fungus used for traditional Chinese medicine with high nutritional and medicinal value. Although the genome sequence of T. guangdongense has been reported, relatively little is known about the regulatory networks for fruiting body development and about the metabolite biosynthesis pathways. In order to address this, an analysis of transcriptome and proteome at differential developmental stages of T. guangdongense was performed. In total, 9076 genes were found to be expressed and 2040 proteins were identified. There were a large number of genes that were significantly differentially expressed between the mycelial stage and the stages. Interestingly, the correlation between the transcriptomic and proteomic data was low, suggesting the importance of the post-transcriptional processes in the growth and development of T. guangdongense. Among the genes/proteins that were both differentially expressed during the developmental process, there were numerous heat shock proteins and transcription factors. In addition, there were numerous proteins involved in terpenoid, ergosterol, adenosine and polysaccharide biosynthesis that also showed significant downregulation in their expression levels during the developmental process. Furthermore, both tryptophan and tryptamine were present at higher levels in the primordium stage. However, indole-3-acetic acid (IAA) levels continuously decreased as development proceeded, and the enzymes involved in IAA biosynthesis were also clearly differentially downregulated. These data could be meaningful in studying the molecular mechanisms of fungal development, and for the industrial and medicinal application of macro-fungi.

Background Fungal GATA-type transcription factors (GATA-TFs) are a class of transcriptional regulators involved in various biological processes. However, their functions are rarely analyzed systematically, especially in edible or medicinal fungi, such as Tolypocladium guangdongense, which has various medicinal and food safety properties with a broad range of potential applications in healthcare products and the pharmaceutical industry. Methods GATA-TFs in T. guangdongense (TgGATAs) were identified using InterProScan. The type, distribution, and gene structure of TgGATAs were analyzed by genome-wide analyses. A phylogenetic tree was constructed to analyze their evolutionary relationships using the neighbor-joining (NJ) method. To explore the functions of GATA-TFs, conserved domains were analyzed using MEME, and cis-elements were predicted using the PlantCARE database. In addition, the expression patterns of TgGATAs under different light conditions and developmental stages were studied using qPCR. Results Seven TgGATAs were identified. They were randomly distributed on four chromosomes and contained one to four exons. Phylogenetic analysis indicated that GATA-TFs in each subgroup are highly conserved, especially for GATA1 to GATA5. Intron distribution analyses suggested that GATA1 and GATA3 possessed the most conserved gene structures. Light treatments induced the expression levels of TgGATA1 and TgGATA5-7, but the expression levels varied depending on the duration of illumination. The predicted protein structures indicate that TgGATA1 and TgGATA2 possess typical light-responsive domains and may function as photoreceptors to regulate downstream biological processes. TgGATA3 and TgGATA5 may be involved in nitrogen metabolism and siderophore biosynthesis, respectively. TgGATA6 and TgGATA7 possess unique Zn finger loop sequences, suggesting that they may have special functions. Furthermore, gene expression analysis indicated that TgGATA1 (WC1) was notably involved in mycelial color transformation, while other genes were involved in fruiting body development to some extent. These results provide valuable information to further explore the mechanisms through which TgGATAs are regulated during fruiting body development.

Our previous study found that LeDnaJ07 RNAi decreased Lentinula edodes resistance to heat stress and Trichoderma atroviride infection. In this study, the structure and function of the LeDnaJ07 gene was analyzed by gene cloning and overexpression in L. edodes stress-sensitive strain YS55 via the Agrobacterium-mediated transformation method. Transformants were confirmed by qRT-PCR, fluorescence observation and Southern blotting. Overexpression of LeDnaJ07 in YS55 not only enhanced L. edodes mycelial resistance to heat stress but also facilitated mycelial growth. In the presence of heat stress, the intracellular IAA content showed a significant increase in the two LeDnaJ07 overexpression strains but only a slight change in the YS55 wild type strain. Moreover, the interaction between LeDnaJ07 and LetrpE was demonstrated via Y2H and BiFC assays. These results suggested that LeDnaJ07 may be involved in regulating IAA biosynthesis and the resistance of L. edodes to heat stresses via interacting with LetrpE.

Lentinula edodes (shiitake mushrooms) is heavily affected by the infection of Trichoderma atroviride, causing yield loss and decreases quality in shiitake mushrooms. The selection and breeding of fungal-resistant L. edodes species are an important approach to protecting L. edodes from T. atroviride infection. Herein, a highly resistant L. edodes strain (Y3334) and a susceptible strain (Y55) were obtained by using a resistance evaluation test. Transcriptome analyses and qRT-PCR detection showed that the expression level of LeTLP1 (LE01Gene05009) was strongly induced in response to T. atroviride infection in the resistant Y3334. Then, LeTLP1-silenced and LeTLP1-overexpression transformants were obtained. Overexpression of LeTLP1 resulted in resistance to T. atroviride. Compared with the parent strain Y3334, LeTLP1-silenced transformants had reduced resistance relative to T. atroviride. Additionally, the LeTLP1 protein (Y3334) exhibited significant antifungal activity against T. atroviride. These findings suggest that overexpression of LeTLP1 is a major mechanism for the resistance of L. edodes to T. atroviride. The molecular basis provides a theoretical basis for the breeding of resistant L. edodes strains and can eventually contribute to the mushroom cultivation industry and human health.

Volatile organosulfur compounds are the main components that contribute to the unique aroma of dried Lentinula edodes. They are mainly generated during the hot-air drying process, and cysteine desulfurase is the key enzyme in this process. Temperature may be an essential factor of volatile organosulfur compound production by influencing the expression of the cysteine desulfurase gene. In this study, the promoter sequence of the cysteine desulfurase gene (pCS) was cloned and analyzed using bioinformatics tools. A series of 5′deletion fragments and site-directed mutations of pCS were constructed to identify the element that responds to heat stress. Six heat shock transcription factor (HSTF) binding sites were predicted by SCPD (The Promoter Database of Saccharomyces cerevisiae) and three of the binding sites were predicted by Yeastract (Yeast Search for Transcriptional Regulators and Consensus Tracking) in pCS. The results indicated that pCS was able to drive the expression of the EGFP (Enhanced Green Fluorescent Protein) gene in L. edodes. Moreover, the fluorescence intensity increased after heat stress. The changes in fluorescence intensity of different 5′deletion fragments showed that the heat response region was located between −500 bp and −400 bp in pCS. The site-directed mutation analysis further showed that the heat-inducible element was between −490 bp and −500 bp (TTTCTAGAAT) in pCS. Our results provide molecular insight for studying the formation of volatile organosulfur compounds in dried L. edodes.

Volatile sulfur compounds gradually develop in Lentinula edodes after hot-air drying, and many genes are involved in the generation of these sulfur compounds. The expression stability of reference genes may vary in a particular experimental treatment when analyzing their expressions by quantitative real-time polymerase chain reaction (qRT-PCR). In this study, the expression profile of 17 candidate genes was assessed in L. edodes under treatment at 50 °C for 0, 1, 2, and 3 h, and the expression stability of each reference gene was analyzed by three statistical algorithms, including geNorm, NormFinder, and BestKeeper. Results indicated that the two optimal reference genes for mycelium and fruiting body were CAC and DAHP as well as CAC and NUP, respectively. Additionally, CAC and DAHP were found to be the two most stable reference genes across the mycelium and fruiting body set. Our results will provide a genetic foundation for further research on the metabolism genes of sulfur compounds in L. edodes.

Lentinula edodes, one of the most popular, edible mushroom species with a high content of proteins and polysaccharides as well as unique aroma, is widely cultivated in many Asian countries, especially in China, Japan and Korea. As a white rot fungus with lignocellulose degradation ability, L. edodes has the potential for application in the utilization of agriculture straw resources. Here, we report its 41.8-Mb genome, encoding 14,889 predicted genes. Through a phylogenetic analysis with model species of fungi, the evolutionary divergence time of L. edodes and Gymnopus luxurians was estimated to be 39 MYA. The carbohydrate-active enzyme genes in L. edodes were compared with those of the other 25 fungal species, and 101 lignocellulolytic enzymes were identified in L. edodes, similar to other white rot fungi. Transcriptome analysis showed that the expression of genes encoding two cellulases and 16 transcription factor was up-regulated when mycelia were cultivated for 120 minutes in cellulose medium versus glucose medium. Our results will foster a better understanding of the molecular mechanism of lignocellulose degradation and provide the basis for partial replacement of wood sawdust with agricultural wastes in L. edodes cultivation.

The extracts from thirty plants collected or brought in the market were added to the culture medium to screen their antimicrobial activities in four pathogens of edible fungi fruiting bodies. The results measuring the radial lengths of four pathogen mycelia demonstrated that in the thirty plants tested, the inhibitory rate of Allium sativum extracts on Trichothecium roseum was 99.1%. The inhibitory rate of Taraxacum mongolicum extracts on Mycogone perniciosa was 96.3%. The inhibitory rate of Punica granatum and Solarium nigrum extracts on Lecanicillium fungicola was 85.2% and 83.5%, respectively. However, all the plant extracts showed no significant inhibitory effects on Verticillium fungicola, the inhibitory rates of which were all less than 40.3%. [ABSTRACT FROM AUTHOR]

A new partitivirus named Lentinula edodes partitivirus 1 (LePVl) was isolated from a diseased L. edodes strain with severe degeneration of the mycelium and imperfect browning in bag cultures. The nucleotide sequences of LePVl dsRNA-1 and dsRNA-2 were determined; they were 2,382 bp and 2,245 bp in length, and each contained a single ORF encoding RNA-dependent RNA polymerase (RciRp) and coat protein (CP), respectively. The purified virus preparation contained isometric particles 34 nm in diameter encapsidating these dsRNAs. Phylogenetic analyses showed LePVl to be a new member of Betapartitivirus, with the RdRp sequence most closely related to Grapevine partitivirus. RT-PCR analysis showed that 27 of the 56 Chinese L. edodes core collection strains carry LePVl, with the virus being more common in wild strains than cultivated strains. In addition, qPCR analysis suggested that coinfection with L. edodes mycovirus HKB (LeV-HKB) could increase replication of the RdRp gene of LePVl. This study may be essential for the development of more accurate disease diagnostics and the formulation of control strategies for viral diseases in L. edodes. [ABSTRACT FROM AUTHOR]