Development of Multiple Nucleotide Polymorphism Molecular Markers for Enoki Mushroom (Flammulina filiformis) Cultivars Identification.

The enoki mushroom (Flammulina filiformis) is one of the most important and popular edible mushrooms commercially in China. However, traditional mushroom cultivar identification is challenging due to poor accuracy, heavy workloads, and low reproducibility. To overcome this challenge, we developed a method for identifying F. filiformis strains using multiple nucleotide polymorphism sequencing (MNP-seq). This involved screening 179 universal MNP markers based on whole-genome sequencing data, constructing an MNP sequence library, and performing multiplex PCR amplification and high-sequencing. We further screened 69 core MNP markers and used them to build a neighbor-joining (NJ) phylogenetic tree of 232 cultivated and wild strains. Our analysis showed that all cultivars could be accurately separated by computing genetic similarity values and that the cultivars could be separated into 22 distinct evolutionary pedigrees. The specific value of genetic similarity can be used as the standard to distinguish F. filiformis cultivars, however, it needs to be comprehensively defined by the additional phenotype and biological characteristics of those strains in the future work.

Physiological changes and gene responses during Ganoderma lucidum growth with selenium supplementation.

Ganoderma lucidum basidiomycota is highly appreciated for its health and nutrition value. In the present study, Ganoderma lucidum was cultivated as selenium transformation carrier, and the physiological changes and gene responses by selenium supplementation were revealed through high-throughput RNA-Seq technology. As a result, selenium supplementation increased the stipe length and the cap size, but decreased the cap thickness of G. lucidum. Mineral salt supplementation could greatly promote the formation of triterpene acids and selenium in G. lucidum. The highest yield was gained in the treatment with selenium content of 200 µg/g. Subsequently, the tissues of G. lucidum at budding and mature stages in this treatment group were sampled for transcriptome analysis and compared to those of a control group without selenium supplementation. A total of 16,113 expressed genes were obtained from the transcriptome of G. lucidum, and GO-annotated unigenes were mainly involved in molecular functions and KEGG-annotated ones were highly expressed in ribosomal pathway. Furthermore, genes involved in carbon metabolism pathway were most promoted by selenium at budding stage of G. lucidum, while gene expression was the highest in the pathway of amino acid biosynthesis at mature stage of G. lucidum. Specially, selenium-related genes in G. lucidum, such as GL23172-G, GL29881-G and GL28298-G, played a regulatory role in oxidoreductase, antioxidant activity and tryptophan synthesis. The results provide a theoretical basis for further study of selenium-enriched mushrooms and aid to development of Se-enriched foodstuff and health products made from fungi.

Temperature affects substrate-associated bacterial composition during Ganoderma lucidum hyphal growth.

The growth of the well-known fungus Ganoderma lucidum is influenced by temperature, which has an impact on the associated microbial structure in the substrate. In this study, we analyzed the bacterial diversity of the substrate at different temperatures using next-generation sequencing technology. A total of 513 733 sequences from 15 samples were assigned to 19 bacterial phyla. The samples were dominated by Proteobacteria, followed by Firmicutes; the 2 phyla exhibited opposite changes with elevated temperature. Bacterial genera showed different abundances at different temperatures, in which Sediminibacterium maintained a stable abundance below 40 °C, while Ochrobactrum and Rhodococcus were enriched with elevated temperature and both showed their highest abundances at 40 °C. Functional prediction uncovered 39 identified KEGG pathways, and bacterial genes involved in the membrane transport pathway exhibited the highest abundance subject to heat (40 °C) during the growth of G. lucidum. In general, our findings illustrated the influence of temperatures on G. lucidum mycelial morphology and the bacterial community in the substrate, and the results will facilitate cultivation of this fungus.

Different maturities drive proteomic and metabolomic changes in Chinese black truffle.

Chinese black truffle (Tuber indicum) is rich in nutrition. However, commercial interests lead to the aroma components and nutrients of T. indicum being greatly affected by overexploitation without consideration of their maturity. This study investigated the proteomic and metabolomic profiles of truffle fruiting bodies at different maturities using a meta-proteomic approach. Among the 3007 identified proteins, the most up-expressed protein in the mature ascocarps was involved in the peptidyl-diphthamide biosynthetic process, while thiamine metabolism was the most differentially expressed pathway. Furthermore, a total of 54 metabolites identified upon LC-MS differed significantly, with 30 being up-expressed in the mature ascocarps, including organic acids, carnitine substances and polysaccharides. Additionally, the ash, protein, fat, crude fiber and total sugar contents were all higher in the mature ascocarps. Overall, our findings reveal that mature truffles have a higher nutritional value, providing a basis for further exploring protein functionality of T. indicum at different maturities.

LC-MS-Based Metabolomic Approach Revealed the Significantly Different Metabolic Profiles of Five Commercial Truffle Species.

Truffles are ascomycetous ectomycorrhizal fungi that have elevated status in the culinary field due to their unique aroma and taste as well as their nutritional value and potential biological activities. Tuber melanosporum, T. indicum, T. panzhihuanense, T. sinoaestivum, and T. pseudoexcavatum are five commercial truffle species mainly distributed in Europe or China. In this study, an untargeted metabolomics technology based on an ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was applied to analyze the metabolic profiles and variations among these five truffle species. In our results, a total of 2376 metabolites were identified under positive ion mode, of which 1282 had significantly differential amounts and covered 110 pathways or metabolisms. Principal component analysis (PCA) and partial least squares-discriminant analysis (PLS-DA) revealed a clear separation from each of these five truffles, indicating a significantly different metabolic profile among them, with the biggest difference between T. melanosporum and the other four truffles. The differential metabolites covered various chemical categories, and a detailed analysis was performed for nine metabolic categories, including amino acids, saccharides and nucleosides, organic acids, alkaloids, flavonoids, carnitines, phenols and alcohols, esters, and sulfur compounds. For each of the nine categories, most of metabolites predominantly accumulated in T. melanosporum compared with the other four truffles. Meanwhile, there were significant differences of the average ion intensity in each category among the five truffles, e.g., higher amounts of amino acids was detected in T. panzhihuanense and T. pseudoexcavatum; T. indicum contained significantly more carnitines, while there were more alkaloids in T. melanosporum. Additionally, some metabolites with biological activities were discussed for each category, such as acetyl-L-carnitine, adenine, neobavaisoflavone, and anandamide. Generally, this study may provide the valuable information regarding the variation of the metabolic composition of these five commercial truffle species, and the biological significance of these metabolites was uncovered to explore the metabolic mechanisms of truffles, which would be helpful for further research on the compounds and potential biological functions in truffles that have not yet been investigated.

Validation of reference genes for quantitative gene expression analysis in Auricularia cornea.

Auricularia cornea Ehrenb., previously named A. polytricha (Mont.) Sacc, has become one of the most widely cultivated mushrooms in China. Considerable research has been conducted on its cultivation, pathogen identification, proteomics, and more. However, to the best of our knowledge, no studies have been performed on reference-gene validation in this species. Formerly, reference genes were selected for their expression levels only relied upon from others species, owing to the fact that the gene stability in this species is unknown. In this study, nine candidate genes, including tubulin alpha-1A chain (TUBA1A), ß-tubulin (Btu), phosphoglucomutase (Pgm), actin 1 (Act1), protein phosphatase 2A regulatory subunit (PP2A), polyubiquitin (UBQ), glyceraldehyde-3-phosphate dehydrogenase (Gapdh), 18S ribosomal protein (18S) and 28S ribosomal protein (28S), were evaluated among different strains and developmental stages. Four algorithms (i.e., geNorm, NormFinder, BestKeeper and RefFinder) were used to analyze candidate genes. The results revealed that UBQ was the most stable reference gene, while 18S was the least stable. Despite these results, the candidate genes were largely inadequate and only two were considered suitable. Based on candidate gene stability, PP2A and UBQ were identified as a set of usable interior control genes for future analyses in this species. This is the first systematic study conducted for selecting reference genes in A. cornea, and lays the foundation for identifying genes and quantifying gene expression in this species.

Influence of Temperature on the Bacterial Community in Substrate and Extracellular Enzyme Activity of Auricularia cornea.

Temperature is an important environmental factor that can greatly influence the cultivation of Auricularia cornea. In this study, lignin peroxidase, laccase, manganese peroxidase, and cellulose in A. cornea fruiting bodies were tested under five different temperatures (20 °C, 25 °C, 30 °C, 35 °C, and 40 °C) in three different culture periods (10 days, 20 days and 30 days). In addition, the V4 region of bacterial 16S rRNA genes in the substrate of A. cornea cultivated for 30 days at different temperatures were sequenced using next-generation sequencing technology to explore the structure and diversity of bacterial communities in the substrate. Temperature and culture days had a significant effect on the activities of the four enzymes, and changes in activity were not synchronized with changes in temperature and culture days. Overall, we obtained 487,694 sequences from 15 samples and assigned them to 16 bacterial phyla. Bacterial community composition and structure in the substrate changed when the temperature was above 35 °C. The relative abundances of some bacteria were significantly affected by temperature. A total of 35 genera at five temperatures in the substrate were correlated, and 41 functional pathways were predicted in the study. Bacterial genes associated with the membrane transport pathway had the highest average abundance (16.16%), and this increased at 35 °C and 40 °C. Generally, different temperatures had impacts on the physiological activity of A. cornea and the bacterial community in the substrate; therefore, the data presented herein should facilitate cultivation of A. cornea.

Proteomic Analysis Revealed the Fruiting-Body Protein Profile of Auricularia polytricha.

Auricularia polytricha is one of the most widely cultivated edible mushrooms in China. Many advances have been made to A. polytricha, but there is still no proteomic information of this species. Our current understanding was based upon the translated information of its transcriptome or other relative species. This study presented the proteomic information of fruiting-body proteins by shotgun liquid chromatography and tandem mass spectrometry (LC-MS/MS), which identified 15,508 peptides corresponding to 1850 high-confidence proteins. Of these, 1383 were annotated across the GO subcategories with 829 (44.81%) involved in biological process, 908 (49.08%) in molecular function, and 406 (21.95%) in cellular components. Among these high-confidence proteins, 132 proteins were annotated as carbohydrate-active enzymes, of which 51 were secreted enzymes. Moreover, a number of commercially important enzymes were detected, functioning as auxiliary activity (AA) family 5 glyoxal oxidase, AA5 galactose oxidase, glycoside hydrolase (GH) family 20 hexosaminidase, and GH47 alpha-mannosidase. To the best of our knowledge, this is the first study to characterize A. polytricha proteome, and also fills the gap of our knowledge on the under-developed mushroom species.

A new species of Scytalidium causing slippery scar on cultivated Auricularia polytricha in China.

Slippery scar is one of the most destructive diseases encountered in the cultivation of Auricularia polytricha (hairy wood ear); however, the identity of the pathogenic agent has remained uncertain. This study was designed to identify the causative pathogen of slippery scar in A. polytricha and to investigate the taxonomic classification of the pathogen by morphological observations, in vivo pathogenicity tests, and molecular evidences of ITS and RPB2 sequences. The results showed that the pathogen was a new Scytalidium species, here named Scytalidium auriculariicola. Scytalidium auriculariicola was characterized by its rapid growth rate, the catenate conidia of variable size, and the pale brown to brown chlamydoconidia. Phylogenetic analyses based on internal transcribed spacer regions and RPB2 sequences on the pathogen isolated and related species supported that S. auriculariicola was a true Scytalidium species. It was congeneric with and close to Scytalidium lignicola, the type species of Scytalidium. However, it differed from the latter species in the size of conidia, 33 different nucleotide bases in ITS sequences and 30 different nucleotide bases in RPB2 sequences.

An assessment of the genetic diversity within Ganoderma strains with AFLP and ITS PCR-RFLP.

Ganoderma lucidum is one of the most important medicinal materials and plant pathogens. Because of its specific interhybridization, the genetic background, however, is relatively unclear. It made identification of Ganoderma strains, especially closely related strains difficulty. Amplified fragment length polymorphism (AFLP) using 14 primer combinations and internal transcribed spacer (ITS) PCR-RFLP were used in a comparative study which was designed to investigate the closely related Ganoderma strains genetic relations at molecular level. The analysis of 37 Ganoderma strains showed there were 177 polymorphic AFLP markers and 12 ITS PCR-RFLP markers, and all accessions could be uniquely identified. Among the Ganoderma accessions, similarity coefficients ranged from 0.07692 to 0.99194 in AFLP. The Ganoderma strains formed a tight cluster in nine groups in AFLP whereas seven groups in ITS PCR-RFLP. The cluster analysis revealed that the taxonomical system of subgenus Ganoderma is composed of Sect. Ganoderma and Sect. Phaeonema, and the strain 22 should be a variant form of strain 21. All methods delineated the Ganoderma strains from the different regions seeming to show a greater level of genetic diversity. It indicated that the genotype study at molecular level is a useful complement method to the current classification system of Ganoderma strains based on morphological traits. The congruency of the experiments was analyzed using the biostatistical software DPS V3.01.

[AFLP analysis for genetic diversity of Ganoderma].

OBJECTIVE: To investigate the genetic diversity of Ganoderma cultivars provided for the genuineness study, germ-plasm resource identification, genetic relationship study, breeding, introduction and cultivante of Ganoderma strains. METHOD: With the software of NTSYSpc 2. 1, 24 materials, of G. lucidum and G. sinense, were studied using AFLP to construct the dendrogram. RESULT: There were 177 polymorphic bands with 14 primer combinations. And all materials could be identified with AFLP. CONCLUSION: There actually existed much genetic diversity at the molecular level among the germplasm resources of Ganoderma strains, and all the strains were clustered into G. lucidum group and G. sinense group at the similarity coefficient 0. 676.