Cloning and Expression Analysis of Bioluminescence Genes in Omphalotus guepiniiformis Reveal Stress-Dependent Regulation of Bioluminescence.

Bioluminescence is a type of chemiluminescence that arises from a luciferase-catalyzed oxidation reaction of luciferin. Molecular biology and comparative genomics have recently elucidated the genes involved in fungal bioluminescence and the evolutionary history of their clusters. However, most studies on fungal bioluminescence have been limited to observing the changes in light intensity under various conditions. To understand the molecular basis of bioluminescent responses in Omphalotus guepiniiformis under different environmental conditions, we cloned and sequenced the genes of hispidin synthase, hispidin-3-hydroxylase, and luciferase enzymes, which are pivotal in the fungal bioluminescence pathway. Each gene showed high sequence similarity to that of other luminous fungal species. Furthermore, we investigated their transcriptional changes in response to abiotic stresses. Wound stress enhanced the bioluminescence intensity by increasing the expression of bioluminescence pathway genes, while temperature stress suppressed the bioluminescence intensity via the non-transcriptional pathway. Our data suggested that O. guepiniiformis regulates bioluminescence to respond differentially to specific environmental stresses. To our knowledge, this is the first study on fungal bioluminescence at the gene expression level. Further studies are required to address the biological and ecological meaning of different bioluminescence responses in changing environments, and O. quepiniiformis could be a potential model species.

Draft Genome Sequence for the Symbiotic Pine Mushroom Tricholoma matsutake.

We report the high-quality genome sequence of Tricholoma matsutake strain 2001, which was isolated from a mushroom fruiting body in South Korea. The genome has 80 contigs, a size of 162.6 Mb, and an N50 value of 5,103,859 bp and will provide insight into the symbiotic association between T. matsutake and Pinus densiflora.

Determination and Analysis of Hyper-Variable A Mating Types in Wild Strains of Lentinula edodes in Korea.

The diversity of A mating type in wild strains of Lentinula edodes was extensively analyzed to characterize and utilize them for developing new cultivars. One hundred twenty-three A mating type alleles, including 67 newly discovered alleles, were identified from 106 wild strains collected for the past four decades in Korea. Based on previous studies and current findings, a total of 130 A mating type alleles have been found, 124 of which were discovered from wild strains, indicating the hyper-variability of A mating type alleles of L. edodes. About half of the A mating type alleles in wild strains were found in more than two strains, whereas the other half of the alleles were found in only one strain. About 90% of A mating type combinations in dikaryotic wild strains showed a single occurrence. Geographically, diverse A mating type alleles were intensively located in the central region of the Korean peninsula, whereas only allele A17 was observed throughout Korea. We also found the conservation of the TCCCAC motif in addition to the previously reported motifs, including ATTGT, ACAAT, and GCGGAG, in the intergenic regions of A mating loci. Sequence comparison among some alleles indicated that accumulated mutation and recombination would contribute to the diversification of A mating type alleles in L. edodes. Our data support the rapid evolution of A mating locus in L. edodes, and would help to understand the characteristics of A mating loci of wild strains in Korea and help to utilize them for developing new cultivars.

New findings on the fungal species Tricholoma matsutake from Ukraine, and revision of its taxonomy and biogeography based on multilocus phylogenetic analyses.

Matsutake mushrooms are among the best-known edible wild mushroom taxa worldwide. The representative Tricholoma matsutake is from East Asia and the northern and central regions of Europe. Here, we report the existence of T. matsutake under fir trees in Eastern Europe (i.e., Ukraine), as confirmed by phylogenetic analysis of nine loci on the nuclear and mitochondrial genomes. All specimens from Japan, Bhutan, China, North Korea, South Korea, Sweden, Finland, and Ukraine formed a T. matsutake clade according to the phylogeny of the internal transcribed spacer region. The European population of T. matsutake was clustered based on the ß2 tubulin gene, with a moderate bootstrap value. In contrast, based on analyses of three loci, i.e., rpb2, tef1, and the ß2 tubulin gene, T. matsutake specimens sampled from Bhutan and China belonged to a clade independent of the other specimens of this species, implying a genetically isolated population. As biologically available type specimens of T. matsutake have not been designated since its description as a new species from Japan in 1925, we established an epitype of this fungus, sampled in a Pinus densiflora forest in Nagano, Japan.

Marasmioid and Gymnopoid Fungi of the Republic of Korea. 8. Gymnopus Section Levipedes.

Collections of Gymnopus sect. Levipedes from the Republic of Korea have been studied. Two new species, Gymnopus dryophiloides and G. brunneodiscus , are described based on their macro- and micromorphological and phylogenetic characteristics. Three other species, referred to as Gymnopus spp. 1, 2, and 3, are distinguished as separate taxa without formal descriptions. Taxonomic and phylogenetic positions of all taxa have been inferred and confirmed by analyses of ITS and LSU sequence data. Their detailed descriptions, illustrations and an identification key are provided.

New Cantharellus species from South Korea.

In this third contribution involving new Cantharellus species from South Korea, two new species are introduced. In addition, we document a first report of the recently described Japanese Cantharellus anzutake outside of Japan based on identical ITS sequence data. Cantharellus citrinus sp. nov. is introduced as a new member of subgenus Cinnabarini, to which the closely related Korean C. albovenosus and Chinese C. phloginus also belong. Cantharellus curvatus sp. nov. is introduced as a new member of subgenus Parvocantharellus, in which the Korean C. koreanus was recently placed. The respective placements of the new taxa are significantly supported by a phylogenetic analysis of sequences from the transcription elongation factor (tef-1).

Acaulosproa koreana, a New Species of Arbuscular Mycorrhizal Fungi (Glomeromycota) Associated with Roots of Woody Plants in Korea.

A new species of arbuscular mycorrhizal fungi (Glomeromycota), Acaulospora koreana, was isolated from forest soils in South Korea. This novel fungus was collected from the rhizosphere of Lindera obtusiloba and Styrax obassia in forest and propagated with Sorghum bicolor in pot. Morphological characteristics of spores of A. koreana are rarely distinguished from Acaulospora mellea, which is reported as one of the most abundant mycorrhizal species in Korea. However, molecular evidence of rDNA sequence using improved primers for glomeromycotan fungal identification strongly supported that A. koreana is different from A. mellea but also any other species belonging to the genus Acaulospora. This is the first novel glomeromycatan fungus introduced in South Korea, but it suggests that there is a high possibility for discovering new arbuscular mycorrhizal fungi considering the abundance of plant species and advanced phylogenetic analysis technique.

Development and Molecular Characterization of Novel Polymorphic Genomic DNA SSR Markers in Lentinula edodes.

Sixteen genomic DNA simple sequence repeat (SSR) markers of Lentinula edodes were developed from 205 SSR motifs present in 46.1-Mb long L. edodes genome sequences. The number of alleles ranged from 3-14 and the major allele frequency was distributed from 0.17-0.96. The values of observed and expected heterozygosity ranged from 0.00-0.76 and 0.07-0.90, respectively. The polymorphic information content value ranged from 0.07-0.89. A dendrogram, based on 16 SSR markers clustered by the paired hierarchical clustering' method, showed that 33 shiitake cultivars could be divided into three major groups and successfully identified. These SSR markers will contribute to the efficient breeding of this species by providing diversity in shiitake varieties. Furthermore, the genomic information covered by the markers can provide a valuable resource for genetic linkage map construction, molecular mapping, and marker-assisted selection in the shiitake mushroom.

Community Structures of Arbuscular Mycorrhizal Fungi in Soils and Plant Roots Inhabiting Abandoned Mines of Korea.

In this study, we collected rhizosphere soils and root samples from a post-mining area and a natural forest area in Jecheon, Korea. We extracted spores of arbuscular mycorrhizal fungi (AMF) from rhizospheres, and then examined the sequences of 18S rDNA genes of the AMF from the collected roots of plants. We compared the AMF communities in the post-mining area and the natural forest area by sequence analysis of the AMF spores from soils and of the AMF clones from roots. Consequently, we confirmed that the structure of AMF communities varied between the post-mining area and the natural forest area and showed significant relationship with heavy metal contents in soils. These results suggest that heavy metal contamination by mining activity significantly affects the AMF community structure.

Identification of Single Nucleotide Polymorphism Markers in the Laccase Gene of Shiitake Mushrooms (Lentinula edodes).

We identified single nucleotide polymorphism (SNP) markers in the laccase gene to establish a line-diagnostic system for shiitake mushrooms. A total of 89 fungal isolates representing four lines, including Korean registered, Korean wild type, Chinese, and Japanese lines, were analyzed. The results suggest that SNP markers in the laccase gene can be useful for line typing in shiitake mushrooms.

Determination of Glucan Contents in the Fruiting Bodies and Mycelia of Lentinula edodes Cultivars.

Shiitake mushrooms (Lentinula edodes) containing ß-glucans may be beneficial for human health; they have been used in the treatment of cancer, hypertension, and high cholesterol levels. The objective of this study was to determine the ß-glucan content in different sections of the fruiting bodies and mycelia of ten shiitake mushroom cultivars. The measured ß-glucan content ranged from 20.06 ± 1.76% to 44.21 ± 0.13% in the pileus sections, and from 29.74 ± 1.40% to 56.47 ± 4.72% in the stipe sections. The results of this study indicate that the variance in ß-glucan content dependent on the shiitake cultivar, and that the ß-glucan content is higher in the stipe than in the pileus.

Enhancement of ß-Glucan Content in the Cultivation of Cauliflower Mushroom (Sparassis latifolia) by Elicitation.

The effectiveness of three kinds of enzymes (chitinase, ß-glucuronidase, and lysing enzyme complex), employed as elicitors to enhance the ß-glucan content in the sawdust-based cultivation of cauliflower mushroom (Sparassis latifolia), was examined. The elicitors were applied to the cauliflower mushroom after primordium formation, by spraying the enzyme solutions at three different levels on the sawdust-based medium. Mycelial growth was fully accomplished by the treatments, but the metabolic process during the growth of fruiting bodies was affected. The application of a lysing enzyme resulted in an increase in the ß-glucan concentration by up to 31% compared to that of the control. However, the treatment resulted in a decrease in mushroom yield, which necessitated the need to evaluate its economic efficiency. Although we still need to develop a more efficient way for using elicitors to enhance functional metabolites in mushroom cultivation, the results indicate that the elicitation technique can be applied in the cultivation of medicinal/edible mushrooms.

Diversity of arbuscular mycorrhizal fungi and their roles in ecosystems.

Arbuscular mycorrhizal fungi (AMF) have mutualistic relationships with more than 80% of terrestrial plant species. This symbiotic relationship is ancient and would have had important roles in establishment of plants on land. Despite their abundance and wide range of relationship with plant species, AMF have shown low species diversity. However, molecular studies have suggested that diversity of these fungi may be much higher, and genetic variation of AMF is very high within a species and even within a single spore. Despite low diversity and lack of host specificity, various functions have been associated with plant growth responses to arbuscular mycorrhizal fungal colonization. In addition, different community composition of AMF affects plants differently, and plays a potential role in ecosystem variability and productivity. AMF have high functional diversity because different combinations of host plants and AMF have different effects on the various aspects of symbiosis. Consequently, recent studies have focused on the different functions of AMF according to their genetic resource and their roles in ecosystem functioning. This review summarizes taxonomic, genetic, and functional diversities of AMF and their roles in natural ecosystems.

Morphological and genetic characteristics of newly crossbred cauliflower mushroom (Sparassis latifolia).

Cauliflower mushroom (Sparassis latifolia or S. crispa) is popular for food and medicine. Importance of new varieties of Sparassis was raised and studied widely by protection system of UPOV. In this study, 10 crossbred strains of Sparassis latifolia that specifically expressed distinctive features during basidiocarp formation and mycelium growth were applied to sawdust medium inoculated with S. latifolia mycelia. The 10 crossbred strains were divided into 3 groups on the basis of morphological (size of marginal wave and basidiocarp color) and genetic characteristics. Each phenotype of the parent and crossbred strains represented 3 marginal wave-sizes (large, medium, and small) and 3 color notations (NN155D, 163C, and 8D). Our result suggests that morphological characteristics of cauliflower mushroom can be affected by various environmental and genetic stimuli under artificial conditions such as crossbreed. Also this research showed genetic differences among breeding isolates and their morphological characteristics were correlated with the molecular data within parent and crossed strain.

Phylogenetic relationships of Korean Sparassis latifolia based on morphological and ITS rDNA characteristics.

Recent studies based on morphological characteristics and molecular analyses have revealed that the characteristics of Sparassis crispa from Asia are not concordant with those of collections from Europe and North America. Consequently, the Asian isolate was redefined as Sparassis latifolia. This study is the first report of Sparassis latifolia collected in Korea. The taxonomic relationships and replacement of Sparassis species were inferred from a comparison of the morphological characteristics and by molecular sequence analysis of the internal transcribed spacer (ITS) rDNA regions. In particular, this study focused on the phylogenetic relationships inferred from the biogeographical distribution of isolates within the genus Sparassis.

SIRT1 attenuates palmitate-induced endoplasmic reticulum stress and insulin resistance in HepG2 cells via induction of oxygen-regulated protein 150.

Endoplasmic reticulum (ER) stress has been implicated in the pathology of type 2 diabetes mellitus (T2DM). Although SIRT1 has a therapeutic effect on T2DM, the mechanisms by which SIRT1 ameliorates insulin resistance (IR) remain unclear. In this study, we investigated the impact of SIRT1 on palmitate-induced ER stress in HepG2 cells and its underlying signal pathway. Treatment with resveratrol, a SIRT1 activator significantly inhibited palmitate-induced ER stress, leading to the protection against palmitate-induced ER stress and insulin resistance. Resveratrol and SIRT1 overexpression induced the expression of oxygen-regulated protein (ORP) 150 in HepG2 cells. Forkhead box O1 (FOXO1) was involved in the regulation of ORP150 expression because suppression of FOXO1 inhibited the induction of ORP150 by SIRT1. Our results indicate a novel mechanism by which SIRT1 regulates ER stress by overexpression of ORP150, and suggest that SIRT1 ameliorates palmitate-induced insulin resistance in HepG2 cells via regulation of ER stress.

New species and new records of Crinipellis from tropical and subtropical forests of the east coast of Mexico.

Crinipellis brunneoaurantiaca, C. pallidibrunnea and C. rubella are described as new species and their taxonomic position is discussed. The two former were collected in subdeciduous tropical forest and the latter in the montane cloud forest, all from the east coast of Mexico (central Veracruz). Crinipellis podocarpi, C. pseudostipitaria var. mesites, C. setipes, recorded in montane cloud forest, and C. tucumanensis, collected in subdeciduous tropical forest, also are discussed. Detailed macro- and microscopic descriptions, illustrations of distinctive microscopic characters and plates are presented for each species.

Spore Dispersion of Tricholoma matsutake at a Pinus densiflora Stand in Korea.

The spore of Tricholoma matsutake is considered to be the starting point of the mushroom growth cycle, but the mechanism of mycelial development from the spore stage is not yet clarified. In this study, we tried to measure how far the spores of T. matsutake disperse from a fruiting body located at a Pinus densiflora stand in Korea. We established 16 slide glasses coated with glycerin near a fruiting body in four directions separated by four different distance intervals within a mushroom productive stand after removing all other fruiting bodies from three plots. The number of dispersed spores increased with time from the first day (475 spores/cm(2)) to the fourth day (836 spores/cm(2)) after the pileus opened. The number of spores dispersed downward was about 1.5 times greater than that dispersed toward the ridge. The number of dispersed spores decreased exponentially as the distance from each fruiting body increased. More than 95% of the spores dropped within a meter from the fruiting body, with 75% dropping within 0.5 m. Even so, the number of spores dispersed over 5 m from the fruiting body was more than 50 million when considering the total number of spores produced by a fruiting body is about 5 billion.

Ergothioneine contents in fruiting bodies and their enhancement in mycelial cultures by the addition of methionine.

The levels of ergothioneine (ERG), which have been shown to act as an excellent antioxidant, were determined in both fruiting bodies and mycelia of various mushroom species. We found that ERG accumulated at different levels in fruiting bodies of mushrooms and showed up to a 92.3-fold difference between mushrooms. We also found that ERG accumulated at higher levels in mycelia than in fruiting bodies of economically important mushroom species such as Ganoderma neo-japonicum, G. applanatum and Paecilomyces tenuipes. The addition of 2 mM methionine (Met) to mycelial culture medium increased the ERG contents in most mushroom species tested, indicating that Met is a good additive to enhance the ERG levels in a variety of mushroom species. Taking these results into consideration, we suggest that the addition of Met to the mycelial culture medium is an efficient way to enhance the antioxidant properties in economically important mushroom species.

Factors influencing the production of water-soluble endopolysaccharides and exopolysaccharides from Lentinus lepideus and their effects on immune cytokine production.

An efficient method to produce water-soluble polysaccharides from Lentinus lepideus is described. The productivity of both endopolysaccharides (PPS) and exopolysaccharides (EPS) was compared under various culture conditions. The effect of treating their own PPS and EPS on immune cytokine production was also studied in relation to culture factors. High yield production of EPS required moderate culture temperature (25 degrees ) as well as long culture period (16-20 days). In contrast, PPS production required high culture temperature (30 degrees ) and short culture period ( days). Most of the carbon sources did not affect polysaccharides and mycelial production except for sucrose. Immune cytokine levels in the EPS treatment varied among carbon sources or culture periods. PPS did not appear to affect much on the production of cytokines, regardless of the culturing factors, except for the culture period. These results suggest that the optimal culture conditions for L. lepideus vary according to culture purposes, and different culture conditions should be used for different targets including mycelial biomass, EPS, and PPS. Whereas the immunomodulating activity of EPS appeared to be affected by culture conditions in L. lepideus, that of PPS did not.