The complete mitochondrial genome of the edible and phytopathogenic fungus Desarmillaria tabescens.

Desarmillaria tabescens is one of the most important edible, medicinal, and phytopathogenic basidiomycetes. The complete mitochondrial genome of this species was determined using next-generation sequencing technology. This mitogenome is a circular molecule of 93,439 bp with a GC content of 29.28% and contains 15 protein-coding, two rRNA (rnl and rns), and 24 tRNA genes. Phylogenetic analysis revealed that D. tabescens is genetically closest to Agrocybe aegerita. Desarmillaria tabescens mitogenome can contribute to our understanding of the phylogeny and evolution of this species.

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.

Development of 44 Novel Polymorphic SSR Markers for Determination of Shiitake Mushroom (Lentinula edodes) Cultivars.

The shiitake mushroom (Lentinulaedodes) is one of the most popular edible mushrooms in the world and has attracted attention for its value in medicinal and pharmacological uses. With recent advanced research and techniques, the agricultural cultivation of the shiitake mushroom has been greatly increased, especially in East Asia. Additionally, demand for the development of new cultivars with good agricultural traits has been greatly enhanced, but the development processes are complicated and more challenging than for other edible mushrooms. In this study, we developed 44 novel polymorphic simple sequence repeat (SSR) markers for the determination of shiitake mushroom cultivars based on a whole genome sequencing database of L. edodes. These markers were found to be polymorphic and reliable when screened in 23 shiitake mushroom cultivars. For the 44 SSR markers developed in this study, the major allele frequency ranged from 0.13 to 0.94; the number of genotypes and number of alleles were each 2-11; the observed and expected heterozygosity were 0.00-1.00 and 0.10-0.90, respectively; and the polymorphic information content value ranged from 0.10 to 0.89. These new markers can be used for molecular breeding, the determination of cultivars, and other applications.

Genet Variation of Ectomycorrhizal Suillus granulatus Fruiting Bodies in Pinus strobus Stands.

The genets of Suillus granulatus in a Pinus strobus stand (13 m × 60 m) were identified using random amplified polymorphic DNA molecular markers and the DNA of mushrooms that fruited for two years, and variations in genet size and distribution were analyzed. From a total of 116 mushrooms, 73 genets were identified and were grouped into three locations. The genets of mushrooms in close proximity differed from each other. The genet sizes varied at any of the three locations. The lengths of the identified genets in the pine stand ranged from 0.09 to 2.90 m. The average number of mushrooms per genet was 1.2 to 2.3, and the percentage of genets that were represented by a single mushroom was 44% to 94%. This variation in the genets of mushrooms in close proximity suggests that the ectomycorrhizal mycelial bodies of S. granulatus propagated sexually by fusing haploid spores derived from the mushrooms gills with below-ground mycelia. Therefore, it is necessary further to investigate the formation of new genets through spores in ectomycorrhizal fungal colonies.

Whole genome de novo sequencing and genome annotation of the world popular cultivated edible mushroom, Lentinula edodes.

Lentinula edodes, the popular shiitake mushroom, is one of the most important cultivated edible mushrooms. It is used as a food and for medicinal purposes. Here, we present the 46.1 Mb draft genome of L. edodes, comprising 13,028 predicted gene models. The genome assembly consists of 31 scaffolds. Gene annotation provides key information about various signaling pathways and secondary metabolites. This genomic information should help establish the molecular genetic markers for MAS/MAB and increase our understanding of the genome structure and function.

Effects of Aeration of Sawdust Cultivation Bags on Hyphal Growth of Lentinula edodes.

The effects of aeration through lid filters on the hyphal growth of Lentinula edodes (oak mushroom) in sawdust cultivation bags were investigated. The aeration treatment levels were traditional 27 mm hole cotton plugs, cotton balls and combinations of seven hole sizes × two hole positions (up and under) in the lids covering plastic bags containing 1.4 kg sawdust medium at 63% moisture that had been autoclaved for one hour and inoculated with sawdust spawn of L. edodes strain 921. Aeration treatment effects were measured based on the CO(2) concentration at the 15th wk, as well as the hyphal growth rate and degree of weight loss of bags every 14 days for 15 wk. In bags with traditional cotton plugs, the CO(2) concentration was 3.8 ± 1.3%, daily mean hyphal growth was 2.3 ± 0.6 mm and daily mean weight loss was 0.84 ± 0.26 g. In the bags with 15 mm diameter holes, the CO(2) concentration was 6.0 ± 1.6%, daily hyphal growth was 2.8 ± 0.2 mm and daily weight loss was 0.86 ± 0.4 g. The bags with 15 mm holes had a higher CO(2) concentration and lower water loss than bags with other hole sizes, but the hyphal growth was not significantly different from that of other bags. The weight loss of bags increased proportionally relative to the lid hole sizes. Taken together, these results indicate that traditional cotton plugs are economically efficient, but 15 mm hole lids are the most efficient at maintaining hyphal growth and controlling water loss while allowing CO(2) emissions.

Ergosterol and Water Changes in Tricholoma matsutake Soil Colony during the Mushroom Fruiting Season.

The purpose of this study is to understand spatio-temporal changes of active fungal biomass and water in Tricholoma matsutake soil colonies during the mushroom fruiting season. The active fungal biomass was estimated by analyzing ergosterol content at four different points within four replicated locations in a single circular T. matsutake colony at Ssanggok valley in the Sogri Mt. National Park in Korea during 2003 to 2005. The four points were the ahead of the colony, the front edge of the colony and 20 cm and 40 cm back from the front edge of the colony. Ergosterol content was 0.0 to 0.7 µg per gram dried soil at the ahead, 2.5 to 4.8 µg at the front edge, 0.5 to 1.8 µg at the 20 cm back and 0.3 to 0.8 µg at the 40 cm back. The ergosterol content was very high at the front edge where the T. matsutake hyphae were most active. However, ergosterol content did not significantly change during the fruiting season, September to October. Soil water contents were lower at the front edge and 20 cm back from the front edge of the colony than at the ahead and 40 cm back during the fruiting season. Soil water content ranged from 12 to 19% at the ahead, 10 to 11% at the edge, 9 to 11% at the 20 cm back and 11 to 15% at the 40 cm back. Our results suggest that the active front edge of the T. matsutake soil colony could be managed in terms of water relation and T. matsutake ectomycorrhizal root development.

Relationship between Water Content and Osmotic Potential of Lentinula edodes.

This study was conducted to understand how osmotic potentials in Lentinula edodes tissues are related to water contents and how they change while a mushroom matures. Water content and osmotic potential of L. edodes mushroom tissues from log cultivation and sawdust cultivation were measured and the relationships were analyzed. Osmotic potentials in the tissues were exponentially proportional to their moisture contents and there were strain differences in the potentials. Strain 290 has lower osmotic potential than strain 302, in the tissues at the same water content. As the mushrooms mature, tissue water content maintained ca 94% in head tissues and ca 90% in gills, but significantly decreased from ca 90% to 82% in the stipe tissues. Osmotic potential changes were similar to the tissue water content changes as the mushrooms mature. While osmotic potentials maintained -0.25 to -0.45 MPa in head and gill tissues, the potentials greatly decreased from -0.65 to -1.33MPa in stipe tissues. Our results show that osmotic potentials in L. edodes tissues are exponentially proportional to tissue water contents, that strains differ in osmotic potential related to water, and that stipe tissues can still have nutritional value when they mature.

Effect of Chitosan Acetate on Bacteria Occurring on Neungee Mushrooms, Sarcodon aspratus.

Minimal growth inhibitory concentrations (MICs) of chitosan acetate (M.W. 60 kDa) on heterotrophic bacteria (strains MK1, S, and R) isolated from the soft-rotten tissues of Neungee mushroom (Sarcodon aspratus) were measured. The slimy substance produced by the MK1 strain was responsible for the diseased mushroom's appearance. The S and R strains were members of the Burkholderia cepacia complex. These strains showed different levels of susceptibility toward chitosan acetate. The MIC of chitosan acetate against the MK1 and S strains was 0.06%. The MIC against the R strain was greater than 0.10%. Survival fractions of the MK1 and S strains at the MIC were 3 × 10(-4) and 1.4 × 10(-3) after 24 h, and 2 × 10(-4) and 7 × 10(-4) after 48 h, respectively. Survival fractions of the R strain after 24 and 48 hr at 0.1% chitosan acetate were 1 × 10(-2) and 6.9 × 10(-3), respectively. Compared to the MK1 and S strains, the low susceptibility of the R stain towards chitosan acetate could be due to the ability of the R strain to utilize chitosan as a carbon source. Thirty-eight percent of Neungee pieces treated in a 0.06% chitosan acetate solution for 2~3 second did not show any bacterial growth at 4 days, whereas bacterial growth around untreated mushroom pieces occurred within 2 days. These data suggest that chitosan acetate is highly effective in controlling growth of indigenous microorganisms on Neungee. The scanning electron micrographs of the MK1 strain treated with chitosan revealed a higher degree of disintegrated and distorted cellular structures.

Identification of bacteria isolated from diseased Neungee mushroom, Sarcodon aspratus.

As the first step in an investigation of the problem with quality deterioration seen in the Neungee mushroom (Sarcodon aspratus) due to bacterial overgrowth during its storage, an attempt to isolate bacterial strains was made using infected gills of Sarcodon aspratus. Five bacterial strains were isolated; one phototrophic cyanobacterial species and four heterotrophic Gram negative rods. The four heterotrophic bacterial isolates (strains P, S, R, and MK1) were subjected to identification based on biochemical characteristics using the Biolog system, cellular fatty acid analysis using the MIDI system, cytology by scanning microscopy, and 16s rDNA sequence analysis. A slow grower, the P strain (ca. 0.7 microm x 1.5 microm), which forms pink colonies on Tryptic Soy agar (TSA) and glucose minimal salt medium containing thiamine (MT medium), belongs to genus Methylobacterium, and is likely M. radiotolerans. The methanol-utilizing capacity of the P strain was confirmed by growth on methanol-supplemented medium as a sole carbon source. Both the S and R strains (ca. 0.5 microm x 0.8 microm) produced smooth and slightly rough white colonies, respectively, on TSA, MT, and potato dextrose (PD) agar are members of the Burkholderia cepacia complex. Although both strains showed some differences from each other in colony morphology, nitrogen fixation capacity, and denitrification, they were considered to be Burkholderia stabilis because their 16s rDNA sequences showed 99.93% similarity with those of B. stabilis LMG 14294T (NCBI AF 148554). The MK1 strain, a rod-shaped bacterium with a tapered end (ca. 0.6 microm x 1.8 microm), produces a copious mucoid substance on MT and PD agar, but not on TSA. Despite extensive identification studies, the M strain is not currently identifiable, which suggests that it is a novel bacterium.