Young Woon Lim

Assessment of soil fungal communities using pyrosequencing.

Pyrosequencing, a non-electrophoretic method of DNA sequencing, was used to investigate the extensive fungal community in soils of three islands in the Yellow Sea of Korea, between Korea and China. Pyrosequencing was carried out on amplicons derived from the 5′ region of 18S rDNA. A total of 10,166 reads were obtained, with an average length of 103 bp. The maximum number of fungal phylotypes in soil predicted at 99% similarity was 3,334. The maximum numbers of phylotypes predicted at 97% and 95% similarities were 736 and 286, respectively. Through phylogenetic assignment using BLASTN, a total of 372 tentative taxa were identified. The majority of true fungal sequences recovered in this study belonged to the Ascomycota (182 tentative taxa in 2,708 reads) and Basidiomycota (172 tentative taxa in 6,837 reads). The predominant species of Ascomycota detected have been described as lichen-forming fungi, litter/wood decomposers, plant parasites, endophytes, and saprotrophs: Peltigera neopolydactyla (Lecanoromycetes), Paecilomyces sp. (Sordariomycetes), Phacopsis huuskonenii (Lecanoromycetes), and Raffaelea hennebertii (mitosporicAscomycota). The majority of sequences in the Basidiomycota matched ectomycorrhizal and wood rotting fungi, including species of the Agaricales and Aphyllophorales, respectively. A high number of sequences in the Thelephorales, Boletales, Stereales, Hymenochaetales, and Ceratobasidiomycetes were also detected. By applying high-throughput pyrosequencing, we observed a high diversity of soil fungi and found evidence that pyrosequencing is a reliable technique for investigating fungal communities in soils.

Rapid phylogenetic dissection of prokaryotic community structure in tidal flat using pyrosequencing

Dissection of prokaryotic community structure is prerequisite to understand their ecological roles. Various methods are available for such a purpose which amplification and sequencing of 16S rRNA genes gained its popularity. However, conventional methods based on Sanger sequencing technique require cloning process prior to sequencing, and are expensive and labor-intensive. We investigated prokaryotic community structure in tidal flat sediments, Korea, using pyrosequencing and a subsequent automated bioinformatic pipeline for the rapid and accurate taxonomic assignment of each amplicon. The combination of pyrosequencing and bioinformatic analysis showed that bacterial and archaeal communities were more diverse than previously reported in clone library studies. Pyrosequencing analysis revealed 21 bacterial divisions and 37 candidate divisions. Proteobacteria was the most abundant division in the bacterial community, of which Gamma-and Delta-Proteobacteria were the most abundant. Similarly, 4 archaeal divisions were found in tidal flat sediments. Euryarchaeota was the most abundant division in the archaeal sequences, which were further divided into 8 classes and 11 unclassified euryarchaeota groups. The system developed here provides a simple, in-depth and automated way of dissecting a prokaryotic community structure without extensive pretreatment such as cloning.

Fast and Cost-Effective Mining of Microsatellite Markers Using NGS Technology: An Example of a Korean Water Deer Hydropotes inermis argyropus

Background Microsatellites, a special class of repetitive DNA sequence, have become one of the most popular genetic markers for population/conservation genetic studies. However, its application to endangered species has been impeded by high development costs, a lack of available sequences, and technical difficulties. The water deer Hydropotes inermis is the sole existing endangered species of the subfamily Capreolinae. Although population genetics studies are urgently required for conservation management, no species-specific microsatellite marker has been reported. Methods We adopted next-generation sequencing (NGS) to elucidate the microsatellite markers of Korean water deer and overcome these impediments on marker developments. We performed genotyping to determine the efficiency of this method as applied to population genetics. Results We obtained 98 Mbp of nucleotide information from 260,467 sequence reads. A total of 20,101 di-/tri-nucleotide repeat motifs were identified; di-repeats were 5.9-fold more common than tri-repeats. [CA]n and [AAC]n/[AAT]n repeats were the most frequent di- and tri-repeats, respectively. Of the 17,206 di-repeats, 12,471 microsatellite primer pairs were derived. PCR amplification of 400 primer pairs yielded 106 amplicons and 79 polymorphic markers from 20 individual Korean water deer. Polymorphic rates of the 79 new microsatellites varied from 2 to 11 alleles per locus (He: 0.050–0.880; Ho: 0.000–1.000), while those of known microsatellite markers transferred from cattle to Chinese water deer ranged from 4 to 6 alleles per locus (He: 0.279–0.714; Ho: 0.300–0.400). Conclusions Polymorphic microsatellite markers from Korean water deer were successfully identified using NGS without any prior sequence information and deposited into the public database. Thus, the methods described herein represent a rapid and low-cost way to investigate the population genetics of endangered/non-model species.

Decay fungi from playground wood products in service using 28S rDNA sequence analysis

Abstract In order to establish integrated control strategies of wood degradation, a systematic survey of basidiomycete decay fungi colonizing various wood products in service is a prerequisite to increasing the service life of wood products. As a first step, we initiated a thorough survey of basidiomycete decay fungi colonizing playground wood products. For accurate fungal identification, traditional methods were complemented with molecular methods, including a BLAST search for large-subunit 28S rDNA sequences in Genbank and phylogenetic analysis. A total of 132 basidiomycete fungi, including 30 different fungal taxa, were isolated from 35 playgrounds in 15 different areas. Eight species, Bjerkandera adusta, Ceriporia lacerata, Gloeophyllum trabeum, Peniophora sp., Phanerochaete sordida, Schizophyllum commune, Sisto-trema brinkmannii, and Trametes versicolor, were predominantly isolated. A combination of traditional and molecular tools allowed a more detailed identification and diversity.

Fungal diversity from western redcedar fences and their resistance to β-thujaplicin

The work reported here investigated the fungal community inhabiting western redcedar fence material with a focus on species colonizing wood below the surface, of which little is known. From seven pieces of fence material, twenty-three different fungal species were isolated and characterized using both traditional morphology and molecular identification methods. The species identified included thirteen ascomycetous and ten basidiomycetous fungi. Isolates were tested for their resistance to β-thujaplicin - one of the principle fungicidal agents of western redcedar heartwood extractives. Generally, ascomycetous fungi exhibited greater resistance to β-thujaplicin than basidiomycetous fungi. Interestingly, three ascomycetous and two basidiomycetous species frequently isolated had high tolerance to this compound. These species could be candidate ’pioneer’ species that invade and detoxify western redcedar extractives, paving the way for colonization by decay fungi.

Leptographium bistatum sp. nov., a new species with a Sporothrix synanamorph from Pinus radiata in Korea

Radiata pine (Pinus radiata) lumber from Australia, Chile, and New Zealand is imported into Korea where it represents one of the most important sources of timber. Blue stain of this timber is a serious problem for which an integrated control strategy is being developed. One of the fungi that has been isolated from stained radiata pine sapwood in Korea is a Leptographium sp. that has a distinct Sporothrix synanamorph. The aim of this study was to classify this fungus. Morphological comparisons showed that this fungus is distinct from all other species of Leptographium and especially L. elegans and L. francke-grosmanniae that also have Sporothrix synanamorphs. Comparisons of sequence data for the ITS2 and part of the 28S rDNA genes as well as the beta-tubulin gene also confirmed that this fungus represents an undescribed taxon, described here as Leptographium bistatum sp. nov.

Delimitation of Russula Subgenus Amoenula in Korea Using Three Molecular Markers

Abstract Distinguishing individual Russula species has been difficult due to extensive phenotypic plasticity and obscure morphological and anatomical discontinuities. Due to highly similar macroscopic features, such as the presence of a red-cap, species identification within the Russula subgenus Amoenula is particularly difficult. Three species of the subgenus Amoneula have been reported in Korea. We used a combination of morphology and three molecular markers, the internal transcribed spacer (ITS), 28S nuclear ribosomal large subunit (LSU), and RNA polymerase II gene (RPB2), for identification and study of the genetic diversity of Russula subgenus Amoenula in Korea. We identified only two species in Korea (R. mariae and R. violeipes); these two species were indistinguishable according to morphology and LSU, but were found to be reciprocally monophyletic species using ITS and RPB2. The markers, ITS, LSU, and RPB2, have been tested in the past for use as DNA barcoding markers, and findings of our study suggest that ITS and RPB2 had the best performance for the Russula subgenus Amoneula.

Sphingopyxis marina sp. nov. and Sphingopyxis litoris sp. nov., isolated from seawater

Two yellow-pigmented, Gram-negative, aerobic bacterial strains, designated FR1087(T) and FR1093(T), were isolated from surface seawater off Jeju Island, Republic of Korea. Both strains required sea salts for growth. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the two isolates belong to the genus Sphingopyxis, showing the highest level of sequence similarity with respect to Sphingopyxis flavimaris SW-151(T) (97.9 %). The two isolates shared 98.5 % sequence similarity. DNA-DNA hybridization between the isolates and the type strain of Sphingopyxis flavimaris clearly suggested that strains FR1087(T) and FR1093(T) represent two separate novel species in the genus Sphingopyxis. Several phenotypic characteristics served to differentiate these two isolates from recognized members of the genus Sphingopyxis. The data from the polyphasic study presented here indicated that strains FR1087(T) and FR1093(T) should be classified as representing novel species in the genus Sphingopyxis, for which the names Sphingopyxis marina sp. nov. and Sphingopyxis litoris sp. nov., respectively, are proposed. The type strain of Sphingopyxis marina sp. nov. is FR1087(T) (=IMSNU 14132(T)=KCTC 12763(T)=JCM 14161(T)) and the type strain of Sphingopyxis litoris sp. nov. is FR1093(T) (=IMSNU 14133(T)=KCTC 12764(T)=JCM 14162(T)).

Degrading ability of oligocyclic aromates by Phanerochaete sordida selected via screening of white rot fungi

Seventy-nine white rot strains were screened to determine if they had the potential for use in the degradation of oligocyclic aromates (PAHs) by measuring their dye-decoloration rate. Fourteen strains that were selected based on their dye-decoloration rate were then evaluated for the ability to tolerate various levels of PAHs spiked in agar medium. The ability of white rot fungi to degrade 3- or 4-ring PAHs (anthracene, phenanthrene, fluoranthene, pyrene) was determined. Two strains of Phanerochaete sordida (KUC8369, KUC8370) were possible PAHs degraders, degrading a significantly greater amount of phenanthrene and fluoranthene than the culture collection strain P. chrysosporium (a known PAHs degrader). The production of manganese peroxidase, the only extracellular ligninolytic enzyme detected during the cultivation, was evaluated.

Genetic variation and relationships in Laetiporus sulphureus s. lat., as determined by ITS rDNA sequences and in vitro growth rate

The aim of this study was to characterise the genetic variation and molecular relationships of the brown rot polypore, Laetiporus sulphureus s. lat., from Europe, South America, Africa, and Asia, using ITS sequences of the nu-rDNA and by comparing the growth rate in vitro. In a NJ analysis of the sequences of 130 individuals of L. sulphureus s. lat., eight distinct clusters emerged, supported by BS values of 70-100%. Within each cluster, the ITS rDNA sequence variation was below 3%. The sequences were also analysed together with Laetiporus sequences available from GenBank. Results demonstrated the possible presence of L. huroniensis in Europe (invalidly named L. montanus) and L. gilbertsonii in South America, and provided more information on the Pan-American and European distribution of one of the clades, currently known in North America as L. sulphureus. L. conifericola formed a separate distinct clade. Moreover, the analysis revealed two unknown Laetiporus taxa in Korea, one in South Africa, and one in Europe. As L. sulphureus is described from Europe (France), and we show that more than one taxon exist here, it is presently not possible to define L. sulphureus s. str. Certain biological differences between some of the clades (in vitro growth rates, chemical composition, and pigmentation) were demonstrated and discussed.