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Unreported Fungal Species of Didymosphaeriaceae from Soil in Korea Mahesh Adhikari, Sang Woo Kim, Dil Raj Yadav, Hyun Seung Kim, Yong Hyun Um, Youn Su Lee* Division of Biological Resource Sciences, Kangwon National University, Chuncheon 200-701, Korea *younslee@kangwon.ac.kr ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION INTRODUCTION  Soil samples were collected from different places of Chuncheongbuk-do province of Korea.  Soil samples were taken approximately from (0~15 cm) depth, air dried and stored in plastic bags at 4ºC until use.  Fungi were isolated through serial dilution technique and purified and differentiated according to their morphological and microscopic characteristics.  The strains were inoculated at one point on 9-cm petri dishes and incubated for 7 days at 25 ℃ in dark place.  DNA of purified fungal isolates was extracted using the QIAGEN Plasmid Mini Kit (Qiagen Sciences, USA) following the manufacturer's instructions.  The PCR product was sequenced with an ABI prism 3730 DNA analyzer (Applied Biosystems, Foster City, CA, USA).  Extracted DNA samples were sent to Macrogen, Korea for sequencing.  The sequences were analyzed using BLAST (Basic Local Alignment Search Tool) on the NCBI website (http://www.ncbi.nlm.nih.gov/BLAST) to find the sequences with the highest similarity to studied sequences.  The identified sequences were listed and alignment was conducted to construct a phylogenetic tree by neighbour-joining method, with a bootstrap value of 1,000 replicated, using MEGA 6.0. In total five hundred soil samples were collected and isolated through the serial dilution technique. Among the isolated fungal isolates, Paraphaeosphaeria sporulosa from the Didymosphaeriaceae family was encountered new in Korea on the basis of morphological and molecular analysis. Colonies on PDA were growing very fast and attains 80-90 mm diameter in 7 days at 25 0 C. The colony color was creamy white. Spores are globose to sub-globose and 1.0 to 2.5 µm in diameter. Evolutionary status of the isolate KNU15-2 and previously identified Paraphaeosphaeria sporulosa was evaluated by the phylogenetic analysis. The isolate was most closely related to Paraphaeosphaeria sporulosa and form a monophyletic group with bootstrap value of 99 %. The phylogenetic analysis showed that the study isolate is Paraphaeosphaeria sporulosa. Further studies of this fungal isolate is worthwhile. REFERENCES The Didymosphaeriaceae are a family of fungi in the order pleosporales. Fungi of this family have a worldwide distribution and are saprobic in both woody and herbaceous plants. Some species are parasitic on other fungi. Soil fungi are one of the important components of microbial communities in terrestrial ecosystem and play vital role in decomposition of organic matter and nutrient cycling [1]. In spite of their immense importance in agro-ecosystem, yet they are probably the most poorly studied organisms. The microbial composition of soil is largely affected by edaphic factors and cropping system [2]. Soil fungal diversity study was undertaken to assess the diversity of fungi in the crop field soil of Chuncheongbuk-do, Korea. Out of identified isolates, Paraphaeosphaeria sporulosa was encountered new in Korea. This study presents the comparison of newly recorded species with previously described Paraphaeosphaeria sporulosa with regard to morphological and phylogenetic characteristics. Soil is a diversified micro-habitat for the development of microbial populations. Soil fungi are important integral components in the soil ecosystem and play a crucial role as a source of food for larger organisms, pathogens and maintain beneficial symbiotic relationships with plants or other organisms. Soil fungal diversity study was carried out to check the species diversity in the crop field soil of Chuncheongbuk-do, Korea in 2015. In total five hundred soil samples were collected and isolated through the serial dilution technique. Isolated fungi were then purified and differentiated according to their morphological and microscopic characteristics. Genomic DNA of the isolates was extracted by using QIAGEN® Plasmid Mini Kit(QIAGEN Sciences, USA) and the identification of fungi was carried out by sequence analysis of internal transcribed spacer (ITS) region of the 18S ribosomal DNA (18S rDNA). Morphologically 220 isolates were found distinct. Among them species of Didymosphaeriacea family were encountered namely Paraphaeosphaeria sporulosa, new and unreported in Korea. This species was identified based on the molecular and morphological analysis and have not been reported officially in the past from Korea. Fig.1. Procedures illustrating isolation of fungi from soil samples. Fig.2. Morphology of the Paraphaeosphaeria sporulosa (KNU15-2) on potato dextrose agar (PDA); A, Back view B, Front view C and D. spores of the isolate. Gen regionIsolateGenBank accession NoClosest GenBank library strainSimilarity (%) ITS regions KNU15-2 JX496114Paraphaeosphaeria sporulosa100 JX496091Paraphaeosphaeria neglecta97 KM873041Paraphaeosphaeria arecacerum95 JQ676199Paraphaeosphaeria sporulosum97 Fig. 1. Neighbor-joining phylogenetic analysis of the partial 18S-ITS1-5.8S-28Sr DNA sequence of Paraphaeosphaeria sporulosa (KNU15-2) obtained from soil in Korea. The phylogenetic tree was constructed using the MEGA 6 program. The sequence obtained this study is shown in boldface. Numerical values on branches are the percentage of 1,000 bootstrap replicates that support the branch.The scale bar represents the number of substitutions per site. Table.2. Identification of fungal isolates to species level with reference species based on the analyses of internal transcribed spacer gene sequences. Warcup JH. Soil-steaming: A selective method for the isolation of ascomycetes from soil. Br Mycol Soc 1951:515-18. Hargreaves SK, Williams RJ, Hofmockel KS. Environmental Filtering of Microbial communities in Agricultural Soil Shifts with Crop Growth. PloS ONE 2015;7:2-14.