A New Record of Pseudeurotium bakeri (KNU14-21) from Crop Field Soil in Korea Mahesh Adhikari, Sang Woo Kim, Hyun Seung Kim, Young Hyum Um and Youn Su Lee* Division of Biological Resources Sciences, Kangwon National University, Chuncheon 24341, Korea ABSTRACT Soil fungi are crucial integral components of the soil ecosystem and play a major role as a source of food for larger organisms. Fungi are widely distributed in all the terrestrial ecosystems. A fungal diversity study was carried out to check the species diversity in the crop field soil of Suncheon-si, Jeollanam-do, Korea. 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). ITS region was analyzed and determine the phylogenetic relationship between the isolate KNU14-21 and previously described Pseudeurotium bakeri. The studied fungal isolate was most closely related to Pseudeurotium bakeri and formed a monophyletic group with a bootstrap value of 99%. The neighbor joining phylogenetic analysis showed that the isolate is closely related to Pseudeurotium bakeri CBS (878.71). The nucleotide sequence was deposited in GenBank and assigned accession number KP for the study isolate. In addition, morphology and microscopic analysis of this fugal isolate was also performed in this study. This fungal isolate has not been reported officially in the past from Korea. INTRODUCTION CONCLUSION REFERENCES Soil fungi have vital ecological roles as decomposers, pathogens and Symbionts. [1]. Fungi have a cosmopolitan distribution and grow in a wide range of habitats, including extreme environments. The fungus kingdom have huge diversity of taxa with various ecologies, morphologies and life cycle with unicellular aquatic chytrids to large mushrooms. In spite of its importance in daily life, little is known of the distribution and biodiversity. On the basis of observations of the ratio of the number of fungal species to the number of plant species in selected environments, the fungal kingdom has been estimated to contain about 1.5 million species [2]. The application of molecular tools, such as DNA sequencing and phylogenetic analysis, to study diversity has greatly enhanced the resolution and added robustness to estimates of genetic diversity within various taxonomic groups [3]. Soil fungal diversity study was carried out to screen the diversity of fungi in the crop field soil of Suncheon-si, Jeollanam-do, Korea. Among the isolated fungal isolates, Pseudeurotium bakeri from the Pseudeurotiaceae family was encountered. This fungal isolate has not been reported officially in the past from Korea. This isolate was identified on the basis of morphological and molecular analysis. MATERIALS AND METHODS Collection of soil samples and fungal isolation:  Soil samples were collected from crop field soil of Suncheon-si, Jeollanam-do from (0-15) depth, air dried and stored in plastic bags at 4ºC until used.  The fungi were isolated by using conventional dilution method technique and supplemented with 100μg chloramphenicol per mL potato dextrose agar (PDA; Difco Laboratories, Detroit, USA) and grown for 7 d at 28 ºC until the growth of colonies was observed. Morphological characterization:  Morphological characteristics of isolates KNU was observed on potato dextrose agar (PDA). Colony diameter and degree of sporulation was determined.  Pictures of colony and micromorphology of the fungal isolate was taken with an HK 3.1 CMOS digital camera (KOPTIC Korea Optics, Korea) attached to an Olympus BX50F-3 microscope (Olympus Optical Co., LTD, Japan). Genomic DNA extraction, sequencing and data analysis:  Total genomic DNA was extracted from isolate KNU14-21 using the DNeasy Plant Mini Kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions.  The internal transcribed spacer region (ITS) gene was amplified using primers ITS1 (5'- TCCGTAGGTGAACCTGCG-3') and ITS4 (5'- TCCTCCGCTTATTGATATGC-3') [4].  The sequence was compared with reference ITS sequences of GenBank at National Center for Biotechnology Information (NCBI) using the basic local alignment search tool.  The nucleotide sequence was deposited in GenBank and assigned accession number KP  Phylogenetic relationships were analyzed using molecular evolutionary genetic analysis (MEGA 6) software. RESULTS AND DISCUSSION  Shape size and structure of conidia and conidiophores confirms morphologically that our study isolate is Pseudeurotium bakeri.  Among the isolated isolates, Pseudeurotium bakeri from the Pseudeurotiaceae family was confirmed as a new to Korea.  Base on the morphological and molecular analysis our studied fungal isolate was confirmed as a new record in Korea.  The isolate shown 99 % similarity with Pseudeurotium bakeri (CBS ). Phylogenetic analysis revealed that the isolate was grouped with reference isolates of Pseudeurotium bakeri with 99% bootstrap support (Fig. 1). These results indicate that isolate KNU14-21 is Pseudeurotium bakeri.  Further studies regarding biotechnological importance of this fungal isolate is needed. Fig.1. Map showing the places of soil sample collection. Map source: Cultural Heritage Administration, Korea Fig.3. Morphological characteristics of Pseudeurotium bakeri KNU14-21 grown for 7 days on potato dextrose agar (PDA), (A) reverse view, (B) obverse view, (C) Simple microscopic picture of conidiophores, (D) Simple microscopic picture of conidia, (E-F) simple microscopic pictures of conidiophores and conidia. Fig. 4. Neighbor-joining phylogenetic analysis of the partial 18S-ITS1-5.8S-ITS2-28S rDNA sequences of Pseudeurotium bakeri KNU14-21 obtained from crop field soil in Korea. The phylogenetic tree was constructed using the MEGA 6 program. Sequence obtained in the study is shown in boldface. The mark (T) indicates type strain. Numerical values (>50) on branches are the bootstrap values as percentage of bootstrap replication from 1000 replicate analysis. Amorphotheca resinae was used as outgroup. The scale bar represents the number of substitutions per site. Fig.2 Procedures illustrating isolation of fungi from soil samples. 1. Swift MJ, Heal OW and Anderson JM Decomposition in Terrestrial Ecosystems (Blackwell Scientific Publications. 2. Hawksworth DL The fungal dimension of biodiversity,magnitude, significance and conservation. Mycological Research 95;6: Hibbet DS, Binder M, Bischoff JF, Blackwell M, Cannon PF, Erikson OE A higher level phylogenetic classification of the fungi. Mycological Research 111;5: White TJ, Bruns T, Lee S, Taylor J Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. 315–322. In Innis, M.A. et al. (eds.), PCR protocols: a guide to methods and applications. Academic Press, SanDiego, USA.