J. Microbiol. Biotechnol. (2014), 24(3), 324–333 http://dx.doi.org/10.4014/jmb.1310.10041 Research Article jmb Diversity and Saline Resistance of Endophytic Fungi Associated with Pinus thunbergii in Coastal Shelterbelts of Korea Young Ju Min1, Myung Soo Park1, Jonathan J. Fong1, Ying Quan1, Sungcheol Jung2, and Young Woon Lim1* 1School of Biological Sciences, Seoul National University, Seoul 151-747, Republic of Korea 2Warm-Temperate and Subtropical Forest Research Center, KFRI, Seogwipo 697-050, Republic of Korea Received: October 14, 2013 Revised: November 29, 2013 The Black Pine, Pinus thunbergii, is widely distributed along the eastern coast of Korea and its Accepted: December 4, 2013 importance as a shelterbelt was highlighted after tsunamis in Indonesia and Japan. The root endophytic diversity of P. thunbergii was investigated in three coastal regions; Goseong, Uljin, and Busan. Fungi were isolated from the root tips, and growth rates of pure cultures were First published online measured and compared between PDA with and without 3% NaCl to determine their saline December 9, 2013 resistance. A total of 259 isolates were divided into 136 morphotypes, of which internal *Corresponding author transcribed spacer region sequences identified 58 species. Representatives of each major fungi Phone: +82-2-880-6708; phylum were present: 44 Ascomycota, 8 Zygomycota, and 6 Basidiomycota. Eighteen species Fax: +82-2-871-5191; exhibited saline resistance, many of which were Penicillium and Trichoderma species. Shoreline E-mail: [email protected] habitats harbored higher saline-tolerant endophytic diversity compared with inland sites. This investigation indicates that endophytes of P. thunbergii living closer to the coast may have pISSN 1017-7825, eISSN 1738-8872 higher resistance to salinity and potentially have specific relationships with P. thunbergii. Copyright© 2014 by The Korean Society for Microbiology Keywords: Coastal pine forest, endophyte community, salinity tolerance, shelterbelt and Biotechnology Introduction growth-promoting metabolites, enhancing nutrient uptake, increasing tolerance to harsh environmental conditions, and The Black Pine, Pinus thunbergii Parl., native to South enhancing defenses to herbivores and pathogens [2, 3, 40, Korea and Japan, is adapted to coastal areas and can 55]. Environmental stressors, such as salinity, determine the tolerate high salt environments [33, 47]. The important role fungal community structure and distribution [29]. Endophytes of P. thunbergii forests as a bioshield protecting arable land can confer habitat-adapted tolerance to host plants, while and buildings from large waves and strong winds was the endophyte may receive benefits such as nutrients and highlighted after recent tsunamis in Indonesia [35] and protection from the host plant [40, 41]. A study examining Japan [50]. For this reason, efforts have been made to the mutualistic relationship between a coastal dunegrass understand the ecology of P. thunbergii to aid in recovering (Leymus mollis) and its endophyte (Fusarium culmorum) and maintaining coastal pine forests in Korea [7, 44]. found rapid growth and salt tolerance when together, but Endophytes are broadly defined as microbes that reside slowed growth when grown separately [40, 41]. entirely in plant tissues of stems, leaves, or roots [6]. Isolating and culturing endophytic fungi are essential in Fungal endophytes are ubiquitous, with a worldwide studying their physiological properties and applying them estimate of one million species [12]. This host-endophyte for biotechnological purposes [34, 51]. However, these relationship can take on several forms, including mutualism, methods are difficult for identification because many fungal commensalism, or parasitism. Most endophytes are classified species lack distinguishing morphological characteristics in the fungal phylum Ascomycota [1, 61], but some when cultured [8]. Recently, the use of DNA sequencing Zygomycota [10, 14, 19, 20] and Basidiomycota [40, 41] and DNA databases has enhanced our ability to accurately species have been identified. classify endophytic fungi [16, 28]. We take this combined Endophytic fungi can be beneficial to plants by producing culture-sequence approach to identify the diversity and March 2014 ⎪ Vol. 24⎪ No. 3 325 Min et al. understand the ecological function of endophytic fungi Inland samples were >200 m from the samples adjacent to the associated with P. thunbergii. shoreline. In total, 54 roots from 18 mature trees were collected. This study represents the first work concerning the The average length of fine root samples was approximately 30 cm. endophyte diversity in P. thunbergii roots. Owing to the Additionally, soil from around the roots of all trees was sampled unique microhabitat of a coastal environment, it is (18 total) to test soil properties. Salinity, pH, total nitrogen, and total phosphorus were analyzed for each sample at the National important to identify the fungal community of saline- Instrumentation Center for Environmental Management (Seoul tolerant fungi that have a potential of enhancing the National University, Korea). survival and proliferation of P. thunbergii. To understand the geographical and ecological variation, we compared Endophyte Isolation and Saline Resistance Test across three regions along the eastern coast of South Korea, Root surfaces were sterilized using standard procedures [46]. sampling tree roots adjacent to the shoreline and inland. These roots were cut to approximately 5 mm in length for isolation of endophytic fungi. Under sterile conditions, the root Materials and Methods fragments were plated on potato dextrose agar (PDA) medium with 100 ppm streptomycin, incubated at 20°C in the dark for Study Site and Collection several days, and subcultured from mycelia margins to a new In the fall of 2012, we sampled three regions spanning the plate to obtain pure cultures. All pure culture isolates were eastern coast of Korea: Goseong, Uljin, and Busan (Fig. 1). Except grouped based on culture characters and microscopic features for Busan, where woody and herbaceous plants are syntopic with (Nikon 80i microscope). A representative isolate of each group P. thunbergii, the sites are primarily occupied by P. thunbergii. In was selected for DNA sequencing and evaluating salt resistance. all regions, the P. thunbergii community is composed of mature Testing for salt resistance was performed by comparing growth trees estimated to be ~60 years old, located at least 200 m inland rates on PDA with and without 3% NaCl, each performed in from the shoreline, and several hundred meters wide. A total of 18 triplicate and averaged. We set 3% NaCl for the growth media to root sections were taken at each site: three root sections from six approximate the average seawater salinity of 3.5% [22]. Results trees (three trees adjacent to the shoreline and three trees inland). are expressed in a ratio of mean growth rate with NaCl/mean growth rate without NaCl. Molecular Approach for Identification of Endophytic Fungi DNA was extracted using a modified CTAB extraction protocol [42]. The internal transcribed spacer region (ITS) was amplified using primer set ITS1F/ ITS4 [57]. PCR was performed on a C1000 thermocycler (Bio-Rad, USA) using the Maxime PCR PreMix- StarTaq (Intron Biotechnology Inc., Korea) in a final volume of 20 µl containing 10 pmol of each primer and 1 µl of DNA. The PCR conditions were 95°C for 10 min, followed by 35 cycles of 95°C for 40 sec, 55°C for 40 sec, and 72°C for 1 min, and a final extension step at 72°C for 10 min. The PCR products were electrophoresed through a 1% agarose gel stained with loading STAR (Dyne Bio, Korea) and purified using the Expin PCR Purification Kit (GeneAll Biotechnology, Korea) according to the manufacturer’s instructions. DNA sequencing was performed at the DNA Synthesis and Sequencing Facility, Macrogen (Korea), using an ABI3700 automated DNA sequencer. The sequences were deposited in GenBank (Table 1). To identify representative endophytes, we use BLAST to compare sequences against type strains [25]. In GenBank, we Fig. 1. The three study regions along the eastern coast of Korea searched for sequences that have the identifiers “type strain”, “ex- in this study. type”, “holotype”, or “isotype” in the description. Since type ∆ indicates each collection point. At each study site, root samples were strains available in GenBank are limited, we also included the top collected from six Pinus thunbergii individuals. Three collection points matching BLAST match for each sample. A phylogenetic tree was were located adjacent to the shoreline, while the other three points constructed to assign species names to samples. When a sequence were ~200 m inland. SB =Sandy Beach, PF =Pine Forest, UA =Urban clustered with a single species, either a type sequence or not, this Area, MF =Mixed Forest, C =Cliff. identification was chosen. When the sequence was highly similar J. Microbiol. Biotechnol. Endophytic Fungi Diversity of Pinus thunbergii 326 Table 1. Classification of endophytic fungi isolated from Pinus thunbergii based on the GenBank database. c No. of isolates Closest GenBank GenBank Growth Fungal identity Classificationa Strainb Goseong Uljin Busan match Acc. No. rated S I S I S I (% of Similarity) Acremonium variecolor A/S SFCF20120803-45 KF313108 1 1 HE608648 (99%) 1.6 Aspergillus sydowiie A/E SFCF20120803-11 KF313094 1 AM883160 (99%) 1.4 Bionectria ochroleucae A/S SFCF20120803-24 KF313107 2 EU071701 (99%) 0.3 Chaetomium sp. A/S SFCF20120803-33