Molecular identification of endophytic fungi isolated from needle leaves of conifers in bohyeon mountain, Korea.

Fungal endophytes are microfungi that live in plants without causing apparent symptoms of infection. This study was conducted to identify endophytic fungi isolated from leaves of coniferous trees in Bohyeon Mountain of Korea. We collected leaves of two species of coniferous trees, Pinus densiflora and Pinus koraiensis, from 11 sites in the study area. A total 58 isolates were obtained and identified using molecular and morphological characteristics. Four species of endophytic fungi were isolated from P. densiflora: Lophodermium conigenum, Leotiomycetes sp., Septoria pini-thunbergii, and Polyporales sp., while two fungal species were isolated from P. koraiensis: Eurotiomycetes sp. and Rhytismataceae sp. The most frequently isolated species were L. conigenum and S. pini-thunbergii.

Introduction

Fungal endophytes are microfungi that colonize and live within healthy plant tissues without inducing symptoms of disease [1]. These organisms are thought to be ubiquitous among terrestrial plants, having been recovered from all major plant lineages in a wide range of terrestrial communities. Additionally, numerous investigations over the past three decades have shown that endophytes are abundant in asymptomatic leaves of many conifers [2-6]. Endophytes may offer significant benefits to their host plants by producing a plethora of substances that provide protection and survival value, such as enhancement of stress-, insect-, and disease-resistance, productivity improvement, and herbicide activity [7]. Accordingly, endophytes likely play an important role in ecological communities [8]. However, few studies of the diversity, host plant affinities, and environmental and medicinal effects of fungal endophytes in Korea have been conducted to date [9, 10]. Therefore, the present study was conducted to isolate and identify endophytic fungi from the leaves of coniferous trees on Bohyeon Mountain of Korea.

Materials and Methods

Sampling

The study area was Bohyeon Mountain, Kyeongbuk Province, Republic of Korea. A total of 11 plants of two coniferous species (five Pinus densiflora and six Pinus koraiensis) were randomly selected at various altitudes on the mountain. The healthy leaves of the host plants were collected, packed into polyethylene bags and transported to the laboratory.

Fungal endophyte isolations

The surface of each coniferous leaf was washed with running tap water and treated with 1% NaOCl solution for 3 min and 70% ethanol for 2 min, after which it was washed with sterilized distilled water. Surface-sterilized samples were then cut into 1-cm sections and four segments per plate were cultured in water agar, potato dextrose agar (PDA), yeast mold agar, and malt extract agar. Plate media were sealed and incubated at 25℃ for up to 8 wk, during which time they were checked often for hyphal growth. The mycelia were transferred to PDA for isolation and axenic culture.

DNA extraction and PCR

All isolates were grouped into morphotypes based on colony shape, height, and color of the aerial hyphae, as well as the base color, growth rate, margin characteristics, surface texture, and depth of growth into the medium [11, 12]. One or two isolates of each morphotype were then selected for molecular identification. DNA was extracted using a DNeasy Plant Mini kit according to the manufacturer's protocols (Qiagen Co., Valencia, CA, USA). PCR was then conducted to amplify the internal transcribed spacer (ITS) region of the extracted DNA, including the 5.8s rDNA, using the primers ITS1F and ITS4 [13] under the following conditions: 94℃ for 5 min followed by 30 cycles of 94℃ for 30 sec, 50℃ for 30 sec, 72℃ for 1 min, and final extension at 72℃ for 5 min. In addition, 28s rDNA was amplified using the primers LR0R and LR16 by PCR under the following conditions: 94℃ for 3 min followed by 36 cycles of 94℃ for 30 sec, 54℃ for 30 sec, 72℃ for 1 min, and final extension at 72℃ for 10 min.

Sequencing and phylogenetic analysis

PCR products were sequenced by Solgent (Daejeon, Korea). The sequences were deposited in NCBI GenBank and compared with those available in GenBank via BLAST searches. Phylogenetic analyses were conducted using the neighbor joining (NJ) methods in MEGA5 [14], and a NJ tree was constructed using the Tamura-Nei distance [15]. All characters were equally weighted and unordered. Gaps and missing data were treated as complete deletions. Support for specific nodes on the NJ tree was estimated by bootstrapping 1,000 replications.

Results and Discussion

A total of 58 fungal endophytes were isolated from 132 leaf fragments from 11 plants of the two species of conifers evaluated in this study. All the isolates were grouped into 18 morphotypes based on morphological characteristics such as colony shape, height, and color of aerial hyphae, base color, growth rate, margin characteristics and surface texture. Molecular identification using ITS-5.8S and large subunit (LSU) rDNA sequences from the isolates revealed seven taxa of endophytic fungi: Eurotiomycetes sp., Leotiomycetes sp., Lophodermium conigenum, Polyporales sp., Rhytismataceae, and Septoria pini-thundergii (Table 1). All of the isolated species belonged to Ascomycetes, except for isolate 12A062, which was a basidiomycetous fungi, Poloyporales sp. Only two taxa, L. conigenum and S. pini-thundergii, were identified at the species level with more than 99% sequence identifies based on both the ITS and LSU rDNA. However, four taxa showed sequences related at above the family level of fungi: Eurotiomycetes, Leotiomycetes, Polyporales and Rhytismataceae.

Table 1

Molecular identification of endophytic fungi isolated from needles of Pinus densiflora and Pinus koraiensis in Korea

ITS, internal transcribed spacer; LSU, large subunit.

Based on analysis of the ITS sequences, ten sequences belonged to Leotiomycetes, most of which were closely related to the Lophodermium sp. (Fig. 1). Moreover, isolate 12A026 was grouped with Coccomyces mucronatus and isolate 12A010 was grouped with an undetermined taxon, fungal endophyte EMS38. Six sequences belonged to Dothideomycetes, and these were closely related to Septoria pini-thunbergii with 99% sequence similarity. The sequence of 12A057 was grouped with Eurotiomycetes, while isolate 12A062 was grouped with Polyporales and Agaricomycetes. These results provide evidence that some leaf endophytes are Basidiomycota, even though most endophytes of conifer leaves are filamentous Ascomycota [16, 17].

Fig. 1

Phylogenetic analysis based on the internal transcribed spacer-5.8S sequences showing the relationships of endophytic fungi isolated from conifer leaves with reference taxa. The tree shown was derived using the neighbor joining method.

LSU rDNA sequences analysis and ITS rDNA were amplified and sequenced for ambiguous sequences (Fig. 2). Isolates related to Lophodermium sp. based on the ITS sequence were classified as L. conigenum when the partial LSU rDNA sequence was used; therefore, we identified this organism as L. conigenum. Isolates 12A010 and 12A026 were closely related to fungal endophytes for ITS sequences (Table 1), but grouped with fungi that belonged to Leotiomycetes and Rhytismataceae, respectively, based on their LSU sequences. Thus, these isolates as well as two other isolates, 12A057 and 12A062, could not be identified at the species or genus level.

Fig. 2

Phylogenetic analysis based on partial large subunit rDNA sequences showing the relationships of endophytic fungi isolated from conifer leaves with reference taxa. The tree shown was derived using the neighbor joining method.

Many studies have traditionally used sequence data from the ITS region to identify sterile cultures and evaluate morphotaxon boundaries [18, 19]. ITS data are considered useful for these purposes due to the rapid evolution of the ITS. However, most fungi are not represented in GenBank, and some GenBank records are misidentified or lack taxonomic information [6]. Therefore, BLAST and phylogenetic analyses of other genomic regions should be combined with those of the ITS region to improve the accuracy of identification. In this study, we selected representative isolates from morphotypes, and then conducted phylogenetic analysis based on ITS and partial LSU rDNA sequences. However, four out of six taxa could not be identified at the species level, indicating that these isolates might be new fungal endophytes. Accordingly, further taxonomic study of these organisms is necessary.

A total 58 endophytes were isolated from 132 leaf fragments in this study and the isolation frequency was 43.9%. Overall, 56 fungal endophytes were isolated from 72 leaf fragments of P. densiflora, while only two were isolated from 60 leaf fragments of P. koraiensis (Table 2), giving an isolation frequency of 77.8% for P. densiflora and 3.3% for P. koraiensis.

Table 2

Endophytic fungi isolated from needles of Pinus densiflora and Pinus koraiensis in Korea

Four taxa were isolated from P. densiflora; L. conigenum, Septoria pini-thunbergii, Leotiomycetes sp., and Polyporales sp., while two taxa, Rhytismataceae sp. and Eurotiomycetes sp., were isolated from P. koraiensis. L. conigenum and S. pini-thunbergii were most dominant on the leaves of P. densiflora, and these taxa accounted for the largest proportion of fungal endophytes in the study site. Furthermore, these organisms showed high affinity to P. densiflora, regardless of altitude. These findings are in accordance with the fact that coniferous endophytes are generally transmitted among hosts via contagious spread [6, 16]. Fungal endophytes isolated from P. densiflora were more diverse than those isolated from P. koraiensis, which suggests specificity between host plants and fungal endophytes.