fungal endophytes are microfungi that colonize and live within healthy plant tissues without inducing symptoms of disease .
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 .
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 .
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 .
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 .
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 ] .
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   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 .
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 , and a nj tree was constructed using the tamura - nei distance .
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 .
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 .
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 ] .
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   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 .
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 , and a nj tree was constructed using the tamura - nei distance .
all characters were equally weighted and unordered . gaps and missing data were treated as complete deletions .
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 :
, 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 . based on analysis of the its sequences
, ten sequences belonged to leotiomycetes , most of which were closely related to the lophodermium sp .
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 ] .
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 .
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 . 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 .
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 .
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 .
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 .
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 .
 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
Materials and Methods
Sampling
Fungal endophyte isolations
DNA extraction and PCR
Sequencing and phylogenetic analysis
Results and Discussion