taehwa , danyang - gun , chungcheongbuk - do , korea ( n 3707'01.42 '' , e 12829'05.11 '' ) . healthy leaves were collected from conifer species growing at the site , including 3 juniper trees ( juniperus rigida siebold et zucc . ) , 4 japanese larch trees [ larix kaempferi ( lamb . ) carr . ] , and 5 pine trees ( pinus densiflora siebold et zucc . ) . each tree was growing at a distance of at least 100 m from the other sampled trees between 400~800 m in altitude , and all trees were sampled using a gps , an aluminium tag , and a tagging tape : april ( spring ) , july ( summer ) , and november ( autumn ) . all the 2-yr - old leaves were washed with tap water and then placed for 3 min in 1% naocl solution , 2 min in 70% ethanol , and finally washed twice with distilled water . these surface - sterilized leaves were cut into 4 segments that were 5 mm in length and then placed into 3 types of culture medium : potato dextrose agar ( pda ) , malt extract agar , and water agar . any hyphae that extended from the leaf fragments were used in a successive culture with pda . all isolates were grouped into morphotypes on the colony shape , height , and color of the aerial hyphae , in addition to the base color , growth rate , margin characteristics , surface texture , and depth of growth into the medium . dna was extracted according to the protocol of the dneasy plant mini kit ( qiagen , hilden , germany ) , and pcr was performed to amplify the internal transcribed spacer ( its ) region , including 5.8s rdna , by 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 a final extension at 72 for 5 min . the pcr products were sequenced and compared with reference sequences on ncbi by using blast . taehwa , danyang - gun , chungcheongbuk - do , korea ( n 3707'01.42 '' , e 12829'05.11 '' ) . healthy leaves were collected from conifer species growing at the site , including 3 juniper trees ( juniperus rigida siebold et zucc . ) , 4 japanese larch trees [ larix kaempferi ( lamb . ) carr . ] , and 5 pine trees ( pinus densiflora siebold et zucc . ) . each tree was growing at a distance of at least 100 m from the other sampled trees between 400~800 m in altitude , and all trees were sampled using a gps , an aluminium tag , and a tagging tape : april ( spring ) , july ( summer ) , and november ( autumn ) . all the 2-yr - old leaves were washed with tap water and then placed for 3 min in 1% naocl solution , 2 min in 70% ethanol , and finally washed twice with distilled water . these surface - sterilized leaves were cut into 4 segments that were 5 mm in length and then placed into 3 types of culture medium : potato dextrose agar ( pda ) , malt extract agar , and water agar . finally , the samples were cultivated in an incubator at 25 and in darkness . any hyphae that extended from the leaf fragments were used in a successive culture with pda . all isolates were grouped into morphotypes on the colony shape , height , and color of the aerial hyphae , in addition to the base color , growth rate , margin characteristics , surface texture , and depth of growth into the medium . dna was extracted according to the protocol of the dneasy plant mini kit ( qiagen , hilden , germany ) , and pcr was performed to amplify the internal transcribed spacer ( its ) region , including 5.8s rdna , by 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 a final extension at 72 for 5 min . the pcr products were sequenced and compared with reference sequences on ncbi by using blast . a total of 59 morphotypes were isolated from the host plants ( table 1 ) . only morphotypes with a  97% similarity value   were used for analysis ( fig . depending on host plants , 6 species were identified among the 12 morphotypes from the juniper trees , 8 species among 21 morphotypes from the japanese larch , and 11 species among 26 morphotypes from the pine trees . one species was isolated from both the juniper tree and the japanese larch , 3 species were isolated from both the japanese larch and the pine tree , and 2 species were isolated from both the pine tree and the juniper tree . in addition , n. diffusa was the most abundant species in the juniper tree ; p. papaya , the japanese larch ; and l. pinastri , the pine tree ( table 1 ) . the shannon index ( h ' )   was used to assess the species diversity of the endophytic fungi ( table 1 ) . in the juniper tree , the total h ' was 1.47 , and the highest h ' ( 1.00 ) was observed in april . in the japanese larch , the h ' was 1.74 , and the highest h ' ( 1.33 ) was observed in november . in the pine tree , h ' was 1.58 , and the highest h ' ( 1.43 ) was observed in november . depending on sampling season , 7 species were identified among the 9 morphotypes in april , 8 species among 21 morphotypes in july , and 9 species among 29 morphotypes in november . one species was isolated during both april and july , 4 species were isolated during both july and november , and 1 species was isolated during both april and november ( table 1 ) . more than 600,000 species of endophytic fungi are theorized to exist worldwide , and various scientific approaches have been used to detect endophytic fungi to date . in the present study , h ' was the highest in the japanese larch , with the greatest number of endophytes being isolated during july and november . these results indicate that endophytes do not only exist in the leaf and/or do not use horizontal transfer ; instead , endophytes might extend from the plant tissue like a branchlet . during july and august , the climate in korea is warm and humid ; hence , climate probably affects endophytic dispersal . these results also indicate that the number of morphotypes belonging to endophytic fungi increases across the season . therefore , it is likely that a combination of these factors enhanced the species diversity of the japanese larch . j. rigida is mainly distributed in lower altitude forests , while l. kaempferi is primarily distributed in middle altitude forests . however , p. densiflora is found at higher altitudes , where the forest conditions are cooler and drier in the korean peninsula . thus , most endophytic fungi were obtained from lower to middle altitudes , with only one species of endophytic fungi being discovered at an altitude above 800 m. this observation indicates that endophyte distribution is influenced by the distribution of host plants . surveys for endophytic fungi have been conducted at all altitudes , with specimens being found at all sites ; however , endophytic fungi have an ability to adapt to variations in abiotic and biotic conditions along the altitudinal gradient . thus , standpoints of host specificity and adaptation ability are required , which is only possible through the collection and study of endophytes as ecological components and biological resources .