ginseng has well - known , diverse actions and effects on the human body , such as nonspecific resistance to biochemical and physical stresses , and the improvement of vitality , longevity and mental capacity [ 1 - 6 ] .
generally 4 to 6 years of growth are required to produce high quality ginseng roots and the cultivation of ginseng should be under shade conditions .
consecutive cultivation in the same soil causes severe reduction in production mainly due to pathogenic infection .
heavy use of chemical pesticides has been applied to ginseng fields to control pathogens , which results in the contamination of ginseng roots and the surrounding soil .
the importance of biological control methods is now widely recognized to produce organic ginseng roots and reduce environmental contamination .
endophytes were subsequently described as fungi and bacteria that spend the most or part of their life cycle internally and asymptomatically in the healthy living tissues of plants .
it is believed that fungal endophytes originated from pathogenic fungi which either lost their virulence or exhibit extended latent periods .
many studies have shown that fungal endophytes are ubiquitous in most plants and colonize without apparent harm to their hosts .
various relationship between fungal endophytes and plants has been observed , which ranges from mutualistic or symbiotic to antagonistic or pathogenic .
there are at least one million estimated species of fungal endophytes in plants   as well as in lichens .
most fungal endophytes belong to the ascomycota clade and some of them belong to the basidiomycota 
 .
rodriguez et al .   identified four classes of fungal endophytes based on host range , colonization pattern , transmission , and ecological function .
the function of fungal endophytes might be dependent upon the plant organ - fungus interaction in each plant .
beneficial endophytes have several positive effects on plants , including growth , nutrient uptake , and tolerance to abiotic and biotic stresses .
medicinal plants produce chemicals that can be used as raw material for pharmaceutical , cosmetic , and fragrance industries .
some fungal endophytes produce secondary metabolites that are potential sources for natural products with unique structures and bioactivities .
endophytes in medicinal plants may be involved in the metabolic pathways of medicinal plants and produce analogous or novel bioactive metabolites ( e.g. , taxol ) .
fungal endophytes produce metabolites with potential use for medicinal , agricultural and industrial purposes , such as antibiotics , antimycotics , immuno suppressants , and anticancer compounds [ 28 - 31 ] .
in addition , fungi have advantages over plants , such as short cultivation time and large biomass production . however , there are very few reports on fungal endophytes in ginseng [ 32 - 34 ]
. therefore , an extensive investigation of fungal endophytes in ginseng can increase the opportunities of identifying potential fungi that can be used to produce bioactive compounds and control against pathogens .
this study has been undertaken to study the diversity of fungal endophytes in three cultivars of panax ginseng meyer cultivated in korea .
three - year - old roots of ginseng , p. ginseng meyer , were harvested from gangwon province , korea during sunny days in august 2010 .
four roots of each cultivar ( chunpoong , yunpoong , and gumpoong ; 12 roots in total ) were harvested , and these were stored at 4 before being processed .
fungal endophytes from ginseng roots were isolated according to xing et al .   with modification .
root samples were thoroughly washed with running tap water and cut into 1 cm long segments with a clean razor blade .
the root segments were surface sterilized with 75% ethanol for 1 min , 4% nahco3 for 3 min , and 75% ethanol for 30 s. the root segments were rinsed three times with sterile distilled water and blotted with sterile tissue paper .
three aliquots of 0.1 ml of the water used for the last washing step were inoculated on potato dextrose agar ( pda ) plates with 200 g / ml ampicillin and 200 g/ ml streptomycin to ensure the elimination of ephiphytic microorganisms .
pda plates were incubated at room temperature and checked every day to detect mycelial growth out of the roots .
the fungal mycelial tips of the emerging mycelia from the edges of the root segments were transferred to new pda plates supplemented with ampicillin and streptomycin .
the transfer of emerging mycelia from the root segments was continued for up to 4 wk . mycelia were scraped using a sterile scalpel from 1- or 2-week - old pda fungal cultures .
the harvested fungal mycelia were frozen in liquid nitrogen and stored at -80 until use .
mycelia were ground to a fine powder using a mortar and pestle in liquid nitrogen with sea sand .
dna was extracted using dneasy plant mini kit ( qiagen , ca , usa ) according to manufacturer s recommendation .
amplification of the internal transcribed spacer ( its ) region was carried out using the universal eukaryotic primers of its1 ( 5 tccgtaggtgaa cctgcgg 3 ) and its4 ( 5 tcctccgcttattgatatgc 3 ) .
pcr was performed in 50 l reaction containing 0.5 g of dna , 25 mol of each primer with the following reaction conditions : 2 min initial denaturing step at 95 , followed by 30 cycles of 1 min denaturation at 95 , 1 min primer annealing at 55 , 1 min extension at 72 , and a final 5 min extension at 72. pcr products were analyzed by electrophoresis in 0.8% agarose gel and the pcr products ( approximately 550 bp ) were excised from the gel .
dna was purified using wizard sv gel and pcr clean - up system ( promega , wi , usa ) .
after sequence analysis , its sequences were searched using the ncbi blast program ( http://www.ncbi.nlm.nih.gov ) .
morphological identification was also carried out to confirm the results of molecular identification based on macroscopic and microscopic appearances .
the percentage of colonization frequency ( % cf ) of fungal endophytes was calculated based on previous studies   as follows : % cf=(ncol / nt)100 , where ncol = number of segments colonized by each fungus ; nt = total number of segments .
a phylogenetic tree was constructed from its1 - 5.8sits2 sequences by the maximum parsimony method and mega5 software ( http://www.megasoftware.net ) .
three - year - old roots of ginseng , p. ginseng meyer , were harvested from gangwon province , korea during sunny days in august 2010 .
four roots of each cultivar ( chunpoong , yunpoong , and gumpoong ; 12 roots in total ) were harvested , and these were stored at 4 before being processed .
fungal endophytes from ginseng roots were isolated according to xing et al .   with modification .
root samples were thoroughly washed with running tap water and cut into 1 cm long segments with a clean razor blade .
the root segments were surface sterilized with 75% ethanol for 1 min , 4% nahco3 for 3 min , and 75% ethanol for 30 s. the root segments were rinsed three times with sterile distilled water and blotted with sterile tissue paper .
three aliquots of 0.1 ml of the water used for the last washing step were inoculated on potato dextrose agar ( pda ) plates with 200 g / ml ampicillin and 200 g/ ml streptomycin to ensure the elimination of ephiphytic microorganisms .
pda plates were incubated at room temperature and checked every day to detect mycelial growth out of the roots .
the fungal mycelial tips of the emerging mycelia from the edges of the root segments were transferred to new pda plates supplemented with ampicillin and streptomycin .
the transfer of emerging mycelia from the root segments was continued for up to 4 wk .
mycelia were scraped using a sterile scalpel from 1- or 2-week - old pda fungal cultures .
the harvested fungal mycelia were frozen in liquid nitrogen and stored at -80 until use .
mycelia were ground to a fine powder using a mortar and pestle in liquid nitrogen with sea sand .
dna was extracted using dneasy plant mini kit ( qiagen , ca , usa ) according to manufacturer s recommendation .
amplification of the internal transcribed spacer ( its ) region was carried out using the universal eukaryotic primers of its1 ( 5 tccgtaggtgaa cctgcgg 3 ) and its4 ( 5 tcctccgcttattgatatgc 3 ) .
pcr was performed in 50 l reaction containing 0.5 g of dna , 25 mol of each primer with the following reaction conditions : 2 min initial denaturing step at 95 , followed by 30 cycles of 1 min denaturation at 95 , 1 min primer annealing at 55 , 1 min extension at 72 , and a final 5 min extension at 72. pcr products were analyzed by electrophoresis in 0.8% agarose gel and the pcr products ( approximately 550 bp ) were excised from the gel .
dna was purified using wizard sv gel and pcr clean - up system ( promega , wi , usa ) .
after sequence analysis , its sequences were searched using the ncbi blast program ( http://www.ncbi.nlm.nih.gov ) .
morphological identification was also carried out to confirm the results of molecular identification based on macroscopic and microscopic appearances .
the percentage of colonization frequency ( % cf ) of fungal endophytes was calculated based on previous studies   as follows : % cf=(ncol / nt)100 , where ncol = number of segments colonized by each fungus ; nt = total number of segments .
a phylogenetic tree was constructed from its1 - 5.8sits2 sequences by the maximum parsimony method and mega5 software ( http://www.megasoftware.net ) .
total of 38 fungal endophytes were isolated from 12 ginseng roots ( 184 segments ) of 3 cultivars .
1 ) . chunpoong ( 56 root segments ) , yunpoong ( 74 segments ) , and gumpoong ( 54 segments ) were colonized by 5 , 17 , and 16 fungal isolates , respectively .
only 5 fungal isolates were identified in chunpoong , which may be due to the contamination or outgrowth of endophytic bacteria .
bacterial growth usually inhibited the outcome of fungal endophytes due to the fast colonization of root segments , even on pda plates containing antibiotics .
fungal endophytes isolated from 3-year - old ginseng roots of 3 cultivars in gangwon province in korea four roots of each cultivar were used to isolate fungal endophytes ( 12 roots in total ) .
the analyses of its1 - 5.8s - its2 regions showed 100% identities of phoma radicina ( fj427058 ) , fusarium oxysporum ( hq328030 ) , setophoma terrestris ( jn615482 : synonym phoma trrestris , pyrenochaeta terrestris ) and ascomycota sp .
the highest cf varied among cultivars from 9.4% to 30.6% and the average cf was 21.5% ( table 1 ) , which may also be due to bacterial contamination or growth of bacterial endophytes .
phoma radicina was the most frequent fungal endophyte in three ginseng cultivars : 80% , 52.9% , and 75% of cf in chunpoong , yunpoong , and gumpoong , respectively . in total
, the percentage of dominant endophytes ( de ) of p. radicina was 65.8% , which is the highest percentage among the detected fungal isolates .
the second most dominant species was f. oxysporum : 20% , 23.5% , and 18.7% of de in chunpoong , yunpoong , and gumpoong , respectively .
the number of fungal endophytes found in this study is lower than expected compared with other studies .
this result indicates that distribution of endophytes depends on plant species and geological locations . as mentioned earlier
, there are only a limited number of studies reported on the isolation of fungal endophytes in ginseng species .
reported 134 fungal isolates with 27 taxa in american ginseng ( p. quinquefolium ) , in which 11 species were identified in detail .
four species ( alternaria , collectotrichum , phoma , and xylariale ) were the most common in 3 different tissues ( root , stem and leaf ) of american ginseng . these genera are known as common endophytes [ 23,42 - 44 ] .
xing et al .   reported tissue specificity for the reported endophytes ; cladosporium sp .
were the dominant isolates in roots , but were not detected in stem or leaf tissues .
in addition , cladosporium sp . was not detected in 4-year - old american ginseng roots , but glomerella cingulata was the dominant species instead .
they also found that the diversity of fungal endophytes in american ginseng roots decreased with age .
this may be because of autotoxic or host defense compounds of american ginseng in the rhizosphere .
the genus , phoma sp . , which was the most dominant isolate in ginseng , was detected only in leaf tissues of 1- , 2- and 3-year - old american ginseng , but not in 4-year old ginseng .
tissue specificity of endophytes has also been reported in previous studies [ 48 - 50 ] .
f. oxysporum was the second dominant isolate in ginseng , while it was only detected in 3-year old roots of american ginseng .
the genus phoma is ubiquitously present in the environment and considered an important fungal plant pathogen . in other plant species , fusarium and phoma
were the most frequent genera among 142 fungal species isolated in roots of 24 plant species growing at 12 sites in spain .
the genera of fusarium and phoma have been known as common endophytes in other studies .
concluded that endophytic communities were dependent on the soil type and plant species , but not on location .
the physio - chemical nature of a soil may influence on the colonization of fungal species to plants .
while f. oxysporum is commonly present in legume species as a pathogen , this fungus can colonize other plants without causing symptoms .
in general , nonpathogenic endophytes can turn into pathogenic strains after introduction into potentially new host plants .
2-rnk that was also isolated from the roots and fruits of neem tree ( azadirachta indica a. juss . ) .
2 shows the maximum parsimony tree grouping fungal endophytes into two clades : 1 ) p. radicina and s. terrestris , and 2 ) f. oxysporum and ascomycota sp . 2-rnk .
we isolated a limited number of fungal endophytes in ginseng root , but our findings can increase the potential use and awareness of fungal endophytes for beneficial applications .
therefore , our finding can increase the possibilities of identifying potential fungi that can be used to protect ginseng plants and produce bioactive compounds .
 in order to investigate the diversity of endophytes , fungal endophytes in panax ginseng meyer cultivated in korea were isolated and identified using internal transcribed spacer ( its ) sequences of ribosomal dna . 
 three cultivars of 3-year - old ginseng roots ( chunpoong , yunpoong , and gumpoong ) were used to isolate fungal endophytes . 
 surface sterilized ginseng roots were placed on potato dextrose agar plates supplemented with ampicilin and streptomycin to inhibit bacterial growth . 
 overall , 38 fungal endophytes were isolated from 12 ginseng roots . according to the sequence analysis of the its1 - 5.8s - its2 
 , 38 fungal isolates were classified into 4 different fungal species , which were phoma radicina , fusarium oxysporum , setophoma terrestris and ascomycota sp . 
 2-rnk . 
 the most dominant fungal endophyte was p. radicina in 3 cultivars . 
 the percentage of dominant endophytes of p. radicina was 65.8% . 
 the percentage of colonization frequency of p. radicina was 80% , 52.9% , and 75% in chunpoong , yunpoong , and gumpoong , respectively . 
 the second most dominant fungal endophyte was f. oxysporum . 
 the diversity of the fungal endophytes was low and no ginseng cultivar specificity among endophytes was detected in this study . 
 the identified endophytes can be potential fungi for the production of bioactive compounds and control against ginseng pathogens . 
INTRODUCTION
MATERIALS AND METHODS
Collection of ginseng roots
Isolation of fungal endophytes
Identification of fungal isolates
Phylogenetic analysis
RESULTS AND DISCUSSION