fungal endophytes are considered to be a group of fungi that live asymptomatically inside the tissues of living plants [ 1 - 3 ] .
fungal endophytes do not penetrate plant cells , but instead grow in the spaces between plant cells or within plant cell walls , while utilizing low levels of nutrients in intercellular fluids and causing no obvious damage .
recent studies have reported that fungal endophytes are ubiquitous in most plants and even lichens .
endophytes are potential genetic sources of natural products for pharmaceutical , agricultural , and industrial purposes .
endophytes from medicinal plants such as ginseng might produce important secondary metabolites for bioactive materials .
in general , fungal endophytes are classified into 2 major groups : clavicipitaceous endophytes and nonclavicipitaceous endophytes .
the clavicipitaceous endophytes are transmitted by seeds , but the mechanism for infection into hosts is still speculative .
the nonclavicipitaceous endophytes found in woody and herbaceous plants provide the potential for a wide variety of direct and indirect interactions between plants and herbivores , including increasing resistance to disease , abiotic stress and the enhancement of plant growth .
fungal endophytes are significant because they produce a wide variety of secondary metabolites with potential uses in medicine , agriculture , and industry .
these compounds can be used for growth promotion of host plants , enhancement of the synthesis of secondary metabolites of host plants , and to help plants defend against pathogens [ 5 - 7 ] .
it has been used for thousands of years as an important source of medicinal herbs . in asia
, ginseng is known as the most valuable medicinal plant . in general , ginseng is a shade - loving plant , with one stalk having 5-fingered leaves , white flowers , red to yellow fruits and pale yellow root .
despite the fact that active compounds are present in all parts of the ginseng plant , only the root is used pharmacologically .
ginseng contains triterpene glycosides , commonly referred to as ginsenosides , compared to saponins contained in other plants .
many active ingredients are found in all parts of the plant , including amino acids , phenols , alkaloids , polypeptides , and vitamins .
ginseng has many proven pharmacological effects such as improvement of cerebral function , immune modulation , pain relief , diabetes , and cancer prevention [ 8 - 10 ] .
the components and contents of ginsenosides are influenced by many factors such as the species , age , plant tissue , cultivation environment , and harvest season .
the cultivation of ginseng can be problematic because ginseng is affected by soil conditions during long periods of cultivation .
one of the major problems is diseases caused by pathogenic fungi due to long periods of cultivation in the same soil .
ginseng can be affected by many fungal pathogens such as gray mold produced by botrytis cinerea , alternaria blight produced by alternaria panax , anthracnose produced by colletotrichum gloeosporioides , sclerotinia white rot produced by sclerotinia sp .
, phytophthora blight produced by phytophthora cactorum , and root rot produced by cylindrocarpon destructans .
the aim of this study was to examine the diversity of fungal endophytes obtained from different ages of p. ginseng cultivated in korea .
the results can be further used for protection of ginseng plants and/or production of bioactive metabolites .
ginseng roots of 1- , 2- , 3- , and 4-year - old plants were harvested from the national institute of horticultural and herbal science ( eumseong , korea ) , which is located in latitude 3656n and longitude 12745e .
ginseng plants were cultivated according to the protocol of  ginseng good agricultural practice standard cultivation guide   developed by the rural development administration , korea .
the soil type was sandy loam with ph < 5.8 and electrical conductivity ( ec ) 0.6 ds / m .
whole roots including rhizomes , main roots and hairy roots were used in this study .
six roots of ginseng plants were used for each age of plant ( 24 plants in total ) .
ginseng roots were washed under running tap water for 5 min to remove soil and debris and root samples were cut into 1010 mm segments using a sterilized blade .
all root segments were surface sterilized in 75% ethanol for 1 min , transferred to 4% nahco3 , washed for 10 min , and then placed in 75% ethanol for 30 s. the sterilized root segments were washed 3 times in sterile distilled water for 1 min and blotted onto a sterile paper towel .
root segments were then transferred to potato dextrose agar ( pda ) plates ( difco , sparks , md , usa ) with 200 mg / ml each of ampicillin and streptomycin to inhibit bacterial growth .
plates were incubated at room temperature , and fungal colonies growing from the edges of the root segments were transferred to new pda plates containing ampicillin and streptomycin .
transfer of fungal colonies from the root segments was continued for up to 4 wk , and the fungal isolates were stored in 10% glycerol at -80 for long - term storage morphological identification of isolated fungal endophytes was done by characterization of spores and hyphae using light microscope ( olympus bx51 ; olympus , tokyo , japan ) .
fungal mycelia were scraped from pda plates and ground into a fine powder using a mortar and pestle in liquid nitrogen and sea sand . a dneasy plant mini kit (
qiagen , valencia , ca , usa ) was used to extract dna from the ground mycelia .
universal eukaryotic primers were used to amplify the fungal ribosomal dna internal transcribed spacer ( its ) regions 1 and 2 of all fungal isolates : its1 ( 5-tccgtaggtgaacctgcgg- 3 ) and its4 ( 5-tcctccgc ttattgatatgc- 3 ) .
the conditions used for polymerase chain reaction ( pcr ) reactions have been previously described .
its sequence information was obtained by the sequencing of the pcr products that were purified from the gel using wizard sv gel and a pcr clean - up system ( promega , fitchburg , wi , usa ) .
pcr - amplified its sequences were searched using the ncbi blast ( http://www.ncbi.nlm.nih.gov ) against those in the genbank database and the results of matched accession numbers were included in table 1 .
a phylogenetic tree was constructed using the maximum parsimony method and mega5 software from its1 - 5.8sits2 sequences ( http://www.megasoftware.net ) .
the colonization frequency ( cf ) of endophytic fungi was calculated as follows : cf=(ncol / nt)100 , where ncol = number of segments colonized by each fungus ; nt = total number of segments .
/ nt)100 , where nf = number of each isolated fungus ; nt = total isolated number of fungi .
ginseng roots of 1- , 2- , 3- , and 4-year - old plants were harvested from the national institute of horticultural and herbal science ( eumseong , korea ) , which is located in latitude 3656n and longitude 12745e .
ginseng plants were cultivated according to the protocol of  ginseng good agricultural practice standard cultivation guide   developed by the rural development administration , korea .
the soil type was sandy loam with ph < 5.8 and electrical conductivity ( ec ) 0.6 ds / m .
whole roots including rhizomes , main roots and hairy roots were used in this study .
six roots of ginseng plants were used for each age of plant ( 24 plants in total ) .
ginseng roots were washed under running tap water for 5 min to remove soil and debris and root samples were cut into 1010 mm segments using a sterilized blade .
all root segments were surface sterilized in 75% ethanol for 1 min , transferred to 4% nahco3 , washed for 10 min , and then placed in 75% ethanol for 30 s. the sterilized root segments were washed 3 times in sterile distilled water for 1 min and blotted onto a sterile paper towel .
root segments were then transferred to potato dextrose agar ( pda ) plates ( difco , sparks , md , usa ) with 200 mg / ml each of ampicillin and streptomycin to inhibit bacterial growth .
plates were incubated at room temperature , and fungal colonies growing from the edges of the root segments were transferred to new pda plates containing ampicillin and streptomycin .
transfer of fungal colonies from the root segments was continued for up to 4 wk , and the fungal isolates were stored in 10% glycerol at -80 for long - term storage
morphological identification of isolated fungal endophytes was done by characterization of spores and hyphae using light microscope ( olympus bx51 ; olympus , tokyo , japan ) .
fungal mycelia were scraped from pda plates and ground into a fine powder using a mortar and pestle in liquid nitrogen and sea sand . a dneasy plant mini kit (
qiagen , valencia , ca , usa ) was used to extract dna from the ground mycelia .
universal eukaryotic primers were used to amplify the fungal ribosomal dna internal transcribed spacer ( its ) regions 1 and 2 of all fungal isolates : its1 ( 5-tccgtaggtgaacctgcgg- 3 ) and its4 ( 5-tcctccgc ttattgatatgc- 3 ) .
the conditions used for polymerase chain reaction ( pcr ) reactions have been previously described .
its sequence information was obtained by the sequencing of the pcr products that were purified from the gel using wizard sv gel and a pcr clean - up system ( promega , fitchburg , wi , usa ) .
pcr - amplified its sequences were searched using the ncbi blast ( http://www.ncbi.nlm.nih.gov ) against those in the genbank database and the results of matched accession numbers were included in table 1 .
a phylogenetic tree was constructed using the maximum parsimony method and mega5 software from its1 - 5.8sits2 sequences ( http://www.megasoftware.net ) .
the colonization frequency ( cf ) of endophytic fungi was calculated as follows : cf=(ncol / nt)100 , where ncol = number of segments colonized by each fungus ; nt = total number of segments .
/ nt)100 , where nf = number of each isolated fungus ; nt = total isolated number of fungi .
a total of 81 fungal endophytes were isolated from ginseng roots : 5 , 22 , 22 , and 32 isolates from 1- , 2- , 3- , and 4-year - old ginseng roots , respectively ( table 1 ) .
ginseng roots of 1- , 2- , 3- , and 4-year - old plants were colonized by 2 , 6 , 8 , and 5 species of fungal endophytes , respectively ( table 1 ) .
putative taxonomic affinities and the cf of the isolated fungal endophytes from ginseng roots 
  ni were in parentheses . the closest matched accession numbers were included after blast searches of its ( internal transcribed spacer ) sequences 
 of fungal endophytes from the roots of 1- , 2- , 3- , and 4-year - old ginseng ( panax ginseng ) plants .
cf of endophytic fungi was calculated 
 as follows : cf=(ncol / nt)100 , where ncol = number of segments colonized by each fungus ; nt = total number of segments .
its , internal transcribed spacer ; cf , colonization frequency ; ni , number of isolated fungal endophyte .
individual cfs were 12% , 40% , 31% , and 40% for 1- , 2- , 3- , and 4-year - old ginseng roots , respectively .
there were no significant differences in the dominant fungal endophytes in different aged roots ( table 2 ) .
phoma radicina was the most frequent fungal endophyte in 2- , 3- , and 4-year - old ginseng roots : 15% , 11% , and 21% cf , respectively .
percentages of dominant endophytes ( de ) of p. radician in 2- , 3- , and 4-year - old ginseng were 37.5% , 38.5% , and 52.5% , respectively . among 2 isolates detected , fusarium solani was the dominant endophyte in 1-year - old ginseng roots : 7% cf and 60% de .
percentage contribution by the de to the fungal endophytes 
 isolated from different ages including 1- , 2- , 3- , and 4-year - old 
 ginseng roots de , dominant endophyte .
the percent of de ( de% ) was calculated as follows : de%=(nf/ 
 nt)100 , where nf = number of each isolated fungus ; nt = total isolated 
 number of fungi . in order to examine the biodiversity of the culturable fungal endophytes in ginseng roots
a total of 81 clones were obtained from the fungal endophytes isolated from 4 age groups of ginseng roots .
blast and maximum- parsimony phylogenetic tree analysis placed the cloned sequences into 11 groups ( fig .
the isolated fungal endophytes belong to 11 fungal groups at different taxonomic levels based on its sequences and morphological identification .
the genera of phoma , collectotrichum and fusarium were the most common fungal endophytes found in ginseng roots cultivated in korea , and are also common fungal endophytes found in p. quinquefolium , tropical , and subtropical plants .
we isolated c. destructans , which is one of the most harmful pathogenic fungi of ginseng . however , it is not clear whether c. destructans is pathogenic or nonpathogenic to ginseng .
f. oxysporum is a common pathogenic fungus in plants ; however , there are nonpathogenic strains that compete for organic substrates with the pathogenic strains .
nonpathogenic strains often reduce the germination of chlamydospores , and also germ tube growth , which leads to a decline in the population of pathogenic strains .
while higher numbers of fungal endophytes were detected in 4-year - old ginseng roots compared to 1- , 2- and 3-year - old ginseng roots , a lower diversity of fungal endophytes was found in 4-year - old ginseng roots ( table 1 ) .
this may be due to differences in cultivation sites , age , and/or longer exposure to chemical fungicides in 4-year - old ginseng cultivation sites .
longer exposure to fungicides might reduce the total number of species of fungal endophytes , while resistant fungal species occupy ginseng roots more frequently .
autotoxic compounds in p. quinquefolium which have recently been isolated and characterized cause a reseeding failure .
other factors can influence the community of fungal endophytes , such as fertilizers , water , temperature , soil , and plant defense compounds [ 24 - 26 ] .
fungi have common mechanisms used to detoxify compounds , such as activation of the membrane transporters that pump toxicants out of fungal cells , and enzymatic detoxification .
fungi that have detoxification mechanisms may increase their competitiveness among tolerant species of root endophytes , which leads to higher growth rates than those of less tolerant fungal endophytes in the presence of host toxins [ 28 - 31 ] .
colonization of mycorrhizal and soil fungi will be greatly affected when plant defense compounds are secreted in soil .
only 3 species were common to 2- , 3- , and 4-year - old ginseng roots such as p. radicina , f. oxysporum , and unknown species 1 .
f. solani was the dominant species in 1-year - old ginseng roots , while it was not found in 3-year - old ginseng roots . among 11 species ,
5 were only detected in certain ages of ginseng roots , which included trichoderma citrinoviride ( 1-year old ) , c. panacicola ( 2-year old ) , f. acuminatum ( 3-year old ) , ascomycete sp .
a small number of fungal endophyte species was detected in 1-year - old p. ginseng roots compared to 1-year - old roots of p. quinquefolium ( 2 vs. 8) .
one possible explanation might be that endophytic communities are dependent on the cultivation environment and method as well as plant species .
t. citrinoviride has a high ability to parasitize pathogens , protect host plants , and produce strongly inhibitory compounds .
produce a wide range of bioactive metabolites which contribute to their mycoparasitic and antibiotic actions .
the genera phoma and fusarium are ubiquitous in the environment and are famous as fungal pathogens . in the current study ,
p. radicina was the dominant endophyte in 2- , 3- , and 4-year - old ginseng roots , while f. solani was the dominant endophyte in 1-year - old ginseng roots .
other studies also showed that phoma and fusarium are common fungal endophytes in plant species .
endophytes have been known for their beneficial functions provided to plants regarding protection against abiotic and biotic stress .
for example , taxomyces andreanae , a fungal endophyte isolated from the pacific yew tree , can produce paclitaxel ( taxol ; bristol - myers squibb , new york , ny , usa ) which is an important anticancer drug . in conclusion
, it is proposed that the diversity of fungal endophytes in ginseng roots is dependent on the age of the plant .
further studies are needed to understand the relationships between the host and endophytes at the molecular and genetic levels , and can lead to the better use of endophytes to protect plants and produce potential secondary metabolites .
 fungal endophytes were isolated from 1- , 2- , 3- , and 4-year - old ginseng roots ( panax ginseng meyer ) cultivated in korea . 
 the isolated fungal endophytes were identified based on sequence analysis of the internal transcribed spacer and morphological characterization by microscopic observations . 
 a total of 81 fungal endophytes were isolated from 24 ginseng roots . 
 fungal endophytes were classified into 9 different fungal species and 2 unknown species . 
 ginseng roots that were 1- , 2- , 3- , and 4-years old were colonized by 2 , 6 , 8 , and 5 species of fungal endophytes , respectively . while phoma radicina was the most frequent fungal endophyte in 2- , 3- , and 4-year - old ginseng roots , fusarium solani was the dominant endophyte in 1-year - old ginseng roots . 
 the colonization frequencies ( cf ) varied with the host age . 
 the cf were 12% , 40% , 31% , and 40% for 1- , 2- , 3- , and 4-year - old ginseng roots , respectively . 
 we found a variety of fungal endophytes that were distributed depending on the age of ginseng plants . 
INTRODUCTION
MATERIALS AND METHODS
Collection of ginseng roots
Isolation of fungal endophytes
Identification of fungal endophytes
Data analysis
RESULTS AND DISCUSSION