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field - cultivated ginseng roots were collected 20 different plots growing ginsengs from 1 to 5 year - old plants in geumsan - gun , korea ( n 3603'~05 ' , e 12732'~33 ' ) , with 4 replicate plots for each age .
a root sample was used for each plot in this study , making a total of 20 samples . to examine amf colonization rates ,
the roots were observed under the microscope ( axio imager ; carl zeiss , jena , germany ) and root colonization rates were measured by the previously described magnified intersects technique .
dna was extracted using the dneasy plant mini kit ( qiagen , venlo , netherlands ) from 20 field - cultivated ginseng root samples .
the nested pcr was performed to amplify the 28s rdna using primers lr1 and flr2 for first pcr , and lr1 and flr4 for second pcr as previously described [ 17 , 18 , 19 ] .
the pcr product was purified by using the qiaquick gel extraction kit ( qiagen ) and cloned using the pgem - t easy vector system following the manufacturer 's instructions ( promega co. , madison , wi , usa ) .
typically , twenty clones from each root sample were selected randomly for rflp in order to group for sequencing .
restriction enzymes hinfi and hsp92ii ( both from promega co. ) were used for pcr - rflp analysis .
the nucleotide sequence of one clone from each rflp type was determined using an automated sequencer ( solgent co. , daejeon , korea ) , and submitted to the genbank at ncbi ( accession nos .
using mega 5 , rdna sequences were aligned with other glomeromycota sequences as reference sequences from genbank to test the topological appropriation . from this analysis ,
a phylogenetic tree was constructed by the neighbor - joining method , with a bootstrap value of 1,000 based on tamura - nei model using mega 5 ; geosiphon pyriformis was used as an outgroup .
relative abundances of each fungal phylotypes were estimated based on clone counts for each rflp type .
univariate analysis of variance ( anova ) was used to evaluate the fixed effect of root ages on percent mycorrhizal root colonization rates and species diversity of amf communities , and principle component analysis was performed to compare the compositions of amf communities using a statistical package spss - win ver .
mycorrhizal colonization rates significantly increased with the age of the root ( p < 0.05 ) ( fig .
the mean colonization rate increased gradually from 5% in the 1-year - old ginseng roots to over 25% in the 5-year - old roots .
moreover , the overuse of soil fertilizer containing phosphorus and limited light availability has been shown to reduce the rate of colonization by amf [ 22 , 23 ] .
in addition , root growth at a faster rate than that of the am fungal hyphae , can also decrease the colonization rate .
even if some of the possible causes described above or other complex environmental factors result in low amf colonization rates , this neither implies a low amfdependence of the host plant nor is it a reflection of the effects of amf on the host plant . here
, we found that the amf colonization rate in ginseng roots increased while the species diversity simultaneously decreased with age .
therefore , we found no positive relationship between the colonization rate and species diversity of amf . in total
, 31 rflp types were obtained from the 200 clones and one clone representing each rflp type was sequenced from each group .
these sequences were used to construct a phylogenetic tree based on the large subunit ( lsu ) regions of rdna ( fig .
, all amf clones were divided into 7 amf clades : funnelliformis caledonium , f. mosseae , gigaspora margarita , paraglomus laccatum , p. occultum , rhizophagus irregularis , and scutellospora heterogama ( table 1 , fig .
r. irregularis was the most dominant species in ginseng roots of all ages used in this study .
on the other hand , g. margarita was observed only in the 1-year - old roots .
the relative abundance of r. irregularis increased from 71.3% in 1-year - old ginseng roots to 92.5% in 5-year - old roots .
however , abundance of other species , including f. mosseae , p. laccatum decreased despite widespread presence in cultivated soil ; f. mosseae , which seems to have adapted successfully to agricultural soil conditions did not colonize substantially with the roots of p. ginseng in this study [ 25 , 26 , 27 ] .
it is therefore likely that the am fungal community composition changed as result of this difference in the agricultural system . in our study ,
s. heterogama was observed to colonize the roots of only 5- year - old ginseng at a relatively lower rate ( 1.3  0.2% ) .
overall , the mean colonization rate increased with an increase in the age of ginseng roots .
this species has not been detected in cultivated soil and ginseng roots previously . in our future work , we will study how symbiosis between r. irregularis and p. ginseng affects ginsenoside content of perennial herbaceous plants .
reported two species of amf , p. brasilianum and glomus spurcum from the roots of 2-year - old fieldcultivated ginseng using primers aml1/aml2 .
several other studies have reported that g. intraradices dominates the amf community in other plant roots , while fungi from the acaulosporaceae or gigasporaceae family are lower in abundance .
although several studies have identified g. intraradices or other species that belong to the acaulosporaceae family , these species were not detected in our study .
however , species diversity of amf colonizing the roots significantly decreased with the root age ( fig .
3 ) . the diversity of amf in 1- to 3-year - old ginseng roots was higher than in 4- to 5-year - old ginseng roots .
this was reflected primarily through the decreased species evenness because the richness of amf was not significantly affected by the age of the ginseng roots .
our results are consistent with those of previous studies because the observed increase in the colonization by r. irregularis with the age of the roots and the absence of other species from more mature roots .
pcr analysis used to determine the influence of the age of ginseng roots on amf communities and revealed that the amf communities were able to discern among them ( fig .
this is similar to a previous study based on the data of amf spores in soil from the field of cultivated ginseng . in our study ,
amf community composition changed with the age of ginseng roots . based on the results of amf spore study
, the diversity of amf species was found to be greater in the soil of field - cultivated ginseng than in the ginseng roots
. however , not all the species identified from the soil were isolated from the roots .
, g. intraradices , g. etunicatum , acaulospora longula , archaeospora trappei , p. brasilianum were isolated only from the soil of cultivated ginseng and four amf species : f. caledonium , f. mosseae , p. occultum , s. heterogama were observed both in the soil and the roots . of these , p. brasilianum has also been detected in another study on ginseng and amf symbiosis .
observed that there was a good agreement between the spores isolated from soil and the phylotypes amplified from roots in a survey of five agricultural fields .
however , others have reported a poor species match between amf spores in the soil and amf in the roots of grassland plants . in this study ,
however , with an increase in the root age , species diversity and evenness of amf colonizing the roots decreased , whereas the dominance of r. irregularis increased .
the amf community colonizing ginseng roots became more uniform with an increase in root age indicating that changes in the preferences of amf for ginseng roots were age - dependent .
in this study , we examined arbuscular mycorrhizal fungal ( amf ) community structure colonizing field - cultivated ginseng roots according of different ages , such as 1- to 5-year - old plant , collected from geumsan - gun , korea . 
 a total of seven amf species namely , funnelliformis caledonium , f. moseae , gigaspora margarita , paraglomus laccatum , p. occultum , rhizophagus irregularis , and scutellospora heterogama were identified from the roots using cloning , pcr - restriction fragment length polymorphism and sequence analysis of the large subunit region in rdna . 
 amf species diversity in the ginseng roots decreased with the increase in root age because of the decreased species evenness . 
 in addition , the community structures of amf in the roots became more uniform . 
 these results suggest that the age of ginseng affects mycorrhizal colonization and its community structure .