in 2005 , wood discs were cut from a discolored stump of pinus thunbergii ( fig .
1 ) grown in a forest in taean , chungnam , korea . after washing with tap water
the wood pieces were surface - sterilized with 0.5% chlorine lax solution for 2 min , washed twice with sterile water , and air dried .
the dried wood pieces were placed on mea ( 2% malt extract agar , bd science , usa ) supplemented with cycloheximide ( 200 g / ml ) and incubated for 10 days at room temperature .
fungal isolates were obtained by taking mycelial tips grown out from the incubated wood pieces .
single spore isolates were prepared from the obtained isolates grown on 2% mea at 25 for seven days .
all the isolated fungi were stored at -80 in 10% glycerol for long - term storage and in water at 4 for short - term storage . to identify optimal growth media , pre - cultured isolates
were transferred to the center of each of pda , mea , oa ( oatmeal agar , bd science , usa ) and omea ( 2% oxoid malt extract agar , oxoid , uk ) media , respectively , and grown for 9 days at 25. mycelial growth was recorded by measuring the diameters of the colonies .
light microscopic images of the morphological features of the isolates were examined under a phase - contrast microscope ( karl zeiss , axioskop 40 ) after growing the culture on 2% mea at 25 for 5~7 days . for scanning electron microscopy ( sem ) , the fungal isolate was grown for 5~7 days at 25 on 2% mea plates overlaid with cellophane ( bio - rad laboratories , canada ) .
pieces of cellophane were fixed in 2% glutaraldehyde buffered in 0.1 m cacodylate buffer for 16 h and 1% osmium tetroxide in 0.1 m phosphate for 1 h. samples were subsequently washed in 0.05 m cacodylate buffer , dehydrated in a series of ethanol washes ( 50% for 20 min , 75% for 20 min , 90% for 20 min , 95% for 20 min and 100% for 20 min ) , passed through ethanol - isoamylacetate , dried with a hitachi critical point dryer and coated with platinum - palladium at 25 nm with an hitachi e-1030 ion sputter .
the specimens were examined with a hitachi s-4300 scanning electron microscope operating at 15 kv . for fungal genomic dna ,
fungi were grown for 5~7 days on 2% mea plates overlaid with cellophane ( bio - rad laboratories , usa ) .
fungal genomic dna for pcr was obtained from the mycelium using the drilling method described by kim et al .
the -tubulin gene was amplified using the primers t10 and bt12 ( o'donnel and cigelnik , 1997 ; kim et al . , 2003 ) .
sequences were manually edited using the chromas v2.31 program and aligned using the clustalw 2 program .
the aligned sequences were analyzed with paup 4.0 beta 10 ( swofford , 2003 ) .
in 2005 , wood discs were cut from a discolored stump of pinus thunbergii ( fig .
1 ) grown in a forest in taean , chungnam , korea . after washing with tap water
the wood pieces were surface - sterilized with 0.5% chlorine lax solution for 2 min , washed twice with sterile water , and air dried .
the dried wood pieces were placed on mea ( 2% malt extract agar , bd science , usa ) supplemented with cycloheximide ( 200 g / ml ) and incubated for 10 days at room temperature .
fungal isolates were obtained by taking mycelial tips grown out from the incubated wood pieces .
single spore isolates were prepared from the obtained isolates grown on 2% mea at 25 for seven days .
all the isolated fungi were stored at -80 in 10% glycerol for long - term storage and in water at 4 for short - term storage .
to identify optimal growth media , pre - cultured isolates were transferred to the center of each of pda , mea , oa ( oatmeal agar , bd science , usa ) and omea ( 2% oxoid malt extract agar , oxoid , uk ) media , respectively , and grown for 9 days at 25. mycelial growth was recorded by measuring the diameters of the colonies .
light microscopic images of the morphological features of the isolates were examined under a phase - contrast microscope ( karl zeiss , axioskop 40 ) after growing the culture on 2% mea at 25 for 5~7 days . for scanning electron microscopy ( sem ) , the fungal isolate was grown for 5~7 days at 25 on 2% mea plates overlaid with cellophane ( bio - rad laboratories , canada ) .
pieces of cellophane were fixed in 2% glutaraldehyde buffered in 0.1 m cacodylate buffer for 16 h and 1% osmium tetroxide in 0.1 m phosphate for 1 h. samples were subsequently washed in 0.05 m cacodylate buffer , dehydrated in a series of ethanol washes ( 50% for 20 min , 75% for 20 min , 90% for 20 min , 95% for 20 min and 100% for 20 min ) , passed through ethanol - isoamylacetate , dried with a hitachi critical point dryer and coated with platinum - palladium at 25 nm with an hitachi e-1030 ion sputter .
the specimens were examined with a hitachi s-4300 scanning electron microscope operating at 15 kv .
for fungal genomic dna , fungi were grown for 5~7 days on 2% mea plates overlaid with cellophane ( bio - rad laboratories , usa ) .
fungal genomic dna for pcr was obtained from the mycelium using the drilling method described by kim et al .
the -tubulin gene was amplified using the primers t10 and bt12 ( o'donnel and cigelnik , 1997 ; kim et al . , 2003 ) .
sequences were manually edited using the chromas v2.31 program and aligned using the clustalw 2 program .
the aligned sequences were analyzed with paup 4.0 beta 10 ( swofford , 2003 ) .
initially . several fungal isolates were obtained from the sapwood pieces of a stained stump of japanease black pine .
they all grew well in the cycloheximide - supplemented mea media , indicating that they are cycleheximide tolerant .
they showed the same morphology and growth pattern , thus , one of the fungal isolates coded as dkm 0514 was used for identification in this work .
the isolate formed pale or red synnemata and droplets of conidia at the apex the synnemata ( fig . 2a~2f ) .
the shape and size of conidia on synnema and mononema are given in table 1 .
these features of synnemata are similar to synnema - forming species of the ophiostoma piceae complex .
( 2006 ) compared morphological characters of seven species of the o. piceae complex , including o. breviusculum , o. canum , o. floccosum , o. piceae , o. quercus , o. setosum , and o. subalpinum . among these compared species reported from conifers ,
especially , the red - brown synnema and yellow color of the conidia mass found in dkm 0514 were considered to be distinct features of o. floccosum by harrington et al .
o. floccosum ( mathiesen ) hunt ( anamorph : pesotum aureum ( hedgecock ) mcnew et harrington ) , one of the common sapwood - staining species ( hunt , 1956 ; harrington et al .
a sporothrix synanamorph with denticles and secondary conidia was observed from dkm 0514 by scanning electron microscopy ( fig .
o. floccosum is a heterothallic species , as it can produce perithecia only through cross of two different mating type partners . in this study
dkm 0514 itself did not form a perithecium , suggesting it is a heterothallic species .
consequently , we could not observe a telemorph of dkm 0514 ( table 1 ) .
the differentiation of the o. piceae complex is not easy based only on morphological characters .
therefore , to confirm whether the isolate is the anamorph of o. floccosum , its -tubulin gene sequence was analyzed .
when the determined -tubulin gene sequence was searched for homologous sequences in the genbank dna database , it matched well with the known -tubulin gene sequence of ophiostoma floccosum kuc 2412 ( accession number dq868378 ) ( 100% similarity ) .
the -tubulin gene sequence of the isolate dkm 0514 was deposited in the genbank under accession no .
the phylogenetic analysis based on the -tubulin gene sequences also revealed that dkm 0514 was placed in a clade with o. floccosum ( fig .
the isolate dkm 0514 from this study showed better mycelial growth on mea than on pda and oa ( fig .
the colony color of o. floccosum dkm 0514 incubated at 25 was creamy on pda , creamy grayish on oa , and first white then gradually turning brownish on mea and omea ( fig .
it produced no culture aroma and no mycelium with concentric rings that were observed as distinguishing characteristics of o. floccosum by harrington et al .
3 ) of the  - tublin gene , and physiological properties , the isolate dkm 0514 was identified as the anamorph of ophiostoma floccosum . this is the first report in korea on the detailed mycological properties of the anamorph of o. floccosum isolated from pinus thunbergii .
 an ophiostoma fungus was isolated from a stump of pinus thunbergii in a forest on the west coast of korea . 
 microscopic analysis using a light microscope , a stereo microscope , and a scanning electron microscope revealed that it had morphological features of pesotum and sporothix synanarmorphs . based on the -tubulin gene sequence analysis , 
 the fungus was identified as the anamorph of ophiostoma floccosum . 
 mycological properties of the species including its growth properties on different culture media were described . 
Materials and Methods
Fungal isolation and culture conditions
Observation of cultural and morphological characteristics
Nucleic acid preparation, PCR amplification and nucleotide sequencing
Molecular phylogenetic analysis
Results and Discussion