an earless kingstar cultivar of muskmelon ( cucumis melo l. ) was cultivated in a greenhouse at jeollabuk - do agriculture research and extension services ( jbares ) , iksan ( latitude : 3558 ' n ; longitude : 1272 ' e ) , from april to august 2009 . gummy stem blight was observed on the stem and leaf stalk of muskmelon during the rainy season . no chemicals were used to treat the gummy stem blight ; thus , the disease was allowed to spread naturally . skin from the diseased portion of the plant was surface - sterilized with 1% naocl for 1 min and was washed three times with sterile de - ionized water to remove the naocl . the pathogen was isolated from the inner part of the stem and cultured on potato dextrose agar ( pda ; difco , sparks , md , usa ) in the dark at 25 for 3 days . the end parts of hypha cultured on pda were transferred aseptically onto malt extract agar ( difco ) and v-8 juice agar ( campbell soup co. , camden , nj , usa ) and incubated at 25 in the dark for 10 days . fruiting bodies ( conidia and conidiophores ) from the pathogen were surface - sterilized and placed on pda for growth under a 12-hr photoperiod for an additional wk to induce sporulation . a morphological examination fruiting bodies were placed on a droplet of sterile water on a microscopic slide and crushed to release the contents . the length  width of the conidia from the pathogen was measured at 400 magnification and was microscopically examined for the presence of septae ( nicon 80i ; nikon , tokyo , japan ) . the size of conidia and conidiophores was measured by using nis elements br-300 software ( nikon ) . the morphological data were compared with previously collected data for d. bryoniae . to obtain pure mycelium , a small amount of tissue was taken from the isolate and cultured on pda in the dark at 25 for 1 wk . the pure mycelium was used for a sequence analysis of the rdna its region ( its-1 region , 5.8s gene , and its-2 region ) . genomic dna was extracted using a dneasy plant mini kit ( qiagen , valencia , ca , usa ) according to the manufacturer 's instructions . amplification and sequencing of the isolate ( in the region containing its1 , its2 , and 5.8s rdna ) was performed using a pair of universal primers - its1 ( 5'-tccgtaggtgaacctgcgg-3 ' ) and its4 ( 5'-tcctccgcttattgatatgc-3 ' )   - which were also used as a positive control in a subsequent diagnostic pcr . the amplification was carried out in a 20-l reaction mixture containing 50 ng of genomic dna , 10 mm tris - hcl , 1.5 mm mgcl2 , 50 mm kcl , 0.01% gelatin , 0.2 mm dntp , 200 ng of each primer , and 1 unit of taq dna polymerase ( promega , madison , wi , usa ) . the reaction mixtures were denatured at 94 for 10 min and subjected to 35 cycles of 1 min each at 94 , annealing at 56~60 for 1 min , extension at 72 for 1 min , and a final extension step of 7 min at 72. an amplified pcr product was separated on 1.5% agarose gel and purified with a dna purification kit ( core - one ; core - bio , seoul , korea ) according to the manufacturer 's instructions . the reactions were monitored using bigdye terminator cycle sequencing kits ( applied biosytems , foster city , ca , usa ) as indicated by the manufacturer and run on an abiprism 3130 automated dna sequencer ( applied biosystems ) as described previously . the rdna its sequence data were analyzed using the dnastar program ( dnastar inc . , sequence data from the ribosomal its genes of didymella spp . were used to perform a preliminary phylogenetic analysis ; 16 its sequences were obtained from the genbank database ( http://www.ncbi.nlm.nih.gov/ ) ( table 2 ) . phylogenetic analysis based on parsimony for its , was obtained from the data by using neighbor - joining methods with mega ver . 4.0 software [ 14 , 15 ] , and the sequence distance was calculated using the tamura - nei , parameter model . the megalign program ( dnastar inc . ) was used to compare the percentage sequence identity from the jeonbuk isolate with that of other didymella spp . in genbank . an earless kingstar cultivar of muskmelon ( cucumis melo l. ) was cultivated in a greenhouse at jeollabuk - do agriculture research and extension services ( jbares ) , iksan ( latitude : 3558 ' n ; longitude : 1272 ' e ) , from april to august 2009 . gummy stem blight was observed on the stem and leaf stalk of muskmelon during the rainy season . no chemicals were used to treat the gummy stem blight ; thus , the disease was allowed to spread naturally . skin from the diseased portion of the plant was surface - sterilized with 1% naocl for 1 min and was washed three times with sterile de - ionized water to remove the naocl . the pathogen was isolated from the inner part of the stem and cultured on potato dextrose agar ( pda ; difco , sparks , md , usa ) in the dark at 25 for 3 days . the end parts of hypha cultured on pda were transferred aseptically onto malt extract agar ( difco ) and v-8 juice agar ( campbell soup co. , camden , nj , usa ) and incubated at 25 in the dark for 10 days . fruiting bodies ( conidia and conidiophores ) from the pathogen were surface - sterilized and placed on pda for growth under a 12-hr photoperiod for an additional wk to induce sporulation . a morphological examination fruiting bodies were placed on a droplet of sterile water on a microscopic slide and crushed to release the contents . the length  width of the conidia from the pathogen was measured at 400 magnification and was microscopically examined for the presence of septae ( nicon 80i ; nikon , tokyo , japan ) . the size of conidia and conidiophores was measured by using nis elements br-300 software ( nikon ) . to obtain pure mycelium , a small amount of tissue was taken from the isolate and cultured on pda in the dark at 25 for 1 wk . the pure mycelium was used for a sequence analysis of the rdna its region ( its-1 region , 5.8s gene , and its-2 region ) . genomic dna was extracted using a dneasy plant mini kit ( qiagen , valencia , ca , usa ) according to the manufacturer 's instructions . amplification and sequencing of the isolate ( in the region containing its1 , its2 , and 5.8s rdna ) was performed using a pair of universal primers - its1 ( 5'-tccgtaggtgaacctgcgg-3 ' ) and its4 ( 5'-tcctccgcttattgatatgc-3 ' )   - which were also used as a positive control in a subsequent diagnostic pcr . the amplification was carried out in a 20-l reaction mixture containing 50 ng of genomic dna , 10 mm tris - hcl , 1.5 mm mgcl2 , 50 mm kcl , 0.01% gelatin , 0.2 mm dntp , 200 ng of each primer , and 1 unit of taq dna polymerase ( promega , madison , wi , usa ) . the reaction mixtures were denatured at 94 for 10 min and subjected to 35 cycles of 1 min each at 94 , annealing at 56~60 for 1 min , extension at 72 for 1 min , and a final extension step of 7 min at 72. an amplified pcr product was separated on 1.5% agarose gel and purified with a dna purification kit ( core - one ; core - bio , seoul , korea ) according to the manufacturer 's instructions . the reactions were monitored using bigdye terminator cycle sequencing kits ( applied biosytems , foster city , ca , usa ) as indicated by the manufacturer and run on an abiprism 3130 automated dna sequencer ( applied biosystems ) as described previously . the rdna its sequence data were analyzed using the dnastar program ( dnastar inc . , madison , wi , usa ) and aligned by using the clustal w method . sequence data from the ribosomal its genes of didymella spp . were used to perform a preliminary phylogenetic analysis ; 16 its sequences were obtained from the genbank database ( http://www.ncbi.nlm.nih.gov/ ) ( table 2 ) . phylogenetic analysis based on parsimony for its , was obtained from the data by using neighbor - joining methods with mega ver . 4.0 software [ 14 , 15 ] , and the sequence distance was calculated using the tamura - nei , parameter model . bootstrap analysis was performed with 1,000 replications to determine the support for each clade . the megalign program ( dnastar inc . ) was used to compare the percentage sequence identity from the jeonbuk isolate with that of other didymella spp . in genbank . d. bryoniae spreads during the rainy season ( july to august ) . in early july , less than 5% of the plants examined were infected ( 15 of 300 plants ) ; however , in early august , 18.4% of the plants were infected . gummy stem blight initially attacks the main stem and then spreads to the branches of the muskmelon . disease symptoms include stem necrosis , gummy exudation , angular water - soaked lesions on the leaves , and rotten fruit . the weight of the sticky grayish - brown mucus that oozes from the infected part of the plant eventually causes the branches of the plant to break down ( fig . 1a and 1c ) . after some time , grayish - brown mucus turns dark brown in color ( fig . black pycnidia were observed on the stems , leaves , and fruit of the muskmelon . a preliminary identification of the fungi was based on the content of conidia and conidiophores , as described in the material and methods section . the mycelia of d. bryoniae from the pda culture were white to dark gray on top and black on the bottom . after 15 days , the d. bryoniae produced a conidial mass with a white aerial mycelium at the center of colony ; few pycnidia were observed . after 10 days , the d. bryoniae colonies on the malt extract agar and v-8 juice agar varied in diameter from 8.5 to 10.5 cm and extended to the edge of the petri dish ( fig . 2a ) . the percentage of monosepta in the conidia of d. bryoniae ranged from 0% to 10% , and the mean length  width of the conidia in the pda was 70 (  0.96 )  32.0 (  0.15 ) m ( fig . our jeonbuk isolate had morphological characteristics similar to those reported by keinath et al . , lee , and somai et al . the cultures of d. bryoniae grown on pda produced olive to dark - green or black substrate mycelium and white and hairy aerial mycelium . compared with growth on pda and malt extract agar , growth on v-8 juice agar tended to occur more frequently in concentric zones for both groups . characteristics such as the size of the conidia and the percentage of septa have been used to study the taxonomy of the genus d. bryoniae and phoma spp . . the size of the conidia and the percentage of monosepta in d. bryoniae were about the same as described previously . some reports suggest that the presence of septae may not be a useful characteristic by which to distinguish the anamorph of d. bryoniae from phoma spp . therefore , additional morphological characteristics and more sensitive methods are needed to differentiate between these closely related species . total 17 rdna its sequence data for didymella spp . were compared and analyzed for similarities . a phylogenetic analysis of the jeonbuk isolate , which was isolated from the field of jbabres , eight species of didymella spp . the size of the entire rdna its region , which was amplified using primers its1 and its4 from a d. bryoniae isolate , was 500 bp and yielded 440 aligned nucleotide positions for all species included in the alignment . the nucleotide sequences in the rdna its region of the jeonbuk isolate were compared with those of the other d. bryoniae spp . the blast analysis showed nucleotide gaps of 1/494 , 2/492 , and 1/478 with identities of 485/492 ( 98% ) , 492/494 ( 99% ) , 491/494 ( 99% ) , and 476/478 ( 99% ) . data from the entire rdna its sequence was analyzed using the dnastar program and was aligned using the clustal w mode ( table 2 ) . the similarity in sequence identity between the rdna its region of the jeonbuk isolate and other d. bryoniae from blast searches of genbank was 100% and was 95.0% within the group . the nucleotide sequences in the rdna its region , obtained from pure cultures of the jeonbuk isolate , were 98.2~99.8% similar to those of isolates with the accession numbers ef160074 , ef160076 , ef107641 , ef107642 , and af297228 in genbank , which were similar to the deposited sequences from the d. bryoniae isolates ( table 2 ) . the phylogenetic analysis of species related to d. bryoniae revealed that d. bryoniae is a monophyletic group distinguishable from other didymella spp . , including ascochyta pinodes , mycosphaerella pinodes , m. zeae - maydis , d. pinodes , d. applanata , d. exigua , d. rabiei , d. lentis , d. fabae , and d. vitalbina . the phylogenetic analysis based on rdna its sequences clearly distinguished d. bryoniae and didymella spp . of the other 10 species , the d. bryoniae group had a bootstrap value of 97% , whereas the remaining species had a bootstrap value of 21~99% ( fig . the jeonbuk isolate was identified as d. bryoniae on the basis of morphological characteristics and a comparison of rdna its sequence data with other related data from genbank and previous studies . the nucleotide sequences in the rdna its region of the jeonbuk isolate were 98.2~99.8% similar to the sequences of d. bryoniae isolates in genbank . the phylogenetic analysis showed that d. bryoniae is a monophyletic group distinguishable from other didymella spp . conducted random amplified polymorphic dna ( rapd ) and its sequence analyses to distinguish d. bryoniae from phoma spp . shim et al .   also analyzed the genetic diversity of d. bryoniae on the basis of rapd profiles , which substantiated by sequence characterized amplified regions marker in korea . these results indicated that the genus d. bryoniae was not monophyletic , and d. bryoniae , d. pinodes , d. rabiei , d. lentis , and d. vitalbina were distinct based on a sequence analysis of its1 , its2 , and 5.8s rdna . additional studies are needed to develop biological and eco - friendly methods to control gummy stem blight in muskmelon to prevent damage caused by d. bryoniae . d. bryoniae spreads during the rainy season ( july to august ) . in early july , less than 5% of the plants examined were infected ( 15 of 300 plants ) ; however , in early august , 18.4% of the plants were infected . gummy stem blight initially attacks the main stem and then spreads to the branches of the muskmelon . disease symptoms include stem necrosis , gummy exudation , angular water - soaked lesions on the leaves , and rotten fruit . the weight of the sticky grayish - brown mucus that oozes from the infected part of the plant eventually causes the branches of the plant to break down ( fig . 1a and 1c ) . after some time , grayish - brown mucus turns dark brown in color ( fig . black pycnidia were observed on the stems , leaves , and fruit of the muskmelon . a preliminary identification of the fungi was based on the content of conidia and conidiophores , as described in the material and methods section . the mycelia of d. bryoniae from the pda culture were white to dark gray on top and black on the bottom . after 15 days , the d. bryoniae produced a conidial mass with a white aerial mycelium at the center of colony ; few pycnidia were observed . after 10 days , the d. bryoniae colonies on the malt extract agar and v-8 juice agar varied in diameter from 8.5 to 10.5 cm and extended to the edge of the petri dish ( fig . 2a ) . the percentage of monosepta in the conidia of d. bryoniae ranged from 0% to 10% , and the mean length  width of the conidia in the pda was 70 (  0.96 )  32.0 (  0.15 ) m ( fig . our jeonbuk isolate had morphological characteristics similar to those reported by keinath et al . , lee , and somai et al . the cultures of d. bryoniae grown on pda produced olive to dark - green or black substrate mycelium and white and hairy aerial mycelium . compared with growth on pda and malt extract agar , growth on v-8 juice agar tended to occur more frequently in concentric zones for both groups . characteristics such as the size of the conidia and the percentage of septa have been used to study the taxonomy of the genus d. bryoniae and phoma spp . . the size of the conidia and the percentage of monosepta in d. bryoniae were about the same as described previously . some reports suggest that the presence of septae may not be a useful characteristic by which to distinguish the anamorph of d. bryoniae from phoma spp . therefore , additional morphological characteristics and more sensitive methods are needed to differentiate between these closely related species . total 17 rdna its sequence data for didymella spp . were compared and analyzed for similarities . a phylogenetic analysis of the jeonbuk isolate , which was isolated from the field of jbabres , eight species of didymella spp . the size of the entire rdna its region , which was amplified using primers its1 and its4 from a d. bryoniae isolate , was 500 bp and yielded 440 aligned nucleotide positions for all species included in the alignment . the nucleotide sequences in the rdna its region of the jeonbuk isolate were compared with those of the other d. bryoniae spp . the blast analysis showed nucleotide gaps of 1/494 , 2/492 , and 1/478 with identities of 485/492 ( 98% ) , 492/494 ( 99% ) , 491/494 ( 99% ) , and 476/478 ( 99% ) . data from the entire rdna its sequence was analyzed using the dnastar program and was aligned using the clustal w mode ( table 2 ) . the similarity in sequence identity between the rdna its region of the jeonbuk isolate and other d. bryoniae from blast searches of genbank was 100% and was 95.0% within the group . the nucleotide sequences in the rdna its region , obtained from pure cultures of the jeonbuk isolate , were 98.2~99.8% similar to those of isolates with the accession numbers ef160074 , ef160076 , ef107641 , ef107642 , and af297228 in genbank , which were similar to the deposited sequences from the d. bryoniae isolates ( table 2 ) . the phylogenetic analysis of species related to d. bryoniae revealed that d. bryoniae is a monophyletic group distinguishable from other didymella spp . , including ascochyta pinodes , mycosphaerella pinodes , m. zeae - maydis , d. pinodes , d. applanata , d. exigua , d. rabiei , d. lentis , d. fabae , and d. vitalbina . the phylogenetic analysis based on rdna its sequences clearly distinguished d. bryoniae and didymella spp . of the other 10 species , the d. bryoniae group had a bootstrap value of 97% , whereas the remaining species had a bootstrap value of 21~99% ( fig . the jeonbuk isolate was identified as d. bryoniae on the basis of morphological characteristics and a comparison of rdna its sequence data with other related data from genbank and previous studies . the nucleotide sequences in the rdna its region of the jeonbuk isolate were 98.2~99.8% similar to the sequences of d. bryoniae isolates in genbank . the phylogenetic analysis showed that d. bryoniae is a monophyletic group distinguishable from other didymella spp . conducted random amplified polymorphic dna ( rapd ) and its sequence analyses to distinguish d. bryoniae from phoma spp . shim et al .   also analyzed the genetic diversity of d. bryoniae on the basis of rapd profiles , which substantiated by sequence characterized amplified regions marker in korea . these results indicated that the genus d. bryoniae was not monophyletic , and d. bryoniae , d. pinodes , d. rabiei , d. lentis , and d. vitalbina were distinct based on a sequence analysis of its1 , its2 , and 5.8s rdna . additional studies are needed to develop biological and eco - friendly methods to control gummy stem blight in muskmelon to prevent damage caused by d. bryoniae .