samples of vegetation affected by the typical fusarium wilt were collected from several areas by cutting the infected part of the stem from the zucchini plants .
the infected tissues were surface - sterilized with a 1% naocl solution for 1min and rinsed 2~3 times with sterilized distilled water . after drying , we cultured the samples in 90-mm petri dishes containing potato dextrose agar ( difco , sparks , md , usa ) in the dark at 25 for 1 wk .
kacc47262 ) and used for the pathogenicity test and molecular analysis . for the pathogenicity test , 10 zucchini seedlings ( cv .
taeyang ) were inoculated with the pathogen by dipping the roots into a conidial suspension ( 2  10 conidia / ml ) .
another 10 seedlings serving as controls were processed similarly by dipping the roots into sterilized distilled water .
the plants were retained in a humid chamber for the first 48 hr and then maintained in the greenhouse with 80% relative humidity at 25  2. three days after inoculation , wilt symptoms started to develop in the plants .
this led to etiolation and vascular discoloration seven days post inoculation ; these symptoms were identical to those originally observed in the plastic greenhouse .
for morphological observation , a small piece of living tissue containing fungal structures was mounted in a drop of water .
bright - field and differential - interference contrast light microscopy ( zeiss ax10 microscope ; zeiss , jena , germany ) were used to observe the specimen .
colonies on potato dextrose agar were 35 mm in diameter and pinkish - white ; they felted with cottony and aerial mycelia within a week ( fig .
1f ) . in culture , microconidia formed abundantly on monophialides , while falcate macroconidia formed on ( usually abundant ) pale orange sporodochia .
the macroconidia were short , falcate to almost straight , thin - walled , and usually threeseptate .
generally , abundant microconidia were formed in false - heads on short monophialides on the hyphae and were hyaline , smooth , oval to ellipsoidal , aseptate , oneseptate , or very rarely two - septate , slightly constricted at the septa with a size range of 4~11  2.5~5.0 m ( fig .
differences in gene dna sequence have previously been used to support morphology - based identification of fusarium species .
further , phylogenetic analysis of dna sequences has been used successfully to distinguish and evaluate the genetic relationship among closely related fusarium species .
the dna sequences of the translation elongation factor 1 gene ( tef ) have been widely used for identification of fusarium species . to further confirm our tentative identification based on morphological characteristics ,
the complete internal transcribed spacer ( its ) region of ribosomal dna ( rdna ) and a partial sequence of tef were amplified using primers its1/its4   and ef-1/ef-2 , respectively .
the amplicons were separated by 1.5% agarose gel electrophoresis , followed by purification using a pcr purification kit ( core - one ; core - bio , seoul , korea ) .
reactions were sequenced using the bigdye terminator cycle sequencing kit ( applied biosystems , foster city , ca , usa ) and analyzed on an abi 3130 automated dna sequencer ( applied biosystems ) .
the identity of the isolates was established by comparing their its and tef sequences with those in the genbank database ( national center for biotechnology information [ ncbi ] us national institute of health , bethesda , md , usa ; http://www.ncbi.nlm.nih.gov/blast ) and fusarium i d ( http://isolate.fusariumdb.org/blast.php ) , respectively .
phylogenetic analysis of its and tef was conducted by neighbor - joining methods using mega version 6.0 , and the sequence distance was calculated based on the tamura - nei parameter model .
bootstrap analysis , with 1,000 replications , was performed to determine the percentage support for each clade .
the its and tef sequences from a representative isolate were deposited in genbank with the accession numbers kf372590 ( 535 bp ) and kf372592 ( 679 bp ) . a blast search of the its sequence in genbank yielded 100% nucleotide identity with the f. oxysporum ( kc577181 ) sequence ; in fusarium i d , the tef sequence was also identical ( fd-01376 ) . the phylogenetic trees generated using the neighbor - joining method grouped the three zucchini isolates , including jabres2 and jabres3 , in the same clade ( figs . 2 and 3 ) .
f. oxysporum - induced fusarium wilt in c. pepo has been reported worldwide , such as in mexico , poland , and greece . although there is a previous record of fusarium sp . on cucurbita sp .
, this is the first report of f. oxysporum causing fusarium wilt on zucchini in korea . because this plant is of great economic importance ,
this practice is one potential reason for the relatively rapid spread of these pathogens , which can also be transmitted via infected weeds or survive on decaying plant material in the soil as saprotrophic mycelia and chlamydospores . currently , the best control strategies against fusarium wilt in zucchini are crop rotation and soil disinfection .
hence , there is an urgent need to develop chemical and ecofriendly control methods for reducing the crop damage caused by these fungal pathogens .
 fusarium wilt of zucchini in jeonju , korea , was first noticed in may 2013 . symptoms included wilting of the foliage , drying and withering of older leaves , and stunting of plants . infected plants eventually died during growth . based on morphological characteristics and phylogenetic analyses of the molecular markers ( internal transcribed spacer rdna and translation elongation factor 1 ) , the fungus was identified as fusarium oxysporum . 
 pathogenicity of a representative isolate was demonstrated via artificial inoculation , and it satisfied koch 's postulates . to our knowledge , this is the first report of f. oxysporum causing wilt of zucchini in korea . 
Isolation of the fungi and pathogenicity test.
Identification and characterization of 