• Korean Journal Plant Pathology
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• v.13 no.2
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• pp.105-112
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• 1997

Didymella bryoniae, gummy stem blight fungus of cucurbits, has been known not to produce its pycnidium in vitro without irradiation. Various methods for producing pycnidiospores of the fungus as an inoculum have been used. However, those methods have not been verified in terms of efficiency of the productivity, activity and synchronous maturation of the inoculum. Therefore, a pycnidiospore production method in vitro that is highly reliable and reproducible has to be developed to obtain a large amount of inoculum for screening disease resistant varieties or effective fungicides. Here we standardized a mass-production technique for pycnidiospores of D. bryoniae in vitro by comprehensively finding the optimal conditions such as kinds and thickness of cultural medium, growing temperature, and quality and duration of irradiation as well as examining the activity and pathogenicity of the pycnidiospores reproduced. In brief, mycelial colony on the PDA plate was cultured at 26$^{\circ}C$ for 2 days under the darkness, and then the plate was irradiated under the UV light (12 hr/a day) for 2~3 days at the same temperature(26$^{\circ}C$). Two days after UV irradiation, a great number of pycnidia was simultaneously formed. This plate was subjected to darkness again for 4~5 days to mature pycnidiospores. We could obtain a large amount of inoculum that is synchronously matured in a short period of time through the above procedures.

• Plant Disease and Agriculture
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• v.5 no.1
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• pp.20-26
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• 1999

Ultraviolet light is required for the sporulation of Didymella bryoniae, a gummy stem blight fungus in cucurbits such as watermelon, melon, oriental melon, cucumber and pumpkin. In this experiment, the upper limit of wave length for the production of pycnidia of D. bryoniae was 365 nm - 375 nm. Two plastic houses were covered with either common transparent film (wave length longer than 225 nm is transmitted) or UV-absorbing film ( wave lenght shorter than 388 nm is absorbed). In both houses, seedlings inoculated with D. bryoniae were placed in the center of the house at 30 days after transplantation of watermelon (cv. Whanhoseong), and the disease incidences between the houses were compared until 80 days after transplantation. The number of disease lesions and incidence of pycnidia-producing lesions under the UV-absorbing film were reduced by 90% and 80%, respectively, compared to the common transparent film. The internode lengths of plants grown in the two houses were not significantly different, but the plants grown under the UV-absorbing film had longer vines and more leaves than plants under the common transparent film. However, fruit characters such as weight, length, width, rind thick and brix, were not different between the two houses. Occurrence of aphids was reduced in the UV-absorbing film, but those of mites or diseases (powdery mildew and sooty mold) were not different between the houses. These results suggest that disease incidence of gummy stem blight of watermelon in the greenhouse can be controlled by the use of UV-absorbing film.

• Journal of Plant Biotechnology
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• v.45 no.3
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• pp.217-227
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• 2018

Gummy stem blight (GSB) is one of the most destructive and economically important, soil borne diseases of melon caused by the ascomycete fungus, Didymella bryoniae throughout the world. In Korea, however, no GSB resistant genotype has been reported yet. The study aimed to identify GSB resistant melon germplasm. We screened a total of 60 genotypes including 16 lines and 44 melon cultivars collected from USA and Korea. Among the 16 melon lines, four lines including 'PI482399', 'PI140471', 'PI136170' and 'PI420145', and two Korean cultivars viz. 'Asia Papaya' and 'Supra' showed complete resistance. We were aware that both genotypic and environmental variations could influence the phenotypic screening of resistance and susceptibility. We therefore, further assessed all genotypes using 20 SSR markers. The SSR marker 'CMCT505' linked to Gsb1 in chromosome 1 perfectly grouped resistant and susceptible lines indicating that resistance is probably due to the presence of Gsb1 gene. Cloning and sequencing of resistant and susceptible Gsb1 amplicons showed that there were 32-bp deletions in resistant line and 39-bp deletions in resistant cultivar compared to susceptible one. Thus, the resistant melon lines and cultivars identified in this study could be recommended for the melon breeding program. Furthermore, the SSR marker 'CMCT505' which is tightly linked with Gsb1 could be used for molecular screening of melon germplasm.

• Korean Journal Plant Pathology
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• v.14 no.4
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• pp.339-344
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• 1998

Since the leaf inoculation procedures are time-consuming and require considerable growth chamber space, a rapid dioassay method for screening of pathogenicity of Didymella bryoniae, a casual agent of gummy stem blight in watermelon, was established in this paper. The method produced reliable results within 8 days ( 5 days for growing seedlings and 3 days for rapid disease response in the seedlings). After contaminants in the root of 4~5 day-old seedlings had been washed using sterilized water, 5 seedlings were dipped into a vial containing 12 ml of conidial suspension (106 cells/ml). After the vials were placed in a growth chamber (22$^{\circ}C$, RH 50%, 14hr light/10hr darkness) for 3 days, susceptibility and resistance of cultivars were determined by the degree of disease response on cotyledon. The result of obtained by the dip-inoculation method was well coincided with the results by the leaf inoculation procedures and the result that had been observed for several years in the field. Screening of collected watermelon cultivars by the dip-inoculation method revealed that all the 21 domestic cultivars collected were susceptible and only 3 foreign cultivars (PI 189225, PI 482322 and IT 188207) were resistant among 18 cultivars A cucumber cultivar (Marketer) and bitter cucumber were proven to be resistant against the D. bryoniae among 8 other different cucurbits tested. The SDS-PAGE patterns of total proteins from a susceptible (Keumcheon) and a resistant (PI 189225) watermelon cultivars were compared 0, 12, 24 and 36 hrs after inoculation. The amounts of two distinct protein bands (24 kDa and 70 kDa) were gradually increased after inoculation in both cultivars.

• The Plant Pathology Journal
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• v.19 no.5
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• pp.260-265
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• 2003

Gummy stem blight caused by Didymella bryoniae occurs exclusively in cucurbits. This fungus has been known not to produce its pycnidium in vitro unless irradiated. In this study, cultural conditions for the mass-production of pycnidiospore by Metal Halide (MH) lamp irradiation were maximized. The mycelia were cultured at $26^{\circ}C$ on PDA for 2 days under dark condition, and then the plate was illuminated with MH lamp continuously for 3-4 days at $26^{\circ}C$. Results show that a great number of pycnidia were simultaneously formed. The pycnidiospores produced were then used as immunogen. Fusions of myeloma cell (v-653) with splenocytes from immunized mice were carried out. Two hybridoma cell lines that recognized the immunogen D. bryoniae were obtained. One monoclonal antibody (MAb), Dbl, recognized the supernatant while another MAb, Db15, recognized the spore. Two clones were selected which were used to produce ascite fluid of the two MAb, Dbl and Db15, the immunotypes of which were identified as IgG1 and IgG2b, respectively. Titers of MAb Dbl and MAb Db15 were measured and the absorbance exceeded 0.5 even at a $10^{-5}$ dilution. The MAbs reacted positively with D. bryoniae but none reacted with other viral isolates, Cucumber mosaic virus and Cucumber green mottle mosaic virus. Sensitivity of MAb was precise enough to detect spore concentration as low as $10^{-3}$/well by indirect ELISA. Characterization of the MAbs Dbl, Db15 antigen by heat and protease treatments, which suggests that the epitope recognized by these two MAbs was glycoprotein.

• Research in Plant Disease
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• v.22 no.2
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• pp.72-80
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• 2016

Gummy stem blight, caused by the fungus Didymella bryoniae, is major disease of watermelons worldwide. The objective of the present study was to establish an efficient screening system to identify watermelon resistant to D. bryoniae. An GSB3 isolate was prepared from a watermelon plant showing typical symptoms of gummy stem blight in Haman-gun and identified as D. bryoniae based on molecular analysis of internal transcribed spacer sequence. A simple mass-production technique of inoculum was developed based on spore production of D. bryoniae GSB3 under several incubation conditions and their virulence on watermelon plants. Resistance degrees of 22 commercial watermelon cultivars to the GSB3 isolate were evaluated. Among them, four watermelon cultivars showing different degree of resistance response were selected for further study. Development of disease on the cultivars according to various conditions including inoculum concentrations, incubation periods in dew chamber, and incubation temperatures was investigated. From the results, we suggest an efficient screening method for resistant watermelon cultivars to gummy stem blight. Seeds of watermelon cultivar are sown and grown in a greenhouse until plant stage of 2-fully expanded leaves. Seedlings are inoculated with D. bryoniae by spraying spore suspension of the fungus at a concentration of $5.0{\times}10^5spores/ml$. The infected plants are incubated in humidity chamber at $25^{\circ}C$ for 48 hours and then transferred to a growth chamber at $25^{\circ}C$ and 80% relative humidity with 12-hour light a day. Three to four days after inoculation, disease severity of the plant are measured using percentage of infected leaf area.

• Proceedings of the Korean Society of Plant Pathology Conference
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• 2003.10a
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• pp.133.1-133
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• 2003

Gummy stem blight, caused by Didymella bryoniae occurs exclusively on cucurbits. This fungus has been known not to produce its pycnidium in vitro unless irradiated. Through this study, we optimized cultural conditions for mass-production of pycnidiospore by Metal Halide Lamp irradiation. In brief, the mycelial was cultured at $26^{\circ}C$ on PDA, for 2 days under the darkness, and then the plate was illuminated with MH lamp continuously for 3-4 days at $26^{\circ}C$, a great number of pycnidia was simultaneously formed. Thus produced pycnidiospores were used as immunogen. From fusions of myeloma cell (v-653) with splenocytes from immunifed mice were car ried out. And, two hybridoma cell lines that recognized the immunogen Didymella bryoniae were obtained. One Monoclonal Antibody, Db1, recognized the supernatant and the other monoclonal antibody, Db15, recognized the spore. Two clones were selected which were used to produce ascite fluid two MAb Db1 and Db15, were immunotyped and identified as IgG1 and IgG2b, respectively. Titer of MAb Db1 and MAb Db15 was measured absorbance exceeded 0.5 even at a $10^{-5}$ dilution. The MAbs reacted positively with Didymella bryoniae but none reacted with other of fungi and CMV, CGMMV Sensitivity of MAb was precise enough to detect spore concentration as low as $10^{3}$ well by indirect ELISA characterization of the MAb Db1, Db15 antigen by heat and protease treatments show that the epitope recognized by the MAb Bb1, Db15 were a glycoprotein.