• Korean Journal Plant Pathology
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• v.14 no.4
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• pp.325-330
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• 1998

On the basis of the fact that the pycnidiospore of Botryosphaeria dothidea, the causal fungus of apple white rot is a typical water borne spore, a method for quantitative analysis of pycnidiospore dispersal from the warts produced on the diseased apple tree stem was developed. The warts on which cracks developed either on or around them were cut off at the base, and shaked in the water for 4hours at 2$0^{\circ}C$, in which condition the maximum number of spores were released. The volume of shaking solution was calculated as 1 ml per one wart. At the end of shaking, Trio, a household detergent was added to the shaking solution to the concentration of 0.1%, and shaked for additional 10 minutes at 35$^{\circ}C$ to take off the spores attached on the glass ware. One milliliter of the spore suspension thus prepared were passed through transparent membrane filter (pore size : 3.0 ${\mu}{\textrm}{m}$), and the spores attached on the filter were counted under a microscope ($\times$200) after staining them with lactophenol supplemented with aniline blue. The results thus obtained were statistically consistent when at least 30 warts were used simultaneously in single shaking. This method can be applicable in the elucidation of ecology of sporulation and spore dispersal, and also in the screening of the sporulation inhibitor which can be used in the control of the disease by reducing the inoculum density.

• The Plant Pathology Journal
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• v.15 no.6
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• pp.330-334
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• 1999

Applying the method of quantitative analysis of pycnidiospore from the detached warts produced by the infection of Botryosphaeria dothidea on apple stems, repeated productivity of spores within the detached warts, variations in the amount of spores within the detached warts, variations in the amount of spores by the length of induction time for sporulation, and the effects of temperature and moisture on the sporulation were investigated. In addition to these experiment, the changes in the state of spores within the pycnidia contained in the warts accompanied by the induction of sporulation and dispersal of spores were also investigated. When detached warts were kept in moist conditions, the sporulation and discharge of spores were also investigated. When detached warts were kept in moist conditions, the sporulation and discharge of spores could be repeated several times, and the amount of spores were almost constant after each repeat of sporulation induction and dispersal of spores in a given period. The fact that the pycnidia filled with spores were observed at considerable rates within the warts which were subjected to the shaking in the water to release spores indicated that the spores might never be released until the pycnidia were fully matured. From the high rate of empty pycnidia even in the warts which were kept in moist conditions for induction of sporulation, the pycnidiospores might be produced through the development of new pycnidia. A considerable amount of pycnidiospores were produced at $5^{\circ}$, and the sporulation was accelerated with the rise of temperature until $35^{\circ}$. When the warts were supplied with sufficient moisture, sporulation was further accelerated. The results obtained in these experiment will be applied in developing the method for assessing the inhibitory efficacies of fungicides on the sporulation of this fungus, with which a new control measure would be developed.

• 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.

• 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.

• Korean journal of applied entomology
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• v.16 no.4 s.33
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• pp.211-215
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• 1977

The Sporulation of Didymella bryomiae were observed under diurnal cycles of light/darkness of near ultraviolet light (NUV) and artificial daylight (ADL) and continous darkness in eight isolates growing on PDA and V-8 juice agar. Light stimulated pycindial and perithecial formation of this fungus on potato dextrose agar and V-8 juice agar. Sprulation was poor in darkness, but some isolates were able to produce pycnidia and perithecia in the absence of light. Perithecial formation was much better under artificial daylight (ADL) on V-8 juice agar than those grown under near ultraviolet light (NUV). In general, cultures grown on V-8 juice agar sporulated better than cultures grown on PDA under three setsof light condition. Most of the pycnidiospores obtained from each isolates of this fungus grown on PDA were non-septate and microtype, but macrotype of non-septate and uniseptate pycnidiospores were produced on V-8 juice agar. Pycnidiospore produced on V-8 juice agar were similar to those produced on the radicle of naturally infected seeds. The appearance of perithecia were quite distinctive from pycnidia. The mature perithecia were darker than pycnidia and whitish spore masses formed on the ostiole of perithecia.

• 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.11 no.1
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• pp.16-20
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• 2005

Several time-course experiments were carried out to understand seasonal development of melanose on yuzu trees at koehung area, Jeonnam province, during May to October. The occurrence of dead twigs, known as a source of infection, was much more in older trees, and from June to August, mostly in July. In the experiment of pycnidia development on dead twigs seasonally collected, the number of developed pycnidia was highest on July-collected dead twigs especially with the diameter of 1.1~1.5 cm. In the collection survey of disseminated pycnidiospores, although the collected number of pycnidiospores was affected with amount of precipitation, the number of observed pycnidiospores in rainwater was relatively high from June to August, with highest in early August in 1997 and late July in 1998. In the inoculation tests on 3-year-old trees and fruits in natural condition, disease occurrences were mostly affected on twigs by inocula treatment in June, and on fruits by inocula treatment in July, respectively.

• The Korean Journal of Mycology
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• v.34 no.1
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• pp.22-28
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• 2006

Attempts were made to control Diatrype stigma occurred on the bed-log of Shiitake using wood vinegar, Pinus koraiensis extract, Piper nigrum extract, and fungicides. Mycelial growth of D. stigma was inhibited completely at 35,000 ppm and no ascospore germinated at 25,000 ppm wood vinegar. Inhibition rates of Pinus koraiensis extract (200 ppm), and Piper nigrum extract (1,000 ppm) to ascospore germination were 98.9% and 95.9%, respectively. In fungicide selection, minimum inhibitory concentration (MIC) of benomyl, carbendazim, and thiabendazole ranged $0{\sim}0.4\;{\mu}g\;a.i/m{\ell}$. Difenoconazole at $0.08\;{\mu}g\;a.i/m{\ell}$ inhibited 98.9% of ascospore germination. Inhibition efficacy of fungicides was not highly variable among the low-, middle-, and high-temperature type strains of shiitake. Benomyl, carbendazim, thiabendazole and thiophanate-methyl could not suppress the mycelial growth of Shiitake. Tebuconazole at $0.4\;{\mu}g\;a.i/m{\ell}$ suppressed 80% of the mycelial growth and it was the highest inhibition rate among the fungicides. In field trials, wood vinegar, Pinus koraiensis extract, Piper nigrum extract, and fungicides were sprayed on the bed-logs before or after D. stigma produced pycnidia. Wood vinegar at 150,000 ppm concentration, showed control effect of 72.7% in the treatment before pycnidiospore formation. On the other hand, 70,000 ppm wood vinegar and 1,000 ppm of thiophanate-methyl showed control effects of 58.1% and 52.3% in the treatment after pycnidiospore formation.

• Korean journal of applied entomology
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• v.23 no.2 s.59
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• pp.82-88
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• 1984

Fruit rot and blister canker, a disease of apple occurring severely in Korea has been studied for correct identification of the syndrome In fruit and apple trees. Among the fungi isolated from blister cankers, rough barks or fruits showing rotting of 7 different host species were Botryosphaeria berengeriana (pycnidial stage. Dethiorella mali), Penicillium expansum and Alternaria sp. from apple rots and Phomopsis sp. from pear fruit rots. The most dominant isolates were B. berengeriana. Ten isolates of D. mali were grouped in to two conidial types based up mycelial growth rate, growth habits and mycelial coloration on PDA. None of 10 isolates was chromogenic. Pycnidia in apple stems, stromatic, dark brown, globose or subglobose and the measuring were $103.5-287.5{\mu}\times92.0-287.5\mu$. The pycnidia contained hyaline, nonseptate, fusiform conidia. The sizes of pycnidiospore of isolates obtained from apple twig were $4.3-7.2{\mu}\times20.0-31.5{\mu}(average\;5.9\times25.4\mu)$. Some conidia of this fungus from apple, pear, peach and ornamental cherry showed 1-,2-,3-septate before or during germination. Microconidia were observed in pycnidia on PDA and fruit lesion of inoculated host. Symptoms on leaves and fruits were contoured brown spots when inoculated. Wart-like protuberance were formed on the surface of apple and pear. Canker appeared on branches of peach and ornamental cherry inoculated.

• Current Research on Agriculture and Life Sciences
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• v.5
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• pp.119-126
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• 1987

This study was conducted to elucidate the most appropriate method to obtain auxotrophic mutants from Valsa ceratosperma, the causal fungus of apple canker, which may be used as a gene marker in detecting the transfer of the factors of avirulent strains to virulent strains. Among the 3 kinds of synthetic media tested, each have two formula for minimal and complete, the medium which has been used in study of Endothia parasitica (E. P medium) was turned out to be most appropriate for the growth of V. ceratosperma. A medium for single colony formation from pycnidiospore of this fungus was developed by adding 0.5% L - sorbose to the E. P minimal medium. The period of incubation in dark for preventing the photoreactivation after U. V irradiation was estimated as about 60hrs at which most of the spores become binucleate. Largest number of putative auxotrophs were obtained at about 50second of irradiation to the spores smeared on the medium for single colony formation, at which the survival rate of spores was 5 to 6 percent. With these method developed in this experiment, 161 isolates of putative auxotrophs were detected among which the nutrient requirement for 10 isolates were determined. Five out of 10 mutants were still virulent to apple tree and all but one could not sporulate.