• The Plant Pathology Journal
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• 제19권5호
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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.

• 한국식물병리학회지
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• 제14권4호
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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.

• Animal cells and systems
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• 제9권2호
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• pp.75-78
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• 2005

Fungicides and antagonists were tested for their efficacy in the management of fungal pathogens of watermelon. The fungal species in different genera were isolated from the seeds of watermelon and their vulnerability was assessed against an array of chemicals and bioagents. Among the fungal pathogens, Fusarium species were effectively controlled by Bavistin. Topsin also showed the promising effects against all the fungal pathogens, and Dithane M-45 effectively controlled Didymella bryoniae. Seed treatment with antagonists like Trichoderma harzianum and T. viride improved the seed germination, seedling vigour and reduced the incidence of seed-borne fungal pathogens. Bavistin and Topsin among chemicals increased significantly the seed germination and vigour index. Trichoderma harzianum showed its efficacy against all Fusarium species and even stood effective than Captan and Blitox. However, Pseudomonas fIuorescens also showed promising effect against Didymella bryoniae over fungicides. Under field condition, Topsin and Dithane M-45 showed better yield than Bioagents.

• 한국식물병리학회:학술대회논문집
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• 한국식물병리학회 2003년도 정기총회 및 추계학술발표회
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• pp.130-130
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• 2003

Gummy stem blight pathogen is very difficult not only to monitor the inoculum levels prior to host infection, and also it is destructive and hard to control in field condition. We have applied RAPD technique to elucidate the genetic diversity of the genomic DNA of Didymella bryoniae and also to generate specific diagnostic DNA probe useful for identification and detection. The 40 primers produced clear bands consistently from the genomic DNA of twenty isolates of Didymella bryoniae, and two hundred seventy-three amplified fragments were produced with 40 primers. The combined data from 273 bands was analyzed by a cluster analysis using UPGMA method with an arithmetic average program of NTSYS-PC (Version 1.80) to generate a dendrogram. At the distance level of 0.7, two major RAPD groups were differentiated among 20 strains. RAPD group (RG) I included 8 isolates from watermelon except one isolate from melon. RAPD group (RG) IV included 12 isolates from squash, cucumber, watermelon and melon.. In amplification experiment with SCAR specific primer RG1F-RG1R resulted in a single band of 650bp fragment only for 8 isolates out of 20 isolates that should be designated as RAPD Group 1. However, same set of experiment done with RGIIF-RGIIR did not result in any amplified product.. Our attempts to detect intraspecific diversity of ITS region of rDNA by amplifying ITS region and 17s rDNA region for 20 isolates and restriction digestion of amplified fragment with 12 enzymes did not reveal polymorphic band. In order to develop RAPD markers for RGIV specific primer, a candidate PCR fragment( ≒1.4kb) was purified and Southern hybridized to the amplified fragment RGIV isolates. This promising candidate probe recognized only RGIV isolates

• The Plant Pathology Journal
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• 제17권6호
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• pp.367.5-368
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• 2001

• 한국균학회지
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• 제10권1호
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• pp.7-13
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• 1982

자연상태(自然狀態)에서 Didymella bryoniae에 이병(罹病)된 오이 및 호박 종자(種子)를 습지법(濕紙法)으로 처리(處理)하여 불완전(不完全) 세대(世代)와 완전세대(完全世代)의 특징(特徵)을 조사(調査)하고 Difco PDA, V-8쥬스한천(寒天) 및 water agar leaf medium에서의 특징(特徵)과 비교(比較) 하였다. 병든 종자(種子)가 발아(發芽)할 때 종피(種皮)나 배축(胚軸)에 형성(形成)된 Didymella bryoniae의 병포자(柄胞子)와 Difco PDA, V-8 juice한천(寒天) 및 WALM에 형성(形成)된 본균(本菌)의 병포자(柄胞子)는 형태적(形態的)으로 많은 차이(差異)가 나타난다. 즉 PDA에 형성(形成)되는 병포자(柄胞子)는 무색(無色) 소형단포자(小型單胞子)가 대부분이며, V-8 juice한천(寒天) 및 WALM에 형성(形成)되는 것은 대형단포자외(大型單胞子外)에 격막(隔膜)이 하나 있는 포자(胞子)를 형성하고 또 균주(菌株)에 따라서는 격막(隔膜)이 두개 있는 포자(胞子)를 형성하므로써 마치 병든 종피(種皮)나 배축(胚軸)에 형성(形成)된 것과 흡사하였다. 한편 자양각은 병든 종자(種子)의 표본(標本)에 따라 잘 형성(形成)되는 것도 있었으나 대부분(大部分)의 표본(標本)에서는 형성(形成)되지 않았다. 외부형태적(外部形態的)인 특징(特徵)은 색깔이 진한 흑색(黑色)이며 유두상각공(乳頭狀殼孔) 위에 백색(白色)의 포자괴(胞子塊)가 형성(形成)되는 특징(特徵)을 가지고 있어서 쉽게 병자각(柄子殼)과 구별(區別)할 수 있었다. 불완전(不完全) 세대(世代)인 병자각(柄子殼)이나 병포자(柄胞子)와 같이 자양각의 각방(殼房)과 포자(胞子)의 크기에는 큰 변이(變異)가 없었다.

• 식물병연구
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• 제22권2호
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• pp.72-80
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• 2016

D. bryoniae에 의해 발생되는 덩굴마름병은 세계적으로 수박 재배에 주요한 병으로 알려져 있으며, 본 실험은 D. bryoniae에 대한 수박의 효율적인 저항성 검정법을 확립하기 위해 수행하였다. 함안 지역에서 전형적인 덩굴마름병 병징을 보이는 수박으로부터 GBS3 균주를 분리하였고 ITS 영역의 염기서열 분석을 통해 GBS3 균주는 D. bryoniae로 동정되었다. 다양한 조건에서의 포자 형성량과 형성된 포자의 5가지 생육 시기의 수박 유묘에 대해 병원력 차이를 조사하고, 이로부터 저항성 검정을 위한 간편한 접종원 대량 생산 방법을 확립하였다. 시판 중인 수박 22개 품종의 GSB3 균주에 대한 저항성 정도를 확인하고, 그 결과로부터 저항성 정도가 다른 수박 4개 품종을 선발하였다. 그리고 접종원 농도, 습실 처리 기간, 접종 후 재배온도 등의 발병 조건에 따른 이들 품종의 덩굴마름병 발생을 조사하였다. 이들 결과로부터 덩굴마름병에 대한 효율적인 수박 저항성 검정 방법으로 본엽 2엽이 충분히 전개된 수박 유묘에 $5.0{\times}10^5spores/ml$ 농도의 D. bryoniae 포자현탁액을 분무접종하고, $25^{\circ}C$ 습실에서 48시간 동안 배양 후에 항온항습실($25^{\circ}C$, 상대습도 80%)로 이동하여 하루 12시간씩 광을 처리하면서 재배하고, 접종 3-4일 후에 접종한 잎의 병반면적률을 조사하는 것을 제안하고자 한다.

• 한국식물병리학회:학술대회논문집
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• 한국식물병리학회 2005년도 한국식물병리학회 임시총회 및 춘계학술발표회
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• pp.45.2-46
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• 2005

• Journal of Plant Biotechnology
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• 제45권3호
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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.

• The Plant Pathology Journal
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• 제17권3호
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• pp.174-179
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• 2001

The plant growth-promoting Pseudomonas strains, WR8-3 (Pseudomonas fluorescens), WR9-11 (Pseudomonas sp.) and WR9-16 (P.putida), which induced resistance systematically in watermelon to gummy stem rot were investigated on their induced systemic resistance(ISR)-related characteristics. The pyoverdine production was repressed in the standard succinate medium by increasing the concentration of $\textrm{FeCL}_3$. But the iron-binding ability on chrome azurol S agar media (CAS) was observed only in the strains, WR8-3 and WR9-16. When the two strains were mutated, the resulting iron-binding siderophore-negative mutants, WR8-3m and WR 9-16m, failed to promote the growth of watermelon and to induce resistance. The strains, WR8-3 and WR 9-16, slightly inhibited the growth of Didymella bryoniae at a low concentration of $\textrm{FeCL}_3$ on Kong's medium B, but not to exert control dffect. The strain WR9-11 showed antagonism in the concentration of $\textrm{FeCL}_3$ from 0 to $1,000\mu\textrm{M}$. When the crude lipoplysaccharide of each strain was treated in the rhizosphere of watermelon, mean lesion area was similar to that of the untreated control. The strains, WR9-11 and WR9-16 produced some level of hydrogen cyanide (HCN). Salicylic acid production was not detected in all of the strains.