인삼뿌리썩음병균, Cylindrocarpon destructans(Zins.) Scholten을 접종한 인삼절편으로 부터 얻은 섬유소분해효소 및 펙틴질분해광소의 역가는 그 농도, 시간에 비례하였다. Cellulase (Cx), endo-polygalacturonase(endo-PG), endo-poiymethylgalacturonase (endo-PMG), exo-polygalacturonase (exe-PG)와 exo-polymeth-ylgalacturonase(exo-PMG)의 역가는 접종후 4 일째 최대였으며 endo-PG와 endo-PMG는 1일 및 2일째는 전혀 검출되지 않았으나 exo-PG와 exo-PMG는 검출되었다. 접종후 6 일째에는 모든 펙틴질분해효소는 활성을 완전히 잃었으나 섬유소분해효소는 높은 역가을 유지하였다. Cx, endo-PG, endo-PMG, exo-PG 및 exo-PMG의 최적 pH는 각각 6.0, 5.5, 8.0, 7.0-7.5, 8.5이였다. Cx, endo-PG, endo-PMG, 및 exo-PG의 최적온도는 각각 exo-PMG 였다. 섬유소분해효소는 공시한 16개 이온중에서 0.05M 만이 억제하였다. 펙틴질분해효소는 이온에 따라 상이하였으나 과 이 가장 현저히 억제하였다. 공시한 8개 농약중에서 어느 것도 유효성분으로는 모든 효소작용을 억제하지 못했으며 exo-PG만은 모든 농약의 의하여 상당히 억제되었다. 그 중에서 Difolatan은 모든 펙틴질분해효소를 가장 잘 억제시켰다. 과 은 펙틴질분해효소를 거의 억제시켰으나 섬유소분해효소는 같은 농도에서 각각 에 이르렀다. Formalin은 exo-PG와 endo-PMG를 완전 억제시켰으나 다른 효소 특히 Cx는 그렇지 못했다. Difolatan은 모든 효소를 이상 억제하였으나 Cx는 그 이하였다. C. destructans가 분필하는 섬유소분해효소 및 펙틴질분해효소는 인삼뿌리썩음병과 밀접한 관계를 가지고 있으며 이 병을 효과적으로 방제하기 위하여 억제되어야 하겠다.

Background : Development of new real-time PCR diagnosis method for simultaneous diagnosis of Cylindrocarpon destructans and Fusarium solani, causative fungi of ginseng root rot disease. C. destructans and F. solani are known to be the major pathogens of ginseng root rot disease. Root rot caused by these pathogens is a disease that is difficult to control because the disease progresses slowly and it is difficult to diagnose early and even when symptoms of plant seeding are present, the disease is already spread in the roots. Diagnostic methods to detect the presence or absence of ginseng roots rot fungi in soil before ginseng cultivation are currently being used as a method for controlling. However, commercialized soil extraction kits and PCR diagnostics have cost, diagnostic time, and single diagnostic problems, and need to develop new diagnostic methods. Methods and Results : Primers and probes in the beta-tubulin 2 gene were designed for species-specific detection. In silico analysis, the detection rate of C. destructans was 100% and the detection rate of F. solani was 95%. The multiplex real time PCR optimization conditions including the internal control were established. The analytical sensitivity using positive samples was 10 copies/㎕ for C. destructans and 10 copies/㎕ for F. solani. As a result of performance comparison test with conventional PCR diagnosis methods, it was confirmed that the developed multiplex real time PCR method has the same or better performance in terms of sensitivity. In the developed soil extraction kit, the extraction time was reduced and the extracted DNA quality was improved, compared to the used soil extraction kit. Conclusion : From the above results, we expect that the developed C. destructans / F. solani multiplex real time PCR diagnosis method and soil extraction kit will be useful for real-time monitoring of ginseng root rot pathogenic fungi in the soil of ginseng cultivation area and diagnosis of suitability of ginseng cultivation area.

Background: Cylindrocarpon destructans (Zins) Scholten is an important pathogenic fungus that causes ginseng root rot in many ginseng growing areas in China. Although C. destructans have been studied worldwide, research on its chemotaxis towards ginseng (Panax ginseng C. A. Meyer) root exudates in the rhizosphere remains limited.Methods and Results: In this study, we collected ginseng root exudates with three different polarities from three-year-old ginseng roots, and performed chemotaxis and spore germination assays to investigate the ability of these exudates to induce the response in C. destructans. The results showed that, compared with other conditions, when C. destructans cultivated at 20°C and a pH of 6 exhibited a strong positive chemotactic response toward 2 ㎎/ℓ aqueous phase, 20 ㎎/ℓ butanol phase, and 0.2 ㎎/ℓ petroleum ether from ginseng root exudates, the chemotactic moving indexes were 0.1581, 0.1638 and 0.1441, respectively. In addition, the spore germination rate with optimal chemotactic parameters were 48%, 53%, and 41% in the aqueous phase, butanol phase and petroleum ether groups, respectiviely, which were significantly higher than that in the control group (23%) (p < 0.05). The mycelial growth rate with optimal chemotactic parameters increased with culture time, and the maximum growth rates in the aqueous phase, butanol phase and petroleum ether groups were 0.425, 0.406 and 0.364 respectively, on the 4th day. The optimal chemotactic parameters were 39.73 ㎎/50 ㎎/ℓ , 48.93 ㎎/50 ㎎/ℓ , and 31.43 ㎎/50 ㎎/ℓ , in aqueous phase, butanol phase and petroleum ether respectively, from ginseng root exudates, compared with 5.5 ㎎/50 ㎎/ℓ , in the control group.Conclusions: The present study revealed that certain ginseng root exudates containing chemical attractants act as nutritional sources or signals for C. destructans and support its colonization of ginseng roots.

Background : Ginseng (Panax ginseng C.A. Meyer) is one of the most important medicinal plants in Korea, but its yields are often reduced by a variety of root pathogens. The root rot of ginseng is a destructive soil-borne disease caused by Cylindrocarpon destructans (teleomorph: Ilyonectria radicicola). To monitor contamination with C. destructans in ginseng harvested in 2015 were sampled from 57 different growing fields. The spore number of C. destructans was quantified by use of a specific primers and selective media (radicicol) in soils of ginseng fields. Methods and Results : The ginseng samples were surface-sterilized and placed on potato dextrose agar plates for 7 day incubation at 20℃. Emerging fungal colonies were counted primarily based on colony and conidia morphology. Further species level identification was confirmed by ITS rDNA sequencing. For quantification of the soil-borne C. destructans, the genomic DNA was extracted from the soil using a NucleoSpin soil kit (MN, Germany). Density of C. destructans was determined by species specific real time PCR (qPCR). The qPCR was completed by running a melting curve analysis. Conclusion : The C. destructans associated with root rot disease of ginseng were detected in more than 60% in pyeongtaek-1, pochenon-1, jecheon-1, chungju-1 and jinan-4. As results of the study, the correlation between pathogen density and identification clearly clarified in the soil.

C. destructans는 인삼에서 가장 문제가 되고 있는 뿌리섞음병을 유발하는 매우 중요한 미생물이다. 현재까지 정상적인 인삼포장이나 폐포지에서도 이 병원균의 농도를 조사할 만한 방법이 없어 이를 쉽게 조사함으로서 인삼 예정지 관린시 도움을 줄 수 있는 새로운 방법이 절실이 요구되고 있다. 본 연구에서는 nested PCR이란 분자생물학적 방법을 이용하여 효과적으로 매우 낮은 농도의 C. destructans을 검출할 수 있는 방법을 개발하였다. 2개의 universal ITS primers(ITS5F와 ITS4R)을 사 용 하 여 Cylindrocarpon spp.의 rDNA로부터 ITS영역을 증폭하였다. 이어 C. destructans의 specific primer(Nest 1 과 Nest 2)을 사용하여 최적의 PCR조건으로 재증폭시켜 밴드를 확인하였다. 또한 이런 2번의 과정을 4개의 primer를 동시에 사용함으로서 한번에 확인할 수 있는 방법을 개발하였으며 이에 따른 PCR조건도 확립하였다. 따라서 본 방법에 의해서 인삼포장의 토양에서 채취된 매우 낮은 농도의 wild type C. destructans spore로부터 성공적으로 positive band을 확인함으로써 추후 인삼포장의 선정 및 4년생에서 6년까지(홍삼포) 재배기간등의 예측에 활용 될 것으로 생각된다.