• Research in Plant Disease
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• v.17 no.2
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• pp.169-176
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• 2011

Phytophthora katsurae is the fungus responsible for chestnut ink disease. The objectives of this study were to determine if a single-strand conformation polymorphism (SSCP) analysis of rDNA-ITS region, elongation factor 1 alpha gene and ${\beta}$-tubulin gene could be used for rapid identification and genetic diversity of P. katsurae, and to assess the potential use of the SSCP technique as a diagnostic tool for P. katsurae. Each regions amplified by PCR using primers designed to overlap the genus Phytophthora were characterized for the Phytophthora species. PCR products were denatured and electrophoresed for SSCP analysis. P. katsurae isolates showed an unique pattern in SSCP analysis and were easily distinguished from other Phytophthora species used as the control. This indicates that SSCP analysis is an useful technique for distinguishing Phytophthora species from genetically close relatives, and show that the SSCP analysis of each region is an efficient detection tool for P. katsurae. But PCR-SSCP analysis of single-gene may have difficulty in distinguishing P. katsurae from other Phytophthora species. Therefore, PCR-SSCP analysis of multi-genes can be useful for rapid and effective identification of P. katsurae.

• Research in Plant Disease
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• v.18 no.2
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• pp.73-79
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• 2012

Phytophthora katsurae is a fungal pathogen responsible for chestnut ink disease. We designed two duplex primer sets (SOPC 1F/1R+KatI 3F/5R, SOPC 1-1F/1-1R+KatI 3F/5R) to detect P. katsurae. SOPC 1F/1R and SOPC 1-1F/1-1R primer pairs were designed for sequence characteristic amplification regions (SCAR) marker, and KatI 3F/5R primer pair was used for P. katsurae-specific primer designed from internal transcribed spacer (ITS) region. To assess the sensitivity of duplex PCR, genomic DNA was serially diluted 10-fold to make the final concentrations from 1 mg/ml to 1 ng/ml. The sensitivity for two primer sets were 1 ${\mu}g/ml$ and 100 ng/ml, respectively. To find detection limits for zoospores of P. katsurae, each zoospore suspension was serially diluted 10-fold to make the final concentrations from $1{\times}10^6$ to $1{\times}10^2$ cells/ml, and then DNA was extracted. The limits of detection for all of two primer sets were $1{\times}10^5$ cells/ml. All of two primer sets were specific to P. katsurae in PCR detection and did not produce any P. katsurae-specific PCR amplicons from other 16 Phytophthora species used as the control. This study shows that duplex PCR using two primer sets might be a useful tool for rapid and efficient detection of P. katsurae.

• Journal of Forest and Environmental Science
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• v.23 no.1
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• pp.65-71
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• 2007

In early 2000's, about six Phytophthora species have been newly described leading mortality on coniferous and broad-leaved trees in forests. Also, some species of Phytophthora are responsible for ink disease in chestnut plantation near or within forests. Similar symptoms of ink disease were appeared in some areas of Kyungnam and Jeonnam providences in 2005, and the pathogen was isolated using Phytophthora- selective medium in 2006. Morphological and genetic analysis were performed to identify the isolate. Also, the pathogenicity was conducted to complete $K\ddot{o}ch^{\prime}s$ postulate and compare susceptibility among chestnut cultivars. The molecular analysis between P. katsurae and P. hevae were performed with the isolates obtained from different countries including Korea or the sequences downloaded from Phytophthora webpage. The result showed that the isolated pathogen from chestnut was P. katsurae. There is no report of P. katsurae in Korea until now. P. katsurae was re-isolated from inoculated chestnut cultivars. Also, there was a slight difference in susceptibility among chestnut cultivars. The rDNA sequence of our isolate showed 100% similarity with sequence of the isolate cultured from Japan and New Zealand.

• Mycobiology
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• v.41 no.2
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• pp.86-93
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• 2013

Sequence characterized amplified region (SCAR) markers are one of the most effective and accurate tools for microbial identification. In this study, we applied SCAR markers for the rapid and accurate detection of Phytophthora katsurae, the casual agent of chestnut ink disease in Korea. In this study, we developed seven SCAR markers specific to P. katsurae using random amplified polymorphic DNA (RAPD), and assessed the potential of the SCAR markers to serve as tools for identifying P. katsurae. Seven primer pairs (SOPC 1F/SOPC 1R, SOPC 1-1F/SOPC 1-1R, SOPC 3F/SOPC 3R, SOPC 4F/SOPC 4R, SOPC 4F/SOPC 4-1R, SOPD 9F/SOPD 9R, and SOPD 10F/SOPD 10R) from a sequence derived from RAPD fragments were designed for the analysis of the SCAR markers. To evaluate the specificity and sensitivity of the SCAR markers, the genomic DNA of P. katsurae was serially diluted 10-fold to final concentrations from 1 mg/mL to 1 pg/mL. The limit of detection using the SCAR markers ranged from $100{\mu}g/mL$ to 100 ng/mL. To identify the limit for detecting P. katsurae zoospores, each suspension of zoospores was serially diluted 10-fold to final concentrations from $10{\times}10^5$ to $10{\times}10^1$ zoospores/mL, and then extracted. The limit of detection by SCAR markers was approximately $10{\times}10^1$ zoospores/mL. PCR detection with SCAR markers was specific for P. katsurae, and did not produce any P. katsurae-specific PCR amplicons from 16 other Phytophthora species used as controls. This study shows that SCAR markers are a useful tool for the rapid and effective detection of P. katsurae.

• The Plant Pathology Journal
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• v.25 no.2
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• pp.121-127
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• 2009

Since July 2005, survey of chestnut ink disease was carried out in chestnut stands located at southern parts of Korea. Dead chestnut trees showing inky ooze on necrotic trunks were found in two different locations. In order to isolate and identify the causal fungus, infected tissues and soil samples around dead or dying trees were collected and placed on Phytophthora-selective medium. Rhododendron and chestnut tree leaves were used as a bait to isolate the fungus from soil samples by attracting zoospores in soil suspensions. On V-8 culture medium, the isolates produced homothallic oogonia with protuberances ($34.0-46.2{\times}21.9-26.7{\mu}m$) abundantly, but did not produced sporangia. Mass production of sporangia was possible by immersing agar plugs with actively growing mycelium in the creek water at $18^{\circ}C$ for 3 days. Sporangia were papillate, and ovoid to obpyriform ($17.0-38.9{\times}14.6-29.2{\mu}m$) in shape. Comparison of the ITS sequences revealed that the isolates had 100% identity to the P. katsurae isolates from Japan and New Zealand and 99.6% identity to other P. katsurae isolates. All of the examined isolates from Korea were completely identical to each other in ITS sequence. Numerous sporangia were formed in filtered as well as unfiltered creek water, but no sporangia formed in sterilized distilled water. Light induced sporangia formation, but has no influence on oospore formation. Amendments of ${\beta}$-sitosterol in culture media have no significant effect on mycelial growth but significantly stimulate oospore and sporangia formation.