Abstract
Ustilaginoidea virens is the causal agent of false smut disease of rice. In this study, we developed a real-time polymerase chain reaction (PCR) assay to clarify the relationship between false smut occurrence on rice and quantification of U. virens from soil in Japan. The method here described is sensitive, detecting less than 50 fg of pathogen DNA, and specific to the nuclear ribosomal DNA for U. virens when tested across 27 rice-pathogenic fungi and bacteria, 26 other fungi and bacteria and four plant species. As few as eight chlamydospores of U. virens per gram soil were detected when added to sterilized Gley and Ando soils. The real-time PCR assay for the soil samples was at least 100-fold more sensitive than the conventional and nested-PCR assays tested. By quantification of U. virens with real-time PCR using DNA extracted from naturally contaminated Gley soils and visual assessment of the disease in agricultural fields, a linear correlation between cycle threshold (CT) values and the number of false smut balls was revealed. Therefore, this specific quantitative assay could be a useful tool for optimization of disease control strategies, and for studying the ecology of U. virens.
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References
Ashizawa, T., & Kataoka, Y. (2005). Detection of Ustilaginoidea virens in rice panicles before and after heading in the field using nested-PCR technique with species-specific primers. Japanese Journal of Phytopathology, 71, 16–19.
Bischoff, J. F., Sullivan, R. F., Kjer, K. M., & White, J. F., Jr. (2004). Phylogenetic placement of the anamorphic tribe Ustilaginoideae (Hypocreales, Ascomycota). Mycologia, 96, 1088–1094.
Brooks, S. A., Anders, M. M., & Yeater, K. M. (2009). Effect of cultural management practices on the severity of false smut and kernel smut of rice. Plant Disease, 93, 1202–1208. doi:10.1094/PDIS-93-11-1202.
Cullen, D. W., Lees, A. K., Toth, I. K., & Duncan, J. M. (2001). Conventional PCR and real-time quantitative PCR detection of Helminthosporium solani in soil and on potato tubers. European Journal of Plant Pathology, 107, 387–398.
Cullen, D. W., Lees, A. K., Toth, I. K., & Duncan, J. M. (2002). Detection of Colletotrichum coccodes from soil and potato tubers by conventional and quantitative real-time PCR. Plant Pathology, 51, 281–292.
Deng, G. S. (1989). Present status of research on false smut in China. Plant Protection Beijing, 15, 39–40.
Ikegami, H. (1963). Invasion of chlamydospores and hyphae of the false smut fungus into rice plants. Research Bulletin of the Faculty of Agriculture, Gifu University, 18, 54–60.
Kageyama, K., Komatsu, T., & Suga, H. (2003). Refined PCR protocol for detection of plant pathogens in soil. Journal of General Plant Pathology, 69, 153–160. doi:10.1007/s10327-002-0037-4.
Okubara, P. A., Schroeder, K. L., & Paulitz, T. C. (2005). Real-time polymerase chain reaction: applications to studies on soilborne pathogens. Canadian Journal of Plant Pathology, 27, 300–313.
Osada, S. (1995). Effect of false smut occurrence on yield and quality of rice. Annual Report of the Society of Plant Protection of North Japan, 46, 30–32.
Ou, S. H. (1972). Rice diseases. Kew: Commonwealth Mycological Institute.
Takahashi, Y. (1896). On Ustilago virens Cooke and a New Species of Tilletia parasitic on rice plant. Botanical Magagine, Tokyo, 10, 16–20.
Tanaka, E., & Tanaka, C. (2008). Phylogenetic study of clavicipitaceous fungi using acetaldehyde dehydrogenase gene sequences. Mycoscience, 49, 115–125. doi:10.1007/s10267-007-0401-5.
Tanaka, T., Ashizawa, T., Sonoda, R., & Tanaka, C. (2008). Villosiclava virens gen. nov., com. nov., teleomorph of Ustilaginoidea virens, the causal agent of rice false smut. Mycotaxon, 106, 491–501.
Tsuda, M., Sasahara, M., Ohara, T., & Kato, S. (2006). Optimal application timing of simeconazole granules for control of rice kernel smut and false smut. Journal of General Plant Patholology, 72, 301–304. doi:10.1007/s10327-006-0288-6.
Volossiouk, T., Robb, E. J., & Nazar, R. N. (1995). Direct DNA extraction for PCR-mediated assays of soil organisms. Applied and Environmental Microbiology, 61, 3972–3976.
White, T. J., Bruns, T., Lee, S., & Taylor, J. (1990). Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In M. A. Innis, D. H. Gelfand, J. J. Sninsky, & T. J. White (Eds.), PCR protocols: A guide to methods and applications (pp. 315–322). California: San Diego.
White, J. F., Jr., Sullivan, R., Moy, M., Patel, R., & Duncan, R. (1990). An overview of some current problems in the classification of plant-parasitic Clavicipitaceae. Studies in Mycology, 45, 95–105.
Yaegashi, H., Fujita, Y., & Sonoda, R. (1989). Severe outbreak of false smut of rice in 1988. Plant Protection Tokyo, 43, 311–314.
Zhou, Y.-L., Izumitsu, K., Sonoda, R., Nakazaki, T., Tanaka, E., Tsuda, M., et al. (2003). PCR-based specific detection of Ustilaginoidea virens and Ephelis japonica. Journal of Phytopathology, 151, 513–518.
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This work was supported in part by a Grant-in-Aid under the Integrated Research Project for Developing a Japanese-style Forage Feeding System to Increase the Forage Self-support Ratio from the Ministry of Agriculture, Forestry and Fisheries of Japan.
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Ashizawa, T., Takahashi, M., Moriwaki, J. et al. Quantification of the rice false smut pathogen Ustilaginoidea virens from soil in Japan using real-time PCR. Eur J Plant Pathol 128, 221–232 (2010). https://doi.org/10.1007/s10658-010-9647-4
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DOI: https://doi.org/10.1007/s10658-010-9647-4