A nested-polymerase chain reaction protocol for the detection of Mycosphaerella nawae in persimmon
- 286 Downloads
Mycosphaerella nawae is the causal agent of circular leaf spot of persimmon. A polymerase chain reaction (PCR) based protocol was developed for M. nawae-specific identification from pure culture, or infected symptomatic and asymptomatic persimmon tissues. Variation among the internal transcribed spacer regions (ITS) of the ribosomal DNA (rDNA) sequences of potentially related fungal species in persimmon orchards was analyzed for a primer pair design. Specificity was confirmed using multiple isolates of these species, other fungal pathogens that cause foliar diseases in persimmon and contaminants commonly obtained in the isolation process. The detection threshold for M. nawae DNA was lowered from 50 pg to 500 fg when nested-PCR was evaluated instead of single PCR. The nested-PCR protocol developed in this study showed its suitability to be applied for the specific detection of M. nawae from three types of naturally infected persimmon tissues: from lesions in fresh leaves, from pseudothecia present in lesions in leaf litter, and from infected asymptomatic leaves. The protocol can be useful for routine diagnosis, disease monitoring programs and for epidemiological research.
KeywordsCircular leaf spot Diagnosis Diospyros kaki Nested-PCR
M. Berbegal was a contract holder of the “Campus de Excelencia Internacional” program of the Universitat Politècnica de Valencia. This research was financially supported by Fundación Agroalimed (Consellería de Agricultura, Pesca i Alimentació, Generalitat Valenciana). We thank J. Armengol for critically reading the manuscript prior to submission.
- David, J. C. (2000). Pseudocercospora kaki. IMI descriptions of fungi and bacteria: 144, sheet 1439. Wallingford: CAB International.Google Scholar
- Food and Agriculture Organization of the United Nations (FAO) (2009). Crop production database FAOSTAT. http://faostat3.fao.org/home/index.html. Accessed 14 Sept 2012
- Ikata, S., & Hitomi, T. (1929). Studies on circular leaf spot of persimmon caused by Mycosphaerella nawae. Special Bulletin of the Okayama Prefecture Agricultural Experiment Station, 33, 1–36 (In Japanese).Google Scholar
- Kang, S. W., Kwon, J. H., & Kim, H. K. (1997). High sporulating medium for Cercospora kaki causing persimmon angular leaf spot. Korean Journal of Plant Pathology, 13, 69–71.Google Scholar
- Kang, S. W., Kwon, J. H., Lee, Y. S., & Park, C. S. (1993). Effects of meteorological factors on perithecial formation and release of ascospores of Mycosphaerella nawae from the overwintered persimmon. Rural Development Administration Journal of Agricultural Science, 35, 337–343 (In Korean).Google Scholar
- Kularatne, H. A. G., Lawrie, A. C., Barber, P. A., & Keane, P. J. (2004). A specific primer PCR and RFLP assay for the rapid detection and differentiation in planta of some Mycosphaerella species associated with foliar diseases of Eucalyptus globulus. Mycological Research, 108, 1476–1493.PubMedCrossRefGoogle Scholar
- Kwon, J. H., Kang, S. W., Park, C. S., & Kim, H. K. (1998). Identification of the imperfect stage of Mycosphaerella nawae causing circular leaf spot of persimmon in Korea. Korean Journal of Plant Pathology, 14, 397–401.Google Scholar
- White, T. J., Burns, T., Lee, S., & Taylor, J. W. (1990). Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetic. 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). San Diego: Academics Press.Google Scholar