Abstract
A quantitative PCR method (qPCR) was developed for the detection and quantification of Ramularia beticola causing Ramularia leaf spot in sugar beet. R. beticola specific primers were designed based on the internal transcribed spacer region 2 (ITS2). The assay was applied on DNA extracted from spores trapped on tape from Burkard spore traps placed in an artificially inoculated sugar beet field trial and in two sugar beet fields with natural infections. R. beticola DNA was detected at variable amounts in the air samples 14 to 16 days prior to first visible symptoms. R. beticola DNA was detected in air samples from fields with natural infection at significant and increasing levels from development of the first symptoms, indicating that spore production within the crop plays a major role in the epidemic development of the disease. Sugar beet leaves sampled from the inoculated field trial were also tested with the qPCR assay. It was possible to detect the presence of R. beticola in the leaves pre-symptomatic at least 10 days before the occurrence of the visible symptoms of Ramularia leaf spot. This is the first report of a molecular assay, which allows screening for the presence of R. beticola in plant material and in air samples prior to the appearance of visible symptoms. An early detection has potential as a tool, which can be part of a warning system predicting the onset of the disease in the sugar beet crop and helping to optimise fungicide application.
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References
Ahrens, W. (1987). Feldversuche zum Auftreten der Ramularia-Blattfleckenkrankheit and Zuckerrüben. Gesunde Pfanzen, 39(3), 113–119. In German.
Asher, M. J. C., & Hanson, L. E. (2006). Fungal and bacterial diseases. In A. P. Draycott (Ed.), Sugar Beet (1st ed., pp. 286–315). Blackwell Publishing Ltd.
Carisse, O., Tremblay, D. M., Levesque, C. A., Gindro, K., Ward, P., & Houde, A. (2009). Development of a TaqMan real-time PCR assay for quantification of airborne conidia of botrytis squamosa and management of botrytis leaf blight of onion. Phytopathology, 99(11), 1273–1280.
Crous, P. W., Kang, J. C., & Braun, U. (2001). A phylogenetic redefinition of anamorph genera in Mycosphaerella based on ITS rDNA sequence and morphology. Mycologia, 93(6), 1081–1101.
Directive 2009/128/EC of the European Parliament and the Council establishing a framework for the Community action to achieve the sustainable use of pesticides (2009). European Parliament and the Council of the European Union.
De Coninck, B. M. A., Amand, O., Delauré, S. L., Lucas, S., Hias, N., Weyens, G., Mathys, J., De Bruyne, E., & Cammue, B. P. A. (2012). The use of digital image analysis and real-time PCR fine-tunes bioassays for quantification of Cercospora leaf spot disease in sugar beet breeding. Plant Pathology, 61, 76–84.
Duvivier, M., Dedeurwaerder, G., de Proft, M., Moreau, J.-M., & Legrève, A. (2013). Real-time PCR quantification and spatio-temporal distribution of airborne inoculum of Mycosphaerella graminicola in Belgium. European Journal of Plant Pathology, 137, 325–341.
Eronen, L. (2008). Ramularian överraskade igen. Betfältet, 4, 19–24. In Swedish.
Giesecke, T., Fontana, S. L., & Pidek, I. A. (2010). From early pollen trapping experiments to the pollen monitoring programme. Vegetation History and Archaeobotany, 19, 247–258.
Goodwin, S. B., Ben M’Barek, S., Dhillon, B., Wittenberg, A. H. J., Crane, C. F., Hane, J. K., Foster, A. J., Van der Lee, T. A. J., Grimwood, J., Aerts, A., Antoniw, J., Bailey, A., Bluhm, B., Bowler, J., Bristow, J., van der Burgt, A., Canto-Canche, B., Churchill, A. C. L., Conde-Ferraez, L., Cools, H. J., Coutinho, P. M., Csukai, M., Dehal, P., De Wit, P., Donzelli, B., van de Geest, H. C., Van Ham, R. C. H. J., Hammond-Kosack, K. E., Henrissat, B., Kilian, A., Kobayashi, A. K., Koopmann, E., Kourmpetis, Y., Kuzniar, A., Lindquist, E., Lombard, V., Maliepaard, C., Martins, N., Mehrabi, R., Nap, J. P. H., Ponomarenko, A., Rudd, J. J., Salamov, A., Schmutz, J., Schouten, H. J., Shapiro, H., Stergiopoulos, I., Torriani, S. F. F., Tu, H., de Vries, R. P., Waalwijk, C., Ware, S. B., Wiebenga, A., Zwiers, L. H., Oliver, R. P., Grigoriev, I. V., & Kema, G. H. J. (2011). Finished genome of the fungal wheat pathogen mycosphaerella graminicola reveals dispensome structure, chromosome plasticity, and stealth pathogenesis. PLOS Genetics, 7(6), e1002070. doi:10.1371/journal.pgen.1002070.
Hansen, A. L. (2008). Effekt på bladsvampe. In J. N. Thomsen (Ed.), Dyrkningsforsøg og undersøgelser i sukkerroer 2007 (pp. 25–27). Holeby: Alstedgaard, Fondet for Forsøg med Sukkerroedyrkning.). In Danish.
Hansen, A. L. (2012a). Bladsvampe—midler og doseringer. In J. N. Thomsen & R. Olsson (Eds.), NBR Nordic Beet Research Faglig beretning—Verksamhetsberättelse 2011 (pp. 28–32). Holeby: NBR Nordic Beet Research Foundation.). In Danish.
Hansen, A. L. (2012b). Tidlig bladsvampebekæmpelse. In J. N. Thomsen & R. Olsson (Eds.), NBR Nordic Beet Research Faglig beretning—Verksamhetsberättelse 2011 (pp. 57–62). In Danish: NBR Nordic Beet Research Foundation.).
Hestbjerg, H., & Dissing, H. (1994). Development of Ramularia leaf spot on sugar beet as influenced by temperature and the Age of the host plant. Journal of Phytopathology, 140, 293–300.
Hestbjerg, H., & Dissing, H. (1995). Studies on the concentration of Ramularia beticola conidia in the air above sugar beet fields in Denmark. Journal of Phytopathology, 143, 269–273.
Hirst, J. (1952). An automatic volumetric spore trap. Annals of Applied Biology, 39, 257–265.
Jørgensen, A. M. (2003). Rizomania og Ramularia i sukkerroer–status. Retrieved October 20, 2012, from https://www.landbrugsinfo.dk/Planteavl/Afgroeder/Sukkerroer/Sider/plvaern2003_13.pdf?download=true
Khan, J., Qi, A., & Khan, M. F. R. (2009). Fluctuations in number of Cercospora beticola conidia in relationship to environment and disease severity in sugar beet. Phytopathology, 99(7), 796–801.
McCartney, H. A., Foster, S. J., Fraaije, B. A., & Ward, E. (2003). Molecular diagnostics for fungal plant pathogens. Pest Management Science, 59(2), 129–142. doi:10.1002/ps.575.
Persson, L., & Olsson, Å. (2008). Odlingsystemets inverkan på svamp- och nematodangrepp i sockerbetor 2006–2008—The impact of cropping systems on pathogenic fungi and nematodes in sugar beet 2006–2008. Holeby: NBR Nordic Beet Research Foundation (Fond). In Swedish with English abstract.
Petersen, J., Adams, H., Schaufele, W. R., & Buttner, G. (2001). Untersuchungen zur Schadwirkung von Ramularia beticola in Zuckerrüben und Möglichkeiten zur Differenzierung der Sortenanfälligkeit nach künstlicher Inokulation. Gesunde Pfanzen, 53, 141–147. In German.
Racca, P., & Jorg, E. (2007). CERCBET 3–a forecaster for epidemic development of Cercospora beticola. Bulletin OEPP/EPPO, 37, 344–349.
Racca, P., Jorg, E., Mittler, S., & Petersen, J. (2002). Sugarbeet leaf diseases–forecasting approaches for the optimization of fungicide application. Zuckerindustrie, 127(12), 949–958.
Rittenour, W. R., Park, J.-H., Cox-Ganser, J. M., Beezhold, D. H., & Green, B. J. (2012). Comparison of DNA extraction methodologies used for assessing fungal diversity via ITS sequencing. Journal of Environmental Monitoring, 14(3), 766–74. doi:10.1039/c2em10779a.
Rogers, S. L., Atkins, S. D., & West, J. S. (2009). Detection and quantification of airborne inoculum of Sclerotinia sclerotiorum using quantitative PCR. Plant Pathology, 58(2), 324–331.
Ruppel, E. G. (1986). Foliar diseases caused by fungi. In E. D. Whitney & J. E. Duffus (Eds.), Compendium of beet diseases and insects (pp. 8–17). St. Paul: APS PRESS The American Phytopathological Society.
Schena, L., Nigro, F., Ippolito, A., & Gallitelli, D. (2004). Real-time quantitative PCR: a new technology to detect and study phytopathogenic and antagonistic fungi. European Journal of Plant Pathology, 110, 893–908.
Taylor, J., Paterson, L. J., & Havis, N. D. (2010). A quantitative real-time PCR assay for the detection of Ramularia collo-cygni from barley (Hordeum vulgare). Letters in Applied Microbiology, 50(5), 493–499.
Thach, T., Munk, L., Hansen, A. L., & Jørgensen, L. N. (2013). Disease variation and chemical control of Ramularia leaf spot in sugar beet. Crop Protection, 51, 68–76.
West, J. S., Atkins, S. D., Emberlin, J., & Fitt, B. D. L. (2008). PCR to predict risk of airborne disease. Trends in Microbiology, 16(8), 380–387.
West, J. S., Atkins, S. D., & Fitt, B. D. L. (2009). Detection of airborne plant pathogens; halting epidemics before they start. Outlooks on Pest Management, 20(1), 11–14.
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. 315–321). San Diego: Academic.
Wolf, P. F. J., Weis, F. J., & Verreet, J. A. (2001). Bekämpfungsschwellen als Indikatoren des Fungizideinsatzes zur Kontrolle des Blattbefalls von Cercospora beticola (Sacc.) in Zuckerrüben. Journal of Plant Diseases and Protection, 108(3), 244–257. In German with English abstract.
Zijlstra, C., Lund, I., Justesen, A. F., Nicolaisen, M., Jensen, P. K., Bianciotto, V., Posta, K., Balestrini, R., Przetakiewicz, A., & Van de Zande, J. (2011). Combining novel monitoring tools and precision application technologies for integrated high-tech crop protection in the future (a discussion document). Pest Management Science, 67, 616–625.
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Wieczorek, T.M., Jørgensen, L.N., Hansen, A.L. et al. Early detection of sugar beet pathogen Ramularia beticola in leaf and air samples using qPCR. Eur J Plant Pathol 138, 775–785 (2014). https://doi.org/10.1007/s10658-013-0349-6
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DOI: https://doi.org/10.1007/s10658-013-0349-6