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RAPID DETECTION AND DIAGNOSIS OF SEPTORIA EPIDEMICS IN WHEAT USINGA PCR/PICOGREEN DNA QUANTIFICATION ASSAY
RAPID DETECTION AND DIAGNOSIS OF SEPTORIA EPIDEMICS IN WHEAT USING A PCR/PICOGREEN DNA QUANTIFICATION ASSAY BA FRAAIJE, S BALDWIN, DJ LOVELL, EA ROHEL and DW HOLLOMON IACR-Long Ashton Research Station, University of Bristol, Long Ashton, Bristol BS41 9AF, UK Background and objectives The control of Septoria diseases in wheat relies predominantly on the use of fungicides. For optimum cost-effective control of leaf blotch fungicides are applied to protect the upper three leaves. The timing and choice of a fungicide (e.g. with curative and/or protective mode of action) depends on the inoculum level and disease risk of a crop [1]. However, as soon as pycnidia are initiated, none of the fungicides currently available are effective in protecting the flag leaf. An accurate pre-symptomatic detection and quantification of Septoria tritici(Mycosphaerella graminicola) can therefore improve disease control through better timing, choice and even dose rate of fungicide sprays. With the polymerase chain reaction (PCR) using primers directed against ITS regions Beck and Ligon [2] were able to detect DNA of S. tritici and Stagonospora nodorum in extracts of wheat leaves which did not show any symptoms. However, methods to quantify PCR products, involving DNA capture steps or labeling reactions, are laborious and time-consuming. This study demonstrates the use of a PCR assay based on primers with the beta-tubulin gene of S. tritici as target. The beta-tubulin primers specifically amplify a single DNA fragment from S. tritici. Because DNA fragments from wheat and other micro-organisms are not amplified, PCR products can be quantified directly and rapidly in solution with the fluorescent dye PicoGreen (Molecular Probes, Leiden, The Netherlands). By testing leaves infected with known amounts of spores and/or genomic DNA of S. tritici, calibration curves for an accurate detection and quantification of S. tritici infection levels on wheat were made. Consequently, this allows us to diagnose wheat crops, to study pre-symptomatic levels of S. tritici and to measure the effect of fungicide application on disease development. Materials and methods DNA extraction, PCR carried out with a Perkin Elmer DNA Thermal Cycler 480 and PicoGreen measurements using a Perkin Elmer LS50B fluorimeter were performed as described previously [3]. Primers El (5'-CGGTATGGGAACACTTCTCATCAG-3') and STSP2R (5'-GTAACGACCGTTGCGAAATCGCT-3') were used in the PCR to specifically amplify a 496-bp fragment of the beta-tubulin gene of S. tritici. Results and conclusions With the PCR/PicoGreen assay as few as 1000 spores, or 2-3 pg DNA of S. tritici, per leaf could be detected. No inhibition or cross reactions were observed in PCR using the betatubulin primers. With the calibration curves obtained with artificially inoculated samples we were able to detect and quantify S. tritici levels in wheat leaves rapidly and accurately. The results from the field trials indicate that the PCR/PicoGreen assay has a potential to quantify S. tritici prior to visual symptoms which will enable farmers to optimise fungicide spray applications in order to control leaf blotch. Additionally, the assay can also be used as a research tool for both studies on S. tritici epidemics and fungicide activity. References 1.Royle DJ, Parker SR, Lovell DJ, Hunter T, 1995. In: Hevlitt HG (ed.) A vital role for fungicides in cereal production, Bios Scientific Publisher Ltd, UK, 105-115. 2.Beck JJ, Ligon JM, 1995. Phytopathology 85, 319-324. 3.Hollomon DW, Fraaije BA, Rohel EA, Butters J, Kendall SJ, submitted. Proceedings of the 15th Long Ashton International Symposium: Understanding pathosystems: a focus on Septoria, 1997.