PCR ASSESSMENT OF WHEAT FIELD SAMPLES FROM THE UK, USA, GERMANY AND FRANCE FOR THE DETECTION OF PSEUDOCERCOSPORELLA HERPOTRICHOIDES
L ETIENNE1, JJ BECK2, C BASSIN1, C THOMAS3, SJE WEST3 and U GISI1
1Novartis Crop Protection Limited, Agrobiology Research Station, CH-4108 Witterswil, Switzerland; 2Novartis Crop Protection, Biotechnology and Genomics Center, 3054 Cornwallis Road, Research Triangle Park, North Carolina 27709 USA; 3Novartis Crop Protection UK Limited, Whittlesford, Cambridge CB2 4QT, UK
Background and objectives
Materials and methods
Wheat samples were taken from multiple plots in the UK, US, Germany and France. Samples were processed in core testing labs in the UK, US and Switzerland. A sample represented 20-30 plants taken randomly from a minimum of 25 acres between growth stages 25-77 on the Zadoks scale. Four cm of each stem were cut directly above the basal culm from each plant. The 20-30 stems for each sample were placed in a plastic bag with an equal volume of proprietary extraction buffer per weight of stem tissue. Tissue was macerated using a roller ball bearing homogenizer. Extraction juice was aliquoted into eppendorf tubes. One aliquot was boiled for 5 minutes and diluted 1:10 in dH20.
Diagnostic PCRs were run with 1 µl of 1:10 diluted wheat extract, 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 2.5 mM MgCl2, 0.2 mM each dNTP, 25 pmol each primer and 2.5 units of Taq polymerase (Perkin-Elmer) in a final volume of 50 µl. Reactions were run for 35 cycles, each consisting of 15 s at 94°C and 1 min at 75°C in a Perkin-Elmer Model 9600 thermal cycler. Ten of the 50 µl of PCR products were analyzed on an ethidium bromide-stained gel. A standardized 0-5 scale was used to measure the relative PCR product intensity on an agarose gel. The more fungal DNA present in the sample, the greater the PCR fragment intensity on a gel.
Results and conclusions
PCR tests are performed pursuant to licensing arrangements with the Perkin-Elmer Corporation under patent rights owned by F. Hoffman-LaRoche Limited and Hoffman LaRoche Incorporated.