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
Previously described polymerase chain reaction (PCR) assays [1] were used to characterize field samples for the presence of Pseudocercosporella herpotrichoides, the causative agent of eyespot in wheat. These assays differentiate between the Rye- and Wheat-pathotypes of P. herpotrichoides and do not amplify PCR products from DNA isolated from other common cereal pathogens and healthy wheat. PCR results using these assays have also previously been shown to correlate with visual disease symptoms and ELISA results for eyespot[1]. Using these PCR assays, it was possible to assess wheat field samples for the presence of the R- and W-pathotypes of P. herpotrichoides.

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
P. herpotrichoides-specific PCR assays were used for assessing wheat field samples from the UK, US, Germany and France for the presence of disease. Although both pathotypes were found in all countries, there were differences in the ratio of the pathotypes found within and between countries. In Europe, the disease incidence was higher in France (93% of all sites monitored) as compared to Germany (77%). W-type eyespot was dominant in France, while the R-type was more frequent in Germany. US testing indicated a dominance in the R-type. Our results helped promote disease awareness and provided information on the regional distribution of R- and W- types of eyespot at different growth stages within and across the countries.

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.

References
1. Beck JJ, Beebe JR, Stewart SJ, Bassin C, Etienne L, 1996. Proceedings Brighton Crop Protection Conference: Pests and Diseases, pp.221-226.