DEVELOPMENT OF A SEED HEALTH TEST TO DETECT PYRENOPHORA GRAMINEA BY PCR
EJA BLAKEMORE, EA STEVENS and WC COOPER
National Institute of Agricultural Botany, Huntingdon Road, Cambridge CB3 OLE, UK
Background and objectives
Traditional seed health tests involve identification of the morphological characteristics of the pathogen after incubating barley seed on agar for a week. This technique is time-consuming, labour intensive and requires highly trained personnel. Therefore a need exists to develop a simple, rapid and effective test for the identification of this pathogen.
Primers to detect P. graminea were designed from a RAPD product after screening more than 60 RAPD primers.The aim was then to use these primers for the detection of target template by PCR, directly from barley seed. It is known that P. graminea is found in the pericarp of the seed (CMI Description No. 388). It may therefore be possible to soak infected barley seed in water to release the target template. Optimal soaking time was investigated to maximize the release of template whilst minimizing effects of inhibitory substances.
Materials and methods
200 uninfected Pipkin winter barley seeds were soaked in 20 ml of sterile distilled water at room temperature. Aliquots (100 ml) were removed at t=0, 5, 10, 15, 30, 60, 90 and 120 min. For each time interval, aliquots of 1, 3, 5, 7.5 and 10 ml, respectively, were added to a PCR reaction. In addition, each PCR reaction was spiked with 10 ng of purified P. graminea genomic DNA to assess the level of PCR inhibition. This experiment was repeated three times but with the following modifications: 0.01, 0.05 and 0.1% bovine serum albumin (BSA) to evaluate its potential for overcoming PCR inhibition; boiling of aliquots for 5 min to maximize release of template DNA; a combination of adding BSA and boiling of samples for optimal PCR.
Individual barley (Bere variety) seeds infected with P. graminea were soaked for 5 min in 1 ml water. The seed soaks were then boiled for 10 min before a 2-ml aliquot was taken from each for PCR, with the addition of 0.05% BSA to the reaction mixture.
Results and conclusions