3.3.6
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
Pyrenophora graminea is pathogenic on barley and is responsible for the seed-transmitted disease leaf stripe. It can cause severe economic loss for barley growers worldwide. The most effective method for controlling seedborne disease, apart from breeding resistant varieties, is seed certification. This disease is not directly covered by seed certification, but there are voluntary standards in the UK due to its economic importance. With increasing environmental awareness for the judicious use of pesticides and the high cost of seed treatments, seed health testing provides an effective means of disease control and appropriate use of seed treatment.

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
P. graminea isolates were isolated from UK barley seed and DNA extracted as described by [1]. Each 25-ml PCR reaction was made as previously described by [1] with the following modifications: 0.2 mM of each primer (PGF1 and PGR1) and 1.5 mM MgCl2. PCR cycling conditions were as follows: step 1 = 2 min initial denature at 92C; step 2 = 1 min denature at 94C; step 3 = 1 min anneal at 45C; step 4 = 1 min extension at 72C. 30 cycles of steps 2-4 were carried out before the final extension at 72C for 10 min.

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
PCR inhibition was observed from soaking seed for 5 min and increased with length of soaking time. BSA had the greatest effect in reducing PCR inhibition compared with boiling the samples. A combination of boiling the seed soak aliquots and the addition of 0.05% BSA was shown to be most effective in minimizing PCR inhibition for all soaking times investigated. These conditions were applied successfully to the amplification of P. graminea DNA directly from infected seed. These results will be used for the further development of a seed health test for this pathogen.

References
1. Stevens EA, Alderson J, Blakemore EJA, Reeves JC, 1996. In Marshall G, ed., Diagnostics in Crop Protection, BCPC Symposium Proceedings No. 65. British Crop Protection Council, Farnham, pp. 99-104.