Application of a new diagnostic technique for the decision of seed-borne Pyrenophora species

Abstract

The aim of the project was to develop a diagnostic method for seed-borne Pyrenophora species on barley, based on DNA primers and the polymerase chain reaction (PCR) which was capable of delivering a rapid throughput routine test. The specific objectives were to optimise the PCR system and check the specificity of some of the primer sequences used, quantify the test for seed-borne net blotch and define a suitable extraction procedure of the fungus from seed, and finally to identify any practical problems which might arise in handling large numbers of tests.

Primer sequences derived from previous MAFF funded research were used. Optimal PCR conditions were established for the ITS (internally transcribed spacer region) primers used to detect net blotch, but it was not possible to use these in the same reaction as the specific primers for leaf stripe and separate PCR runs were needed. The ITS primers were checked against a range of other pathogens and saprophytic organisms commonly found on barley seed, and no significant cross-reactivity was detected. An extraction procedure developed for leaf stripe on barley seed also proved sufficiently reproducible and sensitive for extraction of net blotch. The PCR test for net blotch was quantified by establishing a relationship between % infection on agar plates, and the output of the PCR assay. The relationship proved accurate over a range of low to moderate infection levels when used to predict the infection % of over 80 samples of barley seed previously submitted to NIAB for testing.

The PCR test produced no false positive reactions for either leaf stripe or net blotch when no infection was expected from the agar plate test. Internal controls designed to protect against the possibility of false negatives due to DNA extraction failure were included in all tests. The extraction and testing sequence could be completed within one working day. In common with other advanced PCR systems, the equipment used at NIAB can process batches of samples sequentially throughout the day. The flow of barley samples submitted to NIAB for germination and health testing is likely to reflect the overall processing frequency of winter and spring barley seed lots. Large peaks occur in late July and throughout August, with probably in the region of 1000 lots, either farm-processed or certified seed, requiring testing each week during this period. Two or three laboratories operating high-throughput PCR systems would be able to meeting testing requirements for the majority of seed lots giving a test result within a maximum of 48 hours.

The development of a rapid diagnostic technique for these diseases offers growers the opportunity to make decisions on barley seed treatments on the basis of need without incurring impracticable delays to the processing of seed.