Quantitative and qualitative detection of Pyrenophora species on barley seed using PCR in advisory seed health testing
Pyrenophora graminea, the cause of leaf stripe in barley is a solely seed-borne pathogen capable of causing severe crop losses in both spring and winter crops. Pyrenophora teres (net blotch) is also seed-borne and although other sources of inoculum contribute to epidemic development, high levels of seed infection are likely to be significant. Traditionally both these pathogens have been detected using an agar plate test where the pathogens are identified using both colony and spore morphology. Distinguishing between these two pathogens can be difficult due to their similarity and despite of their differing importance as seed-borne pathogens, laboratories in some countries do not distinguish between them.
Quantification of P. teres DNA extracted from both naturally infected seed and samples blended to give target infection levels gave good correlation with the agar plate test. The artificially blended seed gave a gradient of between 0% - 14% infection. A correlation coefficient of r = 0.931 was between the mean of the plate test results and the mean of the PCR results at each infection level. These data sets were used to produce a linear conversion factor for converting pgDNA to percentage of seeds infected.
The 60 advisory samples tested ranged in infection from 0% to 89% seeds infected. Good correlation (r = 0.88) was again found between the two techniques. It is worth noting that above 40% infection the variability increases considerably. Other work on Microdochium nivale has shown that the amount of pathogen DNA extracted from individual seeds can vary by as much as 25-fold. Seed loading could then be affecting the results seen on the more highly infected seed lots were the effect would be greatest.
The PCR test described here used a LightCycler™, which allows quantification of unknown samples by comparison to standards amplified in parallel reactions. The complete test from receipt of sample to final result can be completed in several hours rather than the conventional plate test, which takes seven days. The system can also cope with a much high number of samples with a single LightCycler™ handling up to 300 samples per day. It thus offers a greater opportunity for farmers and seed merchants in the UK to complete seed health testing within a time scale that allows a treatment decision based on knowledge of disease risks. Comparative testing of the primer sequences and methodology using ISTA evaluation protocols will be sought in order to establish the technique as a future standard.
The financial support of the HGCA is gratefully acknowledged.
The PCR test described here used a LightCycler™, which allows quantification of unknown samples by comparison to standards amplified in parallel reactions. The complete test from receipt of sample to final result can be completed in several hours rather than the conventional plate test, which takes seven days. The system can also cope with a much high number of samples with a single LightCycler™ handling up to 300 samples per day. It thus offers a greater opportunity for farmers and seed merchants in the UK to complete seed health testing within a time scale that allows a treatment decision based on knowledge of disease risks. Comparative testing of the primer sequences and methodology using ISTA evaluation protocols will be sought in order to establish the technique as a future standard.
The financial support of the HGCA is gratefully acknowledged.