PCR DETECTION AND QUANTIFICATION OF A FUNGAL PATHOGEN OF CEREALS
V MULHOLLAND Scottish Agricultural Science Agency, Edinburgh EH12 SNJ, UK Background and objectives Microdochium nivale, (Monographella nivalis) is able to cause pre~mergence blight of winter wheat when an infected seed lot is sown without fungicide treatment. The proscription of organo-mercury within the EU in 1992 has led to an increase in the cost of funigicide treatment. This, alongside consumer unease over the use of chemicals in food production, has caused a more strategic approach to cereal seed treatment to be considered, including the use of advisory pathogen testing of seed lots. Currently, advisory testing is performed using an agar plate test which is a lengthy procedure. Application of polymerase chain reaction (PCR) diagnostics in a phytosanitary context is relatively uncommon. We have developed a test for M. nivale which can detect the presence of this pathogen and indicate if the level of infection is above a threshold limit This threshold may be altered if different pathogen tolerances are needed. Results and conclusions PCR primers (one pair specific for M. nivale and another, flanking, pair which should amplify most species of fungi), based on the sequence of the 28S rRNA gene, were designed and tested using two hundred fungal isolates from cereal seed. This sample of fungi included 85 examples of M. nivale. The fungal 285 rRNA-speciflc primers gave the expected amplification product, but the M. nivale-specific primers also gave a band apparently of the correct size. To check this result non-specific and specific primers were used in combination. These amplifications gave a product only with M. nivale for both sets of primers except one primer-pair which gave a PCR product when tested with two isolates of Drechslera spp. The primer-pair which only produced a product with M. nivale was used further. A method for detecting M. nivale by PCR has been devised  but as it was not based on rRNA genes ~resent in 100 to 1000 copies) one would predict that its sensitivity of detection would be markedly lower than the assay described above. This was indeed the case, the published PCR test was able to detect 1 pg of chromosomal DNA, whilst the test based on rRNA genes was able to detect at least 10 fg, a difference of 100-fold. Quantitative experiments were based on the use of a PCR mimic which is co-amplified with target DNA - the concentration of the PCR mimic included in the amplification can be such that when the target DNA is at a threshold concentration an equal molarity of target and mimic is produced in the PCR . This method relies on the production of a piece of DNA which is unrelated to the region of interest except for the presence of the relevant PCR primer-binding sites at the end of the fragment. In the current example we produced a PCR fragment using hybrid primers, the 5' part of each containing one of the fungal priming sites, the 3' end consisting of part of the bacteriophage M13 genome. This allowed the construction of a fragment of DNA which although mostly unrelated to the rRNA genes was capable of being amplified by the same primers. The product from the mimic was slightly larger than the product from fungi to permited identification. A series of experiments was conducted to ensure that the amplification kinetics of the PCR mimic was similar to the fungal amplification. Once this was established, experiments using seed with known levels of M. nivale infection, isolating template with a crude purification method, were conducted to titrate the concentration of the PCR mimic to the level required for equal co-amplification when the infection with M. nivale reached an appropriate. This method is therefore suitable for using in a seed health context and is flexible enough to find application in many situations. References 1. Nicholson P, Lees AK, Maurin N, Parry DW, Rezanoor HN, 1996. Physiol Mol. Plant Pathol. 48, 257-271. 2. Siebert PD, Kellogg DE, 1995. PCR 2: A practical approach. IRL Fress. pp.135-148.