First Report of Polymyxa graminis f. sp. temperata, a Vector of Soilborne Cereal Viruses in the Czech Republic

Polymyxa graminis L. is an eukaryotic, obligate, biotrophic parasite of plant roots (1), which belongs to a poorly studied, discrete, taxonomic unit informally called the ‘plasmodiophorids’. P. graminis has the ability to acquire and transmit a range of soilborne viruses that belong to at least three separate genera and can cause economically significant diseases in cereal crops. For example, the winter barley disease caused by Barley yellow mosaic virus (BaYMV) and/or Barley mild mosaic virus (BaMMV) is widespread in Europe, Japan, and China; yield losses of >50% may occur when susceptible barley cultivars are grown on severely infested soils (2). Monitoring for P. graminis was started in the Czech Republic in May 2008. Fifty-six soil samples were collected from different localities of cereal production (wheat and barley) in the Královéhradecký Region (eastern Czech Republic). Soil from each sample was placed in five replicate pots (12 × 12 cm) in a greenhouse at 22 to 25°C. Seeds of barley cv. Florian were sown into the soil (10 seeds per pot). Negative control soil (noninfested soils from the Czech Republic) and positive control soil (known P. graminis-infested soil from Germany) were also planted to barley in five replicate pots. After 90 days, plants were collected and the roots were washed thoroughly in sterilized water and examined with a light microscope without staining. The fungus was identified as P. graminis on the basis of morphology of resting spores (cystosori) and sporangia and the size of individual cystosori (4 to 5 μm in diameter) according to Thouvenel et al. (3). Cystosori of P. graminis were observed in the roots of plants grown in 20 of the 56 soil samples, especially the samples from Ceské Mezirici. The presence of P. graminis in the roots of plants grown in the soil samples and the positive control sample versus the absence of the vector in roots of plants in the negative control soil was verified by PCR assay with DNA extracts and the Psp1 and Psp2rev primers according to Legrève et al. (Page 40 in: Proceedings of the Fifth Symposium of the International Working Group on Plant Viruses with Fungal Vectors, 2003). The PCR assay included denaturation at 94°C for 2 min, then 35 cycles including denaturation of 30 s at 94°C, annealing at 60°C for 1 min, and elongation at 72°C for 35 s. A final elongation was completed at 72°C for 7 min. To characterize the P. graminis isolates, the amplified PCR product (a DNA fragment of 472 bp) was sequenced and blasted for each of the samples that tested positive. These sequences were aligned with a known sequence (GenBank Accession No. AM259276) for P. graminis. The sequences from P. graminis on barley were 100% homologous to the published sequence of P. graminis f. sp. temperata. To our knowledge, this is the first report of P. graminis f. sp. temperata in the Czech Republic.