IMPACT OF THE SEEDBORNE STAGE OF RAMULARIA COLLOCYGNI IN BARLEY SEED
P. Matusinsky, L. Leisova-Svobodova, J. Gubis, M. Hudcovicova, L. Klcova, M. Gubisova, P. Marik, L. Tvaruzek, V. Minarikova
Ramularia collo-cygni (Rcc) infects spring and winter barley causing Ramularia leaf spot (RLS). During 2009 and 2010, contrasting years for the natural occurrence of RLS in the Czech Republic, infestation intensity was assessed with field experiments at two locations in relation to Ramularia contamination of seeds. A real time PCR assay was designed to quantify the pathogen in barley tissues. PCR primers and a TaqMan probe were designed to target Rcc-specific DNA sequence. The method was optimized using pure fungal DNA and plasmid standard dilutions. After washing, kernels were dissected into lemma, pericarp, testa, endosperm and embryo which were individually tested by real time PCR for quantifying Rcc, whereas the presence of other potential seed-borne pathogens was checked by standard PCR. The level of seed contamination was not the main factor influencing symptom expression. In 2009, seeds with low Rcc contamination were planted in two locations in both of which severe RLS infection had occured. In 2010, seeds with higher Rcc contamination were planted, but the extent of RLS symptoms on the leaves was much lower. Rcc occurred at higher rates in kernels from the 2009 crop, when RLS was severe. Ramularia DNA was highest in the lemma, and occurred in lower amounts in the pericarp and embryo. It was also found in the water used for washing the kernels. The results showed that Rcc does not penetrate through the testa into the endosperm.