Pavel Matusinsky, Leona Svobodova-Leisova, Pavel Marik and Ludvik Tvaruzek
Agrotest fyto, Ltd., Havlickova 2787, Kromeriz, Czech Republic
Ramularia collo-cygni (Sutton & Waller) (RCC) is a fungal pathogen infesting spring and winter barley (Hordeum vulgare L.) and causes the disease known as Ramularia leaf spot (RLS) manifested by necrotic spots on leaves. The symptoms usually appear during late growth phases of the host plants. When severe, the disease progresses by very rapid development of spottiness. In addition to reducing yields, it also decreases grain quality. A real-time PCR assay was designed to quantify RCC in barley tissues. PCR primers and a TaqMan probe were designed to target an RCC-specific DNA sequence. The method was optimized for pure fungal DNA and plasmid standard dilutions. Kernels were dissected into lemma, pericarp, testa, endosperm and embryo, and a suspension was acquired after washing the kernels. All these parts were tested for amounts of RCC, and the presence of other potential seed-borne pathogens was analyzed by standard PCR diagnostics. The highest level of Ramularia DNA was in the lemma, and it occurred in lower amounts in the pericarp and embryo. Ramularia DNA was also evidenced in the suspension after washing the kernels. Our findings indicate that DNA of RCC is present on the emerging leaves of plants sown from infected seed. This confirms that RCC can be seedborne. DNA of RCC is detectable by molecular methods also in cases when visible symptoms are absent. DNA content in leaves need not necessarily correspond to the intensity of RLS symptoms. The work was supported by the Ministry of Agriculture of the Czech Republic, Projects QJ1310091, QJ1310226, QJ1210008 and RO0211.
Keywords: Ramularia leaf spot, molecular diagnostics, cereal diseases.