First Report of Ramularia Leaf Spot Caused by Ramularia collo-cygni on Leaves and Seeds of Barley in Estonia
Authors: Sooväli P. , Tikhonova M. , Matušinsky P. 
Published: February 2014

Ramularia leaf spot (RLS) is a disease of barley (Hordeum vulgare) caused by the fungus Ramularia collo-cygni Sutton & Waller (Rcc). Rcc causes necrotic lesions, premature senescence of leaves, and yield reduction. Under Estonian conditions, there are usually no leaf spots on the upper leaves of barley prior to flowering. In 2009, 2010, and 2012, symptoms similar to those of RLS were observed on leaves of spring and winter barley in several Estonian agricultural regions. Approximately 30% of the plants in affected fields were symptomatic. Symptoms were not observed in 2011, which was a dry and hot year. Initial symptoms were small brown spots, beginning on the upper leaves (flag leaf, F-1 leaf) at the flowering growth stage (4). Later, the spots spread to the sheaths, stems, and awns and became necrotic. The lateral margins of the spots were delimited by the leaf veins and spots are surrounded by a chlorotic halo. During summer 2012, two samples of 15 F-1 leaves were collected from spring barley cv. Maali and line SJ111609 from the Estonian Crop Research Institute in eastern Estonia in late July at growth stage 71 (4). In addition, six grain samples, containing 200 seeds each of the cv. Maali, were collected from different agricultural regions in Estonia, along with one grain sample of SJ111609 from Jõgeva. All samples were collected from untreated plots and leaves were observed under a dissecting microscope, revealing white clusters of conidiophores in rows on the undersides of the leaves. Conidia and conidiophores were scraped aseptically from the leaf surface using a sterile needle under a dissecting microscope and transferred to potato dextrose agar (PDA) containing ampicillin sodium salt (50 mg l−1). Plates were incubated at 18°C in the dark for 20 days until fungal mycelia were produced. The fungus was initially identified as Rcc on the basis of morphological characteristics (3). Colorless, 0- to 3-septate conidiophores were 15 to 17 × 2 to 5 μm, with a strongly curved end. Conidia were 7 to 11 × 3 to 6 μm, solitary, subglobose, single-celled, and of a darkish color. To confirm the presence of Rcc, DNA was extracted from the original barley leaf material, milled seeds, and positive control mycelia of Rcc grown on PDA using DNeasy Plant Mini Kit (Qiagen Gmbh, D-40724 Hilden, Germany) following manufacturer's guides. Rcc specific primers RC3 and RC5 (1) were used. A positive control consisting of 1 ng of purified Rcc DNA was included in the PCR. Standard PCR was conducted in a SEE AMP Seegene cycler. PCR were carried out in 20 μl volumes, containing 2 μl of DNA, 10 μl PCR mix, 0.4 μl each of forward and reverse Rcc primers, and 7.2 μl H2O. Qualitative detection analyzed by standard PCR with primers RC3 and RC5 revealed the presence of Rcc in symptomatic leaves and seeds. To complete Koch's postulates, a pathogenicity test was performed. Twenty-five barley seedlings were grown under controlled conditions (15°C/48 h dark, 16 h light/8 h dark, 70% RH) and spray-inoculated with a suspension of Rcc mycelium fragments as described by Macepeace et al. (2). The pathogen was re-isolated from leaves with necrotic lesions similar to those observed in the field, thus fulfilling Koch's postulates. To our knowledge, this is the first confirmed report of Ramularia leaf spot caused by Ramularia collo-cygni on barley in Estonia. References: (1) P. Frei et al. J. Phytopathol. 155:281, 2007. (2) J. C. Makepeace et al. Plant Pathol. 57:991, 2008. (3) B. C. Sutton and J. M. Waller. Trans. Brit. Mycol. Soc. 90:55, 1988. (4) J. C. Zadoks et al. Weed Res. 14:415, 1974.