Maize stem samples exhibiting symptoms of anthracnose were collected from a field near Zurich, Switzerland, in September of 2012 and were sent to the fungal genetics laboratory, Centro Hispano-Luso de Investigaciones Agrarias (CIALE) at the University of Salamanca, Spain, for further analysis. The stem samples exhibited glossy, black, and irregularly shaped lesions. Tissue samples, approximately 5 mm2, were dissected from below the epidermis. The tissue samples were surface disinfested for 1 min in 20% sodium hypochlorite and cultured on one half strength acidified PDA supplemented with ampicillin (2). Monoconidial isolates from three different stems were grown on potato dextrose agar (PDA) and had dark gray aerial mycelium with orange spore masses. Conidia were falcate, slightly curved, tapered toward the tips with an average length of 31.77 μm and an average width of 4.76 μm and produced in acervuli with setae, consistent with descriptions of C. graminicola Ces. Wils. Conidial suspensions were prepared for each isolate, and were inoculated onto the leaves of 2-week-old maize plants by laying the plants horizontally in a tray (in pots with their root systems intact) and placing 7.5-μl droplets of a 106 conidial suspension on the leaf surface. The trays were covered and plants were incubated overnight at 23°C. The plants were then returned to their upright position and grown in a growth chamber at 25°C with a 12-h light cycle (3). After 6 days, the inoculated plant leaves exhibited lesions that were elongated and irregularly shaped with necrotic centers and chlorotic margins. The water-inoculated controls did not show symptoms. Microscopic examination revealed the production of conidia on the surface of the leaves, identical to the original isolates. Genomic DNA was extracted using the protocol of Baek and Kenerley (1). A region of the ribosomal DNA repeat was amplified and sequenced using the universal primers ITS4 and ITS5. The resulting sequences were 100% identical to each other and 100% identical to C. graminicola sequences in GenBank. One representative sequence was deposited in GenBank under accession no. KF597538. The 100 most similar sequences in GenBank were used to construct a phylogenetic tree using the neighbor-joining method. The phylogenetic analysis revealed that the isolates clustered within the C. graminicola clade, consistent with their identification as C. graminicola. To our knowledge, this is the first report of anthracnose on maize caused by C. graminicola in Switzerland. Previous reports have demonstrated that the pathogen exists in neighboring countries Germany and France.
References: (1) J.-M. Baek and C. M. Kenerley. Fungal Genet. Biol. 23:34, 1998. (2) S. A. Sukno et al. Appl. Environ. Microbiol. 74:823, 2008. (3) W. A. Vargas et al. Plant Physiol. 158:1342, 2012.
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