Archivio Journal of Plant Pathology

Abstract:

Ramularia collo-cygni B. Sutton & J.M. Waller, the causal agent of Ramularia leaf spot, is reported for the first time in South Africa. The fungus was isolated from barley (Hordeum vulgare L.) leaves grown in the Western Cape Province. Infected plants, with typical Ramularia leaf spot symptoms of small brown rectangular lesions, some surrounded by a yellow halo, visible on both sides of the leaf, were first observed in September 2015. Brown spots were also visible on dead leaves. Microscopic investigation revealed immersed mycelium, with hyaline, septate, branched hyphae colonizing the mesophyll. Dense white to pinkish fungal colonies with a vinaceous reverse, were isolated on potato dextrose agar from barley leaves harvested from four fields in the Overberg region. BLAST searches of the Internal Transcribed Spacer (ITS) region of five isolates (GenBank accession Nos. KX156947-KX156951) resulted in 100% similarity with R. collo-cygni reference sequences. The presence of R. collo-cygni DNA was furthermore confirmed in planta in four different symptomatic barley leaf samples, following DNA extractions and PCR reactions as described by Havis et al. (2006). R. collo-cygni isolate IPP 494 (Balz, 2010) DNA, supplied by Proff. von Tiedemann and Karlovsky at the Georg-August University in Göttingen, Germany, was used as positive control. Pyrenophora teres and Alternaria alternata DNA was included as negative controls, because of their association with barley leaves and the similarity in barley leaf symptoms caused by P. teres. DNA from isolate IPP 494 and the positive barley samples resulted in a 426 base pair PCR product when visualised on an agarose gel following gel electrophoresis. No PCR products were observed for P. teres, A. alternata and the no template control. To our knowledge, this is the first report of R. collo-cygni in barley leaves in South Africa.