Direct-PCR detection and epidemiology of Ramularia collo-cygni associated with barley necrotic leaf spots

Ramularia collo-cygni ( Sutton & Waller) is involved in a disease complex of barley characterized by the formation of necrotic spots on leaves. Isolation of this fungus is difficult, which complicates the study of its epidemiology and the aetiology of the disease complex. A new assay based on polymerase chain reaction ( PCR) was developed to detect the presence of R. collo-cygni ( Rcc) without previous isolation of the fungus nor prior purification of DNA. Primers RC3 and RC5 were designed to amplify a 348 bp fragment of the internal transcribed spacer region of this pathogen. These primers were highly specific to Rcc, as no cross-reactions were observed with other barley pathogens or saprobes commonly found on this crop. Amplification was possible from crude extracts ( direct-PCR), circumventing the need for a DNA purification. Detection of 1 fg of target DNA was achieved with a single PCR. This specific and sensitive assay was used to study the epidemiology of Rcc on winter and spring barley at two locations in Switzerland. Rcc was present on winter barley from snow melting until harvest and colonized gradually all leaf layers. It was also found on volunteers, which could, together with weeds, offer a survival possibility to the pathogen between two barley crops. The fungus was detected on spring barley only after it sporulated on winter barley, indicating that Rcc probably spreads from winter to spring barley. The hypothesis of Rcc being a seed-borne pathogen can be tested more easily with this fast and reliable molecular tool, which may also find applications in barley breeding programmes and fungicide trials.