Direct‐PCR Detection and Epidemiology of Ramularia collo‐cygni Associated with Barley Necrotic Leaf Spots

Abstract

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. collocygni (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 crossreactions 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.

Ramularia collo-cygni: the biology of an emerging pathogen of barley

“…4). Recent work by Frei et al . (2007) has shown that winter barley is an important source of inoculum for spring barley and that the pathogen can spread from the winter to the spring crop.…”

“…It is important to note that the symptoms of RLS can easily be confused with those caused by abiotic stresses (physiological leaf spots, PLS; Wu & von Tiedemann, 2002) and by pathogens such as Pyrenophora teres (Sachs et al , 1998). In fact, some authors have observed that R. collo‐cygni was never the single cause of necrotic leaf spotting in experiments carried out under field conditions in Switzerland (Frei et al , 2007). In this work, most necrotic leaf spotting was attributable to PLS.…”

Infection strategy of Ramularia collo‐cygni and development of ramularia leaf spot on barley and alternative graminaceous hosts

“…RLS can lead to yield losses of up to 20% in barley, with an average loss in Scotland of 0.4 tonnes ha −1 (Oxley & Havis, ). The development of PCR‐based methods for detection of the fungus in barley tissue has expanded understanding of the pathogen's life cycle, particularly the importance of seedborne infection in disease aetiology (Havis et al ., , ; Frei et al ., ; Taylor et al ., ). Recent evidence has suggested that Rcc is likely to undergo sexual reproduction (Piotrowska et al ., ); however, there are many unknowns still surrounding the infection process and biology of this organism.…”

Evidence for seed transmission and symptomless growth of Ramularia collo‐cygni in barley (Hordeum vulgare)

“…The development of molecular-based diagnostic tests for Rcc have allowed more detailed studies of the ecology, aetiology and epidemiology of this poorly understood pathogen (Havis et al, 2006a;Frei et al, 2007). Visual crop assessments and diagnostic analysis have indicated that the pathogen moves up the plant during the growing season and also infects awns and ears .…”