Epidemiology and pathogenicity of Ramularia collo-cygni associated with barley necrotic leaf spot disease

摘要

Ramularia collo-cygni has gained increasing importance as the causal agent of a novel leaf spot disease on barley, Ramularia leaf spot (RLS). The fact that R. collo-cygni (Rcc) has been recognized as a major pathogen of barley only relatively recently is due in large part to difficulties in isolating and identifying the fungus. Although this novel barley pathogen has been studied more intensely in recent years its complete life cycle, particularly the sources of inoculum and the spread of the fungus between different fields and seasons still awaits full elucidation. Recently, molecular bioanalytical tools based on PCR have been developed and are now available for the detection of R. collo-cygni in plant tissue (Havis et al., 2006). Investigating the life cycle R. collo-cygni with PCR based diagnostic has indicated that the fungus stays in latent state forextended periods of time during the season. In the field, it can be detected much earlier than the appearance of visible symptoms but the earliest detection date varies between season and variety (Frei et al., 2007). At the end of the growing season the pathogen is widespread in harvested grains and can be transmitted to developing plants (Havis et al., 2006). The present study confirmed the occurrence of the fungus in latent state during all plant growth stages including seeds. Seeds harvested from a severely infected barley field near Eschwege, Germany were analysed with PCR and showed high incidence of latent infestation. The seeds were sown in pots and kept in controlled conditions (20°C and 14 hours daylight). Plants grown from such seeds were analysed at different growth stages from seedling stage to maturity by qualitative PCR. Analyses revealed the transmission of the fungus from seeds into emerging plants and confirmed the further spread of the fungus in adult plants in a symptom-less manner. Under greenhouse conditions, the symptomless stage of RLS persisted throughout the first generation of plants emerging from infested seeds until maturity and grain formation. This is in contrast to plants grown in the field as shown in previous work indicating that the sudden and relatively late outbreak of RLS symptoms in field-grown barley is related to a loss in physiological vigour during senescence (Schutzendubel et al., 2008). It remains to be determined whether this outbreak of symptoms in field-grown barley derives from symptomless internal infestation or from infection with conidia delivered from the environment, or both. The application of seed hot water treatment (48°C for 5 minutes) did not appear to eliminate the presence of R. collo-cygni or impair the transfer of the pathogen into the developing plant. Similarly, experiments were carried out to test whether seed dressing with fungicides would remove Rcc from seeds. Preliminary results show partial efficacy and differences between the active ingredients ngedients used and therefore these studies will be further going on. In a parallel study, the mobility and spread of Rcc inoculum through the air and over large distances is being investigated. The long-range transport of conidia was studied with spore traps placed in autumn and winter either close to a barley field or in a place at long distance from any fields. On one hand, Petri dishes were used containing vegetables juice agar amended with the selective fungicide Acanto (a.i. picoxystrobin) and Ampicillin and installed 1.5 m above ground level near a barley field. All Petri dishes were brought to the laboratory after being evosed outside for 24 hours. In the lab plates were incubated for 10 days at 20°C under UV light and examined periodically for the development of fungal colonies. On the other hand rain samples. were collected on the roof of a building at several kilometres distance from barley fields and about 30 m above ground. Rain and snow samples were first filtered through 0.2 pm filter membranes and then membranes were used directly for DNA extrac