Role of seedborne infection in rhynchosporium and ramularia epidemics in barley (PhD)

Two economically important pathogens of barley, Rhynchosporium secalis and Ramularia collo-cygni, were studied in this project to better understand disease development and resistance. Prior to symptom development, asymptomatic infection plays vital role in both these pathogens and this has been the focus of the research. This thesis describes the development of a stable transformation protocol, which enabled production of transgenic lines of both pathogens expressing fluorescent markers and the use of these transgenic lines, coupled with quantitative real-time PCR, confocal microscopy techniques and development of an image analysis procedure to study the infection of representative barley genotypes.

R. secalis was transformed to express the green fluorescent protein (GFP) and DsRed fluorescent protein using an Agrobacterium-mediated transformation procedure. This produced stable transformants which were used to perform infection assays under controlled environment. A novel transformation protocol combining both protoplast regeneration and Agrobacterium-mediated transformation was developed to transform R. collo-cygni to express GFP and DsRed fluorescent proteins.

The R. secalis infection pattern was studied using confocal microscopy initially on cultivars carrying known resistance gene. Analysis of the infection pattern was performed using an image analysis protocol that was developed during this project. This revealed significantly different infection patterns on near-isogenic lines of barley (Atlas and Atlas 46) and on cultivars Osiris (Rrs1) and Digger (Rrs2) both pairs differing by presence or absence of the Rrs1 resistance gene. This process was then applied on eight other spring or winter barley genotypes with varying levels of field resistance. Results showed that resistance exerted by Rrs1 failed to completely arrest either pathogen growth or sporulation. In total, twelve different barley cultivar interactions with an isolate of R. secalis were monitored at different time points over the course of infection cycle. These data indicate that resistance mechanisms other than those triggered by Rrs1 might be involve in restriction of the fungal growth.

Quantitative PCR (qPCR) was also performed on seeds and seedlings of various barley cultivars that revealed an active transfer of inoculum from seed to seedling from naturally infected seeds but not from artificially infected ones. This suggests that the seed infection may occur systemically during grain maturation, irrespective of the cultivar’s field resistance.

This thesis describes the first report of successful transformation of R. collo-cygni using a novel method that exploits regenerating protoplasts and Agrobacterium. The infection cycle of R. collo-cygni on barley was studied microscopically on barley using the GFP-expressing isolate produced using this method. A time series observation of the entire infection cycle of R. collo-cygni was performed using confocal microscopy. This revealed the basic infection mechanisms of the pathogen, which targets the sub-stomatal cavity in barley and that sporulation could occur during the asymptomatic phase of the infection cycle.