Infection biology and life cycle of Ramularia collo-cygni
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In recent years, a new threat to barley crops has emerged and gained substantial attention due to its rising economic importance. Relatively little is known about the infection strategy and development of Ramularia leaf spot (RLS) disease on barley. Therefore the overall aim of this project was to increase the understanding of fundamental biology and life cycle of the causal agent, R. collo-cygni. Chapter 2 describes the horizontal transmission of the fungus on barley. Both field and transgenic R. collo-cygni isolates expressing GFP and dsRed fluorescent reporter markers were utilised to visualise the infection progression in living host tissues by various light and confocal microscopy. The existence of a previously unknown structure called stomatopodium (infection peg), involved in the penetration of stomata, was demonstrated. The fungus initially exhibited symptomless epiphytic growth, extending above epidermis and connecting the hyphal aggregates inside substomatal cavities and subsequent initial sporulation. However, during the transition into symptomatic phase, the organised intercellular growth of hyphae into the mesophyll was observed. This hyphal network was involved in the production of asexual spores demonstrating that the raprture of epidermal layer was responsible for local necrosis observed for RLS. In addition to barley, several other speculated R. collo-cygni hosts have been used to verify their compatibility to the pathogen. In chapter 3, a whole plant inoculation assay was developed to investigate the mode of the fungal seed-borne transmission by using GFP expressing strain of the fungus. It is shown here for the first time that the vertical transmission is systemic, involving symptomless colonisation of embryo and closely resembled the mode of dissemination observed for Neotyphodium species, mutualistic fungal endosymbionts on grasses.The impact of fungal infection on seed germination ability was also examined that revealed no significant difference between clean, moderate and high levels of R. collo-cygni DNA. Chapter 4 represents an attempt to discover and analyse the sexual development in R. collo-cygni. As a first step to understand the sexual reproduction cycle in this apparently asexual species, the genetic structure of the mating system was characterised by using PCR-based techniques which demonstrated the heterothallic nature of the fungus. The defined population of R. collo-cygni field isolates was then screened for the presence of the discovered mating type idiomorphs (mat) to determine the frequencies of the mating types in the defined R. collo-cygni populations. The segregation ratio of mat1 and mat2 close to 1:1 indicated a frequent sexual reproduction. In order to verify the existence of functional sexual stage in R. collo-cygni, potential sexual development was induced using the potentially compatible isolates and a comprehensive analysis was undertaken by correlative use of light-, confocal- and low temperature scanning electron microscopy. Two types of multicellular bodies were observed and described. First was the speculated Asteromella stage (male donor) that carries spore-like spermatia. The second structure initially resembled sclerotia that in only a few instances developed into perythecium/ pseudothecium that appeared to carry the sexual spores, ascospores enveloped in asci. Chapter 5 demonstrates the role of rubellin toxin in symptom development by using autofluorescence phenomenon. The structure of putative molecular machinery involved in rubellin biosynthesis was addressed by using bioinformatics approaches and the complete R. collo-cygni genome sequence. A gene cluster encompassing several components of other known secondary metabolite biosynthesis pathways, such as that of dothistromin and aflatoxin, was found and putative protein function of the genes is hypothesised.