Infection biology and life cycle of Ramularia collo-cygni
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Date
29/06/2015Author
Kaczmarek, Maciej
Metadata
Abstract
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.