Infection biology of the plant pathogenic fungus Bipolaris sorokiniana
Abstract: Bipolaris sorokiniana is a fungal pathogen causing severe disease mainly in wheat and barley worldwide. In this thesis two aspects of the infection process were investigated: the adhesion mechanism of the fungus and the role of the non-host specific phytotoxin prehelminthosporol (PHL) in the disease. Adhesion of B. sorokiniana to artificial solid surfaces occurred in two stages: the first by conidia and the second by germlings. Conidial adhesion was mediated by weak hydrophobic forces and occurred only on hydrophobic surfaces. The second stage of adhesion was firm, surface unspecific and accompanied with conidial germination. Concomitant with germling adhesion, the fungus produced an extracellular matrix (ECM). Germlings treated with the protease Pronase E were not adhesive and were lacking ECM proteins. This together with the facts that a protein glycosylation inhibitor and several lectins inhibited adhesion indicated that glycoproteins present in the ECM are involved in germling adhesion. The ultrastructure and composition of the ECM were investigated using electron and light microscopy based methods. The ECM labelled intensively for both proteins and polysaccharides but the proportion of these compounds changed with the morphological stage of the fungus. The ECM proteins were labelled with a membrane impermeable biotinylating agent and extracted using a HCl-glycine buffer. Two-dimensional gel electrophoresis revealed the presence of about 40 proteins on the fungal cell surface. Four of the ECM proteins were isolated, in gel digested with the enzyme trypsin and sequenced by MS/MS. No significant sequence similarity to proteins in databases was obtained. To evaluate the contribution of phytotoxic metabolites to the disease, in vitro production of the toxin PHL in different isolates of B. sorokiniana was quantified and compared to the virulence of the isolates towards barley root seedlings. The isolates with low PHL production had lower virulence compared to those with higher production of PHL, indicating that PHL contributes significantly to the disease severity. However, the virulence did not increase with PHL level among isolates producing high levels of the toxin.
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