Abstract:
The ecological significance of mycoviruses is becoming increasingly recognised, not just for their potential as biocontrol agents but also as driving forces in the evolution and diversification of fungi. Therefore, it is important to understand how mycoviruses and fungi interact on the molecular and biochemical level. To this end the interaction between Botrytis cinerea and the mycoviruses Botrytis virus F and Botrytis virus X was studied. Relative and absolute real time PCR protocols were developed for monitoring the titres of BVX and BVF during transfection studies to monitor changes in virus titre in relation to phenotypic and metabolic changes in the fungal host. Phenotypic changes included severe phenotypical alterations, which were associated with extreme up regulation of carbohydrate, amino acid and lipid metabolism, and induction of stress responses (vacuolisation/cell lysis, increased pigmentation). To study the location and distribution of BVX in infected Botrytis the BVX coat protein was recombinantly expressed in E. coli, BVX specific polyclonal antibodies produced, and protocols developed for the serological detection and visualisation of BVX. Immuno-fluorescence microscopy was used to studying the distribution of BVX within growing Botrytis cultures indicated that the virus is present in aggregates located attached to the cell membrane, the septum, in spores, and in hyphal tips. A combination of light and electron microscopy showed that BVX is often closely associated with cell walls, suggesting that the virus may be moving across the cell wall by altering cell wall composition. If this is shown to be the case then it provides an alternative method to transmission via hyphal anastomosis, which is currently considered to be the only method of horizontal transfer.
