Detection, accumulation, and host range of the Botrytis cinerea viral pathogen, BGDaV1

Abstract

Botrytis cinerea is an important fungal pathogen with a host range of over 200 plant species. Chemical fungicides are the main form of control, however, due to B. cinerea developing resistance to chemical controls, alternative methods are needed, namely biocontrols. Botrytis Gemydayvirus 1 (BGDaV1) is a proposed biocontrol of B. cinerea. This virus is a recently discovered ssDNA mycovirus in the family Genomoviridae, which has previously been shown to be mechanically transmissible to B. cinerea, and slow fungal growth. The aims of this thesis were: 1) find a suitable method for BGDaV1 DNA extraction, and optimal conditions for virus propagation, 2) determine the optimum BGDaV1 infection route into new B. cinerea isolates and, 3) determine the host range of BGDaV1. To establish the most suitable DNA extraction method, three extraction techniques were trialled; the DNeasy® plant mini kit, and CTAB method one and two, which differed in reagents used and time to completion. CTAB method two was shown to produce the highest quality DNA at the most consistent rate. To determine the optimal conditions for virus titre several factors were tested; effects of B. cinerea mycelium age, number of fungal passages, and temperature and light exposure. While age of the culture (up to 3 weeks) did not affect BGDaV1 titre, there was a slight reduction as the number of passages increased. Of the temperature and light conditions tested BGDaV1 accumulated the most, and with the most consistency when B. cinerea was grown at 28°C in constant darkness. To determine the optimum route of BGDaV1 infection to B. cinerea, four infection trials were analysed (complete purification and partial purification one and two, and side-by-side infection). The optimum methods for BGDaV1 infection of B. cinerea were the partial purification trial two and the side-by-side infection assay. To determine host range, first infection of B. cinerea grown on minimal media was trialled. The minimal media trial did not increase significantly the BGDaV1 transmission efficiency. BGDaV1 infection was then attempted on ten species and 18 isolates of fungi in the family Sclerotinaceae and genera Botrytis, Sclerotinia and Monilinia, with the partial purification trial and the side-by-side infection assay. These differed in efficiency but showed BGDaV1 likely has a broad host range.