Effects of growth Medium, temperature and mycelium age on CHV-1 accumulation and transmission

Abstract

Some mycoviruses such as Cryphonectria hypovirus 1 (CHV-1) have a potential application for biocontrol of Cryphonectria parasitica, the causal agent of chestnut blight, but little is known about the factors that regulate the fungus-virus interaction, especially when the concentration of the virus is low as it is usual in the wider environment in England. In this study, four CHV-1 infected isolate types of C. parasitica were tested for parameters relating to hypovirulence by inoculating onto potato dextrose agar (PDA), apples, and sweet chestnut branch fragments. Isolate L-6 (CHV-1 subtype I, haplotype E-5), together with the positive control isolate M784 (same haplotype), presented the highest hypovirulence values. The lowest hypovirulence was observed with isolate M784 previously kept in the cold at 8 °C, followed by isolate Db-1 (same haplotype but naturally mutated). The differences in hypovirulence were much more pronounced when inoculations took place using mycelial plugs taken from the edge of plate colonies as opposed to the centre of a colony. The same strains were used to further evaluate the effects of the growth medium (PDA or plant material), optimum and cold temperatures, and mycelium age (centre or edge plugs) on the respective virus concentration. Virus RNA was extracted and amplified by RT-PCR and the concentration of the CHV-1 DNA band (394 bp) measured. Virus concentration decreased when its host was growing on PDA when compared to its host growing on plant material. Virus concentration was reduced further if held in the cold temperature for 7 days, and the virus concentration decreased even further after an additional incubation period at 25 °C. Virus isolate L-6, sub-cultured from the colony centre, was the only one that recovered to its original concentration following incubation for 7 days on sweet chestnut wood fragments. The concentration of the virus was more stable in the centre of the plates than that found at the edge of a plate colony. Together with the expected 394 bp CHV-1 amplicon, another 90 bp band was occasionally simultaneously amplified using the same RT-PCR procedure. This band was quantified, and sequenced, finding no homologs. It is first time this satellite amplicon has been described and its concentration related with hypovirulence attributes of the virus infected C. parasitica, such as reduced fungal growth, and positive correlation with donor and recipient CHV-1 band load in donor-recipient transmission experiments. We discuss our findings in terms of previous parameters related to hypovirulence.