Investigations of mycoviruses from Leptosphaeria species and their effects on pathogenicity

Abstract

The aims of this research were to assess the incidence of dsRNA viruses in five different plant pathogenic fungi, including Hymenoscyphus fraxineus, Dothistroma septosporum, Leptosphaeria species (Leptosphaeria maculans and Leptosphaeria biglobosa) and Pyrenopeziza brassicae, as their incidence has not been reported previously in these species, and then to investigate the effects of mycoviruses on the growth and pathogenicity of the fungi. Hence, 57 L. maculans and 16 L. biglobosa isolates were screened and 11 L. biglobosa isolates possessed dsRNA elements while only one of the 45 D. septosporum isolates was found to contain a mycovirus subsequently identified as a chrysovirus. In contrast, none of the 162 H. fraxineus and 10 Pyrenopeziza brassicae isolates appeared to contain dsRNA elements. Further research was carried out on L. biglobosa because of the excellent recovery of dsRNA elements and the fact that it is also responsible for causing phoma stem canker which is an economically important disease of oilseed rape worldwide. However, not only L. biglobosa but also the more damaging L. maculans causes phoma stem canker. They are closely related and co-existing plant pathogens. Mycoviruses are a specific group of viruses that infect and replicate in fungi. They can be associated with hypovirulence or hypervirulence but are normally cryptic. Three different mycoviral dsRNAs were identified from L. biglobosa isolate C-Rox 12.8.1, ranging in size from ca. 4.0-4.9 kbp. Sequence analysis of LbMV-1 dsRNAs 1 and 2 revealed that they are most closely related to members of the family Totiviridae. However, attempts to characterise LbMV-1 dsRNA 3 failed. On the other hand, four mycoviral dsRNAs were identified from D. septosporum isolate D 752.1, ranging in size from ca. 2.8-3.5 kbp. Molecular characterisation of DsCV-1 showed that it is very similar to the chrysoviruses. To obtain information on the effects of dsRNAs on their host, cycloheximide treatment was used to eradicate dsRNA elements from L. biglobosa isolate W10. Subsequently, comparative growth experiments to assess radial growth and mycelial weight for virus-infected and virus-free W10 isolates were performed. These experiments showed that the LbMV-1 infection increased the growth of the fungus. To investigate the influence of mycoviruses on the pathogenicity of L. biglobosa, pathogenicity tests were performed using isogenic lines of the W10 isolate inoculated onto oilseed rape cotyledons and disease symptoms were analysed at different time periods; mycoviruses were found to increase fungal pathogenicity. In addition to this, effects of pre-treatment of B. napus leaves with conidia of virus-infected or virus-free L. biglobosa on infection by conidia of L. maculans and development of disease (phoma leaf spot) were studied in a controlled-environment conditions. Pre-treatment of first true leaves with virus-infected L. biglobosa decreased the phoma leaf spot lesion area on second true leaves (systemic effect) as compared to pre-treatment with virus-free L. biglobosa.