Molecular studies on the taxonomy, host-associations and viruses of the Diplodia-like anamorphs of the Botryosphaeriaceae

Abstract

The Botryosphaeriaceae is a family of fungi that includes many species, which are well-known as pathogens, saprophytes and endophytes of plants and especially of trees. As a result of their pathogenic nature and potential threat to plantations and agricultural crops, much research has been devoted to their identification. The main focus of studies that make up this thesis has been on the fungal complex referred to as Diplodia pinea sensu lato. These fungi are members of the Botryosphaeriaceae and studies have specifically concentrated on their taxonomy, host associations and mycovirus infections associated with them. Diplodia pinea sensu lato represents a species complex of highly similar morphological types that mainly infect Pinus spp., world-wide. The species complex includes what have in the past been known as the A, B and C morphological types of D. pinea. Multiple gene genealogies based on sequences of partial protein-coding genes and microsatellite markers were used to resolve the species complex into two genera, D. pinea and D. scrobiculata (= B morphotype). Diplodia-like isolates from Australia, Greece and Cyprus were characterized using both morphological and molecular characteristics. Morphologically, these isolates all have dark, thick-walled conidia (Diplodia-like) but phylogenetically, they could belong to three distinct genera of the Botryosphaeriaceae namely Diplodia, Lasiodiplodia and Dothiorella. Results of this study led to the description of Dothiorella casuarini from Casuarina spp. in Australia and they highlight the fact that similar morphological characteristics and disease etiology does not necessarily provide a true reflection of the evolutionary history of a pathogen. Phylogenetic studies on species of the Botryosphaeriaceae with Diplodia-like anamorphs revealed intriguing host association patterns. The availability of sequence data for many species of the Botryosphaeriaceae made it possible to extend the phylogeny to include six of the ten lineages as previously described for the Botryosphaeriaceae. Angiosperms appeared to be the most common, and possibly ancestral, host group of the Botryosphaeriaceae, with the exception of Macrophomina, Guignardia, Saccharata and “Botryosphaeria” quercuum. Infection of gymnosperms most likely occurred more recently, only in specific groups (Diplodia and Lasiodiplodia) via host shifts. Three distinct viruses have now been characterized from isolates of D. pinea sensu lato. Two of these were previously characterized and are known as Sphaeropsis sapinea RNA virus 1 and 2 (SsRV1 and SsRV2). The third dsRNA element more commonly found in association with D. scrobiculata was characterized in this dissertation and named Diplodia scrobiculata RNA virus 1 (DsRV1). It has a genome of 5018 bp with a unique genome organization characterized by two open reading frames (ORFs). One ORF codes for a putative polypeptide similar to proteins of the vacuolar protein-sorting (VPS) machinery and the other one for a RNA dependent RNA polymerase (RdRp). The hypothetical protein probably has a role in transport or protection of this unencapsulated virus into membranous vesicles. Phylogenetically, DsRV1 groups closest to a dsRNA element from Phlebiopsis gigantea (PgV2) and they both group separately from other families in which fungal viruses have been classified. The frequency and distribution of DsRV1, SsRV1 and SsRV2 were determined in a collection of D. pinea and D. scrobiculata isolates using Real-time PCR. Infections with SsRV1 and SsRV2 occurred in both D. pinea and D. scrobiculata, while DsRV1 was mainly found in D. scrobiculata. DsRV1 was also found to always occur in combination with SsRV1 and/or SsRV2. Therefore, DsRV1 probably selected against a coat protein as the result of a fitness trade-off. Although earlier studies indicated that these viruses have no effect on the phenotype or virulence of D. pinea and D. scrobiculata isolates, the presence of specific viruses in their host populations serve as a useful marker in studying movement of fungal pathogens. The ultimate aim of studies making up this dissertation was to expand the base of knowledge regarding species in the D. pinea species complex. This was justified by the fact that D. pinea is one of the most important tree pathogens in South Africa and that an expanded knowledge might contribute to reducing diseases caused by it. Clearly understanding the identity of the fungus must clearly underpin many elements of a management strategy and this was one of the aims of the suite of studies conducted. Furthermore, I attempted to augment the knowledge base regarding dsRNA elements in D. pinea sensu lato. These studies were of a basic nature and relatively far removed from the practical application level. Nonetheless, it is my hope that they have pushed ahead knowledge barriers and that in some way they will contribute to reducing the impact of Diplodia-associated diseases in the future.