Taxonomy and phylogeny of Ophiostoma spp. with Sporothrix anamorphs and their generic relationships in the Ophiostomatales

The ophiostomatoid fungi included more than 450 species of ascomycetes specifically adapted for insect dispersal. Many of these species have a significant economic impact as sapstaining fungi or tree pathogens harmful to forestry industries, but some are also as opportunistic human pathogens. DNA based studies in recent years have shown that the majority of these fungi belonged in either the Ophiostomatales or Microascales (Sordariomycetes), with a few Sporothrix spp. grouping in the Microstromatales (Ustilaginomycetes). However, most phylogenetic studies have focussed on restricted numbers of taxa sharing similar morphology. The aim of the studies in this thesis was to reconsider the taxonomy of all the ophiostomatoid fungi at the order and family levels, and the status of genera and species with sporothrix-like anamorphs in the Ophiostomatales and Microstromatales. All available published sequence data were screened for reliable sequences representing as many species as possible, and new data were generated where necessary for ex-type or other isolates. The resulting phylogenies enabled the formal redefinition of the Ophiostomatales and Ophiostomataceae, and the description of two new families, the Graphiaceae (Microascales) and Quambalariaceae (Microstromatales). Problems relating to the delineation of Ophiostoma s.l., Leptographium s.l., and Raffaelea s.l. were exposed and discussed, 18 species complexes were defined in the Ophiostomatales, and four genera were formally redefined: Sporothrix, Graphium, Graphilbum and Knoxdaviesia. Forty six new combinations were made, primarily in Sporothrix, Ophiostoma, Graphilbum and Knoxdaviesia. One nomen novum was erected in Ceratocystis and one new Quambalaria species was described. A comprehensive nomenclator for 596 ophiostomatoid species including references to all descriptions, synonymies and phylogenetic data was also compiled. This study represents the first comprehensive, all-inclusive assessment of the taxonomy and nomenclature of the ophisotomatoid fungi based on phylogenetic relationships and the one fungus one name principles. Finally, the immediate and indiscriminate application of the one fungus one name principles in Ophiostoma s.l. and Leptographium s.l. might result in many unnecessary name changes. Thus, several recommendations have been made to ensure nomenclatural stability in these genera in the immediate future and until more robust phylogenies become available that can refine the delineation of these genera.PhDMicrobiology and Plant PathologyUnrestricte

DNA sequence comparisons of Ophiostoma spp., including Ophiostoma aurorae sp. nov., associated with pine bark beetles in South Africa

Bark beetles (Coleoptera: Scolytinae) are well-recognized vectors of Ophiostoma species. Three non-native bark beetle species infest various Pinus species in South Africa, and they are known to carry at least 12 different species of ophiostomatoid fungi. Some of these fungi have not been identified to species level. The aim of this study was to determine or confirm the identities of Ophiostoma species associated with bark beetles in South Africa using comparisons of DNA sequence data. Identities of Ophiostoma ips, O. floccosum, O. pluriannulatum, O. quercus and O. stenoceras were confirmed. Ophiostoma abietinum, O. piliferum and Pesotum fragrans are recognised for the first time and the new species, O. aurorae sp. nov., is described from pine-infesting bark beetles in South Africa

DNA-based identification of Quambalaria pitereka causing severe leaf blight of Corymbia citriodora in China

Quambalaria spp. include serious plant pathogens, causing leaf and shoot blight of Corymbia and Eucalyptus spp. In this study, a disease resembling Quambalaria leaf blight was observed on young Corymbia citriodora trees in a plantation in the Guangdong Province of China. Comparisons of rDNA sequence data showed that the causal agent of the disease is Q. pitereka. This study provides the first report of Quambalaria leaf blight from China, and it is also the first time that this pathogen has been found on trees outside the native range of Eucalypts

Exotic Invasive Elm Bark Beetle, \u3cem\u3eScolytus kirschii\u3c/em\u3e, Detected in South Africa

In February 2005, the exotic bark beetle, Scolytus kirschii (Curculionidae: Scolytinae), was detected infesting English elms (Ulmus procera) in Stellenbosch, South Africa. This appears to be the first report of an infestation of Scolytus species in this country. The presence of this beetle is of concern for several reasons. Scolytus kirschii is a serious pest of elms, capable of killing healthy trees, resulting in considerable economic impact. There also exists the possibility that the beetle may undergo a host switch to indigenous trees, with potentially serious ecological consequences. Furthermore, the beetle is capable of being the vector of the pathogens responsible for Dutch elm disease (DED), Ophiostoma ulmi and Ophiostoma novo-ulmi. None of the trees that we inspected in Stellenbosch exhibited symptoms or signs of DED. Isolations from infested host material likewise failed to detect these pathogens. Nonetheless, the damage to the trees by the beetles alone was sufficient to cause tree death. Future directions for research and management of the beetle in its new environment are discussed

Ophiostoma denticiliatum sp. nov. and other Ophiostoma species associated with the birch bark beetle in southern Norway

Ophiostomatoid fungi were isolated from Scolytus ratzeburgi infesting Betula pendula and B. pubescens in Norway. Fungi were identified based on morphology, DNA sequence comparison for two gene regions and phylogenetic analyses. The most abundant fungus was Ophiostoma karelicum, suggesting a specific relationship between the fungus, the vector insect and the host tree. Our results suggest that O. karelicum occurs across the geographic range of S. ratzeburgi and its close relatedness to the Dutch elm disease fungi suggests that it could be important if introduced into other parts of the world. Other fungi, only occasionally isolated from S. ratzeburgi, were identified as O. quercus and a novel taxon, described here as O. denticiliatum sp. nov

Multi-gene phylogenies define Ceratocystiopsis and Grosmannia distinct from Ophiostoma

Ophiostoma species have diverse morphological features and are found in a large variety of ecological niches. Many different classification schemes have been applied to these fungi in the past based on teleomorph and anamorph features. More recently, studies based on DNA sequence comparisions have shown that Ophiostoma consists of different phylogenetic groups, but the data have not been sufficient to define clear monophyletic lineages represented by practical taxonomic units. We used DNA sequence data from combined partial nuclear LSU and β-tubulin genes to consider the phylogenetic relationships of 50 Ophiostoma species, representing all the major morphological groups in the genus. Our data showed three well-supported, monophyletic lineages in Ophiostoma. Species with Leptographium anamorphs grouped together and to accommodate these species the teleomorph-genus Grosmannia (type species G. penicillata), including 27 species and 24 new combinations, is re-instated. Another well-defined lineage includes species that are cycloheximide-sensitive with short perithecial necks, falcate ascospores and Hyalorhinocladiella anamorphs. For these species, the teleomorph-genus Ceratocystiopsis (type species O. minuta), including 11 species and three new combinations, is re-instated. A third group of species with either Sporothrix or Pesotum anamorphs includes species from various ecological niches such as Protea infructescences in South Africa. This group also includes O. piliferum, the type species of Ophiostoma, and these species are retained in that genus. Ophiostoma is redefined to reflect the changes resulting from new combinations in Grosmannia and Ceratocystiopsis. Our data have revealed additional lineages in Ophiostoma linked to morphological characters. However, these species are retained in Ophiostoma until further data for a larger number of species can be obtained to confirm monophyly of the apparent lineages

Phylogenetic analyses of diverse <i>Podaxis</i> specimens from Southern Africa reveal hidden diversity and new insights into associations with termites

<p>Although frequently found on mounds of the grass-cutting termite genus . Trinervitermes, virtually nothing is known about the natural history of the fungal genus . Podaxis (Agaricaceae) nor why it associates with termite mounds. More than 40 species of this secotioid genus have been described since Linnaeus characterised the first species in 1771. However, taxonomic confusion arose when most of these species were reduced to synonymy with . Podaxis pistillaris in 1933. Although a few more species have since been described, the vast majority of specimens worldwide are still treated as . P. pistillaris. Using 45 fresh and herbarium specimens from Southern Africa, four from North America and one each from Ethiopia, and Kenya, we constructed the first comprehensive phylogeny of the genus. Four of the genotyped specimens were more than 100 y old. With the exception of the type specimen of . Podaxis rugospora, all herbarium specimens were labelled as . P. pistillaris or . Podaxis sp. However, our data shows that the genus contains at least five well-supported clades with significant inter-clade differences in spore length, width and wall thickness, and fruiting body length, supporting that clades likely represent distinct . Podaxis species. Certain clades consistently associate with termites while others appear entirely free-living.</p

Actinomadura rubteroloni sp. nov. and Actinomadura macrotermitis sp. nov., isolated from the gut of the fungus growing-termite Macrotermes natalensis

The taxonomic positions of two novel aerobic, Gram-positive actinobacteria, designated strains RB29$^{T}$ and RB68T, were determined using a polyphasic approach. Based on 16S rRNA gene sequence analysis, the closest phylogenetic neighbours of RB29$^{T}$ were identified as Actinomadura rayongensis DSM 102126$^{T}$ (99.2 % similarity) and Actinomadura atramentaria DSM 43919$^{T}$ (98.7 %), and for strain RB68$^{T}$ was Actinomadura hibisca DSM 44148$^{T}$ (98.3 %). Digital DNA–DNA hybridization (dDDH) between RB29$^{T}$ and its closest phylogenetic neighbours, A. rayongensis DSM 102126$^{T}$ and A. atramentaria DSM 43919$^{T}$, resulted in similarity values of 53.2 % (50.6–55.9 %) and 26.4 % (24.1–28.9 %), respectively. Additionally, the average nucleotide identity (ANI) was 93.2 % (94.0 %) for A. rayongensis DSM 102126$^{T}$ and 82.3 % (78.9 %) for A. atramentaria DSM 43919$^{T}$. dDDH analysis between strain RB68$^{T}$ and A. hibisca DSM 44148$^{T}$ gave a similarity value of 24.5 % (22.2–27.0 %). Both strains, RB29$^{T}$ and RB68$^{T}$, revealed morphological characteristics and chemotaxonomic features typical for the genus Actinomadura, such as the presence of meso-diaminopimelic acid in the cell wall, galactose and glucose as major sugar components within whole-cell hydrolysates and the absence of mycolic acids. The major phospholipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol and phosphatidylinositol mannoside. Predominant menaquinones were MK-9(H$_{6}$) and MK-9(H$_{8}$) for RB29$^{T}$ and MK-9(H$_{4}$) and MK-9(H$_{6}$) for RB68T. The main fatty acids were identified as 10-methyloctadecanoic acid (10-methyl C$_{18:0}$), 14-methylpentadecanoic acid (iso-C$_{16:0}$), hexadecanoic acid (C$_{16:0}$) and cis-9-octadecanoic acid (C$_{18:1}$ ω9c). Here, we propose two novel species of the genus Actinomadura: Actinomadura rubteroloni sp. nov. with the type strain RB29$^{T}$ (=CCUG 72668$^{T}$=NRRL B-65537$^{T}$) and Actinomadura macrotermitis sp. nov. with the type strain RB68$^{T}$ (=CCUG 72669$^{T}$=NRRL B-65538$^{T}$)

Phylogeny of the Quambalariaceae fam. nov., including important Eucalyptus pathogens in South Africa and Australia

The genus Quambalaria consists of plant-pathogenic fungi causing disease on leaves and shoots of species of Eucalyptus and its close relative, Corymbia. The phylogenetic relationship of Quambalaria spp., previously classified in genera such as Sporothrix and Ramularia, has never been addressed. It has, however, been suggested that they belong to the basidiomycete orders Exobasidiales or Ustilaginales. The aim of this study was thus to consider the ordinal relationships of Q. eucalypti and Q. pitereka using ribosomal LSU sequences. Sequence data from the ITS nrDNA were used to determine the phylogenetic relationship of the two Quambalaria species together with Fugomyces (= Cerinosterus) cyanescens. In addition to sequence data, the ultrastructure of the septal pores of the species in question was compared. From the LSU sequence data it was concluded that Quambalaria spp. and F. cyanescens form a monophyletic clade in the Microstromatales, an order of the Ustilaginomycetes. Sequences from the ITS region confirmed that Q. pitereka and Q. eucalypti are distinct species. The ex-type isolate of F. cyanescens, together with another isolate from Eucalyptus in Australia, constitute a third species of Quambalaria, Q. cyanescens (de Hoog & G.A. de Vries) Z.W. de Beer, Begerow & R. Bauer comb. nov. Transmission electron-microscopic studies of the septal pores confirm that all three Quambalaria spp. have dolipores with swollen lips, which differ from other members of the Microstromatales (i.e. the Microstromataceae and Volvocisporiaceae) that have simple pores with more or less rounded pore lips. Based on their unique ultrastructural features and the monophyly of the three Quambalaria spp. in the Microstromatales, a new family, Quambalariaceae Z.W. de Beer, Begerow & R. Bauer fam. nov., is described

Comparative Genomics Reveals Prophylactic and Catabolic Capabilities of Actinobacteria within the Fungus-Farming Termite Symbiosis

Actinobacteria, one of the largest bacterial phyla, are ubiquitous in many of Earth’s ecosystems and often act as defensive symbionts with animal hosts. Members of the phylum have repeatedly been isolated from basidiomycete-cultivating fungus-farming termites that maintain a monoculture fungus crop on macerated dead plant substrate. The proclivity for antimicrobial and enzyme production of Actinobacteria make them likely contributors to plant decomposition and defense in the symbiosis. To test this, we analyzed the prophylactic (biosynthetic gene cluster [BGC]) and metabolic (carbohydrate-active enzyme [CAZy]) potential in 16 (10 existing and six new genomes) termite-associated Actinobacteria and compared these to the soil-dwelling close relatives. Using antiSMASH, we identified 435 BGCs, of which 329 (65 unique) were similar to known compound gene clusters, while 106 were putatively novel, suggesting ample prospects for novel compound discovery. BGCs were identified among all major compound categories, including 26 encoding the production of known antimicrobial compounds, which ranged in activity (antibacterial being most prevalent) and modes of action that might suggest broad defensive potential. Peptide pattern recognition analysis revealed 823 (43 unique) CAZymes coding for enzymes that target key plant and fungal cell wall components (predominantly chitin, cellulose, and hemicellulose), confirming a substantial degradative potential of these bacteria. Comparison of termite-associated and soil-dwelling bacteria indicated no significant difference in either BGC or CAZy potential, suggesting that the farming termite hosts may have coopted these soil-dwelling bacteria due to their metabolic potential but that they have not been subject to genome change associated with symbiosis. IMPORTANCE Actinobacteria have repeatedly been isolated in fungus-farming termites, and our genome analyses provide insights into the potential roles they may serve in defense and for plant biomass breakdown. These insights, combined with their relatively higher abundances in fungus combs than in termite gut, suggest that they are more likely to play roles in fungus combs than in termite guts. Up to 25% of the BGCs we identify have no similarity to known clusters, indicating a large potential for novel chemistry to be discovered. Similarities in metabolic potential of soil-dwelling and termite-associated bacteria suggest that they have environmental origins, but their consistent presence with the termite system suggests their importance for the symbiosis