Marieka Gryzenhout

The ascomycota tree of life: A phylum-wide phylogeny clarifies the origin and evolution of fundamental reproductive and ecological traits

We present a 6-gene, 420-species maximum-likelihood phylogeny of Ascomycota, the largest phylum of Fungi. This analysis is the most taxonomically complete to date with species sampled from all 15 currently circumscribed classes. A number of superclass-level nodes that have previously evaded resolution and were unnamed in classifications of the Fungi are resolved for the first time. Based on the 6-gene phylogeny we conducted a phylogenetic informativeness analysis of all 6 genes and a series of ancestral character state reconstructions that focused on morphology of sporocarps, ascus dehiscence, and evolution of nutritional modes and ecologies. A gene-by-gene assessment of phylogenetic informativeness yielded higher levels of informativeness for protein genes (RPB1, RPB2, and TEF1) as compared with the ribosomal genes, which have been the standard bearer in fungal systematics. Our reconstruction of sporocarp characters is consistent with 2 origins for multicellular sexual reproductive structures in Ascomycota, once in the common ancestor of Pezizomycotina and once in the common ancestor of Neolectomycetes. This first report of dual origins of ascomycete sporocarps highlights the complicated nature of assessing homology of morphological traits across Fungi. Furthermore, ancestral reconstruction supports an open sporocarp with an exposed hymenium (apothecium) as the primitive morphology for Pezizomycotina with multiple derivations of the partially (perithecia) or completely enclosed (cleistothecia) sporocarps. Ascus dehiscence is most informative at the class level within Pezizomycotina with most superclass nodes reconstructed equivocally. Character-state reconstructions support a terrestrial, saprobic ecology as ancestral. In contrast to previous studies, these analyses support multiple origins of lichenization events with the loss of lichenization as less frequent and limited to terminal, closely related species.

Ion Torrent PGM as Tool for Fungal Community Analysis: A Case Study of Endophytes in Eucalyptus grandis Reveals High Taxonomic Diversity

The Kingdom Fungi adds substantially to the diversity of life, but due to their cryptic morphology and lifestyle, tremendous diversity, paucity of formally described specimens, and the difficulty in isolating environmental strains into culture, fungal communities are difficult to characterize. This is especially true for endophytic communities of fungi living in healthy plant tissue. The developments in next generation sequencing technologies are, however, starting to reveal the true extent of fungal diversity. One of the promising new technologies, namely semiconductor sequencing, has thus far not been used in fungal diversity assessments. In this study we sequenced the internal transcribed spacer 1 (ITS1) nuclear encoded ribosomal RNA of the endophytic community of the economically important tree, Eucalyptus grandis, from South Africa using the Ion Torrent Personal Genome Machine (PGM). We determined the impact of various analysis parameters on the interpretation of the results, namely different sequence quality parameter settings, different sequence similarity cutoffs for clustering and filtering of databases for removal of sequences with incomplete taxonomy. Sequence similarity cutoff values only had a marginal effect on the identified family numbers, whereas different sequence quality filters had a large effect (89 vs. 48 families between least and most stringent filters). Database filtering had a small, but statistically significant, effect on the assignment of sequences to reference sequences. The community was dominated by Ascomycota, and particularly by families in the Dothidiomycetes that harbor well-known plant pathogens. The study demonstrates that semiconductor sequencing is an ideal strategy for environmental sequencing of fungal communities. It also highlights some potential pitfalls in subsequent data analyses when using a technology with relatively short read lengths.

Distribution of Chrysoporthe Canker Pathogens on Eucalyptus and Syzygium spp. in Eastern and Southern Africa

Chrysoporthe cubensis and C. austroafricana, collectively known as Cryphonectria cubensis in the past, are important canker pathogens of Eucalyptus spp. worldwide. Previous studies have suggested that Chrysoporthe austroafricana occurs only in South Africa, whereas C. cubensis occurs in Australia, Cameroon, Tanzania, Democratic Republic of Congo, Republic of Congo, Southeast Asia, and South, Central, and North America. In South Africa, C. austroafricana is a pathogen on nonnative Eucalyptus (Myrtaceae) and Tibouchina (Melastomataceae) spp., both residing in the order Myrtales. Recently, the fungus also has been found on native Syzygium cordatum trees in the country, leading to the hypothesis that it is native to Africa. In contrast, C. cubensis is thought to have been introduced into Africa and is known only on non-native Eucalyptus spp. and S. aromaticum (clove) in four countries. The aim of this study was to consider the distribution of Chrysoporthe spp. on non-native Eucalyptus spp. as well as on native Myrtales in southern and eastern Africa. Isolates were collected from as many trees as possible and characterized based on their morphology and DNA sequence data for two gene regions. Results show, for the first time, that C. cubensis occurs in Kenya, Malawi, and Mozambique on nonnative Eucalyptus spp. C. austroafricana was found for the first time in Mozambique, Malawi, and Zambia on non-native Eucalyptus spp. and native S. cordatum. The known distribution range of C. austroafricana within South Africa also was extended during these surveys.

Discovery of the Canker Pathogen Chrysoporthe austroafricana on Native Syzygium spp. in South Africa

Chrysoporthe canker is one of the most important diseases of plantation-grown Eucalyptus spp. in tropical and subtropical regions worldwide. For many years, the disease was reported to be caused by the fungal pathogen Cryphonectria cubensis. Recent DNA-based studies have shown that the fungus in South Africa is not conspecific with Chr. cubensis and it was recently described in the new genus Chrysoporthe as Chrysoporthe austroafricana. Chr. austroafricana is known only from South Africa, where it causes severe cankers on Eucalyptus spp. and on ornamental Tibouchina trees, both of which have been introduced into South Africa. The origin of Chr. austroafricana is unknown, but it is possible that it expanded its host range from native trees related to Eucalyptus and Tibouchina spp. to these exotic hosts. Subsequent surveys of some indigenous South African Myrtales led to the discovery of fruiting structures resembling those of Chr. austroafricana on native Syzygium cordatum and S. guineense. The fungus from these Syzygium spp. was identified as Chr. austroafricana based on morphological characteristics and β-tubulin gene sequences. Pathogenicity trials showed that Chr. austroafricana is more virulent on exotic Eucalyptus spp. than on native S. cordatum. This study represents the first report of Chr. austroafricana from native hosts in South Africa and adds credence to the view that the fungus could be native to this country.

Five New Species of the Botryosphaeriaceae from Acacia karroo in South Africa

Abstract The Botryosphaeriaceae represents an important, cosmopolitan family of latent pathogens infecting woody plants. Recent studies on native trees in southern Africa have revealed an extensive diversity of species of Botryosphaeriaceae, about half of which have not been previously described. This study adds to this growing body of knowledge, by discovering five new species of the Botryosphaeriaceae on Acacia karroo, a commonly occurring native tree in southern Africa. These species were isolated from both healthy and diseased tissues, suggesting they could be latent pathogens. The isolates were compared to other species for which DNA sequence data are available using phylogenetic analyses based on the ITS, TEF-1&agr;, &bgr;-tubulin and LSU gene regions, and characterized based on their morphology. The morphological data were, however, useful to make comparisons with other species found in the same region and on similar hosts. The five new species were described as Diplodia allocellula, Dothiorella dulcispinae, Do. brevicollis, Spencermartinsia pretoriensis and Tiarosporella urbis-rosarum. Evidence emerging from this study suggests that many more species of the Botryosphaeriaceae remain to be discovered in the southern Africa.

Cryphonectria canker on Tibouchina in South Africa

Cryphonectria cubensis is an important canker pathogen of plantation Eucalyptus spp. in tropical and sub-tropical areas of the world, including South Africa. It is best known on Eucalyptus spp., but it also occurs on Syzygium aromaticum (clove). In 1998, C. cubensis was found to cause cankers on the non-myrtaceous hosts Tibouchina urvilleana and T. lepidota in Colombia. In this study, we report on a similar canker disease that has recently been found in South Africa on T. granulosa commonly grown as an ornamental tree. The identity of the pathogen was determined through morphological comparisons and phylogenetic analyses of ITS and β-tubulin gene sequences. The pathogenicity of the fungus was also tested on T. granulosa and E. grandis. Morphological as well as DNA sequence comparisons showed that the fungus on T. granulosa is the same as C. cubensis occurring on Eucalyptus spp. in South Africa. Pathogenicity tests on T. granulosa and E. grandis clones showed that the fungus from T. granulosa is able to cause cankers on both hosts.

Conspecificity of Endothia eugeniae and Cryphonectria cubensis : a re-evaluation based on morphology and DNA sequence data

Abstract Cryphonectria cubensis and Endothia eugeniae are fungal pathogens of Eucalyptus and clove that were reduced to synonymy on the basis of results of cross-inoculation studies, isozyme analysis, cultural studies, and morphology. A previous phylogenetic study on Cryphonectria, based on sequence variation in the ITS region of the ribosomal RNA operon, also supported the conspecificity of C. cubensis and E. eugeniae, but was based on only one E. eugeniae isolate. New collections from clove in Brazil and Indonesia have become available, providing the opportunity to reconsider the conspecificity of C. cubensis and E. eugeniae. The occurrence of C. cubensis on clove was confirmed based on morphological comparisons and phylogenetic analyses of ribosomal DNA and β-tubulin gene sequence data. In addition to C. cubensis, other fungi morphologically similar to Cryphonectria species on the basis of their orange stromata were present on some clove specimens, but no isolates were available for these fungi. Furthermore, some isolates, for which no herbarium material exists, grouped separately from the C. cubensis clade and closer to the Cryphonectria clade. The presence of more than one closely related fungus on clove raises questions relating to the legitimacy of the synonymy of E. eugeniae and C. cubensis. Based on the presence of C. cubensis on the type specimen of E. eugeniae, we recognize the synonymy of the two fungi but provide evidence that other fungi, more closely related to Cryphonectria spp. than to C. cubensis, are present on clove.

Characterization of Phytophthora hybrids from ITS clade 6 associated with riparian ecosystems in South Africa and Australia.

Surveys of Australian and South African rivers revealed numerous Phytophthora isolates residing in clade 6 of the genus, with internal transcribed spacer (ITS) gene regions that were either highly polymorphic or unsequenceable. These isolates were suspected to be hybrids. Three nuclear loci, the ITS region, two single copy loci (antisilencing factor (ASF) and G protein alpha subunit (GPA)), and one mitochondrial locus (cytochrome oxidase c subunit I (coxI)) were amplified and sequenced to test this hypothesis. Abundant recombination within the ITS region was observed. This, combined with phylogenetic comparisons of the other three loci, confirmed the presence of four different hybrid types involving the three described parent species Phytophthora amnicola, Phytophthora thermophila, and Phytophthora taxon PgChlamydo. In all cases, only a single coxI allele was detected, suggesting that hybrids arose from sexual recombination. All the hybrid isolates were sterile in culture and all their physiological traits tended to resemble those of the maternal parents. Nothing is known regarding their host range or pathogenicity. Nonetheless, as several isolates from Western Australia were obtained from the rhizosphere soil of dying plants, they should be regarded as potential threats to plant health. The frequent occurrence of the hybrids and their parent species in Australia strongly suggests an Australian origin and a subsequent introduction into South Africa.

Phylogenetic relationships of Cryphonectria and Endothia species, based on DNA sequence data and morphology

The fungal genera Endothia and Cryphonectria include some of the most important pathogens of forest trees. Despite available new technology, no comprehensive comparative study based on DNA sequence data and morphology has been done on the available isolates representing these two genera. The main objectives of this study were to assess the phylogenetic relationships among species of Cryphonectria and Endothia, for which cultures are available, and to establish a taxonomic framework based on DNA sequence and morphological data, which will aid future studies and identification of species in these and related genera. Comparisons were based on sequence variation found in the ITS region of the ribosomal RNA operon and two regions of the β-tu-bulin gene. In addition, the morphology of these species was examined. The phylogenetic data indicated that Endothia and Cryphonectria reside in two distinct phylogenetic clades. Cryphonectria parasitica, C. macrospora, C. nitschkei, C. eucalypti and C. radicalis represented the Cryphonectria clade. Endothia gyrosa and E. singularis were included in the Endothia clade. An isolate representing E. viridistroma grouped outside the Endothia clade and separately from other groups. Other clades outside the one encompassing Cryphonectria were those represented by the C. cubensis isolates and fungi isolated from Elaeocarpus dentatus originating from New Zealand. These clades could be distinguished from Endothia and Cryphonectria, based on anamorph morphology, stromatal structure and ascospore septation. Cryphonectria and Endothia, therefore, appear to be paraphyletic and taxonomic relationships for these fungi need to be revised.

surveys of soil and water reveal a goldmine of Phytophthora diversity in south African natural ecosystems

Phytophthora species are well-known as destructive plant pathogens, especially in natural ecosystems. It is ironic, therefore, how little is known regarding the Phytophthora diversity in South African natural woody ecosystems. In this study, Phytophthora species were isolated using standard baiting techniques from 182 soil and water samples and these were identified based on ITS and coxI sequence data. The 171 resulting Phytophthora isolates resided in 14 taxa including six known species (P. multivora, P. capensis, P. cryptogea, P. frigida, P. cinnamomi, P. cinnamomi var. parvispora), the known but as yet unnamed Phytophthora sp. PgChlamydo, P. sp. emzansi, and P. sp. Kununurra and five novel taxa referred to as P. sp. stellaris, P. sp. Umtamvuna P. sp. canthium, P. sp. xWS, P. sp. xHennops. Four of the new taxa were found exclusively in water and two of these are hybrids. The most commonly isolated species from soil was P. multivora, a species recently described from Western Australia. Phytophthora frigida was isolated for the first time from stream water. With the exception of P. cinnamomi, very little is known regarding the biology, epidemiology or origin of Phytophthora in South Africa.