Martin Petrus Albertus Coetzee

Microsatellite discovery by deep sequencing of enriched genomic libraries

Robust molecular markers such as microsatellites are important tools used to understand the dynamics of natural populations, but their identification and development are typically time consuming and labor intensive. The recent emergence of so-called next-generation sequencing raised the question as to whether this new technology might be applied to microsatellite development. Following this view, we considered whether deep sequencing using the 454 Life Sciences/Roche GS-FLX genome sequencing system could lead to a rapid protocol to develop microsatellite primers as markers for genetic studies. For this purpose, genomic DNA was sourced from three unrelated organisms: a fungus (the pine pathogen Fusarium circinatum), an insect (the pine-damaging wasp Sirex noctilio), and the wasps associated nematode parasite (Deladenus siricidicola). Two methods, FIASCO (fast isolation by AFLP of sequences containing repeats) and ISSR-PCR (inter-simple sequence repeat PCR), were used to generate microsatellite-enriched DNA for the 454 libraries. From the resulting 1.2-1.7 megabases of DNA sequence data, we were able to identify 873 microsatellites that have sufficient flanking sequence available for primer design and potential amplification. This approach to microsatellite discovery was substantially more rapid, effective, and economical than other methods, and this study has shown that pyrosequencing provides an outstanding new technology that can be applied to this purpose.

The root rot fungus Armillaria mellea introduced into South Africa by early Dutch settlers.

Dead and dying oak (Quercus) and numerous other woody ornamental trees and shrubs showing signs and symptoms of Armillaria root rot were identified in the Company Gardens, Cape Town, South Africa, which were established in the mid‐1600s by the Dutch East Indies Trading Company. Nineteen isolates from dying trees or from mushrooms were collected and analysed to identify and characterize the Armillaria sp. responsible for the disease. The AluI digestion of the amplified product of the first intergenic spacer region (IGS‐1) of the rRNA operon of 19 isolates from the Company Gardens was identical to that of some of the European isolates of A. mellea s. s. The IGS‐1 region and the internal transcribed spacers (ITS) were sequenced for some of the Cape Town isolates. Phylogenetic analyses placed the Cape Town isolates in the European clade of A. mellea, which is distinct from the Asian and North American clades of this species. Identification based on sexual compatibility was conducted using A. mellea tester strains in diploid–haploid pairings, which showed some compatibility between the Cape Town isolates and testers from Europe. Somatic compatibility tests (diploid–diploid pairings) and DNA fingerprinting with multilocus, microsatellite probes indicated that the Cape Town isolates were genetically identical and may have resulted from vegetative (clonal) spread from a single focus in the centre of the original Company Gardens (c. 1652). The colonized area is at least 345 m in diameter. Assuming a linear spread rate underground of 0.3 m/year to 1.6 m/year, the genet (clone) was estimated to be between 108 and 575 years old. These data suggest that A. mellea was introduced into Cape Town from Europe, perhaps on potted plants, such as grapes or citrus, planted in the Company Gardens more than 300 years ago.

Geographical diversity of Armillaria mellea s. s. based on phylogenetic analysis

Armillaria mellea was once thought to be a morphologically variable species with a wide distri- bution and a very broad host range. The relatively recent development of an indirect assessment of sex- ual incompatibility in Armillaria has led to identifi- cation of biological species within A. mellea s. 1. Par- tial intergenic spacer region (IGS) sequences of the ribosomal RNA (rRNA) operon have been deter- mined for most of the Northern Hemisphere species of Armillaria, but not for A. mellea s. s. The aim of this study was to determine the phylogenetic rela- tionships among isolates of A. mellea s. s. based on DNA sequences from the IGS as well as the internal transcribed spacer (ITS) regions. The IGS and the ITS sequence data indicate that A. mellea is highly variable but phylogenetically distant from the other species of Armillaria, and there are four separate groups of A. mellea distinguished by their geograph- ical origin: Asia, western North America, eastern North America and Europe. The data suggest that A. mellea populations from different regions are genet- ically isolated and may be in the process of specia- tion.

Phylogenetic relationships among Armillaria species inferred from partial elongation factor 1-alpha DNA sequence data

Armillaria species are important root rot pathogens with a wide host range and a worldwide distribution. The taxonomy of these fungi has been problematic for many years but the understanding of the relationships between them has been substantially improved through the application of DNA sequence comparisons. In this study, relationships between different Armillaria species were determined using elongation factor 1-alpha DNA sequence data for the first time. A total of 42 isolates, representing the majority of Armillaria species, with diverse geographic distributions and hosts, were included in this study. PCR amplification yielded products of 600 bp for all the isolates. Phylogenetic trees resulting from parsimony analysis showed that this gene region is useful for studying relationships between species. Generally, results were similar to those emerging from previous comparisons using ITS and IGS-1 sequence data. Phylogenetic trees generated from the dataset grouped the African taxa in a strongly supported clade, basal to the rest of the Armillaria species included in the study. The Armillaria species originating from the Northern Hemisphere formed a monophyletic group. Within this group, isolates of A. mellea constituted four subclades, representing their geographical origin. The phylogenetic relationships among species from the Southern Hemisphere were not entirely resolved. However, A. pallidula, A. fumosa and A. hinnulea grouped in a strongly supported clade and isolates of A. limonea formed a sister clade with those of A. luteobubalina. This is the first time a single-copy protein coding gene has been used to study phylogenetic relationships in Armillaria, and overall the data support previously held views regarding the relationships between species.

Characterization of Seiridium spp. Associated with Cypress Canker Based on ß-Tubulin and Histone Sequences

Cypress canker is a serious disease that has devastated Cupressus spp. in many parts of the world. In Mediterranean Europe it has caused the deaths of millions of trees. Three species of Seiridium, S. cardinale, S. cupressi, and S. unicorne, are associated with cypress canker. Considerable debate surrounds the taxonomic status of these fungi. They have been viewed as a single morphologically variable species, three distinct taxa; or two species based on the presence or absence of conidial appendages. Studies based on ribosomal DNA (ITS1, ITS2, and 5.8S gene) sequence failed to separate the cypress canker fungi. In an attempt to distinguish between the species associated with cypress canker we used histone and partial ß-tubulin sequences of fourteen isolates of Seiridium spp. from cypress. Analysis of sequence data showed Seiridium isolates from Cupressus spp., residing in two major clades. One clade accommodated S. unicorne isolates from Portugal and South Africa. The other major clade consisted of two subclades containing non-appendaged S. cardinale isolates. We believe the larger second clade, represents the cypress canker pathogens while the other clade contains the less pathogenic S. unicorne, which has a host range beyond Cupressus. This study thus provides strong evidence to support previous morphological data suggesting three distinct species are associated with cypress canker.

Fungal Planet description sheets: 281-319.

Novel species of fungi described in the present study include the following from South Africa: Alanphillipsia aloeicola from Aloe sp., Arxiella dolichandrae from Dolichandra unguiscati, Ganoderma austroafricanum from Jacaranda mimosifolia, Phacidiella podocarpi and Phaeosphaeria podocarpi from Podocarpus latifolius, Phyllosticta mimusopisicola from Mimusops zeyheri and Sphaerulina pelargonii from Pelargonium sp. Furthermore, Barssia maroccana is described from Cedrus atlantica (Morocco), Codinaea pini from Pinus patula (Uganda), Crucellisporiopsis marquesiae from Marquesia acuminata (Zambia), Dinemasporium ipomoeae from Ipomoea pes-caprae (Vietnam), Diaporthe phragmitis from Phragmites australis (China), Marasmius vladimirii from leaf litter (India), Melanconium hedericola from Hedera helix (Spain), Pluteus albotomentosus and Pluteus extremiorientalis from a mixed forest (Russia), Rachicladosporium eucalypti from Eucalyptus globulus (Ethiopia), Sistotrema epiphyllum from dead leaves of Fagus sylvatica in a forest (The Netherlands), Stagonospora chrysopyla from Scirpus microcarpus (USA) and Trichomerium dioscoreae from Dioscorea sp. (Japan). Novel species from Australia include: Corynespora endiandrae from Endiandra introrsa, Gonatophragmium triuniae from Triunia youngiana, Penicillium coccotrypicola from Archontophoenix cunninghamiana and Phytophthora moyootj from soil. Novelties from Iran include Neocamarosporium chichastianum from soil and Seimatosporium pistaciae from Pistacia vera. Xenosonderhenia eucalypti and Zasmidium eucalyptigenum are newly described from Eucalyptus urophylla in Indonesia. Diaporthe acaciarum and Roussoella acacia are newly described from Acacia tortilis in Tanzania. New species from Italy include Comoclathris spartii from Spartium junceum and Phoma tamaricicola from Tamarix gallica. Novel genera include (Ascomycetes): Acremoniopsis from forest soil and Collarina from water sediments (Spain), Phellinocrescentia from a Phellinus sp. (French Guiana), Neobambusicola from Strelitzia nicolai (South Africa), Neocladophialophora from Quercus robur (Germany), Neophysalospora from Corymbia henryi (Mozambique) and Xenophaeosphaeria from Grewia sp. (Tanzania). Morphological and culture characteristics along with ITS DNA barcodes are provided for all taxa.

Molecular identification and phylogeny of Armillaria isolates from South America and Indo-Malaysia

Armillaria root rot is a serious disease, chiefly of woody plants, caused by many species of Armillaria that occur in temperate, tropical and subtropical regions of the world. Very little is known about Armillaria in South America and Southeast Asia, although Armillaria root rot is well known in these areas. In this study, we consider previously unidentified isolates collected from trees with symptoms of Armillaria root rot in Chile, Indonesia and Malaysia. In addition, isolates from basidiocarps resembling A. novae-zelandiae and A. limonea, originating from Chile and Argentina, respectively, were included in this study because their true identity has been uncertain. All isolates in this study were compared, based on their similarity in ITS sequences with previously sequenced Armillaria species, and their phylogenetic relationship with species from the Southern Hemisphere was considered. ITS sequence data for Armillaria also were compared with those available at GenBank. Parsimony and distance analyses were conducted to determine the phylogenetic relationships between the unknown isolates and the species that showed high ITS sequence similarity. In addition, IGS-1 sequence data were obtained for some of the species to validate the trees obtained from the ITS data set. Results of this study showed that the ITS sequences of the isolates obtained from basidiocarps resembling A. novae-zelandiae are most similar to those for this species. ITS sequences for isolates from Indonesia and Malaysia had the highest similarity to A. novae-zelandiae but were phylogenetically separated from this species. Isolates from Chile, for which basidiocarps were not found, were similar in their ITS and IGS-1 sequences to the isolate from Argentina that resembled A. limonea. These isolates, however, had the highest ITS and IGS-1 sequence similarity to authentic isolates of A. luteobubalina and were phylogenetically more closely related to this species than to A. limonea.

Concerted Evolution in the Ribosomal RNA Cistron

Background Gene conversion is the mechanism proposed to be responsible for the homogenization of multigene families such as the nuclear ribosomal gene clusters. This concerted evolutionary process prevents individual genes in gene clusters from accumulating mutations. The mechanism responsible for concerted evolution is not well understood but recombination during meiosis has been hypothesized to play a significant role in this homogenization. In this study we tested the hypothesis of unequal crossing over playing a significant role in gene conversion events within the ribosomal RNA cistron during meiosis, mitosis or both life stages in the fungal tree pathogen Ceratocystis manginecans. Methods Ceratocystis manginecans, a haploid ascomycete, reproduces homothallically and was found to have two distinct sequences within the internally transcribed spacer (ITS) region of the ribosomal RNA cistron. The different ITS types were scored using PCR-RFLP assays and chi-square analyses to determine the level of significance of the changes in the ratios of the ITS types. Results The relative ratios of the two ITS sequence types changed when the fungal isolates were cultured vegetatively or allowed to produced sexual structures and spores. These active changes were shown to occur more frequently during meiosis than mitosis. Conclusion The evidence presented provides concrete support for homogenization in the rRNA gene clusters found in this fungus and that the most reasonable explanation for this process is unequal crossing over.

Phylogenetic analyses of DNA sequences reveal species partitions amongst isolates of Armillaria from Africa

The basidiomycete genus Armillaria causes root rot and death to woody plants in boreal, temperate and tropical regions of the world. Armillaria root rot has been described from various parts of Africa on many different hosts. However, very little is known regarding the evolutionary relationships among Armillaria species in Africa. The aim of this study was to determine the phylogenetic relationships between isolates originating from different regions in Africa using nDNA sequences from two non-coding gene regions. The ITS and the IGS-1 regions of the ribosomal DNA operon were sequenced and analysed using different phylogenetic tree searching methods. Phylogenetic trees grouped the African taxa in two strongly supported clades. One of these represented A. fuscipes and the other an undescribed but distinct species.

Molecular phylogeny of Armillaria from the Patagonian Andes

A number of species in the plant pathogen genus Armillaria are known from South America where they cause root rot disease on a wide variety of hosts. Knowledge pertaining to phylogenetic relationships of these species with those of other Armillaria species is almost non-existent. In addition, very few cultures representing these species are available, making DNA-based phylogenetic analyses impossible. The aim of this study was to characterise a collection of Armillaria isolates from the Patagonian Andes using DNA sequences and to determine their phylogenetic relationships with other Armillaria species. DNA sequences were obtained from the internal transcribed regions (ITS1, 5.8S and ITS4) and ribosomal large subunit (LSU) gene and used in phylogenetic analyses. Phylogenetic trees generated from the sequences separated the Armillaria isolates into four lineages. Lineages I and II represented A. novae-zelandiae and A. luteobubalina, respectively. Isolates belonging to A. novae-zelandiae from Malaysia, New Zealand, Australia and South America showed considerable intra-clade sub-structure. Lineages III and IV are probably distinct species and are most closely related to A. hinnulea and an unnamed species isolated from New Zealand and Kenya. This is the first comprehensive study of the phylogenetic relationships of Armillaria species from Patagonia and it provides a foundation for future research in this region.