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ABSTRACTAmbrosia beetles (Coleoptera: Curculionidae: Scolytinae) are small wood-boring insects that live in an obligate symbiosis with fungi, which serve as their primary food source. Beetles residing in the genus Euwallaceahave evolved a unique association with a clade of Fusariumthat falls within the aptly named Ambrosia FusariumClade (AFC). The discovery of the invasive polyphagous shot hole borer, E. fornicatus, in South Africa, has heightened awareness of ambrosia beetles and their symbionts in the country. In this study, we investigated the Fusariumsymbionts of three species of Euwallaceain South Africa, with a specific focus on those associated with E. xanthopus. Isolations of Fusariumstrains from both living and dissected beetles yielded nearly 100 isolates. Using multigene phylogenetic analyses, these isolates were identified as six different Fusariumspecies. Fusarium hypothenemiand F. euwallaceaehave previously been reported from South Africa. Fusarium pseudensiformeand FusariumAF-6 are new records for the country. The remaining two species are new to science and are described here as F. rufumsp. nov. and F. floriferumsp. nov. Targeted fungal isolation from specific beetle body parts revealed that the AFC species collected were typically associated with the dissected beetle heads and helped us identify the likely nutritional symbiont of E. xanthopus. This study highlights the understudied diversity of fungal associates of ambrosia beetles present in South Africa.

Ralstonia solanacearumand R. pseudosolanacearumare well-known bacterial plant pathogens that cause significant losses to both ornamental and agricultural plants. It has been suggested that they are not the primary cause of bacterial wilt in Eucalyptusspecies, but rather are opportunistic, taking advantage of trees predisposed to infection by abiotic and biotic factors. To test this hypothesis, the bacterial community within the vascular tissue of asymptomatic Eucalyptus grandis x E. urophyllatrees, and those displaying varying stages of infection in China and Indonesia were compared using 16S rRNA profiling. Asymptomatic trees growing in areas where bacterial infections had never previously been reported to occur were included as controls. Ralstoniaspecies were found within the vascular tissue of both asymptomatic and symptomatic trees, in high abundance. In the control samples, bacterial diversity within the vascular tissue was high with a low abundance of Ralstoniaspecies. The presence of Ralstoniaspecies in asymptomatic and control samples supports the hypothesis that these species are latent and/or opportunistic pathogens in E. grandis x E. urophylla trees.

A root disease in plantations of Pinus radiataand Pinus pinaster, where trees died in distinct patches, was present in the Western Cape province of South Africa during the 1970s and 1980s. Phytophthora cinnamomiwas initially believed to be the cause, but the disease was later ascribed to the insect-associated fungus Leptographium serpens, a fungal species residing in the Ophiostomatales. Doubt regarding the cause of the disease was raised in a later study due to the fact that most Leptographiumspp., particularly those that colonise ray parenchyma tissues, which is the case for L. serpens, are not typically primary disease agents. In this study, cultures of an unidentified sterile fungus collected from the dying trees were revived and identified using DNA sequencing methods, which were not available when the disease was first studied. These cultures were identified as the pyrophillic pathogen Rhizina undulata, well-known to cause patch death of conifers in South Africa and elsewhere in the world. While the patches of dying trees no longer exist and the disease cannot be newly studied, it is most likely that the tree death originally thought to be caused by L. serpenswas due primarily to R. undulata. The study provides a vivid example of the value of preserving cultures of fungi for later study and the power of modern techniques to identify fungal pathogens.

The African fungus Ceratocystis albifundusdoes not cause disease symptoms on its wide range of native woody hosts. However, on non-native Acacia mearnsiiand orchard-grown Protea cynaroides, it represents an economically significant pathogen. Because previous studies exploring the biological fitness of C. albifunduswere constrained by small sample sizes, we aimed to determine how commonly used measures of fitness (growth in culture, pathogenicity and sexual fertility status) vary across natural populations. For this purpose, a collection of 58 isolates originating from diverse hosts and geographic locations in South Africa were subjected to growth studies on synthetic culture medium, pathogenicity tests on A. mearnsiisaplings, and sequence-based assays of fertility status. We found that these traits were generally not correlated with one another, although isolates from the Summer rainfall region and from native hosts induced significantly shorter lesions on A. mearnsiithan isolates from the Winter rainfall region and from diseased A. mearnsiiand orchard-grown P. cynaroidestissues. In other words, aggressiveness of C. albifundusto A. mearnsiiwas significantly influenced by the isolates’ geographic origin and host species, irrespective of their fertility status or growth rates. Additionally, the broad lack of correlation among growth, pathogenicity and fertility suggested that these fitness components are likely underpinned by distinct genetic and molecular mechanisms. Our study thus provides a robust foundation for further exploration of the fitness landscape in this important tree pathogen.

ABSTRACTGraphiumspecies form a well-supported monophyletic lineage within the Microascales (Ascomycota). Members of this genus can be found in association with bark beetles, as well as on tree wounds and in soils. During surveys of bark and ambrosia beetle–associated fungi and cavities made by woodpeckers on hardwood trees in Poland, many isolates with an affinity to Graphiumwere recovered. They were identified based on their morphological characters and sequence data for the internal transcribed spacer (ITS), 28S rDNA, β-tubulin (TUB2), and translation elongation factor 1-α (TEF1) gene regions. The results revealed five new species, described here as G. brachiatum, G. longistipitatum, G. polonicum, G. radicatum, and G. trypophloei.

The pace at which Next Generation Sequence data is being produced continues to accelerate as technology improves. As a result, such data are increasingly becoming accessible to biologists outside of the field of bioinformatics. In contrast, access to training in the methods of genome assembly and annotation are not growing at a similar rate. In this issue, we report on a Genome Assembly Workshop for Mycologists that was held at the Forestry and Agricultural Biotechnology Institute (FABI) at the University of Pretoria, South Africa and make available the 12 draft genome sequences emanating from the event. With the aim of making the process of genome assembly and annotation more accessible to biologists, we provide a step-by-step guide to both genome assembly and annotation, intended to encourage and empower mycologists to use genome data in their research.

Contamination in sequenced genomes is a relatively common problem and several methods to remove non-target sequences have been devised. Typically, the target and contaminating organisms reside in different kingdoms, simplifying their separation. The authors present the case of a genome for the ascomycete fungus Teratosphaeria eucalypti, contaminated by another ascomycete fungus and a bacterium. Approaching the problem as a low-complexity metagenomics project, the authors used two available software programs, BlobToolKit and anvi'o, to filter the contaminated genome. Both the de novoand reference-assisted approaches yielded a high-quality draft genome assembly for the target fungus. Incorporating reference sequences increased assembly completeness and visualization elucidated previously unknown genome features. The authors suggest that visualization should be routine in any sequencing project, regardless of suspected contamination.

ABSTRACTThe Ophiostomatales (Ascomycota) accommodates more than 300 species characterized by similar morphological adaptations to arthropod dispersal. Most species in this order are wood-inhabiting fungi associated with bark or ambrosia beetles. However, a smaller group of species occur in other niches such as in soil and Proteainfructescences. Recent surveys of Termitomycesfungus gardens (fungus combs) of fungus-growing termites led to the discovery of characteristic ophiostomatalean-like fruiting structures. In this study, these ophiostomatalean-like fungi were identified using morphological characteristics, conventional molecular markers, and whole genome sequencing. In addition, the influence of the extracts derived from various parts of Termitomycescombs on the growth of these fungi in culture was considered. Based on phylogenomic analyses, two new genera (Intubiaand Chrysosphaeria) were introduced to accommodate these ophiostomatalean species. Phylogenetic analyses revealed that the isolates resided in three well-supported lineages, and these were described as three new species (Intubia macrotermitinarum, I. oerlemansii, and Chrysosphaeria jan-nelii). Culture-based studies showed that these species do not depend on the Termitomycescomb material for growth.

ABSTRACTSeveral species in the Euwallacea fornicatuscomplex have emerged as important pests of woody plants globally, particularly in habitats where they are invasive aliens. These beetles live in obligate symbioses with fungi in the genus Fusarium. In this study, we identified Euwallaceaspp. and their fungal mutualists that have emerged as pests of planted Acacia crassicarpain Riau, Indonesia. Morphological identification and phylogenetic analyses of the mitochondrial cytochrome oxidase csubunit I (COI) gene confirmed that E. similisand E. perbrevisare the most abundant beetles infesting these trees. Multilocus phylogenetic analyses of their fungal mutualists revealed their nonspecific association with six Fusariumspecies. These included F. rekanumand five novel Fusariummutualists within the Fusarium solanispecies complex (FSSC), four of which reside in the Ambrosia FusariumClade (AFC). These new species are described here as F. akasia, F. awan, F. mekan, F. variasi, and F. warna.

ABSTRACTDuring surveys of insect-associated mycobiomes in Norway, Poland, and Russia, isolates with affinity to Graphilbum(Ophiostomatales, Ascomycota) were recovered. In this study, eight known Graphilbumspecies as well as the newly collected isolates were compared based on morphology and DNA sequence data for four gene regions. The results revealed seven new species, described here as G. acuminatum, G. carpaticum, G. curvidentis, G. furuicola, G. gorcense, G. interstitiale, and G. sexdentatum. In addition to these species, G. crescericumand G. sparsumwere commonly found in Norway. All new species were recovered from conifers in association with bark beetles, cerambycid beetles, and weevils and were morphologically similar, predominantly with pesotum-like asexual morphs. Where sexual morphs were present, these were small ascomata with short necks and rod-shaped ascospores having hyaline sheaths. The results suggest that Graphilbumspecies are common members of the Ophiostomatales in conifer ecosystems.

AbstractDuring a survey of putative fungal pathogens infecting oak trees in the Gangwon Province of the Republic of Korea, a fungus resembling a Ceratocystissp. was repeatedly isolated from natural wounds on Quercus variabilis. Morphological comparisons and DNA sequence comparisons based on partial β-tubulin and TEF-1α gene regions showed that the fungus resided in a distinct lineage. This novel Ceratocystisspecies is described here as C. quercicolasp. nov. This is the first novel species of Ceratocystisto be reported from Korea. A pathogenicity test showed that it can cause lesions on inoculated trees but that it had a very low level of aggressiveness. The discovery of this fungus suggests that additional taxa residing in Ceratocystisare likely to be discovered in Korea in the future.

Araucaria araucanais an ancient conifer, native to the mountain ranges in Chile and Argentina. These trees host a large number of organisms, mainly insects, strongly or even exclusively associated with them. The recent emergence of a novel canker disease on A. araucanahas emphasised the importance of fungi associated with these iconic trees and has resulted in the discovery of various new species. In this study, we considered the identity of an unknown calicioid fungus consistently found on resin on the branches of A. araucana. Preliminary phylogenetic analyses placed isolates in the recently described sub-class Cryptocaliciomycetidae, closest to Cryptocalicium blascoi. However, the morphology of the ascomata and its occurrence in a unique niche suggested that the closest relative could be Resinogalea humboldtensis(Bruceomycetaceae, incertae sedis), a fungus with similar sporing structures found on resin of Araucaria humboldtensisin New Caledonia. There are no living cultures or sequence data available for either R. humboldtensisor its supposed closest relative, Bruceomyces castoris,precluding sequence-based comparisons. Morphological comparisons of the sporing structures on A. araucanaconfirmed that the ascomatal morphology of our unknown calicioid fungus and R. humboldtensisare almost identical and resemble each other more so than B. castorisor Cr. blascoi. A phylogenetic analysis based on the small subunit (SSU), internal transcribed spacer (ITS) and large subunit (LSU) rDNA regions resolved our strains into two clades with Cr. blascoias its closest relative. Further analyses applying the Genealogical Concordance Phylogenetic Species Recognition (GCPSR) based on ITS, mini chromosome maintenance protein complex (MCM7), RNA polymerase II second largest subunit (RPB2) and translation elongation factor 1-alpha (TEF) gene regions, confirmed that strains represent two new species. Based on our morphological observations and phylogenetic analyses, we introduce two new Resinogaleaspecies, R. araucanaand R. tapulicola,and reclassify the genus in the subclass Cryptocaliciomycetidae.

ABSTRACTVietnam and Indonesia have rapidly growing and extensive plantation forestry programs, especially of Acaciaspp. and Eucalyptusspp. As these plantations expand, the threat from pests and diseases also increases. Calonectriaspecies are among those pathogens causing diseases of trees in plantations and nurseries in these countries. Extensive surveys were conducted across plantations and nurseries of Vietnam and parts of Indonesia, where a large number of Calonectriaisolates were retrieved from diseased leaves and soils associated with symptomatic trees. The aim of this study was to identify and resolve the phylogenetic relationships among these isolates using DNA sequence comparisons of four gene regions as well as morphological characters. From a collection of 165 isolates, the study revealed five known and 10 undescribed species. The relatively high diversity of Calonectriaspecies found in this study supports the view that many more species in this genus remain to be discovered in other areas of Southeast Asia.

ABSTRACTThe ascomycete genus Huntiella(Microascales) has a cosmopolitan distribution and occurs on a wide range of woody plants. Little is known regarding the identity, diversity, origin, or impact of these fungi in China. Recently, isolates of Huntiellaspp. were collected from stumps of freshly felled trees or wounds on plantation-grown Eucalyptusin Guangdong, Guangxi, Fujian, and Hainan provinces of southern China. Additional isolates were obtained from stumps of Acacia confusanear Eucalyptusplantations in Hainan Province. The aim of this study was to identify these Huntiellaspecies and to test their pathogenicity on Eucalyptusseedlings. Morphology and multigene phylogenies of the nuclear rDNA internal transcribed spacer (ITS1-5.8S-ITS2 = ITS) region and partial β-tubulin (BT1) and translation elongation factor 1α (TEF1α) genes revealed nine previously unknown Huntiellaspecies, eight from Eucalyptusand one from A. confusa. The mating types of these species were determined, showing that seven are heterothallic, one is homothallic, and one is unisexual (MAT1-2-1gene). Pathogenicity tests showed that the nine Huntiellaspecies can produce lesions on Eucalyptusseedlings, larger than wounds caused by controls on these plants. This study provides a basic understanding of the distribution, diversity, and pathogenicity of Huntiellaspecies in southern China.

AbstractIt is challenging to sequence and assemble genomes of obligate plant pathogens and microorganisms because of limited amounts of DNA, comparatively large genomes and high numbers of repeat regions. We sequenced the 1.2 gigabase genome of an obligate rust fungus, Austropuccinia psidii, the cause of rust on Myrtaceae, with a Chromium 10X library. This technology has mostly been applied for single-cell sequencing in immunological studies of mammals. We compared scaffolds of a genome assembled from the Chromium library with one assembled from combined paired-end and mate-pair libraries, sequenced with Illumina HiSeq. Chromium 10X provided a superior assembly, in terms of number of scaffolds, N50 and number of genes recovered. It required less DNA than other methods and was sequenced and assembled at a lower cost. Chromium sequencing could provide a solution to sequence and assemble genomes of obligate plant pathogens where the amount of available DNA is a limiting factor.

The diversity of Phytophthoraspp. in rivers and riparian ecosystems has received considerable international attention, although little such research has been conducted in South Africa. This study determined the diversity of Phytophthoraspp. within a single river in Gauteng province of South Africa. Samples were collected over 1 y including biweekly river baiting with Rhododendron indicumleaves. Phytophthoraisolates were identified with phylogenetic analyses of sequences for the internal transcribed spacer (ITS) region of the ribosomal DNA and the mitochondrial cytochrome oxidase c subunit I (coxI) gene. Eight Phytophthoraspp. were identified, including a new taxon, P. taxon Sisulu-river, and two hybrid species from Cooke’s ITS clade 6. Of these, species from Clade 6 were the most abundant, including P. chlamydosporaand P. lacustris. Species residing in Clade 2 also were encountered, including P. multivora, P. plurivoraand P. citrophthora. The detection of eight species in this investigation of Phytophthoradiversity in a single riparian river ecosystem in northern South Africa adds to the known diversity of this genus in South Africa and globally.

Ophiostomaspecies, some of which cause sapstain in timber and/or are mild pathogens, are common fungal associates of bark beetles (Coleoptera: Scolytinae). Three new Ophiostomataceae from Spain are recognized in the present study based on comparisons of sequence data for three gene regions as well as morphological characteristics. The new taxa are described as Ophiostoma nebularesp. nov., Ophiostoma euskadiensesp. nov. and Graphilbum crescericumsp. nov.

The genus Celoporthewas first described when C. dispersawas discovered in South Africa associated with dieback and cankers on trees in the Myrtales. Four additional species were recently described from Eucalyptusand Syzygium cuminiin China as well as S. aromaticumand Eucalyptusin Indonesia. Inoculation trials have shown that all Celoporthespecies, including those that have not been found on Eucalyptusspecies in nature, are pathogenic to Eucalyptusand they are thus potentially threatening to commercial Eucalyptusforestry. New isolates, morphologically similar to Celoporthe, have been collected from S. legattiin South Africa and S. guineensein Zambia. Multigene phylogenetic analyses based on DNA sequences of the ITS region, TEF1α gene and two areas of the β-tubulin gene revealed additional cryptic species in Celoporthe. Phylogenetic data were supported by morphological differences. These resulted in the description of two previously unknown species of Celoporthe, namely C. fontanaand C. woodiana, for two of these cryptic groups, while the third group represented C. dispersa. These species all can readily infect Eucalyptusas well as several species of Syzygium, the latter of which are native to Africa.

Grosmannia serpenswas first described from pine in Italy in 1936 and it has been recorded subsequently from many countries in both the northern and southern hemispheres. The fungus is vectored primarily by root-infesting bark beetles and has been reported to contribute to pine-root diseases in Italy and South Africa. The objective of this study was to consider the identity of a global collection of isolates not previously available and using DNA sequence-based comparisons not previously applied to most of these isolates. Phylogenetic analyses of the ITS2-LSU, actin, beta-tubulin, calmodulin and translation elongation factor-1 alpha sequences revealed that these morphologically similar isolates represent a complex of five cryptic species. Grosmannia serpenssensu stricto thus is redefined and comprises only isolates from Italy including the ex-type isolate. The ex-type isolate of Verticicladiella alacriswas shown to be distinct from G. serpens, and a new holomorphic species, G. alacris, is described. The teleomorph state of G. alacriswas obtained through mating studies in the laboratory, confirming that this species is heterothallic. Most of the available isolates, including those from South Africa, USA, France, Portugal and some from Spain, represent G. alacris. The remaining three taxa, known only in their anamorph states, are described as the new species Leptographium gibbsiifor isolates from the UK, L. yamaokaefor isolates from Japan and L. castellanumfor isolates from Spain and the Dominican Republic.

Gondwanamycesand its Custingophoraanamorphs were first described from Proteainfructescences in South Africa. Subsequently these unusual fungi were also found on Cecropiain Central America. During an investigation into the decline and death of native Euphorbiatrees in South Africa, several fungal isolates resembling the anamorph state of Gondwanamyceswere obtained from diseased tissues. In this study these isolates are identified based on morphology and comparisons of DNA sequences. Two previously unknown Gondwanamycesspecies were identified, both were associated with damage caused by beetles (Cossonussp.). Inoculation studies showed that the new species of Gondwanamycesare pathogenic on Euphorbia ingensand may contribute to the decline of these trees.

Many species in the Cryphonectriaceae cause diseases of trees, including those in the genera Eucalyptusand Syzygium. During disease surveys on these trees in southern China, fruiting structures typical of fungi in the Cryphonectriaceae and associated with dying branches and stems were observed. Morphological comparisons suggested that these fungi were distinct from the well known Chrysoporthe deuterocubensis, also found on these trees in China. The aim of this study was to identify these fungi and evaluate their pathogenicity to Eucalyptusclones/species as well as Syzygium cumini. Three morphologically similar fungal isolates collected previously from Indonesia also were included in the study. Isolates were characterized based on comparisons of morphology and DNA sequence data for the partial LSU and ITS nuclear ribosomal DNA, β-tubulin and TEF-1α gene regions. After glasshouse trials to select virulent isolates field inoculations were undertaken to screen different commercial Eucalyptusclones/species and S. cuminitrees for susceptibility to infection. Phylogenetic analyses showed that the Chinese isolates and those from Indonesia reside in a clade close to previously identified South African Celoportheisolates. Based on morphology and DNA sequence comparisons, four new Celoporthespp. were identified and they are described as C. syzygii, C. eucalypti, C. guangdongensisand C. indonesiensis. Field inoculations indicated that the three Chinese Celoporthespp., C. syzygii, C. eucalyptiand C. guangdongensis, are pathogenic to all tested Eucalyptusand S. cuminitrees. Significant differences in the susceptibility of the inoculated Eucalyptusclones/species suggest that it will be possible to select disease-tolerant planting stock for forestry operations in the future.

The Cryphonectriaceae accommodates some of the world’s most important tree pathogens, including four genera known from native and introduced Myrtales in Africa. Surveys in the past 3 y in southern Africa have led to the discovery of cankers with fruiting structures resembling those of the Cryphonectriaceae on trees in the Myrtales in Namibia, South Africa, Swaziland and Zambia. These fungi were identified with morphological characteristics and DNA sequence data. For the first time we report Chrysoporthe austroafricanafrom Namibia and on Syzygium guineenseand Holocryphia eucalyptiin Swaziland on a Eucalyptus grandisclone. The host and geographic ranges of Celoporthe dispersaare expanded to include S. legattiin South Africa and S. guineensein Zambia. In addition a monotypic genus, Latruncellus auroraegen. sp. nov., is described from Galpinia transvaalica(Lythraceae, Myrtales) in Swaziland. The present and other recent studies clearly emphasize the limited understanding of the diversity and distribution of fungi in the Cryphonectriaceae in Africa.

There have been several recent reports of Pterocarpus angolensis(kiaat) trees dying in South Africa, Zambia and Zimbabwe, where this tree is used in traditional medicine and is a valuable source of timber for woodcarving and furniture. A survey of material from diseased P. angolensistrees in South Africa yielded isolates of the Botryosphaeriaceae, an important fungal family known to cause a number of tree diseases. The aim of this study was to identify these Botryosphaeriaceae and to determine their pathogenicity to P. angolensiswith branch inoculations. Seven species of the Botryosphaeriaceae were identified based on a combination of morphological characteristics and sequences from the ITS and EF-1α gene regions. Four of these represent undescribed taxa for which the names Pseudofusicoccum violaceum, P. olivaceum, Diplodia alatafructaand Fusicoccum atrovirensare provided. The remaining three species collected include Lasiodiplodia theobromae, L. pseudotheobromaeand L. crassispora. Inoculation trials on tree branches showed that L. pseudotheobromaeand one isolate of D. alatafructadiffered significantly from control inoculations. The high levels of virulence and common occurrence of L. pseudotheobromaesuggest that this species could play a role in tree dieback and death.

Leptographiumspecies provide an ideal model to test the applications of a PCR microcoding system for differentiating species of other genera of ascomycetes. Leptographiumspecies are closely related and share similar gross morphology. Probes designed for a PhyloChip for Leptographiumhave been transferred and tested as primers for PCR diagnostic against Leptographiumspecies. The primers were combined with complementary universal primers to identify known and suspected undescribed species of Leptographium. The primer set was optimized for 56 species, including the three varieties of L. wageneri, then blind-tested against 10 random DNA samples. The protocols established in this study successfully identified species from the blind test as well as eight previously undescribed isolates of Leptographium. The undescribed isolates were identified as new species of Leptographiumwith the aid of the microcoding PCR identification system established in this study. The primers that were positive for each undescribed isolate were used to determine close relatives of these species and some of their biological characteristics. The transfer of oligonucleotides from a micro-array platform to a PCR diagnostic was successful, and the identification system is robust for both known and unknown species of Leptographium.

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 wasp's 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 taxonomy of Armillariain southern South America has received little attention since the work of Singer and others. In this study we examine the morphological traits and cultural features for taxa representing the lineages revealed based on molecular phylogeny, and we link them to previously described taxa based on morphology. Lineages I–IV were identified as Armillaria novaezelandiae, A. montagnei, A. umbrinobrunneacomb. nov. and A. sparreirespectively. They could be differentiated morphologically based on dimension, features of the epicutis, annulus, stipe, hymenophoral trama and flavor and characteristics in culture. Furthermore there was no evidence of host preference for the species recognized. This is the first study integrating the phylogeny and morphology of Armillariaspecies from Patagonia, and it provides a foundation for future research on these fungi in South America.

Neofusicoccum parvumand N. ribisare closely related species whose identities often have been confused. These fungal plant pathogens were identified recently as the most abundant species of Botryosphaeriaceae (Ascomycetes) isolated from native Syzygium cordatumtrees in South Africa. In another study using multiple gene genealogies from five nuclear loci three undescribed cryptic phylogenetic species as well as N. parvumwere identified among 30 of these isolates. The aim of this study was to clarify the identity of the remaining isolates in the N. parvum/N. ribiscomplex from S. cordatumin South Africa, to describe newly identified cryptic species and to test their pathogenicity. Based on the RNA polymerase II subunit (RPB2) sequence comparisons, the isolates were identified as N. parvumor one of three previously recognized phylogenetic species that are described here as N. cordaticola, N. kwambonambienseand N. umdonicola. These species cannot be separated a priori based on morphological characteristics, although a posteriori analysis of variance showed that the differences in conidial length and width between the species were statistically significant. The isolates of the newly described species as well as N. parvumand N. ribiswere tested for pathogenicity on S. cordatumunder greenhouse conditions. Isolates representing the three new species were significantly more aggressive than N. parvumand N. ribiswith N. kwambonambiensebeing the most aggressive. This study resolved long-standing questions of identity of species within N. parvum/N. ribiscomplex and lays a foundation for further studies on this group of pathogens.

After recent changes to the taxonomy of the Botryosphaeriaceae species with diplodia-like (= dark, ovoid, often pigmented) conidia are considered to belong to at least three genera including Diplodia, Lasiodiplodiaand Dothiorella. In a recent molecular phylogenetic study it became apparent that two groups of isolates with diplodia-like conidia required taxonomic revision. One group of isolates originated from Cupressus sempervirensin Greece and Cyprus and had been identified as D. pineaf. sp. cupressibased on morphological characteristics. The other isolates originated from a Casuarinasp. in Australia and were superficially similar to those in the first group based on their morphologically similar diplodia-like conidia. The aim of this study was to resolve the taxonomy of these two groups of isolates by combining the information from the multiple gene genealogies with morphological characters. The results showed that the isolates from C. sempervirensin Greece and Cyprus represent D. cupressi. The isolates from Casuarinain Australia belong to the more distantly related genus Dothiorellaand represent a distinct species that is described here as Do. casuarinisp. nov.

In this study seven new species of the Botryosphaeriaceae are described from baobab (Adansonia gibbosa) and surrounding endemic tree species growing in the Kimberley region of northwestern Australia. Members of the Botryosphaeriaceae were predominantly endophytes isolated from apparently healthy sapwood and bark of endemic trees; others were isolated from dying branches. Phylogenetic analyses of ITS and EF1-α sequence data revealed seven new species: Dothiorella longicollis, Fusicoccum ramosum, Lasiodiplodia margaritacea, Neoscytalidium novaehollandiae, Pseudofusicoccum adansoniae, P. ardesiacumand P. kimberleyense.

Sporothrix, one of the anamorph genera of Ophiostoma, includes the important human pathogen S. schenckiiand various fungi associated with insects and sap stain of wood. A survey of fungi from wood utility poles in South Africa yielded two distinct groups of Sporothrixisolates from different geographical areas. DNA sequence and morphological data derived in this study showed that isolates in these groups represent two novel species in the S. schenckii-O. stenocerasspecies complex. A new species isolated from pine poles and rosebush wood and phylogenetically closely related to S. pallidais described here as Sporothrix stylites. Phylogenetic analyses also confirmed the synonymy of S. albicansand S. niveawith S. pallida. Sporothrix stylitesand S. pallidaalso are related closely to the isolates from soil, previously treated as “environmental” isolates of S. schenckii. Soil isolates are clearly distinct from human isolates of S. schenckii. We describe the former here as Sporothrix humicola. The isolates from eucalypt poles group peripheral to most other species in the S. schenckii-O. stenocerascomplex and are newly described as Sporothrix lignivora. Phylogenetic analyses of sequences of isolates from soil and wood together with those of clinical isolates showed that the human-pathogenic strains form an aggregate of several cryptic species.

Ophiostoma(Ophiostomatales) represents a large genus of fungi mainly known from associations with bark beetles (Curculionidae: Scolytinae) infesting conifers in the northern hemisphere. Few southern hemisphere native species are known, and the five species that consistently occur in the infructescences of Proteaspp. in South Africa are ecologically unusual. Little is known about the vectors of Ophiostomaspp. from Proteainfructescences, however recent studies have considered the possible role of insects and mites in the distribution of these exceptional fungi. In this study we describe a new species of Ophiostomaand a new Sporothrixspp. with affinities to Ophiostoma, both initially isolated from mites associated with Proteaspp. They are described as Ophiostoma gemellussp. nov. and Sporothrix variecibatussp. nov. based on their morphology and comparisons of DNA sequence data of the 28S ribosomal, β-tubulin and internal transcribed spacer (ITS1, 5.8S, ITS2) regions. DNA sequences of S. variecibatuswere identical to those of a Sporothrixisolate obtained from Eucalyptusleaf litter in the same area in which S. variecibatusoccurs in Proteainfructescences. Results of this study add evidence to the view that mites are the vectors of Ophiostomaspp. that colonize Proteainfructescences. They also show that DNA sequence comparisons are likely to reveal additional cryptic species of Ophiostomain this unusual niche.

The majority of Mycosphaerellaspecies from eucalypts (Eucalyptus, Corymbiaand Angophora) in Australia have been recorded only from trees growing in plantations. This illustrates a bias in research in the past two decades toward commercial enterprise, and it emphasises a lack of understanding of the occurrence of these important fungi under natural conditions. Surveys of foliar fungi in native forests in eastern Australia, as well as adjacent plantations, thus have been initiated in recent years. In this study we describe four new species of Mycosphaerellafrom Eucalyptusspp. as well as other Myrtaceae. Mycosphaerella tumulosasp. nov. (anamorph: Pseudocercosporasp.) was found on more than seven species of Eucalyptusand Corymbiain native forests and plantations in northeastern New South Wales and southeastern Queensland and appears to be relatively common, although not damaging to these trees. Mycosphaerella multiseptatasp. nov. was recorded from several locations on species of Angophorain native forests and amenity plantings. Mycosphaerella pseudovespasp. nov. was found in one location in native forest on E. biturbinata. The first species of Mycosphaerellato be described from Syncarpia, M. syncarpiaesp. nov., was found in native forests in numerous locations from Sydney through to northeastern New South Wales and appears to be relatively common.

Botryosphaeria rhodina(anamorph Lasiodiplodia theobromae) is a common endophyte and opportunistic pathogen on more than 500 tree species in the tropics and subtropics. During routine disease surveys of plantations in Australia and Venezuela several isolates differing from L. theobromaewere identified and subsequently characterized based upon morphology and ITS and EF1-α nucleotide sequences. These isolates grouped into three strongly supported clades related to but different from the known taxa, B. rhodinaand L. gonubiensis, These have been described here as three new species L. venezuelensissp. nov., L. crassisporasp. nov. and L. rubropurpureasp. nov. The three could be distinguished easily from each other and the two described species of Lasiodiplodia, thus confirming phylogenetic separations. Furthermore all five Lasiodiplodiaspp. now recognized separated from Diplodiaspp. and Dothiorellaspp. with 100% bootstrap support.

Several recent studies have reviewed the extent of fungal biodiversity, and have used these data as basis for revised estimates of species numbers based on known numbers of plants and insects. None of these studies, however, have focused on fungal biodiversity in South Africa. Coinciding with the 100th anniversary of the National Collection of Fungi (PREM) in South Africa in 2005, it is thus timely to reflect on the taxonomic research that has been conducted in South Africa over the past Century. Information is presented on the extent of fungal collections preserved at PREM, and the associated research publications that have largely resulted from this resource. These data are placed in context of the known plant and insect biodiversity, and used as basis to estimate the potential number of fungi that could be expected in South Africa. The conservative estimate is of approximately 200 000 species without taking into account those associated with a substantial insect biodiversity.

Species of the ascomycete genus Mycosphaerellaare regarded as some of the most destructive leaf pathogens of a large number of economically important crop plants. Amongst these, approximately 60 Mycosphaerellaspp. have been identified from various Eucalyptusspp. where they cause leaf diseases collectively known as Mycosphaerella Leaf Disease (MLD). Species concepts for this group of fungi remain confused, and hence their species identification is notoriously difficult. Thus, the introduction of DNA sequence comparisons has become the definitive characteristic used to distinguish species of Mycosphaerella. Sequences of the Internal Transcribed Spacer (ITS) region of the ribosomal RNA operon have most commonly been used to consider species boundaries in Mycosphaerella. However, sequences for this gene region do not always provide sufficient resolution for cryptic taxa. The aim of this study was, therefore, to use DNA sequences for three loci, ITS, Elongation factor 1-alpha (EF-1α) and Actin (ACT) to reconsider species boundaries for Mycosphaerellaspp. from Eucalyptus. A further aim was to study the anamorph concepts and resolve the deeper nodes of Mycosphaerella, for which part of the Large Subunit (LSU) of the nuclear rRNA operon was sequenced. The ITS and EF-1α gene regions were found to be useful, but the ACT gene region did not provide species-level resolution in Mycosphaerella. A phylogeny of the combined DNA datasets showed that species of Mycosphaerellafrom Eucalyptuscluster in two distinct groups, which might ultimately represent discrete genera.

Ophiostomarepresents a genus of fungi that are mostly arthropod-dispersed and have a wide global distribution. The best known of these fungi are carried by scolytine bark beetles that infest trees, but an interesting guild of Ophiostomaspp. occurs in the infructescences of Proteaspp. native to South Africa. Phylogenetic relationships between Ophiostomaspp. from Proteainfructescences were studied using DNA sequence data from the β-tubulin, 5.8S ITS (including the flanking internal transcribed spacers 1 and 2) and the large subunit DNA regions. Two new species, O. phasmasp. nov. and O. palmiculminatumsp. nov. are described and compared with other Ophiostomaspp. occurring in the same niche. Results of this study have raised the number of Ophiostomaspecies from the infructescences of serotinous Proteaspp. in South Africa to five. Molecular data also suggest that adaptation to the Proteainfructescence niche by Ophiostomaspp. has occurred independently more than once.

Eight pestalotioid fungi were isolated from the Restionaceaegrowing in the Cape Floral Kingdom of South Africa. Sarcostroma restionis, Truncatella megaspora, T. restionacearumand T. spadiceaare newly described. New records include Pestalotiopsis matildae, Sarcostroma lomatiae, Truncatella betulaeand T. hartigii. To resolve generic affiliations, phylogenetic analyses were performed on ITS (ITS1, 5.8S, ITS2) and part of 28S rDNA. DNA data support the original generic concept of Truncatella, which encompasses Pestalotiopsisspecies having 3-septate conidia. The genus Sarcostromais retained as separate from Seimatosporium.

Botryosphaeriais a species-rich genus with a cosmopolitan distribution, commonly associated with dieback and cankers of woody plants. As many as 18 anamorph genera have been associated with Botryosphaeria, most of which have been reduced to synonymy under Diplodia(conidia mostly ovoid, pigmented, thick-walled), or Fusicoccum(conidia mostly fusoid, hyaline, thin-walled). However, there are numerous conidial anamorphs having morphological characteristics intermediate between Diplodiaand Fusicoccum, and there are several records of species outside the Botryosphaeriaceaethat have anamorphs apparently typical of Botryosphaeria s.str. Recent studies have also linked Botryosphaeriato species with pigmented, septate ascospores, and Dothiorellaanamorphs, or Fusicoccumanamorphs with Dichomerasynanamorphs. The aim of this study was to employ DNA sequence data of the 28S rDNA to resolve apparent lineages within the Botryosphaeriaceae. From these data, 12 clades are recognised. Two of these lineages clustered outside the Botryosphaeriaceae, namely Diplodia-like anamorphs occurring on maize, which are best accommodated in Stenocarpella(Diaporthales), as well as an unresolved clade including species of Camarosporium/Microdiplodia. We recognise 10 lineages within the Botryosphaeriaceae, including an unresolved clade (Diplodia/Lasiodiplodia/Tiarosporella), Botryosphaeria s.str. (Fusicoccumanamorphs), Macrophomina, Neoscytalidiumgen. nov., Dothidotthia(Dothiorellaanamorphs), Neofusicoccumgen. nov. (Botryosphaeria-like teleomorphs, Dichomera-like synanamorphs), Pseudofusicoccumgen. nov., Saccharata(Fusicoccum- and Diplodia-like synanamorphs),“ Botryosphaeria” quercuum(Diplodia-like anamorph), and Guignardia(Phyllostictaanamorphs). Separate teleomorph and anamorph names are not provided for newly introduced genera, even where both morphs are known. The taxonomy of some clades and isolates (e.g. B. mamane) remains unresolved due to the absence of ex-type cultures.

Colletogloeopsis zuluensis, previously known as Coniothyrium zuluense, causes a serious stem canker disease on Eucalyptusspp. grown as non-natives in many tropical and sub-tropical countries. This stem canker disease was first reported from South Africa and it has subsequently been found on various species and hybrids of Eucalyptusin other African countries as well as in countries of South America and South-East Asia. In previous studies, phylogenetic analyses based on DNA sequence data of the ITS region suggested that all material of C. zuluensiswas monophyletic. However, the occurrence of the fungus in a greater number of countries, and analyses of DNA sequences with additional isolates has challenged the notion that a single species is involved with Coniothyrium canker. The aim of this study was to consider the phylogenetic relationships amongst C. zuluensisisolates from all available locations and to support these analyses with phenotypic and morphological comparisons. Individual and combined phylogenies were constructed using DNA sequences from the ITS region, exons 3 through 6 of the β-tubulin gene, the intron of the translation elongation factor 1-α gene, and a partial sequence of the mitochondrial ATPase 6 gene. Both phylogenetic data and morphological characteristics showed clearly that isolates of C. zuluensisrepresent at least two taxa. One of these is C. zuluensisas it was originally described from South Africa, and we provide an epitype for it. The second species occurs in Argentina and Uruguay, and is newly described as C. gauchensis.Both fungi are serious pathogens resulting in identical symptoms. Recognising them as different species has important quarantine consequences.

In a survey for Cryphonectriaand Chrysoporthespecies on Myrtalesin South Africa, a fungus resembling the stem canker pathogen Chrysoporthe austroafricanawas collected from native Syzygium cordatumnear Tzaneen (Limpopo Province), Heteropyxis canescensnear Lydenburg (Mpumalanga Province) and exotic Tibouchina granulosain Durban (KwaZulu-Natal Province). The fungus was associated with dying branches and stems on S. cordatum, H. canescensand T. granulosa. However, morphological differences were detected between the unknown fungus from these three hosts and known species of Chrysoporthe. The aim of this study was to characterise the fungus using DNA sequence comparisons and morphological features. Pathogenicity tests were also conducted to assess its virulence on Eucalyptus(ZG 14 clones), H. natalensisand T. granulosa. Plants of H. canescenswere not available for inoculation. Results showed distinct morphological differences between the unknown fungus and Chrysoporthespp. Phylogenetic analysis showed that isolates reside in a clade separate from Chrysoportheand other related genera. Celoporthe dispersagen. et sp. nov. is, therefore, described to accommodate this fungus. Pathogenicity tests showed that C. dispersais not pathogenic to H. natalensis, but that it is a potential pathogen of Eucalyptusand Tibouchinaspp.

Bark beetles (Coleoptera: Scolytinae) are well-recognized vectors of Ophiostomaspecies. Three non-native bark beetle species infest various Pinusspecies 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 Ophiostomaspecies associated with bark beetles in South Africa using comparisons of DNA sequence data. Identities of Ophiostoma ips, O. floccosum, O. pluriannulatum, O. quercusand O. stenoceraswere confirmed. Ophiostoma abietinum, O. piliferumand Pesotum fragransare recognised for the first time and the new species, O. auroraesp. nov., is described from pine-infesting bark beetles in South Africa.

The genus Quambalariaconsists of plant-pathogenic fungi causing disease on leaves and shoots of species of Eucalyptusand its close relative, Corymbia. The phylogenetic relationship of Quambalariaspp., previously classified in genera such as Sporothrixand Ramularia, has never been addressed. It has, however, been suggested that they belong to the basidiomycete orders Exobasidialesor Ustilaginales. The aim of this study was thus to consider the ordinal relationships of Q. eucalyptiand Q. piterekausing ribosomal LSU sequences. Sequence data from the ITS nrDNA were used to determine the phylogenetic relationship of the two Quambalariaspecies 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 Quambalariaspp. and F. cyanescensform a monophyletic clade in the Microstromatales,an order of the Ustilaginomycetes. Sequences from the ITS region confirmed that Q. piterekaand Q. eucalyptiare distinct species. The ex-type isolate of F. cyanescens, together with another isolate from Eucalyptusin 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 Quambalariaspp. have dolipores with swollen lips, which differ from other members of the Microstromatales(i.e. the Microstromataceaeand Volvocisporiaceae) that have simple pores with more or less rounded pore lips. Based on their unique ultrastructural features and the monophyly of the three Quambalariaspp. in the Microstromatales, a new family, QuambalariaceaeZ.W. de Beer, Begerow & R. Bauer fam. nov., is described.

This study records a severe outbreak of a disease on Zizyphus mucronata(Rhamnaceae) in Greater Kruger National Park, South Africa. The causal agent of the disease was found to be Coniodictyum chevalieri, a fungus previously believed to be very rare. Detailed illustrations of the symptoms and fungus are presented in order to facilitate future studies. The known geographical distribution of Coniodictyumis presented in relation to the distribution of its host, and a short review of its systematic history is also given. This also treats an invalidly published species name in South Africa, which has confused the literature. A DNA-based phylogeny is presented for the pathogen and this reflects the unique nature of its geographical distribution and biology.

Cryphonectria havanensisis a fungus associated with Eucalyptusspecies in Cuba and Florida (U.S.A.). Until recently, there have been no living cultures of C. havanensisand it has thus not been possible to assess its taxonomic status. Isolates thought to represent this fungus have, however, emerged from surveys of Eucalyptusin Mexico and Hawaii (U.S.A.). Results of this study showed that these isolates represent C. havanensisbut reside in a genus distinct from Cryphonectria sensu stricto, which is described here as Microthia. Isolates of an unidentified fungus occurring on Myrica fayain the Azores and Madeira also grouped in Microthiaand were identical to other M. havanensisisolates. Cryphonectria coccolobae, a fungus occurring on sea grape (Coccoloba uvifera) in Bermuda and Florida, was found to be morphologically identical to Microthiaand is transferred to this genus, but as a distinct species. Surveys for M. coccolobaeon sea grape in Florida, yielded a second diaporthalean fungus from this host. This fungus is morphologically and phylogenetically distinct from M. coccolobaeand other closely related taxa and is described as Ursicollum fallaxgen. et sp. nov. Phylogenetic analyses in this study have also shown that isolates of C. eucalypti, a pathogen of Eucalyptusin South Africa and Australia, group in a clade separate from all other groups including that representing Cryphonectria sensu stricto. This difference is supported by the fact that Cryphonectria eucalyptihas ascospore septation different to that of all other Cryphonectriaspecies. A new genus, Holocryphia, is thus erected for C. eucalypti.

Species of Eucalyptusare widely planted as exotics in the tropics and Southern Hemisphere and to some extent in southern Europe, for timber and fibre production. Species of Mycosphaerellaare commonly associated with leaves and twigs of Eucalyptusand can result in defoliation, dieback, and even tree death. In the present study, numerous isolates of Mycosphaerellaspecies were collected from leaf litter, living leaves exhibiting leaf spot symptoms or severe Mycosphaerella leaf blotch symptoms. Isolates were compared based on DNA sequence data for the internal transcribed spacer region (ITS1 & ITS2) and the 5.8S gene. These data, together with characteristics of the fungal growth on three different media, morphology of the anamorph and teleomorph structures as well as ascospore germination patterns were used to describe 21 new species.