Irene Barnes

One fungus, which genes? Development and assessment of universal primers for potential secondary fungal DNA barcodes

The aim of this study was to assess potential candidate gene regions and corresponding universal primer pairs as secondary DNA barcodes for the fungal kingdom, additional to ITS rDNA as primary barcode. Amplification efficiencies of 14 (partially) universal primer pairs targeting eight genetic markers were tested across > 1 500 species (1 931 strains or specimens) and the outcomes of almost twenty thousand (19 577) polymerase chain reactions were evaluated. We tested several well-known primer pairs that amplify: i) sections of the nuclear ribosomal RNA gene large subunit (D1–D2 domains of 26/28S); ii) the complete internal transcribed spacer region (ITS1/2); iii) partial β -tubulin II (TUB2); iv) γ-actin (ACT); v) translation elongation factor 1-α (TEF1α); and vi) the second largest subunit of RNA-polymerase II (partial RPB2, section 5–6). Their PCR efficiencies were compared with novel candidate primers corresponding to: i) the fungal-specific translation elongation factor 3 (TEF3); ii) a small ribosomal protein necessary for t-RNA docking; iii) the 60S L10 (L1) RP; iv) DNA topoisomerase I (TOPI); v) phosphoglycerate kinase (PGK); vi) hypothetical protein LNS2; and vii) alternative sections of TEF1α. Results showed that several gene sections are accessible to universal primers (or primers universal for phyla) yielding a single PCR-product. Barcode gap and multi-dimensional scaling analyses revealed that some of the tested candidate markers have universal properties providing adequate infra- and inter-specific variation that make them attractive barcodes for species identification. Among these gene sections, a novel high fidelity primer pair for TEF1α, already widely used as a phylogenetic marker in mycology, has potential as a supplementary DNA barcode with superior resolution to ITS. Both TOPI and PGK show promise for the Ascomycota, while TOPI and LNS2 are attractive for the Pucciniomycotina, for which universal primers for ribosomal subunits often fail.

Redefining Ceratocystis and allied genera.

The genus Ceratocystis was established in 1890 and accommodates many important fungi. These include serious plant pathogens, significant insect symbionts and agents of timber degradation that result in substantial economic losses. Virtually since its type was described from sweet potatoes, the taxonomy of Ceratocystis has been confused and vigorously debated. In recent years, particulary during the last two decades, it has become very obvious that this genus includes a wide diversity of very different fungi. These have been roughly lumped together due to their similar morphological structures that have clearly evolved through convergent evolution linked to an insect-associated ecology. As has been true for many other groups of fungi, the emergence of DNA-based sequence data and associated phylogenetic inferences, have made it possible to robustly support very distinct boundaries defined by morphological characters and ecological differences. In this study, DNA-sequence data for three carefully selected gene regions (60S, LSU, MCM7) were generated for 79 species residing in the aggregate genus Ceratocystis sensu lato and these data were subjected to rigorous phylogenetic analyses. The results made it possible to distinguish seven major groups for which generic names have been chosen and descriptions either provided or emended. The emended genera included Ceratocystis sensu stricto, Chalaropsis, Endoconidiophora, Thielaviopsis, and Ambrosiella, while two new genera, Davidsoniella and Huntiella, were described. In total, 30 new combinations have been made. This major revision of the generic boundaries in the Ceratocystidaceae will simplify future treatments and work with an important group of fungi including distantly related species illogically aggregated under a single name.

Characterization and distribution of mating type genes in the Dothistroma needle blight pathogens

ABSTRACT Dothistroma septosporum and D. pini are the two causal agents of Dothistroma needle blight of Pinus spp. in natural forests and plantations. Degenerate primers amplified portions of mating type genes (MAT1-1-1 and MAT1-2) and chromosome walking was applied to obtain the full-length genes in both species. The mating-type-specific primers designed in this study could distinguish between the morphologically similar D. pini and D. septosporum and between the different mating types of these species. Screening of isolates from global collections of D. septosporum showed that only MAT2 isolates are present in Australian and New Zealand collections, where only the asexual form of the fungus has been found. In contrast, both mating types of D. septosporum were present in collections from Canada and Europe, where the sexual state is known. Intriguingly, collections from South Africa and the United Kingdom, where the sexual state of the fungus is unknown, included both mating types. In D. pini, for which no teleomorph is known, both mating types were present in collections from the United States. These results provided new insights into the biology and global distribution of two of the worlds most important pine pathogens and should facilitate management of the diseases caused by these fungi.

Ceratocystis pirilliformis, a new species from Eucalyptus nitens in Australia

Several species of Ceratocystis have been recorded on Eucalyptus. These include C. fimbriata, C. eucalypti, C. moniliformis and C. moniliformopsis. Of these, only C. fimbriata is known as a pathogen; it recently has been found causing serious wilt diseases in Uganda, Congo and Brazil. This study was undertaken to collect Ceratocystis species, including C. eucalypti, from artificially induced wounds on Eucalyptus nitens near Canberra in southeastern Australia. Trees were wounded in October 2000, and wounds were examined approximately one month later. Ascomata characteristic of a Ceratocystis species were found covering the wounds, and this fungus also was isolated from the wood using carrot baiting. This species of Ceratocystis has hat-shaped ascospores similar to those of C. fimbriata, but it differs from C. fimbriata and all other species of Ceratocystis in that it possesses ascomata with a pyriform base. Comparison of DNA sequences from the ITS and 5.8S rRNA operon confirmed that the fungus from E. nitens in Australia is unique, and we describe it here as a new species, C. pirilliformis.

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.

Ceratocystis fimbriata infecting Eucalyptus grandis in Uruguay

Uruguay has a rapidly growing forestry industry consisting mainly of exotic Pinus and Eucalyptus spp. Recently, there have been reports of individual E. grandis trees wilting and dying rapidly in plantations. The aim of this investigation was to survey the dying E. grandis in the Rivera area of Uruguay and to determine the cause of the Eucalyptus wilt. Sap-staining symptoms were observed on recently pruned E. grandis. Discs of discoloured wood were cut from these pruned trees and from the stems of dying trees. These disks were stored in a moist environment to induce fungal sporulation. Ascomata, typical of a Ceratocystis sp., were found covering the edges of the wood where streaking symptoms occurred. Morphologically, the fungus resembles C. fimbriata. The internal transcribed spacer regions of the ribosomal RNA operon of the Ceratocystis sp. were amplified and sequenced. Sequence data confirmed placement of this fungus amongst other isolates of C. fimbriata. Furthermore, the sequence data showed that the Uruguay isolates are most closely related to those from diseased Eucalyptus spp. in Brazil, Congo and Uganda. C. fimbriata is a well-known pathogen of many woody plants and could constitute a serious threat to intensively managed E. grandis in Uruguay where the fungus was not previously known. The relationship between the pruning of E. grandis and infection by C. fimbriata will, in future, need to be evaluated.

Population structure and diversity of an invasive pine needle pathogen reflects anthropogenic activity.

Dothistroma septosporum is a haploid fungal pathogen that causes a serious needle blight disease of pines, particularly as an invasive alien species on Pinus radiata in the Southern Hemisphere. During the course of the last two decades, the pathogen has also incited unexpected epidemics on native and non-native pine hosts in the Northern Hemisphere. Although the biology and ecology of the pathogen has been well documented, there is a distinct lack of knowledge regarding its movement or genetic diversity in many of the countries where it is found. In this study we determined the global population diversity and structure of 458 isolates of D. septosporum from 14 countries on six continents using microsatellite markers. Populations of the pathogen in the Northern Hemisphere, where pines are native, displayed high genetic diversities and included both mating types. Most of the populations from Europe showed evidence for random mating, little population differentiation and gene flow between countries. Populations in North America (USA) and Asia (Bhutan) were genetically distinct but migration between these continents and Europe was evident. In the Southern Hemisphere, the population structure and diversity of D. septosporum reflected the anthropogenic history of the introduction and establishment of plantation forestry, particularly with Pinus radiata. Three introductory lineages in the Southern Hemisphere were observed. Countries in Africa, that have had the longest history of pine introductions, displayed the greatest diversity in the pathogen population, indicating multiple introductions. More recent introductions have occurred separately in South America and Australasia where the pathogen population is currently reproducing clonally due to the presence of only one mating type.

Microsatellite markers for the red band needle blight pathogen, Dothistroma septosporum

Twelve microsatellite markers were developed for population analyses of the fungal pathogen, Dothistroma septosporum. Intersimple sequence repeat polymerase chain reaction (ISSR‐PCR) and an enrichment protocol (fast isolation by amplified fragment length polymorphism of sequences containing repeats [FIASCO]) were both used to identify 28 unique microsatellite regions in the genome. From 22 primer pairs designed, 12 were polymorphic. These markers, screened on two populations representing 42 isolates, produced 40 alleles across all loci with an allelic diversity of 0.09–0.76 per locus. Cross‐species amplification showed variable success with Dothistroma rhabdoclinis and Mycosphaerella dearnessi and some sequence variation within isolates of Dothistroma pini. These markers will be used to further study the population structure and diversity of D. septosporum.

Ceratocystis manginecans associated with a serious wilt disease of two native legume trees in Oman and Pakistan

A serious wilt disease has recently been found on Prosopis cineraria (Ghaf) in Oman and on Dalbergia sissoo (Shisham) in Pakistan. Disease symptoms on both these native, leguminous hosts include vascular discolouration and partial or complete wilt of affected trees. A species of Ceratocystis was consistently isolated from symptomatic material. Morphological comparisons and analyses of DNA sequence data of the ITS, β-tubulin, and EF 1-α gene regions showed that the Ceratocystis isolates obtained from both tree species represent C. manginecans. This is the same pathogen that is causing the devastating mango sudden decline disease in Oman and Pakistan. This is also the same pathogen that has been reported causing a wilting disease on Acacia mangium in Indonesia. Cross inoculation with C. manginecans isolates from P. cineraria, D. sissoo and mango showed that the fungus can cause disease on all three trees.

Distribution and population diversity of Ceratocystis pirilliformis in South Africa

Ceratocystis pirilliformis was first isolated from wounds on Eucalyptus nitens in Australia and subsequently found in a similar niche on E. grandis in South Africa. Artificial inoculation studies under field conditions in South Africa resulted in bark lesions and sapstain, suggesting that the fungus is a pathogen of potential importance to forestry in South Africa. Because Eucalyptus spp. are native to Australia and C. pirilliformis was first found there in the absence of disease it has been assumed that the fungus is native to Australia. The aim of this study was to expand the base of knowledge regarding the distribution and population diversity of C. pirilliformis in South Africa. Wounds were examined on Eucalyptus spp. growing in seven of the most important forestry areas in South Africa. PCR-based microsatellite markers, developed for the closely related tree pathogen C. fimbriata sensu lato were used to assess the diversity of the isolates collected. Ceratocystis pirilliformis was found in four out of seven areas surveyed, substantially expanding its known distribution in South Africa. Of the 27 available microsatellite markers, 18 amplified the desired loci of C. pirilliformis and of these, seven were polymorphic. Measures of genetic diversity based on gene diversity, genotypic diversity as well as allelic richness indicated that the isolates from South Africa has a low level of genetic diversity and that the fungus is most likely not native to the country. Inclusion of available Australian isolates showed much higher diversity for C. pirilliformis in that country.