Sarah Hambleton

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.

Distribution and molecular characterization of the root endophyte Phialocephala fortinii along an environmental gradient in the boreal forest of Alberta

Phialocephala fortinii is a common root endophytic fungus with a wide geographic distribution and little, if any, host specificity. Little is known about its habitat specificity, although there is evidence to suggest that high water tables may restrict the occurrence of P. fortinii in wetlands. We tested this hypothesis by determining the distribution of P. fortinii along a sand dune – wetland complex. Isolates of P. fortinii, identified on the basis of cultural and morphological characteristics, were obtained from the roots of vascular plants across the moisture gradient. Three ‘cultural groups’ were recognized among these isolates. Thirty-three of these isolates were compared among themselves and to strains of known identity using PCR/RFLP analysis of the ITS region and a portion of the 28S subunit of rDNA. The restriction digest profiles of all isolates were identical to those of P. fortinii for 4 restriction enzymes. DNA sequences, from a subset of these strains, showed a low percent sequence divergence confirming the reliability of the RFLP data. The same analyses were done with two strains of Leptodontidium orchidicola a culturally similar root endophyte, to ensure that this taxon was not among the transect isolates. DNA data showed a clear difference between P. fortinii and L. orchidicola but did not discriminate among cultural groups. Thus, P. fortinii showed no habitat specificity and occurred in both xeric and hydric sites. RFLP profiles and ITS sequences showed little variation among isolates of P. fortinii and among the isolates of L. orchidicola.

Clonal lineages of Sclerotinia sclerotiorum previously known from other crops predominate in 1999-2000 samples from Ontario and Quebec soybean

Twenty-one mycelial compatibility groups (MCGs) and 41 DNA fingerprints were identified from 213 isolates of Sclerotinia sclerotiorum from 10 fields and three performance trials of soybean, and one test plot and one field of edible bean sampled in eastern Ontario and Quebec (1999) and western Ontario (2000). Population structure was predominantly clonal as evidenced by repeated recovery of MCGs and fingerprints in the sample and the association of fingerprint with MCG. Evolution of new fingerprint phenotypes within some clones was indicated by differences of one to three hybridizing fragments from the most frequently sampled fingerprint associated with an MCG. A single MCG represented 46% of the isolates; this was clonal lineage 1, a frequently sampled clone from Ontario canola in 1989, also sampled previously from cabbage in New York State. Five of the 21 MCGs represented 84% of the total sample. Sixty percent of the isolates had fingerprints identical to or differing by one or two hybridizing fragments to fingerprints archived from previous samples from canola, cabbage, edible bean, and other crops. Results indicate that soybean is infected mainly by pathogen genotypes residual from other crop or weed hosts and that a few pathogen genotypes are responsible for a large proportion of infections.

Molecular Characterization of Reptile Pathogens Currently Known as Members of the Chrysosporium Anamorph of Nannizziopsis vriesii Complex and Relationship with Some Human-Associated Isolates

ABSTRACT In recent years, the Chrysosporium anamorph of Nannizziopsis vriesii (CANV), Chrysosporium guarroi, Chrysosporium ophiodiicola, and Chrysosporium species have been reported as the causes of dermal or deep lesions in reptiles. These infections are contagious and often fatal and affect both captive and wild animals. Forty-nine CANV isolates from reptiles and six isolates from human sources were compared with N. vriesii based on their cultural characteristics and DNA sequence data. Analyses of the sequences of the internal transcribed spacer and small subunit of the nuclear ribosomal gene revealed that the reptile pathogens and human isolates belong in well-supported clades corresponding to three lineages that are distinct from all other taxa within the family Onygenaceae of the order Onygenales. One lineage represents the genus Nannizziopsis and comprises N. vriesii, N. guarroi, and six additional species encompassing isolates from chameleons and geckos, crocodiles, agamid and iguanid lizards, and humans. Two other lineages comprise the genus Ophidiomyces, with the species Ophidiomyces ophiodiicola occurring only in snakes, and Paranannizziopsis gen. nov., with three new species infecting squamates and tuataras. The newly described species are Nannizziopsis dermatitidis, Nannizziopsis crocodili, Nannizziopsis barbata, Nannizziopsis infrequens, Nannizziopsis hominis, Nannizziopsis obscura, Paranannizziopsis australasiensis, Paranannizziopsis californiensis, and Paranannizziopsis crustacea. Chrysosporium longisporum has been reclassified as Paranannizziopsis longispora. N. guarroi causes yellow fungus disease, a common infection in bearded dragons and green iguanas, and O. ophiodiicola is an emerging pathogen of captive and wild snakes. Human-associated species were not recovered from reptiles, and reptile-associated species were recovered only from reptiles, thereby mitigating concerns related to zoonosis.

Comparative Morphology and Phylogenetic Placement of Two Microsclerotial Black Fungi from Sphagnum

Capnobotryella renispora and Scleroconidioma sphagnicola form black, irregularly shaped microsclerotia that are indistinguishable in gross morphology on leaves of Sphagnum fuscum. In culture, microsclerotia of these fungi were similar, in that mature component cells possessed thick, highly melanized cell walls, poorly defined organelles, large lipid bodies and simple septa. They were different in morphogenesis, in the way their component cells were organized and in disseminative propagules. Microsclerotia of S. sphagnicola formed phialidic conidiogenous cells on their surface, whereas in C. renispora, adjacent cells in mature microsclerotia often separated from each other by septum schizolysis and formed chlamydospores. The identification of C. renispora from Sphagnum is provisional despite a 100% ITS sequence match with data for a culture derived from the type strain. No holoblastic, reniform conidia typical of the species were formed in nature or in culture, and the SSU sequence for a separately preserved culture of the ex-type strain was markedly divergent. Parsimony analyses of nuclear ribosomal DNA sequences showed that these two fungi were related to separate orders of Dothideomycetes. Both SSU and ITS data supported a close relationship for S. sphagnicola to the Dothideales sensu stricto, while the closest ITS match was to Rhizosphaera spp. In the SSU analyses, C. renispora was nested within the Capnodiales.

False-positive Histoplasma capsulatum Gen-Probe chemiluminescent test result caused by a Chrysosporium species.

ABSTRACT We describe a case in which the Histoplasma capsulatum AccuProbe test displayed cross-reactivity with a respiratory isolate thought to be Histoplasma but not morphologically consistent with H. capsulatum. The isolate was later identified as the Chrysosporium anamorph of Nannizziopsis vriesii by sequence analysis and phenotypic data.

Poplar rust systematics and refinement of Melampsora species delineation

We present a review of previous taxonomic treatments of the Melampsora species occurring on poplar and describe the features associated with each spore stage in a typical poplar rust life cycle. The morphological, biological and ecological characters traditionally used for taxonomy are summarized for all Melampsora taxa, including 17 accepted species, 2 formae speciales and 2 hybrids, currently listed in the literature as pathogenic on Populus spp. We discuss the historical taxonomic decisions that led to nomenclatural and classification complications for this group of rusts, and highlight the lack of type material for three species known only from the original collections: M. cumminsii, M. multa, and M. osmaniensis. Even when all the traditional features are considered, the taxonomy of Melampsora poplar rust species remains uncertain and incomplete. An overview of recent molecular studies suggests the need for a comprehensive revision of species concepts, based on phylogenetic relationships. To that end, we propose a polyphasic approach, including the Genealogical Concordance Phylogenetic Species Recognition method, be used to build robust and meaningful systematic framework for the Melampsora poplar rusts.

Morphology and phylogenetic placement of Endoconidioma, a new endoconidial genus from trembling aspen.

Endoconidioma populi gen. et sp. nov. is described from black subicula on twigs of trembling aspen, Populus tremuloides, in Alberta, Canada. Pycnidium-like conidiomata are produced on twigs and in culture, but, unlike pycnidia, conidiomata of E. populi have a closed peridium and a locule filled with conidiogenous cells that form conidia endogenously. These endoconidia are hyaline, unicellular and released by the dissolution of the peridial cell wall. In addition to endoconidia, mostly two-celled conidia that form blastically from undifferentiated hyphae occur often in culture but are observed only occasionally on Populus twigs. No coelomycetous taxa have been reported to produce endoconidia, and both the morphological features and DNA sequence data demonstrate that Endoconidioma is distinct from the previously established endoconidial genera. Parsimony analyses of portions of the nuclear ribosomal RNA gene (SSU and ITS) suggest that Endoconidioma is closely related phylogenetically to members of the Dothideales and allied anamorphs in Hormonema and Kabatina.

Laying the foundation for a taxonomic review of Puccinia coronata s.l. in a phylogenetic context

Intra-specific classification of Puccinia coronata has been controversial, with previous approaches falling into three major categories: 1. A two-species system, namely P. coronifera and P. coronata; 2. The same two-species system subdivided into many formae speciales, in which the host range of each is restricted to species within one genus of Poaceae; 3. A one-species system, P. coronata, subdivided into a few varieties with host ranges that may overlap. To re-assess these concepts in the context of multigene analyses and comparative morphological assessments, data were generated for a comprehensive set of herbarium and recently collected specimens, representing a broad range of hosts and geographic origins. Phylogenetic analyses of a combined data set of DNA sequences for four loci (BT, COI, ITS, and RPB2) revealed a high degree of genetic variation. Morphological differences among phylogenetic lineages were overlapping but nine lineages were differentiated using calculated means for teliospore and urediniospore length/width as well as measurements for the teliospore hilum and digitation. The taxon infecting Avena also comprises collections from a wide range of other grass hosts while other lineages, such as those on Bromus and Agrostis, were restricted in host association. Type specimen DNA sequences included in the analyses resolved the placement of five previously described varieties. Based on evidence of host specificity, morphology and multigene analyses, we recognized seven species, one of which was further divided into two varieties. Expanded descriptions, illustrations and a synoptic key are provided. A new series, Puccinia Series Coronata, was erected to accommodate all the lineages comprising P. coronata sensu lato.

Detection and identification of selected cereal rust pathogens by TaqMan® real-time PCR

Abstract The rust species Puccinia graminis and P. striiformis sensu stricto are important cereal pathogens, most well-known for causing the diseases stem rust and stripe rust on wheat and generating significant yield losses. Early and accurate detection of the pathogens would facilitate effective control of the diseases. In the present study, we developed real-time PCR assays to detect the specific lineages that include the wheat pathogens for each species complex, as identified using multi-gene DNA sequence analyses. Four DNA loci, for a comprehensive set of target and closely related fungi collected from diverse hosts and geographic regions, were explored to search for suitable lineage-specific probes: β-tubulin (BT), cytochrome c oxidase subunit 1 (COI), rDNA internal transcribed spacer (ITS), and RNA polymerase II second largest subunit (RPB2). Four TaqMan® real-time PCR assays were designed based on either the BT or RPB2 genes: one targeting Puccinia Series Striiformis (PSBT), one targeting P. striiformis sensu stricto (PSstrRPB2), and two targeting the P. graminis lineages on wheat (Pg2+BT and Pg2RPB2). Sensitivities of the assays were determined to be 0.65 pg µL−1 (Pg2) and 5 pg µL−1 (PS). Specificity of each assay was confirmed using a broad diversity of rusts and other selected wheat-associated fungi. The ITS and COI loci were found to be unsuitable for diagnostic assay development but contributed phylogenetic signal to the multi-gene analyses.