Glen R. Klassen

A molecular phylogeny of Pythium insidiosum

Sequence analysis of the ribosomal DNA internal transcribed spacers (ITS) was used to establish phylogenetic relationships among 23 isolates of Pythium insidiosum, the etiological agent of pythiosis in mammals. The isolates were divided into three distinct clades that exhibited significant geographic isolation. Clade I consisted of isolates from North, Central, and South America, while clade II contained isolates from Asia and Australia. Also present in clade II was an isolate from a patient in the USA, but the origin of the infection may have been in the Middle East. Clade III was comprised of isolates from Thailand and the USA. All 23 P. insidiosum isolates were more closely related to each other than to any other Pythium species in this study. Additionally, all Pythium isolates formed a clade separate from both outgroup species, Phytophthora megasperma and Lagenidium giganteum. The ITS sequence results tend to support the existence of geographic variants or cryptic speciation within P. insidiosum. The sequence information obtained also provides an abundance of data for applications in the diagnosis of pythiosis and identification of P. insidiosum from clinical samples.

DO GALEATE-ASCOSPORE MEMBERS OF THE CEPHALOASCACEAE, ENDOMYCETACEAE AND OPHIOSTOMATACEAE SHARE A COMMON PHYLOGENY?

Using partial small subunit ribosomal gene sequences we show that yeast-like genera that produce galeate (hat-shaped) ascospores and similar-spored members of the Ophiostomatales do not form a monophyletic group. Based on distance and parsimony methods Cephaloascusfragrans and Endomyces decipiens failed to form a monophyletic grouping with species of Ceratocystis sensu stricto and Ophiostoma. Instead these yeast species clustered with Saccharomyces cerevisiae, Kluyveromyces lactis, and Torulaspora delbrueckii in >99% of both the distance and parsimony majority-rule consensus trees generated using the statistical method of bootstrapping. Therefore galeate ascospores appear to be an example of convergent evolution in fungi and by itself this trait should not be considered as an indicator of evolutionary relatedness.

Ceratocystiopsis: a reappraisal based on molecular criteria

Analysis of partial rDNA sequences from both the small and large subunit genes of species of Ceratocystiopsis suggests they represent a polyphyletic group. Therefore the production of falcate-like ascospores, the key feature of this genus, is an example of convergent evolution. The partial rDNA sequences also show that the majority of Ceratocystiopsis spp. can be placed in Ophiostoma along with Certocystis ips , a conclusion supported at the 100% confidence level by bootstrap analysis. Therefore we formally reduce Ceratocystiopsis to synonymy with Ophiostoma and provide the necessary new combinations in Ophiostoma . However, C. proteae, C. falcata , and C. alba are shown to have a very distant relationship with Ophisotoma , and their taxonomic position remains unresolved.

Detection of length heterogeneity in the ribosomal DNA of Pythium ultimum by PCR amplification of the intergenic region

SummaryAbout one-half of the ribosomal repeat unit of two isolates of Pythium ultimum was amplified by means of the polymerase chain reaction using one primer pair. The amplified region includes a small part of the large subunit ribosomal RNA gene, about half of the small subunit ribosomal RNA gene, and the entire intergenic region. The intergenic region of both isolates of P. ultimum has length heterogeneity due to the presence of subrepeat arrays (Klassen and Buchko 1990). PCR amplification of the heterogeneous target DNA resulted in sets of fragments which accurately reflect the heterogeneity in the target DNA, although there is a preferential amplification of the smaller targets. PCR product sizes ranged from 4.6 to 5.8 kb.

Evidence for geographic clusters: Molecular genetic differences among strains of Pythium insidiosum from Asia, Australia and the Americas are explored

Twenty-eight isolates of Pythium insidiosum and P. destruens from Asia, Australia and the Americas were compared on the basis of restriction fragment-length polymorphisms of the amplified ribosomal intergenic spacer. Comparison of band profiles yielded three distinct clusters and an isolate that did not fall into any of the clusters. Cluster I consisted of 16 isolates, all from the Americas (Costa Rica, Brazil, Haiti, United States). Cluster II consisted of seven isolates from Asia (India, Thailand, Japan, Papua New Guinea) and Australia, including the two isolates of P. destruens. This cluster also included a United States isolate from a human who might have contracted an infection of P. insidiosum by contact with food from the Middle East. Cluster III was most distantly related to the other two clusters and consisted of two isolates from Thailand and one from the United States. The isolate excluded from all three clusters was from a spectacled bear in a zoo in the United States. These results indicate that all the isolates are more closely related to each other than to any other Pythium species and thus indeed might be one species, but they also point to geographical variants. Cluster III and Isolate M18 are so distant from the others that they might prove to be separate species. Knowledge of intraspecific variability in P. insidiosum might be important for the management of pythiosis in mammals.

Subrepeat structure of the intergenic region in the ribosomal DNA of the oomycetous fungus Pythium ultimum

SummaryRestriction endonuclease site maps of the rDNA repeat in 14 isolates of the oomycetous fungus Pythium ultimum show that the intergenic region (IGR) includes two regions of heterogeneity. A region located about one kilobase downstream of the 3′ end of the large subunit rRNA gene contains a segment present in multiple versions differeing in size by as much as 0.9 kb. Each isolate shows a unique pattern of heterogeneity in this region. The other region of heterogeneity is located near the centre of the IGR and consists of segments which differ by integral multiples of 385 bp. Thus, the IGR in P. ultimum is dominated by an array of uniform subrepeats. The size of the repeat segment is identical in all isolates, but the number of subrepeats (6–12), and the relative abundance of each variant segment is highly polymorphic. Because no other fungus has been reported with this type or organization in its rDNA repeat, these findings support the idea that Oomycetes are phylogenetically distinct from other fungi. They also provide a new system for the study of the role of rDNA subrepeats in transcriptional regulation.

A region of heterogeneity adjacent to the 5s ribosomal RNA gene of cereal rusts.

Total genomic DNA was isolated from three cereal stem rusts, Puccinia graminis f. sp. tritici, f. sp. secalis, f. sp. avenae, and two cereal leaf rusts, P. recondita f. sp. tritici and P. coronata f. sp. avenae, and analyzed for the presence of heterogeneity in the intergenic region of the ribosomal DNA repeat unit. A 1 kb region of the repeat unit between the 26s and the 5s rRNA genes (IGR-1) was amplified by PCR and was found to be heterogeneous within each isolate and variable in size between races and species. The PCR results were confirmed by Southern blot analysis of native DNA. In an isolate of race C36(48), heterogeneity appeared to be due to variable numbers of 0.1 kb subrepeats in IGR-1. Nine wheat stem rust strains representing nine different races produced a unique pattern of heterogeneity while two different isolates of one race were identical, as were five of another. This may provide a rapid method for race identification in wheat stem rust. Heterogeneity and polymorphism in rye stem rust, oat stem rust, wheat leaf rust, and oat crown rust, was less pronounced than in wheat stem rust. In the course of this work, the 5s rRNA gene was located and its position and orientation within the ribosomal repeat unit was established.

Development of a Species-Specific Probe for Pythium insidiosum and the Diagnosis of Pythiosis

ABSTRACT Pythium insidiosum, the only species in the genus that infects mammals, is the etiological agent of pythiosis, a granulomatous disease characterized by cutaneous and subcutaneous lesions and vascular diseases. Accurate diagnosis of pythiosis and identification of its causal agent are often inconsistent with current immunological diagnostic methods. A species-specific DNA probe was constructed by using a 530-bp HinfI fragment from the ribosomal DNA intergenic spacer of P. insidiosum. When the probe was incubated with dot blots of genomic DNA from 104 Pythium species, it hybridized only to the DNA of P. insidiosum and P. destruens—two species that have been considered conspecific. The probe also hybridized to DNA from 22 P. insidiosum isolates in this study, regardless of their geographic origin or animal host. When tested against genomic DNA from other pathogenic organisms (Aspergillus fumigatus, Basidiobolus ranarum, Conidiobolus coronatus, Lagenidium giganteum, Paracoccidioides brasiliensis, and Prototheca wickerhamii), no cross-hybridization of the probe was detected. The specificity of the probe to hybridize to genomic DNA from all isolates of P. insidiosum and not cross-react with DNA from other Pythium species or pathogens that cause symptoms similar to pythiosis in their hosts makes it a powerful tool for the accurate diagnosis of pythiosis. In addition, the probe has the potential for pathological and environmental diagnostic applications.

An inverted repeat comprises more than three-quarters of the mitochondrial genome in two species of Pythiumm

SummaryPhysical maps of mitochondrial DNA from Pythium torulosum (73 kb) and Pythium diclinum (70 kb) reveal that an exceptionally large proportion (75 and 80%, respectively) of the mitochondrial genomes of these oomycetes exists in the form of an inverted repeat. The size of the inverted repeat is more highly conserved between the two species than is the size of the small single-copy region. This indicates that the presence and size of an inverted repeat may be useful in oomycete systematics at the species level.

Double-stranded RNAs in mitochondrial extracts of stem rusts and leaf rusts of cereals

SummaryDouble-stranded ribonucleic acids (dsRNA) were isolated from mitochondria in urediosporelings of three cereal stem rusts, Puccinia graminis f. sp. tritici, f. sp. secalis and f. sp. avenae, and two cereal leaf rusts, P. recondita f. sp. tritici and P. coronata f. sp. avenae, and analyzed by agarose gel electrophoresis. The double strandedness of the RNA molecules was characterized by nuclease treatments (RNase A, DNase 1 and S1 nuclease) and CF-11 cellulose column chromatography. No interspecific variation in multisegments of dsRNA was observed among races of each forma specialis. As to the interspecific variation, although each of three forma specialis of Puccinia graminis had similar dsRNA segments, 4.8, 5.0 and 5.2 kb, wheat leaf rust and oat crown rust had additional dsRNA segments of 2.7, 2.8, 5.8 and 6.0 kb. The presence of a dsRNA segment of 5 kb size in all isolates and species examined indicates that this unique segment can be a molecular marker for the rust family, Uredinales. Dot-blot hybridization indicated that there is no sequence homology between dsRNA segments and mitochondrial DNA.