Dataset Information

DNA barcoding of oomycetes with cytochrome c oxidase subunit I and internal transcribed spacer.

ABSTRACT: Oomycete species occupy many different environments and many ecological niches. The genera Phytophthora and Pythium for example, contain many plant pathogens which cause enormous damage to a wide range of plant species. Proper identification to the species level is a critical first step in any investigation of oomycetes, whether it is research driven or compelled by the need for rapid and accurate diagnostics during a pathogen outbreak. The use of DNA for oomycete species identification is well established, but DNA barcoding with cytochrome c oxidase subunit I (COI) is a relatively new approach that has yet to be assessed over a significant sample of oomycete genera. In this study we have sequenced COI, from 1205 isolates representing 23 genera. A comparison to internal transcribed spacer (ITS) sequences from the same isolates showed that COI identification is a practical option; complementary because it uses the mitochondrial genome instead of nuclear DNA. In some cases COI was more discriminative than ITS at the species level. This is in contrast to the large ribosomal subunit, which showed poor species resolution when sequenced from a subset of the isolates used in this study. The results described in this paper indicate that COI sequencing and the dataset generated are a valuable addition to the currently available oomycete taxonomy resources, and that both COI, the default DNA barcode supported by GenBank, and ITS, the de facto barcode accepted by the oomycete and mycology community, are acceptable and complementary DNA barcodes to be used for identification of oomycetes.

SUBMITTER: Robideau GP

PROVIDER: S-EPMC3195333 | biostudies-literature | 2011 Nov

REPOSITORIES: biostudies-literature

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DNA barcoding of oomycetes with cytochrome c oxidase subunit I and internal transcribed spacer.

Robideau Gregg P GP De Cock Arthur W A M AW Coffey Michael D MD Voglmayr Hermann H Brouwer Henk H Bala Kanak K Chitty David W DW Désaulniers Nicole N Eggertson Quinn A QA Gachon Claire M M CM Hu Chia-Hui CH Küpper Frithjof C FC Rintoul Tara L TL Sarhan Ehab E Verstappen Els C P EC Zhang Yonghong Y Bonants Peter J M PJ Ristaino Jean B JB Lévesque C André CA

Molecular ecology resources 20110620 6

Oomycete species occupy many different environments and many ecological niches. The genera Phytophthora and Pythium for example, contain many plant pathogens which cause enormous damage to a wide range of plant species. Proper identification to the species level is a critical first step in any investigation of oomycetes, whether it is research driven or compelled by the need for rapid and accurate diagnostics during a pathogen outbreak. The use of DNA for oomycete species identification is well ...[more]

PMID: 21689384

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Project description:Free-living amoebae (FLA) are phagocytic protists that play crucial roles in microbial communities as significant microbial grazers. However, our current knowledge of their diversity, ecology, and population genetic structures is marginal due to the shallow and biased sampling of ecosystems and the use of few, poorly resolving molecular markers. Thirty-two FLA were isolated from soil and water samples collected across representative ecosystems of the State of Morelos in Central Mexico, including the drinking water distribution system (DWDS) from the state capital. We classified our isolates as members of Acanthamoeba, Vermamoeba, Naegleria, and Tetramitus by 18S ribosomal DNA (rDNA) sequencing. Vermamoeba isolates were recovered exclusively from the DWDS samples. In contrast, Naegleria strains displayed a broad distribution in soil and water samples across the natural ecosystems. We used a combination of phylogenetic and population genetic analyses of internal transcribed spacer (ITS) and cytochrome oxidase subunit I (COI) sequences from our isolates and a comprehensive set of reference sequences to analyze the currently known diversity of Naegleria spp. Significant associations were uncovered between the most prevalent lineages of Naegleria and Vermamoeba and broad ecological and geographical variables at regional and global levels. The population structure and cryptic diversity within the Naegleria galeacystis-Naegleria americana and Vermamoeba vermiformis species complexes were thoroughly analyzed. Our results prove that the genus Vermamoeba, which was previously thought to consist of only one species, actually encompasses at least seven widely distributed species, as indicated by consistent evidence from Bayesian phylogenetics, two species-delimitation programs, and population genetics analyses. IMPORTANCE Our study sheds new light on the population genetic structure of V. vermiformis and diverse Naegleria species. Using improved molecular markers and advanced analytical approaches, we discovered that N. americana, previously considered a single species, actually contains multiple distinct lineages, as revealed by COI sequencing. These lineages are highly differentiated, with little gene flow between them. Our findings demonstrate that the genus Vermamoeba holds multiple cryptic species, requiring a significant taxonomic revision in light of multilocus sequence analyses. These results advance our understanding of the ecology, molecular systematics, and biogeography of these genera and species complexes at both regional and global scales. This study has significant implications for diagnosing amoebal infections and evaluating health risks associated with FLA in domestic and recreational waters.

Project description:BACKGROUND:Parafilaria bovicola (Nematoda: Filariidae) causes cutaneous bleedings in bovine species. Flies serve as intermediate hosts. In recent years, reports on bovine parafilariosis have become more frequent, corroborating the necessity of reliable diagnostic interventions especially since no molecular or serological test has been available. We aimed to establish a polymerase chain reaction assay to detect DNA of P. bovicola in flies, skin biopsies and serohemorraghic exudates of bleeding spots. METHODS:PCRs targeting the cytochrome c oxidase subunit 1 (cox1) gene and the internal transcribed spacer region (ITS) of the ribosomal RNA gene cluster were evaluated for their diagnostic sensitivity as well as performance and specificity on biopsy and serohemorrhagic exudate samples from P. bovicola-infected cattle. RESULTS:Using serohemorrhagic exudates (n = 6), biopsies (n = 2) and flies (n = 1), the PCR targeting the cox1 gene resulted in a gel band of almost 700 bp. Cloning, sequencing, and removal of primer sequences yielded a 649-bp fragment of the P. bovicola cox1 gene. The PCR targeting the ITS region showed a band of about 1100 bp. Cloning, sequencing, and removal of primer sequences resulted in a 1083 bp stretch of the P. bovicola ITS region. Testing samples from presumably affected animals, the cox1-PCR resulted in bands with the expected size and they were all confirmed as P. bovicola by sequencing. In contrast, the ITS-PCR proved to be less sensitive and less specific and additionally amplified the ITS region of Musca domestica or buttercup DNA. When analysing for sensitivity, the cox1-PCR yielded visible bands up to 2 ng of genomic DNA, whereas the ITS-PCR produced bands up to 3 ng. In a plasmid dilution series, the minimum number of target DNA copies was 102 for the cox1-PCR and 101 in the ITS-PCR. CONCLUSIONS:The evaluated cox1-PCR enables reliable detection of P. bovicola DNA in skin biopsies and serohemorrhagic exudates. This PCR and, to a limited extent, the ITS-PCR, may help evaluate different therapeutic approaches. Furthermore, the cox1-PCR may be useful for epidemiological studies on the geographical distribution of P. bovicola. Further understanding of the epidemiology of this parasite will help develop and implement effective control strategies.

Dataset Information

DNA barcoding of oomycetes with cytochrome c oxidase subunit I and internal transcribed spacer.

Publications

DNA barcoding of oomycetes with cytochrome c oxidase subunit I and internal transcribed spacer.

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