A quantitative TaqMan MGB real-time polymerase chain reaction based assay for detection of the causative agent of crayfish plague Aphanomyces astaci.

Here we present the development and first validation of a TaqMan minor groove binder (MGB) real-time polymerase chain reaction (RT-PCR) method for quantitative and highly specific detection of Aphanomyces astaci, the causative agent of crayfish plague. The assay specificity was experimentally assessed by testing against DNA representative of closely related oomycetes, and theoretically assessed by additional sequence similarity analyses comparing the primers and probe sequences to available sequences in EMBL/GenBank. The target of the assay is a 59 bp unique sequence motif of A. astaci found in the internal transcribed spacer 1 of the nuclear ribosomal gene cluster. A standard curve for quantification was established by setting up a four-fold dilution series with genomic A. astaci DNA. The absolute limit of detection (LOD(abs)), defined as the lowest concentration yielding a false negative probability<5% was found to be approximately 5 PCR forming units (PFU

Molecular detection and genotyping of Aphanomyces astaci directly from preserved crayfish samples uncovers the Norwegian crayfish plague disease history.

Aphanomyces astaci causes crayfish plague in European freshwater crayfish, but most historical epizootics lack agent isolation and identification. Although declared as crayfish plague outbreaks by the Norwegian Competent Authorities, only presumptive diagnoses without agent isolation exist from Norwegian epizootics until 2005. Molecular methods now allow both A. astaci detection and genotype determination from preserved samples. We therefore aimed to (1) investigate molecularly if A. astaci was involved in a selection of mass-mortality events in Norwegian noble crayfish populations from 1971 to 2004, and (2) determine the eventually involved A. astaci genotype groups both from these historical and also more recent mass-mortality events. DNA was extracted directly from presumptively infected crayfish tissues, and screened by A. astaci specific qPCR. A representative selection of positive samples was confirmed by ITS-sequencing. Finally, genotype determination was performed with microsatellite markers that distinguish all known A. astaci genotype groups. The molecular examination detected A. astaci in crayfish materials from all examined mass-mortality events. The first event in 1971-1974 was caused by the A. astaci genotype group A, presumably the first genotype group that entered Europe more than 150 years ago. All later outbreaks were caused by the A. astaci genotype group B which was introduced to Europe by importation of signal crayfish in the 1960s. The results suggest that molecular methods can verify the involvement of A. astaci in the vast majority of observed crayfish mass mortalities in Europe whenever preserved materials exist. Moreover, microsatellite genotyping can reveal at least parts of the underlying epidemiology.

Exploring the species diversity of Trichoderma in Norwegian drinking water systems by DNA barcoding.

A total of 123 Trichoderma strains were isolated from Norwegian surface-sourced drinking water. The water samples included raw water, treated water, and water from private homes and hospital installations. Trichoderma species are difficult to differentiate morphologically, but recent molecular identification tools, including DNA barcoding, successfully distinguish between closely related species. The diversity of Trichoderma spp. was explored by DNA sequencing of internal transcribed spacer (ITS) and translation elongation factor 1 alpha (TEF-1α). Sequence identification was performed in the TrichOKEY version 2.0 barcode program and in the multilocus similarity search database TrichoBLAST, combined with traditional blast searches in the EMBL/GenBank. A total of 11 known Trichoderma/Hypocrea species were identified. In addition, one group of unidentified Trichoderma strains was found to represent a separate, strongly supported subclade within the Pachybasium'A'/Hamatum clade, based on their TEF-1α haplotypes. Trichoderma viride comprised 49% of the identified strains, and was represented by four and eight slightly different ITS and TEF-1α haplotypes, respectively. Approximately 22% of the surface-derived water samples were positive for T. viride, and the species was frequently isolated throughout the surface-sourced drinking water distribution system. The results indicate that a broad range of Trichoderma species are present in Norwegian surface-sourced drinking. Water treatment has minor effect in removing Trichoderma from raw water, and active growth in the water distribution system is likely to occur.

Polyphasic re-examination of Yarrowia lipolytica strains and the description of three novel Candida species: Candida oslonensis sp. nov., Candida alimentaria sp. nov. and Candida hollandica sp. nov.

The type strain of Yarrowia lipolytica and 38 strains identified as Yarrowia lipolytica, four strains of Candida deformans, including the type and two subcultures of the type, two strains of Candida galli and six unidentified strains that resembled Y. lipolytica were examined by PCR fingerprints using primers M13 and (GAC)5. The same strains, together with four strains of the recently introduced Candida yakushimensis nom. inval., were sequenced for the D1/D2 domain of the 26S rRNA gene and parts of the ITS domain and also studied for their physiological properties. Of the strains identified previously as Y. lipolytica, CBS 2076 had the same fingerprint as the type of C. deformans and strain CBS 4855 was distinct from all other strains. The six strains that resembled Y. lipolytica were separated into two groups distinct from any of the other clades. A total of six groups obtained by fingerprint and sequence data were evaluated by performing DNA reassociation reactions. Mating experiments among the 35 strains of Y. lipolytica sensu stricto showed that 15 strains represented one mating type and 16 strains represented the opposite mating type, while four strains were self-sporulating. Teleomorph states were not produced by C. deformans, C. galli or any of the unidentified isolates. However, positive mating reactions were rarely observed in crosses among C. galli and some strains of Y. lipolytica and C. deformans. Consequently, sharing the same mating type system, C. deformans and C. galli could be considered anamorphs of unnamed Yarrowia species. Results from PCR fingerprints, sequencing and mating studies support the grouping of the studied strains into Y. lipolytica, C. galli, C. deformans, C. yakushimensis nom. inval. and three novel species in the Yarrowia clade: Candida oslonensis sp. nov. (type strain CBS 10146(T) =NRRL Y-48252(T); Mycobank number MB 510769), Candida alimentaria sp. nov. (type strain CBS 10151(T) =NRRL Y-48253(T); Mycobank number MB 510770) and Candida hollandica sp. nov. (type strain CBS 4855(T) =NRRL Y-48254(T); Mycobank number MB 510771).

Diversity and significance of mold species in Norwegian drinking water.

In order to determine the occurrence, distribution, and significance of mold species in groundwater- and surface water-derived drinking water in Norway, molds isolated from 273 water samples were identified. Samples of raw water, treated water, and water from private homes and hospital installations were analyzed by incubation of 100-ml membrane-filtered samples on dichloran-18% glycerol agar. The total count (number of CFU per 100 ml) of fungal species and the species diversity within each sample were determined. The identification of mold species was based on morphological and molecular methods. In total, 94 mold species belonging to 30 genera were identified. The mycobiota was dominated by species of Penicillium, Trichoderma, and Aspergillus, with some of them occurring throughout the drinking water system. Several of the same species as isolated from water may have the potential to cause allergic reactions or disease in humans. Other species are common contaminants of food and beverages, and some may cause unwanted changes in the taste or smell of water. The present results indicate that the mycobiota of water should be considered when the microbiological safety and quality of drinking water are assessed. In fact, molds in drinking water should possibly be included in the Norwegian water supply and drinking water regulations.