Real-Time PCR Assays for Rapid Identification of Common Aphanomyces astaci Genotypes

Abstract

The oomycete Aphanomyces astaci is the etiologic agent of crayfish plague, a disease that has seriously impacted the populations of European native crayfish species. The introduction of non-indigenous crayfish of North American origin and their wide distribution across Europe have largely contributed to spread of crayfish plague in areas populated by indigenous crayfish. Tracking A. astaci genotypes may thus be a useful tool for investigating the natural history of crayfish plague in its European range, as well as the sources and introduction pathways of the pathogen. In this study, we describe the development of real-time PCR TaqMan assays aiming to distinguish the five genotype groups of A. astaci (A–E) previously defined by their distinct RAPD patterns. The method was evaluated using DNA extracts from pure A. astaci cultures representing the known genotype groups, and from A. astaci-positive crayfish clinical samples collected mostly during crayfish plague outbreaks that recently occurred in Central Italy and Czechia. The assays do not cross-react with each other, and those targeting genotype groups A, B, D, and E seem sufficiently specific to genotype the pathogen from infected crayfish in the areas invaded by A. astaci (particularly Europe). The unusual A. astaci genotype “SSR-Up” documented from crayfish plague outbreaks in Czechia and chronically infected Pontastacus leptodactylus in the Danube is detected by the group B real-time PCR. The assay originally developed to detect group C (one not yet documented from crayfish plague outbreaks) showed cross-reactivity with Aphanomyces fennicus; the A. astaci genotype “rust1” described in the United States from Faxonius rusticus is detected by that assay as well. Analyses of additional markers (such as sequencing of the nuclear internal transcribed spacer or mitochondrial ribosomal subunits) may complement such cases when the real-time PCR-based genotyping is not conclusive. Despite some limitations, the method is a robust tool for fast genotyping of A. astaci genotype groups common in Europe, both during crayfish plague outbreaks and in latent infections.

Soil-Related Predictors for Distribution Modelling of Four European Crayfish Species

“…Invasive crayfish species may have different abilities to use these soil structures and could benefit from climate change [39]. Thus, the availability of good distributional predictors can be valuable for investigating invasion dynamics, including the carried cray-fish plague pathogen Aphanomyces astaci with the increasing availability of spatial data associated with pathogen strains and virulence [74,75].…”

Long-term changes in the prevalence of the crayfish plague pathogen and its genotyping in invasive crayfish species in Czechia

“…Genotyping of A. astaci was attempted for selected A. astaci-positive DNA isolates only, preferably exceeding 500 PFU. The pathogen was characterised by fragment analysis at microsatellite loci (Grandjean et al 2014), sequencing of mitochondrial small (rnnS) and large (rnnL) ribosomal subunits (Makkonen et al 2018) and by specific TaqMan qPCR genotyping assays (Di Domenico et al 2021). German toponyms are provided in square brackets for transboundary watercourses.…”

“…Three molecular assays allowing to assign A. astaci strains to genotype groups in mixedgenome samples -microsatellite genotyping (Grandjean et al 2014), mtDNA sequencing (Makkonen et al 2018), and qPCR-based genotyping (Di Domenico et al 2021) -were performed on 20 selected chronically infected crayfish individuals from 18 sampling sites (Suppl. material 1: Table S1).…”

“…Infected North American crayfish tend to have relatively low A. astaci agent levels (e.g., James et al 2017a), which may reduce the success of pathogen genotyping. Amplification of the target DNA fragments for available genotyping methods is usually successful for isolates with agent levels A4 and higher (over 1000 PFU as determined in the ITS-based qPCR detection of the pathogen) and for some isolates of the A3 level (Grandjean et al 2014;Makkonen et al 2018;Di Domenico et al 2021). Therefore, we primarily selected DNA isolates from highly infected hosts (agent level A4 and higher) for the genotyping.…”

“…There is an assumption that distinct A. astaci genotype groups known from Europe are linked to their original North American crayfish carrier (for more details, see Ungureanu et al 2020). Thus, various genotyping assays applicable on either axenic A. astaci cultures (Huang et al 1994;Rezinciuc et al 2014) or on mixed genome samples (e.g., Grandjean et al 2014;Makkonen et al 2018;Minardi et al 2019;Di Domenico et al 2021) should allow tracking the source of infection in crayfish plague outbreaks. Although a recent study using mtDNA sequencing has shown that A. astaci haplotypes are not host speciesspecific (Martín-Torrijos et al 2021), these haplogroups likely include multiple strains that may differ from each other in variable nuclear markers targeted by other genotyping methods (e.g., RAPD: Huang et al 1994;microsatellites: Grandjean et al 2014) or in their physiological properties.…”