Development of new genome-informed genotyping tools for Aphanomyces astaci

Abstract

Aphanomyces spp. are water moulds, eukaryotic fungus-like organisms, belonging to the class Oomycota. This genus contains primary pathogens of plants and animals as well as opportunistic and saprotrophic species. One of the animal parasites (A. astaci) is the causal agent of the crayfish plague, a disease listed by the World Organisation for Animal Health (OIE). It is believed that A. astaci was first introduced into Italy from the US in the late 19th century and rapidly spread in Europe causing the decline of native crayfish. It currently threatens to wipe out the UK native white-clawed crayfish (Austropotamobius pallipes). Random amplified polymorphic DNA (RAPD-PCR) on pure isolates of A. astaci distinguished five genotypes (A, B, C, D, and E). This distinction proved to be a useful tool for epidemiological studies aimed at understanding the history and spread of the disease in Europe; furthermore, there are differences in virulence among genotypes. No discriminatory morphological or physiological characters are available and widely used markers such as the internal transcribed spacer (ITS), the divergent domains regions (D1-D2) of nuclear large subunit (LSU) rDNA, and cytochrome c oxidase subunit I (COI) also fail to discriminate between A. astaci genotypes. There are some practical drawbacks to genotype by the currently available genotyping methods. Whole genome sequencing (WGS) was used to catalogue DNA single nucleotide variants and genotype-unique genomic regions that could be exploited as phylogenetic markers. These newly developed molecular markers were tested both on pure cultures and historical samples derived from outbreaks and carrier crayfish available in our laboratories, validating these genotyping methods, which represent new diagnostic tools aiding the detection and prevention of crayfish plague.