Identification of two GH18 chitinase family genes and their use as targets for detection of the crayfish-plague oomycete Aphanomyces astaci

Abstract

BackgroundThe oomycete Aphanomyces astaci is regarded as the causative agent of crayfish plague and represents an evident hazard for European crayfish species. Native crayfish populations infected with this pathogen suffer up to 100% mortality. The existence of multiple transmission paths necessitates the development of a reliable, robust and efficient test to detect the pathogen. Currently, A. astaci is diagnosed by a PCR-based assay that suffers from cross-reactivity to other species. We developed an alternative closed-tube assay for A. astaci, which achieves robustness through simultaneous amplification of multiple functionally constrained genes.ResultsTwo novel constitutively expressed members of the glycosyl hydrolase (GH18) gene family of chitinases were isolated from the A. astaci strain Gb04. The primary amino acid sequence of these chitinase genes, termed CHI2 and CHI3, is composed of an N-terminal signal peptide directing the post-translational transport of the protein into the extracellular space, the catalytic GH18 domain, a proline-, serine-, and threonine-rich domain and a C-terminal cysteine-rich putative chitin-binding site. The A. astaci mycelium grown in a pepton-glucose medium showed significant temporal changes in steady-state CHI2 and CHI3 mRNA amounts indicating functional constraint. Their different temporal occurrence with maxima at 48 and 24 hours of incubation for CHI2 and CHI3, respectively, is in accordance with the multifunctionality of GH18 family members. To identify A. astaci-specific primer target sites in these novel genes, we determined the partial sequence homologs in the related oomycetes A. frigidophilus, A. invadans, A. helicoides, A. laevis, A. repetans, Achlya racemosa, Leptolegnia caudata, and Saprolegnia parasitica, as well as in the relevant fungi Fusarium solani and Trichosporon cutaneum. An A. astaci-specific primer pair targeting the novel genes CHI2 and CHI3 as well as CHI1 - a third GH18 family member - was multiplexed with primers targeting the 5.8S rRNA used as an endogenous control. A species was typed unambiguously as A. astaci if two peaks were concomitantly detected by melting curve analysis (MCA). For sensitive detection of the pathogen, but also for quantification of agent levels in susceptible crayfish and carrier crayfish, a TaqMan-probe based real-time PCR (qPCR) assay was developed. It targets the same chitinase genes and allows quantification down to 25 target sequences.ConclusionThe simultaneous qualitative detection of multiple sequences by qPCR/MCA represents a promising approach to detect species with elevated levels of genetic variation and/or limited available sequence information. The homogenous closed-tube format, reduced detection time, higher specificity, and the considerably reduced chance of false negative detection achieved by targeting multiple genes (CHI1, CHI2, CHI3, and the endogenous control) at least two of which are subject to high functional constraint, are the major advantages of this multiplex assay compared to other diagnostic m...

Saprolegnia species in Norwegian salmon hatcheries: field survey identifies S. diclina sub-clade IIIB as the dominating taxon

“…The analyses conducted with MEGA, PAUP and TNT were largely congruent, with only minor differences in the statistical bootstrap support values for the different clades (data not shown). For a subset of isolates from each clade, sequences from the GH18 domains of chitinase genes generated according to Hochwimmer et al (2009) supported the ITS-based species identification (data not shown).…”

“…Parsimony analyses of the independent dataset of chitinase genes were largely congruent with the ITS phylogeny and supported 2 different genotypes of the S. diclina sub-clade IIIA and IIIB isolates. However, these data were not included due to presently unresolved problems with insufficient sequence accuracy that may relate to the simultaneous amplification and sequencing of the 3 slightly different homologues of chitinase genes CHI1, CHI2 and CHI3 (see Hochwimmer et al 2009). …”

Aphanomyces astaciPsI-genotype isolates from different Finnish signal crayfish stocks show variation in their virulence but still kill fast

“…The differences in PsI-genotype chitinase genes, even though evident, were minor compared to As-genotype (Makkonen et al, 2012b), which has also shown wider virulence variation in previous studies compared to what we observed with PsI-genotype in this study (Makkonen, 2013). Chitinase production has been claimed to be adaptation to parasitic life style and one of the virulence factors (Unestam, 1966;Hochwimmer et al, 2009). The reports of A. astaci adaptation to the European crayfish hosts (Jussila et al, 2011;Viljamaa-Dirks et al, 2011;Kokko et al, 2012;Svoboda et al, 2012;Kušar et al, 2013) are most probably all concerning As-genotype adaptation.…”

Temporal variation in the prevalence of the crayfish plague pathogen,Aphanomyces astaci, in three Czech spiny-cheek crayfish populations

“…Oidtmann et al (2006) first established a molecular method suitable for the detection of A. astaci in American crayfish. Subsequently, DNA-based detection was employed on all three of the most important American crayfish invaders in Europe (the signal crayfish: Johnsen et al, 2007;Dunn et al, 2009;Hochwimmer et al, 2009;Skov et al, 2011; the spiny-cheek crayfish: Schulz et al, 2006;Kozubíková et al, 2006Kozubíková et al, , 2008Kozubíková et al, , 2010; and the red swamp crayfish: Aquiloni et al, 2011). The above-mentioned studies were mostly limited in geographical coverage and the number of analysed populations.…”