Isolates of Oomycetes and related fungi used to validate the molecular assays were either obtained from The Centraalbureau voor Schimmelcultures (CBS) Fungal Biodiversity Centre (Utrecht, The Netherlands), the German Collection of Microorganisms and Cell Cultures (DSMZ) (Braunschweig, Germany), the American Type Culture Collection (ATCC) or cultured from lesioned tissue by standard methods [60, 61]. The A. astaci-types 1 to 4 were purchased from Lage Cerenius (Uppsala University, Uppsala, Sweden). Javier Diéguez-Uribeondo (Real Jardín Botánico CSIC, Madrid, Spain) provided the A. frigidophilus isolate SAP472 . A DNA aliquot of A. frigidophilus NJM 9665 [6, 62] and A. invadans WIC  was obtained from Mark W. Vandersea (Center for Coastal Fisheries and Habitat Research, National Ocean Service, National Oceanic Atmospheric Administration, Beaufort, North Carolina, USA).
The Austrian A. astaci strains Gb04, Z12, and the A. repetans strain Lk29 were isolated from dissected melanised spots found in the integument of signal crayfish . The A. astaci strain GKS07 was grown out of a moribund noble crayfish collected during an acute crayfish-plague outbreak. Melanised necrobiopsies were incubated in peptone-glucose (PG1) medium (3 g/l glucose, 6 g/l peptone, 0.37 g/l KCl, 0.17 g/l MgCl2·6H2O, 0.15 g/l CaCl2·2H2O, 20 mg/l FeCl3·6H2O, 44 mg/l Na2EDTA, 13 mM sodium phosphate buffer (pH 6.3); ) for three days at 18°C  in a humidified chamber and subcultured every two weeks on PG1 agar medium. The same growth and subculturing conditions were applied to the strains obtained from the culture collections.
Fungal contamination of oomycete culture encountered when culturing the A. astaci strain Z12 and the A. repetans strain LK29 were overcome as follows. A piece of agar culture was incubated for one day at 20°C in autoclaved pond water (pH 6.5 to 7) collected at the central biotop of the University campus. This depletion of nutrients induced the sporulation of the oomycete . Under an inverted microscope the swimm spores were aspired into a 100 μL Gilson pipette and re-cultured on PG1 agar medium.
A fungus isolated from horse food was assigned to Aspergillus sp. based on morphological evaluation and added to the strain collection of the Institute of Bacteriology, Mycology and Hygiene (University of Veterinary Medicine, Vienna).
An overview on the biological material used in this work is presented in Table 1.
Species assignment of Austrian Aphanomyces strains
ITS sequences of nuclear rDNA were analysed to allow species assignation of the Austrian A. astaci strains GB04, GKS07, and Z12 as well as of the A. repetans strain LK29 (Table 1, Additional file 1). For this purpose DNA was extracted from 25 mg drop culture mycelium using the DNeasy Tissue Kit (Qiagen, Hilden, Germany). A DNA fragment of about 1,000 bp was amplified and sequenced using the universal primers V9D (5'-TTACGTCCCTGCCCTTTGTA)  and LSU266 (5'-GCATTCCCAAACAACTCGACTC, ). Sequences obtained were compared with reference homologs of Aphanomyces  retrieved from GenBank. For sequence alignment the CodonCode Aligner software (version 3.0.1; CodonCode, Dedham, USA) was used. Molecular phylogenetic relationships were reconstructed using default settings in a program package for quartet-based maximum-likelihood analysis (TREE-PUZZLE, version 5.2 ) and TreeView for graphical illustration .
Additional evidence for species assignation was obtained from sequence analysis of the large subunit ribosomal RNA gene using the primers nuLSU-5' (5'-CGCTGATTTTTCCAAGCCC) and nuLSU-3' (5'-GAGATAGGGAGGAAGCCATGG) for amplification and sequencing.
Thus far A. astaci represents the only species within the genus Aphanomyces known to produce significant amounts of chitinase in chitin-free medium . This unique feature was additionally used for species assignment. In detail, chitinase activity accumulated in broth culture supernatant was measured in a reaction volume of 100 μL containing 5 mM sodium-phosphate buffer (pH 7), 180 μM 4-methylumbelliferyl-β-D-N,N',N''-triacetylchitotrioside (4-MU-chitotrioside; Sigma-Aldrich, Vienna, Austria) as substrate, and 75 μl of supernatant . Following incubation at room temperature for 10 min, the fluorescence intensity was evaluated under UV light.
DNA isolation from mycelium of oomycetes
The mycelium was transferred to a 2 ml-extraction tube containing 0.7 g Precellys® ceramic beads of 1.4 mm diameter (Peqlab Biotechnology, Erlangen, Germany) and 180 μl buffer ATL, the lysis buffer of the DNeasy® Blood & Tissue Kit (Qiagen, Hilden, Germany). Samples were homogenised twice for 15 s at 5000 rpm using the MagNA Lyser (Roche). Further isolation was performed according to the protocol "Purification of Total DNA from Animal Tissues (Spin-Column Protocol)" provided by the manufacturer.
De novo sequencing of partial GH domain using degenerate PCR primers
Partial GH18 domains of chitinases from various A. astaci strains representing all four genotype groups described (A: L1, Sv, Ra; B: Hö, Yx, Ti; C: Kv; D: Pc; ), the Austrian strain Gb04 isolated in this work and six related oomycete species (A. laevis, A. helicoides, A. repetans, A. irregularis, Saprolegnia parasitica, Achlya racemosa, Leptolegnia caudata (Table 1) were amplified using the primers SEQ685F (5'-CCGGAGACTCGTGGAACGAC) and SEQ1159R (5'-TTGCTCCAGCTGCCCGC). Primers targeting the amino acid motifs DSWND and AGSW, respectively, amplified an approximately 475-bp product by qPCR. The 20-μL reaction consisted of 0.4 × EvaGreen™ dye (Biotium, Hayward, USA), 4 mM MgCl2, 200 μM of each dNTP, 375 nM of each primer, 2 μl template DNA, 1 U GoTaq® DNA polymerase - a proprietary formulation of Taq DNA polymerase (Promega, Madison, USA), and 1 × Colorless GoTaq® Flexi Reaction Buffer (Promega) not containing magnesium. Amplification was performed in the Rotor-Gene 6000 (Corbett Life Science, Sydney, Australia) using denaturation for 4 min at 94°C, amplification for 35 cycles (1 min at 94°C, 1 min at 63°C and 1 min at 72°C), and final elongation of 7 min at 72°C followed by MCA.
Amplicons from Fusarium solani and Trichosporon cutaneum, representing fungi, were obtained with the degenerate primer SEQuni-F (5'-CGCCGGAGAYTCTTGGAAYGA, Y = C or T) in combination with the primer SEQuni-R (5'-CCAGCATAGTCGTAGGCCAT) targeting the amino acid motifs xxDSWND and MTDYAG, respectively.
Agarose gel electrophoresis was used to the determine amplicon size. The MSB® Spin PCRapace Kit (Invitek, Berlin, Germany) was used for amplicon purification in case of a single band showing the expected length. Multiple bands were excised from the gel and purified with the Xact DNA Cleanup kit (genXpress, Wiener Neudorf, Austria).
The BigDye® Terminator sequencing chemistry (Applied Biosystems, Foster City, USA) was used for sequence analysis of amplicons performed at VBC Genomics Bioscience Research GmbH (Vienna, Austria).
Identification and phylogenetic analysis of GH18 domains
The GH18 domain in the amino acid sequences of CHI2 and CHI3 were identified using the Reversed Position Specific Blast (rpsblast) search modus and the conserved domain database . Domain sequences were aligned to GH18 domain sequences of related species with the ClustalW alignment program implemented in the graphical multiple sequence alignment editor SeaView version 4 . Quartet-based maximum likelihood analysis for aligned amino acid sequences was performed using TreePuzzle with default settings . The graphical display of the phylogram was generated as described above.
Western blot analysis of A. astaci culture supernatant
The peptides DEFKTLPWKAE and LYEDPNHPPGAKY were selected from the deduced amino acid sequence of the A. astaci gene CHI1 (GenBank:AJ416354). Conjugates of these peptides with bovine serum albumin (BSA) were obtained from PSL GmbH (Heidelberg, Germany). Coupling to BSA was achieved via the SH group of a cysteine residue introduced at the C terminus of the peptide to be synthesised. Conjugates were used for the production of polyclonal rabbit serum antibodies served as primary antibodies. Peroxidase-labelled goat anti-rabbit IgG antibodies (K&P Laboratories, Gaithersburg, USA) were used as secondary antibodies.
Western-immunoblot analysis was performed as follows. The A. astaci strain Hö was grown in broth culure. The culture supernatant was boiled for 5 min in a buffer consisting of 25 mM Tris-HCl (pH 6.8), 2.2% sodium dodecyl sulfate (SDS), 15% glycerol and 0.001% bromophenol blue. Insoluble debris was removed by centrifugation. Proteins were resolved by SDS-polyacrylamide gel electrophoresis on a 12% polyacrylamide Tris-glycine gel and electroblotted onto a polyvinylidene difluoride (PVDF) membrane (Bio-Rad Laboratories, Hercules, USA) using a tank blot system (Bio-Rad). The Opti-4CN™ substrate detection kit (Bio-Rad) was used for colorimetric detection of secondary antibodies conjugated to horseradish peroxidase.
Determination of complete cDNA- and genomic-DNA sequences for CHI2 and CHI3
Mycelium derived from the A. astaci-strain Gb04 was grown in liquid PG1 medium for three days and transferred to fresh medium for another 24 h. Total RNA was isolated from mycelium using the Plant and Fungi Protocol provided with the RNeasy Plant Mini Kit (Qiagen). Treatment with DNase I (Promega, Mannheim, Germany) was performed at 37°C for 40 min according to the supplier's instructions. The complete cDNA sequences of CHI2 and CHI3 were generated by RACE-PCR using the 5'/3' RACE Kit (Roche Applied Science, Vienna, Austria).
To amplify genomic sequences corresponding to the cDNAs determined, we designed primers in the region of the start and stop codons of CHI2 and CHI3. The common forward primer (Chi5'f: 5'-AGCAAACTGCAACAAGCATG) targeting a region immediately upstream of the start codon of putative CHI2 and CHI3 genomic sequences, was combined with a gene-specific reverse primer binding adjacent to the stop codon (Chi2.3'r: 5'-GGGCACCAGATGAACGACGC or Chi3.3'r: 5'-ACTAACATACACAACGAATGCGC for CHI2 and CHI3, respectively). The matching fragment size between cDNA and respective DNA sequences shown by agarose gel electrophoresis, and the identity of genomic and cDNA sequences identified by a primer-walking strategy (data not shown), were considered as experimental demonstration for the absence of intronic sequences within CHI2 and CHI3 genes.
In silico analysis of amino acid sequences deduced from CHI2 and CHI3
Multiple matching subsegments in two protein sequences were identified with the LALIGN program http://www.ch.embnet.org/software/LALIGN_form.html implementing the algorithm of Huang & Miller .
The theoretical isoelectric points for the protein sequences were calculated using the Protein Isoelectric Point menu within the Sequence Manipulation Suite .
The presence and location of signal peptide cleavage sites in the amino acid sequences of CHI2 and CHI3 were predicted with the SignalP 3.0 Server http://www.cbs.dtu.dk/services/SignalP;"). Protein phosphorylation at serine, threonine or tyrosine residues was predicted with the NetPhos 2.0 Server . Putative sites for amidation, N-myristoylation and cell attachment were identified by a protein pattern search against the Prosite database http://www.expasy.org/prosite/; ). O-, N-, and C-glycosylated sites were predicted with EnsembleGly - a web server for prediction of O-, N-, and C-linked glycosylation sites with ensemble learning .
Transcript quantification by real-time reverse transcription PCR (qRT-PCR)
Propagules of the strain Gb04 were grown in PG1 medium for three days, washed in fresh medium for 2 min and transferred to another portion of fresh medium (time point 0). Twelve, 24, 36, 48 or 72 hours later the mycelium was shortly washed with distilled water, quick-frozen in liquid nitrogen and stored at -80°C. RNA was isolated from three independent samples grown per time point.
For quantification of transcript mass expressed from the chitinase genes CHI2 and CHI3 as well as the endogenous positive control NDUFV1, sense strand transcript standards were generated by in vitro transcription from a PCR product template tailed with the T7 phage promoter sequence. In more detail, for template construction a minimum sequence of 19 bases (5'-TAATACGACTCACTATAGG) required for efficient transcription was selected out of the 23 nt T7 phage promoter sequence and added to the 5' end of the respective PCR primer. In vitro transcription was performed with the RNAMaxx™ High Yield Transcription Kit (Stratagene, Amsterdam, The Netherlands) according to the manufacturer's instructions. Transcription was terminated by adding 1 μl DNase I (10 units/μl RQ1 RNase-Free DNase, Promega) and incubation at 37°C for 40 minutes. The amount of the in vitro transcript was determined by UV-absorbance measurement performed at 260 nm on a GeneQuantII RNA/DNA Calculator (Pharmacia Biotech, Cambridge, UK). Ten-fold serial dilutions were used as absolute concentration standards.
The 10-μl one-step qRT-PCR contained 125 nM of each primer (5'-CCATCACGAACCCCCTTGAG and 5'-GGGCACCAGATGAACGACG for CHI2, 5'-GTGGCCCCATCACGAACC and 5'-ACTAACATACACAACGAATGCGC for CHI3, 5'-TCGGCTGTCGCACTTCTACA and 5'-ATCCACCCCGTTCCTTCG for NDUV1), 75 nM TaqMan probe (Hexachloro-6-carboxyfluorescein (HEX)-5'-CTGCGGCCAATGTACCCCTTGCC black-hole quencher 1 (BHQ1) and 6-carboxyfluorescein (FAM)-5'-TTGTTGCCCTTGCACTGGTCGCC-BHQ1 for NDUV1 and CHI2/CHI3, respectively), 0.1 μl of the QuantiTect RT Mix, 5 μl of the 2 × QuantiTect Probe PCR Master Mix (Qiagen) and 50 ng total RNA or 1 μL in vitro transcript. In minus RT controls the QuantiTect RT Mix was replaced by water. Reverse transcription of one-step RT-PCR was conducted at 50°C for 30 min followed by a 15 min-activation of the HotStartTaq DNA polymerase at 95°C and amplification for 35 cycles (94°C for 20 s, 60°C for 1 min).
Qualitative detection of A. astaci using qPCR/MCA
The 20-μl duplex qPCR/MCA contained 2 μl 10 × PCR buffer B (Solis BioDyne, Tartu, Estonia), 200 nM of forward and reverse chitinase gene(s) primers (5'-TCAAGCAAAAGCAAAAGGCT and 5'-CCGTGCTCGCGATGGA), 125 nM of forward and reverse 5.8S rRNA primers (5'-ATACAACTTTCAACAGTGGATGTCT and 5'-ATTCTGCAATTCGCATTACG, Figure 5a), 200 μM of each dNTP (Fermentas, St. Leon-Rot, Germany), 0.4 × EvaGreen™ (Biotium), 3.0 mM MgCl2, 1 U Taq DNA polymerase chemically modified for "hot start" (Hot FirePol®; Solis BioDyne, Tartu, Estonia) and 10 ng DNA template or water in the case of the no-template control. QPCR/MCA was performed on the StepOnePlus™ Real-Time PCR System (Applied Biosystems) run under the StepOne™ software version 2.0. Polymerase activation (95°C for 15 min) was followed by amplification for 35 cycles (95°C for 15 s, 59°C for 15 s and 72°C for 10 s). After an initial denaturation step at 95°C for 15 s, amplicon melting was recorded during a gradual increase of the temperature from 60°C to 95°C.
Oligonucleotides (Sigma-Aldrich, Steinheim, Germany) were designed with Primer Express Software Version 2.0 (Applied Biosystems). The difference between amplicon melting temperatures was calculated using the Nearest Neighbor mode implemented in the online oligonucleotide properties calculator OligoCalc .
Sensitive detection and quantification of A. astaci using TaqMan qPCR
Duplicate TaqMan qPCR was carried out in a total volume of 20 μl containing 2 μl 10 × PCR buffer A2 (Solis BioDyne), 0.2 mM of each dNTP, 4 mM MgCl2, 300 nM of each primer (Chi3-324f20 and AaChi-Tmr), 150 nM TaqMan probe (AaChi-FAM), 1 U HOT FIREPol DNA polymerase (Solis BioDyne), 20 ng template DNA or water in the case of the no-template control.
Reactions were amplified in the StepOnePlus™ Real-Time PCR System (Applied Biosystems) under the StepOne™ software version 2.0 using thermal cycling conditions of 15 min at 95°C, followed by 50 cycles of 15 s at 95°C and 1 min at 64°C. A standard curve was generated by plotting the logarithm of the standards copy numbers versus measured C
Isolation of spike-in DNA for use in serial dilutions
A crayfish sample extracted from the abdomen of Cherax quadricarinatus (Australian red-claw crayfish) was transferred to a 2 ml-extraction tube containing 0.7 g Precellys® ceramic beads of 1.4 mm diameter (Peqlab Biotechnology, Erlangen, Germany) and 180 μl buffer ATL, the lysis buffer of the DNeasy® Blood & Tissue Kit (Qiagen). The MagNA Lyser (Roche) was used for three mechanical lysis cycles consisting of 30 s at 6,500 rpm followed by 60 s on a cooling block held at 4°C. Further isolation was performed according to the protocol "Purification of Total DNA from Animal Tissues (Spin-Column Protocol)" provided by the manufacturer. DNA concentration was determined spectrophotometrically using the Hellma® TrayCell (Hellma, Müllheim/Baden, Germany) on the Eppendorf BioPhotometer 6131.
Generation of copy standards
A DNA template stock consisting of CHI1, CHI2 and CHI3 sequences was generated as follows. Genomic DNA from chitinase sequences were amplified with the primers Chi3-324f20 (5'-TCAAGCAAAAGCAAAAGGCT) and AaChi-Tmr (5'-TCCGTGCTCGCGATGGA). Amplification was evaluated by the signal generated from the TaqMan® probe AaChi-FAM (5'-FAM-TCAACGTCCACCCGCCAATGG-BHQ-1). Amplification was performed in a total volume of 20 μl containing 2 μl 10 × PCR buffer A2 (Solis BioDyne), 0.2 mM of each dNTP, 4 mM MgCl2, 250 nM of each primer, 150 nM TaqMan probe, 1 U HOT FIREPol® DNA polymerase (Solis BioDyne) and 20 ng DNA or water in the case of the no-template control. DNA denaturation and enzyme activation were performed for 15 min at 95°C. DNA was amplified over 50 cycles consisting of 95°C for 15 s, 60°C for 1 min. QPCR was run on StepOnePlus™ Real-Time PCR System (Applied Biosystems) under the StepOne™ software version 2.0.
PCR fragments were purified with the MSB® Spin PCRapace Kit (Invitek, Berlin, Germany).
The copy number of the target template was determined spectrophotometrically using the Hellma® TrayCell (Hellma, Müllheim/Baden, Germany) on the Eppendorf BioPhotometer 6131. Serial dilutions of the target sequence (108 to 102, 50, 25 and 12.5 copies per 2 μl) prepared in 10 ng/μl C. quadricarinatus DNA were used to determine the amplification efficiency and the quantitative detection limit.
Statistical analysis of expression changes
A univariate one-way analysis of variance (ANOVA) with Scheffè's post-hoc test was used to evaluate the significance of changes in temporal mRNA expression. The dependent variable was the log-transformed mRNA amount. The time was considered a fixed effect. A value of p < 0.05 calculated by the Scheffè's post-hoc test was regarded as significant. The normality assumption was tested using the Kolmogoroff-Smirnow test.