Abstract
Phytophthora ramorum is a pathogenic oomycete that causes sudden oak death in the Western USA and sudden larch death in the UK. Until recently, three genetically divergent clonal lineages of P. ramorum were known (EU1, NA1 and NA2), named according to the continent on which they were first detected. In 2009, a fourth lineage named EU2 was discovered in the UK. Sequencing and microsatellite genotyping revealed that the EU2 lineage is genetically distinct from all other lineages. Allele-specific oligonucleotide-PCR (ASO-PCR) assays using real-time PCR were developed in this study, allowing for the identification of the EU2 lineage. Also, a combination of ASO-PCR assays targeting the cellulose binding elicitor lectin (CBEL) locus was validated to rapidly identify all four lineages. Sequencing of the CBEL locus revealed eight single nucleotide polymorphisms (SNPs) that distinguished EU2 from the other three lineages. Two ASO-PCR assays were developed from these SNPs, providing the ability to rapidly identify EU2 individuals relative to EU1, NA1 and NA2 individuals. These new assays were combined with two existing assays targeting the same locus to allow rapid and simple identification of all four lineages. Blind tests performed on a panel of representative samples revealed diagnostic profiles unique to each lineage. These markers can be used with diseased field samples, making them well suited for routine procedures in diagnostic laboratories to identify P. ramorum.
Résumé
Phytophthora ramorum est un oomycète responsable de la mort subite des chênes dans l’ouest des États-Unis et de la mort subite des mélèzes au Royaume-Uni. Jusqu’à tout récemment, trois lignées clonales génétiquement différenciées étaient connues (EU1, NA1 et NA2), chacune étant nommée selon le continent sur lequel elle a d’abord été détectée. En 2009, une quatrième lignée (EU2) a été découverte au Royaume-Uni. Le séquençage et le génotypage par microsatellites ont révélé que la lignée EU2 est génétiquement distincte des autres lignées. Des tests utilisant la technique du PCR en temps réel et des oligonucléotides spécifiques à des allèles cibles (ASO) ont été développés afin de permettre l’identification de la lignée EU2. De plus, une combinaison de tests ASO-PCR ciblant le locus CBEL (‘cellulose binding elicitor lectin’) et permettant l’identification rapide des quatre lignées a été validée. Le séquençage du locus CBEL a révélé huit polymorphismes nucléotidiques (SNPs) spécifiques à la lignée EU2. Deux tests ASO-PCR ont été développés à partir de ces SNPs, permettant l’identification rapide des individus EU2 par génotypage. Ces tests, combinés à deux tests développés précédemment sur le même locus, ont permis l’identification simple et rapide des quatre lignées. Des tests à l’aveugle sur un échantillon représentatif ont révélé un profil unique pour chacune des lignées. Ces marqueurs peuvent être utilisés sur des échantillons environnementaux les rendant ainsi très utiles dans les laboratoires de diagnostic.
Introduction
Phytophthora ramorum Werres, De Cock & Man in’t Veld is the causal agent of sudden oak death (SOD) and sudden larch death, diseases causing extensive mortality of oaks (Quercus spp.) and tanoaks (Notholithocarpus densiflorus) in the Western USA and of Japanese larch (Larix kaempferi) in the UK (Brasier & Webber Citation2010; Webber et al. Citation2010; Grünwald et al. Citation2012). This pathogen is also regularly observed in ornamental nurseries in North America and in Europe, where it causes ramorum blight on woody ornamental plants (Grünwald et al. Citation2012).
Phytophthora ramorum is an oomycete, a fungus-like diploid microorganism reproducing clonally in nature. It is suspected of having been introduced from one or more unknown locations into North America and Europe (Grünwald et al. Citation2008; Mascheretti et al. Citation2008; Mascheretti et al. Citation2009; Goss et al. Citation2009a). Phytophthora ramorum is divided into at least four genetically divergent clonal lineages (EU1, EU2, NA1 and NA2), each named according to the continent on which it was first detected (Ivors et al. Citation2006; Grünwald et al. Citation2009; Goss et al. Citation2009b). The EU1 lineage was first discovered in Europe and is now found in Europe and North America (Ivors et al. Citation2006; Vercauteren et al. Citation2010; Goss et al. Citation2011). The NA1 and NA2 lineages were first discovered in North America, and have not been reported elsewhere (Ivors et al. Citation2006; Goss et al. Citation2009a). NA1 has a widespread distribution in the USA, largely because of its spread with nursery planting material (Goss et al. Citation2009b). NA2 is common in British Columbia, Canada and in Washington State, and has a much smaller distribution than NA1 in the USA (Goss et al. Citation2011). Recently, a fourth P. ramorum lineage was discovered in the UK, during a severe epidemic on Japanese larch (Brasier & Webber Citation2010; Webber et al. Citation2010). Sequencing and microsatellite genotyping revealed that this new lineage, named EU2, is genetically distinct from the three other P. ramorum lineages (Van Poucke et al. Citation2012).
Over the years, a wide range of molecular markers have been developed to describe, distinguish and identify P. ramorum lineages, including AFLP (Garbelotto et al. Citation2002; Ivors et al. Citation2004; Vercauteren et al. Citation2010), ISSR-PCR (Wiejacha et al. Citation2007), PCR-RFLP (Kroon et al. Citation2004; Martin Citation2008; Elliott et al. Citation2009; Van Poucke et al. Citation2012), microsatellites (Prospero et al. Citation2004; Ivors et al. Citation2006; Prospero et al. Citation2007; Mascheretti et al. Citation2008; Goss et al. Citation2009b; Vercauteren et al. Citation2010, Citation2011) and sequencing of mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) loci (Martin et al. Citation2007; Martin Citation2008; Goss et al. Citation2009a). Although these markers allow the distinction between two or more P. ramorum lineages, most of them require relatively large amounts and/or high quality genomic DNA to provide reliable results (Bonin et al. Citation2004; Cooke & Lees Citation2004; Guichoux et al. Citation2011). The use of these markers thus can require culturing of P. ramorum on appropriate media before DNA extraction and molecular testing, which is time-consuming and may at times be unsuccessful.
Real-time PCR has shown potential in sensitive and specific detection of Phytophthora spp., including P. ramorum (Bilodeau et al. Citation2007b; Martin et al. Citation2009, Citation2012). Allele-specific oligonucleotide-PCR (ASO-PCR) assays, using real-time PCR, are very sensitive tests that can be used with samples comprising small amounts of mixed DNA (such as environmental samples), thus making them ideally suited for routine diagnostics in regulatory laboratories (Bilodeau et al. Citation2007a). ASO-PCR assays were recently developed for P. ramorum using the β-tubulin and cellulose binding elicitor lectin (CBEL) gene regions, allowing discrimination between the European and North American lineages (Bilodeau et al. Citation2007a). ASO-PCR assays in the CBEL gene region even allowed distinction of the NA1 and NA2 lineages (Bilodeau Citation2008). However, no ASO-PCR assays can currently distinguish EU2 from the EU1, NA1 and NA2 lineages, or can differentiate and identify all four P. ramorum lineages.
The objectives of this study were to: (1) develop ASO-PCR assays allowing for the identification of P. ramorum EU2 lineage and (2) validate a combination of ASO-PCR assays allowing for the identification of the four P. ramorum lineages. To achieve these objectives, single nucleotide polymorphisms (SNPs) between EU2 and other lineages in the CBEL gene region were used to design primers and test new ASO-PCR assays. Also, a combination of two newly described and two published ASO-PCR assays in the CBEL gene region was tested to allow for the rapid identification of all four P. ramorum lineages.
Materials and methods
Isolates
Phytophthora ramorum isolates representing all four lineages were obtained as listed in . Isolates were assigned to EU1, EU2, NA1 or NA2 lineages based on sequencing, microsatellite and/or SNP genotyping (Bilodeau et al. Citation2007a; Van Poucke et al. Citation2012). For DNA extraction, P. ramorum isolates were either cultured on 5% V8 juice agar (Miller Citation1955) or potato dextrose agar (PDA) at 20 °C under a 12 h photoperiod. For isolates grown on V8, DNA was extracted from agar plugs using the DNeasy Plant Mini Kit (Qiagen Sciences, Germantown, MD), following manufacturer recommendations. For isolates grown on PDA, DNA was extracted with a chloroform method (Möller et al. Citation1992) from mycelia growing on a cellophane membrane (GE Healthcare Bio-Sciences Corp., Piscataway Township, NJ) placed on top of the agar. DNA concentration was measured with Qubit assays (Life Technologies Inc., Grand Island, NY).
Table 1. Phytophthora ramorum samples used for sequencing, development, validation and/or plant material testing of allele-specific oligonucleotide-PCR (ASO-PCR) assays targeting the cellulose binding elicitor lectin (CBEL) gene region.
DNA sequencing and ASO primer design
The CBEL locus was PCR-amplified using the CBEL5U and CBEL6L primers (Bilodeau et al. Citation2007a) on isolates listed in . Amplification reactions were carried out in 25 uL volumes with the following final concentrations: 1 × buffer, 0.2 mm of dNTP, 0.5 mm of each primer, 1.6 mm of MgCl2, 0.04 U of Platinum Taq DNA polymerase (Life Technologies Inc., Grand Island, NY) and between 4.1–14.4 ng of template DNA. PCR conditions were as follows: 1 step of 3 min at 94 °C, 1 step of 35 cycles of 30 s at 92 °C, 30 s at 59 °C, 1 min at 72 °C and 1 step of 10 min at 72 °C.
All PCR products were purified and sequenced in both directions at the ‘Plateforme de séquençage et de génotypage des génomes’ (CHUL Research Center (CRCHUL), Quebec City, QC). The same primers (CBEL5U and CBEL6L) were used for sequencing as for PCR amplification. Electropherograms were visually inspected to ensure proper base calling. Nucleotide positions that showed double peaks were scored as heterozygous. Sequences were aligned using SeqMan Pro version 8.0.2 (DNASTAR Inc., Madison, WI).
A DNA sequence alignment of the CBEL locus that included EU1, EU2, NA1 and NA2 sequences () was used to identify SNPs distinguishing EU2 from the other P. ramorum lineages. Two ASO primers were designed manually for each of four targeted SNPs (). For a given SNP, ASO primers are identical except for the last base at the 3′ position (). All ASO primers were designed to be used with common forward primer CBEL 412 (Bilodeau et al. Citation2007a). Primer selection criteria included a melting temperature of 66 °C, primer lengths of 18–22 base pairs (bp), avoidance of secondary structures and PCR products of less than 200 bp. SNPs were chosen for primer design based on their position within the sequence and primer selection criteria.
Table 2. Genotypes of the four Phytophthora ramorum lineages at eight single nucleotide polymorphisms (SNPs) found in the cellulose binding elicitor lectin (CBEL) gene region, distinguishing EU2 from the other three lineages.
Table 3. Primers used for allele-specific oligonucleotide-PCR (ASO-PCR) assays targeting the cellulose binding elicitor lectin (CBEL) gene region in Phytophthora ramorum.
Optimization of ASO primer pairs
To optimize the assays, real-time PCR was performed using three isolates of the EU1 lineage, two isolates of the other P. ramorum lineages and a negative control (). For each SNP tested, two separate PCR reactions were conducted, one for each allele targeted. All reactions were carried out in 25 uL volumes with the following final concentrations: 1× GoTaq qPCR Master Mix (Promega, Madison, WI), 0.3 um of each primer and approximately 1 ng of template DNA. PCR conditions were as follows: 1 step of 13.5 min at 95 °C, 1 step of 45 cycles of 15 s at 94 °C, 30 s at annealing temperatures between 60 and 68 °C (2°C increment per test), 30 s at 72 °C, 1 step of 15 s at 95 °C followed by a melting curve from 55 °C to 95 °C, with a reading every 1.0 °C and a hold for 6 s measurement. Fluorescence was also measured during the extension phase at 72 °C. All reactions were run on a Rotor-Gene 6000 Real-Time PCR machine (Corbett-Research, Montreal Biotech Inc., Dorval, QC) and data were analysed with Rotor-Gene Q series software version 1.7 (Corbett-Research, Montreal Biotech Inc., Dorval, QC).
Validation of ASO primer pairs
To distinguish and identify all four P. ramorum lineages, a combination of four CBEL ASO-PCR assays () was validated using a blind test. The blind test consisted of 27 samples and included 5–8 isolates of each P. ramorum lineage (), with DNA concentrations ranging from 0.2–2 ng uL−1. Samples were tested with the combination of four ASO primer pairs, and an overall profile was established for each sample based on the genotype obtained at each locus. For each of the four loci tested, PCR reactions were performed at an optimal annealing temperature of 66 °C (), and data analyses were the same as described in the previous section.
Test with infected plant material
Infected plant material from a leaf inoculation experiment performed previously in our lab (see Supplemental Materials and Methods) was used to further test the two optimized ASO-PCR assays developed in this study. Genomic DNA was extracted from leaves of Camellia japonica and Rhododendron sp. infected with two isolates of P. ramorum EU2 lineage and one isolate of P. ramorum NA2 lineage (), as well as from uninfected leaves of each plant species. As neither EU1 nor NA1 lineages were used in the inoculation experiment, DNA extracted from a pure culture of each of these lineages (approximately 8 ng uL−1; see ) was spiked with DNA extracted from uninfected leaves of each plant species (1:1 ratio). For each of the two ASO-PCR assays tested, PCR reactions were performed at an optimal annealing temperature of 66 °C () and data analyses were the same as described in the previous section.
Results
DNA polymorphisms
Amplification of the CBEL locus produced a PCR fragment of 650 bp. Sequencing of this fragment produced a 616 bp sequence for all EU2, EU1, NA1 and NA2 isolates (GenBank accession numbers listed in ) and revealed eight SNPs distinguishing EU2 isolates from the other three P. ramorum lineages (). Isolates within each lineage presented identical sequences. All nucleotide changes were synonymous substitutions, except for CBEL-589, which produced a non-synonymous change. At this position, EU2 isolates had an asparagine amino acid (AAC; Asn/N), while other lineages had a lysine amino acid (AAG; Lys/K).
Optimization of ASO-PCR assays
ASO primers were designed to amplify specifically EU2 or EU1/NA1/NA2 alleles at four of the eight SNPs identified from the sequencing results (). PCR product lengths were 69, 92, 116 and 152 bp for Rev-ASOCBEL-349, -373, -397 and -436, respectively. Real-time PCR assays were developed using these PCR primers coupled with common primer CBEL 412. For a given ASO-PCR assay, samples were considered heterozygous when the difference in Ct values between both primers was ≤ 2 and homozygous when the difference in Ct values was ≥ 8.
For the assays Rev-ASOCBEL-349 and Rev-ASOCBEL-373, P. ramorum isolates presented the expected genotypes at all temperatures tested. Results indicated homozygous states C/C and G/G for EU2 isolates, while for other lineages they indicated heterozygous state C/T and homozygous state A/A at Rev-ASOCBEL-349 and -373, respectively (). The most important Ct differences were obtained at the highest temperatures tested (66 °C and 68 °C), with over a 8 Ct difference observed for homozygous genotypes at both loci (see example in ). Melting curve analyses also showed a single peak, indicating specific amplification at both loci (data not shown).
Table 4. Genotyping example of Phytophthora ramorum isolates with two new allele-specific oligonucleotide-PCR (ASO-PCR) assays targeting the cellulose binding elicitor lectin (CBEL) gene region.
For the assay Rev-ASOCBEL-397, the EU2 isolates did not present the expected genotype at any of the temperatures tested. Results indicated heterozygous state (A/C), while they should have indicated homozygous state (A/A) at this locus. The other lineages presented the expected genotype at this locus (C/C). For the assay Rev-ASOCBEL-436, EU2 isolates presented the expected genotype (A/A) at all temperatures tested, but the other lineages displayed heterozygous state (A/G) in every test, while they should have displayed homozygous state (G/G). Because both Rev-ASOCBEL-397 and -436 gave unreliable results, these markers were not used further.
Combination and validation of CBEL ASO-assays
The combination of two newly developed and two published CBEL ASO-PCR assays () revealed unique profiles for all four P. ramorum lineages, thus allowing their identification with real-time PCR. No false positive assignments were observed in the blind tests and all lineages were identified correctly (). Samples assigned to the EU1 lineage had a characteristic C/G genotype at ASOCBEL-245 whereas samples assigned to the EU2 lineage had characteristic C/C and G/G genotypes at Rev-ASOCBEL-349 and -373, respectively (). Samples assigned to the NA2 lineage only differed from the EU1 isolates in a C/C genotype at the ASOCBEL-245. Finally, samples assigned to the NA1 and EU2 lineages shared C/C genotypes at ASOCBEL-245 and -412. Melting curve analyses showed a single peak at all loci, indicating that all assays resulted in specific amplifications (Supplemental ). Combined analyses of Ct values and melting curves revealed unspecific amplifications for all negative controls showing positive Ct values ( and Supplemental ).
Table 5. Evaluation of assays in blind tests. Ct values, genotypes and profiles obtained for blind-tested samples of Phytophthora ramorum at four allele-specific oligonucleotide-PCR (ASO-PCR) assays targeting the cellulose binding elicitor lectin (CBEL) gene region.
Test with infected plant material
DNA extracted from the plant material infected with P. ramorum isolates as well as DNA extracted from pure cultures of P. ramorum spiked with uninfected leaf DNA presented the expected genotypes at Rev-ASOCBEL-349 and Rev-ASOCBEL-373, for both plant species (Supplemental ). Melting curve analyses showed a single peak for each assay, indicating specific amplification at both loci (data not shown). As expected, DNA extracted from uninfected leaf material was negative for the presence of P. ramorum (Supplemental ).
Discussion
In this study, we identified eight SNPs in the CBEL locus distinguishing EU2 from the other three P. ramorum lineages. Two of these SNPs were used to develop ASO-PCR assays that allowed for the identification of the EU2 lineage. These two assays, combined with two existing ASO-PCR assays at the same locus (Bilodeau et al. Citation2007a), also allowed for the rapid and simple identification of all the P. ramorum lineages. These assays were conducted using real-time PCR, ensuring a high level of sensitivity with low amounts of biological material without the need to obtain pure cultures, as shown by Bilodeau et al. (Citation2007a).
The polymorphisms observed at the CBEL locus revealed an important divergence between EU2 and the other P. ramorum lineages. Eight SNPs were observed between EU2 and the other three lineages, while only two SNPs were detected previously among EU1, NA1 and NA2 lineages in the same gene region (Bilodeau et al. Citation2007a). The divergence observed at the CBEL locus is consistent with sequencing data revealing that the EU2 lineage is more distantly diverged from the other three P. ramorum lineages (Van Poucke et al. Citation2012). Indeed, EU2 samples showed the highest number of lineage-specific segregating sites at mtDNA loci with 11, compared with 4, 0 and 4 for EU1, NA1 and NA2 lineages, respectively (Van Poucke et al. Citation2012). Polymorphism distinguishing EU2 from the other P. ramorum lineages was also observed in the β-tubulin locus, for which ASO-PCR assays were developed prior to the discovery of the EU2 lineage (Bilodeau et al. Citation2007a). In this case, only one SNP was detected between EU2 and the other three lineages, while two SNPs were detected previously among EU1, NA1 and NA2 lineages (Bilodeau et al. Citation2007a). This SNP was not selected for ASO-PCR assay development, as requirements for designing primers were not met.
Four ASO primer pairs were developed from the eight SNPs found in the CBEL gene region distinguishing EU2 from the other lineages. Two of these primer pairs (Rev-ASOCBEL-349 and -373) yielded constant and successful results, allowing for discrimination of EU2 samples relative to EU1, NA1 and NA2 samples (). However, the other two primer pairs (Rev-ASOCBEL-397 and -436) failed to produce consistent results, despite multiple optimization efforts. For the assay Rev-ASOCBEL-397, re-examination of CBEL sequences did not reveal obvious inconsistencies that might explain the erroneous results obtained at this locus. For the assay Rev-ASOCBEL-436, close examination of the DNA sequence alignment of the CBEL locus revealed that the ASO primer sequence specific to EU2 at this locus was repeated just six base pairs upstream of the beginning of the region targeted by our primers. It is highly possible that the ASO primer specific to EU1, NA1 and NA2 samples annealed to this region, explaining the heterozygous genotype (A/G) obtained for these lineages instead of the expected homozygous genotype (G/G).
The combination of four ASO-PCR assays () revealed unique profiles for each of the four P. ramorum lineages (). Thus, these assays allowed rapid and unambiguous identification of EU1, EU2, NA1 and NA2 lineages from small amounts of DNA. A blind test consisting of samples from all four lineages was used to validate our combination of ASO-PCR assays. The genotyping revealed that all samples were correctly assigned to the corresponding lineage. To further validate these assays, a next step could be to include a validation with field samples comprising all lineages, or with plant material inoculated with P. ramorum from all lineages. Tests performed with either DNA extracted from infected plant material or spiked DNA revealed that the two new assays developed in this study allow the identification and discrimination of the EU2 lineage from the other lineages on two different plant species, without interference (Supplemental ).
The profiles obtained over the four ASO-PCR assays also revealed that EU2 samples were most similar to NA1 samples, sharing the same profile in two out of four assays (). These results contrast with those of Van Poucke et al. (Citation2012), who observed that for mtDNA loci, EU2 isolates tend to be closer to NA2 isolates. However, inconsistent phylogenetic placement of EU2 was also observed at nDNA loci in this study (Van Poucke et al. Citation2012). Thus, in agreement with Van Poucke et al. (Citation2012), our results seem to suggest that more data are needed to infer phylogenetic relationships among P. ramorum lineages.
The choice of ASO-PCR assay development in the CBEL region over the design of specific probes (Taqman, MGB or LNA probes) is explained by three reasons. First, common primers were available for this locus, as ASO-PCR assays targeting this region were already developed (Bilodeau et al. Citation2007a). Second, ASO-PCR assays are easier to design than probes. This is especially true for SNP-rich regions such as the CBEL locus, where SNPs are close to each other, decreasing the chances of success in designing probes specific to one SNP only. Third, ASO-PCR assays are relatively cheap compared with probes, making their development as well as their implementation easier by end-users. Of course, the use of probes has advantages over ASO-PCR assays. First, probes ensure a better specificity compared with the SYBR Green stain used in ASO-PCR, where any amplification product will emit a fluorescent signal. However, the use of melting curve analyses ensures that the Ct values obtained with ASO-PCR assays are specific, as demonstrated in this study. Probes also have a multiplexing advantage over ASO-PCR assays. This advantage was not tested in the present study, but it could eventually be worth investigating for end-users expecting a heavy utilization of these markers.
ASO-PCR assays developed for P. ramorum in the β-tubulin and the CBEL loci (Bilodeau et al. Citation2007a) are currently used for the detection and genotyping of Canadian SOD samples by the Canadian Food Inspection Agency (CFIA) laboratories for samples that test positive for P. ramorum. The new ASO-PCR assays presented in this study now offer a complete set of markers to detect and identify the four P. ramorum lineages known, from small amounts of DNA. These tools can be implemented and used easily in other regulatory or research laboratories to help monitor clonal lineages of this pathogen.
Acknowledgements
The authors would like to thank C. Brasier (Forest Research, Forestry Commission, Farnham, UK) for providing European strains of P. ramorum and M. Newton and D. Shearlaw (Pathogen Identification Research Laboratory, CFIA, Ottawa, Canada) for technical help. Also, thanks to S.C. Brière (Plant Pathology Laboratory, CFIA, Ottawa, Canada) for providing material and sharing information. Support was provided in part by NSERC for a postdoctoral fellowship to M.-C. Gagnon and by Genome Canada and Genome BC through the 2010 Large-Scale Applied Research Project for the TAIGA project (Tree Aggressors Identification using Genomic Approaches; http://taigaforesthealth.com/Home.aspx) and CFIA Research Partnership Strategy (RPS) fund for the project OLF-P-1302.
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