554
Soilborne pathogens / Agents pathogènes telluriques
Multiplex real-time polymerase chain reaction for
identifying potato cyst nematodes, Globodera
pallida and Globodera rostochiensis, and the
tobacco cyst nematode, Globodera tabacum
M. Madani, L.J. Ward, and S.H. De Boer
Abstract: Because the potato cyst nematodes (PCNs), Globodera rostochiensis and Globodera pallida, and tobacco cyst
nematode (TCN), Globodera tabacum, may occur in the same geographic regions, their accurate identification is essential
for implementing appropriate regulatory and crop loss mitigating strategies. Molecular methods for differentiating PCNs
based on the internal genomic transcribed spacer (ITS) sequences have been developed but the potential of the many
DNA amplification-based test formats have not yet been fully explored and most published work has not included TCN.
In our study, the two PCN species and TCN could be distinguished by melting curve analysis of ITS amplicons generated
in an EvaGreen-based real-time polymerase chain reaction (PCR) test. However, a multiplex real-time Taqman PCR test,
which is also based on ITS sequences, was developed in this study with primers and probes modified with locked nucleic
acids and proved to be superior for identification of one or more Globodera spp. in samples containing DNA from cysts
and (or) second-stage juveniles. The test was specific for the PCN and TCN species with efficiencies of 0.91, 1.02, and
0.89 for G. rostochiensis, G. pallida, and G. tabacum tabacum, respectively. The multiplex, real-time PCR test was useful
for distinguishing PCNs and TCN from other nematodes as well as their identification to species level in a single assay,
correctly identifying each species in a DNA template from mixed populations.
Key words: potato cyst nematode, golden cyst nematode, pale cyst nematode, tobacco cyst nematode, real-time PCR,
melting curve analysis, locked nucleic acid, molecular identification.
Résumé : Étant donné que les nématodes à kystes de la pomme de terre (NKP) Globodera pallida et G. rostochiensis
ainsi que le nématode à kystes du tabac (NKT) G. tabacum peuvent être présents dans une même zone géographique,
il est essentiel de pouvoir les identifier avec précision afin d’appliquer les mesures adéquates relatives aux stratégies
d’atténuation en cas de pertes de cultures. Des méthodes moléculaires permettant de différencier les NKP, basées sur
les séquences de l’espaceur interne transcrit (ITS), ont été développées, mais le potentiel des nombreux tests
d’amplification de l’ADN n’a pas encore été complètement exploré, et la plupart des travaux qui ont été publiés sur le
sujet n’ont pas encore été faits sur les NKT. Dans notre étude, les deux espèces de NKP et les NKT pouvaient être
caractérisés par l’analyse de la courbe de fusion des amplicons des ITS générée par PCR en temps réel faisant appel
au marqueur EvaGreen. Toutefois, une PCR multiplex en temps réel Taqman basée également sur les ITS, conçue au
cours de l’étude et faisant appel à l’utilisation d’amorces et de sondes modifiées à l’aide d’acides nucléiques bloqués
(LNA), s’est avérée plus efficace quant à l’identification d’une ou de plusieurs espèces de Globodera dans les
échantillons contenant de l’ADN extrait de kystes ou de plantules ayant atteint le deuxième stade de croissance. Le test
était spécifique des espèces de NKP et NKT à des taux d’efficacité de 0,91, 1,02 et 0,89 pour G. rostochiensis,
Globodera pallida et G. tabacum, respectivement. La PCR multiplex en temps réel s’est avérée utile pour distinguer les
NKP et les NKT d’autres nématodes ainsi que pour les caractériser au niveau de l’espèce en effectuant qu’un seul
biotest, tout en permettant d’identifier chaque espèce à partir d’une matrice d’ADN constituée d’un mélange de
populations.
Mots-clés : nématode à kystes de la pomme de terre, nématode doré, nématode blanc, nématode à kystes du tabac,
PCR en temps réel, analyse de la courbe de fusion, acide nucléique bloqué, identification moléculaire.
nematode identification / multiplex real-time PCR / Globodera species / cyst nematodes
564
Madani et al.:
Accepted 19 October 2008.
M. Madani, L.J. Ward, and S.H. De Boer.1 Charlottetown Laboratory, Canadian Food Inspection Agency, 93 Mount Edward
Road, Charlottetown, PE C1A 5T1, Canada.
1
Corresponding author (e-mail: deboers@inspection.gc.ca).
Can. J. Plant Pathol. 30: 554–564 (2008)
Madani et al.: nematode identification / multiplex real-time PCR / Globodera species / cyst nematodes
Introduction
There are three major groups within the nematode genus
Globodera. They are (i) Globodera rostochiensis (Wollenweber)
Behrens and Globodera pallida (Stone) Behrens, which are
commonly referred to as potato cyst nematodes (PCNs), and
are pathogenic to Solanaceae; (ii) Globodera tabacum
(Lownsbery & Lownsbery) Behrens (tobacco cyst nematodes,
TCN), which are a complex of three subspecies (Globodera
tabacum tabacum (Lownsbery & Lownsbery) Behrens,
Globodera tabacum virginiae (Miller & Gray) Behrens, and
Globodera tabacum solanacearum (Miller & Gray) Behrens);
and (iii) the composite cyst nematodes, which are economically less important and are named after the host plant on
which they are found. The latter group also contains undescribed species and species that are limited to a restricted
area. The composite group of cyst nematodes includes such
species as Globodera achilleae (Golden & Klindic) Behrens,
Globodera artemisiae (Eroshenko & Kazachenko) Behrens,
Globodera millefolii (Kirjanova & Krall) Behrens, Globodera
hypolysi Ogawa, Ohshima & Ichinohe, Globodera chaubattia
(Gupta & Edward) Wouts, Globodera mirabilis (Kirjanova)
Mulvery & Stone, Globodera zelandica Wouts, and Globodera
leptonepia Cobb & Taylor) Behrens.
Economically, the most important members of Globodera
are the PCN species G. rostochiensis and G. pallida, which
are found in many potato growing regions. Potato (Solanum
tuberusom L.) is the major host plant for these two species,
and serious damage has been reported in both cool and temperate geographic regions (Marks and Brodie 1998). Other
Solanaceous plants, such as tomato (Lycopersicum esculentum
Mill.) and eggplant (Solanum melongena L.), as well as
some weeds, have also been identified as host plants for
PCNs (Sullivan et al. 2007). Occurrence of PCNs and
serious damage on potato has been reported in almost all
European countries (Bates et al. 2002; Fleming et al. 1998;
Manduric and Andersson 2003; Manduric et al. 2004;
Minnis et al. 2002). PCN has also been reported in Australia, New Zealand, Africa, the Middle East, Japan, and in
North and Central America (Bulman and Marshall 1997; da
Cunha et al. 2004; Ibrahim et al. 2001; Marks and Brodie
1998; Uehara et al. 2005). In Central America, infested areas have been reported in Mexico, Panama, and Costa Rica
and in the United States in Long Island, western New York,
and, most recently, in Idaho (Handoo et al. 2007; Marks and
Brodie 1998). In Canada, one or both PCN species have
been reported in restricted areas. Both G. rostochiensis and
G. pallida occur in Newfoundland (Olsen and Mulvey 1962;
Stone et al. 1977), but only G. rostochiensis occurs on Vancouver Island (Rott et al. 2008) and has recently been reported in the Saint-Amable area of Quebec (Sun et al. 2007)
and in Alberta (A. Boucher, personal communication).
TCN is also of economic concern because of the damage
it inflicts on commercial tobacco (Nicotiana tabacum L.)
production, including shade-grown or broadleaf and fluecured tobacco cultivars (Syracuse et al. 2004). Moreover,
TCN exerts a synergistic effect on wilt disease of broadleaf
tobacco caused by Fusarium oxysporum Schltdl. (LaMondia
1992). The presence of TCN has been reported from France,
Italy, Spain, China, Pakistan, Morocco, Argentina, the United
States (LaMondia 1992; Syracuse et al. 2004; Wang et al.
555
1999), and from the province of Quebec, Canada (Belair
and Miller 2006).
Identification of the genus Globodera is traditionally based
on seven important characteristics that include morphology
of the vulval cone top in the perineal region of female nematodes (number of ridges and Granek’s ratio), and morphometric characteristics (length of body, tail, and stylet and
shape of stylet and knobs) of second-stage juveniles (J2;
Manduric et al. 2004; Skantar et al. 2007). However, in both
cyst and J2 populations, there is a high percentage of variation in morphometric characters and large overlap in morphological features among species that makes unequivocal
identification difficult (Manduric et al. 2004; Subbotin et al.
2000). It is also impossible to distinguish the three subspecies of G. tabacum based on morphology alone (Mota and
Eisenback 1993). Laboratory-based methods such as enzymelinked immunosorbent assays, protein analysis by isoelectric focusing, and techniques based on the polymerase chain
reaction (PCR) have been developed for routine identification, but results have occasionally been equivocal (Ibrahim
et al. 2001).
However, the full potential of PCR-based strategies for
discriminating Globodera spp. identities has yet to be explored. Thiery and Mugniery (1996) initially described a
method using restriction fragment length polymorphism
(RFLP) in ribosomal DNA (rDNA) of Globodera spp., and
subsequently, a conventional PCR assay was developed to
distinguish between the PCN species (Bulman and Marshall
1997). Recently, Skantar et al. (2007) also used conventional PCR to confirm the identity of G. pallida in Idaho.
Conventional PCR methods require postamplification gel
electrophoresis of DNA amplicons to visualize results, making them more time-consuming and laborious than newer
techniques such as real-time PCR. Real-time PCR methods
have the particular advantage of providing results immediately
with neither the need for further postamplification steps nor
the need for opening tubes containing amplified product
and, thereby, decreasing the risk of cross-contamination. In the
presence of double-strand binding dyes such as SYBRGreen
or EvaGreen, it is also possible to characterize amplicons
by analysis of melting curves. This strategy was successfully
applied to differentiating G. rostochiensis and G. pallida in
an Australian study published while this manuscript was in
preparation (Quader et al. 2008). Real-time TaqMan PCR
assays, with added specificity provided by a hybridization
probe, have been described for a wide range of plant
pathogens, including plant parasitic nematodes such as
Meloidogyne spp. (Zijlstra 1997) and Bursaphylenchus spp.
(Leal et al. 2007). Recently, real-time PCR methods were
developed for quantification of G. pallida, Heterodera
schachtii Schmidt, and Ditylenchus dipsaci (Kühn) Filipjev
(Madani et al. 2005; Subbotin et al. 2005), as well as for
G. rostochiensis and Meloidogyne incognita (Kofoid &
White) Chitwood, but these studies did not determine
whether the tests differentiated among related species (Toyota
et al. 2008). Because three species of Globodera occur in
some geographic regions including Canada and the United
States, a multiplex real-time PCR method to identify
Globodera to the species level in a single assay would be
useful.
556
Can. J. Plant Pathol. Vol. 30, 2008
Table 1. Nematode populations examined in this study and the tests used to identify the nematodes in samples.
Sourcea
Location
No. of
subsamplesb
British Columbia
Quebec
Quebec
Newfoundland
Saanich
Field 1
Field 2
Avondale
1
9
1
3
Newfoundland
United States
United States
United States
France
France
France
CL collection
CL collection
Long Pond
New York
Idaho
Connecticut
Ecosse
Chavornay
Agen
United States
United Kingdom
4
1*
1*
1
1
1
1
1
1
Genbank
accession No.
Speciesc
G.
G.
G.
G.
G.
G.
G.
G.
G.
G.
G.
G.
D.
H.
rostochiensis
rostochiensis
rostochiensis
rostochiensis
pallida
rostochiensis
rostochiensis
pallida
tabacum tabacum
rostochiensis
pallida
tabacum tabacuum
dipsaci
schachtii
FJ212167
FJ212162
FJ212166
FJ212164
FJ212165
FJ212163
Testsd
PCR, ITS sequence, TaqMan probe
PCR, ITS sequence, TaqMan probe
PCR, ITS sequence, TaqMan probe
PCR, ITS sequence, TaqMan probe
PCR, ITS sequence, TaqMan probe
PCR, ITS sequence, TaqMan probe
TaqMan probe
TaqMan probe
ITS sequence, TaqMan probe
PCR, ITS sequence, TaqMan probe
PCR, ITS sequence, TaqMan probe
ITS sequence, TaqMan probe
ITS RFLP
ITS RFLP
a
CL collection, Canadian Food Inspection Agency Charlottetown Laboratory nematode collection.
Samples with asterisks consisted of only DNA; other samples consisted of cysts except vermiform D. dipsaci.
Genera for species are Globodera, Ditylenchus, and Heterodera.
d
PCR, polymerase chain reaction using the method of Bulman and Marshall (1997); ITS, internal transcribed spacer sequence giving 100% similarity
using the nucleotide–nucleotide basic local alignment search tool (BLASTN) from the NCBI database; TaqMan probe, hybridization with the speciesspecific probe developed in this study; and ITS RFLP, ITS restriction fragment length polymorphism.
b
c
However, traditional PCR targets, such as the internal
transcribed spacer (ITS) regions, of closely related species
often show a high degree of homology that presents potential challenges for the design of specific probes that are
practical for multiplex applications. Amplified mismatch
discrimination of one to three bases is not always attainable
through changes in reaction conditions alone and may require the use of modified primers and (or) probes. One such
modification that is particularly suitable for improved mismatch discrimination incorporates an altered nucleotide
containing a 2′–4′ O-methyl covalent link in the ribose ring,
which locally both modifies and “locks” the conformation
of the DNA, increasing hybridization affinity and efficiency.
Locked nucleic acids (LNA) containing modifications at
unique and adjacent positions have been shown to discriminate single- nucleotide polymorphisms and are suitable for
the design of probes that target highly homologous regions
of closely related species (You et al. 2006).
The objective of the present study was to evaluate PCR
strategies for differentiating the three Globodera species
that occur in Canada (G. pallida, G. rostochiensis, and
G. tabacum) in a single assay. We explored PCR-based tests
with amplicon melting temperature analysis and TaqMan
technology utilizing LNA-modified primers and probes. To
our knowledge, no assays have previously been published
for simultaneous discrimination among the three Globodera
spp., viz., the two PCN species and TCN.
Materials and methods
Cyst nematodes
Samples of PCN populations were acquired from two locations in Newfoundland, from one location in the Saanich
Peninsula of Vancouver Island in British Columbia, and
from two potato fields in Quebec recently found to be infested with G. rostochiensis (Sun et al. 2007) (Table 1).
DNA from PCN populations in New York and Idaho were
kindly provided by Xiaohong Wang (Cornell University,
Ithaca, N.Y.). Populations of TCNs were not available from
Canadian sources, but samples of G. tabacum tabacum
were provided by J.A. LaMondia (Connecticut Agricultural
Experiment Station, Windsor, Conn.). Samples of TCN and
both PCN species were also received from D. Mugniery,
Institut national de la recherche agronomique Rennes, France.
For controls, samples of D. dipsaci, a vermiform nematode
morphologically similar to second-stage Globodera juveniles, and H. schachtii, another cyst-forming nematode species, were obtained from the nematode collection held at the
Canadian Food Inspection Agency Charlottetown Laboratory. Identity of previously identified PCN cyst populations
were confirmed by PCR using primers PITSp3, PITSp4,
and ITS 5 as described by Bulman and Marshall (1997)
(Table 2). Additionally, ITS regions of Canadian PCN populations, the PCN and TCN samples from France, and TCN
from the United States were sequenced and compared with
Genbank sequences of the same species using the nucleotide–
nucleotide basic local alignment search tool (BLASTN;
Skantar et al. 2007; Subbotin et al. 2000; Szalanski et al.
1997; Uehara et al. 2005). Heterodera schachtii and
D. dipsaci identity were confirmed by RFLP analysis of the
ITS genes (Szalanski et al. 1997; Wendt et al. 1993).
DNA template preparation
Genomic DNA was obtained from nematode cysts and
J2 populations by a method involving lyophilization and
microlysis. Single cysts or J2 nematodes were first lyophilized in Eppendorf tubes containing 20–60 µL of distilled
water with the following thermal profile: 30 min at –40 °C
followed by 10 min intervals at each of –30, –20, –10, and
5 °C, ramping at 0.1 °C/min between each step. Lyophilized
samples were ground directly in the tubes with hand-drawn
glass pestles and then treated with Microlysis Plus buffer
(The Gel Co., San Francisco, Calif.). A volume of 5 µL of
Madani et al.: nematode identification / multiplex real-time PCR / Globodera species / cyst nematodes
557
Table 2. Sequences of primers and probes used in this study.
Codea
Sequence (5′–3′)b
Reference
PITSp3*
PITSp4*
ITS5
rDNA1
rDNA2
Gfor
Grev*
PITS-pall*
Grost*
Gpall*
Gtab*
EvaF
EvaR*
AGC GCA GAC ATG CCG CAA
ACA ACA GCA ATC GTC GAG
GGA AGT AAA AGT CGT AAC AAG G
TTG ATT ACT TCC CTG CCC TTT
TTT CAC TCG GCC GTT ACT AAG G
GTG TAA CCG ATG TTG GTG GCC
GGA CGT AGC ACA CAA GCG CA
AGC GCA GAC ATG CCG CTG
Cy5-GCT TCC TCC GTT GGC G-IBRQ
TEX613-ATC GTC GAG TCA CCC ATT-IBRQ
FAM-ATA TGC CGC GGG GTA CG-IBFQ
TCG TTG AGC GGT TGT TGC GCC TT
ACG GCC ACG GAC GTA GCA CAC A
Bulman and Marshall 1997
Bulman and Marshall 1997
White et al. 1990
Vrain et al. 1992
Vrain et al. 1992
This study
This study
Modified PITSp3, this study
This study
This study
This study
This study
This study
a
Sequences with asterisks are in antisense orientation.
Boldface and underlined bases are locked nucleotides. Cy5, TEX613, and FAM are fluorescent molecules, and
IBRQ and IBFQ are quencher signal molecules.
b
microlysis 5× buffer and 20 µL of water was added to each
tube and incubated for 35 min with the following temperature treatment: 67 °C for 8 min, 96 °C for 3 min, 65 °C for
4 min, 96 °C for 2 min, 67 °C for 1 min, and 96 °C for
1 min. After a brief centrifugation to collect all the liquid,
preparations were suitable for PCR amplification.
Amplification, cloning, and sequencing of ribosomal
DNA
An approximately 1.2 kb fragment of ribosomal DNA including the ITS1, 5.8S gene, and ITS2 regions (Fig. 1) was
amplified from each Globodera spp. with ITS universal primers rDNA1 and rDNA2 (Table 2), as described by Vrain et
al. (1992), using Sprint Advantage Single Shot PCR Premix
(Clontech, Mountain View, Calif.). Primer concentrations
were 0.5 µmol/L and 2 µL of template DNA was used in final 25 µL reaction volumes. The PCR thermal cycling conditions were one cycle at 95 °C for 3 min; followed by 35
cycles at 94 °C, and 55 °C each for 1 min, and 68 °C for
2 min; and a final cycle at 68 °C for 5 min. Amplified product was separated by gel electrophoresis, excised, purified
from the gel with a gel purification kit (MoBio Laboratories
Inc., Carlsbad, Calif.), and cloned into the pUCm-T vector
(UBI, Calgary, Alta.). Plasmids were purified with an
UltraClean Standard Mini Plasmid Prep Kit (Mo-Bio), and
inserts were sequenced using universal vector sequencing
primers (M13F and M13R) by the Core Molecular Biology
Facility of York University (Toronto, Ont.). Both forward
and reverse sequences were obtained from several clones
representing each Globodera spp.
Real-time PCR
The computer software programs Chromas Lite version
2.01 (Technelysium Pty Ltd, Tewantin, Australia;
http://www.technelysium.com.au/chromas_lite.html) and
GeneDoc version 2.7 (Nicholas and Nicholas 1997) were
used to visualize sequence chromatograms and edit DNA
sequences, respectively. AllelleID (Premier Biosoft International, Palo Alto, Calif.) software was then used for alignment of new sequence data generated in this study with
sequences of Globodera spp. in NCBI GenBank and for de-
signing primers and probes. Primer and probe sequences
were evaluated using Clone Manager (Scientific & Educational Software, Cary, N.C.) and AlleleID software on the
basis of the following criteria: predicted hybridization to
closely related species, potential for primer-dimer formation, length of amplicons, guanine–cytosine content, and
melting temperature (Tm).
The primer set EvaF and EvaR (Table 2) was designed
for use in real-time PCR using EvaGreen chemistry to amplify a 131 bp fragment from the ITS region of G. pallida,
G. rostochiensis, and G. tabacum. Real-time PCR with these
primers was carried out in a Rotor-Gene 3000 real-time
thermocycler (Corbett Robotics Inc. San Francisco, Calif.).
PCR reactions (final volume 25 µL) contained 0.5 µmol/L
of each primer, 2 µL of template DNA, and 12.5 µL of
EvaGreen (20× stock) (Biotium, Hayward, Calif.) in Sprint
Advantage Single Shot PCR Premix (Clontech, Mountain
View, Calif.). The reaction profile consisted of an initial
denaturation–activation step for 3 min at 95 °C followed by
35 cycles of amplification using a two-step thermal profile
of 96 °C for 20 s and 68 °C for 30 s and a final extension at
68 °C for 3 min. Fluorescence in the 6-carboxyfluorescein
aminohexyl amidite (FAM) channel was monitored during
the extension–annealing step of the amplification phase. A
postamplification disassociation curve was generated for
amplicons in the EvaGreen-based real-time PCR assay. DNA
melting curve analysis was performed by cooling the samples at 2.5 °C/s to 60 °C and then increasing the temperature 0.2 °C/5 s to 98 °C while monitoring fluorescence in
the FAM channel. The rate of change in fluorescence for
each temperature was used to determine the melting point
of each amplicon.
A second set of primers, Gfor and Grev (Table 2), located
approximately 200 bp downstream of the 5′end of the 28S
rDNA gene, was designed for use in a TaqMan PCR assay
for the three Globodera spp. Subsequently, a third primer,
PITS-pall, was designed as a modification of primer PITSp3
to ensure adequate amplification of G. pallida sequences in
multiplex PCR. Separate TaqMan probes were designed to
hybridize specifically and differentially to amplified sequences
of G. pallida, G. rostochiensis, and G. tabacum and are
558
Can. J. Plant Pathol. Vol. 30, 2008
Fig. 1. Nucleotide sequence alignment of a segment of the ITS region between 18S and 5.8S genes from Globodera pallida (G.p.), G.
rostochiensis (G.r.), G. tabacum solanacearum (G.t.s.), G. tabacum virginiae (G.t.v.), and G. tabacum tabacum (G.t.t.). Open and dotted
arrows show the locations and orientations of new and published primers, respectively; solid lines depict the position of TaqMan
probes. Gfor and Grev are forward and reverse primers used in the TaqMan assay (232 bp); EvaF and EvaR are forward and reverse
primers used in the real-time assay with EvaGreen dye (126 bp). PITSp4 and PITSp3 are specific primers used by Bulman and Marshall (1997). PITS-pall is a modification of primer PITSp3 that was designed in this study. Gpall, Grost, and Gtab are probes for G. pallida,
G. rostochiensis, and G. tabacum, respectively. The 5′ end of the Gfor sequence is 253 bp from 18S gene, and the 3′ end of the Grev
sequence is 258 bp from 5.8S gene.
identified as Gpall, Grost, and Gtab, respectively (Table 2).
To achieve a high level of specificity and probe fidelity in a
genomic region with few base polymorphisms, locked nucleotides were incorporated into each of the probes (Table 2).
All probes were 3′end-labeled with FQ or RQ Iowa Black
quenchers and labelled with different fluorescent tags at the
5′ ends. Texas Red 613, Cy5, and FAM were used as labels
for the Gpall, Grost, and Gtab probes, respectively, to distinguish amplification of DNA from the three Globodera
spp. on separate channels of the real-time PCR thermocycler.
All primers and probes used in this study were ordered
from Integrated DNA Technologies (Coralville, Iowa).
The real-time TaqMan PCR assay was performed using
the Gfor, Grev, and LNA-modified PITS-pall primers in
combination with the three LNA-modified probes: Gpall,
Gros, and Gtab. Amplification reactions were performed in
25 µL volumes by using the Quantitect multiplex PCR kit
(Qiagen Inc. Mississauga, Ont.) containing dNTPs and hotstart Taq polymerase in a Smartcycler 2 (Cepheid Inc.
Sunnyvale, Calif.) real-time PCR thermocycler. Optimal reaction conditions used 0.5 µmol/L of each primer, 135 nmol/L
of the Gpall and Gtab probes, 110 nmol/L of the Grost
probe, and 2 µL of template DNA. The final optimized thermal cycling profile included an initial activation of Taq
polymerase at 95 °C for 15 min followed by 35–45 cycles
of 20 s at 96 °C and 50 s of extension at 72 °C. Fluorescence was measured at the end of each extension step. The
cycle threshold (Ct) of each amplification reaction was reported based on the first PCR cycle at which fluorescence
exceeded a manual threshold of 10. To verify that only one
PCR product specific to each species was amplified, the size
and presence of amplicons were analyzed by gel electrophoresis using Novex 4%–12% precast tris–borate–EDTA (TBE)
polyacrylamide gels (Invitrogen, Canada Inc., Burlington,
Ont.) in 1× TBE buffer and stained with ethidium bromide.
Efficiency and specificity of the PCR assay
Efficiency of the multiplex real-time TaqMan PCR assay
was determined by testing a two-fold dilution series of template DNA for each of the three Globodera spp. in real-time
TaqMan PCR as described above. The log10 of DNA concentrations of each dilution, based on absorbance readings
at 240 nm, were plotted against the Ct values. A regression
line was fitted, and the goodness of fit was determined by
calculating the regression coefficient (R2). The slope of the
regression line was used in the following equation to calculate reaction efficiency (E): E = 10–1/slope – 1.
A specificity of the primers and probes used in the assay
was tested against DNA of the Canadian PCN populations,
TCN, H. schachtii cysts, and vermiform D. dipsaci nema-
Madani et al.: nematode identification / multiplex real-time PCR / Globodera species / cyst nematodes
559
Fig. 2. Melting curve profiles of amplicons generated using PCR with primers EvaF and EvaR: (A) DNA templates derived from
Globodera rostochiensis, G. pallida, and G. tabacum tabacum separately and (B) a mixed DNA template of G. rostochiensis, G.
pallida, and Heterodera schachtii. Melting profile identifications are shown.
todes. Negative controls of nuclease-free water instead of
DNA template were included in each PCR run.
To evaluate species discrimination in multiplex PCR, DNA
templates were prepared by mixing equal volumes (1.25:l)
of DNA from individual cysts and J2 nematodes from
G. rostochiensis, G. pallida, and G. tabacum populations.
PCR reactions were run for 35 cycles, and the Ct values of
fluorescence signals were recorded in each of the three
thermocycler channels for each PCR run.
Results
Identification of nematodes and sequence alignments
The identity of the Quebec PCN populations was confirmed as G. rostochiensis by PCR using the primers
designed by Bulman and Marshall (1997), and both
G. rostochiensis and G. pallida were identified in the cyst
populations from the two Newfoundland locations. Amplification of the ITS regions by conventional PCR using the
general ITS primers rDNA1 and rDNA2 produced distinct
fragments of about 1200 bp from all the PCN populations
utilized in this study. The ITS regions of the samples we sequenced (Table 1) aligned with the sequences of homologous
G. rostochiensis and G. pallida in the Genbank database In
the region of interest, our G. rostochiensis sequences were
98%–99% identical to 33 G. rostochiensis sequence accessions
in Genbank (eg. EF153839, DQ847120, and AB207271).
Similarly, G. pallida was 97%–99% identical to 17 Genbank
accessions (eg. EF153837 and DQ847109)
The identity of the G. tabacum sample obtained from the
United States was also confirmed by sequencing the ITS
region and by comparison with Genbank sequences using
BLASTN. In the region of interest, it was 100% identical
to three Genbank accessions of G. tabacum tabacum
(EF153842, DQ847116, and AB207272) and differed from
two G. tabacum solanacearum accessions (DQ847114 and
EF153841) and two G. tabacum virginiae accessions
(DQ847113 and DQ84712) in Genbank by two or three nucleotide polymorphisms.
Alignment of the majority consensus sequences of the
ITS region from the three Globodera species is shown in
Fig. 1. Oligonucleotides selected to serve as PCR primers
and probes in this study were located within the ITS regions
in which no sequence polymorphisms occurred within a
species but in which polymorphisms did occur among species. Location of primers and probes within the ITS region
are shown in Fig. 1, and actual positive or negative strand
sequences are given in Table 2.
The RFLP patterns of the ITS genes for H. schachtii and
D. dipsaci conformed to published patterns (data not shown).
Real-time EvaGreen-based PCR
Primer pair EvaF–EvaR were designed specifically for use
in a real-time PCR using the EvaGreen chemistry. In real-
560
Fig. 3. Electrophoretogram of an ethidium bromide stained 4%–
12% precast TBE polyacrylamide gel of PCR products after amplification with primers Gfor, Grev, and PITS-pall for 45 cycles.
Lanes R, P, T, and M were loaded with products generated by PCR
using template DNA from Globodera rostochiensis, G. pallida, G.
tabacum tabacum, and a mixture of all three, respectively.
Lanes L and W show the 100 bp DNA ladder and water controls, respectively.
time PCR with EvaGreen, these primers directed the amplification of a 131 bp amplicon from the ITS1 region of G.
pallida, G. rostochiensis, and G. tabacum. The amplicons of
the three species could be differentiated on the basis of melting curve analysis subsequent to the amplification reaction.
The melting temperature peaks were 88.72 ± 0.10 °C (n = 3),
87.77 ± 0.02 °C (n = 3), and 88.09 ± 0.09 °C (n = 5) for the
G. pallida, G. rostochiensis, and G. tabacum tabacum, respectively (Fig. 2A). However, when DNA from two or
more Globodera spp. were mixed, an intermediate melting
peak appeared, possibly because of hybrid molecules, and prevented unequivocal species identification (Fig. 2B). Distinct
melting curves were not obtained when D. dipsaci or H.
schachtii DNA were used as template for amplification.
Real-time TaqMan PCR
A real-time TaqMan assay was designed as a triplex test in
which amplification of each Globodera spp. was independently monitored on different channels of the thermocycler.
Primer pair Gfor–Grev directed amplification of 232 bp
amplicons with template DNA from G. pallida, G.
rostochiensis, and G. tabacum tabacum. However, in mixed
templates, addition of primer PITS-pall was required to ob-
Can. J. Plant Pathol. Vol. 30, 2008
Fig. 4. Fluorescence traces obtained in the three real-time
thermocycler channels (FAM, Texas Red 613, and Cy5) which
detected amplified DNA from Globodera tabacum tabacum,
G. pallida, and G. rostochiensis, respectively, by species-specific
hybridization of labelled TaqMan probes in multiplex PCR. PCR
template for each sample contained DNA from Globodera cysts
and second-stage juvenile (J2) worms as follows: (A) G.
rostochiensis (J2), G. pallida (J2), and G. tabacum tabacum
(J2); (B) G. tabacum tabacum (cyst) and G. rostochiensis (J2);
(C) G. tabacum tabacum (cyst) and G. pallida (J2); (D) G.
rostochiensis (J2) and G. pallida (cyst); (E) G. pallida (J2);
(F) G. pallida (J2) and G. rostochiensis (cyst); and (G) G.
rostochiensis (cyst).
tain equivalent amplification from the G. pallida template
(Fig. 3). In the real-time PCR format with the three LNAmodified, labelled TaqMan probes and separate or mixed
template DNA from the three Globodera spp., fluorescence
was detected and recorded in each of the three channels of
the SmartCycler thermocycler (Fig. 4). Signals in the Texas
Red, Cy5, and FAM channels corresponded consistently
with template DNA from G. pallida, G. rostochiensis, and
G. tabacum tabacum, respectively, when run separately
(Table 3). DNA obtained from PCN occurrences in New
Madani et al.: nematode identification / multiplex real-time PCR / Globodera species / cyst nematodes
561
Table 3. Globodera spp. identity using the Bulman and Marshall (1997) method and confirmation
by real-time TaqMan PCR assay.
Bulman and Marshall methodb
TaqMan probec
Sample identification No.a
G. rostochiensis
G. pallida
Texas Red
Cy5
FAM
BC Saanich
Quebec F1
Quebec F2
NL AD 1
NL AD 2
NL AD 3
NL LP 1
NL LP 2
NL LP 3
NL LP 4
US-G.t.t.
Fr-G.r.
Fr-G.p.
Fr-G.t.t.
Dd
Hs
+
+
+
–
–
+
+
+
+
+
–
+
–
–
–
–
–
–
–
+
+
–
–
–
–
–
–
–
+
–
–
–
–
–
–
+
+
–
–
–
–
–
–
–
+
–
–
–
+
+
+
–
–
+
+
+
+
+
–
+
–
–
–
–
–
–
–
–
–
–
–
–
–
–
+
–
–
+
–
–
a
BC Saanich, Saanich, British Columbia, sample; Quebec F1 and Quebec F2, Quebec field sample 1 and field
sample 2, respectively; NL AD 1, NL AD 2, and NL AD 3, Avondale, Newfoundland, samples; NL LP 1, NL LP
2, NL LP 3, and NL LP 4, Long Pond, Newfoundland samples; US-G.t.t., US sample of Globodera tabacum
tabacum; Fr-G.r., Fr-G.p., and Fr-G.t.t., French samples of G. rostochiensis, G. pallida, and G. tabacum tabacum,
respectively; and Dd and Hs, control samples of Ditylenchus dipsaci and Heterodera schachtii, respectively.
b
PCR method of Bulman and Marshall (1997).
c
Real-time PCR method developed in this study. Detection of fluorescence signals in each channel
corresponds to fluorescence of species-specific TaqMan probes.
York and Idaho were identified as being G. rostochiensis
and G. pallida, respectively, which were consistent with
known PCN infections in the respective states (data not
shown).
Analysis of melting curves of amplicons generated with
the inclusion of EvaGreen in the PCR mix did not adequately discriminate among amplicons generated with these
primers from DNA templates of the three Globodera spp.
The melting peak of the G. pallida amplicon was 89.5 °C,
whereas the amplicons for G. rostochiensis and G. tabacum
tabacum each generated two very similar melting curves
with major peaks at 87.5 °C and 87.9 °C and minor peaks at
89.9 °C and 89.6 °C, respectively. As in the real-time PCR
with primer pair EvaF–EvaR, no amplification was detected
in the TaqMan PCR with DNA templates from H. schachtii
cysts and vermiform D. dipsaci nematodes.
In the real-time TaqMan PCR assays, a direct relationship
was observed between the amount of template DNA and Ct
values (Fig. 5). Correlation coefficients were 0.9989, 0.9898,
and 0.9816 for amplification of DNA from G. rostochiensis,
G. pallida, and G. tabacum tabacum, respectively. The efficiencies of the real-time TaqMan PCR assays were 0.91,
1.02, and 0.89 for G. rostochiensis, G. pallida, and G.
tabacum tabacum, respectively.
The utility of multiplex PCR for discriminating species
was confirmed by assays using mixed cyst and J2 DNA
from the three Globodera spp. (Table 4, Fig. 4). All fluorescence signals corresponded precisely with presence of cyst
or J2 DNA in the template mix as expected. Ct values for
cyst DNA were consistently lower than for J2 DNA because
DNA prepared from single cysts contained higher genomic
copy numbers than DNA prepared from single J2 nematodes.
Discussion
Although populations of Globodera spp. are distinguishable
on the basis of morphological characteristics and morphometric measurements, characteristics of individual nematodes
and cysts from different species may overlap considerably
(Manduric et al. 2004). However, the use of multiple characters allows for reproducible designation of species identifications. Among the various technologies that have been
evaluated for improving and validating the discrimination
among species, those that are based on amplification of
genomic DNA sequences have proven to be the most sensitive
and robust (Ibrahim et al. 2001). Species identification based
on the PCR primers developed by Bulman and Marshall
(1997) has been found to be particularly useful by several
investigators (Bates et al. 2002; Ibrahim et al. 2001; Minnis
et al. 2002; Skantar et al. 2007) and also confirmed the
identity of G. rostochiensis and G. pallida in our study
(Table 1). Modifications of these primers were made recently to amplify G. tabacum DNA (Skantar et al. 2007).
In our work, we wished to develop a PCR strategy in which
individual Globodera juveniles or cysts could be identified
to species by a single amplification reaction. In such an assay, the same primers would need to amplify DNA from the
three species relevant to our work, and the amplicons would
need to be distinguishable. In addition to gel electrophoretic
mobility, RFLP analysis, and sequencing, amplicons can be
distinguished on the basis of differences in melting curve or
by differential hybridization to labelled probe. These latter
562
Fig. 5. Relationship between cycle threshold and DNA concentration of (A) Globodera rostochiensis, (B) G. pallida, and
(C) G. tabacum tabacum in a three-primer (Gfor, Grev, and PITSpall), multiplex, real-time PCR with fluorescently labelled
TaqMan probes.
two methods can be carried out without the need to remove
amplicon from sealed tubes using real-time PCR thermocyclers.
The genomic ITS region has been targeted by others for
discriminatory PCR assays, and the sequences are readily
available (Bulman and Marshall 1997; Toyota et al. 2008;
Uehara et al. 2005). However, polymorphisms in the ITS of
Globodera spp. are limited. In addition, although selection
of primers that would amplify DNA fragments from all
Globodera spp. was relatively uncomplicated, distinguishing among amplicons was more challenging. Nevertheless,
amplicon melting temperature curves differed sufficiently
among Globodera spp. in the EvaGreen-based assay to
allow consistent species identification. However, in the analysis of mixed samples with DNA of two species, hybridiza-
Can. J. Plant Pathol. Vol. 30, 2008
tion among heterologous strands resulted in intermediate
melting curves, which interfered with unequivocal species
identification. The use of instrumentation with capacity for
high-resolution melting curve analysis would probably have
avoided the problem; with such instruments, amplicons
with even a single nucleotide polymorphism can be discriminated. The usefulness of melting curve analysis for
differentiating between G. rostochiensis and G. pallida was
also confirmed by others who used the primers published
by Bulman and Marshall (1997), which amplified a larger
ITS fragment than in our study (Bates et al. 2002; Quader et
al. 2008). These studies did not include G. tabacum, which
is closely related to G. rostochiensis and has a similar ITS
sequence. The melting peak for G. tabacum tabacum at
88.1 °C was intermediate between that of G. rostochiensis
at 87.7 °C and G. pallida at 88.7 °C. Populations of
G. tabacum solanacearum and G. tabacum virginiae were
not available for this study, but it is known from published
sequences that the ITS sequences are identical to
G. tabacum tabacum in critical regions (Subbotin et al.
2000; Szalanski et al. 1997). Skantar et al. (2007) were also
able to identify G. tabacum and PCN species using modification of Bulman and Marshall (1997) primers in a conventional PCR assay and amplicon identification based on
electrophoretic mobility.
Our multiplex real-time TaqMan PCR assay was developed to differentiate Globodera spp. on the basis of specific
hybridization of labelled probes. Specificity of the probes
was enhanced by including LNAs in probe design. Published research has shown that the stability, affinity, and
specificity of oligonucleotide hybridization in real-time
PCR is enhanced by introduction of LNA residues in primers and probes (Simeonov and Nikiforov 2002). LNA residues have the ribose ring fixed in a locked position by a
linkage between the 2′ oxygen and the 4′ carbon atoms. Incorporation of an LNA monomer raises the thermodynamic
stability of the probe or primer by 3–5 °C and enhances the
affinity for complementary target sequences. For example,
they have been used for enhancing stability and discrimination
of specific hybridization targets in microarrays, real-time
PCR assays, and fluorescent in situ hybridization. (Kubota
et al. 2006).
The different fluorescent dye labels used in our study
permitted differentiation of the three Globodera spp. in the
separate channels of the thermocycler. Although it is possible
to combine melting point analysis with real-time TaqMan
PCR strategies (e.g., Smith et al. 2008), the melting curves
of amplicons generated by the Gfor–Grev–PITS-pall primer
set from DNA of the different Globodera spp. were insufficiently discriminatory with our thermocycler. However, based
on the amplicon sequences melting point differences would
likely have been adequate for discriminating among species
using an instrument capable of high-resolution melting curve
analysis. Notwithstanding this shortcoming in our work, the
multiplex real-time TaqMan PCR was most useful for its
rapid identification of Globodera spp. in single and mixed
samples (Table 4, Fig. 4).
In our study, DNA was simply prepared by disrupting
nematode cells by lyophilization and treatment with the components of a commercially available microlysis kit. However, the efficiency of the PCR reaction was at or near
Madani et al.: nematode identification / multiplex real-time PCR / Globodera species / cyst nematodes
563
Table 4. Range of Ct values for fluorescence response (n = 3) in thermocycler channels for real-time
PCR detecting TaqMan probe for Globodera rostochiensis labelled with Cy5, for G. pallida labelled
with Texas Red-613, and for G. tabacum labelled with FAM.
Ct of fluorescence signal per channela
Nematode stage included in sample
PCR run
G. rostochiensis
G. pallida
G. tabacum
Cy5
Texas Red
FAM
1
2
3
4
5
6
7
8
Cyst
J2
Cyst
J2
None
None
J2
Cyst
J2
Cyst
None
None
Cyst
J2
J2
Cyst
None
None
J2
Cyst
J2
Cyst
J2
Cyst
17.2–19.0
24.8–26.3
17.0–19.4
23.5–25.0
—
—
21.5–23.5
17.0–19.0
23.4–25.8
19.3–20.1
—
—
18.0–20.1
24.5–26.0
22.5–23.2
17.0–19.3
—
—
21.4–24.0
17.6–20.7
22.2–24.3
17.5–18.3
21.7–23.9
17.3–19.8
Note: Template DNA for each PCR run was prepared from samples that included DNA from cysts and (or) J2
nematodes as indicated from the three Globodera spp.
a
—, No fluorescence was observed in the channel.
100% as expected for reactions in which DNA copies double during each cycle. Deviations in amplification efficiency below one occur when PCR is inhibited by
contaminating compounds in template DNA, and occasionally exceeds one when more than two copies are generated
per cycle. Although there was no need in our study to quantify template concentrations, it is noted that the linear regression between Ct values plotted against the logarithm of
template DNA concentration was close to one for each reaction, and thus, the assay could be used in a quantitative
manner if required.
In conclusion, we took advantage of available ITS sequence data to design assays useful for rapid and accurate
identification of Globodera juveniles and cysts. In particular, Globodera cysts often need to be identified to establish
the nature of field infections, which has consequences for
regulatory measures and practices to mitigate crop loss. The
cysts are usually obtained after an extensive soil processing
procedure to isolate a cyst-containing organic fraction. Individual cysts can then be isolated for species identification
by morphological characterization and now using PCR. A
further challenge would be to identify cysts directly in the
organic fraction or even in raw soil extracts. Development
of such technologies would provide significant savings in
nematode surveys. Preliminary results suggest that detection
in soil fractions is possible, but whether the PCR procedures
developed in this study are sufficiently robust to permit their
use for routine detection in field samples is uncertain. Further genomic analysis may reveal genomic regions with
greater discriminatory polymorphisms, but that still remains
to be determined.
Acknowledgements
We thank Steve Wood, Guy Belair, and Michael Rott for
providing Globodera populations from Newfoundland,
Quebec, and British Columbia, respectively, and Xiaohong
Wang for providing DNA from Globodera populations in
New York and Idaho. We also thank J.A. LaMondia for providing cysts of G. tabacum tabacum and D. Mugniery for
providing cysts of all three Globodera spp. investigated in
this study. Michael Rott’s helpful review of the manuscript
prior to submission is particularly appreciated.
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