Nematoda 1

Bulletin OEPP/EPPO Bulletin (2013) 43 (1), 119138
ISSN 0250-8052. DOI: 10.1111/epp.12025
European and Mediterranean Plant Protection Organization

enne et Me
diterrane
enne pour la Protection des Plantes
Organisation Europe
PM 7/40 (3)
Diagnostics
Diagnostic
PM 7/40 (3) Globodera rostochiensis and Globodera pallida
Specific scope
Specific approval and amendment
This standard describes a diagnostic protocol for Globodera

rostochiensis and Globodera pallida1.
Approved as an EPPO Standard in 200309. Revision

approved in 200909, 2nd revision approved in 2012-09.
Introduction
Identity
Globodera rostochiensis and Globodera pallida (potato cyst

nematodes) cause major losses in Solanum tuberosum
(potato) crops (van Riel & Mulder, 1998). The infective
second stage juveniles only move a maximum of about 1 m
in the soil. Most movement to new localities is by passive
transport. The main routes of spread are infested seed potatoes and movement of soil (e.g. on farm machinery) from
infested land to other areas. Infestation occurs when the
second-stage juvenile hatches from the egg and enters the
root near the growing tip by puncturing the epidermal cell
walls, and then internal cell walls, with its stylet. Eventually it begins feeding on cells in the pericycle, cortex or
endodermis. The nematode induces an enlargement of root
cells and breakdown of their walls to form a large, syncytial transfer cell. This syncytium provides nutrients for the
nematode. Infested potato plants have a reduced root system and, because of the decreased water uptake, plant death
can eventually occur.
In this diagnostic protocol different tests are presented
which can be used depending on the circumstances. In
some EPPO countries official control is in place and
requires routine testing. In such situations of routine testing
in the country itself molecular techniques can be very useful. In other situations such as the testing of imported material for potential quarantine or damaging nematodes or new
infestations, identification by morphological methods performed by experienced nematologists are more suited (PM
7/76 Use of EPPO diagnostic protocols).
Name: Globodera rostochiensis (Wollenweber, 1923),

Skarbilovich, 1959
Synonyms: Heterodera rostochiensis, Wollenweber, 1923;
Heterodera schachtii solani Zimmerman, 1927; Heterodera
schachtii rostochiensis (Wollenweber) Kemner, 1929;
Taxonomic position: Nematoda, Tylenchida2, Heteroderidae
EPPO code: HETDRO
Phytosanitary categorization: EPPO A2 list No. 125, EU
Annex designation I/A2
Use of brand names of chemicals or equipment in these EPPO Standards implies no approval of them to the exclusion of others that may
also be suitable.
2013 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 43, 119138
Name: Globodera pallida (Stone, 1973)

Synonyms: Heterodera pallida Stone, 1973
Taxonomic position: Nematoda, Tylenchida2, Heteroderidae
EPPO computer code: HETDPA
Phytosanitary categorization: EPPO A2 list No. 124, EU
Annex designation I/A2
Detection
Symptoms
Above-ground symptoms due to potato cyst nematodes are

not specific and often go undetected. General symptoms
include patches of poor growth in the crop, with plants
sometimes showing yellowing, wilting or death of foliage;
tuber size is reduced and roots are extensively branched
with soil stuck to them. However, many other causes can
lead to these symptoms. Plants should therefore be lifted
2
Developments combining a classification based on morphological data

and molecular analysis refer to Tylenchomorpha (De Ley & Blaxter,
2004).
119
120
Diagnostics
Statutory sampling procedures
Recommendations on sampling can be found in the Council

Directive 2007/33/EC of 11 June 2007 on the control of
potato cyst nematode and repealing Directive 69/465/EEC
(EU, 2007).
Extraction procedures
Fig. 1 Potato roots infected by G. rostochiensis (Courtesy: Plant

Protection Service, NL).
There are various processes for extracting cysts from the

soil. Simple methods based on flotation can be as good
as elutriation. For each method, a description is given in
Appendix 1. Globodera cysts are generally round which
distinguishes them from most other types of nematode
cysts. Prior to identification cysts need to be removed
from the floats produced by the methods described in
Appendix 1. This process usually requires examination of
the float by staff trained in separating cyst nematodes
from similar globular bodies in the soil. It can be time
consuming, depending upon the efficiency of extraction
and whether any further clean-up has been used, e.g. acetone flotation. This process is critical to the efficiency of
the diagnosis as false negative results may result if any
Globodera cysts are missed at this stage. The distinction
between potato cyst nematodes and other cysts based on
morphology can only be reliably performed by trained
experts.
When moist soil samples are not immediately processed
and viability tests are envisaged, they should be stored
below 5C as juveniles may hatch above 5C. In addition
soil samples should not be dried at a higher temperature
than 2530C (and not lower than 40% air humidity) as this
might influence the viability as well.
Bioassay
Another procedure to detect the nematodes is the bioassay

(Appendix 2, test A).
Identification
Fig. 2 Broken cyst with eggs of G. pallida (Courtesy: Plant Protection

Service, NL).
for a visual check for the presence of cysts and young

females on the roots, or a soil sample should be taken for
testing. Young females and cysts are just visible to the
naked eye as tiny white, yellow or brown pin-heads on the
root surface (Figs 1 and 2). Detection by lifting plants is
only possible for a short time as females mature into cysts
and then can easily be lost at lifting, and it is time-consuming. Soil testing is therefore the best way of determining
the presence of potato cyst nematodes.
For identification based on morphology, second-stage

juveniles and cysts should be obtained from soil, plant
roots or tubers. The female colour at the appropriate
stage of development can be used as an indication of
species: a female which changes during maturation from
white to yellow, then into a brown cyst, is G. rostochiensis, while one which changes from white directly to
brown is G. pallida. Identification of cysts and other
stages is in general based on a combination of morphological and morphometric characters and/or biochemical
techniques. In well-defined circumstances molecular techniques can be applied solely. For light microscope identification, it is recommended to examine specimens
mounted in fixative on microscope slides. A flow
Globodera rostochiensis and Globodera pallida
121
Sample of soil
Extraction followed by
Isolation of cysts
G. pallida or
G. rostochiensis
not identified
No
Yes
Morphological identification to
genus level
No
Globodera sp. identified
Yes
Mokecular test for

detection and or
identification
Yes
Yes
Empty cyst?
Identification at
species level not
possible
No
Morphological identification based

on perineal patterns and juveniles
(see Table 1)
Negative
Uncertain
(overlap of values)
Positive
Negative
Molecular
detection
(Appendix 3)
Positive
Positive
G. pallida or
G. rostochiensis
not identified
Negative
G. pallida or
G. rostochiensis
identified
G. pallida or
G. rostochiensis
identified
Fig. 3 Flow-diagram for identification of Globodera rostochiensis and G. pallida.
diagram indicating combinations of methods is given in

Fig. 33.
Identification on the basis of morphological features
Identification of cyst and juveniles to genus level

To identify the cysts to genus level the key of Brzeski
(1998), Baldwin & Mundo-Ocampo (1991), Wouts & Baldwin (1998), Siddiqi (2000) or Subbotin et al. (2010) should
3
Tests for identification in soil extracts or floats based on molecular

methods are being developed and will be considered for inclusion in
this protocol when it is next revised.
be used based on form of the cysts and the characteristics

of the vulva-anus region.
Globodera cysts should meet the following characteristics:
Cysts of Globodera are smoothly rounded with small
projecting neck, no terminal cone present, diameter
450 lm, and have a tanned brown skin. Cuticle surface
with zigzag pattern of ridges, a distinct D-layer is present.
The perineal area consists of a single circumfenestration
around the vulval slit, perineal tubercles on crescents near
vulva. Anus subterminal without fenestra, vulva in a vulval
basin, underbridge and bullae rarely present (Fig. 4). Eggs
retained in cyst, no egg-mass present.
122
Diagnostics
Fig. 4 The perineal region of a Globodera cyst (After Fleming and

Powers, 1998).
G. pallida anterior
Globodera juveniles should meet the following characteristics:

The mobile second-stage juveniles of Globodera are vermiform, annulated and tapering at both ends. Within the
genus Globodera, body length ranging from 445 to
510 lm, stylet length 1925 lm, tail length 3755 lm and
a hyaline tail part of 2131 lm.
Juvenile cyst nematodes may be found in soil
with which extraction was performed for the detection of
free-living nematodes. They can be distinguished
from root-knot nematode juveniles (Meloidogyne spp.) by
a more heavily sclerotised lip region, relatively large
stylet, shape of the tail and more robust appearance
(Fig. 5). In such cases, it is advised to perform an additional cyst extraction and to proceed with this diagnostic
protocol.
Identification to species level
The identification of Globodera to species level based on
morphology can be difficult because of the observed variability of key characteristics. Therefore the use of a combination of cyst and second-stage juvenile characteristics is
recommended for reliable identification.
G. rostochiensis and G. pallida are morphologically and
morphometrically closely related (Stone, 1973a,b). Figure 6
presents some drawings of different stages of G. rostochiensis
(Fig. 6a) and G. pallida (Fig 6b). For cysts, the most
important diagnostic differences are in the perineal area, i.e.
number of cuticular ridges between vulva-anus and Graneks
ratio (Fig. 7A,B). The second-stage juvenile characteristics
are stylet length and stylet knob shape (Table 1; Fig. 7C). As
the range of values for each of these characteristics can
overlap between species, care is needed. In such cases,
confirmation with molecular techniques is recommended.
50 m
5 m
5 m
M. fallax anterior
G. pallida habitus
50 m
M. fallax habitus
5 m
10 m
G. pallida tail
M. fallax tail
Fig. 5 Difference between Meloidogyne and Globodera juveniles.

Comparison between Meloidogyne fallax and 6 G. pallida.
123
Fig. 6 (A) G. rostochiensis. A. Entire juvenile. B. Head region of 2nd-stage juvenile. C. 2nd-stage juvenile lateral field, mid-body. D. Pharyngeal
region of 2nd-stage juvenile. E. Pharyngeal region of male. F. Tail of male. G. Lateral field of male, mid-body. H. Entire cysts. I. Head and neck of
female. J. Entire male. (After: C.I.H. Descriptions of Plant-Parasitic Nematodes, Set 2, No. 16). (B) G. pallida n. sp. Larva. A. Entire. B. Anterior.
C. Head. D. Tail. E. Lateral field mid-body region. F. Lateral field tail. G. Head and face at level of lips. H. Head and face at level of base. (After:
Stone, 1972).
When cysts without live content have been found, species identification may not be possible in most cases4.
The three other Globodera species which could cause
confusion during identification of potato cyst nematodes
in Europe are G. millefolii (Subbotin et al., 2010)5,
G. artemisiae (Eroshenko & Kazachenko, 1972) Behrens,
1975, and G. tabacum sensu lato. These first two species
are not parasitic on potato, but recorded on Achillea
millefolium and Artemisia vulgaris, respectively, in
comparable agricultural areas. The G. tabacum species
4
It should be noted that under European conditions especially when
cysts without live content have been detected on fields used for the production of potato in the past, it is highly probable that these cysts
belong to either one of the potato cyst nematode species G. rostochiensis or G. pallida.
5
Krall (1978) considered G. millefolii (Kirjanova & Krall, 1965) Behrens, 1975 as species inquirenda, as the description was based on a single female. Brzeski (1998) reported on G. achilleae: it may be
conspecific with G. millefolii. According to Subbotin et al., 2010,
2011 G. achilleae is a junior synonym of G. millefolii. So from this
point onwards the species name G. achilleae will not be used and
G. millefolii instead.
complex (G. tabacum tabacum (Lownsbery & Lownsbery,

1954) Skarbilovich, 1959; G. tabacum solanacearum
(Miller & Gray, 1972) Behrens, 1975, and G. tabacum
virginiae (Miller & Gray, 1972) Behrens, 1975) is found
in North and Central America. In Southern Europe,
G. tabacum tabacum is also present. It parasitizes
Nicotiana tabacum (tobacco) and some other solanaceous
plants (but not potato). See Table 1 and Fig. 7 for a
morphometric and morphological comparison between
potato cyst nematodes, G. millefolii5, G. artemisiae and
G. tabacum. See also Baldwin & Mundo-Ocampo (1991),
Mota & Eisenback (1993), Brzeski (1998) Wouts &
Baldwin (1998) and Subbotin et al. (2010) for more
detailed information on other members of the Heteroderinae and identification keys.
Biochemical and molecular methods
As G. rostochiensis and G. pallida are morphologically closely related, many different biochemical techniques have
been developed to distinguish the two species. Schots et al.
(1992) were able to differentiate and quantify them using a
124
Diagnostics
Fig. 7 (A) Perineal measurements for Globodera identification. (B) Vulval-anal ridge patterns for four Globodera species. (C) Stylets from four
species of Globodera (After Flemming & Powers, 1998).
set of three monoclonal antibodies. There were, however,

cross reactivity problems between the antibodies for the
two species.
Several scientists have developed PCR-based tests to separate the two potato cyst nematode species (e.g. Mulholland
et al., 1996; Shields et al., 1996; Thiery & Mugniery,1996;

Bulman & Marshall, 1997; Fullaondo et al., 1999; Fleming
et al., 2000; Zouhar et al., 2000; Bates et al., 2002).
Recently real-time PCR assays have been developed and
can be used for identification (e.g. Nakhla et al., 2010;
4055
4955
5053
4451
5056
2326
2330
2627
2027
2128
Rounded
Rounded
Anteriorly projected *
Rounded
Rounded to slightly
anteriorly projected
2124
25
2324
2022
2125
413524
483516
452486
392468
47655
*Shape of J2 stylet knobs may vary from rounded to anteriorly projected, even within one cyst.
2656
2429
4854
5170
4152
8
6
12
1720
79
452493
480639
510675
445690
536767
artemisiae
millefolii
pallida
rostochiensis
tabacum
G.
G.
G.
G.
G.
356461
380479
451648
382559
459558
1.03.2
1.21.6
2.12.5
3.04.5
1.62.8
Shape of stylet knob

Stylet
Body length
Number of
cuticular ridges
Length
Character Species
Width
Graneks ratio
Anus-fenestral
edge distance
J2
Cyst
Stage
Table 1 Morphological and morphometric characters useful for identification of Globodera species (after to Subbotin et al., 2010). Ranges and means (lm) are given
Hyaline region
Tail
125
Madani et al., 2011). This type of test can also be used for
direct detection of target species without cyst morphological detection (Reid et al., 2010). Recommended molecular
tests are described in Appendix 3.
Isoelectric focusing (IEF) has proved to be sensitive
enough to identify samples of potato cyst nematodes. During IEF, proteins are separated in a pH gradient and
focused at the position in the gradient (the isoelectric
point) where they become electrically neutral. Four species-specific proteins were located, pI 5.9 & 8.7 and pI 5.7
& 6.9. These proteins can be used to identify G. rostochiensis and G. pallida (Fleming & Marks, 1982; Karssen
et al., 1995). Other techniques, such as RFLP analysis of
total DNA (Burrows & Boffey, 1986), diagnostic probes
(Marshall & Crawford, 1987; Burrows & Perry, 1988), and
dot-blotting with specific probes (Marshall, 1993) have also
proved to be useful to distinguish the two species. For a
complete treatise on the use of immunology, protein electrophoresis, IEF and DNA, see Fleming et al. (2000). A
comparison of IEF, ELISA and PCR used for the identification of potato cyst nematodes in field samples was made
by Ibrahim et al. (2001). However, these are not recommended in the protocol.
Many techniques have been developed especially to distinguish G. rostochiensis from G. pallida but have not been
tested so far against species such as G. millefolii, G. tabacum or G. mexicana. This limitation should be noted.
Tests that were developed after 2000 generally do not have
these shortcomings. (Nakhla et al., 2010). Specific
identification of G. achillae from G. rostochiensis and
G. pallida is possible following the PCR RFLP test developed by Sirca et al. (2010). There are also differences
between European and non-European populations of the
two species, which might be made visible with sequencing
techniques (Hockland et al., 2012). For practical application
this technique is not available yet.
A sequencing protocol will be included in the EPPO
Standard PM 7/XXX on DNA barcoding as an identification tool for plant pests (in preparation).
Identification of G. rostochiensis and G. pallida should
preferably combine morphological and molecular methods
especially when new introductions are suspected.
Pathotypes
The term pathotype is used by the International PCN

Pathotype Scheme proposed by Kort et al. (1977) but is
now considered too general. Many PCN populations cannot
conclusively be assigned to the pathotypes described in this
scheme. There are differences in virulence within the
two PCN species, in particular between populations of
G. pallida and they are of utmost importance in populations
from South America, but identification at this level is not
adequate at the moment because it is time consuming,
expensive and requires specific analysis (Hockland et al.,
2012). Any population showing signs of a new or unusual
126
Diagnostics
virulence (i.e., overcoming resistance currently available in

potato cultivars in Europe) should be tested as soon as possible. In practice, the virulence of populations can be tested
on a set of cultivars used in each country.
Reporting and documentation

Guidance on reporting and documentation is given in EPPO
Standard PM 7/77 (1) Documentation and reporting on a
diagnosis.
Testing the viability of eggs and juveniles
Testing of the viability of the eggs and juveniles may be

required for regulatory purposes. This can be done by different methods.
(1) Visual morphological determination of viability, a table
with descriptions and figures is provided (Appendix 4).
These observations require trained personnel.
(2) Determination of viability with a bioassay. Two tests
are described in Appendix 2. Such tests require more
time to perform than visual morphological determination of viability and generally more time than determination of viability by hatching tests. Dormancy might
play a role and should be lifted. An additional aspect
of bioassays is the possibility of false negative results
due to very low cyst contents.
(3) Determination of viability by hatching tests. Three tests
are described in Appendix 5. Such tests require more
time to perform than visual morphological determination
of viability. When determining the viability with a hatching test, it should be noted that cysts which have formed
recently may be dormant (e.g. when sampling is performed in autumn after potato harvest). In such situations
cysts should be exposed to +4C for at least 23 weeks.
(4) Determination of viability using trehalose. The test is
described in Appendix 6, based on the publication by
van den Elsen et al., 2012
(5) Determination of viability and identification on basis of
RNA. A test has been developed, validated and
described (unpublished Dutch report), however, it is
not used for routine testing so far.
Morphological determination of viability by staining with
Meldolas Blue is also possible but the chemical is not easily
available so this technique is not described in the protocol.
Reference material
Reference material can be obtained from:
Plant Protection Service, National Reference Laboratory,
P.O. Box 9102, 6700 HC Wageningen (NL).
Food and Environmental Research Agency (FERA), Sand
Hutton, York YO41 1LZ (GB).
Julius Kuhn-Institut (JKI), Federal Research Centre for Cultivated Plants, Messeweg 1112, 38104 Braunschweig (DE).
French National Institute for Agricultural Research
(INRA) Biology of Organisms and Populations for Plant
Protection Domaine de la Motte, BP 35327 35653 Le Rheu
Cedex.
Department of Plant Protection Biology Nematology,
Swedish University of Agricultural Sciences, Alnarp (SE).
Performance criteria
When performance criteria are available, these are provided
with the description of the test. Validation data are also
available in the EPPO Database on Diagnostic Expertise
(http://dc.eppo.int), and it is recommended to consult this
database as additional information may be available there
(e.g. more detailed information on analytical specificity, full
validation reports, etc.).
Further information
Further information on this organism can be obtained from:
den Nijs L & Karssen G, Plant Protection Service, National
Reference Laboratory, PO Box 9102, 6700 HC Wageningen
NL. E-mail: L.J.M.F.den.Nijs@minlnv.nl
Feedback on this Diagnostic Protocol

If you have any feedback concerning this Diagnostic Protocol, or any of the tests included, or if you can provide
additional validation data for tests included in this protocol
that you wish to share please contact diagnostics@eppo.int.
Protocol revision
An annual review process is in place to identify the need
for revision of diagnostic protocols. Protocols identified as
needing revision are marked as such on the EPPO website.
When errata and corrigenda are in press, this will also be
marked on the website.
Acknowledgements
This Standard was originally developed under the EU
DIAGPRO Project (SMT 4-CT98-2252) by partnership of
contractor laboratories and intercomparison laboratories in
European countries. The first draft was prepared by: den
Nijs L & Karssen G, Plant Protection Service, National
Reference Laboratory, PO Box 9102, 6700 HC Wageningen, The Netherlands. It was revised by Anthoine G
(Anses, Plant Health Laboratory, Angers, FR), Kox L and
den Nijs L (Plant Protection Service, National Reference
Laboratory, Wageningen, NL).
Methods performed in Germany and Austria provided by
Kaemmerer D, Bayerische Landesanstalt f
ur Landwirtschaft. Methods performed in Norway provided by Magnusson C. Methods performed in Sweden provided by
Manduric S.
References
Baldwin JG & Mundo-Ocampo M (1991) Heteroderinae, cyst- and noncyst-forming nematodes. In Manual of Agricultural Nematology (Ed.
Nickle WR), pp. 275362. Marcel Dekker, New York, (US).
Bates JA, Taylor EJA, Gans PT & Thomas JE (2002) Determination of
relative proportions of Globodera species in mixed populations of
potato cyst nematodes using PCR product melting peak analysis.
Molecular Plant Pathology 3, 153161.
Brzeski MW (1998) Nematodes of Tylenchina in Poland and
Temperate Europe. Muzeum i Instytut Zoologii PAN, Warsaw (PL).
Bulman SR & Marshall JW (1997) Differentiation of Australasian
potato cyst nematode (PCN) populations using the polymerase chain
reaction (PCR). New Zealand Journal of Crop and Horticultural
Science 25, 123129.
Burrows PR & Boffey SA (1986) A technique for the extraction and
restriction endonuclease digestion of total DNA from Globodera
rostochiensis and Globodera pallida second-stage juveniles. Revue de
N
ematologie 9, 199200.
Burrows PR & Perry RN (1988) Two cloned DNA fragments which
differentiate Globodera pallida from G. rostochiensis. Revue de
N
ematologie 11, 441445.
De Ley P & Blaxter ML (2004) A new system for Nematoda:
combining morphological characters with molecular trees, and
translating clades into ranks and taxa. In: Proceedings of the Fourth
International Congress of Nematology, 813 June 2002 Tenerife (ES)
(Eds Cook R & Hunt DJ), pp. 865. Brill, Leiden (NL).
EU (2007) Council Directive 2007/33/EC of 11 June 2007 on the
control of potato cyst nematode and repealing Directive 69/465/EEC.
Official Journal of the European Communities L156, 1222.
Fleming CC & Marks RJ (1982) A method for quantitative estimation
of Globodera rostochiensis and Globodera pallida in mixed species
samples. Record of Agricultural Research of the Department of
Agriculture for Northern Ireland 30, 6770.
Fleming CC, Rao J, Moreland B, Craig D & Turner SJ (2000)
Diagnostics of cyst nematodes and tephritid fruit flies using
mitochondrial and ribosomal DNA. Bulletin OEPP/EPPPO Bulletin
30, 585590.
Folkertsma RT, van der Voort JNAMR, van Gent-Pelzer MPE, Groot
KE, van den Bos WJR, Schots A, Bakker J & Gommers FJ (1994)
Inter- and intraspecific variation between populations of Globodera
rostochiensis and G. pallida revealed by random amplified
polymorphic DNA. Phytopathology 84, 807811.
Fullaondo A, Barrena E, Viribay M, Barrena I, Salazar A & Ritter E
(1999) Identification of potato cyst nematode species Globodera
rostochiensis and G. pallida by PCR using specific primer
combinations. Nematology 1, 157163.
Golden AM & Klindic O (1973) Heterodera achilleae n. sp.
(Nematoda: Heteroderidae) from yarrow in Yugoslavia. Journal of
Nematology 5, 196201.
Hockland S, Niere B, Grenier E, Blok V, Phillips M, den Nijs L,
Anthoine G, Pickup J & Viaene V (2012) An evaluation of the
implications of virulence in non-European populations of Globodera
pallida and G. rostochiensis for potato cultivation in Europe.
Nematology, 14, 113.
Holterman M, van der Wurff A, van den Elsen S, van Megen H,
Holovachov O, Bakker J & Helder J (2006) Phylum-wide analysis of
SSU rDNA reveals deep phylogenetic relationships among
nematodes and accelerated evolution towards crown clades.
Molecular Biology and Evolution 23, 17921800.
Ibrahim SK, Minnis ST, Barker ADP, Russell MD, Haydock PPJ,
Evans K, Grove IG, Woods SR & Wilcox A (2001) Evaluation of
PCR, IEF and ELISA techniques for the detection and identification
127
of potato cyst nematodes from field soil samples in England and

Wales. Pest Management Science 57, 10681074.
Karssen G, van Hoenselaar T, Verkerk-Bakker B & Janssen R (1995)
Species identification of cyst and root-knot nematodes from potato
by electrophoresis of individual females. Electrophoresis 16, 105
109.
Kort J, Ross H, Rumpenhorst HJ & Stone AR (1977) An international
scheme for identifying and classifying pathotypes of potato cystnematodes Globodera rostochiensis and G. pallida. Nematologica 23,
333339.
Krall E (1978) Compendium of cyst nematodes in the USSR.
Nematologica 23, 311332.
Madani M, Ward LJ & de Boer SH (2011) Hsp90 gene, an additional
target for discrimination between the potato cyst nematodes,
Globodera rostochiensis and G. pallida, and the related species,
G. tabacum tabacum. European Journal of Plant Pathology 130,
271285.
Marshall JW (1993) Detecting the presence and distribution of
Globodera rostochiensis and G. pallida mixed populations in New
Zealand using DNA probes. New Zealand Journal of Crop and
Horticultural Science 21, 219223.
Marshall JW & Crawford AM (1987) A cloned DNA fragment that can
be used as a sensitive probe to distinguish Globodera pallida from
Globodera rostochiensis and other common cyst-forming nematodes.
Journal of Nematology 19, 541.
Mulholland V, Carde L, ODonnell KJ, Fleming CC & Powers TO
(1996) Use of the polymerase chain reaction to discriminate potato
cyst nematode at the species level. In: Diagnostics in Crop
Protection (ed. Marshall G), pp. 247252. British Crop Protection
Council, Farnham (GB).
Nakhla MK, Owens KJ, Li W & Wei G (2010) Multiplex Real-time
PCR assays for the identification of the potato cyst and tobacco cyst
nematodes. Plant disease 94, 959965.
Phillips MS, Forrest JMS & Wilson LA (1980) Screening for resistance
to potato cyst nematode using closed containers. Annals of Applied
Biology 96, 317322.
Reid A, Kenyon DM, Evans FF, Mulholland V, Pickup J, Blok VC,
Paterson A & Phillips MS (2010) Development of a high-throughput
method for the detection and species determination of PCN. Aspects
of Applied Biology 103, 1316.
Sambrook J, Fritsch EF & Maniatis T (1989) Molecular cloning: a
laboratory manual. Cold Spring Harbor Laboratory Press, Cold
Spring Harbor (USA).
Schots A, Gommers FJ & Egberts E (1992) Quantitative ELISA for the
detection of potato cyst nematodes in soil samples. Fundamental and
Applied Nematology 15, 5561.
Shields R, Fleming CC & Stratford R (1996) Identification of potato
cyst nematodes using the polymerase chain reaction. Fundamental
and Applied Nematology 19, 167173.
Siddiqi MR (2000) Key to Genera of Heteroderinae. In: Tylenchida:
Parasites of Plants and Insects (Ed. Siddiqi MR), pp. 395396.
CABI Publishing, Wallingford, Oxon (UK).
Sirca S, Geric Stare B, Strajnar P & Urek G (2010) PCR-RFLP
diagnostic method for identifying Globodera species in Slovenia.
Phytopathologia Mediterranea 49, 361369.
Stone AR (1972) Heterodera pallida n. sp. (Nematoda: Heteroderidae)
a second species of potato cyst nematode. Nematologica 18, 591
606.
Stone AR (1973a) CIH Descriptions of Plant-Parasitic Nematodes
No 16 Globodera rostochiensis. CAB International, Wallingford
(GB).
Stone AR(1973b) CIH Descriptions of Plant-Parasitic Nematodes No
15 Globodera pallida. CAB International, Wallingford (GB).
128
Diagnostics
Subbotin SA, Mundo-Ocampo M & Baldwin GB(2010) Systematics of

cyst nematodes (Nematoda: Heteroderinae), Nematology Monographs
& Perspectives, Vol. 8A. Brill, Leiden (NL), 107177.
Subbotin SA, Cid del Prado Vera I, Mundo-Ocampo M & Baldwin JG
(2011) Identification, phylogeny and phylogeography of
circumfenestrate cyst nematodes (Nematoda: Heteroderidae) as
interfered from analysis of ITS-rDNA. Nematology 13(7), 805824.
Thiery M & Mugniery D (1996) Interspecific rDNA restriction fragment
length polymorphism in Globodera species, parasites of solanaceous
plants. Fundamental and Applied Nematology 19, 471479.
Turner SJ (1998) Sample preparation, soil extraction and laboratory
facilities for the detection of potato cyst nematodes. In: Potato Cyst
Nematodes: Biology, Distribution and Control (Eds Marks RJ &
Brodie BB), pp. 7590. CAB International, Wallingford (GB).
van den Elsen S, Ave M, Schoenmakers N, Landeweert R, Bakker J
& Helder J (2012) A rapid, sensitive and cost-efficient assay to
estimate viability of potato cyst nematodes. Phytopathology 102,
140146.
van Riel HR & Mulder A (1998) Potato cyst nematodes (Globodera
species) in western Europe. In: Potato Cyst Nematodes: Biology,
Distribution and Control (Eds Marks RJ & Brodie BB), pp. 271298.
CAB International, Wallingford (GB).
Vrain TS, Wakarchuk DA, Levesque AC & Hamilton RI (1992)
Interspecific rDNA restriction fragment length polymorphism in the
Xiphinema americanum group. Fundamental and Applied
Nematology 16, 563573.
Winfield AL, Enfield MA & Foreman JH (1987) A column elutriator
for extracting cyst nematodes and other small invertebrates from soil
samples. Annual Applied Biology 111, 223231.
Wouts WM & Baldwin JG (1998) Taxonomy and identification. In:
The Cyst Nematodes (Ed. Sharma SB), pp. 83122. Kluwer,
Dordrecht (NL).
Zouhar M, Rysanek O & Kocova M (2000) Detection and
differentiation of the potato cyst nematodes Globodera rostochiensis
and Globodera pallida by PCR. Plant Protection Science 36, 8184.
Appendix 1 Methods for extracting cysts

of Globodera spp. from soil
of detergent will cause the cysts to move to the edge and

the cysts can be picked out by hand using a brush. Other
ways are carefully decanting, or using a paper strip around
the beaker and raising the water level so cysts can adhere
to it. A variety of methods to isolate the cysts from the
debris are in use (Turner, 1998).
Fenwick can
The Fenwick can (Fig. 8) is an apparatus that has been in
use for many years. The apparatus is a container, tapering
towards the top, with a sloping collar around the outside of
the rim which collects overflow and directs it towards an
outlet. The can has a sloping internal base with a drain plug
at its lowest point. The can is first filled with water and the
soil sample is added by washing through a 1-mm sieve supported on a long-stemmed funnel above the can. The
organic matter will immediately rise and overflow onto the
collar and be collected on two sieves with an aperture of
840 lm and 250 lm. Most of the cysts in the soil sample
will be collected at this stage. The funnel above the can is
then removed and the soil at the base of the can is elutriated by means of water flowing rapidly through a long
glass or metal tube. The tube is inserted deep into the can
to stir the sediment and release any trapped cysts; this is
continued for approximately 1 min. The cysts are collected
on the 250 lm aperture sieve for further processing.
Some automated versions and modifications of this apparatus exist (e.g. carousel).
Schuiling centrifuge
The Schuiling centrifuge is a semi-automatic extraction
method. The air-dried, 200500 mL soil sample is added
to a transparent cylindrical container half-filled with
water. The contents are swirled with a rotating twopronged fork at 450500 rev min 1, creating a vortex and
causing cysts and similar-sized floating particles to be
forced to the centre through a wire-mesh cylinder
(1.5 mm aperture). The mesh cylinder is fixed above a
The methods described here involve basic extraction techniques. Laboratories may improve them (e.g. to automate
them).
If viability tests have to be performed, methods that use
heat to dry samples are not recommended (samples should
not be dried at a higher temperature than 25C and not
lower than 40% air humidity).
Methods for cyst extraction based on flotation
techniques (for dry cysts/dried soil samples only)
Flask and paper-strip methods

These methods are based on the characteristic that dried
cysts float. The dried soil sample is added to a beaker, flask
or white dish that is filled with water. The suspension is
well stirred. After 30 s to some minutes, depending on the
soil type, the water is cleared and the liquid will only
contain the floating organic debris and cysts. Adding a drop
Fig. 8 Vertical-section diagram of Fenwick can.
tube of the same diameter leading down to an outlet.

While swirling, more water is added around the inside of
the main container washing off any adhering debris and
cysts which are channelled to the outlet with the rest.
The apparatus cleans itself after each sample processing.
Further separation of cysts is by a special cleaning process involving the so-called Schuiling can and special
sieves. In some laboratories the Schuiling units have been
modified to suit different soils and conditions: the modifications include additional spinning and cleaning time, larger collecting sieves, an improved plastic cleaning can
for reducing the amount of debris, and removal of the
electrical parts from the apparatus to the wall above for
safety reasons. Cysts (and debris) are collected on a
250 lm aperture sieve for further processing.
Methods for cysts extraction based on elutriation
techniques (for wet soil)
These methods are based on the difference in density of

cyst in comparison to soil particles and can be used for wet
soil. At the base of a (conical) column water enters through
a perforated tube at a constant rate (minimal 0.6 L min 1).
Soil is added into the column using a funnel. A small plate
baffles the outlet of the funnel so that soil does not fall
down the column too quickly. Water containing flows from
the overflows spout and are collected on a pair of sieves
(250 lm aperture).
Alternatively, the suspension containing the cysts and
organic debris can be carefully decanted to separate it from
the rest of the soil contents, or a paper strip can be attached
around the internal rim of the beaker so that when the
water level is raised, the cysts can adhere to it.
Seinhorst elutriator
The equipment is to be used for extraction of cysts both
from dry and wet soil samples (Fig. 9).
The elutriator is first filled with water and the constant

upward water stream entering the base of elutriator is
opened. This stream allows the wet heavy cysts to float
in the suspension. Next the wet soil sample is placed on
the upper sieve (1 mm pore). The sample is washed into
elutriator by moving the sieve up and down. Heavy particles (sand, etc.) will settle in the bottom part of the elutriator. The cysts and some (light) debris are caught on
0.25 mm pore sieve or a bucket with such a sieve on
the bottom. Fine particles pass through the sieve. The
sample is run for 25 min depending on soil type. After
washing is finished the constant upward water stream
entering the base of elutriator is closed and the content
of the apparatus is drawn off through the lower outlet
onto a sieve or bucket. The collected material is destined
for further processing.
Some automated versions of this apparatus exist.
Wye washer
The apparatus (Winfield et al., 1987) is constructed of a
50-cm length of 15-cm diameter clear acrylic tube which,
at its lower end, is held inside two tight-fitting concentric
PVC sleeves (Fig. 9). Water enters through an inlet pipe on
the outer sleeve and is caused to swirl by means of an
arrangement of grooves and angled holes on the inner sleeve
and the acrylic tube. At the top of the tube is a spout which
directs overflow onto sieves of similar size to those used
with the Fenwick can (i.e. aperture of 840 lm and 250 lm).
A soil sample, up to 1000 mL is added to a small quantity
of water in the Wye washer. More water is added as rapidly
as possible, to break up the soil until the rim is reached,
whereupon the flow is briefly stopped and then increased
gradually to about 10 L min 1 for 10 min. The overflow
carries: (i) small soil particles, which will pass through both
sieves; (ii) large organic debris which will be retained by
the upper larger sieve; (iii) Cysts and similar sized organic
particles, collected on the 250 lm aperture sieve.
B
Acrylic tube
Soil sample
Outer PVC sleeve
Inner PVC sleeve

Cyst and debris
overflow
Inlet pipe
Water inlet
Angled hole
Extract
collection
Soil particles
10 cm
Fig. 9 (A) Seinhorst elutriator. (B) Cross section of the base of the Wye washer.
129
130
Diagnostics
Appendix 2 Bioassays
Test A Bioassay (Method performed in Germany and
Austria)
This method relies on the principle that if potato cyst nematodes are present in a soil sample (even in very low numbers) they will multiply when given access to the roots of
growing potato plantlets in a small container. The presence
of developing cysts on the roots can then be observed
through the transparent walls of the special containers
used6.
Depending on the size of the container about 100
200 mL of soil from the sample should be put into each
container, ensuring that the soil remains suitably moist. Prepare as many containers as needed to process the entire
sample. Eyes are cut from well chitted, certified tubers with
a circular blade (diameter approximately 3 cm) and placed
in the containers. Bioassay in autumn/winter requires chitting of tubers (through fumigation or treatment with gibberellic acid). To avoid growth of fungi, eye cuttings should
be left to dry for half a day at room temperature before
they are placed on the soil samples in the containers (eyes
upwards) and covered with nematode-free soil. Control containers with known infestations are used in each test.
The square containers are placed close together on a
planting table shading each other to prevent the growth of
algae on the transparent walls. To allow optimal host-parasite interaction air temperature in the glasshouse is ideally
maintained at 22/16C (day/night) and always kept below
25C (possibly giving additional light in winter) and above
13C. Containers should be watered moderately to achieve
an optimal root penetration of the soil. Watering may be
done manually or by trickle irrigation. Surplus irrigation
water can run off through a hole in the bottom of the containers. The risk of contaminating healthy samples by
means of adjacent infested samples has been shown to be
unlikely. It might be necessary to take measures against
foliar blight during the course of the bioassay. If an individual plant should die, the soil in the container should be
tested for cysts using the Fenwick can or a related method.
Visual observation of females and cysts is done when
cysts are observed in the control containers, generally after
610 weeks of cultivation. Before counting of females and
cysts, potato leaves are cut with pruning shears. New cysts
are visible on the roots through the transparent walls of the
containers, when infection levels are high. To detect low
levels of infestation, it is advised to inspect roots and soil
after removing from the container and to extract cysts from
the soil when no infection is detected visually.
This can also be performed in closed containers kept in a
dark room (Phillips et al., 1980).
6
They can be obtained from Ritter GmbH, Schwabenstrae 5054,
D-86836 Untermeitingen.
Test B Test of reproduction (Method performed in

Norway)
The infective success of PCN is tested on potato plants in

500 mL pots with sand, using nylon bags with cysts (up to
20) as inoculation units. It is recommended to treat tubers
with gibberellic acid in order to induce and synchronize the
germination. Each pot is filled with 1/3 of the total soil volume, a nylon bag with cysts is placed below one potato tuber,
and then filled up with sand. The pots are placed in a randomised fashion in a growth cabinet with approximate day/
night temperatures of 20C/16C and 18 h light period. The
pots should receive mineral nutrients and water as required.
After 3 months the shoots are cut and the soil and roots are
air-dried. The newly formed cysts are extracted from soil (for
instance by the Fenwick can), and collected and counted.
Each new cyst represents a successful infection and hence a
measure of the infection potential of the population.
Appendix 3 Molecular tests

The following PCR tests are recommended for the identification of isolated cysts or individuals from G. rostochiensis
and G. pallida by use of species-specific primers:
Bulman & Marshall, 1997, a multiplex PCR test using
species-specific primers based on ribosomal 18S and
ITS1 sequences (A).
Fleming et al. (2000) (B) or Thiery & Mugniery (1996)
(C), two ITS-RFLP PCR tests based on primers described
by Vrain et al. (1992).
A commercially available test developed by Blgg (www.
blgg.nl), a real-time SYBR-green test based on rDNA
(LSU) sequences based PCR (D).
The PCR test of Bulman & Marshall was evaluated using
269 field samples containing PCN, and proved more sensitive than ELISA and Isoelectric focusing (IEF) (Ibrahim
et al., 2001).
For each method the DNA isolation methods are
described in detail as in reference cited (and used in the
validation process). There are also other extraction methods
available (see Fleming et al., 2000). Alternatively, commercial kits for DNA isolation are available.
A) Multiplex PCR test (Bulman & Marshall,

1997)
1. General information
1.1 Test developed by Bulman & Marshall (1997). The
test can only be used on nematodes morphologically
identified as Globodera sp., as the primers are not
specific for Globodera spp. (cross reaction with
Heteroderidae cysts may be observed).
1.2 Different G. pallida and G. rostochiensis populations, from different pathotype and geographical origin were used. Full cysts are nucleic acid source.
1.3 The test is designed to the 18S rRNA gene and the
internal transcribed spacer ITS1 region.
1.4 Oligonucleotides: One universal primer ITS5 (5GGAAGTAAAAGTCGTAACAAGG-3), and also
specific primers for G. pallida (PITSp4: 5-ACAACAGCAATCGTCGAG-3) and G. rostochiensis
PITSr3: 5-AGCGCAGACATGCCGCAA-3 result
in amplicons of 265 bp for G. pallida and 434 bp
for G. rostochiensis. The primers can be used in a
mixture for a multiplex PCR.
1.5 Taq DNA polymerase (Life technologies, Carlsbad,
CA, US) used for the amplification.
1.6 Nucleotides are used at a final concentration of
0.16 mM each.
1.7 Molecular grade water (MGW) is used to make up
reaction mixes.
2. Methods
2.1 Nucleic acid Extraction and Purification
Twenty cysts were ground in Eppendorf tubes, using
plastic micro-pestles (Treff, Degersheim, CH), with
200 lL of solution containing 5 M guanidine isothiocyanate, 10 mM EDTA, 50 mM Tris-HCl (pH 7.5),
and 8% mercaptoethanol. After room temperature
incubation for up to 1 h, the DNA containing solution was extracted once with equal volumes of phenol and chloroform-isoamyl alcohol (24:1) and once
with chloroform isoamyl alcohol then precipitated
with 0.3 M sodium acetate and two volumes ethanol.
DNA was resuspended in 100 lL of MGW.
2.2 Polymerase Chain reaction
2.2.1 Master Mix
Reagent
Working
concentration
Volume
per reaction
(lL)
MgCl2
Tris HCl (pH 8.3)
KCl
dNTPs
25 mM
500 mM
500 mM
10 mM
2
1
2.5
0.4
Forward Universal
primer
(ITS5)
Reverse Specific
primer
for G. pallida
(PITSp4)
Reverse Specific primer
for G. rostochiensis
(PITSr3)
Taq DNA Polymerase
(Life Technologies)
Molecular grade water
10 lM
0.625
2 mM
20 mM
50 mM
0.16 mM
(of each
dNTP)
250 lM*
10 lM
0.625
250 lM*
10 lM
0.625
250 lM*
0.12
0.6 U
5 U lL
Final
concentration
(continued)
131
Table (continued)
Reagent
Total DNA extract

Total
Working
concentration
Volume
per reaction
(lL)
Final
concentration
To make
up to 24
1
25
* 250 lM of each primer is mentioned in the original publication but

laboratories performing the test use a final concentration ranging from
0.15 to 1 lM.
2.2.2 PCR cycling parameters

Two minutes at 94C, 35 cycles of 30 s at 94C, 30 s at
55C, 30 s at 72C final elongation 7 min at 72C.
2.3 Analysis of DNA fragments
DNA fragments are separated by electrophoresis on agarose gel and visualized under UV light according to standard procedures (e.g. Sambrook et al., 1989).
3. Essential Procedural Information
3.1 Controls
For a reliable test result to be obtained, the following
(external) controls should be included for each series of
nucleic acid isolation and amplification of the target
organism and target nucleic acid, respectively.
Negative isolation control (NIC) to monitor contamination during nucleic acid extraction: clean extraction buffer.
Positive isolation control (PIC) to ensure that nucleic acid
of sufficient quantity and quality is isolated: nucleic acid
extraction and subsequent amplification of the target
organism.
Negative amplification control (NAC) to rule out false
positives due to contamination during the preparation of
the reaction mix: amplification of molecular grade water
that was used to prepare the reaction mix.
Positive amplification control (PAC) to monitor the efficiency of the amplification: amplification of nucleic acid
of the target organism. This can include nucleic acid
extracted from the target organism, whole genome amplified DNA or a synthetic control (e.g. cloned PCR
product).
As alternative (or in addition) to the external positive
controls (PIC and PAC), internal positive controls (IPC)
can be used to monitor each individual sample separately.
Positive internal controls can either be genes present in the
matrix DNA or added to the DNA solutions.
Alternative internal positive controls can include:
Specific amplification or co-amplification of endogenous
nucleic acid, using conserved primers that amplify conserved non-pest target nucleic acid that is also present in
the sample (e.g. plant cytochrome oxidase gene or
eukaryotic 18S rDNA).
132
Diagnostics
amplification of samples spiked with exogenous nucleic

(control sequence) acid that has no relation with the target nucleic acid (e.g. synthetic internal amplification controls) or amplification of a duplicate sample spiked with
the target nucleic acid.
3.2 Interpretation of results
Verification of the controls
NIC and NAC should produce no amplicons
PIC, PAC should produce amplicons of the expected sizes
(265 bp for G. pallida, and 434 bp for G. rostochiensis)
The IPC should produce amplicon(s) of the expected size
When these conditions are met:
A sample will be considered positive if it produces the
expected amplicons for G. pallida (265 bp) and for
G. rostochiensis (434 bp).
A sample will be considered negative, if it produces no
band or a band of a different size.
Tests should be repeated if any contradictory or unclear
results are obtained.
4. Performance criteria available
The following performance criteria were provided by
Anses Plant Health Laboratory (FR) July 2010 with the
following adaptations of the mastermix: primer concentration 0.64 lM and dNTP 0.25 mM each.
4.1 Analytical sensitivity data
One J2.
4.2 Analytical specificity data
The study included 11 populations of G. pallida, 4 populations of G. rostochiensis, 5 populations of G. tabacum, one population of G. mexicana and G. artemisiae.
The populations cover different geographical areas.
4.3 Data on Repeatability
100% for G. pallida
100% for G. rostochensis
4.4 Data on Reproducibility
96% (1 J2) for G. pallida
100% (1 J2) for G. rostochiensis
4.5 Diagnostic Specificity data
91% for G. pallida the test cross reacted with G. tabacum
virginae
100% for G. rostochensis.
ACGAGCCGAGTGATCCACCG-3 result in a
750 bp amplicon for Globodera species.
1.4 Taq DNA polymerase used for amplification and
enzymes AluI and HinfI for amplicon restriction.
reaction mixes.
2. Methods
DNA is extracted from cysts by standard procedures.
Alternatively, crude DNA extract can be prepared by
crushing or boiling a juvenile nematode in 10 lL of
MGW and then used directly in a PCR reaction.
2.2.1 Master Mix
Reagent
Mg-free 109
reaction buffer
MgCl2
dNTPs
Forward Primer
(rDNA2)
Reverse Primer
(rDNA1 5.8s)
Taq DNA
Polymerase
Total DNA
extract
Molecular
grade water
Total
Working
concentration
Volume per
reaction (lL)
Final
concentration
109
2.5
19
25 mM
10 mM
1.5
1.6
20 mM
1.5 mM
0.64 mM (of each
dNTP)
0.8 mM
20 mM
0.8 mM
0.2
1U
5 U lL
1
To make
up to 25
25

35 cycles of 45 s 94C, 1 min 50C, 1 min 72C.
2.3 Restriction of PCR amplicon
2.3.1 Restriction mix (according to suppliers conditions)
B) ITS PCR-RFLP test (Fleming et al., 2000)
Reagent
1.1 Test developed by Fleming et al. (2000). The ITS1
rDNA region is the target region. The test can only
be used on nematodes morphologically identified as
Globodera spp., as the primers are not specific for
Globodera spp. (cross reaction with Heteroderidae
cysts may be observed).
1.2 Full cysts are nucleic acid source. A single cyst can
be used.
1.3 Oligonucleotides: Primers rDNA2 5-TTG ATT
ACG TCC CTG CCC TTT-3 and rDNA1 5.8s 5-
Restriction enzyme
buffer
Restriction enzyme(s)
PCR product (purified
when complete
digestion not observed)
Total
Working
concentration
Volume
per reaction
(lL)
Final
concentration
109
19
0.2
5
2U
10 U lL
To make
up to 20
20
2.3.2 Incubation time/temperature for digestion: 37C for

4 h. It may occur that the PCR product does not
digest to completion. In such cases, overnight incubation or purification of PCR products before restriction
step should be performed using PCR purification kits.
2.4 Analysis of RFLP fragments:
RFLP fragments are separated by electrophoresis on 2%
agarose gel in 19 TAE buffer and visualized under UV
light according to standard procedures (e.g. Sambrook
et al., 1989).
3.1 Controls
organism and target nucleic acid, respectively:
Negative isolation control (NIC) to monitor contamination
during nucleic acid extraction: clean extraction buffer.
organism.
Positive amplification control (PAC) to monitor the efficiency of the amplification: amplification of nucleic acid of
the target organism. This can include nucleic acid extracted
from the target organism, whole genome amplified DNA or
a synthetic control (e.g. cloned PCR product).
can be used to monitor each individual sample separately. Positive internal controls can either be genes present in the matrix DNA or added to the DNA solutions.
Amplification of samples spiked with exogenous nucleic
3.2 Interpretation of results:
Verification of the controls:
NIC and NAC should produce no amplicons (and hence
no restriction fragments).
PIC and PAC should produce amplicons of the sizes
given in Table 2 (after digestion of the PCR product).
The IPC should produce amplicon(s) of the expected size.
133
When these conditions are met:

restriction fragment lengths as given in Table 2

band or a band of a different size
Table 2 Sizes of RFLP fragments
Enzyme
G. rostochiensis
G. pallida
G. tabacum
AluI
HinfI
360, 160, 150, 80

520, 230
510, 160, 80
370, 230, 150
510, 160, 80
520, 230

results are obtained
ry & Mugnie

ry,
C) ITS PCR-RFLP test (Thie
1996)
1.1 Test developed by Thiery & Mugniery (1996). The
method can only be used on nematodes morphologically identified as Globodera sp., as the primers are
not specific for Globodera spp.
1.2 Full cysts or females are nucleic acid source. G. pallida
and G. rostochiensis populations from various sources
(e.g. pathotypes, geographical origin) were tested.
G. mexicana, G. tabacum tabacum, G. tabacum
virginae and G. tabacum solanacearum were also
included in this test.
1.3 Oligonucleotides: Primers 18S and 26S described
by Vrain et al. (1992), 18S primer 5-TTG ATT
ACG TCC CTG CCC TTT-3 and 26S primer 3GGA ATC ATT GCC GCT CAC TTT-5 result in a
1 200 bp amplicon.
1.4 Taq DNA polymerase (MP biomedicals, Illkirch,
FR, ex Appligene Oncor) used for amplification and
enzyme Bsh1236I for amplicon restriction.
1.5 Nucleotides used at a final concentration of
100 lM.
reaction mixes
1.7 Amplification is performed in a thermal cycler, e.g.
Perkin-Elmer Cetius DNA Thermal Cycler 480.
2. Methods
Genomic DNA was isolated according to Folkertsma
et al. (1994). An extraction with 6 M NaCl solution
was done to precipitate the majority of proteins. RNA
was removed by incubation with 20 lg of RNaseA at
37C for 30 min.
134
Diagnostics
2.2.1
Reagent
Working
concentration
Volume
per reaction
(lL)
MgCl2
dNTPs
25 mM
10 mM
4
0.5
Primer (18S)
Primer (26S)
Taq DNA Polymerase
(MP Biomedicals,
ex Appligene Oncor)
Total DNA extract
to a final volume
of 25 lL
Total
10 lM
10 lM
5 U lL
2.5
2.5
0.1
Final
concentration
2 mM
100 lM (of
each dNTP)
0.5 lM
0.5 lM
0.5 U
5
to make
up to 50
50

NIC and NAC should produce no amplicons, and hence
no restriction fragments.
PIC and PAC should produce restriction fragment
lengths as given in Table 3.
The IPC should produce amplicon(s) of the expected size
When these conditions are met
restriction fragment lengths as given in Table 3.
band or a band of a different size than those given in
Table 3.

Thirty cycles of 1 min 94C, 50 s 60C, 1 min
72C.
2.3 Restriction of PCR amplicon
2.3.1 Restriction mix
According to suppliers conditions.
2.3.2 Incubation time/temperature for digestion: overnight at the recommended temperature (see suppliers information).
2.4 Analysis of DNA fragments:
DNA fragments are separated by electrophoresis on
agarose gel (1.5% for RFLP) and visualized under
UV light according to standard procedures (e.g.
Sambrook et al., 1989).
3.1 Controls
Negative isolation control (NIC) to monitor contamination during nucleic acid extraction: clean extraction
buffer.
Positive isolation control (PIC) to ensure that nucleic
acid of sufficient quantity and quality is isolated: nucleic
acid extraction and subsequent amplification of the target organism.
Positive amplification control (PAC) to monitor the efficiency of the amplification: amplification of nucleic acid
of the target organism. This can include nucleic acid
extracted from the target organism, whole genome
amplified DNA or a synthetic control (e.g. cloned PCR
product).

The following performance criteria were provided by Anses Plant Health Laboratory (FR) July 2010.
One J2
The study included 11 populations of G. pallida, 4 populations of G. rostochiensis, 5 populations of G. tabacum, one population of G. mexicana and G. artemisiae.
The populations cover different geographical areas.
4.3 Data on Repeatability
100% for G. pallida
100% for G. rostochensis
Table 3 Restricted fragments sizes in bp (Thiery & Mugniery, 1996)
Species
Bsh1236I RFLP pattern
G.
G.
G.
G.
G.
G.
500, 400,
500,
445,
445,
445,
rostochiensis (European populations)

pallida (European populations)
mexicana
tabacum tabacum
tabacum virginae
tabacum solanacearum
900,
350,
400,
400,
400,
400,
190,
190,
190,
190,
190,
190,
110
110
110
110
110
110
4.4 Data on Reproducibility

90% (1 J2) for G. pallida
93% (1 J2) for G. rostochiensis
4.5 Diagnostic Specificity
91% for G. pallida (1 non-target species detected in 11)
100% for G. rostochiensis.
D) Real-time SYBR-green assay based on

rDNA (LSU) sequences
1.1 This protocol was originally developed by Blgg bv.
(currently ClearDetections) and the Laboratory of
Nematology (Wageningen University, The Netherlands). The development of these species-specific
LSU rDNA primers was a spin-off of a phylumwide phylogenetic study among nematodes (based
on SSU rDNA) conducted by Holterman et al.
(2006). The Globodera primer sequences were
aligned with 400 nematode sequences in a LSU
rDNA sequence database, including 22 rDNA
sequences of 6 different Globodera species (G. pallida, G. rostochiensis, G. tabacum, G. achilleae
(=G. millefolii), G. artemisiae (=G. hypolysi)).
1.2 A DNA extract of fully-lysed nematodes (or cysts)
is needed as starting material. The tests can be performed on single nematode DNA extracts as well as
on cyst nematode DNA extracts. The simplex and/
or oligoplex amplifications can be performed in any
real-time PCR instrument. Alternatively, the primers
can be used for conventional PCR.
Further information and the Globodera detection kit
including the PCR primers can be obtained from ClearDetections in The Netherlands (http://www.cleardetections.
com/en).
2. Methods
2.1 The LSU (28S) detection kit includes primers to be
used for (real-time) PCR detection of G. pallida
and G. rostochiensis.
3. Essential Procedural information
3.1 Controls
Negative isolation control (NIC) to monitor contamination during nucleic acid extraction: clean extraction
buffer.
organism.
135

Positive amplification control (PAC) to monitor the
efficiency of the amplification: amplification of nucleic
acid of the target organism. This can include nucleic acid
extracted from the target organism, whole genome amplified DNA or a synthetic control (e.g. cloned PCR product).
eukaryotic 18S rDNA)
The cycle cut off value for targets Globodera rostochiensis and Globodera pallida is set at 35, and was
obtained on a Biorad MyiQ Real Time PCR detection
system, making use of the SYBRGreen-mix provided
in the ClearDetections Real-time PCR identification kit
for Globodera rostochiensis and G. pallida. The cycle
cut off value needs to be verified in each laboratory
when implementing the test for the first time.
The PIC and PAC amplification curves should be exponential.
NIC and NAC should be negative (Ct > cut off)
PIC, PAC and IC should have a Ct value below the cut
off value.
When these conditions are met
A test will be considered positive if it produces an
exponential amplification curve, a Ct value below the
cut off value.
A test will be considered negative, if it produces no
exponential amplification curve and a Ct value equal or
above the cut off value
For SYBR Green based real-time PCR tests: the Tm
value should be equal to the TM of the positive control,
1C.
Determination of analytical sensitivity and analytical
specificity was performed on a Biorad MyiQ Real Time
PCR detection system.
136
Diagnostics

The analytical sensitivity is one single PCN juvenile or
egg, against a background of 1000 juveniles or eggs of
non-target cyst nematodes.
The analytical specificity of the G. pallida and
G. rostochiensis primer sets was tested with plasmid
DNA and genomic DNA against G. pallida and
G. rostochiensis,
respectively,
and
G. tabacum,
G. achilleae (=G. millefolii), and Punctodera punctata.
Specificity is defined as a DCt > 20 cycle between the
target and the closest non-target. Except for a single
South American G. pallida population (Otuzco, results
from Anses, Fr), no false positive signals are obtained
with these species. Consequently, it is not advised to use
this test for South- and Central-American cyst nematode
samples. In addition it should be noted that sufficient
contrast with the three G. tabacum subspecies
(G. tabacum tabacum, G. tabacum solanacearum, and
G. tabacum virginiae) cannot yet be guaranteed.
Appendix 4 Visual determination*

Characteristics of live and dead eggs and juveniles of the potato cyst
nematodes
Live eggs, Figs 10 & 11
Dead eggs, Figs 15 & 16
a. Whole egg is intact

b. Egg shell is smooth
c. Egg is clear/transparent with
distinct contents or a dark line
down the middle of the egg
d. Curled juvenile fill up
against the egg shell
e. Sometimes clear lip region
and stylet present
a. Egg may be damaged/broken and

empty
b. Egg shell often not smooth
c. Contents have black/grey
granular appearance with no
structure
d. Shrivelled disintegrated juvenile
in egg
e. No clear lip region or stylet
present
Live juveniles (Figs 12, 13

and 14)
Dead juveniles (Figs 17, 18

and 19)
a. Juvenile has clear lip region,

stylet visible
b. Juvenile has strong smooth
cuticle
c. Intestine is filled with grey
granular structure, solid
a. No clear lip region, partly or

completely grey/black structure
b. Cuticle shrivelled or not intact
d. Clear lopsided distinction

between pharynx and intestine
Included in counts: heads
c. Transparent, body with clear

patches or completely
transparent
d. No clear lopsided distinction
between pharynx and intestine
e. Juvenile sharply bent at an
angle or lying in half circle
Not included in counts: tails,
empty shells
*Based on ring testing between laboratories in The Netherlands, L. den

Nijs.
10
137
15
11
16
12
17
18
13
19
14
Figs 1019 Characteristics of live and dead eggs and juneniles of the potato cyst nematodes.
Appendix 5 Hatching Test

A) Hatching test (method performed in Norway)
The hatching medium, potato root diffusate (PRD), is

obtained by passing 1000 mL of tap water through a
500 mL pot with a 3-week-old potato plant growing in
sand. After filtering, the PRD is stored at +3C in the dark
until needed. The PRD is used without dilution in closed
glass vials ( = 23.5 mm; height 34 mm) functioning as

hatching units. Each vial contains one cyst bag made of
nylon net with 20 cysts, which is completely covered by
PRD. Cysts collected in the autumn need to be exposed
+4C for 23 weeks for the contents to hatch. The test normally lasts for 8 weeks, but the hatch is often already high
within 2 weeks. Each week the cyst bags are transferred to
new hatching units with fresh PRD. The number of hatched
juveniles is counted weekly and accumulated to form the
138
Diagnostics
100
% hatch
80
60
40
20
0
1
Week
Fig. 20 Cumulative hatch of Globodera rostochiensis in potato root
diffusate from 3 weeks-old plants (n = 4)
total hatch. At the end of the test the juveniles remaining in

the cysts are counted and the hatching expressed as a percentage of the cyst content (Fig. 20).
B) Hatching test (methods performed in Sweden)
Prior to the exposure to the hatching stimulus, cysts that

have been stored dry should be presoaked in water in Petri
dishes, staining blocks or other suitable containers for
45 days. Whereas non-hydrated cysts trend to float,
hydrated once sink to the bottom of the Petri dish which
gives a good indication of cysts hydration level. During
this hydration phase, the water in the containers is renewed
daily to prevent bacterial and fungal growth. Repeated up
and down pipetting of the cysts helps to get rid of the fungi
and favours the hydration.
Potato plants for production of the hatching medium,
PRD, are grown in small (200 mL) clay pots with silver
sand substrate in a greenhouse. Three weeks old plants
are removed from the substrate and their roots are rinsed
in water after which the plants are transferred one by
one into a 200 mL beakers filled with tap water and
incubated at room temperature under aeration by an
aquarium air pump (Only the root system is immersed in
the water).
After 24 h diffusate is collected, filtered and ready to
use. Hydrated cysts (up to 100, depending on the number
of cysts found in the sample) are soaked in the undiluted
PRD. The PRD is replaced daily with a fresh one.
The test lasts until juveniles start to hatch or in total for
8 weeks.
Potato root diffusate production: sprout tubers are placed

on a funnel put on a plastic (transparent) beaker filled up with
tap water. This assemblage is put at room temperature
(around 1819C) in the dark for 4 weeks. The water with
PRD is filtered, divided in aliquot parts and frozen until use
( 20C). After 48 h freezing time, the PRD is evaluated with
the previous batch of PRD (previous production) and a reference G. pallida and G. rostochiensis populations. If the test
is satisfactory, the PRD can be used for the hatching test.
Hatching test: instead of being rehydrated, cysts are
deposited in a fine sieve (250 lm), placed on a small dish
filled with PRD. One sieve is prepared per sample to be
tested. 20 cysts of G. pallida and G. rostochiensis are put
respectively in two sieves as positive controls. All samples
and controls are left at room temperature in the dark until
they are checked for hatching. Each sample and the controls are checked for hatching every 10 days. If juveniles
have hatched, the test is considered positive and the result
is that the cysts are viable. New PRD is added every
10 days if necessary.
If no hatching occurs after 30 days, the cysts are crushed
and the viability of juveniles is assessed (Appendix 4). If
viable juveniles are detected, the result of the test is positive,
otherwise it is negative. If positive controls do not hatch, the
viability tests are not considered valid.
Appendix 6 Viability test by trehalose

Based on van den Elsen et al., 2012.
Trehalose, a disaccharide, is present in high concentrations in the perivitelline fluid of eggs of cysts and can be
used as viability marker. Pre-soaked cysts (12 days) are
boiled to set free the trehalose that was present in the live
eggs within the cysts. The boiling of the cysts breaks
down the membrane and releases the trehalose into the
solution. The amount of trehalose can be measured directly
using a simple detection kit; the disaccharide is hydrolysed
into two glucose molecules and subsequently the glucose
can be detected. Cutting the cysts before or after boiling
facilitates the release of the trehalose into the solution.
The latter is necessary when the measurement needs to be
quantitative or when few living content is expected. When
species identification is to be performed after the viability
test, lysis buffer should be added after the trehalose measurement. Extraction of DNA of this solution can take
place and a subsequent PCR test can be performed (see
Appendix 3).
C) Hatching test (methods performed in France)
This test is shorter and follows the procedure described in

test B with the following modifications:

Nematoda 1

Загружено:

Сведения о документе

Оригинальное название

Авторское право

Доступные форматы

Поделиться этим документом

Поделиться или встроить документ

Параметры публикации

Этот документ был вам полезен?

Это неприемлемый материал?

Авторское право:

Доступные форматы

Nematoda 1

Загружено:

Авторское право:

Доступные форматы

Bulletin OEPP/EPPO Bulletin (2013) 43 (1), 119138

ISSN 0250-8052. DOI: 10.1111/epp.12025

European and Mediterranean Plant Protection Organization

PM 7/40 (3) Globodera rostochiensis and Globodera pallida

Specific approval and amendment

This standard describes a diagnostic protocol for Globodera

Approved as an EPPO Standard in 200309. Revision

Globodera rostochiensis and Globodera pallida (potato cyst

Name: Globodera rostochiensis (Wollenweber, 1923),

2013 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 43, 119138

Name: Globodera pallida (Stone, 1973)

Above-ground symptoms due to potato cyst nematodes are

Developments combining a classification based on morphological data

Statutory sampling procedures

Recommendations on sampling can be found in the Council

Fig. 1 Potato roots infected by G. rostochiensis (Courtesy: Plant

There are various processes for extracting cysts from the

Another procedure to detect the nematodes is the bioassay

Fig. 2 Broken cyst with eggs of G. pallida (Courtesy: Plant Protection

for a visual check for the presence of cysts and young

For identification based on morphology, second-stage

2013 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 43, 119138

Globodera rostochiensis and Globodera pallida

Globodera sp. identified

Mokecular test for

Morphological identification based

Fig. 3 Flow-diagram for identification of Globodera rostochiensis and G. pallida.

diagram indicating combinations of methods is given in

Identification of cyst and juveniles to genus level

Tests for identification in soil extracts or floats based on molecular

2013 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 43, 119138

be used based on form of the cysts and the characteristics

Fig. 4 The perineal region of a Globodera cyst (After Fleming and

Globodera juveniles should meet the following characteristics:

Fig. 5 Difference between Meloidogyne and Globodera juveniles.

2013 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 43, 119138

Globodera rostochiensis and Globodera pallida

2013 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 43, 119138

complex (G. tabacum tabacum (Lownsbery & Lownsbery,

set of three monoclonal antibodies. There were, however,

et al., 1996; Shields et al., 1996; Thiery & Mugniery,1996;

2013 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 43, 119138

Shape of stylet knob

Globodera rostochiensis and Globodera pallida

2013 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 43, 119138

The term pathotype is used by the International PCN

virulence (i.e., overcoming resistance currently available in

Reporting and documentation

Testing the viability of eggs and juveniles

Testing of the viability of the eggs and juveniles may be

Feedback on this Diagnostic Protocol

2013 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 43, 119138

Globodera rostochiensis and Globodera pallida

2013 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 43, 119138

of potato cyst nematodes from field soil samples in England and

Subbotin SA, Mundo-Ocampo M & Baldwin GB(2010) Systematics of

Appendix 1 Methods for extracting cysts

of detergent will cause the cysts to move to the edge and

Flask and paper-strip methods

Fig. 8 Vertical-section diagram of Fenwick can.

2013 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 43, 119138

Globodera rostochiensis and Globodera pallida

tube of the same diameter leading down to an outlet.