Blackwell Publishing Ltd
European and Mediterranean Plant Protection Organization
Organisation Européenne et Méditerranéenne pour la Protection des Plantes
PM 7/87 (1)
Diagnostics
Diagnostic
Ditylenchus destructor and Ditylenchus dipsaci
Specific scope
Specific approval and amendment
This standard describes a diagnostic protocol for Ditylenchus
destructor and Ditylenchus dipsaci1.
Approved in 2008-09.
Introduction
Identity
Among the more than 80 species presently recognized in the
genus Ditylenchus Filipjev, 1936, only a few are parasites of
higher plants, while the majority of species is mycophagous.
Ditylenchus destructor is one of the few plant-parasites within
the genus Ditylenchus Filipjev, 1936. The species is recorded
from all continents, mainly from temperate regions. The known
host range comprises more than 100 species of plants from a
wide variety of families. Economically important crops are
potato Solanum tuberosum, Iris spp., Tulipa spp., Dahlia spp.,
Gladiolus spp. Rheum rhabarbarum, Trifolium spp. and
Daucus carota. Some weeds (e.g. Cirsium arvense, Mentha
arvensis, Potentilla anserine, Rumex acetosella and Stachys
palustris (Andersson, 1971)) and sugar beet (Beta vulgaris) can
also be hosts and can act as sources of infection to crop plants.
D. destructor is also capable of reproducing on the mycelium
of many fungi. The nematode attacks subterranean parts of
plants (tubers, stolons, bulbs, rhizomes, roots), but may
occasionally also invade above-ground parts, mainly the base of
stem). D. destructor is unable to withstand excessive desiccation
unlike D. dipsaci.
D. dipsaci is among the plant-parasitic nematodes of greatest
economic impact worldwide and widely distributed mainly in
temperate areas. Almost 500 different plant species are known
as hosts for D. dipsaci but the different biological races of this
nematode each have limited host-ranges. D. dipsaci lives
mostly as an endoparasite in aerial parts of plants (stems,
leaves, flowers), but also attacks bulbs, tubers and rhizomes.
D. dipsaci readily withstands desiccation and can be isolated
even from completely dry plant material after moistening
(resistant stage = fourth-stage juveniles).
Name: Ditylenchus destructor Thorne, 1945
Synonyms: About 30 synonyms but none used in recent years
(Sturhan & Brzeski, 1991)
Taxonomic position: Nematoda: Tylenchida2: Anguinidae
EPPO computer code: DITYDE
Phytosanitary categorization: EU Annex designation: II/A2.
Name: Ditylenchus dipsaci (Kühn, 1857) Filipjev, 1936
Synonyms: About 30 synonyms but none used in recent years
(Sturhan & Brzeski, 1991)
Taxonomic position: Nematoda: Tylenchida2: Anguinidae
EPPO computer code: DITYDI
Phytosanitary categorization: Ditylenchus dipsaci: EPPO A2
list no. 174, EU Annex designation: II/A2.
Detection
Symptoms caused by D. destructor :
Common symptoms of infestation of D. destructor are
discoloration and rotting of the plant tissue.
Potatoes
The first symptoms are white spots under the skin. Badly
affected tubers have slightly sunken areas with cracked and
papery skin (Figs 1 and 2); the tissue below the skin is darkened
and may have a mealy or spongy appearance. Symptoms may
be more visible after storage. There is in general a secondary
invasion of fungi, bacteria and free-living nematodes.
2Recent
1Use
of names of chemicals or equipment in these EPPO Standards implies
no approval of them to the exclusion of others that may also be suitable.
© 2008 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 38, 363–373
development combining a classification based on morphological
data and molecular analysis refer to ‘Tylenchomorpha’ (De Ley & Blaxter,
2004).
363
364
Diagnostics
Fig. 1 Ditylenchus destructor damage on potatoes (pictures Crown
copyright; reproduced courtesy of the Central Science Laboratory, York, GB).
Fig. 3 Carrots showing transverse cracks upon infection by Ditylenchus
destructor.
Fig. 2 Ditylenchus destructor damage on potatoes (LNPV Nematology,
Rennes, FR).
Flower bulbs and corms
Infestations usually begin at the base of the bulb and extend up
to the fleshy scales with yellow to dark brown lesions. Secondary
rotting may occur and the bulbs can be destroyed. Specimens of
D. destructor are accumulated on the boundary between
distinctly diseased parts and healthy sections, but are rarely
isolated from completely decayed tissues.
Fig. 4 Carrot disc with white sub-cortical patch caused by Ditylenchus
destructor.
Carrots
Damage to carrots appears as transverse cracks in the skin with
white patches in the sub-cortical tissue (Fig. 3). The patches are
easily seen in a transverse cut (Fig. 4). Infested areas are subject
to secondary infections by fungi and bacteria resulting in decay
and rot.
Symptoms caused by D. dipsaci
Common symptoms of infestation are swelling, distortion,
discolouration and stunting of above-ground plant parts
(Fig. 5), necrosis and rotting of bulbs and tubers.
Fig. 5 Ditylenchus dipsaci damage on bean plants (pictures Crown
copyright; reproduced courtesy of the Central Science Laboratory York, GB).
© 2008 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 38, 363–373
Ditylenchus destructor and Ditylenchus dipsaci
365
Fig. 8 Ditylenchus dipsaci damage on onions.
Fig. 6 Ditylenchus dipsaci symptoms on Vicia faba seeds (Plant Protection
Service, NL).
Fig. 9 Ditylenchus dipsaci damage on potatoes.
Fig. 7 Ditylenchus dipsaci damage on Phaseolus vulgaris seeds.
Seeds
Small seeds generally show no visible symptoms of infestation,
but in larger seeds (e.g. Phaseolus vulgaris and Vicia faba)
the skin may be shrunken and show discoloured spots
(Figs 6 and 7).
Bulbs, tubers (Figs 8 and 9)
Infested tissue is generally necrotic and bulbs show browning
of the scales in concentric circles (seen in transverse sections,
see Fig. 10); entire bulbs often become soft and, occasionally in
the case of garlic, a strong smell may develop.
Carrots and sugar beet
On carrots early symptoms of D. dipsaci attacks are straddled
leaves, multi-bud plant crowns and light discolorations of taproot tops. The portion of the plant most affected by D. dipsaci
is that 2–4 cm below and above ground. Severe symptoms of
attacks by the nematode are leaf death and tap-root rot (Greco
© 2008 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 38, 363–373
Fig. 10 symptoms of D. dipsaci on Narcissus sp. (LNPV Nematology,
Rennes, FR).
et al., 2002). These symptoms are similar to those described on
sugar beet roots (Dunning, 1957) see Fig. 11.
Extraction of the nematodes
Extraction from plant tissue
D. destructor and D. dipsaci can be detected by placing plant
tissue with suspected infestation into water. Any plant material
366
Diagnostics
Fig. 11 Ditylenchus dipsaci symptoms on Beta
vulgaris.
to be tested is cut into pieces or sliced and placed on a
Baermann funnel on a sieve covered with soft filter paper (e.g.
cotton wool filter). These nematode species are very mobile and
will usually emerge from the tissues within 2 to 4 h; the water
from the bottom of the funnel can then checked by microscope
for the presence of nematodes. Leaving the nematodes much
longer in water will result in an increase in mortality. The use
of mistifier extraction can provide active nematodes for a longer
period. In tissues showing early symptoms of attack the
nematodes can be detected by tissue dissection under a
dissection microscope or by enzymatic destruction of the
tissues, but this latter method is rather costly.
Extraction from soil
D. dipsaci and D. destructor can be extracted from soil by a
suitable extraction method for nematodes of this size (see EPPO
Standard PM 7/41: Diagnostic Protocol for Meloidogyne
chitwoodi and Meloidogyne fallax Appendix 1). Nevertheless
it should be noted that these nematodes are rarely found in soil
unless the latter has been associated with an infested host.
Extraction of D. dipsaci from seeds
For extraction of D. dipsaci, seed samples are placed on a
Baermann funnel (preferably on a sieve covered with soft filter
paper) and covered by a small amount of water. The water is
released from the bottom of the funnel after a period of 12 h to
two days (or longer if the infestation is very low), and checked
for the presence of nematodes using a microscope. Large seeds
can be broken in a blender so that extraction time is reduced.
Larger seed samples are best soaked in an abundance of water,
which is poured off after one day (or longer) into a cylinder. The
bottom layer is then examined for nematodes by microscope
after sufficient time has been allowed for sedimentation to
happen. It may be necessary to pour the water through 3 sieves
of 50 μm or one sieve of 20 μm pore size.
Recovery and identification of D. dipsaci from seed samples
does not generally cause problems. When many nematode
specimens of similar appearance are found (exclusively
fourth-stage juveniles) the species is most probably D. dipsaci.
Nevertheless identification should be confirmed by identification
methods described below.
Identification
The identification of D. destructor and D. dipsaci should
always be based on morphological methods first. Molecular
methods have been developed for the identification of D.
destructor and D. dipsaci and these methods may be used when
there is a low level of infestation or when only juvenile stages
are present.
Morphological methods
For identification, individual nematodes or the entire nematode
suspensions are heated (to approximately 60°C) until the
nematodes become immobile.
The body of D. destructor killed by gentle heat is almost
straight (never C-shaped, spiral or with posterior end markedly
bent to the ventral side). Generally all developmental stages
(females, males, 3 juvenile stages, eggs) can be isolated from
infested plant tissues. Contamination of the extract by freeliving mycophagous and bacteriophagous nematodes is
common, in particular in decaying plant material (e.g. potato
tubers).
The body of D. dipsaci killed by gentle heat is straight or
almost so (never C-shaped, spiral or posterior end markedly
bent to the ventral side). In seed samples, nematodes other than
D. dipsaci will rarely be found but contamination by other
nematodes may occur particularly with samples containing
other plant debris. From seeds, generally only fourth-stage
© 2008 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 38, 363–373
Ditylenchus destructor and Ditylenchus dipsaci
367
Table 1 Key to distinguish Ditylenchus spp. from other tylenchid genera (modified from Brzeski, 1998)
1
2
3
4
5
Females mobile
Females swollen, globose or lemon-shaped
Female gonads prodelphic and outstretched
Female gonads paired or reflex when prodelphic
Pharyngeal glands offset from intestine or slightly overlapping it
Pharyngeal glands distinctly overlapping intestine
Metacorpus offset from procorpus, metacorporeal plates short or absent
Procorpus gradually expands into large metacorpus, metacorporeal plates long
Sperm large, head usually low, spermatheca not off-set.
Sperm small, head usually high, spermatheca off-set in most genera
juveniles of D. dipsaci can be isolated (no adults or early
juvenile stages) and the specimens have a similar appearance.
In bulbs, tubers, rhizomes etc. a variety of nematodes may be
present, which invaded the plant tissues from the soil. In rotting
tissue bacteriophagous or mycophagous nematodes are often
dominant. D. dipsaci is mainly found in plant tissues which are
still viable. All stages of D. dipsaci can be isolated from bulbs
etc. (females, males, 3 juvenile stages, eggs), but fourth-stage
juveniles often prevail.
Microscopical examination has to concentrate on
stylet-bearing nematodes (stylet delicate) with slender and
(almost) straight body and a conical pointed tail. To distinguish
Ditylenchus spp. from other tylenchid genera see Table 1.
For precise identification, examination of the specific
morphological characters at high microscopical magnification
is necessary.
In non-related nematode genera the tail ranges from filiform
to dome-shaped, the body of heat-relaxed specimens from
straight to C-shaped and spiral with the posterior end often ventrally curved, a stylet may be absent, the pharynx distinctly offset from intestine, the vulva located at mid-body, the male tail
lacks a bursa, etc. Specimens of Aphelenchoides spp. are mostly
easily distinguished by their well-demarcated rounded median
pharyngeal bulb, long overlap of the pharynx over the intestine,
presence of a mucro on the tail tip and lack of a bursa on male tail.
2
Other genera
3
Other genera
4
Other genera
5
Other genera
Ditylenchus
Other genera
Discriminating morphological characteristics of D. destructor,
D. dipsaci, D. convallariae and D. myceliophagus are presented
in Table 2; see also Figs 12 and 13.
D. dipsaci and D. detructor have a stylet with a cone of the
length of about 50% of total stylet length, whereas most other
fungivorus species have stylets with cones shorter than 50% of
the total stylet length (mainly about 1/3 of its length).
In most species similar to D. dipsaci, the lateral fields have
four incisures, the basal pharyngeal bulb is distinctly off-set
from the intestine and the tail terminus is sharply pointed. A list
of Ditylenchus species with specific characters has been
published by Brzeski (1991) and Sturhan & Brzeski (1991).
Molecular methods
Several molecular techniques have been developed and are in
use now for diagnostics of Ditylenchus species and are
described in Appendices:
A PCR-RFLP of the ITS-rRNA is described in Appendix 1
(this method is the only one that allows the identification of
both D. destructor and D. dispaci).
PCR with species specific primers have been developed for
diagnostics of normal and giant races of Ditylenchus dipsaci.
The use of PCR with specific primers constitutes a major
development in DNA diagnostics and enables the detection of
Table 2 Discriminating morphological characteristics of D. destructor, D. dipsaci, D. convallariae and D. myceliophagus (after Decker, 1969)
A ratio Body length (mm)
Stylet length (μm)
Posterior bulb
Number of lateral lines
Vulva position (%)
Post-vulval sac length
Vulva-anus length
Tail shape
Tail tip
Spiculum length (μm)
Length of cone/total
stylet length
D. destructor
D. dipsaci
D. convallariae
D. myceliophagus
32 (18–41)
1.0 (0.8–1.4)
10–12
short, dorsally overlapping
6
80 (78–83)
2/3–3/4 of vulva-anus distance
1 3/4–2 1/3 tail length
conoid, usually slightly bent to
ventral side in posterior part
finely rounded
9–12
About 50%
37 (36–40)
1.1 (0.9–1.3)*
11–13
not overlapping
4
82 (79 –82)
1/2 of vulva-anus distance
1 3/4–2 1/4 tail length
conoid
42 (32–54)
1.1 (0.9–1.3)
11–13
not overlapping
6
77 (74–79)
1/4–1/2 of vulva-anus distance
2–2 1/4 tail length
conoid
30 (23–44)
0.9 (0.6–1.0)
7–10
short, dorsally overlapping
6
82.5 (74–90)
2–2 1/4 vulva-anus distance
2–2 1/4 tail length
broadly conoid
sharply pointed
10–12
About 50%
sharply pointed
8–11
<< 50%
finely rounded
9
<< 50%
*The ‘giant’ race of D. dipsaci in Faba bean can be up to 2.0 mm long.
© 2008 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 38, 363–373
368
Diagnostics
Fig. 12 Ditylenchus destructor (A) Female, pharyngeal region. (B) Head of
female. (C) Male, spicule region. (D) End of female tail. (E) Posterior
portion of female. (F) Lateral field at midbody. Each unit on bars = 10 μm
(After Sturhan & Brzeski, 1991).
the stem nematode in a mixture with other soil-inhabiting
organisms by a single PCR test, decreasing diagnostic time and
costs compared with PCR-RFLP. Esquibet et al. (2003) developed a multiplex PCR with SCAR (sequence characterized
amplified region) primers for identification of the giant and the
normal races of D. dipsaci (Table 4 in Appendix 2, Fig. 10)
(Appendix 2). Subbotin et al. (2005) described specific primers
and PCR protocol for detection of D. dipsaci sensu stricto
(Appendix 3). Several other specific primers have been proposed
for diagnostics of D. dipsaci sensu lato (Marek et al., 2005
(Appendix 4); Kerkoud et al., 2007 (Appendix 5); Zouhar
et al., 2007).
Reference material
Julius Kühn Institute (JKI), Federal Research Centre for
Cultivated Plants, Institute for Epidemiology and
Pathogen Diagnostics, Toppheideweg 88, 48161 Münster
(DE).
Pest and Disease Identification Team, Central Science
Laboratory, Sand Hutton, York YO41 1LZ (GB).
Plant Protection Service Servizio fitosanitario regionale Via
di Saliceto, n.81, 40128 Bologna (IT).
Plant protection Service, P.O. Box 9102, 6700 HC Wageningen
(NL).
Department of Plant Protection Biology – Nematology,
Swedish University of Agricultural Sciences, Alnarp (SE).
Fig. 13 Ditylenchus dipsaci. (A) Female, pharyngeal region. (B) Head of
female. (C) Male, spicule region. (D) Posterior portion of female. (E) Lateral
field at midbody. Each unit on bars = 10 μm (After Sturhan & Brzeski, 1991).
Reporting and documentation
Guidance on reporting and documentation is given in EPPO
Standard PM 7/77 (1) Documentation and reporting on a
diagnosis.
Further information
Further information on this organism can be obtained from:
Julius Kühn-Institute, Federal Research Centre for Cultivated
Plants, Institute for Epidemiology and Pathogen Diagnostics,
Toppheideweg 88, 48161 Münster (DE); Plant Protection
Service, Diagnostic Centre, P.O. Box 9102, 6700 HC
Wageningen (NL); Istituto per la Protezione delle Piante,
Sezione di Bari, C.N.R., Via G. Amendola, 165/A, 70126 Bari
(IT); Pest and Disease Identification Team, Central Science
Laboratory, Sand Hutton, York YO41 1LZ (GB); Department of
Plant Protection Biology – Nematology, Swedish University of
Agricultural Sciences, Alnarp (SE).
Acknowledgements
This protocol was originally drafted by: D. Sturhan, retired
from formerly Federal Biological Research Centre for
© 2008 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 38, 363–373
Ditylenchus destructor and Ditylenchus dipsaci
Agriculture and Forestry, now Julius Kühn Institute (DE). The
molecular part has been added by S Subbotin, Plant Pest
Diagnostic Center, California Department of Food and
Agriculture, 3294 Meadowview road, Sacramento, CA 95832
(USA) and G Anthoine LNPV-Unité de Nematologie, Domaine
de la Motte au Viconte BP 35327 Le Rheu (FR).
369
Wendt KR, Vrain TC & Webster JM (1993). Separation of three species of
Ditylenchus and some host races of D. dipsaci by restriction fragment
length polymorphism. Journal of Nematology 25, 555–563.
Zouhar M, Marek M, Douda O, Mazáková J & Ryjánek P (2007).
Conversion of sequence-characterized amplified region (SCAR) bands
into high-throughput DNA markers based on RAPD technique for
detection of the stem nematode Ditylenchus dipsaci in crucial plant hosts.
Plant Soil and Environment 53, 97–104.
References
Andersson S (1971) [The potato rot nematode, Ditylenchus destructor
Thorne, as parasite in potatoes] In Swedish with English summary.
Dissertation. Agricultural College of Sweden, Uppsala (SE).
Brzeski MW (1991) Review of the genus Ditylenchus Filipjev, 1936
(Nematoda: Anguinidae). Revue de Nematologie 14, 9–59.
Brzeski MW (1998) Nematodes of Tylenchina in Poland and temperate
Europe. Muzeum i Instytut Zoologii Polska Akademia Nauk, Warsaw (PL).
De Ley P & Blaxter M (2004). A new system for Nematoda: combining
morphological characters with molecular trees, and translating clades into
ranks and taxa. In: Nematology Monographs and Perspectives. (Ed. Cook
R & Hunt DJ), pp. 633–653. E.J. Brill, Leiden (NL).
Decker H (1969) Phytonematologie. VEB Deutscher Landwirtschaftsverlag,
Berlin (DE).
Dunning RA (1957). Stem eelworm invasion of seedling sugar beet and
development of crown canker. Nematologica 2 (Suppl.), 362–8.
Esquibet M, Bekal S, Castagnone-Sereno P, Gauthier JP, Rivoal R & Caubel
G (1998). Differentiation of normal and giant Vicia faba populations of
the stem nematode Ditylenchus dipsaci: agreement between RAPD and
phenotypic characteristics. Heredity 81, 291–298.
Esquibet M, Grenier E, Plantard O, Andaloussi A & Caubel G (2003) DNA
polymorphism in the stem nematode Ditylenchus dipsaci: Development
of diagnostic markers for normal and giant races. Genome 46, 1077–
1083.
Greco N, Brandonisio A & Boncoraglio P. (2002). Investigations on
Ditylenchus dipsaci damaging carrot in Italy. Fourth International
Congress of Nematology, 2002-06-8/13, Tenerife, Canary Islands, Spain.
Nematology 4, 210 (abstract).
Kerkoud M, Esquibet M, Plantard O, Avrillon M, Guimier C, Franck M
et al. (2007). Identification of Ditylenchus species associated with
Fabaceae seeds based on a specific polymerase chain reaction of
ribosomal DNA-ITS regions. European Journal of Plant Pathology 118,
323–332.
Marek M, Zouhar M, Rysanek P & Havranek P (2005). Analysis of ITS
sequences of nuclear rDNA and development of a PCR-based assay for
the rapid identification of the stem nematode Ditylenchus dipsaci
(Nematoda: Anguinidae) in plant tissues. Helminthologia 42, 49–56.
Sambrook J, Fritsch EF & Maniatis T. (1989) Molecular Cloning: A
Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, New York (US).
Sturhan D & Brzeski MW (1991) Stem and bulb nematodes, Ditylenchus
spp. In: Manual of Agricultural Nematology (Ed. Nickle WR), pp.
423–464. Marcel Dekker, Inc., New York (US).
Subbotin SA, Madani M, Krall E, Sturhan D & Moens M. (2005). Molecular
diagnosis, taxonomy, and phylogeny of the stem nematode Ditylenchus
dipsaci species complex based on the sequences of the internal transcribed
spacer-rDNA. Phytopathology 95, 1308–1315.
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, 563–
573.
Webster JM, Anderson RV, Baillie DL, Beckenbach K, Curran J &
Rutherford T (1990). DNA probes for differentiating isolates of the
pinewood nematode species complex. Revue de Nématologie 13, 255–
263.
© 2008 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 38, 363–373
Appendix 1 ITS-RFLP analysis according to
Wendt et al. (1993)
This protocol is a quite old and some of the reagents and
equipments may have changed. It is strongly recommended to
validate this protocol in the operator’s laboratory conditions.
1. General information
1.1 Protocol developed by Wendt et al. (1993)
1.2 Individuals from D. dipsaci (nomal races and giant race),
D. destructor and D. myceliophagous are extracted from plant
material
1.3 The targeted region is the ITS region (including ITS1,
ITS2 and 5.8S rDNA gene). They are not specific regions
1.4 Oligonucleotides: ITS–specific universal primers
described by Vrain et al. (1992): 18S (5′-TTG ATT ACG TCC
CTG CCC TTT-3′) and 26S (5′-GGA ATC ATT GCC GCT
CAC TTT-3′). The amplicon is approximately 900 bp for D.
dipsaci and D. myceliophagus, and 1200 bp for D. destructor
1.5 Amplification mix is provided in a kit containing Taq
DNA Polymerase, nucleotides and reaction buffer (Perkin Elmer)
1.6 Amplification is performed in a thermocycler, e.g. Twin
Block System EC Cycler (Ericomp).
2. Methods
2.1 Nucleic acid Extraction and Purification
2.1.1 Nematodes that had migrated to the lid of a Petri dish
were rinsed off with 0.05 M NaCl, and concentrated by centrifugation at 2000 rpm for 2 min at room temperature. The NaCl
solution was discarded and the nematodes were resuspended in
seven volumes of Proteinase K buffer (0.1 M Tris pH = 8.0,
0.05 M EDTA, 0.2 M NaCl and 1% SDS) containing
1.0 mg mL−1 proteinase K. The solution containing the nematodes was frozen in liquid nitrogen, transferred to a mortar and
ground into a fine powder. After thawing the solution was transferred to a 50 mL Falcon tube and was extracted three times
with TE (10 mM Tris pH = 8.0 and 1.0 mM EDTA) saturated
phenol pH 8.0 and twice with 24:1 chloroform, iso-amyl
alcohol. The clean DNA was precipitated by two volumes of
95% ethanol, pelleted, dried and redissolved in TE with
10 μg mL−1 RNAse A (Webster et al., 1990)
2.1.2 Store overnight at 4°C or at −20°C for longer periods
2.2 Polymerase Chain reaction
2.2.1 Master mix (provided within kit), 1.5 μM of each Vrain
primers and 20 μg of DNA
370
Diagnostics
Table 3 Restriction patterns of D. dipsaci (host races and giant race), D. destructor and D. myceliophagus with different restriction enzymes (−: data not
available) (length of restriction bands in bp)
Unrestricted PCR product
AccI
AluI
BamHI
DdeI
DraI
HaeIII
HincII
HinfI
HpaII
NsiI
PstI
RsaI
Sau3A
TaqI
D. dipsaci (normal races*)
D. dipsaci (giant race)
D. destructor
D. myceliophagus
900
900
900
340, 220, 180
310, 290, 200
340, 250
900
800
440, 350, 150
320, 200, 180
900
650, 400
450, 250, 140
340, 260, 200, 110, 100
340, 230, 130
900
−
−
−
310, 290, 200
−
800, 200
800
350, 150
600, 200
−
650, 400
490, 450
−
−
1200
1200
370, 290
1000
670, 570
1200
450, 170
900, 250
780, 180
1000
1200
850, 400
600, 250, 170
540, 400, 180
640, 200, 150
900
900
900
900
300, 250, 130
900
450, 200
900
630, 310
900
900
620, 400
900
440, 100
320, 260, 160
*Referred to as «host race» in Wendt et al. (1993).
2.2.2 PCR cycling parameters
1 cycle of 1.5 min 94°C, 30 s at 50°C, 4 min at 72°C; 40
cycles of 45 s at 96°C, 30 s at 50°C, 4 min at 72°C; 1
cycle of 45 s at 96°C, 30 s at 50°C, and a final extension
of 10 min at 72°C
2.3 Restriction of PCR amplicon
2.3.1 Restriction conditions
According to supplier’s conditions (Gibco Co., BRL).
3. Essential Procedural Information
3.1 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)
3.2 Identification of species. Restriction patterns for different
species are presented in Table 3.
3.3 A negative control (no DNA target) should be included in
every experiment to test for contamination as well as a positive
control (DNA from a reference strain of the pathogen)
3.4 The method can only be used on nematodes morphologically
identified as Ditylenchus sp., as the primers are not specific for
Ditylenchus spp.
Appendix 2 Specific SCAR-PCR according to
Esquibet et al. (2003)
1. General information
1.1 Protocol developed by Esquibet et al. (2003)
1.2 Individuals from D. dipsaci (host races and giant race)
and D. myceliophagous are extracted from plant material
1.3 The targeted regions are unknown as the primers were
Sequence Characterized Amplified Region (SCAR)
1.4 Oligonucleotides: for D. dipsaci (host races) H05 (5′TCA AGG TAA TCT TTT TCC CCA CT-3′) and H06 (5′-CAA
CTG CTA ATG CGT GCT CT-3′); for D. dipsaci (giant race)
D09 (5′-CAA AGT GTT TGA TCG ACT GGA-3′) and D10
(5′-CAT CCC AAA ACA AAG AAA GG-3′). The amplicon is
approximately 242 bp for D. dipsaci (host race) and 198 bp for
D. dipsaci (giant race)
1.5 Taq DNA Polymerase 5 U μL−1 (Promega) is used for
PCR amplification
1.6 Nucleotides are used at a final concentration of 0.25 mM
each
1.7 MgCl2 final concentration is 1.5 mM
1.8 Molecular grade water (MGW) is used to make op
reaction mixes; this should be purified (deionised or
distilled), sterile (autoclaved or 0.45 μm filtered) and nuclease
free.
2. Methods
2.1 Nucleic acid Extraction and Purification (Esquibet et al.,
1998)
2.1.1 DNA was extracted from 2500–25 000 frozen mixed
larvae and adults prepared using a phenol-chloroform
procedure: the nematodes were rinsed in a microcentrifuge and
ground in a pestle. Total genomic DNA was extracted according
to the phenol-chloroforme procedure (Sambrook et al., 1989).
Following ethanol precipitation, DNA was resuspended in TE
buffer [0.01 M Tris (pH 8.0), 0.001 M EDTA]
2.1.2 Store overnight at 4°C or at −20°C for longer periods
2.2 Polymerase Chain reaction
2.2.1 Master mix include:
1.5 mM MgCl2
250 μM each dNTP
690 nM each primer for duplex PCR (H05-H06) or
(D09-D10)
© 2008 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 38, 363–373
Ditylenchus destructor and Ditylenchus dipsaci
371
Fig. 14 The amplification of two products
(198 bp and 242 bp) in multiplex PCR using
SCAR primers D09, D10, H05, and H06 for 8
giant and 10 normal populations of D. dipsaci
(after Esquibet et al., 2003).
or 500 nM each primer for multiplex PCR (H05-H06D09-D10)
0.5 U Taq DNA polymerase (Promega)
2.2.2 PCR cycling parameters:
30 cycles of 1 min at 94°C, 1 min at 59°C, 1 min at
72°C.
1.5 Taq DNA Polymerase 5 U μL−1 (Core kit,Qiagen) used
for PCR amplification
1.6 Buffer: provided within the Core kit (Qiagen) at 10X
concentration, when used diluted to 1X
1.7 Molecular grade water (MGW) is used to make up
reaction mixes; this should be purified (deionised or distilled),
sterile (autoclaved or 0.45 μm filtered) and nuclease free.
3. Essential Procedural Information
3.1 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)
3.2 Identification of species (Table 4 and Fig. 14)
3.3 A negative control (no DNA target) should be included in
every experiment to test for contamination as well as a positive
control (DNA from a reference strain of the pathogen).
Table 4 SCAR-PCR patterns of D. dipsaci, D. desructor and
D. myceliophagus (Esquibet et al., 2003)
H05-H06
D09-D10
D. dipsaci
(host races)
D. dipsaci
(giant race)
D.
myceliophagus
242 bp
−
−
198 bp
−
−
Appendix 3 Specific PCR according to
Subbotin et al. (2005)
1. General information
1.1 Protocol developed by Subbotin et al. (2005)
1.2 Juveniles and adults from D. dipsaci (sensu stricto), D.
destructor and Ditylenchus sp. are extracted from plant material
1.3 The targeted region is 18S and ITS1 region
1.4 Oligonucleotides: ITS–specific universal primers
described by Vrain et al. (1992) rDNA2 (5′-TTT CAC TCG
CCG TTA CTA AGG-3′) and DitNF1 specific primer (5′-TTA
TGA CAA ATT CAT GGC GG-3′). The amplicon is
approximately 263 bp for D. dipsaci sensu stricto (giant race
not included)
© 2008 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 38, 363–373
2. Methods
2.1 Nucleic acid Extraction and Purification
2.1.1 For each sample, several dozen juveniles or a few adults
were transferred into 8 μL of double distilled water and 7 μL of
worm lysis buffer in an Eppendorf tube and crushed with a glass
stem using a microhomogenizor Vibro Mixer (Zürich,
Switzerland). Three microliters of proteinase K (600 μg mL−1)
(Promega Benelux, Leiden, The Netherlands) was added. The
tubes were incubated at 65°C (1 h) and 95°C (10 min)
consecutively. After centrifugation (1 min; 16 000 g), 3 μL of
the resulting DNA suspension was added to the PCR mix
2.2 Polymerase Chain reaction
2.2.1 Master mix include:
10.8 μL MGW
2.5 μL of 10X PCR buffer (Qiagen)
5 μL of Q solution (Qiagen)
0.5 μL of dNTPs mixture (no indication of initial-final
concentration)
1.5 μL each primer (1.0 μM)
0.2 μL Taq DNA polymerase 5 U μL−1 (Promega)
3 μL of DNA suspension
2.2.2 PCR cycling parameters:
4 min at 94°C; 35 cycles of 15 s at 94°C, 30 s at 57°C,
30 s at 72°C; and a final extension of 10 min at 72°C.
3. Essential Procedural Information
3.1 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)
3.2 Identification of species (Table 5)
3.3 A negative control (no DNA target) should be included in
every experiment to test for contamination as well as a positive
control (DNA from a reference strain of the pathogen).
372
Diagnostics
Table 5 PCR patterns of D. dipsaci, D. desructor and D. myceliophagus
(Subbotin et al., 2005)
D. dipsaci
D. dipsaci D.
Ditylenchus
(sensu stricto) (giant race) myceliophagus sp.
DitNF1 – 18S 242 bp
−
−
−
Appendix 4 Specific PCR according to Marek
et al. (2005)
2.2 Polymerase Chain reaction
2.2.1 Master mix total volume of 25 μL including:
1.5 mM MgCl2
200 μM each dNTP
10 pmol each primer
1.5 U Taq DNA polymerase 5 U μL−1 (Fermentas)
MGW up to 25 μL
2.2.2 PCR cycling parameters:
3 min at 94°C; 30 cycles of 2 min at 94°C, 30 s at 62°C
(PF1-PR1) or 63°C (PF2-PR2), 2 min at 72°C.
3. Essential Procedural Information
1. General information
1.1 Protocol developed by Marek et al. (2005)
1.2 DNA is extracted either from isolated individuals or
contaminated plant materials
1.3 The targeted regions are 5.8S rDNA gene and flanking
ITS spacer regions using specific primers for D. dipsaci
1.4 Oligonucleotides: two different primers sets are available
for Ditylenchus dipsaci specific identification. The first set is
composed of PF1 primer (5′-AAC GGC TCT GTT GGC TTC
TAT-3′) and PR1 primer (5′-ATT TAC GAC CCT GAG CCA
GAT-3′). The amplicon with this primer set is approximately
327 bp for D. dipsaci
The second primer set is composed of PF2 primer (5′-TCG
CGA GAA TCA ATG AGT ACC-3′) and PR2 primer (5′-AAT
AGC CAG TCG ATT CCG TCT-3′). The amplicon with this
primer set is approximately 396 bp for D. dipsaci
1.5 Taq DNA Polymerase 5 U μL−1 (Fermentas) used for
PCR amplification
1.6 Molecular grade water (MGW) is used to make up
reaction mixes; this should be purified (deionised or distilled),
sterilized (autoclaved or 0.45 μm filtered) and nuclease free.
3.1 Analysis of DNA fragments: DNA fragments are
separated by electrophoresis on agarose gel (1.2% agarose) and
visualized under UV light according to standard procedures
(e.g. Sambrook et al., 1989).
3.2 Identification of species (Table 6)
Table 6 PCR patterns of D. dipsaci, D. desructor and D. myceliophagus
(Marek et al., 2005)
PF1–PR1
PF2–PR2
D. dipsaci
Other species tested
327 bp
396 bp
No amplification
No amplification
3.3 A negative control (no DNA target) should be included in
every experiment to test for contamination as well as a positive
control (DNA from a reference strain of the pathogen).
Appendix 5 Specific PCR according to
Kerkoud et al. (2007)
1. General information
2. Methods
2.1 Nucleic acid Extraction and Purification
2.1.1 Approximately 10 individuals or 0.5–1.0 ng of plant
tissue artificially inoculated with 10 individuals were crushed in
liquid nitrogen using mortar and pestle and homogenized in
300 μL of lysis buffer (100 mM Tis-HCl (pH 8.0), 5 mM
EDTA, 200 mM NaCl, 0.2% SDS and 0.4 mg mL−1 proteinase
K). The mixture was incubated for 1 h at 37°C with shaking and
finally denaturated 5 min at 85°C. The homogenate was
mixed 1:1 with phenol (pH 8.0)-chloroform-isoamylalcohol
(25:24:1), vortexed for 15 min and centrifuged at 7000 g. Each
lysate (water phase) was transferred to a new tube, an equal
volume of chloroform was added, and the extraction was
repeated. DNA was precipitated with an equal volume of
isopropanol in −20°C overnight or in liquid nitrogen for 20 min
and centrifuged at 10 000 g for 10 min. The supernatant was
removed and the remaining pellets were vacuum-dried. The
pellet for each sample was resuspended in 50 μL TE (10 mM
Tris, 1 mM EDTA (pH 8.0) or ddH2O). DNA was stored at
−20°C
1.1 Protocol developed by Kerkoud et al. (2007)
1.2 Individuals from D. dipsaci, D. destructor, D.
myceliophagus, and D. africanus and Ditylenchus sp. were
extracted from plant material
1.3 The targeted regions are 5.8S rDNA gene and flanking
ITS spacer regions using specific primers for D. dipsaci
1.4 Oligonucleotides: two different primers sets are available,
one for D. dipsaci specific identification and the other one for
Ditylenchus sp. identification
The first set is composed of DdpS1 primer (5′-TGG CTG
CGT TGA AGA GAA CT-3′) and rDNA2 (5′-TTT CAC TCG
CCG TTA CTA AGG-3′) (Vrain et al., 1992). The amplicon
with this primer set is approximately 517 bp for D. dipsaci
The second primer set is composed of DdpS2 primer (5′CGA TCA ACC AAA ACA CTA GGA ATT-3′) and rDNA2
(5′-TTT CAC TCG CCG TTA CTA AGG-3′) (Vrain et al.,
1992). The amplicon with this primer set is approximately
707 bp for Ditylenchus sp.
1.5 Taq DNA Polymerase 5 U μL−1 (MP Biomedicals) used
for PCR amplification
© 2008 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 38, 363–373
Ditylenchus destructor and Ditylenchus dipsaci
373
Table 7 PCR patterns of D. dipsaci, D. desructor, D. africanus, D. myceliophagus and Ditylenchus sp. (Kerkoud et al., 2007)
DdpS1-Rdna2
DdpS2-Rdna2
D. dipsaci
D. destructor
D. africanus
D. myceliophagus
Ditylenchus sp.
517 bp
707 bp
−
707 bp
−
707 bp
−
707 bp
−
707 bp
1.6 Molecular grade water (MGW) is used to make op
reaction mixes; this should be purified (deionised or distilled),
sterilized (autoclaved or 0.45 μm filtered) and nuclease free.
2. Methods
2.1 Nucleic acid Extraction and Purification
2.1.1 Stock genomic DNA was extracted according to the
phenol-chloroform procedure (Sambrook et al., 1989). Following
ethanol precipitation, DNA was resuspended in TE buffer
[(0.01 M Tris (pH 8.0), 0.001 M EDTA] and stored at –20°C
A fast method was developed to obtain DNA from single
individuals. Single nematodes were hand-picked, transferred
onto a glass slide under a dissection microscope and crushed
when dry by gentle pressure with the tip of a micropipette. The
crushed nematode was recovered carefully with 13.7 μL of
sterile distilled water and transferred into a 0.2 mL Eppendorf
tube. The tube was immediately placed in ice. The crushed
suspension was stored at –20°C and used for further study.
2.2 Polymerase Chain reaction
© 2008 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 38, 363–373
2.2.1 Master mix (total volume of 20 μL) including:
1.5 mM of amplification buffer with final MgCl2
concentration = 5 mM
200 μM of each dNTP
0.5 μM of each primer
1U Taq DNA polymerase 5 U μL−1 (MP Biomedicals)
2.2.2 PCR cycling parameters:
1 min at 94°C;40 cycles of 30 s at 94°C, 30 s at 60°C,
45 s at 72°C and a final extension of 2 min 72°C.
3. Essential Procedural Information
3.1 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)
3.2 Identification of species (Table 7)
3.3 A negative control (no DNA target) should be included in
every experiment to test for contamination as well as a positive
control (DNA from a reference strain of the pathogen).