SHORT COMMUNICATION

A Comparative and Phylogenetic Study of the Ditylenchusdipsaci, Ditylenchus destructor and Ditylenchus gigasPopulations Occurring in PolandArnika Jeszke, Marta Budziszewska, Renata Dobosz, Anna Stachowiak, Dorota Protasewicz,Przemyslaw Wieczorek and Aleksandra Obrezpalska-Stezplowska

Interdepartmental Laboratory of Molecular Biology, Institute of Plant Protection – National Research Institute, 20 Władysława Wezgorka St, 60-318Pozna�n, Poland

Keywords

Ditylenchus destructor, Ditylenchus dipsaci,

Ditylenchus gigas, phylogenetic analysis,

Poland, rDNA

Correspondence

A. Obrezpalska-Stezplowska, Interdepartmental

Laboratory of Molecular Biology, Institute of

Plant Protection – National Research Institute,

Pozna�n, Poland.

E-mail: ao.steplowska@iorpib.poznan.pl

Received: June 7, 2013; accepted: July 15,

2013.

doi: 10.1111/jph.12161

Abstract

The genus Ditylenchus contains more than 80 recognized nematode species

with a very wide host range. The most serious species are Ditylenchus dips-

aci and Ditylenchus destructor. Populations of D. dipsaci species complex

were collected from Allium cepa, Cichorium endivia and Phlox paniculata in

Poland. The Ditylenchus gigas population was collected from Vicia faba

minor, and populations of D. destructor, from Solanum tuberosum spp. tubero-

sum. Analyses of the rDNA sequences spanning both ITS1 and ITS2 frag-

ment regions were carried out on the collected populations. The obtained

DNA sequences were compared with those DNA sequences deposited in

GenBank of populations isolated in other countries. Phylogenetic analysis

was performed using the data obtained from the DNA sequence compari-

sons. The results indicated that there is no clear distinction between Euro-

pean and non-European populations within D. dipsaci. The results also

showed no clear distinction between populations isolated from different

host plant species, including populations found in Poland. The popula-

tions of D. destructor described here constitute a common group together

with American and Chinese populations belonging to the haplotype C of

the D. destructor species. On the other hand, the D. gigas population was

localized separately from those populations that have been described up

until now, from Europe and Africa. This is also the first report on the

occurrence of D. gigas in Poland.

Introduction

The genus Ditylenchus Filipjev, 1936, consists of more

than 80 nematode species and contains mostly

mycophagous species, but also plant parasites. The

most economically important are Ditylenchus dipsaci

(K€uhn, 1857) Filipiev, 1936, and Ditylenchus destruc-

tor Thorne, 1945. Ditylenchus destructor infests more

than 100 host plant species, including Solanum

tuberosum, and occurs worldwide, mostly in temper-

ate regions. Ditylenchus dipsaci is also present world-

wide in areas that have a moderate climate and

infects more than 500 species of host plants (Caubel

and Pedron 1976). Ditylenchus dipsaci is characterized

by extensive intraspecific variations. At least 30 host

races (with limited host ranges) of this pest can be

distinguished (Sturhan and Brzeski 1991). These

variations led some (e.g. Sturhan and Brzeski 1991;

Palmer et al. 1992; Subbotin et al. 2005) to consider

the D. dipsaci species as a ‘species complex’. This

complex has been subdivided into two groups. The

first contains diploid populations characterized by

their so-called ‘normal size’ and named ‘D. dipsaci

sensu stricto’. This group comprises most of the

populations recorded so far. The second group of

polyploids is further grouped into the following

clades: Ditylenchus spp. B (the so-called giant race

from Vicia faba) that was recently singled out as the

� 2013 Blackwell Verlag GmbH 1

J Phytopathol

new species Ditylenchus gigas (Vovlas et al. 2011);

Ditylenchus sp. C, which is now described as

Ditylenchus weischeri (Chizhov et al. 2010); and

Ditylenchus sp. D, E and F associated with plant spe-

cies of the Fabaceae, Asteraceae or Plantaginaceae.

Vicia faba is the host in which both types of races,

those belonging to D. dipsaci and to D. gigas, can be

found. The above information is further confirmed

by phylogenetic data (e.g. Subbotin et al. 2005).

Both D. dipsaci and D. destructor significantly

adversely affect the quality and the quantity of

plants. These two nematode species cause swelling,

distortions, stunting as well as necrosis and rotting

of plant parts. For this reason, D. dipsaci and D.

destructor are under quarantine regulations in the

European and Mediterranean Plant Protection

Organisation (EPPO) region. Ditylenchus gigas is a

serious pest of V. faba. Its presence was reported in

several European and African countries bordering

the Mediterranean Sea.

Our aim was to characterize and subsequently to

study the phylogeny of some nematode popula-

tions found in Poland, specifically 11 D. dipsaci

populations from three different hosts, three D. de-

structor populations from S. tubersosum ssp.

tuberosum and one D. gigas population from V. faba

ssp. minor. The sequences were compared with

each other and with other populations of these

species available in the GenBank database.

Phylogenetic analysis showed that in the case of

D. dipsaci, even populations isolated from the same

host may be clustered separately. On the other

hand, most of D. destructor populations isolated

from the S. tuberosum are grouped separately from

those of Ipomoea batatas.

Materials and Methods

Nematode samples

Samples of the Ditylenchus species populations occur-

ring in Poland are described in Table 1. Adult nema-

todes were used for the analyses, except for D. gigas,

for which only larvae were available. The nematodes

were assigned to the appropriate species, based on the

morphology and morphometrics listed in the EPPO

guidelines (2008).

DNA isolation, PCR amplification, DNA cloning and

sequencing

For each sample, a few adult (or a few dozen larvae)

nematodes were used for DNA isolation, as described

previously (Nowaczyk et al. 2011). Purified DNA was

used as a template in PCR. Primers amplifying the

region composed of 18S rDNA fragment, ITS1, 5.8S

rDNA fragment, ITS2: FDdips1 and RDdips2 for

D. dipsaci and FDdest1 and RDdest2 for D. destructor

were used to perform the PCR in the conditions

described previously (Kierzek et al. 2010). For visuali-

zation of the PCR products, 1% agarose gel was used,

followed by extraction of the products using

QIAquick� Gel Extraction Kit (Qiagen, Hilden,

Germany). Then, they were cloned into pGEM

T-Easy� vector (Promega, Madison, WI, USA) and

transformed to ElectroMAXTM Stbl4TMEscherichia coli

Table 1 List of nematode populations used in this study with their hosts, geographic location and GenBank accession numbers

Species Name Host

Geographic

location

GenBank

accession

number

Ditylenchus dipsaci Dip.1 Allium cepa Poland KC923218

D. dipsaci Dip.2 A. cepa Poland KC923219

D. dipsaci Dip.3 A. cepa Poland KC923220

D. dipsaci Dip.4 Phlox sp. Poland KC923221

D. dipsaci Dip.6 Phlox sp. Poland KC923222

D. dipsaci Dip.Dit1 A. cepa Poland JN376069

D. dipsaci Dip.Dc Cichorium endivia Poland JN376070

D. dipsaci Dip.Dips A. cepa Poland JN376071

D. dipsaci Dip.Dm A. cepa Poland JN376072

D. dipsaci Dip.Dw A. cepa Poland JN376073

D. dipsaci Dip.Dwb A. cepa Poland JN376075

Ditylenchus gigas Gig.S Vicia faba (seeds) Poland JN376074

Ditylenchus destructor Des.D8 Solanum tuberosum Poland JN376068

D. destructor Des.1 S. tuberosum Poland KC923223

D. destructor Des.2 S. tuberosum Poland KC923224

� 2013 Blackwell Verlag GmbH2

Phylogenetic study of Ditylenchus spp. populations in Poland A. Jeszke et al.

cells (Invitrogen, Carlsbad, CA, USA) using electropo-

ration (Micro Pulser electroporation system; Bio-Rad,

Philadelphia, PA, USA), according to the manufac-

turer’s instructions. Plasmids from six recombinant

clones (for each population sample) were isolated

using the QiaPrep Spin Miniprep Kit (Qiagen) and

automatically sequenced.

Phylogenetic and bioinformatics analyses

Multiple sequence alignments (MSA) were

obtained using CLUSTALX (Thompson et al. 1997)

and then edited manually in GeneDoc (Nicholas

et al. 1997). The comparisons of the nucleotide

sequences for all the analysed populations were

performed in BioEdit (Hall 1999), and phylogenetic

analysis, in MEGA4 software (Tamura et al. 2007)

with the neighbour-joining method (NJ; Saitou

and Nei 1987) and in the bootstrap test (1000 rep-

etitions). Genetic distance was estimated by

Kimura 2-parameter distance method (Tamura

et al. 2007). Phylogenetic trees were then drawn

and visualized using MEGA4. Apart from populations

from Poland being used, the populations deposited

in GenBank from other countries were also

included in the phylogenetic analysis: 67 popula-

tions for D. destructor, 47 for D. dipsaci and 20 pop-

ulations described as Ditylenchus sp. B from V. faba

and described as D. gigas according to the new

nomenclature (Vovlas et al. 2011) for D. gigas. Also

populations that were chosen to represent other

polyploidy races and species were used in phyloge-

netic analyses, including D. weischeri, and popula-

tions of Ditylenchus spp. D, E and F.

Results and Discussion

The PCR amplification of DNA isolated from the anal-

ysed Ditylenchus populations gave amplified products

of 707–715 nucleotides in the case of the D. dipsaci

populations, 714 nt in the case of the D. gigas popula-

tions and 902–903 nt in the case of the D. destructor

populations. The subsequently obtained rDNA

sequences for those populations were sent to

GenBank and are annotated under accession numbers

given in Table 1. Pairwise nucleotide sequence

comparison revealed that all D. dipsaci populations

obtained in our study shared a 99–100% identity with

each other as well as with other D. dipsaci populations

deposited previously in databases.

The only population (S) isolated from V. faba spp.

minor was identified as D. gigas. Comparison of the

nucleotide sequence of this population revealed a

99% identity with other D. gigas populations

described so far.

The populations D8, 1 and 2, of D. destructor,

compared with other populations of this species

present in GenBank, showed an identity level

between 68.5 and 99.8%. American and Chinese pop-

ulations described as haplotype C (Subbotin et al.

2011) showed the highest identity level (99.0–99.8%)

with the D8, 1 and 2, of D. destructor populations.

Polish populations of D. destructor analysed share only

a 93% identity with the Polish population (Stu3),

described previously (Marek et al. 2010).

The phylogenetic analysis for D. dipsaci revealed a

phylogenetic tree which was similar to that obtained

by Subbotin et al. (2005). Two separate clades for dip-

loid races and polyploidy races were indicated

(Fig. 1). It is important to note that when looking at

the tree topology, populations isolated from the same

host are rarely grouped together. This can be observed

(e.g. for D. dipsaci populations isolated from Allium

cepa or Cichorium spp.), and it cannot be explained by

host or geographic origin. In the case of D. dipsaci,

there are more than 30 distinguished host races that

are supposed to be at different stages of speciation;

however, some authors indicate that there is ambigu-

ity about how they should be defined (Sturhan and

Brzeski 1991).

Phylogenetic analysis for D. destructor populations

was performed for populations isolated from

S. tuberosum, I. batatas and Astragalus mongholicus.

For D. destructor, length variability of the ITS1 frag-

ment was found, and eight haplotypes were sepa-

rated (Subbotin et al. 2011). The haplotype A

isolated from sweet potato from China is the most

distinct and formed a separate clade. The previously

reported population (Stu3) from Poland was

assigned as haplotype G (Marek et al. 2010; Subbo-

tin et al. 2011). The populations described in this

study (D8, 1, 2), however, grouped together with

haplotype C populations on a phylogenetic tree

(Fig. 2). This indicates that in Poland, there are at

least two haplotypes present. It is worth noting that

most populations isolated from the sweet potato

cluster separately from those isolated from potato.

But at the same cluster with the presently described

D. destructor populations from Poland grouping

members of haplotype C, there is also a Chinese

population from I. batatas (Fig. 2).

Phylogenetic analysis was carried out with D. gigas

found on V. faba minor seeds in Poland. In spite of a

very high identity level with other populations

reported so far from Europe and Northern Africa,

D. gigas from Poland grouped separately from all of

� 2013 Blackwell Verlag GmbH 3

A. Jeszke et al. Phylogenetic study of Ditylenchus spp. populations in Poland

Germany A.sativa AY574293 Poland A.cepa |KC923219|D.dipsaci 2

Germany D.lutea AY574292 Spain A.sativum HQ219249 IasD10 France M.sativa AY703062 Dd.F.1.3 France V.faba AY703059 Dd.F.1.1 Germany B.vulgaris AY574299 Italy Fragaria sp HQ219241 FrIt09

Poland A.cepa |JN376075| Dwb Poland A.cepa |KC923218| D.dipsaci 1

France B.vulgaris AY703061 Dd.F.1.2 Algeria V.faba AY703064 Dd.DZ

Poland Phlox sp. |KC923221| D.dipsaci 4 Greece A.sativum AM232234 DIGR Spain A.sativum HQ219242 Veg07 Germany Z.mays AY574294 France M.sativaAY703063 Dd.F.1.4

Poland A.cepa |JN376072| Dm Estonia M.sativa AF396320

Germany V.gentianoides AY574295 Turkey A.cepa AY703065 DD.TK

Poland A.cepa |KC923220| D.dipsaci 3 Spain B.vulgaris HQ219243 Sug09 USA A.sativum JX123258 CD1012 Australia V.faba AY703060 Dd.A.1.1 Germany M.sativa|AY574297| Czech Republic C.inthybus DQ452957

Poland A.cepa |JN376071| Dips Israel A.sativum AY574298 Spain A.sativum DQ520932 Czech Republic P.lanceolataGQ469496 Pla Spain B.vulgarisDQ520931 Lebrija Spain B.vulgaris DQ520930 Lebrija Germany L.vulgaris AY574296

Czech Republic B.vulgaris GQ469494 Bvu2 Poland A.cepa |JN376073| Dw

Italy V.faba HQ219244 Mon09 Germany P.drummondii AY574291 Germany A.cepa AY574289 Italy V.faba HQ219248 TcnF09 Italy P.sativum HQ219247 TcnP08 Czech Republic M.sativa DQ452958

Czech Republic A.sativum GQ469497 Asa4 Czech Republic A.sativum DQ452956

Poland A.cepa |JN376069| Dit1 Poland C.endywia |JN37607| Dc

Russia Fragaria sp. AF396321 Italy D.carota AY714535 Germany P.lanceolata AY574301 Estonia T.pratense AY574300

Poland Phlox sp. |KC923222| D.dipsaci 6 Estonia T.pratense AF396319 Yemen A.sativum HQ229002

Spain A.cepa HQ219245 Jer79 Czech Republic B.vulgaris GQ469495 Bvu3. Czech Republic B.vulgaris GQ469493 Bvu1

Italy A.sativum AM232235 DIIT Italy V.faba HQ219246 TcnP09

Germany P.maritimaAY574288 sp. G Germany P.maritima AY574287 sp. G Russia C.setosum AF396322 D.weischeri

Poland V. faba var minor |JN376074|Dip S Italy V.faba |HQ219231|D.gigas Ost10

Estonia C.praemorsaAY574303 sp. E Ukraine P.caespitosa AY574304 sp. D

Estonia P.officinarum AY574302 sp. F. Poland JN376068 D.destructor D8

Fig. 1 Phylogenetic tree of populations belonging to the Ditylenchus dipsaci species complex from this study and deposited in the GenBank database

(bootstrap values cut-off = 40) with genetic distance. The Ditylenchus destructor population was used as an out-group. Chosen populations repre-

senting other polyploidy races of Ditylenchus spp. D, E, F and G, as well as single members of Ditylenchus gigas and Ditylenchus weischeri that were

previously considered as Ditylenchus sp. B and C, respectively, were also added, for visualization of their current status on the tree topology together

with the new, reported D. dipsaci populations.

� 2013 Blackwell Verlag GmbH4

Phylogenetic study of Ditylenchus spp. populations in Poland A. Jeszke et al.

CzechRepublic potato|GQ469491|Stu2 HapG CzechRepublic potato|GQ469490|Stu1 HapG

Poland potato|GQ469492|Stu3 HapG Russia potato|HQ235678|CA213cl1 HapG Rusia potato|HQ235679|CA213cl3 HapG Russia potato|HQ235684|CA215cl2 HapG China sweet potato|EF208211|Tongshan HaB

China|FJ911551|ChaBei HapB Russia potato|HQ235681|CA205cl3 HapG Russia potato|HQ235683|CA210cl2 HapG Russia potato|AY987007| HapB China sweet potato|EF208212|Yishui HapB China potato|EF062572|Des1 HapB Russia potato|HQ235682|CA215cl1 HapG China sweet potato|JX145343I|DitySSDFXSY

Poland potato |JN376068|D8 China sweet potato|EF208210|Sihong HapC

Poland potato |KC923224|D.destructor2 China sweet potato|EF208213|Hanguo HapD

Poland potato |KC923223| D.destructor1 China potato|EF062574|Des3 HapC USA potato|HQ235675|CA209cl2 HapC USA potato|HQ235676|CA209cl1 HapC China potato|EF062573|Des2 HapC China sweet potato|HQ235677|Weifang HapF China A.mongholicus|DQ471335|Ch2 HapF

Iran potato|HQ235696|CA206 cl1HapE Iran|JN166693|

Russia potato|HQ235691|CA210cl3HapE Iran potato|HQ235689|CA214 HapE Iran potato|HQ235690|CA206cl2 HapE Russia potato|HQ235686|CA210 HapE Russia potato|HQ235685|CA213cl2 HapE Iran potato|HQ235694|CA211cl1 HapE Iran potato|HQ235692|CA211cl2 HapE Iran potato|HQ235695.1|CA214 HapE Iran potato|HQ235688|CA212cl2 HapE

Russia potato|HQ235693|CA205 HapE Iran potato|HQ235687|CA212 HapE

China sweet potato|EF210370|Luoyang HapA China sweet potato|EF210372|Miyun1 HapA China sweet potato|EF088933|Ch8 HapA China sweet potato|EF088934|Ch9 HapA China sweet potato|EF088929|Ch6 HapA China sweet potato|EF210365|Taihe HapA China sweet potato|EF088935|Ch10 HapA China sweet potato|EF210374|Jinan HapA China sweet potato|EU499915|Baicheng HaA China sweet potato|EF210367 |Zhuozhou HA China sweet potato|HQ235697|Luoyang HapA China sweet potato|EF088937|Ch12 HapA

China sweet potato|AM232228|DEws HapA China sweet potato|DQ471336|Ch3 HapA China sweet potato|EF088936|Ch11 HapA China sweet potato|AM232229|DEwy HapA China sweet potato|EF210371|Sixian2 HapA

China sweet potato|EF210369|Lulong HapA China sweet potato|EF088938|Ch13 HapA China sweet potato |JX040544|DitySSDFX China sweet potato|EF210373|Changli HapA China sweet potato|EF210364|Daxing1 HapA China sweet potato|AM232230|DEhblf HapA

China sweet potato|EF088930|Ch4 HapA China sweet potato|AM232231|DEjl HapA China potato|EF062575|Des5 HapA China sweet potato|EF210368|Luanxian HaA China sweet potato|AM232227|DEsg HapA China sweet potato|EF088931|Ch5 HapA China sweet potato|EF210366|Xinyi HapA

China sweet potato|EF088932| Ch7 HapA Poland A.cepa|JN376071|Ddips1

Fig. 2 Phylogenetic tree of Ditylenchus destructor populations from the current study and other populations whose sequences were deposited in

the GenBank database (bootstrap values cut-off = 40) including their current haplotype assignment. Ditylenchusdipsaci population was used as an

out-group.

� 2013 Blackwell Verlag GmbH 5

A. Jeszke et al. Phylogenetic study of Ditylenchus spp. populations in Poland

them. This separate grouping might result from the

geographic origin of this population as well as from

the host from which it was isolated. Nonetheless, the

phylogenetic distance between those populations is

rather small (Fig. 3). Specimens of the D. gigas species

were found in Poland in the seeds of V. faba ssp. minor

(‘horse bean’), collected in the 1990s. This plant is

usually cultivated for use as animal fodder. The eco-

nomic significance of this pest in Poland is currently

difficult to establish. It was found accidentally, and no

further screening of D. gigas was performed.

In conclusion, D. dipsaci populations are

characterized by low sequence divergence of

approximately 1%. In the case of D. gigas

populations, there is a high identity level. The

population from Poland differs slightly from other

reported populations from the countries

surrounding the Mediterranean Sea. This is also the

first report on the occurrence of D. gigas in V. faba

ssp. minor seeds, in Poland. The D. destructor

populations described previously and in this study

clustered separately, next to haplotypes G and C,

respectively, in phylogenetic analysis. The obtained

results suggest that haplotype diversity in

potato-growing areas may be much greater than is

currently known.

Acknowledgement

This study was supported by the Polish Ministry of

Agriculture and Rural Development, the Long-Term

Programme of IPP-NRI, Project 2.3.

References

Caubel G, Pedron D. (1976) Distribution geographique du

nematode des tiges Ditylenchus dipsaci (Kuhn) Fil., en cul-

ture de legumineuses fourrageres. Sci Agronom Rennes

8:183–188.

Chizhov VN, Borisov BA, Subbotin SA. (2010) A new

stem nematode, Ditylenchus weischeri sp. n. (Nematoda:

Tylenchida), a parasite of Cirsium arvense (L.) Scop. in the

Central Region of the Non-Chernozem Zone of Russia.

Russ J Nematol 18:95–102.

Italy V.faba D.gigas HQ219233 Noc08 cl11 Italy V.faba D.gigas HQ219232 Noc08 cl17 Italy V.faba D.gigas HQ219231 Ost1 Morocco V.faba AY714539 DD.M.1.4 Morocco V.faba AY714537 DD.M.1.2 Italy V.faba D.gigas HQ219236 Noc08 cl8 Syria V.faba AY574286 sp. B Lebanon D.gigas HQ219238LEb07 cl10 Italy V.faba D.gigas HQ219235 Noc08 Lebanon V.faba D.gigasHQ219237 Leb07 Lebanon D.gigas HQ219234 Leb07 France V.faba AY714541 DD.F.2.1 Morocco V.faba AY714538 DD.M.1.1 Morocco V.faba AF396323 Morocco V.faba AY714540 DD.M.1.5 Spain V.faba D.gigas HQ219240 IasG10 UK V.faba AY574284 sp. B Germany V.faba AY574285 sp. B Tunisia V.faba AY714536 DD.T.1

Poland V.faba var minor JN376074 S Lebanon V.faba D.gigasHQ219239 Leb07 Poland JN376068 D.destructor D8

Fig. 3 Phylogenetic tree of the currently reported Ditylenchus gigas (or previously as Ditylenchus sp. B) from Vicia faba populations, in GenBank

database as well as the D. gigas population isolated from horse bean seeds. Ditylenchus dipsaci was used as an out-group.

� 2013 Blackwell Verlag GmbH6

Phylogenetic study of Ditylenchus spp. populations in Poland A. Jeszke et al.

EPPO. (2008) Ditylenchus destructor and Ditylenchus dipsaci.

EPPO Bull 38:363–373.

Hall TA. (1999) BioEdit: a user-friendly biological

sequence alignment editor and analysis Program for

Windows 95/98/NT. Nucleic Acids Symp Ser 41:95–98.

Kierzek D, Dobosz R, Obrezpalska-Stezplowska A. (2010)

Molecular diagnostics of polish Ditylenchus dipsaci

populations. Prog Plant Prot 50:1265–1269.

Marek M, Zouhar M, Douda O, Mazakova J, Rysanek P.

(2010) Bioinformatics-assisted characterization of the

ITS1-5.8S-ITS2 segments of nuclear rRNA gene clusters,

and its exploitation in molecular diagnostics of European

crop-parasitic nematodes of the genus Ditylenchus. Plant

Pathol 59:931–943.

Nicholas KB, Nicholas HB Jr, Deerfield DW II. (1997)

Genedoc: analysis and visualization of genetic variations.

EMBNEW News 4:14.

Nowaczyk K, Dobosz R, Budziszewska M, Kornobis S,

Kamasa J, Obrezpalska-Stezplowska A. (2011) Analysis of

diversity of golden potato cyst nematode (Globodera rosto-

chiensis) populations from Poland using molecular

approaches. J Phytopathol 159:759–766.

Palmer HM, Atkinson HJ, Perry RN. (1992) Monoclonal

antibodies (Mabs) specific to surface expressed antigens

of Ditylenchus dipsaci. Fundam Appl Nematol

15:511–515.

Saitou N, Nei M. (1987) The neighbor-joining method: a

new method for reconstructing phylogenetic trees. Mol

Biol Evol 4:406–425.

Sturhan D, Brzeski MW. (1991) Stem and bulb nematodes,

Ditylenchus spp. In: Nickle WR (ed) Manual of Agricul-

tural Nematology. New York, NY, USA, Marcel Dekker,

Inc., pp 423–464.

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.

Subbotin SA, Demi AM, Zheng J, Chizhov VN. (2011)

Length variation and repetitive sequences of Internal

Transcribed Spacer of ribosomal RNA gene, diagnostics

and relationships of populations of potato rot nematode,

Ditylenchus destructor Thorne, 1945 (Tylenchida:

Anguinidae). Nematology 13:773–785.

Tamura K, Dudlmy J, Nei M, Kumar S. (2007)

MEGA4: molecular evolutionary genetics analysis

(MEGA) software version 4.0. Mol Biol Evol

24:1596–1599.

Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F,

Higgins DG. (1997) The ClustalX windows interface:

flexible strategies for multiple sequence alignment

aided by quality analysis tools. Nucleic Acids Res

25:4876–4882.

Vovlas N, Troccoli A, Palomares-Rius JE, De Luca F,

Liebanas G, Landa BB, Subbotin SA, Castillo P.

(2011) Ditylenchus gigas n. sp. Parasitizing broad bean:

a new stem nematode singled out from the

Ditylenchus dipsaci species complex using a polyphasic

approach with molecular phylogeny. Plant Pathol

4:762–775.

� 2013 Blackwell Verlag GmbH 7

A. Jeszke et al. Phylogenetic study of Ditylenchus spp. populations in Poland