Molecular characterization of Ditylenchus dipsaci on Onion in Turkey

Molecular characterization of

_{Ditylenchus dipsaci on Onion}

in Turkey

E. Yavuzaslanoglu

O. Ates Sonmezoglu

N. Genc

Z. Akar

B. Terzi

Accepted: 11 October 2017 / Published online: 16 October 2017 # Koninklijke Nederlandse Planteziektenkundige Vereniging 2017

Abstract Ditylenchus dipsaci is a species complex

in-cluding diploid and polyploid individuals. The onion

race of D. dipsaci is a sensu stricto group and has a

wide range of host spectrum. Identification of the

D. dipsaci onion race is difficult using morphological

and morphometrical methods. Species specific primers

are mostly used in molecular approaches for

identifica-tion of D. dipsaci populaidentifica-tions. Fifty one

morphological-ly selected Ditylenchus spp. populations from onion

production areas in Turkey were subjected to molecular

identification using four D. dipsaci species specific

primer sets (PF1-PR1, PF2-PR2, DdpS1-rDNA2,

DitNF1- rDNA2, H05-H06) targeting 5.8S and 18S

rDNA, ITS1 and flanking ITS regions. Thirty nine

percent of the nematode samples were positive with four

primers tested, while four of the nematode samples gave

specific bands with H05-H06 primers. Ditylenchus

dipsaci sensu stricto was identified with specific primer

sets in Adana, Hatay, Tekirdag, Bursa, Aksaray,

Karaman, Eskisehir and Ankara provinces in

Mediter-ranean, Trace, Aegean and Central Regions in Turkey.

Keywords Onion . Stem and bulb nematode .

Species specific primer . Molecular characterization

Ditylenchus dipsaci (Kühn 1857) is among the

signifi-cant damaging plant parasitic nematodes on agricultural

production in temperate conditions. Ditylenchus dipsaci

has very high intraspecific variation (Subbotin et al.

2005

). It has more than 30 races multiplying on 500

plant species (Sturhan and Brzenski

1991

). Therefore, it

is named as species complex. Last taxonomical studies

divided D. dipsaci species complex into two groups;

one is

BD. dipsaci sensu stricto^ group including

dip-loid members, the other group includes polypdip-loid

mem-bers, which is also subdivided into 6 groups;

Ditylenchus sp. B from Vicia faba, Ditylenchus sp. C

from Cirsium setosum, Ditylenchus sp. D related to

Pilosella genera plants, Ditylenchus sp. E found on

Crepis praemorsa, Ditylenchus sp. F related to

Pilosella and Leontodon genera plants and Ditylenchus

sp. G subgroup identified on Plantago maritima plant

(Subbotin et al.

2005

). Further studies identified

Ditylenchus sp. C as Ditylenchus weischeri (Chizhov

et al.

2010

) and Ditylenchus sp. B group from Vicia

faba as Ditylenchus gigas (Vovlas et al.

2011

). Diploid

nematode populations showed very close phylogenetic

relationships and were classified as races (Subbotin

et al.

2005

). Janssen (

1994

) described eight races from

https://doi.org/10.1007/s10658-017-1366-7

E. Yavuzaslanoglu (*)

Department of Plant and Animal Production, Technical Sciences Vocational School, Karamanoğlu Mehmetbey University, Karaman, Turkey

e-mail: eyavuzaslanoglu@kmu.edu.tr O. Ates Sonmezoglu (*)

:

Department of Bioengineering, Faculty of Engineering, Karamanoglu Mehmetbey University, Karaman, Turkey e-mail: ozlemsonmezoglu@kmu.edu.tr

N. Genc

:

Institute of Science, Karamanoglu Mehmetbey University, Karaman, Turkey

Medicago sativa, Trifolium pratense, Avena sativa,

Secale cereale, Beta vulgaris, Narcissus spp., Tulipa

spp. and Allium cepa. The onion race of D. dipsaci

was commonly identified by researchers to damage

onion, garlic, pea and bean, but other hosts have

differ-entiating reactions in different studies (Janssen

1994

;

Mennan

2001

). Due to the inconsistency over host

status, control of the nematode using host crop rotation

is difficult. Anabiosis ability of the nematode in the

absence of the host plant makes it also difficult to

control the nematode under agricultural practices.

Host preferences of the nematodes could be

deter-mined by genetically using techniques determining

polymorphism on pathogenicity genes. The

complex-ity and difficulty on determination of host status and

morphological similarities lead to development of

dif-ferent molecular taxonomic approaches for difdif-ferenti-

differenti-ation of the D. dipsaci species complex on different

host plants (Esquibet et al.

1998

; Subbotin et al.

2005

;

Kerkoud et al.

2007

; Zouhar et al.

2007

; Douda et al.

2013

; Jeszke et al.

2014

). Polymorphism on the

inter-nal transcribed spacer (ITS) regions of ribosomal

DNA is the most commonly used approach for

deter-mination of intraspecific variations of D. dipsaci

(Esquibet et al.

2003

; Marek et al.

2005

; Subbotin

et al.

2005

; Kerkoud et al.

2007

; Zouhar et al.

2007

).

Ditylenchus dipsaci has a local distribution in

Eu-rope being listed on the quarantine A2 list by the

European and Mediterranean Plant Protection

Organi-zation (EPPO) (EPPO

2017

). It was first identified on

onion by Yuksel (

1958

) in Turkey. It was recorded on

onion-growing areas in Trace, Central Anatolian

Pla-teau and Black Sea Region in Turkey (Saltukoglu

1974

; Ozturk

1990

; Mennan and Ecevit

2002

;

Yavuzaslanoglu et al.

2015a

). Yield losses of 41.5–

65% on onion was determined in Turkey (Mennan and

Ecevit

2002

; Yavuzaslanoglu et al.

2015b

).

Studies of the distribution of D. dipsaci on onion

in Turkey to-date have used morphological and

mor-phometric techniques. There is no available detailed

information on the species complex distribution in

large scale onion growing areas of Turkey. The aim

of this study was to investigate the D. dipsaci sensu

stricto group distribution on onion, using specific

PCR techniques, for the market-scale onion

produc-tion areas in Turkey.

Materials and methods

Nematode populations

Nematode populations were obtained from onion

fields in ten provinces in Mediterranean, Trace,

Ae-gean and Central Anatolia Regions in Turkey on

April to May 2016. In total, 51 nematode populations

were studied; five from Adana, six from Hatay, seven

from Tekirdag, one from Balikesir, nine from Bursa,

one from Aksaray, six from Karaman, two from

Kon-ya, twelve from Ankara, and two from Eskisehir

provinces where onion production is most practised

in Turkey (Table

2

). The nematodes used for

molec-ular identification were extracted from either plant

tissues or soil using a

‘modified baerman funnel’

technique (Hooper

1986

). Nematodes were identified

morphologically at genus level as Ditylenchus spp.

and collected with a pasteur pipette from samples in a

PCR tube.

Molecular characterization

DNA was extracted from each nematode population

according to Holterman et al. (

2006

) with some

modifications. Five to ten individual nematodes

were transferred with 25

μl sterile distilled water

into an Eppendorf tube and homogenized in 25

μl

lysis buffer (WLB

). WLB+ contained 10

μl

beta-merkaptoetanol, 40

μl 20 mg/ml Proteinase K and

950

μl WLB- buffer (2 ml 1 M NaCl, 2 ml 1 M

Tris-HCl and 5.5 ml of ddH

O). The mixture was

incu-bated for 90 min at 65 °C and finally denatured

5 min at 95 °C. The tubes were centrifuged at

14,000 rpm for 1 min and stored at

−20 °C. DNA

was eluted in 20

μL ddH

O and stored at

−20 °C.

PCR reactions were carried out as follows: an initial

denaturation step of 3 min at 94 °C was followed by

37 cycles of denaturation for 1 min at 94 °C, annealing

for 45 s at 55

–62 °C (depending upon the annealing

temperature of the primers), extension for 2 min at

72 °C, and conclusion with a final extension step for

10 min at 72 °C. Negative control samples containing

only sterile distilled water (no DNA target) were included.

DNA from a morphologically identified D. dipsaci culture

(Yavuzaslanoglu et al.

2015a

) was used as a positive

control. Specific amplifications were repeated three times

to assess the reproducibility of PCR. PCR amplicons were

separated in 1–2% agarose gels. Electrophoresis was

con-ducted at 90 V of constant power for 3 h.

PCR reactions (for all primers except PF1-PR1

prim-er set) wprim-ere carried out in a BIO-RAD (C1000 Touch)

thermal cycler in a total volume of 25

μL using 1.5 U

Taq DNA Polymerase (Thermo), 200

μM each dNTPs,

1.2

μM MgCl

, 0.5

μM each primer, 10 x Tag Buffer

with KCL. PCR amplification for PF1-PR1 primer was

determined in a total reaction volume of 25

μL, with

2 ng DNA as template, 7.5

μL Dream Taq Green MM

(Thermo), 0.5

μM each primer.

The five species-specific PCR primers targeted to

amplify 5.8S, 18S rDNA genes, ITS1 and flanking

ITS regions used for the identification of D. dipsaci

were listed on Table

1

.

Results and discussion

Using the species-specific PCR primers and nematode

DNA extracted from onion plants and soils, PCR

frag-ments were successfully amplified at expected product

size for each primer set in D. dipsaci samples. All of the

primers give present or absent a specific size band in

D. dipsaci. The used primers in the study were

ampli-fying only D. dipsaci sensu stricto group.

Fifteen of the 51 nematode populations showed

ex-pected band size with all primer sets used in the

molec-ular screening. The numbers of these samples are 1, 2

and 4 in Adana Province; 8 and 11 in Hatay Province;

12, 15 and 18 in Tekirdag Province; 25 in Bursa

Prov-ince; 31 in Aksaray ProvProv-ince; 34 and 35 in Karaman

Province; 50 in Eskisehir Province; 54 in Amasya

Prov-ince and 58 in Corum ProvProv-ince. Twenty one of the

samples were negative with all primer sets. Twenty of

the samples gave positive reaction with at least one of

the primers, reactions were differentiating among

primers (Table

2

).

The amplicon of PF1-PR1 primer set targeting

flanking ITS spacer regions was 327 bp for studied

D. dipsaci populations and 20 of the nematode

popula-tions produced specific bands of D. dipsaci sensu

stricto. Similarly, a fragment with 396 bp band size

was amplified with 20 nematode populations using

PF2-PR2 primers. For both primer sets, samples not

including D. dipsaci observed no amplification in

ac-cordance with Marek et al. (

2005

).

Expected PCR product size 517 bp (Vrain et al.

1992

) for D. dipsaci were obtained using

DdpS1-rDNA2 primers targeted 5.8S rDNA gene and flanking

ITS regions. The specific amplicon was observed for

twenty samples.

The amplicon of DitNF1-rDNA2 primer set is 263 bp

for D. dipsaci sensu stricto (Subbotin et al.

2005

). The

primers do not amplify for D. dipsaci giant race, D.

myceliophagus and Ditylenchus sp. Twenty of the tested

populations were found to be D. dipsaci sensu stricto

using this primer set.

The used primer sets can be used successfully for

D. dipsaci specific identification. Total 20 samples

Table 1 SCAR and SSR primers used in identification of D. dipsaci nematodes Primer No Primer Sequence (5′— 3′) PCR Product

Size (bp)

Targeted regions Reference

1 PF1 5′-AAC GGC TCT GTT GGC TTC TAT-3 327 bp Flanking ITS regions Marek et al. (2005) PR1 5′-ATT TAC GAC CCT GAG CCA GAT-3′

2 PF2 5′-TCG CGA GAA TCA ATG AGT ACC-3′ 396 bp Flanking ITS regions Marek et al. (2005) PR2 5′-AAT AGC CAG TCG ATT CCG TCT-3′

3 DdpS1 5′-TGG CTG CGT TGA AGA GAA CT-3′ 517 bp 5.8S rDNA and flanking ITS regions

Vrain et al. (1992) rDNA2 5′-TTT CAC TCG CCG TTA CTA AGG-3′

4 DitNF1 5′-TTA TGA CAA ATT CAT GGC GG-3′ 263 bp 18S and ITS1 regions Subbotin et al. (2005) rDNA2 5′-TTT CAC TCG CCG TTA CTA AGG-3′

5 H05 5′- TCA AGG TAA TCT TTT TCC CCA CT-3′ 242 bp Flanking ITS regions Esquibet et al. (2003) H06 5′-CAA CTG CTA ATG CGT GCT CT-3′

Table 2 Origin and extraction medium of nematode populations tested in the study and PCR results with specific primer sets (set 1: PF1-PR1, set 2: PF2-PR2, set 3: DdPS1-rDNA2, set 4: DitFN1-rDNA2, set 5: H05-H06)

Sample no Geographical region Province Latitude (N) Longitude (E) Nematode extracted from plant (P) / soil (S)

Set 1 Set 2 Set 3 Set 4 Set 5

1 Mediterranean Adana 36,8307 35,20,291 P, S + + + + – 2 Adana 36,9185 35,390,053 P + + + + – 3 Adana 36,93,854 35,42,001 P, S – – – – – 4 Adana 36,97,576 35,484,092 S + + + + – 5 Adana 36,97,264 35,495,122 S – – – – – 6 Hatay 36,241,252 36,588,585 P + + + + + 7 Hatay 36,241,598 36,619,635 S + + + + – 8 Hatay 36,242,611 36,620,965 P + + + + – 9 Hatay 36,308,026 36,540,532 P + + + + – 10 Hatay 36,359,227 36,417,782 S + + + + – 11 Hatay 36,361,299 36,414,779 P + + + + + 12 Trace Tekirdag 40,991,621 27,538,922 P, S + + + + + 13 Tekirdag 40,998,660 27,599,076 P, S – – – – – 14 Tekirdag 40,998,117 27,608,313 S + + + + – 15 Tekirdag 40,991,898 27,559,392 P, S + + + + – 16 Tekirdag 40,929,668 27,074993 P, S + + + + – 17 Tekirdag 40,881,806 27,071530 S – – – – – 18 Tekirdag 40,880,722 26,770,853 P, S + + + + – 19 Aegean Balikesir 39,358,320 27,577,524 S – – – – – 20 Bursa 40,004027 28,225,124 P, S – – – – – 21 Bursa 40,242,432 28,353,534 S – – – – – 22 Bursa 40,249,119 28,268,498 P, S – – – – – 23 Bursa 40,286,003 28,279,575 P, S + + + + + 24 Bursa 40,338,597 28,571,973 P – – – – – 25 Bursa 40,349,687 28,603,553 P, S – – – – – 26 Bursa 40,283,767 29,720,884 S – – – – – 27 Bursa 40,342,871 29,748,120 P, S – – – – – 28 Bursa 40,345,234 29,756,800 S – – – – – 29 Central Anatolia Konya 37,783,331 31,800,980 P, S – – – – – 30 Konya 37,626,381 34,168,439 S – – – – – 31 Aksaray 38,515,619 33,857,822 P, S + + + + – 32 Karaman 37,110,791 33,112,303 P, S – – – – – 33 Karaman 37,103,613 33,115,460 S – – – – – 34 Karaman 37,101,080 33,115,943 P, S – – – – – 35 Karaman 37,098273 33,116,694 P, S + + + + – 36 Karaman 37,033742 33,078365 P, S – – – – – 37 Karaman 37,103,901 33,058728 S – – – – – 38 Ankara 39,182,804 32,053457 S – – – – – 39 Ankara 39,348,611 32,301,648 S – – – – – 40 Ankara 39,267,027 32,246,245 S + + + + – 41 Ankara 39,264,624 32,078559 S – – – – –

(39%) among the 51 nematode samples from onion

plants and soils were commonly identified as

D. dipsaci sensu stricto by molecular characterization.

Nematodes from 31 locations (61%) did not produced

specific bands with any of the primer sets tested.

How-ever, a specific band for at least one of the primer sets

used was observed for 20 nematode samples (39%).

Duo to the complex nature of the D. dipsaci species

complex, it is very useful to use molecular techniques

for the routine identification of the D. dipsaci samples

from different host plants. Vrain et al. (

1992

), Marek

et al. (

2005

) and Subbotin et al. (

2005

) used and

recommended the specific primers to identify

D. dipsaci sensu stricto. It has also been reported that

rDNA ITS regions can be successfully used for

phylo-genetic analyzes (Subbotin et al.

2005

; Marek et al.

2010

; Vovlas et al.

2011

; Pethybridge et al.

2016

). The

precise identification of the nematode is an important

step for effective control of the host plant. For this

purpose, to know the distribution of the sensu stricto

group of D. dipsaci including the onion race on onion

plants and production soils in Turkey is very valuable

and it is first report on distribution of D. dipsaci sensu

stricto in onion-growing areas in Turkey using

molec-ular tools.

Acknowledgements This research has been financially support-ed by Turkish Scientific and Technical Research Council (TUBITAK) (Project No: 215O468).

Compliance with ethical standards The manuscript has not been submitted to any other journal. The manuscript has not been

published previously (partly or in full). Preliminary data was pre-sented in 32nd ESN Symposium, held in Braga, Portugal, in 28th August -1st September 2016;BMolecular identification of stem and bulb nematode (Ditylenchus dipsaci) on onion in Turkey^ by Nimet Genc, Ozlem Sonmezoglu and Elif Yavuzaslanoglu. A single study was not split up into several parts to increase the quantity of submissions and submitted to various journals or to one journal over time. No data have been fabricated or manipulated (including images) to support our conclusions. No data, text or theories by others are presented.

The research does not require any ethical council permission. Consent to submit has been received explicitly from all co-authors, before the work is submitted. Authors whose names appear on the submission have contributed sufficiently to the scientific work and therefore share collective responsibility and accountability for the results.

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