Molecular characterization of Ditylenchus dipsaci on ... - Springer Link

Eur J Plant Pathol (2018) 151:195–200 https://doi.org/10.1007/s10658-017-1366-7

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 including 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 identification of D. dipsaci populations. Fifty one morphologically 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

E. Yavuzaslanoglu (*) Department of Plant and Animal Production, Technical Sciences Vocational School, Karamanoğlu Mehmetbey University, Karaman, Turkey e-mail: [email protected] O. Ates Sonmezoglu (*) : B. Terzi Department of Bioengineering, Faculty of Engineering, Karamanoglu Mehmetbey University, Karaman, Turkey e-mail: [email protected] N. Genc : Z. Akar Institute of Science, Karamanoglu Mehmetbey University, Karaman, Turkey

sets in Adana, Hatay, Tekirdag, Bursa, Aksaray, Karaman, Eskisehir and Ankara provinces in Mediterranean, 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 significant 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 diploid members, the other group includes polyploid members, 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

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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 differentiating 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 determined by genetically using techniques determining polymorphism on pathogenicity genes. The complexity and difficulty on determination of host status and morphological similarities lead to development of different molecular taxonomic approaches for differentiation 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 internal transcribed spacer (ITS) regions of ribosomal DNA is the most commonly used approach for determination 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 Europe being listed on the quarantine A2 list by the European and Mediterranean Plant Protection Organization (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 Plateau 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 morphometric 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 production areas in Turkey.

Eur J Plant Pathol (2018) 151:195–200

Materials and methods Nematode populations Nematode populations were obtained from onion fields in ten provinces in Mediterranean, Trace, Aegean 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 Konya, twelve from Ankara, and two from Eskisehir provinces where onion production is most practised in Turkey (Table 2). The nematodes used for molecular 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 betamerkaptoetanol, 40 μl 20 mg/ml Proteinase K and 950 μl WLB- buffer (2 ml 1 M NaCl, 2 ml 1 M TrisHCl and 5.5 ml of ddH2O). The mixture was incubated 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 ddH2O 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

Eur J Plant Pathol (2018) 151:195–200

control. Specific amplifications were repeated three times to assess the reproducibility of PCR. PCR amplicons were separated in 1–2% agarose gels. Electrophoresis was conducted at 90 V of constant power for 3 h. PCR reactions (for all primers except PF1-PR1 primer set) were 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 MgCl2, 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 fragments 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 amplifying only D. dipsaci sensu stricto group. Fifteen of the 51 nematode populations showed expected band size with all primer sets used in the molecular screening. The numbers of these samples are 1, 2

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and 4 in Adana Province; 8 and 11 in Hatay Province; 12, 15 and 18 in Tekirdag Province; 25 in Bursa Province; 31 in Aksaray Province; 34 and 35 in Karaman Province; 50 in Eskisehir Province; 54 in Amasya Province and 58 in Corum Province. 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 populations 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 accordance with Marek et al. (2005). Expected PCR product size 517 bp (Vrain et al. 1992) for D. dipsaci were obtained using DdpS1rDNA2 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 Targeted regions Size (bp)

Reference

1

PF1 PR1

5′-AAC GGC TCT GTT GGC TTC TAT-3 5′-ATT TAC GAC CCT GAG CCA GAT-3′

327 bp

Flanking ITS regions

Marek et al. (2005)

2

PF2 PR2

5′-TCG CGA GAA TCA ATG AGT ACC-3′ 5′-AAT AGC CAG TCG ATT CCG TCT-3′

396 bp

Flanking ITS regions

Marek et al. (2005)

3

DdpS1 5′-TGG CTG CGT TGA AGA GAA CT-3′ rDNA2 5′-TTT CAC TCG CCG TTA CTA AGG-3′

517 bp

5.8S rDNA and flanking Vrain et al. (1992) ITS regions

4

DitNF1 5′-TTA TGA CAA ATT CAT GGC GG-3′ rDNA2 5′-TTT CAC TCG CCG TTA CTA AGG-3′

263 bp

18S and ITS1 regions

Subbotin et al. (2005)

5

H05 H06

5′- TCA AGG TAA TCT TTT TCC CCA CT-3′ 242 bp 5′-CAA CTG CTA ATG CGT GCT CT-3′

Flanking ITS regions

Esquibet et al. (2003)

198

Eur J Plant Pathol (2018) 151:195–200

Table 2 Origin and extraction medium of nematode populations tested in the study and PCR results with specific primer sets (set 1: PF1PR1, 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 Set 1 Set 2 Set 3 Set 4 Set 5 from plant (P) / soil (S)

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

+

+

+

+

–

Hatay

36,361,299

36,414,779

P

+

+

+

+

+

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

+

+

+

+

–

1

Mediterranean

11 12

Trace

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

–

–

–

–

–

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

–

–

–

–

–

19

29

Aegean

Central Anatolia

Eur J Plant Pathol (2018) 151:195–200

199

Table 2 (continued) Sample no Geographical region Province

Latitude (N) Longitude (E) Nematode extracted Set 1 Set 2 Set 3 Set 4 Set 5 from plant (P) / soil (S)

42

Ankara

39,277,253

32,075229

S

–

–

–

–

–

43

Ankara

39,346,711

31,979,551

S

–

–

–

–

–

44

Ankara

39,434,225

31,985,016

S

–

–

–

–

–

45

Ankara

39,719,689

32,393,649

S

–

–

–

–

–

46

Ankara

39,707,854

32,403,380

S

–

–

–

–

–

47

Ankara

39,838,486

32,293,308

S

–

–

–

–

–

48

Ankara

39,846,889

32,298,189

S

–

–

–

–

–

49

Ankara

40,110,527

31,857,132

S

–

–

–

–

–

50

Eskisehir 39,707,117

30,412,748

S

+

+

+

+

–

51

Eskisehir 39,458,774

31,328,988

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. However, 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 phylogenetic 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 molecular tools. Acknowledgements This research has been financially supported 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 presented 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 coauthors, 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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