Turkish Journal of Agriculture and Forestry Turk J Agric For (2015) 39: 91-98 http://journals.tubitak.gov.tr/agriculture/ © TÜBİTAK Research Article doi:10.3906/tar-1404-56

Morphological and molecular identification of cereal cyst from the eastern Mediterranean region of

1, 2 2 3 4 Mustafa İMREN *, Lieven WAEYENBERGE , Nicole VIAENE , İbrahim Halil ELEKCİOĞLU , Abdelfattah DABABAT 1 Department of Plant Protection, Faculty of Agriculture and Natural Science, Abant İzzet Baysal University, Bolu, Turkey 2 Agricultural Research Centre Burg, Ghent, Belgium 3 Department of Plant Protection, Faculty of Agriculture, Çukurova University, Adana, Turkey 4 International Maize and Wheat Improvement Center, Ankara, Turkey

Received: 10.04.2014 Accepted: 27.09.2014 Published Online: 02.01.2015 Printed: 30.01.2015

Abstract: The morphological and molecular characteristics of 41 populations of cereal cyst nematodes ( avenae group) collected in Adana, Osmaniye, Kahramanmaraş, Hatay, Gaziantep, and provinces in the eastern Mediterranean region of Turkey were studied. The morphological characters and morphometric features of second-stage juveniles and cysts showed the presence of 3 Heterodera species: H. avenae, H. filipjevi, and H. latipons. All morphological values of these distinct populations were very similar to those previously described for these species. Genetic variation was observed among the identified cyst species H. avenae, H. filipjevi, and H. latipons. Intraspecific polymorphism was observed within H. avenae and H. latipons but not in H. filipjevi populations. Molecular analysis using ITS regions of rDNA confirmed the identities of the 3 Heterodera species. According to our results, 75% of isolates were identified as H. avenae, 15% as H. latipons, and 10% as H. filipjevi.

Key words: Cereal cyst nematode, morphology, sequences, phylogenetic

1. Introduction Ha21 and Ha33, respectively (İmren et al., 2013; Toktay Cereal cyst nematodes (CCNs) cause serious economic et al., 2013). damage in cereal crops worldwide, especially in temperate Heterodera species can be differentiated from each regions (Rivoal and Cook, 1993; Evans and Rowe, 1998; other by their morphological and morphometric features Nicol and Rivoal, 2008). Their ability to withstand (Subbotin et al., 1999; Handoo, 2002; Abidou et al., 2005). desiccation in the protective cyst stage enhances their However, the increasing number of species in this group dispersal and survival. The Heterodera avenae group makes reliable morphological identification more difficult consists of 12 valid and several undescribed species that and time consuming (Rivoal et al., 2003; Subbotin et al., infect cereals and grasses (Wouts and Sturhan, 1995; Gabler 2003). DNA-sequence variation in the internal transcribed et al., 2000; Andres et al., 2001). The main cyst nematode spacer (ITS) regions of ribosomal DNA can be used to species that attack cereals are H. avenae Wollenweber, identify many nematode taxa and phylogenetic relations H. filipjevi(Madzhidov) Stelter, and H. latipons Franklin (Subbotin et al., 2001). The ribosomal genes are highly (Rivoal and Cook, 1993; Evans and Rowe, 1998; Nicol et conserved, allowing for the construction of polymerase al., 2002). chain reaction (PCR) primers. However, the ITS regions In Turkey, H. avenae, H. filipjevi, and H. latipons have separating the ribosomal genes are less conserved, which been increasingly detected over the last few years by allows for the detection of polymorphisms (Madani et al., using morphological and molecular tools, and they are 2004; Kumar et al., 2009). recognized as damaging pathogens of wheat and barley The objective of this study was to identify CCN cultivars in different parts of the country, especially in the populations originating from the eastern Mediterranean southeastern Anatolian region and the central Anatolian region of Turkey. This was done based on morphological plateau (Rumpenhorst et al., 1996; Subbotin et al., 2003; and molecular features. The ITS-rDNA region was Abidou et al., 2005; Şahin 2010; İmren et al., 2012; Dababat sequenced and compared with sequences available in the et al., 2014). Additionally, the pathotypes of H. avenae and GenBank database. Finally, the phylogenetic relationships H. filipjeviin Turkey were characterized as pathotypes between these populations were determined. * Correspondence: [email protected] 91 İMREN et al. / Turk J Agric For

2. Materials and methods 2.1. Nematode population A survey was conducted in Adana, Osmaniye, Kahramanmaraş, Kilis, Gaziantep, and Hatay provinces in the eastern Mediterranean region of Turkey in July 2010 and 2011 after cereals were harvested. During the surveys, 252 soil samples, consisting of 10 subsamples representative for each field, were collected in each location. Cysts were extracted from each soil sample using the sieving and floatation method (Shepherd, 1986). Extracted cysts, which were retained on the 250-µm sieve, were handpicked with a needle using a dissecting stereomicroscope. A total of 41 cyst-forming nematode populations were collected from 252 samples. All cysts were stored in dry conditions at room temperature for molecular and morphological identification. 2.2. Morphological characterization The perineal portions of the cysts of 41 populations were cut and prepared for microscopic examination (Hooper, 1986). For each population, vulval cones of several mature cysts were mounted in glycerin jelly. The underbridge structure, the shape of semifenestra in the fenestral area, and the development of bullae were observed under a microscope and, depending on these structures, the identifications were done (Handoo, 2002) (Figure 1). For each population, second-stage juveniles were killed by gentle heat, fixed in TAF, and embedded in glycerol, resulting in a permanent slide (De Grisse, 1969). Four characters of juveniles (J2) known to be important for taxonomic diagnosis in this group (Subbotin et al., 1999) were included in the analysis. Ten second-stage juveniles of selected cyst populations were examined and measured using a LEICA light microscope. 2.3. Molecular characterization 2.3.1. DNA extraction For each population, 1 cyst was transferred into 45 µL of double-distilled water (ddH2O) in an Eppendorf tube and crushed using a microhomogenizer (Vibro Mixer). After centrifugation of the squashed cyst content, 40 µL of the mix was transferred to a PCR tube (0.2 mL) containing 50 µL of worm lysis buffer and 10 µL of proteinase K (20 mg/mL). The tubes were frozen at –80 °C for at least 10 min and then incubated at 65 °C for 1 h and 95 °C for 10 min consecutively in a thermocycler. After incubation, the tubes were centrifuged for 1 min at 14,000 rpm and kept at –20 °C until use (Waeyenberge et al., 2000). 2.3.2. PCR amplification Figure 1. Fenestral region of cysts of Heterodera avenae, H. PCR amplification of the ITS-rDNA region was filipjevi, and H. latipons. done in a 50-µL reaction volume containing 22 µL of ddH2O, 25 µL of 2X Dream Taq PCR Master Mix (Fermentas Life Sciences), 1 µM each of forward primer (5’-TCCTCCGCTAAATGATATG-3’) (Ferris et al., 1994), (5’-CGTAACAAGGTAGCTGTAG-3’) and reverse primer and 1 µL of DNA extract. The PCR thermal cycler program

92 İMREN et al. / Turk J Agric For consisted of 5 min at 95 °C; 40 cycles of 94 °C for 30 s, 48 3. Results °C for 45 s, and 72 °C for 45 s; and a final extension step 3.1. Morphological characteristics of the Heterodera of 8 min at 72 °C. After PCR amplification, 5 µL of each populations PCR product was mixed with 1 µL of 6X loading buffer The morphological characters and morphometric features (Fermentas Life Sciences) and loaded on a 1.5% standard of the juveniles (body length, stylet length, tail length, TAE buffered agarose gel. After electrophoresis (100 V and length of the hyaline part of the tail) as well as cysts for 40 min), the gel was stained with ethidium bromide (fenestral length, semifenestral width, vulval bridge width, (0.1 µg/mL) for 15 min and visualized and photographed and vulval slit length) were measured. Based on these under UV light. The remaining PCR product was stored features, populations were identified as H. avenae, H. at –20 °C (Subbotin et al., 2003; Tanha Maafi et al., 2003). filipjevi, or H. latipons. 2.3.3. Purification and sequencing The cysts of all species were lemon-shaped, partially The remainder (2 × 45 µL) of the PCR product was loaded transparent, between light and dark brown in color, and on 1% agarose gel for electrophoresis (100 V, 40 min). with ridges running in zigzag patterns. No underbridge The purification process was performed as described in (Figure 1) was found in the vulval cone structures of the manufacturer’s instructions (Wizard SV Gel and PCR any of the examined populations of H. avenae, and the Clean-Up System Kit, Promega). DNA from 41 samples, fenestral length and semifenestral widths were 67–75 µm representing 41 populations, was sequenced (Macrogen) and 19–22 µm, respectively. The fenestral length and the in both directions to obtain overlapping sequences of semifenestral width of H. filipjevi were 45–67 µm and 16– both DNA strands. The sequences were then edited and 24 µm, respectively, and the vulval cone was bifenestrate analyzed. with an underbridge. The fenestration of H. latipons cysts shows 2 distinct semifenestrae, which are more than a 2.3.4. Phylogenetic analysis semifenestral width apart. The underbridge is strong and For identification purposes, an alignment of all our the extremities are bi-trifurcate. Bullae are few to absent. obtained sequences together with those of representatives The fenestral lengths and semifenestral widths of H. of all species of the H. avenae group, as well as some of latipons were 64–67 µm and 19–26 µm, respectively (Table 1). the other Heterodera groups available in GenBank, was Second-stage juveniles of these 3 Heterodera species constructed using ClustalX2.0 (Thompson et al., 1997). are cylindrical in shape, with a slightly offset head and a In agreement with Madani et al. (2004), Cryphodera tapering round tail tip. The stylet is strong with shallow brinkmani (AF274418) and Meloidodera alni (AF274419) anteriorly concave basal knobs. The juvenile body length were also incorporated in the phylogenetic analysis. For of the Turkish populations of H. avenae varied from 584 the phylogenetic analysis, H. latipons (Turkey-JX024184; to 601 µm and stylet length was 24–29 µm with anteriorly Syria-JX024179, JX024182; Iran-HM560790; Morocco- concave basal knobs. The juvenile body length ofH. JQ319036), H. hordecalis (Estonia-AY692356), H. filipjevi filipjevi varied from 477 to 547 µm and stylet length was (Iran-AY148404), H. ustinovi (Belgium-AY148407), H. 23–25 µm with moderately concave stylet knobs. Second- pratensis (Germany-AY148384, Russia-AY148351), H. stage juvenile body length of H. latipons varied from 414 to australis (Australia-AY148394, AY148395, AY148396), 599 µm and stylet length was 22–24 µm (Table 1). H. mani (Germany-AY148375, AY148377, AY148378), 3.2. Molecular characteristics of Heterodera populations H. avenae (Iran-HM560755, Syria-JX024197, China- Forty-one Heterodera populations were sequenced, JN657198, HQ450299, EU616697), H. aucklandica excluding 2 populations. Sequenced Heterodera (United Kingdom, AY148380), H. arenaria (United populations were identified using their ITS-rDNA regions. Kingdom-AF274396), H. humuli (Iran-AF498384), Based on ITS-rDNA sequences, the populations from Kilis, H. schachtii (Morocco-AY166436), H. ciceri (Syria- Osmaniye, and Hatay were identified as H. avenae and H. AY045758), and H. goettingiana (Iran-AF498374) were latipons; those from Adana and Gaziantep as H. avenae; used. and those from Kahramanmaraş as H. filipjevi (Table 2). The alignment was imported into MEGA5 (Tamura The Heterodera avenae group investigated in our study et al., 2011) and, after checking 24 different nucleotide was clearly divided into 4 groups in a phylogenetic tree substitution models, the model with the lowest Bayesian based on the ITS-I, II, and 5.8S rDNA sequences. All groups information criterion score and Hasegawa–Kishino– were formed distinctly from each other in the Heterodera Yano value with gamma distribution was selected. The sequences and were added from GenBank (Figure 2), maximum likelihood tree and bootstrap values were supported by a moderate to high bootstrap value. Group calculated with 100 replicates. The analysis involved 41 I included some H. avenae and H. arenaria populations nucleotide sequences with a total of 771 positions in the (AF274396), whereas Group II included other H. avenae final dataset. populations along with H. avenae populations from Iran

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Table 1. Morphometrics of cysts and second-stage juveniles in 41 populations of 3 species of the Heterodera avenae complex in the eastern Mediterranean region of Turkey.

H. avenae H. filipjevi H. latipons 593.6 ± 2.1 516.3 ± 6.2 475.0 ± 16.4 Body length (584–601) (476.8–547) (414.4–598.4) 25.6 ± 0.7 24.2 ± 0.2 23.0 ± 0.24 Stylet length (24–28.8) (23.2–25.6) (22.4–24) 74.6 ± 0.6 53.92 ± 2.3 50.48 ± 1.47 Tail length (72–76.8) (41.6–64) (44.8–60.8) 48.8 ± 0.51 26.4 ± 1.67 26 ± 0.62 Hyaline length (48–51.2) (22.4–40) (23.2–28.8) 71.36 ± 1.64 54.08 ± 4.24 66.3 ± 0.6 Fenestral length (67.2–75.2) (44.8–67.2) (64–67.2) 21.1 ± 0.6 21.0 ± 1.34 23.0 ± 1.3 Semifenestral width (19.2–22.4) (16–24) (19.2–25.6) 12.16 ± 0.64 12.16 ± 1.50 19.5 ± 0.6 Vulval bridge width (11.2–14.4) (7.2–16) (17.6–20.8) 19.9 ± 1 24.6 ± 1.08 24.5 ± 1.1 Vulval slit length (17.6–2.4) (9.6–20.8) (22.4–27.2)

Measurements are given as mean ± standard deviation (range) and are in µm.

(HM560755), Syria (JX024197), and China (HQ450299, by having a strong underbridge and a lack of distinct JN657198, and EU616697). Group III included H. filipjevi bullae in the vulval cone. The absence of an underbridge populations grouped with H. filipjevi populations from Iran and the presence of well-developed bullae surrounding (AY148404). Group IV included H. latipons populations the vulval cone of the cysts of H. avenae are known to from Turkey (JX024184), Syria (JX024179 and JX024182), be useful characters for morphological identification. H. Iran (HM560790), and Morocco (JQ319036). Moreover, avenae and H. latipons could be readily differentiated by in the last group, H. latipons populations 5 and 34 were the underbridge and bullae presence in the vulval cone in separated from the rest of the group, indicating that they H. latipons (Wouts and Sturhan, 1995; Rivoal et al., 2003; were a subgroup of Group IV. Subbotin et al., 2003). The second-stage juveniles of H. avenae have a longer 4. Discussion tail, stylet, and hyaline part of tail compared to similar Since H. avenae, H. latipons, and H. filipjevi may occur characteristics for H. filipjevi and H. latipons. In addition, as mixtures of populations in the eastern Mediterranean H. latipons populations were separated from the other region of Turkey, accurate species identification is 2 Heterodera species by having the shortest body, stylet, required. Molecular and morphological characterization tail, and hyaline tail lengths. Similarly, morphometric indicated that species of the H. avenae complex inside the characters, such as the hyaline part of the tail of the H. avenae group sensu lato represent a paraphyletic taxon, second-stage juveniles and the fenestra length of cysts, including extensive genetic variation between and within were also used to distinguish these species (Madzhidov, the species involved (Subbotin et al., 2003). 1981; Valdeolivas and Romero, 1990; Wouts and Sturhan, Correlations between several morphological characters 1995). of cysts and juveniles allowed for the restriction of the In this study, intraspecific polymorphism based on numbers of characters that differentiate these species of sequence analysis was shown among populations of H. the H. avenae group. In this study, the cysts of H. avenae avenae, as in previous studies (Bekal et al., 1997; Subbotin had heavy prominent bullae that completely surrounded et al., 1999; Handoo, 2002; Rivoal et al., 2003). The H. the vulval cone, and no underbridge. Compared to H. avenae populations in this study consisted of 2 subgroups. avenae, H. filipjevi had less prominent bullae and a clear These were separated from each other by a value of 63%. but thin underbridge. H. latipons differed from the others Therefore, they were referred to as Group 1 and Group 2.

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Table 2. Locations of Heterodera species in the eastern Mediterranean region of Turkey.

Code Province Location Species Accession number 1 Hatay Merkez-Kırıkhan H. avenae KM199803 2 Merkez-Kırıkhan H. avenae KM199804 3 Toki Evleri-Kırıkhan H. avenae KM199805 4 Türbe-Kırıkhan H. avenae KM199806 5 Mazmanlı-Hassa H. latipons KM199807 6 Başpınar-Kırıkhan H. avenae KM199808 7 Şarkhamamı-Kumlu H. avenae KM199809 8 Karakaya-Reyhanlı H. avenae KM199810 9 Müşrüfe-Reyhanlı H. avenae KM199811 10 Beşarslan-Reyhanlı H. avenae KM199812 11 İskan Evleri-Kırıkhan H. avenae KM199813 12 Merkez-Kırıkhan H. avenae KM199814 13 Adana Giriş Gediği-Sarıçam H. avenae KM199815 14 Kepeztepe-Sarıçam H. avenae KM199816 15 Tülüler-Sarıçam H. avenae KM199817 16 Dutluca-Sarıçam H. avenae KM199818 17 Bucatepe-Sarıçam H. avenae KM199819 18 Çabutlu-Sarıçam H. avenae KM199820 19 Karlık-Sarıçam H. avenae KM199821 20 Yazıbaşı-Karaisalı H. avenae KM199822 21 Hacılar-Karaisalı H. avenae KM199823 22 Kilis Deliçay-Musabeyli H. latipons KM199824 23 Acar-Merkez H. latipons KM199825 24 Yığmatepe-Merkez H. latipons KM199826 25 Doğanlı- H. latipons KM199827 26 Gaziantep Türkyurdu-Karkamış H. avenae KM199828 27 Karaman-Oğuzeli H. avenae KM199829 28 Akçaköy-Karkamış H. avenae KM199830 29 Nogaylar-Nurdağı H. avenae KM199831 30 Beydilli-Nurdağı H. avenae KM199832 31 Sakçagözü-Nurdağı H. avenae KM199833 32 Osmaniye Kuzucu-Mustafabeyli H. avenae KM199834 33 İmran-Mustafabeyli H. avenae KM199835 34 Günyazı-Mustafabeyli H. latipons KM199836 35 Adana Yolu-Merkez H. avenae KM199837 36 Cevdetiye-Merkez H. avenae KM199838 37 Mahadinli-Merkez H. avenae KM199839 38 Kahramanmaraş Merkez-Türkoğlu H. filipjevi KM199840 39 Merkez-Afşin H. filipjevi KM199841 40 Santral-Afşin H. filipjevi KM199842 41 Elbistan Yolu-Afşin H. filipjevi KM199843

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Heterodera avenae-Osmaniye-33 H. avenae-Osmaniye-37 H. avenae-Osmaniye-32 H. avenae-Gaziantep-31 H. avenae-Gaziantep-30

H. avenae-Adana-18

H. avenae-Adana-17

H. avenae-Adana-16

H. avenae-Hatay-12

63 H. avenae-Hatay-11

H. avenae-Hatay-10 H. avenae-Hatay-9 H. avenae-Hatay-8 H. avenae-Hatay-6 H. avenae-Hatay-3 H. avenae-Hatay-2

H. avenae-Hatay-1 Heterodera arenaria AF274396 H. avenae-Adana-21 H. avenae-Osmaniye-36 Heterodera aucklandica AY148380 Heterodera avenae JX024197 Heterodera avenae HM560755 H. avenae-Hatay-4 H. avenae-Hatay-7 H. avenae-Adana-13 H. avenae-Adana-14 99 H. avenae-Adana-15 H. avenae-Adana-19

H. avenae-Adana-20 H. avenae-Gaziantep-26 H. avenae-Gaziantep-27 H. avenae-Gaziantep-28 H. avenae-Gaziantep-29 H. avenae-Osmaniye-35 Heterodera mani AY148377 Heterodera mani AY148378 Heterodera mani AY148375 Heterodera australis AY148395 82 Heterodera australis AY148394 Heterodera avenae HQ450299 Heterodera avenae JN657198 Heterodera avenae EU616697 Heterodera pratensis AY148384 Heterodera pratensis AY148351 71 Heterodera australis AY148396 H. filipjevi-Kahramanmaras-38 Heterodera filipjevi AY148404 H. filipjevi-Kahramanmaras-39 99 H. filipjevi-Kahramanmaras-40 H. filipjevi-Kahramanmaras-41 64 Heterodera ustinovi AY148407 Heterodera hordecalis AY692356 99 Heterodera ciceri AY045758 Heterodera schachtii AY166436 98 H. latipons-Hatay-5 H. latipons-Osmaniye-34 Heterodera latipons JX024182 64 69 Heterodera latipons JX024184 H. latipons-Kilis-22 Heterodera latipons JX024179 95 Heterodera latipons HM560790

H. latipons-Kilis-23 84 Heterodera latiponsJQ319036 64 H. latipons-Kilis-24 H. latipons-Kilis-25 Heterodera humuli AF498384 Heterodera goettingiana AF498374 Cryphodera brinkmani AF274418 99 Meloidodera alni AF274419 Figure 2. Phylogenetic relationships between 4 Heterodera populations collected from the eastern Mediterranean region of Turkey and international samples. Populations from the eastern Mediterranean region are designated with codes as given in Table 2.

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More genetic dissimilarities were revealed by using the H. latipons was the prevalent species in cereal fields in same or extra chromosomal (mitochondrial) DNA (De the southeastern Anatolia region, while H. filipjevi was Giorgi et al., 1994). distributed in most wheat-growing areas of the central The results of our study did not show any intraspecific Anatolian plateau in Turkey. polymorphism between H. filipjevi populations. This study highlighted the ecoregional distribution Furthermore, Bekal et al. (1997) did not detect any genetic of CCNs in the eastern Mediterranean region. Further variation among the different populations of this species. detailed surveys and comprehensive pathotype studies The H. latipons populations in this study consisted of 2 on the Heterodera avenae group using populations from subgroups. Similarly, differences in ITS sequences were different regions in Turkey are necessary to determine previously reported for other H. latipons populations the distribution patterns of CCN more accurately, and to (Ferris et al., 1999; Madani et al., 2004). determine whether differences in pathotypes occur and CCNs are the most important pathogens of wheat and thereby cause different resistance responses in small grain other cereals in Turkey, and they have adverse effects on cultivars being developed to improve the management of the production of wheat. Three species of cyst-forming CCNs in Turkey. nematodes belonging to the Heterodera avenae group were identified in cereal fields in Turkey. H. avenae in Turkey Acknowledgment was reported by Yüksel (1973), Subbotin et al. (2003), This study was funded by grant number 110 O 632 of a and Abidou et al. (2005) based on a few samples. İmren TÜBİTAK project (Scientific and Technological Research et al. (2012) and Şahin et al. (2010) also determined that Council of Turkey).

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