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http://journals.tubitak.gov.tr/agriculture/

Turkish Journal of Agriculture and Forestry Turk J Agric For(2015) 39: 91-98© TÜBİTAKdoi:10.3906/tar-1404-56

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

Mustafa İMREN1,*, Lieven WAEYENBERGE2, Nicole VIAENE2, İbrahim Halil ELEKCİOĞLU3, Abdelfattah DABABAT41Department of Plant Protection, Faculty of Agriculture and Natural Science, Abant İzzet Baysal University, Bolu, Turkey

2Agricultural Research Centre Burg, Ghent, Belgium3Department of Plant Protection, Faculty of Agriculture, Çukurova University, Adana, Turkey

4International Maize and Wheat Improvement Center, Ankara, Turkey

* Correspondence: m.imren37@gmail.com

1. IntroductionCereal cyst nematodes (CCNs) cause serious economic damage in cereal crops worldwide, especially in temperate regions (Rivoal and Cook, 1993; Evans and Rowe, 1998; Nicol and Rivoal, 2008). Their ability to withstand desiccation in the protective cyst stage enhances their dispersal and survival. The Heterodera avenae group consists of 12 valid and several undescribed species that infect cereals and grasses (Wouts and Sturhan, 1995; Gabler et al., 2000; Andres et al., 2001). The main cyst nematode species that attack cereals are H. avenae Wollenweber, H. filipjevi (Madzhidov) Stelter, and H. latipons Franklin (Rivoal and Cook, 1993; Evans and Rowe, 1998; Nicol et al., 2002).

In Turkey, H. avenae, H. filipjevi, and H. latipons have been increasingly detected over the last few years by using morphological and molecular tools, and they are recognized as damaging pathogens of wheat and barley cultivars in different parts of the country, especially in the southeastern Anatolian region and the central Anatolian plateau (Rumpenhorst et al., 1996; Subbotin et al., 2003; Abidou et al., 2005; Şahin 2010; İmren et al., 2012; Dababat et al., 2014). Additionally, the pathotypes of H. avenae and H. filipjevi in Turkey were characterized as pathotypes

Ha21 and Ha33, respectively (İmren et al., 2013; Toktay et al., 2013).

Heterodera species can be differentiated from each other by their morphological and morphometric features (Subbotin et al., 1999; Handoo, 2002; Abidou et al., 2005). However, the increasing number of species in this group makes reliable morphological identification more difficult and time consuming (Rivoal et al., 2003; Subbotin et al., 2003). DNA-sequence variation in the internal transcribed spacer (ITS) regions of ribosomal DNA can be used to identify many nematode taxa and phylogenetic relations (Subbotin et al., 2001). The ribosomal genes are highly conserved, allowing for the construction of polymerase chain reaction (PCR) primers. However, the ITS regions separating the ribosomal genes are less conserved, which allows for the detection of polymorphisms (Madani et al., 2004; Kumar et al., 2009).

The objective of this study was to identify CCN populations originating from the eastern Mediterranean region of Turkey. This was done based on morphological and molecular features. The ITS-rDNA region was sequenced and compared with sequences available in the GenBank database. Finally, the phylogenetic relationships between these populations were determined.

Abstract: The morphological and molecular characteristics of 41 populations of cereal cyst nematodes (Heterodera avenae group) collected in Adana, Osmaniye, Kahramanmaraş, Hatay, Gaziantep, and Kilis 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 nematode 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

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

Research Article

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2. Materials and methods2.1. Nematode populationA 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 characterizationThe 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 characterization2.3.1. DNA extractionFor 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 PCR amplification of the ITS-rDNA region was 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’-CGTAACAAGGTAGCTGTAG-3’) and reverse primer

(5’-TCCTCCGCTAAATGATATG-3’) (Ferris et al., 1994), and 1 µL of DNA extract. The PCR thermal cycler program

Figure 1. Fenestral region of cysts of Heterodera avenae, H. filipjevi, and H. latipons.

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consisted of 5 min at 95 °C; 40 cycles of 94 °C for 30 s, 48 °C for 45 s, and 72 °C for 45 s; and a final extension step of 8 min at 72 °C. After PCR amplification, 5 µL of each PCR product was mixed with 1 µL of 6X loading buffer (Fermentas Life Sciences) and loaded on a 1.5% standard TAE buffered agarose gel. After electrophoresis (100 V for 40 min), the gel was stained with ethidium bromide (0.1 µg/mL) for 15 min and visualized and photographed under UV light. The remaining PCR product was stored at –20 °C (Subbotin et al., 2003; Tanha Maafi et al., 2003).2.3.3. Purification and sequencingThe remainder (2 × 45 µL) of the PCR product was loaded on 1% agarose gel for electrophoresis (100 V, 40 min). The purification process was performed as described in the manufacturer’s instructions (Wizard SV Gel and PCR Clean-Up System Kit, Promega). DNA from 41 samples, representing 41 populations, was sequenced (Macrogen) in both directions to obtain overlapping sequences of both DNA strands. The sequences were then edited and analyzed. 2.3.4. Phylogenetic analysisFor identification purposes, an alignment of all our obtained sequences together with those of representatives of all species of the H. avenae group, as well as some of the other Heterodera groups available in GenBank, was constructed using ClustalX2.0 (Thompson et al., 1997). In agreement with Madani et al. (2004), Cryphodera brinkmani (AF274418) and Meloidodera alni (AF274419) were also incorporated in the phylogenetic analysis. For the phylogenetic analysis, H. latipons (Turkey-JX024184; Syria-JX024179, JX024182; Iran-HM560790; Morocco-JQ319036), H. hordecalis (Estonia-AY692356), H. filipjevi (Iran-AY148404), H. ustinovi (Belgium-AY148407), H. pratensis (Germany-AY148384, Russia-AY148351), H. australis (Australia-AY148394, AY148395, AY148396), H. mani (Germany-AY148375, AY148377, AY148378), H. avenae (Iran-HM560755, Syria-JX024197, China-JN657198, HQ450299, EU616697), H. aucklandica (United Kingdom, AY148380), H. arenaria (United Kingdom-AF274396), H. humuli (Iran-AF498384), H. schachtii (Morocco-AY166436), H. ciceri (Syria-AY045758), and H. goettingiana (Iran-AF498374) were used.

The alignment was imported into MEGA5 (Tamura et al., 2011) and, after checking 24 different nucleotide substitution models, the model with the lowest Bayesian information criterion score and Hasegawa–Kishino–Yano value with gamma distribution was selected. The maximum likelihood tree and bootstrap values were calculated with 100 replicates. The analysis involved 41 nucleotide sequences with a total of 771 positions in the final dataset.

3. Results3.1. Morphological characteristics of the Heterodera populationsThe morphological characters and morphometric features of the juveniles (body length, stylet length, tail length, and length of the hyaline part of the tail) as well as cysts (fenestral length, semifenestral width, vulval bridge width, and vulval slit length) were measured. Based on these features, populations were identified as H. avenae, H. filipjevi, or H. latipons.

The cysts of all species were lemon-shaped, partially transparent, between light and dark brown in color, and with ridges running in zigzag patterns. No underbridge (Figure 1) was found in the vulval cone structures of any of the examined populations of H. avenae, and the fenestral length and semifenestral widths were 67–75 µm and 19–22 µm, respectively. The fenestral length and the semifenestral width of H. filipjevi were 45–67 µm and 16–24 µm, respectively, and the vulval cone was bifenestrate with an underbridge. The fenestration of H. latipons cysts shows 2 distinct semifenestrae, which are more than a semifenestral width apart. The underbridge is strong and the extremities are bi-trifurcate. Bullae are few to absent. The fenestral lengths and semifenestral widths of H. latipons were 64–67 µm and 19–26 µm, respectively (Table 1).

Second-stage juveniles of these 3 Heterodera species are cylindrical in shape, with a slightly offset head and a tapering round tail tip. The stylet is strong with shallow anteriorly concave basal knobs. The juvenile body length of the Turkish populations of H. avenae varied from 584 to 601 µm and stylet length was 24–29 µm with anteriorly concave basal knobs. The juvenile body length of H. filipjevi varied from 477 to 547 µm and stylet length was 23–25 µm with moderately concave stylet knobs. Second-stage juvenile body length of H. latipons varied from 414 to 599 µm and stylet length was 22–24 µm (Table 1).3.2. Molecular characteristics of Heterodera populationsForty-one Heterodera populations were sequenced, excluding 2 populations. Sequenced Heterodera populations were identified using their ITS-rDNA regions. Based on ITS-rDNA sequences, the populations from Kilis, Osmaniye, and Hatay were identified as H. avenae and H. latipons; those from Adana and Gaziantep as H. avenae; and those from Kahramanmaraş as H. filipjevi (Table 2).

The Heterodera avenae group investigated in our study was clearly divided into 4 groups in a phylogenetic tree based on the ITS-I, II, and 5.8S rDNA sequences. All groups were formed distinctly from each other in the Heterodera sequences and were added from GenBank (Figure 2), supported by a moderate to high bootstrap value. Group I included some H. avenae and H. arenaria populations (AF274396), whereas Group II included other H. avenae populations along with H. avenae populations from Iran

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(HM560755), Syria (JX024197), and China (HQ450299, JN657198, and EU616697). Group III included H. filipjevi populations grouped with H. filipjevi populations from Iran (AY148404). Group IV included H. latipons populations from Turkey (JX024184), Syria (JX024179 and JX024182), Iran (HM560790), and Morocco (JQ319036). Moreover, in the last group, H. latipons populations 5 and 34 were separated from the rest of the group, indicating that they were a subgroup of Group IV.

4. Discussion Since H. avenae, H. latipons, and H. filipjevi may occur as mixtures of populations in the eastern Mediterranean region of Turkey, accurate species identification is required. Molecular and morphological characterization indicated that species of the H. avenae complex inside the H. avenae group sensu lato represent a paraphyletic taxon, including extensive genetic variation between and within the species involved (Subbotin et al., 2003).

Correlations between several morphological characters of cysts and juveniles allowed for the restriction of the numbers of characters that differentiate these species of the H. avenae group. In this study, the cysts of H. avenae had heavy prominent bullae that completely surrounded the vulval cone, and no underbridge. Compared to H. avenae, H. filipjevi had less prominent bullae and a clear but thin underbridge. H. latipons differed from the others

by having a strong underbridge and a lack of distinct bullae in the vulval cone. The absence of an underbridge and the presence of well-developed bullae surrounding the vulval cone of the cysts of H. avenae are known to be useful characters for morphological identification. H. avenae and H. latipons could be readily differentiated by the underbridge and bullae presence in the vulval cone in H. latipons (Wouts and Sturhan, 1995; Rivoal et al., 2003; Subbotin et al., 2003).

The second-stage juveniles of H. avenae have a longer tail, stylet, and hyaline part of tail compared to similar characteristics for H. filipjevi and H. latipons. In addition, H. latipons populations were separated from the other 2 Heterodera species by having the shortest body, stylet, tail, and hyaline tail lengths. Similarly, morphometric characters, such as the hyaline part of the tail of the second-stage juveniles and the fenestra length of cysts, were also used to distinguish these species (Madzhidov, 1981; Valdeolivas and Romero, 1990; Wouts and Sturhan, 1995).

In this study, intraspecific polymorphism based on sequence analysis was shown among populations of H. avenae, as in previous studies (Bekal et al., 1997; Subbotin et al., 1999; Handoo, 2002; Rivoal et al., 2003). The H. avenae populations in this study consisted of 2 subgroups. These were separated from each other by a value of 63%. Therefore, they were referred to as Group 1 and Group 2.

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

Body length 593.6 ± 2.1(584–601)516.3 ± 6.2 (476.8–547)

475.0 ± 16.4 (414.4–598.4)

Stylet length 25.6 ± 0.7 (24–28.8)24.2 ± 0.2 (23.2–25.6)

23.0 ± 0.24(22.4–24)

Tail length 74.6 ± 0.6(72–76.8)53.92 ± 2.3 (41.6–64)

50.48 ± 1.47(44.8–60.8)

Hyaline length 48.8 ± 0.51 (48–51.2)26.4 ± 1.67 (22.4–40)

26 ± 0.62(23.2–28.8)

Fenestral length 71.36 ± 1.64 (67.2–75.2)54.08 ± 4.24 (44.8–67.2)

66.3 ± 0.6 (64–67.2)

Semifenestral width 21.1 ± 0.6 (19.2–22.4)21.0 ± 1.34 (16–24)

23.0 ± 1.3(19.2–25.6)

Vulval bridge width 12.16 ± 0.64(11.2–14.4)12.16 ± 1.50 (7.2–16)

19.5 ± 0.6(17.6–20.8)

Vulval slit length 19.9 ± 1 (17.6–2.4)24.6 ± 1.08 (9.6–20.8)

24.5 ± 1.1 (22.4–27.2)

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

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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 KM1998032 Merkez-Kırıkhan H. avenae KM1998043 Toki Evleri-Kırıkhan H. avenae KM1998054 Türbe-Kırıkhan H. avenae KM1998065 Mazmanlı-Hassa H. latipons KM1998076 Başpınar-Kırıkhan H. avenae KM1998087 Şarkhamamı-Kumlu H. avenae KM1998098 Karakaya-Reyhanlı H. avenae KM1998109 Müşrüfe-Reyhanlı H. avenae KM19981110 Beşarslan-Reyhanlı H. avenae KM19981211 İskan Evleri-Kırıkhan H. avenae KM19981312 Merkez-Kırıkhan H. avenae KM19981413 Adana Giriş Gediği-Sarıçam H. avenae KM19981514 Kepeztepe-Sarıçam H. avenae KM19981615 Tülüler-Sarıçam H. avenae KM19981716 Dutluca-Sarıçam H. avenae KM19981817 Bucatepe-Sarıçam H. avenae KM19981918 Çabutlu-Sarıçam H. avenae KM19982019 Karlık-Sarıçam H. avenae KM19982120 Yazıbaşı-Karaisalı H. avenae KM19982221 Hacılar-Karaisalı H. avenae KM19982322 Kilis Deliçay-Musabeyli H. latipons KM19982423 Acar-Merkez H. latipons KM19982524 Yığmatepe-Merkez H. latipons KM19982625 Doğanlı-Elbeyli H. latipons KM19982726 Gaziantep Türkyurdu-Karkamış H. avenae KM19982827 Karaman-Oğuzeli H. avenae KM19982928 Akçaköy-Karkamış H. avenae KM19983029 Nogaylar-Nurdağı H. avenae KM19983130 Beydilli-Nurdağı H. avenae KM19983231 Sakçagözü-Nurdağı H. avenae KM19983332 Osmaniye Kuzucu-Mustafabeyli H. avenae KM19983433 İmran-Mustafabeyli H. avenae KM19983534 Günyazı-Mustafabeyli H. latipons KM19983635 Adana Yolu-Merkez H. avenae KM19983736 Cevdetiye-Merkez H. avenae KM19983837 Mahadinli-Merkez H. avenae KM19983938 Kahramanmaraş Merkez-Türkoğlu H. filipjevi KM19984039 Merkez-Afşin H. filipjevi KM19984140 Santral-Afşin H. filipjevi KM19984241 Elbistan Yolu-Afşin H. filipjevi KM199843

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63

99

82

71

99

64

99

98

64 69

84

99

95

64

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 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 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 Heterodera australis AY148394 Heterodera avenae HQ450299 Heterodera avenae JN657198 Heterodera avenae EU616697 Heterodera pratensis AY148384 Heterodera pratensis AY148351 Heterodera australis AY148396 H. filipjevi-Kahramanmaras-38 Heterodera filipjevi AY148404 H. filipjevi-Kahramanmaras-39 H. filipjevi-Kahramanmaras-40 H. filipjevi-Kahramanmaras-41 Heterodera ustinovi AY148407 Heterodera hordecalis AY692356 Heterodera ciceri AY045758 Heterodera schachtii AY166436 H. latipons-Hatay-5 H. latipons-Osmaniye-34 Heterodera latipons JX024182 Heterodera latipons JX024184 H. latipons-Kilis-22 Heterodera latipons JX024179 Heterodera latipons HM560790 H. latipons-Kilis-23 Heterodera latiponsJQ319036 H. latipons-Kilis-24 H. latipons-Kilis-25 Heterodera humuli AF498384 Heterodera goettingiana AF498374 Cryphodera brinkmani AF274418 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 same or extra chromosomal (mitochondrial) DNA (De Giorgi et al., 1994).

The results of our study did not show any intraspecific polymorphism between H. filipjevi populations. Furthermore, Bekal et al. (1997) did not detect any genetic variation among the different populations of this species. The H. latipons populations in this study consisted of 2 subgroups. Similarly, differences in ITS sequences were previously reported for other H. latipons populations (Ferris et al., 1999; Madani et al., 2004).

CCNs are the most important pathogens of wheat and other cereals in Turkey, and they have adverse effects on the production of wheat. Three species of cyst-forming nematodes belonging to the Heterodera avenae group were identified in cereal fields in Turkey. H. avenae in Turkey was reported by Yüksel (1973), Subbotin et al. (2003), and Abidou et al. (2005) based on a few samples. İmren et al. (2012) and Şahin et al. (2010) also determined that

H. latipons was the prevalent species in cereal fields in the southeastern Anatolia region, while H. filipjevi was distributed in most wheat-growing areas of the central Anatolian plateau in Turkey.

This study highlighted the ecoregional distribution of CCNs in the eastern Mediterranean region. Further detailed surveys and comprehensive pathotype studies on the Heterodera avenae group using populations from different regions in Turkey are necessary to determine the distribution patterns of CCN more accurately, and to determine whether differences in pathotypes occur and thereby cause different resistance responses in small grain cultivars being developed to improve the management of CCNs in Turkey.

AcknowledgmentThis study was funded by grant number 110 O 632 of a TÜBİTAK project (Scientific and Technological Research Council of Turkey).

References

Abidou H, El-Ahmed A, Nicol JM, Bolat N, Rivoal R, Yahyaoui A (2005). Occurrence and distribution of species of the Heterodera avenae group in Syria and Turkey. Nematol Mediter 33: 1–18.

Andres M, Melillo T, Delibes A, Romero MD, Bleve-Zacheo T (2001). Changes in wheat root enzymes correlated with resistance to cereal cyst nematodes. New Phytol 152: 343–354.

Bekal S, Gauthier JP, Rivoal R (1997). Genetic diversity among a complex of cereal cyst nematodes inferred from RFLP analysis of the ribosomal internal transcribed spacer region. Genome 40: 479–486.

Dababat A, Orakçı GE, Toktay H, İmren M, Akın B, Braun HJ, Dreisigacker S, Elekcioğlu İH, Morgunov AI (2014). Resistance of winter wheat to Heterodera filipjevi in Turkey. Turk J Agric For 38: 180–186.

De Giorgi C, Sialer ME, Lamberti F (1994). The use of mtDNA for the identification of plant-parasitic nematodes. In: Lamberti F, De Giorgi C, Bird DM, editors. Advances in Molecular Plant Nematology. 1st ed. New York, NY, USA: Plenum Press, pp. 109–118.

De Grisse A (1969). Redescription où modifications de quelques techniques utilisées dans l’étude des nématodes phytoparasitaires. Mededelingen Rijksfaculteit Landbouw Wetenschappen Gent 29: 734–761 (in French).

Evans K, Rowe JA (1998). Distribution and economic importance. In: Sharma SB, editor. The Cyst Nematodes. 1st ed. Dordrecht, the Netherlands: Kluwer, pp. 1–30.

Ferris VR, Ferris JM, Faghihi J, Ireholm A (1994). Comparisons of isolates of Heterodera avenae using 2-D PAGE protein patterns and ribosomal DNA. J Nematol 26: 144–151.

Ferris VR, Subbotin SA, Ireholm A, Spiegel Y, Faghihi J, Ferris JM (1999). Ribosomal DNA sequence analysis of Heterodera filipjevi and H. latipons isolates from Russia and comparisons with other nematode isolates. Russ J Nematol 7:121–125.

Gabler C, Sturhan D, Subbotin SA, Rumpenhorst HJ (2000). Heterodera pratensis sp. n., a new cyst nematode of the H. avenae complex (Nematoda: Heteroderidae). Russ J Nematol 8: 115–126.

Handoo ZA (2002). Key and compendium to species of the Heterodera avenae group (Nematode: Heteroderidae), J Nematol 34: 250–262.

Hooper DJ (1986). Handling, fixing, staining and mounting nematodes. In: Southey JF, editor. Laboratory Methods for Work with Plant and Soil Nematodes. London, UK: CAB International, pp. 59–80.

İmren M, Toktay H, Bozbuğa R, Dababat A, Elekcioğlu İH (2013). Pathotype determination of the cereal cyst nematode, Heterodera avenae (Wollenweber, 1924) in the Eastern Mediterranean Region in Turkey. Turk J Entomol 1: 13–19.

İmren M, Waeyenberge L, Viaene N, Toktay H, Dababat A, Elekcioğlu İH (2012). Molecular characterization of cereal cyst nematodes from South Anatolian Region in Turkey using ITS-rDNA sequences. Turk J Entomol 4: 491–499.

Kumar P, Gupta VK, Misra AK, Modi DR, Pandey BK (2009). Potential of molecular markers in plant biotechnology. Plant Omics 2: 141–162.

Madani M, Vovlas N, Castillo P, Subbotin SA, Moens M (2004) Molecular characterization of cyst nematode species (Heterodera spp.) from the Mediterranean basin using RFLPs and sequences of ITS-rDNA. J Phytopathol 15: 229–234.

http://dx.doi.org/10.1111/j.0028-646X.2001.00258.xhttp://dx.doi.org/10.1111/j.0028-646X.2001.00258.xhttp://dx.doi.org/10.1111/j.0028-646X.2001.00258.xhttp://dx.doi.org/10.1139/g97-064http://dx.doi.org/10.1139/g97-064http://dx.doi.org/10.1139/g97-064http://dx.doi.org/10.1139/g97-064http://dx.doi.org/10.3906/tar-1305-47http://dx.doi.org/10.3906/tar-1305-47http://dx.doi.org/10.3906/tar-1305-47http://dx.doi.org/10.3906/tar-1305-47

98

İMREN et al. / Turk J Agric For

Madzhidov AR (1981). New species of Bidera filipjevi sp. nov. (Heteroderina: Tylenchida) from Tadzhikistan. Izvectija Akademii Nauk Tadzjikiskov SSR, Otdelenie Biologitseskich Nauk 2: 40–44 (in Russian).

Nicol JM, Rivoal R (2008). Global knowledge and its application for the integrated control and management of nematodes on wheat. In: Ciancio A, Mukerji KG, editors. Integrated Management and Biocontrol of Vegetable and Grain Crops Nematodes. 1st ed. Dordrecht, the Netherlands: Springer, pp. 243–287.

Nicol JM, Rivoal R, Bolat N, Aktas H, Braun HJ, Mergoum M, Yildrim AF, Bagci A, Eleckcioglu IH, Yahyaoui A (2002). The frequency and diversity of the cyst and lesion nematode on wheat in the Turkish Central Anatolian Plateau. Nematology 4: 272.

Rivoal R, Cook R (1993). Nematode pests of cereals. In: Evans K, Trudgill DL, Webster JM, editors. Plant Parasitic Nematodes in Temperate Agriculture. Wallington, UK: CAB International, pp. 259–303.

Rivoal R, Valette S, Bekal S, Gauthier JP, Yahyaoui A (2003). Genetic and phenotypic diversity in the graminaceous cyst nematode complex, inferred from PCR-RFLP of ribosomal DNA and morphometric analysis. Eur J Plant Pathol 109: 227–241.

Rumpenhorst HJ, Elekçioğlu IH, Sturhan D, Öztürk G, Eneli S (1996). The cereal cyst nematode Heterodera filipjevi (Madzhidov) in Turkey. Nematol Mediterr 24: 135–138.

Şahin E (2010). Distribution of the important plant parasitic nematodes in wheat production area in Central Anatolian Plateau and studies on the biology and control of cereal cyst nematode Heterodera Filipjevi. PhD, Çukurova University, Adana, Turkey.

Shepherd AM (1986). Extraction and estimation of cyst nematodes. In: Southey JF, editor. Laboratory Methods for Work with Plant and Soil Nematodes. London, UK: Her Majesty’s Stationery Office, pp. 51–58.

Subbotin SA, Waeyenberge L, Molokanova IA, Moens M (1999). Identification of Heterodera avenae group species by morphometrics and rDNA-RFLPs. Nematology 1: 195–207.

Subbotin SA, Sturhan D, Rumpenhorst HJ, Moens M (2003). Molecular and morphological characterisation of the Heterodera avenae species complex (Tylenchida: Heteroderidae). Nematology 5: 515–538.

Subbotin SA, Vierstraete A, De Ley P, Rowe J, Waeyenberge L, Moens M, Vanfleteren JR (2001). Phylogenetic relationships within the cyst-forming nematodes (Nematoda, Heteroderidae) based on analysis of sequences from the ITS region of ribosomal DNA. Mol Phylogenet Evol 21: 1–16.

Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011). MEGA5: Molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28: 2731–2739.

Tanha Maafi Z, Subbotin SA, Moens M (2003). Molecular identification of cyst forming nematodes (Heteroderidae) from Iran and a phylogeny based on ITS-rDNA sequences. Nematology 5: 99–11.

Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997). The Clustal X Windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25: 4876–4882.

Toktay H, İmren M, Bozbuğa R, Erginbaş Orakçı G, Dababat A, Elekcioğlu IH (2013). Pathotype characterization of the cereal cyst nematode Heterodera filipjevi (Madzhidov, 1981) Stelter in Turkey. Turk J Entomol 37: 213–219.

Valdeolivas A, Romero MD (1990). Morphometric relationships of some members of the Heterodera avenae complex (Nematoda: Heteroderidae). Nematologica 36: 292–303.

Waeyenberge L, Ryss A, Moens M, Pinochet J, Vrain TC (2000). Molecular characterization of 18 Pratylenchus species using rDNA restriction fragment length polymorphism. Nematology 2: 135–142.

Wouts WM, Sturhan D (1995). Heterodera aucklandica sp. n. (Nematoda: Heteroderidae) from a New Zealand native grass, with notes on the species of the H. avenae group. New Zeal J Zool 22: 199–207.

Yüksel H (1973). Türkiye’de bulunan Heterodera (Nematoda: Heteroderidae) türlerinin durumu, bunların morfolojik ve biyolojik farklılıkları üzerinde çalışmalar. Atatürk Universitesi Ziraat Fakültesi Dergisi 4: 53–71 (in Turkish).

http://dx.doi.org/10.1023/A:1022838806268http://dx.doi.org/10.1023/A:1022838806268http://dx.doi.org/10.1023/A:1022838806268http://dx.doi.org/10.1023/A:1022838806268http://dx.doi.org/10.1163/156854199508018http://dx.doi.org/10.1163/156854199508018http://dx.doi.org/10.1163/156854199508018http://dx.doi.org/10.1163/156854103322683247http://dx.doi.org/10.1163/156854103322683247http://dx.doi.org/10.1163/156854103322683247http://dx.doi.org/10.1163/156854103322683247http://dx.doi.org/10.1006/mpev.2001.0998http://dx.doi.org/10.1006/mpev.2001.0998http://dx.doi.org/10.1006/mpev.2001.0998http://dx.doi.org/10.1006/mpev.2001.0998http://dx.doi.org/10.1006/mpev.2001.0998http://dx.doi.org/10.1093/molbev/msr121http://dx.doi.org/10.1093/molbev/msr121http://dx.doi.org/10.1093/molbev/msr121http://dx.doi.org/10.1093/molbev/msr121http://dx.doi.org/10.1093/nar/25.24.4876http://dx.doi.org/10.1093/nar/25.24.4876http://dx.doi.org/10.1093/nar/25.24.4876http://dx.doi.org/10.1093/nar/25.24.4876http://dx.doi.org/10.1163/002925990X00284http://dx.doi.org/10.1163/002925990X00284http://dx.doi.org/10.1163/002925990X00284http://dx.doi.org/10.1163/156854100509024http://dx.doi.org/10.1163/156854100509024http://dx.doi.org/10.1163/156854100509024http://dx.doi.org/10.1163/156854100509024http://dx.doi.org/10.1080/03014223.1995.9518034http://dx.doi.org/10.1080/03014223.1995.9518034http://dx.doi.org/10.1080/03014223.1995.9518034http://dx.doi.org/10.1080/03014223.1995.9518034