CC BY
48Russian Journal of Nematology, 2015, 23 (1), 29 - 40
Incidence of cereal cyst nematodes in the East Anatolia Region in Turkey
Halil Toktay1, Mustafa imren2, Atilla Ôcal3, Lieven Waeyenberge4, Nicole Viaene4 and
Abdelfattah Dababat5
1 Nigde University, Ayhan Sahenk Faculty of Agricultural Sciences and Technologies, Department of Plant Production and
Technologies, Central Campus, 51240, Nigde, Turkey 2 Abant Izzet Baysal University, Faculty of Agriculture and Natural Science, Department of Plant Protection,
izzet Baysal Campus, 14280, Golkoy Bolu, Turkey 3 Bati Akdeniz Agricultural Research Institute, Plant Protection Laboratory, 07500, Antalya, Turkey 4 Institute for Agricultural and Fisheries Research (ILVO), Plant, Crop protection, Burg. Van Gansberghelaan 96, 9820,
Merelbeke, Belgium
5 International Maize and Wheat Improvement Centre, P.K. 39 Emek, 06511, Ankara, Turkey
e-mail: h.toktay@nigde.edu.tr
Accepted for publication 31 March 2015
Summary. Cereal cyst nematodes (Heterodera spp., CCN) are major economic plant-parasitic nematodes of wheat grown in the East Anatolian region of Turkey. Identification of CCN species is essential for choosing the right control strategy. The morphological and molecular characteristics of cyst nematodes (Heterodera spp.) were determined for specimens collected from wheat fields from different provinces of the East Anatolia region in Turkey. Fifty-six percent of wheat fields were infested with cereal cyst nematodes. Based on morphological characterisations and molecular techniques, two Heterodera species, H. filipjevi and H. latipons, were identified. Heterodera filipjevi was the dominant species in the wheat fields of Elazig, Malatya, Sivas, Erzurum, Erzincan, Igdir and Kars provinces, while H. latipons was found only in three provinces (Erzincan, Elazig and Malatya). This is the first detection of H. latipons in the East Anatolia region. Genetic dissimilarity was higher within H. filipjevi populations than within H. latipons populations. While intraspecific polymorphism was not observed within H. latipons, it was recorded in H. filipjevi populations. Our results showed a clear separation of the two cyst nematode species using both morphological and molecular tests, and confirmed the link between genetic and morphological traits.
Key words: Heterodera spp., ITS region, nematode, taxonomy, wheat.
Wheat is the most important and strategic cereal crop in the world, ranking second in total production as a cereal crop behind rice (Nicol et al., 2003). Wheat production has steadily increased in Turkey during the last 35 years and recently reached production of 22 million tons/year from 9 million hectares of land (Anonymous, 2013). East Anatolian region of Turkey produces 1.2 million tons of wheat that is 7.1% of total wheat production in the country (Anonymous, 2013).
Plant-parasitic nematodes are distributed worldwide in wheat cultivation areas and cause annual yield loss of around 7% (Sasser, 1987). Among the plant-parasitic nematodes, cereal cyst nematodes (CCN) are widespread worldwide and incur significant economic yield losses in many
countries, especially under rainfed conditions (Nicol et al, 2003).
The genus Heterodera includes 12 species that adversely affect the roots of cereals and grasses. These species of (CCN) are designated as the Heterodera avenae group (Rivoal & Cook, 1993). Among these, three species (H. avenae, H. _ filipjevi and H. latipons) are the most important cyst nematodes attacking wheat, causing economic yield loss worldwide (Handoo 2002; Subbotin et al, 2003, 2010). These three species have been reported from different parts of Turkey. Heterodera avenae was recorded for the first time in 1974 at Erzurum province (Yuksel, 1973) and is now reported in many cereal-producing regions such as South Anatolia and the Eastern Mediterranean regions (imren et al., 2012, 2015). High populations of
Fig. 1. A map of Heterodera populations showing locations in the seven provinces from where the samples were collected.
H. avenae in commercial fields reduced yields of spring wheat up to 27% and occasionally destroyed the second crop of spring wheat in Eastern Mediterranean region of Turkey (imren & Elek^ioglu, 2014). Heterodera_filipjevi was reported for the first time from the Central Anatolian region of Turkey (Rumpenhorst et al., 1996), while H. latipons was observed in different provinces in the south part of Turkey (Dababat et al., 2015; imren et al., 2015). There is no study of the diversity and occurrence of CCN in East Anatolian region.
Heterodera filipjevi is closely related to H. avenae and H. latipons; only minor morphological differences differentiate them (Handoo, 2002; Subbotin et al, 2003). Identification of these species based on morphology is a time consuming process and demands great skill and training by the observer. Sequences analysis based on the internal transcribed spacer (ITS) region of the ribosomal (r) DNA repeat unit has provided a reliable tool for quick and precise identification of cyst nematode species and subspecies (Subbotin et al, 1999, 2003; Tanha Maafi et al, 2003; Madani et al, 2004). Comparative analysis of the ITS-rDNA of unknown nematode species sequences with those of known species published or deposited in GenBank facilitates rapid identification of most cyst nematode species. The information from molecular tests strengthens the microscopic identifications based on
the analysis of differences in morphology (Handoo, 2002; Subbotin et al., 2003), but morphological identification is also needed to support the identification from molecular tests. Genetic differences among species determined from molecular tests generally corresponded with specific morphological characters in the graminaceous cyst nematode complex (Yan & Smiley, 2010).
imren et al. (2015) have reported on occurrence and diversity of CCN species, based on morphological and molecular identification of populations collected from a limited area of the Mediterranean region of Turkey. This area has a warm climate and is mostly coastal, whereas our study included areas from East Anatolian region that has a continental climate and a high altitude far from the Mediterranean region. Therefore, the climate and ecological conditions of the Mediterranean region are very different from those of the Eastern Anatolian region. In addition, our study includes information on population densities and detailed morphological measurements of nematodes. Yuksel (1973) identified H. avenae in the East Anatolian region, but it most probably belonged to another species of H. avenae group since H. filipjevi was known as H. avenae before the 1980s (Madzhidov, 1981).
The objectives of the present study were: i) to investigate population densities and distribution of
cereal cyst nematodes (Heterodera spp.) collected from wheat fields in the East Anatolia Region of Turkey; ii) to distinguish the Heterodera spp. by sequences of the ITS-rDNA and cyst morphological characters; and iii) to examine possible intraspecific variation within Heterodera spp. populations based on the ITS region of their rDNA.
MATERIALS AND METHODS
Nematode samples. A survey of wheat growing areas in East Anatolia Region was conducted during July and August of 2013, just after harvest (Fig. 1). Using a spade, soil samples were collected at 15-20 cm deep. From each field, a subtotal of 2 kg soil was taken containing fine roots and rhizosphere soil of wheat plants. Samples were sent directly to Laboratory of Nematology, Nigde University for further processing. Cysts were extracted from soil using the Seinhorst method (Seinhorst, 1964). Cysts were collected using 20 * magnification of a stereomicroscope and were sterilised with 0.01% NaOCl for 10 min followed by several rinses with sterilised water. Cleaned cysts were stored at 4°C before molecular and morphological identification.
Morphological and morphometric characterisation. The morphological features and morphometric measurements of the vulval region of adult and second-stage juveniles (J2) were used for diagnosis after fixation in glycerin (Seinhorst, 1959).
Species of Heterodera were differentiated from each other by morphological and morphometric features of J2 (body length, stylet length, tail length, hyaline tail length, stylet knob shape and tail terminus shape) as described previously by Subbotin et al. (1999). Other morphological and morphometric features such as the b and c ratios of J2, the length of cysts excluding the neck (L'), the width of cysts (B), the L'/B ratio, the presence, size and shape of bullae inside the vulval cone and cyst color were also determined (Subbotin et al., 1996, 2003; Handoo, 2002).
Fifteen mature cysts from Heterodera latipons and twenty-five cysts from H. filipjevi vulval cones were mounted in glycerin, and examined. Identifications were made based on underbridge structure, shape of semi-fenestra in the fenestral area and bullae. Cyst characters measured were the fenestral length, semi-fenestral width, vulval bridge width and vulval slit length (Handoo, 2002; Subbotin et al., 2010).
Molecular identification and phylogenetic analysis. DNA extraction. Forty-four Heterodera populations from different provinces were selected
for DNA extraction. For each population, one cyst was transferred into 45 ^l of double distilled water (ddH2O) in an Eppendorf tube and crushed using a micro homogeniser. After centrifugation of the crushed cyst content, 40 ^l of the mix was transferred into a PCR tube (0.2 ml). Fifty ^l of worm lysis buffer (WLB) and 10 ^l of Proteinase K (20 mg ml-1) were added to each tube (Holterman et al, 2006); the tubes were frozen at -80°C for at least 10 min. Then the tubes were 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 16400 g and kept at -20°C until use (Tanha Maafi et al., 2003).
PCR amplification. For molecular identification, the ITS-rDNA region was amplified. One ng of DNA was added to the PCR reaction mixture containing 23 ^l ddH2O, 25 ^l 2* DreamTaq PCR Master Mix (Thermo Scientific, Belgium) and 1 ^M of each forward primer (5'-CG TAACAAGGTAGCTGTAG-3') and reverse primer (5 '-TCCTCCGCTAAATGATATG-3') (Ferris et al., 1993). The DNA thermal cycler program consisted of 5 min at 95°C; 40 cycles of 94°C for 30 s, 45°C for 45 s and 72°C for 45 s; followed by a final elongation step of 8 min at 72°C.
Following PCR amplification, 5 ^l of each PCR product was mixed with 1 ^l of 6* loading buffer (Fermentas Life Sciences, Germany) 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 ^g ml-1) for 15 min, visualised and photographed under UV-light. The remaining PCR product was stored at -20°C.
Sequencing and phylogenetic analysis. Ninety ^l of PCR product was loaded on a 1% agarose gel for electrophoresis (100 V, 40 min). The purification was carried out as described in the manufacturer's instructions (Wizard® SV Gel and PCR Clean-Up System Kit, Promega). Purified PCR-product from each sample was sequenced (Macrogen, Amsterdam, The Netherlands) in both directions to obtain overlapping sequences of both DNA strand. Species were identified using the BLAST tool on the NCBI-website (www.ncbi.com).
For phylogenetic analysis H. latipons (Syria, JX024179), H. hordecalis (Estonia, AY692356), H. filipjevi (Iran, AY148404), H. ustinovi (Belgium, AY148407), H. pratensis (Russia, AY148351, Germany, AY148384), H. australis (Australia, AY148394, AY148396), H. mani (Germany, AY148377, AY148378), H. avenae (China, EU616697, HM560755), H. aucklandica (United Kingdom, AY148380), H. ciceri (Syria, AY045758), H. schachtii (Morocco, AY166346)
and H. goettingiana (Iran, AF498374) were added to our obtained sequences. Cryphodera brinkmani (Japan, AF274418) and Meloidodera alni (Belgium, AF274419) were also included in the phylogenetic analysis.
All sequences were aligned with ClustalX 1.64 with default options. The evolutionary history was inferred using the Maximum Likelihood method based on the General Time Reversible model (Nei & Kumar, 2008). The tree with the highest log likelihood (-7010,1904) is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach, and then selecting the topology with superior log likelihood value. A discrete Gamma distribution was used to model evolutionary rate differences
among sites (5 categories (+G, parameter = 1,1014)). The analysis involved 65 nucleotide sequences. There were a total of 1232 positions in the final dataset. Evolutionary analyses were conducted in MEGA5 (Tamiura et al, 2013).
RESULTS
Field infestation. The survey of cyst nematodes showed that 56% of wheat fields in the East Anatolia Region of Turkey were infested with CCN. Based on morphological characters and molecular techniques, two species, H. filipjevi and H. latipons, were identified. Heterodera filipjevi was the dominant species in the wheat fields of Elazig, Malatya, Sivas, Erzurum, Erzincan, Igdir and Kars provinces, while H. latipons had a very limited distribution, being found in only three provinces (Elazig, Malatya and Erzincan). Heterodera latipons was detected for the first time in the East Anatolia Region.
Fig. 2. Light micrographs of the second-stage juveniles of Heterodera latipons from Erzincan in East Anatolia Turkey. A: the second stage juvenile; B: stylet & DGO; C: anus and hyaline tail tip; D: female fenestra, strong underbridge and bifurcated vulval slit; E: light brown cysts.
Table 1. Occurrence of the cyst nematodes Heterodera filipjevi and H. latipons in wheat fields surveyed East
Anatolian Region in Turkey
Sample no. Provinces Number of fields surveyed Number of infested fields Infestation rate (%) Species Infestation level (average number of cysts per 100 g of soil)
1 Elazig 30 21 70 H. filipjevi + H. latipons 10
2 Malatya 35 21 60 H. filipjevi + H. latipons 9
3 Erzurum 40 18 45 H. filipjevi 4
4 Erzincan 40 20 50 H. filipjevi + H. latipons 4
5 Sivas 70 42 60 H. filipjevi. 8
6 Kars 40 15 47,5 H. filipjevi 3
7 Igdir 25 16 64 H. filipjevi 2
Total 280 157 56
Table 2. Morphological and morphometrical characteristics of cysts of Heterodera filipjevi and H. latipons
Species character Heterodera filipjevi Heterodera latipons
25 15
Mean i S.E. Range Mean i S.E. Range
Length (excl. neck) 641.1 i 18.4 490-852 601.6 i 12.9 510-672
Width 449.1 i 12.2 309-620 442.3 i 13.4 364-530
Length/width 1.43 i 0.02 1.18-1.68 1.37 i 0.03 1.18-1.57
Fenestral length 51.81 i 0.74 40-61 62.74 i 1.17 59-70
Fenestral width 25.85 i 0.56 19-40 19.89 i 0.64 17-22
Vulval bridge width 13.37 i 0.28 11.0-17.5 28.04 i 4.77 18-35
Underbridge length 80.90 i 5.50 71.1-94.8 94.50 i 8.70 87-99
Vulval slit length 9.43 i 1.02 7.5-13.1 7.70 i 1.50 5.2-11.0
Cyst nematodes were detected in almost all the fields with monoculture of wheat in Elazig, Malatya, Erzurum, Erzincan, Sivas, Kars and Igdir provinces. Fields in which cyst nematodes were not detected were generally rotated with other crops. High incidence levels were recorded, e.g., 21 of 30 fields were infested in Elazig. The lowest incidence of infested fields was in Kars with 19 of 40 fields infested.
The average number of cysts per 100 g of soil was estimated based on the total samples per province. The greatest numbers were 10 and 9 cysts in Elazig and Malatya provinces, respectively. Low levels of infestation (2 and 3) were recorded in Kars and Sivas as shown in Table 1.
Morphological and morphometric characterisation of Heterodera populations. Cysts of H. filipjevi were bigger than those of H. latipons and were yellow to light-brown in colour. Heterodera filipjevi was distinguished from H. latipons by its prominent bullae and a clear underbridge, which was very narrow in the centre and located close to the vulval bridge. Cysts of H.
latipons had no bullaea with a strong underbridge, and vulval slit prominent and bifurcated in both sides. They differed from H. filipjevi by a strong and deep underbridge without bullae and greater fenestral length (59-70 ^m vs 40-61 ^m), a larger vulval bridge width (18-35 ^m vs 8-13 ^m), longer vulval slit length (5.3-11 ^m vs 7.5-13.1), tail length (44.1-53.8 ^m vs 41.7-64), hyaline part of tail length (24-30 vs 25-39 ^m), ratio B-J2 (hyaline part of tail length/stylet length) (1.2-1.4 vs 1.2-1.8) and ratio C-J2 (total length/tail length) (8.4-10.7 vs 8.2-11.9) (Tables 2 & 3).
Heterodera filipjevi and H. latipons had cysts of different sizes (490-852 ^m vs 510-672), were brown and dark in colour and with a round and thin vulval cone top respectively (Figs 2 & 3).
Molecular identification and phylogenetic analysis. PCR amplification of the ITS regions of rDNA of all 45 Heterodera populations and species tested produced a single fragment of approximately 1.2 kb. Forty populations from Elazig, Malatya, Sivas, Kars, Erzurum, Erzincan and Igdir were identified as H. filipjevi; 4 populations from Elazig,
Malatya and Erzincan were identified as H. latipons (Table 4).
Based on the ITS-rDNA sequences, Heterodera filipjevi populations showed some intraspecific polymorphism. All H. filipjevi populations grouped together and were supported by a bootstrap value of 96%. Heterodera latipons populations also showed little intraspecific polymorphism. The H. latipons populations analysed here grouped with H. latipons populations from Syria (JX024179) (Fig. 4).
DISCUSSION
This survey of CCN from the East Anatolian Region of Turkey showed the presence of both H. latipons and H. filipjevi in wheat cultivating areas. Both nematodes are reported for the first time from this region. Heterodera filipjevi is widely distributed
in Elazig, Malatya, Sivas, Kars, Erzurum, Erzincan and Igdir provinces but H. latipons was found only in Malatya, Elazig and Erzincan. Finding H. latipons in these areas was unexpected, as the majority of fields were in high-elevated land in Turkey and this species is generally found in fields on the Mediterranean coast or having a Mediterranean climate (imren et al, 2015). Heterodera filipjevi has been reported in Central and South of Turkey with high altitude wheats fields (Dababat et al., 2015).
Our results demonstrate that the highest levels of infestation were found in the Sivas, Elazig and Malatya provinces, where the total number of cysts per 100 g of soil reached 8, 9 and 10, respectively. By contrast, the lowest levels of infestation were found in the Igdir and Kars provinces, where the total numbers of cysts per 100 g of soil recorded were 2 and 3, respectively.
Fig. 3. Light micrographs of Heterodera filipjevi from Erzurum in East Anatolia Turkey. A: the second stage juvenile; B: stylet & DGO; C: anus and hyaline tail tip; D: female fenestra, narrow vulval slit & heavy bullae; E: dark brown cysts.
Table 3. Morphological and morphometrical characteristics of second-stage juveniles of Heterodera filipjevi and H. latipons
Heterodera filipjevi Heterodera latipons
Species character 25 15
Mean i S.E. Range Mean i S.E. Range
L 526.6 i 6.20 477-583 459.6 i 5.45 424-489
a 25.30 i 0.59 17.0-29.7 18.95 i 2.20 16.5-24.17
b 4.39 i 0.05 3.8-4.7 3.75 i 0.07 3.4-4.2
c 9.50 i 0.20 8.2-11.9 9.45 i 0.15 8.4-10.7
c' 3.71 i 0.09 3.2-4.3 3.28 i 0.10 2.4-3.6
Stylet length 22.39 i 0.30 20-25 21.92 i 0.31 19.4-24.0
Labial region height 4.04 i 0.15 3.0-5.4 4.16 i 0.16 2.74-5.05
Labial region diam. 9.38 i 0.20 7.9-11.0 9.13 i 0.14 8.7-10.0
DGO 4.82 i 0.14 3.5-5.7 4.87 i 0.19 3.25-6.0
Distance anterior end to median bulb (mb) 73.21 i 0.98 71.2-81.5 66.85 i 0.87 62.5-71.0
Excretory pore 103.8 i 0.86 98-109 102.0 i 1.27 97-107
Body diameter at mid-body 21.05 i 0.54 18.4-22.8 20.97 i 0.50 19.3-24.0
Body diameter at anus 15.19 i 0.37 12-21 14.96 i 0.46 13.0-19.3
Tail length 55.85 i 1.08 41.7-64.0 48.75 i 0.79 44.1-53.8
Hyalin region (H) 33.85 i 0.81 25-39 27.33 i 0.47 24-30
H/stylet length 1.52 i 0.04 1.20-1.84 1.25 i 0.02 1.2-1.4
L/mb 6.85 i 0.08 6.2-7.5 5.98 i 0.07 5.5-6.2
Measurements are in цш.
In these areas, barley and wheat are continuously cultivated on the same land as monoculture. Although, the incidence and impact of cereal cyst nematode depends on the type of host and soil, nematode pathotype and ecotype and climatic conditions of the area (Rivoal & Cook, 1993), growing cereals as a monoculture has resulted in gradually increasing populations of CCN that influence the amount of yield loss in infested fields. It appears that existence of environmental suitable conditions for the completion of the life cycle of these nematodes can be an important factor for posing a threat to cereal production in Sivas, Elazig and Malatya provinces.
To maintain the population densities of these species of nematodes below damaging levels, appropriate management measures, such as rotational schemes and the use of resistant varieties, are necessary. A number of resistance sources for breeding purposes have been found in domestic cereals and their wild relatives, acting against both Heterodera species (Dababat et al., 2015). However, the efficiency of the genes involved differs according to the virulence of the populations (pathotypes) of Heterodera species, although preliminary studies indicate that several resistance genes of barley or wheat, are to some extent also
resistant to populations of H. filipjevi or H. latipons originating from different sites in North Africa, Europe and Asia (Bekal et al., 1997; Mokabli et al, 2002). Further investigations are necessary to identify suitable resistance sources to be used in cereal breeding programmes.
Correlations between morphological characters of cysts and juveniles allow differentiation of H. latipons and H. filipjevi of the Heterodera avenae group. For example, useful characters for distinguishing these species are the presence of distinct underbridge in the vulval cone and small bullae situated below the fenestrae of cysts of H. filipjevi (Subbotin et al, 2003). Morphometric characters, such as the hyaline part of the tail of the J2 and the fenestral length of cysts, are also important characters (Madzhidov, 1981). Differentiation of H. latipons could be readily achieved using either the fenestral length or the vulval bridge width of the cyst and either the lengths of the tail or hyaline part of the tail of the J2, in addition to the underbridge and bullae presence in the vulval cone (Wouts et al., 1995). In our study, the distinguishing of specimens based on the development of bullae and presence of an underbridge was successful at low magnification, including populations with mixed species (the Elazig, Malatya and Erzincan populations).
Table 4. Locations of Heterodera species in East Anatolia Region of Turkey
No. Code Province Location Species Accession no.
1 25б Malatya Malatya Yazihan Yolu H. filipjevi KP708712
2 257 Malatya Yazihan Kirec Ocagi H. filipjevi KP708745
3 2б1 Malatya Fethiye H.latipons KP708713
4 2б4 Malatya Yazihan H. filipjevi KP708714
5 2б8 Malatya Arapkir Yolu H. filipjevi KP708746
б 275 Malatya Arapkir Yolu H. filipjevi KP708715
7 280 Malatya Pötürge Yolu H. filipjevi KP708716
8 284 Malatya Malatya Dogansehir Yolu H. filipjevi KP708717
9 288 Elazig Cevizkemer Yolu H. filipjevi KP708718
10 291 Elazig Elazig Merkez H.latipons KP708719
11 293 Elazig Elazig Sahinkaya Köyü H.latipons KP708720
12 295 Elazig Yazikonak ihl H. filipjevi KP708747
13 29б Elazig Yazikonak H. filipjevi KP708721
14 297 Elazig Molla Kendi H. filipjevi KP708722
15 300 Elazig Güntas H. filipjevi KP708748
1б 301 Elazig Yukaribagi H. filipjevi KP708723
17 302 Elazig Degirmen önü H. filipjevi KP708749
18 303 Elazig Elazig Yolu H. filipjevi KP708750
19 304 Elazig Elazig Yolu H. filipjevi KP708724
20 325 erzurum Oltu Ardahan Yolu H. filipjevi KP708725
21 354 Igdir Aralik Yolu H. filipjevi KP708726
22 3б4 Igdir Kücük ova yolu H. filipjevi KP708727
23 371 Erzincan Erzincan Asagi Carsi H. filipjevi KP708728
24 378 Erzincan Erzincan H. filipjevi KP708728
25 420 Erzurum Cinsuyu köyü H. filipjevi KP708751
2б 421 Erzurum Cinsuyu Anayola 2 km H. filipjevi KP708735
27 425 Erzurum Erzurum-Askale Eski Yol H. filipjevi KP708736
28 430 Erzincan Mercan Otluk Beli Yolu H.latipons KP708737
29 452 Sivas Incetas Yolu H. filipjevi KP708738
30 457 Sivas Yildizeli Sarkisla Yolu H. filipjevi KP708739
31 4б3 Sivas Yilanhöyük Köyü H. filipjevi KP708740
32 4бб Sivas Gürün Kangal Yolu H. filipjevi KP708752
33 472 Sivas Kangal Kus Kayasi Köyü H. filipjevi KP708730
34 475 Sivas Sutasi Köyü H. filipjevi KP708753
35 480 Sivas Sivas Kangal Yolu H. filipjevi KP708741
3б 482 Sivas Ulas Kangal Yolu H. filipjevi KP708731
37 484 Sivas Ulas Kangal Yolu H. filipjevi KP708742
38 488 Sivas Kabak Cetinkaya H. filipjevi KP708743
39 490 Sivas Kangal Divrigi Yolu H. filipjevi KP708732
40 495 Erzurum Erzincan Ilic Boyalik köyü H. filipjevi KP708754
41 49б Erzurum Erzurum Tekman H. filipjevi KP708744
42 514 Igdir Yagli Girisi H. filipjevi KP708755
43 521 Kars Oluklu köyü giris H. filipjevi KP708733
44 550 Kars Cildir Yolu H. filipjevi KP708734
Fig. 4. Phylogenetic tree of Heterodera spp. populations from East Anatolian region of Turkey.
Our results showed an intraspecific polymorphism between populations of H. filipjevi. Similarly, Subbotin et al. (2003) revealed intraspecific polymorphism between H. filipjevi populations. However, Bekal et al. (1997) did not detect any genetic variation among different populations of this species. Also, imren et al. (2015) did not detect any genetic variation among H. filipjevi populations in Mediterranean region of Turkey. Based on our results, differences in ITS sequences were not observed between H. latipons populations and were found to be close to the Syrian population of H. latipons with bootstrap value 68%. Similar results were obtained by imren et al. (2015), who showed intraspecific differentiation between populations in H. latipons in the Mediterranean region of Turkey between Moroccan population and Syrian population of H. latipons. Madani et al. (2004) and Rivoal et al. (2003) showed an intraspecific variation between populations in H. latipons. However, in this study, we did not find any intraspecific variation in our Syrian population of H. latipons.
The sequences of the ITS region of cyst-forming nematode species is necessary in addition to morphological traits for accurate identification of Heterodera spp. attacking crops, especially when estimating pathogenic characterisations and searching for sources of host plant resistance. The present study has indicated there was no polymorphism in the populations of H. filipjevi and H. latipons concerning molecular parameters.
Variation in the cultivated wheat varieties and alternations in agro-ecosystems could cause the development of new races and resistance breaking populations of CCN (Abdollahi, 2008). Recent work on physiological variation using international differentials revealed the occurrence of two biotypes in the East Mediterranean region of Turkey (imren et al, 2013; Toktay et al, 2013). Heterodera filipjevi belonged to Ha33 H. aveae Ha21 pathotype in Turkey (imren et al., 2013; Toktay et al, 2013). There is no report about H. latipons pathotype in Turkey.
Further investigations are necessary to identify the pathotypes of the H. filipjevi and H. latipons populations of the East Anatolian Region of Turkey, as well as suitable resistance sources to be used in cereal breeding programmes.
ACKNOWLEDGEMENTS
This study was funded by TUBITAK project no. 112 O 565 of (Turkey Scientific and Technical Research Council). The author thanks Prof. Halil
Elekqioglu in Cukurova University, Ms. Nancy de Sutter, Dr Fateh Toumi, Dr Fouad Mokrini and other research workers in ILVO, Belgium for their help in carrying out lab experiments.
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Резюме. Цистообразующие нематоды злаков (Heterodera spp., ЦОН) - причина основных потерь урожая пшеницы в регионе Восточной Анатолии в Турции. Определение видов ЦОН представляет собой ключевой фактор для выбора правильной стратегии контроля этого вредителя. Определены морфологические и молекулярные особенности цистоообразующих нематод рода Heterodera, собранных на пшеничных полях в различных провинциях Восточной Анатолии. Показано, что 56% полей региона поражено этими нематодами. Основываясь на морфологических и молекулярных признаках, выявлено доминирование двух видов Heterodera - H. filipjevi и H. latipons. Heterodera filipjevi был доминантным видом на пшеничных полях в провинциях Элязыг, Малатья, Сивас, Эрзурум, Эрзинджан, Ыгдыр и Карс, тогда как вид H. latipons был выявлен лишь в трех провинциях (Эрзинджан, Элязыг и Малатья). Это первое сообщение о присутствии H. latipons в регионе Восточной Анатолии. Уровень генетического разнообразия популяций был выше у H. filipjevi, чем у H. latipons. Внутривидовой полиморфизм был выявлен у H. filipjevi, но не у H. latipons. Наши результаты показывают наличие четких морфологических и молекулярных различий между двумя видами. Подтверждено соответствие между морфологическими и молекулярными признаками этих видов.
