Turkish Journal of Zoology Turk J Zool (2018) 42: 625-636 http://journals.tubitak.gov.tr/zoology/ © TÜBİTAK Research Article doi:10.3906/zoo-1801-41 Morphometric and genetic variability among Mediterranean cereal cyst nematode (Heterodera latipons) populations in Turkey 1, 2 3 1 4 Mustafa İMREN *, Şenol YILDIZ , Halil TOKTAY , Nagihan DUMAN , Abdelfattah A. DABABAT 1 Department of Plant Protection, Faculty of Agriculture and Natural Science, Abant İzzet Baysal University, Bolu, Turkey 2 Department of Wildlife Ecology and Management, Faculty of Agriculture and Natural Science, Abant İzzet Baysal University, Bolu, Turkey 3 Department of Plant Production and Technologies, Faculty of Agricultural Sciences and Technologies, Niğde Ömer Halisdemir University, Niğde, Turkey 4 International Maize and Wheat Improvement Center (CIMMYT), Ankara, Turkey Received: 23.01.2018 Accepted/Published Online: 01.11.2018 Final Version: 12.11.2018 Abstract: The Mediterranean cereal cyst nematode, Heterodera latipons, is an important plant parasitic nematode that negatively impacts cereal production worldwide, particularly in the Mediterranean Basin. Successful control of cereal cyst nematodes requires knowledge of the nematode species/pathotypes and their biology. However, little information is currently available concerning the phenotypic and genotypic variability of H. latipons populations in Turkey. This study analyzed the morphological/morphometric and genetic characteristics of Turkish H. latipons populations to define intraspecific distinctions between them. Morphological and morphometric studies focused on the pattern of the perineal region of cysts and second stage juveniles (J2), while the molecular analyses used sequenced ITS-rDNA regions targeting the internal transcribed spacer (ITS) region of rDNA. The data from these investigations demonstrate that nematode populations from Adana and Hatay provinces differ from the populations collected in Kilis, Gaziantep, and Mardin provinces. These phenotypic and genotypic differences between H. latipons populations may indicate that they are heterogenic, with at least two pathotypes. To date, this study is the most comprehensive analysis identifying H. latipons populations in major wheat- producing areas of Turkey. Key words: Cereals, Heterodera latipons, heterogeneity, ITS region rDNA, morphometrics, nematodes 1. Introduction on knowledge of nematode species/pathotypes and their Plant parasitic nematodes are responsible for annual biological effects on cereals, as well as on the economically economic losses of up to US$ 100 billion worldwide acceptable control options by farmers (Holgado et al., 2004). (Urwin et al., 1997; Bird and Kaloshian, 2003). Cereal Identifying specific CCN species is therefore an essential nematode genera such as Heterodera (cyst), Meloidogyne component of selecting appropriate control strategies (root-knot), Tylenchorhynchus and Merlinius (stunt), (Hyman, 1996). To date, researchers have identified 12 Ditylenchus (stem), Anguina (seed-gall), Pratylenchus species and intraspecific CCN pathotypes infecting cereals (root-lesion), and Paratrichodorus (stubby root) can cause and grasses; among them H. filipjevi, H. latipons, and H. significant yield losses (Rivoal and Cook, 1993; McDonald avenae are the most damaging species (Rivoal and Cook, and Nicol, 2005). Among the cereal nematodes, cereal cyst 1993; McDonald and Nicol, 2005). H. latipons, for example, nematodes (CCNs) are the most widely studied on wheat has been reported on cereal crops such as wheat, barley, and have been reported by many researchers worldwide and oat throughout the Mediterranean region including (Cook and Noel, 2002; Handoo, 2002; Nicol et al., 2003; Cyprus, Italy, Jordan, Israel, Spain, Morocco, Algeria, and Subbotin et al., 2003; Dababat et al., 2014, 2015). Turkey (Franklin, 1969; Cohn and Ausher, 1973; Mor et al., Heterodera spp. are highly heterogeneous in their 1992; Yousef and Jacob, 1994; Philis, 1995; Rumpenhorst virulence on specific wheat (Triticum aestivum), barley et al., 1996; İmren et al., 2012; Haddadi and Mokabli, 2015; (Hordeum vulgare), and oat (Avena sativa) genotypes Mokrini et al., 2017). (Cook and Noel, 2002; McDonald and Nicol, 2005; Accurately identifying plant parasitic nematodes Dababat et al., 2015). CCN management strategies depend requires both morphological and molecular tools. * Correspondence: [email protected] 625 This work is licensed under a Creative Commons Attribution 4.0 International License. İMREN et al. / Turk J Zool Taxonomists previously identified nematodes to the species and morphometric features. Vulval cone slides were level using quantitative and qualitative characteristics, with prepared by fixing eight cysts each population in formalin- heavy reliance on gross morphological and morphometric glycerol fixative mounted on glycerol and detected with characterizations. The perineal region patterns of cysts and a light microscope, according to Hooper (1986). We second stage juveniles (J2) are the most common features measured the length of the vulval slit, width of the vulval used to differentiate species of Heterodera (Handoo, 2002; bridge, width of the fenestra, length of the fenestra, and Subbotin et al., 2003). Morphological and morphometric length and width of the underbridge. The presence or identification has been widely adopted, but morphological absence of underbridge and bullae analysis of cyst perineal differentiation of Heterodera species is quite difficult and pattern were examined (Handoo, 2002). requires specialist skills. However, new molecular diagnostic Ten juveniles obtained from the same cysts were techniques are overcoming the taxonomic limitations gently heated, fixed in triethanolamine formalin solution, associated with conventional species identification (Szalanski embedded in glycerol, and then prepared on permanent et al., 1997; Al-Banna et al., 2004). For example, the internal slides (Handoo, 2002). We measured body length, stylet transcribed spacer region (ITS) is variable and therefore length, distance from anterior region to junction with the useful for H. avenae group identification and phylogenetic esophagus, body width, distance from anterior region to studies at the species level (Subbotin et al., 2003). the base of the esophageal bulb, tail length, tail width, It is important to understand the genetic variation and length of the hyaline portion of the tail. The most within and/or between populations to develop resistant important characters of J2 identification, a, b’, c, and c’ host plant and gene flow information. No research to date ratios and the ratio of hyaline portion to stylet length, has comprehensively studied the phenotypic and genetic were also calculated (Handoo, 2002). CCN populations differences of H. latipons in Turkey. This study therefore were identified using previously established descriptions aimed to compare morphological and morphometric and diagnostic keys for cyst and J2 morphological characteristics of Turkish H. latipons populations with characteristics (Franklin, 1969; Mulvey and Golden, 1983; those previously reported, and to evaluate their genetic Handoo, 2002). Ten J2s and one cyst for each population polymorphism based on ITS-rDNA sequences. It is difficult were observed, photographed, and measured using a Leica to reliably identify H. latipons using conventional methods DFC295 digital camera installed on a Leica DM5000 B because they occur with other Heterodera species in the same optical microscope and Leica Application Suite (LAS) field. This study utilized morphological, morphometric, and software v.4.1.0. molecular identification to distinguish individual H. latipons Data were analyzed using analysis of variance (ANOVA) populations. procedures in SPSS 10.0 for Windows (SPSS Inc., Chicago, IL, USA) to determine any noteworthy differentiations 2. Materials and methods among the 42 populations (P ≤ 0.05). A standard test of 2.1. Heterodera latipons populations means was performed to determine significant variance A total of 42 Heterodera populations were collected from between populations (P ≤ 0.05). wheat and barley fields in Turkey during the 2016 growing 2.3. Molecular identification season. Of these, 26 populations were collected from the 2.3.1. DNA extraction and amplification southeastern area (Gaziantep, Kilis, and Mardin provinces) DNA was extracted from one single cyst for each population and 16 populations from the eastern Mediterranean region using the protocol of Waeyenberge et al. (2000) with some (Adana and Hatay provinces), as shown in Table 1. modifications. PCR reactions were performed in a total The wheat and barley fields surveyed displayed stunted volume of 50 µL containing 1 µL of nematode DNA, 25 patches, poor plant growth, chlorotic lower leaves, and µL of 2X Dream Taq PCR Master Mix (Fermentas Life few or no tillers. Cyst populations were collected from 42 Sciences, USA), 22 µL of ddH2O, and 1 µM each of forward different fields arbitrarily with separation distance of 7–10 primer AB28 (5ʹ-CGTAACAAGGTAGCTGTAG-3ʹ) and km. A representative sample of 2 kg of soil consisting of 5–7 reverse primer TW81 (5ʹ-TCCTCCGCTAAATGATATG- subsamples was prepared. A modified sieving-decanting 3ʹ) (Joyce et al., 1994). method (Fenwick, 1940) was used to extract cysts from the Reaction steps consisted of an initial denaturation at 95 soil. Cysts were identified to genus level under a stereo- °C for 4 min, followed by 35 cycles of denaturation at 94 °C binocular microscope