ANATOLIA, TURKEY
*M. MREN1, H. TOKTAY1, A. ÖCAL2, J. M. NICOL3,5 and . H.
ELEKÇOĞLU4
1Plant Protection Research Institute, Adana, Turkey. 2Plant Protection Research Institute, Diyarbakır, Turkey. 3CIMMYT (International Maize and Wheat Improvement Centre), ICARDA-CIMMYT Wheat Improvement Program, Ankara Turkey. 4Çukurova University, Faculty of Agriculture, Department of Plant Protection, Balcalı Adana, Turkey. 5Correspondence: [email protected]
SUMMARY
During a survey in 2009, 74 soil samples collected from 27 districts from wheat and barley fields in Southeast Anatolia (Gaziantep, Kilis and Şanlıurfa). Cyst-forming nematodes were only found in 11 samples and were identified as Heterodera avenae by PCR–RFLP.
INTRODUCTION
Cereal cyst nematodes cause serious economical damage in cereal crops worldwide especially in temperate regions (Nicol and Rivoal 2008, Rivoal and Cook 1993, Evans and Rowe 1998). Heterodera avenae group consists of 12 valid and several undescribed species that infect cereals and grasses (Wouts et al. 1995, Gabler et al. 2000, Andres et al. 2001).
Recent surveys of cereal fields on the Anatolian Plateau of Turkey showed that Heterodera filipjevi are widely distributed in major wheat and barley cultivating areas (Yıldırım 2006, Şahin 2009). It was first detected in Turkey during 1995 (Rumpenhorst et al. 1996) and being found in 87% of the wheat growing area in the Central Anatolian Plateau (CAP). Yield losses up to 50% were recorded in commonly grown cultivars in CAP in Turkey (Nicol et al. 2006). H. avenae has been found in Turkey by Yüksel (1973) and Subbotin et al. (2003). The information on the occurrence of CCN from other parts of Turkey is limited.
*İmren M, Toktay H, Öcal A, Nicol JM, Elekçioğlu İH (2009) Occurrence of cereal cyst
nematode, Heterodera avenae in Southeast Anatolia, Turkey. In ‘Cereal cyst nematodes: status, research and outlook.’ (Eds IT Riley, JM Nicol, AA Dababat) pp. 82-87. (CIMMYT: Ankara, Turkey)
The objective of this study was to identify species of cereal cyst nematodes from Southeast Anatolia.
METHODS
Surveys of cereal cyst nematodes were conducted over summer in 2009. Samples were taken from 27 sites in the major wheat and barley growing areas in several provinces in the Southeast Anatolian of Turkey. Two kg of randomly sampled soil was collected in each studied field. Soil samples were processed through Kort elutriator (Kort 1960). Extracted cysts caught on the 250-μm-pore sieve were picked with a brush and gathered under a stereomicroscope.
DNA extraction. The total DNA from one or several cysts was extracted using DNA isolation kit (Fermentas Life Sciences).
PCR. 5 µl of extracted DNA was transferred to an Eppendorf tube containing: 2.5 µl 1x Taq incubation buffer, 2 mM of MgCl2 200 µM dNTPs mixture, 0.4 μM of each primer, 0.2 µl of Taq DNA polymerase and double distilled water to a final volume of 25 µl. The forward primer TW81 (5'-GTTTCCGTAGGTGAACCTGC-3') and the reverse primer AB28 (5'-ATATGCTTAAGTTCAGCGGGT- 3') described by Joyce et al. (1994), were used in the PCR. PCR cycles consisted of an initial denaturation step at 94C for 4 min followed by 35 cycles of 1 min at 94C (denaturation), 1.5 min at 60C (annealing), and 2 min at 72C (elongation). The reaction was terminated by a final extension cycle (72C, 10 min), and the PCR product was stored at 4C. The PCR 5 ml of the product was run on a 1% agarose gel and visualised after electrophoresis (120 V) under ultraviolet light (260 nm).
RFLP. Ten microlitres of the PCR product was digested by one of the following restriction enzymes: AluI, Pst I and BsuRI (HaeIII) in the buffer stipulated by the manufacturer (Fermentas). The digested DNA was run on a 2% TAE buffered agarose gel, stained with ethidium bromide and photographed.
RESULTS AND DISCUSSION
The amplification of the ITS region including the flanking parts of the 18S and 28S genes yielded a single fragment of approximately 1200 bp for all studied populations (Figure 1). The information of eleven H. avenae populations found in this survey are also given in Table 1.
Eleven populations of H. avenae were obviously differentiated by the 566 and 483 bp fragments resulting from AluI digestion (Figure 2), 708 and 341 bp fragments resulting from PstI digestion (Figure 3), 420 and 353 bp fragments resulting from BsuRI (HaeIII) digestion (Figure 4).
The RFLP patterns obtained from the studied populations did not reveal any difference with those previously reported for H. avenae (Subbotin et al. 2003, Mandani et al. 2004, Abidou et al. 2005). H. avenae can be distinguished from the other species by the restriction enzyme AluI. This enzyme reveals heterogeneity of the ITS region among several specimens from some populations of H. avenae. These RFLP profiles should be considered for identification of populations of this species.
Table 1. Occcurrence of Heterodera avenae in Southeast Anatolia, Turkey.
Code Location Province Host
21 Elbeyli-Havuzluçam Kilis Wheat 28 Elbeyli-Sınır Karakolu Kilis Barley 30 Elbeyli-Sınır Karakolu Kilis Wheat 31 Elbeyli- Sınır Ardıçlı Kilis Wheat 33 Elbeyli- Sınır Ardıçlı Kilis Barley 50 Karkamış- Kıvırcık-Sınır Gaziantep Wheat 62 Karkamış- Oğuzeli Yolu Gaziantep Wheat 65 Karkamış- Akçaköy Gaziantep Wheat 66 Karkamış- Akçaköy Gaziantep Barley 67 Karkamış- Arıkdere Gaziantep Barley 72 Karkamış- Küçükeşme Gaziantep Wheat 74 Karkamış- Soylu Gaziantep Wheat
Figure 2. Restriction fragments (566 and 483 bp) of amplified ITS regions of Heterodera avenae digested by AluI. For species code see Table 1; M, 100 bp DNA ladder.
21 28 30 31 33 50 62 65 66 67 72 74
Figure 1. All studied populations yielded a single fragment of about 1200 bp. For
species code see Table 1; M, 100 bp DNA ladder.
Other the restriction enzymes PstI and BsuRI (HaeIII) can be distinguished from the other Heterodera avenae group species (Subbotin et al. 2003, Mandani et al. 2004, Abidou et al. 2005).
H. avenae has been reported in Australia, Canada, India, Israel, Japan and most European countries, South Africa and USA (Smiley et al. 1994, Nicol and Rivoal 2008). It is also found in Morocco, Tunisia, Pakistan, Libya, Turkey (Rumpenhorst et al. 1996), and Estonia (Krall et al. 1999). H.filipjevi is widely distributed in major wheat and barley cultivating areas (Şahin 2008) and was found in 78% of the wheat growing areas of the CAP. H. avenae were reported from Turkey by Yüksel (1973), Subbotin et al. (2003) and Abidou (2005) by using only single samples for identifications. Therefore, further comprehensive surveys are needed to more accurately define the distribution of CCN in Turkey.
This study highlights the ecoregional distribution of CCN in Turkey, where H. filipjevi is clearly the dominant species on the CAP (the major winter wheat producing area). However, in the predominantly Mediterranean spring wheat area of southeast Turkey H. avenae is predominant. More work is needed to map population densities and to define potential economic damage.
Figure 3. Restriction fragments (708 and 341 bp) of amplified ITS regions of Heterodera avenae digested by PstI. For species code see Table 1; M, 100 bp DNA ladder.
21 28 30 31 33 50 62 65 66 67 72 74
Figure 4. Restriction fragments (420 and 353 bp) of amplified ITS regions of Heterodera avenae digested by BsuRI (HaeIII). For species code see Table 1; M, 100 bp DNA ladder.
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