Research Article Open Access Fayazi et al., J Plant Pathol Microb 2012, 3:3 DOI: 10.4172/2157-7471.1000123 Volume 3 • Issue 3 • 1000123 J Plant Pathol Microb ISSN:2157-7471 JPPM an open access journal Keywords: DNA; PCR; P. neglectus; P. thorn Introduction Wheat (Triticum aestivum L.) is an important grain in Iran. Iran has been ranked as the 12th producer of wheat, for producing more than 13.5 Mt during 2008-2009 [4]. Khuzestan, which is located in the southwest of Iran, is the second most important cereal producing province in the country due to the total production of 1.2 Mt during 2008-2009 [5]. Pratylenchus nematodes are common endoparasite of plant all around the world. Te mentioned genus cause necrotic lesions due to being migratory and obligating parasites that invade the cortex of roots, tubers, and bulbs of plant. Tis reduces the yields [6,7]. Several cereal and legume crops are damaged by the cereal and legume root- lesion nematodes, namely P. thornei and P. neglectus, in many parts of the world such as the Mediterranean region, America, the Indian Subcontinent and Australia [8,9]. Te two species P. thornei and P. neglectus are the most common species of Pratylenchus in Iran [10]. Ahmadi et al. [11] showed that the population density of P. thornei in wheat root samples ranged from 1-351 nematodes/g of root sample in Khuzestan. Loof (1991) reported currently that the genus Prtylenchus that includes more than 60 species could be diferentiated only by means of minor morphological and morphometric diferences of adult females and males. Several characters can be used in the distinction of various species of this genus from each other. Tey are as follow: the number of annuli in the lip region, the presence or the absence of a spermatheca in females, the presence or the absence of males, the number of lines in the lateral feld, and the shape of the tail [12,13]. Intraspecifc morphological variability within the genus Pratylenchus is well documented for the most of the characters used in species identifcation; hence, causes difculties in identifying species [13]. Te use of molecular diagnostic tools is a practical solution to overcome such a problem. PCR-based methods are relatively rapid and very reliable; the possess high discriminating potentials, do not rely on the expressed products of the genome, and are independent of the environmental infuence and the stage of the nematode life cycle. A PCR-based assay was used for the identifcation of six species of Pratylenchus through forward and reverse species-specifc primers that were designed from the internal variable portion of the D3 expansion region of the 26S rDNA [14]. Al- Banna et al. [14] distinguished P. neglectus and P. thornei along with four other Pratylenchus spp. using PCR and species-specifc primers derived from the internal variable portion of the D3 expansion region of the 28S rDNA. Te detection and identifcation of P. thornei and P. neglectus from soil developed and two species were diferentiated by PCR products of 144 bp for P. neglectus and 288 bp for P. thornei [15]. In the present study, using nucleotide sequences of the D3 expansion region of 28S rRNA, molecular methods were investigated for the aim of identifying P. thornei and P. neglectus. Materials and Methods Forty soil and wheat root samples were collected from Masjed Solleiman, Behbahan, Bagh Mallek and Shoushtar to elucidate disease situation in Khuzestan. Nematodes were extracted from soil samples using the Jenkins [16] method. We examined the standard morphological and morphometric characters of root-lesion nematodes [13]. Adult females were morphologically identifed as either P. thornei or P. neglectus. Root-lesion nematodes were reared on carrot discs [17,18]. Ten nematodes were extracted from carrot discs through Whitehead and Hemming methods [19]. Specimens used in this study were extracted from infested roots 24 to 48 hrs afer the incubation [20]. Tey were then handpicked, *Corresponding author: Fatemeh Fayazi, Department of Plant Protection, College of Agriculture, Shahid Chamran University, Ahvaz, Iran, Tel: +98-09177758271; Fax: +98-0772-4243385; E-mail: fayazi.fatemeh@yahoo.com Received April 27, 2012; Accepted June 29, 2012; Published July 03, 2012 Citation: Fayazi F, Farokhi-Nejd R, Ahmadi AR, Memari HR, Bahmani Z (2012) Molecular and Morphometric Identifcation of P. Thornei and P. Neglectus in Southwest of Iran. J Plant Pathol Microb 3:123. doi:10.4172/2157-7471.1000123 Copyright: © 2012 Fayazi F, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Abstract Root lesion nematodes are considered as important agents of wheat yield reduction in most parts of the wheat growing areas. To elucidate disease situation in Khuzestan, a southwestern province of Iran, 40 soil & wheat root samples were collected. Morphological studies indicated that disease casual agents belong to Pratylenchus thornei and P. neglectus species. Morphometric studies showed that Differences exist in the body length compared with the studies done so far on these two species of nematode. The DNA of the two species, namely Pratylenchus thornei and P. neglectus, were extracted concidering Madani et al. [1], Silva et al. [2] and Waeyenberg et al. [3] plus some modifcations. The quantity and quality of extracted DNA and its ability in DNA amplifcation and clearance of PCR bands were compared and the results showed that modifed methods of Madani et al. [1] and Waeyenberg et al. [3] were the best methods for P. thornei and P. neglectus species. Polymerase chain reaction (PCR) and species- specifc primers were used to identify P. thornei and P. neglectus. Molecular and Morphometric Identification of P. Thornei and P. Neglectus in Southwest of Iran Fatemeh Fayazi 1 *, Reza Farokhi-Nejd 1 , Ali Reza Ahmadi 2 , Hamid Rajabi Memari 3 and Zeynab Bahmani 1 1 Department of Plant Protection, College of Agriculture, Shahid Chamran University, Ahvaz, Iran 2 Department of Plant Protection, Agriculture and Natural Resources Research Centre of Khuzestan, Ahvaz, Iran 3 Department of Agronomy and Plant Breeding, College of Agriculture, Shahid Chamran University, Ahvaz, Iran Journal of Plant Pathology & Microbiology J o u r n a l o f P l a n t P a t h ol o g y & M i c r o b i o l o g y ISSN: 2157-7471