Document Type : Research Article
Department of Plant Protection, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad
Seed and Plant Improvement Research Department, Khorasan Razavi Agricultural and Natural Resources Research and Education Center, AREEO, Mashhad, Iran
Introduction: Pomegranate is one of the most popular fruit trees and one of the most important export productions in Iran. Root-knot nematodes (Meloidogyne spp.) are the most damaging plant-parasitic nematodes of pomegranate, which cause root galls and disrupt the absorption and transfer of water and food, and eventually decrease the fruit growth. Root-knot nematodes (Meloidogyne spp.) are economically important plant parasites affecting a broad range of host plants, and thus far, 100 nominal species have been described. The development of molecular methods to identify the four major Root-knot nematodes including M. incognita, M. arenaria, M.javanica and M. hapla has been the goal of numerous studies. These species are morphologically similar, making identification difficult for the non-specialist. However, distinguishing them is important for utilizing appropriate crop rotations, managing resistance effectively and plant quarantine requirements. Therefore, some molecular methods for identification of Root-knot nematodes (RKN) species have been developed. Recently, a PCR method based on DNA has been widely used for the identification of nematodes. SCAR- Primers (Sequence characterized amplified region) were developed and are used routinely on a large number of samples with high sensitivity and specificity.
Materials and Methods: In order to investigate the infection of pomegranate orchards to Meloidogyne species, 115 soil and root samples were collected from major areas of pomegranate cultivation from Khorasan Razavi, Northern Khorasan and Southern Khorasan provinces, during 2015-2016. The highest area of pomegranate cultivation is located in Torbat Heydarieh, Bardaskan, Kashmar, Khalil Abad, Bajestan and Ferdows cities. After extraction of nematodes from root and surrounding soil, species identification was performed according to morphological and morphometric characteristics of second-stage juveniles, mature females and perineal pattern of females, and also using molecular methods and specific SCAR primers. For DNA extraction, the procedure proposed by Silva et al, who extracted DNA of second stage juvenile, was applied. In addition, the Atkin methods were employed for extracting DNA of mature female. The DNA was added into the PCR reaction with specific primer (SCAR), and then loaded in gel for further analyses.
Results and Discussion: Major species of root knot nematode were M. incognita, M. javanica and M. arenaria in Khorasan provinces identified based on morphological and morphometric studies as well as SCAR primers. M. incognita showed a band about 399 bp with Inc-14 primer, M. javanica exhibited a band about 670 bp with Jav primer and M. arenaria showed a band about 420 bp with a ar primer. Infection of root knot nematode pomegranate was observed in almost all cities but the intensity of infection varied considerably. The highest percentage of infection on root-knot nematodes pomegranate orchards was observed in Bardaskan (Anabad section of Fatemieh village) with 57.5% and Bajestan (Chah Paliz village) with 32%. M. incognita has the highest distribution in pomegranate orchards. The highest percentage of infected orchards was estimated in Bardaskan (19.93%), Bajestan (12.3%), Khalil Abad (6.9%), Kashmar (4.3%), and Torbat Heidariyeh (3.5%) located in Khorasan Razavi provinces, Ferdows (5.4%) and Nehbandan (1.3%) in Southern Khorasan provinces, and Maneh (1.1%) and Jajarm (0.8%) situated in Northern Khorasan. M. incognita has the highest distribution in Khorasan Razavi and Southern Khorasan provinces, and M. javanica has the highest distribution in Northern Khorasan provinces. The differential host shows the race 2 of M. incognita in area.
Conclusion: Infection of root knot nematode pomegranate is growing and there is a need for accurate identification. Using molecular methods especially SCAR primers for identification of major species of root knot nematode is fast, accurate and reliable.