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. |
- 1- Adam M. A. M., Phillips M. S., and Blok V. C. 2007. Molecular diagnostic key for identification of single juveniles of seven common and economically important species of root-knot nematode (Meloidogyne spp.). Plant Pathology, 56: 190 –197
2-Akhyani A. 1987. Important pest and diseases of pomegranates in Yazd and Isfahan provinces. Introduction to the Articles of the First Seminar on Pomegranate Problems in Iran. College of agriculture and natural resource of Tehran, Kraj. (in Persian)
3- Anonymous. 2005. Statistical Book of Agricultural of Iran. Iranian Statistical Centre,
Tehran, Iran. (in Persian)
4-Askariyan H., Sharifnabi B., Oliya M., Mahdikhani Moghadam E., and Akhavan E. 2009. Identification of Meloidogyne javanica with use of morphometric and morphologic characterize and specific primer in Kerman Province. Sciences and Technology of Agriculture and Natural Resources Ju. 47 : 279-289. (in Persian)
5- Atkin, A. 2012.
6- Behzadi Shahrbabaki H. 1997. Genetic diversity of pomegranate genotypes in Iran. Agriculture Education Pub. 265 p.
7- Barker K.R., Carter C.C., and Sasser J.N. 1985. An advanced treatise on Meloidogyne, Plant. pathol. 4: 212- 233
8- Bashiri S., Jamali S., and Gol Mohamadi M.2012. Molecular Identification of Meloidogyne incognita nematode isolated from kiwifruit roots in northern Iran. 3rd Iranian agricultural Biotechnology Conference. Ferdowsi University of Mashhad.
9-De Ggrisse AT. 1969. Rediscription ou modification de quelques techniques utilisees dans letude des nematodes phytoparasitaries. Meddelingen Rijksfauculteit landbouwweteschappen Bull 34:351-356.
10-De Ley I.T., Karssen G., De Ley P., Vierstraete A., Waeyenberge L., Moens M., Vanfleteren J. 1999. Phylogenetic analyses of internal transcribed spacer region sequences within Meloidogyne. Journal of Nematology, 31: 530–531
11-Esser R.P., Perry V.G., Taylor A.L. 1976. A diagnostic compendium of the genus Meloidogyne (Nematoda: Heteroderidae). Proceedings of Helminthological Society of Washington, 43: 138–150.
12- Fourie, H., Zijlstra C., and A. H. Mcdonald. 2001. Identification of root knot nematode species occurring in South Africa using the SCAR-PCR technique. J. Nematol. 3: 675-680.
13- Jain R.K., Mathur K.N., and Singh R.V. 2010. Estimation of losses due to plant parasitic nematodes on different crops in India. Indian J. Nematol, 37(2): 219-221.
14- Janssen T., Karssen G., Verhaeven M., Coyne D., and Bert W. 2016. Mitochondrial coding genome analysis of tropical root-knot nematodes (Meloidogyne) supports haplotype based diagnostics and reveals evidence of recent reticulate evolution. Scientific Reports, 6: 22591.
15- Jepson S. B. 1987. Identification of root-knot nematodes Meloidogyne species.CABI Publishing, 265p.
16- Jeyaprakash A., Tigano M. S., Brito J. A., and Dickson D .W. 2006.Differentiation of Meloidogyne floridensis from M. arenaria using high-fidelity PCR amplified mitochondrial AT-rich sequences. Nematropica 36 (1): 1-12
17- Hatamabadi Farahani M. 2015. Final report of research in determine distribution, severity of infection and identification of root knot nematode species in pomegranate orchards in Saveh city. Natural research and education center of Iran, Tehran. 31 p. (in Persian)
18-Hatamabadi Farahani M.,Ghalandar M., and Hoseininezhad A. 2018. Investigation of Distribution Root knot nematode in pomegranate orchards in Saveh city.23rd Iranian plant protection congress, Gorgan. P 725. (in Persian)
19-Hartman K. M., and Sasser J. N. 1985. Identification of Meloidogyne species on the basis of differential host test and perineal pattern morphology. Pp. 69–77 in J. N. Sasser and C. C. Carter, eds. An advanced treatise on Meloidogyne, vol. II. Biology and control. Raleigh, NC: North Carolina State University Graphics
20- Harris T.S., Sandal L.J., Powers T.O. 1990. Identification of single Meloidogyne juveniles by polymerase chain reaction amplification of mitochondrial DNA. Journal of Nematology, 22: 518–527
21- Hunt D. J., and Hundoo Z. A. 2009. Taxonomy, identification and principal species.
Pp:55-97. In: R. N. Perry, M. Moens and J. L. Starr. Root-knot nematodes (ed.). CABI, Wallingford, UK
22-Kargar Bideh A. 1989. Investigation of survey plant parasitic nematode in fruit trees ( pomegranate, pistachio and almond) in Yazd province. Ms thesis. Tarbiat Modarres University Faculty of Agriculture. 140 p. (in Persian)
23- Kheiry A., and Baruti SH. 1987. Introducing plant parasitic nematodes collected from soil around pomegranate roots. Introduction to the Articles of the First Seminar on Pomegranate Problems in Iran. College of agriculture and natural resource of Tehran, Kraj. P19. (in Persian)
24- Kiewnick S., Holterman M., van den Elsen S., van Megen H., Frey J.E., Helder J. 2014. Comparison of two short DNA arcoding loci (COI and COII) and two longer ribosomal DNA genes (SSU & LSU rRNA) for specimen identification among quarantine root-knot nematodes (Meloidogyne spp.) and their close relatives. European Journal of Plant Pathology, 140: 97–110.
25- Lunt D. H., Kumar S., Koutsovoulos G., and Blaxter M. L.2014. The complex hybrid origins of the root knot nematodes revealed through comparative genomics. Peer j 2,
26-Mireh Ki K., Abdollahi M., Mohaghegh Dolat abadi M., and Ghezelbash N. 2014. Application of Morphological Characters and PCR- SCAR Markers for Identification of Dominant Species of Root-knot Nematode (Meloidogyne spp.) in Glasshouses of Cold Region of Kohgiluyeh and Boyer-Ahmad Province, Iran. Agricultural biotechnology.vol 13. (in Persian with English abstract)
|
