Molecular Characterization and Genetic Variation of Root-knot Nematode (Meloidogyne spp.,) in Selected Legume Production Areas of Eastern Kenya
Keywords:
Phylogenetic relationships between the Meloidogyne species, intergenic region between 18S and 5S genes, SSU rDNA analysis, Mbeere.Abstract
Sequences from the species under study were closely related to sequences retrieved from sequences databases especially those sequences which were less divergent due to less substitutions, deletions and insertions. It can be concluded that SSUrDNA are useful in identification, inferring genetic diversity and phylogenetic relationships between the isolated root knot nematodes. There is need for a rapid and reliable method to identify populations of root-knot nematodes in order to design effective control programs.
Root-knot nematodes are sedentary endoparasites of plant roots and the primarynematode pathogens of most cultivated crops worldwide, includinglegumes. Root- Knot Nematodes of the genus Meloidogyne is the most economically important nematode pests affecting cowpea and pigeon pea in Eastern Kenya. This study sought to identify the Meloidoigyne species of root-knot nematodes on selected legumes in Mbeere district and characterize the genetic diversity of the species using small subunit (SSU) rDNA. PCR amplifications of the extracted purified DNA were carried out using primers specific for the intergenic spacer region between the 5S and 18S ribosomal DNA and the expected size of about 720bp was obtained. Purified PCR products were then sequenced and thirteen 5S-18S rDNA sequences obtained. The sequences were aligned using CLUSTALW2, Sequence statistics, pairwise differences, and estimates of divergence were determined with MEGA5. Nucleotide diversities were estimated in DnaSPv5. Phylogenetic tree was drawn using Phylowin and edited in MEGA5. From the findings of the study it has been established that root knot nematodes affecting the cowpea and pigeon pea in Mbeere district are M. javanica, M. incognita and M. arenaria. Judging from the extent of differences in base composition biases between sequences, it was concluded that the sequences under study have not evolved with the same pattern of substitution.
References
[2] B. B. Singh. “Recent genetic studies in cowpea,” In: Challenges and Opportunities for Enhancing Sustainable Cowpea Production, C.A. Fatokun, S.A. Tarawali, B.B. Singh, P.M. Kormawa, M. Tamo, Ed. Ibadan, Nigeria:IITA,2002,pp. 3–13.
[3] K. Ahenkora, H. K. Adu-Dapaah, and A. Agyemang. “Selected nutritional components and sensory
attributes of cowpea (Vigna unguiculata [L.] Walp.) leaves.” Plant Foods Human Nutrition,vol. 52, pp. 221–229, 1998.
[4] S. S. Nielsen, T. A. Ohler, and C. A. Mitchell. “Cowpea leaves for human consumption: production, utilization, and nutrient composition.” In: Advances in Cowpea Research, B.B. Singh, D.R. Mohan Raj, K.E. Dashiell, L.E.N. Jackai, Ed. Sayce, Devon, UK: Copublication of International Institute of Tropical Agriculture (IITA) and Japan International Research Center for Agricultural Sciences, 1997, 326–332.
[5] K. Barker. “Introduction and synopsis of advancements in nematology” In: Plant Nematode Interactions, K.R. Barker, G.A. Pederson , G.L. Windham, Ed. Madison, WI: American Society of Agronomy, Crop Science Society of America, Soil Science Society of America, 1998, 1–20.
[6] J. D. Eisenback and H. H. Triantaphyllou. “Root-knot nematodes: Meloidogyne species and races” Manual of Agricultural Nematology, pp. 191-274, 1991.
[7] S. B. Jepson. Identification of Root-knot Nematodes (Meloidogyne species). Wallingford,UK: CABI Publishing, 1987, pp. 265.
[8] A. C. Triantaphyllou. “Cytogenetics, cytotaxonomy and phylogeny of root-knot nematodes.” In : An advanced treatise on Meloidogyne, Biology and control vol. 1. J. N. Sasser, C. C. Carter, Ed. Raleigh: North Carolina State University Graphics, 1985, pp. 113 126 .
[9] T. J. Baum, P.M. Gresshoff, S. A. Lewis and R. A. Dean. “Characterization and phylogenetic analysis of four root- knot nematode species using DNA amplification fingerprinting and automated polyacrylamide gel electrophoresis.” Molecular Plant-Microbe Interactions,vol. 7, pp. 39-47,1994.
[10] J. M. Kavuluko, C. Gichuki, J.W. Waceke and S.M. Runo. “Characterisation of root-knot nematodes (Meloidogyne spp.) from selected legumes in Mbeere District Kenya using isoenzyme phenotypes.” In “Transforming Agriculture for improved livelihoods through Agricultural Product Value Chains: The Proceedings of the 12th KARI Biennial Scientific Conference” Nairobi Kenya, 8- 12th Nov 2010, pp. 92- 97
[11] S. Bekal, J.P. Gauthier and R. Rivoal. “Genetic diversity among a complex of cereal cyst nematodes inferred from RFLP analysis of the ribosomal internal transcribed spacer region.” Genome, vol. 40, pp. 479-486, 1997.
[12] O. Randig, F. Leroy, M. Bongiovanni and P. Castagnone-Sereno. “RAPD characterisation for single females of the root-knot nematodes, Meloidogyne spp.” European Journal of Plant Pathology ,vol. 107 pp. 639–643. 2001.
[13] V. C. Blok, M.S. Phillips and M. Fargette. “Comparison of sequences from the ribosomal DNA intergenic region of Meloidogyne mayaguensis and other major tropical root-knot nematodes.” Nematology, vol. 2, pp. 16-21,1997.
[14] Sambrook and Russel. Molecular cloning. A laboratory manual. New York: Cold spring harbour
publishers. 2001
[15] J. D. Thompson, T. J. Gibson, F. Plewniak , F. Jeanmougin and D. G. Higgins. “The Clustal-x Windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools.” Nucleic Acids
Research,vol. 25, pp. 4876–4882, 1997.
[16] K. Tamura, J. Dudley, M. Nei and S. Kumar. “MEGA4: Molecular Evolutionary Genetics Analysis
(MEGA) software version 4.0.” Molecular Biology and Evolution,vol. 24 pp. 1596- 1599, 2007.
[17] P. Librado and J. Rozas. “DnaSP v5: A software for comprehensive analysis of DNA polymorphism data.”Bioinformatics,vol 25 pp. 1451-1452, 2009.
[18] T.A. Hall. “BioEdit: a user-friendly biological sequence alignment editor and analysis program for
Windows 95/98/NT.” Nucleic Acids Symposium Ser,vol. 41 pp. 95-98, 1999.
[19] F. Tajima. “ Statistical methods to test for nucleotide mutation hypothesis by DNA polymorphism.”
Genetics,vol. 123, pp. 585-595, 1989.
[20] M. A. M. Adam, M. S. Phillips and V. C. Blok . “Identification of Meloidogyne spp. from north east Libya and comparison of their inter- and intro-specific genetic variation using RAPDs.” Nematology , vol. 7, pp. 599–609,2000.
[21] S. A. Subbotin, P.D. Halford, A. Warry and R.N. Perry. “Variations in ribosomal DNA sequences and phylogeny of Globodera parasitising solanaceous plants.” Nematology , vol. 2, pp. 591–604, 2000.
[22] M. S. Tigano, R. M. D. G. Carneiro, A. Jeyaprakash, D. W. Dickson and B. J. Adams. “Phylogeny of Meloidogyne spp. based on 18S rDNA and the intergenic region of mitochondrial DNA sequences.” Nematology , vol. 7,pp. 851–862, 2000.
[23] P. De Ley and W. Bert. “Video capture and editing as a tool for the storage, distribution and illustration of morphological characters of nematodes.” Journal of Nematology , vol.34, pp. 296–302,2002.
[24] J. G. Files and D. Hirsh . “Ribosomal DNA of Caenorhabditis elegans.” Journal of Molecular Biology, vol. 149, pp. 223, 1981.
[25] M. L. Blaxter, P. Deley, J. R. Garrey, L. X. Liu, A. V. Scheldeman, J.R. Vanflenten et al. “Molecular evolutionary framework for the phylum nematode.” Nature , vol. 392, pp. 71-75, 1998.
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