Identification of Meloidogyne species by sequencing of internal transcribed spacer regions of ribosomal DNA of juvenile stages

Juan Jaramillo-Pineda, Martha Guerrero-Olazarán, José Antonio Fuentes-Garibay, José MarÃía Viader-Salvadó, José Lorenzo Meza-García, Lilia Hortencia Morales-Ramos

Abstract


Usually the identification of the Meloidogyne species is based on the morphology of adult females, making it difficult to identify juvenile males and females (J2). Nematodes are considered among the most difficult animals to identify; the use of ribosomal DNA (rDNA)-based diagnostic methods have gained acceptance in applications ranging from quarantine determinations to assessments of biodiversity. Nematodes of the genus Meloidogyne are known for their ability to produce physiological changes in the root system of plants and cause losses in the absorption of nutrients. The aim of this study is to determine if the sequencing of internal transcribed spacer (ITS) regions of rDNA can be used as genetic markers for reliable identification of populations of juvenile males and females (J2) for the main species of the genus Meloidogyne. From samples of diseased tomato roots (Solanum lycopersicum L.), larvae of juvenile females and males of Meloidogyne were collected for the DNA extraction. A rDNA region harboring two ITS regions was amplified. For subsequent sequencing, that region was ligated into pGEM®-T vector. Analysis with the BLASTn program showed that the gene region identified 99.8 % with a gene sequence belonging to Meloidogyne incognita Kofoid & White, 1919. This result suggests that the ITS regions can be used as a genetic marker in populations for Meloidogyne species identification.

Keywords


Meloidogyne incognita; ITS; Ribosomal DNA (rDNA)

Full Text:

PDF (Español)

References


Adam MAM, Phillips MS, Blok VC. 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.

Agrios GN, 2005. Plant Pathology. Fifth Edition. Academic Press. New York, USA. 922p.

Blok VC, Philips MS, and Fargette M. 1997. Comparison of sequences from the ribosomal DNA intergenic region of Meloidogyne mayaguensis and other major tropical root-knot nematodes. Journal of Nematology. 29:16-22.

Cenis JL. 1993. Identification of four Major Meloidogyne spp. by Random Amplified Polymorphic DNA (RAPD-PCR). Phytopathology. 83:76-80.

Dong K. 2007. PCR-RFLP Identification of Globodera pallida, G. rostochiensis, Heterodera spp. and Meloidogyne spp. Available at: http://www.cdfa.ca.gov/plant/PPD/nematode_molecular.html. (Consulta, septiembre 2013).

Eisenback JD, Hirschmann H, Sasser JN, and Triantaphyllou AC. 1981. A guide to the four most common species of root-knot nematodes (Meloidogyne species), with a pictorial key. Journal of Nematology. 13: 513-521.

Hall TA. 1999. BioEdit: A user friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp. Ser. 41: 95-98.

Meza-García JL, Eías-Santos M, Cortez-Mondaca E, Guerrero-Olazarán M, Viader-Salvadó JM, Luna-Olvera HA, Maldonado-Blanco MG, Quintero-Zapata I, and Pereyra-Alférez B. 2014. Evaluation of Heterorhabditis indica (Rhabditida: Heterorhabditidae) Nematode Strain from Sinaloa, Mexico, against Bemisia tabaci Immatures under Laboratory Conditions. Southwestern Entomologist. 39(4), en impresión.

Orui Y. 1998. Identification of Japanese species of the genus Meloidogyne (Nematoda: Meloidogynidae) by PCR-RFLP analysis. Journal of Applied Entomology. 33: 43-51.

Powers TO. 2004. Nematode molecular diagnostics: From Bands to Barcodes. The Annual Review of Phytopathology. 42:367–383.

Powers TO, Harris TS. 1993. A polymerase chain reaction method for identification of five major Meloidogyne species. Journal of Nematology. 25:1-6.

Sambrook J, Russell DW. 2001. Molecular cloning: a laboratory manual, Third Edition. Cold Spring Harbor Laboratory Press. New York, USA. 2100p.

SENASICA-CNRF-Departamento de Análisis de Riesgo de Plagas. 2012. Ficha técnica de Meloidogyne incognita. Available at: http://www.encuentra.gob.mx/resultsAPF.html?q=Meloidogyne%20incognita&client=sagarpa&ts=all&geo=0. (Consulta, septiembre 2013).

Stock SP, Campbell JF, and Nadler SA. 2001. Phylogney of Steinernema travassos, 1927 (Cephalobina: Steinernema) inferred from ribosomal DNA sequences and morphological characters. Journal of Parasitology. 87:877-889.

Vovlas N, Mifsud D, Landa BB and Castillo P. 2005. Pathogenicity of the root-knot nematode Meloidogyne javanica on potato. Plant Pathology. 54:657-664.

Williamson VM, Caswell-Chen EP, and Westerdahl FF. 1997. A PCR assay to identify and distinguish single juveniles of Meloidogyne hapla and M. chitwoodi. Journal of Nematology. 29:9-15.

Wishart J, Phillips MS, and Blok VC. 2002. Ribosomal Intergenic Spacer: A Polymerase Chain Reaction Diagnostic for Meloidogyne chitwoodi, M. fallax, and M. hapla. Phytopathology. 92: 884-892.

Zijlstra C, Lever AEM, Uenk BJ and Silfhout CH. 1995. Differences between ITS Regions of Isolates of Root-knot Nematodes Meloidogyne hapla and M. chitwoodi. Phytopathology. 85: 1231-1237.

Zuckerman BM, Mai WF, and Krusberg LR. 1990. Plant Nematology Laboratory Manual Revised Edition 1990. University of Massachusetts Agricultural Experiment Station, Massachusetts. USA. 252 p.


Refbacks

  • There are currently no refbacks.