Morphological, biochemical and molecular approaches to the identification of Meloidogyne incognita, Meloidogyne javanica and Meloidogyne arenaria in Portugal

Abstract

Mestrado em Engenharia Agonómica - Proteção de plantas - Instituto Superior de Agronomia - ULPlant parasitic nematodes are highly damaging pests in many crops of great economic importance. A substantial part of this damage is caused by infestations of root-knot nematodes (RKN), due to their wide geographical distribution as well as a vast range of host plants. The most important and widely distributed root-knot nematode species are Meloidogyne incognita, M. arenaria and M. javanica accounting for almost 95% of the occurrences, and so, an accurate and reliable identification it is primary to establish effective, sustainable and environmentally safe control measures. The main goals of this study were to characterise morphologically, biochemically and molecularly Portuguese isolates and reference material from Netherlands Food and Consumer Product Safety Authority (NVWA), Wageningen, The Netherlands, in order to find the most suitable tools that can be used in the Laboratory of Molecular Genetics and Nematology at the INIAV in Portugal to assist in the identification of the three most common species of Meloidogyne. Morphological studies incorporated the examination of female’s perineal pattern and measurements of second stage juveniles’ features, which presented enough variation to confirm the unreliability and difficulty of morphological identification alone. Biochemical assays with isozymes such as esterases (EST) were performed on females and the PAGE enzymatic patterns obtained match those already described for M. javanica. Molecular analysis included PCRs using universal primers that target the 28S gene, ITS and IGS ribosomal DNA regions (rDNA) and the region between the cytochrome oxidase (COII) of the mitochondrial DNA (mtDNA) and 16S rDNA genes together with speciesspecific SCAR primers and sequencing and cloning. The sequences obtained from sequencing of PCR fragments and from cloned fragments were aligned and compared to those found in GenBank database through BLAST analysis with inconclusive results. Although it was no possible to uncover an efficient tool for Meloidogyne identification this study pinpointed that the three approaches for root-knot nematode identification need to be used if we are to identify this species accurately and effectively. What’s more this research can be used as platform for further studies regarding this species at the Nematology of INIAV PortugalN/