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