Morphological, Biometrical, Biochemical, and Molecular Characterization of the Coffee Root-Knot Nematode Meloidogyne megadora.

Meloidogyne megadora infects coffee trees, an economically important crop worldwide. The accurate identification of M. megadora is essential for the development of preventive measures to avoid the dispersion of this pathogen and establishment of efficient and sustainable integrated pest management programs. One M. megadora isolate was studied by biometrical, biochemical, and molecular characteristics (random amplified polymorphic DNA [RAPD] and PCR of internal transcribed spacer [ITS] region). Biometrical characteristics of M. megadora females, males, and second-stage juveniles were similar to the original description. Biochemical studies revealed a unique enzyme pattern for M. megadora esterases (Me3) that allowed for species differentiation. Three RAPD primers (OPG-4, OPG-5, and OPG-6) produced specific bands to all Meloidogyne spp. studied: M. megadora, M. arenaria, M. incognita, and M. javanica. Molecular analysis of the ITS region resulted in an amplification product of 700 bp. The phylogenetic relationship between M. megadora and several Meloidogyne spp. sequences was analyzed, revealing that M. megadora clearly differs from the most common root-knot nematode species. Based on the studies conducted, isozyme analysis remains a useful and efficient methodology for M. megadora identification when females are available. Further studies will be needed to convert the M. megadora differential DNA fragment obtained by RAPD and develop a species-specific sequence-characterized amplified region PCR assay for its diagnosis based on second-stage juveniles.

Biometrical, Biochemical, and Molecular Diagnosis of Portuguese Meloidogyne hispanica Isolates.

Meloidogyne hispanica infects many economically important crops worldwide. The accurate identification of this pathogen is essential for the establishment of efficient and sustainable integrated pest management programs. Portuguese M. hispanica isolates were studied by biometrical, biochemical, and molecular characteristics. Biometrical characteristics of M. hispanica females, males, and second-stage juveniles were similar to the original description. Biochemical studies revealed a unique enzyme pattern (Hi4) for M. hispanica esterases that allowed for species differentiation. Molecular analysis of the mtDNA region from COII and 16S rRNA genes resulted in amplification products (1,800 bp) similar to M. hispanica, M. ethiopica, and M. javanica, and the described HinfI was unable to discriminate M. hispanica from the other two species. Analysis of the mtDNA sequences revealed altered nucleotides among the isolates that created new restriction sites for AluI and DraIII. The resulting restriction patterns successfully discriminated between the three species, providing a new tool for Meloidogyne identification. Finally, the phylogenetic relationship between M. hispanica and several Meloidogyne spp. sequences was analyzed using mtDNA, confirming the divergence between meiotic and mitotic species and revealing the proximity of M. hispanica to closely related species. Based on the studies conducted, the application of isozyme or polymerase chain reaction restriction fragment length polymorphism analysis would be a useful and efficient methodology for M. hispanica identification.

Molecular Characterization of Meloidogyne hispanica (Nematoda, Meloidogynidae) by Phylogenetic Analysis of Genes Within the rDNA in Meloidogyne spp.

In the past, the distribution of Meloidogyne hispanica, the Seville root-knot nematode, appeared to be restricted to the southern part of Spain and Prunus spp.; however, its distribution has been confirmed to be worldwide because it occurs in all continents (Europe, Africa, Asia, Australia, and North, Central, and South America). Differentiation of M. hispanica from other Meloidogyne spp., mainly M. arenaria, can be very difficult using morphological and biological traits data. These species are quite similar and can be regularly confused in inaccurate taxonomic comparisons. In this study, species-specific polymerase chain reaction (PCR) and phylogenetic analysis of sequences from three ribosomal (r)DNA regions (18S, internal transcribed spacer [ITS]1-5.8S-ITS2, and D2-D3 of 28S) were used to characterize three M. hispanica isolates from different geographical origins (Brazil, Portugal, and Spain). Molecular analyses showed identical sequences for all three isolates for the three rDNA regions. Maximum parsimony analysis of the three rDNA regions and the species-specific PCR demonstrated and supported the differentiation of M. hispanica from M. incognita, M. javanica, and M. arenaria and from all described root-knot nematode species.