Genetic diversity of the root‐knot nematode Meloidogyne ethiopica and development of a species‐specific SCAR marker for its diagnosis

Abstract

Meloidogyne ethiopica is an important nematode pathogen causing serious economic damage to grapevine in Chile. In Brazil, M. ethiopica has been detected with low frequency in kiwifruit and other crops. The objectives of this study were to evaluate the intraspecific genetic variability of M. ethiopica isolates from Brazil and Chile using AFLP and RAPD markers and to develop a species-specific SCAR-PCR assay for its diagnosis. Fourteen isolates were obtained from different geographic regions or host plants. Three isolates of an undescribed Meloidogyne species and one isolate of M. ethiopica from Kenya were included in the analysis. The results showed a low level of diversity among the M. ethiopica isolates, regardless of their geographical distribution or host plant origin. The three isolates of Meloidogyne sp. showed a high homogeneity and clustered separately from M. ethiopica (100% bootstrap). RAPD screenings of M. ethiopica allowed the identification of a differential DNA fragment that was converted into a SCAR marker. Using genomic DNA from pooled nematodes as a template, PCR amplification with primers designed from this species-specific SCAR produced a fragment of 350 bp in all 14 isolates of M. ethiopica tested, in contrast with other species tested. This primer pair also allowed successful amplification of DNA from single nematodes, either juveniles or females and when used in multiplex PCR reactions containing mixtures of other root-knot nematode species, thus showing the sensitivity of the assay. Therefore, the method developed here has potential for application in routine diagnostic procedures.

Mitochondrial genome plasticity among species of the nematode genus Meloidogyne (Nematoda: Tylenchina)

“…Currently, fast and accurate diagnostic tools are available for most of the economically important Meloidogyne spp. (Wishart et al, 2002;Randig et al, 2002;Zijlstra and Van Hoof, 2006;Tigano et al, 2010;Correa et al, 2013Correa et al, , 2014Pagan et al, in press). However, only a few DNA markers have been tested for population studies within a Meloidogyne species.…”

Diversity of Meloidogyne spp. from peri-urban areas of sub-Saharan Africa and their genetic similarity with populations from the Latin America

“…3). The M. ethiopica population (23) was included as an outgroup, and unfortunately the SCAR marker developed for this species by Correa et al [23] did not amplify DNA specific fragments from this species and from the atypical population Meloidogyne sp.1. These two populations with atypical esterase phenotypes appear to be cryptic species, therefore, in order to clarify their identity, their morphological and other molecular characters need to be further investigated.…”

“…[15,16] and Carneiro et al [17,18]. The DNA-based SCAR-PCR technique enabled the accurate identification of five species: M. arenaria, M. incognita and M. javanica, M. izalcoensis and M. enterolobii, but was unable to definitively characterize M. ethiopica [23] and the two cryptic species, in part because the primers developed for M. ethiopica have been ineffective to identify this species or primers are not yet available for these unknown species [14]. Furthermore, the SCAR-PCR erronously identified Meloidogyne sp.…”

“…Over recent decades, biochemical studies have been conducted using soluble proteins, showing that several species of RKN can be differentiated by the enzymatic phenotypes (esterases) obtained through polyacrylamide gel electrophoresis [15][16][17][18][19]. Since then, studies based on DNA analysis have gradually intensified, with speciesspecific primers (SCAR-PCR) now developed to allow the rapid identification of some species [19][20][21][22][23]. The combined use of isozyme esterase and SCAR markers has been advocated as the most reliable way to accurately identify Meloidogyne spp.…”

Methods and Tools Currently Used for the Identification of Plant Parasitic Nematodes

“…Alternatively, species-specific primers have been designed from sequences randomly scattered throughout nematode genomes, e.g., DNA band obtained from random amplified polymorphic DNA (RAPD) or amplified fragment length polymorphism (AFLP) gels, with posterior cloning and sequencing of bands differential across related species and their conversion into species-specific sequence characterized amplified region (SCAR) markers [21,[43][44][45][46]. SCAR-based markers and rDNA-based specific primers have been used to diagnose nematodes with either conventional or real-time PCR (q-PCR) [6,7,10].…”