Comparison of the sequences of the D3 expansion of the 26S ribosomal genes reveals different degrees of heterogeneity in different populations and species of Pratylenchus from the Mediterranean region

Abstract

Plant parasitic nematodes belonging to the genus Pratylenchus, also known as root lesion nematodes, cause serious economic damage to different crop plants. In order to explore genetic structures in different isolates, we investigated several P. thornei, P. neglectus and P. penetrans populations of different geographic origins. The analysis at the species level was also extended to P. penetrans, P. pinguicaudatus, P. vulnus and P. mediterraneus. Sequence analysis of a specific portion of DNA was carried out. In particular, the sequences of the D3 region of the 26S gene were obtained and compared with similar sequences available in databases. The results support the hypothesis that P. penetrans may represent a species complex, while in P. neglectus the intra-species heterogeneity observed is due to intra-individual variability. Furthermore, the specific conservation of some nucleotides in different P. thornei populations indicates their fixation in the rDNA repeats in this species. The presence of these nucleotides, the molecular signature of P. thornei, may assist in determining the nature of nematode infections.

Molecular variability and phylogenetic relationships among different species and populations of Pratylenchus (Nematoda: Pratylenchidae) as inferred from the analysis of the ITS rDNA

“…Each sample was overlaid with a drop of mineral oil and incubated at 60°C for 1 h and then at 95°C for 10 min to deactivate the proteinase K. The crude DNA extracted from each individual nematode was directly amplified by using the primer pairs: 18S-Int (5′-CGTAACAAGGTAGCTGTAGG-3′) and 26S-Int (5′-TCCTCCGCTAAATGATATGC-3′). PCR conditions were the same as described in De Luca et al (2004a). The ITS amplified fragments from two or three individual nematodes for each population were purified from agarose gel and cloned into the PCR 2.1-TOPO plasmid using the TOPO TA cloning kit (Invitrogen), following the manufacturer's recommendations.…”

“…In particular the 28S rDNA gene has been largely used to discriminate among different populations and species of Pratylenchus. However, several authors (Al-Banna et al 1997;Duncan et al 1999;De Luca et al 2004a;Subbotin et al 2008) argued that the D2-D3 expansion segments do not contain sufficient phylogenetic signal to resolve relationships among Pratylenchus nematodes at species level because of the existence of cryptic or complex species, which are morphologically indistinguishable but genetically divergent, as recently reported for members of this genus (De Luca et al 2010). More recent studies using ITS-rDNA demonstrated the usefulness of this approach for identification and phylogenetic reconstruction within the genus Pratylenchus (Waeyenberge et al 2009;Palomares-Rius et al 2010).…”

Characterization of Pratylenchus crenatus and P. neglectus (Nematoda: Pratylenchidae) associated with wheat crop

“…Ribosomal DNA (rDNA) sequence has been shown to be a reliable genetic marker for identifi cation purposes, and a powerful tool to analyze genetic variation. The 28S D2/D3 rDNA fragment has been used frequently to characterise Pratylenchus populations (Handoo et al, 2001;Al-Banna et al, 2004;De Luca et al, 2004;Inserra et al, 2007;. Previously P. neglectus has been found under the rhizosphere of Brassica (Kumari, 2012) and Pratylenchus sp.…”

Identification of Pratylenchus thornei, the cereal and legume root-lesion nematode, based on SCAR-PCR and satellite DNA

“…Additionally, the satDNA sequence identified can be used as a sensitive and reliable probe to separate P. thornei from other closely related species. However, validation of these methods with additional P. thornei populations from other geographic origins, and with other closely related species, particularly P. mediterraneus populations (De Luca et al, 2004), is required.…”

“…However, economic damage caused by root-lesion nematodes to herbaceous and fruit crops throughout the world is attributable mainly to eight of the most common species, including Pratylenchus brachyurus, P. coffeae, P. neglectus, P. penetrans, P. scribneri, P. thornei, P. vulnus and P. zeae (Barker, 1998). Genetic variability among these Pratylenchus species has been analysed based on the comparison of the nucleotide sequences of the 26S rDNA (Al-Banna, Williamsom, & Gadner, 1997;De Luca, Fanelli, Di Vito, Reyes, & Di Giorgi, 2004). Identification of Pratylenchus spp.…”