Intragenomic rDNA Variants Identified in Rotylenchulus borealis and R. macrodoratus Populations Associated with Olive Groves in Italy

Plant-parasitic reniform nematodes of the genus Rotylenchulus are semi-endoparasites of herbaceous and woody plants occurring in regions with Mediterranean, tropical, and subtropical climates. In the present study, the occurrence of reniform nematodes in the rhizosphere of three olive orchards in Central Italy and six in Sicily (Italy) was investigated. Two Rotylenchulus species were recovered in olive groves in Central Italy, and no Rotylenchulus species were found in Sicily. Using the integrative taxonomy approach, combining morphological, molecular and multivariate morphological analyses, the two species were identified as R. borealis and R. macrodoratus. The D2-D3 sequencing of four individual specimens of Italian R. macrodoratus revealed the occurrence of unique haplotypes differing in nucleotide composition each other. Interestingly, the sequencing of different ITS clones from an individual specimen of the Italian R. borealis showed two ITS paralogs differing in length and nucleotide sequence compared with those of other specimens from the same population and showing higher similarity with those from other populations. Phylogenetic analyses, based on D2-D3 expansion domains of the 28S rRNA gene, ITS, and mitochondrial COI, confirmed the high level of ribosomal variability in both species and the occurrence of new mitochondrial haplotypes for the COI. The present study confirms the occurrence of high variability in Rotylenchulus genus and the existence of variant gene copies in the same specimen that could contribute to the survival of these species in different environments.