First Report of the Spiral Nematode Rotylenchus incultus (Nematoda: Hoplolaimidae) from Cultivated Olive in Tunisia, with Additional Molecular Data on Rotylenchus eximius
Authors: Guesmi-Mzoughi Ilhem , Archidona-Yuste Antonio , Cantalapiedra-Navarrete Carolina , Regaieg Haier , Horrigue-Raouani Najet , Palomares-Rius Juan E. , Castillo Pablo 
Published: July 2017

Abstract
Spiral nematode species of the genus Rotylenchus have been reported on olive (Olea europaea L.) in several Mediterranean countries (Castillo et al., 2010; Ali et al., 2014). Nematological surveys for plant-parasitic nematodes on olive trees were carried out in Tunisia between 2013 and 2014, and two nematode species of Rotylenchus were collected from the rhizosphere of olive cv. Chemlali in several localities of Tunisia (Tables 1,2). Twenty-two soil samples of 3 to 4 kg were collected with a shovel from the upper 50 cm of soil from arbitrarily chosen olive trees. Nematodes were extracted from 500 cm3 of soil by centrifugal flotation method (Coolen, 1979). Specimens were heat killed by adding hot 4% formaldehyde solution and processed to pure glycerin using the De Grisse’s (1969) method. Measurements were done using a drawing tube attached to a zeiss III compound microscope. Nematode DNA was extracted from single individuals and PCR assays were conducted as described by Castillo et al. (2003). Moderate-to-low soil populations of these spiral nematodes were detected (5.5–11.5, 1.5–5.0 individuals/500 cm3 of soil, respectively). This prompted us to undertake a detailed morphological and molecular comparative study with previous reported data. Morphological and molecular analyses of females identified these species as Rotylenchus eximius Siddiqi, 1964, and Rotylenchus incultus Sher, 1965. The morphology of R. eximius females (five specimens studied) was characterized by having a hemispherical lip region clearly off set, with four to five annuli, body without longitudinal striations, lateral fields areolated in the pharyngeal region only, stylet 32 to 36 μm long, and broadly rounded tail. The morphology of R. incultus females (51 females and 16 males; Table 2) was characterized by a hemispherical lip region with the basal annulus subdivided by irregular longitudinal striations, with three, rarely four annuli; stylet 21.5 to 27.5 μm long, female tail hemispherical with terminus regularly annulated; phasmids anterior to anus level (3–6 annuli above). The morphology of the isolated nematodes agreed with previous descriptions of R. eximius (Siddiqi, 1964; Castillo and Vovlas, 2005) and R. incultus (Sher, 1965; Castillo and Vovlas, 2005; Vovlas et al., 2008), respectively. A single individual was used for DNA extraction. Primers and PCR conditions used in this research were specified in Cantalapiedra-Navarrete et al. (2013), and a single amplicon of 800, 1,100, and 450 bp was obtained and sequenced for D2 to D3, ITS1, and cytochrome c oxidase subunit 1 (coxI), respectively. Sequence alignments for D2 to D3 (KX669231–KX669233), ITS1 (KX669238–KX669240), and coxI (KX669244–KX669245) from R. eximius, showed 99% to 97%, 98% to 94%, 93% similarity to other sequences of R. eximius deposited in GenBank (EU280794–DQ328741, EU373663–EU373664, JX015401–JX015402, respectively). Similarly, D2 to D3 (KX669234–KX669237), ITS1 (KX669241–KX669243), and coxI (KX669246–KX669249) sequence alignments from R. incultus, showed 99%, 99% to 95%, 99% to 90% similarity, respectively, to other sequences of R. incultus deposited in GenBank (EU280797, EU373672–EU373673, JX015403, respectively). The best fitted model of DNA evolution was obtained using jModelTest v. 2.1.7 (Darriba et al. 2012) with the Akaike information criterion. BI analyses were performed under the general time reversible (GTR) with invariable sites and a gamma-shaped distribution of substitution rates (GTR + I + G) model for ITS1 and coxI. Phylogenetic analyses of ITS1 and coxI using Bayesian inference (BI) placed R. eximius and R. incultus from Tunisia in subclades that included all R. eximius and R. incultus sequences deposited in GenBank (Fig. 1), which agrees with previous results (Cantalapiedra-Navarrete et al., 2013). Morphology, morphometry, and molecular and phylogenetic data obtained from these samples were consistent with R. eximius and R. incultus identification. To our knowledge, this is the first report of R incultus in Tunisia. Consequently, all these data suggest that spiral nematode species of the genus Rotylenchus are predominant in olive as previously reported in other Mediterranean areas (Ali et al., 2014).