title: First Report of the Spiral NematodeRotylenchus incultus(Nematoda: Hoplolaimidae) from Cultivated Olive in Tunisia, with Additional Molecular Data onRotylenchus eximius.
abstract: Spiral nematode species of the genusRotylenchushave been reported on olive (Olea europaeaL.) 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 ofRotylenchuswere collected from the rhizosphere of olive cv. Chemlali in several localities of Tunisia (Tables 1,2 [Table: see text] [Table: see text] ). 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 cm3of 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 cm3of 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 asRotylenchus eximiusSiddiqi, 1964, andRotylenchus incultusSher, 1965. The morphology ofR. eximiusfemales (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 ofR. incultusfemales (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 ofR. eximius(Siddiqi, 1964; Castillo and Vovlas, 2005) andR. 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), andcoxI(KX669244-KX669245) fromR. eximius, showed 99% to 97%, 98% to 94%, 93% similarity to other sequences ofR. eximiusdeposited in GenBank (EU280794-DQ328741, EU373663-EU373664, JX015401-JX015402, respectively). Similarly, D2 to D3 (KX669234-KX669237), ITS1 (KX669241-KX669243), andcoxI(KX669246-KX669249) sequence alignments fromR. incultus, showed 99%, 99% to 95%, 99% to 90% similarity, respectively, to other sequences ofR. incultusdeposited 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 andcoxI. Phylogenetic analyses of ITS1 andcoxIusing Bayesian inference (BI) placedR. eximiusandR. incultusfrom Tunisia in subclades that included allR. eximiusandR. incultussequences deposited in GenBank (Fig. 1Fig. 1Phylogenetic relationships withinRotylenchusspecies found in Tunisia and other species from GenBank. Bayesian 50% majority rule consensus trees as inferred from ITS1 andcoxIsequences alignments under the GTR + I + G model. Posterior probabilities more than 0.70 are given for appropriate clades. Newly obtained sequences in this study are in bold. Scale bar = expected changes per site.), which agrees with previous results (Cantalapiedra-Navarrete et al., 2013). Morphology, morphometry, and molecular and phylogenetic data obtained from these samples were consistent withR. eximiusandR. incultusidentification. To our knowledge, this is the first report ofR. incultusin Tunisia. Consequently, all these data suggest that spiral nematode species of the genusRotylenchusare predominant in olive as previously reported in other Mediterranean areas (Ali et al., 2014).
authors.affiliation: Department of Biological Sciences and Plant Protection, Higher Institute of Agronomy, University of Sousse, Chott Mariem, UR: R13AGR04, Bp47, 4042 Sousse, Tunisia.
authors.forename: Ilhem
authors.initials: I
authors.lastname: Guesmi-Mzoughi
authors.affiliation: Institute for Sustainable Agriculture, CSIC, Campus de Excelencia Internacional Agroalimentario, ceiA3, Avenida Menéndez Pidal s/n, 14004 Córdoba, Spain.
authors.forename: Antonio
authors.initials: A
authors.lastname: Archidona-Yuste
authors.affiliation: Institute for Sustainable Agriculture, CSIC, Campus de Excelencia Internacional Agroalimentario, ceiA3, Avenida Menéndez Pidal s/n, 14004 Córdoba, Spain.
authors.forename: Carolina
authors.initials: C
authors.lastname: Cantalapiedra-Navarrete
authors.affiliation: Department of Biological Sciences and Plant Protection, Higher Institute of Agronomy, University of Sousse, Chott Mariem, UR: R13AGR04, Bp47, 4042 Sousse, Tunisia.
authors.forename: Hajer
authors.initials: H
authors.lastname: Regaieg
authors.affiliation: Department of Biological Sciences and Plant Protection, Higher Institute of Agronomy, University of Sousse, Chott Mariem, UR: R13AGR04, Bp47, 4042 Sousse, Tunisia.
authors.forename: Najet
authors.initials: N
authors.lastname: Horrigue-Raouani
authors.affiliation: Institute for Sustainable Agriculture, CSIC, Campus de Excelencia Internacional Agroalimentario, ceiA3, Avenida Menéndez Pidal s/n, 14004 Córdoba, Spain.
authors.forename: Juan E
authors.initials: JE
authors.lastname: Palomares-Rius
authors.affiliation: Institute for Sustainable Agriculture, CSIC, Campus de Excelencia Internacional Agroalimentario, ceiA3, Avenida Menéndez Pidal s/n, 14004 Córdoba, Spain.
authors.forename: Pablo
authors.initials: P
authors.lastname: Castillo
keywords: Bayesian inference; detection; new geographic record; phylogeny; spiral nematodes