Carlos Gutiérrez-Gutiérrez

Three populations of dagger nematodes (Xiphinema spp.), one recovered from a cassava field (Manihot esculenta) in northern Ecuador and two other from a maize field (Zea mays) in southern Colombia, were characterized through an integrative approach using morphological data and molecular phylogenetic analysis based on the D2–D3 expansion segments of the 28S and ITS1region of the ribosomal RNA (rRNA) and the partial mitochondrial gene cytochrome c oxidase subunit 1 (COI mtDNA) sequences. Integrating morphological hypotheses with genetic data, the pseudomonodelphic population from Colombia was identified as X. krugi and the monodelphic populations from Colombia and Ecuador were identified as X. brasiliense. Bayesian inference (BI) trees for these three molecular markers allowed establishing phylogenetic relationships highlighting the importance of using phylogenetic data for accurate dagger nematode identification at the species level. To our knowledge, this is the first report of X. brasiliense from Ecuador and Colombia, and the first record of X. krugi in a southerly Colombian region, extending the geographical distribution of these species within South America. In this work, morphological information has been updated to distinguish among Xiphinema species within morphospecies Group 1 and 2. New molecular markers were generated and new knowledge on the genetic diversity, molecular evolution and the geographic distribution of monodelphic Xiphinema species is revealed. This research is one of the first systematic studies for Xiphinema detection in Colombia and Ecuador by an integrative taxonomy.

Between 2022 and 2023 ascaridoid nematodes of the genus Hexametra were recovered from two captive central bearded dragons (Pogona vitticeps Ahl) in the Extremadura region of Spain. These nematodes were identified by an integrative approach combining morpho-anatomical and molecular data. We used two rRNA regions (D2–D3 expansion segments of the 28S, and ITS fragments), and the partial region of the cytochrome oxidase I gene (COI mtDNA) sequences. Hexametra angusticaecoides Chabaud & Brygoo, 1960 (Ascarididae) was morphologically characterized and illustrated using light and scanning electron microscopy. Bayesian inference (BI) and Maximum Likelihood (ML) phylogenetic trees for these genetic markers established relationships that highlight the importance of using molecular and phylogenetic data for accurate species identification within the genus Hexametra. Phylogenetic trees indicated that the Spanish isolates of H. angusticaecoides were clustered in a well-supported clade together with other isolates from Central Europe of the same species. Molecular phylogenetic findings also reflect that there was a higher intraspecific variation for ITS genetic markers at the intraspecific level. Phylogenetic results based on 28S rRNA suggest that Ascarididea was divided into three monophyletic major clades. To our knowledge, this is the first report of this species parasitizing bearded dragons, extending the host range of this species. These findings represent the first formal record of H. angusticaecoides in Spain.

A nematological survey was conducted between 2021 and 2022 in banana fields distributed in two major banana-producing regions in the western coast of Syria. As a result, six populations of Xiphinema spp. identified as X. diffusum, X. pachtaicum, X. simile, X. vuittenezi and X. zagrosense were isolated from the rhizosphere of banana plants (Musa sp.) and characterized using morphological and molecular data based on two rRNA genes (D2–D3 expansion segments of the 28S, and ITS fragments) and partial region of the cytochrome oxidase I gene (COI mtDNA) sequences. Moreover, the molecular identification of the endosymbionts of these populations was also performed using the complete 16S rDNA gene. The phylogenetic relationships of the recovered species of nematodes and respective endosymbionts were reconstructed. Candidatus Xiphinematobacter sp. (OR196969; OR196971) and Ca. Xiphinematobacter sp. (OR196970) were detected in X. diffusum and X. simile, respectively, and clustered together with other Ca. Xiphinematobacter sp.A and sp.I respectively. To our knowledge, this is the first report of X. diffusum, X. simile, X. vuittenezi and X. zagrosense parasitizing banana in Syria, extending the geographical distribution of these species within the Mediterranean Basin.

Pin nematodes (Paratylenchus spp.) currently comprise 132 species of polyphagous plant ectoparasites with at least seven species that are plant pathogenic emphasizing the need for correct identification to establish an appropriate management strategy. Sequences of highly conserved regions of ribosomal and mitochondrial RNA genes are a powerful species-level diagnostic tool within Tylenchulidae. A nematological survey was conducted from 2019 to 2021 in commercial vineyards distributed in four major wine-producing regions in the Central and South Portugal. Nine populations of Paratylenchus spp. were isolated from the rhizosphere of grapevines that were characterized from morphological data and molecular phylogenetic analysis using two rRNA genes (D2–D3 expansion segments of the 28S, and partial 18S) and a region partial of the COI mtRNA gene. Contrasting morphological hypotheses with molecular data provided rapid detection of six species, specifically P. goodeyi, P. hamatus, P. pedrami, P. tenicaudatus, P. variabilis, and P. veruculatus. Bayesian inference (BI) phylogenetic trees for these molecular markers established phylogenetic relationships underscore the importance of using genomic and molecular phylogenetic data for accurate pin nematode identification at the species level. To our knowledge, this is the first report of Paratylenchus spp. in Portugal, extending the geographical distribution of these species in the Mediterranean Basin, and the first record of P. goodeyi, P. pedrami, P.variabilis, P. veruculatus and P. tenicaudatus parasitizing grapevine.

An intracellular bacterium, strain IAS^T, was observed to infect several species of the plant-parasitic nematode genus Xiphinema (Xiphinema astaregiense, Xiphinema incertum, Xiphinema madeirense, Xiphinema pachtaicum, Xiphinema parapachydermum and Xiphinema vallense). The bacterium could not be recovered on axenic medium. The 16S rRNA gene sequence of IAS^T was found to be new, being related to the family Burkholderiaceae, class Betaproteobacteria. Fungal endosymbionts Mycoavidus cysteinexigens B1-EB^T (92.9 % sequence identity) and ‘Candidatus Glomeribacter gigasporarum’ BEG34 (89.8 % identity) are the closest taxa and form a separate phylogenetic clade inside Burkholderiaceae. Other genes (atpD, lepA and recA) also separated this species from its closest relatives using a multilocus sequence analysis approach. These genes were obtained using a partial genome of this bacterium. The localization of the bacterium (via light and fluorescence in situ hybridization microscopy) is in the X. pachtaicum females clustered around the developing oocytes, primarily found embedded inside the epithelial wall cells of the ovaries, from where they are dispersed in the intestine. Transmission electron microscopy (TEM) observations supported the presence of bacteria inside the nematode body, where they occupy ovaries and occur inside the intestinal epithelium. Ultrastructural analysis of the bacterium showed cells that appear as mostly irregular, slightly curved rods with rounded ends, 0.8–1.2 µm wide and 2.5–6.0 µm long, possessing a typical Gram-negative cell wall. The peptidoglycan layer is, however, evident only occasionally and not detectable by TEM in most cells. Another irregularly occurring shell surrounding the endosymbiont cells or the cell clusters was also revealed, probably originating from the host cell membrane. Flagella or spore-like cells do not occur and the nucleoid is diffusely distributed throughout the cell. This endosymbiont is transmitted vertically through nematode generations. These results support the proposal of IAS^T as a new species, although its obligate intracellular and obligate endosymbiont nature prevented isolation of a definitive type strain. Strain IAS^T is therefore proposed as representing ‘Candidatus Xiphinematincola pachtaicus’ gen. nov., sp. nov.

The genusLongidoruscurrently comprises 176 species of polyphagous plant ectoparasites, including eight species that vector nepoviruses.Longidorusis one of the most difficult genera to accurately identify species because of the similar morphology and overlapping measurements and ratios among species. Sequences of ribosomal RNA (rRNA)-genes are a powerful level-species diagnostic tool for the genusLongidorus. From 2015 to 2019, a nematode survey was conducted in vineyards and agro-forest environments in Portugal. The populations ofLongidorusspp. were characterized through an integrative approach based on morphological data and molecular phylogenetic analysis from rRNA genes (D2-D3 expansion segments of the 28S, ITS1, and partial 18S), including the topotype ofL. vinearum.Longidorus bordonensissp. nov., a didelphic species recovered from the rhizosphere of grasses, is described and illustrated.Longidorus vineacola, with cork oak and wild olive as hosts, is also characterized. This is the first time thatL. wicuolea, from cork oak, is reported for Portugal. Bayesian inference (BI) phylogenetic trees for these three molecular markers established phylogenetic relationships among the new species with otherLongidorusspp. Phylogenetic trees indicated that i)L. bordonensissp. nov.is clustered together with otherLongidorusspp. and forms a sister clade withL. piniandL. carpetanensis, sharing a short body and odontostyle length, and elongate to conical female tail, and ii) all the other species described and illustrated are phylogenetically associated, including the topotype isolate ofL. vinearum.

The soil-borne nematodeXiphinema indexis closely linked to its main host, the grapevine, and presents a major threat to vineyards worldwide due to its ability to transmitGrapevine fanleaf virus(GFLV). The phylogeography ofX. indexhas been studied using mitochondrial and microsatellite markers in samples from most regions of its worldwide distribution to reveal its genetic diversity. We first used the mitochondrial markerCytBand illustrated the low intraspecific divergence of this mainly meiotic parthenogenetic species. To generate a higher polymorphism level, we then concatenated the sequences ofCytBand three mitochondrial markers,ATP6,CO1andND4, to obtain a 3044-bp fragment. We differentiated two clades, which each contained two well-supported subclades. Samples from the eastern Mediterranean and the Near and Middle East were grouped into three of these subclades, whereas the samples from the western Mediterranean, Europe and the Americas all belonged to the fourth subclade. The highest polymorphism level was found in the samples of one of the Middle and Near East subclades, strongly suggesting that this region contained the native area of the nematode. An east-to-west nematode dissemination hypothesis appeared to match the routes of the domesticated grapevine during Antiquity, presumably mainly dispersed by the Greeks and the Romans. Surprisingly, the samples of the western subclade comprised only two highly similar mitochondrial haplotypes. The first haplotype, from southern Iberian Peninsula, Bordeaux and Provence vineyards, exhibited a high microsatellite polymorphism level that suggests introductions dating from Antiquity. The second haplotype contained a highly predominant microsatellite genotype widespread in distant western countries that may be a consequence of the massive grapevine replanting following the 19^th-century phylloxera crisis. Finally, our study enabled us to draw a first scaffold ofX. indexdiversity at the global scale.