Carolina Cantalapiedra-Navarrete

Prevalence, polyphasic identification, and molecular phylogeny of dagger and needle nematodes infesting vineyards in southern Spain

The occurrence and geographic distribution of longidorid nematode species inhabiting the rhizosphere of grapevine plants in southern Spain were investigated. Nematode surveys were conducted on 77 vineyards during the spring seasons of 2006, 2007 and 2008 in the main Andalusian grapevine-growing areas, including the provinces of Cádiz, Córdoba, and Huelva. Morphological and morphometrical studies identified two Longidorus and nine Xiphinema species, viz.: Longidorus alvegus, L. magnus, Xiphinema adenohystherum, X. hispidum, X. index, X. italiae, X. lupini, X. nuragicum, X. pachtaicum, X. rivesi, and X. turcicum. Overall, frequencies of infestation were, in decreasing order: X. pachtaicum 90.8%, X. index 30.3%, X. italiae 13.2%, L. magnus 11.8%, X. hispidum 7.9%, X. lupini 3.9%, L. alvegus and X. rivesi 2.6%, and X. adenohystherum, X. nuragicum and X. turcicum 1.3%. Xiphinema hispidum, X. lupini, L. alvegus and L. magnus were compared with nematode type specimens and are reported for the first time in Spain. Furthermore, the male of L. alvegus is described for the first time in the literature. Molecular characterisation of these species using D2–D3 expansion regions of 28S rRNA, 18S rRNA and ITS1-rRNA was carried out and maximum likelihood and Bayesian inference analysis were used to reconstruct phylogenetic relationships among these species and with other longidorids. The monophily of the genera Xiphinema and Longidorus was accepted and the genera Paralongidorus and Xiphidorus were rejected by the Shimodaira-Hasegawa test based on tree topologies.

Cryptic species in plant-parasitic nematodes

This paper summarises the current knowledge concerning cryptic species of plant-parasitic nematode and briefly reviews the different methods available for their detection and characterisation. Cryptic species represent an important component of biodiversity, such speciation being common among plant-parasitic nematodes and occurring in diverse groups with different life history traits, including the spiral, virus vector, root-lesion and false root-knot nematodes. Cryptic species are important for a number of reasons, including food security, quarantine, non-chemical management technologies and species conservation, and should not be ignored. The magnitude of the phenomenon is largely unknown, but the available data on plant-parasitic nematodes demonstrate that reliance on morphology alone for species delimitation seriously underestimates the total number of taxa. Future research should focus on appropriately designed case studies using combined approaches, including large-scale, whole sample analyses by next-generation sequencing or proteomics in order to be able to answer the many questions that still remain.

Molecular analysis and comparative morphology to resolve a complex of cryptic Xiphinema species

Gutiérrez‐Gutiérrez, C., Palomares‐Rius, J.E., Cantalapiedra‐Navarrete, C., Landa, B.B., Esmenjaud, D. & Castillo, P. (2010). Molecular analysis and comparative morphology to resolve a complex of cryptic Xiphinema species. —Zoologica Scripta, 39, 483–498.

Phylogeny, diversity, and species delimitation in some species of the Xiphinema americanum-group complex (Nematoda: Longidoridae), as inferred from nuclear and mitochondrial DNA sequences and morphology

During nematode surveys in southern Spain and Italy 14 populations of Xiphinema species tentatively identified as Xiphinema americanum-group were detected. Morphological and morphometrical studies identified three new species and six known Xiphinema americanum-group species, viz.: Xiphinema parabrevicolle n. sp., Xiphinema parapachydermum n. sp., Xiphinema paratenuicutis n. sp., Xiphinema duriense, Xiphinema incertum, Xiphinema opisthohysterum, Xiphinema pachtaicum, Xiphinema rivesi, and Xiphinema santos. The Xiphinema americanum-group is the most difficult Xiphinema species group for diagnosis since the morphology is very conservative and morphometric characters often overlap. This group includes vectors of several important plant pathogenic viruses that cause significant damage to a wide range of agricultural crops. Molecular characterisation of these species using D2-D3 expansion regions of 28S rRNA, 18S rRNA, ITS1-rRNA and the protein-coding mitochondrial gene, cytochrome oxidase c subunit 1 was carried out and maximum likelihood and Bayesian inference analysis were used to reconstruct phylogenetic relationships among these species and with other Xiphinema americanum-group species.

Unravelling the Biodiversity and Molecular Phylogeny of Needle Nematodes of the Genus Longidorus (Nematoda: Longidoridae) in Olive and a Description of Six New Species

The genus Longidorus includes a remarkable group of invertebrate animals of the phylum Nematoda comprising polyphagous root-ectoparasites of numerous plants including several agricultural crops and trees. Damage is caused by direct feeding on root cells as well as by transmitting nepoviruses that cause disease on those crops. Thus, correct identification of Longidorus species is essential to establish appropriate control measures. We provide the first detailed information on the diversity and distribution of Longidorus species infesting wild and cultivated olive soils in a wide-region in southern Spain that included 159 locations from which 449 sampling sites were analyzed. The present study doubles the known biodiversity of Longidorus species identified in olives by including six new species (Longidorus indalus sp. nov., Longidorus macrodorus sp. nov., Longidorus onubensis sp. nov., Longidorus silvestris sp. nov., Longidorus vallensis sp. nov., and Longidorus wicuolea sp. nov.), two new records for wild and cultivate olives (L. alvegus and L. vineacola), and two additional new records for wild olive (L. intermedius and L. lusitanicus). We also found evidence of some geographic species associations to western (viz. L. alvegus, L. intermedius, L. lusitanicus, L. onubensis sp. nov., L. vineacola, L. vinearum, L. wicuolea sp. nov.) and eastern distributions (viz. L. indalus sp. nov.), while only L. magnus was detected in both areas. We developed a comparative study by considering morphological and morphometrical features together with molecular data from nuclear ribosomal RNA genes (D2–D3 expansion segments of 28S, ITS1, and partial 18S). Results of molecular and phylogenetic analyses confirmed the morphological hypotheses and allowed the delimitation and discrimination of six new species of the genus described herein and four known species. Phylogenetic analyses of Longidorus spp. based on three molecular markers resulted in a general consensus of these species groups, since lineages were maintained for the majority of species. This study represents the most complete phylogenetic analysis for Longidorus species to date.

Morphological and molecular characterisation of Pratylenchus oleae n. sp. (Nematoda: Pratylenchidae) parasitizing wild and cultivated olives in Spain and Tunisia.

A new mono-sexual root-lesion nematode species, Pratylenchus oleae n. sp., parasitizing roots of olive plants cv. Koroneiki in commercial fields at Ouled Chamekh (central Tunisia), and wild and cultivated olive (cv. Picual) plants in Agua Amarga (southern Spain) is described. The new species is characterised by the female having a lip region slightly offset and bearing three annuli, stylet 16.5 (14.5-17.0) μm long, with prominent rounded knobs, pharyngeal overlapping rather long (22–36) μm, lateral fields areolated and with four incisures and diagonal lines in middle band, spermatheca rounded but non-functional, tail short, conoid-rounded to subcylindrical, usually annulated terminus, males unknown, and a specific D2-D3, ITS1, 18S-rRNA, hsp90 and COI sequences. Morphologically this species is related to P. cruciferus, P. delattrei, and P. kumamotoensis. The results of the phylogenetic analysis based on sequences of the D2-D3 expansion regions of 28S, partial 18S and ITS rRNA genes confirmed the close relationship of P. oleae n. sp. with P. dunensis, P. penetrans, P. pinguicaudatus, from which was clearly separated. A PCR-based diagnostic assay was also developed for identification of P. oleae n. sp. using the species-specific primers Poleae_fw1_4 and Poleae_rv1 that amplify a 547-bp fragment in the internal transcribed spacer (ITS1) region of ribosomal DNA, which clearly separate from other root-lesion nematodes damaging olive such as P. penetrans and P. vulnus.

Morphological and molecular characterisation of Paralongidorus plesioepimikis n. sp. (Nematoda: Longidoridae) from southern Spain.

This research was supported by grant AGL2009-06955 from ‘Ministerio de Ciencia e Innovacion’ of Spain, grant AGR-136 from ‘Consejeria de Economia, Innvovacion y Ciencia’ from Junta de Andalucia, and the European Social Fund.

Seven new species of Trichodorus (Diphtherophorina, Trichodoridae) from Spain, an apparent centre of speciation.

During a survey for Trichodoridae in cultivated and natural environments, mainly from southern Spain, 7 new Trichodorus species were found. Four of them, T. andalusicus n. sp., T. asturanus n. sp., T. silvestris n. sp. and T. parasilvestris n. sp., belong to the T. lusitanicus morpho-species group characterised in the male by the slightly ventrally curved spicules with a mid-blade constriction with bristles, and in the female by well developed, rounded triangular to quadrangular, vaginal sclerotised pieces. They were mainly differentiated based upon differences in body length, onchiostyle length, spicule shape and number of ventromedian cervical papillae in the male, and size and shape of the vaginal sclerotised pieces in the female. The presence of the T. lusitanicus morpho-species group with 8 species, apparently endemic to the Iberian Peninsula, could point to a recent speciation event. Additionally, 3 new species, T. onubensis n. sp., T. iliplaensis n. sp. and T. paragiennensis n. sp., more closely resembling the morpho-species group of T. sparsus, to which the Spanish species T. giennensis also belongs, were identified. They were differentiated based on body length, length of onchiostyle and position of the secretory-excretory pore and, in the male, by length and shape of the spicules and number of ventromedian cervical papillae and in the female by the vaginal sclerotised pieces. Molecular support to differentiate the new species using the D2-D3 expansion segments of 28S rDNA is also provided. In addition, a new population of T. variabilis from Greece was studied as well as T. lusitanicus type specimens, and a population of T. giennensis and T. lusitanicus were also sequenced.

Heterodera elachista the Japanese cyst nematode parasitizing corn in Northern Italy: integrative diagnosis and bionomics.

The Japanese cyst nematode Heterodera elachista was detected parasitizing corn cv Rixxer in Bosco Mesola (Ferrara Province) in Northern Italy. The only previous report of this nematode was in Asia (Japan, China and Iran) attacking upland rice; being this work the first report of this cyst nematode in Europe, and confirmed corn as a new host plant for this species. Integrative morphological and molecular data for this species were obtained using D2-D3 expansion regions of 28S rDNA, ITS1-rDNA, the partial 18S rDNA, the protein-coding mitochondrial gene, cytochrome oxidase c subunit I (COI), and the heat-shock protein 90 (hsp90). Heterodera elachista identified in Northern Italy was morphologically and molecularly clearly separated from other cyst nematodes attacking corn (viz. H. avenae, H. filipjevi, H. delvii, H. oryzae, H. sacchari, H. sorghi, H. zeae, Punctodera chalcoensis, and Vittadera zeaphila) and rice (H. oryzae, H. sacchari). The phylogenetic relationships of H. elachista from Northern Italy with other cyst-nematodes using rDNA and mtDNA showed a separation of the genus Heterodera in various morphospecies groups based on vulval cone structures. The development and parasitic habit of H. elachista on naturally infected corn cv Rixxer confirmed a typical susceptible reaction, including multinucleate syncytial cells in parenchymatic cells. Under greenhouse conditions, H. elachista successfully reproduced on two crops widely used in Northern Italy, such as corn (cv PR 33) and rice (cv Baldo). Considering the limited host-range of this nematode, that include two of the three world’s most important crops, special attention is needed for avoiding the dispersal of this nematode into new areas, by movement of soil on equipment, water, and contaminated containers infested soil, or agricultural practices.

Molecular and morphological characterisation of Paralongidorus iranicus n. sp. and P. bikanerensis (Lal & Mathur, 1987) Siddiqi, Baujard & Mounport, 1993 (Nematoda: Longidoridae) from Iran.

Paralongidorus iranicus n. sp., a new bisexual species of the genus, is described and illustrated by light microscopy, scanning electron microscopy and molecular studies from specimens collected in the rhizosphere of Scots pine (Pinus sylvestris) from the Caspian (Khazar) seashore, Nour, northern Iran. Paralongidorus iranicus n. sp. is characterised by the large body size (7.8-11.4 mm), a rounded lip region, clearly set off by a collar-like constriction at level of, or slightly posterior to, the amphidial aperture, and bearing a very large, stirrup-shaped, amphidial fovea, with conspicuous slit-like aperture, a very long and flexible odontostyle ca 170 μm long, guiding ring located at 34 μm from anterior end and males with spicules ca 80 μm long. In addition, data from an Iranian population of P. bikanerensis recovered from the rhizosphere of palm (Phoenix dactylifera L.) in Bam, Kerman province, south-eastern Iran, agree very well and are very close to the original description of the species from India. The D2 and D3 expansion regions of 28S rRNA gene, ITS1, and 18S rRNA sequences were obtained for P. iranicus n. sp. and P. bikanerensis. Phylogenetic analyses of P. iranicus n. sp. and P. bikanerensis rRNA gene sequences and of Longidorus spp. sequences published in GenBank were done using Maximum Likelihood and Bayesian inference. Paralongidorus species (including P. iranicus n. sp.) clustered together; however, P. bikanerensis clustered within Longidorus spp. and was clearly separated from all other Paralongidorus spp. in trees generated from the D2-D3 expansion segments of 28S and partial 18S data set, respectively.