Pablo Castillo

Incidence and Population Density of Plant-Parasitic Nematodes Associated with Olive Planting Stocks at Nurseries in Southern Spain

Nematode population densities were determined in 259 soil and root samples collected from 18 olive nurseries in Córdoba, Jaén, and Sevilla provinces (southern Spain), between October 1997 and May 1998. The most important plant-parasitic nematodes detected, in order of decreasing frequency of infestation (percentage of samples), were Mesocriconema xenoplax (39.0%), Pratylenchus penetrans (32.1%), P. vulnus (25.9%), Meloidogyne incognita (14.7%), M. javanica (11.2%), and M. arenaria (2.7%). No disease symptoms were noted on aboveground organs of infected plants. However, population densities of Pratylenchus and Meloidogyne spp. were at potentially damaging levels in most of the olive nurseries surveyed. Histopathological studies of galled roots from the naturally infected olive planting stocks showed a susceptible response to root-knot nematode infection. Large numbers of egg masses were present within the galled root tissues that might contribute to secondary infections. Feeding by root-knot nematodes induced the expected cellular alterations in the cortex, endodermis, pericycle, and vascular system, including giant-cell formation and the alteration of vascular tissues.

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.

Molecular variability and phylogenetic relationships among different species and populations of Pratylenchus (Nematoda: Pratylenchidae) as inferred from the analysis of the ITS rDNA

Sequence comparisons and molecular phylogenetic analyses were used to describe the nucleotide variability of the ITS containing regions of eighteen Pratylenchus species and several populations. Comparative analysis of nucleotide sequences of the rDNA internal transcribed spacers (ITS1 and ITS2) among Pratylenchus species used in the present study demonstrates that ITS sequences can widely vary in primary sequence and length. Alignment of eighty-seven Pratylenchus sequences and one outgroup taxon reveals the presence of ambiguous regions that have the greatest effect on phylogeny reconstruction. Phylogenetic analyses using Bayesian Inference, Neighbour Joining-LogDet, Maximum Likelihood and Maximum Parsimony, distinguished twelve highly or moderately supported major clades within Pratylenchus. Our results support the taxonomic usefulness of the ITS region to identify root-lesion nematode species of the genus Pratylenchus but the high nucleotide variability, sometimes, can preclude its use to resolve relationships among all members of the genus. In addition, the phylogenetic groupings are not congruent with those defined by characters derived by lip patterns and numbers of lip annuli.

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.

Molecular phylogeny of the genus Rotylenchus (Nematoda, Tylenchida) and description of a new species

A description of a new species of plant parasitic nematodes of the genus Rotylenchus from the family Hoplolaimidae is given and a recognition of Rotylenchus jaeni comb. n., previously known as subspecies R. magnus jaeni, as separate species is proposed. Rotylenchus montanus sp. n. is characterized by a hemispherical lip region with six rarely seven annuli, stylet 33–37 µm, female tail rounded, regularly annulated tip with 12–18 annuli and phasmid located 2–9 annuli anterior to anus. Rotylenchus montanus sp. n. is close to species of the monosexual group R. arsenjevi, R. corsicus, R. fragaricus, R. helicus, R. indorobustus and R. neorobustus, by a number of specific characteristics resulting from its specific matrix code: A5, B1, C1, D4, E2, F2, G3, H2, I2, J2, K2. Molecular characterization of R. montanus sp. n. and other Rotylenchus species are provided using D2–D3 expansion segments of 28S and the ITS1 of rRNA genes. The D2–D3 of 28S rRNA and the ITS1–rRNA sequences of R. montanus sp. n. differed in one nucleotide and in 16–20 nucleotides from those of an unidentified Rotylenchus species from Russia, respectively. Molecular analysis of populations of R. magnus and R. jaeni comb. n. demonstrated differences in the D2–D3 and the ITS1–rRNA sequences. These genetic differences together with some minor morphological characters support that both subspecies should be considered as two cryptic sibling species and warranted their elevation to species rank. The result of phylogenetic analysis of Hoplolaimidae for 45 sequences of the D2 and D3 expansion regions of 28S rRNA gene using Bayesian inference analysis under the complex model is presented. Phylogenetic tree of Rotylenchus species represents seven moderate to highly supported lineages. Grouping of Rotylenchus species within other hoplolaimids and analysis of phylogenetic relationships within the genus Rotylenchus using the ITS1 of rRNA gene sequences are also discussed.

Description and molecular characterisation of Paralongidorus litoralis sp. n. and P. paramaximus Heyns, 1965 (Nematoda: Longidoridae) from Spain.

Paralongidorus litoralis sp. n., a new bisexual species of the genus, is described and illustrated by light microscopy, scanning electron microscopy and molecular studies from specimens collected in a coastal sand dune soil around roots of lentisc (Pistacia lentiscus L.) from Zahara de los Atunes (Cadiz), southern Spain. Paralongidorus litoralis sp. n. is characterised by the large body size (7.5-10.0 mm), a rounded lip region, clearly offset from the body by a collar-like constriction, and bearing a very large stirrup-shaped, amphidial fovea, with conspicuous slit-like aperture, a very long and flexible odontostyle ca 190 μm long, guiding ring located at 35 μm from anterior end, and males with spicules ca 70 μm long. In addition, identification data of a Spanish population of P. paramaximus Heyns, 1965 recovered from sandy soil of a commercial citrus orchard at Alcala de Guadaira (Seville), southern Spain, agree very well with the original description of the species from South Africa. The 18S rRNA and D2 and D3 expansion regions of 28S rRNA gene sequences were obtained for P. litoralis sp. n. and P. paramaximus. Phylogenetic analyses of P. litoralis sp. n. and P. paramaximus rRNA gene sequences and of Longidoridae sequences published in GenBank were done using maximum likelihood and Bayesian inference. In trees generated from the 18S data set Paralongidorus clustered as an external clade from Longidorus, and in trees generated from D2-D3 of 28S dataset Paralongidorus was monophyletic and nested within Longidorus. Maximum likelihood test supported the hypothesis of validity of the Paralongidorus genus.

Molecular Characterization of Meloidogyne hispanica (Nematoda, Meloidogynidae) by Phylogenetic Analysis of Genes Within the rDNA in Meloidogyne spp.

In the past, the distribution of Meloidogyne hispanica, the Seville root-knot nematode, appeared to be restricted to the southern part of Spain and Prunus spp.; however, its distribution has been confirmed to be worldwide because it occurs in all continents (Europe, Africa, Asia, Australia, and North, Central, and South America). Differentiation of M. hispanica from other Meloidogyne spp., mainly M. arenaria, can be very difficult using morphological and biological traits data. These species are quite similar and can be regularly confused in inaccurate taxonomic comparisons. In this study, species-specific polymerase chain reaction (PCR) and phylogenetic analysis of sequences from three ribosomal (r)DNA regions (18S, internal transcribed spacer [ITS]1-5.8S-ITS2, and D2-D3 of 28S) were used to characterize three M. hispanica isolates from different geographical origins (Brazil, Portugal, and Spain). Molecular analyses showed identical sequences for all three isolates for the three rDNA regions. Maximum parsimony analysis of the three rDNA regions and the species-specific PCR demonstrated and supported the differentiation of M. hispanica from M. incognita, M. javanica, and M. arenaria and from all described root-knot nematode species.

Plant-parasitic nematodes infecting grapevine in southern spain and susceptible reaction to root-knot nematodes of rootstocks reported as moderately resistant

Incidence and nematode population densities of plant-parasitic nematodes were determined in 64 samples of soil and grapevine roots collected from commercial vineyards in southern Spain between October 2003 and May 2005. In addition, a histopathological study was done of root-stock roots naturally infected by root-knot nematodes (Meloidogyne spp.). Nematodes infecting the rootstocks were identified according to conventional procedures, and the Meloidogyne spp. were furthermore identified by sequence characterized amplified region-polymerase chain reaction (SCAR-PCR) and isozyme esterase analyses. The most important plant-parasitic nematodes detected, in order of decreasing frequency of total soil infestation and root infection (percentage of samples), were Mesocriconema xenoplax (34.4%), Meloidogyne incognita (26.6%), Meloidogyne javanica (14.1%), Xiphinema index (12.5%), Xiphinema italiae (10.9%), Pratylenchus vulnus (6.3%), and Meloidogyne arenaria (1.6%). No disease symptoms were observed on aboveground plant parts of the infected grapevines, except for plants in some fields where soil was infested with the virus-vector nematodes X. index and X. italiae. Those grapevines showed a yellow mosaic pattern in leaves early in the growing season and the internode shortening characteristic of infections by Grapevine fanleaf virus. Rootstocks infected by root-knot nematodes (Meloidogyne spp.) showed distorted feeder roots and large- to moderate-sized root galls, present either singly or in clusters. Histopathology of galled roots showed a typical susceptible response to infection by root-knot nematodes: cellular alterations were induced in the cortex, endodermis, pericycle, and vascular system, including giant-cell formation and severe distortion of vascular tissues. Most Meloidogyne egg masses ocurred on the surface of the galled root tissues, a position that could facilitate dispersion of the nematode eggs and juveniles and the occurrence of secondary infections. Some of the grapevine rootstocks surveyed in this study (Paulsen 1103, Richter 110, Rupestris du Lot, and SO4) had previously been reported to be resistant to Meloidogyne spp.; however, the population densities of these nematodes found in soil and roots sampled in the present study, as well as the compatible host-parasite relationship revealed by histopathology, indicate a susceptible response to Meloidogyne spp. from southern Spain.