Juan E. Palomares-Rius

The genome and life-stage specific transcriptomes of Globodera pallida elucidate key aspects of plant parasitism by a cyst nematode

BackgroundGlobodera pallida is a devastating pathogen of potato crops, making it one of the most economically important plant parasitic nematodes. It is also an important model for the biology of cyst nematodes. Cyst nematodes and root-knot nematodes are the two most important plant parasitic nematode groups and together represent a global threat to food security.ResultsWe present the complete genome sequence of G. pallida, together with transcriptomic data from most of the nematode life cycle, particularly focusing on the life cycle stages involved in root invasion and establishment of the biotrophic feeding site. Despite the relatively close phylogenetic relationship with root-knot nematodes, we describe a very different gene family content between the two groups and in particular extensive differences in the repertoire of effectors, including an enormous expansion of the SPRY domain protein family in G. pallida, which includes the SPRYSEC family of effectors. This highlights the distinct biology of cyst nematodes compared to the root-knot nematodes that were, until now, the only sedentary plant parasitic nematodes for which genome information was available. We also present in-depth descriptions of the repertoires of other genes likely to be important in understanding the unique biology of cyst nematodes and of potential drug targets and other targets for their control.ConclusionsThe data and analyses we present will be central in exploiting post-genomic approaches in the development of much-needed novel strategies for the control of G. pallida and related pathogens.

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.

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.

Description of Pratylenchus hispaniensis n. sp. from Spain and considerations on the phylogenetic relationship among selected genera in the family Pratylenchidae

A new amphimictic species, Pratylenchus hispaniensis n. sp., parasitising the roots of gum cistus in Andujar (Jaen), southern Spain, is described. The new species is characterised by the presence of numerous males and by the female having a lip region with three annuli, a divided face, a robust stylet (14.5-17.0 μm) with rounded knobs, lateral fields with four lines, V = 80-84, a round spermatheca full of sperm, well developed post-vulval uterine sac and an obliquely truncate tail with irregularly annulated terminus. Morphologically this species is related to P. bhatti, P. kralli, P. mediterraneus, P. pseudofallax and P. thornei. A phenetic study of the 25 most useful diagnostic morphological and allometric characters for Pratylenchus species was done using multivariate factor and linear discriminant analyses. In the factor analysis the first seven factors accounted for 71.1% of the total variance of the characters selected. These factors were related to female tail, pharyngeal overlap, reproductive behaviour, stylet length, L/post-vulval uterine sac ratio, body length and number of lip annuli. Discriminant analysis differentiated Pratylenchus spp. from the three valid species of Zygotylenchus. 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

Distribution and evolution of glycoside hydrolase family 45 cellulases in nematodes and fungi

BackgroundHorizontal gene transfer (HGT) has been suggested as the mechanism by which various plant parasitic nematode species have obtained genes important in parasitism. In particular, cellulase genes have been acquired by plant parasitic nematodes that allow them to digest plant cell walls. Unlike the typical glycoside hydrolase (GH) family 5 cellulase genes which are found in several nematode species from the order Tylenchida, members of the GH45 cellulase have only been identified in a cluster including the families Parasitaphelenchidae (with the pinewood nematode Bursaphelenchus xylophilus) and Aphelenchoididae, and their origins remain unknown.ResultsIn order to investigate the distribution and evolution of GH45 cellulase genes in nematodes and fungi we performed a wide ranging screen for novel putative GH45 sequences. This revealed that the sequences are widespread mainly in Ascomycetous fungi and have so far been found in a single major nematode lineage. Close relationships between the sequences from nematodes and fungi were found through our phylogenetic analyses. An intron position is shared by sequences from Bursaphelenchus nematodes and several Ascomycetous fungal species.ConclusionsThe close phylogenetic relationships and conserved gene structure between the sequences from nematodes and fungi strongly supports the hypothesis that nematode GH45 cellulase genes were acquired via HGT from fungi. The rapid duplication and turnover of these genes within Bursaphelenchus genomes demonstrate that useful sequences acquired via HGT can become established in the genomes of recipient organisms and may open novel niches for these organisms to exploit.

Soil Properties and Olive Cultivar Determine the Structure and Diversity of Plant-Parasitic Nematode Communities Infesting Olive Orchards Soils in Southern Spain

This work has studied for the first time the structure and diversity of plant-parasitic nematodes (PPNs) infesting olive orchard soils in a wide-region in Spain that included 92 locations. It aims at determining which agronomical or environmental factors associated to the olive orchards are the main drivers of the PPNs community structure and diversity. Classical morphological and morphometric identification methods were used to determine the frequency and densities of PPNs. Thirteen families, 34 genera and 77 species of PPNs were identified. The highest diversity was found in Helicotylenchus genus, with six species previously reported in Spain and with H. oleae being a first report. Neodolichorhynchus microphasmis and Diptenchus sp., Diphtherophora sp., and Discotylenchus sp., usually considered fungal feeders, were also reported for the first time associated to olive rhizosphere. PPNs abundance ranged from 66 to 16,288 individuals/500-cm3 of soil with Helicotylenchus digonicus being the most prevalent species, followed by Filenchus sp., Merlinius brevidens and Xiphinema pachtaicum. Nematode abundance and diversity indexes were influenced by olive cultivar, and orchard and soil management practices; while olive variety and soil texture were the main factors driving PPN community composition. Soil physicochemical properties and climatic characteristics most strongly associated to the PPN community composition included pH, sand content and exchangeable K, and maximum and minimum average temperature of the sampled locations. Our data suggests that there is a high diversity of PPNs associated to olive in Southern Spain that can exert different damage to olive roots depending on the olive variety and their abundance. Further analysis to determine the resistance levels of most common olive varieties to the prevalent PPNs in Spain will help to choose the most appropriate ones for the establishment of new plantations. This choice will take into consideration the specific soils and environments where those olive varieties will be established.

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.

Comparative morphometrics and ribosomal DNA sequence analysis of Longidorus orientalis Loof, 1983 (Nematoda: Longidoridae) from Spain and Iran

During recent nematode surveys in a muddy soil around undetermined graminaceous plants in El Rocio, Huelva Province, in southern Spain, and from the rhizosphere of date palm associated with graminaceous vegetation from Abadan, in Khuzestan Province, south-west Iran, populations of Longidorus orientalis were identified. Morphological and morphometrical studies on these populations fit the original description and represent the first report from Spain and Europe. Molecular characterisation of L. orientalis from Spain and Iran, using D2-D3 expansion regions of 28S rRNA, 18S rRNA and ITS-rRNA, is provided. Sequences of the D2-D3 expansion regions and partial 18S genes were analysed using maximum likelihood and Bayesian inference to reconstruct phylogenetic relationships within L. orientalis and other Longidorus species. The results revealed a closer phylogenetic relationship with L. goodeyi for the D2-D3 expansion region and with L. vineacola for the partial 18S region.

Assessment of Helminth Biodiversity in Wild Rats Using 18S rDNA Based Metagenomics

Parasite diversity has important implications in several research fields including ecology, evolutionary biology and epidemiology. Wide-ranging analysis has been restricted because of the difficult, highly specialised and time-consuming processes involved in parasite identification. In this study, we assessed parasite diversity in wild rats using 18S rDNA-based metagenomics. 18S rDNA PCR products were sequenced using an Illumina MiSeq sequencer and the analysis of the sequences using the QIIME software successfully classified them into several parasite groups. The comparison of the results with those obtained using standard methods including microscopic observation of helminth parasites in the rat intestines and PCR amplification/sequencing of 18S rDNA from isolated single worms suggests that this new technique is reliable and useful to investigate parasite diversity.