Gracia Liébanas

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 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

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.

Eutylenchus excretorius Ebsary & Eveleigh, 1981 (Nematoda: Tylodorinae) from Spain with approaches to molecular phylogeny of related genera

Nematode surveys in indigenous vegetation in northern Spain revealed the presence of a nematode population of the genus Eutylenchus associated with moist sandy soils in the rhizosphere of common reed (Phragmites sp.) on the banks of the Tera river in Garray (Soria province). Morphological and morphometrical studies on this population fits with Eutylenchus excretorius, representing the first report for Spain and southern Europe and the fifth report in Europe after Germany, Poland, Czech Republic and Russia. SEM studies were carried out for the first time on this species and showed four lips separated by deep grooves. Each lip bears an elongated, flexible, recurved projection (seta) 12 (11-13) μm long, proximal third wide, gradually attenuating, distal end rounded. Molecular characterisation of E. excretorius using several genes is provided. The sequence of D2-D3 expansion segments of 28S rRNA gene of this population was identical to a previously studied sample from Germany. Phylogenetic analysis using D2-D3 of 28S rRNA and partial 18S rRNA gene sequences of tylenchid nematodes revealed that E. excretorius clustered with moderate support with Cephalenchus hexalineatus. The position of E. excretorius on majority consensus Bayesian phylogenetic tree reconstructed using heat shock protein 90 gene sequence was not well resolved.

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.

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.

Parasitism effects on white clover by root-knot and cyst nematodes and molecular separation of Heterodera daverti from H. trifolii

This research carried out an accurate identification of the root-knot and cyst-forming nematode species parasitizing white clover at the Laceno Lake area in Southern Italy. Two species, the root-knot nematode Meloidogyne hapla and the cyst nematode Heterodera daverti were identified by integrative taxonomic approaches (classical, isozyme pattern, and D2-D3 expansion segments of 28S rRNA, ITS rRNA and coxI of mtDNA gene sequences) and found parasitizing white clover roots. These nematodes were detected in stunted plants with a reduced number of rhizobium nodules and the host suitability was confirmed by the high nematode population densities ranging from 53 to 2350 eggs and J2s per g of fresh roots for M. hapla, and 1.36 eggs and J2s/cm3 of soil for H. daverti; and cyst nematode females were also detected on the roots of clover. The studies on the host-parasite relationships of nematode-feeding sites in white clover roots infected by these nematodes showed a high susceptible response. Meloidogyne hapla and H. daverti infections were also observed on nitrogen-fixing root nodules of white clover, where well established feeding sites allowed active nematode reproduction. Histological examination of nitrogen-fixing root nodule tissues revealed that the nematodes established their permanent feeding sites in the vascular bundles of nodules which appeared enlarged deformed and disorganised by the expansion of nematode feeding cells (giant cells and syncytium) and hyperplasia of the nodule cortex. Additionally, coxI of mtDNA gene is an efficient barcoding sequence for discriminating the identification of H. daverti from H. trifolii.

Molecular and morphological characterisation of Rotylenchus vitis n. sp. (Nematoda: Hoplolaimidae) infectinggrapevine in southern Spain.

Rotylenchus vitis n. sp., a new amphimictic species infesting soil and roots of commercial vineyards in southern Spain, is described. Rotylenchus vitis n. sp. is characterised by a truncate lip region with 7-9 annuli and continuous with the body contour, lateral fields areolated at pharyngeal region only, body without longitudinal striations, stylet length of 36-48 μm, vulva position at 47-57%, tail rounded to hemispherical with 13-21 annuli. Morphologically, this species is related to R. cazorlaensis, R. capitatus, R. elegans, R. fabalus, R. iranicus, R. labiodiscus, R. montanus and R. troncapitatus. The results of the phylogenetic analysis based on the sequences of the D2-D3 expansion regions of the 28S and ITS1-rRNA genes confirmed the species differentiation and the close molecular relationship between R. eximius and R. montanus, respectively.

Rotylenchus castilloi n. sp. (Nematoda: Hoplolaimidae), a new species with long stylet from northern Iran.

Rotylenchus castilloi n. sp., a new bisexual species is described and illustrated based on morphological, morphometric and molecular data. The new species is characterised by having a hemispherical, continuous lip region with an irregular corncob-like appearance under SEM, very long stylet (62-68 µm), vulva located at 49.7-62.2% of body length from anterior end, with a protruding double epiptygma, a rounded to convex-conoid (rarely bi-lobed) tail with 8-12 annuli and specific sequences of D2-D3 segments of 28S and ITS1-rRNA genes. Differences between the new species and four other species of the genus (R. mesorobustus, R. cazorlaensis, R. magnus and R. jaeni) are discussed. Morphologically, the new species can be separated from these species mostly by its body length, lip region characters, stylet length and location of phasmid. Phylogenetic analyses using 721 bp partial sequences of D2-D3 expansion segments of the 28S and 590 bp ITS1-rRNA genes revealed the new species forming a clade with two isolates of R. eximius and two isolates of R. unisexus, two morphologically unrelated species.

A new root-knot nematode Meloidogyne spartelensis n. sp. (Nematoda: Meloidogynidae) in Northern Morocco

Low density of an unknown root-knot nematode was found on wild olive soils at Cape Spartel near Tanger city in northern Morocco. Morphometry, esterase and malate dehydrogenase electrophoretic phenotypes, as well as ribosomal DNA (rDNA) and mitochondrial DNA (mtDNA) sequences demonstrated that this nematode species differs clearly from other previously described root-knot nematodes. The species is herein described, illustrated and named as Meloidogyne spartelensis n. sp. This new root-knot nematode can be morphologically distinguished from other Meloidogyne spp. by: (i) roundish perineal pattern, dorsal arch low, with fine, sinuous cuticle striae, lateral field faintly visible; (ii) female excretory pore posterior to stylet knobs, EP/ST ratio 1.4-2.0; (iii) second-stage juveniles with hemizonid located 1 to 2 annuli anterior to excretory pore and long, sub-digitate tail; and (iv) males with lateral field composed of four incisures, with areolated outer bands. Phylogenetic trees based on 18S, ITS1-5.8S-ITS2, D2-D3 of 28S rDNA, and partial coxII-16S rRNA and coxI gene of mtDNA showed that M. spartelensis n. sp. belongs to an undescribed root-knot nematode lineage that is clearly separated from other species with resemblance in morphology, such as M. dunensis, M. kralli, and M. sewelli.