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Comprehensive morphological and molecular analyses revealed that published ITS sequences of the economically important plant-parasitic nematodePratylenchus goodeyiare actually sequences from distantly free-living bacterivorous ‘cephalobids’. We demonstrated that this incorrect labelling resulted in a cascade of erroneous interpretations, as shown by the reports of ‘P. goodeyi’ on banana in China and on cotton in India. This clearly illustrates the risk of mislabelled sequences in public databases. Other mislabelledPratylenchuscases are discussed to illustrate that this is not an isolated case. Herein,P. lentisn. syn. is considered a junior synonym ofP. pratensiswhileP. flakkensiswas for the first time linked to DNA sequences using topotype material. As taxonomic expertise is decreasing and sequence-based identification is growing rapidly, the highlighted problem may yet increase and a strong link between morphology and DNA sequences will be of crucial importance in order to prevent, or at least minimise, sequence-based misidentifications.

Amphimictic populations of root-lesion nematodes with numerous males and females having three lip annuli, a functional spermatheca and non-areolated lateral field occur on sword fern (Nephrolepis exaltata) in Florida. Identified for decades asPratylenchus penetrans, they appeared to be a morphologically separated species on the basis of a longer stylet (17.8-18.3 μm) thanP. penetrans(15-17 μm) and different lip pattern inen faceview (rectangularvsdumb-bell inP. penetrans). Morphologically similar amphimictic root-lesion nematodes have also been detected on flax lily in Costa Rica. Subsequent morphological observations indicated that these amphimictic root-lesion nematodes from fern and flax lily are closely related to the parthenogenetic speciesP. bolivianus, which has areolated lateral fields. In spite of the reproductive and morphological dissimilarities between these populations, their separation into different species was not supported by the results of molecular analyses of their DNA sequences. The populations used in these analyses included those that are amphimictic from Florida and Costa Rica and others that are parthenogenetic from the type locality in Bolivia, and geographically distant localities in Chile, China, Colombia and Europe. Phylogenetic analyses of the ITS and D2-D3 expansion segments of the 28S rRNA gene indicated that they belong to the same species,P. bolivianus, which consists of two morphotypes,P. bolivianus(am) amphimictic andP. bolivianus(pm) parthenogenetic, herein described and illustrated. Contradictory results were obtained by the analyses using a portion of thehsp90gene. The phylogenetic study, which included sequences of other root-lesion nematodes, a topotype and geographical distant populations ofP. zeae, revealed thatP. bolivianusandP. zeaeformed highly supported clades in the majority consensus trees. PCR with species-specific primers for rapid diagnostics ofP. bolivianusandP. zeaewere developed and tested.

Entomopathogenic nematodes belong to the families of Steinernematidae and Heterorhabditidae. They are obligate and lethal parasites of insects that can provide effective control of some important pests of commercial crops. A total of 53 isolates of EPN were molecularly characterised (ITS region-based) in the present study. Most of the studied isolates belong to theSteinernemagenus and only few isolates belong to theHeterorhabditisgenus. The phylogenetic relations ofSteinernemaandHeterorhabditisspecies were analysed by utilising the maximum likelihood method. In theSteinernemaphylogenetic tree, 99 isolates formed five major, moderately or highly reinforced clades: clade I:affine-intermediumgroup; clade II:carpocapsae-siamkayai-tami-scapterisci; clade III:bicornutum-riobrave-thermophilum; clade IV:glaseri-arenarium-karii-longicaudum; and clade V:feltiae-schliemanni-kushidai-kraussei-oregonense. The BLAST analysis of the ITS region of the rDNA of the steinernematid isolate PAL10 showed a rather low similarity of 93% withS. vulcanicum(accession number: GU929442), supporting the possible designation of a new species. In theHeterorhabditisphylogenetic tree, 25 isolates formed three main clades: clade I:bacteriophora-argentinensis-hepialius; clade II:baujardi-sonorensis-amazonensis; and clade III:indica-brevicaudis-hawaiiensis. All five studied isolates ofHeterorhabditiswere identified asH. indicaandH. bacteriophora. In both phylogenetic trees, the intra-specific variability level was different among clades for some species. The description of the new species (PAL10 isolate) would need further morphometric characterisation, morphologically identification and cross-breeding studies.

Root-lesion nematodes (Pratylenchus) have a worldwide distribution and cause severe production constraints on numerous important crops. During a survey of the wheat-growing area of Morocco, 17 populations of root-lesion nematode were collected. They were identified on the basis of their morphological and morphometric characters, and by molecular methods. Microscopic observations of females and males demonstrated the occurrence ofP. penetransin 13 of the 17 samples;P. thorneiandP. pseudocoffeaewere detected in four samples from Zaers and a single sample from Settat, respectively. A duplex PCR primer set was used to confirm the presence ofP. penetranswhile the species-specific forward primer PTHO and the common reverse primer D3B were used forP. thornei. For the remaining populations, the D2-D3 expansion segments of the 28S rRNA gene were amplified and the obtained sequences were compared with those ofPratylenchusspecies in the GenBank database. This comparison confirmed the morphological identifications and revealed a population ofP. pinguicaudatus. The study of the phylogenetic relationship of the MoroccanPratylenchuspopulations showed a high similarity (99-100%) between allP. penetranspopulations. The population dynamics of sixPratylenchuspopulations from Morocco were evaluated on carrot disk cultures at 4, 8 and 12 weeks after inoculation, and at 10, 15, 20 and 25°C. The optimum temperature for reproduction of all populations was 20°C. After 8 weeks at this temperature, nematode numbers increased up to 458-fold, 310-fold and 252-fold for the four populations ofP. penetrans, theP. thorneiand theP. pseudocoffeaepopulation, respectively.

A real-time quantitative PCR assay was developed for the accurate detection and quantification of the root-lesion nematode, Pratylenchus thornei. A qPCR primer set, including two primers and a probe, was designed based on the sequence of the β-1,4-endoglucanase gene. The assay was optimised by using the primers with SYBR green I dye and setting the qPCR program to different annealing temperatures ranging from 62 to 69°C. Based on the Ct values, we retained the program with an annealing temperature of 69°C. The specificity of the qPCR assay including the probe was confirmed by the lack of amplification of DNA from 47 populations belonging to 15 other Pratylenchus species and nine isolates from P. thornei. The assay was very sensitive as it was able to detect a single individual of P. thornei, even when mixed with up to 80 individuals of P. penetrans. DNA was extracted from exactly 80 P. thornei individuals. A dilution series from this DNA resulted in a standard curve showing a highly significant linearity between the Ct values and the dilution rates (; slope = −3.38; ). The qPCR assay developed in this study proved to be specific and sensitive, thus providing a fast and accurate tool for detection and quantification of this pathogen during research, as well as for diagnostic labs.

Several Heterodera species can reduce the yield of wheat and barley, among which H. avenae, H. filipjevi and H. latipons are economically the most important. Their identification, based on morphological characteristics, is not straightforward but can be made easier using molecular techniques. In this study, we developed species-specific primers for the detection of H. latipons. The actin gene of eight Heterodera species was partially sequenced and, after purifying and sequencing the PCR products, all sequences were aligned to find unique sites. The alignment showed moderate to very high similarities between the species. However, a small fragment of the actin gene was suitable for the construction of a potentially useful species-specific primer for H. latipons. The optimised PCR was subsequently tested with several populations of 14 Heterodera species and a single population of Punctodera punctata. Heterodera latipons was represented by 16 populations originating from six different countries. The primer set (Hlat-act), designed using AlleleID 7.73, was shown to be very specific. To test its sensitivity further, the PCR was conducted on DNA extracted from five second-stage juveniles (J2) of H. latipons mixed with five or 100 J2 belonging to H. avenae. The PCR was able to detect up to 1:10 dilution of the DNA obtained from five J2. The results showed that a specific and sensitive H. latipons species-specific PCR was constructed.

A new root-lesion nematode, particularly pathogenic to Musa spp. and causing important plantain losses in Ghana, is described and named Pratylenchus speijeri n. sp. The cryptic status of this species within the P. coffeae species complex has been assessed and confirmed in this study. An extensive comparison of the morphological and molecular characteristics of this new species with those of P. coffeae and other related amphimictic species did not result in an unambiguous separation of this species from P. coffeae because only a few morphological features of diagnostic value were found. Sequence and phylogenetic analyses of the D2-D3 expansion segments of the 28S rRNA gene, the ITS rRNA gene and a portion of the hsp90 gene of P. speijeri n. sp. and P. coffeae species complex populations from different sources generated majority consensus BI trees with three major clades: P. speijeri n. sp. from Musa spp. roots in Ghana; unidentified or putative new Pratylenchus sp. C1 from Colocasia esculenta roots in Japan and P. coffeae with non-homogeneous relationships from different hosts and distant geographical areas. These results confirmed the validity of P. speijeri n. sp. as a new taxon and indicated that P. coffeae populations from Colocasia in Japan also need to be considered as a new species. Sequence differences in the ITS were used to design group- and species-specific primers to detect P. speijeri n. sp. and other species of P. coffeae species complex. The use of these species-specific primers for the separation of P. speijeri n. sp., Pratylenchus sp. C1 and P. coffeae has important practical application in breeding programmes for agriculture in West Africa.

Three isolates (Dero-1, Dero-8 and Mosisa-1) of a new entomopathogenic nematode, S. ethiopiense sp. n., were isolated by baiting soil samples from the Mendi area, Western Wollega, Ethiopia, with last instar wax moth larvae Galleria mellonella. Infective juveniles of S. ethiopiense sp. n. have a body length of 898 (768-1010) μm, a maximum of eight identical ridges (i.e., nine lines) in the lateral field, excretory pore located at mid-pharynx, hyaline layer occupying approximately half of the tail and c′ = 3.2. First generation males lack a caudal mucron, whereas second generation males possess a short spine-like mucron. The spicules are slightly arcuate, golden-brown in colour and have an ellipsoid or oblong manubrium. First generation females lack a postanal swelling and have a minute protuberance on the tail tip whereas second generation females have a postanal swelling and protruding vulva. Based on the morphology, morphometrics and DNA analysis, the new species belongs to the glaseri group. The closest relative species is the afro-tropical S. karii recorded from Kenya. The BLAST analysis of the ITS region of the rDNA revealed a similarity of 93% with S. karii, supporting the validity of S. ethiopiense sp. n. as a new species. In the phylogenetic trees the new species groups together only with S. karii (bootstrap value of 100%), but is also separated from S. karii by a bootstrap value of 100% or 70%.

Abstract A new species of entomopathogenic nematode, Steinernema lamjungense n. sp., was recovered from soil samples collected from Tarku, Lamjung district of Nepal, during a survey in 2007. The analysis of ITS-rDNA sequence placed S. lamjungense n. sp. in the arenarium-glaseri-karii-longicaudum group. The nematode can be separated from other described species of the group by morphological and morphometric characteristics of different stages and by characterisations and phylogeny of DNA sequences of the D2D3 domain of the LSU or ITS regions of rDNA. For infective juveniles, the new species can be recognised by medium-long body length of 832 (690-950) μm, pharynx length of 127 (110-142) μm, H% = 61 (55-69) and E% = 79 (69-97). The lateral field pattern is 2, 6, 7, 8, 3, 2. The males have well curved, yellowish-brown spicules with a prominent velum. The spicule terminus is blunt with a longitudinal ventral notch. Spicule length is 87 (81-94) μm and gubernaculum length is 57 (50-66) μm in the first generation males. A tail mucron is absent in first but present in second generation males. First generation males have a lateral field with a single ridge. A post-anal swelling is not developed in first generation females but is well developed in second generation females. Three other isolates of the same species were recovered from the neighbourhood of the type locality and yet another isolate was collected from Swarketari, Syangja district.

A new species of entomopathogenic nematode, Steinernema everestense n. sp., was recovered from soil samples collected from Pakhribas, Dhankuta district of Nepal, during a survey in 2007. The analysis of ITS-rDNA and D2D3 LSU sequences placed S. everestense n. sp. in the feltiae-kraussei-oregonense group. The nematode can be separated from other described species of Steinernema by morphological and morphometric characteristics and by characterisations and phylogeny inferred from DNA sequences of the D2D3 LSU or ITS regions of rDNA. For infective juveniles, the new species can be recognised by the body length of 775 (705-838) μm, pharynx 119 (110-130) μm long, H% = 61 (47-68) and E% = 78 (60-89). The lateral field pattern is 2, 8, 6, 4, 3. The males have well curved, brownish-yellow spicules with a prominent but short velum, gubernaculum plump, boat-shaped to almost straight in lateral view, cuneus long, needle-shaped, corpus with two wings in ventral view. Spicule and gubernaculum length of the first generation males is 79 (65-87) μm and 50 (39-57) μm, respectively. A tail mucron is present in both male generations. Genital papillae total 23 or 25, comprising 11 or 12 pairs and a single midventral papilla. Specimens with 23 genital papillae have eight precloacal pairs (seven pairs subventral and one pair lateral), two pairs subterminal and one pair subdorsal. The most posterior two pairs are consistently located near the tail tip. The specimens with 25 genital papillae have an extra adcloacal pair of papillae and the anteriormost two pairs are close together. The vulva is slightly protruding, mostly with equal lips, and low epiptygmata. A post-anal swelling is developed in most first and all second generation females.

Bursaphelenchus nematodes, extracted from 12 samples taken from imported packaging wood originating from seven countries and regions at Nanjing, China, during 2003 and 2004, were identified using morphology, morphometric data and ITS-RFLP patterns. Two samples were identified as B. xylophilus: one, from Canada, was the M-form and another, from Japan, the R-form. Five samples were identified as B. mucronatus, three belonging to the European genotype and two to the East-Asian genotype. Two samples (from Germany and South Korea) were recognised as B. rainulfi. Two samples (South Korea and Japan) were identified as B. doui and a sample from Hong Kong was identified as B. thailandae. Additional morphometric data are provided for the identified species, especially with respect to spicule morphometrics. The CDA of morphometric data discriminated the five species by nine male and eight female characters. The characters used for this analysis corresponded partly to the main taxonomic-informative characters for the genus Bursaphelenchus, viz., body length, stylet length, spicule length, a, c and c′ values. The phylogenetic trees constructed from the D2D3 sequence data supported the division of East Asian and European genotype groups of B. mucronatus. Bursaphelenchus doui grouped with B. xylophilus and B. mucronatus, all members of the xylophilus-group, which was separated from B. rainulfi and B. thailandae.

Abstract ITS1, the 5.8S rRNA gene and ITS2 of the rDNA region were sequenced from 20 different Pratylenchus species. Additionally, the same region was sequenced from seven populations of P. penetrans. After purifying, cloning and sequencing the PCR products, all sequences were aligned in order to find unique sites suitable for the design of species-specific primers for P. penetrans. Since ITS regions showed variability between and even within populations of P. penetrans, only three small DNA sequences were suitable for the construction of three potentially useful species-specific primers. New species-specific primers were paired with existing universal ITS primers and tested in all possible primer combinations. The best performing primer set, supplemented with a universal 28S rDNA primer set that served as an internal control, was tested in duplex PCR. The ideal annealing temperature, Mg2+ concentration and primer ratios were then determined for the most promising primer set. The optimised duplex PCR was subsequently tested on a wide range of different Pratylenchus spp. and 25 P. penetrans populations originating from all over the world. To test the sensitivity, the duplex PCR was conducted on DNA extracted from a single P. penetrans nematode mixed with varying amounts of nematodes belonging to another Pratylenchus species. Results showed that a reliable and sensitive P. penetrans species-specific duplex PCR was constructed.

Abstract Bursaphelenchus chengi sp. n. is described and illustrated. Dauer juveniles were isolated from imported wood packaging materials from Taiwan to Nanjing Port, China. Bursaphelenchus chengi sp. n. was reared and maintained on Petri dish cultures of the fungus Botrytis cinerea. The new species is characterised by the medium body size in both sexes, the presence of only two incisures in the lateral field and the robust and strongly curved spicules. The spicule lamina is angular distally, the rostrum digitate and the condylus rounded. The tail is arcuate with a pointed terminus. The bursa is usually truncate with the posterior margin indented in some specimens or rounded with a fine axial point. Females have a small vulval flap formed by a short extension of the cuticle of the anterior lip, and a conical tail that gradually tapers to an almost straight or slightly recurved, pointed or rounded terminus. Because of the presence of two lateral lines, similar spicule shape, tapering female tail and the presence of a small vulval flap, B. chengi sp. n. should be grouped in the abietinus-group sensu Braasch. together with B. abietinus, B. antoniae, B. hellenicus, B. hylobianum and B. rainulfi. ITS-RFLP profiles support the proposal of the new species, and phylogenetic analysis of the 28S rDNA D2/D3 domain sequence places it close to B. antoniae and other species of the abietinus-group.

Larvae of the scarab beetle Hoplia philanthus Fuessly (Coleoptera: Scarabaeidae) are important pest insects of sports turf, lawns, pastures and ornamentals in Belgium (Ansari et al., 2003b). During a routine field survey of white grubs in Belgian turfgrass, a number of third-instar larvae of H. philanthus were found infected by individuals of the genus Steinernema Travassos, 1927 (Rhabditida: Steinernematidae), a genus of entomopathogenic nematodes (EPN) increasingly used as biological control of insect pests (Ehlers, 1998).

Radopholus arabocoffeae sp. n., a new nematode pathogenic on Coffea arabica cv. Catimor, is described from Vietnam. Females of R. arabocoffeae sp. n. are characterised by the broad amphidial apertures with prominent margins. Males are characterised by the bursa extending to one third, rarely middle, of the tail. The new species belongs to the group of species with a long tail in the female. Radopholus arabocoffeae sp. n. is easily distinguished from R. similis by the bursa reaching to only one third of the tail vs extending to the tail terminus. Radopholus arabocoffeae sp. n. is differentiated from R. bridgei by the lateral field having three bands of equal width (vs middle one narrower than others), lateral field completely areolated over whole body vs not areolated except irregularly in neck and tail, hemizonid distinct vs indistinct, four lateral field incisures terminating far behind phasmid vs three incisures terminating at or just behind phasmid, lateral lines fusing at two thirds of tail vs fusing at one third of tail, longer spicule length (18-21 vs 15.5-18.0 μm), and male bursa usually extending to only one third of tail vs mid tail. Radopholus arabocoffeae sp. n. differs from R. colbrani by the rod-like vs round sperm, spicule length (18-21 vs 13-16 μm), tail length to stylet ratio (4.1-4.9 vs more than 5.1) and presence vs absence of a bursa. Radopholus arabocoffeae sp. n. differs from R. duriophilus by the rod-like vs kidney-shaped sperm. Males further differ from R. duriophilus by shorter stylet length (8.2-11.6 vs 11.5-15 μm), smaller distance between dorsal pharyngeal gland orifice and stylet base (1.7-3.4 vs 4-9.5 μm), shorter hyaline tail (2.6-3.4 vs 4-9.5 μm), and bursa extending to one third of tail vs mid-tail. Female R. arabocoffeae sp. n. differ from R. duriophilus by the broad amphidial aperture with prominent margin present vs absent. Males of R. arabocoffeae sp. n. differ from R. musicola by the rudimentary and amalgamated stylet base (vs with knobs), and inner lateral lines fusing at two thirds of the tail vs just posterior to the phasmid. The high level of ITS-rDNA sequence divergence of R. arabocoffeae sp. n. from other Radopholus spp. and the presence of nucleotide autapomorphies support a separate specific status of this new species. On carrot disks, the two species reproduced from 15-30°C; optimum reproduction occurring at 28°C. The reproductive capacity of R. duriophilus was higher than that of R. arabocoffeae sp. n. Radopholus duriophilus reproduced from single juveniles; R. arabocoffeae sp. n. did not. The correlation between initial densities of Pratylenchus coffeae, R. duriophilus and R. arabocoffeae sp. n. and the weight of C. arabica cv. Catimor fitted the Seinhorst model Y = ym for Pi ≥ T, and Y = ym ˙ m + ym(1–m)z(Pi–T). Coffea arabica cv. Catimor was very susceptible for to all three nematode species tested, but especially so to R. arabocoffeae sp. n. The reproductive capacity of R. arabocoffeae sp. n. on C. arabica cv. Catimor was higher than P. coffeae or R. duriophilus.

Abstract Amplified ITS region products of rDNA from 25 valid species and one unidentified species from the genus Heterodera and from Meloidodera alni were digested by 26 restriction enzymes. A combination of seven enzymes clearly separated the agriculturally most important species from each other and from their sibling species. Species specific digestion profiles of ITS regions and a table with approximate sizes of digested fragments for several identification enzymes are given. Heterogeneity of ITS regions was revealed for some cyst forming nematode species. Des fragments amplifiés de la région de l’ITS du rDNA de 25 espèces valides et d’une espèce non identifiée du genre Heterodera et de Meloidodera alni ont été soumis à une digestion par 26 enzymes de restriction. La combinaison de sept enzymes a permis une séparation nette des espèces les plus importantes en agriculture, tant les unes par rapport aux autres que par rapport aux espèces jumelles. Sont donnés les profils spécifiques de digestion des régions de l’ITS et un tableau regroupant les tailles approximatives des fragments digérés pour plusieurs enzymes d’identification. L’hétérogénéité des régions de l’ITS a été révélée chez quelques espèces de nématodes à kyste.

Canonical discriminant analysis of four morphometric characters of juveniles and restriction enzymes analysis of ribosomal DNA sequences were used to distinguish Heterodera arenaria, H. aucklandica, H. avenae, H. filipjevi, H. hordecalis, H. iri, H. latipons, H. litoralis, H. schachtii and an undescribed species from grasslands. The results of unweighted pair group cluster analysis showed that H. avenae populations formed three groups and H. filipjevi two groups at the 80% level of similarity. Intraspecific polymorphism was revealed by rDNA-RFLP studies and two types of ITS regions within H. avenae populations can be distinguished. The pattern of restriction bands obtained with BsuRI, PstI and TaqI clearly distinguished populations of H. filipjevi from other species of the H. avenae group. Further enzymes and their combinations distinguished the other species. There are no enzymes which differentiate European populations of H. avenae from H. arenaria. Morphometrics, restriction endonuclease cleavage maps of ITS regions and a dendrogram of putative phylogenetic relations of several cyst-forming nematode species are given. Identification des especes du groupe Heterodera avenae par la morphometrie et les rDNA-RFLP - L'analyse canonique discriminante sur quatre caracteres morphometriques des juveniles et l'analyse des enzymes de restriction de l'ADN ribosomal ont ete utilisees pour identifier Heterodera arenaria, H. aucklandica, H. avenae, H. filipjevi, H. hordecalis, H. iri, H. latipons, H. litoralis, H. schachtii et une nouvelle espece originaire de prairies. Les resultats de l'analyse UPGMA ont montre que les populations d'H. avenae formaient trois groupes et celles d'H. filipjevi deux groupes a un niveau de similarite de 80%. Le polymorphisme intraspecifique a ete revele par des etudes de rDNA-RFLP et deux types de regions de l'ITS peuvent etre mis en evidence dans les populations d'H. avenae. Les modeles de bandes de restriction obtenus avec BsuRI, PstI et TaqI ont identifie clairement les populations d'H. filipjevi des autres especes du groupe H. avenae. D'autres enzymes et leurs combinaisons ont identifie les autres especes. Aucun enzyme n'a differencie les populations europeennes d'H. avenae de H. arenaria. Les caracteres morphometriques, les cartes de clivage des regions des ITS par l'endonuclease de restriction et un dendogramme des relations phylogenetiques supposees sont donnes.