Lieven Waeyenberge

Molecular characterisation of 18 Pratylenchus species using rDNA Restriction Fragment Length Polymorphism

The RFLP technique was used to establish a reliable diagnostic method for 18 Pratylenchus species: Pratylenchus agilis, P. bolivianus, P. brachyurus, P. coffeae, P. crenatus, P. fallax, P. goodeyi, P. loosi, P. mediterraneus, P. neglectus, P. penetrans, P. pratensis, P. pseudocoffeae, P. scribneri, P. subranjani, P. thornei, P. vulnus and P. zeae. The polymerase chain reaction (PCR) amplified the ITS regions from all species and populations examined and revealed large differences in length, ranging in size from approximately 900 to 1250 bp. The rDNA fragments were digested with five restriction enzymes (CfoI, DdeI, HindIII, HpaII, and PstI). All Pratylenchus species can be differentiated from each other by a combination of at least two enzymes. CfoI differentiated all nematode species with the exception of P. fallax, P. penetrans and P. pseudocoffeae. P. fallax was further separated by a DdeI restriction, and P. pseudocoffeae by a PstI digestion. Intraspecific RFLP were observed. Upon CfoI, DdeI, HindIII, or HpaII digestion, it was possible to separate the three P. coffeae populations studied from each other. La technique RFLP a ete utilisee pour creer une methode fiable de diagnostic pour 18 especes de Pratylenchus: Pratylenchus agilis, P. bolivianus, P. brachyurus, P. coffeae, P. crenatus, P. fallax, P. goodeyi, P. loosi, P. mediterraneus, P. neglectus, P. penetrans, P. pratensis, P. pseudocoffeae, P. scribneri, P. subranjani, P. thornei, P. vulnus et P. zeae. La reaction de polymerisation en chaine (PCR) a amplifie les regions de l’ITS pour toutes les especes et populations etudiees et a mis en evidence de grandes differences dans la taille des gammes de longueur, de 900 a 1250 bp approximativement. Les fragments de rDNA ont ete digeres a l’aide de cinq enzymes de restriction (CfoI, DdeI, HindIII, HpaII, and PstI). Toutes les especes de Pratylenchus ont pu etre differenciees les unes des autres par une combinaison d’au moins deux enzymes. CfoI a differencie toutes les especes a l’exception de P. fallax, P. penetrans et P. pseudocoffeae. P. fallax a ete ulterieurement separe par une restriction DdeI, et P. pseudocoffeae par une digestion PstI. Des RFLP intraspecifiques ont ete observes. Par les digestions CfoI, DdeI, HindIII, ou HpaII, il s’est revele possible de separer les unes des autres les trois populations etudiees de P. coffeae.

Identification of cyst forming nematodes of the genus Heterodera (Nematoda: Heteroderidae) based on the ribosomal DNA-RFLP

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 amplifies de la region de l’ITS du rDNA de 25 especes valides et d’une espece non identifiee du genre Heterodera et de Meloidodera alni ont ete soumis a une digestion par 26 enzymes de restriction. La combinaison de sept enzymes a permis une separation nette des especes les plus importantes en agriculture, tant les unes par rapport aux autres que par rapport aux especes jumelles. Sont donnes les profils specifiques de digestion des regions de l’ITS et un tableau regroupant les tailles approximatives des fragments digeres pour plusieurs enzymes d’identification. L’heterogeneite des regions de l’ITS a ete revelee chez quelques especes de nematodes a kyste.

Identification of Heterodera avenae group species by morphometrics and rDNA-RFLPs

Summary ‐Canonical discriminant analysis of four morphometric charactersof juvenilesand restriction enzymes analysis of ribosomal DNA sequences were used to distinguish Heterodera arenaria , H. aucklandica , H. avenae, H. elipjevi, 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. elipjevi two groups at the 80% level of similarity. Intraspecie c 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. elipjevifrom 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. Resume ‐ Identie cation des espe ces du groupe Heterodera avenae par la morphometrie et les rDNA-RFLP ‐ L’ analyse canonique discriminante sur quatre caracte res morphome triques des juve niles et l’ analyse des enzymes de restriction de l’ ADN ribosomal ont e te utilise es pour identie er Heterodera arenaria , H. aucklandica , H. avenae, H. elipjevi, H. hordecalis , H. iri, H. latipons, H. litoralis , H. schachtii et une nouvelle espe ce originaire de prairies.Lesre sultats de l’ analyse UPGMAont montre que les populations d’H. avenae formaient trois groupes et celles d’H. elipjevi deux groupes a un niveau de similarite de 80%. Le polymorphisme intraspe cie que a e te re ve le par des e tudes de rDNA-RFLP et deux types de re gions de l’ ITS peuvent e tre mis en e vidence dans les populations d’H. avenae. Les mode les de bandes de restriction obtenus avec BsuRI, PstI et TaqI ont identie e clairement les populations d’H. elipjevi des autres espe ces du groupe H. avenae. D’ autres enzymes et leurs combinaisons ont identie e les autres espe ces. Aucun enzyme n’ a diffe rencie les populations europe ennes d’H. avenae de H. arenaria. Les caracte res morphome triques, les cartes de clivage des re gions des ITS par l’ endonucle ase de restriction et un dendogramme des relations phyloge ne tiques suppose es sont donne s.

Molecular phylogeny of slug-parasitic nematodes inferred from 18S rRNA gene sequences

Terrestrial molluscs are diverse and are infected by many nematodes. We propose a phylogeny of slug-parasitic nematodes using 18S rRNA gene sequences from nematodes isolated from slugs collected from six countries. Eight species, representing six families of nematodes were identified and trees inferred placed them within four (I, III, IV and V) out of the five clades of Nematoda, indicating multiple origins of slug parasitism. Five species representing three families formed a monophyletic group in clade V. Although these species are closely related, their morphology has changed greatly, suggesting adaptive radiation to fill different niches within the host.

Distribution of entomopathogenic nematodes in Southern Cameroon

A first survey of entomopathogenic nematodes (EPN) was conducted in three agro-ecological zones of Southern Cameroon in 2007 and 2008. Entomopathogenic nematodes were recovered from 26 of 251 soil samples (10.4%). Three species, Heterorhabditis baujardi, Steinernema sp. A and Steinernema sp. B were found. The two steinernematids were considered unidentified species. Among the positive samples, 23 samples contained only H. baujardi (88.5%), two contained Steinernema sp. A co-occurring with H. baujardi (7.7%), and one sample contained Steinernema sp. B (3.9%). H. baujardi was frequent in forest and fruit crop (cocoa and oil palm plantations). Steinernema sp. A was found in a tree plantation of teak, Steinernema sp. B in a forest habitat. Nematodes were mostly present in acidic soils with pH ranging from 3.7 to 7.0. The highest EPN presence was recorded in sandy loam, sandy clay loam, sandy clay and clay soils. EPNs were not recovered in sand, loamy sand and clay loam soils. Using principal component analysis for elucidating the major variation patterns among sampling sites, four factors explaining for 73.64% of the overall variance were extracted. Factors were a combination of geographical (latitude, longitude, altitude), soil (pH, contents of sand, silt and clay, organic carbon, texture), and moisture (wilting point, field capacity) parameters as well as climatic parameters (mean annual rainfall, mean air temperature). Logistic regression and redundancy analyses (RDA) revealed that soil pH, longitude, available water and altitude were associated with presence and absence of EPN. Both logistic regression and RDA indicated that, increasing soil pH and longitude, associated with decreasing altitude, led to higher percentages of samples containing entomopathogenic nematodes.

Bursaphelenchus chengi sp. n. (Nematoda: Parasitaphelenchidae) isolated at Nanjing, China, in packaging wood from Taiwan

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.

Development of two species-specific primer sets to detect the cereal cyst nematodes Heterodera avenae and Heterodera filipjevi

Twelve Heterodera species are of major economic significance in wheat and barley. Of these, H. avenae, H. filipjevi and H. latipons are among the most important ones, and sometimes coexist. The identification of Heterodera species using morphological characteristics is time consuming, requires specialized skill and can be imprecise, especially when they occur mixed in field populations. Molecular techniques can provide a more accurate way for nematode identification. This study reports the results of experiments targeting the mitochondrial cytochrome oxidase subunit 1 (COI) gene to develop species-specific primers that could be used for the identification of H. avenae and H. filipjevi. The COI gene of 9 Heterodera spp. and Punctodera punctata was partially sequenced and the resultant sequences were aligned to find unique sites suitable for the design of primers. The alignment showed variability between H. avenae, H. filipjevi and other Heterodera species. Two sets of species-specific primers were identified for the identification of both species and the conditions for their use in PCR were optimised. The specificity of the designed primers was checked by comparison with one population of P. punctata and populations of 14 other Heterodera species, nine populations of H. avenae and 10 populations of H. filipjevi originating from different countries. To test the sensitivity, the PCR was run with DNA extracted from five second-stage juveniles (J2) of H. avenae or five J2 of H. filipjevi mixed with DNA extracted from varying numbers of J2 of H. latipons. It was possible to detect as few as five J2 of H. avenae or H. filipjevi among 100 J2 of H. latipons. The two primers sets allow the detection of H. avenae and H. filipjevi where they occur in mixed populations with other Heterodera spp.

Species-specific duplex PCR for the detection of Pratylenchus penetrans.

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.

A new entomopathogenic nematode, Steinernema robustispiculum n. sp (Rhabditida: Steinernematidae), from Chumomray National Park in Vietnam

Steinernemar robustispiculum n. sp. (Rhabditida: Steinernematidae) was isolated from woodland in Chumomray National Park, Sason, Sathay, Kontum, Vietnam. Its morphology, morphometrics, cross-hybridisation and the ITS-rDNA sequence analysis revealed that S. robustispiculum clearly differs from other known Steinernema spp. As in the cases of S. intermedium (Poinar, 1985), S. robustispiculum has very robust spicules, but it can be distinguished by the longer tail of the infective juvenile, lower E%, shorter spicules, the shape of the spicules, the number of genital papillae in the caudal region and the presence of a mucron on the male tail. S. robustispiculum has a lateral field resembling that of S. sangi Phan, Nguyen & Moens, 2001, but can be distinguished by a higher E%, higher D%, smaller length to width ratio of the spicules and the morphology of both the spicule head (manubrium) and the dorsal lobe of the spicule. The morphometrics of infective juveniles of S. robustispiculum are similar to those of S. monticolum Stock, Choo & Kaya, 1997; these species can be distingusihed by the position of the excretory pore, the smaller length to width ratio of the spicules, and the length and morphology of the spicule head (manubrium). The phylogenetic relationships within Steinernema Travassos, 1927, including the newly sequenced Vietnamese species S. robustispiculum n. sp., S. loci Phan, Nguyen & Moens, 2001, S. thanhi Phan, Nguyen & Moens, 2001 and S. sangi, are presented based on analyses of the ITS-rDNA. The ITS RFLP profiles obtained from 17 different restriction enzymes are also presented.

Development of a species-specific PCR to detect the cereal cyst nematode, Heterodera latipons

Summary ‐ 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.