Showing papers in "Nematology in 2009"

A phylogenetic tree of nematodes based on about 1200 full-length small subunit ribosomal DNA sequences.

Abstract: As a result of the scarcity of informative morphological and anatomical characters, nematode systematics have always been volatile. Differences in the appreciation of these characters have resulted in numerous classifications and this greatly confuses scientific communication. An advantage of the use of molecular data is that it allows for an enormous expansion of the number of characters. Here we present a phylogenetic tree based on 1215 small subunit ribosomal DNA sequences (ca 1700 bp each) covering a wide range of nematode taxa. Of the 19 nematode orders mentioned by De Ley et al. (2006) 15 are represented here. Compared with Holterman et al. (2006) the number of taxa analysed has been tripled. This did not result in major changes in the clade subdivision of the phylum, although a decrease in the number of well supported nodes was observed. Especially at the family level and below we observed a considerable congruence between morphology and ribosomal DNA-based nematode systematics and, in case of discrepancies, morphological or anatomical support could be found for the alternative grouping in most instances. The extensiveness of convergent evolution is one of the most striking phenomena observed in the phylogenetic tree presented here - it is hard to find a morphological, ecological or biological characteristic that has not arisen at least twice during nematode evolution. Convergent evolution appears to be an important additional explanation for the seemingly persistent volatility of nematode systematics.

Application of Pluronic gel to the study of root-knot nematode behaviour

Abstract: Pluronic F-127 is a stable, non-toxic, copolymer that forms a gel at room temperature and a liquid at 15°C when the concentration is 20-30%. Root-knot nematode (Meloidogyne spp.) second-stage juveniles can move freely through the gel and display attraction toward roots of tomato, Medicago truncatula, common bean and Arabidopsis. The excellent clarity of the gel allows examination of behavioural changes in the nematode as it reaches and infects its host. Attraction assays showed that Meloidogyne javanica and M. incognita move to roots much more rapidly than does M. hapla. Nematodes form aggregates when in contact with root tips suggesting that the presence of a signal from the nematode or from the root is involved in the attraction. Nematodes suspended in Pluronic gel without roots aggregate into balls after 1 to 2 days. A coverslip placed on the gel accelerates and serves as a focus for the aggregation, suggesting that lower oxygen, or perhaps a volatile attractant, is involved in this behaviour. These observations demonstrate that Pluronic gel is a useful medium for dissecting attraction of root-knot nematodes to their hosts and for studying additional aspects of their behaviour.

ITS-RFLP analysis, an efficient tool for differentiation of Bursaphelenchus species

Abstract: Restriction analysis of amplified ribosomal ITS sequences has provided species-specific fragment patterns for nematodes of several genera, including Bursaphelenchus. We used restriction enzymes RsaI, HaeIII, MspI, HinfI and AluI to produce ITS-RFLP reference profiles of 44 Bursaphelenchus species, including two intraspecific types in each of B. mucronatus and B. leoni. In addition, reference profiles of Aphelenchoides stammeri and Ruehmaphelenchus asiaticus were produced. Reference profiles of six species are shown here for the first time. Identical ITS-RFLP patterns were usually obtained from different isolates and from individual specimens of the same species. However, in the case of B. 'corneolus', B. lini, B. singaporensis and B. sexdentati, additional bands in the patterns of certain isolates or individual nematodes were observed which may be explained by ITS sequence microheterogeneity, i.e., the presence of ITS sequence variants within the number of rDNA tandem repeats. Since these 'extra' bands appeared only with one out of the five restriction enzymes employed, they did not seriously impair identification of species based on the overall reference patterns. ITS-RFLP analysis has proved valuable for differentiation of the pathogenic pine wood nematode, B. xylophilus, from related species. In many recent descriptions of new Bursaphelenchus species, ITS-RFLP profiles have been used as additional species identification criteria. Comparison of profiles from isolates of many different origins has provided new information on intraspecific types or genetically distinct provenances of several Bursaphelenchus species.

Endophytic bacteria from Ethiopian coffee plants and their potential to antagonise Meloidogyne incognita

Abstract: Summary – Endophytic bacteria were isolated from coffee roots in Ethiopia and identified by Fatty Acid Methyl Ester-Gas Chromatography (FAME-GC). A total of 201 and 114 endophytic bacteria were isolated and identified during the wet and dry seasons, respectively. The most abundant genera were Pseudomonas, Bacillus, Agrobacterium, Stenotrophomonas and Enterobacter. Population densities were higher during the wet season than the dry season ranging from 5.2 × 10 3 to 2.07 × 10 6 cfu (g fresh root weight) −1 . Culture filtrates of the bacterial isolates showed nematicidal effects of between 38 and 98%. The most active strains were Agrobacterium radiobacter, Bacillus pumilus, B. brevis, B. megaterium, B. mycoides, B. licheniformis, Chryseobacterium balustinum, Cedecea davisae, Cytophaga johnsonae, Lactobacillus paracasei, Micrococcus luteus, M. halobius, Pseudomonas syringae and Stenotrophomonas maltophilia. Bacillus pumilus and B. mycoides were most effective in reducing the number of galls and egg masses caused by M. incognita by 33 and 39%, respectively.

Identification of putative expansin-like genes from the pine wood nematode, Bursaphelenchus xylophilus, and evolution of the expansin gene family within the Nematoda

Abstract: We report the cloning and characterisation of genes encoding expansin-like proteins from the pine wood nematodes, Bursaphelenchus xylophilus and B. mucronatus. A small family of genes is present in both species and the Bursaphelenchus genes are most similar to expansins and expansin-like proteins from the potato cyst nematode Globodera rostochiensis and root-knot nematodes. Molecular modelling suggests that the genes could encode a protein with a structure similar to that of functionally characterised expansins. Expression analysis showed that the Bursaphelenchus expansin-like genes are expressed solely in the pharyngeal gland cells, implying a role in the host-parasite interaction, most likely in assisting migration through the plant. Some G. rostochiensis and root-knot nematode expansins are composed of a carbohydrate-binding domain coupled to an expansin domain but no carbohydrate binding domain is present on any of the Bursaphelenchus sequences. We suggest a model for evolution of the expansin gene family within the plant-parasitic nematodes of the Tylenchida and Aphelenchida.

A survey of entomopathogenic nematodes of the families Steinernematidae and Heterorhabditidae (Nematoda: Rhabditida) in the north-west of Iran

Abstract: During 2002-2004, a survey of entomopathogenic nematodes was conducted for the first time in Iran throughout the three provinces in the north-west of the country. Soil samples were tested for the presence of steinernematid and heterorhabditid nematodes by baiting with Galleria mellonella larvae. Of the 833 soil samples studied 27 were positive for entomopathogenic nematodes (3.2%), with 17 (2.0%) containing Heterorhabditis and ten (1.2%) Steinernema isolates. Morphological and molecular studies were carried out to characterise isolates. The Heterorhabditis isolates were identified as Heterorhabditis bacteriophora and Steinernema as Steinernema carpocapsae, S. bicornutum and S. feltiae. Heterorhabditis bacteriophora was the most common species, which was isolated from 17 sites across the three provinces. Steinernema feltiae was the most common species of Steinernema, which was isolated from eight sites but in only two provinces. Steinernema carpocapsae and S. bicornutum were each isolated from only one site. Steinernema spp. were isolated mainly from orchards and grasslands but Heterorhabditis was isolated mainly from grasslands and alfalfa fields.

Pseudaphelenchus yukiae n. gen., n. sp. (Tylenchina: Aphelenchoididae) associated with Cylindrotermes macrognathus (Termitidae: Termitinae) in La Selva, Costa Rica

Abstract: A species of aphelenchoidid nematode was isolated from a subterranean termite, Cylindrotermes macrognathus, during a survey of termite-associated nematodes in a conserved forest in La Selva, Costa Rica. The nematode was morphologically intermediate between the families Aphelenchidae and Aphelenchoididae, i.e., the nematode had a true bursa supported by bursal limb-like genital papillae but lacked a clear pharyngeal isthmus. The molecular phylogenetic status of the new nematode among tylenchid, cephalobid, panagrolaimid, aphelenchid and aphelenchoidid genera was analysed based on ca 1.2 kb of SSU ribosomal DNA sequence and the inferred position was basal to the family Aphelenchoididae. It was clearly not part of the clade containing the genus Aphelenchus (=Aphelenchidae). This nematode is described herein as Pseudaphelenchus yukiae n. gen., n. sp., and the family definition of Aphelenchoididae is emended to include the unique morphological characters of this new genus. The molecular phylogenetic analysis supported the paraphyly of the three Aphelenchoidinae genera Aphelenchoides, Laimaphelenchus and Schistonchus and the monophyly of Ektaphelenchinae, Seinura (Seinurinae) and Noctuidonema (Acugutturinae). However, many more representatives are needed to resolve the family-genus level phylogeny of Aphelenchoididae.

Pine wilt disease: a worldwide threat to forest ecosystems

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Arabinogalactan endo-1,4-β-galactosidase: a putative plant cell wall-degrading enzyme of plant-parasitic nematodes

Abstract: Plant-parasitic nematodes secrete a plethora of enzymes to degrade polysaccharides of the recalcitrant plant cell wall. Here we report on the presence of a putative endo-1,4- β -galactosidase (EC 3.2.1.89) in cyst nematodes of the genus Heterodera . This enzyme hydrolyses β -1,4-galactan in the hairy regions of pectin and to our knowledge it is the first report of this class of enzymes in animals. The gene was cloned from H. schachtii and subjected to a detailed molecular characterisation. The deduced protein contains a putative signal peptide for secretion, being in agreement with the presumed extracellular function of the mature protein. It has a molecular mass of 33.78 kDa and folds into an ( α/β) 8 barrel structure typical for glycosyl hydrolases. The two glutamic acids that function as electron donor and acceptor in the active site are conserved. Whole mount in situ hybridisation revealed that the gene is expressed in the subventral pharyngeal glands and the expression was correlated with the onset of parasitism.

Short-term nematode population dynamics as influenced by the quality of exogenous organic matter

Abstract: The food specificity of nematodes, their high number of species and high abundance in every habitat where decomposition takes place, indicate that the structure of the nematode assemblage has a high information content. Since nematodes respond quickly to changes in soil management and since the nematode fauna can be efficiently analysed, the structure of the nematode assemblage offers an interesting instrument to assess changes in soil conditions. We studied the effect of five organic amendments on the short-term nematode population dynamics and compared them to minerally fertilised and unfertilised plots. The experiment was started in 2005, and samples were taken in spring and autumn 2006 and spring 2007. In spring 2006, no clear differences among treatments in the diversity of free-living nematodes were observed, probably since the organic amendments were applied only twice, of which the last application was carried out 7 months before the sampling. At the second and third sampling, the enrichment index in the organically amended plots was higher than in the unamended plots, owing to the organic matter application. However, in plots amended with farmyard manure and cattle slurry the number of bacterivores increased significantly, while in the compost plots the fungivorous nematodes tended to be higher. This resulted in a low channel index for the manure and slurry plots, indicating a predominant bacterial decomposition pathway, and a higher channel index in the compost plots, suggesting a greater proportion of fungal decomposition. These assumptions on the decomposition of the applied organic matter were strongly supported by the composition of the soil microbial community, determined through PLFA analysis: in the compost plots the bacteria to fungi ratio was lower than in the manure and slurry plots. At all sampling occasions there was a remarkably lower abundance of plant-parasitic nematodes in plots amended with slurry and manure, indicating a negative impact of both amendments on plant-parasitic nematodes. We can conclude from this study that the fertiliser regimes affected the nematode assemblage, but that more samplings in the future are certainly necessary to assess adequately the impact of the different organic amendments.

Schistonchus virens sp. n. (Aphelenchoididae) and Parasitodiplogaster australis sp. n. (Diplogastridae) from Ficus virens (Moraceae) in Australia.

Abstract: A new species each of Schistonchus and Parasitodiplogaster were recovered from the sycones of Ficus virens from St Lucia, Brisbane, QLD, Australia. This is also the first record of Parasitodiplogaster from Australia. The species are described here as Schistonchus virens sp. n. and Parasitodiplogaster australis sp. n. Schistonchus virens sp. n. is differentiated from other species of the genus by a combination of morphological characters, including C-shaped females and males, excretory pore opening located near the head, a short post-vulval uterine sac, rose-thorn-shaped spicule, amoeboid sperm, no gubernaculum, three pairs of subventral papillae on the male tail, DNA sequence data; biogeographical range and host wasp and Ficus species affiliation. Parasitodiplogaster australis sp. n. is differentiated from all other species of the genus by having females with only one gonad, males with C-shaped spicules with an arcuate, slender gubernaculum, characteristic arrangement of the male caudal papillae and DNA sequence data. The generic diagnosis of Parasitodiplogaster is emended to include loss of a female gonad.

Species-specific duplex PCR for the detection of Pratylenchus penetrans

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.

Relationship between soil properties, crop management, plant growth and vigour, nematode occurrence and root damage in East African Highland banana-cropping systems: a case study in Rwanda

Abstract: Parasitic nematodes are widespread in Musa cropping systems in African lowlands where they are known to limit crop production. However, their distribution is very poorly known in the large parts of the East African Highland banana ecology. We carried out a survey in 188 fields in Rwanda to assess and understand nematode occurrence and damage under a wide range of agro-ecological conditions. Altitude varied from 900 to 1800 m above sea level and soil types were distinctly different in the five eco-regions sampled and derived from diverse parent materials; i.e., Ruhengeri (Andosol), Gitarama-Butare (Acrisol), Kibungo (Nitisol), Gashonga (Ferralsol) and Bugarama (Fluvisol and Vertisol). Crop management practices, root health parameters and nematode infection in roots were recorded for a single East African highland banana cultivar (Intuntu, AAA-EA). Plant-parasitic nematodes from five genera were identified: Pratylenchus goodeyi, Helicotylenchus multicinctus, Meloidogyne spp., Radopholus similis and Hoplolaimus pararobustus. Pratylenchus goodeyi was the dominant species in all eco-regions except in Bugarama (lowland). Only the presence of P. goodeyi significantly correlated with root necroses. Altitude was strongly correlated with root densities of P. goodeyi and R. similis. A possible negative impact by P. goodeyi on banana yields was masked by the fact that nematode populations were positively correlated with high plant density and/or mulching practices, which led to relatively high plant vigour irrespective of soil type. Therefore, controlled field experiments will be needed to assess whether root necrosis caused by P. goodeyi at high altitude (>1400 m) actually has a detrimental impact on banana yields, similar to that observed for root-lesion nematodes at lower altitudes.

Differential susceptibility of entomopathogenic nematodes to nematophagous fungi from Florida citrus orchards

Abstract: Laboratory experiments were conducted to study the effects of various trapping and endoparasitic nematophagous fungi (NF) isolated from Florida citrus orchards on five entomopathogenic nematode (EPN) species that show various distributions across Florida's citrus industry. Four trapping NF ( Arthrobotrys oligospora , A. dactyloides , A. musiformis and Gamsylella gephyropaga ) and two endoparasitic NF ( Catenaria sp. and Myzocytium sp.) were tested against Steinernema diaprepesi (Sd), S. glaseri (Sg), S. riobrave (Sr), Heterorhabditis zealandica (Hz) and H. indica (Hi). Fungi were added to soil microcosms either as a pure culture on agar plugs (trapping NF) or as fungal-colonised nematodes (endoparasitic NF) on agar plugs, concurrently with 2000 EPN of a given species. After 7 or 14 days exposure, nematodes were recovered from the soil using Baermann funnels. The recovery of all EPN species was reduced between 56-92% by G. gephyropaga . Neither Sd or Sg were affected by any species of Arthrobotrys , whereas A. musiformis reduced recovery of all other EPN and A. oligospora reduced numbers of all other species except Hi. Both endoparasitic NF reduced the recovery of all EPN except Hi by at least 82%. The data are consistent with the hypothesis that NF may play a role in regulating regional patterns of abundance and diversity of EPN species in Florida, which in turn regulate the abundance of a major citrus pest, the Diaprepes root weevil, Diaprepes abbreviatus .

Root-knot nematodes, Meloidogyne spp., on potato in Portugal

Abstract: Root-knot nematodes (RKN), Meloidogyne spp., are among the most economically important nematodes in agriculture and can cause significant losses in potato (Solanum tuberosum L.), in both warm and cool climates, depending upon the nematode species (Brodie et al., 1993; Greco, 1993). RKN have not yet been recognised as serious nematode pests in the potato-growing areas of Portugal. However, during the past 10 years, nematode damage has been found in some areas and it is clear that infestation of potato fields by RKN is becoming more widespread. Some of the RKN species attacking potato, e.g., M. chitwoodi, also have quarantine status and one isolate originating from Portugal has already been included in a molecular study (Powers et al., 2005). The aim of this work was to provide data on the identification of Meloidogyne spp. on potato in Portugal. During a survey for potato cyst nematodes (PCN), Globodera spp., RKN isolates were detected in tubers and in soil collected in 210 potato fields in various districts of Portugal (Table 1). When tubers infected with RKN were detected, egg masses were collected and susceptible tomato plants (Solanum lycopersicum L.) cv. Easypeel were inoculated with ten egg masses/pot. When only soil samples were taken, tomato plants with four true leaves were transplanted into the original soil. All plants were maintained in a glasshouse at 25-30◦C. After 45 days, plants were uprooted, their roots gently washed free of soil and females, when present, collected with forceps under a stereomicroscope. Morphological characterisation was based on a minimum of 20 perineal patterns (Taylor & Netscher, 1974; Hartmann & Sasser, 1985). Their char-

Description of Ektaphelenchoides spondylis n. sp. (Nematoda: Ektaphelenchinae) isolated from Spondylis buprestoides (Coleoptera: Cerambycidae) in Japan

Abstract: Ektaphelenchoides spondylis n. sp. is described and figured from Spondylis buprestoides. The new species is characterised by the bipartite stylet conus, male spicule with rounded condylus, blunt rostrum and plate-like cucullus, long postuterine sac and long and smoothly tapering female tail. The new species is almost morphologically identical to E. compsi, but can be distinguished from it by stylet morphology, female tail shape, male ratio (a) and molecular sequence of the D2/D3 expansion segment of the large subunit of ribosomal DNA. In the molecular phylogenetic analysis, E. spondylis n. sp. formed a well supported clade with E. compsi within Ektaphelenchoides. Based upon molecular phylogenetic analysis with one locus, Ektaphelenchoides spp. formed a well supported clade and Ektaphelenchus and Cryptaphelenchus also formed a well supported clade. However, the molecular and morphological information available from our study are insufficient to revise the generic taxonomy of the subfamily. More sampling and an integrated taxonomic revision of the subfamily Ektaphelenchinae are needed.

Phoretic dispersal of entomopathogenic nematodes by Hylobius abietis

Abstract: Phoresis is a strategy in which one organism (the phoretic) attaches to another (the host) for the implied purpose of dispersal and is valuable for small flightless invertebrates with limited mobility. Previous work has shown that the relatively immobile larvae of Hylobius abietis (the large pine weevil) are highly susceptible to entomopathogenic nematodes (EPN) and support nematode reproduction whereas adults do not. Here, we demonstrate that the infective juveniles (IJ) of two species of EPN, Heterorhabditis megidis and Steinernema carpocapsae, can use the resistant but highly mobile adults of H. abietis for phoretic dispersal. The ability to use adult H. abietis for phoresis depended on nematode species and habitat (sand vs peat) and there was no evidence of sex-biased dispersal in S. carpocapsae for which the IJ are dioecious. We hypothesise that phoretic dispersal may be a significant mechanism to avoid sibling competition and inbreeding in EPN, which produce tens of thousands of offspring at point sources in a relatively impenetrable habitat.

Host plant resistance to Radopholus similis in a diploid banana hybrid population

Abstract: The objective of the research was to study the genetics of host plant resistance to Radopholus similis in a diploid banana population. Screening of a hybrid population and determination of the segregation ratios can provide information on the inheritance and number of alleles controlling this trait in banana. The diploid banana hybrid population used was derived by crossing the diploid hybrids TMB2x 6142-1 and TMB2x 8075-7. The diploid banana hybrid population was evaluated with the individual root inoculation method using an R. similis population from Namulonge, Uganda. Of the 81 hybrids evaluated, 37 hybrids were resistant, 13 hybrids were partially resistant and 31 hybrids were susceptible to R. similis. After chi-square analysis, the results indicated that resistance to R. similis is controlled by two dominant genes, A and B, both with additive and interactive effects, whereby recessive bb suppresses dominant A.

Acrostichus rhynchophori n. sp. (Rhabditida: Diplogastridae): a phoretic associate of Rhynchophorus cruentatus Fabricius and R. palmarum L. (Coleoptera: Curculionidae) in the Americas

Abstract: Acrostichus dauer larvae (JIII) were recovered during dissections of the palmetto weevil, Rhynchophorus cruentatus, from southern Florida, and the palm weevil, R. palmarum, from Colombia, Costa Rica and Trinidad. Based upon morphological and molecular studies, the four isolates are conspecific and are described herein as A. rhynchophori n. sp. Acrostichus rhynchophori n. sp. is characterised by narrow, flap-like dorsal tooth, female gonads not reflexed to the level of the vulva, male spicule and gubernaculum morphology, i.e., spicule with small and indistinct manubrium embedded in lamina/calomus complex, strong expansion just posterior to manubrium and smoothly curved and smoothly tapered lamina/calomus complex, and gubernaculum with claw-like anterior end in lateral view and three distal branches in ventral view. The new species is distinguished from A. superbus by morphology of the spicule and gubernaculum. Type specimens of four other Acrostichus species, originally described from bark beetles from North America, i.e., A. concolor, A. gubernatus, A. ponderosus and A. taedus, were re-examined and photo-documented.

Behavioural response of Meloidogyne incognita to benzyl isothiocyanate

Abstract: Summary – The breakdown of brassicaceous plant material produces nematotoxic isothiocyanates (ITCs). However, after the incorporation of brassicaceous plant material into soil, many nematodes are likely to be exposed only to sublethal concentrations of these compounds. Although unknown, the effect of these low ITC concentrations on the behaviour of plant-parasitic nematodes could play a role in nematode suppression. To address this question, the behaviour of infective second-stage juveniles (J2) of Meloidogyne incognita was evaluated following in vitro exposure to sublethal concentrations of benzyl isothiocyanate (BITC). Behaviour was qualitatively and quantitatively affected. Overall nematode activity in treatment groups scored visually, and individual nematode movement frequencies, quantified by video assay, were each significantly reduced within 2 h of exposure to 0.01 mM BITC. All responses were dose dependent. Infectivity of BITC-treated J2 of M. incognita on soybean (Glycine max) was measured directly by root staining and gall rating, and indirectly by egg production. All experiments showed significantly reduced infectivity after treatment of J2 with 0.01 mM BITC. In addition, egg production was almost completely eliminated (<5% of control) by 0.03 mM BITC. The correlation between the effect of BITC concentrations on J2 activity and infectivity was positive, with decreased J2 activity and infectivity resulting in decreased egg production. BITC concentrations that do not cause mortality significantly affect the behaviour of M. incognita J2, indicating that sublethal concentrations contribute to the overall nematode suppression by brassicaceous green manures.

Ficotylus congestae gen. n., sp. n. (Anguinata), from Ficus congesta (Moraceae) sycones in Australia

Abstract: A new monotypic genus of tylenchid nematode, Ficotylus n. gen., was recovered from sycones of Ficus congesta L. from Mourilyan, Mission Beach and Cairns, Queensland, Australia. The genus is characterised by having slender nematodes with a long stylet, a sub-cylindrical pharynx lacking a terminal bulb, pharyngeal glands overlapping the intestine for a short distance, secretory/excretory pore opening posterior to the nerve ring, female with a single gonad and post-vulval sac and male with slender, arcuate, cephalated spicules with the bursa arising near the proximal level of the retracted spicule and supported by a pair of papilla-like structures at its widest point. This is the first record of a tylenchid occurring within Ficus sycones. Ficotylus congestae gen. n., sp. n. is distinguished by possessing a long stylet (29-39 μm) with conus forming ca 40% of stylet length and three well developed bifid knobs at the base of the shaft, a degenerate pharynx with a vestigial metacorpus (no valve) and no basal bulb, females with a post-vulval uterine sac and males with a characteristic bursa supported by a pair of papilla-like structures at its widest point, then narrowing rapidly and just enveloping tail. Ficotylus congestae gen. n., sp. n. is a putative member of the Anguinata (sensu Siddiqi, 1985), based on molecular phylogeny of near full length SSU and D2/D3 expansion segments of the large subunit (LSU) rDNA. Morphologically, it has affinities with the superfamily Sphaerularioidea, family Neotylenchidae, where it is tentatively placed.

Rapid and accurate prediction of the frequencies of Bursaphelenchus xylophilus and B. mucronatus in mixed nematode samples using real-time species-specific PCR

Abstract: Accurate detection of Bursaphelenchus xylophilus and prediction of its frequency in crude nematode samples is often hindered by the coexistence of related nematode species, such as B. mucronatus, that are morphologically similar but non-pathogenic. To establish a detection system enabling determination of the relative frequencies of B. xylophilus and B. mucronatus from field nematode samples, we developed a real-time species-specific PCR (rtssPCR) protocol which targets the substantial sequence differences in the 5S rRNA marker gene between the two nematode species. Using standard DNA mixtures of B. xylophilus and B. mucronatus in various ratios, plots of percent species proportion vs cycle threshold value (Ct value) were generated for the prediction of species frequency. The rtssPCR protocol enables the detection of target nematode frequencies as low as 0.16% at the 95% confidence level. When nematode DNA samples were extracted from the mixed specimens of B. xylophilus and B. mucronatus in various ratios and analysed by rtssPCR, the semi-log plot was nearly identical to the plot generated from standard mixed DNA samples, demonstrating that field populations of the nematodes can be directly used for rtssPCR analysis. The rapid and accurate determination of B. xylophilus or B. mucronatus frequencies by this rtssPCR protocol makes it ideal for routine monitoring and quarantine of B. xylophilus in the field.

Poikilolaimus floridensis n. sp. (Rhabditida: Rhabditidae) associated with termites (Kalotermitidae)

Abstract: A survey of termite-associated nematodes was done in southern Florida to compare the diversity of such associations with other latitudes in the Neotropics. Six species (15 colonies) of termites (Isoptera) were collected from the field and eight species (15 colonies) from laboratory populations were examined for nematode associates. All six field-collected termite species, Cryptotermes cavifrons, Incisitermes snyderi, Neotermes jouteli, N. castaneus, Prorhinotermes simplex and Reticulitermes flavipes, representing two families (Kalotermitidae and, for the latter two species, Rhinotermitidae), were associated with nematodes. Nematodes were also isolated from laboratory populations of I. snyderi and Coptotermes formosanus. In total, seven putative species of nematodes were discerned using molecular bar-coding and culturing (when successful) including four rhabditids, one diplogastrid, Rhabditis rainai and a nematode that we are describing herein as Poikilolaimus floridensis n. sp. This nematode was isolated as dauer juveniles in the foregut of N. jouteli, N. castaneus and I. snyderi. It was recovered from workers, a soldier and an alate, suggesting internal phoresy. It is characterised by six triangular cuticular flaps covering the stomatal opening, simple tube-like stomatal structure, i.e., absence of teeth and glottoid apparatus, cuticularised and refractile secretory-excretory pore, conical male tail lacking 'bursa' or spike, short and conical female tail and didelphic female reproductive system. Poikilolaimus floridensis n. sp. is morphologically characteristic and does not easily fit the current genus definition of Poikilolaimus which is redefined herein. Molecular phylogenetic analysis based on near full length SSU ribosomal DNA sequence showed that the new species occupies a basal position in the genus.

Interactions between the potato cyst nematodes, Globodera pallida , G. rostochiensis , and soil-borne fungus, Rhizoctonia solani (AG3), diseases of potatoes in the glasshouse and the field

Abstract: This is the first report of a positive interaction between the nematode Globodera pallida and Rhizoctonia solani diseases of potato, and is an important step in the general understanding of interactions between nematodes and fungal diseases. One glasshouse and two field experiments were performed to investigate interactions between G. pallida or G. rostochiensis and R. solani diseases of potatoes. The glasshouse experiment investigated the independent and combined effects of G. pallida or G. rostochiensis and R. solani on the growth of plants and R. solani disease severity. In the glasshouse experiment the combined effects of G. pallida with R. solani or G. rostochiensis with R. solani showed greater R. solani diseases of potatoes compared with only R. solani. The field experiments examined the development of R. solani diseases on potatoes grown in soil with potato cyst nematode population densities ranging from eight to 140 and four to 50 eggs (g soil)–1 in the years 2006 and 2007, respectively. The 2006 field experiment revealed a clear positive relationship between initial population densities of G. pallida and the incidence of stolons infected by R. solani, subsequent stolon pruning and stem canker. In both field experiments a clear positive relationship was found between densities of nematodes within the potato roots and the incidence of infected stolons, stolon pruning and stem canker. Nematicide applied together with R. solani in the 2007 field experiment reduced the effect of G. pallida on the incidence and severity of diseases caused by R. solani.

Tomato culture filtrate stimulates hatching and activity of Meloidogyne incognita juveniles

Abstract: Hydroponic culture media were tested after growing tomato, okra, cucumber and bean for their effect on hatching and mobility of Meloidogyne incognita second-stage juveniles (J2) in laboratory experiments. Tomato and okra culture media increased the numbers of J2 that hatched as compared to those in water or fresh culture medium. The tomato culture medium increased hatching even in the presence of fosthiazate, an organophosphate nematicide, and at a concentration that inhibited hatching in the absence of tomato culture medium. Neither heat treatment of the tomato culture medium nor change of pH abolished its hatching stimulatory activity. When active J2 were incubated in the tomato culture media, the percentages of nematodes that became quiescent were lower than those of nematodes incubated in water or in fresh culture medium for 3 and 7 days in two trials. Moreover, the sigmoid movement of J2 was faster in the tomato culture medium than in water. Quiescent J2 became active more rapidly in culture media of tomato, cucumber and okra than in water or fresh culture medium. In experiments using soil containing quiescent J2, more J2 were extracted with the Baermann funnel method from soil treated with tomato culture medium than from soil treated with water. The results confirm that nematode stimulants, which may serve as a potential means in nematode control, exist in the plant culture media.

Steinernema australe n. sp. (Panagrolaimomorpha: Steinernematidae), a new entomopathogenic nematode from Isla Magdalena, Chile

Abstract: A new species of entomopathogenic nematode, Steinernema australe n. sp., was isolated from a soil sample taken close to the beach on Isla Magdalena, an island in the Pacific Ocean, 2 km from mainland Chile. Morphologically the new species belongs to the glaseri-group and is characterised by morphometrics of the infective juvenile which has a very long body of 1316 (1162-1484) μm, excretory pore located far posterior to the anterior extremity (110 (95-125) μm), exceptionally long tail of 103 (92-114) μm, H% = 51 (42-61), E% = 107 (94-122) and a ratio = 35 (31-38). The first generation male has 72 (55-78) μm long spicules, a 45 (36-51) μm long gubernaculum and SW% = 172 (118-196). The first generation female can be recognised by well developed double epiptygmata, the lack of a prominent postanal swelling, a mucron on the tail tip and (in 60% of individuals) one to two subsidiary mucrons. Sequences of the ITS and D2D3 regions of the ribosomal DNA confirm that S. australe n. sp. is a valid species.

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

Abstract: 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.

Nematicidal activity of compounds extracted from medicinal plants against the pine wood nematode Bursaphelenchus xylophilus

Abstract: To investigate in vitro nematicidal activity against the pine wood nematode, Bursaphelenchus xylophilus, plant methanolic extracts were obtained from 26 medicinal plants and herbs in Vietnam. Of the plant extracts tested, a 10 mg ml−1 concentration of Cinnamomum cassia bark extract showed the highest level of nematicidal activity against juvenile and adult B. xylophilus after treatment for 7 h. The remainder of the plant extracts had little or no effect on juveniles or adults. Four solvents, n-hexane, chloroform, ethyl acetate and n-butanol, were used initially to partition the compounds obtained from C. cassia extracts. The compound (5 mg ml−1) in the n-hexane fraction showed the highest nematicidal activity at 60 min after treatment. Compounds III and IV were then separated from the n-hexane fraction using silica gel plates. These compounds showed very similar activity against the pine wood nematode. However, treatment with different concentrations of these compounds resulted in a significant difference in their effects on the mortality of the pine wood nematode, with greater concentrations leading to increased mortality after the same exposure time. Overall, treatment with 3 mg ml−1 of compounds III and IV resulted in greater than 95% mortality against juvenile and adult B. xylophilus at 50 min after treatment.

Virulence of entomopathogenic nematodes to different stages of the flatheaded root borer, Capnodis tenebrionis (L.) (Coleoptera: Buprestidae)

Abstract: The virulence of 14 Steinernema feltiae isolates, one S. carpocapsae and three Heterorhabditis bacteriophora isolates was compared in the laboratory against larvae, pupae and adults of the flatheaded root borer, Capnodis tenebrionis . Larval mortality ranged from 50 to 100% and pupae mortality from 0 to 70%. Adults were exposed to different nematode concentrations, 50 and 100 infective juveniles/cm 2 . Steinernema feltiae caused the highest mortality at the two doses tested, reaching 66.7% and 100%, respectively. Significant differences in mortality between C. tenebrionis males and females were observed. The differences in virulence observed at each stage of C. tenebrionis vary within a species and can be higher than differences among species. The results of this study suggest that some of the isolate strains have potential for improved biological control of C. tenebrionis .

Hirschmanniella kwazuna sp. n. from South Africa with notes on a new record of H. spinicaudata (Schuurmans Stekhoven, 1944) Luc & Goodey, 1964 (Nematoda: Pratylenchidae) and on the molecular phylogeny of Hirschmanniella Luc & Goodey, 1964.

Abstract: A new species of the genus Hirschmanniella , H. kwazuna sp. n., is described from unidentified grass growing in undisturbed veldt from South Africa. Hirschmanniella kwazuna sp. n. is characterised by having a very irregular heat-relaxed body posture, body 1522-2049 μ m long, lip region low and rounded with four or five lip annuli, stylet 18-22.5 μ m long, lateral field areolated along entire body, spermatheca filled with sperm, tail with 62-81 ventral annuli narrowing to a tip bearing a ventral mucro, angular crystal-like inclusions within body cavity in most of the specimens and phasmid situated 12-24 annuli or 15-26 μ m anterior to tail tip. Males, like females, have crystal-like inclusions with the tail curved strongly dorsad in most specimens. Juveniles are similar to females. Molecular sequence analysis using the D2-D3 expansion segments of 28S, partial 18S and ITS rRNA sequences distinguished H. kwazuna sp. n. from H. loofi and other species of the genus. Hirschmanniella spinicaudata is reported from South Africa for the first time and described. Phylogenetic analyses based on analysis of the D2-D3, 18S and ITS rRNA genes are given for eight, ten and five valid and unidentified Hirschmanniella species, respectively.