The ribosomal intergenic spacer (IGS) in the potato and tobacco cyst nematodes, Globodera pallida, G. rostochiensis and G. tabacum.

The potato cyst nematodes Globodera pallida and G. rostochiensis (PCN), and tobacco cyst nematode (TCN), G. tabacum, are the most important parasitic nematodes of potato and tobacco worldwide. Ribosomal DNA provides useful molecular data for diagnostics, the study of polymorphisms and for evolutionary research in eukaryotic organisms including nematodes. Here we present data on the structure and organization of a rarely studied part of the intergenic spacer (IGS) region of the PCN and TCN genome of cyst nematodes. This region has shown potential for diagnostic purposes and population studies in other organisms including nematodes. In nematodes, the ribosomal RNA gene cluster comprises three genes: 5.8S, 18S and 28S rRNA, which are separated by spacer regions: the intergenic spacer (IGS), non-transcribed spacer (NTS), externally transcribed spacer (EST) and the internally transcribed spacer (ITS). The intergenic spacer (IGS) region consists of an external transcribed spacer (ETS) and a non-transcribed spacer (NTS) which is located between the 28S of one repeat and the 18S gene of the next repeat within the rRNA genes cluster. In this study, the first flanking portion of the IGS was amplified, cloned and sequenced from PCN and TCN. Primers were then designed to amplify the whole IGS sequence. PCR amplification of IGS from G. tabacum, G. pallida, and G. rostochiensis yielded respectively: a single amplicon of 3 kb, three amplicons sized 2.5, 2.6 and 2.9 kb, and two amplicons sized 2.8 and 2.9 kb. Results showed that Globodera spp. has more than one variant copy of the IGS, with both long and short repetitive DNA elements. An approximately 400 bp long region without any internal repetitive elements, were identified in a position between the two repetitive regions suggesting that there is a 5S gene in the IGS of these species.

Characterization in biological traits of entomopathogenic nematodes isolated from North China.

In a series of bioassays, thirty-one isolates that were collected from diverse locations in northern China and the laboratory kept isolate Steinernema carpocapsae All, were compared in order to select superior isolates for biological control of Bradysia odoriphaga. Virulence of the isolates against B. odoriphaga was significantly different among nematode isolates. Tolerance of infective juveniles (IJs) to heat, cold, and desiccation differed significantly among and within species. Strains from S. carpocapsae, S. ceratophorum, S. longicaudum, Heterorhabditis indica, and H. bacteriophora were more heat tolerant than strains from S. feltiae, S. hebeiense, S. monticolum, and H. megidis. Heterorhabditis megidis, H. bacteriophora, and S. carpocapsae showed better cold tolerance than the other species. High desiccation tolerance was recorded for S. carpocapsae, S. hebeiense, and S. ceratophorum. The infectivity of IJ of these species against Galleria mellonella larvae was not significantly different between the treated and non-treated IJ after the nematodes had been exposed to 40°C for 2 h, -5°C for 8 h or 25% glycerin for 72 h. Nematode survival was significantly affected by exposure time and IJ concentration when exposed to 40°C or -5°C. All nematode isolates lost their infectivity against G. mellonella after exposure to -5°C for 16 h, except for H. megidis LFS10, which had a low infectivity of 3.3%. A hierarchical classification analysis classified the isolates in four main clusters. The fourth cluster, composed of 13 isolates, grouped the isolates that scored well for most traits.

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.

Migratory plant endoparasitic nematodes: a group rich in contrasts and divergence.

Species of migratory plant endoparasitic nematodes of three nematode families, Pratylenchidae, Anguinidae, and Aphelenchoididae, show marked variation in life cycles and fascinating contrasts in host-parasite interactions. The necessity for survival for periods in the absence of a host has resulted in some remarkable behavioral and physiological adaptations, especially in relation to anhydrobiotic survival. Many species are of major economic importance, and interactions with other pathogens enhance crop damage and yield loss. No single management strategy for endoparasitic nematodes is possible because control options have to be tailored to the nematode species, crop type, location, and economic returns. In this review, we focus on the contrast in life cycles and the wide spectrum of adaptations to obligate parasitism of most species of plant endoparasitic nematodes and examine how these aspects impinge on management options.

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.

Natural occurrence and distribution of entomopathogenic nematodes (Steinernematidae and Heterorhabditidae) in Nepal.

A survey of entomopathogenic nematodes (EPN) was conducted for the first time in Nepal during June-December 2007. EPN were recovered from 29 of the 276 soil samples (10.50%). Molecular and morphological tools were used to identify the isolates. Among 29 positive samples, seven samples (24.14%) yielded heterorhabditids (Heterorhabditis indica) and 22 samples (75.86%) contained steinernematids. Among steinernematids, seven isolates were identified as one of four described species (Steinernema abbasi, S. cholashanense, S. feltiae and S. siamkayai) whereas 15 isolates did not fit any description. H. indica was recovered from diverse habitats. Distribution of steinernematids has followed ecological niches and localized areas.

Quantitative analysis of the effects of diffusates from plant roots on the hatching of Meloidogyne chitwoodi from young and senescing host plants.

We analyzed the effects of diffusates from six different crops on the egg hatching of Meloidogyne chitwoodi collected from young and senescing tomato plants using a non-linear model. Diffusates only from tomato and carrot significantly delayed the hatching of eggs from young plants, but did not do so from senescing plants, suggesting that delay of hatching depends on the origin of diffusates and host plant age.

Formulation and application of entomopathogenic nematode-infected cadavers for control of Hoplia philanthus in turfgrass.

BACKGROUND: Commercially produced entomopathogenic nematodes (EPNs) are usually applied in an aqueous suspension. However, recent studies demonstrate that they can be successfully applied as infected cadavers. EPN-infected insect cadavers were formulated in various carrier/coating materials, but only infective juveniles (IJs) from kaolin-formulated and non-formulated cadavers were tested for survival, emergence and virulence. The efficacy of IJs from kaolin-formulated and non-formulated cadavers applied against Hoplia philanthus Füessly in greenhouse and field was also assessed. RESULTS: The kaolin-based formulation (kaolin-starch combination) proved to be the most stable. Emergence of Heterorhabditis bacteriophora Poinar CLO51 from cadavers formulated in kaolin was significantly higher than from non-formulated cadavers. In greenhouse conditions, efficacy of freshly formulated (8 days post-infection) cadavers of H. bacteriophora provided significantly higher H. philanthus control (62%) than three-month-old cadavers (31%) or aqueous applications of the same EPN (39% and 43%). Similarly, under field conditions, significantly higher H. philanthus control was achieved with freshly formulated cadavers (39%) than with three-month-old cadavers (21%) or with aqueous applications (24% and 28%) of H. bacteriophora 2 weeks after application. Additionally, after 1 year, cadaver applications provided >90% H. philanthus control, while aqueous applications of H. bacteriophora gave only 55% control. CONCLUSIONS: This study demonstrated that kaolin-starch coating provided superior protection and preservation of EPN-infected cadavers. Use of formulated cadavers may solve several current problems preventing the wider utilisation of EPNs in pest management.

Development of an efficient application of entomopathogenic nematodes in vegetables.

The withdrawal of approvals for agrochemicals on many food crops within Europe is likely to generate an increasing market for bio-pesticide products, including entomopathogenic nematodes (EPN). However, for the EPN to be a success, a cost-effective and reliable usage should be assured. Therefore, progress in areas like application and formulation technology is needed, next to the development of optimal application strategies. A new research project will focus on optimizing the field application of EPN in vegetables. First, both application and formulation technologies will be optimized. Then, these technologies will be tested under semi-field conditions with special attention for the effect of weather conditions and timing of the application. All the acquired knowledge will be implemented and evaluated in field experiments. In this paper, the first results of the experiments on application technology are discussed.

Effect of several centrifugal pump passages on the viability and activity of Steinernema carpocapsae, a biopesticide.

The effect of several passages through a centrifugal pump on the viability and activity of Steinernema corpocapsoe was investigated. A tendency of reduced viability was detected after 10 passages through the centrifugal pump, but the difference was not statistically significant. Activity decreased significantly from 91.9% to 83.6%. The experimental set-up was chosen to eliminate temperature increase. Therefore, the results prove that mechanical pump damage cannot be excluded as the cause of EPN damage during spray application.

Vertical Distribution of the Plant-Parasitic Nematode, Pratylenchus penetrans, Under Four Field Crops.

ABSTRACT The vertical distribution of Pratylenchus penetrans was monitored in four fields cropped with maize, black salsify, carrot, or potato. Soil samples were collected at 21-day intervals from May 2002 until April 2003 from five plots (2 x 5 m(2)) per field. Per plot, 15 cores were taken to a depth of 70 cm and split into seven segments of 10 cm each. Within the plots, segments from corresponding depths were pooled. After mixing, 200-g subsamples were taken and nematodes were extracted by zonal centrifugation from the root fraction and the mineral soil fraction separately. In most crops, the root fraction contained more than 50% of the total number of P. penetrans. Because the ratio between the numbers of nematodes in the root fraction and mineral soil fraction changes during the growing season, numbers of P. penetrans found in the mineral soil fraction cannot be used to estimate the total number in the soil. Therefore, both fractions have to be processed to obtain a reliable estimate of the density. No nematodes were recovered below 50 cm soil depth, except in the maize field where nematodes were found at 70 cm. The optimum sampling depth for maize, black salsify, carrot, and potato was 45, 25, 25, and 35 cm, respectively. The percentage of nematodes per soil layer was independent of the sampling date, indicating that a defined optimum sampling depth will be applicable throughout all seasons. The cumulative vertical distribution, modeled with a logistic equation, can be used to estimate the sampling error when samples are collected at different depths.

Molecular Diagnostics, Taxonomy, and Phylogeny of the Stem Nematode Ditylenchus dipsaci Species Complex Based on the Sequences of the Internal Transcribed Spacer-rDNA.

ABSTRACT The stem nematode Ditylenchus dipsaci is of great economic importance worldwide as a parasite of agricultural crops and horticultural plants. The internal transcribed spacer (ITS) of rDNA from 23 populations of the D. dipsaci complex from various host plants were amplified and sequenced. Seven previously studied populations were also included in the study. The phylogenetic analysis of the full ITS and ITS2 sequence alignments using minimum evolution, maximum parsimony, and Bayesian inference under the complex model of DNA evolution revealed trees with two main clades: (i) D. dipsaci sensu stricto with diploid chromosome numbers and comprising most isolates from agricultural, ornamental, and several wild plants, and (ii) Ditylenchus spp. with polyploid chromosome numbers, reproductively isolated from diploid populations, and subdivided into six subclades ("giant race" from Vicia faba, Ditylenchus species parasitizing various Asteraceae, and a Ditylenchus sp. from Plantago maritima). Using the energy minimization approach and comparative sequence analysis, it has been found that the secondary structure of ditylenchid ITS2 is organized in three main domains. The importance of knowledge on the RNA structure for phylogenetic analysis is discussed. Conventional polymerase chain reaction (PCR) and real-time PCR with SYBR green dye I with a species specific primer have been developed for detection and quantification of D. dipsaci sensu stricto Validation tests revealed a rather high correlation between real numbers of fourth-stage juveniles of the stem nematodes in a sample and expected numbers detected by real-time PCR. Problems of accuracy of quantification are discussed.

Intraspecific variation in Radopholus similis isolates assessed with restriction fragment length polymorphism and DNA sequencing of the internal transcribed spacer region of the ribosomal RNA cistron.

Restriction fragment length polymorphism and direct sequencing of the internal transcribed spacer rDNA region of 19 isolates of Radopholus similis yielded significant diversity, both among isolates and within some individuals. Restriction fragment length polymorphism with HaeIII, AluI and Tru9I yielded two sets of patterns. Digestion with RsaI revealed one or two supernumerary bands in single nematodes from five isolates, and sequencing confirmed microheterogeneity in four of these. Phylogenetic analysis grouped most isolates closely together, except for the five isolates with additional bands for RsaI. Our data reveal more population structure than previously found and lend further support to the synonymy of R. similis and 'Radopholus citrophilus'.

Phylogenetic Analyses of Meloidogyne Small Subunit rDNA.

Phylogenies were inferred from nearly complete small subunit (SSU) 18S rDNA sequences of 12 species of Meloidogyne and 4 outgroup taxa (Globodera pallida, Nacobbus abberans, Subanguina radicicola, and Zygotylenchus guevarai). Alignments were generated manually from a secondary structure model, and computationally using ClustalX and Treealign. Trees were constructed using distance, parsimony, and likelihood algorithms in PAUP* 4.0b4a. Obtained tree topologies were stable across algorithms and alignments, supporting 3 clades: clade I = [M. incognita (M. javanica, M. arenaria)]; clade II = M. duytsi and M. maritima in an unresolved trichotomy with (M. hapla, M. microtyla); and clade III = (M. exigua (M. graminicola, M. chitwoodi)). Monophyly of [(clade I, clade II) clade III] was given maximal bootstrap support (mbs). M. artiellia was always a sister taxon to this joint clade, while M. ichinohei was consistently placed with mbs as a basal taxon within the genus. Affinities with the outgroup taxa remain unclear, although G. pallida and S. radicicola were never placed as closest relatives of Meloidogyne. Our results show that SSU sequence data are useful in addressing deeper phylogeny within Meloidogyne, and that both M. ichinohei and M. artiellia are credible outgroups for phylogenetic analysis of speciations among the major species.

Improving the biocontrol potential of Steinernema feltiae against Delia radicum through dosage, application technique and timing.

BACKGROUND: The potential of the entomopathogenic nematode (EPN) Steinernema feltiae Filipjev as a biocontrol agent against the cabbage maggot Delia radicum (L.), was assessed in three field tests, focusing on EPN dosage, application technique and timing. RESULTS: Spraying cabbage plant trays with different doses of infective juveniles (IJs) (50,000, 100,000 and 200,000 per plant) generated a similar reduction of plant mortality. Spraying plant trays with 200,000 IJs of Steinernema feltiae per plant temporarily reduced the number of maggots around the plants' roots, while neither spraying a lower dose (50,000 IJs/plant) nor soil drenching with 200,000 or 50,000 IJs/plant) reduced maggot numbers. When applied as a plant tray spray, IJs of S. feltiae took 1-2 weeks to spread through the soil surrounding the roots. The pathogenicity of the EPNs, as evaluated by a Galleria mellonella bait test, was highest (up to 100% mortality) until up to five weeks after application, and declined to control levels after 4-7 weeks. Follow-up drench applications with EPNs, applied one and/or two weeks after the first EPN application, did not influence control of Delia radicum. CONCLUSION: Plant tray spraying provides better placement of Steinernema feltiae than soil drench treatments for control of Delia radicum. Plant mortality was not dose-dependent in the presented trials, unlike the reduction of maggot numbers. Further research into timing and application technique of follow-up treatments with S. feltiae is required to increase efficacy to commercial standards.

Improving the biocontrol potential of entomopathogenic nematodes against Mamestra brassicae: effect of spray application technique, adjuvants and an attractant.

BACKGROUND: Steinernema carpocapsae Weiser, an entomopathogenic nematode (EPN), is a potential biological control agent for the cabbage moth (Mamestra brassicae L.). This research aimed to identify a suitable spray application technique, and to determine whether yeast extract added to an EPN spray has an attracting and/or a feeding stimulant effect on M. brassicae. The biological control capabilities of EPN against this pest were examined in the field. RESULTS: Good coverage of the underside of cauliflower leaves, the habitat of young instar larvae (L1-L4) of M. brassicae was obtained using different spray boom configurations with vertical extensions that carried underleaf spraying nozzles. One of the configurations was selected for field testing with an EPN spray. Brewer's yeast extract stimulated larval feeding on leaves, and increased the mortality of these larvae when exposed to EPN. The field trial showed that a spray application with S. carpocapsae, Addit and xanthan gum can effectively lower the numbers of cabbage heads damaged by M. brassicae. Brewer's yeast extract did not significantly increase this field performance of EPN. CONCLUSION: Steinernema carpocapsae, applied with an appropriate spray technique, can be used within biological control schemes as part of a resistance management programme for Bt.

Screening of common bean (Phaseolus vulgaris) for resistance against temperate root-knot nematodes (Meloidogyne spp.).

BACKGROUND: An important part of the production area of common bean (Phaseolus vulgaris L.) in Belgium is located on the sandy soils of the provinces of Antwerp and Limburg where Meloidogyne chitwoodi (Golden), M. fallax (Karssen) and M. hapla (Chitwood) are present. The host plant status of ten bean cultivars for root-knot nematodes was determined by evaluating penetration, development and egg mass formation after inoculation with second-stage juveniles. RESULTS: The tested cultivars were poor to good hosts for M. chitwoodi, non-hosts or bad hosts for M. fallax and excellent hosts for M. hapla. Significantly fewer M. fallax were found in the roots, and their development was delayed. Penetration of M. hapla took place over a longer period than that of M. chitwoodi and M. fallax. The number of mature females of M. chitwoodi in cv. Polder 6 weeks after inoculation was no different from that in other cultivars, although fewer egg masses were found on this cultivar in the screening test. There was no influence of M. chitwoodi on vegetative growth of cv. Polder. CONCLUSION: The differences found in host plant status of bean cultivars stress the importance of a correct diagnosis of the Meloidogyne species in agricultural fields. Cultivar Polder showed potential as a trap crop for M. chitwoodi.

Effect of the spray application technique on the deposition of entomopathogenic nematodes in vegetables.

BACKGROUND: The present study compared entomopathogenic nematode delivery at the base of savoy cabbage and cauliflower, at the lower side of savoy cabbage and cauliflower leaves and in leek stems and the ground deposition using a five-nozzle spray boom equipped with an ISO 08 flat fan, an air induction flat fan and Twinjet spray nozzles. Additionally, an air support system and a row application system were evaluated. RESULTS: Approximately 40% of the applied nematodes did not reach the foot of the cabbage plants. The use of an air support system or a row application system improved nematode deposition at the savoy cabbage base. Relative nematode deposition on the lower side of savoy cabbage leaves was 27.20%, while only 2.64% of the applied nematodes reached the lower side of cauliflower leaves. After spraying leek with a standard boom, a low relative nematode deposition (26.64%) was measured in the leek stem. Nozzle type affected the distribution of nematodes in droplet spots. CONCLUSION: Nozzle type has a minor effect on the number of entomopathogenic nematodes delivered on difficult-to-reach targets. The use of modified spray application techniques directing the spray to the target site are necessary to increase the chances of contact of entomopathogenic nematodes with their target.

Effect of spray volume on the deposition, viability and infectivity of entomopathogenic nematodes in a foliar spray on vegetables.

BACKGROUND: Spray volume can influence the amount of free water on the leaf surface and subsequently the ability of entomopathogenic nematodes (EPNs) to move. In this study, an investigation was made of the effect of spray volume (548, 730 and 1095 L ha(-1) ) on the deposition, viability and infectivity of EPNs against Galleria mellonella on savoy cabbage, cauliflower and leek. RESULTS: Increasing spray volume decreased nematode deposition on 7.1 cm2 leek leaf discs at a 15° angle with the spray nozzle. Although the number of living nematodes observed on leek after 240 min of exposure was not significantly different between the low-volume application (548 L ha(-1) ) and the high-volume application (1095 L ha(-1) ), a greater infectivity was obtained in the latter application. The higher number of droplets deposited on the leek discs in the high-volume application may have stimulated nematode movement. No significant effect of spray volume was observed on the relative deposition of Steinernema carpocapsae on the bottom side of cauliflower and savoy cabbage leaf discs. In spite of the low S. carpocapsae deposition on the bottom side of the savoy cabbage discs, high infectivity was obtained against G. mellonella. Using the lowest spray volume on savoy cabbage, infectivity decreased with increasing exposure time, while infectivity was not affected by exposure time when a spray volume of 730 L ha(-1) or more was used. CONCLUSION: Spray volume is an important application parameter, as it affects nematode infectivity. Future research should investigate the effect of spray volume in the field and its influence on the effect of adjuvants.