Search

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.

The influence of three pesticides on the viability and infectivity of four Beninese isolates of entomopathogenic nematodes (EPN), Heterorhabditis indica Ayogbe1, H. sonorensis Azohoue2, H. sonorensis Ze3, and Steinernema sp. Bembereke, was determined. The impact of both soil temperature and soil moisture on the virulence of these EPN to Trinervitermes occidentalis was investigated in laboratory assays. The effect of EPN-infected Galleria mellonella larvae on underground populations of Macrotermes bellicosus was also examined. All tested Heterorhabditis species were more tolerant to glyphosate and fipronil than the Steinernema species. Heterorhabditis sonorensis Azohoue2, showed the best results with 63.2% termite mortality at a soil temperature of 35°C. The increase of soil moisture to 20% (w/w) did not negatively influence the virulence of tested EPN. The underground populations of 71% or 60% treated nests were controlled by H. sonorensis Azohoue2- or H. indica Ayogbe1-infected G. mellonella larvae, respectively.

The differential susceptibility of two termite species, Macrotermes bellicosus and Trinervitermes occidentalis, to four entomopathogenic nematodes (EPN) isolates from Benin, Heterorhabditis indica Ayogbe1, H. sonorensis Azohoue2, H. sonorensis Ze3 and Steinernema sp. Bembereke, was bio-assayed in laboratory tests. Soldiers of both M. bellicosus and T. occidentalis were similarly susceptible, but more susceptible than workers. Forty-eight h post-exposure of workers of M. bellicosus to 50 infective juveniles (IJ) of H. indica Ayogbe1, H. sonorensis Azohoue2, H. sonorensis Ze3 and Steinernema sp. Bembereke for each termite resulted in 96.3, 87.9, 94.5 and 75.0% mortality, respectively, whereas under the same conditions, these EPN isolates caused 91.7, 98.5, 75.0 and 95.0% mortality of workers of T. occidentalis. Soldiers of M. bellicosus were the most invaded with 13.2-18.6% of applied IJ. Based on concentration-mortality data, the isolates H. indica Ayogbe1 and H. sonorensis Ze3 were more virulent to M. bellicosus with LC50 values of 11 IJ, whereas Steinernema sp. Bembereke was the most virulent to T. occidentalis with LC50 values of 12 IJ. However, none of these isolates showed the highest penetration rate. All tested EPN isolates can recycle in both M. bellicosus and T. occidentalis. Our EPN repellent-dispersing assay did not show evidence that M. bellicosus and T. occidentalis would be able to detect the presence of IJ of any EPN isolates/species. However, it was observed that nematode dispersal occurred by infected termites or phoresis.

Potato cyst nematodes (PCN) are the most economically important nematode pests of potato. Early harvesting is part of a preventive management approach for very early potato varieties. In Belgium, tubers are harvested before 20 June because it is assumed that no cyst formation occurs by that date. However, this assumption might not be valid any more because of climate change, the availability of new cultivars (with new traits), and the increasing prevalence of Globodera pallida. Therefore, pot, microplot and field trials were conducted to study the life cycle of PCN in early potato-growing conditions. The development of populations of G. pallida on three potato cultivars, Eersteling (susceptible to PCN), Première (resistant to G. rostochiensis) and Ambassador (partially resistant to G. pallida) and that of G. rostochiensis on cv. Eersteling was monitored in the growth chamber with simulation of field temperatures of the season. On cvs Eersteling and Première, second-stage juveniles (J2), males, females and cysts of G. pallida populations were found 28, 56, 63 and 77 days after infestation with cysts (DAI), respectively. The number of degree days (DD) for G. pallida to complete its life cycle using a base temperature of 4°C was calculated 450 DD4. On cv. Ambassador, females of G. pallida were never observed. J2, males, females and cysts of G. rostochiensis were detected 42, 70, 70 and 84 DAI, respectively. This species needed 398 DD6 at a base temperature of 6°C to complete its life cycle. Observations in two fields and in microplots under prevailing weather conditions in 2013 revealed that cysts of G. pallida and G. rostochiensis were formed on June 12, when the accumulated degree days were 463 DD4 and 401 DD6, respectively. Our observations show that both species of Globodera develop earlier than was assumed based on previous data. Therefore, harvesting based on the accumulated heat above the basal development temperature required by PCN species can replace the set harvest date.

Meloidogyne minor is a root-knot nematode reported in Belgium, Ireland, The Netherlands, Portugal, United Kingdom, Chile and the United States. It is found in sport fields and golf courses where it causes the yellow patch disease. However, M. minor has also been detected in potato fields in The Netherlands and the UK and may pose a threat for potato cultivation. Therefore, the life cycle and damage of M. minor on potato cv. Bintje were examined under controlled conditions. To assess its life cycle, young potato plants were inoculated with freshly hatched second-stage juveniles (J2). The developmental stages of M. minor were recorded at weekly intervals after inoculation until second generation J2 were detected. One week after inoculation, only vermiform juveniles were found in the roots. All juveniles were swollen after 3 weeks and the first adult females were observed. Egg masses were seen after 6 weeks together with second generation J2. The number of degree days for M. minor to complete its life cycle was calculated using a base temperature of 5°C (DD5); between 606 and 727 DD5 were needed to complete the life cycle. Damage development of M. minor on potato was examined in a pot experiment with different inoculation densities. Symptoms (galling on the tubers) were similar to those caused by M. chitwoodi and M. fallax. At initial population densities () of 10 J2 (100 cm3 soil)−1 and more, tubers showed galls. Severely damaged potato tubers were observed at (100 cm3 soil)−1 and a damage threshold of 41 J2 (100 cm3 soil)−1 was calculated. An in vitro test showed that five, commonly grown, potato cultivars were good hosts for M. minor. Based on our results, M. minor is able to develop on potato and cause severe damage at low initial population densities. Therefore, further spread of this nematode in agricultural fields should be avoided.

The host-finding ability of 28 Heterorhabditis sonorensis isolates and one H. indica isolate from southern Benin and one H. indica isolate from India was examined in vertical migration sand bioassays against workers of a target citrus termite pest, Macrotermes bellicosus. Thereafter, nine selected isolates were subjected to further investigations on virulence. Our results revealed that both H. sonorensis and H. indica isolates exhibit a cruiser type of search strategy and were capable, to various degrees, of migrating, infecting and killing workers of M. bellicosus in sand columns up to 20 cm long over a period of 3 days. However, only three isolates of H. sonorensis caused 100% mortality to M. bellicosus at the greatest depth tested. The exotic H. indica isolate (LN2) did not show strong finding ability compared to the indigenous one. Concerning virulence, differences were observed among isolates for their ability to invade workers of M. bellicosus. After 12 h post exposure, H. sonorensis from Ze (Ze2) and H. sonorensis from Azohoue (Azohoue2) exhibited the lowest invasion time with IT50 = 3.35 and 3.67 h, respectively, and a higher penetration rate (11.4% and 10%, respectively) compared with the other isolates. In the concentration-mortality test, we found that, based on 95% confidence limits, all H. sonorensis and H. indica isolates appeared to be equal, with LC50 values ranging from nine to 16 infected juveniles (IJ) termite−1. Interestingly, 40 IJ termite−1 were enough to cause 80% mortality to M. bellicosus. Contrary to the LC50, the results of our studies clearly demonstrate that M. bellicosus exhibits a time-dependent susceptibility to the tested nematode isolates. So, the lowest LT50 was observed for H. sonorensis Ze2 (LT50 = 23.30 h), the highest for H. sonorensis Yokon (34.76 h). The LT50 of the indigenous H. indica isolate was estimated to 24.07 h. In addition, all selected isolates were able to reproduce in M. bellicosus workers. The highest reproduction potential in M. bellicosus was observed with H. sonorensis Yokon with 20 213 IJ/termite followed by H. sonorensis Ze2 with 19 368 IJ/termites. All tested Beninese EPN isolates were pathogenic to the citrus termite pest M. bellicosus, with H. sonorensis Ze2 being the most virulent.

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.

Although molecular techniques are revolutionising nematode taxonomy, morphological data still form the basis of nematode species descriptions. However, morphological characters show a natural variability that should be taken into account before describing new species. The current study presents the results of an elaborate morphometric study of Halicephalobus cf. gingivalis, including 15 measurements and 13 indices of 540 specimens, the progeny of a single parthenogenetic female and cultured under different temperature and food conditions and measured in different adult age groups, i.e., young adults with a developed vulva but before the onset of oviposition, adults laying eggs, and old, post-reproductive adults near the end of their life cycle. The morphometric characters were analysed using both univariate (analysis of variance) and multivariate (principal components and canonical discriminant analysis) techniques. The main results reveal that the morphometric characters most used in Halicephalobus identification keys have a huge variability within a single progeny, e.g., body length 1.9 times longer than the shortest or ratio VA/tail length 3.9 times larger than the smallest. This variability has a magnitude that has not been observed in nematodes before. Further, by changing the environmental factors, the morphometric characters are influenced to an extent that one could assign – with seemingly ‘statistical support’ – different ‘species’ of the genus to different subpopulations. With this experimental study we provide convincing elements to advocate an integrative taxonomic approach and to discourage the description of new species based only on morphometric differences.

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.

Four isolates of Radopholus arabocoffeae and one isolate of R. duriophilus were collected during a survey of plant-parasitic nematodes on coffee in Vietnam. Radopholus species only occurred in the Western Highland region. Information on the degree of variability in morphometrics, and morphological and molecular characters amongst and within these species are reported. The survey also yielded a Radopholus isolate collected from Robusta coffee roots and soil in Dak Lak, which is herein described as a new species, R. daklakensis sp. n. Within the genus, R. daklakensis sp. n. is most similar to R. arabocoffeae and R. duriophilus in morphology and morphometrics. It differs from R. arabocoffeae by the stylet knobs always directed posteriorly and the conical tail shape with rounded terminus in females. Radopholus daklakensis sp. n. differs from R. duriophilus by the stylet knobs, which are always directed posteriorly vs rounded, and dorsal knob sometimes projected anteriorly, and sperm rod-shaped vs oval and kidney-shaped. Radopholus daklakensis sp. n. differs from R. similis by the absence of a postrectal intestinal sac, four incisures of the lateral fields terminating far posterior to the position of the phasmid, the bursa in male reaching one-third of tail to half, never reaching tail terminus and smaller stylet length in females. The canonical discriminant analysis separated the Radopholus species by characters such as lip height, stylet length, b, b′, c, V and ratio tail length/stylet length. Phylogenetic analysis of Radopholus species using ITS-rDNA sequences or sequences from the D2-D3 expansion region of the 28S rRNA gene also confirm R. daklakensis sp. n. to be distinct.

The sequence of events during the pre-parasitic and parasitic stages of the life cycle of Globodera rostochiensis, starting from hatching until host invasion and infection, is of major importance. Many of the physiological changes are initiated in the pre-parasitic stage in response to the influences exerted by the root diffusates of the host plant. The objectives of this study were to investigate whether root diffusates and extracts of green manures from Brassicaceae, in particular yellow mustard, fodder radish and rapeseed, have a direct effect on the pre-parasitic stage of G. rostochiensis. Unhatched second-stage juveniles (J2) were exposed to root diffusates and extracts from roots or above-ground parts of the green manures to assess: i) changes in eggshell permeability; ii) changes in diameter of the dorsal gland nucleolus; and iii) the activation of transcription in the dorsal and subventral pharyngeal glands. The 24 h exposure of unhatched J2 to the green manures did not indicate an alteration in eggshell permeability. The diameter of the dorsal gland nucleolus increased significantly after exposure to diffusates but not to extracts of the green manures, although the size in that case still did not exceed the measurements in J2 exposed to tomato root diffusates. The expression of the gene IVg9 in the dorsal gland was not detected after any treatment. However, transcriptional activation in the subventral pharyngeal glands was observed. Although the maximum expression of the cellulase gene was found in J2 exposed to tomato root diffusates, the expression in J2 exposed to root extracts of green manures was higher than in J2 exposed to root diffusates and extracts of above-ground plant parts. The potential of the investigated brassicaceous green manures to influence the pre-parasitic stage of G. rostochiensis is discussed.

A new species of Steinernema was isolated from shrub soils in Liaoning province during a survey for entomopathogenic nematodes in north China. This nematode was obtained by the insect-baiting technique using last instar larvae of Galleria mellonella (Lepidoptera: Pyralidae). This new species is described herein as S. tielingense n. sp. It belongs to the ‘feltiae-kraussei-oregonense’ group and is characterised by infective third-stage juvenile (IJ) with a body length of 915 (824-979) μm, distance from head to excretory pore of 69 (64-73) μm, tail length of 81 (74-85) μm, ratio E=88 (85-94)%, lateral field with eight ridges at mid-body, first generation male spicule 88 (79-98) μm and gubernaculum 61 (49-70) μm long, and first generation female with a vulval protrusion and ratio D=41 (32-49)%. The new species distinctly differs from the related species S. kraussei, S. silvaticum, S. oregonense and S. cholashanense in the different number of ridges in the lateral fields and hyaline tail length as % of total tail length of IJ and male body length and distance from head to excretory pore. Cross hybridisation tests showed that these species were reproductively isolated. The sequences analyses of the internal transcribed spacers (ITS) and D2-D3 regions of the ribosomal DNA confirm this to be a new species.

{null=The potato cyst nematode, Globodera rostochiensis, is a quarantine organism. Environmentally benign control measures for this economically important pest are needed. Green manures, in particular plants from the Brassicaceae, suppress some plant-parasitic nematodes and have potential as control agents. This study examined if growing and incorporating cover crops from the Brassicaceae family influenced hatching of G. rostochiensis. The effect of root diffusates and plant extracts, as well as soil incorporation of plant material from three commonly used species of green manures, was studied in in vitro bioassays and pot tests. The results showed that brassica diffusates and plant extracts were not nematicidal. In addition, although they did not cause hatch by themselves, pretreatment with these solutions enhanced subsequent hatch in host root diffusates. The results are discussed in the context of the hatching response of G. rostochiensis and the likely influence on field usage of these green manures., en= The potato cyst nematode, Globodera rostochiensis, is a quarantine organism. Environmentally benign control measures for this economically important pest are needed. Green manures, in particular plants from the Brassicaceae, suppress some plant-parasitic nematodes and have potential as control agents. This study examined if growing and incorporating cover crops from the Brassicaceae family influenced hatching of G. rostochiensis. The effect of root diffusates and plant extracts, as well as soil incorporation of plant material from three commonly used species of green manures, was studied in in vitro bioassays and pot tests. The results showed that brassica diffusates and plant extracts were not nematicidal. In addition, although they did not cause hatch by themselves, pretreatment with these solutions enhanced subsequent hatch in host root diffusates. The results are discussed in the context of the hatching response of G. rostochiensis and the likely influence on field usage of these green manures. }

Abstract A solution consisting of a mixture of glycerol and fortified artificial seawater was used to induce partial anhydrobiosis at 15°C in different strains of Steinernema carpocapsae. All the strains tested were tolerant to the osmotic solution and the survival and infectivity of the nematodes were not influenced. The osmotic treatment greatly improved heat tolerance of all the tested strains but the heat tolerance of S. carpocapsae MR7 was significantly poorer than that of the other strains. This method could be used to induce S. carpocapsae into partial anhydrobiosis and thereby improve storage of the nematodes. Expression patterns of stress-related genes after osmotic treatment were compared in a heat tolerant strain (All) and a more heat sensitive strain (MR7) after induction of anhydrobiosis. Differences in gene expression after induction of anhydrobiosis between strain MR7 and All were observed that may be related to differences in subsequent heat tolerance.

The decline of populations of the plant-parasitic nematodes Radopholus arabocoffeae and Pratylenchus coffeae was studied after the death and removal of severely damaged coffee plants (Coffea arabica cv. Catimor) in a field naturally infested with both nematodes. Soil samples were taken at monthly intervals up to a depth of 70 cm and divided into 10 cm segments from which nematodes were extracted. The numbers of R. arabocoffeae and P. coffeae in soil and in root fragments were determined. Both species were found up to 70 cm depth. Densities of R. arabocoffeae and P. coffeae populations declined exponentially over time in all soil layers. No nematodes were found in soil and root fragments 10 and 11 months after removal of the infected coffee plants for R. arabocoffeae and P. coffeae, respectively. Arabica coffee seedlings were planted in soil collected from the experimental plots after these time periods. The seedlings were grown for 3 months in a glasshouse and then roots and soil were analysed for presence of both nematode species. No R. arabocoffeae and P. coffeae were found, confirming that the population in the field had completely declined. The results of this experiment demonstrate that a replant of coffee is possible 1 year after removal of infected plants from a plantation without any further (chemical) control measures.

In Europe, root-knot nematodes are increasingly important. Out of more than 90 Meloidogyne species currently described, 23 have been found on the continent. In the cooler climates, Meloidogyne hapla, M. naasi, M. chitwoodi and M. fallax are prevalent. Meloidogyne arenaria, M. javanica and M. incognita are the most common species in warmer conditions of southern Europe, but also in glasshouses in northern Europe. Morphological identification of root-knot nematodes is difficult and time consuming; therefore, many research groups have been developing molecular techniques for identification of Meloidogyne species. Meloidogyne chitwoodi and M. fallax are quarantine organisms and subject to regulations, and the highly aggressive M. enterolobii has been added to the EPPO alert list. Differences between temperate and tropical Meloidogyne species and their prevalence in Europe imply the need for different management strategies in south and north Europe. Possible crop rotations for the control of root-knot nematodes are limited due to the wide host range of several important species. The banning of methyl bromide and restrictions on other fumigant pesticides in the EU have increased the application of biofumigation significantly in south Europe. The egg-parasitising fungus Paecilomyces lilacinus is commercialised in Germany and applied as dispersible granules for application in water. Intensive research is conducted on the egg-parasitising fungus Pochonia chlamydosporia, and the obligate parasitic bacterium Pasteuria penetrans. European research has paid much attention to resistance breeding and selection. The Mi gene of tomato is widely used but resistance-breaking populations of M. incognita and M. javanica have been reported in different countries.

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.

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.

Damage caused to entomopathogenic nematodes by spray application is generally assessed by observing the viability of the infective juveniles under the microscope. To improve the quality and speed of this observation we developed an image processing technique and tested the efficacy of acetic acid and sodium chloride as chemical stimulants. Because of the lower standard error on the results obtained (0.7 vs 1.7), sodium chloride was eventually selected for all subsequent observations. The viability as observed with the image processing technique rose significantly with the time after the nematodes were suspended in water; however, viability as observed under the microscope was not influenced by the time. These differences can be attributed to the difference in type of stimulant (mechanical vs chemical) used. After nematodes had been in suspension for 3.5 h, the viability as measured using the image processing system was still significantly lower than the viability as measured under the microscope. This difference did not disappear after 24 h at 4, 15 or 24°C. Maintaining nematodes for 24 h at 35°C significantly decreased the viability to 5.9% (microscope) or 11.0% (image processing technique). The decrease in viability as observed with the image processing system corresponded better with the decrease in infectivity (i.e., 13.8%). Our results support further use of the image processing technique, not only to observe the viability of entomopathogenic nematodes but also to count the mobile or total number of nematodes of any species.

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.

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.

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.

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.

In the Belgian provinces Antwerp and Limburg, the root-knot nematode, Meloidogyne chitwoodi, is widespread. These regions are important production areas of carrot, Daucus carota, for the processing industry. Nineteen carrot cultivars were screened for potential resistance for M. chitwoodi. Egg masses of M. chitwoodi were found on all cultivars. However, there were no egg masses on more than 80% of plants of cvs Berlanda, Bolero, Chantenay, Nantucket and Parmex. By contrast, on cvs ABK, Douceur, Maxi and Merida egg masses were formed on all of the tested plants. To gain information about the damage caused by M. chitwoodi, carrots were grown in soil infected with different densities of nematodes. There was no effect of M. chitwoodi on the length, width and weight of the carrot taproot. Damage caused by M. chitwoodi was manifested by severe galling near the lenticels. Inoculation of nematodes 6 weeks after the carrots emerged resulted in a higher percentage of infected carrots and damaged taproots compared with earlier inoculation times. The effect of the time of harvest on nematode infection and damage was examined. When harvested 100 days after sowing in soil with low nematode densities (two second-stage juveniles (J2)/100 g soil), no damage was reported. Harvesting 120 and 140 days after sowing resulted in 10 and 20% damaged carrots, respectively. With initial M. chitwoodi densities of 25 J2/100 g soil, the percentage of damaged taproots increased from 10% when harvested 100 days after sowing to 70% when harvested 140 days after sowing. In a field trial, 11.5% of the carrots were damaged after a field period of 139 days and the initial M. chitwoodi population increased from 3 to 111 J2/100 g soil. It is recommended that growing carrots in M. chitwoodi-infested fields should be avoided. However, damage can be limited in fields with low initial nematode populations when the growing period is reduced.

Abstract The influence of temperature and root diffusates from different plants on the hatching behaviour of Pratylenchus penetrans was studied. Diffusates were obtained from maize (cv. Husar), carrot (cv. Masei), black salsify (cv. Omega), pea (cv. Alouette), bean (cv. Polder) and marigold (cv. Single Gold) at different plant ages. Soil leachate was used as a control treatment. Hatching was also examined in 10% dilutions of the root diffusates and the soil leachate. Hatching was monitored at 5-day intervals until 60 days at most. Test solutions were refreshed at the same intervals. Pratylenchus penetrans hatched more at 20°C than at 10, 15 or 25°C. Hatching was stimulated by all host diffusates, as well as marigold; it was highest in maize and bean diffusates (ca 50%). Except for carrot diffusate and soil leachate, hatching activity decreased with increasing plant age. It also decreased in 10% diluted root diffusates, except for pea, black salsify and soil leachate. There was no evidence for the presence of hatching inhibitors in the diffusate of any host.

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.

Abstract The influence of both host and temperature on the vertical migration of Pratylenchus penetrans was examined in a series of experiments using maize, carrot, black salsify, pea, bean and potato. Plants were grown in a pot placed on top of a plastic pipe of variable length in a growth chamber. This pipe was planted into a second pot to maintain the humidity in the pipe. Nematodes were inoculated at the bottom of the pipe and were allowed to migrate upwards for 14 days after which the pipe was cut into 5 cm sectors from which the nematodes were extracted and counted. More nematodes migrated at 21°C than at 16 or 11°C. Migration of P. penetrans towards a host decreased with increasing distance between the nematodes and all hosts, with maize and bean eliciting the most attraction. They were the only hosts able to stimulate nematode migration up to the distal end of 31 cm pipes. Potato and carrot were the least influential hosts. Hosts differently influenced the migration only of second-stage juveniles (J2), with a greater percentage of J2 moving under maize and bean than under carrot. Under all hosts, adults migrated in greater proportions with increasing distance than did other stages. The attractiveness of hosts was age dependent. Generally, infectivity decreased with distance travelled by the nematode.

Abstract The pine wood nematode (PWN), Bursaphelenchus xylophilus, which in China and East Asia is vectored by Monochamus alternatus, is the causal agent of pine wilt disease of Japanese black pine, Pinus thunbergii. The vector emergence and the transmission of nematodes through vector feeding on pine twigs were monitored during 2004 and 2005 in Nanjing, China. The emergence started from late April to the end of June and peaked from late May to early June. There were 438 and 927 adults collected in 2004 and 2005, respectively, and approximately 70% of the beetles emerged during the peak period. Visual estimation of the nematode burden on vectors by observation of the atrium of the first abdominal spiracle gave unreliable information. The percentage of beetles carrying PWN of the total number of emerged beetles was between 20 and 30%. Seventy percent of the nematodes were released from beetle cadavers after 3 days of extraction. The sex and longevity of the beetle had no significant relationship with the number of nematodes that remained in cadavers. Transmission of nematodes into pine twigs through beetle feeding started 10 days after emergence of the vector. The period of nematode transmission could last for up to 79 days after beetle emergence. Two types of nematode-transmission curve were found by measuring the number of nematodes transmitted into pine twigs per day. The unimodal pattern peaked between 3 and 6 weeks after adult emergence; the bimodal pattern had two transmission peaks: one between 2 and 3 weeks, and another between 5 to 7 weeks after beetle emergence.

Heterodera sacchari, H. leuceilyma and H. goldeni are closely related members of the H. sacchari species complex, which is mainly characterised and distinguished from all other described Heterodera species by the presence of finger-like projections of the strongly developed underbridge in the vulval cone of the cysts. Males are rare in all three species and are described here in H. goldeni for the first time. Reproduction appears to be parthenogenetic. There are only minor morphological distinctions between the three species, particularly after our present studies have emended their original descriptions from various populations. Heterodera sacchari and H. goldeni showed differences in the ITS-rRNA gene sequences. Heterodera sacchari was described and reliably identified from many tropical African countries, H. leuceilyma is known only from Florida, USA, and H. goldeni has been identified in Egypt, Israel and Iran. All three species have grasses and other Poaceae as hosts, H. sacchari commonly attacking rice and sugarcane, and H. goldeni reproducing successfully on sugarcane ratoon seedlings. Morphological data emending the descriptions of H. sacchari, H. goldeni and H. leuceilyma from various populations are presented and discussed along with their host and distribution. Molecular characterisation of H. sacchari and H. goldeni is provided. An analysis of phylogenetic relationships within species of the sacchari-group using ITS-rRNA gene sequences is also presented.

The root-knot nematodes, Meloidogyne chitwoodi and M. fallax, cause severe damage to economically important crops and, in 1998, both species were listed as quarantine pests in Europe. Comparative studies were made on the effects of root diffusates and host age on the in vitro hatching of M. chitwoodi and M. fallax. There is a marked contrast in the hatching response of the two species. Hatching of second-stage juveniles (J2) of M. chitwoodi produced on young plants did not require host root diffusate stimulus, whereas at the end of the plant growing season, egg masses contained a percentage of unhatched J2 that require host root diffusate to cause hatch. This form of obligate quiescence at the end of the host growing season was not found in M. fallax. This species hatched well in water and did not require hatch stimulation from root diffusate, irrespective of the age of the plant on which the egg masses were produced. The number of eggs per egg mass for M. fallax collected on senescing plants was significantly greater than the number of eggs per egg mass for M. chitwoodi. The number of eggs per egg mass of M. chitwoodi decreased with plant age. The results are discussed in the context of the differing survival strategies of the two species.

Abstract The reproduction and pathogenicity of Helicotylenchus multicinctus, Meloidogyne incognita and Pratylenchus coffeae was studied on Musa AAA cv. Grande Naine pot plants and microplot plants. First, separate experiments using different inoculation numbers (between 218 and 4144) and different exposure times (between 8 and 22 weeks) were executed in pots for each nematode species. Helicotylenchus multicinctus suppressed root growth by 13%, comparing inoculated with uninoculated plants. In contrast, M. incognita stimulated root growth by 6.7%, while P. coffeae had no effect on root growth. The interaction between R. similis and each of these three nematode species was investigated in a concomitancy experiment in pots. Plants without concomitant species yielded the highest R. similis population densities but did not differ from plants previously inoculated with H. multicinctus and P. coffeae. Meloidogyne incognita was the only concomitant species that reduced R. similis population densities. Inoculation with R. similis affected the recovered numbers of P. coffeae albeit with large variability. In the microplot experiment, all four nematode species except H. multicinctus reduced the bunch weight of bananas, compared to uninoculated plants. Pratylenchus coffeae and R. similis increased root damage by 129 and 262%, respectively, while only the latter species suppressed root weight with 66%.

Abstract The virulence of the entomopathogenic nematodes Steinernema glaseri Belgian strain and S. scarabaei against different development stages of the European cockchafer Melolontha melolontha, Hoplia philanthus and Serica brunnea was compared in the laboratory. The virulence of S. glaseri did not differ among first, second and third instars of M. melolontha, H. philanthus or S. brunnea. However, the virulence of S. scarabaei to second and third instars of M. melolontha was greater than to first instars of this insect species, but did not differ significantly between second and third instars of H. philanthus. Our observations, combined with those of previous studies on other nematode and white grub species, show that nematode virulence against white grub developmental stages varies with white grub and nematodes species.

The effect of a sequence of host and non-host crops on the population dynamics of Pratylenchus penetrans was monitored for 2 years in a field. The sequence was pea (Pisum sativum, 25% cv. Colombia and 75% cv. Coca), French marigolds (Tagetes patula nana cv. Sparky); fallow; carrot (Daucus carota, cv. Amsterdamse bak); bean (Phaseolus vulgaris, cv. Avignon) and again fallow. Samples were taken every 21 days during the marigold crop and following fallow, and at planting and harvest for the other crops. From five 2 × 5 m2 plots, 15 cores were taken per plot to a depth of 70 cm and split into seven segments of 10 cm each. Within the plots, segments from corresponding depths were pooled and nematodes were estimated from a 200 g subsample. Densities of P. penetrans increased 2.2-fold under pea, but decreased by 90% after marigolds had been grown for 105 days. The P. penetrans population was reduced by about 90% in the soil layer above 40 cm while the reduction was about 80% below 50 cm after 105 days of marigold culture. Densities of P. penetrans declined exponentially in time under marigold and fallow. The effect of marigold was persistent as no increase in P. penetrans densities over the whole soil profile was noticed after two crop cycles of host plants.

A new species of Steinernema (Rhabditida), S. hebeiense sp. n. was isolated from sandy soil, Hebei Province, China. Diagnostic characters of infective juveniles of S. hebeiense sp. n. include total body length of 658 (610-710) μm, distance from anterior end to excretory pore of 48 (43-51) μm, tail length of 66 (63-71) μm, and E% ratio of 72 (65-80). The lateral field has eight ridges at the mid-body region (two very thin submarginal ridges, poorly discernible under light microscope, but visible in SEM); and the anterior end is slightly offset and flattened. First generation males have a body length of 1177 (1036-1450) μm, spicule length = 57 (51-63) μm, and gubernaculum length = 46 (38-50) μm. The spicules are light brown in colour and slightly curved; the manubrium is longer than wide. A tail mucron is present in second generation males. These morphometrical and morphometric features classify S. hebeiense sp. n. within the ' feltiae-kraussei-oregonense ' group. Molecular data also classify the new species within this group. Within this group, the smallest differences between the sequences of S. hebeiense sp. n. is with S. weiseri – 73 bp in the ITS rDNA and 13 bp in the studied partial sequence of the D2D3 expansion segment of LSU rDNA.

Abstract The Longidoridae are a group of ectoparasitic nematodes including two subfamilies and six genera with hundreds of species. Sequences of the D2 and D3 expansion region of the large subunit (LSU) rRNA nuclear gene were amplified and used to reconstruct the phylogeny of longidorids. Phylogenetic analyses with maximum parsimony (MP), maximum likelihood (ML) and Bayesian inference (BI) were performed with one outgroup taxon and 62 longidorid sequences. Confidence of inferred clades was assessed by non-parametric bootstrapping for MP and Bayesian posterior probability for ML. All analyses placed Paralongidorus species as an inner group within the otherwise monophyletic genus Longidorus. The genus Xiphinema, except for X. americanum-group species, was placed as the sister group of Longidorus with strong support from the ML and BI analyses. The X. americanum-group was strongly supported as an exclusive clade to other genus Xiphinema species. The position of the Xiphidorus clade was not well resolved and the phylogenetic analyses did not support it as a sister group to Longidorus as previously inferred from morphology. Secondary structure models were constructed for the D2/D3 region of LSU rRNA for all studied species. It was found that sequence-based and structural morphometric rRNA phylogenies were incongruent.

The suborder Criconematina is a large group of ecto- and endoparasitic nematodes, including several species of major agricultural importance. The D2-D3 expansion segments of the 28S nuclear ribosomal RNA gene were amplified and sequenced from 23 nominal and six unidentified species from the genera Mesocriconema, Criconemoides, Ogma, Criconema, Xenocriconemella, Hemicriconemoides, Hemicycliophora, Paratylenchus, Tylenchulus, Trophonema and Sphaeronema, together with outgroup taxa from Tylenchidae (Aglenchus) and Atylenchidae (Eutylenchus). A sequence alignment optimised using the secondary structure model was analysed using maximum parsimony, maximum likelihood and Bayesian inference approaches under two models. All analyses yielded a similar topology with differences primarily in the position of poorly supported clades. Although some molecular trees differ from the previous morphologically based hypotheses of criconematid phylogeny, maximum likelihood tests did not yield statistically significant differences between some of the tested classical morphological and molecular topologies. DNA data support monophyly for the genera Mesocriconema, Hemicriconemoides and Criconema and reject the hypothesis of a single origin of criconematids with a cuticular sheath or 'double cuticle'. Application of the complex model of rRNA evolution, considering paired nucleotides for the stem and unpaired nucleotides for the loop region, resulted in a majority rule consensus Bayesian tree with unresolved relationships between main clades. This lack of resolution is expected by the low number of independently evolving nucleotides. Sequence divergence in this DNA segment between populations of Mesocriconema xenoplax, M. sphaerocephalum and Hemicriconemoides cocophillus suggest the presence of several sibling species under these taxa names.

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 Eighty four new and four published ITS1-5.8S-ITS2 sequences of rDNA obtained from different populations of 24 nominal species and 28 isolates of 16 putative Steinernema species were analysed using the maximum parsimony method. In most of the phylogenetic trees obtained from different ITS alignments and phylogenetic procedures, the 84 isolates formed five main, highly or moderately supported, clades, viz Clade I: 'affine-intermedium'; Clade II: 'carpocapsae-scapterisci-tami'; Clade III: 'feltiae-krausseioregonense'; Clade IV: 'bicornutum-ceratophorum-riobrave'; and Clade V: 'arenarium-glaseri-karii-longicaudum'. The ITS rDNA data were found to be of little utility in resolving relationships between these clades, but were useful in studying relationships between species within certain clades. The level of intra-specific variability was different between clades with sequence divergence of 2.4-2.8% of ITS rDNA for some species. Analysis of a combined data matrix of both molecular and morphological features was performed with six qualitative and three quantitative features.

Isoelectrofocusing of superoxide dismutase (SOD) isoforms was carried out on the extracts of 117 nematode populations belonging to the so-called Xiphinema americanum-group. These populations came from the USA (77), Chile (5), Argentina (1), Venezuela (5), Portugal (15), Italy (2), Crete (1), Montenegro (1), Slovakia (4), Hungary (3), Egypt (1) and India (2). A total of 17 bands of enzyme activity were observed in the screening, whilst single enzyme phenotypes showed from two to eight bands. The high degree of SOD polymorphism of this nematode collection was grouped by cluster analysis into seven distinct homogeneous groups characterised by specific combinations of SOD markers. Sub-groups could be discriminated for larger groups. The small Groups 3 and 5 were constituted mostly by populations from USA east coast states (i.e., NY and PA, respectively). The larger Group 1 resulted from the association of populations coming from various and distant North American States. In other large groups, North American populations were associated with South American and European populations. Overall, the data presented here suggest that geographic separation and different hosts do not seem to be the source of genetic diversity for the X. americanum-group. When an adequate number of populations were collected from the same country, the variability expressed by such a sub-sample was comparable to that of the whole nematode collection. For the first time, homogeneous populations of a large collection of X. americanum-group populations were associated by molecular means in order to explore further approaches that may help resolve the recalcitrant taxonomy and phylogeny of this much debated group.

A survey of entomopathogenic nematodes in Vietnam yielded several Heterorhabditis isolates. The majority belonged to H. indica; their morphometrics are given in this paper. Three isolates collected in forests in Backan, Ninhbinh and Kontum provinces, Vietnam are here described as a new species Heterorhabditis baujardi sp. n. The new species is distinguished from the other ten valid Heterorhabditis species by a combination of morphological, morphometrical, and DNA characters. Heterorhabditis baujardi sp. n. is morphometrically similar to H. indica, but can be separated from this species by the shape of the gubernaculum and the number of normal pairs of genital papillae. The gubernaculum of H. baujardi sp. n. with the proximal end ventrally curved resembles that of H. bacteriophora. Heterorhabditis baujardi sp. n. can be separated from this latter species by a shorter body length of infective juveniles and longer spicules, longer gubernaculum, and a higher spicule length to anal body diameter ratio of males. The canonical discriminant analysis of morphometrical characters of both infective juveniles and males failed to discriminate Heterorhabditis baujardi sp. n. from H. indica. However, the new species was easily distinguished from H. downesi, H. marelatus, H. megidis and H. bacteriophora. Heterorhabditis baujardi sp. n. was slightly separated from H. bacteriophora by variables of the infective juveniles, but was clearly distinguished by variables of the males. Cross-breeding tests using isolates of the new species and H.indica did not yield fertile progeny. Analysis of the ITS1 sequence of rDNA of H. baujardi sp. n. revealed substantial differences with other known ITS1 Heterorhabditis sequences. Phylogenetic relationships between Heterorhabditis species and the usefulness of morphological and molecular characters for identification of species from this group are discussed.

Abstract A new species of the genus Radopholus associated with durian (Durio zibetinus M.) in the Western Highland of Vietnam is described as Radopholus duriophilus sp. n. The new species is close to R. similis, but is distinguished from R. similis by the position of the excretory pore located posterior to pharyngo-intestine junction (vs at level of pharyngo-intestine junction), oval shape sperm (vs rod-like), four incisures terminating far behind position of phasmid (vs three incisures terminating at or just behind phasmid), and bursa in male never reaching tail terminus (vs bursa reaching tail terminus). Females of R. duriophilus sp. n. differ from R. nativus females by stylet length (16.5-19 vs 19-23 μm), oval or kidney-shaped sperm (vs rod-like), four incisures at level of phasmid (vs three) and their areolated lateral field (vs not areolated). The position of excretory pore of both female and male is located posterior to pharyngo-intestine junction (vs at level or anterior to pharyngo-intestine junction). Females of R. duriophilus sp. n. differ from R. clarus females by stylet length (16.5-19 vs 19-21 μm) and areolated lateral field (vs no areolation). Females of R. duriophilus sp. n. differ from R. musicola females by their lateral field with equidistant incisures at mid-body (vs two deep outer folds and two faint shallow inner incisures), oval or kidney-shaped sperm (vs rod-like), and rounded terminus tail (vs sharply pointed). The species also sp. n. differ in male stylet length (11.5-15 vs 8.8-12 μm). Females of R. duriophilus sp. n. differ from R. bridgei females by stylet length (16.5-19 vs 15-17.5 μm), median bulb length (11-16.5 vs 11-13 μm), length of hyaline tail (3-11 vs not more than 4 μm), and lateral field areolated for entire body (vs not areolated except irregularly on neck and tail). The male differs by stylet length (11.5-15 vs 10-12 μm) and length of the hyaline portion (4-9 vs 1-4 μm). In addition, the relatively high level of ITS sequence divergence of the new species from R. similis populations and the presence of nucleotide autapomorphies support a separate specific status for these durian populations. Results of surveys revealed that R. duriophilus sp. n. is rather widely distributed in durian orchards and associated with decline and death of trees in many durian nursery gardens. Densities of nematode population reached thousands of individuals per g of root samples.

Abstract RFLP and sequences of ITS-rDNA of 45 populations of cyst-forming nematodes collected from different parts of Iran were analysed and identified as representatives of 21 species. Eight enzymes generated RFLP for all studied populations. Comparison of RFLP profiles and sequences of the ITS regions with published data confirmed the presence of Heterodera avenae, H. filipjevi, H. glycines, H. hordecalis, H. latipons, H. schachtii and H. trifolii in Iran. RFLP patterns and ITS sequences for H. elachista, H. turcomanica, H. mothi and C. cacti were obtained for the first time in this study. Heterodera humuli, H. goettingiana, H. fici, H. elachista, H. turcomanica and Cactodera cacti are recorded for the first time in Iran. These results correspond with morphological and morphometric identification of the populations. Several populations were not identified at the species level and are attributed to Heterodera sp.; some of these may correspond to new species. Twenty-one new sequences from Iranian cyst-forming nematodes and 36 known sequences were used for the phylogenetic analyses. The cyst-forming nematodes formed several clades corresponding to their morphological features. Heterodera mothi and H. elachista clustered with high support with other Cyperi group species and H. turcomanica formed a moderately to highly supported clade with the Humuli group.

Plant-parasitic nematodes are divided according to their feeding strategy into three major groups: sedentary endoparasites, migratory endoparasites and ectoparasites. Compared to what is known about sedentary endoparasitic nematode species, resistant and tolerant relationships between the nematodes from the latter two groups and their hosts are much less documented. However, methods for screening and evaluation of the resistance and tolerance of plants to migratory plant-parasitic nematodes have been well developed and sources of resistance and tolerance to these nematodes have been found. Advances have been made in breeding resistance to migratory plant-parasitic nematodes in rice, alfalfa, banana, pine trees, grape, woody fruits and other crops. Although accessions immune to stem, leaf and bud nematodes are found quite frequently, host resistance to migratory root-parasitic nematodes has been detected less frequently and generally only partly reduces nematode multiplication. Host tolerance to migratory nematodes is important even for resistant varieties and therefore is gaining attention. An insufficient degree of resistance and tolerance, their variability with the environment, and their linkage to undesired agricultural or horticultural characters are commonly observed. Polygenic bases for plant resistance and tolerance to migratory nematodes have been demonstrated by genetic and biochemical observations and make breeding even more complicated than that for resistance to sedentary plant-parasitic nematodes. These factors, with the presence of different nematode species in the field and community and population differences in pathogenicity, hinder the availability of host resistance and tolerance and offer a big challenge.

Abstract A short insert genomic library for Xiphinema index, the natural vector of Grapevine Fanleaf Virus, was constructed from degenerate oligonucleotide primed PCR (DOP-PCR) products. The genomic library was screened for (CA)n microsatellites. Screening of 6200 colonies and comparison of the sequencing results revealed seven (CA)n containing microsatellites, coded here as XIMSL1, XIMSL2, XIMSL3, XIMSL4, XIMSL5, XIMSL6 and XIMSS1. XIMSL prefixed microsatellites were followed by the motif of the same long interspersed element. Microsatellite XIMSS1 has some similarity to the short interspersed element. Except for XIMSL1, all microsatellites were proven to be effective diagnostic tools at species level. Genetic diversity between and within populations was also evaluated for each microsatellite.

When feeding, Xiphinema index induces remarkable cellular modifications at the feeding site in the root tips of its hosts, similar to those induced by root endoparasites such as Meloidogyne spp. The large size of X. index enables direct analysis of their proteins. Following immunisation of a mouse with macerated extracts of X. index bodies, a panel of monoclonal antibodies (MAbs) was obtained against this nematode. The MAbs were used in immunolocalisation studies and antibodies recognising epitopes present at the surface coat, inner cuticle, dorsal gland cell and dorsal duct, body wall muscle fibres, nervous system and reproductive system were identified. These antibodies provide useful tools for studying plant-nematode interactions.

Abstract Species of the Heterodera avenae complex, including populations of H. arenaria, H. aucklandica, H. australis, H. avenae, H. filipjevi, H. mani, H. pratensis and H. ustinovi, obtained from different regions of the world were analysed with PCR-RFLP and sequencing of the ITS-rDNA, RAPD and light microscopy. Phylogenetic relationships between species and populations of the H. avenae complex as inferred from analyses of 70 sequences of the ITS region and of 237 RAPD markers revealed that the cereal cyst nematode H. avenae is a paraphyletic taxon. The taxonomic status of the Australian cereal cyst nematode H. australis based on sequences of the ITS-rDNA and RAPD data is confirmed. Morphometrical and ITS-rDNA sequence analyses revealed that the Chinese cereal cyst nematode is different from other H. avenae populations infecting cereals and is related to H. pratensis. Bidera riparia Kazachenko, 1993 is transferred to the genus Heterodera as H. riparia (Kazachenko, 1993) comb. n. As a consequence, H. riparia Subbotin, Sturhan, Waeyenberge & Moens, 1997 becomes a junior secondary homonym and is renamed as H. ripae nom. nov. Morphological, morphometrical characters and RFLP profiles for identification of the nine species presently placed in the H. avenae species complex are given.

A method for screening the tolerance of Rosa rootstocks and species to Pratylenchus penetrans was developed after observing the effects of nutrition and initial nematode population density on damage to Rosa corymbifera cv. Laxa. The correlation between initial P.penetrans densities and the weight of host plants fitted (R2 > 0.90) the Seinhorst model, Y = ym for Pi ≤ T, and Y = ym · m + ym(1 – m)z(Pi–T). All inoculated plants of R. virginiana and R. eglanteria survived while there were differences in the mortality after inoculation of 12 other Rosa rootstocks or species. Pratylenchus penetrans caused significant reductions in the numbers of the leaves and roots, and in fresh weights of shoots, roots and whole plants of all 14 accessions. Among these species and cultivars, reductions in root number were least for R. virginiana whereas fresh weights of roots, shoots and whole plants were least affected for R. eglanteria, and the greatest reductions were of R. corymbifera cv. Laxa and R. glauca. Plant mortality and the reduction of shoot and plant weights of surviving plants were positively correlated with the reduction in numbers of secondary roots. The numbers of secondary roots at inoculation were negatively correlated with the degree by which plant weight was reduced and with plant mortality observed 50 days later. Differences in tolerance among species as observed by differences in reduction of plant growth parameters were confirmed by differences of the parameters derived from applying the Seinhorst model to the species.

A method for screening Rosa accessions for their host suitability to Pratylenchus penetrans was developed. The best conditions were obtained in growth chambers with plants grown in sand, inoculated with 250 P.penetrans per 50 cm3 pot and fertilised weekly with 0.75 g/dm3 soluble fertiliser (containing 20% N, 20% P2O5, 20% K2O and balanced trace elements). Fifty days after nematode inoculation, these conditions permitted greater nematode multiplication than any other combination tested and also allowed good host development. When used for the screening of 21 Rosa accessions, these conditions revealed a large variation in host suitability. Least multiplication (Pf/Pi) was observed on R. virginiana (1.36) but this did not differ significantly from that on R. multiflora (2.87). The greatest Pf/Pi was on R. canina cv. Superba but this did not differ significantly from that on R. canina cv. Pollmeriana. The correlation of Pf/Pi with the nematode population and the number of eggs within the roots was significant; the percentage of nematodes outside roots was negatively correlated with Pf/Pi. The intermediate host status of R. corymbifera cv. Laxa, one of the most common rootstocks, was confirmed. Differences in host status became statistically significant when intra-accession variation was observed with a larger number of plants.

Two new species of Steinernema (Rhabditida), Steinernema loci sp. n. and S. thanhi sp. n., were isolated from beach soil in Thanhhoa and Hatinh provinces in the northern part of Vietnam. A combination of morphological, morphometric and rDNA-RFLP features indicated the distinctness of both species from other Steinernema spp. Diagnostic characters of Steinernema loci sp. n. include: total body length (896-1072 μm), distance from anterior end to excretory pore (EP = 71-86 μm), tail length (66-83 μm), lateral field in mid-body with eight ridges (nine distinct lines) and rounded, broad and smooth anterior end of the third stage infective juvenile; first generation males were characterised by body width, spicule length and the ratio spicule length : anal body width (SW). Diagnostic characters of Steinernema thanhi sp. n. third stage infective juveniles include: total body length (720-960 μm), EP (68-84 μm), tail length (52-72 μm), lateral field in mid-body with eight ridges (nine distinct lines) and rounded, broad and smooth anterior end. Spicule length, ratio SW and arrangement of genital papillae characterised the first generation males.