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Root-lesion nematodes of the genus Pratylenchus are among the most important nematode pests that limit production of small-grain cereals. Four Pratylenchus species, viz. P. thornei, P. neglectus, P. penetrans, and P. crenatus, are considered of major economic significance in cereals of which P. thornei and P. neglectus are the most important and widely distributed species in cereal crops worldwide. However, P. thornei is more destructive causing estimated yield losses of up to 50% in the USA and 85% in Australia. This paper provides information regarding the global distribution of Pratylenchus species, yield loss due to their attack, their biology and pathogenic relation to plants, the research cutting edges in nematode identification of different Pratylenchus species, and their control through cultural practices and resistant varieties as correct identification of root-lesion nematodes can be difficult to achieve, particularly if a quick diagnosis is needed. In this context, in recent years, several molecular techniques for these Pratylenchus species have been developed such as quantitative PCR assays which are able to produce precise and rapid identification of several root-lesion nematodes species. So far, many global attempts have been made to control root-lesion nematodes in cereals, including cultural practices and development of resistant varieties. The use of resistant accessions is considered the most economically feasible and environmentally sustainable method. Resistance genes in several lines have been identified and are being used in numerous breeding programmes against root-lesion nematodes species.

The root lesion nematodes Pratylenchus penetrans and P. thornei cause high yield losses in rain-fed wheat fields in Morocco, as well as worldwide. Growing resistant varieties is one of the most effective methods for controlling nematodes. Therefore, a collection of 14 lines of spring wheat and 11 lines of winter wheat (Triticum aestivum and T. durum), provided by CIMMYT, were screened for resistance to P. penetrans and P. thornei in tubes (15 × 20 × 120 mm3) under greenhouse conditions. The resistance level was evaluated based on the numbers of nematodes extracted from roots and soil 9 weeks after infestation. Three lines (L9, L12 and L13) were found moderately resistant (reproduction factor

Morphological and molecular diversity among 11 populations of cereal cyst nematodes from different wheat production areas in Morocco was investigated using light microscopy, species-specific primers, complemented by the ITS-rDNA sequences. Morphometrics of cysts and second-stage juveniles (J2s) were generally within the expected ranges forHeterodera avenae; only the isolate from Aïn Jmaa showed morphometrics conforming to those ofH. latipons. When using species-specific primers forH. avenaeandH. latipons, the specific bands of 109 bp and 204 bp, respectively, confirmed the morphological identification. In addition, the internal transcribed spacer (ITS) regions were sequenced to study the diversity of the 11 populations. These sequences were compared with those ofHeteroderaspecies available in the GenBank database (www.ncbi.nlm.nih.gov) and confirmed again the identity of the species. Ten sequences of the ITS-rDNA were similar (99–100%) to the sequences ofH. avenaepublished in GenBank and three sequences, corresponding with one population, were similar (97–99%) toH. latipons.

Small grain cereals, such as wheat, barley and oats are considered among the most important food sources. Plant-parasitic nematodes play a considerable role in decreasing cereal yields. The three-major species of cereal cyst nematodes (CCN) Heterodera avenae, H. latipons, and H. filipjevi are distributed worldwide and cause considerable damage. This review provides information regarding the global distribution of these nematode species, yield loss due to CCN, their biology and pathogenic relation to plants, identification and control through agricultural practices, biological agents and resistance breeding. As morphological identification of CCN is difficult and time-consuming, several molecular techniques for the identification of these CCN species have been developed in recent years. The restrictions on the use of nematicides demand for resistance to CCN. Resistance genes in several lines are known and are used in numerous breeding programmes against CCN; pyramiding these resistance genes into high yielding cultivars that could become commercially available for farmers is progressing.

Potato cyst nematodes (PCN), Globodera rostochiensis and G. pallida, are major constraints to potato crop production. We studied the effects of several soil amendments on PCN survival and reproduction in pot experiments. Pig slurry, cattle slurry, mineral nitrogen fertilizer (NH4NO3), crab shell compost and wood chip compost at 170 kg N ha−1 reduced the number of viable eggs in cysts of both PCN species in the absence of potato. This resulted in fewer second-stage juveniles (J2) hatching from these cysts and penetrating potato roots than from cysts of non-amended soils. When potato was planted, the same amendments resulted in less reproduction than in non-amended soil. Most reduction of reproduction was achieved in soils amended with pig slurry (87%) and wood chip compost (82%). Adding biochar at 0.3 and 1% did not reduce the survival or the reproduction of any of PCN species; moreover, it inhibited the suppressing effect of wood chip compost and pig slurry on PCN reproduction when added together with these amendments. The release of ammonium and changes in soil microbial community, determined by phospholipid fatty acid (PLFA) analysis, are involved in nematode suppression in soil amended with pig and cattle slurries. However, the suppressing effect of wood chip and crab shell compost can only be explained by the changes in soil microbiota, while the effect of mineral nitrogen fertilizer can only be related with the production of ammonium. Ammonium and microorganisms most probably have affected PCN directly by killing the eggs and juveniles or indirectly by changing the physiology of the root as mentioned amendments reduced hatch and movement of J2, penetration of the roots and females’ fecundity.

Three experiments were conducted to find out the feeding type and age structure of nematodes in different soil habitats; to know the efficiency of different extraction techniques of nematodes and to observe the hatching behavior of Meloidogyne juvenile in a Baermann device. Bacterivore, Omnivore, Predator, Fungivore and Plant Parasitic nematodes were recorded in the soil of flower, vegetable, moss, grass, and corn and compost habitats. Bacterivore was the highest feeder (67.5%) in the soil habitats. In comparison of nematode extraction methods, Seinhorst method extracted more cysts (188), but less juvenile (34) and eggs (18) from cysts than stirring (145, 87 and 50, respectively) while AZC was found superior (936) over Cobb method (204) in extracting nematodes. The AZC method also extracted huge juvenile number from roots which was higher than Baermann funnel method. In Baermann funnel, juvenile hatched more in favorable condition than unfavorable condition which led to an increasing and decreasing pattern of hatching.

In a 3-step approach, three series of experiments were carried out to assess the virulence and environmentally-dependent biocontrol potential of entomopathogenic nematodes from Cameroon against two cassava insect pests, Stictococcus vayssierei and its trophobiotic ant, Anoplolepis tenella, under laboratory and screenhouse conditions. In the first series, 20 isolates (18 Heterorhabditis baujardi, 1 Steinernema cameroonense, and 1 S. nyetense) were separately checked for their ability to infect the two pests at a rate of 500 infective juveniles (IJs)/insect for 36 h, and to select the most virulent isolates. All isolates were pathogenic to both pests, and caused 10% to 90% and 0 to 63% mortality of S. vayssierei and A. tenella, respectively. In a second series of virulence bioassays, 2 sets of 4 selected isolates were examined for: (a) concentration-response, (b) time-response, and (c) reproduction. Stictococcus vayssierei mortality ranging from 56 to 62% were observed at doses of 75–100 IJs/insect within 24–36 h. Nematodes at doses of 100–500 IJs/insect caused 51% mortality within 36 h. Nematodes successfully reproduced in both pests. The third bioassay series investigated migration and host preference abilities of selected isolates as well as nematode attachment to ants. The dispersal of IJs as affected by soil moisture and soil type was studied under screenhouse conditions. Nematodes exhibited positive attraction to both pests, yet with no preferential movement of IJs. Nematode dispersal and persistence (up to 89 days) were favored in sandy clay loam soils at pF 3.5 and pF 4.2 (9–15% moistures). The results from this study are sufficiently encouraging to embark on in situ testing. For integrated S. vayssierei control, implications such as the development of an adequate field application method to be used at field conditions are discussed, as even moderate levels of control might be economically profitable.

Integrated management of potato cyst nematodes (PCN; Globodera rostochiensis and G. pallida) relies heavily on the determination of cyst population densities in soil as well as the viability of the eggs inside the cysts. This study aimed to optimize a quantitative method to determine the number of viable eggs of PCN based on trehalose present in live eggs. Trehalose was extracted from cysts and from a dilution series of eggs and quantified. More trehalose was detected when cysts were crushed than when left intact. Reaction volumes were adapted to the number of eggs because small reaction volumes hampered an accurate extraction of trehalose. A maximum of 10.5 eggs/μl of reaction volume should be used to obtain a significant linear relationship between detected trehalose content and egg numbers. The sensitivity of the trehalose-based method was evaluated by determining the lowest egg detection limit and was defined as five viable eggs. The reliability of this method was tested by comparing efficacy with that of two commonly used assays, visual assessment and hatching test. The trehalose-based method gave viability results similar to those of the visual assessment, which is time consuming, requires trained personnel, and can involve some subjectivity. The hatching test identified fewer viable eggs than the other two methods. In addition, the viability of dead eggs (heated and naturally dead) was tested. No false-positive results (dead eggs declared viable) were obtained with the trehalose-based method. The robustness of the test was demonstrated by measuring the viability of eggs of PCN in different experiments repeated in time. The viability assessment method based on trehalose proved to be an objective as well as sensitive, reliable, robust, fast, and cheap technique for assessing the number of viable eggs in PCN cysts.

Potato cyst nematodes (PCN), Globodera rostochiensis and G. pallida, are major constraints to potato production. They are distributed passively with soil adhering to farm machinery and harvested tubers, but also by waste soil from trading and processing plants. Inundation is considered an environmentally save method for disinfestation of waste soil. We investigated if agro-industrial waste products accelerate this disinfestation process by studying PCN survival, hatching and infectivity over time. Experiments were conducted in 2-L containers filled with 1.5 L non-amended soil, soil amended with steamed or fresh potato peels, calcium sulphate, steamed potato peels mixed with calcium sulphate, or leek leaves. In each container, cysts of G. rostochiensis or G. pallida were buried in the soil in retrievable bags and 400 ml water was added. Non-amended and non-inundated soils were the controls. It took 8 weeks of inundation of non-amended soil to reduce nematode survival by 72 %, while in amended soils survival was reduced up to 99.9 % after 4 weeks. After 1 week, fewer second-stage juveniles (J2) hatched and infected roots in inundated amended soil than in inundated non-amended soil. Host finding ability of surviving J2 was not affected. To understand mechanisms of nematode suppression, concentrations of short chain fatty acids (SCFA), gases (O2, CO2, H2S, CH4, and N2) and phospholipid fatty acids (PLFAs) were determined. Higher concentrations of SCFA and carbon dioxide and lower levels of oxygen were detected in amended treatments. H2S was occasionally detected in the leek-amended soil. The microbial biomass changed significantly in potato peels-amended soils.

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.

Cereal cyst nematodes (CCN) are important plant-parasitic nematodes of wheat and barley, and exist in most of the cereal growing regions of the world. As there is limited information on the nature and distribution of CCN species in Syria, a survey was conducted in north-eastern Syria to assess the distribution of CCN in the main wheat and barley growing areas. In the summer of 2009, a total of 167 composite soil samples were collected from 167 wheat and barley fields. Cysts were extracted from soil using the Fenwick can technique, then quantified and identified up to the species level by using both morphological and molecular methods. The study revealed that 62% of the fields were infested with the three Heterodera species: H. avenae, H. filipjevi and H. latipons. However, the most prevalent species was H. latipons, which was present in 76% of the infested samples, while it occurred alone in 67% of those samples. Heterodera avenae was detected singly in 20% of the samples, while 11% consisted of mixed populations with other species. Heterodera filipjevi was never detected alone; it was found mixed with H. avenae and/or H. latipons in 9% of the samples. The high number of extracted cysts (up to 116 cysts × 200 g−1 of soil) is most likely related to the monoculture practiced in that region and the fact that growers are unaware of the existence of resistant varieties.

Twelve Heterodera species are considered of major economic significance in cereals, of which Heterodera avenae, H. latipons and H. filipjevi are the most important. Precise identification and quantification of these nematodes are necessary to develop effective integrated pest control. This study reports on the use of the mitochondrial cytochrome oxidase subunit 1 (COI) gene to develop qPCR assays that could be used for the identification and quantification of H. avenae and H. latipons. Two qPCR primer sets, each comprising two primers and a probe, were designed for each of both species. After optimization, the qPCR assays using a single second-stage juvenile (J2) were able to identify and quantify H. avenae and H. latipons. Their specificity was confirmed by the lack of amplification of J2 of 14 other Heterodera species. A qPCR using DNA extracted from 120 J2 + eggs of H. avenae and H. latipons resulted in steady Ct-values (Ct = 22.33 ± 0.1 and Ct = 21.83 ± 0.12, respectively). Dilution series of DNA extracted from 120 J2 + eggs of the two species were made. The assays for both species resulted in a standard curve showing a highly significant linearity between the Ct-values and the dilution rates (R2 = 0.99; slope = −3.03 and R2 = 0.99; slope = −3.28 for H. avenae and H. latipons, respectively). The two qPCR assays provide a sensitive and valid tool for rapid detection and quantification of the two species whether they occur alone or in mixtures with other species.

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 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.

Twenty-nine Beninese isolates of Heterorhabditis sonorensis and one local isolate of H. indica were screened in bioassays for their pathogenicity against Macrotermes bellicosus and for their tolerance to heat (40 A degrees C), desiccation (25 % glycerine) and hypoxic conditions. The bioassays showed significant differences among isolates for all tested traits. Most of the isolates (73 %) killed more than 80 % of the insects. The greatest survival of infective juveniles to heat (8 h), desiccation (8 h), and hypoxia (72 h) was observed with the H. sonorensis isolates Kassehlo (72.8 %), Setto1 (72.5 %), and Kissamey (81.5 %, respectively). Hierarchical cluster analysis identified six clusters. One of the clusters grouped three isolates of H. sonorensis (Zoundomey, Akohoun and Kassehlo) that scored well for all traits. These latter isolates were similarly effective by suppressing within five days a population of M. bellicosus naturally imprisoned in 1,000 cm(3) containers and inoculated with a single two-weeks-old EPN-infected Galleria larva.

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.

BACKGROUND: The potential of the entomopathogenic nematode (EPN) Steinernema feltiae Filipjev as a biocontrol agent againstthecabbagemaggotDeliaradicum(L.),wasassessedinthreefieldtests,focusingonEPNdosage,applicationtechnique 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 temporarilyreducedthenumberofmaggotsaroundtheplants’roots,whileneithersprayingalowerdose(50000IJs/plant)nor soil drenching with 200000 or 50000 IJs/plant) reduced maggot numbers. When applied as a plant tray spray, IJs of S. feltiae took 1‐2weeks 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 4‐7weeks. Follow-up drench applications with EPNs, applied one and/or two weeks after the first EPN application, did not influence control of Deliaradicum. 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 withS.feltiaeis required to increase efficacy to commercial standards. c � 2013 Society of Chemical Industry

Pratylenchus penetrans is one of the most economically damaging plant-parasitic nematodes and is found on a wide variety of crops. Correct identification and quantification of this nematode are necessary for providing advice to farmers, but are not easily obtained with the traditional way of microscopic observation. We developed a qPCR assay to detect and quantify P. penetrans in a short but accurate manner. A qPCR primer set, including two primers and a TaqMan probe, was designed based on the sequence of the β-1,4-endoglucanase gene. The assay was optimized by using the primers in a qPCR assay with SYBR green I dye and setting the qPCR program to different annealing temperatures ranging from 60 °C to 64 °C. Based on the Ct-values, we retained the program with an annealing temperature of63 °C. The assay with the probe was very sensitive as it was able to detect a single individual of P. penetrans, even when mixed with up to 80 individuals of P. thornei. The specificity of the reaction was confirmed by the lack of amplification of DNA from 28 populations of 18 other Pratylenchus species and from plant-parasitic nematodes from nine other genera. DNA from 21 different isolates from P. penetrans was amplified. DNA extraction from 80 individuals and quantification by qPCR was repeated four times; Ct-values showed consis- tent results (Ct=24.4±0.4). A dilution series from DNA of P. penetrans resulted in a standard curve showing a highly significant linearity between the Ct-values and the dilution rates (R 2 =0.99; slope=�3.23; E=104 %). The tests showed a high correlation between the real numbers of nematodes and the numbers detected by the qPCR. The developed qPCR assay provides a sensitive means for the rapid detection and reliable quantification of individuals of this pest. This method does not require expertise in nematode taxonomy and morphology, and can be used as a rapid diagnostic tool in research, as well as in diagnostic labs and extension services advising farmers for pest management.

BACKGROUND:SteinernemacarpocapsaeWeiser,anentomopathogenicnematode(EPN),isapotentialbiologicalcontrolagent for the cabbage moth (Mamestra brassicae L.). This research aimed to identify a suitable spray application technique, and to determinewhetheryeast extractaddedtoanEPN sprayhas anattractingand/orafeedingstimulanteffectonM.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 obtainedusingdifferentsprayboomconfigurationswithverticalextensionsthatcarriedunderleafsprayingnozzles.Oneofthe configurations was selected for field testing with an EPN spray. Brewer’s yeast extract stimulated larval feeding on leaves, and increasedthemortalityoftheselarvaewhenexposedtoEPN.ThefieldtrialshowedthatasprayapplicationwithS.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. c � 2013 Society of Chemical Industry

This study explores the influence of a selection of adjuvants and of three different nozzle sizes on the foliar application of entomopathogenic nematodes (EPNs). Two EPN species were studied: Steinernema feltiae and Steinernema carpocapsae. A viability test of EPNs suspended in different solutions of adjuvants showed that all selected alcohol ethoxylates and an alkyl polysaccharide have an immobilising effect on the selected nematode species. In a sedimentation test, xanthan gum proved to be the only adjuvant in a broad selection, capable of delaying sedimentation of EPNs in suspension. Without xanthan gum, sedimentation of S. carpocapsae and S. feltiae was noticeable after 20 and 10 minutes, respectively. When xanthan gum (0.3 g/L) was added to the suspension, no signs of sedimentation were noticed after 20 minutes with both EPN species. An ISO 02 flat fan nozzle can clog when spraying S. carpocapsae. A deposition test determined that an ISO 04 standard flat fan nozzle provides a higher relative...

Chive gnat, Bradysia odoriphaga Yang & Zhang (Diptera: Sciaridae), is the most serious pest of Chinese chive, Allium tuberosum. The present study aimed at determining the biocontrol potential of different indigenous entomopathogenic nematode (EPN) isolates for the management of chive gnat in China. The virulence of 145 EPN isolates belonging to 13 species against B. odoriphaga was evaluated in the laboratory, and then the most promising isolates (Steinernema ceratophorum HQA-87; S. hebeiense JY-82; S. feltiae JY-90, JY-17; S. litorale HXY-68; Heterorhabditis indica ZZ-68; H. bacteriophora NY-63, HQ-94; and H. megidis LFS-10) were selected for further experiments. The mortality of fourth instar B. odoriphaga was significantly affected by nematode isolates and concentrations of infective juveniles (IJ). Third and fourth instars of B. odoriphaga were generally more susceptible to the nematodes than first and second instars and pupae. Pot experiments showed no differences in B. odoriphaga population reduction at 50 and 75 IJ/cm2 of different isolates; the tested isolates caused a 78–94 % reduction of the chive gnat population at an application rate of 75 IJ/cm2. In a field experiment, S. feltiae JY-90 generated the strongest pest suppression 14 days after EPN application, whereas after 28 days pest densities in the plots treated with S. feltiae JY-17 and S. hebeiense JY-82 were similar to those in plots treated with phoxim and yielded significant reductions of the B. odoriphaga populations. Our findings indicate that EPN may have good potential for use in the integrated management of B. odoriphaga populations in Chinese chive.

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

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.

The striped flea beetle, Phyllotretastriolata (F.) (Coleoptera: Chrysomelidae) is a key pest of crucifer vegetables in Southern China. The use of entomopathogenic nematodes (EPNs) within an integrated pest management approach may offer an effective and environmentally safe strategy to suppress outbreaks of this pest. In the present study, the efficacy of Steinernema carpocapsae All and Heterorhabditis indica LN2 for the control of P. striolata in the field was evaluated, as well as the combined application of EPNs and azadirachtin against the pest. Both nematode species were capable of reducing populations of the soil-dwelling stages of P. striolata, thus leading to a reduction of the adult populations and the associated shot-hole damage on the leaves. Nematode treatments also increased cabbage yields as compared to the control and azadirachtin treatments alone. Azadirachtin alone was not effective to prevent damage by P. striolata, but it could enhance the control effect of S. carpocapsae shortly after application. Osmotically treated infective juveniles (IJs) of S. carpocapsae All performed as well as untreated IJs.

Green manures from the Brassicaceae family are used to control different soil-borne pests, including plant-parasitic nematodes due to their biofumigant compounds released during the breakdown of the plants. We studied the biofumigant and biological soil disinfestation effects of a yellow mustard green manure on the potato cyst nematode Globodera rostochiensis , a quarantine species with high economic importance for potato growers worldwide, in the field. In addition, we recorded the changes in the composition of the free-living nematode community for a better understanding of the overall ecological effect of this biocontrol method. We followed agricultural practices of sowing the green manure in late summer and incorporation in fall (with or without plastic cover) before planting of potato. Hatching and infectivity of G. rostochiensis juveniles were not consistently affected by growing yellow mustard or its amendments, but the population densities of plant-parasitic nematodes in general were reduced. Changes in the abundance of nematode trophic groups were observed for treatments with yellow mustard amendment. In addition, a beneficial effect was observed when covering the plots with plastic, as the abundance of plant-parasitic nematodes decreased. The amended plots exhibited an increase of the enrichment index while the channel index decreased significantly, indicating a soil food web with a decomposition pathway dominated by bacterial-feeder nematodes.

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 cm 2 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 againstG.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. c � 2012 Society of Chemical Industry

Secretory proteins encoded by genes expressed in the pharyngeal gland cells of plant-parasitic nematodes have key roles in nematode parasitism of plants. A new β-1,4-endoglucanase gene (designated Ha-eng-1a) was isolated from the cereal cyst nematode Heterodera avenae. The cDNA of Ha-eng-1a encoded a deduced 463-amino acid sequence containing a catalytic domain and a cellulose binding module separated by a linker. The genomic DNA of Ha-eng-1a is 2,129-bp long, containing eight introns ranging from 56 bp to157 bp and nine exons ranging from 70 bp to 299 bp. Southern blot analysis revealed that the Ha-eng-1a gene has two copies. In situ hybridization showed that the Ha-eng-1a transcriptsspecifically accumulated in the two subventral gland cells of the second-stage juveniles. There was evidence for cellulase activity of the recombinant protein Ha-eng-1a in vitro. The results indicated that this β-1,4-endoglucanase gene may play a crucial role in plant cell wall-degradation during penetration and migration of nematodes in the host roots.

The generalist predator Macrolophus pygmaeus Rambur (Hemiptera: Miridae) is a key biological control agent in European greenhouses. The influence of acclimation, infection with endosymbiotic bacteria and diet on the cold tolerance of the Mediterranean biocontrol population was assessed by determining the supercooling point, i. e. the temperature at which the insect's body fluids freeze. This parameter provides a first indication of an insect's establishment potential in a new region and of its possible geographical range. Allowing the predatory bugs to adapt to lower temperatures resulted in an increase in supercooling ability. Macrolophus pygmaeus bugs exposed to antibiotics in their artificial diet and hence cured from their infection with the endosymbiotic bacteria Wolbachia pipientis, Rickettsia bellii and R. limoniae were more tolerant to freezing than infected bugs. The diet of the predators also affected the freezing temperature of the body fluids. Predators fed an artificial diet based on egg yolk were less resistant to freezing than those fed Ephestia kuehniella Zeller (Lepidoptera: Pyralidae) eggs. These findings illustrate that several factors may affect the cold hardiness of a biocontrol agent and may thus complicate the evaluation of its establishment potential in the framework of an environmental risk assessment. © 2012 International Organization for Biological Control (IOBC)., SCOPUS: ar.j, info:eu-repo/semantics/published

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.

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. Copyright © 2011 Society of Chemical Industry

Coffee varieties with resistance for the plant-parasitic nematodes Pratylenchus coffeae and Radopholus arabocoffeae are limited in Vietnam. A selection of imported varieties and high yield varieties of Arabica coffee in Vietnam were evaluated for resistance to both plant-parasitic nematode species in Northern Vietnam. The same experiments were carried out with hybrid arabica coffee, three selected clones of Coffea canephora and one clone of Coffea excelsa in the Western Highland of Vietnam. The screened coffee accessions from Ethiopia (KH1, KH13, KH20, KH21, KH29, and KH31) were susceptible and good host for P. coffeae. Also accessions 90P4 (Portugal) and Oro azteca (Mexico) had a reproduction factor Rf > 1. Pluma Hidalgo (Mexico), 90/6 (Vietnam), 90P3 (Portugal), 90P2 (Vietnam), Variedad (Mexico), 90T (Portugal), and Garnica (Mexico) were poor hosts (Rf 1. Pluma Hidalgo, 90/6, 90P3, 90P2, 90T, Oro azteca, and Garnica were poor hosts (Rf

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. Copyright © 2011 Society of Chemical Industry

Holotrichia longipennis Blanch. (Coleoptera: Scarabaeidae) is a serious pest of commercial crops in Siduwa, Dhankuta, Nepal. Seven indigenous isolates of entomopathogenic nematodes (Steinernema lamjungense LMT5, S. lamjungense SS4, S. everestense DKP4, S. abbasi CS1, S. sp. KL1, Heterorhabditis indica CK2 and H. indica CK6) were used in a series of bioassays against the insect. All isolates showed an increased dispersal in response to H. longipennis. Nematodes were more attracted towards third instar larvae than to second instars. Differences in penetration and multiplication in the insect were observed amongst the seven isolates. Steinernema lamjungense LMT5, S. everestense DKP4 and S. abbasi CS1 caused greater mortality than other isolates to different developmental stages. Pupae and second instar larvae were more susceptible than third instar larvae. Significant differences were observed in LT50 values of the isolates against different stages of H. longipennis. Three isolates (S. lamjungense LMT5, S. everestense DKP4 and S. abbasi CS1) along with a commonly used insecticide (chlorpyrifos) were tested against this insect in pot and field experiments. In pot experiments using maize and cabbage as a host crop, S. lamjungense LMT5 and S. everestense DKP4 performed better than S. abbasi CS1 and yielded a mortality comparable with chlorpyrifos. Similar results were observed in field experiments 3 weeks after nematode application. These experiments overall suggest S. lamjungense LMT5 to be a promising biocontrol agent against H. longipennis followed by S. everestense DKP4 and S. abbasi CS1.

Dispersal mechanisms of soil-borne microfauna have hitherto received little attention. Understanding dispersal mechanisms of these species is important to unravel their basic life history traits, biogeography, exchange of individuals between populations, and local adaptation. Soil-borne nematodes and root-feeding nematodes in particular occupy a key position in soil-food webs and can be determinants for plant growth and vegetation structure and succession. However, their dispersal abilities have been scarcely addressed, predominantly focusing on species of agricultural importance. Still, root-feeding nematodes are usually considered as being extremely limited and bound to the rhizosphere of plants. We investigated a mechanism for long distance dispersal of root-feeding nematodes associated to two widespread coastal dune grasses. The nematodes are known to be crucial for the functioning of these grasses. We experimentally tested the hypothesis that root-feeding nematodes are able to move across long distances inside rhizome fragments that are dispersed by seawater. We also tested the survival capacities of the host plants in seawater. Our study demonstrates that root-feeding nematodes and plants are able to survive immersion in seawater, providing a mechanism for long distance dispersal of root feeding nematodes together with their host plant. Drifting rhizome fragments enable the exchange of plant material and animals between dune systems. These results provide new insights to understand the ecology of dune vegetation, the interaction with soil-borne organisms and more importantly, re-set the scale of spatial dynamics of a group of organisms considered extremely constrained in its dispersal capacities.

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.

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.

{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. }

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.

The pathogenicity and reproductive fitness of Pratylenchus coffeae and Radopholus arabocoffeae from Vietnam on coffee (Coffea arabica) seedlings cv. Catimor were evaluated in greenhouse experiments. The effect of initial population densities (Pi = 0, 1, 2, 4, 8, 16, 32, 64, 128, and 256 nematodes per cm3 soil) was studied for both species at different days after inoculation (dai). The data were adjusted to the Seinhorst damage model Y = m + (1-m).zPi-T. Tolerance limit (T) for P. coffeae was zero for the height and the diameter of the coffee plants. For the diameter, the T-value for R. arabocoffeae was 25.6 for 30 and 60 dai and 12.8 for 90 and 120 dai. After 4 months T was zero. The low tolerance limits indicate that Arabica coffee is highly intolerant to both nematode species. At the end of the experiment (180 dai), all plants were infected and most were dead when inoculated with R. arabocoffeae at initial densities of 32, 64, 128 and 256 nematodes/cm3 soil. For P. coffeae plant death was already observed at the lowest inoculation densities. Growth of coffee was reduced at all inoculation levels for both species. Pratylenchus coffeae and R. arabocoffeae caused intense darkening of the roots, leaf chlorosis and a strong reduction of root and shoot growth. It was observed that P. coffeae mainly destroyed lateral roots rather than tap roots, whereas R. arabocoffeae reduced tap root length rather than the lateral roots. At the lowest inoculum densities, the reproduction factor of P. coffeae was 2.38 and 2.01 for R. arabocoffeae, indicating that arabica coffee is a host for both species. Plant growth as expressed by shoot height and shoot and root weight measured 60 dai was negatively correlated with nematode (both species) density as expressed by the geometric mean of nematode numbers at 30 and 60 dai.