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Soil organisms are a crucial part of the terrestrial biosphere. Despite their importance for ecosystem functioning, few quantitative, spatially explicit models of the active belowground community currently exist. In particular, nematodes are the most abundant animals on Earth, filling all trophic levels in the soil food web. Here we use 6,759 georeferenced samples to generate a mechanistic understanding of the patterns of the global abundance of nematodes in the soil and the composition of their functional groups. The resulting maps show that 4.4 ± 0.64 × 1020 nematodes (with a total biomass of approximately 0.3 gigatonnes) inhabit surface soils across the world, with higher abundances in sub-Arctic regions (38% of total) than in temperate (24%) or tropical (21%) regions. Regional variations in these global trends also provide insights into local patterns of soil fertility and functioning. These high-resolution models provide the first steps towards representing soil ecological processes in global biogeochemical models and will enable the prediction of elemental cycling under current and future climate scenarios., This research was supported by a grant from DOB Ecology to T.W.C., a grant from the Netherlands Organization for Scientific Research (grant 016.Veni.181.078) to S.G., grants from NSF (OPP 1115245, 1341736, 0840979) to B.J.A., by a Ramon y Cajal fellow award (RYC-2016-19939) to R.C.H., a grant from UNEP & Global Environment Facility to J.E.C., a grant from NERC (NE/M017036/1) to T.C., a grant from FAPEMIG/FAPESP/VALE S.A.(CRA-RDP-00136-10) to L.B.C., through the strategic programme UID/BIA/04050/2013 (POCI-01-0145-FEDER-007569) awarded to S.R.C., a grant from CNPq PROTAX (562346/2010-4) to J.M.d.C.C., a grant from DFG (CRC990) to V.K. and S.S., a grant from the MSHE of Russia (AAAA-A17-117112850234-5) to A.A.K., grants from the Chinese Academy of Sciences (XDB15010402) and the National Natural Science Foundation of China (41877047) to Q.L., grants from the National Natural Science Foundation of China (31330011, 31170484) to W.L., grants from NERC (NE/M017036/1) to M.M., grants from the Spanish Ministry of Innovation (CGL2009-14686-C02-01/ 02, CGL2013-43675-P) to J.A.R.M., grants from NSF (DEB-0450537, DEB-1145440) to P.M., T.O.P. and K. Powers, grants from the German Academic Exchange Service (PKZ 91540366) and NAFOSTED (106.05 – 2017.330) to T.A.D.N., by an ARC Discovery project (DP150104199) to U.N.N., by the National Key Research and Development Program of China (2016YFC0502101) and the National Natural Science Foundation of China (31370632) to K. Pan, a grant from the Natural Environment Research Council (NERC) to D.G.W., a grant from BAPHIQ (106AS-9.5.1-BQ-B3) J.-i.Y. The James Hutton Institute receives financial support from the Scottish Government Rural and Environment Science and Analytical Services (RESAS) division. Investigations in northwest Russia were carried out under state order for IB KarRC RAS and are partially supported by the Russian Foundation for Basic Research (18-34-00849). We thank E. Clark and A. Orgiazzi for review of the manuscript; and R. Bouharroud, Z. Ferji, L. Jackson and E. Mzough for providing data.

This bookpresents a compendium of methods to characterizesoil and crops from different pedoclimatic regions.The aspects considered in this handbook of methods are: - Experinmental design - Crop growth and quality properties. This part includes the development of the crops, the yield, the incidence of diseases, and the nutritional evaluation of cereals, legumes and vegetables.Types of sampling of the soils (oriented to determine dry bulk density, biodiversity or physicochemical characteristics). - Earthworm sampling. - Physical characterization of the soil including determination of dry bulk density, coarse fragments, water holding capacity, humidity, soil hydraulic conductivity, particle size distribution and aggregates. - Chemical characterization of the soil including determination of pH, salinity, and content of organic matter, organic and inorganic carbon, total and inorganic nitrogen, available phosphorus, potassium, calcium, magnesium, boron, effective exchange capacity, cations (iron, manganese, copper, zinc and molybdenum), and pesticides. - Biological analyzes that include biodiversity measures (of worms, nematodes and microorganisms), using techniques such as DNA extraction and purification, or fatty acid analysis. - Soil greenhouse gas emission. This work was funded by the European Commission Horizon 2020 project SoildiverAgro [grant agreement 817819].

As the most abundant animals on earth, nematodes are a dominant component of the soil community. They play critical roles in regulating biogeochemical cycles and vegetation dynamics within and across landscapes and are an indicator of soil biological activity. Here, we present a comprehensive global dataset of soil nematode abundance and functional group composition. This dataset includes 6,825 georeferenced soil samples from all continents and biomes. For geospatial mapping purposes these samples are aggregated into 1,933 unique 1-km pixels, each of which is linked to 73 global environmental covariate data layers. Altogether, this dataset can help to gain insight into the spatial distribution patterns of soil nematode abundance and community composition, and the environmental drivers shaping these patterns., This research was supported by a grant from DOB Ecology to T.W.C., a grant from the Netherlands Organization for Scientific Research (grant 016.Veni.181.078) to S.G., grants from NSF (OPP 1115245, 1341736, 0840979) to B.J.A., by a Ramon y Cajal fellow award (RYC-2016-19939) to R.C.H., a grant from UNEP & Global Environment Facility to J.E.C., grants from NERC’s Soil Security Programme to R.D.B. (NE/M017028/1) T.C. (NE/M017036/1), a grant from FAPEMIG/FAPESP/VALE S.A.(CRA-RDP-00136-10) to L.B.C., through the strategic programme UID/BIA/04050/2013 (POCI-01-0145-FEDER-007569) awarded to S.R.C., a grant from CNPq PROTAX (562346/2010-4) to J.M.d.C.C., a grant from DFG (CRC990) to V.K. and S.S., a grant from the MSHE of Russia (AAAA-A17-117112850234-5) to A.A.K., grants from the Chinese Academy of Sciences (XDB15010402) and the National Natural Science Foundation of China (41877047) to Q.L., grants from the National Natural Science Foundation of China (31330011, 31170484) to W.L., grants from NERC (NE/ M017036/1) to M.M., grants from the Spanish Ministry of Innovation (CGL2009-14686-C02-01/02, CGL2013- 43675-P) to J.A.R.M., grant from the Spanish Ministry of Innovation (RYC-2016-19939) to R.C.H., grants from NSF (DEB-0450537, DEB-1145440) to P.M., T.O.P. and K. Powers, grants from the German Academic Exchange Service (PKZ 91540366) and NAFOSTED (106.05–2017.330) to T.A.D.N., by an ARC Discovery project (DP150104199) to U.N.N., by the National Key Research and Development Program of China (2016YFC0502101) and the National Natural Science Foundation of China (31370632) to K. Pan, a ERC Research Council Advanced grant (ERC-Adv 323020 SPECIALS) to W.H.v.d.P, a grant from the Natural Environment Research Council (NERC) to D.G.W., a grant from BAPHIQ (106AS-9.5.1-BQ-B3) to J.-i.Y., a grant from the Russian Foundation for Basic Research (18-29-05076) to A.V.T. The James Hutton Institute receives financial support from the Scottish Government Rural and Environment Science and Analytical Services (RESAS) division. Investigations in Northwest Russia were carried out under state order for IB KarRC RAS and are partially supported by the Russian Foundation for Basic Research (18-34-00849).

Soil biodiversity plays a key regulation role in the ecosystem services that underpin regenerative sustainable agriculture. It can be impacted by agricultural management techniques, both positively (through measures such as compost application) and negatively (through, for example, application of synthetic nitrogen). As one of the most numerous members of the soil biota, nematodes are well established as indicators for the soil food web. However, compost application also includes the addition of nematodes present in compost and their subsequent survival in soil is unknown. Nematode communities within the compost applied to soil, and nematode communities in the soil of a multi-year rotational cropping field trial in Melle (Belgium) were studied using morphological and metabarcoding techniques. Compost (C) and nitrogen fertilizer (NF) treated plots were compared. Three replicate plots were investigated for each of the following treatments: C application only; C and NF application; NF only; no C and no NF (control). Plots were sampled six times between 2015-2017, before and after C or NF were added each spring and after crop harvest (except for 2017). NF treatment resulted in a significant decrease of fungal feeding and predatory nematodes, while herbivorous nematodes were positively affected. Remarkably, we did not find compost addition to exert any noticeable effects on the soil nematode community. The morphological and metabarcoding data resulted in different results of the nematode community composition. However, trends and patterns in the two data sets were congruent when observed with NMDS plots and using the nematode maturity index. Metabarcoding of individual compost nematode taxa demonstrated that nematodes originating from compost did not persist in soil.

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

Small grain cereals have served as the basis for staple foods, beverages, and animal feed for thousands of years. Wheat, barley, oats, rye, triticale, rice, and others are rich in calories, proteins, carbohydrates, vitamins, and minerals. These cereals supply 20% of the calories consumed by people worldwide and are therefore a primary source of energy for humans and play a vital role in global food and nutrition security. Global production of small grains increased linearly from 1960 to 2005, and then began to decline. Further decline in production is projected to continue through 2050 while global demand for these grains is projected to increase by 1% per annum. Currently, wheat, barley, and oat production exceeds consumption in developed countries, while in developing countries the consumption rate is higher than production. An increasing demand for meat and livestock products is likely to compound the demand for cereals in developing countries. Current production levels and trends will not be sufficient to fulfill the projected global demand generated by increased populations. For wheat, global production will need to be increased by 60% to fulfill the estimated demand in 2050. Until recently, global wheat production increased mostly in response to development of improved cultivars and farming practices and technologies. Production is now limited by biotic and abiotic constraints, including diseases, nematodes, insect pests, weeds, and climate. Among these constraints, plant-parasitic nematodes alone are estimated to reduce production of all world crops by 10%. Cereal cyst nematodes (CCNs) are among the most important nematode pests that limit production of small grain cereals. Heavily invaded young plants are stunted and their lower leaves are often chlorotic, forming pale green patches in the field. Mature plants are also stunted, have a reduced number of tillers, and the roots are shallow and have a “bushy-knotted” appearance. CCNs comprise a number of closely-related species and are found in most regions where cereals are produced.

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.

WOS:000411576400005 The cereal cyst nematode, Heterodera filipjevi (Madzhidov) Stelter, was detected in Turkey in 1996 and since then it has been increasingly reported to cause losses in wheat yields. Cereal cyst nematode populations have a wide genetic diversity which makes it difficult to be managed. The objectives of this research were to investigate the genetic variation of 23 cyst nematode populations collected from wheat growing areas of Bolu Province, utilising sequenced ITS-rDNA regions, and monitoring main nematode isolate population dynamics in field conditions. The results of ribosomal DNA sequencing revealed that all cyst populations were H. filipjevi and that little genetic variation was present among populations. The highest number of second-stage juveniles was observed between April and beginning of May in a field experiment; white females were visible on the roots in late May. The hatching process was positively correlated with low temperatures, but there were inverse correlations between reproductive factors and initial nematode population densities.

Comprehensive morphological and molecular analyses revealed that published ITS sequences of the economically important plant-parasitic nematodePratylenchus goodeyiare actually sequences from distantly free-living bacterivorous ‘cephalobids’. We demonstrated that this incorrect labelling resulted in a cascade of erroneous interpretations, as shown by the reports of ‘P. goodeyi’ on banana in China and on cotton in India. This clearly illustrates the risk of mislabelled sequences in public databases. Other mislabelledPratylenchuscases are discussed to illustrate that this is not an isolated case. Herein,P. lentisn. syn. is considered a junior synonym ofP. pratensiswhileP. flakkensiswas for the first time linked to DNA sequences using topotype material. As taxonomic expertise is decreasing and sequence-based identification is growing rapidly, the highlighted problem may yet increase and a strong link between morphology and DNA sequences will be of crucial importance in order to prevent, or at least minimise, sequence-based misidentifications.

Nematodes are ideal biological indicators to monitor soil biodiversity and ecosystem functioning. For this reason, they have been receiving increasing attention from a broad range of scientists. The main method to characterize soil nematode communities until at least genus level is still based on microscopic observations of nematode morphology. Such an approach is time-consuming, labor-intensive, and requires specialized personnel. The first studies on the potential use of DNA-metabarcoding to characterize nematode communities showed some shortcomings: under- or overestimation of species richness caused by failure to detect a number of nematode species or caused by intraspecific sequence variants increasing the number of OTUs (operational taxonomic units) or &lsquo, molecular&rsquo, species, and flaws in quantification. We set up experiments to optimize this metabarcoding approach. Our results provided new insights such as the drastic effect of different DNA-extraction methods on nematode species richness due to variation in lysis efficacy. Our newly designed primer set (18S rRNA gene, V4-V5 region) showed in silico an improved taxonomic coverage compared with a published primer set (18S rRNA gene, V6-V8 region). However, results of DNA-metabarcoding with the new primer set showed less taxonomic coverage, and more non-nematode reads. Thus, the new primer set might be more suitable for whole soil faunal analysis. Species-specific correction factors calculated from a mock community with equal amounts of different nematode species were applied on another mock community with different amounts of the same nematode species and on a biological sample spiked with four selected nematode species. Results showed an improved molecular quantification. In conclusion, DNA-metabarcoding of soil nematode communities is useful for monitoring shifts in nematode composition but the technique still needs further optimization to enhance its precision.

In 2011, second-stage juveniles (J2) of an unknown root-knot nematode (Meloidogyne spp.) were detected during a routine survey for root-knot nematodes on arable land in Harveng, Belgium, after a crop of wheat. Most of the loamy soil samples (36 out of 42) contained J2 of the common root-knot nematode M. naasi Franklin, 1965 (1), while 15 of these also contained the unknown species, albeit in lower densities (22 J2/100 ml vs. 157 J2/100 ml soil). After detailed morphological observation of the unknown J2, they were until further notice identified as Meloidogyne artiellia Franklin, 1961 (2), the British root-knot nematode. To confirm the identification, a pure culture of M. artiellia was established by adding nematode suspensions to pots planted with kale (Brassica oleracea var. laciniata), a non-host for M. naasi (3). After 2 months, Meloidogyne spp. females, males, and J2 were isolated from galled kale roots. Morphological characteristics (n = 25) from the perineal pattern (rounded with fine striae, lateral area with coarse ridges, angular dorsal arch) and stylet knobs (small, ovoid, and backwardly sloping) for the females, the head shape (set off with distinct head cap) and stylet knobs (small, ovoid and backwardly sloping) for the males, the hemizonid position (anterior, adjacent to S to E pore), tail shape (conical), and short tail length (18 to 27 μm) for the J2, fit with previous observed populations of M. artiellia (3). Young egg-laying females were used for isozyme electrophoresis, and showed typical malate dehydrogenase (N1b) and esterase (M2-VF1) patterns (3). Additionally, DNA was extracted from single juveniles by incubating them in a lysis buffer (200 mM NaCl, 200 mM Tris-HCl (pH 8), 1% β-mercaptoethanol and 800 μg/ml Proteinase K) during 1.5 h at 65°C and 5 min at 99°C in a thermocycler. One microliter of crude DNA extract was used for PCR. ITS-rDNA sequencing (GenBank Accession Numbers JX393299 and JX393300) confirmed the identity, showing a 98 to 100% homology with other M. artiellia sequences (AY150368 and AF248478). To our knowledge, this is the first report of the root-knot nematode, M. artiellia, in Belgium. This nematode has been reported from the Mediterranean area, where it causes damage on chickpea and wheat (4), as well as from the U.K. Its finding in Harveng, close to the French border, suggests a more extensive geographical distribution. References: (1) M. T. Franklin. Nematologica 11:79, 1965. (2) M. T. Franklin et al. Suppl.:85, 1961. (3) G. Karssen. Pages 93-97 in: The Plant-Parasitic Nematode Genus Meloidogyne Göldi, 1892 (Tylenchida) in Europe, Brill Leiden, The Netherlands, 2002. (4) M. Di Vito and N. Greco. Revue Nématol. 11:223, 1988.

Amphimictic populations of root-lesion nematodes with numerous males and females having three lip annuli, a functional spermatheca and non-areolated lateral field occur on sword fern (Nephrolepis exaltata) in Florida. Identified for decades asPratylenchus penetrans, they appeared to be a morphologically separated species on the basis of a longer stylet (17.8-18.3 μm) thanP. penetrans(15-17 μm) and different lip pattern inen faceview (rectangularvsdumb-bell inP. penetrans). Morphologically similar amphimictic root-lesion nematodes have also been detected on flax lily in Costa Rica. Subsequent morphological observations indicated that these amphimictic root-lesion nematodes from fern and flax lily are closely related to the parthenogenetic speciesP. bolivianus, which has areolated lateral fields. In spite of the reproductive and morphological dissimilarities between these populations, their separation into different species was not supported by the results of molecular analyses of their DNA sequences. The populations used in these analyses included those that are amphimictic from Florida and Costa Rica and others that are parthenogenetic from the type locality in Bolivia, and geographically distant localities in Chile, China, Colombia and Europe. Phylogenetic analyses of the ITS and D2-D3 expansion segments of the 28S rRNA gene indicated that they belong to the same species,P. bolivianus, which consists of two morphotypes,P. bolivianus(am) amphimictic andP. bolivianus(pm) parthenogenetic, herein described and illustrated. Contradictory results were obtained by the analyses using a portion of thehsp90gene. The phylogenetic study, which included sequences of other root-lesion nematodes, a topotype and geographical distant populations ofP. zeae, revealed thatP. bolivianusandP. zeaeformed highly supported clades in the majority consensus trees. PCR with species-specific primers for rapid diagnostics ofP. bolivianusandP. zeaewere developed and tested.

Entomopathogenic nematodes belong to the families of Steinernematidae and Heterorhabditidae. They are obligate and lethal parasites of insects that can provide effective control of some important pests of commercial crops. A total of 53 isolates of EPN were molecularly characterised (ITS region-based) in the present study. Most of the studied isolates belong to theSteinernemagenus and only few isolates belong to theHeterorhabditisgenus. The phylogenetic relations ofSteinernemaandHeterorhabditisspecies were analysed by utilising the maximum likelihood method. In theSteinernemaphylogenetic tree, 99 isolates formed five major, moderately or highly reinforced clades: clade I:affine-intermediumgroup; clade II:carpocapsae-siamkayai-tami-scapterisci; clade III:bicornutum-riobrave-thermophilum; clade IV:glaseri-arenarium-karii-longicaudum; and clade V:feltiae-schliemanni-kushidai-kraussei-oregonense. The BLAST analysis of the ITS region of the rDNA of the steinernematid isolate PAL10 showed a rather low similarity of 93% withS. vulcanicum(accession number: GU929442), supporting the possible designation of a new species. In theHeterorhabditisphylogenetic tree, 25 isolates formed three main clades: clade I:bacteriophora-argentinensis-hepialius; clade II:baujardi-sonorensis-amazonensis; and clade III:indica-brevicaudis-hawaiiensis. All five studied isolates ofHeterorhabditiswere identified asH. indicaandH. bacteriophora. In both phylogenetic trees, the intra-specific variability level was different among clades for some species. The description of the new species (PAL10 isolate) would need further morphometric characterisation, morphologically identification and cross-breeding studies.

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.

Summary Among the recognised species groups of Heterodera, the Avenae group is one of the largest with a total of 12 species. Ten of them, H. arenaria, H. aucklandica, H. australis, H. avenae, H. filipjevi, H. mani, H. pratensis, H. riparia, H. sturhani and H. ustinovi, are morphologically closely related and represent the H. avenae species complex, and the other two, H. hordecalis and H. latipons, are morphologically more distinct from this complex. In this study we provide comprehensive phylogenetic analyses of several hundred COI and ITS rRNA gene sequences from the Avenae group using Bayesian inference, maximum likelihood and statistical parsimony. Some 220 COI and 11 ITS rRNA new gene sequences from 147 nematode populations collected in 26 countries were obtained in this study. Our study showed that the COI gene is a powerful DNA barcoding marker for identification of populations and species from the Avenae group. A putatively new cyst nematode species related to H. latipons was revealed from the analysis of COI and ITS rRNA gene datasets. COI gene sequences allow distinguishing H. arenaria, H. australis and H. sturhani from each other and other species. Problems of species delimiting of these species are discussed. The results of the analysis showed that COI haplotypes corresponded to certain pathotypes of the cereal cyst nematodes. It is recommended that information on COI haplotypes of studied populations be included in research with these nematodes. Based on the results of phylogeographical analysis and age estimation of clades with a molecular clock approach, it was hypothesised that several species of the Avenae group primarily originated and diversified in the Irano-Anatolian hotspot during the Pleistocene and Holocene periods and then dispersed from this region across the world. Different geographic barriers, centres and times of origin might explain current known distribution patterns for species of the Avenae group. Possible pathways, including a long distance trans-Atlantic dispersal, and secondary centres of diversification are proposed and discussed.

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.

We investigated the possible threats of Scutellonema bradys on potato production by evaluating the host suitability and damage symptoms in five potato varieties grown in Europe and Africa. The probability of nematode entry, spread and establishment in Europe was also investigated, through a pest risk analysis (PRA). Inoculation of sprouted potato tubers with S. bradys in pot tests caused varying levels of damage to the different potato varieties, when these were evaluated 8 weeks later. The nematode infected roots and tubers, causing scaly surface appearance of tubers, brown root lesions and tuber rot. The number of nematodes recovered from roots, tubers and soil was greatest on Desiree, Spunta and Nicola, while reproduction was low on Diamant and Draga. The PRA showed that the likelihood of importation of S. bradys from third countries into the EU is high due to the import of contaminated yam tubers from infested African regions. However, the likelihood that nematodes reach susceptible hosts is considered low; therefore, “entry”, as defined in a PRA, is low. Nematode establishment is possible on hosts like yam, potato and tomato in southern parts of the EU. Nematode spread, in the EU and Africa, occurs through several pathways: yam and potato tubers, peels, soil from infested areas attached to plants or equipment, and travellers carrying plant parts or soil. The study demonstrated that S. bradys can damage potato and highlighted the need to reduce the risk of nematode entry and spread in the EU region, as well as in Africa.

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 pine wood nematode (PWN), Bursaphelenchus xylophilus, causes pine wilt disease and is transmitted by Monochamus beetles. An efficient trapping system for these organisms is essential for their early detection in threatened regions. A wet catching method, using monoethylene glycol (MEG) in a collecting cup to preserve captured insects, was suitable for catching longhorn beetles. As a second step in developing this technique, we studied the influence of MEG on nematode detection. When M. galloprovincialis carrying PWN were submerged in MEG, nematodes died within 24 h. The preservative altered certain morphological features of nematodes immersed in solutions of 10, 30 or 60% MEG, impeding their morphological identification. However, molecular identification was possible as long as the MEG concentration was below 70%. At higher concentrations, a rinsing step with water before DNA extraction was enough to allow molecular detection. Wet trapping requires less frequent monitoring than dry trapping, thus reducing maintenance and related costs., SCOPUS: ar.j, info:eu-repo/semantics/published

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.

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.

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.

A new root-lesion nematode, particularly pathogenic to Musa spp. and causing important plantain losses in Ghana, is described and named Pratylenchus speijeri n. sp. The cryptic status of this species within the P. coffeae species complex has been assessed and confirmed in this study. An extensive comparison of the morphological and molecular characteristics of this new species with those of P. coffeae and other related amphimictic species did not result in an unambiguous separation of this species from P. coffeae because only a few morphological features of diagnostic value were found. Sequence and phylogenetic analyses of the D2-D3 expansion segments of the 28S rRNA gene, the ITS rRNA gene and a portion of the hsp90 gene of P. speijeri n. sp. and P. coffeae species complex populations from different sources generated majority consensus BI trees with three major clades: P. speijeri n. sp. from Musa spp. roots in Ghana; unidentified or putative new Pratylenchus sp. C1 from Colocasia esculenta roots in Japan and P. coffeae with non-homogeneous relationships from different hosts and distant geographical areas. These results confirmed the validity of P. speijeri n. sp. as a new taxon and indicated that P. coffeae populations from Colocasia in Japan also need to be considered as a new species. Sequence differences in the ITS were used to design group- and species-specific primers to detect P. speijeri n. sp. and other species of P. coffeae species complex. The use of these species-specific primers for the separation of P. speijeri n. sp., Pratylenchus sp. C1 and P. coffeae has important practical application in breeding programmes for agriculture in West Africa.

Three isolates (Dero-1, Dero-8 and Mosisa-1) of a new entomopathogenic nematode, S. ethiopiense sp. n., were isolated by baiting soil samples from the Mendi area, Western Wollega, Ethiopia, with last instar wax moth larvae Galleria mellonella. Infective juveniles of S. ethiopiense sp. n. have a body length of 898 (768-1010) μm, a maximum of eight identical ridges (i.e., nine lines) in the lateral field, excretory pore located at mid-pharynx, hyaline layer occupying approximately half of the tail and c′ = 3.2. First generation males lack a caudal mucron, whereas second generation males possess a short spine-like mucron. The spicules are slightly arcuate, golden-brown in colour and have an ellipsoid or oblong manubrium. First generation females lack a postanal swelling and have a minute protuberance on the tail tip whereas second generation females have a postanal swelling and protruding vulva. Based on the morphology, morphometrics and DNA analysis, the new species belongs to the glaseri group. The closest relative species is the afro-tropical S. karii recorded from Kenya. The BLAST analysis of the ITS region of the rDNA revealed a similarity of 93% with S. karii, supporting the validity of S. ethiopiense sp. n. as a new species. In the phylogenetic trees the new species groups together only with S. karii (bootstrap value of 100%), but is also separated from S. karii by a bootstrap value of 100% or 70%.

Meloidogyne minor is a small root-knot nematode that causes yellow patch disease in golf courses and severe quality damage in potatoes. It was described in 2004 and has been detected in The Netherlands, England, Wales, Northern Ireland, Ireland and Belgium. The nematode often appears together with M. naasi on grasses. It causes similar symptoms on potato tubers as M. chitwoodi and M. fallax, which are both quarantine organisms in Europe. An accurate identification method therefore is required. This study describes a real-time PCR assay that enables the identification of M. minor after extraction of nematodes from soil or plant samples. Alignments of sequences of rDNA-ITS fragments of M. minor and five other Meloidogyne species were used to design a forward primer Mminor_f299, a specific primer Mminor_r362 and the specific MGB TaqMan probe P_Mm_MGB321. PCR with this primers and probe results in an amplicon of 64 bp. The analytical specificity of the real-time PCR assay was assessed by assaying it on six populations of M. minor and on 10 populations of six other Meloidogyne species. Only DNA from M. minor gave positive results in this assay. The assay was able to identify M. minor using DNA from a single juvenile independent from the DNA extraction method used.

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.

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.

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

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

Quality of biological control products based on entomopathogenic nematodes can be severely damaged due to exposure to high temperature surpassing 40A degrees C. The study screened 36 natural populations and 18 hybrid or inbred strains of Heterorhabditis bacteriophora for their response to high temperature. Nematodes were tested with or without prior adaptation to heat at 35A degrees C for 3 h. Five strains of H. indica and one of H. megidis were also included. Molecular identification using nuclear ribosomal DNA sequences confirmed the designation to the three Heterorhabditis spp. The mean tolerated temperature ranged from 33.3A degrees C to 40.1A degrees C for non-adapted and from 34.8A degrees C to 39.2A degrees C for adapted strain populations. H. indica was the most tolerant, followed by H. bacteriophora and H. megidis. No correlation was recorded between tolerance assessed with and without adaptation to heat, implying that different genes are involved. Correlation between heat tolerance and mean annual temperature at place of origin of the strains was weak. A high variability in tolerance among strains and the relatively high heritability (hA(2)A = 0.68) for the adapted heat tolerance recorded for H. bacteriophora provide an excellent foundation for future selective breeding with the objective to enhance heat tolerance of H. bacteriophora.

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.

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.

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.

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

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 Amplified ITS region products of rDNA from 25 valid species and one unidentified species from the genus Heterodera and from Meloidodera alni were digested by 26 restriction enzymes. A combination of seven enzymes clearly separated the agriculturally most important species from each other and from their sibling species. Species specific digestion profiles of ITS regions and a table with approximate sizes of digested fragments for several identification enzymes are given. Heterogeneity of ITS regions was revealed for some cyst forming nematode species. Des fragments amplifiés de la région de l’ITS du rDNA de 25 espèces valides et d’une espèce non identifiée du genre Heterodera et de Meloidodera alni ont été soumis à une digestion par 26 enzymes de restriction. La combinaison de sept enzymes a permis une séparation nette des espèces les plus importantes en agriculture, tant les unes par rapport aux autres que par rapport aux espèces jumelles. Sont donnés les profils spécifiques de digestion des régions de l’ITS et un tableau regroupant les tailles approximatives des fragments digérés pour plusieurs enzymes d’identification. L’hétérogénéité des régions de l’ITS a été révélée chez quelques espèces de nématodes à kyste.

Canonical discriminant analysis of four morphometric characters of juveniles and restriction enzymes analysis of ribosomal DNA sequences were used to distinguish Heterodera arenaria, H. aucklandica, H. avenae, H. filipjevi, H. hordecalis, H. iri, H. latipons, H. litoralis, H. schachtii and an undescribed species from grasslands. The results of unweighted pair group cluster analysis showed that H. avenae populations formed three groups and H. filipjevi two groups at the 80% level of similarity. Intraspecific polymorphism was revealed by rDNA-RFLP studies and two types of ITS regions within H. avenae populations can be distinguished. The pattern of restriction bands obtained with BsuRI, PstI and TaqI clearly distinguished populations of H. filipjevi from other species of the H. avenae group. Further enzymes and their combinations distinguished the other species. There are no enzymes which differentiate European populations of H. avenae from H. arenaria. Morphometrics, restriction endonuclease cleavage maps of ITS regions and a dendrogram of putative phylogenetic relations of several cyst-forming nematode species are given. Identification des especes du groupe Heterodera avenae par la morphometrie et les rDNA-RFLP - L'analyse canonique discriminante sur quatre caracteres morphometriques des juveniles et l'analyse des enzymes de restriction de l'ADN ribosomal ont ete utilisees pour identifier Heterodera arenaria, H. aucklandica, H. avenae, H. filipjevi, H. hordecalis, H. iri, H. latipons, H. litoralis, H. schachtii et une nouvelle espece originaire de prairies. Les resultats de l'analyse UPGMA ont montre que les populations d'H. avenae formaient trois groupes et celles d'H. filipjevi deux groupes a un niveau de similarite de 80%. Le polymorphisme intraspecifique a ete revele par des etudes de rDNA-RFLP et deux types de regions de l'ITS peuvent etre mis en evidence dans les populations d'H. avenae. Les modeles de bandes de restriction obtenus avec BsuRI, PstI et TaqI ont identifie clairement les populations d'H. filipjevi des autres especes du groupe H. avenae. D'autres enzymes et leurs combinaisons ont identifie les autres especes. Aucun enzyme n'a differencie les populations europeennes d'H. avenae de H. arenaria. Les caracteres morphometriques, les cartes de clivage des regions des ITS par l'endonuclease de restriction et un dendogramme des relations phylogenetiques supposees sont donnes.