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Taxonomy Phylum Nematoda; class Chromadorea; order Rhabditida; suborder Tylenchina; infraorder Tylenchomorpha; superfamily Tylenchoidea; family Heteroderidae; subfamily Heteroderinae; Genus Globodera. Biology Potato cyst nematodes (PCN) are biotrophic, sedentary endoparasitic nematodes. Invasive (second) stage juveniles (J2) hatch from eggs in response to the presence of host root exudates and subsequently locate and invade the host. The nematodes induce the formation of a large, multinucleate syncytium in host roots, formed by fusion of up to 300 root cell protoplasts. The nematodes rely on this single syncytium for the nutrients required to develop through a further three moults to the adult male or female stage. This extended period of biotrophy—between 4 and 6 weeks in total—is almost unparalleled in plant–pathogen interactions. Females remain at the root while adult males revert to the vermiform body plan of the J2 and leave the root to locate and fertilize the female nematodes. The female body forms a cyst that contains the next generation of eggs. Host range The host range of PCN is limited to plants of the Solanaceae family. While the most economically important hosts are potato (Solanum tuberosum), tomato (Solanum lycopersicum), and aubergine (Solanum melongena), over 170 species of Solanaceae are thought to be potential hosts for PCN (Sullivan et al., 2007). Disease symptoms Symptoms are similar to those associated with nutrient deficiency, such as stunted growth, yellowing of leaves and reduced yields. This absence of specific symptoms reduces awareness of the disease among growers. Disease control Resistance genes (where available in suitable cultivars), application of nematicides, crop rotation. Great effort is put into reducing the spread of PCN through quarantine measures and use of certified seed stocks. Useful websites Genomic information for PCN is accessible through WormBase ParaSite., PCN are biotrophic pathogens with a survival stage that allows them to persist in soil in the absence of a host facilitating their spread to all regions where potatoes are grown.

The impact of increasing temperatures on the population dynamics of the soil‐dwelling nematode Globodera pallida, a persistent and economically important pest of potatoes, was investigated. The reproductive factor (final population⁄initial population) and length of life cycle were found to be temperature sensitive. Pot experiments performed over 4 months allowed comparison of the effect on development of G. pallida of two temperature regimes: an average temperature comparable to current field conditions (14.3 °C) and an average temperature above current field conditions (17.3 °C). A larger second generation of juveniles was observed at 17.3 °C compared to 14.3 °C. Multiplication of G. pallida at field sites in Shropshire and East Lothian (average soil temperatures of 15.5 and 14.1 °C, respectively, during potato cropping) was also examined. A quantitative PCR assay and visual examination of roots were used to monitor the dynamics of the G. pallida populations in both field sites at 4‐weekly intervals. Four cultivars, Desiree, Cara, Maris Piper and Estima, were grown with and without nematicide treatments. Nematicide treatments suppressed population increases at both sites. Females were observed on the roots of cvs Cara and Desiree at the end of the growing season in Shropshire, but not at East Lothian, and are likely to represent a second generation.

This work was supported by the Rural & Environment Science & Analytical Services Division of the Scottish Government, the BBSRC, through the joint projects CRF/2009/SCRI/SOP 0929, BB/L008025/1 and BB/K018299/1. Additional funding was obtained through the James Hutton Institute SEEDCORN initiative, AHDB Potato, the Perry Foundation and The Felix Cobbold Trust. Amanpreet Kaur was supported by the Commonwealth Scholarship Commission through a Commonwealth split-site Ph.D. grant. Key message: The nematode resistance gene H2 was mapped to the distal end of chromosome 5 in tetraploid potato. The H2 resistance gene, introduced into cultivated potatoes from the wild diploid species Solanum multidissectum, confers a high level of resistance to the Pa1 pathotype of the potato cyst nematode Globodera pallida. A cross between tetraploid H2-containing breeding clone P55/7 and susceptible potato variety Picasso yielded an F1 population that segregated approximately 1:1 for the resistance phenotype, which is consistent with a single dominant gene in a simplex configuration. Using genome reduction methodologies RenSeq and GenSeq, the segregating F1 population enabled the genetic characterisation of the resistance through a bulked segregant analysis. A diagnostic RenSeq analysis of the parents confirmed that the resistance in P55/7 cannot be explained by previously characterised resistance genes. Only the variety Picasso contained functionally characterised disease resistance genes Rpi-R1, Rpi-R3a, Rpi-R3b variant, Gpa2 and Rx, which was independently confirmed through effector vacuum infiltration assays. RenSeq and GenSeq independently identified sequence polymorphisms linked to the H2 resistance on the top end of potato chromosome 5. Allele-specific KASP markers further defined the locus containing the H2 gene to a 4.7 Mb interval on the distal short arm of potato chromosome 5 and to positions that correspond to 1.4 MB and 6.1 MB in the potato reference genome. Publisher PDF

Quantitative polymerase chain reaction (qPCR) can be used for the accurate quantification of plant-parasitic nematodes from soil samples. Unlike the traditional Baermann funnel and centrifugal flotation techniques followed by visual enumeration, all developmental stages of plant-parasitic nematodes can be detected and quantified by using qPCR. However, little is known about the amount of DNA between different stages of plant-parasitic nematodes. Here, we show that ITS-copy number varies with developmental stage of Meloidogyne chitwoodi and M. hapla. The number of copies of ITS was lower in cell-stage eggs (51 ± 7.4 for M. hapla and 31 ± 6.0 for M. chitwoodi) than in second-stage juveniles (1345 ± 130.6 for M. hapla and 1036 ± 140.3 for M. chitwoodi) and females (19,508 ± 3367.3 for M. hapla and 9049 ± 316.7 for M. chitwoodi).

Authors acknowledge funding from the Agriculture and Horticulture Development Board (KV) and The James Hutton Institute receives funding from the Rural and Environment Science and Analytical Services (RESAS) Division of the Scottish Government. Postprint

Potato cyst nematodes (PCN) are responsible for large losses in potato yields in many of the world’s potato-growing regions. As soil temperatures increase due to climate change, there is potential for faster growth rates of PCN, allowing development of multiple generations in a growing season. We develop a process-based temperature-dependent model representing the life cycle of Globodera pallida, comprising juvenile, adult and cyst/diapause stages. To incorporate variability in the amount of time spent in each stage caused by genetic/environmental variation, the model is based on a mix of ordinary differential equations (ODEs) with sub-stages, and delay differential equations (DDEs). The effect of climate change is incorporated through the influence of soil temperature on the rate of development and survival in the hatching and juvenile stages. The level of the plant resistance to PCN is incorporated via the proportion of juveniles which become adults. After comparing the model with field data we run simulations to explore the effects of temperature and resistance on PCN populations. We find that with higher temperatures and longer growing seasons multiple generations of PCN can develop within a season, provided any required diapause period is short. Despite this, we show that growing resistant potatoes is a very effective control strategy and planting potatoes with even moderate levels of resistance can counter the effects of climate change.

This work was funded through an AHDB PhD award, the USDA GLOBAL Project and by the Rural and Environmental Science and Analytical Services division of the Scottish Government. Bioinformatics and computational analyses for the generation of the new G. pallida genome assembly were supported by the University of St Andrews Bioinformatics Unit, which is funded by a Wellcome Trust ISSF award (grant 105621/Z/14/Z). Although the use of natural resistance is the most effective management approach against the potato cyst nematode (PCN) Globodera pallida, the existence of pathotypes with different virulence characteristics constitutes a constraint towards this goal. Two resistance sources, GpaV (from Solanum vernei) and H3 from S. tuberosum ssp. andigena CPC2802 (from the Commonwealth Potato Collection) are widely used in potato breeding programmes in European potato industry. However, the use of resistant cultivars may drive strong selection towards virulence, which allows the increase in frequency of virulent alleles in the population and therefore, the emergence of highly virulent nematode lineages. This study aimed to identify Avirulence (Avr) genes in G. pallida populations selected for virulence on the above resistance sources, and the genomic impact of selection processes on the nematode. The selection drive in the populations was found to be specific to their genetic background. At the genomic level, 11 genes were found that represent candidate Avr genes. Most of the variant calls determining selection were associated with H3-selected populations, while many of them seem to be organised in genomic islands facilitating selection evolution. These phenotypic and genomic findings combined with histological studies performed revealed potential mechanisms underlying selection in G. pallida. Publisher PDF

Aphelenchoides besseyi and A. fujianensis have been frequently found in mixed populations associated with forage grass seed in Brazil. The morphological similarity between both species has previously led A. fujianensis to be erroneously identified as A. besseyi. A. besseyi is a quarantine pest in many countries that import Brazilian forage seed; however, there is no current evidence suggesting that A. fujianensis is a plant-parasitic species. Two real-time polymerase chain reaction (qPCR) diagnostics were developed to detect each species and an operational envelope was established. A set of primers and hydrolysis probes for each species was designed targeting the large subunit (LSU) region. To assess their specificity, primers and probes sets were tested with samples of nontarget Aphelenchoides and Paraphelenchus sp. also frequently associated with forage seed. Experiments using dilutions of purified plasmid standards underpinned the sensitivity of the qPCR assays, which detected as few as 10 copies of target nematode ribosomal DNA. Thus, the developed diagnostics were sufficiently sensitive to detect DNA extracted from a fragment of a single target nematode. There was a positive correlation between copy number of the target species and nematode abundance, suggesting the potential of this method for quantification. Evidence of intra-individual variability among cloned sequences of the LSU region in a single A. besseyi population is also reported.

There is an urgent need to develop new interventions to manage pests because evolution of pesticide resistance and changes in legislation are limiting conventional control options for farmers. We investigated β-aminobutyric acid (BABA), jasmonic acid (JA) and fructose as possible plant defence activators against grey mould disease, Botrytis cinerea , and root knot nematode, Meloidogyne incognita. We also tested Trichogramma achaeae parasitoid wasps and an antifeedant plant extract for biocontrol of the invasive tomato leafminer, Tuta absoluta . BABA and JA enhanced resistance of tomato plants to B. cinerea but neither treatment provided complete protection and the efficacy of treatment varied over time with BABA being more durable than JA. Efficacy was partly dependent on tomato cultivar, with some cultivars responding better to BABA treatment than others. Furthermore, treatment of tomato with BABA, JA and fructose led to partial suppression of M. incognita egg mass development. Biocontrol agent, T. achaeae , performance against T. absoluta could be enhanced by adjusting the rearing conditions. Both attack rate and longevity were improved by rearing the parasitoids on T. absoluta rather than on other insects. Finally, Ajuga chamaepitys extract was shown to have significant antifeedant activity against T. absoluta . Our findings suggest that there are potential new solutions for protection of crops but they are more complicated to deploy, more variable and require more biological knowledge than conventional pesticides. In isolation, they may not provide the same level of protection as pesticides but are likely to be more potent when deployed in combination in IPM strategies.

Molecular detection of pest and pathogens relies on rapid and dependable methods for their identification as well as an assessment of their abundance. This study describes the development and evaluation of a diagnostic method for detection ofPratylenchus crenatus,P. penetransandP. neglectus, based on a hydrolysis probe qPCR assay. Primer/probe sets were designed targeting the ITS-1 rDNA. In order to assess the specificity, primer/probe sets were tested with samples of non-targetPratylenchusspecies andRadopholus similis. Experiments using dilutions of purified plasmid standards tested the sensitivity of the hydrolysis assay against detection of DNA extracted from individual nematodes. Target DNA was detected in soil samples collected from potato fields and this indicated thatP. crenatus,P. neglectusandP. penetransare widely distributed in Scotland, frequently co-existing in mixed populations, withP. crenatusmore prevalent than eitherP. neglectusorP. penetrans.

The potato cyst nematodes (PCN) Globodera rostochiensis and Globodera pallida are significant pests of potatoes worldwide. The most effective control methods are crop rotation and the deployment of resistant varieties. Complete resistance to G. rostochiensis based on a single resistance gene has successfully been integrated into many varieties. However, resistance to G. pallida has not been as successful to date, with current varieties only exhibiting partial resistance. Combining partially effective quantitative trait loci (QTLs) for resistance can increase the strength and breadth of the resistance. An additive effect on resistance has previously been demonstrated on combining two QTLs from Solanum tuberosum subsp. andigena (GpaIVsadg) and Solanum vernei (Gpa5). However, populations of G. pallida can be quite divergent and it was unclear whether the relative effects of the individual QTLs and the combined additive effect would be consistent across different G. pallida Pa2/3 populations. Using a mapping population segregating for both QTLs, the effect of the QTLs individually and combined was examined on four UK-derived field populations of G. pallida pathotype Pa2/3, and the relative effects of the individual QTLs and the additive effect of the combination found to be consistent across all populations.

Morphologically similarAphelenchoidesspp. populations extracted from rice and forage grass seeds from different geographical regions in Brazil were morphologically and molecularly characterised. Overall, the populations studied separated into two groups based on morphological and phylogenetic analyses, referred to herein as ‘Group-rice’ and ‘Group-forage’. Bayesian phylogenetic analyses of SSU, LSU and mtCOI regions strongly supported the presence of two dichotomous groups with Group-rice and Group-forage populations genetically similar toA. besseyiandA. fujianensis, respectively. This study reports the presence of a morphologically similar species toA. besseyiassociated with seeds of grasses, but genetically distinct based on three genomic regions, which our results strongly suggest to beA. fujianensis, this being a new geographical record for Brazil. Additional information regarding spicule morphology of maleA. besseyiis also reported.

1.ABSTRACTPasteuria penetransis a gram-positive endospore forming bacterial parasite ofMeloidogynespp. the most economically damaging genus of plant parasitic nematodes globally. The obligate antagonistic nature ofP. penetransmakes it an attractive candidate biological control agent. However, deployment ofP. penetransfor this purpose is inhibited by a lack of understanding of its metabolism and the molecular mechanics underpinning parasitism of the host, in particular the initial attachment of the endospore to the nematode cuticle. Several attempts to assemble the genomes of species within this genus have been unsuccessful. Primarily this is due to the obligate parasitic nature of the bacterium which makes obtaining genomic DNA of sufficient quantity and quality which is free from contamination challenging. Taking advantage of recent developments in whole genome amplification, long read sequencing platforms, and assembly algorithms, we have developed a protocol to generate large quantities of high molecular weight genomic DNA from a small number of purified endospores. We demonstrate this method via genomic assembly ofP. penetrans. This assembly reveals a reduced genome of 2.64Mbp estimated to represent 86% of the complete sequence; its reduced metabolism reflects widespread reliance on the host and possibly associated organisms. Additionally, apparent expansion of transposases and prediction of partial competence pathways suggest a high degree of genomic plasticity. Phylogenetic analysis places our sequence within the Bacilli, and most closely related toThermoactinomycesspecies. Seventeen predicted BclA-like proteins are identified which may be involved in the determination of attachment specificity. This resource may be used to developin vitroculture methods and to investigate the genetic and molecular basis of attachment specificity.

Analysis of the genome sequence of the potato cyst nematode, Globodera pallida, has shown that a substantial gene family (approximately 300 sequences) of proteins containing a SPRY domain is present in this species. This is a huge expansion of the gene family as compared to other organisms, including other plant-parasitic nematodes. Some SPRY domain proteins from G. pallida and G. rostochiensis have signal peptides for secretion and are deployed as effectors. One of these SPRYSEC proteins has been shown to suppress host defence responses. We describe further analysis of this gene family in G. pallida. We show that only a minority (10%) of the SPRY domain proteins in this species have a predicted signal peptide for secretion and that the presence of a signal peptide is strongly correlated with the corresponding gene being expressed at the early stages of parasitism. The data suggest that while the gene family is greatly expanded, only a minority of SPRY domain proteins in G. pallida are SPRYSEC candidate effectors. We show that several new SPRYSECs from G. pallida are expressed in the dorsal gland cell and demonstrate that some, but not all, of the SPRYSECs can suppress the hypersensitive response induced by co-expression of the resistance gene Gpa2 and its cognate avirulence factor RBP-1 in Nicotiana benthamiana.

The root-knot nematodes (genus Meloidogyne) are important plant parasites causing substantial agricultural losses. The Meloidogyne incognita group (MIG) of species, most of which are obligatory apomicts (mitotic parthenogens), are extremely polyphagous and important problems for global agriculture. While understanding the genomic basis for their variable success on different crops could benefit future agriculture, analyses of their genomes are challenging due to complex evolutionary histories that may incorporate hybridization, ploidy changes, and chromosomal fragmentation. Here, we sequence 19 genomes, representing five species of key root-knot nematodes collected from different geographic origins. We show that a hybrid origin that predated speciation within the MIG has resulted in each species possessing two divergent genomic copies. Additionally, the apomictic MIG species are hypotriploids, with a proportion of one genome present in a second copy. The hypotriploid proportion varies among species. The evolutionary history of the MIG genomes is revealed to be very dynamic, with noncrossover recombination both homogenizing the genomic copies, and acting as a mechanism for generating divergence between species. Interestingly, the automictic MIG species M. floridensis differs from the apomict species in that it has become homozygous throughout much of its genome.

Potato cyst nematodes (PCNs), Globodera rostochiensis and G. pallida, cause important economic losses. They are hard to manage because of their ability to remain dormant in soil for many years. Although general knowledge about these plant parasitic nematodes has considerably increased over the past decades, very little is known about molecular events involved in cyst dormancy and hatching, two key steps of their development. Here, we have studied the progression of PCN transcriptomes from dry cysts to hatched juveniles using RNA-Seq. We found that several cell detoxification-related genes were highly active in the dry cysts. Many genes linked to an increase of calcium and water uptake were up-regulated during transition from dormancy to hydration. Exposure of hydrated cysts to host plant root exudates resulted in different transcriptional response between species. After 48 h of exposure, G. pallida cysts showed no significant modulation of gene expression while G. rostochiensis had 278 differentially expressed genes. The first G. rostochiensis significantly up-regulated gene was observed after 8 h and was coding for a transmembrane metalloprotease. This enzyme is able to activate/inactivate peptide hormones and could be involved in a cascade of events leading to hatching. Several known effector genes were also up-regulated during hatching., This work was supported by the Agriflex program, Agriculture and Agri-Food Canada (B.M.), the National Institute for Food and Agriculture from the United States Department of Agriculture (award #2015-69004-23634) (B.M. and V.B.), the Education Spanish Ministry under the “Ayudas para la movilidad postdoctoral en centros extranjeros” scheme (J.E.P.-R.) and by the Scottish Government through the The James Hutton Institute (V.B.).

The Root-Knot Nematodes (RKN; genus Meloidogyne) are important plant parasites causing substantial agricultural losses. The Meloidogyne incognita group (MIG) of species, most of which are obligatory apomicts (mitotic parthenogens), are extremely polyphagous and important problems for global agriculture. While understanding the genomic basis for their variable success on different crops could benefit future agriculture, analyses of their genomes pose challenges due to complex evolutionary histories that may incorporate hybridization, ploidy changes, and chromosomal fragmentation. Here we sequence 19 genomes, representing five species of key RKN collected from different geographic origins. We show that a hybrid origin that predated speciation within the MIG has resulted in each species possessing two divergent genomic copies. Additionally, the MIG apomicts are hypotriploids, with a proportion of one genome present in a second copy, and this proportion varies among species. The evolutionary history of the MIG genomes is revealed to be very dynamic, with non-crossover recombination both homogenising the genomic copies, and acting as a mechanism for generating divergence between species. Interestingly, the automictic MIG speciesM. floridensisdiffers from the apomict species in that it has become homozygous throughout much of its genome.

Dagger and needle nematodes included in the family Longidoridae (viz. Longidorus, Paralongidorus, and Xiphinema) are highly polyphagous plant-parasitic nematodes in wild and cultivated plants and some of them are plant-virus vectors (nepovirus). The mitochondrial (mt) genomes of the dagger and needle nematodes, Xiphinema rivesi, Xiphinema pachtaicum, Longidorus vineacola and Paralongidorus litoralis were sequenced in this study. The four circular mt genomes have an estimated size of 12.6, 12.5, 13.5 and 12.7 kb, respectively. Up to date, the mt genome of X. pachtaicum is the smallest genome found in Nematoda. The four mt genomes contain 12 protein-coding genes (viz. cox1-3, nad1-6, nad4L, atp6 and cob) and two ribosomal RNA genes (rrnL and rrnS), but the atp8 gene was not detected. These mt genomes showed a gene arrangement very different within the Longidoridae species sequenced, with the exception of very closely related species (X. americanum and X. rivesi). The sizes of non-coding regions in the Longidoridae nematodes were very small and were present in a few places in the mt genome. Phylogenetic analysis of all coding genes showed a closer relationship between Longidorus and Paralongidorus and different phylogenetic possibilities for the three Xiphinema species., This research was supported by grants P12-AGR 1486 and AGR-136 from ‘Consejeria de Economia, Innvovacion y Ciencia’ from Junta de Andalucia, and Union Europea, Fondo Europeo de Desarrollo regional, ‘Una manera de hacer Europa’, and grant 219262 ArimNET_ERANET FP7 2012–2015 Project PESTOLIVE ‘Contribution of olive history for the management of soilborne parasites in the Mediterranean basin’ from Instituto Nacional de Investigacion y Tecnologia Agraria y Alimentaria (INIA). The James Hutton Institute receives financial support from the Scottish Government, Rural and Environment Science and Analyical Services Division.

Background The evolution of animal mitochondrial (mt) genomes has resulted in a highly conserved structure: a single compact circular chromosome approximately 14 to 20 kb long. Within the last two decades exceptions to this conserved structure, such as the division of the genome into multiple chromosomes, have been reported in a diverse set of metazoans. We report on the two circle multipartite mt genome of a newly described cyst nematode, Globodera ellingtonae. Results The G. ellingtonae mt genome was found to be comprised of two circles, each larger than any other multipartite circular mt chromosome yet reported, and both were larger than the single mt circle of the model nematode Caenorhabditis elegans. The genetic content of the genome was disproportionately divided between the two circles, although they shared a ~6.5 kb non-coding region. The 17.8 kb circle (mtDNA-I) contained ten protein-coding genes and two tRNA genes, whereas the 14.4 kb circle (mtDNA-II) contained two protein-coding genes, 20 tRNA genes and both rRNA genes. Perhaps correlated with this division of genetic content, the copy number of mtDNA-II was more than four-fold that of mtDNA-I in individual nematodes. The difference in copy number increased between second-stage and fourth-stage juveniles. Conclusions The segregation of gene types to different mt circles in G. ellingtonae could provide benefit by localizing gene functional types to independent transcriptional units. This is the first report of both two-circle and several-circle mt genomes within a single genus. The differential copy number associated with this multipartite mt organization could provide a model system for deconstructing mechanisms regulating mtDNA copy number both in somatic cells and during germline development. Electronic supplementary material The online version of this article (doi:10.1186/s12864-016-3047-x) contains supplementary material, which is available to authorized users.

Plant-parasitic nematodes (PPN) need to be adapted to survive in the absence of a suitable host or in hostile environmental conditions. Various forms of developmental arrest including hatching inhibition and dauer stages are used by PPN in order to survive these conditions and spread to other areas. Potato cyst nematodes (PCN) (Globodera pallida and G. rostochiensis) are frequently in an anhydrobiotic state, with unhatched nematode persisting for extended periods of time inside the cyst in the absence of the host. This paper shows fundamental changes in the response of quiescent and diapaused eggs of G. pallida to hydration and following exposure to tomato root diffusate (RD) using microarray gene expression analysis encompassing a broad set of genes. For the quiescent eggs, 547 genes showed differential expression following hydration vs. hydratation and RD (H-RD) treatment whereas 708 genes showed differential regulation for the diapaused eggs following these treatments. The comparison between hydrated quiescent and diapaused eggs showed marked differences, with 2,380 genes that were differentially regulated compared with 987 genes following H-RD. Hydrated quiescent and diapaused eggs were markedly different indicating differences in adaptation for long-term survival. Transport activity is highly up-regulated following H-RD and few genes were coincident between both kinds of eggs. With the quiescent eggs, the majority of genes were related to ion transport (mainly sodium), while the diapaused eggs showed a major diversity of transporters (amino acid transport, ion transport, acetylcholine or other molecules)., The James Hutton Institute receives funding from the Scottish Government. The authors thank the Education Spanish Ministry for the grant provided for the first author under the “Ayudas para la movilidad postdoctoral en centros extranjeros” scheme.

The potato cyst nematodes (PCN) Globodera pallida and G. rostochiensis are major pests of potatoes. The G. pallida (and G. rostochiensis) life cycle includes both diapause and quiescent stages. Nematodes in dormancy (diapause or quiescent) are adapted for long-term survival and are more resistant to nematicides. This study analysed the mechanisms underlying diapause and quiescence. The effects of several compounds (8Br-cGMP, oxotremorine and atropine) on the activation of hatching were studied. The measurements of some morphometric parameters in diapaused and quiescent eggs after exposure to PRD revealed differences in dorsal gland length, subventral gland length and dorsal gland nucleolus. In addition, the expression of 2 effectors (IVg9 and cellulase) was not induced in diapaused eggs in water or PRD, while expression was slightly induced in quiescent eggs. Finally, we performed a comparative study to identify orthologues of C. elegans diapause related genes in plant-parasitic nematodes (G. pallida, Meloidogyne incognita, M. hapla and Bursaphelenchus xylophilus). This analysis suggested that it was not possible to identify G. pallida orthologues of the majority of C. elegans genes involved in the control of dauer formation. All these data suggest that G. pallida may use different mechanisms to C. elegans in regulating the survival stage., The authors thank the Spanish Ministry of Education for funding to the fi rst author under the ‘ Ayudas para la movilidadpostdoctoralen centrosextranjeros ’ scheme. The James Hutton Institute receives funding from the Scottish Government. Statistical advice from Jim McNicol is gratefully acknowledged (BIOSS, UK).

Summary The potato cyst nematodes (PCNs) Globodera pallida and Globodera rostochiensis are important parasites of potato. PCNs undergo complex biotrophic interactions with their hosts that involve gene expression changes in both the nematode and the host plant. The aim of this study was to determine key genes that are differentially expressed in Globodera pallida life cycle stages and during the initiation of the feeding site in susceptible and partially resistant potato genotypes. For this purpose, two microarray experiments were designed: (i) a comparison of eggs, infective second-stage juveniles (J2s) and sedentary parasitic-stage J2s (SJ2); (ii) a comparison of SJ2s at 8 days after inoculation (DAI) in the susceptible cultivar (Desiree) and two partially resistant lines. The results showed differential expression of G. pallida genes during the stages studied, including previously characterized effectors. In addition, a large number of genes changed their expression between SJ2s in the susceptible cultivar and those infecting partially resistant lines; the number of genes with modified expression was lower when the two partially resistant lines were compared. Moreover, a histopathological study was performed at several time points (7, 14 and 30 DAI) and showed the similarities between both partially resistant lines with a delay and degeneration in the formation of the syncytia in comparison with the susceptible cultivar. Females at 30 DAI in partially resistant lines showed a delay in their development in comparison with those in the susceptible cultivar.

The white potato cyst nematode, Globodera pallida, was recently found in the USA and was identified using molecular and morphometric assessment. This paper examined some biological characteristics of the Idaho population of G. pallida. The samples of the original cysts showed that they had low rates of juvenile hatch and, consequently, low rates of reproduction when compared with glasshouse-reared populations. Virulence tests using sources of partial resistance from Solanum vernei and S. tuberosum ssp. Andigena CPC 2802 compared the Idaho population to European and South American populations of G. pallida. The Idaho population showed a virulence pattern comparable to other European populations in the Pa2/3 virulence group. The overall level of reproduction of the Idaho population on the resistant cultivars was the lowest of all the populations tested, suggesting that it is not highly virulent. These results are compatible with molecular characterisation, which indicates that this population is similar to the European gene pool.

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.

International audience; In this article, we describe the analysis of over 9000 expressed sequence tags (ESTs) from cDNA libraries obtained from various life cycle stages of Globodera pallida. We have identified over 50 G. pallida effectors from this dataset using bioinformatics analysis, by screening clones in order to identify secreted proteins up-regulated after the onset of parasitism and using in situ hybridization to confirm the expression in pharyngeal gland cells. A substantial gene family encoding G. pallida SPRYSEC proteins has been identified. The expression of these genes is restricted to the dorsal pharyngeal gland cell. Different members of the SPRYSEC family of proteins from G. pallida show different subcellular localization patterns in plants, with some localized to the cytoplasm and others to the nucleus and nucleolus. Differences in subcellular localization may reflect diverse functional roles for each individual protein or, more likely, variety in the compartmentalization of plant proteins targeted by the nematode. Our data are therefore consistent with the suggestion that the SPRYSEC proteins suppress host defences, as suggested previously, and that they achieve this through interaction with a range of host targets.

Four field populations of the nematode Globodera pallida were subjected to selection pressure for increased reproductive ability by rearing sub-populations continuously on four partially resistant potato genotypes for 12 generations. The resistance was derived from either Solanum vernei or from S. tuberosum spp. andigena CPC2802. After the 12th generation the original and sub-populations of nematodes were assessed for their reproductive ability on a susceptible genotype and on each of the partially resistant genotypes. Selection pressure was shown to have increased reproductive ability but the increases were specific to the source of resistance used. The average increase on the ex S. vernei clones was from 11% reproduction by the unselected populations to 35·5% reproduction after selection. On the clones derived from CPC2802, which had higher levels of resistance, the increases were larger with an average of 6·6% reproduction for the unselected but 47·4% reproduction after selection. The response to selection differed amongst the initial field populations with some rates of reproduction increased to as much as 79%. A RAPD based analysis of the original and sub-populations after selection indicated small but consistent changes in the genetic structure, which could have been a result of the selection pressure per se and/or the bottlenecks that the populations had gone though.

The cellulose-binding protein gene, cbp , of Meloidogyne javanica encodes a protein that contains a signal secretion peptide and is expressed in the eggs and pre-invasive stage J2s but not from adult females. Several highly similar forms were found in the three root knot nematode species, M. javanica , Meloidogyne incognita and Meloidogyne arenaria indicating very limited divergence of the orthologues and paralogues. The genomic sequences included three introns which differed in size between species and between isolates of M. incognita and M. arenaria , however, the coding regions of the various forms were highly similar. In M. javanica only one form of cbp was found. Treatment of pre-invasive stage J2 nematodes with cbp-dsRNA resulted in a reduction in the ability of J2s to penetrate tomato roots and consequently in a reduction in the number of egg masses produced by the cpb - dsRNA treated nematodes compared to the control nematodes, consistent with an important role of CBP in the early stages of penetration and invasion.

Plant-parasitic nematodes are major agricultural pests worldwide and novel approaches to control them are sorely needed. We report the draft genome sequence of the root-knot nematode Meloidogyne incognita, a biotrophic parasite of many crops, including tomato, cotton and coffee. Most of the assembled sequence of this asexually reproducing nematode, totaling 86 Mb, exists in pairs of homologous but divergent segments. This suggests that ancient allelic regions in M. incognita are evolving toward effective haploidy, permitting new mechanisms of adaptation. The number and diversity of plant cell wall-degrading enzymes in M. incognita is unprecedented in any animal for which a genome sequence is available, and may derive from multiple horizontal gene transfers from bacterial sources. Our results provide insights into the adaptations required by metazoans to successfully parasitize immunocompetent plants, and open the way for discovering new antiparasitic strategies

Following the discovery of Globodera pallida in the Uzhhorod region (Zakarpats'ka oblast') of Ukraine, two populations from this region were further examined. Firstly, their virulence was assessed in relation to two sources of resistance to G. pallida and to other G. pallida populations from Europe and South America. The results showed that the Ukrainian populations were very similar in their virulence to the other European populations. One of the test host genotypes was a new cultivar derived from Solanum tuberosum spp. andigena CPC2802, which proved to be more highly resistant than previously available partially resistant cultivars. The second experiment was a comparison of the mitochondrial Cytochrome B gene from the Ukrainian populations with other European and South American populations. The data indicated that the Ukrainian populations were similar to other European populations.

Recently, a multipartite mitochondrial genome was characterized in the potato cyst nematode, Globodera pallida. Six subgenomic circles were detectable by PCR, while full-length genomes were not. We investigate here whether this subgenomic organization occurs in a close relative of G. pallida. We amplified and sequenced one entire mitochondrial subgenome from the cyst-forming nematode, Globodera rostochiensis. Comparison of the noncoding region of this subgenome with those reported previously for G. pallida facilitated the design of amplification primers for a range of subgenomes from G. rostochiensis. We then randomly sequenced five subgenomic fragments, each representative of a unique subgenome. This study indicates that the multipartite structure reported for G. pallida is conserved in G. rostochiensis. A comparison of subgenomic organization between these two Globodera species indicates a considerable degree of overlap between them. Indeed, we identify two subgenomes with an organization identical with that reported for G. pallida. However, other subgenomes are unique to G. rostochiensis, although some of these have blocks of genes comparable to those in G. pallida. Dot-plot comparisons of pairs of subgenomes from G. rostochiensis indicate that the different subgenomes share fragments with high sequence identity. We interpret this as evidence that recombination is operating in the mitochondria of G. rostochiensis.

We sequenced four mitochondrial subgenomes from the potato cyst nematode Globodera pallida, previously characterized as one of the few animals to have a multipartite mitochondrial genome. The sequence data indicate that three of these subgenomic mitochondrial circles are mosaics, comprising long, multigenic fragments derived from fragments of the other circles. This pattern is consistent with the operation of intermitochondrial recombination, a process generally considered absent in animal mitochondria. We also report that many of the duplicated genes contain deleterious mutations, ones likely to render the gene nonfunctional; gene conversion does not appear to be homogenizing the different gene copies. The proposed nonfunctional copies are clustered on particular circles, whereas copies that are likely to code functional gene products are clustered on others.

This study was supported by the Scottish Society for Crop Research and The University of Leeds Sustainable Agriculture Bursary Fund. SE-vdA is supported by BBSRC grant BB/M014207/1. Distinct populations of the potato cyst nematode (PCN) Globodera pallida exist in the UK that differ in their ability to overcome various sources of resistance. An efficient method for distinguishing between populations would enable pathogen-informed cultivar choice in the field. Science and Advice for Scottish Agriculture (SASA) annually undertake national DNA diagnostic tests to determine the presence of PCN in potato seed and ware land by extracting DNA from soil floats. These DNA samples provide a unique resource for monitoring the distribution of PCN and further interrogation of the diversity within species. We identify a region of mitochondrial DNA descriptive of three main groups of G. pallida present in the UK and adopt a metagenetic approach to the sequencing and analysis of all SASA samples simultaneously. Using this approach, we describe the distribution of G. pallida mitotypes across Scotland with field-scale resolution. Most fields contain a single mitotype, one-fifth contain a mix of mitotypes, and less than 3% contain all three mitotypes. Within mixed fields, we were able to quantify the relative abundance of each mitotype across an order of magnitude. Local areas within mixed fields are dominated by certain mitotypes and indicate towards a complex underlying 'pathoscape'. Finally, we assess mitotype distribution at the level of the individual cyst and provide evidence of 'hybrids'. This study provides a method for accurate, quantitative and high-throughput typing of up to one thousand fields simultaneously, while revealing novel insights into the national genetic variability of an economically important plant parasite. Publisher PDF

This report describes a novel diagnostic method for virus-vector trichodorid nematodes and associated Tobacco rattle virus (TRV) based on a real-time fluorogenic 5' nuclease PCR assay (TaqMan). Two independent primer/probe sets were designed targeting the 18S gene of the ribosomal cistron for the trichodorid species, Paratrichodorus pachydermus and Trichodorus similis. Assays using purified plasmid DNA containing clones of the 18S region and genomic DNA extracted from individuals from both nematode species displayed high specificity as no cros s-reaction was observed between the species or with two non-target trichodorid species Paratrichodorus anemones and Trichodorus primitivus. Relative quantification of target DNA present in unknown samples was performed by comparison of the fluorescence signals of the samples to those obtained from plasmid standard dilutions. Three primer/probe sets were also used to target TRV; one set for RNA1 and the two other sets for RNA2 of specific isolates (TRV-PpK20 and TRV-TpO1). Detection of both trichodorid species and TRV RNA1 and RNA2 from a single sample was achieved and field samples were used to demonstrate the potential of this assay to provide rapid, accurate and sensitive molecular information in relation to risk assessment in the field.

Nine populations of Meloidogyne spp. from Greece have been identified as M. javanica or M. incognita using either isozyme phenotypes or the sequence characterized amplified region-polymerase chain reaction (SCAR-PCR) technique. Virulence against the Mi resistance gene was assayed by pot experiments in controlled conditions and revealed the ability of five populations of M. javanica and one population of M. incognita to reproduce on tomato cultivars containing that gene. A resistance-breaking population of M. incognita is reported for the first time in the country; the M. javanica populations constitute new records for the Greek mainland.

Analyse AFLP de la diversité génétique de Meloidogyne chitwoodi et M. fallax, ravageurs importants des cultures. Les populations de M. chitwoodi et M. fallax se regroupent séparément des autres espèces étudiées. La diversité génétique observée chez M. incognita, M. arenaria, M. javanica, M. hapla et M. mayaguensis est corrélée avec les espèces établies. Deux groupes principaux sont identifiés à l'intérieur de l'ensemble M. chitwoodi / M. fallax, le premier comprenant uniquement des populations de M. chitwoodi, et le second des populations de M. chitwoodi et M. fallax. De plus, M. chitwoodi montre une diversité génétique supérieure à celle de M. fallax et se structure en plusieurs sous-ensembles.

The resistance responses present in 15 wild Solanum species to the potato cyst nematode Globodera pallida were investigated, focusing on hatch, invasion, development, sex determination and fecundity. A wide range of resistance responses was observed among the species, which impacted at different points in the life cycle of G. pallida. For most species a reduced rate of development and subsequent shifts towards an increased male to female ratio, indicative of a poor nutritional host, were observed. However, S. acaule ssp. acaule and S. canasense appeared to possess unique resistance responses, and may comprise valuable sources of new genes for incorporation into potato breeding programmes.

The breadth of resistance to populations of the potato cyst nematodes, Globodera pallida and G. rostochiensis, which differed both in virulence level and country of origin, was investigated in 15 wild Solanum species. A range of responses to the populations was noted, with significant interactions observed between the potato genotypes and Globodera populations assayed. Of the 15 wild Solanum species, seven appeared to be potentially valuable sources of wide spectrum resistance with low multiplication rates with all populations recorded. The inclusion of these species into a potato breeding programme is justified by this study.

Multiplex polymerase chain reaction was used to identify the potato cyst nematodes in soil samples from the Ukraine. The results show the occurrence of Globodera pallida in the Uzhhorod region (Zakarpats’ka oblast’), where only G. rostochiensis had been previously reported. In the mixed potato cyst nematode (PCN) populations, G. pallida was less prevalent (2–5%) than G. rostochiensis (95–98%). A phylogenetic analysis based on ribosomal DNA internal transcribed spacer sequences showed that the Ukrainian population of G. pallida had >99% sequence identity with other G. pallida pa2/3 isolates from Europe. This study has demonstrated that polymerase chain reaction-mediated amplification of specific regions of the potato cyst nematode genome is not only highly effective as a species diagnostic tool but is also a sensitive method which can be used for taxonomic purposes with cyst collections which vary in age.

Due to their capability of eliciting a form of posttranscriptional gene silencing (termed virus-induced gene silencing or VIGS), plant viruses are increasingly used as reverse-genetics tools for functional characterization of plant genes. RNA viruses have been shown to trigger silencing in a variety of host plants, including members of Solanacae and Arabidopsis (Arabidopsis thaliana). Several factors affect the silencing response, including host range and viral tropism within the plant. The work presented here demonstrates that a modified tobacco rattle virus (TRV) vector retaining the helper protein 2b, required for transmission by a specific vector nematode, not only invades and replicates extensively in whole plants, including meristems, but also triggers a pervasive systemic VIGS response in the roots of Nicotiana benthamiana, Arabidopsis, and tomato (Lycopersicon esculentum). This sustained VIGS response was exemplified by the silencing of genes involved in root development (IRT1, TTG1 [transparent testa glabra], RHL1 [root hairless1], and β-tubulin), lateral root-meristem function (RML1 [root meristemless1]), and nematode resistance (Mi). Roots of silenced plants exhibit reduced levels of target mRNA and phenocopy previously described mutant alleles. The TRV-2b vector displays increased infectivity and meristem invasion, both key requirements for efficient VIGS-based functional characterization of genes in root tissues. Our data suggest that the TRV helper protein 2b may have an essential role in the host regulatory mechanisms that control TRV invasion.

Several Paratrichodorus and Trichodorus (trichodorid) nematode species are natural vectors of Tobacco rattle virus (TRV) and cause economically important diseases, especially in potato and ornamental bulbous crops. Identification of trichodorid species based on morphological characters is laborious, time-consuming, and requires the services of highly trained personnel. Molecular diagnostics for trichodorid nematodes, using the ribosomal DNA repeat unit, were successfully developed to distinguish two Paratrichodorus and two Trichodorus species. The complete sequences of the 18S genes and the ITS-1 regions for these species were obtained and species-specific primers successfully designed for them. An RT-PCR assay was developed utilizing isolate-specific primers that amplify serologically distinguishable strains of TRV in individual trichodorid nematodes. The primers were based on the highly conserved RNA-1 segment of the bipartite genome and also on different parts of the RNA-2 segment of the virus genome.

Trichodorids are plant-ectoparasitic nematodes, causing economically important diseases in many crops. In this study, we characterised ribosomal DNA (rDNA) of four trichodorid species from two different genera (i.e., Paratrichodorus macrostylus, P. pachydermus, Trichodorus primitivus and T. similis). Sequence data from the 18S and 5.8S genes showed high similarity; in contrast, sequence data from the internal transcribed spacer regions (ITS) were highly variable. Sequence divergence between the two genera included many insertions and deletions. Trichodorus primitivus and T. similis ITS regions of 1537 and 1303 bp, respectively, are the longest reported to date in the Phylum Nematoda. Further exploration of the sequence data indicated perfect and non-homologous repeats which were confined to the middle of the ITS regions.

Abstract Sequence data were obtained for the full length of the ribosomal DNA (rDNA) 18S gene of specimens from three populations of Paratrichodorus christiei from the USA and four populations of P. minor from Brazil and Portugal. Alignment of the sequences and their application in a phylogenetic analysis revealed that the populations of P.christiei and P.minor formed a single cluster that differentiated from a second cluster comprising P.anemones and P.pachydermus. Previously, P.christiei had been proposed as a junior synonym of P.minor, but this synonymy had not been universally accepted, with many reports, particularly from the USA, continuing to refer to P. christiei. The results obtained here with the sequences of the rDNA 18S gene from the various populations confirm the validity of the synonymy of P.christiei with P.minor.

SUMMARY Some plant endoparasitic nematodes are biotrophic and induce remarkable changes in their hosts in order to ensure a continuous supply of food. Proteins secreted from oesophageal gland cells have been implicated in this pathogenic process. A potentially secreted chorismate mutase has been isolated from the potato cyst nematode Globodera pallida. The gene encoding this protein is expressed in the subventral oesophageal gland cells of the nematode, and the mRNA derived from this gene is only present in the early parasitic stages. Sequence analysis of this gene shows that, like other genes involved in the host-parasite interaction of plant parasitic nematodes, it is likely to have been acquired by horizontal gene transfer from bacteria. The presence of a signal peptide in the deduced amino acid sequence of the G. pallida chorismate mutase and its expression in the subventral oesophageal gland cells suggest that it is secreted from the nematode, pointing to a role for the protein in the host-parasite interaction. The shikimate pathway, of which chorismate mutase is normally a part, is not found in animals but is present in plants and bacteria. In plants it gives rise to a variety of compounds which are important in amino acid synthesis and defence signalling pathways, as well as auxins, which have been implicated in the early development of nematode feeding sites. The potential roles of a nematode chorismate mutase are discussed.