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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

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.

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.

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 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.

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.

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.

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.

The mitochondrial DNA region between the COII and lRNA genes and the 63 base pair tandem repeat region have been used to differentiate and characterise Meloidogyne spp. In this study these regions have been amplified from M. mayaguensis, M. javanica, M. arenaria, M. incognita and M. hapla. Meloidogyne mayaguensis produces a unique product of 705 bp from the COII and lRNA region. Also, a product of 322 bp was produced from the 63 bp repeat region of M. mayaguensis unlike M. javanica, M. arenaria, and M. incognita that exhibit hypervariability in this region. Meloidogyne mayaguensis is a widely distributed root-knot nematode with the potential to cause great economic damage. These molecular diagnostics can be used for accurate identification of M. mayaguensis and can be used to monitor the occurrence and spread of this species, and to provide quarantine services tools to limit its dispersal.

Plant-parasitic nematodes are small and extremely difficult to identify. Previous studies have demonstrated the potential for lectin- or antibody-assisted identification of nematodes. We present an extension of this technology, using antibody- or lectin-coated magnetic beads (Dynabeads) to recover target nematodes from mixtures of specimens. Lectins and antisera that bound specifically and reproducibly to the whole surface of second stage juveniles of Globodera rostochiensis and Meloidogyne arenaria were identified. These were then used as probes bound to Dynabeads to recover nematodes from test suspensions. While both types of probe isolated nematodes from suspension, antibody-coated beads recovered them more efficiently than beads coated with lectins. Other factors that affected the efficiency of recovery, such as the age of the nematode samples, were analysed. This study revealed that Dynabeads coated with a probe of suitable specificity could be used to extract nematodes from mixtures of species. This technology may ultimately be useful in 'non-expert systems' for routine detection and quantification of nematode species.

Abstract Expressed sequence tag (EST) projects offer a rapid route to the discovery of novel genes. Genes expressed in a wide range of parasitic nematodes of medical or veterinary importance have been investigated using EST analysis but these techniques have not yet been applied to plant parasitic nematodes. We describe a small scale EST project using cDNA libraries made from the two species of potato cyst nematode, Globodera rostochiensis and G. pallida, and assess the utility of this approach to identify mRNAs encoding abundantly expressed secreted proteins and other proteins present in the nematode at the onset of parasitism. Approximately 1000 sequences were obtained from G. rostochiensis and 100 from G. pallida. A variety of genes was characterised and approximately 11% of the cDNAs sequenced were apparently PCN specific. Secreted proteins identified included a novel PCN homologue of chorismate mutase, a cDNA recently cloned from the gland cells of Meloidogyne javanica. The results obtained justify a much larger scale application of this technology to these parasites. Utilisation de L'Expressed Sequence Tag pour l'analyse de gènes s'exprimant chez les juvéniles de deuxième stade des nématodes à kyste de la pomme de terre Globodera rostochiensis et G. pallida - l'Expressed Sequence Tag (EST) ouvre une voie rapide vers la découverte de nouveaux gènes. Des gènes s'exprimant chez un large éventail de nématodes parasites d'importance médicale ou vétérinaire ont été étudiés par analyse EST alors que cette technique n'a pas encore été appliquée aux nématodes phytoparasites. Nous décrivons ici un projet EST à petite échelle utilisant les banques d'ADNc constituées à partir de deux espèces de nématodes à kyste de la pomme de terre (PCN), Globodera rostochiensis et G. pallida et nous évaluons l'utilité de cette approche pour identifier les ARNs codant les protéines abondamment sécrétées - et les autres protéines - présentes chez les nématodes lors de l'attaque parasitaire. Mille séquences environ ont été obtenues chez G. rostochiensis et 100 chez G. pallida. Des gènes variés ont été caractérisés et environ 11% des ADNc séquencés sont apparemment spécifiques des PCN. Parmi les protéines sécrétées identifiées figurent un nouvel homologue PCN de la chorismate mutase ainsi qu'un ADNc récemment cloné à partir de cellules glandulaires de Meloidogyne javanica. Les résultats ainsi obtenus justifient une utilisation à plus grande échelle de l'EST pour l'étude de ces parasites.

The conclusions of a collaborative study of the occurrence and importance of root-knot nematodes (RKN, Meloidogyne spp.) and of their control agent, Pasteuria penetrans, in parts of Europe, Africa, South America and the Caribbean are presented. Rootknot nematodes were estimated to reduce the yields of a wide range of horticultural crops by > 25% in Ecuador, Malawi and Tanzania, and by ca 10% in Trinidad and Tobago. The greatest proportion of infected crops were observed in Ecuador (205 of 207) and the least in Trinidad and Tobago (70 of 174). The mean gall index was greatest in Ecuador (5.5). Levels of galling were least in Senegal (1.6), even though 89% of crops were infested and virulent M. mayaguensis was widespread. In all countries, M. incognita and M. javanica were the most abundant species, but M. hispanica occurred widely in Burkina Faso, even in newly cultivated areas in the Sahile. Several new esterase phenotypes were detected, especially in Ecuador and Malawi. Juveniles (J2) collected from the soil during the surveys were examined for attached spores of P.penetrans. It was widespread (20 to 60% of RKN populations), except in Malawi and Tanzania (< 10% were infected), and was found for the first time in Crete (Greece). Generally, < 50% of the J2 carried spores. The occurrence of P.penetrans was sometimes correlated with soil type e.g., in Senegal it was least frequent in sandy soils. Laboratory assays of the binding of spores of isolates of P.penetrans to populations of RKN indicated large variations in specificity and substantial interactions; differences between populations within a species of RKN were sometimes almost as great as those between species. In microplot trials in which an "exotic" isolate of P.penetrans was introduced (ca 103 spores per g soil), its incidence was not increased by increasing the frequency or intensity of the growing of RKN-susceptible crops. However, in two such trials at sites in Tanzania and Ecuador naturally infected with P.penetrans, there were large increases in the proportions of spore-encumbered J2 (up to 100% encumbered) and in the yields of spores (up to 3.3 × 106 spores per mg dry root) in those plots amended with an "exotic" isolate. In these plots, numbers of J2 in the soil were decreased and damage by RKN was suppressed; gall indices were decreased (from > 8 to < 3) and yields were increased (by up to 30%). No such changes were observed in the unamended control plots. Increased suppression of RKN was also observed in a field trial, even in plots where RKN-susceptible and non-host crops were alternated. Increased suppression following amendment with the "exotic" isolate of P.penetrans was not observed at sites not previously infected with P.penetrans. Regression analysis of the results from the microplot and field trials indicated that tomato yields were decreased by > 5% for every increase of one in the gall index. Yields were increased by alternating tomato with leguminous crops in some trials, but not in others. It is proposed that, in natural infections, mutual selection produces a dynamic balance between the P.penetrans and the RKN whereby levels of infection are rarely suppressive. However, the introduction of an "exotic" isolate of P.penetrans, with a different attachment profile, can disturb this balance, resulting in a greatly increased proportion of infected J2 and females, increased yields of spores and more suppression of RKN populations. Importance des nématodes à galles tropicaux (Meloidogyne spp.) et facteurs affectant l'utilité de Pasteuria penetrans, agent de contrôle biologique - Ce travail présente les conclusions d'une étude, menée en collaboration par plusieurs équipes de recherche, sur la présence et l'importance des nématodes phytoparasites du genre Meloidogyne et de leur parasite bactérien, Pasteuria penetrans, dans certains pays d'Europe, d'Afrique, d'Amérique du Sud et des Caraïbes. Les réductions de rendement de cultures maraîchères très diverses dues à ces nématodes atteignent 25% en Equateur, Malawi et Tanzanie, et 10% à Trinidad et Tobago. La plus forte proportion de parcelles infestées a été rencontrée en Equateur (205 sur 207) et la plus faible à Trinidad et Tobago (70 sur 174). C'est en Equateur que l'indice de galles moyen (égal à 5,5) était le plus élevé. Même si cet indice était faible en moyenne au Sénégal (1,6), 89% des cultures étaient infestées dans ce pays, en grande partie par l'espèce virulente M. mayaguensis. M. incognita et M. javanica sont les plus répandues dans tous les pays prospectés. Cependant, M. hispanica est très répandu au Burkina Faso, même dans des zones récemment cultivées en maraîchage en région sahélienne. Plusieurs phénotypes estérasiques nouveaux ont été détectés, spécialement en Equateur et au Malawi. Les juvéniles de second stade (J2) extraits des échantillons de sol collectés lors des prospections ont été examinés pour détecter la présence de spores de P.penetrans sur leur cuticule. Trouvé pour la première fois en Crète (Grèce), P.penetrans est très répandu dans les autres pays prospectés, infestant 20 à 60% des populations de Meloidogyne spp., sauf au Malawi et en Tanzanie où moins de 10% des populations sont atteintes. Le plus souvent, moins de 50% de J2 portent des spores bactériennes. Le taux de parasitisme des J2 par P. penetrans est influencé par les types de sols, comme par exemple au Sénégal où il est très faible dans les sols sableux grossiers. Des expériences en laboratoire portant sur l'attachement de spores de divers isolats de P. penetrans à des populations de Meloidogyne spp. ont révélé une grande variation de la spécificité et des interactions; les différences observées pour diverses populations d'une même espèce de Meloidogyne sont parfois presque aussi marquées que celles observées entre espèces. L'incidence parasitaire d'un isolat "exotique" de P. penetrans introduit dans des microparcelles (ca. 103 sporespar g. de sol) n'a pas été accrue par l'augmentation de la fréquence ou de la densité de plantation des cultures sensibles à Meloidogyne spp. employées. Cependant, dans deux microparcelles natullement infestées en P.penetrans, situées en Equateur et en Tanzanie, l'introduction d'un isolat "exotique" s'est traduite par un accroissement de la proportion de J2 infestés (jusqu'à 100%) et de la production de spores ((jusqu'à 3, 3 × 106 spores par mg [poids sec] de racines), d'une diminution de la population de J2 dans le sol, et d'une disparition des dégâts racinaires; les indices de galles moyens ont diminué (de plus de 8 à moins de 3) et les rendements des cultures ont augmenté (jusqu'à 30%). De tels changements n'ont pas été observés dans des sols non amendés en isolats "exotiques" de P.penetrans. Un meilleur contrôle des populations de Meloidogyne spp. a également été observé au champ, même lorsque la séquence culturale alternait des cultures sensibles et des cultures non-hôtes du nématode. Dans des parcelles non infectées en P.penetrans, la baisse des populations de Meloidogyne spp. n'a pas été observée après introduction d'un isolat "exotique" de la bactérie. Des analyses de régression portant sur les données obtenues en microparcelles ou au champ montrent que les rendements en tomate diminuent de plus de 5% chaque fois que l'indice de galle augmente d'une unité. Ces rendements ont parfois été améliorés lorsque des cultures de légumineuses alternaient les cultures de tomate. Ainsi, sur la base des analyses nématologiques et agronomiques faites en fin de cycles culturaux, il est suggéré que, dans les cas d'infestations naturelles en P.penetrans, des sélections mutuelles entraîneraient un équilibre dynamique entre les populations de la bactérie et celles du nématode, représentatif d'une densité-dépendance retardée. En revanche, l'introduction inondative d'isolats "exotiques" de P.penetrans, aux propriétés parasitaires différentes de celles des populations natives, pourraient rompre temporairement cet équilibre en faveur d'un accroissement de la proportion de nématodes (J2 et femelles) infestés et de la production de spores bactériennes, ainsi qu'un meilleur contrôle des populations de Meloidogyne spp. La capacité des populations de P.penetrans à survivre dans les sols et à contrôler durablement les populations de Meloidogyne spp. dépendraient de la spécificité entre les organismes, des propriétés des sols et des systèmes de culture.

Abstract A targeted survey of vegetable production areas in Crete, Greece, showed that the majority of infestations of rootknot nematodes (RKN) were Meloidogyne javanica. M. incognita appeared to be limited to areas growing pepper that is a host for M. incognita but not for M. javanica. Tests with resistant tomato carrying the Mi gene showed that the M. incognita and the majority of the M. javanica populations were avirulent, and that virulent nematodes could not be selected from them. However, three populations of M. javanica, including some previously identified, were virulent. An AFLP study of the DNA from 22 populations of M. javanica produced 264 scorable amplification products and gave a very high mean similarity (99.4%) between populations, indicating that both virulent and avirulent populations probably derived from the same founder population. Mi-resistant tomato and pepper appear to have considerable utility in the integrated management of RKN in Crete, provided care is taken to monitor the species and virulence or reproductive ability of the nematode populations. Die Variation bei Wurzelgallennematoden (Meloidogyne spp.) auf Kreta in Bezug auf ihre Bekampfung mit resistenten Tomaten und Paprika - Eine gezielte Untersuchung von Gemuseanbaugebieten auf Kreta Griechenland, ergab, dass die Mehrzahl der von Wurzelgallennematoden (RKN) befallenen Flachen mit Meloidogyne javanica verseucht war. M. incognita was offentsichtlich auf Gebiete des Paprikaanbaus beschrankt. Paprika ist ein Wirt fur M. incognita aber nicht fur M. javanica . Versuche mit resistenten Tomaten, die das Mi-Gen besassen, zeigten, dass M. incognita und die Mehrzahl der Populationen von M. javanica avirulent waren, und dass aus ihnen keine virulenten Nematoden selektiert werden konnten. Drei Populationen von M. javanica, darunter schon einige schon vorher identifizierte, waren virulent. Eine AFLP-Untersuchung der DNA von 22 Populationen von M. javanica ergab 264 unterscheidbare Amplifikationsprodukte und zeigte eine hohe mittlere Ahnlichkeit (99,4%) zwischen den Populationen. Dies deutete daraufhin, das virulente and avirulente Populationen wahrscheinlich von der gleichen Grunderpopulation abstammten. Mi-resistente Tomaten und Paprika sind offensichtlich von betrachtlichen Nutzen bei der integrierten Bekampfung von RKN auf Kreta. Voraussetzung ist, dass die Arten und die Virulenz oder die Vermehrungsfahigkeit der Populationen sorgfalting festgestell werden.

A series of sub-populations was produced from a population of Globodera pallida by raising single cyst progenies on three susceptible potato cultivars. After completion of each generation, single cysts were taken to produce the next generation. Six generations were completed and then the resulting sub-populations were multiplied on the susceptible cultivar Ddsir6e. A sample of these sub-populations was then used to conduct a virulence test and to investigate the genetic variation among the sub-populations. The virulence test showed that the susceptible cultivar used in the generation of these sub-populations had a marked effect on the general reproductive capability of the sub-populations and that there was a range of variation in virulence on partially resistant clones. A RAPD study revealed that the range of genetic variation among these sub-populations was comparable to that shown between field populations in other studies and that there was a tendency for the sub-populations to be grouped in relation to the susceptible cultivar they were reared on. These results from an artificially fragmented population are discussed in relation to the introduction and spread of potato cyst nematodes in Europe.