Van Chung Nguyen
email: van-chung.nguyen@inra.fr

Professeur M Pierre Frendo

DEMANGEAT, IRHC, HDR M Gérard

IRHC, HDR Daniel Esmenjaud
email: daniel.esmenjaud@inra.fr

PEROS, DR2, HDR M Jean-Pierre

Chercheur, HDR M Benoît Bertrand

Elisabeth Mme

DR2 Dirlewanger

FOURNET, IR1 M Sylvain

Philippe Castagnone-Sereno

Karine Mulet

Nathalie Marteu

Simon Saucet

Cristina Martin

Paal

Gpa2 G Pallida

S Fendleri Rmc2

M Chitwoodi Janssen

Pepper

Me1

M Incognita Djian

Ma M Arenaria

M Incognita

M Javanica

M Hapla Fery

Dukes

Sugar

M Floridensis Tnl

Peach P Persica

Rmia M Arenaria

Tnl Claverie

Yamamoto & Hayashi

Duval

Almond P Dulcis

Rmja M Arenaria

Tnl Kochba

& Spiegel-Roy

V Champinii N

Diversité génétique du nématode vecteur Xiphinema index sur vigne et application pour optimiser la stratégie de résistance

Keywords: Diversité, vigne domestiquée, durabilité, vigne, microsatellite, gène mitochondrial, Muscadinia rotundifolia, résistance des plantes, propagation des plantes, phylogéographie, Xiphinema, X. index, nématode vecteur diversity, domesticated grapevine, durability, grapevine, microsatellite, mitochondrial gene, Muscadinia, plant resistance, plant propagation, phylogeography, Xiphinema, X. index, vector nematode

Titre: Diversité génétique du nématode vecteur Xiphinema index sur vigne et application pour optimiser la stratégie de résistance

Résumé

Du fait du retrait des nématicides, des alternatives de lutte contre les espèces majeures de nématodes parasites des plantes sont urgentes et la résistance naturelle des plantes est une méthode prometteuse. En vignoble, le nématode Xiphinema index a une forte incidence économique en transmettant le Grapevine fanleaf virus (GFLV), la première virose de la vigne à l'échelle mondiale. Face à ce fléau, des porte-greffe résistants dérivés de la muscadine sont en cours de sélection chez la vigne et utilisent Muscadinia rotundifolia comme source de résistance au nématode vecteur, afin d'arrêter ou retarder la transmission du GFLV. Chez les plantes pérennes et la vigne en particulier, l'interaction longue entre la plante et le nématode accroît le risque de contournement de la résistance. Une étude de génétique des populations a montré que cette espèce parthénogénétique méiotique se reproduit aussi (rarement) par voie sexuée au champ, ce qui peut encore accroître ce risque. La durabilité de la résistance est donc un vrai défi qui doit prendre en compte la diversité génétique du nématode à l'échelle mondiale. Une étude phylogénétique préliminaire avait permis de révéler les clades de diversité prédominants, de sélectionner des populations représentatives et de créer, à partir de ces dernières, des lignées isofemelles utilisées dans notre étude de durabilité de résistance.

Dans ce contexte, la première partie de la thèse a confirmé et approfondi l'approche phylogénétique en s'appuyant sur une gamme élargie d'échantillons de nématodes. Cette étude basée sur des séquences mitochondriales (marqueurs maternels) et sur des locus microsatellites (marqueurs nucléaires neutres) a exploité les données de plus de 80 échantillons représentatifs de l'aire mondiale de distribution de X. index, qui s'étend du Moyen et Proche-Orient, à la partie Est, Centrale et Ouest du Bassin Méditerranéen, et jusqu'aux pays occidentaux (Europe et Amérique du Nord et du Sud). Le marqueur mitochondrial CytB a d'abord été utilisé pour comparer X. index avec une espèce vectrice proche mais amphimictique, X. diversicaudatum. Xiphinema index a montré une variabilité moléculaire nettement plus faible que X. diversicaudatum, en accord avec leurs modes de reproduction respectifs. Nous avons ensuite observé que les séquences concaténées de CytB et de trois autres gènes mitochondriaux ATP6, ND4 et CO1, expriment un patron phylogéographique robuste qui définit trois clades regroupant les échantillons de l'Est Méditerranéen, et un seul clade regroupant les échantillons de Méditerranée de l'Ouest, d'Europe et des Amériques. Le polymorphisme mitochondrial le plus élevé est observé chez l'un des clades du Moyen et Proche-Orient qui englobe les Régions Transcaucasienne et Sud de la Mer Caspienne où la vigne a vraisemblablement été domestiquée et incluraient la région native du nématode. La dissémination ultérieure du nématode d'Est en Ouest durant l'Antiquité vraisemblablement par les Grecs et les Romains se superpose avec celle de sa plante-hôte 'vigne domestiquée'. En Méditerranée de l'Ouest, en Europe et en Amérique, seuls deux haplotypes mitochondriaux très proches sont détectés. Le premier, présent dans les vignobles du Sud de la Péninsule Ibérique, de Bordeaux et de Provence, montre un polymorphisme microsatellite élevé et aurait été introduit durant l'Antiquité. En revanche le second haplotype contient un génotype prédominant très largement répandu dans les pays occidentaux et nous émettons l'hypothèse que ceci est dû à sa dispersion récente lors des replantations massives qui ont suivi la crise phylloxérique de la fin du 19 ème siècle.

La deuxième partie de la thèse a abordé la durabilité de la résistance en conditions contrôlées et au champ. En conditions contrôlées, l'étude a été conduite sur des matériels résistants F1 et BC1 dérivés de la muscadine, en comparant du matériel préalablement multiplié sous forme in vitro et du matériel issu de bouturage ligneux classique. Le facteur de reproduction des nématodes a été suivi sur les plants âgés de 3 à 6 ans inoculés avec un mélange en proportions égales de quatre lignées représentatives retenues précédemment. Nos résultats ont montré que les nématodes élevés sur du matériel issu d'in vitro contournent progressivement la résistance alors que le matériel issu de bouturage classique exprime une résistance durable. Pour le matériel issu d'in vitro, l'hypothèse d'un effet sensibilisant, consécutif à sa longue conservation en conditions in vitro avant son sevrage dans le sol pour la mise en place de l'étude, est mise en avant. La différence d'aptitude d'hôte entre les deux types de matériels élimine a priori l'hypothèse d'une adaptation génétique du nématode comme cela a été observé chez les nématodes à galles Meloidogyne spp. vis-à-vis des Solanacées. Par l'analyse de leurs génotypes microsatellites multilocus (MLGs), des individus hybrides entre lignées différentes ont été détectés à un taux faible mais croissant entre 4 et 6 ans, quels que soient le type de multiplication et le niveau de résistance du matériel végétal. Ces résultats confirment en conditions contrôlées la possibilité de reproduction sexuée déjà observée au champ mais écartent le phénomène d'hybridation comme mode d'adaptation permettant d'aboutir à un contournement de la résistance du matériel issu d'in vitro. Par ailleurs, l'analyse des MLGs a aussi montré des différences d'agressivité entre les lignées en mélange même si aucune n'a été exclue ou au contraire est devenue complètement prédominante au cours des années. La multiplication progressive du nématode chez le matériel résistant issu d'in vitro au cours des années doit être mise en relation avec une architecture différente du système racinaire chez les plantes de ce type et pourrait être due à des modifications physiologiques discrètes mais durables des tissus racinaires apicaux aux dépens desquels le nématode se nourrit. Enfin, en complément de l'étude en conditions contrôlées, l'étude de durabilité au champ a analysé les effectifs de nématodes sur le porte-greffe résistant BC1 et sur des témoins sensibles à X. index dans un essai âgé de 16 ans implanté en parcelle initialement très infestée par des nématodes virulifères. A cette date, les nématodes sont quasiment indétectables sur le matériel résistant qui est dans un bon état de développement végétatif malgré les attaques virales alors que leurs effectifs sont plus élevés, tout en restant très faibles, sur les matériels témoins sensibles à X. index et au GFLV qui sont morts ou fortement dépérissants. Pris en compte globalement, nos résultats montrent que la stratégie de résistance au nématode utilisant du matériel multiplié par bouturage ligneux classique apparaît durable. Cette stratégie ciblée sur le contrôle du vecteur contribuera donc à réduire fortement l'impact du GFLV transmis par X. index en vignoble. III. 1.1.1. Creation and molecular characterization of pure lines of X. index ...............................105 III.1.1.2. The different multiplication modes for plant material .................................................. III. 1.2.1. Materials .......................................................................................................................110 III.1.2.2. Methods.........................................................................................................................111 III. 1.3.1. Evaluation of the resistance durability ..........................................................................115 III.1.3.2. Evaluation of the nematode adaptation .......................................................................119 III. 1.4.1. Evaluation of the resistance durability ..........................................................................126 III.1.4.2. Occurrence of hybridization events in the populations . ................................................128 III.1.4.3. Consequences of the partial breakdown of resistance to X. index in plant material obtained from in vitro ..................................................................................................................129 III. 2.2.1. Plant material ................................................................................................................130 III.2.2.2. Experimental design ......................................................................................................132 III. 2.3.1. Plant development ........................................................................................................132 III.2.3.2. ELISA GFLV infection ......................................................................................................132 III.2.4. Evaluation of nematode numbers collected in 2015 (after 16 years) ...............................134 III. 2.4.1. Materials and methods .................................................................................................134 III.2.4.2. Results ........................................................................................................................... III. 2.5.1. Discussion ......................................................................................................................140 III.2.5.2. Conclusion......................................................................................................................141 Chapter 2 (shown as in the article)

III

III

III.2. RESISTANCE DURABILITY BASED ON

III.2.3. State of the art in

List of abbreviations

Table 1. Mitochondrial haplotypes obtained using CytB, ATP6, CO1 and ND4 gene sequences within the subset of 35 samples (43 individuals) of X. index representing the nematode distribution area Table S1. List and characteristics of the Xiphinema index (Xi) and X. diversicaudatum (Xd) samples of the study with their sample code, individual code, origin (country, region, location) and host plant Table S2. Primers designed to amplify the partial sequences of the mitochondrial genes CytB, ATP6, CO1, and ND4 in Xiphinema index and X. diversicaudatum Table S3. Characteristics of the eight microsatellite loci developed for X. index Table S4. Estimates of evolutionary intra-and interspecific divergences between mitochondrial sequences (Kimura-2-Parameters model) of X. index and X. diversicaudatum Table S5. Genotypes obtained from 8 microsatellite markers within the subset of 35 samples of X. index representing the nematode distribution area Chapter 3

List of figures

Chapter 1

Introduction

Grapevine is one of the most economically important crops worldwide by its products i.e. mainly wine but also table grape, grape juice and raisins. Since the Antiquity, it has known successive growth and crisis phases until today and it is now gaining development in new countries. As many ancient crops, it has to face a very wide and increasing array of pests and diseases. Among these pests, plant-parasitic nematodes (PPNs) have a high impact by their attacks on the roots. Major PPNs are the highly polyphagous root-knot nematodes (RKNs), Meloidogyne spp., and the more specific dagger nematode Xiphinema index, the vector of Grapevine fanleaf virus (GFLV). GFLV is the first grapevine virus by its impact at the global scale. The vector nematode is present in all major world vineyards and is mainly recovered in grapevine fields with a long history of grape cultivation. Besides GFLV grape prophylaxis, control of the vector nematode is the more common strategy to limit virus damage. It used to rely on preplant soil treatments with chemical nematicides combined with fallow crops between two successive grapevines. Nevertheless, nematicides are the most toxic active ingredients used in agriculture and, because of this environmental impact, they are being progressively removed from the market. Fallow strategy requires a very long interval of at least seven years for nematode eradication and this makes it economically unacceptable in premium vineyards. Consequently, alternative nematode management strategies are urgently needed in GFLV infected vineyards.

New management strategies are challenged by biological characteristics of the nematode. The nematode is preferentially located in deep soil layers and this is an important limiting factor for its control. Another major limitation is the high affinity between the virus and its vector and the correlative long retention time of GFLV by the nematode in the soil during the fallow period separating two successive vine crops. Against these constraints, selecting grapevine material carrying germplasm resistance to the nematode and using it for breeding nematode-resistant grapevine rootstocks constitute a promising alternative as it may allow to delay significantly viral transmission. However, in grapevine as in other woody perennial crops occupying the land for several decades, the extended plant-nematode interaction is undoubtedly a major drawback by increasing the risk of resistance breaking, and resistance needs to be evaluated over extents that fit with this long plant life.

In general, plant resistance to ectoparasitic migratory nematodes has been poorly studied in comparison with sedentary endoparasitic nematodes. Nevertheless, in grapevine evaluations performed under field and controlled conditions have early illustrated that host suitability of Vitis species to X. index is variable. Clonal accessions from V. riparia, V. rufotomentosa or V. arizonica have shown their higher resistance within Vitis spp. Parallel studies had also searched for resistance in close genera and accessions within Muscadinia material has been reported as carrying a higher level of resistance than Vitis spp. Muscadine grapes (M. rotundifolia) are now recognized as the best potential sources for resistance to X. index. Consequently, muscadine-derived resistant rootstocks are being selected in grapevine in order to arrest or delay GFLV transmission. Among them, some Vitis x Muscadinia hybrids obtained in France display a high level of resistance but unfortunately major cultural defects inherited from their muscadine parent which render necessary at least one supplemental back cross step. This breeding scheme has been followed to select recently the rootstock 'Nemadex Alain Bouquet' to be used in plots highly infected by the nematode. Considering that this rootstock appears adapted to a limited range of edapho-climatic conditions, new rootstocks are needed to cope notably with the diversity of environments throughout the extensive grapevine geographic distribution.

Few resistance sources are available in muscadine vines. The source from where 'Nemadex Alain Bouquet' has been bred appears particularly interesting for future selection projects. Additional resistance studies are needed to characterize fully the advantages and the limits of the strategy based on this source. This knowledge will allow the integration of the resistance within new management strategies in order to guarantee a sustainable control. Optimizing this resistance strategy needs i) to consider in priority the global diversity of the nematode vector and ii) to take it into account for studies aimed at deciphering both the spectrum and the durability of resistance.

A first appraisal of the nematode genetic diversity of X. index has been obtained through a population genetics study at the field and farm scales. It showed that this meiotic parthenogenetic nematode is also able to reproduce sexually (rarely) in the field. A preliminary phylogenetic work had allowed to reveal the predominant diversity clades and to select representative populations to be used in resistance experiments. In this context, the thesis has a first objective that is to confirm and deepen this phylogeographical approach using an extended geographic coverage of the worldwide nematode distribution.

A consistent assessment of the spectrum and durability conferred by the muscadine source is also indispensable. Characterizing the plant host suitability to X. index leans on both short-and long-duration tests. Short durations allow discrimination of highly susceptible plant material before nematode numbers drop down because of plant damage but, amongst resistant material, longer durations are required to separate early resistance responses from those that are more durable. Notably such long evaluations are essential to discriminate the false (or provisional) resistance resulting from poor root development, from truly resistant material that expresses a sustainable antagonistic effect to nematode multiplication. The second objective of the PhD project is to perform a reliable evaluation of the spectrum and durability of muscadine-derived rootstock material. In conjunction with the perennial status of its grapevine host plant, the ability of X. index to undergo sexual reproduction, even if it is rare, theoretically enhances the risk that the resistance is broken. In this second part, the durability study will be conducted with F1 and BC1 resistant rootstock material. Results should establish whether the nematode is able to adapt to resistance over a long duration post-inoculation and, in the positive case, how this adaptation may occur. Taken together these data will contribute to optimize the strategy for resistance to the vector nematode X. index.

CHAPTER 1 GENERAL INFORMATION, WORK BASES AND OBJECTIVES

This page is intentionally left blank The nematodes are roundworms that belong to the phylum Nematoda. They have successfully adapted to nearly every ecosystem on the earth, from marine and fresh water to soil, and from the polar regions to the tropics, as well as from the highest to the lowest altitudes. They are found in every part of the earth's lithosphere [START_REF] Borgonie | Nematoda from the terrestrial deep subsurface of South Africa[END_REF]. The total number of nematode species has been estimated to be about 1 million [START_REF] Lambshead | Recent developments in marine benthic biodiversity research[END_REF]. So far, more than 25,000 species have been described [START_REF] Zhang | Animal biodiversity : An outline of higher-level classification and survey of taxonomic richness[END_REF][START_REF] Zhang | Animal biodiversity: An update of classification and diversity in 2013[END_REF] of which more than half are parasitic nematodes.

The many parasitic forms include pathogens in most plants and animals as well as humans. In natural or cultivated soils, the nematofauna comprises several feeding types.

These are mainly bacterivores, fungivores, omnivores, herbivores and predators. Nematodes are classified along with insects and other moulting animals in the clade Ecdysozoa, and, unlike flatworms, have tubular digestive systems with openings at both ends.

I.1.2. The plant parasitic nematodes

Among the phylum Nematoda, more than 4,100 species of plant parasitic nematodes (PPNs) have been described (Decraemer et al., 2006) causing an economical damage of approximately $US 80 billion per year [START_REF] Nicol | Current nematode threats to world agriculture[END_REF] [START_REF] Riaz | Use of molecular markers to correct grape breeding errors and determine the identity of novel sources of resistance to Xiphinema index and Pierce's disease[END_REF] and Xiphinema [START_REF] Bouquet | Combination of biotechnological and conventional approaches to rootstock selection presenting a sustainable resistance grape fanleaf disease transmission[END_REF]. Sedentary endoparasites (SE): Meloidogyne [START_REF] Martelli | Compendium of grape diseases[END_REF], Heterodera [START_REF] Andret-Link | Grapevine fanleaf virus: still a major threat to the grapevine industry[END_REF] and Nacobbus [START_REF] Mcdonald | Pathogen population genetics, evolutionary potential, and durable resistance[END_REF]. Migratory endoparasites (ME): Pratylenchus [START_REF] Esmenjaud | Host suitability of Vitis and Vitis-Muscadinia material to the nematode Xiphinema index over one to four years[END_REF] and Hirschmanniella [START_REF] Villate | Control of Xiphinema index populations by fallow plants under greenhouse and field conditions[END_REF]. Migratory semi endoparasites (MSE): Rotylenchus (3), Hoplolaimus (4), Helicotylenchus (5) and Hemicycliophora [START_REF] Siddiqi | H Descriptions of Plant Parasitic Nematodes Set[END_REF].

Sedentary semi-endoparasites (SSE): Rotylenchulus [START_REF] Raski | Strategies against Grapevine fanleaf virus and its nematode vector[END_REF] and Tylenchulus (12) (adapted from [START_REF] Hunt | Identification, morphology and biology of plant parasitic nematodes[END_REF]

Sedentary endoparasitic nematodes

The most damaging nematodes in the world have a sedentary endoparasitic life style.

The two main groups belong to the order Tylenchida and are the root-knot nematodes (RKNs) (Meloidogyne spp.) and the cyst nematodes (Heterodera spp. and Globodera spp.).

Root-knot nematodes (Meloidogyne spp.)

The common name of this genus is due to the characteristic symptom of galls (root knots) induced by the mobile second-stage juveniles while infecting plant roots that subsequently disturb plant development and reduce its growth. This genus is prevalent at the world scale. RKNs are one of the three most economically damaging genera of plant-parasitic nematodes on protected and open field crops and are distributed worldwide. The species with the highest economic impact are parthenogenetic and are able to parasitize a very wide range of plants [START_REF] Sasser | Standardization of host suitability studies and reporting of resistance to to Root-knot nematodes[END_REF]. In the genus Meloidogyne, more than 97 species have been described [START_REF] Hunt | Taxonomy, identification and principal species[END_REF]. Four main Meloidogyne species (M. javanica, M. arenaria, M. incognita and M. hapla) are major pests worldwide. A few emerging species (e.g. M. enterolobii and M. chitwoodi) are also gaining interest by their increasing economical impact [START_REF] Castagnone-Sereno | Diversity and evolution of Root-knot nematodes, genus Meloidogyne : New insights from the genomic Era[END_REF].

Meloidogyne spp. have six life stages: the egg, the four juvenile stages (J1-J4) and the adult. They are separated by five moults, the first moult (J1) occurring within the egg. Eggs grouped in egg masses are the survival stage in the soil or in root debris. Second-stage juveniles or J2s are the sole mobile stage and hatch from eggs to penetrate into the plant. The free phase in the rhizosphere of the host plant is very short because J2s have poor energetic reserves. After penetration, J2s migrate between cortical cells of the root elongation region until the central cylinder in which they settle to undergo their sedentary phase. To become sedentary, they select a few cells of the stele to feed from, thus forming a 'feeding site', grouping several 'giant cells'. In this endophytic sedentary phase, they undergo three molts to become adults. When nematode density is high in root tissues and limits feeding, abundant numbers of males may appear. In the mitotic parthenogenetic prevalent species (M. arenaria, M. incognita and M. javanica), progenies are indeed clones, as males do not participate to reproduction and their occurrence only regulates nematode populations. In meiotic parthenogenetic species such as M. hapla or M. chitwoodi, males, by mating with females, may induce the production of hybrid progenies. The life cycle extends from one to three months, depending on the temperature, and many hundreds of eggs can be produced per female and grouped in a single egg mass (Castagnone-Sereno et al., 2013) (Fig. 1.3). Cyst nematodes (Heterodera spp. and Globodera spp.)

These nematodes belong to the Family Heteroderidae and are unique among PPNs because of the ability of the female to transform into a tough cyst that protects the eggs. The "cyst" is the body of a dead female nematode containing hundreds of eggs. Cysts can persist with viable eggs in the soil for many years, withstanding drought and frost [START_REF] Stone | Co-evolution of nematodes and plants[END_REF] and remaining resistant to chemical and biological stresses. Cyst nematodes contain the two major genera Heterodera and Globodera which induce severe damage to the roots and cause growth retardation and early plant senescence at high population densities.

As RKNs, cyst nematodes have six life stages (egg, four juvenile stages, and adult).

Most species have both male and female adult forms and reproduce sexually. As J1s remain in the egg envelope, J2s hatch directly into the soil and migrate on short distances towards host plant roots. After penetration, they reach the stele tissues by migrating intracellularly, contrary to RKNs. They become sedentary and induce a syncytium fusing several cells on which they feed. They complete their life cycle by molting three times until the adult stage (Jones & Northcote, 1972) (Fig. 1.4).

Migratory endoparasitic nematodes

These nematodes spend much of their life cycle migrating through root tissues, destructively feeding on plant cells. Major genera are Pratylenchus, Radopholus and Hirschmanniella (Fig. 1.2) Furthermore, these nematodes may cause extensive wounds in plant roots, that lead to a potential secondary infection by bacteria and fungi [START_REF] Zunke | Observations on the invasion and endoparasitic behavior of the root lesion nematode Pratylenchus penetrans[END_REF].

They live freely in the soil and lay eggs within the root tissue. Nematodes from the genus Pratylenchus are known commonly as lesion nematodes. They are responsible for root lesion disease on many taxa of host plants in temperate regions around the world. Lesion nematodes usually only feed on the cortex of the roots. Root lesion nematodes infect a great variety of hosts. Pratylenchus penetrans alone has over 350 host plants and its distributed widely throughout temperate areas of the world [START_REF] Corbett | Pratylenchus penetrans. CIH Descriptions of plant-parasitic nematodes[END_REF]. Root lesion nematodes can cause significant yield losses.

Semi-endoparasitic nematodes

During their life cycle, nematodes that behave as semi-endoparasites are able to penetrate partially at different points of the root system for feeding. They are either migratory, e.g. the genera Rotylenchus, Hoplolaimus, Helicotylenchus and Hemicycliophora, or sedentary, e.g. the genera Rotylenchulus and Tylenchulus. The most economically-important species belong to this latter group and are Rotylenchulus reniformis and Tylenchulus semipenetrans (Fig. 1.2)

Ectoparasitic nematodes

In this feeding type, nematodes remain outside the plant and use their stylet to feed from the cells of the roots. Ectoparasitic nematodes can be divided into two main groups in function of the type of damage that they cause. The first group, assembling most genera found in cultivated soils, contains species that are only responsible for direct plant damage.

The second group contains a few genera that cause both direct and indirect plant damage by transmitting plant viruses.

Ectoparasitic nematodes causing only direct damage

These nematodes belong to several common genera often present in crop soils such as Ditylenchus, Tylenchorhynchus and Criconemoides. Plants infested with high densities in the soil may become stunted, turn yellow and produce reduced yields. Some Xiphinema species like X. vuittenezi and X. italiae form small galls on their host root system during their feeding and also belong to this group of nematodes.

Ectoparasitic nematodes causing direct and indirect damage

Besides their direct feeding from the plant roots, some nematode species vector plant viruses. Virus vector nematodes belong to two orders. The first order, Triplonchida (Fig. 1.1), includes the genera Trichodorus and Paratrichodorus (stubby root nematodes) that transmit tubular viruses belonging to the genus Tobravirus (type species Tobacco rattle virus) of the family Virgaviridae. The second order, Dorylaimida, includes the genera Xiphinema (dagger nematodes) (Fig. 1.1), Longidorus and Paralongidorus (needle nematodes), all from the family Longidoridae, that transmit viruses from the genus Nepovirus of the family Secoviridae. Because of their ability to transmit viruses, low nematode numbers in these species can be extremely damaging to plants (Hewitt et al., 1958;[START_REF] Fisher | Feeding of Xiphinema index and X. diversicaudatum[END_REF]. The family Longidoridae comprises the longest plant-parasitic nematodes as their size ranges up-to 12 mm [START_REF] Hooper | Virus vector nematodes-Taxonomy and general introduction[END_REF].

Ectoparasitic nematodes have the simplest life cycle among PPNs (Fig. 1.5). Typically, eggs are laid individually in the soil, hatch at the J2 stage and all larval stages (J2-J4) and adults are parasitic. Root feeding nematodes with short stylets feed on the epidermal and outer cortical cells often at the root tips; those with longer stylets feed deeper in the cortex. Their mode of reproduction may be sexual or asexual (Bridge & Starr, 2007). The genus Xiphinema is of economic importance on grape, strawberry, raspberry, hops and a few other crops. Major nematodes are the vectors species of either European origin, X. index and X. diversicaudatum, or of American origin such as X. americanum, X. californicum, X. rivesi and X. bricolensis. Xiphinema spp. have been shown to induce moderate to large amounts of root damage through root penetration, which in most species results in the formation of galls. Efforts to study these nematodes in more details have proved problematic in some species due to difficulties in maintaining populations in a greenhouse environment.

Virus vector nematodes of the genus Xiphinema may be associated with other non-vector Xiphinema species. In European grapevines, X. index and X. diversicaudatum are often accompanied by the relatively ubiquitous species X. pachtaicum. Other non-vector Xiphinema species in grapevine soils are for example X. italiae in Mediterranean coastal regions and X. vuittenezi in regions with a more continental climate.

I.1.2.2. Control

Control of PPNs used to be mainly based on chemical nematicides. Chemical nematicides are now being banned in most developed countries because of their high acute toxicity to the humans and the environment. Therefore, alternative ways must be considered.

These are mainly i) crop rotation by using non-host plants between two susceptible crops, ii) antagonistic plants that have an adverse effect which reduces the nematode densities in the soil, iii) biocontrol based on nematode enemies or toxic plant products, and iv) plant genetic resistance. Among the previous methods, a promising control alternative appears to be the use of resistant plants.

I.1.3. Resistance to plant parasitic nematodes

A plant is considered as resistant if it has the ability to hinder the growth and/or development of the pathogen (Parlevliet, 1979). In nematology, plant resistance has been defined as the ability of host plants to reduce or prevent nematode reproduction [START_REF] Trudgill | Resistance to and tolerance of plant parasitic nematodes in plants[END_REF].

I.1.3.1. Genetics of resistance

The resistance in a host plant can be roughly divided into two types: horizontal resistance and vertical resistance [START_REF] Vanderplank | Disease resistance in plants[END_REF]). Horizontal resistance is not specific to a race, a strain, a population, or an isolate of the pathogen (or pest), and thus it is characterized by the absence of a specific interaction between the host and each pathogen (or pest) genotype. Vertical resistance is race-, strain-, population-, or isolate-specific and is characterized by a particular interaction between the genotypes of the host and the pathogen (or pest) [START_REF] Vanderplank | Disease resistance in plants[END_REF]. In general, horizontal resistance is considered as polygenic and thus more durable while vertical resistance is considered as monogenic and easier to overcome.

Plants have evolved a complex defense system to resist invaders. Preformed elements of defense, such as cell walls and their reinforcements, are the first barrier for any kind of invader [START_REF] Underwood | The plant cell wall: a dynamic barrier against pathogen invasion[END_REF] and are examples of horizontal resistance. The plant defensive arsenal also includes a broad diversity of constitutively produced toxic phytochemicals [START_REF] Broekaert | Antimicrobial peptides from plants[END_REF] that fit with this type of resistance. To counter these defenses, nematodes use their stylet to puncture the cell wall with physical force while simultaneously releasing a mixture of secretions into the host to aid in penetration [START_REF] Bohlmann | The plant cell wall in the feeding sites of cyst nematodes[END_REF]. Besides these, a more sophisticated system of responses is induced upon infection that is based on the capability of plants to recognize, identify and block the invader at the level of a gene (= protein) called a resistance (R) gene. Such a resistance based on a simple genetic basis is closer to the notion of vertical resistance as it generally controls totally the pathogen (or pest). Nevertheless, the ability of the pathogen to vary and evolve genetically may induce its rapid evolution for adaptation to the intense selection pressure exerted by the R gene and may result in the resistance breaking. A breakdown in resistance is due to an increase in the frequency of pathogen strains that harbor a mutation from avirulence to virulence (McDonald & Linde, 2002). The resistance breaking and on the contrary the resistance durability thus depend on this ability of the pathogen (or pest) to adapt and vary in response to the selection pressure (McDonald & Linde, 2002).

The main genes found out and characterized on different plant species for resistance to nematodes are listed in Table 1.1. All of them have been identified towards root-knot or cyst nematode species, i.e. sedentary endoparasitic nematodes, except XiR1 for resistance to the ectoparasitic species X. index. These R genes differ by their resistance spectrum in particular: some R genes are specific to a single species, for example Hs1 pro-1 and Rmc2, while others control a few (Mi, RMia, RMja, Mur1 and N) or many (Ma) species (Table 1.1). R genes finely mapped or cloned have a NB-LRR core structure and belong either to the TIR-NB-LRR (TNL) family (Gro1-4, Ma, RMia and RMja) or to the CC-NB-LRR (CNL) family (Gpa2, Hero and Mi-1.2). The gene XiR1 [START_REF] Xu | Genetic and QTL analysis of resistance to Xiphinema index in a grapevine cross[END_REF] for resistance to X. index is a TNL gene located on the chromosome 19 of the grapevine genome (Table 1.1).

I.1.3.2. Biological and histological mechanisms

Highly specific nematode resistances can be divided into three major types based on their timing in the ontogeny of feeding structures and their characteristic cytological features (see Goverse & Smant 2014 for a review).

The first type is a rapid hypersensitive response (HR) resulting in host cell death during the initiation of a feeding structure. It takes place early (in annuals, within 24 h of host invasion) and arrests the development of the nematode [START_REF] Paulson | Ultrastructure of the hypersensitive reaction in roots of tomato, Lycopersicon esculentum L., to infection by the root-knot nematode, Meloidogyne incognita[END_REF]. This phenomenon was observed in the interaction between RKNs and either the Mi-1-resistant tomato [START_REF] Melillo | Modulation of reactive oxygen species activities and H2O2 accumulation during compatible and incompatible tomato-root-knot nematode interactions[END_REF] or the Ma-and Me3-mediated resistance from plum and pepper, respectively (Fig. 1.6) (Bleve-Zacheo et al., 1998;[START_REF] Khallouk | Histological mechanisms of the resistance conferred by the Ma gene against Meloidogyne incognita in Prunus spp[END_REF]. The second type of resistance response allows for the initiation but restricts the expansion of nematode-induced feeding structures by the formation of a layer of necrotic cells around the feeding structure. This type was observed for the H1 and Hero resistances to PCN in potato and tomato, respectively [START_REF] Rice | Changes in cell structure in roots of resistant potatoes parasitized by potato cyst-nematodes. I. Potatoes with resistance gene H1 derived from Solanum tuberosum ssp. andigena[END_REF][START_REF] Sobczak | Characterization of susceptibility and resistance responses to potato cyst nematode (Globodera spp.) infection of tomato lines in the absence and presence of the broadspectrum nematode resistance Hero gene[END_REF]. A similar inhibition of the expansion of giant cells of RKNs by necrosis also favors the development of males [START_REF] Kouassi | Resistance to the root-knot nematode Meloidogyne fallax in Solanum sparsipilum: analysis of the mechanisms[END_REF].

The third resistance response type occurs later than the expansion of the feeding structures. This response causes insufficient food supply at this stage and blocks further growth and development of the females. This resistance response type can occur with or without HR [START_REF] Das | Histological characterization of root-knot nematode resistance in cowpea and its relation to reactive oxygen species modulation[END_REF][START_REF] Kandoth | The soybean Rhg1 locus for resistance to the soybean cyst nematode Heterodera glycines regulates expression of a large number of stress-and defense-related genes in degenerating feeding cells[END_REF]. For example, in potato plants with Gpa2 resistance to PCN, a layer of dead cells between the syncytium and the vascular bundle was recorded 10 days after the invasion [START_REF] Koropacka | Molecular contest between potato and the potato cyst nematode Globodera pallida: modulation of Gpa2-mediated resistance[END_REF]. Otherwise, the histological evolution of syncytia of the soybean cyst nematode H. glycines, in soybean genotypes harboring the Rhg1 locus, is not characterized by a typical HR but by the collapse of the syncytia after nuclear and cytoplasmic disintegration [START_REF] Kandoth | The soybean Rhg1 locus for resistance to the soybean cyst nematode Heterodera glycines regulates expression of a large number of stress-and defense-related genes in degenerating feeding cells[END_REF].

I.1.3.3. Molecular mechanisms

At the molecular level, a two-layer detection system has evolved in plants for the purpose of pathogen recognition [START_REF] Jones | The plant immune system[END_REF]Dodds & Rathjen, 2010) (Fig. 1.7). ( 1)

The basal defense is characterized by pattern recognition receptors (PRRs) localized at the cell surface and recognizing pathogen or microbial-associated molecular patterns (PAMPs or MAMPs). PAMPs or MAMPs activate host defence responses (PAMP-triggered immunity or PTI) through a complex signalling cascade. ( 2) The second detection system focuses on effectors secreted by pathogens into the host. These effectors may interfere with PTI responses but are not recognized, leading to effector-triggered susceptibility (ETS). Plants may subsequently be able to recognize effectors by members of a family of intracellular proteins called nucleotide-binding leucine-rich repeat receptor proteins (NLRs). Activation of NLRs leads to a stronger and more intense defense response (effector-triggered immunity, ETI), which often culminates in a form of programmed cell death or hypersensitive response (HR). This phenomenon was well studied for Mi-1 in tomato [START_REF] Melillo | Modulation of reactive oxygen species activities and H2O2 accumulation during compatible and incompatible tomato-root-knot nematode interactions[END_REF], Me3 in pepper [START_REF] Bleve-Zacheo | The pepper resistance genes Me1 and Me3 induce differential penetration rates and temporal sequences of root cell ultrastructural changes upon nematode infection[END_REF], Ma in plum [START_REF] Khallouk | Histological mechanisms of the resistance conferred by the Ma gene against Meloidogyne incognita in Prunus spp[END_REF] for resistance to Meloidogyne spp. and for Hero A in tomato [START_REF] Sobczak | Characterization of susceptibility and resistance responses to potato cyst nematode (Globodera spp.) infection of tomato lines in the absence and presence of the broadspectrum nematode resistance Hero gene[END_REF] for resistance to G.

rostochiensis,

I.1.3.4. Interaction of resistance with plant hormones

Laboratory techniques have allowed the development of in vitro culture methods for intensive plant propagation in particular. Grapevine was among the first plants to be cultured in vitro and reproducible micropropagation techniques were first reported in the late 1960s [START_REF] Galzy | Remarques sur la croissance de Vitis rupestris cultivée in vitro sur différents milieux nutritifs[END_REF]. In vitro culture begins with the excision of a small piece of plant tissue, freeing it from contaminating microorganisms and establishing it in sterile culture. In vitro propagation can be obtained by shoot development from nodal cuttings [START_REF] Galzy | Remarques sur la croissance de Vitis rupestris cultivée in vitro sur différents milieux nutritifs[END_REF], shoot apices [START_REF] Harris | Virus elimination and rapid propagation of grapes in vitro[END_REF][START_REF] Goussard | Effects of cytokinins on elongation, proliferation and total mass of shoots derived from shoot apices of grapevine cultured in vitro[END_REF] and apical tips are commonly used to eliminate viruses and considered the best choice as explant type for general micropropagation purposes [START_REF] Hu | Meristem, shoot tip and bud cultures: A handbook of plant cell culture[END_REF]. Several hormones can be used during the in Several studies have reported the effect of hormones widely used in in vitro cultures on plant resistance. For example, it has been shown that exogenously supplied cytokinins shifted the response of resistant tomato plants (S. lycopersicum) carrying the Mi gene to M. incognita towards susceptibility [START_REF] Dropkin | The hypersensitivity reaction of tomatoes resistant to Meloidogyne incognita: reversal by cytokinins[END_REF] presumably because cytokinin activity was lower in resistant than in susceptible cultivars [START_REF] Gheysen | Gene expression in nematode feeding sites[END_REF]. This highlighted for the first time the cytokinin-mediated suppression of resistance and of the cognate hypersensitive response of tomato roots to the nematode. In the wild resistant tomato S.

sisymbriifolium, resistance was lost in an in vitro medium supplemented with growth hormones (mix of auxin and cytokinin) while the sections of stems and leaves rooted in sand without hormones retained resistance. An additional test with a Mi-resistant tomato cultivar, regenerated from callus and thus using hormones, showed that plants retained resistance to M. incognita but not to M. javanica [START_REF] Fassuliotis | Potential of tissue culture for breeding root-knot nematode resistance into vegetables[END_REF]. In perennials, the influence of cytokinin concentration has also been recorded in the resistant peach rootstock Nemaguard grown in in vitro culture. At high concentration of cytokinin (BA) (2mg/L), the plant lost resistance to M. incognita by forming galls, even though the nematode failed to develop to maturity (incomplete life cycle) [START_REF] Huettel | Influence of cytokinin on in vitro screening of peaches for resistance to nematodes[END_REF]. Previously, [START_REF] Kochba | Level of endogenous cytokinins and auxin in roots of nematode resistant and susceptible peach rootstocks[END_REF] had shown that natural cytokinin activity measured in extracts from Nemaguard was significantly lower than in the susceptible control Baladi. This suggests that high cytokinin levels, either endogenous or applied, correlate with plant susceptibility to rootknot nematodes. Moreover, biologically active cytokinins have been shown to be produced by Meloidogyne spp. [START_REF] Bird | Plant parasitic nematodes: habitats, hormones, and horizontally-acquired genes[END_REF]. On Prunus, Esmenjaud et al. (1993) observed that in vitro rooting of P. cerasifera in a medium supplemented with 0.5 ppm IBA did not influence the subsequent resistance of plants inoculated with M. arenaria 70 days after their acclimatization into a sandy substrate. Resistance of these plants was shown later to be conferred by the Ma gene (Esmenjaud et al., 1996). By contrast, treating resistant tomatoes (cv. Nematex) with a combination of NAA (0.05 ppm) and kinetin (1.07 ppm) altered them to susceptible by producing galls when inoculated with M. incognita, while this resistance alteration was not observed with NAA or kinetin applied separately [START_REF] Sawhney | The role of plant growth hormones in determining the resistance of tomato plants to the root-knot nematode, Meloidogyne incognita[END_REF].

I.1.3.5. Resistance durability

The aim of both breeder and grower is to increase as much as possible the resistance durability. Durability may be highly variable and efforts to obtain and use R factors may be neutralized after different time scales, i.e. from the end of a breeding program up-to over 130 years later such as in the case of the phylloxera aphid resistance of grape rootstocks [START_REF] Pouget | Histoire de la lutte contre le Phylloxera de la vigne en France[END_REF][START_REF] Parlevliet | Durability of resistance against fungal, bacterial and viral pathogens; present situation[END_REF]. Theoretically, absolute durability is believed to not exist.

Definition of durable resistance

As reported above, the major limit to a wide use of genetic resistance against pests and pathogens is their ability to adapt to resistance factors. [START_REF] Johnson | Durable resistance: definition of, genetic control, and attainment in plant breeding[END_REF]1983) defined durable resistance as follows: pathogen resistance is sustainable if it remains effective when a variety is deployed in culture for a large time scale and under favorable conditions to the diseases (or pests). The Johnson's definition does not imply the genetic control of the resistance, its mechanism, or its degree of expression. To go beyond this definition, the reflection will move towards the evaluation of the a priori sustainability and resistance management. Therefore, integrated management aims to increase durability by combining management practices that will constrain the pest population to overcome multiple barriers and decrease the probability that a resistance-breaking genotype will appear. Resistance management is envisaged at two levels: i) the management of genes in planta, that is by introgression of genes or combinations of genes that will be a priori sustainable and ii) the management of genes in situ by the successive deployment of cultivars carrying different genes. Reflection can then commit to three scales: at the gene level, the individual level and the population level. One example of durable resistance is the H1 gene for resistance to G.

rostochiensis in potato (Solanum tuberosum). This gene was introduced and widely grown over 30 years but so far, no nematode virulent populations have been recorded [START_REF] Castagnone-Sereno | Genetic variability of nematodes: A threat to the durability of plant resistance genes?[END_REF] Mechanisms of durability [START_REF] Parlevliet | Identification and evaluation of quantitative resistance[END_REF] indicated that quantitative resistance (QR), closely linked to the notion of horizontal resistance according to Vanderplank, is on average more durable than major gene resistance, a resistance that is related to the vertical resistance. The durability of QR is commonly assumed to be due to the number of genes controlling the trait, though arguments to the contrary have been made [START_REF] Vanderplank | Genetic and molecular basis of plant pathogenesis[END_REF][START_REF] Vanderplank | Host-pathogen interactions in plant disease[END_REF]. Certainly, there are mechanisms other than gene number that could contribute to durability of QR. For example, selection coefficients against individual genes controlling QR will be smaller than those against major gene resistance. The degree of host genotype x pathogen genotype specificity may be less for minor gene than major gene resistance. Host × pathogen x environment interaction could also play an important role for genes of minor effect [START_REF] Kulkarni | Enviroment as the cause of differential interaction between host cultivars and pathogenic races[END_REF]. There have long been concerns that QR may select for pathogen virulence and/or aggressiveness [START_REF] Mundt | Durable resistance: A key to sustainable management of pathogens and pests[END_REF].

Pyramiding consists in stacking R genes into a single genotype or cultivar. Pyramiding R genes can be one solution to improve both durability and level of resistance [START_REF] Nelson | Stabilizing racial populations of plant pathogens by use of resistance genes[END_REF], because, in theory, the pathogen would need double or multiple mutations to overcome the resistance. Besides a quantitatively higher level of resistance, the additional value of pyramided R genes could be a prolonged durability and resistance against a broader spectrum of pathogen races. Therefore, pyramiding of several R genes in a single cultivar should be an effective strategy to control diseases [START_REF] Stukenbrock | The origins of plant pathogens in Agroecosystems[END_REF] or nematodes [START_REF] Djian-Caporalino | Pyramiding, alternating or mixing: Comparative performances of deployment strategies of nematode resistance genes to promote plant resistance efficiency and durability[END_REF]. Pyramiding Me1 and Me3 genes increased the resistance durability to M. incognita in pepper (Capsicum annuum) [START_REF] Djian-Caporalino | Pyramiding, alternating or mixing: Comparative performances of deployment strategies of nematode resistance genes to promote plant resistance efficiency and durability[END_REF], but this was not the case for Bph1 and Bph2 on rice against brown plant hopper in which resistance level of the pyramided line was equivalent to that of the Bph1-single gene [START_REF] Sharma | Marker-assisted pyramiding of brown planthopper (Nilaparvata lugens Stal) resistance genes Bph1 and Bph2 on rice chromosome 12[END_REF].

Resistance/virulence in plant-pathogen interaction

In an evolutionary sense, any resistance is ultimately transitory. In the gene-for-gene model [START_REF] Flor | Current status of the gene-for-gene concept[END_REF], the interaction between a pest and its hosts is an endless race in the nature, where the pest attacks the host, which, in its turn, fights back with its own arsenal. This arms race is often supported by the fact that both host R genes and pest Avr genes undergo positive selection, which can lead to a coevolution between host resistance and pest virulence [START_REF] Parlevliet | Coevolution of host resistance and pathogen virulence; possible implications for taxonomy[END_REF][START_REF] Kraaijeveld | The coevolution of host resistance and parasitoid virulence[END_REF]Singh et al., 2018). Knowing how long a resistant plant (carrying R factors) is able to withstand the selection pressure of a pest is a major challenge. In the context of plant pathology, [START_REF] Shaner | Nomenclature and concepts of pathogenicity and virulence[END_REF] defined that the 'virulent' pathogen is the genotype that can develop and reproduce on host plants carrying resistance genes. Hence, we used here 'virulence' in the standard plant pathology sense, meaning the nominal ability of the nematode to cause a susceptible response on a host plant carrying a given resistance gene. In plant-pathogen interactions, virulence has been identified in fungi, bacteria, viruses and nematodes [START_REF] Castagnone-Sereno | Genetic variability of nematodes: A threat to the durability of plant resistance genes?[END_REF][START_REF] Parlevliet | Durability of resistance against fungal, bacterial and viral pathogens; present situation[END_REF][START_REF] Lecoq | Durable virus resistance in plants through conventional approaches: a challenge[END_REF].

The evolution of pathogens and of nematodes in particular may reduce the effectiveness of a resistance and makes it become shorter. According to McDonald & Linde (2002), pathogens with high evolutionary potential are more able to overcome a resistance than those with a lower evolutionary potential. These authors also predicted the risk of a pathogen by estimating the operating parameters of its populations such as the mode of reproduction, the population size, the gene and genotype flow (Fig. 1.8). Whereby, the highest risk belongs to the populations that have a mixed reproductive mode (both asexual and sexual reproductions), a big size, and whose subpopulations exchange many migrants

Nematode virulence

Virulence in sedentary endoparasitic nematodes Virulence has been detected and studied in many sedentary nematodes. Cases of natural occurrence or laboratory selection of virulent Meloidogyne spp. populations have been described towards diverse R genes (Table 1.2).

For example, virulent Meloidogyne populations to the Mi tomato gene have been detected in many tomato growing areas in the world [START_REF] Kaloshian | Resistancebreaking " nematodes identified in California tomatoes[END_REF][START_REF] Eddaoudi | Identification of the resistance breaking populations of Meloidogyne on tomatoes in Morocco and their effect on new sources of resistance[END_REF][START_REF] Ornat | A population of Meloidogyne javanica in Spain virulent to the Mi resistance gene in tomato[END_REF][START_REF] Castagnone-Sereno | Selection and parasite evolution: A reproductive fitness cost associated with virulence in the parthenogenetic nematode Meloidogyne incognita[END_REF][START_REF] Devran | Occurrence of virulent root-knot nematode populations on tomatoes bearing the Mi gene in protected vegetable-growing areas of Turkey[END_REF].

Reproduction of the nematodes was significantly higher on heterozygous than on homozygous tomato genotypes indicating a dosage effect of the Mi gene [START_REF] Jacquet | Variation in resistance to the root-knot nematode Meloidogyne incognita in tomato genotypes bearing the Mi gene[END_REF]. In laboratory experiments from avirulent M. incognita populations in pepper, virulent variants could be selected toward the Me3 gene but not towards the Me1 gene [START_REF] Castagnone-Sereno | Selection for Meloidogyne incognita virulence against resistance genes from tomato and pepper and specificity of the virulence/resistance determinants[END_REF][START_REF] Balmelle | La viticulture antique en Aquitaine[END_REF]. In these RKNs, virulence to mitotic parthenogenetic nematodes is acquired progressively and mutated genes have been associated with the acquisition of resistance [START_REF] Castagnone-Sereno | Genetic variability and adaptive evolution in parthenogenetic root-knot nematodes[END_REF][START_REF] Castagnone-Sereno | Selection and parasite evolution: A reproductive fitness cost associated with virulence in the parthenogenetic nematode Meloidogyne incognita[END_REF]. As expected from their theoretical evolutionary potential (McDonald & Linde, 2002), virulence seems to be acquired quicker in the case of meiotic nematodes such as M. hapla and M. chitwoodi than in mitotic nematodes. In cyst nematodes, the sexual reproduction may allow a rapid adaptation of the nematodes to the major R genes or R QTLs which they are faced to [START_REF] Bird | Signatures of adaptation to plant parasitism in nematode genomes[END_REF][START_REF] Fournet | Phenotypic and genomic modifications associated with Globodera pallida adaptation to potato resistances[END_REF] Virulence in ectoparasitic nematodes Contrary to sedentary endoparasitic nematodes in Solanaceous crops for which the adaptative phenomena underlying virulence to a specific R gene are now better understood, virulence has never been studied in migratory ectoparasitic nematodes. A single R gene (XiR1) has been evidenced to X. index but this gene has not yet been used in breeding and no data are available on its durability [START_REF] Xu | Genetic and QTL analysis of resistance to Xiphinema index in a grapevine cross[END_REF].

Virulence or adaptation are more difficult to study, given that these species remain in the soil while feeding from the plant and are less dependent on the plant state. At their larval or adult stages, dagger nematodes in general and X. index in particular have a higher natural potential to survive than sedentary endoparasitic nematodes. Indeed, these latter nematodes are extremely fragile when they have undergone the fixed endophytic part of their cycle. If they fail to complete it, they cannot survive while migratory ectoparasitic nematodes may remain alive, even though their feeding is unsuccessful. Consequently, even though it has been based on results obtained up-to 6 years, our study on the resistance durability of X.

index developed further will be blurred by the fact that the nematode can survive for several years in the soil.

I.2. THE GRAPEVINE

In the Vitaceae family, the Vitis genus consists of ~ 60 interfertile species that exist almost exclusively in the Northern Hemisphere and out of which approx. 15 have been used

in breeding programs (Table 1.3).

I.2.1.1. Botanical and genetic data

This species is the wine producer and the producer of table grapes and raisins.

Currently, there are more than 10,000 varieties of V. vinifera, but only a few are of commercial significance for wine production (Sechrist, 2017). Cultivars of V. vinifera form the basis of the majority of wines produced around the world. All of the familiar wine varieties belong to V. vinifera, which is cultivated on every continent except Antarctica. Domesticated vines have hermaphrodite flowers.

The grapevine (V. vinifera L.) is diploid and has a genome size of 475-504.6 Mb [START_REF] Thomas | Repetitive DNA of grapevine: classes present and sequences suitable for cultivar identification[END_REF][START_REF] Lodhi | In situ hybridization in Vitis vinifera L[END_REF]Jaillon et al., 2007;[START_REF] Velasco | A high quality draft consensus sequence of the genome of a heterozygous grapevine variety[END_REF]

I.2.1.2. Evolution and history

The grape has an ancient historical connection with the development of human culture. The grapevine (V. vinifera) might have grown wild in the Caucasus foothills of modern Georgia, Azerbaijan and Armenia 100,000 years ago. The domestication of grape seems linked to the discovery of wine. At first, people could not store the wine and consumed it only seasonally. The invention of pottery allowed year-round storage at least 9,000 years ago (Sechrist, 2017). To date, two forms (wild and cultivated) still co-exist in Eurasia and in North Africa: the cultivated form, V. vinifera subsp. vinifera (or sativa) and the wild form V. vinifera subsp. sylvestris (or sylvestris), sometimes referred to as a separate subspecies (See This et al., 2006 for a review). During domestication, the biology of grapes underwent several dramatic changes to ensure greater sugar content for better fermentation, greater yield and more regular production. Although wild grapes were probably present in many places in Europe during the Neolithic period, archaeological and historical evidence suggest that grapevine domestication occurred in the Near-East (McGovern et al., 1996;McGovern, 2003;Sechrist, 2017).

Historical studies (Zohary, 1996;This et al., 2006;Sechrist, 2017) completed by robust genetic markers (chloroplast DNA, microsatellites) [START_REF] Arroyo-García | Multiple origins of cultivated grapevine (Vitis vinifera L. ssp. sativa) based on chloroplast DNA polymorphisms[END_REF][START_REF] Bacilieri | Genetic structure in cultivated grapevines is linked to geography and human selection[END_REF] allow to propose a hypothesis of the dissemination of grapevine from its native area (Fig.

I.2.2. The muscadine grape

The muscadine grape (Muscadinia rotundifolia Michx.) is native to the southern United

States and has been cultivated for over 400 years [START_REF] Lane | Breeding muscadine and southern bunch grapes[END_REF]. In this thesis, the name "muscadine" will be used exclusively for M. rotundifolia. The muscadine grape differs from the familiar bunch grape (V. labrusca, V. vinifera) in several morphological characteristics, including that it has smaller clusters, its berry has a thick skin and a unique fruity aroma.

Muscadinia grapes have 40 somatic chromosomes (2n = 40) and are characterized by fruits borne in many clusters with few berries per cluster. A long-standing goal of both Vitis and M.

rotundifolia breeding programs has been the development of hybrids between these groups, combining fruit quality from V. vinifera with disease resistance and environmental adaptation of muscadines [START_REF] Bouquet | Vitis x Muscadinia hybridization: A new way in grape breeding for disease resistance in France[END_REF][START_REF] Olmo | The potential role of (vinifera x rotundifolia) hybrids in grape variety improvement[END_REF][START_REF] Goldy | Embryo culture as a means of introgressing seedlessness from Vitis vinifera to V. rotundifolia[END_REF].

I.2.3. Grapevine rootstocks

Nowadays, more than 80% of the vineyards worldwide use grafted plants [START_REF] Smith | Investigations into the mechanisms underlying grapevine rootstock effects on scion growth and yield[END_REF]. Those are scions of V. vinifera grafted onto a rootstock that contains one or several desirable features like abiotic or biotic stress resistances.

I.2.3.1. Origin of rootstock use in viticulture

The root aphid phylloxera (Daktulosphaira vitifolia Fitch) was introduced in Europe (1858-1860) through the importation of infected rooted vines from North America [START_REF] Pouget | Le Phylloxera et les maladies de la vigne. La lutte victorieuse des savants et des vignerons français (1850-1900[END_REF]. The pest lead to the complete destruction of the vineyards in southern France at the end of the nineteenth century and, within 20 years, phylloxera had killed virtually every vineyard in France. In 1869, Laliman -a winegrower in Bordeaux-was the first to suggest a genetic control of phylloxera pest by grafting the susceptible wine varieties onto the American phylloxera-resistant rootstock species [START_REF] Pouget | Le Phylloxera et les maladies de la vigne. La lutte victorieuse des savants et des vignerons français (1850-1900[END_REF]. Therefore, the introduction of several indigenous American non-vinifera Vitis species (Table 1.3) in Europe was urgently performed at that time to be used directly as phylloxera-resistant rootstocks or as source materials for the creation of resistant rootstocks. Some of them are still useful now as source material for resistance to grapevine fungi such as downy and powdery mildews. Because it had solved the problem of the phylloxera epidemics, grafting onto 'American' Vitis rootstocks became, from that period, an obligate practice in viticulture. It is common now in all regions where grapevine is the main crop, except in some isolated areas or regions that have not been contaminated by the aphid or that benefit from particular soil conditions (pure sand, winter flooding…).

I.2.3.2. Selection and creation of grapevine rootstocks

The majority of rootstocks used today are hybrids involving several of the three species V. berlandieri, V. riparia and V. rupestris [START_REF] Galet | Grape varieties and rootstock varieties[END_REF]. Because of the rootstock use, the cultivated grapevine is a combination of two genomes. The rootstock genotype represents the root system of the vine, i.e. the link between the edaphic environment and the plant. Rootstocks are responsible for water and mineral uptake and play an important role for storage. The grafting point defines the interface between shoot (above ground organs, i.e. the cultivar of V. vinifera) and root (below ground organs, i.e. the rootstock cultivar). From the first discovery of phylloxera-resistant rootstocks up to now, grafting sensitive V. vinifera varieties onto resistant rootstocks of North American origins has been the only viable long-term and sustainable solution for grapevine phylloxera management. Using resistant rootstocks are also a privileged way to select for grapevine resistance or adaptation to drought, diseases and pests (including nematodes) [START_REF] Ollat | Rootstocks as a component of adaptation to environment[END_REF]. Muscadines exhibit good resistance traits to pests in particular, making them promising components of rootstocks in viticulture but they show poor performances to key abiotic features such as adaptation to dry and calcareous soils. Hybridization with appropriate Vitis species should allow in the future to combine these abiotic features with resistances to pests and diseases. The rootstock resistance to X. index, notably in Muscadinia material, will be discussed in detail further in the paragraph 'I.4.2' entitled 'The resistance against X. index'.

I.3. THE FANLEAF DEGENERATION OR 'COURT-NOUÉ' DISEASE

Currently, approximately 60 viruses belonging to different families have been reported

on grapevines (Vitis spp.) [START_REF] Martelli | Directory of virus and virus-like diseases of the grapevine and their agents[END_REF]. Therefore, grapevine is the crop with the highest number of viral diseases. Among the grapevine viruses, Grapevine fanleaf virus, Grapevine leafroll disease, Grapevine rugose wood disease and Grapevine fleck virus are the four main disorders having a worldwide economic importance (Andret-Link et al., 2004;[START_REF] Naidu | Grapevine leafroll disease and associated viruses: a unique pathosystem[END_REF], and several others such as Red blotch virus or Grapevine pinot gris virus are considered as emerging [START_REF] Maliogka | Control of viruses infecting grapevine[END_REF][START_REF] Basso | Grapevine virus diseases: economic impact and current advances in viral prospection and management[END_REF].

I.3.1. The viruses involved in the 'Court-noué' disease

The fanleaf degeneration of grapevine or 'Court-noué' disease is due to several viruses that all belong to the Nepovirus group. Among them, GFLV, transmitted by X. index, is by far the most frequent. Another major agent of 'Court-noué' is Arabis mosaic virus (ArMV) transmitted by X. diversicaudatum. The other causal agents of fanleaf degeneration are mainly Tomato black ring virus (TBRV) transmitted by L. attenuatus, Tobacco ringspot virus (TRSV), Grapevine chrome mosaic virus (GCMV) and Tomato ringspot virus (ToRSV) [START_REF] Sanfaçon | Secoviridae: a proposed family of plant viruses within the order Picornavirales that combines the families Sequiviridae and Comoviridae, the unassigned genera Cheravirus and Sadwavirus, and the proposed genus Torradovirus[END_REF] (Table 1.4). In 1970, Cohn & Nitzany reported that X. italiae is another vector of GFLV but this result has never been confirmed (Martelli & Taylor 1990;Taylor & Brown 1997).

Thus, it is unlikely that X. italiae acts as a specific vector of GFLV [START_REF] Brown | Transmission of viruses by plant nematodes[END_REF].

Consequently, X. index is admitted as the unique vector for GFLV.

In the thesis, we will simplify by considering GFLV as the causal agent of 'Court-noué'

or grapevine fanleaf degeneration and X. index as its sole vector nematode.

Paralongidorus maximus Raspberry ringspot virus RpRSV Europe

The host range of GFLV includes several species of Chenopodium and Nicotiana [START_REF] Dias | Host range and properties of grapevine fanleaf and grapevine yellow mosaic viruses[END_REF]. Although Cynodon dactylon (Bermuda grass) was reported to be a host [START_REF] Izadpanah | Bermuda grass as a potential reservoir host for Grapevine fanleaf virus[END_REF], the primary host of GFLV in the agroecosystem is grapevine (Andret-Link et al., 2004).

I.3.2. The Grapevine fanleaf virus

GFLV is a plant pathogenic virus that belongs to the genus Nepovirus of the family Secoviridae. It is transmitted from grapevine to grapevine by the ectoparasitic nematode X. index (Hewitt et al., 1958). Fuchs, 2005). Once infected, the grapevine will decrease its productive life to 15-20 years instead of 30-40 years or longer with a healthy plant. That is an enormous and highly worrying problem in premium vineyards. Once grapevine is infected with the virus, along with the presence of X. index, dissemination of the vector nematode with the soil will help to spread it at a wide scale and will increase the impact of the disease.

I.3.2.1. Symptoms and impact

A survey conducted in 2004 by ONIVINS (Office National Interprofessionnel des Vins) indicated that only 30% of French vineyards were uninfected by 'Court-noué' viruses (Fig.

1 .11). 2004). 70% of French vineyards were infected (of which 35% were severely infected) and only 30% were free of virus.

I.3.2.2. The GFLV structure

GFLV particles are polyhedral with a diameter of 28 nm and consist of three serologically indistinguishable density components called top (T), middle (M), and bottom (B) [START_REF] Quacquarelli | Properties of Grapevine fanleaf virus[END_REF][START_REF] Brown | Transmission of viruses by plant nematodes[END_REF][START_REF] Mayo | Nepoviruses: molecular biology and replication[END_REF]. T component particles are empty shells, M component particles contain RNA2, and B component particles contain both RNA species [START_REF] Quacquarelli | Properties of Grapevine fanleaf virus[END_REF]. The GFLV genome consists of two single strands positive sense RNA1 and RNA2, with a molecular weight of 2.4x10 6 for RNA1 and 1.4x10 6 for RNA2. The two genomic RNAs carry a small covalently linked viral protein (VPg) at their 5' extremity and a poly(A) stretch at their 3' end [START_REF] Pinck | A satellite RNA in Grapevine fanleaf virus strain F13[END_REF]. RNA1 is 7,342 nucleotide Infectivity requires both RNA1 and RNA2 [START_REF] Quacquarelli | Properties of Grapevine fanleaf virus[END_REF].

I.3.2.3. Control of the virus

As there is no curative control of Nepovirus-infected grapevines in vineyard situations, the best way to control GFLV disease in a given area is to keep it out of this area through a system of quarantine, inspections and certifications. Once a grapevine plant is infected by the virus, it will be systematically disseminated into that plant (cell-to-cell movement) and it will become impossible to eliminate. Nevertheless, when the vector is not present in this area, prophylaxis using virus-free plant material is sufficient to solve the problem at the next generation. Hence, GFLV infection can be combatted by ( 1) using GFLV-free plants or propagation materials when the vector is not present in the grapevine field, ( 2) performing an effective management of X. index when the vector is present in order to slowdown the viral dissemination. Hereafter, we will focus on the first point about the virus eradication by summarizing i) the methods that allow the sanitation of virus-infected plant material, ii) the subsequent diffusion of this virus-free plant material through regulatory production schemes,

iii) the natural resistance to GFLV in grapevine, and iv) the research approaches based on genetic engineering that may prevent in the future the virus multiplication in the plant.

Sanitation of GFLV-infected clones

GFLV-infected grapevine material, and high-value clonal accessions in particular, has been and can still be freed of viruses by heat therapy and/or meristem culture [START_REF] Torres-Viñals | Large-scale production of somatic embryos as a source of hypocotyl explants for Vitis vinifera micrografting[END_REF][START_REF] Gambino | Elimination of Grapevine fanleaf virus from three Vitis vinifera cultivars by somatic embryogenesis[END_REF]. Somatic embryogenesis has also been used to efficiently eliminate several phloem-limited viruses from grapevine material [START_REF] Goussard | The effectiveness of in vitro somatic embryogenesis in eliminating fanleaf virus and leafroll associated viruses from grapevines[END_REF]. The success rate is close to 100% with this method without using heat therapy [START_REF] Gambino | Elimination of Grapevine fanleaf virus from three Vitis vinifera cultivars by somatic embryogenesis[END_REF].

Multiplication of virus-free plant material

At the national scale, organizations have been created to evaluate the plant material, eradicate the virus if needed, and then multiply or regulate the multiplication of virus-free

Natural resistance to GFLV in grapevine

In California, several wild clonal accessions of V. vinifera, originating from Iran and Afghanistan, have been described as naturally resistant to GFLV [START_REF] Walker | The genetics of resistance to Grapevine fanleaf virus in Vitis vinifera[END_REF][START_REF] Walker | Sources of resistance to Grapevine fanleaf virus (GFV) in Vitis species[END_REF] but the results have not been confirmed and this research direction has been abandoned. In a complementary approach, the search for systematic detection of natural resistance to GFLV in Vitis material, conducted at INRA Colmar in France [START_REF] Lahogue | Investigations on natural resistance genes for two grapevine viruses: The fanleaf degeneration and the leafroll disease[END_REF] has been unsuccessful. Muscadines (M. rotundifolia) are hosts for GFLV even though this species and its hybrids with Vitis materials appear non or weekly affected by the virus (Walker et al., 1994a;Walker et al., 1994b) (A. Bouquet, pers. com.). Consequently despite the high diversity of Vitis and Muscadinia species [START_REF] Myles | Genetic structure and domestication history of the grape[END_REF][START_REF] Reisch | Grape[END_REF], over sixty years of GFLV resistance screening have not conclusively produced proven natural GFLV resistance in any grapevine genotype [START_REF] Oliver | Tolerance and resistance to viruses and their vectors in Vitis sp.: A virologist's perspective of the literature[END_REF].

Research approaches for virus control based on genetic engineering

Genetic engineering is an attractive way to develop GFLV-resistant rootstocks, especially since specific virus resistance traits can be inserted directly into desirable elite material [START_REF] Fuchs | Transgenic resistance: State of the art and perspectives[END_REF]. A highly-delayed infection has been reported in transgenic Nicotiana benthamiana expressing the CP gene of GFLV [START_REF] Bardonnet | Protection against virus infection in tobacco plants expressing the coat protein of grapevine fanleaf nepovirus[END_REF]. From that time many studies reported the obtention of transgenic grapevine lines expressing a GFLV-derived gene, but only a few of them showed the efficiency of the viral resistance induced [START_REF] Jelly | Transient expression of artificial microRNAs targeting Grapevine fanleaf virus and evidence for RNA silencing in grapevine somatic embryos[END_REF][START_REF] Hemmer | Nanobody-mediated resistance to Grapevine fanleaf virus in plants[END_REF]. Another way was using RNA silencing, a potent immune defense against viruses [START_REF] Jardak-Jamoussi | Development and evaluation of a GFLV inverted repeat construct for genetic transformation of grapevine[END_REF][START_REF] Winterhagen | Gene silencing and virus resistance based on defective interfering constructs in transgenic Nicotiana benthamiana is not linked to accumulation of siRNA[END_REF][START_REF] Gambino | Transgene silencing in grapevines transformed with GFLV resistance genes: analysis of variable expression of transgene, siRNAs production and cytosine methylation[END_REF] or microRNAs (miRNAs). This latter approach is based on the use of miRNAs and their tailored modification for virus resistance. Modified miRNAs are referred to as artificial miRNAs promising since they suggest that such transgenic grapevines are likely to be of practical interest for the control of GFLV.

However, only a few reports address the efficiency of these constructs to induce viral resistance in field conditions. Resistance to GFLV in transgenic rootstocks expressing the GFLV CP gene has been reported after a three-year trial in a naturally infected vineyard in France [START_REF] Vigne | Field safety assessment of recombination in transgenic grapevines expressing the coat protein gene of Grapevine fanleaf virus[END_REF]. Nevertheless, until now and because of the non or at least difficult acceptation of GMOs by the civil society, there has not been any transgenic grapevine or rootstock grown at the field scale in French and European vineyards.

I.4. THE VECTOR NEMATODE XIPHINEMA INDEX

I.4.1. General information

I.4.1.1. Systematics of X. index and close grapevine Xiphinema species

Xiphinema index belongs to the order Dorylaimida and the family Longidoridae. The genus Xiphinema was first described by [START_REF] Thorne | A monograph of the nematodes of the superfamily Dorylaimoidea[END_REF] and X. index was first identified and described by Thorne & Allen (1951) (Fig. 1.18).

Systematics of Longidoridae species used to rely on morphological and morphometrical features. There were progressively completed by molecular sequences. Most often both the ribosomal sequences such as ITS1 [START_REF] Vrain | Intraspecific rDNA restriction fragment length polymorphism in the Xiphinema americanum group[END_REF][START_REF] Cherry | The internal transcribed spacer region of Belonolaimus (Nemata: Belonolaimidae)[END_REF], 28S

(D2-D3 fragment) [START_REF] Nunn | Nematode molecular evolution: an investigation of evolutionary patterns among nematodes based upon DNA sequences[END_REF] and 18S (SSU fragment) [START_REF] Holterman | Phylum-wide analysis of SSU rDNA reveals deep phylogenetic relationships among nematodes and accelerated evolution toward crown clades[END_REF], or the mitochondrial sequences such as the gene CO1 (He et al., 2005) were used. Using the 28S (D2-D3 fragment) sequence, [START_REF] Tzortzakakis | Integrative diagnosis and molecular phylogeny of dagger and needle nematodes of olives and grapevines in the island of Crete, Greece, with description of Xiphinema cretense n. sp. (Nematoda, Longidoridae)[END_REF] illustrated the phylogenetic relationships within the Xiphinema species (Fig. 1.14). The phylogenetic tree obtained allows to separate, with a high boostrap support, most species and shows that both virus vectors X. index and X.

diversicaudatum are closely located while non vector species X. vuittenezi and X. italiae are distant and relatively grouped in the basal part of the dendrogram.

I.4.1.2. Morphology and anatomy

The X. index adult length ranges between 2.5 and 3.5 mm with a long protrusible stylet The male, with a similar body length, is rare and has paired spicules close to the anus [START_REF] Thorne | Paratylenchus hamatus n. sp. and Xiphinema index n. sp., two nematodes associated with fig roots, with a note on Paratylenchus anceps Cobb[END_REF].

I.4.1.3. Geographic distribution

Xiphinema index is present in all major grape-growing regions in Europe, Asia, North and South America, Africa and Oceania (CABI source, Fig. 1.15). Its frequency is generally high in vineyards with a long history of grape cultivation. The nematode has most often been found associated with GFLV transmission to grapevine. Xiphinema index probably originated in the Middle East and was later introduced into different Mediterranean areas [START_REF] Brown | Comments on the occurrence and geographical distribution of longidorid nematodes in Europe and the Mediterranean region[END_REF]Villate, 2008).

I.4.1.4. Identification

Besides X. index, several Longidorids species may be present in grapevine fields. These are mainly X. diversicaudatum, X. vuittenezi, X. italiae and X. pachtaicum. Some of them can be found in a same soil sample, particularly in European vineyards, and most often at low densities [START_REF] Esmenjaud | Vertical distribution and infectious potential of Xiphinema index (Thorne et Allen, 1950) (Nematoda: Longidoridae) in fields affected by Grapevine fanleaf virus in vineyards in the Champagne region of France[END_REF]. Therefore, the species identification is the first important step before any control strategy.

Morphological and morphometrical identification

Species identification in Xiphinema used to be based mainly on classical diagnostic features. A combination of about 20 morphometric and morphological features has been established to elaborate dichotomous or polytomous keys for the identification of different nematodes species. The main diagnostic traits are body length, structure and size of the stylet, structure and position of the guide ring, shape and size of the lips, amphidials, and position and size of the pharyngeal gland nuclei. Additional features at the species level are the development and structure of the female reproductive system, tail shape in all developmental stages, and presence or absence of males. Several keys that relied only on the observation of these features have been constructed. The most reliable and complete has been published in 1975 by Luc & Dalmasso and is usable under the form of a lattice, which is easier to handle than classical dichotomous keys. It does not consider the Xiphinema species from the socalled American group that contains many small-sized species of the American continent out of which some are virus vectors (X. americanum sl, X. americanum ss, X. bricolensis, X.

californicum) (Table 1.4).

However, the high degree of intraspecific morphometric variability can lead to considerable overlap of many characteristics among Xiphinema species and increase the risk markers for the identification of a few species that might be sympatric with X. index in grapevine: the vector species X. diversicaudatum and the non-vector species X. vuittenezi, and X. italiae. These markers are sensitive enough to identify a single nematode, regardless of its development stage (larvae or adult), within a nematode community in vineyard soils or within a mixed population consisting of non-vector and vector nematodes (Fig. 1.17).

Nevertheless, because such SCARs can only distinguish the species that have been considered together for their primer design, their use cannot be universal. A much higher resolution identification can reasonably be obtained using sequences of genes or targeted fragments such as the ribosomal or mitochondrial DNAs. Thus, recently, numerous species from Longidorus and Xiphinema were molecularly characterized with such DNA as tools for species identification (Gutiérrez-Gutiérrez et al., 2011;[START_REF] Archidona-Yuste | Remarkable diversity and prevalence of dagger nematodes of the genus Xiphinema Cobb, 1913 (Nematoda: Longidoridae) in olives revealed by integrative approaches[END_REF][START_REF] Lazarova | Morphological and molecular characterisation, and phylogenetic position of X. browni sp. n., X. penevi sp. n. and two known species of Xiphinema americanum-group (Nematoda, Longidoridae)[END_REF]. Blasting on NCBI, the sequences obtained from appropriate primers (Table 1.5) showed that X. index, X. diversicaudatum and the non-vector species X. vuittenezi, X. italiae and X.

pyrenaicum can be unambiguously distinguished from others and between each other. They constitute the basis of a universal barcoding adapted to grapevine nematode species (V.C.

Nguyen and D. Esmenjaud, 2017, unpublished X. italiae; lanes 6 and 7, X. diversicaudatum + X. italiae; lanes 8 and 9, X. vuittenezi + X. italiae + X.

index; lanes 10 and 11, X. diversicaudatum+ X. vuittenezi + X. italiae + X. index (adapted from [START_REF] Wang | Multiplex polymerase chain reaction identification of single individuals of the Longidorid nematodes Xiphinema index, X. diversicaudatum, X. vuittenezi, and X. italiae using specific primers from ribosomal genes[END_REF]. Eggs are laid singly in the soil close to the feeding site [START_REF] Weischer | Feeding behaviour and pathogenicity of Xiphinema index on grapevine roots[END_REF], hatch a few days later to juvenile 1 (J1) and then molt 4 times to reach the adults stage (Fig. 1.18

A). Males are rare. Under controlled conditions, X. index requires approx. 3 months to complete its life cycle [START_REF] Taylor | On the transmission of grape fanleaf by Xiphinema index[END_REF][START_REF] Wyss | Xiphinema index, maintenance and feeding in monoxenic cultures[END_REF] but in the field the life cycle may last one to several years depending on biotic and abiotic factors (Weischer, 1975). Under adverse conditions, including low moisture, low temperature, and the absence of host plants, X. index undergoes a quiescent phase during which physiological functions are limited and development is interrupted [START_REF] Antoniou | Arrested development in plant parasitic nematodes. Helminthological Abstracts[END_REF]. index population increased more rapidly as the soil temperature increased from 16 to 28° C.

According to [START_REF] Cohn | Investigations on the life cycles and host preference of some species of Xiphinema and Longidorus under controlled conditions[END_REF], the life cycle took 3 to 5 months to complete at 28°

C, but slowed down to 7-9 months at 20 -23° C. In Italy, it was found that X. index numbers are lower in winter [START_REF] Coiro | Population changes of Xiphinema index in relation to host plant, soil type and temperature in southern Italy[END_REF][START_REF] Coiro | Possible herbaceous hosts for Xiphinema index in virus transmission experiments[END_REF]. By contrast, in California the X.

index populations peaked in the winter [START_REF] Feil | Effects of seasonal and site factors on Xiphinema index populations in two California vineyards[END_REF] possibly due to more accurate sampling in moist soils. In England, under controlled conditions, X. index egg-laying peaked during summer months and reached maximum populations in autumn [START_REF] Siddiqi | Co-evolutionary interactions between host resistance and pathogen avirulence genes in rice-Magnaporthe oryzae pathosystem[END_REF] All stages, including adult females, move through the soil to find and feed on roots (Nicholas et al., 2007). Xiphinema index feeds near the root tips by inserting its stylet into the root tissues [START_REF] De Klerk | Relationship between grapevine roots and soil-borne pests. The Grapevine Root and Its Environment[END_REF]. The time that X. index stays at one feeding site can vary from several minutes to several days. Root areas already fed on, attract more nematodes and can result in crowding [START_REF] Weischer | Feeding behaviour and pathogenicity of Xiphinema index on grapevine roots[END_REF].

Host plants and symptoms

Xiphinema index has a limited host range. Vitis spp. are known as the main host but fig (Ficus carica) is a better host plant than grapevines [START_REF] Brown | The reproductive capacity and longevity of Xiphinema index (Nematoda: Dorylaimida) from three populations on selected host plants[END_REF]. This justifies the use of fig as the preferred plant for nematode multiplication under controlled conditions.

Xiphinema index has also been recorded as present on roses and citrus (Nicholas et al., 2007), on petunia, tomato, C. amaranticolor and tobacco [START_REF] Coiro | Possible herbaceous hosts for Xiphinema index in virus transmission experiments[END_REF].

Xiphinema index not only causes indirect damage by GFLV transmission but also direct damage on grapevine root tips by inducing terminal swellings and necroses (Fig. 1.20).

Xiphinema index feeding initially causes a swollen club-like gall on root tips. The feeding wound then becomes reddish brown to black and forms slightly sunken lesions on the roots [START_REF] Wyss | Root and cell response to feeding by Xiphinema index[END_REF]. Common symptoms of X. index feeding are plant stunting, chlorosis, galls forming and then root necrosis. This galling has been shown to occur as early as 24 hours after feeding [START_REF] Fisher | Feeding of Xiphinema index and X. diversicaudatum[END_REF]. The combined effect of X. index feeding and its association with GFLV may kill grapevines (Nicholas et al., 2007).

Distribution in the soil

In general, soil texture and structure are critical for the development of soil-inhabiting nematodes [START_REF] Pitcher | Factors influencing the movement of nematodes in soil[END_REF]. Pore space and moisture capacity are physical characteristics that determine soil suitability as a habitat for nematodes. Nevertheless, X. index has been recovered from a wide range of soil types (sandy soil, silt and clay heavy soils, etc.). Therefore, its distribution is mostly limited by adequate moisture and the presence of suitable hosts.

However, it prefers heavy soils that are less subject to drought but is also present in sandy soils if moisture is sufficient [START_REF] Esmenjaud | Vertical distribution and infectious potential of Xiphinema index (Thorne et Allen, 1950) (Nematoda: Longidoridae) in fields affected by Grapevine fanleaf virus in vineyards in the Champagne region of France[END_REF].

The horizontal and vertical distribution of X. index in vineyard soils is generally clustered and closely related to the distribution of roots. Its density is higher within than between vine rows [START_REF] Feil | Effects of seasonal and site factors on Xiphinema index populations in two California vineyards[END_REF], and the aggregated pattern of nematode distribution correlates with GFLV-infected grapevines [START_REF] Villate | Spatial distribution of the dagger nematode Xiphinema index and its associated Grapevine fanleaf virus in French vineyard[END_REF]. Usually absent from shallow soil layers, X. index is located 0.3-1.5 m deep in vineyard soils where fine rootlets are most abundant [START_REF] Esmenjaud | Vertical distribution and infectious potential of Xiphinema index (Thorne et Allen, 1950) (Nematoda: Longidoridae) in fields affected by Grapevine fanleaf virus in vineyards in the Champagne region of France[END_REF][START_REF] Feil | Effects of seasonal and site factors on Xiphinema index populations in two California vineyards[END_REF]Villate et al., 2008) (Fig. 1.21). Specimens have even been detected at a depth of 3.6 m [START_REF] Raski | Survival of Xiphinema index and reservoirs of fanleaf virus in fallowed vineyard soil[END_REF].

The movement of X. index is limited to a few dozens of cm per year [START_REF] Pitcher | Factors influencing the movement of nematodes in soil[END_REF][START_REF] Taylor | The persistence and spread of Xiphinema diversicaudatum in cultivated and uncultivated biotopes[END_REF]. It moves horizontally and vertically in the soil by following the progression of the roots from which it feeds [START_REF] Thomas | Migration of Longidorus elongatus, Xiphinema diversicaudatum and Ditylenchus dipsaci in soil[END_REF]. Xiphinema index are disseminated with the soil through the use of contaminated equipment such as tractors, planters, planting of infested plants/sods, and soil transfer. Additionally, X. index may be dispersed passively by water streams or irrigation canals [START_REF] Roccuzzo | Notes on nematodes found in irrigation water in southern Italy[END_REF].

I.4.1.6. Genetic diversity at the field, local and regional scales

Genetic diversity of X. index has been studied at the field, local and regional scales using species-specific microsatellites (Villate et al., 2009). At the grapevine field scale, microsatellite markers showed no isolation by distance but a differentiation among sampling points within the grapevine field (Villate et al., 2010). This pattern is consistent with the clonal reproductive mode (rate of 95-100%) and the limited capacities of active dispersal of Xiphinema spp., leading to a patchy spatial distribution within grapevine fields [START_REF] Villate | Spatial distribution of the dagger nematode Xiphinema index and its associated Grapevine fanleaf virus in French vineyard[END_REF]2010). Moreover, a small number of hybridization events were identified at the overlapping zones between different genetic clusters, i.e. probably where two independent nematode introduction events and their cognate patches came into contact (Villate et al., 2010). [START_REF] Villate | Spatial distribution of the dagger nematode Xiphinema index and its associated Grapevine fanleaf virus in French vineyard[END_REF] confirmed the preferred mode of nematode dissemination along the row in the grapevine field.

I.4.1.7. Interaction X. index -GFLV

Virus transmission Hewitt et al. (1958) reported that the dagger nematode X. index is the vector of GFLV. In 1963, typical symptoms of fanleaf degeneration were reproduced in grapevines following graft transmission from GFLV-infected C. amaranticolor and the virus was successfully transferred by sap transmission from symptomatic grapevines to C.

amaranticolor [START_REF] Dias | Host range and properties of grapevine fanleaf and grapevine yellow mosaic viruses[END_REF]. It is worth noting that this discovery was controversial in the early 1960's because phylloxera was believed to be involved in fanleaf degeneration.

Virus retention

The transmission of GFLV by X. index is characterized by a specific and complementary association [START_REF] Brown | Specificity exclusivity and complementarity in the transmission of plant viruses by plant parasitic nematodes: An annotated terminology[END_REF]. Xiphinema index feeds on growing root tips and acquires GFLV particles upon feeding (Hewitt et al., 1958;Raski et al., 1983;[START_REF] Wyss | Xiphinema index, maintenance and feeding in monoxenic cultures[END_REF]. The transmission process is mediated by the ability of X. index to ingest GFLV particles from a virus source grapevine, retain virions at specific retention sites within its feeding apparatus, and subsequently infect a recipient vine by release of virus particles from the retention sites.

Specificity determinants

The X. index/GFLV interaction is very specific. GFLV is transmitted exclusively by this nematode which itself cannot transmit any other Nepovirus [START_REF] Demangeat | Transmission competency of single-female Xiphinema index lines for Grapevine fanleaf virus[END_REF]. The nematode-specific basis of Nepovirus transmission is uncertain. Different strains of X. index reproduce at different rates but did not differ in GFLV transmission competencies [START_REF] Demangeat | Transmission competency of single-female Xiphinema index lines for Grapevine fanleaf virus[END_REF]. The transmission specificity of GFLV maps to their respective 2C CP (Andret-Link et al., 2004) and a 11 amino-acid region of the coat protein (in the βB-βC loop of the two-fold axis of the coat protein junction) determines transmission specificities of X.

index [START_REF] Schellenberger | A stretch of 11 amino acids in the betaB-betaC loop of the coat protein of Grapevine fanleaf virus is essential for transmission by the nematode Xiphinema index[END_REF].

Dissemination of GFLV

The dissemination of GFLV on grapevine may be through different ways, such as its vector X. index (from infected plant to healthy plant during feeding (Fig. 1.22)), human activities (grafting and soil transfer) and seeds (occasional) (Andret-Link et al., 2004;Villate et al., 2010). The transfer of soil contaminated with viruliferous X. index and/or GFLV-infected plant material allows GFLV dissemination over short and long distances [START_REF] Villate | Spatial distribution of the dagger nematode Xiphinema index and its associated Grapevine fanleaf virus in French vineyard[END_REF].

However, the main worldwide dissemination of GFLV is via grafting and extensive exchange of propagation material [START_REF] Martelli | Nematode-borne viruses of grapevine, their epidemiology and control[END_REF]. Despite rigorous sanitary controls, producers lack efficient technologies to control the infection since nematicide chemicals tend to be prohibited. Interestingly, GFLV is presumably native to V. vinifera (Hewitt, 1968;Raski et al, 1983) and probably originated from the same area as domesticated grapevines, i.e. the Caucasus regions between the Black and Caspian seas. GFLV is likely to have been disseminated from this center of origin throughout Europe and then to the rest of the World (Andret-Link et al., 2004).

Nevertheless, despite the implementation of effective measures to limit GFLV dissemination over long distances, the virus remains a recurrent problem in established diseased vineyards.

I.4.1.8. The integrated control of X. index

Control strategies against X. index aim to eliminate or reduce X. index numbers in the soil around the grapevine root system. The control of X. index must face two major spatial and temporal limiting factors. These are i) the location of the nematode in deep soil layers and ii) the survival of the nematode and of the virus in the nematode for over 4 years.

Chemical control

Until recently, X. index control strategies relied almost exclusively on soil disinfection to reduce X. index numbers and delay viral transmission. Before applying nematicides, it is recommended to pull out all the root system, carefully remove the root debris in deep soil layers and dry the soil as much as possible [START_REF] Van Zyl | Xiphinema index and its relationship to grapevines: A review[END_REF]. However, chemical treatments have a limited efficiency particularly in grapevine plots because they only diffuse in the upper soil layers (0 to 30-40 cm) and cannot reach the layers beyond where the highest nematode densities are usually found and feed on deep roots [START_REF] Thomason | Chemical control of nematode vectors of plant viruses[END_REF][START_REF] Esmenjaud | Vertical distribution and infectious potential of Xiphinema index (Thorne et Allen, 1950) (Nematoda: Longidoridae) in fields affected by Grapevine fanleaf virus in vineyards in the Champagne region of France[END_REF]Villate et al., 2008) (Fig. 1.21).

Chemical nematicides are being banned in an increasing number of countries, due to their adverse environmental effects [START_REF] Abawi | Impact of soil health management practices on soilborne pathogens, nematodes and root diseases of vegetable crops[END_REF])

Antagonistic fallow plants

An alternative is the use of plants with an antagonistic effect on X. index to be grown in the fallow period between two successive vine crops. Fallow crops such as marigold and hairy vetch have been shown to reduce X. index populations in the vineyard [START_REF] Villate | Control of Xiphinema index populations by fallow plants under greenhouse and field conditions[END_REF].

Rootstock resistance

To combat X. index in grapevine, resistant rootstocks are a promising perspective and this aspect will be discussed in I.4.2 hereafter. This strategy has already long been the best way to control the root-knot nematodes in vineyards. In the US, resistance sources from Vitis spp. have been used early and the first RKN-resistant rootstocks were developed from V.

champinii, a species which carries resistance to common field populations of M. arenaria, M. incognita and M. javanica. Nevertheless, this source was overcome locally by virulent populations of M. arenaria and M. incognita. This led to the selection of new hybrid material, mostly containing R genes from V. champinii, V. mustangensis or M. rotundifolia [START_REF] Saucet | Tansley review Resistance to root-knot nematodes Meloidogyne spp. in woody plants[END_REF] I.4.2. The resistance against X. index

I.4.2.1. Resistance sources

There have been many researches on grapevine aimed to find out resistance sources to X. index and the subsequent resistance conferred to GFLV via the vector nematode, in greenhouse [START_REF] Harris | Resistance of some Vitis rootstocks to Xiphinema index[END_REF][START_REF] Walker | VR 039-16 Grape Rootstock[END_REF]Malan & Meyer, 1993;[START_REF] Mckenry | Eight-year nematode study from uniformly designed rootstock trials in fifteen table grape vineyards[END_REF][START_REF] Esmenjaud | Host suitability of vitis and vitis-muscadinia material to the nematode Xiphinema index over one to four years[END_REF] and in field conditions (Walker, 1994). Host suitability towards X.

index of some rootstock examples is reported in Table 1.6.

Resistance in Vitis spp.

No noticeable resistance has been found in the cultivated grapevine V. vinifera. The highest resistance to X. index, firstly searched in late 20 th century [START_REF] Harris | Resistance of some Vitis rootstocks to Xiphinema index[END_REF], was found in accessions from V. candicans, V. solonis, V. arizonica, V. rufotomentosa and V. smalliana.

Moderate resistance was observed in V. riparia, V. rubra and V. slavinii [START_REF] Ollat | Rootstocks as a component of adaptation to environment[END_REF].

Among commercial rootstocks, 1613C, a complex hybrid involving V. riparia and V. longii in particular, showed moderate resistance and all others, including the V. champinii rootstock Salt Creek (Ramsey), were quite susceptible [START_REF] Kunde | A test of Vitis resistance to Xiphinema index[END_REF]. [START_REF] Harris | Resistance of some Vitis rootstocks to Xiphinema index[END_REF] also found 1613C moderately resistant. Malan & Meyer (1993) with a South African population of X.

index confirmed the resistance of 1613C and the susceptibility of Ramsey. However, ELISA tests detected the presence of GFLV in the roots of all cultivars after four months and the systemic spread of the virus from six to 18 months after inoculation. There was no evidence of resistance to the transmission of GFLV through feeding of X. index in any of the rootstocks studied, even though some had a low reproduction potential for the nematode and no root damage was observed. [START_REF] Boubals | Résistence de certaines vitaces et des porte-greffes usuels en viticulture au nematode Xiphinema index et à l'inoculation du court noué (GFV)[END_REF] concluded similarly when, five years after inoculation, all the accessions tested showed foliar symptoms of GFLV. They confirmed the susceptibility of Salt Creek, but not the resistance of 1613C, and showed a moderate level of resistance in V. riparia 'Gloire de Montpellier'. The results of Boubals and Pistre showed that great differences of susceptibility/tolerance could exist among varieties of the same Vitis species. For example, V. riparia 'Gloire de Montpellier' is moderately resistant but V. riparia 'Messner n°9' is very susceptible. The evidence of poor correlation between the resistance to transmission of GFLV and the resistance to nematode feeding in Vitis species was also observed by [START_REF] Staudt | Resistance to transmission of Grapevine fanleaf virus by Xiphinema index in some Vitis species and hybrids[END_REF]. Though the susceptibility of V. cinerea to X. index was reported by [START_REF] Kunde | A test of Vitis resistance to Xiphinema index[END_REF] and [START_REF] Boubals | Résistence de certaines vitaces et des porte-greffes usuels en viticulture au nematode Xiphinema index et à l'inoculation du court noué (GFV)[END_REF], a high resistance of the rootstock cultivar 'Börner' (V. riparia x V. cinerea) to nematode feeding and GFLV infection was claimed [START_REF] Becker | Rootstocks with immunity to phylloxera and nematode resistance[END_REF]) but is controversial [START_REF] Sopp | Resistance of rootstocks to the virus transmitting nematode Xiphinema index[END_REF][START_REF] Ipach | Transmission of grapevine fanleaf virus by Xiphinema index to different newly bred rootstocks in greenhouse and field trials[END_REF].

V. rupestris S

In France, the rates of GFLV infection of scions of Cabernet-Sauvignon grafted on Börner and the susceptible rootstock SO4 were equivalent six years after planting (A.

Bouquet, unpublished) (see experiment in Domaine du chapitre in Chapter 4, part 2). More recent screening for resistance in clonal accessions of the species V. aestivalis, V. amurensis, V. bayleyana, V. berlandieri, V. bicolor, V. candicans, V. cinerea, V. doaniana, V. flexuosa, V. lincecumii, V. piazeski, V. rubra, V. rupestris, and V. simpsonii, did not allow to detect X. indexresistant plant material [START_REF] Esmenjaud | Host suitability of vitis and vitis-muscadinia material to the nematode Xiphinema index over one to four years[END_REF]. By contrast, the accessions 'Grand Glabre' and '10128' in V. riparia exhibited a good level of resistance in recent evaluations (D.

Esmenjaud & N. Ollat, unpublished). Interestingly in an accession of the species V. arizonica, a resistance was characterized in UC Davis and shown to be monogenic [START_REF] Xu | Genetic and QTL analysis of resistance to Xiphinema index in a grapevine cross[END_REF]. By contrast, another accession, V. arizonica cv. Wetmoore, resulted quite susceptible (D.

Esmenjaud & N. Ollat, unpublished).

Resistance in Muscadinia rotundifolia

Muscadines (M. rotundifolia) have an overall high degree of resistance to pests and diseases, especially Pierce's disease which severely limits production of Vitis species in the south-eastern United States [START_REF] Bouquet | Vitis x Muscadinia hybridization: A new way in grape breeding for disease resistance in France[END_REF]. They are also among the most resistant grape species to the phylloxera aphid, D. vitifolia, which causes devastating root injury to V. vinifera [START_REF] Husmann | Muscadine grapes[END_REF].

Muscadine grapes comprise currently the best potential sources for X. index resistance and induce a delay in GFLV transmission [START_REF] Bouquet | Resistance to Grape fanleaf virus in Muscadine grape inoculated with Xiphinema index[END_REF][START_REF] Bouquet | Recherche de porte-greffes de vigne résistant à la transmission du virus du court-noué (GFV) par le nématode Xiphinema index Thorne & Allen. I. -Application de la méthode ELISA à la réalisation d'un test rapide de sélection[END_REF]). The rotundifolia have been obtained in France [START_REF] Bouquet | Vitis x Muscadinia hybridization: A new way in grape breeding for disease resistance in France[END_REF] and the US [START_REF] Walker | VR 039-16 Grape Rootstock[END_REF].

In the US (UC Davis breeding program), these were the V. vinifera x M. rotundifolia (VR) hybrids, VR039-16 and VR043-43 (Table 1.6), both patented and released as rootstocks highly resistant to X. index and preventing fanleaf degeneration. However, in the field these resistant rootstocks showed resistance to X. index feeding but they had no ability to inhibit the transmission of GFLV by its vector nematode. Nevertheless, although scions grafted onto these rootstocks became infected with GFLV, disease was not expressed (Walker et al., 1994a;1994b). ) to be used in highly nematode-infected plots under certain soil conditions [START_REF] Bouquet | Breeding rootstocks resistant to grape fanleaf virus spread, using Vitis × Muscadinia hybridization[END_REF]2003;[START_REF] Esmenjaud | Advances in biology, ecology and control of Xiphinema index, the nematode vector of grapevine fanleaf virus[END_REF] (Table 1. [START_REF] Raski | Strategies against Grapevine fanleaf virus and its nematode vector[END_REF]. Considering that American VR039-16 and VR043-43 rootstocks have been practically removed from the market, Nemadex Alain Bouquet is currently the sole commercial rootstock which is resistant to X. index and delays GFLV [START_REF] Ollat | Nemadex Alain Bouquet : un nouveau porte-greffe contrôlant la recontamination des vignes par la maladie du court-noué[END_REF].

I.4.2.2. Histological mechanisms of resistance

Preliminary histological studies, conducted on resistant muscadine material [START_REF] Staudt | Resistance to transmission of Grapevine fanleaf virus by Xiphinema index in Vitis rotundifolia and Vitis munsoniana[END_REF], showed that this species displays a hypersensitivity phenomenon.

I.4.2.3. Bases of the INRA breeding project for new Muscadinia-derived resistant rootstocks

Despite being a good source for X. index resistance, M. rotundifolia is not suitable as rootstock because of its poor rooting ability and its graft incompatibility with V. vinifera [START_REF] Bouquet | Vitis x Muscadinia hybridization: A new way in grape breeding for disease resistance in France[END_REF]. Most but not all V. vinifera x M. rotundifolia hybrids (VRH) are resistant to X. index (Walker et al., 1994;[START_REF] Esmenjaud | Host suitability of vitis and vitis-muscadinia material to the nematode Xiphinema index over one to four years[END_REF]. As illustrated in Table 1.7, F1 hybrid material ranges from resistant (VRH 8771, VR 039-16) to susceptible (VRH 8624). When present in F1, the resistance can be inherited in the back cross 1 (BC1) level as in RPG1 [START_REF] Esmenjaud | Host suitability of vitis and vitis-muscadinia material to the nematode Xiphinema index over one to four years[END_REF].

As reported previously, in France, breeding for Muscadinia-derived resistant rootstocks was undertaken by Alain Bouquet approx. thirty years ago [START_REF] Bouquet | Vitis x Muscadinia hybridization: A new way in grape breeding for disease resistance in France[END_REF][START_REF] Bouquet | Recherche de porte-greffes de vigne résistant à la transmission du virus du court-noué (GFV) par le nématode Xiphinema index Thorne & Allen. I. -Application de la méthode ELISA à la réalisation d'un test rapide de sélection[END_REF]) and the first achievement has been the selection and the release of the BC1 rootstock Nemadex Alain Bouquet (Nemadex AB or RPG1) [START_REF] Ollat | Nemadex Alain Bouquet : un nouveau porte-greffe contrôlant la recontamination des vignes par la maladie du court-noué[END_REF]. The project is now being continued at INRA-EGFV in Bordeaux and, considering the limited viticultural characteristics of this rootstock [START_REF] Claverie | Nemadex AB : Bilan des réseaux d'expérimentation en France[END_REF], its objective is to breed new Muscadinia-derived rootstocks that will carry i) resistances to both Phylloxera and X. index and ii) an adaptation to the wide range of pedoclimatic conditions of the French vineyards.

This material will be selected in particular for good rooting ability and vigor, both features expected from its Vitis parentage. Proof-of-concept studies based on F1 and BC1 Muscadiniaderived materials already obtained have illustrated the ability of these resistant accessions to reduce considerably the vector nematode multiplication and to delay the cognate transmission of GFLV [START_REF] Esmenjaud | Host suitability of vitis and vitis-muscadinia material to the nematode Xiphinema index over one to four years[END_REF]2011;2014)

I.4.2.4. State of the art on the studies for resistance to X. index conducted at INRA UMR ISA

Resistance sources

Resistance studies conducted at INRA UMR ISA have considered several sources currently available at INRA-EGFV in Bordeaux (Table 1.7). These resistance sources are F1 or more complex Muscadinia-Vitis intergeneric accessions because their muscadine parents are no longer available. Among these sources, Durability of the muscadine-derived resistance to X. index A preliminary study under greenhouse-controlled conditions has been conducted until 2010 [START_REF] Esmenjaud | Host suitability of vitis and vitis-muscadinia material to the nematode Xiphinema index over one to four years[END_REF]. It considered the Muscadinia-Vitis F1 accession VRH 97-99-79 from the resistant muscadine accession Carlos (Table 1.7) and the BC1 representative RPG1, with V. rupestris du Lot and V. riparia Gloire de Montpellier as Vitis controls. Plant material has been obtained from in vitro, acclimatized in growth chamber for 2 months without hormones and grown in the greenhouse for 5 months before inoculation. An isolate created from a single female of the population X. index Fréjus has been used as inoculum. The reproduction factor has been evaluated after 1 and 4 years. As expected from previous evaluations, the F1 and BC1 materials resulted resistant (RF ≤ 1) after one year. Nevertheless, the RF values for both accessions were significantly higher after 4 years (5.27 in VRH 97-99-79 and 7.57 in RPG1) than after 1 year. These values suggested that the nematode might have adapted progressively to the resistances carried by both accessions (Fig 1 .27). If we hypothesize that their respective muscadine sources, Carlos and NC 184-4, confer an equivalent level of resistance, the higher RF value (even though it is not significant) in the BC1 than in the F1 appears logical.

The results from Esmenjaud et al. ( 2010) suggest, at least in the experimental conditions based on plants obtained from in vitro, an erosion of resistance over years. The limited durability of the resistance evidenced at both F1 and BC1 levels merits further investigations. R resistant, MR moderately resistant, S susceptible (adapted from [START_REF] Esmenjaud | Host suitability of vitis and vitis-muscadinia material to the nematode Xiphinema index over one to four years[END_REF].

Inheritance of resistance to X. index in muscadine

Previous results highlight the need to decipher the genetics of resistance to X. index in muscadine. To alleviate this concern, a greenhouse study on the inheritance of resistance carried out by the muscadine source NC 184-4 has been undertaken. Despite the difficult hybridization between the genera Vitis and Muscadinia, crosses repeated over several years have allowed to obtain a backcross (BC1) progeny of 66 individuals between VRH 8771 and the susceptible (S) V. vinifera cv. Cabernet-Sauvignon (CS). Resistance (R) phenotyping using a one-year test for the selection of major R factors showed a 58 R : 8 S segregation. The ~ 7 R :

1 S ratio suggests the hypothesis of three dominant and independent R factors inherited from the muscadine source NC 184-4. Using a VRH8771 x CS genetic map in progress based on microsatellite and GBS (genotyping by sequencing) markers, a mapping method derived from BSA (bulked segregant analysis) has identified markers linked to resistance at three chromosomal locations that fit this hypothesis [START_REF] Lalanne-Tisné | Preliminary data on genetics and mapping of grapevine resistance from a muscadine source to Xiphinema index[END_REF]. Nevertheless, given the low numbers of available BC1 individuals, complemental studies are needed. Next studies will aim at confirming the polygenic hypothesis by assessing the segregation of each putative single factor in new BC2 progenies. This will be tentatively performed by backcrossing appropriate BC1 individuals putatively carrying a single factor with S (recessive) individuals.

Another study to be conducted consists in evaluating the effect of the R factors on GFLV infection. Segregating individuals representative of the diverse putative combinations of factors will be evaluated for the dynamics of their viral infection under a high pressure of viruliferous X. index nematodes. In this thesis work, it is important to retain the information that resistance appears polygenic and would be conferred by major dominant gene I.5. STUDY PRESENTATION

I.5.1. Context and originality

The overall objective of my study is to get a better knowledge of the genetic variability of X. index in order to consider it in new control approaches. I will first try to increase our knowledge of the nematode genetic diversity at the world scale by a phylogeography approach. Taking into account these data, my second objective will be to assess the durability of the resistance to X. index and the cognate putative adaptation of the nematode facing this plant resistance.

I.5.2. Study objectives

I.5.2.1. Worldwide genetic diversity of the grapevine vector nematode

As the geographical distribution of the variability is key information for diversity studies, my work will aim at determining the native area of X. index and at tracing its dissemination routes. Four mitochondrial markers (CytB, ATP6, ND4 and CO1) completed by 8 microsatellite markers will be used as tools to characterize the genetic diversity at the world scale.

My results should contribute to answer the question: How is shaped the worldwide genetic diversity of X. index?

I.5.2.2. Application to the development of the resistance strategy

As already mentioned, females reproduce by meiotic parthenogenesis but occasional sexual reproduction may also occur even though males are rare (Villate et al., 2010). The reproductive status of the nematode widely affects the nematode variability. In the aim of an optimal use of the resistance strategy based on Muscadiniaderived resistant rootstocks, my objective will be to establish whether the long-life cycle of grapevine resistant material will allow the nematode to adapt over years and thus compromise its control. This question is particularly accurate given that preliminary data suggest a progressive adaptation of plant material obtained from in vitro. I will thus study the plant resistance and the cognate nematode adaptation in classical plant material obtained from cuttings together with plant material obtained from in vitro. For this, several pure and traceable nematode lines created in our lab (chosen as representative of the diversity highlighted in a preliminary phylogeography study) have been mixed to challenge plant resistance. In order to monitor the putative nematode Xiphinema index is present in most vineyards over the world. In the wine industry and in other grape productions like fruit, raisin or juice, X. index is one of the top ten nematode pests together with root-knot nematodes and cyst nematodes. It not only causes direct damage on root tips by forming galls during its feeding but also transmits GFLV which is the major virus causing grapevine degeneration or 'Court-noué'. In France, this disease was recorded in around 70% of the investigated vineyards. A better knowledge of X. index variability at the individual and population levels will be very helpful for control strategies and for the evaluation of the resistant plant material to this nematode in particular. Hypotheses on the expansion of X. index from its putative 'native area' in the Eastern Mediterranean to the today's grape growing regions along with its host plant have been proposed but they lack reliable supporting data. Domestication of grapevine is dated from 5400-5000 BCE supported by archeological discoveries of ancient wine (from the tartaric acid detected on the inner surface of amphoras) together with fossils of grape seeds (from Caucasus, Armenia and Iran mountains). Then domesticated grapevine was disseminated East-to-West by different ancient civilizations such as the Hittites, Phoenicians, Greeks, Romans, and Arabs, etc., through the Middle East, the Near East, the central and Western Mediterranean until Italy, Southern France, and the Iberian Peninsula. Along with the dispersion of its main host, deciphering the Antiquarian spread of X. index up-to Western European vineyards is a challenging task.

The expansion of the domesticated vine to the rest of the world is more recent. The vine was established and spread to the 'New World' (North and South America) in the 16-17 th centuries by the Spanishs. In North America, the local wild Vitis species (Vitis spp. and Muscadinia) had no wine potential and European vine plants were therefore introduced on the East coast of the United States at the end of the 17 th century. One remarkable event was the accidental introduction, in the years 1858-1860, of the phylloxera from the United States into Europe. This root aphid decimated the French and then the European vineyard in a few decades. In the fight against this pest, many non-vinifera Vitis species (out of which several ones were completely resistant) were also introduced from the US to Europe, to be used directly or after hybridization as resistant rootstocks.

In this chapter, we are going to use different sets of markers to study the genetic diversity of X. index at the world scale. The samples used originate either from the INRA-ISA collection or have been obtained during this study through our nematologists network.

The objective of this work is to:

-get a better knowledge of the genetic diversity of X. index at a world scale by using complementary markers: mitochondrial (maternal) genes and microsatellite (nuclear) sequences. We will also consider a few populations of X. diversicaudatum, a species very close to X. index but with a sexual reproduction mode, in order to compare the diversity of these two major Xiphinema species.

-use, as much as possible, an array of samples representative of the X. index geographic distribution in order to trace the dissemination routes of the nematode from its native area and recover the major historical events that paved its dispersal from the Antiquity epoch.

-establish which are the populations to retain as representatives of the global nematode diversity for the evaluation of the control strategies of X. index in the vineyards. In particular, this will allow conducting reliably the evaluation of the resistant material.

The manuscript written from this work has been submitted to the Journal 'Scientific Reports'.

II.2. SUBMITTED MANUSCRIPT

Phylogeography of the soil-borne vector nematode Xiphinema index sheds light into its Eastern origin and its dissemination with domesticated grapevine

Van Chung Nguyen 1,* , Laure Villate 2 , Carlos Gutierrez-Gutierrez 3 , Pablo Castillo 4 , Cyril Van Ghelder 1 , Olivier Plantard 5 , and Daniel Esmenjaud 1,*

INTRODUCTION

More than 4,100 species of plant-parasitic nematodes have been described 1 causing an economical damage of approx. $US 80 billons per year 2 . Among them, the dagger nematode Xiphinema index has a high economic impact in vineyards worldwide, by transmitting in the soil, from plant to plant, the Grapevine fanleaf virus (GFLV) 3-5 . GFLV, the most severe disease of grapevines, is responsible for fanleaf degeneration 6 which occurs in temperate regions of vine cultivation 7,8 . Xiphinema index is an ectoparasitic nematode with a limited host range and grapevine is by far its major host 9 . Another major ectoparasitic nematode of grapevine is X. diversicaudatum, the vector of Arabis Mosaic virus (ArMV), also involved in grapevine degeneration.

The ban of chemical nematicides in an increasing number of countries, due to their adverse environmental effects, has rendered urgent the development of alternative control methods. In grapevine, breeding for nematode-resistant rootstocks is a promising alternative for nematode and subsequent virus controls and resistant accessions are being developed 10-13 . Another alternative is the use of plants with an antagonistic effect on X. index to be grown in the fallow period between two successive vine crops 14 . In order to evaluate reliably of these control approaches, a better knowledge of the nematode genetic variability is requested. This variability depends mainly on the reproduction mode of the parasite 15 . In X. index, females reproduce by meiotic parthenogenesis 16 but occasional sexual reproduction may also occur even though males are rare 17 .

Beside the knowledge of its reproductive mode, the native area of X. index, where it should display its highest genetic diversity, is also key information for diversity studies. For X. index, this information will help to trace its routes of introduction and will provide a better understanding of its dispersal, which is crucial for sustainable management of GFLV disease.

Because GFLV might have been introduced in Western vineyards from the Middle-East with domesticated grapevine 18 , the native origin of the vector nematode X. index in the Eastern Mediterranean Basin has also been evoked 19-23 . Nevertheless, there is still no conclusive argument available today to support this assumption.

We report hereafter diversity studies conducted in this objective using molecular markers.

Mitochondrial genes sequences are maternal markers commonly used as markers for phylogeography studies 24 . We first studied the mitochondrial genetic diversity of X. index, as a meiotic parthenogenetic species, and of the related amphimictic species X. diversicaudatum, and thus compared vector species with different mating systems, using the gene coding for Cytochrome B (CytB). We then focused on X. index and described the phylogeographical patterns among a high number of sampling locations worldwide, using CytB marker together with three other mitochondrial genes, the ATP synthase subunit 6 (ATP6), the Cytochrome c oxydase subunit 1 (CO1) and the NADH dehydrogenase subunit 4 (ND4). Finally, we relied on eight microsatellite markers specific for X. index 25 to complete the new data set with nuclear markers.

Our data set for X. index contributed to i) evaluate its variability in comparison with the panmictic species X. diversicaudatum, ii) establish the relationship between the geographical patterns of genetic variability observed in its current distribution area, iii) define putative nematode dissemination routes from its Eastern Mediterranean native regions and draw hypotheses on the correlative dispersal itineraries of its domesticated grapevine from the very early Antiquity periods, and iv) get a better appraisal of the worldwide nematode genetic diversity for its control. Finally, our data illustrate how the respective genetic variabilities of a pest and its host plant may give back each other key clues to decipher their common dissemination routes.

MATERIAL AND METHODS

Origin of nematode samples and individuals

The two dagger nematodes, X. index and X. diversicaudatum, have been initially obtained from field soil samples (Table S1). Within a same vineyard plot, a sample corresponded to one soil lump obtained from a single or a few clods dug in a same GFLV (X. index) or ArMV (X. diversicaudatum) focus. The total soil weight ranged from 0.25 to 2.0 kg. All 82 samples containing X. index originated from grapevine plots (Table S1). Out of them, 33 originated from greenhouse rearing of a field sample. Rearing was performed in pots on grapevine or fig hosts grown in a greenhouse collection initiated in 1993 at INRA-ISA, Sophia-Antipolis, France (Table S1). Approx. half of the other X. index samples originated from soils received by express mail and directly used on their date of reception for nematode extraction and storage at -80°C. The last half of remaining samples consisted in individuals extracted from soil by their provider, fixed in 70 % ethanol solution and sent to INRA-ISA for direct processing or storage at -80°C. Samples of X. diversicaudatum were obtained from different field crops (Table S1). They were introduced and either reared at INRA-ISA collection in pots on their original host plant or on a set of other host plants or directly extracted from soil on the date of reception and stored at -80°C. In our study, the 82 samples of X. index covered a geographic area ranging from Middle-and Near East, Eastern Mediterranean, to Europe, North and South America, while the 8 samples of X. diversicaudatum originated from France, The Netherlands and Italy. In both Xiphinema species, 3 individuals per sample were amplified for mitochondrial and microsatellite markers. When several haplotypes or genotypes were detected in the 3 individuals from a same sample, 3 additional individuals, when available, were characterized for both markers types, in order to confirm the results and/or detect putative additional variants. Because of their diverse sampling characteristics and histories and of their low numbers, we always designated in our study the individuals from a same origin as samples and not as populations.

Preparation of DNA templates from single individuals

DNA from a single nematode individual was isolated by a simplified procedure: an adult or juvenile was hand-picked, placed in 0.5 ml PCR tube containing 50μl lysis buffer (KCl 50mM, Gelatin 0.05%, Tris pH 8.2 10mM, Tween 20 0.45%, Proteinase K 60μg/ml and MgCl2 2.5mM). Then eppendorf tubes were alternatively moved from liquid nitrogen to 55ºC water bath for 10 times in order to facilitate break down of the nematode body. This step was followed by an incubation at 60ºC for 90 min (tubes were vortexed at least once during incubation to help breaking up tissues) and by a heating at 95ºC for 10 min to inhibit the reaction of Proteinase K. Finally, individuals were cooled at 4ºC, vortexed briefly (2-3 sec) and centrifuged shortly at 6,000 rpm for 30 sec. DNAs were stored at -20 o C until use for PCR or further experiments.

Primer design for CytB, ATP6, ND4 and CO1 genes

We performed BLAST alignments between each of the CytB, ND4, CO1 and ATP6 sequences of X. americanum obtained by He et al. 26 (accession number NC_005928) and the EST database from X. index on nemaBLAST (http://nematode.net/NN3_frontpage.cgi). We found putative partial sequences of the four mitochondrial homologous genes in X. index (contigs XI01293 for CytB, XI01255 for ATP6, XI01185 for ND4 and XI01295 for CO1). For each gene, an alignment was performed between the X. americanum sequence and each of these partial sequences using Clustal W build in Mega software version 6.0. Then, specific primers were designed from these alignments, for each gene, and used to amplify specific fragments from X. index (CytB-852 bp, ND4-644 bp, CO1-998 bp and ATP6-550 bp) and X. diversicaudatum (CytB-559 bp) (Table S2).

PCR amplification, purification and sequencing of mitochondrial genes

The primers designed enabled the amplification of partial sequences of all four mitochondrial genes from X. index, and of CytB gene from X. diversicaudatum. All amplifications were carried out in 50μL reaction mixture containing 3μL of DNA template, 5 μL of reaction buffer, 2 μL of each primer at 10 μM and 0.4 units of Taq polymerase (AmpliTaq, Applied Biosystems/Perkin Elmer). Amplifications were performed on a Hybaid thermocycler with the following steps: (i) 95°C for 3 min; (ii) 35 cycles with 30 sec at 94°C, 1 min at 59°C (52°C for ATP6) and 1 min 30 sec at 72°C; and (iii) ending with 72°C for 5 min. PCR products were run on 1% agarose gel in 0.5X TAE. When the samples gave unspecific bands, 40μL of PCR product was loaded into 1.5% agarose gels in TAE and the band was recovered from the gel with a sterile scalpel and purified using the MinElute Gel Extraction Kit protocol (Qiagen). DNA sequences obtained from purified PCR products were aligned visually. Because all the sequences are protein coding and have no introns or gaps, alignment was straightforward.

Microsatellite genotyping

We used a set of 8 out of the 9 primer pairs (Table S3) designed by Villate et al. 27 . In our study, we used forward primers carrying directly the fluorescent tag instead of the additional fluorescent M13 tail. PCR reactions were carried out in 10 μL simplex reaction containing 2 μL of DNA extract, 2 μL of 5x Taq reaction buffer, 0.15 units of Taq polymerase, 5U/μL Taq DNA polymerase (AmpliTaq, Applied Biosystems/Perkin Elmer), 0.3 μL of each primer (forward primers were fluoro-labelled with a FAM, PET, NED or VIC dye at the 5' end) at 10 μM. For each reaction we mixed 4 pairs of primers in one tube called mix 1 (microsatellites Xi29, Xi04, Xi16 and Xi13) or mix 2 (microsatellites Xi24, Xi22, Xi32 and Xi27) (Table S3).

Reactions were carried out in a PTC-100 thermocycler (MJ Research) with the following amplification conditions: 95°C for 3 min; 30 cycles with 30 sec at 94°C, 1 min 30 sec at 57°C and 1 min 30 sec at 72°C; and ending with 72°C for 10 min. PCR products were stored at -20°C or used directly by adding 1.5 μL of PCR product into 9.5 μL of formamide containing 0.5 μL of the internal lane standard marker (500-LIZ). Samples were read on a R3130XL Genetic Analyzer 16 Capillary system (Applied Biosystems).

Data analysis

For mitochondrial genes, we computed the mean distances between sequences with the Mega software version 6.0 28 using a Kimura-2-Parameters model and the Maximum Likelihood (ML) method to build phylogenetic trees 29,30 . The sequence of X. americanum was included as an outgroup in our phylogenetic analysis 26 . For microsatellite data, we first analyzed the results with the GeneMarker program version 1.75 (Applied Biosystems). Then we used the POPULATION software version 1.2.31 (available at http://bioinformatics.org/populations/) to compute the distance between individuals by Cavalli-Sforza and Edwards distance (CSE) 31 to draw Neighbor-Joining trees.

RESULTS

Mitochondrial diversity in X. index and X. diversicaudatum with CytB gene

The CytB tree grouped in one clade all samples of X. index and in another clade all samples of X. diversicaudatum (Fig. S1 A). Thus, each species is gathered in a monophyletic group. Then we estimated the evolutionary intra-and interspecific divergences between CytB sequences using a representative individual of all mitochondrial haplotypes (see Table S4 for values of all 10 haplotypes including the 6 haplotypes obtained from CytB sequences and 4 additional haplotypes obtained hereafter from concatenated mitochondrial sequences). A very low divergence (0.0 to 0.012) was obtained among X. index haplotypes while divergence was much higher among X. diversicaudatum haplotypes (0.018 to 0.074). A high interspecific divergence among X. index, X. diversicaudatum and X. americanum was revealed (0.336 to 0.357 between X. index and X. diversicaudatum, 0.909 to 0.915 between X. index and X. americanum and 0.897 to 0.937 between X. diversicaudatum and X. americanum) (Table S4). We then generated a simpler tree than in Fig. S1 using a subset of 35 X. index samples and all X. diversicaudatum samples (Fig. 1). This subset of X. index samples contained 43 individuals comprising one representative individual from each CytB mitochondrial haplotype and from each of the 4 additional haplotypes obtained hereafter from concatenated mitochondrial sequences. For the predominant mitochondrial haplotypes, it also included, individuals representative of distant sample locations in order to cover as much as possible the geographical distribution area of X. index (last column of Table S1). In this tree, X. index and X. diversicaudatum individuals confirmed that they belong to a same monophyletic group with 98% and 100% bootstrap values for each species, respectively (Fig. 1). The X. index samples were distributed into several clades.

Phylogeographical pattern in X. index inferred from single and concatenated sequences of mitochondrial genes

We then still considered the subset of 43 X. index representative individuals from 35 samples to amplify the sequences of ATP6, CO1 and ND4 mitochondrial genes. The ML trees (Fig. S2 A, B and C), designed for each gene, displayed the same general topology as for CytB. Several new haplotypes were obtained for ATP6 (e.g. in the Tabriz and Sharekord Iranian samples and in the Alasehir Turkish sample) (Fig. S2A) and for CO1 (i.e. the particular haplotype common to South American samples and to some samples from the South Iberian Peninsula and France) (Table 1) (Fig. S2B). By contrast to CytB (6 haplotypes with 13 substitutions in the 852-bp sequence, i.e. a polymorphism rate of 1.53%), the ATP6 sequence was the most polymorphic by displaying a total of 9 haplotypes (variable sites frequency: 1.82%; 10 substitutions in the 550-bp sequence), preceding CO1 with 5 haplotypes (variable sites frequency: 1.50%; 15 substitutions in the 998-bp sequence) and ND4 with 4 haplotypes (variable sites frequency:

1.40%; 9 substitutions in the 644-bp sequence).

In order to reinforce these congruent topologies, a consensus ML tree was generated using the 3044-bp fragment obtained from the concatenated sequences of the four genes (Fig. 2A).

Xiphinema index individuals were distributed into two major clades, each clade being separated into two subclades (Fig. 2A and Table 1). In the first clade, the first subclade, designated 'Western' (W), gathered all individuals from Western Europe, North and South America, while the second subclade, designated 'Nearand Middle-East' (NME), grouped individuals encompassing, west to east, Crete, Cyprus, Turkey, Egypt and Iran. In the second clade, the first subclade, designated 'Israel and Palestine' (IP), grouped the 3 individuals (3 samples) from Israel and Cis-Jordan that belong to a single haplotype whereas the second subclade, designated 'Samos and South Italy' (SSI), grouped 3 individuals (3 samples) displaying two haplotypes.

The Western subclade can be divided into two groups, designated Western 1 (W1) and Western 2 (W2), that diverged by a single substitution (CO1-438) (Table 1). W1 comprised 14 individuals (from 14 samples) with a same haplotype ranging from the island of Crete and Pecs in Hungary, in its east geographic expansion, to the island of Madeira and Northern California, in its western expansion. By contrast, W2 comprised 12 individuals (from 12 samples) located in three regions: the South Iberian Peninsula (Spain and Portugal), Bordeaux (France) and Provence (France). The 11 individuals (9 samples) from NME displayed 5 total haplotypes. NME revealed 2 specific haplotypes in the sample Tabriz (Iran) and a single specific haplotype in each of the samples Sharekord (Iran) and Alasehir (Turkey) (Fig. 2A and Table 1).

Subclades NME, IP and SSI, from the Central and Eastern Mediterranean, total 8 haplotypes while the widely spread subclade W only totals 2 haplotypes (Fig. 2A and Table 1). These data show that the samples from the Central and Eastern Mediterranean are highly more variable than those from Western countries.

Diversity of X. index inferred from microsatellite markers

We then considered the 35 samples from which the subset of 43 X. index representative individuals had been retained and characterized their multilocus genotypes (MLGs) (3 to 6 individuals per sample). While mitochondrial genes had exhibited 10 total haplotypes, microsatellite data revealed 36 MLGs in these samples (Table S5). The microsatellite diversity was thus higher than the mitochondrial diversity. The unrooted microsatellite tree obtained from this subset had very low bootstrap values and this topology suggests a noticeable level of homoplasy in the data (Fig. 3). Nevertheless, microsatellites allowed revealing a better resolution within the subclades obtained from mitochondrial markers (Fig. 3), which is coherent with their higher mutation rate.

Several limiting factors and in particular the few nematode individuals available per sample and the high heterogeneity of sampling procedures between samples prevented us to perform population genetics statistics such as comparison of allelic and genotypic richness.

Nevertheless, we performed a raw evaluation of the microsatellite diversity of our data. We first analyzed the samples from the Western subclade (W) that were distributed into only 2 mitochondrial haplotypes (groups W1 and W2 differing by a single substitution). Within the individuals from the widely spread haplotype W1 (samples mainly from Europe and North America), microsatellite polymorphism was very limited and we designated them as i) the poorly polymorphic subgroup (W1-PP). The individuals from the haplotype W2 ranged into two contrasting microsatellite subgroups: ii) the polymorphic samples (W2-P) that we detected in three regions (South Iberian Peninsula, Bordeaux and Provence), and iii) the samples from Chile and Argentina (W2-CA) corresponding to a single MLG. The W1-PP subgroup ( 24individuals corresponding to 23 samples) only displayed 5 variant genotypes of which 3 were monophyletic and differed by a single mutation from the dominant genotype (Fig. 3) and 2 were highly different (5 and 6 mutations) (Table S5). These latter genotypes were distantly located from the cluster of predominant and poorly variable genotypes (Fig. 3). By contrast, the W2-P subgroup (14 individuals corresponding to 10 samples) was highly diverse by displaying 13 total MLG of which 11 were monophyletic (10 MLGs) and three were paraphyletic.

Analysis of the diversity of the NME subclade (11 individuals from 9 samples displaying 9 total MLGs in) showed that its individuals were widely dispersed in the tree with the 4 most extreme variant individuals belonging to the Turkish sample Alasehir [START_REF] Decraemer | Plant nematology[END_REF] and to the Iranian samples [START_REF] Hewitt | Nematode vector of soil-borne fanleaf virus of grapevines[END_REF]. The four samples of subclade IP were gathered in sister groups, as expected from their geographical proximity. By contrast, the three samples from subclade SSI were split into several clusters that were not sister groups (Fig. 3 and Table S5).

DISCUSSION

Genetic diversity on CytB gene in X. index and X. diversicaudatum

The 98 and 100% bootstrap values supporting nodes separating the two species of Xiphinema considered here illustrated that CytB is an efficient marker for their specific identification. The nematode X. diversicaudatum displays a higher intraspecific genetic divergence than X. index. This is an expected result as the two species differ by their reproductive mode which is amphimictic in X. diversicaudatum and mainly meiotic parthenogenetic in X. index, even though this latter species is able to reproduce occasionally through a sexual mode 17 .

Genetic diversity of all mitochondrial sequences in X. index

This study reports four mitochondrial genes partially sequenced, including the ATP6 gene that was sequenced here for the first time from X. index. It shows that the mitochondrial DNA diversity obtained from these sequences within X. index samples is very low (from 1.40% in ND4 to 1.82% in ATP6), which confirms previous data obtained with the CO1 gene 32 . Similar data have been reported for the parthenogenetic plant parasitic nematodes Meloidogyne spp. 33 .

Nevertheless, those values are much lower than in the amphimictic species G. pallida which displayed 12% CytB divergence among native Peruvian clades associated to relatively high levels of diversity and gene flow 34 .

The phylogeography of X. index is closely linked to grapevine

Grapevine is the major host 9 of X. index. Given the ectoparasitic status of the nematode, its long-distance dispersal is linked to grapevine dissemination by man through the transport of rooted plants with their soil substrate 17 . This rooted-grapevine 'dependence' of X. index can make it a reliable marker of the dispersion of its host crop and our results may bring interesting data when faced, in particular, to the grapevine history 35-37 . The low mutation rate of the X. index mitochondrial genome and the reduced number of haplotypes highlight how the geographic distribution of nematode genetic variability may support, confirm or even reveal some grapevine dissemination routes.

The few archaeological and historical facts available on the origin of grape cultivation are the seeds of domesticated grapes dated from 6000 BCE found in Georgia and in Turkey and the correlative earliest evidence of wine production has been found in the northern Zagros mountains in North-Western Iran at about 5400-5000 BCE 38-40 . The Middle-East grapegrowing and wine-making area was presumably located in Northern Mesopotamia and may have also encompassed Turkish mountains from eastern Taurus 41 . Grape cultivars should have been transplanted into the Central and Southern Zagros Mountains (3000 BCE) on the Mesopotamian east side and the Jordan Valley until Egypt (circa 4000-3000 BCE) on the Mesopotamian west side 41 . More recent data on grapevine nuclear microsatellite diversity fit with these putative ancestral domestication events occurring in the Caucasus and the Fertile Crescent 42 .

The mitochondrial phylogeography suggests that the Near-and Middle-East is the native area of X. index

In Europe, X. index has never been found in native forests or climax vegetation 43 . By contrast, in the Middle East, its presence has been reported in Iranian natural woodland (Sturhan in Weischer 44 ) where wild grapevines may be common, e.g. in the forests lying along the Caspian Sea 45 . This explains why X. index occurs frequently in cultivated grapevines of Northern and Western Iran 19 . In our results, the highest mitochondrial diversity is found in the Near-and Middle-East subclade: in particular, the evidence of three different and unique haplotypes in Tabriz and Sharekord Iranian samples suggested this region as being the closest to or corresponding to the native area of X. index. Microsatellites, by stating that the NME subclade also displays a high diversity, provide data in agreement with the hypothesis that this area is the cradle of X. index prior to have been dispersed by man through grapevine domestication.

Dissemination of Eastern samples from the Near-& Middle-East subclade

In the hypothesis of an expansion of cultivated grapevine from Southern Caucasus regions (including today's North-West Iran and East Turkey), it appears credible that X. index has been spread with its grapevine host by two different routes: a first route in Western direction that may have generated the subclade NME (and from which subclade W has then emerged) and a second route in the Southern direction (maybe through Southern Mesopotamia) that gave birth to subclades IP and SSI (Fig. 2B). Later grapevine historical data 40,46,47 and chloroplast DNA and microsatellite information 41,42 were used to build the hypothesis that diffusion of viticulture

Dissemination of Eastern samples from IP and SSI subclades

With the grapevine putative historical expansion in mind, we may hypothesize that the samples from Israel and Palestine (subclade IP) are initial steps of the Southern route. Nevertheless, an overlapping of the subclades NME and IP has certainly occurred, at least in Lebanon. This is supported by the fact that, within the CytB haplotype obtained from single individuals recovered in two Lebanese locations (recorded in Table S1 but not used in our results for their limitation to a single gene), the first one (Xi-Le-Le; from non specified origin) had the specific IP haplotype while the second one (Xi-Le-Ke; from Kefraya) belonged to the most common NME haplotype (e.g. the same as Xi-Cy-Cy).

Because they are closely related to this IP subclade, samples from Samos and South Italy (subclade SSI) might originate from the same region, as Lebanon might have been another dispersion center for X. index (Fig. 2B). The fact that, in microsatellite data (Fig. 3), MLGs Xi-Gr-Sa-A and Xi-Gr-Sa-B from Samos on the one hand and from Xi-It-Pa and Xi-It-PC from Southern Italy on the other hand do not belong to sister groups argues for an ancient divergence between them (associated to homoplasy of the markers). In the Antiquity (2000-500 BCE), the Lebanon area was under the authority of the Phoenician kingdom that had also settled many trade harbors in the Southern Mediterranean coast as far as the Southern Iberian Peninsula (Malaga, Cadiz and Lisbon) 40 . Phoenicians have had a great influence in the spread of vine across the Mediterranean Basin 40 but their possession of Northern Mediterranean trade harbors is weakly plausible. Consequently, nematode introduction from the SSI subclade may have been performed by the Greeks and/or Phoenicians through trade or else in the last two millennia (1500-200 BCE). It may also have occurred later during the Roman Empire epoch.

Origin and dissemination of nematodes of the Western subclade

Western samples of X. index are closely related to the NME subclade and we hypothesize that they have originated from it. One hypothesis is that X. index has been introduced by the Romans after the final unification of Mediterranean territories into the Roman Empire. An alternative hypothesis is that the Greeks have even introduced X. index earlier by their distant trading in Northern Mediterranean harbors until Southern Italy and Southern France (1500-200 BCE) (Fig. 2B). Microsatellite data allow to divide European samples from subclade W into i) the poorly polymorphic widely spread samples (W1-PP) on the one hand and ii) the polymorphic Southern Iberian and French (Provence and Bordeaux) samples (W2-P) on the other hand.

Those two groups differ only by a single substitution in their mitochondrial haplotype. Thus, they might originate from the same Near-or Middle-East location and have been carried either independently or together, until an unknown intermediate focus.

Dissemination of nematodes of the Western 2 group (W2-P and W-CA)

The high diversity of MLGs in W2-P samples suggests that repeated introductions from the same mitochondrial haplotype have occurred, during the Roman Empire epoch (100 , posteriorly to the unification of Western Mediterranean territories. Fréjus (Provence), an important port and the capital of the new Roman Narbonensis Province in 22 BCE, was a place of quick development of viticulture at that time. Almost simultaneously in Bordeaux, archaeological data show that cultivated grapevine has intensively developed from 50 to 250 ACE 50 . The clustering of Fréjus, Bordeaux and South Iberian Peninsula samples (for both mitochondrial and microsatellite data) advocates for dissemination events that occurred in a limited time scale and maybe from a common origin. The long and peaceful Roman period in the Western Mediterranean has been certainly a prerequisite for this wide scale diffusion of the vine together with the nematode.

In the Iberian Peninsula (and a fortiori in its Southern part), chloroplast and microsatellite diversity 41,42 support the hypothesis of a much earlier introduction of grapevine, probably by the Phoenicians, making this country a secondary center of domestication with centralization, intermixing and exchange of varieties. If we consider that subclade SSI is linked with the Phoenician grapevine dissemination, our current data do not support the hypothesis of another nematode route of Phoenician origin up-to the Southern Iberian Peninsula. However, it is also actually possible that our data dealing with the nematode do not allow tracking those putative early-introduced Phoenician grapevines into Spain because they were nematode free. This early grapevine introduction in the Southern Iberian Peninsula by the Phoenicians might explain why some grapevine varieties from South Spain are also recovered in France (e.g. the cultivar 'Palomino fino' which is identical to the French cultivar 'Listan'; Thierry Lacombe, INRA, France, pers. com.).

In America, cultivated grapevine has been imported from Europe since the 16th century 47,51 by Christian missionaries. Introduction of X. index into the New World countries certainly happened through the Spanish colonization of most regions of South America until Argentina and Chile. The complete monomorphism of these South American samples (W2-CA) (Table S5), that illustrates a single introduction event into one of these countries, does not allow to establish whether it occurred directly from Spain, or indirectly from the Canary Islands or another intermediate region 51,52 . Interestingly studies of the Chilean and Argentinian grapevine historical cultivars show that their diversity was poor and derived from local crosses mainly between two of them (Muscat of Alexandria and Listán Prieto). This poor local grapevine diversity fits with the lack of nematode polymorphism.

Origin and dissemination of the Western 1 group (W1-PP)

The second Western haplotype (W1-PP) is almost monomorphic with 19 out of the 24 total samples from this group displaying an identical genotype that ranges from Hungary and Crete eastward to Western and South-Western Europe and until Madeira and California westward (Table S5). This establishes that this haplotype has been spread very recently and presumably from a geographically limited monomorphic focus. This recent dissemination might be consecutive to the massive replants of the vineyards in a huge part of the worldwide grapevine distribution area after the phylloxera crisis in the 19 th century 53,54 . The invariant genotype of most W1 samples may originate from a location initially contaminated by a single introduction event and that has provided nematode-infected rootstocks for the stepwise replant of highly distant vineyards. Interestingly, W1-PP samples exhibit four variant genotypes of which two are highly different and originate from Apulia (Terlizzi) and North Crete, respectively, and two others are less variable but originate from the same Apulian sample and from Sardinia. Such detection of polymorphic individuals in Southern Greece and Southern Italy is quite in line with the hypothesis of a Greco-Roman origin of a portion or of the totality of the Western subclade and these latter regions might be or contain the focus from where the predominant W1 genotype originated (Fig. 2B). Moreover, the recent historical route that presumably lead to its massive dispersal in close linkage with the phylloxera crisis would need to be deciphered. Surprisingly, our data also state that, contrary to what occurred in South America, X. index has not been introduced in California by the Spanish colonization from the 16 th century but much later from the 19 th -20 th centuries with vines grafted on phylloxera-resistant rootstocks that compulsorily originated from Europe. Interestingly several locations from the Southern Iberian Peninsula, Bordeaux and Provence (i.e. having W2-P individuals) also harbored W1-PP individuals, which illustrates their double historical infection. Finally, our data illustrated how the respective genetic diversities of a nematode and its host plant may give back each other key clues to decipher their common dissemination routes.

Interest of phylogeographical data for reliable evaluation of control methods against X. index

The ban of nematicides in an increasing number of countries, due to their adverse environmental effects highlights the need for alternative control methods against X. index.

Breeding for nematode-resistant rootstock accessions and selection of antagonistic plants are the major current research fields. For the durability of the resistance sources, together with the sustainable efficiency of antagonistic plants, an accurate evaluation of the genetic variability of the pest is a central issue. Evaluation of these control approaches requires the use of samples representative of the genetic variability of X. index. This is primarily dependent on the part of the overall genetic diversity carried out by these experimental samples among the total diversity of the nematode. Here, our aim has been to get this primary set of data and to draw a picture of the genetic diversity of X. index at the global scale. Our results have shown that, in order to reflect this worldwide diversity, it is indispensable to retain nematode haplotypes/genotypes from both Western and Eastern locations. Thus, choosing a representative nematode haplotype from each of i) the Western most common genotype (W1-PP), ii) the Near-and Middle East subclade, and iii) the Samos and South Italy or Israel and Palestine subclades is recommended. Fr-Vou Fr-Vou Supplemental Tables Table S1. List and characteristics of the Xiphinema index (Xi) and X. diversicaudatum (Xd) samples of the study with their sample code, individual code, origin (country, region, location) and host plant.

A C C C G G G A A C C A C T A A T T A G C A G G C C T C A C T A G C A C T A G C A G C T A A A Fr-Av Fr-Av A C C C G G G A A C C A C T A A T T A G C A G G C C T C A C T A G C A C T A G C A G C T A A A Fr-Cas Fr-Cas A C C C G G G A A C C A C T A A T T A G C A G G C C T C A C T A G C A C T A G C A G C T A A A Fr-Fr Fr-Fr -A A C C C G G G A A C C A C T A A T T A G C A G G C C T C A C T A G C A C T A G C A G C T A A A Fr-Me1 Fr-Me1 A C C C G G G A A C C A C T A A T T A G C A G G C C T C A C T A G C A C T A G C A G C T A A A US-Lo1 US-Lo1 A C C C G G G A A C C A C T A A T T A G C A G G C C T C A C T A G C A C T A G C A G C T A A A Sp-LM Sp-LM A C C C G G G A A C C A C T A A T T A G C A G G C C T C A C T A G C A C T A G C A G C T A A A Sp-JF1 Sp-JF1-A A C C C G G G A A C C A C T A A T T A G C A G G C C T C A C T A G C A C T A G C A G C T A A A Pt-Le Pt-Le-A A C C C G G G A A C C A C T A A T T A G C A G G C C T C A C T A G C A C T A G C A G C T A A A Pt-Si Pt-Si-A A C C C G G G A A C C A C T A A T T A G C A G G C C T C A C T A G C A C T A G C A G C T A A A It-Te It-Te A C C C G G G A A C C A C T A A T T A G C A G G C C T C A C T A G C A C T A G C A G C T A A A It-Do It-Do A C C C G G G A A C C A C T A A T T A G C A G G C C T C A C T A G C A C T A G C A G C T A A A Hu-Pe Hu-Pe A C C C G G G A A C C A C T A A T T A G C A G G C C T C A C T A G C A C T A G C A G C T A A A Gr-NC Gr-NC-B A C C C G G G A A C C A C T A A T T A G C A G G C C T C A C T A G C A C T A G C A G C T A A A Fr-Fr Fr-Fr -B A C C C G G G A A C C A C T A A T T A G C A G G C T T C A C T A G C A C T A G C A G C T A A A Fr-PL Fr-PL A C C C G G G A A C C A C T A A T T A G C A G G C T T C A C T A G C A C T A G C A G C T A A A Fr-Me2 Fr-Me2 A C C C G G G A A C C A C T A A T T A G C A G G C T T C A C T A G C A C T A G C A G C T A A A Fr-SE1 Fr-SE1 A C C C G G G A A C C A C T A A T T A G C A G G C T T C A C T A G C A C T A G C A G C T A A A Fr-SE2 Fr-SE2 A C C C G G G A A C C A C T A A T T A G C A G G C T T C A C T A G C A C T A G C A G C T A A A Sp-JF1 Sp-JF1-B A C C C G G G A A C C A C T A A T T A G C A G G C T T C A C T A G C A C T A G C A G C T A A A Pt-Si Pt-Si-B A C C C G G G A A C C A C T A A T T A G C A G G C T T C A C T A G C A C T A G C A G C T A A A Pt-La Pt-La A C C C G G G A A C C A C T A A T T A G C A G G C T T C A C T A G C A C T A G C A G C T A A A Pt-Le Pt-Le-B A C C C G G G A A C C A C T A A T T A G C A G G C T T C A C T A G C A C T A G C A G C T A A A Ch-SF Ch-SF A C C C G G G A A C C A C T A A T T A G C A G G C T T C A C T A G C A C T A G C A G C T A A A Ch-Ca Ch-Ca A C C C G G G A A C C A C T A A T T A G C A G G C T T C A C T A G C A C T A G C A G C T A A A Ch-Vi1 Ch-Vi1 A C C C G G G A A C C A C T A A T T A G C A G G C T T C A C T A G C A C T A G C A G C T A A A Cy-Cy Cy-Cy A C C C G G C A A C C A C T A A T T G G C A G A C C C C A C T A G C A C T G G T A G C T A A A Gr-SC Gr-SC A C C C G G C A A C C A C T A A T T G G C A G A C C C C A C T A G C A C T G G T A G C T A A A Gr-NC Gr-NC-A A C C C G G C A A C C A C T A A T T G G C A G A C C C C A C T A G C A C T G G T A G C T A A A Gr-Sa Gr-Sa-C A C C C G G C A A C C A C T A A T T G G C A G A C C C C A C T A G C A C T G G T A G C T A A A Tu-Al Tu-Al-A A C C C G G C A A C C A C T A A T T G G C A G A C C C C A C T A G C A C T G G T A G C T A A A Tu-Al Tu-Al-B A C C C G G C A A C C A C T A A T T A G T A G A C C C C A C T A G C A C T G G T A G C T A A A Tu-Ku Tu-Ku A C C C G G C A A C C A C T A A T T G G C A G A C C C C A C T A G C A C T G G T A G C T A A A Eg-Is Eg-Is A C C C G G C A A C C A C T A A T T G G C A G A C C C C A C T A G C A C T G G T A G C T A A A Ir-Sh Ir-Sh A C C C G G C A A C C A C T A A T T G G C A T A C C C C A C T A G C A C T G G T A G C T A A A Ir-Ta Ir-Ta-A A C C T G G C A A C C A C C A A T T G G C A G A C C C C A C T A G C A C T G G T A G C T A A A Ir-Ta Ir-Ta-B A C C T G G C A A C C A C C A A T T A G T A G A C C C C A C T A G C A C T G G T A G C T A A A CJ-Je CJ-Je G T G C A G C T C T T A C T A G A T A G T G G G C C C T G T C G A T G T C A A C T A C C G G G Is-Is Is-Is G T G C A G C T C T T A C T A G A T A G T G G G C C C T G T C G A T G T C A A C T A C C G G G Is-TA Is-TA G T G C A G C T C T T A C T A G A T A G T G G G C C C T G T C G A T G T C A A C T A C C G G G Gr-Sa Gr-Sa G T G C A C C T C T C A C T G A A T A A T G G G T C C T G T C G A T G T C A A C T A T C A G G It-Pa It-Pa G T G C A C C T C T C A T T G A A T A A T A G G T C C T G T C G A T G T C A A C T A T C A G G

It-PC It-PC G T G C A C C T C T C A T T G A A T A A T A G G T C C T G T C G A T G T C A A C T A T C A G G

Near

When, within a same sample, several mitochondrial haplotypes or multilocus genotypes (MLGs) were detected, they were designated by their sample code completed by a supplemental letter (A, B, etc.) (see Table 1 and Table S5). Type corresponds either to individuals obtained from a field sample (F) or individuals obtained from greenhouse rearing of a field sample (G). The provider (and the year of sampling) of the sample is also indicated. EBI accession numbers of the CytB sequences (all individuals) and of the other mitochondrial sequences, ATP6, CO1, and ND4 (43 representative individuals (rep. indiv.) listed in the last column) are reported.

III.1. RESISTANCE DURABILITY BASED ON A 6-YEAR ASSESSMENT UNDER CONTROLLED CONDITIONS

This study reports investigations in a 6-year assessment initiated at UMR ISA in 2012.

Before this work, studies under controlled conditions had only considered resistance on a duration of one to two years [START_REF] Bouquet | Breeding rootstocks resistant to grape fanleaf virus spread, using Vitis × Muscadinia hybridization[END_REF]. An Introduction part will summarize the state of the art before my thesis work, i.e. the prerequisite data on i) the nematode material used in the study and ii) the plant material (two types of propagation) used in the study. The subsequent parts dealing with nematode extraction, counting and characterization from 2015 to 2018 (Material and methods, Results…) will refer to the work conducted during the thesis.

III.1.1. Introduction

Studies from [START_REF] Esmenjaud | Host suitability of vitis and vitis-muscadinia material to the nematode Xiphinema index over one to four years[END_REF] suggested that, over four years post-inoculation of grapevine obtained from in vitro, X. index adapts progressively to the resistant accessions (VRH8771 and RPG1). In this previous study, plants originating from in vitro were challenged with a single female line (Fréjus, France). This preliminary initial result needs to be confirmed in a more in-depth study. It has been conducted by using plant material obtained from in vitro together with classical hardwood cuttings. Moreover, the putative adaptation of the nematode questions the genetic mechanisms that it may set-up: competition between individuals, sexual reproduction… To get a first answer to this question, genetically distant lines have been mixed

and their respective evolution has been monitored over several years.

III.1.1.2. The different multiplication modes for plant material

Previous experiments conducted at UMR ISA had used plant material propagated with different methods. Material multiplication had been based on plants obtained either from in vitro or from cuttings [START_REF] Esmenjaud | Host suitability of vitis and vitis-muscadinia material to the nematode Xiphinema index over one to four years[END_REF]. In this thesis, we have considered and compared a set of accessions propagated alongside with the two modes. We describe hereafter these two propagation methods.

Material obtained from in vitro

The plants obtained from in vitro originated from in vitro cultures first established in the years 2000 at INRA Montpellier from disinfected buds of all appropriate accessions.

Standard media derived from [START_REF] Murashige | A revised medium for rapid growth and bio assays with tobacco tissue cultures[END_REF] for their introduction (introduction medium) and conservation (multiplication medium). They have been provided by Alain

Bouquet to UMR ISA in 2008. At ISA they have been maintained in a dedicated growth room from the UMR facilities (Team M2P2-Modèles et Méthodes pour la Protection des Plantes) at 22°C with a 16-hr photoperiod provided by cool-white fluorescent lamps. Two media, i.e. the multiplication medium (MM) and the rooting medium ( RM), also derived from the MS medium [START_REF] Murashige | A revised medium for rapid growth and bio assays with tobacco tissue cultures[END_REF]) (Table 3.2), were used. Every two months, new pieces of shoots have been cut from the old culture, planted for new culture onto the MM medium for conservation purpose (no plant hormones) (Table 3.2). They have then been kept for several rounds as MM cultures until their rooting onto the RM medium (0.5 mg/l IBA = 0.5 ppm IBA)

for their subsequent acclimatization and use in the durability experiment ( comprising two nodes were cut and planted individually for rooting into trays of 150-ml cells filled with a fine-sand substrate. No rooting or shooting hormones were used. Trays were placed onto benches and regularly humidified with a mist system. After 8 weeks, rooted cuttings were transferred to UMR ISA at Sophia-Antipolis and repotted into 2-liter pots filled with a sterile sand-loam soil. Plants were inoculated after 2 months and gently repotted into 4liter containers after 2 more months (Figs 3.1 & 3.3).

III.1.2. Material and methods

III.1.2.1. Materials

Nematode material

As previously described, we used the pure lines (or single-female descendents) from four geographical origins (Figs 3.1 & 3.2), designated Iran (Xi-Ir-Sh), Samos (Xi-Gr-Sa), Italy (Xi-It-PC), and Spain (Xi-Sp-LM).

Plant material

The study used several clonal accessions representing different levels of resistance inherited from the muscadine source NC184-4 (Fig.

III.1.2.2. Methods

We will first describe the full steps of the method for nematode extraction from the soil because it has been used for the inoculation of plant material, for the subsequent evaluation of nematode numbers and, also, to recover the nematodes from the phylogeography study in Chapter 3. We will then report the inoculation method, the experimental design and the microsatellite genotyping protocol used in the greenhouse durability study.

Extraction of nematodes from soil

Nematodes were extracted from soil using the elutriation method adapted from Oostenbrink [START_REF] Hooper | Extraction of free-living stages from soil[END_REF]. Content of each 4-liter pot was divided in two 2-liter soil samples.

Each 2-liter sample was processed as follows. Soil was firstly suspended and stirred in 5 liters of water; after 15 seconds of decantation, the suspension was poured onto a sieve with a mesh of 2 mm placed over a 10-liter bucket, and organic material and roots retained by the sieve were eliminated. The resulting 10-liter soil suspension was poured through a 63-µm sieve. The sieve was rinsed to collect debris and nematodes. Nematodes were then separated from the debris using the modified Oostenbrink elutriator method for large nematodes. For this step, the water flow at the bottom of the funnel was set at 700 ml/min (Fig. 3.4). After their separation in the elutriator, nematodes were retained using again the 63-µm sieve. The final remains containing nematodes were rinsed with water and placed onto a double cotton wool filter tissue laid on a 1-mm sieve (diameter: 225 mm) in a glazed plate containing 400 ml of water. Active nematodes were recovered from the bottom of the dish after 3 and 7 days.

Nematodes (female adults, male adults and juveniles) were counted from the suspension obtained at these two dates under a binocular.

Inoculation method and experimental design

Mixed inoculations were performed using 200 total individuals comprising 50 individuals from each of the 4 lines. The individuals were suspended in water and deposited into 2 holes, 2-cm deep and 2 cm from the stem. For a same propagation type, pots inoculated with mixed lines were grouped and surrounded by splash screens to avoid cross contaminations with other modalities. Plants of the different accessions were arranged in a randomized design.

One or two additional plants from each clone were inoculated with 200 individuals of each line as controls (verification of identity and purity of X. index lines). Some additional plants of each clone were used as non-inoculated controls.

In the greenhouse, the protocol with mixed lines has been set up for a total duration of 6 years using the 6 clonal accessions (RPG1, 8771, RGM, CS, RdL and 8624) grown either from cuttings or in vitro (Fig. 3.1B). Containers were drip irrigated daily and temperature was regulated to reach a maximum of 30°C. To avoid frost, greenhouse was heated in winter to keep minimal temperatures above 5 °C. Thus, low temperatures permitted winter leaf fall.

Every year, after complete leaf fall, plants were pruned by keeping two eyes on each major shoot.

Analyzer 16 Capillary system (Applied Biosystems). Fig. 3.5 illustrates the signal of some alleles for the different microsatellite loci and their combinations. This ratio was calculated for each container (replicate) and mean of all replicates allowed to evaluate the RF for each accession. Accessions were classified as resistant (R) when their RF was lower than 1 and susceptible (S) when their RF was above 1. Weight of below-ground part of each plant replicate was also measured to evaluate 'nematode numbers per gram of roots'.

Data from each accession were statistically analysed by a one-way analysis of variance (ANOVA)

using XLSTAT software, Paris, France (version 2014.5.03). Accessions were classified for resistance according to their actual RF values but statistical differences were evaluated after a log 10 (x+1) transformation of nematode numbers [START_REF] Noe | Analysis and interpretation of data from nematological experiments[END_REF]. Means of transformed nematode numbers were then compared by the Fisher LSD multiple range test at P ≤ 0.05.

III.1.3. Results

III.1.3.1. Evaluation of the resistance durability

The durability of nematode-resistant rootstocks and the cognate nematode adaptation has been studied in a 6-year assessment under controlled conditions.

Evaluation of resistance using the reproduction factor

Out of the six grapevine clones (8771, RPG1, RGM, 8624, CS and RdL), the S clones 8624 and RdL did not survive the attacks and died during the first two years. Therefore, we finally The dynamics of nematode numbers differed according to the type of plant multiplication (Fig. 3.6). In cutting material, RFs of 8771 and RPG1 remained lower than 1 while they were intermediate in RGM and high in the susceptible control CS. Nevertheless, numbers tended to be higher in RPG1 (BC1 level) than in 8771 (F1 level), which agrees with the expected dilution of resistance factors in BC1 compared to F1 material. RFs of both R clones suggest that resistance is stable. By contrast, in in vitro material, nematode numbers of both resistant clones were higher than 1 after 3 years and increased regularly and significantly beyond (Fig 4 .6B). RFs of the intermediate clone RGM and the susceptible clone CS increased over years and were also much higher than in cutting material. Consequently, the type of material strongly influenced the resistance estimation at both F1 and BC1 levels. This dependence of resistance on the plant multiplication type is an original data that needs to be studied more deeply. The results obtained for in vitro material confirm the trend previously reported in Fig. 1.27 [START_REF] Esmenjaud | Host suitability of vitis and vitis-muscadinia material to the nematode Xiphinema index over one to four years[END_REF] for another F1 representative clone and for RPG1.

Evaluation of resistance using the nematode numbers per gram of roots

To evaluate the capacities of food supply for the development of X. index in different accessions (8771, RPG1, RGM and CS), we also recorded the total number of X. index per gram of roots (Fig. 3.7). It showed that, in cutting plants, numbers per gram of roots were much lower than in in vitro plants. After 6 years, the resistant accessions 8771 and RPG1 harbored 1.9 and 2.2 individuals per gram of roots, respectively. Meanwhile, RGM and CS harbored 6.9 and 39 individuals, respectively (Fig. 3.7A). In in vitro plants, CS had the highest numbers, i.e. 316 and 430 nematodes per gram of roots after 4 years and 6 years, respectively. The lowest numbers were 6.6 individuals (RPG1 after 6 years) and 20.2 individuals (8771 after 6 years) per gram of roots (Fig. 3.7B). For each propagation type, bars with different letters significantly differ according to Fisher LSD test (P≤0.05). In resistant material, there were at least 7 (8771) and 5 (RPG1) replicates. In RGM and CS, there were 3 to 5 replicates.

We clearly observed that nematode densities in the roots of accessions RGM (intermediate) and CS (susceptible) were much higher than in the resistant accessions 8771 and RPG1. Nevertheless, as previously for RF values, densities in in vitro plants were always much higher than in cutting plants. Finally, results expressed from the numbers per gram of roots paralleled those obtained from the RF criterion. It must also be highlighted that, for the susceptible control CS, densities were 6 to 9 times higher in in vitro plants than in cutting plants.

Evolution of the nematode developmental stages

Our countings have evaluated the numbers of females, larva and male stages. We analyze hereafter their evolution over 3 years (4-to 6-year old plants).

Ratio 'female/larva'. In in vitro plants, the ratio was relatively stable whatever the age of the plants and ranged from 38.7 to 63.3 % (Fig. 3.8B). There was no major difference between resistant material, 8771 and RPG1, versus intermediate (RGM) or susceptible material (CS).

Thus, the proportion was in average of 1.74 to 2. The male numbers. We have recorded the male numbers over 4-to 6-year plants and picked out a total number of 13 males out of approx. 66,000 individuals. This represents a mean occurrence of 2 males per 10,000 nematodes (Table 3.4). We showed that there were very few males detected in the different accessions whatever the plant age and multiplication type.

Consequently, it seems that males are found by chance (only 1 or 2 individuals per modality) and we did not evidence an effect of the accession (R or I or S) or the plant age (4 to 6 years)

or the type of multiplication on the frequency of males (Table 3.4).

III.1.3.2. Evaluation of the nematode adaptation

As explained before, the principle of this experiment is to monitor simultaneously the putative adaptation of the nematode to the resistance and to get data about the hypothetic genetic mechanism of this phenomenon.

Evaluation of the relative abundance of nematode lines

For each plant multiplication type and on each year (from 3 to 6 years), we determined the line identity of representative nematode individuals. In each replicate (pot), we sampled randomly 5 individuals in 2015 and 2016 and 8 individuals in 2017 and 2018. We analyzed their multilocus genotypes (see Table 3.3 for total numbers of plants per propagation type, accession and age). All additional nematodes extracted were kept at -20 o C.

Confirmation of the identification of the genotypes from sampled individuals. We could successfully attribute the high majority of individuals to a given inoculated line. In rare cases, we did not obtain the signal in all loci of an individual as the alleles of 1 to 2 loci were poorly expressed (low peak signal) or completely absent (Fig. 3.12). In such cases, the other loci with unambiguous signals allowed to attribute them to a given line. Exceptionally, alleles from 3 loci were lacking and we considered that these individuals are missing data. Individuals that could not be attributed to a line were analyzed with more attention to establish whether they were hybrid individuals.

Evolution of the line frequency in the different accessions over the 3-to 6-year experiment.

We recovered individuals from all four lines (Italy, Spain, Samos and Iran) in the nematodes sampled on plants obtained from both cutting and in vitro plants (Fig. We then focused more precisely on the data obtained with the resistant accessions 8771 and RPG1 after 6 years to highlight the evolution of the line frequencies between the inoculation (25 %) and the end of the experiment. Chi 2 (χ 2 ) tests showed that we cannot retain the hypothesis that frequencies of each line are equivalent (Table 3.5). The predominance of lines Iran and Italy is confirmed as illustrated in Fig. 3.10. The line Iran resulted particularly predominant in the plant material obtained from in vitro. In vitro origin of plant material modifies the resistance to X. index. Our results clearly show that the in vitro initial conditions of the plant material have influenced the plant host suitability to X. index by facilitating the nematode multiplication. It is hypothesized that the previous continuous in vitro culture of the plant material modifies the resistance to X. index. This may be linked to the modification of the root architecture in the plants obtained from in vitro in comparison with plants rooted from cuttings. Roots systems from in vitro have a more compact shape and produce a higher density of rootlets. The use of the rooting exogenous hormone IBA before the experimental set-up in soil might also be involved in the modification of resistance.

Nevertheless, the effect of IBA in the in vitro rooting medium, studied in the interaction between Prunus plants and root-knot nematodes and added at the same rate as in our durability experiment (0.5 ppm), did not alter the behavior of P. cerasifera accessions, carrying the Ma gene for resistance to M. arenaria, 70 days after plant transfer into a sandy soil (Esmenjaud et al., 1993;1996).

Resistance and structure of the population appear related. The ratio 'female/larva' seems to be linked to the resistance phenomenon. We can put forward that, in plants showing a low RF, the female proportion increases, which corresponds to an aging of the population. This can be explained by the long-life duration of the female stage. Indeed, the dagger nematode X. index has been shown to survive for more than 4 years, while starving in the soil without plant host [START_REF] Demangeat | Survival of Xiphinema index in vineyard soil and retention of Grapevine fanleaf virus over extended time in the absence of host plants[END_REF]. In field soils, the actual survival of adult nematodes is estimated to be of 7 years from the work conducted in the field by [START_REF] Vuittenez | Les viroses de la vigne[END_REF]. An at least 5-6year survival may explain why nematodes are still recovered in our experiments in pots containing 6-year old resistant plants obtained from cuttings. In resistant plants obtained from in vitro, adult nematodes are presumably a mix i) of female individuals that were already at this developmental stage at the time of inoculation and ii) of younger female individuals originating from larval stages that have been able to undergo their development and complete their life cycle. Knowing the proportion of ancient-female and neo-female adults would allow a better interpretation of our results. In our experimental conditions, it would also be worth knowing the real survival potential of X. index individuals kept starving (without host plant) in the soil container.

Male occurrence does not appear to be linked with resistance status. We have observed a rare occurrence of males (mean value of approx. 2 per 10.000), whatever the resistance status of the plant material. Even though counting males is difficult and subject to misevaluation when dispersed among high female numbers, their evaluation is more reliable when few nematodes develop such as in resistant plant material obtained from cuttings. Nevertheless, even in this case, rare males were recovered. It thus appears that male occurrence is not linked with the population density. The increase of the male percentage shown in the case of RKNs facing a poor host or having reached very high densities is not observed for the dagger nematode X.

index. Nevertheless, this suggests that a low basic proportion of males exists in the population whatever the plant status. The proportion that we have obtained confirms the very rare occurrence of males previously reported by many authors (Dalmasso, 1970;[START_REF] Luc | The male of Xiphinema index Thorne & Allen, 1950 (Nematoda: Longidoridae)[END_REF][START_REF] Siddiqi | Co-evolutionary interactions between host resistance and pathogen avirulence genes in rice-Magnaporthe oryzae pathosystem[END_REF]. Nevertheless, an exception has been reported in Australia with a proportion of males as high as 1-2 % [START_REF] Harris | A first record of Tylenchulus semipenetrans and males of Xiphinema index in vineyards in North-eastern Victoria[END_REF].

III.1.4.2. Occurrence of hybridization events in the populations

The first occurrence of hybrid individuals in X. index, previously known as being a facultative-meiotic parthenogenetic species [START_REF] Dalmasso | Ovogenese et embryogenese chez Xiphinema index (Nematoda: Dorylaimida)[END_REF] has been reported recently in the field (Villate et al., 2010). In this work conducted in Bordeaux vineyards, some hybrids could be detected in samples located in the area overlapping two genotypically different groups of individuals from presumably distinct introductions in a same field. Our results have shown for the first time the occurrence of such hybridization events under controlled conditions. We have successfully designed an experiment that allowed to evidence this phenomenon. The characterization and rearing of appropriate single-female descendencies has permitted to create lines from which both the line identity and the hybrid occurrence could be traced over several years. Moreover, our numbers of genotyped individuals were high enough for the detection of hybrids.

We analyze hereafter the putative factors that may either have been associated to, or have led to, or have influenced this occurrence.

Relationship between hybrids and the resistance status of the plant. We would have expected that males appear in high numbers in resistant material to facilitate recombination events and favor the occurrence of individuals carrying virulence factors in order to overcome the resistance. Our results do not support this hypothesis and suggest that hybrids are recovered independently of the plant resistance status. It is probable that hybrids might appear in all conditions and that their numbers might be proportional to the population density. This would explain why we have detected a higher number of hybrids in susceptible or intermediate accessions such as CS, RL and RGM than in resistant accessions such as 8771 and RPG1.

Relationship between hybrids and the age of plants. We could show that the occurrence of hybrid events increased from a single individual after 4 years to 9 and 11 individuals after 5 and 6 years, respectively.

Relationship between hybrids and the type of plants. No clear difference has been observed

between plants obtained from cuttings or in vitro. The difference between young and old plants appears independent from the multiplication status of the plant.

Relationship between hybrid and male numbers. We did not evidence any link between hybrid and male numbers. Nevertheless, as it did not aim at studying the male frequency, our experimental design was not adapted to establish the putative relationship between these two features.

III.1.4.3. Consequences of the partial breakdown of resistance to X. index in plant material obtained from in vitro

We can take into account two complemental aspects. We can consider first the plant side and foresee the consequences in the use of resistant plants obtained from in vitro. Then we can consider the nematode side and discuss about the putative adaptation of the nematode facing resistance factors influenced by this in vitro historical background.

Evolution of the interaction viewed from the plant side. The results that we have obtained strongly suggest taking care when using in vitro material in greenhouse or field experiments. It seems that the plant initially grown in vitro has become more susceptible to the nematode for several years. The plant keeps a memory of its initial history and its interaction with the nematode is modified for a long duration. This exhorts the researchers to avoid the use of such plant material for studies on 'natural' or 'field' plant-nematode interactions. Growers should avoid using such material in order to reduce their nematode reservoir in the soil.

Evolution of the interaction viewed from the nematode side. The ratio female/larva fits with the hypothesis that a 'normal' or 'natural' resistance does occur in cutting plants. In 8771 and RPG1 the populations seem to become 'older' by comparison with in vitro plants. This ratio after 6-year inoculation also increased slightly in RGM and CS, which may be consecutive to high nematode densities in the limited space or/and food resource available from containers with aged plants. These results should discard the hypothesis of a phenomenon of nematode genetic adaptation. It appears probable that the plant physiology is durably modified (i.e., over a duration of at least 6 years) in material previously grown in vitro. The better host suitability of the plant may render it more susceptible to the nematode. In this hypothesis, no particular nematode genetic adaptation is expected and, consequently, those nematode individuals raised on material obtained from in vitro would not have acquired new virulence factors.

Nevertheless, demonstrating this would need to compare the reproduction of these putatively resistance-adapted individuals with individuals that have never been faced to resistant plant material.

Hybridization does not appear as a key factor for overcoming resistance. Hybridization can contribute to adaptation and speciation in populations [START_REF] Burke | Genetics and the fitness of hybrids[END_REF]. In our experiment, as hybrid individuals were recorded apparently whatever the nematode line and the plant accession, resistance may not favor the occurrence of hybrid events. Hence, there was no evidence that hybridization is a key feature for the adaptation of X. index to resistance over time. In moderate and susceptible plants, we may hypothesize that hybridization is consecutive to the selection pressure exerted by the rarefaction of food supply from root tips.

Lines apparently express differences in aggressiveness. Previous work conducted at ISA [START_REF] Demangeat | Transmission competency of single-female Xiphinema index lines for Grapevine fanleaf virus[END_REF]) reported variable reproduction rates of X. index among lines that had been obtained from Cyprus, California and Spain and illustrated differences in their aggressiveness. Our experiments are in overall agreement with these results by showing that the two lines Iran and Italy express a higher aggressiveness than Spain and Samos.

Nevertheless, despite the 6-year duration of the experiment, none of the four mixed lines appeared to exclude totally the others.

III.2. RESISTANCE DURABILITY BASED

III.2.2. Plant material and experimental design

III.2.2.1. Plant material

RPG1 and the three rootstocks, SO4, 140 Ruggeri (140Ru) and Borner have been considered. The SO4 rootstock (an hybrid between V. berlandieri and V. riparia) is very sensitive to GFLV and X. index [START_REF] Goldammer | Wine grape rootstocks[END_REF]. It shows a good ability to grafting and to cutting production. The 140 Ruggeri rootstock (V. rupestris x V. berlandieri) is characterized by a good development in calcareous soils and thus by a good adaptation to lime chlorosis [START_REF] Goldammer | Wine grape rootstocks[END_REF]. The Borner rootstock (V. vinifera x V. cinerea) has been selected in Germany as unfavorable to the multiplication of GFLV (Ruhl, 1996). RPG1 (Nemadex AB) has been obtained from the cross between the F1 Vitis-Muscadinia hybrid VRH 8773 and 140Ru (see Fig. Immediately after sampling, the volume of soil collected in each trench was handled gently and placed in an ice box to protect it from heat and desiccation until its storage at 4-5°C at UMR ISA.

Procedure for extraction

Nematode extraction aimed at detecting X. index in priority. It also aimed at detecting X. pachtaicum, a Xiphinema species commonly found in vineyards. Extractions were performed using the method described previously for X. index under controlled conditions.

Statistical analysis

Statistical analysis has been performed as previously mentioned in the first part (controlled conditions) of this chapter i.e. in the paragraph 'Criteria of resistance evaluation and statistical analysis of the data' (IV.1.2.2 Methods). An ANOVA analysis, conducted on CS experiment showed, after a log 10 (x+1) transformation of data, a significant difference between SO4 and Nemadex AB but not between Borner and both other rootstocks (Table 3 .6). Another ANOVA analysis conducted on all rootstock:variety assemblies also confirmed these results after transformation by stating that Nemadex AB:CS had significantly lower numbers than other assemblies except Borner:CS that resulted intermediate and non-significantly different from others (Table 3.7).

III.2.4.2. Results

Detection

Detection of X. pachtaicum

Nematode numbers. We got a total of 389 X. pachtaicum individuals (223 individuals from the first 2-kg sub-sample and 166 individuals from the second 2-kg sub-sample). From the 63 total samples, this represents 6.17 individuals per sample, i.e. a 18-times higher density than X. index. Numbers ranged from none to 35 individuals per sample (Fig. 3.19). Nematode numbers were equivalent in each of the three rootstocks grafted with CS and no significant differences between rootstocks could be evidenced after data transformation (Table 3 .8).

Though 140Ru grafted with Caladoc had lower nematode numbers (presumably due to lower initial numbers in this part of the field), an analysis conducted with all rootstock:variety assemblies only showed significant differences between 140Ru:Cal and the others assemblies except Nem:Cal (Table 3.9).

III.2.5. Discussion and conclusion

III.2.5.1. Discussion

Experimental designs, that aim at studying GFLV control in the field, are classically based on plant development using aerial rating criteria such as pruning and/or bunch weights and on viral symptoms using ELISA from leaf samplings. Because they are costly and labor-intensive, nematode extractions are rarely performed and data about the dynamics of the vector nematode in GFLV control experiments are lacking. In this context, our study, based on a very dense field soil sampling, brings useful complemental data on nematode numbers in the experimental design.

The field plot in which the experiment has been settled had a very high soil GFLV infectious potential. Consequently, as illustrated in Fig. 3.18, the assay has shown early and marked GFLV attacks. Results have clearly illustrated the interest of the rootstock Nemadex AB to delay viral occurrence and symptoms. This experiment is hence considered as the proof of concept of the strategy based on GFLV control through resistance to its vector nematode.

Our results have first confirmed that the spatial distributions of the virus and the nematode are closely related. We illustrated in particular that rows 4 and 5, the most affected by the virus, harbor the highest X. index numbers. Very few or no nematodes were recovered in the first rows, which is in line with the lowest GFLV percentage in this area. The few individuals from X. index have only been recovered in the three rows from the CS part of the assay. With the exception of a single individual in one Nemadex AB replicate, individuals of X.

index were detected in replicates from SO4 and Borner (Fig. 3.19).

We have shown that X. index nematode densities were very low 16 years after planting.

Such low numbers on that plant age may be essentially due to contrasting host suitability among grapevine rootstocks. On the one hand, plants grafted onto SO4 or 140Ru are either very weak, or hardly surviving or dead because of their susceptibility to nematode and to GFLV attacks and, hence, their limited rooting is expected to have led to the decrease of nematode numbers. On the other hand, the rootstock Nemadex AB has also limited nematode numbers that we can attribute to its resistance behavior. The rootstock Borner expressed as expected an intermediate behavior. Statistical data show that nematode numbers, even though they are low, are significantly higher in nematode (and GFLV) susceptible rootstocks SO4 and 140Ru than in the resistant rootstock Nemadex AB in which almost none has been detected. This strongly suggests that Nemadex AB plants, which are highly vigorous in comparison to other rootstocks, do not harbor nematodes and, consequently, that virulent individuals have not adapted to its resistance factors over years.

By contrast, the more common and non viruliferous species X. pachtaicum had much higher numbers and a more dispersed field distribution than X. index. From our data, it is difficult to compare and conclude on the host suitability of X. pachtaicum to the different rootstocks given that they have very variable vigor and cognate root development.

Nevertheless, in our experiment, resistance factors in Nemadex AB did not seem to express on X. pachtaicum an effect as marked as on X. index.

III.2.5.2. Conclusion

Our study illustrated that nematode densities after 16 years were dependent from both the initial nematode distribution in the field and the rootstock effect. Given that the ability of the nematodes to disperse actively in the soil is extremely limited, we confirmed that the spatial distribution of the vector nematode in the soil and of the aerial virus on the grapevine plants are closely related.

Soil transport allows the diffusion of the virus through its vector. As active dispersal of the nematode per se is low, introduction in a new field is most often performed by man. In our experimental field, a single or several introductions may have been performed by soil transport in the entry of rows 4 and 5, from where secondary focuses have passively appeared further along the rows by mechanical soil tillage. This dynamic of the soil infection fits with the data described in Bordeaux vineyards [START_REF] Villate | Spatial distribution of the dagger nematode Xiphinema index and its associated Grapevine fanleaf virus in French vineyard[END_REF].

The rare detection of X. index in the soil from the resistant accession Nemadex AB strongly suggests that the initial nematode population has not been able to adapt to the resistance factors carried by this rootstock. Even though we have recovered higher numbers of the ubiquitous and quickly colonizing species X. pachtaicum, our results did not allow to state whether Nemadex AB also expresses resistance to this nematode. Our results also support the hypothesis that no adaptation of X. index to Nemadex AB resistance factors has occurred in the conditions of the field experiment.

The few individuals of X. index recovered have not been genotyped individually for their multilocus genotype, but we have characterized other individuals from this field population in the manuscript on phylogeography. These individuals belong to the W1 mitochondrial haplotype (Table S1-Sample #8 designated Xi-Fr-VM from Villeneuve-les-Maguelone, Languedoc, France; Cyt B sequence registered as LT996608).

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Both mitochondrial and microsatellite data allow to refine the diversity of X. index

As a complemental tool to mitochondrial markers, we have used eight microsatellite loci (Villate et al., 2009). Even though the 25 to 30 individuals per population needed for microsatellite population genetics study [START_REF] Hale | Sampling for microsatellite-based population genetic studies: 25 to 30 individuals per population is enough to accurately estimate allele frequencies[END_REF] were not available and prevented us from analyzing criteria such as allelic richness, we could successfully refine the low diversity of X. index using the few individuals available. Mitochondrial data from 'samples' (instead of 'populations') permitted us to trace the hypothetic dissemination routes of X. index and to reveal that they were closely related to the dissemination routes of its domesticated grapevine host plant. In particular, using microsatellite diversity has given us the complemental ability to refine our hypotheses on the epoch of the major nematode dissemination events.

Nematode lines used in the study are globally representative of the diversity of

X. index

Several lines had been created in 2011 using preliminary data from mitochondrial diversity [START_REF] Villate | Spatial distribution of the dagger nematode Xiphinema index and its associated Grapevine fanleaf virus in French vineyard[END_REF]. Our results have confirmed these informations and shown that the four lines selected to challenge the resistance can be considered as globally representative Interestingly, geographical coverage of the four lines spans widely distant countries highly affected by GFLV and ranging from Middle East (Iran), to Near-East (Samos), up-to the Western Mediterranean major countries, Italy and Spain.

IV.1.2. Plant resistance and nematode adaptation

One major objective was to answer the question whether the nematode X. index adapts (and how it may adapt) to plant resistance over years. Given that preliminary data, using plant material obtained from in vitro, suggested a progressive adaptation of the nematode to plant resistance, our work aimed first at confirming this putative adaptation. Consequently, we have performed in parallel a comparison of plant material obtained from in vitro with classically propagated plant material, i.e. hardwood cuttings rooted into a sandy soil substrate. Our results have brought major information on the effect of the two types of plant propagation on i) the plant resistance and ii) the nematode adaptation.

The mode of plant propagation affects plant resistance

We have studied the effect of the mode of plant propagation on plant resistance with two criteria related to the nematode population dynamics: reproduction factor and population structure.

Using four mixed representative lines, we have first monitored, regardless of the line identities, the evolution of the nematode reproduction factor (RF) in plants aged 3 to 6 years.

For both F1 and BC1 resistant material, we could establish that materials obtained from in vitro have significantly higher RFs than materials obtained from cuttings. Cuttings expressed a resistance that was durable as shown by their RFs always lower than 1, whatever the plant age.

The hypothesis of a 'susceptibilization' effect of in vitro culture that preceded our experiment is highly plausible. This effect might be also partly due to IBA, the auxin compound used in the rooting medium just before acclimatization. Nevertheless no IBA effect has been shown on resistant Prunus crops towards root-knot nematodes (Esmenjaud et al., 1993). An effect of cytokinins, as observed in tomato (S. lycopersicum, S. sisymbriifolium) [START_REF] Dropkin | The hypersensitivity reaction of tomatoes resistant to Meloidogyne incognita: reversal by cytokinins[END_REF] or Musa spp. [START_REF] Elsen | In vitro screening for resistance to Radopholus similis in Musa spp[END_REF] towards Meloidogyne and Radopholus similis, respectively, is also excluded as multiplication and rooting culture media were free of these hormones. Nematode progressive multiplication in R accessions obtained from in vitro appear linked to a different architecture of the root system in this propagation type that may have induced discrete but putatively durable physiological changes in apical root tissues from where nematodes feed. The effect of in vitro culture has already been evidenced in interactions between plants and endoparasitic nematodes, but it is reported for the first time here for an ectoparasitic nematode species. Furthermore, the unprecedented duration of 'at least 6 years' suggests that the plant has an unsuspected long memory of its in vitro past. For R. similis in Musa spp. for example, the effect has been shown to be temporary (8 weeks) [START_REF] Elsen | In vitro screening for resistance to Radopholus similis in Musa spp[END_REF].

We could also highlight that, besides harboring contrasting nematode numbers, cutting and in vitro resistant materials exhibit different population structures, after 5 and 6 years, characterized by a number of juveniles significantly lower in cutting plants than in in vitro plants. In resistant accessions, this phenomenon logically illustrates the lower reproductive potential of females from cutting plants in comparison with plants obtained from in vitro.

Females and 4 th -stage juveniles have been shown to survive under starving conditions at stable temperatures of 7 and 20°C for over 4 years [START_REF] Demangeat | Survival of Xiphinema index in vineyard soil and retention of Grapevine fanleaf virus over extended time in the absence of host plants[END_REF]. This duration might be the maximal limit for juveniles, but females might have the ability to survive longer, i.e. up-to 6-7 years. Our results for resistant cutting material confirm that the 'eradication' of the nematodes, and mainly of the females, is particularly difficult: it illustrates the need for long duration experiments in studies on population dynamics of X. index faced to resistant accessions as well as to other control methods such as fallow crops.

The differences between in vitro and cutting plant material observed above in the ratio juveniles/adults were not seen in the ratio males/females. Even though the recognition of the rare males among the high numbers of females is not easy and may have generated poorly reliable data, we did not get any evidence that the sex ratio was influenced by the propagation type or the resistance status of the plant. Consequently, our results do not bring conclusive data in these domains. By contrast, in RKNs, high nematode densities or lack of food resources [START_REF] Davide | Influence of the environment on development and sex differentiation of root-knot nematodes[END_REF] or the presence of resistance genes in the host [START_REF] Moura | Post-infectional development of Meloidogyne incognita on susceptible and resistant soybean genotypes[END_REF] are known to induce the development of an increased rate of males.

Despite the evidence of sexual reproduction, its involvement in the adaptation of the nematode is poorly plausible

The putative adaptation of X. index has been studied first with a nematode genetics approach. Our results have confirmed that microsatellites are reliable markers for monitoring and/or tracing nematode line evolution. Characterization of microsatellite MLGs permitted us to detect a few but clearly identified hybrid individuals between different lines at increasing rates between 4 and 6 years. Thus, our results did confirm under controlled conditions the ability of X. index to reproduce sexually as already observed in the vineyard (Villate et al., 2010).

The occurrence of hybrids in plants was detected from both types of plant propagation.

Our results thus suggest that the nematode may alternate sexual or asexual reproduction modes, even though sexual reproduction appears rare and independent of its biotic environment (R or S accession). This supports the hypothesis that the 'decreased' resistance observed from resistant in vitro material is not due to an adaptation of the nematode consecutive to hybridization. To go further, this also reinforces the probability that we have evidenced an apparent but not real resistance 'overcoming', a phenomenon that is independent from the nematode and might be due to a modified physiological status of the plant. However, our results do not discard totally the possibility that nematodes reared on resistant material obtained from in vitro may progressively acquire adaptative (or virulence?) factors during their long interaction with the plant. Nevertheless, these results, by showing that the resistance from cutting material is durable (over 6 years), conversely suggest that such factors are not selected/expressed under the continuous resistance pressure exerted by the plants from this propagation type. Out of the four lines used and whatever the resistance status of the plant material, our results show differences in lines aggressiveness, Nevertheless, these differences did not induce a total elimination or conversely a complete selection of one of the lines over years. Hence, it appears that, despite their variable aggressiveness, only Iran (and eventually Italy) might have undergone the selection of (a) major adaptative factor(s). This also illustrates the absence of a marked genetic drift between populations kept in the ISA nematode that we revealed in Chile and Argentina. Our study could benefit from a deeper investigation dealing with the historical routes of the conquest of South America, and of the Andean regions in particular, by the Spanish colons. In Peru, Bolivia, Ecuador and Columbia, these data might detect introduction events that would be independent from those revealed in the two more Southern Andean countries that we have explored.

In Western European countries, except in the Southern Iberian Peninsula and in the Bordeaux vineyards, we lack samples from European regions in which initial introduction may be attributed to Romans. In Southern France notably, the single haplotype from Fréjus that we have assigned to this late-Antiquity Epoch is certainly not isolated and complemental informative data would be greatly valuable. Another interesting research field could be dedicated to the dispersal of nematode-contaminated plant material consecutive to the phylloxera crisis at the end of the 19 th century. Because of their still recent historical occurrence, these events should be easier to document. This might be a good example to illustrate how a soil-dwelling nematode with a very limited self-mobility and few host plants has become a parasite with a high economical impact. Grapevine has great cultural and societal impacts, notably in Southern European countries. Thus, in the reverse sense, reconstituting the putative initial focus(es) and dissemination route(s) of the resistant grapevine rootstocks selected and diffused at that time, using X. index as a marker, would be quite original. Such data will contribute to understand the putative and unexpectedly quick dispersal of the nematode at this time and its today's major involvement in GFLV-linked grapevine decline (i.e.

current 'dépérissement de la vigne'), particularly in France.

IV.2.2. Diversity data (and nematode lines) will be a baseline for evaluation of control methods

Besides rootstock resistance to X. index, current research against GFLV includes another major approach based on the use of fallow crops with an antagonistic effect against the vector nematode. In this objective, ongoing experiments conducted in France all deal with populations from the Western (W) subclade. A pioneer work, reporting notably the antagonistic effect of hairy vetch, in an experimental field from the Bordeaux vineyard (Villate et al., 2013) has also considered a population that belongs to this subclade. Genetically-distant populations may show different behaviors to antagonistic plants: extending experimental results to countries harboring other X. index subclades would need to take into account populations from these subclades.

IV.2.3. Plant resistance and nematode adaptation

Deciphering how the mode of plant propagation affects plant resistance is a challenging task

Our data do not permit to know how long the 'suceptibilization' phenomenon associated to the in vitro origin of the resistant material may last, beyond the 6 years of our study. As grapevine plants are perennial crops grown for more than 20-30 years, it is highly plausible but not sure that they gradually recover their full resistance. The link between the architecture of the root system in this propagation type and the discrete but putatively durable physiological changes in apical root tissues from where nematodes feed merits further investigations. These changes are advantageously exploited by the ectoparasitic nematode to facilitate its feeding and subsequent reproduction. Deciphering how an in vitro plant environment may induce putatively durable changes underlying the 'susceptible' physiological status expressed by the plant is a challenge.

Our results highlight the need for a better knowledge of the histological mechanisms of resistance to X. index. In a susceptible plant, the nematode triggers a (limited) cell polyploidization and a cognate feeding site. In muscadine-derived resistant plants, studies on the histological mechanisms of resistance show that a hypersensitive response is induced in both in vitro and cuttings plant materials (D. Esmenjaud and M. Y. Banora, unpublished data).

Our data on the dependence of resistance on the type of plant multiplication suggest that, after an initial in vitro propagation step, the cell hypersensitive response might be delayed in a manner that is apparently incipient for the nematode but that would allow its slightly prolonged feeding from apical root tissues. This 'susceptibilization' may act by gently but lastingly 'softening' the cell wall structure and thus by delaying the critical point from where the feeding cells will trigger a hypersensitive response.

Is there indeed an adaptation of the nematode having faced resistance in plants obtained from in vitro?

A definitive proof of the absence of a specific adaptation of the nematode consecutive to its inoculation on resistant material obtained from in vitro is still needed. It might be performed, on a resistant accession obtained from cuttings, by comparing the population dynamics of i) '(pre)adapted' individuals obtained from in vitro on this accession (carrying the R factors) and ii) 'non (pre)adapted' individuals obtained from a susceptible grapevine accession (lacking the R factors). We cannot exclude that nematodes that have faced a 'softened' resistance have acquired (selected) some pathogenicity factors that might facilitate their subsequent ability to overcome a non-modified ('natural') resistance, represented in our study by plants obtained from cuttings.

What are the determinants driving or influencing the nematode shift from a reproduction mode to another?

We still ignore in X. index the determinants of the change from a reproduction mode to another. These factors may be either biotic or/and abiotic. Studies aiming at identifying them appear necessary but very difficult to conceive. Our study failed to reveal that the plant resistance selection pressure was a major determinant. According to McDonald and Linde (2002), pests with a high evolutionary potential have a higher ability to overcome plant resistance. Thus, pest populations that show the highest probability to overcome resistance are those with a dual reproduction mode. Even though sexual reproduction should contribute to the adaptation of X. index to diverse biotic or abiotic environments, our results did not bring evidence of its involvement to face plant resistance.

Muscadine-derived material is a promising strategy to contribute to GFLV control

Durability is closely linked to the genetic determinism of resistance. Our results highlight the need for a better knowledge of the genetics of resistance to X. index in muscadine-derived material. Ongoing research suggests that resistance is conferred by several independent factors that would be major genes or QTLs. Nevertheless, the availability of a progeny of less than hundred individuals from the BC1 segregating cross between the distant Vitis and Muscadinia genera compromise the precise mapping of resistance. However, the durable resistance, revealed after 6 years under controlled conditions and highly suggested after 16 years under field conditions, might be related to this polygenic status. The creation of monogenic BC2 progenies, i.e. carrying a single putative gene, is currently attempted at UMR EGFV. The subsequent evaluation of these BC2 progenies at UMR ISA would allow to confirm the polygenic status of resistance. Such material will also open new opportunities to relate genetic, histological and durability data.

Arresting or delaying GFLV transmission to grapevine by acting on the vector nematode through plant genetic resistance is a promising alternative. The successful control of the vector nematode, by reducing or delaying GFLV transmission in the field, should be a major component of an integrated control of the virus. Our results bring information that will be useful for the grapevine community and in particular for grapevine breeders, nurserists and growers.

Esmenjaud, D., Minot, J. C., Voisin, R., Salesses, G., Poupet, R., & Onesto, J. P. (1993). Assessment of a method using plantlets grown previously in vitro for studying resistance of Prunus cerasifera Ehr. (Myrobalan Plum) to Meloidogyne spp. Nematropica, 23 [START_REF] Decraemer | Plant nematology[END_REF], 41-48. . The reference genetic code is 'Invertebrate mitochondrial'. For the CytB and CO1 genes, as there were several close species sequences available on the database, our sequences matched specific CytB and CO1 domains and revealed these respective superfamilies. For ATP6 and ND4, only non-specific hits were detected, which is due to the absence of other sequences of these nematodes in the database. Nevertheless, our sequences did belong to the superfamilies ATP and ND4, respectively.

CHAPTER 3 :

 3 EVALUATION OF THE DURABILITY OF NEMATODE CONTROL STRATEGY BASED ON MUSCADINIA-DERIVED RESISTANT ROOTSTOCKS ................................................................104 III.1. RESISTANCE DURABILITY BASED ON A 6-YEAR ASSESSMENT UNDER CONTROLLED CONDITIONS ........................................................................................................................................104 III.1.1. Introduction .........................................................................................................................104

108 III. 1 . 2 .

 10812 Material and methods .........................................................................................................110

. 1 . 3 .

 13 Results ..................................................................................................................................115

  .1.4. Discussion and conclusion ...................................................................................................126

  A LONG-TERM ASSESSMENT UNDER FIELD CONDITIONS .............................................................................................................................................................130 III.2.1. Introduction .........................................................................................................................130 III.2.2. Plant material and experimental design ............................................................................130

  June 2015 ...............................................................................................132

135 III. 2 . 5 .

 13525 Discussion and conclusion ...................................................................................................140

CHAPTER 4 :

 4 CONCLUSIONS AND PERSPECTIVES .........................................................................144 IV.1. CONCLUSIONS ............................................................................................................................144 IV.1.1. The global genetic diversity of X. index ..............................................................................144 IV.1.2. Plant resistance and nematode adaptation .......................................................................145 IV.2. PERSPECTIVES .............................................................................................................................148 IV.2.1. The global genetic diversity of X. index still needs to be refined ......................................148 IV.2.2. Diversity data (and nematode lines) will be a baseline for evaluation of control methods .........................................................................................................................................................149 IV.2.3. Plant resistance and nematode adaptation .......................................................................150 REFERENCES ......................................................................................................................................152 ANNEXES ........................................................................................................................................170

Figure 1 . 1 .Figure 1 . 2 .Figure 1 . 3 .Figure 1 . 4 .

 11121314 Figure 1.1. Schematic overview of the phylum Nematoda. Division in 12 major clades is based on SSU rDNA sequence data Figure 1.2. Different types of feeding strategies used by genera of plant parasitic nematodes in plants during parasitism of root tissue Figure 1.3. Life cycle of a mitotic parthenogenetic RKN Figure 1.4. Life cycle of cyst nematodes Heterodera spp. and Globodera spp.

Figure 1 . 5 .Figure 1 . 6 .Figure 1 . 7 .Figure 1 . 8 .Figure 1 . 9 .Figure 1 . 10 .Figure 1 . 11 .Figure 1 .Figure 1 . 13 .Figure 1 . 14 .Figure 1 . 15 .Figure 1 . 16 .Figure 1 . 17 .Figure 1 . 18 .Figure 1 . 19 .Figure 1 . 20 .Figure. 1 . 21 .Figure 1 . 22 .Figure 1 . 23 .Figure 1 . 24 .Figure 1 . 25 .Figure 1 . 26 . 4 Figure 1 . 27 .Figure 1 .Figure 2 .Figure 3 . 3 Figure 3 . 1 .Figure 3 . 2 .Figure 3 . 3 .Figure 3 . 4 .Figure 3 . 5 .Figure 3 . 6 .Figure 3 . 7 .

 151617181911011111131141151161171181191201211221231241251264127123331323334353637 Figure 1.5. The life cycle of species from the Longidoridae family Figure 1.6. Incompatible interaction in resistant accessions (P.2175 and 35) Figure 1.7. Resistance models during plant-nematode interaction Figure 1.8. The impact of three factors (reproduction mode, gene and genotype flow, and population size) on the evolution of the pathogen or nematode (adapted from McDonald & Linde (2002) and Villate (2008)) Figure 1.9. Dissemination of domesticated grapevine from its putative native area Figure 1.10. GFLV symptoms on grapevine Figure 1.11. Infection of French vineyards in a survey conducted by ONIVINS in France (2004) Figure 1.12. GFLV structure Figure 1.13. Technical regulatory scheme leading to the sanitation and approval of grapevine clones in France

Figure 3 . 8 .Figure 3 . 9 .

 3839 Figure 3.8. Evolution of the ratio female/larva of X. index on the 4 rootstock clones, 8771, RPG1, RGM and CS, obtained from cutting plants and in vitro plantsFigure 3.9. Evolution of the proportion of the four nematode lines of X. index during the durability experiment over 3 to 6 years on the grapevine clones, 8771, RPG1, RGM and CS, on cutting and in vitro plants

Figure 3 . 10 .Figure 3 . 11 .Figure 3 . 12 .Figure 3 . 13 .Figure 3 . 14 .Figure 3 . 15 .Figure 3 . 16 .Figure 3 . 17 .Figure 3 . 18 .Figure 3 . 19 .

 310311312313314315316317318319 Figure 3.10. Relative frequency of each line (Italy, Spain, Samos and Iran) of X. index in the two resistant accessions 8771 and RPG1, after 6 years Figure 3.11. Evidence of microsatellite multilocus hybrid genotypes detected from the four X. index pure lines mixed in containers of plant material obtained from cutting and in vitro Figure 3.12. Electropherograms illustrating the detection of a hybridization event Figure 3.13. Design of the field experiment of Nemadex AB in Domaine du Chapitre Figure 3.14. Results of the evolution of GFLV contaminations by ELISA tests Figure 3.15. Differential GFLV attacks in Nemadex AB and SO4 rootstocks grafted with Cabernet-Sauvignon in the field trial Figure 3.16. Soil sampling in the field in trenches, approx. 60 cm deep, 120 cm long and 40 cm wide, dug by a narrow excavator Figure 3.17. Spatial distribution in 15-year-old grapevine plants (2014) of ELISA GFLV-positive, negative and dead plants Figure 3.18. Spatial distribution, along the 5 grapevine rows, of the individuals of X. index recovered from the field experimental design of Domaine du Chapitre Figure 3.19. Spatial distribution, along the grapevine rows, of the individuals of X. pachtaicum recovered from the field experimental design of Domaine du Chapitre

  . Most PPNs are soil-borne root pathogens and only a few species feed primarily upon shoot tissues. PPNs have appeared independently in four clades: Triplonchida (clade 1), Dorylaimida (clade 2), Panagrolaimoidea (clade 10) and Tylenchida (clade 12) (Fig. 1.1).

Figure 1 . 1 .Figure 1 . 2 .

 1112 Figure 1.1. Schematic overview of the phylum Nematoda. Division in 12 major clades is based on SSU rDNA sequence data. Next to the individual branches of each clade number, the order or suborder names (with the endings -ida and -ina, respectively) are reported. Trophic ecologies are visualized by icons, and only the 4 clades containing plant parasitic nematodes are shown (clades 1, 2, 10 and 12)(adapted from[START_REF] Quist | Evolution of plant parasitism in the phylum Nematoda[END_REF]

Figure 1 . 3 .

 13 Figure 1.3. Life cycle of a mitotic parthenogenetic RKN. (a) A root tip with J2s inside. (b) Typical symptoms (i.e. galls) on tomato roots. (c) An infested root showing a female and five giant cells ( * ) constituting the nematode feeding site (Castagnone-Sereno et al., 2013)

Figure 1 . 4 .

 14 Figure 1.4. Life cycle of cyst nematodes Heterodera spp. and Globodera spp. (Bridge & Starr, 2007)

Figure 1 . 5 .

 15 Figure 1.5. The life cycle of species from the Longidoridae family (Bridge & Starr, 2007)

Figure 1 . 6 .

 16 Figure 1.6. Incompatible interaction in a resistant plum accession carrying the Ma gene. The nematode at the vermiform 2nd-stage juvenile (J2) cannot induce the feeding structure (adapted from Khallouket al., 2011)

Figure 1 . 7 .

 17 Figure 1.7. Resistance models during plant-nematode interaction. Nematodes may face two different plant defense layers: (1) basal defense such as WAK1, PG-inhibiting proteins (PGIPs), pattern-triggered immunity (PTI) including the production of reactive oxygen species (ROS), callose, and lignin. (2) Rgenes that recognize effectors either directly or indirectly and initiate effector-triggered immunity (ETI). R genes may be NLRs (nucleotide-binding leucine rich repeat) and non-NLRs type genes.[START_REF] Holbein | Plant basal resistance to nematodes: An update[END_REF]

  vitro culture and the primary classes are auxins and cytokinins. The most important synthetic auxins are 1-naphthaleneacetic acid (NAA), indole-3-butyric acid (IBA), 4-chlorophenoxyacetic acid (CPA), 2,4-dichlorophenoxyacetic acid (2,4-D), 2-naphthoxyacetic acid (NOA) and indole-3-acetic acid (IAA). Main cytokinins include 6-benzyladenine or benzylaminopurine (BAP), kinetin (Kin), 6-(2-isopentenyl) adenine (2iP), zeatin or zeatin riboside (ZR) and 3-(1,2,3,thidiazol-5-yl)-I-phenylurea or thidiazuron (TDZ).

(

  high gene flow). In such a population, recombination may make it possible to create new virulent individuals. During the asexual phase, the absence of recombination allows the survival and the multiplication of the virulent individuals and the establishment of a new virulent population in the environment.

Figure 1 . 8 .

 18 Figure 1.8. The impact of three factors (reproduction mode, gene and genotype flow, and population size) on the evolution of the pathogen or nematode (adapted from McDonald & Linde (2002) and Villate (2008)).

  1.9). That diffusion of viticulture from the Near East has occurred through two ways around the Mediterranean Basin: (1) a Northern way from Eastern to Western Europe by Hittite, Greek and Roman people in the Antiquity(Sechrist, 2017) and (2) a Southern way through Mesopotamia, Egypt and the Iberian Peninsula by Phoenicians in the Antiquity and Arabs later. Domesticated grapevine finally reached the new world (North and South America) in 16-17 th century by the Spanishs (Fig. 1.9).

Figure 1 . 9 .

 19 Figure 1.9. Dissemination of domesticated grapevine from its putative native area. The primary center of domestication is indicated in the Transcaucasian (dark green).

Figure 1 . 10 .

 110 Figure 1.10. GFLV symptoms on grapevine. A & C: Yellowing symptom. B: Leaf malformation GFLV is responsible for fanleaf degeneration called "court-noué", the most severe viral disease of grapevines. This is a worldwide distributed virus, reported in North and South America, Europe (France, Italy, Spain…), Australia, etc. GFLV causes diverse symptoms and the most frequent are leaf yellowing, mottling and vein clearing, leaf distortion, fanleaf shape (the origin of the name 'Grapevine fanleaf virus'), short internodes and stunted aspect of the plants (origin of the French name 'Court-noué') (Fig.1.10). All of them induce lower fruit quality (with grape 'coulure' and 'millerandage' symptoms on bunches) and lower yields(Andret-Link et al., 2004). To the French grape and wine industries, "Court-noué" causes annual losses estimated of ~1 billion[START_REF] Andret-Link | Transmission specificity of plant viruses by vectors[END_REF]. Once infected, the grapevine will decrease its

Figure 1 . 11 .

 111 Figure 1.11. Infection of French vineyards in a survey conducted by ONIVINS in France (2004). 70% of

(

  nt) long and contains a single open reading frame of 6,855 nt, extending from nt 243 to 7097(Ritzenthaler et al., 1991), coding for the 1A, 1B Hel (helicase), 1C VPg , 1D Pro (proteinase) and 1E Pol (RNA-dependent RNA polymerase) proteins. The RNA2 is 3,774 nt long with a single open reading frame of 3,330 nt, extending from nt 233 to 3562[START_REF] Serghini | RNA2 of Grapevine fanleaf virus: sequence analysis and coat protein cistron location[END_REF] coding for the 2A HP (homing protein), 2B MP (movement protein) and 2C CP (coat protein) proteins. The CP is composed of three domains called C, B, and A(Schellenberger et al., 2011) (Fig. 1.12).

Figure 1 . 12 .

 112 Figure 1.12. GFLV structure. (A) GFLV genetic features. The 5' and 3' untranslated regions are denoted by single lines and the VPg is represented by a black circle. Polyproteins encoded by RNA1 and RNA2 are cleaved in five (1A-1E) and three (2A-2C) final maturation products (open boxes), respectively. 1B Hel (helicase); 1C VPg (viral protein genome-linked); 1D Pro (protease); 1E Pol (RNA-dependent RNA polymerase); 2A HP (homing protein); 2B MP (movement protein) and 2C CP (coat protein). Finally, the CP is composed of three domains from left to right called C, B, and A, respectively. (B) Crystal structure of GFLV capsid (adapted from Schellenberger et al., 2011).

  plant material. Examples of such organizations are the National Grape Clean Plant Network (NCPN) in the US and the Canadian Plant Protection Export Certification Program (PPECP) for grapevine nursery stocks in Canada. In the same way in France, ONIVINS, established in 1983, oversees the entire wood and vine-seed industry and, as such, controls and certifies the propagating material of the vine. Below is a simplified flowchart for the sanitary selection of clones in France (revised in 2013) (Fig. 1.13). The aim is to get healthy clones from the infected initial grapevine clones of interest: plant material undergoes several steps with regular sanitary controls using appropriate molecular diagnostics (ELISA, PCR) and visual observations.

Figure 1 . 13 .

 113 Figure 1.13. Technical regulatory scheme leading to the sanitation and approval of grapevine clones in France (from documents of the Official Directive 2005 for the French technical regulation of vine clones (2005/42/EC) modified in 2013).

(

  amiRNAs).[START_REF] Jelly | Transient expression of artificial microRNAs targeting Grapevine fanleaf virus and evidence for RNA silencing in grapevine somatic embryos[END_REF] developed two artificial microRNAs (pre-amiRNAs) targeting the coat protein (CP) gene of GFLV for RNA silencing in grapevine somatic embryos. Very recently,[START_REF] Hemmer | Nanobody-mediated resistance to Grapevine fanleaf virus in plants[END_REF] used the nanobodies (Nb 23) obtained from camelid after 6 weekly injections of GFLV. The transgenic grapevine did not show any symptoms of GFLV or did not reveal the GFLV presence by ELISA tests at 45 days post-acclimatization. These data are very

Figure 1 . 14 .

 114 Figure 1.14. Phylogenetic relationships between Xiphinema spp. based on D2-D3 region of 28S rDNA. The nematode species L. caespiticola (Longidoridae) and Tylencholaimus mirabilis(Dorylaimidae) were used as outgroups (adapted from[START_REF] Tzortzakakis | Integrative diagnosis and molecular phylogeny of dagger and needle nematodes of olives and grapevines in the island of Crete, Greece, with description of Xiphinema cretense n. sp. (Nematoda, Longidoridae)[END_REF]

( 190 -

 190 215 µm) which allows it to reach the vascular tissue of plants (Fig. 1.18B). The stylet is composed of three characteristic basal flanges in its posterior end. The odontostyle is lined with a cuticle and alongside the odontophore and the esophagus serve as a surface for monolayer retention of GFLV particles (Fig. 1.23). Females have a vulva slit about half bodywidth long usually located at 38-40 percent of body length and two ovaries (Fig. 1.16 B).

Figure 1 . 15 .

 115 Figure 1.15. Distribution map of X. index (CABI, www.cabi.org/isc/datasheet/57032)

  of species misidentification. Main morphological and anatomical features of the very close species X. index and X. diversicaudatum are reported in Fig. 1.16 as an example of similarity between species. Consequently, accurate complemental identification tools based on molecular sequences are useful.

Figure 1 . 16 .

 116 Figure 1.16. Morphological and anatomical features of the dagger nematodes X. diversicaudatum (A) and X. index (B). (a) A full body length with the head (arrow 1) and the tail (arrow 2); (b) head region showing a full stylet (arrow 3) and the esophageal bulb (arrow 4) followed by the intestine (arrow 5); (c) lateral view of the vulva (arrow 6); (d) lateral view of a male tail region with paired spicules (arrow 7); (e) lateral view of the female tail. Scale bars are indicated for each element (Andret-Link et al., 2017)

Figure 1 . 17 .

 117 Figure 1.17. Xiphinema spp. identification based on the ITS1 ribosomal fragment located between the conserved sequences of the 18S and 5.8S genes. A: The ITS1 region and location of the 6 markers XD (X. diversicaudatum), XV (X. vuittenezi), XITA (X. italiae), XI (X. index), and control primers (S-ITS1 for sense (located on 18S gene) and A-ITS1 for antisense (located on 5.8S gene) to amplify ITS1 fragment). B: Electrophoresis of the amplification products from DNA isolated from X. diversicaudatum, X. vuittenezi, X. italiae and X. index in a multiplex test. DNAs were obtained from single individuals. Lanes 1 and 12, M (DNA ladder); lanes 2 and 3, 1.1-kbp control ITS1 fragment from X. index; lanes 4 and 5,

5 .

 5 Biology and ecologyLife cycle and reproduction modeXiphinema index primarily feeds on the root tips of grape rootstocks (Vitis spp.). The reproduction of X. index is mainly meiotic parthenogenetic (Fig.1.19 B) and a single juvenile is capable of establishing a population[START_REF] Dalmasso | Ovogenese et embryogenese chez Xiphinema index (Nematoda: Dorylaimida)[END_REF]. Nevertheless, the occurrence of rare sexual reproduction events has been shown in the field(Villate et al., 2010) (Fig.1.19 A).

Figure 1 . 18 .

 118 Figure 1.18. The dagger nematode X. index. A: life cycle. B: Anterior part of esophagus with spear (odontostyle) averaging 126 µm long and the spear extension (odontophore) with average 70 µm in length; A: Drawing by Nemaplex, University of California, Davis (http://nemaplex.ucdavis.edu/Taxadata/G143s3.aspx); B: Adapted from Siddiqi (1986).

Figure 1 . 19 .

 119 Figure 1.19. Schematic representation the reproduction modes of X. index. A: Amphimictic (sexual) reproduction mode (rare); B: Meiotic parthenogenetic reproduction mode (predominant). 2n represents the diploid chromosome number (2n = 20 in X. index) (adapted from Castagnone-Sereno et al., 2013)Temperature is an important modulating factor on the reproduction and life cycle of X. index, which is typically associated with grapevines in warm climates. Xiphinema index has been shown to survive in a wide range of soil temperatures from -11°C to 35°C, but constant temperatures for 10 days of 45°C or -22°C killed the nematodes[START_REF] Van Zyl | Xiphinema index and its relationship to grapevines: A review[END_REF]. The X.

Figure 1 . 20 .

 120 Figure 1.20. Direct damage on grape caused by X. index. A: Terminal swellings ('galls') on grapevine root system of the susceptible cultivar 'Rupestris du Lot', B: Longitudinal section through a swollen root tip of a young grapevine root showing multinucleate cells after X. index feeding. Nuclei are indicated by black arrows. (Photos credit: INRA Sophia Antipolis).

Figure. 1 . 21 .

 121 Figure. 1.21. Densities of X. index in three soil profiles in Champagne vineyard (France) in different soil layers (depths of soil layers are indicated in cm on the Y axis and nematode numbers are indicated per kg of soil on the X axis)[START_REF] Esmenjaud | Vertical distribution and infectious potential of Xiphinema index (Thorne et Allen, 1950) (Nematoda: Longidoridae) in fields affected by Grapevine fanleaf virus in vineyards in the Champagne region of France[END_REF].

  Since then, this nematode has been considered as one of the most important viticultural pests in grape-growing areas throughout the world. In 1962, Hewitt et al. fulfilled Koch's postulates by using viruliferous X. index to transmit GFLV from C. amaranticolor to grapevines in which typical symptoms developed. These experiments demonstrated conclusively that GFLV is the causal agent of fanleaf degeneration disease.

Figure 1 . 22 .

 122 Figure 1.22. Dissemination scheme of Grapevine fanleaf virus by X. index in the field. A: Anterior part and stylet of a nematode feeding at the root tip. B: Scheme illustrating how the nematodes (in circles) vector the virus from plant to plant in the soil. C: GFLV field aerial symptoms (red ellipse) corresponding to a nematode focus in the soil. D: GFLV viral particles under electron microscope.

Figure 1 . 23 .

 123 Figure 1.23. Schematic representation of the morphological structure of the anterior region of Longidorus and Xiphinema showing the feeding apparatus. The retention site of the viral particles is highlighted in red brackets for both genera; odp: odontophore, gsh: guiding sheath, odt: odontostyle (adapted from Andret-Link et al., 2017. GFLV particles photo: Taylor & Robertson, 1970)In vector nematodes, viral particles are adsorbed at specific sites probably in association with a receptor present at the inner cuticle of the digestive system. The

genus

  Muscadinia has a chromosomal number (2n = 40) different from the genus Vitis (2n = 38). Consequently, introducing this resistance into Vitis material by hybridization has been difficult. Despite this limitation, a few resistant intergeneric F1 hybrids V. vinifera x M.

Figure 1 . 24 .

 124 Figure 1.24. The two resistant clones used in the greenhouse experiment of Chapter 3. Upper photos: VRH8771. A: whole plant, B: shoot and leaf. Lower photos: RPG1. C: whole plant, D: shoot and leaf.In France, the F1 hybrid V. vinifera x M. rotundifolia VRH8771 (VRH for Vitis x rotundifolia hybrid) (Fig.1.24 A&B) is a good source for resistance to X. index[START_REF] Esmenjaud | Host suitability of Vitis and Vitis-Muscadinia material to the nematode Xiphinema index over one to four years[END_REF]). An individual called RPG1 and obtained by backcrossing VRH8773 (a brother clone of VRH8771 which is no longer available) with the Vitis rootstock accession 140Ru has shown good resistance and is now registered as a new rootstock (called 'Nemadex Alain Bouquet') (Fig.1.24 C&D; Fig.1.26) to be used in highly nematode-infected plots under certain soil

  At INRA-ISA, these observations were refined on the roots of the resistant Muscadinia F1 hybrid Chapter 1 62 VRH8771 and of the resistant BC1 cross RPG1: attacks induce a hypersensitive-like response with no or limited galling while the susceptible plants form galls in which multinucleated cells are observed (D. Esmenjaud and M. Banora, unpublished) (Fig. 1.25). Therefore, this resistance phenomenon presumably acts as a filter that prevents or at least delays transmission of virus particles to the healthy tissues.

Figure 1 . 25 .

 125 Figure 1.25. Histological responses of some Muscadinia-derived material resistant to X. index at 12 days post-inoculation. (A) VRH8771 and (B) RPG1 roots display hypersensitive-like responses. (C) Uninoculated control, RPG1.

  . The Muscadinia-Vitis F1 hybrid VRH 8771 is the resistance source retained in the current INRA breeding project. The detailed pedigrees of the VRH 8771 and RPG1 resistant materials, derived from the resistant M. rotundifolia accession NC 184-4, and their detailed male/female status are shown in Fig. 1.26.

Figure 1 . 26 .

 126 Figure 1.26. Pedigree of the resistant plant accessions derived from the muscadine resistance source NC 184-4. R, resistant; S, susceptible; RPG1 = Nemadex Alain Bouquet.

  the breeding program has been based and will continue to be based in priority on VRH 8771 as an F1 representative of the original parental muscadine accession NC 184-4. Several studies, presented hereafter, are being conducted to characterize the resistance properties of VRH 8771 together with RPG1 as a BC1 representative. Diverse control or reference materials have also been used: the susceptible Muscadinia-Vitis F1 VRH 8426, from the parental muscadine Trayshed, the susceptible Vitis accessions, V. rupestris du Lot and V. vinifera Cabernet-Sauvignon, and the moderately resistant accession V. riparia Gloire de Montpellier.

Figure 1 . 27 .

 127 Figure 1.27. Reproduction factors (RF) of X. index in VRH 97-99-79 (= Vitis x Muscadinia F1 accession), in RPG1 (= F1 backcrossed by the Vitis rootstock 140Ru), and in two reference control rootstocks V. rupestris du Lot (RL) and V. riparia Gloire de Montpellier (RGM), evaluated after 1 and 4 years. For these data (6 replicates), bars with at least one common letter do not differ according to Fisher's (LSD) multiple range test at p ≤ 0.05. Plant material has been obtained from in vitro plants. * At 4 years in V. rupestris, values are lacking because of plant mortality consecutive to very high attacks after 1 year.

2 CHARACTERIZATION

 2 accessions representing different levels of resistance will be evaluated. This monitoring will aim at characterizing i) whether the nematode adapts and what is the link between this adaptation and the plant resistance status and ii), in the positive case, how this adaptation might take place: dominance of a particular line,occurrence of hybridization phenomena…My results should contribute to answer the question: Does the nematode X.index adapt and, if yes, how does it adapt to resistant grapevine rootstock plants over years?This page is intentionally left blank CHAPTER

  from the Near East occurred through two different routes around the Mediterranean Basin: a Northern way from Eastern to Western Europe (Hittite, Phrygian, Greek and Roman people in the Antiquity 40 ) and a Southern way through Egypt, the Maghreb up-to Gibraltar and the Iberian Peninsula (Phoenicians and then Romans in the Antiquity and Arabs from the 7th century ACE). In the Northern route, domesticated grapevines appeared in Minor Asia, Southern Greece, Crete and Cyprus in 3000-2500 BCE and in the Southern Balkans 48 in 1500-2000 BCE. They finally reached Southern Italy, Southern France, Spain and Portugal in the first millennium BCE 49 . For X. index, subclade NME matches the area of the Greek civilization with samples from Western Turkey, Cyprus, Crete and Egypt that might be a representative area of the North Mediterranean route of grapevine varieties. Nevertheless, the detection of an Egyptian sample that marks the Southern part of this putative Greek dissemination area suggests an introduction of the nematode there during the late Antiquity, i.e. during the reign of Alexander the Great or the subsequent Hellenistic period (356-31 BCE) and even later when Egypt has been integrated into the Roman Empire.

FiguresFigure 1 .

 1 Figures Figure 1. Maximum likelihood phylogenetic tree constructed for CytB sequences of X. index (subset of 43 representative individuals from 35 samples: see text) and X. diversicaudatum (8 samples) using Mega software version 6.0. X. americanum was used as outgroup.

Figure 2 .

 2 Figure 2. Phylogenetic tree and correlative hypothesis on the dissemination routes of X. index from its native area throughout the Mediterranean basin and beyond.

Figure 3 . 3 Table 1 .

 331 Figure 3. Unrooted tree of X. index individuals based on microsatellite multilocus genotypes

Figure 2 .

 2 Figure 2. Phylogenetic tree and correlative hypothesis on the dissemination routes of X. index from its native area throughout the Mediterranean basin and beyond. (A) Maximum likelihood (ML) phylogenetic tree for concatenated mitochondrial sequences of CytB, ATP6, CO1 and ND4 from the X. index representative individuals (35 samples). Bootstrap values based on 2000 interactions are indicated. (B) Putative dissemination routes inferred from mitochondrial data of the individuals shown in (A) and from microsatellite data of individuals from the same samples with identical haplotypes but different MLGs. The dark green zone (Transcaucasia and Southern Caspian Sea regions) encircles the putative native area of domesticated V. vinifera grapevine from where X. index has been first spread westward (black arrows). The hypothetic route from Today's Lebanon (Phoenician origin) for the subclade 'Samos & South Italy' is shown in a separate map.

  to studies on the mean-term and long-term durability of nematoderesistant rootstocks and cognate nematode adaptation. It develops firstly a resistance durability study conducted over 6 years under controlled conditions. Secondly, it is completed by a field study, established in 1999 in the Domaine du Chapitre (Villeneuve-les-Maguelone) of Montpellier SupAgro by Alain Bouquet. In this latter experiment, GFLV evolution in different rootstock modalities has been monitored by INRA Montpellier, Montpellier SupAgro and ITV, and results reported in this thesis deal with the nematode numbers from a soil sampling performed by ISA in 2015.

Figure 3 . 1 .

 31 Figure 3.1. Durability study under controlled conditions. (A) Creation of the X. index lines from which four ones were retained (Spain, Italy, Samos and Iran). (B) Steps and timing of the study using plants obtained from in vitro and from cuttings. At 3, 4, 5 and 6 years, nematodes were extracted and counted; out of them, a sample was genotyped with a set of microsatellite markers.

Figure 3 . 2 .

 32 Figure 3.2. Microsatellite characteristics of the four X. index lines retained for the study. These lines are designated 'Spain' (Xi-Sp-LM), 'Italy' (Xi-It-PC), 'Samos' (Xi-Gr-Sa) and 'Iran' (Xi-Ir-Sh). The multi-locus genotypes, showing the markers or the combination of markers specific for each line, are reported. Red circles highlight the specific alleles (size in bp) for Spain [Xi32 (GTT)-142], Italy [Xi04 (TG)-186], Samos [Xi24 (AC)-207 and Xi27 (GT)-223], and Iran [Xi16 (TC)-113, Xi22 (AAAC)-149 …].

Figure 3 . 3 .

 33 Figure 3.3. Illustration of the initial propagation modes of the plants used for resistance durability experiments. Left: rooted plants in in vitro rooting medium; right: rooted plants obtained from cuttings in fine-sand substrate.

Figure 3 . 4 .

 34 Figure 3.4. Scheme for the extraction of X. index nematodes from soil by the Oostenbrink elutriator method.

Figure 3 . 5 .

 35 Figure 3.5. Electropherogram illustrating the signal of some alleles or combinations of alleles for the different microsatellite loci of X. index. Xi04, Xi13, Xi16, Xi22, Xi24, Xi27, Xi29 and Xi 32 are the names of the loci. Numbers under each peak are the sizes of alleles in base pairs.Preparation of DNA templates from single individualsDNA from a single nematode individual was isolated by a simplified procedure: a single adult or juvenile was hand-picked, placed in 0.5 ml PCR tube containing 50μl lysis buffer (KCl 50 mM, Gelatin 0.05 %, Tris pH 8.2 10 mM, Tween 20 0.45 %, Proteinase K 60 μg/ml and MgCl2 2.5 mM). Then Eppendorf tubes were alternatively moved from liquid nitrogen to 60°C water bath for 10 times in order to facilitate break down of the nematode body. This step was followed by an incubation at 60°C for 90 min (the tubes were vortexed at least once during incubation to help breaking up tissues) and by a heating at 95°C for 10 min to inhibit the reaction of Proteinase K. Finally, individuals were cooled at 4°C, vortexed briefly (2-3 sec) and centrifuged shortly at 6,000 rpm for 30 sec. DNAs were stored at -20 o C until use for PCR or further experiments.

considered the 4

 4 clones, 8771 (R), RPG1 (R), RGM (intermediate, I) and CS (S), for analysis over a 6-year period (3 to 6 years after planting, in cutting and in vitro material). Nematodes were recovered from soil containers and we evaluated their reproduction (RF= final nematode number/initial nematode number) per clone and type of multiplication. Development of the plants, as a complemental criterion, was estimated by the weight of roots.

Figure 3 . 6 .

 36 Figure 3.6. Reproduction factors (RF) to X. index for accessions 8771, RPG1, RGM, and CS after 3 (blue), 4 (orange), 5 (grey) and 6 (yellow) years. A: Cutting plants. B: In vitro plants. For each propagation type, bars with different letters significantly differ according to Fisher LSD test (P≤0.05). Black stars indicate missing data. In resistant material, there were at least 7 (8771) and 5 (RPG1) replicates. In RGM and CS, there were 3 to 5 replicates.

Figure 3 . 7 .

 37 Figure 3.7. Nematode numbers of X. index per gram of roots on cutting plants (A) and in vitro plants (B).

  14 larva per female. By contrast, in cutting plants, we observed a different evolution. Four-year plants had values ranging from 41.0 to 76.8 % when all accessions are confounded while, in 5-year plants, the ratio increased in both resistant accessions and remained low in others. In 6-year plants, values were very high in 8771 and RPG1 (154.3 to 167.1%) and also increased in the other accessions to higher values (77.4 to 93.2 %) than in previous years (Fig. 3.8A). These values clearly showed an increased proportion of the female stage in plants aged of 5 (8771 and RPG1) or 6 years (all accessions).This increase resulted much more marked in the two resistant accessions. As the numbers of individuals recovered in resistant material has remained stable over the 3 years, these values state clearly that their respective populations have become older, which is in line with the resistant status of their accessions. Even though the phenomenon is less marked, the increased proportions of females in RGM and CS suggest that, in these plants, nematode numbers have reached a high and saturating density, which restricts the production of new progenies by the females. Interestingly, this clear aging of the nematode population in the material obtained from cuttings (undeniable at least in the resistant accessions) has not been observed in the material obtained from in vitro.

Figure 3 . 8 .

 38 Figure 3.8. Evolution of the ratio female/larva of X. index on the 4 rootstock clones 8771, RPG1, RGM and CS obtained from cutting plants (A) and in vitro plants (B). Bars with different letters significantly differ according to Fisher LSD test (P≤0.05). Data from A and B have been mixed for statistical analysis.

  3.9) over the 3-to 6-year experiment. Absence of certain lines in replicates was exceptional and presumably due to chance because of low numbers of individuals genotyped and not to a real lack. For example, in the few in vitro RGM plants genotyped, we recorded 2 lines (Italy and Spain) out of 3 individuals after 3 years, 3 lines (Italy, Spain and Samos) out of 15 individuals after 4 years, and the 4 lines out of 23 individuals and 3 lines (Italy, Spain and Samos) out of 20 individuals after 5 and 6 years, respectively (Fig. 3.9B). Nevertheless, in an overall view, Iran and Italy were the lines most frequently recovered and appeared to express an higher aggressiveness than the two others (Fig. 3.9 A&B).

Figure 3 . 9 .

 39 Figure 3.9. Evolution of the proportion of the four nematode lines (Italy, Samos, Spain and Iran) of X. index during the durability experiment over 3 to 6 years on the grapevine clones, 8771, RPG1, RGM and CS. A: Cutting plants. B: In vitro plants. The numbers below each bar indicate the total nematode numbers that were genotyped.

Figure 3 . 10 .

 310 Figure 3.10. Relative frequency of each line (Italy, Spain, Samos and Iran) of X. index in the two resistant accessions 8771 and RPG1 after 6 years. A: Cutting plants. B: In vitro plants. Detection of hybrid individuals Detection in 3-and 4-year old plants. Search of hybrid individuals has begun in plants aged 3 years, i.e. one year earlier than nematode countings for resistance evaluation. A total of 485 individuals (200 individuals in 3-year plants and 285 individuals in 4-year plants) have been genotyped for microsatellites over the two first years for both cutting and in vitro plants (5 individuals for each replicate = pot). From these data, the first (and sole) evidence of a hybridization event was found at 4 years post-inoculation. This hybrid individual has been detected from an in vitro RPG1 plant and is a cross between lines 'Iran' and 'Italy'. This detection of the hybrid individual between Iran and Italy is illustrated in Fig. 3.12. The figure shows the presence of new allelic associations such as alleles of 124 bp and 149 bp in locus Xi22 and alleles of 212 bp and 216 bp in locus Xi24. This figure also demonstrates how we can distinguish reliably this hybridization phenomenon from accidentally mixed DNAs in these lines. Detection in 5-and 6-year old plants. In 5-year old plants, we monitored 488 individuals and obtained 9 hybrid individuals. In 6-year old plants, 11 hybrid individuals were detected among 496 X. index. These 20 hybrids ranged into crosses involving several lines: Iran versus Italy, Samos vs Spain and Italy vs Spain (Fig. 3.11). Interestingly, in 6-year old plants, most hybrids that we detected have the line Spain as one of their parents. In 5-and 6-year old plants, we

Figure 3 . 11 .

 311 Figure 3.11. Evidence of microsatellite multilocus hybrid genotypes detected from the four X. index lines mixed in containers of plant material obtained from cutting and in vitro. '?' indicates the absence of an amplification signal. Synthesis on 3-to 6-year old plants. In our results, we could detect a total number of 21 hybrid individuals. At least one event has been detected in each grapevine variety (8771, RPG1, RGM, 8624, RdL and CS). Hence, this hybridization event does not appear specific to certain clones and is not linked with the resistance status of the material. Even though we have only few hybridization cases, the hypothesis that the resistant plants (e.g. 8771 and RPG1) might induce a selection pressure that favors the production of crossbreds between the lines is a priori not supported by our results. The frequency of the detection of hybrids varied deeply over years: from none out of 200 individuals after 3 years, it hardly increased to 1 out of 285 individuals after 4 years (3.5 per 10,000) and then moved to 9 out of 488 individuals after 5 years (184 per 10,000) and remained relatively stable with 11 out of 496 individuals after 6 years (221 per 10,000). The contribution of each line to the hybridization event was relatively equivalent after 5 years, ranging from 0.2 (Samos and Spain) to 0.3 (Italy and Iran), but Spain became dominant at 6 years (0.45) preceding all other lines with a contribution from 0.14 (Iran and Samos) to 0.23 (Italy).

Figure 3 . 12 . 1 .

 3121 Figure 3.12. Electropherogram illustrating the detection of a hybridization event (C) between single individuals from the lines Italy (A) and Iran (B), respectively. D shows the control DNA mix between single individuals from Italy and Iran. Graphs of all 8 microsatellite loci (named above each locus in green rectangles) considered in the study are represented.

  1.26 for more details). Nemadex AB has been grafted with either Caladoc or Cabernet-Sauvignon; 140Ru has been grafted with Caladoc while SO4 and Borner have been grafted with Cabernet-Sauvignon (Fig. 3.13).

Figure 3 . 13 .

 313 Figure 3.13. Design of the field experiment of Nemadex AB in Domaine du Chapitre (Villeneuve-les-Maguelonne) (Source: INRA-IFV). Four different rootstocks were used Nemadex AB (Nem), 140 Ruggeri (140Ru), SO4 (SO4) and Borner (Borner) grafted on either variety Caladoc (Cal; rows 1 and 2) or variety Cabernet-Sauvignon (CS; rows 3 to 5). Rootstock-variety assemblies were: Nem:Cal, 140Ru:Cal, SO4:CS, Nem:CS and Borner:CS. Each replicate is composed of 6 plants indicated by the same color and is designated by its number placed after the rootstock-variety assembly.

Figure 3 . 14 .

 314 Figure 3.14. Evolution of GFLV contaminations by ELISA tests in the experiment of Domaine du Chapitre planted in 1999. A: Cabernet-Sauvignon part: CS/Nemadex AB and CS/SO4 from 2000 to 2014. B: Caladoc part: Caladoc/Nemadex AB and Caladoc/140 Ru from 2010 to 2014 (Source: INRA-IFV).

Figure 3 . 15 .

 315 Figure 3.15. Differential GFLV attacks in Nemadex AB and SO4 rootstocks grafted with Cabernet-Sauvignon in the field trial, 12 years after planting (Photo credit INRA Sophia Antipolis, 2011).

Figure 3 . 16 .

 316 Figure 3.16. Soil sampling in the field in trenches, approx. 60-cm deep, 120-cm long and 40-cm wide, dug by a narrow excavator in June 2015. Sampling was performed in the middle part of each replicate (between plants 3 and 4 out of the 6 plants). In each trench, 2 sub-samples of 2 kg of soil were taken by hand.

  of X. index Nematode numbers. In 2015, i.e. 16 years after grapevine planting, extractions from soil samples collected 50-60 cm deep allowed to evaluate nematode numbers. Very low numbers were recovered, i.e. 21 X. index individuals in total (14 individuals from the first 2-kg sub-sample and 7 individuals from the second 2-kg sub-sample) when all the rootstock:variety assembly are confounded. Eleven out of the 63 samples contained X. index and numbers ranged from 1 to 6 individuals per 4 kg of soil. No nematodes were recovered in the Caladoc part of the assay (comparison between Nemadex AB and 140Ru). In the CS part, comparison of three rootstocks showed that the highest numbers were recovered in SO4, followed by Borner (on only 5 replicates) and Nemadex AB. A single nematode has been recovered from the 28 total Nemadex AB samples (112 kg of soil) in the experiment.

Figure 3 . 17 .

 317 Figure 3.17. Spatial distribution of ELISA GFLV-positive, negative and dead plants in 15-year-old grapevine plants (2014). GFLV-positive plants are in pink, the GFLV-negative plants in green, and the dead plants in grey (Source: INRA-IFV). The small brown boxes indicate the location of the sampling holes dug along the rows between two plants (plants 3 and 4).

Figure 3 . 18 .

 318 Figure 3.18. Spatial distribution, along the 5 grapevine rows, of the individuals of X. index recovered from the field experimental design of Domaine du Chapitre. Blue dots indicate the sampling points and yellow circles give the nematode numbers (per 4 kg of soil) in positive samples.

Figure 3 . 19 .

 319 Figure 3.19. Spatial distribution, along the grapevine rows, of the individuals of X. pachtaicum recovered from the field experimental design of Domaine du Chapitre. Blue dots indicate the sampling points and green circles give the nematode numbers (per 4 kg of soil) in positive samples.

  of the nematode diversity. Out of them, one ('Spain' obtained from the sample Xi-Sp-LM) belongs to the Western (W) subclade, another one ('Iran' obtained from the sample Xi-Ir-Sh) belongs to the Near-and Middle-East (NME) subclade while the two others ('Samos' obtained from the sample Xi-Gr-Sa, and 'Italy' obtained from the sample Xi-It-PC) belong to the Samos and South Italy (SSI) subclade. These latter lines, though they belong to the same subclade, are quite different by their microsatellite pattern, which justified their retention as traceable lines.

Figure S3 .

 S3 Figure S3. Conserved domains of the four mitochondrial genes CytB (A), ATP6 (B), CO1 (C) and ND4 (D) using BLASTX (nucleotide -> protein). The reference genetic code is 'Invertebrate mitochondrial'. For the CytB and CO1 genes, as there were several close species sequences available on the database, our sequences matched specific CytB and CO1 domains and revealed these respective superfamilies. For ATP6 and ND4, only non-specific hits were detected, which is due to the absence of other sequences of these nematodes in the database. Nevertheless, our sequences did belong to the superfamilies ATP and ND4, respectively.

Table 1 . 1 .

 11 Main plant genes identified for resistance to nematodes

	AILV	Artichoke Italian latent virus
	ArMV	Arabis mosaic virus
	ATP6	ATP synthase subunit 6
	CO1	Cytochrome c oxidase I

Table 1 . 2 .

 12 Main resistance genes for which virulent populations of Meloidogyne spp. have been observed

Table 1 . 3 .

 13 Main Vitis species used in breeding programs

Table 1 . 4 .

 14 Longidoridae nematode vectors and their associated viruses

Table 1 . 5 .

 15 Markers for barcoding identification of the grapevine nematode species (X. index, X. diversicaudatum, X. italiae, X. pachtaicum,X. vuittenezi, P. vulnus, and L. attenuates)

Table 1 . 6

 16

. Host suitability to X. index of some rootstocks. R, resistant; MR, moderately resistant; S, susceptible Table 1.7. Vitis-Muscadinia accessions available at INRA-EGFV, their host suitability to X. index, and their parentage. Vitis-Muscadinia and Vitis susceptible control material are also shown.

Table 3 . 1 .

 31 Characteristics of the eight microsatellite loci developed for X. index

Table 3 .

 3

2.

Composition of the media used at UMR ISA for introduction (IM), multiplication (MM) and rooting (RM) of in vitro propagation of grapevine plant material

Table 3 . 3 .

 33 Distribution of the number of plants inoculated with mixed lines and used for the adaptation experiment on material aged 3 to 6 years

Table 3 . 4 .

 34 Numbers of males of X. index recovered in different accessions at4, 5 and 6 years

Table 3 . 5 .

 35 Distribution of the numbers of individuals of X. index assigned to each line, after 6 years, in the resistant accessions 8771 (F1) and RPG1 (BC1)

Table 3 . 6 .

 36 ANOVA analysis of X. index numbers (individuals per kg of soil) in the rootstocks Börner, Nemadex AB and SO4 grafted with Cabernet-Sauvignon

Table 3 . 7 .

 37 ANOVA analysis of X. index numbers (individuals per kg of soil) in all rootstock:variety assemblies

Table 3 . 8 .

 38 ANOVA analysis of X. pachtaicum numbers in the rootstocks Börner, Nemadex AB and SO4 grafted with Cabernet-Sauvignon

Table 3 . 9

 39

. ANOVA analysis of X. pachtaicum numbers in all rootstock:variety assemblies

Table 1 . 1 .

 11 Main plant genes identified for resistance to nematodes

	Plant species	R gene	Nematode	R gene	References
			species	class
	Tomato	Mi-1.2	M. arenaria	CNL	Ammati et al., 1985
	Solanum		M. incognita,		Roberts & Thomason, 1989
	lycopersicum		M. javanica
	Potato	Hero	G. rostochiensis CNL	Ganal et al., 1995
	Solanum tuberosum
		Gro1-4	G. rostochiensis TNL

Table 1 .2. Main

 1 resistance genes for which virulent populations of Meloidogyne spp. have been observed.

	Plant species	Resistance	Nematode	References
		gene	species
	Lycopersicum	Mi	M. incognita,	Castagnone-Sereno et al., 1996, 2007
	esculentum		M. javanica	Djian-Caporalino et al., 2011
				Eddaoudi et al., 1997
				Kaloshian et al., 1996
	Capsicum	Me1, Me3,	M. incognita	Castagnone-Sereno et al., 1996, 2007
	annum	Me7		Djian-Caporalino et al., 2011
	Vigna	Rk	M. incognita	Petrillo et al., 2006
	unguiculata			Petrillo & Roberts, 2005a, 2005b
				Roberts et al., 1996
	Phaseolus	Single major	M. hapla	Chen & Roberts, 2003
	vulgaris	gene
	Solanum	Rmc2	M. chitwoodi	Janssen et al., 1998
	fendleri

Table 1 .3. Main

 1 Vitis species used in breeding programs (adapted fromThis et al., 2006)

	Species	Used for breeding	Used for breeding	Used for breeding
		cultivars	rootstocks	interspecific hybrids
	Vitis vinifera	+++++	+	++++
	V. aestivalis	-	-	++
	V. amurensis	+	-	++
	V. berlandieri	+	+++	-
	V. candicans	-	+	-
	V. caribaea	-	-	+
	V. champinii	+	+	-
	V. cinerea	-	+	++
	V. cordifolia	-	+	+
	V. labrusca	+++	++	+++
	V. longii	+	++	-
	V. riparia	++	+++	+++
	V. rupestris	++	+++	+++
	V. simpsonii	-	+	-
	Muscadinia rotundifolia ++	+	+
	I

.2.1. The Vitis vinifera grape

Table 1 . 4 .

 14 Longidoridae nematode vectors and their associated viruses (adapted from[START_REF] Andret-Link | Ectoparasitic nematode vectors of grapevine viruses[END_REF]

	Nematode vector species	Virus names	Acronym Main
					geographical
					location
	Xiphinema	americanum	Peach rosette mosaic virus	PRMV	Worldwide
		'sensu lato'	Tobacco ringspot virus	TRSV
			Tomato ringspot virus	ToRSV
		americanum	Tobacco ringspot virus	TRSV	North America
		'sensu stricto'	Tomato ringspot virus	ToRSV
		bricolensis	Tomato ringspot virus	ToRSV North America
		californicum	Tobacco ringspot virus	TRSV	North and
			Tomato ringspot virus	ToRSV	South America
		diversicaudatum Arabis mosaic virus	ArMV	Europe
			Strawberry latent ringspot virus SLRSV
		index	Grapevine fanleaf virus	GFLV	Worldwide
		rivesi	Tobacco ringspot virus	TRSV	North and
					South America
			Tomato ringspot virus	ToRSV Europe
	Longidorus	apulus	Artichoke Italian latent virus	AILV	Italy
		attenuatus	Tomato black ring virus	TBRV	Europe
		diadecturus	Peach rosette mosaic virus	PRMV	North America
		elongatus	Tomato black ring virus	TBRV	Europe
			Raspberry ringspot virus	RpRSV
		macrosoma	Raspberry ringspot virus	RpRSV Europe

Table 1 . 5

 15

	Sequence or gene	Primer	Primer sequence (5'-3')	Reference

. Markers for barcoding identification of the grapevine nematode species X. index, X. diversicaudatum, X. italiae, X. pachtaicum, X. vuittenezi, P. vulnus, and L. attenuates (V.C. Nguyen and D.

Esmenjaud, 2017: unpublished data from the INRA Lycovitis SMaCH project -2015-2017)

Table 1 . 6 .

 16 Host suitability to X. index of some grapevine rootstocks. R, resistant; MR, moderately

	resistant; S, susceptible
	Rootstock	Genetic origin	Host	Reference
			suitability
	Harmony	(V. longii x Othello) x Dog Ridge	R	Harris (1983)
	Freedom	(V. longii x Othello) x Dog Ridge	R
	3309	V. rupestris x V. riparia	R
	1613C	V. longii x [V. vinifera x (V.	R
		riparia x V. labrusca)]
	VR 039-16	V. vinifera x M. rotundifolia	R	Walker et al. (1991)
				McKenry et al. 2004)
	VRH 8771	V. vinifera x (M. rotundifolia cv.	R	Esmenjaud et al. (2010)
		NC184-4)
	Nemadex Alain	VRH 8771 x (V. Berlandieri x V.	R
	Bouquet (RPG1)	rupestris cv. 140Ru)
	NC 35-50	T6-38op x M. rotundifolia	R
	Riparia Gloire de	V. riparia	MR
	Montpellier (RGM)
	Schwarzmann	V. riparia	MR	Harris (1983)
	110R	V. berlandieri x V. rupestris	S	Malan & Meyer(1993)
	Ramsey	V. champinii	S	Ambrosi (1967)
	Dog Ridge	V. rupestris x V. candicans	S
	Jacquez	V. aestivalis x V. cinerea x V.	S
		vinifera
	775 Paulsen	V. berlandieri x V. rupestris	S
	VRH 8624 (T6-38) V. vinifera x M. rotundifolia	S	Esmenjaud et al. (2010)
	Rupestris du Lot
	(RL)

Table 1 . 7 .

 17 Vitis-Muscadinia accessions available at INRA-EGFV, their host suitability to X. index, and their parentage. Vitis-Muscadinia and Vitis susceptible control materials are also shown.

	Cross or species	Accession	Host	Female parent Male parent
		(% Muscadinia)	suitability
	Intergeneric material
	V. vinifera x	VRH 8624	S	F2-35	M. rotundifolia
	M. rotundifolia	(T6-38)		(Carignan x CS)	Trayshed
		(50 %)
		VRH 8771	R	42-36	M. rotundifolia
		(50 %)		(CS x AB)	NC 184-4
		VRH 97-99-79	R	42-28	M. rotundifolia
		(50 %)		(CS x AB)	Carlos
		NC 35-50	R	T6-38	M. rotundifolia
		(37.5 %)		OP seedling	Dixie
	(V. vinifera x	RPG1	R	VRH 8773	V. Berlandieri
	M. rotundifolia) x Vitis rootstock	(25 %)		[=(CS x AB) x NC 184-4]	Rességuier II x V. rupestris du Lot = 140Ru
	Vitis material
	V. riparia	Gloire de	MR
		Montpellier
	V. rupestris	du Lot (RL)	S
	V. vinifera	Cabernet-	S
		Sauvignon

R, resistant; S, susceptible; MR, moderately resistant; CS, Cabernet-Sauvignon; AB, Alicante-Bouschet

Table S5 . Genotypes obtained from 8 microsatellite markers within the subset of 35 samples of X. index representing the nematode distribution area.

 S5 For sample codes, see TableS1. Single letters A, B, or C in an individual code indicate the existence of different haplotypes or MLGs within the same sample. W1-PP, poorly-polymorphic subgroup of Western samples; W2-P, polymorphic subgroup of Western samples; W2-CA, Chilean and Argentinian subgroup of Western samples.

	# individual	# sample	Sam ple code	Individual code	Country	Region	Location	Host plant	Type		Provider (year)	# rep. indiv.
															CytB	ATP6	COI	ND4
	1	1		Xi-Fr-SE1	Xi-Fr-SE1	France	Bordeaux	St Emilion		Grape F	Bordeaux Sciences Agro (2006)	LT996601	LT996690 LT996733	LT996776	1
	2	2		Xi-Fr-SE2	Xi-Fr-SE2	France	Bordeaux	St Emilion		Grape F	Bordeaux Sciences Agro (2006)	LT996602	LT996691 LT996734	LT996777	2
	3	3		Xi-Fr-PL	Xi-Fr-PL	France	Bordeaux	Pessac-Leognan	Grape F	Bordeaux Sciences Agro (2006)	LT996603	LT996692 LT996735	LT996778	3
	4	4		Xi-Fr-Me1 Xi-Fr-Me1	France	Bordeaux	Medoc		Grape F	Bordeaux Sciences Agro (2006)	LT996604	LT996693 LT996736	LT996779	4
	5	5		Xi-Fr-Me2 Xi-Fr-Me2	France	Bordeaux	Medoc		Grape F	Bordeaux Sciences Agro (2006)	LT996605	LT996694 LT996737	LT996780	5
	6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52	6 7 8 9 10 11 12 13 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 35 36 37 38 39 40 41 42 43 44 45 45 46 47 47 48 Group Lagoa Subclade Xi-Fr-Cah Xi-Fr-Cah France South West France Cahors Xi-Fr-Ga Xi-Fr-Ga France South West France Gaillac Xi-Fr-VM Xi-Fr-VM France Languedoc Villeneuve-les-Maguelonne Grape G Grape F Grape G Xi-Fr-Cai Xi-Fr-Cai France Cotes du Rhône Cairanne Grape G Xi-Fr-CP Xi-Fr-CP France Cotes du Rhône Chateauneuf-du-Pape Grape G Xi-Fr-Po Xi-Fr-Po France Provence Ponteves Grape F Xi-Fr-Cas Xi-Fr-Cas France Provence Cassis Grape G Xi-Fr-Fr Xi-Fr-Fr-A France Provence Fréjus Grape G Xi-Fr-Fr Xi-Fr-Fr-B France Provence Fréjus Grape G Xi-Fr-Bi Xi-Fr-Bi France Provence Biot Grape F Xi-Fr-SJ Xi-Fr-SJ France Provence St Jeannet Grape F Xi-Fr-Et Xi-Fr-Et France Burgondy Etiveau Grape F Xi-Fr-PM Xi-Fr-PM France Burgondy Puligny-Montrachet Grape F Xi-Fr-Vol Xi-Fr-Vol France Burgondy Volnay Grape F Xi-Fr-Meu1 Xi-Fr-Meu1 France Burgondy Meursault Grape F Xi-Fr-LS Xi-Fr-LS France Burgondy Ladoix-Serrigny Grape F Xi-Fr-MS Xi-Fr-MS France Burgondy Morey St Denis Grape G Xi-Fr-Vou Xi-Fr-Vou France Burgondy Vougeot Grape G Xi-Fr-Wi Xi-Fr-Wi France Alsace Wintzenheim Grape G Xi-Fr-Ob Xi-Fr-Ob France Alsace Obernay Grape F Xi-Fr-SP Xi-Fr-SP France Alsace St Pierre Grape F Xi-Fr-Be Xi-Fr-Be France Alsace Bergheim Grape F Xi-Fr-Eg Xi-Fr-Eg France Alsace Eguisheim Grape F Xi-Fr-MO1 Xi-Fr-MO1 France Champagne Mesnil-sur-Oger Grape G Xi-Fr-MO2 Xi-Fr-MO2 France Champagne Mesnil-sur-Oger Grape F Xi-Fr-Cr Xi-Fr-Cr France Champagne Cramant Grape F Xi-Fr-Av Xi-Fr-Av France Champagne Avize Grape G Xi-Fr-Li Xi-Fr-Li France Val de Loire Limeray Grape G Xi-Sp-Bo Xi-Sp-Bo Spain Bollullos del Condado Bonares Grape F Xi-Sp-PC Xi-Sp-PC Spain Bollullos del Condado Palma del Condado Grape F Xi-Sp-JF1 Xi-Sp-JF1-A Spain Jerez de la Frontera Jerez de la Frontera Grape F Xi-Sp-JF1 Xi-Sp-JF1-B Spain Jerez de la Frontera Jerez de la Frontera Grape F Xi-Sp-JF2 Xi-Sp-JF2 Spain Jerez de la Frontera Jerez de la Frontera Grape F Xi-Sp-Ag Xi-Sp-Ag Spain Montilla Moriles Aguilar Grape F Xi-Sp-PG Xi-Sp-PG Spain Montilla Moriles Puente Genil Grape F Xi-Sp-Mo Xi-Sp-Mo Spain Montilla Moriles Moriles Grape F Xi-Sp-Mu Xi-Sp-Mu Spain Murcia Murcia Grape F Xi-Sp-Ju Xi-Sp-Ju Spain Murcia Jumilla Grape F Xi-Sp-LM Xi-Sp-LM Spain La Mancha La Mancha Grape G Xi-Pt-TV Xi-Pt-TV Portugal Torres Vedras Torres Vedras Grape G Xi-Pt-AG Xi-Pt-AG Portugal Lisboa Aldea Galega Grape F Xi-Pt-Le Xi-Pt-Le-A Portugal Lisboa Leiria Grape F Xi-Pt-Le Xi-Pt-Le-B Portugal Lisboa Leiria Grape F Xi-Pt-Pi Xi-Pt-Pi Portugal Lisboa Picanceira Grape F Xi-Pt-Si Xi-Pt-Si-A Portugal Algarve Silves Grape F Xi-Pt-Si Xi-Pt-Si-B Portugal Algarve Silves Grape F Xi-Pt-La Xi-Pt-La Portugal Algarve Subgroup Sample code Individual code List of MLG Fr-Vou Fr-Vou 1 113 117 177 179 127 135 196 Virginie Viguès, ITV (2007) Virginie Viguès, ITV (2007) INRA Sophia (1993) Marion Claverie, ITV (2012) Marion Claverie, ITV (2012) Marc Chovelon, ITAB (2012) INRA Sophia (1993) INRA Sophia (1993) INRA Sophia (1993) C. Gutierrez-Gutierrez, CSIC (2010) INRA Sophia (2011) Marion Claverie, ITV (2012) Marion Claverie, ITV (2012) Marion Claverie, ITV (2012) Marion Claverie, ITV (2012) Marion Claverie, ITV (2012) INRA Sophia (1993) INRA Sophia (1993) INRA Sophia (1993) INRA Colmar (2012) INRA Colmar (2012) INRA Colmar (2012) INRA Colmar (2012) INRA Colmar (2012) INRA Colmar (2014) Géraldine Uriel, CIVC (2012) INRA Sophia (1993) INRA Sophia (1993) Carlos Gutierrez-Gutierrez, CSIC (2010) Carlos Gutierrez-Gutierrez, CSIC (2010) Carlos Gutierrez-Gutierrez, CSIC (2010) Carlos Gutierrez-Gutierrez, CSIC (2010) Carlos Gutierrez-Gutierrez, CSIC (2010) Carlos Gutierrez-Gutierrez, CSIC (2010) Carlos Gutierrez-Gutierrez, CSIC (2010) Carlos Gutierrez-Gutierrez, CSIC (2010) Pablo Castillo, CSIC (2017) Pablo Castillo, CSIC (2017) Maria Arias, CSIC (2005) INRA Sophia (1993) Carlos Gutierrez-Gutierrez, UE (2017) Carlos Gutierrez-Gutierrez, UE (2017) Carlos Gutierrez-Gutierrez, UE (2017) Carlos Gutierrez-Gutierrez, UE (2017) Carlos Gutierrez-Gutierrez, UE (2017) Carlos Gutierrez-Gutierrez, UE (2017) Fr-Wi Fr-Wi 113 117 177 179 127 135 196 Fr-Mo Fr-Mo 113 117 177 179 127 135 196 Fr-Av Fr-Av 113 117 177 179 127 135 196 Fr-Li Fr-Li 113 117 177 179 127 135 196 Fr-Me1 Fr-Me1 113 117 177 179 127 135 196 Fr-Ca Fr-Ca 113 117 177 179 127 135 196 Fr-Fr Fr-Fr-A 113 117 177 179 127 135 196 US-Lo US-Lo 113 117 177 179 127 135 196 US-Na US-Na 113 117 177 179 127 135 196 Sp-LM Sp-LM 113 117 177 179 127 135 196 Sp-Ju Sp-Ju 113 117 177 179 127 135 196 Sp-JF1 Sp-JF1-A 113 117 177 179 127 135 196 Pt-TV Pt-TV 113 117 177 179 127 135 196 Pt-AG Pt-AG 113 117 177 179 127 135 196 Pt-Le Pt-Le-A 113 117 177 179 127 135 196 Pt-Si Pt-Si-A 113 117 177 179 127 135 196 Pt-Ta Pt-Ta 113 117 177 179 127 135 196 It-Pi It-Pi 113 117 177 179 127 135 196 Pt-Ma Pt-Ma 2 113 117 177 179 127 135 196 It-Te It-Te-A 3 113 117 177 179 127 135 196 It-Do It-Do 4 115 117 177 179 127 135 196 It-Te It-Te-B 5 109 117 177 179 127 196 220 Gr-NC Gr-NC-B 6 113 117 177 131 135 196 Fr-Fr Fr-Fr-B 7 113 177 179 127 135 196 200 Fr-PL Fr-PL-A 8 109 113 177 179 117 127 200 204 Fr-PL Fr-PL-B 9 113 177 179 127 200 204 Fr-Me2 Fr-Me2-A 10 109 113 177 179 127 200 204 Fr-Me2 Fr-Me2-B 11 109 113 177 117 127 204 Fr-SE1 Fr-SE1 12 109 113 179 127 200 204 212 Fr-SE2 Fr-SE2-A 13 113 177 179 113 127 200 204 Fr-SE2 Fr-SE2-B 14 113 177 179 117 127 200 204 Sp-JF1 Sp-JF1-B 15 113 117 177 127 196 200 Pt-Si Pt-Si-B 16 113 177 127 135 196 204 Pt-La Pt-La-A 17 113 177 179 127 200 204 Pt-La Pt-La-B 18 113 177 179 113 127 200 204 Pt-Pi Pt-Pi 19 109 113 179 127 196 204 Pt-Le Pt-Le-B 109 113 179 127 196 204 Ch-Vi1 Ch-Vi1 20 109 113 177 179 129 135 196 200 Ch-Vi2 Ch-Vi2 109 113 177 179 129 135 196 200	LT996606 LT996607 LT996608 LT996609 LT996610 LT996611 LT996612 LT996613 LT996614 LT996615 LT996616 LT996617 LT996618 LT996619 LT996620 LT996621 LT996622 LT996623 LT996624 LT996625 LT996626 LT996627 LT996628 LT996629 LT996630 LT996631 LT996632 LT996633 LT996634 LT996635 LT996636 LT996637 LT996638 LT996639 LT996640 LT996641 LT996642 LT996643 LT996644 LT996645 LT996646 LT996647 LT996648 LT996649 LT996650 LT996651 216 124 216 124 216 124 216 124 216 124 216 124 216 124 216 124 216 124 216 124 216 124 216 124 216 124 216 124 216 124 216 124 216 124 216 124 216 124 216 124 216 124 216 124 216 124 214 216 124 216 128 216 214 216 128 214 216 128 216 216 128 216 128 214 216 128 216 128 214 216 128 214 216 128 216 128 216 128 216 216	_ _ _ _ _ _ LT996695 LT996738 _ _ _ _ _ _ LT996696 LT996739 LT996697 LT996740 _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ LT996698 LT996741 _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ LT996699 LT996742 _ _ _ _ _ _ LT996700 LT996743 LT996701 LT996744 _ _ _ _ _ _ _ _ _ _ _ _ LT996702 LT996745 _ _ _ _ LT996703 LT996746 LT996704 LT996747 _ _ LT996705 LT996748 LT996706 LT996749 139 148 139 148 136 148 136 139 148 136 139 148 136 139 148 136 139 148 136 140 136 148 136 139 148 136 139 148 136 148 136 140 139 142 LT996781 _ _ _ _ _ _ LT996782 LT996783 _ _ _ _ _ _ _ _ LT996784 _ _ _ _ _ _ _ _ LT996785 _ _ _ LT996786 LT996787 _ _ _ _ _ _ LT996788 _ _ LT996789 LT996790 _ LT996791 LT996792 142 145 142 145 142 145 142 145 142 145 142 145 142 145 142 145 142 145 142 145 142 145 142 145 142 145 142 145 142 145 142 145 142 145 142 145 142 145 145 142 142 145 145 145 145 142 145 142 145 142 145 142 221 142 145 145 140 139 142	6 7 8 9 10 11 12 13 14 15 16 17 223 223 223 227 227 227 227 227 227 227 227 227 227 227 227 227 227 227 227 227 227 227 227 227 227 227 223 223 227 223 227 223 227 223 227 227 223 223 223 223 223 227 227
				Ch-SF	Ch-SF		109 113	177 179		129		135 196 200	216	140	139 142	227
				Ch-CT	Ch-CT		109 113	177 179		129		135 196 200	216	140	139 142	227
				Ch-Ca	Ch-Ca		109 113	177 179		129		135 196 200	216	140	139 142	227
				Ch-LP	Ch-LP		109 113	177 179		129		135 196 200	216	140	139 142	227
				Ch-IM	Ch-IM		109 113	177 179		129		135 196 200	216	140	139 142	227
				Ch-MP	Ch-MP		109 113	177 179		129		135 196 200	216	140	139 142	227
				Ar-VS	Ar-VS		109 113	177 179		129		135 196 200	216	140	139 142	227
				Eg-Is	Eg-Is	21	109	117 177			127	131		196	212 214	124	132	139	223	231
				Cy-Cy	Cy-Cy	22	113	117 177 179		127			196 200	214	124	148	139 142	223
				Gr-NC	Gr-NC-A	23	113	117 177			127	131		200	214	124	148	139	223 227
				Tu-Ku	Tu-Ku	24	109 113	177 179		127	131		196	204	208	216	139	145	223 227
				Gr-SC	Gr-SC	25	113	177			127			196	214 216 124	148	139	145	227
				Gr-Sa-C	Gr-Sa-C		113	177			127			196	214 216 124	148	139	145	227
				Ir-Ta	Ir-Ta-A	26	109		179		127			196	212	132	135 139	221 223
				Ir-Ta	Ir-Ta-B		109		179		127			196	212	132	135 139	221 223
				Ir-Sh	Ir-Sh	27	113		179	113	127			196	220	212	148	139	145 221
				Tu-AL	Tu-AL-A	28	113	117 177		113	127				204	216 124 128	139	223
				Tu-AL	Tu-AL-B	29	113	177	185 113	127			196	208	216	145
				Is-Is	Is-Is-A	30	113	117	179		127			196	208	216	140	142	221	227
				Is-Is	Is-Is-B	31	109 113	177 179		127			196	208	140	135	142	221
				Is-Je	Is-Je	32	109	117 177 179		127			196	208	216 124	135	142	221	227
				Is-TA	Is-TA	33	109 113	177 179		127				228 208	124	140	135	142	221
				Gc-Sa	Gc-Sa-A	34		117 177 179		127		135 196	204	208	216	145	223 227
				Gc-Sa	Gc-Sa-B	35		117 177 179		127			196	214 216	148	145	223
				It-Pa	It-Pa	36	113		179 185		127		135 196	204	216 124	145 221	227
				It-PC	It-PC		113		179 185		127		135 196	204	216 124	145 221	227

Table 3

 3

	Line	Allele	113 117 177

[START_REF] Nicol | Genomics and Molecular Genetics of Plant-Nematode Interactions[END_REF] Figs. 3.1 & 3.3)

.

179 186 113 127 136 197 204 221 207 212 216 124 149 139 142 146 221 223 227

	µsat	Locus 1 Locus 2	Locus 3	Locus 4	Locus 5	Locus 6	Locus 7	Locus 8
		Xi29 (GA)	Xi04 (TG)	Xi16(TC)	Xi13 (CA)	Xi24(AC)	Xi22(AAC)	Xi32(GTT)	Xi27(GT)
	Spain	113 117 177 179	127 136 197			216 124	142 146	227
	Italy	113	179 186	127 136 197 204			216 124	146 221	227
	Samos	117 177 179	127	197 204		207	216 124	146	223 227
	Iran	113	179	113 127	197	221	212		149 139	146 221

  Sauvignon (CS) and V. rupestris cv. du Lot (RL) were susceptible controls. Two other accessions were used as reference plant material: another susceptible F1 representative accession of the cross Vitis x Muscadinia, VRH8624 (8624), and the rootstock accession expressing an intermediate resistance V. riparia cv. Gloire de Montpellier (RGM). For each accession, two types of plant material have been evaluated: plants previously rooted as hardwood cuttings ('cutting' material) and plants acclimatized in soil from material previously propagated in vitro ('in vitro' material). The methods used to obtain in vitro and cutting plants are those described above.

	1.26): the F1 resistant accession VRH8771
	(8771), the BC1 resistant accession Némadex Alain Bouquet (RPG1). The accessions V. vinifera
	cv. Cabernet-

Table 3 . 4 .

 34 Numbers of males of X. index recovered in different accessions at4, 5 and 6 years

	Year	Accession	Males number	Total X. index number	Frequency (per 10,000)
		RPG1-cutting	2	1,088	18.4
		RGM -cutting	2	4,125	4.8
	4 years
		RPG1-in vitro	1	4,804	2.1
		CS-in vitro	1	10,931	0.9
		RPG1-cutting	1	2,354	4.2
	5 years
		RGM -cutting	1	5,160	1.9
		RPG1-cutting	1	2,311	4.3
		8871-cutting	1	1,331	7.5
	6 years	RGM-cutting	1	4,249	2.4
		8771-in vitro	1	28,337	0.4
		8624-in vitro	1	1,317	7.6
	Total		13	66,007	1.97

Table 3 . 5 .

 35 Distribution of the numbers of individuals of X. index assigned to each line, after 6 years, in

	the resistant accessions 8771 (F1) and RPG1 (BC1).
	Lines	8771	RPG1
		MIV	Cuttings	MIV	Cuttings
	Italy	12	19	13	30
	Spain	7	16	2	15
	Samos	2	6	2	14
	Iran	50	21	39	26
	Total number	71	62	56	85
	Expected number*	17.75	15.50	14.00	21.25
	Threshold value (P< 0.01)	11.35	11.35	11.35	11.35
	χ 2 (3 DoF)	80.94	8.58	65.30	8.98
	Probability H0**	P< 0.01	P< 0.05	P< 0.01	P< 0.01

ON A LONG-TERM ASSESSMENT UNDER FIELD CONDITIONS III.2.1. Introduction Resistance

  durability studies are logically long to perform and, in order to provide results, they must be planned early. In the field, a single trial dates back before 2000. This experiment has been installed from 1999 in the experimental unit of Domaine du Chapitre in a grapevine area known as highly infected by X. index and inducing very diverse and marked symptoms of GFLV. In this experiment, GFLV evolution has been monitored by INRA Montpellier, Montpellier SupAgro and ITV.

The experimental design aimed to evaluate the behavior of different rootstocks against GFLV transmitted by X. index. These were the BC1 rootstock RPG1 (Nemadex AB) in comparison with three other rootstocks, SO4, Ruggeri 140 (140Ru) and Borner. Results that we have obtained and reported within this thesis deal with the nematode numbers from a soil sampling performed in June 2015.

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* The number expected for each line is 25 % of the total number in the hypothesis that all lines have an equivalent frequency (aggressiveness) ** H0 hypothesis = no difference in frequency

Acknowledgments

We are particularly thankful to the numerous contributors that rendered possible this study by their nematode sampling and delivery. We do acknowledge John Philis (Cyprus), Mauro Di Vito, Nicola Greco, and Alberto Troccoli (Italy), Andy Walker (USA), David Nevo and Mishael Mor (Israel), Maria Arias, Antonio Bello and Lee Robertson (Spain), Emmanuel Tzortzakakis (Greece), Sokhandan Bashir and Majid Olia (Iran), Peter Nagy (Hungary), Maria Moreno, Enrique Tapia-Vera and Pablo Meza (Chile), Marcelo Doucet (Argentine), Caroline Ojeil (Lebanon), Galip Kaskavalci and Ibrahim Mistanoglu (Turkey), Mohamed Youssef-Banora (Egypt), Marion Claverie and Virginie Vigues (IFV France), Géraldine Uriel (CIVC France), Marc Chovelon (ITAB France) for the samples of X. index; René Renaud and Jean-Claude Devergne (France), Richard Janssen (The Netherlands), and Mauro Di Vito (Italy) for the samples of X. diversicaudatum. Our gratitude also goes to Julia Truch (University of Oxford, UK) for her help in genotyping and in particular for sequences of rare nematode individuals from Lebanon. Particular thanks to Laurent Bouby (CNRS, ISEM, Univ. Montpellier 2, France), Jean-Pierre Lacombe and Roberto Bacilieri (INRA, UMR AGAP, Montpellier, France) for fruitful discussions. We also thank Thomas Guillemaud (INRA, UMR ISA, Sophia Antipolis, France) for his time and invaluable advice.

ZURLETTO, Pierre ABAD, Antoine DALMASSO and Thomas GUILLEMAUD for their suggestions, advices, and questions that helped me to improve my manuscript a lot. I thank Pierre ABAD for believing in me from the first day I came to IPN-INRA and for hosting me in his team. I offer special recognitions to Cyril VAN GHELDER, Ulysse PORTIER and Ariane FAZARI who helped me Argentina, Portugal, Chile, Italy, Spain, Egypt, Crete… Great thanks to the Ministry of Agriculture and Rural Development, the Ministry of Education and Training of the Socialist Republic of Vietnam and the Plant Protection Research Institute (Vietnam Academy of Agriculture Sciences) for the funding of my Ph.D. fellowship.

Table 1. Mitochondrial haplotypes obtained using CytB, ATP6, CO1 and ND4 gene sequences within the subset of 35 samples (43 individuals) of X. index representing the nematode distribution area.

Author Contributions Statement

Additional information

Data availability statement

EBI accession numbers of the CytB, ATP6, CO1, and ND4 mitochondrial genes sequences (LT996593 to LT996818) are reported in Table S1.

Sequences have been uploaded at the URL: http://www.ebi.ac.uk/ena/data/view/PRJEB26007 They will be available upon manuscript acceptance.

Competing interests

The authors declare that they have no competing interests.

Supplemental data

Supplemental Tables Table S1. List and characteristics of the Xiphinema index (Xi) and X. diversicaudatum (Xd) samples of the study with their sample code, individual code, origin (country, region, location) and host plant Table S2. Primers designed to amplify the partial sequences of the mitochondrial genes CytB, ATP6, CO1, and ND4 in Xiphinema index and X. diversicaudatum Table S3. Characteristics of the eight microsatellite loci developed for X. index Table S4. Estimates of evolutionary intra-and interspecific divergences between mitochondrial sequences (Kimura-2-Parameters model) of X. index and X. diversicaudatum Table S5. Genotypes obtained from 8 microsatellite markers within the subset of 35 samples of X. index representing the nematode distribution area bootstraps. The colors (golden: W1; brown: W2; blue: NME; green: IP and dark blue: SSI) correspond to the mitochondrial topology (see Table 1). For sample codes, see Table S1. Xi-Fr-MO2

Supplemental Figures

List of Table and Figures

Xi-Fr-Cr Xi-Fr-MO2

Xi-Fr-Cr For codes, see Table S1.

III.1.1.1. Creation and molecular characterization of pure lines of X. index

Creation of lines

A first round of creation of single female descendencies or isofemale lines (referred hereafter as lines or isolates) had been undertaken in the late 1990's among the populations available from the INRA collection at UMR-ISA. This was performed on the sole basis of their geographical distance among the grapevine worldwide distribution area and without any information on their relative diversity. When preliminary information on the worldwide diversity of the populations from this collection has been obtained (Villate, 2008), new populations have been retained for the creation of new lines. Those lines have been created as follows. Young plantlets of fig previously obtained from in vitro have been grown in a 100 ml pot for 2-3 months in order to allow the development of a dense root system. For each of the 8 populations retained, 10 adult females have been picked-up and inoculated individually in a 2 cm-deep hole (subsequently carefully filled with fine sand) located 1 cm from the fig plantlet stem within the pot. All plantlets were then placed in trays and grown under controlled conditions in a growth chamber at 25°C and 16L: 8D. Three months after inoculation, plantlet roots were cautiously separated from the soil and observed for the presence of small galls indicative of successful nematode attacks on root tips and of subsequent nematode multiplication. When galling was unambiguously observed, the plantlets and the soil content of their containers were repotted into a 4-liter container to allow better nematode multiplication. Out of the 10 individuals per population (pop.) inoculated singly in pots, a sample of 4 lines (pots) have been retained for further multiplication (Fig. 3.1A).

Molecular characterization of the lines

All new lines (5 lines/pop. x 8 pop. = 40 total lines) were characterized for their mitochondrial haplotype using marker CytB and for their microsatellite genotype using the 8 loci available for X. index (Table 3.1) (Villate et al., 2009). Purity of lines was verified from a sample of 10 individuals. A single line per population from 4 populations was finally retained and used in the experiments described hereafter. Those 4 final lines were selected because they had either specific alleles or a specific combination of alleles that allowed to identify them and thus to distinguish them unambiguously from each other as shown in Fig. Ta: optimal annealing temperature After 6 weeks in the RM medium, rooted plantlets were transferred into 50 x 30 x 15 cm trays filled with a perlite substrate without hormones in an acclimatization chamber at 20-22°C for 6 weeks. Plants were then moved to the greenhouse, transplanted individually into 2liter containers filled with a sterile sand-loam soil and inoculated 2 months later. Two months after inoculation, plants were gently repotted into 4-liter containers. Consequently, the delay between the beginning of the acclimatization of plantlets (end of the in vitro steps) and their inoculation has been approx. 3-4 months.

Material obtained from cuttings

Material for the production of rooted cuttings was sampled in spring from dormant grapevine mother plants in the plant repository of INRA EGFV Bordeaux. Hardwood cuttings

The evaluation of nematode numbers has been performed over 6 years (in 3-, 4-, 5-, and 6-year old plant pots) as reported in Table 4.3. The multiplication of nematodes has been evaluated at each date by extraction of nematodes from all the pot soil content.

Microsatellite genotyping

We used a set of 8 out of the 9 primer pairs designed by Villate et al. (2009) (Table 3.1).

In our study, we used forward primers carrying directly the fluorescent tag instead of the additional fluorescent M13 tail. PCR reactions were carried out in 10 μL simplex reaction containing 2 μL of DNA extract, 2 μL of 5x Taq reaction buffer, 0.15 units of Taq polymerase, 5U/μL Taq DNA polymerase (AmpliTaq, Applied Biosystems/Perkin Elmer), 0.3 μL of each primer (forward primers were fluoro-labelled with a FAM or PET or NED or VIC dye at the 5' end) at 10 μM. For each reaction we mixed 4 pairs of primers in one tube called mix-1

(microsatellites Xi04, Xi13, Xi16 and Xi29) or mix-2 (microsatellites Xi22, Xi24, Xi27 and Xi32) (Table 3.1). Reactions were carried out in a PTC-100 thermocycler (MJ Research) with the following amplification conditions: 95°C for 3 min; 30 cycles with 30 s at 94°C, 1 min 30 s at 57°C and 1 min 30 s at 72°C; and ending with 72°C for 10 min. PCR products were stored at -20°C or used directly by adding 1.5 μL of PCR product into 9.5 μL of formamide containing 0.5 μL of the internal lane standard marker (500-LIZ). Samples were read on a R3130XL Genetic

III.2.2.2. Experimental design

The experimental design (Fig. 3.13) contains five parallel grapevine rows planted in 1999 and divided in two parts as follows. i) The first part (Caladoc part) was planted on the two adjacent rows located in the border of the grapevine field in an area which was less contaminated by X. index.

III.2.3.2. ELISA GFLV infection

The distribution of GFLV infection has been monitored by ELISA tests conducted by IFV and the situation on the date of soil samplings is reported in Fig. 3.17

CHAPTER 4 CONCLUSIONS AND PERSPECTIVES

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CHAPTER 4: CONCLUSIONS AND PERSPECTIVES

Our works have relied on a phylogeographical approach of the nematode vector X. index and on an experimental approach based on plant resistance. Our results have contributed to the global knowledge of X. index diversity and to an increased knowledge of its specific behavior in response to the plant resistance pressure. Hence, nematode studies have been conducted

at different scales: i) the worldwide scale (phylogeography), ii) the field scale (evolution in a plot from Domaine du Chapitre), and iii) the greenhouse scale, i.e. under controlled conditions (dynamics and genetics). This combination of approaches and scales lead us to an enhanced understanding of several biological and genetic features of X. index. The species X. index, despite its high economical impact, is poorly studied in particular because of its ectoparasitic status and of its parasitism affecting a perennial crop. In this last chapter, the main results and conclusions from previous chapters will be reminded and discussed. We will then give their implications and perspectives in both the applied agronomic and cognitive research domains.

IV.1. CONCLUSIONS

IV.1.1. The global genetic diversity of X. index

To decipher how is shaped the worldwide genetic diversity of X. index, we have conducted a study with the highest number ever reported of worldwide geographic samples of the nematode. Our results have leant on both collection populations kept in the greenhouse for variable durations and new samples directly obtained from the field.

The mainly meiotic parthenogenetic species X. index has a lower diversity than the amphimictic species X. diversicaudatum

We first used X. diversicaudatum, a sexually-reproducing species, as the amphimictic reference in our study. Mitochondrial data based on the classical CytB gene allowed to confirm that the diversity of a mainly parthenogenetic species such as X. index is lower than that of its amphimictic sister vector species X. diversicaudatum. As the low diversity evidenced with this single gene jeopardized our objective of a reliable picture of the variability of X. index, we have used three additional mitochondrial genes. In particular, the ATP6 gene sequence was shown to be the most polymorphic and thus to bring interesting complemental information. Thus, data based on four mitochondrial genes, generating an unprecedented concatenated sequence of 3044 bp, permitted to draw reliably a basic diversity picture of X. index. Our wide array of geographic samples illustrated that X. index can be separated into four subclades with well supported nodes.

collection (notably between the population 'Italy' introduced in 1993 in the collection of UMR ISA and the others that date back from 2005-2006, i.e. 12 years later).

Muscadine-derived material appears durably resistant

The adaptation of the nematode to the plant resistance has not been observed on F1

and BC1 materials obtained from classical hardwood cutting propagation in our experimental data under controlled conditions over 6 years. The field study dealing with the nematode distribution in a 16-year old experiment has brought complemental information that are in line with that of the greenhouse experiment as nematodes also did not appear to be able to develop on the BC1 rootstock RPG1 (Nemadex AB). Thus, the control of the nematode vector by a resistance strategy based on muscadine-derived material appears durable.

IV.2. PERSPECTIVES

IV.2.1. The global genetic diversity of X. index still needs to be refined

Even though our study has benefitted from a wide range of samples, our sampling is far from exhaustive. Many gaps need to be fulfilled to complete the dissemination view. These are mainly: i) Eastern Mediterranean uncovered areas (Syria, Iraq, Eastern Turkey, Azerbaijan, Georgia, Armenia…), ii) Northern Africa (Egypt, Tunisia, Algeria, Morocco…), iii) Andean countries other than Chile and Argentina (Peru, Ecuador, Columbia…), and iv) Western

European dissemination areas from both the Antiquity and the Modern Era (Phylloxera crisis).

The Eastern Mediterranean harbors the highest diversity and our few geographical sampling points only give a preliminary picture of this region that needs to be completed by a more reliable coverage. These new elements might be obtained in the near future through close collaborations with local researchers in agronomy together with Archaeologists or specialists more particularly interested in Archaeobotanics. These links would be particularly appreciated to decipher i) the Phoenician heritage and its putative routes from Lebanon and ii)

the hypothetic initial focus(es) of Western introductions of grape by the Greco-Romans.

We also lack data from Northern Africa that could show whether a specific introduction and dissemination route by the Phoenicians and/or later by the Arabs exists in Southern Mediterranean countries. We do know that the nematode is present in Maghreb countries [START_REF] Dalmasso | Importance de la transmission par le sol de la dégénérescence infectieuse de la vigne dans l'Algérois[END_REF][START_REF] Siddiqi | Co-evolutionary interactions between host resistance and pathogen avirulence genes in rice-Magnaporthe oryzae pathosystem[END_REF] in areas that had been planted with grapevine by the French in the 19 th and 20 th centuries after the European colonization (A. Dalmasso, A. Cuany and C. Scotto La Massèse, pers. com.) and consequently at least populations from the Western haplotype should be detected there.

In Southern America and in particular in Andean countries, a survey for new populations would be very useful to bring additional information on the apparent marked bottleneck effect

ANNEXES Supporting information -Chapter 2

These annexes concern the four mitochondrial genes used in our study. We first illustrate that the sequences used contain the functional domains of each gene (Fig. S3). The representative haplotype analyzed is Xi-Fr-Fr-A. Then we report the complete 3044-bp sequence obtained from the four concatenated genes (Fig. S4) for a set of 10 representative haplotypes selected from Fig. 2A in the manuscript.

Conserved domains of the four mitochondrial genes CytB, ATP6, CO1 and ND4