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Le retrait des nématicides rend urgent la mise au point de méthodes alternatives de lutte contre les nématodes parasites des cultures et la création de variétés résistantes est une voie prometteuse. En vignoble, le nématode Xiphinema index a un impact économique élevé en transmettant le Grapevine fanleaf virus (GFLV), principal virus du court-noué de la vigne et première virose de la vigne à l’échelle mondiale. Des porte-greffe résistants vis-à-vis du vecteur X. index basés sur la source de résistance muscadine (Muscadinia rotundifolia) sont en cours de sélection chez la vigne afin de stopper ou retarder l’infection. Sur cette culture, une étude antérieure avait montré que ce nématode parthénogénétique méiotique est aussi capable de se reproduire (rarement) de façon sexuée. Un travail préliminaire de phylogéographie avait permis de révéler les groupes prédominants de diversité et de sélectionner des populations représentatives pour la création de lignées monofemelles. La durabilité de la résistance doit prendre en compte la diversité du nématode. Dans ce contexte, la thèse a d’abord complété et approfondi l’approche phylogéographique en utilisant une très large gamme d’échantillons originaires de l’aire mondiale de répartition de la vigne. Nos résultats permettent de proposer des hypothèses fortes afin de localiser l’aire native du nématode X. index au Moyen-Orient et de retracer ses itinéraires de dissémination à partir de l’Antiquité. IIs illustrent également le lien étroit depuis cette époque entre la dissémination du nématode et celle de la vigne domestiquée par l’homme. La deuxième partie de la thèse a évalué la durabilité de la résistance de matériel porte-greffe issu de la muscadine en serre (nématodes non virulifères sur plants entre 3 et 6 ans) et en vignoble (nématodes virulifères sur plants âgés de 16 ans). En serre, des accessions résistantes F1 ou BC1, préalablement obtenues à partir d’in vitro ou de boutures ligneuses, ont été inoculées avec un mélange de 4 lignées représentatives, chaque lignée étant traçable avec des marqueurs microsatellites. Nous avons montré que les nématodes issus de plants obtenus par multiplication in vitro surmontent progressivement la résistance tandis que le matériel issu de boutures exprime une résistance durable. La multiplication progressive des nématodes sur le matériel résistant uniquement dans le cas où il est issu d’in vitro écarte a priori l’hypothèse d’une adaptation génétique du nématode. Elle apparaît liée à une architecture différente du système racinaire chez les plants issus de ce type de multiplication, multiplication qui pourrait induire des changements physiologiques discrets mais durables dans les tissus racinaires apicaux à partir desquels les nématodes se nourrissent. Le génotypage des nématodes par microsatellites a permis de détecter un taux bas mais croissant d’individus hybrides entre lignées sur les plants âgés de 4 à 6 ans, ce qui confirme l’aptitude de multiplication sexuée précédemment observée en vignoble. Du fait que l’observation d’individus hybrides apparaît indépendante du type de propagation et du statut de résistance de la plante, nos résultats écartent l’hybridation comme mode d’adaptation du nématode qui serait à même d’expliquer le contournement de la résistance chez les plants issus d’in vitro. En vignoble, après 16 années, les nématodes ont été quasi-impossibles à détecter sur l’accession résistante BC1 qui est également peu affectée par les attaques virales, tandis que des effectifs de nématodes plus élevés ont été retrouvés sur une accession témoin sensible dont les plants sont par contre très majoritairement morts ou en dépérissement. Considérés globalement, nos résultats montrent que la stratégie de résistance basée sur la muscadine apparaît durable. Cette stratégie ciblée sur le nématode vecteur contribuera à réduire significativement l’impact du GFLV transmis par X. index. / The ban of most nematicides renders urgent control alternatives against plant-parasitic nematodes and breeding for resistant plant varieties is promising. In vineyards, the nematode Xiphinema index has a high economical impact by transmitting Grapevine fanleaf virus (GFLV), the main virus of ‘Court-noué’ disease and the first grapevine viral disease worldwide. Resistant rootstocks are being selected in grapevine, using Muscadinia rotundifolia (muscadine) as a resistance source to the vector, in order to arrest or delay GFLV transmission. In this crop, a previous study had shown that this meiotic parthenogenetic nematode is able to reproduce sexually (rarely) in the field. A preliminary phylogenetic work had allowed to reveal the predominant diversity groups and to select representative populations for the creation of single-female lines. Resistance durability is a real challenge that must consider the key information of the nematode diversity. In this context, the PhD project first completed and deepened our phylogeographical approach using an extended geographic coverage of the worldwide nematode distribution. Our results allow proposing strong hypotheses to locate the native area of X. index in the Middle-East and trace its dissemination routes from the Antiquity. They also highlight the close link since this epoch between dissemination of the nematode and domesticated grapevine by man. The second part of the PhD project has then evaluated the durability of muscadine-derived rootstock material in greenhouse (non viruliferous nematodes on plants aged 3 to 6 years) and field (viruliferous nematodes on plants aged 16 years) conditions. In the greenhouse, F1 and BC1 resistant accessions, previously obtained from both in vitro and hardwood-cutting propagation, were inoculated with 4 mixed representative X. index lines, traceable each with microsatellite markers. We showed that nematodes from plants obtained from in vitro progressively overcame the resistance while the material obtained from cuttings displayed a durable resistance. Nematode progressive multiplication in resistant accessions obtained only from in vitro removes a priori the hypothesis of a nematode genetic adaptation and appears linked to a different architecture of the root system in this propagation type. This type may have induced discrete but durable physiological changes in apical root tissues from where nematodes feed. Nematode microsatellite genotyping allowed detecting a low but increasing rate of hybrid individuals from 4 to 6 years, which confirms data from the vineyard. As the hybrid occurrence appears independent from the propagation type and the resistance status of the plant, our data discard hybridization as the mode of adaptation of the nematode underlying resistance breakdown from in vitro plants. In field conditions, after 16 years, nematodes were almost undetectable on the resistant BC1 accession, also almost unaffected by the viral attacks, while higher numbers were detected on a susceptible control accession, whose plants were by contrast in high majority dead or poorly vigorous. Taken all together, our results show that the muscadine-derived resistance strategy appears durable. This strategy focused on vector control will significantly contribute to reduce the impact of GFLV transmitted by X. index.

Thesis (PhD(Agric))--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Grapevines are one of the most important and diverse crops in the world, but tend to be susceptible for numerous pests and diseases. The dagger nematode, Xiphinema index (X. index) is a well-known soil-borne pest of grapevine and vector of grapevine fanleaf virus. Several Vitis species showed resistance to this pest. Breeding efforts have been underway for several decades to create resistant rootstocks. However, conventional breeding efforts are time consuming due to grapevines being a perennial crop, its heterozygosity, as well as its long growth cycle. Breeding new grapevine varieties are also expensive and work intensive. The development of marker-assisted selection introduced a way to overcome some of the abovementioned problems. The aim of this study was to broaden the genetic evaluation and breeding efforts for improved X. index resistance in grapevine rootstocks. In 2007 several crosses were made in the University of California, Davis vineyards. The background for all these crosses consisted of V. arizonica. These V. arizonica plants are part of a collection obtained by H.P. Olmo during the 1960’s. In recent studies it was established that X. index resistance is controlled by a single dominant gene. The 0701 (R8916-07 (Wichita Refuge x b40-14) x R8916-32), 0704 (161-49C x b40-14) and 0705 (161-49C x R8916-22) populations were created to confirm the homozygous nature of b40-14, a V. arizonica accession. In addition, several V. arizonica species were screened to confirm their resistance or susceptibility towards X. index feeding. The 0705 population was also used to create a genetic map for X. index resistance. In this study a new and improved screening method was developed to inoculate vines under greenhouse conditions. This screening method proved to be quicker and less damaging on the nematodes than traditional systems. Control varieties were used and O39-16, a commercial rootstock showed no damage, even with high nematode pressure, whereas V. rupestris Saint George had severe root damage and decline after eight weeks of exposure. A range of V. arizonica accessions was tested for their resistance to X. index feeding. Of the 18 genotypes tested, half showed resistance and the rest were susceptible. It is possible that these genotypes are not pure V. arizonica genotypes. Genotypes with V. arizonica in the background were also tested. Wichita Refuge was used as a susceptible female parent and the progeny were expected to be heterozygous resistant. Some of the progeny allowed low levels of feeding damage, which may have been the result of the more effective inoculation method described above. The 0701 population confirmed the hypothesized model of 3:1 (Resistance (R):Susceptible (S)) segregation although 13 of the genotypes showed significantly higher gall numbers than the susceptible female parent. The possibility of transgressive segregation exists, but needs to be confirmed. All progeny from the 0704 population should be heterozygous resistant, but a 1:1 (R:S) segregation pattern was observed. The 0705 population was created as a mapping population to study X. index resistance. This population was also tested in the greenhouse for its X. index resistance and was expected to segregate 1:1 (R:S). The X2 analysis did not fully support this model. A genetic map covering all 19 linkage groups, and positioning 175 polymorphic SSR markers was created for the 164 progeny in the 0705 population. MapQTL analysis revealed a major QTL on linkage group 9 and two minor QTL’s on groups 13 and 19. The major QTL placed between markers VMC1c10 and CTG1032918 with a LOD score of 33.4 explaining 70.5% of the phenotypic variance for X. index. This QTL is the second major QTL discovered for X. index resistance. With the discovery of a second major QTL, the two types of resistance can be pyramided. Work is underway to saturate the area around the major QTL on linkage group 9 and to move towards physical mapping of X. index resistance. The b40-14 V. arizonica accession is also known for its resistance to Pierce’s disease and the possibility of simultaneous expression of two types of resistance is created. The 0705 population can also be used to evaluate phenotypical characteristics in the field to determine if useful rootstocks can be selected. Taken together, the results obtained in this study provide improved methods and highly characterized plant populations to support the efforts in obtaining improved X. index resistance in grapevine rootstocks. / AFRIKAANSE OPSOMMING: Wingerde is van die belangrikste en mees diverse gewasse op aarde, maar hulle neig om vir ‘n verskeidenheid plae en siektes vatbaar te wees. Die dolk-aalwurm, Xiphinema index (X. index), is ‘n bekende grondgedraagde plaag van wingerd en ‘n vektor vir wingerd- netelblaarvirus. Verskeie Vitis-spesies toon weerstand teen hierdie plaag. Daar word reeds vir dekades pogings aangewend om weerstandbiedende onderstokke te kweek. Konvensionele kweekpogings is egter tydrowend omdat wingerd ‘n meerjarige gewas is, op grond van die heterosigositeit van die gewas, sowel as die lang groeisiklus. Dit is ook duur en arbeidsintensief om nuwe wingerdvariëteite te kweek. Die ontwikkeling van merkerondersteunde seleksie het dus ‘n metode verskaf om sommige van bogenoemde probleme te oorkom. Die doelwit van hierdie studie was om die genetiese evaluerings- en kweekpogings vir verbeterde X. index-weerstand in wingerd-onderstokke te verbreed. In 2007 is verskeie kruisings in die wingerde by die Universiteit van Kalifornië, Davis gemaak. Die agtergrond vir al hierdie kruisings het bestaan uit V. arizonica. Hierdie V. arizonica-plante vorm deel van ‘n versameling wat in die 1960’s deur H.P. Olmo verkry is. In onlangse studies is daar bepaal dat X. index-weerstand deur ‘n enkele dominante geen beheer word. Die 0701 (R8916-07 (Wichita Refuge x b40-14) x R8916-32), 0704 (161-49C x b40-14) en 0705 (161-49C x R8916-22) bevolkings is geskep om die homosigotiese geaardheid van b40-14, ’n V. arizonicaafstammeling, te bevestig. Daarbenewens is verskeie V. arizonica-spesies gesif om hulle weerstand teen of vatbaarheid vir X. index voeding te bevestig. Die 0705 bevolking is ook gebruik om ‘n genetiese kaart vir X. indexweerstand te skep. In hierdie studie is ‘n nuwe en verbeterde siftingsmetode ontwikkel om wingerdstokke onder glashuistoestande te inokuleer. Daar is gewys dat hierdie siftingsmetode vinniger en minder skadelik vir die aalwurms as tradisionele metodes is. Beheervariëteite is gebruik en O39-16, ‘n kommersiële onderstok, het geen skade getoon nie, selfs met hoë aalwurmdruk, terwyl V. rupestris Saint George ernstige wortelskade en agteruitgang na agt weke se blootstelling getoon het. ‘n Verskeidenheid V. arizonica-afstammelinge is vir hulle weerstand teen X. index-voeding getoets. Van die 18 genotipes wat getoets is, het die helfte weerstand getoon en die res was vatbaar. Dit is moontlik dat hierdie genotipes nie suiwer V. arizonica-genotipes was nie. Genotipes met V. arizonica in hulle agtergrond is ook getoets. Wichita Refuge is as ‘n vatbare vroulike ouer gebruik en die verwagting was dat die nageslag heterosigoties weerstandbiedend sou wees. Sommige van die nageslag het lae vlakke van voedingskade toegelaat, wat moontlik die gevolg was van die meer doeltreffende inokulasiemetode wat hierbo beskryf word. Die 0701 bevolking het die veronderstelde model van 3:1 (Weerstandbiedend (W):Vatbaar (V)) segregasie bevestig, hoewel 13 van die genotipe noemenswaardig hoër galgetalle as die vatbare vroulike ouer getoon het. Die moontlikheid van transgressiewe segregasie bestaan, maar dit moet nog bevestig word. Alle nageslag van die 0704 bevolking behoort heterosigoties weerstandbiedend te wees, maar ‘n 1:1 (W:V) segregasiepatroon is waargeneem. Die 0705 bevolking is as ‘n karteringsbevolking geskep om X. index-weerstand te bestudeer. Hierdie bevolking is ook in die glashuis vir sy X. index-weerstand getoets en daar is verwag dat dit 1:1 (W:V) sou segregeer. Die X2 analise het nie hierdie model ten volle ondersteun nie. ‘n Genetiese padkaart wat al 19 skakelingsgroepe en die posisies van 175 polimorfiese SSR merkers toon, is vir die 164 afstammelinge in die 0705 bevolking geskep. MapQTL analise het ‘n groot kwantitatiewe eienskap lokus (QTL) op skakelingsgroep 9 en twee kleiner QTL’e op groepe 13 en 19 onthul. Die groot QTL is tussen merkers VMC1c10 en CTG1032918 met ‘n LOD telling van 33.4 geplaas en het 70.5% van die fenotipiese variansie van X. index verklaar. Hierdie QTL is die tweede groot QTL wat vir X. index-weerstand ontdek is. Met die ontdekking van ‘n tweede groot QTL, kan die twee soorte weerstand gepiramideer word. Werk word reeds onderneem om die area rondom die groot QTL op skakelingsgroep 9 te versadig en om na die fisiese kartering van X. index-weerstand te beweeg. Die b40-14 V. arizonica-afstammeling is ook bekend vir sy weerstand teen Pierce se siekte en die moontlikheid word geskep vir die gelyktydige uitdrukking van twee soorte weerstand. Die 0705 bevolking kan ook gebruik word om die fenotipiese kenmerke in die veld te evalueer om te bepaal of bruikbare onderstokke geselekteer kan word. In kombinasie behoort die resultate wat in hierdie studie verkry is, verbeterde metodes en hoogs gekarakteriseerde plantbevolkings te lewer wat die pogings sal ondersteun om verbeterde X. index-weerstand in wingerdonderstokke te verkry.

Realizou-se estudo da fauna de fitonematóides em dois municípios do estado de São Paulo (Pariquera-Açu e Cananéia) sob domínio da Mata Atlântica e dois do estado do Mato Grosso (Nova Maringá e Guarantã do Norte) sob domínio da Floresta Amazônica, com o objetivo de conhecer a diversidade de fitonematóides desses biomas e avaliar o efeito do uso agrícola do solo sobre a comunidade de fitonematóides. Para tanto, foram efetuadas amostragens em áreas de vegetação primária e em áreas próximas cultivadas. Para cada amostra, foram extraídos os nematóides de 200 cm³ de solo e em 10 gramas de raízes pela técnica de peneiramento combinada com flutuação em centrífuga com solução de sacarose. Em seguida, os espécimes foram mortos pelo calor e fixados em formalina. Os nematóides foram posteriormente quantificados, para determinação da abundância de cada táxon, sendo as identificações taxonômicas realizadas com base em características morfológicas e morfométricas. Os dados foram analisados com base na abundância, freqüência e índices de similaridade (Jaccard e Bray e Curtis). Assinalaram-se 34 gêneros e as identificações específicas foram efetuadas para 22 táxons. Para a espécie Aorolaimus banoae foram apresentadas novas características morfológicas e morfométricas. Observou-se que, com a retirada da vegetação primária e a implantação dos cultivos de plantas perenes ou anuais, houve intenso processo de redução da diversidade de fitonematóides e da introdução de novos táxons, o que resultou em baixa similaridade entre os locais de vegetação primária e aqueles cultivados, demostrando-se elevada influência da atividade agrícola sobre a comunidade de fitonematóides. / A study about plant parasitic nematode fauna was conducted in two municipalities from São Paulo State (Pariquera-Açu and Cananéia in the Atlantic Forest) and two from Mato Grosso State (Nova Maringá and Guarantã do Norte in the Amazon Forest), Brazil. The aims of this study were to evaluate the diversity of plant-parasitic nematode fauna in locations of primary and to evaluate the effect of agricultural land use on the plant parasitic nematode communities. Nematodes were extracted from 200 cm³ of soil and 10 grams of roots by a sieving and sugar flotations technique and fixed with formalin. Nematodes were counted for determination of abundances of each taxon and the taxonomic identification was based on morphological and morphometric features. The following variables were obtained: abundance, frequency and similarity indexes (Jaccard and Bray e Curtis). A total number of 34 genera were recorded and 22 taxons were identified at species level. Concerning Aorolaimus banoae species, new morphological and morphometric features were presented. It was observed that, after removal of primary vegetation and implantation of perennial or annual crops, occurred a conspicuous reduction of plant parasitic nematode diversity, associated with introduction of exotic species, resulting in low similarity between location of primary vegetation and the cultivated ones. The results demonstrated that the agricultural practices affect strongly the plant parasitic nematode communities.

Realizou-se estudo da fauna de fitonematóides em dois municípios do estado de São Paulo (Pariquera-Açu e Cananéia) sob domínio da Mata Atlântica e dois do estado do Mato Grosso (Nova Maringá e Guarantã do Norte) sob domínio da Floresta Amazônica, com o objetivo de conhecer a diversidade de fitonematóides desses biomas e avaliar o efeito do uso agrícola do solo sobre a comunidade de fitonematóides. Para tanto, foram efetuadas amostragens em áreas de vegetação primária e em áreas próximas cultivadas. Para cada amostra, foram extraídos os nematóides de 200 cm³ de solo e em 10 gramas de raízes pela técnica de peneiramento combinada com flutuação em centrífuga com solução de sacarose. Em seguida, os espécimes foram mortos pelo calor e fixados em formalina. Os nematóides foram posteriormente quantificados, para determinação da abundância de cada táxon, sendo as identificações taxonômicas realizadas com base em características morfológicas e morfométricas. Os dados foram analisados com base na abundância, freqüência e índices de similaridade (Jaccard e Bray e Curtis). Assinalaram-se 34 gêneros e as identificações específicas foram efetuadas para 22 táxons. Para a espécie Aorolaimus banoae foram apresentadas novas características morfológicas e morfométricas. Observou-se que, com a retirada da vegetação primária e a implantação dos cultivos de plantas perenes ou anuais, houve intenso processo de redução da diversidade de fitonematóides e da introdução de novos táxons, o que resultou em baixa similaridade entre os locais de vegetação primária e aqueles cultivados, demostrando-se elevada influência da atividade agrícola sobre a comunidade de fitonematóides. / A study about plant parasitic nematode fauna was conducted in two municipalities from São Paulo State (Pariquera-Açu and Cananéia in the Atlantic Forest) and two from Mato Grosso State (Nova Maringá and Guarantã do Norte in the Amazon Forest), Brazil. The aims of this study were to evaluate the diversity of plant-parasitic nematode fauna in locations of primary and to evaluate the effect of agricultural land use on the plant parasitic nematode communities. Nematodes were extracted from 200 cm³ of soil and 10 grams of roots by a sieving and sugar flotations technique and fixed with formalin. Nematodes were counted for determination of abundances of each taxon and the taxonomic identification was based on morphological and morphometric features. The following variables were obtained: abundance, frequency and similarity indexes (Jaccard and Bray e Curtis). A total number of 34 genera were recorded and 22 taxons were identified at species level. Concerning Aorolaimus banoae species, new morphological and morphometric features were presented. It was observed that, after removal of primary vegetation and implantation of perennial or annual crops, occurred a conspicuous reduction of plant parasitic nematode diversity, associated with introduction of exotic species, resulting in low similarity between location of primary vegetation and the cultivated ones. The results demonstrated that the agricultural practices affect strongly the plant parasitic nematode communities.