O objetivo do trabalho foi estudar uma forma alternativa para o controle do endurecimento dos frutos do maracujazeiro, pela produção de plantas transgênicas contendo o gene da proteína capsidial do Passionfruit woodness virus - PWV. O vetor de expressão foi construído utilizando-se os plasmídeos pCambia 2300 e pCambia 2301, que contêm o gene de seleção nptII, para resistência ao antibiótico canamicina. O plasmídeo pCambia 2301 contém também o gene repórter uidA (GUS). Os plasmídeos foram introduzidos em Agrobacterium tumefaciens, estirpes EHA 105 e LBA 4404, pelo método do choque térmico. Os explantes para transformação genética constituíram-se de discos de folhas jovens (6 mm de diâmetro), das variedades IAC 275 e IAC 277, coletados de plantas mantidas em sob fotoperíodo de 16 h luz, a 27 °C. Os explantes foram inoculados com suspensão bacteriana (5x108 UFC/mL) por 20 min e transferidos para placa de Petri contendo o meio de cultura MS + thidiazuron (TDZ - 0,25 mg/L) + nitrato de prata (AgNO3 - 4 mg/L) + acetoseringona (1 µM/L). O co-cultivo foi realizado à temperatura de 24 °C, em ausência de luz, por um período de 3 dias. Para seleção e regeneração de plantas os explantes foram transferidos para meio de cultura de seleção MS + TDZ (0,25 mg/L) + AgNO3 (4 mg/L) + canamicina (100 mg/L) + cefotaxime (500 mg/L). A incubação foi realizada a 27 °C, em ausência de luz, por um período de 4 - 6 semanas. As gemas adventícias desenvolvidas foram transferidas para o meio de cultura MSM + 10% de água de coco e incubadas sob fotoperíodo de 16 h de luz. A transformação genética foi identificada pelo teste histoquímico GUS e por PCR. Obteve-se um total de 22 plantas PCR positivas. Destas, 8 foram analisadas por Southern blot para confirmação da integração do transgene. A transcrição e expressão do transgene foram analisadas por Northern e Western blot, respectivamente. As plantas transgênicas avaliadas foram multiplicadas e inoculadas com 3 diferentes estirpes do PWV. A linhagem T2 apresentou resistência a infecção dos três isolados utilizados. / The main purpose of this work was to study an alternative way to control the Passionfruit woodiness virus - PWV through the production of transgenic plants which contained the Passionfruit woodness virus coat protein gene. The binary vector was built by using pCambia 2300 and pCambia 2301 plasmids, which contain the selection gene nptII. The pCambia 2301 plasmid also contains the reporter gene uidA (GUS). The plasmids were introduced into Agrobacterium tumefaciens, EHA 105 and LBA 4404 strains, via thermal shock method. The explants for the genetic transformation were young leaf disks (6 mm of diameter) of IAC 275 and IAC 277 varietys, extracted from plants kept under 16 h photoperiod, at 27 °C. The explants were inoculated with a bacterial suspension (5x108 UFC/mL) for 20 min and then transferred to Petri dishes containing cocolture medium MS + thidiazuron (TDZ - 0,25 mg/L) + silver nitrate (AgNO3 - 4 mg/L) + acetosyringone (1 µM/L). The co-culture was performed at 24 °C t, in the dark, for a three-day period. For the selection and regeneration of plants, the explants were transferred to the selection culture medium MS + TDZ (0,25 mg/L) + AgNO3 (4 mg/L) + kanamycin (100 mg/L) + cefotaxime (500 mg/L). The incubation was performed at 27 °C, in dark, for 4 - 6 weeks. The adventitious buds developed were then transferred to the culture medium MSM + 10% coconut water and kept incubated under 16 h photoperiod. The genetic transformation was identified through GUS and PCR tests. There were 22 PCR positive plants. Out of those, 8 were Southern blot analyzed for the confirmation of transgenc integration. The transgene transcription and expression were determined by Northern and Western blot respectively. The transgenic plants were then multiplied and inoculated with 3 different strains of PWV, and the line 2 showed resistance to the three strains used.

Développer des plantes résistantes aux pathogènes nécessite d’identifier des sources de résistance, de les introduire dans le fonds génétique souhaité et de s’assurer de leurs performance et durabilité. Par l’étude de 2 pathosystèmes distincts, ces travaux de thèse m’ont permis d’appréhender ces différentes étapes.Le pathosystème tomate-bégomovirus présente un intérêt économique important. Une étude par transgénèse suggère que la surexpression de SlNAC1 (codant un facteur de transcription) est bien impliquée dans des mécanismes de résistance au bégomovirus, probablement en activant les mécanismes de résistance systémique acquise. Cette surexpression semble s’accompagner d’un retard de croissance, incompatible avec une utilisation en amélioration des plantes. Le pathosystème tomate-potyvirus permet de comparer au sein d’une même espèce des allèles de résistance naturels et induits, reposant sur des mutations affectant un facteur d’initiation de la traduction eIF4E1. Les travaux menés sur ces allèles ont permis de mettre en lumière une redondance au sein de la petite famille multigénique eIF4E vis-à-vis de la résistance, impliquant eIF4E2 dans les mécanismes de sensibilité et sont en faveur de l’utilisation d’allèles de résistance fonctionnels. De plus, l’introgression de l’allèle naturel dans une lignée de tomate possédant déjà d’autres gènes de résistance aux pathogènes s’accompagne d’une érosion du spectre de résistance dû à un contournement massif par la souche PVY-LYE84.Nos travaux soulignent ainsi les précautions à prendre lors de la mise en place de résistances génétiques ainsi que la nécessité de concilier ces approches avec l’étude du développement de la plante. / The development of plants resistant to need to identify sources of resistance, to introduce them into the desired genetic background and subsequently to monitor their performance and durability. By studying two distinct pathosystems, this thesis work has allowed me to address those three stages.The tomato-begomoviruses pathosystem presents a significant economic interest. Transgenic approach suggests that SlNAC1 (coding for a transcription factor) is involved in resistance mechanisms to begomoviruses, probably by triggering systemic acquired resistance. Besides, the SlNAC1 overexpression seems to be associated with growth retardation, a side-effect incompatible with its use in plant breeding.The tomato-potyviruses pathosystem allowed the comparison within the same species of both natural and induced resistance alleles, associated with mutations affecting the translation initiation factor eIF4E1. Work on those alleles highlighted a redundancy effect within the eIF4E small multigenic family toward resistance, affecting eIF4E2 and favouring the use of functional resistance alleles in resistance. In parallel, the introgression of thebroad-spectrum resistance natural allele was introgressed into a tomato line that already possess other pathogen resistance genes is accompanied by an erosion of the resistance spectrum, due to a massive resistance breakdown by the PVY-LYE84 strain.Altogether, this current work highlight cautions to be taken for introducing resistance genes as well as stressing the importance of combining those approaches with the study of plant development.

Ces dernières années, les facteurs du complexe d'initiation de la traduction eucaryote ont été identifiés comme des déterminants essentiels dans la sensibilité des plantes aux virus à ARN, y compris les potyvirus, genre le plus important à la fois de par leur nombre et leur importance économique chez des espèces légumières, fruitières et de grandes cultures.En particulier, les allèles récessifs mo11 et mo12 du gène mo1 précédemment identifié comme codant pour le facteur d'initiation de la traduction eIF4E, sont actuellement utilisés pour protéger les cultures de laitue contre le potyvirus de la mosaïque de la laitue (LMV).Partant des résultats obtenus au laboratoire démontrant que les déterminants du LMV impliqués dans le contournement de la résistance mo1 comprennent non seulement le domaine codant pour la VPg mais aussi, en amont, l’extrémité carboxy-terminale de celui codant pour la CI, mon travail de thèse a consisté à (1) confirmer que le LMV code pour deux facteurs de virulence, la CI et la VPg (2) identifier par mutagenèse dirigée et aléatoire des acides aminés clés de la CI impliqués dans le contournement de la résistance contrôlée par eIF4E (3) entamer une caractérisation fonctionnelle des interactions impliquant les trois partenaires eIF4E, CI et VPg.Les résultats obtenus ont montré que l'échange de la VPg d'un isolat virulent (LMV-E) dans un non virulent (LMV-0) est suffisant pour restituer la compatibilité complète avec les variétés de laitue portant l'allèle mo11, mais pas mo12, alors que la région codant pour la portion C-terminale de la CI et la 6K2, suffit pour contourner les deux allèles de mo1. Le mutant ponctuel dans la CI (LMV-0-S621T) obtenu par mutagenèse dirigée est capable de contourner la résistance conférée par mo12 et partiellement celle conférée par mo11 tandis que le mutant réciproque (LMV-E-T621S) perd sa capacité à contourner les deux allèles de résistance. Il s’agit donc là du premier exemple d'un gène de CI de potyvirus, agissant comme un déterminant de contournement de la résistance récessive contrôlée par eIF4E. En effet, la VPg des potyvirus a été identifiée jusque là comme l’unique facteur de virulence vis-à-vis de eIF4E. Il semble donc que le LMV puisse utiliser deux facteurs viraux (CI et VPg) pour le contournement des allèles de résistance mo1.Cette propriété, associée au fait que des isolats de LMV d’origine sauvage ou ornementale soient capables d’évoluer vers le contournement pourraient donc présenter un risque pour la durabilité des résistances mo1. Des travaux préliminaires ont été menés lors de cette thèse afin de mieux comprendre le rôle exact des mutations des facteurs de virulence dans le processus évolutif d’adaptation du LMV aux pressions imposées par les allèles mo1.Enfin, l’implication de la CI dans le contournement de la résistance mo1 laisse supposer que cette protéine pourrait elle aussi interagir (comme la VPg), directement ou non, avec eIF4E. Nous avons montré pour la première fois in vitro et in vivo l’interaction entre la région C-terminale de la CI et la protéine eIF4E de laitue. De plus, la mutation en position 621 de la CI ayant un rôle clé dans le contournement de la résistance mo1 ne semble pas affecter ces interactions in vitro / In recent years, the factors of the eukaryotic translation initiation complex were identified as essential determinants of plant susceptibility to RNA viruses, including potyviruses, the largest and economically the most important of the plant virus groups. Members of this group are responsible for important virus diseases affecting all types of vegetable, fruit, and field crops.In particular, the recessive alleles mo11 and mo12 of mo1 gene, which previously identified as encoding for the translation initiation factor eIF4E, are currently used to protect lettuce crops against lettuce mosaic potyvirus (LMV).Based on the results obtained in the laboratory showing that the LMV determinants involved in the mo1 resistance overcoming include not only the area encoding for the VPg but also upstream, the C-terminal region of that encoding for the CI, my work of thesis consisted in (1) confirm that LMV encodes for two virulence factors, CI and VPg (2) identified by site-directed and random mutagenesis a key amino acids of the CI protein involved in eIF4E-mediated resistance overcoming (3) initiate a functional characterization of interactions involving the three partners eIF4E, VPg and CI.The results obtained showed that the exchange of VPg of a virulent isolate (LMV-E) in a non-virulent (LMV-0) is sufficient to restore full compatibility with lettuce cultivars carrying mo11 allele, but not mo12, whereas the region encoding for the C-terminal portion of the CI and 6K2, is sufficient to overcoming both mo1 alleles. The point mutation in the CI (LMV-0-S621T) obtained by site-directed mutagenesis is able to overcome the resistance conferred by mo12 and partly that conferred by mo11 while the reciprocal mutation (LMV-E-T621S) loses its ability to overcome both resistance alleles.So this is the first example of a potyvirus CI gene acting as a virulence determinant in overcoming eIF4E-mediated resistance. Indeed, the potyvirus VPg was previously identified as the sole virulence determinant vis-a-vis eIF4E. It seems that LMV can use two viral factors (CI and VPg) to overcome the mo1 resistance alleles.This property, combined with the fact that LMV isolates from the wild or ornamental origin are able to evolve towards the overcoming could therefore pose a risk to the durability of the mo1 resistance. Preliminary works were carried on during this thesis to better understand the exact role of mutations affecting virulence factors in the evolutionary process of adaptation of LMV to the pressures imposed by mo1 alleles.Finally, the involvement of the LMV CI in mo1 resistance breaking suggests that this protein could also interact (such as VPg), directly or indirectly with eIF4E. We have shown for the first time in vitro and in vivo interactions between the C-terminal region of the CI protein and the lettuce eIF4E protein. In addition, the mutation at position 621 of the CI which has a key role in mo1 resistance overcoming does not seem to affect these interactions in vitro.

Ce travail a porté sur l'identification et la caractérisation de nouveaux facteurs de l'hôte nécessaires au cycle infectieux des virus à ARN (genre Potyvirus) chez Arabidopsis thaliana, ces facteurs pouvant constituer des cibles pour la lutte génétique contre les phytovirus. L'exploration de la diversité génétique naturelle d'Arabidopsis a conduit à l'identification d'un gène récessif de résistance au Watermelon mosaic virus (WMV) chez l'accession Cvi-0. Il s'agit du gène rwm1 qui agit à un stade précoce de l'interaction en empêchant l'accumulation du virus au niveau des premières cellules infectées. Les résultats de clonage positionnel de rwm1, de séquençage d'allèles de résistance et de sensibilité et de validation fonctionnelle argumentent en faveur d'un rôle de la phosphoglycérate kinase chloroplastique (cPGK), une enzyme clé de la photosynthèse, dans la résistance conférée par rwm1. En parallèle, une stratégie gène candidat a été mise en œuvre pour étudier le rôle de la protéine kinase Target Of Rapamycin (TOR) dans l'interaction plante-potyvirus. La caractérisation de lignées RNAi pour le gène TOR d'Arabidopsis montre une diminution voire une suppression de l'accumulation du WMV, ainsi qu'une réduction de l'accumulation du Turnip mosaic virus. L'étude comparative de la composition des complexes de liaison à la coiffe entre une lignée sauvage et une lignée RNAi TOR en conditions saines et infectées par le WMV suggère un rôle de TOR dans l'interaction avec les potyvirus par le biais d'une régulation de l'activité du facteur d'initiation de la traduction eIF3. / This work describes the use of forward and reverse genetics in Arabidopsis thaliana to identify and characterize new host factors required for the infectious cycle of plant RNA viruses (genus Potyvirus). The exploration of the Arabidopsis natural genetic diversity led to the identification of the recessive rwm1 gene for resistance to Watermelon mosaic virus (WMV). Rwm1-mediated resistance was identified in the Cvi-0 accession where it acts at an early stage of the infection process by impairing virus accumulation in initially infected tissues. Map-based cloning results, allelic sequence analysis and functional validation experiments, strongly suggest the involvement of chloroplast phosphoglycerate kinase (cPGK), a key photosynthetic enzyme, in rwm1-mediated resistance. In parallel, a candidate gene approach was developed to investigate the potential role of the TOR kinase (Target Of Rapamycin) in the control of susceptibility to potyviruses. Arabidopsis RNAi lines displaying partial TOR inactivation showed a significant decrease of susceptibility to the infection by two potyviruses, namely WMV and Turnip mosaic virus. The results from a comparative proteomic analysis of cap-binding complexes between wild type and TOR RNAi infected and healthy lines suggest that TOR could play a role in plant-potyvirus interactions by regulating the activity of the translation initiation factor eIF3. Overall, this work opens a challenging research area to provide novel insights on the molecular principles underlying viral infection processes and opportunities to improve and optimize the way we tackle plant resistance to viruses through a diversification of genetic targets.

Les facteurs d’initiation de la traduction sont des protéines de plante cruciales pour l’établissement du cycle viral des Potyvirus. Des mutations dans ces facteurs sont la source de résistances récessives chez de nombreuses plantes d’intérêt agronomique. Ces résistances peuvent être déjouées par les Potyvirus, principalement par l’acquisition de mutations dans la VPg (Viral Protein Genome-linked) permettant de rétablir une interaction compatible avec les formes mutées des facteurs eIF4E. Ce modèle simple de co-évolution n’est pas applicable au pathosystème Laitue/LMV (Lettuce mosaic virus). En effet, chez la laitue, deux allèles mo11 et mo1² du gène mo1 codant pour eIF4E confèrent la résistance au LMV. Dans ce pathosystème, la VPg n’a été associée qu’au contournement de l’allèle de résistance mo11. Un deuxième facteur de virulence entre en jeu : la protéine CI (Cylindrical Inclusion, helicase caractéristique des potyvirus), responsable du contournement des résistances mo11 et mo1². L’étude approfondie de la diversité naturelle du LMV a permis d’identifier 4 positions en acides aminés dans la CI, potentiellement impliquées dans le mécanisme de contournement. Celles-ci ont été étudiées par une approche de génétique inverse. Les résultats mettent pour la première fois en évidence l’existence d’un contournement de résistance partiel (toutes les plantes ne sont pas infectées) et graduel (la proportion de plantes infectées augmente avec le nombre de mutations). Par ailleurs, ce contournement est associé au rétablissement du mouvement en systémie du virus en contexte de résistance et est dépendant du fond génétique du virus. / Eukaryotic translation initiation factors are essential host factors required for Potyvirus infection and represent a major component of recessive resistance against potyviruses in several crops. Potyvirus adaptation to eIF4E-mediated resistance usually occurs through mutations in the VPg (Viral Protein Genome-linked), restorating its interaction with the mutated eIF4E factor. Such co-evolution model doesn’t match the Lettuce/LMV (Lettuce mosaic virus) pathosystem. Indeed, two resistance alleles mo11 and mo1² of the mo1 gene encoding eIF4E, confer resistance against LMV in lettuce. The VPg triggers virulence only towards the mo11 allele. Another virulence determinant was identified: the CI (Cylindrical inclusion, potyviral helicase), which was associated with both mo11 and mo1² overcoming. By investigating the LMV natural diversity, we identified 4 amino acid positions in the CI-Cterminus potentially involved in mo1 overcoming. Reverse genetics experiments were carried out, demonstrating that mutations at these positions are associated with a partial (not all the plants are infected) and gradual (increase with the accumulation of mutations) resistance breaking mechanism. Resistance-breaking mutations were shown to restore the systemic movement of the virus and to be influenced by the viral genetic background.

O Papaya ringspot virus - type W (PRSV-W) e o Zucchini yellow mosaic virus (ZYMV) são as duas espécies de potyvírus que mais causam danos em cucurbitáceas no estado de São Paulo e no Brasil. Uma boa alternativa de controle para as doenças causadas por esses vírus é o emprego da premunização. O principal objetivo desse trabalho foi estudar a competição por sítios de replicação como possível mecanismo de proteção entre a estirpe fraca PRSV-W-1 e a estirpe severa PRSV-W-C em plantas de abobrinha-de-moita. Além disso, procurou-se também conhecer o período mínimo necessário para a proteção de plantas de abobrinha-de-moita premunizadas com a estirpe fraca ZYMV-M, só e em dupla premunização com a estirpe fraca PRSV-W-1, para fornecer subsídios para o uso em condições de campo. O estudo da competição por sítios de replicação como mecanismo de proteção foi feito inoculando-se plantas de abobrinha-de-moita ‘Caserta‘ com a estirpe fraca PRSV-W-1 e desafiando-as com a estipe severa PRSV-W-C. As inoculações de proteção foram feitas nas folhas cotiledonares e as de desafio na folha nova verdadeira, e vice-versa, aos 3, 6 e 9 dias após a primeira inoculação. Plantas infectadas com a estirpe fraca e não desafiadas e plantas infectadas com a estirpe severa foram usadas como controles. As avaliações foram feitas com base na manifestação dos sintomas 30 dias após o desafio. Também foi feito teste de recuperação da estirpe desafiante e detecção desta por RT-PCR, com primers específicos, aos 8 dias após o desafio. Os resultados sugerem que, independente do local onde foi realizada a inoculação de proteção (folha cotiledonar ou folha nova expandida), de uma maneira geral parece haver alguns sítios livres para a superinfecção com a estirpe severa. Quando esta foi inoculada aos três dias após a proteção, ela se estabeleceu em algumas plantas, moveu-se sistemicamente e sobrepôs a estirpe fraca, uma vez que as plantas exibiram sintomas severos. Com o passar do tempo (seis e nove dias após a proteção), todas as plantas ficaram protegidas contra a expressão dos sintomas severos da estirpe desafiante, porém esta foi capaz de se estabelecer nas folhas inoculadas e até mesmo mover sistemicamente em algumas plantas. No caso da proteção com a estirpe fraca ZYMV-M, só ou em mistura com a estirpe fraca PRSV-W-1, contatou-se que plantas de abobrinha-de-moita premunizadas e desafiadas sete dias depois ficaram protegidas contra a infecção e/ou manifestação dos sintomas das respectivas estirpes severas usadas no desafio. / Papaya ringspot virus - type W (PRSV-W) and Zucchini yellow mosaic virus (ZYMV) are two potyviruses associated with severe yield losses on cucurbit crops in the State of São Paulo and other parts of the Brazilian territory. Preimmunization with mild strains has proved to be a good alternative for the control of both viruses in susceptible cultivars. The main purpose of this work was to evaluate the competition for infectable sites as a possible mechanism of cross protection between the mild strain PRSV-W-1 and the severe strain PRSV-W-C in zucchini squash (Cucurbita pepo cv. Caserta). Protective inoculation with the mild strain was done at the cotyledon and the challenge inoculation with the severe strain was applied on the first true expanded leaf, and viceversa. Different plants were challenged at three, six and nine days, respectively. No challenge protected plants and healthy plants infected with the severe strain were used as controls. Evaluations were based on the expression of the symptoms at 30 days after challenge inoculation. Attempts to recover the challenge strain from challenge inoculated and new developed leaves were also done at eight days after challenge inoculation. RT-PCR with specific pairs of primers was also used to detect both stains in some of these samples. Regardless the leaf on which the protective strain was applied (cotyledon or first true expanded leaf) there appear to be some infectable sites available for superinfection with the severe strain. When the challenge inoculation was done at three days after preimmunization, the severe strain was able to superinfect some plants, move systemically and overcome the mild strain, since these plants expressed severe symptoms. All plants become protected against the expression of the symptoms induced by the severe strain when the challenge inoculation was done at six and nine days after preimmunization. However, the severe strain was still detected in the inoculated and upper leaves of few test-plants, eight days after challenge inoculation. In addition to this, it was also determined the period of time necessary to protect zucchini squash plants with a mild strain of ZYMV, named ZYMV-M, alone and in mixture with mild strain PRSV-W-1. The results indicated that single and double preimmunized plants were protected against infection and/or expression of the homologous severe strain seven days after protective inoculation.

O vírus do mosaico amarelo da abobrinha (Zucchini yellow mosaic virus - ZYMV) é provavelmente um dos mais dinâmicos e prejudiciais vírus emergentes de plantas. Desde sua primeira detecção na Itália e na França em 1973 e 1979, respectivamente, o ZYMV tem sido reportado em mais de 50 países variando de condições climáticas tropicais a temperadas em todos os continentes, exceto na Antártida. No Brasil esse potyvirus foi constatado primeiramente nos Estados de São Paulo e Santa Catarina, no início da década de 90 e posteriormente nos Estados do Ceará, da Bahia, do Rio Grande do Norte, do Pará, do Mato Grosso do Sul e do Maranhão. É provável que atualmente ocorra em todos os estados brasileiros onde se cultivam cucurbitáceas. Embora ocorra no Brasil há mais de 20 anos, pouco se conhece sobre as características biológicas, sorológicas e moleculares dos isolados até então encontrados no país. Esse projeto de pesquisa teve por objetivo cobrir parcialmente essa lacuna para fornecer subsídios para programas de melhoramento genético. Foram utilizados 11 isolados coletados em diversos estados brasileiros. A caracterização biológica consistiu no estudo da reação de diversas espécies vegetais inoculadas mecanicamente. A caracterização sorológico consistiu na marcação da proteína capsidial dos isolados com o antissoro policlonal contra o vírus. A caracterização molecular foi feita por meio da análise das sequências de nucleotídeos e aminoácidos deduzidos do gene da proteína capsidial dos isolados. A reação das espécies variou de acordo com o isolado inoculado. Análises de RFLP mostraram que a enzima de restrição Hpa II permitiu diferenciar o isolado fraco ZYMV-M da maioria dos isolados severos. O peso molecular da proteína capsidial dos isolados analisados foi em torno de 36 KDa, típico da proteína capsidial desse potyvirus. A identidade de nucleotídeos do gene da proteína capsidial entre os isolados brasileiros do ZYMV variou de 93% a 100% e a de aminoácidos deduzidos variou de 97% a 100%. Comparados com as sequências correspondentes de isolados do ZYMV de diferentes origens geográficas a identidade de nucleotídeos variou de 82% a 99%, enquanto a de aminoácidos deduzidos ficou entre 87% e 99%. Na árvore filogenética os isolados brasileiros do ZYMV pertencem ao grupo A, subdivisões I ou II, indicando uma variação entre os isolados. Nenhum evento de recombinação foi detectado nos isolados brasileiros. / Zucchini yellow mosaic virus (ZYMV) is probably one of the most dynamics and economically important emerging plant viruses. Since it was first report in Italy and France in 1973 and 1979, respectively, ZYMV has been found in over than 50 countries ranging from tropical to temperate climate conditions in all continents, except in Antartida. In Brazil this potyvirus was first reported in the beginning of 90´s in São Paulo and Santa Catarina States, and then in Ceará, Bahia, Rio Grande do Norte, Pará, Mato Grosso do Sul and Maranhão states. Probably it occurs in all Brazilian states which grow cucurbit species. Although ZYMV occurs in Brazil for more than 20 years, there is little information about the biological, serological and molecular characteristics for the isolates found in the country. The purpose of this work was to cover partially this gap to provide subsidies for genetic breeding programs. Eleven isolates collected in several Brazilian states were used. The biological characterization consisted in the study of the reaction of several vegetal species mechanically inoculated with the virus. The serological characterization consisted in the identification of the molecular weight of the coat protein through western blot analysis. The molecular characterization was done by the analyses of nucleotides and deduced amino acids sequences for the coat protein gene. The host reaction showed slight variation according to the virus. RFLP analyses showed that restriction enzyme HpaII allowed differentiate the mild ZYMV-M isolate from the majority of severe isolates. The coat protein molecular weight for all studied isolates was about 36 KDa, typical of the coat protein of this potyvirus. The nucleotide identity of the coat protein gene among the ZYMV Brazilian isolates ranged from 93% to 100%, and the deduced amino acids sequences ranged from 97% to 100%. Compared to corresponding sequences of ZYMV isolates from different geographical locations the nucleotide sequences identity ranged from 82% to 99%, while the deduced amino acids sequences ranged from 87% to 99%. Phylogenetic analysis place the Brazilian isolates of ZYMV into the group A, subdivision I or II, showing some diversity between the isolates. No recombination event was detected in the Brazilian isolates.

A abobrinha de moita (Cucurbita pepo L.), espécie pertencente à família Cucurbitaceae, apresenta significativa participação na produção mundial e brasileira desta família de plantas. Porém, como em toda cultura de importância econômica, as cucurbitáceas apresentam problemas fitossanitários causados por diferentes agentes etiológicos. Na produção brasileira de abóboras, já foi constatada a presença de 8 vírus, dentre eles os potyvirus PRSV-W (Papaya ringspot virus-type W) e ZYMV (Zucchini yellow mosaic virus) e o tospovirus ZLCV (Zucchini lethal chlorosis virus) têm sido considerados os mais importantes pela predominância em diversas regiões produtoras de cucurbitáceas no país e pelos consideráveis prejuízos que geralmente causam à produção. Considerando que a maioria dos estudos epidemiológicos existentes sobre estas três viroses são poucos e fragmentados, percebe-se que não há estudos que abordam em conjunto todos os parâmetros epidemiológicos de tais vírus. Assim, os objetivos deste trabalho foram estudar o progresso temporal e espacial destas três viroses e verificar a relação entre a epidemiologia das mesmas em campo de abobrinha de moita, além de melhor entender o patossistema em que a clorose letal (causada pelo ZLCV) está inserida Para isso, foram conduzidos experimentos com abobrinha de moita Caserta nos campos experimentais dos Departamentos de Fitopatologia e Nematologia (DFN) e Departamento de Genética (DGN) da Esalq/USP. Primeiramente foram conduzidos em 2009 dois experimentos simultâneos nas áreas do DFN e DGN para estudar a epidemiologia isolada da clorose letal acompanhada da dinâmica populacional do tripes Frankliniella zucchini, vetor deste vírus. Em seguida, em épocas diferentes 3 experimentos foram conduzidos em 2010 com o intuito de comparar as epidemiologias da clorose letal e dos mosaicos comum e amarelo, estes últimos causados por PRSV-W e ZYMV, respectivamente. Nos experimentos apenas com a clorose letal no ano de 2009, o modelo monomolecular foi o que melhor se ajustou aos dados de incidência e através das análises espaciais detectou-se agregação da doença ao final dos dois experimentos. Nos 3 experimentos realizados em 2010, variações na incidência, no ajuste do modelo e no comportamento espacial de cada virose foram freqüentes. Para a clorose letal, o modelo monomolecular ofereceu melhor ajuste apenas na 3ª época de plantio. Nas duas primeiras o modelo gompertz apresentou maior coeficiente de determinação. No comportamento espacial, novamente agregação da doença foi detectada ao final do ciclo da cultura. Para o mosaico amarelo, os modelos que melhores se ajustaram na 1ª, 2ª e 3ª épocas de plantio, foram o logístico e o monomolecular (este nas duas últimas), respectivamente. O padrão espacial desta doença foi ao acaso quando da baixa incidência da doença e agregado nos casos em que elevada incidência ocorreu. Já o mosaico comum apresentou a menor incidência nas 3 épocas. O modelo logístico foi o que melhor se ajustou em todas as épocas e a doença apresentou comportamento espacial ao acaso nos três experimentos. Nos levantamentos do tripes vetor do ZLCV, verificou-se preferência do mesmo por plantas sintomáticas e consideráveis correlações do número de insetos coletados com a incidência da clorose letal. / The zucchini squash (Cucurbita pepo L.) belonging to the Cucurbitaceae family, has a good share of world and brazilian output. However, as in every plants of economically important, cucurbits have problems caused by different etiological agents. In the Brazilian production of zucchini squash, already confirmed the presence of 8 viruses, including the potyviruses PRSV-W (Papaya ringspot virus-type W) and ZYMV (Zucchini yellow mosaic virus) and the tospovirus ZLCV (Zucchini lethal chlorosis virus) have been considered the most important viruses by the predominance in several cucurbits producing regions in the Brazil and the considerable damage on production. Whereas the most of the existing epidemiological studies about these three viruses are few and fragmented, it is clear that there are no studies that deal together all the epidemiological parameters of such viruses. The objectives of this work were to study the temporal and spatial progress of these three viruses and the relation between the epidemiology of these viruses in the same field of zucchini squash in addition to better understand the lethal chlorosis pathosystem (caused by ZLCV). Trials were carried out with zucchini squash \'Caserta in the experimental fields of the Departments of Plant Pathology and Nematology (DPP) and Department of Genetics (DGN) at Esalq/USP. The firsts were conducted in 2009 simultaneously in DPP and DGN to study the epidemiology of lethal chlorosis only and to study the population dynamic of thrips Frankliniella zucchini, the vector of this virus. In 2010 three experiments were carried out in different growing seasons in order to compare the epidemiology of the lethal chlorosis, yellow mosaic and common mosaic caused by ZLCV, PRSV-W and ZYMV, respectively. In the experiments with the lethal chlorosis in 2009, the monomolecular model was the best fit to the incidence data and spatial analysis indicated aggregation of the disease at the end of both experiments. In three experiments carried out in 2010, variations in incidence, in the fit of the model and in the spatial distribution of each virus were frequents. For lethal chlorosis, the monomolecular model provided a better fit only in the 3rd growing season. In the first and second growing seasons Gompertz model had the best coefficient of determination. In the spatial distribution, aggregation of disease was detected at the end of the crop cycle again. For yellow mosaic, the models that best fit in the 1st, 2nd and 3rd planting dates were the logistic and monomolecular (this in the last two) respectively. The spatial pattern of this disease were randomly when the disease incidence was low and aggregated when the disease incidence was high. The common mosaic had the lowest incidence in all three seasons. The logistic model was the best fit in all growing seasons and the disease showed a spatial random distribuctions in all experiments. The thrips vector of ZLCV prefer symptomatic plants and good correlations between the number of insects collected with the incidence of lethal chlorosis was found.

A cana-de-açúcar é cultivada em diversos países do mundo. O Brasil é atualmente o maior produtor mundial de cana-de-açúcar e o maior exportador dos produtos derivados dessa espécie, tais como açúcar e etanol. A cultura da cana-deaçúcar apresenta muitos problemas fitossanitários que prejudicam a produção, entre eles o causado pelo vírus do mosaico da cana-de-açúcar (Sugarcane mosaic virus, SCMV). O mosaico da cana-de-açúcar vem sendo controlado com o uso de variedades e híbridos resistentes. No entanto, frequentemente notam-se plantas sintomáticas nas avaliações de novos clones de cana-de-açúcar, em viveiros de mudas e plantios comerciais. Diante desse fato, o presente trabalho teve como objetivo realizar a caracterização biológica e molecular de isolados do SCMV que induzem sintomas variados em plantas de cana-de-açúcar plantadas nos municípios de Assis, Piracicaba e Ribeirão Preto, SP. Nove isolados, identificados por causarem sintomas fraco, intermediário e severo de mosaico foram coletados em cada localidade. Um isolado que causa mosaico listrado também foi coletado em Piracicaba. Todos os isolados foram estabelecidos na variedade SP86155. A caracterização molecular foi feita por meio da analise das sequências de nucleotídeos e de amino ácidos deduzidos do gene da proteína capsidial dos isolados. Também foram diferenciados por meio da análise de polimorfismo de comprimento de fragmentos. A caracterização biológica foi realizada por testes de proteção entre alguns dos isolados. As sequências parciais de nucleotídeos e de aminoácidos deduzidos do gene da proteína capsidial de cada isolado foram comparadas entre si e com outras sequências correspondentes de isolados de diferentes regiões geográficas. As identidades das sequências de nucleotídeos variam de 94 a 100%, entre os isolados estudados. Quando as mesmas sequências foram comparadas com as sequências de nucleotídeos do gene da proteína capsidial de outros isolados do SCMV as identidades variaram de 79 a 98%. Para as sequências de amino ácidos deduzidos as identidades variaram de 94 to 100%, entre os isolados estudados e de 81 to 98% quando comparadas com as de outros isolados do SCMV. Análise filogenética agrupou os 10 isolados com outros isolados brasileiros do SCMV (JAU - 1, RIB - 1 e PIR - 2 e FER - 1) e com os isolados dos E.U.A. originalmente denominados estirpes A, B, D e E. Analises de RFLP mostraram que a enzima de restrição HinfI foi a que melhor diferenciou os isolados e por isso permitiu a análise da proteção. As plantas inoculadas com os isolados fracos de Piracicaba, Ribeirão Preto e Assis ficaram protegidas contra a invasão sistêmica dos isolados severos dos mesmos locais e contra o isolado que causa mosaico listrado de Piracicaba. Falhas na proteção ocorreram em plantas infectadas com o isolado Piracicaba intermediário e desafiadas com os isolados Ribeirão Preto severo e Assis severo. Em conjunto esses resultados indicam que os dez isolados estudados são estirpes do SCMV que induzem sintomas diferenciados. / Sugarcane crop is raised in several tropical countries around the world. Brazil is the largest producer of sugarcane, as well as the largest exporter of ethanol and sugar, which are outputs of sugarcane processing. The sugarcane production faces several sanitary problems and among them is the mosaic disease caused by the potyvirus Sugarcane mosaic virus (SCMV). Traditionally, resistant varieties and hybrids are used for the control of SCMV. However, new cases of symptomatic plants have been reported in commercial crops and in seedling production facilities. Based on this fact, the present work aims to do both molecular and biological characterization of isolates of SCMV which causes different symptoms in sugarcane crops in the regions of Assis (SP), Piracicaba (SP) and Ribeirão Preto (SP). Nine isolates identified as causing mild, intermediate and severe mosaic were collected in each of the three areas. One isolate which causes striped mosaic was also collected in the Piracicaba area. All isolates were established in the variety SP86155 by mechanical inoculation. The molecular characterization was done through the analysis of nucleotide and deduced amino acid sequences for the coat protein gene. Likewise the isolates were differentiated through the analysis of restriction fragment length polymorphism (RFLP) for further use on biological characterization through cross protection tests between the isolates. The partial nucleotide and deduced amino acid sequences for the coat protein gene were compared among themselves and with corresponding sequences for other isolates from different geographical regions. The identity of the nucleotide sequences ranged from 94 to 100% among the studied isolates. When compared with the corresponding nucleotide sequences of other isolates of SCMV the identities ranged from 79 to 98%. The identity of the deduced amino acids sequences ranged from 94 to 100% among the studied isolates, and from 81 to 98% when compared with isolates form different geographical regions. Phylogenetic analysis grouped all 10 isolates with other Brazilian isolates do SCMV (JAU-1, RIB-1 e PIR-2 e FER-1), as well as with isolates from the U.S.A, originally named strains A, B, D e E. RFPL analyses pointed out that the restriction enzyme Hinfl did the best differentiation of the isolates and was further used for the evaluation of the cross protection tests. Plants inoculated with mild isolates remained protected against the severe isolates and also against the isolate which causes the stripped mosaic. Fails on cross protection occurred when plants infected with the isolate Piracicaba intermediate were challenged with isolates Ribeirão Preto and Assis severe. Together these data indicate that the 10 studied isolates are strains of SCMV that induce different symptoms.

A abobrinha de moita (Cucurbita pepo L.), espécie pertencente à família Cucurbitaceae, apresenta significativa participação na produção mundial e brasileira desta família de plantas. Porém, como em toda cultura de importância econômica, as cucurbitáceas apresentam problemas fitossanitários causados por diferentes agentes etiológicos. Na produção brasileira de abóboras, já foi constatada a presença de 8 vírus, dentre eles os potyvirus PRSV-W (Papaya ringspot virus-type W) e ZYMV (Zucchini yellow mosaic virus) e o tospovirus ZLCV (Zucchini lethal chlorosis virus) têm sido considerados os mais importantes pela predominância em diversas regiões produtoras de cucurbitáceas no país e pelos consideráveis prejuízos que geralmente causam à produção. Considerando que a maioria dos estudos epidemiológicos existentes sobre estas três viroses são poucos e fragmentados, percebe-se que não há estudos que abordam em conjunto todos os parâmetros epidemiológicos de tais vírus. Assim, os objetivos deste trabalho foram estudar o progresso temporal e espacial destas três viroses e verificar a relação entre a epidemiologia das mesmas em campo de abobrinha de moita, além de melhor entender o patossistema em que a clorose letal (causada pelo ZLCV) está inserida Para isso, foram conduzidos experimentos com abobrinha de moita Caserta nos campos experimentais dos Departamentos de Fitopatologia e Nematologia (DFN) e Departamento de Genética (DGN) da Esalq/USP. Primeiramente foram conduzidos em 2009 dois experimentos simultâneos nas áreas do DFN e DGN para estudar a epidemiologia isolada da clorose letal acompanhada da dinâmica populacional do tripes Frankliniella zucchini, vetor deste vírus. Em seguida, em épocas diferentes 3 experimentos foram conduzidos em 2010 com o intuito de comparar as epidemiologias da clorose letal e dos mosaicos comum e amarelo, estes últimos causados por PRSV-W e ZYMV, respectivamente. Nos experimentos apenas com a clorose letal no ano de 2009, o modelo monomolecular foi o que melhor se ajustou aos dados de incidência e através das análises espaciais detectou-se agregação da doença ao final dos dois experimentos. Nos 3 experimentos realizados em 2010, variações na incidência, no ajuste do modelo e no comportamento espacial de cada virose foram freqüentes. Para a clorose letal, o modelo monomolecular ofereceu melhor ajuste apenas na 3ª época de plantio. Nas duas primeiras o modelo gompertz apresentou maior coeficiente de determinação. No comportamento espacial, novamente agregação da doença foi detectada ao final do ciclo da cultura. Para o mosaico amarelo, os modelos que melhores se ajustaram na 1ª, 2ª e 3ª épocas de plantio, foram o logístico e o monomolecular (este nas duas últimas), respectivamente. O padrão espacial desta doença foi ao acaso quando da baixa incidência da doença e agregado nos casos em que elevada incidência ocorreu. Já o mosaico comum apresentou a menor incidência nas 3 épocas. O modelo logístico foi o que melhor se ajustou em todas as épocas e a doença apresentou comportamento espacial ao acaso nos três experimentos. Nos levantamentos do tripes vetor do ZLCV, verificou-se preferência do mesmo por plantas sintomáticas e consideráveis correlações do número de insetos coletados com a incidência da clorose letal. / The zucchini squash (Cucurbita pepo L.) belonging to the Cucurbitaceae family, has a good share of world and brazilian output. However, as in every plants of economically important, cucurbits have problems caused by different etiological agents. In the Brazilian production of zucchini squash, already confirmed the presence of 8 viruses, including the potyviruses PRSV-W (Papaya ringspot virus-type W) and ZYMV (Zucchini yellow mosaic virus) and the tospovirus ZLCV (Zucchini lethal chlorosis virus) have been considered the most important viruses by the predominance in several cucurbits producing regions in the Brazil and the considerable damage on production. Whereas the most of the existing epidemiological studies about these three viruses are few and fragmented, it is clear that there are no studies that deal together all the epidemiological parameters of such viruses. The objectives of this work were to study the temporal and spatial progress of these three viruses and the relation between the epidemiology of these viruses in the same field of zucchini squash in addition to better understand the lethal chlorosis pathosystem (caused by ZLCV). Trials were carried out with zucchini squash \'Caserta in the experimental fields of the Departments of Plant Pathology and Nematology (DPP) and Department of Genetics (DGN) at Esalq/USP. The firsts were conducted in 2009 simultaneously in DPP and DGN to study the epidemiology of lethal chlorosis only and to study the population dynamic of thrips Frankliniella zucchini, the vector of this virus. In 2010 three experiments were carried out in different growing seasons in order to compare the epidemiology of the lethal chlorosis, yellow mosaic and common mosaic caused by ZLCV, PRSV-W and ZYMV, respectively. In the experiments with the lethal chlorosis in 2009, the monomolecular model was the best fit to the incidence data and spatial analysis indicated aggregation of the disease at the end of both experiments. In three experiments carried out in 2010, variations in incidence, in the fit of the model and in the spatial distribution of each virus were frequents. For lethal chlorosis, the monomolecular model provided a better fit only in the 3rd growing season. In the first and second growing seasons Gompertz model had the best coefficient of determination. In the spatial distribution, aggregation of disease was detected at the end of the crop cycle again. For yellow mosaic, the models that best fit in the 1st, 2nd and 3rd planting dates were the logistic and monomolecular (this in the last two) respectively. The spatial pattern of this disease were randomly when the disease incidence was low and aggregated when the disease incidence was high. The common mosaic had the lowest incidence in all three seasons. The logistic model was the best fit in all growing seasons and the disease showed a spatial random distribuctions in all experiments. The thrips vector of ZLCV prefer symptomatic plants and good correlations between the number of insects collected with the incidence of lethal chlorosis was found.