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A virus causing chlorosis and leaf distortion in the Western Australian endemic legume Hardenbergia comptoniana was detected by biological indexing to Chenopodium quinoa and Nicotiana benthamiana. Enzyme linked immuno-sorbent assay (ELISA) using general Potyvirus antiserum and amplification by reverse transcription polymerase chain reaction (RT-PCR) with degenerate primers indicated that it was a species of Potyvirus. It was confirmed as an unknown member of the genus Potyvirus by comparing its coat protein sequence with those of other potyviruses. The name Hardenbergia mosaic virus (HarMV) is proposed for this new virus species. Isolates of HarMV were collected from 13 sites, covering much of the natural range of its host. An experimental host range was determined using nine virus isolates tested against plants from 11 species in three families. Its infectivity on three leguminous species important in agriculture (Lupinus angustifolius, L. luteus and Trifolium subterraneum) was established. The nucleotide (nt) sequences of the coat proteins (CP) of 28 isolates determined there was 24.1- 27.6% diversity with the closest known relative, Passion fruit woodiness virus (PWV). Studies of the nucleotide sequences of the CP showed that there was considerable intra-species divergence (mean 13.5%, maximum 20.5%) despite its relatively small geographical distribution and single known natural host. The observed broad diversity strongly suggests long genetic isolation and that HarMV evolved in the region where it was collected. An examination of its phylogeny showed that 28 isolates clustered into eight clades with high bootstrap support (6.2-20.5% inter-clade diversity). Isolates collected at locations distant to the Perth metropolitan area (Margaret River and Seabird) diverged more from isolates collected in the metropolitan area (15.4-21.1% nucleotide sequence diversity). This virus represents the first endemic species to be characterised from Western Australia. Differences in pathogenicity and symptoms induced on key host species were seen between isolates belonging to different phylogenetic clades. Phylogenetic analysis confirmed the inclusion of HarMV within the Bean common mosaic virus group of the potyviruses and also defined a previously unreported subgroup of six previously described Potyvirus species (Clitoria virus Y, Hibbertia virus Y, PWV, Siratro 1 virus Y, and Siratro 2 virus Y), from Australia, which is further evidence for a prolonged period of genetic isolation. Both in relation to detection of strains of HarMV, and considering the broader issues of biosecurity and parallel detection of plant viruses, a microarray based detection system was established. To optimise conditions for the development of microarrays for virus detection poly-L-lysine (PLL) coated microscope slides produced in the laboratory were compared to commercially produced PowerMatrix slides (Full Moon BioSystems). Variables tested for PLL slide production were: choice of printing buffer, probe concentration, method of immobilisation and slide blocking; and in particular the print buffer and immobilisation method had the greatest effect on the quality of PLL microarray slides. Slides printed on PLL surfaces in a high salt buffer (3x Saline sodium citrate) supplemented with 1.5M betaine and immobilised at 42oC overnight retained the highest amounts of probe DNA of the methods tested. Qualitative comparisons of the two showed more probe was retained on PowerMatrix slides which were also more reliable and consistent than the PLL slides. Probes were designed for eight different virus species and six distinct strains of HarMV to test the potential to use microarrays to distinguish between them. Probes were designed to detect potyviruses at the genus, species and strain levels. Although there was evidence of non-specific hybridisation, the Potyvirus array was used to identify six strains of HarMV by hybridisation to species specific probes. Additionally the array was used to identify three other species of Potyvirus: Bean yellow mosaic virus, PWV and Passiflora foetida virus Y, following amplification with polyvalent PCR primers. In further microarray tests, using labelled first strand cDNA of Potato virus X (PVX) and Potato virus Y (PVY) on an array, PVX was strongly detected in leaves known to be infected, but PVY was only weakly detected in infected leaves. Three methods of pre-amplification of virus nucleic acid before hybridisation to the array were investigated to improve the sensitivity of the assay. Two of the methods, Klenow amplification and randomly primed PCR, amplified the target virus; as confirmed by real time PCR. Of the methods tested only randomly primed PCR improved the sensitivity of the microarray. The best amplification method used genus-specific primers with adaptor sequences. This method when tested by real time PCR showed a 3.7Ct reduction for PVX and 16.8Ct for PVY. The microarray correctly identified both viruses. In this work the first virus (HarMV) endemic to Western Australia was identified, and microarray methods were developed both to identify HarMV and other plant viruses of economic importance. The microarray approach, with further development, may be applicable as a means of identifying incursions of new viruses in a biosecurity situation.

Geminiviruses (family Geminiviridae) are characterized structurally by twinned (geminate) morphology of virions (ca. 18-30 nm) and genetically by a genome comprising one or two small circular single stranded DNA (ssDNA) molecules and they are responsible for major crop losses worldwide. The genus Begomovirus (type member Bean golden yellow mosaic virus) is the largest genus of the family Geminiviridae. The members of this genus have either monopartite or bipartite genomes. They are transmitted by whiteflies and infect only dicotyledonous plants. DNA β molecules are symptom modulating single-stranded sat-DNA molecules which are associated with certain monopartite begomoviruses. These molecules are around half the size (approximately 1350 nt in length) of their helper viruses and rely on the helper begomovirus for movement in plant tissues, replication and plant-to-plant transmission by the whitefly (Bemisia tabaci). They contribute to production of symptoms and enhance helper virus accumulation in certain hosts. DNA β molecules encode a single gene, called βC1, on the complementary strand which is important for pathogenicity and suppression of post transcriptional gene silencing. In this study the regulation of βC1 gene expression, a host factor interacting with βC1 and its role in the pathogenicity of DNA β are described. Transient expression studies using Nicotiana tabacum plants and GUS as a reporter gene, identified the sequences important for transcription of βC1 from DNA β associated with Cotton leaf curl Multan virus (CLCuMV). A 68 nt fragment (between -139 to -207), which contains a G-box motif was sufficient for DNA β promoter activity. Deletion of this region also led to loss of DNA β replication capacity. Mutation of the G-box, located at 143 nucleotides upstream of the βC1 start codon, resulted in a two to three times reduction in the DNA β promoter activity. This motif was shown to bind specifically to the nuclear factors isolated from tobacco leaf tissues. Histochemical staining of transgenic tobacco plants expressing the gus gene driven by full length DNA β promoter showed phloem specific localisation patterns. It was concluded that a G-box motif is required for binding of host nuclear factors and is necessary for efficient expression of this phloem specific βC1 gene. An ubiquitin-conjugating enzyme, called SlUBC, was retrieved from screening of a tomato cDNA library, using βC1 encoded by DNA β associated with CLCuMV as the bait. The SlUBC was shown to complement yeast deficient in the ubiquitin-conjugating enzyme. It is thought that this enzyme is a key factor in the ubiquitin proteasome pathway, which plays a central role in many eukaryotic cellular processes. The authenticity and specificity of this interaction was confirmed both in vivo, using a bimolecular fluorescence complementation assay, and in vitro. Domain mapping of βC1 showed that a myristoylation-like motif is required for the interaction with SlUBC in the yeast system and induction of DNA β specific symptoms in host plants. Western blot analysis showed that expression of βC1 in transgenic tobacco plants decreased the level of poly-ubiquitinated proteins as compared with wild type plants. However, the level of expression of homologous SlUBC remained stable in these transgenic plants. These results indicated that interaction of βC1 with the SlUBC is required for DNA β specific symptom induction possibly through down-regulation of the host ubiquitin proteasome pathway. Using GFP transgenic N. benthamiana plants, the βC1 encoded by DNA β associated with CLCuMV showed suppression of post transcriptional gene silencing. This protein inhibited both local and systemic silencing. However, the low level of GFP fluorescence and also the results of RNA analysis in patch co-infiltration assay indicated that βC1 is a weak suppressor of local RNA silencing as compared with P19 protein from Tomato bushy stunt virus. A three-way grafting assay and separate patch infiltration assays showed that βC1 interferes with the activity of GFP silencing signal. Mutation of Gly103 in βC1 which was shown to be required for interaction with SlUBC and induction of DNA β specific symptoms in host plants, had no effect on the silencing suppression activity of βC1 protein. This work has provided a new insight into the importance of a G-box motif in expression of βC1 gene of DNA β and also for binding to the host nuclear proteins. In addition, interaction with a host factor, SlUBC, has been shown to be required for induction of DNA β specific symptoms in experimental plants using ToLCV as a helper virus. However, this interaction was not required for silencing suppression activity of βC1. The results of this study can be adapted to determine the mode of pathogenesis and regulation of expression of βC1 in cotton leaf curl disease. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1337164 / Thesis (Ph.D.) - University of Adelaide, School of Agriculture, Food and Wine, 2008

Geminiviruses (family Geminiviridae) are characterized structurally by twinned (geminate) morphology of virions (ca. 18-30 nm) and genetically by a genome comprising one or two small circular single stranded DNA (ssDNA) molecules and they are responsible for major crop losses worldwide. The genus Begomovirus (type member Bean golden yellow mosaic virus) is the largest genus of the family Geminiviridae. The members of this genus have either monopartite or bipartite genomes. They are transmitted by whiteflies and infect only dicotyledonous plants. DNA β molecules are symptom modulating single-stranded sat-DNA molecules which are associated with certain monopartite begomoviruses. These molecules are around half the size (approximately 1350 nt in length) of their helper viruses and rely on the helper begomovirus for movement in plant tissues, replication and plant-to-plant transmission by the whitefly (Bemisia tabaci). They contribute to production of symptoms and enhance helper virus accumulation in certain hosts. DNA β molecules encode a single gene, called βC1, on the complementary strand which is important for pathogenicity and suppression of post transcriptional gene silencing. In this study the regulation of βC1 gene expression, a host factor interacting with βC1 and its role in the pathogenicity of DNA β are described. Transient expression studies using Nicotiana tabacum plants and GUS as a reporter gene, identified the sequences important for transcription of βC1 from DNA β associated with Cotton leaf curl Multan virus (CLCuMV). A 68 nt fragment (between -139 to -207), which contains a G-box motif was sufficient for DNA β promoter activity. Deletion of this region also led to loss of DNA β replication capacity. Mutation of the G-box, located at 143 nucleotides upstream of the βC1 start codon, resulted in a two to three times reduction in the DNA β promoter activity. This motif was shown to bind specifically to the nuclear factors isolated from tobacco leaf tissues. Histochemical staining of transgenic tobacco plants expressing the gus gene driven by full length DNA β promoter showed phloem specific localisation patterns. It was concluded that a G-box motif is required for binding of host nuclear factors and is necessary for efficient expression of this phloem specific βC1 gene. An ubiquitin-conjugating enzyme, called SlUBC, was retrieved from screening of a tomato cDNA library, using βC1 encoded by DNA β associated with CLCuMV as the bait. The SlUBC was shown to complement yeast deficient in the ubiquitin-conjugating enzyme. It is thought that this enzyme is a key factor in the ubiquitin proteasome pathway, which plays a central role in many eukaryotic cellular processes. The authenticity and specificity of this interaction was confirmed both in vivo, using a bimolecular fluorescence complementation assay, and in vitro. Domain mapping of βC1 showed that a myristoylation-like motif is required for the interaction with SlUBC in the yeast system and induction of DNA β specific symptoms in host plants. Western blot analysis showed that expression of βC1 in transgenic tobacco plants decreased the level of poly-ubiquitinated proteins as compared with wild type plants. However, the level of expression of homologous SlUBC remained stable in these transgenic plants. These results indicated that interaction of βC1 with the SlUBC is required for DNA β specific symptom induction possibly through down-regulation of the host ubiquitin proteasome pathway. Using GFP transgenic N. benthamiana plants, the βC1 encoded by DNA β associated with CLCuMV showed suppression of post transcriptional gene silencing. This protein inhibited both local and systemic silencing. However, the low level of GFP fluorescence and also the results of RNA analysis in patch co-infiltration assay indicated that βC1 is a weak suppressor of local RNA silencing as compared with P19 protein from Tomato bushy stunt virus. A three-way grafting assay and separate patch infiltration assays showed that βC1 interferes with the activity of GFP silencing signal. Mutation of Gly103 in βC1 which was shown to be required for interaction with SlUBC and induction of DNA β specific symptoms in host plants, had no effect on the silencing suppression activity of βC1 protein. This work has provided a new insight into the importance of a G-box motif in expression of βC1 gene of DNA β and also for binding to the host nuclear proteins. In addition, interaction with a host factor, SlUBC, has been shown to be required for induction of DNA β specific symptoms in experimental plants using ToLCV as a helper virus. However, this interaction was not required for silencing suppression activity of βC1. The results of this study can be adapted to determine the mode of pathogenesis and regulation of expression of βC1 in cotton leaf curl disease. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1337164 / Thesis (Ph.D.) - University of Adelaide, School of Agriculture, Food and Wine, 2008

Les approches de métagénomique basées sur l’utilisation des techniques de séquençage haut débit ont ouvert une nouvelle ère pour la découverte non biaisée et la caractérisation génomique des virus. Comme pour les autres virus, de telles études montrent que la diversité des virus phytopathogènes a jusqu’à tout récemment été fortement sous-estimée. Ces virus constituant une composante potentiellement importante des écosystèmes naturels ou des agrosystèmes anthropisés, il est important d’explorer la diversité des virus associés aux populations végétales et de comprendre les forces structurant cette diversité dans le temps et dans l’espace. Dans le même temps, le développement de telles études reste confronté à des questions d’ordre méthodologique concernant, par exemple, le choix des populations d’acides nucléiques à séquencer, la reproductibilité des analyses ou la disponibilité d’une stratégie permettant de comparer de façon fiable la richesse virale dans différents environnements. Dans le présent travail, le virome associé à des populations végétales échantillonnées dans différents écosystèmes, avec un focus sur les adventices et les plantes sauvages, a été caractérisé par des approches de métagénomique par séquençage haut débit. Dans ces travaux, l’analyse bioinformatique de la richesse du virome a été conduite par deux approches, l’une classique basée sur l’annotation Blast pour l’identification des familles virales présentes dans un échantillon, et l’autre, décrite et validée ici, qui permet de classifier les séquences virales métagénomiques en unités taxonomiques opérationnelles (operational taxonomic units, OTUs) utilisées comme proxy des espèces virales. Toujours dans une perspective méthodologique, les résultats obtenus avec des pools complexes de plantes représentatifs de la diversité végétale au site d’échantillonnage (approche « tondeuse à gazon ») ont permis de comparer les performances des deux techniques actuellement utilisées pour enrichir les séquences virales, la purification d’ARN bicaténaires (double-stranded RNA, dsRNA) ou d’acides nucléiques associés aux virions (virion-associated nucleic acids, VANA). Les résultats obtenus par les deux approches ont mis en évidence des viromes riches mais montrent que l’approche dsRNA devrait être préférée pour l’analyse de tels pools complexes car elle permet de façon reproductible une description plus complète du phytovirome, à l’exception des virus ADN. Les viromes caractérisés montrent, pour les populations végétales de milieux cultivés ou non gérés tempérés échantillonnées, une forte incidence virale (jusqu’à 86.5% dans 126 pools monospécifiques collectés sur une période de deux ans) et confirment la prédominance des virus dsRNA qui représentent plus de 70% des OTU identifiés. Alors qu’une proportion significative des virus ssRNA détectés sont déjà connus, plus de 90% des virus dsRNA détectés sont nouveaux et n’avaient pas été caractérisés auparavant. Un effort important en culturomique visant à comparer le phytovirome avec le mycovirome de cultures fongiques obtenues à partir des mêmes échantillons végétaux a révélé un nombre remarquablement faible d’OTUs partagés, renforçant le questionnement sur la nature, phytovirus ou mycovirus, des virus dsRNA identifiés dans les viromes des plantes. La composition en OTU des viromes analysés s’est révélée variable entre sites d’échantillonnage mais aussi très dynamique dans le temps, avec seulement une très faible fraction des OTUs ré-échantillonnés de façon stable dans la même population végétale sur une période de deux ans. Pris dans leur ensemble, ces travaux exploratoires permettent de mieux raisonner les choix méthodologiques pour l’étude des viromes associés aux plantes et étendent notre connaissance de la diversité des phytovirus, en particulier dans des espèces végétales sauvages largement négligées, apportant des points de référence importants pour de nouveaux travaux en écologie et en évolution virale. / Metagenomics based on high throughput sequencing (HTS) has opened a new era of unbiased discovery and genomic characterization of viruses. As for other viruses, such metagenomic studies indicate that the diversity of plant viruses was until recently far underestimated. As potentially important components of unmanaged and cultivated ecosystems, there is a need to explore the diversity of the viruses associated with plant populations and to understand the drivers shaping their diversity in space and time. At the same time, the development of such studies is still faced by methodological questions concerning, for example, the choice of target nucleic acids populations, the reproducibility of the analyses or the implementation of a strategy to accurately compare virus richness in different environments. In the present thesis the phytovirome associated with plant populations sampled in various ecosystems, with an emphasis on wild plant or weed species was characterized using HTS-based metagenomics. In these experiments, the bioinformatic analysis of the virome complexity was performed using two strategies, a classical one based on Blast-based contigs annotation for the identification of the viral families present in a sample and a novel one, described and validated here, and which allows to classify the metagenomic viral sequences into operational taxonomic units (OTUs) as a proxy to viral species. Also from the methodological perspective, the results obtained using complex plant pools such as those used in the “lawn-mower” sampling strategy allowed to compare the performance of the two currently used viral enrichment methods, double-stranded RNA (dsRNA) or Virion-associated nucleic acids (VANA) purification. The results indicate both of approaches uncovered rich viromes and suggest that the dsRNA approach should be preferred when analyzing complex plant pools since it consistently provided a more comprehensive description of the analysed phytoviromes, with the exception of the DNA viruses. The virome characterization results obtained showed, for the temperate plant populations from unmanaged and cultivated sampling sites, a high virus incidence (up to 86.5% in 126 single species pools collected over a two-year period) and confirmed the predominance of dsRNA viruses with greater than 70% of the phytovirome OTUs. While a significant proportion of detected single-stranded RNA (ssRNA) viruses are already known agents, more than 90% of the dsRNA viruses are novel and had not previously been characterized. A large scale culturomics effort to contrast the phytovirome with the mycovirome of fungal cultures obtained from the same plant samples revealed an extremely low number of shared OTUs, further deepening the debate about the phytovirus or mycovirus nature of the dsRNA viruses identified in plant viromes. The OTU composition of the analyzed phytoviromes varied significantly between sampling sites but was also shown to be highly dynamic over time, with a very low proportion of OTUs consistently re-sampled in the same plant population over a 2 years period. Taken together, these exploratory studies allow a more reasoned choice of methodology for the study of plant-associated viromes and expand our knowledge of plant virus diversity especially in neglected wild plant populations, providing important references for the further viral ecology and evolution studies.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第18334号 / 農博第2059号 / 新制||農||1023(附属図書館) / 学位論文||H26||N4841(農学部図書室) / 31192 / 京都大学大学院農学研究科応用生物科学専攻 / (主査)教授 奥野 哲郎, 教授 佐久間 正幸, 准教授 吉田 天士 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

Positive-strand RNA virus group are the most abundant among viruses affecting plants and animals. To successfully achieve replication, these viruses usurp or co-opt host proteins. To facilitate the discovery of host factors involved in Tomato bushy stunt virus (TBSV), yeast has been developed as a surrogate model host. Genome-wide approaches covering 95% of yeast genes, has revealed approximately hundred factors that could affect virus replication. Among the identified host factors, there are fourteen yeast genes, which affect/regulate lipid metabolism of the host. One of the identified host gene is ERG25, which is an important factor for sterol biosynthesis pathway, affecting viral replication. Sterols present in eukaryotes affect the lipid composition of membranes, where tombusviruses, similar to other plus-strand viruses of tobacco, replicate. Since potent inhibitors of sterol synthesis are known, I have tested their effects on tombusvirus replication. We demonstrated that these sterolsynthesis inhibitors reduced virus replication in tobacco protoplasts. Virus replication is resumed to the wild type level by providing phytosterols in tobacco protoplasts confirming the role of sterols in RNA virus replication in tobacco. We have also identified INO2, a transcription factor for many phospholipid biosynthetic genes, reduces virus replication in its deletion background. When we provided this gene product in the mutant background, viral replication was back to normal, confirming the role of Ino2p in tombusvirus replication. Further biochemical assays showed that the viral inhibition is because of alteration in the formation of the viral replicase complex. Using confocal microscopy, we showed that the viral replication protein, termed p33, is forming large and few punctate structures rather than the small and many by overexpressing Ino2p in the wild type yeast cells. Over-expression of Opi1, an inhibitor of Ino2p led to greatly reduced viral replication, further supporting the roles of the phospholipid pathway in tombusvirus replication. One of the phospholipid, which is regulated by this pathway, is cardiolipin an important component of the mitochondrial as well as peroxisomal membranes. We further characterized how cardiolipin is playing an important role for tombusvirus replication by using different biochemical approaches.

Un paradigme actuel en virologie est que les virus de plantes ne peuvent pas affecter les animaux vertébrés et ne sont pas pathogènes pour l'homme. Cependant, plusieurs éléments remettent en question ce paradigme. L'objectif de cette Thèse a été d'étudier si les tobamovirus peuvent être pathogènes chez l'homme. Ce manuscrit comprend :1) une revue de la littérature portant sur la présence et l'impact potentiel des virus de plantes chez les animaux dont l'homme. 2) une étude sur l'exposition humaine aux tobamovirus et les effets possibles Dans la région de Marseille, nous avons montré la présence du PMMoV dans 7.2% des selles de patients et dans 57 % des produits alimentaires à base de piment. De plus, la présence du PMMoV dans les selles de patients a pu être corrélée des signes cliniques. De plus, nous avons montré que le TMV infectieux était présent dans le tabac de toutes les cigarettes testées mais aussi dans 45 % des salives de fumeurs testées. 3) une étude in vivo. Suite à une inoculation intra-trachéal des souris avec du TMV, nous avons constaté que le virus persiste jusqu'à 14 jours dans le tissu pulmonaire et les macrophages pulmonaires. De plus, nous avons détecté du TMV dans le cytoplasme des macrophages murins jusqu'à 15 jours après inoculation. 4) une étude sur le mode de transmission du TMV à l'homme. Le TMV détecté dans la salive des fumeurs ne serait pas véhiculé par la fumée de cigarette, mais par contact avec du tabac infecté. Nos résultats suggèrent que la frontière entre virus de plantes et virus d'animaux n'est pas aussi stricte qu'il est communément admis et incitent à réévaluer l'éventuelle pathogénicité des phytovirus pour l'homme. / A current paradigm in virology is that plant viruses cannot affect vertebrates and are not pathogenic for humans. However, several recent findings challenge this paradigm. The aim of this thesis was to investigate whether tobamoviruses may be pathogenic in humans. This manuscript contains: 1) A review of the literature of the reasons why plant viruses may cross the border from plants to vertebrates and the possible impact of plant viruses in animals including humans. 2) A study on exposure to tobamovirus and possible effects of these viruses for humans. In the Marseille geographical area, we showed the presence PMMoV in 7.2% of stools from patients tested and in 57% of food products containing hot peppers. In addition, the presence of PMMoV in the stools of patients could be significantly correlated with clinical symptoms. Then, we showed that infectious TMV was present in the tobacco of all cigarettes tested and TMV RNA was detected in 45% of smokers' saliva tested. 3) In vivo study: mice intra-tracheal inoculation with TMV. The results showed the persistence until 14 days of viruses in the lung tissue and in lung alveolar macrophages of mice. In addition, we detected TMV in the cytoplasm of murine macrophages up to 15 days after inoculation. 4) A study of TMV transmission to humans through cigarette smoking. TMV does not seem to be vehicled by cigarette smoke to smoker's saliva but by direct contact with infected tobacco. Our results suggest that the boundary between plant viruses and animal viruses is not as strict as it is commonly accepted and prompt to reassess the potential pathogenicity of these plant viruses for humans.

O mosaico rugoso, causado pelo begomovirus ToSRV, é uma das principais doenças do tomateiro. Neste trabalho avaliou-se a eficácia de quatro inseticidas, ciantranilliprole foliar, ciantraniliprole solo, espiromesifeno e tiametoxam no controle das infecções primária e secundária do ToSRV, em tomateiros, transmitido por Bemisia tabaci biótipo B. Os tratamentos, confinados separadamente em gaiolas a prova de insetos, foram: controle, representado por tomateiros sadios e infectados, pulverizados com água, mais insetos avirulíferos; infecção primária, simulada com tomateiros sadios pulverizados com inseticida, mais insetos virulíferos e infecção secundária, simulada com tomateiros sadios e infectados com o ToSRV, pulverizados com inseticida, mais insetos avirulíferos. Nenhum inseticida foi eficiente no controle da infecção primária. No caso da simulação da infecção secundária, 4% e 16% respectivamente, dos tomateiros tratados com os inseticidas ciantraniliprole solo e ciantraniliprole foliar foram infectados, contra 84% e 74% de tomateiros infectados nos respectivos controles. Para os tratamentos com os inseticidas tiametoxam e espiromesifeno, na simulação da infecção secundária, 58% e 62% dos tomateiros foram infectados, respectivamente, contra 66% e 74% de plantas infectadas nos respectivos controles. O uso racional de inseticidas que reduzem a infecção secundária associado com a eliminação de fontes externas de inóculo poderá contribuir para o manejo da doença. / The severe mosaic, caused by ToSRV begomovirus, is a major disease of tomato. This study evaluated the efficacy of four insecticides, sprayed cyazypyr, drench cyazypyr, sprayed spiromesifen and thiamethoxam on controlling primary and secondary infections by ToSRV to tomato plants, transmitted by Bemisia tabaci biotype B. Treatments were confined separately in proof insects cages, which were: control, represented by healthy and infected tomato plants sprayed with water and aviruliferous insects releasing; primary infection, simulated by healthy tomato plants with insecticide spraying and viruliferous insects releasing, and secondary infection, simulated with healthy tomato plants and ToSRV infected tomato plants, sprayed with insecticide, and aviruliferous insects releasing. None of the insecticides was effective in controlling primary infection. In the case of secondary infection simulation, 4% and 16% of the tomato plants treated with soil and foliar cyazypyr insecticides, respectively, were infected; against 84% and 74% of infected tomato plants in their respective controls. For treatments with thiamethoxam and spiromesifen insecticides spraying, in secondary infection simulation, 58% and 62% of tomato plants were infected, respectively, versus 66% and 74% of infected plants in their respective controls. The rational use of insecticides to reduce secondary infection associated with elimination of external sources of inoculum may contribute to disease management.

As cucurbitáceas no Brasil podem ser infectadas por diferentes vírus, tais como o Papaya ringspot virus - type W (PRSV-W); o Zucchini yellow mosaic virus (ZYMV) e o Cucumber mosaic virus (CMV). Os dois primeiros pertencem ao gênero Potyvirus e no geral ocorrem com maior freqüência do que o CMV, que é uma espécie do gênero Cucumovirus. Os dois potyvirus e o cucumovirus são transmitidos por afídeos de maneira não persistente. O principal objetivo desse trabalho foi o de obter subsídios que possam explicar a menor incidência do CMV em espécies de cucurbitáceas, estudando: (a) a interferência dos potyvirus PRSV-W e ZYMV na transmissão do CMV por Aphis gossypii e Myzus persicae para plantas de abobrinha de moita (Cucurbita pepo ‘Caserta’) e (b) o efeito de isolados do CMV provenientes de maracujazeiro (Passiflora edulis f. flavicarpa), de pimentão (Capsicum annuum), de pepineiro (Cucumis sativus), de meloeiro (Cucumis melo) e de trapoeraba (Commelina virginica) na infectividade de plantas de abobrinha de moita por meio da transmissão por afídeos. Para avaliar a possível interferência dos potyvirus na transmissão do CMV, as plantas de abobrinha de moita foram inoculadas com afídeos que adquiriram cada um dos vírus isoladamente; o CMV simultaneamente com cada um dos potyvirus; um dos potyvirus seguido pelo CMV e vice-versa. Os resultados mostraram, na maioria das vezes, que a transmissão dos vírus isoladamente foi mais eficiente do que em mistura, tanto através de aquisição simultânea como seqüencial. Os potyvirus no geral foram mais eficientemente transmitidos por ambas espécies de afídeos. Quando em mistura (aquisição simultânea ou sequencial), de uma maneira geral, houve uma redução na taxa de transmissão do CMV e do potyvirus presente na mistura. As avaliações sobre o efeito da origem dos isolados do CMV na infectividade de abobrinha de moita mostraram que apenas o isolado de pimentão não infectou plantas de abobrinha de moita quando transmitido por meio dos afídeos A. gossypii e M. persicae. Também não houve infecção quando inoculado mecanicamente. Os demais isolados infectaram abobrinha de moita através da transmissão por ambas espécies de afídeos. Análise da proteína capsidial dos diferentes isolados do CMV indicaram que todas apresentaram a mesma mobilidade em gel de SDS-PAGE. A origem do isolado o CMV, a eficiência da espécie de afídeo na sua transmissão e a interferência dos potyvirus PRSV-W e ZYMV podem explicar em parte a menor incidência desse cucumovirus em cucurbitáceas no país. / The cucurbits in Brazil can be infected by different viruses, such as Papaya ringspot virus - type W (PRSV-W); Zucchini yellow mosaic virus (ZYMV) and Cucumber mosaic virus (CMV). The first two belong to the genus Potyvirus and in general they occur more frequently than CMV, which is a species of the genus Cucumovirus. The two potyviruses and the cucumovirus are transmitted by means of aphids in a non persistent way. The main objective of this work was to obtain subsidies that can explain the lower incidence of CMV in cucurbit species, studying: (a) the interference of the potyviruses PRSV-W and ZYMV in the transmission of CMV by means of Aphis gossypii and Myzus persicae to zucchini squash plants (Cucurbita pepo 'Caserta') and (b) the effect of isolates of CMV from passion flower (Passiflora edulis f. flavicarpa), bell pepper (Capsicum annuum), cucumber (Cucumis sativus), melon (Cucumis melo) and Commelina virginica in the infectividade of zucchini squash plants through the transmission by aphids. To evaluate the possible interference of the potyvirus in the transmission of CMV, zucchini squash plants were inoculated with aphids that acquired each one of the viruses separately; CMV simultaneously with each one of the potyvirus; one of the potyvirus follow by CMV and vice-versa. The results showed that the transmission of PRSV-W, ZYMV and CMV separately was more efficient than in mixture. The potyviruses in general were more efficiently transmitted by both species of aphids than CMV. When in mixture (simultaneous or sequential acquisition), there was a reduction in the rate of transmission of CMV as well as that of the potyvirus present in the mixture. The evaluation on the effect of the origin of the isolate of CMV in the infectivity of zucchini squash showed that only the isolate from bell pepper did not infected the plants when inoculated by means of A. gossypii and M. persicae. This isolate also did not infecte zucchini squash when inoculated mechanically. The others isolate infected zucchini squash when transmitted by both species of aphids. Analysis of the capsidial protein of the different isolates of CMV indicated that all presented the same mobility in SDS-PAGE. The origin of the isolate of CMV, the efficiency of the species of aphid and the interference of the potyviruses PRSV-W and ZYMV on its transmission can partly explain the lower incidence of this cucumovirus in cucurbits species in Brazil.