Thesis (Ph. D.)--Swedish University of Agricultural Sciences, 2001. / Includes bibliographical references.

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.

Neste trabalho foram comparados os valores de absorbância dos extratos, severidade dos sintomas e biomassa das plantas de abobrinha-de-moita (Cucurbita pepo L) cv. Caserta, de abóbora (C. moschata Duch.) cv. Menina Brasileira e de melancia (Citrullus lanatus (Thunb.) Matsum. & Nakai) cv. Crimson Sweet infectadas com três estirpes fracas (PRSV-W-1 J PRSV-W-2 e PRSV-W-3) e uma estirpe severa (PRSV-W-C) do Papaya ringspot virus - type W (PRSV-W). As plantas-teste foram inoculadas em estádio cotiledonar e mantidas em condições de casa de vegetação. Aos 7, 14, 21, 28 e 35 dias após a inoculação (DAI), amostras de discos foliares foram coletadas das plantas, maceradas em tampão PBS, na proporção de 1:20 (p:v) e armazenadas a -20°C. Testes preliminares comparando as técnicas PTA-ELlSA ("Plate Trapped Antigen"-"Enzyme Linked Immunosorbent Assay") e DAS-ELISA ("Double Antibody Sandwich"- ELISA), realizados com diferentes concentrações do PRSV-W purificado, entre 32 e 4096 ng mL-1, mostraram alta correlação entre os valores de absorbância a 405 nm obtidos e as concentrações do vírus para os dois testes. Diante desse resultado, o PTA- ELISA foi adotado nos experimentos de estimativa da concentração viral. A severidade dos sintomas foi avaliada por uma escala dê notas de 1 (sem mosaico) a 5 (mosaico com bolhas, deformações foliares intensas e atrofiamento da planta). A biomassa da parte aérea das plantas foi avaliada através da pesagem das massas fresca e seca aos 40 DAI. Os resultados obtidos mostraram que as concentrações das estirpes fracas, baseadas nos valores de absorbância do PTA-ELlSA, foram menores do que a observada com a estirpe severa nas três espécies estudadas. Houve também uma variação nos picos de concentração virais das estirpes fracas e severa entre os dois experimentos realizados. As estirpes fracas não causaram sintomas de mosaico nas plantas-teste das três espécies estudadas (nota 1). A estirpe PRSV-W-C causou sintomas extremamente severos nas plantas de abobrinha-de-moita e melancia (nota 5) e sintomas relativamente fracos de mosaico nas plantas de abóbora 'Menina Brasileira' (nota 2). Os valores de biomassa das plantas de abobrinha-de-moita e melancia infectadas pelas estirpes fracas sofreram reduções que variaram de 1,7 % a 12,4 %, quando comparados aos das plantas sadias. Já os valores de biomassa das plantas infectadas pela estirpe severa sofreram reduções mais drásticas, variando de 29 % a 74 %. Os valores de biomassa das plantas de abóbora 'Menina Brasileira' infectadas com as estirpes fracas e severa foram ligeiramente superiores aos das plantas sadias. / not available

Neste trabalho foram comparados os valores de absorbância dos extratos, severidade dos sintomas e biomassa das plantas de abobrinha-de-moita (Cucurbita pepo L) cv. Caserta, de abóbora (C. moschata Duch.) cv. Menina Brasileira e de melancia (Citrullus lanatus (Thunb.) Matsum. & Nakai) cv. Crimson Sweet infectadas com três estirpes fracas (PRSV-W-1 J PRSV-W-2 e PRSV-W-3) e uma estirpe severa (PRSV-W-C) do Papaya ringspot virus - type W (PRSV-W). As plantas-teste foram inoculadas em estádio cotiledonar e mantidas em condições de casa de vegetação. Aos 7, 14, 21, 28 e 35 dias após a inoculação (DAI), amostras de discos foliares foram coletadas das plantas, maceradas em tampão PBS, na proporção de 1:20 (p:v) e armazenadas a -20°C. Testes preliminares comparando as técnicas PTA-ELlSA ("Plate Trapped Antigen"-"Enzyme Linked Immunosorbent Assay") e DAS-ELISA ("Double Antibody Sandwich"- ELISA), realizados com diferentes concentrações do PRSV-W purificado, entre 32 e 4096 ng mL-1, mostraram alta correlação entre os valores de absorbância a 405 nm obtidos e as concentrações do vírus para os dois testes. Diante desse resultado, o PTA- ELISA foi adotado nos experimentos de estimativa da concentração viral. A severidade dos sintomas foi avaliada por uma escala dê notas de 1 (sem mosaico) a 5 (mosaico com bolhas, deformações foliares intensas e atrofiamento da planta). A biomassa da parte aérea das plantas foi avaliada através da pesagem das massas fresca e seca aos 40 DAI. Os resultados obtidos mostraram que as concentrações das estirpes fracas, baseadas nos valores de absorbância do PTA-ELlSA, foram menores do que a observada com a estirpe severa nas três espécies estudadas. Houve também uma variação nos picos de concentração virais das estirpes fracas e severa entre os dois experimentos realizados. As estirpes fracas não causaram sintomas de mosaico nas plantas-teste das três espécies estudadas (nota 1). A estirpe PRSV-W-C causou sintomas extremamente severos nas plantas de abobrinha-de-moita e melancia (nota 5) e sintomas relativamente fracos de mosaico nas plantas de abóbora 'Menina Brasileira' (nota 2). Os valores de biomassa das plantas de abobrinha-de-moita e melancia infectadas pelas estirpes fracas sofreram reduções que variaram de 1,7 % a 12,4 %, quando comparados aos das plantas sadias. Já os valores de biomassa das plantas infectadas pela estirpe severa sofreram reduções mais drásticas, variando de 29 % a 74 %. Os valores de biomassa das plantas de abóbora 'Menina Brasileira' infectadas com as estirpes fracas e severa foram ligeiramente superiores aos das plantas sadias. / not available

Viruses are ubiquitous in natural growth environments and cause severe losses to crop yields, globally. Approximately 30% of plant viruses described to date are grouped within the family Potyviridae, making it one of the largest plant virus families. Furthermore, certain potyvirus species can cause devastating diseases in several agriculturally and economically important crops. Hence, gaining insight into potyvirus resistance and recovery mechanisms in plants is an important research focus. This thesis firstly explores how environmental cues can modulate the activity of a central form of viral defence, namely RNA silencing. Specifically, high temperatures and low light intensities were found to increase the efficacy of viral RNA silencing in Arabidopsis, resulting in recovery from infection by Turnip Mosaic Virus. The biological context and potential for agricultural exploitation of these phenomena are discussed. Secondly, this thesis explores the ability to engineer resistance alleles using the latest genome editing techniques. Specifically, resistance to Turnip Mosaic Virus was successfully engineered in Arabidopsis by CRISPR/Cas9-induced deletion of a known susceptibility factor eIF(iso)4E. Biotechnological methods to implement this proof of concept research in crop species were also investigated.

In the U.S., soybean mosaic virus (SMV) is classified into seven strain groups, designated G1 to G7, based on their different responses on resistant soybean [Glycine max (L.) Merr.] cultivars. These responses are: symptomless or resistant (R), necrotic (N), and mosaic or susceptible (S). The gene-for-gene model has been proposed for SMV-soybean interactions. In the majority of cultivars, a single dominant gene, Rsv1, confers both the R and N responses. In the first part of this study, the coat protein (CP) genes of two SMV strains, G1 and G6 were isolated, cloned, and sequenced. Gene isolation was done by reverse transcription-polymerase chain reaction (RT-PCR) on partially purified virus preparation without prior RNA extraction. Amplified products were blunt-end ligated into pNoTA/T7 vector and transformed into competent cells. Sequencing was performed in both directions on heat-denatured double-stranded plasmids. The predicted 265 amino acid sequence of the CP of G1 and G6 strains were 98.9% identical, with only two amino acid differences. Correlating the CP sequences of G1, G2, G6, and G7, with their virulence on resistant soybean cultivars indicated that the CP is not likely to be the R- and/or N-determinant in the SMV-soybean system. The second part of the study involved studying the pathogenesis of G1, G6, and G7 strains on inoculated leaves of R, N, and S soybean cultivars by leaf imprint immunoassay. Results indicated four types of reactions: i) susceptible, showing unrestricted replication and spread; ii) immune, where no virus was detected; iii) systemic spread, showing unrestricted replication but limited spread along the veins; and iv) restricted replication and spread, where infection was restricted to few foci along the veins. Results of this study indicated that Rsv1-mediated resistance is a multicomponent type of resistance that involves both inhibition of virus replication as well as cell-to-cell movement. The third part of the study aimed at investigating Rsv1-mediated resistance at the cellular level. For this purpose, an SMV-soybean protoplast system was developed. Protoplast isolation was based on a combined cellulase-pectolyase Y-23 digestion and metrizamide-sorbitol gradient purification protocol. Virus inoculation of protoplasts was facilitated by either polyethelene glycol (PEG) or poly-L-ornithine (PLO), and method of detection was by Western blotting using antiserum to whole virus. Inoculation by PEG was successful, but results were irreproducible because of the adverse effect of PEG on protoplast viability. Inoculation by PLO was inconclusive because of the high background from residual inoculum. Additional research is needed before a protoplast system can be used to study the mechanism of Rsv1 resistance to SMV at the cellular level. / Ph. D.

Malgré l’importance agronomique des cucurbitacées au Venezuela et le fort impact des maladies virales sur la production, le pathosystème viral y a été peu étudié. Cinq virus ont été décrits par des travaux souvent anciens: le cucumovirus Cucumber mosaic virus (CMV), les potyvirus Papaya ringspot virus (PRSV), Zucchini yellow mosaic virus (ZYMV) et Watermelon mosaic virus (WMV), le comovirus Squash mosaic virus (SqMV), et un begomovirus partiellement caractérisé, le Melon chlorotic mosaic virus (MeCMV). Pour lutter contre ces agents pathogènes, il est nécessaire de bien connaître le pathosystème viral présent localement. Nous avons donc caractérisé les principaux virus provoquant des maladies sur ces cultures dans le pays, en vue de comprendre l’évolution du pathosystème et de développer des méthodes de lutte adaptées. Nos études épidémiologiques ont montré que le begomovirus MeCMV représente la principale menace sur melon et pastèque, les potyvirus ZYMV et PRSV étant la principale menace sur courge. Les isolats Vénézuéliens de ZYMV se sont révélés génétiquement homogènes et biologiquement très variables comme cela a été observé dans d'autres régions du monde. La résistance au ZYMV conférée par le gène Zym chez le melon PI 414723 est surmontée par certains isolats, alors que le concombre TGM représente une source stable de résistance au ZYMV. Les types W et P de PRSV sont présents au Venezuela, mais seul le PRSV-W été trouvé sur cucurbitacées cultivées et sauvages. Une autre souche virale, initialement appelée PRSV-T et détectée au Venezuela, constitue une espèce différente du PRSV d’après ses propriétés moléculaires et biologiques établies dans ce travail. / In Venezuela, cucurbits viruses are among de major constraints for cucurbit production. Five viruses have been described infecting cucurbits in the country: Cucumber mosaic virus (CMV, Cucumovirus), Papaya ringspot virus (PRSV, Potyvirus), Zucchini yellow mosaic virus (ZYMV, Potyvirus), Squash mosaic virus (SqMV, Comovirus), and Melon chlorotic mosaic virus (MeCMV, Begomovirus).The current frequency and impact of these viruses is Venezuela is not well known. In this work, the major cucurbit viruses were identified and characterized in order to estimate the viral pathosystem affecting cucurbit production in the country. The begomovirus MeCMV appears to be the major constraint for melon and watermelon production, while the potyviruses ZYMV and PRSV were the most important viruses infecting squash crops in this survey. Molecular characterisation of ZYMV isolates revealed a low genetic diversity of this virus in Venezuela. In contrast, ZYMV isolates were biologically variable as observed in several countries worldwide. Two types of PRSV, P and W, are present in Venezuela. PRSV-W is the only type naturally infecting cucurbits in Venezuela. Another type of PRSV, formerly referred as PRSV-T, was detected. Its molecular and biological characterisation revealed that it is indeed a new species related to but distinct from PRSV. Therefore, the name zucchini tigré mosaic virus (ZTMV) is proposed for this virus.

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.

Johnsongrass mosaic virus (JGMV) é uma espécie do gênero Potyvirus. A sua distribuição geográfica, até o início da década de 1990, estava limitada à Austrália e aos Estados Unidos, onde causa doença em sorgo, milho e várias gramíneas. Em 2001, o JGMV foi detectado pela primeira vez no Brasil em amostras de híbridos e variedades de milho provenientes da região de Ribeirão Preto, SP mediante análise sorológica (DAS-ELISA), e em 2013 foi detectado mediante RT-PCR em amostras de Pennisetum purpureum provenientes do Estado da Bahia. Em Fevereiro de 2012 a Clínica Fitopatológica da ESALQ/USP recebeu amostras de Panicum maximum cv. Mombaça, com sintomas de mosaico, de São Luiz do Paraitinga, SP. Exames preliminares de contrastação negativa em microscópio eletrônico de transmissão indicaram a presença de partículas virais características de potyvirus. Diante disso, o principal objetivo deste trabalho foi caracterizar o agente etiológico associado às plantas doentes de capim Mombaça mediante testes biológicos, sorológicos e moleculares. Extratos foliares de plantas sintomáticas de capim Mombaça foram inoculados mecanicamente em 69 genótipos da família Poaceae. As avaliações foram feitas com base nos sintomas e por PTA-ELISA usando antissoro policlonal contra a proteína capsidial do potyvirus produzido nesse trabalho, após purificação do isolado viral. As espécies susceptíveis foram Brachiaria brizantha, B. decumbens, B. plantaginea, Cenchrus echinatus, Echinochloa colona, E. crus-galli, E. cruspavonis, Melinis minutiflora, Panicum maximum cv. Colonião, Pennisetum setosum, Rhynchelytrum repens, Rottboellia exaltata, Sorghum bicolor BRS 332, S. bicolor BRS 509, S. bicolor x S. sudanense BRS 802 e S. verticilliflorum. Espécies cultivadas como arroz, aveia, cana-de-açúcar, centeio, milho e trigo não foram infectadas com esse isolado. O peso molecular da proteína capsidial deste potyvirus foi estimado em cerca de 33 kDa por meio de Western blot. Sequência de nucleotídeos do genoma completo (9.885 nt) obtida neste estudo revelou identidade de 82,03% com a única sequência completa do genoma de um isolado do JGMV da Austrália, depositada no GenBank. A partir dessa sequência foram obtidos oligonucleotídeos iniciadores específicos para a detecção do isolado de SP do JGMV mediante RT-PCR. / Johnsongrass mosaic virus (JGMV) is a species of the genus Potyvirus. The geographical distribution, until the early 1990s, was limited to Australia and the United States, where it causes disease in sorghum, corn and various grasses. In 2001, JGMV was first detected in Brazil in samples of hybrids and varieties of corn from the region of Ribeirão Preto, São Paulo State by serological analysis (DASELISA), and in 2013 it was detected by RT-PCR in samples of Pennisetum purpureum from the State of Bahia. In February 2012, the Disease Diagnostic Clinic ESALQ/USP received samples of Panicum maximum cv. Mombaça, exhibiting mosaic symptoms, from the region of São Luiz do Paraitinga, SP. Preliminary examination of negatively stained sap in a transmission electron microscope indicated the presence of potyvirus-like particles. Therefore, the main objective of this study was to characterize the etiologic agent associated with P. maximum cv. Mombaça diseased plants by biological, serological and molecular tests. Leaf extract from Mombaça infected plants was mechanically inoculated in 69 genotypes of the Poaceae family. Evaluations were done based on symptoms expression and PTAELISA using polyclonal antiserum against the capsid protein of the potyvirus produced in the preset work virus purification. Susceptible species were Brachiaria brizantha, B. decumbens, B. plantaginea, Cenchrus echinatus, Echinochloa colona, E. crus-galli, E. crus-pavonis, Melinis minutiflora, Panicum maximum cv. Colonião, Pennisetum setosum, Rhynchelytrum repens, Rottboellia exaltata, Sorghum bicolor BRS 332, S. bicolor BRS 509, S. bicolor x S. sudanense BRS 802 and S. verticilliflorum. Cultivated species such as rice, oats, sugarcane, rye, corn and wheat were not infected with this isolate. The molecular weight of the coat protein of this potyvirus was estimated at about 33 kDa by Western blot. The nucleotide sequence of the complete genome (9885 nt) obtained in this study showed 82.03% identity with an unique sequence for the complete genome of an isolate of JGMV from Australia, deposited in GenBank. From this nucleotide sequence, specific pair of primers was designed for the detection of the São Paulo isolate of JGMV by RT-PCR.

Uma das espécies que atualmente vem despertando interesse econômico por seu elevado valor de mercado é o maracuzajeiro doce (Passiflora alata Curtis). Entretanto, a cultura é afetada por diferentes doenças que prejudicam a produtividade e a qualidade dos frutos, sendo a doença causada pelo Cowpea aphid-borne mosaic virus (CABMV) a que mais afeta a cultura do maracujazeiro no Brasil. O presente trabalho teve como objetivo a obtenção de plantas transgênicas de P. alata visando resistência ao CABMV. O processo de transformação genética utilizado foi via Agrobacterium tumefaciens, estirpe EHA105, contendo o cassete de expressão com um fragmento do gene da proteína capsidial do CABMV, numa construção tipo hairpin e o gene de seleção nptII que confere resistência ao antibiótico canamicina. Para os experimentos de transformação genética foram utilizados como explantes segmentos de hipocótilo e segmentos internodais. Após 2 a 3 dias de co-cultivo em meio de cultura MS (MURASHIGE; SKOOG, 1962) contendo acetosseringona (100 mM), os explantes foram transferidos para meio de cultura de seleção e regeneração constituído de sais minerais e vitaminas de MS, suplementado com benzilaminopurina (BAP - 1mg/L) + thidiazuron (TDZ - 0,5 mg/L) + canamicina (100 mg/L) + cefotaxima (500 mg/L) + nitrato de prata (4,0 mg/L), pH 5,8. Após 4 a 6 semanas de incubação, determinou-se o número de explantes responsivos e as gemas adventícias desenvolvidas foram transferidas para meio de cultura de alongamento MSM + GA3 (1,0 mg/L) + cefotaxima (500 mg/L) + nitrato de prata (4,0 mg/L). As plantas desenvolvidas foram aclimatizadas e analisadas por PCR, utilizando primers específicos para a detecção do fragmento do gene da proteína capsidial do CABMV e do gene de seleção (nptII). Foram identificadas 47 plantas transgênicas PCR positivas para do gene nptII. Até o momento, a integração do gene nptII foi confirmada por Southern blot em 9 plantas / One species that is currently attracting interest due to its high economic value is the sweet passionflower (Passiflora alata Curtis). However, the culture is affected by different diseases that harm the productivity and fruit quality. The disease caused by Cowpea aphid-borne mosaic virus (CABMV) is the one that more affect the culture of passionflower in Brazil. This work aimed to obtain transgenic plants of P. alata resistant to the CABMV. The genetic transformation process was via Agrobacterium tumefaciens, strain EHA105, containing the expression cassette with a fragment of the coat protein gene of CABMV, in a hairpin construct and the selection gene nptII, which confers resistance to the antibiotic kanamycin. In the experiments of genetic transformation hypocotyl segments and internodal segments were used as explants. After 2-3 days of co-cultivation in MS medium (MURASHIGE; SKOOG, 1962) containing acetosyringone (100 mM), the explants were transferred to the selection and regeneration culture medium consisting of mineral salts and vitamins of MS medium supplemented with benzylaminopurine (BAP - 1 mg/L) + thidiazuron (TDZ - 0.5 mg/L) + kanamycin (100 mg/L) + cefotaxime (500 mg/L) + silver nitrate (4.0 mg /L), pH 5.8. After 4-6 weeks of incubation, it was determined the number of responsive explants. Shoots developed were transferred to elongating culture medium MSM + GA3 (1.0 mg/L) + cefotaxime (500 mg/L) + nitrate silver (4.0 mg/L). The developed plants were acclimatized and analyzed by PCR using specific primers to detect the fragment of CABMV and the selection gene (nptII). It was identified 47 transgenic plants PCR positive for the gene nptII. Until this moment, the integration of the nptII gene was confirmed by Southern blot in 9 plants