Caracterización de un geminivirus que afecta al cultivo de soja

El cultivo de soja es uno de los más importantes en nuestro país y las exportaciones de soja han llegado a aportar un monto superior al 50% de los ingresos por exportaciones que genera el sector agrícola, lo que se traduce en una entrada de divisas al país superior a los 2.500 millones de dólares anuales. Entre el 8 y 10% de la producción de este cultivo puede llegar a perderse debido a distintas enfermedades, entre las que se incluyen los virus. Durante las últimas campañas agrícolas, se detectaron en lotes de producción de soja de plantas con síntomas de enanismo, mosaico, severo ampollado y deformación de hojas. Se pudo comprobar que los mismos eran producidos por un virus transmitido por moscas blancas y pertenecientes al grupo de Geminivirus. Objetivos generales: * Aislamiento y caracterización biológica y molecular del geminivirus que afecta la soja. * Evaluación de la incidencia del virus en cultivos de soja y de su efecto sobre el rendimiento, estudios de la epidemiología y evaluación del comportamiento de cultivares y líneas precomerciales de interés frente a infecciones naturales. Objetivos específicos: * Evaluación de la incidencia del virus en cultivos de soja. * Caracterización biológica a través de Rango de hospedantes. * Caracterización de la partícula viral: purificación del virus, determinación de la forma y tamaño de la partícula, del peso molecular de la cápside proteica, aislamiento y caracterización del ácido nucleico. * Identificación de malezas y especies cultivadas o silvestres que puedan actuar como reservorios del mismo. * Evaluar el comportamiento de cultivares y líneas precomerciales de interés frente a las infecciones virales.

Estudio del patosistema geminivirus-moscas blancas, Bemisia tabaci (Hemiptera Aleyrodidae) en cultivos de soja. Study of the geminivirus-whiteflies Bemisia tabaci (Hemiptera Aleyrodidae) pathosystem in soybean crops.

La soja es la especie oleaginosa de mayor importancia y consumo en el mundo. Su cultivo se efectúa en aproximadamente 50 países, aunque el 90 por ciento de la producción está concentrada en: Estados Unidos (40 por ciento), Brasil (24 por ciento), Argentina (18 por ciento) y China (8 por ciento). En nuestro país durante la campaña agrícola 2006/07 se alcanzaron 15.981.264 ha y 47.482.784 t. Actualmente, ocupa una amplia zona ecológica, desde los 23º a los 39º de latitud sur y la región pampeana, con cerca del 90 por ciento de la superficie sembrada en las provincias de Santa Fe (28.5 por ciento), Córdoba (28 por ciento) y Buenos Aires (23 por ciento) es la principal productora. Entre los factores que limitan la producción de este cultivo se encuentra el patosistema begomovirus - mosca blanca (Bemisia tabaci Gennadius), que causa pérdidas económicas, al reducir los rendimientos y calidad de los productos obtenidos y aumentar los costos de producción por el uso intensivo de insecticidas. La continua aplicación de agroquímicos para el control del insecto conlleva la selección de individuos resistentes en la población, que puede verse acelerada si se tiene en cuenta que, durante el ciclo de cultivo, se desarrollan varias generaciones. Además, formas inmaduras y adultos se alojan en el envés de las hojas y la acción de los insecticidas es menos eficaz. Ante esto, los agricultores aumentan el número y frecuencia de las aplicaciones, con lo que se incrementa la presión de selección en las poblaciones, favoreciendo el surgimiento de estirpes resistentes. El control químico resulta antieconómico y genera un severo peligro ambiental, con perjuicios para la salud humana y animal. Por lo tanto, resulta imprescindible la búsqueda de formas alternativas de control de la plaga y de los virus que transmite, a través del manejo integrado sustentable, dentro del cual se destacan la resistencia de la planta hospedante (HPR) y el empleo de métodos culturales. Con respecto a los geminivirus, como primer paso en la búsqueda de resistencia, es de gran importancia identificar las especies y/o razas que se encuentran infectando al cultivo de soja. Además, se presupone la existencia de fuentes de resistencia a moscas blancas en germoplasma de soja y, por ende, a los begomovirus que transmiten y que es posible la caracterización de los mecanismos que confieren dicha resistencia. En base a la problemática expuesta se proponen los siguientes objetivos de trabajo:- Aislar y caracterizar molecularmente a las nuevas especies y/o razas de begomovirus detectadas en cultivos de soja y ajustar las técnicas que permitan su identificación.- Detectar y caracterizar los mecanismos de resistencia, antixenosis, antibiosis o ambas, frente a la mosca blanca B. tabaci en distintos cultivares de soja. Para cumplimentar tales objetivos se efectuará el clonado y secuenciación del genoma completo de los diferentes begomovirus detectados; serán ajustadas técnicas de hibridación molecular con marcado no radioactivo y producidos clones infecciosos que, posteriormente, se emplearán para inocular distintas especies (rango de hospedantes). Por otro lado, se tratará de detectar y caracterizar fuentes de resistencia a B. tabaci en germoplasma de soja (cultivares comerciales y líneas en proceso avanzado de mejoramiento). Como primer paso se efectuará la cría de la mosca blanca bajo condiciones controladas.Se medirán variables indicadoras de antibiosis (en condiciones de no elección o alimentación forzada): número de huevos, ninfas y adultos, tasa de mortalidad, duración del ciclo biológico, longevidad y peso de insectos adultos. Complementariamente se determinará antixenosis (en condiciones de libre elección): índice de atracción y preferencia para la oviposición y su correlación con el número de tricomas foliares. Los resultados del proyecto serán de inmediata transferencia a fitotecnistas, semilleros y sector productivo en general, permitiendo la reducción de pérdidas significativas ocasionadas por begomovirus y por su vector.

Genetic control of characters associated with bean golden mosaic geminivirus resistance in Phaseolus vulgaris L.

Bean golden mosaic is the most important viral disease of the bean crop (Phaseolus vulgaris L.) in Latin America. The genetics of resistance to a Brazilian strain of bean golden mosaic virus (BGMV), was studied in a 4 x 4 diallel cross without reciprocals, among the parental genotypes DOR 303, EMGOPA 201 Ouro, Carnaval, and Redlands Greenleaf C. Seedlings of the four parents, six F1 hybrids, 12 backcrosses, and F2 generations for each combination were inoculated on the eighth day after sowing by exposure to a viruliferous whitefly (Bemisia tabaci Genn.) population for 24 h, in a glasshouse, prior to transplantation to field conditions. The full set of two parents, F1, F2 and respective backcrosses for each combination was considered to be a family. Data were recorded and analyzed for foliar yellowing, plant dwarfing, and pod malformation, using a randomized block design, with two replications. Weighted generation mean analysis was performed for each of the six families. An additive gene action model was significant for the three characteristics evaluated. On the other hand, non-additive gene action had greater absolute value in most cases. Resistance to foliar yellowing conferred by genes from DRO 303 was highly heritable and was expressed equally well in the different genetic backgrounds evaluated. Such resistance may be oligogenic. Broad- and narrow-sense heritabilities were relatively high for all response traits. The three traits studied were all positively correlated, indicating that they can be simultaneously selected for enhancement. The highest correlation coefficient was obtained for dwarfing x pod malformation.

Rep protein of tomato yellow leaf curl geminivirus has an ATPase activity required for viral DNA replication.

The Rep protein of geminiviruses is the sole viral protein required for their DNA replication. The amino acid sequence of Rep protein contains an NTP binding consensus motif (P-loop). Here we show that purified Rep protein of tomato yellow leaf curl virus expressed in Escherichia coli exhibits an ATPase activity in vitro. Amino acid exchanges in the P-loop sequence of Rep causes a substantial decrease or loss of the ATPase activity. In vivo, mutant viruses carrying these Rep mutations do not replicate in plant cells. These results show that ATP binding by the Rep protein of geminiviruses is required for its function in viral DNA replication.

Geminivirus Rep Protein Interferes with the Plant DNA Methylation Machinery and Modifies the Host Epigenome

The apparent simplicity of viruses hides the complexity of their interactions with their hosts. Viruses are masters at circumventing host defenses and manipulating the cellular environment for their own benefit. The replication of the largest known family of single-stranded DNA viruses, Geminiviridae, is impaired by DNA methylation and Arabidopsis mutants affected in cytosine methylation are hypersusceptible to geminivirus infection. This implies that plants might use methylation as a defense against geminiviruses and that the viral genome is a target for plant DNA methyltransferases. We have found a novel counter-defense strategy used by geminiviruses, that reduces the expression of the plant maintenance DNA methyltransferases, MET1 and CMT3, in both, locally and systemically infected tissues. Furthermore, we demonstrated that the virus-mediated repression of these two maintenance DNA methyltransferases is widely spread among different geminivirus species. Additionally, we identified Rep as the geminiviral protein responsible for the repression of MET1 and CMT3, and another viral protein, C4, as an ancillary player in MET1 downregulation. The presence of Rep, suppresses TGS of an Arabidopsis transgene and of host loci whose expression is strongly controlled by CG methylation. Bisulfite sequencing analyses showed that the expression of Rep caused a substantial reduction in the levels of DNA methylation at CG sites. Our findings suggest that Rep, the only viral protein essential for geminiviral replication, displays TGS suppressor activity through a mechanism distinct from the one thus far described for geminiviruses.

Characterization and Experimental Host Range of a Brazilian Tomato Isolate of Tomato severe rugose virus

We report the complete molecular characterization of the DNA-A and DNA-B of a Brazilian tomato isolate of Tomato severe rugose virus (ToSRV) and the experimental host range of the virus determined using white-fly transmission tests. Genome analysis showed that ToSRV has a close evolutionary relationship with Tomato rugose mosaic virus. Of 33 plants species inoculated with viruliferous Bemisia tabaci biotype B, 13 species were susceptible to ToSRV, nine asymptomatically. Therefore, ToSRV disease management strategy should include the control of infected weeds close to tomato fields.

Identification and partial characterization of a Carica papaya-infecting isolate of Alfalfa mosaic virus in Brazil

A Carica papaya plant with severe yellow leaf mosaic, leaf distortion, and systemic necrosis was found in the municipality of Piracicaba, state of So Paulo, Brazil. Transmission electron microscopy (TEM) analysis revealed the presence of potyvirus-like particles and bacilliform particles similar to those of the Alfamovirus genus. The potyvirus was identified as Papaya ringspot virus-type P (PRSV-P). Biological, serological, and molecular studies confirmed the bacilliform virus as an isolate of Alfalfa mosaic virus (AMV). Partial nucleotide and amino acid sequences of the coat protein gene of this AMV isolate shared 97-98% identity with the AMV isolates in the GenBank database. This report is the first of the natural infection of papaya plants by AMV.

Evaluation of the genetic variability of orchid fleck virus by single-strand conformational polymorphism analysis and nucleotide sequencing of a fragment from the nucleocapsid gene

The variability of a fragment of the nucleocapsid gene of orchid fleck virus (OFV) was investigated by single-strand conformational polymorphism (SSCP) analysis and nucleotide sequencing. Forty-eight samples of 18 genera of orchids were collected from Brazil, Costa Rica and Australia. The SSCP analysis yielded six different band patterns, and phylogenetic analysis based on the nucleotide fragment sequence obtained in this work and six available in GenBank showed two different groups, one with isolates 023Germany and So-Japan, and other with the rest of the isolates. None of the analyses showed geographic correlation among the Brazilian strains. The data obtained in this study showed a low genetic variation in this region of the genome; the d(N)/d(S) ratio of 0.251-0.405 demonstrated a negative selective pressure that maintains the stability of the analyzed fragments.

New species emergence via recombination among isolates of the Brazilian tomato infecting Begomovirus complex

Partial nucleotide sequences of five tomato infecting Begomovirus isolates were determined from DNA-A fragments, corresponding to the 5' region of the replication associated protein gene, the intergenic region and the 5' region of the coat protein gene. Isolate DFM shared 95% identity with Tomato mottle leaf curl virus (TMoLCV), isolates 34, PA-05, and Ta4 were 88% identical to Tomato yellow vein streak virus and isolate DF-BR3 shared 77% identity with TMoLCV. Recombination analysis indicated that isolate DF-BR3 was a chimaera, and it provided evidence that there is a complex and actively recombining population of tomato infecting begomoviruses in Brazil.

Characterization of Tomato yellow spot virus, a novel tomato-infecting begomovirus in Brazil

The objective of this work was the biological and molecular characterization of a begomovirus detected in São Joaquim de Bicas, Minas Gerais, Brazil, named TGV-[Bi2], by determining its host range, complete nucleotide sequence and phylogenetic relationships with other begomoviruses. Biological characterization consisted of a host range study using either sap inoculation or particle bombardment as inoculation methods. The yellow spot virus can infect plants in Solanaceae and Amaranthaceae, including economically importat crops as sweet pepper, and weeds as Datura stramonium and Nicotiana silvestris. For the molecular characterization, the full-length genome (DNA-A and DNA-B) was amplified, cloned and completely sequenced. Sequence comparisons and phylogenetic analyses indicated that TGV-[Bi2] constitutes a novel begomovirus species named Tomato yellow spot virus (ToYSV), closely related to Sida mottle virus (SiMoV).

Traditional and transgenic strategies for controlling tomato-infecting begomoviruses

Viruses of to the family Geminiviridae are considered some of the most important pathogens in tropical and subtropical regions of the world. Members of one Geminiviridae genus, Begomovirus, have been causing severe losses, particularly in tomato (Lycopersicon esculentum) production in the Americas and the Caribbean. Several new begomoviruses have been reported in the region and, at least one, Tomato yellow leaf curl virus (TYLCV), has been brought in from the Old World via infected transplants. In addition, the recombination events that are playing an important role in Begomovirus diversity have increased the complexity of their control. This scenario has led to the search for control measures that go beyond traditional host genetic resistance, chemical controls and cultural practices. In this review, besides the recommended classical control measures, transgenic approaches will be discussed, as well as the mechanisms involved in their successful control of viruses.

Genetic diversity of begomoviruses infecting soybean, bean and associated weeds in Northwestern Argentina

The subtropical Northwestern region of Argentina (provinces of Tucumán, Salta, Jujuy, Santiago del Estero and Catamarca) suffers from a high incidence of the whitefly Bemisia tabaci, and the detection of begomoviruses is also common. The Northwest is the main bean-growing region of the country, and approximately 10% of Argentina's soybean crop is grown in this area. We have used a PCR-based assay to establish the identity and genetic diversity of begomoviruses associated with bean and soybean crops in Northwestern Argentina. Universal begomovirus primers were used to direct the amplification of a fragment encompassing the 5' portion of the capsid protein gene. Amplified fragments were cloned, sequenced and subjected to phylogenetic analysis to determine the sequence identity to known begomoviruses. The data indicated the presence of four distinct begomoviruses, all related to other New World begomoviruses. The prevalent virus, which was present in 94% of bean and soybean samples and also in two weed species, is closely related to Sida mottle virus (SiMoV). A virus with high sequence identity with Bean golden mosaic virus (BGMV) was found in beans. The two remaining viruses displayed less than 89% identity with other known begomoviruses, indicating that they may constitute novel species. One of these putative novel viruses was detected in bean, soybean and tomato samples.

Further molecular characterization of weed-associated begomoviruses in Brazil with an emphasis on Sida spp

Begomoviruses are whitefly-transmitted, single-stranded DNA viruses that are often associated with weed plants. The aim of this study was to further characterize the diversity of begomoviruses infecting weeds (mostly Sida spp.) in Brazil. Total DNA was extracted from weed samples collected in Viçosa (Minas Gerais state) and in some municipalities of Alagoas state in 2009 and 2010. Viral genomes were amplified by RCA, cloned and sequenced. A total of 26 DNA-A clones were obtained. Sequence analysis indicated the presence of 10 begomoviruses. All viral isolates from Blainvillea rhomboidea belonged to the same species, Blainvillea yellow spot virus (BlYSV ), thereby suggesting that BlYSV may be the only begomovirus present in this weed species. Four isolates represent new species, for which the following names are proposed: Sida yellow blotch virus (SiYBV), Sida yellow net virus (SiYNV), Sida mottle Alagoas virus (SiMoAV) and Sida yellow mosaic Alagoas virus (SiYMAV). Recombination events were detected among the SiYBV isolates and in the SiYNV isolate. These results constitute further evidence of the high species diversity of begomoviruses in Sida spp. However, the role of this weed species as a source of begomoviruses infecting crop plants remains to be determined.

Adult attractiveness and oviposition preference of Bemisia tabaci (Genn.) (Homoptera: Aleyrodidae) B-biotype in cotton genotypes

Considerada importante praga do algodoeiro, a mosca-branca Bemisia tabaci biótipo B pode através de sua alimentação, diminuir o vigor das plantas, trasmitir vírus e prejudicar a qualidade da fibra. Visando avaliar a resistência de genótipos de algodoeiro, Gossypium hirsutum (L.), à mosca-branca Bemisia tabaci biótipo B, realizaram-se testes de atratividade e não-preferência para oviposição, com e sem chance de escolha, em telado, a temperatura ambiente. Verificou-se baixa atratividade das plantas dos genótipos Fabrika, CNPA Ita 90, Makina, Coodetec 407 e IAC 01-639 CPA 02-24 a adultos dessa mosca-branca, o que pode representar um componente de resistência destes materiais genéticos ao inseto. Os genótipos BRS Aroeira, Coodetec 406, Fabrika e Coodetec 401 apresentaram resistência do tipo não-preferência para oviposição, nos testes com e sem chance de escolha. Os números de tricomas e de glândulas de gossipol por cm² não foram adequados para se avaliar a não-preferência para oviposição de adultos da mosca-branca em genótipos de algodoeiro.

Evaluation of resistance to Tomato severe rugose virus (ToSRV) in Capsicum spp. genotypes

Tomato severe rugose virus (ToSRV) is the predominant species of begomovirus in São Paulo State, Brazil, and infects primarily tomato and pepper plants. There is no information about genetic resistance of pepper to this virus, so in this work the reaction of 29 genotypes of Capsicum spp. was evaluated by inoculation of two ToSRV isolates: ToSRV-Sk (isolated from a tomato plant) and ToSRV-PJU (isolated from a pepper plant). For both isolates, two C. annuun genotypes (Catarino Cascabel - México and Silver) showed no symptoms 30 days after inoculation (d.a.i). In a second experiment, these two genotypes were evaluated for 150 d.a.i and, again, no symptoms could be observed. However, the virus was detected by RCA-PCR, indicating that both genotypes are susceptible, but less affected by ToSRV infection. Catarino Cascabel - México and Silver can be indicated for use in breeding programs for resistance of pepper to ToSRV.