Resistance of bean genotypes to bean golden mosaic virus (VMDF) / Resistência de genótipos de feijoeiro ao vírus do mosaico dourado do feijoeiro (VMDF)

The common bean (Phaseolus vulgaris L.) is a herbal annual plant, which belongs to Fabaceae family, it is grown by small and large farmers alike, in several production systems. It has much economic and social importance. However, the plant is a host to uncounTable fungal, bacterial and viral diseases. Among the viral pathologies there is one known as Bean golden mosaic virus (BGMV), currently spread to virtually every bean production region in Brazil and over the world, causing huge losses season after season. This research's aim was to evaluate the resistance degree presented by fifteen different bean genotypes as to symptomology shown due to infection by the virus on the plants. The experimental design was randomized blocks, with 15 treatments and a witness, Carioca-Pérola cultivar, and compound by four repetitions. Each parcel was compound by four lines of 3,0 meters in length and 0,5m apart, accounting for a total of 60 parcels of 6,0m². The analyzed characteristics were: Emergency, incidence, severity, thousand-bean weight, productivity, and chlorophyll content in the leaves. For characteristic incidence, at both 28 and 38 DAS, the genotypes did not prove to be resistant to the disease. For severity the strains PL 38, from the breeding between IAPAR 57 and IAPAR 72, and 93, from the breeding between ESAL-589 e IAPAR 57, stood out with superior results. For a thousand-bean weight, the genotype UFU Roxo 12, IAPAR 57 X ESAL 589 - PL 48, followed for ESAL 589 X IAPAR 57 - PL 148-1, UFU Preto 29, ESAL 589 X IAPAR 57 - PL 93, IAPAR 57 X IAPAR 72 - PL 25, IAPAR 57 X IAPAR 65 - PL194, CARIOCA PÉROLA-WITNESS, UFU Carioca 14 and ESAL 589 X IAPAR 72 - PL 129 was the one that showed the best result. The highest productivity per hectare was from the genotype IAPAR57 x ESAL 589 ­ PL48 from carioca group. Concerning the parameter chlorophyll content at 28 DAS, the genotypes ESAL 589 X IAPAR 57 - PL 93, ESAL 589 X IAPAR 72 - PL 129, IAPAR 72 X ESAL 589 ­PL49, UFU Roxo 12, ESAL 589 X IAPAR 57 - PL 28, CARIOCA PÉROLA-Witness, UFU Carioca 14 showed less infected area and the highest chlorophyll content, and at 38 DAS there was no significant difference among the genotypes.

O feijão comum (Phaseolus vulgaris L.) é uma planta anual que pertence à família Fabaceae, é cultivado por pequenos e grandes agricultores em diversos sistemas de produção no Brasil. Ele tem muita importância econômica e social. No entanto, a planta é um hospedeiro para muitas doenças fúngicas, bacterianas e virais. Entre as patologias virais tem-se vírus do mosaico dourado do feijoeiro (VMDF-BGMV), que atualmente se encontra em todas as regiões de produção de feijoeiro no Brasil e no mundo. A doença causa enormes prejuízos em todas estações de cultivo, notadamente no outono- inverno. Objetivo deste trabalho foi avaliar o grau de resistência apresentado por quinze diferentes genótipos de feijoeiro quanto à infecção (incidência be severidade) pelo vírus nas plantas. O delineamento experimental foi em blocos casualizados, com 15 tratamentos e uma testemunha, cultivar Carioca-Pérola, e composto por quatro repetições. Cada parcela foi composto por quatro linhas de 3,0 metros de comprimento e 0,5m de distância, correspondendo a um total de 60 parcelas de 6,0m². As características analisadas foram: emergência, incidência, severida de sintomas, peso de mil de mil grãos, produtividade e teor de clorofila nas folhas (índice SPAD). A incidência não se mostrou como variável inadequada para avaliar a resistência aos 28 e 38 dias após a semeadura (DAS). Para severidade a linhagem PL 38 se destacou e foi obtida apartir do cruzamento IAPAR 57 e IAPAR 72. Também apresentou menor severidade a linhagem 93, obtida do cruzamento entre ESAL-589 e IAPAR 57. Para peso de mil grãos, destacaram-se os genótipos UFU Roxo 12 (IAPAR 57 X ESAL 589 - PL 48), seguido por ESAL 589 X IAPAR 57 - PL 148-1, UFU Preto 29 (ESAL 589 X IAPAR 57 - PL 93), IAPAR 57 X IAPAR 72 - PL 25, IAPAR 57 X IAPAR 65 - PL194, CARIOCA PÉROLA-testemunha, UFU Carioca 14 e ESAL 589 X IAPAR 72 - PL 129. A maior produtividade por hectare foi do genótipo IAPAR57 x ESAL 589 - PL48 do grupo carioca. Quanto ao índice SPAD (clorofila aos 28 DAS), os genótipos ESAL 589 X IAPAR 57 - PL 93, ESAL 589 X IAPAR 72 - PL 129, IAPAR 72 X ESAL 589 -PL49, UFU Roxo 12, ESAL 589 X IAPAR 57 - PL 28, CARIOCA PÉROLA, UFU Carioca 14 apresentaram maior teor de clorofila aos 38 DAS (maior média numérica), más não houve diferença significativa entre os genótipos pelo teste de Scott & Knott a 5 % de probabilidade.

Optimización de las condiciones de inoculación por biobalística de un Begomovirus en tomate y tabaco / Optimizing conditions for biolistic inoculation of Begomovirus in tomato and tobacco

La transmisión experimental de Begomovirus es problemática. La mayoría de estos virus se pueden transmitir de planta a planta por su vector biológico, Bemisia tabaci. Las inoculaciones experimentales con mosca blanca son problemáticas debido a sus hábitos de alimentación, requerimiento de una planta viva infectada e instalaciones de contención para el vector. Por su parte la inoculación mecánica de Begomovirus es posible, pero generalmente a tasas bajas y no en todos los casos. Por esta razón el bombardeo de partículas (biobalística) de DNA viral como una estrategia de inoculación fue desarrollada. La posibilidad de utilizar el dispositivo de mano Helios Gen Gun System (Biorad®), un equipo de biobalística, para la transmisión de un Begomovirus bipartita a plantas de tomate y tabaco fue ensayado y optimizado. Los parámetros evaluados fueron: número de disparos (1-2), presión de helio (220 y 320 psi) y diámetro de las partículas de oro (0.6 y 1.6µm). Los síntomas característicos de la enfermedad viral (clorosis, mosaico y deformación de la hoja) aparecieron 3 semanas después del bombardeo en las hojas jóvenes no inoculadas. La replicación del DNA viral en las plantas se confirmó por Reacción en cadena de la polimerasa. Plantas infectadas en un 100 se obtuvieron cuando en el bombardeo se emplearon partículas de oro de 1.6 µm recubiertas con DNA viral a una presión de 320psi. A nuestro entender este es el primer reporte en Colombia de la inoculación directa de plantas de tomate y tabaco con un Begomovirus bipartita usando un dispositivo portátil de biobalística.

Experimental transmission of Begomovirus is problematic. Most Begomoviruses can be transmitted readily from plant to plant by the whitefly vector, but this also requires a live infected plant and extensive facilities to maintain the insect. Whitefly inoculations can also be problematic because of their preferential feeding habits on certain plants. Mechanical inoculation of Begomovirus is possible but generally at low rates and for others not at all. For this reason particle bombardment (biolistic) of DNA viral as an inoculum was developed. The possibility of using the Helios Gen Gun System (Biorad®), a biolistic hand-held device, for transmitting Begomovirus bipartite to tomato and tobacco plants was assayed and optimized. Biolistic inoculation was carried out with the hand held device at 220 or 320 psi, applying 1 or 2 shots /plant and using gold particles of 0.6 or 1.6µm in size. Characteristic symptoms of viral disease (chlorosis, mosaic and leaf deformation) appeared 3 weeks post-inoculation in the newly developing leaves. Replication of the viral DNA in plants was confirmed by Polymerase Chain Reaction. All bombarded plants became infected when biolistic inoculation was carried out with the hand held device at 320psi and using 1.6 µm gold particles in size. To our knowledge this is the first report in Colombia of successful direct inoculation of tomato and tobacco plants with Begomovirus bipartite geminivirus using a biolistic hand-held device.

Distribución y diversidad genética de Begomovirus que infectan tomate (Solanum lycopersicum L) en Colombia / Distribution and genetic diversity of tomato –infecting Begomovirus in Colombia

Las enfermedades causadas por los begomovirus, (Familia Geminiviridae) constituyen una serie limitante para la producción del tomate en Colombia. Sin embargo, la caracterización de estos virus no ha sido realizada al momento. Aquí­ presentamos los resultados de un muestreo a nivel nacional que buscaba conocer la distribución y diversidad genética de los geminivirus que están afectando el cultivo de tomate en Colombia. Los virus fueron detectados mediante PCR, empleando primer universales específicos para el género Begomovirus. Los fragmentos amplificados por PCR fueron sometidos a un análisis tipo RFLP cuyos resultados evidenciaron presencia de infecciones mixtas e individuales de geminivirus en la mayoría de las muestras recolectadas en campo. Los fragmentos amplificados por PCR fueron clonados y secuenciados. El analísis de secuencia y filogenético mostró que los aislados begomovirales colombianos eran gemininivirus bipartitas típicos del Hemisferio Occidental y que algunos eran variantes de PYMV y otros de ToTEV. Mediante el análisis de los elementos cis-regulatorios (iterones) presentes en el promotor del gen de la proteína asociada a replicación (Rep) de los begomovirus aislados es posible postular eventos de pseudorecombinacién que podrían suceder entre ellos durante la ocurrencia de infecciones mixtas en tomate.

Diseased caused by begomoviruses (Family Geminiviridae) constitute a serious constraint to tomato production in Colombia. However characterization of these new viruses had not been carried out so far. Here we report a large scale survey on the distribution and genetic diversity of tomato infecting geminiviruses which are affecting mayor growing area of this crop in the country. Viruses were detected by PCR using universal primers for members of genus Begomovirus. The RFLP analysis of PCR-amplified fragments showed individual and mixed infections of several geminiviruses in many of the samples. PCR-amplified fragments were cloned and sequenced. Based on sequence comparations and phylogenetic analysis, the Colombian geminivirus isolates were new world bipartite geminiviruses showing close relationship with PYMV and ToVEV. By means of bioinformatic analysis of cis-acting elements (iterons) involved in DNA replication of rep gene of Colombian geminivirus isolates was possible to postulate possible pseudorecombinación events that could occur between them but also confirm the occurrence of mixed infections.

Molecular characterization of cotton leaf Curl Multan virus and its satellite DNA that infects Hibiscus rosa-sinensis / 病毒学报

Virus isolate G6 was obtained from Hibiscus rosa-sinensis showing yellow and leaf curl symptoms in Guangzhou, Guangdong Province. The complete nucleotide sequence of DNA-A was determined to be 2 737 nucleotides encoding six potential ORFs. Comparison showed that G6 DNA-A had more than 89% sequence identify with all isolates of Cotton leaf curl Multan virus (CLCuMV) and shared the highest sequence identify (96.1%) with CLCuMV isolate 62. G6 DNA-A had 87.1%-89.8% sequence identity with those of CLCuRV isolates, while less than 87% identities with other begomoviruses. Phylogenetic analysis of G6 DNA-A and selected begomoviruses showed that G6 was most closely related to CLCuMV isolates, and they clustered together as a separate branch. Satellite DNA molecule (G6 DNAbeta) was found to be associated with G6 using the primers beta01 and beta02. G6 DNAbeta contains 1346 nucleotides, with a potential functional ORF (C1) in complementary sense DNA. Pairwise comparison indicated that G6 DNAbeta had the highest sequence identities with CLCuMV DNAbeta (92.1%) and CLCuRV DNAbeta (88.7%), but less than 80% sequence identities with other reported satellite DNA molecules. Phylogenetic analysis indicated that G6 DNAbeta was most closely related to CLCuMV DNAbeta and the two DNAbetas clustered together as a separate branch, and formed the main branch with DNAbeta of CLCuRV and MYVV-Y47. It is concluded that G6 infecting Hibiscus rosa-sinensis is an isolate of CLCuMV.

Sequence and recombination analyses of the geminivirus replication initiator protein.

The sequence motifs present in the replication initiator protein (Rep) of geminiviruses have been compared with those present in all known rolling circle replication initiators. The predicted secondary structures of Rep representing each group of organisms have been compared and found to be conserved. Regions of recombination in the Rep gene and the adjoining 5' intergenic region (IR)of representative species of Geminiviridae have been identified using Recombination Detection Programs. The possible implications of such recombinations on the increasing host range of geminivirus infections are discussed.

Bemnisia tabaci feeding induces pathogenesis-related proteins in cassava (Manihot esculenta Crantz).

Cassava (Manihot esculenta Cranzts) plants fed upon by whitefly Bemisia tabaci showed increased levels of pathogenesis-related (PR) proteins, such as beta-1, 3-glucanase, peroxidase and chitinase activities, as compared to uninfested plants. The enzymes increased in specific activities from 2 to 7 fold and protein content in leaf extracts decreased in whitefly-infested plants, compared to uninfested plants. Among the three PR proteins, B. tabaci feeding induced significantly higher beta-1, 3-glucanase activities, when compared with other two PR proteins. Study also discussed the possible application of PR proteins in whitefly control program.

Expression of a begomoviral DNAbeta gene in transgenic Nicotiana plants induced abnormal cell division / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

An increasing number of monopartite begomoviruses are being identified that a satellite molecule (DNAbeta) is required to induce typical symptoms in host plants. DNAbeta encodes a single gene (termed betaC1) encoded in the complementary-sense. We have produced transgenic Nicotiana benthamiana and N. tabacum plants expressing the betaC1 gene of a DNAbeta associated with Tomato yellow leaf curl China virus (TYLCCNV), under the control of the Cauliflower mosaic virus 35S promoter. Transgenic plants expressing betaC1 showed severe developmental abnormalities in both species. Microscopic analysis of sections of both transgenic and non-transgenic N. tabacum leaves showed abnormal outgrowths of transgenic N. tabacum to be due to disorganized cell division (hyperplasia) of spongy and palisade parenchyma. Immuno-gold labeling of sections with a polyclonal antibody against the betaC1 protein showed that the betaC1 protein accumulated in the nuclei of cells. The possible biological function of the betaC1 protein was discussed.

Infectivity analysis of two variable DNA B components of Mungbean yellow mosaic virus-Vigna in Vigna mungo and Vigna radiata.

Mungbean yellow mosaic virus-Vigna (MYMV-Vig), a Begomovirus that causes yellow mosaic disease, was cloned from field-infected blackgram (Vigna mungo). One DNA A clone (KA30) and five different DNA B clones (KA21, KA22, KA27, KA28 and KA34) were obtained. The sequence identity in the 150-nt common region (CR) between DNA A and DNA B was highest (95%) for KA22 DNA B and lowest (85.6%) for KA27 DNA B. The Rep-binding domain had three complete 11-nt (5'-TGTATCGGTGT-3') iterons in KA22 DNA B (and KA21, KA28 and KA34), while the first iteron in KA27 DNA B (5'-ATCGGTGT-3') had a 3-nt deletion. KA27 DNA B, which exhibited 93.9% CR sequence identity to the mungbean-infecting MYMV, also shared the 3-nt deletion in the first iteron besides having an 18-nt insertion between the third iteron and the conserved nonanucleotide. MYMV was found to be closely related to KA27 DNA B in amino acid sequence identity of BV1 (94.1%) and BC1 (97.6%) proteins and in the organization of nuclear localization signal (NLS), nuclear export signal (NES) and phosphorylation sites. Agroinoculation of blackgram (V. mungo) and mungbean (V. radiata) with partial dimers of KA27 and KA22 DNA Bs along with DNA A caused distinctly different symptoms. KA22 DNA B caused more intense yellow mosaic symptoms with high viral DNA titre in blackgram. In contrast, KA27 DNA B caused more intense yellow mosaic symptoms with high viral DNA titre in mungbean. Thus, DNA B of MYMVVig is an important determinant of host-range between V. mungo and V. radiata.

Detection of tomato leaf curl geminivirus in its vector Bemisia tabaci.

Geminiviruses are single-stranded DNA plant infecting viruses that cause major losses in important crops in tropical and subtropical countries. Tomato leaf curl virus (TLCV) belonging to the genera Begomovirus, is a whitefly-transmitted geminivirus that causes a severe leaf curl disease in tomato (Lycopersicon esculentum). The importance of this disease has prompted a great need for a rapid identification of TLCV in its hosts and vector. Polymerase chain reaction (PCR) is the most sensitive approach to detect a minute amount of viral nucleic acid. It is the most ideal method to amplify geminiviruses as they replicate via a double-stranded, circular DNA form. In this study, geminivirus specific degenerated primers were employed to detect TLCV occurring in its vector whitefly Bemisia tabaci by PCR based approach. One primer pair, amplified TLCV DNA fragment of about 1.1 Kb representing partly replicase gene, intergenic region and partly coat protein gene was used. When a set of primer targeted to the core region of the coat protein gene of geminivirus was used, a PCR amplified fragment of about 0.5 Kb was obtained. This approach is highly useful for an early detection of TLCV occurring in very small amount in the vector B. tabaci. Its implications in geminivirus management strategies and their differentiation and being discussed.

Association of geminivirus infection with yellow green mosaic disease of Cucumis sativus: diagnosis by nucleic acid probes.

Geminivirus association with yellow mosaic disease of C. sativus has been investigated by dot/slot blot hybridization tests using nucleic acid probe derived from DNA-B of Indian Tomato Leaf Curl geminivirus. The disease was transmitted experimentally on C. sativus by whitefly, Bemisia tabaci. Some weeds were also found to harbour geminivirus infection in dot/slot blot hybridization tests.