Quantitative detection of cassava common mosaic virus for health certification of cassava (Manihot esculenta Crantz) germplasm using qPCR analysis.

Cassava (Manihot esculenta Crantz) is a crop of global economic and food safety importance, used for human consumption and in various industrial applications. The genebank of the Genetic Resources Program of the Alliance of Bioversity International and CIAT currently holds the world's largest cassava collection, with 5965 in vitro accessions from 28 countries. Managing this extensive collection involves indexing quarantine pathogens as a phytosanitary certification requirement for safely distributing cassava germplasm. The study therefore aimed to optimize a quantitative diagnostic protocol to detect cassava common mosaic virus (CsCMV) using quantitative PCR (qPCR) as a better alternative to other molecular techniques. This was done through designing primers and a probe in the RdRP region of CsCMV, and optimizing the qPCR conditions of the diagnostic protocol using primer concentration assays, and reaction amplification conditions such as volume and reaction time. We also evaluated the qPCR protocol by comparing the results of 140 cassava accession evaluations using three diagnostic methodologies (DAS-ELISA, end-point PCR, and qPCR) for CsCMV. Our protocol established that qPCR technique analysis is ten-times more sensitive in detecting CsCMV compared to end-point PCR, showing a maximum detection level of 77.97 copies/µL of plasmid, with 76 min of reaction time. The comparison allowed us to verify the level of CsCMV detection through the techniques evaluated, concluding that qPCR was more sensitive and allowed the quantification of viral concentration. The optimized qPCR protocol will be used to accelerate diagnostic screening of cassava germplasm for the presence or absence of CsCMV to ensure safe movement and distribution of disease-free germplasm.

Adhesion factors and antimicrobial resistance of Escherichia coli strains associated with colibacillosis in piglets in Colombia.

Background and Aims: The pathogenicity of Escherichia coli is determined by the presence of genes that mediate virulence factors such as adherence capacity and toxin production. This research aimed to identify the adhesion factors and antibiotic resistance capacity of E. coli strains associated with diarrhea in piglets in Colombia. Materials and Methods: Presumptive E. coli strains were isolated from the rectal swabs of piglets in swine farms between 4 and 40 days of age with evidence of diarrhea. Presumptive E. coli strains were tested for antibiotic resistance. The hemolytic capacity of presumptive E. coli strains was measured and molecularly identified. Strains confirmed as hemolytic E. coli was evaluated for the presence of five adhesion factors (F4, F5, F6, F18, and F41) and resistance to 11 antibiotics. Results: Fifty-two putative E. coli strains were isolated, six of which showed a hemolytic capacity. The hemolytic strains were molecularly identified as E. coli. Adhesive fimbriae were found in five of six ß-hemolytic E. coli isolates. Combinations of the adhesion factors F6-F18 and F6-F41 were linked to antibiotic resistance capacity. Conclusion: The phenomenon of E. coli strains resistant to multiple antibiotics on pig farms represents a constant risk factor for public health and pig production.

Detección molecular de Cucumber mosaic virus y Pepper severe mottle virus en arvenses asociadas al cultivo de ají (Capsicum spp.)

En los últimos años, el ají (Capsicum chinense, Capsicum frutescens y Capsicum annuum var. Acuminatum), cultivado en el Valle del Cauca, se ha visto afectado por enfermedades virales causadas por Cucumber mosaic virus (CMV-ají) y Pepper severe mottle virus (PepSMoV). Pese a que estos dos virus son limitantes para producción del cultivo de ají, en la actualidad, pocos estudios han identificado los hospederos alternos de CMV-ají y PepSMoV. En este trabajo, se evaluó la presencia de CMV-ají y PepSMoV, mediante RT-PCR, en muestras de tejido foliar, de 121 plantas arvenses, asociadas al cultivo de ají, en el Valle del Cauca, Colombia. El análisis molecular indicó la presencia de CMV-ají, en el 21,4 % de las plantas recolectadas y de PepSMoV, en el 20,6 %. Se identificaron las especies arvenses Amaranthus viridis, Parthenium hysterophorus, Hippobroma longiflora, Commelina diffusa, Clitoria ternatea, Crotalaria incana, Desmodium tortuosum, Desmodium intortum, Macroptilium lathyroides, Anoda acerifolia, Boerhavia erecta, Bougainvillea glabra, Rivina humilis, Browallia americana, Capsicum rhomboideum, Solanum americanum y Lantana camara, como hospederas de CMV-ají o PepSMoV. Se presentó infección mixta de CMV-ají y PepSMoV, en 57 % de las arvenses positivas a virus, las cuales, están distribuidas en zonas productores de ají, localizadas en seis municipios del Valle del Cauca. Estos resultados brindan información sobre la distribución de estos virus en el Valle del Cauca, contribuyen al conocimiento de la epidemiología viral y servirán para diseñar medidas de manejo, orientadas a prevenir las infecciones virales en los cultivos de ají.

In recent years, chili pepper (Capsicum chinense, Capsicum frutescens y Capsicum annuum var. Acuminatum) grown in Valle del Cauca has been affected by viral diseases caused by Cucumber mosaic virus (CMV-chili pepper) and Pepper severe mottle virus (PepSMoV). Although these two viruses are limiting to the production of the chili pepper crop, at present, few studies have identified the alternate hosts of CMV-chili pepper and PepSMoV. In this work, the presence of CMV-chili pepper and PepSMoV were evaluated by RT-PCR in leaf tissue samples from 121 weed plants associated with chili pepper cultivation in Valle del Cauca, Colombia. Molecular analysis indicated the presence of CMV-chili pepper in 21.4 % of the collected plants and PepSMoV in 20.6 %. Weed species Amaranthus viridis, Parthenium hysterophorus, Hippobroma longiflora, Commelina diffusa, Clitoria ternatea, Crotalaria incana, Desmodium tortuosum, Desmodium intortum, Macroptilium lathyroides, Anoda acerifolia, Boerhavia erecta, Bougainvillea glabra, Rivina humilis, Browallia americana, Capsicum rhomboideum, Solanum americanum and Lantana camara, as hosts of CMV-chili pepper or PepSMoV. Mixed infection of CMV-chili pepper and PepSMoV was present in 57 % of the weeds positive for viruses, which are distributed in chili pepper producing areas located in six municipalities of Valle del Cauca. These results provide information on the distribution of these viruses in Valle del Cauca. Contribute to the knowledge of viral epidemiology and will serve to design management measures aimed to prevent viral infections in chili pepper crops.

Aislamiento y caracterización de bacterias endémicas colombianas con capacidad de degradar tolueno / Isolation and characterization of Colombian endemic bacteria capable of degrading toluene

RESUMEN Los hidrocarburos aromáticos monocíclicos: benceno, tolueno, etilbenceno y xileno (BTEX), presentes en crudo y refinados de petróleo, hacen parte de los compuestos con más impacto en el medio ambiente y la salud humana, debido a su naturaleza cancerígena, mutagénica y altamente tóxica. Esta investigación tuvo como objetivo obtener y caracterizar bacterias capaces de degradar tolueno. Se realizaron tres muestreos de suelo contaminado con hidrocarburos del Valle del Cauca en tres condiciones: gasolinería, derrame accidental y taller mecánico. Se aislaron bacterias capaces de crecer en tolueno fase vapor como única fuente de carbono y se caracterizaron a nivel morfológico, bioquímico y molecular. Para la caracterización molecular se amplificó, secuenció y analizó con herramientas bioinformáticas el gen ribosomal 16S. Se evaluó la utilización de tolueno directo con concentración al 1% como única fuente de carbono. Se logró aislar 29 bacterias con capacidad de metabolizar tolueno. La caracterización bioquímica y molecular identificó a las bacterias aisladas de suelo contaminado como Pseudomonas y Stenotrophomonas. Las bacterias aisladas en el taller mecánico resultaron ser los microorganismos con mejor crecimiento en tolueno como fuente de carbono, poseen un gran potencial para ser utilizadas para fines de biorremediación de suelos y aguas contaminadas con tolueno.

ABSTRACT Monocyclic aromatic hydrocarbons: benzene, toluene, ethylbenzene, and xylene (BTEX), present in crude oil and refined petroleum products, they are the compounds with a big impact on the environment and human health, due to their carcinogenic, mutagenic, and highly toxic. This research aimed to obtain and characterize bacteria capable of degrading toluene. Three samples of soil contaminated with hydrocarbons were carried out in three different conditions: oil station, accidental oil spill, and mechanical workshop. Bacteria capable of growing in toluene vapor as the unique carbon source were isolated and characterized at a morphological, biochemical, and molecular level. For molecular characterization, the 16S ribosomal gene was amplified, sequenced, and analyzed with bioinformatic tools. The use of direct toluene with 1% concentration as the sole carbon source was evaluated. It was possible to isolate 29 bacteria with the capacity to metabolize toluene. Biochemical and molecular characterization identified bacteria isolated from contaminated soil as Pseudomonas and Stenotrophomonas. The isolated strains in the mechanical workshop were the microorganisms with the best growth in toluene as a carbon source, they have great potential to be used for the bioremediation of soils and waters with toluene pollution.

Physicochemical and physiological changes during the ripening of Banana (Musaceae) fruit grown in Colombia.

The physicochemical and physiological attributes of three contrasting commercial varieties of Musaceae, Dominico Harton (plantain), Guineo (cooking banana) and Gros Michel (dessert banana), were evaluated and statistically analysed during post-harvest ripening. Quality attributes differed markedly among varieties, both in fresh fruits and during ripening. Variety (V) had a significant effect (P < 0.001) on all attributes except total soluble solids (TSS), carotenes and total chlorophyll. Storage time (ST) had a significant effect on all attributes except colour parameter b* and total carotenes. Starch levels decreased significantly (P < 0.001) during ripening, with nearly complete hydrolysis in Gros Michel, followed by Guineo and Dominico Harton. Discriminant analysis showed that central diameter, TSS of the pulp, colour parameter a* and total starch had the highest weight in the differentiation among varieties. These results point out which parameters may help improve current methods for monitoring ripening of bananas, in particular the commercially important varieties in this study.

PRIMER REPORTE DE BEGOMOVIRUS INFECTANDO CULTIVOS DE AJÍ (Capsicum spp.) EN COLOMBIA / First report of begomoviruses infecting pepper (Capsicum spp.) crops in Colombia

RESUMEN Virus del género Begomovirus infectan cultivos de importancia económica en todo el mundo, incluyendo ají. A la fecha, en Colombia no hay reportes de la presencia de begomovirus infectando este cultivo, por lo que el objetivo de esta investigación fue identificar la presencia de virus de este género en ají empleando estrategias moleculares. Se colectaron 197 muestras de ají en diez municipios del Valle del Cauca. Se extrajo el DNA genómico total vegetal y mediante PCR se detectó la presencia de begomovirus. Para establecer la identidad molecular del virus se amplificaron fragmentos de 1,4 kb de muestras colectadas en Palmira y Vijes. Los fragmentos fueron clonados, secuenciados y analizados. Se encontró que el 85,7 % de las muestras de ají evaluadas fueron positivas para begomovirus. Los análisis de secuencia de los fragmentos virales de 1,4 kb arrojaron una identidad de 91,8 % entre ellos y los de secuencia de nucleótidos de los virus aislados en Vijes y Palmira mostró que éstos presentan los valores de identidad más altos (87,2 % y 86,6 %) con el virus de la distorsión de la hoja de maracuyá, un begomovirus aislado de maracuyá en Colombia. Estos análisis estarían indicando que este begomovirus aislado de ají podría ser una nueva especie. De acuerdo con la literatura, este es el primer reporte de un begomovirus infectando cultivos de ají en Colombia.

ABSTRACT Viruses of the genus Begomovirus infect crops of economic importance around the world, including pepper. To date, in Colombia there are no reports of the presence of begomoviruses infecting this crop; therefore, this research work aimed to identify the presence of viruses of this genus in pepper using molecular strategies. Around 197 pepper samples were collected in ten municipalities in Valle del Cauca. Total plant genomic DNA was extracted, and the presence of begomoviruses was detected by using PCR. In order to establish the molecular identity of the virus, fragments of 1.4 kb were amplified from samples collected in Palmira and Vijes municipalities. The fragments obtained were cloned, sequenced, and analyzed. The results show that about 85.7 % of the pepper samples evaluated were positive for begomoviruses. Sequence analysis of the viral fragments of 1.4 kb showed an identity of 91.8 % among them. The nucleotide sequence analysis of the begomoviruses isolated in Vijes and Palmira showed its highest identity values (87.2 % and 86.6 %) with the passion fruit leaf distortion virus, a begomovirus that is affecting passion fruit crops in Colombia. These sequences analyze would indicate that this begomovirus isolated from pepper could be a new species that has not been reported worldwide. To our knowledge, this is the first report of a begomovirus infecting pepper crops in Colombia.

Molecular characterization of a new Begomovirus isolated from five weeds species collected in tomato crops in Valle del Cauca / Caracterización molecular de un nuevo begomovirus aislado de cinco especies de arvenses colectadas en cultivos de tomate en valle del cauca

RESUMEN Las arvenses son hospederos alternos de begomovirus (Geminiviridae), los cuales facilitan su persistencia y propagación a cultivos de interés agronómico, como el tomate. El objetivo de esta investigación fue obtener el genoma completo de un begomovirus bipartita encontrado en Amaranthus dubius, Rivina humilis, Rhynchosia minima, Desmodium sp. y Caesalpinia sp., las cuales fueron colectadas en cultivos de tomate en Ginebra y Cerrito, Valle del Cauca. El genoma del begomovirus fue obtenido utilizando amplificación por círculo rodante y digestión con las enzimas EcoRI y EcoRV, las cuáles cortan el componente genómico A y B, respectivamente. Estos fragmentos fueron clonados, secuenciados y analizados. Finalmente, se verificó la presencia de este begomovirus en todas las arvenses mediante PCR específico. Se obtuvieron tres clonas EcoRI y cinco clonas EcoRV. Los fragmentos que portan los componentes A y B presentan un tamaño de 2 584 y 2 543 nt, respectivamente. El análisis de secuencia de nucleótidos del genoma begomoviral A con otros begomovirus previamente reportados, mostró la mayor identidad (90,9 %) con el virus del mosaico dorado de Rhynchosia de Yucatán. Tomando como base el criterio de demarcación actual para las especies de Begomovirus establecido por el Comité Internacional de Taxonomía de Virus, el geminivirus aislado de las arvenses A. dubius, R. humilis, R. minima, Desmodium sp. y Caesalpinia sp., constituye una nueva especie begomoviral. Con base en la sintomatología observada en campo, se propone el nombre de Virus del mosaico dorado de Rhynchosia de Colombia para designar a esta nueva especie.

ABSTRACT The weeds are alternative hosts of begomoviruses (Geminiviridae), which facilitate their persistence and propagation to crops of agronomic interest, such as tomatoes. The objective of this investigation was to obtain the complete genome of a bipartite begomovirus found in Amaranthus dubius, Rivina humilis, Rhynchosia minima, Desmodium sp. and Caesalpinia sp., which were collected in tomato crops in Ginebra and Cerrito, Valle del Cauca. The genome of the begomovirus was obtained using rolling circle amplification and digestion with the enzymes EcoRI and EcoRV, which cut the genomic component A and B, respectively. These fragments were cloned, sequenced, and analyzed. Finally, the presence of this begomovirus in all weeds was verified by specific PCR. Three EcoRI clones and five EcoRV clones were obtained. The fragments carrying components A and B have a size of 2 584 and 2 543 nt, respectively. The analysis of the nucleotide sequence of the begomoviral A genome with other previously reported begomoviruses showed the highest identity (90.9 %) with the Rhynchosia golden mosaic virus Yucatán. Based on the current demarcation criterion for the Begomovirus species established by the International Committee on Taxonomy of Viruses, the geminivirus isolated from the weeds A. dubius, R. humilis, R. minima, Desmodium sp. and Caesalpinia sp., constitutes a new begomoviral species. Based on the symptoms observed in the field, the name of the Rhynchosia golden mosaic Colombia virus is proposed to designate this new species.

Evaluación del potencial biotecnológico de un promotor derivado del virus de la distorsión de la hoja de maracuyá (PLDV), un begomovirus que infecta maracuyá / Evaluation of the biotechnological potential of a promoter derived from passion fruit leaf distortion virus (PLDV), a begomovirus that infects passionfruit

RESUMEN Los avances biotecnológicos en plantas requieren la bioprospección de nuevos promotores para la expresión de genes de interés agronómico, en particular, es necesario caracterizar nuevos promotores con expresión tejido específica. El objetivo de esta investigación fue evaluar la actividad de expresión del promotor del gen AV1 que codifica para la proteína de la cápside (CP) del virus de la distorsión de la hoja de maracuyá (Passion fruit leaf distortion virus, PLDV) mediante ensayos transitorios de biobalística de baja presión. Se realizó un análisis de la región promotora del gen AV1 empleando herramientas bioinformáticas. Se construyó una fusión traduccional (CP-PLDV-GUS), que porta la región promotora del gen AV1 de PLDV fusionada al gen reportero uidA (GUS). CP-PLDV-GUS fue bombardeado sobre hojas de plántulas de tabaco cultivadas in vitro empleando una pistola de genes. Como control positivo se utilizó el plásmido pBI121 que porta el gen GUS bajo el control del promotor 35S de CaMV. Se llevaron a cabo 11 repeticiones, donde la unidad experimental fue la hoja y la variable de respuesta, la expresión transitoria del gen GUS representado por el número de puntos azules observados en las hojas bombardeadas. Como resultado, el análisis estadístico no paramétrico demostró que existe evidencia muestral suficiente para confirmar que, tanto el promotor AV1 del PLDV y 35S de CaMV presentan una actividad de expresión semejante. Finalmente, el promotor del gen AV1 de PLDV mostró una fuerte actividad de expresión del gen reportero en las células del mesófilo de las hojas, el cual podría ser usado para conferir expresión tejido específica en plantas transgénicas.

ABSTRACT Biotechnological advances in plants require the bioprospecting of new promoters for the gene´s expression of agronomic interest, in particular, it is necessary to characterize new promoters with tissue-specific expression The objective of this research was to evaluate the expression activity of the AV1 gene promoter that codes for the capsid protein (CP) of the Passion fruit leaf distortion virus (PLDV) by means of transient tests of low pressure biobalistics. An analysis of the promoter region was carried out using bioinformatics tools. A CP-PLDV-GUS translational fusion was constructed, which carries the promoter region of the AV1 gene of PLDV fused to the uidA reporter gene (GUS). CP-PLDV-GUS was bombarded on leaves of tobacco seedlings grown in vitro using a gene gun. As a positive control pBI121 carrying the GUS gene under the control of the 35S promoter of CaMV was used. It was carried out 11 repetitions where the experimental unit was the leaf and the response variable the transient expression of the GUS gene represented by number of blue dots observed in the bombarded leaves. As a result, the non-parametric statistical analysis showed that there is sufficient sample evidence to confirm that both the AV1 promoter of PLDV and 35S of CaMV exhibit similar expression activity. Finally, the promoter of the AV1 gene of PLDV showed a strong activity of expression of the reporter gene in the leaf mesophyll cells, which could be used to confer tissue-specific expression in transgenic plants.

Croton golden mosaic virus: a new bipartite begomovirus isolated from Croton hirtus in Colombia.

A new begomovirus infecting Croton hirtus (Euphorbiaceae) from Colombia has been characterized. The complete DNA-A and DNA-B components were determined to be 2613 and 2551 nt in length, respectively, showing the typical genome organization of bipartite New World begomoviruses. DNA-A showed the highest nucleotide sequence identity (81.2%) to sida yellow mottle virus (JN411687), a begomovirus isolated from Sida rhombifolia in Cuba. Based on the current ICTV species demarcation criterion for the genus Begomovirus, we report a new member of this genus infecting C. hirtus. We propose that it be named Croton golden mosaic virus (CroGMV), based on the symptoms observed in the weed. Phylogenetic analysis revealed that CroGMV segregates into a single clade and has some relationship with viruses from Central America and the Caribbean. CroGMV is the first Croton-infecting bipartite begomovirus reported in the world.

Molecular identification of a new begomovirus infecting yellow passion fruit (Passiflora edulis) in Colombia.

The complete genome sequence of a bipartite begomovirus (genus Begomovirus, family Geminiviridae) infecting yellow passion fruit (Passiflora edulis) in the state of Valle del Cauca (Colombia) has been determined. The complete DNA-A and DNA-B components were determined to be 2600 and 2572 nt in length, respectively. The DNA-A showed the highest nucleotide sequence identity (87.2 %) to bean dwarf mosaic virus (M88179), a begomovirus found in common bean crops in Colombia, and only 77.4 % identity to passion fruit severe leaf distortion virus (FJ972767), a begomovirus identified infecting passion fruit in Brazil. Based on its sequence identity to all other begomoviruses known to date and in accordance with the ICTV species demarcation criterion for the genus Begomovirus (≥91 % sequence identity for the complete DNA-A), the name passion fruit leaf distortion virus is proposed for this new begomovirus. To our knowledge, this is the first report of a bipartite begomovirus affecting passion fruit in Colombia and the second report of a geminivirus affecting this crop worldwide.

Primer reporte de un begomovirus presente en maracuyá amarillo [Passiflora edulis f. flavicarpa (Degener)] en Valle del Cauca, Colombia / First report of a begomovirus presents in yellow passionfruit [Passiflora edulis f. flavicarpa (Degene)] in Valle del Cauca, Colombia

El maracuyá amarillo es un cultivo de importancia económica para el Valle del Cauca, pero en los últimos años ha presentado una reducción de hasta un 80% en la producción debido a problemas virales. El objetivo del presente trabajo fue detectar y caracterizar parcialmente begomovirus que podrían estar afectando los cultivos de maracuyá amarillo localizados en dos zonas productoras del Valle del Cauca (La Unión y Palmira). Se colectaron hojas de maracuyá con síntomas típicos de enfermedad viral, se purificó su DNA genómico y se detectó la presencia de begomovirus bipartitas mediante PCR empleando cebadores específicos para el componente A y B, respectivamente. Cinco fragmentos de DNA correspondientes al genoma geminiviral A y B, fueron clonados, secuenciados y analizados con herramientas bioinformáticas. Los resultados evidencian por primera vez en Colombia de la presencia de un begomovirus en maracuyá amarillo. El análisis de la secuencias de nucleótidos indica que este begomovirus está más relacionado con el virus del mosaico enano del frijol, un geminivirus que afecta frijol en Colombia, y no con el virus de la distorsión severa de la hoja de maracuyá, el cual se reportó afectando maracuyá amarillo en Brasil. A la fecha, este resultado constituiría el primer reporte de un begomovirus (perteneciente a la familia Geminiviridae) que afecta maracuyá amarillo en Colombia y que podría ser diferente de otros begomovirus previamente reportados a nivel mundial afectando este cultivo.

The yellow passion fruit is a crop of economic importance to the Valle del Cauca, but in recent years has been a reduction of up to 80% in production due to viral problems. The aim of this study was to detect and characterize partially begomovirus that could be affecting crops of yellow passionfruit located in two growing areas of the Valle del Cauca (La Union and Palmira). Passionfruit leaves with typical symptoms of viral disease were collected, purified genomic DNA and its bipartite begomovirus were detected by PCR using primers specific for component A and B, respectively. Five DNA fragments corresponding to geminiviral A and B genome were cloned, sequenced and analyzed with bioinformatics tools. The results show for the first time in Colombia the presence of a begomovirus in yellow passionfruit. The analysis of the nucleotide sequences indicates that this begomovirus is more related to bean BDMV, a geminivirus affecting beans in Colombia, not PSLDV, which was reported affecting yellow passionfruit in Brazil. To date, this result would be the first report of a begomovirus (belonging to the family Geminiviridae) affecting yellow passion fruit in Colombia, and that might be different from other begomovirus previously reported worldwide affecting this crop.

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.

Detección, identificación y localización geográfica de Begomovirus que afectan al tomate en Colombia / Detection, identification and geographical localization of tomato-infecting Begomovirus in Colombia

En la actualidad, los Begomovirus (Familia Geminiviridae) se han convertido en la mayor amenaza para los cultivos de interés agrícola ubicados en las zonas tropicales y templadas del planeta. Estos virus son transmitidos por la mosca blanca Bemisia tabaci, la cual en los últimos años en Colombia ha tenido un aumento significativo en sus poblaciones y se ha asociado con la aparición de síntomas virales en cultivos de tomate. Muestras de tomate con síntomas virales típicos fueron recolectadas en las cinco principales zonas productoras de esta solanácea en el país. Los Begomovirus fueron detectados por medio de la técnica de hibridación de ácidos nucleicos tipo Dot blot así como por medio de la reacción en cadena de la polimerasa (PCR) en todas las muestras colectadas. Con la excepción de un reporte previo en el Valle del Cauca, este es el primer reporte de Begomovirus afectando cultivos de tomate en los departamentos de Antioquia, Santander, Boyacá y Cundinamarca. Asimismo, es la primera vez que se informa sobre Begomovirus que afectan cultivos de tomate localizados por encima de 1500 msnm en Colombia.

The begomoviruses (Family Geminiviridae) have recently emerged as samples with typical Begomovirus symptoms were collected in five different departments, comprising the mayor tomato growing areas of the country. Begomovirus were detected by Polymerase Chain Reaction (PCR) or Dot Blot Hybridization in all tomato samples collected in whole tomato growing areas of the country. With exception for Valle del Cauca, this is the first report of tomato-infecting Begomovirus in Antioquia, Santander, Boyacá and Cundinamarca departments. Also this is the first report of tomato-infecting Begomovirus crops located above 1500 masl in Colombia.

Functional characterization of the gene cluster from Pseudomonas syringae pv. phaseolicola NPS3121 involved in synthesis of phaseolotoxin.

Pseudomonas syringae pv. phaseolicola is the causal agent of halo blight disease of beans (Phaseolus vulgaris L.), which is characterized by water-soaked lesions surrounded by a chlorotic halo resulting from the action of a non-host-specific toxin known as phaseolotoxin. This phytotoxin inhibits the enzyme ornithine carbamoyltransferase involved in arginine biosynthesis. Different evidence suggested that genes involved in phaseolotoxin production were clustered. Two genes had been previously identified in our laboratory within this cluster: argK, which is involved in the immunity of the bacterium to its own toxin, and amtA, which is involved in the synthesis of homoarginine. We sequenced the region around argK and amtA in P. syringae pv. phaseolicola NPS3121 to determine the limits of the putative phaseolotoxin gene cluster and to determine the transcriptional pattern of the genes comprising it. We report that the phaseolotoxin cluster (Pht cluster) is composed of 23 genes and is flanked by insertion sequences and transposases. The mutation of 14 of the genes within the cluster lead to a Tox(-) phenotype for 11 of them, while three mutants exhibited low levels of toxin production. The analysis of fusions of selected DNA fragments to uidA, Northern probing, and reverse transcription-PCR indicate the presence of five transcriptional units, two monocistronic and three polycistronic; one is internal to a larger operon. The site for transcription initiation has been determined for each promoter, and the putative promoter regions were identified. Preliminary results also indicate that the gene product of phtL is involved in the regulation of the synthesis of phaseolotoxin.

The global arginine regulator ArgR controls expression of argF in Pseudomonas syringae pv. phaseolicola but is not required for the synthesis of phaseolotoxin or for the regulated expression of argK.

In Pseudomonas syringae pv. phaseolicola the enzyme ornithine carbamoyltransferase (OCTase), encoded by argF, is negatively regulated by argR, similar to what has been reported for Pseudomonas aeruginosa. However, production of the phaseolotoxin-resistant OCTase encoded by argK, synthesis of phaseolotoxin, and infectivity for bean pods occur independently of the ArgR protein.

In Pseudomonas syringae pv. phaseolicola, expression of the argK gene, encoding the phaseolotoxin-resistant ornithine carbamoyltransferase, is regulated indirectly by temperature and directly by a precursor resembling carbamoylphosphate.

Pseudomonas syringae pv. phaseolicola synthesizes a non-host-specific toxin, phaseolotoxin, and also synthesizes a phaseolotoxin-resistant ornithine carbamoyltransferase (ROCT) to protect itself from its own toxin. ROCT is encoded by argK, which is expressed coordinately with phaseolotoxin synthesis at 18 degrees C. To investigate the regulatory mechanisms of this system, null mutants were constructed for argK, argF (encoding the phaseolotoxin-sensitive OCTase [SOCT]), and amtA (encoding an amidinotransferase involved in phaseolotoxin synthesis). The argF mutant did not exhibit arginine auxotrophy when grown in M9 medium at 28 degrees C, because under this condition SOCT was replaced by ROCT. This loss of thermoregulation of argK was apparently caused by accumulation of carbamoylphosphate, one of the substrates of SOCT. Carbamoylphosphate, which has a structure similar to that of the inorganic moiety of phaseolotoxin, was used in induction assays with wild-type P. syringae pv. phaseolicola and was shown to be able to induce argK expression in M9 medium at 28 degrees C. These results indicate that argK expression is independent of temperature and is regulated directly by a compound resembling the inorganic moiety of phaseolotoxin.