Growth performance, hematology parameters and Aeromonas hydrophila challenge of pirarucu (Arapaima gigas) in response to dietary protein levels.

The pirarucu (Arapaima gigas) is a fast-growing Amazonian species of high commercial value. The present study aimed to determine the dietary crude protein (CP) level to promote maximum zootechnical performance for pirarucu fingerlings and as their resistance to Aeromonas hydrophila, as well as evaluate their hematological parameters. Pirarucu fingerlings (2.4 ± 0.08 g, 6.8 ± 0.52 cm) were distributed in 18 tanks (140 L, 40 fish per tank, n = 3) and fed six experimental diets consisting of increasing levels of CP: 300, 400, 450, 500, 550 and 650 g kg-1 in a completely randomized design. Data were submitted to one-way ANOVA (p < 0.05) and the ideal CP level for weight gain was determined using polynomial regression analysis. The dietary CP levels were evaluated using a quadratic polynomial regression and the level of 595 g kg-1 was determined for the best weight gain. The hematocrit of fish fed 300 g kg-1 was higher than in the other groups. No mortalities were observed after the 15-day bacterial challenge; however, number of pirarucu with bacterial damage on the pirarucu caudal fin was higher in the group that was fed the diet with 300 g kg-1. A dietary protein level of 618 g kg-1 is therefore recommended for providing maximum weight gain and immunological resistance in pirarucu fingerlings weighing 2.4-112.5 g.

ARABIDOPSIS MUESTRA RESISTENCIA NO-HOSPEDERO CONSTITUTIVA CONTRA Xanthomonas phaseoli pv. manihotis / Arabidopsis shows non-host constitutive resistance against Xanthomonas phaseoli pv. Manihotis

RESUMEN La bacteriosis vascular de la yuca, causada por la bacteria gram negativa Xanthomonas phaseoli pv. manihotis (Xpm), anteriormente conocida como Xanthomonas axonopodis pv. manihotis, es la principal enfermedad bacteriana que compromete su producción. Con la meta de generar una resistencia durable y de amplio espectro a la bacteriosis es posible explotar los mecanismos naturales presentes en plantas no-hospedero. Arabidopsis es una planta modelo extensamente estudiada, la cual es no-hospedero de Xpm. La meta de este estudio fue determinar si la resistencia no-hospedero de Arabidopsis es consecuencia de la presencia de barreras físicas o si esta depende de determinantes genéticos. En este trabajo se evaluó la capacidad de plantas de Arabidopsis de responder a la inoculación con Xpm. Ninguno de los ocho ecotipos de Arabidopsis evaluados mostraron una respuesta hipersensible a la inoculación con ocho diferentes cepas de Xpm. Aunque no se identificó la presencia de especies reactivas de oxígeno si se encontró un bloqueo en el crecimiento de Xpm en las plantas de Arabidopsis. En conjunto, los resultados aquí presentados sugieren que Arabidopsis no está activando una respuesta contra Xpm y que la resistencia observada puede ser consecuencia de las barreras físicas presentes en Arabidopsis que Xpm no es capaz de superar.

ABSTRACT Cassava bacterial blight (CBB), caused by the gram-negative bacteria Xanthomonas phaseoli pv. manihotis (Xpm), previously known as Xanthomonas axonopodis pv. manihotis, is the main bacterial disease compromising cassava production. With the aim to generate durable and broad-spectrum resistance to CBB is possible to exploit the natural mechanism present in non-host plants. Arabidopsis is an extensively studied model plant, which is a non-host of Xpm. The aim of this study was to determinate if the Arabidopsis non-host resistance is a consequence of physical barriers or if it depends on genetic determinants. In this work we evaluated the ability of Arabidopsis plants to respond after Xpm inoculation. None of the eight Arabidopsis ecotypes showed a hypersensitive response after inoculation with eight different Xpm strains. Although reactive oxygen species (ROS) production was not present, impairment in Xpm proliferation was found. These results suggest that Arabidopsis is not activating an immunity response against Xpm and the resistance might be a consequence of physical barriers present in Arabidopsis that Xpm is not able to overcome.

Caseous stomatitis caused by Pseudomonas aeruginosa in Boa constrictor amarali

Background: Pseudomonas aeruginosa is a bacterium that belongs to the microbiota of snakes, but it may also be anopportunistic pathogen and contaminate humans through fecal contact, bites, and injuries. In snakes, this microorganismmay present high pathogenicity at certain conditions and have been associated with high morbidity and mortality. Reportsof infection of Boa constrictor by this pathogen are rare. Thus, this study aimed to describe the P. aeruginosa oral infection in a snake specimen (Boa constrictor amarali), approaching the isolation and identification of the infectious agentsinvolved, the antimicrobial sensitivity and resistance, and the therapeutic protocol adopted.Case: A free-living adult female specimen of Boa constrictor amarali (Amarals boa), with no described previous history was rescued in an urban area by the Environmental Police. Clinical evaluations showed structures of caseous aspectin the oral cavity, with hyperemia spots in the mucosa. Samples of these lesions were sent for mycological examination,and fungal forms were not found. Samples were collected for isolation and culture. The antimicrobial susceptibility of theisolated microorganisms was determined by the modified Kirby-Bauer disk diffusion method. P. aeruginosa was isolatedand showed susceptibility to amikacin, gentamicin, and polymyxin-B; intermediate susceptibility to azithromycin, and ciprofloxacin; and resistance to cephalexin, ceftiofur, chloramphenicol, and enrofloxacin. The treatment consisted of cleaningof the oral cavity, local infiltration of lidocaine for debridement of the caseous area that were later cauterized with iodine.Systemic antibiotic therapy was used, with intramuscular administration of amikacin (5 mg/kg) for the first dose and (2.5mg/kg) for the other doses with intervals of 72 h, and oral administration of metronidazole...(AU)

Prevalence of Xanthomonas euvesicatoria (formally X. perforans) associated with bacterial spot severity in Capsicum annuum crops in South Central Chihuahua, Mexico.

BACKGROUND: Xanthomonas spp. causes bacterial spot disease, which reduces quality and yield of pepper crops in Mexico. Identification of phytopathogen species is necessary to implement more effective control strategies. OBJECTIVE: The aim of this study was to isolate and identify infecting Xanthomonas species in South Central Chihuahua pepper-producing areas. METHODS: Diseased plants were collected from 30 cultivation lots and bacteria were isolated from damaged tissues. Potential causative agents were isolated, identified, and characterized by biochemical and molecular analysis. Pathogenicity tests from each isolate were then performed on 30-d-old pepper seedlings, exposing five leaves to 10 µL of 1 × 108 CFU/mL bacterial suspensions of each isolate, using sterile distilled water as a control. Disease severity was determined after 10 d by calculating leaf damage percentage. Furthermore, we evaluated the susceptibility of the highest bacterial spot severity-causing isolates (13 isolates) to copper sulphate (CuS), copper gluconate (CuG), copper oxychloride + oxytetracycline hydrochloride (Cu + Ox), gentamicin + oxytetracycline hydrochloride (Gen + Ox), and gentamicin sulphate (GenS). Copper-resistance genes (copLAB) were detected by PCR analysis among isolates. RESULTS: Thirty-seven foliage isolates were identified as Xanthomonas euvesicatoria (14%), which were associated with bacterial spot disease in jalapeño pepper. Tested Xanthomonas isolates were resistant to Cu-based compounds, but susceptible to Cu + Ox. All isolates were susceptible to Gen + Ox and GenS. CopLAB genes were detected in all but one strain. CONCLUSIONS: X. euvesicatoria (formally X. perforans) may be considered as an emerging pathogen of bacterial spot pepper in Mexico. Among disease management strategies, alternatives to copper should be taken into consideration.

Caseous stomatitis caused by Pseudomonas aeruginosa in Boa constrictor amarali

Background: Pseudomonas aeruginosa is a bacterium that belongs to the microbiota of snakes, but it may also be anopportunistic pathogen and contaminate humans through fecal contact, bites, and injuries. In snakes, this microorganismmay present high pathogenicity at certain conditions and have been associated with high morbidity and mortality. Reportsof infection of Boa constrictor by this pathogen are rare. Thus, this study aimed to describe the P. aeruginosa oral infection in a snake specimen (Boa constrictor amarali), approaching the isolation and identification of the infectious agentsinvolved, the antimicrobial sensitivity and resistance, and the therapeutic protocol adopted.Case: A free-living adult female specimen of Boa constrictor amarali (Amaral’s boa), with no described previous history was rescued in an urban area by the Environmental Police. Clinical evaluations showed structures of caseous aspectin the oral cavity, with hyperemia spots in the mucosa. Samples of these lesions were sent for mycological examination,and fungal forms were not found. Samples were collected for isolation and culture. The antimicrobial susceptibility of theisolated microorganisms was determined by the modified Kirby-Bauer disk diffusion method. P. aeruginosa was isolatedand showed susceptibility to amikacin, gentamicin, and polymyxin-B; intermediate susceptibility to azithromycin, and ciprofloxacin; and resistance to cephalexin, ceftiofur, chloramphenicol, and enrofloxacin. The treatment consisted of cleaningof the oral cavity, local infiltration of lidocaine for debridement of the caseous area that were later cauterized with iodine.Systemic antibiotic therapy was used, with intramuscular administration of amikacin (5 mg/kg) for the first dose and (2.5mg/kg) for the other doses with intervals of 72 h, and oral administration of metronidazole...

The STK domain of the cassava bacterial blight resistance protein RXam1 interacts with an E3 Ubiquitin Ligase / El dominio stk de la proteína de resistencia a la bacteriosis vascular de yuca rxam1 interactúa con una e3 ubiquitin ligasa

RESUMEN La yuca (Manihot esculenta) representa el pilar de la seguridad alimentaria para cerca de mil millones de personas, principalmente en las zonas tropicales. Uno de los factores limitantes de la producción de yuca es la bacteriosis vascular causada por la bacteria Xanthomonasaxonopodis pv. manihotis (Xam). Recientemente se identificó el gen RXaml el cual confiere resistencia parcial de yuca a cepas de Xam. RXaml codifica una proteína con un dominio LRR (Leucine Rich Repeats) extracelular y un dominio STK (Serina Treonina Kinasa) citoplasmático; estas proteínas son conocidas como RLKs (Receptor Like Kinases). En este estudio se realizó el tamizaje de una librería de ADNc de yuca mediante doble híbrido de levadura para identificar las posibles proteínas que interactúan con el dominio STK de RXam1. El tamizaje de 3x108 clones permitió identificar y confirmar cinco clones de ellos los cuales corresponden al mismo gen, el cual codifica para una proteína que presenta un dominio central de dedos de zinc CHY, seguido por un dominio C-terminal "RING finger" y un "Zinc ribbon" el cual fue denominado CRFE3-1 (Cassava RING Finger E3 ligase). La interacción entre STK y CRFE3-1 fue altamente especifica ya que se demostró también por doble híbrido que STK no interactúa con una E3 ligasa de Arabidopsis, altamente similar a CRFE3-1, así como tampoco CRFE3-1 interactúa con el dominio STK de un RLK de lechuga similar a RXam1. La identificación de CRFE3-1 sugiere que mecanismos de degradación proteica son importantes para regular la actividad de RXam1.

ABSTRACT Cassava (Manihot esculenta) represents food security support for nearly one billion people, mainly in the tropics. One of the limiting factors of cassava's production is cassava bacterial blight, caused by the bacterium Xanthomonas axonopodis pv. manihotis (Xam). Recently, the RXam1 gene was identified, which confers partial resistance to some Xam strains. RXam1 encodes a protein with an extracellular LRR (Leucine Rich Repeats) domain and a cytoplasmic STK (Serine Threonine Kinase) domain; these proteins are known as RLK (Receptor-like Kinases). In this study, a cassava cDNA library was screened using a yeast Two-hybrid assay to identify possible proteins interacting with the STK domain of RXam1. Screening of 3x108 clones allowed identifying and confirming five of them, which correspond to the same gene, and code for a protein that has a core domain of zinc fingers CHY, followed by a C-terminal "RING finger" domain and a "Zinc ribbon". This gene was called CRFE3-1 (Cassava RING Finger E3 ligase). It was also demonstrated by yeast Two-hybrid that STK does not interact with an E3 ligase of Arabidopsis that is highly like CRFE3-1. CRFE3-1 did not show interaction with the STK domain of an RLK of lettuce related to RXam1, indicating a highly specific interaction between cassava RXam1 STK and CRFE3-1. The identification of CRFE3-1 suggests that protein degradation mechanisms are important to regulate the activity of RXam1.

ESTUDIO DE LA EXPRESIÓN DE GENES QUE CODIFICAN PARA PUTATIVAS PROTEÍNAS PR EN YUCA (Manihot esculenta Crantz) / Studying the Expression of Genes Coding for Putative PR Proteins in Cassava (Manihot esculenta Crantz)

RESUMEN Posterior al reconocimiento de agentes patógenos las plantas activan una serie de cascadas de señalización que culminan con la activación de factores de transcripción. Esto genera una concomitante reprogramación de la expresión génica que incluye la activación de la transcripción de los genes PR (relacionados con patogenicidad). Las proteínas PR son conocidas por poseer actividad antimicrobiana y evitan la posterior colonización del patógeno. En este estudio se empleó una aproximación bioinformática para identificar el repertorio de posibles proteínas PR en el genoma de yuca. Adicionalmente, se evaluó la expresión de nueve genes PR a lo largo del tiempo en variedades de yuca resistentes y susceptibles en respuesta a la inoculación con la bacteria Xanthomonas axonopodis pv. manihotis (Xam) mediante RT-PCR. Se encontró que varios genes PR fueron inducidos producto de la herida que se realiza durante el proceso de inoculación. Con el fin de evaluar cuantitativamente la contribución real de la infección bacteriana en la expresión de estos genes, se llevó a cabo una RT-PCR en tiempo real (QRT, Quantitative Real-Time PCR). Se encontró que en la variedad resistente el gen que codifica para MePR1 (Manes06G026900.1) presentó una inducción en su expresión a diferentes tiempos post-inoculación, lo cual no se observó en la variedad susceptible. De esta manera, este gen se constituye en un excelente marcador para evaluar la respuesta molecular de resistencia en plantas de yuca.

ABSTRACT Once pathogens are perceived by plants a signal transduction pathway is activated leading to the induction of transcription factors, which in turn reprogram the host gene expression including the transcription of PR (Pathogenesis-Related) genes. The PR proteins are well known for their antimicrobial activity and for contributing to arrest the invasion of pathogens. In this work, a bioinformatics approach was used to identify the repertoire of possible PR proteins in the cassava genome. Additionally, the expression of nine PR genes was evaluated over a time course in resistant and susceptible cassava varieties in response to inoculation with the bacterium Xanthomonas axonopodis pv. manihotis (Xam) by semiquantitative RT-PCR. It was found that several PR genes were induced as a result of the wound that is made during the inoculation process. In order to evaluate quantitatively the real contribution of the bacterial infection in the expression of the genes, a Real Time RT-PCR (qRT, quantitative Real-Time PCR) was carried out. In the resistant variety the gene coding for MePR1 (Manes06G026900) was induced at different post-inoculation times, which was not observed in the susceptible variety. Therefore, this gene constitutes an excellent marker to evaluate the molecular resistance response in cassava plants.

Diseases and pests noxious to Pleurotus spp. mushroom crops / Enfermedades y plagas nocivas en el cultivo del hongo Pleurotus

The Pleurotus genus is one of most extensively studied white-rot fungi due to its exceptional ligninolytic properties. It is an edible mushroom that possesses biological effects, as it contains important bioactive molecules. It is a rich source of nutrients, particularly proFun gal di se a ses; teins, minerals as well as vitamins B, C and D. In basidiomycete fungi, intensive cultivations of edible mushrooms can often be affected by some bacterial, mold and virus diseases that rather frequently cause dramatic production loss. These infections are facilitated by the particular conditions under which mushroom cultivation is commonly carried out such as warm temperatures, humidity, carbon dioxide (CO2) levels and presence of pests. There is not much bibliographic information related to pests of mushrooms and their substrates. The updated review presents a practical checklist of diseases and pests of the Pleurotus genus, providing useful information that may help different users.

El Pleurotus es uno de los hongos de la podredumbre blanca más extensamente estudiados debido a sus excepcionales propiedades lignocelulolíticas. Es un hongo comestible y también tiene varios efectos biológicos, ya que contiene importantes moléculas bioactivas. Es una fuente rica de nutrientes, particularmente de proteínas y minerales, así como de vitaminas B, C y D. Los cultivos intensivos de hongos comestibles del tipo basidiomicetos a menudo son afectados por enfermedades bacterianas, fúngicas y virales, lo que con frecuencia produce pérdidas significativas en la producción. Estas infecciones son facilitadas por las condiciones particulares bajo las cuales comúnmente se cultivan los hongos, tales como temperaturas cálidas y elevada humedad. Esta revisión presenta una lista práctica y actualizada de enfermedades y plagas frecuentes durante el cultivo del hongo comestible Pleurotus, y proporciona información que puede ser de utilidad para los productores.

Diseases and pests noxious to Pleurotus spp. mushroom crops.

The Pleurotus genus is one of most extensively studied white-rot fungi due to its exceptional ligninolytic properties. It is an edible mushroom that possesses biological effects, as it contains important bioactive molecules. It is a rich source of nutrients, particularly proteins, minerals as well as vitamins B, C and D. In basidiomycete fungi, intensive cultivations of edible mushrooms can often be affected by some bacterial, mold and virus diseases that rather frequently cause dramatic production loss. These infections are facilitated by the particular conditions under which mushroom cultivation is commonly carried out such as warm temperatures, humidity, carbon dioxide (CO2) levels and presence of pests. There is not much bibliographic information related to pests of mushrooms and their substrates. The updated review presents a practical checklist of diseases and pests of the Pleurotus genus, providing useful information that may help different users.

Streptococcus agalactiae infection in Nile tilapia (Oreochromis niloticus) / Infecção por Streptococcus agalactiae em tilápias do Nilo (Oreochromis niloticus)

This review aims to provide greater understanding of estreptococosis in reared tilapia and thereby assist in choosing the best method of prevention and control of this disease. Streptococcus agalactiae is a cosmopolitan pathogen and the main etiologic agent of septicaemia and meningoencephalitis in teleost. Outbreaks caused by this bacteria lead to high rates of morbidity and mortality of fish, resulting in serious economic losses, especially in the cage tilapia culture that is a branch of aquaculture which produces most in Brazil. This paper presents the main estreptococosis characteristics in Nile tilapia and its prevention by using intraperitoneal vaccines. Further studies should be conducted with the intention of clarifying its pathogenesis and improving the prevention and control.

Esta revisão tem por objetivo proporcionar maior entendimento sobre a estreptococose na criação de tilápias e com isso auxiliar na escolha do melhor método de profilaxia e controle desta enfermidade. Streptococcus agalactiae é um patógeno cosmopolita e o principal agente etiológico de septicemia e meningoencefalite em teleósteos. Surtos provocados por essa bactéria levam a altas taxas de morbidade e mortalidade de peixes, resultando em sérios prejuízos econômicos, especialmente na tilapicultura que é o ramo da aquicultura que mais produz no Brasil. Este trabalho traz as principais características da estreptococose em tilápias do Nilo e sua prevenção pelo uso de vacinas intraperitoneais. Novos estudos devem ser realizados com intuito de esclarecer ainda mais sua patogênese e aperfeiçoar os métodos profiláticos e de controle.

Streptococcus agalactiae infection in Nile tilapia (Oreochromis niloticus) / Infecção por Streptococcus agalactiae em tilápias do Nilo (Oreochromis niloticus)

This review aims to provide greater understanding of estreptococosis in reared tilapia and thereby assist in choosing the best method of prevention and control of this disease. Streptococcus agalactiae is a cosmopolitan pathogen and the main etiologic agent of septicaemia and meningoencephalitis in teleost. Outbreaks caused by this bacteria lead to high rates of morbidity and mortality of fish, resulting in serious economic losses, especially in the cage tilapia culture that is a branch of aquaculture which produces most in Brazil. This paper presents the main estreptococosis characteristics in Nile tilapia and its prevention by using intraperitoneal vaccines. Further studies should be conducted with the intention of clarifying its pathogenesis and improving the prevention and control.(AU)

Esta revisão tem por objetivo proporcionar maior entendimento sobre a estreptococose na criação de tilápias e com isso auxiliar na escolha do melhor método de profilaxia e controle desta enfermidade. Streptococcus agalactiae é um patógeno cosmopolita e o principal agente etiológico de septicemia e meningoencefalite em teleósteos. Surtos provocados por essa bactéria levam a altas taxas de morbidade e mortalidade de peixes, resultando em sérios prejuízos econômicos, especialmente na tilapicultura que é o ramo da aquicultura que mais produz no Brasil. Este trabalho traz as principais características da estreptococose em tilápias do Nilo e sua prevenção pelo uso de vacinas intraperitoneais. Novos estudos devem ser realizados com intuito de esclarecer ainda mais sua patogênese e aperfeiçoar os métodos profiláticos e de controle.(AU)

Isolamento de Streptococcus agalactiae em diferentes órgãos de tilápias do nilo (Oreochromis niloticus) criadas em tanques-rede / Isolation of Streptococcus agalactiae in different organs of nile tilapia (Oreochromis niloticus) reared in net cages

A estreptococose é uma das principais causas de mortalidade na criação de tilápias no Brasil, causando grandes perdas econômicas. Assim, o estudo objetivou determinar a frequência de isolamento e identificação por PCR de Streptococcus agalactiae em diferentes órgãos de tilápias do Nilo (Oreochromis niloticus) naturalmente infectadas, oriundas de oito pisciculturas da região norte do estado do Paraná que apresentavam sinais clínicos característicos de infecção estreptocócica. Para tanto, coletaram-se amostras de sangue e fragmentos de rim, fígado, baço, coração e encéfalo. Essas foram semeadas em meio ágar infusão de cérebro e coração (BHI) adicionado 5% de sangue de ovino e incubados à 29ºC, durante 7 dias em aerofília. Após o crescimento bacteriano e a partir das características macro e microscópicas, foram selecionadas colônias compatíveis com as do gênero Streptococcus sp.. As espécies foram identificadas através de PCR e confirmadas por meio do sequenciamento do gene 16S rDNA. Os resultados demonstraram que em tilápias do Nilo infectadas com S. agalactiae, o isolamento é mais frequente em encéfalo, rim e fígado em ordem decrescente.

Streptococcosis is one of the major causes of mortality in tilapia's creation in Brazil, inducing great economic losses. As soon, the study objectived to determinate the frequency of isolation and identification the Streptococcus agalactiae in organs different of Oreochromis niloticus naturally infected, derived from eight fish farms in the northern region of the state of Paraná, that presented clinical signs characteristics of streptococcal disease. However, blood samples and fragments (kidney, liver, spleen, heart and brain) were collected. These all samples were plated on solid medium of brain and heart infusion (BHI) added 5% ovine blood and incubated at 29ºC for 7 days in aerophilic conditions. Behind, the bacterial growth and from the macro and microscopic features, colonies compatibles with Streptococcus sp. gender, were selected. The species were identified by PCR reaction and confirmed by sequencing of 16S rDNA gene. The results exhibited that in tilapia of Nile infected with S. agalactiae the isolation is more common in brain, kidney and liver in descending order.

Análisis genómioo-funcional de proteínas con dominios tir en yuca / Functional Genomic Analysis of Cassava Proteins with TIR domains

Dentro de las proteínas implicadas en inmunidad de plantas y animales se encuentran aquellas que poseen un dominio TIR (Toll Interleukin Receptor). El objetivo de este trabajo fue realizar un análisis genómico global de las proteínas que presentan un dominio TIR en yuca y discernir su posible función en la resistencia a la bacteriosis vascular. En el proteoma de yuca se logró identificar 46 proteínas con dominios TIR, los cuales fueron divididos en cuatro categorías según la presencia o no de otros dominios: TIR (T), TIR- NB (TN), TIR-LRR (TL) y TIR-NB-LRR (TNL). El 56,5 % de las 46 proteínas corresponde a la categoría TNL. Mediante alineamientos múltiples se encontró que no todos los dominios TIR de yuca presentan la región aE implicada en la dimerización y activación de las respuestas de inmunidad. Tres de las cuatro categorías de proteínas (T, TNL y TN) presentan un mayor número de sustituciones sinónimas, sugiriendo que no están implicadas en procesos de reconocimiento. Por medio de doble híbrido de levadura y agroinfiltración se analizaron dos dominios TIR que no presentan la región aE, encontrando que ambos son capaces de formar homo y heterodímeros pero no desencadenan respuestas de defensa. Con este trabajo se pudo concluir que algunas proteínas que poseen dominios TIR pueden funcionar como adaptadores en la transducción de la señal con otras proteínas de resistencia. Además, se puso en evidencia que no siempre la región aE es importante para la dimerización, pero sí para activar las señales de respuestas de defensa.

Proteins containing a TIR domain (Toll Interleukin Receptor) are involved in plant and animal immunity. The aim of this work was to carry out an overall genomic analysis of cassava proteins with a TIR domain and discern their possible role in resistance to cassava bacterial blight. In total 46 proteins with a TIR domain were identified in the cassava proteome and were classed in four categories according the presence or absence of other domains: TIR (T), TIR-NB (TN), TIR-LRR (TL) and TIR-NB-LRR (TNL). 56.6 % of these 46 proteins have TIR, NB and LRR domains. Using multiple alignments it was possible to demonstrate that not all cassava TIR domains contain the aE region, involved in dimerization and activation of immune responses. Three of the four proteins categories (T, TNL and TN) presented a higher number of synonymous substitutions suggesting that they are not involved in recognition process. Two TIR domains not presenting the aE region were analyzed by yeast two hybrid assays and by agroinfiltration, finding that both are able to form homo and heterodimers, but they do not trigger defense responses. With this study it was possible to conclude that TIR domains can function as adaptors in the signal transduction with other resistance proteins. In addition, it became clear that not always the aE region is important for TIR dimerization but it seems necessary to activate defense responses signals.

LA PROTEÍNA PTHB DE Xanthomonas axonopodis pv. Manihotis ES AUTOACTIVA EN ENSAYOS DE DOBLE HÍBRIDO / The PthB Protein from Xanthomonas axonopodis pv. Manihotis is an Autoactive in Yeast Two-Hybrid Assays

La bacteriosis vascular de yuca producida por la bacteria Xanthomonas axonopodis pv. manihotis (Xam) es una enfermedad limitante para la producción de yuca. Dentro de los primeros factores de patogenicidad identificados en esta bacteria se encuentra el gen PthB. La proteína PthB pertenece a la familia de efectores PthA/AvrBs3, que se caracterizan por presentar dominios NLS (Nuclear Localization Signal) y un dominio AAD (Acidic Activation Domain), lo cual sugiere que estas proteínas actúan como factores de transcripción. La identificación de las proteínas de yuca que interactúan con PthB permitiría dar luces sobre la función de esta proteína en la patogenicidad de esta bacteria. En este trabajo se clonó PthB en una fusión traduccional con el BD (Binding Domain) del factor de transcripción GAL4. Después de transformar este constructo en una cepa de levadura, se observó autoactivación de los genes reporteros, incluso a concentraciones altas de 3-AT. La eliminación del primer, segundo o de los dos NLS y del AAD no eliminaron la capacidad de autoactivación de los genes reporteros mediada por PthB. Estos resultados indican la imposibilidad de su utilización en un tamizaje de una librería de ADNc de yuca para identificar las proteínas que interactúan con PthB.

Cassava bacterial blight disease is caused by the gram-negative bacteria Xanthomonas axonopodis pv. manihotis (Xam), and constitutes one of the most important constraints for cassava production. One of the first determinants of pathogenicity identified in this bacterium is the PthB gene. The PthB protein belongs to the PthA/AvrBs3 family, characterized by the presence of Nuclear Localization Signal (NLS) and Acidic Activation (AAD) domains, suggesting that these proteins are transcription factors. The identification of cassava proteins interacting with PthB could give insights about the function of this protein in the pathogenicity of this bacterium. In this work we cloned PthB in the yeast two hybrid expression vector pLAW10, generating a fusion protein with the Binding Domain (BD) of the transcription factor GAL4. In this work, PthB was cloned in a translational fusion with Gal4-BD (DNA Binding Domain). After transforming this construct into a yeast strain, autoactivation of the reporter genes was observed, even at the highest concentrations of 3-AT. The deletion of the first, second or both NLS and the AAD did not eliminate the ability of autoactivation of PthB. These results show the impossibility of using PthB to screen a cassava cDNA library to identify the proteins interacting with PthB.

IDENTIFICACIÓN DE miARNs CONSERVADOS EN YUCA (Manihot esculenta) / Indentification of Conserved miRNAs in Cassava (Manihot esculenta).

Los microARNs (miARNs) son moléculas pequeñas de ARN utilizadas por los eucariotas como un mecanismo de control de la expresión génica. En plantas los miRNAs están implicados en la regulación de distintos aspectos del crecimiento y desarrollo, así como en la tolerancia a estrés biótico y abiótico. Muchos miARNs de plantas se encuentran conservados en todos los grupos de embriófitos, sin embargo aún existen muchas plantas para las que no se conoce el reportorio de miARNs. Asimismo se desconoce el papel que algunos miARNs pueden tener en procesos como defensa contra patógenos. En este trabajo se construyó una librería de ARNs pequeños a partir de muestras de tejidos de Manihot esculenta (yuca) inoculados con la bacteria fitopatógena Xanthomonas axonopodis pv. manihotis (Xam), y se secuenciaron utilizando técnicas de secuenciación de nueva generación (Solexa/Illumina). Se identificaron en la librería 47 familias de miARNs de yuca conservados en otras plantas. Se cuantificó la expresión de estos miARNs, encontrándose similitudes con perfiles de expresión en otras plantas. Se encontró la secuencia de los precursores para algunos miARNs en secuencias de ESTs y GSSs de yuca. Asimismo se predijeron los blancos de estos miARNs en el set de ESTs encontrándose que muchos miARNs están dirigidos contra factores de transcripción, y que existe un gran porcentaje de posibles blancos con función desconocida. Este trabajo es el primer paso hacia entender cómo la vía de miARNs puede estar implicada en la interacción planta-patógeno en el sistema M. esculenta-Xam.

microRNAs (miRNAs) are small RNA molecules used by eukaryotes as a control mechanism for gene expression. In plants, miRNAs play a regulatory role in the expression of various genes involved in growth and development, as well in biotic and abiotic stress tolerance. Many plant miRNAs are conserved in all land plants; however the repertoire of miRNAs is still unknown for many plant species. Likewise, the role for some plant miRNAs in pathogen defense is also unknown. In this work we constructed a library of small RNA from tissues of Manihot esculenta (Cassava) infected with the pathogenic bacteria Xanthomonas axonopodis pv. manihotis (Xam). The small RNAs were sequenced using next-generation sequencing (Solexa/Illumina). We identified 47 conserved miRNAs families in Cassava and quantified their expression, finding similarities with expression profiles from other plants. We found sequences for the precursors of some of these families in sets of ESTs and GSSs. We also predicted targets for these miRNAs in a set of ESTs, finding many miRNAs targeting transcription factors, and regions with unknown function. This work constitutes a first step towards understanding the role of the miRNA pathway in plant-pathogen interaction in the M. esculenta-Xam pathosystem.

Estrategia de sllenciamiento génico en yuca para la validación de genes de resisteniaa / Strategy of Gene Silencing in Cassava for Validation of Resistance Genes.

La yuca (Manihot esculenta) constituye la base de la alimentación para mas de 1.000 millones de personas en el mundo considerándose por esta razón un cultivo primordial para la seguridad alimentaria. La bacteriosis vascular ocasionada por la bacteria gram negativa Xanthomonas axonopodis pv. manihotis (Xam) es uno de los factores más limitantes para la producción en este cultivo. Un gen de resistencia candidato de yuca a la bacteriosis vascular, denominado RXam1, ha sido previamente identificado. En este trabajo se empleó la estrategia de silenciamiento génico mediado por el geminivirus ACMV (del inglés African Cassava Mosaic Virus) para validar la función del gen RXam1. Como control positivo se utilizó el gen su, cuyo silenciamiento produce blanqueamiento en las hojas. Plantas de la variedad SG10735 fueron bombardeadas con las construc-ciones ACMV-A-SU + ACMV-B y ACMV-A-RXam1 + ACMV-B. La eficiencia de silenciamiento empleando el gen su fue baja, observándose un fenotipo de blanqueamiento en solo una de siete plantas. En las plantas posiblemente silenciadas en el gen RXam1, no se logró identificar siRNAs correspondientes a este gen, aunque si se observó una leve disminución en la expresión de RXam1 en una de las plantas evaluadas. Las curvas de crecimiento para la cepa Xam CIO136 en plantas de yuca inoculadas mostraron una leve pero no significativa diferencia en la susceptibilidad de las plantas silenciadas con respecto a las no silenciadas.

Cassava (Manihot esculenta) is a major source of food for more than 1000 million people in the world and constitutes an important staple crop. Cassava bacterial blight, caused by the gram negative bacterium Xanthomonas axonopodis pv. manihotis, is one of the most important constraints for this crop. A candidate resistance gene against cassava bacterial blight, named RXam1, has been identified previously. In this work, we employed the gene silencing approach using the African Cassava Mosaic Virus (ACMV) to validate the function of the RXam1 gene. We used as positive control the su gen, which produce photoblanching in leaves when is silenced. Plants from the SG10735 variety were bombardment with the ACMV-A-SU+ACMV-B y ACMV-A-RXam1+ACMV-B constructions. The silencing efficiency employing the su gene was low, only one of seven plants showed photoblanching. In the putative silenced plants for the RXam1 gene, no presence of siRNAs corresponding to RXam1 was observed; although a low diminution of the RXam1 gene expression was obtained. The growth curves for the Xam strain CIO136 in cassava plants inoculated showing a little but no significance difference in the susceptibility in the silenced plants compared to not silenced.

CONSTRUCCIÓN DE UNA LIBRERÍA DE ADNc EN YUCA: UNA HERRAMIENTA PARA EL DESARROLLO BIOTECNOLÓGICO DEL CULTIVO / Cassava cDNA Library Construction: One Tool for Biotechnological Development of the Crop

La yuca es un cultivo de importancia en la seguridad alimentaria mundial ya que constituye la base de la alimentación de más de 600 millones de personas en el mundo. También es un alto productor de almidón con niveles que oscilan entre 73,7 y 84,9% de su peso seco total en raíces (FAO, 2007). El almidón de yuca puede utilizarse en una gama amplia de industrias (textil, cosmética, alimentaria, etc). Además, es empleado en la producción de biocombustibles. Una de las principales limitantes en la producción de yuca es la bacteriosis vascular producida por la bacteria Xanthomonas axonopodis pv. manihotis (Xam). Esta enfermedad puede comprometer no solo el suministro de almidón para las plantas industriales productoras de bioetanol sino que también puede amenazar la seguridad alimentaria. El mejoramiento genético convencional de yuca es complicado dado su largo ciclo reproductivo, su alta heterocigocidad y su naturaleza tetraploide. Por estas razones se deben buscar alternativas que involucren desarrollos en biotecnología que permitan un mejoramiento eficaz y rápido. En la actual era genómica y postgenómica muchos de los experimentos dependen de la posibilidad de contar con la secuencia de transcritos clonados. Dado que estos clones provienen de librerías de ADNc contar con este tipo de recursos de excelente calidad es un paso esencial. En este artículo reportamos la construcción de una librería de ADNc empleando el sistema Gateway® a partir de plantas de yuca que han sido inoculadas con la cepa CIO151 de Xam. La librería presentó un título de 1 x 10(7) unidades formadoras de colonias (ufc)/ml y el tamaño de los insertos osciló entre 600-1.500 pb. Los análisis de secuencia de 14 clones al azar confirmaron que se trata de genes expresados y mostraron similitud con genes previamente reportados en especies estrechamente relacionadas a yuca. Esta librería se convierte en un excelente recurso para identificar novedosos genes y para el estudio de su función a través de la identificación y la interacción entre proteínas.

Cassava is one of the most important crops for global food security and provides food and livelihood for 600 million people in the developing world. It is also good source of starch, with levels between 73.7 y 84.9% of total dry weight in roots (FAO, 2007). Cassava starch can be used in a wide range of industries (textile, cosmetic, nourishing, etc) and it has a high potential for the production of biofuel. Cassava bacterial blight, caused by Xanthomonas axonopodis pv. manihotis (Xam), is one of the most important diseases that affects cassava. This disease can compromise the starch supply not only for bioetanol production but also affect global food security. The long reproductive cycle, high heterozigosity and tetraploid character of cassava are characteristics that have complicated the genetic breeding for this crop. For these reasons new alternatives based on biotechnology are necessary to accelerate its improvement. In the postgenomic era many experiments rely on the availability of transcript sequences for cloning. As these clones usually originate from cDNA libraries, the quality of these libraries is crucial. In this article we report the construction of the first cassava cDNA library employing the Gateway® system. For this, in vitro grown plants were inoculated with the Xam strain CIO151. The expression library shows a high titer of 1 x 10(7) cfu/ml, with inserts ranging between 600 and 1500 bp. The sequence analyses from 14 random clones confirmed that these are expressed genes and showed similarity with previously cloned genes from species related to cassava. This library is an excellent resource for the identification of novel genes and for functional studies through the identification of their interactions with other proteins.

IDENTIFICACIÓN DEL AGENTE CAUSAL DE UNA BACTERIOSIS EN ÑAME (Dioscorea alata L.) / ISOLATION AND IDENTIFICATION OF A PHYTOPATHOGENIC BACTERIA FROM WATER YAM (Dioscorea alata L.) / IDENTIFICAÇÃO DO AGENTE CAUSAL DE UMA BACTERIOSE NO INHAME (Dioscorea alata L.)

El ñame (Dioscorea alata L.), ampliamente usado como fuente de calorías en África, Asia y el Caribe, es una planta herbácea y trepadora que se reproduce generalmente de forma vegetativa y produce un tubérculo comestible de gran tamaño con alto contenido de almidón. Varios microorganismos fitopatógenos, incluyendo virus, hongos y bacterias, afectan al cultivo. Plantas enfermas de la zona de Guarataro, estado Bolívar, Venezuela, fueron colectadas en el 2003, año en que hubo ~80% de pérdidas en la producción de ñame de la zona a causa de una enfermedad desconocida. Hojas colectadas de plantas enfermas con lesiones acuosas y necróticas fueron esterilizadas superficialmente y transferidas a solución salina para permitir la difusión de bacterias desde los bordes del tejido y ser luego sembradas en medio LB agar. Como controles se emplearon agua y medio LB. Colonias amarillas, brillantes y lisas, fueron visibles a las 24-48h de incubación a 30ºC. Varios aislados fueron inoculados en las pruebas de hipersensibilidad en tabaco y de patogenicidad en plántulas de ñame cultivadas in vitro. Varios grados de respuesta de hipersensibilidad en tabaco fueron observados. Las plántulas de ñame inoculadas mostraron lesiones acuosas en la lámina foliar, las cuales no se observaron en los controles. Aislados bacterianos fueron observados mediante microscopía de luz, revelando bacterias bacilares Gram negativas. Varios flagelos peritricos fueron observados por microscopía electrónica. Los resultados de las pruebas bioquímicas y fisiológicas indicaron que los aislados corresponden a Pantoea agglomerans (Enterobacteriacea). Se discuten diferentes metodologías para la identificación de bacterias.

Water yam (Dioscorea alata L.), a staple crop in Africa, Asia and the Caribbean, is a herbaceous twining vine plant generally reproduced by vegetative sections and highly affected by several phytopathogenic organisms including virus, fungi and bacteria. Diseased plants were collected from Guarataro, Bolívar State, Venezuela, in 2003. That year an unknown disease led to ~80% loss of water yam production. The leaves from diseased plants have watery or necrotic lesions on the leaf lamina. Pieces of necrotic tissue were surface sterilized, then transferred to saline solution and the bacteria allowed to diffuse into the solution from the edge of the dissected tissue. The bacteria were isolated on LB agar media. Yellow, shining, smooth colonies with regular margins were visible within 24-48h of incubation at 30ºC. Several isolates were inoculated on tobacco plants. Water and LB media were used as negative controls. Several degrees of hypersensitivity response resulted from the inoculation of tobacco. However, inoculation of in vitro water yam plants showed watering lesions on the leaf lamina inoculated with bacteria cultures and no lesions on water and media inoculated leaves. Bacterial cells observed by light microscopy showed Gram-negative regular rods. Several peritricous flagella were observed under EM. Data from biochemical and physiological tests indicated that the isolates belong to the Enterobacteriaceas family specifically to Pantoea agglomerans. Different methodologies for bacterial identification are discussed.

O inhame (Dioscorea alata L.), amplamente usado como fonte de calorias na África, Ásia e no Caribe, é uma planta herbácea e trepadeira que se reproduz geralmente de forma vegetativa e produz um tubérculo comestível de grande tamanho com alto conteúdo de amido. Vários microorganismos fitopatógenos, incluindo vírus, fungos e bactérias, afetam ao cultivo. Plantas enfermas da zona de Guarataro, estado Bolívar, Venezuela, foram coletadas em 2003, ano em que houve ~80% de perdas na produção de inhame dessa zona a causa de uma enfermidade desconhecida. Folhas coletadas de plantas enfermas com lesões aquosas e necróticas foram esterilizadas superficialmente e transferidas a uma solução salina para permitir a difusão de bactérias desde as bordas do tecido e ser logo plantadas em meio LBA. Como controles foram empregados água e meio LBA. Colônias amarelas, brilhantes e lisas, foram visíveis às 24-48h de incubação a 30ºC. Vários isolados foram inoculados nas provas de hipersensibilidade em tabaco e de patogenicidade em plântulas de inhame cultivadas in vitro. Vários graus de resposta de hipersensibilidade em tabaco foram observados. As de plântulas de inhame inoculadas mostraram lesões aquosas na lâmina foliar, as quais não se observaram nos controles. Isolados bacterianos foram observados mediante microscopia de luz, revelando bactérias bacilares Gram negativas. Vários flagelos perítricos foram observados por microscopia eletrônica. Os resultados das provas bioquímicas e fisiológicas indicaram que os isolados correspondem a Pantoea agglomerans (Enterobacteriacea). Discutem-se diferentes metodologias para a identificação de.