ABSTRACT
In this study, a new Bacillus velezensis strain Bv-303 was identified and its biocontrol effect against rice bacterial-blight (BB) disease caused by Xanthomonas oryzae pv. oryzae (Xoo) was investigated. Cell-free supernatant (CFS) of strain Bv-303 under different growth conditions were prepared to test the antagonistic activity and stability against Xoo by the Oxford-cup method in vitro. The antibacterial effect of strain Bv-303 to BB disease in rice were further analyzed in vivo by spraying the cell-culture broth (CCB), CFS and cell-suspension water (CSW), respectively, on the rice leaves inoculated with Xoo. Additionally, rice seeds germination rate and seedling growth under the strain Bv-303 CCB treatment were tested. The results showed that the strain Bv-303 CFS significantly inhibited Xoo growth by 85.7%‒88.0% in vitro, which was also stable under extreme environment conditions such as heat, acid, alkali and ultraviolet light. As tested in vivo, spraying the CCB, CFS or CSW of strain Bv-303 on the Xoo-infected leaves enhanced rice plant resistance to BB disease, with CCB showing the highest increase (62.7%) in disease-resistance. Notably, CCB does not have negative effects on rice seed germination and seedling growth. Therefore, strain Bv-303 has great potential for biocontrol of the rice BB disease.
Subject(s)
Oryza , Fatigue Syndrome, Chronic , Bacillus , Xanthomonas , Plant Diseases/microbiologyABSTRACT
Xanthomonas axonopodis pv. punicae (Xap) is a bacterial pathogen wreaking havoc in pomegranate cultivation. It causes bacterial blight disease dwindling yield and making fruit unfit for consumption. Physiological and histological investigations during host-pathogen interaction are prerequisite to assess the onset of defense mechanism in plants. Therefore, we tried to compare the pomegranate resistant (IC 318734) and highly susceptible (Ruby) genotypes challenged with Xap. The bacterial suspension containing Xap cells of 0.3 OD600 (~106 to 107CFU mL?1) was used for challenge inoculation. Uniformly grown resistant and highly susceptible plants were selected, the surface of leaves was pricked and spray-inoculated with bacterial suspension using native strain IIHR1 (NCBI Gen Bank ID: KT 222897). Simultaneously, the control plants were also sprayed with only distilled water and observed. A total of three replications with five plants per replication were maintained and evaluated under completely randomized design. Physiological investigations were recorded using Portable photosynthesis system (LCpro+, ADC BioScientific limited, UK) for one cycle of disease progression viz., 0, 1, 5, 10, 15 and 20 days after bacterial spray inoculation (DAI). Significant changes in gas exchange parameters were witnessed on pathogen inoculation. Higher reduction in mean percent change of photosynthetic and transpiration rate, instantaneous water use efficiency, internal CO2 content, stomatal conductance and relative water content were noticed in highly susceptible genotype than resistant one. On contrary, an increased percent mean change of intrinsic water use efficiency, carboxylation capacity and lignin was documented in resistant genotype. Relative injury caused due to bacterial infection was found high in highly susceptible genotype than resistant one. Histological investigations in highly susceptible and resistant genotype were studied on 20th day of Xap inoculation using Scanning Electron Microscopy. Highly susceptible genotype exhibited maximum deformed cells, tissues and other visible abnormalities upon Xap inoculation. Thus, this study forms a basis for effective disease management and breeding programmes in pomegranate.
ABSTRACT
Bacterial blight caused by Xanthomonas oryzae pv. oryzae (Xoo), is one of the devastating diseases of riceworldwide. The pathogen reported to cause 70% crop loss in some of the susceptible genotypes under diseasefavoring environments, viz., temperature ranging between 25 to 34C and relative humidity more than 70%. InXoo, about 245 genes govern the pathogenicity and host specificity. The hypersensitive response andpathogenicity (hrp) genes responsible for disease occurrence were clustered in the pathogenicity island of 31.3Kb. The protein secreted through type three secretory system and type one secretory system mediates infectionand establishment of the pathogen inside the host. However, elicitor molecules from Xoo triggered the resistantresponse in rice against the pathogen. An array of resistant genes (R genes) was known to be invoked by thehost to combat the bacterial infection. To date, of the 45 Xa genes in rice, nine were cloned and characterized.The evolution of new races has made the task of developing resistant rice genotypes more challenging as itdemands a comprehensive breeding strategy involving the best use of R genes from the existing gene pool.Thus, to combat the infection from the existing races and to slow down the emergence of new Xoo races,pyramiding two or more R genes was found to be effective against bacterial blight disease. In India, thesuccessfully commercialized example includes the development of rice genotypes, viz., Improved Pusa Basmati-1, Improved Samba Mahsuri, PR106, Type 3 Basmati, and Mahsuri with selected R genes, viz., xa5, Xa4,xa13 and Xa21 against bacterial blight resistance. This review primarily portray Xoo-rice interactions andprovides opportunities for its effective management through sustainable technologies.
ABSTRACT
Bacterial blight of pomegranate caused by X. axonopodis pv. Punicae (XAP) assumed epidemic form and resulted in economic burden on farmers. In the current study the pathogen infected samples were collected and the isolated XAP was identity and confirmed through the morphological, biochemical characterization and Pathogenicity test. Bacterium was reisolated from infected plant to prove Koch’s postulates. Efficacy of different chemicals and oils were tested by disc diffusion assay and turbidometrically. Bronopol 3000 ppm (25.6±1.6 mm) and Clove oil (18.0±0.7 mm) formed highest zone of inhibition Turbidometri showed the highest O.D. (0.908 nm) by Copper oxy chloride and Neem oil showed maximum inhibition of growth with O.D. (0.842 nm). Biotic stress (pathogen) induced protein response was studies by using SDS-PAGE method after protein extraction from XAP, healthy P. granatum L. and infected P. granatum L. The protein band pattern showed the unique band no. 2 (Mol.Wt.66000 Da) in infected P. granatum L. as compared to the banding pattern of XAP and healthy P. granatum L. The over expressed protein due to biotic stress could be useful as a marker for detection of the disease at the early stage and for control of the diseases after knowing the biochemical significance of the protein.
ABSTRACT
BACKGROUND: A total of 62,591 cowpea expressed sequence tags (ESTs) were BLAST aligned to the whole-genome sequence of barrel medic (Medicago truncatula) to develop conserved intron scanning primers (CISPs). The efficacy of the primers was tested across 10 different legumes and on different varieties of cowpea, chickpea, and pigeon pea. Genetic diversity was assessed using the same primers on different cowpea genotypes. Singlenucleotide polymorphisms (SNPs) were detected, which were later converted to length polymorphism markers for easy genotyping. CISPs developed in this study were used in tagging resistance to bacterial leaf blight disease in cowpea. RESULTS: A total of 1262 CISPs were designed. The single-copy amplification success rates using these primers on 10 different legumes and on different varieties of cowpea, chickpea, and pigeon pea were approximately 60% in most of the legumes except soybean (47%) and peanut (37%). Genetic diversity analysis of 35 cowpea genotypes using 179 CISPs revealed 123 polymorphic markers with PIC values ranging from 0.05 to 0.59. Potential SNPs identified in cowpea, chickpea, and pigeon pea were converted to PCR primers of various sizes for easy genotyping. Using the markers developed in this study, a genetic linkage map was constructed with 11 linkage groups in cowpea. QTL mapping with 194 F3 progeny families derived from the cross C-152 × V-16 resulted in the identification of three QTLs for resistance to bacterial leaf blight disease. Conclusions: CISPs were proved to be efficient markers to identify various other marker classes like SNPs through comparative genomic studies in lesser studied crops and to aid in systematic sampling of the entire genome for well-distributed markers at low cost
Subject(s)
Genome, Plant , Genomics/methods , Medicago truncatula/genetics , Polymerase Chain Reaction , Chromosome Mapping , Expressed Sequence Tags , Polymorphism, Single Nucleotide , Genomics , Quantitative Trait Loci , Fabaceae/geneticsABSTRACT
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.
ABSTRACT
ABSTRACT: The aim of this study was to evaluate the ethanolic extract of green propolis (EEP) in the protection of common bean plants against two main bacterial cultures, bacterial blight (Xanthomonas axonopodis pv. phaseoli) and wildfire (Pseudomonas syringae pv. tabaci). Experiments on antimicrobial activity were performed, inducing phytoalexins, defense-related enzymes, and disease severity, under concentrations of 0, 0.5, 1.0, 2.5, and 5.0%. The EEP presented antimicrobial activity on both phytobacteria, causing a decrease in their development. It has also promoted a linear accumulation of phaseolin in bean hypocotyls according to the EEP concentration used. There was a reduction in the lesion area, which was caused by bacterial blight on bean leaves treated with EEP, and local and systemic effect were observed. Polyphenoloxidase was activated with 5% EEP, reaching the maximum activation time 62.5 h after application. An increase was observed in the activity of phenylalanine ammonia-lyase in plants treated with EEP, with local and systemic effect. Results indicated the potential of EEP in the control of these diseases.
RESUMO: Este trabalho teve como objetivo avaliar o extrato etanólico de própolis verde (EEP) na proteção de plantas de feijoeiro contra as duas principais bacterioses da cultura, crestamento bacteriano (Xanthomonas axonopodis pv. phaseoli) e fogo selvagem (Pseudomonas syringae pv. tabaci). Foram realizados experimentos sobre atividade antimicrobiana indutora de fitoalexinas, de enzimas relacionadas à defesa, e na severidade da doença, usando as concentrações de 0, 0,5, 1,0, 2,5 e 5,0%. O EEP apresentou atividade antimicrobiana sobre ambas fitobactérias, causando uma redução em seu desenvolvimento. O EEP também promoveu um acúmulo linear de faseolina em hipocótilos de feijoeiro conforme a concentração usada. Houve uma redução na área lesionada pelo crestamento bacteriano em folhas de feijoeiro tratadas com EEP, com efeito local e sistêmico. A enzima polifenoloxidase foi ativada pelo EEP a 5%, com ponto máximo de ativação 62,5 horas após aplicação. Houve um aumento na atividade de fenilalanina amônia-liase em plantas tratadas com EEP, com efeito local e sistêmico. Os resultados indicam o potencial do EEP no controle dessas doenças.
ABSTRACT
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.
ABSTRACT
ABSTRACT: Erwinia psidii causes bacterial blight of guava ( Psidium guajava ), an important disease of this crop in Brazil. The pathogen affects branches and twigs of guava trees, reducing yield significantly. Bacterial dissemination often occurs through contaminated but asymptomatic propagating plant material. The objectives of this research were to evaluate the use of BIO-PCR and conventional PCR to detect E. psidii in inoculated guava plants grown in a greenhouse and in symptomatic and asymptomatic trees from guava orchards. Erwinia psidii strain IBSBF 1576 was inoculated (107CFU mL-1) into young guava shoots and plant tissue was analysed at 0, 5, 10, and 15 days after inoculation. Symptoms were observed after 5 days and all inoculated shoots were PCR positive at all times, by both BIO-PCR and conventional PCR. Under natural infection conditions, 40 samples were tested by BIO-PCR from each of three guava orchards, 20 showing symptoms and 20 asymptomatic. PCR was positive for 58 out of 60 symptomatic samples (96.7%) and for 6.7% of asymptomatic samples, showing that the method can be used to detect the pathogen at early stages of infection. This PCR method may be used as a diagnostic tool to assess bacterial survival, dissemination and disease outbreaks.
RESUMO: Erwinia psidii é o agente causal da seca dos ponteiros da goiabeira ( Psidium guajava ), uma importante doença dessa cultura no Brasil. O patógeno afeta folhas, frutos, ramos e brotações, reduzindo significativamente a produtividade da cultura. A disseminação do patógeno ocorre por meio de material propagativo contaminado, porém assintomático. Os objetivos do trabalho foram avaliar o uso da BIO-PCR e da PCR convencional para detectar E. psidii em plantas inoculadas em casa de vegetação e em plantas sintomáticas e assintomáticas em pomares de goiabeira. A estirpe IBSBF 1576 de E. psidii foi inoculada (107UFC mL-1) em brotações novas de mudas de goiabeira e o tecido foi analisado nos tempos 0, 5, 10, e 15 dias após a inoculação. Sintomas foram observados após 5 dias e todas as plantas inoculadas foram positivas por PCR em todos os tempos avaliados, pelos dois métodos (BIO-PCR e PCR convencional). Sob condições de infecção natural em campo, três pomares foram avaliados por BIO-PCR. De cada pomar, foram coletadas 40 amostras, sendo 20 com e 20 sem sintomas. PCR foi positiva para 58 das 60 amostras sintomáticas (96,7%) e para 6,7% das amostras assintomáticas, demonstrando que o método pode ser usado para detectar o patógeno nos estágios iniciais da infecção. Este método poderá ser útil como uma ferramenta para a diagnose e para monitorar a sobrevivência e disseminação da bactéria e, consequentemente, novos focos da doença.
ABSTRACT
Common and fuscous blights of bean are diseases widely distributed in the world. The most commonly observed symptoms are spots on leaves, stems, pods and seeds. In December 2009, bean plants cv. Uirapuru showing symptoms of wilt similar to those induced by Curtobacterium flaccumfaciens pv. flaccumfaciens were observed in a commercial crop located in the county of Itararé, State of São Paulo, Brazil. The plants were at the last cycle stage with mature pods and these symptoms were noted in the majority of the growing area. Optical microscopic observations of discolored vascular tissue from diseased stems revealed the presence of bacterial masses oozed from infected tissue, indicating that the disease was caused by bacterial pathogen. Isolations on nutrient agar showed circular, convex, yellow colonies with smooth edges. The causal bacterium was Gramnegative and produced a dark brown pigment in culture medium. Biochemical, cultural and physiological tests confirmed its identity as Xanthomonas fuscans subsp. fuscans(syn. Xanthomonas campestris pv. phaseoli"var. fuscans"). The pathogenicity of the isolates was confirmed by artificial inoculations. Systemic infection has been reported in the literature but these kinds of symptoms are not currently observed in Brazilian fields. Bacterial strains were deposited on the Phytobacteria Culture Collection of Instituto Biológico (IBSBF - www.biologico.sp.gov.br/bacterias/php) under accession numbers 2813 and 3028.(AU)
O crestamento bacteriano comum do feijoeiro é uma doença amplamente distribuída no mundo e os sintomas comumente observados são manchas nas folhas, hastes, vagens e sementes. Em dezembro de 2009, plantas de feijoeiro cv. Uirapuru com sintomas de murcha similares aos produzidos por Curtobacterium flaccumfaciens pv. flaccumfaciens foram observadas em campos comerciais localizados no município de Itararé, estado de São Paulo, Brasil. As plantas encontravam-se no final do ciclo, com as vagens já formadas. Os sintomas foram notados em quase a totalidade da área cultivada. Observações ao microscópio óptico de fragmentos de tecido do sistema vascular das hastes de plantas doentes evidenciaram intenso fluxo bacteriano, confirmando tratar-se de doença bacteriana. Isolamentos realizados em meio nutriente ágar produziram colônias de coloração amarelada, brilhantes, convexas, lisas. A bactéria agente causal era Gram-negativa e produtora de pigmento marrom escuro em meio de cultura. Testes bioquímicos, culturais e fisiológicos confirmaram sua identidade como Xanthomonas fuscans subsp. fuscans (sin. Xanthomonas campestris pv. phaseoli "var. fuscans"). A patogenicidade dos isolados foi confirmada por inoculações artificiais em mudas de feijão cv. Carioca e os reisolamentos efetuados resultaram em colônias semelhantes às originais. Embora descrita na literatura, a infecção sistêmica não é usualmente observada nos plantios de feijoeiro em nosso país. Linhagens bacterianas encontram-se depositadas na Coleção de Culturas do Instituto Biológico (IBSBF) sob os números 2813 e 3028.(AU)
Subject(s)
Bacterial Growth , Xanthomonas axonopodis , Infections , FabaceaeABSTRACT
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.
ABSTRACT
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.
ABSTRACT
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.
ABSTRACT
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.
ABSTRACT
Erwinia psidii causes bacterial disease of guava in Brazil. Phenotypic and molecular characterization through rep-PCR fingerprinting of 42 strains from different geographical regions showed that E. psidii populations in Brazil have a low level of genetic diversity and suggest that contaminated plant material is the main source for pathogen dissemination in the country.
Erwinia psidii é o agente causal da seca-dos-ponteiros ou bacteriose da goiabeira no Brasil. A caracterização fenotípica e molecular através de rep-PCR de 42 estirpes patogênicas de diferentes regiões mostrou que as populações de E. psidii no Brasil têm um baixo nível de diversidade genética e sugere que material de propagação infectado é a principal fonte de disseminação do patógeno para novas áreas no país.
ABSTRACT
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.
ABSTRACT
A mandioca apresenta-se como importante fonte de carboidratos, principalmente nos trópicos. A bacteriose causada por Xanthomonas axonopodis p.v. manihots é a doença mais importante desta cultura e seus danos podem chegar a 30% ou mais na produção. Este trabalho objetivou avaliar, em condições de casa de vegetação, a reação de germoplasma de mandioca mansa e de brava à dois isolados de Xanthomonas axonopodis p.v. manihots. Os ensaios foram desenvolvidos no Instituto de Ciências Agrárias da Universidade Federal de Uberlândia. As plantas foram inoculadas pelo corte de três folíolos centrais em três folhas novas completamente abertas, seguindo pela inserção de um palito à altura da axila da folha mais velha, utilizando-se uma suspensão bacteriana contendo 2x109 u.f.c. mL-1 A inoculação ocorreu aos 42 dias de plantio e a avaliação aos 41 dias de inoculação. Os critérios de avaliação foram notas de Sintomas visuais na parte aérea, Porcentagens de desfolha e de Infecção sistêmica do caule. Os resultados mostraram que os critérios de avaliação utilizados foram eficientes no estudo da virulência dos isolados, sendo o isolado obtido na região de Uberlândia mais virulento para mandioca mansa e o de Lavras para mandioca brava, caracterizando a necessidade de utilização de isolados provenientes da região onde o germoplasma será cultivado. Considerando-se a análise da reação da resistência de germoplasma, os critérios porcentagem de infecção sistêmica e de desfolha mostraram-se bastante efetivos. Destacaram-se nesse trabalho as variedades Vassoura, Amarela, Vermelha e Castelinho e o clone CPAC88-11.
The cassava is presented as an important stach source, mainly in the tropics. The bacteriosis diseasecaused by Xanthomonas axonopodis p.v. manihots is the most important disease of this culture and itsdamage can achieve 30% of the production, or even more. This work objectified to evaluate, in greenhouse condition the reaction of "mandioca mansa" and the "mandioca brava" cassava's germoplasma tothe two isolates of Xanthomonas axonopodis p.v. manihots. Trials were developed at the Instituto deCiências Agrárias da Universidade Federal de Uberlândia. The plants had been inoculated by seasor'scuttings of three central leaflets in three completely opened new leaves, following by insertion of a littlewood stick at the oldest leaf's axel, using a bacterial suspension at 2x109u.f.c. mL-1. The inoculation happened at the 42nd day after planting and the evaluation at the 41st day after inoculation. Theevaluation criteria were: notes of visual symptoms in the aerial part, percentages defoliation and systemicinfection of the stalk. The results showed the efficiency of the evaluation criteria applied at this work forthe isolates virulence study. The Uberlândia isolate was more virulent to "mandioca mansa" cassavacultivars and the Lavras isolate was more virulent to "mandioca brava" cassava cultivars. That indicatesthe need of using isolates from the region where the germoplasm will be cultivated. Considering thegermoplasm resistance reaction analysis, both the sistemic infection precentage and the defoliationcriteria presented as very effective. Oustanding behavior was observed for the Vassoura, Amarela,Vermelha and Castelinho cultivars and for the CPAC88-11 clone
Subject(s)
Manihot , Virulence , XanthomonasABSTRACT
Avaliou-se o controle in vitro de Xanthomonas axonopodis pv. manihotis mediante o uso de extrato aquoso de quatro genótipos de cúrcuma provenientes de cultivos de Jaboticabal-SP, Mara Rosa-GO, Maringá-PR e Mercedes-PR, bem como o efeito curativo, através do tratamento de manivas de mandioca infectadas com o patógeno e plantio em condições de campo. No experimento in vitro, o extrato de cúrcuma causou inibição total do crescimento da bactéria, na concentração de 10, para o material proveniente de Mercedes, enquanto que, para a cúrcuma de Jaboticabal, houve controle total a 15 e o de Mara Rosa a 20. A cúrcuma proveniente de Maringá não inibiu totalmente o crescimento, em nenhuma das concentrações utilizadas. No experimento in vivo, o brotamento das plantas foi pouco, devido ao grau de infecção das manivas. O extrato bruto de cúrcuma a 10 proveniente de Mercedes foi prejudicial para a mandioca em condição de campo, reduzindo o estande em relação aos tratamentos controle. Possivelmente, houve ação tóxica direta sobre a fisiologia da planta ou indução de suscetibilidade. Na concentração de 1 da cúrcuma proveniente de Maringá, não houve diferença estatística em relação ás testemunhas, para o parâmetro estande de plantas. O controle químico utilizado não foi eficiente, pois se comportou igual à testemunha água. Com relação à severidade e à produtividade não se conservaram diferenças significativas entre os tratamentos. Os resultados indicam que, embora haja atividade antibacteriana a X. axonopodis pv. manihotis, os extratos de cúrcuma, nas concentrações utilizadas, não apresentam efeito curativo em manivas de mandioca infectadas pelo patógeno
medicinalAbstractThe control of Xanthomonas axonopodis pv. manihotis was evaluated in vitro by using aqueous extractof four turmeric genotypes from Jaboticabal-SP, Mara Rosa-GO, Maringá-PR and Mercedes-PR, as wellas in vivo, by treatment of infected cassava stems and their cultivation at field conditions. The resultsshowed that in vitro experiment, turmeric extract inhibited completely the bacteria growth in theconcentration of 10% for the genotype from Mercedes, while for the Jaboticabal's turmeric there was a total control at 15% and for Mara Rosa at 20%. Turmeric genotype from Maringá did not show fullinhibition of the bacterial growth in none of the extract concentrations used. At field conditions, sproutingswere extremely low, due to the stems infection degree. Turmeric extract at 10%, from Mercedes, washarmful for the cassava, reducing stand regarding the control treatments. Possibly there was a directtoxic action on the plant physiology or susceptibility induction. But in the concentration of 1% ofturmeric from Maringá, there was no statistical difference in relation to the control treatment for the plantstand. The chemical control was not completely efficient. There was no statistical difference amongtreatments for both severity and productivity. The results indicate that, although presenting antibacterialactivity to X. axonopodis pv. manihotis, the turmeric extracts, in the used concentrations, did notpresent curative effect in cassava stems infected with the pathogen.Key words: Bacterial blight of cassava, alternative control, medicinal plant
Subject(s)
/prevention & control , Manihot/microbiology , Plants, MedicinalABSTRACT
In order to investigate whether defense genes PAL, LOX, PBZ1, PR 1 a and Cht 1 participate in APR(adult plant resistance)to rice bacterial blight, their expression were analyzed using RT-PCR. Enzymatic activities of PAL and LOX were also measured. Results indicated that PAL was induced by pathogen and wounding in adult plants while it only induced by pathogen at the seedlings, and the expression of PAL was stronger in adult plants than that in seedlings. Expression of LOX was induced by pathogen both in seedlings and adult plants and it was stronger in adult plants than that in seedlings. Expression PBZ1 was induced by both pathogen and wounding in both seedlings and adult plants and it is earlier and stronger in adult plants than that in seedlings. No expected fragments were obtained for PR 1 a and Cht 1. Enzymatic activities of PAL and LOX were consistent with their mRNA accumulations, respectively. It is probable that activation of PAL,LOX, and PBZ1 play crucial roles in APR to rice bacterial blight.