Biochemical and histological investigations of viral localisation in the hypersensitive reaction of Phaselous vulgaris L. var Pinto to tobacco mosaic virus infection

STOBBS, Lorne,W ABSTRACT Biochemical and Histological Investigations of viral localisation in the hypersensitive reaction of Phaseolus vulgaris L. var Pinto to tobacco mosaic virus infection. The infection of Phaseolus vulgaris L. var Pinto with tobacco mosaic virus (TMV) results in the production of distinct necrotic lesions confining the virus to restricted areas of the leaf surface. Biochemical and histological changes in the leaf tissue as a result of infection have been described. Trace accumulations of fluorescent metabolites, detected prior to lesion expression represent metabolites produced, by the cell in response to virus infection. These substances, are considered to undergo oxidation and in diffusing into adjacent cells, react with cellular constituents causing the death of these cells. Such cellular necrosis in advance of infection effectively limits virus spread. Chromatographic studies on extracts from TMV infected Pinto bean leaf tissue suggests that a number of extra-fluorescent metabolites produced on lesion'expression represent end products of phenolic oxidation r,eactionsoccurring earlier in these cells. Inhibition of phenolic oxidation by ascorbate infiltration or elevated temperature treatment resulted in the absence of extra-fluorescent metabolites and the continued movement of virus in the absence of necrosis. Further studies with i ascorbate infiltration indicated that irreversible necrotic events were determined as early as 12 tci 18 hrs after viral inoculation. Histochemical tests indicated that callose formation was initiated at this time, and occurred in response to necrotisation. Inhibition of necrosis by either ascorbate infiltration or elevated temp8rature treatment resulted in the absence of callose deposition. Scanning electron'micrographs of infected tissue revealed severe epidermal and palisade cell damage. Histochemical tests indicated extensive callose formation in cells bordering the lesion, and suggested the role of callose iTh the blockage of intercellular connections limiting virus movement. The significance of these cellular changes is discussed. ii

Dissecting Phaseolus vulgaris Innate Immune System against Colletotrichum lindemuthianum Infection

Background: The genus Colletotrichum is one of the most economically important plant pathogens, causing anthracnose on a wide range of crops including common beans (Phaseolus vulgaris L.). Crop yield can be dramatically decreased depending on the plant cultivar used and the environmental conditions. This study aimed to identify potential genetic components of the bean immune system to provide environmentally friendly control measures against this fungus. Methodology and Principal Findings: As the common bean is not amenable to reverse genetics to explore functionality and its genome is not fully curated, we used putative Arabidopsis orthologs of bean expressed sequence tag (EST) to perform bioinformatic analysis and experimental validation of gene expression to identify common bean genes regulated during the incompatible interaction with C. lindemuthianum. Similar to model pathosystems, Gene Ontology (GO) analysis indicated that hormone biosynthesis and signaling in common beans seem to be modulated by fungus infection. For instance, cytokinin and ethylene responses were up-regulated and jasmonic acid, gibberellin, and abscisic acid responses were down-regulated, indicating that these hormones may play a central role in this pathosystem. Importantly, we have identified putative bean gene orthologs of Arabidopsis genes involved in the plant immune system. Based on experimental validation of gene expression, we propose that hypersensitive reaction as part of effector-triggered immunity may operate, at least in part, by down-regulating genes, such as FLS2-like and MKK5-like, putative orthologs of the Arabidopsis genes involved in pathogen perception and downstream signaling. Conclusions/Significance: We have identified specific bean genes and uncovered metabolic processes and pathways that may be involved in the immune response against pathogens. Our transcriptome database is a rich resource for mining novel defense-related genes, which enabled us to develop a model of the molecular components of the bean innate immune system regulated upon pathogen attack.

Variability in the indument of Phaseolus vulgaris var. aborigineus (Fabaceae)

The indument of Phaseolus vulgaris L. var. aborigineus (Burkart) Baudet, a native bean from Argentina, potential source for improvement of cultivated varieties, was compared between different genetic lines. Seeds collected from wild, weedy forms and presumptive hybrid individuals in two distant latitudes were sown in two sites of Buenos Aires University Campus (field and greenhouse). Two type of trichomes were found, which differenciate in length and shape. The pubescence density was significantly different between wild specimens from different latitudes and between culture sites for plants of a same procedence. This character could be involved in physical defences of these plants.

Inoculaci?n y establecimiento endof?tico de cepas de los hongos entomopat?genos beauveria bassiana, Metarhizium anisopliae y lecanicillium lecanii en plantas de fr?jol (Phaseolus vulgaris) [recurso electr?nico]

Los hongos end?fitos han recibido una creciente atenci?n en los a?os recientes, ya que han demostrado tener un potencial econ?mico en la agronom?a, debido a su posible uso como agentes de control biol?gico. El establecimiento endof?tico de hongos entomopat?genos en cultivos de inter?s, ofrece un campo amplio y prometedor para comprender las relaciones entre plantas y hongos y los mecanismos de defensa que estos ?ltimos pueden inducir en las plantas. El presente estudio busc? inocular y evaluar la colonizaci?n endof?tica de tres especies de hongos entomopat?genos en plantas de fr?jol (Phaseolus vulgaris). Para ello, se emplearon dos cepas de cada especie, una comercial y la otra de la colecci?n de entomopat?genos del Centro Internacional de Agricultura Tropical; cada cepa constituy? un tratamiento individual. A partir de la siembra y desarrollo de las cepas, se generaron in?culos de 108 conidios/mL que fueron inoculados en las plantas de fr?jol Diacol, variedad Calima. Se encontr? que Beauveria bassiana, Metarhizium anisopliae y Lecanicillium lecanii colonizaron individualmente las hojas de fr?jol. Los porcentajes de colonizaci?n promedio para las cepas de la colecci?n del CIAT fueron de 16% (?= 18,84) para B. bassiana, 13% (?= 4,96) en M. anisopliae y 4% (?= 4,64) para L. lecanii. Con respecto a las cepas comerciales, estos valores fueron 4% (?= 4,64) para B. bassiana; 26% (?= 32) para M. anisopliae y 44% (?= 13,02) para L. lecanii. Todas las cepas de hongos inoculadas pudieron ser recuperadas de tejido vegetal, lo que sugiere su posible uso como agentes de control biol?gico al menos para el cultivo del fr?jol.

Identification of novel drought-tolerant-associated SNPs in common bean (Phaseolus vulgaris)

Common bean (Phaseolus vulgaris L.) is a leguminous in high demand for human nutrition and a very important agricultural product. Production of common bean is constrained by environmental stresses such as drought. Although conventional plant selection has been used to increase production yield and stress tolerance, drought tolerance selection based on phenotype is complicated by associated physiological, anatomical, cellular, biochemical, and molecular changes. These changes are modulated by differential gene expression. A common method to identify genes associated with phenotypes of interest is the characterization of Single Nucleotide Polymorphims (SNPs) to link them to specific functions. In this work, we selected two drought-tolerant parental lines from Mesoamerica, Pinto Villa, and Pinto Saltillo. The parental lines were used to generate a population of 282 families (F3:5) and characterized by 169 SNPs. We associated the segregation of the molecular markers in our population with phenotypes including flowering time, physiological maturity, reproductive period, plant, seed and total biomass, reuse index, seed yield, weight of 100 seeds, and harvest index in three cultivation cycles. We observed 83 SNPs with significant association (p < 0.0003 after Bonferroni correction) with our quantified phenotypes. Phenotypes most associated were days to flowering and seed biomass with 58 and 44 associated SNPs, respectively. Thirty-seven out of the 83 SNPs were annotated to a gene with a potential function related to drought tolerance or relevant molecular/biochemical functions. Some SNPs such as SNP28 and SNP128 are related to starch biosynthesis, a common osmotic protector; and SNP18 is related to proline biosynthesis, another well-known osmotic protector.

Development of super early genotypes for the dry bean (Phaseolus vulgaris) as affected by nitrogen management.

The super early genotypes (SEG) of dry bean (Phaseolus vulgaris L.) have a shorter life cycle (65-75 days) when compared with the season length of traditional cultivars (90-100 days). Timing of nitrogen top-dressing fertilization could be different because of this reduction in length of the SEG life cycle. This study aimed at characterizing, by using growth analysis and vegetation index, super early genotypes of dry bean development as affected by timing of nitrogen application. Field experiments were conducted in the 2014 and 2015 growing seasons in central Brazil with a randomized block experimental design with split plots scheme and four replicates. The plots comprised the dry bean genotypes (Colibri ? check cultivar, CNFC 15873, CNFC 15874, and CNFC 15875), and subplots comprised applications of N at different timings: 90 kg of N at sowing, 90 kg N at top-dressing; 45 kg of N at sowing plus 45 kg at top-dressing, with urea as the source of N. We also used a control treatment without N application. The CNFC 15874 super early genotype of dry bean had the higher grain yield (2776 kg ha-1) and differed from the CNFC 15873 genotype (2492 kg ha-1). Nitrogen fertilization allowed higher grain yield (2619 kg ha-1, when applied N at sowing, 2605 kg ha-1, when applied N at sowing and at top-dressing, and 2680 kg ha-1, when applied N at top-dressing) than the control, 2360 kg ha-1 (no N fertilization). The time of N fertilization in super early genotype of dry bean did not affect grain yield.

Deposição e distribuição de calda de pulverização em plantas de feijoeiro (Phaseolus vulgaris) e capim-braquiária (Brachiaria decumbens)

Com o objetivo de avaliar a deposição e distribuição de solução traçante em plantas de feijoeiro e capim-braquiária, foi conduzido experimento no NuPAM-FCA/ UNESP, campus de Botucatu-SP, utilizando um simulador de pulverização em ambiente controlado. Os tratamentos utilizados foram: bico jato plano XR Teejet (XR110.02VS) + surfactante Aterbane BR (0,5% v/v); bico jato cônico Conejet (TXVK-4) + surfactante Aterbane BR (0,5% v/v); bico jato plano XR Teejet (XR110.02VS); e bico jato cônico Conejet (TXVK-4). Em todos os tratamentos foi aplicada uma solução de NaCl (0,5% p/v) + corante Poliglow laranja (0,5% p/v) + mancozeb (0,5% p/v). As unidades experimentais constituíram-se de vasos com duas plantas-alvo de capim-braquiária posicionadas sob uma planta-alvo de feijoeiro. A visualização da distribuição das gotas nas folhas de capim-braquiária foi efetuada com auxílio de luz negra, e o depósito da calda na superfície das plantas foi quantificado através da condutividade elétrica da solução aplicada e coletada por meio de lavagem de ambos os alvos. Os bicos de pulverização, jato plano (XR Teejet) e cônico (Conejet), não apresentaram diferença no depósito nos folíolos totais de feijoeiro quando submetidos a mesma condição de calda de pulverização. No entanto, o bico jato plano XR Teejet (XR110.02VS) e o bico jato cônico Conejet (TXVK-4) proporcionaram aumento de 67,1 e 61,5% na deposição da calda em relação a área foliar e 106,4 e 66,9% para matéria seca, respectivamente, em relação ao bico jato plano XR Teejet (XR110.02VS) + surfactante Aterbane BR (0,5% v/v) e ao bico jato cônico Conejet (TXVK-4) + surfactante Aterbane BR (0,5% v/v). Para o capim-braquiária, o bico cônico Conejet proporcionou deposição superior e distribuição mais uniforme em relação ao jato plano XR Teejet.

Qualidade de sementes de feijão (Phaseolus vulgaris) após aplicação do carfentrazone-ethyl em pré-colheita

Objetivou-se neste trabalho avaliar os efeitos da dessecação de plantas de feijão (cultivar Talismã) com carfentrazone-ethyl sobre a qualidade das sementes. Realizou-se a dessecação das plantas utilizando cinco doses (0, 10, 30, 60 e 120 g ha-1) de carfentrazone associadas a três épocas de aplicação: 25, 30 e 35 dias após o florescimento (DAF). Em intervalos de dois dias após cada aplicação, quantificou-se a evolução da perda de umidade das sementes. Oito dias após cada aplicação, realizou-se a colheita, sendo determinados o peso de 100 sementes e a proporção de sementes classificadas como "pequenas" (que passaram por peneira de crivo 14/64"), "médias" (retidas entre as peneiras de crivo 14/64" e 16/64") e "maiores" (que ficaram retidas na peneira de crivo 16/64"). A viabilidade das sementes foi avaliada pelo teste de germinação (TG). A aplicação de carfentrazone-ethyl em doses superiores a 60 g ha-1 aos 25 DAF acelerou a perda de umidade das sementes, afetando, o tamanho e o peso de 100 sementes. Melhores resultados foram obtidos com aplicação do dessecante nas doses entre 10 e 30 g ha-1, aos 30 DAF, proporcionando melhor rendimento e qualidade das sementes, sem efeito negativo sobre a germinação.

Purification and partial characterization of Phaseolus vulgaris seed aminopeptidase

The aminopeptidase activity of Phaseolus vulgaris seeds was measured using L-Leu-p-nitroanilide and the L-aminoacyl-ß-naphthylamides of Leu, Ala, Arg and Met. A single peak of aminopeptidase activity on Leu-ß-naphthylamide was eluted at 750 µS after gradient elution chromatography on DEAE-cellulose of the supernatant of a crude seed extract. The effluent containing enzyme activity was applied to a Superdex 200 column and only one peak of aminopeptidase activity was obtained. SDS-polyacrylamide gel electrophoresis (10%) presented only one protein band with molecular mass of 31 kDa under reducing and nonreducing conditions. The aminopeptidase has an optimum pH of 7.0 for activity on all substrates tested and the highest Vmax/KM ratio for L-Leu-ß-naphthylamide. The enzyme activity was increased 40% by 0.15 M NaCl, inhibited 94% by 2.0 mM Zn2+, inhibited 91% by sodium p-hydroxymercuribenzoate and inhibited 45% by 0.7 mM o-phenanthroline and 30 µM EDTA. Mercaptoethanol (3.3 mM), dithioerythritol (1.7 mM), Ala, Arg, Leu and Met (70 µM), p-nitroaniline (0.25 mM) and ß-naphthylamine (0.53 mM) had no effect on enzyme activity when assayed with 0.56 mM of substrate. Bestatin (20 µM) inhibited 18% the enzyme activity. The aminopeptidase activity in the seeds decayed 50% after two months when stored at 4oC and room temperature. The enzyme is leucyl aminopeptidase metal- and thiol group-dependent.

Effect of an aqueous extract of Phaseolus vulgaris on the properties of tail tendon collagen of rats with streptozotocin-induced diabetes

Changes in the structural and functional properties of collagen caused by advanced glycation might be of importance for the etiology of late complications in diabetes. The present study was undertaken to investigate the influence of oral administration of aqueous pod extract (200 mg/kg body weight) of Phaseolus vulgaris, an indigenous plant used in Ayurvedic Medicine in India, on collagen content and characteristics in the tail tendon of streptozotocin-diabetic rats. In diabetic rats, collagen content (117.01 ± 6.84 mg/100 mg tissue) as well as its degree of cross-linking was increased, as shown by increased extent of glycation (21.70 ± 0.90 µg glucose/mg collagen), collagen-linked fluorescence (52.8 ± 3.0 AU/µmol hydroxyproline), shrinkage temperature (71.50 ± 2.50ºC) and decreased acid (1.878 ± 0.062 mg hydroxyproline/100 mg tissue) and pepsin solubility (1.77 ± 0.080 mg hydroxyproline/100 mg tissue). The alpha/ß ratio of acid- (1.69) and pepsin-soluble (2.00) collagen was significantly decreased in streptozotocin-diabetic rats. Administration of P. vulgaris for 45 days to streptozotocin-diabetic rats significantly reduced the accumulation and cross-linking of collagen. The effect of P. vulgaris was compared with that of glibenclamide, a reference drug administered to streptozotocin-diabetic rats at the dose of 600 µg/kg body weight for 45 days by gavage. The effects of P. vulgaris (collagen content, 64.18 ± 1.97; extent of glycation, 12.00 ± 0.53; collagen-linked fluorescence, 33.6 ± 1.9; shrinkage temperature, 57.0 ± 1.0; extent of cross-linking - acid-soluble collagen, 2.572 ± 0.080, and pepsin-soluble collagen, 2.28 ± 0.112) were comparable with those of glibenclamide (collagen content, 71.5 ± 2.04; extent of glycation, 13.00 ± 0.60; collagen-linked fluorescence, 38.9 ± 2.0; shrinkage temperature, 59.0 ± 1.5; extent of cross-linking - acid-soluble collagen, 2.463 ± 0.078, and pepsin-soluble collagen, 2.17 ± 0.104). In conclusion, administration of P. vulgaris pods had a positive influence on the content of collagen and its properties in streptozotocin-diabetic rats.

Early changes in apoplast composition associated with defence and disease in interactions between Phaseolus vulgaris and the halo blight pathogen Pseudomonas syringae pv. phaseolicola

The apoplast is the arena in which endophytic pathogens such as Pseudomonas syringae grow and interact with plant cells. Using metabolomic and ion analysis techniques, this study shows how the composition of Phaseolus vulgaris leaf apoplastic fluid changes during the first six hours of compatible and incompatible interactions with two strains of Pseudomonas syringae pv. phaseolicola (Pph) that differ in the presence of the genomic island PPHGI-1. Leaf inoculation with the avirulent island-carrying strain Pph 1302A elicited effector-triggered immunity (ETI) and resulted in specific changes in apoplast composition, including increases in conductivity, pH, citrate, γ-aminobutyrate (GABA) and K+, that are linked to the onset of plant defence responses. Other apoplastic changes, including increases in Ca2+, Fe2/3+ Mg2+, sucrose, β-cyanoalanine and several amino acids, occurred to a relatively similar extent in interactions with both Pph 1302A and the virulent, island-less strain Pph RJ3. Metabolic footprinting experiments established that Pph preferentially metabolizes malate, glucose and glutamate, but excludes certain other abundant apoplastic metabolites, including citrate and GABA, until preferred metabolites are depleted. These results demonstrate that Pph is well-adapted to the leaf apoplast metabolic environment and that loss of PPHGI-1 enables Pph to avoid changes in apoplast composition linked to plant defences.

Free Cyclitol, Soluble Carbohydrate and Protein Contents in Vigna unguiculata and Phaseolus vulgaris Bean Sprouts

Seeds sprouts have been used as a good source of basic nutrients and nutraceutical compounds. The high nutritional value of seeds derives from the deposition of compounds during development. However some of these molecules are used in metabolic processes like germination, which leads to a considerable variation in their concentrations once these events are completed. In this work, we investigate the levels of inositols (myo-inositol, D-pinitol and ononitol), soluble carbohydrates and proteins in cotyledons of Phaseolus vulgaris and Vigna unguiculata sprouts. Sprouting increased myo-inositol and glucose content and reduction of raffinose and ononitol was observed. The protein levels increased in P. vulgaris and decreased in V. unguiculata sprouting. The level of sucrose was maintained in both sprouts. D-Pinitol was detected only in quiescent seeds. Our results suggested that bean sprout is an important source of proteins, sucrose, glucose and myo-inositol. Additionally, bean sprouts have low levels of raffinose, an antinutritional compound.

Deposição e distribuição de calda de pulverização em plantas de feijoeiro (Phaseolus vulgaris) e capim-braquiária (Brachiaria decumbens)

Com o objetivo de avaliar a deposição e distribuição de solução traçante em plantas de feijoeiro e capim-braquiária, foi conduzido experimento no NuPAM-FCA/ UNESP, campus de Botucatu-SP, utilizando um simulador de pulverização em ambiente controlado. Os tratamentos utilizados foram: bico jato plano XR Teejet (XR110.02VS) + surfactante Aterbane BR (0,5% v/v); bico jato cônico Conejet (TXVK-4) + surfactante Aterbane BR (0,5% v/v); bico jato plano XR Teejet (XR110.02VS); e bico jato cônico Conejet (TXVK-4). em todos os tratamentos foi aplicada uma solução de NaCl (0,5% p/v) + corante Poliglow laranja (0,5% p/v) + mancozeb (0,5% p/v). As unidades experimentais constituíram-se de vasos com duas plantas-alvo de capim-braquiária posicionadas sob uma planta-alvo de feijoeiro. A visualização da distribuição das gotas nas folhas de capim-braquiária foi efetuada com auxílio de luz negra, e o depósito da calda na superfície das plantas foi quantificado através da condutividade elétrica da solução aplicada e coletada por meio de lavagem de ambos os alvos. Os bicos de pulverização, jato plano (XR Teejet) e cônico (Conejet), não apresentaram diferença no depósito nos folíolos totais de feijoeiro quando submetidos a mesma condição de calda de pulverização. No entanto, o bico jato plano XR Teejet (XR110.02VS) e o bico jato cônico Conejet (TXVK-4) proporcionaram aumento de 67,1 e 61,5% na deposição da calda em relação a área foliar e 106,4 e 66,9% para matéria seca, respectivamente, em relação ao bico jato plano XR Teejet (XR110.02VS) + surfactante Aterbane BR (0,5% v/v) e ao bico jato cônico Conejet (TXVK-4) + surfactante Aterbane BR (0,5% v/v). Para o capim-braquiária, o bico cônico Conejet proporcionou deposição superior e distribuição mais uniforme em relação ao jato plano XR Teejet.

Evaluación de la patogeneicidad de la microbiota fúngica asociada a cinco variedades autóctonas españolas de judión (Phaseolus coccineus L.)

Se caracterizó la micoflora presente en las semillas de 5 cultivares de judión (Phaseolus coccineus L.) procedentes de una explotación dedicada a la agricultura ecológica de Oteruelo del Valle (Parque natural de Rascafría, Madrid) y de 5 variedades incluidas en el Catálogo Común de variedades Comerciales de la Unión Europea. Se analizaron un total de 3200 semillas de todos los lotes cosechadas en la campaña 2005-2006. Para ello se colocaron 5 semillas de cada muestra en placas de Petri con medio agar de patata glucosado (PDA) y 6 semillas por muestra en cámara húmeda, realizándose 20 repeticiones en cada caso, analizándose de esta manera al menos 220 semillas por muestra. Para conocer la presencia del género Fusarium se realizaron análisis específicos con todas las muestras, utilizando para ello medio selectivo para Fusarium realizándose lecturas periódicas y anotando el número de especies presentes en cada semilla. Se identificaron un total de 11 especies fúngicas diferentes. la presencia de los diferentes géneros varió entre los cultivares estudiados, siendo mucho menor, aunque no ausente en las semillas comerciales. Entre la microbiota fúngica aislada cabe destacar, por su potencial patogeneicidd o por su capacidad para la producción de micotoxinas o metabolitos secundarios, especies de los géneros Aspergillus, Alternaria o Rhizoctonia. En una segunda parte del estudio se evaluó el efecto que dichos hongos tienen sobre la germinación y nascencia de las semillas, realizándose pruebas de patogeneicidad sobre un total de 200 semillas de Phaseolus vulgaris variedad Calgary. Las inoculaciones se realizaron con cada uno de los dos aislados de los seis géneros de mayor importancia cuantitativa del inventario(Aspergillus, Penicillium Ulocladium, Rhizopus, Cladosporium y Alternaria) Los resultados de las inoculaciones muestran efectos negativos sobre los porcentajes de germinación en todos los tratamientos estudiados y muestran la diferente capacidad parasitaria de cada una de las especies estudiadas sobre Phaseolus vulgaris.