Anatomical and Biochemical Changes Induced by Gluconacetobacter diazotrophicus Stand Up for Arabidopsis thaliana Seedlings From Ralstonia solanacearum Infection.

Nowadays, fertilization and pest control are carried out using chemical compounds that contaminate soil and deteriorate human health. Plant growth promoting bacteria endophytes (PGPBEs), are a well-studied group of bacteria that offers benefits to the host plant, such as phytostimulation, biofertilization, and protection against other microorganisms. The study of Gluconacetobacter diazotrophicus-which belongs to PGPBEs-aids the development of alternative strategies of an integrated approach for crop management practices. Ralstonia solanacearum is responsible for bacterial wilt disease. This phytopathogen is of great interest worldwide due to the enormous economic losses it causes. In this study the action of G. diazotrophicus as a growth promoting bacterium in Arabidopsis thaliana seedlings is analyzed, evaluating the antagonistic mechanisms of this beneficial endophytic bacterium during biotic stress produced by R. solanacearum. Effective colonization of G. diazotrophicus was determined through bacterial counting assays, evaluation of anatomical and growth parameters, and pigments quantification. Biocontrol assays were carried out with Ralstonia pseudosolanacearum GMI1000 model strain and R. solanacearum A21 a recently isolated strain. Inoculation of A. thaliana (Col 0) with G. diazotrophicus Pal 5 triggers a set of biochemical and structural changes in roots, stems, and leaves of seedlings. Discrete callose deposits as papillae were observed at specific sites of root hairs, trichomes, and leaf tissue. Upon R. pseudosolanacearum GMI1000 infection, endophyte-treated plants demonstrated being induced for defense through an augmented callose deposition at root hairs and leaves compared with the non-endophyte-treated controls. The endophytic bacterium appears to be able to prime callose response. Roots and stems cross sections showed that integrity of all tissues was preserved in endophyte-treated plants infected with R. solanacearum A21. The mechanisms of resistance elicited by the plant after inoculation with the endophyte would be greater lignification and sclerosis in tissues and reinforcement of the cell wall through the deposition of callose. As a consequence of this priming in plant defense response, viable phytopathogenic bacteria counting were considerably fewer in endophyte-inoculated plants than in not-inoculated controls. Our results indicate that G. diazotrophicus colonizes A. thaliana plants performing a protective role against the phytopathogenic bacterium R. solanacearum promoting the activation of plant defense system.

Suppression of Reactive Oxygen Species Accumulation in Chloroplasts Prevents Leaf Damage but Not Growth Arrest in Salt-Stressed Tobacco Plants.

Crop yield reduction due to salinity is a growing agronomical concern in many regions. Increased production of reactive oxygen species (ROS) in plant cells accompanies many abiotic stresses including salinity, acting as toxic and signaling molecules during plant stress responses. While ROS are generated in various cellular compartments, chloroplasts represent a main source in the light, and plastid ROS synthesis and/or elimination have been manipulated to improve stress tolerance. Transgenic tobacco plants expressing a plastid-targeted cyanobacterial flavodoxin, a flavoprotein that prevents ROS accumulation specifically in chloroplasts, displayed increased tolerance to many environmental stresses, including drought, excess irradiation, extreme temperatures and iron starvation. Surprisingly, flavodoxin expression failed to protect transgenic plants against NaCl toxicity. However, when high salt was directly applied to leaf discs, flavodoxin did increase tolerance, as reflected by preservation of chlorophylls, carotenoids and photosynthetic activities. Flavodoxin decreased salt-dependent ROS accumulation in leaf tissue from discs and whole plants, but this decline did not improve tolerance at the whole plant level. NaCl accumulation in roots, as well as increased osmotic pressure and salt-induced root damage, were not prevented by flavodoxin expression. The results indicate that ROS formed in chloroplasts have a marginal effect on plant responses during salt stress, and that sensitive targets are present in roots which are not protected by flavodoxin.

Novel insights into the Citrus sinensis nonhost response suggest photosynthesis decline, abiotic stress networks and secondary metabolism modifications.

Plants are constantly exposed to stress factors. Biotic stress is produced by living organisms such as pathogens, whereas abiotic stress by unfavourable environmental conditions. In Citrus species, one of the most important fruit crops in the world, these stresses generate serious limitations in productivity. Through biochemical and transcriptomic assays, we had previously characterised the Citrus sinensis (L.) Osbeck nonhost response to Xanthomonas campestris pv. vesicatoria (Doidge), in contrast to Asiatic citrus canker infection caused by Xanthomonas citri subsp. citri (Hasse). A hypersensitive response (HR) including changes in the expression of several transcription factors was reported. Here, a new exhaustive analysis of the Citrus sinensis transcriptomes previously obtained was performed, allowing us to detect the over-representation of photosynthesis, abiotic stress and secondary metabolism processes during the nonhost HR. The broad downregulation of photosynthesis-related genes was correlated with an altered photosynthesis physiology. The high number of heat shock proteins and genes related to abiotic stress, including aquaporins, suggests that stresses crosstalk. Additionally, the secondary metabolism exhibited lignin and carotenoid biosynthesis modifications and expression changes in the cell rescue GSTs. In conclusion, novel features of the Citrus nonhost HR, an important part of the plants' defence against disease that has yet to be fully exploited in plant breeding programs, are presented.

The LOV protein of Xanthomonas citri subsp. citri plays a significant role in the counteraction of plant immune responses during citrus canker.

Pathogens interaction with a host plant starts a set of immune responses that result in complex changes in gene expression and plant physiology. Light is an important modulator of plant defense response and recent studies have evidenced the novel influence of this environmental stimulus in the virulence of several bacterial pathogens. Xanthomonas citri subsp. citri is the bacterium responsible for citrus canker disease, which affects most citrus cultivars. The ability of this bacterium to colonize host plants is influenced by bacterial blue-light sensing through a LOV-domain protein and disease symptoms are considerably altered upon deletion of this protein. In this work we aimed to unravel the role of this photoreceptor during the bacterial counteraction of plant immune responses leading to citrus canker development. We performed a transcriptomic analysis in Citrus sinensis leaves inoculated with the wild type X. citri subsp. citri and with a mutant strain lacking the LOV protein by a cDNA microarray and evaluated the differentially regulated genes corresponding to specific biological processes. A down-regulation of photosynthesis-related genes (together with a corresponding decrease in photosynthesis rates) was observed upon bacterial infection, this effect being more pronounced in plants infected with the lov-mutant bacterial strain. Infection with this strain was also accompanied with the up-regulation of several secondary metabolism- and defense response-related genes. Moreover, we found that relevant plant physiological alterations triggered by pathogen attack such as cell wall fortification and tissue disruption were amplified during the lov-mutant strain infection. These results suggest the participation of the LOV-domain protein from X. citri subsp. citri in the bacterial counteraction of host plant defense response, contributing in this way to disease development.

Characterization of Citrus sinensis transcription factors closely associated with the non-host response to Xanthomonas campestris pv. vesicatoria.

Plants, when exposed to certain pathogens, may display a form of genotype-independent resistance, known as non-host response. In this study, the response of Citrus sinensis (sweet orange) leaves to Xanthomonas campestris pv. vesicatoria (Xcv), a pepper and tomato pathogenic bacterium, was analyzed through biochemical assays and cDNA microarray hybridization and compared with Asiatic citrus canker infection caused by Xanthomonas citri subsp. citri. Citrus leaves exposed to the non-host bacterium Xcv showed hypersensitive response (HR) symptoms (cell death), a defense mechanism common in plants but poorly understood in citrus. The HR response was accompanied by differentially expressed genes that are associated with biotic stress and cell death. Moreover, 58 transcription factors (TFs) were differentially regulated by Xcv in citrus leaves, including 26 TFs from the stress-associated families AP2-EREBP, bZip, Myb and WRKY. Remarkably, in silico analysis of the distribution of expressed sequence tags revealed that 10 of the 58 TFs, belonging to C2C2-GATA, C2H2, CCAAT, HSF, NAC and WRKY gene families, were specifically over-represented in citrus stress cDNA libraries. This study identified candidate TF genes for the regulation of key steps during the citrus non-host HR. Furthermore, these TFs might be useful in future strategies of molecular breeding for citrus disease resistance.

Multivariate statistical analysis of morpho-anatomical data of nine sect. Caulopterae species (Baccharis - Asteraceae) used in folk medicine

Abstract Baccharis species belonging to sect. Caulopterae are difficult to identify. Most countries are controlling the quality of herbal medicines destined for the internal market or export. "Carquejas" are used arbitrarily for the same medicinal purposes and only three species of sect. Caulopterae are official herbal medicines. In the present study, a morpho-anatomical and statistical analysis was performed with nine species of sect. Caulopterae: Baccharis articulata, B. crispa, B. gaudichaudiana, B. microcephala, B. penningtonii, B. phyteumoides, B. sagittalis, B. triangularis and B. trimera, emphasizing the importance of anatomy as a taxonomic tool. A total of 114 populations of these nine species were examined. The first three principal components of morphoanatomical data provided relevant information to classify the species (75.04% of the total variability). The most discriminatory variable in this issue was the stomatal index (1.0530). We determined the qualitative and quantitative variables in order to differentiate the species by using principal components analysis and ANOVA tests. Stomata type, uniseriate trichome type and presence/absence of collenchyma in the wing margin are the qualitative variables that should be analyzed. Regarding quantitative variables, the epidermal ones in superficial view are more important and discriminatory than those of alate stem cross section and they must be considered for proper quality control of the species of this work.

Contribution of a harpin protein from Xanthomonas axonopodis pv. citri to pathogen virulence.

Xanthomonas axonopodis pv. citri (Xac), the bacterium that causes citrus canker, contains a gene in the hrp [for hypersensitive response (HR) and pathogenicity] cluster that encodes a harpin protein called Hpa1. Hpa1 produced HR in the nonhost plants tobacco, pepper and Arabidopsis, whereas, in the host plant citrus, it elicited a weak defence response with no visible phenotype. Co-infiltrations of Xac with or without the recombinant Hpa1 protein in citrus leaves produced a larger number of cankers in the presence of the protein. To characterize the effect of Hpa1 during the disease, an XacΔhpa1 mutant was constructed, and infiltration of this mutant caused a smaller number of cankers. In addition, the lack of Hpa1 hindered bacterial aggregation both in solution and in planta. Analysis of citrus leaves infiltrated with Hpa1 revealed alterations in mesophyll morphology caused by the presence of cavitations and crystal idioblasts, suggesting the binding of the harpin to plant membranes and the elicitation of signalling cascades. Overall, these results suggest that, even though Hpa1 elicits the defence response in nonhost plants and, to a lesser extent, in host plants, its main roles in citrus canker are to alter leaf mesophyll structure and to aggregate bacterial cells, and thus increase virulence and pathogen fitness. We expressed the N-terminal and C-terminal regions and found that, although both regions elicited HR in nonhost plants, only the N-terminal region showed increased virulence and bacterial aggregation, supporting the role of this region of the protein as the main active domain.

Caracteres micrográficos analíticos de hojas, tallos, inflorescencias-flores de Tagetes lucida Cav. (Asteraceae ­ Helenieae) / Micrographic analytical characters of leaves, stems, inflorescences-flowers of Tagetes lucida Cav. (Asteraceae - Helenieae)

Tagetes lucida Cav. (Asteraceae) es una especie nativa de América tropical, comúnmente usada en medicina vernácula y también como medicamento fitoterápico. La infusión se emplea como tónico para combatir la tos, dolores de cabeza, fiebre, cólicos, dolores abdominales, enfermedades gastrointestinales, dolor corporal y como emenagoga. Los extractos etanólicos de hojas de T. lucida presentan actividad bactericida. El objetivo de este trabajo fue realizar la micrográfica analítica de las partes usadas de esta especie a los fines de la estandarización de la droga cruda. Para ello, se estudió la morfología foliar, la epidermis de la hoja, los estomas, la arquitectura foliar, la estructura interna de hojas, tallos, inflorescencias y flores. En los tallos se han determinado dos tipos de estructuras secretoras coexistentes, conductos y cavidades secretoras de origen esquizógeno; mientras que en las hojas y brácteas involucrales solo encontramos cavidades. Los conductos secretores se caracterizan por presentar escaso diámetro, epitelio secretor uniestratificado y vaina parenquimática. En tanto, las cavidades secretoras son de diámetro conspicuo y epitelio secretor pluriestratificado sin la vaina parenquimática. Esta información es requerida no solo para los procedimientos de identificación que garanticen la utilización de la droga vegetal, sino para el control de calidad exigido en la producción de medicamentos fitoterápicos. (AU)

Tripanocide and antibacterial activity ofAlvaradoa subovata Cronquist extracts / Actividad tripanocida y antibacteriana de extractos de Alvaradoa subovata Cronquist

We studied antioxidant, antibacterial and tripanocide activities of Alvaradoa subovata extracts. The ethanolic extracts showed the greatest DPPH radical scavenging capacity, especially that of bark with an IC50 = 4.7 +/- 0.18 ug/mL. Wood dichloromethane extract displayed growth inhibition of the phytopathogenic bacteria Xanthomona axonopodis in the disk diffusion assay and showed a MIC value of 100 ug/ml. It also showed growth inhibition of Trypanosoma cruzi (IC50 = 0.063 +/- 0.003 mg/mL). A fraction of this extract, which has emodin as the main component, showed tripanocide activity (60 percent of growth inhibition at 100 ug/mL). The main compounds in wood dichloromethane extract were anthraquinones, identified as chrysophanol and emodin, and coumarins, of which scopoletin was identified. These three compound s could serve as analytical markers of the extract. The results of this study show that wood extract of A. subovata constitute a source of bioactive compounds such as antiparasitic and pesticides agents.

En el presente trabajo se estudió la actividad antioxidante, antibacteriana y tripanocida de extractos de Alvaradoa subovata. La mayor actividad depuradora de radicales libres se observó en el extracto etanólico de corteza (CI50 = 4.7 +/- 0.18 ug/mL). El extracto en diclorometano de madera inhibió el crecimiento de la bacteria fitopatógena Xanthomona axonopodis con una CIM = 100 ug/mL. El mismo extracto mostró inhibición del crecimiento de Trypanosoma cruzi (CI50 = 0.063 +/- 0.003 mg/mL). Una fracción de este extracto (100 ug/mL), cuyo componente mayoritario es emodina, inhibió en un 60 por ciento el crecimiento del parásito. Los compuestos mayoritarios detectados en el extracto de madera fueron antraquinonas, entre las cuales se identificaron emodina y crisofanol, y la cumarina escopoletina. Estos tres compuestos podrían servir como marcadores analíticos del extracto. Los resultados de este trabajo muestran que los extractos de A. subovata constituyen una fuente de compuestos bioactivos con potencial como antiparasitarios y plaguicidas.

Parámetros micrográficos para la identificación de leño, corteza y hoja de Quassia amara L. (Simaroubaceae) / Micrographic parameters for identification of Quassia amara L., wood, bark and leaf

Quassia amara L. popularly known as “quasia”, is a shrubby plant from Tropical America. The wood, bark and leave are used in either folk medicine or in procuring phytotherapeutic drugs. The aim of the present work was to analize morphoanatomical and micrographic features which might provide assistance in the identification, analysis and standardization of Quasia amara L wood, bark and leaves. Results. Anatomical study showed white yellowish and diffuse porous wood, confluent paratracheal parenchyma. Rays width 1 cell wide and 8-30 cells high. CaOx crystals are absent. Cortex, 1-4 mm thick, a periderm up to 12 layers phellem cells. Leaf, hipostomatic with dorsiventral mesophyll and high number of sclerosed idioblasts.

Quassia amara L. popularmente conocida como “quasia” es un planta arbustiva de América Tropical. El leño, corteza y hojas son usadas tanto en medicina popular como en la obtención de drogas fitoterapéuticas. El objetivo del presente trabajo es analizar características morfoanatómicas y micrográficas las cuales provean asistencia en la identificación, análisis y estandarización de la madera, corteza y hojas de Quassia amara L. Resultados. El estudio anatómico mostró leño, blanco amarillento, de porosidad difusa. Parénquima paratraqueal confluente. Radios de 1 célula de ancho y 8-30 hileras de alto. Faltan cristales CaOx. Corteza, 1-4 mm de espesor, una peridermis de hasta 12 estratos de células de súber. Hoja, hipoestomática, con mesófilo dorsiventral, con elevado número de idioblastos esclerosados.

Morrenain b I, a papain-like endopeptidase from the latex of Morrenia brachystephana Griseb. (Asclepiadaceae).

A new cysteine endopeptidase (morrenain b I) has been purified and characterized from the latex of stems and petiols of Morrenia brachystephana Griseb. (Asclepiadaceae). Morrenain b I was the minor proteolytic component in the latex but showed higher specific activity than morrenain b II, which was the main active fraction. Both enzymes showed similar pH profiles and molecular masses, but kinetic parameters and N-terminal sequences were quite distinct, demonstrating that they are different enzymes instead of different forms of the same enzyme.