Rhizobial Nod factors are required for cortical cell division in the nodule morphogenetic programme of the Aeschynomeneae legume Arachis

Nod factors are among the best-studied molecules implicated in the signal exchange that leads to legume-rhizobia symbiosis. The role of these molecules in symbiosis development has been primarily studied in legumes invaded through infection threads. In these plants, Nod factors generate several responses required for nodulation, including the induction of cortical cell division to form the nodule primordium. Arachis hypogaea L. (peanut) exhibits a specific mode of rhizobial infection and nodule morphogenetic programme in which infection threads are never formed. The role of Nod factors in this particular mechanism is unknown. In this work, a peanut symbiont mutant strain unable to produce Nod factors was obtained and characterised. The strain Bradyrhizobium (Arachis) sp. SEMIA 6144V2 is altered in the nodC gene, which encodes an N-acetylglucosaminyl transferase involved in the first step of the Nod factor biosynthetic pathway. Further research revealed that, although its ability to colonise peanut roots was unaffected, it is not capable of inducing the division of cortical cells. The results obtained indicate that rhizobial Nod factors are essential for the induction of cortical cell division that leads to nodule primordium formation. © 2011 German Botanical Society and The Royal Botanical Society of the Netherlands.Fil: Ibáñez, Valeria. Universidad Nacional de Río Cuarto; ArgentinaFil: Fabra, Adriana Isidora. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Instituto de Investigaciones Agrobiotecnológicas. - Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones Agrobiotecnológicas; Argentin

Starting points in plant-bacteria nitrogen-fixing symbioses: intercellular invasion of the roots

Agricultural practices contribute to climate change by releasing greenhouse gases such as nitrous oxide that are mainly derived from nitrogen fertilizers. Therefore, understanding biological nitrogen fxation in farming systems is benefcial to agriculture and environmental preservation. In this context, a better grasp of nitrogen-fxing systems and nitrogen-fxing bacteria-plant associations will contribute to the optimization of these biological processes. Legumes and actinorhizal plants can engage in a symbiotic interaction with nitrogen-fxing rhizobia or actinomycetes, resulting in the formation of specialized root nodules. The legume-rhizobia interaction is mediated by a complex molecular signal exchange, where recognition of different bacterial determinants activates the nodulation program in the plant. To invade plants roots, bacteria follow different routes, which are determined by the host plant. Entrance via root hairs is probably the best understood. Alternatively, entry via intercellular invasion has been observed in many legumes. Although there are common features shared by intercellular infection mechanisms, differences are observed in the site of root invasion and bacterial spread on the cortex reaching and infecting a susceptible cell to form a nodule. This review focuses on intercellular bacterial invasion of roots observed in the Fabaceae and considers, within an evolutionary context, the different variants, distribution and molecular determinants involved. Intercellular invasion of actinorhizal plants and Parasponia is also discussed.Fil: Ibañez, Fernando Julio. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas, Fisicoquímicas y Naturales. Departamento de Ciencias Naturales; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; ArgentinaFil: Wall, Luis Gabriel. Universidad Nacional de Quilmes; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Fabra, Adriana Isidora. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas, Fisicoquímicas y Naturales. Departamento de Ciencias Naturales; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; Argentin

Induced systemic resistance -like responses elicited by rhizobia

Rhizobia are soil bacteria that engage into a mutualistic symbiosis with plants and benefit the host by fixing atmospheric N. In addition, rhizobia can be considered as biocontrol agents, contributing to plant health through direct inhibition of a wide range of pathogens. More recently, it became evident that rhizobial invasion of plant roots can also trigger an increased systemic resistance state in the host, a process resembling the Induced Systemic Resistance (ISR) mechanism. However, this indirect biocontrol property of rhizobia was relatively less explored. Scope: In this review article, we present an overview of the current knowledge of ISR -like responses induced by rhizobia, considering general characteristics of this phenomenon, discussing the molecular pathways leading to this response and highlighting potential links between ISR -like responses and the nodulation signaling pathway. Conclusions: A more detailed knowledge of these responses can result in development of biotechnological tools for sustainable crop production, through optimization of the systemic protective effect conferred by rhizobia.Fil: Tonelli, Maria Laura. Universidad Nacional de Rio Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Instituto de Investigaciones Agrobiotecnológicas - Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones Agrobiotecnológicas; ArgentinaFil: Figueredo, María Soledad. Universidad Nacional de Rio Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Instituto de Investigaciones Agrobiotecnológicas - Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones Agrobiotecnológicas; ArgentinaFil: Rodríguez, Johan. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas, Fisicoquímicas y Naturales. Departamento de Ciencias Naturales; ArgentinaFil: Fabra, Adriana Isidora. Universidad Nacional de Rio Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Instituto de Investigaciones Agrobiotecnológicas - Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones Agrobiotecnológicas; ArgentinaFil: Ibañez, Fernando Julio. Universidad Nacional de Rio Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Instituto de Investigaciones Agrobiotecnológicas - Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones Agrobiotecnológicas; Argentin

Identification of miRNAs linked to peanut nodule functional processes

microRNAs (miRNAs) are non-coding small RNAs that regulate gene expression at post-transcriptional level. Thousands of miRNAs have been identified in legumes, but studies about miRNAs linked to peanut nodule functionality are scarce. In this work we analyzed transcriptional changes in peanut nodules to identify miRNAs involved in functional processes of these organs. We found 32 miRNAs precursors differentially expressed in nodules compared with roots, and predicted the potential targets of their corresponding mature miRNAs. Among them, 20 belong to 14 conserved miRNAs families and 12 are Arachis hypogaea-specific miRNAs. Expression levels of 3 miRNAs (ahy-miR399, ahy-miR159 and ahy-miR3508) were confirmed experimentally by qPCR. We also demonstrated that the expression of these miRNAs was not affected by inoculation of a biocontrol bacterium or a fungal pathogen. The catalogue of differentially expressed miRNA precursors and the expression of the corresponding mature miRNA potential targets in the nodules of A. hypogaea obtained in this work is a database of strong candidates, including A. hypogaea-specific miRNAs, for the regulation of the nodule functionality. The analysis of their role in this process will certainly lead to the characterization of essential regulators in these particular aeschynomenoid nodules.Fil: Figueredo, María Soledad. Universidad Nacional de Rio Cuarto. Facultad de Cs.exactas Fisicoquimicas y Naturales. Instituto de Investigaciones Agrobiotecnologicas. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Cordoba. Instituto de Investigaciones Agrobiotecnologicas.; ArgentinaFil: Formey, Damien. Universidad Nacional Autónoma de México; MéxicoFil: Rodríguez, Johan. Universidad Nacional de Rio Cuarto. Facultad de Cs.exactas Fisicoquimicas y Naturales. Instituto de Investigaciones Agrobiotecnologicas. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Cordoba. Instituto de Investigaciones Agrobiotecnologicas.; ArgentinaFil: Ibañez, Fernando Julio. Universidad Nacional de Rio Cuarto. Facultad de Cs.exactas Fisicoquimicas y Naturales. Instituto de Investigaciones Agrobiotecnologicas. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Cordoba. Instituto de Investigaciones Agrobiotecnologicas.; ArgentinaFil: Hernández Delgado, Georgina. Universidad Nacional Autónoma de México; MéxicoFil: Fabra, Adriana Isidora. Universidad Nacional de Rio Cuarto. Facultad de Cs.exactas Fisicoquimicas y Naturales. Instituto de Investigaciones Agrobiotecnologicas. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Cordoba. Instituto de Investigaciones Agrobiotecnologicas.; Argentin

Development and field evaluation of liquid inoculants with native Bradyrhizobial strains for peanut production

A critical process in the leguminous crops cycles is biological nitrogen fixation (BNF). Application of inoculants with N fixing bacteria is economically and environmentally favourable. The aim of this work was to select competitive native peanut microsymbionts, evaluate their survival in inoculant support and assess their impact on peanut (Arachis hypogaea L.) production under field conditions at Córdoba province in Argentina. The efficient N fixing Bradyrhizobium sp. J-81 and Bradyrhizobium sp. J-237, previously obtained from peanut nodules in the region of Cordoba, Argentina, were evaluated. In microcosm assays, plants inoculated with these isolates demonstrated better symbiotic parameters than those inoculated with reference strains. Different bacterial growth media and inoculant stabiliser solutions were evaluated. Balanced medium and arabic gum stabilising solution had optimal bacterial growth and the highest bacterial concentration and viability, respectively. Inoculation with either inoculants resulted in 44% greater peanut pod yield at Pizarro compared to the non-inoculated plants, although no significant differences were found with respect to commercial inoculants treatments.La fixation biologique de l’Azote (FBA) est un processus important dans le cycle de vie des légumineuses. L’application d’inoculum de bactéries fixatrices d’azote est favorable au double plan économique et environnemental. Le but de cette étude était de sélectionner des bactéries symbiotiques de l’arachide natives et compétitives, évaluer leur temps de survie dans support d’inoculum et évaluer leur impact sur la production en plein champ de l’arachide (Arachis hypogaea L.) dans la province de Córdoba en Argentine. Les bactéries fixatrices d’azote Bradyrhizobium sp. J-81 et Bradyrhizobium sp. J-237, extraites de nodules collectés sur des plants cultivés dans la région de Cordoba en Argentina, ont été évaluées. Dans des essais de microcosme, des plants inoculés avec ces isolats ont exhibés de meilleurs paramètres symbiotiques que les plants non inoculés. Différents média de culture bactérienne et supports inoculums ont été testés. Medium mixte et solution stabilisée à la gomme arabique ont respectivement exhibés la croissance optimale des bactéries et la meilleure conservation et viabilité des bactéries. L’application de n’importe quel inoculum produisit 44% plus de rendement en gousses d’arachides à Pizarro par rapport aux plantes non-inoculées, et ceci bien qu’aucune différence significative n’a été observée en comparaison avec les traitements à l’inoculum du commerce.Fil: Valetti, Lucio. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquimicas y Naturales. Departamento de Ciencias Naturales. Cátedra de Biologia General; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; ArgentinaFil: Angelini, Jorge Guillermo. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquimicas y Naturales. Departamento de Ciencias Naturales. Cátedra de Biologia General; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; ArgentinaFil: Taurian, Tania. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquimicas y Naturales. Departamento de Ciencias Naturales. Cátedra de Biologia General; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; ArgentinaFil: Ibañez, Fernando Julio. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquimicas y Naturales. Departamento de Ciencias Naturales. Cátedra de Biologia General; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; ArgentinaFil: Muñoz, Vanina Laura. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquimicas y Naturales. Departamento de Ciencias Naturales. Cátedra de Biologia General; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; ArgentinaFil: Anzuay, María Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; Argentina. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquimicas y Naturales. Departamento de Ciencias Naturales. Cátedra de Biologia General; ArgentinaFil: Ludueña, Liliana Mercedes. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquimicas y Naturales. Departamento de Ciencias Naturales. Cátedra de Biologia General; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; ArgentinaFil: Fabra, Adriana Isidora. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquimicas y Naturales. Departamento de Ciencias Naturales. Cátedra de Biologia General; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; Argentin

Evolution of LysM-RLK Gene Family in Wild and Cultivated Peanut Species

In legumes, a LysM-RLK perception of rhizobial lipo-chitooligosaccharides (LCOs) known as Nod factors (NFs), triggers a signaling pathway related to the onset of symbiosis development. On the other hand, activation of LysM-RLKs upon recognition of chitin-derived short-chitooligosaccharides initiates defense responses. In this work, we identified the members of the LysM-RLK family in cultivated (Arachis hypogaea L.) and wild (A. duranensis and A. ipaensis) peanut genomes, and reconstructed the evolutionary history of the family. Phylogenetic analyses allowed the building of a framework to reinterpret the functional data reported on peanut LysM-RLKs. In addition, the potential involvement of two identified proteins in NF perception and immunity was assessed by gene expression analyses. Results indicated that peanut LysM-RLK is a highly diverse family. Digital expression analyses indicated that some A. hypogaea LysM-RLK receptors were upregulated during the early and late stages of symbiosis. In addition, expression profiles of selected LysM-RLKs proteins suggest participation in the receptor network mediating NF and/or chitosan perception. The analyses of LysM-RLK in the non-model legume peanut can contribute to gaining insight into the molecular basis of legume–microbe interactions and to the understanding of the evolutionary history of this gene family within the Fabaceae.Fil: Rodriguez Melo, Johan Stiben. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; ArgentinaFil: Tonelli, Maria Laura. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Instituto de Investigaciones Agrobiotecnológicas. - Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones Agrobiotecnológicas; ArgentinaFil: Barbosa, María Carolina. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Instituto de Investigaciones Agrobiotecnológicas. - Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones Agrobiotecnológicas; ArgentinaFil: Ariel, Federico. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; ArgentinaFil: Zhao, Zifan. University of Florida; Estados UnidosFil: Wang, Jianping. University of Florida; Estados UnidosFil: Fabra, Adriana Isidora. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Instituto de Investigaciones Agrobiotecnológicas. - Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones Agrobiotecnológicas; ArgentinaFil: Ibañez, Fernando Julio. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Instituto de Investigaciones Agrobiotecnológicas. - Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones Agrobiotecnológicas; Argentin

Contribution of phytochelatins to cadmium tolerance in peanut plants

Cadmium (Cd) is a well known heavy metal considered as one of the most toxic metals on Earth, affecting all viable cells that are exposed even at low concentration. It is introduced to agricultural soils mainly by phosphate fertilizers and causes many toxic symptoms in cells. Phytochelatins (PCs) are non-protein thiols which are involved in oxidative stress protection and are strongly induced by Cd. In this work, we analyzed metal toxicity as well as PCs implication on protection of peanut plants exposed to Cd. Results showed that Cd exposure induced a reduction of peanut growth and produced changes in the histological structure with a deposit of unknown material on the epidermal and endodermal cells. When plants were exposed to 10 μM Cd, no modification of chlorophyll, lipid peroxides, carbonyl groups, or hydrogen peroxide (H2O2) content was observed. At this concentration, peanut leaves and roots glutathione (GSH) content decreased. However, peanut roots were able to synthesize different types of PCs (PC2, PC3, PC4). In conclusion, PC synthesis could prevent metal disturbance on cellular redox balance, avoiding oxidative damage to macromolecules.Fil: Bianucci, Eliana Carolina. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; ArgentinaFil: Sobrino Plata, Juan. No especifíca;Fil: Carpena Ruiz, Ramón O.. Universidad Autónoma de Madrid; EspañaFil: Del Carmen Tordable, María. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales; ArgentinaFil: Fabra, Adriana Isidora. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; ArgentinaFil: Hernández, Luis E.. No especifíca;Fil: Castro, Stella Maris. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; Argentin

The biocontrol agent Bacillus sp. CHEP5 primes the defense response against Cercospora sojina

Glycine max (soybean) production can be dramatically affected by frogeye leaf spot (FLS) caused by Cercospora sojina Hara. The inoculation of biocontrol agents may be an alternative strategy for C. sojina control. The native biocontrol bacterium Bacillus sp. CHEP5 reduced the severity of FLS in soybean by inducing systemic resistance. We suggest that the defense response was primed since the expression of the defense related gene GmAOS was enhanced in induced plants treated with both methyl jasmonate and C. sojina. Furthermore, as GmAOS is related to jasmonic acid biosynthesis, we assume that this phytohormone is involved in induced systemic resistance signaling defense pathway in soybean against C. sojina.Fil: Tonelli, Maria Laura. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales; ArgentinaFil: Fabra, Adriana Isidora. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales; Argentin

Production of reactive oxygen species in peanut nodules

In this study, it was investigated the reactive oxygen species (ROS) production in peanut nodules at three peanut growth stages (R): R1 (flowering), R4 (full pod) and R6 (full seed). Analysis of ROS production showed that superoxide anion and hydrogen peroxide contents decreased while lipid peroxidation and protein oxidation remained unchanged throughout nodule development. Furthermore, it was found that the inside of formed nodules was 100% red at (R1), 54% at (R4) and 39% at (R6), respectively. The total soluble protein content decreased while leghemoglobin content remained unaltered at different growth stages. Thus, our findings suggest that ROS production is not involved in the peanut nodule senescence indicating that this process would not be accompanied by an oxidative burst. It is possible to suggest that the antioxidant system would play an important role in the protection of peanut nodules against ROS production.Fil: Carlier, Evelin. Universidad Nacional de Rio Cuarto; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Castro, Stella Maris. Universidad Nacional de Rio Cuarto; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Fabra, Adriana Isidora. Universidad Nacional de Rio Cuarto; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Symbiotic performance and induction of systemic resistance against Cercospora sojina in soybean plants co-inoculated with Bacillus sp. CHEP5 and Bradyrhizobium japonicum E109

Soybean is an economically very important crop throughout the word and particularly in Argentina. Soybean yield may be affected by many factors such as the lack of some essential nutrients or pathogens attack. In this work we demonstrated that the co-inoculation of the native biocontrol bacterium Bacillus sp. CHEP5 which induces resistance against Cercospora sojina in soybean and the nitrogen fixing strain Bradyrhizobium japonicum E109, was more effective in reducing frog leaf spot severity than the inoculation of the biocontrol agent alone. Probably, this is related with the increase in the ability to form biofilm when both bacteria are growing together. Furthermore, Bacillus sp. CHEP5 inoculation did not affect Bradyrhizobium japonicum E109 symbiotic behavior and flavonoids composition of root exudates in pathogen challenged plants. These results suggest that co-inoculation of plants with rhizobia and biocontrol agents could be a strategy to improve soybean production in a sustainable system.Fil: Tonelli, Maria Laura. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; ArgentinaFil: Magallanes Noguera, Cynthia Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; Argentina. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales; ArgentinaFil: Fabra, Adriana Isidora. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; Argentin