Molecular identification of Azospirillum spp.: limitations of 16S rRNA and qualities of rpoD as genetic markers

Since their discovery, plant-growth promoting rhizobacteria from the genus Azospirillum have been sub-jected to intensive research due to their biotechnological potential as crop inoculants. Phylogeneticanalysis of Azospirillum spp. is carried out by 16S rRNA sequencing almost exclusively, but inconsis-tencies and low confidence often arise when working with close species. In this work, it was observedthat these difficulties might be explained by a high number of rRNA operons with considerable inter-genic variability within Azospirillum genomes. To search for alternative genetic markers from a list ofhousekeeping genes, the correlation between pairwise gene and whole-genome similarities was exam-ined. Due to its good performance, rpoD was selected for further analyses. Genus-specific primers for thePCR-amplification and sequencing of rpoD from Azospirillum spp. were designed and tested on 16 typestrains of different species. The sequences obtained were used for inferring a phylogenetic tree of thegenus, which was in turn used as a reference to successfully identify a collection of 31 azospirilla iso-lated from many different locations of Argentine. In addition, several strains that might represent novelspecies were detected. The results indicate that the sequencing of rpoD is a suitable alternative methodfor a confident molecular identification in Azospirillum spp.Inst. de Microbiología y Zoología Agrícola IMyZAFil: Maroniche, Guillermo Andrés. Unidad Integrada Balcarce-Universidad Nacional de Mar del Plata. Facultad de Ciencias Agrarias-Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Garcia, Julia Elena. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Microbiología y Zoología Agrícola; ArgentinaFil: Salcedo, Florencia. Universidad Nacional de Mar del Plata. Facultad de Ciencias Agrarias; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Creus, Cecilia Mónica. Universidad Nacional de Mar del Plata. Facultad de Ciencias Agrarias; Argentin

Friends or foes in the rhizosphere: traits of fluorescent Pseudomonas that hinder Azospirillum brasilense growth and root colonization

Bacteria of the Azospirillum and Pseudomonas genera are ubiquitous members of the rhizosphere, where they stimulate plant growth. Given the outstanding capacity of pseudomonads to antagonize other microorganisms, we analyzed the interaction between these two bacterial groups to identify determinants of their compatibility. We could establish that, when in direct contact, certain Pseudomonas strains produce lethality on Azospirillum brasilense cells using an antibacterial type 6 secretion system. When analyzing the effect of Pseudomonas spp. diffusible metabolites on A. brasilense growth on King´s B medium, we detected strong inhibitory effects, mostly mediated by siderophores. On Congo Red medium, both inhibitory and stimulatory effects were induced by unidentified compounds. Under this condition, Pseudomonas protegens CHA0 produced a Gac/Rsm-regulated antibiotic which specifically inhibited A. brasilense Sp7 but not Sp245. This effect was not associated with the production of 2,4-diacetylphloroglucinol. The three identified antagonism determinants were also active in vivo, producing a reduction of viable cells of A. brasilense in the roots of wheat seedlings when co-inoculated with pseudomonads. These results are relevant to the understanding of social dynamics in the rhizosphere and might aid in the selection of strains for mixed inoculants.Fil: Maroniche, Guillermo Andrés. Universidad Nacional de Mar del Plata. Facultad de Ciencias Agrarias; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata; ArgentinaFil: Diaz, Pablo Rafael. Universidad Nacional de Mar del Plata. Facultad de Ciencias Agrarias; ArgentinaFil: Borrajo, María Paula. Universidad Nacional de Mar del Plata. Facultad de Ciencias Agrarias; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata; ArgentinaFil: Valverde, Claudio Fabián. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes; ArgentinaFil: Creus, Cecilia Mónica. Universidad Nacional de Mar del Plata. Facultad de Ciencias Agrarias; Argentin

Reference gene selection for gene expression studies using RT-qPCR in virus-infected planthoppers

<p>Abstract</p> <p>Background</p> <p>Planthoppers not only severely affect crops by causing mechanical damage when feeding but are also vectors of several plant virus species. The analysis of gene expression in persistently infected planthoppers might unveil the molecular basis of viral transmission. Quantitative real-time RT-PCR (RT-qPCR) is currently the most accurate and sensitive method used for quantitative gene expression analysis. In order to normalize the resulting quantitative data, reference genes with constant expression during the experimental procedures are needed.</p> <p>Results</p> <p>Partial sequences of the commonly used reference genes actin (ACT), α1-tubulin (TUB), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), elongation factor 1 alpha (EF1A), ribosomal protein S18 (RPS18) and polyubiquitin C (UBI) from <it>Delphacodes kuscheli</it>, a planthopper capable of persistently transmitting the plant fijivirus <it>Mal de Río Cuarto virus </it>(MRCV), were isolated for the first time. Specific RT-qPCR primers were designed and the expression stability of these genes was assayed in MRCV-infective and naïve planthoppers using geNorm, Normfinder and BestKeeper tools. The overall analysis showed that UBI, followed by 18S and ACT, are the most suitable genes as internal controls for quantitative gene expression studies in MRCV-infective planthoppers, while TUB and EF1A are the most variable ones. Moreover, EF1A was upregulated by MRCV infection.</p> <p>Conclusions</p> <p>A RT-qPCR platform for gene expression analysis in the MRCV-infected planthopper vector <it>Delphacodes kuscheli </it>was developed. Our work is the first report on reference gene selection in virus-infected insects, and might serve as a precedent for future gene expression studies on MRCV and other virus-planthopper pathosystems.</p

AcMNPV Core Gene ac109 Is Required for Budded Virion Transport to the Nucleus and for Occlusion of Viral Progeny

The Autographa californica multiple nucleopolyhedrovirus (AcMNPV) ac109 core gene has been previously characterized as an essential late gene. Our results showed that budded virions could be detected in supernatants of infected Sf-9 cells, even when ac109 knockout viruses displayed a single-cell infection phenotype. Moreover, confocal microscopy analysis revealed that budded virions can enter the cytoplasm but are unable to enter the cell nucleus. This defect could be repaired by complementing ac109 in trans. In addition, polyhedra of normal size could be detected in Sf-9 nuclei infected with ac109 knockout viruses. However, electron microscopy demonstrated that these occlusion bodies were empty. Altogether, these results indicate that ac109 is required for infectivity of both phenotypes of virus.Instituto de BiotecnologíaFil: Alfonso, Victoria. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; ArgentinaFil: Maroniche, Guillermo Andrés. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Microbiología y Zoología Agrícola; ArgentinaFil: Reca, Sol Rita. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; ArgentinaFil: Lopez, Maria Gabriela. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina.Fil: Del Vas, Mariana. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; ArgentinaFil: Taboga, Oscar Alberto. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentin

In vivo subcellular localization of Mal de Río Cuarto virus (MRCV) non-structural proteins in insect cells reveals their putative functions

The in vivo subcellular localization of Mal de Río Cuarto virus (MRCV, Fijivirus, Reoviridae) non-structural proteins fused to GFP was analyzed by confocal microscopy. P5-1 showed a cytoplasmic vesicular-like distribution that was lost upon deleting its PDZ binding TKF motif, suggesting that P5-1 interacts with cellular PDZ proteins. P5-2 located at the nucleus and its nuclear import was affected by the deletion of its basic C-termini. P7-1 and P7-2 also entered the nucleus and therefore, along with P5-2, could function as regulators of host gene expression. P6 located in the cytoplasm and in perinuclear cloud-like inclusions, was driven to P9-1 viroplasm-like structures and co-localized with P7-2, P10 and α-tubulin, suggesting its involvement in viroplasm formation and viral intracellular movement. Finally, P9-2 was N-glycosylated and located at the plasma membrane in association with filopodia-like protrusions containing actin, suggesting a possible role in virus cell-to-cell movement and spread.Instituto de BiotecnologíaFil: Maroniche, Guillermo Andrés. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; ArgentinaFil: Mongelli, Vanesa Claudia. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; ArgentinaFil: Llauger, Gabriela. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; ArgentinaFil: Alfonso, Victoria. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; ArgentinaFil: Taboga, Oscar Alberto. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; ArgentinaFil: Del Vas, Mariana. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentin

In vitro PGPR properties and osmotic tolerance of different Azospirillum native strains and their effects on growth of maize under drought stress

Osmotic variations in the soil can affect bacterial growth diminishing the number ofinoculated bacteria. In a scenario of water deficit having tolerant bacteria would bebeneficial to achieve a better response of the plant to stress. Thus, selection of moreresistant bacteria could be useful to design new inoculants to be used in arid zones. In thissense, a group of Azospirillum isolates deposited in INTA collection was characterized inorder to select strains tolerant to osmotic stress. The results obtained demonstrated that Az19 strain has similar in vitro PGPR characteristics to Az39, the most used strain inArgentina for inoculants industries, with the advantage of a better tolerance to osmotic andsalt stress. Inoculation of maize plants with this strain resulted in a better response againstwater deficit compared to Az39 strain, encouraging us to further study the behavior of thisstrain in greenhouse and field trials in view of developing new inoculants suitable for areaswith water deficit.Fil: García, Julia E.. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronomicas. Instituto de Microbiología y Zoología Agrícola; ArgentinaFil: Maroniche, Guillermo Andrés. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires Sur. Estación Experimental Agropecuaria Balcarce; ArgentinaFil: Creus, Cecilia Mónica. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires Sur. Estación Experimental Agropecuaria Balcarce; ArgentinaFil: Suarez Rodriguez, Ramón. Universidad Autónoma del Estado de Morelos; MéxicoFil: Ramirez Trujillo, José Augusto. Universidad Autónoma del Estado de Morelos; MéxicoFil: Groppa, María Daniela. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Genome Sequence of Bradyrhizobium japonicum E109, One of the Most Agronomically Used Nitrogen-Fixing Rhizobacteria in Argentina

We present here the complete genome sequence of Bradyrhizobium japonicum strain E109, one of the most used rhizobacteria for soybean inoculation in Argentina since the 1970s. The genome consists of a 9.22-Mbp single chromosome and contains several genes related to nitrogen fixation, phytohormone biosynthesis, and a rhizospheric lifestyle.Fil: Torres, Daniela Soledad. Universidad Nacional de Rio Cuarto; ArgentinaFil: Revale, Santiago. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Agrobiotecnología de Rosario; ArgentinaFil: Obando, Melissa. Universidad Nacional de Rio Cuarto. Facultad de Cs.exactas Fisicoquimicas y Naturales. Departamento de Cs.naturales. Laboratorio de Fisiologia Vegetal y de la Interaccion Planta-microorganismo; ArgentinaFil: Maroniche, Guillermo Andres. Instituto Nacional de Tecnología Agropecuaria. Centro Nacional de Investigaciones Agropecuarias. Centro de Investigación de Ciencias Veterinarias y Agronómicas. Instituto de Microbiología y Zoología Agrícola; ArgentinaFil: Paris, Gastón. Fundación Instituto Leloir; ArgentinaFil: Perticari, Alejandro. Instituto Nacional de Tecnología Agropecuaria. Centro Nacional de Investigaciones Agropecuarias. Centro de Investigación de Ciencias Veterinarias y Agronómicas. Instituto de Microbiología y Zoología Agrícola; ArgentinaFil: Vazquez, Martin. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Agrobiotecnología de Rosario; ArgentinaFil: Wisniewsk Dyé, Florence. Universite Lyon 2; FranciaFil: Martínez Abarca Pastor, Francisco. Consejo Superior de Investigaciones Científicas. Estación Experimental del Zaidin; EspañaFil: Cassan, Fabricio Dario. Universidad Nacional de Rio Cuarto; Argentin

Interspecific cooperation: enhanced growth, attachment and strain-specific distribution in biofilms through Azospirillum brasilense-Pseudomonas protegens co-cultivation.

Plant-growth-promoting bacteria belonging to Azospirillum and Pseudomonas genera are major inhabitants of the rhizosphere. Both are increasingly commercialized as crops inoculants. Interspecific interaction in the rhizosphere is critical for inoculants aptness. The objective of this work was to evaluate Azospirillum and Pseudomonas interaction in mixed biofilms by co-cultivation of the model strains Azospirillum brasilense Sp245 and Pseudomonas protegens CHA0. The results revealed enhanced growth of both strains when co-cultured in static conditions. Moreover, Sp245 biofilm formed in plastic surfaces was increased 2-fold in the presence of CHA0. Confocal microscopy revealed highly structured mixed biofilms showing Sp245 mainly on the bottom and CHA0 towards the biofilm surface. In addition, A. brasilense biofilm was thicker and denser when co-cultured with P. protegens. In a colony-colony interaction assay, Sp245 changed nearby CHA0 producing small colony phenotype, which accounts for a diffusible metabolite mediator; though CHA0 spent medium did not affect Sp245 colony phenotype. Altogether, these results point to a cooperative interaction between A. brasilense Sp245 and P. protegens CHA0 in which both strains increase their static growth and produce structured mixed biofilms with a strain-specific distribution

Virus infection elevates transcriptional activity of miR164a promoter in plants

Background: Micro RNAs (miRs) constitute a large group of endogenous small RNAs that have crucial roles in many important plant functions. Virus infection and transgenic expression of viral proteins alter accumulation and activity of miRs and so far, most of the published evidence involves post-transcriptional regulations. Results: Using transgenic plants expressing a reporter gene under the promoter region of a characterized miR (P-miR164a), we monitored the reporter gene expression in different tissues and during Arabidopsis development. Strong expression was detected in both vascular tissues and hydathodes. P-miR164a activity was developmentally regulated in plants with a maximum expression at stages 1.12 to 5.1 (according to Boyes, 2001) along the transition from vegetative to reproductive growth. Upon quantification of P-miR164a-derived GUS activity after Tobacco mosaic virus Cg or Oilseed rape mosaic virus (ORMV) infection and after hormone treatments, we demonstrated that ORMV and gibberellic acid elevated P-miR164a activity. Accordingly, total mature miR164, precursor of miR164a and CUC1 mRNA (a miR164 target) levels increased after virus infection and interestingly the most severe virus (ORMV) produced the strongest promoter induction. Conclusion: This work shows for the first time that the alteration of miR pathways produced by viral infections possesses a transcriptional component. In addition, the degree of miR alteration correlates with virus severity since a more severe virus produces a stronger P-miR164a induction.Instituto de BiotecnologíaFil: Bazzini, Ariel Alejandro. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Almasia, Natalia Ines. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Manacorda, Carlos Augusto. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; ArgentinaFil: Mongelli, Vanesa Claudia. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Conti, Gabriela. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; ArgentinaFil: Maroniche, Guillermo Andrés. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Rodriguez, Maria Cecilia. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Distefano, Ana Julia. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Hopp, Horacio Esteban. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Del Vas, Mariana. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Asurmendi, Sebastian. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Complete Genome Sequence of the Model Rhizosphere Strain Azospirillum brasilense Az39, Successfully Applied in Agriculture

We present the complete genome sequence of Azospirillum brasilense Az39, isolated from wheat roots in the central region of Argentina and used as inoculant in extensive and intensive agriculture during the last four decades. The genome consists of 7.39 Mb, distributed in six replicons: one chromosome, three chromids, and two plasmids.Fil: Rivera Botia, Diego Mauricio. Universidad Nacional de Rio Cuarto; ArgentinaFil: Revale, Santiago. Instituto de Agrobiotecnología de Rosario; ArgentinaFil: Molina, Romina Micaela. Universidad Nacional de Rio Cuarto; ArgentinaFil: Gualpa, Jose. Universidad Nacional de Rio Cuarto; ArgentinaFil: Puente, Mariana. Instituto Nacional de Tecnología Agropecuaria. Centro Nacional de Investigaciones Agropecuarias. Centro de Investigación de Ciencias Veterinarias y Agronómicas. Instituto de Microbiología y Zoología Agrícola; ArgentinaFil: Maroniche, Guillermo Andres. Instituto Nacional de Tecnología Agropecuaria. Centro Nacional de Investigaciones Agropecuarias. Centro de Investigación de Ciencias Veterinarias y Agronómicas. Instituto de Microbiología y Zoología Agrícola; ArgentinaFil: Paris, Gastón. Fundación Instituto Leloir; ArgentinaFil: Baker, David. The Genome Analysis Centre; Reino UnidoFil: Clavijo, Bernardo. The Genome Analysis Centre; Reino UnidoFil: McLay, Kirsten. The Genome Analysis Centre; Reino UnidoFil: Spaepen, Stijn. Katholieke Universiteit Leuven; Bélgica. Max Planck Institute for Plant Breeding Research; AlemaniaFil: Perticari, Alejandro. Instituto Nacional de Tecnología Agropecuaria. Centro Nacional de Investigaciones Agropecuarias. Centro de Investigación de Ciencias Veterinarias y Agronómicas. Instituto de Microbiología y Zoología Agrícola; ArgentinaFil: Vazquez, Martin Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular; ArgentinaFil: Wisniewski Dyé, Florence. Université Lyon. Ecologie Microbienne; FranciaFil: Whatkins, Christopher. The Genome Analysis Centre; Reino UnidoFil: Martínez Abarca, Francisco. Consejo Superior de Investigaciones Científicas. Estación Experimental del Zaidin; EspañaFil: Vanderleyden, Jos. Katholieke Universiteit Leuven; BélgicaFil: Cassan, Fabricio Dario. Universidad Nacional de Rio Cuarto; Argentin