Nitric oxide synthase expression in Pseudomonas koreensis MME3 improves plant growth promotion traits.

The development of novel biotechnologies that promote a better use of N to optimize crop yield is a central goal for sustainable agriculture. Phytostimulation, biofertilization, and bioprotection through the use of bio-inputs are promising technologies for this purpose. In this study, the plant growth-promoting rhizobacteria Pseudomonas koreensis MME3 was genetically modified to express a nitric oxide synthase of Synechococcus SyNOS, an atypical enzyme with a globin domain that converts nitric oxide to nitrate. A cassette for constitutive expression of synos was introduced as a single insertion into the genome of P. koreensis MME3 using a miniTn7 system. The resulting recombinant strain MME3:SyNOS showed improved growth, motility, and biofilm formation. The impact of MME3:SyNOS inoculation on Brachypodium distachyon growth and N uptake and use efficiencies under different N availability situations was analyzed, in comparison to the control strain MME3:c. After 35 days of inoculation, plants treated with MME3:SyNOS had a higher root dry weight, both under semi-hydroponic and greenhouse conditions. At harvest, both MME3:SyNOS and MME3:c increased N uptake and use efficiency of plants grown under low N soil. Our results indicate that synos expression is a valid strategy to boost the phytostimulatory capacity of plant-associated bacteria and improve the adaptability of plants to N deficiency. KEY POINTS: • synos expression improves P. koreensis MME3 traits important for rhizospheric colonization • B. distachyon inoculated with MME3:SyNOS shows improved root growth • MME3 inoculation improves plant N uptake and use efficiencies in N-deficient soil.

Azospirillum baldaniorum Sp245 exploits Pseudomonas fluorescens A506 biofilm to overgrow in dual-species macrocolonies.

Biofilms are essential for plant-associated bacteria to colonize their host. In this work, we analysed the interaction of Azospirillum baldaniorum Sp245 and Pseudomonas fluorescens A506 in mixed macrocolony biofilms. We identified certain culture conditions where A. baldaniorum Sp245 exploits P. fluorescens A506 to boost its growth. Azospirillum growth increased proportionally to the initial number of pseudomonads building the biofilm, which in turn were negatively affected in their growth. Physical contact with P. fluorescens A506 was essential for A. baldaniorum Sp245 growth increase. Biofilm ultrastructure analysis revealed that Pseudomonas produces a thick structure that hosts Azospirillum cells in its interior. Additional experimentation demonstrated that Azospirillum growth boost is compromised when interacting with biofilm-deficient Pseudomonas mutants, and that a low oxygen concentration strongly induce A. baldaniorum Sp245 growth, overriding Pseudomonas stimulation. In this line, we used a microaerophilia reporter strain of A. baldaniorum Sp245 to confirm that dual-species macrocolonies contain a higher number of cells under microaerophilic conditions. Taking all the results into consideration, we propose that A. baldaniorum Sp245 can benefit from P. fluorescens A506 partnership in mixed biofilms by taking advantage of the low oxygen concentration and scaffold made up of Pseudomonas-derived matrix, to expand its growth.

Potential of rhizobacteria native to Argentina for the control of Meloidogyne javanica.

Biocontrol of the nematode Meloidogyne javanica was studied using the Argentinean strains Pseudomonas fluorescens MME3, TAE4, TAR5 and ZME4 and Bacillus sp. B7S, B9T and B19S. Pseudomonas protegens CHA0 was used as a positive control. Egg hatching and juvenile mortality were evaluated in vitro by exposure of nematodes to bacterial suspensions or their cell-free supernatants (CFS). The effect of bacteria on nematode infestation of lettuce was also studied. results showed that most of the tested strains and CFS reduced egg hatching and juvenile survival in vitro. The bacterial suspension of Bacillus sp. B9T produced the lowest hatching of eggs. Juvenile mortality was higher when M. javanica was exposed to Bacillus sp. than to Pseudomonas spp. suspensions. Except for CFS of B9T, all filtrates inhibited hatching at levels similar to or higher than the biocontrol strain P. protegens CHA0. The CFS of CHA0 showed the highest level of juvenile mortality followed by Bacillus sp. strains and P. fluorescens TAE4. None of the inoculated rhizobacteria reverted the negative effect of infestation on the aerial dry weight of lettuce plants. However, inoculation impacted on reproduction of M. javanica by reducing the development of galls and egg masses on roots and diminishing the number of individuals both on roots and in the substrate, as well as the reproduction factor. These results show that most of the analyzed native strains can control the nematode M. javanica. Among them, P. fluorescens TAE4 and Bacillus sp. B9T showed the most promising performances for the biocontrol of this pathogen and have a potential use in the formulation of commercial products.

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

BACKGROUND: 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. RESULTS: 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 Delphacodes kuscheli, a planthopper capable of persistently transmitting the plant fijivirus Mal de Río Cuarto virus (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. CONCLUSIONS: A RT-qPCR platform for gene expression analysis in the MRCV-infected planthopper vector Delphacodes kuscheli 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.

Signs of a phyllospheric lifestyle in the genome of the stress-tolerant strain Azospirillum brasilense Az19.

Azospirillum brasilense Az19 is a plant-beneficial bacterium capable of protecting plants from the negative effects of drought. The objective of this study was to determine and analyze the genomic sequence of strain Az19 as a means of identifying putative stress-adaptation mechanisms. A high-quality draft genome of ca. 7 Mb with a predicted coding potential of 6710 genes was obtained. Phylogenomic analyses confirmed that Az19 belongs to the brasilense clade and is closely related to strains Az39 and REC3. Functional genomics revealed that the denitrification pathway of Az19 is incomplete, which was in agreement with a reduced growth on nitrate under low O2 concentrations. Putative genes of the general stress response and oxidative stress-tolerance, as well as synthesis of exopolysaccharides, carotenoids, polyamines and several osmolytes, were detected. An additional poly-beta-hydroxybutyrate (PHB) synthase coding gene was found in Az19 genome, but the accumulation of PHB did not increase under salinity. The detection of exclusive genes related to DNA repair led to discover that strain Az19 also has improved UV-tolerance, both in vitro and in planta. Finally, the analysis revealed the presence of multiple kaiC-like genes, which could be involved in stress-tolerance and, possibly, light responsiveness. Although A. brasilense has been a model for the study of beneficial plant-associated rhizobacteria, the evidence collected in this current study suggests, for the first time in this bacterial group, an unexpected possibility of adaptation to the phyllosphere.

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.

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.

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 subjected to intensive research due to their biotechnological potential as crop inoculants. Phylogenetic analysis of Azospirillum spp. is carried out by 16S rRNA sequencing almost exclusively, but inconsistencies and low confidence often arise when working with close species. In this work, it was observed that 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 of housekeeping genes, the correlation between pairwise gene and whole-genome similarities was examined. Due to its good performance, rpoD was selected for further analyses. Genus-specific primers for the PCR-amplification and sequencing of rpoD from Azospirillum spp. were designed and tested on 16 type strains of different species. The sequences obtained were used for inferring a phylogenetic tree of the genus, which was in turn used as a reference to successfully identify a collection of 31 azospirilla isolated from many different locations of Argentine. In addition, several strains that might represent novel species were detected. The results indicate that the sequencing of rpoD is a suitable alternative method for a confident molecular identification in Azospirillum spp.

Potential of rhizobacteria native to Argentina for the control of Meloidogyne javanica / Potencial de rizobacterias nativas de Argentina para el control de Meloidogyne javanica

Abstract Biocontrol of the nematode Meloidogyne javanica was studied using the Argentinean strains Pseudomonas fluorescens MME3, TAE4, TAR5 and ZME4 and Bacillus sp. B7S, B9T and B19S. Pseudomonas protegens CHA0 was used as a positive control. Egg hatching and juvenile mortality were evaluated in vitro by exposure of nematodes to bacterial suspensions or their cell-free supernatants (CFS). The effect of bacteria on nematode infestation of lettuce was also studied. results showed that most of the tested strains and CFS reduced egg hatching and juvenile survival in vitro. The bacterial suspension of Bacillus sp. B9T produced the lowest hatching of eggs. Juvenile mortality was higher when M. javanica was exposed to Bacillus sp. than to Pseudomonas spp. suspensions. Except for CFS of B9T, all filtrates inhibited hatching at levels similar to or higher than the biocontrol strain P. protegens CHA0. The CFS of CHA0 showed the highest level of juvenile mortality followed by Bacillus sp. strains and P. fluorescens TAE4. None of the inoculated rhizobacteria reverted the negative effect of infestation on the aerial dry weight of lettuce plants. However, inoculation impacted on reproduction of M. javanica by reducing the development of galls and egg masses on roots and diminishing the number of individuals both on roots and in the substrate, as well as the reproduction factor. These results show that most of the analyzed native strains can control the nematode M. javanica. Among them, P. fluorescens TAE4 and Bacillus sp. B9T showed the most promising performances for the biocontrol of this pathogen and have a potential use in the formulation of commercial products.

Resumen Se estudiaron las cepas argentinas Pseudomonas fluorescens MME3, TAE4, TAR5 y ZME4 y Bacillus sp. B7S, B9T y B19S para el control del nematodo Meloidogyne javanica. Pseudomonas protegens CHA0 se utilizó como control positivo. La eclosión de huevos y la mortalidad de juveniles se evaluaron in vitro al exponerlos a suspensiones bacterianas y a sus sobrenadantes libres de células (SLC). Asimismo, se estudió la inoculación bacteriana sobre la infestación del nematodo en lechuga. Los resultados in vitro indicaron que la mayoría de las cepas, así como sus SLC redujeron la eclosión y la supervivencia de M. javanica. La suspensión de Bacillus sp. B9T produjo los menores niveles de eclosión. La mortalidad de juveniles fue mayor al exponerlos a suspensiones de Bacillus sp. respecto de Pseudomonas spp. Los SLC inhibieron la eclosión de huevos en niveles similares o superiores a P. protegens CHA0, excepto por el de B9T. La exposición a SLC de CHA0 registró la mayor mortalidad, seguido por las cepas de Bacillus sp. y P. fluorescens TAE4. La inoculación bacteriana no revertió el efecto de la infestación sobre el peso seco aéreo de las plantas, sin embargo, afectó la multiplicación de M. javanica lo que redujo el desarrollo de agallas y las masas de huevos, y disminuyó el número de individuos presentes tanto en la raíz como en el sustrato, así como el factor de reproducción. Los resultados indican que la mayoría de las cepas nativas evaluadas son capaces de controlar a M. javanica. Entre ellas, P. fluorescens TAE4 y Bacillus sp. B9T, se presentan como las más promisorias para el control de este patógeno, con potencialidad para ser utilizadas en la formulación de productos biológicos.

Plant-associated fluorescent Pseudomonas from red lateritic soil: Beneficial characteristics and their impact on lettuce growth.

Fluorescent Pseudomonas are ubiquitous soil bacteria that usually establish mutualistic associations with plants, promoting their growth and health by several mechanisms. This makes them interesting candidates for the development of crop bio-inoculants. In this work, we isolated phosphate-solubilizing fluorescent Pseudomonas from the rhizosphere and inner tissues of different plant species growing in red soil from Misiones, Argentina. Seven isolates displaying strong phosphate solubilization were selected for further studies. Molecular identification by rpoD genotyping indicated that they belong to different species within the P. fluorescens and P. putida phylogenetic groups. Screening for in vitro traits such as phosphate solubilization, growth regulators synthesis or degradation, motility and antagonism against phytopathogens or other bacteria, revealed a unique profile of characteristics for each strain. Their plant growth-promoting potential was assayed using lettuce as a model for inoculation under controlled and greenhouse conditions. Five of the strains increased the growth of lettuce plants. Overall, the strongest lettuce growth promoter under both conditions was strain ZME4, isolated from inner tissues of maize. No clear association between lettuce growth promotion and in vitro beneficial traits was detected. In conclusion, several phosphate solubilizing pseudomonads from red soil were isolated that display a rich array of plant growth promotion traits, thus showing a potential for the development of new inoculants.

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.

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.

Functional and biochemical properties of Mal de Río Cuarto virus (Fijivirus, Reoviridae) P9-1 viroplasm protein show further similarities to animal reovirus counterparts.

Mal de Río Cuarto virus (MRCV) is a plant virus of the genus Fijivirus within the family Reoviridae that infects several monocotyledonous species and is transmitted by planthoppers in a persistent and propagative manner. Other members of the family replicate in viral inclusion bodies (VIBs) termed viroplasms that are formed in the cytoplasm of infected plant and insect cells. In this study, the protein coded by the first ORF of MRCV segment S9 (P9-1) was shown to establish cytoplasmic inclusion bodies resembling viroplasms after transfection of Spodoptera frugiperda insect cells. In accordance, MRCV P9-1 self-associates giving rise to high molecular weight complexes when expressed in bacteria. Strong self-interaction was also evidenced by yeast two-hybrid assays. Furthermore, biochemical characterization showed that MRCV P9-1 bound single stranded RNA and had ATPase activity. Finally, the MRCV P9-1 region required for the formation of VIB-like structures was mapped to the protein carboxy-terminal half. This extensive functional and biochemical characterization of MRCV P9-1 revealed further similarities between plant and animal reovirus viroplasm proteins.