Immunocytochemical characterisation of endophytic bacteria Azospirillum brasilense, Herbaspirillum seropedicae, Burkholderia ambifaria and Gluconacetobacter diazotrophicus

In the present work an immunocytochemical characterisation of four endophytic bacterial species has been made by using polyclonal antiserum produced against each of the four bacterial strains previously heated at 60 degrees C. The aim of this research is to identify common elements among bacteria associated with their endophytic behaviour. Analysis of extracts of each strain by immunoblotting and ELISA confirmed the presence of proteins from different bacterial strains made up of common epitopes. However, antisera produced against Herbaspirillum seropedicae and Burkholderia ambifaria show a high number of bands recognised on each extracts, while antisera against Azospirillum brasilense and Gluconacetobacter diazotrophicus show a low number of bands recognised on each extract. Immunogold labelling showed that epitopes are located both on the cell wall and in the cytoplasm; most likely they could be precursor cell wall proteins synthesized inside the cytoplasm and subsequently transported onto cell wall. Finally, the common bands among bacterial strains revealed by immunoblotting could play a role as active hydrolases involved in host tissue penetration

Localization of Azospirillum brasilense Cd in inoculated tomato (Lycopersicon esculentum Mill.) roots

Tomato (Lycopersicon esculentum Mill.) seeds inoculated with Azospirillum brasilense Cd were cultured in vitro on solid medium with and without combined nitrogen. Inoculated and control roots from 30-day-old plants were examined by light microscopy (LM), scanning electron microscopy (SEM) and transmission electron microscopy (TEM), to monitor the adhesion, penetration, and localization of bacteria on and within the plant. We also studied the first colonisation steps of three-day-old plants under the microscope and bacteria in culture. Azospirillum brasilense Cd was identified by immunogold or immunofluorescence labelling. In culture, this bacterium has a thick polar flagellum and many thin peritrichous flagella. In aged dark cultures, small cells appeared within cyst-like forms. The bacteria used the polar flagellum to adhere to the epidermis, root hair and root cap areas of tomato roots. They also penetrated the epidermis, root hairs and outer cortex cells. Within the root, A. brasilense formed cyst-like cells that did not divide inside and were rich in poly-β-hydroxybutyrate granules and glycogen. Bacteria within the root had a thick capsule, many granules of different types, and high levels of SOD activity, suggesting that they can fix nitrogen in the intercellular spaces of tomato roots

Localization of Azospirillum brasilense Cd in inoculated tomato (Lycopersicon esculentum Mill.) roots

Tomato (Lycopersicon esculentum Mill.) seeds inoculated with Azospirillum brasilense Cd were cultured in vitro on solid medium with and without combined nitrogen. Inoculated and control roots from 30-day-old plants were examined by light microscopy (LM), scanning electron microscopy (SEM) and transmission electron microscopy (TEM), to monitor the adhesion, penetration, and localization of bacteria on and within the plant. We also studied the first colonisation steps of three-day-old plants under the microscope and bacteria in culture. Azospirillum brasilense Cd was identified by immunogold or immunofluorescence labelling. In culture, this bacterium has a thick polar flagellum and many thin peritrichous flagella. In aged dark cultures, small cells appeared within cyst-like forms. The bacteria used the polar flagellum to adhere to the epidermis, root hair and root cap areas of tomato roots. They also penetrated the epidermis, root hairs and outer cortex cells. Within the root, A. brasilense formed cyst-like cells that did not divide inside and were rich in poly-β-hydroxybutyrate granules and glycogen. Bacteria within the root had a thick capsule, many granules of different types, and high levels of SOD activity, suggesting that they can fix nitrogen in the intercellular spaces of tomato roots

Ion determinations within Azolla leaf cavities by microelectrodes

Calcium, potassium, sodium and chloride microelectrodes directly assembled were used for the in situ measurements of the concentrations of various ions within the Azolla leaf cavities. Moreover, pH was measured using a micro pH electrode. The results showed that: apical leaf cavities (apex and group I) contained 2–4 × 10−5 M calcium, 1.5 × 10−4 M sodium, 3 × 10−3 M potassium, 0.45 M chloride and had pH 6.0; median leaf cavities (groups II and III) which evidenced the highest nitrogenase activity, showed 1.5 × 10−4 M calcium, 2.5–3.5 × 10−4 M sodium, 1–2 × 10−3 M potassium, 0.45-0.35 M chloride and pH 6.5; basal leaf cavities (group IV), in which nitrogenase activity declined, contained 3 × 10−3 M calcium, 1 × 10−3 M sodium, 4 × 10−3 M potassium, 0.25 M chloride and had pH 5.7. These data suggest an active utilization of calcium, sodium and potassium in median leaf cavities from the Azolla-Anabaena system that could promote the high nitrogenase activity of the cyanobacterium. In basal leaf cavities, the increase of the various ion concentrations could be due to a decreased ability in their uptake from the association