Table 1.
Determination of Azoarcus sp. CIB nitrogenase activity.
Figure 1.
Azoarcus sp. CIB produces IAA.
Production of IAA by Azoarcus sp. CIB (white bars) and R. leguminosarum bv. trifolii TT-7C (grey bars) along the growth curve. Growth of Azoarcus sp. CIB (black squares) and R. leguminosarum bv. trifolii TT-7C (grey rhombus) is also represented. IAA was quantified as detailed in Materials and Methods. Graphed values of are the average from three independent experiments +/− S. D.
Figure 2.
Azoarcus sp. CIB is a non-pathogenic bacterium.
Pathogenicity test on tobacco leaves. Bacteria were infiltrated into the intercellular spaces of a tobacco leaf, which was inspected for a visible necrosis after 2 days. Necrosis is visible as light spots (arrows) on leaves infiltrated with Pseudomonas syringae pv. syringae (right) and not visible on leaves infiltrated with Azoarcus sp. CIB (left).
Figure 3.
Quantification of the bacterial endophytic population within inoculated rice roots.
Rice seedlings were inoculated with Azoarcus communis Swub3 (A. communis), Azoarcus sp. CIB (Azoarcus sp. CIB), and E. coli S17-1λpir (E. coli) containing plasmid pSEVA23GFP) that expresses the gfp gene as indicated in Materials and Methods. Plants were grown at 25°C for 5 days, and the kanamycin-resistant bacteria within the root tissue were determined as detailed in Materials and Methods. Graphed values of CFU per g of fresh root (FW) are the average from three independent experiments +/− S. D.
Figure 4.
Images of rice roots inoculated with Azoarcus sp. CIB.
Epifluorescence microscopy images of rice roots after 5 days (A) or 10 days (B) of inoculation with Azoarcus sp. CIB (pSEVA23GFP). A detail of each image is included (lower panels) to illustrate the morphological change of the cells.
Figure 5.
Colonization of rice roots by Azoarcus sp. CIB observed by confocal microscopy.
Confocal images of rice roots after seven days colonization with Azoarcus sp. CIB (pSEVA23GFP) cells. (A) Bacteria are observed as single cells or as clusters of cells attached to the inner intercellular space of the exodermis. (B) XYZ projection of bacterial cells distributed on the inner part (In) or the surface (Out) of the rice root.
Figure 6.
Light micrographs of transversal sections of rice roots inoculated with Azoarcus sp. CIB (pSEVA23GFP) cells and incubated for 7 days.
Colonization of root surface and intercellular spaces under rhizodermis and the first layer of exodermis (A); intercellular colonization of the second and third layers of the exodermis (B); intercellular colonization of the deeper layer of the exodermis, just in contact with the parenchyma (C). IS, intercellular space; Ex, exodermis, Rh, rhizodermis. Red arrows indicate the localization of the bacterial cells.
Figure 7.
Electron microscopy observation of rice roots colonized by Azoarcus sp. CIB.
Electron micrographs showing immunogold localization of NifH epitopes in rice roots inoculated with Azoarcus sp. CIB (pSEVA23GFP) cells for 7 days. Surface colonization (A), intercellular colonization of the second and third layers of the exodermis (C), intercellular colonization of the deeper layer of the exodermis, just in contact with the parenchyma (E). CW, cell wall; Ex, exodermis; IS intercellular space; Rh, rhizodermis; P, parenchyma. In order to distinguish gold particles, the framed areas in A, C and E are magnified in panels B, D and F, respectively.