The effect of boron deficiency on symbiotic nitrogen fixation in pea (Pisum safivum) was examined. l h e absence of boron in the culture medium resulted in a decrease of the number of nodules and an alteration of nodule development leading to an inhibition of nitrogenase activity. Examination of boron-deficient nodules showed dramatic changes in cell walls and in both peribacteroid and infection thread membranes, suggesting a role for boron in the stability of these structures. These results indicate that boron is a requirement for normal nodule development and functionality.

Summary• The infection of white lupin ( Lupinus albus ) roots and the early stages in organogenesis of the lupinoid nodule are characterized in detail in this work.• Immunolabelling of Bradyrhizobium sp. ( Lupinus ) ISLU16 and green fluorescent protein labelling of Mesorhizobium loti NZP2037, two strains that induce nodulation in L. albus , allowed us to monitor the infection and morphogenesis process. Light and transmission electron microscopy, low-temperature scanning electron microscopy, fluorescence and confocal microscopy were employed.• Rhizobia penetrated the root intercellularly at the junction between the root hair base and an adjacent epidermal cell. Bacteria invaded the subepidermal cortical cell immediately beneath the root hair through structurally altered cell wall regions. The newly infected cell divided repeatedly to form the central infected zone of the young nodule. Bacteria seemed to be equally distributed between the daughter cells.• A new mode of direct epidermal infection and an unusual morphogenesis for indeterminate nodules lead to the formation of the lupinoid nodule with unique characteristics.

Vegetative inoculum of Amanita ovoidea (Bull.) Link and three isolates of Suillus collinitus (Fr.) Kuntze, as well as spore inoculum of Rhizopogon roseolus (Corda) Th. M. Fr. and S. collinitus, were evaluated for the production of Pinus halepensis Mill. in nursery and for the establishment of seedlings in a degraded gypsum soil. In nursery, most of the fungi significantly improved the height of seedlings and modified the accumulation of nutrients in needles. The percentage of ectomycorrhizas (ESR) per seedling ranged from 25 to 78%, depending on the fungi. One and 2 years after planting in the field, the survival of seedlings was significantly improved by inoculation with two isolates of S. collinitus and with spores of the same fungus. Inoculation with A. ovoidea had no significant effect on seedling survival, whilst R. roseolus caused a significant mortality of seedlings. Seedling height was significantly improved by inoculation with all fungi except R. roseolus and isolate CCMA-1 of S. collinitus. One year after planting, mycorrhization of control seedlings was negligible, and percentages of ESR were under 38% for the rest of treatments. In spring of the second year, seedlings in all treatments, including the control, became highly mycorrhizal (60-77% of ESR). Low ectomycorrhizal diversity (five morphotypes described) and seasonal variation on morphotype composition were detected 2 years after plantation. From a perspective of soil restoration management under limiting environmental conditions, nursery inoculation with selected fungi can be a key advantage for tree seedlings to surmount the initial transplant stress, assuring their establishment in the field. Our results emphasise the importance of selecting compatible fungal-host species combinations for nursery inoculation and sources of inoculum adapted to the environmental conditions of the transplantation site.

The effect of co-inoculation with Pisolithus tinctorius and a PGPR belonging to the genus Bacillus (Bacillus licheniformis CECT 5106 and Bacillus pumilus CECT 5105) in enhancing growth of Pinus pinea plants and the changes that occurred in rhizosphere microbial communities and the degree of mycorrhization were evaluated. Both bacterial strains of Bacillus promote the growth of Pinus pinea seedlings, but this biological effect does not imply a synergic effect with mycorrhizal infection. However, the positive response to mycorrhiza in a longer-term experiment it could be expected. The introduction of both inocula causes an alteration in the microbial rhizosphere composition, despite the low levels of inocula that were found at the end of the assay.

Lupin nodule cells maintain their ability to divide for several cycles after being infected by endosymbiotic rhizobia. The conformation of the cytoskeletal elements of nodule cells was studied by fluorescence labelling, immunocytochemistry, and laser confocal and transmission electron microscopy. The dividing infected cells showed the normal microtubule and actin patterns of dividing plant cells. The clustered symbiosomes were tethered to the spindle-pole regions and moved to the cell poles during spindle elongation. In metaphase, anaphase, and early telophase, the symbiosomes were found at opposite cell poles where they did not interfere with the spindle filaments or phragmoplast. This symbiosome positioning was comparable with that of the organelles (which ensures organelle inheritance during plant cell mitosis). Tubulin microtubules and actin microfilaments appeared to be in contact with the symbiosomes. The possible presence of actin molecular motor myosin in nodules was analysed using a monoclonal antibody against the myosin light chain. The antigen was detected in protein extracts of nodule and root cytosol as bands of approximately 20 kDa (the size expected). In the nodules, an additional polypeptide of 65 kDa was found. Immunogold techniques revealed the antigen to be localized over thin microfilaments linked to the cell wall, as well as over the thicker microfilament bundles and surrounding the symbiosomes. The pattern of cytoskeleton rearrangement in dividing infected cells, along with the presence of myosin antigen, suggests that the positioning of symbiosomes in lupin nodule cells might depend on the same mechanisms used to partition genuine plant cell organelles during mitosis.

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Nodulated bean plants were exposed to mild salt stress or water deficit in such a way that the nodule's nitrogen-fixing activity was reduced to about 25-30% that of controls. Water-deprived plants showed a slight decrease in the weight of the aerial part, whereas the photosynthetic parameters were not significantly affected. In contrast, saltstressed plants displayed a reversible decrease in the quantum yield of photosystem II photochemistry. Five waterdeficit responsive cDNA clones encoding one lipid transfer protein, two late-embryogenesis abundant (LEA) proteins and two proline-rich proteins (PRPs) showed different organ-specific expression patterns depending on the kind of stress applied. PRPs and one LEA protein, PvLEA-18, exhibited the highest expression in nodules. Anti-PvLEA-18 antibodies were used to immunolocalize the protein in the nodule. PvLEA-18 was localized in the cytoplasm and nucleus of nodule cortex cells, and preferentially in cells of the vascular bundles, showing enhanced accumulation under water deficit. To our knowledge, this is the first time that a LEA protein has been identified in legume nodules.