Inoculation of Pinus halepensis Mill. with selected ectomycorrhizal fungi improves seedling establishment 2 years after planting in a degraded gypsum soil.

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.

Immunolocalization of alkaloids and X-ray microanalysis of elements in lupin seeds.

Immunolocalization of alkaloids in lupin seeds (Lupinus spp.) has been performed by cryofixation and conventional methods. Alkaloids were localized in the protein bodies of the cotyledon cells. Some immunogold particles in the walls of these cells were also observed. There were no differences in the sites of localization between the two mentioned methods. X-ray microanalysis of elements showed the presence of P, Mg, S, and K in the protein bodies of cotyledon cells in lupin seeds. The role of K+ in alkaloids transport is discussed.

Effects of Inoculation with PGPR Bacillus and Pisolithus tinctorius on Pinus pinea L. Growth, Bacterial rhizosphere Colonization, and Mycorrhizal Infection.

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.

Characterization of a Rhizobium meliloti proline dehydrogenase mutant altered in nodulation efficiency and competitiveness on alfalfa roots.

Rhizobium meliloti strain GRM8 is able to transform ornithine into proline by means of an ornithine cyclodeaminase and, therefore, has the ability to use either of these amino acids as its sole carbon and nitrogen source. By Tn5 insertion mutagenesis we obtained a GRM8 mutant derivative strain (LM1) unable to catabolize either ornithine or proline. DNA hybridization studies showed that the LM1 mutant carries a single Tn5 insertion within a chromosomally located gene that, as deduced from a partial nucleotide sequence, encodes a proline dehydrogenase (ProDH). Enzymatic assays confirmed the lack of ProDH activity in cell extracts of strain LM1 and revealed that production of this enzyme is inducible in the parental strain by proline and ornithine. Ultrastructural nodule microscopy analysis, acetylene reduction assays, and dry-weight determinations of nodulated alfalfa plants showed no obvious defect in the nitrogen fixation process of the ProDH- mutant LM1. However, nodulation tests and competition assays demonstrated that in R. meliloti ProDH is required for nodulation efficiency and competitiveness on alfalfa roots.

Essentiality of Boron for Symbiotic Dinitrogen Fixation in Pea (Pisum sativum) Rhizobium Nodules.

The effect of boron deficiency on symbiotic nitrogen fixation in pea (Pisum sativum) was examined. The 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.

Functional expression of the Erwinia uredovora carotenoid biosynthesis gene crtl in transgenic plants showing an increase of beta-carotene biosynthesis activity and resistance to the bleaching herbicide norflurazon.

Among the enzymes involved in carotenoid biosynthesis, phytoene desaturase is considered to be a rate-limiting enzyme in this pathway and is also the target of many bleaching herbicides. This enzyme shows diversity concerning its function and amino acid homology among various organisms. The phytoene desaturase gene crtl of Erwinia uredovora was expressed, the 5'-region of which was fused to the sequence for the transit peptide of a pea Rubisco small subunit, in tobacco plants under the control of the CaMV 35S promoter. This chimeric gene product was targeted into chloroplasts and processed in the transgenic plants. The production and processing of the corresponding protein could be demonstrated by Western blotting. Immunogold localization showed that the location of the gene product Crtl was preferentially in the thylakoids. A radioactive labeling study using the leaves demonstrated enhanced activity for the synthesis of beta-carotene. In addition, the transgenic tobacco acquired elevated resistance to the bleaching herbicide norflurazon.

Leghemoglobin in Lupin Plants (Lupinus albus cv Multolupa).

Leghemoglobin was localized by immunogold techniques in nodules of Lupinus albus cv Multolupa inoculated with Bradyrhizobium sp. (Lupinus) strain ISLU 16. The protein localization was performed in nodules embedded in Spurr's and Araldite epoxy resins and Lowycryl K4M. A very good preservation of both the ultrastructure and antigenicity was obtained with the tissues embedded in Araldite following glutaraldehyde fixation and unpostfixed in osmium tetroxide. Lupin leghemoglobin is a stable and abundant protein which allows a conventional method to be safely used for localization of leghemoglobin. Labeling of leghemoglobin was specifically confined to the cytosol matrix and nuclei. Gold particles were never observed in the peribacteroidal spaces nor in the cytoplasmic organelles of the infected cells. Decrease of leghemoglobin was observed when the plants were grown with 10.7 micromolar and 21.4 micromolar of nitrate.

Immunogold localization of photosynthetic fructose-1,6-bisphosphatase in pea leaf tissue.

An enriched IgG serum fraction obtained from rabbits immunized against pea chloroplast fructose-1,6-bisphosphatase (FBPase) was used, coupled to colloidal gold (15 nanometer particles) goat anti-rabbit IgG, to analyze by electron microscopy the location of photosynthetic FBPase in pea (Pisum sativum L.) leaf ultrathin sections. In accordance with earlier biochemical studies on distribution of FBPase activity, the enzyme was visualized both in the stromal space and bound to the chloroplast membranes. Some gold particles also appear in the cytoplasm, which can be related to the presence in the cytosol of a high molecular weight precursor of this nuclear coded enzyme.