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Within tree communities, the differential use of soil N mineral resources, a key factor in ecosystem functioning, may reflect functional complementarity, a major mechanism that could explain species coexistence in tropical rainforests. Eperua falcata and Dicorynia guianensis, two abundant species cooccurring in rainforests of French Guiana, were chosen as representative of two functional groups with complementary N uptake strategies (contrasting leaf δ (15)N signatures related to the δ (15)N of their soil N source, NO3 (-) or NH4 (+)). The objectives were to investigate if these strategies occurred under contrasted soil N resources in sites with distinct geological substrates representative of the coastal rainforests. Results showed that species displayed contrasting leaf δ (15)N signatures on both substrates, confirming their complementary N uptake strategy. Consequently, their leaf (15)N can be used to trace the presence of inorganic N-forms in soils (NH4 (+) and NO3 (-)) and thus to indicate the capacity of soils to provide each of these two N sources to the plant community.

Eperua falcata (Aublet), a late-successional species in tropical rainforest and one of the most abundant tree in French Guiana, has developed an original strategy concerning N-acquisition by largely preferring nitrate, rather than ammonium (H. Schimann, S. Ponton, S. Hättenschwiler, B. Ferry, R. Lensi, A.M. Domenach, J.C. Roggy, Differing nitrogen use strategies of two tropical rainforest tree species in French Guiana: evidence from (15)N natural abundance and microbial activities, Soil Biol. Biochem. 40 (2008) 487-494). Given the preference of this species for nitrate, we hypothesized that root exudates would promote nitrate availability by (a) enhancing nitrate production by stimulating ammonium oxidation or (b) minimizing nitrate losses by inhibiting denitrification. Root exudates were collected in situ in monospecific planted plots. The phytochemical analysis of these exudates and of several of their corresponding root extracts was achieved using UHPLC/DAD/ESI-QTOF and allowed the identification of diverse secondary metabolites belonging to the flavonoid family. Our results show that (i) the distinct exudation patterns observed are related to distinct root morphologies, and this was associated with a shift in the root flavonoid content, (ii) a root extract representative of the diverse compounds detected in roots showed a significant and selective metabolic inhibition of isolated denitrifiers in vitro, and (iii) in soil plots the abundance of nirK-type denitrifiers was negatively affected in rhizosphere soil compared to bulk. Altogether this led us to formulate hypothesis concerning the ecological role of the identified compounds in relation to N-acquisition strategy of this species., (Copyright © 2013 Elsevier Masson SAS. All rights reserved.)

The Frankia sp. strain ACN14a superoxide dismutase SodF was previously shown to be induced in response to Alnus glutinosa root exudates, and its gene was sequenced. We report here the sequence of the 9-kb genomic segment surrounding the sodF gene and further characterize this gene and its product. Nine ORFs coding for various proteins, such as regulators, acetyl-CoA transferases, and a bacterioferritin A next to the sodF gene, were found. Northern blot analysis showed that the sodF gene was expressed as a major 1-kb transcript, which indicates that it has its own promoter. The sodF gene strongly complemented an Escherichia coli triple mutant (sodA sodB recA), restoring aerobic growth when the gene was expressed from the synthetic tac promoter but when expressed from its own promoter showed only slight rescue, suggesting that it was poorly recognized by the E. coli RNA polymerase. It is noteworthy that this is the first time that a Frankia gene has been reported to complement an E. coli mutant. The superoxide dismutase activity of the protein was inactivated by hydrogen peroxide, indicating that the metal ligand is iron, which is supported by analysis of the protein sequence. Thus, the SodF protein induced in Frankia by root exudates is an iron-containing enzyme similar to the one present in the nodules.

Three series of hopanoids, differing by their configurations at C-17 and C-21, have been identified in several Frankia spp. and other related soil bacteria. The widespread bacterial hopanoids of the 17beta(H),21beta(H) series were accompanied by their isomers of the 17beta(H),21alpha(H) (moretane) and 17alpha(H), 21beta(H) series. The latter series has not previously been found in living organisms and is considered to be a result of the abiotic isomerization of the thermodynamically less stable 17beta(H),21beta(H) hopanoids. This simultaneous presence of three isomeric hopanoid series highlights intriguing problems in the biogenesis of the bacteriohopane skeleton and partly questions the significance of hopanic biomarkers in sediments.

Two-dimensional (2-D) polyacrylamide gel electrophoresis was used to detect proteins induced in Frankia sp. strain ACN14a-tsr by root exudates of its symbiotic host, Alnus glutinosa. The 5 most prominent proteins were purified from 2-D gels and characterized by N-terminal sequencing. All of these proteins had a high percentage of similarity with known stress proteins. One protein match was the Fe superoxide dismutase (Fe-SOD), another was a tellurite resistance protein (Ter), the third was a bacterioferritin comigratory protein (Bcp); and two matches, differing only by their isoelectric point, were the same small heat shock protein (Hsp), a major immune reactive protein found in mycobacteria. This suggests that the symbiotic microorganism Frankia, first responds with a normal stress response to toxic root products of its symbiotic host plant. To confirm its identity, the gene corresponding to the Fe-SOD protein, sodF was isolated from a genomic library by a PCR-approach and sequenced. It is the first stress response gene characterized in Frankia.

Vesicles are specific Frankia structures which are produced under nitrogen-limiting culture conditions. Hopanoids are the most abundant lipids in these vesicles and are believed to protect the nitrogenase against oxygen. The amounts and quality of each hopanoid were estimated in different Frankia strains cultivated under nitrogen-depleted and nitrogen-replete conditions in order to detect a possible variation. Studied Frankia strains nodulating Eleagnus were phylogenetically characterized by analysis of the nifD-K intergenic region as closely related to genomic species 4 and 5. Phylogenetically different strains belonging to three infectivity groups were cultivated in the same medium with and without nitrogen source for 10 d before hopanoid content analysis by HPLC. Four hopanoids together accounted for 23-87% and 15-87% of the total lipids under nitrogen-replete and nitrogen-depleted culture conditions, respectively. Two of the hopanoids found, bacteriohopanetetrols and their phenylacetic acid esters, have previously been described in Frankia Two new hopanoids, moretan-29-ol and a bacteriohopanetetrol propionate, have also been identified. The moretan-29-ol and bacteriohopanetetrols were found to be the most abundant hopanoids whereas the bacteriohopanetetrol propionate and phenylacetates were present at a concentration close to the limit of detection. The ratio of (bacteriohopanetetrols + moretan-29-ol)/(total lipids) varied in most of the strains between nitrogen-depleted and nitrogen-replete culture conditions. In most of the strains, the hopanoid content was found to be slightly higher under nitrogen-replete conditions than under nitrogen-depleted conditions. These results suggest that remobilization, rather than neosynthesis of hopanoids, is implicated in vesicle formation in Frankia under nitrogen-depleted conditions.

Effects of three forage harvesting regimes-total removal of foliage and branches once (T-12) or twice a year (T-6) and 50% removal every 2 months (P-2)-on growth and biological dinitrogen fixation of Gliricidia sepium (Jacq.) Walp were studied under subhumid tropical conditions in Guadeloupe, French Antilles. Gliricidia sepium was grown in association with the perennial C(4) grass Dichantium aristatum (Poir) C.E. Hubbard in a two-storied fodder production system. The medium-term effects of pruning on N(2) fixation were assessed by the (15)N natural abundance method. Gmelina arborea Roxb. was used as the non-fixing reference. The trees in the T-12 regime followed the natural phenological cycle, and flowering and podfilling at the beginning of the dry season reduced both foliage and nodule biomass. The T-6 regime impeded flowering, and only a few flowers, on older branches, were produced in the P-2 regime. In trees in the T-12, T-6, and P-2 regimes, fixed N comprised 54-87, 54-92, and 60-87%, respectively, of the total N in aboveground biomass, depending on sampling date. Total annual accumulation of N in harvestable aboveground biomass was highest in trees in the T-6 regime at 313 kg ha(-1), of which 204 kg ha(-1) of N was fixed from the atmosphere. In all treatments, about 70% of the N exported per year from the plot in the fodder harvest came from N(2) fixation. Thus, N(2) fixation makes an important contribution to the N economy of the G. sepium-D. aristatum forage production system, and greatly reduces the need for fertilizer application.

Effects of four N sources and two defoliation treatments on growth and nitrogenase activity of Gliricidia sepium (Jacq.) Walp seedlings were studied in a greenhouse. All nutrients were supplied in irrigation water to the sterile growing medium. The N sources were: (1) 100 mg l(-1) of N supplied as NO(3) (-) (high-NO(3) (-)), (2) 50 mg l(-1) of N supplied as NO(3) (-) and inoculation with Rhizobium spp. medium-NO(3) (-)), (3)100 mg l(-1) of N supplied as NH(4)NO(3), and (4) inoculation with Rhizobium spp without mineral N (N(2)). At 35 weeks after sowing, mean total biomass was 130.5, 50.5, 22.9 and 17.4 g seedling(-1) in the NH(4)NO(3), N(2), medium-NO(3) (-) and high-NO(3) (-) treatments, respectively. The root/shoot ratio was high in all of the N treatments (1.73-2.77) because the seedlings had big taproots. The medium-NO(3) (-) treatment completely inhibited nodulation, whereas seedlings in the N(2) treatment were profusely nodulated. At 32 weeks after sowing, groups of seedlings in the N(2) and high-NO(3) (-) treatments were subjected to 50 or 100% defoliation. Closed-chamber acetylene reduction assays of intact root systems were conducted to compare nitrogenase activity at 7, 14 and 28 days after defoliation (DAD). At 7 and 14 DAD, nitrogenase activity of completely and partially defoliated seedlings was about 10 and 60%, respectively, of that of undefoliated controls. At 28 DAD, nitrogenase activity of completely defoliated seedlings was twice the predefoliation value, whereas nitrogenase activity of partially defoliated seedlings was only 87% of the predefoliation value. Recovery of nitrogenase activity was strongly correlated with foliage regrowth in the completely defoliated seedlings, but not in the partially defoliated seedlings. Abundant belowground C and N reserves in the large taproot probably contributed to the rapid recovery from defoliation. Accumulation of belowground biomass may also improve defoliation tolerance of mature trees.

Symbioses between the root nodule-forming, nitrogen-fixing actinomycete Frankia and its angiospermous host plants are important in the nitrogen economies of numerous terrestrial ecosystems. Molecular characterization of Frankia strains using polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP) analyses of the 16S rRNA-ITS gene and of the nifD-nifK spacer was conducted directly on root nodules collected worldwide from Casuarina and Allocasuarina trees. In their native habitats in Australia, host species contained seven distinctive sets of Frankia in seven different molecular phylogenetic groups. Where Casuarina and Allocasuarina trees are newly planted outside Australia, they do not normally nodulate unless Frankia is introduced with the host seedling. Nodules from Casuarina trees introduced outside Australia over the last two centuries were found to contain Frankia from only one of the seven phylogenetic groups associated with the host genus Casuarina in Australia. The phylogenetic group of Frankia found in Casuarina and Allocasuarina trees introduced outside Australia is the only group that has yielded isolates in pure culture, suggesting a greater ability to survive independently of a host. Furthermore, the Frankia species in this group are able to nodulate a wider range of host species than those in the other six groups. In baiting studies, Casuarina spp. are compatible with more Frankia microsymbiont groups than Allocasuarina host spp. adapted to drier soil conditions, and C. equisetifolia has broader microsymbiont compatibility than other Casuarina spp. Some Frankia associated with the nodular rhizosphere and rhizoplan, but not with the nodular tissue, of Australian hosts were able to nodulate cosmopolitan Myrica plants that have broad microsymbiont compatibility and, hence, are a potential host of Casuarinaceae-infective Frankia outside the hosts' native range. The results are consistent with the idea that Frankia symbiotic promiscuity and ease of isolation on organic substrates, suggesting saprophytic potential, are associated with increased microsymbiont ability to disperse and adapt to diverse new environments, and that both genetics and environment determine a host's nodular microsymbiont.

The suitability of the natural 15 N abundance and of total N concentration of leaves as indicators of the type of plant N nutrition in a rain forest of French Guiana were tested. Leaf samples from primary legume species, non-legumes (pioneer species) and from the non-N 2 -fixing species Dicorynia guianensis were analyzed. Both δ 15 N and total leaf N varied widely (-1 ?δ 15 N (‰) ? 7 and 1 ? leaf N(%) ? 3.2) suggesting possible distinctions between diazotrophic and non-fixing plants. The δ 15 N also revealed two statistically distinct groups of non-N 2 -fixing species (δ 15 N = 5.14 ± 0.3 vs δ 15 N = 1.65 ± 0.17) related to the different ecological behaviors of these species in the successional processes. We conclude that the δ 15 N signature of plant leaves combined with their total N concentration may be relevant indicators for identifying functional groups within the community of non-N 2 -fixing species, as well as for detecting diazotrophy. Despite the variability in the δ 15 N of the non-N 2 -fixing species, N 2 -fixing groups can still be identified, provided that plants are simultaneously classified taxonomically, by their leaf δ 15 N and total N concentration and by the presence or absence of nodules. The variability in the δ 15 N of the non-fixing species is discussed.

Functional aspects of biodiversity were investigated in a lowland tropical rainforest in French Guyana (5°2'N, annual precipitation 2200 mm). We assessed leaf δ 15 N as a presumptive indicator of symbiotic N 2 fixation, and leaf and wood cellulose δ 13 C as an indicator of leaf intrinsic water-use efficiency (CO 2 assimilation rate/leaf conductance for water vapour) in dominant trees of 21 species selected for their representativeness in the forest cover, their ecological strategy (pioneers or late successional stage species, shade tolerance) or their potential ability for N 2 fixation. Similar measurements were made in trees of native species growing in a nearby plantation after severe perturbation (clear cutting, mechanical soil disturbance). Bulk soil δ 15 N was spatially quite uniform in the forest (range 3-5‰), whereas average leaf δ 15 N ranged from -0.3‰ to 3.5‰ in the different species. Three species only, Diplotropis purpurea, Recordoxylon speciosum (Fabaceae), and Sclerolobium melinonii (Caesalpiniaceae), had root bacterial nodules, which was also associated with leaf N concentrations higher than 20 mg g -1 . Although nodulated trees displayed significantly lower leaf δ 15 N values than non-nodulated trees, leaf δ 15 N did not prove a straightforward indicator of symbiotic fixation, since there was a clear overlap of δ 15 N values for nodulated and non-nodulated species at the lower end of the δ 15 N range. Perturbation did not markedly affect the difference δ 15 N soil  -   δ 15 N leaf , and thus the isotopic data provide no evidence of an alteration in the different N acquisition patterns. Extremely large interspecific differences in sunlit leaf δ 13 C were observed in the forest (average values from -31.4 to -26.7‰), corresponding to intrinsic water-use efficiencies (ratio CO 2 assimilation rate/leaf conductance for water vapour) varying over a threefold range. Wood cellulose δ 13 C was positively related to total leaf δ 13 C, the former values being 2-3‰ higher than the latter ones. Leaf δ 13 C was not related to leaf δ 15 N at either intraspecific or interspecific levels. δ 13 C of sunlit leaves was highest in shade hemitolerant emergent species and was lower in heliophilic, but also in shade-tolerant species. For a given species, leaf δ 13 C did not differ between the pristine forest and the disturbed plantation conditions. Our results are not in accord with the concept of existence of functional types of species characterized by common suites of traits underlying niche differentiation; rather, they support the hypothesis that each trait leads to a separate grouping of species.

The nifD-K intergenic spacer (IGS) of ArI3 and ACoN24d were found to have a length 265 and 199 nucleotides, respectively. They are markedly less conserved than the two neighbouring genes and have, in some instances, a repeated structure reminiscent of an insertion event. The repeated sequence and the IGSs have no detectable homology with sequences in DNA databanks. The IGS has a stem-loop structure with a low folding energy, lower than that between nifH and nifD. No convincing alignment of IGS sequences could be obtained among Frankia strains. Only between ACoN24d and ArI3, which belong to the same genomic species, was the alignment good enough to permit detection of a doubly repeated structure. No promoter could be detected in the IGSs. The putative nifK open reading frame (ORF) in Frankia strain ArI3 has a length of 1587 nucleotides, starting with a GTG codon, preceded by a ribosome binding site of a structure similar to that of nifH (GGAGGN7). The codon usage was similar to that of previously sequenced Frankia genes with a strong bias toward G- and C-ending codons except in the case of glycine where GGT is frequent. Alignment of the three Frankia nifK sequences (EUN1f; ArI3 and ACoN24d) with those of other nitrogen-fixing bacteria permitted detection of a sequence conserved among the three Frankia strains but absent in the other sequences. A primer targeted to that region in combination with FGPD807-85 amplified the nifD-KIGS sequences of all Frankia strains (except the non-nitrogen-fixing Frankia strains CN3 and AgB1-9) and yet failed to amplify DNA of all other nitrogen-fixing bacteria.(ABSTRACT TRUNCATED AT 250 WORDS)