Rhizophagus intraradices and Azospirillum brasilense improve growth of herbaceous plants and soil biological activity in revegetation of a recovering coal-mining area.

We assessed, in a field experiment, the effects of arbuscular mycorrhizal fungi (Rhizophagus intraradices) and plant growth-promoting bacteria (Azospirillum brasilense) on the soil biological activity and the growth of key pioneer species used in the revegetation of coal-mining areas undergoing recovery. We applied four inoculation treatments to the pioneer plant species (Lablab purpureus, Paspalum notatum, Crotalaria juncea, Neonotonia wightii, Stylosanthes guianensis, Andropogon gayanus and Trifolium repens) used in the recovery process: NI (Control - Non-inoculated), AZO (A. brasilense), AMF (R. intraradices), and co-inoculation of AZO and AMF. On the 75th and 180th days, we measured plant dry mass, mycorrhizal colonization, N and P concentration, and accumulation in plant tissue. We collected soil to quantify glomalin content and soil enzyme activity. After 180 days, we did a phytosociological characterization of the remaining spontaneous plants.The both microorganisms, singly or co-inoculated, promoted increases in different fractions of soil glomalin, acid phosphatase activity, and fluorescein diacetate activity at 75 and 180 days. The inoculation was linked to higher plant biomass production (62-89%) and increased plant P and N accumulation by 34-75% and 70-85% at 180 days, compared with the non-inoculated treatment. Among the pioneer species sown Crotalaria juncea produced the highest biomass at the 75th and 180th days (67% and 76% of all biomass), followed by Lablab purpureus (3% and 0.5%), while the other species failed to establish. At 180 days, we observed twenty spontaneous plant species growing in the area, primarily from the Poaceae family (74%). That suggests that the pioneer species present in the area do not hinder the ecological succession process. Inoculation of R. intraradices and A. brasilense, isolated or combined, increases soil biological activity, growth, and nutrient accumulation in key pioneer plant species, indicating the potential of that technique for the recovery of lands degraded by coal mining.

The radiation of nodulated Chamaecrista species from the rainforest into more diverse habitats has been accompanied by a reduction in growth form and a shift from fixation threads to symbiosomes.

All non-Mimosoid nodulated genera in the legume subfamily Caesalpinioideae confine their rhizobial symbionts within cell wall-bound 'fixation threads' (FTs). The exception is the large genus Chamaecrista in which shrubs and subshrubs house their rhizobial bacteroids more intimately within symbiosomes, whereas large trees have FTs. This study aimed to unravel the evolutionary relationships between Chamaecrista growth habit, habitat, nodule bacteroid type, and rhizobial genotype. The growth habit, bacteroid anatomy, and rhizobial symbionts of 30 nodulated Chamaecrista species native to different biomes in the Brazilian state of Bahia, a major centre of diversity for the genus, was plotted onto an ITS-trnL-F-derived phylogeny of Chamaecrista. The bacteroids from most of the Chamaecrista species examined were enclosed in symbiosomes (SYM-type nodules), but those in arborescent species in the section Apoucouita, at the base of the genus, were enclosed in cell wall material containing homogalacturonan (HG) and cellulose (FT-type nodules). Most symbionts were Bradyrhizobium genotypes grouped according to the growth habits of their hosts, but the tree, C. eitenorum, was nodulated by Paraburkholderia. Chamaecrista has a range of growth habits that allow it to occupy several different biomes and to co-evolve with a wide range of (mainly) bradyrhizobial symbionts. FTs represent a less intimate symbiosis linked with nodulation losses, so the evolution of SYM-type nodules by most Chamaecrista species may have (i) aided the genus-wide retention of nodulation, and (ii) assisted in its rapid speciation and radiation out of the rainforest into more diverse and challenging habitats.

High-precision 14C measurements of parenchyma-rich Hymenolobium petraeum tree species confirm bomb-peak atmospheric levels and reveal local fossil-fuel CO2 emissions in the Central Amazon.

Atmospheric radiocarbon (14C) recorded in tree rings has been widely used for atmospheric 14C calibration purposes and climate studies. But atmospheric 14C records have been limited along tropical latitudes. Here we report a sequence from 1938 to 2007 of precisely measured 14C dates in tree rings of the parenchyma-rich Hymenolobium petraeum tree species (Porto Trombetas, 1°S, 56°W) from the Central Brazilian Amazon. H. petraeum has discernible growth ring boundaries that allow dating techniques to be employed to produce calendrical dates. Bomb-peak tree-ring 14C reconstruction coincides with the broader changes associated with reported values of the Southern Hemisphere atmospheric 14C curve (SH zone 3; values within the ±2σ interval), suggesting that inter-hemispheric air-mass transport of excess-14C injected into the stratosphere during intensive atmospheric nuclear tests is relatively uniform across distinct longitudinal regions. From the early 1980s onwards, H. petraeum had lower 14C values than other pantropical 14C records. Through 14C-based estimation, we found a strong influence of fossil-fuel CO2 contributions from Porto Trombetas mining operations and shipping traffic on inland waterways. An increase of at least 6.3 ± 0.8 ppm of fossil-fuel CO2 has been detected by 14C. Our findings invite further 14C analyses using tree rings of tropical tree species as a potential tracer for a wide range of environmental sources of atmospheric 14C-variability.

Nodulation of the neotropical genus Calliandra by alpha or betaproteobacterial symbionts depends on the biogeographical origins of the host species.

The neotropical genus Calliandra is of great importance to ecology and agroforestry, but little is known about its nodulation or its rhizobia. The nodulation of several species from two restricted diversity centres with native/endemic species (Eastern Brazil and North-Central America) and species widespread in South America, as well as their nodule structure and the molecular characterization of their rhizobial symbionts based on phylogeny of the 16S rRNA, recA and nodC gene, is reported herein. Species representative of different regions were grown in Brazilian soil, their nodulation observed, and their symbionts characterized. Calliandra nodules have anatomy that is typical of mimosoid nodules regardless of the microsymbiont type. The rhizobial symbionts differed according to the geographical origin of the species, i.e. Alphaproteobacteria (Rhizobium) were the exclusive symbionts from North-Central America, Betaproteobacteria (Paraburkholderia) from Eastern Brazil, and a mixture of both nodulated the widespread species. The symbiont preferences of Calliandra species are the result of the host co-evolving with the "local" symbiotic bacteria that thrive in the different edaphoclimatic conditions, e.g. the acidic soils of NE Brazil are rich in acid-tolerant Paraburkholderia, whereas those of North-Central America are typically neutral-alkaline and harbour Rhizobium. It is hypothesized that the flexibility of widespread species in symbiont choice has assisted in their wider dispersal across the neotropics.

Mycorrhizal networks facilitate the colonization of legume roots by a symbiotic nitrogen-fixing bacterium.

Arbuscular mycorrhizal fungi (AMF) absorb and translocate nutrients from soil to their host plants by means of a wide network of extraradical mycelium (ERM). Here, we assessed whether nitrogen-fixing rhizobia can be transferred to the host legume Glycine max by ERM produced by Glomus formosanum isolate CNPAB020 colonizing the grass Urochloa decumbens. An H-bridge experimental system was developed to evaluate the migration of ERM and of the GFP-tagged Bradyrhizobium diazoefficiens USDA 110 strain across an air gap compartment. Mycorrhizal colonization, nodule formation in legumes, and occurrence of the GFP-tagged strain in root nodules were assessed by optical and confocal laser scanning microscopy. In the presence of non-mycorrhizal U. decumbens, legume roots were neither AMF-colonized nor nodulated. In contrast, G. formosanum ERM crossing the discontinuous compartment connected mycorrhizal U. decumbens and G. max roots, which showed 30-42% mycorrhizal colonization and 7-11 nodules per plant. Fluorescent B. diazoefficiens cells were detected in 94% of G. max root nodules. Our findings reveal that, besides its main activity in nutrient transfer, ERM produced by AMF may facilitate bacterial translocation and the simultaneous associations of plants with beneficial fungi and bacteria, representing an important structure, functional to the establishment of symbiotic relationships.

Mimosa caesalpiniifolia Benth. adapts to rhizobia populations with differential taxonomy and symbiotic effectiveness outside of its location of origin.

Mimosa caesalpiniifolia Benth. is a legume native to the semi-arid region of Brazil, in the Northeast. Its successful adaptation to other locations, such as the Atlantic Forest in the Southeast region, may be related to its ability to establish symbiosis with nitrogen-fixing bacteria, especially ß-rhizobia of the genus Paraburkholderia. The objective of this work was to determine whether M. caesalpiniifolia adapted to bacterial symbionts in locals where it was introduced. Bacteria were recovered from nodules of M. caesalpiniifolia and characterized at the genetic level by BOX-PCR and sequencing of the 16S rRNA, recA, nifH, and nodC genes. Their symbiotic effectiveness was assessed under axenic conditions. M. caesalpiniifolia nodulated mainly with Paraburkholderia sabiae and a few strains of Rhizobium in the Southeast. On the other hand, the symbionts found in the Northeast were, predominantly, Paraburkholderia diazotrophica. Regardless of its origin, P. diazotrophica promoted a superior accumulation of plant biomass than other bacterial species. The results presented here demonstrate the ability of M. caesalpiniifolia to adapt to bacterial populations outside its location of origin, and indicate that, in this case, the symbiotic effectiveness was associated with the taxonomical classification of the strains.

Two herbicides, two fungicides and spore-associated bacteria affect Funneliformis mosseae extraradical mycelium structural traits and viability.

The extraradical mycelium (ERM) produced by arbuscular mycorrhizal fungi is fundamental for the maintenance of biological fertility in agricultural soils, representing an important inoculum source, together with spores and mycorrhizal root fragments. Its viability and structural traits, such as density, extent and interconnectedness, which are positively correlated with the growth and nutrition of host plants, may be affected by different agronomic practices, including the use of pesticides and by different mycorrhizospheric communities. This work, carried out using a whole-plant experimental model system, showed that structural traits of ERM, such as length and density, were strongly decreased by the herbicides dicamba and glufosinolate and the fungicides benomyl and fenhexamid, while anastomosis frequency and hyphal branching were differentially modulated by singly inoculated mycorrhizospheric bacteria, depending on their identity.

Genetic diversity of symbiotic Paraburkholderia species isolated from nodules of Mimosa pudica (L.) and Phaseolus vulgaris (L.) grown in soils of the Brazilian Atlantic Forest (Mata Atlântica).

Some species of the genus Paraburkholderia that are able to nodulate and fix nitrogen in symbiosis with legumes are called ß-rhizobia and represent a group of ecological and biotechnological importance. We used Mimosa pudica and Phaseolus vulgaris to trap 427 rhizobial isolates from rhizospheric soil of Mimoseae trees in the Brazilian Atlantic Forest. Eighty-four representative strains were selected according to the 16S rRNA haplotypes and taxonomically characterized using a concatenated 16S rRNA-recA phylogeny. Most strains were assembled in the genus Paraburkholderia, including Paraburkholderia sabiae and Pa. nodosa. Mesorhizobium (α-rhizobia) and Cupriavidus (ß-rhizobia) were also isolated, but in smaller proportions. Multilocus sequence analysis and BOX-PCR analyses indicated that six clusters of Paraburkholderia represent potential new species. In the phylogenetic analysis of the nodC gene, the majority of the strains were positioned in the same groups as in the 16S rRNA-recA tree, indicative of stability and vertical inheritance, but we also identified horizontal transfer of nodC in Pa. sabiae. All α- and ß-rhizobial species were trapped by both legumes, although preferences of the host plants for specific rhizobial species have been observed.

Rhizobial characterization in revegetated areas after bauxite mining.

Little is known regarding how the increased diversity of nitrogen-fixing bacteria contributes to the productivity and diversity of plants in complex communities. However, some authors have shown that the presence of a diverse group of nodulating bacteria is required for different plant species to coexist. A better understanding of the plant symbiotic organism diversity role in natural ecosystems can be extremely useful to define recovery strategies of environments that were degraded by human activities. This study used ARDRA, BOX-PCR fingerprinting and sequencing of the 16S rDNA gene to assess the diversity of root nodule nitrogen-fixing bacteria in former bauxite mining areas that were replanted in 1981, 1985, 1993, 1998, 2004 and 2006 and in a native forest. Among the 12 isolates for which the 16S rDNA gene was partially sequenced, eight, three and one isolate(s) presented similarity with sequences of the genera Bradyrhizobium, Rhizobium and Mesorhizobium, respectively. The richness, Shannon and evenness indices were the highest in the area that was replanted the earliest (1981) and the lowest in the area that was replanted most recently (2006).

The complete genome of Burkholderia phenoliruptrix strain BR3459a, a symbiont of Mimosa flocculosa: highlighting the coexistence of symbiotic and pathogenic genes.

BACKGROUND: Burkholderia species play an important ecological role related to xenobiosis, the promotion of plant growth, the biocontrol of agricultural diseases, and symbiotic and non-symbiotic biological nitrogen fixation. Here, we highlight our study as providing the first complete genome of a symbiotic strain of B. phenoliruptrix, BR3459a (=CLA1), which was originally isolated in Brazil from nodules of Mimosa flocculosa and is effective in fixing nitrogen in association with this leguminous species. RESULTS: Genomic comparisons with other pathogenic and non-pathogenic Burkholderia strains grouped B. phenoliruptrix BR3459a with plant-associated beneficial and environmental species, although it shares a high percentage of its gene repertoire with species of the B. cepacia complex (Bcc) and "pseudomallei" group. The genomic analyses showed that the bce genes involved in exopolysaccharide production are clustered together in the same genomic region, constituting part of the Group III cluster of non-pathogenic bacteria. Regarding environmental stresses, we highlight genes that might be relevant in responses to osmotic, heat, cold and general stresses. Furthermore, a number of particularly interesting genes involved in the machinery of the T1SS, T2SS, T3SS, T4ASS and T6SS secretion systems were identified. The xenobiotic properties of strain BR3459a were also investigated, and some enzymes involved in the degradation of styrene, nitrotoluene, dioxin, chlorocyclohexane, chlorobenzene and caprolactam were identified. The genomic analyses also revealed a large number of antibiotic-related genes, the most important of which were correlated with streptomycin and novobiocin. The symbiotic plasmid showed high sequence identity with the symbiotic plasmid of B. phymatum. Additionally, comparative analysis of 545 housekeeping genes among pathogenic and non-pathogenic Burkholderia species strongly supports the definition of a new genus for the second branch, which would include BR3459a. CONCLUSIONS: The analyses of B. phenoliruptrix BR3459a showed key property of fixing nitrogen that together with genes for high tolerance to environmental stresses might explain a successful strategy of symbiosis in the tropics. The strain also harbours interesting sets of genes with biotechnological potential. However, the resemblance of certain genes to those of pathogenic Burkholderia raise concerns about large-scale applications in agriculture or for bioremediation.

Burkholderia species are the most common and preferred nodulating symbionts of the Piptadenia group (tribe Mimoseae).

Burkholderia legume symbionts (also called α-rhizobia) are ancient in origin and are the main nitrogen-fixing symbionts of species belonging to the large genus Mimosa in Brazil. We investigated the extent of the affinity between Burkholderia and species in the tribe Mimoseae by studying symbionts of the genera Piptadenia (P.), Parapiptadenia (Pp.), Pseudopiptadenia (Ps.), Pityrocarpa (Py.), Anadenanthera (A.) and Microlobius (Mi.), all of which are native to Brazil and are phylogenetically close to Mimosa, and which together with Mimosa comprise the "Piptadenia group". We characterized 196 strains sampled from 18 species from 17 locations in Brazil using two neutral markers and two symbiotic genes in order to assess their species affiliations and the evolution of their symbiosis genes. We found that Burkholderia are common and highly diversified symbionts of species in the Piptadenia group, comprising nine Burkholderia species, of which three are new ones and one was never reported as symbiotic (B. phenoliruptrix). However, α-rhizobia were also detected and were occasionally dominant on a few species. A strong sampling site effect on the rhizobial nature of symbionts was detected, with the symbiont pattern of the same legume species changing drastically from location to location, even switching from ß to α-rhizobia. Coinoculation assays showed a strong affinity of all the Piptadenia group species towards Burkholderia genotypes, with the exception of Mi. foetidus. Phylogenetic analyses of neutral and symbiotic markers showed that symbiosis genes in Burkholderia from the Piptadenia group have evolved mainly through vertical transfer, but also by horizontal transfer in two species.

Nodulation in Dimorphandra wilsonii Rizz. (Caesalpinioideae), a threatened species native to the Brazilian Cerrado.

The threatened caesalpinioid legume Dimorphandra wilsonii, which is native to the Cerrado biome in Brazil, was examined for its nodulation and N(2)-fixing ability, and was compared with another, less-threatened species, D. jorgei. Nodulation and potential N(2) fixation was shown on seedlings that had been inoculated singly with five bradyrhizobial isolates from mature D. wilsonii nodules. The infection of D. wilsonii by two of these strains (Dw10.1, Dw12.5) was followed in detail using light and transmission electron microscopy, and was compared with that of D. jorgei by Bradyrhizobium strain SEMIA6099. The roots of D. wilsonii were infected via small transient root hairs at 42 d after inoculation (dai), and nodules were sufficiently mature at 63 dai to express nitrogenase protein. Similar infection and nodule developmental processes were observed in D. jorgei. The bacteroids in mature Dimorphandra nodules were enclosed in plant cell wall material containing a homogalacturonan (pectic) epitope that was recognized by the monoclonal antibody JIM5. Analysis of sequences of their rrs (16S rRNA) genes and their ITS regions showed that the five D. wilsonii strains, although related to SEMIA6099, may constitute five undescribed species of genus Bradyrhizobium, whilst their nodD and nifH gene sequences showed that they formed clearly separated branches from other rhizobial strains. This is the first study to describe in full the N(2)-fixing symbiotic interaction between defined rhizobial strains and legumes in the sub-family Caesalpinioideae. This information will hopefully assist in the conservation of the threatened species D. wilsonii.

Complete genome sequence of Burkholderia phenoliruptrix BR3459a (CLA1), a heat-tolerant, nitrogen-fixing symbiont of Mimosa flocculosa.

The genus Burkholderia represents a challenge to the fields of taxonomy and phylogeny and, especially, to the understanding of the contrasting roles as either opportunistic pathogens or bacteria with biotechnological potential. Few genomes of nonpathogenic strains, especially of diazotrophic symbiotic bacteria, have been sequenced to improve understanding of the genus. Here, we contribute with the complete genome sequence of Burkholderia phenoliruptrix strain BR3459a (CLA1), an effective diazotrophic symbiont of the leguminous tree Mimosa flocculosa Burkart, which is endemic to South America.

Nitrogen-fixing legume tree species for the reclamation of severely degraded lands in Brazil.

The main challenges faced in the reclamation of severely degraded lands are in the management of the systems and finding plant species that will grow under the harsh conditions common in degraded soils. This is especially important in extremely adverse situations found in some substrates from mining activities or soils that have lost their upper horizons. Under these conditions, recolonization of the area by native vegetation through natural succession processes may be extremely limited. Once the main physical and chemical factors restrictive to plant growth are corrected or attenuated, the introduction of leguminous trees able to form symbioses with nodulating N2-fixing bacteria and arbuscular mycorrhizal fungi constitutes an efficient strategy to accelerate soil reclamation and initiate natural succession. These symbioses give the legume species a superior capacity to grow quickly in poor substrates and to withstand the harsh conditions presented in degraded soils. In this article we describe several successful results in Brazil using N2-fixing legume tree species for reclamation of areas degraded by soil erosion, construction and mining activities, emphasizing the potential of the technique to recover soil organic matter levels and restore ecosystem biodiversity and other environmental functions.

Nodulation of Crotalaria podocarpa DC. by Methylobacterium nodulans displays very unusual features.

Crotalaria are plants of the Fabaceae family whose nodulation characteristics have been little explored despite the recent discovery of their unexpected ability to be efficiently nodulated in symbiosis with bacteria of the genus Methylobacterium. It has been shown that methylotrophy plays a key role in this unusual symbiotic system, as it is expressed within the nodule and as non-methylotroph mutants had a depleting effect on plant growth response. Within the nodule, Methylobacterium is thus able to obtain carbon both from host plant photosynthesis and from methylotrophy. In this context, the aim of the present study was to show the histological and cytological impacts of both symbiotic and methylotrophic metabolism within Crotalaria podocarpa nodules. It was established that if Crotalaria nodules are multilobed, each lobe has the morphology of indeterminate nodules but with a different anatomy; that is, without root hair infection or infection threads. In the fixation zone, bacteroids display a spherical shape and there is no uninfected cell. Crotalaria nodulation by Methylobacterium displayed some very unusual characteristics such as starch storage within bacteroid-filled cells of the fixation zone and also the complete lysis of apical nodular tissues (where bacteria have a free-living shape and express methylotrophy). This lysis could possibly reflect the bacterial degradation of plant wall pectins through bacterial pectin methyl esterases, thus producing methanol as a substrate, allowing bacterial multiplication before release from the nodule.

Multi-host ectomycorrhizal fungi are predominant in a Guinean tropical rainforest and shared between canopy trees and seedlings.

The diversity of ectomycorrhizal (ECM) fungi on adult trees and seedlings of five species, Anthonotha fragrans, Anthonotha macrophylla, Cryptosepalum tetraphyllum, Paramacrolobium coeruleum and Uapaca esculenta, was determined in a tropical rain forest of Guinea. Ectomycorrhizae were sampled within a surface area of 1600 m(2), and fungal taxa were identified by sequencing the rDNA Internal Transcribed Spacer region. Thirty-nine ECM fungal taxa were determined, of which 19 multi-hosts, 9 single-hosts and 11 singletons. The multi-host fungi represented 92% (89% when including the singletons in the analysis) of the total abundance. Except for A. fragrans, the adults of the host species displayed significant differentiation for their fungal communities, but their seedlings harboured a similar fungal community. These findings suggest that there was a potential for the formation of common mycorrhizal networks in close vicinity. However, no significant difference was detected for the δ(13)C and δ(15)N values between seedlings and adults of each ECM plant, and no ECM species exhibited signatures of mixotrophy. Our results revealed (i) variation in ECM fungal diversity according to the seedling versus adult development stage of trees and (ii) low host specificity of ECM fungi, and indicated that multi-host fungi are more abundant than single-host fungi in this forest stand.

Occurrence of nodulation in unexplored leguminous trees native to the West African tropical rainforest and inoculation response of native species useful in reforestation.

Despite the abundance and diversity of timber tree legumes in the West African rainforest, their ability to form nitrogen-fixing nodules in symbiosis with rhizobia, and their response to rhizobial inoculation, remain poorly documented. In the first part of this study the occurrence of nodulation was determined in 156 leguminous species growing in six natural forest areas in Guinea, mostly mature trees. In the second part, an in situ experiment of rhizobial inoculation was performed on eight selected tree species belonging to three genera: Albizia, Erythrophleum and Millettia. Of the 97 plant species and 14 genera that had never been examined before this study, 31 species and four genera were reported to be nodulated. After 4 months of growing in a nursery and a further 11 months after transplantation of plants to the field, we observed a highly significant (P < 0.001) and positive effect of inoculation with Bradyrhizobium sp. strains on the growth of the eight tree species tested. The importance of determining the nodulation ability of unexplored local trees and subsequently using this information for inoculation in reforestation programmes was demonstrated.