Sergio Miana de Faria

Burkholderia species are ancient symbionts of legumes

Burkholderia has only recently been recognized as a potential nitrogen-fixing symbiont of legumes, but we find that the origins of symbiosis in Burkholderia are much deeper than previously suspected. We sampled 143 symbionts from 47 native species of Mimosa across 1800 km in central Brazil and found that 98% were Burkholderia. Gene sequences defined seven distinct and divergent species complexes within the genus Burkholderia. The symbiosis-related genes formed deep Burkholderia-specific clades, each specific to a species complex, implying that these genes diverged over a long period within Burkholderia without substantial horizontal gene transfer between species complexes.

Proof that Burkholderia Strains Form Effective Symbioses with Legumes: a Study of Novel Mimosa-Nodulating Strains from South America

ABSTRACT Twenty Mimosa-nodulating bacterial strains from Brazil and Venezuela, together with eight reference Mimosa-nodulating rhizobial strains and two other β-rhizobial strains, were examined by amplified rRNA gene restriction analysis. They fell into 16 patterns and formed a single cluster together with the known β-rhizobia, Burkholderia caribensis, Burkholderia phymatum, and Burkholderia tuberum. The 16S rRNA gene sequences of 15 of the 20 strains were determined, and all were shown to belong to the genus Burkholderia; four distinct clusters could be discerned, with strains isolated from the same host species usually clustering very closely. Five of the strains (MAP3-5, Br3407, Br3454, Br3461, and Br3469) were selected for further studies of the symbiosis-related genes nodA, the NodD-dependent regulatory consensus sequences (nod box), and nifH. The nodA and nifH sequences were very close to each other and to those of B. phymatum STM815, B. caribensis TJ182, and Cupriavidus taiwanensis LMG19424 but were relatively distant from those of B. tuberum STM678. In addition to nodulating their original hosts, all five strains could also nodulate other Mimosa spp., and all produced nodules on Mimosa pudica that had nitrogenase (acetylene reduction) activities and structures typical of effective N2-fixing symbioses. Finally, both wild-type and green fluorescent protein-expressing transconjugant strains of Br3461 and MAP3-5 produced N2-fixing nodules on their original hosts, Mimosa bimucronata (Br3461) and Mimosa pigra (MAP3-5), and hence this confirms strongly that Burkholderia strains can form effective symbioses with legumes.

Plant-associated symbiotic Burkholderia species lack hallmark strategies required in mammalian pathogenesis

Burkholderia is a diverse and dynamic genus, containing pathogenic species as well as species that form complex interactions with plants. Pathogenic strains, such as B. pseudomallei and B. mallei, can cause serious disease in mammals, while other Burkholderia strains are opportunistic pathogens, infecting humans or animals with a compromised immune system. Although some of the opportunistic Burkholderia pathogens are known to promote plant growth and even fix nitrogen, the risk of infection to infants, the elderly, and people who are immunocompromised has not only resulted in a restriction on their use, but has also limited the application of non-pathogenic, symbiotic species, several of which nodulate legume roots or have positive effects on plant growth. However, recent phylogenetic analyses have demonstrated that Burkholderia species separate into distinct lineages, suggesting the possibility for safe use of certain symbiotic species in agricultural contexts. A number of environmental strains that promote plant growth or degrade xenobiotics are also included in the symbiotic lineage. Many of these species have the potential to enhance agriculture in areas where fertilizers are not readily available and may serve in the future as inocula for crops growing in soils impacted by climate change. Here we address the pathogenic potential of several of the symbiotic Burkholderia strains using bioinformatics and functional tests. A series of infection experiments using Caenorhabditis elegans and HeLa cells, as well as genomic characterization of pathogenic loci, show that the risk of opportunistic infection by symbiotic strains such as B. tuberum is extremely low.

Nodulation and nitrogen fixation by Mimosa spp. in the Cerrado and Caatinga biomes of Brazil

*An extensive survey of nodulation in the legume genus Mimosa was undertaken in two major biomes in Brazil, the Cerrado and the Caatinga, in both of which there are high degrees of endemicity of the genus. *Nodules were collected from 67 of the 70 Mimosa spp. found. Thirteen of the species were newly reported as nodulating. Nodules were examined by light and electron microscopy, and all except for M. gatesiae had a structure typical of effective Mimosa nodules. The endosymbiotic bacteria in nodules from all of the Mimosa spp. were identified as Burkholderia via immunolabelling with an antibody against Burkholderia phymatum STM815. *Twenty of the 23 Mimosa nodules tested were shown to contain nitrogenase by immunolabelling with an antibody to the nitrogenase Fe- (nifH) protein, and using the delta(15)N ((15)N natural abundance) technique, contributions by biological N(2) fixation of up to 60% of total plant N were calculated for Caatinga Mimosa spp. *It is concluded that nodulation in Mimosa is a generic character, and that the preferred symbionts of Brazilian species are Burkholderia. This is the first study to demonstrate N(2) fixation by beta-rhizobial symbioses in the field.

Burkholderia sabiae sp. nov., isolated from root nodules of Mimosa caesalpiniifolia

Two rhizobial strains, Br3407(T) and Br3405, were isolated from nitrogen-fixing nodules on the roots of Mimosa caesalpiniifolia, a legume tree native to Brazil. On the basis of 16S rRNA gene sequence similarities, both strains were shown previously to belong to the genus Burkholderia. A polyphasic approach, including DNA-DNA hybridizations, pulsed-field gel electrophoresis of whole-genome DNA profiles, whole-cell protein analyses, fatty acid methyl ester analysis and extensive biochemical characterization, was used to clarify the taxonomic position of these strains further; the strains are here classified within a novel species, for which the name Burkholderia sabiae sp. nov. is proposed. The type strain is strain Br3407(T) (=LMG 24235(T) =BCRC 17587(T)).

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.

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.

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 β 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.

Evaluating the nodulation status of leguminous species from the Amazonian forest of Brazil

Numerous leguminous species are used or have potential uses for timber production, pharmacological products, or land reclamation. Through N(2)-fixation, many leguminous trees contribute to the N-balance of tropical wetlands and rainforests. Therefore, studies of the N(2)-fixation ability of leguminous species appear to be crucial for the better use and conservation of these resources. The global nodulation inventory in the Leguminosae family is constantly being enriched with new records, suggesting the existence of undiscovered nodulated species, especially in tropical natural ecosystems and other hot spots of biodiversity. In this respect, the nodulation of leguminous species from the Amazonian forest of Porto Trombetas (Brazil) was surveyed. Overall, 199 leguminous species from flooded and non-flooded areas, were examined for their nodulation status by combining field observations, seedling inoculations, and screening of N(2)-fixing bacterial strains from the collected nodules. The results revealed a tendency for a higher relative frequency of nodulation in the species from the flooded areas (74%) compared with those from the non-flooded areas (67%). Nodulation was observed in the Caesalpinioideae, Mimosoideae, and Papilionoideae, with 25, 88, and 84% of the examined species in each subfamily, respectively. Of the 137 nodulated leguminous species, 32 including three Caesalpinoideae, 19 Mimosoideae, and 10 Papilionoideae are new records. One new nodulated genus (Cymbosema) was found in the Papilionoideae. Twelve non-nodulating leguminous species were also observed for the first time. The results are discussed based on the systematics of the Leguminosae family and the influence of available nutrients to the legume-bacteria symbiosis.

Role of Methylotrophy During Symbiosis Between Methylobacterium nodulans and Crotalaria podocarpa

Some rare leguminous plants of the genus Crotalaria are specifically nodulated by the methylotrophic bacterium Methylobacterium nodulans. In this study, the expression and role of bacterial methylotrophy were investigated during symbiosis between M. nodulans, strain ORS 2060T, and its host legume, Crotalaria podocarpa. Using lacZ fusion to the mxaF gene, we showed that the methylotroph genes are expressed in the root nodules, suggesting methylotrophic activity during symbiosis. In addition, loss of the bacterial methylotrophic function significantly affected plant development. Indeed, inoculation of M. nodulans nonmethylotroph mutants in C. podocarpa decreased the total root nodule number per plant up to 60%, decreased the whole-plant nitrogen fixation capacity up to 42%, and reduced the total dry plant biomass up to 46% compared with the wild-type strain. In contrast, inoculation of the legume C. podocarpa with nonmethylotrophic mutants complemented with functional mxa genes restored the symbiotic wild phenotype. These results demonstrate the key role of methylotrophy during symbiosis between M. nodulans and C. podocarpa.