Phylogenomics reveals the evolution of root nodulating alpha- and beta-Proteobacteria (rhizobia).

The symbiosis between legumes and nodulating Proteobacteria (so-called rhizobia) contributes greatly to nitrogen fixation in terrestrial ecosystems. Root nodulating Proteobacteria produce nodulation (Nod) factors during the initiation of rhizobial nodule organogenesis on the roots of legumes. Here, we screened the Nod factor production capacity of the previously reported nodule inducing Proteobacteria genera using their genome sequences and assessed the evolutionary history of symbiosis based on phylogenomics. Our analysis revealed 12 genera as potentially Nod factor producing taxa exclusively from alpha- and beta-Proteobacteria. Based on molecular clock analysis, we estimate that rhizobial nitrogen-fixing symbiosis appeared for the first time about 51 Mya (Eocene epoch) in Rhizobiaceae, and it was laterally transferred to multiple symbiotic taxa in alpha- and beta-Proteobacteria. Coevolutionary tests conducted for measuring the phylogenetic congruence between hosts and symbionts revealed only weak topological similarity between legumes and their bacterial symbionts. We conclude that frequent lateral transfer of symbiotic genes, facultative symbiotic nature of rhizobia, differential evolutionary processes of chromosome versus plasmids, and complex multispecies coevolutionary processes have shaped the rhizobia-host associations.

Paraburkholderia spp. are the main rhizobial microsymbionts of Mimosa tenuiflora (Willd.) Poir. in soils of the Brazilian tropical dry forests (Caatinga biome).

Mimosa tenuiflora (Willd.) Poir. is widespread in southern and central American drylands, but little information is available concerning its associated rhizobia. Therefore, this study aimed to characterize M. tenuiflora rhizobia from soils of the tropical dry forests (Caatinga) in Pernambuco State, Brazil, at the molecular and symbiotic levels. Soil samples of pristine Caatinga areas in four municipalities were used to grow M. tenuiflora. First, the bacteria from root nodules were subjected to nodC/nifH gene amplification, and the bacteria positive for both genes had the 16S rRNA gene sequenced. Then, ten strains were evaluated using recA, gyrB, and nodC gene sequences, and seven of them had their symbiotic efficiency assessed. Thirty-two strains were obtained and 22 of them were nodC/nifH positive. Twenty strains clustered within Paraburkholderia and two within Rhizobium by 16S rRNA gene sequencing. The beta-rhizobia were similar to P. phenoliruptrix (12) and P. diazotrophica (8). Both alpha-rhizobia were closely related to R. miluonense. The recA + gyrB phylogenetic analysis clustered four and five strains within the P. phenoliruptrix and P. diazotrophica branches, respectively, but they were somewhat divergent to the 16S rRNA phylogeny. For Rhizobium sp. ESA 637, the recA + gyrB phylogeny clustered the strain with R. jaguaris. The nodC phylogeny indicated that ESA 626, ESA 629, and ESA 630 probably represented a new symbiovar branch. The inoculation assay showed high symbiotic efficiency for all tested strains. The results indicated high genetic diversity and efficiency of M. tenuiflora rhizobia in Brazilian drylands and included P. phenoliruptrix-like bacteria in the list of efficient beta-rhizobia in the Caatinga biome.

Inoculation of Mimosa Pudica with Paraburkholderia phymatum Results in Changes to the Rhizoplane Microbial Community Structure.

Nitrogen fixing symbiosis between rhizobia and legumes contributes significant amounts of N to agricultural and natural environments. In natural soils, rhizobia compete with indigenous bacterial communities to colonize legume roots, which leads to symbiotic interactions. However, limited information is currently available on the effects of the rhizobial symbiont on the resident microbial community in the legume rhizosphere, rhizoplane, and endosphere, which is partly due to the presence of native nodulating rhizobial strains. In the present study, we used a symbiotic system comprised of Paraburkholderia phymatum and Mimosa pudica to examine the interaction of an inoculant strain with indigenous soil bacteria. The effects of a symbiont inoculation on the native bacterial community was investigated using high throughput sequencing and an analysis of 16S rRNA gene amplicons. The results obtained revealed that the inoculation induced significant alterations in the microbial community present in the rhizoplane+endosphere of the roots, with 13 different taxa showing significant changes in abundance. No significant changes were observed in the rhizospheric soil. The relative abundance of P. phymatum significantly increased in the rhizoplane+endosphere of the root, but significant decreased in the rhizospheric soil. While the rhizosphere, rhizoplane, and root endosphere contained a wide diversity of bacteria, the nodules were predominantly colonized by P. phymatum. A network analysis revealed that the operational taxonomic units of Streptomyces and Phycicoccus were positively associated with P. phymatum as potential keystone taxa. Collectively, these results suggest that the success of an inoculated symbiont depends on its ability to colonize the roots in the face of competition by other soil bacteria. A more detailed understanding of the mechanisms by which an inoculated strain colonizes its plant host is crucial for realizing the full potential of microbial inoculants in sustainable agriculture.

Paraburkholderia youngii sp. nov. and 'Paraburkholderia atlantica' - Brazilian and Mexican Mimosa-associated rhizobia that were previously known as Paraburkholderia tuberum sv. mimosae.

Previous studies have recognized South and Central/Latin American mimosoid legumes in the genera Mimosa, Piptadenia and Calliandra as hosts for various nodulating Paraburkholderia species. Several of these species have been validly named in the last two decades, e.g., P. nodosa, P. phymatum, P. diazotrophica, P. piptadeniae, P. ribeironis, P. sabiae and P. mimosarum. There are still, however, a number of diverse Paraburkholderia strains associated with these legumes that have an unclear taxonomic status. In this study, we focus on 30 of these strains which originate from the root nodules of Brazilian and Mexican Mimosa species. They were initially identified as P. tuberum and subsequently placed into a symbiovar (sv. mimosae) based on their host preferences. A polyphasic approach for the delineation of these strains was used, consisting of genealogical concordance analysis (using atpD, gyrB, acnA, pab and 16S rRNA gene sequences), together with comparisons of Average Nucleotide Identity (ANI), DNA G+C content ratios and phenotypic characteristics with those of the type strains of validly named Paraburkholderia species. Accordingly, these 30 strains were delineated into two distinct groups, of which one is conspecific with 'P. atlantica' CNPSo 3155T and the other new to Science. We propose the name Paraburkholderia youngii sp. nov. with type strain JPY169T (= LMG 31411T; SARCC751T) for this novel species.

Serratia sp., an endophyte of Mimosa pudica nodules with nematicidal, antifungal activity and growth-promoting characteristics.

In the present study, the nematicidal activity of an isolated strain of Mimosa pudica nodules was evaluated against the Nacobbus aberrans (J2) phytonymatodes with a mortality of 88.8%, while against the gastrointestinal nematode Haemonchus contortus (L3) and free-living Panagrellus redivivus was 100%. The ability to inhibit the growth of phytopathogenic fungi Fusarium sp., and Alternaria solani, as well as the oomycete Phytophthora capsici, this antifungal activity may be related to the ability to produce cellulases, siderophores and chitinases by this bacterial strain. Another important finding was the detection of plant growth promoter characteristics, such as auxin production and phosphate solubilization. The strain identified by sequences of the 16S and rpoB genes as Serratia sp. is genetically related to Serratia marcescens and Serratia nematodiphila. The promoter activity of plant growth, antifungal and nematicide of the Serratia sp. strain makes it an alternative for the biocontrol of fungi and nematodes that affect both the livestock and agricultural sectors, likewise, candidate as a growth-promoting bacterium.

Complete genome sequence and analysis of Alcaligenes faecalis strain Mc250, a new potential plant bioinoculant.

Here we present and analyze the complete genome of Alcaligenes faecalis strain Mc250 (Mc250), a bacterium isolated from the roots of Mimosa calodendron, an endemic plant growing in ferruginous rupestrian grasslands in Minas Gerais State, Brazil. The genome has 4,159,911 bp and 3,719 predicted protein-coding genes, in a single chromosome. Comparison of the Mc250 genome with 36 other Alcaligenes faecalis genomes revealed that there is considerable gene content variation among these strains, with the core genome representing only 39% of the protein-coding gene repertoire of Mc250. Mc250 encodes a complete denitrification pathway, a network of pathways associated with phenolic compounds degradation, and genes associated with HCN and siderophores synthesis; we also found a repertoire of genes associated with metal internalization and metabolism, sulfate/sulfonate and cysteine metabolism, oxidative stress and DNA repair. These findings reveal the genomic basis for the adaptation of this bacterium to the harsh environmental conditions from where it was isolated. Gene clusters associated with ectoine, terpene, resorcinol, and emulsan biosynthesis that can confer some competitive advantage were also found. Experimental results showed that Mc250 was able to reduce (~60%) the virulence phenotype of the plant pathogen Xanthomonas citri subsp. citri when co-inoculated in Citrus sinensis, and was able to eradicate 98% of juveniles and stabilize the hatching rate of eggs to 4% in two species of agricultural nematodes. These results reveal biotechnological potential for the Mc250 strain and warrant its further investigation as a biocontrol and plant growth-promoting bacterium.

Phylogeny of symbiotic genes reveals symbiovars within legume-nodulating Paraburkholderia species.

Bacteria belonging to the genus Paraburkholderia are capable of establishing symbiotic relationships with plants belonging to the Fabaceae (=Leguminosae) family and fixing the atmospheric nitrogen in specialized structures in the roots called nodules, in a process known as biological nitrogen fixation (BNF). In the nodulation and BNF processes several bacterial symbiotic genes are involved, but the relations between symbiotic, core genes and host specificity are still poorly studied and understood in Paraburkholderia. In this study, eight strains of nodulating nitrogen-fixing Paraburkholderia isolated in Brazil, together with described species and other reference strains were used to infer the relatedness between core (16S rDNA, recA) and symbiotic (nod, nif, fix) genes. The diversity of genes involved in the nodulation (nodAC) and nitrogen fixation (nifH) abilities was investigated. Only two groups, one containing three Paraburkholderia species symbionts of Mimosa, and another one with P. ribeironis strains presented similar phylogenetic patterns in the analysis of core and symbiotic genes. In three other groups events of horizontal gene transfer of symbiotic genes were detected. Paraburkholderia strains with available genomes were used in the complementary analysis of nifHDK and fixABC and confirmed well-defined phylogenetic positions of symbiotic genes. In all analyses of nod, nif and fix genes the strains were distributed into five clades with high bootstrap support, allowing the proposal of five symbiovars in nodulating nitrogen-fixing Paraburkholderia, designated as mimosae, africana, tropicalis, atlantica and piptadeniae. Phylogenetic inferences within each symbiovar are discussed.

Plant growth-promoting bacteria isolated from wild legume nodules and nodules of Phaseolus vulgaris L. trap plants in central and southern Mexico.

Central southern Mexico contains highly diverse legumes. In this study, nodule-associated bacteria (NAB) were isolated from wild legume nodules and from nodules on Phaseolus vulgaris plants used as a plant-trap in soils from the same areas as the wild legumes. The bacteria were identified through the 16S rRNA gene sequence analysis, tested for plant growth-promoting (PGP) activities and the production of antimicrobial compounds, and analyzed for potential nodulation by amplifying the nodC gene. Several genera with PGP activity were isolated from legume nodules, including Achromobacter, Acinetobacter, Bacillus, Brevibacillus, Brevibacterium, Burkholderia, Cupriavidus, Dyella, Ensifer, Enterobacter, Herbaspirillum, Kosakonia, Labrys, Microbacterium, Moraxella, Paraburkholderia, Pseudomonas, Rhizobium, Stenotrophomonas; and Aeromonas, Marinococcus Pseudarthrobacter and Pseudoxanthomonas were found in plant legume nodules for the first time. Pseudomonas was the most common bacteria, and Mimosa pudica was colonized by the largest number of genera (6 different genera). A Burkholderia strain from the Burkholderia cepacia complex and a firmicutes strain harbor the nodC gene, identifying them as potential novel nodulating bacteria and showing that most of the strains isolated in this study were NAB. The most frequent PGP activity identified among the strains isolated from wild legumes was IAA synthesis. Two bacteria, Stenotrophomonas sp. and Rhizobium sp., synthesized more than 250 µg/ml, which is more than the level of synthesis reported in this study for Azospirillum brasilense Sp7 (59.77 µg/ml). Nitrogen fixation and antimicrobial compound production were not common, but the production of siderophores was frequently found among all the strains. This study shows that diverse NAB with PGP activity are very common in the legume nodules from central southern Mexico.

Streptomyces mimosae sp. nov., an endophytic actinomycete isolated from the root of Mimosa pudica in Thailand.

An endophytic actinomycete, strain 3MP-10T, isolated from the root of Mimosa pudica was taxonomically studied based upon polyphasic approaches. This strain formed spiral spore chains on aerial mycelia. ll-Diaminopimelic acid, glucose and ribose were found in the whole-cell hydrolysates. It belonged to the genus Streptomyces and was closely related to Streptomyces zhaozhouensis DSM 42101T (98.9 %) and Streptomyces sedi JCM 16909T (98.6 %) based on 16S rRNA gene sequence analysis results. The major menaquinones were MK-10(H8), MK-10(H6) and MK-9(H8). The predominant cellular fatty acids were iso-C16 : 0, anteiso-C15 : 0 and anteiso-C17 : 0. The detected phospholipids were diphosphatidylglycerol, phosphatidylinositol mannoside, phosphatidylinositol, phosphatidylethanolamine and phosphatidylglycerol. Strain 3MP-10T had a genome size of 7.2 Mb with a genome G+C content of 73.4 mol%. Results of in silico genome-based similarity analysis revealed ANIb values of 84.94 and 84.77 %, ANIm values of 88.01 and 87.92 %, and dDDH values of 29.9 and 29.6 % when compared with S. zhaozhouensis DSM 42101T and S. sedi JCM 16909T, respectively. Based on the polyphasic approach, digital DNA-DNA relatedness and average nucleotide identity, we propose that the novel actinomycete represents a novel species, Streptomyces mimosae, with type strain 3MP-10T (=JCM 33328T=TISTR 2646T).

Novel heavy metal resistance gene clusters are present in the genome of Cupriavidus neocaledonicus STM 6070, a new species of Mimosa pudica microsymbiont isolated from heavy-metal-rich mining site soil.

BACKGROUND: Cupriavidus strain STM 6070 was isolated from nickel-rich soil collected near Koniambo massif, New Caledonia, using the invasive legume trap host Mimosa pudica. STM 6070 is a heavy metal-tolerant strain that is highly effective at fixing nitrogen with M. pudica. Here we have provided an updated taxonomy for STM 6070 and described salient features of the annotated genome, focusing on heavy metal resistance (HMR) loci and heavy metal efflux (HME) systems. RESULTS: The 6,771,773 bp high-quality-draft genome consists of 107 scaffolds containing 6118 protein-coding genes. ANI values show that STM 6070 is a new species of Cupriavidus. The STM 6070 symbiotic region was syntenic with that of the M. pudica-nodulating Cupriavidus taiwanensis LMG 19424T. In contrast to the nickel and zinc sensitivity of C. taiwanensis strains, STM 6070 grew at high Ni2+ and Zn2+ concentrations. The STM 6070 genome contains 55 genes, located in 12 clusters, that encode HMR structural proteins belonging to the RND, MFS, CHR, ARC3, CDF and P-ATPase protein superfamilies. These HMR molecular determinants are putatively involved in arsenic (ars), chromium (chr), cobalt-zinc-cadmium (czc), copper (cop, cup), nickel (nie and nre), and silver and/or copper (sil) resistance. Seven of these HMR clusters were common to symbiotic and non-symbiotic Cupriavidus species, while four clusters were specific to STM 6070, with three of these being associated with insertion sequences. Within the specific STM 6070 HMR clusters, three novel HME-RND systems (nieIC cep nieBA, czcC2B2A2, and hmxB zneAC zneR hmxS) were identified, which constitute new candidate genes for nickel and zinc resistance. CONCLUSIONS: STM 6070 belongs to a new Cupriavidus species, for which we have proposed the name Cupriavidus neocaledonicus sp. nov.. STM6070 harbours a pSym with a high degree of gene conservation to the pSyms of M. pudica-nodulating C. taiwanensis strains, probably as a result of recent horizontal transfer. The presence of specific HMR clusters, associated with transposase genes, suggests that the selection pressure of the New Caledonian ultramafic soils has driven the specific adaptation of STM 6070 to heavy-metal-rich soils via horizontal gene transfer.

Paraburkholderia atlantica sp. nov. and Paraburkholderia franconis sp. nov., two new nitrogen-fixing nodulating species isolated from Atlantic forest soils in Brazil.

A polyphasic study was conducted with 11 strains trapped by Mimosa pudica and Phaseolus vulgaris grown in soils of the Brazilian Atlantic Forest. In the phylogenetic analysis of the 16S rRNA gene, one clade of strains (Psp1) showed higher similarity with Paraburkholderia piptadeniae STM7183T (99.6%), whereas the second (Psp6) was closely related to Paraburkholderia tuberum STM678T (99%). An MLSA (multilocus sequence analysis) with four (recA, gyrB, trpB and gltB) housekeeping genes placed both Psp1 and Psp6 strains in new clades, and BOX-PCR profiles indicated high intraspecific genetic diversity within each clade. Values of digital DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI) of the whole genome sequences were of 56.9 and 94.4% between the Psp1 strain CNPSo 3157T and P. piptadeniae; and of 49.7% and 92.7% between the Psp6 strain CNPSo 3155T and P. tuberum, below the threshold for species delimitation. In the nodC analysis, Psp1 strains clustered together with P. piptadeniae, while Psp6 did not group with any symbiotic Paraburkholderia. Other phenotypic, genotypic and symbiotic properties were evaluated. The polyphasic analysis supports that the strains represent two novel species, for which the names Paraburkholderia franconis sp. nov. with type strain CNPSo 3157T (= ABIP 241, = LMG 31644) and Paraburkholderia atlantica sp. nov. with type strain CNPSo 3155T (= ABIP 236, = LMG 31643) are proposed.

Use of plant materials for the bioremediation of soil from an industrial site.

Bioremediation is one of the existing techniques applied for treating oil-contaminated soil, which can be improved by the incorporation of low-cost nutritional materials. This study aimed to assess the addition of two low-cost plant residues, sugarcane bagasse (SCB) and leaf litter (LL) of the forest leguminous Mimosa caesalpiniifolia plant (sabiá), either separately or combined, to a contaminated soil from a petroleum refinery area, analyzed after 90 days of treatment. Individually, both amounts of SCB (20 and 40 g kg-1) favored the growth of total heterotrophic bacteria and total fungi, while LL at 20 g kg-1 better stimulated the hydrocarbon-degrading microorganism's activity in the soil. However, no TPH removal was observed under any of these conditions. Higher microbial growth was detected by the application of both plant residues in multicontaminated soil. The maximum TPH removal of 30% was achieved in amended soil with 20 g kg-1 SCB and 20 kg-1 LL. All the experimental conditions revealed changes in the microbial community structure, related to the handling of the soil, with abundance of Alphaproteobacteria. This study demonstrates the effectiveness of the plant residues SCB and LL as low-cost nutritional materials for biodegradation of hydrocarbon in real oil contaminated soil by indigenous populations.

noeM, a New Nodulation Gene Involved in the Biosynthesis of Nod Factors with an Open-Chain Oxidized Terminal Residue and in the Symbiosis with Mimosa pudica.

The ß-rhizobium Cupriavidus taiwanensis is a nitrogen-fixing symbiont of Mimosa pudica. Nod factors produced by this species were previously found to be pentameric chitin-oligomers carrying common C18:1 or C16:0 fatty acyl chains, N-methylated and C-6 carbamoylated on the nonreducing terminal N-acetylglucosamine and sulfated on the reducing terminal residue. Here, we report that, in addition, C. taiwanensis LMG19424 produces molecules where the reducing sugar is open and oxidized. We identified a novel nodulation gene located on the symbiotic plasmid pRalta, called noeM, which is involved in this atypical Nod factor structure. noeM encodes a transmembrane protein bearing a fatty acid hydroxylase domain. This gene is expressed during symbiosis with M. pudica and requires NodD and luteolin for optimal expression. The closest noeM homologs formed a separate phylogenetic clade containing rhizobial genes only, which are located on symbiosis plasmids downstream from a nod box. Corresponding proteins, referred to as NoeM, may have specialized in symbiosis via the connection to the nodulation pathway and the spread in rhizobia. noeM was mostly found in isolates of the Mimoseae tribe, and specifically detected in all tested strains able to nodulate M. pudica. A noeM deletion mutant of C. taiwanensis was affected for the nodulation of M. pudica, confirming the role of noeM in the symbiosis with this legume.

Plant-soil feedback of two legume species in semi-arid Brazil.

Positive feedback between arbuscular mycorrhizal fungal (AMF) and vascular plants can contribute to plant species establishment, but how this feedback affects plant invasion by Prosopis juliflora SW. (DC.), or resistance to invasion by Mimosa tenuiflora (Willd.) Poir in Brazilian semi-arid region is not well known. In this work, we tested how modified and native AMF communities affect the establishment of P. juliflora and M. tenuiflora plants. We examined the effects of inoculation with modified and native AMF communities on number of AMF spores, root colonization, number of N-fixing nodules, plant dry biomass, plant phosphorous concentration, and plant responsiveness to mycorrhizas of P. juliflora and M. tenuiflora. We found that the modified AMF community enhanced the root colonization, plant dry biomass, and plant phosphorous concentration of invasive P. juliflora, whereas native AMF enhanced M. tenuiflora. Our results demonstrate that the invasive P. juliflora alters soil AMF community composition, and this change generates positive feedback to the invasive P. juliflora itself and decreases AMF associations with native M. tenuiflora.

Paraburkholderia guartelaensis sp. nov., a nitrogen-fixing species isolated from nodules of Mimosa gymnas in an ecotone considered as a hotspot of biodiversity in Brazil.

A polyphasic approach was used to infer the phylogenetic position of six nitrogen-fixing symbiotic bacteria isolated from Mimosa gymnas nodules grown in an ecotone between the Brazilian biomes of Atlantic Forest and Cerrado, considered as a hotspot of biodiversity. The 16S rRNA gene phylogeny indicated the highest similarity with Paraburkholderia oxyphila (98.7-98.9%), but similar values were found with other Paraburkholderia species. The multilocus sequence analysis (MLSA) of five (recA, gyrB, trpB, gltB, and atpD) housekeeping genes indicated that the CNPSo strains represent a novel lineage, sharing less than 95.7% of nucleotide identity (NI) with other Paraburkholderia species, being more closely related to P. nodosa. Genome parameters were analyzed for strain CNPSo 3008T, and DNA-DNA hybridization revealed a maximum of 55.9% of DNA-DNA relatedness with P. nodosa, while average nucleotide identity with the two closest species was of 93.84% with P. nodosa and of 87.93% with P. mimosarum, both parameters confirming that the strain represents a new species. In the analysis of the nodulation nodC gene, all CNPSo strains showed the highest similarity with P. nodosa, and nodulation tests indicated host specificity with Mimosa. Other phylogenetic, physiological, and chemotaxonomic properties were evaluated. All data obtained support the description of the novel species Paraburkholderia guartelaensis sp. nov., with CNPSo 3008T (= U13000T = G29.01T) indicated as the type strain.

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.

ACC deaminase plays a major role in Pseudomonas fluorescens YsS6 ability to promote the nodulation of Alpha- and Betaproteobacteria rhizobial strains.

Ethylene acts as a major regulator of the nodulation process of leguminous plants. Several rhizobial strains possess the ability to modulate plant ethylene levels through the expression of the enzyme 1-aminocyclopropane-1-carboxylate (ACC) deaminase; however, rhizobia present low enzymatic activities. One possible alternative to this problem resides on the use of free-living bacteria, such as Pseudomonas, presenting high levels of ACC deaminase activity that may be used as adjuvants in the nodulation process by decreasing inhibitory ethylene levels. Nevertheless, not much is understood about the specific role of ACC deaminase in the possible role of free-living bacteria as nodulation adjuvants. Therefore, this work aims to study the effect of ACC deaminase in the plant growth-promoting bacterium, Pseudomonas fluorescens YsS6, ability to facilitate alpha- and beta-rhizobia nodulation. The ACC deaminase-producing P. fluorescens YsS6 and its ACC deaminase mutant were used in co-inoculation assays to evaluate their impact in the nodulation process of alpha- (Rhizobium tropici CIAT899) and beta-rhizobia (Cupriavidus taiwanensis STM894) representatives, in Phaseolus vulgaris and Mimosa pudica plants, respectively. The results obtained indicate that the wild-type P. fluorescens YsS6, but not its mutant defective in ACC deaminase production, increase the nodulation abilities of both alpha- and beta-rhizobia, resulting in an increased leguminous plant growth. Moreover, this is the first report of the positive effect of free-living bacteria in the nodulation process of beta-rhizobia. The modulation of inhibitory ethylene levels by free-living ACC deaminase-producing bacteria plays an important role in facilitating the nodulation process of alpha- and beta-rhizobia.

A nurse plant benefits from facilitative interactions through mycorrhizae.

Plant facilitation promotes coexistence by maintaining differences in the regeneration niche because some nurse species recruit under arid conditions, whereas facilitated species recruit under more mesic conditions. In one Mexican community, 95% of species recruit through facilitation; Mimosa luisana being a keystone nurse for many of them. M. luisana individuals manifest greater fitness when growing in association with their facilitated plants than when growing in isolation. This observation suggests that nurses also benefit from their facilitated plants, a benefit thought to be mediated by mycorrhizal fungi. Under field conditions, we experimentally tested whether mycorrhizal fungi mediate the increased fitness that M. luisana experiences when growing in association with its facilitated plants. We applied fungicide to the soil for nurse plants growing alone and growing in association with their facilitated plants in order to reduce the mycorrhizal colonisation of roots. We then assessed the quantity and quality of seed production of M. luisana in four treatments (isolated-control, isolated-fungicide, associated-control and associated-fungicide). Fungicide application reduced the percentage root length colonised by mycorrhizae and reduced fitness of M. luisana when growing in association with their facilitated plants but not when growing in isolation. This reduction was reflected in the total number of seeds, number of seeds per pod, seed mass and seed viability. These results suggest that nurses benefit from the presence of their facilitated plants through links established by mycorrhizae, indicating that both plants and belowground mutualistic communities are all part of one system, coexisting by means of intrinsically linked interactions.

Isolation and characterization of endophytes from nodules of Mimosa pudica with biotechnological potential.

Legumes establish symbiotic relationships with different microorganisms, which could function as plant growth promotion microorganisms (PGPM). The finding of new PGPM strains is important to increase plant production avoiding or diminishing the use of industrial fertilizers. Thus, in this work we evaluated the plant growth promotion traits of ten strains isolated from Mimosa pudica root nodules. According to the 16S rDNA sequence, the microorganisms were identified as Enterobacter sp. and Serratia sp. To the best of our knowledge this is the first report describing and endophytic interaction between Mimosa pudica and Enterobacter sp. These strains have some plant growth promoting traits such as phosphate solubilization, auxin production and cellulase and chitinase activity. Strains identified as Serratia sp. inhibited the growth of the phytopathogenic fungi Fusarium sp., and Alternaria solani and the oomycete Phytophthora capsici. According to their biochemical characteristics, three strains were selected to test their plant growth promoting activity in a medium with an insoluble phosphate source. These bacteria show low specificity for their hosts as endophytes, since they were able to colonize two very different legumes: Phaseolus vulgaris and M. pudica. Seedlings of P. vulgaris were inoculated and grown for fifteen days. Enterobacter sp. NOD1 and NOD10, promoted growth as reflected by an increase in shoot height as well as an increase in the size and emergence of the first two trifolia. We could localize NOD5 as an endophyte in roots in P. vulgaris by transforming the strain with a Green Fluorescent Protein carrying plasmid. Experiments of co-inoculation with different Rhizobium etli strains allowed us to discard that NOD5 can fix nitrogen in the nodules formed by a R. etli Fix- strain. The isolates described in this work show biotechnological potential for plant growth promoting activity and production of indoleacetic acid and siderophores.

Mycorrhizal inoculation as an alternative for the sustainable production of Mimosa tenuiflora seedlings with improved growth and secondary compounds content.

In this study, we report the effects of arbuscular mycorrhizal fungi (AMF) and increasing doses of phosphorus (P) on the growth and production of secondary metabolites in Mimosa tenuiflora, a medicinal species native to Brazil. We used a completely randomized design with four inoculation treatments: Control not inoculated (1); Claroideoglomus etunicatum (2); Gigaspora albida (3); and C. etunicatum + G. albida (4) and four doses of P; P0 - baseline dose, P8, P16 and P32. After 70 d in a greenhouse, growth, mycorrhizal variables, biochemical and phytochemical parameters were evaluated. Compared to non-mycorrhizal plants, mycorrhized M. tenuiflora seedlings showed greater: growth, greater photosynthetic performance and content of soluble carbohydrates and secondary metabolites, with the most significant benefits occurring in soil with low to moderate P content (up to 16 mg kg-1). The plant growth is severely restricted at low P levels, but the addition of AMF appears to remove this limiting factor. Although M. tenuiflora responds to levels of phosphate fertilization, it responds well to mycorrhizal inoculation, especially with G. albida, which promotes benefits for the initial growth and secondary metabolite content in this plant species of medical and potential commercial interest and may be used instead of phosphate fertilizer.