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The ecological restoration of nickel mining-degraded areas in New Caledonia is strongly limited by low availability of soil mineral nutrients, metal toxicity, and slow growth rates of native plant species. In order to improve plant growth for restoration programs, special attention was paid to interactions between plant and soil microorganisms. In this study, we evaluated the influence of inoculation with Curtobacterium citreum BE isolated from a New Caledonian ultramafic soil on arbuscular mycorrhizal symbiosis and growth of Tetraria comosa, an endemic sedge used in restoration programs. A greenhouse experiment on ultramafic substrate was conducted with an inoculum comprising two arbuscular mycorrhizal fungi (AMF) species isolated from New Caledonian ultramafic soils: Rhizophagus neocaledonicus and Claroideoglomus etunicatum. The effects on plant growth of the AMF and C. citreum BE inoculated separately were not significant, but their co-inoculation significantly enhanced the dry weight of T. comosa compared with the non-inoculated control. These differences were positively correlated with mycorrhizal colonization which was improved by C. citreum BE. Compared with the control, co-inoculated plants were characterized by better mineral nutrition, a higher Ca/Mg ratio, and lower metal translocation. However, for Ca/Mg ratio and metal translocation, there were no significant differences between the effects of AMF inoculation and co-inoculation.

In New Caledonia, one third of the main island is covered by ultramafic soils characterized by high levels of metals and low levels of plant nutrients. In these soils, metal tolerant bacteria may play a role in plant adaptation to the edaphic constraints. Recently, two new bacteria species belonging to the genus Burkholderia sensu lato were isolated from rhizospheric roots of pioneer plant growing in New Caledonian ultramafic soils and were able to tolerate high metal concentrations. The purpose of this study was to determine the major mechanisms by which these two bacteria were able to tolerate metals and if they were able to reduce metal absorption by plants from which they were isolated. Bioaccumulation, adsorption on cell surface, ability to form biofilm and production of exopolysaccharides were assessed. A focus was carried out on Ni, generally the most toxic metal in New Caledonian ultramafic soils. All experiments were performed in comparison with a reference strain isolated from a non-ultramafic habitat, Paraburkholderia caribensis MWAP64T. The two ultramafic bacterial species showed a high tolerance to Ni with a Ni–IC50 of 5 mM for Caballeronia novacaledonica STM10272T and 30 mM for Paraburkholderia ultramafica STM10279T, values significantly higher than those of P. caribensis MWAP64T (2 mM). The major mechanism involved in the bacterial Ni tolerance was related to biofilm formation for C. novacaledonica STM10272T and to acidic exopolysaccharide production for P. ultramafica STM10279T. Both species alleviated metal contents in roots of Tetraria comosa, an endemic pioneer herbaceous species growing on New Caledonian ultramafic soils.

The present study focused on the characterization of 10 Curtobacterium citreum strains isolated from the rhizosphere of pioneer plants growing on ultramafic soils from New Caledonia. Taxonomic status was investigated using a polyphasic approach. Three strains (BE, BB, and AM) were selected in terms of multiple-metal resistance and plant-growth-promoting traits. They were tested on sorghum growing on ultramafic soil and compared with the reference strain C. citreum DSM20528T. To better understand the bacterial mechanisms involved, biosorption, bioaccumulation, and biofilm formation were investigated for the representative strain of the ultramafic cluster (strain BE) versus C. citreum DSM20528T. The polyphasic approach confirmed that all native isolates belong to the same cluster and are C. citreum. The inoculation of sorghum with strains BE and BB significantly reduced Ni content in shoots compared with inoculation with C. citreum DSM20528T and control values. This result was related to the higher Ni tolerance of the ultramafic strains compared with C. citreum DSM20528T. Ni biosorption and bioaccumulation showed that BE exhibited a lower Ni content, which is explained by the ability of this strain to produce exopolysaccharides involved in Ni chelation. We suggested that ultramafic C. citreum strains are more adapted to this substrate than is C. citreum DSM20528T, and their features allow them to enhance plant metal tolerance.

Acacia spirorbis subsp. spirorbis Labill. is a widespread tree legume endemic to New Caledonia that grows in ultramafic (UF) and volcano-sedimentary (VS) soils. The aim of this study was to assess the symbiotic promiscuity of A. spirorbis with nodulating and nitrogen-fixing rhizobia in harsh edaphic conditions. Forty bacterial strains were isolated from root nodules and characterized through (i) multilocus sequence analyses, (ii) symbiotic efficiency and (iii) tolerance to metals. Notably, 32.5% of the rhizobia belonged to the Paraburkholderia genus and were only found in UF soils. The remaining 67.5%, isolated from both UF and VS soils, belonged to the Bradyrhizobium genus. Strains of the Paraburkholderia genus showed significantly higher nitrogen-fixing capacities than those of Bradyrhizobium genus. Strains of the two genera isolated from UF soils showed high metal tolerance and the respective genes occurred in 50% of strains. This is the first report of both alpha- and beta-rhizobia strains associated to an Acacia species adapted to UF and VS soils. Our findings suggest that A. spirorbis is an adaptive plant that establishes symbioses with whatever rhizobia is present in the soil, thus enabling the colonization of contrasted ecosystems.

This study aims to characterize the ectomycorrhizal (ECM) communities associated with Acacia spirorbis, a legume tree widely spread in New Caledonia that spontaneously grows on contrasted edaphic constraints, i.e. calcareous, ferralitic and volcano-sedimentary soils. Soil geochemical parameters and diversity of ECM communities were assessed in 12 sites representative of the three mains categories of soils. The ectomycorrhizal status of Acacia spirorbis was confirmed in all studied soils, with a fungal community dominated at 92% by Basidiomycota, mostly represented by/tomentella-thelephora (27.6%), /boletus (15.8%), /sebacina (10.5%), /russula-lactarius (10.5%) and /pisolithus-scleroderma (7.9%) lineages. The diversity and the proportion of the ECM lineages were similar for the ferralitic and volcano-sedimentary soils but significantly different for the calcareous soils. These differences in the distribution of the ECM communities were statistically correlated with pH, Ca, P and Al in the calcareous soils and with Co in the ferralitic soils. Altogether, these data suggest a high capacity of A. spirorbis to form ECM symbioses with a large spectrum of fungi regardless the soil categories with contrasted edaphic parameters.

Acacia spirorbis subsp. spirorbis Labill. is a legume tree that grows on calcareous, metalliferous and siliceous soils and is endemic to New Caledonia. The present study assesses the nitrogen-fixing potential of A. spirorbis in its contrasted natural environments. Soil samples, the leaves of A. spirorbis and of co-occurring non-N2-fixing reference plant species were collected from nine study sites across New Caledonia. Soil properties were analyzed by ICP-AES. Nitrogen (N) content and natural 15N abundance (δ15N) were measured in A. spirorbis and in reference plants. The percentage of N derived from the atmosphere (%Ndfa) fixed by A. spirorbis was also assessed and correlated with soil parameters. Remarkably, mean N contents in A. spirorbis showed no significant differences whatever the soil categories (20.2 ± 3.5 g kg−1) whereas major differences were found in N contents in the reference plants. The average δ15N value of A. spirorbis was close to zero (+0.29‰), while that of the reference species ranged from −4.83 to +7.05‰. In eight of the nine sites studied, A. spirorbis %Ndfa was above 70%, regardless of the soil category. Unlike in the reference species, no statistical correlations were found between A. spirorbis N content and δ15N abundance and soil parameters. Despite the difficulty of using the δ15N abundance method in such contrasted environments, the resulting data show that A. spirorbis has the highest N2-fixing potential yet recorded in the genus Acacia and its nitrogen fixation is effective almost throughout its natural range.

Nickel (Ni)-tolerant ectomycorrhizal Pisolithus albus was isolated from extreme ultramafic soils that are naturally rich in heavy metals. This study aimed to identify the specific molecular mechanisms associated with the response of P. albus to nickel. In presence of high concentration of nickel, P. albus Ni-tolerant isolate showed a low basal accumulation of nickel in its fungal tissues and was able to perform a metal efflux mechanism. Three genes putatively involved in metal efflux were identified from the P. albus transcriptome, and their overexpression was confirmed in the mycelium that was cultivated in vitro in the presence of nickel and in fungal tissues that were sampled in situ. Cloning these genes in yeast provided significant advantages in terms of nickel tolerance (+ 31% Ni EC50) and growth (+ 83% u) compared with controls. Furthermore, nickel efflux was also detected in the transformed yeast cells. Protein sequence analysis indicated that the genes encoded a P-type-ATPase, an ABC transporter and a major facilitator superfamily permease (MFS). This study sheds light on a global mechanism of metal efflux by P. albus cells that supports nickel tolerance. These specific responses to nickel might contribute to the fungal adaptation in ultramafic soil. (Resume d'auteur)

International audience; Ectomycorrhizal fungi (ECM) isolates of Pisolithus albus (Cooke and Massee) from nickel-rich ultramafic topsoils in New Caledonia were inoculated onto Acacia spirorbis Labill. (an endemic Fabaceae) and Eucalyptus globulus Labill. (used as a Myrtaceae plant host model). The aim of the study was to analyze the growth of symbiotic ECM plants growing on the ultramafic substrate that is characterized by high and toxic metal concentrations i.e. Co, Cr, Fe, Mn and Ni, deficient concentrations of plant essential nutrients such as N, P, K, and that presents an unbalanced Ca/Mg ratio (1/19). ECM inoculation was successful with a plant level of root mycorrhization up to 6.7%. ECM symbiosis enhanced plant growth as indicated by significant increases in shoot and root biomass. Presence of ECM enhanced uptake of major elements that are deficient in ultramafic substrates; in particular P, K and Ca. On the contrary, the ECM symbioses strongly reduced transfer to plants of element in excess in soils; in particular all metals. ECM-inoculated plants released metal complexing molecules as free thiols and oxalic acid mostly at lower concentrations than in controls. Data showed that ECM symbiosis helped plant growth by supplying uptake of deficient elements while acting as a protective barrier to toxic metals, in particular for plants growing on ultramafic substrate with extreme soil conditions. Isolation of indigenous and stress-adapted beneficial ECM fungi could serve as a potential tool for inoculation of ECM endemic plants for the successful restoration of ultramafic ecosystems degraded by mining activities.

New Caledonian serpentine (ultramafic) soils contain high levels of toxic heavy metals, in particular nickel, (up to 20 g kg−1) and are deficient in essential elements like carbon, nitrogen and phosphorus while having a high magnesium/calcium ratio. Although previous studies showed that ectomycorrhizal symbioses could play an important role in the adaptation of the endemic plants to ultramafic soils (FEMS Microbiol Ecol 72:238–49, 2010), none of them have compared the diversity of microbial communities from ultramafic vs non-ultramafic soils in New Caledonia. We explored the impact of edaphic characteristics on the diversity of ectomycorrhizal (ECM) fungi associated with different endemic species of Tristaniopsis (Myrtaceae) growing under contrasting soil conditions in the natural ecosystems of New Caledonia. ECM root tips were thus sampled from two different ultramafic sites (Koniambo massif and Desmazures forest) vs two volcano-sedimentary ones (Arama and Mont Ninndo). The molecular characterization of the ECM fungi through partial sequencing of the ITS rRNA gene revealed the presence of different dominant fungal genera including, both soil types combined, Cortinarius (36.1%), Pisolithus (18.5%), Russula (13.4%), Heliotales (8.2%) and Boletellus (7.2%). A high diversity of ECM taxa associated with Tristaniopsis species was found in both ultramafic and volcano-sedimentary soils but no significant differences in ECM genera distribution were observed between both soil types. No link could be established between the phylogenetic clustering of ECM taxa and their soil type origin, thus suggesting a possible functional—rather than taxonomical—adaptation of ECM fungal communities to ultramafic soils.

International audience; This study focuses on the chemotaxis, colonization and rice growth promoting ability of indole acetic acid (IAA) and 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase-producing rhizobacteria Rhizobium leguminosarum bv. trifolii SN10, previously isolated from root nodules of Trifolium alexandrium L. We show here that R. leguminosarum bv. trifolii SN10 promote the growth of four different rice varieties grown in India in terms of biomass, root branching and N content. In addition, using scanning electron microscopy and viable cell counts, we provide evidence that the bacteria successfully colonize the root surface of the rice variety which showed maximum growth promotion upon inoculation. Not only this, R. leguminosarum bv. trifolii SN10 exhibit a strong chemotaxis response towards the rice seed and root exudates despite the presence of a bacteriostatic phenolic compound, 7-hydroxycoumarin (umbelliferone). Further, R. leguminosarum bv. trifolii SN10 secretion of phytohormones such as IAA and ACC deaminase suggest the potential of the plant growth promoting rhizobacteria to be used as biofertilizer to enhance rice crop production in the subcontinent.

The taxonomic status of eleven rhizospheric bacterial strains belonging to the genus Burkholderia and isolated from roots of Costularia (Cyperaceae), tropical herbaceous pioneer plants growing on ultramafic soils in New Caledonia, was investigated using a polyphasic taxonomic approach. The genetic analyses (16S rRNA genes, gyrB, recA, nreB and cnr) confirmed that all strains are Burkholderia and cluster into two separated groups. The DNA hybridization results showed low relatedness values to the closest relatives Burkholderia species. The phenotypic analyses confirmed that the two groups of strains could be differentiated from each other and from other known Burkholderia species. This polyphasic study revealed that these two groups of strains represent each a novel species of Burkholderia, for which the names Burkholderia novacaledonica sp. nov. (type strain STM10272(T)=LMG28615(T)=CIP110887(T)) and B. ultramafica sp. nov. (type strain STM10279(T)=LMG28614(T)=CIP110886(T)) are proposed, respectively. These strains of Burkholderia presented specific ecological traits such as the tolerance to the extreme edaphic constraints of ultramafic soils: they grew at pH between 4 and 8 and tolerate the strong unbalanced Ca/Mg ratio (1/19) and the high concentrations of heavy metals i.e. Co, Cr, Mn and Ni. Noteworthy B. ultramafica tolerated nickel until 10mM and B. novacaledonica up to 5mM. The presence of the nickel (nreB) and cobalt/nickel (cnr) resistance determinants encoding for protein involved in metal tolerance was found in all strains of both groups. Moreover, most of the strains were able to produce plant growth promoting molecules (ACC, IAA, NH3 and siderophores). Such ecological traits suggest that these new species of Burkholderia might be environmentally adaptable plant-associated bacteria and beneficial to plants.

Eight Frankia-Gymnostoma nitrogen-fixing symbioses endemic to New Caledonia were studied using a range of techniques including molecular typing of Frankia , infectivity/effectivity and host spectrum of the isolates. The study was conducted on 128 Frankia isolates from the eight Gymnostoma species. The RFLP analysis of the rRNA 16S-23S ITS allowed us to cluster the strains in four groups. Symbiotic characterization of the four groups failed to reveal any host specificity both in term of infectivity or effectivity. The characteristics of the strains were those of Eleagnaceae -infective Frankia strains. Co-inoculation of two Gymnostoma species with mixtures of strains evidenced 11 % of mixed RFLP profiles. In such controlled conditions, this was interpreted as co-infections. This work evidenced original features of these Gymnostoma isolates: (i) they were closer to Eleagnaceae -infective Frankia than Casuarina -infective Frankia ; (ii) closer to atypical Frankia from Casuarina ; (iii) poorly host-specific; and (iv) they had a tendency of co-infection. They could represent a primitive group of Frankia able to survive in a wide range of habitats.

Rhizosphere bacteria were isolated from Costularia spp., pioneer sedges from ultramafic soils in New Caledonia, which is a hotspot of biodiversity in the South Pacific. Genus identification, ability to tolerate edaphic constraints, and plant-growth-promoting (PGP) properties were analysed. We found that 105colony-forming units per gram of root were dominated by Proteobacteria (69%) and comprised 21 genera, including Burkholderia (28%), Curtobacterium (15%), Bradyrhizobium (9%), Sphingomonas (8%), Rhizobium (7%), and Bacillus (5%). High proportions of bacteria tolerated many elements of the extreme edaphic conditions: 82% tolerated 100 μmol·L–1chromium, 70% 1 mmol·L–1nickel, 63% 10 mmol·L–1manganese, 24% 1 mmol·L–1cobalt, and 42% an unbalanced calcium/magnesium ratio (1/16). These strains also exhibited multiple PGP properties, including the ability to produce ammonia (65%), indole-3-acetic acid (60%), siderophores (52%), and 1-aminocyclopropane-1-carboxylate (ACC) deaminase (39%); as well as the capacity to solubilize phosphates (19%). The best-performing strains were inoculated with Sorghum sp. grown on ultramafic substrate. Three strains significantly enhanced the shoot biomass by up to 33%. The most successful strains influenced plant nutrition through the mobilization of metals in roots and a reduction of metal transfer to shoots. These results suggest a key role of these bacteria in plant growth, nutrition, and adaptation to the ultramafic constraints.

International audience; The complete and assembled genome sequences were determined for six strains of the alphaproteobacterial genus Methylobacterium, chosen for their key adaptations to different plant-associated niches and environmental constraints.

International audience; Crotalaria are plants of the Fabaceae family whose nodulation characteristics have been little explored despite therecent discovery of their unexpected ability to be efficiently nodulated in symbiosis with bacteria of the genusMethylobacterium. It has been shown that methylotrophy plays a key role in this unusual symbiotic system, as it isexpressed 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 frommethylotrophy. In this context, the aim of the present study was to show the histological and cytological impacts ofboth symbiotic and methylotrophic metabolism within Crotalaria podocarpa nodules. It was established that ifCrotalaria nodules are multilobed, each lobe has the morphology of indeterminate nodules but with a differentanatomy; that is, without root hair infection or infection threads. In the fixation zone, bacteroids display a sphericalshape and there is no uninfected cell. Crotalaria nodulation by Methylobacterium displayed some very unusualcharacteristics such as starch storage within bacteroid-filled cells of the fixation zone and also the complete lysis ofapical nodular tissues (where bacteria have a free-living shape and express methylotrophy). This lysis could possiblyreflect the bacterial degradation of plant wall pectins through bacterial pectin methyl esterases, thus producingmethanol as a substrate, allowing bacterial multiplication before release from the nodule.

International audience; In New Caledonia, a hot spot of biodiversity, plants from the Cyperaceae family are mostly endemic and considered pioneers of the nickel-rich natural serpentine ecosystem. The aim of the study was to highlight the mycorrhizal status of these Cyperaceae and to bring new insights into the role of this symbiosis in plant tolerance to ultramafic soils. Nine Cyperaceae species were studied and presented evidence of root colonization by arbuscular mycorrhizas (AMs), with frequencies ranging from 8% to 57%. The highest level of AM colonization was observed in plants from the endemic dominant genus Costularia. Molecular evidence demonstrated the presence of Glomus species inside the roots. In a controlled greenhouse assay, AM inoculation of Costularia comosa grown under ultramafic conditions significantly enhanced plant growth, with an increase in biomass by up to 2.4-fold for shoots and 1.2-fold for roots, and also reduced nickel content in roots by 2.5-fold, as compared with the controls. All these data support our hypotheses (i) that a relationship exists between the mycorrhizal status of Cyperaceae and their habitat, and (ii) that AM have a positive role in plant tolerance to ultramafic soils (mineral nutrition and metal tolerance), suggesting the use of these pioneer plants with AM management as potential tools for nickel mine site rehabilitation in New Caledonia.; En Nouvelle-Calédonie, un point chaud de la biodiversité, les plantes appartenant à la famille des Cypéracées sont principalement endémiques et sont considérées comme pionnières de l’écosystème naturel de la serpentine riche en nickel. Cette étude visait à éclaircir le statut mycorhizien de ces Cypéracées et d’apporter un nouvel aperçu de rôle de cette symbiose dans la tolérance des végétaux aux sols ultrabasiques. Neuf espèces de Cypéracées ont été étudiées et la colonisation des racines par des mycorhizes arbusculaires (MA) a été mise en évidence avec des fréquences variant de 8 % à 57 %. Le niveau de MA le plus élevé a été noté chez les plantes du genre endémique dominant Costularia. Les données moléculaires ont démontré la présence de Glomus sp. à l’intérieur des racines. Lors d’un essai en serre contrôlée, l’inoculation de MA chez Costularia comosa cultivée en conditions ultrabasiques a significativement accru la croissance des plants, avec une augmentation de la biomasse des pousses de 2,4 fois et des racines de 1,2 fois, réduisant aussi le contenu en nickel des racines de 2,5 fois, tout cela par rapport au contrôle. Toutes ces données plaident en faveur de l’hypothèse (i) d’une relation entre le statut en mycorhize des Cypéracées et leur habitat, et (ii) du rôle positif des MA dans la tolérance aux sols ultrabasiques (nutrition minérale et tolérance aux métaux), suggérant que ces plantes pionnières parallèlement à la gestion des MA puissent constituer des outils potentiels de réhabilitation des sites miniers de nickel en Nouvelle-Calédonie.

International audience; Ectomycorrhizal (ECM) Pisolithus albus (Cooke & Massee), belonging to the ultramafic ecotype isolated in nickel-rich serpentine soils from New Caledonia (a tropical hotspot of biodiversity) and showing in vitro adaptive nickel tolerance, were inoculated to Eucalyptus globulus Labill used as a Myrtaceae plant-host model to study ectomycorrhizal symbiosis. Plants were then exposed to a nickel (Ni) dose-response experiment with increased Ni treatments up to 60 mg kg (- 1) soil as extractable Ni content in serpentine soils. Results showed that plants inoculated with ultramafic ECM P. albus were able to tolerate high and toxic concentrations of Ni (up to 60 mu g g (- 1)) while uninoculated controls were not. At the highest Ni concentration tested, root growth was more than 20-fold higher and shoot growth more than 30-fold higher in ECM plants compared with control plants. The improved growth in ECM plants was associated with a 2.4-fold reduction in root Ni concentration but a massive 60-fold reduction in transfer of Ni from root to shoots. In vitro, P. albus strains could withstand high Ni concentrations but accumulated very little Ni in its tissue. The lower Ni uptake by mycorrhizal plants could not be explained by increased release of metal-complexing chelates since these were 5- to 12-fold lower in mycorrhizal plants at high Ni concentrations. It is proposed that the fungal sheath covering the plant roots acts as an effective barrier to limit transfer of Ni from soil into the root tissue. The degree of tolerance conferred by the ultramafic P. albus isolates to growth of the host tree species is considerably greater than previously reported for other ECM. The primary mechanisms underlying this improved growth were identified as reduced Ni uptake into the roots and markedly reduced transfer from root to shoot in mycorrhizal plants. The fact that these positive responses were observed at Ni concentrations commonly observed in serpentinic soils suggests that ultramafic ecotypes of P. albus could play an important role in the adaptation of tree species to soils containing high concentrations of heavy metals and aid in strategies for ecological restoration.

Contact: philippe.jourand@ird.fr; International audience; Isolates of ectomycorrhizal Pisolithus albus were sampled from both ultramafic and volcano-sedimentary soils in New Caledonia, a tropical hotspot of biodiversity, to investigate the relationships between genetic diversity and edaphic constraint through tolerance to nickel (Ni). Carpophore description, spore morphology and phylogenetic analysis based on internal transcribed spacer (ITS) rDNA sequences confirmed that all isolates belong to P. albus and are closely related to other Australasian specimens. Using molecular tools, ITS-restriction fragment length polymorphism and amplified fragment length polymorphism markers, we showed the existence of two distinct genetic clusters within P. albus: ultramafic and volcano-sedimentary. Mycelia response to Ni toxicity supports such a population structure. Pisolithus albus from ultramafic soils included isolates with a high diversity of in vitro Ni tolerance, with both Ni-tolerant isolates (average Ni EC(50) at 575 mu M) and Ni-sensitive isolates (average Ni EC(50) at 37 mu M). In contrast, all isolates from volcano-sedimentary soils were found to be Ni sensitive (average Ni EC(50) at 32 mu M). We highlight that (1) P. albus population from ultramafic soils of New Caledonia are genetically structured in ecotype, and that (2) Ni tolerance among ultramafic isolates suggests an adaptive physiological response to Ni toxicity.

Some legume species of the Crotalaria genus are specifically nodulated by methylotrophic bacteria belonging to the Methylobacterium nodulans species. The feature of this symbiotic bacterium is its ability to oxidize methanol, a property based on the presence of a methanol dehydrogenase enzyme. Despite a good knowledge of this property and its implication in symbiosis, the molecular dialogue between M. nodulans and crotalaria podocarpa leading to symbiosis is largely unknown, except the presence of a nodA nodulation gene in the genome of M. nodulans ORS 2060. To investigate if M. nodulans ORS 2060 produces Nod factors, molecules considered as the major bacteria-to-plant signals essential for the establishment of rhizobia–legume symbiosis, we identified and sequenced a nodDABCUIJHQ cluster from a genomic library of ORS 2060. Phylogenetic analyses of nod genes revealed that M. nodulans ORS 2060 form a branch together with Burkholderia tuberum STM678 and a strain of Methylobacterium sp. (4-46) isolated from Lotononis , and distinct from all the other rhizobia. To analyse the regulation of ORS 2060 nod genes, we constructed a nodA–LacZ promoter fusion to monitor the nod gene expression with various flavonoids. The flavone apigenin was found to be the strongest inducer of nod gene expression in M. nodulans ORS 2060. This latter flavonoid was used to induce ORS 2060, and Nod factors were purified by high-performance liquid chromatography (HPLC) and further characterized by mass spectrometry. One major Nod factor structure was identified as a pentamer of chitin substituted by C18:1 or C16:0 acyl chains on the non-reducing end and 6-O-sulphated on the other end, suggesting a classic symbiotic dialogue between M. nodulans and C. 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.

Aerobic photosynthetic bacteria possess the unusual characteristic of producing different classes of carotenoids. In this study, we demonstrate the presence of two distinct crt gene clusters involved in the synthesis of spirilloxanthin and canthaxanthin in a Bradyrhizobium strain. Each cluster contains the genes crtE, crtB, and crtI leading to the common precursor lycopene. We show that spirilloxanthin is associated with the photosynthetic complexes, while canthaxanthin protects the bacteria from oxidative stress. Only the spirilloxanthin crt genes are regulated by light via the control of a bacteriophytochrome. Despite this difference in regulation, the biosyntheses of both carotenoids are strongly interconnected at the level of the common precursors. Phylogenetic analysis suggests that the canthaxanthin crt gene cluster has been acquired by a lateral gene transfer. This acquisition may constitute a major selective advantage for this class of bacteria, which photosynthesize only under conditions where harmful reactive oxygen species are generated.

Abstract An aqueous extract from leaves of Crotalaria virgulata subsp. grantiana was assayed for its effects on second-stage juveniles of Meloidogyne incognita. The biological activity was nematostatic; nematodes were not killed but were completely paralysed in a 1 mg/ml (w/v) extract: the LD50 equivalent was estimated to be 0.5 mg/ml. The effect was reversible: juveniles previously paralysed by C. grantiana extract recovered complete mobility in water and were able to infest a susceptible tomato plant. Freeze-dried aqueous extract from C. grantiana leaves added to a sterile sandy substrate at 1 mg/ml protected susceptible tomato plants from M. incognita infestation. This suggests a promising use of C. grantiana as both a green manure and natural alternative to synthetic chemicals in nematode population control, especially in integrated pest management for vegetable crops in organic agriculture of tropical and temperate areas.

Data on 72 non-pigmented bacterial strains that specifically induce nitrogen-fixing root nodules on the legume species Crotalaria glaucoides, Crotalaria perrottetii and Crotalaria podocarpa are reviewed. By SDS-PAGE analysis of total protein patterns and by 16S rRNA PCR-RFLP, these strains form a homogeneous group that is separate from other legume root-nodule-forming bacteria. The 16S rRNA gene-based phylogeny indicates that these bacteria belong to the genus Methylobacterium. They can grow on C1 compounds such as methanol, formate and formaldehyde but not methylamine as sole carbon source, and carry an mxaF gene, encoding methanol dehydrogenase, which supports their methylotrophic metabolism. Presence of a nodA nodulation gene, and ability to nodulate plants of Crotalaria species and to fix nitrogen are features that separate the strains currently included in this group from other members of the genus Methylobacterium. The present study includes additional genotypic and phenotypic characterization of this novel Methylobacterium species, i.e. nifH gene sequence, morphology, physiology, enzymic and carbon source assimilation tests and antibiotic resistance. The name Methylobacterium nodulans sp. nov. (type strain, ORS 2060T=CNCM I 2342T=LMG 21967T) is proposed for this group of root-nodule-forming bacteria.

The in vitro paralysis of second-stage juveniles of Meloidogyne incognita, M. javanica and M. mayaguensis by aqueous extracts of 15 West African Crotalaria species was analysed. A multivariate analysis distinguished four groups, based on their nematostatic activity: i) C. glaucoides extracts were not active; ii) the root extracts from C. goreensis, C. lathyroides and C. perrottetii were more active than the shoot extracts; iii) the shoot extracts from C. comosa and C. cylindrocarpa were more active than the root extracts; iv) both shoot and root extracts of C. atrorubens, C. barkae, C. grantiana, C. hyssopifolia, C. pallida, C. podocarpa, C. retusa, C. senegalensis and C. sphaerocarpa have nematostatic effects depending on either the Crotalaria species or plant tissue from which the extracts were derived. However, considering the greater biomass contribution of the leaves and stems compared to the roots when the plants are used as green manure, C. barkae, C. grantiana, C. pallida and C. podocarpa are the most efficient Crotalaria species, whatever the Meloidogyne species targeted.

Rhizobia described so far belong to three distinct phylogenetic branches within the α-2 subclass of Proteobacteria . Here we report the discovery of a fourth rhizobial branch involving bacteria of the Methylobacterium genus. Rhizobia isolated from Crotalaria legumes were assigned to a new species, “ Methylobacterium nodulans ,” within the Methylobacterium genus on the basis of 16S ribosomal DNA analyses. We demonstrated that these rhizobia facultatively grow on methanol, which is a characteristic of Methylobacterium spp. but a unique feature among rhizobia. Genes encoding two key enzymes of methylotrophy and nodulation, the mxaF gene, encoding the α subunit of the methanol dehydrogenase, and the nodA gene, encoding an acyltransferase involved in Nod factor biosynthesis, were sequenced for the type strain, ORS2060. Plant tests and nodA amplification assays showed that “ M. nodulans ” is the only nodulating Methylobacterium sp. identified so far. Phylogenetic sequence analysis showed that “ M. nodulans ” NodA is closely related to Bradyrhizobium NodA, suggesting that this gene was acquired by horizontal gene transfer.