The cepacian-like exopolysaccharide of Paraburkholderia ultramafica STM10279T enhances growth and metal adaptation of Tetraria comosa on New Caledonian ultramafic soil.

Paraburkholderia ultramafica STM10279T is a metal-tolerant rhizobacterium that promotes plant growth. It was isolated from the roots of Tetraria arundinaceae, a pioneer endemic tropical herb growing on ultramafic soils in New Caledonia. We have recently shown that the main mechanism of metal tolerance of P. ultramafica is related to the production of an acidic exopolysaccharide (EPS). To explore the potential role of this EPS in the plant's environmental adaptation, we first elucidated its structure by employing a combination of chromatography and mass spectrometry techniques. These analyses revealed that the EPS is highly branched and composed of galactosyl (35.8%), glucosyl (33.2%), rhamnosyl (19.5%), mannosyl (7.2%), and glucuronosyl residues (4.4%), similar to the EPS of the Burkholderia cepacia complex known as cepacian. We subsequently conducted greenhouse experiments on Tetraria comosa plantlets inoculated with P. ultramafica or a solution of its EPS during transplanting onto ultramafic substrate. The data showed that the dry weight of T. comosa shoots was 2.5 times higher in the plants treated with the EPS compared to the unexposed plants. In addition, inductively coupled plasma-optical emission spectrometry (ICP-OES) analysis revealed that exposure to the EPS significantly increased Ca, Mg, K, and P uptake as well as K content in roots. In vitro experiments using the Pikovskaya method showed that the EPS was able to solubilize phosphorus. Consistent with the retention of metals in roots and a reduction in shoots, our data revealed a significant decrease in metal translocation factors (TFs) in the plants inoculated with the EPS. These results suggest a beneficial effect of the rhizobacterial EPS on plant growth and abiotic stress mitigation. In addition, the data suggest that the reduced levels of trace metals in plants exposed to P. ultramafica STM10279T are due to metal chelation by the EPS. Further investigations are needed to firmly demonstrate whether this EPS could be used as a biostimulant for plant growth and adaptation to ultramafic soils.

Co-inoculation with a bacterium and arbuscular mycorrhizal fungi improves root colonization, plant mineral nutrition, and plant growth of a Cyperaceae plant in an ultramafic soil.

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.

New Caledonian ultramafic conditions structure the features of Curtobacterium citreum strains that play a role in plant adaptation.

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.

Is a mixture of arbuscular mycorrhizal fungi better for plant growth than single-species inoculants?

Inoculation of arbuscular mycorrhizal fungi (AMF) as plant growth promoters has mostly been conducted using single-species inoculum. In this study, we investigated whether co-inoculation of different native AMF species induced an improvement of plant growth in an ultramafic soil. We analyzed the effects of six species of AMF from a New Caledonian ultramafic soil on plant growth and nutrition, using mono-inoculations and mixtures comprising different numbers of AMF species, in a greenhouse experiment. The endemic Metrosideros laurifolia was used as a host plant. Our results suggest that, when the plant faced multiple abiotic stress factors (nutrient deficiencies and high concentrations of different heavy metals), co-inoculation of AMF belonging to different families was more efficient than mono-inoculation in improving biomass, mineral nutrition, Ca/Mg ratio, and tolerance to heavy metals of plants in ultramafic soil. This performance suggested functional complementarity between distantly related AMF. Our findings will have important implications for restoration ecology and mycorrhizal biotechnology applied to ultramafic soils.

Arbuscular mycorrhizal fungi and sewage sludge enhance growth and adaptation of Metrosideros laurifolia on ultramafic soil in New Caledonia: A field experiment.

The ecological restauration of nickel mining-degraded areas in New Caledonia is strongly limited by soil mineral nutrient deficiencies, heavy metal toxicity and slow growth rate of the native plant species. The improvement of revegetation technics needs multidisciplinary knowledge. A field experiment with relocated topsoil was assessed to test plant inoculation with a mix of three selected arbuscular mycorrhizal fungi (AMF) combined with sewage sludge amendment of the soil. Metrosideros laurifolia seedlings, an endemic Myrtaceae, were inoculated with the mixed AMF isolates and grown in a nursery for 18 months before being planted. 528 days after plantation, the dry weight of inoculated plants was 4 times higher than non-inoculated ones. AMF inoculated plants growing in sewage sludge amended soil showed a dry weight more than seven times higher than control plants. These differences were positively correlated with mycorrhizal colonization. However at this stage, AMF inoculated isolates were reduced in roots of M. laurifolia and replaced by several AMF indigenous species. This AMF diversity was higher in inoculated plants and in non-amended plots. Inoculated plants were characterized by a better mineral nutrition, a higher Ca/Mg ratio and a lower heavy metal translocation. In conclusion, this study showed that AMF inoculation combined with sewage sludge amendment of soil can improve ecological restoration of ultramafic mine-degraded areas.

New method for the identification of arbuscular mycorrhizal fungi by proteomic-based biotyping of spores using MALDI-TOF-MS.

Arbuscular mycorrhizal fungi (AMF, Glomeromycota) are mutualistic symbionts associated with majority of land plants. These fungi play an important role in plant growth, but their taxonomic identification remains a challenge for academic research, culture collections and inoculum producers who need to certify their products. Identification of these fungi was traditionally performed based on their spore morphology. DNA sequence data have successfully been used to study the evolutionary relationships of AMF, develop molecular identification tools and assess their diversity in the environment. However, these methods require considerable expertise and are not well-adapted for "routine" quality control of culture collections and inoculum production. Here, we show that Matrix-Assisted Laser Desorption Ionisation Time of Flight Mass Spectrometry proteomic-based biotyping is a highly efficient approach for AMF identification. Nineteen isolates belonging to fourteen species, seven genera and five families were clearly differentiated by MALDI biotyping at the species level, and intraspecific differentiation was achieved for the majority. AMF identification by MALDI biotyping could be highly useful, not only for research but also in agricultural and environmental applications. Fast, accurate and inexpensive molecular mass determination and the possibility of automation make MALDI-TOF-MS a real alternative to conventional morphological and molecular methods for AMF identification.

Burkholderia novacaledonica sp. nov. and B. ultramafica sp. nov. isolated from roots of Costularia spp. pioneer plants of ultramafic soils in New Caledonia.

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.

The metal transporter PgIREG1 from the hyperaccumulator Psychotria gabriellae is a candidate gene for nickel tolerance and accumulation.

Nickel is an economically important metal and phytotechnologies are being developed to limit the impact of nickel mining on the environment. More than 300 plant species are known to hyperaccumulate nickel. However, our knowledge of the mechanisms involved in nickel accumulation in plants is very limited because it has not yet been possible to study these hyperaccumulators at the genomic level. Here, we used next-generation sequencing technologies to sequence the transcriptome of the nickel hyperaccumulator Psychotria gabriellae of the Rubiaceae family, and used yeast and Arabidopsis as heterologous systems to study the activity of identified metal transporters. We characterized the activity of three metal transporters from the NRAMP and IREG/FPN families. In particular, we showed that PgIREG1 is able to confer nickel tolerance when expressed in yeast and in transgenic plants, where it localizes in the tonoplast. In addition, PgIREG1 shows higher expression in P. gabriellae than in the related non-accumulator species Psychotria semperflorens. Our results designate PgIREG1 as a candidate gene for nickel tolerance and hyperaccumulation in P. gabriellae. These results also show how next-generation sequencing technologies can be used to access the transcriptome of non-model nickel hyperaccumulators to identify the underlying molecular mechanisms.

Mycorrhizal status of Cyperaceae from New Caledonian ultramafic soils: effects of phosphorus availability on arbuscular mycorrhizal colonization of Costularia comosa under field conditions.

Plants from the Cyperaceae family (sedges), usually considered as non-mycorrhizal, constitute almost exclusively the herbaceous stratum of the ultramafic maquis in New Caledonia. These plants are pioneers and are important for the ecological restoration of mined areas. Costularia comosa, one of the most common sedges in this environment, was grown under field conditions on ultramafic soil, fertilized or not with phosphate and/or nitrogen. Results showed that the addition of phosphate to the soil induced a clear increase in mycorrhizal colonization of C. comosa and an increase in arbuscule abundance, reflecting the establishment of a functional mycorrhizal symbiosis. Significant positive correlations were found among mycorrhizal parameters and plant or soil phosphorus concentrations. Nitrogen fertilization did not affect mycorrhizal colonization of C. comosa. The improvement in mycorrhizal colonization by phosphate fertilization did not influence significantly nickel concentrations in the roots and shoots of plants. This study demonstrated that phosphate fertilization of ultramafic soil improved mycorrhizal colonization of C. comosa, with formation of a functional symbiosis under field conditions.

Arbuscular mycorrhizal fungi from New Caledonian ultramafic soils improve tolerance to nickel of endemic plant species.

In order to improve knowledge about the role of arbuscular mycorrhizal fungi (AMF) in the tolerance to heavy metals in ultramafic soils, the present study investigated the influence of two Glomus etunicatum isolates from New Caledonian ultramafic maquis (shrubland), on nickel tolerance of a model plant species Sorghum vulgare, and of two ultramafic endemic plant species, Alphitonia neocaledonica and Cloezia artensis. In a first step, plants were grown in a greenhouse, on sand with defined concentrations of Ni, to appreciate the effects of the two isolates on the alleviation of Ni toxicity in controlled conditions. In a second step, the influence of the AMF on A. neocaledonica and C. artensis plants grown in a New Caledonian ultramafic soil rich in extractable nickel was investigated. Ni reduced mycorrhizal colonization and sporulation of the fungal isolates, but the symbionts increased plant growth and adaptation of endemic plant species to ultramafic conditions. One of the two G. etunicatum isolates showed a stronger positive effect on plant biomass and phosphorus uptake, and a greater reduction in toxicity symptoms and Ni concentration in roots and shoots. The symbionts seemed to act as a barrier to the absorption of Ni by the plant and reduced root-to-shoot Ni translocation. Results indicate the potential of selected native AMF isolates from ultramafic areas for ecological restoration of such degraded ecosystems.

Rhizosphere bacteria of Costularia spp. from ultramafic soils in New Caledonia: diversity, tolerance to extreme edaphic conditions, and role in plant growth and mineral nutrition.

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 10(5) colony-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(-1) chromium, 70% 1 mmol·L(-1) nickel, 63% 10 mmol·L(-1) manganese, 24% 1 mmol·L(-1) cobalt, 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.

New insights into the mycorrhizal status of Cyperaceae from ultramafic soils in New Caledonia.

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 sp. 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.

Reticulate evolution on a mosaic of soils: diversification of the New Caledonian endemic genus Codia (Cunoniaceae).

We reconstructed the evolutionary history of Codia, a plant genus endemic to the New Caledonia biodiversity hotspot in the southwest Pacific, using three single-copy nuclear genes. It seems likely that more than half of Codia species have a hybrid origin, but in the absence of cytological information, it is not known whether polyploids occur. Adaptation to ultramafic soils is possibly a plesiomorphic character for the entire genus. We found that species of hybrid origin can have some morphological characters absent in putative parental species, that is, they exhibit transgressive phenotypes. There is evidence of considerable range alteration post-origin in several species because some likely parental species of hybrid taxa no longer co-occur and are confined to putative rainforest refugia; in some cases, hybrid species do not now co-occur with either of their parental species. These results have implications for the design of conservation strategies, for example, prioritization of parental species for ex-situ conservation and preservation of the contact zones between soil types where hybridization is more likely to occur (i.e. conserving the possibility for the process to continue rather than trying to conserve taxa).

Tolerance and induction of tolerance to Ni of arbuscular mycorrhizal fungi from New Caledonian ultramafic soils.

The influence of Ni on arbuscular mycorrhizal fungi (AMF) has not been studied yet. We tested the tolerance to Ni of five AMF isolates from New Caledonian ultramafic soils. Spore germination indicated that these isolates were clearly more tolerant to Ni than three other isolates from non-ultramafic soils. They were able to germinate at 30 microg g(-1) Ni, whereas spores of the non-ultramafic isolates were totally inhibited at 15 microg g(-1) Ni. Among the ultramafic isolates, two were obtained from roots of Ni-hyperaccumulating plants. Their tolerance to Ni was clearly higher than all the other isolates. The proportion of germinated spores of the different isolates in contact with ultramafic soils showed the same tendencies as those observed with Ni solutions. Tolerance to Ni increased when spores were produced from mycorrhiza on plants grown on sand containing 20 microg g(-1) Ni, in comparison with those produced on sand without Ni. These results indicate that the tolerance to Ni of AMF spores can be induced by the presence of this metal in the substrate.

Occurrence of mycorrhizal symbioses in the metal-rich lateritic soils of the Koniambo Massif, New Caledonia.

The occurrence of arbuscular mycorrhiza (AM) was surveyed in ten endemic plant species of the Koniambo Massif (New Caledonia) and associated metal-enriched ultramafic soils along a topographic sequence ranging from a plateau at 900 m altitude to a valley at 700 m. In the four different plant formations (Araucaria group on the plateau, ligno-herbaceous maquis, Tristaniopsis maquis and Nothofagus forest in the valley), all plants were consistently colonised by AM fungi, even the sedges Costularia arundinacea, C. nervosa and Lepidosperma perteres and the nickel-hyperaccumulating plant Phyllanthus favieri. Dual (AM and ectomycorrhiza EM) colonisation was observed in the two plant formations dominated by the ectomycorrhizal plants Nothofagus balansae for the forest (site 4) and Tristaniopsis guillainii and T. calobuxus for the Tristaniopsis maquis (site 3). In the soils, there are strong positive correlations between microbial activity, black AM spore abundance and concentrations of available metals indicating the role of the biotic component in the release of metals. These results suggest that these symbioses are important in the adaptation of the endemic plants to these soils, and may be relevant to ecological restoration of the ancient nickel mines.

Amycolatopsis plumensis sp. nov., a novel bioactive actinomycete isolated from a New-Caledonian brown hypermagnesian ultramafic soil.

The taxonomic position of an actinomycete isolated from a brown hypermagnesian ultramafic soil was examined using a polyphasic approach. The organism, which was designated SBHS Strp1(T), was found to have chemical and morphological properties typical of Amycolatopsis strains. It was most closely associated with Amycolatopsis kentuckyensis, Amycolatopsis lexingtonensis, Amycolatopsis rifamycinica, Amycolatopsis pretoriensis and Amycolatopsis tolypomycina on the basis of 16S rRNA gene sequence data, and showed a unique pattern of phenotypic properties that distinguished it from the type strains of these taxa. The combined genotypic and phenotypic data show that the organism merits description as a novel species of Amycolatopsis. The name proposed for the novel species is Amycolatopsis plumensis sp. nov.; the type strain is SBHS Strp1(T) (=DSM 44776(T)=NRRL B-24324(T)).

Streptomyces ferralitis sp. nov., a novel streptomycete isolated from a New-Caledonian ultramafic soil.

The taxonomic position of an actinomycete isolated from an ultramafic soil in New Caledonia was determined using a polyphasic approach. The isolate, which was designated SFOp68(T), was shown to have chemical and morphological properties typical of streptomycetes. An almost complete 16S rRNA gene sequence of the isolate was generated and compared with sequences of representative streptomycetes. The 16S rRNA data not only supported the classification of the strain in the genus Streptomyces, but also showed that it formed a distinct phyletic line that was most closely related to one composed of the type strain of Streptomyces rimosus. The two organisms can be readily separated using a diverse range of phenotypic properties. It is proposed that strain SFOp68(T) (=DSM 41836(T)=NCIMB 13954(T)) be classified in the genus Streptomyces as Streptomyces ferralitis sp. nov.

Nocardia neocaledoniensis sp. nov., a novel actinomycete isolated from a New-Caledonian brown hypermagnesian ultramafic soil.

The taxonomic position of an actinomycete isolated from a hypermagnesian ultramafic soil was examined using a polyphasic approach. The strain, designated SBH(R) OA6(T), was shown to have chemical and morphological properties typical of members of the genus Nocardia. The organism was most closely associated with Nocardia asteroides using 16S rRNA gene sequence data. It showed a distinctive set of phenotypic properties that distinguished it from representatives of all species with validly published names classified in the genus Nocardia. The combined genotypic and phenotypic data show that strain SBH(R) OA6(T) (=DSM 44717(T)=NCIMB 13955(T)) merits description as the type strain of a novel Nocardia species, Nocardia neocaledoniensis sp. nov.