Ecological aspects and relationships of the emblematic Vachellia spp. exposed to anthropic pressures and parasitism in natural hyper-arid ecosystems: ethnobotanical elements, morphology, and biological nitrogen fixation.

MAIN CONCLUSION: Emblematic Vachellia spp. naturally exposed to hyper-arid conditions, intensive grazing, and parasitism maintain a high nitrogen content and functional mutualistic nitrogen-fixing symbioses. AlUla region in Saudi Arabia has a rich history regarding mankind, local wildlife, and fertility islands suitable for leguminous species, such as the emblematic Vachellia spp. desert trees. In this region, we investigated the characteristics of desert legumes in two nature reserves (Sharaan and Madakhil), at one archaeological site (Hegra), and in open public domains et al. Ward and Jabal Abu Oud. Biological nitrogen fixation (BNF), isotopes, and N and C contents were investigated through multiple lenses, including parasitism, plant tissues, species identification, plant maturity, health status, and plant growth. The average BNF rates of 19 Vachellia gerrardii and 21 Vachellia tortilis trees were respectively 39 and 67%, with low signs of inner N content fluctuations (2.10-2.63% N) compared to other co-occurring plants. The BNF of 23 R. raetam was just as high, with an average of 65% and steady inner N contents of 2.25 ± 0.30%. Regarding parasitism, infected Vachellia trees were unfazed compared to uninfected trees, thereby challenging the commonly accepted detrimental role of parasites. Overall, these results suggest that Vachellia trees and R. raetam shrubs exploit BNF in hyper-arid environments to maintain a high N content when exposed to parasitism and grazing. These findings underline the pivotal role of plant-bacteria mutualistic symbioses in desert environments. All ecological traits and relationships mentioned are further arguments in favor of these legumes serving as keystone species for ecological restoration and agro-silvo-pastoralism in the AlUla region.

Revealing human impact on natural ecosystems through soil bacterial DNA sampled from an archaeological site.

Human activities have affected the surrounding natural ecosystems, including belowground microorganisms, for millennia. Their short- and medium-term effects on the diversity and the composition of soil microbial communities are well-documented, but their lasting effects remain unknown. When unoccupied for centuries, archaeological sites are appropriate for studying the long-term effects of past human occupancy on natural ecosystems, including the soil compartment. In this work, the soil chemical and bacterial compositions were compared between the Roman fort of Hegra (Saudi Arabia) abandoned for 1500 years, and a preserved area located at 120 m of the southern wall of the Roman fort where no human occupancy was detected. We show that the four centuries of human occupancy have deeply and lastingly modified both the soil chemical and bacterial compositions inside the Roman fort. We also highlight different bacterial putative functions between the two areas, notably associated with human occupancy. Finally, this work shows that the use of soils from archaeological sites causes little disruption and can bring relevant information, at a large scale, during the initial surveys of archaeological sites.

Engineering carbon sequestration on arid lands.

To limit the effects of global warming, arid lands, which constitute approximately one-third of terrestrial surfaces and are not utilized for agriculture, could serve as an effective method for long-term carbon (C) storage. We propose that soil-plant-microbiome engineering with oxalogenic plants and oxalotrophic microbes could facilitate C sequestration on a global scale.

A leguminous species exploiting alpha- and beta-rhizobia for adaptation to ultramafic and volcano-sedimentary soils: an endemic Acacia spirorbis model from New Caledonia.

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.

Microbial island biogeography: isolation shapes the life history characteristics but not diversity of root-symbiotic fungal communities.

Island biogeography theory is one of the most influential paradigms in ecology. That island characteristics, including remoteness, can profoundly modulate biological diversity has been borne out by studies of animals and plants. By contrast, the processes influencing microbial diversity in island systems remain largely undetermined. We sequenced arbuscular mycorrhizal (AM) fungal DNA from plant roots collected on 13 islands worldwide and compared AM fungal diversity on islands with existing data from mainland sites. AM fungal communities on islands (even those >6000 km from the closest mainland) comprised few endemic taxa and were as diverse as mainland communities. Thus, in contrast to patterns recorded among macro-organisms, efficient dispersal appears to outweigh the effects of taxogenesis and extinction in regulating AM fungal diversity on islands. Nonetheless, AM fungal communities on more distant islands comprised a higher proportion of previously cultured and large-spored taxa, indicating that dispersal may be human-mediated or require tolerance of significant environmental stress, such as exposure to sunlight or high salinity. The processes driving large-scale patterns of microbial diversity are a key consideration for attempts to conserve and restore functioning ecosystems in this era of rapid global change.

Ectomycorrhizal Communities Associated with the Legume Acacia spirorbis Growing on Contrasted Edaphic Constraints in New Caledonia.

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.

Ectomycorrhizal fungal diversity associated with endemic Tristaniopsis spp. (Myrtaceae) in ultramafic and volcano-sedimentary soils in New Caledonia.

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.

Transfer to forest nurseries significantly affects mycorrhizal community composition of Asteropeia mcphersonii wildings.

Mycorrhizal symbiosis is extremely important for tree growth, survival and resistance after transplantation particularly in Madagascar where deforestation is a major concern. The importance of mycorrhizal symbiosis is further increased when soil conditions at the planting site are limiting. To identify technical itineraries capable of improving ecological restoration in Madagascar, we needed to obtain native ectomycorrhizal (ECM) saplings with a wide diversity of ECM fungi. To this end, we transplanted ECM seedlings from the wild (wildlings) to a nursery. Using molecular characterisation of internal transcribed spacer (ITS) rDNA, we tested the effect of transplanting Asteropeia mcphersonii wildlings on ECM communities after 8 months of growth in the nursery. With or without the addition of soil from the site where the seedlings were sampled to the nursery substrate, we observed a dramatic change in the composition of fungal communities with a decrease in the ECM infection rate, a tremendous increase in the abundance of an operational taxonomic unit (OTU) taxonomically close to the order Trechisporales and the disappearance of all OTUs of Boletales. Transplanting to the nursery and/or to nursery conditions was shown to be incompatible with the survival and even less with the development in the nursery of most ECM fungi naturally associated with A. mcphersonii wildings.

Rhizobial characterization in revegetated areas after bauxite mining

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

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.

Rhizobial characterization in revegetated areas after bauxite mining.

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

Ectomycorrhizal Pisolithus albus inoculation of Acacia spirorbis and Eucalyptus globulus grown in ultramafic topsoil enhances plant growth and mineral nutrition while limits metal uptake.

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.

Evidence of nickel (Ni) efflux in Ni-tolerant ectomycorhizal Pisolithus albus isolated from ultramafic soil.

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% µ) 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.

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.

Biodiversity of Mimosa pudica rhizobial symbionts (Cupriavidus taiwanensis, Rhizobium mesoamericanum) in New Caledonia and their adaptation to heavy metal-rich soils.

Rhizobia are soil bacteria able to develop a nitrogen-fixing symbiosis with legumes. They are taxonomically spread among the alpha and beta subclasses of the Proteobacteria. Mimosa pudica, a tropical invasive weed, has been found to have an affinity for beta-rhizobia, including species within the Burkholderia and Cupriavidus genera. In this study, we describe the diversity of M. pudica symbionts in the island of New Caledonia, which is characterized by soils with high heavy metal content, especially of Ni. By using a plant-trapping approach on four soils, we isolated 96 strains, the great majority of which belonged to the species Cupriavidus taiwanensis (16S rRNA and recA gene phylogenies). A few Rhizobium strains in the newly described species Rhizobium mesoamericanum were also isolated. The housekeeping and nod gene phylogenies supported the hypothesis of the arrival of the C. taiwanensis and R. mesoamericanum strains together with their host at the time of the introduction of M. pudica in New Caledonia (NC) for its use as a fodder. The C. taiwanensis strains exhibited various tolerances to Ni, Zn and Cr, suggesting their adaptation to the specific environments in NC. Specific metal tolerance marker genes were found in the genomes of these symbionts, and their origin was investigated by phylogenetic analyses.

Tracking nickel-adaptive biomarkers in Pisolithus albus from New Caledonia using a transcriptomic approach.

The fungus Pisolithus albus forms ectomycorrhizal (ECM) associations with plants growing on extreme ultramafic soils, which are naturally rich in heavy metals such as nickel. Both nickel-tolerant and nickel-sensitive isolates of P. albus are found in ultramafic soils in New Caledonia, a biodiversity hotspot in the Southwest Pacific. The aim of this work was to monitor the expression of genes involved in the specific molecular response to nickel in a nickel-tolerant P. albus isolate. We used pyrosequencing and quantitative polymerase chain reaction (qPCR) approaches to investigate and compare the transcriptomes of the nickel-tolerant isolate MD06-337 in the presence and absence of nickel. A total of 1,071,375 sequencing reads were assembled to infer expression patterns of 19,518 putative genes. Comparison of expression levels revealed that 30% of the identified genes were modulated by nickel treatment. The genes, for which expression was induced most markedly by nickel, encoded products that were putatively involved in a variety of biological functions, such as the modification of cellular components (53%), regulation of biological processes (27%) and molecular functions (20%). The 10 genes that pyrosequencing analysis indicated were induced the most by nickel were characterized further by qPCR analysis of both nickel-tolerant and nickel-sensitive P. albus isolates. Five of these genes were expressed exclusively in nickel-tolerant isolates as well as in ECM samples in situ, which identified them as potential biomarkers for nickel tolerance in this species. These results clearly suggest a positive transcriptomic response of the fungus to nickel-rich environments. The presence of both nickel-tolerant and nickel-sensitive fungal phenotypes in ultramafic soils might reflect environment-dependent phenotypic responses to variations in the effective concentrations of nickel in heterogeneous ultramafic habitats.