Ecole Doctorale

M Jean Garbaye

Mlle L Fraissinet-Tachet Maître

M Bernard Ollivier

Claude Mme

Plassard

M Marc Ducousso

M Antoine Galiana

Biodiversité et stratégies adaptatives des bactéries mycorhizosphériques associées à Tristaniopsis dans les écosystèmes ultramafiques

Keywords: Ultramafiques, Ectomycorhizes, Bacteries Mycorhizosphériques, Endémiques, Nickel, Diversité, Tristaniopsis, Ecosystèmes Ultramafic, Ectomycorrhizas, Mycorrhizosphere bacteria, Tristaniopsis, Nitolerance, MHB, New Caledonia

Biodiversity and adaptive strategies of mycorrhizosphere bacteria associated to Tristaniopsis spp. in New Caledonian ultramafic ecosystems

Table of Contents

GenBank accession numbers 200

Table S2. Supplementary data containing MB Isolates obtained from this study with

GenBank accession numbers

General Introduction

1

General Introduction

The continuous world development is the driving force for increasing demands of raw material and energy, resulting in the transformation of more than 40% of vegetated surface of our earth through anthropogenic activities such as grazing, agriculture, forestry, mining, urbanization and industrialization [START_REF] Daily | Restoring value to the world's degraded lands[END_REF]Lagrange 2009). To fulfil this demand, one of very important role has been played by mining activities; which essentially involves the increased access to mineral resources located in the lateritic weathering mantles of tropics, representing one of the major sources of metal on this planet [START_REF] Colin | Equatorial rain forest lateritic mantles: A geomembrane filter[END_REF]). As a result, these activities have affected considerable areas in some countries like USA, China, Australia, New Caledonia, South Africa, Canada, and Brazil. The mining industry has been considered to be involved in the creation of serious environmental hazards like abandoning of many rock dumps, pits, and other opening; which are not only against the clean and safe environment but also strongly disagree with concept of sustainable development. Sustainable development involves the rehabilitation and regulation of such ecosystems with the collaboration of specific rehabilitation companies, industries exploiting the resources, local area people and research [START_REF] Aronsona | Ecological restoration: A new frontier for nature conservation and economics[END_REF]. Therefore, these ecosystems are very interesting models to study in order to conserve their biodiversity, limiting the pollutions generated by exploited large surfaces and to increase our knowledge of complex ecological processes for the reconstruction of the original interface.

Overview

New Caledonia, a French overseas territory in the Southwest Pacific ocean, about 1,500 km east of Australia (Perrier et al. 2006), is an archipelago of around 19,000 km 2 , and consists of a large and small islands, the Loyalty and Huon groups (Fig. 1). New Caledonia is one of the smallest hotspots of biodiversity in the world (the size of New Jersey, Fig. 2) (Myers 2003).

The Main Island of New Caledonia is known as "La Grande Terre" which has rich mineral deposits of nickel, iron ore, manganese, chromium and cobalt. It is stretched from northwest to southeast on nearly 450km long and 40-70km wide (Lagrange 2009) and almost completely surrounded by a coral reef (Fig. 1). New Caledonia is divided into three provinces i.e. North

Province, South Province, and Islands Province. The central mountainous range "Mont Panié"

divides the island into two distinct regions: the "East Coast" exposed to trade winds, wetter, with dense vegetation along with steep slopes; and the "West Coast" downwind, drier,

showing broad plains suitable for growing and alternating with rich mining massive (Lagrange 2009).

The reserves of nickel are among the third largest in the world. Nickel mining activity constitutes the main economic resource of New Caledonia, and makes it the world's third largest producer of nickel. However, agriculture also contributes about 2% of the gross domestic product (GDP), although only 1% of the land is arable. More than one-seventh of the labour force is employed in agriculture to grow certain important crops such as coffee, copra, potatoes, vegetables and maize. Yam and taro are the Melanesians' staple foodstuffs.

Pastures occupy about 15 percent of the land, and cattle, pigs and goats are raised for domestic needs. Commercial fishing includes aquaculture like shrimps, oyster and trochus besides sea fishing. About three-quarters of the vegetation are unique to the main islands "La

Grande Terre" and to some extent, are drought resistant (FAO on-line statistics).

Soil

New Caledonia is characterized by the presence of ultramafic rocks, including peridotites and serpentines (Fig. 1). Serpentine (ultramafic) outcrops are distributed all over the world and,

for their natural geological origin, are characterized by presence of higher levels of heavy metals such as nickel, iron ore, manganese and chromium (Perrier et al. 2006;[START_REF] Sabrié | Mines et environnement[END_REF]Héry et al. 2003). The New Caledonian soils have been resulted from the weathering of peridotites under humid and hot temperature (L 'Huillier et al. 2010). In fact the ferrallitization of massive began on the peridotites rocks in the late Miocene (20MY) and it was quickly followed by leaching of silica and magnesium along with accumulation of iron, chromium and nickel (Latham 1974). These rocks make up one-third of the main island area, whereas they represent only 1% of the earth's surface-emerged lands (Brooks 1987;Héry et al. 2003).

These soils are deficient in essential elements like carbon, nitrogen, phosphorous, and have high Mg-Ca ratio, along with very high level of heavy metals, particularly nickel which is 250 times more to the average level of nickel in normal soils (Héry et al. 2003).

Climate

New Caledonia is located between 19°-22° S and 163°-168° E. The climate of east coast is tropical semi-humid and that of west coast is tropical dry. However, strong seasonal irregularities have been observed resulting year-to-year variation in rainfall patterns in New Caledonia. Overall, following different seasons can be recognized as (Lagrange 2009); a) Hot (tropical) season (mid-November to mid-April) along with frequent tropical depressions and cyclones producing large amounts of precipitation (2,000 mm at sea level, more than 5,000 mm at altitude). e) The mountain climate generally found in the valleys of Grande Terre with high rainfall (up to 6,000 mm), with frequently occurring mists and low temperatures (below 10°C) (FAO on-line statistics).

The annual average temperature of Noumea is 23.5 • C with varying monthly average temperature ranging from 28 • C (in February) to 20 • C (in August) (L 'Huillier et al. 2010).

Similarly in case of rainfall, this varies considerably from one part of country to other e.g. less than 1000 mm per annum for west coast to about 4,000 mm per year around the Mont Panié

(1,628 m altitude). However, the overall climate of New Caledonia is similar to those found on many other tropical islands but it has evolved highly diverse flora because of edaphic constraints and extremely selective nature of ultramafic soils which results in the higher level of plant endemism in this environment.

Plant endemism

Geographic location of New Caledonian ultramafic ecosystem, along with presence of high concentrations of heavy metals, and low fertility level of soil have encouraged the evolution of an ecosystem which is highly adapted to these extreme environmental conditions. It has been classified as "Hotspot of Biodiversity" (Myers et al. 2000) representing world's richest and unusual flora. It is also included in the list of world's few "Magadiverse Country" a concept developed by Mittermeier in 1988 which account for the country with tropical climate and world's highest biodiversity (Fig. 2). Due to high levels of metals along with low nutrients levels and scarcity or at least insufficiency of animal population involved in plant seed dispersal, these soils have evolved a specific and unique plant biodiversity; as only certain number of plant species, endemic to metalliferous soils could be capable of tolerating these exceptional concentrations of heavy metals, (Khan 2005;Khan et al. 2000) e.g. the vascular plants have been shown higher degree of endemism. It has been observed that New Caledonia contains about 3,250 different species of flowering plants out of which more than 74% (roughly 2,430 species) are endemic to this specific environment (Jaffré et al., 2001a). It includes 108 endemic genera, and a remarkable five endemic plant families: 6

Paracryphiaceae, Amborellaceae, Strasburgeriaceae, Phellinaceae and Oncothecaceae. Due to this extraordinary floristic endemism one may consider the New Caledonia flora as one of distinct subkingdom of flora such as ultramafic flora e.g. Koniambo massif. I would say that the Koniambo massif is itself a hotspot of biodiversity with in a hotspot which represents only 2% of New Caledonian's surface that contain 650 different plant species and account for almost 15% of known plant species of the archipelago (Perrier et al. 2006).

Mycorrhizal status of New Caledonian's plant species

It is the generally held view that the majority of plants growing under natural conditions have mycorrhizas (Pillon et al. 2010;Ramanankierana et al. 2007;Smith andRead 1997, 2008).

Symbiotic mycorrhizal fungi such as arbuscular mycorrhizal (AM) fungi or ectomycorrhizal 7 (ECM) fungi are ubiquitous component of most ecosystems throughout the world and form a key component of soil microbiota influencing plant growth and uptake of nutrients (Smith and Read 1997). Symbioses, (both arbuscular mycorrhizal and ectomycorrhizal) are known to play key roles in these processes in allowing the plant nutrition with major elements like N and P, in facilitating access and use of water, in stabilizing the soils through hyphal mats and possibly buffering metal toxicity. Mycorrhizas have also been reported in plants growing on heavy metal contaminated sites [START_REF] Shetty | Effects of mycorrhizae and fertilizer amendments on zinc tolerance of plants[END_REF][START_REF] Pawlowska | The mycorrhizal status of plants colonizing a calamine spoil mound in southern Poland[END_REF][START_REF] Chaudhry | Colonization of iron and zinc-contaminated dumped filtercake waste by microbes, plants and associated mycorrhizae[END_REF] describing that these fungi have evolved a heavy metals (HM) tolerance and that they may play a vital role in the phytoremediation of the contaminated site (Smith and Read 2008;Khan et al. 2000). Since heavy metal uptake and tolerance depend on both plant and soil factors including soil microbes (Khan et al. 2000), we require information on interactions between plant roots and their symbionts such as mycorrhizal fungi and their rhizosphere bacteria, known as mycorrhizopshere bacteria.

In addition to their known direct effect on plant growth, mycorrhizal symbionts could positively act on host plant development through a selective effect on bacterial communities involved in soil functioning and soil fertility because both abiotic and biotic stresses are considered to affect the zone of influence around plant roots called as rhizosphere, which serves as harbour for the multitude of microorganisms. The increase or loss of activity of mycorrhizal fungi has often been detected [START_REF] Bethlenfalvay | Arbuscular mycorrhizas and agrosystem stability[END_REF]. Hence, management of soil mycorrhizal potential is of great importance since mycorrhizal symbiosis determines plant biodiversity, ecosystem variability and productivity directly from its influence on plant mineral nutrition, but also indirectly from its impact on soil microbial functioning (Duponnois et al. 2008).

In this study, we have chosen the plant from Tristaniopsis spp., which belongs to Myrtaceae family. The Myrtaceae is a family of at least 133 genera and > 3,800 species. It has centres of diversity in Australia, Southeast Asia, and tropical to southern temperate America, but has little representation in Africa (Wilson et al. 2001). Recently Perrier and his colleagues (2006) has identified the existence of ectomycorrhizal (ECM) symbioses with Tristaniopsis spp. in the New Caledonian heavy metal enriched soils and suggested that mycorrhizal symbioses could play an important role in the adaptation of these endemic plants to these extreme soils.

We selected Tristaniopsis spp. (Myrtaceae) as a model plants for our study because of: a) Their growing ability on both metalliferous (ultramafic) and nonmetalliferous soils (volcano-sedimentary) and b) Their taxonomic proximity close to ECM model plant i.e. Eucalyptus

In this study, we have studied and carried out the sampling of ectomycorrizal root tips from rhizosphere of five different Tristaniopsis species, which includes Tristaniopsis calobuxus, T. glauca, T. macphersonii, T. guillainii, T. ninndoensis. This last species is endemic to Mont Ninndo while T. calobuxus can be found in both south and north provinces of New Caledonia.

Mycorrhizosphere bacteria

The mycorrhizosphere named the zone influenced by both the plant root and the mycorrhizal fungus; and soil bacteria found there are termed as mycorrhizosphere bacteria (Sanon et al. 2005;Duponnois and Kisa 2006;Garbaye 1994). It is well documented that mycorrhizal symbioses could strongly influenced by the mycorrhizosphere bacteria because of their intraspecific relationship in the mycorrhizosphere (Garbaye 1994). Moreover, there is increasing evidence that rhizosphere bacteria contribute to the metal extraction process, but the mechanisms of this plant-microbe interaction are not yet fully understood [START_REF] Kuffner | Rhizosphere bacteria affect growth and metal uptake of heavy metal accumulating willows[END_REF]. It has been demonstrated that some soil bacteria, could enhance the development of the mycorrhizal symbiosis, which have been named as MHB i.e. Mycorrhiza Helper Bacteria (Garbaye 1994). This bacterial-growth-promoting effect has been shown with different host plants, including herbaceous species [START_REF] Meyer | Selective influence on populations of rhizosphere or rhizoplane bacteria and actinomycetes by mycorrhizas formed by Glomus fasciculatum[END_REF][START_REF] Paula | Synergistic effects of vesiculararbuscular mycorrhizal fungi and diazotrophic bacteria on nutrition and growth of sweet potato[END_REF]Requena et al. 1997) and tree species (Duponnois et al. 1993;Dunstan et al. 1998). The MHB strains that have been identified to date belong to many bacterial groups and genera, such as gramnegative Proteobacteria (Agrobacterium, Azospirillum, Azotobacter, Burkholderia, Bradyrhizobium, Enterobacter, Pseudomonas, Klebsiella and Rhizobium), gram-positive Firmicutes (Bacillus, Brevibacillus and Paenibacillus) and gram-positive Actinomycetes (Rhodococcus, Streptomyces and Arthrobacter) (Frey-Klett et al. 2007).

This study was carried out by keeping in mind that the mycorrhizosphere bacteria (MB) living in the New Caledonian's ultramafic soils could represent an interesting model for the evolution of metal-resistant communities, (a) because they are in contact with one another since millions of years, (b) completely different from that of artificially contaminated soils since these soils are characterized by high levels of naturally occurring metal such as nickel, iron, manganese, cobalt and chromium with low levels of essential nutrients along with a high Mg/Ca ratio (Brooks 1987). Therefore, these serpentine (or ultramafic) soils could provide new strains and new genetic determinants for heavy metal resistance or tolerance that could be exploited in bioremediation practices (Mengoni et al. 2001) of contaminated soils.

Moreover, this study will give us a deep insight into the relationships between mycorrhizosphere bacteria (MB) and ECM associated to the rhizosphere of the Tristaniopsis spp., which can grow on both soils types i.e. ultramafic and volcano-sedimentary in these extreme environmental conditions.

Statement of problem and objectives of study

Although Ni is an essential metal for proper functioning of several microbial enzymes, such as CO dehydrogenase, urease, methyl-coenzyme M-reductase, and hydrogenase (Hausinger 1987). However it is toxic even at low concentrations and can change the microbial community structure (Sandaa et al. 1999). The beneficial effects of ectomycorrhizal symbioses (ECM) and rhizosphere bacteria in nutrient poor environments are well known [START_REF] Vecchio | Negative effects of scale insect herbivory on the ectomycorrhizae of Juvenile pinyon pine[END_REF]Leyval et al., 1997;Smith and Read, 1997;Glick 2003;Zhuang et al., 2007;Mayak et al., 2004;Jourand et al., 2010b). ECM improves the plant nutrition through increased nutrient uptake and exploiting the large nutrient pools that would be unavailable to plant (without mycorrhizas). Further advantages include increase water uptake, carbon transfer between the plants and resistance to pathogen (Smith and Read, 1997). They can also provide protection to the plants from toxic elements such as heavy metals. This protection mechanism varies with the type of the metal pollutants present in the soil and on the relationship of host-fungus. This particular characteristic or relationship is of keen importance in order to establish a vegetative cover on metal polluted sites such as New Caledonian ultramafic soils. Similarly, rhizosphere bacteria are most abundant and diverse in soil and play a major role in improving the plant nutrition (through N-fixation and release of P), transformation of organic matter, improvement of the soil fertility, plant productivity [START_REF] Dey | Growth promotion and yield enhancement of peanut (Arachis hypogaea L.) by application of plant growth-promoting rhizobacteria[END_REF]) and resistance to plant pathogen. They can increase or reduce transfer or absorption of nutrients and certain metal ions . They have different mechanisms of heavy metal tolerance and detoxification [START_REF] Gadd | Microorganisms and heavy metal toxicity[END_REF] and still by the production of chelating agents (Nowack, 2008) that bound metals and reduce their phytotoxicity.

In recent years, many researchers have focused their attention on nickel resistant bacteria communities of ultramafic soils in Italy (Mengoni et al. 2001), India (Pal et al. 2004, 2007) and New Caledonia [START_REF] Saintpierre | Streptomyces yatensis sp. nov., a novel bioactive streptomycete isolated from a New-Caledonian ultramafic soil[END_REF]Héry et al. 2004;Stoppel and Schlegel 1995).

Therefore, New Caledonian soils represent an interesting model for studying the adaptation of mycorrhizosphere bacterial communities to heavy metals, especially the nickel, on long term because of the age of their inter-relationship. Thus we can assume that certain new metal resistant bacterial strains could possibly be isolated and could be characterised from these extreme environment. New Caledonia is also a different case of study in term of type of pollution e.g. most of sites that has been studied involve the study of contaminated or artificially polluted site / soils but here, in New Caledonia, it is natural ecosystem due to specific geologic origin of these soils. In order to evaluate and understand the capacity and level of adaptation of New Caledonian microbial communities, which are associated to Tristaniopsis spp. to these naturally, polluted soils, this study was carried out by exogenous application of Nickel as NiCl 2, and was compared with the MB communities associated to Tristaniopsis spp. from volcano-sedimentary soils of New Caledonia.

Indeed, the New Caledonian soil bacteria, naturally in contact with high levels of toxic metals, have developed specific adaptations to protect themselves from their toxic effects. Some of them are very resistant to nickel and new genetic determinants of resistance were also identified (Schlegel et al. 1991;Stoppel and Schlegel 1995). The symbiotic association could therefore be beneficial for Tristaniopsis spp. if both ECM and MB also have specific capabilities to tolerate high levels of heavy metals, such as nickel in our case. Thus, there could possibly be a synergistic effect of MB and ECM symbioses regarding plant nutrition and resistance to nickel. Therefore, MB-ECM symbiosis could be the one of the most effective process to increase heavy-metal / Ni tolerance and re-vegetation of the Tristaniopsis spp. in ultramafic soils of New Caledonia.

This study which involves the characterization of microbial (ECM and MB) communities associated to Tristaniopsis spp. will not only increase our knowledge about the effect of ultramafism of these soils on concerned microbial communities but also help us to improve our understanding of microbial biodiversity in the rhizosphere of Tristaniopsis spp. Because it is generally held view that the microbes are more sensitive to the heavy metals and heavy metals could alter their activities, modify their physiology and affect their diversity as well (Héry 2004). Thus, this study hypothesizes that ultramafic soils could not only affect the ECM-MB diversity but also select efficient genetic determinants of resistance/adaptation in MB associated to Tristaniopsis spp.

Thus the present study will be carried out with the following objectives:

1) Description of the diversity and specificity of ectomycorrhizal fungi (ECM) associated to various Tristaniopsis spp. in ultramafic vs volcano-sedimentary soils;

2) Characterization of mycorrhizosphere bacterial populations associated to these ECM in the same soil types;

3) Highlighting the role of genetic determinants of Ni-resistance in MB populations of ultramafic soils through analysis of their Ni-tolerance ability and the presence of Ni resistance genes, in comparison with MB from non-ultramafic soils 13 4) Investigating the effect of MB strains on fungal growth of a reference Pisolithus albus strain through in vitro co-culture tests, using selected MB strains isolated from New Caledonia, to assess their Mycorrhiza Helper Bacteria potential.

This study mainly involves the investigation of a soil type's effect on MB-ECM biodiversity and the adaptation of the isolated MB to this extreme environment. This manuscript is divided into three chapters:

Publications and communications

The

Introduction

Approximately 450,000 to 1 million sites in USA and about 750,000 sites in Europe are considered or suspected to be polluted with a diverse group of pollutants such as fuel hydrocarbons, polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), chlorinated aromatic hydrocarbons (CAHs), detergents, and pesticides. On the other hand, the main inorganic pollutants mainly comprise the heavy metal like nickel, cadmium, lead and mercury, nitrate and phosphate, and radio nucleotides [START_REF] Bücking | Ectomycoremediation: An eco-friendly technique for the remediation of polluted sites[END_REF][START_REF] De-Sousa | Contaminated sites: the canadian situation in an international context[END_REF]. Typical techniques used for the decontamination or remediations of polluted sites involve: Some studies [START_REF] Kuiper | Rhizoremediation: A beneficial plant-microbe interaction[END_REF][START_REF] Bücking | Ectomycoremediation: An eco-friendly technique for the remediation of polluted sites[END_REF] suggested that these techniques are not always as efficient as expected, very costly, very invasive and sometimes could be a cause of possible release of pollutants into air or ground water through leaching.

Phytoremediation is another cost-effective and sustainable technique for remediation of contaminated or/ polluted sites [START_REF] Bücking | Ectomycoremediation: An eco-friendly technique for the remediation of polluted sites[END_REF][START_REF] Cunningham | Phytoremediation of contaminated water and soil. Paper presented at the Phytoremediation of soil and water contaminants Washington DC[END_REF]. It has several advantages over conventional techniques such as cheaper: about 10-50% of cost of conventional techniques, normally a useful by-product in the form of wood, pulp or bio-energy is produced, non-invasive and is more environment friendly (Bucking 2011). The remediation of contaminated sites through phytoremediation, that is well documented [START_REF] Tsao | Overview of Phytotechnologies[END_REF], generally involves the re-vegetation or plantation of fast-growing plant species due to their deep root system and high biomass production. Most of tree species used in phytoremediation are believed to live naturally in symbiosis either arbuscular mycorrhizal (AM) fungi (Smith and Reads 2008) or ectomycorrhizal (ECM) fungi. Symbiotic mycorrhizal fungi are ubiquitous component of most ecosystems throughout the world and form a key component of soil micro-biota influencing plant growth and nutrients uptake as shown in Fig 2 .1 [START_REF] Finlay | Integrated nutrient cycles in boreal forest ecosystemsthe role of mycorrhizal fungi[END_REF][START_REF] Bethlenfalvay | Mycorrhizae and crop productivity[END_REF]) e.g.

poplar forms ECM interaction with more than 60 different fungal species. In addition to that, in some cases, about 60% of plant root system is colonized by ECM fungi which can increase its seedling growth up to 400% [START_REF] Cripps | Native mycorrhizal fungi with aspen on smelter-impacted sites in the Northern Rocky Mountains: occurrence and potential use in reclamation[END_REF]. However, ECM contributions to remediation of polluted soils have often been ignored and not fully understood despite its well documented and widely acknowledged importance for plant growth and its microbial community structuring ability in different environments. In our study, we have selected Tristaniopsis spp. as model plant, an ectomycorrhizal tree or shrub genus, to study the adaptation of microbial communities, more particularly ECM and MB, to ultramafic soils.

This part of dissertation is further divided into three parts; first part deals with the geology of New Caledonian soils, second one describes the adaptation of ECM fungi to toxic and/or polluted soils while the third and last section emphasizes on the adaptation of mycorrhizosphere bacteria to these extreme soils. (3) or through dissolution/weathering induced by roots, (4,5) mineralization, (6) Nutrient exchange through symbiotic mycorrhizal hyphae, (7) carbon allocation, (8) transfer of inorganic nutrients from soil solution via hyphae, or via weathering (9), or by direct mobilization of organic nutrients through decomposition (10), and interactions with saprotrophic fungi (11), soil fauna (12) and bacteria ( 13), The filamentous nature of the mycorrhizal mycelium permits interchange of carbon and nutrients between different substrates ( 14) and allocation of photoassimilatederived compounds to microsites within inorganic (15) and organic (16) substrates, (17) C-removal through respiration, (18) mycorrhizal fungi, and other soil organisms (19), may be accompanied by transfer of carbon from mycorrhizal hosts via mycelial connections to mycoheterotrophic hosts (20). Transfer of water via hydraulic lift from roots to mycorrhizal fungi (21) may help to maintain mycelial integrity in dry soil and to condition the environment round hyphal tips, allowing more stable conditions for microbial interactions at this interface [START_REF] Finlay | Integrated nutrient cycles in boreal forest ecosystemsthe role of mycorrhizal fungi[END_REF]).

New Caledonian Soils

Origin

New Caledonia is located, northwest to southeast, roughly between latitudes 20°-23° S and 164°-167° E, with an area of about 16,890 km 2 (Héry 2004;Perrier et al. 2006). The island is 1,220 km from Australia and approximately 400 km from the islands of Vanuatu. It is an area, with an elongate shape 450km long and 40-70km wide and almost completely surrounded by a coral reef known as "Grande Terre". Mountain ranges are complex and dissected by many rivers, with tablelands and peaks reaching elevations of more than 1600 m (Fig. 2.2). Most of the island is covered by wet evergreen forests with anthropogenic savannahs dominating at low elevations. A few small fragments of sclerophyll dry forest remain on the western coast and shrubby vegetation ('maquis minier') occurs on metalliferous soils, mostly in the south of "Grande Terre" and smaller islands extending northward and southward are emerging parts of a folded arc (Fig. 2.3), east to this are Loyalty Islands.

Grande Terre arc mainly composed of sedimentary, volcanic and ultrabasic rocks from Permian (225-280 million years) to Tertiary (1.5 -65 MY) (Héry 2004). The ultrabasic rocks, which are one of important group of Tertiary age, relate to a mantle sheet; a thick layer located under the earth crust and above its core. A large West Coastal area of volcanic rocks is associated to ultrabasic rocks. They are residues of an oceanic floor obducted before the overthrust of ultrabasics rocks, occurred during Eocene to Oligocene (26-53 MY) and created the actual island due to the collision of plates (Cluzel et al. 2001). Temperatures and pressures generated due to these plate collisions, led to a transformation of sediments and volcanic rocks (Héry 2004;Web1). A large sheet of mantle was upraised and over-thrust above other terrains, generating the Western slope Eastern slope last New Caledonian folding, which is further divided into several massifs by diverse abiotic factors such as erosion and temperature (Héry 2004).

Several models have been proposed and debated from more than 30 years in order to address the origin of the New Caledonian ophiolites (consisting in the succession of rocks produced from the melting and cooling, from bottom to top, i.e. peridotite (modified mantle), gabbro, and basalt, as described by [START_REF] Rajakaruna | Serpentine geoecology of eastern north America: a review[END_REF][START_REF] Orstom | Effects of ectomycorrhizal and vesicular-arbuscular mycorrhizal fungi on drought tolerance of four leguminous woody seedlings[END_REF]) and suggested that it was resulted from the middle to late Eocene age from the obduction of the ophiolites, in response to roughly northsouth shortening between the Pacific and Australian Plates (DeMets et al. 1994). However, the age of Loyalty Basin, the geometry of the subduction and obduction, remain controversial.

Relief and nature of bedrock

The Grande Terre is the largest island of the New Caledonia and represents three major types of sedimentary rocks.

Siliceous rocks

Basic, volcanic or sedimentary and calcareous rocks

Ultramafic rocks

Siliceous rocks

As indicated by its name, these rocks are rich in silica and relatively poor in ferromagnesian minerals. It includes diverse set of sedimentary rocks (such as graphite, sandstone, shale and pelites), igneous rocks (granites and rhyolites) and metamorphous rocks (sericite schist, mica and gneiss). These rocks are often quite rich in potash, because of the presence of mica as their constituent or feldspath but they are poor in calcium and phosphorus (L 'Huillier et al. 2010;[START_REF] Becquer | Mobilité du nickel dans les sols ferrallitiques ferritiques du Sud de la Nouvelle-Calédonie[END_REF].

The soils are mostly fersiallitic or ferrallitic according to their climatic regime, however, on most siliceous rocks (siliceous aphanites and alluvium), we observe directly development of podzols.

On the Western Slope, soils are fersiallitic (clay and iron) with strong leaching.

Basic rocks volcanic or sedimentary and calcareous rocks

These rocks are dominated by ferromagnesian minerals and alkaline earth cations. We can classify them as calcareous sedimentary rocks, basic igneous rocks (basaltic tuffs, gabbro), and some as metamorphic rocks especially glaucophanites. These soils are rich in calcium, but very poor in potassium and phosphorus. These clays restrict drainage and development of weathering.

In wet weather (on the east coast), montmorillonite is rapidly transformed to kaolinite which strongly predominates in the upper profile. In drier conditions, calcium released by weathering is rapidly remobilized to form calcareous crusts on the lower slopes and the foothills (Lagrange 2009; [START_REF] Brooks | Some New Zealand and New Caledonian plant accumulators of nickel[END_REF][START_REF] Jaffré | Composition chimique et conditions de l'alimentation minérale des plantes sur roches ultrabasiques (Nouvelle-Calédonie)[END_REF]).

Ultramafic rocks

Magnesium is the dominant element in these rocks, with high contents in silicon and iron and very little aluminum. These are mainly peridotites (dunite and harzburgite) and serpentines. They are very poor in potassium, calcium and phosphorus but they contain relatively high quantities of nickel, chromium and cobalt. During weathering, there is no formation of kaolinite, due to very low levels of aluminum in the rock. There is formation of iron-bearing smectites in the driest areas while it contains oxides and hydroxides of iron in wettest areas (eastern slope) (Latham 1986;[START_REF] Latham | Etude des sols de la Nouvelle-Calédonie: Nouvel essai sur la classification, la caractérisation, la pédogenèse et les aptitudes des sols de NouvelleCalédonie[END_REF]Lagrange 2009).

The soils originated from these ultramafic rocks are of two types:

I. Brown eutrophic soils: mainly composed of iron-bearing smectites in areas rejuvenated by erosion and subjected to a dry climate, vertisols and brown magnesium soils in the plains of the West Coast.

II.

Lateritic soil: Lateritic ferrallitic soil comprising the areas regularly subjected to humid or wet climate along the east coast.

Formations and composition of lateritic ultrabasic massif

The peridotites (mainly the harzburgites and dunites) and serpentines are composed of silicates of ferromagnesian and are characterized by concentration of silica less than 45%, very little Al, Ca, and K while very high quantities of Mg (40%), Fe (6-8%), as well certain metal such as Ni (0.3%), Cr, Co and Mn which are generally trace element in normal soils (L 'Huillier et al., 2010). The soil analyses available to date have characterized the worldwide serpentine soils as soils having extremely high concentrations in certain heavy metals such as Ni, near-neutral pH values and high Mg:Ca ratio [START_REF] Rajakaruna | Serpentine geoecology of eastern north America: a review[END_REF].

A typical soil profile of ultramafic soils is shown in Fig. 2.4. It represents an undisturbed upper horizon, friable or fragile gritty texture, depleted in fines particles. It consists of well crystallized iron oxides (hematite) and oxy-hydroxides (goethite). Because of its color, this horizon is called "red laterite" which gradually changes into "yellow laterites" in the lower horizon. The lateral, clay-silty texture consists of very fine or poorly crystallizes clusters of iron oxydes and hydrooxydes (mainly goethite) (Héry 2004;L'Huillier et al. 2010).

Beneath the yellow laterites, saprolites are located partially decomposed bed-rock called peridotites. The main constituents are oxide and hydro-oxides of iron which are very fine clusters of goethite with poor crystallization. It also contains small quantities of Mg and Fe. On average, pH of these soils is acidic in upper horizon and around neutral as we move down towards the base of profile. Due to the presence of these ultramafic rocks, the plants on this environment are extremely specific and exhibit high degree of endemism (Khan et al. 2000;Brooks 1987). Although it represents only about 1% of submerged surface of earth crust (Brooks 1987), ultramafic rocks occupy an area of around 5500 km 2 in New Caledonia, which constitute almost 30% of its total area and about one third of the "Grande Terre" (Latham 1986;L'Huillier et al. 2010).

Occurrence of mycorrhizal fungi and their effects on plant growth

Generalities

In 1879, Giuseppe Gibelli discovered mycorrhizal symbiosis while he was working at the Botanical Institute of the University of Torino. Albert Bernard Frank used the Greek term 'mycorrhiza' which literally means 'fungus roots' for very first time in 1885 [START_REF] Frank | On the nutritional dependence of certain trees on root symbiosis with belowground fungi (an English translation of A.B. Frank's classic paper of 1885)[END_REF]) during his study on soil microbial-plant relationships. Since then, mycorrhizas have been extensively

studied, characterized and proved as beneficial for plant nutrition and health. Mycorrhizas thus play a key role in the growth and productivity of vascular plants in all types of ecosystems around the world since most terrestrial plants have symbiotic relationships with mycorrhizal fungi, through the formation of specialized and mixed structures between these fungi and plant roots called mycorrhizae (Garbaye 1994;Smith and Read 1997).

Mycorrhizas are an integral part of most plants in nature and occur on 83% of dicotyledonous and 79% of monocotyledonous plant investigated [START_REF] Wilcox | Mycorrhizae[END_REF]. All gymnosperms are reported as being mycorrhizal [START_REF] Newman | The distribution of mycorrhizas among families of vascular plants[END_REF]. Infection of the root system of the plant by these fungi creates a symbiotic (beneficial) relationship between plant and fungus. After root colonization and infection, mycorrhizal fungi develop an external mycelium which is a bridge connecting the root with the surrounding soil (Requena et al. 1997). One of the most significant effects of infection by mycorrhizal fungi on the host plant is the increase in nutrient uptake [START_REF] Koide | Nutrient supply, nutrient demand and plant response to mycorrhizal infection[END_REF]) mainly due to the capacity of the mycorrhizal fungi to absorb nutrients from soil and transfer it to the host roots [START_REF] Asimi | Influence of increasing soil phosphorus levels on interactions between vesicular-arbuscular mycorrhizae and Rhizobium in soybeans[END_REF]. In return, the plants provide the fungus with photosynthetic fixed carbon for its proper physiological functioning (Smith and Read 2008).

Mycorrhizas can be classified into two primary types: arbuscular mycorrhizas (AM formerly named as endomycorrhizas) and ectomycorrhizas, both being the most abundant and widespread as shown in table 2.1 [START_REF] Allen | Ecology of mycorrhizae: A conceptual framework for complex interactions among plants and fungi[END_REF]Smith and Read 1997). Other minor types of mycorrhizae have been classified as ectendo-, arbutoid, monotropoid, ericoid and orchidaceous mycorrhizas . 2.1) which are the main sites of nutrient transfer between the two symbiotic partners (Smith and Read 1997). In fact, the term endomycorrhizae includes a wide variety of shapes and partners (Table 2.1). AM fungi colonize plant roots and penetrate into surrounding soil, extending beyond the root depletion zone and improving water absorption and nutrient uptake through the increase of root surface. However, majority of plant families are symbiotically associated with arbuscular mycorrhizae as compared to ectomycorrhizae. On the other hand, ectomycorrhizas, which are formed by a large number of fungal species belonging mainly to Basidiomycetes but also Ascomycetes, are found on ligneous plant species in most of temperate countries and in many tropical ecosystems. Spores produced by the sporocarps of ectomycorrhizal fungi germinate and colonize the lateral roots which appear as fine and short roots. The fungus forms a mantle which encloses the root. Mycelial hyphae extend to the surrounding soil while others penetrate the epidermal and superficial layers of cortical cells to form the Hartig net, the site for nutritional exchange between the fungal and plant cells [START_REF] López | Increased trehalose biosynthesis in Hartig net hyphae of ectomycorrhizas[END_REF]Smith and Read 1997).

Currently as many as 5000-6000 species of fungi are known to be ectomycorrhizal [START_REF] Bentivenga | Ecology and evolution of arbuscular mycorrhizae[END_REF]. Ectomycorrhizal fungi establish the symbiotic relationship with the roots of certain types of forest trees which includes the members of several plant families such as the Betulaceae, Fagaceae, Pinaceae, Dipterocarpaceae and Myrtaceae [START_REF] Abler | Trace metal effects on ectomycorrhizal growth, diversity, and colonization of host seedlings[END_REF]Smith and Read 2008;Perrier et al. 2006). Basically it is a "give and take" relationship which has been existed between fungus and plant for millions of years, in which the fungus provides mineral nutrients, most importantly nitrogen and phosphorus, to the host plant, and improves plant water uptake in dry environments. In return, the plant provides the fungal symbiont with vital carbohydrates such as glucose and important vitamins such as biotin and thiamine [START_REF] Abler | Trace metal effects on ectomycorrhizal growth, diversity, and colonization of host seedlings[END_REF]). However, in some cases, this interaction could be antagonistic as well. Smith and Read (1997) suggested that the ectomycorrhizal fungi can affect plant growth by increasing its overall biomass production, extra development of rootlets, and improved nutrition through higher nutrients (P and N) uptake compared to non-inoculated control plants.

It is well documented that the ectomycorrhizal symbioses play an important role in improving the successful growth and health of plants especially in nutrient poor environments [START_REF] Vecchio | Negative effects of scale insect herbivory on the ectomycorrhizae of Juvenile pinyon pine[END_REF][START_REF] Tam | Heavy metal tolerance by ectomycorrhizal fungi and metal amelioration by Pisolithus tinctorius[END_REF][START_REF] Howe | Copper-binding proteins in ectomycorrhizal fungi[END_REF][START_REF] Abler | Trace metal effects on ectomycorrhizal growth, diversity, and colonization of host seedlings[END_REF]Smith and Read 1997;[START_REF] López | Increased trehalose biosynthesis in Hartig net hyphae of ectomycorrhizas[END_REF]Duponnois et al. 2008). In addition, plants displaying ECMs also exhibit increased tolerance to pathogens [START_REF] Marx | Ectomycorrhizae as biological deterrents to pathogenic root infections[END_REF] or drought [START_REF] Alvarez | Effect of ectomycorrhizal colonization and drought on reactive oxygen species metabolism of Nothofagus dombeyi roots[END_REF][START_REF] Davies | Non-nutritional stress acclimation of mycorrhizal woody plants exposed to drought[END_REF][START_REF] Orstom | Effects of ectomycorrhizal and vesicular-arbuscular mycorrhizal fungi on drought tolerance of four leguminous woody seedlings[END_REF] and increased carbon transfer between plants [START_REF] Simard | Net transfer of carbon between ectomycorrhizal tree species in the field[END_REF]. Moreover, the ectomycorrhizal fungi are considered to protect the plants against toxic effects of heavy metals (Leyval et al. 1997a;Smith and Read 2008;Jourand et al. 2010;[START_REF] Howe | Copper-binding proteins in ectomycorrhizal fungi[END_REF][START_REF] Tam | Heavy metal tolerance by ectomycorrhizal fungi and metal amelioration by Pisolithus tinctorius[END_REF].

This particular interaction is of great importance not only for re-vegetation of mines spoils but also to re-establish a vegetative cover on naturally occurring heavy metals rich soils of New Caledonia. Although the mechanism and extent of protection varies with the type of host-fungus system as well as the metal pollutants present in the soil. In this study we will try to identify and characterize the different ECM fungi which are associated to Tristaniopsis spp. (Fig. 2.5 and 2.6) from the New Caledonian soils and that could be used in the bioremediation and re-vegetation of nickel rich mine spoils of New Caledonia. In this regard, we will discuss first different examples from literature of bioremediation or detoxification of different pollutants by using ECM.

Role of ectomycorrhizas in the adaptation and tolerance of plant species to polluted and toxic environments

The mycorrhizosphere represents in most soils an even larger surface for the establishment of microbial communities than the rhizosphere. The mycorrhizosphere can be defined as "the zone of soil where the physical, chemical and microbiological processes are influenced by plant roots and their associated mycorrhizal fungi" (Duponnois et al. 2008;[START_REF] Giri | Mycorrhizosphere: strategies and functions[END_REF]. Indeed the extra-radical mycelium of ECM acts as an extension of the plant root system and increase the surface area between hyphae and soil for the absorption and exchange of nutrients between plant and fungus e.g. the extra-radical mycelium of Pisolithus tinctorius represents 99% of the nutrient-absorbing surface length of pine roots [START_REF] Rousseau | Contribution of ectomycorrhiza to the potential nutrient-absorbing surface of pine[END_REF].

ECM fungi were able to grow by using complex organic molecules as a source for reduced carbon [START_REF] Durall | Allocation of 14C-Carbon in Ectomycorrhizal Willow[END_REF][START_REF] Tinker | Carbon Use Efficiency in Mycorrhizas: Theory and Sample Calculations[END_REF] or other nutrients such as nitrogen or phosphate [START_REF] Perez-Moreno | Mobilization and transfer of nutrients from litter to tree seedlings via the vegetative mycelium of ectomycorrhizal plants[END_REF]. Similarly, ECM fungi also produced a variety of lignin-lytic or cell wall-degrading enzymes [START_REF] Read | Mycorrhizas and nutrient cycling in ecosystems -a journey towards relevance?[END_REF] that could also enable them to degrade certain organic pollutants with structural similarities to lignin, such as PAHs, PCBs, 2,4,6-trinitrotoluene (TNT), or DDT [START_REF] Bücking | Ectomycoremediation: An eco-friendly technique for the remediation of polluted sites[END_REF]. Due to these enzymatic activities, which enable ECM fungi to successfully degrade organic pollutants in the soil, ECM fungi may be favored to be used in the bioremediation of soils contaminated with hydrocarbons or other organic pollutants.

Hydrocarbon pollution

It has been reported that plants can successfully be used for the phytoremediation or dendroremediation of soils contaminated with a variety of pollutants, such as Fuel hydrocarbons, polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), chlorinated aromatic hydrocarbons (CAHs), detergents, and pesticides which are the most dominant organic pollutants in soils [START_REF] Wang | Phytoremediation of petroleum polluted soil[END_REF][START_REF] Gunderson | Impact of ectomycorrhizal colonization of hybrid poplar on the remediation of diesel-contaminated soil[END_REF][START_REF] Kuiper | Rhizoremediation: A beneficial plant-microbe interaction[END_REF][START_REF] Tsao | Overview of Phytotechnologies[END_REF][START_REF] Joner | Rhizosphere gradients of polycyclic aromatic hydrocarbon (PAH) dissipation in two industrial soils and the impact of arbuscular mycorrhiza[END_REF][START_REF] Cunningham | Phytoremediation of contaminated water and soil. Paper presented at the Phytoremediation of soil and water contaminants Washington DC[END_REF][START_REF] Cunningham | Phytoremediation of contaminated water and soil. Paper presented at the Phytoremediation of soil and water contaminants Washington DC[END_REF]). The rhizosphere is often considered as the main contributor in the bioremediation by controlling and altering the microbial activities in the vicinity of roots. For example, three to four times more petroleum hydrocarbons (PHC) were degraded in the rhizosphere as compared to unplanted soils [START_REF] Wang | Phytoremediation of petroleum polluted soil[END_REF]) and same effect has been observed for other pollutants [START_REF] Gunderson | Impact of ectomycorrhizal colonization of hybrid poplar on the remediation of diesel-contaminated soil[END_REF]).

However the mechanisms of this degradation in the rhizosphere are very complicated and remain poorly understood (Bucking 2011). Root exudates which are low molecular weight carbon compounds such as sugars, sugar alcohols, amino acids, fatty acids, and proteins represent a significant carbon source (10-20% of photo-synthetically fixed carbon) and promote microbial proliferation and diversity in the rhizosphere (Duponnois et al. 2008). They are also considered to shape the microbial community composition and its capabilities to degrade the recalcitrant compounds.

Petroleum Hydrocarbon

Petroleum hydrocarbons (PHCs) are mainly composed of various aliphatic, alicyclic, and aromatic compounds and such as paraffins, aromatics, and naphthenics in different proportions.

It is very difficult to assess the toxicity of PHCs because of varying chemical composition and lack or limited knowledge about additive, synergistic, or antagonistic effects of the various compounds [START_REF] Bücking | Ectomycoremediation: An eco-friendly technique for the remediation of polluted sites[END_REF][START_REF] Robertson | Petroleum hydrocarbon contamination in boreal forest soils: a mycorrhizal ecosystems perspective[END_REF]) which make it hard to do the remediation of soils contaminated with such pollutants. [START_REF] Nicolotti | Soil contamination by crude oil: impact on the mycorrhizosphere and on the revegetation potential of forest trees[END_REF] reported that for the crude oil, a concentration up to 50 g.kg -1 did not affect the growth of ECM fungal colonization of Populus and only slightly reduced the ECM colonization of spruce after a longer exposure. Similar results over ECM colonization of Populus were observed by [START_REF] Gunderson | Impact of ectomycorrhizal colonization of hybrid poplar on the remediation of diesel-contaminated soil[END_REF] for the diesel oil pollution. Moreover, ECM fungi increase the plant resistance against PHCs although the PHCs can alter the ECM community composition [START_REF] Nicolotti | Soil contamination by crude oil: impact on the mycorrhizosphere and on the revegetation potential of forest trees[END_REF]. Other studies [START_REF] Sarand | Microbial biofilms and catabolic plasmid harbouring degradative fluorescent pseudomonads in Scots pine mycorrhizospheres developed on petroleum contaminated soil[END_REF][START_REF] Heinonsalo | Effects of Pinus sylvestris root growth and mycorrhizosphere development on bacterial carbon source utilization and hydrocarbon oxidation in forest and petroleum-contaminated soils[END_REF][START_REF] Bücking | The fungal sheath of ectomycorrhizal pine roots: an apoplastic barrier for the entry of calcium, magnesium, and potassium into the root cortex?[END_REF][START_REF] Gunderson | Impact of ectomycorrhizal colonization of hybrid poplar on the remediation of diesel-contaminated soil[END_REF]) also suggested the positive effect of ECM colonization on plant resistance against various PHCs pollutants. [START_REF] Gunderson | Impact of ectomycorrhizal colonization of hybrid poplar on the remediation of diesel-contaminated soil[END_REF] reported the reduce uptake of PHCs to the host plant. He showed that three times more PHC was accumulated in mycorrhizal roots which suggested that PHC can be adsorbed by hydrophobins in the fungal sheath. Hydrophobins are fungal cell wall proteins that are involved in cell to cell-surface contact in fungal mycelia and can reduce the apoplastic permeability of the fungal sheath (Bucking et al. 2002;Bucking 2011), and thereby could also reduce the bioavailability of PHCs for the mycorrhizal host plant. [START_REF] Sarand | Microbial biofilms and catabolic plasmid harbouring degradative fluorescent pseudomonads in Scots pine mycorrhizospheres developed on petroleum contaminated soil[END_REF] reported that ECM fungi Suillus bovinus or Paxillus involutus were able to grow in forest humus containing microcosms contaminated with petroleum hydrocarbon (PHC) contaminated soil.

Transmission electron microscopy (TEM) revealed a microbial biofilm at the PHC soil-fungal interface (mycorrhizosphere) composed of morphologically diverse bacterial populations which were able to utilize the organic pollutants such as m-toluate and m-xylene as sole carbon source.

They also expressed marker genes that are involved in the degradation of mono-aromatics e.g.

BTEX (benzene, toluene, ethylbenzene, and o-, m-and p-xylenes), and low molecular mass polycyclic aromatic hydrocarbons (PAHs) [START_REF] Sarand | Microbial biofilms and catabolic plasmid harbouring degradative fluorescent pseudomonads in Scots pine mycorrhizospheres developed on petroleum contaminated soil[END_REF]).

Polycyclic Aromatic Hydrocarbons (PAH)

PAHs are composed of diverse groups of soil pollutants and are recalcitrant to degradation, toxic, mutagenic, or carcinogenic in nature. Therefore, they pose an extremely dangerous threat to environment and human health. PAHs exhibit a wide range of chemical properties (depending on the number of aromatic rings, molecular weight, and structural configuration), water solubility, and volatility [START_REF] Kuiper | Rhizoremediation: A beneficial plant-microbe interaction[END_REF]. PAHs contamination in soils occurs mainly due to leakage from old storage tanks or natural oil reservoirs, oil spills, road surfaces, domestic waste, incomplete fossil fuel combustion, former gas plant facilities, and tanker accidents [START_REF] Morgan | Hydrocarbon degradation in soils and methods for soil biotreatment[END_REF][START_REF] Kuiper | Rhizoremediation: A beneficial plant-microbe interaction[END_REF]).

Many microbes including bacteria, fungi and algae can utilize PAHs as sole carbon source through ring fission [START_REF] Joner | Rhizosphere gradients of polycyclic aromatic hydrocarbon (PAH) dissipation in two industrial soils and the impact of arbuscular mycorrhiza[END_REF][START_REF] Kuiper | Rhizoremediation: A beneficial plant-microbe interaction[END_REF][START_REF] Joner | Rhizosphere effects on microbial community structure and dissipation and toxicity of polycyclic aromatic hydrocarbons (PAHs) in spiked soil[END_REF][START_REF] Bücking | Ectomycoremediation: An eco-friendly technique for the remediation of polluted sites[END_REF][START_REF] Leyval | Potential of arbuscular mycorrhizal fungi for bioremediation[END_REF]. For example, Braun-Lullemann et al. (1999) tested the ability of 27 ECM fungi to degrade different PAHs such as benzo(a) pyrene, phenanthrene, pyrene, and chrysene and found that the majority of ECM fungal species were able to degrade these pollutants (70% of the fungal species degraded phenanthrene, 65% of the species pyrene). These results were further confirmed by [START_REF] Gramss | Conversion rates of five polycyclic aromatic hydrocarbons in liquid cultures of fifty-eight fungi and the concomitant production of oxidative enzymes[END_REF] who showed that Hebeloma crustuliniforme, H. hiemale, and

Lactarius deliciosus showed the highest removal rates of the 22 examined ECM fungi for the degradation of different PAHs pollutants. However, there were inter-strains and inter-species differences in the PAHs degradation capabilities of the different ECM fungi. Furthermore, this degradation effect of ECM for various PAHs varied considerably (from reduced to no degradation) according to the experimental conditions, i.e. in soil compared to pure liquid culture [START_REF] Gramss | Conversion rates of five polycyclic aromatic hydrocarbons in liquid cultures of fifty-eight fungi and the concomitant production of oxidative enzymes[END_REF][START_REF] Genney | Degradation of the polycyclic aromatic hydrocarbon (PAH) fluorene is retarded in a Scots pine ectomycorrhizosphere[END_REF]Joner et al. 2006). This was more particularly due to adsorption of PAHs by soil particles, to the soil structure and pH, low water solubility with a high number of fused rings [START_REF] Kuiper | Rhizoremediation: A beneficial plant-microbe interaction[END_REF]) and a reduced microbial activity due to the competition for limited nutrients.

Heavy Metal pollution and toxicity

Ligneous plant species growing on contaminated soils were shown to be symbiotically associated with the ECM fungi (Perrier et al. 2006) which can develop by improving their nutrient and water supply and also by alleviating heavy metal (HM) toxicity. Heavy metals are metals with a density above 5gcm -3 [START_REF] Nies | Microbial heavy-metal resistance[END_REF]. Of the 90 naturally occurring elements of the Periodic Classification, 21 are non-metals, 16 are light metals and the remaining 53 (with As included) are heavy metals. For the re-vegetation of the areas polluted with HM, the ECM fungi are regarded as positive factors influencing significantly site phytostabilization [START_REF] Vosátka | Mycorrhizal fungi as helping agents in phytoremediation of degraded and contaminated soils[END_REF] 40 by improving plant nutrition in such areas and a strong inter-and intra-specific variation in HM tolerance has been reported for the ECM fungi [START_REF] Muller | AFLP-based assessment of the effects of environmental heavy metal pollution on the genetic structure of pioneer populations of Suillus luteus[END_REF][START_REF] Jentschke | Ectomycorrhizas and cadmium toxicity in Norway spruce seedlings[END_REF][START_REF] Vosátka | Mycorrhizal fungi as helping agents in phytoremediation of degraded and contaminated soils[END_REF]Hartley et al. 1999). Two major groups (Fig. 2.7) of heavy metals are supposed to be extreme threat to human and environmental health [START_REF] Raskin | Phytoremediation of metals: using plants to remove pollutants from the environment[END_REF]) e.g.

(1) Non-radioactive metals which include arsenic (As), cadmium (Cd) copper (Cu), mercury (Hg), Nickel (Ni), lead (Pb), and zinc (Zn) ;

(2) Radioactive metals especially strontium (Sr), cesium (Cs), and uranium (U).

The isolates of ECM fungi isolated from polluted soils were repeatedly shown to perform better in the presence of increasing concentrations of heavy metals when compared to isolate from unpolluted soils [START_REF] Jentschke | Ectomycorrhizas and cadmium toxicity in Norway spruce seedlings[END_REF][START_REF] Vosátka | Mycorrhizal fungi as helping agents in phytoremediation of degraded and contaminated soils[END_REF]. For example, experiments screening HM tolerance of ectomycorrhizal strains of Amanita muscaria, Paxillus involutus, Pisolithus tinctorius, Suillus bovinus, S. luteus and Thelephora terrestris isolated from polluted or unpolluted soils revealed that the strains originating from unpolluted soils were strongly inhibited by HM amendment into growth media, whereas most of the strains isolated from sporocarps growing in polluted soils exhibited tolerance to HMs [START_REF] Vosátka | Mycorrhizal fungi as helping agents in phytoremediation of degraded and contaminated soils[END_REF].

Due to this the positive effect of ECM fungi on plant HM tolerance, ECM can be utilized to develop a mycorrhizal system for the phytoremediation of heavy metal polluted soils so that ECM fungi can alter the HM uptake and its transfer to shoot by plant roots [START_REF] Bücking | Ectomycoremediation: An eco-friendly technique for the remediation of polluted sites[END_REF]Jourand et al. 2010;L'Huillier et al. 2010;[START_REF] Lenczewski | Microbial communities of ultramafic soils in maquis and rainforest at Mont Do, New Caledonia[END_REF]Smith and Read 2008;[START_REF] Howe | Copper-binding proteins in ectomycorrhizal fungi[END_REF][START_REF] Tam | Heavy metal tolerance by ectomycorrhizal fungi and metal amelioration by Pisolithus tinctorius[END_REF]Hartley et al. 1999) and therefore, could be essential for the reforestation of old mine sites especially in the New Caledonia where soils contains high concentrations of heavy metal of natural origin or from anthropogenic activity and exhibit a considerable threat to exposed biota. Most of these metals like Cu, Fe, Mn, Ni and Zn are essential as micronutrients or oligoelements but become toxic when present in excess.

A wide range of ECM fungal communities have been reported to be able to colonize the plant roots in heavy metal contaminated soils [START_REF] Bücking | Ectomycoremediation: An eco-friendly technique for the remediation of polluted sites[END_REF]Jourand et al. 2010;Perrier et al. 2006;[START_REF] Abler | Trace metal effects on ectomycorrhizal growth, diversity, and colonization of host seedlings[END_REF][START_REF] Melanie | Ectomycorrhizal fungal communities in young forest stands regenerating after clearcut logging[END_REF][START_REF] Joner | Rhizosphere gradients of polycyclic aromatic hydrocarbon (PAH) dissipation in two industrial soils and the impact of arbuscular mycorrhiza[END_REF]Joner et al. 2006;[START_REF] Jentschke | Ectomycorrhizas and cadmium toxicity in Norway spruce seedlings[END_REF]Hartley et al. 1999;[START_REF] Sell | Contribution of ectomycorrhizal fungi to cadmium uptake of Poplars and Willows from a heavily polluted soil[END_REF]). Among ECM fungi, ascomycetes have been reported to be more tolerant to heavy metals as compared to basidiomycetes. Likar et al. ( 2009) observed a large diversity of basidiomycetes in less polluted and control plots and the dominancy of ascomycetes in the most polluted plots along a gradient of Pb concentration. ECM phytoextraction of heavy metals could be linked mainly to the type of heavy metal and to the fungal and the plant species involved e.g. Cd. phytoextraction capability of Populus canadensis when co-cultured with Paxillus involutus was increased two fold but the same fungus was unable to extract Cd when grown with Salix viminalis [START_REF] Sell | Contribution of ectomycorrhizal fungi to cadmium uptake of Poplars and Willows from a heavily polluted soil[END_REF]).

Ectomycorrhizal fungi on serpentine soils

In general, we have very limited knowledge on the biodiversity of ECM fungi in metal-polluted environments. This possibly could be due to considerable lack of correspondence between the above-and belowground communities of ectomycorrhizal partners [START_REF] Jentschke | Ectomycorrhizas and cadmium toxicity in Norway spruce seedlings[END_REF] or also very limited number of studies have been carried out (Urban et al. 2008), even though they are the primary candidates for the evolution of adaptations to heavy metal toxicity (Urban et al.

2008).

Serpentinic sites of New Caledonia, as discussed above, composed of serpentinite which is a metamorphic rock, composed partly of the phyllosilicate serpentine {(Mg,Fe) 3 Si 2 O 5 (OH) 4 , magnesium iron silicate hydroxide}. Due to its origin from earth mantle material, the chemistry of serpentine is unlike that of other minerals in the earth's crust. Serpentine has low contents in the plant nutrients K + and Ca 2+ with a high unbalanced Ca/Mg ratio but contains high levels of potentially toxic elements such as Ni 2+ , Cr 3+ and Cr 6+ . However, Ni 2+ is more bio-available than Cr 3+ (Barceloux 1999) and appears to be the most toxic element in ultramafic soils [START_REF] Snavely | Magnesium transport in Salmonella typhimurium: 28 Mg 2+ transport by CorA, MgtA, and MgtB systems[END_REF][START_REF] Nies | CHR, a novel family of prokaryotic proton motive force driven transporters probably containing chromate/sulfate transporter[END_REF]). These soils have evolved a specific and unique plant biodiversity, as only certain number of plant species, endemic to metalliferous soils, could be capable of tolerating these exceptional concentrations of heavy metals (Khan 2005;Khan et al. 2000), in particular the vascular plants that have shown high degrees of endemism. Moser et al., (2009) have studied the communities of ectomycorrhizal (ECM) fungi on serpentine soils in southwestern Oregon (USA) and found no significant difference between the ECM mycorrhizal communities on serpentine and non-serpentine soils. A wide range of ECM fungal communities were shown to be able to colonize the plant roots in heavy metal contaminated soils (Perrier et al. 2006;Urban et al. 2008;Moser et al. 2009;Jourand et al. 2010;[START_REF] Colpaert | Heavy metal pollution and genetic adaptations in ectomycorrhizal fungi[END_REF]). It has also been suggested that there is little evolutionary adaptation towards elevated tolerance in certain ECM fungi (Journad et al. 2010). On the other hand, highly adapted ECM fungi have not been reported so far from metalliferous soils.

Apart from Ascomycetes (discussed earlier) which are considered to be abundant in the metalliferous sites, these sites could also control the diverse populations of Basidiomycetes ECM fungi. Some examples include Hebeloma sp., Pisolithus sp. (Leyval et al. 1997a;Jourand et al. 2010), Rhizopogon sp. [START_REF] Eitinger | Secondary transporters for nickel and cobalt ions: theme and variations[END_REF], Scleroderma sp. [START_REF] Delorme | Influence of the zinc hyperaccumulator Thlaspi caerulescens J. & C. Presl. and the nonmetal accumulator Trifolium pratense L. on soil microbial population[END_REF]) and Amanita muscaria [START_REF] Gast | Heavy metals in mushrooms and their relationship with soil characteristics[END_REF]. It is remarkable that some organisms seem to be pre-destined to evolve through specific metal-tolerance mechanisms e.g. adaptive metal tolerance has been suggested for a few higher fungi, including Pisolithus tinctorius and Pisolithus albus [START_REF] Egerton-Warburton | Differential responses of Pisolithus tinctorius isolates to aluminum in vitro[END_REF]Jourand et al. 2010), Suillus species (Krznaric et al. 2009;[START_REF] Colpaert | Genetic variation and heavy metal tolerance in the ectomycorrhizal basidiomycete Suillus luteus[END_REF], Cenococcum geophilum [START_REF] Gonçalves | Evidence of adaptive tolerance to nickel in isolates of Cenococcum geophilum from serpentine soils[END_REF]) along with some other

Ascomycetes, these ecotypes being specifically adapted to high levels of Al, Ni, Zn, Cd or Cu.

Mechanisms of metal tolerance in ectomycorrhizae

The mechanisms of protection against heavy metals have not yet been fully determined. Many suggestions have been made so far in order to conclude how the fungi reduce metal contents of their plant host. Regarding uptake, ECM fungi can decrease the uptake of different heavy metals to host plants, e.g. aluminum (L 'Huillier and Edighoffer 1996), cadmium (Hartley et al. 1999), copper (Amir et al. 2007), lead [START_REF] Joner | Rhizosphere gradients of polycyclic aromatic hydrocarbon (PAH) dissipation in two industrial soils and the impact of arbuscular mycorrhiza[END_REF] and zinc (Leyval et al. 1997b) and thus alleviate heavy metal toxicity to the host plants. Metal tolerance of higher plants may be due to a range of potential processes as shown in Fig. 2.8. (Jentschke and Godbold 2000). These various processes may include: The significance of these processes may vary as a function of the metal involved, its concentration, and the location of the primary lesion caused by the metal. The exploration of the ECM in the rhizosphere of different Tristaniopsis spp. and its co-associated microbial diversity of a naturally metalliferous soil may reveal useful organisms not only for bioremediation but also to use them as potential model organisms for population and functional studies on their adaptation particularly to high concentrations of Ni in New Caledonian soils and serpentine soils in general.

Role of mycorrhizosphere bacteria (MB) in improving the plants growth

Generally, it is believed that two major groups of bacteria can be found in soil e.g. either (1) free-living or (2) associated with other microbes or plant roots in the rhizosphere. Beneficial free-living soil bacteria which improve the plant growth are known as plant-growth-promoting rhizobacteria [START_REF] Kloepper | Free-living bacterial inocula for enhancing crop productivity[END_REF]). These strains belong mainly to the genera Azotobacter, Pseudomonas, Burkholderia, Acetobacter, Herbaspirillum and Bacillus [START_REF] Glick | Promotion of plant growth by ACC deaminaseproducing soil bacteria[END_REF][START_REF] Hampp | Interaction between soil bacteria and ectomycorrhiza-forming fungi[END_REF][START_REF] Probanza | The influence of native rhizobacteria on european alder Alnus glutinosa (L.) Gaertn.growth[END_REF][START_REF] Glick | The enhancement of plant growth by free-living bacteria[END_REF].

Under natural conditions, most of plant roots exhibit mycorrhizal association with soil fungi (Smith and Read 1997), creating the concept of "Mycorrhizosphere" [START_REF] Linderman | Mycorrhizal interactions with the rhizosphere microflora: The mycorrhizosphere effect[END_REF]) and the designation of mycorrhizosphere bacteria for soil bacteria living nearby (Sanon et al. 2005;Duponnois and Kisa 2006;[START_REF] Linderman | Mycorrhizal interactions with the rhizosphere microflora: The mycorrhizosphere effect[END_REF]). Since microbial activity may differ in mycorrhizosphere, rhizosphere and bulk soils [START_REF] Andrade | Bacteria from rhizosphere and hyphosphere soils of different arbuscular-mycorrhizal fungi[END_REF]), we will here use the term mycorrhizosphere bacteria (MB) to refer to soil bacteria surrounding ectomycorrhizal roots, since only bacteria associated to ECM roots of Tristaniopsis spp. were isolated and characterized in our study.

Soil bacteria may have many advantages in terms of improvement of plant nutrition and growth, nitrogen fixation, improved P-absorption, production of antibiotics, inhibition of the growth of pathogens, help in the mycorrhizal formation, elicitation of tolerance to abiotic stresses, such as drought, salt, heavy metals, nutrient deficiency or excess and the production of phytohormones.

However, here, we will discuss only about MB associated to Tristaniopsis spp. which are related to heavy metal pollution in different soils. These MBs associated to ECM of Tristaniopsis spp.

may play an important role in the biogeochemical cycling of toxic heavy metals present in New Caledonia soils and they could also be potentially used in cleaning up or re-mediating other metal-contaminated environments.

Nickel transport in bacteria

Nickel commonly occurs in nature as Ni (II) and therefore, Ni transport [START_REF] Navarro | The nik operon of Escherichia coli encodes a periplasmic binding-protein-dependent transport system for nickel[END_REF][START_REF] Wu | The hydC region contains a multi-cistronic operon (nik) involved in nickel transport in Escherichia coli[END_REF][START_REF] Seshadri | Roles of nickel binding proteins in Helicobacter species. The University of Georgia[END_REF].

The nickel specific permeases such as HoxN are present in many gram-negative and grampositive bacteria, and in archaea and many fungi [START_REF] Eitinger | Nickel transport systems in microorganisms[END_REF][START_REF] Eitinger | Secondary transporters for nickel and cobalt ions: theme and variations[END_REF]. They are characterized by the presence of eight trans-membrane domains and carry out high affinity Ni 2+ and/or Co 2+ transport into the cell [START_REF] Eitinger | Nickel transport systems in microorganisms[END_REF][START_REF] Seshadri | Roles of nickel binding proteins in Helicobacter species. The University of Georgia[END_REF][START_REF] Eitinger | Secondary transporters for nickel and cobalt ions: theme and variations[END_REF]. However, the source of energy for the transport of Ni 2+ via the permeases still remains unknown [START_REF] Seshadri | Roles of nickel binding proteins in Helicobacter species. The University of Georgia[END_REF]. Examples of other nickel specific permease found in bacteria are NixA and NhlF (Ni-and cobalt-specific permease) [START_REF] Eitinger | Nickel transport systems in microorganisms[END_REF][START_REF] Seshadri | Roles of nickel binding proteins in Helicobacter species. The University of Georgia[END_REF]).

Effect of heavy metals on bacteria

A variety of environmental and health problems have been caused because of the heavy metal contamination of soil. Likewise other living organisms, bacteria also required certain heavy metals for their enzymatic and growth activities such as Ni, Zn, Cu, Mn, etc. (Hausinger 1987;Abou-Shanab et al. 2007). The concentration of these metals needed by bacterial cells is generally very low (Mergeay 1997; Table 2.2). However, high concentrations of these metals are often cytotoxic (Abou-Shanab et al. 2007). This pollution affects the morphology, metabolism and growth of microorganisms in soil through functional disturbance, protein denaturation or destruction of integrity of cell membranes. Various mechanisms of resistance have been reported

in bacteria in response to heavy metals [START_REF] Nies | Efflux-mediated heavy metal resistance in prokaryotes[END_REF]. These mechanisms may be encoded by chromosomal genes, but more usually loci conferring resistance are located on plasmids [START_REF] Cervantes | Copper resistance mechanisms in bacteria and fungi[END_REF][START_REF] Wuertz | The impact of heavy metals on soil microbial communities and their activities[END_REF]. There is also evidence of a correlation between tolerance to heavy metals and antibiotic resistance, a global problem currently threatening the treatment of infections in animals and humans [START_REF] Ziha-Zarifi | In vivo emergence of multidrugresistant mutants of Pseudomonas aeruginosa overexpressing the active efflux system MexA-MexB-OprM[END_REF].

MIC is defined as the minimum concentration of a heavy metal at which microbial growth is completely inhibited by toxicity of heavy metal ions [START_REF] Yilmaz | Heavy metal concentrations in surface soils of Izmit Gulf Region, Turkey[END_REF]. [START_REF] Mergeay | Alcaligenes eutrophus CH34 is a facultative chemolithotroph with plasmid-bound resistance to heavy metals[END_REF] tested the minimal inhibitory concentrations (MICs) of several different metal ions for Escherichia coli on agar medium, and the most toxic metal (with the lowest MIC) was mercury, whereas the least toxic metal tested was manganese as shown in Table 2.2.

Mechanisms of heavy metal tolerance or resistance in bacteria

Bacteria have developed several types of mechanisms to tolerate the uptake of heavy metal ions.

These mechanisms include: metal exclusion by permeability barriers, active transport of the metal away from the cell organism, intracellular sequestration of the metal by protein binding, extracellular sequestration, enzymatic detoxification of the metal to a less toxic form and reduction in metal sensitivity of cellular targets [START_REF] Bruins | Microbial resistance to metals in the environment[END_REF][START_REF] Nies | Ion efflux systems involved in bacterial metal resistances[END_REF]Silver 1996). These resistance mechanisms could be either chromosomal or plasmid based. However, there are some difference between them as well. For example, resistance systems which involved essential metals are usually chromosome-based and are more complex than plasmid systems. On 50 the other hand, plasmid-encoded systems, usually involve toxic-ion efflux mechanisms (efflux is a mechanism responsible for extrusion of toxic substances and antibiotics outside the cell by using energy).

Although scientific community does not differentiate clearly tolerance and resistance to heavy metals and use these terms alternatively to indicate the ability of microbes to grow in HM contaminated soils, we may define them as follows:

Tolerance or Avoidance is a term used to describe a situation in which a validated preservative system ceases to control microbial growth. Generally it involves the metal exclusion by permeability barriers, active transport of the metal away from the cell organism [START_REF] Hall | Cellular mechanisms for heavy metal detoxification and tolerance[END_REF][START_REF] Shoeb | Genetic basis of heavy metal tolerance in bacteria. University of Karachi, Karachi Silver S[END_REF]). Tolerance can have many causes such as changes in environmental conditions, presence of contaminants, or it can be the result of microorganisms which have developed resistance to the preservative system in use.

Resistance is a broader term and may be defined as "a reduction in the susceptibility of the microbe as a result of physiological or genetic changes". It involves several potential mechanisms as described above including mechanisms for tolerance or avoidance along with the intracellular or extracellular sequestration of the metal by protein binding, enzymatic detoxification of the metal to a less toxic form and reduction in metal sensitivity of cellular targets [START_REF] Bruins | Microbial resistance to metals in the environment[END_REF][START_REF] Nies | Ion efflux systems involved in bacterial metal resistances[END_REF]Silver 1996).

However heavy metal ions have to enter the cell in order to cause the toxic effects to the living organisms. Generally, on the basis of energy source, the bacteria have two types of uptake/efflux system for heavy-metal ions (Table 2.3): one is fast, unspecific, used for variety of substrates, and usually driven only by the chemiosmotic gradient across the cytoplasmic membrane of bacteria (Fig 2.9b). The second uptake system has a high substrate specificity, is slower and often uses ATP hydrolysis (Fig. 2.9a) as the energy source, sometimes in addition to the chemiosmotic gradient, which make this uptake systems energetically expensive. Therefore, they are only used by the cell in times of need, starvation or a special metabolic situation [START_REF] Nies | Ion efflux systems involved in bacterial metal resistances[END_REF][START_REF] Nies | CHR, a novel family of prokaryotic proton motive force driven transporters probably containing chromate/sulfate transporter[END_REF][START_REF] Nies | Microbial heavy-metal resistance[END_REF].

RND protein family

RND stands for the resistance nodulation cell division (RND) protein family. This family involved in heavy metal resistance (Ralstonia metallidurans), nodulation (Mesorhizobium loti)

and cell division (E. coli) [START_REF] Vosátka | Mycorrhizal fungi as helping agents in phytoremediation of degraded and contaminated soils[END_REF][START_REF] Nies | Efflux-mediated heavy metal resistance in prokaryotes[END_REF]. Seven other protein families have been included in this family that can be found in all major kingdoms of life [START_REF] Muller | AFLP-based assessment of the effects of environmental heavy metal pollution on the genetic structure of pioneer populations of Suillus luteus[END_REF][START_REF] Nies | Efflux-mediated heavy metal resistance in prokaryotes[END_REF]. It can be classified in two major subfamilies e.g. in bacteria they transport heavy metals, hydrophobic compounds, amphiphiles (soaps and detergents), nodulation factors, termed as HAE-RND protein family.

However, in eukaryotes RND superfamily transport sterols or serve as receptors termed as the heavy metal efflux or HME-RND family (Fig. 2.9b). Cyanobacteria and proteobacteria contained many RND proteins [START_REF] Nies | Efflux-mediated heavy metal resistance in prokaryotes[END_REF]) except symbiotic bacterium Buchnera with no RND protein and three pathogenic bacteria with only one RND (Rickettsia prowazekii, Haemophilus influenzae, Pasteurella multocida) [START_REF] Nies | Efflux-mediated heavy metal resistance in prokaryotes[END_REF]. On the other hand, R. metallidurans strain CH34 has most of RND proteins (20 putative or candidate genes, some of them not complete), followed by its phytopathogenic relative Ralstonia solanacearum (15 genes), Magnetococcus (14 genes) and

Pseudomonas aeruginosa (13 genes). However, R. metallidurans contains more RND proteins than every other bacterium in the analyzed group [START_REF] Nies | Efflux-mediated heavy metal resistance in prokaryotes[END_REF].

CDF protein family

Cation Diffusion Facilitators (CDF family) serve as secondary cation filters in bacteria [START_REF] Nies | Efflux-mediated heavy metal resistance in prokaryotes[END_REF] after RND proteins. This protein family of metal transporters can be found in all three domains of life [START_REF] Nies | Efflux-mediated heavy metal resistance in prokaryotes[END_REF]. Primary substrates of CDF proteins are Zn 2+ , along with Co 2+ , Ni 2+ , Cd 2+ , and Fe 2+ . Metal is transported by CDF family by a concentration gradient, a chemiosmotic gradient, ∆pH or K + gradient [START_REF] Nies | Efflux-mediated heavy metal resistance in prokaryotes[END_REF]. 54

The number of CDF-encoding genes increases in parallel with evolution from yeasts to animals and plants contrary to that of bacteria where one or a few CDF-encoding genes are present.

Generally these genes are located on chromosomes [START_REF] Nies | Efflux-mediated heavy metal resistance in prokaryotes[END_REF] although the function of the proteins has only been characterized in a few bacteria. However, in higher animals, all well known CDF proteins are part of zinc transport systems such as ZnT1, ZTL1 (ZnT-like protein,

P-type ATPases

This protein family utilized energy from ATP hydrolysis to carry out heavy metal transport [START_REF] Nies | Efflux-mediated heavy metal resistance in prokaryotes[END_REF]. They can be found in plants, bacteria and animals (Fig. 2.11). They basically involve the 55 transportation of inorganic cations like H + , Na + , K + , Mg 2+ , Ca 2+ , Cu + , Ag + , Zn 2+ and Cd 2+ [START_REF] Nies | Efflux-mediated heavy metal resistance in prokaryotes[END_REF]Saier 1994). This important protein family can be involved either in import or export of heavy metals depending on the type of heavy metal homeostasis (Fig. 2.12), and thus comprises importing P-type ATPase able to transport its substrate from the outside or from the periplasm to the cytoplasm and exporting P-type ATPase in the reverse way [START_REF] Snavely | Magnesium transport in Salmonella typhimurium: 28 Mg 2+ transport by CorA, MgtA, and MgtB systems[END_REF][START_REF] Nies | Efflux-mediated heavy metal resistance in prokaryotes[END_REF].

Other protein family in this wide transport family includes soft metal-transporting ATPases also known as CPx-type ATPases. This is involved in the transport of Cu + and Ag + , but not Cu 2+ [START_REF] Nies | Efflux-mediated heavy metal resistance in prokaryotes[END_REF]. Zn-CPx-type ATPases which is another subfamily of P-type ATPases are generally involved in the transport of Zn 2+ , Cd 2+ and Pb 2+ . These are the other heavy metal export system which are linked to resistance determinants and may allow a bacterium to grow in specific nickel, cobalt and chromate-rich serpentine environment such as in our case of the New Caledonian soils [START_REF] Baker | Metal tolerance in plants[END_REF]. ChrA-like proteins of the CHR protein family [START_REF] Nies | CHR, a novel family of prokaryotic proton motive force driven transporters probably containing chromate/sulfate transporter[END_REF]) detoxify the oxyanion chromate, while NreB and CnrA are nickel efflux systems.

Chromate detoxification by CHR proteins

CHR proteins serve probably as chromate efflux pumps [START_REF] Alvarez | Chromate efflux by means of the ChrA chromate resistance protein from Pseudomonas aeruginosa[END_REF][START_REF] Pimentel | Efflux of chromate by Pseudomonas aeruginosa cells expressing the ChrA protein[END_REF] which utilized the energy produced by chemiosmotic gradient. Generally they export an anion in the direction of the electric field of the proton motive force, ∆ψ alone is sufficient to drive chromate efflux [START_REF] Nies | Efflux-mediated heavy metal resistance in prokaryotes[END_REF]. [START_REF] Nies | Efflux-mediated heavy metal resistance in prokaryotes[END_REF] compared CHR proteins of 64 prokaryotic genomes and found that one fourth of the bacterial genomes contained at least one putative CHR member with majority of genes involved in heavy metal export by using efflux pumps like Ralstonia metallidurans.

NreB-like proteins

The NreB like protein has been characterized in R. metallidurans strain 31A (Grass et al. 2001).

Nies (2003) reported that CHR protein was accompanied by the presence of NreB-like proteins in the genome of certain bacterial species, for example Synechocystis sp., Nostoc sp., Sinorhizobium meliloti. Ralstonia metallidurans contain two distinct nickel resistance determinants on its native megaplasmids pTOM8 and pTOM9: ncc (Schmidt and Schlegel 1994) and nreB (Grass et al. 2001). Nickel induced specifically the expression of the nreB gene and conferred nickel resistance in both strain 31A and E. coli. Cells with expressed nreB genes 57 resulted in increased removal or decreased accumulation of Ni 2+ , suggesting that NreB like proteins mediates nickel efflux [START_REF] Nies | Efflux-mediated heavy metal resistance in prokaryotes[END_REF]) in bacterial cells.

CnrA-like proteins

The cnrA gene is located directly downstream of the cnr cobalt-nickel resistance determinant in plasmid pMOL28 of R. metallidurans and encodes nickel resistance by itself (Grass et al. 2000;[START_REF] Nies | Efflux-mediated heavy metal resistance in prokaryotes[END_REF]). CnrA-like proteins exhibit 12 or 14 trans membrane spans and, like NreB proteins, a histidine-rich carboxy-terminus [START_REF] Nies | Efflux-mediated heavy metal resistance in prokaryotes[END_REF]. Both CnrA and NreB-like proteins are probably driven by the proton motive force, like CDF proteins and therefore, may work as secondary filter proteins for Ni 2+ similar to CDF proteins that work as secondary filter proteins for Zn 2+ , Co 2+ and Cd 2+ (Nies 2003).

Conclusion

Generally, the export or removal of heavy metals is accomplished at least by CDF2, Zn-CPx and HME1-RND proteins. [START_REF] Nies | Efflux-mediated heavy metal resistance in prokaryotes[END_REF] reported that only a few number of bacterial species have exhibited the collection of all three types of proteins families i.e. an ATPase plus a HME1-RND protein, a CDF2 plus a HME1-RND or exclusively an RND protein. [START_REF] Nies | Efflux-mediated heavy metal resistance in prokaryotes[END_REF] suggested that the presence of a HME1-RND protein is the exception and is co-related to higher levels of resistance to zinc, cobalt and cadmium, while possession of a CDF2 and/or a Zn-CPx-type ATPase is the general case and therefore could be a part of the cellular homeostasis system.

Mycorrhizae Helper Bacteria and mycorrhizosphere bacteria

Mycorrhizae Helper Bacteria (MHB)

Bacterial communities differ among different host plants and different surrounding soil environments [START_REF] Mogge | Bacterial community structure and colonization patterns of Fagus sylvatica L. ectomycorrhizospheres as determined by fluorescence in situ hybridization and confocal laser scanning microscopy[END_REF][START_REF] Timonen | Bacterial community structure at defined locations of Pinus sylvestris -Suillus bovinus and Pinus sylvestris -Paxillus involutus mycorrhizospheres in dry pine forest humus and nursery peat[END_REF]). In addition, bacterial community may also differ depending on ECM formed by each fungal species. Garbaye (1994) reported that the mycorrhizosphere bacteria could strongly influence mycorrhizal symbioses through their intraspecific relationship in the mycorrhizosphere of associated plants. He proposed that some of them these bacteria may consistently promote mycorrhizal development, and termed them as mycorrhizae helper bacteria or MHB under the abbreviated name.

Properties of MHB and taxonomic affiliation

De Oliveira (1988) reported that several bacterial taxa (un-identified) isolated from forest soils stimulated mycorrhiza formation by Hebeloma crustuliniforme with beech seedlings under controlled conditions. Garbaye and Bowen (1989) suggested that mycorrhiza formation was independent of the taxonomy of the bacteria. In a study performed on Pinus radiata seedlings inoculated with Rhizopogon luteolus, they tested the effect of different bacteria previously isolated from ectomycorrhizae of the same fungus species on the formation of mycorrhizal roots in comparison with seedlings inoculated with R. luteolus but grown on sterilized soil. They observed that these bacteria, mostly Gram negative bacteria increased significantly the mycorrhizal biomass (fresh weight) and 80% of them displayed a significant positive effect on mycorrhiza establishment, while only 20% were neutral or inhibitory. Similar significant results

were reported in further studies testing different symbiotic partners [START_REF] Garbaye | Pourquoi et comment observer l'état mycorhizien des plants forestiers[END_REF][START_REF] Garbaye | Pourquoi et comment observer l'état mycorhizien des plants forestiers[END_REF]Duponnois and Garbaye 1991).

MHBs have been isolated from a variety of ectomycorrhizal symbioses, although the nature of the systems (host plants and environments), which have been used to identify MHB, may vary.

The MHB strains that have been identified to date belong to many bacterial groups and genera, as illustrated in An important issue which relates to the understanding of MHB mechanisms is whether MHBfungal interactions are specific? [START_REF] Aspray | Mycorrhization helper bacteria: a case of specificity for altering ectomycorrhiza architecture but not ectomycorrhiza formation[END_REF]). However, Duponnois and Plenchette (2003) suggested that not all fungal-MHB interactions are specific. Basically, it requires a comparative study of MHB from different ectomycorrhizal systems because the concept of MHB is generic. It depends neither on the type of the mycorrhizal symbiosis nor on the taxonomy of the MHB strains. Although many MHBs strains have been isolated and characterized so far, MHBs have not shown any preference for particular plant species. In contrast, some MHBs have various specificities for mycorrhizal fungus species [START_REF] Aspray | Mycorrhization helper bacteria: a case of specificity for altering ectomycorrhiza architecture but not ectomycorrhiza formation[END_REF]). To date, many bacterial strains have been reported (Table 2.5) able to promote either ectomycorrhizal or arbuscular mycorrhizal symbioses (Duponnois and Kisa 2006;Duponnois and Garbaye 1991;Garbaye 1994;Frey-Klett et al. 2007;Duponnois et al. 2008).

Interaction mechanisms between MHB and ectomycorrhizae

Some mycorrhizosphere bacteria can induce strong changes to the morphology of hyphae, also able to develop biofilm-like structures (Fig 2 .13), similar to those observed in the early stages of mycorrhization (Frey-Klett et al. 2007). Some also stimulate apical growth of mycelium [START_REF] Deveau | The mycorrhiza helper Pseudomonas fluorescens BBc6R8 has a specific priming effect on the growth, morphology and gene expression of the ectomycorrhizal fungus Laccaria bicolor strain S238N[END_REF]. A physical contact between MHB and plant roots may be required for the promotion of mycorrhizal symbiosis, as stated by [START_REF] Aspray | Mycorrhization helper bacteria: a case of specificity for altering ectomycorrhiza architecture but not ectomycorrhiza formation[END_REF] for the MHB Paenibacillus sp. strain EJP73. These results showed that strain EJP73 released substances related to the MHB effect only when in contact with the roots; in addition to that, these substances are attached to the bacterial cell wall; and/or that these substances are short-lived and therefore have to be produced continuously [START_REF] Aspray | Mycorrhization helper bacteria: a case of specificity for altering ectomycorrhiza architecture but not ectomycorrhiza formation[END_REF].

61

With a few exceptions, the change of behavior to the fungus occurs even when bacterial cells are not yet in physical contact with their partner. This implies that signals are issued by bacteria and recognized by the fungi. Few of these signals have been isolated so far but preliminary studies suggested that they are likely variable: some are volatile and others are soluble. The best characterized is "auxofurane" a molecule structurally close to auxin and produced by a bacterial strain Streptomyces sp. Adding this molecule to a culture of Amanita muscaria can cause the fungus morphological changes identical to those caused by the presence of the bacteria. The same phenomenon was observed in case of "trehalose", a disaccharide accumulated by many ectomycorhizal fungi and preferentially consumed by bacteria from the mycorrhizosphere. Duponnois and Kisa (2006) reported that a Pseudomonas fluorescens strain perceived the presence of the trehalose at distance and then moves up it through its flagella and utilized it.

Similarly the presence of trehalose increased the mycelium growth in the presence of a strain Pseudomonas monteilii. Fungal growth was increase only when they co-culture the fungi with Pseudomonas monteilii on M9 medium plus trehalose.

Interactions between mycorrhizosphere bacteria (MB) and ectomycorrhizas

Several indirect beneficial effects of MB have already been reported, such as their ability to produce antibiotics. For example, Schelkle and Peterson (1997) reported a synergetic effect between MHB and ECM fungi in Laccaria proxima through the inhibition of root pathogens by the bacterial partner. In addition to preventing pathogen attacks, bacteria can also support ECM development directly. This has been shown for different host/fungus combinations (Garbaye 1994;[START_REF] Frey-Klett | Location and Survival of Mycorrhiza Helper Pseudomonas fluorescens during Establishment of Ectomycorrhizal Symbiosis between Laccaria bicolor and Douglas Fir[END_REF]. In general, the effect ascribed to the presence of bacteria consists of a significantly increased number of infected root tips (Dunstan et al. 1998).

Beneficial effects of MB on ECM formation could also be due to competition for nutrients in the rhizosphere (Fig. 2.14). The siderophores formation could be an example of such mechanism.

Siderophores are iron chelators which make iron available for uptake by the bacteria. These compounds are species-specific and can only be taken up by those bacteria that are able to produce them [START_REF] Hampp | Interaction between soil bacteria and ectomycorrhiza-forming fungi[END_REF]. Therefore, Neidhardt et al. (1990) proposed that protective bacteria synthesizing them could thus out-compete pathogens with regard to Fe in Felimiting soil conditions. Watteau and Berthelin (1990) suggested similar mechanisms for ECM fungi. Similar results were obtained from bacteria isolated from heavy metal contaminated soils. In Glomus mosseae, some MBs had a strong positive impact on germination of spores and on presymbiotic fungal growth under toxic concentrations of heavy metals, i.e. reduced damage on hyphae, increased mycelium growth and improved mycorrhizal formation [START_REF] Vivas | Brevibacillus brevis isolated from Cadmium-or Zinccontaminated soils improves in vitro spore germination and growth of Glomus mosseae under high Cd or Zn concentrations[END_REF]Frey-Klett et al. 2007). Mycorrhizosphere bacteria, more particularly Pseudomonas fluorescens in co-culture with ECM (Suillus bovinus), were able to degrade the hydrocarbon petroleum compounds and improved the survival and growth of fungi [START_REF] Sarand | Tolerance and biodegradation of m-toluate by Scots pine, a mycorrhizal fungus and fluorescent pseudomonas individually and under associative conditions[END_REF]. This opens the possibility for bioremediation of environmental contaminants by using the co-culture of MB and ECM.

Mycorrhizosphere bacteria from serpentine soils

Bacteria present in serpentine soils and their interaction with hyper-accumulating plants have focused the attention of several investigators in past years [START_REF] Delorme | Influence of the zinc hyperaccumulator Thlaspi caerulescens J. & C. Presl. and the nonmetal accumulator Trifolium pratense L. on soil microbial population[END_REF]Abou-Shanab et al. 2003;Abou-Shanab et al. 2007;[START_REF] Gremion | Comparative 16S rDNA and 16S rRNA sequence analysis indicates that Actinobacteria might be a dominant part of the metabolically active bacteria in heavy-metal-contaminated bulk and rhizosphere soil[END_REF]Héry et al. 2003;Héry et al. 2005a;Herrera et al. 2007;[START_REF] Héry | Nickel mine spoils revegetation attempts: effect of pioneer plants on two functional bacterial communities involved in the N-cycle[END_REF][START_REF] Saintpierre | Streptomyces yatensis sp. nov., a novel bioactive streptomycete isolated from a New-Caledonian ultramafic soil[END_REF]). These authors found that bacterial communities originating from serpentine soil tolerated especially high concentrations of metals, such as nickel, and more than those from unpolluted soils. Moreover, the presence of hyperaccumulating plants as Sebertia acuminata, Thlaspi caerulescens or Alyssum bertolonii led to an increase in the proportion of metal-resistant bacteria in the plant rhizosphere. Moreover, metalresistant bacteria present in the plant rhizosphere may play an important role in regulating the availability of metal for the plant (Héry et al. 2003).

The majority of strains reported from serpentine soils belonged to genera Arthrobacter, Bacillus, Microbacterium and Staphylococcus [START_REF] Turgay | Isolation and characterization of metal resistant-tolerant rhizosphere bacteria from the serpentine soils in Turkey[END_REF]Herrera et al. 2007). Sheng et al. Proteobacteria in the mine spoils contrary to that with vegetation where Proteobacteria were in majority. Furthermore, the quality and quantity of root exudates among different plant species could induce modifications in the bacterial diversity and activity in the polluted soils (Herrera et al. 2007;Héry 2004). Thus, nickel mining could degrade the soils structure through further decrease in carbon, nitrogen contents and increasing nickel concentrations (Herrera et al. 2007), this resulting in the selection of certain specific bacterial populations like Bacteroidetes, Actinobacteria and Chloroflexi.

Conclusion

There are various types of interactions between ECM and MB in soils of most of the ecosystems (Fig. 2.14; Frey-Klett et al. 2007). These interactions could be either synergetic or inhibitory [START_REF] Hampp | Interaction between soil bacteria and ectomycorrhiza-forming fungi[END_REF]Frey-Klett et al. 2007) . Direct interactions occur at different levels, such as plant-fungus recognition, stimulation of mycelium growth or establishment of symbiosis, along with indirect ones like reduced soil conduciveness or root receptiveness to the fungus. Mycorrhizosphere bacteria may also be involved in the bioremediation of contaminated sites. On the other hand, the fungus and root could select bacterial populations in the rhizosphere which are preferentially beneficial to them (Frey-Klett et al. 2007) in that particular ecosystem or environmental conditions. The use of both ECM and MB partners as inoculants could be an important strategy for the bioremediation of contaminated soils around the world particularly in the case of New Caledonian soils studied here.

Tristaniopsis spp. from New Caledonia

Tristaniopsis belongs to plant family Myrtaceae which contains about 133 different plant genera and 3800 species (Wilson et al. 2001). It is a large and highly diversified family present in Australia, Southeast Asia, and tropical to southern temperate America, but has little representation in Africa. It is characterized by the presence of combination of the following features: entire leaves containing oil glands, ovary half inferior to inferior, stamens usually numerous, internal phloem, and vestured pits on the xylem vessels (Wilson et al. 2001).

One notable character of the family is that the phloem is located on both sides of the xylem, not x Tristaniopsis calobuxus Brongn. & Gris

x Tristaniopsis capitulata Brongn. & Gris

x Tristaniopsis glauca Brongn. & Gris

x Tristaniopsis guillainii Brongn. & Gris

x Tristaniopsis jaffrei J.W. Dawson

x Tristaniopsis lucida J.W. Dawson

x Tristaniopsis macphersonii J.W. Dawson

x Tristaniopsis minutiflora J.W. Dawson

x Tristaniopsis ninndoensis J.W. Dawson

x Tristaniopsis polyandra P.G. Wilson & Waterhouse

x Tristaniopsis reticulata J.W. Dawson

x Tristaniopsis vieillardii Brongn. & Gris

x Tristaniopsis yateensis J.W. Dawson

Tristaniopsis guillainii

The plants in this species are mostly shrubs or small trees up to 15 m height, with dense branching and first branches covered with short hairs rusty or hairless, and trunk with light grey bark. They are also characterized by the presence of a highly developed root system. This species is found mainly on all massif north of line Boulouparis Canala, with a few or limited representation in the south (Fig. 2.17; http://www.endemia.nc/flore/fiche1408.html).

Tristaniopsis ninndoensis

This group contains bushy shrubs up to 3 m in height with first hairy branches, then greyish, glabrous and reddish, gray to reddish smooth bark peeling off in shreds. This species is endemic and found in extreme north of Grande Terre on Mont Ninndo (Arama). Arama is represented by the presence of volcano-sedimentary soils which are highly affected by erosion (Fig. 2.19; http://www.endemia.nc/flore/fiche1407.html).

Introduction

New Caledonia, a French overseas territory in the Southwest Pacific ocean, about 1,500 km east of Australia, is an archipelago of around 19,000 km 2 (Perrier et al. 2006). The Main

Island of New Caledonia is known as "La Grande Terre" (Fig. 1) and has rich mineral deposits of nickel, iron, manganese, chromium and cobalt (Lagrange 2009; Perrier et al. 2006;Amir et al. 2007). The huge reserves of nickel makes it the world's third largest producer of this metal (Perrier et al. 2006).

New Caledonian soils have derived from the weathering of ultramafic bedrocks including peridotites and serpentines (Fig. 1) and low available concentrations of major plant nutrients (N, P, K), unbalanced high Ca/Mg ratio and high levels of phytotoxic heavy metals, in particular Ni, are regarded as typical characteristics of these soils (Héry et al. 2003;Brooks 1987;Latham 1986). Ni content may reach up to 20 g.kg -1 in ultramafic soils while an average of 50 mg kg -1 is generally measured in cultivated soils while it is one of the most bio-available and phytotoxic element [START_REF] Echevarria | Assessment and control of the bioavailability of nickel in soils[END_REF][START_REF] Wenzel | Accumulation of heavy metals in plants grown on mineralised soils of the Austrian Alps[END_REF]. In addition, low organic matter contents and poor water retention is often associated to these soils resulting in an environment with depressed nutrient and water levels (Brooks 1987). Together, the chemical, physical, and biotic components of the edaphic factor produce cumulative effects of these components called "serpentine syndrome," to which a plant and its associated microbes must have to adapt [START_REF] Jenny | The soil resource: origin and behavior[END_REF]. This particular adaptation of plant and its associated microbes resulted in the evolution of ecosystems with specific and unique plant biodiversity (Héry et al. 2003;Pillon et al. 2010;Amir et al. 2007;Perrier et al. 2006), ectomycorrhizal fungi (Moser et al. 2009;Jourand et al. 2010a) and other microorganisms (Herrera et al. 2007). The major reason for this high level of diversity and endemism in New Caledonia is the presence of naturally Ni-rich ultramafic soils that cover one-third of the main island (L 'Huillier et al. 2010;Jaffré et al. 1987) and classified it as one of the smallest hotspots of biodiversity in the world representing the richest and most unusual flora of world (Myers 2003). More than 74% species of flowering plants (roughly 2,430 species) are endemic to this specific environment (Jaffré et al. 1987;Perrier et al. 2006).

The majority of plants growing under natural conditions are symbiotically associated with mycorrhizas (Pillon et al. 2010;Ramanankierana et al. 2007;Smith andRead 1997, 2008) which are ubiquitous components of most ecosystems throughout the world and form a key component of soil microbiota influencing plant growth and uptake of nutrients (Smith and Read 1997). In New Caledonian soils, a specific flora (Jaffré et al. 1987) as well as adapted fungi and bacterial populations have been reported (Navarro et al. 1999;Héry et al. 2003;Chaintreuil et al. 2007;Colwell et al. 2004;Colwell 2009). The association of trees with ectomycorrhizal fungi tolerant to high levels of toxic metals in soil might be essential for the establishment of plants sensitive to metal-polluted environments (Krznaric et al. 2009;Jourand et al. 2010a). The exploration of ECM fungi and their associated microbial flora in naturally metalliferous soils may reveal useful organisms for bioremediation but could also lead to the identification of potential model organisms for population and functional studies on the adaptation of microorganisms to serpentine soils, more particularly to high concentrations of Ni, in New Caledonian as in other ultramafic soils.

In this study, we selected different species of the ectomycorrhizal Tristaniopsis genus (Perrier et al. 2006;Jourand et al. 2010a) which belongs to Myrtaceae family. The Myrtaceae is a family of at least 133 genera with more than 3800 species (Wilson et al. 2001). It has centers of diversification in Australia, Southeast Asia, and tropical to southern temperate America, but has a few representatives in Africa (Wilson et al. 2001). It was suggested that ectomycorrhizal symbioses could play an important role in the adaptation of these endemic plants to ultramafic soils (Perrier et al. 2006). Tristaniopsis (Myrtaceae) were preferred as model plants among other ectomycorrhizal species native to New Caledonia since (a) they had the ability to grow on both metalliferous (ultramafic) and non-metalliferous (volcanosedimentary) soils and (b) they were taxonomically close to Eucalyptus spp., considering the advanced studies on mycorrhizas and plant genome that have been implemented on this model species.

In this work, our objectives were (1) to characterize the ECM fungal diversity associated to

Tristaniopsis spp. from both ultramafic and volcano-sedimentary soils in New Caledonia by internal transcribed spacer (ITS) sequencing and ( 2) to explore the effect of soil type on ECM fungal diversity.

Materials and methods

Site description and soil characterization

A map location of New Caledonia archipelago including studied sites is presented in Fig. 1.

Study sites located on "Grande Terre" belong mainly to two major categories of soil types i.e. the archipelago irrespective of soil type. On the other hand, T. glauca and T. guillainii were exclusive to ultramafic soils. Three trees at least, separated by 10-15 m, were sampled in each plot. In total, three plots per site were selected for the sampling of ECM roots tips (Table 1).

ITS sequencing and phylogenetic analysis

Total genomic fungal DNA was extracted and purified from ECM roots tips using the PCR products were separated by electrophoresis in 1% w/v agarose gel in 1 x TAE with ethidium bromide at 10 mg/ mL in the running buffer (Moyersoen et al. 2003). Amplified ITS products were purified using the Qiagen Gel extraction Kit (Qiagen). DNA sequencing was performed by Macrogen, using ABI chemistry and an ABI3730 capillary electrophoresisbased genetic analyzer (Macrogen, Seoul, Korea). All ITS sequences were then deposited in the GenBank nucleotide database for assignment of accession numbers (Table S1). Each set of sequences was considered as a different fungal taxon and the consensus sequence was saved and compared with the NCBI database using the BLAST algorithm (Altschul et al. 1997). All sequences matched fungal genera, however only the ones confidently assigned to an ECM genus (with at least the first 5 BLAST hits belonging to one genus) were retained for analyses. ITS sequences (including ITS1-f, 5.8S rRNA gene and ITS4) from this study were then aligned together with reference ITS sequences using ClustalX (Thompson et al. 1997) and corrected manually using Genedoc (Nicholas et al. 1997) The phylogenetic tree based on ITS sequences was constructed with MEGA v5.05 (Tamura et al. 2011) computer program by using neighbor joining method. The tree was bootstrapped with 100 replications of each sequence to evaluate the reliability of tree topology and the GTR+G model was used to calculate the genetic distances. The nucleotide sequences of ITS region amplified with ITS1-f and ITS4 primers were deposited in GenBank nucleotide database under a set of accession numbers as follows: JN847446-JN847504 and JN872841-JN872862 (Table S1, see Annex).

Statistical analysis

The relative abundance of each ECM taxon (as identified by ITS sequence) was estimated based on the number of successfully sequenced ECM root tips obtained from ultramafic and volcano-sedimentary soils. To compare species accumulation and richness estimates between soil types, rarefaction curves (Colwell et al. 2004) including 95% confidence intervals curves were calculated by using EstimateS program (v. 8.2; Colwell 2009) for all sample sets. This corrects for differences in sampling size and allows virtual reduction of the size of all samples to that of the smallest one. The Shannon index was also calculated to estimate the fungal diversity sampled from ultramafic and volcano-sedimentary soils, using the EstimateS software. To check whether the distribution of the various ECM taxa was influenced by soil type, we used the F 2 independence test applied to the number of occurrences of each ECM taxon in each soil (at p = 0.95).

Results

Soil analyses

More complete geochemical soil analyses were reported by Jourand et al. (2010). All tested parameters were significantly different between ultramafic and volcano-sedimentary soils.

Soils from Koniambo massif and Desmazures forest derived from ultramafic bedrocks and showed typical properties of serpentine soils (Table 3) such as low calcium to magnesium ratio, high level of magnesium, low contents of major nutrient elements such as N, P and K.

Iron and nickel contents were significantly higher on serpentine as compared to volcanosedimentary soils. In contrast, the latter soils contained lower amounts of toxic metals such as Ni. DTPA (diethylene triamine penta acetic acid)-Ni concentrations which represent available

Ni being 83-190 times higher in ultramafic soils than in volcano-sedimentary soils (Table 3).

Ectomycorrhizal fungal diversity

From the four Tristaniopsis species investigated, 76 root samples were collected (Table 2).

From these samples, 150 ECM tips were obtained and 97 were successfully amplified and sequenced (Table 1). All sequences obtained were searched in Genbank database in order to assign them a major ECM group, when possible. Overall proportion of the different ECM genera in both soils is reported in Fig. 2. Our study revealed very diverse ECM fungal communities, with almost similar fungal richness and community structure in both ultramafic and volcano-sedimentary soils. Seventy-four distinct ECM taxa (i.e. diverging sequences as described in Table S1) belonging to 10 ECM genera were identified from the ultramafic soils vs twenty-three taxa from 8 genera from volcano-sedimentary habitats. As shown in Fig. 2, in ultramafic soils, Cortinarius genus appears to be the most abundant one (>39%), followed by Pisolithus (19%) and Russula (11 %). Seven other genera were also present but in lower proportions (<10%): Boletellus, Scleroderma, Tomentella, Lactarius, Helotiales, Tricholoma and Thelephorales. Almost similar fungal ECM taxa trend was observed in volcanosedimentary soil (Fig. 2). As attested by F 2 independence test applied to the number of occurrences of each ECM taxon in each soil type, the different ECM genera did not tend to associate with particular soil type (observed F 2 = 6.32 for df = 9 at p = 0.95). Furthermore, when considering all ECM taxa and the four Tristaniopsis species combined, the Shannon index showed that ultramafic soils had a larger diversity than volcano-sedimentary soils, although the latter were less intensively sampled (2.70 vs 2.42, respectively). Despite the unbalanced sampling intensity between both soils, as 3 times more sequenced ECM root tips originated from ultramafic soils, species accumulation curves obtained from the ultramafic and volcano-sedimentary exhibited similar shapes, without signs of saturation (Fig. 3a,b)

suggesting that diversity did not greatly vary between cohorts (Table 4) and increased sampling effort would result in the detection of other ECM genera (Fig. 3a,b). Estimators of species richness gave a rather wide range of values for species numbers: for ultramafic soils, Chao2 estimates a total of 16 species, with the 95% confidence interval from 12 to 52. The means of other estimators also tend to converge in this value range, with the Bootstrap estimator giving the lowest value ( 12), the Jackknife estimators converging at around 16 and the Michaelis-Menten estimator (MM-Means) suggesting the presence of about 12 species. In case of volcano-sedimentary soils, Chao2 estimates a total of 14 species (ranging from 9-50 at α = 0.05) while 14, 10, and 13 species were estimated by Jackknife Bootstrap and MM-Means estimators respectively.

Composition of the ECM community according to host species

Although a few differences were observed in ECM diversity of the four Tristaniopsis host species (Table 4), they displayed similar fungal communities overall. Three out of 10 ECM genera, which corresponded to the most represented ones, occurred on the four host plant species as in both soils. However, a comparison of the frequency of each ECM genus on the four host species could suggest that some ECM genera displayed a preferential occurrence, as

Cortinarius that was more frequent (22 occurrences) on T. calobuxus, all sites combined.

Similar trend was also found for Pisolithus and Russula associated with T. calobuxus. At opposite, Tricholoma was only found once while Thelephora shared double host species respectively (Table 4). However, F 2 independence test applied to the number of occurrences of each ECM taxon in each host plant species did not show any ECM specificity towards a particular host species in ultramafic and/or in volcano-sedimentary soils (F 2 = 21.51 for df = 36 at p = 0.95).

An ITS-based phylogenetic tree was constructed from the most representative isolates as shown in Fig. 4. The phylogenetic analysis revealed that the uncultured fungal isolates obtained from the different soil and plant species origins were affiliated to a variety of ECM lineages, more particularly in Cortinarius, Pisolithus, and Scleroderma, and, to a lesser extent, in Russula, Boletellus and Lactarius. Only Cortinarius and Pisolithus lineages included isolates from both origins here.

Discussion

This study is the first detailed report on the ECM populations associated to Tristaniopsis spp.

from New Caledonia, which is considered as a hotspot of biodiversity (Perrier et al. 2006;Myers et al. 2000). The ultramafic sites at both Koniambo massif and forêt Desmazures are strongly serpentinic and represented high contents of Mg, Cr, and Ni. These sites provide extremely contrasted differences with volcano-sedimentary soils (L 'Huillier et al. 2010;Brooks 1987), with higher concentration of toxic heavy metal such as Ni ranging from 2.8-0.5 to 3.5-0.7 mg.g -1 , contrary to volcano-sedimentary soils, where they ranged from 0.08-0.02 to 0.14-0.02 mg.g -1 (Table 3).

If most studies reported highly diversified ECM fungal communities from a variety of environments and geographic locations (Perrier et al. 2006;Smith and Read 2008;Branco and Ree 2010;Jourand et al. 2010a;Joner et al. 2006;Leyval et al. 1997), extreme environments generally are considered to host lower diversity (Rothschild and Mancinelli 2001). Low ECM fungal diversity has been hypothesized by some authors under harsh environmental conditions [START_REF] Horton | The molecular revolution in ectomycorrhizal ecology: peeking into the black-box[END_REF].

Our study revealed some interesting findings, in particular that the high ECM fungal diversity observed was independent to soil type. We reported equally complex communities from both ultramafic and volcano-sedimentary soils (Fig. 2) and did not find a less diversified serpentine community, even though we were not able to fully describe the ECM fungal communities across the different sites (Table 4). The presence of all major and representative fungal taxa including Cortinarius, Pisolithus, Russula and Thelephora, as well as other genera within the Ascomycota and Basidiomycota (Table 4) in both soils suggests that these ECM may have widespread ability to cope with such extreme edaphic conditions. To our knowledge, this is the first detailed study describing the ECM communities associated to Tristaniopsis in both ultramafic and volcano-sedimentary soils. However, earlier works related to other plantmicrobes associations on ultramafic soils have suggested that these soils act not only as an extreme environment but also as a selective agent on several groups of organisms (Amir et al. 2007;Perrier et al. 2006;Jourand et al. 2010a;Rothschild and Mancinelli 2001). Analyses based on studies from the ultramafic (serpentine) soils had previously indicated that ECM communities from serpentine soils were similar to those from non-ultramafic environments (Moser et al. 2009 The wide phylogenetic distributions of species within ECM fungal communities are illustrated in Fig. 4. While phylogenetic clustering is evidence of habitat filtering when functional traits are conserved (Cavander- [START_REF] Cavander-Bares | Phylogenetic over dispersion in Floridian oak communities[END_REF]Jourand et al. 2010a), we did not find such phylogenetic clustering in ECM from serpentine soils (Fig. 4) suggesting that possibility of habitat filtering associated with trait conservatism may be ruled out. Therefore, more information is needed for a thorough understanding of ECM fungal evolutionary patterns on serpentine soils.

In addition, although ultramafic soils, which contain high concentrations of toxic heavy metals, are generally deficient in essential plant nutrients, the New Caledonian ones studied here contain higher levels of nitrogen and phosphorus than volcano-sedimentary soils.

Moreover, the host plants, that provide carbohydrates to ECM fungi through the symbiosis, could also act as protecting niches for survival and development of ECM fungi and thus mitigating adverse edaphic constraints found in extreme environments [START_REF] Gehring | Ectomycorrhizal fungal community structure of pinyon pines growing in two environmental extremes[END_REF]).

This increased availability in nutritive elements in New Caledonian ultramafic soils might explain the high diversity observed as well as the apparent absence of fungal specialization to ultramafic conditions (Branco and Ree 2010).

This study raised some important queries about how the symbiotic relationships influence the microbial diversity in ultramafic soils. It suggests that this could possibly be the host plant, which might play a key role in buffering the selection pressure of ultramafic soils on microbial communities [START_REF] Gehring | Ectomycorrhizal fungal community structure of pinyon pines growing in two environmental extremes[END_REF]). On the other hand, ECM fungal tolerance to heavy metals could be a driving force for possible plant colonization on ultramafic soils.

Although the mechanisms of this fungal tolerance are still obscure and should be explored (Jourand et al. 2010a), these ultramafic soils could provide new strains and new genetic determinants for heavy metal tolerance. These new mechanisms of tolerance could be exploited in bioremediation and re-vegetation practices of New Caledonian soils. Table 3 Soil properties 1 of the study sites.

1 All data are mean (r standard deviation) of five collected soil samples. E-mail address: waseem.lstm@gmail.com

Introduction

There are many factors either environmental like drought frost…etc or anthropogenic such as industrial pollution or agricultural exploitation that can possibly influence microbial community structure (Herrera et al. 2007;Navarro et al. 1999). Similarly, the presence of higher concentration of naturally occurring heavy metals in soils such as Ni, is one of the major threat in natural environments which effect negatively to associated the microbes (Sandaa et al. 1999). These toxic effects in terms of low microbial biomass and diversity are well known for all microorganisms and promote certain species probably more adapted than others in these extreme environments (Sandaa et al. 1999;[START_REF] Val | Biodiversity of arbuscular mycorrhizal fungi in heavy metal contaminated soils and its implications for bioremediation[END_REF]Khan et al. 2000). Therefore, such extreme soils tend to host low diversity and contain organisms specifically adapted to these edaphic conditions [START_REF] Val | Biodiversity of arbuscular mycorrhizal fungi in heavy metal contaminated soils and its implications for bioremediation[END_REF]. Thus the study of microbial diversity of such habitat would be crucial to understand the adaptive strategies of soil microbes (soil bacteria in our case) to cope with such extreme environments.

The New Caledonian soils are characterized by the presence of ultramafic rocks (Fig. 1), including peridotites and serpentines (Héry et al. 2003). These are deficient in essential elements like C, N, P, and have unbalanced Mg-Ca ratio, along with very high level of toxic heavy metals, particularly nickel which is sometimes 250 times more to the average level of nickel in normal soils (Latham 1986;Héry et al. 2003;Barceloux 1999;[START_REF] Amir | Influence of plants and cropping on microbiological characteristics of some New Caledonian Ultramafic soils[END_REF]. Due to its isolated geographic location, lower fertility level of soils and presence of high concentrations of heavy metals, the ecosystems of New Caledonia have evolved into a specific and unique plant biodiversity (Héry et al. 2003;Perrier et al. 2006) that make it a hotspot of biodiversity (Myers et al. 2000). It represents world's richest and unusual flora (Myers 2003), with a high level of endemism, more than 74% (roughly 2,430 species) plant species present in New Caledonia being endemic to this specific environment (Jaffré et al. 1987;Perrier et al. 2006). In the present study, we have chosen an ectomycorrhizal plant Tristaniopsis spp. (Perrier et al. 2006) from the Myrtaceae plant family (Wilson et al. 2001).

These plants species have the characteristic to grow on both ultramafic and volcanosedimentary soils of New Caledonia. Perrier et al. (2006) suggested that ectomycorrhizal symbioses could play an important role in the adaptation of these endemic plants to these extreme soils (Jourand et al. 2010b).

The interactions between ectomycorrhizae ECM and mycorrhizosphere bacteria (MB) in different soils could be either synergetic or antagonistic (Frey-Klett et al. 2007). Garbaye (1994) reported that the mycorrhizosphere bacteria could strongly influence mycorrhizal symbioses through their intra-specific relationship in the mycorrhizosphere of associated plants such that improvement in the mycelium growth and establishment of symbiosis and called them as 'mycorrhiza helper bacteria' (MHB). Indeed to date, most of MHB have also reported from natural ecosystems: able to improved plant nutrition through nitrogen fixation, siderophores production and phosphorus-solubilizing (Garbaye 1994). But most of these ecosystems do not involve heavy metal stress (Frey-Klett et al. 2007) and we have very limited knowledge about ECM-MB associations in naturally occurring ultramafic soils and their role in plant establishment in these extreme soils as in this case (present study) of New Caledonian. Therefore, combination of co-inoculation of ECM and MB could be important candidate for the re-vegetation of heavy metal contaminated soils around the world particularly in New-Caledonians soils.

Previous studies have indicated that soil bacteria isolated from ultramafic soils tolerated high concentrations of heavy metals, particularly nickel, when assessed on selective medium supplemented with different concentrations of NiCl 2 , as compared to that isolated from nonultramafic soils (Abou-Shanab et al. 2003;[START_REF] Abou-Shanab | Behaviour of bacterial populations isolated from rhizosphere of Diplachne fusca dominant in industrial sites[END_REF]. Other studies showed that such Ni-tolerant strains were playing an important role in regulating the availability of metal for the plant (Héry et al. 2003). In addition, Abou-Shanab et al. (2007) reported that the rhizospheric soil of hyper-accumulating plants, such as Sebertia acuminate and Thlaspi caerulescens had a higher proportion of metal-resistant bacteria than bulk soil. However, it is not fully clear yet whether the ultramafic soils do have some selective effects on bacterial communities and favor only certain bacterial taxonomic groups while disadvantage others.

Certain studies carried out in New Caledonian have reported a specific flora (Jaffré et al. 1987) as well as adapted fungal and bacterial populations in these soils (Herrera et al. 2007;Jourand et al. 2010a;Héry et al. 2003). There are only few papers describing microbial communities of such extreme soils. Among rare studies, Mengoni et al. (2001) and Héry et al. (2003) have found high genetic diversity of bacterial isolates in serpentine soils of Italy and New Caledonia respectively, regardless of selective nature of these extreme environments.

For instance, Héry et al. (2003) found that majority of Actinobacteria were presented in the mine spoils as in sito comparison to natural soils and suggested that these bacteria can be important candidates for ecological restoration of mine spoils. However, our survey is more complete, since we have investigated mycorrhizosphere bacterial communities associated to different Tristaniopsis spp throughout all New-Caledonia.

Consequently, the New-Caledonian context appears as a unique and optimal model to investigate the long-term impact of extreme Ni-rich soils on the composition of MB communities associated to ectomycorrhizal species from Tristaniopsis spp. For this reason, our study focused on two types of soils representative of range of soils found in New Caledonia (Héry et al 2003): ultramafic soils with high concentrations of heavy metals, particularly Ni (3.5 mg.g -1 ) and volcano-sedimentary soils with far lower contents of Ni (0.08 mg.g -1 ).

Thus, the main objectives of this study were: i) to explore the diversity of mycorrhizosphere

Materials and Methods

Site description and soil characterization

A map location of New Caledonia archipelago including studied sites is presented in Fig. 1.

As described above, study sites belong mainly to two soil types i.e. of the Grande Terre. Soil properties of both soil types are described in Table 1.

Site selection, host plant species and sampling

Samples were collected in cool wet season (June 2009) characterized by with minimum temperature and more occasional rain fall (mean annual rainfall 1500-2500 mm) from both ultramafic and volcano-sedimentary soils (Fig. 1). Over all three ultramafic sites including:

Koniamb massif, Forêt Desmazure and Yaté while two volcano-sedimentary soils i.e. Mont

Ninndo and Col d'Arama were chosen for sampling. However Konimabo massif was further divided into three sub-sites which include TG-15, Camps de Chinois and Sommet (Table 2).

Moreover, we restricted our study to the sites dominated by five Tristaniopsis host species i.e.

T. calobuxus, T. glauca, T. guillainii, T. macphersonii and T. nindoensis (Table 2).

Tristaniopsis glauca, T. guillainii and T. macphersonii are only found in ultramafic soils.

Tristaniopsis nindoensis, endemic to Mont Ninndo exclusively grows on volcano-sedimentary soils while, at the opposite, T. calobuxus is ubiquist and is indifferently found on both ultramafic and volcano-sedimentary soils. Within each stand of Tristaniopsis sp., a 900 m 2 (30×30 m) study plot was delineated. To minimize variability in disturbance due to edge effects, plots were centered on a randomly selected point such that the plot boundary was at least 30 m from any road or other abrupt edge as described by Dunham et al. (2007). In addition, only stands dominated by Tristaniopsis species were targeted and sampled for diversity estimations. All plots were searched for ectomycorrhizal (ECM) roots tips by gently removing the upper layers of moss and litter. Two to three ECM root tips per plant and three to four plants per plot were sampled. Overall, three plots per site were selected for sample collection and were searched for ECM roots tips.

Soil properties of studied sites

As shown in Table 1, the ultramafic soils from the study sites Koniambo massif, Forêt

Desmazures and Yaté belong to ferralsol according to the soil classification (FAO UNESCO and as reported by both Latham (1981) and Gillespie and Jaffre 2003). They showed typical characteristics of ultramafic soils such as lower contents of essential elements like carbon, nitrogen, phosphorus, and have high Mg-Ca ratio, along with very high level of heavy metals particularly nickel. In contrast, soils from Col d'Arama and Mont Ninndo were mainly composed of ferric acrisol representing the volcano-sedimentary soils with lower Mg-Ca ratio but containing higher K contents as compared to ultramafic soils (Table 1). Moreover, these soils contained lower amounts of toxic metals such as total Ni contents which ranged from 0.08 ± 0.02 to 0.14 ± 0.02 mg.g -1 of soil, contrary to ultramafic soils where they ranged from 2.8 ± 0.5 to 3.5 ± 0.7 mg/g. Similar trend was observed for extractable DTPA (diethylene triamine penta acetic acid)-Ni concentrations in ultramafic soils which were 83-190 times higher to that of volcano-sedimentary soils. In addition, Jourand et al. (2010) has reported complete geochemical soil analyses of the study sites previously.

Isolation of mycorrhizosphere bacteria, media and culture conditions

The collected ECM root tips were observed under microscope for morphotyping and the number of ECM root tips per morphotype was determined. The ECM root tips were transferred into Eppendorf tubes containing 50µl of distilled water (one representative ECM per morphotype). After crushing, the ECM root tip homogenates were used for fungal DNA extraction and parallel mycorrhizosphere bacteria (MB) isolation. In later case, aliquots of crushed root tips were transferred onto Petri dishes containing Trypcase agar medium (Difco) and incubated at 28°C for 24 to 48 h. One to 2 bacterial isolates were obtained from each crushed ECM root tip. A collection of about 200 bacterial isolates was constituted according to colony morphology. Isolates were checked for purity and then conserved into glycerol (60% V/V) at -80°C for further analyses.

Bacterial 16S rRNA sequencing and phylogenetic analysis

Total genomic DNA from bacteria pure culture was extracted as described previously by Pitcher et al. (1989). A nearly full-length 16S rRNA gene was amplified using primers FGPS-6 (5'-GGACAGTTAGATCTTGGCTACG-3') and FGPS-1509 (5'-AAGGAGGGGATCC-AGCCGCA-3') (Normand et al. 1992). PCR reactions were prepared in a total volume of 25 µL containing aliquots of 1 µL of genomic DNA at 50 ng mL -1 , 1 mM of each primer, 1.5 U of Taq DNA polymerase (Promega, Charbonnières, France), Promega Taq polymerase buffer, 2 mM MgCl 2 and 200 mM dNTP. Amplification was performed with a Thermo Electron Px2

Thermal Cycler programmed as follows: one cycle for 3 min at 95 ethidium bromide at 10 mg mL -1 in the running buffer (Moyersoen et al. 2003). Amplified 16S rRNA products were purified using the Qiagen Gel extraction Kit (Qiagen, Courtaboeuf, France). DNA sequencing was performed by Macrogen, using ABI chemistry and an ABI3730 capillary electrophoresis-based genetic analyzer (Macrogen, Seoul, Korea). All 16S rRNA gene sequences were then deposited in the GenBank nucleotide database for assignment of accession numbers. Each set of sequences was considered a different bacterial species and the consensus sequence was saved and compared with the NCBI database using the BLAST algorithm (Altschul et al. 1997). GenBank accession numbers are reported in Table S2. These sequences were then aligned together with reference 16S rRNA sequences using ClustalX (Thompson et al. 1997) and corrected manually using Genedoc (Nicholas et al. 1997). Molecular phylogeny trees of 16S rRNA sequences of Pseudomonas genus was constructed with MEGA v5.05 (Tamura et al. 2011) computer program by using Maximum Likelihood method. The 16S rRNA sequences (<1400bp) of the reference Pseudomonas species described by [START_REF] Anzai | Phylogenetic affiliation of the pseudomonads based on 16S rRNA sequence[END_REF] were obtained from the GenBank databases for construction of the phylogenetic tree. The tree was bootstrapped with 100 replications of each sequence to evaluate the reliability of tree topology and to calculate the genetic distances; the GTR+G model was used. The nucleotide sequences of 16S rRNA gene amplified with FGPS-6 and FGPS-1509 primers were deposited in GenBank under a set of accession numbers (Table S1).

Characterization of ECM fungal taxa through ITS sequencing

Total DNA was isolated from each ECM tip using a DNAeasy Plant mini-kit following the manufacturer's recommendations (Qiagen, Courtaboeuf, France). Identification of ECM fungi was performed for all ECM root tips by amplifying and sequencing the nuclear rDNA ITS with the fungus-specific primer pair ITS1-F (5'-CTTGGTCATTTAGAGGAAGTAA-3') (Gardes and Bruns 1993) and ITS4 (5'TCCTCCGCTTATTGATATGC-3') (White et al., 1990). PCR reactions were carried out by using a Thermo Electron Px2 Thermal Cycler. A PCR reaction was prepared in a total volume of 25 µL using 1 µL of extracted DNA at 50 ng mL -1 , 1 mM of each primer, 1.5 U of Taq DNA polymerase (Promega, Charbonnières, France), Promega Taq polymerase buffer, 2 mM MgCl 2 and 200 mM dNTP by using the following programme: one cycle for 3 min at 95 • C followed by 40 cycles at 95 • C for 30 s, 53 • C for 1 min, 2 min at 72 • C and a final extension for 10 min at 72 • C. PCR products were separated by electrophoresis in 1% (w/v) agarose gel in 1 x TAE with ethidium bromide at 10 mg.mL -1 in the running buffer (Moyersoen et al., 2003). Remaining purification and sequencing of the ITS amplified PCR product was same to that described for bacteria.

Statistical analyses

For species richness comparisons, the frequency of MB species was defined as the number of sequence type (species) per sample and presence and/or absence data were used in order to calculate species accumulation curves with 95% confidence intervals (Colwell et al. 2004) by using the program EstimateS-v.8.2 (Colwell 2009) for all sample sets. Multiple

Correspondence analysis (MCA) was also carried out to visualize the relationships among soil types, host Tristaniopsis specie, ECM and their MB population using XLSTAT (Addinsoft 2010). F 2 tests were conducted to determine whether frequencies of MB taxa were distributed independently of soil origins, i.e. ultramafic vs volcano-sedimentary ones.

Results

Diversity of mycorrhizosphere bacteria according to soil type

A total of 198 bacterial isolates were obtained from Tristaniopsis spp. root tips from which 16S rRNA was sequenced. All sequences obtained were compared with those available in the GenBank database (using the BLAST algorithm) in order to assign them a major bacterial group. The analysis of 16S rRNA gene sequences showed the presence of 10 genera among the mycorrhizosphere bacterial population (Fig 2). The ultramafic soils were represented by a highly diverse bacterial community contrary to that obtained from volcano-sedimentary soils, with 10 and 6 genera detected respectively. However, four genera exclusively found in ultramafic soils, i.e. Rhizobium, Methylobacterium, Paenibacillus and Ewingella, were present in very low proportions. The species accumulation curves (Fig. 3 and4) obtained from our MB sampling showed that ultramafic soils were more diverse in MB genera than volcano-sedimentary ones but more importantly that, despite a more intensive sampling performed on ultramafic soils, MB diversity found in the latter was not saturated after more than hundred individuals isolated. By contrast, MB diversity of volcano-sedimentary soils was saturated after a far less sampling intensity. In addition, when considering all MB genera exclusively and the five host plant species combined, the Shannon index showed that ultramafic soils had a larger diversity than volcano-sedimentary ones (5.45 vs 4.49, respectively). In the ultramafic soils, mycorrhizosphere bacterial population was slightly dominated but not significantly by Pseudomonas which contributes to more than 30.4% percent of total bacterial population. In terms of abundance, Pseudomonas was followed by Burkholderia (20.8 %), Bacillus (19.2%) and Enterobacter (17.6%), these four genera covering about 88% of total bacterial population. Six other genera were also present but in lower proportions (i.e. <10%): Lysinibacillus, Brevibacillus, Rhizobium, Methylobacterium, Paenibacillus and Ewingella. In contrast, in volcano-sedimentary soils, most of the MB population belonged to Burkholderia which represented more than 42% of the total population, while Pseudomonas (24.4%) and Bacillus (13.3%) were found in significantly smaller proportions (according to F 2 test at 5%). Among the less represented bacterial genera, only Lysinibacillus was found in significant proportions as it reached about 5.6% and 8.9% in ultramafic and volcano-sedimentary soils respectively.

Phylogenetic analysis, applied to the most represented MB genera, i.e. Pseudomonas (Fig. 5), revealed an overall phylogenetically diverse population of Pseudomonas in both soil types.

We selected Pseudomonas only for our phylogenetic analysis because of its equal representation in both ultramafic and volcano-sedimentary soils and its well known role as MHB in other ecosystems [START_REF] Frey-Klett | Location and Survival of Mycorrhiza Helper Pseudomonas fluorescens during Establishment of Ectomycorrhizal Symbiosis between Laccaria bicolor and Douglas Fir[END_REF]Frey-Klett et al. 2007;Garbaye 1994). A

phylogenetic tree constructed by the Maximum Likelihood method had eight main clusters as Pseudomonas syringae, P. fluorescens I and II, P. chlororaphis, P. putida, P. stutzeri, P.

aeruginosa, and P. pertucinogena as shown in Fig. 5. Depending on the sequences that could be retrieved from GenBank, isolates appeared to be related to various Pseudomonas clades.

However, majority of isolates (about 82%) clustered within one clade with strains from P.

fluorescens group II, while 13% of isolates were related to P. chlororaphis group. On the other hand, only one isolate was associated to P. putida and P. fluorescens group I respectively.

Composition of the MB community among host plant species

As reported in Table 4, the contribution of the five major MB genera in the total number of isolates varied according to soil type and Tristaniopsis host species. A comparison of the frequency of each MB genus in the five Tristaniopsis species analyzed revealed that these host plants did not display similar fungal communities (Table 4). However, in some species like the ultramafic-endemic ones T. macphersonii and T. glauca, it was not possible to observe any significant trend of preferential association with MB, considering the too low number of isolates obtained from them (Table 4). Some MB taxa tended to display a preferential occurrence for a particular host plant, such as Bacillus in ultramafic soils that exhibited the highest frequency, i.e. 30%, in T. calobuxus, while they occurred in very low proportion with other host species like T. guillainii, i.e. 4%. Similar trend was also observed in the case of Enterobacter mainly associated to T. guillainii in ultramafic soils (35%) while found in very low proportions or nil in other situations. One of the major preferential MB-host plant association occurred between Pseudomonas and T. calobuxus (27%) or T. guillainii (39%), both from ultramafic soils, although Pseudomonas isolates were always present in proportions higher than 20% (except in the poorly represented T. glauca). By contrast, in volcano-sedimentary soils, Burkholderia were most frequent than in ultramafic soils but did not appear as specifically associated with one of the two Tristaniopsis host species. Moreover, F 2 independence tests showed that MB taxon frequency was significantly influenced: i) by the Tristaniopsis host species (observed F 2 = 31.41 at P>0.95, d.f. = 20); and ii) by the soil type, regardless of the host plant species (F 2 at P>0.95, d.f. = 4).

Diversity and specificity of ectomycorrhizae-mycorrhizosphere bacteria associations according to soil types

The different ECM taxa associated to the studied MB and obtained from both ultramafic and volcano-sedimentary soils are reported in Table 4. The ECM community was found to be composed of members from several fungal genera, particularly Cortinarius which was the most represented in ultramafic soils, contributing to about 30% of total ECM population in this soil type. The other well-represented fungal genera in ultramafic soils were Pisolithus (19% of total root tips) and Russula (11%), while minor ones, including Scleroderma, Tomentella, Boletellus and Tricholoma, contributed each to less than 10% of total ECM population (Table 4). In volcano-sedimentary soils, a similar fungal ECM genera structure was observed (Table 4) as statistically confirmed by F 2 test (at 5%).

In order to investigate potential association and specificity among; host plant species, ECM taxa and their associated MB in both soil types; a multiple correspondence analysis was carried out (Fig. 6). We observed three types of such associations i.e. among host plant, ECM and MB, between host plant specie and their associated MB, and finally between an ECM fungi and its associated mycorrhizosphere bacteria. For example in ultramafic soils, we marked that Pseudomonas were strongly affiliated with Pisolithus and T. callobuxus.

Similarly, a strong close relationship between Burkholderia and Russula was evident as shown in Fig. 6. Also Bacillus appeared to be closely oriented towards T. glauca while Enterobacter were more or less closely related to T. guillainii in ultramafic soils indicating host plant species have influence on overall microbial population in ultramafic soils.

Discussion

This is the first detailed study carried out on molecular identification and characterization of MB originating from serpentine soils. Our results showed that the New-Caledonian ultramafic soils, when compared to volcano-sedimentary soils, do not represent a strong physiological barrier to MB communities associated to Tristaniopsis spp. and are not composed of specific bacterial species. We found a highly diverse MB community associated to different Tristaniopsis spp. growing on these extreme soils and even richer than that found on nonultramafic soils as confirmed by the number of MB genera identified and the rarefaction curves. In contrast, earlier studies suggested that ultramafic soils act not only as an extreme environment for the establishment and evolution of plants and their associated microbes but also constitute a selective (negatively) agent on several groups of organisms (Amir et al. 2007;Rothschild and Mancinelli 2001). This selective effect may result in serpentine adaptive radiation as it was already observed for Pisolithus isolates from New Caledonia (Jourand et al. 2010). However, we were able here to report a highly diverse and complex MB community from ultramafic soils.

Our results showed that Proteobacteria represent more than 62% of the total flora, these results being in agreement with previous data that also showed a predominance of this phylum in rhizospheric bacteria from ultramafic soils [START_REF] Rajkumar | Biotechnological applications of serpentine soil bacteria for phytoremediation of trace metals[END_REF]. Herrera et al. (2007) reported similar results for rhizospheric bacterial communities from ultramafic soils of New Caledonia, more especially mine spoils revegetated by Gymnostoma webbianum, that were characterized through 16S rRNA cloning-sequencing. These authors suggested that this increase in the proportion of Proteobacteria in the plant rhizosphere could be due to nitrogen and carbon supply from the plants, thus creating a similar situation as in a natural soil environment and able to favor rhizosphere bacterial diversity.

Moreover, the majority of MB obtained from New-Caledonian ultramafic soils were almost similar to those described in the literature whatever the environments and geographic locations studied. For example, Bacillus was a dominant genus isolated from roots of the metal hyper accumulator Alyssum bertolonii in Italy [START_REF] Barzanti | Isolation and characterization of endophytic bacteria from the nickel hyperaccumulator plant Alyssum bertolonii[END_REF]). Other rhizobacterial genera like Burkholderia, Pseudomonas, Bacillus, and Paenibacillus were also characterized from Ni-hyper accumulator plants from Oregon serpentine soils in USA (Abou-Shanab et al. 2003;Abou-Shanab et al. 2007) and in New Caledonia (Héry et al. 2003).

The 16S rRNA-based phylogenetic tree obtained from Pseudomonas isolates, which was the most represented MB genus in our study, revealed two main clusters along with the 71

Pseudomonas reference species described by [START_REF] Anzai | Phylogenetic affiliation of the pseudomonads based on 16S rRNA sequence[END_REF]. However, 82% i.e. majority of isolates belonged to P. fluorescens group II cluster while 13% grouped in the neighbour P.

chlororaphis group. This phylogenetic analysis showed a highly homogeneous population of Pseudomonas strains overall, despite their various origins, thus validating their grouping into a single unit, i.e. at genus level, in the multidimentional analyses. Moreover, such phylogenetic homogeneity among MB isolates strengthens the assumption that MB-ECM associations are specific. Pseudomonas fluorescens group includes several groups of bacteria associated with fungi and plants, such as Mycorrhization Helper Bacteria (MHB), EMAB (Ectomycorrhiza Associated Bacteria), and Plant Growth Promoting Rhizobacteria (PGPR) [START_REF] Kozdrój | Mycorrhizal fungi and ectomycorrhiza associated bacteria isolated from an industrial desert soil protect pine seedlings against Cd(II) impact[END_REF]Frey-Klett et al. 2007;[START_REF] Frey-Klett | Location and Survival of Mycorrhiza Helper Pseudomonas fluorescens during Establishment of Ectomycorrhizal Symbiosis between Laccaria bicolor and Douglas Fir[END_REF]. [START_REF] Frey-Klett | Location and Survival of Mycorrhiza Helper Pseudomonas fluorescens during Establishment of Ectomycorrhizal Symbiosis between Laccaria bicolor and Douglas Fir[END_REF] suggested that the presence of P. fluorescens in soils can be selectively stimulated in the vicinity of the mycorrhizas through the release of certain chemical compounds by mycorrhiza, in particular Trehalose used as carbon source. Both arbuscular mycorrhizas [START_REF] Becard | Identification and Quantification of Trehalose in Vesicular-Arbuscular Mycorrhizal Fungi by in vivo 13C NMR and HPLC Analyses[END_REF][START_REF] Van Laere | Trehalose, reserve and/or stress metabolite?[END_REF] and ectomycorrhizas [START_REF] Niederer | Trehalose synthesis in mycorrhiza of Norway spruce: an indicator of vitality[END_REF][START_REF] Frey | Metabolic and genotypic fingerprinting of Fluorescent pseudomonads associated with the Douglas Fir-Laccaria bicolor Mycorrhizosphere[END_REF]) are able to excrete Trehalose, known to be involved in drought resistance [START_REF] Müller | Trehalose and trehalase in plants: recent developments[END_REF]. Like the results obtained in an efficient P. fluorescens MHB strain isolated from a Laccaria bicolor sporocarp [START_REF] Frey | Metabolic and genotypic fingerprinting of Fluorescent pseudomonads associated with the Douglas Fir-Laccaria bicolor Mycorrhizosphere[END_REF], the selection of adapted and efficient MHB from our MB collection could have practical outcome in improving the establishment of ECM symbioses on ultramafic soils in New Caledonia. Work is in progress to assess in vitro in a first step the helper effect of some isolates from this study and their ability to stimulate the growth of Pisolithus albus in co-culture on selective medium.

The availability of growth-limiting nutrients plays a key role in shaping the composition of biotic rhizosphere communities. With change in the plant diversity, composition of organic compounds or root exudates released into rhizosphere are also altered, which potentially affects the rhizosphere bacterial diversity particularly in extreme environments such as ultramafic soils (Abou-Shanab et al. 2007;Herrera et al. 2007;Héry et al. 2005). Moreover, Herrera et al. (2007) reported that different plant species have specific effects on microbial diversity due to the variability in quality and quantity of root exudates produced [START_REF] Zak | Plant diversity, soil microbial communities, and ecosystem function: are there any links?[END_REF]. These previous studies are corroborated by our findings highlighting variations in MB taxa composition according to the different Tristaniopsis species investigated. As clearly established by the multiple correspondance analysis, we observed that Pisolithus and

Pseudomonas were more closely associated to T. calobuxus in ultramafic soils while Enterobacter seemed to be preferentially associated with, T. guillainii. These close ECM-MB-plant species affinities suggest that not only extreme soils but also the host plant species could be strong evolutionary agents for selection of particular MB taxa.

In the case of ECM fungal communities from which the MBs were isolated, we found almost similar fungal communities in both ultramafic and volcano-sedimentary soils. These results are in accordance with those obtained by Moser et al. (2009) who found alike and/or indistinguishable ECM community structures, in terms of abundance and diversity, associated with Q. garryana on both serpentine and non-serpentine soils. Moreover, the most abundant and diverse ECM genera associated with Tristaniopsis were identical to those found in association with other plants on serpentine soils worldwide such as Cortinarius, Russula, Pisolithus, Tomentella, Boletellus (Moser et al. 2009;Branco 2010;Jourand et al. 2010b).

These results, corroborated by previous reports describing hyper-diverse ECM communities in serpentine soils (Urban et al. 2008;Moser et al. 2009), suggest a general pattern (in terms of high diversity) of ECM fungal diversity common to all types of ultramafic ecosystems and indicate at least that serpentine soils do not constitute an environmental barrier for their development.

These results suggest that plant roots may support diverse mycorrhizosphere bacterial communities able to interact with the ECM symbionts through a variety of mechanisms.

Therefore, the size and distribution of MB communities could play an important role in determining the nature and extent of bacterial effects on mycorrhiza formation and growth of their associated plant. Further research is needed to explore the physiological mechanisms involved in MB ultramafic tolerance which would help to better understand the role of symbioses in the maintenance of biodiversity in stressful habitats. Subsequently, metagenomic analyses could also provide additional functional information in the form of genomic sequences and the expression of traits involved in the adaptation of particular microorganisms in these soils. Cr DTPA (µg/g) 0.7 ± 0.1 to 0.28 ± 0.02 0.04 ± 0.005

1 All data are mean (r standard deviation) of five soil samples tested.

2 DTPA: dimethylene triaminopentaacetic acid associations and its role in the adaptation of plants to ultramafic soils would help in the understanding of plant functioning on New Caledonian mine sites.

Keywords: Serpentine, Helper bacteria, Nickel-tolerance, Ectomycorrhizas, Tristaniopsis, Mycorrhizosphere

Introduction

The ultramafic or serpentine soils are considered as highly disadvantageous for most plant species due to their extreme edaphic constraints (Brooks 1987). Basically the soils derived from the weathering of serpentine bedrocks: are deficient in essential plant nutritive elements such as N, P and K along with unbalanced high Ca-Mg ratios while containing very high proportions of toxic heavy metals, particularly nickel (Brooks 1987). In the naturally Ni-rich ultramafic soils of New Caledonia, that cover one-third of the total island area, these highly selective edaphic factors have resulted in the evolution and adaptation of unique plant families and species, 74% of the later being endemic (L 'Huillier et al. 2010;Jaffré et al. 1987). Thus, many Ni hyper-accumulator plant species as well as serpentine-endemic ones are found to New Caledonia [START_REF] Reeves | Nickel Hyperaccumulation in the Serpentine Flora of Cuba[END_REF]. In parallel with their host plants, microbial communities adapted to ultramafic soils have developed various mechanisms of metal-tolerance involving mostly metal efflux membrane transporters. Several studies have shown that, ectomycorrhizal or bacterial strains isolated from metal-rich serpentine soils had a higher tolerance to heavy metals in vitro, particularly nickel, than the ones isolated from non-ultramafic soils (Amir et al. 2007;Jourand et al. 2010b;Abou-Shanab et al. 2003). The nreB determinant which belong to the major facilitator superfamily, was also shown to be metal-ion specific and induced exclusively by nickel but not by zinc or cobalt (Grass et al. 2001). Similarly, the cnr determinant which mediates resistance based on cation efflux, was considered to be involved in both Co and Ni resistance in Ralstonia sp. strain CH34 (Grass et al. 2000). Garbaye (1994) reported that the mycorrhizosphere bacteria could strongly influence mycorrhizal symbioses through an intra-specific relationship involving a range of physiological functions, such as nitrogen fixation, siderophores production and phosphorussolubilizing, that ultimately contribute to plant nutrition. To date many bacterial species have been reported as promoting ectomycorrhizal symbioses, such as Pseudomonas, Bacillus or Streptomyces (Frey-Klett et al. 2007;Garbaye 1994). The so-called Mycorrhizae Helper Bacteria (MHB) strains have a promoting effect on the formation of ectomycorrhizae as described for the first time in the tripartite association between Pseudomonas fluorescens, Laccaria laccata and Douglas-fir (Duponnois and Garbaye 1991). Rather than plant-specific (Garbaye and Bowen 1989), MHB are known as fungal specific since their promoting effect on mycorrhizal formation varies according to the ECM fungal taxa or strains considered (Duponnois et al. 1993). In a first step, the MHB potential of bacterial strains must be assessed in co-culture with ECM fungi to highlight a fungal growth-promoting effect. To date, most of MHB as well as MB-ECM associations described originated from unconstrained forest ecosystems but none from metal-rich soils.

Different species of the Tristaniopsis genus, belonging to the Myrtaceae plant family, were chosen in this study due to their ectomycorrhizal status and their ability to grow on both metalliferous and non-metalliferous soils throughout New Caledonia (Perrier et al. 2006).

Recent studies showed that ectomycorrhizal fungi had a primordial role in the establishment and adaptation of certain plant species to ultramafic soils (Jourand et al. 2010b;Perrier et al. 2006).

Although the diversity of mycorrhizosphere bacteria (MB) associated to Tristaniopsis spp.

was already described as well as close affinities between certain MB and ECM fungal taxa in an earlier study (Waseem et al. 2011a), no data are available about functional characteristics of MB communities associated to Tristaniopsis plants and mutual interactions between MB and ECM, such as MHB properties. More precisely, the ability of MB strains and taxa to tolerate high Ni-concentrations was not investigated, neither the research of Ni-resistance determinants in the different bacterial taxa.

The immediate and middle-term objectives of this study were, first, to isolate and characterize

Ni-tolerant MB associated to Tristaniopsis in Ni-rich ultramafic soils and volcanosedimentary ones; second, to screen highly Ni-tolerant MHB strains which might be used jointly with ECM inoculants for further field experiments on ultramafic soils in the framework of revegetation programs of ultramafic sites in New Caledonia.

More specific objectives of this study were: i) to explore the Ni-related functional diversity of MB associated with Tristaniopsis spp. in ultramafic soils in comparison with that found in volcano-sedimentary soils, in order to highlight a possible adaptation of MB to such environments at two levels: phenotypic characterization through in vitro Ni-tolerance assays and genotypic characterization focused on the detection of nreB and cnrT genes, known to confer Ni-tolerance, throughout all MB origins; ii) to identify possible specificities between MB taxa, ECM fungal taxa and their Tristaniopsis host plant species, the later being either ubiquitous and found in both soil types (T. calobuxus) or specific to one of both soil types (i.e.

the four other Tristaniopsis species studied); iii) to evaluate the MHB potential of MB isolates from different taxa and origins through in vitro co-culture tests.

Materials and methods

Geographic location and host plant species of study sites

A complete map describing the geographic location and soil characteristics of the five studied sites was presented in a previous publication [START_REF] Waseem | Ectomycorrhizal fungal diversity associated to Tristaniopsis spp[END_REF]). Koniambo massif (21°00.412S, 164°49.888E), forêt Desmazures (22°12.161S, 166°37.484E) and Yaté (22°15.459S, 167°00.248E), which are composed of ferralsol (Jourand et al. 2010a), are located on ultramafic soils while col d'Arama and Mont Ninndo (ferric acrisol; Jourand et al. 2010a) were two study sites located on volcano-sedimentary soils. Ectomycorrhizal root tips were collected from five Tristaniopsis species having different soil adaptations: T. macphersonii, T. glauca and T. guillainii were specific to ultramafic soils while T.

ninndoensis, endemic to Mont Ninndo, was exclusively found on volcano-sedimentary soils.

By contrast, the ubiquitous T. calobuxus was found on both ultramafic and volcanosedimentary soils (Table 1).

Soil properties of the study sites

The detailed soil properties of the study sites were described in Waseem et al. (submitted paper, 2011a). The ultramafic sites, i.e. Koniambo massif, forêt Desmazures and Yaté, had very high levels of heavy metals, particularly nickel which ranged from 2.8r 0.5 to 3.5r 0.7 mg.g -1 . These sites also had high contents in Mg (4.1 to 8.7 mg.g -1 ) and Fe (370 to 429 mg.g - 1 ) while they were deficient in essential plant nutrients such as N (0.36 to 0.40 %), P (0.07 to 0.17 mg.g -1 ), and K (0.07 to 0.09 mg.g -1 ). In contrast, volcano-sedimentary soils contained much lower levels of heavy metals, such as nickel (0.08r 0.02 to 0.14r 0.02 mg.g -1 ), were rich in Fe (0.5 mg.g -1 ) and Mg (0.47 mg.g -1 ) while containing higher levels of N (0.24 %), P (0.45 mg.g -1 ), and K (0.32 mg.g -1 ) than ultramafic sites. In the same way as total nickel concentrations, extractable diethylene triamine pentaacetic acid (DTPA)-Ni concentrations, representing the potentially bio-available Ni, was 83 to 190 times lower in non-ultramafic soils than in ultramafic ones.

Sampling of mycorrhizas and isolation of mycorrhizosphere bacteria

Ectomycorrhizal root tips of Tristaniopsis were sampled in June 2008 from the five Tristaniopsis host species distributed in the five sites (Table 1). Samplings were performed in and then conserved into glycerol (60% w/v) at -80°C before molecular analyses and phenotypic studies.

Identification of mycorrhizosphere bacteria through 16S rRNA sequencing

Total genomic DNA of all isolates was extracted from 5 ml cultures grown overnight from

Trypcase broth as described previously by Pitcher et al. (1989). Amplification of 16S rRNA gene sequence was performed by polymerase chain reaction (PCR) with the conserved primers pair: FGPS-6 (5'-GGACAGTTAGATCTTGGCTACG-3') and FGPS-1509 (5'-A-AGGAGGGGATCCAGCCGCA-3') (Normand et al. 1992). Reaction conditions were as follows: one cycle for 3 min at 95 • C followed by 40 cycles at 95 • C for 30 s, 56 • C for 1 min, 2 min at 72 • C and a final extension for 7 min at 72 • C. Each PCR mixture (5 µl) was analyzed by agarose gel (1% w/v) in 1 x TAE with ethidium bromide at 10 mg mL -1 in the running buffer (Moyersoen et al. 2003). For further sequencing reaction, the amplified DNA was purified from salts and primers using the PCR purification kit (Qiagen Inc., Chatsworth, CA, USA)

according to the manufacturer's instructions. DNA sequencing was performed by Macrogen, using ABI chemistry and an ABI3730 capillary electrophoresis-based genetic analyzer (Macrogen, Seoul, Korea). The partial 16S rRNA sequences obtained were matched against nucleotide sequences present in the GenBank database using the BLASTn program (Altschul et al. 1997). All sequences matched bacterial genera although only those confidently assigned to a genus (with at least the first 5 BLAST hits belonging to one genus) were retained for analyses. All 16S rRNA gene sequences were then deposited as environmental bacterial isolates from ectomycorrhizas in the Genebank nucleotide database for assignment of accession numbers (Table S2, see annex at the end).

Bacterial tolerance to nickel

For the estimation of Ni-tolerance of the isolated bacterial isolates, analytical grade salt was used to prepare 0.5 M stock solution of NiCl 2 . 6H 2 O. Each stock solution was filter-sterilized and added to Trypcase agar medium (Difco) to get final concentrations of 1, 2, 3, 5, 10 and 20 mM of Ni following the agar plate method as described in previous studies (Chaintreuil et al. 2007;Abou-Shanab et al. 2007) Only isolates able to grow in both Trypcase agar and liquid culture media containing 20 mM Ni were finally considered as Ni-tolerant.

PCR analysis of Ni-Tolerance determinants in bacterial isolates

Partial amplification of genes nreB and cnrT, known to confer Ni-tolerance in bacteria, was carried out using the following primers: nreB-425F (CCTTCACGCCGACTTTCCAG) and nreB-1179R (CGGATAGGTAATCAGCCAGCA) for amplification of nreB gene; and: cnrT-F (AACAAGCAGGTSCAGATCAAC) and cnrT-R (TGATCAGGCCGAAGTCSAGCG) (Chaintreuil et al. 2007) for amplification of cnrT gene.

Identification of Mycorrhiza Helper Bacteria through in vitro co-culture of mycorrhizosphere bacteria and ECM fungus

ECM-MB in vitro co-culture tests were performed on an aseptic piece of the pileus trama dry tissue as described by Perrier et al. (2006). Stock cultures from each sporocarp were obtained by transferring aseptically a piece of the pileus trama to a solid modified Melin-Norkrans (MMN) medium (Marx, 1969) containing KH 2 PO 4 (0.5 g.L -1 ), (NH 4 ) 2 ,HPO 4 (0.25 g.L -1 ), CaCl 2 (0.05 g.L -1 ), NaCl (0.025 g.L -1 ), MgSO 4 ,7H 2 O (0.15 g L -1 ), thiamine hydrochloride (100 mg.L -1 ), FeCl 3 ,6H 2 O (0.03 g L -1 ), glucose (10 g.L -1 ), malt extract (3 g.L -1 ), and Difco agar (14 g.L -1 ). The pH was adjusted to 5.6 with 1 M HCl and the medium was autoclaved for 20 min at 120°C. All ECM-MB co-cultures were maintained at 28°C on the same medium.

The detail of MB isolates tested and used for co-culture with Pisolithus albus isolate P-278 is reported in Table S1. Pisolithus albus isolate P-278 was isolated from volcano-sedimentary soil of New Caledonia (Jourand et al. 2010a). In total, 13 MB isolates including Pseudomonas

(5 isolates) Burkholderia (4 isolates), Bacillus (3 isolates) and one isolate of Lysinibacillus were tested. The Pisolithus albus isolate P-278 was grown in Petri dishes on MMN agar at 28°C for 2 weeks before using for co-culture with MB. Bacterial strains were cultured overnight in Trypcase Broth (Difco Laboratories) and 10µl of each pure MB culture was transferred onto Petri dish containing Pisolithus albus isolate P-278 on MNM agar medium.

For all isolates tested, a set of five Petri dishes were prepared, sealed with tape to avoid drying and then incubated at 28°C. The control treatment was prepared from a Petri dish containing the same agar medium with fungi but without bacteria. Fungal growth measurements were made by using the foot ruler, and the mean radial growth (mm 2 ) in two perpendicular directions was calculated.

Statistical analyses

We used F 2 independence tests to evaluate the equality of proportions between bacterial populations.

The data obtained from the in vitro co-culture tests between MB strains and Pisolithus albus P-278 reference strain were subjected to a one-way analysis of variance (ANOVA) and the MB strains were ranked into homogenous mean groups according to the Bonferroni's multiple range test at P < 0.05 (Dagnélie, 1975), using XLSTAT¥ (see reference above).

Multiple Correspondence Analysis was performed to examine the relationships between the soil type (ultramafic and volcano-sedimentary soils), ECM fungal genera, and Mycorrhizosphere Bacteria genera, presence of nreB and cnrT genes and in vitro Ni-tolerance of Mycorrhizosphere Bacteria. Computations and graphical display were made using the XLSTAT¥ software package (version 2010.5.04, Addinsoft, Paris, France, http://www.xlstat.com). In this MCA analysis, the ECM fungal genera from which the MB isolates originated were characterized in a previous study (Waseem et al. 2011a, submitted).

Results

Diversity of mycorrhizosphere bacteria

About 200 Mycorrhizosphere Bacteria (MB) isolates were obtained and characterized overall from the ECM root tips of the 5 Tristaniopsis species found in both ultramafic and volcanosedimentary soils. As detailed in Table S2, the 16S rRNA gene sequence analysis allowed to distinguish 10 MB genera overall, including four dominant ones i.e. with number of isolates in proportions higher than 10% for both soils combined: Pseudomonas (30.6%), Burkholderia (28.1%), Bacillus (18.7%) and Enterobacter (15.6%). The ultramafic soils exhibited a more diverse MB community than volcano-sedimentary ones (10 vs 6 genera respectively) with Rhizobium, Methylobacterium, Paenibacillus and Ewingella exclusively found in ultramafic soils though represented in very low proportions (Table S2). In the ultramafic soils, the MB community was slightly dominated, although not significantly, by Pseudomonas, which contributed to 30.4% percent of total isolates, followed by Burkholderia (20.8%), Bacillus (19.2%) and Enterobacter (17.6%), these four genera together covering about 88% of the total bacterial population. The other MB genera detected in proportions lower than 10% were:

Lysinibacillus, Brevibacillus, Rhizobium, Methylobacterium, Paenibacillus and Ewingella. In volcano-sedimentary soils, Burkholderia was the most represented genus with 42.2% of the total number of isolates, followed by Pseudomonas (24.4%), Bacillus (13.3%) and

Enterobacter (6.7%). Lysinibacillus was represented by less than 10% of isolates in both ultramafic and volcano-sedimentary soils, i.e. 5.6 and 8.9% respectively.

Tolerance of Mycorrhizosphere Bacteria to nickel in vitro

As shown in Figure 1, based on the ability of MB isolates to grow on Trypcase medium containing up to 20 mM Ni, the proportion of Ni-tolerant isolates originating from ultramafic soils was much higher than in volcano-sedimentary soils, 62 and 24% respectively, all MB genera combined (significant soil effect according to F 2 independence test at p>0.999).

Among the Ni-tolerant MB isolates from ultramafic soils, 32% were affiliated to Pseudomonas, 26% to Burkholderia, 19% to Bacillus and 16% to Enterobacter as illustrated in Figure 2. The contribution of the different genera was similar among the Ni-susceptible MB isolates (Fig. 2). This showing, that Ni-tolerance was not taxon-dependent, at least at genus level (not significant differences between the proportions of the different genera among

Ni-tolerant and Ni-susceptible populations according to F 2 independence test at p>0.95).

Concerning the MB originating from volcano-sedimentary soils, a majority of Ni-tolerant isolates belonged to Burkholderia and Pseudomonas (data not shown) (observed F 2 = 5.122 at P > 0.95 for df =4).

Detection of nreB and cnrT genes in Mycorrhizosphere Bacteria

As reported in independence test) between the total proportion of cnrT + isolates from ultramafic soils and that from non-ultramafic ones, 12 and 7% respectively.

Association among isolated mycorrhizosphere bacteria and ectomycorrhizas

As illustrated in Figure 3, the multiple correspondence analysis highlighted tight proximities between ultramafic soils, presence of nreB gene and Ni-tolerance, along with a dominance of Pseudomonas and Pisolithus. By contrast, the three other major MB genera tested, i.e.

Bacillus, Burkholderia and Enterobacter, do not appear to be strongly correlated to any factor, except Burkholderia that contained a higher proportion of Ni-susceptible MB isolates in volcano-sedimentary soils. Among the ECM genera, only Pisolithus is correlated to other parameters, as seen above, but also Cortinarius, which isolates harboured MB isolates with low proportions of nreB and cnrT genes compared to other ECM genera. Actually, the MCA graph represented in Figure 3 integrates and reflects the unbalanced proportions between MB isolates from ultramafic soils and those from volcano-sedimentary ones, since about 3 times more isolates originated from ultramafic soils. This results in a concentration of many factors and parameters of the analysis towards the "Soil-UM" barycentre of the graph. Thus, as illustrated in the graph, we observe correlations between the Ni-susceptibility of MB, the absence of cnrT and nreB genes in MB isolates, although the barycentre of these parameters are not tightly grouped and correlated together as for ultramafic soils. Among MB genera, only Burkholderia appears to be closely associated to volcano-sedimentary soils, which is confirmed by the results reported in Table 2 showing proportions of Burkholderia isolates 2 times higher than in ultramafic soils.

For clarity, the different host Tristaniopsis species were not taken into consideration in the MCA analysis since we did not observe any specificity between them and their respective MB isolates in preliminary multidimensional analyses, especially as the MB isolates originated in large majority from the ubiquitous T. calobuxus host.

Effect of mycorrhizosphere bacteria on in vitro Pisolithus albus growth

As shown in Table 4, there was a significant effect of the in vitro co-culture of MB strains on the growth of Pisolithus albus strain P278, when measured in terms of increase in diameter of the fungal hyphae culture in Petri dishes, as attested by the Anova analysis at P < 0.05. Based on the homogeneous mean groups obtained from the Newman & Keuls' multiple range test at P < 0.05, we were able to rank the MB isolates into three distinct groups with reference to the control treatment without any MB isolate. Therefore, from the 13 total MB isolates tested, four of them stimulated significantly the fungal growth while three had no effect on mycelial growth and six had an inhibitory effect. Among the four MB strains that promoted fungal growth, one representative of each MB genus tested was found, i.e. Pseudomonas, Lysinibacillus, Bacillus and Burkholderia. On the other hand, Pseudomonas and Bacillus strains were found as neutral while Pseudomonas and Burkholderia strains had a negative effect on hyphal growth of Pisolithus albus. Among the four fungal growth-promoting MB strains, Pseudomonas isolate G7 was the most efficient one since the mycelial growth of Pisolithus albus strain P-278 was 49% more higher than that of controls (without co-bacterial culture) when co-cultivated together. The three other efficient strains increased the fungal growth by 25 to 29%.

Discussion

Although many studies have been performed so far on the adaptation of microorganisms to metal-rich or -toxic soils, this is the first one carried out on the functional characterization of Mycorrhizosphere Bacteria associated with an ectomycorrhizal tree. This is particularly interesting in the context of naturally metal-rich ultramafic soils as found in New Caledonia since these original conditions led to the development of high degree of endemism and specific adaptations in plant species such as Tristaniopsis spp. (Myrtaceae) and in their associated symbiotic fungi. Although several authors (Moser et al. 2009;Amir et al. 2007;Branco and Ree 2010) did not find any significant difference in the diversity and composition of ECM fungi in such extreme ultramafic soils, even lower in some cases [START_REF] Sheets | Diversity of ectomycorrhizal fungal communities and Cenococcum geophilum populations from serpentine and nonserpentine soils[END_REF], our previous study (Waseem et al, 2011a, submitted) focused on the diversity of ectomycorrhizas associated to Tristaniopsis spp. in the same context revealed a higher fungal species richness in ultramafic soils than in volcano-sedimentary ones. Similarly, the present study showed a higher diversity of Mycorrhizosphere Bacteria (MB) in ultramafic soils than in volcano-sedimentary soils. However, in both studies, the supplementary MB and ECM taxa found in ultramafic soils constituted a minority in terms of proportion of isolates while the major genera present in the two soil types were identical. Thus, the majority of MB isolates were affiliated to four different genera represented by Pseudomonas, Bacillus, Burkholderia and Enterobacter, which represent together 87 and 89% of the total number of isolates in each of the two sites. The presence of these genera in serpentine soils has already been reported in previous studies, as in the case of serpentine soils in Oregon (Abou-Shanab et al. 2003;Abou-Shanab et al. 2007) from which Burkholderia, Pseudomonas, Rhizobium and Bacillus represented the majority of isolates. Surprisingly, Actinobacteria constituted the majority of isolates among bacterial communities isolated from ultramafic New-Caledonian mining sites in a study by Héry et al. (2003) although they originated from non-vegetated bulk soil.

Although all three serpentine sites of our survey shared the four major taxonomic groups, Pseudomonas isolates were more abundant than those of other genera. Similarly, Pseudomonas isolates were dominant in the rhizosphere of the nickel hyper-accumulating plant Alyssum bertolonii whereas Streptomyces were predominant in bulk soil samples (Mengoni et al. 2001).

Overall, Proteobacteria constitute about more than 62% of the total mycorrhizosphere bacterial flora associated to Tristaniopsis spp. in ultramafic soils, i.e. including all major genera except the Firmicute Bacillus. These results are in accordance with those obtained by Abou-Shanab et al. (2003) who reported a majority of rhizospheric bacteria belonging to Proteobacteria. In the same way, Turgay and his colleagues (2011) found a majority of Proteobacteria in the rhizospheric bacterial population isolated associated to Isatis pinnatiloba and Alyssum dasycarpum naturally growing on serpentine soils from Turkey, as well as Mengoni et al. (2004) in the nickel hyper-accumulating plant Alyssum bertolonii from serpentine soils.

Despite the high diversity of MB taxa found, we have not observed any particular specialization of a given bacterial genus in the Ni-tolerance ability since the proportions of Ni-tolerant isolates, as tested under in vitro conditions, were similar in all MB genera within each soil origin. However, it was clearly demonstrated that the overall proportion of Nitolerant isolates from ultramafic soils was much higher than in volcano-sedimentary soils, 62% and 24% respectively i.e. about 2.5 times more. Interestingly, a majority of MBs isolated from ultramafic soils tolerated extreme Ni concentrations ranging from 10 to 20 mM Ni, which were considerably higher than those indicated in earlier studies. For example, Pal et al.

(2004) reported that bacteria obtained from natural serpentine soils of Andaman were able to grow in the presence of 5-7 mM NiCl 2 in vitro. Similarly, certain Pseudomonas strains isolated from the rhizosphere of the nickel hyper-accumulating plant Alyssum bertolonii from serpentine soils in Italy were able to grow up to 10 mM Ni (Mengoni et al. 2001). By contrast, Schlegel et al. (1991) who characterized the rhizobacterial community of nickel hyperaccumulating plants from ultramafic soils of New Caledonia found some strains tolerating up to 10-20 mM Ni, suggesting that these high levels of Ni-tolerance in vitro could be attributed to the high contents of soils in Ni. In the same way, specific nickel-tolerant ECM fungal strains from a specific ecotype of Pisolithus albus (Jourand et al. 2011b) have already been isolated from ultramafic nickel mines in New Caledonia.

Similarly, we found a much higher proportion of MB isolated from ultramafic soils that harboured the nreB gene, known to confer Ni-tolerance to microorganisms, compared to MB issued from non-ultramafic soils, 30% vs 9.5% respectively. By contrast, the occurrence of cnrT gene was generally low in all MB genera and both soils except in Bacillus from ultramafic soils (29%). It had been reported previously that loci nre and cnr played an important role in bacterial resistance to high concentrations of Ni. Chaintreuil et al. (2007) found a direct correlation between high nickel resistance of the Bradyrhizobium sp. bacterial strain STM2464 and the presence of the cnrT gene among a diversity of strains isolated from the legume host species Serianthes calycina in New Caledonia. Similarly, nreB genes were shown to confer nickel resistance in Achromobacter xylosoxidans 31A; a well studied Niresistant bacteria isolated from heavy-metal-contaminated sites, through a metal efflux mechanism that reduced the accumulation of nickel when grown on Tris-minimal medium (Grass et al. 2001). Abou-Shanab et al. (2007) found that the presence of nreB and cnrT genes in both gram-positive and gram-negative bacterial communities (from rhizosphere of Alyssum murale grown on Ni-rich serpentine soil) was strongly correlated to bacterial Ni-resistance. In our study, only the presence of nreB gene, found in much higher proportions (i.e. 3 times more) in ultramafic soils than in volcano-sedimentary ones, was correlated with the Nitolerance ability of MB isolates while cnrT appeared as insensitive to soil conditions.

However, the proportion of nreB + isolates remained a minority among the total MB population from ultramafic soils, i.e. 30%. This could be explained by the fact that nreB gene is needed but not sufficient to confer Ni-tolerance it might act in synergy with other genetic determinants involved in Ni-tolerance. Actually, more than seven different determinants encoding resistance to toxic heavy metals have been reported in the Ralstonia lineage, including nre and cnr genes conferring Ni-tolerance (Schmidt and Schlegel 1994). The expression of Ni-tolerance genes in soils, which have never been studied so far, remains to be assessed through the construction of cDNA libraries with use of specific primers since no correlation, particularly in this study, has been established so far between such gene expression and the occurrence of their corresponding genes.

The multiple correspondence analysis allowed to visualize clearly at the ecosystem level the relationships between the soil type (ultramafic and volcano-sedimentary soils), ECM fungal genera, MB genera, presence of nreB and cnrT genes and in vitro Ni-tolerance of MBs.

Considering the complexity of these associations and the unbalanced representativeness of isolates from each site and soil type, the corresponding MCA graph summarizes accurately the main trends observed throughout this study. Thus, five parameters from the five factors tested were tightly correlated: the most frequent and affine MB-ECM association was represented by the Pseudomonas-Pisolithus couple, which was dominant in ultramafic soils.

Correlatively, MB isolates from ultramafic soils (with high proportions of Pseudomonas isolates) contained the highest proportions of Ni-tolerant MB, themselves harbouring the highest proportions of nreB + gene. Such preferential associations between MB and ECM taxa have already been reported in many cases. For example, 2.2-fold increase in the ectomycorrhizal infection Acacia holosericea roots was observed when Pisolithus albus was co-inoculated with Pseudomonas strains HR13 and HR26 (Founoune et al. 2002;Frey-Klett et al. 2007;Garbaye 1994;Duponnois et al. 1993).

The "Mycorrhiza Helper Bacteria" (MHB) potential of the MB isolated in our study, that was evaluated according to the in vitro co-culture experiment between 13 MB isolates and Pisolithus albus strain P278, was found in the four genera tested, i.e. Pseudomonas, Lysinibacillus, Burkolderia and Bacillus. Despite a low number of isolates tested, the proportion of fungal growth-promoting MB reached about one third (31%) of the total number, which can be considered as a high ratio. To date many bacterial species have been reported as promoting ectomycorrhizal symbioses (Frey-Klett et al. 2007;Garbaye 1994) such as Pseudomonas, Burkholderia, Enterobacter, Bacillus, and Streptomyces. Rather than plantspecific (Garbaye and Bowen 1989), MHB strains are known as fungal specific since the fungal growth-promoting effect varies according to the co-cultivated ECM taxa or strains (Duponnois et al. 1993). This was observed by Duponnois and Garbaye (1991) who showed a significant promoting effect of Pseudomonas fluorescens BBc6 strain on the formation of ectomycorrhizas by an American Laccaria laccata isolate in Douglas-fir, while the same MHB strain associated to a L. laccata strain native to Australia inhibited ectomycorrhizal formation on the same host plant species (Dunstan et al. 1998). Similar results were obtained with Streptomyces sp. AcH 505 MHB strain which had growth-promoting effects on Amanita muscaria and Suillus bovinus, while inhibiting the growth of Hebeloma cylindrosporum (Frey-Klett et al. 2007) due to the production of antibiotics by Streptomyces sp. (Keller et al., 2006). Actually, our data did not show any specificity between MB and ECM taxa in terms of MHB effect since, for example, the 6 Pseudomonas strains originating from ultramafic soils that were co-cultivated with Pisolithus albus P-278 strain had either positive, neutral or negative effects on mycelial growth. Moreover, despite originating from various sites, all Pseudomonas isolates were closely related phylogenetically as shown earlier (Waseem et al. 2011b). Therefore, the specificity between MB and ECM fungi can be rather considered as strain specific as already reported.

Finally, our results on MB diversity showing a preferential association or affinity between Pseudomonas and Pisolithus in the ultramafic sites suggest that the specificity between MB and ECM fungal partners could originate from a co-evolutionary process that would result in a synergistic action of the metal-tolerance mechanisms. b nreB + and cnrT + : occurence of these genes in Mycorrhizosphere Bacteria ; nreB -and cnrA -:

genes not detected)

General discussion and conclusions

The results obtained in the different studies permitted to reply the objectives of thesis. These In this study we have investigated the effect of both ultramafic and volcano-sedimentary soils on ECM fungal diversity associated to various Tristaniopsis spp. Although low microbial diversity was hypothesized by some researchers (Rothschild and Mancinelli 2001;Héry et al. 2005) in such soils, we found highly diverse ECM fungal communities across both ultramafic and volcano-sedimentary soils, and even a higher number of ECM genera in the former. We failed to detect a depauperate ECM community specific to ultramafic soils and marked the presence of all major genera within the Ascomycota and Basidiomycota such as Cortinarius, Pisolithus, Russula and Thelephora etc. in both soil types, suggesting that the ultramafic soils may not be a strong evolutionary agent for selection of particular ECM fungal community. In fact, it has been reported by various scientists from other parts of the world that ECM fungal communities from serpentine sites followed similar patterns of diversity as communities from non-serpentine sites [START_REF] Moser | Comparison of ectomycorrhizas of Quercus garryana (Fagaceae) on serpentine and non-serpentine soils in southwestern Oregon[END_REF]Moser et al. 2009;Branco and Ree 2010). For instance, Branco and Ree (2010) who explored the ECM fungal communities associated to an oak tree (Q. garryana) from adjacent serpentine and non-serpentine soils in Northeastern Portugal, reported the presence of various and similar genera within Ascomycota across both serpentine and non-serpentine soils. The abundant ECM fungal lineage they observed belonged to Agaricales, Boletales, Russulales, Thelephorales etc. They proposed that the ultramafic soils may not be strong evolutionary agent for selection of particular ECM fungal community (Moser et al. 2009). Indeed, the major potential threats that degraded ultramafic soils may pose to ECM fungi could be the higher heavy metal contents and nutrient deficiency which may adversely affect the fungi in variety of ways. However in the last decade, it was described that ECM fungi could tolerate high concentrations of heavy metals [START_REF] Blaudez | Cadmium uptake and subcellular compartmentation in the ectomycorrhizal fungus Paxillus involutus[END_REF]Jourand et al. 2010b;Jourand et al. 2010a) and revealed several mechanisms and physiological strategies in ECM for coping with heavy metals (Hartley et al. 1999).

Therefore, ECM fungi from ultramafic soils can be invulnerable to these chemical threats exerted by ultramafic soils and might be able to tolerate high concentrations of heavy metals present in these soils. On the other hand, we observed that the studied ultramafic soils contained higher N and P compared to the volcano-sedimentary soils. This increased fertility level could explain the high ECM fungal diversity found in these soils (Branco and Ree 2010).

b) Soil effects on the mycorrhizosphere bacterial diversity

In the next step of our study, we tried to figure out the soil effect on mycorrhizosphere bacterial distribution associated to the different ECM-Tristaniopsis spp. symbioses. It is well known that certain metals are essential for proper functioning of several microbial enzymes, such as Ni for CO dehydrogenase, urease, and hydrogenase (Hausinger 1987). However they are toxic at high concentrations and can change the microbial community structure (Sandaa et al. 1999). The beneficial effects of ectomycorrhizal symbioses (ECM) and mycorrhizosphere bacteria (MB) for plants in nutrient-poor environments are well known, such as N 2 -fixation and bio-availability of P, transformation of organic matter, improvement of the soil fertility etc... [START_REF] Vecchio | Negative effects of scale insect herbivory on the ectomycorrhizae of Juvenile pinyon pine[END_REF]Leyval et al. 1997;Smith and Read 1997;Garbaye 1994).

However, the New Caledonian soils are an interesting case study since MB are in contact with metal, particularly Ni, which have been present in these substrates for millions years (Hery (Héry et al. 2003). Our results showed that the MB population in Tristaniopsis mycorrhizosphere was mainly composed of Proteobacteria (69% of isolates) and Firmicutes (29% of isolates). Proteobacteria where mainly represented by Pseudomonas, Burkholderia, and Enterobacter while Bacillus was the most numerous in the Firmicutes. Moreover, a higher richness in MB genera was found in ultramafic soils compared to volcano-sedimentary soils, although they were represented by low proportions of isolates, Very limited research has been carried out, to our knowledge, to explore the bacterial community composition of New Caledonian soils. Our results were in accordance with those obtained by Herrera et al. (2007) who worked on similar constrained ecosystems. These authors who compared the microbial diversity of bare mine spoils with that of re-vegetated ones by Gymnostoma webbianum, suggested that the introduction of plants would allow the increase of Proteobacteria populations due to carbon and nitrogen inputs in the form of root exudates from the plants, and thus creating a situation similar to that of a natural soil environment. We were in a similar situation in exploring the diversity of the mycorrhizosphere bacteria associated to Tristaniopsis plant species. We suggest that plant roots may support diverse bacterial communities with the potential to interact with the ectomycorrhizal symbionts in a variety of mechanisms and therefore, bacterial communities' size and distribution could play an important role in determining the nature and extent of bacterial effects on mycorrhiza formation and its associated plant growth.

c) MB and ECM associations in Tristaniopsis rhizosphere

The elevated levels of heavy metals generally inhibit plant growth and metabolism [START_REF] Sheoran | Effect of cadmium and nickel on photosynthesis and the enzymes of the photosynthetic carbon reduction cycle in pigeonpea (Cajanus cajan L.)[END_REF]) and New-Caledonian soils are marked with the presence of not only higher metal contents such as nickel but also the rarity of essential nutritive elements (Héry et al. 2003;Cluzel et al. 2001). These environmental effects are evident and have been expressed through differences and changes in plant composition, turning the archipelago into a biodiversity hotspot (Myers et al. 2000). Mostly both plant and soil microbes play a vital role in the adaptation to soil conditions mainly to heavy metal uptake and tolerance (Khan 2005). It has been reported that most of bacteria tolerant to high concentrations of heavy metals in plant rhizosphere belong Proteobacteria group (Abou-Shanab et al. 2007;Herrera et al. 2007).

They are also known to have specific genetic determinants involved in heavy metal tolerance.

For example, one of the most studied Proteobacteria model for its high level of metal resistance is Ralstonia eutropha strain CH34 (Schmidt and Schlegel 1994) in which at least seven different determinants encoding resistance to toxic heavy have been found [START_REF] Taghavi | Genetic and physical maps of the Alcaligenes eutrophus CH34 megaplasmid pMOL28 and its derivative pMOL50 obtained after temperature-induced mutagenesis and mortality[END_REF]). Interestingly, we found higher proportions of Ni-tolerant bacteria in Pseudomonas and Burkholderia genera along with some Bacillus isolates (Firmicutes). Moreover, they harboured significantly higher proportions of nreB and cnrT genes, previously reported to confer Ni-tolerance in bacteria. Our results showed that Ni-tolerance determinants were independent of bacterial genus and appeared to be shared equally between gram-positive and -negative bacteria. By contrast, a soil effect on the occurrence of Ni-tolerance determinants was highlighted, nreB and cnrT genes being found in higher proportions in MB from ultramafic soils than in those from volcano-sedimentary ones, thus confirming the selective effects of ultramafism on the evolution of metal-resistance genes as demonstrated in other microorganisms in the literature.

Although the concept of mycorrhiza helper bacteria (MHB) is very well established (Frey-Klett et al. 2007;Garbaye 1994), our results obtained from co-culture tests between MB and Pisolithus albus could be regarded to our knowledge as the first study characterizing the helper effect or mutual associations between MB and ECM from ultramafic soils. We observed that certain MB were more closely associated to particular ECM such as Pseudomonas with Pisolithus in ultramafic soils dominated by Tristaniopsis calobuxus. The possible reason behind this interaction could be either fungal specificity for particular bacteria or vice a versa, although there are several hypotheses for possible mechanisms of interactions between fungal symbiont and their associated bacteria (Garbaye 1994). However, it is not yet clear whether MHB lives preferentially in association with ECM rather than in other soil compartements without ECM.

d) Perspectives

The conclusion derived from this work, interrogates various important aspects that should be focused in the future research plan in these soils. Firstly, some of the results of the present study are partial such as phylogenetic studies that should be completed on priority basis.

Secondly, continuation of characterization of microbial diversity in other plant species in order to have a clearer idea about the plant species effect on overall microbial diversity in such extreme soils.

In the first chapter, we observed that the soil type did not affect strongly the ECM diversity, but we did not know what molecular mechanism could be involved in the adaptation of ECM fungi to these soils. Therefore, the characterization of these mechanisms may provide us new determinants useful to understand the adaptation of these organisms to extreme environments which can be exploited in re-vegetation of these soils. Moreover, genotypic studied should be carried out to identify specific ECM ecotypes highly adapted to these soils (Jourand et al. 2010a) based on ITS-RFLP and AFLP analyses in order to establish a correlation between soil types (ultramafic vs volcano-sedimentary) and fungal ecotypes.

The alternative strategy to identify more accurately the genes involved in Ni-tolerance of the studied ECM fungi and/or their associated MB could be the construction of cDNA libraries using RNA extracted from ectomycorrhizal roots. cDNA libraries are commonly used to track genes of interest. Specific primers can be used from these highly conserved sequences into specifically amplified Ni-tolerance genes present in the cDNA library. Cloning and subsequent sequences of complete genes and their comparison with published sequences should enable us to assess the gene variability as a function of soil type and to identify new determinants of Ni-tolerance in both ECM and MB associated to these soils.

Moreover, the study of MHB is essential in improving the knowledge of how mixed microbial communities stimulate the formation of mycorrhizae. Little is known about the molecular mechanisms induced by MHB and involved in promoting growth of mycobiont. A deeper study on MHB could generate a model for genomic analysis of bacteria-fungus interactions, Further research is needed to explore the MHB effect on associated ECM particularly in ultramafic soils and more specifically in the rhizosphere of plant species other than Ni hyper-accumulators that have the ability to grow on both metalliferous and non-metalliferous soils, which is a prerequisite for their use in strategies for ecological restoration of mine sites.

Figure 1 Figure 1 . 2 .Figure 2 . 2 .Figure 2 Figure 2 . 4 .Figure 2 . 5 .Figure 2 . 6 .Figure 2 . 7 .

 11222224252627 Figure 1.1. Map of New Caledonia; Ultramafic soils covering almost 5500 sq. km of ''La

Figure 2 . 8 .Figure 2 .

 282 Figure 2.8. Different mechanisms of heavy metal tolerance in ECM

Figure 2 .Figure 2 .Figure 2 .Figure 2 . 14 .Figure 2 .Figure 2 .Figure 2 .Figure 2 .Figure 2 .Figure 1 .Figure 2 .Figure 1 .Figure 2 .Figure 1 .Figure 2 .Figure 3 .

 222214222221212123 Figure 2.11. Example of CDF protein

Figure 1 :

 1 Figure 1: Map of New Caledonia; Ultramafic soils covering almost 5500 sq. km of ''La Grande Terre''.

  b) Transition season (mid-April to mid-May) when rainfall decreases. c) Cool season (mid-May to mid-September) with minimum temperature (sometimes below 10°C), more occasional rainfall during the cooler season (1,500 -2,500 mm annually) resulting from cold fronts associated with polar low pressure areas and then d) Another Transition period (mid-September to mid-November) with generally clear weather and increasing southeast trade winds along with high temperature and minimum rainfall.

Figure 2 :

 2 Figure 2: Hot spots of world's biodiversity (source Conservation International)

  Biodiversity and molecular characterization of ectomycorrhizas (ECM) associated to Tristaniopsis spp. in ultramafic and volcano-sedimentary soils; Biodiversity and molecular characterization of mycorrhizosphere bacteria (MB) isolated in different Tristaniopsis spp. from both soil types; Adaptation of isolated MB to Ni through: i) the analysis of their Ni-tolerance ability in pure culture; and ii) the research of cnrT and nreB genes, considered to be involved in Nitolerance.

  results obtained during this research work are actually under evaluation and are being reviewed by International Journal reviewers. However, to date, two manuscripts have already been submitted to International Journals and proposed list of articles is as follows: International Congress on Mycrrhizal Symbiosis, Ecosystems and Environment of Mediterranean area (Mycomed 2010) October 10-13, 2010, Marrakech, Morocco. Galiana A, Lebrun M, Domergue O, Duponnois R, Prin Y, Ducousso M, 2010. Biodiversity of ultramafic soils; for successful re-vegetation of mining sites in New Caledonia. In: 4 th Congress of European Microbiologists FEMS 2011, June 26-30, 2011, Geneva, Switzerland. Waseem M, Galiana A, Lebrun M, Domergue O, Duponnois R, Prin Y, Ducousso M, 2010. Biodiversity and adaptive strategies of mycorrhizosphere bacteria associated to endemic Tristaniopsis species. In: International Symposium on Microbial Ecology (ISME13), August 21-27, 2010, Seattle, USA. Waseem M, Ducousso M, Lebrun M, Domergue O, Duponnois R, Prin Y, Galiana A, 2010. Biodiversity of mycorrhizosphere bacteria associated to endemic Tristaniopsis species. In: 9 the International Mycological Congress (IMC9: The Biology of Fungi), August 1-6, 2010, Edinburgh, UK. Domergue O, Waseem M, Prin Y, Plassard C, Guinberteau J, Bakker M, Augusto L, Trichet P, Saur E, Hinsinger P, Jaillard B, M'Balla J, Duponnois R, Galiana A. 2008. Diversity and Psolubilizing ability of mycorrhizosphere bacteria associated with Pinus pinaster in the Landes forest ecosystem. In: 21 st New Phytologist Symposium, December 10-12, 2008, Montpellier, France (http://www.newphy-tologist.org/mycorrhizal/21stNPS.pdf).

  (a) In case of soil pollution: The soil excavation, transport, washing and metal extraction of contaminated soils. (b) For water pollution: Pumping and treating of contaminated water and addition of chemical reactant like H 2 O 2 or potassium permanganate and incineration.

Figure 2 . 1

 21 Figure 2. 1 Schematic diagram of possible abiotic and biotic interactions in the mycorrhizosphere of plants in forest soil ecosystems (after Finlay et al. 2006) Dashed black arrows indicate C flow while flow of nutrients is represented by solid grey arrows and flow of water by dotted arrows. (1) Nutrient uptake involve direct uptake by roots from dissolved inorganic nutrient sources, (2) or from mineral substrates via physicochemical weathering

Figure 2 . 2 Figure 2 . 3

 2223 Figure 2. 2 Hypsographic curve of the two sides of the "Grande Terre" (Orstom 1981)

Figure 2 . 4

 24 Figure 2. 4 Schematic diagram of typical profile of soil from ultramafic rocks

Figure 2 Figure 2 . 6

 226 Figure 2.5 Microscopic view with transversal cut of a mycorrhizal root from Tristaniopsis plant

Figure 2 . 7

 27 Figure 2.7 Spiral view of Periodic Table (http://umbbd.msi.umn.edu/periodic/spiral.html)

  a. Reduction of metal exposure by excretion of chelating substances; b. Extracellular sequestration (e.g. by mucilage, pH gradients in the rhizosphere); c. Modified uptake systems at the plasma-lemma level; d. Intracellular detoxification.

Figure 2 . 8 .

 28 Figure 2.8. Different mechanisms of heavy metal tolerance in ECM (Jentschke and Godbold 2000).

  2+ is usually the substrate for transport. When present in excess, Ni can enter cells indiscriminately via Mg 2+ transport systems or via Co 2+ transport systems. However, when concentration of Ni is limited, it is imported into the cells via proteins that have a high specificity and affinity for Ni. There are two major types of Ni transporters in bacteria. One is an ATP-binding cassette (ABC) type transporter, such as NikABCDE characterized in Escherichia coli while the other is a Ni-specific integral membrane protein, a permease (HoxN/NixA)[START_REF] Navarro | The nik operon of Escherichia coli encodes a periplasmic binding-protein-dependent transport system for nickel[END_REF]. In NikABCDE (a five-component system), NikA is the soluble, periplasmic protein that binds Ni 2+ , while NikB and NikC form a hydrophobic trans-membrane pore for Ni transport into the cytoplasm and NikD and NikE are involved in ATP hydrolysis and couple the energy generated from ATP hydrolysis to Ni 2+

Figure 2 . 9 .

 29 Figure 2.9. Molecular models of e ux (a) P-type ATPase and (b) RND chemiosmotic antiporter (After Silver and Phung 2005)

Fig 2 .

 2 Fig 2.10 and 2.11), ZnT6 and ZnT7 and in higher plants ZAT, etc. (Nies 2003).

Figure 2 .

 2 Figure 2.11 Example of CDF protein (Haney et al. 2005) Figure 2.10 Model of zinc transport in animal cell (Devis et al. 2009)

Figure 2 .

 2 Figure 2.12 Interplay of transporters of di erent metal e ux families in metal detoxification (Haney et al. 2005) (High-level metal resistance is accomplished by the interplay of at least three families of metal e ux transporters in a two-step process. Cation di usion facilitator (CDF) proteins translocate substrates across the cytoplasmic membrane utilizing the proton-motive force for energization. Likewise, CPx-type or P-type ATPases transport metal cations under ATP-hydrolysis from the cytoplasm to the periplasm. Tripartite HME-RND e ux complexes couple the antiport of protons with the e ux of periplasmic cations to the outside. Re-entry of metal cations under high metal concentration might be prevented by the expression of selective porins in the outer membrane)

Figure 2 .

 2 Figure 2.13 The mycorrhiza helper bacteria (MHB) strain Pseudomonas fluorescens BBc6R8 develops biofilm-like structures on the hyphal surface of the ectomycorrhizal fungus Laccaria bicolor S238N in vitro (from Frey-Klett et al. 2007)

Figure 2 .

 2 Figure 2.14 Simplified representation of interactions between MHB, ECM fungus, plant root and soil during the establishment of the root-fungus symbiosis (after Frey-Klett et al. 2007) Solid arrows (1-4) represent specific helper functions. (1) The bacterium improves soil conductiveness to the fungus; (2) the bacterium improves root receptiveness to the fungus; (3) the bacterium interacts with plant-fungus recognition and symbiosis establishment; (4) the bacterium promotes germination of fungal propagules and survival and growth of the mycelium; (5) the fungus selects bacterial populations in the rhizosphere; (6) the soil supports pre-symbiotic fungal growth; (7) the soil determines root receptiveness to the fungus; (8) roots select bacterial populations in the rhizosphere.

(

  2008) isolated Microbacterium from the roots of Brassica napus. The genera Stenotrophomonas, Arthrobacter and Microbacterium were also isolated from Oregon serpentine soils in U.S.A. (Abou-Shanab et al. 2003). Herrera et al. (2007) observed a significant modifications in the phylogenetic composition of bacterial communities following nickel mining activities, such as the decreased proportion of

  just outside as in most other plants. The leaves are evergreen, alternate to mostly opposite, simple, and usually with an entire (not toothed) margin. The flowers have a base number of five petals, though in several genera the petals are minute or absent. The stamens are usually very conspicuous and brightly colored. Tristaniopsis Brongn.& Grise is a genus of 40 species of shrubs and trees in the family Myrtaceae. They have a wide distribution, ranging from Burma, Thailand, Malaysia, New Caledonia and Australia. Thirteen endemic Tristaniopsis species are found in New Caledonia:

  Perrier and his colleagues (2006) has identified the existence of ectomycorrhizal (ECM) symbioses with Tristaniopsis spp. in the New Caledonian heavy metal enriched soils and suggested that mycorrhizal symbioses could play an important role in the adaptation of these endemic plants to these extreme soils. We selected Tristaniopsis spp. (Myrtaceae) as a model plants for our study because of: a) Their growing ability on both metalliferous (ultramafic) and volcanosedimentary (with lesser Ni contents) for certain species: b) Their taxonomic proximity with the ECM model plant Eucalyptus In this study, we have studied and isolated the microbes (ECM and MBs) from the rhizosphere of Tristaniopsis calobuxus, T. glauca, T. guillainii, T. macphersonii, T. ninndoensis. Tristaniopsis calobuxus This species appears as a shrub or small tree, up to 4 m high, branching dense young branches covered with whitish hairs, bark smooth, light gray. Generally they grow up in groups that are close together but not densely clustered or matted. Tristaniopsis calobuxus have shown the antimalarial activity in vitro due to ellagic acid and a newly identified compound, the 3,4,5trimethoxyphenyl-(6 'galloylation-O-)-O-beta-D-glucopyranoside (Verrot et al. 2001).This species is widely spread on the Grande Terre, most commonly on all the ultramafic soils including ferralitic soils which are more or less acid soils. It can also be found on the metamorphic Northern Province as shown in Fig.2.15 (http://www.endemia.nc-/flore/fiche1402.html).

Figure 2 2

 22 Figure 2.15 Geographic distribution of Tristaniopsis calobuxus in New Caledonia

Figure 2 .Figure 2 .

 22 Figure 2.16 Geographic distribution of Tristaniopsis glauca in New Caledonia

Figure 2 .

 2 Figure 2.18 Geographic distribution of Tristaniopsis macphersonii New Caledonia

Figure 2 .

 2 Figure 2.19 Geographic distributions of Tristaniopsis ninndoensis in New Caledonia

2

 2 ultramafic and non-ultramafic or volcano-sedimentary soils.Jourand et al. (2010a) andPerrier et al. (2006) have already reported a full description, including climate, geology, and vegetation structure of these ultramafic sites. The two ultramafic sites which mainly composed of ferralsol(Journad et al. 2010a) include (i) Koniambo massif (21°00.412S, 164°49.888E) located in the Northern Province and (ii) Forêt Desmazures (22°12.161S, 166°37.484E) found in the south of the main island (Fig.1). On the other hand, two volcano-sedimentary mainly ferric acrisol,[START_REF] Gillespie | Tropical dry forests in New Caledonia[END_REF] were Mont Ninndo (20°16.649S, 164°11.576E) and Col d'Arama (20°16.756S, 164°11.618E); situated in the extreme North of archipelago (Fig. Site selection and sampling ECM root samples were collected in June 2008 from Tristaniopsis stands growing on both ultramafic and volcano-sedimentary soils. Only stands dominated by Tristaniopsis spp. were selected for root collections. ECM root tips were collected by gently removing the upper layers of moss and litter on two to three individuals per plot. Each sample unit consisted of a total volume of 20 to 100 g roots from one plant. Root samples were collected with their adhering soil from four Tristaniopsis species including: T. calobuxus, T. glauca, T. guillainii and T. ninndoensis. Tristaniopsis ninndoensis was exclusively found on volcano-sedimentary soils i.e. Mont Ninndo, contrary to T. calobuxus that was ubiquitous and found almost all over

DNAeasy

  Plant Qiagen Kit (Qiagen, Courtaboeuf, France). Approximately 650-base pair (bp) fragment of nuclear ribosomal ITS rDNA containing the 5.8S region was amplified with specific primers ITS1-f (5'-CTTGGTCATTTAGAGGAAGTAA-3')(Gardes and Bruns 1993) and ITS4 (5'TCCTCCGCTTATTGATATGC-3')(White et al., 1990). PCR reactions were prepared in a total volume of 25 mL containing aliquots of 1 mL of genomic DNA at 50 ng mL-1, 1 mM of each primer, 1.5 U of Taq DNA polymerase (Promega, Charbonieres, France), Promega Taq polymerase buffer, 2 mM MgCl 2 and 200 mM dNTP. Amplification was performed with a DNA thermocycler Mastercyclers Eppendorf system (Eppendorf, Hamburg, Germany) programmed as follows: one cycle for 3 min at 95 • C followed by 40 cycles at 95 • C for 30 s, 53 • C for 1 min, 2 min at 72 • C and a final extension for 10 min at 72 • C.

Figure 1 .

 1 Figure 1. General map of "Grande Terre" indicating the study sites and location of ultramafic massifs (in red). Ultramafic sites were represented by Koniambo massif and Forêt Desmazures; Volcanosedimentary sites, by Col d'Arama and Mont Ninndo.

Figure 2 .

 2 Figure 2. Proportions of isolates in the different ECM genera collected from ultramafic and volcano-sedimentary (Volc.sed) soils.

  bacteria (MB) associated to Tristaniopsis spp. (Myrtaceae) grown on Ni-rich ultramafic soils in comparison with that found on volcano-sedimentary soils; ii) to identify possible specificities, affinities or preferential associations between MB and ECM fungal taxa associated to different Tristaniopsis spp., these host plant species being ubiquitous (T. calobuxus) or specific to one of both soil types (i.e. the four other Tristaniopsis species studied).

  ultramafic and volcanosedimentary soils. The three ultramafic sites include (i) Koniambo massif (21°00.412S, 164°49.888E in Northern Province), (ii) Forêt Desmazures (22°12.161S, 166°37.484E) and Yaté (22°15.459S, 167°00.248E) which are situated in the Southern Province of New Caledonia (Fig. 1). While two volcano-sedimentary sites i.e. Mont Ninndo (20°16.649S, 164°11.576E) and Col d'Arama (20°16.756S, 164°11.618E) are located in the extreme north

Figure 1 .Figure 2 .Figure 3 .Figure 6 .

 1236 Figure 1. General map of New Caledonia describing the

  three plots per site dominated by Tristaniopsis and the roots were collected from at least three individuals (separated by 10-15 m) per plot. The roots of each tree were sampled at the base of trunks by tracing after removal of the upper layers of moss and litter, giving a total volume of 20 to 100 g of roots kept with their adhering soil. All root samples were stored in an ice box at 4°C until arrival at the laboratory 1-3 days after field collection. The roots were then gently separated from surrounding soil and rinsed with distilled water. The ECM root tips obtained from dissection of the mycorrhizal roots were individually transferred to Eppendorf tubes containing 50µl of sterile distilled water. The homogenates obtained from the crushed root tips were separated in two halves. One half of homogenate was used for further DNA extraction and molecular identification of the ECM fungus. The other half of homogenate was transferred onto Petri dishes containing Trypcase agar medium (Difco) before incubation at 28°C for 24 to 48h according to growth rate of the bacterial isolates. A mean of 1 to 2 bacterial isolates were obtained from each crushed root tip. After 3 successive subcultures of purification on Trypcase medium, a collection of about 168 bacterial isolates was obtained,

Figure 1 .Figure 2 .

 12 Figure 1. Proportions of Ni-tolerant a and Ni-susceptible a mycorrhizosphere bacteria according to their soil origin b , all bacterial taxa combined, a Bacterial isolates were considered as Ni-tolerant when they were able to grow on Trypcase medium supplemented with 20mM Ni, and Ni-susceptible when they were unable to grow on medium with 20mM Ni concentrations.

Figure 3 .

 3 Figure 3. Multiple correspondence analysis between soil type, mycorrhizosphere

  were first to characterize the different populations of ectomycorrhizal fungal (ECM) associated to various Tristaniopsis spp. in ultramafic vs volcano-sedimentary soils. Secondly, it was to explore the mycorrhizosphere bacteria (MB) diversity associated to these ECM in both soils types. Thirdly, to analyze the genetic determinants of Ni-tolerance in MB isolated from the different Tristaniopsis species and the specificity of MB-ECM associations in both ultramafic and volcano-sedimentary soils. The main results obtained in these different studies are summarized in this section. a) Ectomycorrhizal fungal diversity associated to Tristaniopsis spp.

Table 2 .

 2

		2. Minimal inhibitory concentrations (MICs) of several heavy metals in Escherichia
	coli	49

Table 2 .

 2 3. Various important protein families involved in heavy metal transport in bacteria 52 Table 2.4. Bacterial heavy metal resistance systems and mechanisms 52

Table 2 .

 2 5. Examples from the literature of mycorrhiza helper bacteria (MHB) enhancing

	mycorrhiza formation

Table 1 .

 1 Geographic locations of sampling sites and four host plant species studied 106

Table 2 .

 2 Soil origin, number of root samples, ECM tips and ITS sequences obtained from four

	Tristaniopsis host plant

Table 3 .

 3 Soil chemical properties of study sites: major element contents 108

Table 4 .

 4 Number of ECM taxa associated to Tristaniopsis plants obtained from different stu-

	dy sites

Table 1 .

 1 Soil properties of the sampling sites 139

Table 2 .

 2 Soil type and host plant species of the sampling sites 140.

Table 3 .

 3 Number of isolates obtained in the major MB genera according to soil type and

	Tristaniopsis host species

Table 4 .

 4 Total number of ECM root tips observed per fungus genus in both soil types

	142

Table 1 .

 1 Sampling sites, host plant species and total number of MB isolated 177

Table 2 .

 2 Proportions (in %) of the different Mycorrhizosphere Bacteria genera present in ultramafic and volcano-sedimentary soils 178

Table 3 .

 3 Effect of soil type on the occurrence of nreB and cnrT genes a in mycorrhizosphere bacteria isolates of the five most represented genera 179

Table 4 .

 4 Effect of Mycorrhizosphere Bacteria on growth of Pisolithus albus co-cultivated in

	Petri dishes

Table S1 .

 S1 Supplementary data containing ECM Isolates obtained from this study with

Table 2 .1. Description of different types of mycorrhizas (adapted from Smith and Read 1997)

 2

	Mycorrhizal	Arbuscular	Ecto	Ectendo	Arbutoid	Monotro-	Ericoid	Orchid-
	type					poid		aceous
	Fungal family	Glomeromycetes Basidiomycetes	Basidiomycetes	Basidiomycetes Basidiomycetes Asco	Basidio
			Ascomycetes	Ascomycetes			mycetes	mycetes
			Zygomycetes
	Plant taxa	Bryophytes	Gymnosperms	Gymnosperms	Ericales	Monotr-	Ericales	Orchid
		Pteridophytes	Angiosperms	Angiosperms		opoideae	Bryophytes	aceae
		Gymnosperms
		Angiosperms
	Intracellular	+	-	+	+	+	+	+
	colonization
	Fungal sheath	-	+	+/-	+/-	+	-	-
	Hartig net	-	+	+	+	+	-	-
	Vesicles	+/-	-	-	-	-	-	-
	Achlorophylly	-	-	-	-	+	-	+
	+ = present; -= absent

Table (http://umbbd.msi.umn.edu/periodic/spiral.html)

Table 2 .2. Minimal inhibitory concentrations (MICs) of several heavy metals in Escherichia coli (Mergeay et al. 1985)

 2

	Heavy Metal	MIC (mM)
	Mercury	0.01
	Silver, Gold,	0.02
	Chromium (Cr(VI)), Palladium	0.2
	Platinum, Cadmium	0.5
	Cobalt, Nickel, Copper, Zinc,	1
	Thallium, Uranium, Lanthanum, Yttrium, Scandium, Ruthenium,	2
	Aluminium,
	Lead, Iridium, Osmium, Antimony, Indium, Rhodium, Chromium (Cr(II)),	5
	Gallium, Vanadium, Titanium, Beryllium
	Chromium (Cr(III))	10
	Manganese	20

(Minimal inhibitory concentrations refer to the smallest concentration necessary to inhibit growth; thus, lower MIC values indicate more toxic metals and higher MICs indicate less toxicity)

Table 2 .3. Various important protein families involved in heavy metal transport in bacteria

 2

	Family	Direction of transport	Energy	Metal ions
	ABC	Uptake	ATP	Mn 2+ , Zn 2+ , Ni 2+ , Fe 2+
		Efflux	ATP
	P-type	Both	ATP	Mg 2+ , Mn 2+ , Ca 2+ , K + , Cu 2+ , Zn 2+ , Cd 2+ ,
				Pb 2+ , Ag +
	A-type	Efflux	ATP	Arsenite
	RND	Efflux	Proton gradient	Co 2+ , Cu 2+ , Ag + , Cd 2+ , Zn 2+ Ni 2+
	HoxN	Uptake	Chemiosmotic	Co 2+ , Ni 2+
	CHR	Antiport	Chemiosmotic	Chromate
	MIT	Uptake	Chemiosmotic	Most Cations
	CDF	Efflux	Chemiosmotic	Cd 2+ , Co 2+ , Fe 2+ , Zn 2+
	(Adapted from Nies 1999)

Table 2 .4. Bacterial heavy metal resistance systems and mechanisms

 2

	Toxic ions	Gene mnemonic	Protein function
	Hg 2+ and organo-mercurials	mer	Mercuric reductase and transport
	AsO 4 3 to and As(OH)3	ars	Arsenate reductase and transport
	As(OH) 3 3À	aso	Arsenite oxidase and transport
	AsO 4	arr	Respiratory arsenate reductase
	Cd 2+	cad	P-type efflux ATPase
	Cd 2+ , Zn 2+ , Co 2+	czc	CBA e ux permease
	Co 2+ , Ni 2+	cnr	CBA e ux permease
	Ni 2+ , Co 2+ , Cd 2+	ncc	CBA e ux permease
	Ni 2+	nre	CBA e ux permease
	Cu 2+ , Cu +	cop and pco	Copper resistance and transport
	CrO 4	chr	Chromate e ux permease
	(Silver and Phung 2005)

Table 2

 2

.5, such as gram-negative Proteobacteria (Agrobacterium, Azospirillum, Azotobacter, Burkholderia, Bradyrhizobium, Enterobacter, Pseudomonas, Klebsiella and Rhizobium), gram-positive Firmicutes (Bacillus, Brevibacillus, and Paenibacillus) and grampositive Actinomycetes (Rhodococcus, Streptomyces, and Arthrobacter).

Table 2 .5 Examples from the literature of mycorrhiza helper bacteria (MHB) enhancing mycorrhiza formation

 2

	ECM	MHB isolates	Host plant	Ecological origin of	Reported mycorrhiza helper	References
				the MHB isolates	effect
	Amanita muscaria,	Streptomyces	Picea abies,	A. muscaria -	1.2-1.7-fold increase in the	(Schrey et al. 2005)
	Suillus bovinus		Pinus sylvestris	containing spruce	second-order root mycorrhizal
				stand	rate
	Hebeloma	Unidentified bacterial	Fagus sylvatica	Soil	1.3-1.7-fold increase in the	(de Oliveira 1988)
	crustuliniforme	isolates			ectomycorrhizal infection
	Laccaria	Pseudomonas fluorescens	Pseudostuga	L. laccata sporocarps	1.2-1.4-fold increase in the	(Duponnois and
	bicolor/laccata	Pseudomonas sp.,	menziesii	and mycorrhizas	ectomycorrhizal infection	Garbaye 1991)
		Bacillus sp.
	Laccaria fraterna,	Bacillus sp.,	Eucalyptus	Sporocarps and	1.8-3.9-fold increase in the	(Dunstan et al. 1998)
	Laccaria laccata	Pseudomonas sp.	diversicolor	ectomycorrhizas of	ectomycorrhizal infection
				L. fraterna
	Lactarius rufus	Paenibacillus sp.,	Pinus sylvestris	L. rufus	1.9-2.4-fold increase in the	(Poole et al. 2001)
		Burkholderia sp.		ectomycorrhizas	ectomycorrhizal infection
	Pisolithus alba	Pseudomonas monteilii ,	Acacia holosericea	Rhizosphere	2.2-fold increase in the	(Founoune et al.
		Pseudomonas resinovorans			ectomycorrhizal infection	2002)
	Pisolithus sp.	Fluorescent pseudomonads Acacia holosericea	Rhizosphere	1.7-2.3-fold increase in the	Founoune et al.
				mycorrhizosphere,	ectomycorrhizal infection	2002)
				galls
	Rhizopogon luteolus	Unidentified bacterial	Pinus radiata	Rhizopogon luteolus	1.2-2.3-fold increase in the	(Garbaye and Bowen
		isolates		ectomycorrhizas	ectomycorrhizal infection	1989)
	Rhizopogon vinicolor	Arthrobacter sp.	Pinus sylvestris	Culture collection	1.2-1.3-fold increase in the	(Rozycki et al. 1994)
	Laccaria laccata				ectomycorrhizal infection
	Different species of	Pseudomonas monteilii	Different Acacia	Rhizosphere	1.4-2.8-fold increase in the	Duponnois and
	Scleroderma and		species		ectomycorrhizal infection	Plenchette 2003)
	Pisolithus
	Suillus luteus	Bacillus	Pinus sylvestris	S. luteus	2.1-fold increase in the	(Bending et al. 2002)
				ectomycorrhizas	mycorrhizal rate for first-order
					roots

(Restricted to ectomycorrhizal associations only, adapted from

Frey-Klett et al. 2007)

  Quercus garryana roots on serpentine and non-serpentine soils in southwestern Oregon, USA. They found abundant and almost similar ECM fungal communities on both soils with, as the most dominant taxa, Coenococcum geophilum, Tuber candidum, Genea harknessii, Tomentella sp., Sebacina sp. and Inocybe sp. Here we just corroborated these findings, suggesting that the ultramafic soils might have not been a strong evolutionary agent for the selection of particular ECM fungal community. Other studies carried out on arbuscular mycorrhizal fungi also reported similar community patterns and high specific richness in both serpentine soils and non-serpentine soils, suggesting that this phenomenon is widespread across the fungal kingdom[START_REF] Fitzsimons | Serpentine soil has little influence on the root-associated microbial community composition of the serpentine tolerant grass species Avenula sulcata[END_REF][START_REF] Schechter | Serpentine and non-serpentine ecotypes of Collinsia sparsiflora associate with distinct arbuscular mycorrhizal fungal assemblages[END_REF].

	(2010) compared ECM fungal communities associated to oak tree from adjacent serpentine
	and non-serpentine soils in northeastern Portugal by sequencing rDNA Internal Transcribed
	Spacer (ITS) fragment. They revealed the presence of several ECM fungal lineages such as
	Agaricales, Boletales, Russulales, Thelephorales along with several genera within
	Ascomycota, across serpentine and non-serpentine soils suggesting that serpentine soils

;

Urban et al. 2008; Branco and Ree 2010)

. For instance, Branco and Ree support fungal communities as rich and complex than those of non-serpentine ones. Similar results were obtained by

[START_REF] Moser | Comparison of ectomycorrhizas of Quercus garryana (Fagaceae) on serpentine and non-serpentine soils in southwestern Oregon[END_REF]

while comparing the ECM fungal communities associated to

In addition, the most abundant ECM genera associated with Tristaniopsis obtained during this study, were identical to that reported previously in association with other ECM plants on serpentine soils worldwide

(Moser et al. 2009; Branco 2010; Jourand et al. 2010b)

. For example,

Branco and Ree (2010)

recently reported the presence of Cortinarius, Russula, Tomentella, Boletellus on serpentine soils while studying the ECM fungal communities associated to evergreen oak tree in Portugal. Likewise, occurrence of Pisolithus

(Jourand et al. 2010a)

, Lactarius

(Moser et al. 2009)

were reported on ultramafic soils in other parts of the world.

Table 2 Soil origin, number of root samples, ECM tips and ITS sequences obtained from four Tristaniopsis host plants

 2

	Soil Type	Host Plant	No. of trees	No. of	No. of	No. of ECM	No. of
			sampled	root	ECM tips	tips	ECM
				samples	used for	successfully	genera
				collected	DNA	sequenced	obtained
					analysis
	Ultramafic	T. calobuxus	20	27	64	44	10
	Ultramafic	T. guillainii	9	18	22	15	7
	Ultramafic	T. glauca	9	10	22	15	7
	Vocano-	T. calobuxus	9	12	20	13	7
	sedimentary
	Vocano-	T. ninndoensis	9	9	22	10	6
	sedimentary

Table 4 Number of root tips successfully sequenced in each ECM genus obtained from the different study sites ECM Genera Study site Host Plant Russula Scleroderma Boletellus Cortinarius Tomentella Pisolithus Lactarius Thelephora Tricholoma Helotiales

 4

	Desmazures	T. calobuxus	3	2	3	8	1	4	0	1	0	2
	(UM 1 )
	Desmazures	T. glauca	2	2	1	4	0	3	1	0	0	2
	(UM)
	Koniambo	T. guillainii	2	0	1	6	1	3	1	0	0	1
	(UM)
	Koniambo	T. calobuxus	2	0	1	10	1	5	0	0	1	0
	(UM)
	Col d'Arama	T. calobuxus	3	0	0	4	2	1	1	1	0	1
	(VS 2 )
	M. Ninndo	T.	1	1	1	3	0	2	0	0	0	2
	(VS)	ninndoensis
	1 UM= Ultramafic soil; 2 VS= Volcano-sedimentary soil.

Table 1 . Soil properties of the sampling sites 1

 1

		Ultramafic soils	Volcano-sedimentary soils
	pH (H 2 0)	5.2 ± 0.2 to 5.3 ± 0.3	4.0 ± 0.2
	N (%)	0.36 ± 0.05 to 0.40 ± 0.05	0.24 ± 0.05
	P (mg/g)	0.07 ± 0.02 to 0.17 ± 0.02	0.045 ± 0.005
	K (mg/g)	0.07 ± 0.01 to 0.09 ± 0.01	0.32 ± 0.06
	Ca (mg/g)	0.26 ± 0.04 to 0.49 ± 0.04	0.009 ± 0.001
	Mg (mg/g)	4.1 ± 0.05 to 8.7 ± 0.3	0.47 ± 0.05
	Fe (mg/g)	370 ± 35 to 429 ± 32	0.5 ± 0.1
	Ni (mg/g)	2.8±0.3 to 3.5 ±0.7	0.14 ± 0.02
	Mn (mg/g)	9 ± 1 to 22 ± 6	0.003 ±0.001
	Cr (mg/g)	17 ± 1 to 18 ± 2	0.09 ± 0.03
	Fe DTPA * (µg/g)	100 ± 9 to 161 ± 20	41 ± 5
	Ni DTPA (µg/g)	19 ± 3 to 25 ± 2	0.3 ± 0.05
	Mn DTPA (µg/g)	1097 ± 60 to 1098 ± 45	0.68± 0.05

Table 3 . Number of isolates obtained in the major MB genera according to soil type and Tristaniopsis host species

 3

	Soil type	Host plant	Burkholderia	Pseudomonas	Bacillus	Lysinibacillus	Enterobacter
		species
	Ultramafic	T. macphersonii	2	2	3	0	2
	Ultramafic	T. calobuxus	24	29	32	13	9
	Ultramafic	T. guillainii	5	9	1	0	8
	Ultramafic	T. glauca	3	1	2	1	2
	Volcano-	T. callobuxus	8	7	3	2	3
	sedimentary
	Volcano-	T. ninndoensis	10	4	3	2	0
	sedimentary

Table 4 . Total number a of ECM root tips observed per fungus genus in both soil types

 4

	ECM fungus	Ultramafic	Volcano-
	genus	soil	sedimentary
			soil
	Russula	9	4
	Scleroderma	4	1
	Boletellus	6	1
	Cortinarius	30	7
	Tomentella	2	2
	Pisolithus	15	3
	Lactarius	2	1
	Thelephora	1	1
	Tricholoma	1	0

a Only ECM root tips associated with successfully characterized MB isolates were considered in this table

  . Liquid pre-cultures were grown overnight in Trypcase medium before transferring 10µl of each culture onto Petri dish containing the different NiCl 2 concentrations. All the Petri dishes were prepared in triplicates for all isolates tested and then incubated at 28°C. Each Petri dish was regularly checked for growth at 2-day intervals for at least one week. Petri dishes without heavy metals were used as controls. In a second step, the Ni-tolerant isolates obtained from the agar plate method were grown in Microwell plate containing Trypcase broth at different Ni concentrations, i.e. 1, 2, 3, 5, 10, and 20 mM of Ni in triplicates. After 48-72 h of growth at 28°C on a shaker at 150 rpm, optical density (600 nm) of the bacterial cultures was measured and compared to that of controls without bacteria in order to verify the Mycorrhizosphere Bacteria ability to tolerate high Ni concentration.

  Table3, we found a much higher proportion of MB isolated from ultramafic soils that harboured the nreB gene compared to MB issued from non-ultramafic soils, i.e. 30 vs 9.5% respectively (significantly different according to F 2 independence test). The highest proportion of nreB + isolates was observed in the Firmicutes: Bacillus and Lysinibacillus with 42 and 57% of total isolates respectively. Almost 30% of nreB + isolates were identified in both Proteobacteria genera Pseudomonas and Burkholderia and only 9% in Enterobacter. By contrast, the occurrence of cnrT gene was generally low in all MB genera and both soils, i.e. from 0 to 20% except in Bacillus from ultramafic soils with 29% of cnrT + isolates. However, and in contrast with nreB gene, there was not significant difference (as attested by F 2

Table 1 . Sampling sites, host plant species and total number of MB isolated

 1

	(*Each differing sequence was considered as taxon; UM= Ultramafic soil ; VS= volcano-
	sedimentary soil)

Table 2 . Proportions (in %) of the different Mycorrhizosphere Bacteria genera present in ultramafic soils (UM) and volcano-sedimentary soils (Volc-sed.), all Tristaniopsis species combined.

 2 As attested by the F 2 independence test (at p > 0.95) applied to the four most represented MB genera, their proportions differed significantly between both soil types. Percentages were calculated from 119 isolates for ultramafic soils and 42 isolates for volcano-sedimentary soils.

	Soil Origin	Pseudomonas (%)	Burkholderia (%)	Bacillus (%)	Enterobacter (%)	Lysinibacillus (%)	Brevibacillus (%)	Rhizobium (%)	Methylobacterium (%)	Paenibacillus (%)	Ewingella (%)
	Ultramafic	30.40	20.80	19.20	17.60	5.60	1.60	0.80	0.80	1.60	1.60
	Volcano-sedimentary	24.4	42.2	13.3	6.7	8.9	4.4	0.0	0.0	0.0	0.0

Table 3 . Effect of soil type on the occurrence of nreB and cnrT genes a in mycorrhizosphere bacteria isolates of the five most represented genera Soil type Pseudomonas Burkholderia Bacillus Lysinibacillus Enterobacter nreB

 3 + cnrT + nreB + cnrT + nreB + cnrT + nreB + cnrT + nreB + cnrT +

	Ultramafic	30.8	7.7	29.6	11.1	41.7	29.2	57.1	0	9.1	4.5
	Volcano-	20.0	20.0	10.5	5.3	0	0	0	0	0	0
	sedimentary

a cnrT + , nreB + : Percentage of isolates harbouring cnrT and nreB genes as detected by PCR analysis.

As attested by the F 2 independence test (at p > 0.95), the proportions of nreB genes in MB isolates from ultramafic soils differed significantly with those from volcano-sedimentary soils. Percentages were calculated from 119 isolates for ultramafic soils and 42 isolates for volcano-sedimentary soils.

.

Table 4 . Effect of Mycorrhizosphere Bacteria on growth of Pisolithus albus co-cultivated in Petri dishes.

 4 Means followed by different letters are significantly different according to the Newman &

	MB strain's	Soil origin a	Bacterial genus	Mean diameter
	name			of fungal
				hyphae culture
				(mm 2 ) b, c
	G7	UM	Pseudomonas	a
	BD36	UM	Lysinibacillus	a
	b29	UM	Bacillus	a
	B10	Volc-sed.	Burkholderia	a
	B123	UM	Bacillus	ab
	-	-	MB-free control	abc
	B22	Volc-sed.	Pseudomonas	abc
	B46	UM	Burkholderia	abc
	B38	UM	Pseudomonas	bcd
	B39	UM	Pseudomonas	cd
	B47	UM	Burkholderia	d
	B41	UM	Pseudomonas	d
	B12	Volc-sed.	Burkholderia	d
	UB38	UM	Pseudomonas	d

a UM: ultramafic soils ; Volc-sed. : volcano-sedimentary soils.

b Means were obtained from three replicates per MB strain tested. c

Table S2 Mycorrhizosphere bacteria isolates obtained from ultramafic and volcano-sedimentary soils with Genbak accession number (see annex at end)

 S2

nreB + nreB - Ni-Tolerant Ni-Susceptible Soil-UM Soil-VS Bacillus Burkholderia Enterobacter Pseudomonas Boletellus Cortinarius Pisolithus Russula Scleroderma cnrT + cnrT -

			Symmetric variable plot
			(axes F1 and F2: 73.48 %)
		1.2
	F2 (22.52 %)	0.2
		-0.8
		-1.8
		-1	0	1
			F1 (50.96 %)

Table S2 MB isolates included in the study with GenBank accession number, and closest BLAST hit

 S2

	B10 B38 BD23	Burkholderia sp. Pseudomonas sp. Ewingella americana	JN975048 Burkholderia sp. GMX2 AM992535 JN975164 Pseudomonas sp. MR14 JN082731 JN975205 Ewingella americana strain CH6 EU708318	100 99 99	2663 2500 2486	0.0 0.0 0.0	This study This study This study
	B12 TCD3-b1-YT BD42	Burkholderia sp. Pseudomonas sp. Burkholderia sp.	JN975049 Burkholderia sp. YXA3-27 JF701944 JN975165 Pseudomonas sp. II4X HQ727967 JN975200 Burkholderia sp. AZ11 AB636680	100 99 99	2589 2514 2109	0.0 0.0 0.0	This study This study This study
	Isolate B16 BD11 B145	Concensus MB Taxon Burkholderia sp. Pseudomonas sp. Bacillus sp.	Genbank JN975050 Burkholderia sp. AZ11 AB636680 Closest vouchered BLAST search JN975166 Pseudomonas fluorescens strain Bi60 HQ336318 JN975219 Bacillus sp. IHB B 4033 HM233997	% 100 99 99	Max 2239 2365 2466	E value 0.0 0.0 0.0	Reference This study This study This study
	B25 BD48 DC3	Burkholderia sp. Pseudomonas sp. Bacillus sp.	Accession JN975051 Burkholderia sp. KM-F5-1 AB622682 JN975167 Pseudomonas sp. II4X HQ727967 JN975220 Bacillus sp. NRS-400 AF169538	Identity 99 99 87	Score 1526 2437 884	0.0 0.0 0.0	This study This study This study
	b17 B19 B23 B28 B61 b68 B108 B118 BG7 B123 B125 B151 BD10 BD12 BD18 BD33 Tb2D1-c1 B33 BD52 B149 B42 BD8 TCD1-B1_YT B43 B4 BD32 B44 B134 BD57 B46 B40 3B7 B50 B1 BTN3D1-f1 b55 TB4D2-e1-CDC CB1 B56 b60 CB2 B64 e1_b-TCD3-YT TCD3-b1-YT B73 YT-TCD3-A1 D4-a2-YT B77 TCD2-A1_YT BD11 B102 B2 BD58 BD43 B3 G12 BD46 5 B40 B-TN3D3-d3 B6 O4 MN-TN3D3-E2 B24 B51 O2 b26 S15 B32 S16	Bacillus sp. Bacillus sp. Bacillus sp. Bacillus sp. Bacillus sp. Bacillus sp. Bacillus sp. Bacillus sp. Bacillus sp. Bacillus sp. Bacillus sp. Bacillus sp. Bacillus sp. Bacillus sp. Bacillus sp. Bacillus sp. Bacillus sp. Burkholderia sp. Pseudomonas sp. Paenibacillus sp. Burkholderia sp. Pseudomonas sp. Bacillus sp. Burkholderia sp. Pseudomonas sp. Enterobacter sp. Burkholderia sp. Pseudomonas sp. Enterobacter sp. Burkholderia sp. Pseudomonas sp. Lysinibacillus sp. Burkholderia sp. Pseudomonas sp. Bacillus sp. Burkholderia sp. Pseudomonas sp. Bacillus sp. Burkholderia sp. Pseudomonas sp. Bacillus sp. Burkholderia sp. Pseudomonas sp. Pseudomonas sp. Burkholderia sp. Pseudomonas sp. Bacillus sp. Burkholderia sp. Pseudomonas sp. Pseudomonas sp. Burkholderia sp. Enterobacter sp. Pseudomonas sp. Burkholderia sp. Enterobacter sp. Pseudomonas sp. Burkholderia sp. Enterobacter sp. Pseudomonas sp. Burkholderia sp. Enterobacter sp. Pseudomonas sp. Burkholderia sp. Enterobacter sp. Lysinibacillus sp. Burkholderia sp. Enterobacter sp. Enterobacter sp. Enterobacter sp. Bacillus sp.	Number JN975081 Bacillus cereus strain GGBSTD2 GQ857475 JN975082 Bacillus weihenstephanensis AB592543 JN975083 Bacillus weihenstephanensis AB592543 JN975084 Bacillus cereus strain HKS GU329911 JN975085 Bacillus sp. JG-TB10 FR849922 JN975086 Bacillus thuringiensis FJ393313 JN975087 Bacillus cereus strain bp2 HQ645936 JN975088 Bacillus cereus strain IHB GU186119 JN975089 Bacillus cereus strain IHB GU186119 JN975090 Bacillus sp. M3. JN089354 JN975091 Bacillus weihenstephanensis AM747230 JN975092 Bacillus cereus strain BC JN676165 JN975093 Bacillus sp. Pc3T GU391505 JN975094 Bacillus thuringiensis solate LDC-391 EU625359 JN975095 Bacillus sp. PG-5-9 JF820122 JN975096 Bacillus sp. M3.TYA JN089354 JN975097 Bacillus thuringiensis TCCC11336 FJ393313 JN975168 Pseudomonas sp. OT6 HM747953 JN975221 Paenibacillus sp. UXO5-11 DQ522106 JN975052 Burkholderia sp. MED-7 AB438046 JN975053 Burkholderia sp. AZ11 AB636680 JN975169 Pseudomonas sp. P12(2011) JF430807 JN975222 Bacillus thuringiensis strain Bi51 HQ336298 JN975054 Burkholderia sp. SBH-9 AB366331 JN975170 Pseudomonas fluorescens Pf0-1 CP000094 JN975206 Enterobacter sp. AR19 HM027905 JN975055 Burkholderia sp. CCGE1003 CP002218 JN975171 Pseudomonas sp. II4X HQ727967 JN975207 Enterobacter aerogenes strain PSB28 FJ360760 JN975056 Burkholderia sp. CNW17 HQ231926 JN975172 Pseudomonas fluorescens strain Bi60 HQ336318 JN975223 Lysinibacillus sp. L2 STR GQ202674 JN975057 Burkholderia sp. ANL2 HM125148 JN975173 Pseudomonas fluorescens strain d3 HQ166099 JN975224 Bacillus cereus strain p2 HM004555 JN975058 Burkholderia sp. AR05 HM027900 JN975174 Pseudomonas sp. OT6 HM747953 JN975225 Bacillus sp. K8SC-2 JF799969.1 JN975059 Burkholderia sp. JV3 CP002986 JN975175 Pseudomonas sp. II4X HQ727967 JN975226 Bacillus cereus strain E22 GU826149 JN975060 Burkholderia sp. JV3 CP002986 JN975176 Pseudomonas sp. TS2 HM747951 JN975165 Pseudomonas sp. II4X HQ727967 JN975061 Burkholderia sp. BAR061 JF962420 JN975177 Pseudomonas sp. 2l-40(2010) HQ403196 JN975227 Bacillus cereus strain XJU-1 EF185296 JN975062 Burkholderia sp. AZ11 AB636680 JN975178 Pseudomonas sp. LCB28 JN650571 JN975166 Pseudomonas fluorescens strain Bi60 HQ336318 JN975063 Burkholderia sp. TSO11-3 AB545645 JN975117 Enterobacter aerogenes strain 1-WCH FJ811873 JN975208 Pseudomonas sp. OT6 HM747953 JN975064 Burkholderia sp. T132 GU573900 JN975118 Enterobacter sp. AR19 HM027905 JN975201 Pseudomonas sp. P12(2011) JF430807 JN975065 Burkholderia sp. AZ11 AB636680 JN975119 Enterobacter aerogenes strain PW161 JF494822 JN975227 Pseudomonas sp. NZ096 AY014817 JN975066 Burkholderia sp. PMS-6 GQ468397 JN975120 Enterobacter aerogenes strain PW161 JF494822 JN975229 Pseudomonas fluorescens strain Bi60 HQ336318 JN975067 Burkholderia sp. PMS-6 GQ468397 JN975121 Enterobacter aerogenes strain PSB28 FJ360760 JN975209 Lysinibacillus sp. L2 STR GQ202674 JN975068 Burkholderia sp. CCGE1003 CP002218 JN975122 Enterobacter sp. AR19 HM027905 JN975230 Enterobacter sp. MTH17 HQ202542 JN975123 Enterobacter sp. AR15 HM027902.1 JN975231 Bacillus sp. 3541BRRJ JF309223	100 99 99 100 98 97 98 100 100 100 98 99 100 100 100 100 97 97 98 99 99 99 99 99 99 100 96 98 99 100 99 99 100 99 99 100 99 97 100 100 99 100 99 99 100 99 99 100 99 99 99 95 98 98 100 99 97 98 98 99 100 99 99 96 100 96 100 100 100 99	2318 2486 2460 2545 2104 2181 1581 2733 2695 1864 1526 1636 2578 2480 2587 1864 2336 2246 2408 2381 2380 2612 2526 2342 2507 2526 2376 2380 2522 2515 2349 2684 1618 2385 2455 2387 2509 2408 1779 2489 2621 1687 2498 2524 2033 1963 2578 2239 2361 2365 1819 2219 2390 2161 2475 2628 1997 2390 2383 2567 2570 2349 1891 2217 2717 2376 2475 2408 2385 2675	0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0	This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study
	CDC-c D3-a1-YT S9 B34 S5 B54 S2	Bacillus sp. Bacillus sp. Burkholderia sp. Enterobacter sp. Lysinibacillus sp. Enterobacter sp. Bacillus sp.	JN975098 Bacillus weihenstephanensis AM747230 JN975099 Bacillus sp. IHB B 4020 HM233992 JN975069 Burkholderia sp. SWF66029 FJ648691 JN975124 Enterobacter sp. EC_1_4 GU272339 JN975232 Lysinibacillus sp. GO6(2011) JF825994 JN975125 Enterobacter sp. NCCP-240 AB665214 JN975233 Bacillus cereus strain 1 FJ435213	99 99 97 99 100 100 100	2599 2338 2408 2365 2654 2601 2643	0.0 0.0 0.0 0.0 0.0 0.0 0.0	This study This study This study This study This study This study This study
	TG-YT-L2D2-F1 BD33 S10 B58 B1 BD22 TCDCB4D2-e1	Bacillus sp. Bacillus sp. Burkholderia sp. Enterobacter sp. Bacillus sp. Enterobacter sp. Pseudomonas sp.	JN975100 Bacillus cereus strain PPB11 HM771666 JN975101 Bacillus sp. I7 HQ600994 JN975070 Burkholderia sp. R4M-O GQ478268 JN975126 Enterobacter sp. B1-2 HQ189496.1 JN975234 Bacillus thuringiensis AM747225 JN975127 Enterobacter aerogenes strain PSB28 FJ360760 JN975235 Pseudomonas fluorescens strain d3 HQ166099	100 100 96 98 99 100 99	2791 2571 2246 2495 2946 2616 2385	0.0 0.0 0.0 0.0 0.0 0.0 0.0	This study This study This study This study This study This study This study
	BD108 BC11 CB7 BD54 TGL2D2-c1-DZ BD59 TN3D1-a1	Bacillus sp. Bacillus sp. Burkholderia sp. Enterobacter sp. Pseudomonas sp. Enterobacter sp. Bacillus sp.	JN975102 Bacillus cereus strain bp2 HQ645936 JN975103 Bacillus cereus strain BC-shrimp-39 JN676165 JN975071 Burkholderia sp. SBH-7 AB366329 JN975128 Enterobacter aerogenes strain PSB28 FJ360760 JN975236 Pseudomonas sp. OT6 HM747953 JN975129 Enterobacter sp. AR15 HM027902.1 JN975237 Bacillus thuringiensis strain TCCC11336 FJ393313	100 99 97 97 99 98 99	1581 1671 2385 2125 2509 2243 2551	0.0 0.0 0.0 0.0 0.0 0.0 0.0	This study This study This study This study This study This study This study
	G9 S5 CB9 b6 b29 G13 b_29D	Bacillus sp. Bacillus sp. Burkholderia sp. Enterobacter sp. Bacillus sp. Bacillus sp. Bacillus sp.	JN975104 Bacillus cereus strain BC-shrimp-39 JN676165 JN975105 Bacillus cereus strain EKS4 DQ648274 JN975072 Burkholderia sp. MED-7 AB438046 JN975130 Enterobacter aerogenes strain 11-WCH FJ811872 JN975236 Bacillus cereus strain X5 FJ763651 JN975212 Bacillus sp. K8SC-2 JF799969 JN975237 Bacillus sp. MB1(2011) HQ600985	99 99 97 99 100 96 99	1626 1680 2296 1427 2578 1468 2596	0.0 0.0 0.0 0.0 0.0 0.0 0.0	This study This study This study This study This study This study This study
	5 O3 BC3 b60 BD73 B21 e1_b-TCD3-YT S6 S13 BC2 B90 G2 3B8 BD25 S17 CB4 CB5 CB6 C6 ZDB-Tb2D1-b1 DZB-Tb2D1-c2 B8 O5 B120 B47 C10 C9 G15 C4 C1 C11 A1 G7 O7 YT-TCD3-A1 G5 A4 TCD3-A1-b_YT	Bacillus sp. Bacillus sp. Burkholderia sp. Lysinibacillus sp. Enterobacter sp. Burkholderia sp. Pseudomonas sp. Bacillus sp. Bacillus sp. Burkholderia sp. Paenibacillus sp. Pseudomonas sp. Lysinibacillus sp. Burkholderia sp. Bacillus sp. Bacillus sp. Bacillus sp. Bacillus sp. Bacillus sp. Bacillus sp. Bacillus sp. Burkholderia sp. Burkholderia sp. Bacillus sp. Burkholderia sp. Burkholderia sp. Bacillus sp. Streptomyces sp. Burkholderia sp. Bacillus sp. Bacillus sp. Burkholderia sp. Pseudomonas sp. Burkholderia sp. Bacillus sp. Enterobacter sp. Rhizobium sp. Pseudomonas sp.	JN975106 Bacillus cereus strain EKS4 DQ648274 JN975073 Burkholderia sp. BAR061 |JF962420 JN975194 Lysinibacillus fusiformis JF970585 JN975192 Enterobacter sp. MTH17 HQ202542.1 JN975198 Burkholderia ambifaria AB568317 JN975193 Pseudomonas sp. II4X HQ727967 JN975107 Bacillus cereus strain IMAUB1030 FJ641013 JN975108 Bacillus cereus WSBC10206 Z84590 JN975109 Bacillus sp. DU53(2010) HM567109 JN975074 Burkholderia sp. MED-7 AB438046 JN975195 Paenibacillus glebae AM745264 JN975202 Pseudomonas sp. TS2 HM747951 JN975196 Lysinibacillus sphaericus FN433026 JN975199 Burkholderia sp. KM-F5-1 AB622682 JN975110 Bacillus cereus strain WYLW1 HM003212 JN975111 Bacillus sp. K8SC-2 JF799969 JN975112 Bacillus sp. DU49(2010) HM567104 JN975113 Bacillus sp. SYW15 FJ601645 JN975114 Bacillus thuringiensis strain 2110 JF947357 JN975115 Bacillus thuringiensis strain TCCC11336 JN975116 Bacillus sp. Na2 GU391509 JN975047 Burkholderia sp. isolate N-APS2 EU035638 JN975079 Burkholderia sp. S3. HM063917 JN975218 Bacillus mycoides strain B20 JN377666 JN975199 Burkholderia sp. BAR061 JF962420 JN975078 Burkholderia sp. S2.1 AF247494 JN975217 Bacillus sp. BAM295 AB330406 JN975204 Streptomyces sp. MJM8568 EU603367 JN975215 Bacillus cereus strain CG-T2 GQ342295 JN975077 Burkholderia sp. SWF66029 FJ648691 JN975214 Bacillus weihenstephanensis EF210283 JN975076 Burkholderia sp. LUP AY307923 JN975213 Pseudomonas sp. GmFRB093 AB369355 JN975075 Burkholderia sp. SBH-7 AB366329 JN975197 Bacillus sp. YC EF068266 JN975204 Enterobacter aerogenes strain 1-WCH FJ811873 JN975203 Rhizobium sp. mpig9.6 AY528711 JN975177 Pseudomonas sp. 2l-40(2010) HQ403196	98 95 99 92 100 96 100 100 97 97 96 99 84 100 90 98 96 100 99 97 97 100 97 96 95 98 98 97 98 89 93 98 96 98 93 100 95 99	1621 1371 1204 1296 2520 1332 2489 2657 1689 1752 1553 2498 641 2223 1151 2280 1645 2482 2464 2336 2336 2581 1611 1530 1405 2540 2459 1483 2484 1067 2116 1658 2106 1746 1721 2520 2039 1963	0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0	This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study

UM= Ultramafic soils; VS= Volcano-sedimentary soils.

Acknowledgements

Acknowledgements This research work was financially supported by the Agence Nationale de la Recherche, ANR 07 BIODIV ULTRABIO. We are thankful to Higher Education Commission of Pakistan for supporting this work through a PhD fellowship. Acknowledgements This research work was financially supported by the Agence Nationale de la Recherche, ANR 07 BIODIV ULTRABIO. We are thankful to Higher Education Commission of Pakistan for supporting this work through a PhD fellowship.

Acknowledgements

This research work was financially supported by the Agence Nationale de la Recherche, ANR 07 BIODIV ULTRABIO. We are thankful to Higher Education Commission of Pakistan for supporting this work through a PhD fellowship.

". I also thank the staff of Laboratoire des Symbioses I have made it through this process, thank you. Financial support was made possible through the CIRAD and ANR (Agence Nationale de Recherche) for research work and for assisting conferences within and/or out of France. I am also thankful to Higher Education Commission of Pakistan for awarding the PhD fellowship

Supplementary data Table

 S1……………………………………………………………….200 Supplementary data Table S2……………………………………………………………… 204

List of Figures

List of Tables

Review of Literature

Table 2.1. Description of different types of mycorrhizae

Chapter III

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

Abstract

New Caledonian serpentinic (ultramafic) soils contain high levels of toxic heavy metals particularly nickel (until 20 g.kg -1 ). They are deficient in essential elements like carbon, nitrogen, phosphorus, and have high Mg-Ca ratio. This work aims at exploring the impact of such edaphic characteristics on soil microbial communities, by analysing the diversity of ectomycorrhizal fungi associated to several species of Tristaniopsis naturally growing on contrasted soils conditions including ultramafic and volcano-sedimentary ones. For this, about 150 ectomycorrhizas were sampled from two different ultramafic sites (Koniambo and Desmazures forest) vs two volcano-sedimentary ones (Arama, Mont Ninndo). Molecular characterization of fungi (through partial sequencing of the ITS rRNA gene) revealed the presence of different dominant fungal genera such as Cortinarius, Pisolithus, Russula, and Boletellus. We could not detect any phylogenetic clustering linked to soil origins maybe due to possible habitat selection on associated ECM fungal communities. Instead, we found that ultramafic soils supported large ECM diversity, suggesting possible ECM fungal adaptation to ultramafic soils that can probably be exploited in the understanding of plant functioning on these sites. Keywords: Ultramafic, Ectomycorrhizal fungi, Nickel, Diversity, Endemic Chapter V

Adaptation of mycorrhizosphere bacteria to extreme nickel contents of New Caledonian ultramafic soils

Adaptation of mycorrhizosphere bacteria to extreme nickel contents of New

Caledonian ultramafic soils

Muhammad WASEEM 1 , Marc DUCOUSSO 1,2 , Clemence CHAINTREUIL 3 , Robin DUPONNOIS 3 , Michel LEBRUN 1,4 , Antoine GALIANA 1 ( in progress)

1 CIRAD, UMR113 LSTM, F-34398 Cedex 5, Montpellier, France