G Godillot

L Guerlou-Demourgues

L Croguennec

K M Shaju

C Delmas

Effect of Temperature on Structure and Electronic Properties of Nanometric Spinel-Type Cobalt Oxides

Temperature is shown to have a huge influence on the electronic properties of nanometric spinel type cobalt oxides precipitated at low temperature in alkaline media. The initial phase, with formula H x Li y Co 3-δ O 4 , contains hydrogen, lithium, cobalt vacancies, and a mixed valence Co 4+ /Co 3+ within the structure, leading to an electronic conductivity higher than that of stoichiometric Co 3 O 4 . Its structural evolution under thermal treatment was studied by X ray diffraction and chemical analysis, which reveal modifications in structure and compositions, involving water release, increase of the Co/O atomic ratio, and modification of the Co 4+ /Co 3+ ratio. The RT to 300 °C range is particularly interesting as a single phase domain and the materials obtained in this temperature range were investigated by chemical analysis, electronic conductivity and specific surface area measurements. Upon increasing temperature, the enhancement of the Co 4+ /Co 3+ ratio, together with cationic redistribution in the spinel framework, results in an improvement of the electronic conductivity (more than 2 orders of magnitude for materials heated above 150 °C). Finally, the systematic thermal study of electronic conductivity and specific surface area of the materials allows to determine an optimal heat treatment temperature leading to an optimized active electrode material for electrochemical energy storage applications, especially in supercapacitors. Such a solid state chemistry approach combining many material characterization techniques to reach a complete knowledge of the material is quite rare in the literature concerning oxides for supercapacitors.

■ INTRODUCTION

Co 3 O 4 has been extensively studied in a wide range of applications such as catalysis (mainly for the treatment of waste gases), [START_REF] Grillo | Low Temperature Oxidation of Carbon Monoxide: the Influence of Water and Oxygen on the Reactivity of a Co 3 O 4 Powder Surface[END_REF][START_REF] Wang | Preparation and Catalytic Performance of Co 3 O 4 Catalysts for Low Temperature CO Oxidation[END_REF][START_REF] Irfan | Co 3 O 4 Based Catalysts for NO Oxidation and NO x Reduction in Fast SCR Process[END_REF] sensing, [START_REF] Li | Co 3 O 4 Nanomaterials in Lithium Ion Batteries and Gas Sensors[END_REF][START_REF] Cao | Hierarchically Structured Cobalt Oxide (Co 3 O 4 ): the Morphology Control and Its Potential in Sensors[END_REF] or magnetism. [START_REF] Makhlouf | Magnetic Properties of Co 3 O 4 Nanoparticles[END_REF][START_REF] Ichiyanagi | Magnetic Study on Co 3 O 4 Nanoparticles[END_REF] It is also particularly considered in electrochemical energy storage, as negative electrode material in lithium batteries [START_REF] Thackeray | Lithium Insertion into Co 3 O 4 : a Preliminary Investigation[END_REF][START_REF] Larcher | The Electrochemical Reduction of Co 3 O 4 in a Lithium Cell[END_REF] or in supercapaci tors. [START_REF] Lin | Characterization of Sol Gel Derived Cobalt Oxide Xerogels as Electrochemical Capacitors[END_REF][START_REF] Xiong | Controllable Synthesis of Mesoporous Co 3 O 4 Nanostructures with Tunable Morphology for Application in Supercapacitors[END_REF] In this last field, increasing capacities, which are highly dependent on specific surface area and pore size distribu tion, [START_REF] Lin | Characterization of Sol Gel Derived Cobalt Oxide Xerogels as Electrochemical Capacitors[END_REF][START_REF] An | Supercapacitors Using Single Walled Carbon Nanotube Electrodes[END_REF] requires to prepare very small particles. One way to achieve this goal is the "chimie douce" approach, low energy consuming, and which enables to prepare nanometric powders with controlled texture and morphology. It is also known to promote the precipitation of kinetically metastable phases, including materials with many defects that can exhibit original and interesting properties.

Co 3 O 4 crystallizes in a normal spinel structure (space group Fd3m), which can be described as a three dimensional [Co 2 O 4 ] framework of Co 3+ ions in edge sharing octahedral sites (16d), with empty channels in the three space directions. Co 2+ ions are located in tetrahedral sites (8a), at the intersection of the channels, and share only corners with the octahedra. The electronic transfer between Co 2+ ions in tetrahedral sites and Co 3+ ions in octahedral sites is impossible since Co 3+ ions are not stabilized in tetrahedral environment, and occurs only between Co 3+ ions (t 2g [START_REF] Makhlouf | Magnetic Properties of Co 3 O 4 Nanoparticles[END_REF] LS) across the shared edges of the CoO 6 octahedra. Therefore, Co 3 O 4 is a semiconductor with a poor electronic conductivity. [START_REF] Appandairajan | Lithium Substituted Cobalt Oxide Spinels Li x M 1-x Co 2 O 4 (M = Co 2+ , Zn 2+ ; 0 ≤ x ≤ 0.4)[END_REF] The spinel structure is well known to accommodate a wide range of metal cations with different oxidation states. Substituted cobaltites were already explored by Holgersson et al. [START_REF] Holgersson | U ̈ber einige Neue Kobaltite vom Spineltypus[END_REF] More recently, Pyke et al. studied the substitution of many transition elements (Li, Cu, Al, Ga, Mn, Ti, Sn, Ge) for cobalt in Co 3 O 4 and concluded to the existence of solid solutions in all cases. [START_REF] Pyke | Surface and Bulk Phases in Substituted Cobalt Oxide Spinels[END_REF] Ni doped [START_REF] Tareen | Hierarchically Porous Ni Co Oxide for High Reversibility Asymmetric Full Cell Supercapacitors[END_REF] and K doped [START_REF] Asano | Potassium Doped Co 3 O 4 Catalyst for Direct Decomposition of N 2 O[END_REF] Co 3 O 4 were also inves tigated, but most of works focus on Li doping, showing a significant improvement in electronic conductivity. [START_REF] Thackeray | Lithium Insertion into Co 3 O 4 : a Preliminary Investigation[END_REF][START_REF] Appandairajan | Lithium Substituted Cobalt Oxide Spinels Li x M 1-x Co 2 O 4 (M = Co 2+ , Zn 2+ ; 0 ≤ x ≤ 0.4)[END_REF][START_REF] Rasiyah | A Mechanistic Study of Oxygen Evolution on Li Doped Co 3 O 4[END_REF][START_REF] Zhecheva | Doping of Co 3 O 4 with Lithium by a Solid State Reaction in Air. I. Oxidation Degree and Coordination of Cations[END_REF][START_REF] Antolini | Formation of Lithium Cobalt Spinel Oxide (Li x Co 3-x O 4 ) by Solid State Reaction of a Li 2 CO 3 -Co 3 O 4 Powder Mixture[END_REF] This property was mainly explained by the presence of Co 4+ to ensure charge balance when lithium is substituted for Co 2+ or Co 3+ , thus, allowing delocalization of holes in the octahedral network, and leading therefore to conductivities higher than that of stoichiometric Co 3 O 4 . Depending on synthesis conditions (precursors, temperature), Li ions were determined to occupy 8a sites, [START_REF] Rasiyah | A Mechanistic Study of Oxygen Evolution on Li Doped Co 3 O 4[END_REF] 16d sites, [START_REF] Appandairajan | Lithium Substituted Cobalt Oxide Spinels Li x M 1-x Co 2 O 4 (M = Co 2+ , Zn 2+ ; 0 ≤ x ≤ 0.4)[END_REF] and interstices, 8 but were also identified to be distributed in several spinel sites. [START_REF] Zhecheva | Doping of Co 3 O 4 with Lithium by a Solid State Reaction in Air. I. Oxidation Degree and Coordination of Cations[END_REF][START_REF] Zhecheva | Low Temperature Preparation of a Lithium Cobalt Spinel[END_REF] In particular, micrometric spinel type cobalt oxides containing H and Li in the structure were prepared in our group by electrochemical oxidation of CoO. [START_REF] Tronel | New Spinel Cobalt Oxides, Potential Conductive Additives for the Positive Electrode of Ni MH Batteries[END_REF] Such materials were shown to contain hydrogen and lithium ions, as well as cobalt vacancies in both tetrahedral and octahedral sites. It was demonstrated that cobalt vacancies are compensated by H + and Li + ions, but also by the presence of Co 4+ ions. As a result, very good electronic properties could be achieved, with electronic conductivity higher by 4 or 5 decades (at room temperature), as compared with stoichiometric Co 3 O 4 . [START_REF] Tronel | New Spinel Cobalt Oxides, Potential Conductive Additives for the Positive Electrode of Ni MH Batteries[END_REF] It has also been highlighted that a moderate thermal treatment leads to an additional increase of conductivity, due to modifications in structure/composition, involving water release and increase of the Co/O atomic ratio. [START_REF] Douin | Effect of Thermal Treatment on the Electronic Conductivity Properties of Cobalt Spinel Phases Synthesized by Electro Oxidation in Ternary Alkaline Electrolyte (KOH, LiOH, NaOH)[END_REF][START_REF] Douin | Improvement by Heating of the Electronic Conductivity of Cobalt Spinel Phases, Electrochemically Synthesized in Various Electrolytes[END_REF] More recently, materials with similar composition but nanometric size were prepared. [START_REF] Shaju | Strategies for Synthesizing Conductive Spinel Cobalt Oxide Nanoparticles for Energy Storage Applications[END_REF] These materials, denoted as nano Co 3 O 4 in the following, are synthesized by precipitation at low temperature in various alkaline media. They also show high electronic conductivity, as well as the presence of lithium, hydrogen, and cobalt vacancies. According to the first results we recently reported, these nano Co 3 O 4 phases are promising candidates as electrodes materials for hybrid supercapacitors, [START_REF] Godillot | Original Conductive Nano Co 3 O 4 Investigated as Electrode Material for Hybrid Supercapacitors[END_REF] thanks to a high conductivity and a high specific surface induced by their nanometric size. The present paper will study on the effect of temperature, focusing on the relationships between structure, chemical composition, and electronic properties.

■ EXPERIMENTAL SECTION Material Preparation. As described in detail in a previous paper, [START_REF] Shaju | Strategies for Synthesizing Conductive Spinel Cobalt Oxide Nanoparticles for Energy Storage Applications[END_REF] the initial nano Co 3 O 4 type phase was prepared by precipitation at 90 °C of a cobalt salt in a 8 M KOH + 0.5 M NaOH + 0.5 M LiOH alkaline solution. Cobalt nitrate hexahydrate (Fluka) was thus dissolved into distilled water, mixed with hydrogen peroxide (Fluka; with a Co(NO 3 ) 2 /H 2 O 2 molar ratio of 3:2), and then added dropwise to the ternary alkaline solution to induce precipitation of the nanometric cobalt oxide. The suspension obtained was allowed to ripen for 4 h at 90 °C. Afterward, the precipitate was centrifugated and washed with distilled water until neutrality and then dried at 60 °C for 24 h. The resulting powder was finally ground in a pestle and mortar. As nanometric materials, all the synthesized samples tend to adsorb large amounts of molecules contained in air (especially water) and were, therefore, stored in a desiccator to keep a constant and low amount of adsorbed water. Reference high temperature Co 3 O 4 (HT Co 3 O 4 ) was obtained from the thermal treatment of cobalt carbonate (Alfa Aesar), at 450 °C, under an oxygen flow for 15 h.

Heat Treatment. The initial nano Co 3 O 4 powder was subjected to heat treatments, under air, at temperatures ranging from 100 to 650 °C (100, 150, 200, 250, 300, 400, and 650 °C). These temperatures were determined on the basis of the X ray diffraction in situ analysis, taking into account that thermal analysis shows a continuous loss of water from room temperature and a decomposition of Co 3 O 4 into CoO after 650 °C, as evidenced elsewhere. [START_REF] Godillot | Promising Nanometric Spinel Cobalt Oxides for Electrochemical Energy Storage: Investigation of Li and H Environments by NMR[END_REF] The temperature was increased at the rate of 2 °C/min up to the target temperature, held for a 4 h isothermal stage and then decreased down to room temperature at 2 °C/min. All the heat treatments were performed ex situ on the same starting material to investigate accurately the effect of temperature on the parameters studied, especially on the chemical composition. The starting fresh precipitated material is denoted as SP initial ("SP" for spinel), while the heated samples are called SP 100, SP 150, ..., SP 650, depending on the treatment temperature.

Characterization. Both in situ and ex situ analysis were carried out on nano Co 3 O 4 . In situ analysis consisted of studying the evolution of the X ray diffraction pattern of SP initial sample with temperature. For ex situ analysis, SP initial and the seven heat treated samples (SP initial to SP 650) were investigated, at room temperature, by means of X ray diffraction, chemical analysis, specific surface area and electronic conductivity measurements.

To carry out in situ analysis, heat treatment was coupled with X ray diffraction analysis by connecting an Anton Paar HTK 1200 high temperature furnace to the diffractometer. The sample was placed in a variable temperature chamber under air, and a first acquisition was performed at room temperature. Then, the temperature of the chamber was gradually increased up to 650 °C (3 °C/min), and X ray data acquisition was performed during the 4 h isothermal stages (every 50 °C). Then, a symmetrical process was applied during the cooling down of the chamber, and a last X ray diagram was recorded at room temperature.

In thermal in situ analysis, X ray diffraction (XRD) data were collected with a Philips PANalytical X'Pert Pro diffractometer using the cobalt Kα radiation. The powder diffraction patterns were recorded for around 4 h (2.41 s per step) in the 10-110°( 2θ) angular range, with a 0.0167°(2θ) step size and a 2.122°( 2θ) active width in the detector. Similar conditions were used to record ex situ XRD patterns but with longer acquisition time (11 h, 6.44 s per step).

Structure refinements were based on the Rietveld 28 method using the FullProf program. [START_REF] Rodriguez Carvajal | FULLPROF: a Program for Rietveld Refinement and Pattern Matching Analysis[END_REF] The diffraction peaks were fitted with a pseudo Voigt function PV = ηL + (1 -η)G, η being the Lorentzian contribution to the Gaussian peak shape. Lattice parameters, parameters of the profile function (η, U, V, W), position of oxygen, cobalt occupancies, and isotropic atomic displacement parameters (B) were refined. Moreover, the materials studied are poorly crystallized and exhibit a strong and angular dependent background. A polynomial function with six refined parameters was therefore chosen to describe it. Finally, using the Scherrer's formula, the instrumental width was neglected, compared to the broad peaks exhibited by the nanometric materials studied.

Specific surface area measurements were carried out on a Quantachrome Instruments Autosorb 1. For each sample, around 30 mg of material was degassed at 150 °C for 15 h under helium, and an adsorption/desorption isotherm was recorded under nitrogen for relative pressures ranging from 0.02 to 0.99. All the isotherms obtained revealed to be type IV isotherms, [START_REF] Sing | Reporting Physisorption Data for Gas/Solid Systems with Special Reference to the Determination of Surface Area and Porosity[END_REF] that is, characteristics of a mesoporous material. Consequently, for every material, specific surface area could be determined using the BET theory [START_REF] Emmett | The Use of Low Temperature Van der Waals Adsorption Isotherms in Determining the Surface Area of Iron Synthetic Ammonia Catalysts[END_REF][START_REF] Brunauer | Adsorption of Gases in Multimolecular Layers[END_REF] on adsorption isotherm for P/P 0 < 0.35 (identical results were obtained from desorption isotherms).

Chemical analysis was performed by Inductive Coupled Plasma (on a Varian 720 ES ICP OES) for Li, Na, K, and Co titration, and by elementary microanalysis (on a Thermo flash EA 1112 Series CHNS analyzer) for H and C titration. The average oxidation state of cobalt was established by the iodometric titration method. Cobalt ions at an oxidation state higher than 2 in the material can be reduced to Co 2+ by I -ions (from KI solution), I -being oxidized into I 2 . Back titration of previously formed I 2 by S 2 O 3 2-(0.1 M Na 2 S 2 O 3 ) allows the average oxidation state of cobalt to be deduced. Around 30 mg of material was introduced in a PTFE container with 5 mL of H 2 O, 10 mL of 10 g/L KI, and 5 mL of 12 M HCl. A second container, constituting a reference sample, was prepared with the same solutions but no powder. Both containers were sealed and heated in an oven at 60 °C for 20 h to complete dissolution and reduction of the material. Such a method is particularly useful to dissolve cobalt oxide powders while avoiding evaporation of I 2 at high temperatures. However, it must be noticed that Cl -ions from the hydrochloric acid solution (which is used for the dissolution of the material) can also reduce Co n+ ions (n > 2). The 5 mL of HCl were consequently added carefully at the very end of the container filling, to avoid direct contact with the material. Back titration was finally performed after the cooling of the containers. The titrated volume of the reference sample was subtracted in order to consider the oxidizing effect of dissolved oxygen. With the aim of following the evolution of cobalt average oxidation state upon temperature, efforts have been made to minimize as much as possible the uncertainty on the measurement by carrying out a large number of analyses; especially to limit the influence of the titrated volume of the reference sample which was found to vary among experiments.

Temperature dependent electronic conductivity measure ments were carried out on 8 mm diameter pellets with the four probe technique, [START_REF] Laplume | Bases Theóriques de la Mesure de Reśistivitéet de la Constante de Hall par la Methode des Quatre Pointes[END_REF] using a direct current. Each value of electronic conductivity is determined from the measurement of the potential resulting from the application of a DC current in both positive and negative sense (according to the four probe technique) in order to avoid stray voltages and therefore to get rid of the diode effect. The temperature was progressively raised to 400 °C and then cooled down to room temperature at a rate of 1 °C/min. The pellets were obtained by compacting around 200 mg of powder at 8 t/cm 2 (thickness close to 1.2 mm and density close to 3.3 g/cm 3 ). Because of the low temperature synthesis of the materials, the pellets could not be sintered.

■ RESULTS AND DISCUSSION

1. Structure and Morphology. Starting Material. The X ray diffraction diagram of the fresh precipitated material (SP initial) is compared in Figure 1 with that of a reference HT Co 3 O 4 phase. It shows the presence of a pure phase, with spinel structure, isotypic with the HT Co 3 O 4 one. The pattern of SP initial exhibits very broad diffraction peaks, which confirm the presence of nanometric coherent domains. By applying the Scherrer's formula on the most intense four peaks ((220), (311), (511), and (440)), their size has been estimated to stay close to 5 nm, in agreement with that determined by TEM measurements. [START_REF] Godillot | Original Conductive Nano Co 3 O 4 Investigated as Electrode Material for Hybrid Supercapacitors[END_REF] In addition, an inversion of the (220)/(111) line intensity ratio can be noticed in Figure 1 between SP initial and the reference phase (on the contrary to the HT Co 3 O 4 diffraction pattern, the (220) line is more intense than the (111) one in nanometric material). Such an inversion has already been observed in similar micrometric materials and was explained by the presence of cobalt vacancies in the structure, mainly in the tetrahedral sites. [START_REF] Tronel | New Spinel Cobalt Oxides, Potential Conductive Additives for the Positive Electrode of Ni MH Batteries[END_REF][START_REF] Douin | Effect of Thermal Treatment on the Electronic Conductivity Properties of Cobalt Spinel Phases Synthesized by Electro Oxidation in Ternary Alkaline Electrolyte (KOH, LiOH, NaOH)[END_REF] Thus, by means of XRD simulations, Tronel et al. have demonstrated that the (220)/ (111) line intensity ratio is strongly connected to the cobalt occupancy in tetrahedral sites of such Co 3 O 4 spinel type materials [START_REF] Tronel | New Spinel Cobalt Oxides, Potential Conductive Additives for the Positive Electrode of Ni MH Batteries[END_REF] (the former decreases exponentially with the latter).

In Situ High-Temperature X-ray Diffraction Study. The structural evolution of SP initial was followed by X ray diffraction during heat treatment, as described in the Experimental Section, and the evolution with temperature of the resulting X ray diffraction patterns is displayed in Figure 2. The initial and final diagrams, recorded at room temperature, are respectively denoted as RT i and RT f. Upon the temperature increase, the following modifications can be highlighted. (i) The width of the diffraction lines decreases beyond 200 °C, indicating an increase of the size of the crystalline domains in the material. (ii) The inversion of the (220)/(111) line intensity ratio evidenced in the starting material tends to break up, leading to a ratio similar to that of reference HT Co 3 O 4 . This suggests a modification of the structure, as well as of the cobalt sites occupancy. (iii) Beyond 400 °C, new diffracted lines emerge and can be assigned to LiCoO 2 . The appearance of LiCoO 2 is particularly highlighted by the splitting of the peak at 22.1°(2θ) in two lines: the (003) line of the LiCoO 2 phase and the (111) line of the Co 3 O 4 one. Indeed, at room temperature, the (003) line of LiCoO 2 is hidden by the (111) line of the Co 3 O 4 (RT f), but the two phases can be distinguished at higher temperature (400 to 650 °C) due to different thermal dilatation coefficients. Thus, the (003) line of LiCoO 2 is shifting toward low angles with increasing temperature (increase of the cell parameters with thermal dilatation), while the (111) line of the Co 3 O 4 is almost not displaced. On the contrary, two new lines appear in the last pattern (RT f) at 43.7 and 53.0°(2θ), which can be respectively assigned to the (101) and (104) lines of LiCoO 2 . They were hidden by those of Co 3 O 4 between 400 and 650 °C, but enable the identification of the LiCoO 2 phase in the RT f diffraction pattern.

To clarify this evolution, diffraction data refinements with the Rietveld method were performed on the diffraction patterns recorded during the in situ thermal treatment (see the Experimental Section for details). The comparison of both experimental and calculated diagrams of SP initial at room temperature (RT i) is displayed in Figure 3a and confirms that the material crystallizes in a normal cubic spinel structure. Oxygen ions (32e) form a cubic close packed arrangement, where Co 2+ ions occupy the tetrahedral sites (8a) and Co 3+ the octahedral sites (16d). In our model, the oxygen occupancy was fixed to 100% (no significant variation was observed when refining it). Results of the refinement are reported in Table 1. They underline the presence of cobalt vacancies in both tetrahedral and octahedral sites. Indeed, compared to the ideal Co II [Co III ] 2 O 4 phase where 8a and 16d sites are fully occupied, SP initial exhibits a structure where only 67 and 82% of the latter sites are, respectively, occupied, as proposed to explain the inversion of the (220)/(111) line intensity ratio. Despite the rather good reliability factors obtained for this refinement, the high values of isotropic atomic displacement (between 2 and 3 Å 2 ) must be pointed out. They could be explained by the nanometric dimensions of the material, which induce strong surface effects, [START_REF] Weirich | Rietveld Analysis of Electron Powder Diffraction Data from Nano crystalline Anatase, TiO 2[END_REF] and thus can lead to a distribution of the Co-O bond lengths. One has to keep in mind that in a Ø5 nm spherical particle of an oxide exhibiting a compact lattice, 1/3 of the oxygen atoms belong to the first surface layer. [START_REF] Shaju | Strategies for Synthesizing Conductive Spinel Cobalt Oxide Nanoparticles for Energy Storage Applications[END_REF] Anyway, occupancy and atomic displacement parameters are strongly correlated and such high isotropic atomic displacement values suggest that the real uncertainty on occupancy values is much higher than the one estimated in the Rietveld refinement. Consequently, in the following of the study, absolute values of occupancy will be considered with caution and we will rather focus on their evolution with temperature.

XRD patterns of SP initial heated at different temperatures were refined in the same way, except for those beyond 400 °C, for which an additional LiCoO 2 phase was considered, as discussed above. For example, the experimental and calculated diagrams of the material heated at 650 °C, displayed in Figure 3b, show that three diffractions lines of LiCoO 2 can be detected, which enables only a refinement of its cell parameters. Let us note that LiCoO 2 exhibits two different structures depending on the synthesis conditions. A HT LiCoO 2 layered phase (hexagonal cell, R3m space group) is usually obtained at high temperature, whereas a LT LiCoO 2 spinel type phase (Fd3m space group) is generally formed below 400 °C. [START_REF] Antolini | LiCoO 2 : Formation, Structure, Lithium and Oxygen Nonstoichiometry, Electrochemical Behaviour and Transport Proper ties[END_REF] For comparison with the high temperature phase, the XRD pattern of LT LicoO 2 can be indexed in a hexagonal cell. The two phases exhibit very similar XRD diagrams, but can be distinguished thanks to the c hex /a hex ratio parameter; it is equal to 2√6 (4.90) for LT LiCoO 2 and close to 4.99 for HT LiCoO 2 . [START_REF] Gummow | Structure and Electrochemistry of Lithium Cobalt Oxide Synthesized at 400[END_REF][START_REF] Shao Horn | Structural Features of Low Temperature LiCoO 2 and Acid Delithiated Products[END_REF] In our case, the c hex /a hex ratio was determined to be 4.99 for the material cooled down to room temperature (RT f). Consequently, based on the value of the c hex /a hex ratio and on the results of Douin et al. in a similar study, [START_REF] Douin | Effect of Thermal Treatment on the Electronic Conductivity Properties of Cobalt Spinel Phases Synthesized by Electro Oxidation in Ternary Alkaline Electrolyte (KOH, LiOH, NaOH)[END_REF] we show that the HT LiCoO 2 phase may be formed during the XRD in situ analysis. Thus, the HT LiCoO 2 structure is described in the hexagonal system (R3m space group), with oxygen in the 6c position (0, 0, 0.2559), cobalt ions in the 3a sites (0, 0, 0), and lithium ions in the 3b sites (0, 0, 1/2). For this secondary phase, only the lattice parameters (a hex and c hex ) and the parameters of the profile function were refined because of its presence in small amounts. In particular, the atomic positions were fixed to values reported in the literature. [START_REF] Douin | Effect of Thermal Treatment on the Electronic Conductivity Properties of Cobalt Spinel Phases Synthesized by Electro Oxidation in Ternary Alkaline Electrolyte (KOH, LiOH, NaOH)[END_REF] The results of pattern refinements upon temperature increasing are summar ized in Table 2. Due to the lack of information about the HT LiCoO 2 phase (low number of visible peaks), no quantitative analysis could be undertaken. Finally, one can notice that in the case of similar micrometric materials studied by Douin et al., [START_REF] Douin | Effect of Thermal Treatment on the Electronic Conductivity Properties of Cobalt Spinel Phases Synthesized by Electro Oxidation in Ternary Alkaline Electrolyte (KOH, LiOH, NaOH)[END_REF] the HT LiCoO 2 phase appears only beyond 500 °C (vs 400 °C in our case). Such a discrepancy can be explained by the nanometric dimensions of our material, that may lower the energy required to achieve the phase separation.

Figure 4a presents the evolution of the cubic cell parameter for the spinel phase obtained during the in situ thermal treatment (full squares). The values obtained for materials annealed ex situ and cooled down to room temperature are also reported (empty squares) and will be commented on in the following. As expected, the cubic lattice parameter globally increases with temperature and decreases upon cooling due to the thermal dilatation of the unit cell. However, two peculiar phenomena are observed. The lattice parameter (i) decreases with temperature increasing from 25 to around 200 °C, and (ii) does not recover its initial value after the cooling down (it rises from 8.0758 to 8.0805 Å). Those two effects suggest modifications in the structure/composition of the material and will be explained in the chemical composition section. The formation of LiCoO 2 has been evidenced beyond 400 °C. Similarly, the temperature dependence of the LiCoO 2 cell parameters is plotted in Figure 4b. Both a hex and c hex lattice parameters exhibit a linear variation with temperature, fully explained by thermal dilatation.

The thermal evolution of the cobalt occupancies within the spinel structure is displayed in Figure 5a. As mentioned before, occupancy values are associated with a large uncertainty due to high atomic displacement values. Nevertheless, we can observe that the cobalt occupancy in the 8a tetrahedral sites is subjected to huge modifications with temperature, rising from 67% in the starting material to 93% in the final material (at 650 °C). The evolution in the 16d octahedral sites occupancy is less obvious due to uncertainties and could be assumed to remain quite constant. Consequently, upon increasing temperature, there is a continuous material reconstruction with a decrease of the number of cobalt vacancies, leading to a structure close to that of ideal Co 3 O 4 . Such a behavior is in agreement with the constant increase of the Co/O ratio displayed in Figure 5b and can be explained by structure reorganization through the departure of oxygen atoms from the spinel structure.

Ex Situ High-Temperature X-ray Diffraction Study. The thermal in situ X ray diffraction performed on SP initial has highlighted enhancement of crystallization upon temperature increasing from room temperature to 650 °C. Moreover, the 100-300 °C range seems to be very interesting since additional phenomena appear (decrease of the cubic cell parameter, increase of Co/O ratio) and since the material is monophasic (HT LiCoO 2 appears beyond 400 °C). Thus, the SP initial material has been heat treated ex situ at 100, 150, 200, 250, 300, 400, and 650 °C, to recover easily the material and look further in the mechanisms induced by temperature effect. These ex situ heat treated materials will be denoted as SP 100, SP 150, ..., SP 650 in the following.

X ray patterns of the synthesized phases (not shown here, but presented in Figure S1 in the Supporting Information or in

= ηL + (1 η)G η = 0.91(2) half-width parameters H 2 = U tan 2 θ + V tan θ + W U = 6.9(8) V = 10(9) W = 0.3(2) Conventional Rietveld R-Factors with Bragg Contribution (%) R wp = 1.11 R B = 1.44 χ 2 = 1.44
ref 27 have been recorded at room temperature and refined using the Rietveld method. Just as for the thermal in situ analysis, results of the refinements are reported in Table 3. The evolution of the cubic cell parameter with the synthesis temperature is compared in Figure 4a with the in situ results. The same trend, that is, decrease of a cub up to 200 °C, followed by an increase, is observed for both in situ and ex situ analysis.

For the latter, no thermal dilatation is involved since the XRD diagrams have been recorded at room temperature, and the evolution evidenced can be only explained by irreversible modifications of the structure and the chemical composition. In particular, the a cub parameter value at the highest temperature is observed to get closer to that of stoichiometric Co 3 O 4 (8.088 Å). [START_REF] Roth | The Magnetic Structure of Co 3 O 4[END_REF] It is also interesting to note that the spinel phase heated in situ and ex situ at 650 °C leads to the same a cub parameter (8.081 Å) at room temperature. Then, the cobalt occupancy values detailed in Table 3 are in good agreement with those obtained by means of in situ analysis (Table 2). A huge increase can be noticed for cobalt occupancy in tetrahedral sites (from 61% in SP initial to 93% in SP 650), while the occupancy in octahedral sites seems to remain constant at around 85%. Finally, it must be added that HT LiCoO 2 is not detected in SP 400 for the reasons detailed previously, that is, the HT LiCoO 2 lines are hidden by those of Co 3 O 4 due to the broadness of the latter at room temperature.

Particle Size Evolution with Temperature. Figure 6 displays the evolution of average particle sizes (estimated using Sherrer's formula on ex situ XRD patterns) and of specific surface areas as a function of heating temperatures. SP initial shows particles of around 5 nm, whose size remains stable up to 200 °C and then grows. The specific surface area is stable below 200 °C (less than 10% loss) and a huge drop off can be noticed between 200 and 300 °C where it is divided by two. The two evolutions are in good agreement and lead to the same conclusion: the increasing temperature has no effect on particle size up to 200 °C and grain growth can be observed beyond.

2. Chemical Composition. Chemical compositions of SP initial and heat treated samples are summarized in Table 3; data of ideal Co 3 O 4 are also reported as reference. Since a pure spinel type "Co 3 O 4 " phase has been demonstrated to exist only until 300 °C included (an additional HT LiCoO 2 phase emerges beyond 400 °C), the chemical analysis will focus on the study of materials prepared up to 300 °C.

The chemical analysis results show that (i) the amounts of C, N, Na, and K are very low (ratios over Co below 0.01), so they can be neglected. (ii) Significant amounts of hydrogen and lithium were detected in these samples and can be assigned to ions in the spinel structure, since a single phase has been evidenced. The Li/Co ratio remains constant (0.05-0.06) over the considered temperature range. As far as hydrogen is concerned, its amount gradually decreases with the increasing treatment temperature (H/Co = 0.85 for SP initial against 0.23 after heating at 300 °C). Hydrogen can be a priori attributed to protons within the spinel structure and to water adsorbed at the surface of the nanometric particles. The departure of protons is necessarily coupled with that of oxygen from the structure, since TGA analysis under argon coupled with mass spectrometry shows a continuous release of H 2 O in the RT to 650 °C range (see Figure S2 in the Supporting Information). This behavior is in accordance with the increase of the Co/O ratio evidenced in the X ray diffraction section. (iii) The Co content in the studied materials is lower than that in ideal Co 3 O 4 (73.42 mass%), which is in good agreement with the presence of cobalt vacancies in the spinel structure, but which can also be explained by the presence of additional species in the spinel structure (protons, lithium) or adsorbed water. The Co amount tends anyway to increase with temperature, in agreement with the release of oxygen and the increase of the Co/O ratio. The average oxidation state of cobalt is higher than that expected in an ideal Co II Co III 2 O 4 spinel (2.67), which strongly supports the presence of Co 4+ ions. It remains quite stable up to 250 °C and then slightly decreases, getting closer to that of ideal spinel.

Formulas were thus established for each ex situ heat treated sample on the basis of chemical analysis results (chemical composition and cobalt oxidation state titration) and crystal refinement data, established in the X ray analysis section (cobalt occupancies in tetrahedral and octahedral sites). For this purpose, Co 2+ ions are considered to occupy partially the 8a tetrahedral sites, while Co 3+ and Co 4+ are supposed to be localized in the 16d octahedral sites. The stoichiometric coefficient of each cobalt ion is evaluated from the occupancies calculated by structural refinement and of the cobalt average oxidation state. Let us remind that the oxygen sites (32e) were supposed to be fully occupied in this model. The overall charge balance is then ensured by all the lithium ions and an adjusted amount of protons. Remaining hydrogen atoms allow finally to determine the amount of adsorbed water molecules. Consid ering these hypotheses, such a typical formula can be written for SP initial: Li ions are supposed to occupy the 8a tetrahedral sites, according to results obtained on micrometric cobalt oxides, [START_REF] Tronel | New Spinel Cobalt Oxides, Potential Conductive Additives for the Positive Electrode of Ni MH Batteries[END_REF] even if its location in other sites of the spinel structure (16d, 16c, 8b, 48f) cannot be totally excluded. As shown in the above formula, protons are too numerous to be only located in the cobalt tetrahedral vacancies and might also occupy some other sites of the spinel structure, which are not determined at the present time. Formulas of all heat treated samples have been established in the same way and are reported in Table 3. Their evolution shows, as expected, a decrease in the amounts of structural protons and adsorbed water molecules.

The variation of the Co 4+ /Co 3+ ratio in the octahedral sites as a function of the heat treatment temperature can be deduced from the above formulas; it is displayed in Figure 7. The Co 4+ / Co 3+ ratio passes through a maximum, reached at 200 °C (0.23), and then goes down. This evolution has to be related to the variation of the cubic cell parameter of the spinel phase (see the X ray diffraction ex situ analysis and Figure 4a) with temperature. Indeed, the general tendency shows an increase of the cell parameter when the synthesis temperature increases from RT up to 650 °C, with a peculiar decreasing phenomenon in the 100-200 °C range. Comparing this evolution with that of the cell parameter, it can be observed that the increase of the Co 4+ /Co 3+ ratio is linked to a slight contraction of the lattice.

Partial Discussion. In summary, as reported in the previous sections, upon increasing temperature from room temperature to 650 °C, the following structural and composition modifications have been evidenced: (i) a continuous release of hydrogen, (ii) an increase of the amount of cobalt in the tetrahedral sites while the octahedral occupancy remains stable, (iii) a stabilization of the average oxidation state of cobalt below 200 °C, followed by a decrease, and (iv) an increase of the Co 4+ /Co 3+ ratio up to 200 °C, which then decreases. All these effects can be explained as already reported for similar micrometric materials. [START_REF] Douin | Effect of Thermal Treatment on the Electronic Conductivity Properties of Cobalt Spinel Phases Synthesized by Electro Oxidation in Ternary Alkaline Electrolyte (KOH, LiOH, NaOH)[END_REF] When temperature increases, the departure of protons from the lattice entails a reorganization within the spinel structure, with a filling of the tetrahedral sites, where only Co 2+ ions are stable from crystal field consid erations, and an increase of the Co/O ratio. The departure of protons is finally balanced by an increase of the Co 4+ /Co 3+ ratio in the octahedral network.

3. Electronic Properties. Electronic properties of the ex situ heat treated materials were investigated under air in the 25 to 300 °C range, which corresponds to the spinel single phase domain. Figure 8 compares the temperature dependence of electronic conductivity for HT Co 3 O 4 , SP initial, SP 200, and SP 300. The curves corresponding to the materials synthesized at 100, 150, and 250 °C are not reported for more clarity. Nevertheless, Figure 9 presents the conductivity values for all the synthesis temperatures. As shown by the arrows in Figure 8, the measurements were performed by increasing temperature from RT to 400 °C, and then decreasing down to RT, giving rise to a singular curve shape, involving two different domains: a strong and irreversible conductivity increase up to 400 °C, followed by a quasi linear conductivity decrease when temper ature goes down to 25 °C. The curve parts that correspond to decreasing temperatures are completely reversible and fully reproducible when increasing-decreasing temperature cycles are performed afterward. They characterize the electronic behavior of the materials after heating at 400 °C, whatever the initial synthesis temperature. These behaviors correspond to a hopping type mechanism, with activation energies in the 0.01-0.04 eV range, as expected for semiconductor materials. On the contrary, the curve parts that correspond to increasing temperatures are irreversible and are likely to result from three different contributions, occurring at different scales, and all leading to an increase of conductivity with temperature. (i) The first effect results from the fact that, at the microscopic scale, increasing temperature induces a sintering of the pellet, as suggested by the increase of the size of coherent domains, The electronic conductivity, which results from a delocalization effect in the octahedral network, linked to the presence of Co 4+ ions, tends therefore to follow the Co 4+ /Co 3+ ratio. The materials that are heat treated at temperatures between 150 and 300 °C exhibit the highest conductivity at room temperature, around 10 -1 S•cm -1 , against about 10 -4 S•cm -1 , for the pristine spinel phase.

As we recently reported, [START_REF] Godillot | Original Conductive Nano Co 3 O 4 Investigated as Electrode Material for Hybrid Supercapacitors[END_REF] the electrochemical behavior of this pristine material as positive electrode of supercapacitor is quite interesting, leading to capacity of 320 F/g in 8 M KOH, with a very small amount of carbon black added (5%). The similar heat treated materials, which exhibit a RT conductivity that is 3 orders of magnitude higher, appear therefore as promising candidates for the same application. Nevertheless, the design of supercapacitor materials requires finding the best compromise between electronic conductivity, which is at the top for the material annealed at 250 °C, and specific surface area, which shows highest BET areas up to 200 °C. The material that is heat treated at 200 °C appears therefore to be a good candidate (σ RT = 0.083 S•cm -1 , 180 m 2 /g) as electrode material and will be investigated in hybrid supercapacitor systems in future studies.

■ ASSOCIATED CONTENT

* Supporting Information

Two additional figures are given in Supporting Information. The first one (Figure S1) shows the X ray diffraction patterns of SP initial and the heat treated materials (SP 100 to SP 650). The second one (Figure S2) is the TGA measurement (coupled with mass spectroscopy) of the SP initial compound which evidence a continuous proton departure in the 25-650 °C range. This material is available free of charge via the Internet at http://pubs.acs.org.

■ AUTHOR INFORMATION

Corresponding Author *Phone: +33 (0) 5 40 00 27 25. Fax: +33 (0) 40 00 27 61. E mail: guerlou@icmcb bordeaux.cnrs.fr.

Notes

Figure 1 .

 1 Figure 1. X ray diffraction patterns of (a) reference HT Co 3 O 4 and (b) the fresh precipitated material (SP initial), with their indexation in the cubic cell (Fd3m space group).

Figure 2 .

 2 Figure 2. Evolution of the X ray diffraction patterns obtained in situ during thermal treatment of the SP initial material from room temperature (RT i) to 650 °C, with a final cooling down to room temperature (RT f).

Figure 3 .

 3 Figure 3. Rietveld refinements of the X ray diffraction patterns of SP initial phase, (a) at room temperature and (b) after a thermal treatment in situ at 650 °C. Each picture shows the experimental (red line) and calculated (black line) X ray powder diffraction patterns, the difference curve between the two profiles (blue line), and the positions of diffraction lines for the Co 3 O 4 and LiCoO 2 phases (green lines).

Figure 4 .

 4 Figure 4. Variations of the cell parameters of (a) the spinel type Co 3 O 4 phase and (b) the HT LiCoO 2 phase, as deduced from the thermal X ray diffraction in situ analysis (temperature increases up to 650 °C (red square) and then decreases to room temperature (blue square)). The values obtained for materials that were ex situ heat treated (SP initial to SP 650), that is, cooled down to room temperature, are also displayed (green open square). The HT LiCoO 2 has been evidenced to appear irreversibly below 400 °C and the superposed points between 400 and 650 °C account for the hexagonal cell parameters upon increasing and decreasing temper atures.

Figure 5 .

 5 Figure 5. Thermal evolutions of (a) the cobalt occupancy in the 8a tetrahedral sites and in the 16d octahedral sites and (b) the Co/O ratio, as deduced from the Rietveld refinement of the XRD diagrams resulting from the thermal X ray diffraction in situ analysis (from 25 to 650 °C).

Figure 6 .

 6 Figure 6. Evolution of average sizes of coherent domains (deduced from Scherrer formula; blue circle) and of specific surface areas (BET measurements; red triangle) for spinel type materials, heat treated at temperatures ranging from 25 to 300 °C.

Figure 7 .

 7 Figure 7. Variation of the Co 4+ /Co 3+ ratio (deduced from Rietveld refinement and chemical analysis) in spinel type materials, heat treated at temperatures ranging from 25 to 650 °C.

Table 1 .

 1 Structural and Profile Parameters, Determined from the Rietveld Refinement of the X ray Diffraction Pattern Recorded for the SP Initial Material

			SP-Initial, Co 3 O 4 Type Phase, Fd3̅ m, a cub = 8.076(1) Å
	atom	site		Wyckoff positions	occupancy	B/Å
	Co	8a	1/8	1/8	1/8	0.67(2)	2.7(2)
	Co	16d	1/2	1/2	1/2	0.82(4)	2.4(1)
	O	32e	0.2603(4)	0.2603(4)	0.2603(4)	1	2.7(3)
				Conditions of the Run
		T (K)			300
		angular range			10 ≤ 2θ ≤ 110°s
		tep scan increment (2θ)		0.0167°z
		ero point			0.05(1)
		number of fitted parameters		19
				Profile Parameters
		pseudo-Voigt function		PV

Table 2 .

 2 Structural Parameters of the X ray Diffraction Patterns, Obtained During the In Situ Analysis of the SP Initial Sample

			Co 3 O 4 phase, Fd3̅ m				LiCoO 2 phase, R3̅ m
				cobalt occupancy
	T/°C	a cub /Å	oxygen position	8a sites	16d sites	Co/O	a hex /Å	c hex /Å
	RT-i	8.076(1)	0.2603(4)	0.67(2)	0.82(2)	0.58
	50	8.077(1)	0.2601(4)	0.67(2)	0.82(2)	0.58
	100	8.076(1)	0.2605(4)	0.70(1)	0.85(2)	0.60
	150	8.069(1)	0.2596(4)	0.68(2)	0.83(2)	0.59
	200	8.075(1)	0.2594(4)	0.74(1)	0.85(2)	0.61
	250	8.0870(7)	0.2594(3)	0.82(1)	0.87(1)	0.64
	300	8.0937(4)	0.2603(3)	0.87(1)	0.90(1)	0.67
	350	8.0984(3)	0.2607(2)	0.88(1)	0.91(1)	0.67
	400	8.1041(3)	0.2610(2)	0.90(1)	0.90(1)	0.68	2.828(4)	14.175(3)
	450	8.1100(2)	0.2610(2)	0.91(1)	0.90(1)	0.68	2.831(3)	14.191(2)
	500	8.1151(2)	0.2614(2)	0.94(1)	0.92(1)	0.70	2.834(4)	14.205(2)
	550	8.1204(2)	0.2613(2)	0.93(1)	0.92(1)	0.69	2.836(3)	14.221(2)
	600	8.1269(2)	0.2614(3)	0.94(1)	0.92(1)	0.69	2.838(3)	14.244(2)
	650	8.1350(1)	0.2616(3)	0.94(1)	0.93(1)	0.70	2.840(3)	14.272(2)
	RT-f	8.0805(1)	0.2627(3)	0.93(1)	0.91(1)	0.69	2.816(4)	14.059(4)

Table 3 .

 3 Structural Parameters and Chemical Compositions Determined for Heat Treated Materials, SP Initial to SP 650 (Obtained by Heating Ex Situ the SP Initial Material at Temperatures Ranging from 25 to 650 °C) a Co 3 δ O 4 + LiCoO 2 " Co 3 δ O 4 + LiCoO 2 " Corresponding formulae were established on the basis of cobalt occupancies determined by Rietveld refinement and chemical analysis. Values for ideal Co 3 O 4 are also reported.

			Rietveld analysis				chemical analysis
				Co occupancy
											avg
									Li/Co	H/Co	oxidation
	synth.		oxygen			Co	Li	H	atomic	atomic	state of
	T/°C	a hex /Å	position	8a sites	16 sites	wt %	wt %	wt %	ratio	ratio	cobalt	formula
	initial	8.077(2)	0.2628(6)	0.61(2)	0.86(4)	68.85	0.47	1.00	0.06	0.85	2.80	H 1.34 Li 0.14 Co II 0.61 [Co III 1.58 Co IV 0.14 ]O 4 •0.32H 2 O
	100	8.073(2)	0.2612(7)	0.66(3)	0.84(4)	67.13	0.42	0.97	0.05	0.84	2.83	H 1.25 Li 0.13 Co II 0.66 [Co III 1.42 Co IV 0.26 ]O 4 •0.36H 2 O
	150	8.067(3)	0.2604(8)	0.68(3)	0.83(5)	66.97	0.42	0.93	0.05	0.81	2.83	H 1.28 Li 0.12 Co II 0.68 [Co III 1.37 Co IV 0.28 ]O 4 •0.31H 2 O
	200	8.064(2)	0.2588(6)	0.69(2)	0.81(3)	68.14	0.42	0.85	0.05	0.73	2.83	H 1.37 Li 0.12 Co II 0.69 [Co III 1.31 Co IV 0.30 ]O 4 •0.16H 2 O
	250	8.066(2)	0.2596(5)	0.75(2)	0.84(3)	68.13	0.42	0.57	0.05	0.49	2.81	H 1.04 Li 0.13 Co II 0.75 [Co III 1.39 Co IV 0.29 ]O 4 •0.07H 2 O
	300	8.071(2)	0.2597(6)	0.83(3)	0.86(3)	73.40	0.45	0.29	0.05	0.23	2.77	H 0.80 Li 0.13 Co II 0.83 [Co III 1.48 Co IV 0.24 ]O 4 •0.00H 2 O
	400 "Li x 650 8.0775(5) 0.2622(3) 0.84(2) 0.90(2) 73.42 0.45 0.08 0.05 0.06 2.75 8.0815(1) 0.2620(2) 0.93(1) 0.92(2) 73.66 0.46 0.02 0.05 0.02 2.75 "Li x ideal 0.2631 1 1 73.42 2.67 Co II [Co III 2 ]O 4
	Co 3 O 4
	a

The authors declare no competing financial interest.

■ ACKNOWLEDGMENTS

The authors would like to thank C. Denage and P. Dagault for technical assistance, and the French National Agency for Research (ANR, Stock E 2008 program, SupECCO project) for financial support.


Arry Morel

Jérémy Guinard

Fabien Lonjon

Lakshmi Sujeeun

Patrick Barberis

Stéphane Genin

Fabienne Vailleau

Marie-Christine Daunay

Jacques Dintinger

Stéphane Poussier

Jérémy Guin A Rd

L Ak

Shmi Sujeeun

Fabienne Vaille Au

M Arie-Christine Daunay

Nemo Pee Ters
email: nemo.peeters@inra.fr

A Nd

Emm A Nuel

W Ick Er
email: wicker@cirad.fr

The eggplant AG91-25 recognizes the Type III-secreted effector RipAX2 to trigger resistance to bacterial wilt (Ralstonia solanacearum species complex)

niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

SUMMARY

To deploy durable plant resistance, we must understand its underlying molecular mechanisms. Type III effectors (T3Es) and their recognition play a central role in the interaction between bacterial pathogens and crops. We demonstrate that the Ralstonia solanacearum species complex (RSSC) T3E ripAX2 triggers specific resistance in eggplant AG91-25, which carries the major resistance locus EBWR9. The eggplant accession AG91-25 is resistant to the wild-type R. pseudosolanacearum strain GMI1000, whereas a ripAX2 defective mutant of this strain can cause wilt. Notably, the addition of ripAX2 from GMI1000 to PSS4 suppresses wilt development, demonstrating that RipAX2 is an elicitor of AG91-25 resistance. RipAX2 has been shown previously to induce effector-triggered immunity (ETI) in the wild relative eggplant Solanum torvum, and its putative zinc (Zn)binding motif (HELIH) is critical for ETI. We show that, in our model, the HELIH motif is not necessary for ETI on AG91-25 eggplant. The ripAX2 gene was present in 68.1% of 91 screened RSSC strains, but in only 31.1% of a 74-genome collection comprising R. solanacearum and R. syzygii strains. Overall, it is preferentially associated with R. pseudosolanacearum phylotype I. RipAX2 GMI1000 appears to be the dominant allele, prevalent in both R. pseudosolanacearum and R. solanacearum, suggesting that the deployment of AG91-25 resistance could control efficiently bacterial wilt in the Asian, African and American tropics. This study advances the understanding of the interaction between RipAX2 and the resistance genes at the EBWR9 locus, and paves the way for both functional genetics and evolutionary analyses.

Keywords: bacterial wilt, disease resistance, functional genetics, host-pathogen interactions, plant immunity, plant-pathogenic bacteria, Solanum melongena

I N T RO D U C T I O N

In agrosystems, plants are constantly challenged by various pathogens, such as bacteria, viruses, fungi and nematodes. Monoculture as it is currently practised in modern agroecosystems not only leads to a reduction in plant genetic variability, but also to the selection of highly specialized and aggressive pathogens [START_REF] Stukenbrock | The origins of plant pathogens in agro-ecosystems[END_REF]. The use of plant resistant germplasm appears to be the best strategy to control plant diseases, as it is cost-effective and environmentally friendly (Boyd et al., 2013). However, an increasing number of studies have regularly reported cases of resistance breakdown (Daverdin et al., 2012; Kiyosawa, 1982; Montarry et al., 2006; Moury, 2010; Palloix et al., 2009; Zeigler et al., 1994), which have questioned the durability of plant resistance. The prediction of this durability can be achieved by deciphering the evolutionary potential of the pathogen [START_REF] Mcdonald | Pathogen population genetics, evolutionary potential, and durable resistance[END_REF]. However, first, we need to understand the molecular bases of plant-microbe interactions in order to assess the evolutionary constraints on their stability and dynamics. Bacterial type III effectors (T3Es) are key components in this interaction as they can both subvert and trigger the plant immune system [START_REF] Buttner | Behind the lines-actions of bacterial type III effector proteins in plant cells[END_REF]. As such, these molecules are players in the identification, description and therefore development of sustainable resistance strategies [START_REF] Clarke | Genome-enabled phylogeographic investigation of the quarantine pathogen Ralstonia solanacearum Race 3 Biovar 2 and screening for sources of resistance against its core effectors[END_REF][START_REF] Vleeshouwers | Effectors as tools in disease resistance breeding against biotrophic, hemibiotrophic, and necrotrophic plant pathogens[END_REF].

Bacterial wilt affects more than 54 botanical families, including several crops of major interest (tomato, potato, banana, groundnut, eucalyptus) [START_REF] Hayward | Biology and epidemiology of bacterial wilt caused by Pseudomonas solanacearum[END_REF][START_REF] Hayward | The hosts of Pseudomonas solanacearum. In: Bacterial wilt -the disease and its causative agent, Pseudomonas solanacearum[END_REF]. Its causative agent is the soil-borne Betaproteobacterium Ralstonia solanacearum, a species complex composed of four phylotypes of specific geographical origins: Asia and Africa for phylotype I, Africa for phylotype III, America for phylotype II and Indonesia for phylotype IV [START_REF] Fegan | Bacterial wilt disease and the Ralstonia solanacearum species complex[END_REF][START_REF] Wicker | Contrasting recombination patterns and demographic histories of the plant pathogen Ralstonia solanacearum inferred from MLSA[END_REF]. A recent taxonomic update [START_REF] Safni | Polyphasic taxonomic revision of the Ralstonia solanacearum species complex: proposal to emend the descriptions of Ralstonia solanacearum and Ralstonia syzygii and reclassify current R. syzygii strains as Ralstonia syzygii subsp. syzygii subsp.nov., R. solanacearum phylotype IV strains as Ralstonia syzygii subsp. indonesiensis subsp. nov., banana blood disease bacterium strains as Ralstonia syzygii subsp. celebesensis subsp. nov. and R. solanacearum phylotype I and III strains as Ralstonia pseudosolanacearum sp[END_REF] has proposed that R. solanacearum should be renamed the Ralstonia solanacearum species complex (RSSC), subdivided into three distinct species: R. pseudosolanacearum (phylotypes I and III), R. solanacearum (phylotypes IIA and IIB) and R. syzygii (phylotype IV and BDB).

The search for and breeding for plant resistance against RSSC are critical for food security in numerous countries. However, the genetic bases of plant resistance to this major plant pathogen still remain unknown, except for the model species Arabidopsis thaliana (Bernoux et al., 2008; Deslandes and Genin, 2014; Deslandes et al., 2002; Le Roux et al., 2015; Tasset et al., 2010) and Medicago truncatula (Ben et al., 2013; Lohou et al., 2014; Turner et al., 2009; Vailleau et al., 2007). Interactions between RSSC strains and Solanaceae genotypes (tomato, eggplant and pepper) have already been investigated [START_REF] Lebeau | Résistance de la tomate, l'aubergine et le piment à Ralstonia solanacearum: interactions entre les géniteurs de résistance et la diversité bactérienne, caractérisation et cartographie des facteurs génétiques impliqués chez l'aubergine[END_REF][START_REF] N'guessan | Phylogénie, structure génétique et diversité de virulence de Ralstonia solanacearum, agent du flétrissement bactérien, en Côte d'Ivoire[END_REF] and formalized into six patterns, called pathoprofiles [START_REF] Lebeau | Bacterial wilt resistance in tomato, pepper, and eggplant: genetic resources respond to diverse strains in the Ralstonia solanacearum species complex[END_REF]. This screening established that the highest resistance levels against RSSC were found in the eggplant germplasm. The resistance of the AG91-25 accession (Ano et al., 1991(Ano et al., , 2002) ) has been mapped and led to the identification of the major resistance locus ERs1 [START_REF] Lebeau | Genetic mapping of a major dominant gene for resistance to Ralstonia solanacearum in eggplant[END_REF], later named EBWR9 [START_REF] Salgon | Eggplant resistance to the Ralstonia solanacearum species complex involves both broad-spectrum and strainspecific quantitative trait loci[END_REF]. In order to characterize the durability of AG91-25 resistance, it is necessary to understand the molecular basis of the interaction between RSSC and the AG91-25 eggplant immune system.

In this work, we validated the contribution of the GMI1000 allele of ripAX2 to the control of disease by AG91-25. We showed that the immunity triggered by RipAX2 does not require a putative zinc (Zn)-binding motif, previously shown to be critical to trigger immunity on Solanum torvum leaves [START_REF] Nahar | Ralstonia solanacearum type III secretion system effector Rip36 induces a hypersensitive response in the nonhost wild eggplant Solanum torvum[END_REF]. Finally, we investigated the prevalence and allelic variability of ripAX2 alleles in pathogen populations in order to predict the durability of EBWR9 resistance if it were to be deployed in specific R. solanacearum-infested areas.

R E S U LT S

RipAX2 GMI1000 triggers AG91-25 resistance Disease control by a single resistance (R) gene is often caused by the specific recognition of a single pathogen effector, which elicits plant defences [START_REF] Buttner | Behind the lines-actions of bacterial type III effector proteins in plant cells[END_REF]. In order to investigate whether AG91-25 resistance is associated with an effector from GMI1000, 41 single mutants of effectors described in [START_REF] Cunnac | Inventory and functional analysis of the large Hrp regulon in Ralstonia solanacearum: identification of novel effector proteins translocated to plant host cells through the type III secretion system[END_REF] were screened for disease appearance on a few GMI1000-resistant AG91-25 plants, after inoculation by soil drenching [10 8 colony-forming units (CFU)/mL, 10 mL per plant]. Susceptible MM738 plants were also included in the tests. From this screen, only the ripAX2 mutant GMI1000 ripAX2::pCZ367 was found to cause disease on AG91-25 plants. To confirm the role of RipAX2 in AG91-25 disease resistance, a complemented strain was made by introducing a wild-type copy of ripAX2 in the mutant (GMI1000 ripAX2::pCZ367::ripAX2). The GMI1000 wild-type strain and the GMI1000 ripAX2::pCZ367::ripAX2 strain were not able to trigger disease on AG91-25, whereas the ripAX2 mutant caused disease 4 days after inoculation (Fig. 1). The strains GMI1000, GMI1000 ripAX2::pCZ367 and GMI1000 ripAX2::pCZ367::ripAX2 were still able to induce disease on the susceptible control MM738, with similar wilting rates (Fig. 1). Biological replicates performed on AG91-25 and on the MM738 susceptible control gave similar results (Figs S1 and S2, see Supporting Information).

The eggplant pathogenic strain PSS4 expressing ripAX2 GMI1000 becomes avirulent on AG91-25

The PSS4 strain is pathogenic on AG91-25 and its genome lacks any ripAX2 allele [START_REF] Guinard | Draft genome sequences of nine strains of Ralstonia solanacearum differing in virulence to eggplant (Solanum melongena)[END_REF]. In order to investigate whether the presence of ripAX2 is sufficient for AG91-25 to control disease, a PSS4 strain expressing ripAX2 GMI1000 was generated, and inoculated by soil drenching as mentioned above. This strain was not able to cause disease on AG91-25, but still able to wilt the MM738 susceptible control (Fig. 2). Thus, we established that the expression of ripAX2 GMI1000 in an AG91-25 pathogenic strain is sufficient to trigger AG91-25 resistance. Biological replicates performed on AG91-25 and on the MM738 susceptible control gave similar results (Figs S1 andS2).

RipAX2-triggered AG91-25 resistance restricts plant colonization only after root infection

From the soil, R. solanacearum can enter the root system by wounds or lateral root emerging points. It then colonizes the cortex, invades the xylem and spreads to aerial parts, where it causes wilting [START_REF] Denny | Plant pathogenic Ralstonia species[END_REF]. This multistep infection process makes it challenging to determine where resistance actually occurs.

In order to better characterize AG91-25 resistance, bacterial multiplication in the stem was quantified after root inoculation and stem injection. After root inoculation, the bacterial load (log 10 CFU/g fresh weight) reached high levels in AG91-25 infected with PSS4 and GMI1000 ripAX2::pCZ367, 5 days after inoculation (means of 10.3 and 9.7, respectively; Fig. 3). No bacteria were detected in the stems of plants inoculated with PSS4 expressing ripAX2 and with GMI1000 ripAX2::pCZ367::ripAX2 at 5 or 10 days post-inoculation. A few plants inoculated with GMI1000 showed some bacterial multiplication in their stem at 5 or 10 days post-inoculation (Fig. 3); one of Fig. 1 The RipAX2 type III effector of Ralstonia pseudosolanacearum is necessary for resistance in AG91-25 eggplants. (A) Photographs taken at 6 days post-inoculation. The R. pseudosolanacearum GMI1000 wild-type strain and the GMI1000 ripAX2::pCZ367::ripAX2 complemented strain are not able to trigger disease on AG91-25, whereas the GMI1000 ripAX2::pCZ367 mutant is able to do so. (B) Survival curves representing the wilting of AG91-25 eggplants after root inoculation with the GMI1000 wild-type strain, GMI1000 ripAX2::pCZ367 mutant and the complemented strain [10 8 colony-forming units (CFU)/mL, 10 mL per plant, 18 plants per strain]. Log-rank (Mantel-Cox) test P values compared with GMI1000 are shown below the graph. (C) Photographs taken at 6 days post-inoculation. The strains GMI1000, GMI1000 ripAX2::pCZ367 and GMI1000 ripAX2::pCZ367::ripAX2 are all able to wilt MM738 susceptible eggplants. these plants showed wilting symptoms, suggesting that resistance did not occur in some rare cases. After stem injection, a high bacterial load was detected after 3 days in AG91-25 inoculated with GMI1000, GMI1000 ripAX2::pCZ367 and GMI1000 ripAX2::p-CZ367::ripAX2 (Fig. 4). The levels of bacteria were not significantly different between plants injected with GMI1000 and GMI1000 ripAX2::pCZ367 and high in all plants sampled, contrasting strongly with the results after root inoculation shown in Fig. 3. Furthermore, we observed wilting symptoms at 3 days after stem injection by all strains on some of the plants before destructive sampling (33% of AG91-25 inoculated with GMI1000, 53% of AG91-25 inoculated with GMI1000 ripAX2::pCZ367 and 40% of AG91-25 inoculated with GMI1000 ripAX2::pCZ367::ripAX2), indicating that the bacteria had reached, colonized and clogged the xylem vessels of the stem after injection.

Collectively, these results indicate that the key biological process inducing resistance to bacterial multiplication most probably occurs before the stem multiplication stage.

The conserved putative Zn-finger domain is not critical for RipAX2-triggered immunity

The putative Zn-finger domain has been shown to be required for the immunity triggered by RipAX2 RS1000 (initially named Rip36) on S. torvum [START_REF] Nahar | Ralstonia solanacearum type III secretion system effector Rip36 induces a hypersensitive response in the nonhost wild eggplant Solanum torvum[END_REF]. RipAX2 RS1000 and RipAX2 GMI1000 have exactly the same amino acid sequence (Fig. S3, see Supporting Information). We generated the same point mutant (E149A) and showed that the integration of this mutated version (through the integrative plasmid pAM123) in both GMI1000 ripAX2::pCZ367 and PSS4 backgrounds (inoculated by soil drenching) restored the avirulence phenotype on AG91-25 eggplant (Fig. 5A,B), unlike on S. torvum [START_REF] Nahar | Ralstonia solanacearum type III secretion system effector Rip36 induces a hypersensitive response in the nonhost wild eggplant Solanum torvum[END_REF].

Biological replicates performed on AG91-25 and on the MM738 susceptible control gave similar results, as detailed in Figs S4 and S5 (see Supporting Information), respectively.

ripAX2 is highly prevalent in RSSC and is preferentially associated with phylotype I

The ripAX2 gene was successfully amplified and sequenced in 68.1% of the world RSSC collection considered (n = 91). This proportion was lower (31.1%) in the RSSC genomes available in the RalstoT3E database (n = 74), most probably as a result of the difference in phylotype composition: our collection contains a majority of R. pseudosolanacearum phylotype I, whereas the RalstoT3E database contains a majority of R. solanacearum phylotype II and R. syzygii phylotype IV (Table 1). Based on the 15 strains common to both collections, polymerase chain reaction (PCR)-based and genome sequencing-based screenings gave highly concordant results (Table S3, see Supporting Information).

Because both collection compositions were unbalanced across phylotypes, we compared ripAX2-per-phylotype distributions using χ 2 tests. We observed that ripAX2 was significantly Log 10 CFU/g fresh weight Log 10 CFU/g fresh weight more present in R. pseudosolanacearum phylotype I than expected in both collections, whereas it was significantly more absent in R. syzygii phylotype IV (Table 1). ripAX2 was less commonly present in R. solanacearum phylotypes IIA and IIB, but this difference was not statistically supported. Hence, ripAX2 seemed to be preferentially associated with phylotype I. Focusing on phylotype I, in which sample sizes were highest, we assessed whether the ripAX2 distribution could be associated with region of origin (defined as Africa, Asia or South America). Despite the low sample size, RipAX2 seemed to be significantly less prevalent in South America (six strains of 12; χ 2 P = 0.004**).

Among 19 RipAX2 identified alleles, RipAX2 GMI1000 is the most prevalent Although most ripAX2 DNA sequences were 657 base pairs in length (219 amino acids), four were highly divergent (608 to 666 amino acids) and could not be aligned with the others. After the first 103 base pairs, the sequence amplified in RUN1546, RUN1740 and RUN1743 contained a very long insertion and several early stop codons, suggesting that these alleles code for non-functional proteins (Fig. S6, see Supporting Information). Meanwhile, the RUN1994 sequence was incomplete (206 amino acids), with also several early stop codons. Fifteen other RipAX2 alleles were identified (Fig. 6), among which seven could be considered as non-functional because of early stop codons generating a truncated protein by more than 20% of the reference effector protein (RipAX2 GMI1000 ). Amongst these 19 alleles, the 11 non-functional alleles were not considered in the analysis below.

Amongst the eight probably functional alleles, RipAX2 GMI1000 (coded HapAA22; Table S5, see Supporting Information) was the most prevalent, and found in both R. pseudosolanacearum phylotype I (n = 40) and R. solanacearum phylotype IIA (n = 1). Apart from RipAX2 GMI1000 , all seven alleles were phylotype specific: three were specifically found in phylotype I (HapAA02, HapAA07, HapAA20), whereas two alleles were phylotype III specific (HapAA05 and HapAA08) and two were phylotype IIA specific ( HapAA03 andHapAA06).

The analysis of the geographical distribution of the different RipAX2 alleles was hampered by the large sample imbalances between countries. To take into account these sample unbalances, we looked not only at the number of alleles per region, but also at the genotype-per-strain rate (also named G/N). Across the three regions represented in the collection, Africa displayed the highest G/N (meaning that, with a set amount of strains, more different alleles were found), followed by Asia and then South America (Fig. S7, see Supporting Information). The RipAX2 GMI1000 allele was prevalent within each region, and its distribution was not significantly associated with any country or region (Table S6, see Supporting Information). Apart from RipAX2 GMI1000 , no other allele was found to be present in more than one region. We have demonstrated that ripAX2 GMI1000 is the determinant triggering resistance to RSSC in the AG91-25 eggplant line. Moreover, the naturally virulent PSS4 strain no longer triggers disease once it expresses the RipAX2 GMI1000 allele, without significantly affecting its aggressiveness on the susceptible control MM738.

Our research was supported by previous results indicating that RipAX2 is associated with resistance in solanaceous plants (eggplant AG91-25, tomato Hawaii 7996; [START_REF] Pensec | Towards the identification of type III effectors associated with Ralstonia solanacearum virulence on tomato and eggplant[END_REF]. Furthermore, we showed that plants recognizing RipAX2 largely excluded bacteria from entering the stems after root colonization, suggesting that the resistance occurs in the early stages of infection. This resistance could occur anywhere from the infected roots to further root and lower stem colonization.

Recently, an orthologue of ripAX2 GMI1000 in the RS1000 strain (ripAX2 RS1000 ) has been shown to induce a strong hypersensitive response (HR) in the leaves of a wild relative of eggplant (S. torvum cv. torubamubiga), but not on eggplant (Solanum melongena cv. senryo-nigou) [START_REF] Nahar | Ralstonia solanacearum type III secretion system effector Rip36 induces a hypersensitive response in the nonhost wild eggplant Solanum torvum[END_REF]. However, as no R gene has been characterized to date in these two cultivars, the R gene involved in the recognition of RipAX2 RS1000 has not yet been identified. In this model, the recognition of the effector was dependent on the putative Zn-binding domain HEXXH. More precisely, the glutamic acid at position 149 was critical for the recognition of RipAX2 by the S. torvum immune system. We observed that this HEXXH domain was conserved within all the likely 'functional' alleles (i.e. predicted to code for a 219-amino-acid protein) identified in the RSSC, whereas the corresponding strains differed in virulence on AG91-25 eggplant.

In addition, we experimentally demonstrated that the mutation E149A did not change its ability to trigger resistance on AG91-25 eggplant, indicating that this residue was not critical for this phenotype. Collectively, these results suggest that the RipAX2 elicitation of defences may involve different mechanisms in S. torvum leaves and S. melongena root infection. It will be interesting to study RipAX2 residues under selective pressure in order to test which protein features are critical for S. melongena AG91-25 resistance.

RipAX2 is the second known RSSC effector triggering eggplant resistance, the first being RipP2 (formerly named PopP2) recognized by RE-bw (Xi'ou et al., 2014).

It remains to be determined which plant proteins specifically recognize RipAX2. It is tempting to speculate that the direct or indirect recognition of RipAX2 involves one of the genes located within the major resistance locus EBWR9 [START_REF] Salgon | Eggplant resistance to the Ralstonia solanacearum species complex involves both broad-spectrum and strainspecific quantitative trait loci[END_REF]. Indeed, strains controlled by the EBWR9 locus tested so far (GMI1000, CMR134) carry a functional allele of ripAX2, whereas all EBWR9-uncontrolled strains carry no ripAX2 (CFBP2957, CFBP3059, PSS4), or a non-functional copy of ripAX2 (To10). EBWR9 is located in a 3-cM zone, corresponding to an approximately 3-Mb [START_REF] Lebeau | Genetic mapping of a major dominant gene for resistance to Ralstonia solanacearum in eggplant[END_REF] interval, which has been reduced to 1.77 Mb and located on the long arm of chromosome 9 [START_REF] Salgon | Eggplant resistance to the Ralstonia solanacearum species complex involves both broad-spectrum and strainspecific quantitative trait loci[END_REF]. Five candidate R genes have been identified in this region. The fine genetic mapping of this quantitative trait locus (QTL) is currently in progress [START_REF] Salgon | Déterminisme génétique de la résistance au flétrissement bactérien chez l'aubergine et application en sélection variétale[END_REF] to pave the way to gene cloning. Another way to determine the Table 1 Distribution of ripAX2 within the Ralstonia solanacearum species complex (RSSC) world collection as estimated by polymerase chain reaction (PCR) sequencing (A), and within the RalstoT3E database of genomic sequences as estimated by automatic annotation (B). The gene was considered to be present if annotated as functional (PROT), frameshifted (FS) or pseudogene (PS). Results are expressed in strain numbers (percentage). Sample distributions per state (presence/absence) and phylotype were compared using a χ 2 test. The H0 tested is: 'Respective frequencies of ripAX2 presence and absence within each phylotype are equal to their expected values based on the total frequencies'. ‡

The H0 tested is: 'Respective distributions of phylotypes within each ripAX2 state (presence, absence) are equal to their expected values based on the total frequencies'.

NS, not significant; *significant (0.05 > P ≥ 0.01); **highly significant (0.01 > P ≥ 0.001); ***very highly significant (P < 0.001).

candidate genes involved in the recognition of RipAX2 would be to use reverse genetics: virus-induced gene silencing (VIGS) on eggplant [START_REF] Liu | Virus-induced gene silencing in eggplant (Solanum melongena)[END_REF] and RNA interference (RNAi) have been used successfully to find the R genes involved in effector recognition in Nicotiana benthamiana [START_REF] Brendolise | A novel hairpin library-based approach to identify NBS-LRR genes required for effector-triggered hypersensitive response in Nicotiana benthamiana[END_REF]. ripAX2 is a rather highly prevalent effector in the RSSC, as reflected by the observation that 68.1% of our strain collection carried an allele of this gene, but seems to be preferentially associated with phylotype I, absent in phylotype IV and rare in phylotype II. It is important to stress that future screenings of much larger collections, balanced across the different phylotypes infecting the Solanaceae, will allow a much more precise assessment of the distribution of ripAX2 in natural diversity. Moreover, RipAX2 GMI1000 is the dominant allele in every region probed, suggesting that a large proportion of the pathogen population is controlled by AG91-25 eggplant. One may thus speculate that the deployment of the AG91-25 line or derived eggplant cultivars would provide efficient control of the prevalent populations. However, seven other potentially functional alleles have been found that may modulate the plant defence response. From our data, one strategy built by RSSC strains to bypass RipAX2-triggered plant resistance includes the loss of ripAX2 (the PSS4 case) and the inactivation of the gene through frameshifts (the 11 probably non-functional alleles identified), but it is plausible that point mutations within the gene coding sequence or the alteration of gene expression through mutations in the promoter region could also lead to a loss of RipAX2 recognition. A deeper understanding of the RipAX2-eggplant interaction is needed: (i) to identify the critical residues for AG91-25 recognition and resistance, taking into account the natural variability detected; and (ii) to assess the possible variations in effector expression. Moreover, a global surveillance of RSSC populations will help to assess the respective frequencies of the different functional alleles. As this global screening considered RSSC populations that were not challenged by AG91-25 resistance, future research should address the evolution of RipAX2-containing populations submitted to AG91-25 resistance selection pressure.

To conclude, we have shown that RipAX2 triggers resistance on AG91-25 resistant eggplant, whose recognition occurs in the Fig. 6 Proteic alignment of 15 RipAX2 alleles (of 19) identified within the world Ralstonia solanacearum species complex (RSSC) collection. Each allele number is linked to its representative strain and to the number of strains in which it was found. Proteic sequences were aligned (MUSCLE with default settings) and clustered using the FastTree method, with optimization of the Gamma20 likelihood at 20 rate categories of sites. The zinc (Zn)-binding motif (HEXXH) described by [START_REF] Nahar | Ralstonia solanacearum type III secretion system effector Rip36 induces a hypersensitive response in the nonhost wild eggplant Solanum torvum[END_REF] is marked by dark shading (positions 152-158). The effector sequence is considered to be non-functional (NF) if its length is less than 80% of the GMI1000 reference sequence (219 amino acids). [Colour figure can be viewed at wileyonlinelibrary.com] early infection stages and may be mediated by an R gene present in the ERs1/EBWR9 locus. This latter statement needs to be investigated further thanks to the generation of near-isogenic lines (NILs) carrying (or not) EBWR9, or by reverse genetic methods. Both approaches could allow the cloning of this gene and a better characterization of AG91-25 resistance to the RSSC.

E X P E R I M E N TA L P R O C E D U R E S

Plant material

Two eggplant accessions (S. melongena) were used: the resistant eggplant AG91-25, also called MM960, and the susceptible eggplant MM738 (Ano et al., 1991, 2002; Doganlar et al., 2002; Wu et al., 2009). Eggplant seeds were sown 4 weeks before inoculation and were then transplanted into 7 × 7 × 6-cm 3 black plastic pots at 1 week after sowing. Four-week-old plants were transferred to a growth chamber at 90% relative humidity, with a day/night thermoperiod of 28 °C/24 °C and a 12-h light/12-h dark photoperiod.

Bacterial strains

The distribution of the ripAX2 coding sequence within the RSSC was assessed on a world collection of 91 bacterial strains, gathering representatives of the species R. pseudosolanacearum (phylotypes I and III) and R. solanacearum (phylotypes IIA and IIB) (see Table S1 in Supporting Information). Because of its limited epidemiological importance in Solanaceae, R. syzygii ssp. indonesiensis (phylotype IV) was not considered in this screening. Strains were chosen following two criteria: (i) they were sampled from 13 geographical areas of agronomic importance, and/ or containing international breeding stations, in Africa (Burkina Faso, Cameroon, Ivory Coast, South Africa), Asia and Indian Ocean (Reunion Island, India, Indonesia, Thailand, Philippines, Taiwan, Australia), and Caribbean and South America (French Guiana, Martinique); and (ii) they were mostly isolated from the Solanaceae. Some of these strains were screened for pathogenicity on MM738 and AG91-25, following the methodology described in N'Guessan et al (2012). All were able to trigger disease on MM738, but displayed different interaction profiles on AG91-25 resistant eggplant, ranging from being unable to colonize and wilt AG91-25 to highly pathogenic strains (Table S1).

Bacterial cultures and DNA extraction

From stock tubes stored at -80 °C, strains were revived in nutrient broth (one to two beads per 3 mL of broth) for 48 h at 28 °C, and then quadrant streaked (50 µL of broth) on Kelman's medium. Cultures were then multiplied from a single colony and suspended (one 10-µL loopful) in 1 mL of NaCl (0.5 m) for rinsing. Suspensions were then centrifuged (6000 g for 3 min) and pellets were stored at -20 °C until DNA extraction. Genomic DNA was extracted using the Wizard ® Genomic DNA Purification Kit (Promega, Charbonnières, France) following the manufacturer's recommendations. DNA concentration and quality were assessed using a NanoDrop ® 8000 device. Bacterial DNAs were then diluted to 2 ng/μL and stored at -20 °C until use.

ripAX2 cloning and complementation constructs

The strain GMI1000 ripAX2::pCZ367 has been obtained previously (Cunnac et al., 2004). The complementation was generated by a double crossing-over insertion using a plasmid derived from the pRC toolkit [START_REF] Monteiro | A chromosomal insertion toolbox for promoter probing, mutant complementation, and pathogenicity studies in Ralstonia solanacearum[END_REF]. The pNP329 plasmid [START_REF] Wang | Functional assignment to positively selected sites in the core type III effector RipG7 from Ralstonia solanacearum[END_REF] was modified (Acc65i-BsrGI fragment was removed) to generate pNP380. The ripAX2 gene [coding sequence (CDS) flanked in 5′ by 300 bp and in 3′ by 949 bp] was subcloned from GMI1000 genomic DNA into pNP380 to generate the pNP384 plasmid. Starting from pNP384, the ripAX2 CDS was point mutated to generate the equivalent of Rip36E149A [START_REF] Nahar | Ralstonia solanacearum type III secretion system effector Rip36 induces a hypersensitive response in the nonhost wild eggplant Solanum torvum[END_REF] by site-directed mutagenesis using the following oligonucleotides: oAM277 (GGGCATGGATCAGTGCATGGGCGAGGACA) and oAM278 (TGTCCTCGCCCATGCACTGATCCATGCCC). This generated the plasmid pAM123. These plasmids were used to transform both GMI1000 ripAX2::pCZ367 and the PSS4 strain.

Plant infection

Bacterial strains were grown overnight in BG medium and then diluted to an optical density at 600 nm (OD 600 ) of 0.1 (~10 8 CFU/ mL) with water to obtain the inocula. For each plant, a deep 'L'shaped cut was made in the soil with a sharp scalpel in order to cut the roots. Ten millilitres of inoculum were poured into the 'L'-shaped open soil surface. The symptoms were scored daily on a 0-4 scale (no wilting to complete wilting). For stem bacterial load quantification, 1 cm of stem was sampled above the cotyledons, weighed and the surface was disinfected in a 70% alcohol solution, followed by rinsing in water. It was then cut into six parts and placed into 1 mL of water for a minimum of 30 min to allow the bacteria to diffuse. Appropriate dilutions were then plated in order to evaluate the bacterial load.

For stem injection, 10 µL of a bacterial suspension at 10 6 CFU/ mL were injected above the cotyledons with a microsyringe. The microsyringe was sterilized between each plant by pumping up and down with ethanol and then four times with water. For sampling, 1 cm of stem surrounding the injection point was taken, weighed, surface sterilized, cut into pieces and placed into 1 mL of water to diffuse. Appropriate dilutions were plated.

ripAX2 prevalence and allele diversity in RSSC

The prevalence and diversity of the ripAX2 gene were addressed on a 91-strain RSSC collection described above, taking advantage of the available genomic sequences (Ailloud et al., 2015;[START_REF] Guinard | Draft genome sequences of nine strains of Ralstonia solanacearum differing in virulence to eggplant (Solanum melongena)[END_REF][START_REF] Remenant | Genomes of three tomato pathogens within the Ralstonia solanacearum species complex reveal significant evolutionary divergence[END_REF][START_REF] Salanoubat | Genome sequence of the plant pathogen Ralstonia solanacearum[END_REF], as well as using PCR screening.

The distribution of the ripAX2 coding sequence within the RSSC was also assessed on a world collection of 74 whole-genome sequences (Table S2, see Supporting Information), publicly available and harboured in the RalstoT3E database (https://iant.toulouse. inra.fr/bacteria/annotation/site/prj/T3Ev3/). ripAX2 was assessed as 'present in the strain as one copy', 'present but frameshifted in the strain', 'missing in the strain' or 'present as a pseudogene copy in the strain'.

Design of ripAX2 PCR primers

The ripAX2 GMI1000 coding sequence was downloaded from the website 'RalstoT3E' (https://iant.toulouse.inra.fr/bacteria/annotation/site/prj/T3Ev3/) and BLAST-queried on the MaGe interface (Magnifying Genomes, https://genoscope.cns.fr/microscope/ mage) to search for gene orthologues and to extract the 400bp upstream and downstream regions. Orthology search was performed using the following parameters: (i) sequence identity above 80%; and (ii) minimal (MinLrap) and maximal (MaxLrap) ratios of alignment lengths above 90%. Gene and flanking regions of ripAX2 GMI1000 , ripAX2 Y45 and ripAX2 MOLK2 were concatenated using Geneious v5.5 (https:// www.geneious.com, [START_REF] Kearse | Geneious Basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data[END_REF] and then aligned using ClustalW [START_REF] Thompson | CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice[END_REF]. External primers were designed on the consensus sequences of the flanking regions of ripAX2 GMI100 and ripAX2 Y45 only (phylotype I) using the Primer3 algorithm under Geneious, with the following parameters: annealing temperature around 60 °C and primer sizes ranging from 18 to 25 nucleotides. Details of the primer sequences are given in Table S4 (see Supporting Information).

Gene amplification and sequencing

For each strain, putative effectors were PCR amplified on 20 ng of sample DNA template. PCRs (total volume of 25 µL) consisted of 1 U of Red Goldstar Taq DNA polymerase, 25 pmol of each primer, 1 × PCR buffer, 1.5 mm MgCl 2 , 0.2 mm of each deoxynucleotide (dNTP) and 1 × Q-solution. The reaction was cycled in an Eppendorf (Montesson, France) Mastercycler Gradient thermocycler or an Applied Biosystems (Villebon-sur-Yvette, France) GenAmp PCR System 9700 thermocycler with a first denaturation step at 96 °C for 5 min, followed by 30 cycles of 30 s at 95 °C, 60 s at 55-57 °C (see Table S2) and 60 s at 72 °C, and a final elongation step of 10 min at 72 °C. All PCR products were resolved on a 2% agarose gel and visualized with UV light after ethidium bromide staining (5 µg/mL); fragment sizes were estimated in comparison with a 100-bp DNA ladder (New England BioLabs, Evry, France).

PCR products were vacuum dehydrated and sent to Beckman Coulter Genomics (Takeley, Essex, UK) for further purification and double-strand sequencing using PCR primers as sequencing primers. Raw sequences from both strands were assembled using Geneious v5.5 to generate consensus sequences; all ambiguous sequences were reamplified and resequenced. Alignments were carried out using the ClustalW alignment tool under Geneious, together with the reference sequences (GMI1000, RS1000, MOLK2).

Sequence extremities were trimmed on the basis of the sequence quality and reading frame, and to match the gene entire length. In some cases, the entire gene sequence was obtained by assembling sequences obtained from both external and internal primers. Each strain was amplified at least twice.

ACC E SSI O N N U M B E R S

The DNA sequences described in this article were deposited in GenBank under the numbers MF055715-MF055746. Details are given in Table S3.

SU PP O R T I N G I N F O R M AT I O N

Additional supporting information may be found in the online version of this article at the publisher's web site: Fig. S1 Kaplan-Meier survival curves representing the wilting of AG91-25 plants inoculated with the strains GMI1000, GMI1000 ripAX2::pCZ367, GMI1000 ripAX2::pCZ367::ripAX2, PSS4 and PSS4::ripAX2 in two supplemental replicates (12 and 22 plants per strain, respectively). Log-rank (Mantel-Cox) tests are given with P value. Fig. S2 Kaplan-Meier survival curves observed on MM738 plants (susceptible control) inoculated with strains GMI1000, GMI1000 ripAX2::pCZ367, GMI1000 ripAX2::pCZ367::ripAX2, PSS4 and PSS4::ripAX2 in one supplemental replicate. Log-rank (Mantel-Cox) tests are given with P value. Fig. S3 Alignment of the RipAX2 protein alleles from GMI1000, RS1000 and MOLK2, and their percentages of identity. Fig. S4 Kaplan-Meier survival curves observed on AG91-25 plants inoculated with strains GMI1000 ripAX2::pCZ367, GMI1000 ripAX2::pCZ367::ripAX2, GMI1000 ripAX2::pCZ367:: ripAX2-E149A, PSS4, PSS4::ripAX2 and PSS4::ripAX2-E149A in two supplemental replicates (22 plants and 18 plants per strain for each respective replicate). Log-rank (Mantel-Cox) tests are given with P value. Fig. S5 Kaplan-Meier survival curves observed on MM738 plants inoculated with strains GMI1000 ripAX2::pCZ367, GMI1000 ripAX2::pCZ367::ripAX2, GMI1000 ripAX2::pCZ367:: ripAX2-E149A, PSS4, PSS4::ripAX2 and PSS4::ripAX2-E149A in three replicates. Log-rank (Mantel-Cox) tests are given with P value. Fig. S6 DNA alignment of the reference ripAX2 alleles (GMI1000 and RS1000) with the long ripAX2 alleles amplified within the phylotype I Ivorian strains RUN1546 (sequevar 13), RUN1740 and RUN1743 (sequevar 31), and in the Guianese phylotype IIA RUN1994 (sequevar 41). Alignments were performed using MUSCLE under GENEIOUS v5.5. Each strain is represented by its nucleotide sequence (upper line) and proteic sequence (lower line). Black squares indicate stop codons. Fig. S7 Geographical distribution of the 19 RipAX2 alleles, either functional (PROT) or non-functional (NF), across regions within the phylotype I strains. The heatmap was built with the R package pheatmap, based on Euclidean distances between observations; the clusters were calculated using the Ward method. For each region, the number of RipAX2 alleles (G) and phylotype I strains (N) are presented. 'Africa' gathers Burkina-Faso, Cameroon, Ivory Coast, Reunion Island and South Africa; 'Asia' gathers Australia, India, Indonesia, Philippines, Taiwan and Thailand; 'South America' gathers French Guiana and Martinique. The heatmap values correspond to the numbers of strains. Table S1 Bacterial strains of the Ralstonia solanacearum species complex (RSSC) tested for the presence of the ripAX2 coding sequence. Phylotype I and III strains belong to the species R. pseudosolanacearum, and phylotype IIA and IIB strains belong to the species R. solanacearum.

Table S2 Distribution of ripAX2 within the 74 Ralstonia solanacearum species complex (RSSC) genomes harboured in the RalstoT3E database. Strains are ranked by phylotype and code. Phylotypes I and III are grouped within R. pseudosolanacearum, phylotypes IIA and IIB within R. solanacearum and phylotype IV within R. syzygii. The ripAX2 type III effector is present as a complete sequence (PROT), frameshifted (FS), pseudogenized (PS) or absent (NO). Table S3 Comparison of ripAX2 distribution, assessed by polymerase chain reaction (PCR) amplification or genome sequencing, in the 15 strains common to our collection and the RalstoT3E database. The gene was present (1), frameshifted (FS) or absent (0). Table S4 External and internal primers designed to amplify the ripAX2 coding sequence. Both external and internal primers were designed on the alignment of GM1000 and RS1000 ripAX2 gene and neighbouring regions. Table S5 Bacterial strains of the Ralstonia solanacearum species complex (RSSC) with their host of isolation, nucleotidic and proteic RipAX2 alleles, and GenBank accession numbers. Table S6 Specificities in distributions of the RipAX2 protein alleles (Hap_AA) across regions, as assessed by χ 2 tests. (A) Distribution of each allele across regions. Status is 'absence' (ABS), 'functional' (PROT) or 'non-functional' (NF). (B) Distribution of each region across alleles.

Fig. 2

 2 Fig.1The RipAX2 type III effector of Ralstonia pseudosolanacearum is necessary for resistance in AG91-25 eggplants. (A) Photographs taken at 6 days post-inoculation. The R. pseudosolanacearum GMI1000 wild-type strain and the GMI1000 ripAX2::pCZ367::ripAX2 complemented strain are not able to trigger disease on AG91-25, whereas the GMI1000 ripAX2::pCZ367 mutant is able to do so. (B) Survival curves representing the wilting of AG91-25 eggplants after root inoculation with the GMI1000 wild-type strain, GMI1000 ripAX2::pCZ367 mutant and the complemented strain [10 8 colony-forming units (CFU)/mL, 10 mL per plant, 18 plants per strain]. Log-rank (Mantel-Cox) test P values compared with GMI1000 are shown below the graph. (C) Photographs taken at 6 days post-inoculation. The strains GMI1000, GMI1000 ripAX2::pCZ367 and GMI1000 ripAX2::pCZ367::ripAX2 are all able to wilt MM738 susceptible eggplants. (D) Survival curves representing the wilting of MM738 eggplants after root inoculation. Log-rank (Mantel-Cox) test P values compared with GMI1000 are shown below the graph. [Colour figure can be viewed at wileyonlinelibrary.com]

Fig. 3

 3 Fig. 3 In planta growth of Ralstonia pseudosolanacearum strains GMI1000, GMI1000 ripAX2::pCZ367, PSS4, GMI1000 ripAX2::pCZ367::ripAX2 and PSS4::ripAX2 inoculated on AG91-25 eggplants at 5 days (A) and 10 days (B) post-inoculation. Each strain was inoculated by soil drenching [~10 8 colony-forming units (CFU)/mL, 10 mL per plant]. Each point corresponds to the value obtained from a single plant. The three biological replicates are shown in different colours. If bacterial colonies were counted on plates, but their number was inferior to 10, the points were taken off as the actual bacterial quantity could not be assessed precisely. Statistical analyses for grouping were performed by Kruskal-Wallis test with α = 0.05. [Colour figure can be viewed at wileyonlinelibrary.com]

Fig. 4

 4 Fig.4In planta growth of Ralstonia pseudosolanacearum after stem injection of strains GMI1000, GMI1000 ripAX2::pCZ367 and GMI1000 ripAX2::pCZ367::ripAX2 on MM738 and AG91-25 eggplants, just after injection (A) and 3 days after injection (B). Each strain was inoculated by injection into the stem [10 6 colony-forming units (CFU)/mL, 10 µL per plant]. Each point corresponds to the value obtained from a single plant. The three biological replicates are shown in different colours. Statistical analyses for grouping were performed by Kruskal-Wallis test with α = 0.05. No statistical differences were found just after injection by the different strains in MM738 and AG91-25. No statistical difference was found 3 days after injection in MM738. [Colour figure can be viewed at wileyonlinelibrary.com]

Fig. 5

 5 Fig. 5 RipAX2-triggered immunity does not require the conserved putative zinc-finger domain. (A) Photographs taken at 14 days post-inoculation for GMI1000 ripAX2::pCZ367, GMI1000 ripAX2::pCZ367::ripAX2 and GMI1000 ripAX2::pCZ367::ripAX2-E149A, and at 15 days post-inoculation for PSS4, PSS4::ripAX2 and PSS4::ripAX2-E149A, inoculated by soil drenching [~10 8 colony-forming units (CFU)/mL, 10 mL per plant]. Strains complemented with RipAX2 mutated in the putative zinc-finger domain (point mutation E149A) were not able to trigger disease on AG91-25 eggplants. (B) Survival curves representing the wilting of AG91-25 eggplants after Ralstonia pseudosolanacearum inoculation. Log-rank (Mantel-Cox) test shows that the wilting rates of plants inoculated with the GMI1000 ripAX2::pCZ367 mutant and the two complemented strains (with RipAX2 or with RipAX2-E149A) are significantly different (P values of 0.004 and 0.009, respectively). The wilting rates of the plants inoculated with PSS4 and the two trans-complemented strains (with RipAX2 or with RipAX2-E149A) are also significantly different (P < 0.0001 and 0.0003, respectively). Each strain was inoculated by soil drenching on three replicates of nine plants. [Colour figure can be viewed at wileyonlinelibrary.com]

© 2018 BSPP AND JOHN WILEY & SONS LTD MOLECULAR PLANT PATHOLOGY (2018) 19(11), 2459-2472

AC K N OW L E D G E M E N T S

Edith Lallemand-Mamosa, Sylvain Lebon and Jean-Michel Baptiste are greatly acknowledged for their excellent technical support. We wish to thank Professors Yuki Ichinose (Okayama University, Okayama, Japan) and Takafumi Mukaihara [Research Institute for Biological Sciences (RIBS), Okayama, Japan] for sharing their anti-RipAX2 antibody. We also thank Sebastien Cunnac for critical reading of the first version of this article.

J.G.'s PhD grant was financially supported by a fellowship from both CIRAD and the University of la Réunion, by the projects 'GENETOM3' and 'BSV2.1' (FEDER project financed by the European Union and Région Réunion), by the CASDAR project RESAUBER ('Sustainable management of eggplant resistances to bacterial wilt', French Ministry of Agriculture) and by the Agreenium Foundation through the EIR-A program. A.M. and F.L. were funded by a grant from the French Ministry of National Education and Research. This work was also supported by the French Laboratory of Excellence project 'TULIP' (ANR-10-LABX-41).

CO N F L I C T O F I N T E R E ST

The authors declare no conflicts of interest.

R E F E R E N C E S