Marie Fabre

Pascale Chavet

Théo Fornerone

Benjamin Juan

Olivier Abossolo

Fabrice Pardo

Jean Blouin

Lionel Dany

Laurence Mouchnino

Somatosensory cortical facilitation during step preparation restored by an improved body representation in obese patients

Keywords: Anticipatory postural adjustments, obesity, plantar sole afferents, step initiation, body representation, EEG

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Introduction

Gait is initiated by anticipatory postural adjustments (APA) which are largely under feedforward control [START_REF] Massion | Movement, posture and equilibrium: interaction and coordination[END_REF]. Such mode of control for gait initiation relies on the use and the quality of an internal representation of the body position in space [START_REF] Assaiante | Updating process of internal model of action as assessed from motor and postural strategies in young adults[END_REF] which is built up and updated by multiple sensory modalities. For example, somatosensory inputs provide crucial information on body position for setting the APA, notably the forces exerted on the ground that shift the body weight forward and laterally towards the supporting side to unload the stepping leg [START_REF] Mouchnino | Online control of anticipated postural adjustments in step initiation: evidence from behavioral and computational approaches[END_REF].

Importantly, several studies have pointed out notable impairments in the APA of obese patients [START_REF] Cau | Center of pressure displacements during gait initiation in individuals with obesity[END_REF] and in their balance stability in general [START_REF] Hue | Body weight is a strong predictor of postural stability[END_REF]. These impairments are interpreted to be a consequence of a reduced tactile sensitivity stemming from the over-compression of the plantar mechanoreceptors in obese individuals [START_REF] Wu | Impaired plantar sensitivity among the obese is associated with increased postural sway[END_REF]. A recent somatosensory-evoked potential (SEP) study performed with non-obese standing participants wearing a weight-vest (i.e., ~27% increased body weight) showed the impact of extra weight on plantar sensory transmission [START_REF] Lhomond | Neural Consequences of Increasing Body Weight: Evidence from Somatosensory Evoked Potentials and the Frequency-Specificity of Brain Oscillations[END_REF].

The first response recorded over the somatosensory cortex to electric stimulation of the foot sole was reduced when the participants wore the loaded vest. It should be noted that body oscillations also increased in this situation.

While the assumption that APA setting and balance control in obese individuals are impaired by a decreased tactile sensitivity appears undisputed, the question remains as to whether such impairments are also linked to an altered body internal representation (BIR) in this population. It is indeed well known that perceptual distortion of the body representation is the core feature of obesity and eating disorders [START_REF] Cash | The nature and extent of body-image disturbances in anorexia nervosa and bulimia nervosa: A meta-analysis[END_REF]. An impaired body representation in obese patients could have in turn detrimental effects on the processing of foot cutaneous inputs and on the preparation of the APA associated to step initiation. The importance of the BIR in such sensorimotor processes is suggested by results showing that the enhanced processing of foot cutaneous inputs prior to step initiation coincides with an increase in the activity of the extrastriate body area (EBA) [START_REF] Mouchnino | Facilitation of cutaneous inputs during the planning phase of gait initiation[END_REF]; a key region for implementing and updating multisensory representations of the body in humans [START_REF] Arzy | Neural basis of embodiment: distinct contributions of temporoparietal junction and extrastriate body area[END_REF][START_REF] Limanowski | Integration of visual and proprioceptive limb position information in human posterior parietal, premotor, and extrastriate cortex[END_REF]. The BIR would be critical for the top-down signal that increases the response to tactile foot stimulation relevant for step initiation [START_REF] Duysens | Gating of sensation and evoked potentials following foot stimulation during human gait[END_REF].

Based on the presumed altered BIR in obese individuals, using SEP techniques we first tested our prediction that the neural mechanisms reflecting enhanced processing of cutaneous inputs reported in non-obese individuals during the planning of a step forward [START_REF] Limanowski | Integration of visual and proprioceptive limb position information in human posterior parietal, premotor, and extrastriate cortex[END_REF] will be absent in obese individuals.

The goal of the present study was to further test the impact of the body representation on the sensorimotor processes associated with step preparation. This was achieved by comparing the response of obese patients to foot cutaneous stimulations before and after a 15-day intervention program composed of motor and cognitive activities involving body representation (e.g., proprioceptive training, dance and acting classes, mindfulness-based stress reduction (MBSR)). For instance, dance or dance-like training is known to improve the accuracy of body representation [START_REF] Golomer | Influence of vision and motor imagery styles on equilibrium control during whole-body rotations[END_REF]14] and also to change the APA setting [15]. It enables dancers to control to a greater extent the APA associated with leg raising with feedforward processes when compared to non-dancers who make greater use of sensory-based feedback processes [15]. On the other hand, focusing essentially on self-awareness and body sensation, the MBSR is known to impact body representations as suggested by the increased in grey matter concentration in temporoparietal area (TPJ) of MBSR participants [START_REF] Hölzel | Mindfulness practice leads to increases in regional brain gray matter density[END_REF]. We hypothesized that after the 15-day intervention program, obese participants will show greater activities over cortical areas involved in body representation during the planning of stepping movements. This increased activity should be associated with an enhancement of the processing of foot cutaneous inputs and changes in the APA. Finally, to gauge whether our program had an impact on the BIR, we compared the participants' responses provided before and after the intervention program to 2 standardized body image questionnaires [START_REF] Koleck | The bodyimage questionnaire: an extension[END_REF]. Indeed, body image perception may constitute a perceptual outcome of sensorimotor processes according to the embodied theories [START_REF] Pezzulo | The mechanics of embodiment: A dialog on embodiment and computational modeling[END_REF] which propose that the conceptual representations are grounded in sensory and motor systems. 1). Note that weight loss was not an objective of the program and was only marginal (averaged body mass loss: 2.6 ± 1.8 %). We also tested a control group of 5 participants (mean age: 50 ± 13 years, 3 women; mean BMI: 35 ± 3) who did not follow the training program. The experiment was conducted with the understanding and written consent of each participant, and the local research ethics committee at Saint-Christophe Clinic approved all procedures. The protocol and procedure were in accordance with the 1964 Declaration of Helsinki.

Methods

Task and Stimulating system

The experiment was performed before (D1) and after (D15) the intervention program and was part of a clinical package. For the control group, participants were tested at D1 and 2weeks later (D15). Participants standing on a force platform performed a "Standing" and a "Stepping" tasks. Before each trial of each task, participants were asked to self-select a comfortable side-by-side foot position and to stand still with the eyes closed and the arms alongside the body. In the Stepping task, participants were instructed to produce with their right leg, a small step forward at a comfortable speed, and to land on the same force platform surface on which they were standing. They were asked to synchronize the onset of their step with the third auditory signal (i.e., the last of three 1-s interspaced 100-ms tones, Fig 1). For reasons of homogeneity, the same tones were played in the Standing task. Each standing or stepping trial recording lasted 5s.

In both tasks, four electrical stimulations were delivered to the plantar sole (i.e., tibial nerve innervating the cutaneous receptors) of the left foot (see for details Fig. 1 legend). Each

Behavioral recordings and analyses

Participants stood on a 46 × 51 cm force plate (AMTI OR6-6, Watertown, MA, USA).

Ground reaction forces (mediolateral, ML and anteroposterior, AP) and torques were recorded at a sampling rate of 1000 Hz. The ML shear force was used to identify the APA initiation (Fig. 1). To determine if the APA were modified at D15, we analyzed the lateral force exerted onto the ground (i.e. thrust, from onset to peak, Fig. 1). To assess the effect of the training program on balance control, we computed for both the Standing and Stepping tasks, the integral of the horizontal forces (i.e. impulse) in both AP and ML directions over a 1.5s-period starting at the first tone, i.e. when participants had to stand still (for the stepping task, this time-window was virtually free of step-related muscular activity). In order to analyse the forward step execution, the motion of the right foot was recorded with a Flock of Birds (Ascension Corporation) (Fig. 1).

Electrophysiological recordings and analyses

Electroencephalographic (EEG) activity was continuously recorded from 64 Ag/AgCl surface electrodes embedded in an elastic cap (BioSemi ActiveTwo system: BioSemi, Netherlands). The signal was pre-amplified at the electrode sites, post-amplified with DC amplifiers, and digitized at 1024 Hz. Off-line filtering with a 50 Hz digital notch filter and a 0.5-45 Hz band-pass digital filter (48 dB/octave) implemented in BrainVision Analyzer 2 software (Brain Products, Germany) was applied. We measured the SEPs (P1N1, Fig. 3A) over the Cz electrode which overlays the sensorimotor cortices.

The neural source of the SEPs was estimated using the averaged preprocessed data with the dynamic statistical parametric mapping (dSPM) implemented in the Brainstorm software [START_REF] Tadel | Brainstorm: a user-friendly application for MEG/EEG analysis[END_REF]. The forward model was computed using a three-shell sphere boundary element model (BEM) on the anatomical MRI brain MNI Colin 27 template (15000 vertices), a predominant volume conductor model. We contrasted the absolute EEG activity computed in the source space between St1 and St0 during three 40-ms time-windows from the stimulation onset: 15-55 ms (preceding P1), 55-95 ms (encompassing P1N1), and 95-135 ms (after N1). For comparison, the same computations were performed at D1.

Body schema/image questionnaires

Currently, there is no test that can be used to directly assess the quality of an internal body representation. In the present study, this assessment was performed using two standardized questionnaires: the Body-Image Questionnaire (BIQ) [START_REF] Koleck | The bodyimage questionnaire: an extension[END_REF] and an adaptation of the 1983 Stunkard's Figure Rating Scale (Fig. 6B).

Statistical analyses

All behavioral (APA and balance stability) and EEG dependent variables showed normal distributions (p > 0.05, Shapiro-Wilk test). The Standing task (St0) was used as a baseline reference for assessing SEP modulation during the preparation phase of the stepping movement and was included in the factor Epoch along with St1 and St2. The amplitudes and latencies of the SEPs were submitted to 2 (Day: D1, D15) x 3 (Epoch: St0, St1, St2) repeatedmeasures analyses of variance (ANOVA). Significant effects were further analyzed using the Tukey's HSD post hoc test. Wilcoxon's test was used to compare the scores of the questionnaires. Since the sample size of the control group was small, partial Eta-Squared values for the effect size were given for the ANOVA. The threshold of significance was set at 5% for all analyses.

Results

Motor task performance and equilibrium

The time to peak force significantly decreased at D15 relative to D1 (t17 = 3.29, p = 0.004, Fig. 2A) while no significant difference in the peak force amplitude was observed (t17 = -0.73, p = 0.48). As a consequence, the amount of lateral force exerted onto the ground (i.e. the area under the force-time trace) significantly decreased in D15 (t17 = -2.52, p = 0.02). The latency of these lateral forces did not significantly differ between D1 and D15 (t17 = -1.75, p = 0.1). The timing of the onset of the forces relative to the Go signal varied between participants, ranging between -229 ms and +252 ms (average: 7.4 ± 130 ms). Because St1 was triggered 600 ms before the Go signal, this indicates that for all participants, St1 epoch was free of motor activity and occurred during the planning of the movement. The amplitude and the duration of the forward step movement did not significantly differ between D1 and D15 and were on average 26 ± 5 cm and 938 ± 203 ms, respectively (amplitude: t17 = 0.39, p = 0.69; duration: t17 = 0.66, p = 0.52).

Variables related to balance control (Fig. 2B) were analyzed to verify whether the stability varied across the experimental sessions (D1, D15). The ANOVAs did not show significant effect of Day (D1 and D15) on the force integrals in both the Standing (F1,17 = 0.28; p = 0.59; p = 0.91) and Stepping tasks (F1,17 = 0.01; p = 0.91). Importantly, these behavioural results indicate that the control of the patients' APA was modified after the program but not the control of their static balance.

Brain activity

The ANOVA revealed a significant Epoch x Day interaction on the amplitude of the P1N1 (F2,34 = 3.49; p = 0.04) (Fig. 3B). The decomposition of the interaction indicated that solely after the program (i.e. D15), the P1N1 amplitude was greater during the early preparation phase of the stepping movement (St1) than in both St0 (p = 0.0017) and St2 (p = 0.0003). Note that in the standing task (St0) similar P1N1 amplitudes were observed at D1 and D15 (p > 0.05).

The ANOVA did not show significant effect of Epoch (F1,17 = 2.38; p = 14) or Day (F1,17 = 0.2; p = 0.63) on P1 latency (overall mean 66 ± 9 ms). Similarly, the latency of N1 (overall mean 92 ± 11 ms) was not significantly impacted by the factors Epoch (F1,17 = 0.8; p = 0.36) and Day (F1,17 = 0.18; p = 0.68).

The source analyses estimated the left posterior parietal cortex (PPC) as the generator of the increased SEP at D15 (see statistical maps of Fig. 4). No such significant effect of the APA preparation on the PPC activity was found at D1. Interestingly, we also observed an increased activity of left premotor cortex (BA6, PMC) during the preparation phase at D15 which culminated 95-135 ms after St1 stimulation and which was not observed at D1. Being first observed 95-135 ms post St2 stimulation (see Fig. 5), the premotor activity was delayed in D1 relative to D15.

Importantly, the control group did not show significant effect of Epoch (St0, St1; F1,4 = 0.66; p = 0.55, ηP 2 = 0.14) or Day (D1, D15; F1,4 = 0.47; p = 0.53, ηP 2 = 0.11) or Epoch x Day interaction (F2,8 = 2.19; p = 0.17, ηP 2 = 0.35) on the amplitude of the P1N1. In addition, none of the 3 analyzed time-windows showed significant neural sources activity (e.g., PPC, PMC) differences between St0 and St1, both at D1 and at D15. Moreover, the latency of the left PMC activity did not significantly differ between D1 and D15 (t4 = 0.08; p = 0.93). On average, the activity of the PMC increased 115 ms ±10 after St2. This latency is similar to that observed in the experimental group, but only at D1. Overall, similarities of the SEP and brain sources variables suggest that the changes observed at D15 in the experimental group resulted from the intervention program rather than from uncontrolled time-related effects.

Body representation/image questionnaires

The BIQ score was greater at D15 than at D1 (z = 2.11; p = 0.03), suggesting a change in the obese patients' body image at the end of the program (Fig. 6A). Moreover, the patients perceived their BMI (Fig. 6B) as being smaller at D15 than at D1 (z = 2.66; p = 0.007).

Discussion

Through behavioural (APA) and EEG (SEP and source localisation) analyses, the present study showed an enhancement of somatosensory processes during step preparation after a two-week intervention program that aimed at improving obese patients' BIR.

Previous studies have shown that all markers of postural stability improve when obese individuals lose weight (~20% weight loss in Teasdale et al. [START_REF] Teasdale | Reducing weight increases postural stability in obese and morbid obese men[END_REF]) and that the transmission of sensory inputs to the cortex is greater in individuals not carrying extra weight [START_REF] Lhomond | Neural Consequences of Increasing Body Weight: Evidence from Somatosensory Evoked Potentials and the Frequency-Specificity of Brain Oscillations[END_REF]. In the present study, weight loss after the intervention program was ~3% only and was not associated with balance improvement. Therefore, the changes in the sensorimotor processes observed here unlikely resulted from the patients' decreased body weight. In addition, because it was not observed in the Standing task at D15, the increased SEP at D15 cannot merely stem from a greater sensitivity of cutaneous receptors per se (note this is also suggested by the same perceptual threshold to cutaneous stimulations between D1 and D15). One could argue that the increase in early SEP could have resulted from uncontrolled time-related effects rather than genuine changes in sensorimotor processing due to the program. However, the absence of increased negativity at D15 relative to D1 in the control group (corroborated by high Etasquared values), composed of obese patients who did not follow the intervention program, does not support this hypothesis. Rather, we hypothesize that the sensorimotor changes observed after the program, which enhanced relevant foot cutaneous information for step preparation [START_REF] Morasso | Can Muscle Stiffness Alone Stabilize Upright Standing?[END_REF], were linked to a modified BIR or a more efficient use of this representation by the patients.

Several key elements of the present study support this hypothesis. The various modules of the program taken by the patients are known to engage cognitive and sensorimotor dimensions of the BIR [15,14,[START_REF] Hölzel | Mindfulness practice leads to increases in regional brain gray matter density[END_REF]; for review, see Fabre et al. [START_REF] Fabre | Enhancing the internal representation of the body through sensorimotor training in sports and dance improves balance control[END_REF]. Indeed, activities emphasizing body awareness such as dance, Tai Chi Chuan or MBSR improve balance sensorimotor control and BIR. For example Marmeleira et al. [14] has shown that sedentary participants over 55 years of age trained in creative dance (i.e., 54 h over 12 weeks) show improvements in the sense of joint position and in the perception of segment orientation relative to space, suggesting enhanced proprioceptive sensory processes. Similarly McGibbon et al. [START_REF] Mcgibbon | Tai Chi and vestibular rehabilitation effects on gaze and whole-body stability[END_REF] reported in participants older than 50 years that a 10-h training in Tai Chi Chuan leads to an improvement of balance control through vestibular mechanism. On the other hand, mindfulness practice, in addition to improving balance [24], has been associated with neural structural changes as observed by [START_REF] Hölzel | Mindfulness practice leads to increases in regional brain gray matter density[END_REF] in the temporo-parietal junction (TPJ) after a 8 week-program (total of 26 h). As reviewed by [25], this cortical region is known to host a spatial representation of the self [START_REF] Arzy | Neural basis of embodiment: distinct contributions of temporoparietal junction and extrastriate body area[END_REF], and to be involved in the perception of body parts ( [START_REF] Ora | Arm crossing updates brain functional connectivity of the left posterior parietal cortex[END_REF]) in the mental imagery of one's body [START_REF] Ionta | Multisensory mechanisms in temporo-parietal cortex support self-location and firstperson perspective[END_REF] The increased activity of the inferior parietal lobules (BA 39/40, i.e. TPJ) prior to, and during the P1N1 at D15, but not at D1 suggests a stronger involvement of the BIR during the planning of the stepping movement after the program. This representation would play an important role in the setting of the APA as suggested by studies showing that the APA of individuals involved in dance and dance-like training are highly accurate and do not necessitate online adjustments [15]. APA setting is known to be achieved by adjusting their temporal dimension [START_REF] Mille | Are human anticipatory postural adjustments affected by a modification of the initial position of the center of gravity ?[END_REF] and was reflected here in a decreased duration of the shear forces exerted on the ground.

The premotor activities (i.e. BA6) preceding step initiation were observed earlier at D15 than at D1, while the APA onset time and the step length were not modified by the program.

The dorsolateral PMC is known for contributing to the motor command of self-paced stepping movements and for generating feedforward signals for triggering the APA [START_REF] Massion | Movement, posture and equilibrium: interaction and coordination[END_REF][START_REF] Fabre | Enhancing the internal representation of the body through sensorimotor training in sports and dance improves balance control[END_REF]. The PMC is also considered as being crucial in situations where the mapping between sensory inputs and motor actions is important [START_REF] Miller | An integrative theory of prefrontal cortex function[END_REF]. In this light, changes in the premotor activities noted here at D15 and not present in the control group at D1 or D15, are consistent with an enhanced preparation of the focal leg movement after the program. This preparation could have beneficiated from BIR-related information stored in the PPC which has dense connections with the premotor cortex [START_REF] Ora | Arm crossing updates brain functional connectivity of the left posterior parietal cortex[END_REF]. Then by improving the BIR is also a means to improve the motor preparation as previously shown for dancers [15] resulting here in modification of premotor activations at D15.

A limitation of the present study is that it does not provide information as to whether the behavioral and neurophysiological changes observed during step preparation last over time after the intervention program. Another limitation is that it is still not possible to differentiate between the various contributions to these changes of the different modules of the program and of the different sensory modalities involved. Even if some training element are performed with eyes closed (e.g., MBSR and relaxing session), it is possible that visual information during training still contributes to the changes of BIR observed since it is constructed with multisensory information [START_REF] Arzy | Neural basis of embodiment: distinct contributions of temporoparietal junction and extrastriate body area[END_REF][START_REF] Limanowski | Integration of visual and proprioceptive limb position information in human posterior parietal, premotor, and extrastriate cortex[END_REF]. Also even with eyes closed, as in this experiment, it is possible for the participants to use a visual representation of the body during the preparation of a movement with the lower limb. Indeed Mouchnino et al. [START_REF] Mouchnino | Facilitation of cutaneous inputs during the planning phase of gait initiation[END_REF] have shown that an increase activation of the extrastriate body area (EBA) is a part of the step preparation mechanism with eyes closed. EBA is known to host visual representation of the body, like Limanowski [START_REF] Limanowski | Integration of visual and proprioceptive limb position information in human posterior parietal, premotor, and extrastriate cortex[END_REF] showed that this brain areas integrate visual and proprioceptive information into a body representation. Downing et al. [START_REF] Downing | A cortical area selective for visual processing of the human body[END_REF] also showed that EBA is specialized in visual processing of the human body.

In conclusion, our results show that a two-week program improves the BIR of obese patients, which in turn, leads to an enhanced processing of foot cutaneous inputs prior to step initiation. Despite the limitations of the protocol, the results of the present study suggest that the implementation of intervention programs which incorporate tasks targeting different dimensions of the BIR, could be part of a strategy to improve the dynamic postural control in obese individuals. by an isolated bipolar constant current stimulator (DS5 Digitimer, Welwyn Garden City, UK); using 5 × 9 cm Platinum Foam electrodes located under the metatarsal region (cathode) and underneath the heel (anode). The stimulation consisted of a single rectangular 10-ms pulse. The stimulation intensity was set at 25% higher than the lowest intensity producing a constant perception of the stimulation while standing still. For each participant, the minimal intensity required for the stimulation to be constantly perceived was similar for both D1 and D15. Therefore, the stimulation intensity was kept identical for both sessions (mean amplitude: 8.2 ± 1.7 mA). For all participants, this intensity was below the motor threshold (i.e., no visible evoked response in the EMG traces).

(

  10 ms square pulses) stimulation was separated by 500ms. For the Stepping task, the stimulation occurred twice during the preparation phase of the APA (St1 and St2, respectively 600 ms and 100 ms before the GO signal) and twice during the execution phase of the APA (St3 and St4) (Fig.1). The same 4 stimulations were delivered relative to the third tone in the Standing task (hereafter all referred to as St0). As the present paper focused on the sensorimotor processes related to APA preparation, only St1 and St2 were included in the analyses, St3 and St4 permitting the same number of stimulations in both tasks. Forty stimulations were delivered for each condition. Ten trials were necessary in the Standing task to obtain 40 stimulations (4 stimulations per trial). In the Stepping task, because there was only one St1 and St2 stimulations per trial, 40 trials were necessary to have 40 stimulations for the St1 and St2 epochs. The tasks were presented in a random order.

  [START_REF] Ionta | Multisensory mechanisms in temporo-parietal cortex support self-location and firstperson perspective[END_REF]). The structural change observed over the TPJ region by[START_REF] Hölzel | Mindfulness practice leads to increases in regional brain gray matter density[END_REF] [START_REF] Hölzel | Mindfulness practice leads to increases in regional brain gray matter density[END_REF] is then consistent with an improvement of the BIR after the mindfulness program.

Figure 1 :

 1 Figure 1: Left panel: Position of stimulation electrodes under the left foot. The stimulus was delivered

Figure 2 :Figure 3 :

 23 Figure 2: A. Left panel: Difference for each participant between the mean time to peak force recorded at D15 and at D1 (D15-D1). Negative values indicate that time to peak force was shorter at D15. Right panel: Mean amplitude for all participants of the averaged thrust computed between the APA onset and the lateral peak force in the Stepping task in D1 and D15 (error bars represent standard error across participants) (* p < 0.05). B. Mean impulse in AP and ML directions computed during the Standing task and prior to the APA in the Stepping task (left and right panels, respectively) when the participants were still motionless (error bars represent standard deviation across participants)

Figure 4 :Figure 5 :

 45 Figure 4: Statistical source estimation maps (top views) for the Stepping (St1) vs Standing (St0) tasks contrast averaged over three time windows for both D1 and D15: preceding the P1 component [15;55 ms], encompassing the P1N1 [55;95 ms], and following the N1 component [95;135 ms]. The superior (BA 7) and inferior (BA 39/40) PPC showed greatest activities before, during and after the SEP when participants prepared a step at D15 (as indicated by the shade of red)

Figure 6 :

 6 Figure 6: A. Mean averaged scores across all participants to the 19-item (bipolar) questionnaire [23] at D1 and D15. The aim of this questionnaire is to assess obese individuals' perception, satisfaction, and attitudes towards their bodily appearance. For each item, participants rated themselves on fivepoint scales, the sum of which providing a favourable body image index (scores of 19 and 95 corresponding to the least and most favourable scores, respectively). Error bars represent standard error across participants. B. Adaptation of the Stunkard's Figure Rating Scale where participants had to select the figurine (among 7 figurines, BMI ranging from 25 to 37, see left panel) with the silhouette that they deemed to be closest to their usual appearance. The histogram shows the mean average of perceived BMI in percentage of the real BMI for all participants at D1 and D15 (error bars represents standard error across participants). (*, p < 0.05; **, p < 0.01).

Acknowledgements

The authors are deeply grateful to the health care team and all participants from the Saint Christophe clinic who participated to the pre and post-program evaluation. We also thank Didier Louber for his technical assistance and Franck Buloup for developing the software Docometre used for behavioral data processing and acquisition.

This work was supported by the DISREMO CNRS project.


domain: sdv sde

text:
José Silvestre

Maria Isabel Ferreira

Evapotranspiration and water relations from a vineyard in central Portugal during spring-summer periods

published or not. The documents may come   L'archive ouverte pluridisciplinaire


Gérard Dedieu

Pascale Cayrol

Laurent Kergoat

Sophie Moulin

The coupling of process models and remotely sensed data assimilation for regional

Informus Workshop

VITO -septembre 1998 -Antwerp, Belgique

The coupling of process models and remotely sensed data assimilation for regional modeling (upscaling)

Gérard DEDIEU 1 , Pascale CAYROL 1 , Laurent KERGOAT 2 , and Sophie MOULIN 1 1 CESBIO, Unité mixte de recherche 56390 (CNES-CNRS-UPS), 18, avenue Edouard Belin, 31401 Toulouse Cedex-4 France 2 Laboratoire d'Ecologie Terrestre, Unité mixte de recherche 5522 (CNRS-UPS), 13 av Colonel Roche, bp 4403, 31405 Toulouse cedex 4 Vegetation process models are being developed to describe vegetation functioning under various environmental constraints, such as water ressources and climate change. These models provide satisfactory results at the local scale but are difficult to extend at landscape and regional scales. Our talk will address the combined use of process models and remotely sensed data in order to perform this extension.

We will present the concept and examples of results of remotely sensed data assimilation in coupled models of vegetation growth and water budget. Models are used to predict satellite radiances in solar and thermal infrared spectral bands. Over a given time period, the distance between predicted and observed radiances is minimized by tuning some model parameters. This approach allows to correct model parameters over large regions Results we obtained for semi-arid grasslands will be presented. The assimilation technique was applied over one season to daily 1km resolution NOAA/AVHRR data. Shortwave radiances, combined as NDVI, allowed to adjust the partitioning of photosynthesis products between shoots and roots. Thermal infrared data were used to derive soil thermohydric properties. Tuning model parameters against satellite radiances leads to improved model outputs, including Leaf Area Index time profile and latent heat fluxes.

In addition, we will present preliminary results obtained when using NOAA/AVHRR data to monitor the phenology of deciduous forests. Finally, the various possible uses of assimilation techniques for vegetation studies will be summarized and discussed.

Key words : vegetation functioning, modeling, remote sensing, assimilation technique


est destinée au dépôt

  Effect of shortened photoperiod on the annual blood levels of GTH1 and GTH2 in rainbow trout (Oncorhynchus mykiss) F. Le Menn, E. Bon, Marina Govoroun, Bernard Breton To cite this version: F. Le Menn, E. Bon, Marina Govoroun, Bernard Breton. Effect of shortened photoperiod on the annual blood levels of GTH1 and GTH2 in rainbow trout (Oncorhynchus mykiss). 10. Conference of European Comparative Endocrinologists from Molecular to Ingrative Biology, Sep 1996, Rouen, France. Masson, Annales d'Endocrinologie, 57 (4) Supplément, 1996. hal-02770361


cantons in France), a third of the cantons are experiencing a decrease in population. These cantons represent 50% ofthe territory but only 2% of the total population (Houée, 1996). This urbanisation has two consequences: on the one hand, the spatial expansion of the cities conflicts with other uses of the surrounding land, destroying many valuable landscapes. On the other hand it creates a strong demand for nature. The expansion of the cities and the correlated land consumption by transportation infrastructures has been very important in France. From 1982 to 1990 the developed areas rose from 3,271,000 ha to 3,762,000 ha (61,000 ha per year). The major part of this increase, about three quarters, corresponds to a loss of agricultural land (Cavailhès and Norrnandin, 1994). This leads to landscape deterioration as well as lirnited access to open space and countryside. While the arnount of surface area involved in the quarry industry and mining is small compared to other land-uses, the impact on the landscape is significant. Every year 500 million tons are extraeted from the French subsoil (lFEN, 1994). When the entire land surface is removed there is a large arnount of "tailings" or residual rock, which create visual pollution. The extraction of rnaterials from the rivers is an important cause of wetland loss.

With respect to the demand for nature, it is clear that as people get richer their demand for countryside arnenities increases, since the income elasticity for this type of goods is high. That the countryside is more and more popular is observable through the increase in the number of people involved in hiking. Occasional hikers numbered 1,900,000 in 1984 and 3,900,000 in 1989, whereas the number ofregular hikers grew from 840,000 to 2,200,000 during the sarne period (lFEN, 1994). ln 1993 the number of national park visitors was around 2 million a year. Farming tourism, which concerned only 2% of the farms in the 1988 agricultural census, is a growing activity reflecting the increased demand for space.

Another significant indicator is the arnount offarm property purchased by non-farmers, which rose in the 1980s. It represents nowadays almost a third of the area sold on the land market and well over half of the property value that changes hands. The purchase of second homes makes up another part of the market and represents an important demand, since one fifth of rural dwellings are second homes. The countryside that attracts these urban people is located in regions characterized by their rural arnenities and the quality of their landscape: Vosges, Morvan and Northen Massif Central, Limousin, Périgord, Pyrénées, Cévennes, Alps, as well as Sologne and parts of the mixed wood1and and pastures of Western France (Cavailhès et al., 1994). Amenities alone cannot explain this location in the country; accessibility, budgetary decisions, etc are also influential, but demand for the landscape is clearly invol\'ed.

Deterioration of landscape by agriculture

Historically agriculture has made a significant contribution to the landscape in large areas of France, even if it has been a major modifier of the rural environmént, and if ecosystem change is embedded in agricultural processes. However, when in the sixties agriculture becarne a sector closely linked with input suppliers strengthening the productivity of the soil, a disruption to the generations of farmers previously practising agricultural techniques in harmony with the environment occurred.

The ways in which intensification has damaged the landscape may be analysed under a number of headings. First of all as indicated in table 1, there have been very important changes in land-use. Regarding cropping patterns, the most significant aspect for the environment was the shift between arable land and permanent grassland. The area used for agriculture decreased between 1970 and 1994. If we consider the principal categories of crops there is a dramatic increase in wheat from 3.7 million ha in 1970 to 4.3 million in 1994 and to a lesser extent in maize (from lA to 1.7 million ha). The development of monoculture has created a landscape which most people do not find attractive, and an increased intensity of land-use with its consequences being the destruction of biotopes and the contamination of surface and ground waters.

Comparable developments have taken place in the livestock sector characterised by a dramatic increase in intensive animal husbandry, notably of pigs, poultry and cattle. Local effects of this intensive farming concem visual amenity since high-quality rurallandscapes are damaged by the erection of grain silos and modem agricultural buildings for animals. However, the quantity of manure produced by intensive animal husbandry, combined with the inorganic fertilisers used on crops, cause large quantities of nitrates and phosphorus to run off into rivers. lbis over-supply of nitrogen leads to the eutrophication of slow-running and surface waters. Eutrophication also occurs in estuaries and marine waters. lbis results in a disruption of the ecosystems, with algae growth disturbing the oxygen balance, and this subsequently results in the death of fish. In addition, algae can emit bad odours.

Otherwise, intensification and specialisation have come about through major structural changes, i.e. a rapid increase in the average farm size (from 13 ha in 1955 to 35 ha in 1993) involving the substitution of machines for manual labour. The number and the power of machines used per square kilometre of arable land has continued to rise leading to soil compaction and erosion. Furthermore, the ever-increasing size of machines calls for wider plots which are obtained through land amalgamation and plot consolidation. Since 1945 over 15 million hectares have been consolidated, this represents half of the area used for agriculture. As indicated in map 1, the consolidation of landholdings has been very important in Northem France, mainly in the Bassin Parisien where cereal farming dominates, but also in the East (Alsace) and in the West (Bretagne and especially Morbihan). Puy-de-Dôme in the Massif Central has also seen significant land consolidation. The most important effect of plot consolidation on the landscape and nature is the eliminationof features such as hedgerows, groves and isolated trees. The best index of this loss of quality is the rate of hedgerow removal. The national survey of forestry, which is carried out every 12 years, gives detailed information. Between the two last surveys 536,000 km of hedgerows had been removed (pointerau and Bazile, 1995). Figures 2 and3, which give the prevalence of hedgerows at the time of the first survey (1963)(1964)(1965)(1966)(1967)(1968)(1969)(1970)(1971)(1972)(1973)(1974)(1975)(1976)(1977)(1978)(1979)(1980) and the rate of removal respectively, indicate that regions with the longest hedgerows have the highest rate of removal. Five "départements", alliocated in the western part of the country, contained 25% of the total hedgerow length. There are Côtes d'Armor (102,000 km), Finistère (79,120 km) Vendée (51,100 km), Manche (46,736 km) and Loire-Atlantique (41,160 km). In these five "départments" the total removal between the two surveys reached 200,000 km.

Fig: 3. Hedgerow removal between the two surveys (1963-1980/1977-1990) D <lO,OOO km a 10,000 to 20,000 km _ 20,000 to 40,000 km

_ >40,OOOkm

Source: [START_REF] Pointereau | Arbres des champs: haies, alignements, prés vergers ou l'art du bocage[END_REF] 3 The legal context

Whilst demand for leisure use of the countryside increased, intensification and urbanisation have damaged the quality of the landscape. These changes have recently been observed and a specific conservation and management policy was implemented in July 1993 with the Countryside Act. In fact, there is an important history of French regulation which started at the end of the 19th century introducing the protection of particular sites. This point is considered in the first subsection. Following this, the regulation was extended to protect natural sites and historical monuments in the middle of the century. This aspect is exarnined in the second subsection. Finally a more generallegal framework is defined which covers large parts of the territory. Using [START_REF] Rousso | Le droit du paysage[END_REF] we present this evolution from specific to ecological units, and from protection-oriented regulations towards a more comprehensive approach involving management of the whole countryside. The second half of the 19th century is characterized by the romanticism of the French elites, with a pronounced interest in cultural inheritance through archeology, ancient monuments and sites of particular interest. For instance the frrst conservation land purchase by the public sector took place in 1861 with the purchase of 1,000 ha of the forest of Fontainebleau to preserve the countryside which was irnrnortalised by the Barbizon school in their paintings.

However, the role played by the voluntary bodies, in particular the Touring Club de France and a range of associations concemed with recreation in mountain areas was important. Their lobbying activities resulted in the law of 1906 conceming the protection of natural sites which enlarged the 1aw of 1887 protecting artistic and historic monuments [START_REF] Baraque | Le paysage et l'administration[END_REF]. In Deeember 1913 the aet on historic buildings was passed This act, in force ever since, is used today to proteet not only historic casties but all kinds of interesting buildings sueh as ancient factories, railway stations and hotels.

According to the level of the monument's historie interest, two levels of protection are possible. In the most demanding case, the monument is classified as a place of historic interest ("le classement'~. Any changes require the agreement of the ministry in charge of historie buildings. Less restrictive is registration ("l'inscription'~where changes are possible if the agreement of the regional director of cultural affairs is obtained.

The law of 1930 proposes the same type of protection for natural monuments and sites of "artistic, scientific, legendary or picturesque" interest with classification and registration. Farming without any agreement is possible for registered sites only. In June 1993 the number of classified sites was 2,564 whereas registered sites were twice as numerous [START_REF] Ifen | L'environnement en France[END_REF].

Originally, these laws aimed at protecting "small-scale" nature: particular rock formations, waterfalls, unusual trees and so forth. They were based on a type-of-monument approach. Nowadays this type of regulation is used to prevent the destruction of green areas, as weIl as flora and fauna in general. For instance, the Massif des Maures, which stretches for several tens of thousands of hectares, is c1assified. During the first half of 1993, 18 sites were regulated corresponding to Il,627 ha. AIl these sites are specific, but ail unique (surroundings of the city of Carcassonne, the pond of Aigues-Mortes, the gorge of Héric in Hérault, are sorne examples).

3.2 Extended protection orthe sites and the monuments in the middle orthe 20th century

As mentioned above, existing laws have been used in a more extensive way to protect the surroundings of the sites, but the February 1943 act aims at protecting the area surrounding historic monuments for up to a 500m radius. ln 1992 there were 40,450 protected areas [START_REF] Ifen | L'environnement en France[END_REF]. The Malraux Act (4th August 1962) enlarges the area of protection in urban zones in which there are architectural sectors presenting a cultural, artistic or historic interest.

The Malraux Act proposes a management system for entire sectors designated by the authorities as being in need of safeguarding. It corresponds to a land-use plan which inc1udes the restriction and the rehabilitation of large parts of urban zones.

However, the most important conservation initiative is the setting up of the National Parks (2nd July 1960). National Parks are initially designated because of their value as "important sanctuaries ofnature ", but an important feature of these areas is that they cover land which is inhabited, largely privately owned and used econornically. Therefore it is necessary for their management to take into account their value for conservation, amenity and recreation to society as a whole on the one hand, and to recognize an indigenous "human" element on the other hand. Consequently there is a compelling regulation which distinguishes two types of areas : => a central area, characterized by wildemess, generally uninhabited, with strict protection. Sometimes, the core is a reserve in full, where fauna and flora are protected for scientific reasons ; => around this central area, there is a peripheral zone used econornically by an indigenous population. The regulation has implications for the objectives and operations of such designated areas, placing particular emphasis on integrating rural conservation with other land-use, particularly agriculture.

France has seven National Parks covering a total area of 12,758 square kilometres which corresponds to 2,2 %ofthe territory. Table 3 gives more information about these parks. The National Parks were centrally initiated and approved, in consequence they are managed by an administration created with a "top-down" hierarchical frarnework under the control of the Ministry of the Environrnent.

In the sarne spirit as with the central areas of the National Parks there are also natural reserves which are older since they were set up by the 2nd May 1930 Act. These reserves concem areas of high ecological value with scarce or threatened resources. Nowadays, there are 114 reserves with an average size smaller than the National Parks.

Furthermore a set of 27 Regional parks covering 8% of the territory exists. These parks are generally situated in economically disadvantaged areas with a relatively high percentage of employment in farming, low population density, and difficult natural conditions, but they are characterized by their "natural beauty".

These Regional Parks originated in the legislative period marking the normalisation of the post-war decentralisation movement, with a fust wave during the seventies (20 parks established from 1969 to 1980), and a second wave during the eighties (7 other parks established from 1985 to 1991). Consequently they have a goveming Park Committee oflocal and regional representatives and a permanent technical staff funded by central and local govemment. The Regional Parks receive the majority of their funds directly from local and regional bodies, and national and European contributions are channelled through these bodies.

In contrast to National Parks, uninhabited in their central areas, the Regional Parks aim at the maintenance of communities and the reversal of rural dec1ine. For this reason conservation is considered as a practice which requires active management rather than the static protection of valued land. This involves an integrated approach to conservation and development issues. Schemes are developed through a process of consultation between farmers, agricultural and environmental administrators, academic researchers, and elected pe,ople.

Due to coordination problems between the local and central govemment in setting park objectives and allocating resources, a decree of 25th April 1988 imposed greater national scrutiny of park funding and park performance. Funds are only given for specific park activities, and all Regional Park designation is subject to appraisal and review every ten years by the Ministry of the Environrnent.

A more comprehensive approach

Legislation has made progress in setting up a more comprehensive approach to the countryside through the Coastal Protection Act and the Mountain Act on the one hand, and the Landscape Act on the other hand.

Coastal Protection Act

ln order to safeguard threatened coasts and lake shores a national government agency, the Coast and Lake Shore Conservancy Authority (Conservatoire de l'Espace Littoral et des Rivages Lacustres) was established in the spirit of the British National Trust. It was set up by the Law of 10th July 1975, and this administrative body is defined by the Rural Code (act L 243, R 243). Its sole task is to purchase land to maintain the natural inheritance and to preserve it for future generations. This act applies to the 277 continental coastal "cantons" and the overseas coastal "cantons" (the Départements d'Outre Mer). It is not only shoreline that is considered, since sectors bordering large stretches of water are also taken into account in order to manage ecological units. These units represent a lenght of 6,933 km and an area of 5,141,000 ha The Conservancy receives an annual state budget, and land held by the organisation is inalienable. By mid 1993, in 314 transactions, a total of 580 km of shoreline were purchased corresponding to a total area of 41,779 ha. Concerning the size of the sites, 172 are under 50 ha, 46 between 50 and 100 ha, 81 between 100 and 500 ha, and 4 over 1,000 ha. If we consider the type of purchased shoreline, it appears that 48.3% are rocky coasts, 32.4% sandy coasts, 13.8% wetlands and 5.5% various areas [START_REF] Ifen | L'environnement en France[END_REF].

The Conservancy does not manage any of the sites it owns. There are management agreements with local authorities (communities and départements) and sometimes this role is delegated to voluntary organisations or farmers. To preserve or re-establish traditional landuse, the Conservancy proposes covenants which place legal restrictions on lan-use. According to the state of the ecosystems, the management is oriented towards either simple conservation or restoration. The choice between these two options is done following an eco-audit when such assessment is possible.

There is coordination between the national and local levels. Specifically, the Conservancy makes decisions on its purchasing progranune ciosely together with the départements and regions through councils composed of elected members which examine the operations of the Conservancy. Progranunes of coordination are then drawn up which define the sites where responsibility is reserved for the départements.

Mountain Act

The Mountain Act of 9th January 1985 is based on the same principles : there are specific problems for these spaces and they need a comprehensive approach. Such an approach must take into account aesthetic aspects but also the historical, economic and sociological elements that define a mountainous massif. The "Mountain Act" deals with 5,600 communities and airns at controlling the impact of economic development on the landscape.

Landscape Act

The Landscape Act of 8th January 1993 is more general than the Coastal Protection and the Mountain Acts. It gives the state possibility to intervene in order to protect any noteworthy landscape. It aims at integrating landscape in land-use plans, in areas of protection of architectura1 sectors, and the entrance to towns. Nowadays any programme of land development or land management has to take into account the countryside. These programmes include land consolidation even if there are still problems with the removal of hedgerows and ploughing up of permanent pastures, or destruction of woodlands and scrub. The underlying philosophy of this law is its extension from the protection of interesting natura1 features to more ordinary areas. For instance, building licences must take into account information about landscape in the land-use-plans (plans d'Occupation des Sols, POS), which define the zones for urbanisation and those which are reserved for other uses such as agriculture, mineral exploitation and recreation. The building itself and the surroundings must be integrated with the environment. Finally, the new legal developments permit better management of the landscape through the protection of sites, ecosystems and the countryside. Furthermore, specific agreements with important public bodies were set up in 1993. French Telecom has undertaken to bury any new extension or renewal of its network. lt is foreseen that in the year 2000 about two-thirds of the lines surrounding ancient monuments or sites of particular interest will be hidden. Electricité de France, the public body producing and distributing electricity, will bury 55,000 km of electric lines within a 4 year period.

Implementation of EC Regulation 2078/92

The application of Regulation 2078/92 in France has given rise to numerous and specific measures (Boisson and Buller, 1996). Two types of measures can be detailed : those that apply to the entire French area, and those that are focused on regionallevel or local zones.

3. The grassland premiwn is a nationwide measure aimed at protecting low density grazing lands. This 300 FFr premimwn per hectare of permanent grassland 1995 is granted to farmers who comply, for five years, with the following prescriptions:

• no decrease of permanent grassland acreage (both in absolute and relative terms),

• LSU/ha < 1.4 if the share of permanent grassland in total acreage is smaller than 75%; LSU/ha < 1 otherwise, where LFU is livestock unit, • maintain permanent grassland, hedges, field gutters and ditches, • permanent grassland acreage ~3 ha, • non eligible for a retirement scheme.

The maximwn eligible acreage is 100 ha (maximwn total premiwn is FFr 30,000). The full arnount per hectare (x) is : FFr 200 (1993), FFr 250 (1994), FFr 300 (1995[START_REF] Houee | Les politiques de développement rural[END_REF], 1997).

Fig 1 :Fig 2 :

 12 Fig 1: Land consolidation as a percentage ofthe land-usedfor agriculture (results by "département'?

Table 1 :

 1 Changes in main categories ofland-use in mil/ion ha(1970 -1994)

		1970	1994
	Area used for agriculture	32.5	30.6
	ofwhich arable land	16.8	18.0
	ofwhich permanent grassland	14.1	12.6
	Wooded area	14.2	14.6
	Source: Agreste, 1996

  Public purchase of 1,000 ha of the forest of Fontainebleau

	1887	Protection of artistic and historic monuments
	1906	Protection of natural sites
	1913	Act for historic buildings
	1930	Protection of natural monuments and sites of "artistic, scientific, legenclary or
		picturesque" interest
	1943	Act protecting the nearby surroundings ofhistoric monuments
	1960	Setting up ofNational Parks
	1975	Coastal Protection Act
	1985	Mountain Act
	1993	Landscape Act

Source: Own compilation 3.1 The protection of specifie sites at the end of 19th century

Table 3 :

 3 The French National Parks

	Name of the Park	Location		Central area
	(established in)	(départements)		(km2)
	Cévennes (2nd September, 1970)	Lozère, Gard, Ardèche	3,115.0	840.0
	Guadeloupe (7th March, 1989)	Guadeloupe	296.7	173.8
	Les Ecrins (23rd March, 1973)	Hautes-Alpes, Isère	2,704.0	918.0
		74

Table 4 .

 4 4.1 The E8A scheme Regulation 2078/92 represents an endorsement of the original ESA approach included in Article 19 of EC Regulation 797/85. After an initially slow start, the number of designated ESAs bas grown rapidly from 22 in 1991, to 61 by 1993. Potential target zones were identified by the four types of environmental issue to be addressed : those threatened by water pollution, those of particular interest for rare and threatened species, those of extensive pastoral agriculture threatened by farm abandonment, and those subject to forest fires. Table 4 presents the 61 ESAs accepted for central government funding. Of this total 34 only have been accepted by the EC for cofunding. Characteristics ofFrench ESAs by type (1994) Considerable differences exist among the various ESA types as indicated by table 5 which gives the characteristics of the agreements.

		nwnbof	Total amount of	Surface area of	Eligible area (ha)
		ESASs	annual payments	ESAs (ha)
			(million FFr)l
	water pollution	3	2.2	7,400	5,700
	sensitive ecosystems	30	52.5	220,700	83,700
	farming retreat	24	33.1	492,000	106,600
	forest fire prevention	4	4.7	17,200	9,200
	total	61	92.5	737,300	205,200
	Source: CNASEA, 1995.

Table 5 :

 5 Characteristics ofthe agreements according to type ofESAs(1994)

	Nwnberof	Area under	Average area	Average payment
		contracts	agreement (ha)	under agreement	per contract
				per contract (ha)	(1000FFR)
	water pollution	131	4,356	33.25	14,214
	sensitive ecosystems	4,152	71,163	17.14	Il,587
	farming retreat	1,266	33,780	26.68	Il,374
	forest fue prevention	70	4,648	66.40	35,957
	total	5,619	113,947	35.87	Il,900
	Source: CNASEA, 1995.
	3.4.2 National measures

ECU l=FFr 7.18 (1994)

Amount per hectare is adjusted to take into account the nurnber of LSU/ha ( y ). Real arnount per hectare equals: o x if 0.6 < y < l, o x if 1 < y < 1.4 and the share of pennanent grassland in total acreage is greater than 75%, o x *y / 0.6 if y < 0.6.

Since the introduction ofthis measure 177,084 applications have been accepted by the end of 1994 corresponding to sorne 5.7 million hectares (CNASEA, 1995). The applications are concentrated in the Massif Central, an area characteristised by extensive animal husbandry.

A specific policy conceming a sustainable deve10pment scheme has been implemented in France, although this measure is not explicit1y in Regulation 2078/92. TIùs new apparoach is based on "sustainable farm deve10pment plans" (plans de Developpement Durable: PDD). The PDDs are voluntary farm management plans aimed at ensuring good countryside practices to improve agriculture's role in countryside management. They target individual farms regrouped in particular sites where a partnership between farmers, local po1icy makers, and representatives of the local population is possible.There are 750 farms located in 60 sites (CNASEA, 1995) for which development scenarios are approved after an environmental audit. Farmers then receive subsidies for a frrst five-year period. After an assessment the contract can be carried on for a new five-year period.

Regional and local measures

Regional and local measures are in fact the core of Regulation 2078/92. Ali these measures oilly apply within identified spatial areas defined at the regionallevel. Table 6 sunrrnarizes the package of the regional and local programmes. The authors consider a lirnited portion of land on the outskirts of a metropolitan area. The WTP of the resident population for landscape preservation is estimated and used to assess a conservation programme. Bonnieux F, Le Goffe P, 1996. Cost-benefit analysis oflandscape restoration : a case study in Western France. EU Workshop on "Landscape and Nature Conservation" Stuttgart, (Germany), September 26-29, 1996.

This paper is based on a case study related to a project of restoration of hedgerows. Over a 100-year time horizon this project would be profitable since its internai rate of return would be around 5% . Nevertheless private benefits are not enough to carry out restoration. It is required to take amenities and recreational values into account to justify public expenditure.

Coison F, Stenger A, 1996. Interpretation of an application of the contingent valuation method to agricultural landscapes: the problern of embedding effects. Unpublished Paper, INRA, Nantes (France).

The main contribution of the paper is to address the issue of geographicai part-whole bias. The test is not conclusive. The main drawback, however, is related to the survey itself since it is a mail survey.

There is a very lirnited number of hedonic priee studies whose objective is to value landscape and countryside amenities. The first one encompasses all of France with the dependent variable equalling the rent for a vacation house.


V Decognet

P Nicot

Y Trottin-Caudal

C Fournier

BIOCONTROL OF BOTRYTIS CINEREA STEM INFECTION ON GREENHOUSE TOMATOES WITH AN ANTAGONISTIC STRAIN OF FUSARIUM

INTRODUCTION

In heated greenhouses of southern France, the most devastating gray mold symptom on tomato is the development of stem lesions resulting from infection of pruning wounds by Botrytis cinerea. Stem infections are very difficult to avoid although the incidence of the disease can be reduced by several cultural practices, such as cutting the leaves close to the stem [START_REF] Martin | Perspectives de lutte intégrée contre la pourriture grise de la tomate sous serre[END_REF], removing the leaves on bright and sunny days and irrigating the plants early in the day rather than late [START_REF] Jarvis | Disease escape. Chapter 7 In: Managing diseases in greenhouse crops[END_REF], by avoiding excess humidity in the greenhouse, or by the use of fungicides [START_REF] Nicot | Integrated control of Botrytis cinerea on greenhouse tomatoes[END_REF].

With the objective of developing a biological control method, 175 isolates were collected from the microflora of pruning wounds on tomato plants (Nicot et al., 1996). In a screening procedure, two bacteria (a pseudomonad and an enteric bacterium) and a fungus (provisionally identified as Fusarium sp.) were selected for their efficiency in protecting the pruning wounds on tomato plants. This paper presents the results of greenhouse trials with the fungal antagonist and the evaluation of several factors of its efficiency.

MATERIALS AND METHODS

Inoculum production: Shake cultures of the fungal antagonist were prepared in yeast-malt extract broth. Prior to utilization, the cell suspensions were filtered to remove mycelium fragments, centrifuged and resuspended in water. B. cinerea (BC1 strain) was grown on Potato Dextrose Agar. After 10 days of incubation, spores were obtained by washing the cultures with sterile water. Spore concentrations were determined with a haemocytometer and adjusted as desired. Tests in growth chamber: The effect of spore density on the efficacy of the antagonist was tested on 9-week old tomato plants, cv "Rondello". B. cinerea and the antagonist were applied simultaneously on fresh pruning wounds. Leaf removal was conducted either by cutting the petioles flush to the stems or by leaving 5-cm long stubs attached to the stems.

The progression of lesions on the petiole stubs or on the stems was measured daily. The areas under the disease progress curves (AUDPC) were used to calculate an index of antagonism [START_REF] Nicot | Evaluation of effectiveness of selected microbial strains for the control of grey mold tomato[END_REF]. Tests in experimental greenhouses: Three tests were conducted between 1994 and 1996 on plants (cv. "Rondello ") approximately 5 months old in a glasshouse at INRA. The efficiency of the strain of Fusarium sp. was compared to that of a fungicide (carbendazime + diethofencarb). Treatments were arranged in a block design with four replicates (10 plants per replicate, 5 leaves per plant).

A similar test was conducted in 1996 in a glasshouse at CTIFL (cv "Trust") on plants 3 months old. The treatments were distributed randomly in a block design with three replicates (10 plants per replicate, 5 leaves per plant).

The cultural practices were kept as close as possible to a commercial situation. The antagonist, the fungicide and B. cinerea were applied once, immediately after leaf removal on petioles cut at 5 cm from the Comment citer ce document : Decognet, V., Nicot, P., Trottin-Caudal, Y., Fournier, C. (1997). Biocontrol of Botrytis cinerea stem infection on greenhouse tomatoes with an antagonistic strain of Fusarium. In: 10. Congress of the Mediterranean Phytopathological Union (p. 695-699). Presented at 10. Congress of the Mediterranean Phytopathological Union, Montpellier, FRA (1997-06-01 -1997-06-05). FRA :

2 stem. They were sprayed in a localized fashion to ensure that the treatment was evenly distributed on all wound surfaces. Different levels of inoculum of B. cinerea were used in the different trials to obtain different conditions of disease severity. The antagonist was applied as a suspension containing 3-8 x 10 5 spores per ml and the fungicide was dosed at 20 ml commercial product (Sumico) per liter. Stem infection was monitored weekly after inoculation until the end of the growing season. The results were expressed as percentages of pruning wounds developing stem lesions and the statistical were performed on the AUDPC's. The efficiency of the control methods was estimated by the reduction in AUDPC for treated plants relative to the AUDPC of the untreated plants.

Survival of Fusarium sp. on plant tissue: During one of the experiments conducted in the glasshouse at INRA, petiole stubs were inoculated with the antagonist to study its survival in greenhouse conditions. Petiole stubs were collected weekly and ground in phosphate buffer. Serial dilutions were then plated on synthetic agar medium amended with streptomycin sulfate (50 mg/l) and the results were expressed as colony forming units per gram of fresh plant tissue.

RESULTS

Factors of efficacy of the antagonist on young potted plants: The incidence of infections on petiole stubs was reduced significantly when 10 3 and 10 4 spores of Fusarium sp. were applied on the pruning wounds simultaneously with 10 5 spores of B. cinerea (Table 1a). Approximately, 90-95% protection was provided by the antagonist. Efficiency rates were reduced when only 100 or 10 spores were applied on the pruning wounds. High rates of protection were observed when the antagonists were applied on pruning wounds from leaves removed flush to the stem (Table 1b).

Efficacy of the antagonist in greenhouse trials: In the experiment conducted at CTIFL, the first stem lesions were recorded 14 days after inoculation (DAI) on the plants not protected by the antagonist or the fungicide (Figure 1). During the following 2 weeks the incidence of stem lesions increased sharply on these plants, then stabilized at approximately 40 %. For the plants protected by the antagonist, disease incidence increased slowly during 40 days and then stabilized at approximately 9%. No stem lesions were observed on the fungicide treated plant. The evolution of the disease was comparable in the trials conducted at INRA (see Fig. 1; data shown for 1996). The incidence of stem lesions was very high in all trials on the plants inoculated with B. cinerea alone, and it was variable according to the level of inoculum of the pathogen (Table 2-a). In all trials, both the antagonist and the fungicide significantly limited disease development (Table 2-b). The efficacy of the antagonist was slightly less affected by the overall severity of the tests than that of the fungicide (Table 2).

Survival of the antagonist in wounds on tomato plants: Two tests were conducted in greenhouse conditions and the population of Fusarium sp. was monitored for 3 weeks after a spore suspension was applied on petiole stubs. Only slight changes in population size were observed for the duration of the experiment (Figure 2), indicating a steady survival of the fungus within the pruning wounds.

DISCUSSION

The antagonistic strain of Fusarium sp. isolated from the epiphytic microflora of greenhouse tomatoes significantly reduced the incidence of stem lesions on tomatoes in four greenhouse trials conducted between 1994 and 1996. Its efficiency in protecting pruning wounds was consistent for two cultivars of tomatoes, and over a variety of fluctuating environmental conditions and levels of disease pressure.

The antagonist survived well in the pruning wounds, with population densities remaining close to those introduced. It appeared to retain efficiency over several months even in the most severe of the tests, where disease incidence reached nearly 80% on the control plants. Work is in progress to further evaluate the potential of this micro-organism as a biocontrol agent.

Comment citer ce document : Decognet, V., Nicot, P., Trottin-Caudal, Y., Fournier, C. (1997). Biocontrol of Botrytis cinerea stem infection on greenhouse tomatoes with an antagonistic strain of Fusarium. In: 10. Congress of the Mediterranean Phytopathological Union (p. 695-699). Presented at 10. Congress of the Mediterranean Phytopathological Union, Montpellier, FRA (1997-06-01 -1997-06-05). FRA :

Comment citer ce document : Decognet, V., Nicot, P., Trottin-Caudal, Y., Fournier, C. (1997). Biocontrol of Botrytis cinerea stem infection on greenhouse tomatoes with an antagonistic strain of Fusarium. In: 10.

Figure 1 :

 1 Figure 1: Protection of pruning wounds on tomatoes against Botrytis cinerea in greenhouse trials in 1996: () non protected control; () fungicide; ( ) antagonist.

Figure 2 :

 2 Figure 2: Survival of the antagonist Fusarium sp. in pruning wounds on tomatoes. Error bars indicate the standard error of the mean.

Table 1 :

 1 Efficiency of a strain of Fusarium sp. to protect pruning wounds on 9-week old tomato plants against infection by Botrytis cinerea. : AUDPC: « area under the disease progress curve » over 7 days or 13 days after inoculation; the values followed by different letters differ significantly at 5% level (Newman and Keuls test).

	A: Pruning by cutting leaves 5 cm from the stem
	Treatment (Number of spores/wound)	AUDPC *		Percentage of
		(7 days)		protection
	B. cinerea (10 5 )	143.8 a
	B. cinerea (10 5 ) + antagonist (10 4 )	13.7 b	90
	B. cinerea (10 5 ) + antagonist (10 3 )	6.9 b	95
	B. cinerea (10 5 ) + antagonist (10 2 )	108.1 a	25
	B. cinerea (10 5 ) + antagonist (10)	123.1 a	14
	B: Pruning by cutting leaves flush to the stem
	Treatment (Number of spores/wound)	AUDPC *		Percentage of
		(13 days)		protection
	B. cinerea (10 4 )	294.6	a
	B. cinerea (10 4 ) + antagonist (10 6 )	15.0	b	95
	B. cinerea (10 4 ) + antagonist (10 5 )	32.1	a	89

Congress of the Mediterranean Phytopathological Union (p. 695-699). Presented at 10. Congress of the Mediterranean Phytopathological Union, Montpellier, FRA (1997-06-01 -1997-06-05). FRA : 4 *

Table 2 :

 2 Efficacy of a strain of Fusarium sp. to protect pruning wounds of tomato against Botrytis cinerea in Percentage of pruning wounds developping stem lesions at the end of the growing season.

	greenhouse conditions.
	___________________________________________________________________________
	Year	1994-1995	1995-1996	1996	1996 (CTIFL)
	B. cinerea inoculum 1	500	50	airborne	50
	___________________________________________________________________________
	A. Botrytis cinerea	78,5 a 2	56,0 a	43,3 a	42,3 a
	+ Fusarium sp.	41,2 b	30,0 b	16,8 b	9,0 b
	+ Sumico	23,8 c	7,0 c	8,0 b	0	c
	___________________________________________________________________________
	B. Rate of protection of the pruning wounds (%), computed on the basis of the AUDPC's over the growing
	season
	Botrytis cinerea	a 2	a	a		a
	+ Fusarium sp.