F Boulard

G Rohaut

D Dupré

F Barbe Barrailh

A Person

M Leclerc

E Jacquot

L Glais

Impact of the potato inoculation date on potato virus Y load and viral distribution in daughter tubers at harvest

Keywords: PVY, early/late infection, mature plant resistance, virus translocation, tubers virus accumulation, daughter tubers

Aged plants are more difficult to infect than young plantlets. This modification of susceptibility is described as mature plant resistance (MPR). For potato virus Y (PVY), MPR is known to lead to i) low infection rates of plants inoculated at post-flowering stage and ii) decrease of the number of infected daughter tubers. However, the impact of inoculation date on the capacity of PVY to accumulate in daughter tubers has not been studied so far. Field and greenhouse experiments were carried out to better understand PVY epidemiology and to help potato growers to evaluate consequences of early/late infections on the quality of their crops. In field trials, potato plants (cv. Bintje) were covered by insect proof nets from planting to harvest except for a 14-day period to expose plants to natural PVY infections. Under controlled conditions, potato plants were mechanically inoculated with PVY at different dates from preflowering stages (early inoculations) to post-flowering stage (late inoculations). At harvest, daughter tubers were individually collected and analysed to define proportions and viral load of infected tubers according to the time between virus inoculation and harvest. Our results showed that although the age of plants at the time of inoculation can modify their susceptibilit y to PVY infection, in return, early and late PVY inoculations lead to similar i) rates of infected tubers at the plant scale and ii) equivalent viral accumulation in infected tubers. All together, these data revealed that both early/late infections are high risks for the sanitary quality of potato tubers.

INTRODUCTION

Potato virus Y (PVY) [START_REF] Shukla | The Potyviridae[END_REF] is the type-member of the genus Potyvirus [START_REF] Brunt | The general properties of potyviruses[END_REF]. PVY infects a wide range of host plants from the family Solanaceae and is transmitted in a non-persistent manner by more than 65 aphid species, including the green peach aphid (Myzus persicae) which is the most efficient PVY vector [START_REF] Lacomme | Transmission and epidemiology of Potato virus Y[END_REF]. PVY is the most economically damaging virus on potato. Indeed, in the absence of protection against aphids (e.g. sprays of mineral oil), yield losses due to PVY infection can reach up to 80.0% [START_REF] Valkonen | Viruses: economical losses and biotechnological potential[END_REF]. In the 1960s, PVY isolates were first divided into two main strain groups, PVY N and PVY O , based on their ability to induce (PVY N ) or not (PVY O ) tobacco vein necrosis on Nicotiana tabacum cv Xanthi and on their pathogenicity on potato (Solanum tuberosum) [START_REF] Singh | Discussion paper: The naming of Potato virus Y strains infecting potato[END_REF]. Then, since PVY variants with particular properties have been reported between late 1980s and early 2000s, PVY species are now classified into seven main groups: PVY O , PVY N , PVY C , PVY N-Wi , PVY NT N , PVY Z and PVY E [START_REF] Kehoe | Improving Potato virus Y strain nomenclature: lessons from comparing isolates obtained over a 73-year period[END_REF]. Members of PVY NT N , PVY Z and PVY E strain groups have been described as the most damaging PVY isolates on potato crops because of their ability to induce necrotic symptoms on tubers of susceptible cultivars [START_REF] Beczner | Studies on the etiology of tuber necrotic ringspot disease in potato[END_REF][START_REF] Piche | Diversity among Potato virus Y isolates obtained from potatoes grown in the United States[END_REF][START_REF] Galvino-Costa | A novel type of Potato virus Y recombinant genome, determined for the genetic strain PVY E[END_REF].

When a viruliferous aphid inoculates PVY to a host plant, the infection starts at the inoculated site. After a viral multiplication/accumulation phase in the first infected cells, the virus moves locally from cell-to-cell, crosses different cell layers of the inoculated organ and reaches the phloem tissues [START_REF] Rupar | Fluorescently tagged Potato virus Y: a versatile tool for functional analysis of plant-virus interactions[END_REF]. Afterwards, viruses spread into the whole plant leading to a systemic infection of the host [START_REF] Otulak | Ultrastructural events during hypersensitive response of potato cv. Rywal infected with necrotic strains of Potato virus Y[END_REF]. This process that drives the within-host spatio-temporal dynamics of PVY is generic. However, it has been demonstrated that its magnitude depends on several biotic (e.g. host genotype and viral strain) and environmental (e.g. temperature) factors. Indeed, it has been shown that the proportions of infected organs (leaves, stems and tubers) were higher with PVY N than with PVY O [START_REF] Basky | Differences in aphid transmissibility and translocation between PVY N and PVY 0 isolates[END_REF] and that the genetic background of potato cultivars significantly impacts the dynamics of systemic infections. According to the presence of R (ER, extreme resistance) or Ny (HR, hypersensitive resistance) genes, the movement of virus within the infected tissues is blocked (ER) or restricted (HR) to cells near the inoculation site [START_REF] Szajko | The novel gene Ny-1 on potato chromosome IX confers hypersensitive resistance to Potato virus Y and is an alternative to Ry genes in potato breeding for PVY resistance[END_REF]. Finally, the age of the plant at the time of inoculation also influences within-host virus developme nt.

Indeed, it has been shown that old plants are more difficult to infect than young plantlets. This change in the susceptibility of the host with its age is common for infections with plant pathogens and has been described as ontogenic resistance, age-related resistance, developmental resistance, or as in our specific case study, mature plant resistance (MPR). MPR has been described for several viral pathosystems (e.g. wheat dwarf virus/winter wheat [START_REF] Lindblad | Temporal spread of Wheat dwarf virus and mature plant resistance in winter wheat[END_REF]; potato virus S (PVS)/potato and potato virus X (PVX)/potato [START_REF] Venekamp | Mature plant resistance of potato against some virus diseases. Concurrence of development of mature plant resistance against Potato virus X and decrease of ribosome and RNA content[END_REF]) but remains poorly understood. Previous studies suggest that MPR does not result from the expression of any resistance gene, but is rather a consequence of i) a drastic decrease of ribosome and RNA contents [START_REF] Venekamp | Mature plant resistance of potato against some virus diseases. Concurrence of development of mature plant resistance against Potato virus X and decrease of ribosome and RNA content[END_REF] and ii) an increase of callose deposition at plasmodesmata [START_REF] Kumar | Dynamics of plant responses during compatible and incompatible PVY-potato interactions[END_REF]. MPR has already been reported in several studies for the PVY/potato pathosystem [START_REF] Beemster | Virus translocation in potato plants and mature-plant resistance[END_REF][START_REF] Gibson | The development of mature plant resistance in four potato cultivars against aphidinoculated potato virus Y O and Y N in four potato cultivars[END_REF][START_REF] Shrestha | Interactions among potato genotypes, growth stages, virus strains, and inoculation methods in the Potato virus Y and green peach aphid pathosystem[END_REF][START_REF] Dupuis | The movement of Potato virus Y (PVY) in the vascular system of potato plants[END_REF]. These studies suggest that the main observed developmental resistance is associated with the flowering stage when foliage has stopped to grow and when tubers has started to grow, i.e. about 70 days after planting (DAP). In addition, [START_REF] Dupuis | The movement of Potato virus Y (PVY) in the vascular system of potato plants[END_REF] demonstrated that part of the MPR phenomenon can be due to the decay of the mother tuber which impedes the movement of PVY from one stem to the other. MPR in potato plant, associated with a decrease in the number of infected tubers on the total tuber harvested, is known to depend on both host genotype and virus isolate. [START_REF] Shrestha | Interactions among potato genotypes, growth stages, virus strains, and inoculation methods in the Potato virus Y and green peach aphid pathosystem[END_REF] used a semiquantitative ELISA approach to show that PVY load in potato leaf was similar within plants inoculated before and after the flowering stage. However, no data are available on translocatio n efficiency of PVY within infected tubers at the scale of the plant submitted to early or late PVY inoculations. In this study, we addressed the effects of plant age at infection for the within-ho st dynamics of PVY in infected plants. We used both field and greenhouse experiments to investigate PVY accumulation in plants and the translocation of virus to daughter tubers using several PVY isolates naturally and mechanically inoculated to potato plants. The main aim of this work is to provide new insights into the translocation of PVY to daughter tubers, and therefore into the epidemiology of the disease. This knowledge is particularly important for growers to evaluate the risk on the quality of potato tubers according to the timing of infectio ns (early/late) along the cropping season.

MATERIALS AND METHODS

Field trials

Field trials were carried out in an experimental potato field located in the North of France. PVYsusceptible cv. Bintje potato seeds were used. For each year of experimentation, tubers came from a single certified super-elite class (less than 1.0% of virus-infected tubers) seed lot supplied by the French seed potato growers (FN3PT, Paris, France). The layout of the trial consisted of a 50-meters long and 5-meters wide plot with a single row of 120 potato seeds (plot A) and seven extra plots (plots B to H) of 5-meters long and 1-meter wide (Figure 1a). These extra plots were planted with 2 rows of 10 potato seeds. Aphid populations present in the trials were monitored twice a week from planting to harvest using yellow water pan traps.

In plot A, potato plants were exposed to aphids during the whole experiment. In plots B to H, an insect proof net (800µx1000µ, Biotex, Saint Wandrille Rançon, France) was used to protect plants from planting to harvest, except for a 14 day period during which the plants were exposed to natural aphid populations potentially presents at the trial site. This window of net removal (WNR) was different for each plot and corresponded to the 28 th -42 nd (i.e. D28/42) day after planting (DAP) for plot B. To allow aphid exposure at the different potato physiological stages (Supplementary Figure 1), the net-free periods were scheduled at D42/56, D56/70, D70/84, D84/98, D98/112 and D112/126 for plots C, D, E, F, G and H, respectively (Figure 1a). To determine the sanitary status of planted material, plants from plots B to H were individually sampled when removing the insect-proof net and leaves were analysed using ELISA as described below.

Likewise, plants in plot A were sampled at 28 DAP. PVY-infected plants were maintained in the experiment, but data associated with these plants and their tubers were removed from the analysis. At the end of each WNR period, plants were treated by an insecticide (Mandarin, Proactive agent pyrethrinoid, 0.25L/ha, Philagro, Saint Didier au Mont d'Or, France) to prevent any further infections. To determine the sanitary status of plants after each WNR period, at 133 DAP, 20 plants evenly distributed in plot A and all plants from plots B to H were manually and individually harvested. Then, 15 daughter tubers were randomly collected for each plant, immediately transferred to the laboratory. Dormant tubers were directly analysed by real-time RT-PCR, and growing-on ELISA were applied on plantlets from replanted daughter tubers. The status (PVY-infected or healthy) and the serotype of PVY isolate(s) infecting mother plants of the different plots were defined using cumulated ELISA results from each of the 15 collected daughter tubers per plant. A mother plant was assigned to PVY-infected status when at least one of its 15 tested daughter tubers was infected. The infected mother plant was considered as having a mixed infection when either i) at least one of its daughter tubers was infected with PVY isolates belonging to different serotypes or ii) different PVY serotypes were individua lly detected in different daughter tubers. This experimental procedure was repeated during two consecutive growing seasons between 3 May and 16 September 2016, and 20 April and 31 August 2017.

Greenhouse experiment

Greenhouse experiments based on mechanical inoculation of cv. Bintje were carried out in 2016 and in 2017. Potato seeds, initially stored at 4°C, were placed at 20°C in the dark for few weeks to break the tuber dormancy. As soon as sprouts emerged, 108 tubers were individually planted in an insect-proof greenhouse at 20°C ± 2°C with natural light. At 12 DAP, all except one emerging stem were removed from each plant. Sets of 12 plants were mechanically inoculated with the referent isolate PVY N -605 [START_REF] Jakab | Infectious in vivo and in vitro transcripts from a full-length cDNA clone of PVY-N605, a Swiss necrotic isolate of Potato virus Y[END_REF]. Each set of plants was inoculated according to [START_REF] Glais | The amino acid 419 in HC-Pro is involved in the ability of PVY isolate N605 to induce necrotic symptoms on potato tubers[END_REF]. Briefly, crude sap produced from an PVY N -605-infected source plant was rubbed on the apical 2-3 true youngest potato leaves of each plant of a set. Inoculation of a set was performed at 20,47,54,61,68,75,82,89 and 100 DAP (Figure 1b) which corresponds to different developmental stages of potato (Supplementary Figure 2). Each potato plant was individually harvested at 140 DAP and the sanitary status of tubers were directly analysed by real-time RT-PCR, and an ELISA was applied on plantlets from replanted daughter tubers.

Plant sampling and PVY detection

Potato leaves collected from either one stem (in greenhouse experiments) or multiple stems of the same plant (in field trials) were placed in a plastic bag, afterwards transferred to the laboratory for ELISA analysis. Tubers were stored at 4°C for two months. Then, one eye-plug was sampled on the sprout rose-end on each tuber. Eye-plugs were placed into a gibberellic acid solution (4mg/L) for 1 h and dried overnight at room temperature before being planted in greenhouse at 20°C ± 2°C with natural light. Two months later, potato plantlets were sampled as mentioned above and tested by ELISA for the presence of PVY.

Samples were tested by DAS-ELISA as described by [START_REF] Glais | The amino acid 419 in HC-Pro is involved in the ability of PVY isolate N605 to induce necrotic symptoms on potato tubers[END_REF] with a mixture of monoclonal and polyclonal antibodies raised against PVY (M. Guillet, FN3PT/INRAE, Rennes, France). Then, PVY-infected samples were tested with a PVY N -monoclonal antibody (Bioreba, Reinach, Switzerland) and with a PVY O -monoclonal antibody (15C10) provided by FN3PT/INRAE. Samples were considered infected when ELISA values (optical density at 405 nm) were higher or equal to three times ELISA values for healthy control samples.

In field trials, for each plot/year combination, a maximum of two samples infected by a PVY serotype (i.e. by either -O, -N or -U serotypes avoiding mixed infections) were selected for partial molecular characterization of viral genome. A total of 26 and 22 infected samples collected from potato plantlets grown from single infected daughter tubers were selected in 2016 and 2017, respectively (Supplementary Table 1). An immunocapture (IC)-RT-PCR procedure was applied according to [START_REF] Glais | RFLP mapping of the whole genome of ten viral isolates representative of different biological groups of Potato virus Y[END_REF] for the fixation of PVY particles present in plant sap and the amplification of two regions of the PVY genome. The regions coding for HC-Pro/P3 proteins (nt 1672-2666, according to [START_REF] Jakab | Infectious in vivo and in vitro transcripts from a full-length cDNA clone of PVY-N605, a Swiss necrotic isolate of Potato virus Y[END_REF] and coat protein (CP) (nt 8572-9373, according to [START_REF] Jakab | Infectious in vivo and in vitro transcripts from a full-length cDNA clone of PVY-N605, a Swiss necrotic isolate of Potato virus Y[END_REF], where hot spots of recombination can be used to identify PVY strain group members, were amplified and sequenced through Genoscreen platform (France) by direct sequencing of amplification products in forward and reverse directions. The acronym and the sequence of primers used for the different steps (RT, PCR, sequencing) are described in Supplementary Table 2. Nucleotide sequences were edited by the BioEdit software [START_REF] Hall | BioEdit: an important software for molecular biology[END_REF] and compared with 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]

Potato tuber sampling, nucleic acid extraction and real-time RT-PCR diagnosis

A peel of approximately 20 mg was taken from the heel end of each washed tuber directly after the harvest. For potato plants from field trial, peels of 15 tubers per plant were pooled and ground with a Pollähne press (Meku, Wennigsen, Germany). Potato plants from the greenhouse experiments produced fewer and smaller daughter tubers. Thus, peels from 2 to 4 tubers per plants were pooled. The tuber sap was diluted (v/v) with 500 µl of Mortimer buffer (0.2% BSA, 0.5% polyvinyl pyrrolidone, 0.02 M PBS, 0.05% Tween 20). After centrifugation for 2 min at 10 000 rpm, the extraction of nucleic acids was performed on 200 µl of the supernatant using InviMag virus RNA Kit (Stratec Molecular, Berlin, Germany) following manufacture r's recommendations. This method associated with the KingFisher T M mL System (ThermoFis her Scientific, Waltham, USA) allows the automatic efficient handling of RNA on magnetic particles and by a series of three washes in different buffers to obtain purified nucleic acids.

Briefly, a cell lysis was performed by adding 400 µl of Lysis buffer in the presence of 20 µl proteinase K and 20 µl carrier-RNA from the extraction kit to the 200 µl of the supernatant from tuber sap. This solution was incubated, while being agitated, at 65°C for 10 min. Then, the mixture was transferred into the KingFisher mL instrument, mixed with 260 µl of binding solution and 20 µl of solution containing the magnetic beads. Three automatic washing steps of the particles fixed to RNA were then performed and after 15 min, 100 µl of pure nucleic acids were eluted. An aliquot of 2 µl of viral RNA (± 70 ng/µl) was analysed by multiplex RT-qPCR in a total volume of 25 µl using the AgPath-IDTM One-Step RT-PCR kit (ABI, Foster, CA, USA) according to the manufacturer's instructions, except for the primers (Univ F, Univ R) and probe (Univ) used at concentrations according to [START_REF] Kogovsek | Single-step RT real-time PCR for sensitive detection and discrimination of Potato virus Y isolates[END_REF], i.e. 300 nM and 200 nM, respectively. In addition, an endogenous control based on the amplification of the plant cytochrome oxidase gene (Cox) was used to validate the quality of each extraction.

Concentrations of primers (COX F, COX RW) and probe (COXSOL 1511T) were used according to [START_REF] Boonham | Direct detection of plant viruses in potato tubers using realtime PCR[END_REF]. Real-time thermal cycling conditions were as follows: 10 min at 45°C for the reverse transcription (RT) of the viral RNA, then 15 min at 95°C for the RT inactivation and initial denaturation of nucleic acids, followed by 40 cycles of amplifica tio n with 45 sec at 60°C and 15 sec at 95°C. Data were analysed with the LightCycler ® 480 software v.1.5.0 (Roche). Viral quantification was obtained by comparison with standard fractions containing the targeted PVY sequence at different concentration (from 10 8 to 10 2 copies per reaction) according to [START_REF] Balme-Sinibaldi | Improvement of Potato virus Y (PVY) detection and quantitation using PVY N -and PVY O -specific real-time RT-PCR assays[END_REF]. This standard corresponds to a plasmid (p3'end) in which PVY N -605 nucleotide sequence from nt 4065 to 9801 has been introduced [START_REF] Tribodet | Characterization of Potato virus Y (PVY) molecular determinants involved in the vein necrosis symptom induced by PVY N isolates in infected Nicotiana tabacum cv. Xanthi[END_REF]. In our experimental conditions with tuber extracts, a sample is considered infected by PVY once the CT value, defining the amplification cycle number at which the fluorescence exceeds the reporting threshold, is below 30. This CT threshold corresponds to the RT-qPCR detection limit fixed under our experimental conditions at 10 3 copies of the targeted PVY RNA per sample. This CT cut-off was validated in our laboratory by comparing qPCR assays carried out on tubers and ELISA tests carried out on plantlets after replanting the corresponding tubers. Based on this CT threshold, more than 99.0% of correlation was found between both diagnostic techniques (data not shown).

Statistical analyses

Prevalence data were analysed using Generalised Linear Models with a Binomial distributio n:

RESULTS

Vegetative development of potato plants in trials

In the field trials, the physiological stages of the plant were noted at the beginning of each of the 7 WNR (Supplementary Figure 1). In the greenhouse trials, the mechanical inoculation was performed at nine dates after planting.

At 20 DAP, for both repetitions, potato plants were shorter than 30 cm, while they reached a height of about 80 cm and full flowering at 47 and 54 DAP (Supplementary Figure 2). At 61 DAP, plants were still green, but some flowers have started to fall. At 68 DAP, some leaves at different stages in plants became yellow, and only few flowers were left. At 75 and 82 DAP, yellowing was more pronounced on leaves and generalized with the fall of all flowers. At 89 DAP, the whole plants were yellow. Finally, at 100 DAP the yellowing of plants was noted with the drying and the fall of basal leaves.

Monitoring of aphid flights

In 2016, a total of 550 aphids were trapped during the monitored period (i.e. from the planting to the harvest of potato). Given the WNR periods, different aphid pressures were recorded (Figure 2a and Table 1). For WNR that corresponds to plot B, 54 aphids were collected. Indeed, in 2017 the percentage of infected plants was 100.0%, 80.0%, 100.0%, 100.0%, 100.0%, 52.6%, 25.0% and 15.0% in plots A (20/20), B (16/20), C (20/20), D (20/20), E (20/20), F (10/19), G (5/20) and H (3/20), respectively. Under these experimental conditio ns, potato plants were i) highly infected by PVY from 42 to 98 DAP and ii) mostly uninfected when exposed to natural infections occurring from 98 DAP, when the number of trapped aphids was low. Statistical analyses highlighted that the period at which the plant was infected was significant and explained 73.4% of the observed infection rate (p=2. 2E-16). In addition, a significant effect of the number of caught aphids according to the WNR period was also observed (p=2.2E-16) and explained 22.5% of the total deviance.

For PVY inoculations carried out under greenhouse conditions, similar infection pattern was observed in experiments in 2016 and 2017. Infection rates above 75.0% were obtained for inoculations carried out between the 20 th and the 61 st DAP while later inoculations were associated with infection rates below 60.0% (Figure 2b). The last two inoculations performed at 89 and 100 DAP were all unsuccessful. [START_REF] Lacroix | Biological characterization of French Potato virus Y (PVY) isolates collected from PVY-susceptible or -resistant tobacco plants possessing the recessive resistance gene va[END_REF]. These particular PVY U isolates are characterized by a positive detection with the non-specific antibody raised against PVY, but a lack of detection with PVY N and PVY O monoclonal antibodies. Based on these serological properties, PVY U isolates can only be identified in single infected daughter tubers but not in mixed infection. The proportions of each type of infection (i, ii and iii) were different between plots (Figure 3a).

Virus diversity within plots

Conversely to 2016, infections with PVY of serotype-O were more frequent in 2017. It was observed in plot A where 95.0% of potato plants (19/20) were in mixed infection with PVY isolates of serotype-O and -N. This trend was mainly observed in the first half of growing potato season (plots B to D) (Figure 3b). Indeed, we noted in plot B a high proportion (55.0%, 11/20) of plants infected only by PVY of serotype-O, and a high prevalence of mixed infections in plots C (65.0%, 13/20) and D (100.0%, 20/20). Plots E to H were mainly single-infected by PVY of serotype-N.

A partial molecular characterization of up to 2 isolates/serotype/plot was performed by sequencing fragments in the HC-Pro/P3 (994 bases from nt 1672 to 2666) and in the CP (801 bases from nt 8572 to 9373) genes (Supplementary Table 1). These two regions of the PVY genomes are known to be hot spots of recombination events between PVY N and PVY O sequences [START_REF] Glais | Genomic variability in Potato potyvirus Y (PVY): evidence that PVY N W and PVY NTN variants are single to multiple recombinants between PVY O and PVY N isolates[END_REF]. In 2016 experiment, 15 out of the 16 isolates of serotype-N displayed recombination junctions (RJ) in HC-Pro/P3 (at nts 2414-2418) and in CP (between nts 9163-9182) regions. These molecular features are characteristic of PVY NT N isolates. The isolate without RJ displayed a N-type nucleotide sequence and should correspond to a PVY N isolate or a non-recombinant PVY NT N isolate [START_REF] Glais | PVY N -605, the reference PVY N isolate, displays a PVY NTN non-recombinant genome[END_REF]. Eight serotype-O PVY isolates were sequenced. Three isolates showed no RJ and displayed an O-type nucleotide sequence (not shown) suggesting their assignment to PVY O strain and five isolates displayed a RJ in HC-Pro/P3 region (at position 2392-2393 or 2395-2396) and a O-type nucleotide sequence in the CP region (not shown). According to these molecular characteristics, these five isolates belong to PVY N-Wi strain. Two PVY U isolates were included in the sequence analyses. One presents the two RJ described for PVY NT N strain whereas the other PVY U isolate has PVY N-Wi molecular pattern (i.e. a RJ in the HC-Pro/P3 region). In 2017, the 13 sequenced serotype-N isolates displayed PVY NT N type RJs in HC-Pro/P3 and CP regions. Eight out of nine sequenced serotype-O isolates corresponded to PVY O isolates because they have no recombinatio n junction and displayed an O-type nucleotide sequence. One serotype-O isolate showed a single RJ in the HC-Pro/P3 region located at position 2392-2393 leading to its assignment to the PVY N-Wi strain.

Virus translocation in daughter tubers

Due to a low number of harvested tubers from experiments carried out under controlled conditions, translocation of PVY from inoculated leaves to daughter tubers was analyzed only under field conditions. In 2016, half of infected plants from plot A had between 25.0% and 92.8% infected daughter tubers (63.2% on average) (Figure 4a), with only two plants showing an infection of 100.0% of their progeny tubers (Table 2). In 2017, 50.0% of infected plants from plot A displayed between 92.3% and 100.0% of infected daughter tubers (97.2% on average) (Figure 4b), with 15 out of 20 infected plants showing an infection of 100.0% of their daughter tubers (Table 2). Except this difference, the distribution of the percentage of infected daughter tubers per plants in plots B to H showed the same trend in both years, although a low interannual variation of this distribution was observed between 2016 and 2017 experiments (1.9% of the deviance, p=2.26E-05). In return, the effects of the WNR period (19.3% of the deviance, p=1.17E-12), and the WNR x year interaction (8.0% of the deviance, p=1.4E-13) were significant. Indeed, the pairwise comparisons of the percentage of infected tubers in plants between the different plots in 2016 highlighted that plots D and E (88.0% on average) showed a higher percentage of infected tubers than in plots B, C, F, G, H (65.0% on average), and this was not different from plot A (63.2% on average) (Table 2). In 2017, statistical analyses also highlighted a significant influence of the WNR period on virus translocation to tubers (40.1% of the deviance, p=2.2e-16). Plants infected in plots B, C, E, F, G, H had 58.0% on average percentage of infected tubers, whereas infected plants from plot D showed 96.3% of infected tubers, as in plot A (Table 2).

Viral accumulation in daughter tubers

PVY load in infected samples was estimated for each studied potato plant by bulking the peels of daughter tubers. In field conditions, the change of the distribution of PVY titer in daughter tubers with the date of infection plants showed similar pattern in both years, although significa nt effect, but low influential, of the year (6.3% of the deviance, p=0.0010) or of the year x WNR period (5.4% of the deviance, p=0.023) was found. In pooled data of 2016 and 2017, 12.6% of the deviance of the PVY titer in infected tubers was explained by the WNR period (p=3.86E-06). In 2016, tubers from half of infected plants in plot A had a PVY titer between 1.28E05 (Log=11.76) and 1.28E06 (Log=14.06) copies of viral RNA/2 µl of total RNA extract with an average virus load at 6.9E05 PVY copies (Figure 5a). As plot A was continuously exposed to natural viral infection along the growing period, viral load in infected plants from this plot should correspond to the maximum of virus accumulation for this trial. Pairwise comparisons analyses highlighted that the PVY load was not different in tubers from infected plants of plots A, B, C, F, G and H (3.94E5 PVY copies on average) (Figure 5a). In return, a significa nt ly higher PVY titer was observed in tubers from infected plants from plots D and E (9.99E5 PVY copies on average). In 2017 experiment, the period from which the plant was infected also explained 32.0% of the variability of PVY titer in progeny. Indeed, contrary to plants of plots B, E, F and H where no significant difference in PVY titer in infected tubers was noted (4.1E05 PVY copies on average), a higher PVY load was statistically observed in infected plants from plot D (1.01E06 copies on average) which is close to that of plants from plot A (7.1E05) (Figure 5b). Plants of plots C and G displayed an intermediate PVY load between these two groups.

To complement the analyses of viral load carried out at the plot scale, in 2016 experiment virus quantifications were carried out at plant scale in order to study PVY load in the daughter tubers from an infected mother plant. Two plants each from plots B, D, F, and H were randomly selected, and their daughter tubers were individually tested in real-time RT-PCR. Statistica l analyses revealed that PVY load was not significantly different between infected daughter tubers from the same mother plant, nor between the two studied plants in each plot (0.059<p(Tuber)<0.84; 0.10<p(Plant)<0.82) (Supplementary Figure 3).

For the greenhouse trials, a significant low inter-annual variation (7.27% of the deviance, p=0.012) in PVY load was observed. Indeed, an average PVY titer of 9.05E04 copies of viral RNA/2 µl of total RNA extract was obtained in 2016 experiment (Figure 5c), and 2.74E04 in 2017 experiment (Figure 5d). Nevertheless, both trials highlighted the same trend, with no significant effect of the date of inoculation (p >0.05).

DISCUSSION

The age of plants at the time of inoculation is known to be one of the factors that determines the susceptibility of potato to PVY [START_REF] Beemster | Virus translocation in potato plants and mature-plant resistance[END_REF][START_REF] Venekamp | Mature plant resistance of potato against some virus diseases. Concurrence of development of mature plant resistance against Potato virus X and decrease of ribosome and RNA content[END_REF][START_REF] Gibson | The development of mature plant resistance in four potato cultivars against aphidinoculated potato virus Y O and Y N in four potato cultivars[END_REF][START_REF] Shrestha | Interactions among potato genotypes, growth stages, virus strains, and inoculation methods in the Potato virus Y and green peach aphid pathosystem[END_REF][START_REF] Chikh-Ali | Effects of the age-related resistance to Potato virus Y in potato on the systemic spread of the virus, incidence of the potato tuber necrotic ringspot disease, tuber yield, and translocation rates into progeny tubers[END_REF]. However, impact of mature plant resistance (MPR) on PVY accumulation in daughter tubers produced by infected potato has been poorly described in the literature. Such new insights into the PVY/potato pathosystem are important i) for researchers to better understand the epidemiology of this viral disease and ii) for growers to evaluate the consequences of early/late infections on the quality of their potato production. In the present study, field and greenhouse experiments were designed to assess how the dynamics of viral accumulation in tubers changes with the timing of inoculation during the growing season. Our study confirmed an ontogenetic change in plant resistance. This is characterised by an increase in host susceptibility followed by a strong decrease until near complete resistance, with the flowering stage as the benchmark of the appearance of MPR. This pattern of plantvirus interactions was observed in field and greenhouse conditions, and is consistent with published studies that pointed out a MPR switch about 60 DAP (i.e. 30 days after potato plant emergence) and the complete MPR about 90 DAP (i.e. 60 days after potato emergence) [START_REF] Venekamp | Mature plant resistance of potato against some virus diseases. Concurrence of development of mature plant resistance against Potato virus X and decrease of ribosome and RNA content[END_REF][START_REF] Gibson | The development of mature plant resistance in four potato cultivars against aphidinoculated potato virus Y O and Y N in four potato cultivars[END_REF][START_REF] Dupuis | The movement of Potato virus Y (PVY) in the vascular system of potato plants[END_REF]. In our study this evolutio n towards less susceptible plants was observed earlier in greenhouse conditions (61 DAP) than in field conditions (from 84 to 98 DAP). The difference in controlled conditions, and the time lag of almost 15 days of the potato flowering could explain this observed difference. In field trials, we observed a clear decrease of plant susceptibility which is statistically mainly due to the plant age at the infection, and to a lesser extent, due to the number of caught aphids at the differe nt WNR periods. However, at the plant scale we noted that this phenomenon had no significa nt effect on viral translocation in daughter tubers. This means that if the infection occurred 28/42 (plot B) or 112/126 (plot H) days after planting the virus infected on average 58.0-65.0% of daughter tubers per infected plant. Some previous studies reported that the appearance of the MPR phenomenon occurs together with a decrease in the proportion of infected progeny tubers [START_REF] Beemster | Virus translocation in potato plants and mature-plant resistance[END_REF][START_REF] Venekamp | Mature plant resistance of potato against some virus diseases. Concurrence of development of mature plant resistance against Potato virus X and decrease of ribosome and RNA content[END_REF][START_REF] Gibson | The development of mature plant resistance in four potato cultivars against aphidinoculated potato virus Y O and Y N in four potato cultivars[END_REF][START_REF] Dupuis | The movement of Potato virus Y (PVY) in the vascular system of potato plants[END_REF][START_REF] Chikh-Ali | Effects of the age-related resistance to Potato virus Y in potato on the systemic spread of the virus, incidence of the potato tuber necrotic ringspot disease, tuber yield, and translocation rates into progeny tubers[END_REF]. Moreover, [START_REF] Beemster | Virus translocation in potato plants and mature-plant resistance[END_REF] stated that the longer the period is between inoculation of PVY O and harvest, the more tubers are infected. However, all these studies compared the number of infected tubers to the total number of tuber tested in trials, and not to the total progeny tubers from infected plant, as in our study. These two levels of analysis could explain the discrepancy with our results. In return, for the two consecutive years a significantly higher percentage of infected daughter tubers was observed in plants infected from 56 to 84 days after planting (plots D/E-2016-88.0% on average; or D-2017-96.0% on average). In 1991, similar results were reported with the aphid inoculation of PVY O at different weeks after the emergence of potato plants [START_REF] Gibson | The development of mature plant resistance in four potato cultivars against aphidinoculated potato virus Y O and Y N in four potato cultivars[END_REF]. Indeed, this study showed that at 56 DAP, the PVY aphid inoculation of potato cultivar King Edward led to 62.0% of infected tubers from PVYinoculated plants, whereas at 35, 42, 70 and 84 DAP, the percentages were 45.0%, 54.0%, 46.0% and 33.0%, respectively. To a lesser extent the same trend was also observed with the potato cv. Record.

For both field experiments, PVY load in daughter tuber lead to similar conclusions to those obtained for the percentage of infected tubers. Firstly, between early (plot B) or late infectio ns (plot H) no significant differences were observed in PVY load in daughter tubers. This result is consistent with [START_REF] Shrestha | Interactions among potato genotypes, growth stages, virus strains, and inoculation methods in the Potato virus Y and green peach aphid pathosystem[END_REF] who revealed that mean PVY titer estimated by semiquantitative ELISA in potato leaf sap was not statistically different between i) aphid-mediated or mechanical inoculations and ii) early and late inoculations. Our results highlight that if plant infection occurrs in the period 7 to 21 or 91 to 105 days before harvesting, the virus accumula tes at the same level in daughter tubers. Secondly, a higher PVY titer was also observed in progeny when mother plants were infected 56 to 84 DAP (plots D and E, 2016) or 56 to 70 DAP (plot D, 2017). The difference of PVY translocation and accumulation in tubers in these plots compared to all other plots could not be explained by the presence of particular PVY isolates within these WNR periods because similar PVY isolates (NTN, N, O, and W isolates) were also identified in other plots. These results suggest that a plant infected between 56 and 84 DAP may favour viral translocation and accumulation in daugther tubers rather than when infect io ns occur earlier or later. In our experiments, this time window overlaps a period from full flowering to drop of flowers. The flowering physiological stage seems to be associated with the tuber growth with an influx of water and starch to the tubers [START_REF] Ellissèche | Aspects physiologiques de la croissance et du développement[END_REF]. This transfer of nutrients to tubers at this period could explain the promoted spread and load of PVY in daughter tubers.

In addition, our results pointed out the expected effect of the number of caught vectors in field that was significant to explain both prevalence and load of PVY in daughter tubers. The most interesting point of this study is the fact that accumulation of PVY in infected daughter tubers had similar level even if mother plant was infected during its active growth or senescence phases. This result was obtained in field conditions (where potato PVY infectio ns can result from multiple aphid-mediated viral inoculations) and in greenhouse conditio ns (where potato PVY infection resulted from a single mechanical inoculation). That means that similar PVY load in infected tubers was reached soon after (i.e. a few days) an inoculation (e.g. plots H, inoculated 7/21 days before harvest/diagnosis) and was maintained at this level even for long infection period (e.g. plot B, inoculated 91/105 days before harvest/diagnosis). [START_REF] Mehle | Spread of potato virus Y NTN in potato cultivars (Solanum tuberosum L.) with different levels of sensitivity[END_REF], who studied the spread of PVY NT N in various potato organs, demonstrated that i) the PVY NT N isolate reaches the roots in 9 days (or less according to the potato cultivar) and ii) accumulates in infected plant at a level which does not increase between the ninth and the twentieth day after inoculation.

In our field study, PVY accumulated in infected tubers from cv. Bintje at a level above 10E05 copies/2 µL of total RNA extracts. It would be worth investigating PVY translocation and accumulation for a range of cultivars with contrasted level of resistance to confirm or reject that PVY load in tubers remains above the threshold sensitivity of molecular detection tool. Early detection of PVY in dormant tubers through real-time RT-PCR method recently became a standard in testing procedure for several certified European laboratories [START_REF] Schumpp | Large-Scale RT-qPCR Diagnostics for Seed Potato Certification[END_REF].

It is therefore essential to guarantee the reliability of this molecular tool to detect PVY in dormant tubers of any potato cultivar that has been subjected to either early or late inoculatio n.

Nowadays, seed potato growers still neglect the impact of late PVY infections, typically after flowering, on tuber production. However, our work demonstrates that late infections still represent a risk regarding vertical transmission of PVY in potato. Thus, growers may consider that PVY remains a risk to their crop from planting to harvest, and in the case where tubers are produced for replanting, monitoring, control and protection of the crop is essential during the whole growing season. Anoecia corni 0 1 4 0 1 0 0 0 0 0 1 2 0 0 Anuraphis farfarae 0 0 0 1 0 0 0 0 0 0 0 0 0 0 Cavariella theobaldi 0 0 0 0 0 0 0 0 0 0 1 0 0 0 Eriosoma ulmi 0 1 0 0 0 0 0 0 0 1 0 0 0 0 Hyalopterus pruni 0 1 0 0 0 0 0 0 0 3 1 0 0 0 Myzus sp 0 0 3 0 0 0 0 0 0 3 0 0 0 0 Pterolcallis alni 0 0 0 0 0 0 0 0 0 1 0 1 0 2 Rhopalosiphum minsertum 0 0 0 0 1 0 0 0 0 0 0 0 0 0

  to three published PVY sequences: PVY N 605 (accession number X97895), PVY O 139 (accession number U09509) and PVY NT N H (accession number M95491).

  ni~Binomial [Ni,p], where n is the number of infected plants (or tubers) among a sample of N tested plants (or tubers) and p the probability of infection, with a logit link function. The effects of experimental year, the plant age at infection or exposure to vectors were tested with Wald tests. PVY load in daughter tubers was analysed by considering its natural log and a Generalised Linear Model with a Gaussian distribution to test the effects of experimental year and the plant age at infection with Wald tests. For each statistical model pairwise comparisons and Tukey tests were used to compare and rank the levels of each tested variable. All the statistical analyses were performed with the R software (R core team, 2016).

  For both 2016 and 2017 experiments, at the WNR associated with plot B (28-42 DAP), potato plants reached 30-40 cm. At the following observations of growing stage, plants were in flower bud (plot C (42-56 DAP)), in full flowering (plot D (56-70 DAP)), flowers had fallen (plot E (70-84 DAP)), still well green (plot F (84-98 DAP)) and with a pronounced yellowing (plot G (98-112 DAP)). Then, potato plants were senescent with the drying of stems at the observation carried out on plot H (112-126 DAP).

  For the following WNR applied to plots C to H, 128, 105, 61, 33, 8 and 1 individuals were trapped. In 2017, a total of 1533 aphids were collected throughout the potato cultivation period. During the different WNR (corresponding to exposure of plots B to H) 93, 312, 767, 173, 37, 17 and 21 aphids were trapped, respectively. Whatever the date of the experiment, the higher number of trapped aphids was obtained in plot C (128 and 312 aphids for 2016 and 2017, respective ly) and in plot D (105 aphids for 2016 and 767 for 2017), corresponding to 42-70 DAP. In return, in 2016 this number was much lower in plot G (8 aphids) and plot H (1 aphid), corresponding to 98-126 DAP. In both experiments, Myzus persicae constituted 48.0-80.0% of trapped aphid species according to the considered plot.Monitoring of the percentage of infected plantsIn 2016 field trial, only 3 out of the 258 tested plants (each of the plots D and E contained only 19 plants) were initially PVY-infected. These three plants belonged to the plot A. In 2017, 2 plants (1 from plot A, and 1 from plot F) were found infected at the beginning of the experime nt.The infected status of these plants resulted from seed-borne PVY infections. Data from these PVY-infected plants were not considered in the analyses. Plot A was exposed to natural infections during the whole potato growing season. In 2016, 19 of the 20 randomly selected plants of plot A were infected by PVY at the end of the experime nt (Figure 2a). ELISA performed on plantlets from replanted daughter tubers revealed that potato plants from plots B to H displayed different infection rate according to the WNR period. Indeed, the percentage of infected plants was 35.0% (7/20) in plot B (WNR = 28-42 DAP) and increased steadily to 85.0% (17/20) and 100.0% (19/19) in plot C (WNR = 42-56 DAP) and plot D (WNR = 56-70 DAP), respectively. This rate remained high (90.0%-95.0%) for plots E (18/19; WNR = 70-84 DAP) and F (18/20; WNR = 84-98 DAP) but decreased to low values (10.0%-25.0%) for plots G (5/20; WNR = 98-112 DAP) and H (2/20; WNR = 112-126 DAP). Similar results were noted in 2017, although a low inter-annual variation of the percentage of infected plants was observed between these two biological repetitions (1.60% of the deviance, p=1.11E-06).

  ELISA characterization of PVY present in field infected plants showed that in 2016 plot A was infected by PVY of serotype-N (19/19) while some of these infected plants were also infected by PVY of serotype-O (4 Y N /Y O mixed infected plants). Plots B to H with different WNR periods had different patterns of PVY serotype. Indeed, plots B, G and H were infected by PVY of serotype-N only, whereas plants from plots C, D, E and F were infected either by PVY of serotype i) -N, ii) -N and O or iii) -N, -O and isolate(s) from the unconventional serotype-U

  Nevertheless, the ontogenetic change of plant susceptibility with age should be another factor interfering with virus infection because the same number of vectors has contrasted effects on the rate of infected daughter tubers and PVY load in infected hosts when the age of plant at infection changes. Results in plot A demonstrated this interaction, because a low aphid flight activity as in 2016 (550 individuals) led to a lower percentage of infected daughter tubers and PVY titer, than with a high aphid flight activity as in 2017 (1533 individuals). However, the number of aphids may not be the only factor affecting PVY translocation and accumulation in daughter tubers from infected plants. Indeed, even if in 2016 experiment plots B and E shared a similar number of trapped aphids (54 and 60, respectively), a similar number of trapped M. persicae (34 and 39, respectively), and similar PVY isolates in infected plants, a significa nt difference in PVY translocation and PVY titer in daughter tubers was observed.

Figure 1 :

 1 Figure 1: Schematic representation of field trials and description of procedures applied in field

Figure 2 :

 2 Figure 2: Percentage of PVY-infected potatoes of cv. Bintje in field (a) and under greenhouse

Figure 3 :

 3 Figure 3: Proportion of the different PVY serotypes identified in infected potato plants from

Figure 4 :

 4 Figure 4: Boxplots of the distribution of the percentage of infected daughter tubers resulting

Figure 5 :

 5 Figure 5: Boxplots of the distribution of PVY titer in pooled daughter tubers of all infected

Table 2 :

 2 Sanitary status of daughter tubers in field trials (2016 and 2017).

	Other	8 18 27 10 7 8 1	17 36 39 23 7 4 5
	Total number of trapped aphids	54 128 105 60 33 8 1	93 312 767 173 37 17 21

net removal period Average percentage of infected daughter tubers Percentage of mother plants with 100% infected daughter tubers

		A	0/133	63.2	10.5 (2/19)*
		B	28/42	68.6	43.0 (3/7)
		C	42/56	70.0	11.8 (2/17)
	2016	D E	56/70 70/84	88.3 88.1	42.1 (8/19) 44.4 (8/18)
		F	84/98	67.0	0 (0/18)
		G	98/112	71.6	40.0 (2/5)
		H	112/126	49.4	0 (0/2)
		A	0/133	97.2	75.0 (15/20)
		B	28/42	60.3	18.7 (3/16)
		C	42/56	73.7	25.0 (5/20)
	2017	D E	56/70 70/84	96.3 66.0	60.0 (12/20) 20.0 (4/20)
		F	84/98	36.8	0 (0/10)
		G	98/112	69.0	0 (0/5)
		H	112/126	45.0	0 (0/3)

* : number of plants with 100% infected daughter tubers / total number of PVY-infected plants

ACKNOWLEDGEMENTS

This work was supported by the French seed potato growers (FN3PT/inov3PT) in the context of UMT InnoPlant 2 (http://www.umt-innoplant.fr/). We thank Maryse Guillet (FN3PT/inov3PT, Le Rheu) who kindly provided some serum and monoclonal antibodies for PVY DAS-ELISA. All authors disclose any potential sources of conflict of interest: any interest or relationship, financial or otherwise, that might be perceived as influencing the author's objectivity.

The circles correspond to outliers. For each plot, PVY load in individual daughter tubers was analysed by considering its natural log and a Generalised Linear Model with a Gaussian distribution to test the effects of the tuber and the plant with Wald tests.

Supplementary


Frédérique Larrarte
email: frederique.larrarte@univ-eiffel.fr

Hugues Chollet
email: hugues.chollet@univ-eiffel.fr

Franziska Schmidt
email: franziska.schmidt@univ-eiffel.fr

Mohamed Belmokhtar
email: mohamed.belmokhtar@univ-eiffel.fr

Coraline Wintenberger
email: coraline.wintenberger@cerema.fr

Monitoring of Real Sites affected by Scour: Observation, Analysis and Feedback of Field Data

Keywords: Scour, Bridge, Open channels, Profilers, Bathymetry

Infrastructures of public transport networks can be affected by erosion processes. Detecting this risk is thus a major challenge for sustainable development and land use planning and monitoring systems are promising tools in this context.. The current paper reports the experience of monitoring a bridge in a sand bed river affected by incision. After remembering briefly the site identification process, the choice of the scour parameters to be monitored and the choice of the instrumentation, we present the feedback that we can do after about one year of monitoring with a discussion of the positive and negative aspects of the site and the set-up implemented to measure the scour parameters. We make a focus on the analysis of the measurements that have been done with the statistics that can be made and compare with literature data as well as data from the national French data base on water levels. Finally, we discuss the conclusion that can be obtained for structures managers.

INTRODUCTION

With the development of the public transport networks, guidelines [START_REF] Gauthey | Traité de la construction de Ponts[END_REF] were published to explain the phenomena that can affect structures used to cross rivers and wet areas. Those guides provide design rules and dimensioning laws to engineers and practitioners and they continue to be updated to take account of the new knowledges and technical progress [START_REF] Setra | Technical guide[END_REF][START_REF] Pelaez | Control de la erosion fluvial en puentes[END_REF][START_REF] Hoffmans | Scour Manual[END_REF]May et al., 2002;[START_REF] Dtmr | Bridge Scour Manual[END_REF]Sheppard et al., 2011;Arnason et al, 2012;[START_REF] Setra | Application guide of the technical instruction for the monitoring and maintenance of engineering structures -Foundations in aquatic sites[END_REF]. Researches are still active in many countries [START_REF] Ettema | Flow-Field Complexity and Design Estimation of Pier-Scour Depth: Sixty Years since Laursen and Toch[END_REF][START_REF] Chevalier | A review of Soils, Structures and Hydraulics : Expertise and Applied Research (SSHEAR) project[END_REF][START_REF] Guan | Characterization of horseshoe vortex in a developing scour hole at a cylindrical bridge pier[END_REF][START_REF] Bento | Risk-based methodology for scour analysis at bridge foundations[END_REF], just to give some examples.

Despite of everything, the structures can be affected by erosion processes such as the Coarraze railway (France) embankment destroyed in 2013. Thus detecting the risk of scouring of structures, beds or banks due to natural hazards (floods, extreme hydraulic regimes, dam failures) is a major challenge for sustainable development and land use planning and monitoring can be an interesting tool.

This paper relies to a French research project named SSHEAR [START_REF] Chevalier | A review of Soils, Structures and Hydraulics : Expertise and Applied Research (SSHEAR) project[END_REF] which objective is to improve understanding of the scouring process with innovative observation tools and physical and numerical hydraulic modelling. [START_REF] Larrarte | A French Experience of Continuous Scour Monitoring on a Real Site[END_REF] presented the general approach and [START_REF] Larrarte | Hydraulics and bridges : a French case study of monitoring of a bridge affected by scour[END_REF] detailed the monitoring of an abutment. The current paper reports the experience of monitoring a bridge pier. After remembering briefly the site identification process, the choice of the scour parameters to be monitored and the choice of the instrumentation, we present the feedback that we can do after about one year of monitoring with a discussion of the positive and negative aspects of the site, and the set-up implemented to measure the scour parameters. We make a focus on the analysis of the measurements that have been done with the statistics that can be made, and compare with literature data as well as data from the national French data base on water levels. Finally, we discuss the conclusion that can be obtained for structures managers. Experimental site selection was an important first part of SSHEAR project [START_REF] Larrarte | A French Experience of Continuous Scour Monitoring on a Real Site[END_REF]. Several criteria were defined to select the ones that would be monitored during either brief campaigns or for some longer continuous periods. Several criteria were defined to select the sites that would be monitored during a brief campaign and for some longer continuous periods. These criteria were related to:  representative sites on which scouring phenomena have been observed and are in progress;  river conditions (flow conditions, hydro-morphology, nature of sediment…);  geology and geotechnical parameters (foundations type, bedrock or reinforcement presence…);  documented sites for which data are accessible (geometry, type of support, inspection and monitoring report, bathymetric, geotechnical, hydraulic data, etc.);  rules and regulations that have to be respected in the national context.  site accessibility;  staff and materials safety… An inventory of SSHEAR partners sites affected by scouring phenomena was carried out by the network managers (railway and highway), using geographical information system The selection has then been finalized and restricted to 3 sites [START_REF] Larrarte | A French Experience of Continuous Scour Monitoring on a Real Site[END_REF]. In this paper we focus on a motorway bridge. The monitoring of the embankment site is reported by [START_REF] Larrarte | Hydraulics and bridges : a French case study of monitoring of a bridge affected by scour[END_REF].

SITE SELECTION AND INSTRUMENTATION

Site characteristics

The A71 motorway bridge (owner company: Vinci Autoroutes/Cofiroute) over the Loire is representative of large structures crossing a river in France in an oceanic climate. Moreover, the Loire catchment represents 20 % of the metropolitan area and the A71 motorway is a major highway of France connecting Paris area to central France. At the level of the bridge, the catchment area is equal to 37000 km 2 and the river flows through sedimentary rocks of the Paris basin. A severe incision of the main branch of the river, owing to a combined effect of the groynes for navigation (nineteenth to twentieth centuries) and intense sediment extraction led to exposure of the bedrock in this area. Bank erosion and lateral shifting is also constrained by artificial levees built for flood prevention. On the site, the Loire river flows between a hillside on the right-hand side and a levee on the left-hand side. The floodplain extends over a complete distance of 650 m.

The Loire river shows a range of fluvial patterns from single channel (straight or meandering) to anabranching. At the study site, the channel course is straight with a sinuosity index of 1 [START_REF] Latapie | Modélisation de l'évolution morphologique d'un lit alluvial: exemple de la Loire Moyenne[END_REF] and an island split the channel course downstream the bridge between a main channel on the right hand and secondary channel on the other side (Figure 1). The width of its bed is 300 m and the average slope is 0.00035 m/m [START_REF] Latapie | Modélisation de l'évolution morphologique d'un lit alluvial: exemple de la Loire Moyenne[END_REF]. The bedload of the river is mainly composed of siliceous sediments from sand to gravel particles even if some flint cobbles resulting from the weathering of the calcareous bedrock are present. Thus, the bedload is mainly composed of large grainsize range from 0.4 mm up to 4 mm.

The bridge is approximately 400 m long. This structure is based on 6 supports, 4 of which are located in the riverbed and named P2 to P5 from the right bank to the left bank (Figure 1). Each support (except the abutments C1 and C6) are made up of ellipsoidal columns whose height is about 8 m. The French National database "Banque Hydro" (http://www.hydro.eaufrance.fr/) has a point located 5 km upstream (Orléans). This station records Loire flow rates since 1964. The water level, about 90 m, is presented in the NGF reference. The Nivellement Général de la France (NGF) is a network of altimetric landmarks spread over the French continental metropolitan territory, as well as in Corsica. This network is currently the official levelling network in metropolitan France. Water level and monthly flow rate fluctuations, depicted on Figure 2a, are characteristic of the Loire river hydrologic regime determined by two climatic influences: rainfall coming from the Atlantic Ocean (mainly during winter) or rainy storms in the upper mountainous reaches of Massif Central that occur during spring [START_REF] Dacharry | Les grandes crues historiques de la Loire (The high historical floods of the Loire)[END_REF]. 3) underlying, on one hand, that this is an active site for erosion/deposition and, on an other hand, that punctual surveys are not sufficient to follow the phenomena. The monitoring was thus decided to be implanted close to support P4 (black dashed circles on Figure 3).

Instrumentation

A first major influence on scour is related to hydraulics acting on the structure. A huge consequence can be the generation of scour holes close to the piers. So the choice has been made to have continuous measurements of both hydraulics properties and bathymetry. In parallel to scour monitoring, accelerometers have been installed on the bridge to monitor the sites through the responses of the bridge structure. Indeed, using structures responses makes it possible to monitor the scour at the bottom of the pier [START_REF] Boujia | Using Rocking Frequencies of Bridge Piers for Scour Monitoring[END_REF].

We have tried to look at what exists in countries whose context seems similar to the French hydro-geological context, that ranges from oceanic to Mediterranean climate through mountainous one, if we limit our present study to the metropolitan area, for practical reasons. After a long discussion process to precise monitoring expectations and technical specifications and taking account of the financial constraints, the choice has been to implement:  a Valeport VRS-20 radar level sensor on the bridge to measure the water level relatively to a fix point;  a Kongsberg Dual Axis sonar to scan the bathymetry;  a Teledyne RDI Monitor 1200kHz ADCP to measure the 3D velocity field;  three accelerometers (one tri-axial PCB 629A11 and two mono-axial PCB 393B31).

The sonar and the profiler have been implemented on a raft made of two boards (Figures 4,5a,). The sensors are connected to a laptop and measurements are transmitted to a data server. The international standards and recommendations have been followed in collaboration with sensor suppliers.

RESULTS

The site instrumentation started on October 2020 and was supposed to end 18 months later in order to handle both a full range of annual seasons and 2 examples of the most rainy period of a hydrological year. However, the process has faced various problems from raft cap-sizing to intrusions into the bridge leading to acts of vandalism. Nevertheless, it was possible to acquire a first experience of site monitoring.

Water levels

Figure 6 presents an extract of the water level chronicles recorded with the limnimeter every level every 5 minutes, with the sonar every 60 minutes and data from the banque hydro. It is interesting to notice that the dynamic remain the same for all the data. However a gap can be observed with the banque hydro data, this is attributed to the strong difference between sections. There is no floodplain at the gauging station, the riverbed of 330 m large is constrained by the hillside on the right-hand and the levee on the left-hand side. In addition, the flow is influenced by an old navigation groyne. At the monitored bridge section, on the left-hand side there is 250 m large of floodplain between riverbed and levees. Moreover, island could influence flow levels. It can be noticed that the water level reacts to flow events but with a quite slow behavior attributed to the wide area of the catchment, unlike the highly reactive Aurence watershed [START_REF] Larrarte | Hydraulics and bridges : a French case study of monitoring of a bridge affected by scour[END_REF] where the other site with a continuous monitoring in the SSHEAR project.

Velocity

Figure 7 presents an extract of the water level chronicles, in local reference, recorded with the limnimeter every level every 5 minutes as well as the upper velocity chronicle. Here we call upper velocity the mean value of the velocities measured in a distance between 0.60 and 1.20m from the surface. We can observed that the dynamic of both data are similar with a decrease during the first 15 days and fluctuations after, the almost steady water level correspond to low water level of the end of the hydrological year.

Dynamic behavior of the structure

In order to monitor the scour depth and the hydraulic speed, a solution is to measure their consequences on the dynamic behavior of the bridge: indeed, many researchers show the interest of vibration-based monitoring in order to monitor external phenomenon as scour [START_REF] Peter Carden | Vibration based condition monitoring: A review[END_REF][START_REF] Foti | Influence of foundation scour on the dynamic response of an existingbridge[END_REF]. In our case study, the accelerations of both the deck and the pier have been recorded for a long time (over 320 days), and can be accessed remotely. These time series (Figure 9) are the answer on one vibration recording, and are directly linked to the scour conditions. Many information are available through a frequency analysis.

By averaging signals all over a day, the subspace procedure for eigenfrequency extraction showed that a 3 rd subspace let us monitor the scour phenomenon. In fact, this space is close to the bridge pier responses in Figure 10 and some natural frequencies change over time (see Figure 12).

When combining these results with the other monitoring data (from the other monitoring techniques), the frequency changes due to scour and/or hydraulic flow can be discriminated from the changes due to a damage of the structure. Nevertheless, if we consider that the bridge remains -during a given time-in the same structural A new method developed by Belmokhtar et al. ( 2021) makes it possible to monitor the natural frequencies every day. In Figure 10, Fast Fourier Transform has been applied to the time series showed (Figure 9). health, this procedure makes it possible to calculate and monitor the scour depth at the supports of the bridge.

CONCLUSIONS

Infrastructures of public transport networks can be affected by erosion processes thus detecting this risk is a major challenge for sustainable development. Monitoring of the affected structures could help to prevent scour related failures. The current paper reports the experience of monitoring a real bridge located near a French city. The in situ monitoring device presented in this paper shows that such complex instrumentation is now possible thanks to the progress of sensors. The apparatus allow following the various hydraulic conditions (flow depth, velocity profile…), the local response of the sediments (scour/erosion or deposit) and the dynamical behavior of the bridge (vibration measurements) with a high acquisition frequency, especially in comparison with annual survey.

Other teachings can be drawn from this project. First of all, whatever its technology, a sensor will not give any results if it can not be implemented and maintained properly. Doing measurements in a river proved means that all the set-up and instrumentation has to support drought and floods. Moreover, human interactions can disrupt the experimental campaign. In this site, a low cost raft has been selected to minimize the cost as well as to not draw too much attention to the sensors.

Anyway, these first observations are very encouraging for the future data exploitation as flow characteristic and dynamical properties of the bridge can be extracted from the measurements. The crossed study of all these parameters in relationship with the specific site environment characteristics will then allow increasing our knowledge of in situ scour processes and their link to structural dynamical properties of structure.

Proceedings of the 39 th IAHR World Congress 19-24 June 2022, Granada, Spain ©2022 IAHR. Used with permission / ISSN-L 2521-7119

FUNDING

This research was funded by Agence Nationale de la Recherche (French National Research Agency) within the project SSHEAR ANR-2014-CE03-0011.

Figure 1 .

 1 Figure 1. A71 motorway double viaduct close to Orléans (France) over Loire river

  The hydrographic year lasts from September to August with floods occurring during winter high flow stage or in spring and low flow stage during summer. Strong fluctuations are seen but cannot explain the observed evolution of the bathymetry in a simple way. The monthly average flow rate fluctuations of Loire river at Orléans station are detailed on Figure 2b that shows strong inter annual variations. a) Water level and flowrate chronicles b) Monthly flow rate fluctuations (Banque hydro data) 5 km upstream of the bridge.

Figure 2 .

 2 Loire river (Orléans) site characteristics. Cerema has a long term partnership with the highway company Vinci Autoroutes/Cofiroute and evaluates scour on various bridges for many years. Sonar bathymetries were carried out in 1993, 2004 and 2011 and significant local erosions in the vicinity of bridges piers were identified especially on the left bank of support P4. But the next survey, in 2016, has shown accretion phenomenon close to these pier (Figure

Figure 3 .

 3 Figure 3. Results of comparison between 3 bathymetric surveys (2004, 2011 and 2016) near the support P4 (see Figure 1) of the A71 highway viaduct over the Loire river. Black dashed circles: expected place of the monitoring set-up.

Figure 4 .

 4 Figure 4. General view of bridge and instrumentation location. The first accelerometer has been installed in the deck (internal walls of the box-girder) and is recording the vibrations of the bridge in the two directions (vertical and in the direction of the hydraulic flow). The second ones are installed at the top of pier P4, and monitors the accelerations of the pier in the same directions (Figure 4, 5b, 5c).

Figure 6 .

 6 Figure 6. Water level chronicles (in local reference).

Figure 7 .

 7 Figure 7. Water level (in local reference) and upper velocity chronicles.

Figure 8 .

 8 Figure 8. Example of observed bathymetric.

Figure 9 :

 9 Figure 9: Example of the recorded time series (30 oct. 2020, 16ℎ44).

Figure 10 :

 10 Figure 10: FFT applied to the 4 recorded time series.

Figure 12 :

 12 Figure 12: Evolution of natural frequencies every day with method developed by Belmokhtar et al. (2021).

  Proceedings of the 39 th IAHR World Congress 19-24 June 2022, Granada, Spain

©2022 IAHR. Used with permission / ISSN-L 2521-7119

  Proceedings of the 39 th IAHR World Congress 19-24 June 2022, Granada, Spain

©2022 IAHR. Used with permission / ISSN-L 2521-7119

ACKNOWLEDGEMENTS

The authors thank the members of GERS-SRO unit from Univ Gustave Eiffel Marne-la-Vallée (France) and the members of the "Groupe Risques Inondations -Surveillance des Ouvrages et des Milieux" of the Direction territoriale Normandie-Centre/Agence de Blois of the Cerema for their contribution to the experiments and Sidoine de la Roque and Souleymane Diallo of Vinci Autoroute for their support related to Loire river experiments.