X Guan Ab

S Essakhi

H Laloue

P Nick

C Bertsch

J Chong

Mining new resources for grape resistance against Botryosphaeriaceae: a focus on Vitis vinifera subsp. sylvestris

Keywords: botryosphaeria dieback, defence responses, grapevine, grapevine trunk diseases, Vitis vinifera subsp. sylvestris

Botryosphaeria dieback is an important grapevine trunk disease with global impact. Susceptibility differences between grape varieties manifest as different expression of canopy symptoms in the field. However, the cause of these dieback symptoms and their relation with wood necrosis remain only partially understood. As a first step towards future strategies for resistance breeding, wood necrosis was investigated over a large selection of the Vitaceae family members following artificial inoculation of the Botryosphaeriaceae fungi Neofusicoccum parvum and Diplodia seriata into woody internodes. Large variation of resistance levels was found, with good performance in several accessions from V. vinifera subsp. sylvestris, the ancestor of cultivated grapevine. To get insight into the mechanisms of this apparent resistance, expression of defence genes was studied in V. vinifera cv. Chardonnay, Gew€ urztraminer and different V. vinifera subsp. sylvestris genotypes, in both green and necrotic areas of inoculated woods. Resistance to Botryosphaeriaceae in V. vinifera subsp. sylvestris correlated with earlier and higher induction of some defence genes, both in green and necrotic wood. Moreover, leaves of several V. vinifera subsp. sylvestris accessions were also less susceptible to necrosis induced by treatment with a culture filtrate of Botryosphaeriaceae, compared to commercial cultivars of V. vinifera. The results show that V. vinifera subsp. sylvestris provides interesting genetic resources for breeding new varieties with enhanced resistance to botryosphaeria dieback.

Introduction

Botryosphaeria dieback, esca and eutypa dieback are three economically important grapevine trunk diseases causing severe yield reduction and affecting the viability of plants in vineyards worldwide. The incidence of trunk diseases has increased considerably over the past few decades and, so far, no efficient curative treatment is available to control these diseases [START_REF] Grosman | Maladies du bois de la vigne. Synth ese des dispositifs d'observation au vignoble, de l'observatoire 2003-2008 au r eseau d' epid emiosurveillance actuel[END_REF][START_REF] Bruez | Overview of grapevine trunk diseases in France in the 2000s[END_REF]. Grapevine infection by fungal trunk pathogens results in the formation of cankers and wood discolouration. It is believed that infection takes place primarily via pruning wounds, although contamination of nursery stock does occur [START_REF] Bertsch | Grapevine trunk diseases: complex and still poorly understood[END_REF].

Botryosphaeria dieback, one of the major trunk diseases, is associated with the development of fungi from the Botryosphaeriaceae family, especially Neofusicoccum parvum and Diplodia seriata, in the wood of diseased grapevines. Botryosphaeriaceae species are well known pathogens causing dieback in apples, pine trees and grapevines. These fungi are latent pathogens in many woody hosts and are characterized by a quiescent passive life phase, followed by an active pathogenic phase [START_REF] Slippers | Botryosphaeriaceae as endophytes and latent pathogens of woody plants: diversity, ecology and impact[END_REF]. There are more than 22 different species in the Botryosphaeriaceae family, which have been identified in vineyards from different countries based on morphological and taxonomic studies, as well as on analysis of nucleotide sequences from multiple genes (for reviews see [START_REF] Urbez-Torres | The status of Botryosphaeriaceae species infecting grapevines[END_REF] and [START_REF] Bertsch | Grapevine trunk diseases: complex and still poorly understood[END_REF]). Species of Botryosphaeriaceae infecting grapevines can be divided into three different virulence rankings and Neofusicocum spp. belong to the highly virulent species, whereas Diplodia spp. are moderately virulent [START_REF] Urbez-Torres | The status of Botryosphaeriaceae species infecting grapevines[END_REF]. The first description of botryosphaeria dieback disease of grapevine was reported in 1974 in Hungary and was associated with Diplodia mutila [START_REF] Lehoczky | Black dead-arm disease of grapevine caused by Botryosphaeria stevensii infection[END_REF]. This disease was originally called 'black dead arm' (BDA) and was characterized by diffuse chlorosis of the leaves followed by wilting and black streaking of the wood in the xylem [START_REF] Lehoczky | Black dead-arm disease of grapevine caused by Botryosphaeria stevensii infection[END_REF]. It was shown thereafter that Botryosphaeriaceae species are associated with a broad number of different grapevine trunk disease symptoms including bud necrosis, shoot dieback, leaf spots, fruit rots, vascular discol-ouration of wood and perennial cankers, leading to a loss in economic production, and eventually culminating in the death of the whole plant.

Urbez-Torres (2011) thus proposed the disease name 'botryosphaeria dieback' to include all the symptoms caused by Botryosphaeriaceae species on grapevine. It is known that Botryosphaeriaceae produce cell wall-degrading enzymes and phytotoxic metabolites whose synergistic action may cause the disease symptoms of botryosphaeria dieback (dead spurs, shoot dieback, stunted shoots) [START_REF] Andolfi | Phytotoxins produced by fungi associated with grapevine trunk diseases[END_REF]. However, even when pathogens do infect the vascular system successfully, this does not necessarily lead to the expression of dieback symptoms in the first season. Moreover, it can happen that in the subsequent year, the same vine shows reduced or even no symptoms at all [START_REF] Bertsch | Grapevine trunk diseases: complex and still poorly understood[END_REF]. In the search for fungal toxins, four dihydroisocoumarins (mellein, 4-hydroxymellein, 7-hydroxymellein and 4,7-dihydroxymellein) were isolated from a culture filtrate of D. seriata [START_REF] Andolfi | Phytotoxins produced by fungi associated with grapevine trunk diseases[END_REF]. In addition, analysis of the metabolites produced by 13 isolates of N. parvum revealed that these compounds belong to four different families (dihydrotoluquinones, epoxylactones, dihydroisocoumarins and hydroxybenzoic acids; [START_REF] Abou-Mansour | Phytotoxic metabolites from Neofusicoccum parvum, a pathogen of Botryosphaeria dieback of grapevine[END_REF]. A recent study also highlighted the role of secreted proteins for the cytotoxicity of culture filtrates from N. parvum [START_REF] B Enard-Gellon | Toxicity of extracellular proteins from Diplodia seriata and Neofusicoccum Plant Pathology (2015) parvum involved in grapevine Botryosphaeria dieback[END_REF].

Studies on botryosphaeria dieback have been conducted at multiple levels: in naturally infected vineyards, under controlled conditions using greenhouse cuttings, and using in vitro grapevine models that were artificially infected [START_REF] Bruez | Overview of grapevine trunk diseases in France in the 2000s[END_REF][START_REF] Travadon | Susceptibility of cultivated and wild Vitis spp. to wood infection by fungal trunk pathogens[END_REF][START_REF] Ram Irez-Suero | Extracellular compounds produced by fungi associated with Botryosphaeria dieback induce differential defence gene expression patterns and necrosis in Vitis vinifera cv. Chardonnay cells[END_REF]. The physiological and histochemical alterations associated with Botryosphaeriaceae infection include blocked xylem vessels by induction of tyloses or production of gums (such as black goo), as well as by physical impedance by the pathogen itself [START_REF] Bertsch | Grapevine trunk diseases: complex and still poorly understood[END_REF]. Moreover, pathogen-derived macromolecules can plug the pits, reducing translocation of water [START_REF] Edwards | Impairment of grapevine xylem function by Phaemoniellea chlamydospora infection is due to more than physical blockage of vessels with 'goo[END_REF][START_REF] Bertsch | Grapevine trunk diseases: complex and still poorly understood[END_REF]. Not surprisingly, the expression of symptoms caused by Botryosphaeriaceae in grapevines is accentuated by water stress [START_REF] Niekerk | Influence of water stress on Botryosphaeriaceae disease expression in grapevines[END_REF]. Grapevines exposed to water stress developed more severe disease symptoms (lesion lengths) in response to inoculation with Botryosphaeriaceae.

Defence responses to trunk diseases have been studied at the leaf level in the case of esca, another major grapevine trunk disease. The main causal agents of esca are the tracheomycotic agents Phaemoniella chlamydospora and Phaeoacremonium aleophilum, as well as several basidiomycete species, with Fomitiporia mediterranea being the most common [START_REF] Bertsch | Grapevine trunk diseases: complex and still poorly understood[END_REF]. Symptoms associated with esca are brown wood streaking and leaf discolouration resulting in a 'tiger stripe' appearance [START_REF] Surico | Older and more recent observations on esca: a critical overview[END_REF]. The leaves of esca-affected plants are characterized by high tannin contents [START_REF] Valtaud | Systemic damage in leaf metabolism caused by esca infection in grapevines[END_REF]. Resveratrol and other phenolic compounds could also be detected in leaves and berries before the appearance of esca symptoms [START_REF] Letousey | Early events prior to visual symptoms in the apoplectic form of grapevine esca disease[END_REF] and were accompanied by induction of defence genes such as PAL and STS (encoding phenylpropanoid biosynthesis enzymes), and the accumulation of pathogenesis-related (PR) proteins [START_REF] Letousey | Early events prior to visual symptoms in the apoplectic form of grapevine esca disease[END_REF]. At the wood level, analysis of the proteome of black-streaked trunks lends further support for the induction of proteins involved in defence [START_REF] Magnin-Robert | Proteomic insights into changes in grapevine wood in response to esca proper and apoplexy[END_REF]. Defence responses to botryosphaeria dieback were less studied. However, PR proteins, members of the enzymatic antioxidant system, total phenolics and stilbenes were found to be more abundant in the brown-striped wood of botryosphaeria dieback-affected grapevines [START_REF] Spagnolo | Differential responses of three grapevine cultivars to Botryosphaeria dieback[END_REF].

Introgression of resistance factors from wild grape into cultivated varieties has been a successful strategy to contain important grapevine diseases such as downy or powdery mildews [START_REF] Eibach | The use of molecular markers for pyramiding resistance genes in grapevine breeding[END_REF]. However, so far, no genetic resources for a similar strategy have been identified in the case of fungal trunk diseases. In the field, sensitivity of Vitis vinifera against fungal trunk diseases seems to vary according to the cultivar [START_REF] Grosman | Maladies du bois de la vigne. Synth ese des dispositifs d'observation au vignoble, de l'observatoire 2003-2008 au r eseau d' epid emiosurveillance actuel[END_REF][START_REF] Bruez | Overview of grapevine trunk diseases in France in the 2000s[END_REF], suggesting that the resistance level might be under genetic control. For instance, Chardonnay infected by the causal agents of trunk diseases presents fewer leaves with symptoms compared to other cultivars. However, so far, no totally resistant cultivar has been reported [START_REF] Surico | Older and more recent observations on esca: a critical overview[END_REF]. Several studies with artificial inoculation of cultivated grapevines or rootstocks with N. parvum and D. seriata demonstrated a differential susceptibility of different accessions, but again no complete qualitative resistance was found [START_REF] Travadon | Susceptibility of cultivated and wild Vitis spp. to wood infection by fungal trunk pathogens[END_REF][START_REF] Billones-Baaijens | Susceptibility of common rootstock and scion varieties of grapevines to Botryosphaeriaceae species[END_REF].

In an attempt to identify novel genetic resources for resistance against trunk diseases, the current study conducted a broad screen to evaluate the susceptibility levels of a large selection of accessions from the Vitaceae family to N. parvum and D. seriata. The leaf and wood symptoms of V. vinifera subsp. sylvestris, the ancestor of V. vinifera, were also studied. Differential defence gene expression was examined in wood symptoms in a variety of accessions.

Materials and methods

Plant material

The members of the Vitaceae family used in this study are presented in Figure 1.

Inoculation experiments were conducted on detached canes, which consisted of 0Á8 9 10 cm sections between two nodes of 1-year-old dormant lignified canes collected from two non-Vitis genera (Parthenocissus tricuspidata and Ampelopsis japonica), one subgenus (Muscadinia rotundifolia cv. Regale), four Vitis species (V. rupestris, V. amurensis, V. riparia, V. aestivalis), 16 V. vinifera commercial cultivars (Cabernet sauvignon, Chardonnay, Chasselas, Chenin, Colombard, Danlas, Fantasy seedless, Gamay, Gew€ urztraminer, Grenache, Muscat d'Alexandrie, Pinot noir, Riesling, Savagnin blanc, Sultanine, Trousseau) and two rootstocks (41B (V. vinifera 9 V. berlandieri), 110R (V. berlandieri 9 V. rupestris). All accessions were obtained from a collection from INRA Colmar, France (December 2012and 2013, February 2014). Inoculation experiments with V. vinifera subsp. sylvestris were conducted on 1-year-old internodes (December 2012 and 2013) collected from V. vinifera subsp. sylvestris accessions Ke 33, Ke 53, Ke 74, Ke 83, Ke 84, Ke 94, Ke 95 and H€ o 29. The Ke accessions originate from the largest sylvestris population in Germany at the Ketsch peninsula, and the H€ ordt accession from an isolated individual in the H€ ordt alluvial forest (Upper Rhine valley, Rheinland-Pfalz). These accessions are part of a complete genetic copy for the sylvestris genotypes still available in Germany that was established for an ex situ conservation project in the Botanical Garden of the Karlsruhe Institute of Technology, and characterized genetically by microsatellite markers [START_REF] Nick | Sch€ utzen und n€ utzenvon der Erhaltung zur Anwendung. Fallbeispiel Europ€ aische Wildrebe[END_REF].

For the study of leaf response to culture filtrates of N. parvum and D. seriata, the third to sixth fully expanded leaves beneath the apex from the above-cited accessions were collected in May 2013 and May 2014.

Fungal isolates and inoculation experiments

Diplodia seriata strains 99.7 (Rhône-Alpes, France) and 98.1 (Pyr en ees Orientales, France) have been described by [START_REF] Larignon | Observation on Black Dead Arm in French vineyards[END_REF]. Neofusicoccum parvum strain Bt 67 and N. parvum Bourgogne S-116 were isolated from vineyards in Estremadura (Portugal) and Bourgogne (France), respectively. All strains were grown on solid medium in Petri dishes containing 26Á5 g L À1 potato dextrose broth (PDB; Laboratorios Conda) and 15 g L À1 Bacto agar (Kalys SA Vitro) at 26°C in the dark.

For wood inoculation experiments, internodes were collected from dormant canes. A power drill no. 5 (Bosch PSB 500R) was used to wound the woody stems (5 mm in diameter, 3 mm in depth) at the centre point between nodes. Cuttings were inoculated by filling the wound with a 6 mm diameter plug collected from a 14-day-old fungal culture on potato dextrose agar (PDA), and then sealing this inoculation site with Parafilm. Control internodes were mock inoculated with plugs of non-colonized PDA. After inoculation, detached internodes were incubated in saturating humidity at 28°C in darkness. After 21 days of incubation for D. seriata 98.1 and 7-14 days in the case of N. parvum Bt 67, the stems were debarked and necrotic areas were quantified from digital images using IMAGEJ software (http://imagej.nih.gov/ij/). The difference in sampling time is due to a slower growth of D. seriata compared to N. parvum in the wood.

To validate the inoculation method used in this work, the agar plug inoculation method was compared with the suspension mycelium inoculation developed by [START_REF] Travadon | Susceptibility of cultivated and wild Vitis spp. to wood infection by fungal trunk pathogens[END_REF]. To produce the starter culture, 10 plugs of 2 9 2 cm from a 7day culture on PDA were inoculated in a 250 mL Erlenmeyer flask containing 100 mL bacteriological malt extract and incubated at 27°C and 150 rpm for 3 days. Agar with mycelia attached was collected into a 50 mL Falcon tube and malt extract medium was added to reach a final volume of 20 mL. Five millilitres of glass beads with a diameter of 3 mm were added into the suspension mycelia and the tube was agitated with a vortexer at full speed to disperse the mycelia. Two millilitres of this homogenized starter culture were then inoculated into a 125 mL Erlenmeyer flask containing 40 mL of bacteriological malt extract. After incubation at 28°C and 150 rpm for 3 days, the entire 40 mL liquid culture was homogenized and the concentration of mycelial fragments, which were primarily < 200 lm in length, was estimated with a haemocytometer. The final concentration of inoculum was adjusted with sterile water to 2 9 10 5 fragments mL À1 .

For the leaf treatment with fungal extracellular compounds, 4 9 2 cm agar plugs with 10-day-old fungi grown on PDA were cultured into 250 mL malt extract (20 g L À1 ) liquid cultures in 1 L Erlenmeyer flasks at 220 rpm, 28°C in darkness. After 14 days, fungal culture medium was centrifuged for 8 min at 15 000 g, 25°C, and the supernatant was collected and filtered through membranes with 0Á20 lm pore size (Sartorius Stedim Biotech GmbH) to eliminate spores and sterilize the solution. As it was previously shown that the quantity of extracellular proteins reflects the fungal biomass (M. B enard-Gellon, Universit e de Haute-Alsace, Colmar, France, personal communication) and to ensure that the different culture filtrates were comparable, total proteins were quantified in the different experiments by measuring the 280 nm absorbance with a NanoDrop ND-1000 spectrophotometer (Thermoscientific).

Leaf discs of 1 cm diameter were excised using a cork borer and placed on wet filter paper in Petri dishes with the adaxial side up. Before immersion into sterilized fungi culture filtrate solution, the leaf discs were washed with 70% ethanol for 30 s, and rinsed in water. Subsequently, leaf discs were immersed in fungi culture filtrate solution for 24 h, at 25°C in darkness. Necrosis was monitored for three independent experimental series of 5-10 leaf discs for each genotype.

Measurement of necrosis in wood and leaves

For experiments with all the accessions from the Vitaceae family, the necrotic area and the total internode area were quantified using IMAGEJ software (http://imagej.nih.gov/ij). Specifically, after setting the scale for each image, the 'threshold colour' function was used to highlight the necrotic areas by adjusting 'hue and brightness' parameters. Then the wand (tracing) tool (setup: mode, 8-connected; tolerance, 2Á0) and 'ROI manager' were used to precisely select the individual necrotic area from each internode. The percentage of internode necrotic area was calculated by dividing the necrotic area by the total internode area. Data were obtained for three independent experimental series of 7-8 internodes measured for each genotype. For experiments with V. vinifera cvs Chardonnay, Gew€ urztraminer and subsp. sylvestris, the necrotic areas were relatively smaller than in the inoculation experiments with the Vitaceae family. The percentage of internode necrotic area in inoculated wood was thus divided by the percentage of internode necrotic area in controls inoculated with PDA. The results are expressed in relative units. For experiments using leaf discs, the percentage of necrotic area was calculated for each leaf disc as described for the woody internodes.

Statistical analysis

The results obtained for wood inoculation were compared for each genotype by using a multifactorial ANOVA followed by a multiple comparison of means using Duncan's test (at P ≤ 0Á05, STATGRAPHICPLUS; Manugistics, Inc.).

Gene expression analysis by real-time quantitative RT-PCR

After inoculation, small pieces of internode wood were frozen in liquid nitrogen at 12 h, 3 days and 7 days post-inoculation (pi) for N. parvum Bt 67, and 12 h, 3 days and 21 days pi for D. seriata 98.1. The green wood section (GW) and the necrotic wood section (NW) immediately surrounding the necrosis were separated. Total RNA was extracted using the RNeasy Plant Mini kit (QIAGEN) following the manufacturer's instructions and RNA concentration was determined with a Qubit fluorometer (Invitrogen). One microgram of total RNA was incubated with one unit of RNase-free DNase I (Euromedex) for 30 min at 37°C according to the manufacturer's instructions. cDNA was synthesized from RNA with the SuperScript II reverse transcriptase (Invitrogen).

Quantitative PCR was performed on the CFX 96 Real Time PCR System with a C1000 thermal cycler (Bio-Rad). Primers described in Ram ırez-Suero et al. (2014) were used for phenylalanine ammonia lyase (PAL), the first enzyme of the phenylpropanoid pathway; stilbene synthase (STS) and resveratrol Omethyltransferase (ROMT), two enzymes involved in the synthesis of stilbene phytoalexins; superoxide dismutase (SOD), involved in scavenging of superoxide; lipoxygenase (LOXC), the first committed enzyme of the jasmonic acid pathway; the pathogenesis-related proteins PR1 and PR6; and HSR1, a cell death marker (B ezier et al., 2002).

Two biological replicates each comprising three technical replicates were conducted for each gene in a total volume of 20 lL containing 10 lL SYBR Green MasterMix (Euromedex), 1 lL each primer at 10 lM and 10 ng cDNA, with the following thermal cycling conditions: 3 min at 95°C; then 40 cycles of 15 s at 95°C, 15 s at 60°C, 20 s at 72°C, 10 s at 77°C. The specificity of the individual PCR amplification was checked using a heat dissociation curve from 55 to 95°C following the final cycle of the PCR and by sequencing the final PCR products. The results obtained for each gene of interest were normalized to the expression of two reference genes (VvACT1 (AF369524) and VvEF1a (CB977561)) and relative expression (fold induction) compared to mock-inoculated controls was calculated as described by [START_REF] Pfaffl | A new mathematical model for relative quantification in real-time RT-PCR[END_REF]. Mean values and standard deviations were obtained from three technical and two biological replicates.

Results

Susceptibility of members of the Vitaceae family to N. parvum and D. seriata

In preparation for this study, two inoculation methods were compared, i.e. infection using a suspension of mycelia (adapted from [START_REF] Travadon | Susceptibility of cultivated and wild Vitis spp. to wood infection by fungal trunk pathogens[END_REF] and infection using a colonized agar plug. Inoculation with the agar plug produced lower variability compared to inoculation with the mycelial suspension, as seen from smaller standard errors between technical replicates (Fig. 2). However, inoculation with the mycelial suspension produced a somewhat higher spread of necrosis. This may be due to the faster spreading rate in the vascular tissues of fungi grown in liquid medium compared to the fungi grown on solid agar plates.

It was therefore decided to use agar plug inoculation to compare susceptibility across different members of the Vitaceae family after inoculation into 1-year-old detached canes. Necrosis was quantified 7-21 days after inoculation with N. parvum Bt67 and D. seriata 98.1. Although no genotype was completely resistant to either N. parvum (Fig. 3a) or D. seriata (Fig. 3b), the necrotic area was significantly different (P < 0Á05) and allowed classification of the accessions into four levels of susceptibility.

With respect to inoculation with N. parvum (Fig. 3a), Parthenocissus tricuspidata was the least susceptible among the tested genotypes. Some genotypes, e.g. Muscadinia rotundifolia cv. Regale and the 110R rootstock, known for their resistance to other grapevine pathogens, showed high levels of susceptibility. Grapevine cultivars cultivated in the French Jura, known for a high incidence of canopy symptoms in vineyards (V. vinifera cv. Trousseau, V. vinifera cv. Savagnin blanc, V. vinifera cv. Colombard [START_REF] Grosman | Maladies du bois de la vigne. Synth ese des dispositifs d'observation au vignoble, de l'observatoire 2003-2008 au r eseau d' epid emiosurveillance actuel[END_REF]), also showed a serious level of necrosis after N. parvum inoculation. Interestingly, some cultivars like V. vinifera cv. Grenache, characterized by a low incidence of esca/BDA canopy symptoms in vineyards, in this experiment showed a high level of susceptibility to N. parvum. Finally, V. vinifera cv. Chardonnay, Cabernet sauvignon and Pinot noir were among the less susceptible genotypes.

Similarly for inoculation with D. seriata 98.1, no completely resistant genotype was identified (Fig. 3b). Again, P. tricuspidata showed the smallest necrosis surface, as for N. parvum inoculation. Together with V. vinifera cv. Fantasy seedless and Ampelopsis japonica, this accession fell into the less susceptible group. The accessions 41B and 110R, which have been used as disease-resistant rootstocks, showed a high level of necrosis after D. seriata inoculation. Again, the cultivars from the French Jura, which are known for a high incidence of canopy symptoms in vineyards (V. vinifera cv. Trousseau, V. vinifera cv. Savagnin blanc, V. vinifera cv. Chenin, V. vinifera cv. Colombard) also exhibited a high level of susceptibility to D. seriata inoculation. Interestingly, A. japonica that had been highly susceptible to N. parvum, showed a low level of susceptibility to D. seriata.

Susceptibility of V. vinifera subsp. sylvestris to N. parvum and D. seriata

In the next step, the study was extended to V. vinifera subsp. sylvestris, the ancestor of cultivated grapevine. After inoculation of detached canes with N. parvum, all of the tested V. vinifera subsp. sylvestris accessions were significantly less susceptible than V. vinifera cvs Chardonnay and Gew€ urztraminer (Fig. 4a). Among them, the sylvestris genotypes Ke 33 and Ke 95 were ranked as the less susceptible genotypes. In terms of inoculation with D. seriata, the less susceptible genotypes were V. vinifera subsp. sylvestris H€ o 29 and V. vinifera subsp. sylvestris Ke 83 and Ke 84 (Fig. 4b). Surprisingly, V. vinifera cv. Gew€ urztraminer, which had been found to be sensitive to N. parvum, exhibited a low level of necrosis upon inoculation with D. seriata (similar to Ke 94), whereas Chardonnay was significantly more susceptible. The sylvestris Ke 74, which had performed relatively well under challenge by N. parvum, exhibited the highest level of susceptibility to D. seriata, similar to V. vinifera cv. Chardonnay.

Defence gene expression in inoculated internodes

To determine whether the lower susceptibility observed in some V. vinifera subsp. sylvestris accessions is associated with a differential induction of defence responses, four sylvestris accessions with good performance under inoculation of both pathogens were selected (H€ o 29, Ke 33, Ke 53 and Ke 83) along with two cultivars (Chardonnay and Gew€ urztraminer). The expression of several defence genes was evaluated by quantitative RT-PCR in internodes from detached canes 12 h, 3 days and 7 days post-N. parvum inoculation (Fig. 5a), and 12 h, 3 days and 21 days post-D. seriata inoculation (Fig. 5b), both in necrotic wood and in green wood surrounding the necrotic lesion. Following inoculation with both fungi, defence gene induction began as soon as 12 h pi, and was maximal at 3 days pi (Fig. 5).

The expression of two genes, SOD and HSR1, correlated with susceptibility to both fungi. The expression of SOD was significantly higher both in green and necrotic wood in V. vinifera cvs Chardonnay 12 h pi with D. seriata and Gew€ urztraminer 3 days pi with N. parvum. The expression of VvHSR1, a cell death marker, was also more stimulated in V. vinifera cv. Chardonnay 12 h and 3 days pi with D. seriata (Fig. 5b).

After N. parvum inoculation, the expression of STS was activated earlier (from 12 h pi) and to a higher extent in the resistant genotypes compared to a later activation in the sensitive genotypes (Fig. 5a). However, earlier expression of STS was not observed in the resistant genotypes following D. seriata inoculation (Fig. 5b). LOXC expression was also stimulated earlier (from 12 h pi with N. parvum) in the resistant V. vinifera subsp. sylvestris genotypes compared to V. vinifera cv. Chardonnay or Gew€ urztraminer and again this was not found with D. seriata. The overall pattern of PR protein expression did not correlate well with the resistance levels after infection with both fungi, although there was a trend for an earlier expression of PR6 12 h after N. parvum inoculation in V. vinifera subsp. sylvestris, except for Ke 53 (Fig. 5a).

Necrosis symptoms on leaf discs treated with culture filtrate of N. parvum and D. seriata It is believed that Botryosphaeriaceae produce phytotoxic metabolites that could be translocated via the xylem sap and responsible for the canopy disease symptoms, including wilting and chlorosis of the leaves [START_REF] Bertsch | Grapevine trunk diseases: complex and still poorly understood[END_REF]. The necrotic response to extracellular compounds produced by N. parvum and D. seriata was therefore examined in leaf discs of the selected four sylvestris accessions compared to the cultivars Chardonnay and Gew€ urztraminer. Foliar discs were incubated with a liquid culture filtrate from two N. parvum (Bt 67 and Bourgogne S-116) and two D. seriata (98.1 and 99.7) strains. There was no significant difference in leaf necrosis among the different sylvestris accessions; however, the necrosis of cv. Gew€ urtztraminer treated with extracellular compounds from D. seriata 99.7, and of cv. Chardonnay treated with culture filtrate from N. parvum Bourgogne S-116, were significantly higher compared to the other genotypes (Fig. 6). Overall, the two V. vinifera subsp. sylvestris accessions Ke 33 and Ke 53 developed smaller necrosis after treatment with Botryosphaeriaceae extracellular compounds. (Duncan, 1955). The genotypes are divided into four groups by dotted lines according to their susceptibility.

Discussion

In an attempt to identify potential sources for resistance breeding, this study screened the susceptibility to Botryosphaeriaceae across different members of the Vitaceae, with a focus on V. vinifera subsp. sylvestris. The results show that genetic variability exists in the response to both N. parvum and D. seriata, although none of the genotypes showed complete resistance. In general, N. parvum was found to be more aggressive than D. seriata after artificial inoculation of internodes. This observation is in agreement with the results obtained by [START_REF] Amponsah | Identification, potential inoculum sources and pathogenicity of botryosphaeriaceous species associated with grapevine dieback disease in New Zealand[END_REF], indicating that N. parvum produced longer lesions than D. seriata. Also [START_REF] Martos | Production of phytotoxic metabolites by five species of Botryosphaeriaceae causing decline on grapevines, with special interest in the species Neofusicoccum luteum and N. parvum[END_REF] showed a greater phytotoxic activity of culture filtrate from N. parvum compared to D. seriata on leaves of V. vinifera cv. Tempranillo, and Ram ırez-Suero et al.

(2014) further reported that culture filtrate from N. parvum Bourgogne S-116 is more aggressive than culture filtrate from D. seriata 98.1 on grapevine calli. Although none of the genotypes was completely resistant to any of the pathogens, two sylvestris genotypes performed best with respect to reduced necrosis. For N. parvum, genotype Ke 33 showed the least necrosis symptoms, whereas genotype H€ o 29 was the most resistant to D. seriata (Fig. 4). Also, after treatment with the Botryosphaeriaceae extra-cellular compounds, two sylvestris genotypes, Ke 33 and Ke 53, produced the smallest necrotic areas.

Concerning rootstock sensitivity to Botryosphaeriaceae, 110R was more sensitive to both N. parvum and D. seriata, whereas 41B was relatively less susceptible to N. parvum but was highly susceptible to D. seriata. The results are in agreement with the study of [START_REF] Billones-Baaijens | Susceptibility of common rootstock and scion varieties of grapevines to Botryosphaeriaceae species[END_REF] showing that rootstocks generally produced significantly longer lesions than scions. Rootstocks 110R and 140 Ruggeri were also greatly affected by fungi associated with Petri disease and esca [START_REF] Gramaje | Field evaluation of grapevine rootstocks inoculated with fungi associated with Petri disease and esca[END_REF]. The relative sensitivity of rootstocks is not surprising because rootstock varieties were not selected for resistance to wood-destroying diseases but for other agronomic traits, such as soil type adaptation, vigour of growth and tolerance or resistance to root parasites. In the present experiments, grapevines originating from North America (Muscadinia rotundifolia, V. riparia and V. aestivalis), which have developed resistance to pathogens such as the biotrophs Erysiphe necator and Plasmopara viticola, are nevertheless highly susceptible to N. parvum. However, V. riparia and M. rotundifolia exhibited rather low susceptibility to D. seriata. In another study susceptibility of genotypes, originating from crosses of North American Vitis (V. berlandieri, V. riparia, V. rupestris), to fungi associated with esca has been examined in controlled conditions and in the field. All the genotypes exhibited a wide range of susceptibility to P. alelophilum without any resistance [START_REF] Eskalen | Rootstock susceptibility to 'Phaeomoniella chlamydospora' and 'Phaeoacremonium' spp[END_REF][START_REF] Gramaje | Field evaluation of grapevine rootstocks inoculated with fungi associated with Petri disease and esca[END_REF]. One of the main questions arising from this work is whether results from the sensitivity of different grapevine cultivars in vineyards (canopy symptoms) can be inferred from results of sensitivity levels after artificial inoculation. This study gives evidence for a correlation: for instance, cultivars characteristic of the French Jura region (Trousseau, Savagnin blanc) are characterized by a high incidence in esca/BDA leaf symptoms (Grosman & Doublet, 2012), and these cultivars also range among the most sensitive in the wood inoculation tests for both N. parvum and D. seriata performed here. Gew€ urztraminer and Savagnin blanc belong to the same family of 'Traminer', and Gew€ urztraminer has been selected from an aromatic mutation that occurred in Savagnin rose in the 19th century [START_REF] Rusjan | The preliminary study of grape quality of some clones 'Gew€ urztraminer' (Vitis vinifera L.) in Slovenia[END_REF]. Similar to Savagnin blanc and Trousseau, Gew€ urztraminer shows high expression of canopy symptoms in vineyards [START_REF] Grosman | Maladies du bois de la vigne. Synth ese des dispositifs d'observation au vignoble, de l'observatoire 2003-2008 au r eseau d' epid emiosurveillance actuel[END_REF]. In contrast, Chardonnay and Pinot noir, which are among the less susceptible genotypes (present study [START_REF] Travadon | Susceptibility of cultivated and wild Vitis spp. to wood infection by fungal trunk pathogens[END_REF]), also show a low incidence of dieback symptoms in vineyards. The similar level of susceptibility between these two cultivars could be related to their parental relationship, Chardonnay being the progeny of a single pair of parents, Pinot and Gouais blanc [START_REF] Bowers | Historical genetics: the parentage of Chardonnay, Gamay, and other wine grapes of northeastern France[END_REF]. Although the assay system here reflects in many cases the differences observed in vineyards with respect to symptom expression, it is difficult to draw a strict parallel between field observations and inoculation tests, because the esca/BDA canopy symptoms are observed in vineyards and wood necrosis is measured in inoculation tests. For example, Chasselas has a low incidence of leaf symptoms in vineyards but is among the more susceptible cultivars after wood inoculation with both N. parvum and D. seriata (Fig. 3). Mechanisms of plant defence in wood and canopy may differ, because leaf symptoms are believed to result from the translocation of phytotoxic compounds, whereas wood symptoms result from enzymatic degradation of the wood. Moreover, a complex of fungi is associated with esca and botryosphaeria dieback, which may be one reason why data collected under labo-ratory conditions are not always correlated with field data [START_REF] Murolo | Effects of grapevine cultivar, rootstock and clone on esca disease[END_REF]. In addition, field assays are influenced by environmental factors such as climatic and soil conditions. For example, an overview of grapevine trunk diseases in France showed that the same V. vinifera cultivar could be significantly more affected in one region than in another [START_REF] Bruez | Overview of grapevine trunk diseases in France in the 2000s[END_REF].

Of course, infection of freshly cut canes represents an artificial system, but at this stage, only experimental reduction will allow mechanisms of infection and defence to be addressed. Two levels of reduction have therefore been administered to screen different genotypes for differences in their response: (i) instead of the naturally occurring variable pathogen communities comprising different fungi, artificial inoculation with single fungal strains has been used; and (ii) instead of working with entire plants that are very difficult to standardize and are limited in number (V. vinifera subsp. sylvestris is at the verge of extinction), freshly cut canes were used. To what extent the data from this experimental model mirrors the situation in the field will be further tested by future studies.

Overall, this work confirms that the sensitivity of grapevine to botryosphaeria dieback depends on genetic factors. Despite the caveat on the predictability of field performance by laboratory assays, these data on the susceptibility within the Vitaceae family are important readouts for molecular marker-assisted breeding as a strategy for sustainable viticulture. Figure 3 divides the varieties into four groups according to their susceptibility to Botryosphaeriaceae. Based on these data plots, genomic information from the more resistant genotypes could be used to identify molecular markers for marker-assisted breeding. In addition, defence signalling transduction comparing genotypes with different susceptibilities might identify molecular or cellular events that are relevant for the interaction between host and pathogen. The tested genotypes of V. vinifera subsp. sylvestris developed significantly smaller lesions to both N. parvum and D. seriata compared to cv. Chardonnay or Gew€ urztraminer. As the wild European grapevine is the ancestor of cultivated V. vinifera, it is possible to introduce such genetic factors through introgression by simple crosses. In fact, a small crossing population for V. vinifera subsp. sylvestris H€ o 29 9 V. vinifera cv. Augster weiß (a male-sterile ancient variety) has already been successfully established as a first step to launch a breeding strategy for higher tolerance to botryosphaeria dieback. However, the results from the assay system have to be confirmed by whole plant inoculation and monitoring of necrosis.

To know if the lower susceptibility to Botryosphaeriaceae could be associated with changes in defence gene expression, the expression of several defence markers were monitored after infection. No significant differences in defence gene expression were found between green and necrotic wood, suggesting that signals originating from the necrotic part rapidly migrate to induce defence responses in the green wood surrounding the lesion. The higher expression of SOD in susceptible genotypes such as Gew€ urztraminer might be a consequence of a higher oxidative stress associated with a stronger progression of necrosis in this cultivar.

Several defence genes (STS, LOXC and PR6) were expressed earlier (12 h) after inoculation with N. parvum in most of the V. vinifera subsp. sylvestris accessions compared to V. vinifera cv. Chardonnay and Gew€ urztraminer. At 3 days pi, considering the relatively resistant genotypes V. vinifera subsp. sylvestris Ke 33 and Ke 53, the expression pattern of STS was higher compared to the other genotypes following N. parvum inoculation. During a screen for stilbene synthesis in response to stress (using a short UV-C pulse at 254 nm on leaves for induction), the sylvestris genotypes H€ o 29, Ke 33, Ke 53 and Ke 83 were found to accumulate a much higher level of the antimicrobially active stilbenes trans-resveratrol and their oxidative oligomers, the viniferins, than the tested cultivated grape varieties as well as many of the sylvestris genotypes [START_REF] Duan | Genetic diversity of stilbene metabolism in Vitis sylvestris[END_REF]. The same genotypes also showed partial resistance to downy and powdery mildew, indicating that stilbene accumulation might contribute to the performance under Botryosphaeriaceae challenge. Stilbenes, which are associated with higher resistance of grapevine to pathogens (reviewed in [START_REF] Chong | Metabolism and roles of stilbenes in plants[END_REF]), are also toxic to fungi associated with botryosphaeria dieback [START_REF] Lambert | Phenolics and their antifungal role in grapevine wood decay: focus on the Botryosphaeriaceae family[END_REF] and a comparative analysis of cultivars with different susceptibility to esca showed that lower susceptibility correlated with more rapid accumulation of STS transcripts [START_REF] Lambert | A faster and a stronger defense response: one of the key elements in grapevine explaining its lower level of susceptibility to esca?[END_REF].

However, timely accumulation of stilbenes should not be seen as the only relevant factor for the response to Botryosphaeriaceae. First, it should be noted that the correlation between early induction of STS transcripts and partial resistance holds for N. parvum, but not for D. seriata, which might indicate that this pathogen can silence basal immunity. Secondly, xylem anatomy might modulate the progression of infection. In a recent article, [START_REF] Pouzoulet | Can vessel dimension explain tolerance toward fungal vascular wilt diseases in woody plants? Lessons from Dutch elm disease and esca disease in grapevine[END_REF] provide evidence that susceptibility of three V. vinifera commercial cultivars to esca disease is correlated to large vessel diameter. Vessel dimensions could play a role in the compartmentalization of the pathogen and at the same time may influence the sensitivity of the plant to drought. Study of the xylem structure of V. vinifera subsp. sylvestris could help to evaluate whether higher tolerance is related to the diameter of xylem vessels. Thirdly, other secondary metabolites, such as flavonoids, might also contribute to resistance.

In conclusion, this study shows that different accessions from the Vitaceae family are differentially susceptible to wood necrosis caused by Botryosphaeriaceae fungi. It is thus likely that the genotype of the plant plays a role in the resistance to botryosphaeria dieback. Moreover, several accessions of V. vinifera subsp. sylvestris, the ancestor of V. vinifera, are more resistant to artificial inoculation than cultivars such as Chardonnay and Gew€ urztraminer. These findings suggest that the possibility of creating new grapevine varieties with enhanced resistance to trunk pathogens has realistic potential. Further work will be aimed at elucidating the mechanisms of low susceptibility in some of the V. vinifera subsp. sylvestris cultivars such as the earlier induction of defence responses linked with basal immunity, secondary metabolite contents, and also wood structure.

Figure 1

 1 Figure 1 Partial classification of Vitaceae adapted from Galet (1967).

Figure 2

 2 Figure 2 Comparison of the inoculation methods of mycelial suspension versus mycelia on agar tested on Vitis vinifera cv. Chardonnay inoculated with Neofusicoccum parvum. Necrosis symptoms on V. vinifera cv. Chardonnay woody internodes 7 days after N. parvum Bt 67 inoculation. Relative units represent the percentage of internode necrotic area in inoculated wood divided by the percentage of internode necrotic area in controls inoculated with potato dextrose agar. Data are expressed as a box-and-whisker plots showing median and interquartile range (IQR). The insert image is the mycelia used for inoculation in suspension medium. Mean AE SE was calculated from three biological replicates each calculated from 7-8 internodes.

Figure 3

 3 Figure 3 Susceptibility of the Vitaceae family to Neofusicoccum parvum Bt 67 (a) and Diplodia seriata 98.1 (b). Box plots illustrate the distribution of percentage of internode necrotic area measured within 25 genotypes at day 14 after inoculation with N. parvum and at day 21 after inoculation with D. seriata. The percentage of internode necrotic area was calculated by dividing the necrotic area by the total internode area. Mean AE SE was calculated from three independent experiments from 2013 to 2014, each calculated from the mean of 7-8 internodes. Data are expressed as a box-and-whisker plots showing median and interquartile range (IQR). The same letter above columns indicates no significant difference at P < 0Á05(Duncan, 1955). The genotypes are divided into four groups by dotted lines according to their susceptibility.

Figure 4 Figure 5

 45 Figure 4 Susceptibility of Vitis vinifera and V. vinifera subsp. sylvestris to Neofusicoccum parvum Bt 67 (a) and Diplodia seriata 98.1 (b). Box plots illustrating the distribution of necrosis relative units was measured for eight V. vinifera subsp. sylvestris accessions (Ke 33, 53, 74, 83, 84, 94, 95 and H€ o 29) and two V. vinifera cultivars (Chardonnay (CH) and Gew€ urztraminer (GW)) at day 7 after inoculation with N. parvum, and at day 21 after inoculation with D. seriata. Relative units represent the percentage of internode necrotic area in inoculated wood divided by the percentage of internode necrotic area in controls inoculated with potato dextrose agar (PDA). Mean AE SE was calculated from three independent experiments from 2012 to 2013, each calculated from the mean of 7-8 internodes. Internodes inoculated with PDA were used as control. Data are expressed as a box-and-whisker plots showing median and interquartile range (IQR). The same letter above columns indicates no significant difference at P < 0Á05 (Duncan, 1955).

  Figure 5 (continued).

  Figure 6 Necrosis symptoms on grapevine leaf discs treated with culture filtrate from Neofusicoccum parvum and Diplodia seriata.

	Four V. vinifera subsp. sylvestris accessions
	(Ke 33, Ke 53, Ke 83 and H€ o 29) and two
	V. vinifera cultivars (Chardonnay and
	Gew€ urztraminer) were studied. Percentages
	of necrosed leaf area at 24 h after
	inoculation are compared within six
	genotypes. Mean AE SE was calculated from
	three biological replicates from year 2012 to
	2013 each calculated from 5-10 leaf discs
	per genotype.

PlantPathology (2015)

Acknowledgements

This work was supported by BACCHUS Interreg project IV Oberrhein/Rhin sup erieur and by the European COST action COST-FA1303. The authors would like to thank Joachim Daumann for collecting the wood and leaves in the Botanical Garden of the Karlsruhe Institute of Technology, Germany. They are grateful to D. Merdinoglu and V. Dumas for taking samples of wood of the different Vitaceae accessions in INRA Colmar.


Po-Keng Cheng
email: ansd39@gmail.com

Fundamentalists in the cryptocurrency markets

Keywords: Bitcoin, Agent-based model, Noise traders, Fundamentalists, Speculative bubbles, Heavy tails

may come

Fundamentalists in the cryptocurrency markets

Introduction

Bitcoin is a type of cryptocurrency based on decentralization and uses peer-to-peer networking, open source, and blockchain as the underlying technology. Due to the limited supply, anonymity of accounts, lack of supervision by governments, and difficulty in tracing it, Bitcoin has become a popular electronic currency, with a lower transaction fee than that found in the general financial system. The value of Bitcoin has rapidly risen, from one Bitcoin being equivalent to 0.06 U.S. dollars in 2010 to more than 10,000 U.S. dollars in 2020. This growth has greatly exceeded that of other financial investment products. Some economists, including several Nobel Prize winners, describe this phenomenon as a speculative bubble. Moreover, Bitcoin is not seen as a currency but as a speculative commodity [START_REF] Baek | Bitcoins as an investment or speculative vehicle? A first look[END_REF][START_REF] Bouoiyour | What does Bitcoin look like?[END_REF]. [START_REF] Kristoufek | What are the main drivers of the Bitcoin price? Evidence from wavelet coherence analysis[END_REF] stated that the price of Bitcoin will be affected by the following factors: law of supply and demand, transactions, public discussion, and financial hedging tools. Some studies have discussed the informational efficiency of the Bitcoin market. For example, [START_REF] Urquhart | The inefficiency of Bitcoin[END_REF] argued that the Bitcoin market is inefficient. However, as more investors analyze and trade Bitcoin, the Bitcoin market may be moving toward becoming efficient. [START_REF] Nadarajah | On the inefficiency of Bitcoin[END_REF] continued the research of [START_REF] Urquhart | The inefficiency of Bitcoin[END_REF] and converted the Bitcoin rate of return. Their results suggested that the converted Bitcoin rate of return is actually market efficient. [START_REF] Tiwari | Informational efficiency of Bitcoin-An extension[END_REF] reexamined the information efficiency of the Bitcoin market and stated that the Bitcoin market is generally efficient.

The application of heterogeneous agent models (HAMs) to Bitcoin and digital currency is still a new field. [START_REF] Cocco | Modeling and Simulation of the Economics of Mining in the Bitcoin Market[END_REF], [START_REF] Cocco | Using an artificial financial market for studying a cryptocurrency market[END_REF][START_REF] Cocco | An Agent-Based Artificial Market Model for Studying the Bitcoin Trading[END_REF] have designed a HAM model to simulate the Bitcoin mining process and buying and selling (or Bitcoin market) transactions. In this paper, we follow the method of [START_REF] Cheng | Speculative bubbles and crashes: Fundamentalists and positivefeedback trading[END_REF] and estimate the parameters of the cryptocurrency markets. We explore the reasons for the rise in the value of cryptocurrency and seek to understand fundamentalist and chartist trading behavior with regard to cryptocurrency over the years. That is, this study attempts to understand the effect of different types of investors on the value of cryptocurrency in different periods.

The rest of the paper is organized as follows. Section 2 presents the methodology. Section 3 provides the results. Section 4 concludes.

We adopt the model (Case 1) with t-distributed shock terms from [START_REF] Cheng | Speculative bubbles and crashes: Fundamentalists and positivefeedback trading[END_REF] and estimate the key parameters. The model is briefly reviewed in Appendix 1.

We set the window size equal to 500 trading days and the interval between each window equal to 20 trading days. Here fundamentalists' eagerness towards profits ( f α ), positive-feedback traders' funding rate ( c r ), and noise traders' reaction strength (γ ) are the chosen key parameters.

Within the chosen parameters space, we run the simulations and apply the two-sample Kolmogorov-Smirnov test (KS test) and Cramer-von Mises test (CM test) with historical returns as the benchmark for estimating the coefficients of the parameters. Our null hypothesis is that the distribution of asset returns in the simulated data has the same continuous distribution as those for the historical data. We set the confidence interval equal to 95%, and then ran 500 trials. We then chose the set of parameters that minimizes the number of trials that reject the null hypothesis.

Results

We present the results of parameter estimations f α , c r , γ here. We use the Bitcoin adjusted closing price, the Ethereum adjusted closing price, and the Litecoin adjusted closing price from Yahoo! Finance. 1 Figure 1 depicts the estimated f α for Bitcoin. From Cheng and Kim (2017), we know that f α is related to fundamentalists' risk aversion. If f α is larger, fundamentalists are more willing to take risks and the market prices tend toward their fundamental values. We could observe in the figure 1 α is generally low, c r is generally high, and γ is generally high for Ethereum and Litecoin. The low f α tells us that fundamentalists are risk averse to burst bubbles in the Ethereum market and the Litecoin market. In addition, we could also observe that f α is higher in 2017 while Litecoin was rising, in which fundamentalists drive up the prices. The high c r in the Ethereum market and the Litecoin market imply that we could easily observe crashes, and the high γ in the Ethereum market and the Litecoin market show that these two markets are always agitated. In this paper, we investigate cryptocurrency appreciation and estimate the parameters of the cryptocurrency markets. We determine that fundamentalists are more willing to take risks, thus pushing up the prices of Bitcoin from 2017 to 2018, which indicates that the increase in Bitcoin prices may not represent a speculative bubble. Moreover, our results suggest that the cryptocurrency markets are instable and agitated, with crashes easily occurring.

Appendix 1 Model review

In this appendix, we briefly review the model. A single stock market exists that is populated with three types of traders: fundamentalists, positive-feedback traders, and noise traders. All traders in the market are short-sighted and possess beliefs regarding the next period's price for the stock. Fundamentalists believe stock prices will move back to the stock's fundamental values. They form their expected price based on the difference between the fundamental value and the current market price,

1 f t t p p + -,
and they adjust their expected price each period. The fundamentalists' demand for stock f t x and adaptive process 1 f t p + are shown in equations ( 1) and ( 2).

(

)

1 1 exp 1 1 f f t t f t f f t t p p x r p r p α +     -     = --           (1)
( )

* * * 1 2, 1
, where

t f t t t t t t t p p p p p p ν ε + = =+ - = + ∑ (2)
where f α indicates the eagerness of fundamentalists towards profits, f r is the funding rate fundamentalists face to finance their positions, * p denotes the initial fundamental value of the stock, ν captures the speed at which fundamentalists expect the market price to move back to the fundamental value, and 2,t ε represents the shock terms resulting from changes of policies and rare events.

Positivefeedback traders chase market trends. They form their expected price based on the differences between their previous expected price and the current market price, )

1 1 1 exp 1 1 c c c t t t c t c c t t p p p x r p r p α β + +     - +     = --           (3)
( )

1 c c c t t t t p p p p µ + = + - (4)
where c α represents positive-feedback traders' eagerness towards profits, β is the coefficient of the wealth effect, and c r is the funding rate positive-feedback traders pay in financing their positions, µ is the error correction coefficient representing how sensitive positive-feedback traders correct their expected stock price for the next period.

The accumulated noise traders' demand for stock n t

x is as follows:

1, 2,
, where

n t t t t t x γε ε ε ε = = + (5) ( ) ( ) ( ) ( ) ( ) 1, 2, 1, 2, 1, 2, , . . .; 0; 0 0 t t t t t t t i i d E E E E E ε ε ε ε ε ε ε = = = + = ( 6
)
where γ is the reaction strength of noise traders to noisy information and 1,t ε comes from the demand for the stock from liquidity traders and traders with biased belief or sentiments; 1,t ε is normally distributed, and 2,t ε varies with different probability distributions.

A market maker exists whose role is collecting orders, announcing execution prices, and executing transactions. The market price each period is determined by the demand for the stock:

+ + + = = - = = - = - + - ∑ ∑ (8)
where ϕ is the intensity of choice to switch strategies, and

0 ϕ > ; , f c t t π π
are the cumulative profits realized by fundamentalists and positivefeedback traders, respectively, each period.

  that the appreciation and depreciation of Bitcoin prices in 2013 to 2014 and the rising of Bitcoin prices from 2017 to 2018 were driven by fundamentalists. The rising of Bitcoin prices from 2017 to 2018 especially indicate that fundamentalists believe that one Bitcoin is valued over than 10,000 U.S. dollars. This is in line with the finding by Lee et al.(2020) that technological innovations are also a reason for the sharp rise in Bitcoin prices instead of being caused by speculative behaviors.

Figure 2

 2 Figure 2 presents the estimated c r . From Cheng and Kim (2017), c r also indicates the stability of the market. If c r is larger, we would observe

Figure 1 :

 1 Figure 1: Estimated f α by CM test

Figure 4 :Figure 5 :

 45 Figure 4: Comparison between CM test and KS test, f α

Figure 9 :

 9 Figure 9: Estimated c r of Ethereum by CM test

  and adjust their expected price each period. The demand of positive-feedback traders for stock c shown in equations (3) and (4).

(

  θ is the market sensitivity corresponding to changes in the demand for stocks, n is the total number of traders in the market, and t κ reflects the population fraction of positive-feedback traders in the market at each period. Noise traders exist in the market with a fixed weight of ξ , and the proportion of fundamentalists in the market equals 1 t

	t p	1 +	t p -=	θ	n	 	( 1	t κ ξ --	)	t x	f	+	κ	c t t x	+	ξ	n t x	 			(7)
																														κ ξ --. The
	population fractions of fundamentalists and positive-feedback traders are
	updated each period as follows:
	κ	t	1	1 exp	(	( ϕ π π 1 f t ξ	c t	)	)	;	π	t	f		t	t	1	( x p f t t	1	t p	) ;	π	c t	t	t	1	( x p c t t	1	t p	)

where


Eunice Phd Cascant
email: eunice.cascant@univ-lyon3.fr

OPPORTUNTIY RECOGNITION, AN INFLUENCE ON REFUGEES AND ASYLUM SEEKERS SOCIAL ENTREPRENEURSHIP IN HOST COUNTRIES: FROM THE LENS OF THE MIXED EMBEDDEDNESS AND EFFECTUAL FRAMEWORKS

. This paper bridges the conceptual and contextual gaps of female refugee social entrepreneurship literature (Czinkota. Khan & Knight, 2021; Elo,Aman & Täube,2018; Sinkovic & Reuber, 2021;Zucchella, 2021).It also addresses the scarcity of research on the relevance of gender in social entrepreneurship. Our main question is: How does opportunity recognition as a construct influence female refugees' social entrepreneurship in the host country? We draw upon the mixed embeddedness (Kloosterman, 2010; Kloosterman, Rusinovic & Yeboah,2016) and effectual lenses (Sarasvathy, 2008) to understand how female refugees become social entrepreneurs, and what makes them different.

Theoretical background

With the current geopolitical unrest, over 3 million people have left Ukraine and crossed into neighbouring countries (IOM, 2022). This is in addition to the recent statistics that show that, 82.4 million people were forcibly displaced world-wide at the end of 2020.

However, the phenomenon of refugees' inclusion in host countries and across the globe still remains a paradox for host governments due to their heterogeneity [START_REF] Harima | Co-Creation of Social Entrepreneurial Opportunities with Refugees[END_REF]. Therefore, to overcome unemployment and under employment many of them resort to entrepreneurship. To promote social cohesion through tackling societal challenges through their social entrepreneurial ventures with "a social goal" (Short, Moss & Lumpkin, 2009, p. 162).

The "hybrid nature of social entrepreneurship" influences female refugee social entrepreneurs to succeed since they adhere to both social goals even with stiff financial constraints. The transnational social entrepreneurs take advantage of being multiply embedded in different institutional environments an element that shapes their understanding of themselves and their environment [START_REF] Bolzani | Exploring the multi-level processes of legitimacy in transnational social enterprises[END_REF]. Despite the impact of the endogenous and exogenous factors could undermine the existence of their social ventures. Female refugee social entrepreneurs are able to collaborate with services providers to meet untapped economic and social needs through co-creation and co-production while responding to the needs of marginalised segments of the society.

The first opportunity creation theory; asserts that opportunities do not exist independently from the entrepreneur. They are created by the actions, reactions, and enactment of entrepreneurs as they explore new ways to generate new products or services [START_REF] Eckhardt | Opportunities and Entrepreneurship[END_REF].

According to this view, entrepreneurs cannot search for opportunities, but rather act and observe the effects of their actions [START_REF] Short | Research in social entrepreneurship : Past contributions and future opportunities[END_REF] an argument supported by the effectual and mixed embeddedness frameworks. However, social entrepreneurship opportunities are different from those found in profit ventures especially for the female refugees.

Methodology

We conducted a qualitative and exploratory study in order to develop a deep understanding of a complex social phenomenon [START_REF] Eisenhardt | Building theories from case study research[END_REF]. We conducted semi-structured interviews with 22 female refugee entrepreneurs (i.e. with a defined legal status and are beneficiaries of international protection) and 3 service providers working with exiled people's social entrepreneurship in France. Data has been collected from April-July 2021 and January-February 2022 and analysed through a content analysis. The interviews were conducted both in French and English languages. The duration of the interviews ranged from 45 minutes to one hour.

Results

We identified that female refugee social entrepreneurs recognise opportunities, through transformation of their human and social capital to be able to grasp the opportunities available within the host country market environments an aspect referred as "agency".

Secondly, most of the female refugee social entrepreneurs recognise their liability of foreignness but devise means through effectual decision-making process to create social ventures.

Thirdly, we aggregated our results also by highlighting the role of networks in creating opportunities for female refugee social entrepreneurs. Our target group acknowledges the importance of growing strategic networks as an important factor. Although some of them did not rely on the diaspora networks (as they differed in their entrepreneurial visions).

It worth noting that, while effectual approaches open up and create new markets with a low failure cost, expert social entrepreneurs who choose to build large ventures have to become good at using both casual and effectual tool boxes.

Fourthly, social value creation as another aggregated construct, there is an untapped opportunity for service providers and refugee social entrepreneurs to work together to achieve both business and social impact goals. Refugee social enterprises do bring inclusion and sustainability into value chains and create impact.

Finally, innovation and digital (skills and tools) appeared to play a crucial role. Most female refugee social entrepreneurs acknowledged to using digital pathways to unlock markets that were out of their reach, develop strategic networks and gain visibility. The digital tools also avoiding stereotypes and marginalization that could impact refugees' social ventures. Moreover, their ventures became more resilient during the global COVID-19 pandemic due to their online presence.

Results from the service providers such as refugee incubators, accelerators and NGOs confirm that are entrepreneurial in nature despite the difficulties encountered in their migration trajectories.

Additionally, entrepreneurship is a way of promoting integration and diversity especially through co-production and co-creation initiatives.

Contribution to theory and policy

This study is a novel attempt in providing a longitudinal analysis on how extant resources determine the opportunity spaces explored by the female refugees in turbulent host country business environments. The study makes specific contributions. Conceptually, female refugee social entrepreneurs are not only recognized for their contributions towards economic growth but also creating impactful social ventures aimed at wealth creation hence eradicating poverty and income inequality. We make theoretical contribution by enriching literature on opportunity recognition and its influence of female refugee social entrepreneurship through the lenses of the mixed embeddedness and effectual frameworks [START_REF] Ram | What happens to refugeeorigin entrepreneurs? Combining mixed embeddedness and strategy perspectives in a longitudinal study[END_REF]. Empirically, our research unveils the quasi systematic inclusion of sustainability in business models to promote social value. Managerial wise, to female refugee entrepreneurs, it is relevant to take into consideration the indicators of social entrepreneurship so as to measure their social impact. This could act as a tool for policy makers and stakeholders to work towards building inclusive entrepreneurial ecosystems and provide guidance in designing policies enabling female refugee to venture in social enterprises and create social impacts.


est destinée au dépôt

  Eunice Cascant. The development of entrepreneurial orientation among female migrants and its influence on venture internationalization: a qualitative studybased on the French Context.. 47 TH EIBA 10 th Danny Van Den Bulcke Doctoral Symposium(DVDB-DS) on December 10 th in Madrid,Spain, Dec 2021, Madrid, Spain. hal-03679292