Brigitte Maisonneuve

Michel Pitrat

Patrick Gognalons

Benoît Moury
email: benoit.moury@inra.fr

L Virosa

Growth stage-dependent resistance to the potyviruses lettuce Italian necrotic virus and Lettuce mosaic virus displayed by Lactuca sativa introgression lines carrying the Mo3 locus from L. virosa

Keywords: lettuce, linkage, Mo3 locus, pleiotropy, Potyvirus, resistance

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

Introduction

Lettuce (Lactuca sativa, family Asteraceae) crops are affected by several potyviruses (genus Potyvirus; family Potyviridae), including Lettuce mosaic virus (LMV), Turnip mosaic virus (TuMV), Bidens mottle virus (BiMoV), Endive necrotic mosaic virus (ENMV) and lettuce Italian necrotic virus (LINV) [START_REF] Christie | Characterization and electron microscopy of a virus isolated from Bidens and Lepidium[END_REF][START_REF] Zink | Relationship of a non-lethal reaction to a virulent isolate of lettuce mosaic virus and turnip mosaic susceptibility in lettuce[END_REF][START_REF] Zitter | Incidence of lettuce mosaic and bidens mottle viruses in lettuce and escarole fields in Florida[END_REF][START_REF] Provvidenti | Sources of resistance to viruses in the Potyviridae[END_REF][START_REF] Blancard | A Color Atlas of Diseases of Lettuce and Related Salad Crops. Observation, Biology and Control[END_REF][START_REF] Ciuffo | Molecular identification and biological characterization of a new potyvirus in lettuce[END_REF][START_REF] Desbiez | Molecular and biological characterization of two potyviruses infecting lettuce in southeastern France[END_REF]. LINV was recently described in lettuce crops in Italy and southeast France and induces severe necrosis and plant wilting, leaf distortion and/or vein clearing. Its natural host range seems to be limited to L. sativa cultivars. However, experimental inoculations showed that it can infect plants of other species in the family Asteraceae (Calendula spp., Cichorium spp., Helianthus annuus, Tragopogon pratensis and Zinnia elegans) and Nicotiana benthamiana [START_REF] Desbiez | Molecular and biological characterization of two potyviruses infecting lettuce in southeastern France[END_REF]. Transmission by Myzus persicae (Homoptera: Aphididae) was demonstrated experimentally [START_REF] Ciuffo | Molecular identification and biological characterization of a new potyvirus in lettuce[END_REF][START_REF] Desbiez | Molecular and biological characterization of two potyviruses infecting lettuce in southeastern France[END_REF]. LINV is phylogenetically related to LMV in the genus Potyvirus. No source of resistance to LINV was found in L. sativa accessions carrying known potyvirus resistance genes such as the mo1 1 or mo1 2 alleles conferring LMV resistance, or the Tu gene conferring TuMV resistance [START_REF] Desbiez | Molecular and biological characterization of two potyviruses infecting lettuce in southeastern France[END_REF]. However, a few accessions of the wild species L. virosa and L. perennis were found to be resistant to a French isolate of LINV (LINV-Fr). The L. virosa accession PIVT1398 appears particularly interesting because it has a broad resistance spectrum including to LMV and ENMV-Fr isolates [START_REF] Desbiez | Molecular and biological characterization of two potyviruses infecting lettuce in southeastern France[END_REF] and because of the possibility of obtaining fertile intercrosses between L. virosa and L. sativa [START_REF] Maisonneuve | Sexual and somatic hybridization in the genus Lactuca[END_REF]. The LMV resistance of PIVT1398 was shown to be conferred by the single gene Mo3 in F1, F2 and F3 progenies derived from crosses with the LMV-susceptible accession L. virosa PIVT280 [START_REF] Maisonneuve | New resistance against downy mildew and Lettuce mosaic potyvirus in wild Lactuca spp[END_REF]. Mo3 was inherited as a dominant gene after inoculation with LMV-0 but as an incompletely dominant gene after inoculation with LMV-13, the heterozygous plants inoculated with LMV-13 being often necrotic [START_REF] Maisonneuve | New resistance against downy mildew and Lettuce mosaic potyvirus in wild Lactuca spp[END_REF]. LMV-0 is avirulent towards the mo1 1 or mo1 2 resistance alleles that are present in many cultivars of L. sativa, whereas LMV-13 is virulent towards both alleles [START_REF] Dinant | Lettuce mosaic virus[END_REF]. Later on, L. sativa introgression lines (ILs) carrying the Mo3 locus were developed in several genetic backgrounds via a backcross programme [START_REF] Maisonneuve | Lactuca virosa, a source of disease resistance genes for lettuce breeding: results and difficulties for gene introgression[END_REF]. The results of inoculation with either LMV-0 or LMV-9 (an mo1 1 virulent isolate; [START_REF] Dinant | Lettuce mosaic virus[END_REF], depending on the recurrent L. sativa cultivar (carrying the mo1 + or mo1 1 gene, respectively), fit to the hypothesis of one gene and strong necrosis associated with heterozygous plants. Some F3 and F4 populations tested with LMV-0 or LMV-9 displayed uniform resistance reactions with neither symptoms nor virus detection in apical, uninoculated leaves. The objectives of the present study were to analyse the phenotypic reaction of L. virosa PIVT1398 to LINV and LMV inoculation and to check if the Mo3 locus could also be involved in the LINV resistance using data obtained from the reaction of ILs carrying this genetic factor(s).

Materials and methods

Plant material

The Mo3 gene from L. virosa PIVT1398 (= CGN09365) was independently introduced by backcrosses into two butterhead lettuce cultivars, Girelle and Mantilia, producing ILs Girelle-Mo3 and Mantilia-Mo3, respectively (Tables S1 &S2). Two other L. sativa cultivars, Columbus and Mariska, were used for the backcrosses (Tables S1 &S2). Mantilia carries the mo1 1 gene, that confers resistance to LMV-0. The three other L. sativa cultivars (Columbus, Girelle and Mariska) are susceptible to LMV (mo1 + ). Girelle-Mo3 was developed from the F1 interspecific cross Columbus × PIVT1398, followed by seven backcrosses to L. sativa and three generations of selfing (= BC7S3). Mantilia-Mo3 was developed from the interspecific cross PIVT1398 × Columbus followed by nine backcrosses to L. sativa cultivars and three generations of selfing (= BC9S3). Eight and four generations of selfing were also inserted between the backcrosses in the genealogies of Girelle-Mo3 and Mantilia-Mo3, respectively (Tables S1 &S2). For both ILs, selection for LMV resistance was made in different generations, with LMV-13 in the first generations and with LMV-0 or LMV-9 from BC2. The phenotypes of Girelle-Mo3 and Mantilia-Mo3 were similar to those of Girelle and Mantilia, respectively, in terms of plant appearance, growth and development. LMV-susceptible accession L. virosa LS238 was also used as control.

Virus isolates and inoculation procedure

The origins of the French isolate LINV-Fr and of isolates LMV-9 and LMV-13 are described in [START_REF] Desbiez | Molecular and biological characterization of two potyviruses infecting lettuce in southeastern France[END_REF] and [START_REF] Dinant | Lettuce mosaic virus[END_REF], respectively. LMV-9 overcomes the mo1 1 resistance allele, whereas LMV-13 overcomes both mo1 1 and mo1 2 resistance alleles.

The inoculation procedure was as described in [START_REF] Desbiez | Molecular and biological characterization of two potyviruses infecting lettuce in southeastern France[END_REF], with virus being propagated in Mantilia. After sowing on paper in Petri dishes (2 days at 6 °C without light, then 5-7 days in a climate chamber at 24/18 °C and 16 h light), the germinated seeds were transplanted in soil in 0.5 L pots in an insect-proof greenhouse for growing and tests. The seedlings were inoculated 16-34 days after sowing to compare virus infection and symptoms in different growth stages: from young plants with two to three leaves to older plants with up to six to seven leaves. Whatever the stage at inoculation, the two oldest leaves were rubinoculated. A comparison of four growth stages at inoculation (performed from 18 to 34 days post-sowing) was made on Girelle-Mo3 in December/January; then comparisons of two inoculation stages (16 vs 28 or 20 vs 28 days post-sowing) were made in April/May and August, respectively.

Virus detection

Symptoms were recorded regularly and the infection status of the plants was confirmed by enzyme-linked immunosorbent assay (ELISA), at 7-10 days post-inoculation (inoculated leaves), or at 2-6 weeks post-inoculation (apical, uninoculated leaves). LINV-Fr was detected by antigen-coated plate (ACP)-ELISA with a generic potyvirus-group kit (Agdia). LMV was detected by double antibody sandwich (DAS)-ELISA with a specific polyclonal antiserum as described in [START_REF] Maisonneuve | New resistance against downy mildew and Lettuce mosaic potyvirus in wild Lactuca spp[END_REF]. Virus detection was arbitrarily considered positive when the absorbance at 405 nm (A405) was more than three times that of the healthy controls.

LINV was also detected by reverse transcription-polymerase chain reaction (RT-PCR). Total RNAs were purified from 200 mL of the leaf extracts used for ELISA using the Tri-Reagent kit (Merck) according to the manufacturer's instructions. Two-step RT-PCRs were performed.

Results

Resistance to LINV-Fr

Following inoculation with LINV-Fr at 20 days after sowing, all plants of Girelle-Mo3 and Mantilia-Mo3 (278 in total) showed necrotic lesions on inoculated leaves, similar to hypersensitive reactions (HR), at 5-7 days post-inoculation (dpi). The donor of the Mo3 resistance gene, L. virosa accession PIVT1398, showed rare and tiny necrotic lesions on inoculated leaves at the same time. In contrast, the L. sativa parental lines Girelle and Mantilia as well as the L. virosa susceptible control accession LS238 did not show any symptoms on inoculated leaves. At 6-12 dpi, Girelle and Mantilia showed systemic vein clearing and leaf curling, progressively evolving into a general wilting and the death of the plants one month after inoculation (Table 1; Fig. S1). Lactuca virosa LS238 showed milder mosaic symptoms on apical, uninoculated leaves, which did not induce death of plants. No symptoms were observed at the systemic level in L. virosa PIVT1398 or in a large majority (81%) of plants belonging to Girelle-Mo3 and Mantilia-Mo3. However, chlorotic/necrotic lesions (CNL) developed on the first two apical leaves of 22% and 14% of the plants belonging to Girelle-Mo3 and Mantilia-Mo3, respectively, from 6 to 14 dpi (Table 1; Fig. S2). Most of the plants showing these CNL symptoms at the systemic level continued to grow and newly developed leaves did not show any symptoms. However, 7.2% of the total Girelle-Mo3 plants stayed stunted with limited growth, and new leaves also showed CNLs. Overall, 95% of IL plants did not show symptoms on apical leaves at 38 dpi (Table 1). ACP-ELISA was performed using samples from the apical leaves with the aim of detecting virus infection. All Girelle, Mantilia

Resistance phenotypes of L. sativa Mo3 introgression lines against LINV-Fr and LMV

In the third experiment, the resistance to LINV-Fr was compared to that expressed against two LMV isolates (Table 2). The resistance phenotypes observed upon inoculation with LMV-13 were similar to those observed with LINV-Fr except that symptoms were more severe with LMV-13 (Fig. S2). In Girelle-Mo3 plants inoculated at the youngest stage (20 days after sowing), necrotic lesions were observed on inoculated and apical leaves for all plants. They were larger than those observed with LINV-Fr and the plants remained stunted afterwards with no recovery. Strong positive reactions were observed in ELISA in these plants. At the oldest stage (28 days after sowing), only one plant of 22 was infected at the systemic level with necrotic lesions and evidence of virus infection in ELISA (Table 2). LMV-13 infected all inoculated Girelle plants (Fig. S1) but none of the L. virosa PIVT1398 plants at the systemic level. In PIVT1398, necrotic lesions in inoculated leaves were more visible than with LINV-Fr. In relation to LMV-9, there was no evidence of infection in Girelle-Mo3 (Fig. S2) or in L. virosa PIVT1398 in inoculated leaves or at the systemic level, and based either on symptom recording or ELISA, whatever the inoculation stage. However, LMV-9 infected all Girelle plants with mosaic symptoms (Fig. S1) and strong ELISA reactions at the systemic level.

Discussion

All results are in agreement that the region encompassing the Mo3 locus (or the Mo3 gene itself) determines resistance against several LMV isolates in L. virosa PIVT1398 (Maisonneuve et al., 1999 and this study) and is also involved in resistance to LINV-Fr. Indeed, all plants belonging to Girelle-Mo3 and Mantilia-Mo3 showed a very high resistance level to LINV-Fr compared to the Girelle and Mantilia recurrent parents: (i) no dead plants were observed in L. sativa Mo3 ILs at 38 dpi, while 100% Girelle and Mantilia plants were dead at this evaluation time; (ii) most of the inoculated IL plants did not show symptoms on apical leaves; (iii) none of them showed clear evidence of virus infection at the systemic level based on ELISA, even in the presence of CNL; and (iv) a frequent recovery was observed from 14 to 38 dpi in plants showing early CNL symptoms. Moreover, IL plants resulting from two independent interspecific crosses and with two different genetic backgrounds showed a similar high proportion of symptomless plants at the systemic level at 14 dpi.

Necrotic lesions on inoculated leaves and CNLs on some apical leaves of plants of the ILs combined with low virus titres in these leaves suggest that the resistance is linked to HR mechanisms, consistent with its dominant inheritance for LMV [START_REF] Maisonneuve | New resistance against downy mildew and Lettuce mosaic potyvirus in wild Lactuca spp[END_REF].

Pleiotropic effect of the Mo3 gene conferring resistance to both LMV and LINV-Fr is supported by the fact that the Girelle-Mo3 and Mantilia-Mo3 ILs have undergone 17 potential rounds of meiosis, which could have separated two potential resistance genes by recombination. There are other examples of large spectrum resistance genes or loci conferring resistance to several potyvirus species, like the Pvr4 locus in Capsicum annuum conferring resistance to at least six potyviruses [START_REF] Janzac | Phenotype and spectrum of action of the Pvr4 resistance in pepper against potyviruses, and selection for virulent variants[END_REF] and the I locus in bean (Phaseolus vulgaris) conferring resistance to at least 10 potyviruses [START_REF] Meziadi | Genetic resistance against viruses in Phaseolus vulgaris L.: state of the art and future prospects[END_REF]. However, the analysed ILs result from interspecific crosses between L. virosa and L. sativa, for which recombination could have been suppressed in parts of the genome as observed in other plant interspecific crosses [START_REF] Frary | Molecular mapping of the centromeres of tomato chromosomes 7 and 9[END_REF][START_REF] Wenzl | A high-density consensus map of barley linking DArT markers to SSR, RFLP and STS loci and agricultural traits[END_REF][START_REF] Ganal | A large maize (Zea mays L.) SNP genotyping array: development and germplasm genotyping, and genetic mapping to compare with the B73 reference genome[END_REF][START_REF] Sim | Development of a large SNP genotyping array and generation of high-density genetic maps in tomato[END_REF]. Hence, it is concluded that the single gene Mo3 could have a pleiotropic effect, conferring resistance to both LMV and LINV-Fr. However, it is not possible to exclude categorically the hypothesis of two distinct loci, one (Mo3) conferring resistance to LMV and the other conferring resistance to LINV-Fr. Under this alternative hypothesis, the two loci would be located in the same genome region and recombination between them could be impaired because of the incompatibility between the L. virosa and L. sativa genomes. The L. virosa PIVT1398 accession is also resistant to a third potyvirus, ENMV-Fr, but the link with Mo3 using the L. sativa ILs could not be tested because the recipient parents, Girelle and Mantilia, are also resistant to ENMV-Fr [START_REF] Desbiez | Molecular and biological characterization of two potyviruses infecting lettuce in southeastern France[END_REF].

The phenotype of the resistance was found to be dependent on the virus species, on the virus isolate (for LMV) and on the plant growth stage at inoculation. With the LMV-9 isolate, the phenotype resembles an extreme resistance, with no manifestation of an HR in inoculated leaves of L. virosa PIVT1398 or of the tested Mo3 ILs, and no virus detected in inoculated or apical leaves. Growth stage-dependent resistance was observed with LINV-Fr and LMV-13.

At the youngest stage for inoculation, the resistance was not sufficient to block systemic infection. A proportion of plants showed systemic infection, as attested by ELISA, RT-PCR and inoculation tests. For these two viruses, resistance was associated with expression of necrotic lesions in inoculated leaves and, in some cases, in apical leaves. Hence, the resistance phenotype resembles an HR, but resistance does not completely hamper the virus movement at the systemic level when plants are young and/or when the virus is highly aggressive, like LMV-13. The CNLs observed at the systemic level following inoculation with LINV-Fr may therefore correspond to a secondary induction of plant resistance. As the plant grows, these successive inductions of plant defences at different leaf levels may ultimately confine the virus, which would explain the recovery phenotype observed in most of the plants. These differences of resistance level of Mo3 have been observed previously in the L. virosa genetic background between LMV-0 and LMV-13 [START_REF] Maisonneuve | New resistance against downy mildew and Lettuce mosaic potyvirus in wild Lactuca spp[END_REF]. Furthermore, a recovery phenotype was observed for LINV-Fr plants inoculated at a young stage and expressing symptoms at the systemic level. Later on, in most of the plants, newly emerged leaves were devoid of symptoms and no virus was detected.

The comparison between the resistance phenotypes of L. virosa PIVT1398 and the L. sativa Mo3 ILs also suggests an effect of the genetic background of the Mo3 resistance locus (or its genome region). Indeed, the L. virosa PIVT1398 resistance to LINV-Fr and LMV-13 is almost complete, with a lack of infection at the systemic level and very small necrotic lesions on inoculated leaves, whereas frequent systemic infections were observed for the ILs in the two L. sativa backgrounds when inoculated at a young stage and local lesions were much larger.

That could be due to a loss of some complementary genes efficient against LINV-Fr or instability of the expression of Mo3 in the L. sativa background. Additional studies analysing the resistance reaction of the F2 progeny of PIVT1398 × LS238 would be necessary for more conclusive evidence. a in days after sowing.

b mean absorbance values at 405 nm in ELISA of symptomatic leaves relative to mockinoculated plants (mean of at least five replicas).

c chloro-necrotic lesions.

d RNA was purified from 8 of these 13 plants to check virus presence by RT-PCR.

e necrosis.

  Primer 7091CP-R (5′-GCTAGCTGATTCTTGGTGGC-3′) and Avian myeloblastosis virus reverse transcriptase (Promega) were used for RT. Primers 7091CP-R and 7091Nib-F (5′-TGGTTCATGTCTCATAGGGG-3′) and Taq DNA polymerase (Promega) were used for PCR. The primers were designed based on the sequence of isolate LINV-Fr (accession number KU941945) to amplify a region of c. 650 nucleotides (nt) at the junction of the NIb and coat protein cistrons. Mock-inoculated plants of the same genotype(s) were used as negative controls and plants with symptoms, of the susceptible genotypes Mantilia or Girelle, as positive controls. The PCR programme was 5 min at 95 °C; 40 cycles at 95 °C for 30 s, 57 °C for 30 s and 72 °C for 45 s; and 72 °C for 10 min.

  and L. virosa LS238 plants reacted positively, whereas none of the plants of either L. virosa PIVT1398 or L. sativa Mo3 ILs were positive, regardless of the presence of CNLs. A few plants with CNLs showed absorbance values (A405) close to twice those of negative controls, which may indicate very low virus accumulation or replication.Resistance to LINV-Fr and plant growth stageThe resistance status of Girelle-Mo3 plants inoculated at different growth stages was compared in three independent experiments (Table2) in order to gain insight into the peculiar range of LINV-Fr resistance phenotypes observed in the ILs (showing either complete resistance to systemic infection, limited systemic invasion followed by a recovery, or systemic invasion leading to plant stunting). For all experiments, plants had two expanded leaves at the youngest growth stage. This stage corresponded to 16-20 days after sowing, depending on the season in which the experiment was conducted. In all experiments, 100% of the Girelle susceptible controls showed infection, and 100% of the L. virosa PIVT1398 controls showed resistance at the systemic level, based on symptom occurrence and ELISA. All plants belonging to the ILs showed necrotic HR-like lesions on inoculated leaves. The systemic infections in IL plants were stage-dependent in the three experiments. Overall, CNLs were observed at the systemic level only on plants inoculated with LINV-Fr at young stages, from 16 to 22 days after sowing. No plant showed these kind of symptoms when inoculated 28 days after sowing or later (Table 2). The percentage of plants showing CNLs at the systemic level varied greatly among different experiments (2.4% for Girelle-Mo3 plants inoculated 22 days after sowing in experiment 1 to 96% for plants inoculated 20 days after sowing in experiment 3), which could be related to the climatic conditions (lowest percentage in winter, intermediate in spring and highest in summer) or to the inoculum concentration. As previously, no evidence of virus detection in leaves showing CNLs was obtained by ELISA. The presence of LINV-Fr was also checked by RT-PCR in plant samples corresponding to experiment 2 (Fig. 1). A strong PCR signal was obtained for the Girelle susceptible control on inoculated leaves, and no evidence of virus presence was obtained by RT-PCR on the inoculated leaves of L. virosa PIVT1398 showing tiny necrotic lesions. On Girelle-Mo3 inoculated leaves showing larger necrotic lesions, a large number of samples reacted positively in RT-PCR (10/13 tested plants inoculated 16 days after sowing; 1/5 tested plants inoculated 28 days after sowing), although only faint DNA bands were visible, indicative of low virus titres (Fig. 1). For Girelle-Mo3 plants, positive PCR signals were also observed for apical leaves in six of 10 tested plants showing CNLs at the systemic level (four of these six plants are shown in Figure 1). In contrast, no signal was observed in apical leaves of the other, symptomless plants (0/10 tested plants). In symptomless apical leaves collected on plants that showed the CNLs, LINV-Fr was only rarely detected by PCR (1/6 tested plants with a faint DNA band; Fig. 1). Leaves of the plants belonging to Girelle-Mo3 that showed CNLs were also used to inoculate plants of Lactuca spp. genotypes, either LINV-Fr susceptible or resistant. For this, a pool of leaves of the four plants that showed CNLs in experiment 1 were used. Plants of the susceptible genotypes developed symptoms at a rate varying from 44% (L. virosa LS238; 11/25 plants) to 56% (L. sativa 'Girelle'; 28/50 plants). In addition to the absence of virus detection in ELISA and the absence of, or weak signals in RT-PCR, this confirmed the low virus titre in leaves showing CNLs. In contrast, no infection was obtained in inoculated plants of the resistant genotypes L. virosa PIVT1398 and Girelle-Mo3 (0/25 each), suggesting the absence of resistance-breaking LINV-Fr mutant in leaves showing CNLs. As a control, backinoculations from Girelle plants with symptoms to Girelle led to 100% (10/10) of plants with symptoms.

Figure 1

 1 Figure 1 Detection of LINV-Fr by RT-PCR. Each lane corresponds to leaf RNA extracts from one individual plant. The arrow indicates a faint band corresponding to LINV-Fr. Gi: Girelle; HC: healthy control; IL: introgression line Girelle-Mo3; apic1: apical leaf showing chlorotic/necrotic lesions; apic2: apical leaf with no chlorotic/necrotic lesions; inoc: inoculated leaf; PIVT: Lactuca virosa PIVT1398.

Table 1

 1 Symptoms observed on apical uninoculated leaves in Lactuca spp. genotypes inoculated with LINV-Fr, 20 days after sowing. indicate significant differences between the introgression lines Girelle-Mo3 and Mantilia-Mo3 for a given date (Fisher exact test; p=0.005).

	Plant genotype	Symptoms 14 dpi a	Symptoms 38 dpi
	L. virosa PIVT1398	0/14	0/14
	L. virosa LS238	14/14 Mo b , vein clearing	14/14 Mo
	L. sativa 'Girelle'	14/14 Mo, stunting	14/14 dead
	Girelle-Mo3	39/180x c CNL d (21.7%)	13/180y stunting (7.2%)
	L. sativa 'Mantilia'	14/14 Mo, stunting	14/14 dead
	Mantilia-Mo3	14/98x CNL (14.3%)	0/98x

a days post inoculation. b mosaic. c x and y d chloro-necrotic lesions.

Table 2

 2 Symptoms observed on apical uninoculated leaves in the introgression line Girelle-Mo3 inoculated with LINV-Fr, LMV-9 or LMV-13 at different growth stages. In each experiment, L. sativa 'Girelle' susceptible controls showed 100% infection, either with mosaic and wilting (with LINV-Fr) or mosaic only (with LMV). In contrast, L. virosa PIVT1398 plants did not show any infection at the systemic level.

	Experiment	Virus	Plant stage at	Plants showing symptoms	A405 values b
			inoculation a	on apical leaves / total
	1	LINV-Fr	18	3 / 36 CNL c	1.6
	1	LINV-Fr	22	1 / 41 CNL	1.1
	1	LINV-Fr	28	0 / 38
	1	LINV-Fr	34	0 / 38
	2	LINV-Fr	16	13 / 30 CNL d	1.5
	2	LINV-Fr	28	0 / 30
	3	LINV-Fr	20	24 / 25 CNL	Not tested
	3	LINV-Fr	28	0 / 22
	3	LMV-9	20	0 / 25
	3	LMV-9	28	0 / 22
	3	LMV-13	20	25 / 25 Nec e	32.0
	3	LMV-13	28	1 / 22 Nec	6.9

Acknowledgements

The authors thank Joël Béraud, Nathalie Truglio and Michel Pascal in the Plant Pathology Unit of INRA for their efficient help in plant growing. They would also like to thank Hervé Lot and Sylvie Souche of the Plant Pathology Unit of INRA and Yannick Bellec and Eric Martin of the Plant Breeding Unit of INRA for production of L. sativa Mo3 introgression lines, and Anders Kvarnheden and two anonymous reviewers for the thorough review of the manuscript.

Supporting Information


B A Bolou Bi

B Moury

K Abo

F Sorho

M Cherif

G Girardot B , N'dri

P Kouassi

D Kone

Survey of viruses infecting open-field pepper crops in Côte d'Ivoire and diversity of Pepper veinal mottle virus and Cucumber mosaic virus

Keywords: pepper, Capsicum spp., Potyvirus, Pepper veinal mottle virus, Cucumovirus, Cucumber mosaic virus, resistance

The prevalence of viruses in pepper crops grown in open fields in the different agro-ecological zones (AEZs) of Côte d'Ivoire was surveyed. Pepper veinal mottle virus (PVMV; genus Potyvirus) and Cucumber mosaic virus (CMV; genus Cucumovirus) were the most frequent viruses among those surveyed, while tobamoviruses (genus Tobamovirus) were detected at low frequency. PVMV showed a high heterogeneity across AEZs, which may be related to climatic, ecological or agronomical conditions, whereas CMV was more homogeneously distributed.

The molecular diversity of CMV and PVMV were analyzed from partial genome sequences. In spite of a low number of CMV isolates characterized, two molecular groups were revealed, one corresponding to IA subgroup and the other to reassortants between IA and IB subgroups.

RNAs 1 and 3 of the reassortants clustered with the IB subgroup of CMV isolates whereas their RNA 2 clustered with the IA subgroup. Importantly, RNA 1 of CMV isolates of the IB subgroup has been shown to be responsible for adaptation to pepper resistance. The diversity of PVMV in the VPg-and coat protein-coding regions revealed multiple clades. The central part of the VPg showed a high level of amino acid diversity and evidence of positive selection, which may be a signature of adaptation to plant recessive resistance. As a consequence, for efficient deployment of resistant pepper cultivars, it would be desirable to examine the occurrence of virulent isolates in the CMV or PVMV populations in Côte d'Ivoire and to follow their evolution as the resistance will become more largely deployed.

Introduction

Pepper belongs to the family Solanaceae and the genus Capsicum. It is grown in all regions of Côte d'Ivoire throughout the year. Pepper crops are generally rainfed but irrigation is necessary during the dry season spanning from November to May. The best yields are obtained with plantations in March-April for rainy season production and in September-October for dry season production [START_REF] Fondio | Bien cultiver le piment en Côte d'Ivoire. Fiche technique du Centre National de Recherche Agronomique (CNRA)[END_REF]. The latest official reports of FAO showed that the pepper production in Côte d'Ivoire could reach 32,900 tons/year and is therefore an important cash crop for growers (FAOSTAT, 2012).

Diseases, including viral ones, are one of the major limitations to pepper production throughout the world by reducing fruit quality and quantity. In Côte d'Ivoire, four pepper viruses have been described: Cucumber mosaic virus (CMV; genus Cucumovirus, family Bromoviridae), Pepper veinal mottle virus (PVMV; genus Potyvirus, family Potyviridae), Tobacco mosaic virus (TMV) and Pepper mild mottle virus (PMMoV; genus Tobamovirus, family Virgaviridae) [START_REF] Fauquet | Maladies virales des plantes en Côte d'Ivoire[END_REF]. Recently, Pepper vein yellows virus (PepVYV; genus Polerovirus, family Luteoviridae) was found in association with PVMV and CMV on pepper in the East of Côte d'Ivoire [START_REF] Bi | First report of Pepper vein yellows virus in field-grown pepper in Ivory Coast[END_REF].

PVMV, which is the most devastating pepper virus in West Africa, was found for the first time in Ghana [START_REF] Brunt | Pepper veinal mottle virus: a new member of the Potyvirus group from peppers (C. annuum and C. frutescens) in Ghana[END_REF], then in Nigeria, the African country with the highest pepper production [START_REF] Lana | An unusual new virus, possibly of the potyvirus group, from pepper in Nigeria[END_REF], and in Côte d'Ivoire on Nicotiana tabacum [START_REF] Wijs | Pepper veinal mottle virus in Ivory Coast[END_REF], the weed Physalis angulata and Capsicum spp. [START_REF] Fauquet | Maladies virales des plantes en Côte d'Ivoire[END_REF]. In Côte d'Ivoire, 100% losses of marketable fruits due to PVMV infection have been reported, causing field abandonment before harvest and making cultivation of pepper not profitable in some areas [START_REF] Fauquet | Maladies virales des plantes en Côte d'Ivoire[END_REF]. Pepper plants infected by PVMV show various symptoms depending on the isolate agressiveness, age of plants at the time of infection and pepper cultivar.

The symptoms exhibited on leaves include mild mottle, mosaic, vein banding, ringspots, various types of necrosis, discoloration and deformation. Severe stunting of the whole plant is also frequently observed [START_REF] Green | Characteristics and control of viruses infecting peppers. A literature review. Asian Vegetable Research and Development Centre[END_REF]. In addition, symptoms may appear on leaves, stems, flowers and fruits. In Côte d'Ivoire, PVMV was shown to be transmitted in nonpersistent and non-circulative manner by aphids of the species Aphis gossypii, Aphis spiraecola and Toxoptera citricidus [START_REF] Wijs | Pepper veinal mottle virus in Ivory Coast[END_REF]. CMV has a very broad host range, including over 1000 species in more than 85 botanical families. As PVMV, it is transmitted from plant to plant in natural epidemic conditions in a non-permanent and non-circulative manner by aphids. TMV, in addition to pepper, infects tomato, tobacco, eggplant and some wild species in Côte d'Ivoire [START_REF] Fauquet | Maladies virales des plantes en Côte d'Ivoire[END_REF]. PMMoV, in contrast, has a more restricted natural host range that includes mostly members of the genus Capsicum [START_REF] Brunt | Plant Viruses Online: Descriptions and Lists from the VIDE Database[END_REF]. Like other tobamoviruses, TMV and PMMoV are highly contagious and the primary sources of infection are contaminated seeds and soil (Toyoda et al., 2004). Secondary infections occur through contact between plants and plant handling. Because of the non-persistent mode of transmission of PVMV and CMV, insecticide spraying is often inefficient to control them in the field [START_REF] Afouda | Occurrence and distribution of viruses infecting tomato and pepper in Alibori in northern Benin[END_REF]. Therefore, control methods include mostly prophylaxis and plant genetic resistance [START_REF] Caranta | A complementation of two genes originating from susceptible Capsicum annuum lines confers a new and complete resistance to pepper veinal mottle virus[END_REF][START_REF] Fajinmi | Evaluation of pepper intercropped with tall companion crop in the management of Pepper veinal mottle virus potyvirus disease and its vectors or cultivated pepper (Capsicum annuum L.) in Nigeria[END_REF].

The goal of the present study was to estimate the prevalence and diversity of the main viruses infecting pepper crops in Côte d'Ivoire in order to evaluate the possibility to control them with adequate resistance genes. sampled per field, depending on the field acreage. Immediately after collection, samples were placed in a plastic bag and stored in a mobile refrigerator. They were subsequently kept desiccated on calcium chloride and stored at 4°C.

Materials and Methods

Sample collection

Virus detection

Samples were tested by double antibody sandwich-enzyme-linked immunosorbent assay (DAS-ELISA) for infection by PVMV, CMV, PVY, TMV and PMMoV with polyclonal antisera produced by INRA Pathologie Végétale unit [START_REF] Moury | Serological, molecular, and pathotype diversity of Pepper veinal mottle virus and Chili veinal mottle virus[END_REF][START_REF] Janzac | Characterization of a new potyvirus infecting pepper crops in Ecuador[END_REF]. Samples were considered positive when absorbance values at 405 nm (A405) were at least three times greater than the mean absorbance value of five healthy control samples.

Total RNAs were extracted from 5 mg of dried leaf tissue from samples that reacted positively to PVMV or CMV in DAS-ELISA using the Tri-Reagent kit (Molecular Research Center, Cincinnati, USA) according to the manufacturer's instructions. Two-step reverse transcriptionpolymerase chain reactions (RT-PCR) were then performed using PVMV-or CMV-specific primers (Supplementary Table 2) as described by [START_REF] Moury | Serological, molecular, and pathotype diversity of Pepper veinal mottle virus and Chili veinal mottle virus[END_REF]. For PVMV, two different genome regions were targeted, encoding either the viral genome-linked protein (VPg) or coat protein (CP). For CMV, three different regions corresponding to the three genomic RNAs were targeted. RT-PCR amplicons were sequenced directly with primers PVMV-VPg-rv, PVMV-CP-rv, 1Cons-AS1, G2-CrFLP-AS or G3-CrFLP-AS (Supplementary Table 2) by Genoscreen (Lille, France).

Molecular variability of PVMV and CMV isolates

Sequence quality was checked using ChromasPro version 1.7.6 (Technelysium Pty Ltd, Australia). Sequence alignments were performed using the ClustalW program implemented in the MEGA7 software [START_REF] Kumar | MEGA7: Molecular Evolutionary Genetics Analysis version 7.0 for bigger datasets[END_REF] and gaps, only present at the beginning or end of the sequence alignments, were removed. Phylogenetic analyses were performed using the neighbour-joining (NJ), maximum-likelihood (ML) and maximum-parsimony (MP) methods implemented in MEGA7 and the robustness of the tree topology was evaluated with 500 bootstrap resamplings. These methods were run with the optimal nucleotide-substitution model selected by MEGA7. Sequences obtained from isolates from Côte d'Ivoire were also compared to sequences from reference isolates or to additional sequences retrieved from sequence databases using the BLASTn program [START_REF] Altschul | Basic local alignment search tool[END_REF].

To identify codon sites with positive selection, i.e. sites showing a dN/dS ratio between the nonsynonymous (dN) and the synonymous (dS) substitution rates in an alignment of amino acid-coding sequences significantly higher than 1.0, ML approaches implemented in the HyPhy (Kosakovsky Pond & Frost, 2005a) package were performed using the NJ phylogenetic tree obtained previously. In HyPhy, the SLAC (single-likelihood ancestor counting), FEL (fixedeffects likelihood), IFEL (internal fixed-effects likelihood), REL (random-effects likelihood) and FUBAR (fast unbiased Bayesian approximation) methods (Kosakovsky Pond & Frost, 2005a, 2005b;[START_REF] Pond | Adaptation to different human populations by HIV-1 revealed by codon-based analyses[END_REF][START_REF] Murrell | FUBAR: a fast, unconstrained Bayesian approximation for inferring selection[END_REF] were used to detect individual codon positions subjected to positive selection. In addition, the Branch-site REL method [START_REF] Pond | A random effects branch-site model for detecting episodic diversifying selection[END_REF] was used to detect episodic positive selection along particular branches of the phylogenetic tree and the MEME (mixed-effects model of evolution) method [START_REF] Murrell | Detecting individual sites subject to episodic diversifying selection[END_REF] was used to detect episodic positive selection affecting individual codon positions.

Results

Virus prevalence

From all samples showing virus-like symptoms, 27.4% (142/525) were positive for at least one virus (Table 1). There was no clear association between symptoms observed in the field and the detected viruses, maybe because of coinfection of plants with several viruses or because of the confounding effect of other biotic or abiotic stresses. This may also be due to the presence of other, untested viruses like poleroviruses [START_REF] Bi | First report of Pepper vein yellows virus in field-grown pepper in Ivory Coast[END_REF], which detection is inefficient on desiccated leaf material, or begomoviruses. Begomoviruses have not been described in pepper crops in Côte d'Ivoire but are responsible for yellow vein disease in pepper in neighboring countries like Burkina Faso and Mali (Tiendrébéogo et al., 2011). The most prevalent virus was PVMV with 21.2 % (92/525) positive samples, followed by CMV detected in 11% (52) of samples. PMMoV and TMV were detected at low frequencies, with respectively 2.5% (13) and 1.3% (7) positive samples. Since serological cross-reactivity occurs between TMV and other tobamoviruses like ToMV (Tomato mosaic virus), the precise species to which belongs the virus detected by DAS-ELISA remains to be determined. No sample was found infected by PVY. Overall, the prevalence of PVMV was significantly higher than that of CMV, and both PVMV and CMV showed highly significantly higher prevalence than TMV, PMMoV or PVY (Table 1).

A statistical analysis of virus prevalence among AEZs was performed for the four AEZs (I, II, IV and VI) where more than 12 plants were sampled. The highest virus prevalence (combining the four detected viruses) was recorded in AEZ II (52.2%). It was significantly higher than virus prevalence in AEZ IV (33.3%), and both were significantly higher than virus prevalence in AEZs I (18.1%) and VI (11.8%). The prevalence of PVMV was highly heterogeneous among AEZs. PVMV was not detected in AEZ VI, whereas it reached a maximum of 36.7% in AEZ II. In contrast, the prevalence of CMV was more homogeneous among AEZs, varying from 5.7 (AEZ I) to 14.0% (AEZ IV), if we consider only the AEZs with substantial sampling effort (n>11). Consequently, climate and/or ecological differences in the different AEZs seem to play a major direct or indirect role in determining the distribution of PVMV infection, and to a lower extent of CMV infection on pepper in open fields in Côte d'Ivoire.Molecular variability of CMV isolates Partial sequences, 307 to 521 nucleotide long, were obtained for the three RNAs of Ivorian CMV isolates. Some sequences showed a large number of ambiguous nucleotides (double peaks) in the sequence chromatograms and were consequently discarded in further analyses.

These ambiguities result most probably from mixed infections of plants by several isolates. However, sequences were kept when there was a marked difference in the peak heights and at these positions, the predominant nucleotide in the virus population was considered in further analyses. Sequence accession numbers are MG334359 to MG334384. Similar tree topologies were obtained with the three different phylogenetic methods NJ, ML and MP. Concerning RNAs 1 and 3, two groups of Ivorian isolates could be distinguished, clustering either with group IA or IB reference isolates (Fig. 2A and2C). Since the IB subgroup of CMV is not monophyletic for RNAs 1 and 3 (Tepfer et al., 2016), we looked for the IB CMV isolates that shared the highest nucleotide identity with the Ivorian isolates using BLASTn. For RNA 1, the most similar isolate was from Egypt (accession number KT921314, 97% nucleotide identity) followed by isolates from India, Malaysia and Italy (Vir isolate) (89-94% identity). For RNA 3, the most similar isolates were from China and Thailand (98% identity), followed by a large number of isolates from South-east Asia and Tunisia (96-98% identity).

In contrast, all RNA 2 sequences clustered with the subgroup IA reference isolate (Fig. 2B). These groups were supported by high bootstrap values (≥76%). No Ivorian isolate clustered with the group II reference isolate. Sequences of the three viral RNAs could be analyzed for four Ivorian isolates only, named 350, 406, 563 and 566. Isolates 350 and 406 had a typical genome structure of IA group, whereas isolates 563 and 566 had a reassortant type of genome structure, clustering with group IB for RNAs 1 and 3 and with group IA for RNA 2. All five isolates clustering with group IB for one or the other genome segment were collected in the region of Bongouanou (AEZ IV, central-Western part of Côte d'Ivoire; Fig. 1). In contrast, isolates of group IA are distributed in most agro-ecological zones.

Molecular variability of PVMV isolates

As with CMV, a number of VPg-and CP-coding sequences of PVMV isolates could not be exploited because of a large number of nucleotide ambiguities, most likely the result of nucleotide variation within the virus population. A 479-nucleotide-long alignment of VPgcoding sequences was obtained with 42 Ivorian isolates collected for this study (accession numbers MG334290 to MG334331), one older Ivorian reference isolate (accession number DQ009807; date and location of collection unknown) and one Ghanaian isolate (accession number DQ645484). Using the best-fit nucleotide substitution model (Tamura 3 + ) in the MEGA7 software, the mean pairwise distance between sequences was 0.15 (maximum pairwise distance 0.41). Similar tree topologies were obtained with the different phylogenetic methods.

Based on bootstrap supports ≥69% in the maximum-likelihood tree, five PVMV groups could be distinguished, one (group 3) including the older Ivorian isolate (Fig. 3A). The Ghanaian For the CP-coding region, a 588-nucleotide-long sequence alignment was obtained with 26

Ivorian isolates collected for this study (accession numbers MG334332 to MG334357), the older Ivorian reference isolate (accession number MG334358) and 13 sequences from African or Asian isolates available in GenBank. Using the best-fit nucleotide substitution model (Tamura 3 + ) in the MEGA7 software, the mean pairwise distance between sequences was 0.08 (maximum pairwise distance 0.29). Again, similar tree topologies were obtained with the different phylogenetic methods. Based on bootstrap supports ≥62% in the maximum-likelihood tree, three PVMV groups could be distinguished (Fig. 3B). The majority of Ivorian isolates (15/26) clustered in group 3. This group contains isolates from different parts of Côte d'Ivoire.

Four isolates collected in Gonaté, in the central-Western region of Côte d'Ivoire, clustered in group 1, which is related to a group of isolates collected in Ghana and Asia. Four isolates collected in the regions of Bongouanou or Gonzagueville (Eastern or South-eastern Côte d'Ivoire) clustered in group 2. One isolate (number 447) collected in the region of Bongouanou clustered with an isolate from Senegal. Finally, two Ivorian isolates also collected in the region of Bongouanou (isolates 563 and 573) did not cluster with additional isolates based on a threshold of 50% bootstrap support.

Several isolates belonged to different phylogenetic groups for the VPg-and CP-coding regions (Fig. 3). For example, isolate 465 clustered in group 5 with isolates 516, 517 and 518 for the VPg-coding region, whereas these isolates were split into two separate groups for the CP-coding region (group 3 for isolate 465 and group 1 for isolates 516, 517 and 518). Similarly, isolates 228, 438 and 439 clustered together in group 2 for the CP-coding region, whereas they were split into group 3 (isolate 228), group 1 (isolate 439) or were not related to any group (isolate 438) for the VPg-coding region. This suggests that recombination events may have affected PVMV evolution. An alternative hypothesis is that some plants were mixed-infected by several PVMV variants and that the primers used amplified preferentially one or another variant depending on the genome region. The tree of the CP-coding region, which includes a set of non-Ivorian PVMV isolates, is also indicative of long-range dissemination of PVMV. This is suggested by the clustering of Ivorian isolate 447 with an isolate from Senegal and by the proximity between group 1 of Ivorian isolates and a group containing all Asian isolates characterized to date. This group includes also a Ghanaian isolate.

The sequences of the VPg-coding region of PVMV were analyzed for amino acid polymorphism and evidence of positive selection. The region spanning from amino acid position 103 to 125 of PVMV VPg is of particular relevance since it corresponds to the region of PVY VPg (amino acid positions 101 to 123) which determines adaptation to pepper recessive resistance mediated by the eukaryotic translation initiation factor 4E (eIF4E) encoded by alleles of the pvr2 gene [START_REF] Ruffel | A natural recessive resistance gene against potato virus Y in pepper corresponds to the eukaryotic initiation factor 4E (eIF4E)[END_REF][START_REF] Ayme | Different mutations in the genome-linked protein VPg of Potato virus Y confer virulence on the pvr2 3 resistance in pepper[END_REF][START_REF] Charron | Natural variation and functional analyses provide evidence for co-evolution between plant eIF4E and potyviral VPg[END_REF] (Fig. 4A).

Moreover, alleles of the pvr2 gene and of the pvr6 gene, encoding the eIFiso4E isoform, were shown to confer resistance to PVMV [START_REF] Caranta | A complementation of two genes originating from susceptible Capsicum annuum lines confers a new and complete resistance to pepper veinal mottle virus[END_REF]Ruffel et al., 2006). Ten different PVMV haplotypes were observed based on the amino acid diversity in this VPg region for the Ivorian isolates collected in this study (Fig. 4B). The older Ivorian isolate and the Ghanaian isolate (accession DQ645484) represent two more haplotypes. One haplotype was predominant (24/42 isolates), one had an intermediate prevalence (7/42) and the others were represented once or twice only. A large amino acid diversity was observed at positions 117 and 122, with four and five different amino acids, respectively. Since these positions correspond to positions in PVY VPg that are virulence determinants and/or are subjected to positive selection (Fig. 4A), we performed an analysis of positive selection for PVMV as well, using the Hyphy software suite (Table 2). Among the six algorithms used, two (SLAC and FUBAR) failed to detect positive selection in the VPg-coding sequence. Position 82 of the VPg was detected by the FEL and IFEL methods. Positions 117 and 119 were detected by one method only (REL and MEME, respectively). The strongest evidence of positive selection was obtained for position 122 that was detected by three different methods and, for two of them, with strong statistical support (pvalue ≤10 -4 ). Interestingly, positions 117 and 122 of PVMV VPg aligned with positions 115 and 120 of PVY VPg that were shown to be responsible for adaptation to eIF4E-mediated recessive resistance in pepper [START_REF] Ayme | Different mutations in the genome-linked protein VPg of Potato virus Y confer virulence on the pvr2 3 resistance in pepper[END_REF]. In addition, the Branch-site REL algorithm identified four internal branches in the phylogenetic tree that corresponded to episodic positive selection (Fig. 3A). These branches linked groups 2, 3 and 4 of PVMV isolates, and a subgroup of group 1 excluding isolate 419, to the rest of the tree.

Discussion

In this survey of viruses infecting open-field pepper crops in Côte d'Ivoire, PVMV and CMV were found most prevalent. Prevalence of PVMV was highly heterogeneous. It was absent from AEZ VI corresponding to Sudanian savanna and reached 36.7% of samples in AEZ II (Table 1). Environmental conditions, including climate and soil, may directly or indirectly affect virus prevalence. AEZ VI was the driest sampled zone, even though it corresponds to a humid tropical savanna area. Indirect environmental effects on virus prevalence include vegetation type (different crops and weeds that may host the virus) and structure (more or less isolated patches of host plants), aphid populations that are virus vectors and agricultural practices. Contrasting with PVMV, prevalence of CMV was more homogeneous between AEZs, spanning from 5.7% to 14.0% for AEZs significantly sampled (>11 samples). CMV has a much larger host range than PVMV, including many alternative hosts to pepper. By ensuring the possibility for CMV to persist in alternative hosts during the period of absence of pepper crops, this difference may explain the observed relative homogeneity of CMV prevalence.

Control of CMV or PVMV epidemics relies mainly on prophylaxis and plant genetic resistance.

Polygenic resistance to CMV, combining several QTLs (quantitative trait loci) acting on different steps of plant infection, has been used to create highly-resistant pepper cultivars, including F1 hybrids (Caranta et al., 1997a[START_REF] Caranta | QTLs involved in the restriction of cucumber mosaic virus (CMV) long-distance movement in pepper[END_REF][START_REF] Palloix | Advanced breeding for virus resistance in plants[END_REF]. However, a recent epidemiological survey in Tunisia showed that several CMV isolates were adapted to the resistant pepper cultivar 'Milord' [START_REF] Tamarzizt | Cucumber mosaic virus populations in Tunisian pepper crops are mainly composed of virus reassortants with resistance-breaking properties[END_REF]. One common property shared by all CMV isolates virulent towards pepper resistance was the fact that they had a IB-type RNA 1. Noticeably, RNA 1 of IB-type CMV isolates determines also virulence towards on plants carrying the Cmr1 resistance gene that restricts the systemic movement of CMV group IA in pepper [START_REF] Kang | Helicase domain encoded by Cucumber mosaic virus RNA1 determines systemic infection of Cmr1 in pepper[END_REF]). As a consequence, since several Ivorian CMV isolates are reassortants with a IB-type RNA 1 (Fig. 2), their virulence towards pepper resistance should be carefully examined before resistance deployment.

Resistance in pepper towards PVMV has also been studied extensively. A bigenic recessive resistance to PVMV has been identified [START_REF] Caranta | A complementation of two genes originating from susceptible Capsicum annuum lines confers a new and complete resistance to pepper veinal mottle virus[END_REF], which results from the combination of resistance alleles at the pvr2 locus encoding the eIF4E and a natural knock-out allele of the pvr6 gene encoding the eIFiso4E isoform (Ruffel et al., 2006). The phenotype is a total resistance, with no virus detection in the inoculated organs or at the systemic level. Among resistance alleles at the pvr2 locus, only alleles pvr2 1 , pvr2 2 and pvr2 7 were shown to complement pvr6 for PVMV resistance [START_REF] Rubio | Allele mining in the pepper gene pool provided new complementation effects between pvr2-eIF4E and pvr6-eIF(iso)4E alleles for resistance to pepper veinal mottle virus[END_REF]. PVMV-resistant pepper cultivars (with the pvr2 1 + pvr6 or pvr2 2 + pvr6 combinations) are being released in Africa but are yet poorly deployed in Côte d'Ivoire (Bolou Bi A, personal observations). A polygenicresistance against potyvirus E, that belongs to a particular strain of PVMV [START_REF] Moury | Serological, molecular, and pathotype diversity of Pepper veinal mottle virus and Chili veinal mottle virus[END_REF], has also been studied in pepper. It has a quantitative phenotype, with symptoms at the systemic level being milder and expressing later, and it includes two QTLs encompassing the pvr2 and pvr6 loci (Caranta et al., 1997b). This resistance has not been included in breeding programs. The central part of the VPg of PVY shows a large amino acid diversity and evidence of positive selection at some amino acid positions [START_REF] Moury | Interaction patterns between Potato virus Y and eIF4E-mediated recessive resistance in the Solanaceae[END_REF][START_REF] Khalifa | Tunisian Potato virus Y isolates with unnecessary pathogenicity towards pepper: support for the matching allele model in eIF4E resistance-potyvirus interactions[END_REF]. The high rate of amino acid substitutions in this small region was explained by the selection of virulence properties against eIF4E-mediated recessive resistance in several crops belonging to the family Solanaceae including pepper [START_REF] Ayme | Different mutations in the genome-linked protein VPg of Potato virus Y confer virulence on the pvr2 3 resistance in pepper[END_REF][START_REF] Moury | Interaction patterns between Potato virus Y and eIF4E-mediated recessive resistance in the Solanaceae[END_REF][START_REF] Khalifa | Tunisian Potato virus Y isolates with unnecessary pathogenicity towards pepper: support for the matching allele model in eIF4E resistance-potyvirus interactions[END_REF].

The observation of a similarly high level of amino acid diversity and evidence of positive selection in the corresponding region of PVMV VPg (Table 2; Fig. 4) is surprising because of the rarity of grown PVMV-resistant pepper cultivars. Several hypotheses can be raised to explain this evolution pattern of PVMV VPg. First, virulence towards the pvr2 + pvr6 combination of resistance genes in pepper involving amino acid substitutions in PVMV VPg could have been selected in spite of the low frequency of grown resistant pepper cultivars.

Second and more plausibly, amino acid substitutions in PVMV VPg may have been responsible

for PVMV adaptation to other, unstudied plant resistances in pepper, other crops that are hosts of PVMV like tomato or African eggplant (Solanum aethiopicum) or wild plants. Third, the high amino acid substitution rate in the central part of VPg may not exclusively be related to plant host adaptation. This protein region was shown to show a high level of intrinsic disorder [START_REF] Hébrard | Intrinsic disorder in viral proteins genome-linked: experimental and predictive[END_REF], which may allow the virus to tolerate a high amino acid substitution rate.

Whatever the reason, this high diversity raises some threats about the future deployment of PVMV-resistant pepper cultivars, since some VPg mutations may be responsible for the occurrence of virulent variants in PVMV populations or may facilitate the future acquisition of virulence properties through additional mutations, which would impair resistance durability.

Surveys of PVMV diversity and evolution as the resistance will be deployed would therefore be highly desirable to predict potential resistance breakdowns.

  Leaf samples were collected from plants showing symptoms of possible viral etiology from 2014 to 2015 in the different agro-ecological zones (AEZs) of Côte d'Ivoire (Fig. 1; Supplementary

  isolate and two Ivorian isolates did not cluster with additional isolates based on this bootstrap threshold or a lower stringency threshold of 50% bootstrap support. The largest group 1 contained isolates from different regions of Côte d'Ivoire. Group 2 contained two isolates from central Côte d'Ivoire (Yamassoukro and Gonaté). Group 3 contained three isolates from the South-East (Gonzagueville) and one isolate from central Côte d'Ivoire. Group 4 contained three isolates from central (Yamassoukro) or south-western (Gagnoa) part of Côte d'Ivoire and group 5 contained four isolates from central Côte d'Ivoire (Yamassoukro and Gonaté). Consequently, there was no clear association between geography and genetic clustering of PVMV isolates.

Figure 1

 1 Figure 1 Sampling sites in the different agro-ecological zones ("Zones agro-écologiques"-

Figure 2

 2 Figure 2 Unrooted maximum-likelihood phylogenetic trees of partial sequences

Figure 3 Figure 4

 34 Figure 3 Unrooted maximum-likelihood phylogenetic trees of partial sequences of Ivorian

Table 1

 1

  ). A total of 525 pepper plants were collected in open fields, including 73 Capsicum annuum and 452 C. chinense plants. Two to five plants with symptoms were

Table 2

 2 Amino acid positions of the VPg of PVMV isolates significantly affected by positive

	selection a
						Algorithm
	Amino acid	FEL	IFEL		REL		MEME
	position in VPg	dN/dS	P	dN/dS	P	dN/dS	P	dN/dS	P
	82	∞ b	0.084	∞ b	0.017
	117					2.5	<10 -5
	119							NA c	0.077
	122			5.7	0.030	2.6	10 -4	NA	5×10 -6
	a The analysis was performed with the HyPhy software suite (Kosakovsky Pond and Frost
	2005a) and conducted on 38 non-redundant sequences containing the eight 3'-proximal
	codons of the 6K2-coding region and the 151 5'-proximal codons (on a total of 191) of the
	VPg-coding region. When available, the estimated dN/dS value is indicated for detected
	codon positions. A p-value threshold of 0.10 was considered.

b Estimate of dN/dS was infinite because of the lack of synonymous substitutions (dS=0). c NA, not available.

Acknowledgments

Thank is due to ECOWAS (Economic Community of West African States) through the PACER program and to colleagues at the Plant Physiology laboratory in Félix Houphouët-Boigny University and at INRA Pathologie Végétale in Montfavet for their support.

CP cistron a Y: C or T; W: A or T; R: A or G; H: A, C or T; N: A, C, G or T.

b Referring to CMV isolate I17F (accession numbers HE793683, HE793684 and Y18137 corresponding to RNAs 1, 2 and 3, respectively). c Referring to PVMV isolate ns1 (accession number FJ617225).