# Serological, molecular, and pathotype diversity of pepper veinal mottle virus and chili veinal mottle virus

Benoît Moury, Alain Palloix, Carole Caranta, Patrick Gognalons, Sylvie

Souche, Kahsay Gebre Selassie, Georges Marchoux

► To cite this version:

Benoît Moury, Alain Palloix, Carole Caranta, Patrick Gognalons, Sylvie Souche, et al. Serological, molecular, and pathotype diversity of pepper veinal mottle virus and chili veinal mottle virus. Phytopathology, 2005, 95 (3), pp.227-232. 10.1094/PHYTO-95-0227. hal-02683216

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# Serological, Molecular, and Pathotype Diversity of *Pepper veinal mottle virus* and *Chili veinal mottle virus*

Benoît Moury, Alain Palloix, Carole Caranta, Patrick Gognalons, Sylvie Souche, Kahsay Gebre Selassie, and Georges Marchoux

First, fourth, fifth, sixth, and seventh authors: Station de Pathologie Végétale, Institut National de la Recherche Agronomique, F-84143 Montfavet cedex, France; and second and third authors: Unité de Génétique et d'Amélioration des Fruits et Légumes, Institut National de la Recherche Agronomique, F-84143 Montfavet cedex, France.
Accepted for publication 26 October 2004.

## ABSTRACT

Moury, B., Palloix, A., Caranta, C., Gognalons, P., Souche, S., Gebre Selassie, K., and Marchoux, G. 2005. Serological, molecular, and pathotype diversity of *Pepper veinal mottle virus* and *Chili veinal mottle virus*. Phytopathology 95:227-232.

Variability within the pepper-infecting potyviruses *Pepper veinal mottle virus* (PVMV) and *Chili veinal mottle virus* (ChiVMV) in Africa and Asia was investigated. Coat protein (CP) gene sequence diversity revealed three clades that corresponded to three geographic locations and there was no evidence of presence of the ChiVMV/Asian group in western or central Africa. These clades included closely related isolates that potentially belong to two viral species, which is consistent with current nomenclature. These clades could not be unambiguously identified with poly-

clonal antisera; however, reverse transcription-polymerase chain reactions allowed differentiation of the isolates into two species based on a large indel in the CP gene. PVMV and ChiVMV isolates were classified into three and two pathotypes, respectively, in relation to pepper genotypes carrying different resistance factors. Specificity of resistance only partially corresponded to molecular diversity of the isolates. Only one isolate of PVMV could infect pepper genotypes carrying the two recessive genes *pvr6* and *pvr2<sup>2</sup>*; however, these genotypes were not infected by PVMV in field trials in Senegal, despite a high prevalence of PVMV in the surrounding pepper plants.

*Additional keyword: etiology.*

Five *Potyvirus* spp. commonly affect pepper crops (*Capsicum* spp.). *Potato virus Y* (PVY) is distributed worldwide and is the only *Potyvirus* sp. affecting pepper crops in Europe. *Tobacco etch virus* (TEV) and *Pepper mottle virus* (PepMoV) are prevalent in America (25,30), whereas *Pepper veinal mottle virus* (PVMV) and *Chili veinal mottle virus* (ChiVMV) are common in Africa and Asia, respectively (5). These geographic distributions are not absolute because TEV was isolated sporadically in Turkey (42), Sudan (23), India (4), and China (B. Moury, data not shown) and ChiVMV has been isolated in eastern Africa (27).

PVMV first was isolated in pepper (*Capsicum* spp.) and petunia (*Petunia × hybrida* (Hook.) Vilm.) in eastern Ghana in 1971 (5). Since then, PVMV was isolated in several African countries, mostly in sub-Saharan regions (17,18) but also in Tunisia (15) and the Near East (40). The natural hosts of PVMV include pepper, tomato (*Lycopersicon esculentum* Mill.), tobacco (*Nicotiana tabacum* L.), eggplant (*Solanum melongena* L.), tomato eggplant (*S. integrifolium* Poir.), petunia, *S. nigrum* L., *Datura metel* L., *D. stramonium* L., *Physalis angulata* L., *P. micrantha* Link, and *Telfairia occidentalis* Hook. F. (1,3,5,14). ChiVMV (synonyms include *Pepper vein-banding mosaic virus* [39], *Pepper vein-banding virus* [20], or *Chili vein-banding mottle virus* [34]), is the most important virus in pepper crops throughout eastern Asia.

The use of conventional phytosanitary practices is often inefficient against these potyviruses because they spread rapidly in the field through nonpersistent transmission by aphids. Thus, resistant

cultivars remain the most economical and reliable method of control. In pepper, few sources of resistance to PVMV have been characterized. Combination of the *pvr6* gene, originating from the Indian cv. Perennial, and of the *pvr2<sup>1</sup>* or *pvr2<sup>2</sup>* alleles confers high level resistance to PVMV (7; data not shown), whereas neither *pvr6*, *pvr2<sup>1</sup>*, or *pvr2<sup>2</sup>* alone confer any resistance to this virus. Perennial also is resistant to potyvirus E (a virus related to PVMV isolated from pepper in Ethiopia in 1977; K. Gebre Selassie, unpublished data) and this resistance involves six additive or epistatic quantitative trait loci (QTLs) (6).

A network program, including breeders and geneticists from Africa, Asia, and West Indies, was initiated to create pepper cultivars resistant to the most damaging diseases (29). To facilitate breeding of pepper cultivars with wide-spectrum *Potyvirus* sp. resistance and deployment on a regional scale, we provide here an evaluation of the serological, molecular, and pathotype diversity of PVMV and ChiVMV in relation to sources of resistance.

## MATERIALS AND METHODS

**Virus isolates.** Isolates related to PVMV, ChiVMV, or both (Table 1) were used to develop serological and molecular diagnostic methods and to study host range in solanaceous hosts. PVY isolate To-72 (13) also was used for serological tests and represented a more distantly related *Potyvirus* sp. In addition, two surveys of pepper production fields or experimental trials were conducted in Senegal in May 2001 (in the Dakar, Thiès, and Saint Louis du Sénégal areas) and May 2002 (in the Dakar and Mboro areas). In the Dakar area, 60 different pepper genotypes, including 15 breeding lines homozygous for both the *pvr2<sup>2</sup>* and *pvr6* alleles, were grown and surveyed for virus infection. Leaf and fruit samples were collected from plants showing symptoms of possible viral etiology and further tested by enzyme-linked immunosorbent

<footnote>Corresponding author: B. Moury; E-mail address: moury@avignon.inra.fr</footnote>

<footnote>DOI: 10.1094/PHYTO-95-0227
This article is in the public domain and not copyrightable. It may be freely re-
printed with customary crediting of the source. The American Phytopathological
Society, 2005.</footnote>
assay (ELISA) for infection by PVMV, ChiVMV, PVY, *Tomato spotted wilt virus* (TSWV), *Tobacco mosaic virus* (TMV), *Pepper mild mottle virus* (PMMoV), and *Cucumber mosaic virus* (CMV).

**Serological tests.** Several polyclonal antisera were produced in our laboratory or were gifts from other researchers (26) (Table 2). Double-antibody sandwich (DAS)-ELISAs were performed on samples from infected pepper plants (cv. Yolo Wonder) as described previously (24). Samples were considered positive when absorbance values were at least three times greater than the mean absorbance value of five healthy control samples.

**Sequence analyses.** Three degenerate oligonucleotide primers (Table 3, A, B, and C) were defined in regions of the NIB and coat protein (CP) genes where conservation was observed at the amino acid level among PVY, PepMoV, TEV, and ChiVMV. RNA was extracted from infected plant material (Tri Reagent kit; Molecular Research Center Inc., Cincinnati, OH) and a reverse transcription (RT) step was performed with the polyT primer (Table 3) and

*Avian myeloblastosis virus* reverse transcriptase (Promega Corp., Madison, WI). Two overlapping cDNA fragments covering the 3'-proximal terminus of the NIB gene, the entire CP gene, and the 3' nontranslated region (NTR) of PVMV were further amplified by polymerase chain reaction (PCR) using *Taq* DNA polymerase (Promega Corp.) and primers A and B or primers C and polyT. These RT-PCR fragments were purified, treated with the Klenow fragment of DNA polymerase I, and cloned into the *SmaI* site of pUC13. For each cDNA fragment, nucleotide sequence reactions of two clones were performed by Genome Express (Meylan, France). The sequence of the CP gene of PVMV isolates CAC2, F-Bot, S31, potyvirus E, and Y90/34 were deposited in GenBank under accession nos. AJ780966 to AJ780970. CP sequences from potyviruses infecting solanaceous crops (Table 1) were aligned using the ClustalW program, version 1.8 (37). These alignments were used to define PCR primers D and E (Table 3) for specific detection and differentiation of PVMV and ChiVMV. Phyloge-

<table><caption>TABLE 1. *Potyvirus* isolates compared</caption><thead><tr><th>Virus<sup>a</sup></th><th>Isolate</th><th>Country</th><th>Collected by donor or reference</th><th>GenBank accession no.</th></tr></thead><tbody><tr><td>PVMV</td><td>CAC2</td><td>Senegal</td><td>A. Palloix</td><td>AJ780966</td></tr><tr><td>PVMV</td><td>CAC3</td><td>Senegal</td><td>H. Laterrot</td><td>...</td></tr><tr><td>PVMV</td><td>CAC4</td><td>Senegal</td><td>H. Laterrot</td><td>...</td></tr><tr><td>PVMV</td><td>CAC94</td><td>Senegal</td><td>H. Laterrot</td><td>...</td></tr><tr><td>PVMV</td><td>F-Bot</td><td>Cameroon</td><td>R. Nono Womdim</td><td>AJ780967</td></tr><tr><td>PVMV</td><td>S23</td><td>Ghana</td><td>A. A. Brunt</td><td>...</td></tr><tr><td>PVMV</td><td>S31</td><td>Ghana</td><td>A. A. Brunt</td><td>AJ780968</td></tr><tr><td>PVMV</td><td>Y90/34</td><td>Yemen</td><td>D. Walkey</td><td>AJ780969</td></tr><tr><td>PVMV</td><td>Potyvirus E</td><td>Ethiopia</td><td>K. Gebre Selassie</td><td>AJ780970</td></tr><tr><td>PVMV</td><td>IC</td><td>Ivory Coast</td><td>J. J. De Wijs; 15</td><td>...</td></tr><tr><td>PVMV</td><td>TU2</td><td>Tunisia</td><td>15</td><td>...</td></tr><tr><td>ChiVMV</td><td>Taiwan</td><td>China (Taiwan)</td><td>S. K. Green</td><td>...</td></tr><tr><td>ChiVMV</td><td>Beijing</td><td>China</td><td>B. Moury</td><td>...</td></tr><tr><td>ChiVMV</td><td>India</td><td>India</td><td>20</td><td>AJ237843</td></tr><tr><td>ChiVMV</td><td>Japan</td><td>Japan</td><td>...</td><td>AB012221</td></tr><tr><td>ChiVMV</td><td>Thai</td><td>Thailand</td><td>8</td><td>U72193</td></tr><tr><td>PVY</td><td>To-72</td><td>France</td><td>13</td><td>...</td></tr><tr><td>PVY</td><td>SON41</td><td>France</td><td>24</td><td>AJ439544</td></tr><tr><td>PVY</td><td>N</td><td>France</td><td>32</td><td>D00441</td></tr><tr><td>PVY</td><td>O</td><td>Canada</td><td>33</td><td>U09509</td></tr><tr><td>PepSMV</td><td>...</td><td>Argentina</td><td>31</td><td>X66027</td></tr><tr><td>PepYMV</td><td>...</td><td>Brazil</td><td>19</td><td>AF348610</td></tr><tr><td>PepMoV</td><td>C</td><td>United States (California)</td><td>38</td><td>M96425</td></tr><tr><td>PepMoV</td><td>FL</td><td>United States (Florida)</td><td>41</td><td>AF501591</td></tr><tr><td>PTV</td><td>PPK13</td><td>Peru</td><td>35</td><td>AJ437280</td></tr><tr><td>PVV</td><td>M97</td><td>United Kingdom</td><td>28</td><td>AJ253123</td></tr><tr><td>PVV</td><td>PA10</td><td>Peru</td><td>35</td><td>AJ516021</td></tr><tr><td>WPMV</td><td>...</td><td>Peru</td><td>36</td><td>AJ437279</td></tr><tr><td>TVMV</td><td>...</td><td>...</td><td>11</td><td>X04083</td></tr><tr><td>PVA</td><td>HER</td><td>Finland</td><td>21</td><td>AJ131400</td></tr><tr><td>PVA</td><td>U</td><td>United States</td><td>21</td><td>AJ131402</td></tr><tr><td>TEV</td><td>HAT</td><td>...</td><td>2</td><td>M11458</td></tr><tr><td>TEV</td><td>NW</td><td>United States (Louisiana)</td><td>9</td><td>L38714</td></tr></tbody></table>

<sup>a</sup> PVMV, *Pepper veinal mottle virus*; ChiVMV, *Chili veinal mottle virus*; PVY, *Tomato virus Y*; PepSMV, *Pepper severe mosaic virus*; PepYMV, *Pepper yellow mosaic virus*; PTV, *Peru tomato virus*; PVV, *Potato virus V*; WPMV, *Wild potato mosaic virus*; TVMV, *Tobacco vein-mottling virus*; PVA, *Potato virus A*; TEV, *Tobacco etch virus*.

<table><caption>TABLE 2. Reactivity of *Pepper veinal mottle virus* (PVMV), *Chili veinal mottle virus* (ChiVMV), and *Potato virus Y* (PVY) in infected plant extracts with polyclonal antibodies by double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA)</caption><thead><tr><th rowspan="2">Virus species/isolate</th><th colspan="6">Sources of polyclonal antibodies<sup>a</sup></th></tr><tr><th>F-Bot<br>INRA</th><th>PVMV 374/94<br>H. J. Vetten</th><th>Y90/34<br>D. G. A. Walkey</th><th>Potyvirus E<br>INRA</th><th>ChiVMV<br>Japan</th><th>PVY To-72<br>INRA</th></tr></thead><tbody><tr><td>PVMV/CAC2, CAC3, CAC4, CAC94, F-Bot, IC, S23, S31</td><td>++</td><td>+</td><td>++</td><td>+</td><td>+</td><td>0</td></tr><tr><td>PVMV/Y90-34</td><td>+</td><td>+</td><td>++</td><td>++</td><td>++</td><td>0</td></tr><tr><td>PVMV/potyvirus E</td><td>+</td><td>++</td><td>++</td><td>++</td><td>++</td><td>0</td></tr><tr><td>ChiVMV/Taiwan, Beijing, Thai</td><td>0</td><td>0</td><td>+</td><td>+</td><td>++</td><td>0</td></tr><tr><td>PVY/To-72</td><td>+</td><td>+</td><td>0</td><td>+</td><td>0</td><td>++</td></tr></tbody></table>

<sup>a</sup> Results of DAS-ELISA: 0 = no serological reaction (absorbances at 405 nm [$A_{405}$] <3 times the healthy control); + = weak serological reactions ($A_{405}$ between 3 and 5 times the healthy control); ++ = strong serological reactions ($A_{405}$ >20 times the healthy control). There were no reactions between 6 and 19 times the healthy control.
<figure><img src="image_2.png" /><figcaption>**Fig. 1.** Unrooted neighbor-joining phylogenetic tree representing an alignment of 708 nucleotides at the 3' end of the coat protein gene of potyviruses infecting solanaceous crops. Bootstrap analysis was applied using 1,000 bootstrap samples. Bootstrap values >70% at internal nodes are reported. The scale bar represents the relative genetic distance.</figcaption></figure>

tree topology was obtained (Fig. 1). Bootstrap values associated to the branches of the tree supported the existence of two major clades (Fig. 1). The first clade includes PVY, PepMoV, *Pepper yellow mosaic virus*, *Pepper severe mosaic virus*, *Peru tomato virus*, *Potato virus V*, and *Wild potato mosaic virus*, as mentioned by Spetz et al. (35). The second one includes PVMV and ChiVMV. Viruses within the first clade do not share common geographic distributions or plant host ranges. Thus, the evolutionary forces that drove speciation within this group are as yet unknown and may be complex. The PVMV/ChiVMV clade comprises three subgroups: the east Asian ChiVMV isolates, the Ethiopian and Yemeni PVMV isolates, and the west and central African PVMV isolates, which correspond to the groups obtained with pairwise similarities (Fig. 1; Table 4). It is possible that geographic isolation was responsible for diversification within this group.

**Specific detection of PVMV and ChiVMV by RT-PCR.**

Based on the sequences of the N1b and CP genes available for PVMV, ChiVMV, PVY, PepMoV, and TEV, we defined primers D and E (Table 3) to specifically detect viruses of the PVMV/ChiVMV group and to distinguish PVMV from ChiVMV. Primer D is a degenerate primer that corresponds to the 'GKAPYI' amino acid motif common to the N1b protein of PVMV, ChiVMV, PVY, and PepMoV but which is slightly different from the 'GKAPYL' motif in TEV. Primer E corresponds to the 'TDMSLAR' amino acid motif conserved in the CP of PVMV, ChiVMV, and TEV but not in PVY and PepMoV. Therefore, RT-PCR experiments conducted with primers D and E are expected to give a 737-bp DNA fragment with PVMV isolates, a 788-bp DNA fragment with ChiVMV, and no DNA amplification with PVY or PepMoV. Based on sequences available in GenBank, TEV is expected to differ at one or two nucleotide positions in the 3' end of primer D. Consequently, RT-PCR on TEV RNA with primers D and E could

<figure><img src="image_3.png" /><figcaption>**Fig. 2.** Reverse transcription-polymerase chain reaction products obtained with primers D and E (Table 3) using RNA extracts from pepper plants infected with individual potyviruses or potyvirus combinations. Lanes 1 to 3, *Pepper veinal mottle virus* (PVMV) isolates; lanes 4 and 5, *Chili veinal mottle virus* (ChiVMV) isolates; lane 6, size marker; lane 7, PVMV and ChiVMV mixed infection; lane 8, healthy control; and lane 9, *Potato virus Y*.</figcaption></figure>

give a 737-bp DNA fragment or no amplification at all, depending on the sequence and PCR conditions. RT-PCR experiments conducted on RNA extracts from PVMV isolates F-Bot, S31, CAC2, Y90/34, and potyvirus E gave a DNA product of the expected size. The specificity of primers D and E was evaluated further with PVMV isolates CAC3, CAC4, CAC94, CAF1, and S23; with ChiVMV isolates Taiwan, Beijing, and Thai; and with a limited number of reference isolates of PVY, TEV, and PepMoV (Table 1). Only isolates from the PVMV/ChiVMV group amplified a DNA product. The ChiVMV and PVMV isolates produced DNA fragments of expected size (Fig. 2). RT-PCR experiments conducted with primers D and E on RNA extracted from a Yolo Wonder pepper plant infected with both isolates F-Bot of PVMV and Taiwan of ChiVMV produced the same two fragments obtained separately from each isolate (Fig. 2). Consequently, these primers could be used to reveal mixed infections. Moreover, the single-fragment profiles obtained by RT-PCR from the PVMV and ChiVMV isolates under study indicated that these isolates are not mixtures of PVMV and ChiVMV.

**Host range of PVMV.** In all, 10 PVMV and 3 ChiVMV isolates revealed few differences in host range among several solanaceous species (data not shown). All 13 isolates induced systemic mosaic symptoms in *N. benthamiana*, *N. glutinosa*, and *Petunia × hybrida* whereas none infected *S. melongena*. Y90/34 was the only isolate able to systemically infect *D. stramonium*. Symptomless infections were observed in *N. tabacum* with the four PVMV isolates from Senegal (CAC2, CAC3, CAC4, and CAC94) and the three ChiVMV isolates. Symptomless infection of tomato with PVMV isolates CAC2, CAC4, S23, S31, and IC also was observed.

Five pepper genotypes known to possess resistance factors against potyviruses were evaluated (Table 5). Yolo Wonder and Florida VR2 were infected by all isolates tested and exhibited systemic mosaic symptoms. CM334 was susceptible to all isolates of PVMV and ChiVMV but exhibited a delay in onset and reduced intensity of systemic symptoms after inoculation with PVMV isolates Y90/34 and potyvirus E and ChiVMV isolate Taiwan. CM334 carries the two *Potyvirus* sp. resistance genes *Pvr4* and *pvr5*. *Pvr4* confers a high level of resistance to PVY and PepMoV isolates, with no virus detection in the inoculated or apical leaves, whereas *pvr5* confers a phenotypically similar resistance to PVY pathotype 0 (10). The partial resistance of CM334 to potyvirus E was found to be linked to *pvr5* (A. Palloix, unpublished data). However, we do not know if the same gene or genes also confer partial resistance to PVMV isolate Y90/34 and to ChiVMV isolate Taiwan. We also do not know if the same gene in the *pvr5* region confers the high-level resistance to PVY 0 and the partial resistance to potyvirus E.

The DH801 line possessing the *pvr2*² and *pvr6* genes gave the broadest resistance and did not support systemic infection by PVMV or ChiVMV (Table 5). This was confirmed during multiple inoculation tests performed in the breeding program for resis-
<table><caption>TABLE 5. Pathogenicity of *Pepper veinal mottle virus* (PVMV) and *Chili veinal mottle virus* (ChiVMV) isolates on pepper genotypes carrying various resistance systems<sup>a</sup></caption><thead><tr><th rowspan="2">C. annuum genotype</th><th rowspan="2">Potyvirus resistance genes<sup>b</sup></th><th colspan="3">PVMV isolates</th><th colspan="2">ChiVMV isolates</th></tr><tr><th>CAC2</th><th>CAC3</th><th>CAC4</th><th>CAC94</th><th>IC</th><th>F-Bot</th><th>S23</th><th>S31</th><th>Y90/34</th><th>potyvirus E</th><th>Beijing</th><th>Thai</th><th>Taiwan</th></tr></thead><tbody><tr><td>Yolo Wonder</td><td>...</td><td>S</td><td></td><td></td><td>S</td><td>S</td><td></td><td></td><td></td><td></td><td>S</td><td>S</td><td>S</td><td>S</td></tr><tr><td>Florida VR2</td><td><i>pvr2</i><sup>22</sup></td><td>S</td><td></td><td></td><td>S</td><td>S</td><td></td><td></td><td></td><td></td><td>S</td><td>S</td><td>S</td><td>S</td></tr><tr><td>CM334</td><td><i>Pvr4</i>, <i>pvr5</i></td><td>S</td><td></td><td></td><td>r</td><td>S</td><td></td><td></td><td></td><td></td><td>S</td><td>r</td><td></td><td></td></tr><tr><td>Perennial</td><td>Several QTLs, <i>pvr6</i></td><td>S</td><td></td><td></td><td>R<sup>c</sup></td><td>R</td><td></td><td></td><td></td><td></td><td>R</td><td>R</td><td></td><td></td></tr><tr><td>DH801<sup>d</sup></td><td><i>pvr2</i><sup>22</sup>, <i>pvr6</i></td><td>R<sup>c</sup></td><td></td><td></td><td>R</td><td>R</td><td></td><td></td><td></td><td></td><td>R</td><td>R</td><td></td><td></td></tr></tbody></table>

tance to potyviruses (10 tests performed between 1994 and 2004). However, during the introgression of the digenic resistance from DH801 into bell pepper lines, 1 of 120 plants displayed delayed symptoms on fruit following inoculation with the IC isolate of PVMV. Back-inoculations of DH801 plants with virus extracted from this fruit showed 100% (45 of 45 inoculated plants) systemic infection. This adapted PVMV isolate showed the same host range and symptom expression as isolate IC, except for DH801 plants.

‘Perennial’ showed resistance to East African and Asian PVMV or ChiVMV isolates but not to West African PVMV isolates. The phenotype of the resistance was similar to that of DH801. Consequently, Perennial could be used to breed pepper cultivars resistant to PVMV and ChiVMV in Asia. However, more isolates in these groups should be evaluated for pathogenicity on Perennial. Characterization of as few as three ChiVMV isolates with five pepper genotypes revealed pathogenicity differences (Table 5), suggesting that much variability exists within ChiVMV. The resistance of Perennial to potyvirus E was shown to be conferred by four additive and two epistatic QTLs (6). It remains to be determined if the same QTLs (or some of them) are effective against PVMV isolate Y90/34 and especially against ChiVMV isolates, which are more distant. Perennial is the only known pepper genotype with broad resistance corresponding to the geographic distribution and phylogenetic grouping of PVMV and ChiVMV isolates. In spite of the relatively small number of isolates tested, an explanation could be that acquisition or loss of pathogenicity toward Perennial was more ancestral than acquisition or loss of pathogenicity toward CM334 or DH801.

**Incidence of PVMV in pepper in Senegal.** During surveys in 2001 and 2002, severe mosaic symptoms in pepper were observed on more than 50% of the plants. More rarely, necrosis on leaves and an irregular ripening of bell pepper fruit were observed. In 2001 and 2002, 14 of 28 and 25 of 26 samples, respectively, from plants showing virus symptoms reacted positively for at least one virus in DAS-ELISA. Detection of the viruses was efficient in both leaves and fruit. Each year, positive serological reactions to PVMV or ChiVMV were more frequent than any other tested virus and recorded for all locations sampled. CMV also was detected in 2001 (three samples) and PMMoV was detected in 2002 (one sample). RT-PCR experiments with primers D and E were conducted on 17 isolates that reacted positively both to PVMV and ChiVMV in DAS-ELISA. Only DNA fragments with the specific size corresponding to PVMV were obtained. This suggested that ChiVMV was not present in the collected samples and confirmed that serological cross reactions occurred between ChiVMV and PVMV.

The DH801 pepper genotype and 15 breeding lines homozygous for both the *pvr2*<sup>2</sup> and *pvr6* alleles tested negative for PVMV and ChiVMV in DAS-ELISA despite a high prevalence of the virus in the surrounding plants in the trials. Therefore, either

appearance of virulent isolates is a rare event or virulent isolates are less fit and do not spread efficiently in the agroecosystem. However, it should be emphasized that no pepper cultivars with resistance to PVMV presently are grown on a large scale in the region. If PVMV variants able to overcome the resistance conferred by DH801 can be generated, their frequency may be expected to increase rapidly if cultivars with resistance derived from DH801 are deployed.

## ACKNOWLEDGMENTS

We thank C. Duranton and R. N. Womdim (Technisem-Tropicam, Senegal) and Z. Baoxi and W. Lihao (IVF-CAAS, China) for assistance in collecting virus samples, and D. Fargette for reading the manuscript before its submission.

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<footnote><sup>a</sup> S = accumulation of virus and symptom expression at the systemic level; r = accumulation of virus but few symptoms at the systemic level; R = no virus accumulation at the systemic level.</footnote>

<footnote><sup>b</sup> According to Kyle and Palloix (22).</footnote>

<footnote><sup>c</sup> Polygenic resistance to potyvirus E (6).</footnote>

<footnote><sup>d</sup> Doubled-haploid lines derived from the F<sub>1</sub> hybrid between Florida VR2 and Perennial.</footnote>

<footnote><sup>e</sup> Digenic resistance due to a complementation between *pvr2*<sup>2</sup> and *pvr6* characterized against S23, F-Bot, and IC (7).</footnote>
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