relative incidence, spatial distribution and genetic diversity of cucurbit viruses in eastern spain

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Annals of Applied Biology ISSN 0003-4746 RESEARCH ARTICLE Relative incidence, spatial distribution and genetic diversity of cucurbit viruses in eastern Spain M. Juarez 1 , P. Legua 1 , C.M. Mengual 2 , M.A. Kassem 2 , R.N. Sempere 2 , P. G ´ omez 2 , V. Truniger 2 & M.A. Aranda 2 1 Escuela Polit ´ ecnica Superior de Orihuela, Universidad Miguel Hern ´ andez de Elche, Orihuela, Alicante, Spain 2 Departamento de Biolog´ ıa del Estr ´ es y Patolog´ ıa Vegetal, Centro de Edafolog´ ıa y Biolog´ ıa Aplicada del Segura (CEBAS)-CSIC, Espinardo, Murcia, Spain Keywords Cucurbit; epidemiology; evolution; genetic diversity; mixed infections; virus. Correspondence Miguel A. Aranda, Centro de Edafolog´ ıa y Biolog´ ıa Aplicada del Segura (CEBAS), Apdo. Correos 164, 30100 Espinardo, Murcia, Spain. Email: [email protected] Received: 3 December 2012; revised version accepted: 25 March 2013. doi:10.1111/aab.12029 Abstract Viral diseases that could cause important economic losses often affect cucurbits, but only limited information on the incidence and spatial distribution of specific viruses is currently available. During the 2005 and 2006 growing seasons, systematic surveys were carried out in open field melon (Cucumis melo), squash and pumpkin (Cucurbita pepo), watermelon (Citrullus lanatus) and cucumber (Cucumis sativus) crops of the Spanish Community of Valencia (eastern Spain), where several counties have a long standing tradition of cucurbit cultivation and production. Surveyed fields were chosen with no previous information as to their sanitation status, and samples were taken from plants that showed virus-like symptoms. Samples were analysed using molecular hybridisation to detect Beet pseudo-yellows virus (BPYV), Cucurbit aphid-borne yellows virus (CABYV), Cucumber mosaic virus (CMV), Cucumber vein yellowing virus (CVYV), Cucurbit yellow stunting disorder virus (CYSDV), Melon necrotic spot virus (MNSV), Papaya ring spot virus (PRSV), Watermelon mosaic virus (WMV) and Zucchini yellow mosaic virus (ZYMV). We collected 1767 samples from 122 independent field plots; out of these, approximately 94% of the samples were infected by at least one of these viruses. Percentages for the more frequently detected viruses were 35.8%, 27.0%, 16.5% and 7.2% for CABYV, WMV, PRSV and ZYMV, respectively, and significant deviations were found on the frequency distributions based on either the area or the host sampled. The number of multiple infections was high (average 36%), particularly for squash (more than 57%), with the most frequent combination being WMV + PRSV (12%) followed by WMV + CABYV (10%). Sequencing of WMV complementary DNA suggested that ‘emerging’ isolates have replaced the ‘classic’ ones, as described in southern regions of France, leading us to believe that cucurbit cultivation could be severely affected by these new, emerging isolates. Introduction Cucurbits include crop species of utmost importance for Mediterranean agriculture. Spain is among the main producers of cucurbit fruits in the world, with cucurbits production having a high economic value due to Spain’s specialisation in high quality and out of season production for export (FAO, 2007). Several counties of the Valencian Community (eastern Spain) have a longstanding tradition of cucurbits cultivation, in particular watermelon, melon, pumpkin, cucumber and squash. In these growing areas, as in others, virus-induced diseases represent an ever- increasing problem for cucurbits cultivation. Indeed, more than 35 different viruses have been described infecting cucurbits (Provvidenti, 1996), and at least ten of them have been identified in Spanish cucurbit crops causing infections with serious economic repercussions (Luis- Arteaga et al., 1998; Kassem et al., 2007). Among them are Cucumber mosaic virus (CMV), Cucurbit aphid-borne yellows virus (CABYV), Papaya ring spot virus (PRSV), Watermelon mosaic virus (WMV) and Zucchini yellow mosaic virus (ZYMV), transmitted by aphids; Beet pseudo-yellows 362 Ann Appl Biol 162 (2013) 362 – 370 © 2013 Association of Applied Biologists

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Annals of Applied Biology ISSN 0003-4746

R E S E A R C H A R T I C L E

Relative incidence, spatial distribution and genetic diversityof cucurbit viruses in eastern SpainM. Juarez1, P. Legua1, C.M. Mengual2, M.A. Kassem2, R.N. Sempere2, P. Gomez2, V. Truniger2 & M.A. Aranda2

1 Escuela Politecnica Superior de Orihuela, Universidad Miguel Hernandez de Elche, Orihuela, Alicante, Spain

2 Departamento de Biologıa del Estres y Patologıa Vegetal, Centro de Edafologıa y Biologıa Aplicada del Segura (CEBAS)-CSIC, Espinardo, Murcia, Spain

KeywordsCucurbit; epidemiology; evolution; genetic

diversity; mixed infections; virus.

CorrespondenceMiguel A. Aranda, Centro de Edafologıa y

Biologıa Aplicada del Segura (CEBAS), Apdo.

Correos 164, 30100 Espinardo, Murcia, Spain.

Email: [email protected]

Received: 3 December 2012; revised version

accepted: 25 March 2013.

doi:10.1111/aab.12029

Abstract

Viral diseases that could cause important economic losses often affect cucurbits,but only limited information on the incidence and spatial distribution of specificviruses is currently available. During the 2005 and 2006 growing seasons,systematic surveys were carried out in open field melon (Cucumis melo), squashand pumpkin (Cucurbita pepo), watermelon (Citrullus lanatus) and cucumber(Cucumis sativus) crops of the Spanish Community of Valencia (eastern Spain),where several counties have a long standing tradition of cucurbit cultivationand production. Surveyed fields were chosen with no previous informationas to their sanitation status, and samples were taken from plants that showedvirus-like symptoms. Samples were analysed using molecular hybridisationto detect Beet pseudo-yellows virus (BPYV), Cucurbit aphid-borne yellows virus

(CABYV), Cucumber mosaic virus (CMV), Cucumber vein yellowing virus (CVYV),Cucurbit yellow stunting disorder virus (CYSDV), Melon necrotic spot virus (MNSV),Papaya ring spot virus (PRSV), Watermelon mosaic virus (WMV) and Zucchiniyellow mosaic virus (ZYMV). We collected 1767 samples from 122 independentfield plots; out of these, approximately 94% of the samples were infected byat least one of these viruses. Percentages for the more frequently detectedviruses were 35.8%, 27.0%, 16.5% and 7.2% for CABYV, WMV, PRSV andZYMV, respectively, and significant deviations were found on the frequencydistributions based on either the area or the host sampled. The number ofmultiple infections was high (average 36%), particularly for squash (morethan 57%), with the most frequent combination being WMV + PRSV (12%)followed by WMV + CABYV (10%). Sequencing of WMV complementary DNAsuggested that ‘emerging’ isolates have replaced the ‘classic’ ones, as describedin southern regions of France, leading us to believe that cucurbit cultivationcould be severely affected by these new, emerging isolates.

Introduction

Cucurbits include crop species of utmost importance for

Mediterranean agriculture. Spain is among the main

producers of cucurbit fruits in the world, with cucurbits

production having a high economic value due to Spain’s

specialisation in high quality and out of season production

for export (FAO, 2007). Several counties of the Valencian

Community (eastern Spain) have a longstanding tradition

of cucurbits cultivation, in particular watermelon, melon,

pumpkin, cucumber and squash. In these growing areas,

as in others, virus-induced diseases represent an ever-increasing problem for cucurbits cultivation. Indeed, morethan 35 different viruses have been described infectingcucurbits (Provvidenti, 1996), and at least ten of themhave been identified in Spanish cucurbit crops causinginfections with serious economic repercussions (Luis-Arteaga et al., 1998; Kassem et al., 2007). Among themare Cucumber mosaic virus (CMV), Cucurbit aphid-borneyellows virus (CABYV), Papaya ring spot virus (PRSV),Watermelon mosaic virus (WMV) and Zucchini yellow mosaicvirus (ZYMV), transmitted by aphids; Beet pseudo-yellows

362 Ann Appl Biol 162 (2013) 362–370© 2013 Association of Applied Biologists

M. Juarez et al. Cucurbit viruses in Valencia

virus (BPYV), Cucurbit yellow stunting disorder virus (CYSDV)and Cucumber vein yellowing virus (CVYV), transmittedby whiteflies; and Melon necrotic spot virus (MNSV),transmitted by a soil fungus. Previous reports haveemphasised the relative importance of CABYV and WMVin cucurbits in Spain in areas different from those in theValencian Community. For instance, CABYV was foundto be the prevalent virus infecting cucurbit crops of theMurcia region (South-eastern Spain), with incidencesreaching 100% of the plants in affected fields (Kassemet al., 2007), and WMV was found to have a veryhigh relative incidence in this and other Spanish areas(Luis-Arteaga et al., 1998; Moreno et al., 2004). Asregards to WMV, recent molecular epidemiology studiesof French isolates have suggested the introduction of new,more aggressive, ‘emerging’ (EM) isolates distant at themolecular level from the ‘classic’ (CL) isolates present inFrance for more than 30 years (Desbiez et al., 2009); thedata suggested that WMV-EM isolates did not spread inFrance over long distances, but rapidly replaced the pre-existing WMV-CL isolates in all sites where both groupsoccurred (Desbiez et al., 2009).

Here we describe the results of a systematic surveythat was carried out during two consecutive growingseasons (2005 and 2006) in cucurbit (watermelon,melon, pumpkin, cucumber and squash) crops of theValencian Community (Spain). Out of the nine differentviruses tested, CABYV was the most prevalent one.We also conducted an analysis for the partial geneticcharacterization of WMV isolates, showing that EMisolates have replaced the CL ones, as described for certainregions of France (Desbiez et al., 2009).

Materials and methods

Surveys and sample collection

Surveys were performed in open field melon (Cucumismelo), watermelon (Citrullus lanatus), cucumber (Cucumissativus), squash and pumpkin (Cucurbita pepo) crops ofthe Valencian Community during the 2005 and 2006growing seasons. Four main sampling zones were definedaccording to their agro-ecological conditions (Fig. 1):Zone A, including Vega Baja del Segura, Pilar de laHoradada and Campo de Elche. Zone B, including Vallede la Albaida and Alto Vinalopo counties, where a coolerclimate temporally displaces seasons with respect to otherzones. Zone C, including Campo del Turia, Ribera Bajaand Ribera Alta del Jucar, Huerta Norte and Sur ofValencia, where watermelon is the predominant cucurbitcultivated. Zone D, including Bajo Maestrazgo and PlanaBaja de Castellon, where intensive melon cultivationis predominant (Fig. 1). Samples were obtained fromrandomly distributed fields in these areas, and included

305 melon, 323 watermelon, 84 squash, 60 cucumberand 36 pumpkin samples from 59 fields for 2005, and406 melon, 397 watermelon, 84 squash, 48 cucumberand 24 pumpkin samples from 63 fields for 2006 (Fig. 1).Each field was visited and sampled at least twice, beforeand at the beginning of the harvest. Viral symptomsobserved included yellowing, mosaic, mottling, necrosisof the leaves and stems, vein clearing and leaf laminadistortions. Incidence of yellowing symptoms was visuallyevaluated in each field by examining 300–900 plantsfollowing a W-shaped itinerary, and expressed as thepercentage of total plants. A maximum of 20 samples perfield from symptomatic plants were collected. A sampleconsisted of two symptomatic leaves per plant.

Virus detection

Samples were analysed to determine the presence ofBPYV, CABYV, CMV, CVYV, CYSDV, MNSV, PRSV, WMVand ZYMV. Samples were used immediately after collec-tion for the detection of viruses by dot-blot hybridisation.Samples from plants showing yellowing symptoms wereadditionally analysed by tissue print hybridisation todetect the phloem-associated viruses BPYV, CABYV andCYSDV. Virus detection was performed as described byKassem et al. (2007). Note that the sum of the frequen-cies expressed in percentages exceeded 100 due to thepresence of multiple infections (see below).

Partial sequencing of WMV isolates

RNA extracts from 39 WMV-infected samples, preparedas in Gonzalez-Ibeas et al. (2012), were used as inKassem et al. (2007) to synthesize complementaryDNA (cDNA) to a region of 408 nt in the NIb andCP WMV cistrons. Samples were randomly pickedamong the whole collection of WMV-infected samples.Primers used were 5′-GGCTTCTGAGCAAAGATG-3′ and5′-CCCAYCAACTGTYGGAAG-3′ (Desbiez et al., 2009).RT-PCR products were purified from agarose gels afterelectrophoresis by using Geneclean turbo columns (MPBiomedicals, Illkrich, France) and sequenced (Secugen,Madrid, Spain) using the same primers.

Sequences from worldwide isolates were retrieved fromGenBank (http://www.ncbi.nlm.nih.gov/). The accessionnumbers of the corresponding sequences are listed in thecorresponding figures or tables.

Analysis of WMV nucleotide sequences

Nucleic and amino acid sequences were aligned usingClustalX (Thompson et al., 1997) and BioEdit (Biologicalsequence alignment editor, Tom Hall, Ibis biosciences,

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Cucurbit viruses in Valencia M. Juarez et al.

Figure 1 Areas surveyed in the Valencian Community (Spain). Four

main sampling zones were defined according to their agro-ecological

conditions: Zone A, including Vega Baja del Segura, Pilar de la Horadada

and Campo de Elche. Zone B, including Valle de la Albaida and Alto

Vinalopo counties, where a cooler climate temporally displaces seasons

with respect to other zones. Zone C, including Campo del Turia, Ribera

Baja and Ribera Alta del Jucar, Huerta Norte and Sur of Valencia, where

watermelon is the predominant cucurbit cultivated. Zone D, including

Bajo Maestrazgo and Plana Baja de Castellon, where intensive melon

cultivation predominates.

Carlsbad, CA USA) programmes. Putative recombina-tion events were evaluated using the RDP3 pack-age (http://darwin.uvigo.es/rdp/rdp.html) using defaultdetection thresholds. Phylogenetic analyses were per-formed with the MEGA 4 package (Tamura et al., 2007).Phylogenetic trees shown in this paper were constructedusing the minimum-evolution (ME) method with dis-tances estimated by the Kimura 2-parameter (K2P)method. A bootstrap value for each internal node of

the tree was calculated using 1000 random pseudorepli-cates. The ratio of non-synonymous (dN) to synonymous(dS) nucleotide substitutions (ω = dN/dS) was calculatedusing the Pamilo–Bianchi–Li method (Li, 1993). Toassess selection pressures, a maximum likelihood (ML)approach was used. In this case, non-synonymous andsynonymous substitution rates were estimated using theHyPhy package (http://www.hyphy.org) and the ratioof non-synonymous/synonymous substitution rates (ω)was calculated under the MG94 model as implementedin HyPhy.

Other statistical analyses

The distribution frequency of each virus was analysedusing a generalised linear mixed (GLM) model with bino-mial error structure and logit link function on binary datatesting for associations between the presence/absence ofvirus infection and the possible explanatory variables ofzone and year. Note that some crop species were not avail-able for all zones during 2005 and 2006 and therefore thisdata set was not fitted as a factor level when interactionterms were analysed. Analysis of potential associationsamong viruses in multiple viral infections was carriedout using a contingency table approach (Gomez et al.,2010), comparing the observed virus frequencies (pres-ence/absence) with those that would be expected if therewere no association between viruses of the correspondingsingle infection with a chi-squared test. All analyses werecarried out using JMP software, considering P-values lessthan 0.05 significant.

Results

Virus incidence per year, zone or host

We surveyed a total of 122 commercial fields in fourregions of the Valencian Community (eastern Spain) thathave distinct agro-ecological conditions (Fig. 1). Surveyswere conducted during two consecutive years, providing808 and 959 samples for 2005 and 2006, respectively. Wesampled 1767 symptomatic plants, of which 1658 (94%)were infected by at least one virus. CABYV was the mostprevalent virus, detected in about 35% of the infectedsamples, closely followed by WMV, which infected about27% of the samples (Table 1). PRSV followed in relativeimportance, with a frequency of around 16%. We foundthat ZYMV, MNSV, CMV, CVYV and CYSDV infected lessthan 10% of the total plants, and BPYV was not presentin any sample (Table 1). The presence of viruses changedsignificantly among agro-ecological zones (χ2 = 27.09,P < 0.001, d.f. 3) and crop species (χ2 = 52.08, P < 0.001,d.f. 4), and virus occurrence was not affected by theinteraction between year and zone (χ2 = 3.63, P = 0.304,

364 Ann Appl Biol 162 (2013) 362–370© 2013 Association of Applied Biologists

M. Juarez et al. Cucurbit viruses in Valencia

Table 1 Occurrence of virusesain cucurbits grown in different agro-ecosystems of the Valencian Community, Spain

Infected Samples (%)

Yearb Zonec Hostd

2005 2006 A B C D Melon Watermelon Cucumber Squash Pumpkin

Virus CABYV 251 (39.5) 342 (33.4) 294 (39.5) 71 (40.3) 137 (31.0) 91 (30.7) 259 (41.3) 190 (31.8) 46 (34.3) 62 (26.8) 36 (54.5)WMV 187 (29.4) 261 (25.5) 175 (23.5) 44 (25.0) 151 (34.2) 78 (26.4) 149 (23.8) 191 (31.9) 22 (16.4) 62 (26.8) 24 (36.4)PRSV 85 (13.4) 189 (18.5) 122 (16.4) 28 (15.9) 76 (17.2) 48 (16.2) 85 (13.6) 134 (22.4) 0 (0.0) 53 (22.1) 2 (3.0)ZYMV 33 (5.2) 86 (8.4) 36 (4.8) 12 (6.8) 32 (7.2) 39 (13.2) 22 (3.5) 36 (6.0) 33 (24.6) 28 (12.1) 0 (0.0)MNSV 29 (4.6) 62 (6.1) 42 (5.6) 0 (0.0) 16 (3.6) 33 (11.1) 75 (12.0) 16 (2.7) 0 (0.0) 0 (0.0) 0 (0.0)CMV 30 (4.7) 48 (4.7) 27 (3.6) 14 (8.0) 30 (6.8) 7 (2.4) 37 (5.9) 14 (2.3) 11 (8.2) 12 (5.2) 4 (6.1)CVYV 0 (0.0) 29 (2.8) 22 (3.0) 7 (4.0) 0 (0.0) 0 (0.0) 0 (0.0) 17 (2.8) 4 (3.0) 8 (3.5) 0 (0.0)CYSDV 20 (3.1) 6 (0.6) 26 (3.5) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 18 (13.4) 8 (3.5) 0 (0.0)BPYV 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0)

aViruses analysed were: Beet pseudo-yellows virus (BPYV), Cucurbit aphid-borne yellows virus (CABYV), Cucumber mosaic virus (CMV), Cucumber vein

yellowing virus (CVYV), Cucurbit yellow stunting disorder virus (CYSDV), Melon necrotic spot virus (MNSV), Papaya ring spot virus (PRSV), Watermelon

mosaic virus (WMV) and Zucchini yellow mosaic virus (ZYMV). Viruses were diagnosed by molecular hybridisation either in tissue prints or dot-blots

as described in Kassem et al. (2007). The total number of samples analysed was 1767 from 122 different fields in four agro-ecological regions (Fig. 1).

Samples were from plants showing virus-like symptoms.bPercentages of infection are relative to the number of infected samples, which were 635 and 1023 for years 2005 and 2006, respectively.cPercentages of infection are relative to the number of infected samples, which were 744, 176, 442 and 296 for Zones A, B, C and D, respectively.dPercentages of infection are relative to the number of infected samples, which were 627, 598, 134, 233 and 66 for melon, watermelon, cucumber,

squash and pumpkin, respectively.

d.f. 3; Table 1). Note that some crops were not alwaysavailable for all zones in both years, and therefore, thedata ‘host’ could not be included in this interactionanalysis. In contrast, when the distribution of virusfrequencies was compared, no significant differences werefound over the 2 years (χ2 = 2.88, P < 0.26, d.f. 8), whilevirus distribution appeared to be uneven among zones(χ2 = 198.62, P < 0.001, d.f. 24) and hosts (χ2 = 479.63,P < 0.001, d.f. 32). For instance, we did not detect MNSVin zone B, or CVYV in zones C and D, or CYSDV inzones B, C and D. Furthermore, the prevalent virusin Zone C was not CABYV (31%), but WMV (34%)(Table 1). However, CABYV was the prevalent virusfor all hosts except for watermelon, and WMV wasthe second most important virus in all hosts exceptcucumber, were ZYMV had significantly more relevance.Similarly, in squash, the incidence of PRSV seemed to beparticularly high, equalling that of CABYV; in cucumber,the two whitefly-transmitted viruses, CVYV and CYSDV,had higher incidences than in other hosts (Table 1).

During surveys, the proportion of plants showingyellowing symptoms was visually estimated for eachplot. Among the viruses studied, CABYV, CYSDV andBPYV are the three that are able to induce yellowingsymptoms. Given the scarce importance of CYSDV andBPYV deduced from the above results, an estimation ofyellowing incidence could provide an estimation of theCABYV incidence in the plots considered. Thus, in 36%and 76% of the plots, the estimated incidence of CABYVwas greater than 20% in 2005 and 2006, respectively;

CABYV was present in all plots, while in three and two ofthem, 100% of the plants showed yellowing symptomsduring 2005 and 2006, respectively. Symptoms of plantsinfected by CABYV resembled those described by Kassemet al. (2007) and Juarez et al. (2004).

Multiple infections

The number of multiple infections was approximately36% of the total number of infected samples. Interest-ingly, this proportion rose up to 57% for squash. Theanalysis of the distribution of frequencies for the differentviral combinations showed that there were significantdifferences among hosts (Table 2). For instance, in melon,watermelon and squash, the most frequent double infec-tion was WMV/PRSV, followed by WMV/CABYV andWMV/ZYMV. For these three crop species, the mostfrequent triple infection was WMV/PRSV/CABYV. Bycontrast, in cucumber, the prevalent combination wasWMV/ZYMV, and in pumpkin WMV/CABYV (Table 2).These differences may be a consequence of the differentfrequency distributions for the individual viruses (seeabove), or may be due to interactions among viruses thatare somehow selected by nature. In order to characteriseany potential bias in the association among the viruses,we followed a contingency table approach to assesswhether the presence of each virus in multiple infectionswas independent of the presence of the other(s), since theobserved frequencies did not deviate from those expectedin the case of no association. This statistical analysis

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Table 2 Multiple infections in cucurbits grown in the Valencian Community, Spain

Infected Samples (% of the Total Number of Infected Samples)b

Infectiona Melon Watermelon Cucumber Squash Pumpkin Total

DoubleWMV + PRSV 88 (14.0) 75 (12.5) 0 34 (14.6) 2 (3.0) 199 (12.0)WMV + CABYV 78 (12.4) 39 (6.5) 7 (5.2) 32 (13.7) 11 (16.7) 167 (10.0)WMV + ZYMV 32 (5.1) 25 (4.2) 13 (9.7) 23 (9.9) 0 93 (5.6)WMV + CMV 10 (1.6) 22 (3.7) 7 (5.2) 7 (3.0) 1 (1.5) 47 (2.8)

TripleWMV + CABYV + PRSV 19 (3.0) 11 (1.8) 0 19 (8.1) 0 49 (2.9)WMV + CABYV + ZYMV 3 (0.5) 3 (0.5) 5 (3.7) 16 (6.9) 0 27 (1.6)WMV + CVYV + PRSV 0 1 (0.2) 4 (3.0) 2 (0.8) 0 7 (0.4)WMV + CABYV + MNSV 6 (1.0) 0 0 0 0 6 (0.4)

More than threeWMV + CABYV + PRSV + ZYMV + MNSV 3 (0.5) 0 0 0 0 3 (0.2)

Total 239 (38.1) 176 (29.4) 36 (26.9) 133 (57.1) 14 (21.2) 598 (36.1)

aOnly the most frequent combinations are indicated.bThe total number of melon, watermelon, cucumber, squash and pumpkin samples infected by at least one of the viruses considered was 627, 598, 134,

233 and 66, respectively.

showed that there was a strong association betweenseveral viruses for multiple infections (Table 2), includingthe following combinations: for melon, WMV + PRSV(χ2 = 48.337, P < 0.001), WMV + ZYMV (χ2 = 37.907,P < 0.001), and WMV + CABYV + MNSV (χ2 = 20.838,P < 0.001); for watermelon, WMV + CABYV (χ2 = 19.09,P < 0.001), WMV + ZYMV (χ2 = 10.42, P < 0.001), andWMV + CMV (χ2 = 13.972, P < 0.001); for cucumber,WMV + CVYV + PRSV (χ2 = 28.57, P < 0.001); andfor squash, WMV + CABYV + ZYMV (χ2 = 47.273,P < 0.001).

WMV genetic diversity

The genetic diversity of the prevalent virus, CABYV,will be described elsewhere (M.A. Kassem, P. Gomez& M. A. Aranda, unpublished data). In this work,we decided to do a first approach to the diversity ofWMV, the second most important virus after CABYV.Almost a decade ago, Moreno et al. (2004) studied thediversity and evolution of WMV in Spain. They found ahighly homogeneous population in which recombinationseemed to have played a major role in diversitymaintenance (Moreno et al., 2004). Interestingly, a setof the WMV isolates characterised by Moreno et al.(2004) were from the geographic area surveyed in thisstudy; our preliminary analysis suggested that all theirisolates belonged to the CL group described by Desbiezet al. (2009). To test whether the structure of WMVpopulations sampled in this area had changed with time,we sequenced, as in Desbiez et al. (2009), a region of408 nt in the replicase and coat protein cistrons of 39 ofour WMV isolates. Before phylogenetic reconstruction,

recombination occurrence was tested using the suiteof programmes included in RDP3; no recombinationamong isolates was detected. Phylogenetic relationshipsamong WMV isolates were inferred by various methods,including minimum evolution, maximum parsimony andML; topologies inferred by the different methods were allsimilar. French isolates from each of the clades identifiedby Desbiez et al. (2009) were included in the analysis asreference. Interestingly, Valencian isolates clustered intofour main groups, three of them containing the referenceisolates of EM groups 1, 2 and 3, and one of themcontaining the reference isolates of CL group (Fig. 2A).EM clades contained most Valencian isolates except forfive, suggesting that EM types prevail in the WMVValencian populations. To test whether a replacement oftypes had occurred over time, sequences of WMV isolatesfrom the Mediterranean coastal area determined byMoreno et al. (2004) were included in a new phylogeneticanalysis. As shown in Fig. 2B, old Mediterranean isolatescluster with CL Valencian isolates, and none of themcluster with the new EM isolates (Fig. 2B). This resultsuggests a recent introduction of EM isolates in this areaand a partial replacement of CL by EM types.

We also estimated the relative rates of change atnon-synonymous (dN) and synonymous positions (dS)by ω = dN/dS to assay the direction and strength ofthe selection pressure acting on coding regions withinWMV populations. We found a ratio ω < 1, indicatingthat purifying selection was restricting variability in thepopulation. An alignment of the amino acid sequencesputatively encoded by the nucleotide sequences deter-mined in this study showed that CL isolates have thetypical KE insertion in the N-terminal part of the CP

366 Ann Appl Biol 162 (2013) 362–370© 2013 Association of Applied Biologists

M. Juarez et al. Cucurbit viruses in Valencia

Figure 2 Phylogenetic trees for WMV isolates constructed using the minimum-evolution method. Bootstrap values (1000 pseudoreplicates) above 50%

are shown. (A) NIb-CP partial sequences of 39 Spanish WMV isolates sequenced in this study in addition to eight Genbank isolates from different origins,

corresponding to distinct phylogenetic groups as described in Desbiez et al. (2009) and marked by (�). (B) CP partial sequences of Spanish WMV isolates:

39 isolates from 2005–2006 (this study) and 26 isolates from 1995 and 1999 (Moreno et al., 2004). The groups’ names are indicated on the right side of

the tree.

Ann Appl Biol 162 (2013) 362–370 367© 2013 Association of Applied Biologists

Cucurbit viruses in Valencia M. Juarez et al.

protein. Codons under selection were detected usingHyPhy. We found no significant evidence of positiveselection on any codon, but several codons were foundto be under negative selection (data not shown).

Discussion

In this article we have analysed a large number of cucurbitsamples to determine the presence of nine virusesbelonging to six different genera. The data presentedhere showed that CABYV was the most prevalent andwidespread virus in cucurbit crops of the Valencia region(Spain) during 2005–2006. Since its first detection inFrance (Lecoq et al., 1992), CABYV has been shownto be one of the most common cucurbit viruses in awide variety of areas and environments (Lecoq, 1999;Lecoq et al., 2003). Indeed, our previous surveys showedthat CABYV was also the prevalent virus in the Murciaregion (Spain) (Kassem et al., 2007). In contrast with thisobservation, the incidence of CABYV in Valencia did notseem to be as high as in Murcia, where it was close to 80%during the 2003–2004 seasons. Yearly differences mayexplain this contrasting data, but other ecological factorsmay play a role (Tomlinson, 2008): whereas the Murciaregion was rather homogeneous in agro-ecological terms,favouring the epidemic expansion of diseases, the regionsthat we have analysed in this work were more variedin terms of prevalent crop species, timing of cultivationand other agro-ecological factors. These differences can beused to explain the lesser incidence of CABYV in Valenciathan in Murcia, and also the observed differences amongthe distribution of frequencies of individual virusesamong zones within Valencia. Not surprisingly, therewere also differences among the distribution of virusfrequencies for different hosts. For example, the secondmost important virus in cucumber was ZYMV instead ofWMV; also for cucumber, the frequencies of whitefly-transmitted viruses were particularly high. This may bea consequence of a number of aspects intrinsic to thecrop species, including virus and vector susceptibility andpropensity, but also cultural practices, since in the caseof cucumber, most of the samples were from greenhousecrops, where higher incidences of whiteflies are knownto occur.

Data of significant importance refer to the number ofmultiple infections. On average for all species, we detectedmultiple infections in more than 36% of the infectedsamples, but for squash this figure increased up to approx-imately 57%. The most common double infection wasWMV + PRSV, closely followed by WMV + CABYV. Inter-estingly, a similar study performed during the same periodof time in Iran, showed that CABYV + WMV was also themost frequent combination in this country (Bananej &

Vahdad, 2008). However, surveys in other areas havepointed to lower percentages of multiple infections. In1998 in Lebanon, approximately 13% of melon and30% of squash plants showed multiple infections withZYMV/CABYV being almost the only virus combinationfound (Abou-Jawdah et al., 2000). In other studies inwhich CABYV was not included, double infections rangedbetween 0 and 15%. These studies included virusessuch as ZYMV, WMV, CMV and PRSV, and cucum-ber, melon and watermelon as hosts. The highest value(15%) was found for cucurbits in Turkey doubly infectedwith ZYMV and WMV (Luis-Arteaga et al., 1998; Sevik& Arli-Sokmen, 2003). In agreement with our data fromValencia, our survey in Murcia (Kassem et al., 2007) foundeven a higher proportion of multiple infections, reaching60% on average of the infected samples. We believe that itis unlikely that the number of multiple infections is higherfor the southeastern part of Spain than for other areas ofthe world, but rather believe that in our work a highernumber of viruses was tested per sample, giving us theopportunity of detecting more multiple infections thanin other surveys where a lesser number of viruses weretested. In any case, this leads to a subject of significant rel-evance, the consequences in epidemiological and diseasedevelopment in terms of a high frequency of multipleinfections. For example, enhanced symptom expressionhas been associated with mixed infections (Gomez et al.,2010), although interactions driving virulence in otherdirections may also occur in nature (Gomez et al., 2009).Enhanced symptom expression may be due to synergisticeffects occurring in a number of virus combinations andis probably related to the silencing suppression ability ofthe viruses involved (Hull, 2002). For viruses in the fam-ily Luteoviridae, synergistic effects have been described(Savenkov & Valkonen, 2001). The high incidence ofCABYV observed here, together with the high numberof double and multiple infections (especially the dou-ble infections of CABYV and potyviruses), suggests thatCABYV may become an important threat to cucurbitcrops in the future. An aspect that deserves particularattention refers to the bias found for more frequent thanexpected occurrence of specific combinations of viruses.Perhaps the sample was not big enough as to have cer-tainty about this bias but, if true, it must have a biologicalmeaning. Interestingly, frequent non-fortuitous associa-tions appeared to occur among viruses transmitted byaphids, so perhaps there is some aspect related to trans-mission responsible for these observations. In any case,further research is needed to understand causes and con-sequences of specific virus associations.

Our analysis of genetic diversity showed that WMVisolates can be divided into four genetic groups, threeof them related to French EM isolates, and another

368 Ann Appl Biol 162 (2013) 362–370© 2013 Association of Applied Biologists

M. Juarez et al. Cucurbit viruses in Valencia

containing just five isolates out of 39, related to CLisolates (Desbiez et al., 2009). Thus, CL isolates seem tobe a minority within the WMV populations in easternSpain. This contrasted with information available onWMV populations sampled in this geographical areabefore 2004 (Moreno et al., 2004), and with the observedstability of populations of other viruses in areas closeto the area studied here (Sanchez-Campos et al., 2002;Marco & Aranda, 2005), suggesting a recent introductionof EM isolates followed by a rapid expansion and dis-placement of CL types. Indeed, our phylogenetic analysisseemed to confirm this hypothesis, as all isolates fromthe Mediterranean area in Moreno et al. (2004) groupedwith our five isolates belonging to the CL group. Besides,our unpublished results suggest that EM isolates werethe most frequently isolated types for the 2007–2012period in these and other areas of cucurbit cultivation inSouthern Spain, leading to confirm the replacement ofCL isolates. The WMV-EM isolates are more virulent thanWMV-CL isolates, at least in squash: the symptoms theyinduce and the virus accumulation are higher than for CLisolates (M. Juarez & M.A. Aranda, unpublished data).Higher virus accumulation may be associated with highertransmission probabilities, explaining the rapid expansionof EM isolates. Interestingly, this scenario resemblesthat described in southeastern Spain in the replacementof Pepino mosaic virus isolates of the European type byisolates of the Chilean type, which are more aggressive intomato (Gomez et al., 2009, 2012), and might be relatedto the instability of industrial agro-ecosystems in thecurrent context of global change (Canto et al., 2009).

In conclusion, both CABYV and WMV-EM seem to betwo emerging viruses of utmost importance for cucurbitcultivation in southeastern Spain and other geographicareas, with PRSV being another potyvirus of significantimportance. Other viruses analysed in this work seem tohave a more local or specific relevance, as can be thecases of the two whitefly-transmitted viruses CVYV andCYSDV, relevant in areas or cultivation conditions weretheir vectors are particularly abundant.

Acknowledgements

This work was supported by grants AGL2009-07552/AGR(Ministerio de Economıa y Competitividad, Spain) andGV 05/060 (Generalitat Valenciana, Spain). We thank M.Fon ([email protected]) for checking the English andM.C. Montesinos and B. Gosalvez for technical assistance.

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