Evaluation of apricot (Prunus armeniaca L.) resistance to Applechlorotic leaf spot virus in controlled greenhouse conditions

Ana García-Ibarra & Federico Dicenta &

Pedro Martínez-Gómez & Manuel Rubio

Accepted: 21 May 2012 /Published online: 1 June 2012# KNPV 2012

Abstract Apple chlorotic leaf spot virus (ACLSV)seems to be the causal agent of apricot viruela disease.This disease has become an important problem forapricot production in Spain, mainly affecting the‘Búlida’ cultivar, although no information is availableabout the behaviour of other cultivars with regards toACLSV. In this study, the behaviour of 29 apricotcultivars against ACLSV (Apr 62 isolate) was evalu-ated under controlled conditions in an insect-proofgreenhouse. Three different rootstocks, ‘GF305’peach, ‘Real Fino’ apricot and ‘Adesoto’ plum, werefirst inoculated by grafting ACLSV-infected bark andwere later grafted with the apricot cultivar to be eval-uated. Apricot cultivars were evaluated during threecycles of study. ACLSV was asymptomatic on theleaves of all cultivars and rootstocks, so level of suscep-tibility or resistance was determined by virus detectionthrough RT-PCR. ‘GF305’ rootstock showed a greatersusceptibility level than ‘Real Fino’ and ‘Adesoto’.Most of the cultivars were susceptible to ACLSV withdifferent levels of susceptibility, and only ‘Bergeron’and ‘Mauricio’ were resistant.

Keywords ACLSV. Breeding . Disease .

Susceptibility . Viruela

Introduction

Spain is one of the biggest producers and exporters ofapricots (Prunus armeniaca L.) in the European Union,with an annual production of 121,616 t in 2009 (http://faostat.fao.org). The Region of Murcia (SoutheastSpain) is the most important apricot area, with 68 % ofnational production (http:/ /www.marm.es/es/agricultura/estadisticas/). In this region, apricot produc-tion is affected by different viral diseases includingsharka, caused by Plum pox virus (PPV), and the lesswell known viruela. From the early 1970s, a substantialexpansion of viruela has occurred (Peña-Iglesias 1968),and it has become one of the main restrictive factors inthe production of this species (Peña-Iglesias and Ayuso1975; Peña-Iglesias 1988; Cañizares et al. 2001).Viruela disease causes spots and deformations in fruitsand mainly affects the principal cultivar: ‘Búlida’(Peña-Iglesias and Ayuso 1970). In addition, studiesunder field conditions have described viruela diseaseon other cultivars, including ‘Real Fino’, ‘Galta Roja’or ‘Moniquí’ (Peña-Iglesias 1968). Llácer (1973) alsodescribed different degrees of susceptibility in fieldconditions, observing severely affected varieties(‘Arrogante’) and other less affected varieties (‘MoniquíFino’ and ‘Velazquez’).

Apple chlorotic leaf spot virus (ACLSV) seems tobe the causal agent of viruela disease (Peña-Iglesiasand Ayuso 1975; Cañizares et al. 2001; García-Ibarraet al. 2010a). ACLSV belongs to the Betaflexiviridaefamily (Carstens 2010), included in the Trichovirus

Eur J Plant Pathol (2012) 133:857–863DOI 10.1007/s10658-012-0009-2

A. García-Ibarra : F. Dicenta : P. Martínez-Gómez :M. Rubio (*)Departament of Plant Breeding, CEBAS-CSIC,PO Box 164, 30100 Espinardo, Murcia, Spaine-mail: mrubio@cebas.csic.es

genus (Martelli et al. 1994). This virus infects woodyspecies of the Rosaceae family, causing differentsymptoms that depend on the species infected andthe virus isolate. ACLSV is also considered to be thecausal agent of plum bark split (Dunez et al. 1973) andapple top-working disease (Yanase 1974). The defin-itive control of this virus should be carried out byobtaining resistant cultivars. However, informationregarding the susceptibility or resistance of apricotcultivars to ACLSV is quite scarce. In field conditions,Desvignes and Boyé (1988) classified apricot cultivarsinto three susceptibility categories according to theirresponse to ACLSV: symptomatic cultivars in whichACLSV was detected by ELISA (‘Luizet’, ‘Bergeron’,‘Hatif de Colomer’, ‘Piraña’); symptomatic cultivarswith an irregular viral distribution (‘Polonais’,‘Houcall’); and asymptomatic cultivars with presenceof ACLSV detected by ELISA (‘Canino’, ‘Rouge duRoussillon’, ‘Tardif de Bordaneil’).

At CEBAS-CSIC in Murcia, a study to evaluate theresistance/susceptibility of the main apricot cultivars toACLSV was carried out in controlled conditions in anisolated, insect-proof greenhouse, incorporating molec-ular detection methods (RT-PCR). As far as we know,this study is the first to evaluate the behaviour of apricotgenotypes against ACLSV in controlled conditions.

The aim of this work was to evaluate the resistanceof different apricot cultivars grown in Spain toACLSV in controlled greenhouse conditions.

Material and methods

Plant material

The plant material assayed included 29 apricot culti-vars of different origins (Table 1). In addition, threedifferent rootstocks were assayed for grafting the cul-tivars to be evaluated: ‘GF305’ peach (P. persica L.Batsch) seedlings, ‘Real Fino’ apricot seedlings and‘Adesoto’ plum (P. insititia L.) obtained by in vitroculture. Five replications of each cultivar were graftedonto each rootstock.

ACLSV isolate

The ACLSV isolate used was Apr 62 (AJ586635),provided by Dr. Arben Myrta of the MediterraneanAgronomic Institute of Bari (Italy). Apr 62 is a

representative isolate among the nine Spanish ACLSVisolates characterized to date, which have a high degreeof homology (94–98 %) (Al Rwahnih et al. 2004). Apr62 was selected because of its higher infection andmultiplication ratio on GF305 and Real Fino rootstocks(García-Ibarra et al. 2010b). All Spanish isolates studiedwere asymptomatic on apricot leaves.

Resistance evaluation in controlled conditions

The behaviour of the apricot cultivars was evaluatedunder controlled conditions in an insect-proof green-house located in the experimental field of CEBAS-CSIC in Murcia (Spain). The evaluation method wassimilar to that used to evaluate PPV resistance(Rubio et al. 2009). Two-month-old rootstocks wereinoculated by grafting a piece of bark from other plantsinfected with Apr-62 isolate. One month later, fivereplications of each rootstock were grafted by cultivar,and the plants were submitted to artificial dormancy in acool chamber at 7°C, in darkness, for 2 months. Theplants were then transferred to the greenhouse for4 months. Three cycles of study were carried out (eachseparated by a 2-month cold period). During each cycle,presence of ACLSV was detected in cultivars and root-stock leaves by RT-PCR. Two specific primers (5′-CCATCTTCGCGAACATAGC-3′ and 5′-GTCTACAGGCTATTTATTATAAG-3′) within the coat proteinsequence were assayed (Sánchez-Navarro et al. 2005).

The presence of symptoms of “dark green sunkenmottle” on infected GF305 (Desvignes and Boyé1988) was scarce and very difficult to observe. Forthis reason, visual observation of symptoms was dis-carded and RT-PCR was used in all rootstocks andcultivars in which the isolate Apr 62 was alwaysasymptomatic. Cultivars and rootstocks were classi-fied according to RT-PCR detection as susceptible(RT-PCR positive) or resistant (RT-PCR negative).The degree of susceptibility of each cultivar was esti-mated as function of the percentage of RT-PCR posi-tive replications. Only plants with their rootstockinfected with ACLSV (RT-PCR positive) were consid-ered for the evaluation process.

Results

Among the 145 plants initially prepared by rootstock,only those with the apricot sprouted were considered

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for the analysis. The success of the grafting was high-est for ‘Real Fino’ apricot (130 out of 145 rootstocksgrafted) and lower for ‘GF305’ (64) and ‘Adesoto’(84) (Table 2). In addition, during the first cycle ofstudy, most of the analysed rootstocks infected withACLSV (RT-PCR positive) were ‘GF305’ peach, with38 % inoculation efficiency. The other rootstocksshowed a very low percentage of infection (7 % for

‘Real Fino’ apricot and 1 % ‘Adesoto’ plum). Thenumber of plants in which the virus was detectedincreased considerably in cycle 2 and was stable incycle 3, and ‘GF305’ always showed a higher percent-age of infected plants than ‘Real Fino’ and ‘Adesoto’.

Table 1 shows the results of the evaluation ofACLSV resistance of the 29 apricot cultivars assayedduring the three cycles of study. The number of plants

Table 1 Evaluation of resistance of apricot cultivars againstACLSV grafted onto three different rootstocks. Number oftested plants (n) and number of plants RT-PCR positive inthe three cycles studied. In addition, total number of

observations for all cycles were recorded as the numberof susceptible replications/total number of replications eval-uated (S/T) and the percentage of susceptible replications(S)

Cultivar Pedigree Origin Cycle 1 Cycle 2 Cycle 3 Total

n RT-PCR+ n RT-PCR+ n RT-PCR+ S/T (n) S (%)

Búlida Precoz Unknown Spain 1 1 5 5 5 5 11/11 100

Murciana Orange Red×Currot Spain 0 0 3 3 3 2 5/6 83

Mitger de Castelló Unknown Spain 2 1 1 1 2 2 4/5 80

Búlida Unknown Spain 1 1 8 8 7 3 12/16 75

Búlida de Arques Unknown Spain 2 1 6 6 3 1 8/11 73

Dorada Bergeron×Moniquí Spain 2 1 2 1 2 2 4/6 67

Canino Unknown Spain 1 1 5 4 5 2 7/11 64

Lito SEO×Tyrinthos Greece 1 1 5 4 5 2 7/11 64

Real Fino Unknown Spain 3 1 5 3 5 4 8/13 61

Bebeco Unknown Greece 1 0 5 5 4 1 6/10 60

Rojo Pasión Orange Red×Currot Spain 1 1 2 2 2 0 3/5 60

Goldrich Unknown USA 3 1 2 2 2 1 4/7 57

Palstein Blehein×Canino South Africa 2 0 6 6 6 2 8/14 57

Moniquí Unknown Spain 1 0 4 4 4 1 5/9 55

Tyrinthos Unknown Greece 1 1 4 3 4 1 5/9 55

Selene Goldrich×A2564 Spain 3 1 7 7 7 1 9/17 53

Bergarouge Unknown France 0 – 2 2 2 0 2/4 50

Rosa Orange Red×Palstein Spain 0 0 4 2 4 2 4/8 50

Tardif de Bordaneil Unknown France 2 0 3 3 2 0 3/7 43

Z308-6 Goldrich×Lito Spain 2 1 4 3 4 0 4/10 40

Estrella Orange Red×Z211-18 Spain 0 0 3 1 3 1 2/6 33

Sublime Orange Red×Z211-18 Spain 1 0 1 1 1 0 1/3 33

Bergeron Unknown France 3 0 7 0 6 0 0/16 0

Mauricio Unknown Spain 1 0 6 0 6 0 0/13 0

Orange Red Lasgerdi Mashad×NJA2 USA 1 0 2 0 2 0 0/5 0

Pepito del Rubio Unknown Spain 0 0 1 0 1 0 0/2 0

San Castrese Unknown Italy 0 0 3 0 3 0 0/6 0

Valorange Orange Red×Currot Spain 1 0 0 0 0 0 0/1 0

Velázquez Unknown Spain 0 0 2 0 2 0 0/4 0

Total 36 13 108 76 102 33 122/246 45 %

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finally evaluated (rootstock RT-PCR positive and apri-cot bud sprouted) was very low during the first cycle(36 replications out of 278), and higher during thesecond (108 replications) and third cycle (102 repli-cations). The number of susceptible apricots (RT-PCRpositive) was 13, 76 and 33 during the three cycles ofstudy, respectively.

During the first cycle of study, at least one replica-tion of 13 of the 29 cultivars tested were RT-PCRpositive for ACLSV, including ‘Búlida Precoz’,‘Mitger de Castelló’, ‘Búlida’, ‘Búlida de Arques’,‘Dorada’, ‘Canino’, ‘Lito’, ‘Real Fino’, ‘Rojo Pasión’,‘Goldrich’, ‘Tyrinthos’, ‘Selene’ and ‘Z308-6’.

During the second cycle, the number of infectedplants increased considerably, with ACLSV detectedin 76 apricot plants. In this cycle, nine additionalcultivars were added to the list of susceptible cultivars,including ‘Murciana’, ‘Bebeco’, ‘Palstein’, ‘Moniquí’,‘Bergarouge’, ‘Rosa’, ‘Tardif de Bordaneil’, ‘Estrella’and ‘Sublime’.

Finally, during the third cycle, only 33 apricotplants were ACLSV positive, meaning we did notdetect the virus in 43 repetitions which had beenACLSV positive in the previous cycle. No new culti-vars were classified as susceptible in this third cycle.

In summary, after three cycles of evaluation, 22 ofthe 29 cultivars tested showed susceptibility to Apr-62ACLSV isolate and were RT-PCR positive in some ofthe replications evaluated. On the other hand, onlyseven varieties (‘Bergeron’, ‘Mauricio’, ‘OrangeRed’, ‘Pepito del Rubio’, ‘San Castrese’, ‘Valorange’and ‘Velázquez’) behaved as resistant, since they didnot test ACLSV positive by RT-PCR in any replication(Table 1, Fig. 1). Among these varieties, only Ber-geron and Mauricio had a reliable number of replica-tions evaluated.

Discussion

Our results showed that ACLSV can infect apricot,peach and plum species of the Prunus genus. The highsusceptibility of peach ‘GF305’ rootstock to ACLSVagrees with the previous description of this rootstock asa woody indicator of different Prunus viruses (Bernhardet al. 1969). Regarding apricot, Peña-Iglesias (1968)described the great susceptibility of ‘Real Fino’ apricotrootstock, which is in agreement with our findings.Finally, the susceptibility observed in ‘Adesoto’ plumis also consistent with previous studies in other plumspecies in field conditions evaluated using the ELISAtest (Dunez et al. 1973).

During the first cycle after inoculation, mostinfected rootstocks were ‘GF305’, showing the rela-tively fast diffusion of ACLSV in this rootstock. Thisbehaviour was described by Cambra et al. (1986), whoobserved a higher translocation rate in this peachrootstock compared to other apricot rootstocks. Inaddition, the increase in number of infected plantsbetween the first and the second cycle suggests thatACLSV needs time to multiply and infect rootstocks.This slow multiplication rate in comparison with otherviruses such as PPV or PNRSV has been described inother works on ACLSV (Bernhard et al. 1969; Cambraet al. 1986; Dosba et al. 1986; García-Ibarra et al.2011). Furthermore, the low success of grafting ofinfected plants in our study is consistent with theprevious results of Desvignes and Boyé (1988), whodescribed a decrease of more than 50 % in graftcompatibility in different Prunus rootstocks after in-oculation with ACLSV.

In the case of ‘Real Fino’, the lower rate of infec-tion (only 7 %) resulted in a higher percentage ofgrafting success. The high compatibility of this root-stock with apricot cultivars (in fact, this is the mostused apricot rootstock in Spain) likely also favouredgrafting success. The behaviour of ‘Adesoto’ wasdistinct, for despite the low initial rate of infection(1 %), grafting success was also low (somewhat higherthan GF305). This can be explained by the low graftcompatibility of this rootstock with apricot cultivarsunder our evaluation conditions in controlled condi-tions (Rubio et al. 2009).

The high variability found between replications andbetween different cycles for the same plant follows thesame pattern that has been described for PPV in Pru-nus species (Rubio et al. 2005). One example is the

Table 2 Number of rootstocks infected with ACLSV analysed(N) and percentage of replications RT-PCR positive (+) in eachcycle. Among the 145 replications (by rootstock) initially assayed,only rootstocks with the cultivar bud developed were analysed

Rootstock N Cycle 1 Cycle 2 Cycle 3RT-PCR+ RT-PCR+ RT-PCR+

GF305 64 38 61 61

Real Fino 130 7 32 29

Adesoto 84 1 33 32

Total 278 13 39 37

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significant drop in the number of infected plants in thethird cycle, probably due to uncontrolled factors (stateof plants, time of sampling, temperature) during thiscycle. Furthermore, recent studies in field conditionson the detection of ACLSV in leaves and fruits bydifferent methods have clearly shown the sink effect offruit, whose tissues accumulate large concentrations ofACLSV in comparison with leaf tissues (García-Ibarraet al. 2010a, 2011).

Our results showed a generalized susceptibility toACLSV in apricot species, which agrees with previousstudies on ACLSV susceptibility (Desvignes andBoyé 1988) and viruela susceptibility in field condi-tions (Peña-Iglesias 1968; Llácer 1973). However, inour controlled greenhouse conditions, we were able todetect different levels of susceptibility as a function ofpercentage of plants in which the virus was detectedby RT-PCR. These percentages varied between 100 %in the case of ‘Búlida Precoz’ and 33 % in ‘Sublime’and ‘Estrella’ cultivars. This data may be related to thesusceptibility level of each cultivar to ACLSV.

Although the pedigree of most of the evaluatedcultivars is unknown, some descendants and theirprogenitors were evaluated in this study. ‘Selene’ and‘Z-308-06’, as with their progenitor ‘Goldrich’, werefound to be susceptible to ACLSV. On the other hand‘Murciana’, ‘Rojo Pasión’, ‘Rosa’, ‘Estrella’ and‘Sublime’, descendants of ‘Orange Red’ (whose resis-tance would have to be checked), were susceptible tothe virus. Likewise, ‘Dorada’ (descendant of the resis-tant ‘Bergeron’) was susceptible.

The most susceptible cultivars were the Spanishcultivars ‘Búlida Precoz’, ‘Murciana’, ‘Mitger deCastelló’, ‘Búlida’ and ‘Búlida de Arques’, with ahigh percentage of susceptible replications (RT-PCRpositive), between 100 % and 73 %. In the case of thetraditional cultivar ‘Búlida’ and the related cultivars‘Búlida Precoz’ and ‘Búlida de Arques’, this high

susceptibility to ACLSV may be related to the suscep-tibility of these cultivars to viruela disease in fieldconditions (Peña-Iglesias 1968; Peña-Iglesias andAyuso 1970; Llácer 1973). In addition, other Spanishcultivars classified as susceptible were ‘Canino’,‘Real Fino’ and ‘Moniquí’. This is also the case for‘Palstein’, a descendant of ‘Canino’. In agreementwith these results on ACLSV, ‘Moniquí’ was de-scribed by Peña-Iglesias (1968) and Mañas et al.(2000) as susceptible to viruela, although with a lowersusceptibility than ‘Búlida’. In addition, ACLSV sus-ceptibility in ‘Canino’ was described by Dosba et al.(1986). These results again support the relationshipbetween viruela disease and ACLSV previously de-scribed by different authors (Peña-Iglesias and Ayuso1975; Cañizares et al. 2001; García-Ibarra et al.2010a).

The Greek cultivars ‘Bebeco’, ‘Lito’ and ‘Tyrinthos’were also classified as susceptible. In agreement withthese findings, the susceptibility of ‘Tyrinthos’ to viruelawas described by Alioto et al. (1995), who observedsymptoms of this disease in the Italian apricot cultivar‘Cafona’ (initially virus-free) grafted onto ‘Tyrinthos’trees infected by ACLSV (ELISA positive). Two out ofthree French cultivars studied (‘Tardif de Bordaneil’ and‘Bergarouge’) were classified as susceptible. Regardingthese cultivars, Desvignes and Boyé (1988) indicatedthat ‘Tardif de Bordaneil’ did not show ACLSV symp-toms, although the virus was detected by ELISA. TheNorth American cultivar ‘Goldrich’was also susceptibleto ACLSV.

The virus was never detected in only in seven out ofthe 29 cultivars evaluated. However, in five of theseseven cultivars, the low number of replications evalu-ated does not allow us to confirm their real resistance,which would require evaluating a larger number ofplants. This low number of replications in some culti-vars was due to the successive loss of plants during

632 bp

M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

Fig. 1 Agarose gel showing the band corresponding to ACLSV(632 bp) obtained by RT-PCR in the apricot cultivars analysed.Lanes 1–24. 1: ‘Selene’, 2: ‘Tyrinthos’, 3: ‘Rosa’, 4: ‘RojoPasión’, 5: ‘Palstein’, 6: ‘Orange Red’, 7: ‘Estrella’, 8: ‘Canino’,9: ‘Murciana’, 10: ‘Z308-6’, 11: ‘Goldrich’, 12: ‘San Castrese’,

13: ‘Sublime’, 14: ‘Búlida’, 15: ‘Moniquí’, 16: ‘Bergarouge’,17: ‘Valorange’, 18: ‘Velázquez’, 19: ‘Pepito del Rubio’, 20:‘Lito’, 21: ‘Bergeron’, 22: ‘Mauricio’, 23: ‘Búlida de Arques’,24: ‘Real Fino’. Lane M: molecular weight marker 1 Kb plus(Invitrogen)

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different steps of the evaluation (inoculation, grafting,artificial cycles, etc.) as described in PPV resistanceevaluation (Rubio et al. 2009).

These five cultivars have different origins. Three areSpanish(‘PepitodelRubio’, ‘Velázquez’and ‘Valorange’,a descendant of ‘Orange Red’ and ‘Currot’), one Ameri-can (‘OrangeRed’) and one Italian (‘SanCastrese)’. Con-versely, ‘Velazquez’ was classified as susceptible toviruela by Llácer (1973), although the fruit symptoms heobserved were much lower than in ‘Búlida’. This contra-dictoryresultcouldbedueto the lownumberof repetitionsanalysed in our studyor the inconsistency of the field dataincludingonly fruit symptoms.The resistanceobserved inthese cultivarsmust be considered as a preliminary result,as it would be necessary to continue evaluation with ahigher number of repetitions.

The only resistant cultivars, with a reliable number ofreplications evaluated, were ‘Bergeron’ (French) and‘Mauricio’ (Spanish). These cultivars are traditionalcultivars from France and Spain respectively with anunknown pedigree. In agreement with these results,‘Mauricio’ has never shown viruela symptoms in fieldconditions in more than 30 years of cultivation in theRegion of Murcia (Soler and Cano 2004). However,Desvignes and Boyé (1988) described that ‘Bergeron’manifested ACLSV symptoms (such as rosette andspots in stem), but never detected the virus by ELISA,which could be explained by confusion of symptoms(the virus was not really present), or by the low sensi-tivity of the technique applied (ELISA).

In our opinion, there is a relationship betweenACLSVand viruela disease, although this has not beenexperimentally confirmed. García-Ibarra (2011) couldnot conclude that viruela disease was caused solely byACLSV, since most fruits with or without viruelasymptoms had both ACLSV and HSVd (Hop stuntviroid). Furthermore this work showed that the expres-sion of viruela symptoms was influenced by environ-mental conditions, and was stronger in a cold and wetenvironment in the pre-harvest phase.

New studies are in progress to test the resistance of‘Bergeron’ and ‘Mauricio’ to ACLSVon fruits of adulttrees in order to validate the results obtained on leavesin controlled conditions.

Acknowledgments This study was supported by a project ofthe Seneca Foundation of the Region of Murcia (08672/PI/08):“Importance, transmission and resistance sources of the mainviruses affecting stone fruits in the Region of Murcia.”

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