Eur J Plant Pathol (2012) 133:857–863 DOI 10.1007/s10658-012-0009-2
Evaluation of apricot (Prunus armeniaca L.) resistance to Apple chlorotic 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 for apricot production in Spain, mainly affecting the ‘Búlida’ cultivar, although no information is available about the behaviour of other cultivars with regards to ACLSV. In this study, the behaviour of 29 apricot cultivars against ACLSV (Apr 62 isolate) was evaluated under controlled conditions in an insect-proof greenhouse. Three different rootstocks, ‘GF305’ peach, ‘Real Fino’ apricot and ‘Adesoto’ plum, were first inoculated by grafting ACLSV-infected bark and were later grafted with the apricot cultivar to be evaluated. Apricot cultivars were evaluated during three cycles of study. ACLSV was asymptomatic on the leaves of all cultivars and rootstocks, so level of susceptibility or resistance was determined by virus detection through RT-PCR. ‘GF305’ rootstock showed a greater susceptibility level than ‘Real Fino’ and ‘Adesoto’. Most of the cultivars were susceptible to ACLSV with different levels of susceptibility, and only ‘Bergeron’ and ‘Mauricio’ were resistant. Keywords ACLSV . Breeding . Disease . Susceptibility . Viruela A. García-Ibarra : F. Dicenta : P. Martínez-Gómez : M. Rubio (*) Departament of Plant Breeding, CEBAS-CSIC, PO Box 164, 30100 Espinardo, Murcia, Spain e-mail:
[email protected]
Introduction Spain is one of the biggest producers and exporters of apricots (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 (Southeast Spain) is the most important apricot area, with 68 % of national production (http://www.marm.es/es/ agricultura/estadisticas/). In this region, apricot production is affected by different viral diseases including sharka, caused by Plum pox virus (PPV), and the less well known viruela. From the early 1970s, a substantial expansion of viruela has occurred (Peña-Iglesias 1968), and it has become one of the main restrictive factors in the production of this species (Peña-Iglesias and Ayuso 1975; Peña-Iglesias 1988; Cañizares et al. 2001). Viruela disease causes spots and deformations in fruits and mainly affects the principal cultivar: ‘Búlida’ (Peña-Iglesias and Ayuso 1970). In addition, studies under field conditions have described viruela disease on other cultivars, including ‘Real Fino’, ‘Galta Roja’ or ‘Moniquí’ (Peña-Iglesias 1968). Llácer (1973) also described different degrees of susceptibility in field conditions, observing severely affected varieties (‘Arrogante’) and other less affected varieties (‘Moniquí Fino’ and ‘Velazquez’). Apple chlorotic leaf spot virus (ACLSV) seems to be the causal agent of viruela disease (Peña-Iglesias and Ayuso 1975; Cañizares et al. 2001; García-Ibarra et al. 2010a). ACLSV belongs to the Betaflexiviridae family (Carstens 2010), included in the Trichovirus
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genus (Martelli et al. 1994). This virus infects woody species of the Rosaceae family, causing different symptoms that depend on the species infected and the virus isolate. ACLSV is also considered to be the causal agent of plum bark split (Dunez et al. 1973) and apple top-working disease (Yanase 1974). The definitive control of this virus should be carried out by obtaining resistant cultivars. However, information regarding the susceptibility or resistance of apricot cultivars to ACLSV is quite scarce. In field conditions, Desvignes and Boyé (1988) classified apricot cultivars into three susceptibility categories according to their response to ACLSV: symptomatic cultivars in which ACLSV was detected by ELISA (‘Luizet’, ‘Bergeron’, ‘Hatif de Colomer’, ‘Piraña’); symptomatic cultivars with an irregular viral distribution (‘Polonais’, ‘Houcall’); and asymptomatic cultivars with presence of ACLSV detected by ELISA (‘Canino’, ‘Rouge du Roussillon’, ‘Tardif de Bordaneil’). At CEBAS-CSIC in Murcia, a study to evaluate the resistance/susceptibility of the main apricot cultivars to ACLSV was carried out in controlled conditions in an isolated, insect-proof greenhouse, incorporating molecular detection methods (RT-PCR). As far as we know, this study is the first to evaluate the behaviour of apricot genotypes against ACLSV in controlled conditions. The aim of this work was to evaluate the resistance of different apricot cultivars grown in Spain to ACLSV in controlled greenhouse conditions.
Material and methods Plant material The plant material assayed included 29 apricot cultivars of different origins (Table 1). In addition, three different rootstocks were assayed for grafting the cultivars to be evaluated: ‘GF305’ peach (P. persica L. Batsch) seedlings, ‘Real Fino’ apricot seedlings and ‘Adesoto’ plum (P. insititia L.) obtained by in vitro culture. Five replications of each cultivar were grafted onto each rootstock.
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representative isolate among the nine Spanish ACLSV isolates characterized to date, which have a high degree of homology (94–98 %) (Al Rwahnih et al. 2004). Apr 62 was selected because of its higher infection and multiplication ratio on GF305 and Real Fino rootstocks (García-Ibarra et al. 2010b). All Spanish isolates studied were asymptomatic on apricot leaves. Resistance evaluation in controlled conditions The behaviour of the apricot cultivars was evaluated under controlled conditions in an insect-proof greenhouse located in the experimental field of CEBASCSIC in Murcia (Spain). The evaluation method was similar to that used to evaluate PPV resistance (Rubio et al. 2009). Two-month-old rootstocks were inoculated by grafting a piece of bark from other plants infected with Apr-62 isolate. One month later, five replications of each rootstock were grafted by cultivar, and the plants were submitted to artificial dormancy in a cool chamber at 7°C, in darkness, for 2 months. The plants were then transferred to the greenhouse for 4 months. Three cycles of study were carried out (each separated by a 2-month cold period). During each cycle, presence of ACLSV was detected in cultivars and rootstock leaves by RT-PCR. Two specific primers (5′CCATCTTCGCGAACATAGC-3′ and 5′-GTCTA CAGGCTATTTATTATAAG-3′) within the coat protein sequence were assayed (Sánchez-Navarro et al. 2005). The presence of symptoms of “dark green sunken mottle” on infected GF305 (Desvignes and Boyé 1988) was scarce and very difficult to observe. For this reason, visual observation of symptoms was discarded and RT-PCR was used in all rootstocks and cultivars in which the isolate Apr 62 was always asymptomatic. Cultivars and rootstocks were classified according to RT-PCR detection as susceptible (RT-PCR positive) or resistant (RT-PCR negative). The degree of susceptibility of each cultivar was estimated as function of the percentage of RT-PCR positive replications. Only plants with their rootstock infected with ACLSV (RT-PCR positive) were considered for the evaluation process.
ACLSV isolate Results The ACLSV isolate used was Apr 62 (AJ586635), provided by Dr. Arben Myrta of the Mediterranean Agronomic Institute of Bari (Italy). Apr 62 is a
Among the 145 plants initially prepared by rootstock, only those with the apricot sprouted were considered
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Table 1 Evaluation of resistance of apricot cultivars against ACLSV grafted onto three different rootstocks. Number of tested plants (n) and number of plants RT-PCR positive in the three cycles studied. In addition, total number of Cultivar
Pedigree
Origin
Spain
observations for all cycles were recorded as the number of susceptible replications/total number of replications evaluated (S/T) and the percentage of susceptible replications (S) Cycle 1
Cycle 2
Cycle 3
Total
n
RT-PCR+ n
RT-PCR+ n
RT-PCR+ S/T (n)
S (%)
1
1
5
5
100
Búlida Precoz
Unknown
5
5
11/11
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
for the analysis. The success of the grafting was highest for ‘Real Fino’ apricot (130 out of 145 rootstocks grafted) and lower for ‘GF305’ (64) and ‘Adesoto’ (84) (Table 2). In addition, during the first cycle of study, most of the analysed rootstocks infected with ACLSV (RT-PCR positive) were ‘GF305’ peach, with 38 % inoculation efficiency. The other rootstocks showed a very low percentage of infection (7 % for
36 13
108 76
102 33
122/246 45 %
‘Real Fino’ apricot and 1 % ‘Adesoto’ plum). The number of plants in which the virus was detected increased considerably in cycle 2 and was stable in cycle 3, and ‘GF305’ always showed a higher percentage of infected plants than ‘Real Fino’ and ‘Adesoto’. Table 1 shows the results of the evaluation of ACLSV resistance of the 29 apricot cultivars assayed during the three cycles of study. The number of plants
860
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Table 2 Number of rootstocks infected with ACLSV analysed (N) and percentage of replications RT-PCR positive (+) in each cycle. Among the 145 replications (by rootstock) initially assayed, only rootstocks with the cultivar bud developed were analysed Rootstock
N
Cycle 1 RT-PCR+
Cycle 2 RT-PCR+
Cycle 3 RT-PCR+
GF305
64
38
61
61
Real Fino
130
7
32
29
Adesoto
84
1
33
32
Total
278
13
39
37
finally evaluated (rootstock RT-PCR positive and apricot bud sprouted) was very low during the first cycle (36 replications out of 278), and higher during the second (108 replications) and third cycle (102 replications). The number of susceptible apricots (RT-PCR positive) was 13, 76 and 33 during the three cycles of study, respectively. During the first cycle of study, at least one replication of 13 of the 29 cultivars tested were RT-PCR positive 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 infected plants increased considerably, with ACLSV detected in 76 apricot plants. In this cycle, nine additional cultivars 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 apricot plants were ACLSV positive, meaning we did not detect the virus in 43 repetitions which had been ACLSV positive in the previous cycle. No new cultivars were classified as susceptible in this third cycle. In summary, after three cycles of evaluation, 22 of the 29 cultivars tested showed susceptibility to Apr-62 ACLSV isolate and were RT-PCR positive in some of the replications evaluated. On the other hand, only seven varieties (‘Bergeron’, ‘Mauricio’, ‘Orange Red’, ‘Pepito del Rubio’, ‘San Castrese’, ‘Valorange’ and ‘Velázquez’) behaved as resistant, since they did not test ACLSV positive by RT-PCR in any replication (Table 1, Fig. 1). Among these varieties, only Bergeron and Mauricio had a reliable number of replications evaluated.
Discussion Our results showed that ACLSV can infect apricot, peach and plum species of the Prunus genus. The high susceptibility of peach ‘GF305’ rootstock to ACLSV agrees with the previous description of this rootstock as a woody indicator of different Prunus viruses (Bernhard et al. 1969). Regarding apricot, Peña-Iglesias (1968) described the great susceptibility of ‘Real Fino’ apricot rootstock, which is in agreement with our findings. Finally, the susceptibility observed in ‘Adesoto’ plum is also consistent with previous studies in other plum species in field conditions evaluated using the ELISA test (Dunez et al. 1973). During the first cycle after inoculation, most infected rootstocks were ‘GF305’, showing the relatively fast diffusion of ACLSV in this rootstock. This behaviour was described by Cambra et al. (1986), who observed a higher translocation rate in this peach rootstock compared to other apricot rootstocks. In addition, the increase in number of infected plants between the first and the second cycle suggests that ACLSV needs time to multiply and infect rootstocks. This slow multiplication rate in comparison with other viruses such as PPV or PNRSV has been described in other works on ACLSV (Bernhard et al. 1969; Cambra et al. 1986; Dosba et al. 1986; García-Ibarra et al. 2011). Furthermore, the low success of grafting of infected plants in our study is consistent with the previous results of Desvignes and Boyé (1988), who described a decrease of more than 50 % in graft compatibility in different Prunus rootstocks after inoculation with ACLSV. In the case of ‘Real Fino’, the lower rate of infection (only 7 %) resulted in a higher percentage of grafting success. The high compatibility of this rootstock with apricot cultivars (in fact, this is the most used apricot rootstock in Spain) likely also favoured grafting success. The behaviour of ‘Adesoto’ was distinct, for despite the low initial rate of infection (1 %), grafting success was also low (somewhat higher than GF305). This can be explained by the low graft compatibility of this rootstock with apricot cultivars under our evaluation conditions in controlled conditions (Rubio et al. 2009). The high variability found between replications and between different cycles for the same plant follows the same pattern that has been described for PPV in Prunus species (Rubio et al. 2005). One example is the
Eur J Plant Pathol (2012) 133:857–863 M
1 2
3
4
5
861 6 7
8
9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
632 bp
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: ‘Rojo Pasión’, 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)
significant drop in the number of infected plants in the third cycle, probably due to uncontrolled factors (state of plants, time of sampling, temperature) during this cycle. Furthermore, recent studies in field conditions on the detection of ACLSV in leaves and fruits by different methods have clearly shown the sink effect of fruit, whose tissues accumulate large concentrations of ACLSV in comparison with leaf tissues (García-Ibarra et al. 2010a, 2011). Our results showed a generalized susceptibility to ACLSV in apricot species, which agrees with previous studies on ACLSV susceptibility (Desvignes and Boyé 1988) and viruela susceptibility in field conditions (Peña-Iglesias 1968; Llácer 1973). However, in our controlled greenhouse conditions, we were able to detect different levels of susceptibility as a function of percentage of plants in which the virus was detected by 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 the susceptibility level of each cultivar to ACLSV. Although the pedigree of most of the evaluated cultivars is unknown, some descendants and their progenitors were evaluated in this study. ‘Selene’ and ‘Z-308-06’, as with their progenitor ‘Goldrich’, were found to be susceptible to ACLSV. On the other hand ‘Murciana’, ‘Rojo Pasión’, ‘Rosa’, ‘Estrella’ and ‘Sublime’, descendants of ‘Orange Red’ (whose resistance would have to be checked), were susceptible to the virus. Likewise, ‘Dorada’ (descendant of the resistant ‘Bergeron’) was susceptible. The most susceptible cultivars were the Spanish cultivars ‘Búlida Precoz’, ‘Murciana’, ‘Mitger de Castelló’, ‘Búlida’ and ‘Búlida de Arques’, with a high percentage of susceptible replications (RT-PCR positive), between 100 % and 73 %. In the case of the traditional cultivar ‘Búlida’ and the related cultivars ‘Búlida Precoz’ and ‘Búlida de Arques’, this high
susceptibility to ACLSV may be related to the susceptibility of these cultivars to viruela disease in field conditions (Peña-Iglesias 1968; Peña-Iglesias and Ayuso 1970; Llácer 1973). In addition, other Spanish cultivars classified as susceptible were ‘Canino’, ‘Real Fino’ and ‘Moniquí’. This is also the case for ‘Palstein’, a descendant of ‘Canino’. In agreement with these results on ACLSV, ‘Moniquí’ was described by Peña-Iglesias (1968) and Mañas et al. (2000) as susceptible to viruela, although with a lower susceptibility than ‘Búlida’. In addition, ACLSV susceptibility in ‘Canino’ was described by Dosba et al. (1986). These results again support the relationship between viruela disease and ACLSV previously described by different authors (Peña-Iglesias and Ayuso 1975; Cañizares et al. 2001; García-Ibarra et al. 2010a). The Greek cultivars ‘Bebeco’, ‘Lito’ and ‘Tyrinthos’ were also classified as susceptible. In agreement with these findings, the susceptibility of ‘Tyrinthos’ to viruela was described by Alioto et al. (1995), who observed symptoms of this disease in the Italian apricot cultivar ‘Cafona’ (initially virus-free) grafted onto ‘Tyrinthos’ trees infected by ACLSV (ELISA positive). Two out of three French cultivars studied (‘Tardif de Bordaneil’ and ‘Bergarouge’) were classified as susceptible. Regarding these cultivars, Desvignes and Boyé (1988) indicated that ‘Tardif de Bordaneil’ did not show ACLSV symptoms, although the virus was detected by ELISA. The North American cultivar ‘Goldrich’ was also susceptible to ACLSV. The virus was never detected in only in seven out of the 29 cultivars evaluated. However, in five of these seven cultivars, the low number of replications evaluated does not allow us to confirm their real resistance, which would require evaluating a larger number of plants. This low number of replications in some cultivars was due to the successive loss of plants during
862
different steps of the evaluation (inoculation, grafting, artificial cycles, etc.) as described in PPV resistance evaluation (Rubio et al. 2009). These five cultivars have different origins. Three are Spanish (‘Pepito delRubio’, ‘Velázquez’ and ‘Valorange’, a descendant of ‘Orange Red’ and ‘Currot’), one American (‘Orange Red’) and one Italian (‘San Castrese)’. Conversely, ‘Velazquez’ was classified as susceptible to viruela by Llácer (1973), although the fruit symptoms he observed were much lower than in ‘Búlida’. This contradictoryresult could bedueto the low number of repetitions analysed in our study or the inconsistency of the field data including only fruit symptoms. The resistance observed in these cultivars must be considered as a preliminary result, as it would be necessary to continue evaluation with a higher number of repetitions. The only resistant cultivars, with a reliable number of replications evaluated, were ‘Bergeron’ (French) and ‘Mauricio’ (Spanish). These cultivars are traditional cultivars from France and Spain respectively with an unknown pedigree. In agreement with these results, ‘Mauricio’ has never shown viruela symptoms in field conditions in more than 30 years of cultivation in the Region of Murcia (Soler and Cano 2004). However, Desvignes and Boyé (1988) described that ‘Bergeron’ manifested ACLSV symptoms (such as rosette and spots 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 sensitivity of the technique applied (ELISA). In our opinion, there is a relationship between ACLSV and viruela disease, although this has not been experimentally confirmed. García-Ibarra (2011) could not conclude that viruela disease was caused solely by ACLSV, since most fruits with or without viruela symptoms had both ACLSV and HSVd (Hop stunt viroid). Furthermore this work showed that the expression of viruela symptoms was influenced by environmental conditions, and was stronger in a cold and wet environment in the pre-harvest phase. New studies are in progress to test the resistance of ‘Bergeron’ and ‘Mauricio’ to ACLSV on fruits of adult trees in order to validate the results obtained on leaves in controlled conditions. Acknowledgments This study was supported by a project of the Seneca Foundation of the Region of Murcia (08672/PI/08): “Importance, transmission and resistance sources of the main viruses affecting stone fruits in the Region of Murcia.”
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