Determination of resistance to sharka (plum pox) virus in apricot

Scientia Horticulturae 91 (2001) 59±70

Determination of resistance to sharka (plum pox) virus in apricot Tarek A. Moustafa, MarõÂa L. Badenes*, Jose MartõÂnez-Calvo, Gerardo LlaÂcer Instituto Valenciano de Investigaciones Agrarias (IVIA), Apartado O®cial, 46113 Moncada, Valencia, Spain Accepted 22 January 2001

Abstract The Mediterranean basin countries account for more than 50% of world apricot production. However, the spread of plum pox virus in this area is causing important losses and represents a limitation for the crop. In 1993, at IVIA, Valencia, a breeding program to develop cultivars resistant to sharka was initiated and an ef®cient procedure for the determination of sharka resistance in the progenies was needed. A methodology described previously in France, that uses apricot seedlings grafted on peach was developed to improve reliability. The germination and growing of `GF-305' peach seedlings was improved, the subsequent grafting protocol was optimised, and a better source of inoculum was identi®ed. A revised procedure is presented, which is more ef®cient than that reported previously. # 2001 Elsevier Science B.V. All rights reserved. Keywords: Apricot; Plum pox virus; Prunus armeniaca; Sharka; Resistance

1. Introduction The `sharka' virus (plum pox virus or PPV) is the most important virus affecting stone fruit crops in Europe. It causes important losses of fruit, mainly in apricot and European plum. Some characteristics of the disease make its control very dif®cult: the virus is spread by aphids in a non-persistent way, which allows ef®cient spread of the disease through orchards before its symptoms are expressed. The best PPV vectors are not pests for the crops; therefore spraying *

Corresponding author. E-mail address: [email protected] (M.L. Badenes). 0304-4238/01/$ ± see front matter # 2001 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 2 3 8 ( 0 1 ) 0 0 2 3 6 - 9

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with pesticides is not an ef®cient practice. Additionally, the problem of disease detection before symptom expression has not been solved (Roy and Smith, 1994; Nemeth, 1994). In Spain, PPV was detected on Japanese plum (Prunus salicina Lindl.) in 1984. Since 1987, the virus has spread rapidly on apricot, causing fruit deformations that have resulted in important crop losses (LlaÂcer et al., 1985; LlaÂcer and Cambra, 1986; LlaÂcer, 1987). A project based on eradication of infected source plants did not stop the disease in Valencia and PPV is now the most important limiting factor for the crop (LlaÂcer and Cambra, 1998). Effective control of the disease will require cultivars resistant to PPV. The Mediterranean basin countries account for more than 50% of world apricot production (FAO, 1999). Breeding programs aimed at obtaining new cultivars resistant to PPV with good agronomic and commercial quality are in progress in Greece, France, Italy and Spain (Karayiannis et al., 1991; Audergon et al., 1994; Bassi et al., 1995; Egea et al., 1999). In 1993, an apricot breeding program was initiated at IVIA, Valencia, based on crosses between North American cultivars resistant to PPV and native cultivars from Valencia, susceptible to the virus (Badenes et al., 1996). The screening of the trait on thousands of seedlings requires that an ef®cient screening methodology be developed. The methodology to determine sharka resistance was described by Audergon and Morvan (1990). A seedling of `GF-305' peach, which is very susceptible to PPV and develops symptoms quickly, is used as an indicator of the disease. The apricot to be studied is grafted on the `GF-305' peach seedling. Once the apricot bud grows out, the virus is inoculated by grafting. After inoculation, symptoms are observed on apricot and peach and the presence of the virus is analyzed by ELISA-DASI and/or RT-PCR. If symptoms are observed on apricot and peach, the apricot is considered to be susceptible; if symptoms are not observed on apricot but the virus is detected, the apricot is considered to be tolerant; if symptoms are observed only on peach and the virus is not detected on apricot then the apricot is resistant, allowing translocation but not multiplication of the virus; if there are no symptoms at all and no presence of the virus, then the apricot is considered to be resistant (Table 1). Since the apricot and peach are temperate species, a chilling treatment is necessary after grafting to break dormancy and to promote shoots where symptoms can be scored. To con®rm the results, a second chilling treatment for promoting new shoots is recommended. It is possible that seedlings that tested negative for the presence of the virus after the ®rst chilling treatment could be escapes and might score as susceptible after a second chilling treatment. This procedure has been used frequently in France (Dosba et al., 1991, 1992; Audergon et al., 1994, 1995). However, many of the details affecting the different steps are not published. At IVIA, screening of progenies for resistance showed that the technique was not suitable for large scale screening. Several factors concerning `GF-305' growth and apricot grafting needed to be addressed. Every

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Table 1 Classi®cation of genotypes Apricot

Peach `GF-305'

Trait

Symptoms

ELISA

Symptoms

ELISA

‡

‡ ‡

‡ ‡ ‡

‡ ‡ ‡

a b

Susceptible Toleranta Resistantb Resistant

Virus multiplication occurs but there are no symptoms. Virus multiplication is prevented.

step of the grafting±inoculation-expression of symptoms procedure must be done correctly to get a plant response, a failure in any one of those steps will result in no symptom expression and could lead to an incorrect conclusion that the tested plant is resistant when it is not. The dif®culty of screening effectively may explain the limited progress towards resistance achieved by many of the breeding programs. A standard determination not dependent on environmental conditions and genotypes analyzed would be most desirable as it could be applied in different climatic areas by different teams and the results would be comparable. This study was conducted to develop an ef®cient, simple and short technique to determine resistance to sharka. Several factors involved in the procedure were evaluated:  methods of germination of `GF-305' peach aimed at increasing germination percentage and obtaining homogeneous seedlings;  best season for grafting apricot on peach to obtain the maximum percentage of plants available for screening;  comparison of sources of inoculum in terms of infection by PPV;  comparison of two techniques varying in sequence of grafting and inoculation. A new procedure to determine sharka resistance is presented. 2. Material and methods Seeds of peach `GF-305' came from an orchard located at IVIA. Buds from apricot came from the breeding program in progress at IVIA. The plant materials used in this study were grown in a greenhouse at 188C night average and 288C day average and 50±80% humidity in pots …16  16 cm†. A commercial substrate (Vriezenveen Potgrond PP2) was used. The strain of sharka used for inoculation was a Dideron isolate (the most common in Spain) described and characterized by Asensio (1996). It is the isolated 3.3 RB from a Japanese plum `Red Beaut',

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transmitted to `GF-305' by grafting and later transmitted to apricot `Canino' by aphids, Myzus persicae species (Avinent et al., 1994). 2.1. Optimization of seed germination After harvesting, the stones were removed from the `GF-305' fruits and dried at room temperature. A control lot was kept for standard strati®cation. In another lot, the seeds were excised by scissors and kept at room temperature. To prepare embryos, the testa was removed after 24 h soaking in 0.2% bleach. The embryos were soaked in 0.2% fungicide for 10 min. The following chilling treatments were studied: four lots of 100 embryos (endocarp and testa removed) were strati®ed for 20, 30, 40 and 50 days, respectively, three lots of 100 seeds (endocarp removed) were strati®ed for 60, 75 and 90 days. A standard strati®cation of stones for 120 days was used as reference. All lots were strati®ed in damp vermiculite, in a dark cold room at 48C. After each treatment the seeds were put into a chamber at 268C, 70±80% humidity and 16 h daylight photoperiod. Percentage of germination and level of grouped emergence (the extent to which all seeds germinated at the same time) were recorded. A seed was considered germinated when radicle measured more than 3 mm. After germination, seedlings were transplanted to pots and kept in a greenhouse. Plant length was recorded weekly during a 2-month period. Results were studied by ANOVA and multiple regression, using statgraphics software plus V 2.1. 2.2. Determination of best season for grafting Grafting was tested in autumn, winter and spring. Buds from nine different cultivars and one selection were tested per season. Three-month-old seedlings of `GF-305' were used for grafting. Two buds were grafted per peach seedling. The number of plants used per treatment and cultivar is shown in Table 3. Buds grafted in autumn came from shoots harvested in October and kept in a cold room for 30 days. Buds grafted in winter were harvested at the end of February and grafted without cold treatment. Buds grafted in spring were harvested in May and kept in a cold room for 30 days before grafting. After grafting, the plants were kept for 3±4 weeks in a greenhouse and later they were moved to a dark cold room at 58C for 2 months. After chilling treatment the plants were pruned to promote new shoots. Those plants that broke at least one bud were recorded as successfully grafted. Results were studied by ANOVA; factors studied were season and accession. 2.3. Comparison of peach and apricot as source of inoculum Two sources of inoculum were tested, peach `GF-305' and apricot `Canino'. Two lots of 30 plants each from `GF-305' were used as indicator plants. They

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were inoculated with PPV from peach `GF-305' and apricot `Canino', respectively, by grafting. Once the buds had taken, the plants were kept in a dark cold room for 2 months. After the chilling treatment the plants were pruned to promote new shoots for observation of symptoms. Later, the presence/absence of virus was con®rmed by ELISA-DASI. Results were analyzed by ANOVA. 2.4. Comparison of two techniques varying in inoculation±grafting sequence Technique A, previously described by Audergon and Morvan (1990) and technique B described by MartõÂnez-GoÂmez and Dicenta (1999) were compared. For technique A, the apricots to be tested were grafted on `GF-305' and later the apricots were inoculated with PPV. For technique B, the apricots were grafted onto a `GF-305' rootstock previously infected with PPV (Fig. 1). In both cases, 30 selections and four apricot cultivars were used and grafting was done in spring, the complete procedure being as described in Section 2.3. Plants on which the

Fig. 1. Procedure A: (1) grafting of the apricot to be studied on a peach `GF-305'; (2) inoculation of apricot by grafting; (3) chilling treatment of 2 months in a dark cold room; (4) production of new shoots and observation of symptoms. Procedure B: (1) inoculation of peach `GF-305' by grafting; (2) grafting of the apricot to be studied; (3) chilling treatment of 2 months in a dark cold room; (4) production of new shoots and observation of symptoms.

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apricot graft on `GF-305' reached a convenient size for determining sharka resistance were recorded. A third technique, similar to B, was studied, consisting of grafting the apricot and inoculating the PPV on peach, simultaneously. 3. Results 3.1. Optimization of seed germination Table 2 shows results of the different strati®cation procedures. Variables measured were: percentage of germination; the period of time during which seeds emerged; terminal shoot length. The best germination percentages were obtained when embryos (seed coat removed) were strati®ed for 20 or 30 days. The period of emergence was very short (less than 3 days from beginning to end). Longer chilling treatments of embryos resulted in contamination. The best terminal seedling growth was obtained by the standard procedure (chilling stones for 120 days ); however, emergence was delayed by 2±3 months. Chilling of embryos for 20±30 days resulted in the best treatment producing a high percentage of seed germination, seed emergence grouped, and seedlings which were large enough for grafting apricots. 3.2. Determination of best season for grafting Table 3 shows results obtained per cultivar and season of grafting. This experiment included cultivars with different origins. The best result (90% of plants grafted were available for testing) was obtained when buds were harvested in October and kept in a dark cold room for 30 days. Signi®cant differences were obtained between autumn and the other seasons. No signi®cant differences were Table 2 Germination percentage, emergence and terminal shoot length of `GF-305' seedlings obtained with different chilling treatments Chilling treatment

Germination (%)

Emergencea

TSL (cm)b

20 days embryos 30 days embryos 60 days seeds 75 days seeds 90 days seeds 120 pits

91.2 96.6 48.5 68.0 59.2 77.3

1 1 2 2 2 3

42.0 37.7 41.9 47.6 47.5 53.8

a

a* a a b b c

Emergence: 1 Ð all seedlings emerged within 3 days; 2 Ð emergence lasted 2 weeks; 3 Ð emergence lasted 2±3 months. b Terminal shoot length Ð measured 2 months after emergence. * Different letters indicate signi®cant differences at p  0:05:

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Table 3 Number of plants where grafting of apricot on `GF-305' was successful, according to the seasona Cultivar

Number of plants with budbreak of apricot/number of plants grafted Autumn

Winter

Spring

Goldrich Harcot Ginesta G  G 93-10 Lito Katy Sunglo Palau Pandora Mitger

13/14 12/13 16/16 14/16 4/6 8/8 7/7 4/6 6/6 6/7

4/8 8/8 2/8 2/8 3/8 7/8 3/8 1/8 8/8 5/8

5/6 5/6 3/6 5/6 5/6 5/6 ± 2/6 3/6 2/6

Total (%)

90/99 (90.9 %)

43/80 (53.7%)

35/54 (64.8 %)

a

Signi®cant differences were found between autumn and the other seasons. No signi®cant differences were observed between grafting in winter or spring. No signi®cant interaction between genotype and season was observed.

obtained between winter and spring. Autumn was the best season for grafting all cultivars and is recommended as the best time for setting up the test plants. No interaction between accession and season was observed which indicated that autumn was the best season for grafting all apricot genotypes. 3.3. Comparison of peach and apricot as source of inoculum Table 4 shows the percentage of plants infected after inoculation when infected buds were obtained from peach `GF-305' and apricot `Canino', respectively. There are signi®cant differences with respect to inoculum source. More infected plants were obtained when apricot was used as the source of inoculum and when test trees were evaluated both 2 and 4 weeks after chilling. No additional increase in the number of infected plants was observed beyond 4 weeks from inoculation. The high percentage of infection obtained using apricot indicated that this source should be used in routine tests for sharka resistance. Table 4 Percentage of `GF-305' seedlings infected by PPV according to the source of inoculuma Source of inoculum

Two weeks after chilling

Four weeks after chilling

Peach Apricot

18 33

63 96

a

No additional increase in the number of infected plants was observed beyond 4 weeks from inoculation.

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3.4. Comparison of two techniques varying in inoculation and grafting sequence Table 5 presents results obtained using both techniques A and B. The numbers indicated those plants that showed good characteristics, successful grafting and Table 5 Comparison of two techniques A and B. Numbers indicate those plants raised in good condition for determining resistance to sharka virus Genotypes tested SEO  P 93-1 SEO  P 93-2 SEO  P 93-3 SEO  P 93-4 SEO  P 93-5 SEO  P 93-6 SEO  P 93-8 G  G 93-1 G  G 93-5 G  G 93-7 G  G 93-8 G  G 93-10 G  G 93-12 G  G 93-13 G  G 93-15 G  G 93-17 G  G 93-18 G  G 93-19 G  G 93-20 G  G 93-21 G  G 93-23 G  G 93-24 G  G 93-25 G  G 93-26 G  G 93-27 G  G 93-30 G  G 93-32 G  G 93-33 G  G 93-34 G  G 93-35 Canino Harcot Goldrich Stark Early Orange Total a b

Six plants per genotype. Five plants per genotype.

Aa

Bb

0 0 1 3 2 2 5 3 4 1 0 3 0 2 2 3 4 1 3 2 2 5 2 6 1 0 1 4 1 0 2 5 1 0

4 1 4 4 4 4 2 3 2 3 5 4 4 2 3 5 3 4 4 2 5 5 5 5 5 3 2 5 1 2 2 3 1 1

71

112

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size for determining sharka resistance. Thirty-four different genotypes were used. Using technique A, six plants per genotype were grafted (204) and 35% of the plants grew large enough to analyze the trait. However, seven accessions had no plants that could be evaluated although six plants were grafted. On the other hand, technique B used ®ve plants per accession (170) and 66% reached the minimum conditions for PPV resistance determination. Technique B always gave plants for analysis. The variant of technique B, grafting simultaneously the apricot to be tested and the bud infected by PPV, resulted in similar numbers of plants (data not shown). The advantage of this last procedure is to shorten the sharka resistance determination by 1 month. 4. Discussion Evaluation of sharka virus resistant apricot is based on a biological test where peach seedling `GF-305' is used as an indicator of susceptibility. The procedure requires vigorous `GF-305' plants on which to graft the apricots and to score the symptoms. Seedling growth depends on overcoming the seed endodormancy that inhibits seed germination. Low temperatures are necessary to achieve the balance of inhibitors and promoters (Bewley and Black, 1983). Several authors (Chao and Walker, 1966; Zigas and Coombe, 1977; Frisby and Seeley, 1993a±c) studied the effect of chilling treatments in apricot and peach on seed germination and seedling growth. Seeds that received minimal strati®cation developed into abnormal plants, exhibiting dwar®sm. This is the reason that different chilling treatments on seeds and embryos were tested and compared with respect to germination percentage, emergence and seedling growth. Our results with embryos showed that a chilling treatment of 20±30 days gives a high percentage of germination, with good seedling growth and all the seedlings emerged at the same time which simpli®ed enormously the ®rst step of the procedure. In the previous studies, we tested treatments of seeds with growth regulators (gibberellic and cytokinin) on embryos. They produced high percentages of germination (higher than 90%) but produced abnormal plants that were dwarfed with deformed leaves and aborted apices (data not shown). When the best season for grafting was studied, we found that by chilling apricot buds and the `GF-305' rootstock, determination of sharka resistance can be accomplished at any time during the year. The greatest number of plants obtained per season was obtained in autumn. Results obtained in the autumn from this study were much better than those obtained in spring or winter or reported by other authors (MartõÂnez-GoÂmez and Dicenta, 1999). `GF-305' infected by PPV has been extensively used as a source of inoculum. `GF-305' is a good indicator of PPV, expressing sharka symptoms quickly and clearly. This suggested that high titres would be found in this peach; however,

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previous observations at IVIA (data not published) indicated that PPV replication in peach was less than in apricot, at least for Spanish strains. Our results show that apricot `Canino' infected by PPV is a better source of inoculum than `GF-305' peach. This is not unexpected, since the Dideron strain which has spread through Spain is better adapted to apricot than to peach (Quiot et al., 1995). Our results indicate that procedure B (described by MartõÂnez-GoÂmez and Dicenta, 1999) gives higher numbers of plants for testing than that of the former procedure described by Audergon and Morvan (1990), procedure A. Additionally, technique B is quicker than technique A. We developed a variant of procedure B, consisting of grafting the apricot and inoculating PPV on `GF-305' simultaneously, shortening the procedure by 1 month. Determination of sharka resistance includes steps of grafting, inoculation of plants, growth of plants in greenhouses, chilling treatments and pruning to promote new shoots. Every step needs to be accomplished successfully in order to complete the whole procedure. 5. Conclusion Our recommendation for determination of PPV resistance in apricots is as follows: (1) Obtain `GF-305' seedlings by germination of embryos after 20±30 days of chilling treatment; (2) 10±12 weeks later, inoculate with PPV, Dideron strain, by grafting buds from an apricot already infected; (3) simultaneously graft the apricots to be tested on to peach `GF-305', ideally in the autumn, the budsticks for grafting being kept in a cool room at 48C for 1 month; (4) apply a chilling treatment of 2 months, 4 weeks from grafting when the bud has `taken', and prune the chilled plants to promote new shoots for scoring symptoms; (5) analyze for the presence of virus by ELISA-DASI and/or RT-PCR . We recommend two cycles of chilling and observations and screening 5±6 plants per genotype. Using this procedure, about 500 seedlings from different crosses and populations (F1 and F2) obtained from our breeding program have been tested successfully. Screening of progenies by this protocol will allow inheritance studies of the trait and mapping of loci, which would make molecular assisted selection possible. Acknowledgements This research was supported by a grant from the ComisioÂn Interministerial de Ciencia y TecnologõÂa (AGF95-0737-C02-02). We thank Dr. Par®tt and Dr. Remington for revision and improvement of the manuscript. T.A.M. was funded by a fellowship from la Agencia EspanÄola de CooperacioÂn Internacional.

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