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Sharka of Stone Fruits (Plum pox virus): A Destructive Diseases of Mediterranean Fruit Tree Species

Jifara Teshale

Department of Agribusiness and value chain management, Ambo University, College of Agriculture, Ambo, Ethiopia

Abstract: A brief account is given on the situation of Sharka disease in the Mediterranean countries. The recent developments in the field of virus characterization and diagnosis, epidemiology, genetic resistance and control measures are briefly reviewed and analyzed. The necessity to intensify and extend the territory monitoring activity and the use of certified plant propagating material of stone fruits is strongly recommended.

Keywords: PPV, diagnosis, epidemiology, control, resistance

Received March25, 2014; Revised April 24, 2014; Accepted May 09, 2014

Citation: Jifara Teshale, Sharka of Stone Fruits (Plum pox virus): A Destructive Diseases of Mediterranean Fruit Tree Species. Research in Plant Sciences, 2 (2). 45-49. January 2014. doi:10.12691/plant-2-2-3

1. Introduction

Sharka disease, caused by pox virus (PPV), is considered the most devastating viral disease of stone fruit trees, affecting mainly apricots, peaches and plums. Since its first detection in in 1917 (Atanasoff, 1932), the disease spread progressively to all , and then to the rest of the world. A part for very few exceptions (, and ), all the world’s important “stone fruit” industries are seriously threatened by this disease. The intense trade and movement of plants and fruit products, along with the ease with which sharka disease spreads in nature, have made global a problem that was initially only local, and the dissemination of the disease throughout the world now seems only a matter of time.

Susceptible hosts. All Prunus species can be infected by pox virus (PPV), although the presence and intensity of symptoms vary according to species and cultivar, virus strain and sanitary status of the host. The most important susceptible hosts are apricots (P. armeniaca), European and Japanese plums (P. domestica and P. salicina), peaches (P. persica) and their rootstocks (P. persica and P. domestica seedlings, P. cerasifera, P. insititia, P. marianna, etc.). Sweet and sour cherry (P. avium and P. cerasus) can be also affected, although with low incidence and limitedly by the PPV-C strain.

Many other wild and ornamental Prunus species can host PPV: P. spinosa, P. blireana, P. serotina, P. japonica, P. persica f. atropurpurea, P. glandulosa, P. mandshurica, P. mexicana, P. laurocerasus, P. mahaleb, P. davidiana, etc. (James and Thompson, 2006). They may act as symptomatic or, even more dangerously, as symptomless reservoirs of PPV from which the virus can spread to the cultivated crops.

The agent of the disease. The causal agent of Sharka disease is pox virus (PPV), a member of the Potyvirus genus in the Potyviridae family, whose genome has been completely sequenced and characterized (Salvador et al. 2006). PPV virions are long, flexuous and rod-shaped of 660–750 nm in length, formed by a single coat protein (CP) of about 36 kDa arranged helicoidally around one molecule of ssRNA of positive polarity (Brunt et al., 1996).

The PPV genomic RNA has a protein (VPg) linked to its 5’-end (Riechmann et al., 1989) and a long poly(A) tail at its 3’-end (Laín et al., 1988). The genome contains a long open reading frame (ORF) that is translated upon infection, into a large polyprotein of 355.5 kDa (Riechmann et al., 1991). The PPV polyprotein is co- and post-translationally cleaved by three virus-encoded proteinases to produce at least 10 mature protein products. From 5’ to 3’ termini the following proteins can be recognised: P1, HCPro (helper component protein), P3, 6K1, CI (cytoplasmic inclusion), 6K2, NIa-VPg (nuclear inclusion a VPg), NIaPro (nuclear inclusion a protein), Nib (nuclear inclusion b protein) and CP (coat protein). The 5’- and 3’- untranslated regions (UTR) of PPV consist of 147 and 222 nts, respectively. For a complete description of the PPV genome and of characteristics and functions of the single proteins it is remanded to Salvador et al. (2006).

Based on biological, serological and molecular information available, seven different types or strains of PPV can be recognised: D (Dideron), M (Marcus), EA (El Amar), C (Cherry), W (Winona), T (Turkey) and Rec (Recombinant between D and M), with D and M largely prevailing (Candresse and Cambra, ì--2006; Serçe et al., 2009).

The correct identification of PPV strains can be helpful in establishing the control strategies, since, in general, it can be indicative of the different biological characteristics of the virus, i.e. pathogenicity, host range and epidemiological behaviour. Even if this correlation is not always easy to demonstrate, on the basis of previous observations and reports it could be in general affirmed that:

•  PPV-M isolates are spread more readily by aphids to peach cultivars than PPV-D isolates, causing faster epidemics and more severe symptoms in peach trees;

•  PPV-D isolates are naturally able to infect apricot and plum, and rarely spread from these hosts to peach cultivars;

•  PPV-C is the only PPV strain able to infect and systemically invade cherry hosts;

•  PPV-Rec isolates can have intermediate characteristics between M and D isolates, with pathogenic and epidemiological behaviour more similar to D strains.

2. Detection and Characterization of Plum Pox Virus

Biological methods. Sap inoculation to herbaceous hosts and graft-transmission onto woody indicators represent the most ancient and traditional techniques for PPV and sharka detection. Among the several herbaceous indicators of PPV, Chenopodium foetidum, Nicotiana clevelandi, N. benthamiana and Pisum sativum are the most recommended for diagnosis and characterization studies (Gentit, 2006).

The preferred woody indicator for PPV biological indexing is the peach seedling GF305. P. tomentosa and P. persica cv. Elberta are also recommended as alternative to GF305. Grafting onto woody indicators requires long time for answers if compared with other diagnostic techniques; nevertheless, it remains compulsory to integrate other diagnostic techniques in the application of certification programs of nursery materials.

Serological methods. A decisive impulse to the diagnosis of PPV at a large scale has been given by ELISA. Today, together with polyclonal antisera, many monoclonal antibodies are available as commercial kits. Their use allows to overcome numerous problems associated with polyclonal antibodies, i.e. poor specificity, lack of homogeneity among different batches, strong dependence on the individual immunised animals, low sensitivity. The 5B-IVIA monoclonal antibody of Cambra et al. (1994) has been widely recognized and validated for the universal detection of any PPV isolate. Many strain-specific monoclonal antibodies have been also produced, i.e. 4DG5 for PPV-D (Cambra et al., 1994), AL for PPV-M (Boscia et al., 1997), EA24 for PPV-EA (Myrta et al., 1998) TUV and AC for PPV-C (Nemchinov et al., 1996; Myrta et al., 2000).

Molecular methods. Since their initial description, molecular methods for PPV detection have been increasingly used for their higher sensitivity than ELISA. An eight-fold increase of sensitivity can be obtained with molecular hybridization assay (Varveri et al., 1988; Wetzel et al., 1990), but the use of radioactivity limits the application of such technique for routine analysis. This problem is overcome by the use of polymerase chain reaction (PCR), and in particular by reverse transcriptase-PCR (RT-PCR) which expands the application of the technique by allowing the detection of RNA targets. Although several primers have been described as recognising all PPV isolates, those described by Wetzel et al. (1991) and Levy and Hadidi (1994) are considered as reference for general PPV detection by PCR methods. Sequence data from a number of PPV isolates have permitted the clustering of PPV strains into different groups and the development of group- or strain-specific detection assays (Candresse et al., 1998).

A substantial increase in sensitivity of PPV detection (ca. 250 times) is obtained by the implementation of an immunocapture step prior to RT-PCR (IC-RT-PCR) that simultaneously decreases the concentration of inhibitors (Wetzel et al., 1992).

To eliminate the time-consuming plant material homogenization in extract preparation, tissue prints of plant material (fresh section of tender shoots, leaves, flowers or fruits) and squash capture of single aphids on paper (Whatman 3MM) have been also performed (Olmos et al., 1996; 1997; Cambra et al., 1997). A disadvantage of this technique is the small amount of the sample which can be loaded on paper pieces.

Further improvements in the sensitivity of PPV detection have been obtained with several variations of the classical PCR method: heminested-PCR, PCR-ELISA, nested-PCR in a single closed tube, and Co-PCR using a universal probe for post-PCR hybridization detection (Olmos et al., 1997; 1999; 2002).

A significant advance in the detection, quantification and differentiation of PPV strains is by real-time PCR using either SYBR Green I or Taqman chemistry (Schneider et al., 2004; Olmos et al., 2005; Varga and James, 2005; Capote et al., 2006). These fluorescent techniques provide a more rapid, sensitive and reliable diagnosis of PPV disease, allow the quantification of PPV viral targets and does not require post-PCR electrophoresis or colorimetric detection.

The use of four easy sample preparation methods (dilution, spot, squash and tissue-print) coupled with highly sensitive real-time RT-PCR has been proposed for routine diagnosis of PPV at a large scale and for the analysis of samples collected during the winter (Capote et al., 2009).

An integrated approach, which includes biological indexing, serological and molecular assays using validated reagents and methods, has been recommended in the EPPO protocol for PPV detection and characterization of its strains (OEPP/EPPO, 2004).

Epidemiology of Sharka. PPV is a non-persistently transmitted aphid-borne virus. Many aphid species are known vectors of this virus. Several Prunus colonizing species, i.e. Myzus persicae, Brachycaudus helychrisi, Phorodon humuli, Hyalopterus pruni were detected as PPV vectors (Kassanis and Šutić, 1965; Jordović, 1963; Minoiu, 1973). Then, as more species were studied, it was demonstrated that more than 20 species belonging either to Prunus colonizing or no colonizing species were able to transmit the virus (Labonne et al., 1995).

The disease spreads rapidly in orchards but the rate of progression may vary according to the identity of the PPV strain and the Prunus species. Sharka disease spreads more rapidly in peach orchards infected by PPV-M than in apricot orchards infected by PPV-D. Based on more detailed observations and modelling studies, it was estimated that with no disease control measures, 100% disease incidence could be reached in 5 or 6 years in peach orchards infected by PPV-M, whereas in apricots infected by PPV-D, it would take 16 years to reach the same level of incidence (Adamolle et al., 1994; Hubert et al., 1988). Slight differences in biological properties related to host–vector–virus interactions could explain those distinct epidemic behaviours. Among those are: (1) the better efficiency of PPV-M isolates to infect peach after aphid inoculation (M. persicae) (2) the higher rate of transmission by aphids from leaves infected with PPV-M and (3) a longer period of virus availability for aphids with PPV-M infected peach leaves than with apricot leaves infected either by PPV-M or PPV-D (Quiot et al., 1995).

No PPV isolate is currently recognized to be seed-transmitted, so vertical transmission of PPV from infected mother plants to their progeny does not occur. The virus was detected in seed coats and cotyledons, but embryonic tissue and seedlings obtained from germinated seeds never showed symptoms, and gave negative results for PPV with both ELISA and PCR assays (Pasquini and Barba, 2006).

PPV control and monitoring. Control strategies in different countries can vary according to the absence or presence of the disease.

In countries where sharka has never been detected in fruit orchards (, , , etc) control strategies are based on measures that minimize the risk of entry/establishment of the disease. Important pre-import measures consist of i) limiting the importation of germplasm and/or of fresh fruits of Prunus only from countries where the disease is known not to occur; exception can be done for Prunus plants obtained in the framework of official Certification programs; ii) strengthening of entry (x-ray equipment, detector dogs, well-trained inspectors, etc.) and post-entry quarantine measures (maintenance of imported Prunus plants under observation in isolated conditions for a minimum of 1-2 years); iii) providing a capability for fast and accurate diagnosis of the virus; iv) increasing awareness of sharka disease within local stone fruit industry; v) operating a national surveillance system in fruit orchards and nurseries (official monitoring program); vi) encouraging the use of “virus free” materials.

In countries where Sharka is present only in restricted areas and is under official control (, , , etc.), type and entity of control measures should consider the importance of the fruit industry in the country, the distribution of fruit cultivation in the area, the existing level of PPV infection, the PPV strain involved, etc. For a successful disease management the information and the direct involvement of all producers and actors is essential. In general, once detected the presence of infected trees in the field, the following measures have to be taken: i) removal of all infected trees (and eventually of surrounding trees to the infected ones); ii) removal of the entire block when infection is higher than 10% (even less, in the case of PPV-M in peach orchards); iii) heavy limitation to nursery activity in infected areas. Heavier eradication measures can be justified in the case of early introduction of the disease in important fruit growing areas, when the complete eradication of the disease is considered still possible.

In countries where Sharka disease is endemic and its eradication is considered no more possible (many centre-eastern European countries, Balkan area), the use of sharka tolerant/resistant varieties seems to be the only realistic possibility for the survival of the local fruit industry. To this aim different strategies can be pursued:

•  selection of tolerant/resistant varieties;

•  conventional breeding of native varieties with tolerant/resistant varieties;

•  use of genetically modified plants (OGM).

In general, for all susceptible stone fruit species, the presence of tolerance/resistance characters in most commonly cultivated varieties is rare. Furthermore, the breeding with PPV resistant, but not well performing varieties (low fruit quality, low/high chilling requirement, early or late production, etc.) appears necessary.

In apricot (P. armeniaca) most of the PPV-resistance sources are North American varieties with Asiatic ancestors (Badenes et al., 1996). Stark Early Orange, Goldrich, Stella, Sunglo, Orange Red, Harlayne, Harcot, Veecot and Lito are some of the most commonly donors used in international breeding programs. To improve the quality of the new resistant hybrids, in some cases the selected first generation seedlings have been used for a second generation of crossing with native cultivars. A monogenic control of resistance trait, with the gene of resistance being heterozygous for the locus, has been hypothesised for “Goldrich” and “Stark Early Orange” donors, since a segregation of 1:1 susceptible/resistant seedlings is obtained (Dicenta et al., 2000), and being homozygous for “Stella”, since 100% of obtained seedlings is resistant (Dicenta and Audergon, 1998). Two genes control has been hypothesised in other experiments, where segregation was 3:1 susceptible/resistant (Dosba et al., 1991; Moustafa et al., 2001).

In peach (P. persica) the most promising sources for PPV resistance are some hybrids of P. persica x P. davidiana obtained in France (Decroocq et al., 2005) and some hybrids of P. persica x P. dulcis obtained in California (Martínez-Gómez et al., 2004).

In plum (P. domestica and P. salicina) new varieties tolerant to PPV were initially obtained by selecting hybrids from crossing between tolerant old varieties (Stanley, President, Gerstetter, Cacanska lepotica, Cacanska najbolja). However the tolerance character of these new hybrids was only ephemeral since it was loose in presence of unfavourable environmental conditions (high temperature, etc.). Since the lack of immune genotypes among plum varieties the attention of breeders was paid to the “hypersensitivity” character. The extremely sensitive cv. Ortenauer to PPV infection was therefore used in hybridization programs with Stanley and gave interesting new hybrids, among which the complete PPV resistant cv. Jojo. The use of hypersensitive genotypes has interesting application for the nursery activity in regions with sharka problems, since when they are grafted onto PPV-infected rootstocks, the developing young shoot will die off. Similar results can be obtained when hypersensitive rootstocks are used. In this case latently PPV-infected scions grafted onto the rootstocks will die off. As a result, only healthy plants will leave the nursery.

A limiting factor for the progress of breeding work is the laborious and long time requiring procedure for screening sharka resistance. PPV susceptibility assessment is done by symptom observation, ELISA test and PCR (when ELISA is negative) on both GF305 rootstock and grafted scion. GF305 can be top-grafted onto “ELISA-negative” scion to establish if the hybrid is resistant or immune (appearance or absence of symptoms onto top-grafted GF305, respectively).

The efficiency in the selection of progenies in the first steps of development of the seedlings can be significantly increased by molecular assisted selection (MAS). Very important results have been recently obtained in the identification of molecular markers co-segregating with genes/QTLs controlling the resistance to PPV and in the production of STS (Sequence Tagged Site) markers suitable for MAS (Decroocq et al., 2005; Lambert et al., 2007; Dondini et al., 2010).

In addition to the classical approaches for producing resistant cultivars, genetic transformation offers a promising alternative. An example of successful application of this approach is the plum clone C5 containing the PPV-CP transgene insert (Scorza et al., 1994). This clone (today named ‘HoneySweet’) has shown durable, stable and high level of PPV-resistance, which can be easily transferred by breeding programs to other varieties (Scorza and Ravelonandro, 2006). In countries where legislative obstacles do not exist this clone is ready to be cultivated in open field (Scorza et al., 2010).

3. Conclusions

In several Mediterranean countries, mainly those of the south coast, Sharka disease is not yet present or is restricted only in limited areas. In recent years, in these countries, stone fruits are becoming strategic for the economic and social development of local populations; therefore, the import of Prunus materials is consistently increasing to compensate the weakness of local nursery production. Standing this situation (increasing importation of stone fruit plants, widespread of Sharka disease in many exporter countries and absence of adequate phytosanitary laws), the risk of introducing infected plants has now become very high. It is therefore urgent and priority for the Mediterranean countries to implement preventive phytosanitary measures, such as to strengthen the Quarantine service, intensify PPV monitoring on the territory and establish Certification programs of plant propagating material. Furthermore, major efforts must be devoted to the intensification of research network activities and cooperative projects within Mediterranean countries to harmonize legislative measures and enforce national capacity buildings in the plant health sector.

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