Breeding for resistance to plum pox potyvirus in the Czech Republic1

Breeding for plum cultivars resistant to plum pox potyvirus (PPV) is in progress in the Czech Republic, while projects for apricot and peach have started in 1991. The aim is to create an assortment of apricot and peach cultivars for northern regions of Europe in which PPV is widely distributed.     

Identification of Plum pox virus Determinants Implicated in Specific Interactions with Different Prunus spp

ABSTRACT The characterization of pathogenic properties of two infectious clones of Plum pox virus (PPV) isolates, pGPPV (D group) and pGPPVPS (M group), was investigated in their woody hosts (seedlings of Prunus spp.). The two clones differed in their ability to infect plum and peach cultivars, from no infection to local and systemic infection. The phenotype determinants were located with a set of chimeric viruses from the two clones. In plum, determinants of systemic infection were located in a genomic fragment encoding the P3 and 6K1 proteins, which might influence genome amplification or virus movement. The capacity of pGPPVPS to induce stable local and systemic infections in peach was not located accurately and might be influenced by multiple determinants carried by different regions of the genome, excluding those encoding the protein 1, the majority of helper component, nuclear inclusions a and b, and coat protein. We conclude that PPV infections of plum and peach are governed by different determinants.     

Susceptibility of apricot cultivars to artificial inoculation with plum pox potyvirus and use of ELISA for its evaluation1

The susceptibility to plum pox potyvirus (PPV) of 13 apricot cultivars and two hybrids was studied under isolated conditions in the experimental orchard of the Research Breeding Station at Vesele from 1989 to 1993 after artificial inoculation of the trees. The plants were inoculated in the nursery in 1988 by chip-budding with a local source of virus and then planted into the field trial. First symptoms of plum pox appeared on the leaves in 1990. Reinoculation of symptomless trees was done in summer 1991. By 1993, plum pox had totally invaded some apricot trees of the most susceptible cultivars (Madarska, Velkopavlovicka, Kisinevskij rannij, Ligeti orias, Bergeron, Vesna and Vestar). Uninoculated control trees remained symptomless despite the proximity of diseased trees and the fact that the orchard was not sprayed with insecticides during the period of experiment. Cvs Julskij, Veecot, Vegama and Veharda remained free from plum pox symptoms on leaves and fruits. In 1993-05/06, the trees were assayed for the presence of the PPV antigen by ELISA. Leaf samples from 33 trees showing symptoms were positive. Samples prepared from the healthy tissues of leaves with symptoms were negative. Other trees (indexed by ELISA) growing in the neighbourhood of diseased trees in the period of 5 years were visually and ELISA negative.     

Sensitivity of the detection of plum pox potyvirus by molecular assays1

Recently, plum pox potyvirus (PPV) has been found in Basilicata, southern Italy, on plum, apricot and peach. In 1992-09, we started a large-scale survey to verify the effectiveness of diagnostic methods used during seasons when it is difficult to reveal any presence of the virus. The assays were carried out by dot-blot hybridization on stone-fruit cultivars normally planted in this area. The virus was found, by dot-blot hybridization, to be present in seven cultivars of peach, four of apricot and one of plum. All plants were 8–10 years old and, except for two apricot cultivars, were not displaying any apparent symptoms in spring 1992. Five peach cultivars, intended for use as primary sources of propagation material, were then selected for further study, and assayed in 1992–11 by ELISA and RT-PCR. ELISA tests on these selected peach cultivars were consistently negative, while PCR tests were consistently positive. However ELISA tests gave positive results when repeated in 1993–05. These results not only suggest that primary propagation material should be tested by techniques more sensitive than ELISA, but also question the usefulness of carrying out tests during any phenological phases of the plant.     

Resistance to plum pox potyvirus in apricots1

The susceptibility of 115 apricot cultivars to plum pox potyvirus (PPV) has been examined, since 1981, in the experimental plots of the Pomology Institute at Naoussa and Skydra, Makedonia (GR). Inoculation was assured by aphids, transmitting strain PPV-M (Marcus) from naturally infected trees in adjacent peach orchards. For each cultivar, four to six trees were examined for at least 4 years. Observations on symptoms were made on leaves early in May and on fruits at maturity. Most cultivars expressed severe disease symptoms. Those without symptoms were inoculated by grafting onto heavily infected old apricot trees. The grafted shoots were tested for PPV in the following year by ELISA and on the woody indicator GF305. The cultivars which were rated as resistant after artificial inoculation and ELISA came from North America: Early Orange, Stella, NJA2, Sunglo, Veecot, Harlayne, Goldrich and Henderson. Most of these have been crossed with quality cultivars for the creation of resistant hybrids. The PPV resistance of large numbers of these apricot hybrids is now under investigation.     

History and importance of plum pox in stone-fruit production1

Plum pox potyvirus (PPV), which first appeared in Bulgaria in 1915, has spread to nearly every European country, and can today also be found in various eastern Mediterranean countries. The pest is of great economic importance especially in some southern and central European countries, causing considerable losses in production of primarily plum, apricot and peach. The extent of losses varies between countries of different climates, depending on cultivars and virus strains. In joint infections with other viruses, PPV provokes synergistic effects, enhancing the economic importance of the pest. Since PPV is transmitted by insects, spread of the virus can be reduced only by eradicating sources of infection and use of virusfree propagating material. Thus introduction of more reliable detection techniques is essential, facilitating mass testing of trees. Close collaboration in studying strain-related problems of PPV and in searching for resistant and tolerant cultivars is similarly important.     

Control and monitoring: monitoring and eradication of sharka in south-east Italy over 15 years

When the first foci of sharka were discovered in Puglia region (south-east Italy) in the late 1980s, the regional agricultural authorities launched a programme for Plum pox virus (PPV) monitoring and disease eradication. The infecting virus strain was identified as PPV-D. From 1989 to 1993, a strong eradication campaign was successfully carried out involving 13 plum and 2 apricot orchards with different levels of infection. During 1994–2000, besides plum, apricot and peach, monitoring was extended to sweet cherry. At that time, surveys and testing did not reveal any new PPV focus, but the eradication of infected trees continued in a couple of orchards. In 2001–05, particular attention was paid to peach, as devastating PPV-M outbreaks had developed in other areas of the country. A new PPV focus was found in apricot, caused by PPV-Rec, which was promptly eradicated. In the following two years, surveys in the once infected orchard and surrounding peach plantings did not detect any virus spread. The endeavour has taken 15 years making this PPV monitoring and eradication programme the longest in Italy. Its overall results indicate that the fruit tree industry in Puglia region can now be regarded as essentially PPV-free.     

Current status of sharka disease in Puglia (Italy) and implementation of an eradication programme1

Plum pox potyvirus (PPV) was introduced into Puglia (IT) a few years ago with infected propagative material coming from nurseries outside the region. Infections were detected in commercial plum and apricot orchards, but not in local nurseries. Extensive surveys were carried out in young apricot, plum and peach orchards to assess the distribution, incidence and spread of PPV infections, with a view to possibly enforcing an eradication programme. Surveys were based on visual inspections of about 300 commercial orchards and nurseries (covering an area of more than 550 ha) in the whole of Puglia, by ELISA and IBM tests, and by biological testing on GF305 of field samples. A total of 23,000 plants were individually checked by ELISA and IEM. The eradication involved 13 plum (35 ha) and 2 apricot (5 ha) orchards showing different levels of PPV infection. Six of the plum plantings showed infection exceeding 30% and were completely uprooted; in the remaining orchards, infection did not exceed 10% and uprooting was limited to infected trees. PPV was never detected in peach. The high incidence of PPV in some plum orchards was due to the heavy initial contamination of propagating material. Secondary virus spread was monitored in apricot orchards and found to occur at a low rate.     

Recent results on resistance to plum pox potyvirus in plum in Romania,and on the epidemiology of the disease1

Of 50 plum cultivars investigated for infection by plum pox potyvirus (PPV), three had the virus in the latent state (Mansan, Twilight and Frontier), 19 showed very slight or slight symptoms on leaves, 16 showed moderate symptoms and 12 severe symptoms. No symptoms were seen on fruits of cvs Diana, Ialomita, Abundance, Silvia, Mont Royal, Red Glow, Locale de Turt, Krikon and Blue Free. Slight symptoms present only on the fruit skin were noted on cvs Gras ameliorat, Seneca, Rosior de Zalau, Prun protuberat, Anna Späth, Early Rivers, LU, Pitestean, Pamiat Timiriazeva, Dunarea Albastra (Blue Danube), Gras romanesc and others. Few plum cultivars had sensitive or very sensitive fruit. Most hybrids obtained in Romania are tolerant in terms of symptoms on fruits. Of the rootstocks, Red Dwarf Myrobalan, Buburuz, Marianna and P. besseyi showed no or very slight symptoms. Plum plantations located close to a PPV infection source for 6–9 years were 13.2–45.5% infected. Trees planted in an infected orchard were up to 74% infected in 6 years.     

Susceptibility of some North American and Yugoslav plum cultivars to plum pox potyvirus in Poland1

All North American plum cultivars investigated in Poland for their reaction to plum pox potyvirus appeared to be susceptible, showing severe symptoms on the leaves. Fruits of cvs Valor and Empress were also heavily damaged. Of the Yugoslav cultivars evaluated, Cacanska Najbolja was most tolerant whereas Cacanska Rodna appeared to be most sensitive to PPV.     

Viruses of stone fruits in Albania

Surveys were carried out in the main stone-fruit growing areas of Albania to assess the phytosanitary status of Prunus in conimercial orchards and varietal collections. The presence of virus and virus-like diseases and their identification was ascertained through field observations, sap transmission to herbaceous hosts, graft transmission to woody indicators, ELISA and IEM tests. The mean infection level was 42%. In particular, infections in apricot and almond were 12 and 16%, respectively, i.e. lower than in plum and cherry (47 and 56%, respectively). The following viruses were identified: plum pox potyvirus (PPV). apple chlorotic leaf spot trichovirus (ACLSV). prunus necrotic ringspot (PNRSV) and prune dwarf (PDV) ilarviruses. PPV infection was very severe in plum, and limited in apricot and peach. Apple mosaic ilarvirus (ApMV), and six nepoviruses tested for (SLRV, TBRV, RRV, CLRV, ArMV and ToRSV) were not encountered in Primus.     

Detection of plum pox virus in Spain

Until recently, plum pox (sharka) virus (PPV) was never detected in Spain on any of the material analysed by the enzyme-linked immunosorbent assay (ELISA). It was only in June 1984 that the virus was first detected by two different antiscra in Japanese plum trees ( Prunus salicina ), cv. Red Beaut, showing typical symptoms of the disease. The detection was later confirmed by graft-transmission to GF-305 peach seedlings, and also by immunoelectron microscopy. The PPV was experimentally transmitted from GF-305 to GF-305 by aphids and from GF-305 to herbaceous plants by mechanical inoculation. Thus far (January 1985), PPV has been detected basically in Japanese plum trees in Sevilla, Murcia, Valencia and Castellón, in apricot in Castellón, and in peach trees in Sevilla and Lérida.
De nombreuses analyses par la méthode ELISA n'ont, pendant longtemps, pas permis de détecter le plum pox virus (agent de la sharka) en Espagne. Ce n'est qu'en juin 1984 que la présence du virus a été confirmée, par l'utilisation de deux antiséra différents, chez des pruniers japonais ( Prunus salicina ) cv. Red Beaut qui manifestaient des symptôines typiques de la maladie. La détection a été confirmée par greffage sur des plants du pêcher GF-305, ainsi que par microscopie immuno-électronique. La transmission du PPV de GF-305 à GF-305 a été réalisée à l'aide de pucerons et de GF-305 à des plantes herbacées mécaniquement. A cette date (janvier 1985). le PPV n'a été détecté quc dans des prunicrs japonais dans, les provinces de Sevilla, Murcia, Valencia et Castellón, ainsi que dans des abricotiers en Castellón et dans des pêchers en Sevilla et en Lérida.     

Control of Plum pox virus through the use of genetically modified plants

Genetic resistance to Plum pox virus (PPV) is the most viable alternative for long-term control of sharka disease. In addition to the classical approaches to producing resistant germplasm and cultivars, genetic transformation offers a promising genetic approach to resistance. We show, using the example of C5 plum, that genetically engineered resistance can provide durable, stable and high levels of PPV resistance. A review of the results of work with C5 including molecular analyses of resistance and long-term field testing is presented.     

A preliminary account of the sanitary status of stone-fruit trees in Morocco

Field surveys were carried out in the main stone-fruit growing areas of Morocco to evaluate the sanitary status of commercial orchards, varietal collections and nurseries. The presence of virus and virus-like diseases was checked by ELISA, sap transmission to herbaceous hosts, testing on woody indicators and molecular hybridization (dot-blot and tissue-printing). 1211 samples (382 almond, 339 peach, 291 plum, 150 apricot and 49 cherry) were tested by ELISA for the presence of Prunus necrotic ring spot virus (PNRSV), Prune dwarf virus (PDV), Apple chlorotic leaf spot virus (ACLSV), Apple mosaic virus (ApMV) and Plum pox virus (PPV). The overall average of virus infection rate was 16.4%, whereas that of single species was: 22.6% for almond, 17.8% for plum, 15% for peach, 10.2% for cherry, and 2.7% for apricot. The following viruses were detected: PNRSV, PDV, ACLSV and ApMV. 565 samples were tested by dot-blot and tissue-printing hybridization for the presence of Peach latent mosaic viroid (PLMVd) and Hop stunt viroid (HSVd). 48 samples were infected, 41 by PLMVd and 7 by HSVd. In addition, nested-PCR tests identified Plum bark necrosis and stem-pitting associated virus (PBNSPaV) in a few almond trees affected by stem pitting.     

Epidemiology of sharka disease in Spain

PPV was first detected in Spain in 1984 in Japanese plum ( Prunus salicina Lindl) cv. Red Beaut and spread very quickly to other Japanese and European plums and apricot cultivars but left peach cultivars unaffected. In the years following the detection of PPV, the predominant aphid species visiting Prunus orchards in Mediterranean areas were Aphis gossypii followed by Aphis spiraecola , the latter being the main aphid species found at present. Both species are considered to be the main vectors of PPV in Spanish early Prunus growing areas. Spatial analysis of the spread of PPV-D in Japanese plum and apricot trees confirmed the lack of significant association between immediately adjacent trees. The observed spatial pattern of sharka suggests a lack of movement of PPV-viruliferous aphid vectors to immediately adjacent trees and indicates their preferential movement to trees several tree spaces away. PPV-D is the only type currently present in Spain, with the exception of a PPV-M outbreak that was detected in and successfully eradicated from Aragón in 2002. The short-distance spread of PPV-M infection occurred as far as 12 m along the rows of peach trees. However, PPV-D has not been observed to spread through peach cultivars, despite being grown in the vicinity of heavily infected plots of apricot or Japanese plum trees.     

Serological characterization of Italian isolates of plum pox potyvirus1

An Italian isolate of plum pox potyvirus (PPV) from apricot, Ispave 17, was used as antigen for production of monoclonal antibodies. Six clones secreting specific antibodies to PPV were obtained. All these monoclonal antibodies were used to test a collection of different Italian PPV isolates, collected from plum, apricot and peach orchards, and other European isolates (including PPV-D and PPV-M serotypes), using DAS-ELISA, SDS-PAGE, western blot and GIEM. In western blot analysis, the PPV-M and PPV-D coat protein, detected directly from crude peach GF305 extracts, showed different electrophoretic mobility, the coat protein of PPV-M being slightly larger than that of PPV-D. ELISA tests, performed with fixed dilutions of antibodies and limiting dilutions of clarified samples, showed with some monoclonal antibodies a marked difference between PPV-M and PPV-D strains, at ratios greater than 1:40 (w/v). Also in GIEM some monoclonal antibodies gave a good labelling reaction only with PPV-D serotype. With the help of this differentiation, it was found that all Italian isolates tested were of the D serotype and none of the severe M strain of PPV, which has not been reported in Italy.