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.     

Diversity of Plum pox virus isolates in Bosnia and Herzegovina

Sixteen Plum pox virus (PPV) isolates from several stone fruit cultivars, host species, orchards and geographical areas of Bosnia and Herzegovina were selected for typing, using serotype-specific monoclonal antibodies (MAbs) and PCR–RFLP, targeting the 3' terminal region of the coat protein (CP) and P3-6K₁ with restriction enzymes Rsa I and Dde I. Four PPV isolates were identified as PPV-M by serology and PCR; eight isolates were identified as PPV-D based on PCR–RFLP on both genomic regions, but were not recognized by the D-specific MAb4DG5. Four isolates from plum were identified as natural D/M recombinants (PPV-Rec), based on conflicting results of CP and P3-6K₁ typing. To investigate the genetic diversity of Bosnian PPV isolates in more detail, five isolates (three PPV-Rec, one PPV-M and one PPV-D) were partially sequenced in the region spanning the 3' terminal part of the NIb gene and the 5'-terminal part of the CP gene, corresponding to nucleotides 8056–8884. Nucleotide sequence alignment of recombinant isolates showed that they were closely related at the molecular level to previously characterized recombinants from other European countries, and shared the same recombination break point in the 3' terminal part of the NIb gene. This is the first report of naturally infected Prunus trees with PPV-M, PPV-D and PPV-Rec in Bosnia and Herzegovina. The high variability of the Bosnian PPV isolates fits with the presence of this virus in the country over a long period.     

Existence of two serological subclusters of Plum pox virus, strain M

A large-scale serological characterisation of Plum pox virus (PPV) isolates was carried out with 19 monoclonal antibodies (MAbs), including the universal MAb5B and the following strain-specific MAbs: AL (specific to PPV-M), 4DG5 (specific to PPV-D), TUV and AC (specific to PPV-C), and EA24 (specific to PPV-EA). The study involved 108 PPV isolates of different geographical origin (Albania, Bulgaria, Cyprus, Czech Republic, Egypt, France, Germany, Greece, Italy, Hungary, Moldova, Romania, Slovakia, Spain, Turkey and Yugoslavia) and hosts (almond, apricot, peach, plum and cherry). The inter- and intra-strain serological relationships of PPV isolates were evaluated by DASI-ELISA. High serological variability was detected, not only between strains, but also among isolates of the same strain. Computer-assisted analysis of serological data support the hypothesis of the existence of two distinct subclusters, denoted PPV-M₁ and PPV-M₂, which seem to prevail in Mediterranean and Eastern–Central European countries, respectively.     

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.     

Development of an on-site plum pox virus detection kit based on immunochromatography

Sharka disease, caused by plum pox virus (PPV), is the most serious viral disease of stone fruit trees. Among the eight known strains of the virus, PPV-D is the most important due to its recent global spread. Although enzyme-linked immunosorbent assay (ELISA) is the most common approach for diagnosing sharka, it involves time-consuming steps and requires expensive equipment and trained technicians. In this study, an on-site PPV detection kit based on immunochromatography was developed using polyclonal antibodies against the coat protein (CP) of a PPV-D isolate. The immunochromatographic (IC) assay kit was as sensitive as a commercial ELISA system for detecting Japanese PPV-D isolates. Moreover, it was easy to use (a one-step procedure), and results could be obtained on-site within 15 min without special laboratory equipment. The IC assay kit detected the virus from every aerial part of symptomatic Japanese apricot trees. In a detailed study of viral localization in leaves, the most suitable plant parts for use in the IC assay were symptomatic mesophyll tissues and the region from the petiole to the main vein. A positive reaction was also observed using the CP of other major (PPV-M and PPV-Rec) and minor (PPV-EA, PPV-W, and PPV-T) strains.     

Detection of plum pox potyvirus using monoclonal antibodies to structural and non-structural proteins1

Eleven monoclonal antibodies specific to plum pox potyvirus (PPV) coat protein were obtained by hybridoma technology from Spanish PPV isolates. In addition, two monoclonal antibodies specific for PPV cylindrical inclusions (CIP non-structural proteins) were obtained. The monoclonal antibodies specific for PPV coat protein were assayed by DASI ELISA against 81 PPV isolates. At least nine different epitopes were found and 21 distinct serological patterns of reaction (serogroups) were established using nine selected monoclonal antibodies against the collection of PPV isolates, indicating the high variability of coat protein among PPV isolates. Changes in epitope composition were observed after aphid and mechanical transmission, indicating the occurrence of mixtures of isolates in field trees. Monoclonal antibody 5B reacted with all PPV isolates assayed, with very high affinity, using DASI ELISA. This method was compared with immunocapture-PCR on field samples in spring, and showed very good coincidence of results. The efficiency of PPV detection can be slightly increased using monoclonal antibodies specific to cylindrical inclusions mixed with monoclonal antibodies against structural proteins, and using mixtures of monoclonal antibodies against different epitopes of coat protein. ELISA-I and immunoprinting-ELISA were able to detect CIP and PPV in extracts and tissue section, respectively, of woody plants. Two monoclonal antibodies offer the possibility of distinguishing between Marcus and Dideron PPV types (M or D). These D-specific monoclonal antibodies can be used in routine tests with high affinity.     

First report of plum pox virus infecting Japanese apricot (<Emphasis Type="Italic">Prunus mume</Emphasis> Sieb. et Zucc.) in Japan

For the first time, plum pox virus (PPV) has been detected in commercial Japanese apricot (Prunus mume) trees in Tokyo, Japan. These trees had ringspot or mottle on leaves, color breaking of petals and, occasionally, mild ringspots and malformation on fruits. The virus was identified based on the morphology of virus particles, serology, and RT-PCR. The amplified nucleotide fragment shared 100% identity with a partial coat protein gene of PPV-D isolates.     

Epidemiology of sharka disease in France

Plum pox virus was first detected in France in the 1960s. Both PPV-D and PPV-M strains are present but epidemics related to the PPV-M strain detected in the late 1980s are the most problematic. The two PPV strains have unequal distributions in peach and apricot orchards and different prevalences. More than 20 different aphid species have been identified as vectors of PPV but most of them do not colonize Prunus species. Thus, aphids involved in the spread of PPV in orchards are essentially visiting aphids. The main sources of inoculum for the vectors are leaves and fruits of infected stone-fruit trees. Spontaneous, wild and ornamental Prunus species such as Prunus dulcis , P. spinosa or P. pissardii are susceptible to PPV isolates found in France but their role as a reservoir in sharka epidemics is probably negligible. 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. Analysis of spatial patterns of disease has shown that secondary spread by aphids frequently occurs over short distances in the orchards (aggregated patterns) but also that dissemination at longer distances (of several hundred metres) is a common event.     

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.     

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.     

Cross reactions of an antiserum to plum pox potyvirus1

In the early spring of 1992, plum pox-like viruses (PPLVs) were detected by standard ELISA in some Prunus species. The isolates reacted positively with plum pox potyvirus (PPV) antisera in immunosorbent electron microscopy and Western blot analysis. In Western blot analyses, bands associated with the coat protein subunits of the PPLVs were 48–56 kDa, whereas bands associated with the coat protein subunits of known PPV isolates were 32–37 kDa in size. Also, the PPLVs differed from known PPV isolates in their symptoms on woody and herbaceous indicators, and in their herbaceous host range. None of these PPLVs appears to be an isolate of PPV.     

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.     

Significance of the heterologous encapsidation of zucchini yellow mosaic potyvirus in transgenic plants expressing plum pox potyvirus capsid protein1

Transgenic Nicotiana benthamiana plants expressing the coat protein of an aphid-transmissible strain of plum pox potyvirus (PPV-D) were infected with an aphid non-transmissible strain of another potyvirus, zucchini yellow mosaic potyvirus (ZYMV-NAT). Non-viruliferous Myzus persicae could acquire and transmit ZYMV-NAT from these plants but not from infected N. benthamiana control plants (not transformed, or transformed by the vector alone). Immunosorbent electron microscopy experiments using the decoration technique revealed that ZYMV-NAT virus particles in the infected transgenic plants expressing the PPV coat protein could be coated not only with ZYMV antibodies but also, on segments of the particles, with PPV antibodies. This suggests that aphid transmission of ZYMV-NAT occurred through heterologous encapsidation, and reveals a potential risk of releasing genetically engineered plants expressing viral coat proteins into the environment.     

Detection and characterization of Plum pox virus: serological methods

Tremendous progress has been made in the research and development of Plum pox virus (PPV) serological reagents and methods in recent years. Two facts have revolutionised the serological detection and characterization of the virus: the development of the ELISA method in 1977, and the later emergence of specific monoclonal antibody technology. The availability of commercial kits has popularised PPV diagnosis, now making diagnosis possible at large scale for quarantine purposes, eradication programmes and control of the disease in nurseries. The use of the universal monoclonal antibody 5B-IVIA, used in DASI-ELISA, is the most accurate system for routine PPV detection. Likewise, the use of typing monoclonal antibodies gives exact characterization of the main PPV types described: 4DG5 for PPV-D, AL for PPV-M, EA24 for PPV-EA, and TUV and AC for PPV-C. There is, in general, an excellent correlation between serological data obtained with PPV specific monoclonal antibodies and data obtained by molecular PCR based methods. ELISA using a single or a mixture of monoclonal antibodies will remain the preferred method for universal detection and routine screening of PPV for years to come. Today, other serological methods and reagents are also recommended in the EPPO Diagnostic Protocol, increasing the number of reliable tests available for PPV detection. These developments have helped to control sharka disease in recent years. International co-operation in this field has been crucial to the improvement and validation of serological tools for PPV detection and characterization.     

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.     

Current situation of sharka disease in Ankara,Turkey

Sharka disease has a limited distribution in Turkey and does not present a problem for stone fruit production. However, sharka is the most common virus disease of apricots, plums and peaches in Ankara, although it is not a common disease in other cities in Turkey. In different parts of Ankara, 212 private gardens in 21 locations were surveyed forPlum pox virus (PPV) incidence. All together 935 trees of apricot, plum, peach, sweet cherry and sour cherry were sampled and tested for the presence ofPPV by double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA).PPV was identified in numerous trees,viz. 286 apricots, 172 plums and 65 peaches. Strain differentiation ofPPV was accomplished using double-antibody sandwich indirect enzyme-linked immunosorbent assay (DASI-ELISA). These assays confirmed the presence of isolates belonging toPPV-M, PPV-D and mixed infections ofPPV-M+D. This is the first report of the presence ofPPV-D and mixed infections ofPPV-M+D in Turkey. http://www.phytoparasitica.org posting July 14, 2004.     

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.