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.     

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.     

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.     

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.     

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.     

Causal agent of sharka disease: new and emerging events associated with Plum pox virus characterization

Plant RNA viruses have a high genetic variation potential due to the absence of proofreading activity in their RNA replicase. In addition to mutation, recombination is generally thought to be an important source of variability. Both evolutionary processes have contributed to the diversity of Plum pox virus (PPV). There are now six recognized subgroups, strains or serotypes of PPV (D, M, Rec, EA, C and W). Isolates belonging to the PPV-Rec subgroup are derived from RNA recombination between PPV-D and PPV-M and occur frequently in various central and eastern European countries. The divergent isolate W3174 is a new and distinct strain of PPV, identified as PPV-W. It is quite conceivable that, with time, other groups will be defined and that the present classification will need revision to accommodate additional PPV variability.     

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.     

Plum pox potyvirus on sour cherry in Moldova

Plum pox potyvirus (PPV) was identified in six cultivars of sour cherry in the collection orchard of the Moldavian Horticultural Research Institute. Study of biological properties of the sour cherry isolate in herbaceous indicators showed its similarity or identity with the PPV isolate of Van Oosten and a significant difference from isolates widespread in Moldova. A purified viral preparation was used to develop antiserum with a working titre of 1:1024. Comparative serological examination of the sour cherry and conventional plum PPV isolates using ELISA, ISEM and SDS-PAGE of the protein capsid could not differentiate these isolates. The sour cherry isolate was transferred to plum resulting in weak but distinctive PPV symptoms in susceptible cv. Sopernitsa.     

Genetic variability of <Emphasis Type="Italic">Plum pox virus</Emphasis> isolates in the Czech Republic

Plum pox virus (PPV), the causal agent of Sharka disease, is an important pathogen of stone fruit trees. In this study, 24 new Czech PPV isolates from five different orchards were collected and characterized, molecularly. PPV-D isolates were identified in all orchards studied; whereas PPV-Rec isolates were identified in only two of them. A phylogenetic analysis on (Cter) NIb-(Nter) CP was performed. Three Czech PPV-D isolates BOH11CZ, BOH12CZ, and BOH13CZ diverged into a significantly separated cluster. PPV-Rec isolates formed a fairly homogenous group. However, the Bohutice and the Lipov PPV-Rec isolates clustered in two significantly separated branches.     

A Natural Population of Recombinant Plum Pox Virus is Viable and Competitive under Field Conditions

The isolate BOR-3, collected in Slovakia in 1996, was recently identified as a natural recombinant between an M and D type of Plum pox virus (PPV). Biological assays demonstrated its capacity to be aphid- and graft-transmitted to various Prunus spp. hosts. A study was carried out to determine the further presence of PPV recombinants in two epidemiologically distinct areas – Slovakia and France. Tools based on PPV-M and D subgroup typing, targeting P3–6K₁, CI and CP regions of the PPV genome were used for recombinant identification. Closely related recombinant variants were detected in different Prunus spp. during a survey conducted in Slovakia in 2001, but not within a set of selected PPV isolates from France collected between 1985 and 2001. Sequence analysis of the (Cter)NIb–(Nter)CP region of 10 recombinant isolates from Slovakia showed their high homology, reaching more than 98%. All the recombinant isolates shared the same recombination breakpoint situated in the C terminus of the NIb gene. Our study demonstrates that the PPV recombinants are viable and competitive with conventional PPV-M and D isolates. The present work indicates that the occurrence of recombinants within PPV isolates might be more common than previously assumed.     

Breeding for resistance: breeding for Plum pox virus resistant apricots (Prunus armeniaca L.) in the Czech Republic

Plum pox virus (PPV) causes serious damage in apricots grown in the Czech Republic and other countries where it is present. To study PPV resistance in apricot, three backcrosses between apricot cultivars or selection resistant to PPV and apricot cultivars or selections susceptible to PPV ('LE-3218' × 'Stark Early Orange', 'LE-3241' × 'Vestar' and 'LE-3246' × 'Vestar') were performed. The seedlings were repeatedly inoculated with PPV-M strain by an infected chip. The resistance of the plants was evaluated by visual inspection for symptoms and ELISA in three consecutive growth periods. The Chi-square (χ²) test was used to analyse the data. It was found that two independent dominant complementary genes conditioned PPV resistance in apricot in the case of the backcrosses. The mode of inheritance for resistance to PPV in cv. Harlayne was determined from F1 progenies. Resistance to PPV in 'Harlayne' was controlled by three independent complementary dominant genes. This knowledge will help us in effective planning of apricot breeding programmes.     

Plum pox virus detection in dormant plum trees by PCR and ELISA

An immunocapture polymerase chain reaction (IC-PCR) protocol and ELISA were compared for their effectiveness in detecting plum pox virus (PPV) in dormant plum material. Although the IC-PCR was about one thousand times more sensitive than ELISA, PPV was detected by ELISA in 71–80% of bark samples collected in December, January and March 1996/97 from pot-grown rootstock trees inoculated with PPV the previous March, compared with 85–86% detection in the same samples by IC-PCR. In similar samples from one-year-old shoots taken from infected branches of orchard trees, 66–81% were positive by ELISA compared with 81–87% by IC-PCR. With bulked samples taken from the fibrous roots of the pot-grown trees, PPV was detected in 92–100% of samples by IC-PCR in winter compared with only 38–65% by ELISA. These results were confirmed in samples from the roots and shoots of the same trees in 1997/98. Three samples per shoot would have been sufficient to detect PPV by ELISA in 87 of the 88 infected shoots tested during the two winters. However, infected shoots are irregularly distributed in diseased trees and PCR assays of root samples offer the potential for improving the reliability of identifying trees infected with PPV.     

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.     

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.     

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.     

Genetic analysis suggests a long and largely isolated evolutionary history of plum pox virus strain D in Turkey

Plum pox virus (PPV) strain D is globally distributed and causes serious losses in stone fruits in over 40 countries. Here, full-length genomic sequences were analysed for 44 PPV-D isolates from all regions of Turkey, together with partial sequences for a larger number of isolates. PPV-D isolates from Turkey are similar to other PPV-D isolates in all major genomic features. However, the majority of Turkish PPV-D isolates form separate phylogenetic clusters from all other isolates and show a geographical clustering tendency, suggestive of limited movement between regions. In particular, PPV-D isolates from Thrace and Central Anatolia formed a monophyletic sister cluster to the cluster that includes all previously known PPV-D isolates. Two isolates with strong evidence of recombination with the PPV-T strain were identified, together with two isolates with weaker evidence for intra-D strain recombination. The genetic diversity of PPV-D was found to be particularly high in Turkey (0.017 ± 0.001%), close to that observed for PPV-D world diversity once the over-represented isolates from Japan, the USA and Canada have been excluded (0.020 ± 0.001%). Taken together, these results suggest a long and largely isolated evolutionary history of PPV-D in Turkey and further extend knowledge of the diversity of this highly successful strain. The high diversity of PPV-D in Turkey, together with the basal phylogenetic position of Turkish isolates, are compatible with a hypothesis making Turkey the centre of origin of the D strain.