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 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.     

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.     

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.     

Rapid detection of plum pox virus by reverse transcription recombinase polymerase amplification

Journal of Plant Diseases and Protection - Plum pox virus (PPV) is one of the most destructive viral pathogens infecting stone fruit trees worldwide. As PPV causes a viral disease that requires...     

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.     

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.     

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.     

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 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.     

Spatial Pattern Analysis of Sharka Disease (Plum pox virus Strain M) in Peach Orchards of Southern France

ABSTRACT The spatial pattern of Sharka disease, caused by Plum pox virus (PPV) strain M, was investigated in 18 peach plots located in two areas of southern France. PPV infections were monitored visually for each individual tree during one to three consecutive years. Point pattern and correlation-type approaches were undertaken using the binary data directly or after parsing them in contiguous quadrats of 4, 9, and 16 trees. Ordinary runs generally revealed a low but variable proportion of rows with adjacent symptomatic trees. Aggregation of disease incidence was indicated by the theta parameter of the beta-binomial distribution and related indices in 15 of the 18 plots tested for at least one assessment date of each. When aggregation was detected, it was indicated at all quadrat sizes and tended to be a function of disease incidence, as shown by the binary form of Taylor's power law. Spatial analysis by distance indices (SADIE) showed a nonrandom arrangement of quadrats with infected trees in 14 plots. The detection of patch clusters enclosing quadrats with above-average density of symptomatic trees, ellipsoidal in shape and generally extending from 4 to 14 trees within rows and from 4 to 10 trees perpendicular to the rows, could be interpreted as local areas of influence of PPV spread. Spatial patterns at the plot scale were often characterized by the occurrence of several clusters of infected trees located up to 90 m apart in the direction of the rows. When several time assessments were available, increasing clustering over time was generally evidenced by stronger values of the clustering index and by increasing patch cluster size. The combination of the different approaches revealed a wide range of spatial patterns of PPV-M, from no aggregation to high aggregation of symptomatic trees at all spatial scales investigated. Such patterns suggested that aphid transmission to neighboring trees occurred frequently but was not systematic. The mechanism of primary virus introduction, the age and structure of the orchards when infected, and the diversity of vector species probably had a strong influence on the secondary spread of the disease. This study provides a more complete understanding of PPV-M patterns which could help to improve targeting of removal of PPV-infected trees for more effective disease control.     

李痘病毒单克隆抗体及TAS-ELISA试剂盒的研制

将纯化的李痘病毒(Plum pox virus,PPV)制剂免疫BALB/c小鼠,用SP2/0骨髓瘤细胞与经李痘病毒免疫的BALB/c小鼠的脾细胞融合,有限稀释法克隆和间接ELISA法筛选出2株稳定分泌李痘病毒单克隆抗体的杂交瘤细胞株3F1,7A8。用间接ELISA方法对所获得的2个杂交瘤细胞株进行亚型鉴定分别为IgG1、IgG3。间接ELISA方法测定腹水效价分别为3F1:1.0×106,7A8:1.0×105。以多克隆抗体为包被抗体、单克隆抗体为检测抗体的TAS-ELISA试剂盒与李痘病毒的D株系、M株系的病毒分离物均有反应,与同属的马铃薯A病毒、莴苣花叶病毒、西瓜花叶病毒2号、马铃薯Y病毒坏死株系不发生交叉反应。     

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.