Spread Of Plum Pox Virus Research Articles

Determination of fruit chemical contents of two plum cultivars grafted on four rootstocks

The study was aimed at assessing chemical composition of fruits of plum cultivars ʻČačanska Lepoticaʼ and ʻJojoʼ grafted on four rootstocks with different mechanisms of resistance to Plum pox virus (PPV): sensitivity, ʻWavitʼ and ʻWeiwaʼ and hypersensitivity, ‘Docera 6’ and ‘Dospina 235’. The cultivar significantly influenced on content of sugars and organic acids, with the highest values in cultivar ʻJojoʼ. The highest content of sucrose, sorbitol and total sugars were found in ʻWeiwaʼ, and all individual and total acids were higher in fruits from trees grafted on ʻWavitʼ. On the one hand ‘Čačanska Lepotica’ had a high content of malic acid, but on the other hand a low content of citric and fumaric acids, regardless of the rootstock. The most abundant phenolic compounds, phenolic acids and flavanols, were shown to be at the highest level in the combinations ʻČačanska Lepoticaʼ/ʻDocera 6ʼ and ʻJojoʼ/ʻWavitʼ, whereas less abundant phenolic compounds, flavonols and anthocyanins, were the highest in the combinations ʻČačanska Lepoticaʼ/ʻWavitʼ and ʻJojoʼ/ʻWeiwaʼ. In order to improve nutritive fruit quality and the technology of growing, as well as to delay the spread of PPV, rootstocks ʻWavitʼ and ʻWeiwaʼ can be recommended for further promotion in modern plum plantations.

Spread and competitiveness ofPlum Pox Virus:Rec and -D strains in experimental plum orchard

Sharka disease caused by Plum pox virus (PPV) is endemic in Bulgaria. The speed and mode of spread of PPV-Rec and PPV-D isolates, molecularly characterised in advance and introduced in an experimental plum orchard to serve as permanent sources of infection was studied from 2008 to 2015. The trees were visually inspected for symptoms development and tested by DASI-ELISA twice each year. The positive samples were serologically (-M and -D specific MAbs) and molecularly (IC-RT-PCR) strain-typed, and sequenced in (Cter)NIb-(Nter)CP coding region. Artificially inoculated trees were found infected in the spring of 2009. After 7 years 43 trees (11.2%) became naturally infected. The majority of the trees were infected by PPV-Rec (65%), while only one tree (2.5%) by PPV-D. Nevertheless PPV-M was not introduced in the orchard, this strain was found in single and mixed infections with PPV-Rec strain. None of the isolates in naturally infected trees was identical to PPV-Rec and PPV-D inoculums, while several of them were a 100% identical to the respective PPV-Rec, PPV-D or PPV-M isolates from neighbourhood-infected trees. After the initial random pattern of PPV spread, aggregation of the new infections around the trees infected in previous years was observed. The presence of only Phorodon humuli and Hyalopterus pruni complex, known as PPV vectors and their poor abundance (5.0% from all caught aphids), together with some specific features of the landscape influenced the slow natural development of PPV in the experimental orchard.

Perspective on strategies for controlling the spread of Plum pox virus, causal agent of sharka/plum pox disease

Plum pox virus (PPV), the causal agent of sharka/plum pox disease, is considered the most harmful virus that threatens safe and successful cultivation of stone fruits (Prunus spp.; almond, apricot, cherry, nectarine, peach, and plum). Cambra et al. (2006) estimated that financial losses associated with sharka disease globally were approximately 3.6 billion (Bn) Euros for apricot for the preceding 30 years, 5.4 Bn Euros for plum for the preceding 30 years, and 0.58 Bn Euros for peach for the preceding 20 years. A considerable amount of research has been carried out on characterizing PPV, hence its identification as one of the top 10 plant viruses in molecular plant pathology (Scholthof et al., 2011). In spite of, and maybe because of, these research activities, the virus has been detected in almost every country where any significant commercial stone-fruit cultivation occurs, with the exception perhaps of Australia, New Zealand and South Africa. Are very strict plant quarantine programs or remoteness the keys to preventing the spread of PPV? Strategies for effectively controlling the spread of PPV must include simple, accurate and effective diagnostics (with PPV-appropriate sampling) that should be part of any quarantine program, survey activity, or phytosanitary certification program. Accurate information about the spread of PPV, better understanding of the various pathways for movement of the virus and taking the necessary steps to obstruct or remove these pathways are essential for preventing further spread of this very harmful virus.

Using geographical information system (GIS) technology to control the spread of Plum pox virus in Odessa region, Ukraine

Viral diseases are among the main causes for declines in the productivity of stone-fruit plantations and their adaptability to abiotic factors. Virus-infected plants are often more susceptible to adverse ambient conditions and more sensitive to fungal and bacterial diseases. To date, Plum pox virus (PPV) is subject to quarantine list A2 of the European Plant Protection Organization (EPPO). The virus is on the list of regulated pests common in Ukraine, and is subject to internal quarantine. In Ukraine, the areas of PPV spread amount to 40,132,764 ha. In Odessa region, districts totaling 18.5 ha are in PPV quarantine. Six hotbeds of PPV infection totaling 28 ha were found in Odessa region. For the first time in Odessa region, PPV was found on cherry trees. PPV-D was the only strain of the virus detected in every hotbed in Odessa region. To conduct phytosanitary zoning and determine potential areas for the progress of the disease in Odessa region, we have used the modern method of geographical information system (GIS) technology. GIS-aided maps were constructed showing PPV spread in Odessa region. To our knowledge, this is the first attempt to employ GIS technology to control plant pests in Ukraine. Provided it is properly maintained, the geospatial data, and the ability to generate detailed maps with it, is key to the success of PPV containment.

Evaluation of the Production Traits and Fruit Quality of German Plum Cultivars

Due to the substantial damage caused in Hungarian plum orchards by the spread of plum pox virus (PPV), high-yielding, often self-fertile German, Serbian and Romanian cultivars with better resistance to PPV have been introduced. Observations on growth characteristics, flowering and harvest time, yield potential and PPV resistance have been made in commercial plum orchards and gene bank collections since 2006, and these were supplemented with laboratory analysis on the fruit. German cultivars have spread widely in recent years, so it is particularly important to investigate the production value of these cultivars. In addition to field observations, a detailed analysis has been made over the last two years of the physical and chemical properties of the fruit of the most promising German cultivars (‘Topfive’, ‘Toptaste’, ‘Tophit’, ‘Topend Plus’ and ‘Jojo’) and of the control cultivars ‘Cacanska rodna’ and ‘Stanley’. The use of the German cultivars led to a lengthening of the harvest period, as fruit could still be harvested from ‘Topend Plus’ even in the middle of October. The onset of bearing was early for these cultivars, which had good yield potential, producing large numbers of buds not only on the spurs, but also on the long shoots. With the exception of ‘Jojo’ the cultivars exhibited greater initial vigour and the scaffold branches grew at a steeper angle, so greater care was required to form a favourable crown structure. The cultivar most resistant to PPV was ‘Jojo’, and ‘Topend Plus’ exhibited moderately severe leaf symptoms, while the other cultivars had only mild leaf symptoms. Among the cultivars included in the study, ‘Topend Plus’ and ‘Tophit’ had the largest fruit size, while ‘Toptaste’ had outstanding chemical properties, especially as regards the soluble solids and polyphenol contents.

GISTECHNOLOGY FOR THE MONITORING OF SHARKA DISEASE IN THE ODESSA REGION

Plant virus causes many important plant diseases and are responsible for huge losses in crop production and quality in all parts of the world, and consequently, agronomists and plant pathologists have devoted considerable effort toward controlling virus diseases. One the most important virus on many Prunus species, causing great economic losses is Plum pox virus (PPV),casual agent of Sharka disease. Since its discovery, Sharka has been considered as a calamity in stone orchards. The virus has been detected in almost every country where any significant commercial stone fruit cultivation occurs [1]. The virus is entered into the list of regulated pests common in Ukraine. In Ukraine, the total area of PPV spread totals 4013,2764 ha. In Odessa region, 18.5 ha districts are in PPV quarantine. Six hotbeds of PPV infection totalling 28 hectares were found in Odessa region. For the first time in Odessa region, PPV was found on cherry trees. Peach and plum trees are hit equally. In this study, we use geographic information systems technology to identify potential locations in a Odessa region for controlling the spread of Plum pox virus. To our knowledge, this is the first attempt to employ GIS technology for controlling plant diseases in Ukraine. Provided it is properly maintained, the geospatial data, and the ability to generate detailed maps with it, is key to the success of PPV containment. Information management will be a key to improving for controlling the spread of Plum pox virus.

Aphid Transmission of the Ontario Isolate of Plum Pox Virus.

Utilization of timed virus acquisition access probes in studies of plum pox virus (PPV) transmission by aphids demonstrated that endemic species transmitted the virus readily from plum, Prunus domestica (L.) Batsch; peach, P. persica (L.); or dwarf flowering almond, P. glandulosa Thunberg., to peach seedlings. The green peach aphid, Myzus persicae (Sulzer), was shown to be the most efficient vector. Acquisition of virus by green peach aphids from infected peach leaves resulted in 18-28% infected peach seedlings, while aphids previously fed on infected leaves of plum transferred virus to 36% of peach seedlings. Although the spirea aphid, Aphis spiraecola (Patch), was a less efficient vector than M. persicae it is perhaps more important for the spread of PPV due to its greater abundance and occurrence earlier in the season when peach trees are thought to be more susceptible to infection. Virus transmission rates varied depending on the virus source and healthy test plant species. In contrast to many previous studies, aphid inoculation of the experimental host Nicotiana benthamiana Domin occurred at a low rate, never exceeding 4%. Acquisition of PPV by M. persicae from infected peach fruit was greatly reduced compared with acquisition from leaves. The results of this research indicate that the Ontario isolate of PPV-D is readily transmissible by aphids to peach and natural spread of the virus needs to be considered in future management or eradication programs.

The efficiency of RNA interference for conferring stable resistance to plum pox virus

Plum transformed with an intron hairpin RNA CP (ihpRNA-CP) was resistant to plum pox virus (PPV) infection through the specific process of RNA silencing involving both small interfering-RNA (siRNA) and a methylated virus transgene. Silencing specifically targeted the PPV genome and led to the degradation of viral RNA in the model plant species Nicotiana benthamiana and the natural Prunus domestica host. Plums inoculated with the five major PPV strains, three widespread PPV strains (D, M, and Rec), and the atypical EA strain did not allow systemic spread of PPV in greenhouse-grown transgenic ihRNA-CP plum over multiple cycles of vegetative growth and cold-induced dormancy. PPV ihRNA-CP N. benthamiana displayed an immunity reaction and also allowed for the testing of PPV-C, a strain that was unable to infect P. domestica. This stable resistance demonstrated in plum based on the accumulation of siRNA can prevent PPV infection and can also act as a “curative” when PPV is inoculated through graft inoculation, through a recovery reaction. Regardless PPV strain variability based on geography, host species, epidemiology and serotypes of the CP protein and substitutions of nucleotides at the NH2-terminus of CP of the major five PPV strains tested, we show that the use of a PPV-CP intron hairpin (ihp) RNA is an effective strategy to specifically target the PPV genome. We provide methods and tools that demonstrate a reliable path towards developing PPV resistance suitable for protecting stone fruit orchards.

Biotech/GM crops in horticulture: plum cv. HoneySweet resistant to Plum pox virus

Commercialisation of Biotech/GM (Biotech) crops started in 1995. Not only field crops, but also horticultural transgenic crops are under development and are beginning to be commercialised. Genetic engineering has the potential to revolutionise fruit tree breeding. The development of transgenic fruit cultivars is in progress. Over the past 20 years an international public sector research team has collaborated in the development of HoneySweet plum which is highly resistant to Plum pox virus (PPV) the most devastating disease of plums and other stone fruits. HoneySweet was deregulated in the USA in 2010. HoneySweet (aka C5) has been evaluated for eleven years (2002–2012) in a regulated field trial in the CzechRepublic for the resistance to PPV, Prune dwarf virus (PDV), and Apple chlorotic leaf spot virus (ACLSV), all of them being serious diseases of plum. Even under the high and permanent infection pressure produced through grafting, PPV has only been detected in HoneySweet trees in several leaves and fruits situated close to the point of inoculum grafting. The lack of infection spread in HoneySweet demonstrates its high level of PPV resistance. Co-infections of PPV with PDV and/or ACLSV had practically no influence on the quantity and quality of HoneySweet fruit which are large, sweet, and of a high eating quality. In many respects, they are superior to the fruits of the well-known cultivar Stanley. Many fruit growers and fruit tree nurseries in the CzechRepublic are supportive of the deregulation of HoneySweet plum to help improve the plum production and control the spread of PPV.

SILENCING IN GENETICALLY ENGINEERED PRUNUS DOMESTICA PROVIDES DURABLE AND SAFE RESISTANCE TO PLUM POX VIRUS (SHARKA DISEASE)

Originally identified in Bulgaria in 1915, Plum pox virus (PPV) is the most damaging virus of stone fruit trees, including apricot, plum, peach and cherry. PPV steadily spread throughout Europe over the years since its discovery and at the turn of the century (1999-2000) it reached North America (USA and Canada). While many strategies to control the spread of PPV have been undertaken over the decades and many studies have contributed to the characterization of the virus isolates there has been relatively little progress in the development of resistant varieties. With the paucity of natural resistance, transgenic technology, based on the engineering of the virus capsid gene, was investigated as a useful source of resistance. This work identified the C5 plum clone as highly resistant to PPV infection. These findings were supported by detailed molecular studies indicating that post-transcriptional gene silencing (PTGS) is the resistance mechanism with resistance being mediated through the production of small interfering RNA (siRNA). The durability of PPV resistance in C5 (named 'HoneySweet') is reflected through more than 10 years of field tests. In total, over 15 years of research with 'HoneySweet' have demonstrated that this clone and the resistance mechanism that it represents is: i) an important tool to demonstrate the successful deployment of biotechnology against a quarantine pest; ii) a safe product of biotechnology; and iii) a useful strategy for avoiding the use of pesticides to control natural aphid vectors of PPV. The deregulation of 'HoneySweet' in the USA by USDA/APHIS (Federal Register Doc. E7-13649, July 12 2007) and clearance by the U.S. Food and Drug Administration (FDA) corroborate these findings.

Salicylic acid‐mediated and RNA‐silencing defense mechanisms cooperate in the restriction of systemic spread of plum pox virus in tobacco

Plum pox virus (PPV) is able to replicate in inoculated leaves of Nicotiana tabacum, but is defective in systemic movement in this host. However, PPV produces a systemic infection in transgenic tobacco expressing the silencing suppressor P1/HC-Pro from tobacco etch virus (TEV). In this work we show that PPV is able to move to upper non-inoculated leaves of tobacco plants expressing bacterial salicylate hydroxylase (NahG) that degrades salicylic acid (SA). Replication and accumulation of PPV is higher in the locally infected leaves of plants deficient in SA or expressing TEV P1/HC-Pro silencing suppressor. Accumulation of viral derived small RNAs was reduced in the NahG transgenic plants, suggesting that SA might act as an enhancer of the RNA-silencing antiviral defense in tobacco. Besides, expression of SA-mediated defense transcripts, such as those of pathogenesis-related (PR) proteins PR-1 and PR-2 or alternative oxidase-1, as well as that of the putative RNA-dependent RNA polymerase NtRDR1, is induced in response to PPV infection, and the expression patterns of these defense transcripts are altered in the TEV P1/HC-Pro transgenic plants. Long-distance movement of PPV is highly enhanced in NahG x P1/HC-Pro double-transgenic plants and systemic symptoms in these plants reveal that the expression of an RNA-silencing suppressor and the lack of SA produce additive but distinct effects. Our results suggest that SA might act as an enhancer of the RNA-silencing antiviral defense in tobacco, and that silencing suppressors, such as P1/HC-Pro, also alter the SA-mediated defense. Both an RNA-silencing and an SA-mediated defense mechanism could act together to limit PPV infection.

Plum Pox Virus Mixed Infection Detected on Apricot in Pakistan.

Sharka, caused by Plum pox virus (PPV), is the most detrimental viral disease of stone fruit trees. First reported from Bulgaria in 1917, the virus is now widespread in Europe, the Mediterranean Basin, and Asia Minor and is sporadically present in North and South America. On the basis of molecular and serological properties, six PPV subgroups are recognized, from which PPV-D, PPV-M, and PPV-Rec are the most common (1,2). Several apricot trees (Prunus armeniaca) showing mild, pale green rings and diffuse chlorotic spots on leaves were found in a small orchard in the Baltistan District in northern Pakistan at approximately 2,400 m above sea level. Dried leaf samples from one symptomatic tree randomly selected from the orchard were positive for PPV using double-antibody sandwich enzyme-linked immunosorbent assay with antisera prepared in the laboratory, immunoblot analysis, and reverse transcription-polymerase chain reaction (RT-PCR) targeting the capsid protein (CP) gene using standard procedures (1). To check the subgroup affiliation and evaluate the molecular variability, the 562-bp variable region spanning the C-terminus of NIb and the N-terminus of the CP was amplified, the RT-PCR product was cloned into the pGEM-T Easy vector (Promega, Madison, WI), and positive clones were analyzed by restriction and sequence analyses. Interestingly, sequence analysis of four clones revealed mixed infection, i.e., the presence of two different PPV isolates in the apricot sample. One isolate belonged to PPV-D (GenBank Accession No. DQ422147) and the other belonged to the PPV-Rec subgroup (GenBank Accession No. DQ422148). Multiple alignment of the sequenced genome portion of the Pakistan PPV-D isolate indicated 96 to 99% nt identity with various PPV-D isolates without unique, clear-cut differences. Similarly, the PPV-Rec isolate had 98 to 99% identity with European PPV-Rec isolates and retained the cross-over at nucleotide position 8450 in the 3' terminus of NIb. This sequence had the amino acid signature at the N-terminus of the CP typical of the PPV-Rec subgroup (2). Moreover, no particular clustering of the Pakistan isolates within PPV-D and PPV-Rec could be observed after phylogenetic analysis. The DAG motif, essential for aphid transmission, was present in both sequences. To our knowledge, this is the first indication of PPV occurrence in Pakistan and first identification of the PPV-Rec isolate outside Europe. Together with previous reports on the PPV presence in China and Kazakhstan (3,4), this report indicates the need for more detailed epidemiological studies focusing the PPV spread and its molecular diversity in Asia.

POTENTIAL USE OF TRANSGENIC PLUMS RESISTANT TO PLUM POX VIRUS FIELD INFECTION

Research to date indicates that post-transcriptional gene silencing (PTGS) is an effective strategy for the development of stable high-level resistance to Plum pox virus in plum. Field tests in Poland, Romania, and Spain show that after 5–6 years of natural aphid vectored inoculation, trees of the PTGS clone C5 remain virus-free. The effectiveness of transgene-based PTGS for imparting potyvirus resistance and the relatively rapid incorporation of this trait into Prunus germplasm suggests continued exploration and employment of this strategy for producing virus resistant stone fruits and other woody perennial tree fruits. INTRODUCTION Plum pox virus (PPV), the causal agent of sharka disease, is one of the most serious viruses affecting stone fruits, a group of commercially-produced Prunus species that includes peach, plum, apricot, and cherry (Nemeth, 1994). PPV is spread by aphids and through the use of infected budwood. Symptoms of plum pox infection include leaf chlorosis, fruit chlorosis and deformation, premature fruit drop, and tree decline, particularly in the presence of other common Prunus viruses (Roy and Smith, 1994). Originally reported from Bulgaria in 1932, PPV has spread throughout Europe where it has caused the destruction of over 100 million stone fruit trees. The scarcity of natural resistance and the lack of efficient control measures have made it difficult to stop the spread of PPV. Quarantine, plant sanitation, and eradication are the only available control strategies. Serious consequences result when these measures fail. Recent outbreaks of PPV in Chile, U.S., and Canada illustrate the potential for spread of this disease and the devastating consequences to the fruit industry. For example, during the first 2.5 years following the appearance of PPV in a limited area of Pennsylvania over 550 ha of stone fruit orchards were destroyed in order to contain and ostensibly eradicate the virus. Important and useful work is underway on the development of resistant cultivars of stone fruits utilizing naturally occurring resistance genes (Hartmann, 2002; Kegler et al., 1998; Polak et al., 2002). Yet there are few highly resistant genotypes, and the number of genes affecting resistance, their inheritance, and their effectiveness against various strains of PPV are areas of inquiry that require further study. It is clear that a multifaceted approach to developing resistant plants is necessary. Using plum (Prunus domestica L.) as a model, we have shown that through genetic transformation, stone fruits highly resistant to PPV can be developed (Scorza et al., 1994; Ravelonandro et al., 1997). This resistance is based on post-transcriptional gene silencing (PTGS) (Turner and Schuch, 2000) induced by the PPV coat protein transgene that was inserted into plum Proc. XXVI IHC – Genetics and Breeding of Tree Fruits and Nuts

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.

Natural Spread of Plum Pox Virus in Ankara, Turkey

AbstractPlum pox virus (PPV) has a limited distribution in Turkey, so is not a serious problem for stone fruit production. However, it is widespread in apricot, plum and peach trees in the Ankara region where its natural spread was studied between 1995 and 2002 in 10 home gardens planted with apricot, plum and peach trees. The presence of the virus was determined by visual observation of symptoms in leaves and fruits and by double antibody sandwich enzyme‐linked immunosorbent assay (DAS‐ELISA); strains were identified by double antibody sandwich indirect enzyme‐linked immunosorbent assay (DASI‐ELISA) and immunocapture reverse transcription‐polymerase chain reaction (IC‐RT‐PCR) tests. Within 8 years, PPV incidence in the gardens near to, and 0.5 km from an infection source was 85.7–100 and 25–29.4%, respectively. This difference in incidence was attributed to the distance from the infection source. All PPV isolates were identified as PPV‐M. It was found that sharka disease was introduced to the healthy gardens by mealy plum aphids (Hyalopterus pruni), the only vector of PPV found in Ankara. This is the first report of PPV spread in Turkey.

SEOPA-1 and GOLGI-2 Apricot Seedlings Are Resistant to Plum Pox Virus

SEOPA-1 and GOLGI-2 Apricot Seedlings Are Resistant to Plum Pox Virus

Systemic spread of Plum pox virus (PPV) in Mariana plum GF 8‐1 in relation to shoot growth

Infection of Prunus spp. by Plum pox virus (PPV) is characterized by an uneven distribution of the virus within the tree and branches. In order to gain a better understanding of this distribution, a method for modelling tree growth was used. PPV spread was followed within susceptible Mariana plum clone GF 8‐1 shoots for 4 months after inoculation. Shoot growth was unaffected by the presence of the virus. Symptoms appeared on leaves produced in the most actively growing parts of the shoots, i.e. at the beginning of the season. PPV was detected in leaves other than those showing symptoms. The proportion of leaves with detectable virus decreased from the zone showing symptoms, with 100% ELISA‐positive responses, to the shoot tip with no detectable virus in leaves produced between 111 and 127 days after inoculation. Furthermore, a higher proportion of positive ELISA results was obtained below the zone showing symptoms (77%) compared with 50% above. PPV was detected in 95% of the most vigorous shoots 71 days after inoculation compared with 37% of slower‐growing, later‐produced shoots.

Determination of resistance to sharka (plum pox) virus in apricot

The Mediterranean basin countries account for more than 50% of world apricot production. However, the spread of plum pox virus in this area is causing important losses and represents a limitation for the crop. In 1993, at IVIA, Valencia, a breeding program to develop cultivars resistant to sharka was initiated and an efficient procedure for the determination of sharka resistance in the progenies was needed. A methodology described previously in France, that uses apricot seedlings grafted on peach was developed to improve reliability. The germination and growing of ‘GF-305’ peach seedlings was improved, the subsequent grafting protocol was optimised, and a better source of inoculum was identified. A revised procedure is presented, which is more efficient than that reported previously.

TRANSGENE-BASED RESISTANCE TO PLUM POX VIRUS (SHARKA DISEASE) IS TRANSFERRED THROUGH INTERSPECIFIC HYBRIDIZATION IN PRUNUS

Plum pox virus (PPV) resistant rootstocks would be useful to control the spread of PPV in nurseries and orchards. The PPV-resistant transgenic Prunus domestica plum clone C-5 was used as a parent with Prunus spinosa to develop PPV resistant hybrid progeny. This work showed that C-5 plum is sexually compatible with the Prunus rootstock species P. spinosa. Resistance assays of chip-bud inoculated plants following two cycles of growth and cold-induced dormancy demonstrated that the transgenic hybrid clones were symptomless and DAS-ELISA negative. Further, immuno-blotting failed to detect PPV CP in inoculated transgenic hybrids. These results demonstrated the usefulness of C-5 for developing PPV resistant rootstocks with P. spinosa.

Transformation of Plum with the Papaya Ringspot Virus Coat Protein Gene and Reaction of Transgenic Plants to Plum Pox Virus

Transgenic plum plants expressing the papaya ringspot virus (PRV) coat protein (CP) were produced by Agrobacterium-mediated transformation of hypocotyl slices. Hypocotyl slices were cocultivated with Agrobacterium tumefaciens strain C58/Z707 containing the plasmid pGA482GG/CPPRV-4. This plasmid carries the PRVCP gene construct and chimeric NPTII and GUS genes. Shoots were regenerated on Murashige and Skoog salts, vitamins, 2% sucrose, 2.5 μm indolebutyric acid, 7.5 μm thidiazuron, and appropriate antibiotics for selection. Integration of the foreign genes was verified through kanamycin resistance, GUS assays, polymerase chain reaction (PCR), and Southern blot analyses. Four transgenic clones were identified. Three were vegetatively propagated and graft-inoculated with plum pox virus (PPV)-infected budwood in a quarantine, containment greenhouse. PPV infection was evaluated over a 2- to 4-year period through visual symptoms, enzyme-linked immunosorbent assay, and reverse transcriptase PCR assays. While most plants showed signs of infection and systemic spread of PPV within l-6 months, one plant appeared to delay the spread of virus and the appearance of disease symptoms. Virus spread was limited to basal portions of this plant up to 19 months postinoculation, but, after 32 months symptoms were evident and virus was detected throughout the plant. Our results suggest that heterologous protection with PRVCP, while having the potential to delay PPV symptoms and spread throughout plum plants, may not provide an adequate level of long-term resistance.