A Single Amino Acid Mutation in the Plum pox virus Helper Component-Proteinase Gene Abolishes Both Synergistic and RNA Silencing Suppression Activities

ABSTRACT The effects on symptom expression of single amino acid mutations in the central region of the Plum pox virus (PPV) helper component-proteinase (HC-Pro) gene were analyzed in Nicotiana benthamiana using Potato virus X (PVX) recombinant viruses. PVX recombinant virus expressing the wild-type variant of PPV HC-Pro induced the expected enhancement of PVX pathogenicity, manifested as necrosis and plant death. Recombinant virus expressing a variant of PPV HC-Pro containing a single point mutation ( HCL(134)H) was unable to induce this synergistic phenotype. The RNA silencing suppressor activity of PPV HC-Pro was demonstrated in a transient silencing suppression assay. In contrast, the HCL(134)H mutant showed no such activity. These results indicate that a unique point mutation in PPV HC-Pro impaired its ability to suppress RNA silencing and abolished its capacity to induce synergism, and clearly shows for the first time the link between these two functions in potyvirus HC-Pro. Additionally, we compared the effects on virus accumulation in N. benthamiana plants infected with either the PVX recombinant constructs or with native viruses in double infection experiments. PVX (+) and (-) strand genomic RNA accumulated at similar levels in plants infected with PVX recombinants, leading to an increase in PVX pathology, compared with plants infected with PVX alone. This finding confirms that the enhancement of pathogenicity associated with synergistic interaction is not a consequence of more efficient PVX replication due to RNA silencing suppression by PPV HC-Pro.     

The question of seed transmissibility of Plum pox virus

For many years, Plum pox virus (PPV) was considered to be transmissible by seed, increasing the fear of long-distance spread of the disease. In the late 1970s, it was claimed on the basis of biological transmission of the virus to herbaceous indicator plants and the development of serological diagnosis based on polyclonal antibodies, that PPV was seed-transmitted, with a different infection rate according to the plant species and part of the seed which was tested. In the 1990s, PPV was characterized into four different types, and specific monoclonal antibodies were produced for them. These new and more sensitive diagnostic techniques, together with RT-PCR with different sets of specific primers, were used to approach once again the problem of PPV transmission through seeds. The virus was detected in seed coats and cotyledons, but embryonic tissue and seedlings obtained from germinated seeds never showed symptoms, and gave negative results for PPV with both ELISA and PCR assays. No PPV isolate is currently recognized to be seed transmitted, so vertical transmission of PPV from infected mother plants to their progeny does not occur. Hypothetically, the only possibility of seed transmission would arise from a mutation in the helper component of the virus, associated with high susceptibility of the infected Prunus cultivar.     

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.     

Hosts and symptoms of Plum pox virus: Herbaceous hosts

Plum pox virus (PPV) is polyphagous and epidemic. Apart from cultivated and wild Prunus species, a large number of herbaceous plants can be hosts of the virus. New herbaceous host species are continuously being reported following artificial inoculation studies. Some of these herbaceous hosts, Chenopodium foetidum , Nicotiana clevelandii , N. benthamiana and Pisum sativum are very useful for concentrating and purifying the virus. The list of plants that have been found to be infected with PPV in their natural environment is shorter than the list of plants which can be experimentally infected. The role of weed species in PPV survival and spread in orchards is poorly understood. It is widely accepted that annual plants or weeds are not important in the epidemiology of PPV.     

Unravelling the Prunus/Plum pox virus interactions

Plum pox virus (PPV) belongs to the genus Potyvirus that contains the largest number of virus species infecting plants. Its virus genome has been extensively characterised and sequenced. However, few data are available on its interactions with woody host plants. We therefore focused, in the past 4 years, both on the cellular and molecular aspects of the compatible and incompatible Prunus /PPV interactions. GFP (Green fluorescent protein)-tagged PPV and in situ hybridisation were used to compare the localization of viral particles in stems and leaves of susceptible and resistant apricot cultivars. In parallel, molecular tools were developed through the cloning and characterization of polymorphic, homologous resistance genes and of candidate genes involved in the expression of Prunus /PPV interactions. Candidate genes are currently used to target genomic regions involved in resistance or susceptibility and to identify molecular markers indispensable for further marker assisted selection for resistance to sharka disease.     

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...     

Comparison of four techniques for plum pox virus detection

Journal of Plant Diseases and Protection - Twenty-four stone fruit trees showing typical symptoms of plum pox virus (PPV) were tested for PPV using ELISA, RT-PCR, real-time RT-PCR and RT-LAMP. The...     

Hosts and symptoms of Plum pox virus: woody species other than fruit and ornamental species of Prunus

The presence of Plum pox virus (PPV) in woody hosts other than in fruit and ornamental Prunus species was evaluated. PPV symptoms, and their variability and intensity, are described in leaves and fruits of Prunus cerasifera , Prunus spinosa , Ligustrum vulgare and Euonymus europaea . Juglans regia is not confirmed as a new host of PPV.     

Epidemiology of sharka disease in North America

The history of North American (USA and Canada) epidemics of Plum pox virus (PPV), as well as hypotheses about the general North American epidemiology of PPV, are summarized. The hierarchical sampling method, combined with reliable DASI-ELISA detection/confirmation procedures, were successfully used for extensive PPV surveys of stonefruit-producing areas in both countries. The influence of epidemiological analyses on eradication policy is described.     

Evaluation of peach and nectarine cultivars in Bulgaria for their resistance to plum pox potyvirus

In spring and summer 1989, it was established that plum pox potyvirus (PPV) was present in certain peach cultivars in Bulgaria. At the same time, we started to investigate the distribution of PPV in naturally infected 4–5 year-old peach and nectarine cultivars and hybrids in order to optimize PPV detection. Over 160 peach and nectarine cultivars and hybrids were evaluated. In about 40% of the genotypes, typical plum pox symptoms were observed. The latter were estimated and divided into groups depending on their susceptibility to PPV. Observations were made on the population density of seven aphid species established in the peach orchards. Five proved to be vectors of the virus. Myzus persicae was the vector that played the main role in spreading the virus on peach.     

Potential role of almond in sharka epidemics: susceptibility under controlled conditions to the main types of plum pox potyvirus and survey for natural infections in France 1

The changes in cropping conditions of almond and the development of efficient tools for the detection and characterization of plum pox potyvirus (PPV) populations have led us to reassess the potential susceptibility of this species to sharka disease and its role as a virus reservoir. The susceptibility of almond cv. Aï to nine isolates, representative of the known diversity among PPV populations, was assessed under controlled conditions. Most isolates were able to infect almond, by graft or aphid inoculation, causing generalized stable infections without any obvious sharka symptoms. These infected almonds were found to constitute a potential source of virus for aphid vectors, mainly in the case of M isolates. Surveys were carried out in the south of France, in foci of M and D strains of PPV, to evaluate the presence of natural infections of almond. No typical symptoms were observed and the virus was never detected. It can be assumed that the actual limited prevalence of PPV in France does not lead to a sufficiently high inoculum pressure to allow almond trees to be infected.     

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.     

Detection of plum pox potyvirus and analysis of its molecular variability using immunocapture-PCR1

We have developed an immunocapture-PCR (IC-PCR) detection technique for plum pox potyvirus (PPV) which is both simple and highly sensitive. This single-day assay can detect about 2000 virus particles (200 fg of virus) diluted in 100 μl of crude plant sap, which is equivalent to a sensitivity about 2000 times better than that of a standard ELISA assay. RFLP analysis and sequencing of the amplified cDNA fragment indicate that three groups of strains with limited intra-group variability can be discriminated. Two of these groups correspond to the previously described D and M serotypes of PPV. The third group contains, so far, only the El Amar Egyptian isolate. Strains belonging to the D or M serotypes can easily be discriminated by Rsal polymorphism in the amplified cDNA fragment. Synthetic oligonucleotides allowing specific amplification of PPV strains belonging either to the D or to the M serotypes have also be designed.     

Plum pox in north america: identification of aphid vectors and a potential role for fruit in virus spread

ABSTRACT Thirteen aphid species were tested for their ability to transmit Pennsylvania isolates of Plum pox virus (PPV) collected in Columbia (PENN-3), Franklin (PENN-4), and York (PENN-7) Counties, PA. Four species, Aphis fabae, A. spiraecola, Brachycaudus persicae, and Myzus persicae, consistently transmitted PPV in preliminary transmission tests. Two species, Metopolophium dirhodum and Rhopalosiphum padi, were occasional inefficient vectors. Toxoptera citricida, from Florida, also was an effective vector but it does not occur in major stone-fruit-growing states. Species not transmitting PPV in parallel tests included Acyrthosiphon pisum, Aphis glycines, Aulacorthum solani, Macrosiphum euphorbiae, Rhopalosiphum maidis, and Sitobion avenae. When given a 3-day probing access period simultaneously on PPV-infected peach seedlings and healthy peach seedlings, Myzus persicae, Aphis spiraecola, A. fabae, and B. persicae transmitted PPV to 63, 31, 38, and 32% of the healthy peach seedlings, respectively. When given a similar probing period on PPV-infected peach fruit and healthy peach seedlings, the same aphid species transmitted PPV to 50, 35, 0, and 0% of seedlings, respectively. Results support the hypothesis of secondary PPV spread by indigenous aphids in Pennsylvania, and suggest that PPV-infected fruit has the potential to function as a virus source for long-distance dispersal.     

Causal agent of sharka disease: Plum pox virus genome and function of gene products

Our understanding of the molecular biology of Plum pox virus (PPV) and of the interactions between the virus and its hosts has advanced notably in recent years. Complete genome sequences have been obtained showing up the existence of new subgroups. Functions related to replication, symptom induction, virus movement, and interference with host defense mechanisms have been assigned to the different protein products derived from the proteolytic processing of PPV polyprotein. Moreover, the use of new approaches to define protein–protein interactions between the different viral proteins and between these and the host proteins has begun to yield the first results improving our understanding of how the virus and its host interact during infection.     

Control and monitoring: quarantine situation of Plum pox virus in New Zealand

Biosecurity New Zealand recognizes that Plum pox virus (PPV) is one of the most destructive diseases of stone fruit and aims to keep New Zealand free from it. PPV has never been detected in New Zealand orchards during national surveys. but it has been detected in post-entry quarantine. Biosecurity New Zealand has analysed the potential pathways for PPV to enter the country, and developed phytosanitary regulations and prevention control measures to reduce the risks. A contingency plan, a response programme, and diagnostic protocols have been developed and simulation exercises based on a hypothetical PPV incursion have been carried out.     

Production of plants of Nicotiana benthamiana and Prunus domestica transgenic for plum pox potyvirus coat protein,and demonstration of PPV resistance in transformed N. benthamiana1

Leaf discs of Nicotiana benthamiana plants were transformed with Agrobacterium tumefaciens and transgenic plants expressing plum pox potyvirus (PPV) coat protein (CP) were generated. Homozygous R₂ progeny from these plants were inoculated with PPV. Plants were scored for the appearance of symptoms and tested for infection by DAS-ELISA. Various levels of resistance were obtained after an initial stage in which PPV was able to multiply in all the transgenic plants. Within 2–3 weeks after inoculation, the transgenic resistant plants fully recovered from virus infection. Conversely, control and susceptible transgenic lines developed severe symptoms and high virus titres. Prunus domestica (plum) was transformed by inoculating hypocotyl slices with A. tumefaciens containing a binary plasmid which included the NPTII, GUS, and PPV CP genes within its T-DNA region. Transgenic shoots were rooted and established in the glasshouse. Analysis of selected transformants by PCR showed that the engineered foreign genes had been integrated, including that for PPV CP. Histological assays on young leaves of these putative transformants gave a positive reaction. This suggests that all genes transferred are expressed in these transformed plums.     

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.     

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.