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.     

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.     

Control and monitoring: Post-Entry Quarantine System for stone fruits in Chile

In Chile a Post-Entry Quarantine System for propagating plant material has been established to prevent the introduction of quarantine diseases and to maintain good national phytosanitary status. Four types of quarantine facilities are in place and may be used for Prunus spp. and hybrids: absolute quarantine, filter quarantine and farm quarantines levels 1 and 2. All imported Prunus material is submitted to biological, serological and molecular analysis for Plum pox virus (PPV) detection.     

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.     

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.     

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.     

Quantitative resistance to Plum pox virus in Prunus davidiana P1908 linked to components of the eukaryotic translation initiation complex

A complex, polygenic resistance to Plum pox virus (PPV) was previously described in a wild peach-related species, Prunus davidiana clone P1908. In the current study, an analysis of quantitative trait loci (QTL) was performed on an F₂ population comprising 99 individuals obtained by selfing the F₁ individual #40 of an interspecific cross between susceptible nectarine cv. Summergrand and the resistant P. davidiana clone P1908. Six QTL were identified using both parametric and non-parametric methods of detection, individually explaining 5–28% of the phenotypic variance. The total phenotypic variation explained ranged from 29 to 58%. Alignment of the genetic map of the F₂ cross with the P. davidiana parent map showed consistency of QTL over generations, with three of the six QTL co-localizing at the 1-LOD interval and another one at the 2-LOD interval. Two of the QTL were mapped onto linkage group one, where resistance to PPV was previously mapped in apricot. Development and mapping of new microsatellite markers linked to candidate genes revealed a striking co-localization of three of the detected QTL with gene copies coding for eukaryotic translation initiation factors eIF4E and eIF(iso)4G. As co-localization of one QTL with candidate gene eIF(iso)4E was previously reported in the F₁ population, the results reported here strongly reinforce the idea that components of the eukaryotic translation initiation complex are correlated with resistance to PPV in P. davidiana P1908.     

Characterization of Plum Pox Virus PPV-BT-H Isolated from Naturally Infected Blackthorn (Prunus spinosa L.) in Hungary

Plum pox virus (PPV) was found naturally infecting blackthorn (Prunus spinosa L.) plants in different regions in Hungary. The virus was identified on the basis of reactions with indicator plants, by DAS–ELISA tests and by RT–PCR. PPV isolated from blackthorn (PPV-BT-H) caused yellow lesions with a necrotic centre on Chenopodium foetidum L. indicating that it belongs to the intermediate pathotype. The coat protein gene of the blackthorn isolate was cloned, sequenced and compared with other PPV sequences. The BT-H isolate and the Hungarian plum isolate SK68 belong to different subclusters of the M group in contrast to the Hungarian almond isolate (PA) which belongs to the D group. Detecting PPV on blackthorn demonstrated that this plant may be an important source and reservoir for PPV in Hungary.     

Hosts and symptoms of Plum pox virus: ornamental and wild Prunus species

Several ornamental and wild Prunus species have been identified as natural and/or experimental hosts for Plum pox virus (PPV). The significance of natural vs. experimental hosts, graft or bud-transmitted infections vs. aphid-transmitted infections in ornamental or wild Prunus hosts, and their relevance in the field situation, are not clearly understood. However, since PPV is aphid-transmitted, any host in the field or nursery serves as a potential reservoir and source of inoculum and must be monitored and controlled in any PPV eradication or management programme.     

Breeding for resistance: breeding for resistance to Plum pox virus in Italy

Plum pox virus (PPV) resistance in apricot and peach in Italy has been researched in several Italian institutions, including the Institutes for Fruit Tree Culture and Plant Pathology in Rome, which are assessing resistance, and the Universities of Bari, Bologna and Milan, examining breeding techniques, both by cross-breeding and genetic transformation approaches. A reliable protocol for resistance assessment has been set up, in which biological indexing, ELISA and PCR techniques are used. No source of resistance has yet been found in peach. Hybridization with Prunus davidiana could be investigated given the promising results obtained within the INRA programme in France. Almost 6000 apricot seedlings from controlled crosses between resistant parents have been investigated in search of possible molecular markers, but no positive results have been obtained so far, probably due the fact that resistance to both D and M strains is sought. The transformation of cultivars has proven difficult in apricot where the regeneration step is particularly critical, while in peach a reliable regeneration protocol still remains to be set up.     

Advances and prospects in potato virology with special reference to virus resistance

Knowledge of the nucleotide sequences in the genomic nucleic acid of several potato viruses has enabled the open reading frames to be identified. These open reading frames are expressed by a variety of strategies, to produce proteins with functions in virus nucleic acid replication, virus particle production, cell-to-cell transport of virus and virus transmission by vectors. The activity of such proteins depends on their interactions with other viral or non-viral materials.Several other biological properties of plant viruses can also be related to individual viral gene products. For example, in plants co-infected with a specific pair of unrelated viruses, one virus can benefit from an ability to use the gene product of the second virus in replication, cell-to-cell transport or transmission by vectors. Similarly, different host resistance genes are targeted against viral replicase, movement protein or coat protein. Thus it is becoming possible to relate gene-for-gene (or more accurately, viral gene domain-host gene) interactions to events at the molecular level. Genetically engineered resistance to plant viruses likewise can be targeted against individual viral genes, and probably also against viral regulatory sequences. Such transgenic resistance seems likely to be as durable as conventional host resistance but durability should be improved by producing plants with combinations of resistances of different kinds, either conventional or genetically engineered, or both.     

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.     

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.     

Breeding for resistance: breeding for Plum pox virus resistant plums (Prunus domestica L.) in Germany

In German plum breeding programmes new varieties tolerant to Plum pox virus (PPV) were initially obtained by selecting hybrids originating from crossings between tolerant old varieties while, later on, sharka tolerant varieties bred in Čačak, Serbia were used as crossing partners. Varieties released from these programmes replaced the sensitive ones in all sharka infected regions in Germany. While all the new tolerant varieties can be infected by the virus, PPV symptoms on the fruits are acceptable. However, environmental factors can weaken the plant, causing them to suffer more from PPV infections and to display worse symptoms on fruits, as did occur during some recent very dry and warm years. A breeding programme at the University of Hohenheim is tackling this problem with genotypes that show a hypersensitive response after infection. These genotypes are completely field resistant to PPV, remaining virus free in the field since they cannot be infected via aphid transmission. They are able to isolate the virus after infection. The first hypersensitive variety was 'Jojo'. Many seedlings originating from crossings with at least one hypersensitive parent are under evaluation. Since the inheritance of the trait 'hypersensitivity against PPV' is very good, combining hypersensitivity with excellent fruit quality and good cropping capacity will be possible soon. In 2005, a breeding programme for hypersensitive Prunus genotypes began at the Technical University of Munich.     

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.     

First detection and complete genome characterization of a Cherry (C) strain isolate of plum pox virus from sour cherry (Prunus cerasus) in Germany

Journal of Plant Diseases and Protection - An isolate of plum pox virus (PPV) was detected in sour cherry (Prunus cerasus) in the Eastern region of Germany. Symptoms observed on infected sour...