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.     

Approaches to pathogen-mediated resistance breeding against plum pox potyvirus in stone-fruit trees1

In a programme for developing systems which allow the transfer of foreign genes into apricot cultivars, we have tested cotyledons of immature embryos, somatic embryos and leaf discs. Apricot plants have been transformed, and then regenerated, with Agrobacterium tumefaciens strain LBA 4404 containing various binary plasmids: pBinGUSint, carrying the marker gene β-glucuronidase (GUS), and pBinPPVm, carrying the coat-protein gene of plum pox potyvirus (PPV). The marker gene GUS was used for visual evaluation of the efficiency of the transformation system. The coat-protein gene was used in the hope of introducing coat protein-mediated resistance to one of the most important stone-fruit pathogens in Europe and the Mediterranean area.     

Transmission of plum pox potyvirus in Spain1

Transmission tests were conducted in the laboratory to determine which are the aphid species responsible for the great natural spread of plum pox potyvirus (PPV) observed in the field in Spain. Woody hosts were used in these tests and different transmission techniques were compared. The aphid species tested were Myzus persicae, Aphis gossypii, A. spiraecola, A. fabae, Hyalopterus pruni and Brachycaudus prunicola. Although the transmission rates obtained were, in general, quite low, it can be stated that, except for B. prunicola (pending confirmation of results), all species tested transmitted PPV under the conditions of the trial.     

Cross reactions of an antiserum to plum pox potyvirus1

In the early spring of 1992, plum pox-like viruses (PPLVs) were detected by standard ELISA in some Prunus species. The isolates reacted positively with plum pox potyvirus (PPV) antisera in immunosorbent electron microscopy and Western blot analysis. In Western blot analyses, bands associated with the coat protein subunits of the PPLVs were 48–56 kDa, whereas bands associated with the coat protein subunits of known PPV isolates were 32–37 kDa in size. Also, the PPLVs differed from known PPV isolates in their symptoms on woody and herbaceous indicators, and in their herbaceous host range. None of these PPLVs appears to be an isolate of PPV.     

Recent results on resistance to plum pox potyvirus in plum in Romania,and on the epidemiology of the disease1

Of 50 plum cultivars investigated for infection by plum pox potyvirus (PPV), three had the virus in the latent state (Mansan, Twilight and Frontier), 19 showed very slight or slight symptoms on leaves, 16 showed moderate symptoms and 12 severe symptoms. No symptoms were seen on fruits of cvs Diana, Ialomita, Abundance, Silvia, Mont Royal, Red Glow, Locale de Turt, Krikon and Blue Free. Slight symptoms present only on the fruit skin were noted on cvs Gras ameliorat, Seneca, Rosior de Zalau, Prun protuberat, Anna Späth, Early Rivers, LU, Pitestean, Pamiat Timiriazeva, Dunarea Albastra (Blue Danube), Gras romanesc and others. Few plum cultivars had sensitive or very sensitive fruit. Most hybrids obtained in Romania are tolerant in terms of symptoms on fruits. Of the rootstocks, Red Dwarf Myrobalan, Buburuz, Marianna and P. besseyi showed no or very slight symptoms. Plum plantations located close to a PPV infection source for 6–9 years were 13.2–45.5% infected. Trees planted in an infected orchard were up to 74% infected in 6 years.     

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.     

Susceptibility of some North American and Yugoslav plum cultivars to plum pox potyvirus in Poland1

All North American plum cultivars investigated in Poland for their reaction to plum pox potyvirus appeared to be susceptible, showing severe symptoms on the leaves. Fruits of cvs Valor and Empress were also heavily damaged. Of the Yugoslav cultivars evaluated, Cacanska Najbolja was most tolerant whereas Cacanska Rodna appeared to be most sensitive to PPV.     

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.     

Sensitivity of the detection of plum pox potyvirus by molecular assays1

Recently, plum pox potyvirus (PPV) has been found in Basilicata, southern Italy, on plum, apricot and peach. In 1992-09, we started a large-scale survey to verify the effectiveness of diagnostic methods used during seasons when it is difficult to reveal any presence of the virus. The assays were carried out by dot-blot hybridization on stone-fruit cultivars normally planted in this area. The virus was found, by dot-blot hybridization, to be present in seven cultivars of peach, four of apricot and one of plum. All plants were 8–10 years old and, except for two apricot cultivars, were not displaying any apparent symptoms in spring 1992. Five peach cultivars, intended for use as primary sources of propagation material, were then selected for further study, and assayed in 1992–11 by ELISA and RT-PCR. ELISA tests on these selected peach cultivars were consistently negative, while PCR tests were consistently positive. However ELISA tests gave positive results when repeated in 1993–05. These results not only suggest that primary propagation material should be tested by techniques more sensitive than ELISA, but also question the usefulness of carrying out tests during any phenological phases of the plant.     

Expression of the mature form of plum pox potyvirus helper component1

Polyclonal antibodies raised to the plum pox potyvirus (PPV) helper component (HC) have been produced for the specific detection of HC protein in PPV-infected plants. These experiments suggested that PPV HC is a single soluble protein of 48 kDa. The independent expression of the first two and half cistrons of the PPV genome in transgenic plants suggests that the P1 and P2 (HC-Pro) proteases are involved in the processing of the mature form of HC.     

Detection of plum pox potyvirus using monoclonal antibodies to structural and non-structural proteins1

Eleven monoclonal antibodies specific to plum pox potyvirus (PPV) coat protein were obtained by hybridoma technology from Spanish PPV isolates. In addition, two monoclonal antibodies specific for PPV cylindrical inclusions (CIP non-structural proteins) were obtained. The monoclonal antibodies specific for PPV coat protein were assayed by DASI ELISA against 81 PPV isolates. At least nine different epitopes were found and 21 distinct serological patterns of reaction (serogroups) were established using nine selected monoclonal antibodies against the collection of PPV isolates, indicating the high variability of coat protein among PPV isolates. Changes in epitope composition were observed after aphid and mechanical transmission, indicating the occurrence of mixtures of isolates in field trees. Monoclonal antibody 5B reacted with all PPV isolates assayed, with very high affinity, using DASI ELISA. This method was compared with immunocapture-PCR on field samples in spring, and showed very good coincidence of results. The efficiency of PPV detection can be slightly increased using monoclonal antibodies specific to cylindrical inclusions mixed with monoclonal antibodies against structural proteins, and using mixtures of monoclonal antibodies against different epitopes of coat protein. ELISA-I and immunoprinting-ELISA were able to detect CIP and PPV in extracts and tissue section, respectively, of woody plants. Two monoclonal antibodies offer the possibility of distinguishing between Marcus and Dideron PPV types (M or D). These D-specific monoclonal antibodies can be used in routine tests with high affinity.     

Non-structural plum pox potyvirus proteins detected by immunogold labelling

Plum pox potyvirus (PPV) induces in infected Nicotiana clevelandii cells characteristic crystalline inclusions known as nuclear inclusions (NI) when located in the nucleus and as dense material (Dm) when located in the cytoplasm. Crystalline inclusions contain protease (NIa) and RNA-dependent RNA polymerase (NIb) proteins. It is now well established for all potyviruses that cylindrical inclusions contain CI helicase ATPase protein (Martin et al., 1992). The intracellular location of other non-structural PPV proteins remains unknown. Using Escherichia coli expression vectors, specific antibodies were obtained against P1, P3, 6K2 and NIb PPV proteins for which antibodies were not yet available. As expected, NIb antiserum labelled crystalline inclusions. P1, P3 and 6K2 proteins were present in both types of crystalline inclusions found in the nucleus and in the cytoplasm of PPV-infected leaves of N. clevelandii, suggesting that nuclear inclusions and dense material were composed of the same proteins. This composition is discussed.     

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.     

Significance of the heterologous encapsidation of zucchini yellow mosaic potyvirus in transgenic plants expressing plum pox potyvirus capsid protein1

Transgenic Nicotiana benthamiana plants expressing the coat protein of an aphid-transmissible strain of plum pox potyvirus (PPV-D) were infected with an aphid non-transmissible strain of another potyvirus, zucchini yellow mosaic potyvirus (ZYMV-NAT). Non-viruliferous Myzus persicae could acquire and transmit ZYMV-NAT from these plants but not from infected N. benthamiana control plants (not transformed, or transformed by the vector alone). Immunosorbent electron microscopy experiments using the decoration technique revealed that ZYMV-NAT virus particles in the infected transgenic plants expressing the PPV coat protein could be coated not only with ZYMV antibodies but also, on segments of the particles, with PPV antibodies. This suggests that aphid transmission of ZYMV-NAT occurred through heterologous encapsidation, and reveals a potential risk of releasing genetically engineered plants expressing viral coat proteins into the environment.     

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.     

Susceptibility of apricot cultivars to artificial inoculation with plum pox potyvirus and use of ELISA for its evaluation1

The susceptibility to plum pox potyvirus (PPV) of 13 apricot cultivars and two hybrids was studied under isolated conditions in the experimental orchard of the Research Breeding Station at Vesele from 1989 to 1993 after artificial inoculation of the trees. The plants were inoculated in the nursery in 1988 by chip-budding with a local source of virus and then planted into the field trial. First symptoms of plum pox appeared on the leaves in 1990. Reinoculation of symptomless trees was done in summer 1991. By 1993, plum pox had totally invaded some apricot trees of the most susceptible cultivars (Madarska, Velkopavlovicka, Kisinevskij rannij, Ligeti orias, Bergeron, Vesna and Vestar). Uninoculated control trees remained symptomless despite the proximity of diseased trees and the fact that the orchard was not sprayed with insecticides during the period of experiment. Cvs Julskij, Veecot, Vegama and Veharda remained free from plum pox symptoms on leaves and fruits. In 1993-05/06, the trees were assayed for the presence of the PPV antigen by ELISA. Leaf samples from 33 trees showing symptoms were positive. Samples prepared from the healthy tissues of leaves with symptoms were negative. Other trees (indexed by ELISA) growing in the neighbourhood of diseased trees in the period of 5 years were visually and ELISA negative.     

Production and characterization of a monoclonal antibody specific to the M serotype of plum pox potyvirus

A monoclonal antibody to an Albanian isolate of plum pox potyvirus (PPV) was obtained (MAbAL), that specifically recognized strain M of this virus. The specificity of MAbAL, assessed by comparative ELISA on 130 PPV isolates of different geographical origin, 22 of which were also tested by comparative IC-PCR, gave consistent and highly reproducible results. MAbAL seems to be elicited by a stable surface determinant that makes it particularly suitable for successful use under a wide range of conditions. MAbAL is an useful addition to the panel of PPV-specific MAbs available to date.