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Sharka caused by Plum pox virus (PPV) is the most harmful disease of stone fruits in Europe and elsewhere in the world. There is no highly PPV resistant cultivar of plum. Biotech approach has led to the development of resistance through genetic engineering. In this study, we evaluated a transgenic plum Prunus domestica L., clone C5 (cv. HoneySweet), where the PPV resistance is based on RNA interference (RNAi). Resistance in C5 plums has been evaluated for PPV, Prune dwarf virus (PDV), and Apple chlorotic leaf spot virus (ACLSV) in a regulated field trial in the Czech Republic for 12 years (2002–2013). Even under high and permanent infection pressure introduced through graft inoculation of the viruses, PPV has been detected in C5 trees only in several leaves situated close to the point of inoculum grafting in the first nine years. Mild symptoms of PPV disappeared year by year. No PPV symptoms were observed in the last three years and results of ELISA detection were negative. Similar results were obtained, when RT-PCR was used for PPV detection. No natural infection of PPV by aphids was recorded in C5 plums, even if PPV infected plants were growing in the same experimental field in close vicinity. Co-infections of PPV with PDV and/or ACLSV had practically no influence on stability of resistance in C5 trees. Twelve years of field testing in the Czech Republic clearly demonstrated the high level of resistance of C5 either to PPV infection by graft inoculation or natural infection through aphid vectors.

'HoneySweet' is a transgenic plum protected against Plum pox virus (PPV). Its behaviour to natural PPV infection was the subject of different field trials undertaken in several endemic European countries. The first experiment in Romania was performed between 1996-2006 and no 'HoneySweet' tree was found infected under high natural PPV infection pressure. To further asses the durability of resistance to PPV of 'HoneySweet', a new field trial was established in 2013. As inoculum sources of PPV were used plants, previously artificially inoculated with D or Rec strains, grown in pots and placed inside of the blocks. Limited treatments with insecticides were made within the plot in order to stimulate the virus spread by aphids. The monitoring of PPV spreading was made by serological and molecular assays. Strains discrimination was made by RT-PCR. The temporal spread of PPV revealed a continual evolution of infection in conventional plums. No trees of transgenic plum 'HoneySweet' expressed PPV symptoms or were found infected, by serological and molecular assays, confirming its durable resistance to natural PPV infection with D or Rec strains. [ABSTRACT FROM AUTHOR]

The plum seed wasp (Eurytoma schreineri Schr.) is a polyphagous pest that can cause significant damages to the plum crop. A transgenic plum variety, resistant to Plum pox virus, was evaluated to the attack frequency of E. schreineri in the context of reduced number of insecticide treatments against aphids, in comparison with two conventional plum varieties. The most affected variety by plum seed wasp, for two consecutive years, was ‘Stanley’, followed by ‘Reine Claude d’Althan’, and then ‘HoneySweet’ transgenic plum. The differences between variants were statistically assured. [ABSTRACT FROM AUTHOR]

Transgenic plums, Prunus domestica L. clone C5, were inoculated by bud grafting with Plum pox virus (PPV-Rec, recombinant strain originated from plum), PPV-Rec + Apple chlorotic leafspot virus (ACLSV), PPV-Rec + Prune dwarf virus (PDV), and PPV-Rec + ACLSV + PDV. Non-inoculated transgenic plums served as controls. Plants were grown in an open field for 5 years. They were evaluated by visible symptoms, by DAS-ELISA and RT-PCR. Mild PPV symptoms, diffuse spots or rings appeared two years after inoculation in some leaves of plants artificially inoculated with PPV-Rec, PPV-Rec + ACLSV, PPV-Rec + PDV, and PPV-Rec + ACLSV + PDV. Severe PPV symptoms appeared in leaves of shoots growing from infected buds used for inoculation. During the following three years, further weakening of PPV symptoms was observed in transgenic plants. In 2007, very mild PPV symptoms were found in only a few leaves, and over 60%, resp. 70% of the C5 trees showed no PPV symptoms. The presence of PPV was confirmed by ELISA, ISEM and RT-PCR. No difference in PPV symptoms was observed between PPV-Rec and combinations PPV-Rec + ACLSV, PPV-Rec + PDV, PPV-Rec + ACLSV + PDV. No symptoms of ACLSV appeared in combinations of ACLSV with PPV-Rec and PPV-Rec + PDV during 2004-2007, but the presence of ACLSV in leaves of transgenic plants clone C5 was proved by ELISA and RT-PCR. Neither synergistic nor antagonistic effects of ACLSV on PPV-Rec were observed. No symptoms of PDV appeared in combinations of viruses with PDV during 2004-2007. PDV was not detected by ELISA, and the presence of PDV was uncertain by RT-PCR in most of inoculated trees in 2006 and 2007. The results of RT-PCR will be further confirmed by sequence analysis and discussed. These results suggest a possible antagonistic interaction between PPV-Rec and PDV in plum clone C5.

Transgenic plums of Prunus domestica L. transformed with the Plum pox virus coat protein gene (PPV-CP) were the subjects of three experiments undertaken in Romania. In the first experiment PPV-CP transgenic clones C2, C3, C4, C5, C6 and PT3 were evaluated for Sharka resistance under high natural infection pressure. Transgenic clone C5, subsequently named 'HoneySweet', showed high resistance to PPV. None of the C5 trees became naturally infected by aphids for more than ten years. The resistance of C5 was based on post-transcriptional gene silencing (PTGS). In the second experiment we assessed the effect of two heterologous viruses (Prunus necrotic ringspot virus and Prune dwarf virus) on the efficacy and stability of PTGS - mediated resistance to PPV displayed by the C5 plum. The engineered resistance to PPV in C5 transgenic plums was stable and was not suppressed by the presence of the assayed heterologous viruses. Some PPV-CP transgenic plum clones that are susceptible to PPV including C2, C3, C4 and PT3 display a constitutive transcription of PPV-CP sequence. In the third experiment we used these plants to assess the environmental safety issues related to potential hazards concerning the emergence of PPV variants. The serological and molecular variability of PPV detected in transgenic and in conventional plums revealed that the transgenic plums do not affect the diversity of indigenous PPV populations. We suggest that the safety and efficacy data developed in Romania and other European countries and in the United States of America over the last 15 years justifies an expansion of the field tests in Romania and in other countries that are experiencing the damages to plum production caused by PPV. [ABSTRACT FROM AUTHOR]

Transgenic clones C2, C3, C4, C5, C6, PT3 and PT5 of Prunus domestica L. transformed with the Plum pox virus coat protein gene (PPV-CP) were evaluated for Sharka resistance under high infection pressure in field natural conditions in Romania. Transgenic clone C5, recently named "HoneySweet", showed high resistance to PPV. None of the C5 trees becames naturally infected by aphids for more than ten years. Known to develop the post-transcriptional gene silencing (PTGS) mechanism, we assessed the effect of heterologous viruses on the efficacy and stability of PTGS displayed by the C5 plum against PPV. In this way, C5 trees were graft-inoculated with different combinations of Prunus necrotic ringspot virus (PNRSV), Prune dwarf virus (PDV) and PPV-D strain. The potential for suppression of the silencing mechanism mediated by these ilarviruses was evaluated in orchard and nursery. Both trials showed that, the engineered resistance to PPV in C5 transgenic plums was stable and was not suppressed by the presence of the assayed heterologous viruses over a three-year experimental period. [ABSTRACT FROM AUTHOR]

Transgenic clones C2, C3, C4, C5, C6, and PT-6, of plum (Prunus domestica L.) transformed with the coat protein (CP) gene of Plum pox virus (PPV), PT-23 transformed with marker genes only, and nontransgenic B70146 were evaluated for sharka resistance under high infection pressure in field trials in Poland and Spain. These sites differed in climatic conditions and virus isolates. Transgenic clone C5 showed high resistance to PPV at both sites. None of the C5 trees became naturally infected by aphids during seven (Spain) or eight (Poland) years of the test, although up to 100% of other plum trees (transgenic clones and nontransgenic control plants) grown in the same conditions showed disease symptoms and tested positively for PPV. Although highly resistant, C5 trees could be infected artificially by chip budding or via susceptible root-stock. Infected C5 trees showed only a few mild symptoms on single, isolated shoots, even up to 8 years post inoculation. These results clearly indicate the long-term nature and high level of resistance to PPV obtained through genetically engineered resistance. [ABSTRACT FROM AUTHOR]

To assess the durability of resistance to PPV of 'HoneySweet' transgenic plum harbouring plum pox virus (PPV) capsid gene, a new field trial was recently established in Romania at Fruit Research & Development Station Bistrita. 'Stanley' and 'Reine Claude d'Althan' cvs. were used as control. PPV inoculum source was secured within the experimental plot. PPV monitoring was made by visual observations of symptoms development on leaves and by serological (DAS-ELISA) and molecular (IC/-RT-PCR) testings. Three years after tree planting PPV was observed and detected only on 'Stanley'. 'HoneySweet' and 'Reine Claude d'Althan' cultivars remained PPV free. [ABSTRACT FROM AUTHOR]