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Jean-Michel Hily, Michel Ravelonandro, Vern Damsteegt, Carole Bassett, Cesar Petri, Zongrang Liu, and Ralph Scorza

lines grew normally, developed normal flowers, and set viable seeds. Screening for plum pox virus resistance in Nicotiana benthamiana T 1 generation. To evaluate the effect of siRNA accumulation on resistance to PPV we selected for resistance

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P. Martínez-Gómez, M. Rubio, and F. Dicenta

The resistance to a Dideron isolate of Plum pox virus, which causes sharka disease, of four apricot (Prunus armeniaca L.) cultivars from North America (`Harlayne', `Henderson', `Sunglo', and `Veecot') and a Greek cultivar Lito (a cross of American cultivar Stark Early Orange × Greek cultivar Precoce Tirynthos) was evaluated. `Stark Early Orange' and `Canino', previously rated as resistant and susceptible respectively, were included as controls. Resistance, herein, was defined as inability to infect plants by graft-inoculation and negative assays by enzyme-linked immunosorbent assay. Cultivars found to be resistant were: `Harlayne', `Henderson', `Sunglo', `Lito', and `Stark Early Orange'. Cultivars Veecot and Canino were susceptible.

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P. Martínez-Gómez, M. Rubio, F. Dicenta, and T.M. Gradziel

Sharka [(plum pox virus (PPV)] mainly affects Prunus species, including apricot (Prunus armeniaca L.), peach (Prunus persica L.), plum (Prunus salicina Lindl., Prunus domestica L.), and, to a lesser degree, sweet (Prunus avium L.) and sour cherry (Prunus cerasus L.). Level of resistance to a Dideron isolate of PPV in seven California almond [P. dulcis (Miller) D.A. Webb], five processing peach cultivars, and two peach rootstocks was evaluated. In addition, almond and peach selections resulting from interspecific almond × peach hybridization and subsequent gene introgression were tested. Evaluations were conducted in controlled facilities after grafting the test genotypes onto inoculated GF305 peach rootstocks. Leaves were evaluated for PPV symptoms during three consecutive cycles of growth. ELISA-DASI and RT-PCR analysis were also employed to verify the presence or absence of PPV. Peach cultivars and rootstocks showed sharka symptoms and were ELISA-DASI or RT-PCR positive for some growth cycles, indicating their susceptibility to PPV. Almond cultivars and almond × peach hybrids did not show symptoms and were ELISA-DASI and RT-PCR negative, demonstrating resistance to PPV. Two (almond × peach) F2 selections as well as two of three backcrossed peach selections also showed a resistant behavior against the PPV-D isolate. Results demonstrate a high level of resistance in almond and indicate potential for PPV resistance transfer to commercial peach cultivars.

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Zongrang Liu, Ralph Scorza, Jean-Michel Hily, Simon W. Scott, and Delano James

Stability of gene silencing-based resistance to plum pox virus in transgenic plum ( Prunus domestic a L.) under field conditions Transgenic Res. 13 427 436 Hily, J.M. Scorza, R. Webb, K. Ravelonandro, M

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Jean-Michel Hily, Ralph Scorza*, and Michel Ravelonandro

We have shown that high-level resistance to plum pox virus (PPV) in transgenic plum clone C5 is based on post-transcriptional gene silencing (PTGS), otherwise termed RNA silencing (Scorza et al. Transgenic Res. 10:201-209, 2001). In order to more fully characterize RNA silencing in woody perennial crops, we investigated the production of short interfering RNA (siRNA) in transgenic plum clones C3 and C5, both of which harbor the capsid protein (CP) gene of PPV. We used as a control, plum PT-23, a clone only transformed with the two marker genes, NPTII and GUS. We show in the current report that C5 constitutively produces two classes of siRNA, the short (21-22 nucleotides) and long (≈27 nucleotides) species in the absence of PPV inoculation. Transgenic susceptible clone C3 and the control clone PT-23, when healthy, produce no siRNA. Upon infection, these clones produce only the short siRNA (21-22 nt). This siRNA production suggests that plum trees naturally respond to virus infection by initiating PTGS or PTGS-like mechanisms. This study also suggests that high-level virus resistance in woody perennials may require the production of both the short and long size classes of siRNA, as are produced by the resistant C5 plum clone.

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Ralph Scorza, Laurene Levy, Vern Damsteegt, Luz Marcel Yepes, John Cordts, Ahmed Hadidi, Jerry Slightom, and Dennis Gonsalves

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.

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Ralph Scorza, Michel Ravelonandro, Ann Callahan, Ioan Zagrai, Jaroslav Polak, Tadeuz Malinowski, Mariano Cambra, Laurene Levy, Vern Damsteegt, Boris Krška, John Cordts, Dennis Gonsalves, and Chris Dardick

, M. 2004 Stability of gene silencing-based resistance to Plum pox virus in transgenic plum ( Prunus domestica L.) under field conditions Transgenic Res. 13 427 436 Jefferson, R.A. 1987 Assaying chimeric genes in plants: The GUS gene fusion system

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Jose Martínez-Calvo, Gerardo Llácer, and Marisa Luisa Badenes

‘Rafel’ and ‘Belgida’ are mid- to early-ripening apricot cultivars ( Prunus armeniaca L.) with good yield, excellent fruit quality, self-compatibility, and resistance to Sharka, a disease caused by the Plum pox virus , a serious limiting factor

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Jose Martínez-Calvo, Gerardo Llácer, and Maria Luisa Badenes

‘Moixent’ is a self-compatible, early-ripening apricot cultivar ( Prunus armeniaca L.) with excellent fruit quality and resistance to sharka (plum pox virus), a serious disease limiting apricot fruit production in affected areas. ‘Moixent’ fruits

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Maria L. Badenes, Jose Martínez-Calvo, Helena Gómez, and Elena Zuriaga

. Comparative analysis of tree and fruit characteristics of ‘Dama Taronja’, ‘Dama Rosa’, ‘Ginesta’, ‘Katy’, and ‘Goldrich’ in an experimental plot at Instituto Valenciano de Investigaciones Agrarias, Valencia, Spain. Plum pox virus resistance. Sharka disease