scholarly journals Monitoring Prunus Necrotic Ringspot Virus Infection by Hybridization with a cRNA Probe after in vitro Micrografting

1995 ◽  
Vol 120 (6) ◽  
pp. 928-931 ◽  
Author(s):  
Kathleen Heuss-LaRosa ◽  
Rosemarie Hammond ◽  
James M. Crosslin ◽  
Christine Hazel' ◽  
Freddi A. Hammerschlag
Keyword(s):  
Prunus Persica ◽  
Blot Hybridization ◽  
Ringspot Virus ◽  
Shoot Cultures ◽  
Crna Probe ◽  
Elisa Testing ◽  
Prunus Necrotic Ringspot Virus ◽  
Coat Protein Mediated Resistance ◽  
In Vitro Micrografting

In vitro micrografting was tested as a technique for inoculating peach [Prunus persica (L.) Batsch] shoot cultures with Prunus necrotic ringspot virus (PNRSV). Cultured `Suncrest' shoots derived from a naturally infected tree (as indicated by ELISA testing) maintained virus in vitro, with virus concentrations in growing tips and folded leaves being several times those of fully expanded leaves. Infected shoots served as graft bases and source of the virus. Grafted tips were derived from `Suncrest' trees that had tested negative for the virus. Leaf samples were collected from the tips following grafting and analyzed for the presence of virus by slot-blot hybridization with a (DIG)-labeled cRNA probe derived from PNRSV RNA 3. Rates of successful grafting ranged from 55% to 73% in three trials and PNRSV was found in all tips analyzed. Virus concentrations approximated those found in source shoots, suggesting that in vitro micrografting should be useful for screening transformed peach shoots for coat protein-mediated resistance to PNRSV. Chemical name used: digoxigenin (DIG).

scholarly journals Monitoring Prunus Necrotic Ringspot Virus Infection by Hybridization with a CRNA Probe following in Vitro Grafting

HortScience ◽  
1995 ◽  
Vol 30 (4) ◽  
pp. 876B-876
Author(s):  
K. Heuss-La Rosa ◽  
R. Hammond ◽  
J.M. Crosslin ◽  
C. Hazel ◽  
F. Hammerschlag
Keyword(s):  
Prunus Persica ◽  
Blot Hybridization ◽  
Ringspot Virus ◽  
Crna Probe ◽  
Slot Blot Hybridization ◽  
Elisa Testing ◽  
Prunus Necrotic Ringspot Virus ◽  
Coat Protein Mediated Resistance ◽  
In Vitro Micrografting

In vitro micrografting was tested as a technique for inoculating peach [Prunus persica (L.) Batsch] with prunus necrotic ringspot virus (PNRSV). Cultured `Suncrest' shoots derived from a naturally infected tree (as indicated by ELISA testing) maintained virus in vitro, with virus concentrations in growing tips and folded leaves being several times those of fully expanded leaves. The infected shoots served as graft bases and the source of virus. Grafted tips were derived from `Suncrest' trees that had tested negative for the virus. Leaf samples were collected from the tips following grafting and analyzed for the presence of virus by slot-blot hybridization with a digoxigenin-labeled cRNA probed derived from PNRSV RNA 3. Rates of successful grafting were 55% and 73% in three trials and PNRSV was found in all tips analyzed. Virus concentrations approximated those found in the source shoots, suggesting that this method should be useful for screening transformed peach shoots for coat protein-mediated resistance to PNRSV.

scholarly journals A cRNA Probe Detects Prunus Necrotic Ringspot Virus in Three Peach Cultivars after Micrografting and in Peach Shoots following Long-term Culture at 4 °C

HortScience ◽  
1999 ◽  
Vol 34 (2) ◽  
pp. 346-347 ◽  
Author(s):  
K. Heuss ◽  
Q. Liu ◽  
F.A. Hammerschlag ◽  
R.W. Hammond
Keyword(s):  
Prunus Persica ◽  
Blot Hybridization ◽  
Ringspot Virus ◽  
Shoot Cultures ◽  
Crna Probe ◽  
Reading Frame ◽  
Prunus Necrotic Ringspot Virus ◽  
Repeated Use ◽  
Graft Success

As part of a program to develop transgenic peach (Prunus persica L. Batsch) cultivars with resistance to Prunus necrotic ringspot virus (PNRSV), we are testing a system for measuring virus in peach shoot cultures. Micrografting in vitro is used for inoculation and slot-blot hybridization, with a digoxigenin (DIG)-labeled cRNA probe complementary to the 5′ open reading frame (ORF) of PNRSV RNA 3, for detection. In this study, we investigated whether infected shoots maintain virus infection over long periods of culture at 4 °C and if PNRSV-infected `Suncrest' shoot cultures can serve as graft bases to transmit virus equally well into cultivars Nemaguard, Springcrest, and Suncrest. The results of RNA hybridization analysis showed that virus was present in extracts of leaf samples from 2-year-old PNRSV-infected `Suncrest' shoots that had been subjected to varying lengths of incubation at 4 °C in the dark, suggesting that infected shoots can be maintained for repeated use. Rates of graft success were higher in heterografts between `Suncrest' bases and tips of `Springcrest' or `Nemaguard' than in autografts between `Suncrest' and `Suncrest', and there was equal efficacy of graft inoculation from `Suncrest' into these three cultivars.

scholarly journals Transmission of Prunus Necrotic Ringspot into Three Peach Scion Cultivars, Measured by RNA Hybridization following in Vitro Micrografting

HortScience ◽  
1996 ◽  
Vol 31 (4) ◽  
pp. 678d-678
Author(s):  
Kathleen Heuss ◽  
Qingzhong Liu ◽  
Rosemarie Hammond ◽  
Freddi Hammerschlag
Keyword(s):  
Prunus Persica ◽  
Hybridization Analysis ◽  
Shoot Cultures ◽  
Success Rates ◽  
Virus Transfer ◽  
Significant Difference ◽  
Prunus Necrotic Ringspot Virus ◽  
Repeated Use ◽  
Graft Success

As part of our program to develop transgenic peach cultivars with improved disease resistance, we showed that grafting of in vitro cultured `Suncrest' peach [Prunus persica (L.) Batsch] tips `onto decapitated stems of Prunus necrotic ringspot virus (PNRSV) infected `Suncrest' shoot cultures, resulted in consistent transfer of virus across grafts as demonstrated by RNA hybridization analysis, suggesting that such a system could be useful for measuring resistance to PNRSV in peach shoot cultures. We have extended these studies to include grafts of `Springcrest' and `Nemaguard' test tips onto `Suncrest' stocks. RNA hybridization analysis showed that PNRSV persists in shoot cultures for 18 months after initiation from PNRSV-infected `Suncrest' trees and after 16 weeks of treatment of 4°C in the dark, suggesting that a supply of infected shoot cultures could be maintained for repeated use. Graft success rates for grafts of `Springcrest' onto `Suncrest' and `Nemaguard' onto `Suncrest', equaled or exceeded success rates for `Suncrest' onto `Suncrest'. Virus was transmitted from infected stocks into `Suncrest', `Springcrest', and `Nemaguard' test tips by 2 weeks in most successful micrografts. There was no significant difference in the virus concentrations among the three scions at 2, 4, and 6 weeks after grafting, suggesting that there is equal efficacy of virus transfer through grafts from `Suncrest' to the three cultivars, and that no differences in resistance to PNRSV exist among these cultivars.

scholarly journals Prunus necrotic ringspot virus Early Invasion and Its Effects on Apricot Pollen Grain Performance

2007 ◽  
Vol 97 (8) ◽  
pp. 892-899 ◽  
Author(s):  
Khalid Amari ◽  
Lorenzo Burgos ◽  
Vicente Pallas ◽  
María Amelia Sanchez-Pina
Keyword(s):  
Developmental Stages ◽  
Generative Cell ◽  
Pollen Grains ◽  
Growth Zone ◽  
Pollen Tubes ◽  
Climatic Conditions ◽  
Pollen Grain ◽  
Ringspot Virus ◽  
Prunus Necrotic Ringspot Virus

The route of infection and the pattern of distribution of Prunus necrotic ringspot virus (PNRSV) in apricot pollen were studied. PNRSV was detected both within and on the surface of infected pollen grains. The virus invaded pollen during its early developmental stages, being detected in pollen mother cells. It was distributed uniformly within the cytoplasm of uni- and bicellular pollen grains and infected the generative cell. In mature pollen grains, characterized by their triangular shape, the virus was located mainly at the apertures, suggesting that PNRSV distribution follows the same pattern as the cellular components required for pollen tube germination and cell wall tube synthesis. PNRSV also was localized inside pollen tubes, especially in the growth zone. In vitro experiments demonstrated that infection with PNRSV decreases the germination percentage of pollen grains by more than half and delays the growth of pollen tubes by ≈24 h. However, although PNRSV infection affected apricot pollen grain performance during germination, the presence of the virus did not completely prevent fertilization, because the infected apricot pollen tubes, once germinated, were able to reach the apricot embryo sacs, which, in the climatic conditions of southeastern Spain, mature later than in other climates. Thus, infected pollen still could play an important role in the vertical transmission of PNRSV in apricot.

scholarly journals A Rapid Protocol for Evaluating Prunus Germplasm for Tomato Ringspot Virus Resistance

HortScience ◽  
1994 ◽  
Vol 29 (9) ◽  
pp. 1068-1070
Author(s):  
J.M. Halbrendt ◽  
E.V. Podleckis ◽  
A. Hadidi ◽  
R. Scorza ◽  
R. Welliver
Keyword(s):  
Prunus Persica ◽  
Blot Hybridization ◽  
Control Plant ◽  
Chenopodium Quinoa ◽  
Ringspot Virus ◽  
Prunus Domestica ◽  
Dot Blot ◽  
Tomato Ringspot Virus ◽  
Leaf Tissues ◽  
And Control

Rooted cuttings of `Halford' and `Redhaven' peaches [Prunus persica (L.) Batsch] and `Stanley' (Prunus domestica L.) and `Marianna 2624' (P. cerasifera × P. munsoniana) plums were planted in soil containing ≈38 tomato ringspot virus-(TomRSV) infested nematodes (Xiphinema americanum sensu lato Cobb) per 100 cc. Test- and control-plant sap extracts were made from root and leaf tissues after 10, 22, and 34 weeks. Aliquots of these samples were assayed by mechanical inoculation to Chenopodium quinoa Willd. Total nucleic-acid extracts prepared from the remainder of each sample were analyzed by dot blot hybridization using a cRNA probe for TomRSV. The bioassay identified one `Stanley' and two `Redhaven' infected plants. Hybridization results indicated that two of two `Stanley', three of three `Halford', five of five `Redhaven', and zero of six `Marianna 2624' were infected. Our results demonstrate the sensitivity of molecular hybridization for TomRSV detection in Prunus and substantiate the TomRSV resistance of `Marianna 2624'.

scholarly journals First Report of Prunus necrotic ringspot virus in Peach in Mexico

Plant Disease ◽  
2008 ◽  
Vol 92 (3) ◽  
pp. 482-482 ◽  
Author(s):  
R. De La Torre-Almaraz ◽  
J. V. Montoya-Piña ◽  
S. Alcacio-Rangel ◽  
G. Camarena-Gutiérrez ◽  
M. Salazar-Segura
Keyword(s):  
Prunus Persica ◽  
Rna Extraction ◽  
Ringspot Virus ◽  
Plum Pox Virus ◽  
Rt Pcr ◽  
First Report ◽  
Field Samples ◽  
Prunus Necrotic Ringspot Virus ◽  
San Martín ◽  
Das Elisa

Peach (Prunus persica (L.) Batsch) is one of the most important fruit crops in the temperate regions of Mexico. In 2006, during a survey conducted in commercial peach orchards in Puebla, Mexico for viral diseases, many trees were observed with foliar symptoms that included yellow mottle, ringspot, line patterns, and mosaic. Samples (flowers, young shoot tips, and leaves) were collected from 120 symptomatic trees in three locations (San Martin Texmelucan, Domingo Arenas, and Tepetzala). All samples were tested using double-antibody sandwich (DAS)-ELISA kits (Agdia, Inc., Elkhart, IN) for the presence of the following viruses: Apple mosaic virus, Plum pox virus, Prune dwarf virus, and Prunus necrotic ringspot virus (PNRSV). Sap extracts from young symptomatic leaves and shoots were used to mechanically inoculate Chenopodium quinoa, C. amaranticolor, Gomphrena globosa, Nicotiana tabacum cv. Xanthi, N. glutinosa, N. clevelandii, N. benthamiana, Datura stramonium, Capsicum annuum, and Solanum lycopersicum. Plants were kept in a greenhouse with approximate temperatures of 25 to 35°C, humidity of 70%, and 12 h of light. Sap extracts were also used for dsRNA extraction and analyses (2) and RNA extraction for use in reverse transcription (RT)-PCR with the Access RT-PCR system (Promega, Madison, WI) and primers that annealed to a conserved region in the PNRSV coat protein gene (1). The expected size amplicons of approximately 450 bp were generated from all field-collected samples. The PCR products from three geographically distinct PNRSV isolates (Domingo Arenas [Accession No. DQ979004], Tepetzala [Accession No. DQ979005], and San Martin Texmelucan [Accession No. EF456771]) were directly sequenced with a Genetic Analyzer 3100 (Applied Biosystems, Foster City, CA) and their nucleotide and deduced amino acids sequences were more than 93% identical to corresponding sequences of PNRSV available in the NCBI/GenBank database. PNRSV was the only virus detected by DAS-ELISA in flowers and young shoots from 60 of the symptomatic field samples tested from the three locations. DsRNA banding patterns were obtained from 40 field-collected symptomatic samples; all showed three bands of approximately 3.6, 2.5, and 1.8 kb, the expected sizes for RNAs 1, 2, and 3 of PNRSV, respectively. DsRNAs were not detected in asymptomatic plants. PNRSV transmission by mechanical inoculation induced mosaic symptoms in N. tabacum cv. Xanthi and necrotic local lesions in G. globosa. Although G. globosa is reported to be a systemic host of PNRSV and N. tabacum is not reported to be a host, symptomatic plants were positive for PNRSV in DAS-ELISA tests. It is possible that there was an additional virus not detected in our assays that was responsible for the unexpected reactions in the host range studies. To our knowledge, this is the first report of PNRSV in peach in Mexico. References: (1) D. J. MacKenzie et al. Plant Dis. 81:222, 1997. (2) R. A. Valverde et al. Plant Dis. 74:255,1990.

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