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Mutagenic Analysis and Localization of a Highly Conserved Epitope Near the Amino-Terminal End of the Citrus Tristeza Closterovirus Capsid Protein

  • Pappu, H. R.
  • Pappu, S. S.
  • Kano, T.
  • Koizumi, M.
  • Cambra, Mariano
  • Moreno, Pedro
  • Su, H. J.
  • Garnsey, S. M.
  • Lee, R. F.
  • Niblett, C. L.
The monoclonal antibody (MAb) 3DF1 is the first commercially available citrus tristeza closterovirus (CTV)-specific MAb. It detects a broad spectrum of CTV isolates from various parts of the world. To precisely map the antigenic determinant recognized by 3DF1, the capsid protein (CP) genes of four 3DF1-nonreactive isolates were cloned as complementary DNA and their nucleotide sequences determined. Comparison of the deduced CP sequences of the four nonreactive isolates with those of previously sequenced 3DF1-reactive isolates revealed differences at three positions near their amino terminal ends. The amino acids Asp-2, Lys-13, and Phe-28 were conserved in all the 3DF1-reactive isolates, but they were replaced by Gly, Thr/Asp, and Tyr, respectively, in the CPs of the nonreactive isolates. Site-specific mutations were introduced into the cloned CP genes of the 3DF1-nonreactive isolate B215 and the 3DF1-reactive isolate T36. The serological reactivities of the wild-type and mutant CPs of B215 and T36 expressed as recombinant fusion proteins in Escherichia coli were evaluated by Western blot analysis. A point mutation (A-->G) resulting in an Asp-->Gly change at amino acid position 2 of the CP of isolate T36 abolished the reactivity with the MAb, whereas a reverse mutation resulting in a Gly-->Asp change at the same position conferred reactivity on the CP of the nonreactive B215 isolate. The implications of the observed antigenic diversity on virus detection are discussed.
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Agrobacterium-Mediated Transformation of Sweet Orange and Regeneration of Transgenic Plants

  • Pena, Leandro
  • Cervera, M.
  • Juárez, José
  • Navarro, A.
  • Pina, José A.
  • Durán-Vila, Núria
  • Navarro, Luis
Transgenic sweet orange (Citrus sinensis L. Osbeck) plants have been obtained by Agrobacterium tumefaciens-mediated gene transfer. An hypervirulent A. tumefaciens strain harboring a binary vector that contains the chimeric neomycin phosphotransferase II (NPT II) and beta-glucuronidase (GUS) genes was cocultivated with stem segments from in vivo grown seedlings. Shoots regenerated under kanamycin selection were harvested from the stem segments within 12 weeks. Shoot basal portions were assayed for GUS activity and the remaining portions were shoot tip grafted in vitro for production of plants. Integration of the GUS gene was confirmed by Southern analysis. This transformation procedure showed the highest transgenic plant production efficiency reported for Citrus.
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High-Efficiency Agrobacterium-Mediated Transformation and Regeneration of Citrus

  • Pena, Leandro
  • Cervera, M.
  • Juárez, José
  • Ortega, C.
  • Pina, José A.
  • Durán-Vila, Núria
  • Navarro, Luis
A procedure for increased efficiency of production of transgenic citrus plants was developed by extending the exposure of the explants to the selection agent and by grafting in vitro the regenerated shoot apices onto seedling rootstocks. Carrizo citrange (Citrus sinensis L. Osbeck x Poncirus trifoliata L. Raf.) stem segments from in vitro grown seedlings were cocultivated with Agrobacterium tumefaciens EHA 105 carrying the binary vector p35SGUSINT. The intron-containing beta-glucuronidase (GUS) gene in the T-DNA served as reporter in the histochemical assay and the neomycin phosphotransferase II (NPT II) gene provided resistance to kanamycin and was used as selectable marker. Regenerated shoots were harvested from the stem segments within 5 to 6 months. Extended time periods of selection greatly improved recovering of transformed shoots and reduced the occurrence of escapes. However, prolonged exposure to kanamycin favoured the regeneration of chimeric shoots. Shoot basal portions were GUS-assayed for screening transformants and the remaining portions were shoot tip grafted in vitro for production of whole plants. Citrus genetic transformation was confirmed by polymerase chain reaction (PCR), Southern and Northern analysis. The transformation efficiencies obtained are the highest reported so far for citrus.
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A Simple Imprint-Hybridization Method for Detection of Viroids

  • Romerodurban, J.
  • Cambra, Mariano
  • Durán-Vila, Núria
An imprint-hybridization method has been designed to simplify the processing of samples during routine viroid indexing. The method requires minimal sample manipulation and has been evaluated for detection of viroids in 11 viroid-host combinations including 4 viroids (CEVd, CSVd, HSVd, ASBVd) and 7 hosts (chrysanthemum, citron, cucumber, Gynura, tomato, peach and avocado). The method is fast and sensitive, and provides additional information on the sites of viroid accumulation.
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Characterization and detection of plum pox virus using monoclonal antibodies, ACTA HORTICULTURAE

  • Asensio, M.
  • Gorris, María T.
  • Sanz, A.
  • Camarasa, E.
  • Perez, E.
  • Carbonell, Emilio A.
  • Cambra, Mariano
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Factors Affecting Citrus Tree Isozyme-Gene Expression

  • Asins, María J.
  • HERRERO, R.
  • Navarro, Luis
Ten enzymatic systems of Citrus species and cultivars have been evaluated for identification purposes and for genetic variability studies. The following factors that could affect their expression were studied: season of sampling, location, rootstock, position of the branch, infection, and age of the tree. Differences involving the presence-absence of the Cu/Zn SOD within the same tree were found. This difference is mainly related to the position of the leaf relative to the sunlight. No change was observed at any of the ten enzymatic systems assayed regarding the location, the rootstock, the growing condition, the season, or the infection with most virus and virus-like pathogens. Viroids induced noticeable changes on 6PG and PRXa zymograms in C. medica. A new peroxidase (not present in healthy plants) was detected that could be related to appearance of symptoms. This may induce errors when trees without sanitary control are characterized by this enzymatic system. On the other hand, it provides a new possibility for studying the plant response to the presence of viroids. An effect of age, from 3 months up to 12 years, was observed on citrange Troyer and mandarin Cleopatra PRX, MDH and 6PG patterns. An important change occurs around the first year, most likely related to the end of the seedling stage. This is followed by a long transition phase, the end of which (around 9 years later) coincides with a change in the PRX pattern. These age-related changes seem to involve post-translational mo difications of pre-existing isozymes.
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Serological Characterization of Potato Isolates of Erwinia-Carotovora Subsp Atroseptica and Subsp Carotovora using Polyclonal and Monoclonal-Antibodies

  • Alarcon, B.
  • Gorris, María T.
  • Cambra, Mariano
  • López, María M.
Serological, biochemical and physiological characteristics of 81 strains of Erwinia carotovora subsp. atroseptica (Eca) and 67 strains of subsp. carotovora (Ecc) from potato, isolated in Spain and from several international collections, have been studied. Ouchterlony double diffusion (ODD), indirect immunofluorescence (IIF) and indirect enzyme-linked immunosorbent assay (ELISA) were the methods used. The antibodies were polyclonals from eight antisera prepared with Era serogroup I and Ecc serogroup III and tao monoclonal antibodies (MAbs), 4G4 from Spain and 4F6 from Canada, both prepared with Era strains of serogroup I. Serogroup I for Era and several serogroups for Ecc were the most commonly found in the collection studied. Serological relationships between Era and Err independently of the serogroups were observed by IIF and ELISA using polyclonal antibodies. Common epitopes between all Era and Err studied were detected. Both MAbs recognized epitopes in Eca strains of serogroups I and XXII in IIF and ELISA but they did not react with strains of other serogroups nor Err strains. The pattern of reaction against the strains assayed was rather similar but not identical indicating that they represent two different and well conserved epitopes. This study confirms the serological complexity of Err and Era and gives information about the serological probes for detection of both subspecies.
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Analysis of the Spatial Spread of Sharks (Plum Pox Virus) in Apricot and Peach Orchards in Eastern Spain

  • Gottwald, T. R.
  • Avinent, L.
  • Llácer, Gerardo
  • Hermoso-De-Mendoza, Alfonso
  • Cambra, Mariano
Spatial patterns of sharka disease, caused by plum pox virus (PPV) and vectored by several species of aphid, were determined by double antibody sandwich-enzyme-linked immunosorbent assay using polyclonal antibodies in newly infected, mature apricot and peach orchards in eastern Spain. Among yearly assessments of plots examined for within- and across-row aggregation of adjacent sharka-diseased trees, only a few transects were found to have aggregation by ordinary runs analyses. Analyses, using beta-binomial index of dispersion (I-beta) to determine if spatial aggregation was present in each plot for data partitioned into quadrats of different spatial dimension, demonstrated occasional aggregation and results were generally inconclusive. Significant (I-beta) values, when present, were generally found associated with plots with higher disease incidence. No disease gradients were discernible for any of the plots and years. More rigorous spatial analyses were used to test for spatial relationships over longer distances. Two-dimensional distance class analyses indicated a spatial dependency of PPV-infected stone-fruit trees over distance, a general scarcity of significant distance classes near the origin, and the presence of significant distance classes occasionally comprising small loose clusters at distances near the center or distal end of the proximity matrices especially during the initial stages of the epidemics. Geostatistical analysis confirmed the lack of significant associations among immediately adjacent trees and the trend for higher order spatial associations in semivariograms for distances corresponding to the center and distal ends of the proximity matrices. This trend in semivariance over distance was best described by linear or exponential increase models compared with transitional models commonly used in geostatistics. Correlation analysis indicated a significant conservation of orientation of localized systemic infections in scaffold branches over years. The spatial patterns of sharka suggest the lack of movement of PPV-viruliferous aphid vectors to immediately adjacent trees and their preferential movement to trees several tree spaces away.
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Characterization of Monoclonal-Antibodies Against Erwinia-Carotovora Subsp Atroseptica Serogroup-i - Specificity and Epitope Analysis

  • Hyman, L. J.
  • Wallace, A.
  • López, María M.
  • Cambra, Mariano
  • Gorris, María T.
  • Perombelon, M. C. M.
The characteristics of two monoclonal antibodies (Mabs), A23/1221.59.44.d.3 (1221) and A23/1239.36.64.e.2 (1239), against Erwinia carotovora subsp, atroseptica serogroup I produced in this study were compared with those of two other independently obtained Mabs, 4G4 in Spain and 4F6 in Canada, using different strains as immunogen and different screening procedures. The reaction pattern of Mabs 1221 and 1239 determined by indirect ELISA on over 200 bacterial strains including five E.c. atroseptica and 36 E.c, carotovora serogroups, seven Erw. chrysanthemi biovars, 23 other plant bacterial pathogens and 33 saprophytic bacteria from potato was similar to that of 4G4. Specificity for E.c. atroseptica serogroup I was improved, especially when skimmed milk (Marvel) was used instead of bovine serum albumin as blocking agent. Mabs 1221, 1239 and 4G4 reacted positively with all 22 E.c. atroseptica serogroup I, the dominant E.c. atroseptica serogroup on potato, strains tested and only with two out of five E.c. atroseptica serogroup XXII strains, one E.c. carotovora serogroup XXI strain and one strain of a saprophytic bacterium, Comamonas sp. Essentially similar results were obtained when examined by immunofluorescence. Characterization of the four Mabs showed that they were IgG, and SDS-PAGE/immunoblot results suggested that they were probably against the O-side chain of bacterial cell wall lipopolysaccharides. In competition ELISA between biotin-labelled and unlabelled Mabs, the competition pattern of the four Mabs was similar. Transformation of the data obtained by titrating the concentration of Mabs 1221, 1239 and 4G4 against a constant bacterial density using the direct linear plot method of Eisenthal and Cornish-Bowden (1974), assuming an analogy between antibody-antigen interaction and Michaelis-Menten enzyme kinetics, indicated that Mab affinity (K-m) and avidity (V-max) were both highest for 4G4 and lowest for 1239.
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