Field Trials of Plum Clones Transformed with the Plum pox virus Coat Protein (PPV-CP) Gene.

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 rootstock. 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.

Incidence and epidemiology of Citrus tristeza virus in the Valencian community of Spain.

The first outbreak of citrus tristeza disease in Spain caused by Citrus tristeza virus (CTV) was recorded in 1957 in the Valencian Community (VC). In total c. 40 million trees, mainly of sweet orange and mandarin grafted on sour orange rootstocks, declined due to CTV. Large-scale surveys in different municipalities of the VC indicated that the disease spread very fast. Incidence increased from 11% in 1989 to 53% in 1998. Toxoptera aurantii and Aphis spiraecola (inefficient aphid vectors of CTV) predominated before 1985-87. Since then the relatively efficient vector Aphis gossypii has become dominant and induced an epidemic that has been modelled. The large number of A.gossypii that visited each clementine tree (estimated to exceed 97000 per year) explained the difference between the temporal pattern of spread of CTV in clementine which followed the Gompertz model and that in sweet orange (logistic model). The susceptibility of the different citrus species to CTV infection by aphids seems to depend on the number of young, succulent shoots produced. The epidemiological data allowed specific recommendations to be made to growers in order to facilitate a change to a modern citrus industry based on the use of selected varieties grafted on tristeza-tolerant rootstocks produced within a certification scheme. This has been done already in almost 90% of the VC citrus-growing area. The tristeza problem has been solved unless more aggressive isolates are introduced and become prevalent.

Simultaneous detection and typing of plum pox potyvirus (PPV) isolates by heminested-PCR and PCR-ELISA.

Two techniques for simultaneous detection and typing of plum pox potyvirus (PPV) isolates belonging to the D or M serotypes, heminested PCR (H-PCR) and PCR-ELISA, have been developed. Ten PPV isolates typed using PPV-D and PPV-M specific monoclonal antibodies by ELISA-DASI were used to validate these two methods. The results obtained show a complete coincidence of the nucleic acid-based techniques with the serological data. When serial dilutions of infected plant extracts were assayed, H-PCR and PCR-ELISA were found to be 100 times more sensitive than the more conventional immunocapture-PCR (IC-PCR) assay. Testing of 228 PPV-infected fruit tree samples coming from different hosts and locations indicated that so far only PPV type D appears to be present in Spain and in Chile. Coupled with print-capture sample preparation (Olmos et al., Nucl. Acids Res. 24, 2192-2193, 1996) the increased sensitivity provided by heminested-PCR allowed the detection of PPV targets of D and M types, in wingless individuals of the aphid vector Aphis gossypii.

Single-step multiplex RT-PCR for simultaneous and colourimetric detection of six RNA viruses in olive trees.

A single-step multiplex RT-PCR was developed for the simultaneous and colourimetric detection of six RNA viruses (Cucumber mosaic virus, Cherry leaf roll virus, strawberry latent ringspot virus, Arabis mosaic virus, Olive latent-1 virus and Olive latent-2 virus) which infect olive trees. Six compatible primer set for one-step RT-PCR amplification in a single closed-tube and 3' digoxigenin labelled probes were designed in optimal, specific and conserved regions. The method has been assessed with 195 Spanish field olive trees, suggesting that approximately 1.5% of the tested material was infected by Cucumber mosaic virus and 0.5% by Cherry leaf roll virus. This method saves time and reagent costs compared with monospecific RT-PCR which needs several reactions for the same number of tests. Using colourimetric detection, it is possible to analyse many samples, it increases sensitivity 10-fold, and whilst facilitating the interpretation of results, it avoids the use of gels and the toxic ethidium bromide. The method could be used routinely for sanitary and certification programmes.

Comparison of monoclonal antibodies and polymerase chain reaction assays for the typing of isolates belonging to the d and m serotypes of plum pox potyvirus.

ABSTRACT Plum pox potyvirus (PPV) isolates may be divided into four groups separated by serological, molecular, and epidemiological differences. Monoclonal antibodies specific for the two major groups of isolates, represented by the D and M serotypes of the virus, have been obtained. Polymerase chain reaction (PCR)-based assays allowing the direct detection and differentiation of PPV isolates have also been developed. We now report on a large-scale comparison of these two typing approaches. The results obtained show an overall excellent correlation between the results obtained in indirect double-antibody sandwich enzyme-linked immunosorbent assay using PPV-D- and PPV-M-specific monoclonal antibodies and those derived from either specific PCR assays or restriction fragment length polymorphism analysis of PCR fragments. Without exception, all isolates reacting positively with the PPV-M-specific monoclonal antibody were found to belong to the M serotype using the PCR-based assays, while 51 out of 53 isolates recognized by the D-specific monoclonal antibodies belonged to the D serotype according to the PCR typing results. However, failure to react with a specific monoclonal antibody did not prove as effective a predictor of the serotype of the isolate analyzed. In a few cases, the results obtained with the various techniques diverged, indicating low level variability of the epitopes recognized by the serotype-specific monoclonal antibodies. Isolates belonging to the two minor groups of PPV (El Amar and Cherry) also gave divergent results, indicating that the current typing assays are not suited for the analysis of such isolates.

Evidence of Biological Control of Agrobacterium tumefaciens Strains Sensitive and Resistant to Agrocin 84 by Different Agrobacterium radiobacter Strains on Stone Fruit Trees.

The effectiveness of Agrobacterium radiobacter K84, 0341, and a K84 non-agrocin-producing mutant (K84 Agr) in biological control of crown gall on rootstocks of stone fruit trees was determined in three experiments. In experiment 1, K84 and 0341 controlled crown gall on plum plants in soil inoculated with two strains of Agrobacterium tumefaciens resistant to agrocin 84. In experiment 2, K84 controlled crown gall on peach plants in soils inoculated with strains of A. tumefaciens sensitive or resistant to agrocin 84 or with a mixture of both. However, the effectiveness of K84 was higher against the sensitive strain than against the resistant strain. There was a residual effect of K84 from one year to another in soil inoculated with the sensitive strains. In experiment 3, K84 and K84 Agr controlled crown gall on plum and peach plants in soils inoculated with strains of A. tumefaciens sensitive or resistant to agrocin 84. The control afforded by K84 was higher than that provided by K84 Agr against the sensitive strain but was similar against the resistant strain.

Characterization of Monoclonal Antibodies Specific for Erwinia carotovora subsp. atroseptica and Comparison of Serological Methods for Its Sensitive Detection on Potato Tubers.

Seven monoclonal antibodies (MAbs) to Erwinia carotovora subsp. atroseptica have been produced. One, called 4G4, reacted with high specificity for serogroup I of E. carotovora subsp. atroseptica, the most common serogroup on potato tubers in different serological assays. Eighty-six strains belonging to different E. carotovora subsp. atroseptica serogroups were assayed. Some strains of serogroup XXII also reacted positively. No cross-reactions were observed against other species of plant pathogenic bacteria or 162 saprophytic bacteria from potato tubers. Only one strain of E. chrysanthemi from potato cross-reacted. A comparison of several serological techniques to detect E. carotovora subsp. atroseptica on potato tubers was performed with MAb 4G4 or polyclonal antibodies. The organism was extracted directly from potato peels of artificially inoculated tubers by soaking or selective enrichment under anaerobiosis in a medium with polypectate. MAb 4G4 was able to detect specifically 240 E. carotovora subsp. atroseptica cells per ml by indirect immunofluorescence and immunofluorescence colony staining and after soaking by ELISA-DAS (double-antibody sandwich enzyme-linked immunosorbent assay) after enrichment. The same amount of cells was detected by using immunolectrotransfer with polyclonal antibodies, and E. carotovora subsp. atroseptica and subsp. carotovora were distinguished by the latter technique. ELISA-DAS using MAb 4G4 with an enrichment step also efficiently detected E. carotovora subsp. atroseptica in naturally infected tubers and plants.

Lipopolysaccharides as Determinants of Serological Variability in Pseudomonas corrugata.

The variation in biochemical and serological features of 128 isolates of Pseudomonas corrugata has been studied with 56 isolates from Spain and 72 isolates from other countries. Isolates were analyzed with common diagnostic tests and with the AP150CHE system. Variability among isolates for some standard tests usually listed as positive or negative for this species, such as arginine dihydrolase and gelatin hydrolysis, lipase and lecithinase activities, pigment production, and wrinkled colony morphology, was observed. Three antisera were raised against the type strain and two Spanish isolates from tomato and pepper plants. Serological reactions were studied by indirect immunofluorescence and indirect enzyme-linked immunosorbent assay. Eighty-three isolates reacted with a single antiserum, 6 reacted with two antisera, and none reacted with three antisera. Thirty-nine isolates did not react with any of the three antisera. These results suggest that serology will not be a useful method for routine diagnosis of P. corrugata unless common antigens can be identified. Electrophoresis and immunoelectrotransfer were used to study the antigens involved. Each antiserum reacted with whole-cell lysates, giving two common bands for P. corrugata isolates and other Pseudomonas species and a ladder-like pattern characteristic of lipopolysaccharides (LPS). Common bands were not observed after proteinase K treatment. More than 10 LPS patterns were distinguished in 98 isolates after silver staining of polyacrylamide gels. There was no correlation between the geographical origin or host of the isolates and the LPS patterns. A correlation between LPS groups and serological reaction was observed.

New device and method for capture, reverse transcription and nested PCR in a single closed-tube.

A device and improved method based on the use of a compartmentalized Eppendorf tube that allows capture, reverse transcription and nested-PCR in a single closed-tube has been developed and patented. The main advantages of the system are the high sensitivity obtained, the simplicity, the low risk of contamination and the easy establishment of adequate conditions for nested-PCR. The method has been successfully applied to the detection and characterization of citrus tristeza closterovirus and plum pox potyvirus isolates in plant tissues and single aphids squashed on paper. This device and methodology could be easily adapted to the detection of other targets.

Characterization of monoclonal antibodies against Erwinia carotovora subsp. atroseptica serogroup I: specificity and epitope analysis.

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 IgG3 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.(ABSTRACT TRUNCATED AT 250 WORDS)

Production and characterization of monoclonal antibodies to plum pox virus and their use in differentiation of Mediterranean isolates.

Monoclonal antibodies (MAbs) specific to plum pox virus (PPV) were prepared by fusing myeloma cell lines to spleen cells of mice immunized with purified virus, including virus prepared with protease inhibitors to preserve the integrity of the coat protein (CP). The characterized MAbs could be used in ELISA to differentiate several Mediterranean PPV isolates differing in their geographical origin and CP size. At least seven antigenic sites could be established based on the recognition pattern and competition binding analysis, and the epitopes could be classified in three groups by Western blot analysis of intact and trypsin digested virus particles. By means of electron microscopy the epitopes could be seen to be located on the surface of the virus particles.