Detection and identification of the fire blight pathogen, Erwinia amylovora, can be accurately done by polymerase chain reaction (PCR) analysis in less than 6 h. Two oligomers derived from a 29-kb plasmid which is common to all strains of E. amylovora were used to amplify a 0.9-kb fragment of the plasmid. By separation of the PCR products on agarose gel, this fragment was specifically detected when E. amylovora DNA was present in the amplification assay. It was not found when DNA from other plant-pathogenic bacteria was used for the assay. A visible band specific to the 0.9-kb fragment was produced with DNA from fewer than 100 E. amylovora cells. A signal of similar strength was also obtained from E. amylovora cell lysates in the presence of the mild detergent Tween 20. Signals were weaker when bacteria were added to the PCR mixture without the detergent. As with results obtained from hybridization experiments using pEA29 DNA, the PCR signal was obtained with E. amylovora isolates from various geographic regions. This technique could also be used for detection of the fire blight pathogen in extracts of tissue obtained from infected plant material.
To cure the fireblight pathogen Erwinia amylovora of a 29 kb plasmid (pEA29) that is common in strains of these bacteria, restriction fragments of this plasmid were inserted into plasmids based on replication functions of bacteriophage fd, which cannot replicate in bacteria without expression of viral gene 2-protein. A 4.4 kb PstI fragment including the unique BamHI site of pEA29 allowed pfd plasmids to be propagated in E. amylovora. Furthermore, selection for these plasmids removed the natural 29 kb plasmid, apparently because of plasmid incompatibility. The pfd plasmids with the 4.4 kb insertion had a high tendency to segregate in E. amylovora when grown without selective pressure. Plasmid-free E. amylovora strains were virulent in standard tests on slices of immature pears, but symptom development was delayed compared to the wild-type. When assayed on pear seedlings a deficiency in pathogenicity was observed. Furthermore, strains without pEA29 spread more slowly on a lawn of pear cells than did wildtype strains. Plasmid-free strains of E. amylovora were not auxotrophic, but synthesis of extracellular polysaccharide was altered under certain growth conditions.
Erwinia amylovora, the causal agent of fire blight, carries the common plasmid pEA29 of 29 kb. To screen for occurrence of natural strains without plasmid pEA29, we applied PCR analysis with primers from the plasmid and the chromosomal ams region. In addition, a described TaqMan probe from pEA29 and newly designed primers from the amsregion were used for identification by real-time PCR. One strain isolated in Iran, one strain from Spain and two strains from Egypt lacked plasmid pEA29. From a recent screening series in southern Germany, in 123 E. amylovora strains from necrotic fire blight host plants, one strain was found without the common plasmid. The strains without pEA29 were virulent in assays with immature pears and on apple seedlings, but showed a reduced growth level in minimal medium without amino acids and thiamine. Transposon-labelled pEA29 was transformed into the plasmid-free strains resulting in restoration of this growth deficiency. The plasmid was stably maintained in these E. amylovora cells. The newly designed chromosomal primers for conventional and for realtime PCR identified E. amylovora strains in field samples lacking pEA29. These variants are apparently rare, but were detected in isolates from different regions in the world with fire blight.
Eighty one isolates of Ralstonia solanacearum -like bacteria on triphenyl tetrazolium chloride (TTC) medium were collected from different Solanaceae crops (i.e. potato, tomato and pepper plants and potato tubers) at various sites in Ethiopia. Of these, 62 strains were identified as R. solanacearum based on their cultural characteristics on TTC medium, tomato pathogenicity bioassay, carbon source utilisation patterns and a specific PCR-based assay. By Hayward's classification method, based on carbon source utilisation, 19 of the 62 R. solanacearum strains were identified as biovar I and 43 strains were identified as biovar II. The biovar I strains exhibited a high growth rate at high temperatures (37 degrees C). Whereas the growth rate of biovar II strains was greatest at lower temperatures (22 degrees C). Biovar I strains had broader host range than biovar II strains, which were limited to potato, tomato, and eggplant. To our knowledge, this is the first report of R. solanacearum biovar I in Ethiopia. The existence of biovar I strains in Ethiopia raises concerns because they have a broader host range than biovar II strains.
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