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Reliability of diagnostic techniques forErwinia amylovora, the causative agent of fire blight disease

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Abstract

A total of 20 putative strains ofErwinia amylovora originating from 11 samples of host plants with symptoms of fire blight were analyzed in detail using commercial polyclonal antibodies in immunochemical tests. Fourteen strains reacted negatively in all tests; 6 strains reacted positively with a polyclonal antibody for PTA-ELISA (plate-trapped antigen-enzyme linked immunosorbent assay) at a concentration corresponding toA 620=0.1, while atA 620 readings of 0.01 and 0.001 the results were negative. Five strains reacted positively with a polyclonal antibody for indirect immunofluorescence test at all tested concentrations. Three of those strains were positive in the PCR test with AMSbL and AMSbR primers designed for detection ofE. amylovora. In hypersensitivity test in tobacco and in immature pear fruit assay, all putative strains were negative while a known reference strain ofE. amylovora gave a typical hypersensitive-reaction response. On a medium with 5% sucrose the reference strain ofE. amylovora produced levan while putative strains did not. After modification of the PCR protocol, 3 putative strains reacted as negatives. Optimization of PCR test was achieved by finding the optimum annealing temperature and time for primers. The recommended annealing temperature (49 °C) for these primers was increased to 55 °C and the annealing time was reduced from 2 min to 30 s. Using the microbial identification systemBiolog those 3 strains were identified asPantoea dispersa (1 strain) andPantoea agglomerans (2 strains). The strains are supposed to be white variants of the speciesP. dispersa andP. agglomerans occurring less frequently than the yellow variants. Since there were positive reactions in our immunochemical tests these strains could cause false positives in routine screening of plant samples.

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Abbreviations

HR:

hypersensitive reaction

IF:

immunofluorescence

PTA-ELISA:

plate-trapped antigen-enzyme linked immunosorbent assay

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Authors and Affiliations

  1. Department of Bacteriology, Research Institute of Crop Production, 161 06, Prague-Ruzyně, Czechia

    B. Kokošková

  2. Department of Plant Virology, Institute of Plant Molecular Biology, Academy of Sciences of the Czech Republic, 370 05, České Budějovice, Czechia

    I. Mráz

Authors
  1. B. Kokošková
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  2. I. Mráz
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Correspondence to B. Kokošková.

Additional information

This work was supported by project M01-01-03 of theMinistry of Agriculture of the Czech Republic.

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Kokošková, B., Mráz, I. Reliability of diagnostic techniques forErwinia amylovora, the causative agent of fire blight disease. Folia Microbiol 50, 217–221 (2005). https://doi.org/10.1007/BF02931569

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  • DOI: https://doi.org/10.1007/BF02931569

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