SIMULTANEOUS DETECTION OF ERWINIA AMYLOVORA STRAINS WITH AND WITHOUT PLASMID pEA29 AND OF ANTAGONISTIC BACTERIA BY REAL-TIME PCR

Most natural Erwinia amylovora strains from plants with fire blight contain plasmid pEA29. We have screened many isolates from the lab collection and 125 additional strains from diseased plants isolated 2005 in Germany. One strain among the recent isolates was devoid of plasmid pEA29. From the other strains, we found two strains from Egypt, one strain from Iran and one strain from Spain lacking pEA29. The Spanish strain contained a different plasmid larger than pEA29. In minimal medium without thiamine, these strains showed growth retardation, which was relieved after transfer of a transposon-labelled plasmid pEA29 into the cells. In conventional PCR, all these strains did not produce a signal with plasmid primers, but with primers from the chromosomal ams region of E. amylovora. From plasmid and chromosomal DNA sequences PCR primers were designed to detect E. amylovora strains with and without plasmid pEA29. Strains of the antagonistic species Erwinia tasmaniensis and Erwinia billingiae were detected with PCR primers from EPS encoding genes and E. tasmaniensis also with primers from the hrpL region. From these regions, TaqMan probes were designed, which allowed simultaneous screening of E. amylovora and E. tasmaniensis in the same environment. After inoculation of apple flowers with E. amylovora and the antagonistic strains Et1/99 and Eb661, these bacteria were monitored with real-time PCR.