Detection of Erwinia species from the apple and pear flora by mass spectroscopy of whole cells and with novel PCR primers

Abstract

To detect the apple and pear pathogens Erwinia amylovora and Erwinia pyrifoliae as well as the related epiphytes Erwinia tasmaniensis and Erwinia billingiae, we created novel PCR primers and also applied them to a series of other plant-associated bacteria as control. To facilitate fast diagnosis, we used matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS). The PCR primers were deduced from the pstS-glmS regions, which can include the gene for levansucrase, and also from regions encoding capsular polysaccharide synthesis. All primer combinations were specific for their associated Erwinia species to detect them with conventional PCR, also in mixed cultures from necrotic plant tissue. Other primers designed for quantitative PCR with SYBR Green or together with TaqMan probes were applied for real-time detection to determine growth of Erw. amylovora, Erw. billingiae, Erw. pyrifoliae and Erw. tasmaniensis in apple blossoms. From whole-cell protein extracts, profiles were generated using a Bruker microflex machine and Erwinia strains classified according to a score scheme. The designed PCR primers identified the Erwinia species unambiguously and can be applied to qualitative and quantitative tests. MALDI-TOF MS data were in agreement with the PCR assays. The applied diagnosis methods allow fast and precise monitoring of two pathogenic and two epiphytic Erwinia species. They are valuable for population studies with apple and pear flowers and with diseased plant material.