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Contribution of Erwinia amylovora Exopolysaccharides Amylovoran and Levan to Biofilm Formation: Implications in Pathogenicity

First author: Department of Plant Biology and Department of Plant Pathology, Michigan State University, East Lansing 48824; second and fourth authors: Department of Plant Pathology, Michigan State University; and third author: Department of Crop Sciences, University of Illinois at Urbana-Champaign, Urbana 61801.

Erwinia amylovora is a highly virulent, necrogenic, vascular pathogen of rosaceous species that produces the exopolysaccharide amylovoran, a known pathogenicity factor, and levan, a virulence factor. An in vitro crystal violet staining and a bright-field microscopy method were used to demonstrate that E. amylovora is capable of forming a biofilm on solid surfaces. Amylovoran and levan production deletion mutants were used to determine that amylovoran was required for biofilm formation and that levan contributed to biofilm formation. In vitro flow cell and confocal microscopy were used to further reveal the architectural detail of a mature biofilm and differences in biofilm formation between E. amylovora wild-type (WT), Δams, and Δlsc mutant cells labeled with green fluorescent protein or yellow fluorescent protein. Scanning electron microscopy analysis of E. amylovora WT cells following experimental inoculation in apple indicated that extensive biofilm formation occurs in xylem vessels. However, Δams mutant cells were nonpathogenic and died rapidly following inoculation, and Δlsc mutant cells were not detected in xylem vessels and were reduced in movement into apple shoots. These results demonstrate that biofilm formation plays a critical role in the pathogenesis of E. amylovora.