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Liberibacter crescens, a presumed bacterial plant pathogen, forms biofilm in vitro

Eber Naranjo: Department of Entomology and Plant Pathology, Auburn University

<div>The <em>Liberibacter</em> genus includes heretofore unculturable plant pathogens that cause considerable economic losses worldwide. The only culturabed species of the genus, <em>Liberibacter crescens</em> (Lcr), has been used as a surrogate for pathogenic <em>Ca.</em> Liberibacter species. The replacement of fetal bovine serum (FBS) by methyl-β-cyclodextrin (MBC) in BM7 medium (modified medium: mBM7) resulted in a significant increase in Lcr cell attachment. MBC and FBS concentration gradients showed 0.75g/l was the optimum MBC concentration for Lcr viability and biofilm formation, and demonstrated that FBS prevented Lcr biofilm formation. Assays comparing Lcr cell attachment force in microfluidic chambers (MC) for both culture media confirmed that FBS presence in BM7 prevents Lcr cell-surface attachment. Cell adhesion assays showed that Lcr cells aggregated significantly less in BM7 compared with mBM7. Calcofluor white staining of two-week old GFP-Lcr cultures on each media exhibited qualitative differences in cell aggregation and revealed the presence of exopolysaccharides in Lcr biofilm extracellular matrix. Time-lapse microscopy assays in MC with mBM7 showed Lcr cells are non-motile, and form aggregates by remaining joined after cell division. Staining of these aggregates with the LIVE/DEAD® BactLight Bacterial viability kit in MC revealed a high proportion of live cells in Lcr biofilms. This study demonstrates the potential of Lcr to form biofilms under specific growth environments.</div>