Biochemical characterization and identification of ice-nucleation-active (INA) willow pathogens by means of BIOLOG® MicroPlate, INA gene primers and PCR-based 16S rRNA-gene analyses

Detection and identification of ice-nucleation active (INA) bacteria, was carried out in several independent investigations from diseased willow plants in different regions in Sweden and Estonia. Many of these bacteria, alone or together, cause serious bacterial disease problems in willow (Salix spp.) plants in combination with frost leading to dieback in plantations for energy forestry purposes. Methods used for identification were BIOLOG (R) MicroPlates, biochemical tests including growth in different media and pathogenic tests, designing and using selective INA primers, and 16S rRNA gene analyses. The taxonomic tools, especially phylogenetic analysis derived from 16S rRNA gene sequences, clearly distinguished many bacteria. The identified strains from willows (20 clones) belonged to at least eight different genera and 12 species showing variable levels of aggressiveness and ice-nucleation activity under laboratory and greenhouse conditions. Diseased willows were found associated with the presence of Agrobacterium tumefaciens, Bacillus spp., Clavibacter spp., Erwinia rhapontici, Frigoribacterium. faeni, Pseudomonas brenneri, P. fluorescens, P. frederiksbergensis, P. graminis, P. syringae, P. veronii, Sphingobacterium/ Pedobacter, Sphingomonas/non-fluorescent P. fluorescens (different biotypes), Xanthomonas campestris and related species.