Identification of Erwinia amylovora by Growth Morphology on Agar Containing Copper Sulfate and by Capsule Staining with Lectin

Abstract

Erwinia amylovora strains formed yellow colonies on minimal agar medium MM2 containing asparagine and copper sulfate (MM2Cu), in contrast to a white morphology on minimal agar without copper salt. Additionally, the colonies were mucoid to various extents. The yellow color was characteristic for the fire blight pathogen, including strains from raspberry and from other unusual host plants, and was used to establish a novel plating technique for identification of E. amylovora. The new identification method was especially superior to semi-selective media with sucrose when natural levan-deficient strains were assayed. No growth of E. amylovora was observed for the similar medium MM1 containing 2 mM CuSO4, due to its low content of as paragine. Identification by colony morphology on MM2 agar with copper was confirmed by staining the bacterial capsules with FITC-labeled lectin from Abrus precatorious, a compound which has a high affinity for galactose residues, the main sugar in the capsular exopolysaccharide amylovoran of E. amylovora. Other plant-associated bacteria usually did not produce the typical colony morphology of E. amylovora on MM2 agar with copper. Furthermore, those cells were not stained with the Abrus lectin. Capsule staining was also observed for weakly mucoid strains of E. amylovora, but not for strains with mutations affecting amylovoran synthesis. The secretion of fluorescent compounds by Pseudomonas syringae pathovars and even growth of any other bacterial colonies adjacent to E. amylovora could interfere with the formation of typical yellow colonies on MM2Cu, which could be white in case of dense plating. After screening for white colonies on LB agar, E. amylovora was identified in extracts from Cotoneaster leaves and in bark from apple trees with fire blight symptoms by its yellow growth pattern on MM2Cu agar and by capsule staining. The proposed selective medium gives a clear signal, is easy to prepare, does not contain dyes or any compounds toxic to humans, and can also detect E. amylovora strains deficient in levan synthesis.