Testing a Rapid Diagnostic Medium for Erwinia amylovora and Development of a Procedure for Sampling Blossoms in Pear Orchards

Abstract

The coliform agar produced by Merck was tested for rapid diagnosis of Erwinia amylovora (the causal agent of fire blight) in pear blossoms. The medium enabled the diagnosis to be completed within 36 h. Diagnoses performed with the medium were confirmed by the BIOLOG and the fatty-acid profile methods. The diagnostic medium was used to determine the spatial distribution of colonized blossoms in the orchards and it was found that E. amylovora may be distributed both in clusters and at random. These findings were used in the development of a statistical model for sampling blossoms in the orchard. The model determines the number of trees to be sampled in the orchard and the number of blossoms be taken from each tree, which would enable the true colonization incidence of blossoms in the orchard to be estimated at desired levels of accuracy and confidence. Parameters included in the model are: the total number of trees in the orchard (T), the number of trees to be sampled in the orchard (t), the number of blossoms to be sampled from each tree (n), the true colonization incidence of blossoms (pi), a coefficient of aggregation (rho), the required level of confidence (1 - alpha), and the required level of accuracy (L). Sensitivity analyses revealed that the parameter governing sample size is the required level of accuracy. Sampling of 20 blossoms from each of several hundred trees is required to achieve an accuracy of +/-1%, but only a few single trees are needed for an accuracy level of +/-10%. A sampling procedure then was developed, validated with an independent data set, and found to be accurate. It was concluded that sampling of pear blossoms and estimation of the incidence of blossom colonization by E. amylovora could improve fire blight management, but not in all cases.

Characterisation of naturally occurring Erwinia amylovora strains lacking the common plasmid pEA29 and their detection with real-time PCR

“…Due to the intermediate copy number of plasmid pEA29, primers derived from the plasmid will be more sensitive than chromosomal primers and might be preferred for screening of low levels of the fire blight pathogen. On the other hand, disease detection in orchards is often based on fire blight symptoms such as wilt, necrosis and sometimes ooze formation, so identification of E. amylovora is usually done on MM2Cu agar (Bereswill et al 1998): ng, no growth; ym, colonies yellow, mucoid; MM1 agar (Bereswill et al 1998): muc, mucoid; but, butyrious; Cas-medium (Schwyn and Neilands 1987): +, halos indicating release of siderophores; Cf, coliform agar, Rapid Diagnosis medium of Kritzman et al (2003). The first three strains were isolated in Germany 2005 from diseased plants, isolates four to seven are part of strains summarised in Table 4 a high bacterial level.…”

Unexpected Diversity during Community Succession in the Apple Flower Microbiome

“…Fire blight prevention strategies include application of antibiotics, such as streptomycin, and bacterial antagonists that compete with E. amylovora on floral stigmata and nectaries. E. amylovora resistance to antibiotics (19) and mixed effectiveness of antagonists (20–22) make fire blight a persistent problem. A thorough understanding of the flower microbiome may reveal new antagonists as well as insights about the identities, dynamics, and interplay of commensal microorganisms with the plant host, pathogens, and pollinators.…”

Characterization of Erwinia amylovora Strains from Croatia

“…The Fe-CAS-HDTMA agar was prepared according to Schwyn and Neilands (1987) with glucose as the carbon source and a low phosphate medium instead of MM9 salts. The rapid diagnosis (RD) medium (Kritzman et al, 2003) was the commercial preparation of the coliform agar (Merck, Germany). Only strain EaCro16K did not show the typical pink colony colour on Cf agar.…”