Detection and identification of the fire blight pathogen, Erwinia amylovora, can be accurately done by polymerase chain reaction (PCR) analysis in less than 6 h. Two oligomers derived from a 29-kb plasmid which is common to all strains of E. amylovora were used to amplify a 0.9-kb fragment of the plasmid. By separation of the PCR products on agarose gel, this fragment was specifically detected when E. amylovora DNA was present in the amplification assay. It was not found when DNA from other plant-pathogenic bacteria was used for the assay. A visible band specific to the 0.9-kb fragment was produced with DNA from fewer than 100 E. amylovora cells. A signal of similar strength was also obtained from E. amylovora cell lysates in the presence of the mild detergent Tween 20. Signals were weaker when bacteria were added to the PCR mixture without the detergent. As with results obtained from hybridization experiments using pEA29 DNA, the PCR signal was obtained with E. amylovora isolates from various geographic regions. This technique could also be used for detection of the fire blight pathogen in extracts of tissue obtained from infected plant material.

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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.

Transposon Tn5, on a mobilizable ColEl plasmid, on a Ti plasmid derepressed for bacterial transfer, and on the bacteriophage fd genome, was used to construct pathogenicity mutants of the fire blight pathogen Erwinia amylovora. Eleven nonpathogenic mutants were isolated from 1600 independent mutants screened. These mutants were divided into three types: auxotrophs, exopolysaccharide (EPS)-deficient mutants and a mutant of the dsp phenotype. According to their insertion sites the Tn5 mutants were mapped into several classes. Some of the mutants could be complemented with cosmid clones from a genomic library of the parent strain for EPS production on minimal agar. EPS-deficient mutants and the dsp mutant could complement each other to produce virulence symptoms on pear slices.

Galactose metabolism mutants of Erwinia amylovora were created by transposon insertions and characterized for their growth properties and interaction with plant tissue. The nucleotide sequence of the galE gene was determined. The gene, which encodes UDP-galactose 4-epimerase, shows homology to the galE genes of Escherichia coli, Neisseria gonorrhoeae, Rhizobium meliloti, and other gram-negative bacteria. Cloned DNA with the galE and with the gaiT and galK genes did not share borders, as judged by the lack of common fragments in hybridization with chromosomal DNA. These genes are thus located separately on the bacterial chromosome. In contrast to the gal operon of E. coli, the galE gene of E. amylovora is constitutively expressed, independently of the presence of galactose in the medium. The function of the galE gene but not of the galT or galK gene is required for bacterial virulence on pear fruits and seedlings. In the absence of galactose, the galE mutant was deficient in amylovoran synthesis. Subsequently, the galE mutant cells elicited host defense reactions, and they were not stained by fluorescein isothiocyanate-labelled lectin, which efficiently binds to amylovoran capsules ofE. amylovora. The mutation affected the side chains of bacterial lipopolysaccharide, but an intact 0 antigen was not required for virulence. This was shown with another mutant, which could be complemented for virulence but not for side chain synthesis of lipopolysaccharide.The gram-negative bacterium Erwinia amylovora is the causal agent of fire blight, a necrotic disease of rosaceous plants (49). To escape plant defense mechanisms and to cause symptoms, E. amylovora requires the capsular exopolysaccharide amylovoran (4, 20, 44). Amylovoran synthesis mutants are nonpathogenic (6, 47). A chromosomal cluster of ams genes necessary for amylovoran synthesis has been characterized (7). Cloned E. amylovora genes were able to complement mutants in the cps genes of the maize pathogen Erwinia stewartii, which are involved in exopolysaccharide (EPS) synthesis. The galE gene is adjacent to the cps gene cluster in E. stewartii (14). The dominant sugar in amylovoran is galactose (45). Consequently, disruption of the galactose metabolism affects the capsule synthesis and virulence of E. amylovora, and growth in the presence of galactose restores the ability to produce EPS (8). The genetics of gal genes in E. amylovora have not been resolved.Enterobacteria utilize galactose by the LeLoir pathway (1). The first reaction in this pathway is catalyzed by galactokinase (EC 2.7.1.6), which phosphorylates free galactose to galactose-1-phosphate. In the next steps, galactose-1-phosphate uridylyltransferase (EC 2.7.7.10) transfers the UDP residue from UDP-glucose to galactose-1-phosphate, and UDP-galactose 4-epimerase (EC 5.1.3.2) catalyzes the reversible conversions of UDP-galactose and UDP-glucose. In Escherichia coli, the structural genes galK, galT, and galE are organized in an operon which is induced by galactose (1). Two adjacent promoters are regulat...