In the spring of 2012 and 2013, symptoms similar to those of fire blight were observed on pear trees (Pyrus communis cv. Alexandrine, Williams) in Tunisia at flowering stages. Disease symptoms appeared in 2012 in the region of Mornag and in the following year extended to the regions of Manouba and Tebourba. More recently, the disease was observed in the regions of Bizerte, Zaghouan, and Beja. The percentages of orchard areas that had infected plants varied from 10 to 40%. Some orchards in Mornag region exhibited more than 75% disease incidence. Symptoms were observed on flowers and young shoots. Blighted blossoms appeared wilted, shriveled, and brown, and dead flowers remained on the stems. Infected shoots wilted rapidly and often formed shepherd's crooks at their tips. Samples of diseased young shoots and flowers were subjected to pathogen isolation and identification. Bacteria were isolated from washed tissues on King's B medium (KB) (1). Colonies with morphology similar to that of Erwinia amylovora were purified by sub-culturing on KB. The strains were first characterized based on morphology and biochemical tests (1). Sixteen strains produced white colonies on KB, were gram-negative, did not produce a fluorescent pigment on KB, did not grow at 35°C, and induced a hypersensitive reaction when infiltrated into tobacco leaves (cv. Xanthi). These strains were identified as E. amylovora by double-antibody sandwich indirect-ELISA and immunofluorescene microscopy using a polyclonal antibody (2) and nested PCR targeting the pEA29 plasmid (3). Pathogenicity was tested using a detached-fruit assay (1). Each strain was inoculated onto three pear fruit (cv. Alexandrine) wounded with a scalpel dipped in a 109 CFU/ml bacterial suspension. The inoculated fruit were incubated at 25°C and 80% relative humidity in plates with sterile 1% agar. Negative controls consisted of fruit wounded with a scalpel dipped in sterile distilled water. Seven days after inoculation, symptoms of discoloration, browning, and production of bacterial ooze appeared at the inoculated points. No symptoms developed on negative controls. Reisolation of bacteria yielded colonies with characteristics of E. amylovora. Purified amplicons from nested PCR were sequenced (KF302525, KF302526) and a BLAST search of the GenBank database revealed 98% homology with E. amylovora strain HF560643.1.
References: (1) Anonymous. OEPP/EPPO Bull. 34:159, 2004. (2) M. T. Gorris et al. Acta Hortic. 411:41, 1996. (3) P. Llop et al. Appl. Environ. Microbiol. 66:2071, 2000.