Fire blight is a destructive and sporadic disease of crabapple (Malus floribunda) and other plants in the Rosaceae in many areas of the world. From 2007 to 2010, sudden wilting, shriveling of flowers, leaf and shoot blight, and cankers with brown discoloration on twigs of crabapple were observed in residential landscapes of Konya Province, Turkey. Disease incidence ranged from 20 to 40% in different areas of this province, and surveys showed that ~163 ha were infested. Isolations were made from sections of symptomatic leaves, shoots, and cankers using 70% ethanol for 1 s to surface-sterilize the tissue sections, followed by rinsing three times in sterilized distilled water (SDW). Then, a 1 g subsample of each tissue section was homogenized in 10 ml phosphate buffered saline (PBS), and a 10-fold serial dilution of each homogenate prepared for six dilutions. From each homogenate, an aliquot of each dilution was plated onto 5% nutrient sucrose agar and King's B agar media, and the plates incubated for 2 to 3 days at 27°C (3). Bacterial strains were identified on the basis of biochemical, physiological (2), and molecular tests (1). Twenty-seven representative bacterial strains were each gram negative, rod-shaped, mucoid, fermentative, yellow-orange on Miller and Scroth agar medium, positive for levan formation and acetoin production, and showed no growth at 36°C. The strains were also positive for gelatin hydrolysis and negative for esculin hydrolysis, indole, urease, catalase, oxidase, arginine dihydrolase, reduction of nitrate, and acid production from lactose and inositol (2). Two reference strains of Erwinia amylovora (EaP28 and NCPPB 2791) from a culture collection at Selcuk University were used as positive control strains. All strains induced a hypersensitive response in tobacco (Nicotiana tabaccum cv. White Burley) plants within 24 h after inoculation with a 108 CFU/ml bacterial suspension in SDW (~50 μl), and the strains produced ooze on inoculated immature pear fruit slices cv. Ankara. All strains were identified as E. amylovora using the species-specific primers A/B (1), which amplified a 1 kb DNA fragment by PCR assay. Pathogenicity was confirmed by inserting a suspension (108 CFU/ml SDW) of each of the 27 bacterial strains and two reference strains, EaP28 and NCPPB 2791, into actively growing shoot tips of 3-year-old plants of M. floribunda cv. Hilleri, using a 0.46 mm-diameter hypodermic needle. Leaf and shoot blight symptoms typical of fire blight were observed within 2 weeks. SDW was injected similarly as a negative control treatment, and no symptoms were observed. All tests were repeated three times with the same results. Re-isolations were done from the control plants as well as shoots and leaves inoculated with the two reference strains and the 27 bacteria identified as E. amylovora. Bacteria isolated from inoculated plants were identified as E. amylovora using the biochemical, physiological, and molecular tests described above, but this bacterium was not isolated from the control plants. To our knowledge, this is the first report of E. amylovora on crabapple in Turkey.
References: (1) S. Bereswill et al. Appl. Environ. Microbiol. 58:3522, 1992. (2) A. L. Jones and K. Geider. Laboratory Guide for Identification of Plant Pathogenic Bacteria, pp. 40-55, American Phytopathological Society, St. Paul, MN, 2001. (3) R. A. Lelliott and D. E. Stead. Methods for Diagnosis of Bacterial Diseases of Plants (Methods in Plant Pathology). Oxford, UK, 1987.