K. K. Bastas, Selcuk University, Faculty of Agriculture, Department of Plant Protection, Campus, TR-42075 Konya, Turkey;
F. Sahin, Yeditepe University, Faculty of Engineering and Architecture, Department of Genetics and Bioengineering TR-34755 Istanbul, Turkey; and
R. Atasagun, Agriculture District Directorate of Sarayonu, Konya, Turkey
During the summers of 2008 and 2010, leaf and shoot blight, wilting of the tips of young infected shoots, and cankers with brown discoloration on twigs were observed on six dog rosehip (Rosa canina) plants from four different private orchards in Eregli district of Konya Province, Turkey. Disease incidence was estimated to be approximately 0.5% on rosehips over 2 years within all survey areas, and surveys showed that ~4 ha was infested. Bacteria isolated from diseased leaf and shoot tissues was macerated and streaked on nutrient sucrose agar (NSA) and King's medium B (KB). Typical light cream, levan-positive colonies developed on NSA medium after a 2-day incubation at 25°C. Colonies on KB were white and non-fluorescent (1). Bacterial strains were identified on the basis of biochemical, physiological (2), and molecular tests (3). Eleven representative bacterial strains isolated were gram-negative, rod-shaped, mucoid, fermentative, yellow-orange on Miller & Scroth medium, positive for levan formation and acetoin production, no growth at 36°C, positive for gelatin hydrolysis, and negative for esculin hydrolysis, indole, urease, catalase, oxidase, arginine dehydrolase, reduction of nitrate, and acid production from lactose and inositol. Two reference strains of Erwinia amylovora (Burr.) Winslow et al. (Ea43b and NCPPB 2791) obtained from culture collection of Selcuk University, Department of Plant Protection, Turkey, were used as positive controls. 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 sterilized distilled water (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 set A/B (A: 5′ CGGTTTTTAACGCTGGG 3′ and B: 5′ GGGCAAATACTCGGATT 3′) (3) by PCR assay to generate a 1-kb DNA fragment, and fatty acid methyl ester (FAME) profiles determined by Sherlock Microbial Identification System software with similarity indices ranging from 84 to 97%. Pathogenicity was tested by inserting a suspension (108 CFU/ml SDW) of each of the 11 bacterial strains and two references strains into actively growing shoot tips and petioles of 4-year-old plants of Rosa canina using a 0.46-mm-diameter hypodermic needle. Leaf and shoot blight symptoms resembling the natural infection were developed on the inoculated plants 7 to 10 days after inoculation. SDW was injected similarly as a negative control treatment, and no symptoms were observed on the control plants. All tests were repeated three times. Re-isolations were done from shoots and leaves of inoculated plants with the two reference strains and the 11 bacteria, and control plants. Obtaining bacteria 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 rosehip in Turkey.
References: (1) R. A. Lelliott and D. E. Stead. Methods for Diagnosis of Bacterial Diseases of Plants (Methods in Plant Pathology). Oxford, UK, 1987. (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) S. Bereswill et al. Appl. Environ. Microbiol. 58:3522, 1992.