International Journal of Agriculture and Forestry 2014, 4(5): 408-413 DOI: 10.5923/j.ijaf.20140405.10 Association of Xanthomonas sp. with Apple Trunk Canker Disease in an Apple Orchard from Iran Nadjmieh Shaki 1,* , Nader Hasanzadeh 1 , Mansoureh Keshavarsi 2 , Eisa Nazerian 3 1 Department of Plant Pathology, Faculty of Agriculture and Natural Resources, Science and Research Branch, Islamic Azad University, Tehran, Iran 2 Seed and Plant Improvement Institute, Karaj, Iran 3 Plant Protection Department, National Research Station of Ornamental Plants, Mahallat, Iran Abstract During a survey of fruit orchards in northern part of Iran, some apple trees with trunk canker symptoms were observed. The infected trees lacked any visible symptoms on leaves and fruits. On NA and YDC media a Gram-negative, yellow-pigmented bacteria were consistently isolated. Based on pathogenicity, biochemical and molecular tests and fatty acid analysis, the bacterial agent was identified as Xanthomonas sp. The isolates were pathogenic on apple cv. prime gold detached leaves in laboratory conditions and in one-year old seedlings of cv. golden delicious in glasshouse condition. A typical canker symptoms were observed in apple trunks after seven months from artificial inoculation and the agent was re-isolated and Koch's hypothesis were fulfilled. DNA sequences were matched with Xanthomonas sp. sequence deposited in to NCBI GenBank. The bacterium could only survive and infect in high relative humidity and at temperature ranging 26-28°C. This is the first report of naturally occurring Xanthomonas sp. from apple canker disease symptoms. Keywords Trunk canker, Apple, Xanthomonas sp 1. Introduction Apple (Malus domestica Borkh) is the third horticulture products in Iran, accounting 1662430 tons fruit product per year. Also, Iran is the 8th apple producer in the world (FAOSTAT, 2010). Many common bacterial diseases such as fire blight, blister spot, crown gall and hairy roothave been reported on apple worldwide (Vanneste, 2000 and Bradley et al., 2010) but natural association of apple trunk canker with Xanthomonas sp. has not been yet reported. Only a rare occurrence of an unusual Xanthomonas campestris strain with apple was observed on symptomatically apple tissue in vitro (Mass et al., 1985). Xanthomonas is a genus of Gammaproteobacteria that includes numerous phytopathogenic species, each characterized by a narrow host range. However, as a whole, the genus members are able to infect a broad range of plants, distributed over 124 monocotyledonous and 268 dicotyledonous plant species (Albuquerque et al., 2011). Some of the Xanthomonads could cause severe damage on economically important crops. Most of these pathogens have a well-defined and narrow plant host range (Maes, 1993). The genus also includes strains which may be associated with plant material but are not pathogenic * Corresponding author: nadjmieh@yahoo.com (Nadjmieh Shaki) Published online at http://journal.sapub.org/ijaf Copyright © 2014 Scientific & Academic Publishing. All Rights Reserved (Brenner et al., 2005). In this study, bacterial isolates associated with natural trunk cankers on apples were isolated and investigated using some standard techniques. 2. Material and Methods 2.1. Isolation and Preliminary Characterization of Bacterial Isolates During spring and summer 2010, an orchard located in the northern part of Iran was surveyed and about 30 canker samples on local apple cultivars were collected. Small pieces of trunk and branches were cut and immersed in 5 ml saline solution (0.80% NaCl) for 20 min and 30µl of suspension was cultured on nutrient agar (NA) and incubated at 26°C for 3-5 days. Yellow pigment colonies were purified and subjected to key physiological and biochemical tests including gram reaction (Suslow et al., 1982), fluorescent pigment production (King et al., 1954), growth at 25°C, 27°C and 37°C, oxidase (Kovacs, 1956), catalase (Bradbury, 1986), Oxidative ⁄ Fermentative (O/F) (Hugh and Leifson, 1953), levan production from sucrose (Lelliott and Stead, 1987), gelatin and starch hydrolyses (Fahy and Persley, 1983), nitrate reduction, production of arginine dihydrolase, ability to produce acid from carbohydrates (arabinose, glucose and mannose), growth in presence of 5% NaCl, proteolytic, lipolytic and amylolytic capability, aesculin hydrolysis (Schaad et al., 2001), Xanthomonadin production