Reactive Oxygen Species Levels of Some Apple Cultivars after Fire Blight Infection

Erwinia amylovora, the causal agent of fire blight of Maloideae, induces in its susceptible host plants an oxidative burst as does an incompatible pathogen. The oxidative burst, a rapid production of reactive oxygen species (ROS) released into the apoplast, is described as one of the earliest responses to pathogen infection and is generally associated with hypersensitive reaction (HR). In this work, we examined the possible involvement of ROS in the initiation of infection of different apple cultivars by E. amylovora. The experiments were performed on apple seedlings (six to eight leaves) from apple cultivars (Malus domestica cv. Gala, cv. Golden, cv. Scarlet and cv. Braeburn) with M9 rootstock. Plants were grown in individual pots in the greenhouse at 25 °C ± 2 °C in 60% to 65 % relative humidity under natural photoperiod. Completely folded the youngest leaves of plants were inoculated with highly virulent wild-type strain Ea 43b suspensions adjusted to a concentration of 10⁸ cfu·ml^-1 and sampled at various times for 24^th and 72^nd hours. Control plants were sprayed with sterile distilled water. Leaf tissues (0.5 g fresh weight) were homogenized in 1 ml of ice-cold 50 mm sodium phosphate buffer (pH 7.5) containing 1 mm polyethyleneglycol, 1 mm phenylmethylsulfonyl fluoride, 8% (w/v) polyvinylpolypyrolydone, and 0.01% (v/v) Triton X-100. Homogenates were centrifugated at 16,000g for 20 min at 4°C and supernatants were immediately measured by a spectrophotometric method and determined hydrogene peroxidase (H₂O₂), ascorbate peroxidase (AsPOX), super oxide-dismutase (SOD), catalase (CAT), peroxidase (POX), proline accumulation and chlorophyll concentration levels. All experiments were performed with a minimum of three tissue sample replicates per time point. The data were statistically evaluated by ANOVA variance analyze and Duncan multiple range tests. Our results showed that a sustained production of ROS and its immediate consequences in plant tissues and the level of enzyme activities showed variability in different apple cultivars in comparison with control plants.