Histology of Reactive Oxygen Species Generation in Oxidative Burst of Interaction between Quince (Cydonia oblonga Mill.) Genotypes with Causal Bacteria of Fire Blight Disease

This research was conducted to identify the fire blight resistance and related oxidative responses in quince genotypes.  KVD4, NB2, PH2 and KVD3 genotypes and Quince C rootstock were inoculated in vitro with Erwinia amylovora Ea273 and necrosis progress and ROS generation, including O2-, H2O2 and OH- in control and infected shootlets was compared. Results showed three types of oxidative responses in genotypes. In NB2 and Quince C rootstock, with a higher level of disease resistance, they had a fast O2- increase, a delay in H2O2 generation, and a faster increase in OH- severity. KVD4 had a moderate susceptibility level and similarly moderate behavioral in generation of three ROS. In third group, more susceptible genotypes, PH2 and KVD3, showed low O2- generation, faster H2O2 increase and slower rate of increase of OH- level. It seems that defensive mechanism of quince against the fire blight disease is completely different from pear tree, which mainly accumulates H2O2 as a barrier. 

Fire blight (Erwinia amylovora) is considered the most important limiting factor for pome fruit trees such as apples, pears and quinces (Van der Zwet, and Keil, 1979). Numerous factors are effective for E. amylovora’s pathogenicity and molecular interaction with host plants, but the oxidative burst is the critical factor that results in host cell degradation and pathogen growth (Azarabadi et al., 2016). Among various hosts of fire blight causal agent, the quince trees are the most susceptible one and the disease usually causes large disease symptoms such as bloom blight, shoot blight, canker and trunk necrosis that leads to entire decline of the tree in the outbreak years (Van der Zwet, and Keil, 1979). Quince tree also is native to Iran and this country is among the main producers of quince fruits in the world (Abdollahi, 2023). Due to importance of quince tree in Iran and high damages of fire blight disease on it, the current research was conducted to evaluate the mechanisms of resistance to this disease in level of oxidative burst and severity of generation of various reactive oxygen species in the different quince genotypes.
 

Quince genotypes including KVD4, NB2, PH2 and KVD3, along with Quince C rootstock were established in in vitro conditions on modified QL medium and inoculated with Erwinia amylovora type strain Ea273. Following this step, the progress of disease necrosis and generation of reactive oxygen species (ROS), including superoxide radical (O2-), hydrogen peroxide (H2O2) and hydroxyl radical (OH-); as the main species of active oxygen during interaction of E. amylovora with the host plants (Venisse et al., 20002), were compared in three separate experiments in control and infected in vitro shootlets. Severity of the generation of each reactive oxygen species evaluated by the index of red color in the tissues of quince genotypes from score 0 (without any trace of ROS) to 5 (the highest severity of each individual ROS) following the colorimetric method described by of Azarabadi et al. (2016). Traces of superoxide radical, hydrogen peroxide and finally hydroxyl radical were determined by nitro blue tetrazolium (NBT), 3, 3´-Diaminobenzidine-HCl (DAB)-HCl and Dimethyl sulfoxide (DMSO), respectively, Following Azarabadi et al. (2016). Data were collected in 120 hours time course after inoculation of in vitro shootlets with with Erwinia amylovora type strain Ea273, and the curves were depicted using Microsoft Excel.
 

The results showed three types of oxidative stress and ROS generation patterns in responses to fire blight disease progress in the tissues of studied quince genotypes. Quince C and NB2 genotypes, with higher levels of disease resistance, demonstrated faster increase in the superoxide generation, delay in the generation of hydrogen peroxide, and faster increase in severity of the hydroxyl radical. Also, KVD4 genotype had moderate susceptibility level to the disease and similarly moderate behavioral of the amount of the generation of three reactive oxygen species including superoxide, hydrogen peroxide and hydroxyl. In the third group, the more susceptible genotypes, PH2 and KVD3, showed fast necrosis progress, low generation of superoxide, faster increase of hydrogen peroxide and slower rate of increase of hydroxyl radical level. Considering the various patterns of ROS generation in the evaluated quince genotypes, it can be concluded that the defensive mechanisms of the quince tree against fire blight is completely different from pear tree in which the accumulation of hydrogen peroxide is the main barrier for resistance to the fire blight disease (Azarabadi et al., 2016).