Application of novel image base estimation of invisible leaf injuries in relation to morphological and photosynthetic changes of Phaseolus vulgaris L. exposed to tropospheric ozone

This study aimed to evaluate the degree of Phaseolus vulgaris L. (bean) leaf tissue injury caused by tropospheric ozone. To validate O3 symptoms at the microscopic level, Evans blue staining together with an image processing method for the removal of distortions and calculation of dead leaf areas was applied. Net photosynthetic rate (PN), stomatal conductance (gs) and intercellular CO2 concentration (Ci) were determined to evaluate leaf physiological responses to ozone. It was found that both resistant and sensitive varieties of bean were damaged by ozone; however, the size of necrotic and partially destroyed leaf area in the sensitive genotype (S156) was bigger (1.18%, 2.18%) than in the resistant genotype (R123), i.e. 0.02% and 0.50%. Values of net photosynthetic rates were lower in the sensitive genotype in ambient air conditions, than in the resistant genotype in ambient air conditions. We further found that there was a correlation between physiological and anatomical injuries; net photosynthetic rate (PN) was negatively correlated with percentage of necrotic area of both genotypes, while stomatal conductance (gs), intercellular CO2 concentration (Ci) were positively correlated with percentage of necrotic tissue of both genotypes. Moreover, visible injures in both genotypes were positively correlated with percentage of anatomical injures. In conclusion, the presented combinations of morphological, anatomical and physiological markers allowed differential diagnosis of ozone injury.