Spatial referencing of chlorophyll fluorescence images for quantitative assessment of infection propagation in leaves demonstrated on the ice plant: Botrytis cinerea pathosystem.

BACKGROUND: Chlorophyll fluorescence analysis is one of the non-invasive techniques widely used to detect and quantify the stress-induced changes in the photosynthetic apparatus. Quantitative information is obtained as a series of images and the specific fluorescence parameters are evaluated inside the regions of interest outlined separately on each leaf image. As the performance of photosynthesis is highly heterogeneous over a leaf surface, the areas of interest selected for generating numeric data are crucial for a reliable analysis. The differences in intact leaf physio-morphological characters and in the structural effects of stress between leaves increase the risk of artefacts. RESULTS: The authors propose a new enhanced method for precise assessment of stress-induced spatiotemporal changes in chlorophyll a fluorescence exemplified in the leaves of common ice plants infected with a fungal pathogen. The chl a fluorescence leaf image series obtained with Imaging-PAM fluorometer are aligned both by affine and nonlinear spline transforms based on the set of control points defined interactively. The successive readings were taken on the same leaf and this image sequence registration allows to capture quantitative changes of fluorescence parameters in time and along selected directions on the leaf surface. The time series fluorescence images of attached leaf, aligned according to the proposed method, provide a specific disease signature for an individual leaf. The results for C3 and Crassulacean Acid Metabolism (CAM) plants have been compared with respect to the type of photosynthetic metabolism and the image alignment accuracy has also been discussed. CONCLUSIONS: The image alignment applied to the series of fluorescence images allows to evaluate the dynamics of biotic stress propagation in individual plant leaves with better accuracy than previous methods. An important use of this method is the ability to map the fluorescence signal horizontally in one leaf during disease development and to accurately compare the results between leaves which differ in morphology or in the structural effects of stress. This approach in analysing chlorophyll fluorescence changes can be used to receive spatial and temporal information over a sample area in leaves infected by different pathogenic fungi and bacteria.