An Optimized in situ Quantification Method of Leaf H(2)O(2) Unveils Interaction Dynamics of Pathogenic and Beneficial Bacteria in Wheat

Hydrogen peroxide (H(2)O(2)) functions as an important signaling molecule in plants during biotic interactions. However, the extent to which H(2)O(2) accumulates during these interactions and its implications in the development of disease symptoms is unclear. In this work, we provide a step-by-step optimized protocol for in situ quantification of relative H(2)O(2) concentrations in wheat leaves infected with the pathogenic bacterium Pseudomonas syringae pv. atrofaciens (Psa), either alone or in the presence of the beneficial bacterium Herbaspirillum seropedicae (RAM10). This protocol involved the use of 3-3′diaminobenzidine (DAB) staining method combined with image processing to conduct deconvolution and downstream analysis of the digitalized leaf image. The application of a linear regression model allowed to relate the intensity of the pixels resulting from DAB staining with a given concentration of H(2)O(2). Decreasing H(2)O(2) accumulation patterns were detected at increasing distances from the site of pathogen infection, and H(2)O(2) concentrations were different depending on the bacterial combinations tested. Notably, Psa-challenged plants in presence of RAM10 accumulated less H(2)O(2) in the leaf and showed reduced necrotic symptoms, pointing to a potential role of RAM10 in reducing pathogen-triggered H(2)O(2) levels in young wheat plants.