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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.

An Optimized in situ Quantification Method of Leaf H2O2 Unveils Interaction Dynamics of Pathogenic and Beneficial Bacteria in Wheat