Exogenous melatonin orchestrates multiple defense responses against Botrytis cinerea in tomato leaves

Infection of tomato leaves and fruits by necrotrophic fungal pathogen Botrytis cinerea causes considerable economic loss. Melatonin has multiple physiological functions and is considered a potential biocontrol agent for its positive effects on plant innate immunity. In tomato, melatonin reduces Botrytis-caused post-harvest fruit decay by activating jasmonic acid (JA) signaling. In this study, we analyzed the effects of melatonin on resistance to Botrytis and associated defense mechanisms in tomato leaves. Treatment of detached tomato leaves with melatonin at a concentration as low as 100 mu M induced resistance to Botrytis based on reduced lesion size and fungal growth. Melatonin-induced resistance to gray mold was associated with activation of a complex set of defense mechanisms in tomato leaves. First, melatonin suppressed Botrytis-induced cell death and accumulation of reactive oxygen species (ROS), which was correlated with increased activities of ROS-scavenging enzymes and enhanced expression of their corresponding genes. Second, melatonin treatment induced callose synthase genes and promoted rapid and strong callose deposition in tomato leaves. Melatonin also induced other anti-microbial genes encoding chitinases, beta-1,3-glucanases, phenylalanine ammonialyases and polyphenol oxidases. Third, melatonin increased Botrytis-induced expression of genes encoding SlWRKY33, SlMYC2 and SlERF transcription factors, which activate defense-related genes expression through JA and ethylene (ET) signaling pathways. By contrast, melatonin suppressed salicylic acid (SA) signaling based on reduced expression of the SlPR1 marker gene, which is known to antagonize defense against Botrytis. These results collectively indicate that exogenous melatonin orchestrates multiple signaling pathways to activate a complex set of defense mechanisms against Botrytis in tomato leaves.