Humic Substances from Different Sources Modulate Salicylic Acid-Mediated Defense in Plants Infected by Powdery Mildew

Modern agriculture relies heavily on chemical inputs to sustain productivity, yet their intensive use poses environmental and health risks. Sustainable strategies based on biostimulants have emerged as promising alternatives to reduce agrochemical dependence. Among these compounds, humic substances (HS) stand out for their ability to modulate plant growth and activate defense responses. This study aimed to evaluate the effects of HS from different sources-vermicompost (Vc) and peat (Pt)-on the salicylic acid (SA)-mediated defense pathway in tomato plants (<i>Solanum lycopersicum</i> cv. Micro-Tom) infected with <i>Oidium</i> sp. The HS were characterized by solid-state ¹³C CPMAS NMR to determine the relative distribution of carbon functional groups and structural domains, including alkyl, O-alkyl, aromatic, and carbonyl carbon fractions, as well as hydrophobicity-related indices. Enzymatic activities of lipoxygenase, peroxidase, phenylalanine ammonia lyase, and beta 1,3-glucanase were determined spectrophotometrically, and RT-qPCR quantified gene transcription levels involved in SA signaling and defense (<i>MED25</i>, <i>MED16</i>, <i>MED14</i>, <i>NPR1</i>, <i>ICS</i>, <i>PAL</i>, <i>LOX1.1</i>, <i>MYC2</i>, <i>JAZ</i>, <i>jar1</i>, <i>CAT</i>, <i>POX</i>, <i>SOD</i>, <i>APX</i>, <i>ERF</i>, <i>PR-1</i>, <i>PR-2</i>, <i>PR-4 e PR-5</i>). Both HS significantly reduced disease severity and activated key SA-related defense genes, including the regulatory gene <i>NPR1</i> and the effector genes <i>PR1</i>, <i>PR2</i> and <i>PR5</i>, with Pt providing greater protection. Notably, HS amplified defense-related gene expression and enzymatic activities specifically under infection, showing a stronger induction than in non-infected plants. These results demonstrate that structural differences among HS drive distinct and enhanced defense responses under pathogen challenge, highlighting their potential as sustainable tools for improving plant immunity in agricultural systems.