Synergistic curative effects of Trichoderma hamatum and Rumex dentatus against Alternaria alternata, the causal agent of tomato leaf spot disease

The current investigation identified and characterized <i>Alternaria alternata</i> as the causal agent of tomato leaf spot through morphological traits and multilocus phylogenetic analysis [internal transcribed spacer (ITS), translation elongation factor 1-alpha (<i>tef1</i>-α), and the RNA polymerase II second-largest subunit (<i>RPB2</i>)], thereby confirming its classification within the <i>A. alternata</i> complex. Four <i>Trichoderma</i> isolates (Ham34, Ham35, Ham36, and Ham37) were recovered from loamy rhizosphere soil associated with healthy tomato plants. Among them, Ham34 (<i>Trichoderma hamatum</i>) showed the most significant antagonistic activity in dual-culture assays. Ethyl acetate extracts of all isolates exhibited concentration-dependent antifungal effects, with Ham34 showing the most potent inhibition (57.8% at 2000 µg/mL). Gas chromatography-mass spectrometry (GC-MS) profiling of the Ham34 extract identified bioactive metabolites, including (-)-spathulenol (28.2%) and glycerol 1,2-diacetate (21.4%). Among the four tested plant extracts, <i>Rumex dentatus</i> (RD) showed superior activity, achieving 68.5% inhibition at 2000 µg/mL, followed by moderate inhibition from <i>Cichorium intybus</i>, <i>Conium maculatum</i>, and <i>Capsicum annuum</i>. High-performance liquid chromatography (HPLC) profiling of <i>Rumex dentatus</i> extract revealed high levels of phenolic and flavonoid compounds, with a total concentration of 108.63 µg/mL. The primary identified constituents were rutin (33.1 µg/mL), gallic acid (22.7 µg/mL), and chlorogenic acid (17.9 µg/mL). Under greenhouse conditions, tomato plants (<i>Solanum lycopersicum</i> cv. Super Strain B) were treated with Ham34 ethyl acetate extract, RD extract, and their combination (Ham34 + RD). The combined treatment significantly reduced disease incidence (11.1%) and severity (16.7%), outperforming Ridomil Gold^® in disease suppression. Growth parameters and chlorophyll content (SPAD) were significantly enhanced in the combined treatment, with increased shoot and root biomass and length. Biochemical analyses revealed that the combined Ham34 + RD treatment enhanced antioxidant enzyme activities, including catalase (CAT) and peroxidase (POD), and increased total soluble protein content to 342.8 µg/mL compared to 331.1 µg/mL in the untreated control. Moreover, the treatment elevated total phenolic content to 256 mg GAE/g, indicating improved stress mitigation and enhanced plant vigor. This eco-friendly approach provides a low-cost, chemically stable, and safe alternative to synthetic fungicides, highlighting the synergistic potential of integrating <i>R. dentatus</i> and <i>T. hamatum</i> for sustainable management of <i>A. alternata</i>-induced tomato leaf spot.