The Gram-negative bacterium Dickeya solani causes potato soft rot and blackleg, and effective control measures remain limited. We evaluated the agricultural potential of Aspergillus sulphureoviridis , a recently described and chemically unexplored fungus. Bioactivity-guided molecular networking of extracts that inhibited D. solani led to the isolation of six metabolites: cladobotrin IV ( 1 ), two new analogs cladobotrin VII and cladobotrin VIII ( 2 and 3 ), and the orsellinic acid-derived metabolites F9775A ( 4 ), F9775B ( 5 ), and violaceol I ( 6 ). Among these, the polyketide F9775B ( 5 ) inhibited both Bacillus subtilis and D. solani (IC ₅₀ 89.4 and 59.3 µg/mL, respectively), while cladobotrin VIII ( 3 ) was active against B. subtilis (IC ₅₀ 95 µg/mL). Furthermore, co-culture with the beneficial rhizobacterium B. subtilis revealed an additional interaction: A. sulphureoviridis induced a pronounced antifungal defense in B. subtilis . MALDI-MSI showed that this interaction spatially enriched canonical Bacillus lipopeptides (surfactins and fengycins) at the confrontation zone, whereas the shortened fengycins variants were detected only intracellularly within the Bacillus colony. This indicates that A. sulphureoviridis triggers B. subtilis to mobilize and secrete bioactive lipopeptides for defense while retaining the less active short-chain fengycin-like compounds intracellularly. Together, these results establish A. sulphureoviridis as both a source of antimicrobial compounds and an inducer of beneficial bacterial defenses, providing a rationale for further in-planta validation and development as a sustainable biocontrol agent. IMPORTANCE: Potato soft rot and blackleg caused by Dickeya solani are difficult to control, creating a need for sustainable biocontrol strategies. Here, we show that the recently described fungus Aspergillus sulphureoviridis has a dual function relevant to crop protection. Bioactivity-guided molecular networking enabled the targeted discovery of six antibacterial metabolites, including two new cladobotrin analogs and the first cladobotrins reported from Aspergillus , and identified F9775B as the main metabolite active against D. solani . Using MALDI mass spectrometry imaging, we further demonstrate that A. sulphureoviridis does not act only through its own metabolites but also triggers Bacillus subtilis to enrich and deploy surfactin and fengycins at the interaction zone. These findings expand the known chemical diversity of A. sulphureoviridis and support its potential as a dual-function contributor to sustainable microbe-based biocontrol.

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