Abstract
Zucchini yellow mosaic virus [Potyvirus cucurbita flavitesselati, (ZYMV-Z)] is a major pathogen causing severe yield losses in global Squash (Cucurbita pepo L). production, with limited sustainable control strategies available. This study aimed to elucidate the biocontrol potential of whey, a by-product of the dairy industry with recognized biofertilizer and biostimulant properties, in integration with arbuscular mycorrhizal fungi (AMF-M) symbiosis, and to uncover the underlying multilayered defense mechanisms in ZYMV-infected squash plants. ZYMV-infected and non-infected plants were treated with whey (applied-foliar-Wf or via the rhizosphere-Wr) and M, either individually or in combination (WfMZ, WrMZ). Plant growth traits, physiological indicators, biochemical defense responses, soil properties, and mycorrhizal colonization parameters were comprehensively evaluated. ZYMV infection markedly suppressed plant growth, while the integration of whey and AMF significantly mitigated these adverse effects. Notably, the WfMZ combination maintained chlorophyll content and substantially reduced disease severity even under viral stress. This synergistic interaction was associated with a multifaceted activation of the plant’s defense machinery: WfMZ and WrMZ treatments induced the highest total antioxidant activity, phenolic content, proline accumulation, and PAL/CAT enzyme activities. whey acted as a microbial biostimulant by enhancing AMF spore density and root colonization, whereas AMF improved plant-phosphorus uptake and buffered ZYMV-induced rhizosphere acidification. Overall, the integration of whey and AMF represents an effective and sustainable biocontrol strategy against ZYMV. This efficacy stems from the synergy of multiple mechanisms, including direct biostimulation, physiological buffering, rhizosphere engineering, and systemic defense induction along the root–shoot axis. The findings highlight a promising approach for converting agricultural by-products into high-value biocontrol agents for managing viral diseases in plants. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s00572-026-01262-7.