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In this study, ZYMV was isolated from symptomatic squash plants in Kafr El-Sheikh Governorate and confirmed by RT-PCR and sequencing (GenBank Accession No. PV833355). The antiviral efficacy of two microbial biocontrol formulations-Bio-Nagi (\n                    <jats:italic>Trichoderma asperellum<\/jats:italic>\n                    ) and Bio-4 (a\n                    <jats:italic>Bacillus<\/jats:italic>\n                    spp. consortium)-was assessed through foliar application and seed priming at pre-, concurrent-, and post-inoculation timings. Disease severity analyses showed that Bio-4 applied concurrently reduced symptom development by 62%, while Bio-Nagi pre-treatment resulted in a 48% reduction. Transmission electron microscopy revealed substantial protection of chloroplast and nuclear ultrastructure in Bio-4\u2013treated plants compared with infected controls. Antioxidant enzyme activities (SOD and POD) increased by 1.8\u20132.4-fold following microbial treatments, indicating enhanced physiological defense responses. qRT-PCR profiling demonstrated strong differential induction of defense genes: PR1 was upregulated 5.6-fold in Bio-Nagi pre-treated plants, whereas PR3 and PR5 increased by 7\u20139-fold in Bio-4 concurrent and post-inoculation treatments. Overall, Bio-4 and Bio-Nagi mitigated ZYMV-associated symptoms and activated host defense pathways in squash. These findings underscore the importance of application timing and support microbial biocontrol agents as sustainable strategies for ZYMV management in squash production systems.\n                  <\/jats:p>","DOI":"10.1186\/s12866-026-05161-x","type":"journal-article","created":{"date-parts":[[2026,6,30]],"date-time":"2026-06-30T06:16:07Z","timestamp":1782800167000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Molecular and enzymatic insights into biocontrol-mediated resistance against Zucchini yellow mosaic virus in squash (Cucurbita pepo L.)"],"prefix":"10.1186","volume":"26","author":[{"given":"Radwa M.","family":"Shafie","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ahmed A.","family":"kheder","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mohamed A.","family":"Abou-Attia","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Noher A.","family":"Mahmoud","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sawsan M.","family":"Saied","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Amira A.","family":"Mazyad","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0121-7829","authenticated-orcid":false,"given":"Hayam S.","family":"Abdelkader","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2026,6,30]]},"reference":[{"key":"5161_CR1","unstructured":"Abd El-Shafi S, Abd El Gwad S, Sleem E. 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