Physiological and Multi-Omics Insights into Trichoderma harzianum Alleviating Aged Microplastic Stress in Nicotiana benthamiana

Microplastics derived from biodegradable PBAT film, widely used in agriculture, pose ecological and biological hazards. This study explores how <i>Trichoderma harzianum</i> T4 mitigates this microplastic-induced stress in <i>Nicotiana benthamiana</i>. Using five experimental setup-control (CK), low/high-dose aged microplastics (MP80/MP320), and their co-treatments with <i>T. harzianum</i> T4 (MP80+T4/MP320+T4), multi-omics analyses reveal the microplastic stress-alleviating mechanisms of <i>T. harzianum</i> T4. Aged microplastics significantly inhibit plant growth, promote reactive oxygen species (ROS) and malondialdehyde (MDA) accumulation, and disrupt metabolic homeostasis. Conversely, <i>T. harzianum</i> T4 activates the plant antioxidant defense system, reducing ROS/MDA levels and upregulating superoxide dismutase (SOD)/peroxidase (POD) activities, and promotes biomass. Transcriptomic analysis shows <i>T. harzianum</i> T4 reverses gene expression patterns disrupted by microplastics, particularly in DNA replication and pentose-glucuronic acid pathways. Metagenomic sequencing indicates <i>T. harzianum</i> T4 restores soil microbial diversity, increases the abundance of <i>Bacteroidota</i> and <i>Myxococcota</i>, downregulates antibiotic resistance genes (e.g., <i>tetA5</i>, <i>MDR</i>), and upregulates carbohydrate-active enzymes (CAZys), thereby enhancing carbon metabolism. In conclusion, <i>T. harzianum</i> T4 alleviates microplastic stress through a tripartite mechanism: activating plant stress-response gene networks, reshaping soil microbial communities, and modulating functional gene expression, offering a promising bioremediation strategy.