An eco-friendly approach harnessing Trichoderma lixii ORT2 for reducing chemical phosphatic fertilizer dependency and groundwater phosphorus management through integrated in silico, in vitro and omic studies

Groundwater pollution has become an escalating global issue, with excessive application of phosphorus (P) fertilizers emerging as a major contaminant. The overuse of chemical phosphatic fertilizers has intensified the problem of P infiltration into water table, compromising soil health and exacerbating groundwater pollution. This underscores the urgent need to explore sustainable alternatives to chemical fertilizers to safeguard groundwater and soil health. The enzyme alkaline phosphatase (ALP) plays important role in efficient P-solubilization achieved by Trichoderma sp. a soil fungus known for its gradual transportation of P from fixed sources to plants. This mechanism not only minimizes P leaching into groundwater but also reduces dependency on fertilizers. To investigate the active role of fungal ALP in P-solubilization, amino acid/gene sequences of enzyme from 20 Trichoderma strains were retrieved from NCBI. Molecular docking revealed strong catalytic interaction (−6.93 kcal/mol) between complex-bound P and the ALP, involving two hydrogen bonds and key residues Gln286 and Arg340. To confirm ALP-mediated P-solubilization, 12 newly isolated Trichoderma spp., were assessed in vitro using media amended with tri-calcium phosphate (TCP), zinc phosphate (Zn₃(PO₄)₂), or ferric phosphate (FePO₄) as sole phosphorus source. Trichoderma lixii ORT2 demonstrated highest P-solubilization, yielding 547.40 μg/mL phosphate from TCP, 538.6 μg/mL from Zn₃(PO₄)₂, and 423.45 μg/mL from FePO_4, in 72 h. Solubilization was accompanied by simultaneous expression of ALP and siderophore in the same aliquot. TCP induced maximum expression of ALP (9.47 U/mL), followed by Zn₃(PO₄)₂ and FePO₄ showing 8.65 and 5.64 U/mL, respectively, in culture filtrates at 72h, that was further confirmed through proteomic analysis. FESEM micrographs confirmed P binding and biosorption into fungal mycelia as P-globules. Furthermore, Cicer arietinum inoculated with Trichoderma lixii ORT2 bio-formulations showed significant enhancement in growth parameters in TCP amended soil as compared to di-ammonium phosphate (DAP). The integrated approach of microbial P-solubilization and further utilization by plants for their growth as major nutrients merges microbial and phyto-remediation to address sustainability issues related to the management of soil health and phosphorus contamination. This strategy aims to substitute chemical phosphatic fertilizers, which are significant contributors to rising dissolved total phosphorus (DTP) in groundwater. This approach directly supports SDG 6 by reducing P leaching and improving groundwater quality, ensuring sustainable water and sanitation management for all.