Harnessing the phytobiome for resilience: Biofilm-forming PGPR mediate physiological adaptations through enhanced seed colonization and oxidative regulation to mitigate drought stress in Cyamopsis tetragonoloba.
Global climate change and the escalating drought cycles have severely compromised agricultural productivity in marginalized agroecosystems worldwide. This has necessitated a deeper understanding of the role of the phytobiome in the host plant's resilience. Although seed biopriming with plant growth-promoting bacteria (PGPB) is widely acknowledged for alleviating abiotic stress, the role of PGPB biofilms in enhancing the efficiency of biopriming remains a critical knowledge gap. In this study, the biofilm-forming capacity of nine thermohalotolerant, ACC-deaminase-producing PGPB strains previously isolated from the rhizosphere of Cyamopsis tetragonoloba grown in arid regions of India was evaluated. Strains were classified into biofilm-forming and biofilm-deficient groups based on pellicle formation, wrinkle formation by macrocolony, safranin staining quantification (OD492 > 2.5 v/s OD492 < 0.5), FTIR spectroscopy, and FESEM analyses. Selected representatives of both groups, Bacillus altitudinis C-17 and Bacillus subtilis J-35 (biofilm-forming), and Enterobacter cloacae C-35 (biofilm-deficient), were subjected to comparative seed biopriming assays, carried out on a drought-susceptible variety of C. tetragonoloba under simulated drought (-1.5 MPa). Results revealed that simulated drought completely arrested seed germination in the unprimed control group, whereas biopriming with the biofilm-forming strain J-35 restored germination by >70% (p < 0.005). The biofilm-deficient strain, C-35, showed a non-significant recovery (p = 0.476). In pot assay conducted under water deficit, biopriming with biofilm-forming PGPBs significantly improved seedling vigor, as evidenced by improved root length, shoot length, leaf area, and wilting index. Statistical analyses revealed that biofilm formation strongly correlated with enhanced seed colonization efficiency (R2 = 0.953, p < 0.005). Furthermore, biofilm-forming strains induced robust modulation of host oxidative homeostasis, as evident from ∼15.8-fold increase in proline content, a 3- to 5-fold increase in superoxide dismutase activity, and a negative correlation with harmful H2O2 accumulation (R = -0.816). These observations clearly demonstrate that enhanced biopriming by biofilm-forming PGPBs stems from the underlying mechanism in which biofilms act as biological anchors, enhancing seed colonization and triggering robust modulation of the host plant's antioxidant machinery. These findings provide a fundamental understanding necessary to exploit the social microbial behavior in developing next-generation bioinoculants for crops grown in climate-vulnerable agroecosystems, including drought-prone arid and semi-arid regions. The core hypothesis tested in this study is that biofilm formation by PGPB serves as more than a microbial survival strategy; it is a critical functional determinant of phytobiome resilience that enhances host physiological plasticity and abiotic stress resilience through persistent host-microbe interaction and the systemic modulation of oxidative stress.