Competitive Confrontation or Cooperative Coexistence? Environmental Drivers Shaping Interactions Between Endophyte Bacillus atrophaeus and Epiphyte Pseudomonas chlororaphis O6 in Wheat Rhizosphere

Background: Both endophytes and epiphytes play a critical role in shaping microbial community structure and plant health, yet the mechanisms underlying antagonism between co-occurring beneficial microbes and effects on the host plant remain poorly understood. In this study, we investigated antagonistic interactions between Pc O6, a root colonizing plant beneficial Pseudomonas soil isolate, and JunSE1L, a Bacillus endophyte isolated from winter wheat ( Triticum aestevum , var. Juniper). The endophyte/epiphyte interactions were evaluated on the wheat roots and using agar-based and liquid assays to precisely control nutrient conditions, cell density, and environmental factors, like temperature. Results: The JunSE1L Bacillus endophyte emerged from roots and colonized the rhizoplane for wheat grown in sterile sand; however, in a chemotaxis assay, Pc O6 migrated through wet sand to the wheat root and inhibited JunSE1L emergence. Zone-of-inhibition assays revealed that Pc O6 strongly inhibited JunSE1L growth under biofilm-promoting conditions on both rich (LB) and defined minimal (MM) media, with significantly larger inhibition zones observed on MM agar. The extent of inhibition increased as JunSE1L background cell density decreased, indicating a strong density-dependent response. In contrast, reciprocal assays showed no inhibition of Pc O6 by JunSE1L, even at low Pc O6 densities, demonstrating a unidirectional antagonistic interaction. On LB agar at 22°C Pc O6 produced characteristic orange colored antimicrobial phenazines that were absorbed by surrounding JunSE1L colonies without any signs of inhibition. On MM agar, Pc O6 ceased to produce phenazines, while also more strongly inhibiting JunSE1L, suggesting that phenazine-associated compounds are not the primary inhibitory agents under nutrient-limited conditions. On rich LB agar at 37°C and Pc O6 failed to produce phenazines or show any inhibition of JunSE1L. In liquid MM culture, Pc O6 cell-free supernatant suppressed JunSE1L growth without eliminating viability, consistent with a bacteriostatic effect in MM. Inhibitory activity persisted following Proteinase K treatment and heat exposure, ruling out proteinaceous toxins or enzymes. Diffusion rates of the inhibitory compound(s) in agar were consistent with sub 5 kDa metabolites. Conclusions: Together, these results demonstrate that Pc O6 exerts a temperature-, nutrient-, and density-dependent inhibitory effect on JunSE1L mediated by a non-proteinaceous, low-molecular weight compound. At ambient temperatures under rich nutrient conditions the endophyte and epiphyte co-exist; however, on defined nutrient media, which may reflect the competition for nutrients in the rhizoplane, the Pc O6 epiphyte strongly suppresses the endophyte in vivo and in vitro . While the chemical identity of the inhibitory factor(s) remains unknown, the findings narrow the range of candidate molecules and highlight the importance of environmental context in shaping bacterial antagonism. Further chemical characterization will be required to identify the compound and elucidate its role in microbial community assembly and benefits to the host plant.