Unlocking the Power of Pseudomonas : Network-Guided Isolation Enhances Plant Growth on Achnatherum Inebrians

The tripartite interplay among plants, bacteria, and fungal endophytes is crucial for maintaining host plant fitness. However, how Epichloë endophytes influence plant-associated bacterial communities in cool-season grasses, particularly in ecologically important yet understudied species, such as Achnatherum inebrians, remains unclear. Using phylogenetic molecular ecological network analysis, we determined that seed-borne (seed epiphytic) and phyllosphere bacterial communities of Epichloë-infected (EI) A. inebrians exhibited reduced network complexity compared to Epichloë-free (EF) plants. Across all samples, Proteobacteria and Firmicutes dominated the keystone taxa, with Pseudomonas (OTU744 and OTU8264) consistently identified as a hub genus in both seed-borne and phyllosphere networks. Culture-based analysis revealed that endophyte-infected plants had a significantly (P < 0.05) higher relative abundance of Pseudomonas and Bacillus than EF A. inebrians, especially Pseudomonas comprised 13, 35, and 33% of isolates from the seed, leaf, and rhizosphere of A. inebrians, respectively. To capture potential functional diversity, we selected two phylogenetically distant Pseudomonas strains from each of the three ecological niches for further analysis. Inoculation of A. inebrians seedlings with these strains consistently promoted plant growth, enhanced forage quality (total nitrogen content), and improved nutritional value (ether extract). Whole-genome sequencing combined core-genome phylogenetic tree of the six Pseudomonas strains and confirmed that five strains belong to P. atacamensis, whereas Pse19 was P. cucumis. Our findings reveal that Epichloë endophytes modulate bacterial network stability and enrich plant-associated Pseudomonas, which synergistically enhance host performance. Collectively, this study provides a mechanistic framework for manipulating keystone taxa and beneficial isolates to improve grass productivity in grassland agricultural ecosystems.IMPORTANCEAlthough the tripartite interplay between plants, bacteria, and fungal endophytes, such as Epichloë, is recognized as vital for host fitness, the specific mechanisms through which these endophytes shape associated bacterial communities, particularly in ecologically significant grasses, such as Achnatherum inebrians, remain poorly understood. This study provides crucial mechanistic insights by revealing that Epichloë endophytes reconfigure the structure and stability of both seed-borne and phyllosphere bacterial networks in A. inebrians, leading to reduced complexity but enrichment of specific keystone taxa. We identify Pseudomonas as a consistently dominant hub genus across these niches. Notably, functional validation shows that diverse Pseudomonas isolates, representative of those enriched by the endophyte, significantly enhance host plant growth, forage quality, and nutritional value. Our findings reveal a synergistic mechanism where Epichloë endophytes modulate bacterial network stability to favor beneficial Pseudomonas populations, collectively boosting host performance.