Salicaceae endophyte inoculation alters root biomass allocation and architectural traits in Populus across nitrogen environments

Abstract

Salicaceae endophytes have demonstrated capacity to promote growth in Populus, and several strains are diazotrophic, yet their effects on root functional and architectural traits remain poorly characterized. Understanding how these endophyte inoculation and nitrogen regimes shape below-ground functional traits is critical for evaluating their potential as biological inoculants in nitrogen-limited plant production and bioenergy systems. We hypothesized that endophyte inoculation would reduce root-to-shoot ratio and shift biomass allocation toward coarse roots, consistent with relaxed nitrogen foraging if the dominant mechanism were alleviation of nitrogen limitation. Because many of these endophytes also produce auxin, we further considered an alternative, nitrogen-independent prediction in which inoculation directly stimulates root proliferation and deeper rooting irrespective of nitrogen supply. We therefore evaluated root architectural responses across contrasting nitrogen environments to distinguish these competing expectations. Three complementary experiments were conducted using tissue cultured Populus plants. Two pot-based experiments examined biomass allocation and root functional class responses to endophyte inoculation. A hydroponic experiment using a rhizobox imaging system and image-based phenotyping with RhizoVision Explorer was conducted across two independent experimental rounds to characterize root system architectural responses. Twelve architectural traits were extracted from final harvest images and analyzed using two-way ANOVA with experimental round and block as fixed covariates. Endophyte inoculation significantly increased net photosynthetic rate, total plant biomass, and coarse root biomass under low nitrogen, while reducing root-to-shoot ratio across contrasting nitrogen environments, indicating effects that were not solely dependent on nitrogen availability. The same nitrogen-independence was evident in the rhizobox experiment, where inoculated plants developed deeper, more extensive, and more vertically oriented root systems than mock-inoculated controls regardless of nitrogen level. Across both growth systems, therefore, inoculation shifted plants toward greater root growth and altered biomass allocation independently of nitrogen supply. Principal component analysis of shoot and root functional traits showed that endophyte inoculation drove a coordinated whole-plant transition from nitrogen acquiring toward biomass-accumulating phenotypes, explaining 60.9% of total trait variance along a single multivariate axis. Our results indicate that inoculation with Salicaceae endophyte consortia can alter root architectural traits and shift above- and below-ground carbon partitioning in Populus. The inoculation-driven reduction in root-to-shoot ratio was statistically consistent across high and low nitrogen, with no significant interaction. This pattern aligns more closely with the alternative, nitrogen-independent prediction we proposed. Because nitrogen fixation was not directly measured, this distinction remains an inference. These findings position the endophyte consortia as candidates for improving performance in Populus production systems where nitrogen inputs are constrained. Future studies are recommended to examine endophyte colonization persistence under field soil conditions, quantify nitrogen fixation and phytohormone contributions in planta to determine whether the responses documented here are reliably expressed at ecologically relevant scales.