Abstract
Biofertilizers offer a promising avenue to reduce phosphate fertilizer reliance. However, inconsistent lab-to-field conversion undermines confidence in the technology. Increasingly, initial screens of bacteria are conducted in silico, based on the presence of a widely accepted set of phosphate solubilization-associated "canon" genes. However, these genes capture only a subset of the mechanisms observed experimentally in microbe-mediated solubilization. This study uses a transcriptomic systems approach to compare the transcriptional response of three novel Pantoea rara strains with divergent phosphate solubilization indices (PSI): a wild type (PSI = 1.74), an enhanced efficiency mutant (PSI = 4.13), and a null mutant (PSI = 0), which are indistinguishable using canonical gene markers alone, to phosphate limitation. Soluble phosphate limitation triggered robust but transient transcriptional responses across 80 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Despite its presence, the gcd gene, the most commonly used marker for phosphate solubilization functionality, was not upregulated in either of the solubilizing strains. Instead, each strain appeared to rely on a unique combination of organic acids, including acetic, citric, formic, and malic acid. Notably, the divergent phenotypes of both mutants were linked to a rewiring of the phosphate limitation response, namely a heightened limitation response in the enhanced mutant and an apparent "phosphate blindness" in the null mutant. These findings suggest that system-level characterization of phosphate solubilizers can reveal auxiliary pathways and genes overlooked by current annotation frameworks. Integration of auxiliary contributors with the existing canon could improve the predictive power of pre-field phosphate solubilization screens and subsequently help bridge the lab-to-field gap in biofertilizer development.IMPORTANCEDespite the potential benefits to the productivity of agricultural systems and the health of local ecosystems, enthusiasm for phosphate-solubilizing biofertilizers has been dampened by a significant performance gap that occurs when candidates are transitioned from the lab to the field. This inconsistency is partially attributable to narrow screening strategies that rely on simplified in vitro assays and the broad use of a narrow set of canonical genes, such as gcd, as functional proxies. This study leveraged a rare set of three Pantoea rara strains that differ phenotypically in their phosphate solubilization capacity but are identical using standard genetic markers. This system enabled the identification of overlooked genetic and regulatory contributors. These findings reveal limitations in current screening methods and underscore the need for a more comprehensive molecular framework to guide biofertilizer discovery.