Often in silico, rarely in vivo: characterizing endemic plant-associated microbes for system-appropriate biofertilizers

The potential of phosphate-solubilizing microbes (PSMs) to enhance plant phosphorus uptake and reduce fertilizer dependency remains underutilized. This is partially attributable to frequent biofertilizer-farming system misalignments that reduce efficacy, and an incomplete understanding of underlying mechanisms. This study explored the seed microbiomes of nine Australian lucerne cultivars to identify and characterize high-efficiency PSMs. From a library of 223 isolates, 94 (42%) exhibited phosphate solubilization activity on Pikovskaya agar, with 15 showing high efficiency (PSI > 1.5). Genomic analysis revealed that the "high-efficiency" phosphate-solubilizing microbes belonged to four genera (<i>Curtobacterium</i>, <i>Pseudomonas</i>, <i>Paenibacillus</i>, <i>Pantoea</i>), including novel strains and species. However, key canonical genes, such as <i>pqq</i> operon and <i>gcd</i>, did not reliably predict phenotype, highlighting the limitations of <i>in silico</i> predictions. Mutagenesis of the high-efficiency isolate <i>Pantoea rara</i> Lu_Sq_004 generated mutants with enhanced and null solubilization phenotypes, revealing the potential role of "auxiliary" genes in downstream function of solubilization pathways. Inoculation studies with lucerne seedlings demonstrated a significant increase in shoot length (<i>p</i> < 0.05) following treatment with the enhanced-solubilization mutant, indicating a promising plant growth-promotion effect. These findings highlight the potential of more personalized "system-appropriate" biofertilizers and underscore the importance of integrating genomic, phenotypic, and <i>in planta</i> analyses to validate function. Further research is required to investigate links between genomic markers and functional outcomes to optimize the development of sustainable agricultural inputs.