Digging into the Lettuce Cold-Specific Root Microbiome in Search of Chilling Stress Tolerance-Conferring Plant-Growth-Promoting Bacteria

Growth of lettuce ( Lactuca sativa) is severely hampered by low temperatures, even when cultivated under greenhouse conditions. Root-associated bacteria might promote plant growth under stressful conditions. Therefore, we analyzed the effect of low temperatures on the lettuce root-associated microbiome to evaluate whether microbiome-based selection aids in identiying bacteria that stimulate plant growth in the cold. 16S ribosomal RNA gene amplicon sequencing was used to examine the compositional differences in the lettuce root-associated microbiome when grown under low- and control-temperature conditions. Chilling temperatures significantly altered the lettuce root endosphere composition, whereas their effects were less severe in the rhizosphere and absent in the bulk soil. Several cold-enriched families were found in both the rhizosphere and the root endosphere, nine of which were Oxalobacteraceae, Pseudomonadaceae, Flavobacteriaceae, Microscillaceae, Sphingobacteriaceae, Comamonadaceae, Devosiaceae, Methylophilaceae and env.OPS_17. Concurrently, a collection of lettuce root-colonizing bacteria was established and, based on correlation with these families, representative isolates were screened. None of the lettuce root isolates showed growth-promoting effects but three growth-promoting Flavobacterium strains from an available collection of grass root-colonizing bacteria were identified. Amplicon sequence variant (ASV) annotation of the lettuce and grass strains revealed that strains matching cold-enriched or highly abundant ASVs in at least one soil promoted growth in the cold. Overall, our data demonstrate that microbiome analyses, combined with high-throughput bacterial isolations might be a helpful tool to isolate effective cold growth-promoting strains.