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 the identification of bacteria that stimulate plant growth in the cold. 16S rRNA 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 its effects were less severe in the rhizosphere and absent in the bulk soil. Several cold-enriched families were found, of which nine, the Oxalobacteraceae, Pseudomonadaceae, Flavobacteriaceae, Microscillaceae, Spingobacteriaceae, Comamonadaceae, Devosiaceae, Methylophilaceae and env.OPS_17, in both the rhizosphere and the root endosphere. 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.