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Plants are increasingly considered as secondary reservoirs for commensal and pathogenicEscherichia colistrains, but the ecological and functional factors involved in this association are not clear. To address this question, we undertook a comparative approach combining phenotypic and phylogenetic analyses ofE. coliisolates from crops and mammalian hosts. Phenotypic profiling revealed significant differences according to the source of isolation. Notably, isolates from plants displayed higher biofilm and extracellular matrix production and higher frequency of utilization of sucrose and the aromatic compoundp-hydroxyphenylacetic acid. However, when compared with mammalian-associated strains, they reached lower growth yields on many C-sources commonly used byE. coli. Strikingly, we observed a strong association between phenotypes andE. coliphylogenetic groups. Strains belonging to phylogroup B1 were more likely to harbour traits indicative of a higher ability to colonize plants, whereas phylogroup A and B2 isolates displayed phenotypes linked to an animal-associated lifestyle. This work provides clear indications thatE. coliphylogroups are specifically affected by niche-specific selective pressures, and provides an explanation on whyE. colipopulation structures vary in natural environments, implying that different lineages inE. colihave substantially different transmission ecology.