Technical Abstract: Contaminated fresh produce has become the number one vector of non-typhoidal salmonellosis to humans. However, Salmonella enterica genes essential for the life cycle of this organism outside the mammalian host are for the most part unknown. Screening deletion mutants led to the discovery that an aroA mutant had a significant alfalfa seedling colonization defect due to a failure to replicate. AroA is part of the chorismic acid biosynthesis pathway, a central metabolic node involved in aromatic amino acid and siderophore production. Addition of tryptophan or phenylalanine to alfalfa root exudates did not restore the aroA mutant replication. However, addition of ferrous sulfate restored in planta growth of the aroA mutant, as well as alfalfa colonization. Tryptophan and phenylalanine auxotrophs had minor plant colonization defects suggesting that sub-optimal tryptophan and phenylalanine concentrations in root exudates were not major limiting factors for Salmonella replication. An entB mutant defective in siderophore biosynthesis had similar colonization and growth defects as the aroA mutant and the defective phenotype was complemented with addition of ferrous sulfate. Biosynthetic genes of each Salmonella siderophore, enterobactin and salmochelin, were up-regulated in alfalfa root exudates; yet, only enterobactin is sufficient for plant survival and persistence. Similar results in lettuce, indicate that siderophore biosynthesis is a widespread or perhaps universal plant colonization fitness factor for Salmonella, similar to other plant-associated bacteria.