Abstract Onion center rot is caused by at least four species of Pantoea ( P. ananatis, P. agglomerans, P. allii, and P. stewartii subsp. indologenes ). Critical onion pathogenicity determinants for P. ananatis were recently described but whether those determinants are common among other onion-pathogenic Pantoea species remains unknown. In this work, we report onion pathogenicity determinants in P. stewartii subsp. indologenes and P. allii . We identified two distinct secondary metabolite biosynthetic gene clusters in different strains of onion pathogenic P. stewartii subsp. indologenes . One cluster is similar to the previously described HiVir phosphonate biosynthetic cluster of P. ananatis and another is a novel putative phosphonate biosynthetic gene cluster, which we name “Halophos”. The Halophos gene cluster was also identified in P. allii strains. Both clusters are predicted to be phosphonate biosynthetic clusters based on the presence of a characteristic phosphenolpyruvate phosphomutase ( pepM ) gene. The deletion of pepM gene from either the P. stewartii subsp. indologenes HiVir or Halophos clusters caused loss of necrosis on onion leaves and red onion scales, and resulted in significantly lower bacterial populations compared to the corresponding wildtype and complemented strains. Seven ( halB - halH ) out of eleven genes ( halA - halK ) in the Halophos gene cluster are required for onion necrosis phenotypes. The onion non-pathogenic strain PNA15-2 gained the capacity to cause foliar necrosis on onion via exogenous expression of a minimal seven gene Halophos cluster ( halB -halH) . Furthermore, cell-free culture filtrates of PNA14-12 expressing the intact Halophos-gene cluster caused necrosis on onion leaves consistent with the presence of a secreted toxin. Together, these observations indicated that pepM genes in both phosphonate biosynthetic gene clusters (HiVir and Halophos) are important for Pantoea spp. onion pathogenicity and the biosynthetic product of the Halophos cluster causes necrosis on onion leaf tissue. Overall, this is the first report of onion pathogenicity determinants in P. stewartii subsp. indologenes and P. allii . Author summary Onion center rot is caused by multiple Pantoea species including P. stewartii subsp. indologenes and P. allii . We identified two distinct secondary metabolite biosynthetic clusters associated with onion pathogenic strains, the validated HiVir phosphonate cluster and a putative phosphonate biosynthetic cluster that we named as Halophos based on the associated “halo” phenotype on the red onion scales. We found that pepM genes from each cluster (HiVir and Halophos) are required for onion infection by P. stewartii subsp. indologenes and P. allii but not for millet infection by P. stewartii subsp. indologenes . Conversely, the T3SS was important for millet infection by P. stewartii subsp. indologenes but not onion infection. Induction of the intact Halophos cluster was associated with the accumulation of a necrosis-inducing factor in culture, which suggests it might be a secreted phytotoxin. Seven of the eleven Halophos cluster genes are required for onion necrosis phenotypes and expression of this minimal cluster conferred a limited onion necrosis phenotype to an onion-non-pathogenic Pantoea strain. We provide evidence of a Halophos biosynthetic gene cluster to be associated with onion pathogenicity in strains of P. stewartii subsp. indologenes and P. allii .

A novel biosynthetic gene cluster across the Pantoea species complex is important for pathogenicity in onion