Bacterial heart rot of pineapple reported in Hawaii in 2003 and reoccurring in 2006 was caused by an undetermined species of Dickeya. Classification of the bacterial strains isolated from infected pineapple to one of the recognized Dickeya species and their phylogenetic relationships with Dickeya were determined by a multilocus sequence analysis (MLSA), based on the partial gene sequences of dnaA, dnaJ, dnaX, gyrB and recN. Individual and concatenated gene phylogenies revealed that the strains form a clade with reference Dickeya sp. isolated from pineapple in Malaysia and are closely related to D. zeae; however, previous DNA-DNA reassociation values suggest that these strains do not meet the genomic threshold for consideration in D. zeae, and require further taxonomic analysis. An analysis of the markers used in this MLSA determined that recN was the best overall marker for resolution of species within Dickeya. Differential intraspecies resolution was observed with the other markers, suggesting that marker selection is important for defining relationships within a clade. Phylogenies produced with gene sequences from the sequenced genomes of strains D. dadantii Ech586, D. dadantii Ech703 and D. zeae Ech1591 did not place the sequenced strains with members of other well-characterized members of their respective species. The average nucleotide identity (ANI) and tetranucleotide frequencies determined for the sequenced strains corroborated the results of the MLSA that D. dadantii Ech586 and D. dadantii Ech703 should be reclassified as Dickeya zeae Ech586 and Dickeya paradisiaca Ech703, respectively, whereas D. zeae Ech1591 should be reclassified as Dickeya chrysanthemi Ech1591.
Recently, species clustering within Dickeya zeae has been identified as complex, encompassing validly published names, including D. oryzae and D. parazeae, with some strains potentially delineating new species. In this study, genomes of strains isolated from a bacterial heart rot outbreak in pineapple (Ananas comosus var. comosus) on Oahu, Hawaii, along with two strains from pineapple in Malaysia, were sequenced. Orthologous average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values among the sequenced genomes ranged from 98.93-99.9% and 91.8-99.9%, respectively, supporting the classification of seven strains within the same species. Comparisons of ANI and dDDH values between these seven strains and type strains of D. zeae, D. parazeae, and D. oryzae ranged from 94.4-95.9% and 57.2-66.5%, respectively. These values fall below the proposed boundaries for new species designation, supporting the delineation of a novel species. Phylogenetic analyses, including 16S rRNA, gapA, multi-locus sequence analysis (MLSA) of 10 housekeeping genes, whole-genome, and pangenome analyses, were concordant and revealed a distinct monophyletic clade, separating these strains from other members of the D. zeae complex, with D. oryzae as the closest relative. Notably, a nitrogen fixation gene cluster comprising 28 genes, similar to the Klebsiella spp. nitrogenase gene cluster, was found in the genome of the seven pineapple strains. Based on polyphasic approaches, including ANI, dDDH, biochemical, physiological, and phylogenomic analyses, we propose the reclassification in a new species of the five pineapple strains from Hawaii A5391, A5410T, A5611, A6136, and A6137, together with the two pineapple strains from Malaysia CFBP 1272 and CFBP 1278, previously classified as D. zeae. We propose the name Dickeya ananae sp. nov. for this taxon, represented by the type strain A5410T (= ICMP 25020T = LMG 33197T).