The genus Herbaspirillum comprises 13 species, the majority of which are plant colonizers. However, some species are occasionally isolated from environmental sources, including water and polluted soil, while others are opportunistic human pathogens. Four novel bacterial strains were isolated from diseased foliage of tomato and Boston fern in Florida, USA. Phylogenetic analysis based on the 16S rRNA gene sequence placed all strains into the genus Herbaspirillum. The Gram-negative strains produced opaque, creamy white, mucoid colonies, which is typical of the genus Herbaspirillum. Biolog biochemical profiling also identified those strains as members of Herbaspirillum. The strains were subjected to whole-genome sequencing, and their genomes were compared with those of reference strains of Herbaspirillum spp. using average nucleotide identity (ANI). The two strains isolated from Boston fern shared 99% pairwise ANI, as did the two strains isolated from tomato. Among all reference genomes tested, the novel strains shared the highest ANI to Herbaspirillum huttiense subsp. huttiense (G21-1742 and NC 40101, 96.76%; SE1, 97.23%; F1, 97.16%) and to H. huttiense subsp. putei. These values are above the established 95% threshold for species delineation based on ANI. As the ANI between members of the two currently described subspecies of H. huttiense, i.e. huttiense and putei, is also ~97%, it can be inferred that the two groups of novel strains described in this study should be considered as candidates for classification as two new subspecies of H. huttiense, given that the current H. huttiense subspecies also have ~97% with the fern and tomato strains. In silico DNA–DNA hybridization results were consistent with those of ANI; comparison of G21-1742 and NC 40101 with H. huttiense subsp. putei IAM 15032Tand H. huttiense subsp. huttiense LMG 2199T produced DNA–DNA hybridization (DDH) values of 66.1 and 73.6 %, respectively. Similarly, SE1 and F1 had DDH values of 68.9 and 68.8% with H. huttiense subsp. putei IAM 15032T and 77.1 and 76.7% with H. huttiense subsp. huttiense LMG 2199T, respectively. The genomes of all novel isolates carry genes involved in plant pathogenesis, including those of the type III secretion system, which are not present in other H. huttiense strains. Based on genomic and phenotypic data, we conclude that these strains represent the first phytopathogenic subspecies within H. huttiense and the names proposed are H. huttiense subsp. nephrolepidis for the two strains isolated from Nephrolepis exaltata (designated strain, G21−1742=LMG 33362=NCPPB 4765) and H. huttiense subsp. lycopersici (designated strain, SE1=LMG 3361=NCPPB 4764) for the two strains isolated from Solanum lycopersicum.
