Taxonomic of Pantoea associated with bacterial blight of Eucalyptus

Abstract

The genus Pantoea has seven species and two sub-species, isolated from diverse geographical and ecological sources. The majority of Pantoea species are plant-associated and cause a wide variety of diseases on a range of hosts. P. ananatis causes disease on many agricultural crops including onion, maize, sudangrass, honeydew melon and pineapple. P. ananatis has been identified as the causal agent of a serious bacterial blight and dieback of Eucalyptus in South Africa. Bacterial isolates have also been recovered from Eucalypts in South America and Uganda exhibiting Pantoea ananatis-like symptoms. These isolates have not been identified. Identification of P. ananatis, based on phenotypic analysis, is difficult due to similarities in phenotypic characteristics between Pantoea species and related Enterobacteriaceae. Regular isolations of P. ananatis have highlighted the need for a rapid, molecular-based identification technique for the pathogen. A PCR assay, based on amplification of a partial region of the 16S-23S ITS gene with species-specific primers, was evaluated for the rapid identification of P. ananatis. Authentic strains of P. ananatis were included in the study, along with the unidentified isolates from South America and Uganda, and authentic strains of all species of the genus Pantoea. All authentic strains of P. ananatis produced a single PCR product of 398 bp following amplification. Only one unidentified isolate from South America produced the 398 bp PCR product. The only other species to be detected by the primers was P. stewartii subsp. indologenes. For the 16S-23S ITS-PCR assay to successfully detect only P. ananatis, the species-specific primers will have to be modified to increase their specificity. Little is known concerning the genetic relatedness between species and strains of Pantoea, and no standardised molecular typing system exists for the genus. The entire 16S-23S ITS gene was evaluated for a genetic relatedness study for the genus Pantoea. Universal primers were used to amplify the entire spacer region. Multiple amplification products were visible for all Pantoea strains and the unidentified isolates. This indicated that the Pantoea genome contains multiple copies of the rRNA operon and a high degree of similarity exists among the rRNA operons of species of the genus Pantoea. Therefore it is not possible to determine the genetic relatedness of Pantoea species based on a typing technique targeting the 16S-23S ITS region. The entire genome was then screened by AFLP analysis to examine the genetic relatedness of the genus Pantoea. The AFLP technique was found to be successful and distinct clusters were visible for each Pantoea species in the dendrogram. The majority of the South American and Ugandan isolates formed three separate clusters from P. ananatis. Representative strains were chosen from among the unidentified isolates for 16S rRNA sequencing. Based on the resulting phylogram, it is clear that at least two new Pantoea species or subspecies exist among the South American and Ugandan isolates, and more than one Pantoea species may be associated with bacterial blight and dieback of Eucalyptus. DNA-DNA hybridisations will be performed on these isolates to determine their correct taxonomic position within the genus Pantoea.