Detection of Burkholderia in the seeds of Psychotria punctata (Rubiaceae) - Microscopic evidence for vertical transmission in the leaf nodule symbiosis

Background and aims The bacterial leaf nodule symbiosis is a close interaction between endophytes and their plant hosts, mainly within the coffee family. The interaction between Rubiaceae species and Burkholderia bacteria is unique due to its obligate nature, high specificity, and predominantly vertical transmission of the endophytes to the next generation of host plants. This vertical transmission is intriguing since it is the basis for the uniqueness of the symbiosis. However, unequivocal evidence of the location of the endophytes in the seeds is lacking. The aim of this paper is therefore to demonstrate the presence of the host specific endophyte in the seeds of Psychotria punctata and confirm its precise location. In addition, the suggested location of the endophyte in other parts of the host plant is investigated. Methods To identify and locate the endophyte in Psychotria punctata, a two-level approach was adopted using both a molecular screening method and fluorescent in situ hybridisation microscopy. Key results The endophytes, molecularly identified as Candidatus Burkholderia kirkii, were detected in the leaves, vegetative and flower buds, anthers, gynoecium, embryos, and young twigs. In addition, they were in situ localised in leaves, flowers and shoot apical meristems, and, for the first time, in between the cotyledons of the embryos. Conclusions Both independent techniques detected the host specific endophyte in close proximity to the shoot apical meristem of the embryo, which confirms for the first time the exact location of the endophytes in the seeds. This study provides reliable proof that the endophytes are maintained throughout the growth and development of the host plant and are transmitted vertically to the offspring.