Background and aimsThe 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.MethodsTo 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 resultsThe 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.ConclusionsBoth 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.

Background & aims The bacterial leaf nodule symbiosis is an interaction where bacteria are housed in specialised structures in the leaves of their plant host. In the Rubiaceae plant family, host plants interact with Burkholderia bacteria. This interaction might play a role in the host plant defence system. It is unique due to its high specificity; the vertical transmission of the endophyte to the next generation of the host plant; and its supposedly obligatory character. Although previous attempts have been made to investigate this obligatory character by developing Burkholderia -free plants, none have succeeded and nodulating plants were still produced. In order to investigate the obligatory character of this endosymbiosis, our aims were to develop Burkholderia- free Psychotria umbellata plants and to investigate the effect of the absence of the endophytes on the host in a controlled environment. Methods The Burkholderia- free plants were obtained via embryo culture, a plant cultivation technique. In order to analyse the endophyte-free status, we screened the plants morphologically, microscopically and molecularly over a period of three years. To characterise the phenotype and growth of the in vitro aposymbiotic plants, we compared the growth of the Burkholderia -free plants to the nodulating plants under the same in vitro conditions. Key results All the developed plants were Burkholderia -free and survived in a sterile in vitro environment. The growth analysis showed that plants without endophytes had a slower development. Conclusions Embryo culture is a cultivation technique with a high success rate for the development of Burkholderia -free plants of P . umbellata . The increased growth rate in vitro when the specific endophyte is present cannot be explained by possible benefits put forward in previous studies. This might indicate that the benefits of the endosymbiosis are not yet completely understood.

Organisms are increasingly facing multiple, potentially interacting stressors in natural populations. The ability of populations coping with combined stressors depends on their tolerance to individual stressors and how stressors interact, which may not be correctly captured in controlled laboratory settings. One largely unexplored reason for this is that the microbial communities in laboratory settings often differ from the natural environment, which could result in different stressor responses and interaction patterns. In this study, we investigated the impact of single and combined exposure to a toxic cyanobacterium and an oomycete-like parasite on the performance of three Daphnia magna genotypes. Daphnia individuals were first sterilized and then experimentally given a natural or a laboratory-derived microbial inoculum. Survival, reproduction and body size were monitored for three weeks and gut microbiomes were sampled and characterized at the end of the experiment. Our study confirmed that natural and laboratory microbial inocula and gut microbiomes are differently structured with natural microbiomes being more diverse than laboratory microbiomes. Our results showed that exposure to the stressors reduced D. magna performance compared to the controlD. magna not exposed to any stressor. An antagonistic interaction between the two biotic stressors was revealed with respect to D. magna survival when Daphnia individuals were exposed to the laboratory microbial inoculum. This effect was consistent across all three genotypes. In Daphnia exposed to a natural microbial inoculum this antagonistic interaction could not be detected and the genotype x exposure interaction was genotype-dependent. Our results indicate that host-stressor interactions depend on the microbial inoculum and that the gut microbiome potentially has a strong role in this, thereby providing a largely unexplored dimension to multiple-stressor research.