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Thesis objectivesThe main objective of this thesis was to study the diversity and evolution of bacterial leaf endophytes in African Rubiaceae. Endophytic bacteria were found in six rubiaceous species that cause gousiekte in South Africa: two species of the tribe Pavetteae have nodulated endophytes and four species of the Vanguerieae tribe have non-nodulated endophytes. This second type of endosymbiosis was not studied before. Firstly, a phylogenetic and taxonomic study of Vanguerieae had to be carried out before the evolution of bacterial leaf symbiosis could be studied. Secondly, the bacterial endophytes in the gousiekte-inducing species were identified. Thirdly, the diversity and evolution of the endophytic bacteria in the tribe Vanguerieae was studied. Fourthly, the geographic distribution of the plant-bacteria association in Africa was investigated. Finally, a study on Burkholderia caledonica was carried out to test the genetic diversity within this species. Revision of Cuviera and Globulostylis (Vanguerieae)Generic circumscriptions within the tribe Vanguerieae (Rubiaceae) have been under discussion for a long time. Recent molecular studies, while providing new insights, have not yet solved all the problems. In this part the taxonomy and phylogeny of the Vanguerieae tribe are investigated, with a focus on the genus Cuviera s.l. A new and updated phylogenetic tree of the tribe is presented. On both molecular and morphological evidence, Cuviera is restricted to a group of ten West and Central African species. Globulostylis, previously included in Cuviera, is reinstated as a distinct genus, with eight species from Central Africa. Both genera are revised; the latter includes three new species (Globulostylis dewildeana, G. rammelooana, G. robbrechtiana) and two new combinations (G. leniochlamys and G. uncinula). The close relationship of both Cuviera s.s. and Globulostylis to Vangueriella is shown. Six aberrant species (most of them from EastAfrica) are excluded from Cuviera, but further work is needed before they can be confidently assigned to other genera. Endophytic bacteria in toxic South African plantsSouth African plant species of the genera Fadogia, Pavetta and Vangueria (all belonging to Rubiaceae) are known to cause gousiekte (literally ‘quick disease’), a fatal cardiotoxicosis of ruminants characterized by acute heart failure four to eight weeks after ingestion. Noteworthy is that all these species harbour endophytes in their leaves: nodulated bacteria in specializednodules in Pavetta and non-nodulated bacteria in the intercellular spaces between mesophyll cells in Fadogia and Vangueria. Isolation and analyses of these endophytes reveal the presence of Burkholderia bacteria in all plant species implicated in gousiekte. Although the nodulatedand non-nodulated bacteria belong to the same genus, they are phylogenetically not closely related and fall in different bacterial clades. P. harborii and P. schumanniana have their own specific endophyte – Candidatus B. harborii and Candidatus B. schumannianae – while the non-nodulatedbacteria found in the other gousiekte-inducing plants show high similarity to B. caledonica. In this group, the bacteria are host specific at population level. Investigation of gousiekte-inducing species from other African regions resulted in the discovery of the same endophytes. Several other plants of the genera Afrocanthium, Canthium, Keetia, Psydrax, Pygmaeothamnusand Pyrostria were studied and were found to lack bacterial endophytes. The discovery and identification of Burkholderia bacteria in gousiekte-inducing species open new perspectives and opportunities for research not only into the cause of this economically important disease, but also into the evolution and functional significance of bacterial endosymbiosis in Rubiaceae. The same bacteria are consistently found in gousiekte-inducing species from different regions indicating that these species will also be toxic to ruminants in other African countries, if the endophytes play a role in the disease.Endophytic Burkholderia in RubiaceaeSymbiotic ß-proteobacteria do not only occur in root nodules of legumes but are also found in leaves of certain Rubiaceae. The discovery of bacteria in plants formerly not implicated in endosymbiosis suggests a wider occurrence of plant-microbe interactions. Several ß-proteobacteria of the genus Burkholderia are detected in close association with tropical plants. This association is present in three separate phylogenetic clades, which suggests a recent and open plant-bacteria association. The presence or absence of Burkholderia endophytes is consistent at generic level and therefore implies a predictive value for the discovery of bacteria. Only a single Burkholderia species is found in association with a given plant species. However, the endophyte species are promiscuous and can be found in association with several plant species. Most of the endophytes are part of the plant-associated beneficial and environmental (PBE) group, but others are closely related to B. glathei. These soil bacteria, together with related nodulated and non-nodulated endophytes, are transferred to a newly defined and larger PBE group within the genus Burkholderia. Vanguerieae-Burkholderia association in Sub-Saharan AfricaIn Rubiaceae, certain species are known to be closely associated with endophytic leaf bacteria. These endophytes are either found in specialized leaf nodules or are freely distributed among the mesophyll cells. This second non-nodulated type of endosymbiosis was discovered in a few representatives of the Vanguerieae tribe of Rubiaceae and is especially known from South Africa. The identity of the endophytes was designated as Burkholderia, a genus known for its pathogens but also for its plant-associated representatives. For this part, our aim was to further document the Burkholderia diversity associated with Rubiaceae host plants and to establish whether the interaction is widespread in sub-Saharan Africa. Many representatives of the Vanguerieae tribe were investigated for the presence of endophytes. Special attention was paid to collect plants from different African regions in order to study the distribution range of the plant-bacteria association. The association is found in five different genera (Fadogia, Fadogiella, Globulostylis, Rytigynia, Vangueria) and is restricted to three clades. The endophytic bacteria belong to the genus Burkholderia and are part of the plant-associated beneficial and environmental (PBE) group. Some endophytes are very similar to B. caledonica, B. graminis, B. phenoliruptrix or B. phytofirmans, while others are classified in new OTUs that show no similarity with any previously named Burkholderia species. The association is not obligate for the bacterial partner and is considered a loose and recent interaction, which is demonstrated by the fact that the endophytescan be cultivated and that no coevolution occurs. The geographical distribution of the association is restricted by the distribution range of the host plants and comprises the whole of sub-Saharan Africa.Intraspecific variation in Burkholderia caledonicaThe best-known interaction between bacteria and plants is the legume-Rhizobium symbiosis, but other plant-bacteria interactions exist, such as between Rubiaceae and Burkholderia. It was demonstrated that a number of bacterial endophytes in Rubiaceae are closely related to the soil bacterium Burkholderia caledonica. This intriguing observation is here explored further by investigating B. caledonica isolates from different geographic regions and with different niches, namely free-living bacteria in soil and endophytic bacteria in host plants. By applying multilocus sequence analysis, we found that all these isolates belong to the species B. caledonica, but two genetically different groups are identified. Group A holds only European soil isolates and group B holds soil isolates from Africa, with the exception of one European soil isolate. This indicates a strong trend of biogeographic separation. Besides soil-dwelling bacteria, endophytic isolates of B.caledonica are also found in certain members of African Rubiaceae, but only in group B. These endophytes are closely related to the African soil isolates, which indicates a possible exchange of bacteria between soil and host plant.

582.972 --- 632 --- Academic collection --- Rubiaceae. Madder. Bedstraw (Lady's bedstraw etc.) --- Plant damage, injuries. Plant diseases. Pests, organisms injurious to plants. Plant protection --- Theses --- 632 Plant damage, injuries. Plant diseases. Pests, organisms injurious to plants. Plant protection --- 582.972 Rubiaceae. Madder. Bedstraw (Lady's bedstraw etc.)