Epsilon Open Archive

Abstract

The non-infecting, plant-associated bacteria have attracted increased attention for stimulating plant growth and as environmentally friendly plant protection agents. However, further elucidation of these characters and of the modes of action involved is dependent on availability of suitable isolation, culturing and screening procedures. In this thesis, a Pantoea agglomerans strain, selected for its induction of cytokinin-like effects in test plants and its stimulation of plant growth, was found to secret different cytokinins in its culture filtrate as demonstrated by IAC, HPLC and GC-MS. In addition to the known cytokinins, isopenenyladenine, isopentenyladenosine and their 2-methylthiol derivates, it was shown tro also produce the cytokinin, 5'-Deoxyisopentenyladenosine, being isolated from a natural source for the first time. A different group of plant-associated bacteria belonging to the genus Methylobacterium was studied concerning their natural populations, which were followed for two growing seasons in red clover and winter wheat. The populations were higher in red clover than in wheat and changed regularly over the growing season. This pattern was clear and statistically validated in red clover, but was less obvious in winter wheat due to high variability in population sizes. Colonization studies conducted in the greenhouse with gfp-tagged mutatns revealed that phyllosphere colonization occurs by direct contact with the leaf surface. However, in some plants species, like red clover, phyllosphere colonization also occurred via seeds. In addition to their ability to colonize the phyllosphere, members of Methylobacterium were also able to colonize the rhizosphere of the tested plant species. Upon visualization of gfp-tagged bacterial mutants sprayed on the aerial part of red clover, bacterial cells were localized in the grooves between the leaf epidermal cells, which may give them protection and access to methanol. The bacterial cells colonized red clover phyllosphere by forming large aggregates, a feature which is known to increase survival of epiphytic bacteria in the phyllosphere. Several Methylobacterium spp. produce cytokinins, e.g.zeatin riboside, and we demonstrated that three out of 16 isolates were able to produce also indole-3-acetic acid in culture filtrates. Group specific PCR primers, designed as a first step for describing community structure of Methylobacterium spp., were tested for specificity in type strains of different bacterial species. One primer set (404f and 1123r), successfully amplified the 16SrRNA of Methylobacterium spp but not the other tested bacterial genera. T-RFLP profiles of these bacteria were fairly similar in the two tested plant species. However, more differences were observed in T-RFLP profiles of red clover environmental samples than from winter wheat samples. The samples were collected from different locations and at different times during the growing season.