Browsing by Subject "Motility assay"

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    Interactions between non-symbiotic N2-fixing bacteria and plant roots in plant-microbial associations
    (2009) Calvo Alegre, Olga-Cristina; von Wirén, Nicolaus
    The development of biofertilizers on the basis of plant growth promoting rhizobacteria (PGPR) may be a promising approach to partially substitute costly and energy-consuming mineral fertilizers in agricultural plant production and to support agriculture in developing countries. A successful and competitive rhizosphere colonization of PGPR strains has been identified as a prerequisite for the expression of plant growth promoting effects. Apart from a wide range of external factors with an impact on the colonization process, such as soil properties, temperature, soil moisture and fertilization levels, in particular plant-microbial interactions may play an important role for the successful establishment of compatible associations. In this context, certain plant root exudates may act as signals to mediate bacterial responses with importance for root colonisation (e.g. motility and chemotaxis, production of extracellular polysaccharides). On the other hand, the induction of bacterial plant growth promotion may also depend on ability of the host plant to respond to the presence and the activity of the associated bacteria. It was therefore the aim of this thesis to investigate the contribution of putative PGPR to growth and N uptake in wheat plants and characterize the underlying mechanisms in root-bacterial associations. In the first part of this thesis, the contribution of various non-symbiotic diazotrophic rhizobacteria to plant growth promotion and N nutrition has been studied in a series of greenhouse pot inoculation experiments with wheat (Triticum aestivum L.). Different bacterial inoculants, plant genotypes, soil properties, water regimes and N fertilization levels have been varied as factors with potential impact on plant growth promotion by diazotrophs. The contribution of biological nitrogen fixation was assessed by the 15N dilution method. Plant growth and grain yield were influenced by the different N fertilization levels but no stimulation of growth or N uptake was note upon bacterial inoculation. These observations suggested a high degree of specificity or limiting factors, determining a successful plant-microbial association. The second part describes possible mechanisms that may be involved in the establishment of diazotrophs in the rhizosphere of suitable host plants. As an initial step of the colonization process, a targeted movement of the bacteria to the root surface is required and root exudates may act as attractants. Since dicarboxylic acids are known to exert chemotactic activity on diazotrophic bacteria, seed and root exudates of two graminaceous crops (Triticum aestivum L. and Zea mays L.) and for comparison also of a non-graminaceous plant species (Phaseolus vulgaris L.) were collected in hydroponic culture with and without N supply, and organic acid profiles in these root exudates were analysed. Bacterial motility assays were conducted with the major carboxylates detected in the root exudates of the selected plant species and compared to glucose and water, using Brevibacillus reuszeri as a model bacterium. Pure malate, which was found at high levels in root exudates of bean and wheat, and particularly malonate (bean) and t-aconitate (maize) stimulated the motility of Brevibacillus reuszeri as compared with glucose or water. A particularly intense promotion of bacterial motility was recorded in the presence of crude root exudates of wheat and maize plants grown under N limitation, which was not observed for root exudates of bean. However, this was not related with comparable changes of malate or t-aconitate concentrations in the root exudates. In wheat exudates, malate concentrations even decreased in response to N limitation. These findings suggest the presence of specific factors released in root exudates of N-deficient cereals, promoting the rhizosphere colonisation with B. reuszeri. For an identification of the respective factors, a more comprehensive profiling of the root exudates is necessary. In associations with diazotrophic bacteria, host plants are supplied with ammonium by the bacterial partner. This raised the question whether plant ammonium uptake systems have an impact on the efficiency of the association. To address this problem, an antisense approach was conducted with tomato, with characterised ammonium transporters (LeAMT1;1 and LeAMT1;2). The final goal was the inhibition of the ammonium transporters by production of LeAMT antisense lines to study their putative role in plant associations with diazotrophic bacteria. Northern blot analysis revealed a strong repression of LeAMT1;2 expression in three independent antisense lines associated with a lower ammonium uptake capacity under N-sufficient and N-deficient growth conditions. In contrast, LeAMT1,1 expression was only weakly repressed in antisense lines and there was no impact on N uptake. A faster decline of chlorophyll in older leaves indicates a physiological function of LeAMT1;1 and LeAMT1;2 in ammonium uptake and retrieval in shoot and root cells. The absence of consistent effects on N acquisition of the investigated antisense lines limited the suitability of this approach for studies on associations with diazotrophic bacteria.