Techniques and Consequences of GMM Soil Inoculations

In all three experimental setups it was intended to start the experiment with approx. 106 cells of the strain g-1 soil in the upper soil horizon (plough layer; 0 to 25 cm depth). In greenhouse experiments, batch culture grown cells were harvested, washed in potassium phosphate buffer (50 mM, pH 7.2), and mixed with sterile peat and soil taken from the upper 4 cm of the soil columns. This mixture was loaded onto the soil columns and alfalfa seeds were added. A similar technique was applied to inoculate the field lysimeters. However, we abstained from utilizing peat as the carrier, since laboratory studies did not show any beneficial effect of peat on the establishment of S. meliloti in the field soil. Also, to study the survival and vertical transport of inoculated cells we decided to exclude peat as a material, since peat would have provided additional nutrients and would have protected the inoculated cells against predation. Finally, vertical transport was a monitoring parameter in this study and adsorption of the inoculated cells to the peat would have prevented translocation of such cells into deeper soil horizons. Due to the size of the field plots that had to be inoculated, the surface soil could not be removed and inoculated in the laboratory. Instead, a spraying machine, normally used to apply pesticides onto experimental field plots, was utilized and bacterial suspensions were directly sprayed onto the soil surface in the field. In contrast to the other systems, aerial spread of the inoculant into neighboring field plots could not be excluded even though the spraying machine was modified by utilizing a rather low pressure (1 bar). Also, a specifically developed box only allowed bacterial cells to move downwards but not sidewards (see Fig. 9.2, right side). During the inoculation period the wind strength was low (0.5-3 ms-1).

The concentration of inoculated cells (106 cfu g-1 in the plough horizon) was one to three orders of magnitude above the upper limit of rhizobial populations found in well colonized soils.6,7 Compared to the total number of bacterial cells commonly found in soil, the inoculant was less than 1% of the population. With the three experimental settings used in this study (soil columns, field lysimeters, field plots), the titers of inoculated cells, as determined immediately after the beginning of each experiment varied by one order of magnitude (Table 9.1). However, as shown by the survival of both strains, S. meliloti L1 and L33, the fate of the release strain was not dramatically influenced by the number of

Fig. 9.2. Three model systems used to study the survival and microbial ecology of bioluminescent S. meliloti strains (GMMs) in soil. Left, soil columns in the greenhouse, allowing to study bacterial colonization of different soil horizons; field lysimeters (middle) of the same design, and field plot inoculation (right).

Fig. 9.2. Three model systems used to study the survival and microbial ecology of bioluminescent S. meliloti strains (GMMs) in soil. Left, soil columns in the greenhouse, allowing to study bacterial colonization of different soil horizons; field lysimeters (middle) of the same design, and field plot inoculation (right).

cells initially introduced into each model system. Due to the relatively low amount of microbial cells added, it was not surprising to see that general parameters, like microbial biomass, organic carbon or total nitrogen (with variances of at least ± 10%), did not respond to this treatment at all. Other parameters, like population sizes of culturable heterotrophic bacterial communities on cellulose, glucose or aromatic compounds, also did not respond to the inoculation procedure. Thus, these parameters, which were suspected to be indicative for nonintended, dramatic changes within the soil microbial community, were only monitored during the first two stages of this investigation (soil columns and field lysimeters).

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