Experimental Design What Can Be Achieved with Each System

Three different successive experimental setups were used at the FAL (Braunschweig) to study the survival and ecological fitness of tagged strains, S. meliloti L1 and L33 (Fig. 9.2). The first experimental setup consisted of soil columns in the greenhouse, the second were field lysimeters of the same size as the greenhouse soil columns and the third setup were field plots which were inoculated with the respective S. meliloti strains. Characteristics of the three setups are shown in Table 9.1. The experiments were started in three successive years (1993 to 1995).

ft, meliloti 2011

ft, meliloti 2011

1011 bp

1011 bp

Fig. 9.1. Genetic maps of the chromosomal recA regions in the S. meliloti (former name: Rhizo-bium meliloti) strains 2011 (wild-type), L33 and L1 and positions of primers, which allow to differentiate L1 and L33 by size of the PCR product. Reprinted with permission from: Dammann-Kalinowski T, Niemann S, Keller M et al. Appl Microbiol Biotechnol 1996; 45:509-512.

Table 9.1. Comparison of microcosms, lysimeters and field plots used to study the survival and ecological interactions of Sinorhizobium meliloti strains in soil

Parameter

Greenhouse soil columns

Field lysimeters

Field plots

Size

diameter: 30 cm depth: 65 cm

diameter: 30 cm depth: 65 cm

3 m x 3 m squares no depth limitation

soil horizons

reconstructed, equilibrated for 2 month

reconstructed, equilibrated for 1 year

natural horizons (plough layer 25 cm depth)

number of replicates for each treatment

3

4

5

total surface area inoculated with each strain

0.4 m2

0.6 m2

45 m2

total number of genetically engineered cells released

approx. 3 x 1011

approx. 1 x 1011

approx. 2 x 1013

inoculation technique

mixing of bacterial cell suspension and peat into the upper 4 cm of the soil

mixing of bacterial cell suspension into the upper 4 cm of soil in the laboratory and subsequent transfer onto field lysimeters

spraying of cell suspension onto the soil surface

Monitoring parameters

* survival of GMMs

* recombinant gene persistence

* nodulation efficiency

* GMMs colonization of different soil horizons

* effect on plant growth

* effect on organic carbon and nitrogen concentrations in soil

* microbial biomass

* quantification of selected culturable bacterial populations

* immediate metabolic response ("Biolog-method")

* survival of GMMs * survival of GMMs;

* nodulation efficiency * horizontal

* GMMs colonization transport of of different soil horizons GMMs

* vertical transport of * rood nodule GMMs occupancy in the

* effect on plant growth field

* effect on organic car- * nodulation bon and nitrogen con- efficiency centrations in soil * colonization of

* microbial biomass rhizospheres from

* quantification of selec- host plants and ted culturable bacterial weed populations * impact of GMMs and the microbial diversity found in plant rhizospheres * ingestion and transport of GMMs by soil invertebrates

Parameter

Greenhouse soil columns

Field lysimeters

Field plots

stability of ecological parameters

Low

Medium

High

maximum length of monitoring

1.5 years

2 years

> 3 years

* crevices in soil columns and rim effects

* limited amount of sampling dates

* limited amount of sampling dates

* refilling holes after auger insertions and its impact on soil structure

* large scale production of inoculants

* inoculation of field plots without aerial spread of GMMs

* spread into neighboring plots with host plants

Reference

Schwieger et al23

Schwieger et al24

Dresing et al25 Schwieger and Tebbe26

The major difference between soil columns in the greenhouse and field lysimeters were the environmental factors acting on both systems. In the greenhouse, soil columns were wetted to allow plant growth. However, in order to study active movement of the GMM strains, columns were not saturated with water and, thus, no flow-through water could be collected. In order to protect the columns from frost damage, the minimum temperature in the greenhouse was not permitted to be below 4°C. Moreover, the maximum temperature was not above 30°C. In contrast, field lysimeters were exposed to the natural conditions, including periods with temperatures below 0°C and above 30°C. Wind exposure in the field resulted in a rather quick drying of the soil in the lysimeters, even after heavy rain periods. On the other hand, large amounts of flow-through rain water could be collected; 42.5 l per lysimeter over a period of 18 months. The soil surface inoculated in the field plot investigation was approx. 100 times larger than that of the greenhouse columns or field lysimeters. This allowed the removal of more material for analyses during this investigation. Also, holes created by auger insertions in the plot experiment during sampling were not refilled, as in greenhouse or lysimeter studies, since vertical transport was not analyzed and such holes were not much different in size compared to those, naturally produced by mice in the field.

The selection of parameters monitored along with the assessment of the survival of the inoculated S. meliloti strains L1 and L33, respectively, depended on the characteristic specificities of each model system. Since the model systems were studied subsequently, we could optimize our monitoring methods and omit parameters that proved to be insensitive or remained unaffected. For instance, the immediate metabolic response of soil extracted microbial communities20,21 (community level physiological profiles, CLPP) did not detect any differences between the treatments (inoculation, noninoculated controls). Therefore, this parameter was not further included in the field monitoring. Transport by rain water could not be analyzed on the field plots, but on the other hand, the plots were ideal for study of the colonization of rhizospheres of host plants and weed plants growing between the host plants. Also, only from the field plots, a large variety of soil insects could be collected during the growing season. The gut contents of several of these insects were analyzed for the occurrence of marker gene tagged, bioluminescent cells.22

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