and modelling studies on the fate in soil of introduced biologically-contained
Successful and effective biological control of plant pathogens often requires
that genetically modified micro-organisms (GMMs) be introduced into the
environment (i.e. soil). This practice carries with it several biosafety
issues, and the implications of these for both the environment and the
community need to be considered. The potential bacterial biocontrol agent
Pseudomonas fluorescens, carrying genes producing anti-Tipula
proteins, was studied as a GMM model organism in this project. To achieve
optimal effectiveness and to minimise risks of the application, the fate
of the GMM in the target ecosystem, i.e. the soil, needs to be understood
and, if possible, managed. The research required in this area was performed
using enclosed environments, such as soil microcosms, and preceded any
large-scale field trials.
The objectives of this project were firstly to develop strategies for
the optimal tracking of GMMs introduced into the soil. The second aim
was to study the ecology of introduced genetically modified organisms
(GMOs) in the soil. Thirdly, the project aimed to design strategies for
active or passive containment. The final objective was to develop a predictive
model for the fate of GMOs on the basis of realistic soil parameters.
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Approach and methodology
Pseudomonas fluorescens was used as a model organism for the insertion
of insect biocontrol (anti-Tipula) genes. It then served as a gene
carrier vehicle for the delivery of the beneficial genes into the rhizosphere
(the environment surrounding the plant root) of gramineous plants. The
organism was also modified with promoter probe insertions in order to
pinpoint genes responsive to the rhizosphere and soil environments, with
the aim to use these signals for biological containment. In addition,
information was gathered with other non-differentiating bacteria in order
to pinpoint genes or triggers in their starvation stress response programmes,
which could potentially be useful for biological containment. Finally,
a mathematical model predicting inoculant fate in soil, based upon realistic
soil parameters, was developed.
Click to view graphic (pdf)
Main findings and outcome
Tracking methods, including selective plating, immunofluorescence and
polymerase chain reaction (PCR)-assisted detection using specific primers
and probes for the GMMs under study, were developed and successfully applied,
to monitor the Pseudomonas fluorescens gene delivery vehicles,
and the 'foreign' DNA, in soil microcosms. Persistence of the gene in
the absence of detectable surviving cells was found, which indicated the
importance of further monitoring of the inserted gene in addition to that
of the cells. Promoter probe insertions identified gene expression which
is regulated by environmental triggers, i.e. by the rhizosphere (from
root exudation) versus the bulk soil (carbon starvation). The rhizosphere
responsive operons were found to react to the presence of proline which
was abundantly present in the exudates of three gramineous plants. Several
starvation-induced genes were further identified and their use in passive
containment systems was indicated, whereas active containment using two
selected host killing genes was not successful. The starvation response
in non-differentiating bacteria was unravelled using two-dimensional gel
electrophoresis as well as stress survival testing.
and in situ studies revealed that Pseudomonas fluorescens
carrier bacteria quickly developed generalised cellular stress resistance
upon starvation. This occurred via the programmed induction of a myriad
of stress responsive genes. Testing in soil showed that introduced Pseudomonas
species variants quickly responded to soil oligotrophy (nutrient scarcity,
mainly organic carbon) by producing stress resistant cells. A mathematical
model was developed that adequately described the dynamics of introduced
Pseudomonas fluorescens populations in the soil.
This project uncovered important results concerning potential biosafety
hazards to both the community and the environment relating to the safety
of the use of GMOs in crops. The study showed that the Pseudomonas-based
potential biocontrol agents persisted for a limited time in the soil and
wheat rhizospheres, however associated heterologous DNA persisted for
a more extended period of time.
Environmentally-responsive genes were detected in the Pseudomonas-based
biocontrol organisms. These are useful for environmentally-regulated expression
of beneficial genes as well as biological containment. The model biocontrol
agents responded to carbon starvation in soil by forming cells with enhanced
stress resistance. This study also showed the potential for developing
a mathematical model which can adequately describe the fate of inoculant
bacteria in soil.
Givskov M., Eberl L., Møller S., Poulsen L.K. and Molin S.,
Responses to nutrient starvation in Pseudomonas putida
strain KT2442: Analysis of general cross-protection, cell morphology,
and macromolecular content.
J. Bacteriol., 176, 1994, pp. 7-14.
Molin S., Kjelleberg S., Release of engineered micro-organisms:
biological containment and improved predictability for risk assessment.
AMBIO, 22, 1993, pp. 242-245.
Van der Hoeven N., Van Elsas J.D., Heijnen C.E., A model based
on soil structural aspects describing the fate of genetically modified
bacteria in soil.
Ecol. Modelling, 89, 1996, pp. 161-173.
Van Overbeek L.S., Eberl L., Givskov M., Molin S, Van Elsas J.D.,
Surival of, and induced stress resistance in, carbon-starved
Pseudomonas fluorescens cells residing in soil.
Appl Environ Microbiol, 61, 1995, pp. 4202-4208.
Van Overbeek L.S., Van Veen J.A., Van Elsas J.D., Induced
reporter gene activity, enhanced stress resistance, and competitive
ability of a genetically modified Pseudomonas fluorescens
strain released into a field plot planted with wheat.
Appl Environ Microbiol, 63, 1997, pp. 1965-1973.
October 1991 - April 1994
J.D. van Elsas
Plant Research International (formerly IPO-DLO)
Technical University of Denmark
University of New South Wales
N. van der Hoeven
TNO Delft (NL)