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Risk Assessment
Record information and status
Record ID
11963
Status
Published
Date of creation
2006-03-27 09:06 UTC (mette.svejgaard@naturvardsverket.se)
Date of last update
2019-02-15 10:27 UTC (Melanie.Josefsson@naturvardsverket.se)
Date of publication
2019-02-15 10:28 UTC (Melanie.Josefsson@naturvardsverket.se)

General Information
Country
  • Sweden
Title of risk assessment
Non-resident microorganisms used for biocontrol of fungal pathogens
Date of the risk assessment
2005-04-05
Competent National Authority(ies) responsible for the risk assessment
Swedish Chemicals Agency
Box 2
Sundbyberg
Sweden, SE 172 13
Phone:+46 8 519 41100
Fax:+46 8 735 7698
Email:kemi@kemi.se
Url:Swedish Chemicals Agency
Contact details of the main responsible risk assessor
Ph.D Mette Svejgaard
Swedish Ministry of the Environment
Stockholm
Sweden
Phone:+46 (0)70 374 2604
Email:mette.svejgaard@regeringskansliet.se
Risk assessment details
Living modified organism
Pseudomonas fluorescens strain SBW25 modified for biocontrol of fungal pathogens
Selectable marker genes and reporter genes
Scope of the risk assessment
Risk assessment report / Summary
Methodology and points to consider
Potential adverse effects identified in the risk assessment
The effects on non-target microorganisms in this project will most probably be transient. One aim of this field trial is to investigate the possible impacts on non-target fungi and bacteria. SBW25:tgl will interact with other microorganisms in the environment. One of the aims of
this study is to evaluate the impact of the inoculum on the ecologically important bacteria
and fungi. The possible effects are most likely to be transient but it still could affect the soil
environment and thus the growing plants.
Likelihood that the potential adverse effects will be realized
The effects on non-target
microorganisms in this project will most probably be transient. One aim of this field trial is to
investigate the possible impacts on non-target fungi and bacteria. None of these possible
effects is thought to be irreversible. No difference between the SBW25:tgl and the wild-type
organism is expected.
A few bacterial cells may spread by wind and water from the release site. Rain
splashes could carry the organism short distances (dm) from leaves and maybe from
the soil. SBW25:tgl could disperse through surface contact with colonized plants.
Studies on sugar beet demonstrate that flying insects can be contamined with GMMs
after landing on colonized leaves. Thus any organism (birds, mammals etc) could act
as a vector of the GMM (or any other microorganism).
It is however unlikely that these ways will result in large, active populations of
SBW25:tgl.
Possible consequences:
None of the inserted genes produce substances that
are harmful to humans, animals or plants. The wild-type bacterium is not pathogenic to
humans, animals or plants.
A few bacterial cells may spread by wind and water from the release site. It is however
unlikely that this will result in established, active populations of SBW25:tgl.
Estimation of the overall risk
Effects will be transient: No effects that are not reversible
Recommendation(s)
Management strategy - Immediately after sowing the plots will be covered with a plastic cover to protect the
seeds from birds and to retain the moisture after the sowing by hand. The plastic will
be removed as soon as germinating has begun. The plastic will be incinerated. A fence
will protect the plots from large herbivores and from intrusion by unauthorized
individuals. A bird net will protect the seeds from birds.
Boots etc will be dipped for decontamination in a 1% solution of hypochlorite after
leaving the release site as part of the normal experimental practice, to prevent unnatural
spread to the surrounding environment. Excess sample material dilution series, agar
plates, wash water, enrichment broth etc. will be autoclaved.
Receiving environment(s) considered
The release will take place in the municipality of Uppsala, Sweden. The release site of the GMMs are 3 squaremeters.
The fauna of the sites is typical of agricultural land. The surrounding habitats include
mixed agriculture (meadows, arable crops and woodland). A fence will protect the
plots from large herbivores. Birds and insects. A bird net will protect the seeds from
birds.
LMO detection and identification methods proposed
(a) Techniques used to detect the GMO in the environment
Selective plating (resistance to potassium tellurite): The resistance to potassium
tellurite gives a way of quantifying the amount of SBW25:tgl cells by plating on agar
plates containing potassium tellurite. TelR-positive bacterial colonies appears as black
colonies due to the conversion of TeO2(-2)  to metallic tellurium. Resistance to tellurite
has been developed as a way of avoiding antibiotic resistance genes in environmental
field trials.
Flow cytometry (green fluorescent protein): allows fluorescent cells to be easily
detected in complex environmental samples. Flow cytometry has earlier been used to
monitor SBW25:gfp/lux in soil samples.
Fluorescence stereomicroscopy (green fluorescent protein): is used to visualize
fluorescent bacteria by eye under blue-light illumination as micro-colonies on plant
material and on agar plates.
Epifluorescence microscopy (green fluorescent protein: is used to study gfp-tagged
cells with a magnification up to 1000 X.
Confocal scanning laser microscopy (green fluorescent protein): is an imaging
technique based on fluorescence that provides greater resolution than standard
fluorescence microscopy. CSLM scans one focus layer at a time and if these layers are
saved, software can be used to create three-dimensional images of the plant tissues thus
displaying the gfp-tagged bacteria inside or on the surface of plants.
Luminometry (bioluminescence): is used to measure luciferase activity from
metabolically active cells. The phenotype of the expressed luxAB genes, encoding
luciferase, is light production, bioluminescence. Bioluminescence, which requires ndecanal
as a substrate, is quantified in a luminometer. No bioluminescence is produced
without addition of n-decanal. Detectors measure the emitted light and light intensity is
proportional to the number of cells in the sample and their metabolic activity.

(b) Techniques used to identify the GMO
PCR amplification: of introduced genes or by using 16S specific primers for SBW25.
Fluorescence stereomicroscopy (green fluorescent protein): due to the specificity of the
gfp gene this method can also be used for identification.
Epifluorescence microscopy (green fluorescent protein: is used to study gfp-tagged
cells with a magnification up to 1000 X.
Flow cytometry (green fluorescent protein): due to the specificity of the gfp gene this
method can also be used for identification.
Confocal scanning laser microscopy (green fluorescent protein): is an imaging
technique based on fluorescence that provides greater resolution than standard
fluorescence microscopy. CSLM scans one focus layer at a time and if these layers are
saved, software can be used to create three-dimensional images of the plant tissues thus
displaying the gfp-tagged bacteria inside or on the surface of plants.
Luminometry (bioluminescence): due to the specificity of the luxAB genes this method
can also be used for identification.
Additional Information
Additional Information
The organism Pseudomonas fluorescens is a natural member of the rhizosphere bacterial communities on
wheat and other plants. The strain SBW25 was originally isolated from sugar beet in the UK.
SBW25:tgl is expected to behave identically to the wild type SBW25 on field grown plants.
The marker genes give no selective advantages to the GMM. SBW25 is sensitive to cold and
will probably not survive well in the Swedish winter. The effects on non-target
microorganisms in this project will most probably be transient. One aim of this field trial is to
investigate the possible impacts on non-target fungi and bacteria. None of these possible
effects is thought to be irreversible. No difference between the SBW25:tgl and the wild-type
organism is expected.
The bacterium will be used as a plant growth-promoting strain on wheat by seed treatment.
The telAB/kilA/gfp/luxAB genes have been inserted into the chromosome of P. fluorescens
SBW25:tgl for monitoring purposes. This combination of marker genes gives an ideal
combination for observing released bacteria in field trials. SBW25 is an efficient rhizosphere
colonizer, which is a necessary trait for a biocontrol agent. Pseudomonas spp. are not known
to produce long term survival structures. The bacterium SBW25 has been extensively studied
in laboratory, greenhouse and field trials. None of the inserted genes produce substances that
are harmful to humans, animals or plants. The wild-type bacterium is not pathogenic to
humans, animals or plants.
A few bacterial cells may spread by wind and water from the release site. It is however
unlikely that this will result in established, active populations of SBW25:tgl.

Research monitoring
The bacterium will be used as a plant growth-promoting strain on wheat by seed treatment.
The telAB/kilA/gfp/luxAB genes have been inserted into the chromosome of P. fluorescens
SBW25:tgl for monitoring purposes.

The whole risk assessment is available in the PDF document attached hereby.
Other relevant website address or attached documents