the risks involved in the release of genetically manipulated micro-organisms
Rhizobia are common soil bacteria that form nitrogen-fixing root nodules
in symbiotic association with compatible host legumes. They have been
used as legume inoculants for a century, and as such, have been and are
still released in agricultural soils throughout the world. Since they
are also the most intensely studied and the best known soil micro-organisms,
we have chosen them as model bacteria to investigate inoculant survival
and gene transfer between introduced and native bacteria in the field
environment. Improved strains of agriculturally important bacteria can
be obtained through genetic manipulation, and implies their subsequent
release into the open environment. However, our knowledge of the fate
and dispersal of bacterial inoculants after field release is limited so
it is difficult to predict if a given organism can become established
and exchange genetic information with native soil bacteria. Circumstantial
evidence that genetic exchanges between strains of rhizobia occur in a
field environment has been provided by population studies. However, information
on the time scale and on the conditions in which these exchanges take
place, is still missing.
The project aimed at increasing our knowledge in this area by introducing
genetically marked bacteria into agricultural soils, and studying the
survival of these bacteria and the transfer of marker genes to native
strains in a field environment.
Approach and methodology
Genetically marked derivatives of Rhizobium leguminosarum bv. viciae,
the microsymbiont of pea, that harbour symbiotic genes on a conjugative
plasmid (pSym) were released in fields in the three collaborative countries.
The strains were marked with genes conferring antibiotic resistance on
the chromosome by selecting naturally-occurring chromosomal mutations,
and on the pSym by insertion of transposon Tn5. By permitting direct
isolation and enumeration of the released bacteria from soils, these markers
allowed the assessment of the survival of the inoculant bacteria after
their soil release. The transfer of pSym from the released rhizobia to
indigenous strains was investigated by screening pea nodule isolates for
the acquisition of the antibiotic resistances borne by Tn5. Gene
transfer from rhizobial inoculants and from a nitrogen-fixing bacterium
suitable for release (Enterobacter agglomerans) to a range of soil
bacterial genera, was studied in the laboratory, in soil and in agar media.
Click to view graphic
Main findings and outcome
Differences in the survival of the strain released in field soils of the
three countries were observed. At Rothamsted (UK) the strain became an
established part of the microflora while in Bayreuth (Germany) and in
Dijon (France) the numbers of viable cells dropped below the limit of
detection after ten and two months respectively. A second strain released
in Dijon became established one year later. Environmental parameters therefore
appear to play an important part in the establishment and behaviour of
a bacterial inoculant after its release.
At the Rothamsted and Dijon sites, where the native rhizobial population
is 104 to 105 cells g-1 soil, the survival
of the established inoculant strains has been followed since their release,
13 and 12 years ago respectively. These strains presently account for
one to ten percent of the native R. leguminosarum bv. viciae
In the two sites, the released rhizobia could be established in the absence
of the host plant. Nevertheless, the host plant had a significant effect
in increasing the survival of the inoculant rhizobia when these rhizobia
show a strong selective advantage for nodulation. This increase was limited
to the period of plant vegetation.
Spread of inoculant from the site of application may be mediated by migration
along extending roots over short distances, over moderate distances by
water and more significantly by bulk soil movement due to mechanical cultivation.
Under laboratory conditions the inoculant strains could transfer the Tn5-marked
plasmid, at frequencies varying from 10-1 to the limit of detection
10-9, to other rhizobia on both agar media and in sterile soils.
However, soil transfer was only detected when each parent was present
at 106, a level seldom reached by rhizobial populations in
field soils. The narrow host-range plasmid present in E. agglomerans
could only be transferred to very closely related strains.
In the field there do not appear to be special factors that greatly increase
the frequency of genetic interactions above those observed in laboratory
conditions, since no transfer of Tn5 to the more than 104
rhizobia isolated from nodules was observed.
The use of genetically modified rhizobia in field experiments, facilitated
by the Tn5 marker, generated widespread interest and provided data on
factors affecting survival and spread of inoculants. No transfer of marker
genes was observed in the field, probably because levels reached by the
field population are too low to support detectable frequencies of transfer
during the time period that the experiments could monitor. The release
sites have proved useful for subsequent experiments on inoculant spread
and survival in the field.
Hirsch P.R. and Spokes J.R., Rhizobium leguminosarum
as a model for investigating gene transfer in soil, in Risk
assessment for deliberate releases: the possible impact of genetically
engineered micro-organisms on the environment, W. Klingmüller
(ed.), Springer-Verlag, Berlin, Heidelberg, 1988, pp. 10-17.
Amarger N., Hirsch P. and Klingmüller W., Assessing the
risk involved in the release of genetically manipulated micro-organisms,
in Biotechnology R. and D. in the E.C. Biotechnology Action Programme,
A. Vassarotti and E. Magnien (eds.), Elsevier, Amsterdam, Vol. II,
1990, pp. 411-415.
Amarger N., and Delgutte D., Monitoring genetically manipulated
Rhizobium leguminosarum bv. viciae released in the
field, in Biological Monitoring of Genetically Engineered
Plants and Microbes, D.R. MacKenzie and S.C. Henry (eds.), Agricultural
Research Institute, Bethesda, Maryland, USA, 1990, pp. 221-228.
Klingmüller W., Plasmid transfer in natural soil: a case
by case study with nitrogen-fixing Enterobacter.
FEMS Microbiology Ecology, 85, 1991, pp. 107-115.
Hirsch P.R. and Spokes J.D., Survival and dispersion of genetically
modified rhizobia in the field and genetic interactions with native
FEMS Microbiology Ecology, 15, 1994, pp. 147-159.
July 1986 December 1989
INRA - Dijon (FR)
Universität Bayreuth (DE)