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EC-sponsored Research on Safety of Genetically Modified Organisms - A Review of Results
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image Assessing the risks involved in the release of genetically manipulated micro-organisms

Background and objectives

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.
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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.
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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 population.

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.
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Conclusions

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.

 

Major publications

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 strains”.
FEMS Microbiology Ecology, 15, 1994, pp. 147-159.
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imageResearch project
 
Contract number
BAP-0024/0107/0108

Period
July 1986 – December 1989

 


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Partners


N. Amarger
INRA - Dijon (FR)

P. Hirsch
IACR-Rothamsted
Harpenden (UK)

W. Klingmüller
Universität Bayreuth (DE)

 
 
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