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EC-sponsored Research on Safety of Genetically Modified Organisms - A Review of Results
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image Exploitation of Chinese biodiversity resources in sustainable crop production, using a biotechnological approach for rhizobial diversity evaluation, strain improvement and risk assessment

Background and objectives

Symbiotic nitrogen fixation, which occurs in the rhizome of many legumes, is of major importance for sustainable food production. China as a region offers one of the broadest spectra of rhizobial biodiversity in the world, but its natural resources are as yet underexplored and underexploited for sustainable production of agricultural products. By characterising Chinese rhizobial biodiversity resources, especially by using molecular methods for bacterial characterisation and identification, and testing strains for nitrogen fixation capacity in controlled and in field conditions, we aimed to create collections of rhizobial strains to be used as inoculants or to serve as gene pools for future inoculant development. At the same time, we developed methods for monitoring genetically modified inoculated bacteria in the field, such as marker genes and molecular fingerprints.

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The picture shows the effect of inoculation in the field experiment with Astragalus sinicus in Hubei. Courtesy Dr.
Ping Wang.

Approach and methodology

The marker genes lux and gusA were used to tag wild-type, efficient strains of Mesorhizobium huakuii, which nodulates Astragalus sinicus. The strains were first tested in greenhouse experiments for nitrogen fixation ability, competitiveness and persistence, and good, marked strains were used for inoculation in the field. The genomic fingerprinitng techniques, AFLP and rep-PCR, were applied to Mesorhizobium huakuii and Bradyrhizobium sp. (Arachis hypogaea), that nodulate peanut. Both methods gave fingerprints which could be used to distinguish single strains out of collections of several hundred isolates from different parts of China.

Main findings and outcome

Field experiments with Astragalus sinicus showed a range of effects from good to remarkable of inoculation on nodulation and nitrogen fixation. The results indicated that Rhizobium inoculation could greatly increase the plant biomass of Astragalus sinicus. Since there were few indigenous rhizobia able to nodulate A. sinicus, the nodule occupancy was very high and could reach 100% within 90 days. The four superior strains, 2524, 2689, 2746, and 2644, had good symbiotic performance, and exhibited 10% higher yields than the strain Tr2 in the Hubei, Shanxi, and Jilin experiments. Using the genetically marked strains, the nodule occupance could be followed with A. sinicus.

In order to monitor the inoculant strain in pot and field experiments, strain JS5A16 was successfully marked with the luxAB and gusA genes and the resulting strains were designated as JS5A16L and JS5A16G, respectively. The marking was achieved by introducing suicide plasmids pDB30 and pCAM111, which carry Tn5-luxAB and mini Tn5-gusA genes, respectively, into strain JS5A16 through conjugative transfer. The necessary detecting methods were established.

Luminescent colonies on plates were viewable in the dark and could be recorded by using either x-ray or colour camera film. Nodules formed by JS5A16L could be detected directly by a CCD camera, or by PCR amplification of the luxA gene. The gusA marked strain, JS5A16G, formed blue colonies on plates containing 50 mg/ml X-GlcA. In general, it was difficult to detect the luxAB marked Rhizobium strains in the small nodules. In that case, gusA marked strains gave better results. Both marker genes however give reliable detection of Rhizobium recovered from soil, rhizosphere and nodules.

Inoculation experiments in several Chinese provinces showed that there was a significant inoculant strain x plant cultivar interaction with peanut (Arachis hypogaea), which means that it is important to consider both partners when improving the use of biological nitrogen fixation by inoculation with efficient rhizobia.

Both the rep-PCR and the AFLP genomic fingerprinting methods could be considered as identification methods for following inoculant strains in peanut nodules. However, the AFLP method was more reproducible and will therefore be used in future field experiments by the partners.


Both approaches (marker genes and molecular fingerprinting) allowed monitoring of rhizobia in greenhouse and field experiments. The use of marker genes in controlled experiments like the ones described has a very low risk when considering biosafety aspects. Replacement of antibiotic resistance with other selectable markers when introducing the marker genes into the inoculant strains should, however, be considered. The molecular fingerprints serve as intrinsic markers and are thus safe from a GMO safety point of view. Both methodologies developed are suitable for monitoring genetically modified rhizobia released into the environment.


Major publications

Zhang X.-X., Guo X.-W., Terefework Z., Cao Y.-Z., Hu F.R., Lindström K. and Li F.-D., “Genetic diversity among rhizobial isolates from field-grown Astragalus sinicus of Southern China”.
Systematic and Applied Microbiology, 22, 1999, pp. 312-320.

Zhang X.P., Kaijalainen S., Nick G., Terefework Z., Paulin L. and Lindström K., “Biodiversity of Chinese peanut bradyrhizobia”.
Systematic and Applied Microbiology, 22, 1999, pp. 378-386.

Tas É. and Lindström K., “Detection of bacteria by their intrinsic markers”, in Jansson J.K., van Elsas J.D. and Bailey M. (eds.), Tracking Genetically Engineered Micro-organisms: Method Development from Microcosms to the Field, R.G. Landes Company, Austin, Texas, USA, 2000, pp. 53-68.
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imageResearch project

Contract number

September 1996 – August 1999

K. Lindström
University of Helsinki (FI)

Project website address



F-D. Li
Huazhong Agricultural University
Wuhan (CN)

G. Cheng
Soils and Fertilizers Institute
Beijing (CN)

C. Wenxin, S-S. Yang
China Agricultural University
Beijing (CN)

X. Zhang
Sichuan Agricultural University
Yaan (CN)

P. Young
University of York (UK)

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