of Chinese biodiversity resources in sustainable crop production, using
a biotechnological approach for rhizobial diversity evaluation, strain improvement
and risk assessment
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.
picture shows the effect of inoculation in the field experiment
with Astragalus sinicus
in Hubei. Courtesy Dr.
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.
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.
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.
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.
September 1996 August 1999
University of Helsinki (FI)
Huazhong Agricultural University
Soils and Fertilizers Institute
C. Wenxin, S-S. Yang
China Agricultural University
Sichuan Agricultural University
University of York (UK)