Page 2 Plant-Bacteria Interactions Edited by Iqbal Ahmad, John ...

170j 9 Rice–Rhizobia Association: Evolution of an Alternate Niche
soils rather than in monoculture within flooded soils, highlighting a long-term
benefit of rice–legume rotation in promoting this intimate plant–microbe
association.
Standard microbiological techniques were used to isolate into pure culture these
rhizobial nodule occupants representing the numerically dominant endophytes of
rice roots, verify that they were authentic rhizobia by testing their ability to nodulate
berseem clover in gnotobiotic culture and evaluate their nitrogen-fixing activities
on their natural clover host. These symbiotic performance tests (plus 16S rDNA
sequencing) confirmed that the rice-adapted isolates were authentic strains of R.
leguminosarum bv. trifolii capable of nodulating berseem clover under axenic conditions
and that both effective and ineffective rhizobial isolates were included in the
culture collection [1,7].
To fulfill Koch s postulates, these endophytic rhizobial strains were cultured on
roots of rice plants under microbiologically controlled conditions, and then reisolated
from surface-sterilized roots, 32 days after inoculation. Strain identification
tests using plasmid profiling and BOX-PCR genomic fingerprinting showed that
these reisolates were the same as the original inoculant strains, fulfilling Koch s
postulates and confirming that they can form intimate endophytic associations with
rice roots without requiring the assistance of other soil microorganisms [1]. Using
a similar experimental approach, the existence of endophytic, bean-nodulating
rhizobia in Indian cultivated rice was also verified [30].
We have used various molecular approaches such as 16S rDNA PCR-RFLP, BOX-
PCR, plasmid profiling and SDS-PAGE to reveal the genomic diversity of riceborne
rhizobial isolates. These studies helped to define the breadth of this ecological
niche for rhizobia and guided our selection of isolates that can represent the genomic
diversity in various studies of this association. It also indicated that our culture
collections of rice-adapted rhizobia contained different strain genotypes that vary in
their ability to evoke growth responses and that diverse populations of rhizobia
colonize rice root interiors in different agroecosystems of Egypt and India [7,31].
9.4
Association of Rhizobia with Other Cereals Like Wheat, Sorghum, Maize and Canola
Since this discovery of a third ecological niche for Rhizobium (Figure 9.2), we have
created an international network of collaborators to expand the intrinsic scientific
merit of this project in both basic and applied directions of beneficial plant–microbe
interactions. As an outcome, many tests of the generality of endophytic, plant growth
promoting rhizobia within cereals have indicated that this type of association is
widespread worldwide rather than being restricted to a particular crop (rice) and
place (Nile delta). Other natural associations of endophytic plant growth promoting
rhizobia within field-grown roots of wheat, barley, sorghum, canola, millet, rice and
maize rotated with legumes have now been described in Canada, Mexico, Morocco,
South Africa, Venezuela, China, India and elsewhere [32–36] (Y. Jing, personal
communication). Thus, despite some initial reservations about this novel finding