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

9.2 Landmark Discovery of the Natural Rhizobia–Rice Associationj167
New Frontier Project (1994) was developed by the IRRI to coordinate worldwide
collaborative efforts among research centers to explore natural rice–bacteria associations
to reduce the dependence of rice on synthetic mineral nitrogen resources.
The long-term objective of that project was to enable self-fertilization of rice plants.
The working group of the project concluded that exploratory research is primarily
needed to assess the feasibility of nitrogen fixation in rice by an international
multidisciplinary group. In fact, this research was considered to be scientifically
risky because it was entering unfamiliar territory, but likely to have an enormous
impact on agricultural productivity if successful. One of the research directions
recommended at that workshop was to determine if rhizobia naturally colonize the
interior of rice roots when this cereal grows in rotation with a legume crop, and if so,
to assess the potential impact of this novel plant–microbe association on rice production.
This idea is derived from the general concept that roots of healthy plants
grown in natural soil eventually develop a continuum of root-associated microorganisms
extending from the rhizosphere to the rhizoplane and even deeper into the
epidermis, cortex, endodermis and vascular system [18–21]. Typically, the presence
of these beneficial microorganisms within roots does not induce any disease symptom.
These microorganisms are described as endophytes or internal root colonists
since they can intimately colonize the interior of living plant tissue [22,23]. In the
mid-1990s, a multinational collaborative project was initiated to search for natural,
intimate associations between rhizobia and rice (Oryza sativa L.), assess their impact
on plant growth and exploit those combinations that can enhance grain yield with
less dependence on inputs of chemical nitrogen fertilizers.
An important development in the exploration of rice–endophytic rhizobia associations
took place when a natural plant growth promoting association was discovered
between rice and Rhizobium leguminosarum bv. trifolii, which is the root-nodule
nitrogen-fixing symbiont of berseem clover (Trifolium alexandrinum L.) commonly
cultivated in the Nile delta region of Egypt. This intimate association is believed to
have evolved as a result of rice being rotated successfully with berseem clover for
between 700 and 1400 years [1].
The guiding hypothesis was that the natural endophytic associations between
rhizobia and cereal roots would most likely occur where cereals are successfully
rotated with a legume crop that could enhance the soil population of the corresponding
rhizobial symbionts. Such natural Rhizobium–cereal associations would be perpetuated
if they were mutually beneficial. If this hypothesis was correct, the cereal
roots growing at these sites should harbor, along with other microbes, a high
population density of endophytic rhizobia that are already adapted to be highly
competitive for colonization of the interior habitats of crop roots, being protected
from stiff competition with other soil–rhizosphere microorganisms under field
conditions. This is where endophytic rhizobia are strategically located, because a
more rapid and intimate metabolic exchange is possible within host plant tissues
rather than just on their epidermal surface. An ideal location to test this hypothesis
was in the Egyptian Nile delta where rice has been rotated with the forage legume,
Egyptian berseem clover (T. alexandrinum L.) since antiquity. In this area, japonica
and (more recently) indica and hybrid rice cultivars are cultivated by transplantation in