9

BIOLOGICAL NITROGEN FIXATION AND

FUTURE CHALLENGES OF AGRICULTURE
The Endophytic Connection

Federico Sanchez,· Luis Cardenas, and Carmen Quinto

Departamento de Biologia Molecular de Plantas

Instituto de Biotecnologia
Universidad Nacional Aut6noma de Mexico
Apartado Postal 510-3, Cuemavaca Morelos, 62271, Mexico

ABSTRACT

Feeding the growing global population, anticipated to be 8 billion by the year 2020,
is one of the most important recent challenges of agriculture. The increase in cereal grain
yield, to cope with this demand, directly implies a dramatic increase in the use of nitro-
gen-based fertilizers and agrochemicals. Some of these intensive agricultural practices
have progressive detrimental effects on the environment. This review is focused on some
novel insights gain<::d into the understanding of associative and symbiotic interactions of
plants with nitrogen-fixing organisms that makes Biological Nitrogen Fixation (BNF) a vi-
able answer to this compelling dilemma.

INTRODUCTION
Food production must be markedly increased during the next decades, since human
population is expected to increase by about 95 million annually. It is estimated that there
will be 8 billion people by the year 2020 (Chrispeels and Sadava, 1994).
Both striking increases in cereal grain yields in the developed countries and "green
revolution" success in agriculture in the developing world, during the last few decades, is
a direct consequence of dramatic increases in the use of nitrogen-based fertilizers (Vance,

• Corresponding author; telephone 52-73291653; fax 52-73-136600; e-mail: federico@ibt.unam.mx

107
F. Shahidi et al. (eds.), Chemicals via Higher Plant Bioengineering
© Kluwer Academic /Plenum Publishers, New York 1999
108 F. Sanchez et at.

1998) with concomitant negative effects on the environment. However, there is not a sim-
ple answer to this predicament; the fundamental problem of feeding the growing popula-
tion and the progressive environmental degradation of the ecosystems associated with
some intensive agricultural practices demands effective solutions to these problems.
After photosynthesis, biological nitrogen fixation (BNF) is probably the most impor-
tant metabolic process on Earth. Whereas photosynthesis supplies carbon and oxygen nec-
essary for life on our planet, BNF results in the reduction of nitrogen gas (approximately
80% of our atmosphere) into ammonia. Although all living organisms require reduced am-
monia for the synthesis of essential biochemicals such as nucleic and amino acids, only
certain prokaryotes are capable of "fixing nitrogen" due to the synthesis of the enzyme ni-
trogenase which catalyzes the conversion of atmospheric nitrogen into ammonia.
The association between grasses and endophytic diazotrophs and the interaction be-
tween legume crops and endosymbiotic nitrogen-fixing bacteria give to some plants the
selective ability to grow in nitrogen-poor soils. These remarkable associations and symbi-
otic interactions are especially important for natural ecosystems, forestry and for sustain-
able and intensive agriculture. The use of BNF, as compared to fertilizer application, has
the advantage of putting all the N into the plant and spares the use of fossil fuel, which
are, and will continue to be in the future, major points to consider for the protection of the
environment. On the other hand, most food production in the developing countries of the
tropics is based on small farms, often family operated, especially in the Central and South
America, and Africa where the use of fertilizer and agro-chemicals is minimal. Improving
of nitrogen-fixing traits for grasses and cereal-associations through molecular tools and
genetic manipulations for better inocula, and breeding for high yield grain legume crops in
such circumstances, provides excellent opportunities for the production of green manure,
and high protein food sources, at the same time, helping to protect the soil organic matter
and enhance soil fertility (Rolfe et aI., 1998b).

ENDOPHYTIC DIAZOTROPHIC ASSOCIATIONS WITH

CEREALS
Research on nitrogen fixation in cereals started with the pioneering work ofDobere-
iner (von Bulow and Dobereiner, 1975) and the isolation and description of bacteria of the
genus Azospirillum (Tarrand et aI., 1978). This genus comprises free-living N2 fixing rhi-
zosphere bacteria that have been isolated from different soil types and from the roots of
numerous wild and cultivated plants all over the world. Field trials, carried out at multiple
locations, have shown that under certain environmental and soil conditions, inoculation
with Azospirillum has beneficial effects on plant yields (see the review by Okon and La-
bandera-Gonzalez, 1994). However, in general it is not N2 fixation, but bacterial phytohor-
mone biosynthesis, that has been proposed for being responsible for the observed plant
growth promotion upon Azospirillum inoculation, since proliferation of root mass is al-
ways beneficial to the plant in absorbing extra nutrient (Okon and Vanderleydeo, 1997).
Regarding direct plant growth promotion, the ability of Azospirillum brasilense to produce
indol-3-acetic acid (IAA) is now intensively studied. IAA biosynthesis in A. brasilense has
proven to be very complex, since at least three biosynthetic pathways are operative
(Costacurta and Vanderleyden, 1995). This property is exploited with the commercializa-
tion of inoculants such as Azogreen by the Lipha Company in France.
One of the restrictions with the inoculation of diazotrophic bacteria, like Azospiril-
tum, is that they only fix nitrogen under nitrogen-limiting, microaerobic conditions. There-