Chairs Comments Biological Nitrogen Fixation And Sustainable Agriculture

CIFN/UNAM, Res. Program Plant Mol. Biol., Cuernavaca, Morelos, México

Nitrogen is the major limiting nutrient for most crop species. The acquisition and assimilation of nitrogen for plant growth and development is second in importance only to photosynthesis (Vance 1997). Biological nitrogen fixation constitutes the main natural input of nitrogen into the biosphere. This represents around 50% of the total fixed nitrogen, the other half is fixed through chemical processes. The chemical reduction of nitrogen for fertilizers production, mainly via the Haber-Bosch process, has been a fundamental process for mankind's development since it has made it possible to feed around 40% of the total world population. The striking rise in cereal yields in developing countries during the last half of the century is directly attributable to a ten-fold increase in nitrogen fertilizer use. The "Green Revolution" favored the selection of crops that respond favorably to chemical fertilizers. The great increase in the production and use of chemical fertilizers for agricultural production during the last decades has resulted in serious ecological alterations, such as: the volatilization of nitrogen oxides to the atmosphere that causes the depletion of ozone, the depletion of non-renewable resources, an imbalance in the global nitrogen cycle and leaching of nitrate into groundwater (Kinzing, Socolow 1994).

Sustainable agriculture is broadly defined as agriculture that is managed towards greater resources efficiency and conservation while maintaining an environment favorable for the evolution of all species. One of the driving forces behind agricultural sustainability is the effective management of nitrogen in the environment. Successful manipulation of nitrogen inputs through the use of biological fixed nitrogen result in farming practices that are economically viable and environmentally prudent.

The primary source of biologically fixed nitrogen for agricultural system is through the Rhizobium (and related genera) - legume symbiosis (Vance 1997). The amount of nitrogen fixed by legumes is quite amazing. Legumes provide 25-35% of the worldwide protein intake. The agronomic use of symbiotic nitrogen fixers used as inoculants or "biofertilizers" is a good alternative to chemical fertilization. An important goal for sustainable agriculture, which will result in humanitarian and economic benefits, is to enhance the use and to improve the yield of biologically fixed nitrogen by legumes. Environmental and management limitations to legume growth are the major factors regulating nitrogen fixation, although practices that either limit the presence of effective rhizobia in the soil or enhance soil nitrate can also be critical (Peoples et al. this volume).

In the Nitrogen Fixation Research Center (CIFN/UNAM), in Cuernavaca, Mexico, a global project on the research on biological nitrogen fixation for sustainable agriculture has been carried out for several years. An important specific project on this subject includes the production and evaluation of Rhizobium biofertilizers for beans. Common bean (Phaseolus vulgaris) is the second most important crop in Mexico; it constitutes the main protein source for Mexicans' diet. Dr Jaime Mora, is the scientist from CIFN/UNAM responsible for the Rhizobium biofertilizers project, which is being done in collaboration with INIFAP, the agricultural research institute from the Agriculture Ministry from the Mexican government. Native R. etli or R. tropici strains isolated from different regions of Mexico and Central America, as well as genetically engineered strains improved for symbiotic nitrogen fixation, have been tested as bean biofertilizers in different experimental fields. Also, different agricultural technologies for watering and adding the biofertilizer, such as the dripping technology, were tested. The best results obtained in the field trials gave around 80% crop yield using biofertilizer as compared to the yield obtained following the addition of chemical fertilizer. Besides the ecological benefits, the latter may represent around a 10-fold saving in agricultural costs to Mexican farmers.

The group of Mariangela Hungria and Diva de S. Andrade in Londrina, Brazil have studied the role of biological nitrogen fixation in the two most important legume crops: bean and soybean (Glycine max). They are characterizing the biodiversity of indigenous rhizobial populations and their effect in inoculation with introduced improved strains. In both crops selected strains usually increase grain yield. There are other limiting factors from improving crop yield such as soil conditions (temperature, moisture, acidity).

Mpepereki et al. (this volume) emphasize the benefits that biological nitrogen fixation can bring to the cultivation of soybean in poor and marginalized communities of Sub-Saharan Africa. They are developing a research-extension model for promoting biological nitrogen fixation among peasant farmers of Zimbabwe. Potential to improve food security and alleviate poverty among the rural poor is tremendous.

Biological nitrogen fixation for cropping systems is also important for industrialized countries. Martin H. Entz is studying this issue for the prairie provinces of Canada. Integrated agricultural systems that include both ruminant livestock and crop production, arguably provide the best opportunities for capturing biological nitrogen fixation benefits in food production. The advantages of integrated food production systems as compared to monocultures, due to the role of biological nitrogen, are being analyzed.

Another important aspect of the research of biological nitrogen fixation towards sustainable agriculture includes associative nitrogen fixers. These microorganisms, including genera such as Azospirillum, Herbaspirillum, and Acetobacter, may associate as endophytes or may colonize the rhizosphere of important cereal crops and may be advantageous for crop production. Crop growth promotion by associative microorganisms is not always provided through biological nitrogen fixation. It is well documented that the growth promotion of maize by Azopirillum, a root colonizer, is due to the excretion of auxins and other phytohormones that promote root growth and allow a better capacity for absortion of nutrients from the soil. At CIFN in Mexico, a project is being carried out with Dr Jesus Caballero-Mellado as responsible in collaboration with INIFAP, with the aim of developing, producing and distributing biofertilizers, based in Azospirillum, for cereals crops such as maize, wheat, sorghum. Azospirillum biofertilizer was used in around 2 million ha in crop fields from different states of Mexico during 1999 and 2000. An average increase of 26% in the production of basic grains was obtained in 75% of the fields inoculated with the biofertilizer. This also represents great saving for farmers.


Kinzing AP, Socolow RH (1994) Physics Today 47, 24-35

Vance CP (1997) In Legocki A, Bothe H, Piihler A (eds) Biological Fixation of Nitrogen for

Ecology and Sustainable Agriculture, pp. 179-186, Springer-Verlag, Berlin, Germany


'IAPAR, Cx. Postal 481, 86001-970, Londrina, PR, Brazil

2Embrapa Soja, Cx. Postal 231, 86001-970, Londrina, PR, Brazil

1. Introduction

Soybean (Glycine max L.) and common bean (Phaseolus vulgaris L.) are the main legume crops grown in Brazil and in some South America countries. In Brazil, 32 million soybean grains are produced in 12.8 million hectares with average yield of 2500 kg ha"1. Farmers desiring high profits employ high technology and large land areas destined for crop exportation. A similar situation is verified in neighboring countries like Argentina and Paraguay. Unlike soybean, common beans are mainly cropped for food by smallholders with a low level of technology resulting in average yield of only 700 kg ha"1 in 4.5 million hectares. Biological nitrogen fixation (BNF) plays an important role in the successful management of both crops.

2. Rhizobial Soil Population and Diversity

Soybean was introduced in Brazil 120 years ago and several experiments have shown that uncropped soils are void of bradyrhizobia able to establish an effective symbiosis with this legume. The crop expanded in the 1960s and has been intensively inoculated since then, so that today most soils where this legume is grown show a very high population of soybean bradyrhizobia, estimated in 103 to 106 cells g"1 of soil. In areas cropped to soybean for the first time, the few nodules formed were identified as Bradyrhizobium japonicum and B. elkanii strains used in commercial inoculants and dispersed from other cropped fields, as well as some fast-growing indigenous rhizobia (Ferreira et al. 2001). Sequencing of the 16S rRNA genes of those fast-growing strains has detected similarity with Rhizobium tropici and Rhizobium genomic species Q (unpublished data). Furthermore, several strains resembling agrobacteria that effectively nodulate soybeans were also isolated from soybean nodules in both Brazil and Paraguay (Figure 1, Chen et al. 2000). However, although many Brazilian soybean cultivars are effectively nodulated by those fast-growing strains, and those bacteria are usually found in high number in soils, they compete poorly with B. japonicum and B. elkanii (Hungría et al. 2001).

Soybean seeds, even when harvested in an area with a very high population of naturalized strains, usually carry very few viable cells. As an example, in 28 field experiments performed from 1996 to 2001 in areas cropped with soybean for the first time, nodule number varied from 0 to 3 nodules per plant, with an average of 0.15 (unpublished data).

A different situation is found with the bean crop, since almost all soils, even when they have never been cropped before with this legume, show a very high population of indigenous rhizobia, estimated in 103 to 106 cells g"1 soil depending on crop and soil management practices (Andrade 1999; unpublished data). Furthermore, a high level of rhizobial diversity is also found in soil. For example, in a survey of soils from seven Brazilian states, 38 different RFLP-PCR profiles were detected. When 207 strains from two of those States (Pernambuco and Paraná) were characterized, Pernambuco, with alkaline soils and semi-arid climate, and Paraná, with acid soils and tropical and subtropical climates, a very high number (90% of the isolates) of unique strains was shown, as revealed by the BOX-PCR analysis (Grange 2001). There was no effect of either the bean cultivars (black or colored seeds) used as trap plants, or of the ecosystem on the rhizobial diversity. The sequencing of the 16S rRNA genes of some of those strains has shown that bean plants had the capacity to trap several rhizobial species, as shown in Figure 2 (Grange 2001).

Another difference from soybean is that common bean seeds usually carry many rhizobial cells, probably due to the harvesting method. However, this is a questionable hypothesis since little is known of survival ability of rhizobia on seeds. For example, when non-sterilized seeds from thirty different sites in Brazil (Paraná and Minas Gerais) were used, independent of whether they came from smallholders or if they were certified seeds produced with high technology, nodules formed in 37% of the plants and the number per plant varied from 2 up to 45 (unpublished data).


Cropped areas rC

PRY 65 LM G 11936 LMG11915 PRY 62 HAMBI 540 CIAT899 USDA 205 SEMIA 5080 USDA 110 PRY 1 SEM IA 566 PRY 42 USDA 6 PRY 40 SEMIA 587 SEMIA 5019 USDA 31 PRY 52 PRY 49 USDA 76


B. japonicum

B. elkanii

Figure 1. Dendrogram built with the UPGMA algorithm with the aligned 16S rRNA partial sequences of isolates from field-grown soybean nodules in Paraguay (PRY), from areas cropped for several years with this legume and of fourteen reference strains belonging to three genera. See Chen etal. (2000).

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