The International Consortium on Agricultural Biotechnology Research (ICABR)

 

Biotechnology as an Alternative to Chemical Nitrogen Fertilizers Use:
improving Biological Nitrogen Fixation


Roberto Defez , Serena Camerini,   Beatrice Senatore, Carmen Bianco, Emma Miraglia, Esther Imperlini
International Institute of Genetics and Biophysics, CNR
Italy

Angelo Spena, Simona Di Gregorio, Tiziana Pandolfini,
Faculty of Science, Verona
Italy

Giuseppe L. Rotino
ISPORT
Italy

Legumes (with a few minor additions) are the only living organisms self-autonomous for organicating Carbon and Nitrogen. The carbon organication depends on photosynthesis and it is common to any plant. Nitrogen prototrophy derives from the unique association of leguminous plants with Rhizobia, a nitrogen fixing soil bacteria. Agriculture in the developed world has become increasingly dependent on chemical fertilizers for achievement and maintenance of the high yields possible with modern crop types. The world request for nitrogen fertilizers grew up from 3 to 100 million tons in the last 55 years. The coupling of
such a simple technology for the chemical synthesis of ammonia with the reduced prices of fuels resulted in low prices for nitrogen fertilizers and in a widespread industrial production, mostly located in developing countries, with limited rules on the environmental impact of the production. Remarkably, the world leader of this sector holds only 6% of the total market of nitrogen fertilizers.

The environmental injuries related to chemically produced nitrogen fertilizers are largely underestimated. At present in the world from 2% to 5% of all types of fuels are burned in order to produce nitrogen fertilizers. Leaching by rain or watering, excessive use by farmers and a limited capability of plants to take up nitrogen fertilizers ultimately result in the oxidation of ammonia to nitrate, an endemic pollutant of drinking, ground and surface water in Europe. Finally, up to 10% of total nitrate produced is naturally converted in nitrous oxide, a gas causing greenhouse effects with a refracted power 180-times higher than that of carbon dioxide. The intensive agricultural productions currently necessary to sustain the market strongly depend on the use of nitrogen fertilizers. Our goal is to replace chemically synthesised nitrogen compounds by biological nitrogen fixation is based on the recent finding that at least two Rhizobium strains are able to increase biological nitrogen fixation and plant biomass production in association with a competent legume plant. Rhizobium-legume symbiosis accounts for amounts of nitrogen available for other organisms that are similar to those of chemical nitrogen fertilizers. The main difference consists in the source of energy in the two cases: instead of burning fuel, the symbiosis uses the photosynthates to provide carbon molecules to the endosymbiont (the bacteria), which is able to break the triple bound of N2 to reduce it to ammonia. The latter is taken up by the plant and converted into amino acids allowing plant growth. In addition, differences exist in slow and local release of nitrogen in the case of legumes, compared to the high concentration that are delivered in reduced periods of time when chemical nitrogen fertlisers are applied. This is an important point considering the increasing pollution of soil and water by chemical nitrogen fertilizers. Legumes are important components of many cropping systems from the beginning of agriculture and are grown commonly in areas where cereals such as maize, rice, wheat and barley are the main crops that deplete the soils from nitrogen. Legumes are not only grown in pure stands, but often are interplanted with other species, e.g. in vineyards or mais. Inter-cropping two or more species more effectively exploits environmental resources, a system well known from agriculture in underdeveloped countries which are to be adapted for intensive agriculture. Estimates of nitrogen fixation by food legumes reach up to more than 300 kg N/ha/year. The level of fixation depends on species, cultivar, water supply, inoculation, crop management practices, soil conditions, and fertility, and for these conditions optimised systems could be developed. We have genetically modified Rhizobia in order to convert them into producers of plant phytohormones. ("Method to control gene expression in bacteria, namely Rhizobiaceae, to improve root nodule development, nitrogen fixation and plant biomass production". Inventors: Defez R., Spena A. Patent Holders C.N.R. & G.IN.E.ST.R.A. PCT24190 deposited on november the 9th 1999). Consequently, phytohormones are locally delivered to root nodules, the new plant organ derived from the symbiotic association of legume root cell and bacteria. Root nodules generated by modified rhizobia are morphologically and functionally improved when compared to the controls. Legume plants nodulated by modified Rhizobia show an increase in plant biomass of at least 50-75%, measured as dry weight. We have mesured even a consistent (50%) increase in seed production and in nitrogen fixation ability. Seed and stem composition do not show any relevant alteration in nitrogen (protein) content. Soil sample analysis show that plants inoculated with the genetically modified strain do not deprivate the soil more than plant inoculated with the control bacterial strain. Economic exploitation and strategic impact. The exploitation and dissemination plans relate to the applications that can be envisaged from the data obtained. Basically the applications can be grouped into a) improved Rhizobium genetic material, b) improved legume genetic material, c) improved agricultural and environmental practices. The applications that we foresee are detailed below. a) Rhizobium inoculants is a world-wide business which is currently quite depressed in Europe. It received a boost during the 80s' because of the arrival in Europe of intensive cultivation of soybean that required the simultaneous inoculation of the competent rhizobia (Bradyrhizobium japonicum). Then the cultivation was reduced in Europe and with so the production of inoculants for nodulating legume seeds. However it is considered that there is a sizable market for strains with improved characteristics able to compete with endogenous strains, which has been relatively unexplored. It should be underlined that legumes occupy 5% of total European arable soils while such rate raised up to 25% in USA and Canada. b) Forage legumes. Our work is directly relevant to the production of improved forage legume in all climatic zones. With the need in the EU to find new proteinaceous sources for animal nutrition, legumes will become more important, both as forage and as seed (see below) legumes. i) Seed legumes. On a global scale, legumes provide 33% of total protein for human diet. They may also be grown for animal feed as high-protein containing fodder. Genes identified to improve nitrogen fixation in few species may be used in strategies to create corresponding improvements in other legume species. ii) Plant biomass production as an alternative renewable source of energy. New strategies of production of electricity are now based on recycling organic matters. The potential market of energy coming from recycling biomass is huge. At present the energy available coming from biomass is worth 17% of the total energy available in Sweden, 15% in Finland, 13% in Austria and 7% in Denmark. The European Union plans are to reach soon 10% in the average consumption. Cutting of plants is a valid source of renewable biomass and an arbustive nitrogen-fixing legume such as Acacia, which is a classical pioneer plant used to reduce desertification of the land and for bioremediation of polluted area (mines, heavy metals rich soils, etc.) satisfies many of the criteria of an efficient biomass producer.  c) A main application of our work is foreseen in the use of legumes as fertilizer plants for other agriculturally important plants. We wish to investigate the use of legumes as cover crops above fruit trees, olive or vineyard. We will investigate the best cultivation practices in order that a legume "fertilizer" plant should provide sufficient nitrogen to support the growth and productivity of fruit, olive or grape. Finally legumes are well-known colonisers of poor soils and disturbed habitats. The use of these nitrogen fixing plants could be envisaged in areas where ground cover is required for soil stabilisation, arrest of soil erosion and disidratation.

Acknowledgements

This work has been partially financed by the program "Biotecnologie II" of the C.N.R. and is part of G.IN.E.ST.R.A. (Genetic INitiative in European STrategical Research in Agriculture) research activities supervised and executed by the AgroBiotechNetwork. 

Acknowledgements

This work has been partially financed by the program "Biotecnologie II" of the C.N.R. and is part of G.IN.E.ST.R.A. (Genetic INitiative in European STrategical Research in Agriculture) research activities supervised and executed by the AgroBiotechNetwork.

 

Home Page Program Registration Hotel accommodation Ravello and surroundings Social program