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Biological Nitrogen Fixation of Microorganisms |
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Biological Nitrogen Fixation
Biological fixation of atmospheric nitrogen was the topic for investigations by many workers. Fred, Baldwin and McCoy in 1932 from the U.S.A. put forth their exhaustive work on root nodule bacteria. At Rothamsted in England, Thornton in 1947 studied nodule bacteria from clovers.
Jensen's method devised in 1942 for studying nodulation of plants on agar in test tubes helped in furthering investigations in this field. The German bacteriologist Bortels demonstrated in 1936 that an adequate supply of molybdenum was essential for accelerating nitrogen fixation by nodulating legumes.
The Japanese scientist Kubo found out in 1939 that root nodules of legumes could be effective only in the presence of a red pigment in them. At Helsinki, Virtanen and his school in 1947 studied leghaemoglobin and the chemistry and mechanism of nitrogen fixation.
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The discovery of consistent nitrogen fixation in cell-free extracts of Clostridium pasteurianum by Carnahan and others at the Du Pont Laboratory in the U.S.A. in 1960 was a landmark in the field of biological nitrogen fixation. Of equal importance was the development of the isotope method to quantify the amount of nitrogen fixed by the use of 15N and mass spectrometer developed by Burris and Wilson in 1957.
The enzyme nitrogenase was isolated, characterized and most of its biochemical properties understood except the nature of intermediates between nitrogen and ammonia. Applying the knowledge obtained with asymbiotic nitrogen fixers, Bergersen in Australia elaborated the biochemistry of nitrogen fixation in legume root nodules in the sixties. Nutman in England from 1948 onwards gave us an insight into the hereditary mechanisms behind nodulation in legumes.
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At the same time, he proposed the theory of microinvagination of root hairs as an explanation for the origin of infection threads in root hairs of clovers.
The work on nitrogen fixation in nodulated plants other than legumes was taken up by Bond in the U.K. and Quispel, Silver and Becking in Europe who made considerable headway on this aspect between 1950 and 1970. During the same period, Fogg and Stewart in the U.K. intensified the work on nitrogen fixing blue-green algae.
One of the significant advances with regard to non-leguminous root nodulation has been the isolation of an actinomycetous endophyte (Frankia sp.) from root nodules of Comptonia peregrina in 1978 by Callaham, Del Tredici and Torrey.
The isolated endophyte was slow growing and could re-infect and produce nodules on the roots of the same host plant.
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Another important observation by Trinick from Australia in 1973 was the isolation of Rhizobium from the root nodules of the genus Trema (Parasponia) which has highlighted a unique association of Rhizohil1m with non-leguminous plants resulting in the formation of root nodules.
Bacteria containing fertilizers such as 'Azotobacterin' and 'Phosphobacterin' were extensively used in Russia to improve soil fertility. Krasilnikov and Mishustin in 1937 were busy in Russia on several problems relating to interactions between plants and soil microorganisms. The English translation of 'Mikrobiologiya' opened the door of Russian microbiology to the English-speaking world.
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The Australian group led by
Vincent in 1954 was very active in the study of all aspects of nodulation, particularly in. understanding the environmental factors in legume root nodulation as evidenced by the work of Gibson in 1965.
While other Australian workers like Date, Brockwell and Roughley in 1962 were busy in the development of techniques involved in inoculant production and application to seed, Burton in 1950 in the U.S.A., was chiefly responsible for the establishment of legume inoculant research in North America with all its industrial implications.
The extensive use of the assay procedure utilizing the nitrogenase-catalyzed reduction of acetylene to ethylene coupled with the sensitive gas chromatographic analysis proposed by Hardy and his associates in 1968 of the Du Pont Laboratory in the U.S.A. may be regarded as a turning point in the field of biological nitrogen fixation.
The ease and rapidity with which this technique could be used gave an impetus to many investigators to measure nitrogenase activity in situ in many natural ecosystems.
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The Brazilian group of workers headed by Dobereiner showed how Azotobacter paspali could specifically inhabit the roots of the grass Paspalum notatum and fix abundant amounts of nitrogen, an observation which was later confirmed by the acetylene reduction reaction.
Dobereiner also introduced the concept of 'associative symbiosis' or 'diazotrophic biocoenocis' after the rediscovery of Azospirillum within plant cells of many graminaceous plants. Blue-green algae and Azolla received greater attention in developing countries as a nitrogen supplement in rice cultivation.
Rapid advances have been made in the U.K. and the U.S.A. in our knowledge of the physiology, biochemistry and kinetics of the enzyme nitrogenase. Those active in this field are Eady and his associates in England and Burris and Orme-Johnson in Wisconsin. Pate and his associates in Australia have quantified the interrelationship between photosynthates (energy) and nitrogen fixation in legumes.
The rapid expansion in the field of bacterial genetics had its impact on the study of nitrogen-fixing microorganisms. Presently, our understanding of the genetic loci involved in the process of biological nitrogen tixation in Azotobacter, Azospirillum, Rhizobium and blue-green algae (cyanobacteria) has reached new heights due to the efforts of many groups of workers in Europe, Australia and the U.S.A.
We are now aware that rhizobial genes control infection, nodulation and host range in legumes. Extrachromosomal genetic particles known as plasmids are known to carry nitrogen fixing traits. Other nodulation genes have been identified that are functionally and structurally conserved in rhizobia. In short, it has become clear that establishment of legume nodule symbiosis is the result of a multitude of communications and biochemical interactions between the legume host and the rhizobial symbiont.
This area of plant-microorganisms relationships has been enlarged due to the efforts of several molecular biologists such as Postgate, Beringer, Dixon, Johnston, Kennedy, Brill, Ausubel, Helinksy, Fisher, Long, Denarie, Downie, Kondorosi, Pueppke, Rolfe, Gershoff, Broughton, Spanik, Stacey, Verma, Krishnan, Elmerich, Nuti and others all over the world.
Dommergues and .associates from France and Senegal have discovered the occurrence of nodules on stems of Sesbania rostrata which fix nitrogen by virtue of which, the legume serves as an excellent green manure in low-land rice cultivation. Similarly, the group has also discovered nitrogen-fixing Stem nodules on Casuarina spp. caused by Frankia.
It is now known that rhizobia can fix nitrogen independently in cultures and this fact has been substantiated by 15N as well as acetylene reduction methods. The question whether rhizobia can establish endogenously within the cells of plant roots other than those of legumes and Parasponia, and induce the formation of nodules has been tackled by a team of workers headed by Cocking in England who have attempted to induce nodules on roots of rice, wheat and rape seedlings by inoculating the roots with the stem nodulating bacterial species, Azorhizobium caulinodans.
Nodule-like structures capable of fixing nitrogen have no doubt been demonstrated in rice and rape seedlings under laboratory conditions but field applications of any of these laboratory findings are yet to be realised.
Studies on soil microorganisms in relation to soil fertility were enlarged in several agricultural universities in India through applied research programmes under the All India coordinated research project funded by the Indian Council of Agricultural Research and the Department of Science and Technology.
These projects were related to the field application of nitrogen fixing microorganisms and the role in the decomposition of organic matter. The main emphasis has been to minimize the use of chemical fertilizers in the cultivation of rice, legumes and millets.
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