(1), (2) Department of Soil Biology, Faculty of Soil Science, Moscow Lomonosov State University, Moscow , Russia

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1 Scientific registration n : 182 Symposium n : 10 Presentation : oral The dual role of nitrogen-fixing microbial consortia in transformation of nitrogen in plant rhizosphere La role double de la consortia microbienne fixatrice d azote dans la transformation de l azote a la rhizosphere de la plant GLAGOLEVA Olga (1), UMAROV Marat (2) (1), (2) Department of Soil Biology, Faculty of Soil Science, Moscow Lomonosov State University, Moscow , Russia SUMMARY A connection between nitrogen fixation and denitrification of 150 natural nitrogen-fixing bacterial consortia (mixed cultures) isolated from the rhizosphere of different nonleguminous plants has been studied. The consortia included mainly representatives of the genera Arthrobacter, Azospirillum, Bacillus, Cellulomonas, Corynebacterium, Klebsiella and Pseudomonas. The overwhelming majority of investigated consortia was able to carry out both nitrogen fixation and denitrification. The maximums of nitrogenase and denitrifying activities of the same mixed cultures did not coincide but successively changed each other in time. Under laboratory conditions, consortia showing the highest nitrogen-fixing activity were also the most active denitrifiers, whereas in the rhizosphere, approximately 30% of the mixed bacterial cultures fulfilled only one of these processes. Only 12% of the consortia were active nitrogen fixers in association with plants, while 18% of the cultures showed denitrifying activity considerably exceeded their nitrogenase activity in the rhizosphere. These differences in the behaviour of diazotrophs in the rhizosphere can result in significant changes in their influence on plant growth and yield. Key words: microbial consortium, rhizosphere, associative nitrogen fixation, denitrification Mots cles: consortium microbien, rhizosphere, fixation d azote associative, denitrification INTRODUCTION Nitrogen is one of the most important factors limiting crop yield. The potential exists to satisfy the needs of nearly all crop plants for fixed nitrogen through the use of nitrogen-fixing bacteria. In recent years, there have been numerous attempts to use various nitrogen-fixing 1

2 bacteria for the enhancement of associative nitrogen fixation in the rhizosphere of nonleguminous plants. A promising method to intensify nitrogen fixation in the root zone of plants is an application of artificial and natural diazotrophic microbial consortia (mixed cultures), as these consortia produce a stronger and more stable effect on cultivated plants than pure cultures of nitrogen fixers (Umarov et al., 1993; Hoflich et al., 1994; Kaiser, 1995). However, even in such a case the inoculation of plants with diazotrophic microorganisms does not always result in a desirable effect, as many of nitrogen-fixing bacteria carry out not only nitrogen fixation, but also denitrification and nitrification (Umarov, 1990). Nevertheless, in world agricultural practice microbial cultures for the inoculation of plants are usually selected considering only their high nitrogen-fixing activity, even though the positive effects of increased nitrogen fixation in the root zone can be counteracted by considerable losses of nitrogen, either through the leaching of nitrates resulting from nitrification or through the loss of N gases produced by denitrification. Consequently, the interdependence of nitrogen fixation and other processes of the nitrogen cycle both on an organismic and an ecological level, should be considered. The present study was designed to investigate the interrelations between nitrogen fixation and denitrification carried out by microbial consortia under laboratory conditions and in the rhizosphere of several nonleguminous plants inoculated with the same cultures in model experiments. MATERIALS AND METHODS A collection of 150 nitrogen-fixing bacterial consortia (mixed cultures) was isolated from samples of fresh rhizosphere soil of different nonleguminous plants on a nitrogen-free Dobereiner medium (Dobereiner, 1988). The selection criteria were 1)high nitrogenase activity, and 2)stability of the consortia during prolonged laboratory cultivation. The stability of the mixed cultures was tested by six sequential platings on solid and two passages in liquid nitrogen-free medium. By the stability we meant constant taxonomic composition and invariable structure (i.e. relative content of each bacterial component in the consortia), persistence of high nitrogenase activity, and constant total cell count in the mixed cultures. The consortia were mostly binary, although some consisted of three or more components. These included mainly representatives of the genera Arthrobacter, Azospirillium, Bacillus, Cellulomonas, Corynebacterium, Klebsiella and Pseudomonas. Identification was performed in accordance with The Prokaryotes (1981) and Bergey's Manual (1989). Nitrogen-fixing (nitrogenase) activity of the mixed cultures was measured by the acetylene reductase assay (ARA) using a gas chromatograph with a flame-ionization detector. Denitrifying activity was estimated by emission of nitrous oxide (N 2 O) using a gas chromatogragh with a thermal conductivity detector (Methods of Soil Microbiology and Biochemistry, 1991). In experiments with plants, rape (Brassica napus var. napus) and barley (Hordeum vulgare var. polystichum) were used. Seeds were sterilized in 30% H 2 O 2 for 20 min, washed 6 times with sterile water and placed on the surface of agarized nitrogen-free Dobereiner medium in Petri dishes. These were incubated for 72 h at 28. Germinated sterile seeds were placed in 60-ml tubes containing 15 ml of sterile semi-solid nitrogen-free Hellriegel mixture 2

3 (Glagoleva et al., 1994) (two seedlings per tube). Suspensions of each consortium were introduced into the tubes at a total concentration of 10 8 cells/ml. Seedlings inoculated with consortia killed by autoclaving heating were used as controls. The tubes were incubated for 24 days at 25 in a luminostat (10000 lx, 10 h illumination/day). When measuring denitrifying activity, a solution of KNO 3 (0.1 mg/ml) was added to the tubes. All measurements were performed with 5 replications. RESULTS AND DISCUSSION The taxonomic composition, structure and functional characteristics of the consortia studied remained constant over a long period of laboratory cultivation (50 days), indicating the stability of the interspecific relations of the microorganisms in these systems. Furthermore, the nitrogenase activity of the mixed bacterial cultures was 3-15 times higher than that of pure cultures isolated from the original consortia (data are not shown). Therefore, the use of natural diazotrophic mixed cultures may be a promising method for the increasing of nitrogen fixation in the rhizosphere of nonlegumes. Studying of the dynamics of nitrogen-fixing activity of mixed cultures showed that in most consortia the maximum of the activity fell on the first or the second day of bacterial cultivation; after that, nitrogenase activity decreased and reached a plateau on a certain sufficiently high level (Fig.1). The same kinetics of nitrogen fixation were observed with pure cultures of bacteria isolated from the original consortia, although the nitrogenase activity of the separated partners was significantly lower than that of the mixed cultures, as mentioned above. Mixed cultures with another type of kinetics of nitrogen fixation were found considerably seldom (5% from the total number of the consortia). In such a case, the highest nitrogenase activity was settled on the 5-6th day of bacterial growth. Our interest has been to examine if some of the diazotrophic consortia from our collection can carry out the process of denitrification. Essentially all nitrogen-fixing consortia investigated were able to denitrify under laboratory conditions. We followed the dynamics of denitrifying activity of the mixed cultures and found that in most cases the maximal denitrifying activity was observed on the 4th-5th day of growth, after which the activity decreased to a constant level (Fig.2). Thus, it has been revealed that the maximal activities of nitrogen fixation and denitrification of the same mixed cultures do not coincide, but successively change each other in time. Primary activity one of these processes was observed on different stages of bacterial growth, while the activity of the other was reduced. One can suppose that mixed microbial cultures are able to carry out these two main processes of nitrogen cycle on different stages of their development. A comparative analysis of the nitrogen-fixing and denitrifying activities of the consortia under laboratory conditions showed a positive correlation between the activities of these processes. The consortia showing the highest nitrogenase activity were also the most active denitrifiers. Investigation of interdependence of nitrogen fixation and denitrification processes in the rhizosphere of plants inoculated with mixed cultures of diazotrophic bacteria became the next 3

4 step of our research. For the experiments on plant inoculation 50 the most active consortia from our collection have been selected. Rape and barley were used as test-plants. The results of our measurements of nitrogenase and denitrifying activities in the root zone of rape and barley indicated that the plants stimulated both nitrogen fixation and denitrification in the rhizosphere. However, the activity of these processes among the consortia differed. Based on these differences, it was possible to divide our collection of mixed cultures into three groups: 1)consortia showing high nitrogenase activity ( nm C 2 H 4 /plant/h) and low denitrifying activity in association with plants (about 12% from total number of the consortia); 2)mixed cultures displaying high denitrifying activity ( µm N 2 O/plant/h) under plant influence, while their nitrogen-fixing activity is low (18%); 3)consortia with intermediate levels of nitrogen fixation and denitrification in the rhizosphere (20-40 nm C 2 H 4 /plant/h, and µm N 2 O/plant/h, respectively) (70%). Important new information provided by this study is that approximately 18% of the diazotrophic consortia studied possessed high nitrogenase activity under laboratory conditions but became active denitrifiers in association with plants. These differences between the activities of nitrogen fixation and denitrification in the rhizosphere probably reflect the most common features of the behaviour of natural populations of diazotrophic bacteria when interacting with plants. Fine mechanisms responsible for the selective stimulation of nitrogen fixation or denitrification in plant rhizosphere are an open question to be addressed in future investigations. However, the results of this study indicate that it is necessary to consider the dual role of nitrogen-fixing bacteria introduced in the rhizosphere when selecting microbial cultures for practical use. The dual behaviour of diazotrophs in the rhizosphere can significantly change the expected positive results of inoculation with nitrogen-fixing microorganisms in terms of plant growth and yield. REFERENCES Bergey's Manual of Systematic Bacteriology (1989) (Williams S.T., Sharpe M.E. and Holt J.G., Eds.) 2298pp. Williams and Wilkins, Baltimore, MD. Dobereiner J. (1988) Isolation and identification of root associated diazotrophs. Plant and Soil 110, Glagoleva O.B., Umarov M.M. and Zlotnikov A.K. (1994) Nitrogenase activity of rhizosphere nitrogen-fixing bacteria in pure and mixed cultures Microbiology (Tr. from Mikrobiologiya) 63(2), Hoflich G., Wiehe W. and Kuhn G. (1994) Plant growth stimulation by inoculation with symbiotic and associative rhizosphere microorganisms. Experientia 50, Kaiser P. (1995) Diazotrophic mixed cultures of Azospirillum brasilense and Enterobacter cloacae. NATO ASI Series. V.637 Azospirillum V1 and Related Microorganisms (Fendrik I. et al., Eds.) Springer-Verlag Berlin Heidelberg. Methods of Soil Microbiology and Biochemistry (1991) (in Russian) (Zvyagintsev D.G., Ed.) pp Moscow State University press, Moscow. The Prokaryotes. A handbook on habitats, isolation and identification of bacteria (1981) 4

5 Vols.1,2 (Starr M.P., Stolp H. et al., Eds.) 2284 pp. Springer-Verlag Berlin Heidelbarg. Umarov M.M. (1990) In Soils and the Greenhouse Effect (Bouwman A.F., Ed.) P.263, John Wiley and Sons Ltd. Umarov M.M., Shabayev V.P., Smolin V.Yu. and Mamedov N.M. (1993) Nitrogenase activity in the rhizosphere and Triticale yield with mixed nitrogen-fixing microbial cultures applied Eurasian Soil Science 9, Keywords : nutritional competition, bacterial straims, carbon source Mots clés : compétition nutritionnelle, souches bactériennes, source carbonée Figure 1. The dynamics of nitrogen-fixing activity of nitrogen-fixing consortia under laboratory conditions. 5

6 Figure 2. The dynamics of denitrifying activity of nitrogen-fixing consortia under laboratory conditions. 6

STUDIES ON THE EXPLORATION OF BIOFERTILIZERS IN TEA

STUDIES ON THE EXPLORATION OF BIOFERTILIZERS IN TEA Synopsis of the thesis to be presented in candidature for the award of the Ph.D. degree of the Calicut University By N. Tensingh Baliah Under the Guidance