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Use of Root Exometabolites by Associative Nitrogen-Fixing Microorganisms
USE OF ROOT EXOMETABOLITES BY ASSOCIATIVE NITROGEN-FIXING MICROORGANISMS Kravchedko L.V., Makarova N.M. The All-Union Research Institute of Agricultural Microbiology, 188 620 Leningrad-Pushkin, USSR
ABSTRACT The model experiments have shown’that at growing two nitrogen fixing strains on root exometabolites the carbon amount used by bacteria is 64-93 % and 36-74 % for rice. At the growth of maize the similar values are 67-69 % and 51-59 %. The efficiency of nitrogen fixation depends on the composition o f root exudates and trophic interactions with non-fixing microflora.
INTRODUCTION Root exometabolites are the major substrates used to compensate for the increased energy expenses of associative nitrogen-fixing bacteria in the rhizosphere (Umarov, 1982). They include water-soluble organic compounds, released by growing roots, and volatile metabolites possessing high reactivity and permeability in soil. The soluble root exudates consist mostly of aminoacids, organic acids and sugars readily metabolized by microorganisms. Basically volatile metabolites are composed of ethanol and acetaldehyde. However up to now there is no clear understanding of what portion of the energy available fmnroot exometabolites is directly consumed by the biological nitrogen fixation process. This paper deals with the determination of the factors affecting the efficiency of substrate utilization by associative N2- fixing microorganisms.
MATERIALS AND METHODS Strains of associative nitrogen-fixing bacteria released from cereal roots and samples of rhizosphere soil were used in this study. The effect of rice and maize root exudates on the growth of microorganisms has been examined under model conditions in liquid medium contained in flasks with plants where root exometabolites were the sole energy and carbon source (VanEura et al., 1977).
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The influence of the substrata composition on the efficiency of its utilization by diazotrophs has been studied by continuous flow cultivation of microorganisms in columns filled with soil (Macura, 1961). The carbon source was varied but the amount of the carbon substrate supplied to the columns was the same. In the first three treatments the amount of mineral nitrogen in the substrate supplied to the columns was replaced by the same concentration of aminoacid nitrogen. The period of continuous cultivation of soil samples was 8 days. Numbers of diazotrophs were enumerated on non-nitroqenous malate medium, and nitrogenase activity was determined by the acetylene-reduction method.
RESULTS AND DISCIJSSION Observations on the dynamics of two nitrogen-fixing bacteria i.~. Azospirillum brasilense and Mycobacterium rubeacearum grown on rice root exudates are given in the Table 1. Determination of N2-fixers numbers shows that prolongation of the cultivation period results in the stepwise increase of 5. brasilense in the solution and on the roots while the amount of M. rubeacerum drops. A similar pattern was observed in the experiments with maize (Table 2). The total amount Table 1 The influence of rice root exudates on the growth of nitrogen-fixing bacteria Incubation period, d
Bacteria
Azospirillum brasilense ~~~~~~~
0-6 6-12 12-18
Plant Cell number, dry weight, 10-6 per plant Mineralized C, % g Solution Root surface
0.26
73.7 121.8 258.5
49.0 72.3 69.1
92.6 63.8 84.4
0.14 0.21 0.27
645.3 130.5 113.3
85.3 58.3 36.8
74.1 44.4 35.7
0.15 0.22
~
Mycobacterium rubeacearum
0-6
6-12 12-18
of bacteria of both kinds using maize root exudates was approximately a factor of 3.5 higher than the amount on rice exudates. However, though the numbers of Mycobacterium in the rhizosphere of the both plants were twice the numbers of Azospirillum, 9. brasilense used more carbon from root exometabolites. Thus the amount of carbon consumed by bacteria was defined on the one side by the composition of the
-278-
organic compounds released by roots, and on the other by trophic demands of microorganisms studied. Table 2
The influence of maize root exudates on the growth of nitrogen-fixing bacteria
Bacteria
Azospirillum brasilense Mycobacterium rubeacearum
Incubation period, d
Plant Cell number, dry weight, 10-6 per plant Mineralized C, g Solution Root % surface
0-6 6-12 12-18
1.24 1.66 1.54
0-6 6-12 12-18
1.10 1.77
1-52
318
210
194
172
960
278
66.8 68.5 68.5
1200 1820 602
129 160 65
58.6 50.5 51.4
The microbiological analysis results show that the amount of diazotrophs considerably varied with the substrate composition (Table 3 ) . Replacement of mineral nitrogen by aminoacid nitrogen in nutrient solution resulted in the variation of the amount of soil diazotrophs. The maximum level of nitrogenase activity in the examined treatments was observed when serine and alanine were added. Despite the fact that the substrate amount added to the columns containing soil was the same, nitrogenase activity differed between the treatments by a factor of 5 and more. Volatile organic root exometabolites can also influence the nitrogen metabolism of the soil diazotrophs. Table 4 summarizes the results of replacing the malate used in liquid mineral medium for the growth of two strains of A. brasilense, by the same amount of ethanol or 25 % the amount of acetaldehyde. Ethanol and acetaldehyde drastically inhibited the nitrogenase activity by a factor of 50-100. Volatile metabolites, especially ethanol, were an adequate substrate for the growth of diazotrophs numbers. The efficiency of the energy substrate used by associative nitrogen fixers depends to a great extent on the nature of interactions between microbial complexes in the rhizosphere. During combined cultivation of nitrogen fixing and non-fixing microorganisms a stimulatory effect on nitrogen fixing activity may be observed (Tahle 5 ) . During the growth
-279-
Table 3
Influence of water-soluble root exudates components on diazotrophic associations and nitrogenase activity in soil Cell number, g-’ soil
Nitrogen source
Carbon source
Nitroqenase activity , / m o l e s C2H4. d-1. q-1 soil
.
,sa&mmse
NH4Cl NH4Cl NH4Cl Glutamic acid Alanine Serine
Malate saccharose+lllalate
saccharoee+malate saccharose+malab .Sa-+mala*
Table 4
1.44 1.55 2.44 0.72 3.69 4.99
Influence of volatile root exometabolites on cell numbers and nitrogenase activity of nitrogen-fixing bacteria Microorganism sp. 7 Sp.25 Cellnumbera, Nitroqenase Cell numbers, Nitroqenase 10-6.m1-1 activity, 10-6.m1-1 activity ,umles C2H4 .d-’ per 108 cells
Compound
Malate Ethanol Acetaldehyde
Table 5
28 123 144 35 236 269
218 632 60
70.5 1 .o 0.8
188 1020 16
91.1 1.9 1.5
Dynamics of nitrogenase activity (nmoles C2H4 .ml-’. h-l) in pure and mixed cultures of nitrogen-fixing bacteria
Incubation period, h
Microorganisms 4
22
46
70
~
sp. 7 Sp.7 Sp.7
+ +
0
44.1 92.8
30.6 50.7
0.86 1.00
0.10 0.12
0
100.7
35.2
2.57
0.26
0
Pseudomonas putida Arthrobacter globi fonni s
94 ~~~~
-280-
of Sp. 7 strain and nitrogen non-fixing rhizospheric bacteria the nitrogenase activity was twice that of the pure culture. The protein amount in the treatments studied remained at the same level. Thus the complicated trophic interactions between N2-fixing microorganisms and non-fixing complexes, occuring in the root zone, define the ability of the associative nitrogen fixers to function actively in the rhizosphere. The efficiency of associative nitrogen fixation is stimulated to a great extent by the composition of individual components in root exometabolites.
REFERENCES MACURA, J.: Continuous flow method in soil microbiology. 1.Apparatus. Folia Microbiol. 6: 328-334, 1961. UMAROV, M.M.: Significance of asymbiotic nitrogen fixation in the nitrogen balance in soil. Izv. AN SSSR. Ser. Biol. No 1: 92-105, 1982. VANCURA, V., PRIKRYL, Z., KALACHOVh, L., WURST, M.: Some quantitative aspects of root exudation. In: Soil Organisms as Components of Ecosystem. Ecol. Bull. (Stockholm) 25: 381-386, 1977.
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81-
ABSTRACT The model experiments have shown’that at growing two nitrogen fixing strains on root exometabolites the carbon amount used by bacteria is 64-93 % and 36-74 % for rice. At the growth of maize the similar values are 67-69 % and 51-59 %. The efficiency of nitrogen fixation depends on the composition o f root exudates and trophic interactions with non-fixing microflora.
INTRODUCTION Root exometabolites are the major substrates used to compensate for the increased energy expenses of associative nitrogen-fixing bacteria in the rhizosphere (Umarov, 1982). They include water-soluble organic compounds, released by growing roots, and volatile metabolites possessing high reactivity and permeability in soil. The soluble root exudates consist mostly of aminoacids, organic acids and sugars readily metabolized by microorganisms. Basically volatile metabolites are composed of ethanol and acetaldehyde. However up to now there is no clear understanding of what portion of the energy available fmnroot exometabolites is directly consumed by the biological nitrogen fixation process. This paper deals with the determination of the factors affecting the efficiency of substrate utilization by associative N2- fixing microorganisms.
MATERIALS AND METHODS Strains of associative nitrogen-fixing bacteria released from cereal roots and samples of rhizosphere soil were used in this study. The effect of rice and maize root exudates on the growth of microorganisms has been examined under model conditions in liquid medium contained in flasks with plants where root exometabolites were the sole energy and carbon source (VanEura et al., 1977).
-277-
The influence of the substrata composition on the efficiency of its utilization by diazotrophs has been studied by continuous flow cultivation of microorganisms in columns filled with soil (Macura, 1961). The carbon source was varied but the amount of the carbon substrate supplied to the columns was the same. In the first three treatments the amount of mineral nitrogen in the substrate supplied to the columns was replaced by the same concentration of aminoacid nitrogen. The period of continuous cultivation of soil samples was 8 days. Numbers of diazotrophs were enumerated on non-nitroqenous malate medium, and nitrogenase activity was determined by the acetylene-reduction method.
RESULTS AND DISCIJSSION Observations on the dynamics of two nitrogen-fixing bacteria i.~. Azospirillum brasilense and Mycobacterium rubeacearum grown on rice root exudates are given in the Table 1. Determination of N2-fixers numbers shows that prolongation of the cultivation period results in the stepwise increase of 5. brasilense in the solution and on the roots while the amount of M. rubeacerum drops. A similar pattern was observed in the experiments with maize (Table 2). The total amount Table 1 The influence of rice root exudates on the growth of nitrogen-fixing bacteria Incubation period, d
Bacteria
Azospirillum brasilense ~~~~~~~
0-6 6-12 12-18
Plant Cell number, dry weight, 10-6 per plant Mineralized C, % g Solution Root surface
0.26
73.7 121.8 258.5
49.0 72.3 69.1
92.6 63.8 84.4
0.14 0.21 0.27
645.3 130.5 113.3
85.3 58.3 36.8
74.1 44.4 35.7
0.15 0.22
~
Mycobacterium rubeacearum
0-6
6-12 12-18
of bacteria of both kinds using maize root exudates was approximately a factor of 3.5 higher than the amount on rice exudates. However, though the numbers of Mycobacterium in the rhizosphere of the both plants were twice the numbers of Azospirillum, 9. brasilense used more carbon from root exometabolites. Thus the amount of carbon consumed by bacteria was defined on the one side by the composition of the
-278-
organic compounds released by roots, and on the other by trophic demands of microorganisms studied. Table 2
The influence of maize root exudates on the growth of nitrogen-fixing bacteria
Bacteria
Azospirillum brasilense Mycobacterium rubeacearum
Incubation period, d
Plant Cell number, dry weight, 10-6 per plant Mineralized C, g Solution Root % surface
0-6 6-12 12-18
1.24 1.66 1.54
0-6 6-12 12-18
1.10 1.77
1-52
318
210
194
172
960
278
66.8 68.5 68.5
1200 1820 602
129 160 65
58.6 50.5 51.4
The microbiological analysis results show that the amount of diazotrophs considerably varied with the substrate composition (Table 3 ) . Replacement of mineral nitrogen by aminoacid nitrogen in nutrient solution resulted in the variation of the amount of soil diazotrophs. The maximum level of nitrogenase activity in the examined treatments was observed when serine and alanine were added. Despite the fact that the substrate amount added to the columns containing soil was the same, nitrogenase activity differed between the treatments by a factor of 5 and more. Volatile organic root exometabolites can also influence the nitrogen metabolism of the soil diazotrophs. Table 4 summarizes the results of replacing the malate used in liquid mineral medium for the growth of two strains of A. brasilense, by the same amount of ethanol or 25 % the amount of acetaldehyde. Ethanol and acetaldehyde drastically inhibited the nitrogenase activity by a factor of 50-100. Volatile metabolites, especially ethanol, were an adequate substrate for the growth of diazotrophs numbers. The efficiency of the energy substrate used by associative nitrogen fixers depends to a great extent on the nature of interactions between microbial complexes in the rhizosphere. During combined cultivation of nitrogen fixing and non-fixing microorganisms a stimulatory effect on nitrogen fixing activity may be observed (Tahle 5 ) . During the growth
-279-
Table 3
Influence of water-soluble root exudates components on diazotrophic associations and nitrogenase activity in soil Cell number, g-’ soil
Nitrogen source
Carbon source
Nitroqenase activity , / m o l e s C2H4. d-1. q-1 soil
.
,sa&mmse
NH4Cl NH4Cl NH4Cl Glutamic acid Alanine Serine
Malate saccharose+lllalate
saccharoee+malate saccharose+malab .Sa-+mala*
Table 4
1.44 1.55 2.44 0.72 3.69 4.99
Influence of volatile root exometabolites on cell numbers and nitrogenase activity of nitrogen-fixing bacteria Microorganism sp. 7 Sp.25 Cellnumbera, Nitroqenase Cell numbers, Nitroqenase 10-6.m1-1 activity, 10-6.m1-1 activity ,umles C2H4 .d-’ per 108 cells
Compound
Malate Ethanol Acetaldehyde
Table 5
28 123 144 35 236 269
218 632 60
70.5 1 .o 0.8
188 1020 16
91.1 1.9 1.5
Dynamics of nitrogenase activity (nmoles C2H4 .ml-’. h-l) in pure and mixed cultures of nitrogen-fixing bacteria
Incubation period, h
Microorganisms 4
22
46
70
~
sp. 7 Sp.7 Sp.7
+ +
0
44.1 92.8
30.6 50.7
0.86 1.00
0.10 0.12
0
100.7
35.2
2.57
0.26
0
Pseudomonas putida Arthrobacter globi fonni s
94 ~~~~
-280-
of Sp. 7 strain and nitrogen non-fixing rhizospheric bacteria the nitrogenase activity was twice that of the pure culture. The protein amount in the treatments studied remained at the same level. Thus the complicated trophic interactions between N2-fixing microorganisms and non-fixing complexes, occuring in the root zone, define the ability of the associative nitrogen fixers to function actively in the rhizosphere. The efficiency of associative nitrogen fixation is stimulated to a great extent by the composition of individual components in root exometabolites.
REFERENCES MACURA, J.: Continuous flow method in soil microbiology. 1.Apparatus. Folia Microbiol. 6: 328-334, 1961. UMAROV, M.M.: Significance of asymbiotic nitrogen fixation in the nitrogen balance in soil. Izv. AN SSSR. Ser. Biol. No 1: 92-105, 1982. VANCURA, V., PRIKRYL, Z., KALACHOVh, L., WURST, M.: Some quantitative aspects of root exudation. In: Soil Organisms as Components of Ecosystem. Ecol. Bull. (Stockholm) 25: 381-386, 1977.
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81-