World Journal of Microbiology and Biotechnology 6, 340-342
Short Communication
Resistance of Bradyrhizobiumjaponicum strains to selected antibiotics
A.C. Borges, W.V. Guimarfies, R.M.C. Muchovej and G. de Los Reyes Gonz~lez
The majority of the 50 Bradyrhizobium japonicum strains tested were resistant to ampicillin, kanamycin, streptomycin and tetracycline in concentrations below 100 ltglml but resistant to chloramphenicol in concentrations equal to or above 100 Fg/ml. Two strains had high levels of resistance to ampicillln and to streptomycin and six strains were very sensitive to several antibiotics. La majorit6 de la cinquantaine de souches de Bradyrhizobium japonicum s'est r~v~16e r6sistante/t I'ampicilllne, /, la kanamycine, la streptomycine et /t la t6tracycline aux concentrations inft~rieures & lO0~.g/ml mats au chloramphenicol aux concentrations 6gales ou sup6rieures /i 100 rtg/ml. Deux souches pr6sentalent un haut degr(~ de rt~sistance ~ I'amplcilline et tJ la streptomycine. Six souches 6talent tr6s sensibles /t plusleurs antlblotiques. A.C. Borges, W.V. Guim&res and R.M.C. Muchovej are with the Departamento de Biologia Geral, Universidade Federal de Vir Vi~osa, MG, 36570, Brasil; G. de Los Reyes Gonz~.lez is with INIFAP-SARH, Veracruz, 91700, Mexico. W.V. Guimar&es is the Corresponding Author.
Introduction In areas where soybeans are cultivated, the association with Bradyrhizabium japonicum is responsible for a great part of the nitrogen incorporated in the soil. In inoculation programmes, it is necessary to utilize strains that are capable of establishing and persisting in the soil and also have an elevated efficiency of nitrogen fixation. The establishment of the symbiosis depends, among other factors, on the ability of the rhizobium to withstand anti-microbial substances present in the soil. It has been observed that B. japonicum strains isolated from acid soils are more tolerant to streptomycin and speetinomycin than those isolated from more neutral soils (Ayanaba & Wong 1982). Also slow-growing rhizobia are more resistant to anti-microbial agents than are faster growing strains (Graham 1963). Resistance to streptomycin has been one of the most frequently used markers in genetic studies of rhizobia (Brockwell et aL 1977). Mutants of B. japonicum resistant to kanamycin, streptomycin and viomycin are unaltered in their nodulation and nitrogen-fixation capacities (Levin & Montgomery 1974). However, B. japonicum mutants resistant to high concentrations of streptomycin lose their capacity to nodulate plants (Alves & Azevedo 1975). The use of mutagenic agents allows the production of mutants with high resistance to antibiotics. There is, however, the risk of altering the beneficial properties of the bacterium. The utilization of natural resistance to the antibiotics increases the possibility of obtaining strains that are more adapted to the soil conditions without compromising the nitrogen fixation capacity of the bacterium. The objective of this study was to determine the level of resistance of several B. japonicum strains to selected antibiotics.
Materials and Methods
(~ 1990 Rapid Communications of Oxford Ltd.
340
Fifty strains of B.japonicum, obtained from the Culture Collection of the Instituto de Pesquisas Agron6micas (IPAGRO-Secretaria da Agricultura do Rio Grande do Sul, Brazil), were tested. Forty-three of these were isolated from Brazil, five from the USA and two from Australia. The cultures were grown on Medium 79 (Fred & Waksman 1932), pH 6.6. The
Antibiotic resistance of Bradyrhizobium japonicum conservation of the cultures was done on M79 agar slants under sterilized mineral oil. The resistance level of each strain to ampicillin (Ap), kanamycin (Kin), chloramphenicol (Cm), streptomycin (Sm) and tetracycline (Tc) was tested by spreading serial dilutions of the culture on the surface of the M79 agar with up to 500 #g of each antibiotic per ml. The strains of B. japonicum were incubated in 5 ml of M79 broth until the growth phase had started. The cells were then transferred to the plates with M79 plus antibiotics, using a multiple replicator with 17 needles (Azevedo et al. 1982). After 8 days at 28~ the presence or absence of cellular growth was recorded. The level of resistance was defined as the highest antibiotic concentration that permitted cellular growth.
Results and D i s c u s s i o n The majority of the B. japonicum strains were resistant to Ap, Km, Sm and Tc below 100 #g/ml of medium, but 96% were resistant to Cm at or above 100 #g/ml (Fig. 1). The levels of resistance of strains to Ap, Km, Cm and Tc followed a unimodal distribution (Trabulsi et al. 1970): that is the number of strains increased with increasing concentration of the antibiotic, reached a maximum then decreased as the concentration of antibiotics increased further. In the presence of Sm there was a different pattern: two maxima, one at 10 #g/ml the other at 100 #g/ml, were apparent. According to Trabulsi et al. (1970) this is a bimodal distribution where the first group of strains with lower resistance can be considered as sensitive and the second group with greater resistance as resistant. Microbial antagonists present in the rhizosphere of leguminous plants can influence the competition and survival
50
AMPICILLIN
Z < n,los
I
KANAMYCIN
CHLORAMPHENICOL
STREPTOMYCIN
TETRACYCLINE
30-
b_
0 n~ W
20-
00 Z
0
o o o o o o
o o o o o o
o o o o o o
o o o o o o
o o o o o
o
LEVELS OF RESISTANCE (pug/ml) Figure 1. Frequency of levels of natural resistance to antibiotics of 50 Bradyrhizobium japonicum strains grown in medium 79 for 8 days at 28~ (average of three replications).
341
A.C. Borges et al. of rhizobia, as much as their level of resistance to anti-microbial compounds and their capacity to establish the symbiosis (Schreven 1964). The knowledge of the levels of resistance of B. japonicum strains to antibiotics can aid in tracing the strains introduced into the soil, in studies of competitivity for nodule infection sites and in ecological studies (Beynon & Josey 1980). Chloramphenicol and streptomycin are probably the markers of choice for genetic studies of the B. japonicum strains tested.
Acknowledgements This study was funded by grants from the Conselho Nacional de Desenvolvimento Cientifico e Tecnol6gico (CNPq) and Coordenag~o de Aperfeigoamento de Pessoal de Nivel Superior (CAPES), Brazil.
References ALVES, M.F. & AZEVEDO,J.L. 1975 Isolamento e caracterizaggo de mutantes de Rhizobiura japonicum resistentes ~ estreptomicina. Summa Phytopato/ogica 1, 205-212. AYANABA,A. & gONG, A.L. 1982 Antibiotic-resistant mutants identified from nodules of uninoculated soybeans grown in a strongly acidic soil. Soil Biologyand Biochemistry 14, 139-143. AZEVEDO, J.L., CASSINI,S.T.A. & OL1VEIRA,A. 1982 Replicador multialqa para uso geral em bacteriologia. Po/iagro 4, 11-18. BEYNON,J.L. & JOSEY,D.P. 1980 Demonstration of heterogeneity in a natural population of Rhizobium phaseo/i using variation in intrinsic antibiotic resistance. Journal of Genera/ Microbiology 118, 437-442. BROCKWELL, J., SCHWINGHAMER, E.A. & GAULT, R.R. 1977 Ecological studies of root-nodule bacteria introduced into field environment~V. A critical examination of the stability of antigenic and streptomycin resistance markers for identification of strains of Rhizobium trifo/ii. Soil Biology and Biochemistry 9, 19-24. FRED, E.B. & WAKSMAN, S.A. 1932 Laboratory Manual of General Microbiology. New York: McGraw-Hill. GRAHAM, P.H. 1963 Antibiotic sensitivities of root nodule bacteria. Australian Journal of BiologicalScience 16, 557-559. LEVIN, R.A. & MONTGOMERY,M.P. 1974 Symbiotic effectiveness of antibiotic-resistant mutants of Rhizobiumjapon#um. Plant and Soil 41, 669-676. SCHREVEN,D.A. VAN 1964 The effect of some actinomycetes on rhizobia and Agrobacterium radiobater. Plant and Soil 21, 283-302. TRABULSI, L.R., ZULIANI,M.E. & TOLEDO,M.R.F. 1970 Resistance to nine drugs of Shigel/a strains isolated in S~o Paulo between 1963-1968. Revistade Microbio/ogia1, 71-72.
(Received 9 January 1990; revised and accepted 11 May 1990)
842
Short Communication
Resistance of Bradyrhizobiumjaponicum strains to selected antibiotics
A.C. Borges, W.V. Guimarfies, R.M.C. Muchovej and G. de Los Reyes Gonz~lez
The majority of the 50 Bradyrhizobium japonicum strains tested were resistant to ampicillin, kanamycin, streptomycin and tetracycline in concentrations below 100 ltglml but resistant to chloramphenicol in concentrations equal to or above 100 Fg/ml. Two strains had high levels of resistance to ampicillln and to streptomycin and six strains were very sensitive to several antibiotics. La majorit6 de la cinquantaine de souches de Bradyrhizobium japonicum s'est r~v~16e r6sistante/t I'ampicilllne, /, la kanamycine, la streptomycine et /t la t6tracycline aux concentrations inft~rieures & lO0~.g/ml mats au chloramphenicol aux concentrations 6gales ou sup6rieures /i 100 rtg/ml. Deux souches pr6sentalent un haut degr(~ de rt~sistance ~ I'amplcilline et tJ la streptomycine. Six souches 6talent tr6s sensibles /t plusleurs antlblotiques. A.C. Borges, W.V. Guim&res and R.M.C. Muchovej are with the Departamento de Biologia Geral, Universidade Federal de Vir Vi~osa, MG, 36570, Brasil; G. de Los Reyes Gonz~.lez is with INIFAP-SARH, Veracruz, 91700, Mexico. W.V. Guimar&es is the Corresponding Author.
Introduction In areas where soybeans are cultivated, the association with Bradyrhizabium japonicum is responsible for a great part of the nitrogen incorporated in the soil. In inoculation programmes, it is necessary to utilize strains that are capable of establishing and persisting in the soil and also have an elevated efficiency of nitrogen fixation. The establishment of the symbiosis depends, among other factors, on the ability of the rhizobium to withstand anti-microbial substances present in the soil. It has been observed that B. japonicum strains isolated from acid soils are more tolerant to streptomycin and speetinomycin than those isolated from more neutral soils (Ayanaba & Wong 1982). Also slow-growing rhizobia are more resistant to anti-microbial agents than are faster growing strains (Graham 1963). Resistance to streptomycin has been one of the most frequently used markers in genetic studies of rhizobia (Brockwell et aL 1977). Mutants of B. japonicum resistant to kanamycin, streptomycin and viomycin are unaltered in their nodulation and nitrogen-fixation capacities (Levin & Montgomery 1974). However, B. japonicum mutants resistant to high concentrations of streptomycin lose their capacity to nodulate plants (Alves & Azevedo 1975). The use of mutagenic agents allows the production of mutants with high resistance to antibiotics. There is, however, the risk of altering the beneficial properties of the bacterium. The utilization of natural resistance to the antibiotics increases the possibility of obtaining strains that are more adapted to the soil conditions without compromising the nitrogen fixation capacity of the bacterium. The objective of this study was to determine the level of resistance of several B. japonicum strains to selected antibiotics.
Materials and Methods
(~ 1990 Rapid Communications of Oxford Ltd.
340
Fifty strains of B.japonicum, obtained from the Culture Collection of the Instituto de Pesquisas Agron6micas (IPAGRO-Secretaria da Agricultura do Rio Grande do Sul, Brazil), were tested. Forty-three of these were isolated from Brazil, five from the USA and two from Australia. The cultures were grown on Medium 79 (Fred & Waksman 1932), pH 6.6. The
Antibiotic resistance of Bradyrhizobium japonicum conservation of the cultures was done on M79 agar slants under sterilized mineral oil. The resistance level of each strain to ampicillin (Ap), kanamycin (Kin), chloramphenicol (Cm), streptomycin (Sm) and tetracycline (Tc) was tested by spreading serial dilutions of the culture on the surface of the M79 agar with up to 500 #g of each antibiotic per ml. The strains of B. japonicum were incubated in 5 ml of M79 broth until the growth phase had started. The cells were then transferred to the plates with M79 plus antibiotics, using a multiple replicator with 17 needles (Azevedo et al. 1982). After 8 days at 28~ the presence or absence of cellular growth was recorded. The level of resistance was defined as the highest antibiotic concentration that permitted cellular growth.
Results and D i s c u s s i o n The majority of the B. japonicum strains were resistant to Ap, Km, Sm and Tc below 100 #g/ml of medium, but 96% were resistant to Cm at or above 100 #g/ml (Fig. 1). The levels of resistance of strains to Ap, Km, Cm and Tc followed a unimodal distribution (Trabulsi et al. 1970): that is the number of strains increased with increasing concentration of the antibiotic, reached a maximum then decreased as the concentration of antibiotics increased further. In the presence of Sm there was a different pattern: two maxima, one at 10 #g/ml the other at 100 #g/ml, were apparent. According to Trabulsi et al. (1970) this is a bimodal distribution where the first group of strains with lower resistance can be considered as sensitive and the second group with greater resistance as resistant. Microbial antagonists present in the rhizosphere of leguminous plants can influence the competition and survival
50
AMPICILLIN
Z < n,los
I
KANAMYCIN
CHLORAMPHENICOL
STREPTOMYCIN
TETRACYCLINE
30-
b_
0 n~ W
20-
00 Z
0
o o o o o o
o o o o o o
o o o o o o
o o o o o o
o o o o o
o
LEVELS OF RESISTANCE (pug/ml) Figure 1. Frequency of levels of natural resistance to antibiotics of 50 Bradyrhizobium japonicum strains grown in medium 79 for 8 days at 28~ (average of three replications).
341
A.C. Borges et al. of rhizobia, as much as their level of resistance to anti-microbial compounds and their capacity to establish the symbiosis (Schreven 1964). The knowledge of the levels of resistance of B. japonicum strains to antibiotics can aid in tracing the strains introduced into the soil, in studies of competitivity for nodule infection sites and in ecological studies (Beynon & Josey 1980). Chloramphenicol and streptomycin are probably the markers of choice for genetic studies of the B. japonicum strains tested.
Acknowledgements This study was funded by grants from the Conselho Nacional de Desenvolvimento Cientifico e Tecnol6gico (CNPq) and Coordenag~o de Aperfeigoamento de Pessoal de Nivel Superior (CAPES), Brazil.
References ALVES, M.F. & AZEVEDO,J.L. 1975 Isolamento e caracterizaggo de mutantes de Rhizobiura japonicum resistentes ~ estreptomicina. Summa Phytopato/ogica 1, 205-212. AYANABA,A. & gONG, A.L. 1982 Antibiotic-resistant mutants identified from nodules of uninoculated soybeans grown in a strongly acidic soil. Soil Biologyand Biochemistry 14, 139-143. AZEVEDO, J.L., CASSINI,S.T.A. & OL1VEIRA,A. 1982 Replicador multialqa para uso geral em bacteriologia. Po/iagro 4, 11-18. BEYNON,J.L. & JOSEY,D.P. 1980 Demonstration of heterogeneity in a natural population of Rhizobium phaseo/i using variation in intrinsic antibiotic resistance. Journal of Genera/ Microbiology 118, 437-442. BROCKWELL, J., SCHWINGHAMER, E.A. & GAULT, R.R. 1977 Ecological studies of root-nodule bacteria introduced into field environment~V. A critical examination of the stability of antigenic and streptomycin resistance markers for identification of strains of Rhizobium trifo/ii. Soil Biology and Biochemistry 9, 19-24. FRED, E.B. & WAKSMAN, S.A. 1932 Laboratory Manual of General Microbiology. New York: McGraw-Hill. GRAHAM, P.H. 1963 Antibiotic sensitivities of root nodule bacteria. Australian Journal of BiologicalScience 16, 557-559. LEVIN, R.A. & MONTGOMERY,M.P. 1974 Symbiotic effectiveness of antibiotic-resistant mutants of Rhizobiumjapon#um. Plant and Soil 41, 669-676. SCHREVEN,D.A. VAN 1964 The effect of some actinomycetes on rhizobia and Agrobacterium radiobater. Plant and Soil 21, 283-302. TRABULSI, L.R., ZULIANI,M.E. & TOLEDO,M.R.F. 1970 Resistance to nine drugs of Shigel/a strains isolated in S~o Paulo between 1963-1968. Revistade Microbio/ogia1, 71-72.
(Received 9 January 1990; revised and accepted 11 May 1990)
842
