- Email: [email protected]
Antibiotic-resistant mutants identified from nodules of uninoculated soybeans grown in a strongly acidic soil
5011 tiwl Bio~hcm. Vol. 14. pp. I39 to 143. IYX? Printed in Great Brilnin. All nghts reserved
Copyright
003%0717/82/02013Y-05SO3.00/0 0 1982 Pergamon Press Ltd
ANTIBIOTIC-RESISTANT MUTANTS IDENTIFIED FROM NODULES OF UNINOCULATED SOYBEANS GROWN IN A STRONGLY ACIDIC SOIL
fnternational
A. AYANAEAand A. L. WONG Institute of Tropical Agriculture, PMB 5320 Ibadan, Nigeria (Accepted 10 August 1981)
Summary-Two hypotheses that antibiotic-resistant nodule isolates from uninoculated soybeans grown in a strongly acidic soil were naturally occurring rhizobia which had acquired resistance to spectinomytin and streptomycin or were contaminants from adjacent, inoculated treatments, were tested in laboratory experiments. Soybean nodule isolates (166) as well as 48 cowpea and 89 Rhizobiumjaponicum strains were used in tests of resistance to six concentrations (O-500 pg ml- ‘) of kanamycin, spectinomycin and streptomycin, tolerance of stresses of pH 4.6, with or without 50 PM Al, and serological cross-reactivity. More strains from the strongly acidic soil were resistant to the antibiotics than from slightly acidic soils, but resistance to antibiotics did not necessarily entail resistance to pH 4.6 or to 50 PM Al. Twenty-three nodule isolates which were resistant to spectinomycin or streptomycin cross-reacted with antisera of the inoculum strains, indicating that they were contaminants. None of 59 antibioticsensitive nodule isolates from uninoculated plants and none of 31 from inoculated plants cross-reacted with the antisera. All 53 antibiotic-resistant isolates from nodules of plants in inoculated plots crossreacted with test antisera, indicating stability of the antibiotic markers. Cowpea rhizobia were generally more resistant to the antibiotics and more tolerant of pH 4.6 and 50~~ Al than were R. japonicum. Among strains of R. japonicum, slow growers were more resistant to antibiotics than moderately fast growers.
tal sites, cross-contamination from inoculated plots was greater at the strongly acidic soil site. We report experiments to test the two hypotheses.
INTRODUCTION
The suitability of the antibiotic-resistance marker method for ecological studies of rhizobia has been demonstrated by Brockwell er ul. (1977). When a single antibiotic is the marker for rhizobia introduced into field environments, subsequent identification of nodule isolates may not be unequivocal, particularly, if they are from nodules of uninoculated plants. Although cross-contamination is often the cause, an alternative possibility is soil-induced genetic changes of naturalized populations. The latter is seldom given serious consideration. We used the antibiotic-resistance marker method to study the persistence of singly-labeled spectinomycin and streptomycin mutants of Rhizobium juponicum introduced into a strongly acidic (pH 4.6, HzO) Ultisol at Onne, Nigeria, and in a slightly acidic (pH 6.5, HxO) Alfisol at Ibadan (Bromfield and Ayanaba, 1980). With successive croppings over 2 yr, we found that over 91% of nodule isolates from plants grown in the uninoculated, control plots on the strongly acidic soil grew on antibiotic media; the corresponding values for the less acidic soil remained low at 5% (A. Ayanaba, A. L. Wong and E. S. P. Bromfield, unpublished). We formulated two hypotheses in an attempt to identify the rhizobia. First, it was possible that naturalized cowpea or soybean rhizobia had acquired resistance to the antibiotics. More generally, we supposed that rhizobia resistant to extreme acidity factors also could be resistant (cross-resistant) to the antibiotics. An alternative hypothesis was that in spite of similar precautionary measures at both experimen-
MATERIALSAND METHODS Rhizobia
A description of the broad groupings of the 136 strains used in these experiments is presented in Table 1; their identities are available upon request. Antibiotic resistance assays
Petri plates containing the yeast-mannitol agar medium (YMA) of Vincent (1970) with 0, 5, 10, 50, 100, 250 or 500 pg ml- ’ of filter-sterilized streptomycin sulfate, spectinomycin sulfate or kanamycin sulfate were prepared. Week-old cultures of 47 cowpea and 86 soybean rhizobia (wild type and mutants; Table 1) were washed with sterile water from the surface of YMA plates and the resulting suspension was used to inoculate plates at each concentration of antibiotic with sterile cotton swabs. Inoculated plates were incubated at 25-28°C for 7-10 days and scored for growth by comparison with growth on YMA without antibiotics. Tolerance to low pH and high aluminum
The defined media of Keyser and Murms (1979) solidified with agar, with and without 50~ Al, at pH 4.6 were used. Cell suspensions wete prepared (Keyser and MUMS, 1979) and the plates were inoculated with sterile cotton swabs. A selection of 44 “cowpea” rhizobia and 31 R. japonicum strains was
139
140
A.
AVANABA
and A. L. WONC;
Table 1. Description of 136 rhizobial strains used in the tests Description
Group
Cowpea rhizobia, isolated from Vigna unguiculatn grown in slightly acidic soils Same as Group A, but from strongly acidic Ultisol from Onne “Cowpea” miscellany rhizobia, non-
Vigna isolates Antibiotic-resistant mutants of rhizobia in Groups A and B Sub-total for “Cowpea” rhizobia Indigenous rhizobia isolated from Glycine mux; they nodulate both G. max and I! unguiculata. Slow growers Same as Group E but moderately fast growers Authentic R. japonicum from culture collections Antibiotic-resistant and other mutants of strains in Groups E, F and G Sub-total for R. japonicum
Number
21 19 I
2 49 39 10 18 20 82
used; all were resistant to > lOO~gml_ ’ of either kanamycin, spectinomycin or streptomycin. Serological
tests
Antisera of the two antibiotic resistance mutants IRj 2lOlspc’ (resistant to 2OOpg ml-’ spectinomycin; wild type is strain 46 from M. N. Zayed of Zambia) and IRj 2114str’ (resistant to 200 pg ml- ’ streptomycin; wild type is strain RCR 3407 = CB 1809 from R. J. Roughley of Rothamsted Experimental Station, UK) were produced and used as described by Vincent (1970) for somatic agglutination. Antisera were diluted 1:800 and 0.5 ml mixed with 0.5 ml washed ceil suspension of whole cells to provide a final dilution of 1: 1600. Twenty-three rhizobia isolated from randomly-picked nodules of uninoculated plants and 53 from inoculated plants which grew in antibioticcontaining media were transferred onto plates of YMA, allowed to grow for 7 days and washed with 0.W~; saline before testing. Ninety nodule isolates which grew in control but not antibiotic media were similarly tested. RESULTS
Antibiotic resistunre USSLI~S
The 114 wild-type rhizobia showed three types of response to the three antibiotics (Table 2). First, at antibiotic concentrations < 100 pg ml-t, the proportion of strains resistant to kanamycin was least and to spectinomycin most. Resistance to the antibiotics was not related to their molecular weights. Second, cowpea rhizobia of Groups A, B and E were generally more resistant to high concentrations of the antibiotics than were R. japonicum strains (Groups F and G). Third, with the exception of the 9 strains resistant to kanamycin, cowpea strains from the strongly acidic soil were more resistant to high concentrations (100-500 fig ml- t) of the antibiotics than strains from less acidic soils.
Antibiotic-resistant Table 3. Cross-resistance
Source of resistance*
141
mutants
of mutants of “cowpea” and soybean rhizobia to antibiotics at the three highest concentrations Number of strains growing at concentration Kanamycin(~g ml- ‘) of ~tibiotic shown Spectmomycin Streptomycin
No. of strains tested
100
250
500
100
250
500
100
250
500
1 5 7 5 1
1 0 0 1 0
1 0 0 0 0
1 x
0 5 0 5 0
‘0 5 0
0
0 0
0 0 :
0 0 7
:
:
0
;
kan’ spc’ str’ spc’/str’ cry’
x
5 0
* spc’/str’ denote resistance mutants labeled in the sequence spectinomycin, then streptomycin. each at 250 fig ml- ‘. cry’ = erythromycin-resistant (> 200 pg ml-t).
Antibiotic-resistant mutants failed to grow in media containing the three antibiotics unless they were de-
rived from parents resistant to the antibiotics (Table 3). However, one mutant resistant to both spectinomycin and streptomycin grew in media with 1OOpg ml- ’ kanamycin.
strains (Table 5). Isolates which could not grow in antibiotic media were not agglutinated, whether they were from the strongly acidic soil or not. A similar result was obtained with the isolates from inoculated plots. DISCUS!SION
Tolerance to Iow pH und high Al
Not all wild type strains which were resistant to high concentrations of antibiotics tolerated pH 4.6 or 50~~ Al (Table 4). Sixteen of the 63 strains tolerated acidity and 10 of these 16 also tolerated the Al stress. A greater proportion (9 of 17) of cowpea strains of acid soil origin tolerated acidity than did strains of non-acid soil origin. Three of the 12 antibiotiGr~istant mutants tolerated low pH or high Al; all were derived from parents tolerant of these stresses. On stress media, some strains formed micro-colonies which, if grown for 10 days, coalesced to form confluent colonies. On non-stress media such strains always formed single, macro-colonies. It seems that such strains, if scored for growth on stress media after prolonged incubation or if grown in liquid media, would be incorrectly considered as stress-tolerant. Serological tests
All antibiotic-resistant- isolates from uninoculated plots cross-reacted with antisera of the inoculum
Our first hypothesis was that naturally-occurring rhizobia had acquired resistance to spectinomycin and streptomycin. If so, a factor present in the strongly acidic soil but not the slightly acidic one, had rendered rhizobia in the former soil more prone to acquire antibiotic resistance. The major difference between the two soils is acidity. The principal source of acidity in the strongly acidic soil is exchangeable aluminum (Juo and Ballaux, 1977). Therefore, we reasoned that indigenous rhizobia which tolerated acidity or aluminum could acquire resistance to the antibiotics used and their growth in antibiotic media would have accounted for the higher proportion of antibiotiG~sitive nodule isolates from control plots in the strongly acidic soil. Although cross-resistance between antibiotic tolerance and acidity tolerance is not known, cross-resistance to antibiotics among rhizobia is known. Thus, Schwinghamer (1967) noted that mutants which were selected for resistance to streptomycin showed a weak
Table 4. Tolerance of “cowpea” and soybean rhizobia to acidity (pH 4.6) and high aliminum (50 PM Al at pH 4.6) stresses. All were resistant to 3 100 fig ml- 1 of either kanamycin, spectinomycin or streptomycin
Description Wild type “cowpea” strains Acid soil origin* Non-acid soil origin Wild type soybean strains R. japonicum from acid soil R. ja~nicum from non-acid soiit Antibiotic resistance mutants Cowpea strains Soybean strains
Number examined ::
l&O) 2 10
No. of rhizobia growing in stress medium Aluminum Acid 9 2
6 0
0 S(3)
0 4(3)
:
0 2
* Acid soil refers to the strongly acidic soil of Gnne; non-acid soil refers to slightly acid soils. t Numbers in parentheses are of “cowpea” type rhizobia; see Table 1, Group E.
A. AYANABAand A. L. WONG
142
Table 5. Somatic agglutination reactions of nodule isolates by antisera produced against R. japonicum strains IRj 2lOlspc’ and IRj 2114str’ Responses of isolates to pH of antibiotics soil of shown* isolation str’ spc’ str”/spc” stP/spcs
4.6 4.6 4.6 6.5
isolates from uninoculated Isolates from plots inoculated plots No. Reaction No. Reaction tested tested (%I (%I 13 10 2 57
100 100 0 0
31 22 3:
100 100 0 0
* str = streptomycin and spc = spectinomycin. The superscripts r and s denote resistance and sensitivity, respectively, to the antibiotics shown.
cross-resistance to kanamycin, and he found crossresistance among mutant strains to kanamycin, neomycin and viomycin. To test our first hypothesis, we compared intrinsic antibiotic resistance of wiid-type strains from the strongly acidic soil with those from less acidic soils and, examined the ability of rhizobia which grew in media containing high concentrations of antibiotics to also tolerate the acidity stresses of low pH and high Al. The results showed that a higher proportion of wild-type strains from the strongly acidic soil were tolerant of the three highest concentrations of spectinomycin and streptomycin than strains of less acidic soil origin. However, a selection of the wild type rhizobia which tolerated high concentrations of antibiotics did not necessarily tolerate low pH or high Al. Moreover. none of the antibiotic resistance mutants tolerated acidity unless their parents were tolerant. Thus, our data do not show cross-resistance between the antibiotics and pH or Al, nor do they show that antibiotic resistance was acquired. To test the second hypothesis. antigenic character of the two inoculum strains was used as a second marker to each antibiotic. The results strongly suggested that all the isolates from uninoculated plots which tolerated high concentrations of antibiotics were the inoculum strains. We were surprised at the result because several precautions had been taken to minimize rhizobial cross-contamination at both iocations (Bromtield and Ayanaba, 1980). The experimental design at the acidic soil site was split-split plot with four replications; inoculation treatments were main plots, lime treatments were sub-plots and cultivars were sub-sub plots. Main plots were 2 m apart, with bunds and drainage channels around all plots. Uninoculated seed was sown before inoculated seed, and in subsequent operations clean polyethylene boots were worn prior to entry into plots and all implements were rinsed in 707; alcohol before use. Nodules were collected from plants harvested from the second row, free of edge effects. But because the site was over 5OOkm from Ibadan, animal and human movement over the plots could not be controlled as rigorously as at Ibadan. Such movement could have caused rhizobial cro~~nt~inaiion. It is also very likely that the greater amount of rainfall on the acidic soil (> 2.5 m as against 1.25 m on the less acidic soil) may have caused rhizobial movement to uninoculated
plots. When Brockwell et al. (1977) identified inoculum strains in uninoculated plants, they suggested water movement or human agency as the cause of contamination. The propensity for genetic transfer among Rhhbium in soil is low {Schwinghamer and Dudman, 1973); so also is mutation in soil (Brockwell ef al., 1977). Our finding that the antigenic character was neither gained nor lost and that antibiotic resistance was not acquired. confirm these views. Moreover, it seems that even in a soil with severe stresses as exist in the strongly acidic soil (pH 4.6 and exchangeable Al concentrations of up to 2.0m-equiv lOOg_’ soil) these processes of selection are unaffected in the short term. We could not corroborate the observation of Schwinghamer (1967) concerning cross-resistance between streptomycin and kanamycin resistance; this could have been due to differences in rhizobial species or concentrations of antibiotics. Schwinghamer (1967) employed mutants of R. leg~minos~r~m, R. trzyofii, R. p~aseoli and R. rne~~iot~and less than IOO~g ml-’ antibiotics, We employed R. ,~a~on~curnand cowpea rhizobia, and because of our interest in the likelihood of cross-resistance occurring under natural field conditions, we examined “ecological concentrations (2 100 pg ml- ‘)” of the antibiotics. Also, there was no cross-resistance between spectinomycin and streptomycin resistance, even though these antibiotics have similar molecular structure and mode of action. This is in agreement with the report by Schwinghamer and Dudman (1973). It was interesting to observe that cowpea rhizobia were more tolerant of high concentrations of antibiotics than R. j~~~~c~rn. Graham (1963) reported that slow-growing rhizobia of the cowpea and soybean groups were more tolerant of antibiotics than fast growers of the clover-pea group. Pankhurst (1977) found slow-growing Lotus rhizobia to be. more resistant to antibiotics, including spectinomycin and strep tomycin. than fast-growing Lotus rhizobia. Our results show similar effects of antibiotics on cowpea and soybean rhizobia and suggest that an “ecological concentration” of the antibiotics can be used to further distinguish among slow-growing soybean rhizobia. Thus, the 49 indigenous soybean strains which also noduiate cowpea (Table 2) and the 47 cowpea strains (Table 2) responded similarly. Keyser and Munns (1979) reported that cowpea rhizobia were more tolerant of acidity and Al than soybean rhizobia; moreover, strains which tolerated Al also tolerated acidity but not vice versa. Our observations agree with theirs. In conclusion, contamination of control plots by inoculum strains accounted for the recovery of antibiotic resistance mutants from uninoculated plants. But the fact that a second marker had to be resorted to to prove this strengthens the call by Brockwell er al. (1977) and others for the use of secondary or tertiary markers as an insurance measure. In this regard, our observation that antibiotic resistance mutants tolerated soil acidity stresses suggests that rhizobial toierance of 50~~ Al at low pH has some potential. Arknowledgzmenrs-IITA journal series paper no. 171. This work was supported in part by the United Nations
Antibiotic-resistant Environment Programme. Spectinomycin sulfate was a gift from the Upjohn-Co.. US& and D; G. H. Elkan. USA. nrovided the rhizobial mutants of kanamvcin and ervthromycin.
REFERENCES BROCKWELLJ., SCHWINGHAMER E. A. and GAULT R. R. (1977) Ecological studies of root-nodule bacteria introduced into field environments-V. A critical examination of the stability of antigenic and streptomycin-resistance markers for identification of strains of Rhizobium trifolii. Soil Biology & Biochemistry 9, 19-24. BROMFIELD E. S. P. and AYANABAA. (1980) The efficacv of soybean inoculation on acid soil in iropical Africa. Plant & Soil 54,95-K%. GRAHAMP. H. (1963) Antibiotic sensitivities of the root nodule bacteria. Australian Journal of Biological Sciences 16, 557-559.
mutants
143
Juo A. S. R. and BALLAUXJ. C. (1977) Retention and leaching of nutrients in a limed ultisol under cropping. Journul Soil Science Society of America 41. 757-761. KEYSERH. H. and MUNI& b. N. (1979) Tolerance of rhizobia to acidity, aluminum and phosphate. Journul Soil Science Socierv of America 43. 519-523. PANKHURST C. e. (i977) Symbiotic effectiveness of antibiotic-resistant mutants of fast and slow-growing strains of Rhizohium nodulating Lotus species. Cunudiun Journul of Microbiology 23, 10261033. SCHWINGHAMER E. A. (1967) Effectiveness of Rhizobium as modified by mutation for resistance to antibiotics. Antonie van Leeuwenhoek 33, 121-136. SCHWINGHAMER E. A. and DUDMANW. F. (1973) Evaluation of spectinomycin resistance as a marker for ecological studies with Rhizohium spp. Journul of Applied Bacreriology 36, 263-272. VINCENT.J.M. (1970) A Manual for the Pracrical Study of Root Nodule Bacteria. IBP Handbook 15. Blackwells. Oxford.
Copyright
003%0717/82/02013Y-05SO3.00/0 0 1982 Pergamon Press Ltd
ANTIBIOTIC-RESISTANT MUTANTS IDENTIFIED FROM NODULES OF UNINOCULATED SOYBEANS GROWN IN A STRONGLY ACIDIC SOIL
fnternational
A. AYANAEAand A. L. WONG Institute of Tropical Agriculture, PMB 5320 Ibadan, Nigeria (Accepted 10 August 1981)
Summary-Two hypotheses that antibiotic-resistant nodule isolates from uninoculated soybeans grown in a strongly acidic soil were naturally occurring rhizobia which had acquired resistance to spectinomytin and streptomycin or were contaminants from adjacent, inoculated treatments, were tested in laboratory experiments. Soybean nodule isolates (166) as well as 48 cowpea and 89 Rhizobiumjaponicum strains were used in tests of resistance to six concentrations (O-500 pg ml- ‘) of kanamycin, spectinomycin and streptomycin, tolerance of stresses of pH 4.6, with or without 50 PM Al, and serological cross-reactivity. More strains from the strongly acidic soil were resistant to the antibiotics than from slightly acidic soils, but resistance to antibiotics did not necessarily entail resistance to pH 4.6 or to 50 PM Al. Twenty-three nodule isolates which were resistant to spectinomycin or streptomycin cross-reacted with antisera of the inoculum strains, indicating that they were contaminants. None of 59 antibioticsensitive nodule isolates from uninoculated plants and none of 31 from inoculated plants cross-reacted with the antisera. All 53 antibiotic-resistant isolates from nodules of plants in inoculated plots crossreacted with test antisera, indicating stability of the antibiotic markers. Cowpea rhizobia were generally more resistant to the antibiotics and more tolerant of pH 4.6 and 50~~ Al than were R. japonicum. Among strains of R. japonicum, slow growers were more resistant to antibiotics than moderately fast growers.
tal sites, cross-contamination from inoculated plots was greater at the strongly acidic soil site. We report experiments to test the two hypotheses.
INTRODUCTION
The suitability of the antibiotic-resistance marker method for ecological studies of rhizobia has been demonstrated by Brockwell er ul. (1977). When a single antibiotic is the marker for rhizobia introduced into field environments, subsequent identification of nodule isolates may not be unequivocal, particularly, if they are from nodules of uninoculated plants. Although cross-contamination is often the cause, an alternative possibility is soil-induced genetic changes of naturalized populations. The latter is seldom given serious consideration. We used the antibiotic-resistance marker method to study the persistence of singly-labeled spectinomycin and streptomycin mutants of Rhizobium juponicum introduced into a strongly acidic (pH 4.6, HzO) Ultisol at Onne, Nigeria, and in a slightly acidic (pH 6.5, HxO) Alfisol at Ibadan (Bromfield and Ayanaba, 1980). With successive croppings over 2 yr, we found that over 91% of nodule isolates from plants grown in the uninoculated, control plots on the strongly acidic soil grew on antibiotic media; the corresponding values for the less acidic soil remained low at 5% (A. Ayanaba, A. L. Wong and E. S. P. Bromfield, unpublished). We formulated two hypotheses in an attempt to identify the rhizobia. First, it was possible that naturalized cowpea or soybean rhizobia had acquired resistance to the antibiotics. More generally, we supposed that rhizobia resistant to extreme acidity factors also could be resistant (cross-resistant) to the antibiotics. An alternative hypothesis was that in spite of similar precautionary measures at both experimen-
MATERIALSAND METHODS Rhizobia
A description of the broad groupings of the 136 strains used in these experiments is presented in Table 1; their identities are available upon request. Antibiotic resistance assays
Petri plates containing the yeast-mannitol agar medium (YMA) of Vincent (1970) with 0, 5, 10, 50, 100, 250 or 500 pg ml- ’ of filter-sterilized streptomycin sulfate, spectinomycin sulfate or kanamycin sulfate were prepared. Week-old cultures of 47 cowpea and 86 soybean rhizobia (wild type and mutants; Table 1) were washed with sterile water from the surface of YMA plates and the resulting suspension was used to inoculate plates at each concentration of antibiotic with sterile cotton swabs. Inoculated plates were incubated at 25-28°C for 7-10 days and scored for growth by comparison with growth on YMA without antibiotics. Tolerance to low pH and high aluminum
The defined media of Keyser and Murms (1979) solidified with agar, with and without 50~ Al, at pH 4.6 were used. Cell suspensions wete prepared (Keyser and MUMS, 1979) and the plates were inoculated with sterile cotton swabs. A selection of 44 “cowpea” rhizobia and 31 R. japonicum strains was
139
140
A.
AVANABA
and A. L. WONC;
Table 1. Description of 136 rhizobial strains used in the tests Description
Group
Cowpea rhizobia, isolated from Vigna unguiculatn grown in slightly acidic soils Same as Group A, but from strongly acidic Ultisol from Onne “Cowpea” miscellany rhizobia, non-
Vigna isolates Antibiotic-resistant mutants of rhizobia in Groups A and B Sub-total for “Cowpea” rhizobia Indigenous rhizobia isolated from Glycine mux; they nodulate both G. max and I! unguiculata. Slow growers Same as Group E but moderately fast growers Authentic R. japonicum from culture collections Antibiotic-resistant and other mutants of strains in Groups E, F and G Sub-total for R. japonicum
Number
21 19 I
2 49 39 10 18 20 82
used; all were resistant to > lOO~gml_ ’ of either kanamycin, spectinomycin or streptomycin. Serological
tests
Antisera of the two antibiotic resistance mutants IRj 2lOlspc’ (resistant to 2OOpg ml-’ spectinomycin; wild type is strain 46 from M. N. Zayed of Zambia) and IRj 2114str’ (resistant to 200 pg ml- ’ streptomycin; wild type is strain RCR 3407 = CB 1809 from R. J. Roughley of Rothamsted Experimental Station, UK) were produced and used as described by Vincent (1970) for somatic agglutination. Antisera were diluted 1:800 and 0.5 ml mixed with 0.5 ml washed ceil suspension of whole cells to provide a final dilution of 1: 1600. Twenty-three rhizobia isolated from randomly-picked nodules of uninoculated plants and 53 from inoculated plants which grew in antibioticcontaining media were transferred onto plates of YMA, allowed to grow for 7 days and washed with 0.W~; saline before testing. Ninety nodule isolates which grew in control but not antibiotic media were similarly tested. RESULTS
Antibiotic resistunre USSLI~S
The 114 wild-type rhizobia showed three types of response to the three antibiotics (Table 2). First, at antibiotic concentrations < 100 pg ml-t, the proportion of strains resistant to kanamycin was least and to spectinomycin most. Resistance to the antibiotics was not related to their molecular weights. Second, cowpea rhizobia of Groups A, B and E were generally more resistant to high concentrations of the antibiotics than were R. japonicum strains (Groups F and G). Third, with the exception of the 9 strains resistant to kanamycin, cowpea strains from the strongly acidic soil were more resistant to high concentrations (100-500 fig ml- t) of the antibiotics than strains from less acidic soils.
Antibiotic-resistant Table 3. Cross-resistance
Source of resistance*
141
mutants
of mutants of “cowpea” and soybean rhizobia to antibiotics at the three highest concentrations Number of strains growing at concentration Kanamycin(~g ml- ‘) of ~tibiotic shown Spectmomycin Streptomycin
No. of strains tested
100
250
500
100
250
500
100
250
500
1 5 7 5 1
1 0 0 1 0
1 0 0 0 0
1 x
0 5 0 5 0
‘0 5 0
0
0 0
0 0 :
0 0 7
:
:
0
;
kan’ spc’ str’ spc’/str’ cry’
x
5 0
* spc’/str’ denote resistance mutants labeled in the sequence spectinomycin, then streptomycin. each at 250 fig ml- ‘. cry’ = erythromycin-resistant (> 200 pg ml-t).
Antibiotic-resistant mutants failed to grow in media containing the three antibiotics unless they were de-
rived from parents resistant to the antibiotics (Table 3). However, one mutant resistant to both spectinomycin and streptomycin grew in media with 1OOpg ml- ’ kanamycin.
strains (Table 5). Isolates which could not grow in antibiotic media were not agglutinated, whether they were from the strongly acidic soil or not. A similar result was obtained with the isolates from inoculated plots. DISCUS!SION
Tolerance to Iow pH und high Al
Not all wild type strains which were resistant to high concentrations of antibiotics tolerated pH 4.6 or 50~~ Al (Table 4). Sixteen of the 63 strains tolerated acidity and 10 of these 16 also tolerated the Al stress. A greater proportion (9 of 17) of cowpea strains of acid soil origin tolerated acidity than did strains of non-acid soil origin. Three of the 12 antibiotiGr~istant mutants tolerated low pH or high Al; all were derived from parents tolerant of these stresses. On stress media, some strains formed micro-colonies which, if grown for 10 days, coalesced to form confluent colonies. On non-stress media such strains always formed single, macro-colonies. It seems that such strains, if scored for growth on stress media after prolonged incubation or if grown in liquid media, would be incorrectly considered as stress-tolerant. Serological tests
All antibiotic-resistant- isolates from uninoculated plots cross-reacted with antisera of the inoculum
Our first hypothesis was that naturally-occurring rhizobia had acquired resistance to spectinomycin and streptomycin. If so, a factor present in the strongly acidic soil but not the slightly acidic one, had rendered rhizobia in the former soil more prone to acquire antibiotic resistance. The major difference between the two soils is acidity. The principal source of acidity in the strongly acidic soil is exchangeable aluminum (Juo and Ballaux, 1977). Therefore, we reasoned that indigenous rhizobia which tolerated acidity or aluminum could acquire resistance to the antibiotics used and their growth in antibiotic media would have accounted for the higher proportion of antibiotiG~sitive nodule isolates from control plots in the strongly acidic soil. Although cross-resistance between antibiotic tolerance and acidity tolerance is not known, cross-resistance to antibiotics among rhizobia is known. Thus, Schwinghamer (1967) noted that mutants which were selected for resistance to streptomycin showed a weak
Table 4. Tolerance of “cowpea” and soybean rhizobia to acidity (pH 4.6) and high aliminum (50 PM Al at pH 4.6) stresses. All were resistant to 3 100 fig ml- 1 of either kanamycin, spectinomycin or streptomycin
Description Wild type “cowpea” strains Acid soil origin* Non-acid soil origin Wild type soybean strains R. japonicum from acid soil R. ja~nicum from non-acid soiit Antibiotic resistance mutants Cowpea strains Soybean strains
Number examined ::
l&O) 2 10
No. of rhizobia growing in stress medium Aluminum Acid 9 2
6 0
0 S(3)
0 4(3)
:
0 2
* Acid soil refers to the strongly acidic soil of Gnne; non-acid soil refers to slightly acid soils. t Numbers in parentheses are of “cowpea” type rhizobia; see Table 1, Group E.
A. AYANABAand A. L. WONG
142
Table 5. Somatic agglutination reactions of nodule isolates by antisera produced against R. japonicum strains IRj 2lOlspc’ and IRj 2114str’ Responses of isolates to pH of antibiotics soil of shown* isolation str’ spc’ str”/spc” stP/spcs
4.6 4.6 4.6 6.5
isolates from uninoculated Isolates from plots inoculated plots No. Reaction No. Reaction tested tested (%I (%I 13 10 2 57
100 100 0 0
31 22 3:
100 100 0 0
* str = streptomycin and spc = spectinomycin. The superscripts r and s denote resistance and sensitivity, respectively, to the antibiotics shown.
cross-resistance to kanamycin, and he found crossresistance among mutant strains to kanamycin, neomycin and viomycin. To test our first hypothesis, we compared intrinsic antibiotic resistance of wiid-type strains from the strongly acidic soil with those from less acidic soils and, examined the ability of rhizobia which grew in media containing high concentrations of antibiotics to also tolerate the acidity stresses of low pH and high Al. The results showed that a higher proportion of wild-type strains from the strongly acidic soil were tolerant of the three highest concentrations of spectinomycin and streptomycin than strains of less acidic soil origin. However, a selection of the wild type rhizobia which tolerated high concentrations of antibiotics did not necessarily tolerate low pH or high Al. Moreover. none of the antibiotic resistance mutants tolerated acidity unless their parents were tolerant. Thus, our data do not show cross-resistance between the antibiotics and pH or Al, nor do they show that antibiotic resistance was acquired. To test the second hypothesis. antigenic character of the two inoculum strains was used as a second marker to each antibiotic. The results strongly suggested that all the isolates from uninoculated plots which tolerated high concentrations of antibiotics were the inoculum strains. We were surprised at the result because several precautions had been taken to minimize rhizobial cross-contamination at both iocations (Bromtield and Ayanaba, 1980). The experimental design at the acidic soil site was split-split plot with four replications; inoculation treatments were main plots, lime treatments were sub-plots and cultivars were sub-sub plots. Main plots were 2 m apart, with bunds and drainage channels around all plots. Uninoculated seed was sown before inoculated seed, and in subsequent operations clean polyethylene boots were worn prior to entry into plots and all implements were rinsed in 707; alcohol before use. Nodules were collected from plants harvested from the second row, free of edge effects. But because the site was over 5OOkm from Ibadan, animal and human movement over the plots could not be controlled as rigorously as at Ibadan. Such movement could have caused rhizobial cro~~nt~inaiion. It is also very likely that the greater amount of rainfall on the acidic soil (> 2.5 m as against 1.25 m on the less acidic soil) may have caused rhizobial movement to uninoculated
plots. When Brockwell et al. (1977) identified inoculum strains in uninoculated plants, they suggested water movement or human agency as the cause of contamination. The propensity for genetic transfer among Rhhbium in soil is low {Schwinghamer and Dudman, 1973); so also is mutation in soil (Brockwell ef al., 1977). Our finding that the antigenic character was neither gained nor lost and that antibiotic resistance was not acquired. confirm these views. Moreover, it seems that even in a soil with severe stresses as exist in the strongly acidic soil (pH 4.6 and exchangeable Al concentrations of up to 2.0m-equiv lOOg_’ soil) these processes of selection are unaffected in the short term. We could not corroborate the observation of Schwinghamer (1967) concerning cross-resistance between streptomycin and kanamycin resistance; this could have been due to differences in rhizobial species or concentrations of antibiotics. Schwinghamer (1967) employed mutants of R. leg~minos~r~m, R. trzyofii, R. p~aseoli and R. rne~~iot~and less than IOO~g ml-’ antibiotics, We employed R. ,~a~on~curnand cowpea rhizobia, and because of our interest in the likelihood of cross-resistance occurring under natural field conditions, we examined “ecological concentrations (2 100 pg ml- ‘)” of the antibiotics. Also, there was no cross-resistance between spectinomycin and streptomycin resistance, even though these antibiotics have similar molecular structure and mode of action. This is in agreement with the report by Schwinghamer and Dudman (1973). It was interesting to observe that cowpea rhizobia were more tolerant of high concentrations of antibiotics than R. j~~~~c~rn. Graham (1963) reported that slow-growing rhizobia of the cowpea and soybean groups were more tolerant of antibiotics than fast growers of the clover-pea group. Pankhurst (1977) found slow-growing Lotus rhizobia to be. more resistant to antibiotics, including spectinomycin and strep tomycin. than fast-growing Lotus rhizobia. Our results show similar effects of antibiotics on cowpea and soybean rhizobia and suggest that an “ecological concentration” of the antibiotics can be used to further distinguish among slow-growing soybean rhizobia. Thus, the 49 indigenous soybean strains which also noduiate cowpea (Table 2) and the 47 cowpea strains (Table 2) responded similarly. Keyser and Munns (1979) reported that cowpea rhizobia were more tolerant of acidity and Al than soybean rhizobia; moreover, strains which tolerated Al also tolerated acidity but not vice versa. Our observations agree with theirs. In conclusion, contamination of control plots by inoculum strains accounted for the recovery of antibiotic resistance mutants from uninoculated plants. But the fact that a second marker had to be resorted to to prove this strengthens the call by Brockwell er al. (1977) and others for the use of secondary or tertiary markers as an insurance measure. In this regard, our observation that antibiotic resistance mutants tolerated soil acidity stresses suggests that rhizobial toierance of 50~~ Al at low pH has some potential. Arknowledgzmenrs-IITA journal series paper no. 171. This work was supported in part by the United Nations
Antibiotic-resistant Environment Programme. Spectinomycin sulfate was a gift from the Upjohn-Co.. US& and D; G. H. Elkan. USA. nrovided the rhizobial mutants of kanamvcin and ervthromycin.
REFERENCES BROCKWELLJ., SCHWINGHAMER E. A. and GAULT R. R. (1977) Ecological studies of root-nodule bacteria introduced into field environments-V. A critical examination of the stability of antigenic and streptomycin-resistance markers for identification of strains of Rhizobium trifolii. Soil Biology & Biochemistry 9, 19-24. BROMFIELD E. S. P. and AYANABAA. (1980) The efficacv of soybean inoculation on acid soil in iropical Africa. Plant & Soil 54,95-K%. GRAHAMP. H. (1963) Antibiotic sensitivities of the root nodule bacteria. Australian Journal of Biological Sciences 16, 557-559.
mutants
143
Juo A. S. R. and BALLAUXJ. C. (1977) Retention and leaching of nutrients in a limed ultisol under cropping. Journul Soil Science Society of America 41. 757-761. KEYSERH. H. and MUNI& b. N. (1979) Tolerance of rhizobia to acidity, aluminum and phosphate. Journul Soil Science Socierv of America 43. 519-523. PANKHURST C. e. (i977) Symbiotic effectiveness of antibiotic-resistant mutants of fast and slow-growing strains of Rhizohium nodulating Lotus species. Cunudiun Journul of Microbiology 23, 10261033. SCHWINGHAMER E. A. (1967) Effectiveness of Rhizobium as modified by mutation for resistance to antibiotics. Antonie van Leeuwenhoek 33, 121-136. SCHWINGHAMER E. A. and DUDMANW. F. (1973) Evaluation of spectinomycin resistance as a marker for ecological studies with Rhizohium spp. Journul of Applied Bacreriology 36, 263-272. VINCENT.J.M. (1970) A Manual for the Pracrical Study of Root Nodule Bacteria. IBP Handbook 15. Blackwells. Oxford.