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Acid production, acid tolerance and growth rate of Lotus rhizobia in laboratory media
Soil Viol. Biochm. Vol. 14. pp. 127 10 131. 1982 Printed III Great Britain. All rlghts reserved
0038~717/82/020127-05fo3.00/0 CopyrIght 0 1982 Pergamon Press Ltd
ACID PRODUCTION, ACID TOLERANCE AND GROWTH RATE OF LOTUS RHIZOBIA IN LABORATORY MEDIA J.
E.
COOPER
Agricultural and Food Bacteriology Research Division, Department of Agriculture for Northern Ireland, Newforge Lane, Belfast BT9 5PX, Northern Ireland (Accepted 10 September 1981) Summary-Twenty-seven strains of Lotus rhizobia were tested for acid tolerance in yeast-extract mannitol broth (pH 4.6) by multiplication from low initial cell densities. Acid production on unbuffered yeast-extract mannitol agar slopes incorporating bromothymol blue indicator was also determined. Ail slow-growing, alkali-producing strains were acid sensitive and four of the six fast-growing, acidproducing strains were acid tolerant as was one fast-growing, alkali-producing strain. The method of testing for acid tolerance proved suitabte for fast-growing strains and results are discussed in relation to the ecological impIications of acid and alkali production by rhizobia.
INTRODUCTION
Effectiveness
The ability of rhizobia to tolerate low pH values and associated acidity stresses in laboratory media has been shown to be of predictive value in selecting strains which form effective symbioses with host plants in acid soils (Keyser er al., 1979). Tolerance of this type is a consistent and stable strain property, uninfluenced by previous cultural conditions (Munns and Keyser, 1981). Tolerance of low pH has been tested by two methods which allow a strain to multiply at a constant low pH value. Date and Halliday (1979) monitored the growth of an isolate from Stylosunthes in a defined medium containing arabinose or galactose at a constant pH of 4.5. Keyser and Munns (1979) found that the growth of alkali-producing, slow-growing strains from low numbers ( <104ml-‘) in acid, mannitol-based broths was not accompanied by any pH change before the attainment of turbidity (ca. 10’ cellsml-‘). This approach has not been applied to fast-growing, acid-producing strains commonly found in temperate zones. Norris (1965) considered slowgrowing strains to be generally more acid tolerant than the fast-growing types. Rhizobia from the genus Lotus were used in my study since they comprise both fast- and slow-growing types which can nodulate the same host. Tests of acid tolerance were applied to 27 strains of Lotus rhizobia and the relationships between growth rate, acid production and acid tolerance within the group were investigated. MATERIALS AND METHODS
Rhizobia
Twenty slow-growing and 7 fast-growing strains of rhizobia were used (Table 1). All strains were maintained at 4°C on yeast~xtract mannitol agar (YEMA) slopes incorporating 3 g C!aCOs I- ’ (Vincent, 1970). totus
tests
All strains were tested for effectiveness on Lotus pedunculatus growing in N-free rooting solution (PH
6.7) in 150 x 19 mm test tubes with seedlings sup ported by a roll of filter paper (Cooper, 1978). Plants were grown for 8 weeks in a Shearer CEL 255-6 growth cabinet with a 17 h, 20°C light period and a 7 h. 16°C dark interval. Maximum growth response was obtained with strain ~~814s while strain 1Ola was used as as ineffective control. Dry weight of tops provided the measure of effectiveness. Acid and alkali production
All strains were checked for net acid or alkali production on slopes of an unbuffered yeast-extract mannitol agar incorporating bromothymol blue indicator (Norris, 1965). For the fast-growing strain NZP2037 pH changes were also determined in a variety of liquid media which included yeast-extract mannitol broth (YEMB, Vincent, 1970) and defined broths in which mannitol, galactose or arabinose were used as C sources (Date and Ha&day, 1979). pH was measured by glass electrode on subsamples taken aseptically from flasks shaken at 150rev. min-’ and 28°C. Viable cells were also counted on the same samples by surface plating of dilutions on YEMA. Growth at low pH
All tolerance tests were based on multiplication of strains from low initial cell densities (~lO~rnl_~) in YEMB acidified to pH 4.6 with HCl. A selection of strains was studied in detail by monitoring viable counts and pH values throughout a Uday incubation. This was examined in duplicate 20ml amounts of YEMB in 50 ml flasks inoculated with dilutions of young cultures and shaken at 150 rev. min - f and 22°C. Periodically samples were withdrawn aseptically and. changes in pH and viable count were determined by methods previously de-
127
128
J. E.
COOPER
Table 1. Details of
Strain number
Speed of growth
Host plant
Rhizobium
strains Acid/alkali production on medium of Norris (1965)
Effectiveness on Lotus
pedunculatus
Source of strains
FL F2, F3, F4, F5, Fl, F44, F55, F79, F89, F90, F97, F98, FlOl, F114, F124 F129
L. pedunculatus
Slow
Effective except F44 and F129: ineffective
All alkali producers
cc806 cc807 ~~814s
L. pedunculatus L pedunculatus L. hispidus
Slow Slow Slow
Ineffective Effective Effective
Alkali Alkali Alkali
Division of Plant Industry, CSIRO, Canberra, Australia
396 391 421
Lotus Lotus Lotus
Fast Fast Fast
Ineffective Ineffective Ineffective
Alkali Acid Acid
Unidad Symbiosis, CICA, Castellar. Buenos Aires, Argentina
1Ola 102a
L. corniculatus L. corniculatus
Fast Fast
Ineffective Ineffective
Acid* Acid*
Baljvlxt Laboratoriet Sveriges Lantbruks Universitet, Uppsala, Sweden
NZP2037
Lotus
Fast
Effective
Acid
DSIR, Palmerston North, New Zealand
3EOa7
L. corniculutus
Fast
Ineffective
Acid
US Department of Agriculture, Beltsville, Maryland, U.S.A.
spp spp spp
spp
Northern Ireland pasture soils via plant-infection dilution assays
* Poor growth on Norris’ medium and weak acid production in YEMB
10 9
(a)
C
-6 . S-
vrn
0
2
4
6
6
IO0
2
4
6
-5 -4
6
-3 IO
Titno, days Fig. I. Growth of strain NZP2037 and pH changes in YEMB (a) and defined media with mannitol (bk galactose (c) or arabinose (d) as carbohydrate source. W----W pH; +O viable count.
129
Acid tolerance of Lotus rhizobia Growth in YEMB adjusted to pH 6.7 was monitored for comparative purposes. The remaining strains were screened in a test in which tolerance of acidity was indicated by visual turbidity within 10 days for fast-growing strains and 20 days for slow-growers. scribed.
RESULTS
Growth rate, acid and af~fj ~roduct~o~ All slow-growers were net alkali-producers on Norris’s medium whereas all fast-growers except strain 396 were net acid-pr~u#rs (Table 1). In symbiosis with L. pedunculutus the trend was for slow-growers to be effective and fast-growers ineffective. Of the latter group only strain NZP2037 was effective on this host. In the acid and alkali production tests on Norris’s medium all strains demonstrated unidirectional changes in pH. However, it was clear from the assess-
Acid tolerance
Figure 2 shows pH trends and viable counts for five
PH
.
lor
ment of pH trends in liquid media using strain NZP 2037 that the direction of change of pH was influen~d by the com~sition of the medium (Fig. lf In YEMB, the medium which most closely resembled the medium of Norris (1965) for determination of acid or alkali production, change in pH was consistently downwards with a marked fall only occurring at stationary phase. A similar pattern was observed in defined arabinose medium. In defined mannitol and galactose broths, pH actually increased during the logarithmic phase of growth and then fell sharply as stationary phase was reached. In the three defined media carbon was present in both the ~bohydrate component and glutamic acid. One slow-growing strain (cc 807) was cultured in YEMB and defined mannitol and galactose broths: in the three media the pH increased.
-8
8-
-7 -6 -5 k -4 -I $4
4-
Z 3
3
2-
iE Q
lO--8
4
0
46
0
4
6
12
16
20
24
20
24
3 0
4
6
12
I6
20
24
l2162?024
2L.
t 8
12
mn%dayr
I6
2 0
4
8
12
I6
20
24
Time, days
Fig. 2. Viable counts and pH trends of five fast-growing and two slow-growing strains of Lotus rhizobia in YEMB adjusted to pH 4.6 or 6.7. O---O viable counts at pH 6.7; O---O viable counts at pH 4.6; A-A pH trends from initial value of 6.7; A-A pH trends from initial value of 4.6.
J. E. COOPER
130 Table
2. Growth
of strains
at pH 4.6 in YEMB
from low initial cell densities
0 Strain Slow-growers,
no growth
Fast-growers,
growth
at
at pH 4.6
pH 4.6
Days 10* 2ot (Viable cells ml- ‘)
Fl F2 F3 F4 F5 F7 F44 F55 F89 F90 F97 F98 FlOl F114 F120 F129 cc 806 cc 807
78 364 146 170 112 104 150 210 238 146 120 200 274 174 100 168 50 340
IOla 102a
900 900
50 <50 <50 75 50 50 <50 50 75 <50 50 75 100 150 100 <50 50 t50
>lO’ > 10’
* Not-determined for slow-growers. t Not determined for fast-growers.
fast-growing and two slow-growing strains in YEMB adjusted to pH 4.6 or 6.7. Three of the fast-growers multiplied at the lower pH value, making between 104- and lo’-fold increases in number before altering the reaction of the medium. Of these. two were acidproducers and one was an alkali-producer. Neither slow-growing strain multiplied at pH 4.6 and the number of viable cells declined to undetectably low levels after 3 weeks. Results for the remainder of the strains, using the simpler screening test (Table 2) indicate none of the slow-growing strains multiplied at pH 4.6 whereas the two fast-growers multiplied readily. A count of viable cell numbers after 3 weeks indicated that populations of most of the sensitive strains had declined from the immediate post-inoculation level while a few were recovered at densities similar to those originally added. DISCUSSION
All fast-growing strains used in my study were, according to the records of the various supplying laboratories. effective on the hosts from which they were originally isolated. Their performance in the present effectiveness tests supports previous observations that dual effectiveness of strains, particularly on L. pedunculatus and L. corniculatus, is rare (Erdman and Means, 1949; Jensen and Hansen, 1968). Results from my study lend no support to the view that acid production in culture media is an indication of acid sensitivity (Norris, 1965; Brockwell et al., 1966; Bromfield and Jones, 1980). Indeed strain NZP 2037, a strong acid-producer in Norris’s medium, grew well at pH 4.6 whereas all the slow-growing, alkali-producing strains were acid sensitive. Acid or alkali production is a markedly variable strain characteristic and many studies have shown
that it is influenced by changes in composition of the growth medium (Lange, 1961; Jarvis et al., 197’7; Parker et al., 1977; Date and Halliday. 1979). My work shows, additionally, that acid or alkali can be generated in a single medium by one strain at different phases of the growth cycle (Fig. 1). On these grounds alone it would be unwise to attribute an ecological role to acid production by rhizobia. Whether rhizobia ever effect a change of pH in soils is open to serious doubt. Parker et al. (1977) reported no change in the pH of the rhizosphere of hosts or water extracts of soils with a variety of fast- and slowgrowing rhizobia. In this connection it is important to note that, in my study, pH changes were only associated with relatively high cell densities, even in unbuffered media (Fig. 2). The method of testing for acid tolerance by growth from low cell densities proved entirely satisfactory for fast-growing strains of Rhizobium and this type of test could be of practical value if it can be shown that the behaviour of organisms in laboratory media is similar to that in acid soils. Acknowledgements-1 thank the curators of the various culture collections (noted in Table 1) for supplying the strains of Rhi,-obium and Mr D. Campbell for isolating strains from Northern Ireland pasture soils.
REFERENCES S. K. and REA GERALDINEA. (1966) Acid production by rhizobia from the genera Trifolium and Lotus. Journal of the Australian Institutr of Agricultural Science 32, 295-297. BROMFIELDE. S. P. and JONESD. G. (1980) Studies on acid tolerance of Rhi;obium trifolii in culture and soil. Journal qf Applied Bacteriology 48, 253-264.
BROCKWELL J.. Asuo
Acid tolerance of Lotus rhizobia CARPERJ. E. (1978) A method for testing Rhizohium
tiveness at low pH.
Soil
Biology &
Biochemistry
eEecIO.
81-83.
DATE R. A. and HALLIDAYJ. (1969) Selecting Rhizohium for acid infertile soils of the tropics. Nature 277, 62-64. ERDMANL. W. and MEANSU. M. (1949) Strains of Rhizobium effective on the trefoils, Lotus corniculatus and Lotus uliginosus. Proceedings. Soil Science Society of America. 14, 170-175. JARVISB. D. W.. MACLEANT. S., ROBERXSON I. G. C. and FANNINGG. R. (1977) Phenetic similarity and DNA base sequence homology of root nodule bacteria from New Zealand native legumes and Rhizobium strains from agricultural olants. New Zealand Journal of Aaricultural Research’U),
235248.
JENSENH. L. and HANSENA. L. (1968) Observations on host plant relations in root nodule bacteria of the LotusAnthyllis and the Lupinus-Ornithopus groups. Acta Agric’ulturae
Scandinavica
18, 135-142.
KEYSERH. H. and MUNNSD. N. (1979) Tolerance or rhizo-
131
bia to acidity, aluminium and phosphate. Science Society
of America
Journal.
Soil
43, 519-523.
KEYSER H. H., MUNNS D. N. and HOHENBERG J. S. (1979) Acid tolerance of rhizobia in culture and symbiosis with cowpea. Journal. Soil Science Society of America 43, 719-722. LANGER. T. (1961) Nodule bacteria associated with indigenous Leguminosae of South-Western Australia. Journal of General
Microbiology
26, 351-359.
MUNNSD. N. and KEYSERH. H. (1981) Responses of Rhizohium strains to acid and aluminium stress. Soil Biology &Biochemistry 13, 115118. NORRISD. 0. (1965) Acid production by Rhizohium. A unifying concept. Plant and Soil. 22. 143166. PARKERC. A., TRINICK M. J. and CHATELD. L. (1977) Rhizobia as soil and rhizosphere inhabitants. in A Treatise on Dinitrogen Fixation logy (R. W. F. Hardy and 31 l-352. Wiley, New York.
VINCENTJ. M. (1970) A Root-Nodule
Bacteria.
Manual
IV:
Agronomy
and Eco-
A. H. Gibson, Eds), pp. for
the Practical
Blackwell. Oxford.
Study
of
0038~717/82/020127-05fo3.00/0 CopyrIght 0 1982 Pergamon Press Ltd
ACID PRODUCTION, ACID TOLERANCE AND GROWTH RATE OF LOTUS RHIZOBIA IN LABORATORY MEDIA J.
E.
COOPER
Agricultural and Food Bacteriology Research Division, Department of Agriculture for Northern Ireland, Newforge Lane, Belfast BT9 5PX, Northern Ireland (Accepted 10 September 1981) Summary-Twenty-seven strains of Lotus rhizobia were tested for acid tolerance in yeast-extract mannitol broth (pH 4.6) by multiplication from low initial cell densities. Acid production on unbuffered yeast-extract mannitol agar slopes incorporating bromothymol blue indicator was also determined. Ail slow-growing, alkali-producing strains were acid sensitive and four of the six fast-growing, acidproducing strains were acid tolerant as was one fast-growing, alkali-producing strain. The method of testing for acid tolerance proved suitabte for fast-growing strains and results are discussed in relation to the ecological impIications of acid and alkali production by rhizobia.
INTRODUCTION
Effectiveness
The ability of rhizobia to tolerate low pH values and associated acidity stresses in laboratory media has been shown to be of predictive value in selecting strains which form effective symbioses with host plants in acid soils (Keyser er al., 1979). Tolerance of this type is a consistent and stable strain property, uninfluenced by previous cultural conditions (Munns and Keyser, 1981). Tolerance of low pH has been tested by two methods which allow a strain to multiply at a constant low pH value. Date and Halliday (1979) monitored the growth of an isolate from Stylosunthes in a defined medium containing arabinose or galactose at a constant pH of 4.5. Keyser and Munns (1979) found that the growth of alkali-producing, slow-growing strains from low numbers ( <104ml-‘) in acid, mannitol-based broths was not accompanied by any pH change before the attainment of turbidity (ca. 10’ cellsml-‘). This approach has not been applied to fast-growing, acid-producing strains commonly found in temperate zones. Norris (1965) considered slowgrowing strains to be generally more acid tolerant than the fast-growing types. Rhizobia from the genus Lotus were used in my study since they comprise both fast- and slow-growing types which can nodulate the same host. Tests of acid tolerance were applied to 27 strains of Lotus rhizobia and the relationships between growth rate, acid production and acid tolerance within the group were investigated. MATERIALS AND METHODS
Rhizobia
Twenty slow-growing and 7 fast-growing strains of rhizobia were used (Table 1). All strains were maintained at 4°C on yeast~xtract mannitol agar (YEMA) slopes incorporating 3 g C!aCOs I- ’ (Vincent, 1970). totus
tests
All strains were tested for effectiveness on Lotus pedunculatus growing in N-free rooting solution (PH
6.7) in 150 x 19 mm test tubes with seedlings sup ported by a roll of filter paper (Cooper, 1978). Plants were grown for 8 weeks in a Shearer CEL 255-6 growth cabinet with a 17 h, 20°C light period and a 7 h. 16°C dark interval. Maximum growth response was obtained with strain ~~814s while strain 1Ola was used as as ineffective control. Dry weight of tops provided the measure of effectiveness. Acid and alkali production
All strains were checked for net acid or alkali production on slopes of an unbuffered yeast-extract mannitol agar incorporating bromothymol blue indicator (Norris, 1965). For the fast-growing strain NZP2037 pH changes were also determined in a variety of liquid media which included yeast-extract mannitol broth (YEMB, Vincent, 1970) and defined broths in which mannitol, galactose or arabinose were used as C sources (Date and Ha&day, 1979). pH was measured by glass electrode on subsamples taken aseptically from flasks shaken at 150rev. min-’ and 28°C. Viable cells were also counted on the same samples by surface plating of dilutions on YEMA. Growth at low pH
All tolerance tests were based on multiplication of strains from low initial cell densities (~lO~rnl_~) in YEMB acidified to pH 4.6 with HCl. A selection of strains was studied in detail by monitoring viable counts and pH values throughout a Uday incubation. This was examined in duplicate 20ml amounts of YEMB in 50 ml flasks inoculated with dilutions of young cultures and shaken at 150 rev. min - f and 22°C. Periodically samples were withdrawn aseptically and. changes in pH and viable count were determined by methods previously de-
127
128
J. E.
COOPER
Table 1. Details of
Strain number
Speed of growth
Host plant
Rhizobium
strains Acid/alkali production on medium of Norris (1965)
Effectiveness on Lotus
pedunculatus
Source of strains
FL F2, F3, F4, F5, Fl, F44, F55, F79, F89, F90, F97, F98, FlOl, F114, F124 F129
L. pedunculatus
Slow
Effective except F44 and F129: ineffective
All alkali producers
cc806 cc807 ~~814s
L. pedunculatus L pedunculatus L. hispidus
Slow Slow Slow
Ineffective Effective Effective
Alkali Alkali Alkali
Division of Plant Industry, CSIRO, Canberra, Australia
396 391 421
Lotus Lotus Lotus
Fast Fast Fast
Ineffective Ineffective Ineffective
Alkali Acid Acid
Unidad Symbiosis, CICA, Castellar. Buenos Aires, Argentina
1Ola 102a
L. corniculatus L. corniculatus
Fast Fast
Ineffective Ineffective
Acid* Acid*
Baljvlxt Laboratoriet Sveriges Lantbruks Universitet, Uppsala, Sweden
NZP2037
Lotus
Fast
Effective
Acid
DSIR, Palmerston North, New Zealand
3EOa7
L. corniculutus
Fast
Ineffective
Acid
US Department of Agriculture, Beltsville, Maryland, U.S.A.
spp spp spp
spp
Northern Ireland pasture soils via plant-infection dilution assays
* Poor growth on Norris’ medium and weak acid production in YEMB
10 9
(a)
C
-6 . S-
vrn
0
2
4
6
6
IO0
2
4
6
-5 -4
6
-3 IO
Titno, days Fig. I. Growth of strain NZP2037 and pH changes in YEMB (a) and defined media with mannitol (bk galactose (c) or arabinose (d) as carbohydrate source. W----W pH; +O viable count.
129
Acid tolerance of Lotus rhizobia Growth in YEMB adjusted to pH 6.7 was monitored for comparative purposes. The remaining strains were screened in a test in which tolerance of acidity was indicated by visual turbidity within 10 days for fast-growing strains and 20 days for slow-growers. scribed.
RESULTS
Growth rate, acid and af~fj ~roduct~o~ All slow-growers were net alkali-producers on Norris’s medium whereas all fast-growers except strain 396 were net acid-pr~u#rs (Table 1). In symbiosis with L. pedunculutus the trend was for slow-growers to be effective and fast-growers ineffective. Of the latter group only strain NZP2037 was effective on this host. In the acid and alkali production tests on Norris’s medium all strains demonstrated unidirectional changes in pH. However, it was clear from the assess-
Acid tolerance
Figure 2 shows pH trends and viable counts for five
PH
.
lor
ment of pH trends in liquid media using strain NZP 2037 that the direction of change of pH was influen~d by the com~sition of the medium (Fig. lf In YEMB, the medium which most closely resembled the medium of Norris (1965) for determination of acid or alkali production, change in pH was consistently downwards with a marked fall only occurring at stationary phase. A similar pattern was observed in defined arabinose medium. In defined mannitol and galactose broths, pH actually increased during the logarithmic phase of growth and then fell sharply as stationary phase was reached. In the three defined media carbon was present in both the ~bohydrate component and glutamic acid. One slow-growing strain (cc 807) was cultured in YEMB and defined mannitol and galactose broths: in the three media the pH increased.
-8
8-
-7 -6 -5 k -4 -I $4
4-
Z 3
3
2-
iE Q
lO--8
4
0
46
0
4
6
12
16
20
24
20
24
3 0
4
6
12
I6
20
24
l2162?024
2L.
t 8
12
mn%dayr
I6
2 0
4
8
12
I6
20
24
Time, days
Fig. 2. Viable counts and pH trends of five fast-growing and two slow-growing strains of Lotus rhizobia in YEMB adjusted to pH 4.6 or 6.7. O---O viable counts at pH 6.7; O---O viable counts at pH 4.6; A-A pH trends from initial value of 6.7; A-A pH trends from initial value of 4.6.
J. E. COOPER
130 Table
2. Growth
of strains
at pH 4.6 in YEMB
from low initial cell densities
0 Strain Slow-growers,
no growth
Fast-growers,
growth
at
at pH 4.6
pH 4.6
Days 10* 2ot (Viable cells ml- ‘)
Fl F2 F3 F4 F5 F7 F44 F55 F89 F90 F97 F98 FlOl F114 F120 F129 cc 806 cc 807
78 364 146 170 112 104 150 210 238 146 120 200 274 174 100 168 50 340
IOla 102a
900 900
50 <50 <50 75 50 50 <50 50 75 <50 50 75 100 150 100 <50 50 t50
>lO’ > 10’
* Not-determined for slow-growers. t Not determined for fast-growers.
fast-growing and two slow-growing strains in YEMB adjusted to pH 4.6 or 6.7. Three of the fast-growers multiplied at the lower pH value, making between 104- and lo’-fold increases in number before altering the reaction of the medium. Of these. two were acidproducers and one was an alkali-producer. Neither slow-growing strain multiplied at pH 4.6 and the number of viable cells declined to undetectably low levels after 3 weeks. Results for the remainder of the strains, using the simpler screening test (Table 2) indicate none of the slow-growing strains multiplied at pH 4.6 whereas the two fast-growers multiplied readily. A count of viable cell numbers after 3 weeks indicated that populations of most of the sensitive strains had declined from the immediate post-inoculation level while a few were recovered at densities similar to those originally added. DISCUSSION
All fast-growing strains used in my study were, according to the records of the various supplying laboratories. effective on the hosts from which they were originally isolated. Their performance in the present effectiveness tests supports previous observations that dual effectiveness of strains, particularly on L. pedunculatus and L. corniculatus, is rare (Erdman and Means, 1949; Jensen and Hansen, 1968). Results from my study lend no support to the view that acid production in culture media is an indication of acid sensitivity (Norris, 1965; Brockwell et al., 1966; Bromfield and Jones, 1980). Indeed strain NZP 2037, a strong acid-producer in Norris’s medium, grew well at pH 4.6 whereas all the slow-growing, alkali-producing strains were acid sensitive. Acid or alkali production is a markedly variable strain characteristic and many studies have shown
that it is influenced by changes in composition of the growth medium (Lange, 1961; Jarvis et al., 197’7; Parker et al., 1977; Date and Halliday. 1979). My work shows, additionally, that acid or alkali can be generated in a single medium by one strain at different phases of the growth cycle (Fig. 1). On these grounds alone it would be unwise to attribute an ecological role to acid production by rhizobia. Whether rhizobia ever effect a change of pH in soils is open to serious doubt. Parker et al. (1977) reported no change in the pH of the rhizosphere of hosts or water extracts of soils with a variety of fast- and slowgrowing rhizobia. In this connection it is important to note that, in my study, pH changes were only associated with relatively high cell densities, even in unbuffered media (Fig. 2). The method of testing for acid tolerance by growth from low cell densities proved entirely satisfactory for fast-growing strains of Rhizobium and this type of test could be of practical value if it can be shown that the behaviour of organisms in laboratory media is similar to that in acid soils. Acknowledgements-1 thank the curators of the various culture collections (noted in Table 1) for supplying the strains of Rhi,-obium and Mr D. Campbell for isolating strains from Northern Ireland pasture soils.
REFERENCES S. K. and REA GERALDINEA. (1966) Acid production by rhizobia from the genera Trifolium and Lotus. Journal of the Australian Institutr of Agricultural Science 32, 295-297. BROMFIELDE. S. P. and JONESD. G. (1980) Studies on acid tolerance of Rhi;obium trifolii in culture and soil. Journal qf Applied Bacteriology 48, 253-264.
BROCKWELL J.. Asuo
Acid tolerance of Lotus rhizobia CARPERJ. E. (1978) A method for testing Rhizohium
tiveness at low pH.
Soil
Biology &
Biochemistry
eEecIO.
81-83.
DATE R. A. and HALLIDAYJ. (1969) Selecting Rhizohium for acid infertile soils of the tropics. Nature 277, 62-64. ERDMANL. W. and MEANSU. M. (1949) Strains of Rhizobium effective on the trefoils, Lotus corniculatus and Lotus uliginosus. Proceedings. Soil Science Society of America. 14, 170-175. JARVISB. D. W.. MACLEANT. S., ROBERXSON I. G. C. and FANNINGG. R. (1977) Phenetic similarity and DNA base sequence homology of root nodule bacteria from New Zealand native legumes and Rhizobium strains from agricultural olants. New Zealand Journal of Aaricultural Research’U),
235248.
JENSENH. L. and HANSENA. L. (1968) Observations on host plant relations in root nodule bacteria of the LotusAnthyllis and the Lupinus-Ornithopus groups. Acta Agric’ulturae
Scandinavica
18, 135-142.
KEYSERH. H. and MUNNSD. N. (1979) Tolerance or rhizo-
131
bia to acidity, aluminium and phosphate. Science Society
of America
Journal.
Soil
43, 519-523.
KEYSER H. H., MUNNS D. N. and HOHENBERG J. S. (1979) Acid tolerance of rhizobia in culture and symbiosis with cowpea. Journal. Soil Science Society of America 43, 719-722. LANGER. T. (1961) Nodule bacteria associated with indigenous Leguminosae of South-Western Australia. Journal of General
Microbiology
26, 351-359.
MUNNSD. N. and KEYSERH. H. (1981) Responses of Rhizohium strains to acid and aluminium stress. Soil Biology &Biochemistry 13, 115118. NORRISD. 0. (1965) Acid production by Rhizohium. A unifying concept. Plant and Soil. 22. 143166. PARKERC. A., TRINICK M. J. and CHATELD. L. (1977) Rhizobia as soil and rhizosphere inhabitants. in A Treatise on Dinitrogen Fixation logy (R. W. F. Hardy and 31 l-352. Wiley, New York.
VINCENTJ. M. (1970) A Root-Nodule
Bacteria.
Manual
IV:
Agronomy
and Eco-
A. H. Gibson, Eds), pp. for
the Practical
Blackwell. Oxford.
Study
of