Screening clover and Lotus rhizobia for tolerance of acidity and aluminium

Soil

Bid.

Printed

Biochem.

in Great

Vol. 17. No. 4, pp. 493-497, Britain. All rights reserved

1985 Copyright

0

003%0717/85 $3.00 + 0.00 1985 Pergamon Press Ltd

SCREENING CLOVER AND LOTUS RHIZOBIA FOR TOLERANCE OF ACIDITY AND ALUMINIUM M. WOOD’* and J. E. COOPER’~~ ‘Department of Agricultural and Food Bacteriology, The Queen’s University of Belfast and 2Agricultural and Food Bacteriology Research Division, Department of Agriculture for Northern Ireland, Newforge Lane, Belfast BT9 5PX, Northern Ireland (Accepted 30 December

1984)

rhizobia (55 strains) were screened for tolerance of acidity and Al, using the technique of Keyser and Munns (1979). Assessment of visible turbidity after 14 days indicated three strains tolerant of pH 4.5 (although growth rate was reduced), 25 strains tolerant of 5 PM Al and no strains tolerant of

Summary-Clover

50p~Al at pH 5.5. 50p~Al caused a decrease in the numbers of acid-tolerant strains at pH 4.5. Tolerance of acidity or Al was not associated with the pH or Al status of the soil from which a strain was isolated. Screening of eight strains of clover rhizobia and nine strains of Lotus rhizobia using turbidity assessment and viable counts indicated seven strains of clover rhizobia with different degrees of tolerance of 20 PM Al but none tolerant of 50 PM Al at pH 5.5. All Lotus rhizobia (both slow- and fast-growers) were tolerant of 20 and 5OpM Al at pH 5.5, with 50pMAI causing a reduction in growth rate. Subculturing of strains in non-stressed and stressed media had no effect on the response to 50 PM Al at pH 5.5.

INTRODUCTION

of clover rhizobia for tolerance of acidity and Al, using turbidity assessments, (b) screen clover and Lotus rhizobia for tolerance of Al at pH 5.5 using turbidity assessments and viable counts, (c) attempt to improve the Al tolerance of clover and Lotus rhizobia by repeated subculturing in stressed media.

The accurate assessment of pH limits for multiplication of Rhizobium requires precise control of pH during the course of an experiment (Munns, 1977). Inclusion of the major acid-related stress, aluminium, requires additional control of factors determining the solubility of Al, in particular the concentration of phosphate (Munns, 1977; Cooper et al., 1983). Keyser and Munns (1979) used a low phosphate liquid medium to screen strains of R. japonicum and cowpea rhizobia for the ability to multiply and produce visible turbidity from a low initial inoculum density, at constant pH with a known concentration of Al; more detailed studies used viable counts. This technique has been used to screen Lotus rhizobia (Cooper, 1982) cowpea rhizobia (Hartel and Alexander, 1983) R. phaseoli (Lowendorf and Alexander, 1983a), R. meliloti (Lowendorf and Alexander, 1983b) and R. trijolii strains from Trifolium subterraneum (Thornton and Davey, 1983) for tolerance of acidity and Al. We have reported that strains of R. trifolii from T. repens respond similarly to acidity and Al in modified Munns and Keyser (1981) medium and in the rhizosphere of the host legume (Wood et al., 1984; Wood and Cooper, 1985). Strains were sensitive to acidity alone, therefore no effect of Al could be detected at pH < 5. However, Al inhibited multiplication and nodulation in the pH range 5-6. Slow-growing Lotus rhizobia (Bradyrhizobium sp.) are also sensitive to acidity, whereas fast-growing Lotus rhizobia (R. loti) are tolerant (Cooper, 1982). The technique of Keyser and Munns (1979) was used in the present study to (a) screen a large number

*Present address: Department Reading, London Road.

MATERIALS AND METHODS General

Strains of Rhizobium trifolii comprised 55 isolated in our laboratory from Northern Ireland pasture soils (BEL strains, including 1130, 1192, 1205, 1214, 1240, 1241, 1309, 1312), and HP3 described by Wood and Cooper (1984). Strains of Lotus rhizobia comprised BEL strains 0003, 0055 and 0079 (formerly F79) isolated in our laboratory, NZP strains 2014, 2037, 2042, 2182, 2241 (obtained from DSIR, Palmerston North, New Zealand) and CC814s (obtained from Division of Plant Industry, CSIRO, Canberra, Australia). Strains were maintained at 4°C on yeast extract-mannitol (YEM) agar slopes incorporating 3 g CaCO, 1-l (Vincent, 1970). The screening medium was modified Munns and Keyser (1981) medium with the following composition (PM): CaCl,.6H,O, 1000, MgS0,.7H,O, 500; KCl, 50; FeEDTA, 25; KH2P0,, 10; H,BO,, 10; MnS04.4H20, 1; ZnS04.7H20, 0.5; CuSO,.SH,O, 0.1; Na,MoO,. 2H,O, 0.025; CoCI, .6H,O, 0.005. After autoclaving, glutamic acid (1.8 g 1-l ), arabinose (0.3 g I-‘), galactose (0.3 gl-‘), thiamine-HCl (lOOpgl_‘) and biotin (25OpgII’) were added as filter-sterilized solutions. pH was adjusted with concentrated hydrochloric acid and, where required, Al was then added as a filter-sterilized solution of AIK(SO,),. 12H20. Medium was dispensed as either 22 ml aliquots into 50 ml Erlenmeyer flasks, or 5 ml aliquots into plastic screw-capped test tubes (Ster-

of Soil Science, University of Reading RG1 5AQ, England. 493

494

M. Woon and J. E. C~OPEK

ilin). After 24 h, the medium was inoculated with a dilution of a culture of Rhizobium in YEM broth (27°C for 4 days) to give IO*-IO3 cfu ml-‘, and shaken at 15’ or 22°C. The medium was checked daily for visible turbidity and, where appropriate, viable counts were made on YEM agar. Two replicates were included treatment-‘. Screening and Al

clover

rhizobiu

for

tolerance

clover strain to Al

Multiplication of strain BEL1214 was studied at pH 5.5 with 0 Al and at pH 4.5 with 0 or 50 PM Al. Flasks were incubated at 15°C and viable counts made over 7 days. Screening of clover and Lotus rhizobia for tolerance of Al at pH 5.5 R. trjfolii strains HP3, BELl130, 1205, 1214, 1240, 1241, 1309, 1312 and Lotus rhizobia strains BEL003, 0055, 0079, NZP2014, 2037, 2042, 2182. 2242 and CC814s were screened in tubes at pH 5.5 with 0, 20 or 50p~Al. The time taken for visible turbidity to appear and where no visible turbidity appeared, viable counts after 21 days at 22°C were the criteria for tolerance. In addition, the growth rate of strains on YEM agar was assessed by the time taken for 1-2 mm dia colonies to appear at 25°C. Subculturing media

clover and Lotus

rhizobia

in stressed

R. trtfolii strains BEL1241, 1309, 1312 and Lotus rhizobia strains BEL0079, NZP2014, NZP2037 and CC8914s were used. Turbid media from the previous experiment, pH 5.5 with 0 or 20~~ Al for clover rhizobia and pH 5.5 with 0 or 50 /rM Al for Lotus rhizobia, were diluted in 4 strength Ringer’s solution to give 102-10’ cfu ml-’ when inoculated into fresh medium containing 0, 20 or 50 PM Al at pH 5.5. The time taken for the appearance of visible turbidity, and, where no visible turbidity appeared, viable counts after 21 days at 22°C were the criteria for tolerance. This procedure was repeated for a second subculture. The final subculture studied strains BEL 13 12 (clover strain) and BEL0079 (Lotus strain) at pH 5.5 with OAl and 10 or lOOOpMP, and pH 5.5 with 50~~ Al and 10pMP. Inoculum sources comprised turbid media from the second subculture experiment, 0 or 20 p M Al for BEL13 12 and 0 or 50 PM for BEL0079, and also 4-day old YEM broth cultures of the two strains. Viable counts were made over 18 days at 22°C and after transformation to log (N + l), were analysed by analysis of variance. RESULTS AND

55 0

PH Al (/CM)

54 I

No. tolerant strains No. sensitive strains

5.5 5

55 50

25 29

0 54

4.5 0 3 51

of ucidity

Fifty-five strains of R. tr$olii were selected at random from a collection of 178 isolates and screened in flasks at pH 4.5 with 0 Al and at pH 5.5 with 0, 5 or 50pMAl. The appearance of visible turbidity within 14 days at 22’C was the criterion for tolerance. Response of an acid-tolerant

Table I. Screenmg of 55 strains of clover rhizobta for tolerance of acidity and Al, based upon the presence or absence of visible turbidity within 14 days at 22 C

three strains of clover rhizobia (6%) were tolerant of pH 4.5. Thornton and Davey (I 983) found a similar proportion (10%) of clover strains for T. subterraneum tolerant of pH 4.2. Twenty-five strains (467; of our isolates) were tolerant of 5 PM Al at pH 5.5, confirming our report of variation in the response by strains of clover rhizobia to low concentrations of Al (2-10~~) at pH 5.5 (Wood and Cooper, 1984). Thornton and Davey (1983) reported strains of R. phaseoh sensitive to 5 PM Al at pH 4.4, but Al tolerance was increased at higher pH values. All of our strains were sensitive to 50 PM Al at pH 5.5. The three acid-tolerant strains were isolated from soils of pH 5.7, 6.0 and 6.3, indicating no association between acid-tolerance of a strain under laboratory conditions and the pH of its natural environment. Table 2 shows a similar lack of association between tolerance of 5 PM Al at pH 5.5 and either the pH or the Al status (exchangeable Al) of the soil from which a strain was isolated. This may be due to the importance of microsite conditions, particularly in the rhizosphere, in the ecology of Rhizobium. Such variations in soil properties remain undetected by bulk soil analyses. It is impossible to relate exchangeable Al values for such a wide range of soils to concentrations of Al in soil solution because of the complex nature of Al exchange reactions in soil. Figure 1 shows that the rate of multiplication of the acid-tolerant strain BEL 1214 was lower at pH 4.5 than at pH 5.5 and that 50 PM Al caused a decrease in numbers. This strain was, therefore, only partially tolerant of acidity, and the addition of 50~~ Al imposed a further severe stress. This was also shown for another acid-tolerant strain. Multiplication of acid-tolerant strains of R. trifolii from T. subterraneum (Thornton and Davey, 1983). R. ,juponicum and cowpea rhizobia (Keyser and Munns, 1979; Hartel and Alexander, 1983) and R. phaseoh (Lowendorf and Alexander, 1983a) was also reduced or inhibited by 5550 PM Al. Tolerance

of Al

at pff 5.5 in clover and Lotus rhizobiu

Screening based solely upon turbidity assessment indicated no clover rhizobia tolerant of 50 ELMAl at Table 2. The relationship between tolerance of 5 PM Al at pH 5.5 by clover rhizobia and either the pH or Al status of the soils from which they were isolated (Al data available for only 38 strum) Soil pH 4.s4.9 No. tolerant strains No. sensitive strains

2 2

DISCUSSION

6.0-6.9

~7.0

10 Y

I2 I5

I 3

Soil .41 (mequiv kg ‘1

Tolerance of acidity and AI in clover rhizobia The screening procedure based solely upon visible turbidity assessment (Table 1) indicated that only

S.&S 9

No. tolerant strains No. sensitive strains

0

0 10.

5 7

14 II

>I0 I 0

495

Acid- and Al-tolerance af clover and Lotus rhizobia

T

pH 5.5, no Al

‘.-

pH 4.5,50pMAl - _._ _ _,.,_ -.

Time

(days)

Fig. 1. Response of clover strain BEL1214 to pH and (2 observations

Al

per mean).

pH 5.5, but a large number tolerant of 5p~ Al. However, significant muhiplication could have occurred without achieving the cell density necessary to produce visible turbidity (approx. IO6cfu ml.-‘). In addition, previous experiments have shown that R. trzyolii strain HP3 could multiply with 20p~AI at pH 5.5, although at a rate lower than in the absence of Al (Wood and Cooper, 1984). Screening based upon visible turbidity assessment and viable counts (Table 3) indicated that all strains of Lotus rhizobia were tolerant of 20 and 50 /*MAl at pH 5.5; multiplication was unaffected by 20 PM Al and slowed by 50pMAl. There was variation in the response by clover rhizobia to 20 PM Al at pH 5.5 both in the time taken for visible turbidity to appear (14-2 L days) and in the final ceN density of strains not producing turbidity (3.0 x lo*-75 x 105cfuml-‘). All strains multiplied, although at a reduced rate, with 20 PM Al except BELll30 which declined in numbers. There was no variation in the response of the clover rhi-

Table

3. Screening

of clover

and

zobia to 50 pM Al at pH 5.5; all strains declined in numbers. The differences between strains in growth rate on YEM agar, important in the taxonomy of the Rhizobiaceae (Jordan, 1984), were reflected in the growth rates in non-stressed Liquid medium. Slowgrowing Lotus rhizobia ~~r~~~rhi~ob~urnsp.) are sensitive to acidity (pH 4.5) whereas fast-growing Lorus rhozibia (R. loti) are tolerant (Cooper, 1982). However, both the slow-growers (7-8 days for colony formation) and the fast-growers (4-5 days for colony formations were tolerant of Al at pH 5.5. (Table 3). Slow-growers were more tolerant of 50 PM Al than the fast-growers. The clover rhizobia (sensitive to Al) were confirmed as fast-growers, although there was variation in their growth rate (3-6 days for colony formation). There was, therefore, no relationship between growth rate and Al tolerance. Subculture experiments

Subculturing the clover and Lotus rhizobia in stressed media produced no increase in growth rate in non-stressed and stressed media (Table 4). In particular, numbers of clover rhizobia declined at pH 5.5 with 50 pM Al irrespective of previous treatment. There was a general trend for growth rates in stressed media to be slower after subculturing (time taken for visible turbidity to appear was increased). This may have been due to exhaustion of reserves of P in the cells after subculturing in a low P (10 PM) medium. P stress would reduce both the growth rate and final cell density (Keyser and Munns, 1979; Beck and Munns, 1984). Similar results were also observed for clover and Lotus rhizobia su~ultured in non-stressed (no Al) medium. In order to study further the response of these rhizobia to subculturing, and also the role of P, a final subculture experiment was carried out using a high concentration of P (1OOO~M) and viable counts.

Lotus rhizobia for tolerance of Al at pH 5.5 within (2 observations per mean)

21 days

at 22-C

Al (IIM) 0

Strain

Growth rate on YEMA” (days)

initial No. (cfu ml-‘)

Clover rhizobia HP3 II30 1205 1214 1240 1241 1309 1312

3 6 5 3 5 5 4 3

x x x x 90 3.0 x 5.6 x 1.0 x

Lotus rhizobia 0003 0055 0079 CC8 14s NZP2014 NZP2037 NZP2042 NZP2 I82 NZP2241

8 7 7 7 4 4 5 7 4

ND 1.6 x 10’ 1.3 x 10’ 1.8 x 10’ 4.0 x 10’ 4.0 x 103 4.0 x 103 6.0 x LO* 4.0 x 103

*Yeast extract-mannitol ND = not determined.

1.1 1.1 2.4 2.1

aear. -

103 10’ 10” 10’ 102 lo2 to-’

Turbidity ------.-__----

5 8.5 9 4.5 9 7 4 4.5 8 I ;s

i 8 4

_____-_ (days)

50

20 Final No. (cfu&‘) 7.5 3.0 1.5 1.3

x x x x

Turbidity (days)

< 10
10s 102 10s 10s

21 17 17 14 11.5 1.5 8.5 8.5 4 4 4 9 4

Final No. (cfu ml-‘)

I2 10.5 11.5 8.5 8 10 8 10 10.5

496

M.

WOOD

and J. E. CAPER

Table 4. Screening

of clover and Lotus rhizobia for tolerance of Al at pH 5.5 after one (a) or two (b) subcultures in defined medium at pH 5.5 with 20 JLMAl, or after one (c)or two(d) subcultures in defined medium at pH 5.5 with 50 FM Al (2 observations per mean) within

21 days

at 22”C,

Al (w) 0 Turbidity Strain

20 (days)

Initial No. (CfUrnl~‘)

Clover rhizobia 1241a 1241b 1309a 1309b 1312a 1312b

6.0 x lo’ 6.0 x 10’ 8.0 x lo’ 70 7.0 x IV 4.0 x IO”

Lotusrhlzobia 0079c 0079d CC814sc CC814sd NZP2014c NZP2014d NZP2037c NZP2037d

1.2 x 90 3.4 x 1.5 x 2.7 x 4.0 x 5.0 x 1.6 x

50 Final No. (cfu ml-‘)

7 8 5 5.5 4.5 6

102

18 15 20 8 5 5 4 5

IO’ IO’ IO’ IO’ 10’ 10’

11.5 20 12 14 7 II

< 10 < IO
ND ND ND ND ND ND ND ND

14 15 14 II 8 14 8 I2

ND = not determined.

shown

by the higher final cell density in the pH 5.5, The Lotus strain did not respond to increased P concentration, indicating that the maximum cell density was limited by some factor other than P supply (counts after 22 days showed that stationary phase had been reached by 18 days). The clover strain responded in the same way to P irrespective of previous treatment, indicating that the reduction in growth rate after subculturing in stressed medium, observed in the previous experiment, may

Analysis of variance of the results for BEL1312 (clover strain) and BEL0079 (Lotus strain) showed significant (P -C 0.001) interactions for both strains between previous treatment, present treatment and time, although these are not readily explained by examination of the growth curves (Fig. 2). In all treatments without Al, the clover strain yielded a higher final cell density than the Lotus strain under both P treatments. Multiplication of the clover strain in the pH 5.5, 10 PM P treatment was P-limited as IcJr

l -•---•

. 1000

1OOOpM P treatment.

r13128

:

__-.-.-.

1312C

P, OAI

. lOP,50Al

Ii-

0

‘i., I

..__-.----

‘\ ,,.--...

-.._--

B0079A

Time

(days)

Fig. 2. Response of clover strain BEL1312 and Lotus strain BEL0079 to P and Al at pH 5.5 after one subculture in YEM broth (A), or three subcultures in defined medium at pH 5.5 with no Al (B), 20 PM Al (C) or 50 pM Al (D) (2 observations per mean; SE of difference 0.2).

Acid-

and Al-tolerance

have been due to experimental error rather than P depletion. The response of the Lotus strain to the different treatments was unaffected by previous treatments. There was no evidence, therefore, of variation within the strains in their response to Al at pH 5.5, nor of spontaneous mutation giving rise to genotypes with increased tolerance during the course of these experiments (70-80 generations). We conclude that our results demonstrate the tolerance of Lotus rhizobia and the sensitivity of clover rhizobia to 50 pM Al at pH 5.5, and show that repeated subculturing under stressed conditions does not alter these properties. Bromfield and Jones (1980) found that the stepwise subculture of strains of clover rhizobia on solid media of decreasing pH did not alter tolerance of acidity, and Munns and Keyser (1981) reported that repeated subculture of cowpea rhizobia using both solid and liquid media incorporating acidity and Al did not alter tolerance of these stresses. The sensitivity of clover rhizobia (fast-growers) to acidity (pH 4.5) shown here contrasts with the acid tolerance of fast-growing Lotus rhizobia, but is similar to the sensitivity of slow-growing Lotus rhizobia to acidity (Cooper, 1982). Strains of Lotus rhizobia tolerant of acidity in liquid culture media have been shown to form the majority of nodules on Lotus pedunculatus when in competition with sensitive strains in low pH solution culture (Cooper et al., 1985). This result, together with those from soil inoculation experiments with acid-tolerant cowpea rhizobia (Keyser et al., 1979; Hartel and Alexander, 1983) suggests that the strains of clover rhizobia reported here tolerant of 20 PM AI at pH 5.5, and the Lotus rhizobia tolerant of 50 pM Al at pH 5.5 may also be useful for achieving nodulation of clover and Lotus spp under conditions of acid- and Al-stress. Acknowledgements-We thank the Agricultural and Food Research Council for providing financial support and Miss C. P. Liddane. DSIR. Palmerston North. New Zealand. for supplying cultures of fast-growing Lotus rhizobia

REFERENCES Beck D. P. and Munns D. N. (1984) Phosphate nutrition of Rhizobium spp. Applied and Environmental Microbiology 47, 278-282. Bromfield E. S. P. and Jones D. G. (1980) Studies on acid

of clover

and Lorus rhizobia

497

tolerance of Rhizobium trifolii in culture and soil. Journal of Applied Bacteriology 48, 253-264. Cooper J. E. (1982) Acid production, acid tolerance and growth rate of Lotus rhizobia in laboratory media. Soil Biology & Biochemistry 14, 127-131. Cooper J. E., Wood M. and Holding A. J. (1983) The influence of soil acidity factors on rhizobia. In Temperate Legumes: Physiology, Genetics and Nodulution (D. G. Jones and D. R. Davies, Eds), pp. 319-335. Pitman, London. Cooper J. E., Wood M. and Bjourson A. J. (1985) Nodulation of Lotus pedunculatus in acid rooting solution by fast- and slow-growing rhizobia. Soil Biology & Biochemistry 17, 487492. SIartel P. G. and Alexander M. (1983) Growth and survival of cowpea rhizobia in acid, aluminium-rich soils. Soil Science Society of America Journal 47, 502-506. Jordan D. C. (1984) Family Rhizobiaceae. In Berge.l;‘s Manual qfsystematic Bacteriology, Vol. 1 (J. G. Holt and N. R. Krieg, Eds), pp. 234-256. Williams & Wilkins, London. Keyser H. H. and Munns D. N. (1979) Tolerance of rhizobia to acidity, aluminium and phosphate. Soil Science Society of America Journal 43, 519-523. Keyser H. H., Munns D. N. and Hohenberg J. S. (1979) Acid tolerance of rhizobia in culture and symbiosis with cowpea. Soil Science Society of America Journal 43, 7 19-722. Lowendorf H. S. and Alexander M. (1983a) Identification of Rhizobium phaseoli strains that are tolerant or sensitive to soil acidity. Applied and Environmental Microbiology 45, 737-742. Lowendorf H. S. and Alexander M. (1983b) Selecting Rhizobium meliloti for inoculation of alfalfa planted in acid soils. Soil Science Society of America Journal 47, 935-938. Munns D. N. (1977) Soil acidity and related factors. In Exploiiing the Legume-Rhizobium Symbiosis in Tropical Agriculture (J. M. Vincent, A. S. Whitney and J. Bose, Eds), pp. 211-236. University of Hawaii College of Tropical Agriculture Miscellaneous Publications 145. Munns D. N. and Keyser H. H. (1981) Responses of Rhizobium strains to acid and aluminium stress. Soil Biology & Biochemistry 13, 115-l 18. Thornton F. C. and Davey C. B. (1983) Acid tolerance of Rhizobium trifolii in culture media. Soil Science Society of America Journal 47, 496-50 1. Vincent J. M. (1970) A Manual for the Practical Study of Root-Nodule Bacteria. Blackwell, Oxford. Wood M. and Cooper J. E. (1984) Aluminium toxicity and multiplication of Rhizobium trifolii in a defined growth medium. Soil Biology & Biochemistry 16, 571-576. Wood M., Cooper J. E. and Holding A. J. (1984) Aluminium toxicity and nodulation of Tr(folium repens. Plant and Soil 78, 381-391.