Jurnal Mikrobiologi Indonesia, Februari 2000, 24-28 ISSN 0853-358X
Root Colonization and Symbiotic Effectiveness of
Acid Tolerant and Acid Sensitive Strains of Bradyrhizobia
in Acid Soils
A. INDRASUMUNAR', P. J. DARD & N. W. MENZIES2
`Research Institute for Food Crops Biotechnology, .i'alan Tentara Felajar No. 3A, Bogor 16111 `School ofLand and Food, TheUniversityof Queensland; St Lucia 4072,QLD, Australia
Vol. 5, No. I
Acid soil is characterized by numerous growth limIting factors. Low pH may reduce the availability of element such as Ca, Mg, P and Mo, and increase the availability of Al and Mn, limiting plant growth and survival and effectiveness of bradyrhtzobia. The formation of symbiosis between legumes and bradyrhizobia is affected by acid soil stresses factors. A pot ezperiment was conducted to study the usefulness of screening of Bradyrhizobiunt japonicum in acidic agar media In predicting their performance in acid soils. Four strains ofB. Japonkumthat were similar In their effectiveness at neutral pH sand culture bnt different in their tolerance of acid soil stress factors in agar media were used for testing their ability to colonize Al tolerant soybean P1 416937 rhizosphere and their effectiveness in three acid soils differing in their chemical characteristics. The treatments were arranged in a completely randomized block design with 4 replicates. The results showed that soybean Inoculated with the acid tolerant strains had significantly greater nodulatlon and symbiotic effectiveness than plants Inoculated with the acid sensitive strain. This result confirmed that the performance of B. japonicumin acid soils can be predicted from their perfonnance in acidic agar media.
Keywords:Bra dyrhizobiumjaponicutn,acid sensitive, acid toierant, root colonization, symbiotic effectiveness
Much agricultural land has acid infertile soil. There are several potentially growth-limiting factors associated with acid soils. Low pH soils often have high levels of soluble Al, Mn, and reduced availability of Ca, Mg, P and Mo, affecting plant growth and survival and symbiotic effec tiveness of Bradyrhizobium. Amelioration of these prob lems, especially in the subsoil, is difficult and generally not economically feasible Foy et al. 1974. An alternative approach, which may be more cost effective, is to select varieties Sartain & Kamprath 1978 and screen Brady rhizobium strains Taylor et a!. 1991 tolerant of acid soil stresses.
Strains differ in their ability to grow on agar media formulated to simulate stresses associated with acid soils. Studies have been reported by Hartel & Alexander 1983 with cowpea; Ayanaba et a!. 1983 with cowpea & soybean; Karanja & Wood 1988 with beans. Few authors have extended their laboratory findings to the field. Those that did had varying results. Most reports are of only a few strains tested in soil, which either confirm or place doubt on the prescreening process in defined media.
With strains of rhizobia for clover, Thornton & Davey 1983 found a positive correlation between growth at low pH in laboratory media and nodulation in acid soils, whereas Bromfield & Jones 1980 found no relationship. Similarly Vargas& Graham 1989 demonstrated a positive relationship with strains of Rhizobiurn !eguminosarum by
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phaseo!i, whereas Lowendorf & Alexander 1983 found no relationship.
This experiment compares the performance of B. japonicum strains which differ in their tolerance of acidity on agar medium Indrasumunar & Dart 1999, as inocula for soybean in three acid soils which had different levels of Ca, Al and Mn and contained no indigenous population of B. japonicum nodulating soybean. The hypothesis is that strains which grew on a laboratory agar containing cationic imbalances which simulated those of an acid soil would survive and nodulate soybean better than an acid sensitive strain in acid soils.
MATERIALS AND METHODS
This experiment was conducted in the glasshouse of the University of Queensland Australia from 26/03/1997 to 21/ 05/1997.
Bradyrhizobium Strains. Fifteen strains of B. japonicum obtained from Bogor Research Institute for Food Crops Biotechnology, which had been classified as either tolerant or sensitive to mineral imbalances associated with acid soils and to low pH on agar medium Indrasumunar & Dart 1999, were tested for their symbiotic effectiveness in sand culture neutral pH. Four strains of B. japonicum, which have different tolerance to acid agar media but equal effectiveness in sand culture, were chosen for this experiment. These four were FCB 152 and CB 1809 acid tolerant, FCB 230 moderately tolerant and FCB 179 acid sensitive.
Vol.5,2000 J. Mikrobiol. Indon. 25
from South East Queensland. Soil I Yellow Kurosol, 12% clay, 1.4% organic C, soil 2 Yellow Kandosol, 23% clay, 2.2% organic C, and soil 3 Sapric Organosol, 12% caly, 15% organic C. Soils were ground and sieved to pass 2 mm dia. Before being analyzed for soil pH 1:2.5 H20; ex changeable bases extracted by 0.2 M BaC10.2 M NH4CI Oilman & Sumpter 1986; exchangeable Al and Mn extracted by 2 M KC1 soil: solution = 1:10. Elemental
concentrations in the extracts were determined using ICPAES. Ammonium and nitrate extracted by 2M KC1 was measured by steam distilation method Bremner & Keeney 1965. Destructive harvests were undertaken for symbiotic effectiveness parameters at days 28 and 56 and the soil subsanipled for chemical analysis. Chemical characteristics of the soilsarepresented in Table 1.
Naturalized Bradyrhizobium populations nodulating soybean were absent from the soils as determined by a soil dilution, plant infection assay using Glycine ussuriensis
grown in 150 x 25 mm test tubes.
Bradyrhizobia colonization and symbiotic effective ness. One kg of soil was placedin 10-cm dia. pots for the root colonization study and 1.2 kg of soil was placed in
14-cm dia. pots for the effectiveness trial. Root coloniza tion and symbiotic effectiveness of theB. japonicum strains
was evaluated utilizing the acid tolerant soybean cultivar from Japan Plant introduction P1 416937. The seeds were sterilized in 90% ethanol for 30 seconds and 1.2% HC1O4 for 4 miii, and rinsed 7 times with sterile deionized water, and imbibed for 2 h. Five seeds were planted in each pot and inoculated with I ml broth culture of acid tolerant or acid sensitive strains at the rate of 10' cfis seed-l. Uninoculated seeds were included as a control. At 14 days after planting seedlings were thinned to 3 per pot. Pot
watering was applied three times a day using an
automatic irrigation system to bring the soils to field
capacity. Four replicates were used pertreatment, and pots
were kept in a glasshouse with 14-15 h daylight and
temperature range 25-33°C. The plants were harvested at days 7 and 14 for root colonization and at days 28 and 56
for the assessment of nodulation and N2-fixation.
Root colonization. The most probable number of
Bradyrhizobium in the rhizosphere capable of nodulating soybean were counted using a dilution/nodulationfrequency plant infection test Brockwellet a!. 1975. The test plant was G. ussuriensis Regel and Maack. Samples for serial dilution and inoculation of test plants were prepared by
Table I. Some chemical properties of acid soils used in this trial.
Soil
Exchan geable ca tions cmo I,,kg' Micro nuirien
mgkg ts
pt-1
Types Ca Mg Na K' Al Mn"
Soil I 0.24 002 0.16 0.44 148 7.65 4.62 Soil 2 0.84 0.52 0.07 0.28 1.84 4.95 4.58
Soil 3 1.63 3.69 1.79 0.53 1.84 9.05 4.38
suspensioninsterile, 1/4 strength Fahraeus, N-free nutrient media Fahraeus 1957 and agitation in a vortex mixer. Samples consisted of the root of the three plants shaken free of excess soil from each pot. The roots were initially suspended in 30 mL followed by tenfold dilution.
Effectiveness trial. Nodule number and nodule dry
weight per plant were measured. The harvested samples were dried at 80°C for 7 days, ground to fme powder and analyzed for N concentration using a Leco 2000 CNS analyser. The amount of N fixed was estimated by the N difference method, in which the N yield of the shoot of uninoculated soybean was subtracted from that of the inoculated soybean and the difference assumed to be derived from N2-fixation.
Treatment of data. The data were statistically evalu ated by analysis of variance for a completely randomized block design with each treatment replicated four times. Duncan's Multiple Range Test at 5% probability levels
were used for mean separation. Numbers of Brady
rhizobiumwas transformed using log10 MPN, where MPN is most probable number.
RESULTS
Root colonization. The MPN determination on the 3
soils revealed that there were no indigenous Brady rhizobiumcapable of nodulating soybean present; therefore, rhizobia counts indicate populations of Bradyrhizobium
introduced into the soil. All strains were inoculated at
approximately the same rate of106 cfia seed', but at day 7
after inoculation there was a significant difference among
strains in their ability to colonize soybeanroots. The acid sensitive strain FCB 179 colonized soybean roots more
than acid tolerantstrains FCB 152 and CB1809 Table 2. The population of the acid sensitive straindecreased at day 14, particularly in soil 3, while the acid tolerant strain numbers tended to increase. In general, root colonization by
Bradyrhizobium was better in soils 1 and 2 than in soil 3. Soil 3 had a lower pH and contained a high level of both exchangeable Al and Mn, while soil 1 only had high Mn
andsoil 2 high Al Table 1. The lower pH and the presence of both Al and Mnmay have caused the lower survival of
B.japonicum insoil 3 than in the other soils.
Nodulation. There was a significant P<O.05 interac
tion between B. japonicum strains and acid soil types on nodule number Figure 1 and nodule day weight Figure 2 at 28 and 56 da:'s after planting. At day 28 nodulationin
soil 1 was very poor. The acid tolerant strains CB 1809 and
FCB 152 averaged about 1 nodule çr plant, while the
moderately tolerant strain FCB 230 and the acid sensitive
strain FCB 179 formed less than 0.3 nodules per plant. Nodulation in soil2and3 was better thaninsoil 1. Insoil
2,the number of nodules formed by the acid sensitivestrain
FCB 179was significantly P<0.05 less than for CB 1809 and FCB 152. In soil 3, the acid tolerant strains CB 1809, FCB 152 and the moderately tolerant strain FCB 230 had
26 INDRASUMIJNARET.4L. J. Mikrobiol. Indon.
Table 2. Colonization ofthesoybean rhizosphere in three acid soils by B.japonicurn at days land 14.
Log cell number per 3 soybean rhizospheres'
Days Day 7 Day 14
Soil Soil 1
Soil Soil
2 3
Averange Soil Soil
1 2
Soil Averange 3
Bradyrhizobi urnStrains
FCB 179 5.93 6.11 5.54 5.86 5.89 5.85 4.82 5.52b AS
FCB 152 5.50 5.31 5.52 5.44 6.08 6.22 6.18 AT
Cl8O9 5.34 5.81 5.05 5.40 6.56 6.62 6.91 6.7a AT
In each column or row, values followed by the same letters are not significantly P<0.05 different.
significantly P<0,05 larger nodule number and dry weight than the acid sensitive strain FCB 179. In general the acid tolerant strains had a better nodulation than the acid sensitive strain and nodulation in soils 2 and 3 was better than in soil 1.
Nodulation at day 56 was much better than at day 28. Nodules continued to form between day 28 and day 56, with a 2-3 fold increase in number for soils 2 and 3 and 5-6 fold increase in nodule weight per plant. For soil I, virtu ally all nodulation occurred in this period and although the nodules developed rapidly, nodule weight per plant was still a third less than for the other two soils. There was a significant P<0.05 interaction between strains of Brady rhizobium and soil types in both nodule number Figure 1 and nodule dry weight Figure 2 at day 56. In soil 1, the acid tolerant strains FCB 152 and CB18O9, and the moder ately tolerant strain FCB 230 had significantly P<0.05 more nodule number and weight per plant than did the acid sensitive strain FCB 179. While in soil 2 even though the acid sensitive strain had a lower nodule number than the acid tolerant strains, there was no significant P<0.05 Averange 5.48 5.67 5.05
a a b
6.12 6.09 5.47
a a b
6. 16 ab Figure 2. Dry weight of nodules formed by B.japonicurnstrains at days 28 and 56.
difference in nodule dry weight between strains. In soil 3, the acid tolerant strains FCB 152 and CB 1809 had significantly P<0.05 larger nodule number than the acid sensitive strain FCB 1.79, but they had similar nodule weight per plant.
Presumptive Nitrogen fixation. At day 28 there was no significant P<0.05 interaction between strains and soil types in presumptive N,-fixation, but comparison among strains shows that the acid tolerant strains CB 1809, FCB 152, and the moderately tolerant strain FCB 230 tended to have presumptive N2-fixation Figure 3 than uninoculated soybean, while the acid sensitive strain FCB 179 tended to have a lower presumptive N2-fixation than uninoculated soybean. At day 56, no interaction was found between Bradyrhizobium strains and soil types in presumptive N,-fixation, but comparison among strains shows that the acid tolerant strains FCB 152 and CB 1809 fixed sig nificantly P<0.05 more N than the moderately tolerant strain FCB 230 arid the acid sensitive strain FCB 1 79.
DISCUSSION
Root colonization. The rhizosphere population of the acid sensitive strain at day 7 was significantly P<0.05 greater than that of the acid tolerant strains, but by day 14 the acid tolerant strain numbers were 4-fold FCB 152 and 15-fold CB 1809 larger than the acid sensitive strain FCB 179 Table 2. The correlation between soybean nodulation at days 28 and 56 Figure 1 with the number of rhizobia at day 7 was poor, but with the number of rhizobia at day 14 was strong. Figure 1 shows that there was a significant P<0.05 increase in nodulation of all strains from day 28 to day 56. This means that the nodulation process continued during the soybean growth. The increase in rhizosphere colonisation by the acid tolerant strains and the decrease of rhizosphere colonisation by the acid sensitive strain from day 7 to day 14 Table 2 seems related to the better nodulation of the acid tolerant strains than the acid sensitive strain at days 28 and 56.
The lack of a pH effect on the rhizosphere population of the acid sensitive strain at day 7 may be due to the large
Ce,.wd Oi'C9176UFcB2 SF?1050c6 1606
$Co,mo WcB 179 *rce 230 UC0 192 OCO 1909
Vol. 5, 2000 J. Mikrobiol. Indon. 27
Figure 3. Effect of B. japonicuin inoculation on presumptive N2-fixationofsoybean grown in three acid soils.
moculum application of 106 cells seed. Lovato et a!. 1985 reported that when high concentrations >106 cells seed-I of bean rhizobia were applied, soil acidity affected bean nodulation but had no effect on the survival of rhizobia in the bean rhizosphere. Rhizobia population may be protected from the bulk soil solution acidity by rhizosphere materials such as the mucigel.
The poor relationship between numbers of rhizobia in the rhizosphere at day 7 and nodulation may reflect the timing of such counts. The early stages of the infection process immediately following germination may be critical to subsequent nodulation events. A measure of the speed of root colonization rather than the maximum number reached may be more important in determining a strain's success for nodulation Gemell & Roughley 1993. Vargas & Graham 1988 found that at 2 to 5 days after inoculation, multipli cation and adhesion of acid sensitive R. legurninosarum by. phaseoli to root hairs of bean was strongly affected by acid pH. By 8 days however both acid sensitive and acid tolerant strains had a similar rhizosphere population even though their nodule number was similar. By contrast, Frey & Blum 1994 found that the nodule number `ere not related to the relative numbers of strains in the first five days of bean rhizosphere prior to nodulation.
Effectiveness trial. The poor nodulation in soil I at day 28 might be caused by the decrease in pH of this soil after planting. Table 3 shows that the pH of this soil decreased from 4.62 at planting to 4.01 at day 28. The decrease in pH is likely to be due to the rapid uptake of NH4' by the plants. At planting this soil contained 148 sg Nl-14-N g' Table 3, as it had just been fertilized by the farmer just before soil collection. This uptake would be balanced for charge by the plant by a net efflux of protons resulting in a lowering of the soil pH. Some of the NH: would be nitrified to NO3 with a consequent generation of protons. When subse quently this NO., is taken up by the plant, the efflux of balancing hydroxyl ions would result in an increase in soil pH, as evidenced by the increase to pH 4.52 at day 56 Table 3. The p1-I increased slightly in the other soils also.
The relatively small change in NH4-N concentration in soils 2 and 3 over time suggests that the KCI extractant was detaching NH4 from clays that was not plant available.
The low level of Ca and high level of mineral N is likely to be the main cause of poorer nodulation in soil 1. The calcium level in soils 2 and 3 was higher than soil 1 Table 1, while mineral N of these soils was significantly lower than that of soil I Table 3. Calcium is essential for nodulation Munns 1970, and it can slightly alleviate the Al mediated inhibition of induction of one of the nod genes in R. leguniinosarum by. trfo1ii Richardson et cii. 1988. Soil 1 had the lowest exchangeable Ca and high mineral N 160 .`g g', and these might have contributed to the poorer nodulation. Brockwell et cii. 1989 and Bergersen et a!. 1989 found that a high level of mineral N 37.6 p.g g5 caused reduction in seedling rhizosphere colonization, and slower and poorer nodulation which led to reduced N2-fixation of soybean.
Correlation between strain tolerance of acidity and performance in acid soils. The response of the soybean-B. japonicurn symbiosis seemed related to the infecting strain's tolerance of acidity in agar media. Soybean inocu lated with the acid tolerant strains had significantly P<0.05 greater symbiotic effectiveness than plants inocu lated with the acid sensitive strain. Similar results were reported by Graham et a!. 1982 and Vargas & Graham 1989 for R. phaseoli and Thornton & Davey 1983 for R. irfo1ii. Graham et a!. 1982 found that strain C1AT899 which was tolerant of low pH, high Al and Mn in agar medium and performed better in N,-fixation with beans and survived better in acid soil than the acid sensitive strain CL&T640. Similarly Thomton & Davey 1983 showed R. trfolii strains which grew in liquid medium at pH 4.2 and containing 151.iM Al survived in greater numbers than acid sensitive strains when inoculated into sterile or non-sterile acid soil.
By contrast, Bromfield & Jones 1980 for R. trfb1ii and Howieson el a!. 1988 for R. meliloti, found poor cone lation between tolerance of strains in acid media and in acid soils. Bromfield & Jones 1980 reported that R. trfoIii strain l6strr which was .tolerant of acidic media pH 4.6 Table3.Changes in pH, ammonium and nitrate concentration of acid soils during soybean growth.
Soilffime NH-N .1g!g soil Nl1,-N j.g/g soil p11
Soil Day 0 477 13.8 4.2
Day 28 16.6 49 4.01
Day 56 4.4 2.3 4.52
Soil Day 0 7.7 2.3 4.58
Day 28 5.8 3.3 4.66
Day 56 6.3 2.9 4.74
Soil DayO 10.1 8.8 4.35
Day 28 13.5 1.8 4.44
Day 56 13.6 4.0 4.68
28 INDRASUMUNAR ETAL. J. Mikrobiol. Indon.
REFERENCES
Ayanaba, A., S. Asanuma & D. N. Munns. 1983. An agar plate method for rapid screening of Rhizobium for tolerance to acid-aluminum stress. J. Soil. Sci. Soc. Am. 47:256-258.
Bergersen, F. J., J. BrockweH, R. R. Gault, L. J. Morthorpe, M. B. Peoples & C. L. Turner. 1989. Effects of available soil nitrogen and rates of inoculation on nitrogen fixation by irrigated soybean and evaluation of&`N methods for measurement. Aust. J. Agric. Res. 40:763-780.
Bremner, J. M. & D. R. Keeney. 1965. Steam distilation methods for determination of ammonium, nitrate and nitrite. Analytica Chimica Acte. 32:485-496.
Brockwell, J., A. Diatloff, A. Grassia & A. C. Robinson. 1975. Use of wild soybean Glycine ussuriensis Regel and Maack as a test plant in dilution nodulation frequency test for counting Rhizobium japonicwn. Soil Biol. Biochent 7:305-311.
Brockwell, J., R. K. Cault, L. J. Morthorpe, M. B. Peoples, G. L. Turner & F. Bergersen. 1989. Effects of soil nitrogen status and rate of inoculation on the establishment of populations of Bradyrhizobium japonicum and on the nodulation of soybeans. Aust. J. Agric. Res.
40:753-762.
Bromfield, E. S. P. & D. G. Jones. 1980. Studies on acid tolerance of Rhizobium trifolii in culture and soil. J. App!. Bacteriol. 48:253-264. Fahraeus, G. 1957. The infections of clover root hairs by nodule bacteria
studied by a simple glass slide technique. J. Ge,,. Microbiol. 16:374-381.
Foy, C. D., H. H. Lalever, J. W. Schwartz & A. L. Fleming. 1974. Aluminium tolerance of wheat cultivars related to region of origin. Agron.J. 66:123-126.
Frey, S. D. & L. K. Blum. 1994. Effect of pH on competition for nodule occupancy by type I and type 11 strains of Rhizobium leguminosarum by. phaseoli. Plant Soil. 163:157-164.
Gemell, L. G. & R. J. Roughley. 1993. Field evaluation in acid soils of strains of Rhizobium legwninosarum by. trifolli selected for their tolerance or sensitivity to acid soil factors in agar medium. Soil Biol. Biochem. 25:1447-1452.
Gemell, L. G., R. J. Roughley, M. L. Reed & E. J. Hartley. 1993. Screening of Riiizobium leguminosarom by trifolii for adaptation to acid and neutral soils using a selective agar medium. Soil Biol. Biochem. 25:1463-1464.
Glilman, G. P. & E. A. Sumpter. 1986. Modification to the compulsive exchange method for measuring exchange characteristics of soils. Aust. J. Soil Res. 24:61-66.
Graham, P. H., S. F. Viteri, F. Mackie, A. T. Vargas & A. Palacios. 1982. Variation in acid soil tolerance among strains of Rhizobium phaseoli. Field Crops Res. 5:121-128.
Hand, P. G. & M. Alexander. 1983. Growth and survival of cowpea rhizobia in acid, aluminum-rich soils. J. Soil Sci. Soc. Am. 47:502-506.
Howleson, J. G., M. A. Ewing & M. F. D'Antuono. 1988. Selection for acid tolerance in Rhizobium meliloti. Plant Soil 105:179-188.
lndrasumunsr, A. & P. J. Dart. 1999. Laboratory prescreening of Bradyrhizobium japonicum for acid pH tolerance to predict their survival in acid soils. J. Mikrobiol. Indon. 4:40-45.
Karanja, N. K. & M. Wood. 1988. Selecting Rhizobium phaseoli strains for use with beans Phaseolu.s vzdgaris L. in Kenya: Infectiveness and tolerance of acidity and aluminium. Plant Soil 112:7-13.
Lovato, P. F., J. C. Pereira & C. Vidor. 1985. Population changes in Rhizobium phaseoli strains in bean rhizosphere. R. Bras. Ci. Solo. 9:211-218.
Lowendorf, H. S. & M. Alexander. 1983. Identification of Rhizobium phaseoli strains that are tolerant or sensitive to soil acidity. App!. Environ. Micro biol. 45:737-742.
Munns, D. N. 1970. Nodulation of Medicago sativa in solution culture. 1. Acid sensitive steps. PlantSoil28:129-l46.
Richardson, A. E., R, J. Simpson, M. A. Djordjevic & B. 6. Rolfe. 1988. Expression of nodulation gens in R.hizobium leguminosarum by. trifolii as affected by. low pH and by Ca and Al ions. App!. Environ. Microbiol. 54:2541-2548.
Roselam, C. A., S. J. Bicudo & 0. M. Marubayashi. 1995. Soybean yield and root growth as affected by lime rate and quality. Di dalam: R. A. Date, N. J. Grundon, G. E. Rayment & M. E. Probert ed. Plant-Soil Interactions at Lw pH: Principles and Management. Him. 543-547. Dordrecht: Kluwer Academic Publishers.
Sartain, .1. B. & E. J. Kamprath. 1978. Aluminium tolerance of soybean cultivars based on root elongation in solution culture compared with growthin acid soil. Agron. J. 70:17-20.
Taylor, R. W., M. L. Williams & K. K. Sistani. 1991. N2 fixation by soybean.Bradyrhizobium combinations under acidity, low P and high Al stresses. Plant Soil 131:293-300.
Thornton, F. C. & C. B. Davey. 1983. Response of the clover-Rhizobium symbiosis to soil acidity and Rhizobiunt strain Trifolium subterraneum, nitrogen fixation. Agron. J. 75:557-560.
Vargas, A. A. T. & P. H. Graham. 1988. Phaseolus vulgaris cultivar and Rhizobium strain variationin acid-pH tolerance and nodulation under acid conditions. Field Crops Res. 19:91.101.
Vargas, A. T. & P. H. Graham. 1989. Cultivar and pH effect on competition for nodule sites between isolates of Rhizobium in beans. Plant Soil 117:195.200.
was less persistentthan
three acid soils tested Howieson et a!. 1988
the acid sensitive strain 7Ast,' in
pH 3.9, 4.7 and 5.1. Similarly found low correlation r = 0.16
between the field ratings and the laboratory ratings for the 20 strains ofR. melioti.
The acidified medium used in the assessment of acid tolerance by Bromfield & Jones 1980 and Howieson et al. 1988 did not contain high levels of Al or Mn which are often associated with acid soil stresses. It mightbe neces sary to include Al and Mn in the test medium to mimic the soil stresses. Gemeil et a!. 1993 suggested that acidified medium for screening acid tolerant rhizobia should contain a combination of acidity stresses, such as low pH, Ca and P, plus high Al and Mn.
Results from this experiment confirm that laboratory prescreernng of Bradyrhizobium for acid, Al and Mn
tolerance in acid media was successful in selecting strains which were symbiotically competent in low pH soils, and
those which were less so. Moreover, these experiments suggest that laboratory prescreening of Bradyrhizobium for acid tolerance can assist in the selectionofstrains suitable
