Root Colonization and Symbiotic Effectiveness of Acid Tolerant and Acid Sensitive Strains of Bradyrhizobia in Acid Soils
Jurnal Mikrobiologi Indonesia, Februari 2000, 24-28
ISSN 0853-358X
Vol. 5, No. I
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, The University of Queensland; St Lucia 4072, QLD, Australia
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 of B. Japonkum that 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. japonicum in acid soils can
be predicted from their perfonnance in acidic agar media.
Key words: Bradyrhizobiumjaponicutn, 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
* Penuiis untuk korespondensi, Tel. +62-251-337975,
Fax. ±62-251-338820, E-mail: rifcb@indo.net.id
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.
Soil characteristics. The A horizon of three acid soils
with no previous history of soybean cropping was collected
Vol.5,2000
J. Mikrobiol. Indon.
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 soils are presented 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 placed in 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 the B. 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 per treatment, 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 Brockwell et 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.
Exchangeable ca tions cmoI ,, kg'
Soil
Types
Micro nuirien ts
pt-1
mg kg
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
3.69
1.79
0.53
1.84
9.05
4.38
Soil 3
*
1.63
mg kg'
0.2 NI BaCI,INI-i,Cl; `2M KCI
25
suspension in sterile, 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
rhizobium was 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
rhizobium capable of nodulating soybean present; therefore,
rhizobia counts indicate populations of Bradyrhizobium
introduced into the soil. All strains were inoculated at
approximately the same rate of 106 cfia seed', but at day 7
after inoculation there was a significant difference among
strains in their ability to colonize soybean roots. The acid
sensitive strain FCB 179 colonized soybean roots more
than acid tolerant strains FCB 152 and CB1809 Table 2.
The population of the acid sensitive strain decreased 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
and soil 2 high Al Table 1. The lower pH and the presence
of both Al and Mn may have caused the lower survival of
B.japonicum in soil 3 than in the other soils.
Nodulation. There was a significant P
ISSN 0853-358X
Vol. 5, No. I
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, The University of Queensland; St Lucia 4072, QLD, Australia
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 of B. Japonkum that 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. japonicum in acid soils can
be predicted from their perfonnance in acidic agar media.
Key words: Bradyrhizobiumjaponicutn, 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
* Penuiis untuk korespondensi, Tel. +62-251-337975,
Fax. ±62-251-338820, E-mail: rifcb@indo.net.id
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.
Soil characteristics. The A horizon of three acid soils
with no previous history of soybean cropping was collected
Vol.5,2000
J. Mikrobiol. Indon.
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 soils are presented 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 placed in 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 the B. 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 per treatment, 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 Brockwell et 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.
Exchangeable ca tions cmoI ,, kg'
Soil
Types
Micro nuirien ts
pt-1
mg kg
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
3.69
1.79
0.53
1.84
9.05
4.38
Soil 3
*
1.63
mg kg'
0.2 NI BaCI,INI-i,Cl; `2M KCI
25
suspension in sterile, 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
rhizobium was 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
rhizobium capable of nodulating soybean present; therefore,
rhizobia counts indicate populations of Bradyrhizobium
introduced into the soil. All strains were inoculated at
approximately the same rate of 106 cfia seed', but at day 7
after inoculation there was a significant difference among
strains in their ability to colonize soybean roots. The acid
sensitive strain FCB 179 colonized soybean roots more
than acid tolerant strains FCB 152 and CB1809 Table 2.
The population of the acid sensitive strain decreased 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
and soil 2 high Al Table 1. The lower pH and the presence
of both Al and Mn may have caused the lower survival of
B.japonicum in soil 3 than in the other soils.
Nodulation. There was a significant P