Original scientific paper DOI: /10.5513/JCEA01/20.3.

2118
Journal of Central European Agriculture, 2019, 20(3), p.938-952

Incubation of soil with agricultural lime and phosphorus enhances biological

nitrogen fixation and yield of soybean (Glycine max (L.) Merrill) in an ultisol

Hamisi J. TINDWA1 (✉), Alinafe KACHIGUMA1, 2, Jerome P. MREMA1

1
Department of Soil and Geological Sciences, Sokoine University of Agriculture, P. O. Box 3008, Morogoro, Tanzania
2
Soils and Agricultural Engineering Section, Chitedze Agricultural Research Station, P. O. Box 158, Lilongwe, Malawi

✉ Corresponding author: [email protected]

ABSTRACT
Low pH and phosphorus are among major soil chemical constraints to Biological Nitrogen Fixation (BNF) and soybean
production in sub-Saharan Africa. A screenhouse pot experiment was conducted during the 2016/2017 season at
Sokoine University of Agriculture- Morogoro Tanzania, to examine the ameliorating effect of liming to pH, phosphorus
uptake, BNF and yield of soybean grown on an ultisol. The experiment was a 33 factorial-laid out as a split-plot with
two replications. Source of nitrogen (inoculation, non-inoculation and applied inorganic nitrogen) was main plot factor,
while the subplots received combinations of lime and phosphorus. The levels of lime used for the incubation experiment
were 0, 5 and 10 tons/ha while those of P were 0, 50 and 100 kg/ha. A commercial inoculant-Legumefix, was used for
inoculation. Results indicated that liming significantly (P<0.05) raised the soil pH and that the interaction of lime and
phosphorus showed significant (P<0.05) effect on nodulation and N2 fixation. Overall, application of 5 tons of lime/ha
and 100 kg P/ha gave the best-case scenario in terms of pH amelioration, P availability and BNF enhancement by the
soybean-Bradyrhizobium symbiosis.

Keywords: BNF, lime, pH, phosphorus, ultisol

INTRODUCTION produce it (Singh and Shivakumar, 2010; Aniekwe and

Mbah, 2014).
Soybean (Glycine max (L.) Merrill) is a leguminous
vegetable plant of the Pea family, Fabaceae (Singh and Agronomically, soybean is a good rotation and/or inter-
Shivakumar, 2010), which grows under a wide range of mixed crop especially with cereals, where in addition to
environments notably from tropical and subtropical to reducing pathogen infestation rates, it improves overall
temperate climates. It is a rich source of inexpensive soil fertility mainly through its capacity to symbiotically fix
proteins (Binang et al., 2013), dietary fiber, minerals and atmospheric nitrogen to plant available forms of nitrogen
other bioactive compounds such as isoflavones (Macák (Akibode and Maredia, 2011; Chianu et al., 2011).
and Candráková, 2013). Soybean has multiple economic Biological nitrogen fixation by a soybean-rhizobia
uses as it can be sold as food grain and/or in processed association, can however, be affected by a myriad of
forms like soy nuts, soy milk, soy pulp and oil (Akibode factors including the soil reaction, soil N content, soil
and Maredia, 2011). Its other product lines include edible phosphorus, and the native rhizobial population in a
oils, printing ink and biodiesel. As a result, soybean serves particular soil (Giller and Wilson, 1991). For example,
as a major foreign exchange earner for countries that both saline-sodic, sodic (pH≥9) and very strongly acidic

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Kachiguma et al.: Incubation of soil with agricultural Lime and Phosphorus enhances Biological...

(pH≤5) soil conditions reduce the survival of rhizobia thus Morogoro municipality, Morogoro region, Tanzania. The
inhibit nodulation and biological N fixation (Mabrouk and farm lies between latitude 06° 50' 24.7" S and longitude
Belhadj, 2012) mainly because rhizobia are neutrophils 37° 38' 59.8" E and at an elevation of 526 m above sea
in nature preferring slightly acidic to slightly alkaline (pH level (Msanya et al., 2003). The rainfall distribution is
5.1-8.9) conditions for their optimum growth and activity bimodal with the first rainy season running from March to
(Slonczewski et al., 2009). On the other hand, soils rich May while the second rainy season runs from November
in plant available nitrogen (≥30 mg N/kg in the 0-30 cm to January. Twenty soil sample portions of 10 kg were
layer) have been found to subdue the biological nitrogen collected randomly from 3 ha area at the rooting depth of
fixation process through retardation of root infection 0-30 cm, thoroughly mixed, air dried, ground and sieved
by rhizobia, nodule development and the extent of through a 6 mm sieve and mixed again to make one
nodulation (Singh and Shivakumar, 2010; Chianu et al., composite sample.
2011; Weisany et al., 2013). Furthermore, in very acidic
From the sieved composite sample, about one-kilogram
pH conditions such as those found in ultisols, another
soil was sieved again through a 2 mm sieve for laboratory
important plant nutrient, phosphorus tends to be fixed
analysis on chemical, physical and biological properties
to plant unavailable forms making supplemental P
of the soil. Particle size distribution was determined
fertilization a necessity to enhance N fixation.
by the hydrometer method after dispersion with 5%
Since ultisols, which are characterized by low soil sodium hexametaphosphate (Gee and Bauder, 1986)
fertility attributed to low pH, are widespread in Africa and soil textural class was determined using the United
(Giller and Wilson, 1991), such soils are, therefore, State Department of Agriculture (USDA) soil textural
associated with pronounced low yields of important class triangle (United State Department of Agriculture,
crops such as soybean (Chianu et al., 2011). Liming and 1975). Soil pH was determined electrometrically in 1:2.5
phosphorus application have been shown to enhance BNF soil:water and soil:0.01 M CaCl2 suspensions (Thomas,
in ultisols (Chianu et al., 2011; Binang et al., 2013; Njeru 1996). CEC and exchangeable bases (Ca2+ Mg2+, K+ and
et al., 2013). However, information on the optimum rates Na+) were determined by the 1 M (pH 7) NH4-acetate
of liming materials and phosphorus for BNF and soybean saturation method followed by displacing adsorbed NH4+
production in Sub Saharan Africa (SSA) is scanty, hence using 1 M KCl. Exchangeable Ca2+ and Mg2+ were quantified
need to generate such information is both urgent and using an atomic absorption spectrophotometer (AAS) and
crucial. In the current study, conducted during the year exchangeable K+ and Na+ by the use of a flame photometer
2016/2017, optimal amounts of phosphorus and calcite (Thomas, 1996). Organic carbon was determined by the
that should be applied to an ultisol for enhanced BNF Walkely and Black wet oxidation method (Nelson and
and soybean yield at the Magadu section of the Sokoine Sommers, 1996). Total N was determined by the Kjeldahl
University of Agriculture (SUA) farm were determined. digestion-distillation method as described by Bremner
(1996). Available P was extracted by the Bray-1 method
MATERIALS AND METHODS (Kuo, 1996) and quantified spectrophotometrically at 884
nm. Plant available micronutrients (Fe, Cu, Zn and Mn)
Description of study area, soil sampling and analysis
were extracted by the DTPA reagent method (Lindsay and
This study was designed and conducted as a Norvell, 1978) while boron was determined by hot water
glasshouse pot experiment during 2016/2017 growing extraction method (Bremner, 1996) and exchangeable
season based on an extremely acidic (pH<4.5) ultisol from acidity by the use of titration method (Thomas, 1996).
Magadu section of the Sokoine University of Agriculture
Indigenous rhizobial presence was checked by
(SUA) farm. The Magadu section of the SUA farm is
conducting the Most Probable Number (MPN)-plant
located at the foot slopes of the Uluguru Mountain in

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Original scientific paper DOI: /10.5513/JCEA01/20.3.2118
Kachiguma et al.: Incubation of soil with agricultural Lime and Phosphorus enhances Biological...

infection technique (Giller and Wilson, 1991) to determine in paper envelopes and kept in ventilated oven at 60-70
the potential of the soybean crop to form effective °C to constant weight prior to dry matter determination
soybean-rhizobia symbiosis with indigenous populations using an electronic balance. The dried plant samples
or that inoculation would be required. were milled into powder and wet digested by Kjeldahl
method and the total N accumulation was determined by
Screenhouse pot experiment and total N determination distillation-titration method as described by Peoples et
The study was a factorial experiment laid down in al. (1989). N2 fixed was estimated as total N balance as
a split plot design with two replications. The main plot follows:
factor was the source of nitrogen at three levels namely Total N balance = (final N in soil + N in plant material) –
(i) inoculation with legumefix commercially available (initial N in soil)
inoculant, (ii) non-inoculation where BNF if any would To recover the below-ground biomass, the growth
only have resulted from indigenous rhizobial species and medium containing the roots and associated nodules was
(iii) inorganic N applied at a rate of 200 kg N/ha. The carefully placed in small sized sieve enough to trap any
sub-plots factor involved the different combinations of dry matter from being washed away. The roots were then
lime and phosphorus also at three levels of 0, 5 and 10 washed thoroughly with water to remove soil particles and
tons/ha and 0, 50 and 100 kg/ha of lime and phosphorus, organic debris. From the clean root system, nodules were
respectively. Pure calcite (CaCO3) was used as the carefully plucked out and both their numbers and fresh
agricultural liming material and triple super phosphate volumes determined. Nodule numbers were determined
(Ca(H2PO4)2·H2O) was used as the source of phosphorus by manual counting per plant while fresh nodule volumes
and urea (CO(NH2)2 as source of N were determined by the volume displacement technique
Experimental growth medium was prepared by mixing as explained by Solomon et al. (2012). Briefly, all nodules
four kg of composite soil which had previously passed from a single plant were cleaned, blotted dry and then
through a 6 mm sieve with desired combinations of lime placed in a 10 ml capacity plastic measuring cylinder half
and P and placed into 5-liter capacity plastic pots. Several filled with water. The volume of water displaced by the
holes were made at the bottom of each pot and loosely nodules obtained from the sampled plants was recorded,
plugged with cotton wool to facilitate drainage. The pots and the average was considered as nodule volume per
were maintained at field capacity moisture status and plant. Nodule dry weight was determined by measuring
incubated for 40 days. The post-incubation pH of each weight of the nodules using an electronic balance after
pot was measured electrometrically in 1: 2.5 soil: water drying the nodules at 70 °C to constant weight.
suspension as described by Thomas (1996). Two pre-
Quantification of soybean content of Ca, P and total dry
germinated soybean seeds were then sown at a depth of
matter yield
about 15 mm in each pot. To appropriate pots, LegumeFix
was used to inoculate the germinated seedlings by Following 40 days of vegetative growth, soybean
pipetting 1 ml of the inoculant to each seedling two days plants were carefully harvested, processed and subjected
after emergence. With all the recommended agronomic to total plant analysis for determination of phosphorus
practices observed, the plants were allowed to grow for and calcium contents. Briefly, samples were oven-dried,
40 days post-planting. ground and sieved through a 0.5 mm sieve. The sample
powder was further subject to dry combustion (ashing)
Following the 40 days period of vegetative growth, a
method as described by Anderson and Ingram (1989).
sharp knife was used to cut off each plant at the base
Phosphorus was determined by spectrophotometer and
just above the soil level. The above ground shoots of
atomic adsorption spectrometer analysis was used for Ca
the sampled plants were cleaned of any debris, packed
determination.

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Original scientific paper DOI: /10.5513/JCEA01/20.3.2118
Kachiguma et al.: Incubation of soil with agricultural Lime and Phosphorus enhances Biological...

For dry matter yield estimates, soybean plants reported elsewhere that while high concentrations of
were sampled at flowering stage of growth and the mineral N either as nitrate or ammonium or ammonium
above ground shoot of the sampled plants was used to nitrate significantly suppress nodule numbers, nodule
determine dry matter yields (DMY). Plant samples were dry weight and total N2 fixed per plant of nodulated
cleaned of any debris and then packed in envelopes and soybeans (Gulden and Vessey, 1997; Gan et al., 2004),
dried in ventilation oven at 60-70 °C to constant weight. too low concentrations of nitrogen in growth media are
The dry matter yields were determined using an electron equally detrimental to BNF especially at initial stages of
balance. growth before the plant can reliably fix its own nitrogen
by symbiotic BNF.
Statistical analysis
The experimental soil had low available phosphorus
The data collected were subjected to analysis of (P) (<15 mg/kg soil) which necessitated application of
variance (ANOVA) using GenStat 14 edition. Treatment
th
P fertilizer to enhance soybean N2 fixation. P is highly
means were separated using New Duncan’s Multiple required in energy transfer processes involved in BNF
Range Test (NDMRT) at 0.05 probability level. (Rotaru and Sinclair, 2009a, b) and both abrupt removal
of P supply and suboptimal presence of P in a growth
RESULTS AND DISCUSSION
medium have been shown to severely arrest nodule
growth and substantially reduce nitrogenase activity
Selected properties of the soil used in this study
leading to ultimate reduction in N2 fixation (Hog-Jensen
Table 1 shows selected properties of the soil used in et al., 2002).
this study. Accordingly, the textural class of the soil from
Exchangeable calcium (Ca) of the soil under study
the study area was loam (United State Department of
was low (<4 cmol/kg soil). However, in this study,
Agriculture, 1975), which would enhance the growth and
the calcium problem was addressed by the type of
production of soybean due to its good aeration, moisture
phosphatic fertilizer used (Ca(H2PO4)2·H2O) as it would
and nutrient retention capabilities among others.
supply Ca in addition to phosphorus in the equation. Both
However, the pH of this soil (pH 4.6), which corresponds
exchangeable magnesium and exchangeable potassium
to very strongly acidic soil reaction, is not suitable for
readings were high (>0.5 cmol/kg soil and ≥0.4 cmol/
growth and development of most annual crops including
kg soil, respectively) according to the categorization by
soybean. This is because low soil pH impairs plant
Landon (1991) hence needed no corrective measures for
development and affects availability of various plant
the current study.
nutrients such as phosphorus and exchangeable bases
(Marschner, 1995). Various studies have reported more Overall, based on the soil analytical data presented in
than 90% reduction in nodule numbers and up to 50% Table 1, the Ultisol of the study area is rated as of low
reduction in nodule weight due to low soil pH (Alva et al., fertility status for soybean production. The soil fertility
1990; Evans et al., 1990). Furthermore, the strongly acidic limitations being very low soil pH, low total N and low
soil reaction has been shown, elsewhere, to adversely available P among others. Enhanced and sustainable
impair BNF as most N2-fixing symbionts prefer slightly soybean production at the study site (Magadu section of
acidic (pH 5.1) to slightly alkaline (pH 8.9) soil conditions SUA farm) can be attained if the following are taken on
(Bhagwat and Apte, 1989; Graham et al., 1994). board, namely liming the soil, application of phosphorus
fertilizer and inoculation of soybean seeds before
The total N of the soil was very low (<0.1%), according
planting. Liming the soil to increase the soil pH to at least
to Landon (1991), indicative of requisite levels of soil
5.5 can enhance cation exchange capacity of the soil and
N for the legumes to amply fix nitrogen. It has been
increase mineralization of soil N through decomposition

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Kachiguma et al.: Incubation of soil with agricultural Lime and Phosphorus enhances Biological...

Table 1. Chemical, physical and biological properties of the Raising pH of the ultisol through liming led to enhanced
soil (Ultisol) from the study area calcium and phosphorus content in soybean plant shoots
Soil parameter Value
Results on pH level, calcium and phosphorus content
Clay (%) 25 of the soil following incubation of the acidic soil with a
Silt (%) 30 mixture of agricultural lime (calcite) and phosphorus for
forty days maintained at field capacity are presented in
Sand (%) 45
Tables 2 and 3. Soil pH increased significantly (P<0.05)
Soil textural class Loam
as the rate of liming increased from 0 to 5 to 10 tons/
pH in water (H2O) 4.6 ha regardless of the level of phosphorus applied. pH
pH in CaCl2 (0.01 M CaCl2) 4.2 increased from 4.6 to 6.8 when lime applied was increased

Organic carbon (%) 0.64

from 0 to 10 tons/ha. Similar results on pH increase
due to lime application have been reported elsewhere
Organic matter (%) 1.1
(Bekere, 2012; Ayodele and Shittu, 2014). Furthermore,
Total N (%) 0.08 there was a general tendency for the pH to decrease, but
Available P Bray 1 (mg/kg) 7.42 non-significantly, with increasing amounts of P applied at
Cation exchange capacity (cmolc/kg) 13.51 a constant liming rate (Table 2).

Ca
2+
1.6 Results show that calcium content of soybean shoots,
2+ was significantly (P<0.05) different among the main plot
Mg 0.81
+
treatments (Table 3). Further analysis of the interaction
K 0.4
between lime and phosphorus application revealed that
+
Na 0.07 at any level of P applied, the calcium content of soybean
Base saturation (%) 21.32 shoots increased with increasing liming rate from 0 to 5 to
10 t/ha in inoculated, non-inoculated and N applied plots.
Indigenous rhizobia count (2 to 79 cells/g soil) = low
The inorganic N applied plants had higher Ca content
followed by inoculated plants while the non-inoculated
of organic matter as the microbial activities increase. plants had the least shoot Ca content at nearly all levels
Application of phosphorus fertilizer can also enhance and of combinations of lime and P applied (Figure 1).
sustain soybean production through BNF (Mahamood, Following concurrent application of lime and
2008). Phosphorus acts as an energy source for N2 phosphorus and subsequent growing of soybean plants,
fixation because it is required in biosynthesis of no meaningful trend on the levels of shoot P content
adenosine triphosphate (ATP). Up to 16 molecules of ATP was observed among the three main plot treatments of
are converted to adenosine diphosphate (ADP) as each inoculated, non-inoculated and inorganic N applied (Table
molecule of N2 is reduced to NH3. 3), regardless of the level of lime and/or phosphorus
The observed low numbers of indigenous rhizobia applied. This observation was also supported by the
capable of nodulating soybean could be attributed to results of the analysis of the interaction effects of lime
the fact that the Magadu section of the SUA farm has and phosphorus application on P content of soybean
not been under soybean or other related legumes for a shoots (Figure 2).
long time. (Morel et al., 2012). This called for inoculation This could be attributed to the fact that P was also
of soybean seeds as soybean will likely respond to used for energy storage and transfer from the leaves
inoculation if the number of Bradyrhizobia in the soil is to the roots for the symbionts in the inoculated plot
greater than 50 cells/g soil (Mishra et al., 2013). (Weisany et al., 2013). However, this observation is not

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Kachiguma et al.: Incubation of soil with agricultural Lime and Phosphorus enhances Biological...

Table 2. Effect of lime and phosphorus on pH and Ca content soybean shoots

Water parameters and bulk density

% Ca in the main plots

Lime (tons) Phosphorus (kg) pH
Inoculated Inorganic N Non-inoculated

0 0 4.6a 0.212a 0.326abcde 0.25abcd

50 4.6a 0.236ab 0.33abcdef 0.354abcdefg

100 4.5a 0.297abcde 0.312abcde 0.245abc

5 0 5.9b 0.515cdefghij 0.605ghijk 0.373abcdefgh

50 6b 0.638hijk 0.652ijk 0.397abcdefghi

100 5.7b 0.491bcdefghij 0.695jkl 0.402abcdefghi

10 0 6.8c 0.558efghijk 0.913l 0.425abcdefghij

50 6.8c 0.52defghijk 0.913l 0.468abcdefghij

100 6.7c 0.6fghijk 0.785kl 0.686jkl

CV (%) = 50.5

Values in the same column followed by different letters are significantly different according to Duncan's New Multiple Range
a, b, c, d, e, f, g, h, i, j, k, l

Test at P=0.05.

Table 3. Effect of lime and phosphorus on P content in soybean shoots

Water parameters and bulk density

Shoot P (%) in the main plots

Lime (tons) Phosphorus (kg) pH
Inoculated Inorganic N Non-inoculated

0 0 4.6 0.0778ab 0.2991fg 0.1257abcd

50 4.6 0.113abcd 0.464h 0.1779abcdef

100 4.5 0.2272cdefg 0.4696h 0.1708abcdef

5 0 5.9 0.0553a 0.2723efg 0.1116abcd

50 6 0.1018abc 0.3033fg 0.1511abcde

100 5.7 0.1342abcde 0.2794efg 0.0919abc

10 0 6.8 0.1807abcdef 0.2554defg 0.1765abcdef

50 6.8 0.1891abcdef 0.2526defg 0.2216bcdef

100 6.7 0.3681gh 0.1891abcdef 0.2737efg

CV (%) = 50.5

a, b, c, d, e, f, g, h
Values in the same column followed by different letters are significantly different according to Duncan's New Multiple Range
Test at P=0.05.

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Kachiguma et al.: Incubation of soil with agricultural Lime and Phosphorus enhances Biological...

Figure 1. Profile plots showing interaction effect of liming and phosphorus application on Ca content of soybean shoots (For the
determination of Ca content of the soybean plants, samples were oven-dried, ground and sieved through a 0.5 mm sieve. The sam-
ple powder was further subjected to dry combustion (ashing). Ca content of the dry ash sample was determined using an atomic
adsorption spectrometer (Varian).)

Figure 2. Profile plots showing interaction effects of lime and P application on P content of soybean plant shoots (Plant samples
were oven-dried, ground and sieved through a 0.5 mm sieve. The sample powder was further subjected to dry combustion (ashing),
allowed to cool, and dissolved in dilute HCl prior to analysis. For Phosphorus quantification, a colour development method using
the ammonium molybdate/ vanadate mixed reagents was followed as described by Okalebo et al. (2002). The colour intensity of the
deep blue complex was determined using a UV- visible spectrophotometer (Biomate6, Thermoscientific).)

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Kachiguma et al.: Incubation of soil with agricultural Lime and Phosphorus enhances Biological...

consistent with the findings by Basu et al. (2008) who Furthermore, results on effects of phosphorus and lime
reported that non-inoculated control plants accumulated application on nodule dry weights are presented in Figure
more P than the inoculated treatments. 5. Consistently higher dry nodule weights were recorded
in inoculated plants compared to non-inoculated plants
Liming and phosphorus application enhanced nodulation
(Figure 5, A). Like with both nodule numbers and nodule
of soybean
fresh volume per plant, profile plot analysis indicated
Effects of incubating the acidic ultisol with agricultural that highest nodule dry weights per plant were obtained
lime (calcite) and phosphorus on nodulation was examined when the soil was limed at 5 tones/ha and P applied at
in terms of nodule numbers, nodule fresh volume and 100 kg/ha in the inoculated plants followed by the non-
nodule dry weight in a potted soil experiment for plants inoculated plots. Plants grown in pots receiving inorganic
grown on inoculated, non-inoculated and inorganic N N gave negligible and inconsistent nodule dry weight
applied main plot treatments. values (Figure 5, B). The observed negative influence of
Results on nodule numbers are presented in Figure 3 inorganic N to nodulation and N2 fixation by soybeans
(A). At all treatment combinations, there were significantly is not surprising as numerous studies have shown that
(P<0.05) higher nodule numbers in inoculated legumes except for starter N applied at sowing, N fertilization of
than either inorganic N applied or non-inoculated legume N2-fixing legumes such as soybeans does depress BNF
pots. Results showed further that there was a clear (Gan et al., 1997; Gan et al., 2004; Salvagiotti et al., 2009).
interaction effect of the applied phosphorus and lime
on nodule numbers. Accordingly, the best-case scenario Liming and phosphorus application increased the
was observed when the soil was limed at 5 tons/ha and amount of biologically fixed N and dry matter yield of
P applied to 100 kg/ha under both inoculated and non- soybean on an ultisol
inoculated plots (Figure 3, B).
Figure 6 shows data for effects of lime and phosphorus
The interaction in inorganic N applied plot on nodule on the amounts of N2 fixed by the soybean-rhizobia
numbers was inconsistently small, probably due to high symbiosis tested under inoculated and non-inoculated
plant available N content in the soil solution which could conditions. Total N measurements were done as a proxy
have retarded root infection by the bradyrhizobia and to the amount of biologically fixed N per plant. Results
subsequent nodule development. In a previous study indicated that highest amounts of N2 fixed were obtained
Gan et al. (2004) reported that high concentrations of when the soil was limed at 5 tons/ha and P applied at 100
mineral N (≥10 mM), either as nitrate or ammonium or kg/ha. Overall, inoculated plots had higher amounts of
ammonium nitrate could significantly suppress nodule fixed N2 compared to non-inoculated plot (Figure 6). This
numbers, nodule dry weight and total N2 fixed per plant observation is also consistent with other studies such as
of nodulated soybeans. that reported by Mahamood (2008) who observed an
A similar trend was also observed with nodule fresh increase in N2 fixed in inoculated plants with application
volume per plant. There were significantly higher fresh of lime, P and the presence of adequate numbers of
nodule volume values in inoculated legumes than either Bradyrhizobium japonicum. However, Giller (2001)
non-inoculated or inorganic N applied plants (Figure 4, A). reported non-significant results and concluded that the
Profile plot analysis indicated that in both inoculated and ability to form nodules is not always enough to obtain an
non-inoculated pots, the highest fresh nodule volume effective nitrogen fixation symbiosis.
values were recorded when the soil was limed at 5 tones/
Dry matter content of plants was taken as a measure
ha and P applied at 100 kg/ha while values of nodule
of yield as influenced by improvements in pH and
fresh volume per plant from the inorganic N applied plots
subsequently P availability and N fixation. Results on
were negligible and inconsistent (Figure 4, B).

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Kachiguma et al.: Incubation of soil with agricultural Lime and Phosphorus enhances Biological...

Figure 3. Main plot effects (A) and profile plots showing interaction effects (B) of the application of lime and phosphorus on nodule
numbers of soybean plants (Following five weeks of growth, plants were carefully uprooted, roots gently washed to detach the soil
from the root system and nodules counted manually. Nodule numbers were estimated per plant. No significant differences in nodule
numbers were observed in inorganic N applied and non-inoculated pots.). Error bars indicate mean ± standard deviation. Bars of the
same colour marked by same letters are not significantly (P≤0.05) different according to New Duncan’s multiple range test.

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Kachiguma et al.: Incubation of soil with agricultural Lime and Phosphorus enhances Biological...

Figure 4. Main plot effects (A) and profile plots showing the interaction effects (B) of lime and phosphorus application on nodule
fresh volume of soybean plants grown on a potted soil. Error bars indicate mean ± standard deviation. Bars of the same colour
marked by same letters are not significantly (P≤0.05) different according to New Duncan’s multiple range test.

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Kachiguma et al.: Incubation of soil with agricultural Lime and Phosphorus enhances Biological...

Figure 5. Main plot effects (A) and profile plots showing the interaction effects (B) of lime and phosphorus application on nodule
dry weight of soybean plants grown on potted soil. Error bars indicate mean ± standard deviation. Bars of the same colour marked
by same letters are not significantly (P≤0.05) different according to New Duncan’s multiple range test.

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Kachiguma et al.: Incubation of soil with agricultural Lime and Phosphorus enhances Biological...

Figure 6. Effect of lime and phosphorus on the amount of N2 fixed (For quantification of N, above ground biomass samples of soy-
bean plants were dried, milled into powder, sieved to pass through a 0.5 mm sieve and wet digested by Kjeldahl digestion procedure.
Total N accumulation was determined by distillation-titration method as described by Peoples et al. (1989). N2 fixed was estimated
as total N balance using the formula: Total N balance = (final N in soil + N in plant material) – (initial N in soil): Error bars indicate
mean ± standard deviation. Bars of the same colour marked by same letters are not significantly (P≤0.05) different according to New
Duncan’s multiple range test.

shoot dry matter yield are presented in Figure 7. While application at 100 kg/ha gave the best advantage in terms
no significant (P≤0.05) differences were observed among of total dry matter yield in all the main plot treatments
the inoculated, non-inoculated and inorganic N applied of inoculated, non-inoculated and inorganic N applied
plants, profile analysis showed existence of interaction (Figure 4 and Figure 7). Overall, under these conditions,
between lime and P application inoculated conditions gave better dry matter yields than
both the non- inoculated and inorganic N applied plots.
Accordingly, liming the soil at 5 tons/ha coupled with P

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Kachiguma et al.: Incubation of soil with agricultural Lime and Phosphorus enhances Biological...

Figure 7. Main plot treatment effects (A) and profile plots showing the interaction effects (B) of liming and phosphorus application
on shoot dry weights (Dry matter content of plants was taken as a measure of yield as influenced by improvements in pH. The dry
matter yields were determined using an electron balance by measuring the weight in (g) of oven-dry plant shoot samples at repre-
sentative samples for each treatment combination. Error bars indicate mean ± standard deviation. Bars of the same colour marked
by same letters are not significantly (P≤0.05) different according to New Duncan’s multiple range test.

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Original scientific paper DOI: /10.5513/JCEA01/20.3.2118
Kachiguma et al.: Incubation of soil with agricultural Lime and Phosphorus enhances Biological...

CONCLUSIONS Basu, M., Bhadoria, P.B.S. (2008) Performance of groundnut (Arachis

hypogea linn) under nitrogen fixing and phosphorus solubilizing
The soil used in this study had a low population of the microbial inoculants with different levels of cobalt in alluvial soils of
eastern India. Agronomy Research, 6 (1), 15-25.
native Bradyrhizobium, therefore, sustainable soybean Bekere, W., Endalkachew, W., Tesfu, K. (2012) Growth and nodulation
production at the Magadu section of SUA farm requires response of soybean (Glycine max L.) to Bradyrhizobium inoculation
and phosphorus levels under controlled condition in South Western
inoculation of the seeds with Bradyrhizobium jopanicum Ethiopia. African Journal of Agricultural Research, 7 (30), 4266-
if one needs to capitalize on BNF. The soil pH of the 4270. DOI: https://dx.doi.org/10.5897/AJAR12.1095
Bhagwat, A.A., Apte, S.K. (1989) Comparative analysis of proteins
experimental soil (Ultisol) increased with increase in the
induced by heat shock, salinity, and osmotic stress in the nitrogen-
rate of liming. Liming at 10 tons per hectare increased the fixing cyanobacterium Anabaena sp. strain L-31. Journal of
Bacteriology, 171, 5187–5189.
pH of the soil from 4.6 to about 6.8, in which microbial
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activity for nodulation and N fixation were enhanced. Binang, W.B., Ojikpong, T.O., Garjila, Y.A., Esang, D.M., Okpara,
D.A. (2013) Evaluation of liming materials and Bradyrhizobium
No advantage is obtained on BNF by applying P on inoculation on the productivity of soya bean in the humid tropical
this soil in the absence of liming. Increasing liming rate ultisols of south-eastern Nigeria. Journal of Agriculture and
Sustainability, 4 (2), 141-153.
to 5 tons/ha and phosphorus to the rate of 100 kg/ha Bremner, J.M. (1996) Nitrogen total. In: Sparks, D.L., ed. Methods of soil
improved the nodulation parameters namely nodule analysis, Part 3: Chemical methods. SSSA Book series 5. Madison,
WI: Soil Science Society of America, 1085-1122.
numbers, nodule fresh volume and nodule dry weight as Chianu, J.N., Nkonya, E.M., Mairura, F.S., Chianu, J., Akinnifesi, F.K.
well as N2 fixed by soybean plants. (2011) Biological nitrogen fixation and socioeconomic factors for
legume production in sub-Saharan Africa. Agronomy for Sustainable
Therefore, it is recommended that lime and Development 31 (1), 139-154.
phosphorus application at the rate of 5 tons/ha and 100 DOI: https://dx.doi.org/0.1051/agro/2010004
Evans, J., Dear, B., O’Connor, G. (1990) Influence of an acid soil on
kg/ha respectively, combined with inoculation should be the herbage yield and nodulation of five annual pasture legumes.
employed for optimum BNF on the study soil at Magadu Australian Journal of Experimental Agriculture, 30 (1), 55 – 60.
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section of SUA farm that would ultimately increase Gan, Y., Peoples, M.B., Rerkasem, B. (1997) The effect of N fertilizer
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Field Crops Research. 51 (3), 221-229.
required to augment observations made in the current
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study. Gan, Y., Stulen, I., Van Keulen, H., Kuiper, P.J.C. (2004) Low concentrations
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inhibiting specific nodulation (nodule DW g−1 root dry weight) and
ACKNOWLEDGEMENTS
specific N2 fixation (N2 fixed g−1 root dry weight) in soybean. Plant
and Soil, 258, 281–292.
This work was funded in full by the Alliance for Green
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Revolution in Africa (AGRA) partly through availing lab Gee, G.W., Bauder, J.W. (1986) Particle size analysis. In: Klute, A., ed.
and field research funds. Methods of soil analysis, Part 1: Physical and mineralogical methods.
Madison, WI: Soil Science Society of America, American Society of
Agronomy.
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