Introduction
Bradyrhizobium are beneficial symbionts that are highly present in
terms of its distribution as a free-living bacteria in different habitats and
in association with leguminous plants (Sprent et
al. 2017) unlike other N2-fixing microsymbionts, it exhibits a life cycle (bipartite)
which varies between the free-living state in soils and as a symbiotic partner
inside root nodules of legumes (Jaiswal and
Dakora 2019). The inoculation of the Bradyrhizobium
strains often leads to the supply of the nitrogen needed by plant through (Hungria and Mendes 2015) biological nitrogen
fixation (BNF) without the addition of chemical N fertilizer which eventually
leads to economic savings (Hungria and Mendes
2015), decrease in emission of greenhouse gases, and reduce risk of
contamination of surface and ground water with nitrate (Hungria and Mendes 2015; Moraes et al. 2017).
Bradyrhizobium are major
components of soil microbiota which enhanced the improvement of soil fertility,
plant growth nutrition and
contributes an important role in organic agriculture which eventually led to
the reduced application of inorganic fertilizers and agrochemicals. it usually
colonize roots of leguminous plants and form symbiotic association that leads
to utilization of water and adequate nutrient uptake (Gitonga et al. 2021). The use of
beneficial soil microorganisms in organic farming, is an improving and
promising smart-technology that could be used to reduce the intensive use of
inorganic fertilizers to amend leguminous crops (Fasusi
et al. 2021). The roles of the these bacteria have been marginalized in modern agriculture
since microbial communities in conventional farming systems have been modified
due to tillage (Kerry et al. 2018; Yadav et
al. 2018) and high inputs of inorganic fertilizers, herbicides and
pesticides (Malhotra et al. 2015; Prashar
and Shah 2016).
Bambara groundnut (Vigna subterranea (L.) Verdc.)
is an indigenous and underutilized African leguminous crop, grown for human and
animal consumption. It is ranked third in importance after other legumes like
groundnut (Arachis hypogea) and cowpea (V. unguiculata) in
Africa (Mubaiwa et al. 2017).
Bambara groundnut seed contains carbohydrate (58.3%), protein (23.7%) and fat
(4.3%) with high amount of methionine content than other leguminous crop (Oyeyinka et al.
2018). It has the ability to produce high yields even in low nutrient
soils and where there is drought stress due to low soil moisture (Ibny et al. 2019).
Bambara groundnut can fix about 4 to 200 kg N ha−1
(Mohale et
al. 2014) with symbiotic relationship with soil bacteria called
‘rhizobia (Puozaa et al. 2017). As
rhizobial species nodulating, most legumes can vary between geographical locations,
it is important to continuously explore new geographic regions to identify
rhizobia that are capable of effectively nodulating and promoting the growth of
Bambara groundnut (Ibny et al. 2019).
Some of the most promising strains for inoculation of Bambara groundnuts established
were; BAMKis12, BAMKis8, BAMKis4, BAMKar3 (Bradryrhizobium spp.), BAMKbay8 and BAMsp3 (Burkholderia
spp.) which produced highly
effective nodules and high plant biomass value, (Benson and Fredrick 2019).
The deficiency of N in soil often result to leaf
senescence, lower yield production, and biomass in plants (Kant et al. 2011). High cost of
procuring the inorganic N fertilizer by the tropical farmers is a major
challenge and most farmers that can afford usually apply below manufacturer
recommendation. Very little information is available on the biodiversity of
rhizobia nodulating Bambara groundnut in African soils, except for a few
studies which have shown that Bambara groundnut is nodulated by species of the
genus Bradyrhizobium. Therefore, this study was conducted to evaluate
the effect of inoculation of B. japonicum
strains with reduced cost to amend and to improve the growth traits,
nutrient uptake, and yield of different accessions of Bambara groundnut that differs
in genetic composition.
Materials and Methods
Seed
scarification
The seeds planted in the glasshouse were scarify using
scalpel, the seeds of the accessions were introduced into a beaker containing
3% w/v (Sodium hypochloride), the solution was decanted and rinse with sterile
water and 90% of ethanol was added for 30 sec and was decanted and also rinse
after 1 min with sterile water (Davis et al.
1991).
Authentication
of the strains
Seeds were sterilized, using 3% w/v sodium hypochloride,
planted in sterile carrier materials and allowed to grow for 10 days before
inoculation with broth containing about 1 × 109 cfu/mL of rhizobia
cell. Plants were allowed to grow for about six to eight weeks to check for
infectivity of rhizobia isolates. Most effective isolates were selected and used
for pot experiments and field experiment (Vincent
1970).
Broth
preparation
To prepare 1 liter of solution, mannitol 10 g,
yeast extract powder 0.5 g, potassium phosphate 0.5 g, magnesium sulphate 0.2
g, sodium chloride 0.1 g, agar powder 15 g. The mixture was dissolved in a
conical flask with 1000 mL of distilled water and stir to homogenize the
solution. The pH was adjust to 6.8 and sterilize using the autoclave at 121°C
for 15 min at a pressure of 15 psi, place in the water bath to adjust
temperature to 47°C and was poured in sterile petri dishes (Vincent 1970).
Determination
of most probable number
Rhizobia were isolated from
nodules on Congo red agar (Woomer 1994)
using spread plate method. Two undamaged nodules samples were picked from each
plant of Bambara groundnut and placed in sterile water for about 15 to 20 min
to rehydrate them after which they were surface sterilized using 3% sodium
hypochlorite for 3 min. They were then rinsed with sterile water after which
they were further sterilized with 95% ethanol and then rinsed with six changes
of sterile water (Woomer 1994). The
nodules were then transferred into sterilize petri-dishes, crushed with flamed
glass rod and mixed with a few drops of sterile water. A loop full of the crushed
nodule were streaked on Congo red agar and then incubated at 28°C for 5–7 days
isolates were purified and identified.
Nutrient solution preparation
To prepare 1 L of macronutrient, 100 g of calcium phosphate, 20 g of
potassium phosphate, 20 g of hydrated magnesium sulphate, 20 g of sodium
chloride, 10 g of iron (11) chloride are dissolve in 100 mL of distilled water
and stir till the solution is homogenous. To prepare 1 L of micronutrient
solution, 2.86 g of orthoboric acids, 1.81 g of magnesium chloride, 0.22 g of
zinc sulphate, 0.025 g of sodium molybdate were dissolve in 1000 mL of
distilled water and stir (Steiner and Soil 1961).
Pre-sowing
soil analysis used in pot and field experiments
The pH of the
soils used in the glasshouse in both seasons was neutral, the pH of the soil in
Ikenne was acidic in nature in both seasons while in Ibadan, pH was slightly acidic. The %
organic carbon ranged from 0.25 to 0.40 in both glasshouse and on the field
which shows the soil used is normal. The phosphorus in the soil used in the
glasshouse is low, higher % of P was recorded in the soil used in the field in
both locations and seasons. The % of K
present in the soil used in both glasshouse and in the field in both seasons shows availability in moderate quantities. The
calcium in the soil used in the glasshouse were extremely high, while the
calcium in the field in Ibadan were considered to be normal (Table 1). The Mg present in the soils used for the glasshouse
experiments were high in both sterile and non-sterile soil in both seasons.
Higher Mg values were obtained in Ikenne in both season while lower quantities
of Mg were obtained in Ibadan in both seasons. The particle size analysis was
determined by the (hydrometer method). The soil textural class shows that both
the soil used in the glasshouse and on the field is a loamy sand (Table 1).
Pot
experiment
An experiment was carried out in the glasshouse in 2018
and 2019 at the International Institute of Tropical Agriculture (IITA)
headquarter, Ibadan, Nigeria [Latitude (Lat) 7° 22´ 30 N and Longitude (long)
3° 45´ 54 E] to determine the growth and nutrient uptake of different
inoculated accessions. The soil used in the glasshouse was carefully sieve
through 3 mm sieve and was sterilized at 121°C for 1 h using the autoclave.
Ten
accessions of Bambara groundnut namely TVSu-378, TVSu-506, TVSu-787, TVSu-1606,
TVSu-1698, TVSu-1739, TVSu-710, TVSu-365, TVSu-475 and TVSu-305 were selected
from the International Institute of tropical Agriculture (IITA) Gene bank and
were inoculated with each B. japonicum
strains: FA3, RACA6, USDA110 and IRJ2180A containing 2.8 ×
107, 7.2 × 106, 4.3 × 107, 1.4 × 107 cfu/mL
respectively, N fertilizer (Urea, 20 kg/ha) and an uninoculated control. The
choice of the B. japonicum strains
depend on the availability and authencity (ability to
nodulate bambara groundnut) and were inoculated to the seedlings of the ten
accessions of Bambara groundnut in the glasshouse (Argaw 2014).
The
experiment was laid out in a completely randomized design (CRD) with factorial
arrangement using sterile and nonsterile soil and was replicated three time.
The seeds were surface sterilized and two seeds were sown into 10 kg pot
containing the sterile and nonsterile soil in the glasshouse. N free nutrient
solution and sterile water was added to each pot at regular interval (twice a
week) (Afzal et al. 2010). Data
regarding growth traits and nutrient uptake were collected at 10 WAP while
biomass yield was recorded at 50% flowering after drying at 72°C for 48 h (Argaw 2014).
Data collection
All data collected on growth parameters and biomass yield were taken
base on the descriptor of Bambara groundnut, International Plant Genetic
Resources Institute, (IPGRI) Rome
(Italy); International Institute of Tropical Agriculture; International Bambara
Groundnut Network (BAMNET). Vegetative traits recorded in
this study from the descriptor include (Peduncle length (cm), number of leaves,
terminal leaflet length (cm), terminal leaflet width (cm), petiole length,
plant spread (cm), plant height (cm), numbers of stem and branches per plant.
Determination of nitrogen in plant: preparation
of digest
A 0.2 g of plant sample was weighed into a digestion tube, then 2.5 mL
of acid mixture and 3 mL of hydrogen peroxide was added to the plant samples
and was stirred and place on the digesting block at 150°C for 30 min in the
fume cabinet and temperature was increased to 330°C. The sample was digested
until extract turn colourless, sample was removed from the fume cabinet and
place in the rack to cool and top up with 50 mL of distilled water (Anderson and Ingram 1989). All digest were diluted 1:9 (w/v) with distilled water and 0.2 mL
of sample digested was taken with a micropipette and was place in a clearly
labelled test tube and 5.0 mL of the reagent N1 and N2
was added, vortex and allow to stay for 2 h and absorbency was measured at 650
nm. The blue is stable for 10 h and the concentration of N in the solution was
measured. Nitrogen concentration in the sample materials expressed in %N is calculated as:
% N = (A-B) x V x K
%2017-24_files/image001.png){kind=small}
H2 x W x al
Where A = Concentration of N in the solution (n/mol), B = concentration
of N in the blank (n/mol), V = total volume at the end of analysis procedure (mL),
W = weight of dried samples (g), H= 1000, K = 100 and al = aliquot of the
solution taken (mL).
N1= 34g sodium salicylate, 25 g sodium
citrate and 25 g sodium tartrate in 75 mL water
N2= 30 g sodium hydroxide,10 mL sodium
hypochlorite and make up to 1 L (Lindner and
Harley 1942).
Field experiments
Field experiments was carried out at two different geographical
locations: Ibadan (7° 38’N, 3° 89’E) and Ikenne (6° 86’N,
3° 71’E), Nigeria in 2019 and 2020 cropping seasons to determine the growth,
nutrient uptake, and yield of inoculated accessions of Bambara groundnut. Each
field were prepared using a tractor driven plough and harrowed to remove plant
debris and 2 m plot were made with a spacing of 25 cm between plot and 1m
between each rep. The experiments were arranged in randomize complete block
design (RCBD) on the field in both locations and cropping season and was
replicated three (3) times.
Ten accessions
of Bambara groundnut: TVSu-378, TVSu-506, TVSu-787, TVSu-1606, TVSu-1698,
TVSu-1739 TVSu-710, TVSu-365, TVSu-475 and TVSu-305 used in the glasshouse were
also used for field studies and were randomly selected from the International
Institute of Tropical Agriculture (IITA) Gene bank. Six seeds from each
accession were coated with each of the B.
japonicum strains FA3, RACA6, USDA110, and IRJ2180A containing 2.8×107,
7.2×106, 4.3×107, 1.4×107 Cfu/mL respectively,
N fertilizer (Urea, 46%) and an uninoculated control. Seed of accessions were
coated with the B. japonicum strains
using gum arabic and allowed to dry with the seeds (Nodumax IITA) before
planting on the field at both locations and cropping seasons. Regular weeding
was done manually using hoe to remove weeds.
Data were
collected at 12 weeks after planting (WAP) on growth traits and at 50%
flowering on nutrient uptake. Data recorded on growth traits
include peduncle length (cm), terminal leaflet length (cm), terminal leaflet
width (cm), petiole length, plant spread (cm) and plant height (cm). Data was
taken using a well calibrated meter rule, while the data on the numbers of
stem, number of leaves, number of branches and on flowering recorded was
obtained by counting. Data on chlorophyll content of accessions of Bambara
groundnut was obtained using the spad meter. Also, data on yield was obtained
using the weighing balance.
Statistical analysis
Data collected were subjected to four-way ANOVA
(Analysis of Variance): pot (Accessions, strains, soil status and season),
field (Accessions, strains, locations and seasons) statistical analysis system
(SAS) package 9.4 and means were separated by using Ducan Multiple Range Test
(Dmrt) at P < 0.05 (Zatybekov et al.
2017).
Results
Microbial
population of the soil used for both pot and field experiment
The
non-sterilize soils used in the glasshouse experiments, based on microbial
analysis, indicated the presence of low rhizobia population during 2018 season
(1.28 × 102) and high in 2019 (1.53 × 109) season (Table 2). However, in Ikenne field, rhizobia population was
high i.e., 2.03 × 109
cfu/mL and 2.47 × 107 cfu/mL in 2019 and 2020 season,
respectively. In Ibadan field, the rhizobial population was also high i.e., 1.83×105 cfu/mL and 3.05 × 107 cfu/mL in 2019 and 2020 season, respectively (Table 2).
Growth
traits and nutrient uptake of inoculated accession under glasshouse conditions
At 10 weeks after planting (WAP), analysis of variance
(ANOVA) indicated that significant differences were recorded among accessions,
soil status (sterile and non-sterile soil) and seasons in the growth traits
recorded (Table 3). These significant differences among the accessions, soil
status and seasons was due to the inoculation of B. japonicum strains. While non-significant difference was recorded
among the strains inoculated in all the growth traits under glasshouse
conditions. The non-significant differences recorded among strains inoculated
under glasshouse conditions showed that the B.
japonicum strains were nearly equal in performance. Furthermore,
non-significant difference was recorded in growth traits regarding the
interaction of accessions with soil status, strains with soil status, and
accessions with strains with exception to chlorophyll content of accessions
(Table 3).
ANOVA
indicated that interactive effect of accessions and seasons had significant
effect on the number of stem (NOS) and number of leaf (NOL) (779.31** and
6041.09*). Likewise, interaction of strains and seasons (620.32* and 5978.13*),
accessions × strains × soil status × season (368.90** and 8737.41**) had
significant effect on NOS and NOL of Bambara groundnut
(Table 3). However, all two-way and three-way interactions were non-significant
on entire growth-related traits (plant height, canopy spread, and chlorophyll)
of Bambara groundnut (Table 3).
ANOVA
indicated that B. japonicum strains had significant effect on Bambara
groundnut accessions on days to 50% flowering (399.45**) and nutrient uptake (%N and %P) at flowering and at
harvest (Table 4). Moreover, non-significant difference was recorded among the
strains inoculated in the number of days to 50% flowering (261.39ns), % P uptake at flowering (0.005ns) and at harvest (0.004ns) under glasshouse
conditions (Table 4). However, significant difference was recorded among the
strains inoculated regarding N uptake at flowering (20.87**) and at
harvest (16.07**) under glasshouse conditions due to the inoculation of B. japonicum strains (Table 4).
Moreover, significant difference was recorded in Pflw and PHvst in the soil
status (0.17** and 0.19**) and season (1.06** and 0.23**) due to the inoculation of B. japonicum strains. Nevertheless, the interactions of accessions
with soil status, strains with soil status, and accessions with strains, soil
status and season had non-significant effect on nutrients uptake recorded at
50% flowering under glasshouse conditions (Table 4). However, significant
difference was also recorded regarding interaction of accessions with strains
on number of days to 50% flowering (282.45**), accessions
with season on the P uptake at flowering (0.01**) and strains with
season on N uptake at flowering (0.78**) and at harvest (Table 4).
Growth traits, nutrient uptake and
yield of inoculated accessions on the field
Significant difference was recorded in Ibadan and Ikenne
in both seasons in TVSu-1739 in the plant height (25.53a), terminal
leaf length (13.89a), terminal leaf width (2.75a), and
petiole length (2.29a) compared to other inoculated accessions
(Table 5). Likewise, TVSu-378 strain recorded higher number of branches, number
of stems and number of leaves per plant at both locations and seasons compared
to other inoculated accessions. Moreover, strains TVSu-506 and TVSu-305
observed higher leaf area and chlorophyll contents, respectively (Table 5).
Table 1: Physiochemical properties of soil
used for the study in the glasshouse and on the field
|
Experiment |
Pot
Experiment |
Field Experiment |
||||||
|
Year |
2018 |
2018 |
2019 |
2019 |
2019 |
2020 |
2019 |
2020 |
|
Soil
status/ Location |
Sterile
Soil |
Non
sterile Soil |
Sterile
soil |
Nonsterile
soil |
Ikenne
|
Ikenne
|
Ibadan |
Ibadan |
|
pH (H2O) |
7.43 |
7.39 |
7.1 |
6.81 |
4.46
± 0.08 |
4.91
± 0.09 |
6.84
± 0.1 |
7.13
± 0.11 |
|
N
(%) |
0.05 |
0.04 |
0.05 |
0.10 |
0.073
± 0.02 |
0.121
± 0.01 |
0.150
± 0.013 |
0.119
± 0.02 |
|
OC
(%) |
0.38 |
0.40 |
0.03 |
0.25 |
0.329
± 0.07 |
0.297
± 0.01 |
0.407
± 0.10 |
0.336
± 0.06 |
|
Bray
P (mg kg-1) |
3.30 |
3.71 |
2.55 |
3.71 |
13.23
± 4.26 |
22.46
± 3.43 |
15.352
± 3.74 |
52.84
± 6.67 |
|
Sand
(%) |
81.00 |
83.00 |
77.0 |
81.00 |
76.00
± 1.16 |
76.00
± 1.16 |
83.00
± 1.16 |
82.00 ± 0.0 |
|
Clay
(%) |
13.00 |
11.00 |
19.00 |
11.00 |
16.00
± 1.16 |
20.00
± 2.00 |
10.00
± 0.00 |
8.00 ± 0.00 |
|
Silt
(%) |
6.00 |
6.00 |
4.00 |
8.00 |
6.00
± 1.15 |
3.00
± 1.15 |
7.00
± 1.15 |
10.00 ± 0.0 |
|
Ca
(Cmol/kg) |
6.74 |
5.92 |
8.53 |
8.63 |
3.530
± 2.50 |
1.505
± 0.24 |
1.216
± 0.10 |
1.101
± 0.30 |
|
Mg
(Cmol/kg) |
6.74 |
5.92 |
0.20 |
0.20 |
0.80
± 0.37 |
0.404
± 0.02 |
0.075
± 0.01 |
0.158
± 0.05 |
|
K
(Cmol/kg) |
0.24 |
0.22 |
0.27 |
0.29 |
0.560
± 0.24 |
0.242
± 0.06 |
0.137
± 0.02 |
0.201
± 0.02 |
|
Na
(Cmol/kg) |
0.06 |
0.06 |
0.08 |
0.08 |
0.076
± 0.01 |
0.082
± 0.00 |
0.055
±0.02 |
0.057
± 0.01 |
|
ECEC
(Cmol/kg) |
13.79 |
12.12 |
9.08 |
9.21 |
4.96
± 1.56 |
2.23
± 0.57 |
1.29
± 0.56 |
1.47
± 0.48 |
|
Zn
(mg kg-1) |
3.24 |
2.66 |
9.91 |
9.39 |
1.964
± 1.67 |
1.196
± 0.19 |
1.19 ± 0.10 |
0.66
± 0.04 |
|
Cu
(mg kg-1) |
0.95 |
0.49 |
0.64 |
0.70 |
1.167
± 0.62 |
2.045
± 0.19 |
0.86 ± 0.11 |
0.97
± 0.22 |
|
Mn
(mg kg-1) |
266.0 |
286.2 |
214.7 |
222.1 |
12.15
± 10.5 |
116.7
± 3.31 |
244.20
± 18.4 |
383.6
± 33.4 |
|
Fe
(mg kg-1) |
17.19 |
17.19 |
85.54 |
94.01 |
25.58
± 7.47 |
88.29
± 4.08 |
133.33
± 3.33 |
114.4
± 3.85 |
|
Glomus |
- |
159 |
- |
145 |
106 |
117 |
135
|
152 |
|
acaulospora |
- |
211 |
- |
200 |
192 |
185 |
202 |
208 |
|
enthrophospora |
- |
30 |
- |
36 |
25 |
28 |
34 |
32 |
|
Spore/100gdwt |
- |
401 |
- |
406 |
380 |
392 |
412 |
432 |
|
Soil textural class |
Loamy
sand |
Loamy
sand |
Loamy
sand |
Loamy
sand |
Loamy
sand |
Loamy
sand |
Loamy
Sand |
Loamy
sand |
Table 2: Microbial population of the soil
used for both pot and field experiment
|
Experiment
site |
year |
Soil
status/Location |
colony
forming unit |
|
Glasshouse |
2018 |
Non
sterile soil |
1.28
× 102 |
|
|
2019 |
Non
sterile soil |
1.53
× 109 |
|
Field |
2019 |
Ibadan |
1.83
× 105 |
|
|
2019 |
Ikenne |
2.03
× 109 |
|
|
2020 |
Ibadan |
3.05
× 107 |
|
|
2020 |
Ikenne |
2.47
× 107 |
Table 3: Analysis of variance (ANOVA) of
accessions of Bambara groundnut inoculated with B. japonicum strains on the growth traits at 10WAP under glasshouse
conditions in both seasons
|
Source
of variation |
DF |
PLH (cm) |
CS (cm) |
NOS/plant |
NOL/plant |
CHPY |
|
Accessions |
9 |
407.97** |
92.31**
|
1845.74**
|
17593.64** |
417.89** |
|
Strains |
5 |
12.017ns |
1.69ns |
274.76ns |
526.42ns |
95.11ns |
|
Soil
Status |
1 |
841.42** |
159.41** |
15797.87** |
160776.32** |
384.79ns |
|
Season |
1 |
4969.64** |
2134.50** |
5037.88** |
36155.86** |
6896.92** |
|
Rep |
2 |
28.48ns |
17.25ns |
1673.01** |
8792.17* |
82.23ns |
|
Accessions*Strains |
45 |
30.38ns |
8.90ns |
319.78ns |
2905.64ns |
232.18** |
|
Accessions*Soil
status |
9 |
26.95ns |
10.50ns |
293.01ns |
3224.99ns |
175.16ns |
|
Accessions*season |
9 |
33.19ns |
5.79ns |
779.31** |
6041.09* |
148.13ns |
|
Strains*Soil
Status |
5 |
6.08ns |
4.47ns |
280.94ns |
3314.61ns |
240.49ns |
|
Strains*seasons |
5 |
52.34ns |
14.37ns |
620.32* |
5978.13* |
254.50ns |
|
Acc*Strain*soil*seas |
131 |
32.04
ns |
7.51ns |
368.90** |
8737.41** |
150.86ns |
*= Significant; ** = Highly
significant; ns = Non-significant, DF = Degree of freedom; NOB = number of
branches, NOL= Number of leaves, CS = Canopy spread; PLH; Plant height; NOS =
Number of stem; CHPY= Chlorophyll; WAP = Weeks after planting
Analyzed data indicated that different B. japoniocum strains inoculation had
significant effect on entire growth-related traits of Bambara
groundnut on the field in both locations and seasons. Among different strains tested, FA3 observed higher
plant height, number of branches and stems/ plant, leaf area, terminal leaf
length and width, number of leaves/plant and chlorophyll contents compared with
all other strains and control (Table 6).
Inoculation of different B. japoniocum strains
significantly improved the nutrient uptake and yield of inoculated accessions
of Bambara groundnut. Higher N contents were recorded at flowering and harvest
in TVSu-1739, TVSu-378 and TVSu-787 accessions respectively in Ibadan and Ikenne in both seasons and were
significantly higher than other inoculated accessions of Bambara groundnut
(Table
7). Likewise, high P uptake was recorded at flowering and at harvest in TVSu-787 and
TVSu-1606, TVSu-378 and TVSu-787, respectively
(Table 7). Among the inoculated
accessions, higher yield was recorded by TVSu-1698 (1205.5 kg ha-1) compared to other inoculated accessions at both locations and seasons due to the inoculation of B. japonicum strains that enhanced the
yield components (Table 7).
Table 4: Analysis of variance reflecting
accession of Bambara groundnut inoculated with B. japonicum strains on the number of days to 50% flowering, %N and
%P at flowering and harvest under glasshouse conditions in both season
|
Sources
of variations |
DF |
Days
to 50% Flw |
P@Flw
(%) |
N@Flw
(%) |
N@Hvst
(%) |
P@Hvst
(%) |
|
Accessions |
9 |
399.45** |
0.012** |
0.98**
|
0.67** |
0.02**
|
|
Strains |
5 |
261.39ns |
0.005ns |
20.87** |
16.07** |
0.004ns |
|
Soil
Status |
1 |
53.06ns |
0.17** |
0.28ns |
3.01** |
0.19** |
|
Seasons |
1 |
792.23** |
1.06** |
1.28*
|
0.019ns |
0.23** |
|
Rep |
2 |
196.78ns |
0.012** |
0.17ns
|
0.56ns |
0.02** |
|
Accessions*Strains |
45 |
282.45** |
0.003ns |
0.38ns
|
0.24ns |
0.002ns |
|
Accessions*Soil
Status |
9 |
226.80ns |
0.003ns |
0.17ns |
0.16ns |
0.004ns |
|
Accessions*season |
9 |
226.80ns |
0.01** |
0.78**
|
0.25ns
|
0.003ns |
|
Strains*soil status |
5 |
99.12ns |
004ns |
0.43ns |
0.21ns |
0.01ns |
|
Strains*season |
4 |
189.71ns |
0.003ns |
0.48ns |
0.85** |
0.003ns
|
|
Acc*Strain*soil*season |
131 |
163.25ns |
0.002ns |
0.29ns
|
0.003ns
|
0.003ns
|
*
= Significant; ** = Highly significant; ns = Non-significant; DF = Degree of
freedom; Flw = Flowering; Hvst = Harvesting; %P = Phosphorus; %N = Nitrogen; @ =
at
Table
5: Effect of the
inoculation of B. japonicum strains on the growth traits of accessions
of Bambara groundnut at 12WAP in (Ibadan and Ikenne) in both seasons
|
Accessions |
PLH (cm) |
NS/plant |
NB/plant |
LA (cm2) |
NOL/plant |
TLL (cm) |
TLW (cm) |
PEL (cm) |
CPHY |
|
TVSu-506 |
24.01ab |
55.08d |
50.90e |
21.05a |
160.47f |
5.75b |
2.44b |
1.66c |
31.85d |
|
TVSu-1739 |
25.53a |
50.04e |
45.28f |
18.82b |
147.75g |
13.89a |
2.75a |
2.29a |
36.57b |
|
TVSu-305 |
20.42c |
69.64c |
62.76c |
14.89c |
209.36c |
5.75b |
2.64ab |
1.45d |
41.99a |
|
TVSu-787 |
21.76bc |
79.43b |
72.39b |
12.15d |
234.18b |
5.51bc |
2.00d |
1.78b |
33.13c |
|
TVSu-378 |
18.84d |
95.00a |
79.75a |
12.68d |
278.75a |
5.10cd |
2.20cd |
1.43d |
31.83d |
|
TVSu-1698 |
22.70b |
70.79c |
65.09c |
15.98c |
202.89d |
5.89b |
2.69a |
1.47d |
35.04b |
|
TVSu-710 |
18.77d |
39.40g |
34.94h |
12.76d |
121.68g |
5.35bcd |
2.22cd |
1.41d |
31.05d |
|
TVSu-475 |
21.29bc |
62.90d |
56.61d |
13.92d |
183.75e |
5.83b |
2.34c |
1.38e |
31.70d |
|
TVSu-365 |
18.85d |
78.62b |
65.27c |
10.20e |
210.12c |
4.45d |
1.74e |
1.39e |
27.84f |
|
TVSu-1606 |
17.23e |
47.01f |
39.71g |
12.61d |
133.51h |
4.89d |
2.08d |
1.22f |
29.38e |
Mean
with the same letter are not significantly different at P < 0.05 level of probability according to DMRT
Table 6: Effect of the inoculation of B. japonicum strains on the growth
traits of accessions of Bambara groundnut at 12WAP in (Ibadan and Ikenne) in
both season
|
Strains |
PLH
(cm) |
NB/plant |
NS/plant |
LA
(cm2) |
NOL/plant |
TLL
(cm) |
TLW
(cm) |
PEL
(cm) |
CS
(cm) |
CPHY
units |
PL
(cm) |
|
FA3 |
22.71a |
62.73a |
70.11a |
15.74ab |
209.25a |
6.00b |
2.56a |
1.73a |
12.21a |
34.53a |
12.69a |
|
USDA110 |
20.23b |
54.68bc |
60.83b |
12.93b |
177.48b |
5.44c |
2.22c |
1.49c |
10.37bc |
33.26ab |
9.89b |
|
N |
18.99c |
48.20d |
53.59c |
13.02b |
156.83c |
5.18c |
2.21c |
1.47c |
9.97c |
31.48b |
9.03a |
|
RACA6 |
20.78b |
55.64b |
61.15b |
17.09a |
178.01b |
5.41c |
2.36b |
1.56bc |
10.99b |
33.35ab |
9.88b |
|
IRJ2180A |
20.38b |
51.08cd |
58.84b |
13.45b |
167.07bc |
9.75a |
2.22c |
1.45c |
10.46bc |
30.96b |
9.46a |
|
Control |
21.07b |
52.18bcd |
58.03bc |
14.83ab |
167.44bc |
5.90b |
2.46ab |
1.64ab |
11.06b |
34.09a |
9.48b |
Means
with the same letter are not significantly different at P < 0.05 level of probability according to DMRT
PLH
= Plant height; NS = Number of stem; NB = Number of branches; LA = Leaf area;
NOL = Number of leaves; TLL = Terminal leaf length; TLW = Terminal leaf width;
PEL = Petiole length; CHPY = Chlorophyll; PL = Peduncle length
Table 7: Mean separation reflecting nutrient uptake of accessions of
Bambara groundnut inoculated with B.
japonicum strains at flowering, harvest and yield (Ibadan and Ikenne) in
both season
|
Accessions |
N@Flw (%) |
P@Flw (%) |
N@Hvst (%) |
P@Hvst (%) |
Yield/plot
(g) |
Yield
(kg ha-1) |
|
TVSu-1739 |
2.09a |
0.19b |
1.67bc |
0.12b |
58.33ab |
784.3bcd |
|
TVSu-378 |
2.05ab |
0.21ab |
1.80a |
0.15a |
27.43de |
403.1def |
|
TVSu-305 |
2.03abc |
0.17d |
1.62cd |
0.12bc |
64.15ab |
825.2abc |
|
TVSu-710 |
2.02a-d |
0.17d |
1.68b |
0.12bcd |
30.06cde |
364.7ef |
|
TVSu-365 |
2.01bcd |
0.17d |
1.48f |
0.11d |
60.89ab |
812.7abc |
|
TVSu-1698 |
2.01bcd |
0.19bc |
1.53ef |
0.11d |
80.92a |
1205.5a |
|
TVSu-787 |
2.00bcd |
0.21a |
1.75a |
0.14a |
39.99b-e |
669.6b-e |
|
TVSu-475 |
1.99bcd |
0.17cd |
1.52ef |
0.11d |
56.31b |
871.1ab |
|
TVSu-506 |
1.96cd |
0.19b |
1.56ed |
0.12bcd |
42.42bcd |
523.3c-f |
|
TVSu-1606 |
1.95d |
0.21a |
1.53ef |
0.11cd |
51.08bc |
733.5b-e |
Means with the
same letter are not significantly different at 5% level of probability using
DMRT
N
= Nitrogen; P = Phosphorus; Flw = Flowering; Hvst = Harvest; @ = at
Discussion
In general,
the results obtained in this study, eventually revealed the relevance of B. japonicum strains to improve the
growth traits and nutrient uptake of accessions of Bambara groundnut under glasshouse (sterile and non-sterile soil) conditions.
Also, inoculation of B. japonicum
strains enhanced growth traits, nutrient uptake and yield of accessions of
Bambara groundnut in the field in both locations and seasons over the N
fertilizer application (Table 6 and 7).
Significant differences were recorded among the
inoculated accessions in the growth traits and nutrient uptake in the
glasshouse and on the field on yield components in both locations and seasons.
Among the B. japonicum strains
inoculated to accessions, FA3 showed higher significant difference compare to
other strains, N fertilizer application, and uninoculated control which shows
that significant differences exist among the B. japonicum strains (Table 6). The result obtained in this study
is related to the result recorded by (Tarekegn et al. 2017) when cowpea
upon inoculation with Bradyrhizobium on
the field, significantly enhanced the growth traits, nodulation, and yield of
cowpea when compared with the uninoculated control.
Furthermore, the nutritional benefit recorded in both studies was as a result of easy translocation of nutrients
from the soil by the strains to a point where the root can intercept the
nutrients for optimum growth and developments. The result obtained on nutrition
is similar to the findings of (Biswas et
al. 2000) when inoculation with rhizobia resulted to larger amount of N uptake when
compared with the uninoculated control. Superior performance was recorded among
inoculated accessions of Bambara groundnut, as the response to the B japonicum strains varies probably, due
to genetic compositions of accessions that differs. The variability recorded
among inoculated accessions of Bambara groundnut correlates with the findings
of (Nyoki and Ndakidemi 2014) where
varying responses were observed among varieties of soybean that were inoculated
on the field.
It seemed apparent from the responses obtained in this study that inoculation of FA3 strains
mostly supported the growth, nutrition and yield of accessions of Bambara
groundnut. FA3 strains perform better compare to other B. japonicum strains inoculated, N fertilizer applied, and
uninoculated control in Ibadan and Ikenne in both seasons (Table 6). The results obtained correlates with the result
of the research study recorded by Kaschuk et al. (2016), in which neither basal nor topdressing of N application improved
yields of determinate and in determinate
soybean cultivars, but the sole inoculation of Bradyrhizobium was enough to supply all N
required by soybean plants. The result of this study also agrees with the
research conducted by (Hungria and
Mendes 2015), that embraced rhizobia inoculation and zero N fertilizer application to enhance legume production.
Our findings reveals that the inoculation of the B. japonicum strains can help to improve the growth, nutrient uptake and yield of Bambara groundnut with reduced cost
as against the use of inorganic N fertilizers
which is very expensive to procure and also contaminate the soil after use (Cordeiro and Echer 2019). Also, in another
study conducted inoculating five cowpea varieties with five Bradyrhizobium
isolates, result revealed that Bradyrhizobium inoculation improve the growth, biomass, and nodulation
performance of the varieties of cowpea, Ayalew and Yoseph (2020). Bambara
groundnut in this study, refuses to pod in the glasshouse in both seasons, which also
therefore, limits it production in most research to growth traits and nutrition
under glasshouse conditions.
Conclusion
Results
of this study unveiled that the inoculation of B. japonicum strains can help to improve the growth traits,
nutrient uptake and
yield of Bambara groundnut accessions under glasshouse and field conditions. Resultantly it can
reduce reliance on inorganic N fertilizer which most tropical farmers cannot
afford and for those that can afford apply at low rate than recommended. Much
effort is needed to introduce the inoculant to the farming community (farmers).
It is therefore important to introduce and recommend the cheap and friendly
technology to the poor farmer’s communities of the nation. FA3 therefore, can
be introduce to the farmers to enhance optimum production of Bambara groundnut.
Acknowledgements
The authors are grateful to the genetic resources center
of the International Institute of Tropical Agriculture, Ibadan, Nigeria. For
funding the project.
Author
Contributions
TDB
and MA planned the experiments, TDB and OOB
interpreted the results, MA, OOB and OO supervision and TDB statistically
analyzed the data and write up.
Conflict of Interest
All
authors declare no conflict of interest.
Data
Availability
Data presented
in this study will be available on a fair request to the corresponding author.
Ethics Approval
Ethics
Approval number: N W U - 0 1 2 1 7 - 1 9 - A 9
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