EFFECTS OF COPPER TOXICITY ON NODULATION AND BELOW-GROUNDBIOMASS OF Centrosema pubescens BENTH (BUTTERFLY PEA) ON ARABLE LANDS IN SOUTH EASTERN NIGERIA

Abstract

The use of chemicals in tree crop production accounts for over 35% toxicity in arable land and agro-ecological system. The effect of copper toxicity on nodulation and ground biomass was studied. The experiment was organized in a Randomized complete Block Design using (3) different soil media (copper toxic soil, copper toxic soil amended with organic manure and a control soil) as treatments. Centrosema pubescens were grown for 10 weeks in the 3 different plots and the parameters used in measuring effect of cu toxicity were number of root nodules and fresh root weight. Data were analyzed using Analysis of Variance (ANOVA) and Least Significant Difference (LSD) at 0.05% level of significance. Results showed that there was a 32.44% and 55.08% increase in the mean root fresh weight and aerial weight respectively of centrosema after 10 weeks of growing on cu contaminated soil amended with organic manure (CTO) when compared with the copper toxic soil. Centrosema grown on control soil (CS) has the highest mean number of root nodules (160/plant). Growing centrosema in cu toxic soil (CT) reduced levels of some nutrients, this may be as a result of constant spraying of the plantation with Bordeaux mixture (field survey report). The levels of potassium, iron, lead and copper increased in the centrosema roots when grown in (CT) soil compared with the control, also the amendment of Cu toxic soil with organic manure increased the level of Nitrogen at the rate of 2.21 % Centrosema grown in soils with elevated content of copper should be avoided as forage for Livestock in order not to allow the buildup of Cu in the food chain.

Full text

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EFFECT
S
OF COPPER TOXICITY ON NODULATION AND BELOW
-
GROUND
BIOMASS
OF
Centrosema pubescens
BENTH (BUTTERFLY PEA)
ON ARABLE LANDS IN SOUTH
EASTERN
NIGERIA
1
Ekwugha
E.U.
, 1O
nyema
M.C.,
2N
zegbule E.C.
,
and
3P
eter
O
noh
C.A.
.
1
Department of
F
orestry and Wildlife
Technology, Federal University of Technology Owerri, Nigeria
2
Department of Forestry and Environmental Management, Michael Okpara University of Agriculture
Umu
dike, Nigeria
.
3
Department of Crop Science Technology, Federal University of Technology Owerri, N
igeria.
ABSTRACT
The use of chemicals in tree crop production accounts for over 35% toxicity in arable land and agro-
ecological system. The effect of copper toxicity on nodulation and ground
biomass
was studied. The
experiment was organized in a Randomized complete Block Design using (3)
different
soil media
(copper toxic soil, copper toxic soil amended with organic manure and a control soil) as treatments.
Centrosema pubescens were grown for 10 weeks in the 3 different plots and the parameters used in
mea
suring effect of cu toxicity were number of root nodules and fresh root weight. Data were
analyzed using Analysis of Variance (
ANOVA
) and Least Significant Difference (
LSD
) at 0.05%
level of significance. Results showed that there was a 32.44% and 55.08% increase in the mean root
fresh weight and aerial weight respectively of centrosema after 10 weeks of growing on cu
contaminated soil amended with organic manure (CTO) when compared with the copper toxic soil.
Centrosema grown on control soil
(CS)
has the highest mean number of root nodules (160/plant).
Growing centrosema in cu toxic soil
(CT)
reduced levels of some nutrients, this may be as a result of
constant spraying of the plantation with Bordeaux mixture (field survey report). The levels of
potassiu
m, iron, lead and copper increased in the centrosema roots when grown in (CT)
soil
compared with the control, also the amendment of Cu toxic soil with organic manure increased the
level of Nitrogen at the rate of 2.21 %
Centrosema
grown in soils with elevated content of copper
should be avoided as forage for Livestock
in order not to allow the build
up of Cu
in the food chain
.
Keywords
:
Toxicity, Bordeaux, Nodulation, Belowground, Accumulation.

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NTRODUCTION
Copper is an essential element required for normal plant growth and metabolism. It plays a significant
role in a number of physiological
processes
such as the photosynthetic and respiratory electron
transport chain, BNF, protein metabolism, antioxidant activity, cell wall metabolism, hormone
percept
ion and fruit sweetening. (Younis, 2007).
Toxic levels of copper arely occur naturally in soil. However, copper may accumulate due to
persistent use of copper fungicides by farmer’s. Copper toxicity is a problem of both agricultural and
environmental significance. Whist the farmer’s objective is to apply fungicides to agricultural
crop/plants to control fungal disease in plantation; inevitably a proportion of the sprayed chemical will
affect non
-
target species. Much of the lost chemicals will enter the soi
l surface where it will persist for
a long period of time and potentially migrate to non-target areas due to leaching. The metals enter the
root in the form of dissolved ions and move with the inflow of water apoplastically through the root
hairs and into the cortex and are conducted to other parts of the plants. (Wightwick and Allinson,
2007).
Concerns have been raised over the long term use of copper based fungicides which can result in
accumulation of copper in the soil. This in turn can have adverse effect on soil organisms (e.g.
bacteria, fungi, earthworm, microorganisms) and potentially pose a risk to long term fertility of soil
(Wightwick and Allinson, 2007). It has been found that increase in total copper concentration were
correlated (R2=0.50) with decrease in earthworm biomass (Paollet et al., 1998). Copper has been
shown to have toxic effect on a variety of metabolic processes essential for plant growth and
development, including photosynthesis, transpiration, DNA synthesis, and mitotic activity; of these
processes, photosynthesis was found to be the most sensitive to copper contamination (Anne-
Noelle,
et al 2012)
.
Shallow rooted plants like Centrosema pubescens are easily affected by metal soil
contamination particularly because of the metal’s high affinity to solid phase organic matter
(Sheldona and Menzies, 2005).
Centrosema pubescens is a native plant commonly found in wet tropical areas of the world. It is one
of the most widely distributed of the entire tropical legume where it grows naturally in farms
and
secondary bushes. It is a vigorous perennial herb and has a climbing twinning habit when support is
available.
(Nworgu and Egbunike, 2013). It has the tendency to root at the nodes. Nodulation
continues throughout the active growth period of the plant (Bowen, 1959a) with the nodules
remaining small but active during slow growing phase
(zacharia, 2005)
.
This
character needs to be re-evaluated especially in the light of changing environmental and
ecological situations across different regions of the world. Given the importance of legumes as food
and cash crops as well as forage in the livestock industry and the wide usage on copper containing
compounds in agriculture and crop farming activities, the physiological effect on these
plants
deserves t
o be deeply
evaluated.

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MATERIALS AND
METHOD
Study Area
The study was conducted at the nursery unit of the Department of Crop Science Technology
in
Federal University
of
Technology
, Owerri,. The area lies within Latitudes 50 20¹ and 6
0 02
¹
N and
Longitude
s
60 30
¹
and
7
0 32
¹E. the area is a rain forest vegetation with relative humidity between 70
and 80%.
( Imo State planning and Economic Commission, 2000).
METHODS
Seeds
of
Centrosema pubescens
used
for the
research
were collected from fallow plots wit
hin
Federal University of Technology Owerri. Viability of the seeds was tested by floatation in water and
only viable seeds were used for the research.
Viable seeds of
Centrosema pub
e
scens
were planted in a germination box
es
and left in the open at the
nur
sery.
The seeds were maintained by watering the boxes daily for two weeks before transplanting.
The
Centrosema
seedlings were transplanted two weeks after germination into the experimental pots.
Three
(3) soil samples (A,B, C) were separately used to fill 12 pots each. A 3.5kg weight of each soil
sample
was used in filling the pots. The first soil sample (Soil Sample A) was collected from
a
Gmelina arborea plantation that was not affected by Bordeaux mixture. The soil sample was used as
control to obtain t
he natural growth rate and nodulation of
Centrosema pubescens.
The second soil sample (Soil Sample B) was collected from a cocoa plantation located
a
distance
away from Gmelina arborea. The continuous spaying of the plantation to control blackpod disease
on the cocoa as part of the management practice have contributed to elevated level of toxic metal
concentrations in this site (
Nzegbula, 2003)
.
The third soil sample (Soil Sample C) was a copper contaminated soil amended with organic manure.
The poultry
ma
nure used was collected from the poultry unit of Federal University of Technology
Owerri. The poultry manure (0-5g) was used to amend a portion of the soil sample B collected from
the copper toxic location in each pot. In all 48 pots were used for the experiment. The pot experiment
was laid out in a randomized Complete Block Design with four (4) replicates.
Baseline soil chemical properties (N, P, K, Cu, Pb and Fe) of these plots with the above formulations
were analyzed prior to the experiment in line with standard methods (AOAC, 1990)
At
ten
(1
0)
weeks after transplanting, the growing
C.
pubscens
seedlings were carefully
upro
oted,
washed under tap
water and labeled. A hand lens was used to count the nodules formed
per seedling of
the plant. Again, the weight of the belowground biomass (root mass) was determined using an
electronic weighing balance (Tonimax model), oven-dried at 600C, digested
while
the
heavy metal
(Cu, Fe and Pb)
composition
were determined using standard laboratory methods and procedures.
Data
were obtained
based on number of nodules per plant.

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The mean number of root nodules in each of the three treatments was calculated and recorded
accordingly. Data on cumulative fresh root weight were
taken;
this was done by cutting off the root
of
the
freshly uprooted plant
and weighed using electronic weighing balance
Different levels of some
micro,
macronutrients and heavy metals within the root Zone of centrosema
were analyzed at (10 weeks) of growth on Cu
conta
minated soil, Cu contaminated soil amended with
poultry manure and the control.
Analysis of variance was used to determine variations in the parameters obtained from the plots.
Means were separated using least significant difference
(LSD) at
5
% confidence level.
RESULTS AND DISCUSSIO
N
The three soil samples (CS, CT and CTO) which was analyzed for physio-chemical properties and
heavy metals before the trials shows that the copper level in CT and CTO soils were more than
that
in
the control soil. (Table 1 ). This indicates that the cocoa plantation have excessive build-up of copper.
Earlier studies in this plantation had shown that the soil has heavy build-up of copper substances
(Nzegbule,
2003). Consequently, farmers in the plantation find it difficult to re-establish cocoa
seedlings in the plantation. “Field survey showed that the plantation was sprayed with Bordeaux
mixture at least three times per annum” This was attributed to frequent spraying of the plantation with
Bordeaux mixture
. L
evels of copper (
Cu
) rarely occur naturally in s
oils, copper may
accumulate
due to
application of sewage sludge, pig slurries or mine slag or more commonly, through persistent use of
copper containing fungicides or fertilizers (
O’ Sollivan
et al.
, 1997).
There was no significant difference in fresh root weight of C
entrosema
planted in the different soil
samples. Table 2. (Table 2a and 2b) Copper toxicity affected root nodule development of C
entrosema
.This was shown by the decline in the mean number of root nodules in
Centrosema
grown on copper
contamin
ated soil when compared to that of control and Cu-contaminated soil amended with poultry
manure
(F
ig.
1), table 3. T
he
repeated use of Bordeaux mixture (CUSO4
.5H
20+Ca (OH)2) as a
fungicides on potatoes, snap beans and orchard crops has led to instances of copper toxicity.
The
major effect of heavy metals on seeds of arable crops are manifested by overall abnormalities and
decrease in germination, reduced root and shoot elongation, dry weight, total salable protein level.
(Wang
et al.,
2003)
. Oxidative damage, membrane alteration, altered sugar and protein
metabolisms,
nutrient loss (Ahmad and Asharaf et al., 2011, and
Pourrut
et al,.
2011)
all contributing to seed
toxicity and productivity
loss
.
However, addition of organic manure minimized the negative impact of cu-toxicity on nodulation of
C
entrosema
.There is no doubt that higher crop production responds to organic manure application,
which results in greater root exudates and more crop residues. Different bacterial genera are vital
components of soils. They are involved in various biotic activities of the soil ecosystem to make it
dynamic for nutrient turn over and sustainable for crop production (
Ah
mad
and
Khan
2009d
and
Chandler
et al., 2008). They stimulate plant growth through mobilizing nutrients in soils,
producing numerous plant growth regulators, protecting plants from phytopathogens by controlling or

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inhibiting them, improving soil structure and bioremediating the polluted soils by sequestering toxic
heavy metal species and degrading xenobiotic compounds (like pesticides) (
Ah
mad 2012, Hayat et al.,
2010,
Rajkumar
et al., 2010
and
Braud
et al.,
2009
). Indeed, the bacteria lodging around/in the plant
roots (rhizobacteria) are more versatile in transforming, mobilizing, solubilizing the nutrients
compared to those from bulk soils (
Hayat
et al., 2010). Therefore, the rhizobacteria are the dominant
deriving fo
rces in recycling the soil nutrients and consequently, they are crucial for soil fertility (
Glick,
2012
).
In
the
same vein, copper availability decreases as organic matter in soil increases, organic
matter binds copper more tightly than any other micronutrients and as well reduces its ability in the
soil. Table 4 shows significant difference on the phosphorus, iron, copper content of centrosema roots
grown in the cu-contaminated soil amended with organic manure and the control
treatment
(Cs)
. The
plant root analysis showed that copper toxicity inhabits chemisorptions of calcium and magnesium
nutrients in the
centrosema
roots which were amendable using poultry manure. Copper toxicity
facilitates the chemisorptions of potassium, iron, lead and copper nutrients and phosphorus level in
centrocema
roots. Consequently, nitrogen and phosphorus level in centrosema roots were enhanced by
the amendment of cu-contaminated soil with poultry manure. Significant
increase
were recorded
in
nutrient content,
pH
values, available water and decreased availability of heavy metal using organic
manure. (Baker, et al.,
2011).
CONCLUSION
Centrosema pubescens
is a forage crop and nitrogen f
ixing plant which also grows in cocoa plantati
on.
Elevated level of copper (Cu
) in the soil
had no significance
on fresh root
weight of
centrosema
. There
were significant
at
decli
ne
(P=0.05)
in the number of nodules of Centrosema pubesc
en
s because of
coppe
r toxicity. The study shows that the amendment of copper toxic soil by poultry manure
minimized the impact of cu-toxicity on number of root nodules.
Growing
centrosema in copper toxic
soil reduced the content of calcium and magnesium nutrient in the roots
of
centrosema
. On the other
hand, there was an increase in the levels of potassium, iron, lead in the roots of copper toxic soil.
Centrosema
responds to increased copper and therefore should be avoided as forage for livestock. The
presence
of copper in the soil affects root nodulation of centrosema, hence may not be efficient in
fertility restoration of soils degraded by high concentration of copper.
Table
1:Soil Micro and Macro Elements Analysis
CS
CT
CTO
Nitrogen
(%)
0.09 0.144 0.304
Organic c
arbon(%)
2.185 3.325 7.030
Phosphorus
(mg/kg)
39.16 24.38 39.16
ELEMENT

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Potassium
(Cmol/kg)
0.78 0.76 1.74
Magnesium (Cmol/kg)
1.60 2.88 4.10
Calcium
(Cmol/kg)
3.84 8.64 12.80
Sodium
(Cmol/kg)
0.33 0.23 0.35
Heavy metals
Copper
(mg/g)
6.00
15.60
15.20
Lead
(mg/g)
1.90 3.00 3.60
Iron (m
g/g)
900.48 1386.34 1280.37
Table 2: Fresh root weight and Fresh aerial weight of Centrosema pubesces at 10 weeks of
grow
th
Treatments
Mean fresh root weight
Mean fresh aerial weight (g)
CT
/PLOT A 9.28 56.64
CTO
/ PLOT B
12.29 87.84
CS
/PLOT C 8.53 50.30
Table 2a:
One
-way ANOVA for the assessment of the effect of copper toxicity on the number of
leaves of
Centrosema pubescens
Number of leaves:
Sample
CT
CTO
CS
B1 71.25 60.75 160.75
B2 113.50 121.75 168.00
B3 40.25 77.00 86.00
B4 47.25 57.00 140.75

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Total
272.25
316.50
555.50
Mean
68.06c 79.13
bc
138.88a
Anova: Single Factor
SUMMARY
Groups
Count
Sum
Average
V
ariance
CT
4
272.25
68.0625
1093.807
CTO
4
316.5
79.125
882.8542
CS
4
555.5
138.875
1375.354
LSD =
53.46505
ANOVA
Source of
Variation
SS Df
MS
F P-
value
F crit
Between Groups
11609.14
2
5804.568
5.194995
0.031622
4.256495
Within
Groups
10056.05
9
1117.339
Total
21665.18
11
Decision:
Since
F>F
crit
we can reject HO and conclude that there is a significant difference in the
number of leaves of Centrosema pubescens planted in the different soil samples.Since Analysis of
Variance has provided statistical evidence to reject the null hypothesis, Fisher’s LSD procedure will be
used to determine where the differences occur.
Interpretation:
The means superscript overlaps (i.e the mean values for the effect of copper toxi
city
on the number of leaves of
Centrosema pubescens
are not allsignificantly different from each other).

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Table 2b:
One
-
way Anova for assessment of the effect of copper toxicity on the fresh root weight
of
Centrosema pubescens
after planting
Fresh root w
eight
Sample
CT
CTO
CS
B1 10.13 9.15 3.73
B2 8.00 6.00 6.00
B3 13.00 16.00 20.40
B4 6.00 18.00 4.00
Total
37.13
49.15
34.13
Mean
9.28
12.29
8.53
Anova: Single Factor
SUMMARY
Groups
Count
Sum
Average
Vari
ance
CT
4
37.13
9.2825
8.985892
CTO
4
49.15
12.2875
31.93063
CS
4
34.13
8.5325
63.61956
NO
LSD
ANOVA
Source of
Variation
SS
df
MS
F P-
value
F crit
Between
Groups
31.59007
2
15.79503
0.453289
0.649284
4.256495

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Within Groups
313.6082
9
34.84536
Total
345.1983
11
Decision
: Since
F<F
crit
we accept H
O
and conclude that there is no significant difference in fresh root
weight of
Centrosema pubescens
planted in the different soil samples.
Table 3:
One
-
way A
nova for assessment of the effect of copper toxicity on the fresh aerial weight
of
Centrosema pubescens
after planting
Fresh aerial weight
Sample
CT
CTO
CS
B1 60.54
85.37
29.19
B2 80.00
46.00
58.00
B3 54.00
86.00
88.00
B4 32.00
134.00
26.00
Total
226.54
351.37
201.19
Mean
56.64
87.84
50.30
Anova: Single Factor
SUMMARY
Groups
Count
Sum
Average
Variance
CT
4
226.54
56.635
391.6662
CTO
4
351.37
87.8425
1296.939
CS
4
201.19
50.2975
838.9007
NO L
SD

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ANOVA
Source of
Variation
SS Df
MS
F P-
value
F crit
Between Groups
3231.599
2
1615.799
1.917858
0.202395
4.256495
Within Groups
7582.518
9
842.5021
Total
10814.12
11
Decision:
Since
F<F
crit
we can accept HO
and conclu
de that there is no significant difference in the
fresh aerial weight of
Centrosema pubescens
planted in the different soil samples.
Figure 1
: Mean number of root nodules of
Centrosema pubescens
after ten (10) weeks of
study
180
160
140
120
100
80
60
40
20
0
CT
CTO
CS

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T
able
4: Levels of some macronutrients and heavy metals in the root
of
Centrosema pubescens
after (10) weeks of growing on Cu-contaminated soil, Cu-contaminated soil amended poultry
manure and control soil.
Treatments
Levels of some macronutrients and heavy metals in
the plant root
Fe
(mg/kg)
Cu
(mg/kg)
Pb
(mg/kg)
N
(%)
P
(mg/kg)
CT
462.94a 4.61
ab
2.27a 1.95 329.3b
CTO
354.57c 4.29
bc
2.08
bc
2.21 344.b
ab
CS
364.28
bc
3.71c 2.05c 1.71 194.3c
LSD @5%
15.99 0.65 0.16 - 64.94
Mean values within column with different superscript are significantly different at 0.05 level of
significance.
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