American-Eurasian J. Agric. & Environ. Sci.

, 15 (5): 944-956, 2015

ISSN 1818-6769
IDOSI Publications, 2015
DOI: 10.5829/idosi.aejaes.2015.15.5.12634

Mycorrhizae Inoculation Did Not Influence the

Response of Cocoa Seedlings to Water Stress
G.U. Chibuike and 2A.J. Daymond

1,3

Department of Geography and Environmental Science,

The University of Reading, Whiteknights, Reading, UK
2
School of Agriculture, Policy and Development,
The University of Reading, Whiteknights, Reading, UK
3
Department of Soil Science and Land Resources Management,
University of Nigeria, Nsukka, Nigeria
1

Abstract: A greenhouse experiment was conducted to determine whether mycorrhiza reduces the impact of
water stress on cocoa seedlings. The effects of two factors mycorrhizae and watering on growth and
physiology of cocoa seedlings were monitored for 10 weeks. Four treatments were derived from combinations
of these factors as follows: -M-W (without mycorrhizae + water stress); -M+W (without mycorrhizae + regular
watering); +M-W (with mycorrhizae + water stress) and +M+W (with mycorrhizae + regular watering). Watering
was done with 500 ml of water every 3 days and 6 days for the regularly watered and water-stressed treatments,
respectively. Results show that mycorrhizae did not colonize roots of cocoa seedlings and hence did not
influence their response to water stress. High P content of the soil was likely to have been the main reason for
absence of mycorrhizal colonization. Water stress negatively influenced most plant growth and physiological
parameters, though significance (p < 0.05) of its effect was more noticeable in seedling height. A soil with low
or moderate P content would be required to study whether mycorrhiza reduces the impact of water stress on
cocoa seedlings.
Key words: Mycorrhizae

Water stress

Cocoa seedlings

INTRODUCTION
Water is essential for the proper growth and
functioning of all plants. It is particularly necessary
during the early stage of plant growth. Shortage of water
during this stage results in growth reduction, increased
mortality and susceptibility to diseases [1]. For plants
which will later be transplanted, it is necessary that they
are not subjected to severe water stress as this will affect
their establishment in the field and in turn affect their
performance.
Cocoa (Theobroma cacao) requires a relatively
high amount of water during the early stage of its growth
and hence is very sensitive to water stress [2]. Carr &
Lockwood [3] reported that water-stressed cocoa
seedlings quickly reach a point of no return from which

they cannot recover. Cocoa seedlings growing under

water stress conditions have been shown to exhibit a
reduction in vegetative growth compared to those
receiving an adequate supply of water [4, 5]. This
reduction is mainly as a result of changes in physiological
processes going on in the plant [1].
In general, plants respond to water stress in two main
ways drought avoidance and drought tolerance [6].
Drought avoidance enables plants to maintain high
tissue water potential under water stress. It involves
changes in plant morphology such as a decrease in leaf
area, reduction in stomatal conductance and chlorophyll
content, extended root systems and reduced shoot/root
ratio [6-8]. Drought tolerance on the other hand is the
ability of plants to continue their normal functions even
at low tissue water potentials. This is usually achieved

Corresponding Author: G.U. Chibuike, Department of Geography and Environmental Science, The University of Reading,
Whiteknights, Reading, UK.

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Am-Euras. J. Agric. & Environ. Sci., 15 (5): 944-956, 2015

MATERIALS AND METHODS

either through the accumulation of organic and inorganic

solutes such as soluble sugars, amino acids (especially
proline), Ca2+, Mg2+ and K + or through the activation of
antioxidant systems [6, 9].
Mycorrhiza - a symbiotic association between fungi
and the roots of vascular plants - has been used to
combat water stress in various plants [9-11]. Although the
mechanisms employed by mycorrhiza in the alleviation of
water stress are not fully understood [9], these
mechanisms (possible mechanisms) can be grouped into
direct and indirect mechanisms. Mycorrhiza can directly
alleviate water stress in plants by enhancing both drought
avoidance and drought tolerance mechanisms. When
subjected to water stress, plants inoculated with
mycorrhiza showed higher stomatal conductance,
photosynthetic rate, transpiration rate, leaf water
potential, relative water content, CO2 exchange rate and
hence increased growth than those without mycorrhiza
[9, 12]. This method of improving plant growth may be
particularly beneficial to cocoa as most tree crops are
known to exhibit drought avoidance mechanisms when
subjected to water stress [1, 6]. Mycorrhiza has also been
showed to increase osmotic adjustment in tree crops
growing in water stressed soils via increasing the
concentrations of organic solutes (sugars, proteins,
flavonoid and proline) and/or inorganic solutes
(Ca2+, Mg2+ and K+) in these plants [9, 10]. Improved
osmotic adjustment in the inoculated plants significantly
improved their growth under the stressed environment.
By increasing the surface area of plant roots through
mycorrhizal fungi hyphae, mycorrhiza increases the
amount of mineral nutrient (especially P, N, Zn and Cu)
and water the plant is able to acquire from the soil [13-15].
This indirectly improves the growth and performance of
plants in water-stressed environments [10, 11]. The ability
of plants to acquire more nutrients under water stress
condition also builds their immunity and hence increases
their ability to withstand pest and diseases [16].
Furthermore, the modified rhizosphere of mycorrhizal
colonized plants favours the activities of various
beneficial soil microorganisms which may not only make
more nutrients available to water-stressed plants but also
improve their health status via antagonistic behaviour to
harmful soil organisms [16].
Although a number of studies have examined the
effect of mycorrhizae on the growth and physiology of
crops growing in water-stressed environments, such
studies for cocoa seedlings are rare in literature. Thus, the
aim of this study was to determine whether mycorrhiza
reduces the impact of water stress on cocoa seedlings.

Experimental Design: John Innes loam-based compost JI No. 2 (John Innes Manufacturers, Theale, Reading, UK)
- was used in this study. Properties of the soil before and
after cultivation are contained Table 3. Four months old
cocoa seedlings (Amelonado variety) were transplanted
into plastic pots (18 cm wide and 15 cm deep) and grown
for 10 weeks in a greenhouse with average maximum and
minimum temperatures of 34.7C and 19.8C respectively.
Average relative humidity of the greenhouse ranged from
41.9% (minimum) to 80.6% (maximum). Four treatments
were applied to these seedlings based on combination of
2 factors (mycorrhizae and watering) as follows:
Treatment 1 (-M-W) = without mycorrhizae inoculum
+ water stress
Treatment 2 (-M+W) = without mycorrhizae inoculum
+ regular watering
Treatment 3 (+M-W) = with mycorrhizae inoculum +
water stress
Treatment 4 (+M+W) = with mycorrhizae inoculum +
regular watering
Each treatment had 9 replicates making a total of 36
sampling units. The treatments were arranged in the
greenhouse using the Completely Randomized Design.
Treatments were watered with 500 ml of water every
3 days (regularly watered) and 6 days (water-stressed).
During the seventh week of the experiment, cocoa nutrient
solution was applied to all the treatments - via soil
application - because the seedlings were chlorotic.
Application of Mycorrhizal Inoculum: ROOTGROW
(PlantWorks Ltd., Sittingbourne, Kent, UK) a commercial
mycorrhizal inoculum recommended for woody plants was
applied to the mycorrhizal treatments as follows: first, a
dipping gel which came with the inoculum was dissolved
in 10 L of water. After 5 minutes, mycorrhizal granules
(composed of 5 species of arbuscular mycorrhizal (AM)
fungi and 2 species of ectomycorrhizal (ECM) fungi of UK
origin) were added to the liquid and stirred until it turned
into a thick liquid. Roots of the selected seedlings were
dipped into the solution ensuring even coverage before
they were transplanted into the plastic pots.
Plant Growth and Physiological Parameters Monitored:
Plant height was measured every week using a measuring
ruler. New leaf appearance was equally monitored every
week; tags were used to demarcate new leaves from older
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Statistical Analyses: In order to satisfy the assumptions

of parametric test, all data obtained were checked for
normality using Anderson-Darling test and for equal
variances using Levenes and Bartletts tests. They were
then analysed with Analysis of Variance (ANOVA).
General Linear Model (GLM) was used to examine
differences between factors and their interaction, while
one-way ANOVA was used to compare soil properties
before and after cultivation. Turkeys family error rate was
used to detect differences between treatment means both
for GLM and one-way ANOVA. Paired t-test was used to
compare differences in photosynthetic activities taken at
two intervals. All analyses were carried out on Minitab 16
software.

ones. Leaf chlorophyll content of the youngest fully

expanded leaf for each treatment was
measured
fortnightly with Hansatech Model CL-01 chlorophyll
content meter (Hansatech Instruments, Kings Lynn, UK).
Meter readings were converted to actual values using the
linear regression equation described by Daymond et al.
[17].
Photosynthetic activities such as stomatal
conductance (gs), transpiration rate (E), photosynthetic
rate (PN) and instantaneous water use efficiency
(WUEinst = PN/E) were also measured on the youngest
(or second youngest) fully expanded leaf during Weeks
4 and 8 using Lcpro portable infra-red gas analyser
(ADC Bioscientific, Great Amwell, Herts., UK) fitted with
a light source to ensure that light was saturating.
These measurements were taken in the morning during
Week 4 and in the afternoon during Week 8.
At the end of the experiment, seedlings were
harvested and leaf area was measured with a leaf area
machine (Delta-T Devices, UK). Plant biomass was
determined by taking the fresh and oven-dry weights
(oven set at 70C) of above and below ground seedling
components.

RESULTS
Plant Growth and Physiological Parameters
Seedling Height: Fig. 1 shows that there was a steady
trend (continuous increase) in seedling height among all
the treatments from Week 1 to Week 10. The effect of
mycorrhizae was not significant (p > 0.05) throughout the
experiment unlike the effect of watering which was
significant (p < 0.05) from Week 4 to Week 10 - with the
regularly watered seedlings (-M+W and +M+W)
performing better i.e. having greater heights than the
water-stressed seedlings (-M-W and +M-W). Midway
through the experiment (Week 5), an increase in height
was observed of more than 38% in the regularly watered
seedlings and about 17% in the water-stressed seedlings.
By the end of the experiment (Week 10), increases of
more than 81% and 67% were recorded in the regularly
watered and water-stressed seedlings, respectively.
The interaction between mycorrhizae and watering was
not significant (p > 0.05).

Root Staining and Examination for Mycorrhizal Fungi:

After harvest, roots of 3 replicates from each treatment
were physically examined for signs of ECM fungi. These
replicates were preserved overnight in a mixture of acetic
acid and ethanol (1:4) and examined for mycorrhizal
colonization (by both AM and ECM fungi) using a
modified version of the method described by Koske and
Gemma [18]. Percentage mycorrhizal colonization was
calculated using the formula below:
Number of root
segments colonized
Percentage mycorrhizal colonization =
x 100
Number of root

New Leaf Appearance and Leaf Area: The trend in new

leaf appearance was not consistent in the course of the
experiment (Fig. 2). The number of leaves produced by the
various treatments gradually increased from Week 2 to
Week 5 when there was a mean increase of about 3.23
leaves in the water-stressed treatments and about 1.72
leaves in the regularly watered treatments. However, this
trend changed in Week 6 and by Week 7, the number of
leaves produced by both the regularly watered and waterstressed treatments was less than 1. By Week 9, the trend
changed to a mean increase of about 2.83 leaves in the
water-stressed treatments and 3.73 leaves in the regularly

segments examined

Soil Analyses: After the seedlings were harvested, soils

samples from each treatment were air-dried, sieved with a
2 mm sieve and analysed for some chemical properties.
These properties were compared to the initial soil
properties before cultivation. The soil analyses carried out
were: soil pH (with deionized water) and organic matter
(by loss on ignition method) as described by Rowell [19];
and available phosphorus by the Olsen P method as
described by Ministry of Agriculture, Fisheries and Food
[20].
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Table 1: Effects of watering and mycorrhizae on total number of new leaves
and total leaf area
Treatment

Total no. of new leaves

Total leaf area (cm2)

Regular watering

12.06a0.48

2105a87.3

Water stress

12.00a0.46

2015a80.3

Without mycorrhizae

11.67A0.43

2044A93.3

With mycorrhizae

12.39A0.49

2076A74.7

Watering

Mycorrhizae

Numbers represent meanstandard error of the mean (n = 18). Means with

the same letters - small letters for watering and capital letters for
mycorrhizae - are not significantly different (p > 0.05). Interaction between
watering and mycorrhizae was not significant (p > 0.05).

Fig. 1: Variations in height of cocoa seedlings. -M-W =

without mycorrhizae + water stress; -M+W =
without mycorrhizae + regular watering; +M-W
= with mycorrhizae + water stress; +M+W =
with mycorrhizae + regular watering. Bars
represent standard error of the mean (n = 9).
N.B. The effect of mycorrhizae was not
significant (p > 0.05). The effect of watering was
significant (p < 0.05) from Week 4 to Week 10.
Interaction between watering and mycorrhizae
was not significant (p > 0.05).

Results of the ANOVA for new leaf appearance

indicate that mycorrhizae had no significant effect
(p > 0.05) on the seedlings throughout the experiment.
However the effect of watering was significant (p < 0.05)
for 4 weeks: the regularly watered treatments produced
more leaves during Weeks 3 and 4 while the waterstressed treatments produced more leaves during Weeks
5 and 6. Interaction between the factors was not
significant (p > 0.05) throughout the experiment.
Table 1 shows that both mycorrhizae and watering
had no significant effect (p > 0.05) on the total number of
new leaves produced throughout the experiment, this was
also true for the total leaf area. Interaction between these
factors was not significant (p > 0.05) in both cases.
Leaf Chlorophyll Content: Leaf chlorophyll content
fluctuated throughout the experiment (Fig. 3). An increase
of about 19% to 21% was observed among the treatments
during Week 4. However, this change was not significant
until Week 6 when a significant (p < 0.05) relative change
(from Week 4) of approximately -29% was recorded in the
water-stressed treatments as opposed to -42% in the
regularly watered treatment. The water-stressed
treatments continued to show significantly higher leaf
chlorophyll content than the regularly watered treatment
from Week 6 to Week 10, although the difference was
more pronounced in Weeks 8 and 10. The effect of
mycorrhizae on leaf chlorophyll content together with its
interaction with watering was not significant (p > 0.05)
throughout the experiment.

Fig. 2: Variations in new leaf appearance of cocoa

seedlings. -M-W = without mycorrhizae + water
stress; -M+W = without mycorrhizae + regular
watering; +M-W = with mycorrhizae + water
stress; +M+W = with mycorrhizae + regular
watering. Bars represent standard error of the
mean (n = 9). N.B. The effect of mycorrhizae was
not significant (p > 0.05). The effect of watering
was significant (p < 0.05) from Week 3 to Week
6.
Interaction
between
watering
and
mycorrhizae was not significant (p > 0.05).

Photosynthetic Activities: Fig. 4 shows that mycorrhizae,

watering and their interaction had no significant effect
(p > 0.05) on stomatal conductance (gs) of the seedlings
during Weeks 4 and 8. Furthermore, the results of paired
t-test indicate that gs recorded in Week 4 was not
significantly different (p > 0.05) from that of Week 8 even

watered treatments. Again, this dropped by Week 10

when the number of leaves produced by both the nonstressed and water-stressed treatments was not greater
than 1.
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Fig. 3: Variations in leaf chlorophyll content of cocoa seedlings. -M-W = without mycorrhizae + water stress; -M+W
= without mycorrhizae + regular watering; +M-W = with mycorrhizae + water stress; +M+W = with
mycorrhizae + regular watering.. Bars represent standard error of the mean (n = 9). N.B. The effect of
mycorrhizae was not significant (p > 0.05). The effect of watering was significant (p < 0.05) from Week 6 to
Week 10. Interaction between watering and mycorrhizae was not significant (p > 0.05).

Mean Stomatal Conductance (mol/m2/s)

0.04

Aa
Aa

Aa

Aa

Aa

Aa

0.03

Aa

Aa

0.02

0.01

0.00
Treatment

-M-W

-M+W +M-W +M+W

Week 4

-M-W

-M+W +M-W +M+W

Week 8

Fig. 4: Stomatal conductance (gs) of cocoa seedlings taken at two intervals. -M-W = without mycorrhizae + water stress;
-M+W = without mycorrhizae + regular watering; +M-W = with mycorrhizae + water stress; +M+W = with
mycorrhizae + regular watering. Bars are one standard error from the mean (n = 9). Means with the same
letters - small letters for watering and capital letters for mycorrhizae - are not significantly different (p > 0.05).
Interaction between watering and mycorrhizae was not significant (p > 0.05) at both intervals.
though there was 26% increase in the water-stressed
treatments (-M-W and +M-W) and reduction of about 6%
in the regularly watered treatments (-M+W and +M+W).
Similarly both factors and their interaction had no
significant effect (p > 0.05) on transpiration rate (E) during
Weeks 4 and 8 (Fig. 5). However, when E of both weeks
were compared using paired t-test, a significant reduction
(p < 0.05) was recorded in Week 8 i.e. a relative change of
-11%, -36%, -33% and -40% was observed in -M-W, M+W, +M-W and +M+W, respectively.
For photosynthetic rate (PN), the effect of watering
was significant (p < 0.05) during Week 4 when the average
PN of the regularly watered seedlings was 3.89 mol m 2

s 1 compared to 2.02 mol m 2 s 1 in the water-stressed

seedlings (Fig. 6). The effect of watering was not
significant (p > 0.05) in Week 8. Similarly, the effects of
mycorrhizae and the interaction between both factors
were not significant (p > 0.05) at both intervals. Results of
paired t-test show that PN at the two intervals were not
significantly different (p > 0.05) from each other even
though there was a relative change of -38% and 25% in
the regularly watered and water-stressed seedlings,
respectively.
The effects of mycorrhizae, watering and their
interaction were not significant (p > 0.05) on
instantaneous water use efficiency (WUEinst) of the
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Mean Transpiration Rate (mmol/m2/s)

1.6

Aa
Aa

1.4

Aa

Aa

1.2
1.0

Bb

Bb

Bb

0.8

Bb

0.6
0.4
0.2
0.0
Treatment

-M-W

-M+W +M-W +M+W

Week 4

-M-W

-M+W +M-W +M+W

Week 8

Fig. 5: Transpiration rate (E) of cocoa seedlings taken at two intervals. -M-W = without mycorrhizae + water stress; M+W = without mycorrhizae + regular watering; +M-W = with mycorrhizae + water stress; +M+W = with
mycorrhizae + regular watering. Bars are one standard error from the mean (n = 9). Means with the same
letters - small letters for watering and capital letters for mycorrhizae - are not significantly different (p > 0.05).
Interaction between watering and mycorrhizae was not significant (p > 0.05) at both intervals

Mean Photosynthetic Rate (umol/m2/s)

Aa

Aa

Aa

Ab

Aa

Aa

Aa

Ab

0
Treatment

-M-W

-M+W +M-W +M+W

Week 4

-M-W

-M+W +M-W +M+W

Week 8

Fig. 6: Photosynthetic rate (PN) of cocoa seedlings taken at two intervals. -M-W = without mycorrhizae + water stress;
-M+W = without mycorrhizae + regular watering; +M-W = with mycorrhizae + water stress; +M+W = with
mycorrhizae + regular watering. Bars are one standard error from the mean (n = 9). Means with the same
letters - small letters for watering and capital letters for mycorrhizae - are not significantly different (p > 0.05).
Interaction between watering and mycorrhizae was not significant (p > 0.05) at both intervals.
seedlings during Week 4, even though WUEinst of the
water-stressed was lower than that of the regularly
watered seedlings (Fig. 7). Similarly, the effects of both
factors and their interaction were not significant on
WUEinst of the seedlings during Week 8. However, during
this period, the WUEinst of the water-stressed seedlings
increased by 41% and was thus slightly higher than that
of the regularly watered seedlings. The results of paired
949

t-test analysis show that there was a significant reduction

(p < 0.05) in the WUEinst recorded in Week 8 compared to
that recorded in Week 4.
Plant Biomass: Table 2 shows that the effects of
mycorrhizae and watering on seedling biomass were not
significant (p > 0.05). The interaction between these
factors was also not significant (p > 0.05). However, it can

Mean Instantaneous WUE (umol CO2/mmol H2O)

Am-Euras. J. Agric. & Environ. Sci., 15 (5): 944-956, 2015

Aa
Bb

Bb
Bb
Bb

Aa
Aa

Aa

-M-W

-M+W +M-W +M+W

Week 4

0
Treatment

-M-W

-M+W +M-W +M+W

Week 8

Fig. 7: Instantaneous water use efficiency (WUEinst) of cocoa seedling taken at two intervals. -M-W = without
mycorrhizae + water stress; -M+W = without mycorrhizae + regular watering; +M-W = with mycorrhizae +
water stress; +M+W = with mycorrhizae + regular watering. Bars are one standard error from the mean (n
= 9). Means with the same letters - small letters for watering and capital letters for mycorrhizae - are not
significantly different (p > 0.05). Interaction between watering and mycorrhizae was not significant (p > 0.05)
at both intervals.

Fig. 8: Compound microscope images showing absence of mycorrhizal colonization. (a) -M-W = without mycorrhizae
+ water stress; (b) -M+W = without mycorrhizae + regular watering; (c) +M-W = with mycorrhizae + water
stress; (d) +M+W = with mycorrhizae + regular watering. Magnification = 10x.
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Table 2: Effects of watering and mycorrhizae on cocoa seedling biomass
Treatment

Shoot biomass (g)

Root biomass (g)

Total biomass (g)

Shoot to root ratio

Regular watering

14.32a0.80

5.28a0.26

19.60a1.00

2.72a0.10

Water stress

12.97 0.31

5.42 0.46

18.38 0.65

2.53a0.16

Without mycorrhizae

13.69A0.69

5.25A0.30

18.94A0.91

2.64A0.11

With mycorrhizae

13.60A0.58

5.44A0.43

19.04A0.82

2.61A0.16

Watering
a

Mycorrhizae

Numbers represent meanstandard error of the mean (n = 12). Means with the same letters - small letters for watering and capital letters for mycorrhizae are not significantly different (p > 0.05). Interaction between watering and mycorrhizae was not significant (p > 0.05).
Table 3: Differences between initial soil properties and properties of soil at end of experiment
Treatment

pH

Organic matter (%)

Available P (mg kg 1)

Initial soil

5.51 0.06

23.82 0.66

80.59a3.41

Soil with -M-W

4.89 0.15

8.21 0.27

58.68b2.00

Soil with -M+W

5.61a0.15

9.13b0.44

63.16b2.21

Soil with +M-W

5.22ab0.05

9.81b0.50

68.11ab2.94

Soil with +M+W

5.59 0.09

8.45 0.46

64.71ab7.23

-M-W = without mycorrhizae + water stress; -M+W = without mycorrhizae + regular watering; +M-W = with mycorrhizae + water stress; +M+W = with
mycorrhizae + regular watering. Numbers represent meanstandard error of the mean (n = 5). Means with the same letters are not significantly different
(p > 0.05).

DISCUSSION

be seen that shoot and total biomass of the regularly

watered treatments were higher than that of the waterstressed treatments. On the other hand, the root biomass
of the water-stressed treatments was higher than that of
the regularly watered treatments.

Absence of Mycorrhizal Colonization: The lack of

mycorrhizal colonization of cocoa seedling roots explains
why the effect of mycorrhizae was not significant in this
experiment. To a large extent, it also explains why the
interaction between watering and mycorrhizae was not
significant throughout the experiment. The main reason
for the absence of colonization may be attributed to the P
content of the soil which was approximately 80 mg kg 1
before cultivation and ranged from about 59 mg kg 1 to 68
mg kg 1 in the various treatments after cultivation.
According to Rosen et al. [21], soils with more than 18
mgP kg 1 (determined through Olsen P method) can be
classified as having very high amounts of available P.
It is widely known that mycorrhizal colonization of plant
roots is greatly hindered by high amounts of soil P [22-24]
this may be due to the detrimental effect of P on fungi
hyphae and other fungal structures [25]. Thus, the P
content of the soil may have prevented the colonization
of the seedling roots since even after cultivation soil P
was still very high.
The P requirement of the plant to be colonized also
affects mycorrhizal colonization. Abbot & Robson [26]
reported that at soil P levels greater than that required by
a plant, growth of vesicles of AM fungi is hindered thus
affecting colonization. At a soil P level of about 51 mg
kg 1, 36% to 45% AM colonization occurred in Asperula

Mycorrhizal Colonization: There was 0% colonization in

both the inoculated and non inoculated seedling roots. In
other words, the roots showed no signs of AM fungi (i.e.
absence of arbuscules and vesicles) and ECM fungi
(absence of Hartig net) when they were viewed under a
compound microscope after staining with Trypan blue
(Fig. 8). Other signs of fungi such as hyphae and spores
were also not present. In addition, when the roots were
physically examined before staining with Trypan blue,
there were also no signs of ECM fungi (i.e. no
pigmentation and mantle).
Soil Properties: Results in Table 3 show that pH of the
initial soil was similar to pH of the cultivated soils except
for the soil with -M-W which had a significantly lower (p
< 0.05) pH compared to the initial soil. Organic matter in
the initial soil was significantly higher (p < 0.05) than that
in the cultivated soils (for all the treatments). Available P
in the initial soil was significantly higher (p < 0.05) than
that in the treatments without mycorrhizae (-M-W and M+W) but did not differ significantly (p > 0.05) with the
treatments with mycorrhizae (+M-W and +M+W).
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odorata [25]. Root colonization of onion plants by AM

fungi also occurred at a soil P level of 50 mg kg 1 [22].
Since mycorrhizal colonization occurred in these plants, it
can be assumed that their P requirements were not met
even though the P content of the soil was high.
Colonization of cocoa seedling roots by AM fungi at an
initial soil P level of 7.82 mg kg 1 and 27.5 mg kg 1 have
been reported by Cuenca et al. [13] and Chulan and Ragu
[27]. Thus, it can be deduced that the P requirement of
cocoa seedlings used in this study was already met by the
high P content of the soil and thus the seedlings were not
colonized by mycorrhizal fungi.
The organic matter content of the soil used in this
study may have also influenced the absence of
mycorrhizal colonization. John Innes soil used for this
study can be classified as an organic soil because it
contains more than 19% organic matter [21]. Since the
organic matter content of this soil is high, it can be
assumed that organic forms of essential nutrients such
as N will also be high in this soil [28]. Studies have
shown that soil N can indirectly influence mycorrhizal
colonization. For instance, Sylvia and Neal [29] reported
that under high soil N conditions, mycorrhizal
colonization of Allium cepa was limited by the addition of
soil P. However, when soil N was limiting, addition of P
did not hinder mycorrhizal colonization. Although it was
thought that mycorrhizal fungi - especially AM fungi - do
not utilize organic forms of soil nutrients [30], recent
research has proved this wrong [31, 32]. Thus, it can be
deduced that the high organic matter (high N content) of
the initial soil indirectly contributed to the absence of
mycorrhizal colonization.
The application of a nutrient solution during the
seventh week of the experiment may have also affected
mycorrhizal
colonization.
However,
this
was
unavoidable since most of the seedlings were
chlorotic
at
this stage (there was also visible
improvement in the seedlings after application).
Furthermore, the results of the experiment show that the
effect of mycorrhizae was not significant even before the
solution was applied, thus the effect of applying the
solution may be negligible compared to the nutrient
content of the initial soil.
Another factor that may have affected mycorrhizal
colonization of cocoa roots is specificity of the fungi
species used. Few species of AM fungi are specific to
host plants, though these species are rare and their
specificity largely depends on the ecology of the plant
they colonize [33]. The extensive root structure of cocoa
seedlings may have also contributed to the lack of

mycorrhizal colonization, although this may be negligible.

Brundrett [34] reported that plants with root that are able
to access and absorb immobile forms of soil nutrients are
less dependent on mycorrhizal fungi.
Since there was no mycorrhizal colonization, it can be
deduced that the presence of mycorrhizae did not
influence the response of cocoa seedlings to water stress;
hence, the rest of the paper will focus on the effect of the
water stress on the seedlings growth and physiology.
Effect of Water Stress on Plant Growth and Physiological
Parameters: The effect of water stress on seedling height
became significant from Week 4 to the end of the
experiment - with the water-stressed seedlings showing
reduced height compared to the regularly watered
seedlings. At the end of the experiment, the difference in
height between the regularly watered and water-stressed
seedlings was about 14%. This is similar with the works of
Mohd et al., [5], Zhang et al. [8] and Kirnak et al. [35]
who recorded significant reduction in heights of waterstressed cocoa seedlings, eggplants and oriental lily,
respectively.
Similarly, the effect of water stress was also
significant on new leaf appearance. However, the number
of leaves produced by water-stressed and regularly
watered treatments fluctuated in the course of the
experiment. While the regularly watered treatments
produced more leaves at certain periods, the waterstressed treatments produced more leaves at other
periods (Fig. 2). The reason for this inconsistency may be
attributed to the watering regime. Hutcheon [36] reported
that the end of a water stress period is followed by a
period of synchronized flush of leaf growth in cocoa
seedlings. This period of new leaf growth is usually
independent of the plants water status until the next
period of water stress [3, 37].
Although, the number of new leaves produced by the
treatments did not follow a consistent pattern on a weekly
basis, the total number of new leaves produced at the end
of the experiment show that the regularly watered
treatments produced more leaves than the water-stressed
treatment even though this was not significant. The total
leaf area of the water-stressed treatments was also lower
than that of the regularly watered treatments. The reduced
leaf area of the water-stressed treatments is a common
drought avoidance mechanism exhibited by waterstressed plants to enable them maintain high tissue water
potential during water stress [6, 7]. The absence of a
significant difference between the water-stressed and
regularly watered treatments (in terms of total leaf area)
952

Am-Euras. J. Agric. & Environ. Sci., 15 (5): 944-956, 2015

may have been because the duration of the experiment

was not long enough for the treatment effect to become
significant.
Effect of water-stress on leaf chlorophyll content was
significant from Week 6 to Week 10. However, unlike in
most studies where water-stressed plants showed lower
chlorophyll content compared to regularly watered plants
[8, 35, 38], the water-stressed cocoa seedlings showed
significantly higher chlorophyll content compared to the
regularly watered treatments. The reason for the higher
chlorophyll content of the water-stressed plants may have
been because of their higher N content (though this was
not assessed). Sanchez et al. [38] reported that the N
content of water-stressed maize plants was not affected
by the water stress condition. The marked decline in leaf
chlorophyll content for all treatments during Week 6 was
due to leaf chlorosis which occurred during that period.
However, with the application of cocoa nutrient solution
in Week 7, chlorophyll content of the treatments
increased significantly (Fig. 3).
Results of photosynthetic activities show that gs, E
and PN of the water-stressed seedlings were lower than
that of the regularly watered seedlings during Week 4,
even though significant effect was observed only on PN.
This corresponds to results of most studies where
photosynthetic activities in various water-stressed plants
were monitored [5, 8, 35, 38]. The reduction in
photosynthetic activities in the water-stressed treatments
may be attributed to partial stomatal closure which
reduces photosynthesis in water-stressed plants [1, 3].
Although water stress is known to increase WUEinst
in plants [1, 39], results of this study show that WUEinst of
the water-stressed seedlings was lower than that of the
regularly watered seedlings during Week 4 (though the
reverse occurred during Week 8). The reason for this
lower WUEinst in the stressed seedlings may have been
because WUEinst is dependent on the severity of water
stress. Under severe water stress conditions, reduced
WUEinst was recorded in wheat [40, 41]; while, under
moderate water stress condition, increases in WUEinst was
recorded in the same plant [42, 43]. This is because, at
severe water stress conditions, photosynthesis may be
hindered by non-stomatal factors [44].
There was a general decline in photosynthetic
activities (except for WUEinst) during Week 8 compared to
Week 4 especially for transpiration rate. Sharkey [45]
reported that moderate heat stress resulting from elevated
temperatures reduces photosynthesis. Thus, difference
between the two intervals may be due to the different
measurement time morning for Week 4 and afternoon for

Week 8 since afternoon temperatures are usually higher

than morning temperatures. Furthermore, relative humidity
is usually lower in the afternoon which results in partial
stomatal closure and hence reduced photosynthesis [3].
This is similar to the work by Rajendrudu and Naidu [46]
who observed that gs, E and PN of water-stressed Tectona
grandis were reduced during afternoon period.
Water stress reduced seedlings shoot and total
biomass but increased their root biomass; this resulted to
a lower shoot to root ratio in the stressed seedlings.
These results correspond to that of similar studies [5, 35].
The increased root biomass of the water-stressed
seedlings may be attributed to the extended root systems
typical of water-stressed plants due to their foraging for
water [6].
CONCLUSIONS
Results of this study show that water stress reduced
the growth of cocoa seedlings mainly as a result of
changes in physiological processes i.e. reduction in
photosynthetic activities in the stressed seedlings.
Mycorrhizae did not reduce the impact of water stress on
cocoa seedlings. This was due to the absence of root
colonization by mycorrhizal fungi. This highlights the fact
that several factors may influence mycorrhizal
colonization even after inoculation. The high P content of
the soil may have been the main reason for this lack of
colonization. High nutrient content (especially N) of the
soil evident from its high organic matter content may have
also prevented colonization. Specificity of mycorrhizal
fungi and the extensive root system of cocoa seedlings
are other factors (though negligible) that may have also
prevented colonization. Based on the above, in order for
definite conclusions to be drawn as to whether the
presence of mycorrhizae influences the response of cocoa
seedlings to water stress, it is recommended that future
work utilises a less fertile soil or a soil with low to
moderate P content. It is also important to identify the
exact species of mycorrhizal fungi in the inoculum so as to
establish that they actually colonize cocoa plant.
ACKNOWLEDGEMENT
This research was an integral part of a Masters
degree programme funded by the Felix Trust. Dr. G.P.
Warren and Dr. E.J. Shaw, both of Soil Research Centre,
Department of Geography and Environmental Science,
The University of Reading, assisted the authors during
mycorrhizal analysis. This assistance is gratefully
acknowledged.
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Am-Euras. J. Agric. & Environ. Sci., 15 (5): 944-956, 2015

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