micorrhizae inoculation did not influence the response of cocoa seedlings to water stress

7/25/2019 Micorrhizae Inoculation Did Not Influence the Response of Cocoa Seedlings to Water Stress

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American-Eurasian J. Agric. & Environ. Sci., 15 (5): 944-956, 2015ISSN 1818-6769 IDOSI Publications, 2015DOI: 10.5829/idosi.aejaes.2015.15.5.12634

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

Whiteknights, Reading, UK.

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Mycorrhizae Inoculation Did Not Influence the

Response of Cocoa Seedlings to Water Stress

G.U. Chibuike and A.J. Daymond1,3 2

Department of Geography and Environmental Science,1

The University of Reading, Whiteknights, Reading, UKSchool of Agriculture, Policy and Development,2

The University of Reading, Whiteknights, Reading, UKDepartment of Soil Science and Land Resources Management,3

University of Nigeria, Nsukka, Nigeria

Abstract:A greenhouse experiment was conducted to determine whether mycorrhiza reduces the impact ofwater stress on cocoa seedlings. The effects of two factors mycorrhizae and watering on growth andphysiology 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 + regularwatering); +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 notinfluence their response to water stress. High P content of the soil was likely to have been the main reason forabsence of mycorrhizal colonization. Water stress negatively influenced most plant growth and physiologicalparameters, though significance (p < 0.05) of its effect was more noticeable in seedling height. A soil with lowor 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 they cannot recover. Cocoa seedlings growing under

Water is essential for the proper growth and reduction in vegetative growth compared to those

functioning of all plants. It is particularly necessary receiving an adequate supply of water [4, 5]. Thisduring the early stage of plant growth. Shortage of water reduction is mainly as a result of changes in physiologicalduring this stage results in growth reduction, increased processes going on in the plant [1].mortality and susceptibility to diseases [1]. For plants In general, plants respond to water stress in two main

which will later be transplanted, it is necessary that they ways drought avoidance and drought tolerance [6].are not subjected to severe water stress as this will affect Drought avoidance enables plants to maintain high

their establishment in the field and in turn affect their tissue water potential under water stress. It involvesperformance. changes in plant morphology such as a decrease in leaf

Cocoa (Theobroma cacao) requires a relatively area, reduction in stomatal conductance and chlorophyllhigh amount of water during the early stage of its growth content, extended root systems and reduced shoot/rootand hence is very sensitive to water stress [2]. Carr & ratio [6-8]. Drought tolerance on the other hand is theLockwood [3] reported that water-stressed cocoa ability of plants to continue their normal functions even

seedlings quickly reach a point of no return from which at low tissue water potentials. This is usually achieved

water stress conditions have been shown to exhibit a


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

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either through the accumulation of organic and inorganic MATERIALS AND METHODSsolutes such as soluble sugars, amino acids (especiallyproline), Ca , Mg and K or through the activation of Experimental Design:John Innes loam-based compost -2+ 2+ +

antioxidant systems [6, 9]. JI No. 2 (John Innes Manufacturers, Theale, Reading, UK)

Mycorrhiza - a symbiotic association between fungi - was used in this study. Properties of the soil before andand the roots of vascular plants - has been used to after cultivation are contained Table 3. Four months oldcombat water stress in various plants [9-11]. Although the cocoa seedlings (Amelonado variety) were transplantedmechanisms employed by mycorrhiza in the alleviation of into plastic pots (18 cm wide and 15 cm deep) and grownwater stress are not fully understood [9], these for 10 weeks in a greenhouse with average maximum andmechanisms (possible mechanisms) can be grouped into minimum temperatures of 34.7C and 19.8C respectively.direct and indirect mechanisms. Mycorrhiza can directly Average relative humidity of the greenhouse ranged fromalleviate water stress in plants by enhancing both drought 41.9% (minimum) to 80.6% (maximum). Four treatmentsavoidance and drought tolerance mechanisms. When were applied to these seedlings based on combination ofsubjected to water stress, plants inoculated with 2 factors (mycorrhizae and watering) as follows:mycorrhiza showed higher stomatal conductance,photosynthetic rate, transpiration rate, leaf water Treatment 1 (-M-W) = without mycorrhizae inoculum

potential, relative water content, CO exchange rate and + water stress2hence increased growth than those without mycorrhiza Treatment 2 (-M+W) = without mycorrhizae inoculum[9, 12]. This method of improving plant growth may be + regular wateringparticularly beneficial to cocoa as most tree crops are Treatment 3 (+M-W) = with mycorrhizae inoculum +known to exhibit drought avoidance mechanisms when water stresssubjected to water stress [1, 6]. Mycorrhiza has also been Treatment 4 (+M+W) = with mycorrhizae inoculum +showed to increase osmotic adjustment in tree crops regular wateringgrowing in water stressed soils via increasing theconcentrations of organic solutes (sugars, proteins, Each treatment had 9 replicates making a total of 36flavonoid and proline) and/or inorganic solutes sampling units. The treatments were arranged in the(Ca , Mg and K ) in these plants [9, 10]. Improved greenhouse using the Completely Randomized Design.2+ 2+ +

osmotic adjustment in the inoculated plants significantly Treatments were watered with 500 ml of water every

improved their growth under the stressed environment. 3 days (regularly watered) and 6 days (water-stressed).By increasing the surface area of plant roots through During the seventh week of the experiment, cocoa nutrientmycorrhizal fungi hyphae, mycorrhiza increases the solution was applied to all the treatments - via soilamount of mineral nutrient (especially P, N, Zn and Cu) application - because the seedlings were chlorotic.and water the plant is able to acquire from the soil [13-15].This indirectly improves the growth and performance of Application of Mycorrhizal Inoculum:ROOTGROWplants in water-stressed environments [10, 11]. The ability (PlantWorks Ltd., Sittingbourne, Kent, UK) a commercialof plants to acquire more nutrients under water stress mycorrhizal inoculum recommended for woody plants wascondition also builds their immunity and hence increases applied to the mycorrhizal treatments as follows: first, atheir ability to withstand pest and diseases [16]. dipping gel which came with the inoculum was dissolvedFurthermore, the modified rhizosphere of mycorrhizal in 10 L of water. After 5 minutes, mycorrhizal granulescolonized plants favours the activities of various (composed of 5 species of arbuscular mycorrhizal (AM)beneficial soil microorganisms which may not only make fungi and 2 species of ectomycorrhizal (ECM) fungi of UKmore nutrients available to water-stressed plants but also origin) were added to the liquid and stirred until it turnedimprove their health status via antagonistic behaviour to into a thick liquid. Roots of the selected seedlings wereharmful soil organisms [16]. dipped into the solution ensuring even coverage before

Although a number of studies have examined the they were transplanted into the plastic pots.effect of mycorrhizae on the growth and physiology ofcrops growing in water-stressed environments, such Plant Growth and Physiological Parameters Monitored:studies for cocoa seedlings are rare in literature. Thus, the Plant height was measured every week using a measuringaim of this study was to determine whether mycorrhiza ruler. New leaf appearance was equally monitored everyreduces the impact of water stress on cocoa seedlings. week; tags were used to demarcate new leaves from older


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Number of root

segments colonizedPercentage mycorrhizal colonization = x 100

Number of root

segments examined


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ones. Leaf chlorophyll content of the youngest fully Statistical Analyses:In order to satisfy the assumptions

expanded leaf for each treatment was measured of parametric test, all data obtained were checked for

fortnightly with Hansatech Model CL-01 chlorophyll normality using Anderson-Darling test and for equal

content meter (Hansatech Instruments, Kings Lynn, UK). variances using Levenes and Bartletts tests. They were

Meter readings were converted to actual values using the then analysed with Analysis of Variance (ANOVA).linear regression equation described by Daymond et al. General Linear Model (GLM) was used to examine

[17]. differences between factors and their interaction, while

Photosynthetic activities such as stomatal one-way ANOVA was used to compare soil properties

conductance (g ), transpiration rate (E), photosynthetic before and after cultivation. Turkeys family error rate wassrate (P ) and instantaneous water use efficiency used to detect differences between treatment means bothN(WUE = P /E) were also measured on the youngest for GLM and one-way ANOVA. Paired t-test was used toinst N(or second youngest) fully expanded leaf during Weeks compare differences in photosynthetic activities taken at

4 and 8 using Lc portable infra-red gas analyser two intervals. All analyses were carried out on Minitab 16pro(ADC Bioscientific, Great Amwell, Herts., UK) fitted with software.

a light source to ensure that light was saturating.

These measurements were taken in the morning during RESULTS

Week 4 and in the afternoon during Week 8.

At the end of the experiment, seedlings were Plant Growth and Physiological Parameters

harvested and leaf area was measured with a leaf area Seedling Height: Fig. 1 shows that there was a steady

machine (Delta-T Devices, UK). Plant biomass was trend (continuous increase) in seedling height among all

determined by taking the fresh and oven-dry weights the treatments from Week 1 to Week 10. The effect of

(oven set at 70C) of above and below ground seedling mycorrhizae was not significant (p > 0.05) throughout the

components. experiment unlike the effect of watering which was

Root Staining and Examination for Mycorrhizal Fungi: regularly watered seedlings (-M+W and +M+W)

After harvest, roots of 3 replicates from each treatment performing better i.e. having greater heights than the

were physically examined for signs of ECM fungi. These water-stressed seedlings (-M-W and +M-W). Midway

replicates were preserved overnight in a mixture of acetic through the experiment (Week 5), an increase in heightacid and ethanol (1:4) and examined for mycorrhizal was observed of more than 38% in the regularly watered

colonization (by both AM and ECM fungi) using a seedlings and about 17% in the water-stressed seedlings.

modified version of the method described by Koske and By the end of the experiment (Week 10), increases of

Gemma [18]. Percentage mycorrhizal colonization was more than 81% and 67% were recorded in the regularly

calculated using the formula below: watered and water-stressed seedlings, respectively.

not significant (p > 0.05).

Soil Analyses:After the seedlings were harvested, soils experiment (Fig. 2). The number of leaves produced by the

samples from each treatment were air-dried, sieved with a various treatments gradually increased from Week 2 to2 mm sieve and analysed for some chemical properties. Week 5 when there was a mean increase of about 3.23

These properties were compared to the initial soil leaves in the water-stressed treatments and about 1.72

properties before cultivation. The soil analyses carried out leaves in the regularly watered treatments. However, this

were: soil pH (with deionized water) and organic matter trend changed in Week 6 and by Week 7, the number of

(by loss on ignition method) as described by Rowell [19]; leaves produced by both the regularly watered and water-

and available phosphorus by the Olsen P method as stressed treatments was less than 1. By Week 9, the trend

described by Ministry of Agriculture, Fisheries and Food changed to a mean increase of about 2.83 leaves in the

[20]. water-stressed treatments and 3.73 leaves in the regularly

significant (p < 0.05) from Week 4 to Week 10 - with the

The interaction between mycorrhizae and watering was

New Leaf Appearance and Leaf Area:The trend in new

leaf appearance was not consistent in the course of the


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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. Barsrepresent 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).

Fig. 2: Variations in new leaf appearance of cocoaseedlings. -M-W = without mycorrhizae + waterstress; -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).

watered treatments. Again, this dropped by Week 10when the number of leaves produced by both the non-stressed and water-stressed treatments was not greaterthan 1.

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 (cm )2

Watering

Regular watering 12.06 0.48 2105 87.3

a a

Water stress 12.00 0.46 2015 80.3a a

Mycorrhizae

Without mycorrhizae 11.67 0.43 2044 93.3A A

With mycorrhizae 12.39 0.49 2076 74.7A A

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).

Results of the ANOVA for new leaf appearanceindicate 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 producedmore leaves during Weeks 3 and 4 while the water-stressed treatments produced more leaves during Weeks5 and 6. Interaction between the factors was notsignificant (p > 0.05) throughout the experiment.

Table 1 shows that both mycorrhizae and wateringhad no significant effect (p > 0.05) on the total number ofnew leaves produced throughout the experiment, this wasalso true for the total leaf area. Interaction between thesefactors was not significant (p > 0.05) in both cases.

Leaf Chlorophyll Content: Leaf chlorophyll contentfluctuated throughout the experiment (Fig. 3). An increaseof about 19% to 21% was observed among the treatmentsduring Week 4. However, this change was not significantuntil Week 6 when a significant (p < 0.05) relative change(from Week 4) of approximately -29% was recorded in thewater-stressed treatments as opposed to -42% in theregularly watered treatment. The water-stressedtreatments continued to show significantly higher leafchlorophyll content than the regularly watered treatmentfrom Week 6 to Week 10, although the difference wasmore pronounced in Weeks 8 and 10. The effect of

mycorrhizae on leaf chlorophyll content together with itsinteraction with watering was not significant (p > 0.05)throughout the experiment.

Photosynthetic Activities:Fig. 4 shows that mycorrhizae,watering and their interaction had no significant effect(p > 0.05) on stomatal conductance (g of the seedlingss)during Weeks 4 and 8. Furthermore, the results of pairedt-test indicate that g recorded in Week 4 was notssignificantly different (p > 0.05) from that of Week 8 even


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Treatment

Week 8Week 4

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

0.04

0.03

0.02

0.01

0.00

MeanStomatalConductance(mol/m2/s)

Aa

Aa

Aa

Aa

Aa

Aa

AaAa


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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).

Fig. 4: Stomatal conductance (g ) of cocoa seedlings taken at two intervals.-M-W = without mycorrhizae + water stress;s-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 s compared to 2.02 mol m s in the water-stressedtreatments (-M-W and +M-W) and reduction of about 6% seedlings (Fig. 6). The effect of watering was notin the regularly watered treatments (-M+W and +M+W). significant (p > 0.05) in Week 8. Similarly, the effects of

Similarly both factors and their interaction had no mycorrhizae and the interaction between both factorssignificant effect (p > 0.05) on transpiration rate (E) during were not significant (p > 0.05) at both intervals. Results ofWeeks 4 and 8 (Fig. 5). However, when E of both weeks paired t-test show that P at the two intervals were notwere compared using paired t-test, a significant reduction significantly different (p > 0.05) from each other even(p < 0.05) was recorded in Week 8 i.e. a relative change of though there was a relative change of -38% and 25% in-11%, -36%, -33% and -40% was observed in -M-W, - the regularly watered and water-stressed seedlings,M+W, +M-W and +M+W, respectively. respectively.

For photosynthetic rate (P , the effect of watering The effects of mycorrhizae, watering and theirN)was significant (p < 0.05) during Week 4 when the average interaction were not significant (p > 0.05) onP of the regularly watered seedlings was 3.89 mol m instantaneous water use efficiency (WUE ) of theN

2

1 2 1

N

inst


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Treatment

Week 8Week 4


1.6

1.4

1.2

1.0

0.8

0.6

0.4

0.2

0.0

MeanTranspirationRate(mmol/m2/s)

Bb

Bb

BbBb

Aa

Aa

Aa

Aa

Treatment

Week 8Week 4


4

3

2

1

0

MeanPhotosyn

theticRate(umol/m2/s)

AaAaAa

Aa

Aa

Ab

Aa

Ab


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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 = withmycorrhizae + regular watering. Bars are one standard error from the mean (n = 9). Means with the same


Interaction between watering and mycorrhizae was not significant (p > 0.05) at both intervals

Fig. 6: Photosynthetic rate (P ) of cocoa seedlings taken at two intervals.-M-W = without mycorrhizae + water stress;N-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


Interaction between watering and mycorrhizae was not significant (p > 0.05) at both intervals.

seedlings during Week 4, even though WUE of the t-test analysis show that there was a significant reductioninstwater-stressed was lower than that of the regularly (p < 0.05) in the WUE recorded in Week 8 compared towatered seedlings (Fig. 7). Similarly, the effects of both that recorded in Week 4.factors and their interaction were not significant onWUE of the seedlings during Week 8. However, during Plant Biomass: Table 2 shows that the effects ofinstthis period, the WUE of the water-stressed seedlings mycorrhizae and watering on seedling biomass were notinstincreased by 41% and was thus slightly higher than that significant (p > 0.05). The interaction between theseof the regularly watered seedlings. The results of paired factors was also not significant (p > 0.05). However, it can

inst


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Treatment

Week 8Week 4


4

3

2

1

0MeanInstantaneousWUE(umolCO

2/mmolH2O)

Bb

Bb

Bb

Bb

Aa

Aa

Aa

Aa


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Fig. 7: Instantaneous water use efficiency (WUE ) of cocoa seedling taken at two intervals. -M-W = withoutinstmycorrhizae + 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

Watering

Regular watering 14.32 0.80 5.28 0.26 19.60 1.00 2.72 0.10a a a a

Water stress 12.97 0.31 5.42 0.46 18.38 0.65 2.53 0.16a a a a

Mycorrhizae

Without mycorrhizae 13.69 0.69 5.25 0.30 18.94 0.91 2.64 0.11A A A A

With mycorrhizae 13.60 0.58 5.44 0.43 19.04 0.82 2.61 0.16A A A A

Numbers represent meanstandard error of the mean (n = 12). Means with the same letters - small letters for watering and capital le tters 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 80.59 3.41a a a

Soil with -M-W 4.89 0.15 8.21 0.27 58.68 2.00b b b

Soil with -M+W 5.61 0.15 9.13 0.44 63.16 2.21a b b

Soil with +M-W 5.22 0.05 9.81 0.50 68.11 2.94ab b ab

Soil with +M+W 5.59 0.09 8.45 0.46 64.71 7.23a b ab

-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).

be seen that shoot and total biomass of the regularly DISCUSSIONwatered treatments were higher than that of the water-stressed treatments. On the other hand, the root biomass Absence of Mycorrhizal Colonization: The lack ofof the water-stressed treatments was higher than that of mycorrhizal colonization of cocoa seedling roots explainsthe regularly watered treatments. why the effect of mycorrhizae was not significant in this

Mycorrhizal Colonization:There was 0% colonization in interaction between watering and mycorrhizae was notboth the inoculated and non inoculated seedling roots. In significant throughout the experiment. The main reasonother words, the roots showed no signs of AM fungi (i.e. for the absence of colonization may be attributed to the Pabsence of arbuscules and vesicles) and ECM fungi content of the soil which was approximately 80 mg kg(absence of Hartig net) when they were viewed under a before cultivation and ranged from about 59 mg kg to 68compound microscope after staining with Trypan blue mg kg in the various treatments after cultivation.(Fig. 8). Other signs of fungi such as hyphae and spores According to Rosen et al. [21], soils with more than 18were also not present. In addition, when the roots were mgP kg (determined through Olsen P method) can bephysically examined before staining with Trypan blue, classified as having very high amounts of available P.there were also no signs of ECM fungi (i.e. no It is widely known that mycorrhizal colonization of plantpigmentation and mantle). roots is greatly hindered by high amounts of soil P [22-24]

Soil Properties: Results in Table 3 show that pH of the hyphae and other fungal structures [25]. Thus, the Pinitial soil was similar to pH of the cultivated soils except content of the soil may have prevented the colonizationfor the soil with -M-W which had a significantly lower (p of the seedling roots since even after cultivation soil P< 0.05) pH compared to the initial soil. Organic matter in was still very high.the initial soil was significantly higher (p < 0.05) than that The P requirement of the plant to be colonized alsoin the cultivated soils (for all the treatments). Available P affects mycorrhizal colonization. Abbot & Robson [26]in the initial soil was significantly higher (p < 0.05) than reported that at soil P levels greater than that required bythat in the treatments without mycorrhizae (-M-W and - a plant, growth of vesicles of AM fungi is hindered thusM+W) but did not differ significantly (p > 0.05) with the affecting colonization. At a soil P level of about 51 mgtreatments with mycorrhizae (+M-W and +M+W). kg , 36% to 45% AM colonization occurred inAsperula

experiment. To a large extent, it also explains why the

1

1

1

1

this may be due to the detrimental effect of P on fungi

1


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odorata[25]. Root colonization of onion plants by AM mycorrhizal colonization, although this may be negligible.fungi also occurred at a soil P level of 50 mg kg [22]. Brundrett [34] reported that plants with root that are able1

Since mycorrhizal colonization occurred in these plants, it to access and absorb immobile forms of soil nutrients arecan be assumed that their P requirements were not met less dependent on mycorrhizal fungi.

even though the P content of the soil was high. Since there was no mycorrhizal colonization, it can beColonization of cocoa seedling roots by AM fungi at an deduced that the presence of mycorrhizae did notinitial soil P level of 7.82 mg kg and 27.5 mg kg have influence the response of cocoa seedlings to water stress;1 1

been reported by Cuenca et al. [13] and Chulan and Ragu hence, the rest of the paper will focus on the effect of the[27]. Thus, it can be deduced that the P requirement of water stress on the seedlings growth and physiology.cocoa seedlings used in this study was already met by thehigh P content of the soil and thus the seedlings were not Effectof Water Stress on Plant Growth and Physiologicalcolonized by mycorrhizal fungi. Parameters:The effect of water stress on seedling height

The organic matter content of the soil used in this became significant from Week 4 to the end of thestudy may have also influenced the absence of experiment - with the water-stressed seedlings showingmycorrhizal colonization. John Innes soil used for this reduced height compared to the regularly wateredstudy can be classified as an organic soil because it seedlings. At the end of the experiment, the difference in

contains more than 19% organic matter [21]. Since the height between the regularly watered and water-stressedorganic matter content of this soil is high, it can be seedlings was about 14%. This is similar with the works ofassumed that organic forms of essential nutrients such Mohd et al., [5], Zhang et al. [8] and Kirnaket al. [35]as N will also be high in this soil [28]. Studies have who recorded significant reduction in heights of water-shown that soil N can indirectly influence mycorrhizal stressed cocoa seedlings, eggplants and oriental lily,colonization. For instance, Sylvia and Neal [29] reported respectively.that under high soil N conditions, mycorrhizal Similarly, the effect of water stress was alsocolonization ofAllium cepawas limited by the addition of significant on new leaf appearance. However, the numbersoil P. However, when soil N was limiting, addition of P of leaves produced by water-stressed and regularlydid not hinder mycorrhizal colonization. Although it was watered treatments fluctuated in the course of thethought that mycorrhizal fungi - especially AM fungi - do experiment. While the regularly watered treatmentsnot utilize organic forms of soil nutrients [30], recent produced more leaves at certain periods, the water-

research has proved this wrong [31, 32]. Thus, it can be stressed treatments produced more leaves at otherdeduced that the high organic matter (high N content) of periods (Fig. 2). The reason for this inconsistency may bethe initial soil indirectly contributed to the absence of attributed to the watering regime. Hutcheon [36] reportedmycorrhizal colonization. that the end of a water stress period is followed by a

The application of a nutrient solution during the period of synchronized flush of leaf growth in cocoaseventh week of the experiment may have also affected seedlings. This period of new leaf growth is usuallymycorrhizal colonization. However, this was independent of the plants water status until the nextunavoidable since most of the seedlings were period of water stress [3, 37].chlorotic at this stage (there was also visible Although, the number of new leaves produced by theimprovement in the seedlings after application). treatments did not follow a consistent pattern on a weeklyFurthermore, the results of the experiment show that the basis, the total number of new leaves produced at the endeffect of mycorrhizae was not significant even before the of the experiment show that the regularly wateredsolution was applied, thus the effect of applying the treatments produced more leaves than the water-stressedsolution may be negligible compared to the nutrient treatment even though this was not significant. The totalcontent of the initial soil. leaf area of the water-stressed treatments was also lower

Another factor that may have affected mycorrhizal than that of the regularly watered treatments. The reducedcolonization of cocoa roots is specificity of the fungi leaf area of the water-stressed treatments is a commonspecies used. Few species of AM fungi are specific to drought avoidance mechanism exhibited by water-host plants, though these species are rare and their stressed plants to enable them maintain high tissue waterspecificity largely depends on the ecology of the plant potential during water stress [6, 7]. The absence of athey colonize [33]. The extensive root structure of cocoa significant difference between the water-stressed andseedlings may have also contributed to the lack of regularly watered treatments (in terms of total leaf area)


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may have been because the duration of the experiment Week 8 since afternoon temperatures are usually higherwas not long enough for the treatment effect to become than morning temperatures. Furthermore, relative humiditysignificant. is usually lower in the afternoon which results in partial

Effect of water-stress on leaf chlorophyll content was stomatal closure and hence reduced photosynthesis [3].

significant from Week 6 to Week 10. However, unlike in This is similar to the work by Rajendrudu and Naidu [46]most studies where water-stressed plants showed lower who observed that g , E and P of water-stressed Tectonachlorophyll content compared to regularly watered plants grandiswere reduced during afternoon period.[8, 35, 38], the water-stressed cocoa seedlings showed Water stress reduced seedlings shoot and totalsignificantly higher chlorophyll content compared to the biomass but increased their root biomass; this resulted toregularly watered treatments. The reason for the higher a lower shoot to root ratio in the stressed seedlings.chlorophyll content of the water-stressed plants may have These results correspond to that of similar studies [5, 35].been because of their higher N content (though this was The increased root biomass of the water-stressednot assessed). Sanchez et al. [38] reported that the N seedlings may be attributed to the extended root systemscontent of water-stressed maize plants was not affected typical of water-stressed plants due to their foraging forby the water stress condition. The marked decline in leaf water [6].chlorophyll content for all treatments during Week 6 was

due to leaf chlorosis which occurred during that period. CONCLUSIONSHowever, with the application of cocoa nutrient solution Results of this study show that water stress reducedin Week 7, chlorophyll content of the treatments the growth of cocoa seedlings mainly as a result ofincreased significantly (Fig. 3). changes in physiological processes i.e. reduction in

Results of photosynthetic activities show that g , E photosynthetic activities in the stressed seedlings.sand P of the water-stressed seedlings were lower than Mycorrhizae did not reduce the impact of water stress onNthat of the regularly watered seedlings during Week 4, cocoa seedlings. This was due to the absence of rooteven though significant effect was observed only on P . colonization by mycorrhizal fungi. This highlights the factNThis corresponds to results of most studies where that several factors may influence mycorrhizalphotosynthetic activities in various water-stressed plants colonization even after inoculation. The high P content ofwere monitored [5, 8, 35, 38]. The reduction in the soil may have been the main reason for this lack ofphotosynthetic activities in the water-stressed treatments colonization. High nutrient content (especially N) of the

may be attributed to partial stomatal closure which soil evident from its high organic matter content may havereduces photosynthesis in water-stressed plants [1, 3]. also prevented colonization. Specificity of mycorrhizalAlthough water stress is known to increase WUE fungi and the extensive root system of cocoa seedlingsinst

in plants [1, 39], results of this study show that WUE of are other factors (though negligible) that may have alsoinstthe water-stressed seedlings was lower than that of the prevented colonization. Based on the above, in order forregularly watered seedlings during Week 4 (though the definite conclusions to be drawn as to whether thereverse occurred during Week 8). The reason for this presence of mycorrhizae influences the response of cocoalower WUE in the stressed seedlings may have been seedlings to water stress, it is recommended that futureinstbecause WUE is dependent on the severity of water work utilises a less fertile soil or a soil with low toinststress. Under severe water stress conditions, reduced moderate P content. It is also important to identify theWUE was recorded in wheat [40, 41]; while, under exact species of mycorrhizal fungi in the inoculum so as toinstmoderate water stress condition, increases in WUE was establish that they actually colonize cocoa plant.instrecorded in the same plant [42, 43]. This is because, atsevere water stress conditions, photosynthesis may be ACKNOWLEDGEMENThindered by non-stomatal factors [44].

There was a general decline in photosynthetic This research was an integral part of a Mastersactivities (except for WUE ) during Week 8 compared to degree programme funded by the Felix Trust. Dr. G.P.instWeek 4 especially for transpiration rate. Sharkey [45] Warren and Dr. E.J. Shaw, both of Soil Research Centre,reported that moderate heat stress resulting from elevated Department of Geography and Environmental Science,temperatures reduces photosynthesis. Thus, difference The University of Reading, assisted the authors duringbetween the two intervals may be due to the different mycorrhizal analysis. This assistance is gratefullymeasurement time morning for Week 4 and afternoon for acknowledged.

s N


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