Embed Size (px)
Citation preview
This article was downloaded by: [The Aga Khan University]On: 09 October 2014, At: 15:56Publisher: Taylor & FrancisInforma Ltd Registered in England and Wales Registered Number: 1072954 Registered office: MortimerHouse, 37-41 Mortimer Street, London W1T 3JH, UK
Acta Agriculturae Scandinavica, Section B — Soil &Plant SciencePublication details, including instructions for authors and subscription information:http://www.tandfonline.com/loi/sagb20
Arbuscular mycorrhiza has limited effects onyield and quality of tomatoes grown under soillesscultivationMartin Makgose Maboko a b , Isa Bertling b & Christian Phillipus Du Plooy aa Agricultural Research Council – Vegetable and Ornamental Research Institute ,Pretoria , South Africab Horticultural Science , University of KwaZulu-Natal , Pietermaritzburg , South AfricaAccepted author version posted online: 21 Feb 2013.Published online: 11 Apr 2013.
To cite this article: Martin Makgose Maboko , Isa Bertling & Christian Phillipus Du Plooy (2013) Arbuscular mycorrhiza haslimited effects on yield and quality of tomatoes grown under soilless cultivation, Acta Agriculturae Scandinavica, SectionB — Soil & Plant Science, 63:3, 261-270, DOI: 10.1080/09064710.2012.755219
To link to this article: http://dx.doi.org/10.1080/09064710.2012.755219
PLEASE SCROLL DOWN FOR ARTICLE
Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) containedin the publications on our platform. However, Taylor & Francis, our agents, and our licensors make norepresentations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose ofthe Content. Any opinions and views expressed in this publication are the opinions and views of the authors,and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be reliedupon and should be independently verified with primary sources of information. Taylor and Francis shallnot be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and otherliabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to orarising out of the use of the Content.
This article may be used for research, teaching, and private study purposes. Any substantial or systematicreproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in anyform to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://www.tandfonline.com/page/terms-and-conditions
ORIGINAL ARTICLE
Arbuscular mycorrhiza has limited effects on yield and quality oftomatoes grown under soilless cultivation
MARTIN MAKGOSE MABOKO1,2, ISA BERTLING2, & CHRISTIAN PHILLIPUS DU
PLOOY1
1Agricultural Research Council � Vegetable and Ornamental Research Institute, Pretoria, South Africa and 2Horticultural
Science, University of KwaZulu-Natal, Pietermaritzburg, South Africa
AbstractA study was conducted investigating the possible utilization of mycorrhiza to enhance yield and quality of tomatoes grown ina soilless culture system using sawdust or coir as growing media. The experiment was carried out in temperature-controlledand non-temperature-controlled (NTC) tunnels. Fertigation was applied at three levels (100%, 75%, and 50%) of therecommended nutrient concentration. MycorootTM, containing four arbuscular mycorrhizal species (Glomus etunicatum,Paraglomus occultum, Glomus clarum, and Glomus mosseae), was applied at seeding, as well as transplanting. Growingtomatoes under reduced nutrient supply reduced the total soluble solids in the juice of the fruits, but improved total andmarketable yield, as well as the number of marketable fruits. This effect was more substantial in the temperature-controlledtunnel than in the NTC tunnel. Fruit firmness and leaf chlorophyll concentrations were significantly higher in plants grownin the temperature-controlled tunnel. Growing tomatoes in sawdust improved the leaf Mn and Ca concentration over that oftomato plants grown in coir. Mycorrhiza colonization did not have a beneficial effect on tomato yield and quality. Furtherstudies, including different media, nutrient composition, and concentration need to be carried out to investigate the possibleeffect of AMF failing to improve yield, despite AMF root colonization, and to reveal the cause of the poor beneficial effect ofAMF on tomato plants grown under soilless culture.
Keywords: Chlorophyll, coir, fertigation, fruit firmness, sawdust, temperature, yield.
Introduction
Arbuscular mycorrhizal fungi (AMF) are able to
form mutualistic relationships with 80% of all
terrestrial plants, including most agricultural, parti-
cularly horticultural crops, as well as certain forestry
species (Pozo & Azcon-Aguilar 2007). Such interac-
tions result in the transfer of carbon (sugars) from
the host plant to the fungi while the fungi improve
the uptake of water and nutrients by the root system
(Tahat et al. 2008). The intra-radical colonization of
plant roots by AMF results in the formation of
arbuscules, specialized fungal structures (for the
exchange of nutrients with the host plants), and
vesicles (storage organelles), which can significantly
enhance the absorbing capacity of the root for water
and nutrients (Kaya et al. 2003). Many improve-
ments can be achieved by this AMF�host plant
interaction, such as better plant establishment and
growth, enhanced water and nutrient uptake, and
improved resistance to biotic and abiotic stresses
(Davies et al. 1992; Smith & Read 1997; Muok &
Ishii 2006; Sawers et al. 2008), ultimately leading to
increased growth and yield. The resultant improved
productivity of AMF-inoculated plants has been
attributed to enhanced acquisition of nutrients of
low mobility, such as P, Zn, and Cu (Lambert et al.
1979; Ortas et al. 1996; Liu et al. 2002; Kaya et al.
2003; Ryan & Angus 2003). The transport and
absorption of such nutrients in soils low in P, Ca,
and Mg (Liu et al. 2002) result in increased root and
shoot biomass, as well as enhanced yield.
Previous studies have shown that the mycorrhizal
colonization of tomato plants had beneficial effects
on plant growth (Al-Karaki et al. 2001) and can
Correspondence: M. M. Maboko, Agricultural Research Council �Vegetable and Ornamental Research Institute, Private Bag X 293, Pretoria 0001, South
Africa. E-mail: [email protected]
Acta Agriculturae Scandinavica Section B � Soil and Plant Science, 2013
Vol. 63, No. 3, 261�270, http://dx.doi.org/10.1080/09064710.2012.755219
(Received 5 September 2012; revised 27 November 2012; accepted 28 November 2012)
# 2013 Taylor & Francis
Dow
nloa
ded
by [
The
Aga
Kha
n U
nive
rsity
] at
15:
56 0
9 O
ctob
er 2
014
cause yield increases in field-grown crops. Host plants
benefit from AMF through the enhanced production
of growth-regulating substances, increased photo-
synthesis, and improved osmotic adjustment under
water and salinity stress (Al-Karaki 2006). However,
Mueller et al. (2009) did not observe beneficial
effects of AMF on growth and nutrient uptake when
plants were grown in peat or sand.
Plant cultivation under soilless conditions sup-
presses the AMF�plant root association (Linderman
& Davis 2003), possibly due to high rates of
fertilizers applied to plants grown under this system.
Mycorrhiza colonization is also influenced by the
growing medium (Corkidi et al. 2004); certain
soilless media, like redwood shavings, and certain
barks contain high concentrations of phenols which
have an inhibitory effect on mycorrhizal colonization
(Biermann & Linderman 1983; Graham & Timmer
1984; Johnson & Hummel 1986). Mycorrhiza for-
mation has been successful when soil was added to
the soilless medium (Linderman & Davis 2003).
Alternatively, the use of slow release fertilizers (Colt-
man et al. 1988) or reduced phosphorus fertilization
(Caron & Parent 1988, Peters & Habte 2001) also
allows the establishment of AMF in the medium.
The beneficial effects of AMF on crops grown in soil
have been reported; however, there is limited re-
search on the benefits of mycorrhiza for plants grown
under soilless conditions (Al-Karaki 2006, Dasgan et
al. 2008, Abak et al. 2010).
In southern Africa, as well as in many other
countries in tropical/subtropical climates the poten-
tial for high productivity of tomatoes exists, due to the
high solar radiation received. However, constraints
such as excessive heat, especially during the summer
season, can reduce the productivity. Under such
conditions, farmers in South Africa tend to produce
tomatoes under protection in greenhouses or tunnels
that rely on natural ventilation (Maboko et al. 2012).
However, during the hot summer season in non-
temperature-controlled (NTC) tunnels, there is a
tendency towards poor plant growth, low yield, and
poor quality (Maboko et al. 2012).
Due to the beneficial effects of mycorrhiza on plant
growth, particularly under environmental stress, this
study was carried out to investigate the effects of
mycorrhiza, growing media, and strength of supplied
nutrient concentration on tomatoes grown in tem-
perature-controlled (TC) and NTC tunnels.
Materials and methods
Application of treatments
Experiments were conducted in NTC and TC
tunnels at the Agricultural Research Council-Roo-
deplaat Vegetable and Ornamental Plant Institute
(ARC-Roodeplaat VOPI), Roodeplaat, South Africa
(25859?S; 28835?E, altitude 1200 m a.s.l.) from
October 2010 to February 2011.
Five-week-old fresh-market tomato seedlings (cul-
tivar ‘FA593,’ Sakata seed, Southern Africa, Pty.
Ltd) were transplanted into 10 L plastic bags
containing sawdust or coir as a growing medium.
The growing media were washed thoroughly with tap
water (three times) before filling the bags and
moistened again before the transplanting of seed-
lings. Arbuscular mycorrhiza fungi were applied at
seeding and transplanting, as early inoculation
results in the rapid spread of mycorrhiza to new
roots during germination (Ikiz et al. 2009), con-
tributing to higher yields following transplantation to
the field (Stewart et al. 2005, Douds et al. 2008).
MycorootTM, containing four arbuscular mycorrhiza
species (Glomus etunicatum, Paraglomus occultum,
Glomus clarum, and Glomus mossea), was applied at
seeding and transplanting. MycorootTM was applied
at a rate of 1 g L�1 Hygromix† (commercial
seedling growth medium, Hygrotech) and it was
thoroughly mixed before seeding. One teaspoon (7
g) of MycorootTM granules (1 g MycorootTM contains
approximately 100 propagules, with a minimum of
10 spores per gram) was applied to the planting holes
at the time of transplanting. The root system of the
seedling was placed on top of the MycorootTM
granules and covered with growing medium.
The composition and chemical concentration of
fertilizers used for tomato production were: Hygro-
ponic† (comprising N (68 mg/kg), P (42 mg/kg), K
(208 mg/kg), Mg (30 mg/kg), S (64 mg/kg), Fe
(1.254 mg/kg), Cu (0.022 mg/kg), Zn (0.149 mg/kg),
Mn (0.299 mg/kg), B (0.373 mg/kg), and Mo (0.037
mg/kg)); calcium nitrate [Ca(NO3)2] (comprising N
(117 mg/kg) and Ca (166 mg/kg)); and potassium
nitrate (KNO3) (comprising K (38.6 mg/kg) and N
(13.8 mg/kg)). Plants were also subjected to three
fertigation treatments, i.e., 100%, 75%, and 50% of
the recommended nutrient concentration (Table I).
The different fertilizer regimes were applied to
tomato plants grown in a TC tunnel equipped with
two fans and a pad (1.1 kW fans, 1300 mm diameter)
cooling system, and a NTC tunnel which relied on
natural ventilation by means of a flap and door
system that could be opened on each side. Tunnels
(10 m width�30 m length) were covered with a 200
mm light-diffusive plastic (Evadek green tint). The
floor of the plastic tunnel was covered with 200 mm
white plastic. Plants were planted at a density of 2.5
plants m�2, with eight data plants in each replicate
per treatment. The treatments consisted of three
fertigation, two growing media, and two mycorrhiza
treatments in each tunnel. Plants were irrigated, one
262 M. M. Maboko et al.
Dow
nloa
ded
by [
The
Aga
Kha
n U
nive
rsity
] at
15:
56 0
9 O
ctob
er 2
014
dripper per plant (discharge rate of 35 mL min�1) at
two hourly intervals, seven times a day. The irrigation
volume was gradually increased as the plants en-
larged to ensure that 10�15% of the applied water
leached out to reduce salt build-up in the growing
medium (total daily irrigation during the growing
season ranged from 735 to 2205 mL per plant
equivalent to three to nine minutes, respectively)
(Maboko et al. 2012). The physical properties of
sawdust and coir used in this study were % moisture,
pH, bulk density (g mL�1), water-holding capacity
(%), and air porosity (%) which was 8.39 and 13.30,
6.30 and 6.60, 0.059 and 0.0619, 51.5 and 71.7, and
24.69 and 6.93, respectively. The chemical proper-
ties of sawdust and coir (mg L�1) was, respectively,
NO3� (0.3 and 1.0), NO2� (0.2 and B0.6), Cl�(30.1 and 154.6), SO4
� (24.4 and 489.0), PO4�
(5.9 and 12.5), Na� (5.1 and 109.5), Ca2� (13.5
and 9.1), Mg2� (5.8 and 13.7), Fe� (1.60 and 1.45),
Mn2� (0.69 and 0.10), and Cu2� (0.13 and 0.05).
The electrical conductivity of sawdust and coir was
0.23 and 1.41 dS m�1, respectively.
The pH of the nutrient solution was maintained
within a range of 5.8�6.1. Maximum, minimum, and
mean monthly ambient temperatures for the experi-
mental sites during the experimental period were
recorded using data-loggers (Tinyview, Gemini data
loggers (UK) Ltd), which were placed at a height of 1.5
m and covered with a Stevenson-type screen ACS-500.
Plants were trained to a single stem by twisting
trellis twine around the main stem and fixing it to a
stay wire 2 m above the ground surface to support
the plant. Side branches were removed weekly to
maintain a single stem system. When the plants had
reached the horizontal wire at 2 m, the growing point
was removed to stop further plant growth.
Mycorrhiza colonization
At the end of the experiment, two plants per replicate
per treatment were used to determine the percentage
of AMF colonization. Roots were rinsed carefully
with tap water, with root clearing and staining
procedures performed according to Koske and
Gemma (1989). Colonization by AMF was examined
microscopically to determine the percentage of root
segments containing arbuscules and vesicles using the
gridline intercept method (Giovannetti & Mosse
1980).
Plant growth and fruit yield measurements
Fruit were harvested weekly at the breaker stage in
mid-summer from December to February. Yield data
were collected from six plants per treatment, and the
performance of the treatments evaluated using total
yield, marketable and unmarketable yield, as well as
physiological and pathological disorders as para-
meters. Fruit were regarded as unmarketable when
they exhibited cracking, zippering, rotting, blossom-
end rot, rain-check, cat-face, or fell into the extra
small size category (less than 40 mm fruit diameter)
(Maboko et al. 2011). Fruit firmness was measured
using an Effegi-type Bishop FT 327 firmness tester
with an 11.3 mm diameter plunger. Six ripe fruits of
larger size (60�70 mm diameter) per treatment and
replicate were collected, and readings were taken at
four areas in the equatorial region of the fruit. The
percentage of total soluble solids (oBrix) and the
electrical conductivity (EC) of the tomato juice were
determined in five fruit per replicate and treatment
obtained from the fifth truss. Fruit were placed in a
blender and the resultant puree filtered through
cheese cloth, to determine oBrix and EC of the
tomato juice using a pocket refractometer PAL-1
(ATAGO†) and an EC meter, respectively.
Leaf analysis
The leaf chlorophyll concentration was measured at
the leaf tip of the fourth leaf from the growing point.
Four plants were selected per replicate per treatment
to determine the leaf N, P, K, Ca, Mg, Mn, Zn, and
B concentrations in the fourth leaf from the growing
point. Tomato leaves were oven-dried at 708C for
48 h and, subsequently, ground using a mill with a
1 mm sieve. Nitrogen was determined on dry-milled
material using a Carlo Erba NA 1500 C/N/S
Table I. Amount of fertilizer applied (g L�1) for fertigation treatments at different growth stages of the tomato plant.
Application time
Fertilizer
(g L�1)
100% full nutrient
concentration
75% nutrient
concentration
50% nutrient
concentration
Transplanting to first flower
truss
Hygroponic 1.0 0.75 0.5
Ca(NO3)2 0.8 0.6 0.4
1st flower truss to 3rd flower
truss
Hygroponic
Ca(NO3)2
1.2
0.5
0.9
0.375
0.6
0.25
3rd flower truss to end Hygroponic
Ca(NO3)2
KNO3
1.2
0.8
0.3
0.9
0.6
0.225
0.6
0.4
0.15
Effect of growing media, nutrient and mycorrhiza 263
Dow
nloa
ded
by [
The
Aga
Kha
n U
nive
rsity
] at
15:
56 0
9 O
ctob
er 2
014
Analyzer. An aliquot of the digest solution was used
for the ICP-OES (Inductively Coupled Plasma
Optical Emission Spectrometry) for the determina-
tion of Ca, Mg, P, K, Zn, Mn, and B. All nutrient
analyses were expressed on a dry mass basis.
Statistical procedures
A complete randomized block design was used for
each of the two tunnel facilities (TC and NTC
tunnels). A 3�2�2 factorial design was used, with
three factors (fertigation treatment, i.e., 100%, 75%,
or 50% nutrient concentration, growing media, i.e.,
sawdust or coir, and mycorrhiza, i.e., AMF applied
or no AMF applied) randomly replicated within each
of the four block replicates.
The data obtained from the two tunnels were
tested for homogeneity of variances using Levene’s
test. In cases where the variability in the observations
of the two tunnel facilities were of comparable
magnitude, an analysis of the two tunnels observa-
tions together could be validly carried out. In cases
where there was strong evidence against homogene-
ity, a weighted analysis of the two tunnel facilities’
observations together was carried out using the
inverse of the pooled variances of each tunnel as
weight (John & Quenonille 1977). The Shapiro�Wilk test was performed to test for normality
(Shapiro & Wilk 1965). Student’s t-Least Significant
Differences were calculated at the 5% level to
compare treatment means of significant effects. All
data analyses were carried out using GenStat†
version 11.1 (Payne et al. 2008).
Results
In cases where there were no significant interaction
effects among the treatments, only the main factors
were discussed.
Percentage of AMF root colonization
There was no significant interaction between nutri-
ent concentration, growing medium, and tunnel
facilities on root colonization (Table II). There was
a tendency towards increased root colonization in
the TC tunnel, as compared with the NTC tunnel,
although not significant. Similarly, neither the grow-
ing medium nor the nutrient concentration had a
significant effect on root colonization.
Effects of growing medium
Analysis of physical and chemical properties of
sawdust and coir indicated a better water-holding
capacity and higher salt concentration of coir com-
pared with sawdust. The colonization of tomato
roots by AMF was not affected by the growing
medium; in both media, three-quarters of the roots
were colonized by AMF (Table II). The chlorophyll
concentration of leaves increased in plants grown in
sawdust as compared with coir, although only
significantly 84 DAT (Table III). Plants grown in
coir produced firmer fruit than those grown in
sawdust (Table III). There was a tendency towards
an increase in marketable yield, number of market-
able fruit, and total yield of plants grown in coir
compared with sawdust (Table IV). Neither 8Brix
nor EC of the tomato juice were significantly
influenced by the growing medium (Table IV).
Sawdust improved the Mn and Ca leaf concentration
compared with coir (Table V). Other elements
were not significantly affected by the growing
medium, although Zn and B concentrations showed
a tendency towards an increase with sawdust as
medium.
Effects of mycorrhiza
Mycorrhiza treatment did neither affect leaf chlor-
ophyll concentration (Table III), nor yield, nor
quality of hydroponically grown tomatoes signifi-
cantly (Table IV); neither was the concentration of
selected mineral nutrients in leaf tissues altered by
the AMF inoculation (Table VI). Moreover, neither
8Brix nor EC of tomato juice were significantly
affected by AMF (Table IV); however, there was a
tendency towards higher leaf chlorophyll and leaf
mineral concentrations in plants inoculated with
AMF compared with non-inoculated plants.
Table II. Effect of tunnel facility, nutrient concentration, and
growing medium on AMF root colonization in tomatoes.
Treatment % AMF root colonization
Tunnel facility
NTC 76.9
TC 79.0
LSD0.05 ns
Nutrient concentration (%)
100 75.6
75 77.1
50 81.1
LSD0.05 ns
Growing medium
Coir 78.2
Sawdust 77.7
LSD0.05 ns
NTC, non-temperature-controlled tunnel; TC, temperature-
controlled tunnel; AMF, arbuscular mycorrhiza fungi; ns, non-
significant difference; LSD, least significant difference.
264 M. M. Maboko et al.
Dow
nloa
ded
by [
The
Aga
Kha
n U
nive
rsity
] at
15:
56 0
9 O
ctob
er 2
014
Effects of nutrient concentration
The nutrient concentration did not have a significant
influence on AMF root colonization; however, there
was a tendency towards a decrease in AMF root
colonization with an increase in nutrient concentra-
tion (Table II). The leaf chlorophyll concentration
was highest 70 DAT in plants fertigated at 100%,
compared with fertigation at 50% and 75% of the
recommended nutrient concentration (Table III).
Fruit firmness was not significantly affected by
nutrient concentration (Table III). Plants fertigated
with the recommended nutrient concentration had a
significantly higher N, K, and Mn leaf concentration
than plants fertigated with 50% or 75% of the
recommended nutrient solution (Table VI).
Effects of tunnel facility
The tunnel facilities, i.e., TC and NTC tunnel, did
not influence AMF root colonization significantly
(Table II). Temperature differences were observed
between the two tunnel facilities, with higher max-
imum temperatures in the NTC tunnel than the TC
tunnel (Table VII). Plants in the TC tunnel con-
tained higher leaf chlorophyll concentrations, as well
as firmer fruit than plants grown in the NTC tunnel
(Table III). However, in the NTC tunnel, the tomato
leaves had higher Mg, Ca, and Zn concentration
than plants grown in the TC tunnel (Table VI).
Interaction effects of tunnel facilities and fertigation
Independent of fertigation level, leaf chlorophyll
concentrations were highest 70 DAT in the TC
tunnel, followed by the 100% nutrient concentration
in the NTC tunnel (Figure 1). Tomato leaves of
plants in the TC tunnel had a higher chlorophyll
concentration than those in the NTC tunnel. Sur-
prisingly, the chlorophyll concentration was not
affected by the applied nutrient concentration in
the TC tunnel, while in the NTC tunnel, leaf
chlorophyll concentration increased when plants
were supplied with 100% compared with 50% or
75% of the recommended nutrient concentration.
The highest total yield, marketable yield, and
number of marketable fruits were obtained from
plants fertigated at 50% and 75% of the recom-
mended nutrient concentration in the TC tunnel
(Table VIII). Unmarketable yield was significantly
lower at all fertigation treatments in the TC tunnel,
Table III. Effects of tunnel facility, nutrient concentration,
growing medium, and arbuscular mycorrhiza on tomato leaf
chlorophyll concentration (SPAD) of the fourth youngest, fully
developed leaf, and fruit firmness.
56 DAT 70 DAT 84 DAT Fruit firmness (N)
Tunnel facility
NTC 44.2b 42.1b 37.8b 12.40b
TC 58.3a 62.9a 61.4a 15.05a
LSD0.05 5.4 6.8 5.8 0.67
Nutrient concentration (%)
50 50.4 58.8a 53.8 13.73
75 50.9 47.9b 45.9 13.43
100 52.3 50.7b 49.1 14.02
LSD0.05 ns 3.9 ns ns
Growing medium
Sawdust 52.1 54.1 52.3a 13.51b
Coir 50.4 50.8 46.9b 13.94a
LSD0.05 ns ns 3.7 0.41
Mycorrhiza
�AMF 51.6 53.4 50.7 13.90
�AMF 50.8 51.6 48.5 13.55
LSD0.05 ns ns ns ns
Note: Figures within a column followed by the same letter are not
significantly different (p �0.05) from another, using Fishers’
protected t-test. DAT, days after transplanting; NTC, non-
temperature-controlled tunnel; TC, temperature-controlled
tunnel; �AMF, plants inoculated with arbuscular mycorrhiza;
�AMF, plants without arbuscular mycorrhiza inoculation; ns,
non-significant difference; LSD, least significant difference.
Table IV. Effects of growing medium and mycorrhiza on tomato yield and quality.
Treatment
Marketable yield
g plant�1
Number of marketable
fruit plant�1
Unmarketable yield
(g plant�1)
Total yield
(g plant�1) 8Brix
EC
(dS.m�1)
Growing media
Sawdust 4037 33.46 847 4883 4.50 3.94
Coir 4182 34.06 873 5067 4.56 3.95
LSD0.05 Ns ns ns ns ns ns
Mycorrhiza
�AMF 4113 33.57 852 4965 4.55 3.98
�AMF 4106 33.95 868 4985 4.51 3.90
LSD0.05 ns ns ns ns ns ns
Note: Figures in a column followed by the same letter are not significantly different (p �0.05), using Fishers’ protected t-test.
ns, non-significant difference; LSD, least significant difference; �AMF, plants inoculated with arbuscular mycorrhiza fungi; �AMF, plants
without arbuscular mycorrhiza fungi inoculation; EC, electrical conductivity.
Effect of growing media, nutrient and mycorrhiza 265
Dow
nloa
ded
by [
The
Aga
Kha
n U
nive
rsity
] at
15:
56 0
9 O
ctob
er 2
014
whereas plants in the NTC tunnel, fertigated at
50% and 75% of the recommended nutrient con-
centration, had the highest unmarketable yield
(Table VIII).
Plants fertigated at 100% of the recommended
nutrient concentration in the TC tunnel, as well as
plants fertigated at 75% and 100% of the recom-
mended nutrient concentration in the NTC tunnel,
had the highest oBrix, compared with other treat-
ments (Table VIII)); NTC fruit showed a tendency
toward higher oBrix than TC ones.
The Ca leaf concentration was significantly higher
in leaves of tomato plants fertigated at 50% and 75%
of the recommended nutrient concentration and
grown in the NTC tunnel than leaves from all plants
in the TC tunnel (Figure 2). The 75% and 100%
fertigation treatments resulted in the highest Ca leaf
concentration in the NTC and TC tunnel facilities,
respectively. The NTC tunnel also significantly
increased the Mg concentration when plants were
supplied with 50% and 75% of the recommended
nutrient concentration, while no such difference
was found when plants were supplied with the
recommended nutrient concentration of 100%
(Figure 3).
Interaction effects of growing media and mycorrhiza, and
growing media, fertigation, and tunnel facilities
Plants grown in sawdust and inoculated with AMF
showed a significant increase in leaf Ca concentra-
tion in contrast to plants grown in coir (Figure 4).
Generally, leaf chlorophyll concentration was sig-
nificantly higher in plants grown in sawdust and coir
at all nutrient concentrations in the TC tunnel,
compared with plants grown in the NTC tunnel,
with the lowest leaf chlorophyll concentration re-
corded for plants grown in sawdust, and fertigated at
50% and 75% of the recommended nutrient con-
centration (Figure 5).
Discussion
Organic growing media are commonly colonized by
fungi (Koohakan et al. 2004); however, in this
study, AMF colonization in coir and sawdust was at
78.2% and 77.7%, respectively. Despite this rela-
tively high AMF root colonization of tomato plants
(Table II) compared with reports by Abak et al.
(2010), Ikiz et al. (2009), Dasgan et al. (2008), and
Al-Karaki et al. (2001), a significant improvement
of tomato yield could not be detected (Table IV).
Table V. Effects of growing media on tomato leaf nutrient concentration (% DM basis).
Growth media K (%) Ca (%) Mg (%) Zn (mg kg�1) B (mg kg�1) Mn (mg kg�1) 8Brix EC (dS.m�1)
Sawdust 3.76 1.93a 0.47 35.07 49.9 99.4a 4.50 3.94
Coir 3.72 1.72b 0.50 31.97 44.9 46.8b 4.56 3.95
LSD0.05 Ns 0.18 ns ns ns 15.8 ns ns
Note: Figures in a column followed by the same letter are not significantly different (p �0.05), using Fisher’s protected t-test.
ns, non-significant difference; LSD, least significant difference.
Table VI. Effects of tunnel facilities, nutrient concentration, and mycorrhiza on nutrient concentration of tomato leaf tissues (% on dry
weight basis).
Treatment N (%) P (%) K (%) Ca (%) Mg (%) Zn (mg kg�1) B (mg kg�1) Mn (mg kg�1)
Tunnel facilities
NTC 3.76a 0.48 3.85 2.20a 0.57a 36.65 54.3 80.4
TC 3.21b 0.46 3.62 1.46b 0.40b 30.38 40.5 65.8
LSD0.05 0.44 ns ns 0.23 0.13 ns ns ns
Nutrient concentration/fertigation
100% 3.67a 0.46 4.12a 1.85 0.50 35.31 48.3 99.2a
75% 3.29b 0.50 3.66b 1.89 0.47 33.93 47.9 67.7b
50% 3.50a 0.46 3.44b 1.75 0.47 31.31 46.0 52.4b
LSD0.05 0.26 ns 0.43 ns ns ns ns 23.25
Mycorrhiza
�AMF 3.54 0.47 3.81 1.87 0.51 34.15 49.2 73.2
�AMF 3.44 0.47 3.67 1.78 0.46 32.89 45.6 73.0
LSD0.05 ns ns ns ns ns ns ns ns
Note: Figures in a column followed by the same letter are not significantly different (p �0.05), using Fisher’s protected t-test.
TC, temperature-controlled tunnel; NTC, non-temperature-controlled tunnel; �AMF, plants inoculated with arbuscular mycorrhiza;
�AMF, plants without arbuscular mycorrhiza inoculation; ns, non-significant difference; LSD, least significant difference.
266 M. M. Maboko et al.
Dow
nloa
ded
by [
The
Aga
Kha
n U
nive
rsity
] at
15:
56 0
9 O
ctob
er 2
014
Previous investigations in soilless growing medium
(perlite) on various plant species, such as muskmelon
(Abak et al. 2010) and pepper (Ikiz et al. 2009), have
shown that AMF colonization can increase plant
growth and yield; however, in our study, mycorrhiza
colonization did neither have a significant influence
on tomato yield nor quality. This effect might be
aligned to the choice of growing medium as organic
growing media (sawdust and coir) might release
phenolics, lignin, and other organic compounds,
thereby reducing mycorrhizal development that could
have otherwise improved yield and quality of toma-
toes under soilless conditions.
The higher water-holding capacity of coir com-
pared with sawdust might have contributed to firmer
fruit and the tendency towards increased tomato
yield in the former medium by allowing uninter-
rupted water uptake resulting in more turgid cells
(Jones & Corlett 1992). The reduced Mn and Ca
concentrations of leaf tissues of plants grown in coir
(Table V) might be explained by the high Na, S, K,
and Cl concentrations of the medium, possibly
suppressing the uptake of other nutrients, particu-
larly Ca through cationic competition (Table V).
This finding supports observations by Adams and
Ho (1995) that high soil salinity can result in
reduced Ca uptake into tomato fruit. Inoculation
with AMF only increased the leaf Ca concentration
of plants grown in sawdust, but not of plants grown
in coir (Table V). Several reports indicate that in
soils with low mineral content, AMF colonization
improves acquisition of low mobile nutrients, such as
P, Zn, and Cu (Lambert et al. 1979; Ortas et al.
1996; Liu et al. 2002; Kaya et al. 2003; Ryan &
Angus 2003), as well as improving transport and
absorption of P, Ca, and Mg (Liu et al. 2002).
Our study also found a tendency towards higher
leaf mineral concentrations in AMF-inoculated
plants, compared with non-AMF-inoculated plants;
Figure 1. Interaction effects of tunnel facilities and fertigation on
tomato leaf chlorophyll concentration at 70 days after transplanting.
NTC, non-temperature-controlled tunnel; TC, temperature-
controlled tunnel; 50%, 75%, and 100%, percentage of nutrient
concentration; LSD, least significant difference; values marked with
the same letter are not significantly different (p �0.05).
Table VII. Maximum, minimum, and mean monthly ambient temperature for the experimental sites during the experimental period.
Non-temperature-controlled tunnel (NTC) Temperature-controlled tunnel (TC)
Month Max Min Average Max Min Average
October 51.9 15.8 29.0 39.1 13.5 23.4
November 51.9 13.8 30.8 39.0 11.0 23.9
December 51.9 16.3 29.9 36.6 13.8 22.9
January 50.5 17.2 27.9 32.7 15.5 21.4
February 48.5 16.3 26.4 32.3 14.1 20.5
March 45.4 14.33 24.7 32.7 11.6 19.5
Max, maximum air temperature; Min, minimum air temperature.
Table VIII. Interaction effects of tunnel facility and nutrient concentration on tomato yield and quality.
Treatment (%)
Marketable yield
(g plant �1)
Number of marketable
fruit plant�1
Unmarketable yield
(g plant�1)
Total yield
(g plant�1) 8Brix
EC
(dS.m�1)
TCx100 3951b 35.3b 694c 4644b 4.9a 4.0ab
TCx75 5252a 40.0a 539cd 5825a 4.2c 3.9ab
TCx50 5447a 41.4a 497d 5944a 4.2c 3.9ab
NTCx100 3275cd 28.8cd 1009b 4284b 4.8a 4.2a
NTCx75 3593c 30.5c 1171ab 4764b 4.7ab 3.8b
NTCx50 3140d 26.7d 1249a 4389b 4.5b 3.9ab
LSD0.05 347.1 2.9 178.4 427 0.25 0.3
Note: Figures in a column followed by the same letter are not significantly different (p �0.05), using Fishers’ protected t-test.
NTC, non-temperature-controlled tunnel; TC, temperature-controlled tunnel; 50%, 75%, and 100%, percentage of nutrient
concentration; EC, electrical conductivity; LSD, least significant difference.
Effect of growing media, nutrient and mycorrhiza 267
Dow
nloa
ded
by [
The
Aga
Kha
n U
nive
rsity
] at
15:
56 0
9 O
ctob
er 2
014
however, AMF did not enhance the uptake of any of
the analyzed nutrients into leaf tissue, except for
higher Ca, resulting in a higher Ca leaf concentration
of tomato plants grown in sawdust (Figure 4).
Surprisingly, fertilizers with the recommended
nutrient concentration (100%) resulted in a lower
total and marketable yield than using the reduced
nutrient concentrations (50% and 75% of recom-
mended fertilizer amount); this indicates that either
the recommended nutrient concentration is exces-
sive or the EC of the nutrient solution was too high
for optimal root development. In future studies, the
EC of the 10% drainage water should be measured
to confirm such assumption. The colonization of
tomato roots with AMF was effective; however, the
rate was not significantly affected by the nutrient
concentration, indicating that inoculating tomato
plants with AMF cannot be used as a tool to reduce
the quantity of fertilizer application. The highest
marketable and total yield was obtained in plants
fertigated with 50% or 75% of the recommended
nutrient concentration; however, the oBrix of the
tomato juice was significantly higher in fruit from
plants cultivated at 100% of the recommended
nutrient concentration. Seemingly, the higher TSS
of fruit from plants grown in the recommended
nutrient concentration compromised yield, an in-
dication that the recommended nutrient concentra-
tion is not optimal. The high nutrient concentration
was also reported by another author to reduce yield
(Adams 1991). The 100% nutrient concentration
might have restricted water transportation to fruits
and thus increased oBrix (Adams 1991; Cornish
1992; Lin & Glass 1999).
Growing conditions in the TC tunnel resulted in a
higher leaf chlorophyll concentration of tomato
plants, compared with those in the NTC tunnel.
The higher chlorophyll concentration in plants
grown in the TC tunnel (Figure 1) indicates that
plants were able to photosynthesize more effectively
than those in the NTC tunnel, and, thereby, supply
assimilates for fruit development and plant growth
contributing to the higher total and marketable yield
obtained in the TC tunnel.
The reduced fruit firmness and the lower chlor-
ophyll concentration in the NTC tunnel could be
due to extremely high temperatures (45.4�51.98C)
in the NTC tunnel, compared with the TC tunnel
(32.7�39.18C) (Table II). Such reduction in fruit
firmness and leaf chlorophyll concentration under-
line the importance of the high maintenance re-
quired in protected cultivation when aiming at
producing high-quality fruit, as the high tempera-
tures in the TC tunnel were due to the unusual
failure of electricity and malfunctioning of the wet
walls. Poor fruit firmness could also be the conse-
quence of the processes that involve biochemical
changes in cell wall structure, resulting in flesh
softness due to high temperature. Such effects
caused by high air temperatures have been reported
as mainly associated with the reduction of the
photosynthetic activity (Ciu et al. 2006).
The poor yield in the NTC tunnel could be
explained by the prevailing high air temperature,
which can increase unmarketable yield and reduce
Figure 2. Interaction effects of fertigation and tunnel facility on
Ca concentration of tomato leaf tissues.
NTC, non-temperature-controlled tunnel; TC, temperature-
controlled tunnel; 50%, 75%, and 100%, percentage of nutrient
concentration; LSD, least significant difference; values marked
with the same letter are not significantly different (p �0.05).
Figure 3. Interaction effects of fertigation and tunnel facility on
Mg concentration of tomato leaf tissues.
NTC, non-temperature-controlled tunnel; TC, temperature-
controlled tunnel; 50%, 75%, and 100%, percentage of nutrient
concentration; LSD, least significant difference; values marked
with the same letter are not significantly different (p�0.05).
Figure 4. Interaction effects of growing medium and mycorrhiza
on leaf Ca concentration of tomatoes.
AMF plants, plants inoculated with arbuscular mycorrhiza; Non-
AMF plants, plants without arbuscular mycorrhiza inoculation.
LSD, least significant difference; values marked with the same
letter are not significantly different (p�0.05).
268 M. M. Maboko et al.
Dow
nloa
ded
by [
The
Aga
Kha
n U
nive
rsity
] at
15:
56 0
9 O
ctob
er 2
014
fruit set (Maboko et al. 2012). This is in accordance
with Peet et al. (1997) who reported decreased fruit
number and fruit weight per plant as well as a
decrease in seed number per tomato fruit at an air
temperature of 298C, compared to an air tempera-
ture of 258C. In tomatoes, temperatures above 258Ccause nonlinear yield reductions (Peet et al. 1997).
Similarly, Sato et al. (2000) and Saeed et al. (2007)
reported that impairment of pollen and anther
development by high temperatures contribute to a
poor fruit set in tomatoes, while high ambient air
temperature in NTC tunnels seems to have no
significant influence on AMF root colonization.
AMF root colonization is reported to improve plant
resistance to biotic and abiotic stresses. Zhu et al.
(2010) reported that inoculation of maize roots with
AMF protects plants against high temperature stress
(408C) by improving photosynthesis and plant water
status. It was, therefore, expected that mycorrhiza
colonization would improve yield in the NTC tunnel
due to avoiding heat stress and improving leaf
chlorophyll concentration, water status, and nutrient
uptake. Although there was high AMF colonization in
this study, this did not improve yield under NTC
conditions, possibly due to the high ambient tem-
perature or the choice of growing media not appro-
priate to AMF colonization. Relying on natural
ventilation to reduce the heat load inside the tunnel
was seemingly insufficient to gain benefits from AMF
inoculation; however, the leaf mineral concentration
in the NTC tunnel was higher than in the TC tunnel.
Data presented indicate that TC tunnels can
significantly increase total as well as marketable yield
over that what can be achieved in NTC tunnels; AMF
root colonization was not able to establish significant
influence on tomato yield and quality. Further studies,
including different media, nutrient composition, and
concentration need to be carried out to investigate the
possible effects of AMF failing to improve yield,
despite successful AMF root colonization, and to
reveal the cause of the poor beneficial effects of AMF
on tomato plants grown under soilless conditions.
Acknowledgments
The authors acknowledge the financial support from
Agricultural Research Council-Vegetable and Orna-
mental Plant Institute (ARC-VOPI). The assistance
by Ms Maphefo Wendy Sekgota from the ARC-
PPRI in determining mycorrhiza colonization and
Ms Liesl Moorey from the ARC-Biometry Unit in
advising on methods of statistical analysis are grate-
fully acknowledged.
References
Abak K, Dasgan HY, Rehber Y, Ortas I 2010. Effect of vesicular
arbuscular mycorrhizas on plant growth of soilless grown
muskmelon. Acta Hortic. 871:301�306.
Adams P. 1991. Effects of increasing the salinity of the nutrient
concentration with major nutrients or sodium chloride on
the yield, quality and composition of tomato grown in
rockwool. J Hortic Sci. 66:201�207.
Adams P, Ho LC. 1995. Uptake and distribution of nutrients in
relation to tomato fruit quality. Acta Hortic. 412:374�385.
Al-Karaki GN. 2006. Nursery inoculation of tomato with
arbuscular mycorrhizal fungi and subsequent performance
under irrigation with saline water. Sci Hortic. 109:1�7.
Al-Karaki GN, Hammad R, Rusan M. 2001. Response of two
cultivars differeng in salt tolerance to inoculation with
mycorrhizal fungi under salt stress. Mycorrhiza. 11:43�47.
Biermann B, Linderman RG. 1983. Effect of container plant
growth medium and fertilizer phosphorus on establishment
and host growth response to vesicular- arbuscular mycor-
rhizal fungi. J Am Soc Hortic Sci. 108:962�971.
Caron M, Parent S. 1988. Definition of a peat-lite medium for the
use of vesicular arbuscular mycorrhizae in horticulture. Acta
Hortic. 221:289�294.
Figure 5. Interaction effects of growing media, fertigation and tunnel facility on leaf chlorophyll concentration of tomato at 84 days after
transplanting.
TC, temperature-controlled tunnel; NTC, non-temperature-controlled tunnel; Sa, sawdust; 50%, 75%, 100% percentage of nutrient
concentration; LSD, least significant difference; values marked with the same letter are not significantly different (p�0.05).
Effect of growing media, nutrient and mycorrhiza 269
Dow
nloa
ded
by [
The
Aga
Kha
n U
nive
rsity
] at
15:
56 0
9 O
ctob
er 2
014
Ciu L, Li J, Fan Y, Xu S, Zhang Z. 2006. High temperature
effects on photosynthesis, PII functionality and antioxidant
activity of two Festuca arundinacea cultivars with different
heat susceptibility. Bot Stud. 47:61�69.
Coltman RR, Waterer DR, Huang RS. 1988. A simple method for
production of Glomus aggregatum inoculum using controlled-
release fertilizer. Hort Science. 23:213�215.
Corkidi L, Allen EB, Merhaut D, Allen MF, Downer J, Bohn J,
Evans M. 2004. Assessing infectivity of commercial mycor-
rhiza inoculants in plant nursery conditions. J Environ
Hortic. 22:149�154.
Cornish PS. 1992. Use of high electrical conductivity of nutrients
solution to improve the quality of salad tomatoes grown in
hydroponic culture. Aust J Exp Agric. 32:513�520.
Dasgan HY, Kusvuran S, Ortas I. 2008. Response of soilless
growth tomato plant arbuscular mycorrhizal fungal (Glomus
faciculatum) colonization in recycling and open system. Afr J
Biotechnol. 7:3606�3613.
Davies FY, Potter Jr JR, Linderman RG. 1992. Mycorrhiza and
repeated drought exposure affect drought resistance and
extraradical hyphae development of pepper plants indepen-
dent of plant size and nutrient content. J Plant Physiol.
139:289�294.
Douds DD, Nagahashi G, Reider C, Hepperly RR. 2008.
Choosing a mixture ratio for the on-farm production of
AM fungus inoculum in mixtures of compost and vermicu-
lite. Compost Sci Util. 16:52�60.
Giovannetti M, Mosse B. 1980. An evaluation of techniques for
measuring vesicular arbuscular mycorrhizal infection in
roots. New Phytol. 84:489�500.
Graham JH, Timmer LW. 1984. Vesicular-arbuscular mycorrhizal
development and growth response of rough lemon in soil and
soilless media: effect of phosphorus source. J Am Soc Hortic
Sci. 109:118�121.
Ikiz O, Abak K, Dasgan HY, Ortas I. 2009. Effects of mycorrhizal
inoculation in soilless culture on pepper plant growth. Acta
Hortic. 807:533�540.
John JA, Quenouille MH. 1977. Experiments: design and
analysis. London and High Wycombe: Charles Griffin.
Chapter 13:232�248.
Johnson CR, Hummel RL. 1986. Influence of media on endo-
mycorrhizal infection and growth response of Severinia
buxifolia. Plant Soil. 93:35�42.
Jones HG, Corlett JE. 1992. Current topics in drought physiology.
J Agric Sci. 119:291�296.
Kaya C, Higgs D, Kirnak H, Tas I. 2003. Mycorrhizal coloniza-
tion improves fruit yield and water use efficiency in water-
melon (Citrullus lanatus Thunb.) grown under well- watered
and water-stressed conditions. Plant Soil. 253:287�292.
Koohakan P, Ikeda H, Jeanaksorn T, Tojo M, Kusakari SI, Okada
K, Sato S. 2004. Evaluation of the indigenous microorgan-
isms in soil-less culture: occurrence and quantitative char-
acteristics in the different growing systems. Sci Hortic.
101:179�188.
Koske RW, Gemma JN. 1989. A modified procedure for staining
roots to detect VA mycorrhizas. Mycol Res. 92:486�505.
Lambert DH, Baker DE, Cole H Jr. 1979. The role of mycor-
rhizae in the interactions of phosphorus with zinc, copper
and other elements. Soil Sci Soc Am J. 43:976�980.
Lin WC, Glass ADM. 1999. The effects of NaCl addition and
macronutrients concentration on fruit quality and flavor
volatiles of greenhouse tomatoes. Acta Hortic. 481:487�491.
Linderman RG, Davis EA. 2003. Soil amendment with different
peatmesses affects mycorrhizae of onion. HortTechnology.
1:285�289.
Liu A, Hamel C, Elmi A, Costa C, Ma B, Smith DL. 2002.
Concentrations of K, Ca and Mg in maize colonized by
arbuscular mycorrhizal fungi under field conditions. Can J
Soil Sci. 82:271�278.
Maboko MM, Du Plooy CP, Bertling I. 2011. Comparative
performance of tomato cultivars cultivated in two hydro-
ponic production systems. S Afr J Plant Soil. 28:97�102.
Maboko MM, Du Plooy CP, Bertling I. 2012. Comparative
performance of tomato cultivars in temperature and non
temperature controlled plastic tunnel. Acta Hortic.
927:405�411.
Mueller A, Franken P, Schwarz D. 2009. Nutrient uptake and
fruit quality of tomato colonized with Mmycorrhizal fungus
Glomus mosseae (BEG) under deficient supply of nitrogen
and phosphorus. Acta Hortic. 807:383�388.
Muok BO, Ishii T. 2006. Effect of arbuscular mycorrhizal fungi on
tree growth and nutrient uptake of Sclerocarya birrea under
water stress, salt stress and flooding. J Japan Soc Hort Sci.
75:26�31.
Ortas I, Harries PJ, Rowell DI. 1996. Enhanced uptake of
phosphorus by mycorrhizal sorghum plants as influenced
by form of nitrogen. Plant Soil. 184:255�264.
Payne RW, Murray DA, Harding SA, Baird DB, Soutar DM.
2008. GenStat for Windows† (11th Edition) Introduction.
Hemel Hempstead, UK: VSN International.
Peet MM, Willits DH, Gardner RG. 1997. Response of ovule
development and post-pollen production processes in male-
sterile tomatoes to chronic, sub-acute high temperature
stress. J Exp Bot. 48:101�111.
Peters SM, Habte M. 2001. Optimizing solution P concentration
in a peat-based medium for producing mycorrhizal seedlings
in containers. Arid Land Res Manag. 15:359�370.
Pozo MJ, Azcon-Aguilar C. 2007. Unraveling mycorrhiza-
induced resistance. Curr Opin Plant Biol. 10:393�398.
Ryan MH, Angus JF. 2003. Arbuscular mycorrhizae in wheat and
field pea crops on a low P soil: increased Zn uptake but no
increase in P-uptake or yield. Plant Soil. 250:225�239.
Saeed A, Hayat K, Khan AL, Iqbal S. 2007. Heat tolerance
studies in tomato (Lycopersicon esculentum Mill.). Int J Agric
Biol. 9:649�652.
Sato S, Peet MM, Thomas JF. 2000. Physiological factors limit
fruit set of tomato (Lycopersicon esculentum Mill.) under
chronic, mild heat stress. Plant Cell Environ. 23:719�726.
Sawers RJH, Gutjahr C, Paszkowski U. 2008. Cereal mycorrhiza:
an ancient symbiosis in modern agriculture. Trends Plant
Sci. 13:93�97.
Shapiro SS, Wilk MB. 1965. An analysis of variance test for
normality (complete samples). Biometrika. 52:591�611.
Smith SE, Read DJ. 1997. Mycorrhizal symbiosis. San Diego
(CA): Academic Press.
Stewart LI, Hamel C, Hogue R, Moutoglis P. 2005. Arbuscular
mycorrhizal inoculated strawberry plant responses in a high
soil phosphorus rotation crop system. Mycorrhiza. 15:612�619.
Tahat MM, Kamaruzaman S, Radziah O, Kadir J, Masdek HN.
2008. Response of (Lycopersicum esculentum Mill.) to differ-
ent arbuscular mycorrhizal fungi species. Asian J Plant Sci.
7:479�484.
Zhu X, Song F, Xu H. 2010. Influence of arbuscular mycorrhiza
on lipid peroxidation and antioxidative enzyme activity of
maize plants under temperature stress. Mycorrhiza. 20:325�332.
270 M. M. Maboko et al.
Dow
nloa
ded
by [
The
Aga
Kha
n U
nive
rsity
] at
15:
56 0
9 O
ctob
er 2
014
