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Journal of
Agricultural Science
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Volume 3, Number 5, May 2013 (Serial Number 25)
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Journal of Agricultural Science
and Technology A
Volume 3, Number 5, May 2013 (Serial Number 25)
Contents
Review
331 Plant Enzymes, Root Exudates, Cluster Roots and Mycorrhizal Symbiosis are the Drivers of P
Nutrition in Native Legumes Growing in P Deficient Soil of the Cape Fynbos in South Africa
Sipho Thulane Maseko and Felix Dapare Dakora
Research Papers
341 Effect of Ripening Stages on Basic Deep-Fat Frying Qualities of Plantain Chips
Ogan Mba, Jamshid Rahimi and Michael Ngadi
349 Production and Nutritive Value of Shrub Legumes in West Timor, East Nusa Tenggara Province,
Indonesia
Sophia Ratnawaty, Hartutik, Soebarinoto and Siti Chuzaemi
356 Study of Sandy Soil Compaction
Andrea Formato, Gian Pio Pucillo and Antonio Abagnale
368 Potential Impacts of Various Land Use Forms on Land Degradation in Arid Regions (Case Study:
Kuwait)
Raafat Misak and Adeeba Al-Hurban
380 Effects of Using Different Percentages of Fenugreek Flour to Improve the Sensory, Rheological
Properties and Keeping Quality in Maize Dough to Produces Gluten-free Breads
Abdulsalam Abdulrahman Rasool, Dana Azad Abdulkhaleq and Dlir Amin Sabir
385 Applicability of Cytoplasmic Male Sterility (CMS) as a Reliable Biological Confinement Method
for the Cultivation of Genetically Modified Maize in Germany
Heidrun Bückmann, Alexandra Hüsken and Joachim Schiemann
404 Evaluation of Fungicides for Controlling Stem Rust Race Ug99 on Bread Wheat
Joseph Kinyoro Macharia, Ruth Wanyera and Samuel Kilonzo
410 Occurrence and Effects of Pineapple Mealybug Wilt Disease in Central Uganda
Bosco Bua, Jeninah Karungi and Geoffrey Kawube
417 Electronic Identification of Livestock to Improve Turkey’s Animal Production System
Sezen Ocak, Sinan Ogun and Zuhal Gunduz
Journal of Agricultural Science and Technology A 3 (2013) 331-340 Earlier title: Journal of Agricultural Science and Technology, ISSN 1939-1250
Plant Enzymes, Root Exudates, Cluster Roots and
Mycorrhizal Symbiosis are the Drivers of P Nutrition in
Native Legumes Growing in P Deficient Soil of the Cape
Fynbos in South Africa
Sipho Thulane Maseko1 and Felix Dapare Dakora2
1. Department of Crop Sciences, Faculty of Science, Tshwane University of Technology, Pretoria 0001, South Africa
2. Chemistry Department, Faculty of Science, Tshwane University of Technology, Pretoria 0001, South Africa
Received: January 30, 2013 / Published: May 20, 2013. Abstract: The Cape fynbos is characterised by highly leached, sandy, acidic soils with very low nutrient concentrations. Plant-available P levels range from 0.4 μg P g-1 to 3.7 μg P g-1 soil, and 1-2 mg N g-1 soil. Despite these low nutrient concentrations, the fynbos is home to 9,030 vascular plant species with 68.7% endemicity. How native plant species survive such low levels of available P is intriguing, and indeed the subject of this review. In the fynbos soils, P is easily precipitated with cations such as Fe and Al, forming Al-P and Fe-P in acidic soils, or Ca-P in neutral-to-alkaline soils. The mechanisms for promoting P availability and enhancing P nutrition include the development of mycorrhizal symbiosis (with 80%-90% of higher plants, e.g., Cyclopia, Aspalathus, Psoralea and Leucadendron etc.) which exhibits 3-5 times much greater P acquisition than non-mycorrhizal roots. Formation of cluster roots by the Leguminosae (Fabaceae) and their exudation of Kreb cycle intermediates (organic acids) for solubilizing P, secretion of root exudate compounds (organic acids, phenolics, amino acids, etc.) that mobilize P. The synthesis and release of acid and alkaline phosphatase enzyme that catalyze the cleavage of mineral P from organic phosphate esters in acidic and alkaline soils, and the development of deep tap roots as well as massive secondary roots within the uppermost 15 cm of soil for capturing water and nutrients. Some fynbos legumes employ all these adaptive mechanisms for enhancing P nutrition and plant growth. Aspalathus and Cyclopia species typically form mycorrhizal and rhizobial symbiosis for improving P and N nutrition, produce cluster roots and acid phosphatases for increasing P supply, and release root exudates that enhance P solubilisation and uptake.
Key words: Cape fynbos, Cyclopia, Aspalathus, phosphorus, mycorrhiza, phosphatases.
1. Introduction
The Cape fynbos is part of the Cape Floristic
Region, and is characterised by strongly leached,
acidic sandy soils with very low nutrient
concentrations, especially phosphorus (P) [1, 2]. That
not with standing, it has the most diverse flora
comprising about 9,030 vascular plant species with
68.7% endemicity [3, 4]. Although P is an important
nutrient for plant growth, it remains one of the most
Corresponding author: Felix Dapare Dakora, professor,
research fields: agrochemurgy and plant symbioses. E-mail: dakorafd@tut.ac.za.
limiting elements in soils. For example,
plant-available P levels in the Cape fynbos soils in
particular are very low, range between 0.4 μg P g-1
and 3.7 μg P g-1 [5]. How the native plant species
survive on such low levels of available P is intriguing,
and indeed the subject of this review. Although many
soils contain about 20%-80% P in the organic form [6]
and fynbos soils in particular contain about 58%-77%
organic P [7], most of it is unavailable for plant uptake
due to complexion with Ca, Fe and Al [8], and/or
microbial incorporation into organic matter following
decomposition [9].
DAVID PUBLISHING
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Plant enzymes, Root Exudates, Cluster Roots and Mycorrhizal Symbiosis are the Drivers of P Nutrition in Native Legumes Growing in P Deficient Soil of the Cape Fynbos in South Africa
332
Under acidic conditions, phosphate ions easily
precipitate with cations such as Fe and Al, thereby
forming bound-P which is unavailable to plants [10].
As a result, P is generally present in only micromolar
(μΜ), or lesser concentrations in soils, even though
most mineral nutrients occur in millimolar (mM)
levels in the soil solution. Because of its low
micromolar concentrations, P uptake during water
absorption by plants typically accounts for only about
1%-5% of the plant’s P demand [11], an amount too
little to drive the multitude of P-requiring metabolic
processes in cells. Furthermore, P mobility in soils is
very low, and this adds to its limiting status in
ecosystems. Although the application of P-fertilizers
could potentially increase P concentration in soils, the
efficiency of these fertilizers is low in cropping
systems, with only 10%-20% of fertilizer-P being
used by crops in the year of application [12]. This
reduced efficiency of applied P is due to rapid
conversion to bound forms such as Al-P and Fe-P in
acidic soils, or Ca-P in neutral-to-alkaline soils [8,
13].
Where P is low in natural ecosystems, native and
endemic plant species must have mechanisms for
coping with low P stress. Plants that are native to low
P (soils such as those of the fynbos) develop various
strategies that are directed at enhancing P acquisition,
especially sparingly soluble P [14, 15]. The fact that
the Cape fynbos soils are very low in P, and yet this
biome is home to some 9,030 plant species with
68.7% endemicity, suggests the presence of
plant-evolved mechanisms for coping with low P
stress. For example, in addition to deep tap roots,
fynbos plant species develop lots of secondary roots
that are concentrated within the uppermost 15 cm of
soil, and decrease exponentially with depth [16]. A
recent review has described the strategies used by
cultivated crop species to acquire P under low nutrient
conditions [17]. This report addresses the biological
features of plant adaptation to low-P stress in the Cape
fynbos of South Africa. These adaptations include the
role of mycorrhizal symbiosis, cluster root formation,
root exudates, secretion of P-solubilizing enzymes and
root architecture/function.
2. Plant Secretion of Phosphatase Enzymes
Secretion of enzymes such as phosphatases is
another way by which plants and microbes respond to
P deficiency in soils [10, 18]. There are two types of
phosphatases: acid phosphatases (APases), which are
of plant origin and display an optimum activity below
pΗ 7 [19], and alkaline phosphatases, which are
secreted by bacteria, fungi and earthworms and
function catalytically above pΗ 7 [20]. Although a
large proportion of P occurs as organic P in soil [21],
plants can only utilize P in the inorganic form [22]. In
low P soils, phosphatase enzymes serve to catalyze the
cleavage of mineral P from organic phosphate esters
in both acidic and alkaline soils [23], and thus make P
more available in the rhizosphere for root uptake.
Usually, an increase in phosphatase activity in
rhizosphere soil implies increased hydrolysis of soil
phosphate esters, and elevated bioavailability of P for
plant uptake [24]. Thus, acid and alkaline phosphatase
activity is often used as a biological indicator of P
mineralization and P availability in the rhizosphere
[18].
Rhizosphere soil therefore usually has higher
phosphatase activity than bulk soil (Fig. 1A). Three
fynbos legumes, (namely Cyclopia genistoides,
Aspalathus caledonensis, Aspalathus aspalathoides
and the non-legume Leucadendron strictum)
co-occurring at the same fynbos site showed
significant variation in acid phosphatase activity. The
Cyclopia species exhibited significantly higher APase
activity in its rhizosphere, followed by A.
caledonensis, Aspalathus aspalathoides and L.
strictum, which recorded a much lower APase activity
(Fig. 1B). This might suggest that these legumes
exhibit different degrees of tolerance to low P in the
Cape fynbos. No doubt, the synthesis of
macromolecules such as enzymes is very costly to the
Plant enzymes, Root Exudates, Cluster Roots and Mycorrhizal Symbiosis are the Drivers of P Nutrition in Native Legumes Growing in P Deficient Soil of the Cape Fynbos in South Africa
333
0
100
200
300
400
500
600
700
Cyclopiagenistoides
Aspalathuscaledonensis
Aspalathusaspalathoides
Leucadendronstrictum
Ac p
hosphata
se
(μg p
-nitro
phenol.g
-1soil Fwt.h
-1)
a
b
c c
B
0
100
200
300
400
500
600
700
Ac
phosp
hat
ase
(μg p
-nitro
phen
ol.g
-1so
il F w
t.h
-1)
Cyclopia genistoides
Rhizosphere soil
Bulk soil
a
b
A
Fig. 1 A) Acid phosphatase activity in rhizosphere and bulk soils of Cyclopia genistoides; B) Acid phosphatase activity in rhizosphere soil of Cyclopia genistoides, Aspalathus caledonensis, Aspalathus aspalathoides and Leucadendron strictum sampled from Koksrivier farm. Data are mean for enzyme activity and error bars show one standard error; columns with different letters are significantly different at P ≤ 0.05.
C economy of plants, hence their formation in large
quantities can only occur when there is a strong
biological demand, such as enhancing P acquisition in
low-P environments [8, 25].
However, the data in Fig. 1B could also be
interpreted to mean that A. aspalathoides and L.
strictum probably have other mechanisms for
increasing P availability in the rhizosphere for plant
uptake. Interestingly, excavation of these plants at
Koksrivier frequently revealed the presence of a large
mass of cluster roots on A. aspalathoides and L.
strictum, and few (or none) on C. genistoides and A.
caledonensis. This could imply that A. aspalathoides
and L. strictum obtain more P from other sources such
as cluster root formation than from secretion of
APases. Whatever the case, the lower APase activity
of A. aspalathoides and L. strictum and the higher
activity in C. genistoides and A. caledonensis (which
all co-occur naturally at the same site in the fynbos)
suggest adaptation to the low P conditions by the
former and sensitivity to low P by the latter [26, 27].
Although one study found no relationship between
organ APase activity and plant adaptation to low-P
soil [28], other studies have suggested that APase
activity in plant organs serve to mobilize and
translocate P from senescing plant parts to P sinks
[29], thus maintaining optimal P metabolism in
species growing in low P soils [30]. A bioassay of
APase activity in organs of the non-legume,
Leucadendron strictum and two Aspalathus species
from different locations in the Cape fynbos revealed
marked variation in enzyme functioning. Aspalathus
cordata sampled from the Silvermine area of the Cape
fynbos exhibited a significantly greater APase activity
in leaves and roots compared to that in stems and
nodules (Fig. 2A). Aspalathus aculeata, however,
showed decreased APase activity from leaves to roots
(Fig. 2B), in a manner similar to Leucadendron
strictum from the Kanetberg Mountains, which also
exhibited a decrease in enzyme activity from leaves
and stems to roots (Fig. 2C). Assuming APase activity
of organs is a measure of P pool size, P metabolism,
Plant enzymes, Root Exudates, Cluster Roots and Mycorrhizal Symbiosis are the Drivers of P Nutrition in Native Legumes Growing in P Deficient Soil of the Cape Fynbos in South Africa
334
0
20
40
60
80
100
120
140
160
Leaves Stems Roots Nodules
Ac/
ph
osp
hat
ase
(μg
p-n
itro
ph
eno
l.g-1 F
wt.
h-1)
aa
bb
A. cordata
Silvermine: APase
A
0
100
200
300
400
500
600
Leaves Stems Roots
Ac /p
hosphata
se
(μg p
-nitro
phenol.g
-1 F
wt.h
-1)
a
a
b
A. aculeata
Malmesbury: APase
B
0
2
4
6
8
10
12
14
Leaves Stems Roots
Ac/
ph
osp
hat
ase
(μg
p-n
itro
ph
eno
l.g-1 F
wt.
h-1)
a
ab
c
Kanetberg: APase
L. strictum (non-legume)
C
Fig. 2 Acid phosphatase activity in organs of A) Aspalathus cordata; B) Aspalathus aculeata and C) Leucadendron strictum. Data are mean of five replicates for enzyme activity and error bars show one standard error. Columns with different letters are significantly different at P ≤ 0.05.
and potential P supply to sinks, then the stems of A.
cordata, as well as the roots of A. aculeata and L.
strictum exhibited the lowest P-supplying capacity.
More of these studies are needed to understanding P
acquisition in the low nutrient fynbos.
3. Root Exudates
Plant root exudates consist of simple
polysaccharides, amino acids, fatty acids, organic
acids, phenolic compounds, inorganic ions, enzymes
such as acid phosphatases and gaseous molecules
produced by root hairs [8, 10, 22, 31, 32]. There are
also high-molecular weight components such as
mucilage (polysaccharides), root border cells and
proteins produced by roots of some plants [33]. Root
exudation represents a significant carbon cost to the
plant [34], with the amount of photosynthate secreted
as root exudates varying with plant age and
physiological state, as well as soil type [32, 35, 36].
The concentration of organic acids such as lactate,
acetate, oxalate, succinate, fumarate, malate, citrate,
isocitrate and aconiate is reported to increase in the
Plant enzymes, Root Exudates, Cluster Roots and Mycorrhizal Symbiosis are the Drivers of P Nutrition in Native Legumes Growing in P Deficient Soil of the Cape Fynbos in South Africa
335
root exudates of many plant species with P deficiency
[37-39]. Of the organic acids exuded in the
rhizosphere, citric, malic and oxalic acids are the most
efficient in solubilising unavailable soil P due to their
rapid complexation with metals such as Al, Ca, Fe and
trace metals, to form chemically-bound forms like
Al-P, Ca-P and Fe-P [37, 40, 41]. Citric acid is
reported to be the dominant organic acid exuded by
legume species [42] and alfalfa [10], and may well be
the major acid used by fynbos legumes to mobilize P.
Citrate secretion by P-deficient common bean was
found to be effective in mobilizing P from Al-P and
Fe-P compounds [43].
In contrast, graminaceous plant species secrete
phytosiderophores (amino acids) which form much
more stable complexes with Fe, Zn and Cu than Krebs
cycle intermediates [40]. The uptake of these
phytosiderophore-P complexes by plants results in a
desorption of the P ions via a ligand exchange reaction,
and eventually in an increased bioavailability of P in
the rhizosphere [10].
4. Cluster Roots
Cluster roots (also known as proteoid roots)
represent a major adaptation for P acquisition by
terrestrial plants belonging to the families Fabaceae,
Proteaceae, Casuarinaceae and Myricaceae [31, 44].
Cluster root formation is common in the Cape fynbos
of South Africa and in the southwestern part of
Western Australia, and plays an important role in
nutrient acquisition (especially P) in these
nutrient-poor environments [44]. Cluster roots are
hairy rootlets formed during the early rains of the wet
season, and are usually concentrated in the uppermost
10 cm of the soil [16].
Cluster roots formation is reported to be triggered
by P deficiency [9, 31]. However, the fact that
Aspalathus linearis easily forms cluster roots in the
acidic sands of the Cederberg Mountains, but not in
acid-washed sand without P, suggests that there might
be other unknown factors eliciting cluster root
formation in fynbos species. Once formed, cluster
roots can occupy 60%, or more, of the whole root
system [45], in the subsoil containing the highest
concentration of nutrients [46]. Cluster roots enhance
P acquisition, in that, they have over five times the
surface area of an equivalent non-cluster root dry mass
[16].
According to Vorster and Jooste [47], cluster roots
exhibit a more effective P absorption compared to
non-cluster roots. Kreb cycle intermediates released
by cluster roots are probably the most effective in
mobilizing P [11]. These organic acids are able to
release P from strongly bound forms by complexing
with Al or Fe of Al-P and Fe-P in acid soils, or the Ca
of Ca-P in alkaline soils [8, 45]. In so doing, P is
mobilized for uptake by plant roots. Cluster roots also
release phenolics, which may be involved in the
mobilization of P [8]. White lupin (Lupinus albus L.)
is reported to secrete large amounts of organic acids,
H+, isoflavonoids and acid phosphatases [31, 48] for
enhancing P uptake and assimilation.
5. Root Architecture and Function
Plant roots do not only function as organs for water
and nutrient uptake, but also serve to mobilize nutrient
elements in the rhizosphere. The spatial configuration
of plant roots in soil (otherwise called root
architecture) represent a nutritional response to
variations in the supply and distribution of nutrient
elements in a given environment [49, 50]. With
nutrient-poor soils, the fine and long root hairs of
plants are well distributed throughout the soil, thus
covering a large surface area which enables plants to
optimize water and nutrient uptake, especially during
periods of deficit. That way, localized depletion of
minerals, and/or uneven distribution in space and time,
are addressed by root architecture [51, 52]. With
legumes, P availability is greater in surface or
near-surface horizons than in the subsoil due to P
deposition by decaying leaves, roots and nodules, as
well as by biological, chemical and physical processes
Plant enzymes, Root Exudates, Cluster Roots and Mycorrhizal Symbiosis are the Drivers of P Nutrition in Native Legumes Growing in P Deficient Soil of the Cape Fynbos in South Africa
336
in the topsoil. The levels of P are therefore generally
greater at the sub-surface and decreases with soil depth
[53]. Ancient soils such as those in the fynbos are
acidic with high concentrations of Al and Fe, which
form unavailable compounds with P (e.g., Al-P and
Fe-P), thus resulting in very low P levels. Plants in
such low-P environments such as the fynbos therefore
tend to have highly branched root architecture in order
to maximize P acquisition. Furthermore, the rate of
diffusion of P in soil is reported to be very low (10-12
to 10-15 m2 s-1) [54]. In low-P soils, this triggers native
plant species to expand their root surface area and thus
provide greater root-soil contact for an effective P
uptake [55].
While little is known about roots in the fynbos, in
general, an increased root length and decreased root
diameter tends to increase the amount of absorptive
surface area of the root relative to the volume of soil
[54, 56]. Whether this mechanism is used by fynbos
legumes (and non-legumes) to enhance P acquisition
remains to be determined. However, root hair density
of the model plant, Arabidospsis thaliana was found
to increase significantly in plants exposed to low-P
compared to high P, being five times greater in low P
than high P [57]. This suggests that in the low-P soils
of the Cape fynbos, native legumes are likely to have
greater root hair density in response to the low P
status of the ecosystem.
6. Mycorrhizal Symbiosis
The formation of fungal symbiosis with native plant
species is one way of enhancing P nutrition in low
nutrient environments [58, 59]. In this mutualistic
association, soil fungi penetrate plant roots and supply
the host plant with nutrients such as P in return for
fixed C. It is therefore not surprising that this fungal
symbiosis is mostly found under nutrient-limiting
conditions such as those of the Cape fynbos. About
80%-90% of higher plants are reportedly infected by
mycorrhizal fungi [60, 61]. In the fynbos, the species
with known mycorrhizal status include those of
Cyclopia [62-64], Aspalathus [63, 65], Leucadendron
[16, 63, 65] and Psoralea [66, 67]. More must be
waiting to be discovered.
There are six types of mycorrhizal symbioses, and
these include arbuscular, arbutoid, ecto, ericoid,
monotropoid, and orchid mycorrhizae [68, 69]. Of
these, arbuscular mycorrhizae and ectomycorrhizae
are the most common in natural ecosystems such as
the fynbos. Colonisation of plant roots by both
arbuscular and ectomycorrhizae are widespread in
nutrient-poor soils [33, 70], with ectomychorrhizae
being more abundant in N-limited ecosystems and
arbuscular mycorrhizae in drier low-P soils [71] of the
Cape fynbos. In these nutrient-poor environments, P
acquisition by mycorrhizal roots can be 3 to 5 times
higher than non-mycorrhizal roots [53, 72]. Over 80%
of terrestrial plant species enhance P uptake from
arbuscular mycorrhizal symbiosis with Zygomycete
fungi of the Order Glomales [73]. Once plant roots are
infected, the fungi proliferate their hyphae to provide
a much greater surface area for water and nutrient
absorption. This increase in surface area together with
the presence of high-affinity P uptake and transporters
in the external hyphae enhance P acquisition and
promote growth of mycorrhizal plants in P-deficient
soils [74, 75].
Both arbuscular mycorrhizae and ectomycorrhizae
produce mycelia that can extend beyond the nutrient
depletion zone of immobile nutrients in the
rhizosphere and form a complex architecture with an
efficient nutrient-collecting network [70]. These
mycelia thus enable mycorrhizal plants to explore a
larger volume of soil, thus decreasing the distance for
diffusion of absorbed phosphate ions. In so doing,
mycorrhizal hyphae can transport P over a distance
exceeding 40 cm to the fungus-root interface, where P
transfer to the host plant is effected [76]. Fungal
mycelia also have smaller diameters (2-4 μm), which
enable the hyphae to penetrate soil pores too small for
plant root hairs to enter [77]. That way, a relatively
larger soil volume is explored for delivering P per unit
Plant enzymes, Root Exudates, Cluster Roots and Mycorrhizal Symbiosis are the Drivers of P Nutrition in Native Legumes Growing in P Deficient Soil of the Cape Fynbos in South Africa
337
surface area than uncolonized roots [78]. According to
Ref. [79], arbuscular mycorrhizal fungi belonging to
the genera Acaulospora and Glomus, are the most
dominant in P acquisition in the Cape fynbos. In
addition to mycelial activity, mycorrhizal fungi also
synthesise and release acid and alkaline phosphatase
enzymes that further promote increased P acquisition
by plants in nutrient-poor environments such as the
fynbos [80].
7. Economic Importance of Native Plant Species in the Cape Fynbos
Although no inventory currently exists on the
economic importance of the 9,030 plant species
occurring in the Cape fynbos, it is clear that its
uniqueness and biodiversity of flora is major attraction
for tourists. Additionally, species such as Aspalathus
linearis, which is the source of Rooibos tea, a
beverage with many health benefits, contributes about
R500 million annually to the South African economy.
Cyclopia is also a fynbos legume with 25 species that
are used to make Honeybush tea, another herbal
beverage that contributes about R12 million per year
to the South African economy [81, 82]. Other legume
and non-legume species such as Leucadendron, and
Psoralea are used in the horticultural industry as fresh
or dried cut flowers [83]. Many fynbos species,
including Sutherlandia, are used as medicinal plants
for treating various physiological disorders in rural
communities [84]. Despite the significant contribution
of fynbos flora to the South African economy, so little
is known about their adaptation to the low nutrients
and limiting soil moisture of their environment.
8. Future Research
To overcome constrains to P nutrition, which
decreases crop yields, efforts must be made to
understand how native plants in natural ecosystems
adapt to low-P conditions. Knowing these
mechanisms is likely to provide solutions for breeding
agricultural crops with tolerance of low P.
Furthermore, increasing our knowledge of P
acquisition through the activity of acid and alkaline
phosphatases in naturally low-P-tolerant native plant
species could provide a platform for designing crop
plants with tolerance of P deficiency, which is
prevalent in many agricultural soils of Africa.
Most low-P soils tend to have high Al and Fe or
high Ca. It is therefore important to understand the
biology of P mobilization in soils dominated by Al-P,
Fe-P or Ca-P, as this could provide an opportunity for
identifying crop species for biomes with similar soils.
Finally, improved P nutrition in agricultural crops
in Africa is a necessity for increased food security.
Therefore, any new knowledge added to our current
understanding of P nutrition using native legumes of
the Cape fynbos can only increase agricultural crop
yields.
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Journal of Agricultural Science and Technology A 3 (2013) 341-348 Earlier title: Journal of Agricultural Science and Technology, ISSN 1939-1250
Effect of Ripening Stages on Basic Deep-Fat Frying
Qualities of Plantain Chips
Ogan Mba, Jamshid Rahimi and Michael Ngadi
Department of Bioresource Engineering, Macdonald Campus of McGill University, Lakeshore Road, Ste-Anne-de-Bellevue, Quebec
H9X 3V9 21111, Canada
Received: December 19, 2012 / Published: May 20, 2013. Abstract: Plantains (Musa paradisiaca L.) are a major food staple in West Africa and are cooked in various forms. The objective of this work was to evaluate the frying characteristics of plantains at different stages of ripening. The plantains used in the study were at the unripe stage through four different ripening stages. The samples were peeled and sliced into 2 mm thickness and blanched in hot water at 70 °C for 3 min. The slices were then deep fried in canola oil at 180 °C. The result showed that ripening stage significantly affected moisture loss and fat absorption profiles of the plantain chips. Fully ripened plantain absorbed up to 34% (db) oil during 4 min frying, much higher than unripe plantain. The oil uptake and moisture loss during frying of the plantain chip samples were modelled using 1st order kinetics. The kinetic parameters including rates of moisture loss and oil uptake varied according to the different stages of ripening. Ripening had a significant effect on the colour lightness (L) of the chips. Similarly, the redness (a), the yellowness (b) and textural characteristics were significantly affected by ripening stage.
Key words: Plantain ripening, plantain chips, deep-frying, oil uptake, moisture loss, kinetics.
1. Introduction
Plantain (Musa paradisiaca L.) is native to
Southeast Asia, Africa and South America. It is one of
the world’s most important food crops. At harvest, the
pulp and peel of plantain make up about 62% and
38%, respectively. The pulp of matured green unripe
fruit is rich in starch, dietary fiber, potassium,
magnesium, iron, phosphorus, calcium, vitamin C and
carotene. The iron content of plantain is 100% usable
for human consumption [1, 2]. Plantain is a
climacteric fruit. Its ripening process continues even
after harvesting from the parent plant due to evolution
of ethylene. Plantain ripening involves irreversible
series of physical, physiochemical, biochemical and
organoleptic changes in the fruit. During ripening, the
peel changes from green to yellow and finally to dark
brown. At the same time, pulp color changes from
Corresponding author: Michael Ngadi, Ph.D., research
fields: food and bioprocess engineering. E-mail: michael.ngadi@mcgill.ca.
creamy white to yellow or yellow-orange. The change
in the colour of pulp/peel is due to the activity of
polyphenol oxidase which is abundant in plantain [3,
4]. Ripe plantain can be eaten fresh on account of its
content of vitamin C and other essential minerals.
Plantain ripening changes the osmotic balance
between pulp and peel. A significant increase in the
sugar content of the ripe pulp has been reported [5].
There are many different ways of processing and
consuming plantain in West Africa. Deep-frying is a
widespread method of plantain chips production.
Plantain chips are a popular snack food item in Africa
[6]. Plantains are cooked and dried by immersing in
hot edible oil (between 130 °C and 190 °C at
atmospheric pressure) for a few minutes. This results
in simultaneous heat and mass transfer. As heat is
transferred from the oil to the food, moisture is
evaporated and oil is absorbed. The frying process
results in unique flavor, color and texture attributes
which are the main drivers of consumer acceptability
D DAVID PUBLISHING
Effect of Ripening Stages on Basic Deep-Fat Frying Qualities of Plantain Chips
342
of the products [7]. Some models that describe the
characteristics of moisture loss and fat uptake, as well
as the relationship between moisture loss and fat
uptake in deep-fried food products have been
developed [8, 9]. The kinetics of the frying process,
including the oil diffusion and absorption has been
studied for a range of fried products including
potatoes and Musa species [10-12].
The combined effect of high rate of metabolism,
tropical humid conditions, inadequate postharvest
handling systems and poor marketing conditions have
resulted in postharvest losses of plantains. Plantain
becomes very perishable on ripening, resulting in
many fruits being discarded. Hence, the need to
process ripe plantain into other finished products such
as plantain chips. While there are many studies on
fried products and oil deterioration, little attention has
been paid to the effect of the variability of the raw
material on its frying behaviour and the
physicochemical properties of the final product [12].
Therefore, the objective of this work was to evaluate
the frying characteristics of stored plantains at
different stages of ripening. The result will provide
useful information to the millions throughout the
world of whom the plantain fruit is critical to their
nutritional and economic well-being.
2. Materials and Methods
2.1 Preparation of Samples
Mature Columbian Plantain fingers at unripe green
stage and Canola oil, were purchased from a local
grocery store in Montreal, Canada. The plantains were
stored and allowed to ripen slowly under ambient
conditions of 25 °C and 80%-85% RH until they
reached the desired different stages of ripeness. The
guide was the banana ripening chart [13]. The samples
and the stages of ripening used in this work are
described in Table 1. On each frying day, the plantain
sample was washed and manually peeled. The pulp
was sliced to a thickness of 2 ± 0.1 mm using Rival
Precision Slicer model C1105. The diagonal slices
Table 1 Description of plantain ripening stages.
Ripening stage
Storage day
Peel color index number
Peel color
A 1 1 Green
B 3 3 Green-yellow
C 7 6 All yellow
D 8-9 7 Yellow flecked with brown
were blanched at 70 °C for 3 min in a water bath. Paper
towel was used to dry the slices and allowed to
completely dry at room temperature.
2.2 Deep-Frying Process
Four liters of Canola oil was preheated to 180 ±
2 °C in a temperature controlled bench top fryer
(model no: D24527DZ, Délonghi Inc. USA). The
blanched plantain slices was divided into 5 different
portions and fried for 1, 2, 3 and 4 min. The fifth
portion was not fried (0 min) which served as control.
Ten slices per sampling were put into sample holders
that ensured complete immersion in the hot oil. After
each frying time, the chips were well drained of
adhering surface oil by shaking and blotted with paper
towel. The chips were allowed to cool at room
temperature. All the experiments were carried out in
triplicates. Polyethylene self-sealing storage bags were
used to pack the chip samples before the analyses that
followed.
2.3 Moisture and Fat Analysis
Fried plantain chips were weighed, and then dried
at 105 °C to constant weight in a forced air convention
oven (Isotemp 700, Fischer Scientific, Pittsburgh,
USA). Moisture content was determined
gravimetrically [14]. AOAC [15] standard procedure
and Soxhlet method were used for fat extraction and
analysis. The dried samples were ground using
Sumeet electric grinder. Approximately 5.0 g of each
ground sample was used for fat extraction. The fat was
extracted using petroleum ether in a Soxhlet extractor
(SER 148, Velp Scientifica, Usmate, Italy). The fat
content was obtained on dry weight bases for each
plantain chip sample as the ratio of mass of oil
Effect of Ripening Stages on Basic Deep-Fat Frying Qualities of Plantain Chips
343
extracted to mass of dried samples.
2.4 Colour Measurements
The colour parameters (L, a and b) of the plantain
chips samples were measured by reflectance using a
Konica Minolta colorimeter (model no: CR-300,
Konica Minolta Sensing Inc. Osaka, Japan). The
instrument was standardized each time before reading
measurements using a white ceramic plate. Samples
were scanned at 5 different locations to determine the
L, a and b values as the average of the five
measurements.
2.5 Texture Evaluation
Textural properties were evaluated instrumentally at
room temperature by a compression/puncture test
using an Instron Universal Testing Machine (Model
4502, Canton MA, USA). Force vs displacement (F/D)
curves were generated with the puncture test at
different ripening stages and frying times by mounting
the sample on a flat rigid support where the distance
between the support and a cylindrical punch was 15
mm [16]. The punch diameter and the crosshead speed
were 5 mm min-1 and 25 mm min-1, respectively. The
textural parameters Hardness (equals Maximum
Force/Maximum deformation) and Crispiness (equals
slope of the linear section of the curve) [16, 17] were
obtained from the F/D curves. Each measurement was
repeated five times and their mean values were used
as indicators of the parameters.
2.6 Statistical Analysis/Kinetics Model
The SAS system software (Version 9.2, SAS
Institute, Inc., 1999, Cary, NC, USA) was used for
statistical analysis. Duncan’s multiple range test
(DMRT) was used to estimate significant differences
among means at a 5% probability level. All analyses
were conducted in triplicate. Similarly, a first order
kinetics was used to describe oil uptake and moisture
loss during deep-fat frying [8]. Recognizing that there
is a saturation oil content beyond which minimal oil is
absorbed into the product during frying, the following
rate equation was used. d
- ( )ou e
ok O O
dt (1)
where, O is oil uptake in the sample, Oe is the
equilibrium oil content, Kou is the oil uptake rate
constant and t is the frying time. Integrating, the
equation can be adjusted and expressed in the form of
oil uptake as follows:
(1 )ouk tu usO O e (2)
where, Ou is the fat uptake, (i.e., the difference
between the oil content at a given time and the initial
oil content), Ous is saturation oil content (i.e., the
difference between equilibrium oil content and the
initial oil content).
Since frying was done at high temperature (above
boiling point of water), equilibrium moisture content
was assumed to be zero (i.e., moisture was completely
evaporated). Although in reality equilibrium does not
exist in deep fat frying, the system behaves as if there
is equilibrium. This is due to the physical changes in
the product during frying which restrict moisture and
oil transfers. Hence the model described by Debnath
et al. [18] was used to describe the kinetics of
moisture loss ratio: * 1 mlkM e t (3)
where, *M is the moisture ratio (i.e., the ratio of moisture at a given time and initial moisture) and Kml
is the moisture loss rate constant.
The kinetic parameters were obtained using
non-linear regression in MATLAB (Version 7.6.0.324
R2008a, The Mathworks, Inc., Natick, MA, USA).
3. Results and Discussion
3.1 Fat Uptake/Moisture Loss
The overall influence of plantain ripening on the fat
uptake and moisture content of the plantain chips is
presented in Figs. 1 and 2. The initial moisture content
of mature green plantain was 67.126% ± 0.53% which
decreased gradually to a significant 62.274% ± 0.62%
by the yellow-brown stage of ripeness. The initial
moisture content of the green plantain sample is in
Effect of Ripening Stages on Basic Deep-Fat Frying Qualities of Plantain Chips
344
Fig. 1 Fat uptake trend of unripe and ripe plantain chips during deep-fat frying.
◊ = experimental result plot for unripe green plantain; □ = experimental result plot for plantain at green-yellow stage of ripening; ∆ = experimental result plot for plantain at all yellow stage of ripening; × = experimental result plot for plantain at yellow/fleck of brown stage of ripening; Continuous line = Predicted values.
Fig. 2 Moisture loss trend of unripe and ripe plantain chips during deep-fat frying.
◊ = experimental result plot for unripe green plantain; □ = experimental result plot for plantain at green-yellow stage of ripening; ∆ = experimental result plot for plantain at all yellow stage of ripening; × = experimental result plot for plantain at yellow/fleck of brown stage of ripening; Continuous line = Predicted values.
Effect of Ripening Stages on Basic Deep-Fat Frying Qualities of Plantain Chips
345
line with the values reported by Agunbiade et al. [19]
and Avallone et al. [20]. The decrease in moisture
content of the un-fried samples could be due to the
reported increase of sugar during ripening from nearly
zero at green stage to around 30% in overripe plantain
[21]. It was observed that the moisture content of the
fried plantain chips continued to decrease as ripening
progressed. There was a significant difference in the
mean moisture content of each of the fried chips at
different ripening stages. As shown in Fig. 1, ripening
increases the fat uptake of the plantain chips. There
was a significant increase in fat absorption from the
initial 0.3% (db) in mature green sample that was not
fried reaching up to 34% (db) oil in fully ripened
plantain chips after 4 min frying. The increased oil
absorption during deep frying of ripe plantains may be
attributed to changes in porosity and molecular size
redistribution as sugar content of plantains increases.
Moreira et al. [22] had shown that porosity and
particle size of masa flour used to produce tortilla
chips had an effect on the oil uptake during the deep
fat frying process.
The average moisture content of the unripe plantain
used in this work was 65.0% and was significantly
higher than the mean moisture content of 8.01% found
in the chips after 4 min of frying. The moisture
changes during frying showed the typical extensive
decrease with increasing frying time [9, 23]. The trend
showed that frying at 1 min led to a rapid moisture
decrease (33.78%). This means that the rate of
moisture loss of the fried chips increased as ripening
progressed and the fruit became softer (Fig. 2). The
moisture loss rate was similar as frying time
progressed. Generally, the results agree with literature
in terms of the moisture content values, oil content
values and the effect of temperature on heat and mass
transfers. The parameters of the first order kinetic
model of fat uptake and moisture loss for different
ripening stages of the fried plantain chips is presented
in Table 2.
It has been reported that there is a rapid linear
Table 2 Parameters of first-order kinetic model of fat uptake and moisture loss for different ripening stages of fried plantain chips.
Ripening stage Ous (% db) Kou (min-1) Kml (min-1)
A 6.611 ± 0.097 0.652 ± 0.493 0.745 ± 0.041
B 10.46 ± 0.388 2.361 ± 0.491 0.773 ± 0.024
C 15.50 ± 0.16 2.015 ± 1.388 0.819 ± 0.028
D 32.93 ± 0.092 1.021 ± 3.357 0.794 ± 0.027
increase in temperature during sensible heating of
chips. The crisp was heated from its initial
temperature to the boiling point of water. As the oil
content plateaus, the moisture content decreased. A
threshold moisture value was achieved when the
moisture evaporation rate became negligible and the
chips were sensibly heated to the frying oil
temperature [24, 25]. Diaz et al. [12] reported that oil
uptake is determined by the variety of plantain used,
irrespective of processing time and temperature. Oil
uptake in the case of plantain does not appear to be
correlated to the raw material water content as it was
previously described for potato chips [26, 27] and
cassava chips [28]. Nevertheless, a positive correlation
was observed in this work between the amount of oil
uptake and the weight of moisture removed. Oil
uptake was influenced by stage of ripening and frying
time. This result might be explained by an indirect
effect associated with the rheological behaviour of
ripe plantain, the firmness of which decreased as
ripening progressed. Previous studies have also shown
that oil uptake was related to the adhesion of oil to the
chip surface after removal from the hot oil [22, 29].
3.2 Colour/Texture Properties
The physical characteristics of the plantain chips,
namely colour is shown in Table 3, while texture
parameters are shown in Figs. 3a and 3b. In Table 3,
the lightness (L), redness (a), yellowness (b), and
metric chroma (C*) values are shown. It was observed
that at all the ripening stages evaluated the chips had
bright colours (L > 50). However, the lightness of the
fried chips significantly decreased as ripening
progressed. The mature “green” samples (A) had a
Effect of Ripening Stages on Basic Deep-Fat Frying Qualities of Plantain Chips
346
Table 3 Colour characteristics of the plantain chips.
Ripening Stages L a b C*
A 68.01 ± 1.49a 4.57 ± 1.08c 42.13 ± 2.14b 42.74 ± 1.03b
B 65.63 ± 3.46b 6.32 ± 1.73b 42.57 ± 3.08b 43.40 ± 0.90b
C 62.57 ± 4.09c 8.60 ± 1.83a 45.79 ± 3.66a 46.92 ± 0.68a
D 60.99 ± 3.26d 8.78 ± 1.15a 44.78 ± 2.87a 46.09 ± 0.49a
Each value represents the mean ± standard deviation; Means within each column with different letters are significantly different
(P > 0.05). C* = Metric Chroma =2 2a b .
0
2
4
6
8
10
12
14
0 1 2 3 4 5
H*
Frying Time (mins.)
Normalized Hardness
A
B
C
D
0
10
20
30
40
50
60
70
80
0 1 2 3 4 5
CR*
Frying Time (mins)
Normalized Crispiness
A
B
C
D
Fig. 3 Effect of ripening on (A) hardness and (B) crispiness of plantain chips.
H* = Ratio of hardness at any time and hardness at time zero; CR* = Ratio of crispness at any time and crispness at time zero; ◊ = experimental result plot for unripe green plantain; □ = experimental result plot for plantain at green-yellow stage of ripening; ∆ = experimental result plot for plantain at all yellow stage of ripening; × = experimental result plot for plantain at yellow/ fleck of brown stage of ripening.
mean L value of 68.01 ± 1.49 while the “yellow/trace
of brown” samples (D) had a mean score 60.99 ± 3.26.
The redness of the chips samples were significantly
increasing as ripening progressed up to the fully ripe
“yellow” stage (sample C). There was no significant
difference in the redness of chips produced from
ripening stages C and D which had redness mean
score of 8.60 ± 1.83 and 8.78 ± 1.15, respectively. In
general, an increase in redness is negative for colour
of fried products [9]. The redness of the plantain chips
was within the acceptable range (a < 10). The more
desired color of fried chips is the yellowness. The data
also showed that this parameter was significantly
affected by ripening. The result further showed that
yellowness of the chips from samples A and B
(“green” and onset of ripening) were similar but
significantly different from (P > 0.05) samples C and
D (45.79 ± 3.66 and 44.78 ± 2.87). The mean metric
chroma values confirmed this trend in colour change
and indicated a tendency toward browning with
increased frying time. The colour changes also
indicated more Maillard reaction with frying time
which utilized the abundant reducing sugars in ripened
plantain [30]. The increase in colour intensity is in
agreement with the studies that related the colour of
French fries and potato chips [9] and plantains [6] to
their reducing sugar content.
The hardness and crispiness of the plantain chips
were significantly affected by ripening (P > 0.05).
Texture of plantain chips is an important sensory
parameter that determines acceptability and shelf
stability. The data showed a progressive decrease in
the mean values of hardness, 16.99 ± 2.53 N mm-1
(sample A) to 4.90 ± 0.15 N mm-1 (sample D). As
shown in Fig. 3a, hardness increased at all ripening
stages as frying time increased due to crust
A B
Effect of Ripening Stages on Basic Deep-Fat Frying Qualities of Plantain Chips
347
development [8]. The same trend was observed in the
crispiness values which came down from 20.13 ± 4.28
(sample A) to 2.08 ± 0.09 (sample D). Fig. 3b showed
that mature green plantain chips had a much higher
crispness than the ripe plantains. There was no
significant difference in the crispness of chips
produced from ripening stages C and D. This could be
due to increased oiliness as ripened plantain chip
samples had much higher oil uptake. The observed
textural changes is in agreement with published data
which shows that during fruit ripening, the fruit
changes from hard to pulpy soft due to breakdown of
cell wall structure and polysaccharides and moisture
loss due to respiration [3]. The degradation of pectin
from cell wall and middle lamella seems to be
responsible for tissue softening of plantain. Kajuna et
al. [31] also pointed out a decrease of the modulus of
elasticity over the ripening period of plantains as the
increase in osmotic pressure led to a reduction in
turgor pressure in the pulp tissue. This is attributed to
the hydrolysis of starch to sugar.
4. Conclusions
This work evaluated the influence of ripening on
the fat uptake and water content of stored plantain.
The result showed that ripening had a major effect on
fat uptake. The fat content rose from the initial 0.36%
to 34% after 4 min of frying fully ripened samples.
The moisture loss positively correlated with the fat
uptake. The L, a and b color changes of plantain chips
were affected by ripening especially from the fully
ripe stage. Ripening also significantly affected the
textural parameters evaluated. This work established a
relationship between plantain ripening and behaviour
during frying. This enables utilization of the ripened
plantain for chips production rather than allow ripened
fruits to waste.
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Journal of Agricultural Science and Technology A 3 (2013) 349-355 Earlier title: Journal of Agricultural Science and Technology, ISSN 1939-1250
Production and Nutritive Value of Shrub Legumes in
West Timor, East Nusa Tenggara Province Indonesia
Sophia Ratnawaty1, 2, Hartutik3, Soebarinoto3 and Siti Chuzaemi3
1. Postgraduate Program, Faculty of Animal Husbandry, University of Brawijaya, Malang, East Java 65145, Indonesia
2. Assesment Institute of Agriculture Technology East Nusa Tenggara, Kupang, East Nusa Tenggara Province 85362, Indonesia
3. Department of Animal Nutrition, Faculty of Animal Husbandry, University of Brawijaya, Malang, East Java 65145, Indonesia
Received: March 4, 2013 / Published: May 20, 2013. Abstract: This study aimed to identify the potential of shrub legumes as protein feed in West Timor. Eight shrub legumes, i.e., Clitoria ternatea Q5455 (CT Q5455), Clitoria ternatea millgara (CT millgara), Centrosema pascuorum bundey (CP bundey), Centrosema pascuorum molle (CP molle), Macroptilium bracteatum juanita (MB Juanita), Macroptilium bracteatum cadaarga (MB cadaarga), Dolichos lablab (DL) and Stylossanthes seabrana (SS) were evaluated for their yield and nutritive value in Randomized Block Design. Each legume was cultivated in four plots of different soil fertility as block (5 × 5 m2 per plot). Phosphorus (P) fertilizer was applied once just before planting at level of 50 kg ha-1. Watering was done three times per week using sprinkler. The legumes were harvested at 120 days after planting (dap) in three sub-plots of 1 × 1 m2 size for their yield measurement. Samples of each legume was taken for Dry Matter (DM), Organic Matter (OM), Crude Protein (CP), cellulose, hemi-cellulose and lignin content and in vitro DM, OM and CP digestibility test. Data were statistically analyzed using Genstat release 12.2. The results showed that the legumes showed significantly different (P < 0.01) in biomass yield. The highest biomass yield was shown by SS (6,739 kg DM ha-1; 6,120 kg OM ha-1 and 1,224 kg CP ha-1) and the lowest was by DL (1,294 kg DM ha-1; 1,157 kg OM ha-1 and 242 kg CP ha-1). In general, there was no significant difference (P > 0.05) of DM digestibility of the eight legumes, except for DL which showed significantly highest (P < 0.01) DM and OM digestibility compared to the other. Key words: Biomass, digestibility, feed, shrub legumes, yield.
1. Introduction
East Nusa Tenggara (NTT) is one of important beef
cattle centre in Indonesia, especially in respect of its
land area for feed resources. Land size of NTT is
2,962,571 ha, 60% of this area is natural savanna,
which is the largest area of savanna in Indonesia.
Population of beef cattle in this area is 720,000 heads
with stocking rate of 4-5 cattle km-2 [1]. Forage
production of the savanna in the rainy season is 1.7
ton Dry Matter (DM) (ha year)-1; which is three times
higher than the production in the dry season (0.54 t
DM (ha year)-1). The carrying capacity of beef cattle is
1.4-2.8 AU (ha year)-1 [2]. The carrying capacity is
still in the range of those reported by McIlroy [3]
Corresponding author: Sophia Ratnawaty, M.Sc., research
field: animal nutrition. E-mail: sophiaratnawaty@yahoo.com.
which is 0.2-7 AU ha-1.
Classical problems of beef cattle production in East
Nusa Tenggara in particular in West Timor district is
lack of feed during the dry season. During this season,
feeds are available in low quantity and quality. These
conditions have caused cattle to loss of weight and
high mortality of calves, especially less than one year
old calves. During dry season, the loss weight reached
up to 20%-25% of the cattle weight [4].
To overcome the problems, it is urgent to improve
the condition of the natural savanna. The improvement
can be done by introduction or planting annual shrub
legumes in the savanna. Legumes have higher
potential advantages in this case compared to natural
grass. Legumes are able to return soil fertility back by
catching natural air nitrogen and deposit it into the soil.
DAVID PUBLISHING
D
Production and Nutritive Value of Shrub Legumes in West Timor, East Nusa Tenggara, Province, Indonesia
350
Legumes with their steep roots have higher chance to
get deeper soil water and other nutrients for their
growth and maintain soil humidity. With those
capacities, legumes have higher potential to survive
and produce forage even during the dry season than
grasses. Legumes contain higher crude protein which
can be used as feed supplement during the harsh
season. The characteristic advantage of legumes was
summarized by Gutteridge and Shelton [5]. Bamualim
et al. [4] reported that protein content of natural grass
in June-November declined to below 7%. Rubianty et
al. [6] reported that crude protein content of Clitoria
ternatea and Centrosema pascuorum hay during the
dry season was 21.32% and 19.56%, respectively.
Therefore, in this research, it was to evaluate the
potential of some variety of shrub legumes in East
Nusa Tenggara in terms of their production and
nutritive value.
2. Materials and Methods
This study was conducted for 12 months from April
2011 to March 2012 in Field Laboratory of
Assessment Institute of Agriculture Technology
(AIAT) Kupang district East Nusa Tenggara province.
Eight shrub legumes were used and evaluated in
this study, i.e., Clitoria ternatea Q5455 (CT Q5455),
Clitoria ternatea Millgara (CT millgara), Centrosema
pascuorum Bundey (CP bundey), Centrosema
pascuorum Molle (CP molle), Macroptilium
bracteatum Juanita (MB Juanita), Macroptilium
bracteatum Cadaarga (MB cadaarga), Dolichos
lablab (DL) and Stylossanthes seabrana (SS).
The research was done in randomized block design
of eight treatments and four blocks as replication. The
treatments were the eight shrub legumes and the
blocks were based on the soil fertility. The legumes
were planted as monoculture in plots for each legume
and block. Each plot size was 5 × 5 m2.
The legumes were planted using seeds in rows of
holes in each plot, at distance of 50 cm between rows
and 25 cm between holes. Five seeds were planted in
each hole.
Phosphorus fertilizer (P) at level of 50 kg ha-1 was
applied once just before planting and watering using
sprinkler was regularly applied three times per week.
Data collection was done on agronomy parameters,
including seedling percentage, plant height and
biomass yield. Seedling percentage was observed
twice within seven days after planting. Plant height
was measured above the ground until the tip of the
longest leaf on day 14th, 28th, 42nd and 56th after
planting. The measurement was done in 1 × 1 m2
sub-plot of each plot. Biomass yield measurement was
done by harvesting legumes in three 1 × 1 m2
sub-plots on each plot at 120 days after planting.
Sample of biomass from each plot was taken for
analysis of DM, Organic Matter (OM), Crude Protein
(CP), content [7]; Neutral Detergent Fiber (NDF),
Acid Detergent Fiber (ADF), cellulose, hemi-cellulose
and lignin content [8]; and in vitro DM, OM and CP
digestibility test [9]. Data were statistically analyzed
using Genstat release 12.2 [10].
3. Results and Discussion
3.1 Growth Parameters and Biomass Yield
Growth parameters and biomass yield of the eight
legumes evaluated in this research are presented in
Table 1.
Data on Table 1 showed that among legumes
showed significantly different of growth parameters
(P < 0.01). All shrub legumes evaluated in this
research showed high seedling percentage, except for
CP bundey. Low seedling percentage of CP bundey
was caused by poor seed quality used in supporting
seedling and also related with type of soil, in which
CP bundey would need medium until high soil fertility.
Previous study done by Ratnawaty and Fernandez at
farmers’ field [11] showed different results from this
study. It was reported that average plant height of
Clitoria ternatea was 12.3 cm; Centrosema
pascuorum (9.93 cm) and Dolichos lablab (4.6 cm) at
the age of 60 days after planting. This was due to
shrub legumes grown in farmers’ fields were not given
Production and Nutritive Value of Shrub Legumes in West Timor, East Nusa Tenggara, Province, Indonesia
351
Table 1 Growth parameters and biomass yield of eight legumes harvested on 120 days after planting (Average ± SD).
Shrub Legumes Seedling percentage (%)
Plant height (cm)Biomass yield on 120 days after planting
(kg DM ha-1) (kg OM ha-1) (kg CP ha-1)
CT Q5455 67.5b ± 8.5 22.9b ± 7.9 5,691d ± 294.0 4,892bc ± 437.1 1,029d ± 68.6
CP bundey 47.5a ± 10.3 13.6ab ± 4.4 3,924bc ± 601.3 3,690b ± 732.8 713bc ± 141.0
DL 90.0c ± 7.1 38.8c ± 13.7 1,294a ± 148.0 1,157a ± 180.1 242a ± 35.8
MB juanita 97.5c ± 2.5 12.7ab ± 4.4 2,528ab ± 286.1 2,277a ± 328.7 451ab ± 65.9
MB cadaarga 100.0c ± 0 16.04ab ± 6.6 1,477a ± 262.2 1,343a ± 309.4 237a ± 62.6
CT millgara 95.0c ± 5 24.3b ± 8.9 5,328cd ± 127.9 4,880bc ± 167.3 972cd ± 30.1
CP molle 82.5bc ± 6.3 10.2a ± 2.9 2,273a ± 308.1 2,116a ± 371.5 408a ± 71.4
SS 70.0c ± 7.1 5.4a ± 2.3 6,739d ± 774.0 6,120c ± 902.6 1,224d ± 181.6 a-dValues in the same column with different superscript are significantly different (P < 0.01).
watering and fertilizer as in this study.
Reksohadiprojo [12] suggested that legumes growth
was highly dependent on seed quality and soil type
where the plants grow. Centrosema was known quite
slow related with resistance and adaptation toward
environmental condition for period of 8-9 months [13].
Centrosema growth would be stunted in environment
temperature of 18-24 °C [14]. Low land is more
appropriate for Centrosema growth [15].
In addition, among legumes species showed
significantly different of biomass yield on 120 days
after planting (P < 0.01). The highest biomass yield
was shown by SS, CT Q5455 and CT Millgara, while
the lowest one was shown by DL. This was as also
reported in several previous research that forage
production varied between shrub legumes species
[14-16]. This was happening due to physiological
difference such as leaf wide index, relationship of
leaf-water, carbohydrates reservation, and water
utilization efficiency [17]. Morphological difference
between shrub legumes species, such as bud residue
after trimming, plant height, roots length and surface
width, bud length and diameter that re-grow [14, 18].
Data on Table 1 showed that there was no likely
consistent relationship between seedling percentage,
plant height or length and biomass yield. Biomass
yield was more likely affected by morphological
differences rather than by plant growth parameters. SS
with moderate seedling percentage but the lowest
plant height showed the highest biomass yield,
however, DL with the highest seedling percentage and
plant height showed the lowest biomass yield.
Biomass yields in this research were higher than the
results of Ratnawaty et al. [19] who reported that
biomass yield of shrub legumes planted as
monoculture in Tobu village, Timor Island was in the
range of 1.1-2.2 t DM ha-1. This might be caused by
better forage management applied in this research
mainly fertilizer and regular watering application.
Budisantoso et al. [20] reported that biomass
production of Centrosema pascuorum was 3.3 t DM
ha-1 and Clitoria ternatea was 1.8 t DM ha-1, and
biomass production of Centrosema pascuorum was
the highest after re-growth with 45 days cutting
interval, followed by Clitoria ternatea, Desmanthus
pernambucanus, Macroptilium bracteatum and
Aeschynomene americana.
Lefi et al. [21] found difference in leaf production
and aging between M. areborea and M. citrina, while
Pang et al. [22] reported difference in phosphorus
utilization efficiency, root surface wide and root
length between perennial legumes species including B.
bituminosa. Cox et al. [23] reported forage production
of Lablab, MB juanita and cadaarga also CT millgara
planted in two seasons was 2.1, 2.0 and 1.0 t ha-1 in
first year, respectively, and the production decreased
as much as 0.2, 1.1 and 0.7 t ha-1 in second year.
Whitbread et al. [24] reported biomass production of
CT millgara increased from 1,277 kg DM ha-1 in the
first year become 4,047 kg DM ha-1 in the third year,
Production and Nutritive Value of Shrub Legumes in West Timor, East Nusa Tenggara, Province, Indonesia
352
while MB juanita was relatively stable with 3,382 kg
DM ha-1 in the first year and 4,940 kg DM ha-1 in the
third year.
Result of this research was different from research
done by Ayiyi et al. [25] that biomass production of
Lablab purpureus planted in Limpopo, South Africa
was around 1,000-5,000 kg ha-1 on 87 days after
planting. Result of Clem and Cook [26] measured
forage production of several kind of shrub legumes for
three years (2000-2002) obtained that forage
production of Lablab purpureous decreased from
3,863 kg ha-1 in 2000 to 59 kg ha-1 in 2001. Same case
happened on MB juanita where forage production
tended to decrease for the three consecutive years
cuttings from 2.499, 88 to 687 kg ha-1, however,
Stylosanthes scabra showed forage production to
increase from 2.045 kg ha-1 to 5.529 kg ha-1.
3.2 Nutritive Values
Nutrients content of the eight legumes evaluated in
this research is presented in Table 2.
Data in Table 2 showed that crude protein and other
nutrients content of the eight shrub legumes were
relatively similar. Crude protein content ranged from
17.85% to 18.73%. This CP content was relatively
high, therefore the legumes were potential as protein
feed supplement to substitute for concentrate feed.
Energy content of MB cadaarga, MB juanita and CP
molle is lower compared to CT Q5455, CT millgara,
CP bundey and SS. This is consistent with ash content
in this four shrub legumes. NDF is forage component
which is the biggest part of plant’s wall. This
compound consists of cellulose, hemi-cellulose, lignin,
silica and several fibroses protein [27], which is
mainly utilized by rumen microbes to produce energy
and for its life needs.
Ginting and Tarigan [28] reported that Centrosema
pubescens had some advantages compared to Arachis
pintoi based on its chemical composition (crude
protein, NDF and ADF). Other studies which also
reported similar result was done by Khamseekhiew et
al. [29], Nasrullah et al. [30] and Evitayani et al. [31],
although both legumes were planted in different
condition. Protein content of feed was reported had
positive and significant relationship with dry matter
and organic matter consumption [32, 33], but it is
negatively related with NDF content through physical
effect or filling effect [34, 35].
Digestibility reflects the amount of feed consumed,
digested, absorbed and utilized by animals’ body to
supply nutrient requirement. This is an early
indication of nutrient availability for the animal, after
Table 2 Nutrients content of eight legumes harvested on 120 days after planting.
Nutrient (%) Shrub legumes
CT Q5455
CP bundey
DL MB juanita
MB cadaarga
CT millgara
CP molle SS
DM (%) 91.13 91.47 77.88 90.44 91.51 89.27 92.42 93.14
Ash (% DM) 6.16 9.20 7.84 9.38 10.40 6.87 14.74 9.07
CP (% DM) 18.16 18.24 18.73 18.49 17.85 17.95 18.08 18.17
CF (% DM) 38.03 38.34 32.49 41.27 41.06 37.24 34.51 38.23
EE (% DM) 2.43 2.02 2.66 2.83 2.62 2.77 2.05 2.36
NDF (% DM) 59.22 61.23 61.88 61.86 60.29 55.96 56.68 58.56
ADF (% DM) 42.63 41.22 42.08 44.67 45.50 39.87 40.04 43.80
Cell. (% NDF) 31.17 26.94 30.53 30.55 29.97 28.19 23.73 31.97
Hemi-cell. (% NDF) 16.59 20.10 19.80 17.20 14.79 16.09 16.64 14.76
Silica (% NDF) 0.09 0.92 1.10 0.91 1.30 0.38 4.35 0.52
Lignin (% NDF) 11.37 13.36 10.46 13.21 14.23 11.30 11.95 11.32
GE (kkal kg-1) 3,493 3,725 3,548 3,188 3,099 3,895 3,151 3,361
Laboratory Analysis, Laboratory of Animal Feed and Nutrition, Faculty of Animal Husbandry, University of Brawijaya, Malang, Indonesia, 2011.
Production and Nutritive Value of Shrub Legumes in West Timor, East Nusa Tenggara, Province, Indonesia
353
Table 3 In vitro DM, OM and CP digestibility of shrub legumes harvested on 120 days after planting.
Shrub legumes In vitro digestibility (%)
DM OM CP
CT Q5455 61.1a ± 3.1 63.3c ± 2.3 60.5 ± 5.5
CP bundey 64.3a ± 1.3 60.6bc ± 1.2 70.6 ± 2.5
DL 74.2b ± 0.9 74.7d ± 1.4 57.7 ± 5.0
MB juanita 64.6a ± 3.2 62.9c ± 2.7 62.8 ± 1.1
MB cadaarga 67.1a ± 1.7 64.8c ± 1.9 62.9 ± 1.6
CT millgara 59.9a ± 2.3 55.8ab ± 2.9 67.5 ± 3.4
CP molle 64.7a ± 1.9 61.9bc ± 2.2 62.2 ± 4.0
SS 60.9a ± 1.5 53.7a ± 1.1 59.9 ± 0.9 a-dValues in the same column with different superscript are significantly different (P < 0.01).
consumption. The higher the feed digestibility, the
higher the nutrient supply to the animals’ body. In
vitro, DM, OM and CP digestibility of shrub legumes
harvested on 120 days after planting evaluated in this
research are presented in Table 3.
Data in Table 3 showed that in general there was no
significant difference (P > 0.05) of DM digestibility of
eight shrub legumes, except for DL which showed
significantly the highest (P < 0.01) DM and OM
digestibility compared to the other. DL contained the
highest CP but the lowest CF and lignin. It is well
known that high CP content supports high digestibility,
however, high CF and mainly lignin content suppress
digestibility [27, 35]. In respect of Centrosema
pubescen, DM, OM and CP digestibility in this study
were lower than those reported by Ginting et al. [28]
which were 73.3%, 74.2% and 89.9%, respectively.
Digestibility of Clitoria ternatea and Centrosema
pascuorum cv. cavalcade was higher than Rubianty et
al. [6] who reported that DM digestibility of Clitoria
ternatea and Centrosema pascuorum cv. cavalcade in
hay form is about 50.15% and 53.52%, OM
digestibility is 53.47% and 55.67%. Zhou et al. [36]
reported DM digestibility of Flemingia macrophylla
and Casia bicapsularis was at range 58.18%-71.81%
while OM digestibility ranged from 40.70% to
72.70%. Mlay et al. [37] reported OM digestibility of
Macroptilium atropurpureun cv. siratrio is 65.8%,
which was close to the results in this research.
4. Conclusions
It was concluded that Stylosanthes seabrana,
Clitoria ternatea Millgara, Clitoria ternatea Q5455,
and Centrosema pascuorum Bundey produced the
highest digestible protein compared to the other four
legumes. The earlier four legumes are potential as
protein feed supplement to improve crude protein
availability, which is as the most often deficient
nutrient. Therefore, it has potential to be developed in
West Timor.
Acknowledgments
The authors gratefully acknowledge The Board of
Agricultural Research Development and Higher
Education, Ministry of Agriculture, Jakarta for the
financial support. The authors further appreciate the
Field Laboratory of Assessment Institute of
Agriculture Technology (AIAT) Kupang District,
West Timor for growing the legumes.
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Journal of Agricultural Science and Technology A 3 (2013) 356-367 Earlier title: Journal of Agricultural Science and Technology, ISSN 1939-1250
Study of Sandy Soil Compaction
Andrea Formato, Gian Pio Pucillo and Antonio Abagnale
Department of Agricultural Science, University of Naples, Naples, Italy
Received: February 11, 2013 / Published: May 20, 2013. Abstract: In this paper, a study of sandy soil compaction with different granulometry and moisture content has been performed, and soil mechanical property variations in moisture and granulometry have been investigated. Investigations were performed to compare hydrostatic compression test (HCT) responses and evaluate the compression index, Cc, which is an indicator of the soil’s susceptibility to compaction-induced damage. The experiments have been performed on 24 soil samples typologies. Each sample has been obtained by combining three types of soil granulometry (types A, B and C) with a relative content varying from 0% to 100% in 20% increments. Soil type A had a granulometry ranging between 0.5 mm and 1 mm, type B between 0.25 mm and 0.5 mm, and type C less than 0.25 mm. These samples were representative of a sandy soil, chemically inactive and had various granulometries and initial moisture contents. A cell for HCT has been set up to allow the initial volume measurement of the test pieces and the subsequent changes during HCT with an estimated error less than 0.1 cm3. All samples were pre-compacted and prepared in agreement with the actual standards. The experimental data are reported in diagrams, the data allowed comparison of the mechanical behaviors between the considered unsaturated soils and underlined how soil moisture and granulometry affect soil response during HCT. Furthermore, because of the methodology used, the equipment was very economical. Key words: Compression test cell, hydrostatic soil compression, soil mechanics, unsaturated soils, sandy soil compaction.
1. Introduction
Soil compaction is a phenomenon causing both the
reduction of agricultural production and an increase in
the energy required to perform soil workmanship [1-7].
Undoubtedly, a better understanding of soil response
to applied loads can improve workmanship systems.
In fact, this knowledge allows the design of more
efficient soil workmanship tools and traction devices
[8-13]. The basic subject concerning soil mechanics in
engineering studies can be summarized as follows:
given an initial state, in terms of strains, stresses and
boundary conditions, the study of the dynamics of a
known type of soil deals with the determination of the
consequent deformation of the masses when external
loads are applied [14-16]. Unlike engineering soils,
unsaturated soil mechanics show greater complexity,
as reported in many important studies [17-19], even in
the simplified hypothesis of both continuity and
Corresponding author: Andrea Formato, professor,
research field: agricultural engineering. E-mail: formato@unina.it.
isotropy. Agricultural soils are unsaturated soils with
few proper exceptions, the main difference from
engineering soil is the organic content. Apart from this
difference, some mechanical phenomena studied in
agriculture (such as the soil spontaneous arrangement
and compacting due to surface loads) point out
problems that are very similar to those experienced by
unsaturated soils in engineering. These problems
come from the prominence of the involved masses,
from the slow evolution of the phenomena and from
the important role of capillary stresses. Therefore, due
to the above-mentioned phenomena, it seems logical
to extend the unsaturated soil mechanics to
agricultural soils through proper engineering.
Nevertheless, some consider such a statement
excessive for agricultural soil break-up workmanship,
so following an approximated and less complicated
approach could be worthwhile [20-22]. Supposing an
initial macroscopic homogeneity of the system before
the tool action, we have to consider a large percentage
of water in its liquid state taking up only a part of the
DAVID PUBLISHING
D
Study of Sandy Soil Compaction
357
voids’ volume. Both meniscus formation (capillary
forces) and stresses can occur due to the presence of
water and air. In these conditions, these forces can be
considered almost negligible in comparison with those
forces related to the physical-chemical bonds that act
on the particles and that require soil break-up by
means of mechanical tools [23, 24]. Therefore, we can
postulate that the contribution to the stress state
provided by the pressure difference between the two
fluid phases for the soil breaking-up workmanship can
be negligible in comparison with that contribution
provided by the forces generated by the working tools.
In these terms, the total main measurable stresses may
be sufficient to define the stress state at every point for
the agricultural soil submitted to breaking-up
workmanship considering the homogeneity, isotropy,
continuity of the soil and neglecting the forces due to
the fluid. In those cases, the soil characteristic
relationship is expressed by a spatial diagram,
reporting the strain variable of the void index, e, for
the stress state parameters, analogous to the actual
approach utilized in the theory of the critical state of
saturated soils [25-28]. Therefore, the soil state can be
represented by assuming the voids index as the strain
variable and expressing it as a function of the applied
load. Generally, this scheme is used to study the
soil-tool interaction. The soil-tool interaction
phenomena have been studied by numerous authors
with mathematical models [29-31]. According to the
mechanical behaviour of the considered soil, the
authors used a particular model, and also with the
simplified hypothesis of homogeneity, isotropy and
continuity of the soil, they were able to validate the
models, obtaining satisfying results. The
above-mentioned hypothesis, utilized to model the soil
for the study of the soil-tool interaction, is therefore
not valid, in general, for engineering soils and for the
soil physics in engineering soils. Furthermore, as we
know, hydrostatic compression tests are also
performed to evaluate the soil mechanical behaviour
when necessary. The soil compression characteristic is
a fundamental mechanical property of the soil that
relates the effect of compressive stress on the soil
volumetric parameter [32]. The modulus of the slope
of the virgin compression curve is commonly called
the compression index, Cc. The Cc parameter is an
indicator of both the susceptibility of a soil to damage
by compaction [33, 34] and of compressibility. The
study of compression curves for widely different soils
aimed to relate, the compression curves to properties
of agricultural soils, and to present methods for
predicting and describing the degree of compaction
from an applied stress has been performed by
numerous authors [35, 36], but since this matter is
ample and complex, they are still necessary further
close examinations and tests. Addressed in this way,
in this paper, we will show how the compacting
curves are obtained by hydrostatic compression tests
on loose sandy soil change with both soil
granulometry and soil moisture content (saturation
value minus the moisture value) and also how to
evaluate the compression index Cc based on
laboratory test data. The tests have been performed on
soil test pieces prepared by starting from a loose soil
with a known granulometry and moisture value and
opportunely pre-compacted to simulate a compacted
agricultural soil in workmanship conditions. The
experiments have been performed using a simplified
equipment set-up that is able to perform compression
hydrostatic tests on initially uncompacted sandy soil
(with known composition and granulometry) and for
different moisture conditions.
2. Materials and Methods
To express the strain state, the voids index is
adopted. Denoting the soil sample total volume and
that occupied by the solid with Vc and Vs, respectively,
the void index is:
(1)
we can adopt the normal mean stress, p, and the
Study of Sandy Soil Compaction
358
deviatoric stress, q, as stress state variables. In
particular, denoting as pc the hydrostatic pressure
detected in the test cell and the (measurable)
increase of axial stress acting on the soil test piece
(deviatoric stress), the total stress is:
(2)
On the basis of the above hypotheses, the soil
characteristic can be represented with a spatial
diagram in which the strain state variable, e, depends
on the stress state variables p and q [37-40]. As stated,
the change of the void index according to the
compression pc for a determined soil has been studied
in this research. Therefore, to characterize the soil
response, test equipment has been set up that is able
to detect the hydrostatic compression curves and to
evaluate the changing of the strain variable of the
voids index e for a determined soil.
2.1 Test Equipment
The equipment scheme is shown in Fig. 1. The
equipment includes a cell (6) connected to a
measuring burette (1). The test cell has two flanges (7,
8) that are fixed to a transparent Plexiglas cylinder by
means of brass screws. In addition to the sealing
rings (10), the two flanges have a central conic seat
that avoids air bead formation during cell filling and
emptying operations. The equipment is fully
demountable to allow fast soil test piece preparation.
The upper flange (7) includes a 10 cm3 full scale
burette with 0.1 cm3 resolution that allows accurate
evaluation of soil test piece volume variations. The
burette, however, is able to detect a maximum total
volume variation of only 10 cm3. This problem has
been overcome with an auxiliary reservoir (4), at the
same pressure as the water in the test cell and located
at a higher height. The auxiliary reservoir is connected
by a pipe with an R2 tap to the lower zone of the test
cell. As shown in Fig. 1, the cell and the auxiliary
reservoir are always at the same pressure during the
test. Therefore, opening the tap will send water into
the cell to perform some compensation during the test
when the volume variations are greater than 10 cm3.
During the tests, the cell is submitted to the pressure
imposed by a circuit fed by the reservoir of a
compressor machine. The pressure value is indicated
by a pressure transducer (5) and recorded using a
data acquisition system with a sampling frequency of
1 Hz. The pressure transducer has a 200 kPa full scale
Fig. 1 Equipment scheme.
Study of Sandy Soil Compaction
359
and 5 kPa resolution. The lower flange (8) has a base
(9) where the soil test piece is installed. The base is
connected to the outside through a porous septum.
2.2 Test-cell Setting
Because a Plexiglas cylindrical cell was used, it has
been necessary to evaluate its deformations. Therefore,
after flooding the whole circuit with water, the
equipment was pressurized up to 200 kPa in 5 kPa
increments. The corresponding test cell volume
variations have been recorded.
2.3 Test Piece Preparation
The container for the soil samples is made of 0.5 mm
thick rubber latex that guarantees water-tight sealing for
the soil test piece when it is pressurized. Furthermore,
during the soil filling of the rubber container, because
the working pressure was approximately 1 kPa, the
container elastic reaction on the soil was negligible.
Once the rubber latex container was filled with the
considered soil, the soil test piece was hermetically
sealed by inserting the base, which was shut using a
thread and adhesive tape. Subsequently, the soil test
piece was weighed and then placed in the test cell on
the base (9) to measure the sample volume.
2.4 Soil Volume Measurement
To measure the volume of the soil test piece, it is
necessary to refer to an initial value of the water
volume in the measuring circuit, which is made up of
two branches: one is the test cell with a notched
burette (1) and the other is the burette (2). To
determine the initial value of the water volume, water
was introduced into the system as far as possible (in
both branches), and two lined-up notches were placed
on burettes (1) and (2). The water surfaces of both
branches must coincide with the two lined up notches
when the soil test piece is not in the cell, and in this
condition, the R1 faucet is open and only the
atmospheric pressure works on the water surfaces.
Next, burette (2) is moved downward so that its water
surface is below the flange (8), and the entire volume
of the water circuit moves into burette (2), emptying
the test cell to allow the introduction of the soil test
piece. The R1 faucet is then closed. After installing
the soil test piece, closing and sealing the test cell,
burette (2) of the system (with the R1 faucet closed)
is returned to its initial position where the two
reference notches of the system were lined up.
However, the two water surfaces no longer line up.
By moving all of the circuit water into burette (2), its
water surface is now higher than the reference notches.
Therefore, by opening the R1 faucet, the test cell is
flooded until the water level reaches the preset notch
on burette (1). The R1 faucet is then closed to
interrupt the connection between the two branches.
The level of the water in burette (2) will now be
different from the level in burette (1). The gradient
between the two burettes is equal to the volume of the
soil test piece to be measured. Therefore, the value of
the water volume in burette (2) between the reference
notch and the water surface represents the measure of
the initial volume, Vpi, of the soil test piece. The
volume values of the fitting masses (lower base,
porous septum, rubber membrane) must be deducted
from the initial value of the test piece. Therefore, once
the initial mass of the moisture soil test piece (Mw, its
moisture (w), the density of the solid (s), the initial
volume of the soil test piece (Vpi), and the change in
volume (V) are known, the other characteristic
parameters can be determined as follows: dry mass:
Md = Mw/(1+w); solid phase volume: Vs = Md/s;
soil test piece volume: Vc = Vpi - V; void
volume: Vv = Vc - Vs; void index: e = Vv/Vs.
2.5 Soil Sample Preparation
Starting with three types of soil granulometry,
namely granulometry type A (ranging between 1 mm
and 0.5 mm), B (from 0.5 mm and 0.25 mm) and C (<
0.25 mm), and combining the three types to create a
relative content varying from 0% to 100% in 20%
increments, 24 soil samples were created for the
Study of Sandy Soil Compaction
360
experiments. Each component (types A, B or C) was
present with a content of 0%, 20%, 40%, 60%, 80%
or 100%. The soil sample labels are shown in Table 1.
Additionally, U0, U5 and U10 refer to a moisture
content of 0%, 5% and 10%, respectively. Each
sample was chemically inactive, easily feasible in the
laboratory, and quite representative of a sandy soil.
2.6 Initial Compression of the Test Piece
Because agricultural soils, when subjected to
workmanship, are at a certain level of compaction, all
of the soil test pieces were submitted to 100 kPa
compaction prior to performing the compression tests.
After compaction, the water volume in burette (1) will
decrease to a certain value due to the reduction in
volume of the soil test piece. After the pre-compacting
step, the water level in the burette was re-established
to perform the scheduled tests.
Table 1 Soil granulometry with weight (%) on dry soil.
Soil type
Granulometry type A between 1 mm and 0.5 mm
Granulometry type B between 0.5 mm and 0.25 mm
Granulometrytype C < 0.5 mm
S01 0 0 100
S02 0 20 80
S03 0 40 60
S04 0 60 40
S05 0 80 20
S06 0 100 0
S07 20 0 80
S08 40 0 60
S09 60 0 40
S10 80 0 20
S11 100 0 0
S12 20 80 0
S13 40 60 0
S14 60 40 0
S15 80 20 0
S16 20 60 20
S17 20 40 40
S18 20 20 60
S19 60 20 20
S20 40 20 40
S21 20 20 60
S22 60 20 20
S23 40 40 20
S24 20 60 20
2.7 Test Execution
All of the considered soil test pieces were submitted
to hydrostatic compression following the load cycle
from 0 kPa to 1.5 × 102 kPa in steps of 1 kPa. The
change in volume of the soil test piece was recorded
for every step of pressure increase. During the initial
compression phase, the soil test piece under a constant
pressure continuously changes in volume, resulting in
a time dependence of the volume reached by the soil
test piece during the test. After 5 min, however, the
volume variations of the soil test piece at the same
pressure were almost negligible. Every test has been
repeated three times, and a statistical analysis of the
obtained data has been performed. The results were
processed using Statgraphics Plus Package 4.1
(Statpoint Technologies, INC., Warrenton, Virginia,
USA). A variance and media analysis were performed
to evaluate the percentage error for the obtained
results in the different tests performed.
3. Results and Discussion
Figs. 2-4 show the p-e diagrams obtained
experimentally, where p is the pressure value used for
the soil test pieces and e is the corresponding value of
the void index. Each point represents the mean value
of three experimental data, with a maximum error less
than 5% detected during all of the tests. From the p-e
diagrams, a good correspondence to the well-known
behaviour of soil submitted to compression tests can be
seen. The curves obtained by the hydrostatic
compression tests demonstrate that the unsaturated soil
behaviour is quite similar to that of a saturated soil
during the drained compression tests. Starting from an
initial value of the voids ratio, the soil shows a
decreasing volume with increasing pressure. We have
noted that these data results are in agreement with the
results obtained by previous studies available in
literature. [1, 5, 6, 13, 17-19, 25, 27, 35, 38, 41]. In
fact, this soil behaviour is due mainly to two
phenomena: a re-arrangement of the particles, which
probably remain undeformed and tend to fill the initial
Study of Sandy Soil Compaction
361
0,40
0,50
0,60
0,70
0,80
0,90
1,00
0 0,2 0,4 0,6 0,8 1 1,2 1,4
Pressure - 10 5 Pa
Vo
id in
dex
- e
-
S01_U0
S02_U0
S03_U0
S04_U0
S05_U0
S06_U0
S07_U0
S08_U0
S09_U0
S10_U0
S11_U0
S12_U0
S13_U0
S14_U0
S15_U0
S16_U0
S17_U0
S18_U0
S19_U0
S20_U0
S21_U0
S22_U0
S23_U0
S24_U0
Fig. 2 p-e diagrams of the considered soil samples with moisture content of 0%.
0,50
0,60
0,70
0,80
0,90
1,00
1,10
0 0,2 0,4 0,6 0,8 1 1,2 1,4
Pressure - 10 5 Pa
Vo
id in
dex -
e -
S01_U5
S02_U5
S03_U5
S04_U5
S05_U5
S06_U5
S07_U5
S08_U5
S09_U5
S10_U5
S11_U5
S12_U5
S13_U5
S14_U5
S15_U5
S16_U5
S17_U5
S18_U5
S19_U5
S20_U5
S21_U5
S22_U5
S23_U5
S24_U5
Fig. 3 p-e diagrams of the considered soil samples with moisture content of 5%.
Study of Sandy Soil Compaction
362
0,50
0,60
0,70
0,80
0,90
1,00
1,10
1,20
0,00 0,20 0,40 0,60 0,80 1,00 1,20 1,40
Pressure - 10 5 Pa
Vo
id in
de
x -
e -
S01_U10
S02_U10
S03_U10
S04_U10
S05_U10
S06_U10
S07_U10
S08_U10
S09_U10
S10_U10
S11_U10
S12_U10
S13_U10
S14_U10
S15_U10
S16_U10
S17_U10
S18_U10
S19_U10
S20_U10
S21_U10
S22_U10
S23_U10
S24_U10
Fig. 4 p-e diagrams of the considered soil samples with moisture content of 10%.
interstitial voids, and an elastic volumetric change of
the whole particles considerably larger than the sum of
the deformations of the single particles. In Figs. 2-4,
the compacting curves obtained from the soil samples
with different moisture values (0%, 5% and 10% in
weight of water) are also compared. Obviously, the
different moisture values cause the initial values of the
voids ratio to be different. In particular, the initial voids
ratio increases with the moisture content, which agrees
well with the compacting curves obtained by other
authors, that have studied this phenomena [1, 6, 17, 27,
28, 40, 42].
Because the moisture content is far from the
saturation point, such behaviour is due to the formation
of new aggregates of particles. In fact, inside a particles
system, the water increase causes the particles
rearranging in agglomerations with greater mean
diameter and so the void index increases, as mentioned
earlier. The compacting curves of the investigated soil
samples demonstrate the regular behaviour of the
samples. Three values of moisture content were
considered, and we have noted that every curve
shows a limit value, and this is in agreement with the
data results obtained by numerous authors. [1, 2, 6, 9,
13, 14, 16, 17, 22, 23]. Table 2 summarizes the Cc
values for the various soil sample compositions, which
have been evaluated considering the first four points in
the p-e diagram. In the same table, the A (big), B
(mean) and C (thin) granulometry values for the soil
samples considered are reported. In the following
section, the experimental data have been compared
considering six groups of soil composition:
Group 1: soil samples S01, S02, S03, S04, S05, S06
In this group, the soil samples with granulometry
types C and B have been compared. Examining the
diagrams with u = 0%, where u is the moisture
content, an exponential law for the compaction
curves can be seen, according to that was found by
numerous authors [1, 2, 6, 9, 13, 14, 16, 17, 22, 23, 42,
43]. The lower limit is attained with the S01 type with
Study of Sandy Soil Compaction
363
Table 2 Cc values for the soil samples considered, obtained considering the first four points in the p-e diagram: Mean ± dxm (data average of three samples considered) dxm = standard deviation of the Mean.
U = 0% Mean ± dxm
U = 5% Mean ± dxm
U = 10% Mean ± dxm
S01 -0.2172 ± 0.068 -0.5319 ± 0.049 -0.6398 ± 0.035
S02 -0.2172 ± 0.041 -0.5319 ± 0.061 -0.6398 ± 0.040
S03 -0.2172 ± 0.032 -0.5319 ± 0.039 -0.6398 ± 0.061
S04 -0.2172 ± 0.055 -0.5319 ± 0.058 -0.6398 ± 0.039
S05 -0.2172 ± 0.022 -0.5319 ± 0.042 -0.6398 ± 0.049
S06 -0.2164 ± 0.041 -0.5319 ± 0.053 -0.6398 ± 0.052
S07 -0.3314 ± 0.037 -0.4720 ± 0.060 -0.4040 ± 0.059
S08 -0.3334 ± 0.051 -0.4720 ± 0.057 -0.3066 ± 0.038
S09 -0.3254 ± 0.045 -0.4720 ± 0.049 -0.3066 ± 0.047
S10 -0.3212 ± 0.062 -0.4720 ± 0.055 -0.3860 ± 0.051
S11 -0.1954 ± 0.055 -0.4720 ± 0.066 -0.4160 ± 0.039
S12 -0.3334 ± 0.061 -0.4720 ± 0.058 -0.3066 ± 0.051
S13 -0.2934 ± 0.043 -0.4720 ± 0.049 -0.3066 ± 0.055
S14 -0.3334 ± 0.058 -0.4720 ± 0.056 -0.3066 ± 0.046
S15 -0.3334 ± 0.039 -0.4720 ± 0.045 -0.3066 ± 0.049
S16 -0.1840 ± 0.047 -0.3090 ± 0.042 -0.5737 ± 0.054
S17 -0.1840 ± 0.051 -0.3090 ± 0.046 -0.5537 ± 0.057
S18 -0.1840 ± 0.060 -0.3090 ± 0.054 -0.5537 ± 0.059
S19 -0.1840 ± 0.048 -0.3090 ± 0.051 -0.5537 ± 0.038
S20 -0.1840 ± 0.039 -0.3090 ± 0.048 -0.5737 ± 0.052
S21 -0.1840 ± 0.053 -0.3090 ± 0.039 -0.5937 ± 0.042
S22 -0.1840 ± 0.039 -0.3090 ± 0.045 -0.5937 ± 0.056
S23 -0.1840 ± 0.048 -0.3090 ± 0.061 -0.5737 ± 0.039
S24 -0.1840 ± 0.051 -0.3090 ± 0.053 -0.5737 ± 0.060
100% of granulometry type C. By increasing the
percentage of B, it increases the total voids index for
the soil sample (B, C) and therefore the curves move
upward and the curves’ slopes increase. Then the
curves show higher values of void index and the gap
between the curves decreases (Figs. 2-4). This is in
agreement with the data available in literature. [1, 6,
13, 16, 17, 27, 34-36, 42]. For the soil samples
considered, changing the content of soil granulometry
B and C, the compression index shows low variations
(between -0.2164 and -0.2172 at u = 0%; -0.5319 at u
= 5%; -0.6398 at u = 10%). As shown in Table 2, by
increasing the moisture content, the compression
index increases, ranging between -0.2164 and -0.6398.
This behavior was also found by numerous authors.
[13, 17, 18, 27, 32, 34, 35, 38, 42, 43].
Group 2: soil samples S01, S07, S08, S09, S10, S11
With soil samples composed of C and A soil types,
the data show that at 0% moisture content, by
increasing the percentage of granulometry type A with
big granulometry in the soil sample considered (A and
C), the total voids index increases, and therefore the
curves tend to shift upward. As in the previous case,
when the moisture content increases, the gap between
the curves decreases and the slope increases (Figs.
2-4). This is in accord with numerous authors [1, 6, 13,
16, 17, 27, 34-36, 42]. For the soil samples
considered, by changing the percentage of A and C,
the compression index changes (between -0.1954 and
-0.3334 at u = 0%; between -0.4720 and -0.5319 at u
= 5%; between -0.3066 and -0.6398 at u = 10%).
Excluding the S01 granulometry combination, the
Cc increases when the moisture content changes from
zero to 5% and decreases when u = 10% (Table 2).
Group 3: soil samples S06, S12, S13, S14, S15, S11
In this group, the soil samples with granulometry
Study of Sandy Soil Compaction
364
type B and A have been compared. With 0% moisture
content, the curves are included between those of S06
and S11. With the increasing percentage of
granulometry type A with big granulometry, in the soil
sample (A and B), the total voids index increases, and
therefore the curves shift upward. By increasing the
moisture content, these differences tend to decrease and
the curves’ slopes increase (Figs. 2-4). By varying the
soil composition, the Cc also changes (between -0.1954
and -0.3334 at u = 0%; between -0.4720 and -0.5319 at
u = 5%; between -0.3066 and -0.6398 at u = 10%).
Excluding the S06 composition, the Cc increases when
the moisture content changes from 0% to 5% and
decreases when u = 10% (Table 2).
Group 4: soil samples S12, S16, S17, S18, S07
In this group, the soil samples with 20%
granulometry type A and different percentages of
granulometry type B and C have been compared. With
a moisture content of 0%, the curves of the soil
samples S07 (B = 0%) and S12 (C = 0%) are quite
similar, while the others shift upward. These
differences decrease when the moisture content
increases (Figs. 2-4). That is, the percentage increase
of the component B, in the soil sample (A, B and C),
increases the total voids index, and therefore the
curves move upward. For the soil samples, S16, S17
and S18, the granulometry variations do not
appreciably influence the compression index (between
-0.1840 and -0.1840 at u = 0%; -0.3090 at u = 5%;
-0.5537 at u = 10%), but greater values were found for
S12 and S7 at each moisture content (Table 2).
Group 5: soil samples S15, S19, S20, S21, S02
In this group, the soil samples with 20%
granulometry type B and different percentages of
granulometry type A and C have been compared. With
0% moisture content, the curves of the soil samples
S15 (C = 0%) and S02 (A = 0%) are shifted
downward from the other curves. However, these
differences decrease when the moisture content
increases (Figs. 2-4). In these soil samples with (A, B
and C) components, the influence of the component A,
with big granulometry, is noticed. In fact, when there
is not the component A, the mean granulometry of the
soil sample (A, B and C) is lower than the component
A. For the soil samples S19, S20 and S21, when
changing the soil granulometry, the compression
index Cc shows small variations (-0.1840 at u = 0%;
between -0.3090 and -0.3048 at u = 5%; -0.5737 at u =
10%), but greater values were found for S02 and S15
at each moisture content (Table 2).
Group 6: soil samples S10, S22, S23, S24, S05
In this group, the soil samples with 20%
granulometry type C and different percentages of
granulometry type A and B have been compared.
With 0% moisture content, the curves of the soil
samples S05 (A = 0%) and S10 (B = 0%) are shifted
downward from the other curves. These differences
decrease when the moisture content increases (Figs.
2-4). The behavior of the soil sample (A, B and C) is
influenced by the presence of the components with
granulometry A and B (big and mean), being fixed to
20% the component C. Therefore their presence
moves upward the obtained diagrams. For the soil
samples considered, by changing the soil
granulometry, the compression index shows a small
variation for S22, S23 and S24 (-0.1840 at u = 0%;
-0.3090 for u = 5% and -0.5737 and -0.5937 for u =
10%), but greater values were found for S05 and S10
at each moisture content. Excluding the S10
granulometry combination, the Cc value increases
with moisture content, ranging between -0.1840 and
-0.5937. The data show that for three typology of soil
granulometry combinations, the slope, for a fixed
value of moisture content, is almost insensitive to the
granulometry composition, in the first group, the A
component is present and the B or C components are
absent; in the second, A is absent and B and C are
present; in the last, all the granulometry types are
present. More specifically, among these typologies,
the greatest slope values are obtained in the second
group. The slope’s absolute value for a fixed
granulometry combination increases with moisture
Study of Sandy Soil Compaction
365
content, with the exception of those combinations in
which neither B nor C is present. Moreover, from all
the obtained Cc data, the minimum value is attained
when the granulometry composition is balanced, that
is, all soil types are present. The maximum value is
obtained with A and C granulometries (A = 40%, C =
60%) and A and B granulometries (A = 20%, B = 80%
and A = 60%, B = 40%). From the diagrams obtained
at the same moisture level but in soils with different
granulometries, we can see that the inclination of the
curves depends on the granulometry value and that the
effects due to the different granulometries tend to zero
as moisture increases, i.e., the effects due to the
different granulometries decrease when the moisture
increases. Comparing the graphs that have been
obtained at the same soil granulometry but with
different moisture values, we can see that the curves’
slopes depend on the moisture value. Therefore,
during these tests, the following results have been
obtained:
(1) All the diagrams obtained by submitting the soil
test pieces to hydrostatics compression tests follow an
exponential curve in the p-e plane, which is the curve
of consolidation;
(2) When the moisture increases under the same
pressure, the soil undergoes greater deformation,
becoming more sensitive to the increasing pressure.
All that was also noted by numerous authors [1, 6,
16, 17, 19, 28, 32, 35, 39, 42], therefore, according to
all the obtained comparisons, it was possible to
confirm the validity of the considered equipment.
4. Conclusions
A study of sandy soil compaction with different
granulometry and moisture content has been
performed. In this way, we can characterize the
considered soil and determine more easily the soil
model that could be utilized in the soil-tool interaction
study. Further, simplified equipment to carry out
hydrostatic compression tests on unsaturated soils has
been set up that makes it possible to examine soil test
pieces with different granulometries and with different
moisture values (those soils were unsaturated and far
from the saturation point). The obtained data agree
with the most reliable theories about soil compression
and confirmed the validity of the equipment.
Furthermore, the considered equipment was very
inexpensive compared to the prices of other
equipment of the same type but maintained the same
margin of error.
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[42] A.S.S. Noor, Soil compression index prediction model for fine grained soils, International Journal of Innovations in Engineering and Technology (IJIET) 1 (4) (2012) 34-37.
[43] B.T. Tiwari, B. Ajmera, New correlation equations for compression index of remoulded clays, J. Geotech. Geoenviron. Eng. 138 (6) (2012) 757-762.
Journal of Agricultural Science and Technology A 3 (2013) 368-379 Earlier title: Journal of Agricultural Science and Technology, ISSN 1939-1250
Potential Impacts of Various Land Use Forms on Land
Degradation in Arid Regions (Case Study: Kuwait)
Raafat Misak1 and Adeeba Al-Hurban2
1. Environmental and Urban Development Division, Kuwait Institute for Scientific Research (KISR), Safat 13109, Kuwait
2. Department of Earth and Environmental Sciences, Faculty of Science, Kuwait University, Safat 13060, Kuwait
Received: January 14, 2013 / Published: May 20, 2013. Abstract: During the last 20 years, land use in Kuwait was remarkably changed in various forms. Such changes resulted in positively constructive, as well as, adversely destructive impacts on the local environment and ecology. In 1993-1994, a buffer zone of 15 km wide and more than 200 km long was established between Iraq and Kuwait. This allowed the restoration of biodiversity, enhancement of ecological conditions and stabilization of fragile soils. In 1991, long bund walls were constructed along the Saudi-Kuwaiti borders by Saudi Arabia and in 1993-1994, a ground trench was dug along the Iraqi-Kuwaiti borders by Kuwait. Bund walls are piles of excavated soils, 2-3 m high, 3-5 m wide and several tens of kilometers length. Constructing 1 km length of a bund wall required 2,500-3,000 m3 (1,000 m length × 5 m average width × 0.5 m depth) amount of soil, which disturbed a groundcover strip of an average area of 10,000 m2 (1,000 m length × 10 m average width). Border trenches are ground hollows 2-3 m deep, 3-5 m wide and 220 km long. The amount of excavated soil from digging 1 km long trench ranged from 6,000-8,000 m3. Field work indicated that 1 km long of trench had disturbed a groundcover of an average area of 12,000 m2 (1,000 m length × 12 m average width). Such man-induced land features are closely related to land degradation processes, as they were of adverse environmental impacts on soil, surface hydrologic conditions and natural vegetation. The main objective of this study is to assess the immediate and long term impacts of the introduced land use forms in selected areas in Kuwait, such as Wadi Al Batin (Western part of Kuwait). To achieve such an objective, intensive field program was designed and implemented and the collected data and available information were analyzed and interpreted. Key words: Bund walls, trenches, man-induced land features, surface hydrologic conditions.
1. Introduction
The State of Kuwait occupies a total area of about
118,000 km2. From West and North, it is bordered by
Iraq and from South by Saudi Arabia. The Arabian
Gulf skirts Kuwait from East (Fig. 1). The
Kuwaiti-Iraqi borders are marked by a ground trench
(about 3-5 m wide and 2-3 m deep), established by
Kuwait in 1993-1994 and running along about 212 km.
About 70 km of this trench cut the main course of
Wadi Al Batin, the natural barrier between Iraq and
Kuwait. On the other hand, the border between
Kuwait and Saudi Arabia is marked by a bund wall
Corresponding auther: Raafat Misak, professor, research
fields: desert geomorphology, geomorphological and environmental changes in desert, semi-arid and arid regions. E-mail: rmisak@kisr.edu.kw.
(2-3 m high and 3-5 m wide), established by Saudi
Arabia in 1991. Both the trench and bund wall
introduced significant change to the local
environmental, ecological and hydrological systems
existing in the area.
Precipitation in Kuwait is scanty and irregular [1].
Rainy season extends between October and April. The
average annual precipitation is around 110 mm.
Surface runoff is developed few hours after intensive
rainfall of 30-40 mm in one storm [2]. Flood events of
different intensities and impacts were recorded in
February 1993, December 1995, November 1997,
March 2002, January 2007, November 2009 and
January-February 2011.
Kuwait is located on the downwind side of the high
deflation area of the Mesopotamian flood plain in
DAVID PUBLISHING
D
Potential Impacts of Various Land Use Forms on Land Degradation in Arid Regions (Case Study: Kuwait)
369
Fig. 1 Satellite image of Kuwait showing bordering countries and two main strategic landforms (natural wind corridor and Wadi Al Batin). The arrow indicates the prevailing Northwesterly winds.
southern Iraq. This plain is considered as one of the
most significant sources of aeolian sediments to
Kuwait [3-5]. In Kuwait, the prevailing wind
directions are from the Northwesterly quadrant and to
a lesser extent from the southeast. Winds from other
directions occur less frequently and for shorter
durations [3]. The maximum wind speed ranges from
7.5 m s-1 to 20 m s-1 in June, while in July and August
it ranges from 10 m s-1 to 18 m s-1, and from 9 m s-1 to
19 m s-1, respectively. The average wind speed attains
5.8 m s-1, 5.4 m s-1 and 4.7 m s-1 in June, July and
August, respectively. According to the literature
sourced information and field observations, it was
indicated that the prevailing Northwesterly winds,
during summer and spring times, carry huge amounts
of drifted sand from Iraq to Kuwait.
1.1 Land Degradation
In Kuwait, land degradation processes were studied
by several authors including Khalaf [6], Omar, 1991
[7], Misak et al. [8], Zaman [9], Misak et al. [10],
Shahid et al. [11], Al-Dousari et al. [12], Al-Awadhi
and Misak [4], Al-Awadhi et al. [13], Omar et al. [14],
Misak et al. [15] and Misak et al. [5]. A comparison
between the density of the vegetation cover (mainly
perennial desert shrubs) in some parts of Kuwait
during early 1980s of the last century and Feb. 2010
revealed that vegetation cover has been severely
degraded [16].
As stated by Misak [17], land degradation processes
Potential Impacts of Various Land Use Forms on Land Degradation in Arid Regions (Case Study: Kuwait)
370
prevail in the majority of the terrestrial environment of
Kuwait, which constitutes about 80% of the country.
Land degradation processes include: loss of top soils
(through both wind and water erosion), soil crusting,
sealing and compaction, loss of soil productivity, soil
salinization, vegetation degradation, loss of
biodiversity and hydrological disruption.
Intensive anthropogenic activities (especially during
drought periods), in addition to the military operations
during the Gulf Wars of 1990-1991 and 2003
constitute the main causes of land degradation in the
terrestrial environment of Kuwait. These two main
causes are exacerbated by the inherent fragility of
soils, vegetation cover, micro-landforms and land
misuse. Generally, degradation is obviously
intensified during drought periods, e.g., 1983-1985,
2007/2008 and 2008/2009, whereas rainy periods, e.g.,
1993-1997 and 2003/2004 temporarily restore the
damage caused by land degradation.
The intensity, mechanism and extent of land
degradation exhibit remarkable variations in the
various land use forms [8]. Livestock grazing is the
major land use form in the open desert, which
constitutes about 75% of Kuwait’s surface area. In
these areas, indicators of soil, vegetation and
hydrological degradation, as well as, loss of
biodiversity are prevailing. In the agricultural areas
(about 2.7% of Kuwait), depletion of soil productivity,
water logging and soil salinization are recorded. In the
oil field areas (about 7% of Kuwait) soil
contamination by crude oil and surface deformation
by gatch pits (soil mining sites) are major indicators of
land degradation. The areal extent of oil
contamination in the oil field areas is about 114 km2
and the volume of contaminated soils is close to
4,400,000 m3 [18].
1.2 Interaction between Introduced Land Use Types
and Land Degradation Processes
During the period between 1992 and 2011, Kuwait
experienced remarkable changes in land uses, such as
fencing or protection of wide areas (about 12% of
Kuwait), either due to political demands or to
socioeconomic activities. Under the prevailing
environmental conditions of Kuwait, protection of
desert areas for at least five years resulted in
stabilization of active sandy bodies, enhancement of
physical characteristics of soils, encouragement of
natural recovery of vegetation and wildlife and
rehabilitation of biodiversity. Examples of positively
impacting introduced land use forms are:
establishment of the buffer zone between Iraq
and Kuwait (5 km wide and about 220 km long) in
1994/1995;
fencing of oil fields (about 7% of the country)
during the period 1996-2002;
re-fencing of Sabah Al Ahmad Wildlife Reserve
(about 330 km2) in 2000;
rehabilitation of Liyah degraded terrain (about
200 km2), to the north of Jahra City in 2003-2010;
managing camping and recreational activities in
open desert areas (ongoing);
setting up a plan for establishment of a number of
protected areas in the northwestern and Western parts
of Kuwait (2008);
establishment of Al Quran protected area in the
southeastern part of Kuwait (2011).
Literaturaly, little information is available about
the interaction between land use and land
degradation processes. Based on the intensity of
damage to ground cover (soils and vegetation) [5]
identified three classes of land use: a) extremely
destructive (damage to ground cover extends
between 1.5-5 m from ground surface); b) destructive
(damage to ground cover is restricted to the most
upper 50-75 cm thick of soil); and c) nondestructive
(damage to ground cover is not noticeable, with the
exception of war related damage). Khalaf et al. [16]
stated that bund walls and roads crossing drainage
basins act as dams for surface runoff during heavy
rainfall. The runoff water accumulates at the
upstream sides of the roads and bund walls.
Potential Impacts of Various Land Use Forms on Land Degradation in Arid Regions (Case Study: Kuwait)
371
2. Methods
To assess the impact of land use on land
degradation, the following activities were conducted:
Delineation of major and secondary drainage basins
(Wadis) using ArcGIS 9.3. For this purpose, Landsat
Thematic Mapper bands 2, 4 and 7 color composite
image of Kuwait (March 1995) was used. In this
image, the near infra-red band four was exposed
through a green filter and therefore significant
vegetation in the main channels and tributaries shows
up in shades of green. This image was recorded during
a good rainy season (about 167 mm of rainfall), so
very dense vegetation is shown in the main courses
and tributaries of drainage basins. Therefore,
digitization of these basins (Wadis) was an easy task.
Zones where drainage basins are dissected by human
induced barriers such as bund walls (2-3 m high and
3-5 m wide), trenches (3-5 m wide and 2-3 m depth)
and highways are delineated. These disrupted zones are
observed in images recorded after 1995. The areal
extent of disrupted zones in km2 was calculated using
Arc GIS 9.3. For this purpose landsat image of Kuwait
(2006) and google image of 2010 were applied.
About eight field trips during different seasons were
arranged to the western, southern and northeaster parts
of Kuwait. Frequent field trips aimed to: 1) roughly
estimate the amounts of soils which were excavated to
establish the border trench and bund wall; 2) assess
and record field observations on the mechanisms of
hydrologic disruption/ hydrologic drought; 3) locate
points of intersection between Wadis and surface
hydrological barriers including trenches, bund walls
and highways in at least 20 stations; and 4) to
document field findings through close up field
photographs.
3. Results and Discussion
Under the fragile environment of Kuwait, fluvial
and aeolian land forms and processes are very
sensitive to certain land use changes. At least three
land use types have negative impacts on the
mentioned processes and forms. These are raised
roads, bund walls and trenches. As observed in the
field, a 30-40 km segment of the border trench acts as
a trap for saltating sands coming from Iraq. Moreover,
this trench acts, during heavy rainy seasons, as an
artificial water collector for runoff water. Runoff
water results in considerable soil losses and collapses
of the sides of the trench.
3.1 Inventory and Assessment of Degradation
Processes Connected to Land Use
As indicated from the field survey during the course
of the present study, several mechanisms of land
degradation are observed in wide areas of Kuwait.
Some of these mechanisms are directly related to
current land use types, these include:
soil mining, vegetation degradation and loss of
biodiversity;
surface hydrologic disruption;
interruption of aeolian activities.
3.1.1 Soil Mining, Vegetation Degradation and
Loss of Biodiversity (the case of Iraq-Kuwait border
zone)
The establishment of a trench and a bund wall along
Kuwait-Iraq border resulted in soil mining and
disruption of desert surface. It is estimated that about
12,720,000 m3 and 375,000 m3 of soil were excavated
to establish the trench and the bund wall respectively.
At the same time at least 2,544,000 m2 and 1,500,000
m2 was disturbed by heavy machinery during digging
the trench and establishing the bund wall (Table 1).
The heavy machinery resulted in removal of
vegetation cover (mainly Haloxylon salicornicum and
Stipagro stisplumosa) and degradation of biodiversity.
It is observed that vegetation was completely cleared
from an area of approximately 4 km2 of land adjacent
to the trench and bund wall (Table 1).
3.1.2 Surface Hydrologic Disruption
Before establishment of the border trench between
Kuwait and Iraq in 1993-1994, the runoff water of the
tributaries feeding Wadi Al Batin from Western Iraq
Potential Impacts of Various Land Use Forms on Land Degradation in Arid Regions (Case Study: Kuwait)
372
Table 1 Approximate amount of excavated soils and disturbed areas along Kuwait-Iraq border.
Land use type (introduced form)
Amount of excavated soils (m3 km-1 length)
Total amount of excavated soils (m3)
Disturbed area (m2 km-1 length)
Total disturbed area (m2)
Remarks
Border trench 6,000 12,720,000 12,000 2,544,000 Total length 212 km
Bund wall 2,500 375,000 10,000 1,500,000
Total - 13,095,000 - 4,044,000
Fig. 2 Drainage basins in Kuwait and adjacent parts in Iraq and Saudi Arabia.
and Eastern Kuwait was flowing towards the main
course of the Wadi. Currently, the runoff water does
not drain in the main course of the Wadi. It is
accumulated in the border trench. Moreover, the
runoff water of Wadi Al Rimma (in Saudi Arabia) was
flowing towards Wadi Al Batin. The bund wall
between Saudi Arabia and Kuwait blocks the runoff
water flowing from Saudi Arabia to Kuwait. In
December 1995, December 2002, November 2009 and
January 2011 huge amounts of runoff water were
drained in the border trench.
To identify changes in surface drainage caused by
land use types, a detailed map for drainage basins was
produced by the first author (Fig. 2). Based on this
map, a detailed field survey was conducted on various
drainage basins.
Field survey indicated that almost all the drainage
basins are subjected to different degrees of surface
disruption. Disruption is represented by the following
forms:
loss of runoff water in the border trench;
blocking the surface runoff with its load of out
washed deposits in front of bund walls and raised roads;
modification of the courses of water channels;
fragmentation of drainage basins into portions of
different sizes;
Potential Impacts of Various Land Use Forms on Land Degradation in Arid Regions (Case Study: Kuwait)
373
soil losses through water erosion on both sides of
the border trench.
Figs. 3-6 show several forms of surface hydrologic
disruption.
3.1.2.1 Zones of Surface Hydrological Disruption
Based on the results of the present study, four zones
of surface hydrological disruption are described and
mapped (Fig. 7). These are:
Kuwait-Saudi Arabia border (1,961.7 km2);
Wadi Al Batin (552.2 km2);
Al Ritqa-Abdaly (748.1 km2 );
Raudtain-Um Al Eish (714.2 km2).
In the mentioned zones, hydrological disruption is
represented by interruption of the natural flow of
surface runoff during flooding seasons. Consequently
huge amounts of surface runoff are miss-distributed.
Fig. 3 Significant differences in vegetation cover at both sides of a bund wall, east of the main channel of Wadi Al Batin in 2009 (right photo) and 2011 (left photo).
Fig. 4 Blocking of surface runoff with its load of out washed deposits and development of manmade playa in front of a bund wall at the eastern flank of Wadi Al Batin.
Potential Impacts of Various Land Use Forms on Land Degradation in Arid Regions (Case Study: Kuwait)
374
Fig. 5 Soil losses through surface runoff at Umm Qaser area (Northeastern part of Kuwait). Arrows indicate the direction of runoff which flows towards a trench (February, 2011).
Fig. 6 Accumulation of flood water in the trenches; right photo: border trench at Al Salmi left photo: Umm Qaser trench (Feb. 2011).
The main causes and indicators of surface
hydrological disruption are summarized in Table 2.
3.3 Interruption of Aeolian Activities
The border trench in a stretch of about 35 km length
extending between Al Abraq (South) and Al
Huwaimiliyah (North) acts as a trap for salting sands
coming from Iraq (Figs. 8 and 9). The amount of
sands trapped in the trench between 2005 and 2009
was about 350,000 m3 [19]. After filling the trench
with shifting sands, maintenance operations start to
clear the sands. The removed sands are dumped in
open areas close to the trench. The dumped sands act
as active source of shifting sands and dust.
The supply of sands from Iraq to Al Huwamiliyah
dune fields, at the Northwestern part of Kuwait, is
interrupted by the border trench, which traps salting
sands. As indicated through recent field surveys, the
Potential Impacts of Various Land Use Forms on Land Degradation in Arid Regions (Case Study: Kuwait)
375
Fig. 7 Zones of surface hydrologic disruption.
Table 2 Causes, indicators and potential impact of surface hydrological disruption.
Zone of disruption Area (km2) Main causes of disruption Indicators of disruption Potential impact
Kuwait-Saudi Arabia border
1,961.7 Border bund wall Blocking of runoff water and outwashed sediments at the upstream portions of Wadis
Development of new sources of dust and sands
Wadi Al Batin 552.2
Border trenches and bund walls Road segments (Salmi and Abraq)
Trapping of surface run off in border trenches. Blocking of runoff water and out washed sediments at the upstream portions of Wadis
Hydrologic drought Development of new sources of dust and sands
Al RitqaAbdaly 748.1
Raudtain-Um El Eish 714.2 Abdaly Road North-south bund walls East-West bund walls
Blocking of runoff water and outwashed sediments along segments of Abdaly Road, Ritqa cut and North-South and East-West bund walls
Disturbance of recharge conditions of fresh groundwater aquifers of Rawdatain-Um Al Eish; Development of new sources of dust and sands
rate of deflation of these dunes exceeds that of
deposition. Deflation of sand dunes is manifested by
exposed plant roots and development of fields of
granule ripples. It is recently observed that several
sand dunes at the extreme upwind side of Al
Huwamiliyah dune fields are in their way to disappear
(blow out). They lost their classic morphology
(Barchan forms) and are transformed into low
featureless deflated sand bodies (Fig. 10). The
common Haloxylon salicornicum plant species, which
was prevailing in this area is replaced with invader
plant species (Cornulacae sp.).
4. Conclusions
During the period 1992-2011, Kuwait experienced
remarkable changes in land uses. Some of these
Potential Impacts of Various Land Use Forms on Land Degradation in Arid Regions (Case Study: Kuwait)
376
Fig. 8 Right photo: Tongues of shifting sands from Iraq to Kuwait in 2010; left photo: recent sand dunes crossed the border line between Iraq and Kuwait (pipeline) in Al Huwaimiliyah area, Northwestern part of Kuwait in 2009.
Fig. 9 Filling the border trench with shifting sands coming from Iraq (May 2009).
changes have positive environmental and ecological
impacts, while others have destructive effects.
Construction of a trench and a bund wall at the border
between Kuwait and Iraq has negative impacts on
local fluvial and aeolian processes. These two forms
of land use caused hydrologic disruption and
interruption of aeolian processes. It is estimated that
about 12,720,000 m3 and 375,000 m3 of soil were
mined (excavated) to establish the trench and the bund
wall, respectively. At the same time, at least 2,544,000
m2 and 1,500,000 m2 of land was disturbed by heavy
machinery during digging the trench and building the
bund wall. During heavy rainy season, e.g., 2001/2002
and 2009/2010, millions of cubic meters of runoff
water are lost in the border trench. During dust and
sand storms (almost every month), the trench acts as a
trap for salting sands coming from Iraq. Some 350,000
m3 of shifting sands were trapped in the trench during
Potential Impacts of Various Land Use Forms on Land Degradation in Arid Regions (Case Study: Kuwait)
377
Fig. 10 Deflation and ecological deterioration of sand dunes at Al Huwaimiliyah area (extreme Northwestern part of Kuwait). Note the exposed 30-40 cm of plant roots. Arrow indicates wind direction.
the period from 2005 and 2009. Periodical clearance
of sands outside the trench for maintenance purpose
resulted in sand encroachment problems in parts of
Kuwait. To restore the border zone between Kuwait
and Iraq, it is recommended to design and implement
a watershed management for the Eastern tributaries of
Wadi Al Batin. Currently, huge amounts of runoff are
lost in the border trench. Simple water harvesting
techniques including cisterns and earth dykes are
strongly recommended. To manage the hazards of
shifting sands and in turn to control soil erosion, it is
recommended to protect two highly fragile areas of
total 800 km2 for at least five years.
5. Recommendations
5.1 Proposed Restoration Measures
To restore the border zone between Kuwait and Iraq
(including Wadi Al Batin), the following approaches
are recommended:
watershed management;
managing the hazards of shifting sands.
5.1.1 Watershed Management
To avoid losses of rainwater and runoff and to
protect infrastructures such as the electric security
fence, it is recommended to design and implement a
watershed management for the Eastern tributaries of
Wadi Al Batin. Simple water harvesting techniques
including earth dykes (1-2 m high) are strongly
recommended (Fig. 11). This approach was
successively tested in Kuwait [2]. The harvested water
could be used for irrigation of drought resistance trees
and shrubs as well as other purposes.
5.1.2 Managing the Hazards of Shifting Sands
To manage the hazards of shifting sands, it is
recommended to adopt the following measures:
Establishment of a 25 km green belt of
Ziziphusspinachristi and Tamarixaphlae trees at Al
Huwaimiliyah area (main gate for saltating sands
coming from Iraq). These trees which trap huge
amounts of drift sands, are surviving very well in the
border zone. The number of rows and spacing
between trees and the most appropriate mechanical
measures for mobile sand control can be identified
through wind tunnel experiments.
Controlling human activities, especially grazing and
military maneuvers, for at least five years in areas A
(600 km2) and B (210 km2), which are severely
degraded (Fig. 12). Degradation is represented by soil
losses (mainly through deflation processes), soil
crusting and sealing, surface deformation, vegetation
degradation and loss of biodiversity. Intensive human
pressure during the last two decades including military
Potential Impacts of Various Land Use Forms on Land Degradation in Arid Regions (Case Study: Kuwait)
378
Fig. 11 A simple model for watershed management, eastern tributaries of Wadi Al Batin; note the dissection of catchment areas by tracks and bund walls at the right side.
Fig. 12 Image of Kuwait showing areas proposed for controlling human activities for at least five years; A: Huwaimiliyah-Al Atraf (600 km2) and B: Umm Qaser-Al Maghasel (210 km2).
Potential Impacts of Various Land Use Forms on Land Degradation in Arid Regions (Case Study: Kuwait)
379
operations of the Gulf War (August 1990-February
1991) is the main cause of soil and vegetation
degradation. As in the case of areas of similar
environmental conditions, protection of areas A and B
will result in natural recovery represented by soil
stabilization and enhancement of the vegetation cover.
Pre-allowing the public use and utilization of the areas,
a sustainable land use plan should be adopted to
identify and specify land use plan.
Acknowledgments
The authors are grateful to the land border security
staff, Ministry of Interior, Kuwait, specially Mr. A. Al
Bargas and to the Geo-informatics Centre, KISR for
their assistance and support during the course of the
study.
References
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Journal of Agricultural Science and Technology A 3 (2013) 380-384 Earlier title: Journal of Agricultural Science and Technology, ISSN 1939-1250
Effects of Using Different Percentages of Fenugreek
Flour to Improve the Sensory, Rheological Properties
and Keeping Quality in Maize Dough to Produces
Gluten-free Breads
Abdulsalam Abdulrahman Rasool1, Dana Azad Abdulkhaleq1 and Dlir Amin Sabir2
1. Field Crops Department, Faculty of Agricultural Sciences, University of Sulaimani, Sulaimani, Iraq
2. Food Sciences Department, Faculty of Agricultural Sciences, University of Sulaimani, Sulaimani, Iraq
Received: January 28, 2013 / Published: May 20, 2013. Abstract: Adding different percentages 1%, 3% and 5% of fenugreek flour depending on maize flour base to gluten free bread recipe for making tanoor bread was used to improve the tanoor bread quality, in term of sensory properties like volume, crust, color, symmetry, bake uniformity, texture, grain, aroma and taste. Also some rheological properties like gelatinization temperature and maximum viscosity of dough with bread keeping quality were used. The aim of this work is to produce suitable bread with maintaining the bread quality by selecting the suitable percentage to produce gluten free bread for people who suffering from celiac diseases and its contribution to health benefits. Significant differences were observed by using 5% of fenugreek flour in term of gelatinization temperature, maximum viscosity, breed keeping quality and volume. While using 1% of fenugreek flour significantly improved symmetry value, bread texture, crumb color, aroma and taste. Key words: Maize, Fenugreek, sensory evaluation, rheological properties, keeping quality, gluten-free bread.
1. Introduction
Maize (Zea mays L.) is the most widely grown crop
in the America, with 70-100 million acres grown
annually in the United States alone, accounting for
nearly 40% of the all maize grown in the world. It is a
versatile crop, and can be grown in a number of
different environments; it is grown on every continent
except Antarctica. Maize is also higher yielding than
many other grains (its average yield is 14 t ha-1 but it
can reach over 27 t ha-1), and therefore often less
expensive. Together with wheat and rice, maize is one
of the three most important crops in the world due to
its nutritional qualities and ease of cultivation. Maize
contains high levels of starch and valuable proteins
and oils. Depending on the variety, maize may contain
Corresponding author: Dlir Amin Sabir, Ph.D., research
field: food science. E-mail: dlearsaber@yahoo.com.
a number of important B vitamins, folic acid, Vitamin
C and provitamin A, also rich in minerals and it is a
good source of dietary fiber and protein, while being
very low in fat and sodium [1].
Fenugreek plant (Trigonella foenum graecum L.) is
widely distributed throughout the world and which
belongs to the family Fabacecae. It is a herb recorded
as one of the oldest medicinal herbs, highly esteemed
by both East and West, and has been regarded as a
cure for just about every ailment known to man.
Fenugreek has the most beneficial action on cleansing
the blood. As a diaphoretic, it is able to bring on a
sweat, and help detox the body. Commonly found
growing in the Mediterranean region of the world.
While the seeds and leaves are primarily used as a
culinary spice, it is also used to treat a variety of
health problems in Egypt, Greece, Italy and South
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Asia. Emphasis is put on studying the practical
feasibility of such bread in a regular bakery
environment, and on understanding the underlying
physicochemical principle [2, 3].
The plant contains active constituents such as
alkaloids, flavonoids, steroids, Saponins, etc.. It is an
old medicinal plant. It has been commonly used as a
traditional food and medicine. Fenugreek is known to
have hypoglycemic, and hypocholesterolaemic, effects,
anti-inflammatory effects. Recent research has
identified fenugreek as a valuable medicinal plant
with potential for curing diseases and also as a source
for preparing raw materials of pharmaceutical industry,
like in steroidal hormones [2].
Gluten-free bread was once regarded as a main
product for people with a rare disorder, celiac disease
[3]. However, it has meanwhile been recognized that
celiac disease is much more widespread than
previously thought, and an average worldwide
prevalence of Gluten-free breads based on isolated
starches, or on flour from gluten-free cereals like maize
have been described in the literature for many decades
has long been known that mixtures of maize, starch
and water can form a dough with properties similar to
wheat dough, provided that they are mixed above room
temperature [4]. Use of fenugreek, a food with
demonstrated efficacy in lowering blood sugar, is
limited by its bitter taste and strong flavor. A bread
incorporating fenugreek using a proprietary process
was tested for its taste acceptability and its effect on
carbohydrate metabolism. We developed a fenugreek
bread formula that was produced in a commercial
bakery by incorporating fenugreek flour into a standard
maize bread formula. Whole wheat bread was prepared
by the same formula in the same bakery using wheat
flour. Fenugreek and maize bread samples were
evaluated for sensory attributes and nutrient
composition. Color, firmness, texture and flavor
intensity between the fenugreek added to maize flour
and control. Tanoor bread is single layered flat. Tanoor
bread is popular in Iraq, Khubz and Naan are different
names used for the same product in various parts of the
Middle East. The name comes from the type of oven
used to bake this product, i.e., tanoor, which means
clay oven. Tanoor bread requires 100% flour, 1%
active dry yeast, 0.75%-1.5% salt and various amounts
of water. The processing steps are mixing, dividing,
proofing, sheeting, docking, baking and packaging.
Crust color is reddish brown. High quality tanoor bread
has a uniform thickness with an even distribution of
small blisters on the top crust [5]. The aim of this work
is to produce suitable bread with maintaining the bread
quality by selecting the suitable percentage to produce
gluten free bread for people whom suffering from
celiac diseases and its contribution to their health and
increase bread shelf life stability.
2. Materials and Methods
2.1 Maize Seed
Maize seed cultivar 704 which obtained from
Sulaimani research center was used in this study, then
the seeds were milled by locally miller in city center
then the flour was used alone and mixed with different
percentages 1%, 3% and 5% of fenugreek flour which
is available in the local market.
2.2 Dough Preparation
Measurement of rheological characteristics of
dough bread baking process; the processing steps were
dough mixing to optimum dough development, bulk
fermentation 60 min, dividing and rounding (50 g
pieces), intermediate proofing (10 min), sheeting, final
proofing (30 min), baking (288 °C for 15 min),
cooling. The dough pieces were baked in electric oven.
And then the bread parameters and sensory evaluation
characters like volume crust color, symmetry, bake
uniformity, texture, grain and aroma were evaluated
according to AACC method 10-12.01 baking
guidelines for scoring experimental bread [6].
2.2.1 Rheological Characteristics of Dough
Amylograph procedure was used for rheological
Effects of Using Different Percentages of Fenugreek Flour to Improve the Sensory, Rheological Properties and Keeping Quality in Maize Dough to Produces Gluten-free Breads
382
characteristics according to AACC methods 61-01.01
Amylograph Method for Milled Rice was performed
using a 60 g of sample [7].
2.2.2 Baking Volume
AACC method 22-10.01 was used for Measurement
of Volume by Rapeseed displacement [8].
2.3 Chemical Composition of Maize and Fenugreek
The following methods were used for flour analysis
as shown in Table 1.
AACC method 08-21.01 Prediction of Ash Content
in Wheat Flour—Near-Infrared Method [9], 39-10.01
Near-Infrared Reflectance Method for Protein
Determination in Small Grains [10], AACC method
Grains 0-10.01 Crude Fat in Flour, Bread, and Baked
Cereal Products [11], AACC method 44-01.01
Calculation of Percent Moisture [12], AACC method
76-13.01 Total Starch Assay Procedure Megazyme
Amyloglucosidase/Alpha-Amylase Method [13].
2.4 Method of Statistical Analysis
For the statistical tests of variance analysis, least
significant difference (LSD) test and SPSS software,
version 18, were used.
3. Results and Discussion
Table 2 and Appendix 1 show highly significant
differences regarding bread volume, the biggest
volume was obtained by using 5% fenugreek. This
may be due to the physiochemical properties of the
fenugreek protein which present in the seeds. The
highest symmetry, bread texture, crumb color aroma
Table 1 Chemical composition of maize and fenugreek.
Chemical composition (%)
Maize Fenugreek
Starch 71.5 51
Protein 10.3 26.0
Fat 3.8 8.0
Ash 1.3 3.0
Moisture 13.1 12
and taste were obtained by using 1% Fenugreek, this
may be due to the effect of the emulsion agent which
present in fenugreek seeds which lead to produce a
desirable and uniform air bubbles in the dough and
final product similar results observed by those whom
reported by Maleki et al. [14]. The desirable aroma of
fenugreek seeds came from polysaccharides
(galactomannan), volatile oils and alkaloids, such as
choline and trigonelline, that is why the addition of
1% of fenugreek seeds was more desirable to add in
such cases. Regarding crust color, bake uniformity
and grain, there were no significant differences among
all of the treatments, this results are in agreement with
researchers [15].
As shown in Table 3 and Appendix 2, highly
significant differences were noticed among the
treatments. The addition of 5% Fenugreeks increased
gelatinization temperature and maximum viscosity.
This could be due to the effect of emulation agent that
found in this kind of improver that has been used in
our local area at house levels similar results are
reported by authors [16].
Data in Table 4 and Appendix 3 indicate that there
were significant differences between treatments in
term of adding different percentages of fenugreek
Table 2 Effect of the addition of fenugreek flour on the sensory evaluation characters.
Treatments Volume (15)
Crust color (15)
Symmetry (5)
Bake uniformity (5)
Texture (15)
Crumb color (10)
Grain (10)
Aroma (15)
Taste (20)
Control 8.333 2.333 1.333 2.667 9.333 6.667 5.000 8.667 9.000
1% Fenugreek 13.667 3.333 4.000 3.333 13.333 9.333 8.667 12.667 16.333
3% Fenugreek 13.000 1.667 2.000 2.667 6.667 5.667 7.333 10.000 5.000
5% Fenugreek 14.333 1.667 3.667 2.333 6.333 4.667 5.333 7.333 3.667
L.S.D (P ≤ 0.05) 2.306 n.s 1.803 n.s 2.369 1.960 1.883 2.306 3.216
Effects of Using Different Percentages of Fenugreek Flour to Improve the Sensory, Rheological Properties and Keeping Quality in Maize Dough to Produces Gluten-free Breads
383
Appendix 1 Mean squares of variance analysis for the sensory evaluation characters.
S.O.V d.f MS
Volume (15)
Crust color (15)
Symmetry (5)
Bake uniformity (5)
Texture (15)
Crumb color (10)
Grain (10)
Aroma (15)
Taste (20)
Treatments 3 22.222** 1.444n.s 4.972* 0.528n.s 31.417** 12.083** 8.972** 15.556** 97.222**
Exp. error 8 1.500 0.333 0.917 0.833 1.583 1.083 1.000 1.500 2.917
Table 3 Effect of the addition of fenugreek flour on the rheological properties.
Treatments Gelatinization temp. (°C) Maximum viscosity (Bu)
Control 72.000 620.000
1% Fenugreek 81.000 340.000
3% Fenugreek 82.667 510.000
5% Fenugreek 89.667 713.333
L.S.D (P ≤ 0.05) 3.075 44.490
Appendix 2 Mean squares of variance analysis for the rheological properties.
S.O.V d.f MS
Gelatinization temp. (°C) Maximum viscosity (Bu)
Treatments 3 158.444** 77208.333**
Exp. error 8 2.667 558.333
Table 4 Effect of fenugreek addition on breed keeping quality.
Treatments 24 h 48 h 72 h
Control 5.667 4.667 3.333
1% Fenugreek 6.667 5.333 4.667
3% Fenugreek 7.000 6.333 5.333
5% Fenugreek 8.000 7.000 6.333
L.S.D (P ≤ 0.05) n.s 1.631 1.438
Keeping quality score = 10 degrees.
Appendix 3 Mean squares of variance analysis for breed keeping quality.
S.O.V d.f MS
24 h 48 h 72 h
Treatments 3 2.778n.s 3.222* 4.750**
Exp. error 8 1.167 0.750 0.583
after 48 h and 72 h of shelf life at laboratory
temperature. In this study, the ability to roll and fold
characteristic used for evaluating shelf life stability of
tanoor bread. Breads made with the three levels (1%,
3% and 5%) of fenugreek flour had higher scores for
tearing quality compared with the control, this may be
due to the effect of preservation agent choline and
trigonellin which found in high amount in fenugreek,
this results are in agreement with researchers [15] or
may belong to the emulation properties of fenugreek
protein content and its role in improving bread
keeping quality similar results are observed by
researchers [17].
4. Conclusions
It was concluded that there were significant
differences between treatments in term of adding 1%)
of fenugreek regarding bread volume, bread texture,
crumb color, symmetry and aroma. While using (5%)
of fenugreek increase shelf life stability for 48 h and
72 h which was significantly differed from the other
additions of 1% and 3 % respectively.
Effects of Using Different Percentages of Fenugreek Flour to Improve the Sensory, Rheological Properties and Keeping Quality in Maize Dough to Produces Gluten-free Breads
384
Acknowledgments
Appreciation and highly grateful are also expressed
to Food Science Department, Faculty of Agricultural
Sciences-University of Sulaimani for making this
research possible. Special thanks and appreciations are
due to Mr. Nehad the Chairman of the Quality Control
Department in Sulaimani. The authors’ best gratitude
and thanks are also expressed to Mrs. Jutyar the Head
of Field Crops Department at Bakrajo Research
Station for his help during this research.
References
[1] T.M. Fulton, C.S. Buckler, R.A. Kissel, The Teacher-Friendly Guide to the Evolution of Maize, Paleontological Research Institution, Ithaca, NY, 2011.
[2] H.S. Snehlata, D.R. Payal, Fenugreek (Trigonella foenum-graecum L.): An overview, International Journal of Current Pharmaceutical Review and Research 2 (2012) 169-187.
[3] A. Fasano, C. Catassi, Current approaches to diagnosis and treatment of celiac disease: An evolving spectrum, Gastroenterology 120 (2001) 636-651.
[4] T.J. Schober, S.R. Bean, D.L. Boyle, Gluten-free sorghum bread improved by sourdough fermentation: Biochemical, rheological, and microstructural background, Journal of Agricultural and Food Chemistry 55 (2007) 5137-5146.
[5] H.A. Faridi, Flat breads, in: Y. Pomeranz (Ed.), Wheat Chemistry and Tech-Srinology, St. Paul, 1988, pp. 457-506.
[6] AACC Methods 10-12.01 Baking Guidelines for Scoring Experimental Bread Methods of the American Association of Cereal Chemistry, Am. Assoc. Cereal Chem. Inc., St. Paul, Minnesota, 2000.
[7] AACC Methods 61-01.01 Amylograph Method for Milled Rice Measurement of Alpha-Amylase Activity with the Amylograph Approved Methods of the
American Association of Cereal Chemistry, Am. Assoc. Cereal Chem. Inc., St. Paul, Minnesota, 2000.
[8] AACC Methods 22-10.01 Measurement of Volume by
Rapeseed Displacement Approved Methods of the
American Association of Cereal Chemistry, Am. Assoc.
Cereal Chem. Inc., St. Paul, Minnesota, 2000.
[9] AACC Method 08-21.01 Prediction of Ash Content in
Wheat Flour—Near-Infrared Method, Approved Methods
of the American Association of Cereal Chemistry, Am.
Assoc. Cereal Chem. Inc., St. Paul, Minnesota, 2000.
[10] AACC, 39-10.01 Near-Infrared Reflectance Method for
Protein Determination in Small Grains American
Association of Cereal Chemistry, Am. Assoc. Cereal
Chem. Inc., St. Paul, Minnesota, 2000.
[11] AACC Method Grains 0-10.01 Crude Fat in Flour, Bread,
and Baked Cereal Products. American Association of
Cereal Chemistry, Am. Assoc. Cereal Chem. Inc., St.
Paul, Minnesota, 2000.
[12] AACC Method 44-01.01 Calculation of Percent Moisture.
American Association of Cereal Chemistry, Am. Assoc.
Cereal Chem. Inc., St. Paul, Minnesota, 2000.
[13] AACC Methods 76-13.01 Total Starch Assay Procedure
(Megazyme Amyloglucosidase/Alpha-Amylase Method
the American Association of Cereal Chemistry, Am.
Assoc. Cereal Chem. Inc., St. Paul, Minnesota, 2000.
[14] M. Maleki, J.L. Vetter, W.J. Hoover, The effect of
fenugreek seeds, sugar, shortening and soya flour on the
staling of barbari flat bread, Journal of the Science of
Food and Agriculture 32 (1981) 1209-1212.
[15] N.A. El Nasri, A.H. El Tinay, Functional properties of
fenugreek (Trigonella foenum graecum) protein
concentrate, Food Chemistry 103 (2007) 582-589.
[16] J.F. Qarooni, Governing the quality of bread in the
Middle East, Ph.D. Dissertation, School of Biological
Technologies, University of New South Wales, 1988, pp.
1-215.
[17] M. Maleki, R.C. Hoseney, P.J. Mattern, Effects of loaf volume, moisture content and protein quality on the softness and staling rate of bread, Cereal Chemistry 57 (1980) 138-140.
Journal of Agricultural Science and Technology A 3 (2013) 385-403 Earlier title: Journal of Agricultural Science and Technology, ISSN 1939-1250
Applicability of Cytoplasmic Male Sterility (CMS) as a
Reliable Biological Confinement Method for the
Cultivation of Genetically Modified Maize in Germany
Heidrun Bückmann1, Alexandra Hüsken2 and Joachim Schiemann1
1. Institute for Biosafety in Plant Biotechnology, Julius Kühn-Institut-Federal Research Centre for Cultivated Plants, Quedlinburg
06484, Germany
2. Department of Safety and Quality of Cereals, Max Rubner-Institut, Detmold 32756, Germany
Received: February 7, 2013 / Published: May 20, 2013. Abstract: The cultivation of genetically modified (GM) plants requires the reduction of an unwanted spread of genes (biological confinement). Cytoplasmic male sterility (CMS) inhibits the development of functional pollen, but nuclear restorer (Rf) genes and environmental impacts can restore the fertility. The aim of this study was to verify whether CMS in maize hybrids is a reliable confinement method for the prospective cultivation of GM maize in Germany. Two-year field experiments in three different environments were conducted with three CMS maize hybrids which vary in the CMS stability, one conventional maize variety (all yellow kernels) and white maize as pollen recipient. Tassel characteristics, pollen vitality and cross-pollination rates were investigated. The CMS stability was dependent on the genotype and the specific weather conditions per year and location. In all maize hybrids CMS was unstable. One CMS maize hybrid showed a high level of CMS stability and very low cross-pollination rates in any case (< 1%). The two other CMS maize hybrids developed more fluctuant and fertile tassels with few or many pollen, respectively. Compared with a conventional and fully fertile maize variety, cross-pollination of all CMS maize hybrids was strongly reduced (84%-99%). In conclusion, the CMS trait can be proposed as a useful biological confinement method to reduce pollen-mediated gene flow from GM maize. Key words: CMS, pollen release, biological confinement, cross-pollination reduction, GM maize (Zea mays L.).
1. Introduction
The cultivation of genetically modified (GM) crops
might result in unwanted intermingling with other
agricultural products based on the so-called gene flow.
Gene flow can occur seed-mediated,
vegetative-propagule-mediated or pollen-mediated [1].
The extent of each type of gene flow depends on the
cultivated plant species. Several biological
confinement methods have been developed to prevent
or reduce pollen-mediated gene flow [2-5]. These
methods are based on either naturally occurring or
engineered phenomena, like cleistogamy, plastid
Corresponding author: Heidrun Bückmann, Ph.D.,
research field: biosafety research with genetically modified plants. E-mail: heidrun.bueckmann@jki.bund.de.
transformation, cytoplasmic male sterility (CMS),
transgene excisions or incompatible genomes [1]. An
effective biological confinement method requires a
high level of gene stability and the knowledge of its
reliability. It should meet the requirements of gene
flow reduction for the specific GM crop.
For the cultivation of maize in the Middle-European
climate, gene flow occurs mainly by pollen dispersal.
It is based on the huge amount of pollen, the flowering
time and synchronisation of pollen donor and pollen
recipient plants and the pollen competition between
both. External conditions like wind direction and
speed, temperature and air humidity as well as
distances between different maize fields can influence
the pollen-mediated gene flow significantly [6, 7].
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Biological confinement methods like CMS are not
applied in GM maize cultivation yet but should be
considered and tested for gene flow reduction in
specific applications.
CMS occurs naturally in many plant species [8, 9].
It is a maternally inherited trait that prevents the
development of functional pollen [10, 11]. CMS
results from a loss-of-function mutation in the
mitochondrial genome [12] which causes a
dysfunction of the respiratory metabolism and an
abnormal production of male gametes [13, 14]. Hence,
plants develop no vital pollen while the female
fertility is not affected. Three main CMS-types are
known for maize [15]: CMS-T or Texas cytoplasm
[16, 17], CMS-S or USDA-cytoplasm [18] and
CMS-C or Charrua-cytoplasm [19]. Although the
primary determination of CMS occurs extranuclearly,
certain nuclear genes-restorer of fertility genes
(Rf)-can compensate the CMS effect of the cytoplasm
[11] and can result in fluctuated or fertile tassels with
more or less vital pollen in the first-generation
progeny (F1).
Apart from the internal interactions between
mitochondrial and nuclear genes, fertility of many
CMS plant species can also be restored by
environmental impacts like heavy rain or extreme heat
[7, 20-23]. Sterility decreases with decreasing
temperatures and day length [21, 24, 25]. Correlations
between climatic factors during the 10 days before
anthesis of maize with partial reversion to male
fertility were ascertained by Weider et al. [7] for
several CMS hybrids in different environments. In
most instances, the involved mechanisms are still
unclear.
The aim of this study was to verify whether CMS in
maize might be a reliable method to reduce the
pollen-mediated gene flow. It should be clarified
whether selected CMS maize hybrids are stably sterile
or restored to fertility within different environments in
Germany. In case that (partial) restoration of fertility
occurs, knowledge about out-crossing rates from CMS
maize hybrids into pollen recipient maize is important.
Therefore, field trials were performed to study the
stability of CMS and the extent of gene flow from
unstable maize hybrids as a function of the distance to
recipient maize plants under practical agricultural
conditions.
2. Materials and Methods
2.1 Site Descriptions
In 2009 and 2010, field trials were conducted in
three different environments in Germany. Groß
Lüsewitz is located in Western Pomerania,
North-Eastern Germany, and characterised by an
average annual precipitation of 610 mm with summer
drought and soils of sandy loam. The average
temperature is 6.9 °C (Table 1). Braunschweig in
Lower Saxony (Northern Germany) belongs to a
typical agricultural area with deep-grounded loamy
clay soils and average rain fall rates of 628 mm per
year and temperatures of 9.2 °C. Freising, Bavaria
(Southern Germany), is characterised by a mild
climate with an average annual temperature of 7.5 °C,
an average precipitation of 788 mm and a sandy loam
soil.
2.2 Field Trial Design
The field trials included plots of three different
Table 1 Characteristics of the field trial locations in Germany.
Groß Lüsewitz Braunschweig Freising
Soil type Sandy loam Loamy clay Sandy loam
Altitude 15 m 82 m 480 m
Ø annual temperature 6.9 °C 9.6 °C 7.5 °C
Ø annual precipitation 610 mm 580 mm 793 mm
Applicability of Cytoplasmic Male Sterility (CMS) as a Reliable Biological Confinement Method for the Cultivation of Genetically Modified Maize in Germany
387
CMS maize hybrids and one conventional fully fertile
maize hybrid variety. At the adverse side of each of
these plots, one plot with a white maize variety used
as pollen recipient was located. The distance between
these plots was 3.5 m. Each plot measured 48 m width
to 69 m length (Fig. 1). The trials were designed
downwind with the CMS maize hybrid plots
westwards of the white maize plots at all locations. To
prevent cross-pollination between the test units (CMS
maize hybrid and pollen recipient maize) hemp was
grown as a natural pollen barrier. The trials were
conducted under local agricultural conditions.
2.3 Maize Genotypes
The appropriate CMS hybrids for the field trials
were selected according to the results of a previous
experiment in 2008 when 10 CMS maize hybrids of
the three CMS types [7] were tested in a randomised
block trial with four repetitions at each location. The
plants were tested for the flowering behaviour (tassel
characteristics, pollen shedding, flowering time and
duration), the vitality of pollen is produced, the
flowering synchronism with pollen recipient plants
and the plant height. The hybrids with the highest
out-crossing potential caused by pollen production
and pollen vitality and the lowest out-crossing
potential were selected as well as a CMS hybrid with
a medium out-crossing tendency. The following
hybrids were selected: “DSP2”, a CMS T-type maize
hybrid (Delley Seeds and Plants Company, DSP,
Switzerland) and “Torres” and “Zidane”, both CMS
S-type maize hybrids (Kleinwanzlebener Saatzucht
AG, Germany). These hybrids were grown in
comparison with the conventional fully fertile maize
hybrid “Delitop”. All plants develop yellow kernels.
A white grain maize test hybrid, DSP 17007(Delley
Seeds and Plants Company, Switzerland), was
cultivated as pollen recipient [26].
Fig. 1 Field trial design with assessment and sampling points.
Applicability of Cytoplasmic Male Sterility (CMS) as a Reliable Biological Confinement Method for the Cultivation of Genetically Modified Maize in Germany
388
2.4 Tassel Characteristics and Pollen Vitality
At 12 assessment points (Fig. 1) within one CMS
maize plot always 20 plants were tested on their tassel
characteristics during the male flowering period.
According to the stability of the CMS trait, they were
classified into sterile, restored or fertile tassels as
described by Weider et al. [7]. Sterile tassels do not
develop any anthers and therefore pollen, while partly
restored tassels produce anthers, generally only on the
lateral branches. They can produce different amounts
of pollen that can be vital or sterile. A fertile tassel
produces many anthers on all branches and sheds lots
of vital pollen.
If anthers were developed, 12 self-pollinations per
plot were carried out to investigate the vitality of the
pollen. Within one plot, a minimum number of 12 ears
were isolated with a paper bag before silking. The day
before self-pollination, the tassels were isolated and
the pollen was collected in a paper bag. When
self-pollinated, the ears were carefully fertilised with
the collected pollen and were further isolated until the
end of the pollen shedding period to prevent allogamy.
At harvest, the self-pollinated ears were picked and
the mean kernel set (MKS) was calculated by counting
the kernels developed and compared with a fully
germinated ear of the same CMS maize hybrid. This
hybrid was hand-pollinated with vital pollen of the
white maize. The MKS is an indicator of the vitality
of pollen developed and is expressed in percentage.
2.5 Cross-Pollination
At defined distances (Fig. 1) within the white maize
plots, 20 cobs were harvested and yellow and white
kernels, respectively, were counted to determine the
level of cross-pollination.
The cross-pollination from CMS maize into
conventional maize was tested by a simple colour
detection system. All CMS maize hybrids develop
yellow kernels. If white maize is pollinated by yellow
maize pollen, it produces yellow kernels due to
hereditary dominance. To calculate the
cross-pollination rate of the pollen donor plots, it is
necessary to determine the average grain number per
ear/cob. On this number, the percentage of the
cross-pollination rate can be based and the results are
expressed as percentages from the total number of
kernels per cob. The data of all 20 plants per
assessment point were averaged.
2.6 Weather Data
At all field trial locations, weather data were
collected during the whole flowering period. In
Braunschweig and Freising mobile weather stations
were kindly provided by the Deutsche Wetterdienst
(DWD Braunschweig and DWD Freising). In Groß
Lüsewitz, the data were provided by biovativ GbmH.
Wind speed and direction were measured at 2 m above
soil surface. Air temperature and precipitation as well
as air humidity were recorded.
2.7 Data Analysis
The average results of 20 plants per assessment and
harvest point within each plot in two years were
visualised by using the software Origin 8.1.
3. Results
3.1 Experimental Years
The two experimental years differed tremendously.
2009 was characterised by a higher mean temperature
in Braunschweig (10.0 °C), Freising (8.7 °C) and
Groß Lüsewitz (8.8 °C) compared to the local
long-standing mean temperature (9.3 °C, 7.5 °C,
8.3 °C; Figs. 2a-2c). Temperature peaks appeared in
July and August at each location. The annual
precipitation was similar to the mean values in
Braunschweig (619 mm vs. 628 mm). The rainfall
measured in Freising was about 30 mm above average
(820 vs. 788 mm), whereas in Groß Lüsewitz
Applicability of Cytoplasmic Male Sterility (CMS) as a Reliable Biological Confinement Method for the Cultivation of Genetically Modified Maize in Germany
389
Fig. 2a Long-standing annual precipitations and average temperatures compared with 2009 and 2010 data from Braunschweig (prec.: precipitation; temp.: temperature).
Fig. 2b Long-standing annual precipitations and average temperatures compared with 2009 and 2010 data from Groß Lüsewitz (prec.: precipitation; temp.: temperature).
Fig. 2c Long-standing annual precipitations and average temperatures compared with 2009 and 2010 data from Freising (prec.: precipitation; temp.: temperature).
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rainfall rates were 40 mm below the long-standing
mean.
For the outcome of the field trials, the period
between April and August between sowing and the
end of the flowering period is important. In April
2009, weather conditions were satisfactory in
Braunschweig and Groß Lüsewitz. Temperatures and
precipitations were comparable to the long-standing
means. In Freising, it was about 4 °C warmer and
more humid. The following months were similar to
the long-standing weather conditions. Hence, seed
emergence succeeded and the crop developed equally
at any location.
2010 was generally colder and wetter than in the
long-standing mean which was clearly shown in
Braunschweig with a 1 °C lower mean temperature
and a precipitation increase of 122 mm. Especially in
May, when the maize started to grow, 3 °C lower
temperatures and heavy rainfall rates occurred. Just
before onset of flowering, temperatures raised up to
3.5 °C above the long-standing mean in July. A
similar situation was measured in Groß Lüsewitz with
temperatures of 0.8 °C below, and rainfall of 53 mm
above the long-standing mean. Therefore, vegetation
began late and crops did not develop equally. The
flowering period started about five days later than in
2009, and flowering was generally inhomogeneous.
As in Braunschweig, mean temperatures were 3.5 °C
above the long-standing mean in July.
In Freising, temperatures were comparable to the
mean, and a 62 mm higher precipitation was measured.
The plants developed more equally than in
Braunschweig and Groß Lüsewitz without important
delays. In July, at the onset of flowering, mean
temperatures measured 3 °C above the long-standing
mean.
3.2 Flowering Period
Depending on the mild 2009 climate in Freising,
flowering occurred efficiently. Male flowering of CMS
hybrids and “Delitop” started about 90 days after
sowing and was finished after 97 days (Fig. 3). In the
same year, the tassels of the CMS maize hybrids in
Braunschweig bloomed between 92 and 111 days after
sowing. BBCH 65 (main pollen shedding) laid about
100 days after sowing except for “Zidane” where main
pollen shedding developed faster (95-97 days after
sowing). In Groß Lüsewitz vegetation started later and
tassel development varied according to the genotype.
The CMS hybrids bloomed between 96 and 117 days
after sowing. BBCH 65 was reached after 105 days on
average. Flowering synchronisation between pollen
shedding of the hybrids and silking of the white maize
was generally ascertained. Male flowering of pollen
donor and pollen acceptor plots, respectively,
developed within the same period.
Depending on the weather conditions, plant
development in 2010 was delayed and unbalanced in
Braunschweig and Groß Lüsewitz. Therefore, the
flowering time was long and the development stages of
maize plants differed within one plot. In Braunschweig
tassels of the CMS hybrids as well as the control hybrid
began blooming after 91 days, and flowering lasted up
to 115 days after sowing. The growth stage BBCH 65
was reached after about 101 to 108 days after sowing.
Silking of the white maize began 5 to 7 days later and
ended up to 130 days after sowing. The latest crop
development was observed in Groß Lüsewitz and took
between 97 and 124 days after sowing; the main pollen
shedding occurred between 105 and 115 days after
sowing. The crop was generally badly developed and
extremely irregular grown. Silking of the white maize
was delayed up to 10 days. Therefore, synchronisation
between pollen shedding of donor plants and silking of
receptor plants was weak.
In Freising, flowering conditions were comparable
with 2009. The plants developed more equally than at
the two other locations. Based on the average
temperature and slightly higher precipitation, the male
flowering of the CMS maize hybrids and “Delitop”
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Fig. 3 Main flowering periods of tassels of the CMS maize hybrids (DSP2, Torres and Zidane) and the fully fertile control variety (Delitop) as well as ears of white maize at Braunschweig, Groß Lüsewitz and Freising in 2009 and 2010 (mB: male flower; wB: female flower; FR: Freising; BS: Braunschweig; GL: Groß Lüsewitz).
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took place between 84 and 101 days after sowing. The
main pollen shedding period of the CMS maize
hybrids was between 90 and 93 days, while main
pollen shedding of “Delitop” lasted up to 98 days after
sowing. In general, tassels of the pollen donor and
ears of the recipient occurred synchronously.
3.3 CMS Stability
In both growing seasons, no CMS hybrid was 100%
sterile at any location but differences between the
levels of sterility were identified. In 2009, the highest
level of CMS stability was detected for “Torres”,
CMS S-cytoplasm, within all environments. “Torres”
developed partly restored tassels (99%) with a small
amount of pollen in Braunschweig (Table 2). In Groß
Lüsewitz, 80% partly restored and 20% fertile tassels
were observed but pollen developed very scarce.
Self-pollination of “Torres” resulted in a mean kernel
set (MKS) of less than 1% kernel per cob without
differences between the locations. No environmental
impact on tassels depending on the location was found
for “Zidane” either (CMS S-cytoplasm). Partly
restored and fertile tassels were found in
Braunschweig and Groß Lüsewitz. The pollen
developed was vital, and a relatively large proportion
of kernels developed after self-pollination (MKS Table 2 Tassel characteristics and pollen development and vitality of CMS maize hybrids (DSP2, Torres and Zidane) at Braunschweig, Groß Lüsewitz and Freising in 2009 and 2010.
CMS hybrid Location Tassel (%) Pollen (%)
MKS (%) pollen vitality
2009 2010 2009 2010 2009 2010
DSP2
Braunschweig
Sterile 96 98 No 96 99
8.6 Partly restored 3 1 Few 3 1 0.2
Fertile 1 1 Many 1 0
Groß Lüsewitz
Sterile 5 76 No 10 76
0.0 Partly restored 87 22 Few 78 24 40
Fertile 8 2 Many 12 0
Freising
Sterile n.s. 96 No n.s. 96
1.7 Partly restored n.s. 4 Few n.s. 4 81
Fertile n.s. 0 Many n.s. 0
Sterile 1 7 No 13 39 0.0
Torres
Braunschweig Partly restored 99 92 Few 87 61* 0.7
Fertile 0 1 Many 0 0
Groß Lüsewitz
Sterile 0 1 No 12 3
3.1 Partly restored 80 98 Few 87 96 0.0
Fertile 20 1 Many 1 1
Freising
Sterile n.s. 3 No n.s. 100
0.4 Partly restored n.s. 97 Few n.s. 0 0.2
Fertile n.s. 0 Many n.s. 0
Sterile 0 0 No 0 0 1.6
Zidane
Braunschweig Partly restored 58 59 Few 33 87 20
Fertile 42 41 Many 67 13
Groß Lüsewitz
Sterile 0 0 No 0 0
6.9 Part rest 51 87 Few 57 87 44
Fertile 49 13 Many 43 13
Freising
Sterile n.s. 0 No n.s. 3
4.1 Partly restored n.s. 98 Few n.s. 95 44
Fertile n.s. 2 Many n.s. 2
n.s. = not specified; * = very few.
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20%-40%). Contrary to the results of the previous
experiments, the CMS maize hybrid “DSP2” (CMS
T-cytoplasm) developed different tassels at different
locations. In Braunschweig, the majority of plants
were sterile (96% at the assessment points), except
single plants with fully fertile tassels and a lot of
pollen, equally spread within the plot. These plants
did not occur at the assessment points and were,
therefore, not measured. Nearly no kernel was
produced after self-pollination. Mainly partly
restored tassels (87%) with a small amount of pollen
(78%) but a MKS of 40% was measured in Groß
Lüsewitz. In Freising, the majority of plants
developed fertile tassels containing a lot of vital
pollen (observed but not measured), and a MKS of
80% was measured. To prove whether environmental
impacts of seed impurity caused partly restored or
fertile DSP2 tassels, DNA extractions of leaf
material and qualitative PCRs [27] were carried out.
100 seeds were germinated in the greenhouse, and
leaves were used for analysis. All 100 plants
possessed the CMS T-type of the DSP2 plants and
seed purity could be proven (data not shown).
In 2010, all CMS maize hybrids developed their
tassels similarly at the different locations. “Torres”
developed mainly partly restored tassels (92%-98%).
No pollen was developed at all in Freising while in
Groß Lüsewitz a small amount of pollen was found
(96%). Compared with results from 2009 there was a
difference within the ranking of “few” (Table 2) in
Braunschweig. In 2010, a much smaller amount of
pollen was developed than in 2009. “Zidane”
developed comparably at all locations. The majority of
the tassels expressed fluctuated tassels (59%-98%)
with a small amount of pollen (87%-95%). At all
locations, “DSP2” developed mainly sterile tassels.
Partly restored and sometimes fertile tassels were
observed, equally distributed over the whole plots.
The tassels developed no or only a little pollen. The
pollen vitality of all three CMS maize hybrids resulted
only low rates of MKS compared to 2009.
Summarising the tassel developments in both
experimental years, the highest stability of sterility
was estimated for “Torres”, and the highest variability
could be detected for “DSP2”. The pollen vitality of
“DSP2” and “Zidane” was mainly influenced by the
experimental year.
3.4 Cross-Pollination
In both experimental years, the cross-pollination
of the CMS maize hybrids and the control variety
“Delitop” differed considerably at all locations. In
2009, higher cross-pollination rates were measured
than in 2010, resulting from the weather conditions
in both years. Influenced by the flowering period and
delayed crop development as well as the locally
reduced synchronisation in flowering, combined with
lower wind speeds in 2010 (Braunschweig: 2.5 m s-1,
2009: 2.9 m s-1; Groß Lüsewitz: 2.2 m s-1, 2009: 2.3
m s-1; Freising: 1.5 m s-1; 2009: 1.8 m s-1),
cross-pollination was relatively low (see data for
“Delitop”; Figs. 4a and 4b, Tables 3a and 3b).
The geographical position of the trial sites should
be taken into account when cross-pollination rates are
compared. According to the higher wind speeds in
both years, the highest cross-pollination rates of
“Delitop” (on average of the whole plot: 12.8% in
2009, 7.2% in 2010) were measured in Braunschweig
compared to Freising (mean 2009: 4.8%; 2010: 5.1%)
and Groß Lüsewitz (mean 2009: 4.8%; 2010: 2.6%).
As expected, cross-pollination decreased with higher
distances to the pollen donor field. In Braunschweig,
in 2009, an out-crossing rate from “Delitop” of 62.2%
was measured in the first row and after 3.5 m distance
to the donor plot, respectively, and was already
reduced to 13.6% after further 3 m. At 35.25 m and
68.25 m distance to “Delitop”, cross-pollination rates
of 4.4% and 1.1%, respectively, were found. Lower
cross-pollination rates of “Delitop” were detected in
Freising with 21.6% and Groß Lüsewitz with 27.3%
in the first row of the recipient plot. At 52 m, only
0.5% out-crossing of “Delitop” was found in Freising,
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Fig. 4a Distribution and cross-pollination rates of CMS-maize hybrids (DSP2, Torres and Zidane) and the fully fertile control variety Delitop as well as wind regimes during flowering in Braunschweig, Groß Lüsewitz and Freising in 2009 (WH = wind hours).
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Fig. 4b Distribution and cross-pollination rates of CMS-maize hybrids (DSP2, Torres and Zidane) and the fully fertile control variety Delitop as well as wind regimes during flowering in Braunschweig, Groß Lüsewitz and Freising in 2010 (WH = wind hours).
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Table 3a Mean values and standard deviations of the cross-pollination data in 2009.
Distance to pollen donor (m) 3.50 6.50 11.00 14.75 19.25 30.25 35.75 52.25 68.75
Braunschweig
Delitop
MV 62.20 14.20 14.30 6.65 8.50 4.41 4.60 1.86 1.13
SD 2.07 1.57 3.91 2.84 2.33 2.18 2.59 1.00 0.18
DSP2
MV 12.50 1.46 0.65 0.26 0.24 0.24 0.26 0.35 0.15
SD 2.42 0.56 0.26 0.09 0.03 0.15 0.15 0.09 0.06
Torres
MV 0.89 0.16 0.12 0.21 0.21 0.11 0.11 0.12 0.12
SD 0.17 0.04 0.02 0.10 0.14 0.05 0.02 0.03 0.05
Zidane
MV 6.63 1.02 0.89 0.96 1.68 0.61 0.66 0.59 0.64
SD 1.63 0.34 0.18 0.16 0.62 0.05 0.39 0.27 0.24
Groß Lüsewitz
Delitop
MV 27.30 2.34 7.47 3.01 1.57 0.70 0.57 0.14 0.20
SD 10.10 1.63 2.10 1.03 1.01 0.54 0.25 0.07 0.19
DSP2
MV 6.74 1.16 0.42 0.14 0.18 0.04 0.13 0.07 0.05
SD 1.50 0.81 0.13 0.02 0.13 0.03 0.09 0.05 0.04
Torres
MV 0.49 0.07 0.03 0.08 0.06 0.06 0.04 0.06 0.03
SD 0.08 0.04 0.02 0.03 0.04 0.04 0.03 0.05 0.02
Zidane
MV 2.47 0.32 0.22 0.24 0.14 0.12 0.08 0.09 0.11
SD 0.50 0.21 0.11 0.13 0.13 0.21 0.08 0.08 0.06
Freising
Delitop
MV 21.60 6.95 3.71 3.31 3.10 1.79 1.41 0.50 1.10
SD 5.62 0.49 0.69 0.70 1.07 0.55 0.33 0.12 0.33
DSP2
MV 11.20 1.19 0.72 0.44 0.54 0.25 0.20 0.12 1.10
SD 3.25 0.30 0.08 0.16 0.24 0.03 0.05 0.04 0.03
Torres
MV 0.60 0.18 0.18 0.14 0.20 0.18 0.17 0.16 0.14
SD 0.16 0.09 0.08 0.05 0.03 0.03 0.05 0.06 0.03
Zidane
MV 11.70 1.38 0.99 0.67 0.51 0.34 0.33 0.30 0.23
SD 2.13 0.19 0.20 0.21 0.19 0.04 0.13 0.11 0.08
MV, mean value; SD, standard deviation.
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Table 3b Mean values and standard deviations of the cross-pollination data in 2010.
Distance to pollen donor [m] 3.50 6.50 11.00 14.75 19.25 30.25 35.75 52.25 68.75
Braunschweig
Delitop
MV 45.33 7.49 3.95 2.70 2.55 1.19 0.60 0.42 0.32
SD 3.46 1.75 1.61 0.87 0.46 0.34 0.24 0.11 0.13
DSP2
MV 3.19 0.26 0.21 0.17 0.13 0.15 0.36 0.06 0.15
SD 1.79 0.13 0.08 0.05 0.03 0.11 0.43 0.02 0.06
Torres
MV 0.27 0.11 0.09 0.09 0.10 0.08 0.09 0.05 0.08
SD 0.14 0.09 0.02 0.02 0.06 0.09 0.04 0.04 0.04
Zidane
MV 0.98 0.23 0.31 0.28 0.39 0.28 0.14 0.19 0.20
SD 0.18 0.80 0.11 0.09 0.14 0.09 0.11 0.07 0.17
Groß Lüsewitz
Delitop
MV 16.40 4.34 1.13 0.48 0.18 0.17 0.14 0.05 0.04
SD 4.02 1.14 0.37 0.16 0.02 0.08 0.05 0.02 0.03
DSP2
MV 1.37 0.33 0.15 0.09 0.06 0.08 0.06 0.02 0.05
SD 0.65 0.08 0.05 0.02 0.02 0.04 0.03 0.01 0.04
Torres
MV 0.37 0.32 0.31 0.29 0.16 0.25 0.22 0.09 0.06
SD 0.06 0.16 0.13 0.23 0.06 0.10 0.04 0.11 0.02
Zidane
MV 1.15 0.44 0.28 0.31 0.16 0.89 0.36 0.14 0.08
SD 0.47 0.12 0.14 0.18 0.17 0.89 0.12 0.07 0.04
Freising
Delitop
MV 25.80 7.77 3.51 3.41 3.25 2.26 1.38 1.25 1.01
SD 6.92 2.30 1.12 1.06 0.56 1.09 1.03 0.95 0.78
DSP2
MV 2.07 0.13 0.35 0.40 0.25 0.05 0.14 0.04 0.03
SD 1.11 0. 03 0.42 0.57 0.25 0.05 0.18 0.03 0.03
Torres
MV 0.53 0.15 0.08 0.15 0.15 0.30 0.20 0.22 0.11
SD 0.18 0.15 0.06 0.15 0.09 0.33 0.24 0.33 0.12
Zidane
MV 1.75 0.50 0.30 0.35 0.31 0.25 0.25 0.25 0.24
SD 0.78 0.36 0.30 0.16 0.24 0.14 0.18 0.23 0.18
MV, mean value; SD, standard deviation.
and already after 30.25 m 0.7% cross-pollination was
detected in Groß Lüsewitz. A similar situation but
lower out-crossing rates were measured in 2010. At 35
m distance to the “Delitop” plot the cross-pollination
rate was below 1.0% in Braunschweig and Freising.
This rate was already reached at a distance of 14.75 m
in Groß Lüsewitz.
Comparing the 2009 CMS maize hybrids, the
highest cross-pollination rate was detected for “DSP2”
(on average 1.0%-1.8% of the whole plot), and the
lowest for “Torres” (< 0.2%) at all locations (Fig. 4a
and Table 3a). The highest values were measured in
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398
the first pollen recipient plot of “DSP2” in
Braunschweig (12.6%) with a strong decrease (1.4%
at 6.5 m and < 1% at 11 m). This decrease was also
found in Freising and Groß Lüsewitz and for “Zidane”
at all locations on a lower level. In 2010, the lowest
cross-pollination rates were measured for “Torres”
again (0.1%-0.2%). Differences between “DSP2” and
“Zidane” were small. The values varied between 0.3%
and 0.5%. In Freising and Groß Lüsewitz,
out-crossing from “Zidane” was even slightly higher
than “DSP2” (Fig. 4b and Table 3b). At a distance of
6.5 m to the pollen donor plot, all cross-pollination
rates were below 1% for all CMS maize hybrids.
3.5 Reduction of Cross-Pollination
In relation to the conventional fully fertile maize
hybrid “Delitop” cross-pollination of the CMS maize
hybrids was strongly reduced. Within the first 30 m of
the white maize plots, the area of the highest
out-crossing potential, the cultivation of “Torres”
reduced cross-pollination by 96.5% in 2009 and
88.9% in 2010, respectively, on average of all three
locations (Table 4). With “Zidane”, a reduction of
83.7% (2009) and 84.4% (2010) on average and
“DSP2” of 84.2% (2009) and 89.9% (2010) on
average was realised. Except for “Zidane”, the highest
reductions of out-crossing were found for
Braunschweig in both trial years which can be clearly
seen in the first row at 3.5 m distance to the pollen
donor plot.
Depending on the generally low cross-pollination
rates of Torres, the cross-pollination reduction was
consistently high from 3.5 m to 30.25 m distances and
was mainly above 95% at the different distances
within one plot. The two other CMS maize hybrids
showed an increasing reduction of cross-pollination
from 6.5 m onwards based on the higher
cross-pollination rates at 3.5 m distance to the pollen
donor plots in 2009. These effects were more balanced
in 2010, depending on the lower out-crossing rates.
Although these values are very satisfactory at the
first instant, no CMS maize hybrid could realise a
cross-pollination reduction of 100%.
4. Discussion
The objective of this study was to verify whether
CMS is a reliable confinement tool for the cultivation
of GM maize. The reliability of the CMS trait was
tested in three different environments in Germany and
data on the stability and reliability of the CMS trait for
confinement purposes were collected.
The relatively good growing conditions at all
locations in 2009, closely linked to the long-standing
mean temperatures and precipitations, allowed for
satisfactory crop development in an appropriate and
expected time scale. Contrary, the weather conditions
in 2010 were poor and crop development delayed
resulting in longer flowering periods and an
unsatisfactory synchronisation of male and female
flowers in donor and recipient plots, particularly in
Groß Lüsewitz and Braunschweig. Especially, the
temperature during germination and juvenile
development of the plants is vital. Delays in crop
development are highly expectable if temperatures
decrease below 10 °C for several days [28]. In May
2010, this situation occurred in Braunschweig and
Groß Lüsewitz. Temperatures in Freising were similar
to the long-standing mean temperature. Water
availability was not a critical point even if it was dry
at sowing and wet during the juvenile development in
May at all locations.
In both experimental years, no CMS maize hybrid
developed 100% sterile tassels, but differences in the
restoration of fertility were assessed. Generally, the
CMS effect, which is caused by a mitochondrial
dysfunction, can be compensated by restorer of
fertility genes (Rf genes) [11]. This compensation can
result in partly restored or fertile tassels with more or
less vital pollen. This fact is described in a number of
publications and explained as nucleo-mitochondrial
conflict which leads to gynodioecy, a co-occurrence
of female and hermaphroditic individuals within one
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Table 4 Reduction of cross-pollination by using CMS maize hybrids (DSP2, Torres and Zidane) in comparison to a conventional and fully fertile maize hybrid (Delitop = 100%) in (a) 2009 and (b) 2010 at Braunschweig, Groß Lüsewitz and Freising.
(a) 2009 Distance to pollen donor (m)
3.50 6.50 11.00 14.75 19.25 30.25
DSP2 MW
Braunschweig -79.82 -89.90 -95.42 -93.48 -97.15 -94.65 -91.73
Groß Lüsewitz -74.16 -55.58 -95.18 -94.52 -86.50 -91.69 -82.94
Freising -48.29 -82.92 -80.48 -86.85 -82.72 -85.98 -77.87
MW -67.42 -76.13 -90.36 -91.62 -88.79 -90.77 -84.18
Torres
Braunschweig -98.57 -98.85 -98.90 -96.84 -97.51 -97.51 -98.03
Groß Lüsewitz -98.24 -96.41 -99.60 -97.48 -96.44 -91.74 -96.65
Freising -97.20 -97.41 -95.05 -95.92 -93.58 -89.84 -94.83
MW -98.00 -97.56 -97.85 -96.74 -95.84 -93.03 -96.50
Zidane
Braunschweig -89.33 -92.18 -92.39 -85.48 -80.25 -86.22 -87.64
Groß Lüsewitz -88.73 -81.52 -97.46 -93.63 -96.70 -78.92 -89.49
Freising -45.87 -80.17 -73.30 -79.84 -83.57 -81.22 -74.00
MW -74.64 -84.62 -87.71 -86.32 -86.84 -82.12 -83.71
(b) 2010 Distance to pollen donor [m]
3.50 6.50 11.00 14.75 19.25 30.25
DSP2 MW
Braunschweig -92.96 -96.48 -94.32 -93.22 -94.84 -87.81 -93.27
Groß Lüsewitz -91.66 -92.44 -86.72 -80.46 -67.98 -83.85
Freising -91.64 -98.31 -88.33 -86.96 -92.00 -97.40 -92.44
MW -92.09 -95.74 -89.79 -86.88 -84.94 -92.60 -89.85
Torres
Braunschweig -99.25 -98.52 -97.74 -96.55 -95.87 -91.55 -96.58
Groß Lüsewitz -97.76 -92.60 -72.48 -39.36 -75.55
Freising -97.88 -98.05 -97.38 -94.91 -95.16 -83.21 -94.43
MW -98.29 -96.39 -89.20 -76.94 -95.52 -87.38 -88.85
Zidane
Braunschweig -97.84 -96.95 -92.20 -88.89 -84.54 -76.62 -89.51
Groß Lüsewitz -92.98 -89.75 -75.36 -35.88 -73.49
Freising -93.66 -93.46 -89.90 -88.49 -89.93 -85.65 -90.18
MW -94.82 -93.39 -85.82 -71.09 -87.24 -81.14 -84.39
MV = mean value.
population [14]. Apart from that, environmental
conditions can affect maize with respect to partial
restoration of male fertility [7, 8, 20-23, 29-31]. The
data presented revealed differences in the
development of tassels and the release of pollen
according to the location and the year, which were
dependent on the genotype.
The CMS-T-maize hybrid “DSP2” developed all
forms of tassels, depending on the location and
weather conditions of the years. CMS-T-cytoplasm is
considered to provide the most reliable male sterility
[7] but exceptions are known. Generally, two
dominant and jointly acting Rf genes are essential for
the restoration of fertility, Rf1 and Rf2. Especially, the
Rf1 allel affects the transcription of the responsible
mitochondrial gene (T-urf13) and leads to pollen
fertility [32]. This process can only happen in the
presence of Rf2. Dominant alleles of other Rf genes
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400
like Rf8 and Rf* which were detected by Dill et al. [33]
can compensate the Rf1 gene in the presence of Rf2.
These genes are rare in T-cytoplasms of maize, and
their expression is affected by the environment and
results in a few fertile or partly fertile plants in one
population [7]. The fact that in Braunschweig only a
few individuals of “DSP2” restored to fertility can
lead to the conclusion that the minor Rf8 gene was
involved in this process as described by Dill et al. [33].
Weider et al. [7] who tested “DSP2” as well but on a
smaller scale, described a slightly unstable behaviour
of sterility, but reversion to fertility occurred also only
a few times across all environments. Different to this,
a stronger response of “DSP2” to weather conditions
was found in Freising and Groß Lüsewitz, where
partly restored and fertile tassels appeared in 2009 but
mainly sterile tassels were found in 2010. The related
MKS data of “DSP2” underlined this weather impact
slightly because much higher rates were measured in
2009 than in 2010. The existing pollen is, therefore,
viable depending on the sporophytic T-cytoplasm as
described by Dill et al. [33]. Weider et al. [7] revealed
that the MKS values of “DSP2” decrease when
temperatures increase and, therefore, correspond with
the results from Groß Lüsewitz and Freising, where
MKS values were low and the temperatures increased
a short time before the onset of anthesis.
In both years, the CMS-S-types “Torres” and
“Zidane” developed partly restored and fertile tassels
at all three locations. “Torres” developed the smallest
amount of pollen which was mainly sterile, while
“Zidane” developed fluctuated and fertile tassels and
released explicitly more vital pollen. Hence, the
sterility of this cytoplasmic type is classified as being
unstable [34, 35]. Compared to the T-cytoplasm, the
restoration in S-cytoplasms is generally more complex
which is likely depending on the dominant Rf3
restorer gene and a large number of further
spontaneously occurring Rf genes and more than 60
restoring allele mutations [34, 36, 37]. The Rf3 gene is
not environmentally sensitive [38] compared to the
recently identified Rf9 gene [38]. Especially moderate
temperatures were positively affecting the expression
of Rf9 compared to higher temperatures. Therefore, a
higher level of Rf9 gene expression in “Zidane” could
be expected, especially in 2009 when this hybrid
developed large amounts of pollen per tassel. “Torres”
stayed more sterile, even when partly restored tassels
were detected. Possibly more Rf3 genes were involved
in the restoration process.
At all locations and for all CMS maize hybrids, the
development of vital pollen was depending on the year.
Higher MKS values were detected in 2009 than in
2010. This phenomenon might be explained by the
sensitivity of rf gene activation or inactivation to
environmental impacts and temperatures just before
anthesis [7]. In the first half of July 2010, the air
temperatures raised by 3 °C and 3.5 °C, respectively,
above the long-standing average temperature at all
trial locations. In general, low quantities of pollen
were produced by all CMS maize hybrids and,
consequently, MKS values were very low as well.
This indicates an influence of air temperature just
before anthesis on the restoration of fertility on both
T- and S-cytoplasm. Weider et al. [7] studied the
period of 10 days before anthesis to clarify whether
different climatic factors affect the MKS of several
CMS hybrids. They found positive as well as negative
correlations between the MKS and different climatic
factors, but a rule for interactions could not be
detected. Duvick [29] and Tracy et al. [30] already
assumed that humid and cool conditions cooperate in
restoration of fertility, whereas dry and hot conditions
maintain sterility. The influence of high temperatures
was described for other crops as well. Increasing rates
of sterility of CMS cotton plants were found with
increasing temperatures and day length [21]. This
might not only be caused by Rf expression but also by
toxin production [39] which affects the CMS trait. In
experiments with sorghum, Elkonin et al. [40]
revealed that variations of precipitation up to three
weeks before the beginning of anthesis correlate
Applicability of Cytoplasmic Male Sterility (CMS) as a Reliable Biological Confinement Method for the Cultivation of Genetically Modified Maize in Germany
401
positively in the expression of fertility restoring genes
with water availability. Elkonin et al. [40] described
that following a wet season, the percentage of fertile
and partially fertile plants was significantly higher
than in drought seasons. This suggests that water
limiting conditions prevent the expression of restorer
genes resulting in a stable sterility.
In our study, the stability of the CMS trait of maize
hybrids was tested under large-scale and practical
agricultural conditions for the first time. Thus, no
comparable data of cross-pollination rates are
available in the scientific literature.
Very low cross-pollination rates of the CMS-maize
hybrids were found in both experimental years and at
all locations. The maximum pollen spread was
measured for “DSP2” at Braunschweig in 2009, where
12.6% cross-pollination was found in the first row of
the recipient plot. The values decreased rapidly and
were already below 1% at 11 m distance to the pollen
donor plot. All other cross-pollination rates ranged
below these, particularly for “Torres” based on very
low pollen releases. The data presented are based on a
visual detection system where yellow kernels in white
maize cobs were counted.
In relation to the fully fertile variety “Delitop”, all
CMS maize hybrids provided a strong reduction of
cross-pollination which varied slightly between 2009
and 2010, due to the weather conditions and crop
development. Generally, the reduction was on average
highest for “Torres” (up to 99%) and the lowest for
“Zidane” (84%). The reduction decreased with further
distances to the pollen donor plot. According to these
results, CMS maize hybrids provide efficient
confinement for pollen-mediated gene flow.
The choice of an appropriate CMS maize hybrid
should depend on the trait and the purpose of its
release to the environment. The T-cytoplasm is no
longer used for breeding due to its sensitivity to the
fungal pathogen Bipolaris maydis [7]. Apart from the
CMS-C-type which is the most widely applied form of
CMS today [41], it is also possible to cultivate
CMS-S-types successfully. A number of interesting
S-cytoplasms existed that produce only a very small
amount of viable pollen [7] and could be used for
confinement purposes. Thus, it may be concluded that
CMS can be a useful confinement tool when used in
carefully selected genotypes and accurately tested for
stability. Wang et al. [42] recently developed a system
for producing plant-made pharmaceuticals (PMP) in
CMS maize by using CMS-T-cytoplasm lines. It is not
developed with CMS maize hybrids yet. A successful
cultivation of GM CMS-maize hybrids requires a
sufficient pollination of the plants by admixing a
male-fertile and non-GM pollen donor [43]. If the
CMS maize hybrid and the pollinator plant provide a
different genetic background, yield can be
significantly increased [26, 44, 45]. The so-called
Plus-Hybrid-Effect [43, 46] combines the potential
benefits of CMS and a Xenia effect. Hence, pollen
confinement combined with yield increase offers the
potential for a successful cultivation of GM maize.
Currently, it is recommended to grow 80:20 mixtures
of GM:CMS hybrids and male fertile non-GM hybrids
for obtaining sufficient yields [44]. Studies being
performed in the frame of an EU-funded project
(www.price-coexistence.com) will provide further
data to precise the current recommendations.
Summarising the data presented, it becomes
obvious that efficient confinement strategies can be
developed by exploring the CMS trait. For example,
depending on the CMS maize hybrids explored,
isolation distances between neighbouring GM and
non-GM maize fields could be strongly reduced to a
maximum 10 m to be in line with the European
coexistence requirements based on a labelling
threshold of 0.9%.
5. Conclusions
Cultivation of the CMS maize hybrids tested in our
study can be proposed as a useful tool for
cross-pollination reduction. The out-crossing potential
of traits into neighbouring maize crops can be
Applicability of Cytoplasmic Male Sterility (CMS) as a Reliable Biological Confinement Method for the Cultivation of Genetically Modified Maize in Germany
402
drastically reduced.
The behavior and development under several
environmental conditions should be known before
selecting the appropriate CMS maize hybrid for
combinations with GM traits. More knowledge is
required on terms and conditions of the expression of
Rf genes and their sensitivity towards environmental
factors. Depending on the GM trait and the purpose of
the environmental release, CMS as a reliable
biological confinement method for GM maize might
be combined with other confinement methods, e.g.,
small isolation distances, buffer zones or border zones
of non-GM maize [6, 47-50] or variations in flowering
behavior which could be influenced by growing
varieties of different ripening groups and/or choosing
different sowing dates [51, 52]. The co-cultivation
with a sufficient quantity of a non-related, non-GM
male fertile maize is necessary for gaining sufficient
yield.
Acknowledgments
The project was kindly funded by the Federal
Ministry of Education and Research, BMBF (Project
No.: 0315 210 D).
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Journal of Agricultural Science and Technology A 3 (2013) 404-409 Earlier title: Journal of Agricultural Science and Technology, ISSN 1939-1250
Evaluation of Fungicides for Controlling Stem Rust Race
Ug99 on Bread Wheat
Joseph Kinyoro Macharia1, Ruth Wanyera2 and Samuel Kilonzo2
1. Department of Crops, Horticulture and Soils, University of Egerton, Njoro 536-20115, Kenya
2. Kenya Agricultural Research Institute (KARI)-Njoro, Njoro 20107, Kenya
Received: February 9, 2013 / Published: May 20, 2013. Abstract: Stem rust race Ug99, also designated TTKSK (Puccinia graminis f. sp. tritici) cause stem or black rust, which is a serious disease of wheat worldwide. Field experiments were conducted at two sites during 2008 and 2009 growing seasons to evaluate the effectiveness of two new foliar fungicides: viz. Nativo 300 SC (trifloxystrobin 100 g L-1 + tebuconazole 200 g L-1) and Prosaro 250 EC (prothioconazole 125 g L-1 + tebuconazole 125 g L-1), in controlling stem rust on susceptible wheat cultivar Duma. AmistarXtra 280 SC (azoxystrobin 200 g L-1 + cyproconazole 80 g L-1) and Folicur 250 EC (tebuconazole) were used as checks. The treatment at each site and year included non-treated control and two spray applications of the fungicides at growth stages (GS) 55 (heading) and 65 (flowering). Stem rust severities were scored using the modified Cobb scale at 14-day intervals after application. The data were used to calculate mean rust severity (MRS). Stem rust epidemics were severe at KARI-Njoro in 2008 and the treatment effects on stem rust severities, grain yield and 1,000 kernel weights were significant at both KARI-Njoro and Mau-Narok sites. The fungicide treatments, significantly (P ≤ 0.05) reduced stem rust severity, increased grain yield and 1,000 kernel weight of the susceptible wheat cultivar Duma compared to the non-treated control. Both fungicides: Nativo 300 SC and Prosaro 250 EC applied at the rate of 1.0 L ha-1 were recommended for commercial use.
Key words: Stem rust, wheat (Triticum aestivum L.), fungicide.
1. Introduction
Stem or black rust, caused by Puccinia graminis
Pers. f. sp. tritici Eriks. and E. Henn., is a periodically
devastating disease of wheat (Triticum aestivum L.)
worldwide [1, 2]. The disease had been controlled
effectively through widespread growing of resistant
cultivars [3]. Following the re-emergence of an
aggressive and highly virulent race Ug99 detected in
Uganda in 1999 [4], also designated as TTKSK based
on the North American nomenclature [2, 5] and the
subsequent isolation of other variants within the Ug99
lineage in Kenya, poses a serious threat to the existing
wheat cultivars worldwide. Kenya is designated as a
“hotspot” for stem rust because disease outbreaks
occur regularly due to favorable environmental
Corresponding author: Joseph Kinyoro Macharia, Ph.D.,
research fields: crop pathology and cereal rusts. E-mail: josephkinyoro@gmail.com.
conditions [6]. For over 30 years genetic resistance
has provided adequate protection against stem rust
disease [7, 8]. However, the susceptibility of the most
wheat cultivars and heavy yield losses exceeding 80%
[9] incurred by farmers has entailed the adoption of an
integrated mitigation strategy. Therefore, the
evaluation of the available fungicides for efficacy
against the stem rust pathogen is necessary.
Fungicide application has become an integral part
of wheat production in Kenya since the mutation of
stem rust race Ug99. However, the number of
fungicides recommended for the control of stem rust
are few. This is because the effects of fungicides on
yield loss prevention are poorly understood and data
limited [10]. This paper reports field experiments
conducted under natural infection to determine the
efficacy of two new foliar fungicides, viz. Nativo 300
SC (trifloxystrobin 100 g L-1 + tebuconazole 200 g L-1)
DAVID PUBLISHING
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Evaluation of Fungicides for Controlling Stem Rust Race Ug99 on Bread Wheat
405
and Prosaro 250 EC (prothioconazole 125 g L-1 +
tebuconazole 125 g L-1) on stem rust severity, grain
yield and 1,000-kernel weight.
2. Materials and Methods
2.1 Materials
Field trials were conducted in 2008 and 2009 at
Kenya Agricultural Research Institute (KARI)-Njoro
and Mau-Narok (Purko Ranch), Kenya. The test
cultivar was Duma, which is popular and
recommended for low and medium elevation
growing-areas. The cultivar is highly susceptible to
stem rust, but is fairly resistant to other rusts. Stem
rust epidemics occurred naturally at both sites. A
randomized complete block design with four
replications was used. The cultivar Duma was planted
in 9 m2 plots (Table 1) using an experimental
seed-drill at a seeding rate of 100 g plot-1. A uniform
application of Di-ammonium phosphate fertilizer
(18% N: 46%; P: 0% K) was applied at planting at the
recommended rate of 150 kg ha-1. The plots were
sprayed with Stomp 500 E (pendimethalin), a
pre-emergent herbicide at the rate of 3 L ha-1, to
control grass weeds and Buctril MC (bromoxynil +
MCPA) at the rate of 1.25 L ha-1 at growth stage (GS)
24 (tillering) [11] to control broad leaf weeds.
Metasystox 250 EC (oxy-demeton-s-methyl)
insecticide was applied at the rate of 0.5 L ha-1 to
control cereal aphids. The fungicide treatments
included two new products: Nativo 300 SC
(trifloxystrobin 100 g L-1 + tebuconazole 200 g L-1)
and Prosaro 250 EC (prothioconazole 125 g L-1 +
tebuconazole 125 g L-1), each applied at three rates:
0.6, 0.75 and 1.0 L ha-1. Two standard fungicides:
AmistarXtra 280 SC (azoxystrobin 200 g L-1 +
cyproconazole 80 g L-1) and Folicur 250 EC
(tebuconazole), each applied at the rate of 1.0 L ha-1,
and non-treated control were used for comparison.
2.2 Methods
The fungicides were applied twice at GS 55 and 65
(Table 1) using a 15 L capacity knapsack sprayer and
recommended water volumes of 200 L ha-1. The
applications were on the stems and the flag leaf
canopy. The plots were monitored for the appearance
of stem rust and disease severities were scored on
whole plots using the modified Cobb scale [12] before
fungicide application, and at two 14-day intervals
following the application (Table 1). In 2008, the first
reading prior to fungicide treatment was done at GS
65 (flowering) and the second reading was 14-16 days
after the first treatment at GS 71 (seed water ripe).
The third reading was done at GS 77 (late milk). At
maturity, the plots were harvested using a Hans-Ulrich
Hege plot combine harvester (Type: Hege 125C.
Maschinenbau D 7112 Waldenburg/Wurtt-Hohebuch,
Table 1 Management activities for each of the four trials evaluating fungicides for efficacy against stem rust.
Management activity, GS* Trial
2008-2009 2009-2010
KARI-Njoro Mau-Narok KARI-Njoro Mau-Narok
Planting date: May 30 Sept. 19 May 16 Sept. 29
Fungicide application:
1st date at GS 55 Aug. 4 Nov. 15 Aug. 9 Dec. 15
2nd date at GS 65 Aug. 18 Nov. 30 Aug. 23 Jan. 2
Rust evaluation:
1st date at GS 65 Aug. 9 Oct. 4 July 24 Nov. 28
2nd date at GS 71 Aug. 23 Nov. 8 Aug. 7 Dec. 12
3rd date at GS 77 Sept. 8 Nov. 22 Aug. 21 Feb. 19
Harvesting: Nov. 6 Apr. 4, 2010 Oct. 31 Apr. 1, 2011
GS* = Growth Stage; GS 55 = heading, GS 65 = flowering, GS 71 = seed water ripe and GS 77 = late milk [11].
Evaluation of Fungicides for Controlling Stem Rust Race Ug99 on Bread Wheat
406
West Germany). Grain weight measurements were
taken after harvest and drying to 13% moisture
content. A sample of grain was taken from each plot
for determination of kernel weight based on the weight
of 1,000 grains.
2.3 Data Analysis
Data on disease severity, grain yield and 1,000
kernel weight were analyzed according to the analysis
of variance procedure using the SAS statistical
package (PROC-ANOVA), and differences in
treatment effects were compared at P ≤ 0.05 using the
least significant difference test [13, 14].
3. Results and Discussion
In 2008, stem rust at both KARI-Njoro and
Mau-Narok trials developed early in the growing
season; as a result, the mean rust severity (MRS) in
non-treated plots was 52.5% and 40.8%, respectively
(Table 2). In 2009, the MRS was moderate and the
MRS in non-treated control plots was 23.8% and
16.9%, respectively (Table 3). All the fungicide
treatments reduced stem rust on susceptible cultivar
Duma. Fungicide applications significantly (P ≤ 0.05)
reduced MRS compared to the non-treated control,
with Prosaro at 1.0 L ha-1, Nativo at 0.75 and 1.0 L
ha-1 and the standards (Folicur 250 EC and
AmistarXtra 280 SC), performing better than Prosaro
at 0.6 L ha-1, 0.75 L ha-1 and Nativo 300 SC at the
rate of 0.6 L ha-1 (Tables 2 and 3). The highest
disease severity reduction in 2008 was generally
observed in plots that were sprayed with Prosaro 250
EC at 1.0 L ha-1 (59.4%), Nativo 300 SC and
AmistaXtra 280 SC at 1.0 L ha-1 (55.6% each) at
KARI-Njoro and Prosaro 250 EC at 0.6 L ha-1
(75.7%), Prosaro 250 EC at 1.0 L ha-1 (75.5%) and
Nativo 300 SC at 1.0 L ha-1 (73.5%) at Mau-Narok.
In 2009, the highest disease severity reduction was in
plots sprayed with Folicur 250 EC at 1.0 L ha-1
(85.3%), followed by Prosaro 250 EC at 0.75 L ha-1
(82.6%) and 1.0 L ha-1 (77.8%) at KARI-Njoro and
Prosaro 250 EC at 0.6 L ha-1 (76.5%), Prosaro 250
EC at 1.0 L ha-1 (75.5%) and Nativo 300 SC at 1.0 L
ha-1 (73.47%) at Mau-Narok.
Table 2 Effect of fungicide treatments on stem rust severity, grain yield and 1,000 kernel weight on wheat cultivar Duma KARI-Njoro and Mau-Narok 2008.
KARI-Njoro Mau-Narok
Treatmente Stem rust severity Grain yield 1,000 kernel weight Stem rust severity Grain yield 1,000 kernel weight
Rate (L ha-1)
MRSa Reductionb
(%) (t ha-1)
Increasec
(%) (g)
Increasec
(%) MRSA Reductionb
(%) (t ha-1)
Increasec
(%) (g)
Increasec
(%)
Non-treated - 52.5 a - 0.5 b - 27.9 c - 40.8 a - 1.4 c - 29.4 c - Nativo 300 SC
0.6 31.3 b 40.4 1.1 a 54.5 33.4 b 19.7 14.7 bc 63.9 2.8 ab 50.0 39.3 ab 33.7
Nativo 300 SC
0.75 24.6 bc 53.1 1.2 a 58.3 35.9 a 28.7 11.3 bc 72.3 2.6 b 46.2 41.1 a 39.8
Nativo 300 SC
1.0 23.3 c 55.6 1.3 a 61.5 33.8 b 21.1 10.8 bc 73.5 2.6 b 46.2 38.2 ab 29.9
Prosaro 250 EC
0.6 25.4 bc 51.6 1.2 a 58.3 34.9 ab 25.1 9.9 c 75.7 3.3 b 75.6 41.0 a 39.5
Prosaro 250 EC
0.75 24.2 bc 53.9 1.1 a 54.5 34.7 ab 24.4 14.2 bc 65.2 2.5 b 44.0 37.4 b 27.2
Prosaro 250 EC
1.0 21.3 c 59.4 1.2 a 58.3 35.6 a 27.6 10.0 bc 75.5 2.4 b 41.2 38.4 ab 30.6
AmistaXtra 280SC
1.0 23.3c 55.6 1.1 a 54.5 33.6 b 20.4 15.4 bc 62.3 2.6 b 46.2 37.4 b 27.2
Folicur 250EC 1.0 25.4 bc 51.6 1.2 a 58.3 35.9 28.7 16.7 b 59.1 2.7 b 48.1 38.4 ab 30.6
Meand - 24.9 52.7 1.2 57.3 28.5 24.5 12.9 68.4 2.7 49.7 38.9 25.1
Lsd (0.05) - 7.2 - 0.2 - 1.7 - 6.7 - 0.5 - 3.0 - aMRS = mean rust severity (modified Cobb scale); breduction (%) = (non-treated MRS-fungicide-treated MRS) × 100 non-treated-1; cincrease (%) = (treated – non-treated) × 100 treated-1; dmean = mean of fungicide-treatments; etreatment means within columns followed by the same letter are not significantly different at P ≤ 0.05 according to least significant difference (LSD) test.
Evaluation of Fungicides for Controlling Stem Rust Race Ug99 on Bread Wheat
407
Table 3 Effect of fungicide treatments on stem rust severity, grain yield and 1,000 kernel weight on wheat cultivar Duma KARI-Njoro and Mau-Narok 2009.
KARI-Njoro Mau-Narok
Treatmente
Stem rust severity Grain yield 1,000kernel weight Stem rust severity Grain yield 1,000 kernel weight
Rate (L ha-1)
MRSa Reductionb
(%) (t ha-1)
Increasec
(%) (g)
Increasec
(%) MRSA Reductionb
(%) (t ha-1)
Increasec
(%) (g)
Increasec
(%)
Non-treated - 23.8 a - 1.0 c - 38.7 b - 16.9 ab - 2.2 d - 34.8 b - Nativo 300 SC
0.6 7.8 bc 67.4 1.8 b 44.4 43.0 a 10.0 12.5 abc 26.0 4.3 ab 48.8 46.4 a 25.0
Nativo 300 SC
0.75 6.5 bc 72.6 1.9 b 47.4 42.6 a 9.2 6.7 c 59.2 3.2 c 31.3 44.2a 21.3
Nativo 300 SC
1.0 6.9 bc 71.0 2.3 a 56.5 43.7 a 11.4 8.3 c 50.9 4.4 a 50.0 47.2 a 26.3
Prosaro 250 EC
0.6 8.9 b 62.6 2.1 ab 52.4 43.9 a 11.8 15.6 ab 7.7 3.6 bc 38.9 43.6 a 20.2
Prosaro 250 EC
0.75 4.1 bc 82.6 1.9 ab 47.4 43.2 a 10.4 18.1 a 7.1 4.3 ab 48.8 43.7 a 20.4
Prosaro 250 EC
1.0 5.3 bc 77.8 2.1 ab 52.4 42.9 a 9.8 11.9 bc 29.6 3.9 abc 43.6 46.9 a 25.8
AmistaXtra 280 SC
1.0 8.9 b 62.6 2.3 a 56.5 43.7 a 11.4 11.3 bc 33.1 4.2 ab 47.6 45.2 a 23.0
Folicur 250 EC
1.0 3.5 c 85.3 2.2 ab 54.5 42.7 a 9.2 9.4 c 44.4 4.3 ab 48.8 46.1 a 24.5
Meand - 6.5 72.7 2.1 54.1 43.2 10.4 11.8 32.3 4.0 44.7 45.4 23.3
Lsd (0.05) - 5.2 - 0.4 - 2.5 - 5.9 - 0.7 - 5.3 - aMRS = mean rust severity (modified Cobb scale); breduction (%) = (Non-treated MRS - fungicide-treated MRS) × 100/Non-treated;
cincrease (%) = (treated – Non-treated) × 100 Non-treated-1; dmean = mean of fungicide-treatments; etreatment means within columns followed by the same letter are not significantly different at P ≤ 0.05 according to least significant difference (LSD) test.
The fungicide treatment effect on grain yield and
1,000 kernel weight was significant (P ≤ 0.05) at the
two sites and years. In 2008, the highest grain yield of
1.3 t ha-1 was recorded in plots treated with Nativo
300 SC at 1.0 L ha-1, which was 61.5% higher than the
non-treated control at KARI-Njoro. This was followed
by Nativo 300 SC at 0.75 L ha-1 Prosaro 250 EC at 0.6
L ha-1 and 1.0 L ha-1, and Folicur 250 EC at 1.0 L ha-1,
which were 58.3% higher than the non-treated control.
Plots treated with Nativo 300 SC at 0.75 L ha-1 and
Prosaro 250 EC at 1.0 L ha-1 had the highest
1,000-kernel weights with 28.7% and 27.6% increases,
respectively, over the control. In Mau-Narok in the
same year, Prosaro 250 EC and Nativo 300 SC at the
rate of 0.6 L ha-1 and Folicur 250 EC at 1.0 L ha-1 had
the highest grain yields of 3.3 t ha-1, 2.8 t ha-1 and 2.7 t
ha-1, increases of 75.6%, 50% and 48.1%, over the
untreated control (Table 1). In 2009, treatments of
Nativo 300 SC, AmistarXtra 280 SC and Folicur 250
EC, all at 1.0 L ha-1, had the highest grain yields of
2.3 t ha-1 and 2.2 t ha-1. Plots treated with Prosaro 250
EC at 0.6 L, Nativo 300 SC and AmistarXtra 280 SC
at 1.0 L ha-1 had increased 1,000 kernel of 11.8% and
11.4% over the non-treated control at KARI-Njoro. In
Mau-Narok, treatments of Nativo 300 SC at 1.0 L ha-1,
Prosaro 250 EC at 0.75 L ha-1 and Folicur 250 EC at
1.0 L ha-1 had the highest grain yields of 4.4 t ha-1 and
4.3 t ha-1. The 1,000 kernel weight was the highest
with Nativo at 0.75 L ha-1, 1.0 L ha-1 and Prosaro 250
EC at 1.0 L ha-1.
The average grain yields and 1,000 kernel weights
across the sites varied from one treatment to another,
ranging from 1.2-4.0 t ha-1 and 28.5-45.4 g,
respectively. Significant (P ≤ 0.05) grain yield
increases of 57.3% and 49.7% were obtained at
KARI-Njoro and Mau-Narok in 2008, while 54.1%
and 44.7% increases occurred in 2009, respectively.
Similar increases in 1,000 kernel weight occurred at
both sites, KARI-Njoro (24.5% and 25.1%) in 2008,
and 10.4% and 23.3% in 2009 (Tables 2 and 3).
In Kenya, wheat is grown in many agro-ecological
zones that have different planting dates [15]. These
Evaluation of Fungicides for Controlling Stem Rust Race Ug99 on Bread Wheat
408
staggered plantings provide green crops for most of
the year allowing urediniospores to move from one
area to another. The favorable environmental
conditions, and the presence of host plants year-round,
favor the survival of high levels of inoculum. It is
therefore difficult to prevent or reduce infection of
susceptible cultivars. There is very little published
information on fungicide-use to control wheat stem
rust specifically related to the stem rust race Ug99 and
its variants. The occurrence of stem rust infections and
the onset of epidemics differed from year to year and
site to site. Stem rust levels were high in 2008 at
KARI-Njoro and Mau-Narok (despite early moisture
stress at KARI-Njoro; rain showers at GS 65 initiated
the spread of the disease). In 2009, the epidemics were
moderate to low at both sites. The low disease
pressure could have been due to the heavy rains at the
vegetative growth stage (May-September at
KARI-Njoro and October-November at Mau-Narok)
and later on, drought in Mau-Narok during December,
2009 and January-February, 2010.
In environments, such as those at KARI-Njoro and
Mau-Narok in 2008, where stem rust epidemics began
early, followed by conditions favorable for pathogen
growth and spread, stem rust can greatly reduce grain
yield and 1,000 kernel weight in susceptible varieties.
In that year, the average grain yield and 1,000 kernel
weight loss at KARI-Njoro and Mau-Narok in 2008
were 50.0% and 48.0%, 2.1% and 24.4%, respectively.
In 2009, the losses were 52.4%, 45.0%, 10.4% and
23.3%, respectively, in untreated control plots versus
fungicide-treated plots. These results are consistent
with losses reported in other studies [8, 16, 17].
Pretorius [18] reported that yield losses caused by
stem rust ranged from 7% to 35% depending on
variety. Dill-Macky and Roelfs [17] observed yield
losses of 50% to 58% after inducing severe stem rust
epidemics in barley and wheat, respectively. Mayfield
[19] found a clear relationship between grain yield and
disease severity by demonstrating that prevention of a
1% increase in stem rust severity saved a 2% loss in
grain yield. In the present study, all fungicide
applications resulted in lower disease severities and
higher yields than non-treated control plots.
Loughman et al. [8] reported that Folicur
(tebuconazole) provided more consistent results in
terms of both disease control and yield increases than
Triad (triadimefon) or Impact (flutriafol). In the
current study, the new fungicide treatments: Nativo
300 SC (trifloxystrobin 100 g L-1 + tebuconazole 200
g L-1) and Prosaro 250 EC (prothioconazole 125 g L-1
+ tebuconazole 125 g L-1) and the standards, applied
at 1.0 L ha-1 were generally effective in reducing the
disease and increasing grain yield and 1,000 kernel
weight. The lower rates resulted in more variable
disease control, grain yields and 1,000 kernel weight
responses.
Rust severity was relatively low in 2009 in the trials
at KARI-Njoro and Mau-Narok compared to 2008, yet
grain yields increased in response to fungicide
applications. This yield increase under relatively low
disease pressures may have been due to greening
effects of the fungicides [6]. Such stimulatory effect
of fungicide treatments on growth may enhance grain
yields even in the absence of disease [20, 21] and as
demonstrated in this study, fungicides treatments may,
if applied under high and moderate disease pressure at
critical crop growth stages, increase yields by
suppressing or eliminating the negative effects of stem
rust disease.
4. Conclusions
The impact of fungicide-use in the management of
stem rust was well illustrated at both trial sites. The
fungicide treatments had the ability to suppress
disease development and protect the crop canopy,
which is vital for dry matter accumulation and yield.
The study also displayed that stem rust could severely
reduce wheat grain yield of susceptible cultivars,
therefore, the adoption of foliar fungicides to combat
stem rust disease as a short term control strategy until
resistant cultivars are developed is encouraged in
Evaluation of Fungicides for Controlling Stem Rust Race Ug99 on Bread Wheat
409
Kenya. Consequently, both fungicides: Nativo 300 SC
and Prosaro 250 EC applied at the rate of 1.0 L ha-1
were recommended for commercial use.
Acknowledgments
The authors acknowledge the funding from Bayer
Crop Science, East Africa, and the support of D.
Onyango and P. Kinyanjui during implementation of
the trials.
References
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[2] R. P. Singh, D.P. Hodon, J. Huerta-Espino, Y. Jin, P. Njau, R. Wanyera, et al., Will stem rust destroy the world’s wheat crops?, in: D.L. Sparks (Ed.), Advances in Agronomy, Academic Press, London, UK, 2008, pp. 271-309.
[3] Y. Jin, L.J. Szabo, M.N. Rouse, T. Fetch, Jr., Z.A. Pretorius, R. Wanyera, et al., Detection of virulence to resistance gene Sr36 within the TTKS race lineage of Puccinia graminis f. sp. Tritici, Plant Disease 93 (2008) 367-370.
[4] Z.A. Pretorius, R.P. Singh, W.W. Wagoire, T.S. Payne, Detection of virulence to wheat stem rust gene Sr31 in Puccinia graminis f. sp. tritici in Uganda, Phytopathology 84 (2000) 203.
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[10] R. Wanyera, J.K. Macharia, S.M. Kilonzo, Challenges of fungicide control on wheat rusts in Kenya, in: O. Carisse (Ed.), Fungicides, ISBN: 978-953-307-266-1, Publisher: in Tech., 2010, pp. 123-138.
[11] J.C. Zadoks, T.T. Chang, C.F. Konazak, A decimal code for growth stages of cereals, Weed Research 14 (1974) 415-421.
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[16] R. Dill-Mackey, G. Rees, G.J. Platz, Stem rust epidemics and their effects on grain yield and quality in Australian barley cultivars, Australian Journal of Agricultural Research 41 (1990) 1057-1063.
[17] R. Dill-Macky, A.P. Roelfs, The effect of stand density on the development of Puccinia graminis f. sp. tritici in barley, Plant Disease 84 (2000) 29-34.
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[21] S.N. Wegulo, J.M. Rivera, C.A. Martison, F.W. Nutter, Jr., Efficacy of fungicide treatments for common rust and northern leaf sport in hybrid corn seed production, Plant Dis. 82 (1998) 547-554.
Journal of Agricultural Science and Technology A 3 (2013) 410-416 Earlier title: Journal of Agricultural Science and Technology, ISSN 1939-1250
Occurrence and Effects of Pineapple Mealybug Wilt
Disease in Central Uganda
Bosco Bua1, Jeninah Karungi2 and Geoffrey Kawube2
1. Department of Agriculture, Kyambogo University, Kampala, Uganda
2. Department of Agricultural Production, College of Agricultural and Environmental Sciences, Makerere University, Kampala,
Uganda
Received: October 12, 2012 / Published: May 20, 2013. Abstract: Pineapple mealybug wilt disease (PMWD) is one of the latest outbreaks of diseases attacking pineapple in Uganda. However, its occurrence and effects have not been documented and quantified, yet the disease poses a serious threat to the pineapple industry. Therefore, the objective of this study was to assess the occurrence and effects of PMWD on pineapple in central Uganda. Semi-structured questionnaire was used to solicit information from 82 respondents consisting of farmers, opinion leaders, key informants, political and technical leadership during May 2011. PMWD was observed in all the fields surveyed but with varying incidences and severities. In addition, PMWD was more common during the dry seasons than the rainy seasons where higher incidences were associated with high mealybug populations. PMWD manifested as a syndrome characterized by yellowing of leaves, stunting, wilting and rotting of roots. The effects of PMWD were variable but yield reductions and low plant populations were widely reported. Although, the occurrence of PMWD was reported to the different level of authority in the districts, very little was done to curb its spread.
Key words: Control, effects, perceptions, pineapple mealybug wilt disease, occurrence.
1. Introduction
Pineapple (Ananas comosus (L.) Merr) is an
important horticultural crop in many tropical and
sub-tropical countries including Uganda [1]. In
Uganda, pineapple is widely grown in the central
region in the districts of Mukono, Kayunga, Luwero
and Masaka. The other pineapple growing districts are
Ntungamo and Kabale in South-Western Uganda.
Although, the local and export demand for Pineapple
has steadily increased with the estimated production
and export earnings of 2,426,000 MT and
US$181,000 in 2008, respectively, pineapple
production is still below optimum because of a
diversity of constraints [2]. Pests and diseases, lack of
improved pineapple production technologies and
Corresponding author: Bosco Bua, Ph.D., research fields:
crop science, plant diseases diagnosis, management and epidemiology, pathogens genetic diversity, horticulture. E-mail: bbua@kyu.ac.ug, boscobua@yahoo.com.
declining soil fertility are among the notable
constraints [3, 4]. Pineapple mealy bug wilt disease
(PMWD) also called pineapple wilt or quick wilt, the
latest outbreak of diseases attacking pineapple in
Uganda has been reported and confirmed in two major
pineapple growing districts of Mukono and Kayunga
[5]. Since its first reported outbreak in the two
districts in 2009, PMWD has continued to spread in
many parts of the country and is a serious threat to the
pineapple industry.
Pineapple mealy bug wilt disease manifests itself as
wilting of the central leaves due to loss of turgidity in
the leaves and inability of the roots to grow, collapse
and rotting. According to Gary et al. [6], PMWD is a
widespread and devastating disease in many pineapple
growing areas of the world. However, PMWD is only
reported from areas of the world where members of
the Dysmicoccus mealybugs species complex occur.
In fact, higher incidences of PMWD were associated
DAVID PUBLISHING
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Occurrence and Effects of Pineapple Mealybug Wilt Disease in Central Uganda
411
with high mealybugs populations. However, PMWD
only develops in plants infected with a closterovirus,
designated pineapple mealybug associated virus
(PMaV-1 & PMaV-2) that are also exposed to
mealybugs feeding [7-9]. In fact, a strong association
was found between PMWaV and PMWD [10, 11],
where both grey and pink pineapple mealybugs
(Dysmicoccus neobrevipes and D. brevipes) were
identified as vectors of the virus [10]. PMWaV-1 is
correlated with growth reductions of plant crop [10]
and yield reductions in the ratoon crops [12].
However, PMWaV-2 infection and mealybugs feeding
are necessary for the development of PMWD [13, 14].
Although, the yield effects of PMWD is variable
losses amounting to 35% has been reported [13].
Unless managed, the disease is very destructive and
devastating making commercial growing of pineapple
impossible [6]. Although, PMWD has been reported
in Uganda, the occurrence and effects have not been
documented. Therefore, the objectives of this study
were to 1) assess the occurrence and distribution of
pineapple mealybugs wilt disease in the districts of
Kayunga and Mukono and 2) assess the effects of
pineapple mealybugs wilt disease on pineapple.
2. Materials and Methods
The study was conducted in central Uganda in 2011.
Both Mukono and Kayunga districts located within
the Lake Victoria basin crescent are among the
leading pineapple producers in the country.
Multi-stage random sampling technique was used to
identify the sub counties, parishes, villages and the
respondents in the study sites. The two leading
pineapple growing sub counties identified in each
district were Ntunda and Goma, and Kangulumira and
Kayunga for Mukono and Kayunga, respectively.
From each sub county, two major pineapple growing
parishes were selected depending on the size and
intensity of pineapple production. In general, 2-3
villages were surveyed per parish depending on the
population in the area giving a total of 20 farmers per
sub-county and 40 farmers per district including one
technical staff per sub-county. Semi-structured
questionnaires were used to gather information from
82 respondents in the two districts. For each of the
farmer interviewed, a pineapple field was visited to
assess the incidence and severity of the PMWD using
visual symptoms. Disease incidence was assessed as
the number of plants diseased expressed as a percent
of the total number of plants assessed per field.
Disease severity was visually assessed as the
percentage leaf area affected (PLAA) using a scale of
1-5, where, 1 = no symptoms and 5 = over 75% of the
leaf wilted and dead [15]. Diseased pineapple plant
samples were also collected for laboratory
identification of mealybug species associated with
PMWD and viral characterisation. Detailed
descriptions of the locations of the fields surveyed
were captured by geographical positioning systems
(GPS). All the data collected was edited, coded and
entered into an excel spreadsheet (version 2007). The
data was analyzed using descriptive statistics of the
SPSS computer package (version 14.0).
3. Results
The socio-economic characteristics of the
respondents are presented in Table 1. Over 60% of the
respondents were males compared to the females.
Similarly, over 60% of the respondents were married
as opposed to the unmarried. Additionally, 40% of the
respondents had secondary school education whereas
12% had no formal education. Overall, over 90% of
the respondents grew pineapple as a major of source
of cash income. Besides, pineapple was reportedly
used for a variety of other purposes including home
consumption and processing into juices as well as
solar drying for exports. The other crops grown for
income generation included coffee, maize, cooking
banana and tomatoes, although maize and cooking
banana were also the major staple crops in the areas.
However, beans and cassava were also ranked very
highly as the major staple crops in the area (Table 2).
Occurrence and Effects of Pineapple Mealybug Wilt Disease in Central Uganda
412
Table 1 Social characteristics of the respondents in Mukono and Kayunga districts, Uganda, 2011.
Characteristics Frequency (%)
Sex
Male 66
Female 34
Marital status
Single 18
Married 65
Others (divorce, widowed) 17
Educational level
Not educated 12
Primary 37
Secondary 40
Tertiary 11
Occupation
Farming 92
Business 4
Others 4
Total 100
The scale of area under pineapple production varied
greatly from a minimum of 0.25 to a maximum of 20
acres, respectively (data not shown). Similarly, the
length of involvement in pineapple growing ranged
from 1 to 33 years, respectively (Table 3). However,
the planting of pineapple was done throughout the
year, although over 50% of the pineapple was planted
during the dry seasons compared to the rainy seasons.
Interestingly, a sizeable proportion of the pineapple
was also planted in both seasons (Table 4). Although,
the source of pineapple planting materials was diverse,
close to 60% of the respondents obtained planting
materials from the plural compared to other sources
(Table 5). In fact, 70% of the respondents prefer
growing smooth cayenne to other varieties (Table 5).
Farmers’ perceptions of the constraints to pineapple
production are presented in Table 6. The major
constraints were diseases, drought and pests as
opposed to lack of planting materials. Of the diseases,
the major challenge is the outbreak of PMWD which
has devastated a large expanse of pineapple
plantations in the two districts. Seventy five percent of
the respondents reported PMWD as a widespread and
devastating disease of pineapple (Table 7). However,
the respondents had varied views on the number of
years that PMWD has been observed in pineapple
fields.
PMWD was observed in all the fields surveyed but
with varying incidences and severities on individual
farms. The lowest and highest PMWD incidence was
Table 2 Food and cash crops grown in Mukono and
Kayunga districts, 2011.
Crops Frequency (%)
Food
Maize 80
Banana 73
Beans 67 Potato Cassava
66 59
Cash
Pineapple 96 Coffee Banana Maize Tomato
37 11 7 7
Total 100
Table 3 Years of involvement in Pineapple cultivation in
Mukono and Kayunga districts, 2011.
Involvement Time (years)
Minimum 1
Maximum 33
Table 4 Planting seasons of Pineapple in Mukono and
Kayunga districts, 2011.
Seasons Frequency (%)
Rainy seasons 15
Dry seasons 52
All seasons 33
Total 100
Table 5 Source of Pineapple planting materials and
varieties grown in Mukono and Kayunga districts, 2011.
Characteristics Frequency (%)
Source of planting material
Own field 28
Neighbours 57
Markets 11
Donations 4
Varieties/cultivars
Smooth cayenne 70
Others 30
Total 100
Occurrence and Effects of Pineapple Mealybug Wilt Disease in Central Uganda
413
Table 6 Constraints to Pineapple growing in Mukono and
Kayunga districts, 2011.
Constraints Frequency (%)
Diseases 78
Drought 71
Pests 67
Shortage of land 55
Low price 47
Lack of transport 38 Soil infertility Lack of planting materials
45 17
Table 7 Knowledge of Pineapple mealybugs wilt disease in
Mukono and Kayunga districts, 2011.
Knowledge Frequency (%)
Yes 75
No 25
Total 100
15% and 100%, respectively. Correspondingly, fields
with higher PMWD incidences also recorded higher
severities and vice versa. However, the frequency of
occurrences ranged from 7% to 27% for the 15-30 and
95 and above incidence, respectively (Fig. 1). In
addition, PMWD was more common during the dry
seasons than the rainy seasons (Table 8). Pineapple
mealybugs wilt disease manifested as yellowing of
leaves, wilting, stunting and rotting of the roots (Table
9). Although, various views were advanced on the
causes of PMWD, the agents mentioned included
insects, sunshine and other unknown factors (Table
10). The major effects of PMWD reported were low
yield and low plant populations, respectively (Table
10). Because of the perceived effects of PMWD, the
respondents reported the occurrences of the disease to
the different level of authority in the districts (Table
11). However, the same respondents affirmed that no
actions were being taken to minimize the spread of the
disease (Table 11).
The various methods used to control PMWD by the
respondents are presented in Table 12. Although, over
50% of the respondents rogued off the infected
pineapple plants, 34% did nothing to control PMWD.
Admittedly, the effectiveness and efficacy of the
control measures were not very obvious to the
respondents. To facilitate control and management of
PMWD, the respondents reported that they were
trained on disease recognition, spread and control by a
number of institutions including the district
agricultural extension officials, the national
agricultural research organization (NARO) and
national agricultural advisory services (NAADS)
among others. However, due to the difficulty in
differentiating among the latent infections, other
diseases and selection of clean planting materials, a
number of respondents just used whatever plant
Fig. 1 Incidence and frequency occurrence of PMWD in Mukono and kayunga distrits, 2011.
Incidence
Fre
quen
cy (
%)
Occurrence and Effects of Pineapple Mealybug Wilt Disease in Central Uganda
414
Table 8 Season of occurrences of Pineapple mealybug wilt disease in Mukono and Kayunga districts, 2011.
Season Frequency (%)
Rainy seasons 71
Dry seasons 3
Both seasons 24
Do not know 2
Total 100
Table 9 Symptoms of Pineapple mealybug wilt diseases in Mukono and Kayunga, 2011.
Symptoms Frequency (%)
Wilting 8
Purpling and yellowing of leaves 11
Stunting 1
Yellowing and wilting 57
Rotting 3
Don’t know 20
Total 100
Table 10 Causes and effects of Pineapple mealybug wilt disease in Mukono and Kayunga districts, 2011.
Characteristics Frequency (%)
Causes
Pests 10
Sunlight 2
Do not know 88
Effects
Low yield 48
Low plant population 27
Do not know 25
Total 100
Table 11 Reporting and action on the presence of Pineapple mealybug wilt disease in Mukono and Kayunga districts, 2011.
Characteristics Frequency (%)
Report
Yes 28
No 72
Action
No action 82
Action 18
Total 100
materials were available. Overall, the majority of the
respondents obtained information on the control of
PMWD through friends, progressive farmers and mass
media (data not shown).
Table 12 Control of Pineapple mealybug wilt disease in Mukono and Kayunga districts, 2011.
Method Frequency (%)
Roguing 52
Spraying 3
Roguing and spraying 9
Use of resistant varieties 1
Do nothing 35
Total 100
4. Discussion
Pineapple mealybug wilt disease (PMWD) was
observed in all fields surveyed but the incidences and
severities varied within the individual farms in central
Uganda. However, the frequency of occurrences was
more during the dry seasons than the rainy seasons.
According to National agricultural research
organisation [3], PMWD is a widespread and
devastating disease in many pineapple growing areas
of the world. In fact, the high frequency of occurrence
of PMWD is expected because the mealybugs which
vector PMWD are normally abundant in the dry
seasons [3, 11]. In addition, the high frequency of
occurrences of PMWD may be attributed to the
unknown status of the planting materials. This is
expected because most of the planting materials used
by the respondents were reportedly sourced from own
fields’ and neigbhours within the locality. Yet,
common knowledge indicates that recycling of
planting materials especially vegetatively propagated
crops is risky because the status of the disease is not
easily verifiable [16].
Although, PMWD was observed in all the fields
surveyed, only 20% of the fields had 100% incidence
indicating probably that PMWD is not a fast spreading
disease or low transmission efficiency of the
mealybugs vectors. This could be true because the
mealybugs which vector PMWD are immobile and
therefore requires to be moved from one plant to
another or from field to field. In fact, the strong
association reported between the mealybugs and
PMWD may support these assertions [11]. Similarly,
Occurrence and Effects of Pineapple Mealybug Wilt Disease in Central Uganda
415
studies on the epidemiology of banana streak virus
disease by Kubiriba [17] showed that mealybugs are
important vector of banana streak virus in banana
plantations. However, the causes of PMWD were
unknown to the majority of the respondents though it
was indirectly related to factors such as sunshine,
insects, soil infertility and witchcraft. Although, the
majority of the respondents were not able to pinpoint
the actual causes of PMWD but related it indirectly to
factors such as sunshine, insects, soil infertility and
witchcraft shows that the respondents are aware of the
constraints inflicting their crops. Therefore, there is
need to empower the respondents to be able to
understand the relationships between the host plant,
pathogens or disease causing organisms and the
environment or the disease triangle. Earlier, Awuah
and Adzim [18] also admitted that the causes of red
leaf disease (RLD) assumed to be PMWD is not
known with accuracy. Yet, it has been demonstrated
by many authors that the identification of the causal
organisms, their biology, means of spread and host
range is imperative towards the development of
appropriate control measures against many diseases
[16]. Unless managed, the PMWD is very destructive
and devastating making commercial growing of
pineapple impossible. Accordingly, in this study,
various attempts were reported towards the control
and management of PMWD including roguing of the
infected plants, use of host plant resistance, spraying
or a combination of the above methods. Although, the
majority of the respondents relied on roguing as the
main way of controlling pineapple mealybugs wilt
disease, Gibson et al [19] and Thresh [20] asserted
that the use of phytosanitary practices such as roguing
is only effective for viral infections and diseases
which do not spread very fast. In fact, the consistent
association between mealybugs and PMWD as well as
the strong association between ants and mealybugs
may confirm this assertion [3]. Although, the use of
host plant resistance has been recommended as the
most economical and sustainable way of managing
many plant diseases [20], the use of host plant
resistance in this study was minimal. This is also true
for other countries. According to Sether and Hu [14],
smooth cayenne, the most commonly grown pineapple
variety is very susceptible to PMWD. In fact, no
sources of resistance to PMWD have so far been
reported in Uganda. Besides, the polyploidy and
vegetatively propagated nature makes the
identification of sources of resistance in pineapple
very complicated. The study has also shown that
majority of the respondents had adequate knowledge
of PMWD. Although, roguing is recommended for the
control of PMWD, the use of insecticidal sprays to
control the attendant ants is considered the best option.
In fact, limited varietal resistance was reported for the
control of PMWD. Although, it was reported that
training on disease recognition, means of spread and
control of PMWD was conducted by different
technical groups, the majority of the respondents
continue to seek for more information about PMWD
through friends, mass media and progressive farmers.
This therefore calls for more awareness creation and
sensitisation if PMWD is to be properly managed.
5. Conclusions
Although, production and export statistics show that
pineapple is an important source of livelihoods to the
farming communities in Central Uganda, especially
the districts of Mukono and Kayunga, the outbreak of
PMWD among other constraints is threatening to the
pineapple industry. Unless managed, the disease is
very destructive and devastating making commercial
growing of pineapple impossible. However, the study
has shown that the causes of PMWD were unknown to
the majority of the respondents though indirectly
related to factors such as sunshine, insects and soil
infertility making it very difficult to institute an
appropriate control and management strategy.
Therefore, there is need to empower the respondents
to be able to understand the relationships between the
host plants, pathogens or disease causing organisms
Occurrence and Effects of Pineapple Mealybug Wilt Disease in Central Uganda
416
and the environment so as to take appropriate control
measures. In fact, it has been recommended that the
identification of the causal organisms, their biology,
means of spread and host range is imperative towards
the development of appropriate control measures
against any disease. Therefore, it is imperative that the
search for sources of resistance to PMWD be
undertaken so as promote the sustainable pineapple
production by growing resistant varieties.
Acknowledgments
Fund for this study was a grant awarded to the first
author by the Ministry of Education and Sports
through National Council for Higher Education. The
facilities and permission provided by the collaborating
institutions as well as the information by the
respondents are very gratefully acknowledged.
References
[1] J.W. Purseglove, Tropical Crops. Monocotyledons. English Language Book Society and Longman, England, 1988.
[2] Uganda Investment Authority (UIA), Pineapple Production and Export Statistics, Annual Report, Kampala, Uganda, 2008.
[3] National Agricultural Research Organisation (NARO), Pineapple Production in Uganda, Annual Report, Ministry of Agriculture, Animal Industry and Fisheries, Entebbe, 2003.
[4] National Agricultural Advisory Services (NAADS), Overview of Pineapple Production in Uganda, Annual Report, Ministry of Agriculture, Animal Industry, Entebbe, 2005.
[5] Anonymous, First report of pineapple mealybug wilt disease in Mukono, Annual Report, Ministry of Agriculture, Animal Industry and Fisheries, Entebbe, 2009.
[6] C. Gary, J. Jahn, W. Beardsley, H. González-Hernández, A review of the association of ants with Mealybug wilt disease of pineapple, Proceeding of the Hawiian Entomology Society 36 (2003) 9-28.
[7] U.B. Gunasinghe, T.L. German, Association of virus particles with mealybugwilt of pineapple (Abstr.), Phytopathology 76 (1986) 1073.
[8] U.B. Gunasinghe, T.L. German, Further characterization of
virus associated with mealybug-wilt of pineapple, (Abstr.), Phytopathology 7 (1987) 1776.
[9] U.B. Gunasinghe, T.L. German, Detection of viral RNA in mealybugs associated with mealybug-wilt of pineapple, (Abstr.), Phytopathology 78 (1988) 1584.
[10] D.M. Sether, J.S. Hu, Corollary analyses of the presence of pineapple mealybug wilt associated virus and the expression of mealybug wilt symptoms, growth reduction, and/or precocious flowering of pineapple (Abstr.), Phytopathology 88 (1998) 80.
[11] D.M. Sether, J.S. Hu, A closterovirus and mealybug exposure are both necessary components for mealybugs wilt of pineapple symptom induction (Abstr.), Phytopathology 90 (2000) 71.
[12] D.M. Sether, J.S. Hu, The impact of Pineapple mealybug wilt-associated virus-1 and reduced irrigation on pineapple yield, Australas. Plant Pathol. 30 (2001) 31-36.
[13] D.M. Sether, J.S. Hu, Closterovirus infection and mealybug exposure are both necessary factors for the development of mealybug wilt disease, Phytopathology 92 (2002) 928-935.
[14] D.M. Sether, J.S. Hu, Yield impact and spread of Pineapple mealybug wilt associated virus-2 and mealybug wilt of pineapple in Hawaii, Plant Disease 86 (2002) 867-874.
[15] G. Hughes, S. Samita, Analysis of patterns of pineapple mealybug wilts disease in Sri Lanka, Plant Disease 82 (1998) 885-890.
[16] G.N. Agrios, Plant Pathology, 5th ed., Academic Press, New York, 2005, p. 948.
[17] J. Kubiriba, Epidemiology of banana streak virus (BSV) in East African highland bananas (AAA-EA), PhD Thesis, University of Greenwich, England, 2005.
[18] R.T. Awuah, E. Adzim, Etiological and epidemiological studies of the red leaf disease of pineapple in Ghana, African Crop Journal Science 12 (2004) 153-162.
[19] R.W. Gibson, S.C. Jeremiah, V. Aritua, R.P. Msabaha, I. Mpembe, J. Ndunguru, Sweetpotato virus disease in sub-Saharan Africa: Evidence that neglect of seedlings in traditional farming systems hinders the development of superior landraces, Journal of Phtyopathology 148 (2000) 441-447.
[20] J.M. Thresh, Control of plant virus diseases in sub-Saharan Africa: The possibility and feasibility of integrated approach, African Crop Science Journal 11 (2003) 199-224.
[21] J.E. Vanderplank, Plant Diseases: Epidemics and Control, Academic Press, New York and London, 1963.
Journal of Agricultural Science and Technology A 3 (2013) 417-422 Earlier title: Journal of Agricultural Science and Technology, ISSN 1939-1250
Electronic Identification of Livestock to Improve
Turkey’s Animal Production System
Sezen Ocak, Sinan Ogun and Zuhal Gunduz
Middle East Sustainable Livestock, Biotechnology and Agro-Ecology Research and Development Centre, University of Zirve,
Gaziantep 27260, Turkey
Received: January 22, 2013 / Published: May 20, 2013. Abstract: Major disease outbreaks, increasing demand for animal food products, intensification of animal production systems, increased consumer awareness about food quality and safety, as well as heightened consciousness about animal welfare issues has seen the need for more reliable animal identification in Turkey’s animal production system. Animal identification and traceability systems have seen rapid development in the world’s main livestock producing nations and have been recognised by the main food, health and livestock trading authorities. The benefit of this system affects all the participants in the food chain (farm to fork) by limiting the spread of animal disease, assuring food safety and quality, minimizing the potential trade loss and minimizing government control. Electronic identification (EID) is one of the main technologies adopted, whereby each individual animal is identified and traced through a unique identification number saved on an electronic transponder (eartag or bolus). The present animal identification and registration system in Turkey does not use an electronic identification tool and is administered manually which often causes unreliable and incorrect results. Concerns for animal and human health, as well as food safety assurance, have motivated efforts in Turkey to intensify animal identification system. This paper has provided the basis for how animals can be accurately traced and monitored from their birth until their slaughter, tracking every single parameter that could be of interest: animal health history, disease control, milk/meat/wool production and nutrition. This study summarizes information on EID available from around the world, discusses the advantages and challenges in its application in Turkey and provides recommendations as to the systems suitability to upgrade the present status of the Turkish National Livestock Identification System. Key words: EID, radio-frequency identification (RFID), livestock identification, food safety and traceability, Turkish national livestock identification system.
1. Introduction
The ability to identify and trace livestock from
property of birth to sale or final slaughter is crucial to
the national livestock registration scheme. The
adoption of such a system benefits the nation livestock
sector by limiting the spread of animal disease,
assuring food safety and quality, reducing potential
trade loss and minimising government control.
Livestock registration system in Turkey is carried out
manually and is administered by the Ministry of Food,
Agriculture and Livestock. A government appointed
veterinary officer records the visual eartag number at
Corresponding author: Sezen Ocak, Ph.D., research fields: reproductive biotechnology, small ruminant breeding and animal management systems. E-mail: sezen.ocak@zirve.edu.tr.
each farm manually and then transfers the information
onto the electronic database known as Turk-Vet. A
number of mistakes are often experienced during this
information transfer process due to the manual entry
of each identification number. Human error is a major
factor the more times a piece of data is handled.
To minimize these issues, in many of the world’s
main livestock producing nations including Europe
and Australia, electronic identification systems (EID)
are utilized. EID reduces reading time, errors and
administrative burden via automatic digital reading of
the unique identification number assigned to each
transponder chip. As a result, the system provides
major advantages in disease control, monitoring any
illegal movement or sourcing of livestock within or
DAVID PUBLISHING
D
Electronic Identification of Livestock to Improve Turkey’s Animal Production System
418
entering the country. In addition, one can easily trace
the animal or the animal product, as well as evaluate
the performance of the animal at the production stage
far more easily than if were recorded manually. The
use of a radio frequency regulated ear tag (EID)
allows the automatic reading of individual animal
identification number, to which one can assign
numerous traits or information about the animal whilst
transferring it into a database. It is quick, safe and
reliable. Electronic identification system can also be a
very useful tool for on-farm management. By the use
of electronic tag reading equipment, scales, automatic
drafting systems and computer management software
farmers can correlate the identifying number with all
relevant information to that animal and its
performance. Depending on the farmers management
objectives, records could include: genetics/bloodlines,
breeding history, weight gain, vaccine and disease
control history and relevant withholding periods for
certain pharmaceuticals, AI and pregnancy scanning
data, wool fibre or milk value measurements and the
list goes on [1].
As a result of their research and past experience in
various other livestock producing nations, the authors
strongly feel that EID is an inevitable must for Turkey
livestock industry. It will not only be of great benefit
to the producers in re-establishing their herd but will
be a much needed tool for maintaining the health of
that herd by the government keeping reliable records
for disease control. In most other nations noted for
their livestock industry, the use of EID has allowed
them to eradicate serious diseases such as Brucellosis
and TB. In conclusion, EID system in Turkey, if
adopted, will provide effective disease control, fast
access to accurate information, precise record keeping
for breeding, monitor and minimizing animal health
risks, providing full traceability of produce for
consumers, tracing and recalling unsafe foods rapidly
and finally averting illegal animal imports.
History of EID or RFID and its application to
livestock.
In 1948, a paper entitled “Communication by
Means of Reflected Power” written by Stockman [2]
was published. In 1964, Harrington examined the
electromagnetic theory related to RFID in his paper
“Theory of Loaded Scatterers” [3]. In the late 1960s,
two companies named Sensormatic and Checkpoint
were founded. These companies together with another
company called Knogo developed Electronic Article
Surveillance (EAS) equipment to deter the theft of
merchandise [4]. Large companies, such as Raytheon
and RCA, developed electronic identification systems
in 1973 and in 1975, respectively. During the 1970s,
research laboratories and universities, such as the Los
Alamos Scientific Laboratory and Northwestern
University, were involved in RFID research. The
adoption of the system for vehicle surveillance was
considered. The Los Alamos Scientific Laboratory,
the International Bridge Turnpike and Tunnel
Association (IBTTA) and the United States Federal
Highway Administration organized in 1973 a
conference on RFID which concluded that there was
no national interest in the development of a standard
for vehicle identification. This decision to the
development of a range of RFID systems.
In 1987, the first commercial application of RFID
was developed in Norway and followed by the Dallas
North Turnpike in the United States in 1989. In the
1990s European companies, such as Alcatel, Bosch
and Phillips spin-off companies, such as Combitech,
Tagmaster and Baumer were involved in the
development of a pan-European standard for vehicle
tolling applications. These companies helped develop
a common standard for electronic tolling [4]. In 1996,
Australia’s Department of Agriculture on the
recommendation of a government-industry working
party agreed to utilize the RFID concept and develop
an electronic identification system which has become
known as the National Livestock Identification
System (NLIS) in Australia. The objective was to
improve the existing visually read identification
arrangements in place at the time by identifying cattle
Electronic Identification of Livestock to Improve Turkey’s Animal Production System
419
more permanently on their property of birth with an
electronic ear tag or rumen bolus that could be read
quickly and accurately. A key driver for the NLIS in
Australia was concern about the identification and
tracing of cattle in the event of a major exotic disease
outbreak such as Foot and Mouth Disease (FMD). A
government study estimated that in Australia the
overall economic loss as a result of such an outbreak
would be between 2 to 13 billion dollars. Though an
NLIS would not prevent a disease outbreak, it would
be able to reduce the financial and social impact of a
disease epidemic due to its accurate identification and
rapid traceability capabilities. This concept also
applies directly to Turkey.
Since then, there has been extensive development
and introduction of “whole of life” identification and
tracking systems in major cattle producing and/or beef
consuming countries such as Canada, United States,
Japan, South Korea, New Zealand, Brazil, Uruguay
and Botswana in addition to European Union (EU)
countries, Turkey will need to be following such
examples in order to maintain access to, and remain
competitive in, key overseas and regional markets.
Regulation (EC) No. 1760/2000 of the European
Parliament and of the Council of July 17, 2000
establishes rules for the identification and registration
of bovine animals and labeling of beef and beef
products [5]. Whether Turkey joins the EU or just
remains a trading partner, it needs to adhere to these
regulations by establishing similar standards in it
livestock identification standards.
2. Principles of EID/RFID
Electronic ID is synonymous with RFID. RFID is
accomplished through the use of two main
components: a transponder and a reader. A
transponder (integrated circuit and antenna) or
“microchip” is attached to an animal using a tag, bolus,
subcutaneous implant or electronic mark on the
pastern (leg band). A reader emits energy in the form
of radio waves. The transponder absorbs energy from
waves and transmits a signal containing the unique
number. The reader receives the transponder signal
and displays a unique identifier in the form of a
number [6]. This can be linked to a computer database
(either on a PC or on a handheld computer) which
links the electronic number to a recognized animal
identifier, e.g., the ear tag number for cattle.
In practice this means that when the animal is
“read” by the electronic reader, the animal ID is
displayed or recorded. This means that the animal
does not need to be physically identified, making the
process less stressful for the animal and easier for the
person keeping record. The two most common types
of electronic identifiers are electronic eartags and
ruminal boluses. Electronic eartags can be applied
early in the life of an animal. Eartags are easily visible
to the naked eye where as the bolus is swallowed by
the animal and is finally deposited in the reticulum.
Albeit fairly minimal, there is a certain loss rate with
eartags and the problem with the bolus id that it is
sometimes difficult to get a reading. If the right type
of bolus is applied to the right type of animal, loss
rates are extremely low. There are also other types of
identifiers (injectable transponder, electronic mark on
the pastern = leg band), but they can only be used with
certain limitations due to trade. RFID tags can be
either passive, active or battery assisted passive. An
active tag has an on-board battery and periodically
transmits its ID signal. A battery-assisted passive
(BAP) has a small battery on board and is activated
when in the presence of a RFID reader. A passive tag
is cheaper and smaller because it has no battery.
Instead, the tag uses the radio energy transmitted by
the reader as its energy source. The interrogator must
be close for RF field to be strong enough to transfer
sufficient power to the tag. Since tags have individual
serial numbers, the RFID system design can
discriminate several tags that might be within the
range of the RFID reader and read them
simultaneously [7].
Tags may either be read-only, having a
Electronic Identification of Livestock to Improve Turkey’s Animal Production System
420
factory-assigned serial number that is used as a key
into a database, or may be read/written, where
object-specific data can be written into the tag by the
system user. Field programmable tags may be
written-once, read-multiple; “blank” tags may be
written with an electronic product code by the user.
RFID tags contain at least two parts: an integrated
circuit for storing and processing information,
modulating and demodulating a radio-frequency (RF)
signal, collecting DC power from the incident reader
signal, and other specialized functions; and an antenna
for receiving and transmitting the signal. Fixed readers
are set up to create a specific interrogation zone which
can be tightly controlled. This allows a highly defined
reading area for when tags go in and out of the
interrogation zone. The commonly used frequency
bands can be seen in Table 1. Mobile readers may be
hand-held or mounted on carts or vehicles.
Signaling between the reader and the tag is done in
several different incompatible ways, depending on the
frequency band used by the tag. Tags operating on LF
and HF frequencies are, in terms of radio wavelength,
very close to the reader antenna, less than one
wavelength away. In this near field region, the tag is
closely coupled electronically with the transmitter in
the reader. The tag can modulate the field produced by
the reader by changing the electrical loading the tag
represents. By switching between lower and higher
relative loads, the tag produces a change that the
reader can detect. At UHF and higher frequencies, the
tag is more than one radio wavelength from the reader.
The tag can backscatter a signal. Active tags may
contain functionally separated transmitters and
receivers, and the tag need not respond on a frequency
related to the reader’s interrogation signal [10].
An electronic product code (EPC) is one common
type of data stored in a tag. When written into the tag
by an RFID printer, the tag contains a 96-bit string of
data. The first eight bits are a header which identifies
the version of the protocol. The next 28 bits identify the
organization that manages the data for this tag; the
organization number is assigned by the EPCG lobal
consortium. The next 24 bits are an object class,
identifying the kind of product; the last 36 bits are a
unique serial number for a particular tag. These last two
fields are set by the organization that issued the tag.
Rather like a URL, the total electronic product code
number can be used as a key into a global database to
uniquely identify a particular product or an animal [11].
3. Advantages and Challenges of EID
Electronic identification system is an important tool
for traceability of products (animal and public health,
products, brands etc.), traceability of animals for
disease control (e.g., foot and mouth disease),
management on farm (lambing, milk recording,
feeding etc.), health certificates (national and
international livestock trade), animal welfare (e.g.
transport), herd book (e.g., pedigree, performance),
application of certain medications (hormones, vaccines
etc.), eradication programs (brucellosis, tuberculosis
scrapie) [12]. It provides to make less error and much
Table 1 RFID frequency bands [8, 9].
Band Regulations Range Data speed Remarks
120-150 kHz (LF) Unregulated 10 cm Low Animal identification, factory data collection
13.56 MHz (HF) ISM band worldwide 1 m Low to moderate Smart cards
433 MHz (UHF) Short range devices
1-100 m Moderate Defense applications, with active tags
868-870 MHz (Europe) 902-928 MHz (North America) UHF
ISM band 1-2 m Moderate to high
EAN, various standards
2,450-5,800 MHz (microwave)
ISM band 1-2 m High 802.11 WLAN, bluetooth standards
3.1-10 GHz (microwave) Ultra wide band to 200 m High Requires semi-active or active tags
Electronic Identification of Livestock to Improve Turkey’s Animal Production System
421
faster than data recording manually. Electronic
tagging is therefore an essential tool for automatic
reading of individual identities and recording of
movements. This system can be a very useful tool for
on-farm management. Farmers have the possibility to
record individually management data like weights or
lambing results and could recall data of their animals
from the slaughterhouse. Equipment failure, the
average failure rate of tags (i.e., tags not
communicating with the reader) is very low.
Environment and facility challenges—The working
environment for livestock involves dust, water,
manure and many elements that are unfriendly to
electronic equipment [13], however, most EID
equipment in these days are built to meet the harsh
challenges of the environments, they are required to
perform in. Electromagnetic interference is another
problem source. Several forms of electronic animal ID
use radio signals to send information between the tag
and the reader antenna. In many livestock processing
facilities, there are multiple sources of
electromagnetic interference including: metal, motors,
and other radio frequency signals [6]. As long as the
used is mindful of these issues, they are not overly
burdensome to overcome as locations can be
manipulated. A common concern for producers is the
impact electronic animal ID will have on the
efficiency of working livestock whether sheep, goats
or cattle. Equipment breakdowns can be particularly
frustrating when labor costs increase due to down time.
Issues with how livestock flow past the panel reader to
make sure each ID is captured will require advice
from the technology provider. In these types of
installations, if alleys and gates do not force animals
to walk single file, the system may experience
problems with tag collision. Tag collision occurs
when more than one electronic identification tag
passes by the reader simultaneously [14]. This is a
design issue and can be averted if proper yards or
handling facilities are built with EID readers in mind.
4. Conclusions
Electronic animal identification system is a
developing field in the world. Concerns for animal
and human health, as well as food safety assurance,
have motivated efforts to intensify animal
identification system. This paper has provided the
basis for how animals can be accurately traced and
monitored from their birth until their slaughter,
tracking every single parameter that could be of
interest: animal health history, disease control,
milk/meat/wool production and nutrition. EID systems
used in many countries around the world have had a
high success rate. There is no reason that the same
success rate could not be achieved in Turkey’s
livestock industry.
Acknowledgments
This paper has been presented in the 23rd
International Scientific Expert Congress on
Agriculture and Food Industry, on September 27-29,
2012 in Izmir, Turkey.
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