Hindawi Publishing Corporation

Psyche
Volume 2010, Article ID 958348, 5 pages
doi:10.1155/2010/958348

Research Article
Bioactivity of Powder and Extracts from Garlic, Allium sativum L.
(Alliaceae) and Spring Onion, Allium fistulosum L. (Alliaceae)
against Callosobruchus maculatus F. (Coleoptera: Bruchidae) on
Cowpea, Vigna unguiculata (L.) Walp (Leguminosae) Seeds

Abiodun A. Denloye
Department of Zoology, Lagos State University, P.O. Box 36 LASU Post Office, Badagry Expressway, Lagos 101010, Nigeria

Correspondence should be addressed to Abiodun A. Denloye, bio [email protected]

Received 5 April 2010; Accepted 29 June 2010

Academic Editor: Arthur G. Appel

Copyright © 2010 Abiodun A. Denloye. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited.

Laboratory bioassays were conducted to investigate the bioactivity of powders, extracts, and essential oils from Allium sativum L.
(Alliaceae) and A. fistulosum L. (Liliaceae) against adults, eggs, and larvae of Callosobruchus maculatus F. (Coleoptera: Bruchidae).
On the basis of 48 hr median lethal toxicity (LC50 ), test plant powders and extracts from A. sativum were more toxic to C. maculatus
adults than those from A. fistulosum. The 48 hr LC50 values for the powder against the test insect species were 9.66 g/kg and
26.29 g/kg for A. sativum and A. fistulosum, respectively. Also the 48 hr LC50 values obtained show that aqueous extracts of the test
plant species, 0.11 g/L (A. sativum) and 0.411 g/L (A. fistulosum) were more toxic to C. maculatus than the corresponding ethanol
extracts. There was no significant difference in the toxicity of vapours from the two test plant species against C. maculatus, although
A. sativum gave lower values. The study shows that A. sativum and A. fistulosum have potentials for protecting stored cowpea from
damage by C. maculatus.

1. Introduction Several methods are used in controlling insects in stored

grains, including physical (smoking, sun-drying, heating),
Grain storage has often resulted in quantitative and qualita- cultural, biological (male insect sterilization, natural ene-
tive losses due to physical, chemical, and most importantly mies, resistant grain varieties), and chemical (synthetic and
biological factors such as pests which may be birds, rodents, natural products) methods. The most common and widely
fungi, or insects [1–3]. The most important among storage used is the chemical method involving mainly the use of
pests are insects because apart from their direct damage synthetic insecticides.
they create conditions that allow secondary infestation by rot
organisms mainly fungi [1, 4]. Several workers have reported the successful wide scale
Once infestation is established pest insects cause gradual use of synthetic organic insecticides, commencing with
and progressive damage leading to losses in weight, nutri- the organochlorines in the middle 1940s, followed by the
tional, organoleptic, and aesthetic quality of stored grains. later use of organophosphates, carbamates, pyrethroids,
Osuji [1] listed 40 insects affecting stored grains, the most avermectins, and others. Insecticides most commonly used
important among which is the cowpea weevil, Callosobruchus to protect stored grains from insect pests include aluminium
maculatus F. (Coleoptera; Bruchidae) responsible for up to phosphide, lindane, methyl bromide, ethylene dibromide,
100% infestation of cowpea, Vigna unguiculata (L.) Walp edifenphos, pirimiphos methyl, permethrin, malathion,
(Leguminosae) during storage [1, 3, 5]. These observations sumithion, chlorpyrifos methyl, chlorpyrifos, propoxur,
justify the control of insect pests like C. maculatus in order to fenithrothion, dichlorvos, bromophos, fenvalerate, biores-
reduce losses in stored cowpea. methrin, phenothrin, and deltamethrin [3].
2 Psyche

The observed overreliance on insecticides was mainly, oil was extracted from 500 g of pulverised A. sativum, or
due to their initial quick action, ease of use and general A. fistulosum by hydrodistillation for 7–8 hrs in a Clavenger
efficiency in reducing pest populations and damage. How- apparatus [11], collecting the volatile oil over hexane, which
ever, there are limitations to their use mainly the deleterious was removed by passing it over anhydrous sodium sulphate.
side-effects to nontarget species including humans and the Each of the essential oils was stored in glass vials kept in
development of resistant strains of pests [6, 7]. In addition refrigerator at 4◦ C to reduce evaporative loss until when
to these limitations, there is also the problem of high cost of needed for bioassays.
synthetic insecticides, which is a limiting factor particularly
to the largely peasant farmers of Africa including Nigeria. 2.2. Callosobruchus Maculatus Culture. Cowpea weevil, C.
Due to the foregoing reasons, there has been a need maculatus (F.) starter cultures obtained from the insectary
to search for new insecticides with novel mechanism of of Nigerian Stored Product Research Institute (NSPRI),
action. In this regard, many scientists have reasoned that it Abule-Oja, Lagos, where they have been held in cultures
is advantageous to investigate natural products as a source for decades unexposed to insecticide were used. Fresh
of degradable insecticides that may turn out to be safer to experimental cultures were prepared from the original stocks
humans and the rest of the environment than the synthetics. and maintained at 30 ± 1◦ C temperature and 70 ± 4% relative
In the present study, garlic, Allium sativum (Alliaceae) humidity as described by Denloye et al. [12]. Callosobruchus
and Spring onion, A. fistulosum (Alliaceae) were screened was maintained on cowpea seeds. The grains were disinfested
for their bioactivity against C. maculatus. Members of the by picking those with damage holes and heating in the oven
genus Allium have been known to demonstrate repellent and at 50◦ C for five hours. Disinfested grains were measured into
insecticidal properties against medically important insect clean 1 L Kilner jars with screw caps. Each jar contained 500 g
pest species [8] and a few workers including Stoll [9] and of cowpea into which seven 0-1-d old adult C. maculatus
Oparaeke et al. [10] have reported their potency against (2 ♂, 5 ♀) were introduced. All adult C. maculatus were
other insects. However, there is a dearth of studies on the removed from the culture after seven days for oviposition to
bioactivity of extracts and volatile oils from these plant take place. Fresh cultures were made from this for subsequent
species against C. maculatus, especially their eggs and larvae. tests.
Understanding the toxicity of compounds to adults and
immature stages is very important as it would indicate the
appropriate time to apply them for adequate control of 3. Bioassays
the insect pests. The present study would therefore provide
3.1. Acute Toxicity of Plant Powders. Twenty active 0–3-day-
the needed information on the toxicity of the extracts and
old C. maculatus (mixed sexes) were exposed to disinfested
volatile essential oils from A. sativum and A. fistulousm,
cowpea grains admixed with powdered plant material at
respectively, against adult, larva, and egg of C. maculatus.
concentrations ranging between 5.0 g/kg and 320 g/kg or
without plant material as control in disposable plastic cups
covered with muslin.
2. Materials and Methods
2.1. Test Plant Materials. The cloves of garlic A. sativum and 3.2. Acute Toxicity of Aqueous and Ethanol Extracts. Similar
leaves of Spring Onion A. fistulosum obtained from Iyana Iba sets of experiments as described above were carried out, but
market, Lagos, were the test plant materials used. this time grains were treated by dipping them for approx-
Test plant materials were used against test insect species imately 30 secs in different concentrations (0.5–16 g/L) of
in four formulations, namely, powder, aqueous and ethanol each plant extract.
extract of powders, and essential oils prepared as described
below. To prepare the powder, plant parts were first dried 3.3. Fumigant Toxicity of Volatile Essential Oils
slowly to constant weight in a wooden cabinet (1.0 m ×
0.5 m × 1.0 m) fitted with 100 watts bulb, which provided 3.3.1. Adults. Fumigation bioassays were carried out in 1 L
an average temperature of about 42◦ C for 7–14 days before airtight Kilner jars using the method of Don Pedro [13, 14].
pulverization in a Binatone blender (model No. BLG 400). In this procedure, a 7 cm-diameter Whatmann No. 1 filter
The powders were passed through sieve of 0.1 mm mesh size paper was always impregnated uniformly with a test essential
to standardize particles size. oil at predetermined concentrations, and quickly hung with
Aqueous and ethanol extract were each prepared from a thread in the fumigation chamber already holding 20 adult
the powder. In each case, 500 g of plant powder was steeped test insects. The chamber was then sealed with the cap,
in 1 L of water or ethanol that served as solvent, for 24 hrs. screwing the ring holding a glass lid tightly on to a rubber
The mixture was then passed through Whatman No. 1 filter washer covered with aluminium foil to prevent reaction with
paper (15 cm diameter). The filtrate in each case was stored essential oil. The cap remained tightly screwed to ensure
in a labelled Kilner jar while the residue was reextracted with fumigation in the airtight chamber for 24 hrs. In controls,
water or ethanol, respectively, and all filtrates combined for insects were left in airtight sealed chambers without oil on
each treatment. Each of the combined filtrates was then dried the filter paper. There were four replicates per treatment.
over a water bath at 50◦ C temperature and the resultant After the 24 hr fumigation the chambers were opened and
residue used as crude active ingredient. Volatile essential the insects that were still alive transferred into recovery
Psyche 3

Table 1: Acute (48 h) toxicity of test plant materials against Callosobruchus maculatus.

LC50 LC95
Formulation Test plant species Regression equation DF Slope (±SE)
95% Confidence Limits 95% Confidence Limits
Powder A. sativum 9.661 (7.957–11.691) 70.143 (50.983–96.317) Y = −1.888 + 1.916x 4 1.916 ± 0.031
(g/kg) A. fistulosum 26.293 (20.485–33.632) 501.742 (293.804–854.42) Y = −1.829 + 1.288x 4 1.288 ± 0.018
Aqueous A. sativum 0.110 (0.087–0.137) 1.30 (0.80–2.17) Y = −1.475 + 1.583x 3 1.538 ± 0.03
extracts (g/l) A. fistulosum 0.411 (0.314–0.510) 4.017 (2.788–6.659) Y = 0.643 + 1.667x 5 1.667 ± 0.035
Ethanol A. sativum 0.219 (0.181–0.261) 1.297 (0.959–1.803) Y = 1.409 + 2.134x 3 2.134 ± 0.046
extracts (g/l) A. fistulosum 0.863 (0.687–1.072) 12.955 (7.624–28.913) Y = 0.089 + 1.403x 3 1.403 ± 0.027
DF: Degree of Freedom; SE: Standard Error.

chambers. Mortality counts were taken in the recovery 4.2. Essential Oils. Similar experiments were carried out
chambers every 24 hrs for seven days. using concentrations (0.8 mL/L to 12.80 mL/L) of essential
oil of A. sativum and A. fistulosum, respectively, instead of
aqueous extracts.
3.3.2. Eggs. Fumigation of C. maculatus eggs on cowpea
was carried out in 1 L airtight Kilner jar using 0.5 mL of
A. sativum or A. fistulosum oil, respectively. Twenty seeds 4.3. Assessment of Mortality. In all bioassays insects were
bearing one egg each were assayed against each of the test counted as dead when they failed to move any part of their
oils and replicated four times. A control, also replicated four body after prodding with fine brush bristle.
times, was set up similarly but the filter paper had no oil.
The egg bearing cowpeas were transferred after 24 hours to 4.4. Data Analyses. Quantal responses (mortality) of C.
ventilated plastic cups and later inspected for hatched (or maculatus were subjected to probit analysis [15] using
unhatched) eggs under a stereomicroscope with X 8 objective computer software after correcting for mortality with Abbot
after 12 days. formula [16]. From these analyses, LC50 (the concentration
at which 50% of test insects died at a given time) and LC95
values of test plant materials were computed.
3.3.3. Larvae. Another similar experiment was set up with
the arrangement described above using 6–8-day-old hatched
eggs (i.e, 1-2-day-old larvae) since eggs hatch into larvae after 5. Results
6 days of incubation. A batch of 20 cowpea seeds, each of
which had one 6–8-day-old eggs were placed in fumigation 5.1. Acute Toxicity of Test Plant Powders to C. maculatus. The
chamber having 7 cm diameter filter paper impregnated 48 hr LC50 values of A. sativum (9.66 g/kg) and A. fistulosum
with various concentrations of test oils. After 24 hours of (26.29 g/kg) and their corresponding LC95 values against C.
fumigation, the cowpea seeds were transferred into ventilated maculatus are shown in Table 1. Powdered A. sativum was
plastic cups and left for 21 days. Each treatment and significantly more toxic to the test insect species than A.
control was replicated four times. Mortality was assessed fistulosum (no overlap in 95% confidence limits).
by dissecting each cowpea seeds to recover dead (or living)
larvae. 5.2. Acute Toxicity of Test Plant Extracts to C. maculatus.
The aqueous extracts were more toxic to C. maculatus than
the ethanol extracts. Probit analysis show that the 48 h LC50
4. Persistence of Test Plant Materials values of the A. sativum aqueous extract was 0.11 g/l, a value
4.1. Extracts. Forty undamaged cowpea grains were treated lower than that of A. fistulosum (0.41 g/l). For the ethanol
by dipping for approximately 30 secs in predetermined con- extracts, the A. sativum gave LC50 value of 0.22 g/l which is 4X
centrations (0.5 to 8.0 g/L) of aqueous extracts of either A. lower than the corresponding value for A. fistulosum shown
sativum or A. fistulosum, and allowed to drain on filter paper in the Toxicity Factor column (Table 1).
for 5 minutes before transferring into bioassay containers.
Several sets of treated seeds and two controls were prepared. 5.3. Fumigant Toxicity of Test Essential Oils to C. maculatus
For each set of treated seeds and controls, bioassays were Adult, Eggs, and Larvae. There was no significant difference
started off by introducing 10 adult C. maculatus aged 0– in the toxicity of A. sativum essential oil when compared
3 days at preset times expressed as Hours After Treatment with that of A. fistulosum (no overlap in 95% confidence
(HAT), namely, 0 (immediately after treatment), 12, 24, limits) although A. sativum gave lower LC50 and LC95 values
96, 168, and 336 HAT. Each treatment and control was relative to A. fistulosum essential oil (Table 2) against both
replicated four times. Each set of experiments was assessed the adults and the eggs, respectively. The essential oils of A.
by taking mortality of test insects every 12 hours for sativum and A. fistulosum resulted in mortality of the larvae
336 hours. of C. maculatus in the cowpea grains, though below 20%.
4 Psyche

Table 2: Fumigant toxicity of test essential oils to C. maculatus adults and eggs.

LC50 LC95
Test insect species Test plant species Regression equation DF Slope (±SE)
(95% Confidence limits) (95% Confidence limits)
Adults A. sativum 15.46 (12.44–19.153) 157.122 (104.97–235.058) Y = −1.948 + 1.638x 3 1.638 ± 0.024
A. fistulosum 23.144 (18.403–29.059) 363.125 (205.718–643.59) Y = −1.883 + 1.38x 3 1.38 ± 0.021
Eggs A. sativum 14.536 (11.826–17.953) 142.789 (79.183–262.334) Y = −1.933 + 1.663x 3 1.663 ± 0.032
A. fistulosum 20.844 (15.589–28.232) 335.986 (137.429–858.69) Y = −1.802 + 1.367x 3 1.367 ± 0.031
DF: Degree of Freedom; SE: Standard Error.

25 8
7
6

LC 50 values (g/l)
20
Mean mortlaity (±SD %)

5
4
15
3
2
10 1
0
0 12 24 96 168 336
5
Hours after treatment (HAT)

A. sativum extract
0
Essential oil treatment A. fistulosum extract

Figure 2: Persistence of test plant extracts against C. maculatus

A. sativum oil adult.
A. fistulosum oil

Figure 1: Fumigant toxicity of essential oil of Allium spp against C. 20

maculatus larvae.
18
16
14
A. sativum resulted in a higher number of dead larvae than
LC50 (mL/l)

A. fistulosum oil (Figure 1). 12

10

6. Persistence of Plant Extracts and Oils for 8

Bioactivity against C. maculatus 6

4
6.1. Extracts. The persistence of the toxicity of aqueous 2
extracts of both test plant species is shown in Figure 2. The 0
computed LC50 values for the two test extracts increased 0 12 24 96 168
slightly by 12 hrs and was maintained up to 24 hrs. The Hours after treatment (HAT)
ethanol extract of A. fistulosum was less persistent than that
of A. sativum (Figure 2). A. sativum oil
A. fistulosum oil

6.2. Essential Oils. The potency of the oils from the two test Figure 3: Persistence of test plant essential oils against C. maculatus
plant species remained only for 12 hrs, after which it was lost adult.
rapidly. The potency of A. fistulosum oil was completely lost
by 96 HAT (Figure 3).
against C. maculatus adults with LC50 values of 9.66 g/Kg.
7. Discussion This value show that powdered A. sativum was equally toxic
to C. maculatus as Citrus species in studies carried out by
The results demonstrate that although A. sativum and A. Don-Pedro [17] and Kellouche and Soltan [18].
fistulosum are of the same genus, they showed different The extracts of A. sativum were more toxic to C.
potencies against the adults, eggs, and larvae of C. maculates, maculatus than those of A. fistulosum in this study. This may
respectively. The powder of A. sativum gave high toxicity be because the active principles responsible for the activity of
Psyche 5

the test extracts were present in higher quantities in the A. [8] A. A. Denloye, W. A. Makanjuola, and O. O. Babalola,
sativum than the A. fistulosum. In addition, A.sativum may “Toxicity and repellent effects of crude aqueous extracts
contain other compounds not contained in A. fistulosum. of garlic (Allium sativum) on larval and adult Anopheles
Our study shows that the aqueous extracts were toxic mosquitoes,” African Entomology, vol. 11, no. 2, pp. 287–290,
to C. maculatus, thus reinforcing earlier observations that 2003.
members of the genus Allium are potent against insects. [9] G. Stoll, Natural Crop Protection Based on Local Farm Resources
Denloye and Makanjuola [19] and Denloye et al. [8, 20] have in the Tropics and Subtropics, Josef Margraf, 1987.
reported the insecticidal potency of the aqueous extracts of [10] A. M. Oparaeke, M. C. Dike, and C. I. Amatobi, “Insecticide
A. sativum against Sitophilus zeamais and Anopheles species. potential of extracts of garlic, Allium sativum (Linnaeus) bulb
Our results from the present study agree with these earlier and African nutmeg, Monodora myristica (Gaertn) Dunal
seed for insect control on cowpea. Entomology in nation
reports.
building: the Nigerian experience,” in Proceedings of 30th
The solvent used in extracting plant materials for insecti- Annual Conference of Entomological Society of Nigeria (ESN
cidal potency is highly important as our present study shows. ’99), Kano, Nigeria, October 1999.
Ethanol extracts were less toxic than the aqueous extracts. [11] L. A. Tapondjou, C. Adler, H. Bouda, and D. A. Fontem,
This agrees with earlier reports that aqueous extracts of garlic “Efficacy of powder and essential oil from Chenopodium
A. sativum were more toxic to S. zeamais than the methanolic ambrosioides leaves as post-harvest grain protectants against
extract [20]. This could be because the active principles in six-stored product beetles,” Journal of Stored Products Research,
the test plant materials are more soluble in water. Grieve [21] vol. 38, no. 4, pp. 395–402, 2002.
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ethanol is due to the fact that alkyl compounds present in the A. Makanjuola, “Assessment of the efficacy of actellic and
Alliacea family are readily obtained by distillation with water. sumithion in protecting grains from insect infestation during
Our results in the present study show that the effectiveness storage,” Journal of Entomology, vol. 5, no. 1, pp. 24–30, 2008.
of a natural plant extracts increase with decreasing polarity [13] K. N. Don-Pedro, “Investigation of single and joint fumigant
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reports by Denloye et al. [20] and Ojewole et al. [22]. Science, vol. 46, no. 1, pp. 79–84, 1996.
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exploited for the prevention and control of C. maculatus Cambridge, UK, 1971.
infestation of stored cowpea. Overall, the results obtained [16] W. S. Abbot, “A simple method of computing the effectiveness
from this study portend greater usefulness for A. sativum as a of an insecticide,” Journal of Economic Entomology, vol. 18, pp.
source of bioactive formulations capable of protecting stored 265–267, 1925.
cowpea from infestation by C. maculatus. [17] K. N. Don-Pedro, “Toxicity of some citrus peels to Dermestes
maculatus Deg. and Callosobruchus maculatus (F),” Journal of
Stored Products Research, vol. 21, no. 1, pp. 31–34, 1985.
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