SYNERGISTIC EFFECT OF BALANITES AEGYPTIACA ESSENTIAL OIL AND STORAGE MATERIALS ON COWPEA SEEDS Synergistic effect of Balanites aegyptiaca essential oil and storage materials on cowpea seeds

INTRODUCTION
The cowpea, Vigna unguiculata (L.), is a legume
of the Fabaceae family. It is one of the most important
legume crops in the world. The plant thrives in
temperate climate and requires little agricultural inputs
for growth [1]. Cowpeas are popular and cheap in many
developing countries. In Nigeria, for instance, they are
a major staple crop. Small-holder farmers are the major
producer of cowpea grains in Nigeria [2].
Cowpeas serve as a rich and affordable source of
nutrients, especially protein, in sub-Saharan Africa and
some parts of America and Asia [3]. Cowpea beans
can be cooked, powdered, germinated, or even used as
part of a weaning formula. As a dish, they complement
tubers and cereals. Consequently, cowpea grains are
present in the diet of many developing countries where
population suffers from malnutrition and protein
deficiencies.
Cowpeas are mainly cultivated by the local farmers
for profits and satisfy the basic nutritional needs of the
local population. However, farmers fail to meet the
local demand as a result of drastic post-harvest losses
caused by insects and other pests. In fact, these losses
are considered as one of the underlying causes of food
scarcity and poverty [4].
Insects damage cowpea grains by boring holes,
thus causing weight loss, poor quality, and low market
value Cowpea aphids (Aphis craccivora L.), leafhoppers
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(Empoasca spp.), cowpea weevils (Callosobruchus
maculatus), and witch-weed (Striga gesnerioides) are
the main insect species that feed on cowpea seeds [5, 6].
Callosobruchus maculatus is the main pest that
causes losses in cowpea grains. C. maculatus i s a fieldto-
store pest that typically begins in the field, and the
level of prior-harvest infestation determines the extent
of damage to stored grains [7]. A range of insect pest
control measures have been adopted over decades to
reduce the prevalence of cowpea grain loss in the field
and during storage.
According to Ogunfowokan et al., Nigerian farmers
use Lindane (Gammalin 20EC), Dichlorvos
dichlorodiphenyltrichloroethane (DDT), Chlorpyrifos,
Endosufan, and Aldrin to prevent pest infestation [8].
Synthetic chemicals are very effective in preserving
grains and increasing their production yield. However,
they have major drawbacks as improper use often
results in environmental pollution, pesticide residue in
food, and toxic poisoning of the ecosystem [9]. Hence,
several attempts have been made to test pesticides that
are environmentally friendly, harmless to people, and
inexpensive.
Resourceful African farmers tried to use such
natural preservatives as plant powder, ashes, and cow
dung. Several authors have documented the insecticidal
efficacy of plant products on different types of pests [10].
In fact, plant products with aromatic properties
are known to prevent insect infestation of stored
cowpeas [11]. Ikbal and Pavela made an extensive
research to assess the use of aromatic plants as
pesticides during storage [12]. They reported that
essential oils of plant origin could serve as botanical
insecticides as they contain a lot of bioactive compounds
with insecticidal, nematicidal, larvicidal, and antifeedant
properties that inhibit insect oviposition [13, 14].
Essential oils are natural derivatives from aromatic
plants which contain volatile and phenolic compounds
with unique flavors. Several works have reported
the insecticidal effect of essential oils during grain
storage [15–19].
Balanites aegyptiaca fruits have quite a number
of bioactive compounds with various medicinal
properties [20]. The essential oil extracted from
B. aegyptiaca possesses anticancer, antimicrobial,
antioxidant, anticarcinogenic, antidiabetic, antifeedant,
and antiviral activities [21, 22]. Natural fumigants
developed from plants do not threaten the ecosystem.
Insecticides based on essential oils are sold all over the
world, but their production does not exceed 5% [23]. In
Asia, Europe, and North America, natural extracts have
been used as insecticides for more than a century, much
longer than any synthetic insecticides.
Previous studies have investigated the insecticidal
efficacy of oil extracted from B. aegyptiaca. For
instance, Mokhtar et al. reported a strong effect of
B. aegyptiaca seed oil on the mortality rate of red flour
beetle (Tribolium castaneum Herbst) [17]. Similarly,
Nwaogu and Yahaya investigated the insecticidal
efficacy of oil extracted from B. aegyptiaca in stored
cowpea seeds [18]. The studies provided evidence that
B. aegyptiaca seed oil could be used as an insecticide
against storage pest. However, very few publications
feature the use of B. aegyptiaca essential oils in
controlling insect infestation of stored grains.
Storage materials are also important for seed
treatments and grain quality. Buleti et al. conducted
an experiment in the Northeast of Nigeria to assess the
effect of B. aegyptiaca oil on weevil growth in cowpea
grains stored in various packing materials [15]. The
present research provides some new data on using plant
products as storage pesticides.
STUDY OBJECTS AND METHODS
Plant material. Fruits of Balanites aegyptiaca L.
were acquired from the Gashua market in Yobe State,
Nigeria. The fruits were authenticated at the Department
of Agronomy, Faculty of Agriculture, Federal University
Gashua, Nigeria.
Preparing the seeds. The mesocarp of B. aegyptiaca
was scraped with a clean sharp knife and dried
in an oven at 45°C for 24 h to reduce stickiness. The
endocarp was broken down with a hammer to obtain
seeds. Then, the seeds were dried to constant weight in
an airtight oven (45°C, 72 h). Subsequently, the seeds
were milled into fine particles using an electric blender
and stored in zip lock bags.
Extracting B. aegyptiaca essential oil. The
method developed by Nguefack et al. was used to
extract essential oil from B. aegyptiaca seeds [24]. The
experiment began by placing 500 g of pulverized seeds
of B. aegyptiaca in a 5 L flask. After that, distilled
water was added to cover the sample. Essential oil
was obtained by hydrodistillation using a modified
Clevenger-type apparatus at normal atmospheric
pressure and 96–97°C for 4 h. The resulting essential oil
was collected by drying it out with anhydrous sodium
sulfate and kept at 4°C in Eppendorf tubes until the gas
chromatography-mass spectrometry (GC/MS) analysis.
Evaluating the effect of B. aegyptiaca essential oil
against Callosobruchus maculatus.
Experimental site. The experiment was carried
out in the Agronomy Laboratory of the Department
of Agriculture, Federal University, Gashua, after the
growing season of 2021.
Experimental design and treatment. The
research involved five storage materials (jute bags,
polythene bags, sacks, plastic containers, and glass
bottles), experimental samples (5, 10, and 15 mL of
B. aegyptiaca essential oil diluted in 1 mL of acetone),
a check sample (with 0.125 g of aluminum phosphide),
and control (acetone). The experiment was laid out in a
completely randomized design with three replications of
each treatment.
Insect culture: source and rearing. C. maculatus
was first cultured from a cowpea seed infested at the
local market in Gashua. Weevils multiplied in fresh and
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previously uninfested cowpea varieties in the laboratory
at an ambient temperature (27–30°C) and relative
humidity (70–75%).
Treatment and maintenance of cowpea seeds. Forty
kilograms of pristine cowpea seeds was purchased
directly from the local farmers in Gashua, immediately
after harvest. To destroy and/or prevent any initial
infection, they were placed in a plastic container and
maintained in the freezer below 0°C for five days. After
that, the seeds were taken out of the freezer and placed
on a laboratory bench, covered with a screen, and left to
equilibrate for 72 h [25].
Adult mortality of C. maculatus. Mortality contact
effect of B. agyptiaca essential oil on adult C. maculatus
was determined using the method developed by
Obeng et al. [26]. According to the procedure, 200 g of
cowpea seeds was held in different storage materials
and then thoroughly mixed with: (a) 5, 10, and 15 mL
of essential oil diluted in 1 mL of acetone; (b) 0.125 g
of aluminum phosphide; and (с) acetone (control).
After that, the storage materials were left open for 2 h
at room temperature to disperse acetone. Thereafter,
20 unsexed pairs (10 males and 10 females) of threeday-
old C. maculatus beetles were introduced into the
storage materials. They were kept on laboratory benches.
Dead insects were counted after 24, 48, and 72 h after
infestation using Abbott’s equation [27]. To confirm
mortality, insects were probed three times with a sharp
pin [28]. The data were subjected to Probit analysis [29].
Oviposition. For this part of the experiment, 100 g
of cowpea seeds was held in the varying storage
materials and thoroughly mixed with 5, 10, and 15 mL
of essential oil diluted in 1 mL of acetone, 0.125 g of
aluminum phosphide, and control (acetone). After that,
10 males and 10 females of three-day-old newly sexed
C. maculatus beetles were introduced into the storage
materials, where they paired and laid eggs. Following
egg deposition, 100 seeds were randomly selected on
days 7, 30, 60, and 90, and the number of eggs deposited
on the cowpea seeds was counted and recorded in each
treatment and replicate [30].
Egg hatchability. At this stage, 100 g of cowpea
seeds was infested with 20 (10 males and 10 females)
sexed pairs of five-to-seven-day-old C. maculatus
beetles in a transparent plastic container. The insects
paired and laid eggs for six days. After oviposition,
100 seeds (27 g) bearing eggs were chosen and placed
in various storage materials that contained pure and
uninfested cowpea seeds (73 g). They were thoroughly
mixed with 5, 10, and 15 mL of essential oil diluted in
1 mL of acetone, 0.125 g of aluminum phosphide, and
control (acetone).
In each treatment and replicate, the cowpea
seeds were stored on the laboratory bench until adult
beetles emerged. The number of emerged adults was
recorded on days 30, 60, and 90 after the exposure.
The percentage of adult emergence was calculated
conversely from each of the treatments and replicates
according to the method developed by Adesina and
Ofuya [31], with a slight modification (1):
(1)
Seed perforation. This test included 100 g of
cowpea seeds held in the varying storage materials and
thoroughly mixed with 5, 10, and 15 mL of essential oil,
0.125 g of aluminum phosphide, and control (acetone).
After that, 10 pairs of three-day-old newly sexed C.
maculatus beetles were introduced into the storage
materials and kept in the laboratory for 90 days. Every
four weeks for three months, the number of exit holes
was assessed by counting in each seed from a random
sample of 100 seeds.
The weevil perforation index (WPI), which measured
the protective ability of the storage material, was
calculated according to standard methods. If the weevil
perforation index was ≥ 50%, it indicated an increase in
weevil infestation or a low efficacy of the plant material.
To obtain the percent protection ability (PPA), the weevil
perforation index was subtracted from 100 using the
following equation (2):
where WPI is the weevil perforation index; WPI > 50 is
the negative protectant of plant material, i.e., low antiweevil
activity; WPI < 50 is the positive protectant, i.e.,
high anti-weevil activity.
Seed weight loss. To calculate the seed weight loss,
100 g of cowpea seeds was randomly selected after
30, 60, and 90 days of storage. To obtain the final seed
weight for the sample, all dead insects and other debris
in the cowpea seeds were removed, and the cowpea
seeds were weighed. As described by Sibakwe and
Donga [32], the percentage of seed weight loss was
calculated using the following equation (3):
(2)
(3)
Seed damage. After 30, 60, and 90 days of storage,
100 g of cowpea seeds were randomly selected from
the lots. We divided seeds into two groups, damaged
and undamaged, and counted seeds with exit
holes. Adenekan et al. [30] described how to quantify
the percentage of damaged seed using the following
equation (4):
(4)
Egg hatching =
𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑝𝑝𝑝𝑝𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑝𝑝𝑝𝑝𝑁𝑁𝑁𝑁𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝 𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑒𝑒𝑒𝑒
𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑒𝑒𝑒𝑒 𝑖𝑖𝑖𝑖𝑝𝑝𝑝𝑝 𝑡𝑡𝑡𝑡𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑡𝑡𝑡𝑡
× 100
WPI =
𝑃𝑃𝑃𝑃𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑃𝑃𝑃𝑃𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑖𝑖𝑖𝑖𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒
𝑃𝑃𝑃𝑃𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑃𝑃𝑃𝑃𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑖𝑖𝑖𝑖𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒 + 𝑃𝑃𝑃𝑃𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑃𝑃𝑃𝑃𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑖𝑖𝑖𝑖𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝 𝑆𝑆𝑆𝑆𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁 𝑖𝑖𝑖𝑖𝑝𝑝𝑝𝑝 𝐶𝐶𝐶𝐶𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝐶𝐶𝐶𝐶
× 100
Weight loss =
𝐼𝐼𝐼𝐼𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒 𝑤𝑤𝑤𝑤𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁ℎ𝑡𝑡𝑡𝑡 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒 − 𝐹𝐹𝐹𝐹𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒 𝑤𝑤𝑤𝑤𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁ℎ𝑡𝑡𝑡𝑡
𝐼𝐼𝐼𝐼𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝 𝑤𝑤𝑤𝑤𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁ℎ𝑡𝑡𝑡𝑡 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒
× 100
Seed damage =
𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒
𝑇𝑇𝑇𝑇𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒
× 100
Germination percentage =
𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑒𝑒𝑒𝑒 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒 𝑒𝑒𝑒𝑒 𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡 𝑡𝑡𝑡𝑡ℎ𝑁𝑁𝑁𝑁 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃
𝑇𝑇𝑇𝑇𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒 𝑝𝑝𝑝𝑝𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝑝𝑝𝑝𝑝𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡
× 100
Egg hatching =
𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑝𝑝𝑝𝑝𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑝𝑝𝑝𝑝𝑁𝑁𝑁𝑁𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝 𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑒𝑒𝑒𝑒
𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑒𝑒𝑒𝑒 𝑖𝑖𝑖𝑖𝑝𝑝𝑝𝑝 𝑡𝑡𝑡𝑡𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑡𝑡𝑡𝑡
× 100
WPI =
𝑃𝑃𝑃𝑃𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑃𝑃𝑃𝑃𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑖𝑖𝑖𝑖𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒
𝑃𝑃𝑃𝑃𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑃𝑃𝑃𝑃𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑖𝑖𝑖𝑖𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒 + 𝑃𝑃𝑃𝑃𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑃𝑃𝑃𝑃𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑖𝑖𝑖𝑖𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝 𝑆𝑆𝑆𝑆𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁 𝑖𝑖𝑖𝑖𝑝𝑝𝑝𝑝 𝐶𝐶𝐶𝐶𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝐶𝐶𝐶𝐶
× 100
Weight loss =
𝐼𝐼𝐼𝐼𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒 𝑤𝑤𝑤𝑤𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁ℎ𝑡𝑡𝑡𝑡 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒 − 𝐹𝐹𝐹𝐹𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒 𝑤𝑤𝑤𝑤𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁ℎ𝑡𝑡𝑡𝑡
𝐼𝐼𝐼𝐼𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝 𝑤𝑤𝑤𝑤𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁ℎ𝑡𝑡𝑡𝑡 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒
× 100
Seed damage =
𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒
𝑇𝑇𝑇𝑇𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒
× 100
Germination percentage =
𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑒𝑒𝑒𝑒 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒 𝑒𝑒𝑒𝑒 𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡 𝑡𝑡𝑡𝑡ℎ𝑁𝑁𝑁𝑁 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃
𝑇𝑇𝑇𝑇𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒 𝑝𝑝𝑝𝑝𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝑝𝑝𝑝𝑝𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡
× 100
Egg hatching =
𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑝𝑝𝑝𝑝𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑝𝑝𝑝𝑝𝑁𝑁𝑁𝑁𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝 𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑒𝑒𝑒𝑒
𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑒𝑒𝑒𝑒 𝑖𝑖𝑖𝑖𝑝𝑝𝑝𝑝 𝑡𝑡𝑡𝑡𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑡𝑡𝑡𝑡
× 100
WPI =
𝑃𝑃𝑃𝑃𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑃𝑃𝑃𝑃𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑖𝑖𝑖𝑖𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒
𝑃𝑃𝑃𝑃𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑃𝑃𝑃𝑃𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑖𝑖𝑖𝑖𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒 + 𝑃𝑃𝑃𝑃𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑃𝑃𝑃𝑃𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑖𝑖𝑖𝑖𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝 𝑆𝑆𝑆𝑆𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁 𝑖𝑖𝑖𝑖𝑝𝑝𝑝𝑝 𝐶𝐶𝐶𝐶𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝐶𝐶𝐶𝐶
× 100
Weight loss =
𝐼𝐼𝐼𝐼𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒 𝑤𝑤𝑤𝑤𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁ℎ𝑡𝑡𝑡𝑡 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒 − 𝐹𝐹𝐹𝐹𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒 𝑤𝑤𝑤𝑤𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁ℎ𝑡𝑡𝑡𝑡
𝐼𝐼𝐼𝐼𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝 𝑤𝑤𝑤𝑤𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁ℎ𝑡𝑡𝑡𝑡 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒
× 100
Seed damage =
𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒
𝑇𝑇𝑇𝑇𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒
× 100
Germination percentage =
𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑒𝑒𝑒𝑒 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒 𝑒𝑒𝑒𝑒 𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡 𝑡𝑡𝑡𝑡ℎ𝑁𝑁𝑁𝑁 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃
𝑇𝑇𝑇𝑇𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒 𝑝𝑝𝑝𝑝𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝑝𝑝𝑝𝑝𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡
× 100
Egg hatching =
𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑝𝑝𝑝𝑝𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑝𝑝𝑝𝑝𝑁𝑁𝑁𝑁𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝 𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑒𝑒𝑒𝑒
𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑒𝑒𝑒𝑒 𝑖𝑖𝑖𝑖𝑝𝑝𝑝𝑝 𝑡𝑡𝑡𝑡𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑡𝑡𝑡𝑡
× 100
WPI =
𝑃𝑃𝑃𝑃𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑃𝑃𝑃𝑃𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑖𝑖𝑖𝑖𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒
𝑃𝑃𝑃𝑃𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑃𝑃𝑃𝑃𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑖𝑖𝑖𝑖𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒 + 𝑃𝑃𝑃𝑃𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑃𝑃𝑃𝑃𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑖𝑖𝑖𝑖𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝 𝑆𝑆𝑆𝑆𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁 𝑖𝑖𝑖𝑖𝑝𝑝𝑝𝑝 𝐶𝐶𝐶𝐶𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝐶𝐶𝐶𝐶
× 100
Weight loss =
𝐼𝐼𝐼𝐼𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒 𝑤𝑤𝑤𝑤𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁ℎ𝑡𝑡𝑡𝑡 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒 − 𝐹𝐹𝐹𝐹𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒 𝑤𝑤𝑤𝑤𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁ℎ𝑡𝑡𝑡𝑡
𝐼𝐼𝐼𝐼𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝 𝑤𝑤𝑤𝑤𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁ℎ𝑡𝑡𝑡𝑡 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒
× 100
Seed damage =
𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒
𝑇𝑇𝑇𝑇𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒
× 100
Germination percentage =
𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑒𝑒𝑒𝑒 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒 𝑒𝑒𝑒𝑒 𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡 𝑡𝑡𝑡𝑡ℎ𝑁𝑁𝑁𝑁 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃
𝑇𝑇𝑇𝑇𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒 𝑝𝑝𝑝𝑝𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝑝𝑝𝑝𝑝𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡
× 100
356
Ajayi F.F. et al. Foods and Raw Materials. 2022;10(2):353–364
Seed germinability. After the storage period,
15 seeds were randomly picked from the various
storage materials to test the effect of the essential
oil concentrations on the germinability of cowpea
seeds. A seed from each treatment was placed in 9-cm
Petri dishes with moistened Whatman filter paper on
laboratory benches at room temperature (27–30°C) and
relative humidity (70–75%) [33]. Each treatment was
triplicated. To avoid contamination, the seeds were
watered (23 mL) twice a day (morning and evening)
with distilled water from a wash bottle. According to
Olisa et al. [34], the germination percentage of cowpea
seeds was estimated from germination data on day 7
after sowing according to the following equation (5):
(5)
Data analysis. Natural mortality in the control
samples was corrected using Abbott’s formula [27]. The
acquired numerical data was square root transformed
, and the adjusted mortality and other data in
percentages was transformed arc sine before being
subjected to the analysis of variance using JMP 13
Computer Software (2016). The Student Newman-
Keuls (SNK) test was used to differentiate significant
treatment means at the 5% level of probability. With
the SPSS statistical software (version 19), the data were
subjected to a two-way analysis of variance (ANOVA) at
the 5 % significance level, and Duncan’s Multiple Range
Test was used to separate the means.
RESULTS AND DISCUSSION
Mortality of Callosobruchus maculatus L. exposed
to Balanites aegyptiaca L. essential oil stored in
different storage materials after 24, 48, and 72 h.
The treatment under discussion provided appropriate
protection to cowpea seeds against C. maculatus. Table
1 shows the effect of B. aegyptiaca oil extract and such
storage materials as jute bags, polythene bags, sacks,
plastic containers, and glass bottles on cowpea weevil
mortality.
The experiment showed a statistically significant
correlation between the effects of B. aegyptiaca
essential oil and storage materials (F(16,75) = 41.813,
P = 0.000) after 24 h of exposure. A simple main effect
analysis showed that B. aegyptiaca essential oil had a
statistically significant effect on mortality of cowpea
weevils at 24, 48, and 72 h, respectively (P < 0.000).
Glass bottles, plastic containers, and jute bags
proved to be the most effective storage material, while
samples stored in polythene bags had the lowest beetle
mortality rate at all the B. aegyptiaca essential oil
concentrations. Our results confirmed those obtained
by Buleti et al., who reported a higher weevil mortality
rate in grains stored in glass bottles compared to other
storage materials [15]. On the other hand, the results can
be explained by the techno-functional properties of the
storage materials, e.g., water vapor permeability, the
interaction between the plant extracts and the material,
etc. [35]. Thus, such vapor proof containers as glass
bottles, plastic containers, and jars can provide good
insulation against weevils, thereby inhibiting their
survival: insects suffocate as soon as they run out of
oxygen.
In addition, the abrasive effect and contact toxicity
of essential oils on the pest cuticle interferes with insect
respiratory mechanism, thereby causing a knock down
effect. This study is similar to the research conducted by
Karimzadeh et al., who reported that the abrasive effects
of combined insecticides may cause abrasion of insect
cuticle and dehydration of the insect body, thus leading
to insect mortality [36].
Evidently, the B. aegyptiaca essential oil treatments
had a noticeable effect on the population growth rate and
mortality of the weevils. Low quantity of the essential
oil (5 mL) resulted in a lower mortality rate, while high
quantities (15 mL) provided the highest mortality rate,
irrespective of the storage material used.
Similarly, the population of the weevils decreased
as the treatment intervals progressed from 24 to 72 h.
For instance, at the same essential oil dose, plastic
containers and glass bottles caused 62.5 and 70%
mortality rate, respectively, after 24 h. Likewise, 80 and
90% mortality rate were recorded after 48 h. However,
100% mortality was recorded for glass bottles, plastic
containers, and jute bags after 72 h. Most importantly,
all the three doses and storage materials showed high
mortality rates of C. maculatus after 72 h of exposure, if
compared to the control samples.
The insecticidal efficacy of B. aegyptiaca essential
oil could be attributed to such active compounds as
hexadecanoic acid, (9Z,12Z)-octadeca-9,12-dienoic acid,
(Z)-octadec-9-enoic acid, ethyl hexadecanoate, 3,3-dihydroxypropyl
hexadecanoate, and methyl hexadecanoate.
All these compounds have been reported to possess
repellent and insecticide activities [17]. This result
confirms the findings obtained by Mokhtar et al.,
who observed 100% mortality of C. maculatus after
24 h on cowpeas treated with chloroform extract of
B. aegyptiaca seeds at 1.131 mg·cm–2 [17]. Various
studies have also demonstrated the insecticidal effect
of B. aegyptiaca essential oil against such pests as
C. maculatus, Tribolium casteneum, and khapra
beetle [17,18].
In our research, various doses of B. aegyptiaca
essential oil and storage materials provided an excellent
protection against C. maculatus, both independently
and synergistically. B. aegyptiaca essential oil extract
had a substantial impact on the longevity and survival
of cowpea weevils. In addition, glass bottles, plastic
containers, and jute bags caused 100% mortality at the
highest dose (15 mL) after 72 h of exposure.
WPI =
𝑃𝑃𝑃𝑃𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑃𝑃𝑃𝑃𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑖𝑖𝑖𝑖𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒 + 𝑃𝑃𝑃𝑃𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑃𝑃𝑃𝑃𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑖𝑖𝑖𝑖𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝 𝑆𝑆𝑆𝑆𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁 𝑖𝑖𝑖𝑖𝑝𝑝𝑝𝑝 𝐶𝐶𝐶𝐶𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝐶𝐶𝐶𝐶
× 100
Weight loss =
𝐼𝐼𝐼𝐼𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒 𝑤𝑤𝑤𝑤𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁ℎ𝑡𝑡𝑡𝑡 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒 − 𝐹𝐹𝐹𝐹𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒 𝑤𝑤𝑤𝑤𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁ℎ𝑡𝑡𝑡𝑡
𝐼𝐼𝐼𝐼𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝 𝑤𝑤𝑤𝑤𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁ℎ𝑡𝑡𝑡𝑡 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒
× 100
Seed damage =
𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒
𝑇𝑇𝑇𝑇𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒
× 100
Germination percentage =
𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑒𝑒𝑒𝑒 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒 𝑒𝑒𝑒𝑒 𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡 𝑡𝑡𝑡𝑡ℎ𝑁𝑁𝑁𝑁 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃
𝑇𝑇𝑇𝑇𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒 𝑝𝑝𝑝𝑝𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝑝𝑝𝑝𝑝𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡
× 100
357
Ajayi F.F. et al. Foods and Raw Materials. 2022;10(2):353–364
Table 1 Mortality of cowpea weevils exposed to Balanites aegyptiaca oil extract in different storage materials after 24, 48, and 72 h
Samples Storage materials Mortality ± SE, %
24 h 48 h 72 h
Balanites aegyptiaca essential oil
(5 mL)
Glass bottle 20.00 ± 0.00 46.50 ± 4.79 90.00 ± 0.00
Jute bag 12.50 ± 5.00 25.00 ± 4.08 70.00 ± 0.00
Plastic container 20.00 ± 0.00 60.00 ± 0.00 61.20 ± 2.50
Polythene bag 10.00 ± 0.00 25.00 ± 0.00 60.00 ± 0.00
Sack bag 10.00 ± 0.00 30.00 ± 0.00 75.00 ± 0.00
Balanites aegyptiaca essential oil
(10 mL)
Glass bottle 40.00 ± 0.00 65.00 ± 5.77 100.00 ± 0.00
Jute bag 30.00 ± 0.00 60.00 ± 0.00 83.60 ± 2.50
Plastic container 35.00 ± 0.00 75.00 ± 0.00 90.00 ± 0.00
Polythene bag 23.70 ± 2.50 42.00 ± 5.00 70.00 ± 0.00
Sack bag 32.50 ± 2.89 50.00 ± 0.00 90.00 ± 0.00
Balanites aegyptiaca essential oil
(15 mL)
Glass bottle 70.00 ± 0.00 80.00 ± 0.00 100.00 ± 0.00
Jute bag 50.00 ± 0.00 70.00 ± 0.00 100.00 ± 0.00
Plastic container 62.50 ± 2.89 90.00 ± 0.00 100.00 ± 0.00
Polythene bag 32.50 ± 2.89 55.00 ± 0.00 80.00 ± 0.00
Sack bag 55.00 ± 0.00 70.00 ± 0.00 97.50 ± 5.00
Aluminum phosphide Glass bottle 90.00 ± 0.00 100.00 ± 0.00 100.00 ± 0.00
Jute bag 81.20 ± 2.50 90.00 ± 0.00 100.00 ± 0.00
Plastic container 90.00 ± 0.00 100.00 ± 0.00 100.00 ± 0.00
Polythene bag 85.00 ± 4.08 90.00 ± 0.00 95.00 ± 5.77
Sack bag 90.00 ± 0.00 100.00 ± 0.00 100.00 ± 0.00
Control (acetone) Glass bottle 0 0 20.00 ± 0.00
Jute bag 0 0 0
Plastic container 0 0 02.50 ± 2.89
Polythene bag 0 0 0
Sack bag 0 0 0
A P < 0.05 P < 0.05 P < 0.05
B P < 0.05 P < 0.05 P < 0.05
AB P < 0.05 P < 0.05 P < 0.05
A – essential oil; B – storage material
Oviposition of C. maculatus exposed to
B. aegyptiaca essential oil stored in different
storage materials after 7, 30, and 90 days of
storage. Figures 1–3 show the mean value of
oviposition in both treated and untreated cowpeas.
The analysis of variance showed that a statistically
significant correlation between the effects of
B. aegyptiaca oil extracts and storage materials
on the oviposition of cowpea weevils (F(16,75) =
3346.73, P = 0.000). When compared to the control, the
treated samples showed a much lower oviposition. At a
seven-day interval, all the storage materials showed the
same trend in the total oviposition of cowpea weevils as
the essential oil doses increased, except polythene bags
(Fig. 1).
B. aegyptiaca essential oil significantly reduced the
number of eggs laid by weevils. The highest amount
of eggs was recorded at 5 mL, and then it significantly
decreased at 10 mL. The lowest amount of eggs was
registered at 15 mL. The drastic reduction in the
number of eggs laid by cowpea weevils might have
been caused by the toxicity of the plant material active
components to the weevils rather than by the prevention
of oviposition.
The previous section that the B. aegyptiaca essential
oil caused the highest mortality rate in the experimental
samples, which was associated with its insecticidal
effect. The chemical composition of plant oils and
their phytocompounds is known to produce a toxic and
repellent effect on insects that live in stored grain [37].
Grains stored in glass bottles had the fewest eggs,
while those stored in polythene bags had the maximal
number of eggs. Even though polythene bags had the
lowest effect on weevil oviposition, they also showed
a significant reduction in the number of eggs laid
compared to the untreated samples. Buleti et al. also
reported a reduction in the number of eggs laid in glass
bottles [15]. Furthermore, the experimental samples
revealed just a few eggs on day 90, with a mean
fecundity of 1–4 eggs for glass bottles and 1–14 eggs for
plastic containers. A slightly higher value was recorded
for polythene and sack bags with a mean of 18–45 and
8–30 eggs, respectively. However, the values recorded
358
Ajayi F.F. et al. Foods and Raw Materials. 2022;10(2):353–364
Figure 1 Effect of Balanites aegyptiaca on Callosobruchus
maculatus oviposition at various treatments in different
storage materials after 7 days of storage
Figure 3. Effect of Balanites aegyptiaca on Callosobruchus
maculatus oviposition at various treatments in different
storage materials after 90 days of storage
Figure 2. Effect of Balanites aegyptiaca on Callosobruchus
maculatus oviposition at various treatments in different
storage materials after 30 days of storage
in polythene and sack bags were lower than in the
control samples with a mean fecundity of 256–544 eggs
per female after 90 days, which implied a significant
difference in the oviposition of treated cowpeas.
The synergic treatment of B. aegyptiaca essential
oil and storage materials killed more than 50% of the
total eggs laid at various stages of development from
7 to 90 days. Likewise, the high numbers of eggs laid
in polythene and sack bags could be explained by the
porous surface of these materials that allowed moisture
and air circulation. Such conditions encouraged weevils
present in the seed lot to lay eggs and proliferate
continuously.
The lower oviposition rates observed in this study
suggested that B. aegyptiaca essential oil could be
useful as cowpea protectants. This finding confirms that
made by Alves et al., who discovered that lemon grass
essential oil extract reduced C. maculatus oviposition
significantly [38]. Previous studies by a Nwaogu
and Yahaya and Aous et al. reported the effect of
essential oil of Cymbopogon schoenanthus (L.) on the
development of freshly laid eggs and newborn larvae of
C. maculatus [18, 39]. The extract probably contained
a powerful oviposition deterrent. Also, cowpea seeds
that are packaged in glass bottles and plastic containers
showed a low oviposition, which made them the optimal
ovipositional deterrents in this study.
Egg hatchability of C. maculatus exposed to
B. aegyptiaca essential oil stored in different storage
materials after 24, 48, and 72 h. Table 2 shows the
effect of B. aegyptiaca essential oil on C. maculatus
egg development and hatchability. The main significant
effect of B. aegyptiaca essential oil and storage
materials (P < 0.05) was observed after 30, 60, and
90 days. Similarly, the study revealed no significant
Number of eggs laid
0
Treatment
80
60
40
20
Packaging
100
120
140
Glass bottle Jute bag
Plastic container Polythene bag
Sack bag
Number of eggs laid
0
Treatment
10 mL 15 mL 5 mL Aluminum
phosphide
Control
(acetone)
80
60
40
20
Packaging
Glass bottle
Jute bag
Plastic container
Polythene bag
Sack bag
10 mL 15 mL 5 mL Aluminum
phosphide
Control
(acetone)
Number of eggs laid
0
Treatment
400
300
200
100
600
500
Glass bottle
Packaging
Jute bag
Plastic container Polythene bag
Sack bag
10 mL 15 mL 5 mL Aluminum
phosphide
Control
(acetone)
359
Ajayi F.F. et al. Foods and Raw Materials. 2022;10(2):353–364
Table 2 Callosobruchus maculatus egg hatchability treated with Balanites aegyptiaca essential oil in different storage materials
after 30, 60, and 90 days of storage
Samples Storage materials Mean number of hatched eggs ± SE
30 days 60 days 90 days
Balanites aegyptiaca essential oil
(5 mL)
Glass bottle 4.10 ± 0.17 2.40 ± 0.25 1.00 ± 0.00
Jute bag 7.00 ± 0.00 4.70 ± 0.14 1.30 ± 0.08
Plastic container 4.20 ± 0.68 3.10 ± 0.78 1.10 ± 0.03
Polythene bag 8.60 ± 0.79 7.70 ± 0.17 5.50 ± 0.05
Sack bag 6.40 ± 0.36 4.60 ± 0.09 1.60 ± 0.00
Balanites aegyptiaca essential oil:
(10 mL)
Glass bottle 1.50 ± 0.78 1.00 ± 0.00 1.00 ± 0.00
Jute bag 5.10 ± 0.15 3.60 ± 0.79 1.20 ± 0.02
Plastic container 2.20 ± 0.15 1.20 ± 0.96 1.00 ± 0.01
Polythene bag 5.40 ± 0.34 6.30 ± 0.24 3.30 ± 0.28
Sack bag 3.20 ± 0.00 2.00 ± 0.02 1.00 ± 0.00
Balanites aegyptiaca essential oil:
(15 mL)
Glass bottle 0.80 ± 0.09 1.00 ± 0.00 1.00 ± 0.00
Jute bag 3.00 ± 0.00 2.20 ± 0.11 1.00 ± 0.00
Plastic container 1.10 ± 0.05 1.10 ± 0.10 1.00 ± 0.00
Polythene bag 2.90 ± 0.12 6.10 ± 0.13 1.90 ± 0.01
Sack bag 2.90 ± 0.17 1.90 ± 0.07 1.00 ± 0.00
Aluminum phosphide Glass bottle 0.20 ± 0.06 1.00 ± 0.00 1.00 ± 0.00
Jute bag 1.00 ± 0.13 1.00 ± 0.00 1.00 ± 0.00
Plastic container 1.00 ± 0.00 1.00 ± 0.00 1.00 ± 0.00
Polythene bag 1.10 ± 0.00 1.00 ± 0.00 1.00 ± 0.00
Sack bag 0.50 ± 0.04 1.00 ± 0.00 1.00 ± 0.00
Control (acetone) Glass bottle 16.60 ± 0.00 20.40 ± 0.67 42.10 ± 1.14
Jute bag 23.40 ± 0.84 27.30 ± 1.51 52.60 ± 31.1
Plastic container 18.20 ± 0.56 21.90 ± 0.89 52.00 ± 0.04
Polythene bag 31.30 ± 0.76 38.90 ± 0.93 72.40 ± 1.47
Sack bag 26.80 ± 1.30 26.20 ± 0.46 66.40 ± 0.28
A P < 0.05 P < 0.05 P < 0.05
B P < 0.05 P < 0.05 P < 0.05
AB P > 0.05 P > 0.05 P > 0.05
A – essential oil; B – storage material
(P > 0.05) interactive effect of B. aegyptiaca
essential oil and storage materials on C. maculatus
egg hatchability. The results demonstrated that the
productivity in the experimental samples was extremely
low at all intervals and doses, with the mean values
ranging from 1 to 8.6.
Hence, over 65% of the total eggs laid in the
experimental samples died at different stages of
development in all the trials. B. aegyptiaca oil
extracts obviously had a strong larvicidal effect on the
development of immature weevils. Similarly, the high
mortality of C. maculatus in the experimental samples
implied that the plant had some phytochemical properties,
which reduced egg production [40].
Furthermore, the egg hatchability reduced as the
storage interval progressed from 30 to 90 days. Among
the experimental samples, the highest mean values of
egg hatchability were observed in cowpeas stored in
polythene bags at all the essential oil concentrations.
However, the values observed in polythene bags were
lower in comparison to the control samples. Therefore,
the low egg hatchability was caused by the effectiveness
of B. aegyptiaca essential oil with its poisonous component
and physical properties, which affected the
surface and oxygen tension of eggs.
The essential oils of Borago officinalis, Melissa
officinalis, Carapichea ipecacuanha, and Laurus nobilis
have also been reported to reduce hatchability [41].
Similarly, Piper gaudichaudianum essential oil showed
a better insecticidal activity against Lucilia cuprina
third instar larvae under laboratory conditions [42].
Weevil Perforation Index, weight loss, damage,
and germinability of cowpea seeds treated with B.
aegyptiaca essential oil stored in different storage
materials after 30, 60, and 90 days. These parameters
of cowpea seeds indicate its suitability for consumption
and other aesthetic values because damaged seeds with
holes and flour dust are not marketable. Figures 4 and
5 show the weevil perforation index for cowpea seeds
treated with different concentrations of B. aegyptiaca
essential oil packaged in different storage materials on
days 30, 60, and 90.
After 30 days, the highest weevil perforation
index was observed in polythene and sack bags. When
compared to the control treatment, the cowpea seed
treated with B. aegyptiaca essential oil showed a
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Ajayi F.F. et al. Foods and Raw Materials. 2022;10(2):353–364
Figure 4 % Weevil perforation index of cowpea seeds treated
with Balanites aegyptiaca in different storage materials after
30 days of storage
Figure 5 % Weevil perforation index of cowpea seeds treated
with Balanites aegyptiaca in different storage materials after
90 days of storage
substantial reduction in seed damage. The analysis
of variance showed a statistically significant effect
of B. aegyptiaca oil extracts and storage materials
on weevil perforation index (F(16, 75) = 176.150,
P = 0.000). Exposure of weevil-infested cowpea seeds
to B. aegytiaca essential oil in various storage materials
resulted in a significant reduction in seed weight
(F(16,75 = 311.357, P = 0.000).
The weevil perforation index decreased following
the increase in B. aegytiaca essential oil concentration
for all storage materials. The lowest weevil perforation
index was observed in the seeds packaged in glass
bottles followed by those stored in plastic containers.
The highest weevil perforation index was recorded in the
seeds stored in polythene bags throughout the storage
period (Figs. 4 and 5).
Furthermore, exposure of weevil-infested cowpea
seeds to B. aegytiaca essential oil in various storage
materials resulted in a significant reduction in seed
weight (F(16,75) = 311.357, P = 0.000). After 90 days of
exposure, the seeds treated with B. aegyptiaca essential
oil in all the storage materials showed a significantly
low weight loss at all doses (5, 10, and 15 mL), except
the cowpea seeds stored in polythene bags (Table 3).
For these parameters, interactive treatment of
B. aegyptiaca essential oil and storage materials
had equally significant effects (P < 0.05) on cowpea
weevils.
Taken together, B. aegyptiaca had a more
detrimental effect on the weevils in comparison to
the control. A similar research by Borzoui et al. also
registered a significantly low amount of seed damage
because the oviposition rate was reduced by the
sublethal doses of essential oil [43]. Among all the
storage materials, glass bottles and plastic bags showed
the lowest weevil perforation index, seed weight loss,
and seed damage. The low seed weight loss and damage
observed in the seeds stored in these storage materials
could be attributed to the significant reduction of
weevils that could have caused seed damage.
B. aegyptiaca essential oil and storage materials
demonstrated both individual and interactive effects on
the germination rate of cowpea seeds. The germination
rate of the treated seeds ranged from 46.7 to 93.3%,
which was lower than the values reported for cowpea
seed germination rate by Gad et al. [44].
An increased germination rate of cowpea seeds
was observed with increased B. aegyptiaca essential
oil concentration, which implied a strong relationship
between the treatment and germination rate. Similar
results were described by Bhavya et al. and Harshani
and Karunaratne, who indicated that essential oils and
their principal components affected seed germination.
Storage materials also affected seed germination [45, 46].
In this study, glass bottles proved to be the optimal
storage material as indicated by the maximal seed
germination rate of 88.3, 91.6, and 93.3% after essential
oil treatment of 5, 10, and 15 mL, respectively. Storing
seeds in appropriate storage material retained higher
germination capacity rate.
Number of seeds with holes
0
Treatment
Number of seeds with holes
0
Treatment
20.00
40.00
60.00
Glass bottle
Packaging
Jute bag
Plastic container Polythene bag
Sack bag
20.00
40.00
60.00
80.00
100.00
Glass bottle
Packaging
Jute bag
Plastic container Polythene bag
Sack bag
10 mL 15 mL 5 mL Aluminum
phosphide
Control
(acetone)
10 mL 15 mL 5 mL Aluminum
phosphide
Control
(acetone)
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Table 3 Effect of Balanites aegyptiaca essential oil and storage material on cowpea seed damage, weight loss, and germination rate
caused by Callosobruchus maculatus infestation
Samples Storage
materials
Seed weight loss Seed damage Germination
30 days 60 days 90 days 30 days 60 days 90 days rate
Balanites
aegyptiaca
essential
oil (5 mL)
Glass bottle 8.30 ± 0.00 3.50 ± 0.03 1.00 ± 1.63 1.00 ± 0.00 1.00 ± 0.05 3.00 ± 0.00 88.30 ± 3.30
Jute bag 13.40 ± 0.16 6.00 ± 0.19 1.50 ± 0.25 2.00 ± 1.63 4.50 ± 0.14 7.50 ± 0.21 64.60 ± 4.91
Plastic
container
12.50 ± 0.29 3.90 ± 0.00 1.20 ± 0.19 1.00 ± 0.00 1.90 ± 0.22 3.30 ± 0.23 80.00 ± 0.00
Polythene bag 15.60 ± 0.17 8.50 ± 0.25 3.00 ± 0.16 5.90 ± 0.25 7.00 ± 0.17 11.40 ± 1.49 46.70 ± 0.00
Sack bag 13.60 ±0.47 6.80 ± 0.14 1.40 ± 0.25 2.20 ± 0.00 5.00 ± 0.16 7.70 ± 0.81 60.00 ± 0.00
Balanites
aegyptiaca
essential
oil (10 mL)
Glass bottle 7.80 ± 0.09 2.00 ± 0.08 0.80 ± 0.00 0 1.00 ± 0.00 1.90 ± 0.17 91.60 ± 3.30
Jute bag 11.70 ± 0.04 5.40 ± 0.32 1.50 ± 0.00 2.00 ± 0.00 4.50 ± 0.10 7.00 ± 0.08 60.00 ± 0.00
Plastic
container
10.00 ± 0.13 2.30 ± 0.34 0.90 ± 0.17 0 1.00 ± 0.00 2.00 ± 0.00 80.00 ± 0.00
Polythene bag 13.00 ± 0.17 11.50 ± 0.57 2.50 ± 0.21 3.90 ± 0.41 7.00 ± 0.00 9.10 ± 0.87 55.00 ± 2.01
Sack bag 12.00 ± 0.74 5.70 ± 0.19 1.30 ± 0.28 2.00 ± 0.16 5.40 ± 0.18 6.90 ± 0.01 60.00 ± 0.00
Balanites
aegyptiaca
essential
oil (15 mL)
Glass bottle 2.80 ± 0.16 1.60 ± 0.02 0.30 ± 0.23 0 1.00 ± 0.00 1.50 ± 0.00 93.30 ± 0.00
Jute bag 10.80 ± 0.48 3.10 ± 0.00 0.70 ± 0.19 0 2.00 ± 0.07 5.00 ± 0.16 66.70 ± 0.00
Plastic
container
3.10 ± 0.24 1.90 ± 0.10 0.30 ± 0.19 0 1.00 ± 0.00 2.00 ± 0.71 93.00 ± 0.00
Polythene bag 11.70 ± 0.84 9.70 ± 0.22 1.40 ± 0.16 2.00 ± 0.16 5.00 ± 0.38 6.00 ± 0.00 65.00 ± 1.88
Sack bag 10.50 ± 0.38 3.40 ± 0.13 0.80 ± 0.00 0 4.00 ± 0.16 5.00 ± 0.00 60.00 ± 0.00
Aluminum
phosphide
Glass bottle 0 0 0.10 ± 0.00 0 0 0 100.00 ± 0.00
Jute bag 0 0 0.10 ± 0.00 0 0 0 100.00 ± 0.00
Plastic
container
0 0 0.10 ± 0.00 0 0 0 100.00 ± 0.00
Polythene bag 0 0 0.10 ± 0.00 0 0 0 100.00 ± 0.00
Sack bag 0 0 0.10 ± 0.00 0 0 0 100.00 ± 0.00
Control
(acetone)
Glass bottle 18.60 ± 0.26 37.40 ± 0.13 42.00 ± 0.08 15.70 ± 1.25 28.00 ± 0.16 71.80 ± 0.43 26.70 ± 0.00
Jute bag 28.60 ± 0.36 42.40 ±1.77 48.50 ± 0.00 20.90 ± 0.68 30.80 ± 1.45 79.70 ± 1.39 20.00 ± 0.00
Plastic
container
20.80 ± 0.16 40.30 ± 1.57 45.70 ± 0.22 16.00 ± 0.16 29.20 ± 0.66 77.80 ± 0.44 26.70 ± 0.00
Polythene bag 35.60 ± 0.77 56.00 ± 0.00 56.50 ± 0.25 23.70 ± 0.48 38.60 ± 0.41 91.50 ± 1.07 13.30 ± 0.00
Sack bag 26.40 ± 0.16 44.30 ± 1.80 50.00 ± 1.63 18.90 ± 0.17 34.10 ± 1.57 84.10 ± 1.72 20.00 ± 0.00
A P < 0.05 P < 0.05 P < 0.05 P < 0.05 P < 0.05 P < 0.05 P < 0.05
B P < 0.05 P < 0.05 P < 0.05 P < 0.05 P < 0.05 P < 0.05 P < 0.05
AB P < 0.05 P < 0.05 P < 0.05 P < 0.05 P < 0.05 P < 0.05 P < 0.05
A – Balanites aegyptiaca essential oil; B – storage material
Therefore, the B. aegyptiaca essential oil could be
used to protect cowpea seeds stored in glass bottles
because the values observed were related to the
international germination threshold of 90% required
by seed exportation. This result is similar that obtained
by Buleti et al., who also used glass bottles as storage
containers [15].
On the contrary, cowpea seeds stored in polythene
bags displayed the lowest germination rate, which
indicated a strong relationship between weight loss,
damage score, and germination rate. However, the
germination rates were not as highly related with seed
damage traits. The lowest germination rate observed
could be explained by weevil damage that occurred
due to minimal weevil mortality rate in polythene
bags, which, in its turn, led to nutrient exhaustion [15].
Storing seeds in inappropriate storage materials could
significantly decline their germination rate and resulted
in a rapid loss of seed viability.
Overall, even though B. aegyptiaca essential oil
had a stronger effect on cowpea weevils, they failed to
remove cowpea weevils completely; rather, they only
lowered their numbers. Thus, hermetic storage could
enhance the quality of cowpea seeds during storage.
CONCLUSION
The combination of essential oil of Balanites
aegyptiaca L. and storage materials had significant
effects on weevil proliferation in cowpea seeds
(P > 0.05) during storage. B. aegyptiaca essential oil
proved to possess insecticide properties that can help
control Callosobruchus maculatus L. in stored cowpea
seeds.
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After 90 days of storage, mortality, oviposition, and
egg hatchability fell down, following the increase in
the concentration of B. aegyptiaca essential oil. In
addition, such storage materials as glass bottles, plastic
containers, and jute bags also reduced the population of
cowpea weevils in cowpeas during storage. Hermetic
storage material – glass bottles – had the greatest
effecton weevil infestation and sustained the quality
of cowpeas under storage conditions. These findings
suggest that B. aegyptiaca essential oil could be useful
as a botanical insecticide against cowpea pests. A largescale
trial is required to perform a toxicity assay of
B. aegyptiaca essential oil.
CONTRIBUTION
Feyisola Fisayo Ajayi obtained the funds, designed
the experiment, collected the data, and wrote the
manuscript original draft, as well as performed the
formal laboratory research. Akama Friday Ogori
conducted the formal analysis and research, wrote
the article, reviewed scientific publications, and
edited the manuscript. Vivien O. Orede performed the
formal analysis and research, reviewed and editing
the manuscript. Emmanuel Peters performed the
formal analysis and research, as well as provided the
experimental design. All the authors were equally
involved in reading and approving of the final manuscript
before submission and are equally accountable for any
potential cases of plagiarism.
CONFLICT OF INTEREST
The authors declare no conflict of interests regarding
the publication of this article.