0

DECLARATION

I Mr. Jitendra Kulaste hereby declare that the work entitled

“Biopesticidal activities of BLUMEA ERIANTHA on wheat
weevil pest SITOPHILUS GRANARIUS ” is a record of my
own work carried out from June 2024 to August 2024 at
Biosciences Department, Barkatullah University, Bhopal and
submitted for the partial fulfilment of project work in foutrth
paper of Zoology by Barkatullah University, Bhopal. The matter
embodied in the project is not substantially the same as the
one, which has been submitted to complete our semester/Year
and other academic qualification at any other university in India
or abroad.

(Jitendra Kulaste) Place………………

B.SC.Zoology Date…………….…

Pharmacogenic BARKATULLAH UNIVERSITY

Laboratory
Hoshangabad Road, Bhopal-
Department of 462026
Biosciences
Mobile: +91 9827598990
Dr. Kapil K. Soni
Email: kapil k [email protected]
Assistant Professor

S.No…………..BU/BioSci/2024 Bhopal, dated……….

………….

1
CERTIFICATE

This is to certify that the project work of paper fourth entitled

“Biopesticidal activities of BLUMEA ERIANTHA on wheat weevil
pest SITOPHILUS GRANARIUS" which is being submitted by
JITENDRA KULASTE for the partial fulfillment of the requirements of the
project work on paper fourth ,Zoology in the Department of Biosciences
, Barkatullah University, Bhopal (M.P.). It is a record of candidate's own
work carried out by him from June, 2024 to August 2024 under my
supervision and guidance. The matter embodied in this report has not
been submitted for any other purposes.
(Dr. Kapil K. Soni) Place………………
Supervisor Date………………

Forwarded by
(Dr. Kapil K. Soni)
Assistant Professor
Department of Bioscience

2
 ACKNOWLEDGEMENTS

An academic project marks the beginning of the transition from an

amateur to a professional. It is the forum where theory is put into
perspective. I use this space to express my gratitude to all those who
have either directly or incognito rendered support in my work. I use the
word “Thanks” by sincerely hoping that this will convey my depth of
feelings towards them.

This research would not exist without the strong support of my project
work which only possible due to the generosity of many people
particularly, my director Dr. Kapil Kumar Soni Assistant Professor,
Barkatullah University, Bhopal. I am thankful to him for his diligent
supervision without which this work would never have assumed its
present form.

I express my deep sense of gratitude and indebtedness to the Head of

Department (HOD), Department of Biosciences Prof. R.K.Garg, Barkatullah
University, Bhopal for giving me opportunity to complete my project work
in this renowned institute and for his constant inspiration throughout the
course of this work, which helped me to complete this work smoothly.

My special thanks to the Head of Department (HOD), Department of

Zoology and Applied Aquaculture Prof. Vipin Vyas as he helped me
tremendously by contributing his intellectual views and instruction, which
made my work better and easy.

I also thankful to Research Scholars Mr.Rajendra and my classmates

Kritika Yadav, Maseerah Seikh ,Akash Sharma and Rupali Chaubey ,for
helping in my project work.

Last but not the least, I am thankful to God and all those who helped me
knowingly and unknowingly in various ways in completion of this project
work. I thank them profusely and sincerely.

Closing with regards

(Jitendra kulaste )

B.SC.Zoology

6th semester

Place………………

3
Date…………….…

SUMMARY
One of the outstanding problems in pest control is the extensive use of
synthetic compounds characterized by concerns such as risks to non-
target organisms, slow degradation, and development of resistance. For
these reasons, the interest in more ecofriendly pesticides, such as
botanicals, is progressively increased in the last two decades. In this
regard, having recently found that essential oil obtained by blumea
erientha has biological activity against Sitophilus granarius, here we
checked whether and how different crude extracts obtained by the
blumea erientha also presented toxicity (contact, ingestion, inhalation)
and/or repellent activity against the same insect, which is one of the most
damaging pests of stored products. Results reposted here clearly show
that, in addition to the essential oil, erientha crude extracts (methanol,
and distilled water ) preserve interesting activities against pests.
Moreover, since they can be easily obtained and produce high yields,
erientha crude extracts could represent a valid tool for S.
granarius control.

The use of bioinsecticides, rather than synthetic compounds, appears a

goal to be pursued in pest control, especially for species such
as Sitophilus granarius . which attack stored products. Since blumea
erientha is a remarkable source of bioactive compounds, this study
investigated the bioactivity of blumea erientha flowers extracts against S.
granarius adults by evaluating toxic (contact, inhalation, and ingestion),
repellent, antifeedant, and nutritional effects as well as their
anticholinesterase activity and olfactory sensitivity. Blumea erientha
extracts were obtained by soaking dried and ground blumea in solvents
of decreasing polarity: methanol and distilled water .
Dried
crude extracts were resuspended in each solvent, and used in topical
application, ingestion, and fumigation toxicity assays, as well as in contact
and short-range repellency tests, in vitro anticholinesterase activity
evaluation, and electroantennographic tests. No inhalation toxicity for the
extracts was found. On the contrary, all extracts showed adult contact
toxicity 24 h after treatment (adult for distilled water and methanol
extracts, respectively); negligible variations for these values at 48 h were
found. The anticholinesterase activity shown by all extracts suggested
that the inhibition of this enzyme was one of the mechanisms of action.
Interestingly, flour disk bioassays revealed a significant ingestion toxicity
for the acetone extract and a lower toxicity for the other two extracts.

4
Moreover, all extracts affected insect nutritional parameters, at the
highest dose checked. Filter paper and two-choice pitfall bioassays
showed repellent activity and a strong reduction of insect orientation to a
highly attractive food odor source, with minor differences among extracts,
respectively. Finally, the presence of volatile compounds in the different
extracts that are perceived by insect antennae was confirmed by
electroantennography. All these findings strongly suggest a possible use of
blumea erientha extracts against S. granarius, thus further confirming
this plant as an interesting species for pest control.

5
 INTODUCTION
Botanical pesticides represent a valuable alternative to synthetic
chemicals since their use significantly reduces the risk to non-target
organisms due to their rapid degradation in the environment; moreover,
by providing novel and multiple modes of action, the probability of pest
resistance development is reduced as well. Accordingly, the number of
papers describing plant extracts active against different pests is
progressively increasing, as shown by a simple search using the keyword
couple “plant extract” and “pest” (Scopus database,
https://www.scopus.com, accessed date 19 February 2021).

Contol of stored grains insects relies heavily on the use of synthetic

insecticides which has led to problems such as environment disturbance,
increasing costs of application, pest resurgence, pest resistance to
pesticides and lethal effect on non target organisms including human
being ,in addition to direct toxicity to users . the ancient man had
deployed different method of control ,including prayers ,magic spells,
cultivation system ,mechanical practices as well as application of organic
and inorganic substances to product his crops forms the attack of weed,
diseases, and insect pest (Kulkarni et al ., 2009) . between 500 BC and
the 19th century a number of substances classified as pesticides and
defined as any substances and mixture of substances intended for
preventing ,destroying, repelling or mitigating any pest which were used
to control the pest which are harmful organisms that may be insect or
rodents .These pesticides were sulphur, arsenic, lead and mercury (Shin –
foon chiu et al., 1950 ) . The first synthetic organic pesticide which was
used in later for agriculture purposes (Samarasekera, 1997 ) . There is no
doubt that the use of insecticides has contributed immensely to the
increase in agricultural productivity and to the improvement in human
health, particularly the eradication of malaria in the developed and
developing countries of the world in the 20 th century and beyond .
However , it has been established that the use of synthetic organic
pesticides, mainly the chlorinated hydrocarbons such as DDT and its
derivatives has led to serious environmental pollution which are affecting
human health and causing death of non – organisms including animals,
plants and fish . IN 2001, this problem was put in Stockholm Convention
and eventually DDT was banned in 2004 . However, before banning the
DDT , number of effort were made by many researchers for alternative
sources of pesticides due to some other reasons including non-specificity,
ineffectiveness, unsuccessful marketing of the synthetic pesticides by

6
prospective users, high cost of synthetic insecticides and development of
resistance (Gregson et al., 1978; Okwute et al., 2009)

7
In this regard, extracts and/or essential oils from several plants proved
to exert insecticidal activity against pest of wheat,Recently, Blumea
eriantha essential oil (EO) and its main constituents (α-humulene, β-
myrcene, and β-caryophyllene) proved to exert interesting properties
againstA high contact and a lower inhalation toxicities, as well as a
good repellent activity, were found against adult insects the granary

weevil, Sitophilus granarius (L.) To provide a wider scenario on the hop

biological activity in insects, the insecticidal, electrophysiological, and
behavioral activities of n-hexane, methanol, and acetone crude
extracts from BLUMEA ERIANTHA of Central Italy were investigated

against the granary weevil adults. Contact Toxicity The contact toxicity
of hop extracts to granary weevil adults was determined by topical
application [8,24]. Plant extract samples were prepared by dissolving
the residues of n-hexane, methanol, and acetone extracts in n-hexane,
acetone:methanol (1:1), and acetone, respectively. For each sample,
two-fold serial dilutions (150.00–4.69 μg/μL) were prepared and for
each dilution an aliquot (0.5 µL) was applied on the pronotum of S.
granarius adults in thanatosis using a Hamilton’s syringe (700 series,

MicroliterTM Hamilton Company, Reno, NV, USA) Each dilution was

assayed on 40 unsexed adults of S. granarius divided in 8 replicates
and an equal number of individuals was treated with the respective
solvent as a control. For each replicate, insects were confined in a Petri
dish within a metal rings (Ø 4.0 × 2.5 cm), covered with metallic net
(mesh 1 mm) to prevent insects escape, with 5 wheat kernels, and
maintained in the dark under controlled conditions (26 ± 2 °C and 60 ±
5% r.h.). Insect mortality was recorded after 24 and 48 h. The
percentage mortalities were transformed to arcsine square-root values
for one-way analysis of variance (ANOVA).Treatment means were
compared and separated by Tukey’s HSD test. The lethal dose 50
(LD50) and 90 (LD90) values, the confidence upper and lower limits,
regression equations, and chi-square (χ2) values were calculated using
probit analysis [25]. Statistical analyses were performed with SPSS
(Statistical Package for the Social Sciences) v.23 for Windows (SPSS

Inc., Chicago, IL, USA) . As part of the ongoing research on the

biological activity of hop plant, results reported in this study shows that
n-hexane, methanol, and acetone hop extracts exert different
bioactivities against the pest S. granarius. Topical application of all
three extracts resulted in a high contact mortality, reaching the 100%
value at the highest dose tested (75.00 μg/adult). Among the extracts,

8
the n-hexane was found to be the most active showing significant
mortality starting from 9.37 µg/adult. The high contact toxicity of hop
extracts was supported by the respective LD50 values which were
comparable among them and lower than the values reported for similar
extracts of other plants, such as Scrophularia canina L. [36] and
Dittrichia viscosa (L.) [8], against the same insect species.

On the basis of above mentioned botanical description , the systematic

position of the plant is as under:

 Kingdom – Plantae
 Division – Angiosperm
 Sub – Division – Eudicots
 Order – Asterales
 Family – Asteraceae
 Sub- Family – Asteroideae
 Genus – Blumea

9
Identification and Collection of plant material

A plant material was identified and collected from the campus of

Barkatullah University , Bhopal near to the Department of Zoology and
Applied Aquaculture . Barkatullah university campus are covers 360 acres
of land (1.5 km) located Jabalpur- Jaipur Highway(NH-12) at a distance
3km from the city of Bhopal located in tehsil Huzur of district Bhopal the
area covers the wide variety of plant and animal species in all of these ,
one species is blumea erientha also found in the campus .

Here , some identification and morphological features

are mentione

1. Erect unbranched herbs, to 50 cm high

2. Stems reddish, sparsely hairy.
3. Leaves 3-6 x 1-2 cm, elliptic to obovate,
acuminate at base, scabrid above.
4. Heads 6 x7 mm, in terminal congested leafy panicle; peduncle 5 mm
long; bracts 6 x 0.5 mm, lanceolate, acuminate, brown; outer smaller,
pubescent.
5. Bisexual flowers few, central; corolla 4 mm long, sparsely hairy; lobes
pubescent. Achene 1 mm long, ribbed, sparsely hairy; pappus 4 mm long,
brownish. 6.Genus Blumea is found in the tropical and sub-tropical zones
of Asia, especially the Indian Subcontinent and Southeast Asia. A few
species are found in Australia and still fewer in Africa

10
 METHODOLOGY
3.1 Isolation of extract by Soxhlet extraction
process.
Isolation of the extract from the powdered
plant material was done by using Soxhlet apparatus by applying different
solvents.

Leaves of Blumea erientha

was collected from the campus of Barkatullah university , Bhopal District
(M.P.) and washed thoroughly with distilled water . Plant materials were
dried under shade at room temperature in to the laboratory and after
drying, it was found 72% less in amount . A total 180 gm of pulverized
plant material was defatted in distilled water and then 70gm of plant
material was uploaded in Soxhlet apparatus for extraction indifferent
solvents in increasing order of polarity i.e. Methanol and Distilled water for
3 to 4 days .

Isolated extract were then filtered through Whatman’s filter paper

and the filtrate was then separately condensed to dryness using water
bath . The crude extract was then measured and the percentage yield was
obtained in methanol (4.15%) and distilled water (11.15%) with respect to
dried powder .

3.2 Extraction by
Thin layer chromatography

Chromatography means color or to write which is originally

described by Tswett(1906) . Chromatography is the collective term used
for the separation of mixture in laboratory The mixture is dissolved in a
fluid (solvent) called mobile phase which carries it through a structure
holding another material called the stationary phase Thin layer
chromatography (TLC) is a tried and true method for the separation of
components in a mixture based on the polarity of the individual
components. When a standard is included, this method can also be used
for the identification of each component of the mixture.

The stationary phase for TLC consists of a plate, usually a sheet of

plastic or glass, that is coated with an absorbent material (i.e. silica). The
extracted sample mixture (such as a lipid extract from cells) is applied at
the bottom of the plate, allowing the solid phase to capture the mixture.
The plated sample is then “developed” by placing the TLC plate into a
sealed chamber containing the mobile phase solvent system.

11
 Rf = Distance travelled by solutes
Distance travelled by solvents

3.3 Rearing of testing insect

Adult insects of Sitophilus of order Coleoptera and

were collected from the food grain store house available in local market of
Bhopal (M. P,) .
The beetles were reared on
healthy and cereal grain of wheat Triticum aestivum L. in glass jars .One
hundred insects were released in 2kg of wheat grains capped with the
muslin cloth for ventilation .

Culture were maintained in laboratory, under controlled

temperature (28+-2C), relative humidity(75+-5%RH), and a photoperiods
of 12:12 (L:D) hrs. Then insects were recorded in jar on wheat grains and
each time early age beetles were used for the experiments .

Figure 1. S. granarius was reared in a 300ml wide-mouthed glass jars

containing wheat

12
 3.4 Experimental Bioassay protocol

In bio- assay protocol, mainly 4 groups of insects (10 inches in

each jar) were organized for the treatment of different herbal extract .

Group 1 was served as control in which

insects were reared in maintained laboratory conditions in glass jars of
standard size and no treatment was given to the insects of this group .

Group 2 was
served as vehicle control , in which insects were reared in glass jars
vehicle solvent (1ml) that was used for extraction of plant extract, to
observe the consequence of vehicle solvent on animals .

Group 3 was served as treated, in which

insects were reared by treating it with different concentration of plant
extract in increasing order from 10% (100mg/ml) to (100mg/ml), to
monitor the insecticidal efficacy of different extract of each plant .

Group 4 was served as standard , in which insects were reared

by treating it with standard reference drug Parad Tikdi by dissolving
500mg tablet in distilled water and concentration was maintained 100% ,
to observe efficacy of standard drug with the comparison of herbal
extracts.

For the
efficacy of herbal extracts, purified fractions of the each plant extracts
were tested on the insects /pest flour beetle by applying procedure
mentioned below :

1. Adult of Tribolium castaneum were exposed with

various increasing concentration of selected plant extracts.
2. For this purposes , separate filter
paper strips (1cm) were coated with different concentrations of plant
extract and were placed in the glass culture jar and open ends were
plugged with aluminium foil with holes for aeriation .
3. Then , coated filter paper strips were kept air – dried before
application . 4. Only solvent treated filter paper strips were
used as vehicle control. 5. Ten adult insects were released in
glass culture jar . 6. For insecticidal activity, ten
different concentrations of plant extracts wre used and for each
concentration , three replicates were placed . 7.

13
Mortality in Tribolium castaneum was recorded after 24 hrs in the
presence and absence of plant extracts, separately

14
 8. Finally , values were calculated according to the methods of
finney (1971). 9. All the photographs were taken which is shown
below :

15
16
S. Extract Solvent Fraction Color Active
No used System s characteristi fraction
. Used obtaine cs s
d
1 Entire Methanol Sn-1 Green Dark Bioactive
plant : water green fraction
methano (03:01)
l extract

2 Entire Methanol Sn-2 Yellowish Poorly

plant : water green bioactive
Water (50:50) fraction
extract

17
Result and Conclusion

Essential oils from various plants have shown promise as sources for
insecticides. Earlier attempts to explore the toxicity of plant derivatives
against S. granarius and Sitophilus oryzae have been made by essential
oils.( Aslan et al. ) evaluated essential oil from the plant species Blumea
erientha for their toxicities against the adults of Lasioderma serricorne
and S. granaries In that study, although insecticidal activities against
these pests were achieved with essential oils from all three plant species,
the oil of O. vulgare was found to be the most effective against S.
granaries( Kordalı et al. ) studied the toxicity of essentials oils isolated
from Blumea erientha species to S. granarius. All of the essential oils
tested were found to be toxic to adults of S. granarius. The oils showed
about 80-90 % mortality of granary weevil tested insecticidal properties
of essential oil isolated from Blumea erientha Results showed that the oil
was more toxic against S. granarius.

As part of the ongoing research on the biological activity of

Blumea erientha plant, results reported in this study shows that methanol,
and Distilled water Blumea extracts exert different bioactivities against
the pest S. granarius. Topical application of all three extracts resulted in a
high contact mortality, reaching the 100% value at the highest dose
tested (75.00 μg/adult). Among the extracts, the methanol was found to
be the most active showing significant mortality starting from 9.37
µg/adult.

Efficacy of different plant extracts was evaluated for their

growth inhibiting and grain protecting action against granary weevil, S.
granarius. The reduction of first adult emergence of granary weevil by
using the extract ofleaf of parsley, mint and thyme can control the weevil.
The plant extract will be very economic and non-toxic for human and
environment will remain safer. Considering cost effectiveness, easy
preparation, easy usage technology and environment-friendly advantages
plant extracts can be the most important component of integrated pest
management in controlling granary weevil of storage. On the other hand,
wheat is an important agricultural crop, especially, for solving the world
hunger problem.

18
Experimen Conc. No. Mortali Mortal Mortal %
tal-group of ty ity ity Mortality
Inse
ct
Trial 1 Trial 2 Trial 3
Control - 10 0 0 0 0.00±0.0
0
Vehicle 1 ml 10 0 0 0 0.00±0.0
control 0
(water)
Blumea 10% 10 1 0 1 6.67±5.7
erientha (50mg/ml 7
Water )
Extract
Blumea 20% 10 2 0 1 3.33±5.7
erientha (100mg/ 7
Water ml)
Extract
Blumea 30% 10 0 1 0 0.00±0.0
erientha (150mg/ 0
Water ml)
Extract
Blumea 40% 10 4 0 0 3.33±5.7
erientha (200mg/ 7
Water ml)
Extract
Blumea 50% 10 3 2 1 6.67±5.7
erientha (250mg/ 7
Water ml)
Extract
Blumea 60% 10 5 1 1 6.67±5.7
erientha (300mg/ 7
Water ml)
Extract
Blumea 70% 10 4 3 1 3.33±5.7
erientha (350mg/ 7
Water ml)
Extract
Blumea 80% 10 6 2 4 3.33±5.7
erientha (400mg/ 7
Water ml)
Extract
Blumea 90% 10 7 3 2 3.33±5.7
erientha (450mg/ 7
Water ml)
Extract
Blumea 100% 10 8 5 6 50.00±10
erientha (550mg/ .00
Water ml)

19
Extract

20
References

1. Nerio L.S., Olivero-Verbel J., Stashenko E. Repellent activity of essential

oils: A review. Bioresour. Technol. 2010;101:372–378.
doi: 10.1016/j.biortech.2009.07.048. [PubMed] [CrossRef] [Google Scholar]

2.Rajashekar Y., Gunasekaran N., Shivanandappa T. Insecticidal activity of

the root extract of Decalepis hamiltonii against stored-product insect pests
and its application in grain protection. J. Food Sci. Technol. 2010;47:310–
314. doi: 10.1007/s13197-010-0049-6. [PMC free article] [PubMed]
[CrossRef] [Google Scholar]
3. Jacobs, S. & D. Calvin, (2001). Entomological notes. College of
Agricultural Sciences. Department of Entomology. The Pennsylvania State
University, 2 p
4. Kordalı, S., I. Aslan, O. Calmasur & A. Cakır, (2006). Toxicity of essential
oils isolated from three Artemisia species and some of their major
components to granary weevil, Sitophilus granarius (L.) (Coleoptera:
Curculionidae). Industrial Crops and Products, 23: 162-170

5. Kordalı, S., A. Cakır, H. Ozer, R. Cakmakcı, M. Kesdek & E. Mete, (2008).

Antifungal, phytotoxic and insecticidal properties of essential oil isolated
from Turkish Origanum acutidens and its three components, carvacrol,
thymol and p-cymene. Bioresource Technology, 99: 8788-8795

6. Isman M.B. Commercial development of plant essential oils and their

constituents as active ingredients in bioinsecticides.

21