Investigation of the pesticidal as well as antimicrobial effect of the aqueous leaf extract from naturally available plants

1. Investigation of the pesticidal as well as
antimicrobial effect of the aqueous leaf
extract from naturally available plants
M.Sc. 4th Semester in Biotechnology
Roll- PG/VUOGP57/BIT-IVS No. 034
Registration No. – 02180 of 2020-2021

2. INTRODUCTION
■ Chemical pesticide were found to pollute in every stream, cause different
diseases, some are non- biodegradable and decrease soil diversity.
■ Plant material are safer for environment, less expensive to use as a
pesticide.
■ Plant material contain secondary metabolism that have antimicrobial,
antioxidant, anti infectious and anti tumor activities.

3. AIMS AND OBJECTIVES
■ To prepare a bio pesticide from naturally available leaves.
■ To assay the antimicrobial effect of that bio pesticide against Gram
positive and Gram negative pathogenic bacteria.

4. METHODS
■ Collection of Plant Material and Preparation of aqueous Leaf extract(sample 1,sample 2,sample
3,sample 4,sampl 5,sample 6).
Sample – 1 (S 1) :Vitex negundo, Pongamia pinnata, Datura stramonium.
Sample – 2 (S 2) :Calotropis gigantea, Thevetia peruviana, Ipomoea aquatica.
Sample – 3 (S 3) :Azadirachta indica, Nyctanthes arbor-tristis , Pongamia pinnata.
Sample – 4 (S 4) : Azadirachta indica, Vitex negundo, Clerodendrum infortunatum.
Sample – 5 (S 5) :Carica papaya, Nyctanthes arbor-tristis, Clerodendrum infortunatum
Sample – 6 (S 6) :Azadirachta indica, Pongamia pinnata, Carica papaya.
Three types of leaves were properly washed → each leaf is weighted 16 gram → leaves were
suspended in 150 ml of distilled water → extract was done at 80°C in a water bath for 2hours
→ Filter → Store at 4°C

5. Antimicrobial Assay
Collection and maintenance of test organisms (Escherichia coli, Pseudomonas sp., Staphylococcus
aureus, and Bacillus subtilis and unknown air borne bacteria.)
↓
Chloramphenicol was used as positive control and sterilized distilled water was used as negative
control.
↓
Nutrient agar (NA) was distributed in seven petriplate for each bacteria culture.
↓
Bacterial culture was given in each petriplate and spread it .
↓
Wells were Bored and sample were given.
↓
Petriplate were wrap by paraffin and Plates Were incubated .

6. RESULTS
Agar well diffusion assay:Zone of growth inhibition (mm including well diameter 7 mm ±
SD) showing antimicrobial activity.
Antimicrobial activities on Escherichia coli:
• Inhibition zone of positive control (C+) : 30± 0.4
• Inhibition zone is not detected of negative control (C-)
Concentr
ation (%)
S1 S2 S3 S4 S5 S6
100 16 ±
0.81
13 ± 1 ND 21.6 ±
0.5
ND ND
70 16 ±
0.81
11 ± 1 ND 20 ± 1 ND ND
50 9 ±
0.81
9 ± 1 ND 12.6 ±
0.5
ND ND
30 ND ND ND 9 ± 0.5 ND ND
30 30 30 30 30 30
0 0 0 0 0 0
16
13
0
21.6
0 0
15
11 0
20
0 0
9 9 0
12.6
0 0
0 0 0 9.3 0 0
0
10
20
30
40
S1 S2 S3 S4 S5 S6
Escherichia coli
C+ C- 100 70 50 30
Table: Zone of inhibition of six sample on Escherichia coli, ‘ND’
denotes Not Detected.
Figure: Inhibition zone of six samples on Escherichia coli use
Chloramphenicol as positive control(C+) and sterilized water as
negative control (C-)

7. Pictorial representaion of antimicrobial assay on Gram negative bacteria
Escherichia coli
Control
Well 1 =100%, well 2=70%, well 3=50% and well 4=30%

8. Antimicrobial activity on Staphylococcus aureus:
■ Inhibition zone of positive control (C+) : 28 ± 1
■ Inhibition zone is not detected of negative control (C-)
Concentrat
ion
(%)
S1 S2 S3 S4 S5 S6
100 ND ND ND 12 ± 1 12.6 ±
0.4
16.6
±0.81
70 ND ND ND 11.6 ± 1 10 ±
0.81
ND
50 ND ND ND 11 ±
0.94
9 ±
0.81
ND
30 ND ND ND ND ND ND
28 28 28 28 28 28
0 0 0 0 0 0
0 0 0
12.6 12.3 13
0 0 0
11 10
0
0 0 0
10 9
0
0 0 0 0 0 0
0
5
10
15
20
25
30
35
S1 S2 S3 S4 S5 S6
Staphylococcus aureus
C+ C- 100 70 50 30
Table: Inhibition zone of six sample on Staphylococcus aureus,
ND’ denotes Not Detected.
Figure: Inhibition zone of six samples on Staphylococcus aureus
use Chloramphenicol as positive control (C+) and sterilized water as
negative control (C-)

9. Pictorial representation of antimicrobial assay on Gram positive bacteria
Staphylococcus aureus.
Control
Well 1 =100%, well 2=70%, well 3=50% and well 4=30%

10. Antimicrobial activity on Bacillus subtilis
■ Inhibition zone of positive control (C+) : 28 .6 ± 0.94
■ Inhibition zone is not detected of negative control (C-)
Concentr
ation (%)
S1 S2 S3 S4 S5 S6
100 20 ±
0.81
ND 23 ±
0.81
17 ±
0.81
16 ±
0.9
18 ±
0.4
70 16.6 ±
0.9
ND 20 ±
0.81
16 ±
0.94
14.6 ±
0.4
17 ±
0.4
50 14 ±
0.81
ND 18.6±0
.94
16 ±
0.4
12 ±
0.4
16.3±0
.47
30 12 ±
0.81
ND 15 ±
0.4
ND 12 ±
0.4
15 ±
0.81
28.6 28.6 28.6 28.6 28.6 28.6
0 0 0 0 0 0
20
0
23
16.6 16 17
16.6
0
20
16 14.6
17
14
0
18.6
16
12
16.3
12
0
15
0
11
15
0
5
10
15
20
25
30
35
S1 S2 S3 S4 S5 S6
Bacillus subtilis
C+ C- 100 70 50 30
Table : Inhibition zone of six sample on Bacillus
subtilis, ‘ND’ denotes Not Detected.
Figure : Inhibition zone of six samples on Bacillus subtilis use
Chloramphenicol as positive control (C+) and sterilized water as negative
control (C-)

11. Pictorial representation of antimicrobial assay against Gram positive
bacteria Bacillus subtilis
Control
Well 1 =100%, well 2=70%, well 3=50% and well 4=30%

12. Antimicrobial activities on Pseudomonas sp
■ Inhibition zone of positive control (C+) : 28 ± 0.94
■ Inhibition zone is not detected of negative control (C-)
Conce
ntrati
on (%)
S1 S2 S3 S4 S5 S6
100 18 ±
0.81
22 ±
0.81
ND 11.6 ±
0.4
21.3 ±
0.8
ND
70 17.4 ±
0.47
21.8±0
.47
ND 9.6 ±
0.81
21 ±
0.8
ND
50 16.6±0
.47
20.4 ±
0.9
ND 8.3 ±
0.9
19.6 ±
0.4
ND
30 14 ± 1 16.3 ±
0.4
ND 7.8 ± 1 18.3 ±
0.4
ND
28 28 28 28 28 28
0 0 0 0 0 0
18
22
0
11.6
21.3
0
17.6
21.6
0 9.6
21
0
16.6
20.3
0 8.3
19.6
0
14
16.3
0 8
18.3
0
0
10
20
30
40
S1 S2 S3 S4 S5 S6
Pseudomonas sp.
C+ C- 100 70 50 30
Table: Inhibition zone of six sample on Pseudomonas
sp., ‘ND’ denotes Not Detected.
Figure : Antimicrobial activity of six samples on
Pseudomonas sp use Chloramphenicol as positive control (C+)
and sterilized water as negative control (C-)

13. Pictorial representation of antimicrobial assay against Gram negative
bacteria Pseudomonas sp.
Control
Well 1 =100%, well 2=70%, well 3=50% and well 4=30%

14. Antimicrobial activities on unknown air borne bacteria
■ Inhibition zone of positive control (C+) : 31± 1
■ Inhibition zone is not detected of negative control (C-)
Concentr
ation (%)
S1 S2 S3 S4 S5 S6
100 19.3±0
.81
21.3 ±
0.8
15.3±0
.94
23 ±
0.81
21 ±
0.4
19.6±0
.81
70 18.6 ±
0.4
20.6
± 0.9
13.3 ±
0.4
22.3±0
.81
20 ±
0.4
18.6 ±
0.9
50 17.6 ±
0.4
19.3 ±
0.8
11.6 ±
0.81
19.6 ±
0.9
19.6±0
.81
18 ±
0.4
30 17.3 ±
0.8
18 ±
0.4
9 ± 0.4 18.6 ±
0.8
19 ±
0.4
16.6 ±
0.4
31 31 31 31 31 31
0 0 0 0 0 0
19.3
21.3
15.3
23
21 19.6
18.6
20.6
13.3
22
20 18.6
17.6 19.3
11.6
19.6 19.6 18
17.3 18
9
18.6 19
16.6
0
5
10
15
20
25
30
35
S1 S2 S3 S4 S5 S6
Unknown air born bacteria
C+ C- 100 70 50 30
Table : Inhibition zone of six sample on Unknown
air born bacteria ,‘ND’ denotes Not Detected.
.
Figure : Antimicrobial activity of six samples on Unknown air
born bacteria use Chloramphenicol as positive control (C+) and
sterilized water as negative control (C-)

15. Pictorial representation antimicrobial assay against an unknown air born
bacteria
Control
Well 1 =100%, well 2=70%, well 3=50% and well 4=30%

16. Biopesticidal assay:
Experiment -1: Pesticidal effect of sample 4 on beetle
• The beetles were collected from the maize field.
↓
• Two leaf disc were prepared and both were washed with 70% ethanol.
↓
• One of the leaves was dip in sample-4 and the another one was not dip any solution.
↓
• Then the two leaf disc were placed on two another Petri plate together with four beetles. Petri
plate were filled with 2% agar solution.
↓
• Mortality of beetles were observed in 1hour, 2hour, 4hour, 24hour and 48hour.

17. Results of Pesticidal effect of sample 4 on
beetle :
Pictorial representation of
pesticidal assay on beetle
of control Petri plate
Pictorial representation of
pesticidal assay on beetle
by applying Sample-4
Control
0
2
4
1 2 4 24
48
Time (hour)
NO.
OF
MORTAL
BEETLES
Pesticidal effect on
bettles
Control sample 4

18. Experiment -2: Pesticidal assay of sample 1, sample 4 and sample 5 on fruit fly larva .
• The larva of fruit fly was collected.
↓
• Four fresh tomato leaves were collected and washed with 70% ethanol.
↓
• Three leaves were dip according to sample-4, Sample-1 and Sample -5 and the another
one was not dip any solution which one use as control.
↓
• Then the four leaves were separately placed on four another Petri plate together with
seven larva of fruit fly. Petri plates were filled with 2% agar solution.
↓
• Observed the damage area of three leaves those are dip in sample Compare to the
Control one.

19. Results of Pesticidal effect of sample 1, sample 4
and sample 5 on fruit fly larva :

20. CONCLUSION
■ It is conclude that the aqueous leaf extract of the mixture of Azadirachta indica
(Neem),Vitex nigundo (Nisinda),Clerodendrum infortunatum (Bhat) have good
pesticidal effect. It may be used as biopesticide for long run.
■ Aqueous extract of leaf mixture are inhibit the growth of some pathogenic
bacteria. So the mixture of leaf extract can be used as disinfectant and floor
cleaner.

21. FUTURE PROSPECTS
This project work may have the following future prospects –
• Isolation, purification and characterization of different molecules which are
involved for antimicrobial activity.
• Pesticidal assay can be done on different pests on agricultural field.
• Determination of the effective dose of pesticide.

22. REFERENCES
■ Baswa M, Rath CC, Dash SK, Mishra RK ,2001, Antibacterial activity of Karanj (Pongamia pinnata) and Neem (Azadirachta
indica) Seed oil. J. Microbios 105, 183-189
■ Chandramu C, Manohar RD, Krupadanam DGL, Dashavantha RV ,2003. Isolation, characterization and biological activity of
betulinic Acid and ursolic acid from Vitex negundo L. Phytother. Res. 17:129-134
■ Dr. Garima Gupta, Shirin Sharma and Dr. Neelima Kumar, 2020,Carica papaya aqueous leaf extracts as potential botanical
insecticide against rose aphids (Macrosiphum rosaeformis D.), Journal of Entomology and Zoology Studies, Vol. 8, Issue 3.
■ Kehinde AS, Adelakun KM, Attahiru H, Adedeji AS and Akinade GT,2018 ,Antibacterial Activities of Crude Extract of
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Nat. Prod.,15: 18-20.
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23. THANK YOU