Indian Journal of Experimental Biology Vol. 42, January 2004, pp. 91-95
Bioactivity of non-edible oil seed extracts and purified extracts against Helicoverpa armigera (Hubner)
Pushpa Pawar*, Mary Joseph*, Vijay Tungikar* & Swati Joshi**
*Entomology Department, National Chemical Laboratory, Pune 411 008
**Division of Organic Chemistry: Technology, National Chemical Laboratory Pune 411 008, India Received 19 May 2003; revised 2 September 2003
Extracts and purified extracts of seeds of two plant species , Madhuca latifolia and Calophyllum inophyllwn when evaluated against the 2"d instar larvae of Helicoverpa armigera reared on synthetic diet, exhibited high larval mortality, prolongation of developmental period, morphological deformities and highly significant reduction in adult emergence. The reduction in larval weights in the treatments was also highly significant.
Keywords: Botanical pest control agents, Calophyllum inophyllum, Growth inhibition, Helicoverpa amtigera, Madhuca latifolia
Helicoverpa armigera a polyphagous pest is reported to damage 181 species of host plants belonging to 45 families!.. Although the pest can be controlled by the use of conventional insecticides, but in view of the environmental hazards there is a need to use eco- friendly pesticides derived from plants. Therefore the present work was undertaken to explore the bioactivity of the non-edible oil seed extracts, and purified extracts of Mahua (Madhuca latifolia) and Undi (Calophyllum inophyllum) which have already been characterized for its insecticidae-4, antifeedant5 activity and as surface protectants6 in stored products.
Materials and Methods
Extraction of seeds-Seeds of C. inophyllum and M. latifolia were collected from Western ghat, Maharashtra, India in April 2000, shade dried, powdered and processed as follows:
Powdered seeds of C. inophyllum, 1 kg, were extracted, at room temperature with acetone followed by methanol (2L x 3 each). From the combined extracts, solvent was evaporated separately and dried to yield acetone extract UA (189_6 g, 18.96%) and methanol extract UB (28.8 g, 2_88%).
Powdered seeds of M.latifolia, 1kg, were extracted with acetone, as mentioned above, to yield acetone extract, MA (201.73 g, 20_1 %). Fresh 1kg of
*For correspondence:
E-mail: Pawar@ems.ncl.res.in, pushpapawar@yahoo.com Phone: 0091-20-5893300, 5893400 (Ext. 2302)
Tele/Fax: (0): 0091-20-5893153
M. latifolia seed powder was extracted with methanol, 1 L, and pet-ether (60-80°C), 2 L, by stirring mechanically for 30 min. Solubles were filtered and the residue was extracted two more times with fresh quantities of methanol and pet- ether as mentioned above. The methanol and pet-ether layers were separated from which solvent was evaporated separately to yield, pet-ether extract MB (212 g, 21.2%) and methanol extract MC (88 g, 8.8%).
Purification of extracts
Extract UA,l35g, was partitioned between aqueous methanol (85%), 200 ml, and pet- ether, 400 rnl. The pet-ether solubles and methanol solubles were separated from which solvent was evaporated to yield pet- ether solubles UA~. 118.5 (16.59%) and methanol soluble UA2 13.75 g (1.89%) Extract UB, 28.8g, was treated with acetone 50rnl x 3 at room temperature as mentioned above to yield acetone soluble UB~. 23.2 g (2.32%) and acetone insoluble UB2, 5.6 g (0.56%).
Formulations of extracts and purified extracts
UA~. UA2, UB~. MA and MB, 1g each were formulated as their ECs using tween 20, 400 mg and mixing mechanically at room temperature for 30 min.
which were diluted with 50 ml water, to obtain stable emulsion_ Solutions of UB2 and MC were prepared by dissolving lg of these extracts in 10 ml aqueous ethyl alcohol (50%). Blank controls were also prepared simultaneously.
92 INDIAN J EXP BfOL, JANUARY 2004
Biological Assay
Solutions and emulsions were diluted further with water to obtain required concentration of 5, 1, 0.5, 0.1% of test samples. The synthetic diet was prepared7 and 10 g each impregnated at 30°C with various concentrations of the samples and mixed thoroughly.
Control diet was prepared by using blank formulations. For toxicity studies, the solidified diet was cut into pieces and offered to pre-weighed H.armigera 2"d instar larvae Ten replicates were used for each treatment and the experiment was repeated three times. Untreated controls were run simultaneously. The vials were kept at 27°C and 60- 70% RH, 14:10: (L: D). Mortality count after 24 hr was noted in each replicate. Lethal concentration for 50% larval mortality i.e. LC5a for each sample was calculated. For studies on growth inhibition, early 2"d instar larvae weighing between 10-15 mg were used.
At sub-lethal concentration the larvae were allowed to feed adlibitum on the treated diet throughout their larval period. Every alternate day, they were given their respective treated diet. The weight of larvae on 3rd , 5th an d 7th d ay were note d an d mean wetg . h t gam . of each treated groups were calculated. Comparative growth i.e. ratio of weight relative to control was also calculated. Daily observations on larval mortality (LM), larval-pupal intermediate (LP) malformed pupae (MP), deformed adults (DA)and normal adult emergence was recorded. The adults that were morphologically identical with the counterparts emerging in control were regarded as normal. The effective concentration for inhibition of 50% normal adult emergence i.e. E15a was calculated for each sample. Percent inhibition of emergence of adult i.e.% E.I was calculated by the following formula8.
% El=lOO-T/Cx 100. where Tis the emergence of adults in treatment and C is the emergence of adults in control.
Data was analysed statistically by using Student's t test. Elso and LC5a were calculated by subjecting the data to probit analysis9.
Result
Effect on growth-Early 2"d in star larvae of H. amtigera when exposed to various concentrations of formulations exhibited larval mortality, reduction in growth and development as well as morphological deformities. In case of C. inophyllum seed extracts, UA2 and UB2 exhibited 100% larval mortality at higher concentration of 5%. Extract UA1 and UB1
induced > 70% mortality. However at lower
concentrations in all the extracts of C. inophyllum the morphological deformities were 10-15% (Fig. 1). Larval period was prolonged for 10-13 days in all the extracts.
In case of M.latifolia, all seed extracts exhibited 100% larval mortality at 5%. However, MA and MB were effective at 1% concentration also. At lower concentrations in all the extracts 10-12% larval pupal intermediates and deformed pupae and 10-30%
deformed adults were emerged (Fig. 1). The larval period was prolonged for13-l7 days.
Percent adult emergence was reduced at higher and lower concentrations also. Extracts UA1, UB2 and UA2 inhibited 100% adult emergence only at highest concentration of 5% while at lower concentrations of I and 0.5% the emergence inhibition was only 40- 79%. Extracts MA, MB and MC also inhibited 100%
adult emergence at higher concentration i.e. 5 and 1%.
They were effective at lower concentrations of. 0.5 and 0.1% also (Table 1).
Effect on larval weights-Table 1 reveals the significant reduction in the weights of treated larvae.
Among the extracts of C. inophyllum, in UB2 and UA2
at highest concentration of 5% the larvae did not survive after 24 hr. At 1% concentration the larvae could gain sufficient weight. Percent comparative growth was also significant (P < 0.001 ). Where as in case of UA1 and UB1 the loss in weight was significant at 5%. Percent comparative growth was highly significant (P < 0.005) in UA1 and less significant (P < 0.001) in UA2. Larvae fed on treated diet with concentration of 5 and 1% of extract MA and MB did not survive after 24 hr. However at lowest concentration of 1 and 0.5%, the percent comparative growth was highly significant (P < 0.005) in case of MC and MA respectively.
Toxicity and relative efficacy-Toxicity studies revealed that among the C. inophyllum seed extracts UB2 was found to be most toxic exhibiting LC5a 0.623% and EI50 0.161% and activity wise these extracts were graded as UB2 > U A2 > U A 1 > UB 1
(Table 2).
Among M. latifolia seed extract MA was most active exhibiting LC5a 0.532% and E15a 0.04518%.
Activity wise these extracts were graded as MA>MB >MC (Table 2).
Discussion
The findings reveal that at higher concentration of 5%, M. latifolia and C.inophyllum, inflicted 100%
larval kill. Similar toxic effects have been reported
PA WAR et al.: BIOACTIVITY OF NON-EDIBLE SEED EXTRACTS 93
with N. viresscens when these seed oils were sprayed on plants10 .At sublethal concentration mostly all the extracts retarded the growth and dev~lopment
effectively. Similar results with other plant extracts were reported on C. cephalonica11. The retarded growth of the larvae is directly proportional to the weights of the body. Similar effects of weight loss in case of Callosobruchus chinensis have been reported with P.glabra oil12• Morphological deformities at pupal and adult stage and toxic effects suggest the interference of these extracts with growth and
UA1
100
.~ iii Ill 80 CIGJ
0 ·- 60
_,.,
_g
E
e-.E 40
~~ 20
~ 0
5 0.5 0.1
Cone.%
UA2
iii 100 .!:! In 80
.2~ 60
_g
E
e-.E 40
0 ..
:EO 20 'if!
0
5 0.5
MA
Cone.%100
iii 80
.!::! In CIGJ 60
0 . ,
:g
-~e-.E 40
~2l 20 'if!
0
5 0.5
MC Cone.%
100
iii 80
.~ In
.2~ 60
_g
E
e-.E 40
~2l
~ 20 0
5 0.5
Cone%
0.1
development processes. Since these processes are regulated by morphogenetic hormones, it can be suggested that these extracts interfere with these hormones of the insects. These extracts caused various morphological deformities, thereby inhibiting overall population of adults. Such type of effect where emergence of adults are inhibited have also been reported on C. cephalonica13and T. castaneum14•
In the present work, extracts UB2, M~ and MB was found to be most promising. If these extracts retain their activity in field trials, it may well become
UB1
5 0.5 0.1
Cone.%
UB2
I~
5 It,
I~
0.5 Inr:O
0.1 IMB
Cone.%0.1 5 0.5 0.1
Cone%
CLM DLP liMP ISlDA
0.1
Fig !-Morphological deformities shown by various extracts of C. inophyllum and M. latifolia against 2"d instar larvae of H. annigera LM-Larval mortality, LP-larval pupal intermediate, MP-Malformed pupae, DA-Deformed adult.
94
Extract
UAI
UA2
UBI
UB2
MA
MB
MC
Control
INDIAN J EXP BIOL, JANUARY 2004
Table 1- Effect of various extracts of seeds on growth of 2"dinstar larvae of H. annigera Cone.
(%)
5
1 0.5 0.1 5 I 0.5 0.1 5
I 0.5 0.1 5
I 0.5 0.1 5 1 0.5 0.1 5 I 0.5 0.1 5 1 0.5 0.1
55
---o'rM.:..e:..:a:.;..;n..:.Ia=r_v~al'---w--'t'-'(;rm""g!.!..) - - - - , . , - - - Comparative 3'd day 5'" day 7'h day growth(%) 0 day
13.22± 1.10 1-3.24 ± 0.84 12.99±0.91 12.35±0.62 12.43±0.80 14.05 ±0.52 13.48±0.85 12.34±0.92 12.73±0.84 12.66±0.82 13.12±0.90 13.10±0.62 13.52±0.64 13.35±0.92 14.19±0.80 13.65±0.8 12.11 ±0.84 13.99±0.99 13.72±0.92 12.40±0.80 12.20±0.82 13.90±0.92 13.78±0.86 13.12±0.84 12.53±0.82 12.68±0.64 12.74±0.72 12.79±0.88 12.58±0.82
53.41 ±2.10 81.26±3.10 121.26± 1.4 29.07++
73.41 ±2.82 1 11.25±2.80 152.49±2.8 37.5r 112.20±3.60 132.18±2.92 184.24±3.80 45.20+
128.42±3.10 180.47±3.80 221.33±4.52 52.91
* *
121.26±3.1 201.48±4.1 147.73±3.8 232.83±4.5 192.96±4.1 315.66±4.8 80.60 ± 2.10 142.58 ± 1.64 121.62±2.84 210.11±2.92 151.25 ± 2.92 265.53 ±2.85 195.18± 1.10 321.10±3.6
*
*98.03±2.2 161.02±2.1 I 04.41 ± 1.8 200.35 ± 1.9 163.5±2.1 261.43±1.10
* *
*
*26.54± 1.1 54.72±2.2 75.62± 1.40 120.29±2.8
* *
* *
65.50± 1.64 121.80±2.2 94.67± 1.84 180.90±2.6
35.49 ± 0.92 65.49 ± 1.64 83.54± 1.10 185.83± 1.82 110.84± 1.12 201.84±2.8
163.50 ± 2. 225.08 ±2.8
*
254.85 ±2.8 282.16 ± 3.1 351.20±3.8 212.08 ±3.6 271.12±3.2 326.66±3.9 361.78 ±4.2
*
214.50±3.8 305.1 ±3.2 320.14±3.6
*
*112.72± 1.10 196.90± 1.5
* *
183.07 ± 2.82 309.21 ±2.84
*
121.88± 1.10 209.87±2.10 339.81 ±3.82 383.20±3.8
0 44.97+
62.49 72.91 43.78+
59.73 74.55 83.94 0 39.84+
50.22+
66.85 0 0 26.84++
49.78+
0 0 45.78+
79.89 0 27.01++
53.18+
88.23 100
% EI 100 58.64 40.31 27.34 100 71.14 59.73 22.55 89.84 63.17 48.69 19.77 100 79.06
69.5 37.76
100 100 90.26 59.06 100 100 90.00 59.03 100 100 69.21 48.43
2.5 Values significantly different from control analysed by Student, t test
++P<0.005+P<0.001; *Larval mortality.
UAI: Pet ether soluble of acetone extract; UA2: Methanol soluble of acetone extract; UB1:Acetone soluble of methanol extract and UB2: Acetone insoluble of methanol extract of C.inophylum seeds. MA: Acetone extract ;
MB : Pet ether extract and MC: Methanol extract of M.latifolia seeds.
Fraction
UA1
UA2 UB1
UB2 MA MB MC
Table 2 -Elsa and LCsa values of various seed extracts against 2nd ins tar of H. annigera Regression
equation
-1.0052x + 6.574 -1.3932x + 6.895 -0.9762x + 6.642 -1.5343x + 6.855 -l.l689x + 6.934 -1.543Ix + 7.884 -l.0479x + 7.200
Elsa
0.368 0.229 0.481 0.161 0.0451
0.074 0.125
Fiducial limits 95%
(Upper-lower) 0.4591-0.2080 0.3492-0.1516 0.1403-0.0174 0.3056-0.0996 0.0954-0.0343 0.1182-0.4312 0.3021-0.0986
Regression equation
1.2813x + 2.3458 l.5419x + 2.0875 1.0842x + 2.7079 1.6300x + 2.074 l.518x + 2.3800 1.4560x + 2.3250 l.4429x + 2.2969
LCsa
1.117 0.774 1.308 0.623 0.532 0.687 0.741
Fiducial limits
95%
(Upper-lower) 2.543-0.4449 1.215-0.4130 3.857-0.2722 1.1241-0.3964 0.7840-0.405 0.9460-0.3891 0.9112-0.6654 UA1: Pet ether soluble of acetone extract; UA2: Methanol soluble of acetone extract: UB1: Acetone soluble of methanol extract and UB2: Acetone insoluble of methanol extract of C.inophyllum seeds. MA: Acetone extract; MB: Pet ether extract and MC: Methanol extract of M.latifo/ia seeds.
PAWAR et al.: BIOACTIVITY OF NON-EDIBLE SEED EXTRACTS 95
an efficient and non hazardous substitute for pest controL The foregoing account makes it abundantly clear that new avenues for further work on various plant products/ derivatives and their constituents are very much desired. Identification and development of still more safe and eco friendly compounds will widen the choice and therefore scope and probability of their actual development
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