G T Gujar*, V Kalia, Archana Kumari & TV Prasad. Division of Entomology, Indian Agricultural Research Institute, New Delhi , India

Transcription

1 Indian Journal of Experimental Biology Vol. 42, February 2004, pp Potentiation of insecticidal activity of Bacillus thuringiensis subsp. kurstaki HD-1 by proteinase inhibitors in the American bollworm, Helicoverpa armigera (HUbner) G T Gujar*, V Kalia, Archana Kumari & TV Prasad Division of Entomology, Indian Agricultural Research Institute, New Delhi , India Received 17 July 2003; revised 22 September 2003 The effect of crude proteinase inhibitor extracts from seeds of different crop plants (black gram, chickpea, chickling vetch, finger millet, French bean, green gram, horse gram, lentil, pea and soybean) on the insecticidal activity of B. thuringiensis var. kurstaki HD-1 was investigated against neonate larvae of H. armigera by diet incorporation method. The larval mortality due to crude proteinase inhibitors alone (5% seed weight equivalent) ranged from 4.1 to 19.1%; the maximum mortality with finger millet and the minimum with pea var. DDR-23. A mixture of B. thuringiensis var. kurstaki HD-1 (10 ppm) and proteinase inhibitor (5% seed weight equivalent) was synergistic in larval mortality with respect to proteinase inhibitors of pea var. DMR-16, chickling vetch var. RLK-1098 and , lentil var. ILL-8095 and L-4076, soybean var. PK-1042, PK-416 and Pusa-22, chickpea var. Pusa-413, French bean (Chitra) and black gram; and antagonistic with respect to those of finger millet, horse gram and kidney bean. The larval growth reduction with crude proteinase inhibitors alone ranged from 17.9 to 53.1 %; the maximum growth reduction with soybean var. PK-1042 and minimum with lentil var. L A mixture of B. thuringiensis var. kurstaki and proteinase inhibitor was synergistic in growth reduction with respect to proteinase inhibitors of lentil var. ILL-8095, and L-4626 and antagonistic with respect to that of finger millet. The midgut proteinase inhibition with crude seed extracts (3.3% seed weight equivalent) ranged from 9.3 to 60.9% and was negatively correlated with larval mortality. These results showed that interactive effect of B. thuringiensis var. kurstaki HD- 1 and proteinase inhibitors in the larvae of H. armigera depended upon the quality and quantity of proteinase inhibitors, which vary widely in different plants. Keywords: American bollworm, Bacillus thuringiensis, Helicoverpa armigera, Proteinase inhibitors Plant proteinase inhibitors are mostly polypeptides or proteins, which occur in a wide variety of plants and have been evolved as plant defenses against herbivoresu. The effects of plant proteinase inhibitors on different types of animals including insects have been reported 3-5. These plant proteinase inhibitors inhibit the synthesis of proteins, thereby affecting proteolysis of proteins and consequently affecting amino acid availability for insect growth and development of the insects. Alternately, proteinase inhibitors enhance pernicious hyperproduction of proteinase activity of some kind in a compensatory manner, which leads to scarcity of amino acid supply for insect growth and development 6. These proteinase inhibitors are insecticidal at high concentrations 7 and growth inhibitory at low concentrations 8 9. These are effective against a wide range of insect species. Bacillus thuringiensis is an important soil inhabiting, aerobic bacterium pathogenic to insects. It is used as insecticide for important defoliators *For correspondence : Telefax: gtgujar@iari.res.in belonging to Lepidoptera and Coleoptera. B. thuringiensis has also been found useful for the control of mosquitoes and other public health insects. It is relatively safe to higher animals and is considered environmentally compatible 10. B. thuringiensis produces parasporal proteinaceous crystals, which on digestion in the midgut of insects bind with brush border membrane vesicles and cause pore formation in the cell membrane affecting ionic balance. In the process, the insects show feeding deterrence followed by intoxication. The use of proteinase inhibitors for the control of insects is made possible through transgenic technology involving cloning of genes of proteinase inhibitors in plant. The first transgenic plant to be transformed was tobacco with cowpea trypsin inhibitor (CpTi) gene for the protection against the tobacco horn worm, Manduca sexta 11 Since then, more than 18 crop plants have been transformed with proteinase inhibitor genes 5 12 Li et al. 13 reported high resistance of CpTi gene containing transgenic cotton lines to the American bollworm, Helicoverpa armigera (Hiibner). Further, in recent times, genes of CpTi and Cry 1 A toxins were cloned together in

2 158 INDIAN J EXP BIOL, FEBRUARY 2004 cotton (var. SGK 321) to enhance its ability to protect against a wide range of insect pests 14 Alternately, the proteinase inhibitors could be used as synerfists in B. thuringiensis formulations 15. Zhang et al. 1 reported biochemical basis of synergism between B. thuringiensis and CpTi toxin, which was attributed to more availability of Cry lac toxin due to inhibition of proteolysis of Cry 1 Ac in the midgut of larvae of H. armigera. However, Tabashnik et al. 17 did not find potentiation of B. thuringiensis toxicity with soybean trypsin inhibitors to the diamondback moth, Plutella xylostella. In view of these developments, it is necessary to study interaction of proteinase inhibitor and B. thuringiensis against the important insect pests. This aspect was studied in the present investigation by using proteinase inhibitors from different plant seeds and B. thuringiensis var. kurstaki HD-1 against H. armigera, a polyphagous key pest of cotton, pulses and many other crops. Materials and Methods Proteinase inhibitors and B. thuringiensis var. kurstaki HD-1-Seeds of different plants (black gram, chick pea, chickling vetch, finger millet, French bean, green gram, horse gram, lentil, pea and soybean) were ground to a coarse powder, which was extracted with ice-cold 100 mm Tris-HCI buffer (ph 7, 0.1% ascorbic acid) in the ratio of 1: 10 by mixing overnight. The extract was filtered through the several layers of cheese-cloth 18 Filtrates, referred to as crude proteinase inhibitors, were stored at -20 C till further in use. Acetone powder of spore and crystal complex of B. thuringiensis var. kurstaki HD-1, originally received in the form of culture blot as a gift from Bacillus Genetic Stock Center, OSU, USA, was prepared using procedure described by Dulmage et al 19 The endotoxin and spore counts of B. thuringiensis var. kurstaki HD-1 preparation were estimated as per Mohan and Gujar 20 and found to be Jlg 100 mg- 1 and 89.3 x mg- 1 respectively. Test insect- Larvae of H. armigera used in the present investigations were originally collected from the pigeon pea fields in the Institute, and reared in the laboratory on the chickpea based artificial diet at 27 ± 2 C and 60-70% RH as per Nagarkatti and Prakaash 21. The adults emerging from pupae were offered J 0% honey soluti on fortified with multivitamins throughout their egg-layi ng period. Five to ten pairs of adults were kept in each jar covered with markin cloth. The eggs laid on the markin cloth were kept in separate jar at 27 ± 2 C moistened with water. The newly hatched larvae were transferred to the test diet with a fine camel hair brush for bioassays. Bioassays-The bioassays were carried out by diet incorporation method as per Gujar et a/ 22 The crude proteinase inhibitor extract was mixed at a concentration of 5% of original seed weight in the casein-free artificial diet during diet preparation, when temperature reached below 40"C. The diet was allowed to set in plastic Petri dish (5 em diam. and 2 em height) at 27 ± 2 C. Ten neonate larvae were released in each replicate, with five replicates per treatment of crude proteinase inhibitor. The same diet incorporation assay was also used to examine the effect of B. thuringiensis var. kurstaki HD-1 alone (1 0 ppm), B. thuringiensis var. kurstaki HD-1 (10 ppm) and crude proteinase inhibitor (5 % seed weight equivalent) together. The test diets were prepared fresh every time, taking care of replacing the equal volume of water in the diet for the test solution, thereby, maintaining consistency of diet for all experiments. The lower Petri-dish was covered with a single layer of face-tissue paper to avoid escape of ti ny larvae and ensure trapping of excess water, before fitting the upper Petri-dish. The observations on larval mortality were recorded after 96 hr. The larval mortality in treatment was corrected using Abbott's formula 23. Ten surviving larvae of each treatment were weighed individually to calculate growth reduction. A co-toxicity factor (CTF) was also calculated to determine additive, antagonistic and synergistic effects, first by estimating expected mortality (Oe) from Pa + Pb (1- Pa)/100; where Pa was the observed mortality caused by B. thuringiensis var. kurstaki and Pb was that caused by crude proteinase inhibitor alone, subsequently estimating CTF=(Oc-Oe)x 100/0c; where Oc is the observed percentage mortality produced by the combination of B. thuringiensis var. kurstaki HD-1 with crude proteinase inhibitor, Oe is the expected percentage mortality. A CTF of +21 or more, indicating synergism; -21 or less, antagonism, and -21 to +21, additive effect was used as per Mansour et az2 4 Proteinase inhibition-last instar larvae, hr old, were first starved for 6 hr, immobilized at -20 C and dissected for midgut in ice-cold 20 mm Tris-HCI buffer (ph 8). Five midguts were homogenized in 1 ml Tris-HCI buffer and centrifuged at 19,621 g at 4 C for 15 min. The supernatant was stored in aliquots at -20 C till further analysis.

3 GUJAR et al: POTENTIATION of Bt ACTIVITY WITH PROTEINASE INHIBITORS IN HELICOVERPA 159 Proteinase activity was measured with azocasein as per Marchetti et ap 5 A mixture of 100 J.ll azocasein (2% prepared in 20 mm Tris-HCl-buffer ph 8) and 50 J.ll midgut protease extract was incubated at 30 C for 1 hr and then reaction terminated by precipitating with 100 J.ll of freshly prepared 10% trichloroacetic acid, followed by mixing with 1 ml of 1 N sodium hydroxide in equal volume, which gave orange coloured solution measured at 420 nm. For protease inhibition, 50 J.ll of crude proteinase inhibitor extract (equivalent to 5 mg seed weight) was pre-mixed with equal volume of midgut extract and incubated at 30 C fo r 1 hr and then, 50 J.ll azocasein (4%) was added to the mixture and incubated again for 1 hr. Thereafter that, remaining protease activity measured as above. In control, midgut extract was pre-mixed with equal volume of Tris buffer for 1 hr and processed as above. Three replicates were used for an estimation of protease activity. Proteinase inhibition was estimated by substracting absorbance due to crude proteinase inhibitor from that of control and dividing the same with proteinase activity in the control and expressing it in per cent. Statistical analysis- The data on per cent corrected mortality and growth reduction due to a combination of B. thuringiensis var. kurstaki HD-1 and crude proteinase inhibitor were subjected to analysis of variance for least square design at 5% level of significance by using Genstat software of Indostat Services, Hyderabad. The proteinase inhibition was correlated with % larval mortality and growth reduction due to crude proteinase inhibitors. Results The mortality due to crude proteinase inhibitors at 5% ranged from 4.1 to 19.1% (4.7-fold). The maximum mortality was recorded with finger millet and minimum mortality was recorded with pea var. DDR-23. The larval mortality due to B. thuringiensis var. kurstaki HD-1 ranged from 14.6 to 65.9% (4.5- fold). The combined effect of crude proteinase inhibitor and B. thuringiensis var. kurstaki HD-1 caused larval mortality ranging from 10.6 to 86.9% (8-fold); suggesting possibility of synergism (Table 1). Table I-Effect of proteinase inhibitors on toxicity of B. thuringiensis var. kurstaki HD-1 against neonate larvae of H. armigera (HUbner) S. No. Proteinase inhibitor Corrected m ortalit~ (%) Co-toxicity Combined effect Proteinase Btk PI Btk +PI Expected factor (S/An) inhibition (%) Blackgram (Local) cd s 9.35" 2 Chickpea (Pusa-413) b s 25.96h 3 Chickling vetch ( ) b s 37.66cf 4 Chickling vetch (EC ) c A 15.04k 5 Chickling vetch (RLK-1098) a s 34.77g 6 Finger millet (Local) An 9.77mn 7 French bean (Local-Red) cd A 1 9.7~ 8 French bean (Local-Chitra) d s 35.75fg 9 Green gram (Local) cd A 23.03; 10 Horse gram (Local) An 39.52c II Kidney bean (Local) An Lentil (L-4626) cd A NA 13 Lentil (L-4076) c s 47.99c 14 Lentil (Precoz 98/2008) b A NA 15 Lentil (ILL-8095) b s b 16 Lentil (L4642) a A 21.22j 17 Pea (DMR-11) a s 45.28d 18 Pea (DDR-23) b A 43.55d 19 Pea(DDR-13) b A 11.38'" 20 Pea (P-1542) " A 10.87'" 21 Pea (DMR-7) b A 51.88b 22 Soybean (PK-416) b s 60.94" 23 Soybean (Pusa-16) a A 43.7d 24 Soybean (PK 1042) b s 47.69c 25 Soybean (Pusa-22) c s 24.46hi S =synergistic; A= additive; An= antagonistic Btk Bacillus thuringiensis var. kurstaki HD-1, PI proteinase inhibitor, Figures in the column followed by same alphabet do not differ significantly.

4 160 INDIAN J EXP BIOL, FEBRUARY 2004 On the basis of co-toxicity factor, the synergistic effect of B. thuringiensis var. kurstaki and crude proteinase inhibitor was observed in pea var. DMR- 11, chickling vetch var. RLK 1098 and var. B , lentil var. ILL 8095 and var. L 4076, soybean var. PK 1042, var. PK 416 and var. Pusa 22, chickpea var. Pusa 413, French bean (Chitra) and black gram. The crude proteinase inhibitors of pea var. DMR-7, var. DDR-13, var. DDR-23, and var. P-1542, chickling vetch var. EC , lentil var. L 4626, var. Precoz 98/2008 and var. L 4642, soybean var. Pusa 16, French bean and green gram showed additive effect when assayed with B. thuringiensis var. kurstaki HD-1. The larval mortality was antagonistic in case of crude proteinase inhibitor of kidney bean, finger millet and horse gram combined with B. thuringiensis var. kurstaki HD-1. The per cent growth reduction with crude proteinase inhibitor alone ranged from 17.9 to 53.1% (3-fold). The maximum per cent growth reduction was recorded in soybean var. PK 1042 and minimum per cent growth reduction was recorded in lentil var. L The growth reduction due to B. thuringiensis var. kurstaki HD-1 ranged from 49.3 to 91.6% (<2- fold) and due to both B. thuringiensis var. kurstaki HD-1 and crude proteinase inhibitor from 56.3 to 93.9% (<2-fold). A combined effect of B. thuringiensis var. kurstaki HD-1 and crude proteinase inhibitor on larval growth was additive with respect to pea var. DMR-7, DDR- 13, DDR-23, P-1542 and DMR-11, chickling vetch var. RLK 1098, EC and L 4642, soybean var. Pusa 16, PK 1042, PK 416, and Pusa 22, chickpea var. Pusa 413, horse gram, kidney bean, French bean (red), green gram, French bean (Chilra) and black gram. The effect was synergistic in lentil var. ILL 8095, and L Antagonistic effect was observed in finger millet. In lentil var. L 4626, the combination effect of B. thuringiensis var. kurstaki HD-1 and crude proteinase inhibitor was additive with respect to mortality but synergistic with respect to larval growth reduction (Table 2). The synergistic mortality effect and additive growth reduction effect was found with respect to proteinase inhibitors in pea var. DMR 11, chickling vetch var. RLK 1098 and B , lentil var. L 4076, soybean Table 2-Effect of proteinase inhibitors on growth reduction of Bacillus thuringiensis var. kurstaki HO-I against neonate larvae of Helicoverpa armigera (Hiibner) S. No. Proteinase inhibitor growth reduction(%) Coinhibition Combined effect Btk PI Btk +PI Expected factor (S/An) I Blackgram (Local) 66.0 ± ± ± 1.3c A 2 Chick pea (Pusa-4I3) 82.6± ± ±1.lb A 3 Chickling vetch (B ) 76.3 ± ± ±0.9" A 4 Chickling vetch (EC ) 49.3 ± ± ± A 5 Chickling vetch (RLK-1098) 71.5± ± ± 1.4< A 6 Finger millet (Local) 91.6 ± ± ±6.9b An 7 French bean (Local-Red) 56.8 ± ± ±3.ld A 8 French bean (Local-Chitra) 66.0± ± ±2.2c A 9 Green gram (Local) 56.8± ± ± 1.1 c A 10 Horse gram (Local) ± ±2.0b A II Kidney bean (Local) 91.6± ± ±2.4" A 12 Lentil (L-4626) ± ± ±3.0c s 13 Lentil (L-4076) 49.3± ± ± A 14 Lentil (Precoz 98/2008) 82.6± ± ±0.9" A 15 Lentil (ILL-8095) 49.3 ± ± ±2.0c s 16 Lentil (L4642) 78.0± ± ± 1.7< A 17 Pea (DMR-1 1) 86.9 ± ± ±0.9b A 18 Pea (DDR-23) 82.6± ± ± A 19 Pea (DDR-1 3) 82.6± ± ± A 20 Pea (P-1542) 86.9± ± ±0.9" A 21 Pea (DMR-7) 82.6± ± ± A 22 Soybean (PK-416) 76.3± ± ± 1.7b A 23 Soybean (Pusa-1 6) 78.0± ±5.1 8l.8±2.6b A 24 Soybean (PK 1042) 76.3 ± ± ± A 25 Soybean (Pusa-22) 71.5± ± ±3.5c A S = synergistic; A= additive; An= antagonistic Btk Bacillus thuringiensis var. kurstaki HD-1, PI proteinase inhibitor, Figures in the column followed by same alphabet do not di ffer significantly

5 GUJAR et al: POTENTIATION of Bt ACTIVITY WITH PROTEINASE INHIBITORS IN HELICOVERPA 161 var. PK 1042, PK 416, Pusa 22, Bengal gram var. Pusa 413, French bean (Chitra) and black gram. The mortality effect was antagonistic in finger millet, horse gram and kidney bean but growth and development effect was antagonistic only in finger millet. Out of 25 crude proteinase inhibitors under investigation, 11 extracts synergized insecticidal activity (Table 1), whereas two crude proteinase inhibitors viz., lentil (L-4626) and lentil (ILL-8095) synergized growth reduction due to B. thuringiensis var. kurstaki HD-1 (Table 2). The larval mortality was negatively correlated with proteinase inhibition (r value -0.49). As the proteinase inhibition increased, the mortality decreased at a rate of -1.7%. However, there was no correlation (r -0.04) between growth reduction and proteinase inhibition. Discussion The neonate larvae of H. armigera responded to crude proteinase inhibitors, resulting in growth reduction and mortality. The larval mortality showed a wide variation as compared with growth reduction. It was also observed that some crude proteinase inhibitors synergized larval toxicity of B. thuringiensis var. kurstaki HD-1, while few proteinase inhibitors did so for synergizing growth reduction. It is interesting to note that there was no correlation between growth reduction and proteinase inhibition; but some negative correlation between larval mortality and proteinase inhibition. Our results showed that 25 inhibitors under investigation from different seeds varied greatly in quality. The potency of proteinase inhibitor depended upon the structural compatibility with the proteinases in the target organism, physiological conditions within the midgut (e.g., ph), and dietary quality (e.g., polyphenoloxidase activity, protein quality and quantity) Since dietary quality and physiological conditions within midgut were uniform, the differences in the efficacy of proteinase inhibitors could be attributed to the substrate specificity, variability of proteinases and their ability to proteolytically degrade proteinase inhibitors, and amount of proteinase inhibitor available for action. The six midgut proteases with molecular weights from 30 to 72 kda have been reported in the larvae of H. armigera and most being serine trypsin-kind, few chymotrypsin-like The major protease activity was associated with 24 kda 29 and 29.5 kda 31 The midgut proteinase inhibition in the present study varied from 9.3 to 60.9%, and was similar to that reported with Cajanus proteinase 0.5 mg/rnidgut compared with 100% inhibition with soybean trypsin inhibitor 34 ' and other trypsin inhibitors 35. Johnston et al. 36 reported higher growth reduction of larval H. armigera on a diet containing soybean trypsin inhibitor than on diet containing soybean Bowmann-Birk chymotrypsin inhibitor; with significant reduction in trypsin-like activity in larval midgut with trypsin inhibitor than control. Soybean proteinase inhibitor diet potentiated Heliothis virescens larval mortality due to B. thuringiensis var. kurstaki HD Besides, allelochernicals present in the crude proteinase inhibitor extracts may also influence interaction between proteinase inhibitor and B. thuringiensis. The plant phenolics 37, chlorogenic acid and polyphenol oxidase 38, some furanocoumarins in celeriac (Apium graveolens) 39 and secondary plant metabolites 40 are reported to influence toxicity of B. thuringiensis to insects. The antagonism reported in few cases of crude proteinase inhibitors is an indication of insect ability to synthesize more of proteinases of some kind or other, digest proteinase inhibitors, and incompatibility between proteinase inhibitors and proteinases. Giri et al. 41 reported ability of H. armigera gut proteinases to proteolytically digest chickpea trypsin inhibitor and hence, did not find their inhibition. Similarly, Broadway and Duffel found elevated protease activity in Helicoverpa zea when fed on 1.2% casein and 0.18% either soybean trypsin inhibitor or potato trypsin inhibitor. The growth reduction of H. zea was attributed to pernicious excessive production of proteinases, which depleted amino acid pool. In conclusion, the present studies have shown that proteinase inhibitors have the potential to significantly reduce the growth and development of herbivorous insects and largely potentiate B. thuringiensis var. kurstaki HD-1 toxicity. Acknowledgement Authors are grateful to ICAR, New Delhi for financial assistance. 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