Effect of Seed Proteinaceous Inhibitors on Digestive Protease Activity of Tomato Leaf Miner Tuta absoluta (Meyrick) (Lep.: Gelechiidae)

پژوهش های حفاظت گیاهان ایران

Article 4, Volume 30, Issue 3 - Serial Number 33, September 2017, Pages 449-459   

Document Type: Research Article

DOI: 10.22067/jpp.v30i3.45016

Authors

M. University of Tehran^{* 1}; A. R. Bandani²; Gh. Sabahi²

¹Ph.D. Student Department of Plant Protection

²University of Tehran

Abstract

Introduction: Tomato leaf miner Tuta absoluta Meyrick (Lep.: Gelechiidae) is one of the most important pest of tomato around the world which have been introduced to Iran in recent years. Pesticides can usually reduce the damage caused by the pest, however the extensive use of pesticides substantially increase production costs and lead to development of insecticide resistance. Thus, pest resistance and also adverse effects of pesticides on the human health and the environment make search for alternative control method/s inevitable. The aim of this study was to examine the effect of proteinaceous extracts of Datura (Datura stramonium L.), Bean (Phaseolus vulgaris L.), Wild Oat (Avena fatua L.), Broad bean (Vicia faba L.), Amaranth (Amaranthus retroflexus L.), Mung bean (Vigna radiate L.), Pea (Pisum sativum L.), Canola (Brassica napus L.), Triticale (Triticosecale wittmack) and Sivand, Aflak and Alborz wheat (Triticum aestivum L.) cultivars seeds on protease activity of tomato leaf miner. Also in this study optimum pH for protease activity and effects of pH on inhibitory activity of extracts were determined.
Materials and Methods: For this work fourth instar larvae of Tuta absoluta was used for enzyme extraction. Plant proteinaceous extracts were obtained using 0.1 M NaCl. For protease activity assay 10 μl enzyme extract and 50 μl substrate solution (Azocasein 2%) were mixed with 40 μl of the 20 mM Glycine-NaOH buffer at pH 10.0 After 60 min incubation, 100 μl of 30% trichloroacetic acid (TCA) was added to the reaction mixture, and kept at 4 °C for 30 min, followed by centrifugation at 15,000gfor 15 min to precipitate non-hydrolysis substrate. 100 μl of 1 M NaOH was added to 100μl supernatant and the absorbance at 405 nm was measured. To determine the effect of the seed proteinaceous extracts on the protease activities, enzymes were pre-incubated with each seed extracts for 30 min at 35 °C followed by determination of the residual enzyme activity as described before. To determine the effect of pH on inhibitory activity of the seed extracts, highest concentration of Mung bean, Pea, Canola, Broad bean, wild oat and two wheat cultivars (Alborz and Aflak) seed proteinaceous extracts were incubated along with the enzyme for 30 min at pH set at 6-12, and then enzyme activity was recorded as described before. Protein concentration was measured using bovine serum albumin (Bio-Rad, München, Germany) as a standard.
Results and Discussion: The effect of pH on protease activity showed that the greatest activity was observed at pH 10 and in the other pHs, it was lower, that this result Corresponded with the pH in other lepidopteran larvae. Also greatest activity of protease at alkaline pH indicates high activity of serine proteases in the alimentary canal. The greatest activity of protease was observed at 40 °C that wasn’t significant compared to 35°C. Effects of all seed proteinaceous extracts on protease activity were concentration dependent, so that the results showed that at the highest concentration, extracts of datura, bean, wild oat, broad bean, amaranth, mung bean, pea, canola, triticale and Sivand, Aflak and Alborz wheat cultivars inhibited the enzyme activity of the larvae with the percentage of 26, 25, 24, 40, 37, 33, 57, 53, 44, 43, 50 and 51, and the lowest concentration of seed extracts inhibited enzyme activity with the percentage of 11, 4, 6, 15, 8, 8, 16, 22, 11, 9, 13 and 16, respectively. Also the greatest inhibition of mung bean, pea, canola, broad bean, wild oat, Alborz and Aflak seed extracts were observed at alkaline pH which is the optimum pH for the activity of this enzyme in the in vitro condition. Due to the alkaline pH of lepidopteran larval gut lumen and maximum activity of protease, which occurs at the alkaline pH, it can be expected that in the in vivo condition, the extracts are impressed protease activity. However, complementary studies will be needed to achieve that.
Conclusion: Our results showed that the proteinaceous seed extracts have biological activity against the tomato leaf miner and therefore it may contribute towards the development of new insect pest control strategies. Thus, further study of these inhibitors including their purification, characterization and structure elucidation as well as the feasibility of their expression in transgenic host plants will unveil more detailed feature of the inhibitor.

Keywords

Inhibition; Protease; Proteinaceous extracts; Tomato leaf miner

References

1- Ajamhassani M., Zibaee A., Sendi J.J., Askary H., and Farrar N. 2012. Proteolytic Activity in the Midgut of the Crimson Speckled Moth Utethesia pulchella L.(Lepidoptera: Arctiidae). Journal of Plant Protection Research, 52: 368-373.

2- Baker J.E. 1987. Purification of isoamylases from the rice weevil, Sitophilus orizae L. by HPLC and their interaction with partially purified amylase inhibitor from wheat. Insect Biochemistry, 17: 37- 44.

3- Bandani A.R. 2005. Effect of plant α-amylase inhibitors on sunn pest, Eurygaster integriceps Puton. α-amylase activity. Communications in Agricultural and Applied Biological Sciences, 70(4): 869-873.

4- Bandani A.R., Kazzazi M., and Mehrabadi M. 2009. Purification and characterization of midgut α‐amylases of Eurygaster integriceps. Entomological Science, 12: 25-32.

5- Borzoui E., Bandani A.R., and Goldansaz S.H. 2013. Effect of cereal seed proteinaceous extracts on α-amylase and proteinase activity of salivary glands of Carob moth, Ectomyelois ceratoniae (Lep.: pyralidae). Journal of Crop Protection, 285-296.

6- Bradford M. 1976. A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72: 248-254.

7- Chougule N.P., Doyle E., Fitches and Gatehouse J.A. 2008. Biochemical characterization of midgut digestive proteases from Mamestra brassicae (Lepidoptera: Noctuidae) and effect of soybean Kunitz inhibitor (SKTI) in feeding assay. Journal of Insect Physiology, 54: 563–572.

8- Dastranj M. 2012. Effect of plant inhibitors extracted from wheat, pea and bean on amylase and proteinase activity of Plutella xylostella and Pieris rapae. Msc. Thesis. University of Tehran. (in Persian)

9- Dastranj M., Bandani A.R., and Mehrabadi M. 2013. Age-specific digestion of Tenebrio molitor (Coleoptare: Tenebrionidae) and inhibition of proteolytic and amylolytic activity by plant proteinaceous seed extracts. Journal of Asia-Pacific Entomolog, 16: 309-315.

10- Desneux N., Wajnberg E., Wyskhuys K.A.G., Burgio G., Arpaia S., Narvaez-Vazquez C.A., Cabrera J.G., Ruescas D.C., Tabone E., Frandon J., Pizzol J., Poncet C., Cabello T., and Urbaneja A. 2010. Biological invasion of European tomato crops by Tuta absoluta: ecology, geographic expansion and prospects for biological control. Journal of Pest Science, 83: 197-215.

11- Elpidina E.N., Tsybina T.A., Dunaevsky Y.E., Belozersky M.A., Zhuzhikov D.P., and Oppert B. 2005. A chymotrypsin-like proteinase from the midgut of Tenebrio molitor larvae. Biochimie 87: 771–779.

12- Franco O.L., Rigden D.J., Melo F.R., and Grossi-de- Sa M.F. 2002. Plant α-amylase. Structure, function and potential for crop protection. European Journal of Biochemistry, 269: 397-412.

13- Garcia M.F., and Espul J.C. 1982. Bioecology of the tomato moth (Scrobipalpul absoluta) in Mendoza, Argentine Republic. Revista de Investi Investigation’s Agropecuarias, 17: 135-146.

14- Gatehouse A.M.R., and Gatehouse J.A. 1998. Identifying proteins with insecticidal activity: use of encoding genes to produce insect-resistant transgenic crops. Pesticide Science, 52: 165-175.

15- Gatehouse A.M.R., Norton E., Davison G.M., Babbe S.M., Newell C.A., and Gatehouse J.A. 1999. Digestive proteolytic activity in larvae of tomato moth, Lacanobia oleracea, effects of plant protease inhibitors in vitro and in vivo. Journal of Insect Physiology, 45: 545–558.

16- Chitgar M.G., Ghadamyari M., and Sharifi M. 2013. Identification and Characterisation of Gut Proteases in the Fig Tree Skeletoniser Moth, Choreutis nemorana Hubner (Lepidoptera: Choreutidae). journal of Plant Protection Science, 49: 19-26.

17- Guedes R.N.C., and Picanço M.C. 2012. The tomato borer Tuta absoluta in South America: pest status, management and insecticide resistance. EPPO Bull, 42: 211-216.

18- Heidarvash S. 2013. Study of amylase and proteinase enzymes of tomato leaf miner (Tuta absoluta). Msc. Thesis. University of Tehran. (in Persian)

19- Hilder V.A., Gatehouse A.M.R., Sheerman S.E., Baker R.F., and Boulter D. 1987. A novel mechanism of insect resistance engineered into tobacco. Nature, 300: 160‒163.

20- Isman M.B. 2006. Botanical insecticides, deterrents, and repellents in modern agriculture and an increasingly regulated world. Annual Review Entomology, 51: 45-66.

21- Jamal F., Pandey P.K., Singh D., and Khan M. 2013. Serine protease inhibitors in plants: nature’s arsenal crafted for insect predators. Phytochemistry Reviews, 12: 1-34.

22- Kazzazi M., Bandani A.R., and Hosseinkhani S. 2005. Biochemical characterization of α-amylase of the Sunn pest, Eurygaster integriceps. Entomolical Science, 8: 371-377.

23- Klowden M.J. 2007. Physiological system in insects. Elsevier, UK.

24- Mahdavi A., Ghadamyari M., Sajedi R.H., Sharifi M., and Kouchaki B. 2012. Identification and partial characterization of midgut proteases in the lesser mulberry pyralid, Glyphodes pyloalis. Journal of Insect Science, 1536-2442.

25- Mehrabadi M., Bandani A., Saadati F., and Mahmudvand M. 2011. α-Amylase activity of Stored Products Insects and Its Inhibition by Medicinal Plant Extracts. Journal of Agricultural Science & Technology, 13,1173-1182.

26- Melo F.R., Sales M.P., Silva L.S., Franco O.L., Bloch J.R.C., and Ary M.B. 1999. α-amylase inhibitors from cowpea seeds. Protein and Peptide Letter, 6: 387-392.

27- Picanco M., Leite G.L.D., Guedes R.N.C., and Silva E.A. 1998. Yield loss in trellised tomato affected by insecticidal sprays and plant spacing. Crop Protection, 17: 447-452.

28- Reyes M., Rocha K., Alarcon L., Siegwart M., and Sauphanor B. 2012. Metabolic mechanisms involved in the resistance of field populations of Tuta absoluta (Meyrick)(Lepidoptera: Gelechiidae) to spinosad. Pesticide Biochemistry and Physiology, 102: 45-50.

29- Saadati F., and Bandani A.R. 2011. Effects of serine protease inhibitors on growth and development and digestive serine proteinases of the sunn pest, Eurygaster integriceps. Journal of Insect Science, 2: 72.

30- Silva C.C., Jham G.N., Picanco M., and Leite G.L.D. 1998. Comparison of leaf chemical composition and attack patterns of Tuta absoluta in three tomato species. Agron. Lus, 46: 61–71.

31- Silva C.P., Terra W.R., Samuels R.I., Isejima E.M., Bifano T.D., and Almeida J.S. 2001. Induction of digestive α-amylase in larvae of Zebrotes subfasciatus (Coleoptera: Bruchidae) in response to ingestion of common bean α-amylase inhibitor. Journal of Insect physiology, 47: 1283‒1290.

32- Silva G.A., Picanço M.C., Bacci L., Crespo A.L.B., Rosado J.F., and Guedes R.N.C. 2011. Control failure likelihood and spatial dependence of insecticide resistance in the tomato pinworm, Tuta absoluta. Pest Management Science, 67: 913-920.

33- Tatli I., Bandani A.R., and Moslemi A. 2013. The elm leaf beetle α-amylase and its activity relationship with insect feeding. Archives of Phytopathology and Plant Protection, 46, 917-926.

34- Volpaciella M., Leoni C., Costanza A., De leo F., Gallerani R., and Ceci Lr. 2011. Cystatins, serpins and other families of protease inhibitors in plants. Current protein peptid science, 12: 386-395.