@article { author = {University of Tehran, M. and Bandani, A. R. and Sabahi, Gh.}, title = {Effect of Seed Proteinaceous Inhibitors on Digestive Protease Activity of Tomato Leaf Miner Tuta absoluta (Meyrick) (Lep.: Gelechiidae)}, journal = {Journal of Iranian Plant Protection Research}, volume = {30}, number = {3}, pages = {449-459}, year = {2016}, publisher = {Ferdowsi University of Mashhad, press.}, issn = {2980-8170}, eissn = {2783-5383}, doi = {10.22067/jpp.v30i3.45016}, 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}, title_fa = {تأثیر مهارکننده های پروتئینی بذر گیاهی بر فعالیت پروتئاز گوارشی شب پره مینوز گوجه فرنگی Tuta absoluta Meyrick (Lep.: Gelechiidae)}, abstract_fa = {مینوز گوجه فرنگی Tuta absoluta Meyrick (Lep.: Gelechiidae) از مهم ترین آفات این محصول در جهان محسوب می شود که در سال های اخیر به کشور وارد شده است. هدف این مطالعه تعیین اثر عصاره های پروتئینی استخراجی از دانه های 12 گیاه بر فعالیت پروتئاز گوارشی این حشره است. در این مطالعه اسیدیته بهینه فعالیت پروتئازی و تأثیر اسیدیته بر فعالیت مهارکنندگی عصاره ها نیز تعیین گردید. این بررسی نشان داد که ترکیبات استخراج شده از دانه گیاهان نخود، کلزا و دو رقم گندم (البرز و افلاک) به ترتیب با 57، 53، 51 و 50% مهارکنندگی اثر چشمگیری بر فعالیت پروتئاز لارو مینوز گوجه فرنگی دارند. همچنین مهارکننده های استخراج شده از تریتیکاله، رقم سیوند گندم و باقلا به ترتیب با 44، 43 و 40% مهارکنندگی نیز توانایی خوبی در مهار پروتئاز مینوز گوجه فرنگی نشان دادند. اما مهارکننده های داتوره، لوبیا و یولاف وحشی به ترتیب با 26، 25 و 24% مهارکنندگی نسبت به سایر مهارکننده ها تأثیر کمتری روی پروتئاز لاروی نشان دادند. این مطالعه همچنین نشان داد که بیشینه فعالیت پروتئازی در آفت مذکور در اسیدیته 10 و دمای 40 درجه سلسیوس صورت می گیرد. از طرفی بیشترین مهارکنندگی حاصله از دانه های نخود، کلزا و دو رقم گندم البرز و افلاک در اسیدیته های قلیایی انجام شد. با توجه به قلیایی بودن اسیدیته معده آفت، و اینکه بیشترین فعالیت آنزیم نیز در اسیدیته قلیایی است می توان نتیجه گرفت که این مهارکننده ها قادرند در شرایط طبیعی (In vivo) در مهار پروتئاز آفت مؤثر واقع شوند هر چند برای تحقق این امر انجام آزمایش های تکمیلی ضروری خواهد بود.}, keywords_fa = {پروتئاز، عصاره های پروتئینی,مهارکنندگی,مینوز گوجه فرنگی}, url = {https://jpp.um.ac.ir/article_36334.html}, eprint = {https://jpp.um.ac.ir/article_36334_fd7e6a94802d7ef98ffdee44494a6683.pdf} }