Diyala Journal of Agricultural Sciences (DJAS), 10(2): 12-19, 2018 Abdullah et al. 21 http://www.agriculmag.uodiyala.edu.iq/ IDENTIFICATION AND EFFECTS OF BIO-PRODUCT COMPOUNDS OF Cyperus rotundus ON THE BIOLOGICAL AND QUANTITY DNA IN Tribolium castaneum (Herbst.)(Tenebrionidae: Coleoptera) Suhaira Waleed Abdullah1,2 Mohhamad Hassan Sallow 1 Dilpak Peer Khder1 1Dept. Plant Protection, College of Agric., Sallahddin University, Iraq. 2Corresponding author: suhairwaleed@yahoo.com ABSTRACT The plant pesticides have recently got great importance than the synthetic pesticides, against stored product pests because of their lesser hazards to the environment and other related biomass. The bio-product compounds were isolated after preparation of ethanolic extract from Cyperus rotundus rhizome by using soxhelt apparatus. The analysis of High-Performance Liquid Chromatography (HPLC) also showed identification of 2 compounds; Caffeine and 4-Hydroxy benzoic acid. The longest duration of both the larval stage and the pupal stage were 17 and 7 days, respectively at 2% F2. The mortality rate in larval stage reached 90% at 2% F3. The identification of Tribolium castaneum was carried out via designing specific primers with the use of PCR technique, the presence of more than 100 bp band specific for amplification of beta-tubulin intron gene of T. castaneum found in the negative control were absence in the positive controls helps to detect the effect of bio-product compounds at 2% of fractions on quantity of DNA in this insect. Key words: Tribolium castaneum, Cyperus rotundus, PCR DNA. INTRODUCTION The damage caused by red flour beetle, Tribolium castaneum (Herbst.) (Coleoptera:Tenebrionidae) to various stored and food commodities like grain, flour and dried fruits is recorded to be 15-20% which is capable of measuring losses worth millions of Dolars every year in a developing country observed among damage proteins and fats of wheat, whereas, negative correlation was found in carbohydrate (Wakil et al., 2003). Control of these pests are prime important in order to meet the demands of increasing population. The outbreaks of these pests could be avoided either by protect in and/or by treatment of the stored commodities with chemicals. Protection includes all the prophylactic measures and disinfestation of stores, bins, bags and grains by using benzene hexachloride (BHC), baythion, diazinon, gardonaa and malathion etc. Perveen et al., (2013) reported these chemicals applying before the grains being stored in order to eliminate chances of future infestation of the pests. Treatment of grains, on the other hand, has to be carried out with fumigants when infestation of the Recievied: 5/12/2016 Accepted: 16/7/2017 http://www.agriculmag.uodiyala.edu.iq/ http://www.agriculmag.uodiyala.edu.iq/ mailto:suhairwaleed@yahoo.com Diyala Journal of Agricultural Sciences (DJAS), 10(2): 12-19, 2018 Abdullah et al. 21 http://www.agriculmag.uodiyala.edu.iq/ pests appears during the storage (Perveen and Shah, 2012). Fumigants such as phosphine and methyl bromide were used quick and effective tools for insect control in food commodities. Insect resistance, toxic residues and other factors on stored grain (Perveen and Shah, 2012; Riebeiro et al., 2003). Plant-derived substances, natural plant products and bio-insecticides have recently become of great interest owing to their versatile applications (Baris et al., 2006) for protection of agricultural commodities due to their low mammalian and vertebrate’s toxicity and low persistence, no undesirable effects on animals and human beings (Raja and Albert, 2006). Therefore, development of bio- insecticides has been focused as a viable pest control strategy in recent years (Hashim and Devi 2003; Meena and Prates, 2006). The development of environmentally friendly insecticides, having specificity to insects has captured worldwide attention of scientists (Ishaaya and Degheele, 1998). Plants provide potential alternatives to currently used insect control agents. Photochemical studies have shown that the major chemical components of this herb are essential oils, flavonoids, terpenoids, mono-and sesquiterpenes. The plant contains cyprotena, cypera-2, 4-diene, & cyperene substances. The main objectives of this study were to: 1. Provide useful manage stored product insects via using diagnosed bio- product compounds in the plant extracts of Cyperus rotundus. 2. Identify and effects of these compounds on the test insect DNA quantity via molecular based methods. MATERIALS AND METHODS Extraction of plant The study was carried out during 2015-2016 at Department of Plant Protection of Agriculture, College of Salahaddin University. The rhizome of medicinal herb Cyperus rotundus (200g) were rinsed and dried at 30∓1°C under shed in laboratory. Then were ground to fine powder using an electric grinder, extraction using a soxhelt apparatus for six hrs. and extracted with petroleum ether, ethanol and D.W. Fractionations were made according to (Perveen and Shah, 2012). Laboratory Insects rearing The rearing method was adopted according to (Naqavi and Perveen, 1991) with some modifications. Adults of red flour beetle, Tribolium castaneum (Herbst.) were collected from the culture in Plant Protection Department in College of Agriculture and reared under controlled temperature at 30 ∓ 1˚C, RH 60 ∓ 2 . Ten pairs of adults of T. castaneum were taken in plastic bottles (height: http://www.agriculmag.uodiyala.edu.iq/ http://www.agriculmag.uodiyala.edu.iq/ Diyala Journal of Agricultural Sciences (DJAS), 10(2): 12-19, 2018 Abdullah et al. 21 http://www.agriculmag.uodiyala.edu.iq/ 14 cm; dm: 8 cm) containing 300 g of wheat flour media (fine flour: wheat bran: Brewer’s yeast; 7: 2: 1) and were tied with muslin cloth placed on a stand in laboratory to protect them from pests. They had incubation period 4-6 days, 6-7 larval instars and completed their life cycle in 22-25 days. When pure culture (after 4-5 generations) and sufficient population of uniform age and size were are experiments. HPLC Analysis The HPLC analysis was carried out by using ODSC18 column, 25cm x4.6x5µm. The mobile phase was (acetone 80+20 water), flow rate 0.5 ml/min and (Wave Length 280nm UV light) (Harborne, 1973). Test insects The (ten g) of flour treated with different concentrations (0.5, 1.5 and 2%) of the fractions, then dried overnight. The adults 1-2 days old were used in the experiment. Three replicates each consisted of ten insects were placed inside glass container that include 25g of flour. Each treated replicates were replaced in incubator and covered by muslin cloths. The studied features were as following: 1- Duration of larval stage and % larval mortality. 3- The number of emerged adults. Preparation of controls The positive control of the T. castaneum was prepared by extracting DNA from pure insect samples. The negative control was the DNA extracted from, treatment insects. DNA extraction DNA was extracted from the pure cultures of insects and the flour with a slight modification of a very simple extraction method called the high salt extraction method (Aljanabi and Martinez, 1997). Forty milligrams of the sample were crushed using 400 µL of sterile salt homogenizing buffer containing Tris, NaCl, and EDTA. Forty microliters of SDS (20%) and 8 µL of Proteinase K (20 mg/mL) were added and the samples were incubated at 65ºC overnight when 300 µL of 6M NaCl were added. DNA was recovered using isopropanol at half the volume of the supernatant and centrifuging at maximum speed for 10 s. The DNA was washed using 70% ethanol. http://www.agriculmag.uodiyala.edu.iq/ http://www.agriculmag.uodiyala.edu.iq/ Diyala Journal of Agricultural Sciences (DJAS), 10(2): 12-19, 2018 Abdullah et al. 21 http://www.agriculmag.uodiyala.edu.iq/ Primers Insect nuclear primers were bought from the University of British Columbia, Vancouver, BC (http: //www.michaelsmith. ubc.ca/services/NAPS/Primer Sets/Primers_Oct2015.pdf) to identify insect specific primers. The primer combinations were tried as a screening process to randomly identify insect specific primers. There were 50 primers in the set. About 58 combinations were screened on all six insect species based on the details given about the primers and the genes they amplify. The initial conditions for the PCR for screening were maintained as: 94°C for 1 min, followed by 30 cycles of 1 min at 94°C, 1 min at 56°C, and 2 min at 72°C. Final extension was done at 72°C for 5 min after the 30 cycles. Once specific bands were obtained at low annealing temperature (56°C), the annealing temperature was then increased to 60°C to increase the stringency and to confirm the specificity of the bands. It was found that the primers designed to amplify the protein-coding gene, elongation factor 1-alpha, ATC TCC GGA TGG CAC GG (CT) GAC AA (EFS599); ACG TTC TTC ACG TTG AA (AG) CCA A (EFA923), were specific to T. castaneum. The amplified PCR product was purified with a QIAquick PCR purification kit (Qiagen ®). The assigned accession numbers was AY819656 (T. castaneum). Thermal cycler PCR was performed on a total volume of 25 µL using a Techne thermocycler (Techne, Burkhardtsdorf, Germany). One half microliters of DNA (20 ng/µL) were used with 19.5 µL of PCR mix containing 2.5 µL of 10X PCR buffer, 2 µL of DNTP’s (each at 10 mM), 0.2 µL of Taq polymerase 5 units/µL and 0.75 µL of magnesium chloride 50 mM. Two microliters each of forward and reverse primer (20 pmoles /25 µL) were added to make the final volume to 25 µL. The thermo cycler program used for the specific primers was 94°C for 1 min, followed by 30 cycles of 1 min at 94°C, 1 min at 60°C, and 2 min at 72°C. Final extension was done at 72°C for 5 min after the 30 cycles. The above program was used the insect specific primers was identified. RESULTS The analysis of High-Performance Liquid Chromatography (HPLC) showed identification of two active compounds caffeine, P-Hydroxy benzoic acid 60.45- 79.65%, respectively. Only the P-Hydroxy benzoic acid in F2 was recorded 70.34%, Table 1. http://www.agriculmag.uodiyala.edu.iq/ http://www.agriculmag.uodiyala.edu.iq/ Diyala Journal of Agricultural Sciences (DJAS), 10(2): 12-19, 2018 Abdullah et al. 21 http://www.agriculmag.uodiyala.edu.iq/ 0 50 100 150 200 250 300 350 DATA DATA -50 0 50 100 150 200 250 300 DATA DATA Fig. 1. Peak of Caffeine Fig. 2. Peak of P-Hydroxy benzoi acid Table 1. The compounds which diagnostic via HPLC. The Table 2 shows the significantly effects of the fraction F2 which isolated by (Column Chromatography) of the ethanolic extract of Cyperus rotundus rhizome on duration of larval stage which reached 16 day in treatment with concentration of 2%. The mortality rates were 25, 30 and 50% for 0.5, 1.5 and 2 percent concentrations. The duration of pupal stage with concentration as it was higher in the highest concentration 6, 7 and 7 days at 0.5, 1.5 and 2% concentration. The maximum concentration (2%) caused reduction in number of the adults 9, 8 and 5 at 0.5, 1.5 and 2%, respectively. Table 2. The Effect of F2 on the biological features of T. castaneum Concentration, % The period Larval /day Mortality % in larval stage The period pupal /day Number of the adult 0.5 12 c 25 c 6 c 9 b 1.5 13 b 30 b 7 b 8 b 2 16 a 50 a 7 b 5 c Control 13 b 10 d 13 a 50 a Within a column means followed by a same letter are not significantly different at 5% level. The Table 3 shows the significantly effects of the fraction F3 which isolated by (CC) of the ethanolic extract of Cyperus rotundus rhizome on duration larval stage which reached 14 days in treatment with concentration 2%. The mortality rates were 65, 70 and 90% at 0.5, 1.5 and 2% concentrations. The duration pupal stage varied with concentration as it was higher in the highest concentration 8, 9 Fractions Caffeine P-Hydroxy benzoic acid Rt % Rt % F2 2.33 70.34 F3 2.74 60.45 2.90 79.65 Standard compounds 2.74 2.23 http://www.agriculmag.uodiyala.edu.iq/ http://www.agriculmag.uodiyala.edu.iq/ Diyala Journal of Agricultural Sciences (DJAS), 10(2): 12-19, 2018 Abdullah et al. 21 http://www.agriculmag.uodiyala.edu.iq/ and 7 days at 0.5, 1.5 and 2% concentration. The maximum concentration (2%) caused reduction in the adult's population 2. Table 3. Effect of F3 on the biological features of T. castaneum Concentration, % The period Larval stage/day Mortality % in larval stage The period pupal stage /day Number of the adult 0.5 15 a 65 c 8 b 3 c 1.5 14a 70 b 9 b 4 b 2 14a 90 a 7 c 2 d Control 13 b 10 d 13 a 50 a Within a column means followed by a same letter are not significantly different at 5% level. Fig.1. Gel plate PCR product of pure red flour beetle DNA using elongation factor 1- Alpha primers (EFS599; EFA923) Fig. 2. Gel plate PCR product of (a) positive control, Negative controls of Treatment insect withF2 at 2% (b) Treatment insect withF3 at 2% (c) of T. castaneum. DISCUSSION The bio-products are more significant for controlling the stored grains and agricultural pests due to their lesser harmful effects compared to chemical pesticides. The high salt extraction method (Aljanabi and Martinez, 1997) was adopted to extract DNA from the pure insect DNA extraction using the promega http://www.agriculmag.uodiyala.edu.iq/ http://www.agriculmag.uodiyala.edu.iq/ Diyala Journal of Agricultural Sciences (DJAS), 10(2): 12-19, 2018 Abdullah et al. 21 http://www.agriculmag.uodiyala.edu.iq/ wizard genomic DNA extraction kit which extracted DNA from a single beetle that was successfully amplified by the insect-specific primer. The size of the band was Approximately 100 bp and the repeatability was 100% (Fig.2). The DNA extracted from the insects treated with fractions was amplified by PCR. From Fig (2) the negative controls, insects treated with F2 (b) and F3(c) had bands of sizes varying from 110-3000 bp but the positive control of the pure insect (a) DNA alone had a PCR product at 100 bp. Thus the presences of these bands in the negative control were absence in the positive control helps to detect the effect of bio-product compounds at 2% of fractions. REFERENCES Aljanabi, S.M and I. Martinez. 1997. Universal and rapid salt extraction of high quality genomic DNA for PCR-based techniques. Nucleic Acids Research 25: 4692–4693. Baris, O. M. Gulluce. F. Sahin. H. Ozer. H. Kilic. H. Ozkan. M. Sokmen and T. Ozbek. 2006. Biological activities of the essential oil and methanol extract of Achillea biebersteinii Afan (Asteraceae). Turk. J. of Biol. 30: 65-73. Hashim, M. S. and K. S. Devi. 2003. Insecticidal actions of the polyphenolic fractions from the stem bark of Streblus asper on Dysdercus cingulalus, Fitoterapia, 74(7-8): 670-676. Harborne, J. B. 1973. Phytochemical Methods Aguide to modern technique of plant analysis." 1st ed., Cox & Wyman. London. 52-73. Ishaaya, I. and D. Degheele. 1998. Insecticides with Novel Modes of Action: Mechanisms and Application. Narosa Publishing House. New Delhi. India, 1-289. Meena, R. P. Suhag and H.T. Prates. 2006. Evaluation of ethanolic extract of Baccharis genistelloides against stored grain pests. J. Stor. Prod. Res. 34(4): 243-249. Naqvi, S. N. H and F. Perveen. 1991. Toxicity and residual effect of Nerium indicum crude extract as compared with coopex against adults of Tribolium castaneum (Coleoptera: Tenebrionidae). Pak. J. Entomol. Kar. 6(1-2): 35-44. Naqvi, S. N. H. and F. Perveen. 1993. Toxicity of some plant extracts in comparison to coopex (Bioallethrin: Permethrin) against stored grain pest (Callosobrucus analis) (Coleoptera : Bruchidae). Pak. J. Entomol. Kar., 8(1): 5–15. http://www.agriculmag.uodiyala.edu.iq/ http://www.agriculmag.uodiyala.edu.iq/ Diyala Journal of Agricultural Sciences (DJAS), 10(2): 12-19, 2018 Abdullah et al. 21 http://www.agriculmag.uodiyala.edu.iq/ Perveen, F., S. Zaib, S. Irshad and M. Hassan. 2012. Antioxidant and DNA protection activities of the hill toon, Cedrela serrata (Royle) leaves and its fractions. J. Nat. Prod., 5: 207-213. Perveen, F. and M. Shah. 2013. Reduction in fertility by Nerium indicum leaves extract in adults of red flour beetle. Tribolium castaneum (Coleoptera: Tenebrionidae) compared with Coopex (Bioallethrin:Permethrin). J. Agri. Sc. Tech. USA, 155-160. Riebeiro, B. M., R. N. C. Guedes, E. E. Oliveira and J. P. Santos. 2003. Insecticide resistance and synergism in Brasilian populations of Sitophilus zeamais (Coleoptera: Curculionidae). J. Stor. Prod. Res. 39: 21-31. Raja, N. S. Albert. S. Ignacimuthu and S. Dorn. 2001. Effects of plant volatile oils in stored cowpea Vigna unguiculata L. (Walpers) against Callosobruchus maculatus (F.) (Coleoptera: Bruchidae) infestation. J. Stor. Prod. Res. 37: 127-132. Shaaya, E. and M. Kostyukovsk. 2006. Essential oils: Potency against stored product insects and mode of action. Stewart Postharvest Review 2(4):34-40. Wakil, W. H., M. Javed and S. Anwar. 2003. Comparison of nutritiona losses of insects infested wheat in laboratory and public storages. Pak. J. Arid Agri. 6: 1-6. لخنفساء DNA في حياتية وكمية Cyperus rotundusد ع الس الفعالة لنباتتشخيص المركبات Tribolium castaneum(Herbst.) (Tenebrionidae: Coleoptera) الطحين الحمراء 2خضر دلباك بير 2حمد حسن سلوم 1،2سهيره وليد عبدهللا ، العراق. جامعة صالح الدين -كلية الزراعة -سم وقاية النباتق2 suhairwaleed@yahoo.comالمسؤول عن النشر: المستخلص مبيدات اآلفات ذات األصل النباتي اهتماما كبيرا مقارنة بالمبيدات الصناعية ضد آفات الحبوب لقيت في HPLCالتي شخصت بتقانة الفعالةالمخزونة نظرا ألنها ال تلحق أضرارا كبيرة بالبيئة. المركبات caffeine 4.HydroxyهيCyperus rotundus الكحولي لرايزومات نبات السعد المستخلص benzoic acid. تم اختبار تأثير األجزاء المفصولة في حياتية خنفساء الطحين الحمراءTribolium castaneum حيث أظهر الجزءF2 7و 17تأثير في كال فترتي الدورين اليرقي والعذري حيث بلغ ذري حيث تأثيرا في الدور الع 2%بتركيز F3. أظهر الجزء 2%المعاملة بالتركيز التوالي عند على في bp 100 أكبرمن وظهور حزمة PCRتم تشخيص الحشرة باستخدام تقنية 95% . بلغت نسبة الموت فيها. DNAالمنتجات الطبيعية على كمية لتأثير، كمؤشر السلبيةالمجموعة الضابطة .Cyperus rotundusخنفساء الطحين الحمراء، نبات السعد :حيةالكلمات المفتا http://www.agriculmag.uodiyala.edu.iq/ http://www.agriculmag.uodiyala.edu.iq/ mailto:suhairwaleed@yahoo.com