Testing the effect of amomum subulatum extract on the larvae of pieris rapae (khóa luận tốt nghiệp)

VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTEACHNOLOGY GRADUATE THESIS TESTING THE EFFECT OF AMOMUM SUBULATUM EXTRACT ON THE LARVAE OF PIERIS RAPAE Practicing Student : Nguyen Dinh Thinh Class : K61-CNSHE Tutor : Dr Dang Thi Thanh Tam HA NOI - 2021 COMMITMENT I hereby declare that this thesis is composed of my original works and contains no material previously published or written by another person I commit that the references in the thesis are citied and all the help are thanked Hanoi, January 28th 2021 Student Nguyen Đinh Thinh i ACKNOWLEDGEMENTS First and foremost, I have to express my appreciation and big thanks to the the Director of Vietnam National University of Agriculture , Faculty board and teachers in the Department of Plants Biotechology for creating an interesting learning environment and providing me priceless knowledge through five academic years During the time doing this thesis, many people helped me in bringing this work to existence, especially my supervisor Dr Dang Thi Thanh Tam, who supported and inspired me to accomplish this thesis.Without her precious support it would not possible tocomplete this thesis successfully I would like thank my partner Trinh Thi Huong Tra and all my friends , who were by my side to help and give me all best conditions Last but not least, I am also grateful to thank my parents for supporting and encouraging me spiritually throughout my thesis and my life in general Hanoi, January 28th 2021 Student Nguyen Đinh Thinh ii CONTENTS COMMITMENT i ACKNOWLEDGEMENTS ii CONTENTS iii LIST OF TABLES v LIST OF FIGURE vi PART I INTRODUCTION 1.1 Introduction 1.2 Aims 1.3 Objectives PART II LITERATURE REVIEW 2.1 Overview of Pieris rapae 2.1.1 Scientific Classification 2.1.2 Life Cycle of Pieris rapae 2.1.3 The damage of Pieris rapae 2.1.4 Some research on Pieris rapae 2.2 Overview of Amomum subulatum plant 11 2.2.1 Scientific Classification 11 2.2.2 Morphological characteristics 12 2.2.3 Distribution and Habitat 13 2.2.4 Benefits of Amomum subulatum plant 14 PART III MATERIALS AND METHODS 16 3.1 Location and time of the study 17 3.1.1 Location 17 3.1.2 Time of the the study 17 3.2 Materials 17 3.2.1 Research materials 17 3.2.2 Instruments, chemicals and analytical equipment 17 iii 3.3 Methods 18 3.3.1 Growing cabbage 18 3.3.2 Pieris rapae culture 19 3.3.3 Amomum subulatum extract preparation 21 3.3.4 Experiments 23 PART IV RESULTS AND DISCUSSIONS 27 4.1 Testing the toxicity of Amomum subulatum extract on Pieris rapae larvae 27 4.2 Testing the antifeeding effect of Amomum subulatum extract on P rapae larvae 30 PART V CONCLUSIONS AND SUGGESTION 33 REFERENCE 34 APPENDICES 36 iv LIST OF TABLES Table 3.1 Diluting a starting stock solution to create solutions at various concentrations 21 Table 4.1 Mortality rate of larvae in the treatment with Amomum subulatum 27 Table 4.2 Leaf consumption rate of larvae in the treatment with Amomum subulatum 28 Table 4.3 Deterrency percentage of leaf consumption value of larvae after 24 hours 30 v LIST OF FIGURE Figure 2.1 Life cycle of Pieris Rapae Figure 2.2 Young caterpillars of Pieris rapae were causing damage on cabbage plants Figure 2.3 Morphological characteristics of Amomum subulatum 12 Figure 2.4 Amomum subulatum plants are grown in Vietnam (Ha Giang) 13 Figure 3.1 The process of growing cabbage 18 Figure 3.2 The process of Pieris rapae culture 20 Figure 3.3 The process of reparing for Amomum subulatum extract 22 Figure 3.4 The process of testing the toxicity of Amomum subulatum extract 24 Figure 3.5 The process of testing the antifeeding effect of Amomum subulatum extract on P rapae larvae 26 Figure 4.1 Mortality rate of larvae in the treatment with Amomum subulatum after 48 hours 28 Figure 4.2 Experiment at initial time and after 48 hours 29 Figure 4.3 The consumption area between control and treatment sample at 0.5 % concentration of A.subulatum extract 26 Figure 4.4 Control and treatment sample at initial time and after 24 hours at 0.5 % concentration of A.subulatum extract 32 vi PART I INTRODUCTION 1.1 Introduction In the past 30 years , scientists have been intensively investigated about plant-derived extracts and phytochemicals with the main purpose was an effort to develop alternatives to conventional insecticides with reduced environmental impacts and protected people's heath Synthetic insecticides can leave potentially toxic residues in food products and be deleterious to non-target organisms Every year, the crop losses due to pests may reach 10–20% In Vietnam, the overuse of some chemical classes of insecticides has led to environmental and human health problems.Synthetic chemical insecticides have many harm that endangering human health and the environment such as effects on the health of applicators, farmers, and even consumers; it accidentally kills wildlife ,beneficial insects, including pollinators and espiecial pollutes underground water From the drawbacks of chemical insecticides and their discontinued manufacture have spurred scientist study and search for biological pesticides with the ingredient that are found in products of natural origin The botanicalbased pesticides often good for user, no harmful with other organisms Plantbased bio-pesticides are generally thought to be more selective, be less harmful to nontarget organisms, degrade quickly, and be less phytotoxic Therefore, botanical insecticides may be effective for managing the small white butterfly, Pieris rapae which is an important pest that affects cruciferous crops worldwide A single female lays 300 eggs on average, but clutches may include close to 1000 eggs Each young larvae occupies an outer leaf of a cabbage for feeding Older larvae move onto inner cabbage leaves and into cabbage heads by eating outer leaves of the cabbage head and gnawing into it Plants soiled with excrement rot easily In the world, some scientists have the study using plant extracts to protect some of vegetables from larvae of Pieris rapae such as R XU et al.( 2008) performed a study about the control effects of pumpkin leaf extract on caterpillars of the small cabbage butterfly and the result showed feeding restrain, at concentration 1.4 g/l (0.98± 0.008) , 0.7 g/l (0.61± 0.026), 0.35 g/l (0.41± 0.027) and 0.18 g/l (0.36± 0.022) In the presence of pumpkin extract, feeding of larvae will decrease and the impact on crops will be limited Beside , Hasheminia et al.(2013) conducted a study about effect of Milk Thistle, Silybium marianum extract on the small white butterfly with toxicity tests and the result showed that LC50 and LC25 values were 2.94% and 1.20% With using plant extract helped to reduce number of larvae of Pieris rapae that damaged the plant In Vietnam, farmers used many ways to control larvae of Pieris rapae With manual ways, they used racket to catch butterfly and young caterpillar beside they grown some types of plant that can be ability repel the butterfly.But the most popular way ,they used chemical pesticides to kill young caterpillar that would bring the active effects and effect quickly and in a timely manner It is this way of control P.rapae larvae of Vietnamese farmers, leading to harm to human health, environmental pollution, and ecological impacts and with the benefits pesticides of biological origin will bring us and the environment, I think we should regularly use them From the above bases, I always have the desire to find new directions and especially it can be applied in Vietnam with the following criteria easy to implement, cheap price, abundant raw materials and high efficiency I have learned and knowed that Amomum subulatum plants were used for many different purposes, especially research on insecticide, antibacterial (Satyal et al., 2012), antifungal and antioxidant activities ( Kapoor et al., 2008) Besides Amomum subulatum plants are quite popular in Vietnam, the price is quite cheap, easy to buy and convenient to use As a consequence, I want to find the plant extracts that have highly effective insecticide on Pieris rapae I decide to choose the subject: “Testing the effect of Amomum subulatum extract on the control of larvae of Pieris rapae In this research, the toxicity and antifeeding effects of Amomum subulatum extract on Pieris rapae were tested 1.2 Aims Testing the effect of Amomum subulatum extract on the control of larvae of Pieris rapae 1.3 Objectives Testing the toxicity of Amomum subulatum extract on young caterpillars Testing the antifeeding effect of Amomum subulatum extract on young caterpillars Preparing 3rd instar larvae and hours of starvation Diluting A.subulatum extract at different concentrations Following up and recording results at the time mark Spraying A.subulatum extract over the entire caterpillars and leaves Figure 3.4 The process of testing the toxicity of Amomum subulatum extract 24 Experiment 2: Testing the antifeeding effect of Amomum subulatum extract on P rapae larvae Preparing two cabbage leaf (1 control and treatment) We sprayed 0.5 % concentration of A.subulatum extract over the entire surface of the cabbage leaf and then dried at room temperature for 30 seconds All larvae starved before taking experiment After hours of starvation, 3rd instar larvae were placed onto control leaf and treated leaf placed in plastic box (15 ×7× cm) After 24 hours, leaf consumption was recorded and used software to analyzed The index of feeding deterrence was calculated as (C - T)/(C + T) × 100 where C is the consumption of the control leaf and T is the consumption of the treated leaf Each experiment was repeated three times with 10 larvae each time Step Preparing starvated instar larvae for 4hours and two cabbage leaves Step Weighing the weight of caterpillars and the weight of leaves rd 25 Step Preparing a plant extract and a control methanol sample Step Spraying the plant extract over the entire surface of the leaf and drying at room temperature Step Placing larvae onto control leaf , treatment leaf and following up Figure 3.5 The process of testing the antifeeding efffect of Amomum subulatum extract on P rapae larvae 26 PART IV RESULTS AND DISCUSSIONS 4.1 Testing the toxicity of Amomum subulatum extract on Pieris rapae larvae Testing toxicity of Amomum subulaum extract were conducted on thirty 3rd instar larvae of Pieris rapae and five different concentration After 24 hours and 48 hours, the obtainable results are presented in table 4.1,table 4.2 and figure 4.1 Table 4.1.Mortality rate of larvae in the treatment with Amomum subulatum Mortality rate after 24 hours Concentration of extract (%) No of death larvae/total Mortality rate after 48hours (%) No of death larvae/total 0.5% 0/30 13.33 4/30 1% 0/30 6.66 2/30 2% 3.33 1/30 10 3/30 4% 13.33 4/30 30 9/30 8% 10 3/30 40 12/30 27 Figure 4.1 Mortality rate of larvae in the treatment with Amomum subulatum after 48 hours Table 4.2 Leaf consumption rate of larvae in the treatment with Amomum subulatum Concentration of extract 0.5% 1% 2% 4% 8% 79.28% 74.54% 68.24% 17.87% 14.8% Leaf consumption rate after 24 hours After spraying A.subulatum extract on larvae, at all of concentrations larvae quickly crawled out of leaf position After hours, at all of concentration larvae had the phenomenon of leaf-eating At 4% and 8% concentration began to appear larvae were dead After 24 hours, the result in table 4.2 showed that leaf consumption rate of larvae reduced from 79.28% at 0.5% concentration to 14.8% at 8% concentration The concentration of A.subulatum extract was inversely proportional to the leaf consumption of larvae, at higher concentrations so leaf consumption of larvae was smaller After 48 hours, the result in table 4.1 and figure 4.1 showed that the concentration of A.subulatum 28 extract was proportional to the mortality of the larvae When the concentration of the extract increased from 1% to 8%, the mortality increased from 6.66% to 40% But at 0.5 % concentration had a different point, mortality was 13.33 % and larger than at 1% concentration (6.66%) It could be depend on the size, strength of larvae As announced Hasheminia et al.(2013), LC50 and LC25 values for S marianum extract were 2.94% and 1.20% Beside , Hasheminia et al.(2011), LC50 and LC25 values of Artemisia annua L extract were 9.387% and 4.19% and LC50 and LC25 values of Achillea millefolium L extract were 3.645% and 1.69% The toxicity of A.subulatum extract was lower than other extract Figure 4.2 Experiment at initial time and after 48 hours ( treatment with % concentration of A.subulatum extract) 29 4.2 Testing the the antifeeding effect of Amomum subulatum extract on P rapae larvae Testing the antifeeding effect of A.subulatum on P rapae lavae at 0.5% concentration Twenty-3rd instar P.rapae larvae were used for each treatment There were two control sample and two treatment This experiment was followed after 24 hours The obtained results are presented in table 4.3 and figure 4.3 Table 4.3 Deterrency percentage of leaf consumption value of larvae after 24 hours Formula Mean Control 31.88 Treatment 13.08 Difference between means (B - A) ± SEM df -18.81 ± 2.772 Leaf arae (cm²) 40 30 20 10 C on en t Tr ea tm tr ol Figure 4.3 The comsumption area between control and treatment sample at 0.5 % concentration of A.subulatum extract 30 Results showed that at 0.5% concentrations had large difference between control sample and treatment sample on larvae's leaf consumption The area of leaf consumption of the control samples was 31.88 while the area of leaf consumption of the treatment was 13.08 The effect of toxicity of A.subulatum extract reduced efficiency of the conversion of larvae's ingested food so that in treatment sample larvae would eat less than control sample When the larvae continued to eat the treated cabbage leaves, the larvae appeared deformed, weak vitality, the rate of larvae completing their life cycle did not exceed 30% As announced by R XU et al.( 2008), pumpkin leaf extract showed result that feeding restrain at the lowest concentration(0.18 g/l ) was 0.36 ± 0.022 Beside, that 0.5% concentration of A.subulatum extract showed results that the index of feeding deterrence were 45.9 % and 38.4 % after repeated times Both two extract had effect to reduce efficiency of the conversion of Pieris rapae larvae's ingested food but the effect of toxicity of A.subulatum extract was strongger than the effect of toxicity of pumpkin leaf extract Addition, as announced Hasheminia et al.(2011), the result showed that at 0.625% concentration, the antifeeding effect of Achillea millefolium L extract on P.rapae larvae was 44.185% and Artemisia annua L extract was 29.826% Both two extracts had the low effective with the antifeeding of larvae as well as pumpkin leaf extract 31 Control sample hour Treatment sample 24 hours hour 24 hours Figure 4.4 Control and treatment sample at initial time and after 24 hours at 0.5 % concentration of A.subulatum extract 32 PART V CONCLUSIONS AND SUGGESTION Conclusions - Amomum subulatum extract had toxicity effect on larvae of Pieris rapae, at 8% concentration of Amomum subulatum extract had mortality was 40% after 48 hours - At 0.5% concentration of Amomum subulatum extract, the index of feeding deterrence of P.rapae larvae were 45.9 % and 38.4 % after 24 hours with repeated times Suggestion Due to the limited time and research conditions,we would like to give some suggestions for future direction: -Testing the antifeeding effect of Amomum subulatum extract on P rapae larvae with higher concentrations -.Testing the oviposition effect of A.subulatum extract to P Rapae - Testing the self-defense respone to A.subulatum extract in cabbage plants - Combing A.subulatum extract with some binders to make the extract exist longer in the environment 33 REFERENCE Elshaier, Manal (2018) Effect of some plant extracts on the larvae of Pieris rapae L (Lepidoptera: Pieridae) Biohouse Journal of Research in Science Vol Hasheminia S.M., Sendi J.J., Jahromi K.T and Moharramipour S (2013) Effect of milk thistle, Silybium marianum, extract on toxicity, development, nutrition, and enzyme activities of the small white butterfly, Pieris rapae J Insect Sci Vol 13 Pp 146 XU, R & YANG, S & WU, D & KUANFG, R.-P (2008) Control effects and economic evaluation of pumpkin leaf extract on caterpillars of the small cabbage butterfly The Journal of Agricultural Science Vol 146 Pp 583 589 Satyal P., Dosoky N.S., Kincer B.L and Setzer W.N (2012) Chemical compositions and biological activities of Amomum subulatum essential oils from Nepal Nat Prod Commun Vol Pp 1233-1236 Kapoor, I.P.S & Singh, Bragy & Singh, Gurdip & Isidorov, Valery & Szczepaniak, Lech (2008) Chemistry, antifungal and antioxidant activities of cardamom (Amomum subulatum) essential oil and oleoresins International Journal of Essential Oil Therapeutics Vol Pp 29-40 DUBEY, R.K & KUMAR, RAJESH & CHANSOURIA, J & DUBEY, N.K (2008) Evaluation of Amomum subulatum Roxb oil as a source of botanical fungitoxicant for the protection of mango fruits from fungal rotting Journal of Food Safety - J FOOD SAFETY Vol 28 Pp 400-412 Shen J., Cong Q., Kinch L.N., Borek D., Otwinowski Z and Grishin N.V (2016) Complete genome of Pieris rapae, a resilient alien, a cabbage pest, and a source of anti-cancer proteins F1000Res Vol pp 2631 Seyyedeh Masoumeh Hasheminia., Jalal Jalali Sendi., Khalil Talebi Jahromi., Saeid Moharramipour (2011) The effects of Artemisia annua L and Achillea millefolium L crude leaf extracts on the toxicity, development, feeding 34 efficiency and chemical activities of small cabbage Pieris rapae L (Lepidoptera: Pieridae) Pesticide Biochemistry and Physiology.Vol 99 Pp 244-249 V K Bisht, J S Negi, A K Bhandari and R C Sundriyal (2011) Amomum subulatum Roxb: Traditional, phytochemical and biological activitiesAn overview Herbal Research and Development Institute, Mandal, Gopeshwar – 246 401, District – Chamoli, Uttarakhand, India African Journal of Agricultural Research Vol 6(24), Pp 5386-5390 10 T L LADD, JR., J D WARTHEN, JR., ANDM G KLEIN (1984) Japanese Beetle (Coleoptera: Scarabaeidae): The Effects of Azadirachtin on the Growth and Development of the Immature Forms Agricultural Research Service, U.S Department of Agriculture, Ohio Agricultural Research and Development Center, Wooster, Ohio 44691 J Econ Entomo Vol 77, Pp 903905 11 Y S RA0, ANAND KUMAR, SUJATHA CHATTERJEE, R NAIDU & C K GEORGE (1993) Large cardamom (Amomum subulatum Roxb.)-a review Regional Research Station, Cardamom Research Institute Spices Board, Dzongu House, Bhalvakhani Gangtok - 737101, Sikhim, India Journal of Spices & Aromatic Crops Vol &2, Pp 1-15 12 Harcourt DG (1962) Design of a sampling plan for studies on the population dynamics of the imported cabbageworm, Pieris rapae (L.) (Lepidoptera: Pieridae) University of Florida Canadian Entomologist Vol 94, Pp 849-859 35 APPENDICES Appendix: Statistical analysis Experiment 1: Testing the toxicity of Amomum subulatum extract on Pieris rapae larvae PARAMETER ESTIMATES 95% Confidence Interval Probit Parameter Estimate Std.error Z Sig Lower bound Upper bound Concentration 941 291 3.236 001 371 1.512 Intercept -1.191 479 1.445 148 231 1.171 A Probit model: probit(p) = intercept + bx (covariates x are transformed using the base 10.000 logarithm.) CHI-SQUARE TESTS Probit Pearson Goodness-Of-Fit Test Chi-Square DFB SIG 4.240 237A A Since the significance level is greater than 050, no heterogeneity factor is used in the calculation of confidence limits B Statistics based on individual cases differ from statistics based on aggregated cases 36 CELL COUNTS AND RESIDUALS Number Concentration Number of Observed Expected Residual Probability responses responses subjects Probit -0.301 30 2.106 1.894 0.07 30 3.505 -1.505 0.117 0.301 30 5.461 -2.461 0.182 0.602 30 7.987 1.013 0.266 0.903 30 12 10.999 1.001 0.367 37 Experiment 2: Testing the antifeeding effect of Amomum subulatum extract on larvae of P rapae At 0.5% concentration of A.subulatum extract Table analyzed Column b Vs Column a Treatment Vs Control Unpaired t test P value P value summary Significantly different (p < 0.05)? One- or two-tailed p value? T, df 0.0210 * Yes Two-tailed T=6.785, df=2 How big is the difference? Mean of column a Mean of column b Difference between means (b - a) ± sem 95% confidence interval R squared (eta squared) 31.88 13.08 -18.81 ± 2.772 -30.73 to -6.880 0.9584 F test to compare variances F, dfn, dfd P value P value summary Significantly different (p < 0.05)? Data analyzed Sample size, column a Sample size, column b 2 38