VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY - - GRADUATE THESIS TESTING THE EFFECT OF KAEMPFERIA GALANGA L ON THE LARVAE OF PIERIS RAPAE L HANOI-2021 VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY - - GRADUATE THESIS TESTING THE EFFECT OF KAEMPFERIA GALANGA L ON THE LARVAE OF PIERIS RAPAE L Student : Trinh Thi Huong Tra ID : 610681 Class : K61CNSHE Supervisor : Dr Dang Thi Thanh Tam HA NOI-2021 COMMITMENT I hereby declare that all information in this document has been obtained and presented in accordance with academic rules and ethical conduct I also declare that all the help to complete this thesis has been thanks Trinh Thi Huong Tra i ACKNOWLEDGEMENT This study would not have been possible without the support of many people I would like to express my gratitude to my professors of Department of Plant Biotechnology at Faculty of Biotechnology in Vietnam National University of Agriculture for their support and conducting the research Particular thanks to Dr Dang Thi Thanh Tam for her valuable comments and helpful advice, scientific , and valuable feedback throughout the time studying at the laboratory of the Department of Plant technology I would also like to extend my partner Nguyen Dinh Thinh for helping me to perform some experiments Finally, I would like to express my special thanks to my family for great encouragement as well as my friends for their supports Trinh Thi Huong Tra ii CONTENTS COMMITMENT i  ACKNOWLEDGEMENT ii  CONTENTS iii  LIST OF TABLES v  LIST OF FIGURES vi  PART I INTRODUCTION 1  1.1 Introduction 1  1.2 Purpose and requirement 2  1.2.1 Purpose 2  1.2.2 Requirement 2  1.3 Research location 2  PART II LITERATURE REVIEW 3  2.1 Pieris rapae 3  2.2 Some researchs on Pieris rapae 8  2.3 Kaempferia galanga L 10  PART III MATERIALS AND METHODS 13  3.1.Materials 13  3.1.1.Research materials 13  3.1.2 Instruments and chemicals 13  3.2 Methods 13  3.2.1 Growing cabbage 13  3.2.2 Pieris rapae culture 14  3.2.3 Preparation the plant extracts 15  3.2.4 Toxicity tests 16  3.2.5 Antifeeding test 18  3.3 Experiments 19  iii PART IV RESULTS AND DISCUSSIONS 21  PART V CONCLUSIONS AND SUGGESTIONS 27  5.1 Conclusions 27  5.2 Suggestions 27  REFERENCES 28  iv LIST OF TABLES Table 4.1 Mortality rate of larvae during Kaempferia galanga L treatment 21  Table 4.2 Dose–response parameters of Pieris rapae after 24 hours 23  Table 4.3 Deterrence percentage acreage leaves of Kaempferia galanga extract on Pieris rapae larvae 24  v LIST OF FIGURES Figure 2.1 Butterfly and larvae of Pieris rapae 3  Figure 2.2 Eggs of Pieris rapae 4  Figure 2.3 Five stages of Pieris rapae larvae 5  Figure 2.4 Pupae of Pieris rapae 6  Figure 2.5 Adult of Pieris rapae 7  Figure 2.6 Kaempferia galanga plants 10  Figure 3.1 Cabbage seeds 14  Figure 3.2 The process of growing cabbage 14  Figure 3.3 Pieris rapae culture 15  Figure 3.4 Preperation steps of the Kaempferia galanga L extract 16  Figure 3.5 Steps of toxicity tests 17  Figure 3.6 Steps of antifeeding test 19  Figure 4.1 Mortality rate of larvae after 24 hours of treatment 21  Figure 4.2 Testing toxicity of (4%) Kaempferia galanga extract on Pieris rapae larvae 23  Figure 4.3 The leaf consumption of larvae in the treatment with 0.5% Kaempferia galanga extract 24  Figure 4.4 The leaf consumption of larvae in the treatment with 1% Kaempferia galanga extract 25  Figure 4.5 The effect of Kaempferia galanga extract on the antifeeding of Pieris rapae larvae 26  vi PART I INTRODUCTION 1.1 Introduction In Vietnam, cruciferous vegetables (broccoli, cabbage, cauliflower, kale, collards ) are preferred by many people because of their rich nutrients They have high economic value and are widely grown Cruciferous vegetables are the main crops of many regions such as Sapa, Son La, Đa Lat, However, cruciferous vegetables have a short growth time, so they are planted continuously, harvested in scattered batches Besides, they have the features of stems, soft and weak leaves, containing many nutrients It is suitable with the warm and humid climate of our country, so it is damaged by many kinds of worms such as silkworm, Pieris rapae, click beetles , seriously affecting the productivity and quality of vegetables (Trinh, 1999) In the cruciferous vegetable pest group, Pieris rapae is the most dangerous pest for vegetables Every year they appear from 14-15 times, with high density (Lam, 2008) Pieris rapae is a small to medium-sized butterfly species of the family Pieridae It is believed to have originated in the Eastern Mediterranean and widespread in Europe and Asia The butterfly is recognizable by its white color with small black dots on its wings Young caterpillars gnaw the green matter of vegetable leaves From stage two or more, they gnaw leaf blades and remain only the veins Their waste can spoil the leaves So if the high density is left, the field of vegetables will be bare and frayed To control Pieris rapae, the farmers mainly use chemical methods In vegetable-growing areas, pesticides are used extensively and continuously Chemical control is highly effective but it contains negative risks to organisms, the ecosystem and human health These drawbacks associated have spurred the search for biological pesticides Because biological control is an environmentally friendly method for the environment In this field, plant extract is a solution commonly However, this method still exist some limitations It is more susceptible to environmental conditions, slow action and sometimes is unpredictable Because of the reasons above, the study was performed to find the plant extracts that have highly effective insecticide on Pieris rapae 1.2 Purpose and requirement 1.2.1 Purpose Testing the effect of Kaempferia galanga extract to control Pieris rapae 1.2.2 Requirement  Testing the toxicity of Kaempferia galanga extracts on young caterpillars  Testing the antifeeding effect of Kaempferia galanga extracts on young caterpillars 1.3 Research location The study was performed at Department of Plant Biotechnology ,Faculty of Biotechnology and Institute of Agro- Biology in Vietnam Nation University of Agriculture Fomula Extract concentration Control 0,5% methanol Treated 0,5% extract Control 1% methanol Treated 1% extract 20 PART IV RESULTS AND DISCUSSIONS Experiment Testing toxicity of the Kaempferia galanga extract on Pieris rapae larvae Testing toxicity of the Kaempferia galanga extract were performed on 30 Pieris rapae larvae at stage After 24 hours and 48 hours, the obtained results are presented in table 4.1 and Figure 4.1 Table 4.1 Mortality rate of larvae during Kaempferia galanga L treatment Formula Extract Mortality rate (%) concentration After h After 24 h After 48 h 0.5% (0/30) 10 (3/30) 26.67 (8/30) 1% (0/30) 16.67 (5/30) 33.33 (10/30) 2% 10 (3/30) 23.33 (7/30) 43.33 (13/30) 4% 46.67 (14/30) 60 (18/30) 60 (18/30) 8% 90 (27/30) 100 (30/30) 100 (30/30) 120 y = ‐0.3541x2 + 15.348x + 0.459 R² = 0.9893 Motality rate % 100 80 60 40 20 0 10 Concentration % Figure 4.1 Mortality rate of larvae after 24 hours of treatment 21 After the treatment with Kaempferia galanga extract, at concentrations 0.5%, 1%, 2% and 4%, the larvae crawled out of the leaf Moreover, at 8% of the extract, the alive larvae did not move In the first hours, the control sample and the concentration of 0.5%, 1%, the larvae started eating leaves However, from 2% - 4% of extract, no larvae ate the leaves At 8% concentration the larvae were dead in half After 24 hours, the results showed that the mortality rates increased when the extract's concentration condensed The LC50 values, confidence limit (95%) and regression slope at 24h showed in table 4.2 The LC50 values for Kaempferia galanga were 2.474% at 95% CI The LC50 were low so the toxicity of Kaempferia galanga on Pieris rapae larvae were high The Kaempferia galanga extract and study of Seyedeh et al (2011) were extracted by procedure of Warthen et al (1984) The extracts were extracted equivalent to 30g dried plant in 300 ml of 85% methanol As announced by Seyedeh et al (2011), the LC50 values for A annua extract and A millefolium extract on Pieris rapae larvae were 9.387% and 3.645% Compare with the analytical results in Table 4.2, the LC50 value for Kaempferia galanga extract was lower, so Kaempferia galanga extract was more toxic than A annua and A millefolium extracts Extract of Kaempferia galanga contain ethyl esters of cinnamate and pmethoxycinnamate that are antagonistic toward the polyphagous of Spodoptera littoralis larvae (Pandji, 1993) The hexane fraction, dichloromethane fraction and methanolic fraction of Kaempferia galanga extract effected larvicidal against C quinquefasciatus (Pitassawat,1998) Rhizome extracts of Kaempferia galanga also showed effect against S oryzae at high concentration, the mortality was 100% (at 80 mg/ml concentration) after 24 hour (Dash, 2017) In this study, Kaempferia galanga rhizome extract 22 showed potent activity against Pieris rapae Thus, the Kaempferia galanga extracts has high potential in control pests Table 4.2 Dose–response parameters of Pieris rapae after 24 hours Plant extract N LC50 (95%CI) dfᵇ SE Kaempferia 30 2.474 (1.805-3.143) 0.341 galanga Figure 4.2 Testing toxicity of (4%) Kaempferia galanga extract on Pieris rapae larvae Experiment Testing the effect of Kaempferia galanga extract on the anti-feeding of Pieris rapae larvae Testing the effect of Kaempferia galanga extracts on the antifeeding of Pieris rapae larvae was performed on 30 Pieris rapae larvae at stage There were three control sample and three treatments Leaves were photographed and leaves area would be measured after hours, hours and 24 hours to determind the acreage consumption of leaf by Pieris rapae The obtainable results are presented in table 4.3, figure 4.3 and figure 4.4 23 Table 4.3 Deterrence percentage acreage leaves of Kaempferia galanga extract on Pieris rapae larvae Fomula Concentration Mean Control 0.5% 32.28 Treated 0.5% 25.93 Control 1% 39.24 Treated 1% 41.40 Difference P value between means (B - A) ± SEM 6.350 ± 6.214 0.3646 df -2.164 ± 6.827 0.7671 Figure 4.3 The leaf consumption of larvae in the treatment with 0.5% Kaempferia galanga extract 24 Figure 4.4 The leaf consumption of larvae in the treatment with 1% Kaempferia galanga extract Results showed that at both 0.5% and 1% concentrations there was no significant difference between the control sample and treatment on larvae's leaf consumption The area of leaf consumption of the control samples at the concentrations of 0.5% and 1% were 32.28 and 39.24, while the area of leaf consumption of the treatment at 0.5% and 1% concentrations was 25.93 and 41.40 The P-value of 0.5% and 1% extract treatments were 0.3646 and 0.7671 Neither of the two concentrations was effective for anti-feeding of Pieris rapae larvae As announced by Seyedeh et al in 2011, Achillea millefolium L extract (with deterrence was 44.185%) and Artemisia annua L extract (29.826%) Similarly, Kaempferia galanga extract was low effective Kaempferia galanga extract was not high effectively antifeeding as well as Garlic, Mint, Fenugreek oils with 87.21%, 86.93%, 79.13% as reported by Esmat et al in 2019 25 Figure 4.5 The effect of Kaempferia galanga extract on the antifeeding of Pieris rapae larvae 26 PART V CONCLUSIONS AND SUGGESTIONS 5.1 Conclusions - Kaempferia galanga extract was toxic to Pieris rapae larvae, at 8% concentration the mortality was 100% after 24h The LC50 values for Kaempferia galanga were 2.474% at 95% CI - At 0.5% and 1% concentration, Kaempferia galanga extract did not have antifeeding effect on Pieris rapae larvae 5.2 Suggestions Due to the limited time and research conditions and not have expanded the topic, so we would like to give some suggestions as follows: - Testing toxicity of the Kaempferia galanga extract on Pieris rapae larvae at stage and stage - Testing the effect of Kaempferia galanga extract on oviposition of Pieris rapae - Testing the self-defense respone to Kaempferia galanga extract in cabbage plants - Combine Kaempferia galanga extract with some binders to make the extract exist longer and stable in the environment 27 REFERENCES HASHEMINIA S SEYEDEH M., ET AL (2011) "THE EFFECTS OF ARTEMISIA ANNUA L AND ACHILLEA MILLEFOLIUM L CRUDE LEAF EXTRACTS ON THE TOXICITY, DEVELOPMENT, FEEDING EFFICIENCY AND CHEMICAL ACTIVITIES OF SMALL CABBAGE PIERIS RAPAE L (LEPIDOPTERA: PIERIDAE)." 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JOURNAL OF ETHNOPHARMACOLOGY THOMAS MORDEGREN (2002) "THOMAS NORDEGREN (2002) THE A-Z ENCYCLOPEDIA OF ALCOHOL AND DRUG ABUSE UNIVERSAL-PUBLISHERS." UNIVERSAL-PUBLISHERS HARCOURT DG 1962 DESIGN OF A SAMPLING PLAN FOR STUDIES ON THE POPULATION DYNAMICS OF THE IMPORTED CABBAGEWORM, PIERIS RAPAE (L.) (LEPIDOPTERA: PIERIDAE) CANADIAN ENTOMOLOGIST 94: 849-859 KIUCHI F, NAKKAMURA N, TSUDA Y, KONADA K & YOSHIMURA H (1988) STUDIES ON CRUDE DRUGS EFFECTIVE ON VICERAL LARVA MARGINS II LARVICIDAL PRINCIPLES IN KAEMPFERIA RHIZOMA CHEM PHARM BULL (TOKYO) 36:412-415 PITASSAWAT, B., CHOOCHOTE, W AND KANJANAPOTHI D 1998 SCREENING FOR LARVICIDAL ACTIVITY OF TEN CARMINATIVE PLANTS SOUTHEAST ASIAN JOURNAL OF TROPICAL MEDICINE AND PUBLIC HEALTH 29:660- 662 HUONG, H T (2011) "BIOLOGICAL AND ECOLOGICAL CHARACTER OF PIERIS RAPAE L DAMAGING ON CABBAGE." J.D WARTHEN JR., M STOKES, M.P JACOBSON, KOZEMPEL, ESTIMATION OF AZADIRACHTIN CONTENT IN NEEM EXTRACTS AND FORMULATIONS, J LIQ CHROMATOGR (1984) 591–598 29 Caculate LC50 in SPSS Data Information N of Cases Valid Rejected Missing LOG Transform Cannot be Done Number of Responses > Number of Subjects Control Group 0 Convergence Information Number of Iterations Optimal Solution Found PROBIT 13 Yes Parameter Estimates Parameter Estimate Std Error Z Sig 95% Confidence Interval Lower Bound PROBITa Upper Bound concentration 2.474 341 7.246 000 1.805 3.143 Intercept -1.025 171 -6.000 000 -1.196 a PROBIT model: PROBIT(p) = Intercept + BX (Covariates X are transformed using the base 10.000 logarithm.) Chi-Square Tests Chi-Square dfb Sig 30 -.854 PROBIT Pearson Goodness-of-Fit Test 10.901 012a a Since the significance level is less than 050, a heterogeneity factor is used in the calculation of confidence limits b Statistics based on individual cases differ from statistics based on aggregated cases Cell Counts and Residuals Number concentration Number of Subjects Observed Responses Expected Responses Residual Probability PROBIT -.301 30 1.151 1.849 038 000 30 4.579 421 153 301 30 11.687 -4.687 390 602 30 18 20.363 -2.363 679 903 30 30 26.600 3.400 887 Confidence Limits Probability 95% Confidence Limits for concentration 95% Confidence Limits for log(concentration)b Estimate Lower Bound Bound Upper Bound PROBITa Upper Bound Estimate 010 298 000 858 -.526 -5.068 020 384 000 1.005 -.416 -4.400 002 030 451 000 1.114 -.346 -3.978 047 040 509 000 1.207 -.293 -3.661 082 050 562 000 1.291 -.250 -3.404 111 060 611 001 1.368 -.214 -3.186 136 31 -.067 Lower .070 657 001 1.442 -.182 -2.995 159 080 702 001 1.513 -.154 -2.825 180 090 746 002 1.583 -.128 -2.671 199 100 788 003 1.651 -.104 -2.529 218 150 990 011 1.999 -.005 -1.950 301 200 1.186 032 2.391 074 -1.501 379 250 1.386 074 2.881 142 -1.131 460 300 1.594 153 3.556 202 -.816 551 350 1.814 282 4.575 259 -.550 660 400 2.051 469 6.247 312 -.329 796 450 2.310 707 9.176 364 -.151 963 500 2.597 975 14.546 414 -.011 1.163 550 2.919 1.253 24.735 465 098 1.393 600 3.287 1.532 44.778 517 185 1.651 650 3.717 1.813 86.038 570 258 1.935 700 4.230 2.102 176.312 626 323 2.246 750 4.864 2.412 391.004 687 382 2.592 800 5.683 2.761 966.283 755 441 2.985 850 6.813 3.183 2817.228 833 503 3.450 900 8.559 3.749 10992.215 932 574 4.041 910 9.043 3.894 15299.088 956 590 4.185 920 9.601 4.054 21923.740 982 608 4.341 930 10.255 4.236 32584.190 1.011 627 4.513 940 11.037 4.445 50760.159 1.043 648 4.706 950 12.002 4.692 84225.887 1.079 671 4.925 960 13.244 4.996 152843.691 1.122 699 5.184 970 14.949 5.389 318377.119 1.175 732 5.503 32 .980 17.560 5.950 845936.913 1.245 775 5.927 990 22.632 6.933 3959029.003 a A heterogeneity factor is used b Logarithm base = 10 33 1.355 841 6.598 T-test to compare the consumption of leaf Table Analyzed anti feeding 0,5% after 24h Column A control vs vs, Column B treated Unpaired t test P value 0,3646 P value summary ns No Significantly different (P < 0.05)? One- or two-tailed P value? Two-tailed t, df t=1,022, df=4 How big is the difference? 32,28 Mean of column A Mean of column B 25,93 Difference between means (A - B) ± SEM 6,350 ± 6,214 95% confidence interval -10,90 to 23,60 R squared (eta squared) 0,2070 F test to compare variances F, DFn, Dfd 9,662, 2, P value 0,1876 P value 0,1876 P value summary ns No Significantly different (P < 0.05)? Data analyzed Sample size, column A Sample size, column B Table Analyzed anti feeding 1% after 24h control Column A vs vs, Column B treated Unpaired t test P value 0,7671 ns P value summary Significantly different (P < 0.05)? No One- or two-tailed P value? Two-tailed t, df t=0,3170, df=4 How big is the difference? 39,24 Mean of column A Mean of column B 41,40 -2,164 ± 6,827 Difference between means (A - B) ± SEM 95% confidence interval -21,12 to 16,79 R squared (eta squared) 0,02450 F test to compare variances F, DFn, Dfd 1,300, 2, P value 0,8696 P value summary ns Significantly different (P < 0.05)? No Data analyzed Sample size, column A Sample size, column B 34