Khảo sát tình hình gây hại, đặc điểm sinh học và hóa chất tín hiệu trong quản lý sâu kéo màng, hellula undalis (lepidoptera crambidae) hại cải tại đồng bằng sông cửu long tt tiếng anh

MINISTRY OF EDUCATION AND TRAINING CAN THO UNIVERSITY SUMMARY Specialized discipline: PLANT PROTECTION Code: 62 01 12 TRAN THANH THY STUDY ON DAMAGE SITUATION, BIOLOGICAL CHARACTERISTICS AND SEMIOCHEMICAL FOR THE PEST MANAGEMENT OF Hellula undalis (LEPIDOPTERA: CRAMBIDAE) ON GREEN MUSTARDS IN THE MEKONG DELTA OF VIETNAM Can Tho, 2019 WORRK DONE IN CAN THO UNIVERSITY Main supervisor: Dr Nguyen Loc Hien, Can Tho University Sub supervisor: Associate Prof Dr Le Van Vang, Can Tho University Thesis seminar at:…………………………………………………… Time:………………………………………………………………… Reviewer 1: Reviewer 2: This dissertation is available in following libraries: Learning Resource Center of Can Tho University National Library of Vietnam LIST OF PUBLISHED PAPERS The scientific article Tran Thanh Thy, Le Van Vang, Nguyen Loc Hien and Phan Thi Thanh Tuyen, 2016 Effects of green mustard cultivars and temperature on some biological characteristics of the cabbage webworm, Hellula undalis Fabricius (Lepidoptera: Crambidae) Journal of science, Can Tho University Special issue: Agriculture ISSN 1859-2333 (3): 193-199 Tran Thanh Thy, Le Van Vang, Nguyen Loc Hien and Nguyen Huu Minh Tien, 2016 Semiochemical application for the pest management of Hellula undalis Fabricius (Lepidoptera: Crambidae) on green mustards in the Mekong Delta Journal of science, Can Tho University Special issue: Agriculture ISSN 1859-2333 (3): 200-209 Tran Thanh Thy and Le Van Vang, 2017 Study on mass rearing of cabbage webworm (Hellula undalis Fabricius) Journal of Viet Nam Agriculture science and technology ISSN 18591558 4(77): 76-81 Tran Thanh Thy, Le Van Vang and Nguyen Loc Hien, 2018 Genetic diversity of cabbage webworm (Hellula undalis Fabricius) on green mustard by using ISSR marker in the Mekong Delta Journal of Viet Nam Agriculture science and technology ISSN 1859-1558 2(87), 65-70 Tran Thanh Thy, Le Van Vang Nguyen Loc Hien, 2018 Study on damage situation of the cabbage webworm, Hellula undalis Fabricius (Lepidoptera: Crambidae) on green mustards at the Mekong Delta Journal of science, Can Tho University ISSN 1859-2333 54(3B): 115-124 Chapter 1: INTRODUCTION 1.1 The significance of the dissertation Green vegetables are vital for human health as they contain all important nutrients According to the World Health Organization (WHO), insufficient intake of green vegetables is the main reason causing approximately 2.7 millions people to die each year (Le Hong Phuc, 2010) Vietnam has a long history of vegetable farming (Le Thi Khanh, 2009), and the Brassicaceae family (cabbage, cauliflower, radish, ) is one of the major plant groups that have been cultivated here However, both vegetable farming and production seem quite difficult due to harmful worms such as Hellula undalis Fabricius, Plutella xylostella, Phyllotreta striolata Fabricius, Spodoptera litura, etc…(Ho Thi Thu Giang, 2005; Tran Dang Hoa et al., 2013) The cabbage webworm (Hellula undalis Fabricius) is a moth of the Crambidae family The larvae feed on a wide range of plants, mainly of the Brassicaceae family It is a widespread species which is found in tropical and subtropical regions (Waterhouse and Norris, 1989) or temperate region (Kalbfleisch, 2006) Females lay their eggs on the leaves of cabbage, and the larva of H undalis bores into the heads and leaf petioles (Veenakumari et al., 1995; Sivarpagasam and Chua, 1997) The damage caused by the larvae of H undalis to cabbage severely impacts yield in Hawaii, India, Malasia, Philippines, Taiwan, Egypt, Iraq and Japan (Kalbfleisch, 2006) In Vietnam, H undalis is also a threat to the productivity and quality in autumn-winter crops (Duong Thi Van, 2012) Good management of H undalis requires farmers to use many types of chemicals in high frequency, the most common chemicals being Permethrin, Abamectin, Teflu benzuronm, Mephosfolan, Terbufos, and Carbofuran In Malaysia, farmers usually use chemicals at the seedling stage (Parker et al., 1995) According to Ta Thi Huynh Dao and Nguyen Van Huynh (2008), 95% of farmers in My Xuyen and Ke Sach districts (Soc Trang province) used chemicals to keep H undalis from destroying their cabbage farms However, only 45% agreed that the treatments were good for their crops Nowadays, sustainable agriculture requires the use of biological control Consequently, applying a semiochemical is one of the more beneficial integrated pest management approaches (Srinivasan, 2008) Semiochemicals are chemicals that mediate interactions between organisms, and learning about semiochemical applications is a good strategy for biological control instead of using a large amount of harmful chemicals for decades (Gibb et al., 2005) From this point of view, the dissertation titled “Study on damage situation, biological characteristics and semiochemical for the pest management of Hellula undalis (Lepidoptera: Crambidae) on green mustards in the Mekong Delta of Vietnam” was made to develop a new and environmentally safe method for reducing the risk of H undalis infestations in Mekong Delta fields and replacing the need for harmful chemicals 1.2 Objective and research requirements 1.2.1 Purpose Effective management of H undalis using chemical signals in order to protect green vegetables in the Mekong Delta 1.2.2 Objectives To evaluate damage caused by H undalis to green vegetables in provinces (Vinh Long, Can Tho and Hau Giang) To evaluate genetic diversity in H undalis moth populations in the Mekong Delta To investigate the role of different green vegetable varieties and temperature on H undalis development To determine the chemical structure of the sex pheromones of H undalis To Syntheses of the compound of E11,E13-16:Ald To assess the effect of pheromone on H undalis under field conditions To evaluate the effect of citronella oil and garlic oil on sexual harassment of H undalis in net house and field conditions To apply chemical signals in the management of H undalis in the Mekong Delta 1.3 New contributions of the dissertation Determination of the composition of the new sex pheromone, (Z) -11-tetradecenyl acetate of H undalis It also determines the role of unsaturated hydrocarbons (Z, Z, Z) -tricosatriene) to catch H undalis male in field trials of sex pheromone The results ushered in the promising application of sex pheromone by H undalis Determination of the biological characteristics, genetic diversity, and the influence of different green mustard cultivars and temperature on some biological characteristics, the multipliable index of a generation (R0) and population growth rates (r) of H undalis from three consecutive generations Syntheses of the compound (E, E) -11,13-hexadecadienal (E11,E13-16:Ald), the sex pheromone component of H undalis, by the Wittig reaction An evaluation of the effectiveness of synthetic sex pheromone, Cymbopogon citratus, and Allium sativum in the management of H undalis in the Mekong Delta Chapter 3: MATERIALS AND METHODS 3.1 Duration and location of study Duration: from 11/2014 to 2/2018 Locations: The studies were conducted in the laboratory of Department of Plant Protection, and Department of Genetic and Plant Breeding, College of Agriculture & Applied Biology, Can Tho University; the laboratory of Agricultural Science, Cuu Long University; and the Laboratory of Chemical Ecology, Tokyo University of Agriculture and Technology, Japan Experiments under field conditions were conducted on cabbage farms in Long Ho, Tra On, Binh Minh (Vinh Long), Cai Rang district (Can Tho city), and Chau Thanh A (Hau Giang) 3.2 Methods 3.2.1 Investigation and evaluation of the influence of H undalis on farms in Vinh Long, Can Tho and Hau Giang provinces 3.2.1.1 Farmer survey on the situation of H undalis in Vinh Long, Can Tho and Hau Giang provinces Approach: A survey was conducted in the form of interviews with farmers using a prepared questionnaire A total of 180 farmers who were growing vegetables on a minimum area of 500 m2 were interviewed (one per household) At each farmer's farm, interviews were conducted with the farmer directly Locations: Interviews were carried out in districts: Binh Minh (20 houses), Binh Tan (20 houses) and Long Ho (20 houses) in Vinh Long province; Phong Dien (20 houses), Binh Thuy (20 houses) and Cai Rang (20 houses) in Can Tho City; Long My (30 houses) and Vi Thuy (30 houses) in Hau Giang province 3.2.1.2 Evaluation of the influence of H undalis and others insects on different cabbages From the investigation, we chose 25 cabbage farms (3 farms per district, except farms in Long Ho district) Method: Each investigated cabbage farm, we chose 10 points fixed diagonally, each point equivalent m2 Record: The damage caused by H undalis every days (a total of recordings) The harmful insects which could not be identified in the field were sampled in small boxes, and sent to the laboratory H undalis 's damage rate (%) The prevalence of a specie is based on the incidence of that species on the cabbage farms 3.2.2 Assessment of genetic diversity of H undalis population in Mekong Delta via ISSR markers 3.2.2.1 Collecting samples in the Mekong Delta 260 samples of H undalis were collected in the Mekong Delta (20 samples per province) Collected samples were stored in an Eppendoft tube (10ml) in 70% Ethanol, and sent to the Department of Genetic and Plant Breeding, College of Agriculture & Applied Biology, Can Tho University for DNA extraction and evaluating the genetic diversity via ISSR marker 3.2.2.2 CTAB buffer using for DNA extraction a DNA extraction: DNA of H undalis were extracted via Taylor and Powell (1982) b PCR reaction – ISSR marker 10 ISSR primers were used in this research, followed by Mostafa et al (2011), Latif et al (2013) and Nirmaladevi et al (2016) PCR reactions were prepared in 20 µl: 2,0 µl buffer (10X), 0,4 µl dNTPs (10 mM), 0,5 µl primer ISSR (10 pM), 0,2 µl Taq ADN Polymerase (5U/ µl), 1,0µl ADN (100 ng/ µl ) 15,9 µl H 2O 10 ISSR primers were used in this research, following Mostafa et al (2011), Latif et al (2013) Nirmaladevi et al (2016) The primers were synthesized by Phusa Biochem PCR reactions (20 µl) contain: 2.0 µl buffer (10X), 0.4 µl dNTPs (10 mM), 0.5 µl primer ISSR (10 pM), 0.2 µl Taq ADN Polymerase (5U/ µl), 1.0 µl ADN (100 ng/ µl ) 15.9 µl H 2O There were two melting steps at 94 C, the first step was started for and the second for 30 sec, followed by 40 cycles Annealing step was setup based on Tm of each primer (Table 2) The primer extension step was 0 performed at 72 C for 30 sec and min, as the final step The PCR samples were stored at 10 C for further analysis For gel electrophoresis, we used CompactPAGE-twin AE-7341 polyacrylamide gel (8%) with TBE 0.5X buffer by CompactPAGE-twin AE-7341 Staining the gels with Ethidium bromide (1 mg/l), and the gels were carefully cleared before taking the photos c Genetic diversity analysis To calculate the fragment sizes, we used Gel Analyzer software All amplified bands were recorded in binary data (1 and 0), then NTSYSpc 2.0 was used to construct the relationship tree by using UPGMA method 3.2.3 Survey the influence of host on H undalis development Experiments were arranged in a completely randomized design (CRD) with five treatments The feed was leaf of five varieties including Brassica integrifolia, Brassica juncea, Brassica campetrus, Brassica rapachinensis and Spinacia oleracea and 30 repetitions Each replicate of a treatment was a plastic box for feeding a H undalis larvae that had just hatched from the leaves of green mustards Recording results: size and development time for whole stages of H undalis, number of eggs per female adult per day 3.2.4 Survey the influence of temperature on H undalis development Experiments were arranged in the form of a CRD with four temperature treatments, 16 oC, o 20 C and 25oC and room temperature, each with with 30 repetitions Each replicate was a feed box containing leaves of the selected variety from the results of the treatment examining the host effect (Section 3.2.3) Recording results: development time at each stage and life cycle of H undalis at each survey temperature condition 3.2.5 Investigation of the ability to reproduce within a narrow range of H undalis The survey was conducted according to the Wilson method (1971) and adjusted to the conditions of the laboratory Recording data: number of eggs, incubation period, hatching rate, total larvae, larval survival rate, total number of pupae, survival rate of each stage, of the heir and the number of births after each generation, the multiplier of a generation, the incremental index of nature 3.2.6 Research on pheromone of H undalis 3.2.6.1 Determination of chemical composition a Pheromone extraction Pheromone of H undalis was extracted following Vang et al (2013) b Gas chromatography analysis - Electroantennogram A gas chromatography diagram – electroantennogram (EAG) of samples was constructed via GC-EAD using detectors at the same time, including FID (Flame Ionization Detector) of gas chromatography and EAD (Elecreoantennogram Detector) (Figure 3.1) Signals of pheromone accepted from analysis were amplified before exporting to diagram (EAG) (Le Van Vang, 2016) c Gas chromatography analysis – Mass spectrum A mass spectrum of electroantennogram active components in pheromone samples and standards were recorded via GC HP 6890 and MS (Mass Selective Detector) HP 5973 Ionization was performanced by Electron Impact Mode in 70 eV and 230 C The mass spectrum was set up in m/z ranging from 40-500 Column chromatography was performed using DB-23 The temperature program used for this analysis was similar to GC-EAD analysis The temperature program for samples 0 0 was 80 C (for minute), increasing to 210 C (8 C/min) and held at 220 C in 20 minutes Figure 3.1: Gas chromatography diagram – electroantennogram Pheromone extraction with dimethyl disulfide (DMDS) The double-bond position in the molecule of the pheromone component was determined by GC-MS analysis of the extracted pheromone samples from DMDS according to the method described by Vang et al 3.2.6.2 Synthesis of E11, E13-16: Ald compounds, main sex pheromone component of H undalis E11, E13-16: Ald, the main pheromone component of H undalis, were synthesized according to the method of Le Van Vang and Nguyen Thi Tien (2015) 3.2.6.3 Field evaluation of the attraction of synthetic sex pheromone to adult H undalis * The effect of the attraction of Z3,Z6,Z9-23: H (T23); E11, E13-16:Ald; Z11-16: Ald and Z1114:OAC components to H undalis was evaluated, 28/3/2017 to 30/4/2017, Vinh Long province: The experiment was conducted in a completely randomized block design factor (RCBD) with treatments, which were different mixing ratios of the above four components Each treatment was replicated times corresponding to pheromone traps, in which control treatments used a rubber septum containing 10 μl nhexane and a second one used virgin H undalis 3.2.7 Effect of semiochemical on mating and spawning H undalis 3.2.7.1 The net - house condition * Experiment 1: Harassing effects on mating, 08-15/11/2015, Mekong University The experiment was conducted in a RCD, one choice with treatments (Cymbopogon citratus, Alliums sativum, E10-15:Ald, virgin H undalis and 10 µl n-Hexane) and repetitions Each replicate of a treatment is a net which inside had a trap with two virgin H undalis, one harassment substances (suspended in a trap, above two virgin H undalis trap) and released on 15 H undalis males has just been assimilated Monitoring Targets: The number of males were attracted to the trap after days after releasing males * Experiment 2: Harassing effects on spawning, 20 - 27/11/2015, Mekong University The experiment layout was similar to experiment 1, only difference being to put green mustard pots in the middle of the net, one harassment substances hanging to green mustard pot and released into pairs of H undalis (male / female) = 1) that they are mated in a paper bag Monitoring Target: The number of eggs laid on the green mustards pots after days after release 3.2.7.2 Field conditions * Experiment 3: Harassing effects on mating, 02- 30/1/2016, Can Tho City The experiment layout was similar to the net house, the only difference being that the experiment was conducted in RCBD with treatments and repetitions Each replicate of a treatment was a trap with two virgin H undalis and one harassment substance hanging to the trap Monitoring Target: The number of males caught in traps after days after trapping * Experiment 4: Harassing effects on spawning, 02- 30/1/2016, Can Tho city The experiment layout was similar to the net house, the only difference being that the experiment was conducted in RCBD with treatments and repetitions Each replicate of a treatment was m2 of green mustards inside which hangs one harassment substances at about 30 cm and covered by a cover C citratus, A sativum were renewed once a week Monitoring targets: On each plot, the number of eggs of H undalis laid on at 7, 14, 21 and 28 days Validity of C citratus, A sativum and E10-15: Ald according to the Abbott (1925) 3.2.8 Semiochemical application for the pest management of Hellula undalis The experiment was conducted in RCD, independent, one choice with treatments (C citratus, A sativum, pesticides, uncontrolled) are separate fields from 01-30/3/2016 at Vinh Long province Each treatment was conducted separately in each field to avoid impact Monitoring Target: The number of eggs of H undalis before and after the experiment was set at locations: m to the west, m to the east, m to the north, m above the North, m to the South, m to the South, m to the South, and m to the South from the hang bags of essences and survey of m At the same time, both of pesticides and uncontrolled fields were record the number of eggs H undalis in the similar location as the field management with lemongrass and garlic essences 3.3 Data analysiss All data were recorded in table or graph via Microsoft Excel 2010, and statistical analysis by SPSS 21.0 Data which collected in 25 fields were used to determine the quantity, the frequency and the damage of H undalis Using Duncan to analyze the influence of food, temperature, pheromone effectiveness, signal chemicals on H undalis at signicant level of 5% Chapter 4: RESULTS AND DICUSSIONS 4.1 Results of investigation and assesment the rate of damage caused by H undalis in Vinh Long, Can Tho and Hau Giang province 4.1.1 Rate of H undalis, damage and precaution 4.1.1.1 Rate of H undalis and damage caused by H undalis According to farmers, H undalis mainly caused damage to cabbage sprout (71.88%) in which 100% farmers in Binh Tan district – Vinh Long province, Cai Rang district – Can Tho city Only 18.96% farmers said that H undalis caused damage to cabbage sprouts and leaves, and 9.16% on leaves Most farmers (82.70%) believe that H undalis causes serious damage in the dry season from mid-November to April At the time of damage, 63.13% of farmers recorded serious H undalis damage 10-15 days after planting the green mustards (DAP) when the green mustards had 3-5 leaves suitable for H undalis to lay eggs, larvae hatched to attack All interviewed farmers reported that H undalis didn’t cause damage at the time of harvest (25-30 DAP) (Table 4.1) The results from the farmers showed that the growers were knowledgeable and paid much attention to the damage caused by H undalis 4.1.1.2 The pest management of H undalis Some farmers reported that H undalis caused serious damage (39.38%), while others (38.75%) reported only average damage To deal with H undalis infestations, up to 99.37% of farmers using chemical pesticides, including: Cuc synthesis (Cyperan 10EC, Bulldock 25EC, Serpa 10EC, Cyper-alpha 5ND, Peran 50EC, Sumi-alpha 5SC); Organic phosphate (Selecron 500EC); Carbamate (Padan 95SP) and biocatalysts (NeemNim, Phesoltin 5.5EC, Bihopper 270EC, Vertimec 1.8EC, Atapron 5SC, BT, Biobit 32B.FC) for treatment, most periodically sprayed (65.43%) with concentration recommended on the label (87.93%) 20.84% of farmers thought that the effective use of chemicals was good, while the remaining farmers thought that H undalis was difficult to treat with chemical drugs because it was wrapped inside the weaver with a water-repellent silk At the same time, 91.46% of farmers reported that they had a known set up pesticides for H undalis (Table 4.1) The results of this investigation show that H undalis is a serious pest and not a good target for pesticides 4.1.2 Field survey on the damage situation of H undalis 4.1.2.1 Composition and prevalence of major pests on green mustards The results of the field survey recorded species of insect pests including Phylotreta striolata, Plutelld xylostella, Spodoptera litura, Plusia eriosoma, Pireis rapae, Liriomyza trifolii, Brevicoryne brassicae, Helicoverpa armigera and Hellula undalis, each with a different prevalence and frequency (Table 4.2) Three species, P striolata, P xylostella, H armigera were very prevalent (>50%) and their frequency was 100% (5/5 times); H undalis, S litura were moderately prevalent (> 25-50%) and had a frequency of 100% (5/5 times) Other insect pests such as P eriosoma, P rapae, L trifolii are less prevalent (5-25%) and their frequency was lower 4.1.2.2 Evaluate the incidence of H undalis Field surveys on species of green mustards including Brassica integrifolia, B juncea, Nasturtium officinale, B oleraceae, B campestris, B sinensis and Raphanus satius at times during the growing season at districts in three provinces/cities of Vinh Long, Can Tho and Hau Giang, although farmers controled chemical pesticides (Vertimec 1.8EC, Biotit 32B.FC, Match 50ND, Cyperan 10EC, Karate 2.5EC, Reasgant 3.6EC and Rockest 55EC) for periodic prevention, H undalis still appears and is harmful with the rate of damage on each species of green mustards at each survey stage of each area is not the same When N officinale, B campestris and B oleraceae have a high rate of damage with an average of times 8.34%, 11.34% and 7.40%, respectively, at Binh Minh, Binh Tan and Long Ho (Vinh Long province), B integrifolia and B juncea had high rates of damage at Phong Dien, Binh Thuy and Cai Rang (Can Tho city) and Long My; Vi Thuy (Hau Giang province) (Figure 4.1) Developments of rate of damaging, the seven surveyed species were six including B integrifolia, B juncea, B oleraceae, B campestris, B sinensis and Raphanus satius are similarly, H undalis was damaged at DAP, highest increase to 12 DAP, then slowly decreasing to 18 DAP (excluding B campestris, B integrifolia and B oleraceae), highest decrease until 30 DAP At the same time, the damage caused by H undalis to N officinale was increased continuously until 30 DAP Field survey results show that H undalis was damaged on B juncea, B integrifolia and B campestris (at 12-18 DAP) were higher than those on the other fields On the other hand, excluding for N officinale, those on species were H undalis to harm in 30 DAP were not significant (Figure 4.2) Table 4.1: Rate of H undalis, damage and precaution Rate of farmers (%) Item Harmful way Attack the sprout Attack the leaves Attack the sprout and leaves Damage to cabbage of 5–10 DAP 10–15 DAP 15–20 DAP 20–25 DAP 25–30 DAP Damage to season of Rainy season Dry season Damage of level Serious Average Slowl Prevention method Break your hands Chemical pesticides Other When to use the pesticides Immediately appear Many Periodically Concentration of use As recommended Lower recommended Average Binh Minh Binh Tan Long Ho Phong Đien 71.88 9.16 18.96 75.00 25.00 100 0 80.00 20.00 75.00 25.00 16.25 63.13 18.13 2.50 10.00 65.00 20.00 5.00 35.00 60.00 5.00 0 60.00 30.00 10.00 17.30 82.70 25.00 75.00 10.00 90.00 39.38 38.75 21.88 20.00 60.00 20.00 0.63 99.37 Cai Rang Long My Vi Thuy 45.00 30.00 25.00 100 0 46.70 13.30 40.00 53.30 30.00 16.70 10.00 65.00 20.00 5.00 30.00 65.00 5.00 0 35.00 60.00 50.00 0 16.70 70.00 13.30 0 13.30 60.00 26.70 0 15.00 85.00 25.00 75.00 30.00 70.00 10.00 90.00 6.70 93.30 16.70 83.30 60.00 40.00 85.00 15.00 20.00 60.00 20.00 20.00 20.00 60.00 60.00 40.00 23.30 63.30 13.30 26.70 26.70 46.70 100 0 100 5.00 95.00 0 100 0 100 0 100 0 100 0 100 25.41 9.16 65.43 30.00 15.00 55.00 15.00 85.00 55.00 45.00 30.00 15.00 55.00 25.00 75.00 15.00 85.00 10.00 23.30 66.70 23.30 20.00 56.70 87.93 12.07 100 100 100 100 100 100 46.70 53.30 56.67 43.30 10 Binh Thuy Table 4.8: Effects of different temperatures to the life cycle and developmental period of H undalis on B juncea CV (%) The stage Time (days) on different temperatures Egg Larva: - 1st instar - 2nd instar - 3rd instar - 4th instar Cashew Pupae Adult: - Adult – lay - Adult – prolonged lay - Longevity (Female) - Longevity (Male) Cycle life 160C 6.17 a 200C 4.00 b 250C 3.57 b The lap (30.2 0C) 1.83 b 7.50 a 11.20 a 7.97 a 9.87 a 4.33 a 9.33 a 5.25 b 5.63 b 3.50 b 4.63 b 1.63 b 5.88 c 3.23 c 3.17 c 2.80 bc 4.30 b 1.03 c 6.60 b 2.03 d 2.17 d 2.43 c 2.50 c 1.13 c 4.40 d 5.29 a 2.80 b 10.29 a 9.63 a 61.64 a 1.83 b 2.67 b 6.50 b 6.00 b 32.17 b 1.50 b 2.93 b 6.57 b 6.65 b 25.14 c 1.38 b 3.62 a 6.08 b 4.67 c 17.54 d 45.44** 24.39** 21.87** 29.86** 26.29** 28.76** 12.69** 24.69** 23.12** 10.85** 8.70** 7.30** In the same row, numbers with same letters are not statistically different and by the Duncan test **: significant difference of 1% Table 4.9: Effects of different temperatures to larvae and pupae survival on B juncea CV (%) The stage Rate of survival (%) on different temperatures Larva: - 1st instar - 2nd instar - 3rd instar - 4th instar Cashew Pupae 160C 200C 250C The lap (30,2 0C) 59.52 b 63.,05 c 65.79 b 46.11 c 69.65 52.38 b 68.67 b 75.83 b 73.50 ab 58.45 b 81.81 71.04 a 81.33 a 87.70 a 81.32 ab 74.48 a 92.38 78.33 a 88.33 a 92.61 a 89.07 a 76.97 a 91.70 90.72 a 11.83** 10.94** 14.50* 12.51** 10.41ns 15.58** In the same row, numbers with same letters are not statistically different and by the Duncan test **: significant difference of 1%; *: significant difference of 5% Table 4.10: The effect of different temperatures on the egg laying rhythm of H undalis Days of egg laying Total (eggs/female) Number of eggs (eggs/day) on different temperatures 0 0 16 C 20 C 25 C The lap (30.2 C) 38.00 b 49.36 b 92.85 a 88.47 a 45.67 56.14 52.77 59.33 17.57 c 44.91 ab 38.75 b 55.33 a 0.00 b 24.50 a 25.64 a 35.46 a 0.00 b 0.00 b 0.00 b 18.36 a 97.33 d 144.29 c 203.08 b 247.33 a CV (%) 39.01** ns 42.54 ** 37.36 53.16** ** 13.05 26.39** In the same row, numbers with same letters are not statistically different and by the Duncan test **: significant difference of 1%; ns: non significant difference Similar to the number of eggs laid, temperature also had a significant effect on the rate of egg- hatching, with low hatching rates at low incubation temperatures Specifically, incubation at the lap (30.20C) gave the highest hatching rate (92.08%), followed by at 25 0C (74.16%), 200C (51.00%) and lowest at 160C 33.66% (Table 4.11) The results of the experiment showed that temperature affected the growth time, the larval and pupae mortality, the number of eggs laying and the hatching rate of the eggs Among the temperature conditions, 30.20C is best suited for the development of H undalis population for scientific research 18 Table 4.11: Effects of different temperatures on hatchability of H undalis The rate of hatchability Average Standard Deviation (SD) Variability The rate of hatchability (%) on different temperatures 160C 200C 250C The lap (30.2 0C) CV (%) ** 33.66 d 51.00 c 74.16 b 92.08 a 15.89 12.98 8.62 8.87 5.33 21.67-55.0 40.0-63.33 65.0-83.33 86.67-96.67 In the same row, numbers with same letters are not statistically different and by the Duncan test **: significant difference of 1% 4.3.3 The ability on mass rearing of H undalis from adult 4.3.3.1 The ability on eggs laying of H undalis with three consecutive generations Under laboratory conditions, the number of eggs of H undalis progressively declined over three generations, although this decrease was not statistically significant Specifically, in the 1st generation, the number of eggs was 247.93 eggs, while in the lower nd generation, respectively, 234.33 eggs and 3rd generation were 221.47 eggs (Table 4.12) Table 4.12: The ability on eggs laying of H undalis with three consecutive generations in laboratory conditions at Mekong University Generations (F) The ability on eggs laying of H undalis Eggs (eggs/female) 247.93 1st 234.33 2nd 221.47 3rd Significance ns CV% 13.82 In the same column, numbers with same letters are not statistically different and by the Duncan test ns: no difference 4.3.3.4 The ability in population development of H undalis H undalis's ability to develop populations in the lap on B juncea is demonstrated by survival and egg laying H undalis survival at 16 days of age was 100%, 80%, 86%, corresponding to 1st, 2nd and 3rd generations and then decreased H undalis begins spawning after 12 to 13 days of age, spawning and reaching its peak on days 13, 14 and 12, corresponding to generations 1st, 2nd and 3rd Total of H undalis female was born from mother a very large, variability in from 33.07 to 42.13 (Table 4.13) Table 4.13: The ability in population development of H undalis from three consecutive generations in laboratory conditions at Mekong University Date of age x 1st generations 2nd generations 3rd generations - 12 13 14 15 16 17 18 19 20 21 Total T0C H% lx 1.00 1.00 1.00 1.00 1.00 0.66 0.40 0.13 mx 10.40 9.50 7.80 6.30 5.70 2.43 0 R0 10.40 9.50 7.80 6.30 3.76 0.97 0 42.13 30.67 68.95 38.73 lx 1.00 1.00 1.00 1.00 0.8 0.66 0.46 0.26 0.067 19 mx 7.50 8.10 7.80 6.10 3.40 1.70 1.0 0 35.60 30.46 69.50 R0 7.50 8.10 7.80 4.88 2.24 0.78 0.26 0 31.56 lx 1.00 1.00 1.00 1.00 0.86 0.66 0.33 0.13 mx 9.10 6.30 6.90 4.20 3.30 2.10 1.17 0 33.07 31.02 70.67 R0 9.10 6.30 6.90 4.20 2.84 1.39 0.39 0 31.12 Table 4.14 shows that with suitable green mustard, H undalis was cultivated on B juncea sprouts with the high multipliable index of a generation (variability: 31.12 to 38.73; mean 33, 80) The multiplicity index of each species over the three generations is different, so that in the third generation (T 0C = 31.02; H% = 70.67) was 31.12, while the first generation (T 0C = 30.67; H% = 68.95) was 38.73 The right, population growth rates of H undalis was quite high (variability: 0.56 to 0.57; mean 0.563) These results shows that H undalis has a high population growth rates Table 4.14: Some of biological of H undalis population from three consecutive generations in laboratory conditions at Mekong University Targets Multipliable index of a generation (R0) Population growth rates (r) Date of age (x) T0C H% 1st generations 38.73 0.56 6.47 ± 0.74 30.67 68.95 2nd generations 31.56 0.57 6.07 ± 0.96 30.46 69.50 3rd generations 31.12 0.56 6.13 ± 0.83 31.02 70.67 Average 33.80 0.563 4.4 Sex pheromone of H undalis 4.4.1 Components and chemical structure of sex pheromone of H undalis 4.4.1.1 The GC-EAD analysis of crude extract of pheromone The GC-EAD analysis of crude extract from pheromone glands of virgin female (1,0 FE) H undalis shows the antennae of male moths responded with electroantennogram detector to four component with electroantennogram (EAG), at the retention time (tR) of 15.50 minutes (component 1) with 105 µV, 16.05 minutes (component 2) with 192 µV, 17.65 minutes (component 3) with 320 µV and 18.78 minutes (component 4) with 99 μV (Figure 4.5) It is concluded that sex pheromone of H undalis in the Mekong Delta have four component 4.4.1.2 The GC-MS analysis of crude extract of pheromone Figure 4.5: The mass spectrum GC-EAD of crude extract pheromone gland from H 20 undalis (1.0 FE) Figure 4.6: The mass spectrum GC-MS of crude extract pheromone gland from H undalis (20 FE) The GC-MS analysis of crude extract of pheromone (20 FE) by H undalis is shown in Figure 4.6 Four components of the EAG-active component were present in the GC (Total Ion Chromatogram) analysis of GC-MS at 13.49 minutes, 14.36 minutes , 15.99 minutes and 16.98 minutes respectively with a ratio of 3: 10: 0.5: 4.4.1.3 The extracted form is derivatized with DMDS Figure 4.7 shows the GC-MS analysis of crude extracts of pheromone (20 FE) that have been derivatized with DMDS Component was the DMDS derivative present on the total chromatogram with a retention time at 30.68 minutes, while the retention time of component at 29.79 minutes The mass chromatogram (MC) of component of the ion at m/z 332, 117 and 215, reveal the structure of double bonds in the molecule at C11 of the hexadecenal The mass chromatogram (MC) of component of the ion at m/z 348, 199 and 259, reveal the structure of double bonds in the molecule at C11 of tetradecenyl acetate Components and in the molecule containing the double conjugate, so DMDS for MS with non decay ions, the position of the double bonds in the molecules of these components can be determined by MS of the unconfirmed pheromone component as a basis for the decision to select a synthetic reference substance 4.4.2 The analysis of synthetic standard Compounds: Z11-14:OAc, Z11-16:Ald, E11,E13 -16: Ald and E11, E13-16: OH were chosen as the benchmark to verify the interpretation from the results of MS of crude extract of pheromone and DMDS Figure 4.7: TIC (Top) and the mass spectrum of component 1, treat with DMDS 21 4.4.2.1 The GC-EAD analysis Results of analysis of synthetic standard (10 ng/component) showed that Z1114:OAc, Z11-16:Ald, E11,E13-16:Ald and E11, E13-16:OH were given GC-EAD and have the retention time (tR) similarity with components 1, 2, and 4, respectively (Figure 4.8) It is concluded that components 1, 2, and are Z11-14:OAc, Z11-16:Ald, E11,E1316:Ald and E11,E13-16:OH Figure 4.8: The GC-EAD analysis of synthetic standard Z11-14:OAc, Z11-16:Ald, E11,E1316:Ald E11,E13-16:OH of H undalis 4.4.2.2 The GC-MS analysis (GC – MS) Similar to GC-EAD analysis, GC-MS analysis of synthetic standard yielded the retention time (tR) and MS of Z11-14:OAc, Z11-16:Ald, E11,E13-16:Ald and E11,E13-16:OH (Figure 4.9) are coincident with tR and MS of the pheromone components 1, 2, and This result again confirms the components 1, 2, and are Z11-14:OAc, Z11-16:Ald, E11,E13-16:Ald and E11,E13-16:OH are sex pheromone of H undalis Figure 4.9: The GC-MS analysis of synthetic standard 4.5 Synthesizing (11E,13E)-11,13-hexadecadienal components (11E, 13E) -11,13-hexadecadienal compounds, E11,E13-16:Ald was successfully synthesized by a route using a Wittig reaction with commercial 11-bromo-1-undecanol as the original reactant (Figure 4.10) After protecting a hydroxyl group (OH) of 11-bromo-1undecanol by MOM-ether (82.6% yield) MOM-ether is boiled with PPh at 90 0C for 72 hours, then combined with (E)-2-hexenal by Wittig reaction, in THF and n-butyl lithium (nBuLi) Reactions are obtained compounds of the MOM-ether of (11E, 13Z) -11,13hexadecadiene-1-ol and (11E, 13E) -11,13-hexadecadiene-1-ol (41.2% yield) A mixture of MOM-ether is impregnated with 0.5N HCl in methanol to remove the OH of MOM-ether (86.4% yield) Using a mixture of 15% AgNO3 in Silica gel was used as a stationary phase 22 for choromatography to analysize and purify, (11E, 13E) -11,13-hexadecadiene-1-ol The separate oxygenation on (11E,13E)-11,13-hexadecadienal by PCC with a reaction yield of 70.3% HO a Br MOMO c MOMO MOMO 4a MOMO 4b PPh Br O e d b Br HO f H E11,E13-16:Ald Figure 4.10: Scheme for synthesis of E11,E13-16:Ald a, DMM/LiBr/p-TsOH/24 (85,7%); b, PPh3/90oC/72 giờ; c, (E)-2-hexenal/n-BuLi/THF; d, 0,5N HCl/methanol/24 giờ; e, 15% AgNO3; f, PCC/CH2Cl2 4.6 The effects of synthetic pheromone on H undalis in the field trials Table 4.15: Number of H undalis males were catched by related compounds in experiments on green mustard in Long Ho district, Vinh Long province from 28/3/2017 to 30/4/2017, T0C = 31.1; H% = 66.2 Treatment Pheromone lures (mg/septum) E11 E13-16: Z11-16:Ald Ald 0.35 0.15 0.35 0.15 0.35 0.15 0.35 10 µl n-hexane H.undalis virgin female males/trap/week (Total of males/3 weeks) E-1 0.05 0.22 c (0.67) E-2 0.05 0.11 c (0.33) E-3 2.56 c (7.67) E-4 0.05 56.33 a (169) E-5 0.00 c (0) E-6 42.89 b (128.67) CV (%) 16.07 Significance ** In the same column, numbers with same letters are not statistically different and by the Duncan test **: significant difference of 1% Z11-14: OAc 0.15 0.15 0.15 T23 As expected, the results presented in Table 4.15 show that a number of H undalis males were caught by related compounds of Z11-14:OAc (0.15 mg), Z11-16:Ald (0.15 mg), E11,E1316:Ald (0.35 mg) and T23 (0.05 mg) (treatment E-4, 56.33 males/trap/week) gave differ significantly compare with positive control (treatment E-6, 42.89 males/trap/week) and negative control (treatment E-5, 0.0 males/trap/week) On the other hand, the treatments that had three-component, no different significantly from negative control, sex pheromone of H undalis was a mixture of Z11-14:OAc, Z11-16:Ald, E11,E13-16:Ald and T23 serve as active ingredients The results show that alongside E11,E13-16:Ald, H undalis shows details Z11-14:OAc and Z11-16:Ald with hydrocarbons (T23) are pheromone sex components 23 4.7 Effect of semiochemical on mating and spawning cabbage webworm 4.7.1 Effect of semiochemical on mating Semiochemical: C citratus, A sativum and E10-15:Ald were harassing on mating of H undalis male, very different significantly compare with virgin female and n- hexane in the network and field trials (Table 4.16) Table 4.16: Number of H.undalis males into trap and harassing effect on mating of semiochemical Field trials Net - house Treatment Total adult male into Harassing trap (males/trap/week) effect (%) Total adult male into trap (males/trap/week) Harassing effect (%) 2♀ + E10-15 1.00 b 92.86 a 5.33 b 87.89 a 2♀ + C citratus 0.67 b 95.05 a 4.67 b 92.16 a 2♀ + A sativum 0.33 b 97.62 a 5.67 b 91.29 a 2♀ + n-hexane 13.00 a 7.33 b 55.33 a 6.15 b Virgin female (2♀) 13.67 a 0.00 b 54.33 a 0.00 b CV (%) 17.45 8.96 30,88 15.10 Significance ** ** ** ** In the same column, numbers with same letters are not statistically different and by the Duncan test **: significant difference of 1% Net-house: The number of H.undalis caught in traps on E10-15: Ald (1.00 males/trap/week), C citratus (0.67 males/trap/week) and A sativum (0.33 males/trap/week) were lower and differed significantly from the positive control (Virgin female, 13.67 males/trap/week) H undalis males caught in traps on the positive control and n-hexane (13.00 males/trap/week) were not different significantly, suggesting that n-hexane no effect of caught The results show that E10-15:Ald, C citratus and A sativum have been harassed on affecting the ability to find H undalis female to mating effects of 92.86%, 95.05% and 97.62%, respectively (Table 4.16) Field trials: the same as in net-house, E10-15:Ald, C citratus and A sativum were highly harassing to mating of H undalis male Number of H undalis caught into trap on E10-15:Ald (5.33 males/trap/week), C citratus (4.67 males/trap/week) and A sativum (5.67 males/trap/week) were lower and differed significantly from negative control (nhexane, 55.33 males/trap/week) and positive control (54.33 males/trap/week) (Table 4.16) Effect of harassing of E10-15:Ald, C citratus and A sativum were 87.89%, 92.16% and 91.29% at field trials, lower compare net-house, insignificant 4.7.2 Effect of semiochemical on spawning Net-house: number of eggs layed on green mustard on E10-15:Ald (14.33 eggs), C citratus (5.00 eggs) and A sativum (1.00 egg) were different significantly compare with nhexane (220.67 eggs) and virgin female (222.67 eggs) The results show that E10-15:Ald, C citratus and A sativum have an effect on the ability of H undalis to reach the host to lay eggs Effect of harassing of E10-15:Ald, C citratus and A sativum were 93.40%; 97.66% and 99.58%, respectively (Table 4.17) On the other hand, the number of eggs layed between n-hexane and positive control were not significantly different, suggesting that nhexane did not affect spawning 24 Table 4.17: Number of H.undalis females to eggs laying and harassing effect on spawning of semiochemical Field trials Net - house Total eggs Harassing Total eggs Harassing (eggs/5 effect (%) (eggs/5 pots) effect (%) pots) (3♀ + 3♂) + E10-15 14.33 b 93.40 b 1.00 b 97.62 a (3♀ + 3♂) + C citratus 5.00 b 97.66 a 1.33 b 96.83 a (3♀ + 3♂) + A sativum 1.00 b 99.58 a 1.33 b 96.33 a ( 3♀ + 3♂) + n-hexane 220.67 a 0.92 c 39.33 a - 0.84 b pairs of male and female 222.67 a 0.00 c 38.67 a 0.00 b CV (%) 12.68 2.81 25.25 8.38 Significance ** ** ** ** In the same column, numbers with same letters are not statistically different and by the Duncan test **: significant difference of 1% (1) Field trials: non 3♀ + 3♂ Treatment Field trials: Effect of harassing of E10-15:Ald, C citratus and A sativum were 97.62%; 96.83% and 96.33%, respectively, difference statistically compared to n-hexane and positive control Results in table 4.22 show that the number of eggs laid at survey point (Total 6.0 m2) of E10-15:Ald (1.00 egg/2.0 m2), C citratus (1.33 eggs/2.0 m2) and A sativum (1.33 eggs/2.0 m2) were different significantly compared with n-hexane (39.33 eggs/2.0 m2) and the positive control (38.67 eggs/2.0 m2) Although E10-15 had effectively harassed on mating and spawning of H undalis and similari significantly with C citratus and A sativum, the synthesis of E10-15:Ald difficulties and raw materials are scarce than C citratus and A sativum 4.8 Semiochemical application for the pest management of Hellula undalis 4.8.1 Effectiveness of C citratus and A sativum for the pest management of Hellula undalis 4.8.2.1 Effect of C citratus on spawning at field trials Table 4.18: Influence of C citratus to eggs laying of H undalis female at field trials Location put C citratus bags Before the experiment (eggs/ 2m2) Number of eggs of H undalis female (eggs/ 2m ) st nd rd Total Week Week Week Left m 3.00 1.67c 4.00b 2.00b 7.67b Right m 1.67 2.67bc 3.33b 1.33b 7.33b Above m 2.33 2.00c 5.00b 1.67b 8.67b Above m 0.67 1.67c 4.00b 2.33b 8.00b Above m 1.67 9.00a 14.33a 6.33a 29.67a Below m 2.00 2.67bc 4.33b 1.33b 8.33b Below m 5.00 3.33bc 5.00b 2.33b 10.67b Below m 4.33 7.67ab 12.67a 6.67a 27.00a CV (%) 84.44 42.55 36.05 37.52 15.76 Significance ns * * ** ** In the same column, numbers with same letters are not statistically different and by the Duncan test **: significant difference of 1%; *: significant difference of 5%; ns: no difference Datas were converted to Log (X + 1) before statistical analysis Table 4.18 shows that the number of H undalis eggs on B juncea at distances m and m at located hang C citratus plastic bags were not different significantly, but different 25 significantly with distance m These results show that the effective distance of C citratus bag (2 ml) for H undalis is m and the effective area of C citratus/ bag (2 ml) is about 64 m2 Datas were converted 4.8.2.2 Effect of A sativum on spawning at field trials The same as the effect on C citratus, after weeks of experiments show that eight survey distances to assess the influence of A sativum on harassing effect on spawning of H undalis at the field condition had significant statistical differences At distances above m and st rd below m has the highest number of eggs from week to week , respectively 10.67; 2 15.00; 6.67 eggs/2 m for the distance of above m and 10.67; 13.67; 7.00 eggs/2 m for a distance of below m, statistically significant difference with the remaining distances (