MINISTRY OF EDUCATION AND TRAINING CAN THO UNIVERSITY LE DUY STUDY ON THE RESISTANCE MECHANISM OF BARNYARDGRASS (Echinochloa crus-galli (L.) Beauv.) TO QUINCLORAC IN THE MEKONG DELTA OF VIETNAM DOCTORAL DISSERTATION IN PLANT PROTECTION SUPERVISOR Assoc Prof Dr NGUYEN MINH CHON 2018 ACKNOWLEDGEMENT I appreciate all the support from my dissertation supervisor Associate Professor Dr Nguyen Minh Chon, Deputy Director of Biotechnology Research and Development Institute, Can Tho University, and my mentor Dr Richard K Mann, Research Fellow of Dow AgroSciences, Dr Chon and Dr Mann are the two scientists who have been restlessly supporting my career and always provide the valuable advice for this dissertation I am grateful to all of all fellows and friends with whom I have worked together in this projects I appreciate Mr Nguyen Tan Thuan and Ms Tran Thi Lai who helped on the seed collections and the data collection, also Mr Ngo Thanh Phu who greatly helps to format the document Dr Yerkes, Dr Cicchillo, Staci, Dave, Debbie and Bill of Discovery Center, Dow AgroSciences, the dissertation will never be done without your expertise, my sincere appreciation to all of you This dissertation would not have been done without Dr Hutchin, Dr Bobba, Dr Masters and Sir Taylor Your behind the scene support are unmeasurable And I would never be able to accomplish my goals without my family Tien Giang,…………………… Le Duy i TÓM TẮT Đề tài thực nhằm nghiên cứu tính kháng thuốc cỏ cỏ lồng vực (Echinochloa spp.) ruộng lúa, 78 mẫu hạt cỏ lồng vực sưu tập từ tỉnh Đồng sông Cửu Long (ĐBSCL) Các nghiên cứu luận án tìm thấy quần thể cỏ lồng vực kháng thuốc cỏ thuộc nhóm ALS (bispyribac penoxsulam) nhóm thuốc cỏ auxin tổng hợp (quinclorac), giá trị LD90 trung bình bispyribac, penoxsulam quinclorac 33,1; 15,1 550,2 g/ha Kết thử nghiệm thuốc trừ cỏ rinskor quần thể cỏ lồng vực kháng thuốc cho thấy quần thể kháng thuốc mẫn cảm với rinskor Kết phân tích kiểu hình cỏ lồng vực luận văn cho thấy có nhóm cỏ tương ứng với lồi cỏ lồng vực ĐBSCL Echinochloa crus-galli, Echinochloa oryzoides Echinochloa erecta, cỏ lồng vực nước (Echinochloa crus-galli) loài phổ biến Nhằm làm rõ đa dạng di truyền quần thể cỏ, phương pháp Random amplified polymorphic DNA (RAPD) dùng để phân tích di truyền 13 quần thể Việt Nam quần thể cỏ Mỹ Kết cho thấy đoạn mồi oligonucleotide cho kết 46 băng đa hình 15 quần thể, khoảng cách di truyền quần thể phả hệ 0,09 đến 0,39 Kết phân tích di truyền phương pháp RAPD giúp khẳng định mức độ đa dạng di truyền cao quần thể cỏ lồng vực ĐBSCL, nhiều loài bị nhầm lẫn với giống kiểu hình Nhằm làm rõ chế kháng thuốc quinclorac cỏ lồng vực nước (Echinochloa crus-galli), nghiên cứu tập trung tìm hiểu mức độ phiên mã gen mức độ hoạt động enzym β-cyanoalanine synthase (CAS) quần thể cỏ lúa sau xử lý thuốc Kết cho thấy thời điểm sau phun quinclorac, quần thể kháng thuốc (R) đẩy nhanh q trình phiên mã chuyển hóa thành enzyme CAS, tốc độ trình nhanh so với quần thể mẫn cảm (S) Ở thời điểm ngày sau xử lý , mức độ phiên mã gene CAS quần thể R giảm mức khác biệt khơng có ý nghĩa so với đối chứng, nhiên mức độ hoạt động enzyme CAS mức cao so với đối chứng quần thể S Từ khóa: cỏ lồng vực, cyanoalanide synthase, kháng thuốc cỏ, RAPD, ii SUMMARY The aim of this dissersation was to study the herbicide resistance of the barnyardgrass (Echinochloa spp.) in provinces of the Mekong Delta of Vietnam, seventy-eight seed samples of Echinochloa spp collected from rice field for the study The results found the ALS-resistant and synthetic auxin-resistant E crus-galli were confirmed at several locations in the Mekong Delta The average LD90 value of bispyribac, penoxsulam and quinclorac for assessed weed populations was 33.1, 15.1 and 550.2 g a.i/ha respectively The new herbicide rinskor was tested in weed populations exhibited resistance to current herbicides, results showed that the Echinochloa populations resistant to bispyribac, penoxsulam and quinclorac were susceptible to the rinskor under greenhouse test The morphology analysis indicated there are main groups that corresponding to species of Echinochloa crus-galli, Echinochloa oryzoides and Echinochloa erecta found in Mekong Delta, the Echinochloa crus-galli was the most popular species identified in the study, to extend the study, we used random amplified polymorphic DNA (RAPD) analysis and greenhouse testing to study the genetic diversity of 15 Echinochloa populations in the Mekong Delta, Vietnam, and the state of Arkansas, U.S Six oligonucleotide primers produced 46 bands were polymorphic among the 15 populations The cluster analysis separated the 15 populations into main clusters with the genetic distances within the clusters ranging from 0.09 to 0.39 The results of RAPD are useful to confirm the high diversity of Echinochloa spp populations in Mekong Delta of Vietnam, many Echinochloa species with similar morphology could be confused with the others To focus on the mechanism of quinclorac resistance in barnyardgrass (Echinochloa crus-galli), the research have investigated the transcript and activity of enzyme β-cyanoalanine synthase (CAS) in leaf tissue of barnyardgrass populations and rice One hour post quinclorac treatment, R populations were able to rapidly utilize CAS transcript to possibly fuel increased CAS protein activity, this process is significantly higher than the process in S populations Three days following quinclorac treatment, the utilization effect on CAS transcript levels had ceased, however, CAS protein activity remained higher in every population compared to non-treated controls and S populations Keywords: Echinochloa, cyanoalanine synthase, Herbicide resistance, RAPD iii STATEMENT ON ACADEMIC INTEGRITY The results presented in this dissertation is the sole effort of the author, except where explicitly stated All references related to the studies are acknowledged and properly cited All of data and research results in this document are not published in publications of any different authors Ph.D Student iv TABLE OF CONTENTS Page SUMMARY iii TABLE OF CONTENTS .v CHAPTER .1 INTRODUCTION 1.1 Problem statement 1.2 Targets of dissertation .2 1.3 Studied objectives and limitation of dissertation .2 1.4 Major research of dissertation 1.5 Contributions of dissertation .3 CHAPTER .4 LITERATURE REVIEW 2.1 Overview of the Mekong Delta in Vietnam and rice cultivation .4 2.2 Definitions of weed and herbicide resistance .5 2.3 Overview of Echinochloa spp in the rice field 2.4 Herbicide for barnyardgrass control 10 2.4.1 Overview of herbicidal active ingredient bispyribac 10 2.4.2 Overview of herbicidal active ingredient penoxsulam .11 2.4.3 Overview of herbicidal active ingredient quinclorac 13 2.5 Herbicide resistance and testing methods 15 2.5.1 The importance of herbicide resistance management .15 2.5.2 Target site resistance 17 2.5.3 Non target site resistance 19 2.5.4 Multiple herbicide resistance 23 2.5.5 Popular testing methods for herbicide resistance 24 2.6 Herbicide resistance management strategy 31 2.6.1 Minimize weed seed dispersal .31 2.6.2 Crop rotation 31 2.6.3 Herbicide rotation and herbicide mixture 31 2.7 Reported mechanism of herbicide resistant barnyardgrass (Echinochloa crusgalli) 32 2.7.1 Herbicide resistance research in Echinochloa spp 32 2.7.2 Enhancement of β-CAS synthase (detoxification of cyanide) in quinclorac resistance in Echinochloa spp .34 2.7.3 Modification in the transduction pathway of auxin reception-signal in R and S Echinochloa plant 36 2.7.4 Other factors associated to the resistance mechanisms to quinclorac in barnyardgrass 36 2.7.5 Herbicide resistance via pollen mediated gene flow in barnyardgrass 37 CHAPTER .37 MATERIALS AND METHODS .37 3.1 Conceptual framework diagram .37 3.2 Materials 38 3.3 Research methods .42 3.3.1 Survey on farmer practice in rice cultivation and weed management in the Mekong Delta 42 3.3.2 Classification of the collected Echinochloa spp populations based on plant characteristics 43 3.3.3 Evaluate the herbicide-resistance level in collected Echinochloa spp populations to active ingredients of bispyribac-sodium, penoxsulam and quinclorac by dose-response screening method 44 3.3.4 Evaluate the efficacy of rinskor as new herbicide in herbicide resistance barnyardgrass populations 47 3.3.5 Compare the activity of enzyme β-cyanoalanine (CAS) in quincloracsusceptible and quinclorac-resistant barnyargrass plant to study biochemical mechanism of quinclorac-resistance in barnyardgrass 47 3.3.6 Identify genetic variation among quinclorac-resistant and quincloracsusceptible Echinochloa crus-galli populations in the Mekong Delta 49 3.3.7 Measure mRNA expression level of CAS gene in quinclorac-resistant and quinclorac-susceptible barnyardgrass .52 CHAPTER .57 RESULTS AND DISCUSSION 57 4.1 Herbicide application practice and weed management in rice field at Mekong Delta .57 4.1.1 Rice cultivation practice .57 4.1.2 Important weed species in the rice field at seven provinces of the Mekong Delta 59 4.1.3 Weed management by hand weeding 60 4.1.4 Weed escaped controlling and the cost on weed management in the Mekong Delta 62 4.2 Morphology and distribution of Echinochloa spp in the Mekong Delta 64 4.2.1 Plant characteristics .64 4.2.2 Correlation between biological characteristics of Echinochloa plants 67 4.2.3 Distribution of Echinochloa spp in the Mekong Delta 69 4.3 Herbicide resistant Echinochloa spp in the Mekong Delta .71 4.3.1 Distribution of herbicide resistant Echinochloa spp in provinces of Mekong Delta 71 4.3.2 Herbicide resistance in three weed groups 72 4.3.3 The solo resistance and multiple resistance in Echinochloa spp populations .73 4.3.4 Evaluate multiple herbicide resistance level by resistance score 75 vii 4.3.5 Impact of field size to resistance score under different water management conditions .76 4.3.6 Correlation between field size and hand weeding 77 vii G Activity of enzyme CAS at 3d (control) Summary of Fit Rsqua re Adj Rsqua Root Value 0.987419 0.982177 2.238804 Mean Mean 13.20167 of Obser 18 vation An aSlyource DF Sum of Squares Mean Square F Ratio Prob > F Na < 47 944.133 me 00 20 Er 12 60.14 5.0 ror 69 12 C 17 47 To 80 CV(%)=127.3 G Activity of enzyme CAS at 3d (treated) Summary of Fit Rsqua re Adj Rsqua Root 0.997559 0.996542 1.032831 Mean Mean 16.41889 of Obser 18 vation An aSlyource N a Er 12 ro C 17 To C V( Value DF Sum of Squares < 52 1046.39 00 31 12.80 1.0 09 527 44 Mean Square F Ratio Prob > F 0.9 0.8 0.7 0.6 Series1 0.5 Linear (Series1) 0.4 0.3 0.2 0.1 0 20 40 60 80 100 120 G Na2S Standard Curve EXTR A650 APPENDIX H CAS transcript in R populations barnyardgrass population and Oryza sativa at hour after treated H 1.1 Ech_01 (None-Treated and Quinclorac Treated) Assuming unequal variances Di ffe St 0.33383 0.01750 83.57322 R 0.05242 dUp pe Lo DF 0.17016 0.7 P 57 -0.3 we Co nfi P 78 0.95 0.6 21 H 1.2 Ech_02 (None-Treated and Quinclorac Treated) Assuming unequal variances Di ffe St 1.21267 0.03750 85.38582 R 0.03092 dUp pe Lo DF 0.11531 0.2 P 75 -0.1 we Co nfi P 37 0.95 0.8 62 H 1.3 Ech_03 (None-Treated and Quinclorac Treated) Assuming unequal variances Di ffe St - t0.06329 3.23303 dUp pe Lo DF - 50.0015* P -0.9 we Co nfi P 99 0.0008* 0.95 H 1.4 Ech_04 (None-Treated and Quinclorac Treated) Assuming unequal variances Di ffe St - t0.05443 3.60262 dUp pe Lo DF - 50.0003* P -0.9 we Co nfi P 99 0.0002* 0.95 H 1.5 Ech_05 (None-Treated and Quinclorac Treated) Assuming unequal variances Di ffe St dUp pe Lo we Co nfi - R 3.97095 - 40.0241* P -0.9 P 88 0.0120* 0.95 H 1.6 Rice (None-Treated and Quinclorac Treated) Assuming unequal variances Di ffe St - t0.03506 3.95661 dUp pe Lo DF - 10.0048* P -0.9 we Co nfi P 97 0.0024* 0.95 CAS transcript in R populations Ech_03, Ech_04, Ech_05 and Oryza sativa at day after treated H 2.1 CAS transcript in Ech_01 (None-Treated and Quinclorac Treated) Assuming unequal variances Di ffe St 8.68411 0.13500 25.24532 00.01554 dUp pe Lo 60.17440 20.0003* 60.09559 0.0001* we Co nfi 40.95 0.9 99 H 2.2 CAS transcript in Ech_02 (None-Treated and Quinclorac Treated) Assuming unequal variances Di ffe St - t0.01652 dUp pe Lo DF - < P - 1.0000 we Co nfi P 0.95< H 2.3 CAS transcript in Ech_03 (None-Treated and Quinclorac Treated) Assuming unequal variances Di ffe St - t0.07793 3.56034 dUp pe Lo DF 0.21982 0.9 P 28 -0.5 we Co nfi P 35 0.95 0.4 64 H 2.4 CAS transcript in Ech_04 (None-Treated and Quinclorac Treated) Assuming unequal variances Di ffe St 0.09982 0.01250 R 0.12522 5.91635 dUp pe Lo DF 0.31997 0.9 P 23 -0.4 we Co nfi P 61 0.95 0.5 38 H 2.5 CAS transcript in Ech_05 (None-Treated and Quinclorac Treated) Assuming unequal variances Di ffe St 0.19432 0.00750 53.20814 R 0.03860 dUp pe Lo DF 0.12594 0.8 P 57 -0.4 we Co nfi P 28 0.95 0.5 71 H 2.6 CAS transcript in Rice (None-Treated and Quinclorac Treated at 3d) Assuming unequal variances Di ffe St - t0.04093 5.99866 dUp pe Lo DF 0.00515 0.0 P 59 -0.9 we Co nfi P 70 0.0297* 0.95 APPENDIX I The protocol of High Capacity cDNA Reverse Transcription Kits (Thermo Fisher cat# 4368813) was used to synthesized the cDNA Workflow descriptions: Prepare 2X reverse transcription master mix Add RNA to reverse transcription reactions Perform reverse transcription in a thermal cycler Use the reverse transcription reactions (cDNA) directly for quantitative or other PCR application Store the reverse transcription reactions (cDNA) at: o o C to C for short-term storage -25oC to -15oC for long-term storage A Prepare the 2X RT master mix Allow the kit components to thaw on ice Calculate the volume of components needed to prepare the required number of reactions Note: Prepare the RT master mix on ice C o m WW i i 2 0 0 8 2 0 M 1 ul 0 ti µ µ R N N uc 2 T 10 ot .0 Place the 2X RT master mix on ice and mix gently B Prepare the reverse transcription reactions Pipette 10 μL of 2X RT master mix into each well of a 96-well reaction plate or individual tube Pipette 10 μL of RNA sample into each well, pipetting up and down two times to mix Seal the plates or tubes Briefly centrifuge the plate or tubes to spin down the contents and to eliminate any air bubbles Place the plate or tubes on ice until you are ready to load the C Program the thermal cycling conditions Program the thermal cycler using the conditions below S StepStepStepStep e o T 25 37 84 C m T 1 5∞ m APPENDIX K Triticum aestivum (LOC100682425) beta-cyanoalanine synthase mRNA, complete cds ATGGAGAGGG TGGTGATGAG GCTGGTGAGG AACAAGCAGT CCCTCCGCCA GCTGCAAGGG 61 GCCGCCGCCG GCGGCCTAGC GTCGTCGTCC TCCGCCTCCT CGACCGCCGG CGCCGCCGCG 121 TCCTCCTTCT CCACCCTGCA GCAGCAGCAG GAGGACCACC CCGGAGTGCT CAACATCAGG 181 GACACCGCCG CCCACCTCAT CGGCCGGACG CCGCTGGTGT ACCTGAACAA GGTGACGGAG 241 GGGTGCGGCG CCCGCGTCGC CGCCAAGCTC GAGTTCCTCC AGCCTTCCTT CAGCGTCAAG 301 GACAGGCCAG CAATCTCCAT GATCGAAGAC GCGGAGAAGA AGGGGCTGAT AACCCCAGGC 361 AAGACGACGC TGATCGAGCC CACATCCGGG AACATGGGCA TCGGCCTGGC GTTCATGGCA 421 GCACTCAAAG GGTACGAGCT CGTGCTGACG ATGCCATCAT ACACCAGCCT CGAGAGGAGG 481 GTGGTCATGA AGGCCTTCGG TGCGCAGCTC GTGCTCACTG ACCCGGCCAA GGGGATGGGA 541 GGCACCGTCA GGAAAGCAAC CCAGCTCTAT GAGAACCATC CCAGCGCCTT CATGCTCCAG 601 CAGTTCGAGA ATCCCGCCAA CGTCCAGGTG CACTACGAGA CTACCGGTCC GGAGATATGG 661 GAGGACACGC TCGGGCAGGT CGACATCTTC GTCATGGGAA TCGGCAGCGG CGGCACCGTC 721 ACCGGCGTCG GCAAGTACCT CAAGGAGAAG AACCCCAACG CAAAGATCTA TGGAGTTGAA 781 CCTGCCGAAG CAAATGTCCT GAACGGTGGC AAGCCAGGGC CTCACCTCAT CACTGGAAAT 841 GGAGTTGGAT TTAAGCCAGA TATTTTGGAT ATGGATATAA TGGAGAAGGT TCTAGAGGTG 901 AAAAGCGAGG ACGCCGTTAC GATGGCGCAA CAGTTGGCAC TGCAGGAGGG CCTGCTGGTG 961 GGGATATCAT CAGGAGCCAA CACAGTGGCT GCGATCGAAC TCGCAAAGAG GCCAGAAAAC 1021 AAAGGGAAGC TCATCGTGAC TGTTCATCCG AGCGCGGGAG AGCGATACTT ATCGTCGGCG 1081 CTATTCGAGG GTCTGAGGAA GGAGGCTGAG GCAATGCAGC CAGTGCCAGT GGACTAA Echinochloa crus-galli β-Actin mRNA, partial cds (HQ395760.1) CTCGACTCTG GTGATGGTGT GAGCCACACT GTCCCAATTT ACGAAGGGTA CACGCTTCCT 61 CATGCTATTC TTCGATTGGA CCTTGCTGGT CGTGACCTTA CCGACAACCT GATGAAGATC 121 CTTACCGAGA GGGGTTACTC CTTCACCACA ACCGCCGAGC GAGAAATTGT CCGTGACATC 181 AAGGAAAAGC TTGCCTACAT TGCCCTTGAC TACGAGCAGG AGCTGGAGAC TGCCAGGACC 241 AGCTCCACCG TTGAGAAGAG CTACGAGCTG CCTGATGGAC AGGTCATCAC AATTGGTGCT 301 GAGAGGTTCA GGTGCCCTGA GGTGCTGTTC CAGCCATCGT TCATTGGCAT GGAAGCTCCT 361 GGCATCCATG AAGCCACATA CAACTCCATC ATGAAGTGCG ATGTTGATAT CAGGAAGGAC 421 CTATATGGTA ATGTCGTCCT CAGTGGTGGT TCCACGATGT TCCCAGGTAT TGCTGACCGT 481 ATGAGCAAGG AGATCACTGC GCTTGCACCC AGCAGCATGA AGGTGAAGGT GGTTGCGCCA 541 CCAGAGAGGA AGTACAGTGT CTGGATTGGT GGCTCCATTC TGGCCTCTCT CAGTACCTTC 601 CAGCAGATGT GGATCTCCAA GGCGGAGTAC GA APPENDIX L Photos of the herbicide test 0.25 X 0.5 X 1.0 X 2.0 X 4.0 X 8.0 X L Dose response of a moderate quinclorac-resistant (RR) barnyardgrass sample collected in Mekong delta, photo taken at day after treated S population R? population RR population RRR population L Response of different barnyardgrass populations to the label dose of quinclorac at days after treated L One survival barnyardgrass in the population treated by quinclorac at 3-4 leaf stage, photo taken at 10 day after treatment, the plant stunted but recovered from the quinclorac damage L The seed of E.crus-galli L The seed of E oryzoides L The seed of E erecta Rice seedling Green basal of Echinochloa oryzoides L Seedling of E oryzoides in the rice field Red basal of Echinochloa crus-galli L Seedling of E crus-galli in the rice field APPENDIX M The suggested morphological key by Tabacchi et al (2006) for the classification of the Echinochloa as showed in below description, the author using the Amplified fragment length polymorphism (AFLP) to validate the Echinochloa spp classification, the traits were based on the descriptions of Pignatti’s calssification key and Carretero’s key: Red basal stem section, spikelet length _ 2.5 mm E colona Red basal stem section, spikelet length _ mm and_ 3.6 mm E crus-galli Green basal stem section, spikelet length _ 3.9 mm 2 Leaf sheath hairs present …………………………… E phyllopogon Leaf sheath hairs absent …………………………… 3 Spikelets with awn longer than cm Ratio G/L _0.45 E oryzoides Spikelets awnless or mucronate Ratio G/L _ 0.45 E oryzicola Carretero’s taxonomy (Carretero, 1981) (translated from Spanish): Spikelets less than 2.8 mm in length……………………………………… E colonun Greater spikelets…………………………………………………………………… 2 Large spikelets (at least 3.6 mm in length) Lower glume ½ to 3/5 of the length of the spikelet Sterile with or nerves…………… ……………… …… E oryzicola Without all these characters at once……………………………………………… 3 Most spikelets are less than 3.4 mm in length and abundant and long spines Frequently curved leaves………………………………………………… E crus-galli Many of the spikelets are at least 3.4 mm in length Leaves generally straight…….4 Spikelets easily exceed 3.6 mm in length Inflorescence generally ñútante and aristada Glabrous pods and pulvins Stigmata usually reddish………… E oryzoides Spikelets of 3.2-3.8 mm Inflorescence form erect to unattached, aristada or mocha Glabrous or hairy pods and pulvins Stigmata white or reddish… E hispidula Note: The weed species validation conducted by Tabacchi et al (2006) using Amplified fragment length polymorphism (AFLP) marker identified the E hispidula (described by Carretero, 1981) is the same species of E erecta (described by Pignatti, 1982) The plant characteristics of E erecta also aligned with the descriptions of Heap (2017) Therefore the species name of E erecta was used for the weed found in this study ... nhằm nghiên cứu tính kháng thuốc cỏ cỏ lồng vực (Echinochloa spp.) ruộng lúa, 78 mẫu hạt cỏ lồng vực sưu tập từ tỉnh Đồng sông Cửu Long (ĐBSCL) Các nghiên cứu luận án tìm thấy quần thể cỏ lồng vực. .. thuốc trừ cỏ rinskor quần thể cỏ lồng vực kháng thuốc cho thấy quần thể kháng thuốc mẫn cảm với rinskor Kết phân tích kiểu hình cỏ lồng vực luận văn cho thấy có nhóm cỏ tương ứng với lồi cỏ lồng. .. truyền cao quần thể cỏ lồng vực ĐBSCL, nhiều loài bị nhầm lẫn với giống kiểu hình Nhằm làm rõ chế kháng thuốc quinclorac cỏ lồng vực nước (Echinochloa crus- galli), nghiên cứu tập trung tìm hiểu