Luận án tiến sĩ về cơ chế kháng thuốc quinclorac của cỏ lồng vực nước Echinochloa crus-galli - Le Duy, Can Tho University

MINISTRY OF EDUCATION AND TRAINING CAN THO UNIVERSITY LE DUY STUDY ON THE RESISTANCE MECHANISM OF BARNYARDGRASS (Echinochloa crus-galli (L.) TO QUINCLORAC IN THE MEKONG DELTA OF VIETNAM DOCTORAL DISSERTATION IN PLANT PROTECTION SUPERVISOR Assoc. 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. 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 do 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. 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. Masters and Sir. Your behind the scene support are unmeasurable. And I would never be able to accomplish my goals without my family.

Le Duy i TÓM TẮT Đề tài được thực hiện nhằm nghiên cứu về tính kháng thuốc cỏ của cỏ lồng vực (Echinochloa spp.) trên ruộng lúa, 78 mẫu hạt cỏ lồng vực đã được sưu tập từ 7 tỉnh ở Đồng bằng sông Cửu Long (ĐBSCL). Các nghiên cứu trong luận án tìm thấy các quần thể cỏ lồng vực kháng thuốc cỏ thuộc nhóm ALS (bispyribac và penoxsulam) và nhóm thuốc cỏ auxin tổng hợp (quinclorac), giá trị LD90 trung bình của bispyribac, penoxsulam và quinclorac lần lượt là 33,1; 15,1 và 550,2 g/ha. Kết quả thử nghiệm của thuốc trừ cỏ rinskor trên các quần thể cỏ lồng vực kháng thuốc cho thấy các quần thể kháng thuốc trên đều mẫn cảm với rinskor. Kết quả phân tích kiểu hình cỏ lồng vực trong luận văn cho thấy có 3 nhóm cỏ chính tương ứng với 3 loài cỏ lồng vực tại ĐBSCL là Echinochloa crus-galli, Echinochloa oryzoides và Echinochloa erecta, trong đó cỏ lồng vực nước (Echinochloa crus-galli) là loài phổ biến nhất.

Nhằm làm rõ hơn về đa dạng di truyền trong quần thể cỏ, phương pháp Random amplified polymorphic DNA (RAPD) đã được dùng để phân tích di truyền của 13 quần thể tại Việt Nam và 2 quần thể cỏ tại Mỹ. Kết quả cho thấy 6 đoạn mồi oligonucleotide cho kết quả 46 băng đa hình trong 15 quần thể, khoảng cách di truyền của các quần thể trong cây phả hệ là 0,09 đến 0,39. Kết quả của phân tích di truyền bằng phương pháp RAPD giúp khẳng định mức độ đa dạng di truyền cao trong quần thể cỏ lồng vực tại ĐBSCL, nhiều loài bị nhầm lẫn với nhau do rất giống nhau về kiểu hình. Nhằm làm rõ cơ chế kháng thuốc quinclorac của cỏ lồng vực nước (Echinochloa crus-galli), nghiên cứu đã tập trung tìm hiểu mức độ phiên mã của gen và mức độ hoạt động của enzym β-cyanoalanine synthase (CAS) trong lá của 5 quần thể cỏ và lúa sau khi xử lý thuốc.

Kết quả cho thấy ở thời điểm 1 giờ sau khi phun quinclorac, các quần thể kháng thuốc (R) có thể đẩy nhanh quá trình phiên mã và chuyển hóa thành enzyme CAS, tốc độ của quá trình này nhanh hơn so với các quần thể mẫn cảm (S). Ở thời điểm 3 ngày sau xử lý , mức độ phiên mã của gene CAS trong các quần thể R giảm về mức khác biệt không có ý nghĩa so với đối chứng, tuy nhiên mức độ hoạt động của enzyme CAS vẫn ở mức cao so với đối chứng và 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 7 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. 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 3 main groups that corresponding to 3 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. Six oligonucleotide primers produced 46 bands were polymorphic among the 15 populations. The cluster analysis separated the 15 populations into 2 main clusters with the genetic distances within the clusters ranging from 0. 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 5 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.D Student iv TABLE OF CONTENTS Page SUMMARY. iii TABLE OF CONTENTS .2 Targets of dissertation .3 Studied objectives and limitation of dissertation .4 Major research of dissertation .5 Contributions of dissertation .1 Overview of the Mekong Delta in Vietnam and rice cultivation .2 Definitions of weed and herbicide resistance .3 Overview of Echinochloa spp.

in the rice field. Herbicide for barnyardgrass control .1 Overview of herbicidal active ingredient bispyribac .2 Overview of herbicidal active ingredient penoxsulam .3 Overview of herbicidal active ingredient quinclorac .5 Herbicide resistance and testing methods .1 The importance of herbicide resistance management.2 Target site resistance .3 Non target site resistance .4 Multiple herbicide resistance .5 Popular testing methods for herbicide resistance .6 Herbicide resistance management strategy .1 Minimize weed seed dispersal .3 Herbicide rotation and herbicide mixture .7 Reported mechanism of herbicide resistant barnyardgrass (Echinochloa crus-galli) .1 Herbicide resistance research in Echinochloa spp.2 Enhancement of β-CAS synthase (detoxification of cyanide) in quinclorac resistance in Echinochloa spp.3 Modification in the transduction pathway of auxin reception-signal in R and S Echinochloa plant .4 Other factors associated to the resistance mechanisms to quinclorac in barnyardgrass .5 Herbicide resistance via pollen mediated gene flow in barnyardgrass .37 MATERIALS AND METHODS .1 Conceptual framework diagram .1 Survey on farmer practice in rice cultivation and weed management in the Mekong Delta .2 Classification of the collected Echinochloa spp. populations based on plant characteristics.3 Evaluate the herbicide-resistance level in collected Echinochloa spp. populations to 3 active ingredients of bispyribac-sodium, penoxsulam and quinclorac by dose-response screening method .4 Evaluate the efficacy of rinskor as new herbicide in herbicide resistance barnyardgrass populations .5 Compare the activity of enzyme β-cyanoalanine (CAS) in quinclorac- susceptible and quinclorac-resistant barnyargrass plant to study biochemical mechanism of quinclorac-resistance in barnyardgrass .6 Identify genetic variation among quinclorac-resistant and quinclorac- susceptible Echinochloa crus-galli populations in the Mekong Delta .7 Measure mRNA expression level of CAS gene in quinclorac-resistant and quinclorac-susceptible barnyardgrass .57 RESULTS AND DISCUSSION .1 Herbicide application practice and weed management in rice field at Mekong Delta.1 Rice cultivation practice .2 Important weed species in the rice field at seven provinces of the Mekong Delta .3 Weed management by hand weeding .4 Weed escaped controlling and the cost on weed management in the Mekong Delta .2 Morphology and distribution of Echinochloa spp.

in the Mekong Delta .2 Correlation between biological characteristics of Echinochloa plants.3 Distribution of Echinochloa spp. in the Mekong Delta .3 Herbicide resistant Echinochloa spp. in the Mekong Delta .1 Distribution of herbicide resistant Echinochloa spp. in 7 provinces of Mekong Delta .2 Herbicide resistance in three weed groups .3 The solo resistance and multiple resistance in Echinochloa spp.4 Evaluate multiple herbicide resistance level by resistance score .5 Impact of field size to resistance score under different water management conditions .6 Correlation between field size and hand weeding .7 The impact of hand-weeding to herbicide resistance of Echinochloa spp.

in the Mekong Delta.4 Weed control efficacy of rinskor in Echinochloa spp. in the Mekong Delta .1 Control efficacy of rinskor as a new herbicide against three Echinochloa spp. groups collected in the Mekong Delta .2 Control efficacy of rinskor as new herbicide against Echinochloa spp. populations collected in Mekong Delta .3 Correlation between resistance level of bispyribac, penoxsulam and quinclorac .4 Efficacy of bispyribac, penoxsulam and quinclorac in susceptible E.

crus-galli compared to resistant plants .5 Efficacy of rinskor for control of susceptible or resistant barnyardgrass to bispyribac, penoxsulam and quinclorac .5 Biodiversity study by RAPD analysis in 15 barnyardgrass populations from Vietnam and the U.1 RAPD analysis of 15 barnyardgrass populations .2 The genetic diversity of Echinochloa crus-galli and herbicide resistance level .6 Biochemical mechanism and molecular mechanism of quinclorac resistance in barnyardgrass .1 B-CAS activity in 5 quinclorac resistant barnyardgrass populations .2 CAS transcript abundance in leaf tissue of five barnyardgrass populations .3 Biochemical and molecular mechanism of quinclorac resistance in Echinochloa crus-galli in Mekong Delta .101 CONCLUSIONS AND RECOMMENDATIONS .103 viii LIST OF TABLES Page Table 3.1 List of primers were used for RAPD analysis in the research .2 Steps in PCR reactions .3 RT-qPCR primer sequence detecting β-CAS and β-Actin synthase .1 Rice cultivation practice of farmer in Mekong Delta .2 Farmer perception about important weed species in rice field at 7 provinces of Mekong Delta .3 Farmer response about most escaped weed after herbicide treatments and need hand-weeding to control .4 The three most popular herbicides for escaped Echinochloa spp. control in Mekong Delta .5 The cost for weed management in Mekong Delta.6 Plant characteristic of Echinochloa spp. collected in Mekong Delta .7 Distribution of Echinochloa species in Mekong Delta .8 Herbicide-resistance level in populations to bispyribac, penoxsulam and quinclorac in different provinces .9 Percent of solo-resistance and multiple-resistance herbicides in three groups of Echinochloa spp.10 Percent of barnyardgrass population resistant to single and multiple herbicides of bispyribac, penoxsulam and quinclorac in different provinces .11 Resistance Score of bispyribac, penoxsulam and quinclorac herbicide- resistance of 78 Echinochloa spp.12 Average LD90 of 3 Echinochloa groups to bispyribac, penoxsulam, quinclorac and rinskor .13 Average LD90 of barnyardgrass population to bispyribac, penoxsulam, quinclorac and rinskor .14 Six informative primers in RAPD analysis of Echinochloa crus-galli populations.15 Lethal dose of quinclorac needed to kill 90% of the population (LD 90) and the Resistance level of 15 Echinochloa crus-galli populations collected in Vietnam and U.16 Mortality of herbicide susceptible (S) and herbicide resistant (R) barnyardgrass treated by rinskor at different dose .17 Six informative primers in RAPD analysis of Echinochloa crus-galli populations.