Nghiên cứu tính kháng và cơ chế kháng thuốc của cỏ lồng vực nước (echinochloa crus galli) đối với hoạt chất quinclorac tại đồng bằng sông cửu long

The new herbicide rinskor was tested in weedpopulations exhibited resistance to current herbicides, results showed that the Echinochloa populations resistant to bispyribac, penoxsulam an

MINISTRY OF EDUCATION AND TRAINING

CAN THO UNIVERSITY

LE DUY

STUDY ON THE RESISTANCE MECHANISM OF

BARNYARDGRASS (Echinochloa crus-galli (L.)

2018

ACKNOWLEDGEMENT

I appreciate all the support from my dissertation supervisor Associate Professor Dr.Nguyen Minh Chon, Deputy Director of Biotechnology Research and DevelopmentInstitute, 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 beenrestlessly supporting my career and always provide the valuable advice for thisdissertation

I am grateful to all of all fellows and friends with whom I have worked together in thisprojects I do appreciate Mr Nguyen Tan Thuan and Ms Tran Thi Lai who helped onthe 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 ofDiscovery Center, Dow AgroSciences, the dissertation will never be done without yourexpertise, 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

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 quầnthể cỏ lồng vực kháng thuốc cỏ thuộc nhóm ALS (bispyribac và penoxsulam) và nhómthuốc 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 củathuốc trừ cỏ rinskor trên các quần thể cỏ lồng vực kháng thuốc cho thấy các quần thểkháng 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) đãđược 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 đadạng di truyền cao trong quần thể cỏ lồng vực tại ĐBSCL, nhiều loài bị nhầm lẫn vớinhau 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 quầnthể cỏ và lúa sau khi xử lý thuốc Kết quả cho thấy ở thời điểm 1 giờ sau khi phunquinclorac, các quần thể kháng thuốc (R) có thể đẩy nhanh quá trình phiên mã vàchuyển hóa thành enzyme CAS, tốc độ của quá trình này nhanh hơn so với các quầnthể 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 mứckhác biệt không có ý nghĩa so với đối chứng, tuy nhiên mức độ hoạt động của enzymeCAS vẫn ở mức cao so với đối chứng và quần thể S

Từ khóa: cỏ lồng vực, cyanoalanide synthase, kháng thuốc cỏ, RAPD,

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 ofbispyribac, penoxsulam and quinclorac for assessed weed populations was 33.1, 15.1and 550.2 g a.i/ha respectively The new herbicide rinskor was tested in weedpopulations 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 polymorphicDNA (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 amongthe 15 populations The cluster analysis separated the 15 populations into 2 mainclusters 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 5 barnyardgrass populationsand rice One hour post quinclorac treatment, R populations were able to rapidly utilizeCAS transcript to possibly fuel increased CAS protein activity, this process issignificantly higher than the process in S populations Three days following quincloractreatment, the utilization effect on CAS transcript levels had ceased, however, CASprotein activity remained higher in every population compared to non-treated controlsand S populations

Keywords: Echinochloa, cyanoalanine synthase, Herbicide resistance, RAPD

STATEMENT ON ACADEMIC INTEGRITY

The results presented in this dissertation is the sole effort of the author, exceptwhere explicitly stated All references related to the studies are acknowledged andproperly cited All of data and research results in this document are not published inpublications of any different authors

Ph.D Student

iv

TABLE OF CONTENTS

Page

SUMMARY iii

TABLE OF CONTENTS v

CHAPTER 1 1

INTRODUCTION 1

1.1 Problem statement 1

1.2 Targets of dissertation 2

1.3 Studied objectives and limitation of dissertation 2

1.4 Major research of dissertation 2

1.5 Contributions of dissertation 3

CHAPTER 2 4

LITERATURE REVIEW 4

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 8

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 crus-galli) 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 3 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 3 active ingredients of bispyribac-sodium, penoxsulam and quinclorac by dose-response screening method 44

73.3.4 Evaluate the efficacy of rinskor as new herbicide in herbicide resistancebarnyardgrass populations 47

3.3.5 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 47

3.3.6 Identify genetic variation among resistant and quinclorac-susceptible 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 4 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 7 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

4.3.5 Impact of field size to resistance score under different water management

conditions 764.3.6 Correlation between field size and hand weeding 77

4.3.7 The impact of hand-weeding to herbicide resistance of Echinochloa spp in

the Mekong Delta 78

4.4 Weed control efficacy of rinskor in Echinochloa spp in the Mekong Delta 80

4.4.1 Control efficacy of rinskor as a new herbicide against three Echinochloa spp. groups collected in the Mekong Delta 80

4.4.2 Control efficacy of rinskor as new herbicide against Echinochloa spp populations collected in Mekong Delta 81

4.4.3 Correlation between resistance level of bispyribac, penoxsulam and quinclorac .82

4.4.4 Efficacy of bispyribac, penoxsulam and quinclorac in susceptible E crus-galli compared to resistant plants 84

4.4.5 Efficacy of rinskor for control of susceptible or resistant barnyardgrass to bispyribac, penoxsulam and quinclorac 86

4.5 Biodiversity study by RAPD analysis in 15 barnyardgrass populations from Vietnam and the U.S 87

4.5.1 RAPD analysis of 15 barnyardgrass populations 87

4.5.2 The genetic diversity of Echinochloa crus-galli and herbicide resistance level .92

4.6 Biochemical mechanism and molecular mechanism of quinclorac resistance in barnyardgrass 95

4.6.1 B-CAS activity in 5 quinclorac resistant barnyardgrass populations 95

4.6.2 CAS transcript abundance in leaf tissue of five barnyardgrass populations .97

4.6.3 Biochemical and molecular mechanism of quinclorac resistance in Echinochloa crus-galli in Mekong Delta 99

CHAPTER 5 101

CONCLUSIONS AND RECOMMENDATIONS 101

5.1 Conclusions 101

5.2 Recommendations 102

9REFERENCES 103

LIST OF TABLES

Page

Table 3.1 List of primers were used for RAPD analysis in the research 51

Table 3.2 Steps in PCR reactions 53

Table 3.3 RT-qPCR primer sequence detecting β-CAS and β-Actin synthase

53 Table 4.1 Rice cultivation practice of farmer in Mekong Delta 58

Table 4.2 Farmer perception about important weed species in rice field at 7 provinces of Mekong Delta 59

Table 4.3 Farmer response about most escaped weed after herbicide treatments and need hand-weeding to control 61

Table 4.4 The three most popular herbicides for escaped Echinochloa spp control in Mekong Delta 62

Table 4.5 The cost for weed management in Mekong Delta 63

Table 4.6 Plant characteristic of Echinochloa spp collected in Mekong Delta 65

Table 4.7 Distribution of Echinochloa species in Mekong Delta 70

Table 4.8 Herbicide-resistance level in populations to bispyribac, penoxsulam and quinclorac in different provinces 71

Table 4.9 Percent of solo-resistance and multiple-resistance herbicides in three groups of Echinochloa spp 74

Table 4.10 Percent of barnyardgrass population resistant to single and multiple herbicides of bispyribac, penoxsulam and quinclorac in different provinces 75

Table 4.11 Resistance Score of bispyribac, penoxsulam and quinclorac herbicide-resistance of 78 Echinochloa spp populations 76

Table 4.12 Average LD90 of 3 Echinochloa groups to bispyribac, penoxsulam, quinclorac and rinskor 81

Table 4.13 Average LD90 of barnyardgrass population to bispyribac, penoxsulam, quinclorac and rinskor 82

Table 4.14 Six informative primers in RAPD analysis of Echinochloa crus-galli populations 84

Table 4.15 Lethal dose of quinclorac needed to kill 90% of the population (LD90) and the Resistance level of 15 Echinochloa crus-galli populations collected in Vietnam and U.S 85

the Resistance level of 15 Echinochloa crus-galli populations collected in Vietnam

and U.S 93

LIST OF FIGURES

Page

Figure 2.1 Soil distribution map of Mekong Delta 5

Figure 2.2 Echinochloa crus-galli flower at mature stage 9

Figure 2.3 Infestation of Echinochloa crus-galli in rice field of Long An province, July 2016 9

Figure 2.4 Chemical structure of bispyribac 10

Figure 2.5 Process of manufacturing penoxsulam from N-(triazolo[1,5-a]pyrimidine) sulfonamides 12

Figure 2.6 Molecular structure of quinclorac 13

Figure 2.7 Chronological increase in Resistant Weeds Globally 16

Figure 2.8 GST-catalyzed detoxification of atrazine in plants 21

Figure 2.9 The minimal ABC transporter has four domains Two transmembrane domains (TMDs) bind ligand, and transport is driven by ATP binding and hydrolysis by the two nucleotide binding domains (NBDs)

23 Figure 2.10 Plant nursery for herbicide screening test 26

Figure 2.11 Results of herbicide screening in different weed populations 26

Figure 2.12 Dose response curves for a Susceptible (S) and a Resistant (R) population 27

Figure 3.1 The workflow designation 38

Figure 3.2 The map of sampled barnyardgrass 40

Figure 3.3 Prepare the barnyardgrass seedling for herbicide screening

41 Figure 3.4 Symptom of bispyribac in barnyardgrass leaf 45

Figure 3.5 Symptom of penoxsulam in barnyardgrass leaf

46 Figure 3.6 Symptom of quinclorac in barnyardgrass leaf 46

Figure 3.7 Symptom of rinskor in barnyardgrass leaf 46

Figure 3.8 Ninety-six wells microplate for the spectrophotometer reading at wavelength 650Å 49

Figure 3.9 β-CAS gene mined from the in-house ECHCR transcriptome 54 Figure 4.1 Mosaic plot diagram of hand-weeding practice after herbicide application in

71 survey fields in Mekong Delta 60

Figure 4.2 Flowers of three Echinochloa spp groups in the study 66

Figure 4.3 Correlation between plant height and shoot dry weight of three Echinochloa

group 67Figure 4.4 Correlation between panicle emerged date and grow duration of three

Echinochloa groups 68

Figure 4.5 Resistance level of three Echinochloa groups to bispyribac, penoxsulam and

quinclorac in different provinces 73Figure 4.6 Correlation between Resistance Score and field size under different watermanagement conditions 77Figure 4.7 One way ANOVA t-test for field size and hand-weeding practice 78Figure 4.8 One way ANOVA t-test for the impact of hand-weeding to herbicideresistance in rice field 79Figure 4.9 Correlation between LD90 value of herbicides in barnyardgrass 83

Figure 4.10 Electrophoresis image of 6 primers produced polymorphic bands 89

Figure 4.11 The dendrogram of 15 Echinochloa crus-galli populations from Vietnam

(10, KG-01, TG-03, HG-06, HG-02, 08, HG-03, 04, VL-03, HG-01,

CT-02, CT-01, VL-01) and U.S (A-S, AR) 90

Figure 4.12 Geographic distribution of 13 Echinochloa crus-galli populations collected

in the Mekong Delta, Vietnam 91

Figure 4.13 Mode of action of quinclorac in E crus-galli and the process to measure

the activity of CAS after quinclorac treatment 95

Figure 4.14 LD50 and % Mortality of 5 barnyardgrass populations foliar-treated byquinclorac 95Figure 4.15 Activity of enzyme CAS (nmol H2S/100ug/minute) in barnyardgrass leaftissue treated by quinclorac 97Figure 4.16 CAS transcript abundance was significantly decreased 1 hour after

quinclorac treatment in resistant populations Ech_03, Ech_04, Ech_05 and Oryza sativa

however, remained unchanged in susceptible populations Ech_01 and Ech_02 98

Figure 4.17 Three days following quinclorac treatment, CAS transcript abundance wasnot significantly different than the non-treated controls in all populations except forEch_02 where data is unavailable 99

LIST OF ABBREVIATIONS

ALS Acetolactate synthesis

ACCase Acetyl CoA Carboxylase

CAS Cyanoalanide synthase

PCR Polymerase Chain Reaction

RAPD Random Amplified Polymorphic DNART-qPCR Real-time Polymerase Chain Reaction

CHAPTER 1 INTRODUCTION1.1 Problem statement

According to Moody (1988) weed competition in flooded rice fieldsmight decrease grain yield by 25% In several cases, uncontrolled weeds ondry direct- seeded rice could impact up to 50% yield loss on rice (Chauhan,2012) To date, approximately 388 biotypes of 210 species that showed

resistance to herbicides had been documented (Caseley et al 2013) Among key weeds in the field, the barnyardgrass (Echinochloa crus-galli) had been

considered as the most notorious for a long time, as a C4 plant, the growth rate

of barnyardgrass is far dominant to rice (Caton et al 2004) In addition,

Echinochloa crus-galli and Echinochloa colona can mimic the rice appearance

at the early stage of weed seedlings, the similarity in plant appearance make ithard to control by hand weeding (Chauhan, 2012)

Herbicides still play an important role as the most effective method inweed management on rice fields, however, herbicide resistance is an certainissue From 1994 to 2014, the number of herbicide resistance cases on riceculture is 32 species in 25 countries and 8 Mode of Action (MoA) groups.Among 127 reports over the world, 43 instances were reported on

barnyardgrass (Echinochloa spp.), in which, 12 out of 43 cases resisted to

group B (Acetolactate synthase inhibitors) and 7 out of 43 cases resisted togroup O (Synthetic auxins) These reports also included one case that resisted

to both MoA groups (Heap, 2014)

The barnyardgrass could evolve resistance to several current herbicideactive ingredients, especially to two groups of B and O In this situation,further research about herbicide resistance in barnyardgrass is critical Inrecent years, there are many methods to study herbicide resistance includingbioassay with screening and DNA analysis, such as Rapid Whole-PlantAssay for Post- Applied Herbicides, Seed Germination Assays, Agar-BasedSeedling Assays, Leaf Disc Assays, Pollen Germination Test, DNA-Based

Assays (Burgos et al.,

2013)

Today herbicide testing methods exhibit particular advantages anddisadvantages For multiple study purposes, a single or combination of severalmethods could be utilized Based on targets of research, Rapid Whole-PlantAssay for POST-Applied Herbicides and Seed Germination Assays would besuitable to identify the herbicide resistance level of weed, and DNA-BasedAssays would be appropriate to detect the resistant gene in weed biotypes As

an outcome, the dissertation of “Study on the resistance mechanism of barnyardgrass (Echinochloa crus-galli) to quinclorac in the Mekong Delta of Vietnam” was proposed.

1.2 Targets of dissertation

Evaluate the morphological variability and genetic diversity of

Echinochloa spp populations in Mekong Delta of Vietnam.

Evaluate the herbicide-resistance level of Echinochloa spp to bispyribac,

penoxsulam and quinclorac in rice fields at the Mekong Delta of Vietnam.Explain the biochemical mechanism and the molecular mechanism of

quinclorac-resistance in Echinochloa crus-galli.

1.3 Studied objectives and limitation of the dissertation

This study is limited to rice fields in the 7 provinces of Mekong Delta ofVietnam (Long An, Tien Giang, Vinh Long, Can Tho, An Giang, Hau Giangand Kien Giang)

The targeted weed in the study is Echinochloa spp., the weed was

examined for herbicide-resistance evaluation and mapping purpose The

Echinochloa crus-galli is main species studied for quinclorac-resistance

mechanisms at the biochemical and molecular level

1.4 Major research topics of the dissertation

(1) Survey the rice cultivation and weed management practice in ricefields at the Mekong Delta of Vietnam

(2) Evaluate the diversity of Echinochloa spp population in rice fields at

the Mekong Delta of Vietnam

(3) Evaluate the herbicide-resistance level of the collected Echinochloa

spp samples to bispyribac-sodium, penoxsulam and quinclorac by response screening method

dose-(4) Evaluate the efficacy of rinskor, a new herbicide against the

herbicide- resistant Echinochloa crus-galli to find new effective herbicide for

current herbicide-resistant barnyardgrass

(5) Use RAPD analysis to evaluate the genetic diversity of Echinochloa

crus-galli populations, and the correspondence of quinclorac-resistance and

genetic distance of Echinochloa crus-galli populations in the Mekong delta

(6) Measure the activity of β-cyanoalanine synthase in the leaf tissue of

Echinochloa crus-galli to study the biochemical mechanism of

quinclorac-resistance in barnyardgrass

(7) Measure the expression level of mRNA of CAS gene in barnyardgrass

to study the molecular mechanism of quinclorac-resistance in barnyardgrass

1.5 Contributions of dissertation

This research presents useful information about the diversity of

Echinochloa spp in the rice field of Mekong Delta of Vietnam (Mekong

Delta), which will be important for further study about this weed species.The most important result of this study is data about the herbicide-

resistance of Echinochloa spp., the research has confirmed the existence of herbicide-resistant Echinochloa spp populations in Mekong Delta This

research also evaluates the relationship between farmers’ weed managementpractice and the herbicide-resistance, therefore, the practical solutions forherbicide-resistant weed were also determined and suggested in thedissertation

The mechanisms of quinclorac-resistance in barnyardgrass wereconfirmed and elucidated at enzyme and molecular level, through themeasurement of quinclorac detoxifying enzyme activity and its geneexpression level in barnyardgrass The results establish important informationfor the further study about the mechanism of the herbicide-resistance in weeds

CHAPTER 2 LITERATURE REVIEW2.1 Overview of the Mekong Delta in Vietnam and rice cultivation

Vietnam is one of the top rice exporters globally In 2015, about 45million tons of rice was produced in Vietnam, of which 22.4% was exported,and the remaining was for domestic consumption (USDA, 2017) For severalyears, rice is one of the most important crops in Vietnam with approximate 7.6million hectares cultivated in the country (General statistics office of Vietnam,2017) Moreover, about 55% of rice in the country was grown in the MekongDelta, and the average yield of this region was 5.96 ton/ha (General statisticsoffice of Vietnam, 2017) which was 38% higher than the global average yield(FAOSTAT, 2017)

Mekong Delta, Vietnam is an area of 40,577 km2 This is the mostdownstream part of the Mekong River, the total population is over 17 million

with 3.96 million hectares for agriculture activities (Le Anh Tuan et al., 2007).

Mekong Delta produces more than 50% of cereal food for all of Vietnam Riceexportation from this area is one of the most important income for the country,54% of rice in the Delta was cultivated during the Summer-Autumn season(May to August); resulted in the highest yield harvested during Winter-Springseason (January to April), which could be 20.5% higher than average of the

year (Nguyễn Hoàng Dân et al., 2015).

Average field size per household in the Mekong Delta is 1.29 ha, higherthan average size in the country which is 0.44 ha Although the area of ricegrowing has reduced since 1980, the rice farming system now changes to anintensification model, in 2010 there was 530,000 ha growing rice in triplecrops per year compared to 23,000 ha in 1980 (Nguyễn Đức Thành và ĐinhTuấn Minh, 2015) The average cost of pesticide per season in this area was17-20% (Hồ Cao Việt, 2011)

The Mekong Delta natural condition is divided by 12 soil types (Fig 2.1),and their distribution influences the local agricultural activities and riceproduction (Minh, 2002) 80% of the total surface water used for rice growingareas, the intensive rice growing areas are located in upstream and midstream

provinces (Dang Kieu Nhan et al., 2007).

In recent year, water shortage and the saline water intrusion are one of themajor threats for rice cultivation in Mekong Delta Saline water could intrudefar into the irrigation system of many provinces in the dry season of 2016,

areas of Vam Co River (about 90 km away from the sea)

were

ntrusion (Lê Anh Tuấn, 2016)

igure 2.1 Soil distribution map of Mekong Delta (Minh, 2002)

5

majority impacted

by the i

F

2.2 Definitions of weed and herbicide resistance

There are many definitions for weed, and one of the well-knowndefinitions is “The plant growing where it is not wanted” (Blatchley, 1912;Zimdahl, 2013) The most troublesome aspect of weeds is crop competition,but sometimes some weed species are also prone to cause strong allergies (e.g.hay allergies) and skin dermatitis in sensitive individuals (Molinar, 2002).Weeds are considered as one of the most costly factors in controlling andlimiting crop production globally as yield losses caused by weeds mainly comefrom direct competition with crop plants for water, nutrients, light, and space

(Rosskopf et al., 1999) The peculiar biological traits of weeds, including seed

dormancy, germination, and emergence over long periods of time, long-termsurvival of buried seeds, abundant seed production, rapid populationestablishment, capacity to colonize new sites, and multiple adaptations for

spread, can provide weeds evolutionary advantages over cultural plants andcrops (Molinar, 2002)

In most cropping systems, weeds can significantly reduce crop yields,

plant for animal feeding and also fiber quality (Rosskopf et al., 1999) Besides

the direct impacts, weeds can also serve as alternate hosts to insect pests andpathogens in the field, in consequences lead to higher operating costs andincreased risk of diseases (Wisler and Norris, 2005) There are several reports

about the Echinochloa crus-galli serves as host for virus transmitting brown

plant hoppers in the rice field at the end of the rice season (Hattori, 2001) and

Zhou et al (2008).

Weed control is an important practice for any intensive cropping system.There are several methods for weed management in the field includingmechanical based weeding (weeding by hand, tool, machine or lasertransmitter to burn the plant), chemical based (herbicide), crop competitionmodel (allelopathy), and biological control (parasite insect, fungi, grazingcattle or other herbivores) For crop production in large scale, the chemicalbased method is still the most reliable and cost-effective solution for weed

management (Harker and O’Donovan, 2013) Several bio-herbicides like

pathogen or plant extractions are also studied, but the effect is still limited inlarge scale application (Van-Driesche and Bellows, 1996)

After the herbicide introduced in the market, the evolution of theherbicide-resistance in weeds had already been predicted by Blackman (1950)

The first case of atrazine and simazine resistance Senecio vulgaris was found

in

1968 and first reported in the USA in 1970, this type of weeds had evolvedresistance to herbicides inhibiting the electron transport in photosystem II(PSII- inhibitors) after the herbicides had been applied once or twice annuallyfor 10 years (Ryan, 1970), the herbicide-resistance report was continued by

Radosevich and Appleby (1973) for Amaranthus retroflexus L.

The definition of the herbicide-resistance has been mentioned by several

authors, according to Heap et al (1993), the herbicide-resistance is “the

evolved capacity for a previously herbicide-susceptible weed population towithstand a herbicide and complete its life cycle when the herbicide is used atits normal rate in an agricultural situation”

The Herbicide Resistance Action Committee (HRAC, 2017) definesherbicide resistance as “the naturally occurring inheritable ability of someweed biotypes within a given weed population to survive a herbicidetreatment that

- Herbicide tolerance is the inherent ability of a species to survive andreproduce after herbicide treatment This implies that there was noselection or genetic manipulation to make the plant tolerant; it isnaturally tolerant.

The European and Mediterranean Plant Protection Organization (OEPP/EPPO) describes the herbicide-resistance as “the naturally occurring, in-heritable adjustment in the ability of individuals in a population to survive aplant protection product treatment that would normally give effective control.”The EPPO guidelines introduce a difference between resistance that can beverified at the laboratory level and resistance observed in the field situation,which is referred to as “practical resistance” and defined as “the loss of fieldcontrol due to a shift in sensitivity” According to this distinction, the detection

of herbicide resistance at the laboratory level does not always connect to thereduction of that pest control in the field (EPPO, 1988)

Herbicide-resistant individual plant normally exists in any weedpopulations at a lower level before application of herbicide, but the continuousselection pressure imposed by herbicides on plants allows the resistance to

increase in frequency (Jasieniuk et al., 1996) A number of factors contribute

to the evolution of herbicide resistance in any weed species includingfrequency of resistant alleles in a population, number and mode of herbicideapplications, efficacy of used dosage, seed bank in soil, and other biologicalfactors (Preston and Powles, 2002)

In general, apart from the pre-existing resistant population, the movement

of genes (via seeds or pollen depend on species) from resistant populations innearby fields also becomes the source of new resistant genes in the gene pool

of population The gene mutations associated to the resistance to aspecific herbicide class are not induced by application of the herbicide, but

rather to occur naturally (Jasieniuk et al., 1996).

2.3 Overview of Echinochloa spp in the rice field

The genus Echinochloa includes over 250 species, and most of these are considered as weeds Weed species belonging to Echinochloa are varied in

their growth habit, distribution, and morphology (Barrett and Wilson, 1983)

Barnyardgrass (Echinochloa spp.) is C4 monocots (Caton et al., 2010) This

weed originated from Europe and has been now documented worldwide (Dorie

and McNeill, 1980) The morphology of Echinochloa spp is highly diverse, in

many cases, there are several species confused with one another Many species

have been misidentified as Echinochloa crus-galli because of this species high

popularity In many places of the world, the barnyardgrass is a popular local

name for weeds in Echinochloa family, in fact, many different species that were misnamed as Echinochloa crus-galli (Rutledge et al., 2000)

Echinochloa crus-galli has spread across 61 countries in the world and

been considered as a severe weed in 36 crops (Bajwa et al., 2015) This weed

is highly adapted to a wider range of photoperiods Due to thecontinuous morphological variations among species, the classification of

Echinochloa has remained a serious problem for weed scientists (Damalas et al., 2008).

According to Ampong-Nyarko and Datta (1991), the optimal conditionfor barnyardgrass is high soil moisture (70% to 90%) Its seeds can germinateunder water, however, the germination rate is inversely proportional to thedepth of water During the first stage of vegetative growth, it is nearlyimpossible to distinguish between the rice plants and barnyardgrass This can

be seen as a result of phenotypic evolution, and the pressure for this evolution

is a very long period of hand weeding in rice cultivation

In Asia, rice fields have three main barnyardgrass species, Echinochloa

crus-galli, Echinocloa colona, and Echinochloa glabrescens, which found in

Vietnam (Koo et al., 2005) The barnyardgrass (Echinochloa crus-galli) is a

highly competitive weed in rice field because of large biomass and seedproducing capability as one plant can produce 2000 to 4000 seeds in ideal

condition (Gibson et al., 2002).

Barnyardgrass (Echinochloa crus-galli (L.)) is a problematic weed

worldwide since it possesses a C4 photosynthetic pathway, which isbiologically more advantageous than C3 Poaceae crops such as rice As amore significant competitor to rice, barnyardgrass could spread their seeds at a

tremendous level, causing up to 80% reduction in rice yield (Van-Devender et

al., 1997).

9There are about 400 weed species reported in upland crops and rice fields

of Vietnam; among those weeds, barnyardgrass (Echinochloa crus-galli) is one

of the most critical grass weeds in rice fields as this weed can reduceapproximately 25% rice yield under high infestation condition (Chin, 2001)

Report of Chu Van Hach et al (1998) also mentions a significant negative

relationship between grain yield and barnyardgrass’ dry matter accumulation.Barnyardgrass density at 258 and 340 plants/m2rice caused yield losses wereabout 86.7% in first season and 63.5% in second season

Study results of Hoang Vu Duy et al (2013) showed that photosynthetic

rate and dry matter accumulation rate of barnyardgrass were higher than those

of rice at all growth stages although stomatal conductance, transpiration rateand SPAD value of rice were higher than those of barnyardgrass Thephotosynthetic rate, dry matter accumulation rate and nitrogen content inleaves also increased in both rice and barnyardgrass as nitrogen levelsincreased

Ho Le Thi et al (1998) also stated that Echinochloa crus-galli is one of

the most troublesome weeds in Mekong Delta because it could serverelyreduce rice yield in both quantity and quality Furthermore, 60-80% of thenitrogen from soil and considerable amounts of other macronutrients can beremoved by barnyardgrass (Maun and Barrett, 1986)

Heap (2017) mentioned that the majority of Echinochloa spp were

resistant to almost all available active ingredients in the market 43 out of 127reported cases of herbicide resistance on grass involved barnyardgrass species,

among which Echinochloa crus-galli in Brazil showed resistance to both B and

O MoA

Figure 2.2 The

Echinochloa crus-galli

flower at mature stage,

Long An province, July

2016

Figure 2.3 Infestation of

Echinochloa crus-galli in rice

field of Long An province,

July 2016

Allelopathy has been recognized as a strong mechanism of weed invasion

in the field since the allelochemicals found in the root zone of weed could

inhibit the crop growth (Lorenzo et al., 2013) Echinochloa crus-galli is a

troublesome weed with strong allelopathic competition potential in many

crops (Chung et

al., 2001) In 2006, a study was conducted to identify and isolate 15

allelochemicals from Echinochloa crus-galli root exudates, all of these

allelochemicals belong to different chemical classes that inhibit growth in

various plants (Xuan et al., 2006).

2.4 Herbicide for barnyardgrass control

2.4.1 Overview of herbicidal active ingredient bispyribac

Figure 2.4 Chemical structure of bispyribac (NCBI, 2017)

The Bispyribac-sodium or pyrimidinyl carboxy herbicide bispyribacsodium (sodium 2,6-bis[4,6 dimethoxypyrimidine- 2-yl)oxy]benzoate) is aClass D growth regulator, the herbicidal effect of this molecule was firstinvestigated by Fagerness and Penner in 1998 Bispyribac-sodium is readilyabsorbed by roots and translocated to shoots (Lycan and Hart, 2006) Thebispyribac-sodium was used for selective post emergence control of

Echinochloa crus-galli (L.) Beauv (Williams, 1999) and many other

problematic weeds in rice (Martini et al., 2015a).

Bispyribac-sodium is a systemic herbicide that moves fromphotosynthetically active leaves to meristematic regions of plants, to achieveoptimum weed control efficacy, a certain amount of herbicide needs to reachthese zones, causing the death of susceptible plants (Murata and Los, 1997;

Martini et al., 2015a).

Bispyribac-sodium could damage rice seedling, the herbicide effect rice

by making foliar injury, root growth inhibition, and root dry weight reduction

(Devine, 1989; Devine et al (1990); Shaner, 1991; Dunand and Dilly, 1994;

Braverman and Jordan, 1996) Zhang and Eric (2002) proved that rice plantsare tolerant to bispyribac sodium depending on the cultivars and growth stage

of seedling, bigger rice seedling are more tolerant to the herbicide compared tosmaller plant

Bispyribac-sodium affects the plants by inhibit Acetolactate synthase(ALS), also called Acetohydroxyacid synthase (AHAS), a key enzyme in thebiosynthesis of the branched-chain amino acids isoleucine, leucine, and valine.Plant dies as a result of ALS inhibition and low branched-chain amino acidproduction reduction in leaf tissues, and because the inhibition of amino acidproduction happens gradually, it could take several days to see the symptoms

of damage in weed foliar (LaRossa and Schloss, 1984; Shimizu et al., 1994, 2002; Osuna et al., 2002).

According to Martini et al (2015b), cytochrome P450 monooxygenase

(P450s), a large membrane-bound enzyme family, is the key enzyme thatmetabolize the bispyribac-sodium in plant tissue This enzyme couldmetabolize many herbicides, including bispyribac, propanil and various ALSinhibitors Low temperature promotes membrane rigidification, which willlower the P450 activity, contributing to reduction in herbicide metabolismand increasing herbicide damages on rice plants

Bispyribac-sodium resistance has been reported in barnyardgrass

Echinochloa phyllopogon, Echinochloa oryzicola (Fischer et al., 2000) and a

cross resistance in Echinochloa phyllopogon and Cyperus difformis The

reason is due to the increase of cytochrome P450 monooxygenation by

inhibitors and target site alteration (Osuna et al., 2011) The cytochrome P450

enzyme could be inhibited by the organophosphate pesticide like Malathionbecause application of Malathion could increase the susceptibility ofbispyribac-resistant weed (Murata and Los, 1997)

In Vietnam, bispyribac is also a common herbicide for controllingbarnyardgrass and broadleaf weeds in the rice field This product is widelyused as a solo product or mixture with other herbicides Some brands ofbispyribac- sodium (min 93%) are Nominee (10SC and 100OF manufactured

by Kumiai Chem Ind Co., Ltd), Nonee-cali (10WP and 100SC manufactured

by Cali - Parimex Inc), and Sunbishi (10SC manufactured by Sundat (S) PteLtd) Based on labeled recommendation of Kumiai Chem, bispyribac 100OF

is highly effective against barnyardgrass and broadleaf weeds, and the productshould be used at 2-4 leaf stage of barnyardgrass (Plant Herbicides Permitted

in Vietnam

2013, Ministry of Agriculture and Rural Development)

2.4.2 Overview of herbicidal active ingredient penoxsulam

The penoxsulam is a herbicidal active ingredient in group B (ALSinhibitor) This herbicide was developed from N-(triazolo[1,5-a]pyrimidine)

sulfonamides Initially, reversing the (-SO2NH–) linkage in a]pyrimidine) sulfonamides resulted in Flumetsulam – an herbicidal activeingredient that can inhibit Acetolactate synthase activity in broadleaf and grassweeds Later on, Florasulam, another herbicidal active ingredient for broadleafgrasses was developed by core heterocyclic variation process However, bothFluroslam and Flumetsulam have no efficacy on true grasses (Poaceae) Thesubsequent researches discovered that structural changes in phenyl rings inFlorasulam produced a new active ingredient with effective control in Poaceaeand broadleaf grasses This active ingredient is later registered with the trade

N-(triazolo[1,5-name Penoxsulam Johnson et al (2009).

Figure 2.5 Process of manufacturing Penoxsulam from

N-(triazolo[1,5-a]pyrimidine) sulfonamides (Johnson et al., 2009).

Penoxsulam is a strong herbicidal active ingredient, especially in thepost-emergence stage However, the efficacy of penoxsulam treatment in pre-

emergence stage is limited (Ottis et al., 2003) Study on portioning showed

that Penoxsulam is highly mobile in soil and water, but has difficultyevaporating in air under normal condition The degradation of penoxsulam inthe environment is mainly photo degradation and biodegradation in soil andwater (Jabusch and Ronald, 2006)

Dilpreet et al (2012) have studied herbicide-resistance in three

barnyardgrass populations which confirmed to be resistant weeds toimazethapyr and penoxsulam (ALS inhibitor herbicides) in Mississippi andArkansas The results showed that the herbicide detoxification via theenhanced activity of cytochrome P450 monooxygenase (CYP) was the mainmechanism

in those biotypes, the weeds had evolved herbicide-resistance to two ALSherbicides in this study The Malathion was used in that research to determine

if the organophosphate molecule would help to overcome the resistance, datashowed that additional of Malathion in mixture of imazethapyr andpenoxsulam significantly reduced the dry weight and increased mortality ofresistant weeds, which clearly proved the CYP boosting was a majormechanism for ALS herbicide resistance in those barnyardgrass biotypes

In another research of Dilpreet et al (2011) the similar biotypes from

Arkansas and Mississippi barnyardgrass which have evolved cross-resistance

to imazamox, imazethapyr, penoxsulam and bispyribac-sodium Thesequencing of a 1701 base pair ALS coding sequence found changes in Ala122

to Val and Ala122 to The substitutions in two biotypes that highly resistant toimazamox This is a result of target site resistance, which couldsignificantly reduce herbicide efficacy because the herbicide molecules couldnot bind to targeted enzyme in weed tissue On the other hands, absorption of14C- bispyribacsodium, -imazamox, and -penoxsulam was similar in allbiotypes However, the translocation of 14C-Bispyribac and 14C-Imazamoxwere 31−

43% and 39% less in R compared to S biotypes respectively The resultintroduces a second mechanism of ALS herbicide resistance in barnyardgrasspopulations

2.4.3 Overview of herbicidal active ingredient quinclorac

Figure 2.6 Molecular structure of quinclorac (NCBI, 2017)

Quinclorac was researched and developed by the BASF Company in

1993 Quinclorac is one of the most popular herbicides for grassy weed control

in rice field globally This molecule is well studied and reported in manypublications since it has introduced into the market In the research ofGrossmann and S Florene (1998), quinclorac (quinolinecarboxylic acidquinclorac (3,7-dichloro-

8-quinolinecarboxylic acid) was an artificial synthetic auxin, which isclassified to group O of HRAC

Quinclorac is mostly used as a post-emergent herbicide in the rice field.

It is also used as pre-emergent herbicide due to high stability in soil The longresiduality of this herbicide in soil could control new germinated barnyardgrass

seeds (Hill et al., 1998) and cause phytotoxicity in rotation crops in the next season An application of soil bacteria Bacillus megaterium could speed up the degradation of quinclorac in soil (Liu et al., 2014).

Quinclorac is absorbed through the leave and root systems, and laterdistributed through xylem and phloem systems (Grossmann and Kwiatkowski,

2000) Quinclorac was proved to be safe on rice plants (Oryza sativa) and highly effective on Echinochloa, Digitaria, and Setaria The selectivity can be

explained by a study of Grossmann and Jacek (1993), in which quincloracmetabolic process in rice was different from barnyardgrass When absorbed,Quinclorac would stimulate the activity of ACC synthase Thus, acceleratedthe ACC synthesis (1-aminocyclopropanecarboxylic acid) ACC will later

be accumulated in cells and then metabolized in to Ethylene and cyanide(HCN) HCN caused cell damages in barnyardgrass, but these damages werelimited in rice plants

In rice, a high amount of beta cyanoalanine synthase will be synthesized

to degrade cyanide – the precursor of HCN Therefore, rice plants are safe fromHCN intoxication during Quinclorac application Similar research results were

later recorded by Nguyen et al (2008) The research of Yasuor et al (2012) demonstrated that Echinochloa phyllopogon in California has been developed

quinclorac resistance by at least 2 mechanisms, including enhanced activity ofbeta cyanoalanine synthase and reduced sensitivity of Quinclorac on internalethylene production

In Vietnam, quinclorac is a popular post-emergent herbicide forcontrolling barnyardgrass in a rice field There are several registered products

of quinclorac, including solo herbicide and mixtures Some common brands areFacet 250SC (manufactured by BASF chemical), Clorcet (50WP, 250SC, and300SC manufactured by Cali – Parimex Inc.), Ekill (25 SC, 37WG, and80WG manufactured by Map Pacific PTE Ltd), and Forwacet (50 WP and250SC manufactured by Forward International Ltd)

According to the label rate of BASF Chemical, quinclorac 250 SC should

be treated at 125-250 g a.i/ha for barnyardgrass at the 3-5 leaf stage of seedling(Plant Herbicides Permitted in Vietnam 2013, Ministry of Agriculture andRural Development)

2.5 Herbicide resistance and testing methods

2.5.1 The importance of herbicide resistance management

Weed control by herbicides is one of the most effective method for weedmanagement in large-scale crop production worldwide However, similar toother chemical based solutions, weed management by herbicide has shownboth advantages and disadvantages The herbicide treatment timing for weedcontrol can be categorized by pre-emergence and post-emergence, thedifferentiation based on the mechanism and application timing of theparticular herbicide against target weeds Herbicides should be selected based

on the mode of action and selectivity in different crop models (Talbert andBurgos, 2007)

Cost-effectiveness is the biggest advantage of herbicide application in

crop production Results in a study of Samar et al (2007) showed that the cost

of herbicide application for grassy weeds control in direct seeding rice in Indiawas

17-45%, lower than hand-weeding and mulching method According to Rao et

al (2007), herbicide treatment was the most cost-effective method for direct

seeding rice due to the incensement of labor cost in rural areas of Asiacountries since last decades Therefore, management of herbicide usage is veryimportant for rice production in this area

Research of Jesusa et al (2012) in the Philippines showed that the cost of

herbicide application for non-resistant barnyardgrass control was lower than amanual hand-weeding model in rice On the other hand, the total cost forcontrolling of herbicide-resistant barnyardgrass was significantly higher than

in normal methods because it required both herbicide and hand-weedingpractice to control herbicide-resistant weeds

The compatibility with mechanical and crop production in large scale aretwo other reasons made herbicide become widely adopted in global ricecultivation, also because the manual hand-weeding was limited by small-scaleproduction and the labor shortage was a trend in many rural areas of Asia (Rao

et al., 2007).

However, the herbicide-resistance is the biggest issue of weedmanagement by herbicide Repeating application of herbicide would lead to

herbicide resistance as an undesired consequence Echinochloa spp was one of

the most reported species in the database of Herbicide Resistance ActionCommittee with 80 reported cases (Heap 2017) (Figure 2.7) Herbicide-resistance will increase the cost on management and the evolution of herbicide

resistance would become a serious threat to global crop yield (Powles andHoltum, 1994).

According to Huang and Gressel (1997), over 2 million hectares of rice in

China were infested with Echinochloa crus-galli that evolved target site

cross-resistance and multiple-cross-resistance to butachlor and thiobencarb The trend wasincreasing with the adoption of an intensive crop cultivation system in thecountry

Figure 2.7 Chronological increase in Resistant Weeds Globally

(Heap, 2017)

In the Philippines, although herbicide was the most cost-effective method

for controlling Echinochloa crus-galli, if resistance occurred about $100/ha per

year will be lost Farmers had to apply hand weeding practice, which was quiteexpensive and it increased the cost of production In addition to the costmatter, overused herbicides also led to negative environmental impacts.Herbicides are generally toxic to aquatic organisms in rice field condition.Following proper recommendations and other weed management methods are

very important for sustainable weed management (Jesusa et al 2012)

Herbicide-resistance management is the most important practice in cropproduction It required a higher number of herbicides to control resistant

weeds, and will increase the cost of weed control (Norsworthy et al., 2012).

It also required a very high dose of herbicide to control resistant weedsTherefore,