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 weed populations exhibited resistance to current herbicides, results showed that the Echinochloa populations resistant to bispyribac, penoxsulam a

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 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 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 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

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,

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 33.1, 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 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.S

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 0.09 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 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

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

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

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

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 76

4.3.6 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

REFERENCES 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

Table 4.16 Mortality of herbicide susceptible (S) and herbicide resistant (R) barnyardgrass treated by rinskor at different dose 86

Table 4.17 Six informative primers in RAPD analysis of Echinochloa crus-galli

populations 88

Table 4.18 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 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 67 Figure 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 73 Figure 4.6 Correlation between Resistance Score and field size under different water management conditions 77 Figure 4.7 One way ANOVA t-test for field size and hand-weeding practice 78 Figure 4.8 One way ANOVA t-test for the impact of hand-weeding to herbicide resistance in rice field 79 Figure 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

(CT-10, KG-01, TG-03, HG-06, HG-02, CT-08, HG-03, CT-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 by quinclorac 95 Figure 4.15 Activity of enzyme CAS (nmol H2S/100ug/minute) in barnyardgrass leaf tissue treated by quinclorac 97 Figure 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 was not significantly different than the non-treated controls in all populations except for Ech_02 where data is unavailable 99

LIST OF ABBREVIATIONS

ALS Acetolactate synthesis

ACCase Acetyl CoA Carboxylase

CAS Cyanoalanide synthase

LD90 Lethal Dose of 90% of population

PCR Polymerase Chain Reaction

RAPD Random Amplified Polymorphic DNA

RT-qPCR Real-time Polymerase Chain Reaction

CHAPTER 1 INTRODUCTION

1.1 Problem statement

According to Moody (1988) weed competition in flooded rice fields might decrease grain yield by 25% In several cases, uncontrolled weeds on dry 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 it hard to control by hand weeding (Chauhan, 2012)

Herbicides still play an important role as the most effective method in weed management on rice fields, however, herbicide resistance is an certain issue From 1994 to 2014, the number of herbicide resistance cases on rice culture 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 to group 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 herbicide active ingredients, especially to two groups of B and O In this situation, further research about herbicide resistance in barnyardgrass is critical In recent years, there are many methods to study herbicide resistance including bioassay with screening and DNA analysis, such as Rapid Whole-Plant Assay for Post-Applied Herbicides, Seed Germination Assays, Agar-Based Seedling Assays,

Leaf Disc Assays, Pollen Germination Test, DNA-Based Assays (Burgos et al.,

2013)

Today herbicide testing methods exhibit particular advantages and disadvantages For multiple study purposes, a single or combination of several methods could be utilized Based on targets of research, Rapid Whole-Plant Assay for POST-Applied Herbicides and Seed Germination Assays would be suitable to identify the herbicide resistance level of weed, and DNA-Based Assays 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 of Vietnam (Long An, Tien Giang, Vinh Long, Can Tho, An Giang, Hau Giang and 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 rice fields

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 management practice and the herbicide-resistance, therefore, the practical solutions for herbicide-resistant weed were also determined and suggested in the dissertation The mechanisms of quinclorac-resistance in barnyardgrass were confirmed and elucidated at enzyme and molecular level, through the measurement of quinclorac detoxifying enzyme activity and its gene expression level in barnyardgrass The results establish important information for the

further study about the mechanism of the herbicide-resistance in weeds

CHAPTER 2 LITERATURE REVIEW

2.1 Overview of the Mekong Delta in Vietnam and rice cultivation

Vietnam is one of the top rice exporters globally In 2015, about 45 million tons of rice was produced in Vietnam, of which 22.4% was exported, and the remaining was for domestic consumption (USDA, 2017) For several years, rice

is one of the most important crops in Vietnam with approximate 7.6 million hectares cultivated in the country (General statistics office of Vietnam, 2017) Moreover, about 55% of rice in the country was grown in the Mekong Delta, and the average yield of this region was 5.96 ton/ha (General statistics office 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 most downstream 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 Rice exportation 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-Spring season (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, higher than average size in the country which is 0.44 ha Although the area of rice growing has reduced since 1980, the rice farming system now changes to an intensification model, in 2010 there was 530,000 ha growing rice in triple crops per year compared to 23,000 ha in 1980 (Nguyễn Đức Thành và Đinh Tuấn Minh, 2015) The average cost of pesticide per season in this area was 17-20% (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 rice production (Minh, 2002) 80% of the total surface water used for rice growing areas, 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 the major threats for rice cultivation in Mekong Delta Saline water could intrude far into the irrigation system of many provinces in the dry season of 2016,

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

by the intrusion (Lê Anh Tuấn, 2016)

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

2.2 Definitions of weed and herbicide resistance

There are many definitions for weed, and one of the well-known definitions 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 and limiting crop production globally as yield losses caused by weeds mainly come from 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-term survival of buried seeds, abundant seed production, rapid population establishment, capacity to colonize new sites, and multiple adaptations for

spread, can provide weeds evolutionary advantages over cultural plants and crops (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 and pathogens in the field, in consequences lead to higher operating costs and increased 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 including mechanical based weeding (weeding by hand, tool, machine or laser transmitter

to burn the plant), chemical based (herbicide), crop competition model (allelopathy), and biological control (parasite insect, fungi, grazing cattle or other herbivores) For crop production in large scale, the chemical based 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 in large scale application (Van-Driesche and Bellows, 1996)

After the herbicide introduced in the market, the evolution of the herbicide-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 evolved resistance to herbicides inhibiting the electron transport in photosystem II (PSII-inhibitors) after the herbicides had been applied once or twice annually for 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 to withstand a herbicide and complete its life cycle when the herbicide is used at its normal rate in an agricultural situation”

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

would, under normal use conditions, effectively control that weed population Selection of resistant biotypes may result in control failures”

The WSSA (Weed Science Society of America 1997) also distinguish the herbicide-resistance to herbicide-tolerance as:

- Herbicide resistance is the inherited ability of a plant to survive and reproduce following exposure to a dose of herbicide normally lethal to the wild type In a plant, resistance may be naturally occurring or induced

by such techniques as genetic engineering or selection of variants produced by tissue culture or mutagenesis

- Herbicide tolerance is the inherent ability of a species to survive and reproduce after herbicide treatment This implies that there was no selection or genetic manipulation to make the plant tolerant; it is naturally 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 a plant protection product treatment that would normally give effective control.” The EPPO guidelines introduce a difference between resistance that can be verified at the laboratory level and resistance observed in the field situation, which is referred to as “practical resistance” and defined as “the loss of field control due to a shift in sensitivity” According to this distinction, the detection

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

Herbicide-resistant individual plant normally exists in any weed populations at a lower level before application of herbicide, but the continuous selection 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 including frequency

of resistant alleles in a population, number and mode of herbicide applications, efficacy of used dosage, seed bank in soil, and other biological factors (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 in nearby fields also becomes the source of new resistant genes in the gene pool of population The gene mutations associated to the resistance to a specific 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 the continuous

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 condition for barnyardgrass is high soil moisture (70% to 90%) Its seeds can germinate under water, however, the germination rate is inversely proportional to the depth of water During the first stage of vegetative growth, it is nearly impossible 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 seed producing 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 is biologically more advantageous than C3 Poaceae crops such as rice As a more 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)

There 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 reduce approximately 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 were about 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 rate and SPAD value of rice were higher than those of barnyardgrass The photosynthetic rate, dry matter accumulation rate and nitrogen content in leaves also increased

in both rice and barnyardgrass as nitrogen levels increased

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 serverely reduce rice yield in both quantity and quality Furthermore, 60-80% of the nitrogen from soil and considerable amounts of other macronutrients can be removed 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 127 reported 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 bispyribac sodium (sodium 2,6-bis[4,6 dimethoxypyrimidine- 2-yl)oxy]benzoate) is a Class D growth regulator, the herbicidal effect of this molecule was first investigated by Fagerness and Penner in 1998 Bispyribac-sodium is readily absorbed by roots and translocated to shoots (Lycan and Hart, 2006) The bispyribac-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 from photosynthetically active leaves to meristematic regions of plants, to achieve optimum weed control efficacy, a certain amount of herbicide needs to reach these 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 plants are tolerant to bispyribac sodium depending on the cultivars and growth stage

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

Bispyribac-sodium affects the plants by inhibit Acetolactate synthase (ALS), also called Acetohydroxyacid synthase (AHAS), a key enzyme in the biosynthesis of the branched-chain amino acids isoleucine, leucine, and valine Plant dies as a result of ALS inhibition and low branched-chain amino acid production reduction in leaf tissues, and because the inhibition of amino acid production 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 that metabolize the bispyribac-sodium in plant tissue This enzyme could metabolize many herbicides, including bispyribac, propanil and various ALS inhibitors Low temperature promotes membrane rigidification, which will lower the P450 activity, contributing to reduction in herbicide metabolism and 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 Malathion because application of Malathion could increase the susceptibility of bispyribac-resistant weed (Murata and Los, 1997)

In Vietnam, bispyribac is also a common herbicide for controlling barnyardgrass and broadleaf weeds in the rice field This product is widely used

as a solo product or mixture with other herbicides Some brands of 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) Pte Ltd) Based

bispyribac-on labeled recommendatibispyribac-on of Kumiai Chem, bispyribac 100OF is highly effective against barnyardgrass and broadleaf weeds, and the product should 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 (ALS inhibitor) 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 active ingredient that can inhibit Acetolactate synthase activity in broadleaf and grass weeds Later on, Florasulam, another herbicidal active ingredient for broadleaf grasses was developed by core heterocyclic variation process However, both Fluroslam and Flumetsulam have no efficacy on true grasses (Poaceae) The subsequent researches discovered that structural changes in phenyl rings in Florasulam produced a new active ingredient with effective control in Poaceae and 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 the post-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 difficulty evaporating in air under normal condition The degradation of penoxsulam in the environment

is mainly photo degradation and biodegradation in soil and water (Jabusch and Ronald, 2006)

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

barnyardgrass populations which confirmed to be resistant weeds to imazethapyr and penoxsulam (ALS inhibitor herbicides) in Mississippi and Arkansas The results showed that the herbicide detoxification via the enhanced activity of cytochrome P450 monooxygenase (CYP) was the main mechanism

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

if the organophosphate molecule would help to overcome the resistance, data showed that additional of Malathion in mixture of imazethapyr and penoxsulam significantly reduced the dry weight and increased mortality of resistant weeds, which clearly proved the CYP boosting was a major mechanism 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 The sequencing

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 to imazamox This is a result of target site resistance, which could significantly reduce herbicide efficacy because the herbicide molecules could not bind to targeted enzyme in weed tissue On the other hands, absorption of 14C-bispyribacsodium, -imazamox, and -penoxsulam was similar in all biotypes However, the translocation of 14C-Bispyribac and 14C-Imazamox were 31− 43% and 39% less in R compared to S biotypes respectively The result introduces a second mechanism of ALS herbicide resistance in barnyardgrass

populations

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 many publications since it has introduced into the market In the research of Grossmann and S Florene (1998), quinclorac (quinolinecarboxylic acid quinclorac (3,7-dichloro-8-quinolinecarboxylic acid) was an artificial synthetic auxin, which is classified

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 long residuality 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 later distributed 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 quinclorac metabolic process in rice was different from barnyardgrass When absorbed, Quinclorac would stimulate the activity of ACC synthase Thus, accelerated the 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 were limited

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 from HCN 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 of beta cyanoalanine synthase and reduced sensitivity of Quinclorac on internal ethylene production

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

of quinclorac, including solo herbicide and mixtures Some common brands are Facet 250SC (manufactured by BASF chemical), Clorcet (50WP, 250SC, and 300SC manufactured by Cali – Parimex Inc.), Ekill (25 SC, 37WG, and 80WG manufactured by Map Pacific PTE Ltd), and Forwacet (50 WP and 250SC 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 and Rural 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 weed management in large-scale crop production worldwide However, similar to other chemical based solutions, weed management by herbicide has shown both advantages and disadvantages The herbicide treatment timing for weed control can be categorized by pre-emergence and post-emergence, the differentiation based on the mechanism and application timing of the particular herbicide against target weeds Herbicides should be selected based on the mode of action and selectivity in different crop models (Talbert and Burgos, 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 India was

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 Asia countries since last decades Therefore, management of herbicide usage is very important 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 a manual hand-weeding model in rice On the other hand, the total cost for controlling of herbicide-resistant barnyardgrass was significantly higher than in normal methods because it required both herbicide and hand-weeding practice

to control herbicide-resistant weeds

The compatibility with mechanical and crop production in large scale are two other reasons made herbicide become widely adopted in global rice cultivation, also because the manual hand-weeding was limited by small-scale production 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 weed management 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 Action Committee 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 and Holtum, 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 was increasing with the adoption of an intensive crop cultivation system in the country

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 quite expensive and it increased the cost of production In addition to the cost matter, 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 crop production 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 weeds Therefore,

increased the use of herbicide and caused adverse effects to the environment and

human health (Monaco et al., 2002)

The initial frequency of resistant individuals to a Group B herbicide (sulfometuron-methyl) was 1 in 45,000 to 1 in 8,0000 plants in untreated annual ryegrass, which also suggested that higher initial frequencies would allow rapid evolution of resistance with fewer herbicide applications (Preston and Powles, 2002) In general, the initial frequency of resistance in any population to all herbicides may be 1 in 10,000 to 1 in a billion plants (Storrie, 2007)

There are seven recommended practices for herbicide resistance

management (William et al., 2012), and the most important practice is using

different herbicide MOAs in the annual rotation, tank mixtures, and sequential applications Using multiple MoAs and mixtures is also a vital strategy for herbicide resistance management, because the possibility for an individual weed plant to evolve its herbicide-resistance to multiple herbicides at the same time

is less feasible (Bradley et al., 2014)

2.5.2 Target site resistance

2.5.2.1 Mechanism of resistance to ACCase inhibitor

Each herbicide has a specific site of action (SOA), usually an enzyme or protein in a plant cell Any change or shift in target sites can result in resistance (target-site resistance), and the changes will reduce the binding of herbicide to targeted enzyme or protein (Storrie, 2007)

Fenoxaprop-P-ethyl is classified as ACCase inhibitor herbicide, the molecule is an effective herbicide for grassy weed control in rice The Fenoxaprop inhibits enzyme acetyl-CoA carboxylase (ACCase) in

mitochondria of susceptible plant cell (Phongphitak et al., 2014)

The ACCase is the first enzyme of fatty acid synthesis in cell plastid There are two forms of ACCase in higher plants, the eukaryotic and prokaryotic, in which the eukaryotic cytosolic form is mostly resistant to herbicide and prokaryotic plastidic form is susceptible to herbicide (Konishi and Sasaki, 1994)

The plastidic enzyme is subdivided into two isoforms of dimeric form and unusual multidomain form the dimeric form is found in most monocots and all dicots The multidomain form is found only in graminaceous monocots, and it

is the main target for ACCase inhibiting herbicide The inhibition of ACCase will interrupt the synthesis of phospholipid needed for building cell wall and cell development (Konishi et at 1996)

Fenoxaprop-P-ethyl was used for sprangletop (Leptochloa chinensis (L.)

Nees) control in rice field of Thailand in 1992 However, the

fenoxaprop-resistant sprangletop was first reported in 2002 (Bo et al., 2012), a study in

Saphan-Sung province that found nine of eleven weed samples were fenoxaprop-resistant sprangletop In this study, fenoxaprop was used more than twice per year in the same location for almost 10 years The fenoxaprop-resistant sprangletop in this study could survive at a dosage of 10-25 folds higher than label dose The enzyme ACCase of resistant weed was 10 times less sensitive

to fenoxaprop compared to enzyme of susceptible sample As a result, this study confirmed the phenomenon of target site herbicide resistance or mutation of

target site (Pornprom et al., 2006)

Reducing the sensitivity of target enzyme in Echinochloa crus-galli to fenoxaprop also discovered, in the report of Amany et al (2012), the required

dose to reduce 50% biomass of fenoxaprop resistant barnyardgrass to be 12.07 times higher than the dose for susceptible population An alteration in the gene(s) of ACCase had greatly reduced the binding probability of fenoxaprop,

and resulted in higher survival of resistant weed under herbicide treatment 2.5.2.2 Mechanism of resistance to ALS inhibitor

Group B herbicide or ALS inhibitor is the largest herbicide group today This herbicide group is designed to target Acetolactate synthase (ALS) or Acetohydroxyacid synthase (AHAS), with AHAS as the main enzyme in synthesis of many amino acids in the plant cell, including isoleucine, leucine, valine (LaRossa and Schloss, 1984) Interruption of amino acid synthesis will influence the protein production and result in plant’s death, which means that

an alteration of amino acid coding for the ALS is one of the most pivotal mechanism of ALS inhibitor resistance in weed (Heap, 2017)

Until October 2017, there were 46 of 145 reported species resistant to ALS inhibitor herbicides (Heap, 2017) Similar ALS herbicide resistance due to an

alteration of amino acid coding for the ALS are also reported in Arabidopsis thaliana (Yu and Powles, 2014; Kaundun, 2014)

Penoxsulam is an ALS herbicide used for weed control in rice field

globally, and this herbicide is highly effective towards Echinochloa spp weed

A study on 172 Echinochloa oryzoides populations in Turkey showed 78% of

these populations were resistant to penoxsulam, and 14 populations were ALS and ACCase resistant weed (Emine and Husrev 2011)

The ALS resistant barnyardgrass was also reported in Italy where 90% of herbicides were ALS inhibitors The genetic makeup of five barnyardgrass

populations that were confirmed to become immune to bensulfuronmethyl and imidazolinone was studied The analysis discovered several mutations in the genetic code for ALS The alteration of G by T in the genetic code resulted in a replacement of tryptophan by leucine, and this modification of ALS reduced

herbicide influence in weeds growth (Panozzo et al., 2013)

2.5.2.3 Mechanism of resistance to photosynthesis inhibitor

Photosynthesis inhibitor is one of the most popular herbicides used for weed management in the rice field The herbicide of this group inhibits the photosynthesis at photosystem II (PSII) by binding into the activity site of QB,

a plastoquinone in protein D1 belong to photosystem II, which locates in thylakoid of chlorophyll (Gronwald, 1997)

The binding of herbicide molecules can block electron transfer from QA in protein D2 to QB This interruption is resulted in the failure of CO2 fixation and the production of ATP and NADPH in plant cell (Gronwald, 1997) However,

the resistance mechanism to PSII inhibitor was found in Arabidopsis thaliana This herbicide resistance was related to gene psbA encoded protein D1 in DNA

of chlorophyll Genetic analysis showed an alternation of serine by glycine in location 264 This change decreased the affinity between herbicide molecule and QB because the binding was depended on the hydrogen bond between herbicide molecule and hydroxyl group of serine in QB (Trebst et al., 1991)

Gronwald (1997) also discovered the replacement of eight amino acids near QB

to be responsible for herbicide resistance level in Lolium multiflorum,

depending on the replaced amino acid and location of replacement

The target site mutation was the main mechanism of ametryn and

metribuzin resistance in Echinochloa colona in Iran The molecular analysis in

five resistance samples revealed two mutations in weed DNA This mutated sequence got guanine instead of adenine in locations of 232 and 286 in the gene

psbA encoded for ALS, which resulted in a higher resistant level of weed to

ametryn and metribuzin (Elahifard et al., 2013)

2.5.3 Non target site resistance

2.5.3.1 Enhanced activity of cytochrome P450 in herbicide metabolism

Beside target site resistance, non-target site resistance is another important element of the herbicide-resistance in plants There are several mechanisms of non-target site resistance, one of the most studied mechanism is enhanced activity of cytochrome P450 in plant cell This cytochrome P450 is a family of isozyme, the enzyme responsible for the metabolism of several chemical and

compounds in cell Plant cell can biotransform toxic compounds and also herbicide through two phases: first is oxidation and then followed by conjugation with glutathione Enzyme P450 monooxygenase is the main

enzyme in the first phase of herbicide detoxification in plant cell (Cummins et al., 2013)

The non-target site ALS resistant Echinochloa crus-galli was also reported

in Japan with one population became resistant to three molecules of penoxsulam, propyrisulfuron, and pyriminobac-methy Molecular analysis showed the difference in structures of amino acid coding for ALS did not contribute to the ALS herbicide resistance, instead, the resistance was due to

acceleration of cytochrome P450 metabolism of herbicide (Iwakami et al.,

2015)

Malathion can be used as inhibitor of cytochrome P450 in plant cell

according to a study on bispyribac resistant Echinochloa phyllopogon in the

U.S., which showed the bispyribac-resistant sample was also cross-resistant to bensulfuron methyl In this study, the cytochrome P450 in leaf extract of resistant plant was 3.8 to 6.9 folds higher than enzyme activity of susceptible sample The target site mutation was not responsible for the resistance in this case In addition, the addition of Malathion in the herbicide mixture resulted in 88-97% weed control efficacy in herbicide-resistant population compared to 53% control efficacy of the control sample Inhibition of cytochrome P450 was

the main factor to suppress the detoxification in resistant weed (Fischer et al.,

2000)

Cytochrome P450 is not only effective against ALS inhibitor herbicides but also responsible for detoxification of several ACCase inhibitor herbicides (fenoxaprop-ethyl, cyhalofop-butyl) and lipid synthesis inhibitor (thiobencarb)

in Echinochloa phyllopogon in California, U.S After being treated by

fenoxaprop and thiobencarb, the cytochrome P450 activity in tissue extract of resistant plant was 141% and 300% higher than enzyme activity in susceptible plant respectively It implied a significantly higher rate of detoxification in

herbicide-resistant plant over susceptible plants in this study (Yun et al., 2005)

2.5.3.2 Enhanced activity of glutathione S-transferase in herbicide metabolism

The Glutathione S-transferase (GST) (Figure 2.8) is an enzyme group found in higher plant, fungi, insect and mammal, there are 55 and 79 GST genes

discovered in Arabidopsis and rice (Oryza sp.) respectively (Dixon and Edwards, 2010; Jain et al., 2010) The GST enzymes can catalyze xenobiotics

(alien agents in plant cell) by conjugate the tripeptide glutathione with toxic substances in cell This enzyme group is also responsible for the transformation

of anthocyanin and cinnamic acid While the cytochrome P450 is the major enzyme of the first phase (oxidation) of herbicide detoxification, GST is the main enzyme of the second phase of catalyzation, the process prevents herbicide

to interact with their binding target sites After catalyzation, the glutathione pump in cell wall can excrete the catalyzed substances out of cell, and substances can be isolated in stroma or intercellular (Marrs, 1996)

An important role of GST is detoxification of multiple xenobiotics, including many herbicides used for weed control today The multiple-herbicide

resistance in annual rye-grass (Lolium rigidum) and black-grass (Alopecurus myosuroides) was associated with enhanced activity of GST AmGSTF1 is an

glutathione peroxidase, and this enzyme can increase the activity of GST in black-grass, resulted in multiple herbicide resistance in this weed In addition,

up-regulation of the AmGSTF1 in transgenic Arabidopsis thaliana (multiple

herbicide resistant black-grass as gene donor) also showed similar herbicide

resistance to black-grass (Cummins et al., 2013)

Figure 2.8 GST-catalyzed detoxification of atrazine in plants

(Ryan, 1970) Many herbicides include atrazine and paraquat that induce oxidative reactions in plant cell that can be effectively detoxified by GST in many

herbicide-resistant weeds (Katerova et al., 2010) On the other hand, the

increased expression of GST enzyme is also an essential part of safener-induced

protection in cereal herbicides Arabidopsis seedlings treated by herbicide

safeners of benoxacor, fenclorim and fluxofenim showed enhanced GST activities toward different herbicides, therefore, this mechanism is widely used

to study safener for herbicides (DeRidder et al., 2002) Milner et al (2001) also

reported GST as the main mechanism of herbicide resistant black-grass

(Alopecurus myosuroides) in England

2.5.3.3 ATP-binding cassette (ABC transporters) in herbicide metabolism

The ATP-binding cassette (ABC transporters) (Figure 2.9) are a large family of protein found in all plant species Most of ABC transporters are membrane proteins that transport various substrates in plant cell, including lipids, auxin and pesticide molecules across intracellular membranes These ABC transporters are very important in pesticide resistance mechanism of insect

pest and weed (Andolfo et al., 2015)

Study of auxinic herbicide detoxification of William (2012) on

Arabidopsis thaliana found that the protein PDR9, a ABC transporters in cell

membrane, is a major factor in the detoxifying process This protein was not responsible for transportation of natural auxin (IAA) in leaf, but it was specified for transporting synthetic auxin herbicide like 2,4-dichlorophenoxyacetic acid

(2,4-D), and the mutation pdr9-1 is responsible for resistance to 2,4-D by

enhancement of protein PDR9 in plant

The glyphosate (N-phosphonomethyl glycine) is one of the most used herbicide for non-selective weed control management worldwide today Glyphosate binds into enzyme 5-enolpyruvylshikimate 3-phosphate synthase (EPSPS) EPSPS is a main enzyme in shikimate pathway of plant cell, and it is critical for aromatic amino acid synthesis The binding of glyphosate molecule blocked the phosphoenol pyruvate, and interrupted amino acid synthesis, which resulted in plants’ death within a very short time after herbicide application

(Schönbrunn et al., 2001)

There are various mechanisms of glyphosate-resistance reported including: enzyme EPSPS mutation (target site resistance), up-regulation of

gene EPSPS expression, reduced uptake and transportation of glyphosate, and

mutation in ABC transporter genes (Sammons and Gaines, 2014)

The activity of ABC transporters and expression of their genes in Conyza canadensis were heavily influenced by environmental conditions The genes M10 and M11 (ABC transporter) in the glyphosate resistant Conyza canadensis

under temperature 25-35oC condition showed up-regulation and increased activity of ABC transporter However, under low temperature at 8oC, the expression level of these two genes in resistant plant was not much different to susceptible plants The detoxification also decreased, and the plants’ mortality

was increased as a result (Tani et al., 2016)

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) (Linton, 2007) 2.5.4 Multiple herbicide resistance

Up to 2017, there were 88 weed species reported as multiple-herbicide resistant weeds globally (Heap, 2017) Herbicide application at a lower than recommended dose can increase the risk of herbicide resistance, because of accumulation of individuals that could survive under herbicide selection Those plants were low resistant to the herbicide, however, the next generations could inherit the characteristics to help them survive better under the same selection factor (same herbicide) Resistance to multiple herbicides could be accumulated

in the same way (Gressel, 2009)

The multiple-resistance due to target-site modification and non-target site

evolution had commonly been observed in Lolium rigidum, to both groups A

and B herbicides Target-based resistances occur as the result of different mutations in the target region(s) (Devine and Eberlein, 1997) Multiple

resistance has been documented in Amaranthus hybridus, with altered sites of

action to triazines (C) and 4 0 ALS-inhibiting (imazamox and thifensulfuron)

(B) herbicides (Maertens et al., 2004)

In Amaranthus powellii, the mutations in the target sites of triazine and

imidazolinone, the psbA (substitution of glycine for serine) and ALS genes (substitution of threonine for serine) would cause a multiple-resistance (Diebold

et al., 2003) The resistant weed with multiple-resistance mechanisms in a

population will increase the rate of spread and selection for resistance

Multiple-resistance could be results of different mechanisms of

herbicide-resistance Alopecurus myosuroides were glyphosate-resistant plant, and this

resistance was associated with two mechanisms including reduction of herbicide translocation in leaf and increasing of detoxification of herbicide by cytochrome

P450 in plant cell (Neve and Powles 2005, Mayank et al., 2010)

It was also reported that Echinochloa crus-galli in Arkansas (U.S.) was

quinclorac and propanil-resistance by at least two different mechanisms Plants could metabolize quinclorac by enhancing activity of β-cyanoalanine synthase, and oxidize the propanil by increasing activity of cytochrome P450 in plant

tissue (Mahmood et al., 2016)

Lolium multiflorum is the weed with highest number of cases reported for

multiple herbicide resistance Four genes were found over-expressed in resistant

Lolium multiflorum, including two cytochrome P450s, one nitronate

monooxygenase and one glycosyl-transferase This weed exhibited resistance

to both ACCase inhibitors and ALS inhibitors in this study (Mahmood et al.,

2016) As mentioned in the review of Glutathione-S-transferase, the increased activity of this enzyme also plays an essential role in multiple herbicide

resistance

2.5.5 Popular testing methods for herbicide resistance

Since the first report of herbicide resistant weed, herbicide resistant weed control became an urgent issue worldwide Different resistance testing methods have been used such as pot-seed, pot-plant, petri dish germination, molecular techniques, and radiolabeled assay Among these methods, petri dish germination has the advantages of short duration and low cost However, it cannot detect resistance regardless of mechanism, and it is not suitable for all kinds of weeds and herbicides (Moss, 1999)

On the other hand, pot-seed method is suitable for all weed species as well

as all kinds of herbicides and it can detect resistant mechanism Pot-plant method can give the answer in the same crop year, but field conditions have to

be imitated Molecular assay and radiolabeled assay give fast results in detection, yet the cost is very high and it is limited in mechanism discoveries

(Heap et al., 1993)

2.5.5.1 Greenhouse pot-seed from field screening method

According to Moss (1999), the reliability of results based on plant assays

is mainly dependent on the quality of the seed sample from which they were grown Poor quality seeds will often lead to low germination percent or produce poor plants with consequent variable response to herbicides Best practice is to collect seeds when most of seeds in flower were matured; collecting too early

or too late is likely to lead to samples with low viability Collect ripe seeds by gently rubbing inflorescence over a bag or tray

It is important to collect the seed in an area of at least 50m2 to 100m2 within the main problematic area, unless the problem is confined to one or more smaller, distinctive patches Quality is more important than quantity Collect at least a volume of 250ml of grass-weeds’ seeds The amount of seed to collect

of other weeds will vary with seed size and ease of collection, but the aim must

be to collect an adequate sample (several thousand seeds) of ripe seeds Do not collect in wet conditions Beware of rapid heating of freshly collected samples

- do not store in polyethylene bags Seeds should be kept in paper envelopes for transport and storage (Llewellyn and Powles, 2011)

Air dry seeds as soon as possible after collection Clean samples to remove poor quality seeds The best technique for cleaning samples will vary with species but sieving to remove large pieces of plant debris and air flow to remove

lighter seeds are appropriate for many species (Heap et al., 1993)

This method is suitable for all weed species as well as all kinds of herbicides It can detect resistance regardless of mechanism and the cost is both medium and quite acceptable However, since it depends on seed collection, the answer cannot be expected in the same crop year and field conditions have to be

mimicked The test duration is also slow (Moss et al., 2007) Also according to Moss et al (2012), at least 6 doses are required to obtain good estimation of

LD50 for screening test

Figure 2.10 Plant nursery

for herbicide screening test

Figure 2.11 Results of herbicide screening in different weed populations

According to Moss (1999), the most widely used test for resistance involves growing plants from seeds collected from the suspected field and spraying them with herbicides applied either at a single discriminating dose, or

a range of doses Such assays are usually conducted in a glasshouse or controlled environment chamber

An essential component of all resistance assays is the inclusion of an appropriate susceptible referenced population Statistical advice should be sought to ensure that the experimental design and replication is proper For dosage response experiments (Figure 2.12), use a wide range of different doses

to obtain a response curve The response curve help to estimate the resistance degree and calculate the doses required to produce the same effect in resistant and susceptible populations (Llewellyn and Powles, 2011)

Ratios of these estimates, (variously termed ED50, GR50, LD50 or 150), relative to that of a susceptible population, provide a resistance index (RI) which enables the degree of resistance to be described relatively simply To obtain a good estimate of ED50 the dose range should be relatively wide and at least six doses are needed It is usually best that each dose is twice the preceding dose in the range (e.g 10, 20, 40, 80, 160, 320 g a i./ha) The dose range used should include doses both below and above the recommended rate because herbicides are normally more active under greenhouse conditions (Llewellyn and Powles, 2011)