Evolution and spread of glyphosate resistant barnyard grass (echinochloa colona (l ) link)

Evolution and Spread of Glyphosate Resistant Barnyard Grass Echinochloa colona L.. 75 Chapter 5: Inheritance of Evolved Glyphosate Resistance in Barnyard Grass Echinochloa colona from

Evolution and Spread of Glyphosate Resistant Barnyard Grass

(Echinochloa colona (L.) Link)

By

Thai Hoan Nguyen

This thesis is submitted in fulfilment of the requirements

for the degree of

Doctor of Philosophy

School of Agriculture, Food and Wine

Faculty of Sciences The University of Adelaide Waite Campus

March, 2015

Abbreviations

ACCase: Acetyl-CoA carboxylase

AFLP: Amplified fragment length polymorphism

AGRF: Australian Genome Research Facility

ALS: Acetolactate synthase

EPSP: 5-enolpyruvylshikimate-3-phosphate synthase

HAT: Hour after treatment

LD50: Lethal dosage (dose required to control 50% of individuals in the population) LSD: Least significant different

NSW: New South Wales

PCR: Polymerase chain reaction

QLD: Queensland

R/S: Resistance/susceptibility

RAPD: Randomly amplified polymorphic DNAs

RFLP: Restriction fragment length polymorphism

SA: South Australia

SE: Standard error

SSR: Simple sequence repeats

VIC: Victoria

WA: Western Australia

Table of Contents

Abbreviations i

Table of contents ii

List of tables vii

List of figures ix

Abstract xi

Declaration xiii

Tables Published with Consent from Copyright Holders in this Thesis xiv

Figures Published with Consent from Copyright Holders in this Thesis xiv

Acknowledgements xv

Chapter 1: General Introduction 1

Chapter 2: Literature Review 6

2.1 Introduction 6

2.2 Echinochloa spp 6

2.2.1 Geographical distribution of Echinochloa spp in the world 6

2.2.2 Biology 8

2.2.3 Agronomical features 8

2.2.4 Problems caused by Echinochloa spp in Australia 9

2.2.5 Herbicide resistance in Echinochloa spp 10

2.2.6 Herbicide resistance in weed species in Australia 11

2.3 Causes of resistance evolution 13

2.3.1 Genetic mutations endow resistance 13

2.3.2 Initial frequency of resistant alleles 14

2.3.3 Selection pressure 14

2.3.4 Inheritance of resistance 15

2.3.5 Gene migration 15

2.3.6 Fitness of resistant individuals 16

2.3.7 Characteristics of the seed bank 17

2.4 Glyphosate 17

2.4.1 Properties of glyphosate 17

2.4.2 Mode of action 18

2.4.3 Glyphosate resistant weeds 19

2.4.4 Mechanisms of glyphosate resistance 20

2.5 Molecular markers 22

2.5.1 Scientific basis of the use of molecular makers in determining the spread of resistance evolution in Echinochloa spp 22

2.5.2 DNA fingerprinting techniques 22

2.5.3 DNA sequencing 24

2.6 Conclusions 25

Literature cited 26

Chapter 3: Genetic Diversity of Glyphosate Resistant Junglerice (Echinochloa colona) in New South Wales and Queensland 34

Materials and methods 36

Plant material 36

Glyphosate dose response experiment 38

AFLP analysis 39

Results and discussion 41

Response to glyphosate 41

AFLP analysis 43

Genetic diversity across E colona populations 43

Genetic diversity within populations 45

Acknowledgements 51

Literature cited 51

Chapter 4: Temperature Influences the Level of Glyphosate

Resistance in Barnyard Grass (Echinochloa colona) 56

Abstract 56

1 Introduction 57

2 Materials and methods 58

2.1 Plant material 58

2.2 Temperature response to glyphosate 58

2.3 Identifying target-site mutations 59

2.4 EPSPS gene relative copy number 61

2.5 Shikimate assay 62

2.6 Effect of temperature on absorption and translocation of glyphosate 63

3 Results 64

3.1 Temperature response experiments 64

3.2 Target-site mutations 66

3.3 EPSPS gene copy number 67

3.4 Effect of temperature on shikimate accumulation 67

3.5 14 C-glyphosate absorption and translocation as affected by temperature 68

4 Discussion 70

4.1 Temperature influences glyphosate resistance 70

4.2 Target-site contributes to glyphosate resistance 71

4.3 14 C-glyphosate absorption and translocation as affected by temperature 71

5 Conclusions 74

Acknowledgements 75

References 75

Chapter 5: Inheritance of Evolved Glyphosate Resistance in Barnyard Grass (Echinochloa colona) from Australia 80

Abstract 80

5.1 Introduction 81

5.2 Materials and methods 82

5.2.1 Plant material 82

5.2.2 Gene flow between resistant and susceptible individuals 83

5.2.3 Hand crossing of resistant and susceptible individuals 85

5.2.4 Sequencing of F 1 and F 2 progenies 85

5.2.5 Shikimate accumulation 86

5.2.6 Segregation test 87

5.2.7 Response to glyphosate 88

5.2.8 EPSPS cDNA sequencing 88

5.3 Results and discussion 89

5.3.1 Plant growth of the parental populations 89

5.3.2 Gene flow frequency 91

5.3.3 Detecting EPSPS gene mutation in F 1 and F 2 progenies 93

5.3.4 Shikimate assay 94

5.3.5 Segregation test 95

5.3.6 Dose response to glyphosate of F 2 progenies 97

5.3.7 EPSPS cDNA sequencing 98

5.4 Conclusions 100

Literature cited 101

Chapter 6: General Discussion 107

6.1 Discussion of results 107

6.2 Conclusions 113

6.3 Contributions to knowledge 114

6.4 Future research 114

Literature cited 115

Appendices 121

Appendix 1: Geographical sites of towns where are nearest to origins of 65 E colona populations used in this research (Chapter 3) 121

Appendix 2: Response of eleven E colona populations to different

glyphosate rates in the dose response experiment (Chapter 3) 122

Appendix 3: Distance matrix from AFLPs based on jaccard’s

coefficient in comparison between populations collected across

Queensland and New South Wales, Australia (Chapter 3) 123

Appendix 4: Distance matrix from AFLPs based on jaccard’s

coefficient in comparison within populations collected in

New South Wales, Australia (Chapter 3) 128

Appendix 5: Number and name indexes of E colona

populations in Chapter 3 and Appendices 3 and 4 141

Appendix 6: Dendrogram of the partial sequence of the predicted

amino acid at codon 106 in the EPSPS gene of the susceptible

population (Echi S) and the resistant population (A533.1) (Chapter 4) 142

Appendix 7: The sections of the treated leaf, the non-treated leaves,

the stem and the roots of E colona plants were cut at harvest time points

of 12, 24, 48 and 72 hours after glyphosate application (Chapter 4) 143

Appendix 8: Response of E colona populations to glyphosate

(240 g a.i ha-1) at two different temperature levels (20 and 30oC)

at three weeks after glyphosate application (Chapter 4) 144

Appendix 9: The pair of resistant (A533.1) and susceptible (Echi S)

E colona individuals at flowering, and two single spikes of each

resistant and susceptible individual were bagged with glassine bags

as controls before anthesis in gene flow experiments (Chapter 5) 145

Appendix 10: Survivors of E colona in gene flow frequency

experiment at 30 days after spraying glyphosate (Chapter 5) 145

Appendix 11: Growth time, flower-head number, 100-seed weight

and germinability of the resistant (A533.1) and the susceptible

(Echi S) populations of E colona in gene flow experiments (Chapter 5) 146

Appendix 12: Number of E colona plants before and after spraying

glyphosate in the gene flow frequency experiment (Chapter 5) 147

List of Tables

Chapter 2

Table 2.1 Documented herbicide resistance in weed species in Australia 12

Chapter 3

Table 1 Location and resistance phenotype of E colona populations

used in this study with resistance that was determined by

treating with glyphosate at 270 g a.e ha-1 37

Table 2 AFLP adaptors and primers 40

Table 3 The glyphosate resistance levels of ten E colona populations 42

Table 4 Between-population genetic structure: fragment lengths, total

number of fragments, number and percentage of polymorphic

fragments produced by each primer set used to analyse the

polymorphisms of one individual from each of 62

E colona populations 44

Table 5 Within-population genetic structure: fragment lengths, total

number of fragments, number and percentage of polymorphic

fragments produced by each primer set used to analyse the

polymorphisms of one individual from each of two glyphosate

resistant E colona populations (63 and 64) and one susceptible

population (65) 47

Chapter 4

Table 1 Locations are nearest to origins of E colona populations

used in this study 58

Table 2 The primers and probes used to identify the target-site

mutation and determine the genomic copy number of

EPSPS and ALS using quantitative real-time PCR 60

Table 3 Glyphosate dose required to control 50% (LD50)

(g a.e ha-1) of susceptible and resistant E colona

populations at 20oC and 30oC 66

Table 4 Nucleotide and predicted amino acid sequence of

EPSPS DNA isolated from a susceptible and ten

resistant populations of E colona 67

Chapter 5

Table 1 Growth time (transplanting, flowering and harvest)

of the resistant (A533.1) and the susceptible (Echi S)

populations of E colona in the gene flow experiment 89

Table 2 Flower head number, 100-seed weight and germinability

of the resistant (A533.1) and the susceptible (Echi S)

populations of E colona in the gene flow experiment 90

Table 3 Survival of F1 progenies from four parental susceptible

plants in the gene flow experiment to determine gene flow

frequency between resistant (A533.1) and susceptible

(Echi S) E colona plants 91

Table 4 Segregation of the F2 progenies from selfed F1 survivors of

E colona from the gene flow experiment after glyphosate

treatment at of 240 g a.e ha-1 96

Table 5 Mutation at position 106 of the cDNA EPSPS gene

detected after cloning of resistant and susceptible

plants, and F2 progenies of E colona 99

Figure 1 UPGMA phenogram of the genetic relationship between E colona

populations collected across QLD and NSW, Australia 46

Figure 2 UPGMA phenogram showing the genetic relationship

within two resistant populations (63 and 64 in Table 3.1)

and the susceptible population (65) of E colona collected

from three separate fields in NSW, Australia 49

Chapter 4

Figure 1 Response of E colona populations to glyphosate at 20oC and 30oC 65

Figure 2 Shikimic acid accumulation of leaf discs from two resistant

(A533.1 and A818) and one susceptible (Echi S) E colona

populations at different glyphosate concentrations at 20oC

and 30oC 68

Figure 3 14C-glyphosate absorbed and translocated to plant sections

of two resistant (A533.1 and A818) and one susceptible

(Echi S) E colona populations at 20oC and 30oC at 12,

24, 48 and 72 hours after glyphosate application 69

Chapter 5

Figure 1 Survival percentage at 21 days after glyphosate

treatment of the resistant (A533.1) and the susceptible

(Echi S) populations of E colona in the rate test 90

Figure 2 The morphology of E colona flowers at the

opening of the flower and before pollination,

with pollen grains adhering to the stigmata 92

Figure 3 Shikimate accumulation of parental plants

(A533.1 and Echi S) and the F1 cross of E colona 95

Figure 4 Glyphosate dose response of susceptible and resistant

populations of E colona and the F2 population 98

Abstract

Echinochloa colona is an important summer-growing weed species in northern Australian

cropping regions The intensive use of glyphosate in summer fallow operations has led to the

appearance of glyphosate resistant E colona populations at a large number of sites Studies of

the genetic diversity, resistance mechanisms, inheritance and spread of resistance were

undertaken to better understand the evolution of glyphosate resistance in this species A

survey of 65 barnyard grass populations collected from Queensland and New South Wales

determined 34 populations were resistant to glyphosate with resistance levels ranging from 2

to 11-fold High genetic diversity within and between these populations was identified by the

AFLP technique A total of 99.2% of alleles identified within populations were polymorphic

with a higher percentage of polymorphic alleles within the two resistant populations

compared to the susceptible population The level of glyphosate resistance in populations was

dependent on the ambient temperature Resistant populations showed a noticeably higher

level of resistance at 30oC compared to 20oC whereas there was no effect of temperature on the response of the susceptible population Experiments were carried out on glyphosate

absorption and translocation in resistant and susceptible plants to identify the reason for these

differences and the results showed a considerable decrease in glyphosate absorption into

leaves at 30oC Differences were also identified in glyphosate translocation between the treated leaves and the other sections of plants at the different temperatures There were no

differences in glyphosate absorption or translocation between the susceptible population and

the resistant populations suggesting that differences in absorption and translocation of the

herbicide are not the mechanism of resistance in the studied populations Studies of EPSPS

gene copy number showed gene amplification was not the resistance mechanism either A

mutation was detected at codon 106 (proline substituted by serine) of the EPSPS gene of the

most resistant population, A533.1, indicating the presence of target-site resistance in this

population Gene flow by pollen exchange between the glyphosate resistant population

A533.1 and the susceptible population Echi S occurred at a frequency of 1.38% when progeny

from the susceptible parent was tested at 240 g a.e ha-1 of glyphosate The mutation in the

EPSPS gene was detected in 24 F1 progenies of this population pair Segregation of resistance

in the gene flow experiment between resistant and susceptible individuals occurred at a 3:1

resistance : susceptibility ratio in the F2 generation indicating the trait of glyphosate resistance

is a single dominant trait of E colona Sequencing the EPSPS cDNA of five parental and F2

filial individuals revealed at least two EPSPS genes present in E colona Shikimate

accumulation of the F1 hybrid and the glyphosate response of F2 progenies were intermediate between the two parental populations

Tables Published with Consent from Copyright Holders in this Thesis

Table 2.1 Documented herbicide resistance in weed species

in Australia (Heap, 2014) 12

Figures Published with Consent from Copyright Holders in this Thesis

Figure 2.1 Chemical structure of glyphosate (Franz et al., 1997) 17

Figure 2.2 Activity of glyphosate on reaction catalysed by enzyme

5-enolpyruvylshikimate-3-phosphate synthase (Amrhein et al., 1980) 19

Acknowledgements

I would like to express sincere thanks to my supervisors, Associate Professor Christopher

Preston, Dr Peter Boutsalis and Dr Jenna Malone, for their guidance, help and support

through the whole of my research project Dr Jenna Malone has guided and provided me with

valuable advice on technical aspects, especially on molecular biotechnology The materials

and methods section in my thesis and papers was also mostly corrected by Dr Jenna Dr Peter

Boutsalis gave me technical advice on experiments in shade-house Particularly, I am greatly

indebted to my principal supervisor Associate Professor Christopher Preston, who has given

me invaluable guidance, advice, comments and encouragement throughout this research In

addition, he has helped me in improving skills in experimental data analysis and other

scientific aspects

I am extremely grateful to the staff of the weed science group, Sarah Morran, Mahima

Krishnan, Robin St John-Sweeting, Patrick Krolikowski, Ruwan Lenorage and Geetha

Velappan Sarah Morran guided me through the glyphosate absorption and translocation

experiment Mahima Krishnan made a valuable contribution to the technique of EPSPS

cDNA sequencing Patrick Krolikowski, Ruwan Lenorage and Geetha Velappan gave me

assistance in giving an eye to experiments in shade-house including watering experimental

plants My sincere appreciation is also sent to lab mates, Patricia Adu-Yeboah, Mohammed

Hussein Minati Al-Asklah, Rupinder Haur Saini, Lovreet Singh Shergill and Duc The Ngo,

who shared study experience with me, encouraged me in my PhD program and provided me

with priceless discussions

I would like to express my sincerest gratitude to my external advisor, Dr John Heap, who

gave me the useful advice on experiments on the temperature response of glyphosate resistant

barnyard grass populations I would like to extend my gratitude to post-graduate coordinators

Associate Professor Gurjeet Gill and Dr Matthew Denton for support and helpful advice My

special thanks were also sent to Van Lam Lai, Thanh Dzung Phan, Anh Nghia Nguyen and

my colleagues at the Rubber Research Institute of Vietnam for support and encouragement

I gratefully acknowledge the Ministry of Education and Training, Vietnam for scholarship

funding, the Rubber Research Institute of Vietnam for permission to leave my duties and

study overseas, and the University of Adelaide for access to study facilities

After all, my deepest gratefulness is dedicated to the spirits of my dearly beloved mother, who

had brought up me through the last half of her lifetime and has lately passed away I am

deeply indebted to all members in my big family, my wife and daughters for their constant

love and moral support The patient wait and encouragement of my wife and daughters

throughout four years of my PhD program have motivated me to work diligently and

accomplish this thesis

Chapter 1

General Introduction

High competition for water, sunlight and nutrients with major agricultural crops has made the

barnyard grasses (Echinochloa spp.) major weed species; and they were rated among the 18

most troublesome weeds in agriculture worldwide (Holm et al., 1977) These are annual weed

species often found in paddy fields throughout the rice-growing regions around the world

(Mooney and Hobbs, 2000) In Australia, Echinochloa spp were recognised as common weed

species in summer fallows (Walker et al., 2004), as well as in crops Two barnyard grass

species (Echinochloa colona and Echinochloa crus-galli) were rated in the top three most

harmful weeds in vegetable crops (Holm et al., 1977; Walker et al., 2004)

Many methods of controlling Echinochloa spp have been used including: hand-weeding,

trampling, cultural measures, mechanical weeding and using chemical herbicides (Matsunaka,

1983) Among these methods, herbicides have been the most widely used in recent years in

most countries in the world The intensive use of herbicides over a long period of time has

resulted in the evolution of herbicide resistance in these grass species For example, the

herbicide propanil has been repeatedly used for a long time to control E crus-galli and other

grass weeds, and as a result, resistance to propanil in this grass has occurred (Norsworthy et

al., 1998)

Since the herbicide glyphosate was introduced to world agriculture in 1974, it has become the

world’s most widely used herbicide, especially since the development of genetically modified

crops with resistance to glyphosate However, continued dependence on glyphosate over a

large area ranging from field agricultural systems to inner-city landscapes has increased the

number of weed species, including E colona, that have evolved resistance to this herbicide

(Duke and Powles, 2008) Up to now, glyphosate resistance in E colona has been found in

three countries including Australia, America and Argentina (Heap, 2014) Among these

countries, Australia has been discovered to have the largest area of glyphosate resistant E

colona with three states namely New South Wales (NSW), Queensland (QLD) and Western

Australia (WA) (Heap, 2014) Glyphosate resistance has occurred in many plant species

(Lorraine-Colwill et al., 2003; Powles and Preston, 2006; Michitte et al., 2007; Gaines et al.,

2010) However, glyphosate resistance in E colona, was only recently discovered and is

poorly understood (Storrie et al., 2008; Heap, 2014)

With the aim of improving understanding of glyphosate resistance evolution in E colona, this

research was implemented to evaluate the genetic variability, assess the impacts of

temperatures on the resistance level, and determine the inheritance and spread of glyphosate

resistance in this grass species Initially, a survey was conducted through QLD and NSW, and

the response of E colona populations to glyphosate doses was examined to establish the real

status and herbicide resistance levels of E colona in these two states In the following studies,

the genetic diversity of E colona populations collected from different locations in QLD and

NSW was evaluated to understand the potential spread of glyphosate resistant E Colona

(Chapter 3) The response of glyphosate resistance in E colona to two different temperatures

was also assessed and the reasons causing different responses were elucidated (Chapter 4)

Finally, movement of the glyphosate resistance gene in E colona over the susceptible

population and the pattern of the resistance inheritance were evaluated (Chapter 5) Overall,

the components of this thesis are outlined as follows:

• Chapter 1: General introduction

• Chapter 2: A literature review covered the biological and agronomical characteristics of

Echinochloa spp., geographical distribution and impacts of these grass species, the

properties and action mode of glyphosate, glyphosate resistance evolution and the

mechanisms of resistance, and molecular markers which could be used to research on

genetics of E colona

• Chapter 3: Tested populations for glyphosate resistance, evaluated the response of resistant

E colona populations to different glyphosate rates and investigated the genetic variability within and between E colona populations collected through two states QLD and NSW in

Australia with the AFLP technique

• Chapter 4: Assessed the response of six glyphosate resistant E colona populations to two

temperature regimes (20oC and 30oC), investigated whether glyphosate resistance mechanism played a role in the response to temperature and whether absorption and

translocation of glyphosate was different at the two temperatures

• Chapter 5: Evaluated gene flow due to pollen exchange between the resistant population A533.1 and the susceptible population Echi S, the inheritance of glyphosate resistance

from a hand-cross between the same populations, the presence of mutations within the

EPSPS gene and whether the mutation was present on one or more homeologs

• Chapter 6: General discussion of the research This chapter also includes the principal conclusions of the research, contributions of the research to knowledge and farming

practice, and the potential studies for the future

Several results of this research were presented at the 18th Australasian Weeds Conference organised by Council of Australasian Weed Societies Inc and Weed Society of Victoria at the

Sebel and Citigate Albert Park, Melbourne, Victoria on 8-11 October 2012 Some information

from Chapter 3 and 4have already been also published on the proceedings of this conference

as:

Hoan Nguyen, T., Malone, J., Boutsalis, P and Preston, C (2012) Glyphosate

resistance in barnyard grass (Echinochloa colona) In Valerie Eldershaw, V (ed.),

Proceedings of the 18th Australasian Weeds Conference on Developing Solutions to Evolving Weed Problems, Melbourne, Victoria, Australia, 8-11 October 2012 Weed

Society of Victoria Inc., Batman, Vic 3058, Australia, 237-240

The results in Chapters 3 and 4 were submitted to the two journals as follows:

Nguyen, T.H., Malone, M.J., Boutsalis, P and Preston, C (2015) ‘Genetic diversity of

glyphosate resistant junglerice (Echinochloa colona) in New South Wales and

Queensland’, Weed Science, Manuscript number: WS-D-15-00006

Nguyen, T.H., Malone, M.J., Boutsalis, P., Shirley, N and Preston, C (2015)

‘Temperature influences the level of glyphosate resistance in barnyard grass

(Echinochloa colona)’, Pest Management Science, Manuscript number: PM-15-0127

As submitted to the journals, these Chapters have been prepared in publication format

Literature Cited

Duke, S.O and Powles, S.B (2008) 'Glyphosate-resistant weeds and crops', Pest

Management Science 64 (4), 317-318

Gaines, T.A., Zhang, W.L., Wang, D.F., Bukun, B., Chisholm, S.T., Shaner, D.L., Nissen,

S.J., Patzoldt, W.L., Tranel, P.J., Culpepper, A.S., Grey, T.L., Webster, T.M., Vencill, W.K., Sammons, R.D., Jiang, J.M., Preston, C., Leach, J.E and Westra, P (2010)

'Gene amplification confers glyphosate resistance in Amaranthus palmeri', Proceedings of the National Academy of Sciences of the United States of America 107

(3), 1029-1034

Heap, I (2014) The international survey of herbicide resistant weeds, viewed 30 July 2014,

<www.weedscience.org>

Holm, L.G., Plucknett, D.L., Pancho, J.V and Herberger, J.P (1977) The world's worst

weeds: Distribution and biology The University Press of Hawaii, Honolulu, Hawaii

Lorraine-Colwill, D.F., Powles, S.B., Hawkes, T.R., Hollinshead, P.H., Warner, S.a.J and

Preston, C (2003) 'Investigations into the mechanism of glyphosate resistance in

Lolium rigidum', Pesticide Biochemistry and Physiology 74 (2), 62-72

Matsunaka, S (1983) Evolution of rice weed control practices and research: World

perspective In The Conference on Weed Control in Rice, Los Baños, Laguna,

Philippines, 31 August - 4 September 1981 International Rice Research Institute (IRRI), Los Banos, Philippines, 5-17

Michitte, P., De Prado, R., Espinoza, N., Ruiz-Santaella, J.P and Gauvrit, C (2007)

'Mechanisms of resistance to glyphosate in a ryegrass (Lolium multiflorum) biotype from Chile', Weed Science 55 (5), 435-440

Mooney, H.A and Hobbs, R.J (2000) Invasive species in a changing world Island Press,

Washington, D.C., US

Norsworthy, J.K., Talbert, R.E and Hoagland, R.E (1998) 'Chlorophyll fluorescence for

rapid detection of propanil-resistant barnyard grass (Echinochloa crus-galli)', Weed Science 46 (2), 163-169

Powles, S.B and Preston, C (2006) 'Evolved glyphosate resistance in plants: Biochemical

and genetic basis of resistance', Weed Technology 20 (2), 282-289

Storrie, A., Cook, T., Boutsalis, P., Penberthy, D and Moylan, P (2008) Glyphosate

resistance in awnless barnyard grass (Echinochloa colona (L.) Link) and its implications for Australian farming systems In the 16th Australian Weeds Conference, Cairns Convention Centre, North Queensland, Australia, 18-22 May 2008 Queensland Weed Society, Queensland, Australia, 74-76

Walker, S., Widderick, M., Storrie, A and Osten, V (2004) Preventing glyphosate resistance

in weeds of the northern grain region In the 14th Australian Weeds Conference - Weed management: balancing people, planet and profit, Wagga Wagga, New South Wales, 6-9 September 2004 Weed Society of New South Wales, Sydney, Australia, 428-431

McGillion and Storrie, 2006) These grass species have been commonly found in paddy fields

around the world (Mooney and Hobbs, 2000), in summer fallows and the grain-growing lands

in QLD and NSW (Australia) (McGillion and Storrie, 2006) Lands with moist soil and mild

weather are suitable for the growth of barnyard grasses (Holm et al., 1977) As a result of

using glyphosate, a highly effective and broad spectrum herbicide, over a long period of time

to control weeds, the evolution of herbicide resistance has appeared (Norsworthy et al., 1998)

Glyphosate resistance in Echinochloa colona species was first identified in NSW, Australia in

2007 and was later discovered in the United States in 2008, Argentina in 2009, QLD and WA

(Australia) in 2009 and 2010 respectively (Heap, 2014) This review will cover aspects of

biological and agronomical characteristics of Echinochloa spp., properties and action mode of

glyphosate, glyphosate resistance and causes, mechanisms of resistance in plant species, and

molecular markers involving the studies of resistance evolution Relevant methodologies of

the studies will also be reviewed

2.2 Echinochloa spp

2.2.1 Geographical distribution of Echinochloa spp in the world

Echinochloa spp are widely distributed throughout the world E colona is distributed mainly

in equatorial regions of the world, whereas E crus-galli is more widely distributed in both the

northern and southern hemispheres (Smith, 1983) According to Holm et al (1977), E colona

is distributed through the agricultural areas from 23o N to 23o S, especially in Asia, Australia, Pacific Islands, South America and the Caribbean However, this grass species is less severe

in North Africa and Europe; and its distribution is not yet recorded in temperate regions of the

world E crus-galli is distributed over a larger area from 50o N to 40o S It is indigenous to

India and Europe Similar to E colona, the presence of E crus-galli is negligible in Africa

(Holm et al., 1977) Gonzalez et al (1983) reported that E colona was a common grass weed

in wetland rice, as well as dryland rice in almost all countries of Latin America This is an

extremely serious grass weed species and it is widespread throughout rice fields in Central

America, the Caribbean and tropical South America (Gonzalez et al., 1983) As affirmed by

Michael (1983), E colona is an important grass weed in the subtropical and tropical

rice-growing regions It also occurs on paddy fields in NSW, Australia and in the southern United

States at times

In Australia, Echinochloa spp occur commonly in the grain-growing areas in the north of the

country (Walker et al., 2004) In a survey in 1989, Felton et al (1994) found that Echinochloa

spp were the most important and common grass weeds in northern NSW while McGillion

and Storrie (2006) stated E colona is a troublesome grass for many crops in central and

southern QLD, and in southern, central and northern NSW Friend (1983) also confirmed that,

with the exception of the arid west and Tasmania, this grass species is widely distributed in all

other States of Australia including inland areas and coastal QLD It has been generally

supposed that wild ducks may have introduced Echinochloa spp to Australia; subsequently,

weed seeds were spread over paddy fields by means of the canal system which connects fields

with one another (Holm et al., 1977)

The above information indicates that Echinochloa spp inhabit many large areas worldwide

with a diverse living environment Consequently, due to the problems caused by these grass

species there is a need to develop appropriate control methods in order to minimize damage to

crops and the environment

2.2.2 Biology

Echinochloa spp are summer-growing annual grasses that grow best where there is plenty of available water (Holm et al., 1977) Yabuno (1983) suggested that E colona can also grow in

comparatively arid regions; however, in flooded conditions with 10 cm of water the growth of

seedlings would be halted In the United States, studies by McGillion and Storrie (2006)

showed E crus-galli can survive and grow in areas with temperatures ranging from 6 - 28oC,

an average annual rainfall of 310 - 2500 mm and soil pH of 4.8 - 8.2 Fertile, damp and

nitrogen rich soils are the best conditions for the growth of this grass species However, E

crus-galli can also flourish on sandy and humus soils, and even submerged soil As stated by Holm et al (1977), light is essential for the germination of E crus-galli seeds and flooding

inhibits seeds from germination For seedlings, the best growth is at 30oC, growth is sluggish

at 10oC and stopped at 5oC

In terms of propagation, Echinochloa spp reproduce sexually; however, stem nodes can root

when stems lie prostrate on the ground allowing asexual reproduction under some

circumstances (Holm et al., 1977) The number of seeds produced by mature plants is large

and one mature plant can bear more than a thousand seeds (Holm et al., 1977)

2.2.3 Agronomical features

Morphologically, Echinochloa spp resemble rice Barrett (1983) suggested that rice mimicry

in Echinochloa spp evolved over a long period of time as a result of hand-weeding rice in

Asian countries, because other weeds that are morphologically different from rice were

preferentially removed from paddies by hand weeding, hence Echinochloa spp would

increase in numbers

High densities of Echinochloa spp have dramatic impacts on crop yield Smith (1983)

concluded that on dryland and wetland paddy fields that are direct seeded or transplanted,

these grasses cause a considerable decrease in yield and quality of rice Therefore, their

presence would have a major impact on profitability of farmers (Smith, 1983) In addition to

rice, a wide variety of agricultural crops worldwide were impacted by Echinochloa spp

including vegetables, corn, sugarcane, sugar beets, sorghum, millet, potatoes, peanuts, jute,

citrus, orchards, vineyards, soybeans, bananas, cassava, taro, cowpeas, abaca, pineapples,

sweet potatoes, cotton, tea, tobacco, coffee and coconuts (Holm et al., 1977) According to

McGillion and Storrie (2006), E crus-galli not only caused a significant decrease in the yield

of agricultural crops, but also reduced production of forage crops by removing up to 80% of

available nitrogen in the soil At the same time, the accumulation of nitrates at high levels in

Echinochloa spp could harm cattle that graze the foliage (McGillion and Storrie, 2006) During the early growth stages of rice, competition from Echinochloa spp is particularly

severe compared to later growth stages, because these grasses cause serious effects on the

height, panicle number, grain yield, fertility and straw weight of rice For example, in Taiwan,

E crus-galli caused a decrease of 85% in rice yield (Datta, 1981)

2.2.4 Problems caused by Echinochloa spp in Australia

In Australia, rice was directly sown in both dryland and irrigated systems (Swain, 1973), and

the existence of E crus-galli in large numbers has resulted in a reduction in rice yield of two

to four tons per hectare (McGillion and Storrie, 2006) In sugarcane fields, this grass was

rated as one of three most damaging grass species (Holm et al., 1977) Apart from rice and

sugarcane, E crus-galli decreased yield of a wide range of other crops in Australia, such as

cotton, corn, sorghum and vegetables Similarly, in northern Australia, E colona is a

troublesome grass in rice, cotton, sorghum, sugarcane, corn, linseed, safflower and

vegetables As a result of prostrate growth habit, it is easy for this weed to obtain space below

the shade of other plants so that it can compete with surrounding crops without difficulty

(Holm et al., 1977) According to results of a survey conducted by Walker et al (2005) in

summer fallows of subtropical Australia, 82% of growers rated Echinochloa spp as their

most common weeds and the most difficult to control

2.2.5 Herbicide resistance in Echinochloa spp

As a result of the herbicide use in agriculture with an increasing intensity, herbicide resistance

of weeds is becoming more and more serious According to McGillion and Storrie (2006),

herbicide resistance of weed individuals within a population occurs in three main ways: (1)

pre-existing resistance, (2) importation of genes from resistant populations through crop seed

sources contaminated with weed seeds, animals or machinery, and (3) natural dispersal via

water and wind As a whole, once herbicides of the same group are intensively applied to a

weed population, susceptible individuals will be killed while resistant ones will set seeds

leading to an increase in the number of resistant individuals in the population (McGillion and

Storrie, 2006)

Similar to other weed species, prolonged use of herbicides for the control of Echinochloa

species readily results in the evolution of herbicide resistance An example of this occurred

with the propanil herbicide, an acylanilide herbicide that was introduced into the United

States in the 1960s to control Echinochloa spp and some other weeds in rice Propanil was

widely used on rice paddies in America and in some other countries (Hoagland et al., 2004)

As a consequence of the over-reliance on this herbicide, Hirase and Hoagland (2006)

discovered resistance in Echinochloa spp populations to herbicide at recommended doses of

3.6 to 5.6 kg a.i per hectare In addition, among eleven samples of Echnochloa spp collected

in Texas in 1992, seven samples were found resistance to propanil (Hoagland et al., 2004)

Subsequently, resistance of these grasses to propanil was also discovered at 160 sites through

38 rice-growing counties in Arkansas (Norsworthy et al., 1998) Hoagland et al (2004)

showed that propanil resistance in Echinochloa spp has occurred in a large number of

rice-growing areas in the world including Italy, Sri Lanka, Mexico, Central America nations,

Columbia, Texas, Thailand, Greece, Venezuela and Costa Rica The weeds were resistant to

propanil as a result of increased detoxification by aryl acylamidase (Hoagland et al., 2004) In

another study in California, two Echinochloa species, namely early watergrass (E oryzoides)

and late watergrass (E phyllopogon), were determined to be resistant to a variety of

herbicides including bispyribacsodium, thiobencarb, fenoxaprop-ethyl and molinate although

these herbicides belong to different mode-of-action groups (Fischer et al., 2000) Especially,

E colona has been so far found resistance to a series of herbicides including glyphosate,

atrazine, azimsulfuron, bispyribac-sodium, cyhalofop-butyl, fenoxaprop-P-ethyl,

fluazifop-P-butyl, haloxyfop-P-methyl, imazapic, imazapyr, metribuzin, propanil and quinclorac (Heap,

2014)

2.2.6 Herbicide resistance in weed species in Australia

In Australia, from 1982 to 2014 there have been reports of at least 40 weed species found

resistance to many different herbicides (Heap, 2014) Out of species, Lolium rigidum was

determined as resistant to the most numerous herbicides (10 herbicides) and resistance in

Sisymbrium orientale was found in five states of Australia In E colona, resistance to both

atrazine and glyphosate has been found in NSW, while resistance to glyphosate only has been

reported in three states QLD, NSW and WA (Table 2.1) (Heap, 2014)

Table 2.1 Documented herbicide resistance in weed species in Australia (Heap, 2014)

group

States with confirmed resistant populations

A, B, C1, D, F3, G, K1, K2, K3, N

NSW, VIC, SA,

WA

G, O

NSW, VIC, SA,

WA

F1, O

NSW, QLD, VIC, SA, WA

NSW: New South Wales, QLD: Queensland, VIC: Victoria, SA: South Australia, WA: Western Australia

Factors that influence the resistance evolution of weeds include the initial frequency of

resistance gene(s), herbicide group used, the effectiveness of herbicide, the weed population

size that is treated with herbicides and the biological characteristics of weed (McGillion and

Storrie, 2006)

2.3 Causes of resistance evolution

2.3.1 Genetic mutations endow resistance

Genetic mutations that result in herbicide resistance are unlikely to be caused by herbicides,

but occur naturally in susceptible populations at very low initial frequencies (Gressel and

Segel, 1978; Jasieniuk et al., 1996; Letouze and Gasquez, 2001) According to Merrell

(1981), the frequency of natural mutations in living organisms ranges from 1x10-6 to 1x10-5gametes/locus/generation However, normally due to the higher rates of gene flow compared

to the mutation rates, the initial frequency of herbicide resistant individuals would be high

(Crow, 1983; Jasieniuk et al., 1996) Jasieniuk et al (1996) suggested that in practice the

frequency of mutations to herbicide resistant weeds has not yet been determined, and although

the mutation rate of plants might be actually low, the likelihood of detecting at least one

mutant weed plant that is resistant to a herbicide in fields of dense mono-species weeds is

high Therefore, the formula for determining the probability of appearance of at least one

herbicide resistant plant in a population with various weed density and total weed numbers

was suggested by Jasieniuk et al (1996) as follows:

P = 1 – (1 – p)n

Where, P = probability of occurrence of at least one resistant plant, p = expected frequency of

resistant plants in the population, and n = total number of plants in a population

In general, in a dense weed population, the occurrence likelihood of a herbicide resistant plant

is higher than that in a sparse weed population (Preston and Powles, 2002)

2.3.2 Initial frequency of resistant alleles

The initial frequency of resistant alleles plays a significant role in how fast resistance evolves

When herbicides are applied to a weed population, resistance will occur quickly if the initial

gene frequency of resistant plants is high (Preston and Powles, 2002) Normally, the initial

frequency of resistant alleles in a population is very low For this reason, a large number of

seeds or plants need to be sampled and screened in order that estimates of the frequency of

resistance alleles can obtain high precision (Jasieniuk et al., 1996) The initial frequency of

mutant individuals resistant to a specific herbicide before it was applied to a weed population

has been estimated in several weed species For example, Matthews and Powles (1992)

estimated the frequency of diclofop-methyl resistant rigid ryegrass (Lolium ridigum) plants at

0.02 ± 0.0092 in formerly unsprayed farm populations and at <0.002 in non-farm populations

through southern Australia Before that, Darmency and Gasquez (1990) estimated the

frequencies of triazine resistant individuals within seven common lambsquarters

(Chenopodium album) populations at gardens in France at 1 x 10-4 to 3 x 10-3

2.3.3 Selection pressure

The application of herbicides is a key factor that promotes the rapid evolution of herbicide

resistant weed populations Herbicides eliminate susceptible weed individuals and increase

the proportion of resistant individuals Therefore, the repeated use of herbicides provides a

selective pressure to weed populations (Jasieniuk et al., 1996) However, the number of

herbicide applications for a resistant population to occur varies depending on herbicide group

applied For example, for weed individuals with one resistant gene, herbicide groups that

inhibit photosynthesis at photosystem II (e.g triazines) (Group C) and microtubule assembly

(e.g dinitroanilines) (Group K1) require more herbicide applications than herbicide groups

that inhibit acetyl-CoA carboxylase (Group A) and acetolactate synthase (Group B)

(McGillion and Storrie, 2006)

2.3.4 Inheritance of resistance

Herbicide resistance in weed species can be inherited on the nuclear or cytoplasmic genome

For the majority of modes of action of herbicide, resistance is transmitted to the progeny

through ovules and pollen However, for target-site resistance to the triazine herbicides, most

weed species transmit their resistance via the cytoplasm (Jasieniuk et al., 1996), because the

gene for the target-site resistance to triazine herbicides was found on the chloroplast genome

(Hirschberg et al., 1984) This is a significant distinction that needs to be noted As confirmed

by Powles and Preston (2006), the mechanisms of glyphosate resistance in weed species are

inherited as a single trait and occur on the nuclear genome In most weed species, when the

resistance allele occurs on the nuclear genome, herbicide resistance is controlled by an

allele(s) that is at least partially dominant An allele that is at least partially dominant will

spread far faster within a population compared to a recessive allele, because heterozygotes

will express at least part of the phenotype of the homozygote under selection pressure

(Darmency and Gasquez, 1990; Matthews and Powles, 1992) After herbicide application, a

rare dominant resistance allele will be established more easily in a weed population than a

recessive resistance allele (Merrell, 1981)

2.3.5 Gene migration

While mutation plays a role as an initial origin of herbicide resistant alleles, gene migration

together with the use of herbicides can enhance the spread and expansion of herbicide

resistant weed populations Resistance alleles can be dispersed over adjacent susceptible weed

populations from an original resistant population through seed or pollen If the level of gene

migration is higher than that of mutation, a rapid rise in the number of individuals resistant to

a certain herbicide can occur before the herbicide is used Consequently, gene migration may

shorten the time period of establishing a large herbicide resistant population after application

of the herbicide (Jasieniuk et al., 1996)

Recent studies have shown that gene migration between weed populations was severely

restricted; and the gene flow rates vary from 0.5 to 5.5 individuals per generation (Slatkin,

1985a, 1985b, 1987) Although there was little experimental data on gene migration among

herbicide resistant weed populations, the data on the spread of pollen containing resistance

alleles within populations were recorded by Maxwell (1992) and Stallings et al (1995) for

two weed species with two herbicides: namely resistance to sulfonylurea in kochia (Kochia

scoparia) and resistance to diclofop-methyl in Italian ryegrass (Lolium multiflorum) In

kochia, 1.4% of seeds set on susceptible individuals at a 28.9 m distance were resistant, while

in Italian ryegrass 1% of seeds set on susceptible individuals were resistant at 6.84 m

(Maxwell, 1992; Stallings et al., 1995) These data demonstrate that herbicide resistant genes

can be rapidly spread over short distances in some species

2.3.6 Fitness of resistant individuals

According to McGillion and Storrie (2006), within the same species, herbicide resistant weeds

are often less robust than susceptible weeds Therefore, in the absence of herbicides, resistant

weeds will produce fewer seeds than susceptible individuals Triazine resistant canola

(Brassica napus) can be considered as a typical example for this case because triazine

resistance mechanism occurs in chloroplast and the yield of triazine resistant B napus

varieties is lower than that of other varieties (Beversdorf et al., 1988)

2.3.7 Characteristics of the seed bank

Apart from the factors mentioned above, resistance evolution also depends partly on the

available seed bank In practice, weed species that have a greater number of dormant seeds

will slow down the progress of resistance At the same time, a large number of susceptible

seeds from seed bank will dilute resistance in the population (McGillion and Storrie, 2006)

For Echinochloa spp., photoperiod and several other natural factors affect the production of

dormant seeds and the duration of dormancy For example, the dormancy period of this seeds

is 4 to 8 months in Japan and 4 to 48 months in the United States (Holm et al., 1977)

2.4 Glyphosate

2.4.1 Properties of glyphosate

Glyphosate is common name given to the herbicide N - (phosphonomethyl) glycine Its most

well-known trade name is Roundup™; however, it has many other different trade names

Currently, there were a variety of formulations of glyphosate including trimethylsulfonium,

isopropylamine, potassium or ammonium salts Glyphosate is formulated as a salt because it

is a simple zwitterionic amino acid (Figure 2.1) Its molecular weight varies depending on

formulations For example, isopropylamine salt: 228.19 and trimethylsulfonium salt: 245.23

As a trimethylsulfonium salt, it is clear amber to yellow, mild sulfur odour and 1.23 - 1.25

g/mL in density at 20oC It is stable for 32 days at 25oC and pH 5, 7, or 9 with melting point at

200oC (Franz et al., 1997; Vencill, 2002)

Figure 2.1 Chemical structure of glyphosate (Franz et al., 1997)

The herbicidal function of glyphosate was discovered by Monsanto Agricultural Products

Company in 1970 Since this chemical came onto the market in 1974, it has become the most

widely used herbicide in the world Since glyphosate-resistant agricultural crops have been

introduced and its functionality has been extended, glyphosate has even become a more

important herbicide (Duke et al., 2003) The use of this herbicide has continued to grow in

almost all countries around the world as its price has been decreasing and its patent has

expired In terms of toxicology and environment, glyphosate is comparatively safe On

account of its properties, glyphosate has become a unique global herbicide with no other

herbicide having similar chemical structure or molecular target-site (Franz et al., 1997; Duke

et al., 2003)

As a non-selective herbicide with wide activity spectrum and high efficacy, glyphosate has

been considered as an ideal herbicide (Duke and Powles, 2008) Although it kills weeds

slowly after application, it is translocated to metabolic sites quickly This makes it highly

effective on intractable weeds, particularly monocotyledon grass species such as blady grass

(Imperata cylindrical), quack grass (Agropyron repens), Johnson grass (Sorghum halepense)

and Cyperus species (Duke et al., 2003) Owing to its anionic nature, glyphosate is tightly

bound by soil components and subsequently inactivated by soil microorganisms once it is in

contact with soil (Duke et al., 2003) However, the fertilisation of soil with phosphate

together with the presence of mycorrhiza resulted in the remobilisation of glyphosate residues

into plants and caused the plant’s mortality (Beltrano et al., 2013) Normally, glyphosate is

only applied to weed foliage, and due to high stability, it can be mixed with other additives,

adjuvants and pesticides (Duke et al., 2003)

2.4.2 Mode of action

Glyphosate targets and binds to the enzyme EPSP (5-enolpyruvylshikimate-3-phosphate)

synthase, that catalyses the condensation of shikimate-3-phospate and phosphoenolpyruvate

to form 5-enolpyruvylshikimate-3-phosphate (Figure 2.2) (Amrhein et al., 1980) EPSP

synthase is an important enzyme in the shikimate pathway, facilitating the biosynthesis of

aromatic compounds in plants (Marques et al., 2007) Therefore, when glyphosate is applied

to plants, the shikimate pathway is blocked resulting in inhibition in carbon flow through the

shikimate pathway to aromatic amino acids, flavonoids, anthocyanins and lignins (Harring et

al., 1998) Additionally, glyphosate also inhibits the photosynthesis of several species, such as sugar beet (Beta vulgaris) owing to a decline in stomatal conductance as a secondary effect

(Servaites et al., 1987) Because of its slow activity, glyphosate symptoms in the majority of

weeds usually occur some days after application and consist of epinasty, loss of terminal

domination and anthocyanins, growth delay, wilting and chlorosis (Harring et al., 1998)

Shikimate-3-phosphate Phosphoenolpyruvate 5-enolpyruvylshikimate- Phosphate 3-phosphate

Figure 2.2 Activity of glyphosate on reaction catalysed by enzyme

5-enolpyruvylshikimate-3-phosphate synthase (Amrhein et al., 1980)

2.4.3 Glyphosate resistant weeds

Similar to other herbicides, the continuous application of glyphosate on a large scale in many

countries around the world has led to a noticeable increase in the number of glyphosate

resistant weed species (Powles and Preston, 2006) There are currently 29 weed species

worldwide that have been reported as glyphosate resistant (Heap, 2014), such as L ridigum in

Australia (Powles et al., 1998; Pratley et al., 1999; Lorraine-Colwill et al., 2003) and

California (Simarmata et al., 2003), Italian ryegrass (Lolium multiflorum) in Chile (Perez and

Kogan, 2003), goosegrass (Eleusine indica) in Malaysia (Lee and Ngim, 2000; Ng et al.,

2004), horseweed (Conyza canadensis) in Delaware and Mississippi (VanGessel, 2001; Koger

et al., 2004) and palmer amaranth (Amaranthus palmeri) in Georgia (Culpepper et al., 2006)

E colona was reported as a glyphosate resistant grass species in several countries of the world

including Australia (NSW, QLD and WA), America (California) and Argentina (Santa Fe)

(Heap, 2014) The amount of glyphosate resistant populations in this grass species in

Australia was assessed as part of this study project at the University of Adelaide

2.4.4 Mechanisms of glyphosate resistance

According to Devine and Shukla (2000), mechanisms leading to the phenomenon of herbicide

resistance in weeds include: lowered sensitivity to herbicides of target sites owing to

mutation, increase in the number of target sites, increased activity of detoxifying herbicides,

reduced activity of activating herbicides, decreased herbicide translocation or herbicide

sequestration away from target sites

For glyphosate, there are four main mechanisms of resistance in plants that have been

determined to-date (1) Target-site resistance: A mutation within the target-site prevents the

herbicide from binding as effectively; therefore, the plants will be less affected by the

herbicide As specified by Powles and Preston (2006), a mutation that occurs by changes in

amino acid 106 of EPSPS gene from proline to either serine or threonine results in glyphosate

resistance in weeds; and Funke et al (2009) found that a double mutation (threonine97 to isoleucine and proline101 to serine) in the EPSPS gene caused a glyphosate resistance in Escherichia coli (2) Non-target-site resistance is comprised of a decrease in translocation of

the herbicide (Powles and Preston, 2006) (3) Reduced foliar uptake from the abaxial leaf

surface leads to the lower volume of herbicide reaching the target-sites (Michitte et al., 2007)

(4) Amplification of target-site gene: the genome of glyphosate resistant plants contained

many more copies of the EPSPS gene than that of susceptible plants (Gaines et al., 2010) To

date, five weed species have been found resistance to glyphosate by amplification of the

EPSPS gene including A palmeri, tall waterhemp (Amaranthus tuberculatus), L multiflorum,

K scoparia and spiny amaranth (Amaranthus spinosus) (Sammons and Gaines, 2014) and the first case of this resistance mechanism was discovered in A palmeri in Georgia, USA (Gaines

et al., 2010)

In a study on glyphosate resistance mechanism in L ridigum, Lorraine-Colwill et al (2003)

concluded that differential glyphosate translocation within the plant caused a significant

difference between resistant and susceptible populations According to these authors, after

application, glyphosate was accumulated in the roots of susceptible individuals and in the leaf

tips of resistant ones At the same time, as suggested by these authors, it was likely that a

cellular glyphosate pump was present and pumps glyphosate out of plant cells Moreover,

pumping glyphosate in resistant plants was more active than that in susceptible ones As a

result, glyphosate within resistant plants was transferred to the leaf tips through transpiration

(Lorraine-Colwill et al., 2003) The absorption of glyphosate into plant cells also depends on

a phosphate transporter of the plasma membrane When this phosphate transporter is inhibited

due to mutation, the uptake of glyphosate would be considerably reduced (Denis and Delrot,

1993; Morin et al., 1997; Hetherington et al., 1998; Versaw and Harrison, 2002)

Additionally, several other resistance mechanisms have been proposed such as arresting

glyphosate (sequestration) in laticifers (Foley, 1987) or in vacuole (Ge et al., 2010), cellular

uptake and discharge (Hetherington et al., 1998), plant metabolism of glyphosate (Komoβa et al., 1992), accelerated EPSPS transcription and prolonged half-life (Holländer-Czytko et al.,

1992)

2.5 Molecular markers

2.5.1 Scientific basis of the use of molecular makers in determining the spread of resistance evolution in Echinochloa spp

As mentioned above, once a genetic mutation causing herbicide resistance in a weed

population occurs, resistant genes will be transmitted to the progeny and can be dispersed to

susceptible populations through seeds, pollen or vegetative portions (Levin, 1981;

Christoffers, 1999) Thanks to molecular makers, the level and range of resistance spread

could be determined through studying genetic structure and diversity of populations Initially,

resistant populations are probably very small, but they quickly increase in size with continued

herbicide application Modern genetic techniques have been becoming a useful means of

identifying precise genetic modifications in individual weeds and the spread of these

mutations within and between populations With these techniques, understanding of genetic

diversity, herbicide resistance evolution and spread of this resistance in weed species is

becoming clearer

2.5.2 DNA Fingerprinting techniques

Molecular markers are indispensable in DNA fingerprinting techniques At present, several

different markers are popularly used in genetics including RFLPs (Restriction fragment length

polymorphism), RAPD (Randomly amplified polymorphic DNAs), SSRs (simple sequence

repeats) and AFLPs (amplified fragment length polymorphism) Among these markers, with

the exception of RFLPs, the remaining markers are the most commonly used due to their

quickness, simplicity and usefulness (McGregor et al., 2000)

For RFLP, variations in length of restriction fragments cut from genomic DNA by a

restriction endonuclease are identified between individuals (Williams, 1989) RFLP markers

were first used in the 1980s They are the first generation of DNA markers and considered as

one of the best for plant genome mapping (Acquaah, 2007) RFLP is used as a helpful tool in

determining the genetic variation of plant species An advantage of RFLPs is that it is not

necessary to know the sequence used as a probe However, this marker system is fairly

expensive and it has low throughput (Acquaah, 2007)

RAPD is a PCR (polymerase chain reaction) based marker system that was introduced in

1990 (Williams et al., 1990) In this technique, only a short (around 10 bases), single and

random primer is used to amplify the genomic DNA (Acquaah, 2007) RAPD markers are

appropriate for DNA fingerprinting, gene mapping, and applications of animal and plant

breeding, especially for population genetics (Williams et al., 1990) So far, RAPD markers

have been used in assessing the genetic diversity of Echinochloa millets (Hilu, 1994) as well

as in examining herbicide resistance evolution in E crus-galli (Rutledge et al., 2000)

Currently, RAPDs are considered as an effective assay for polymorphisms, because they

produce high levels of polymorphism that can be rapidly and simply identified The

disadvantage of this marker system is that the information content obtained from an

individual RAPD marker is negligible (Williams et al., 1990)

SSRs (or microsatellites) are also PCR-based markers They are random tandem repeats of 2 -

5 nucleotides such as GT or GACA which appear in microsatellites The copy number of

repeats is a polymorphism source in plants and it is different among individuals Although

this technique is more dependable than the RAPD technique, it is more expensive to

implement Furthermore, to use SSRs technology, it is required to know the sequence of

nucleotides in order to design primers for PCR This technique also requires complicated

electrophoresis systems and computer software for exact separation and count of DNA bands

(Acquaah, 2007)

AFLP was introduced by Vos et al (1995) The procedure for AFLP involves three steps: (1)

restriction of the DNA and ligation of oligonucleotide adapters to the cut ends, (2) selective

PCR amplification of DNA restriction fragments and (3) electrophoretic product analysis of