NON-TARGET-SITE RESISTANCE TO ALS INHIBITORS IN …

NON-TARGET-SITE RESISTANCE TO ALS INHIBITORS IN WATERHEMP

BY

JIAQI GUO

THESIS

Submitted in partial fulfillment of the requirements

for the degree of Master of Science in Crop Sciences

in the Graduate College of the

University of Illinois at Urbana-Champaign, 2014

Urbana, Illinois

Master’s Committee:

Professor Patrick J. Tranel, Chair

Professor Dean E. Riechers

Associate Professor Aaron G. Hager

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ABSTRACT

The acetolactate synthase (ALS) enzyme, or acetohydroxyacid synthase (AHAS) enzyme,

is an essential enzyme in branched-chain amino acid biosynthesis, and is the target site of five

families of herbicides referred to as ALS inhibitors. Waterhemp (Amaranthus tuberculatus) is

considered one of the most problematic weeds in the Midwest cropping region. The evolution of

herbicide resistance and multiple resistance mechanisms within the species is one of the major

properties making it difficult to control, and ALS-resistant waterhemp populations have been

found and studied considerably. A waterhemp population (designated MCR) from Illinois with

resistance to HPPD and atrazine was found to segregate for both high and moderate levels of

resistance to ALS inhibitors. Plants in this population with high-level resistance had the

Trp574Leu ALS mutation, which is present in other waterhemp populations resistant to ALS

inhibitors. Plants from the MCR population that showed only moderate levels of resistance to

ALS inhibitors did not have this mutation. Thus, research was conducted to investigate the

resistance mechanism in the waterhemp plants with moderate resistance to ALS-inhibitors.

Plants with moderate resistance were crossed and the resulting progeny where characterized.

Firstly the ALS gene of the progeny was sequenced and in vitro ALS enzyme assays were

conducted, and results indicated that the plants lacked a target-site mutation. Secondly, a series

of greenhouse dose-response experiments were conducted to evaluate the resistance level across

different chemical families of ALS-inhibitors. Thirdly, malathion, a cytochrome P450-inhibiting

pesticide, was incorporated with ALS-inhibitor application to unveil the possible mechanism of

resistance. Based on the results obtained, it was concluded that both target-site-mutation-based

and metabolism-based ALS resistance mediated by cytochrome P450s exist in the original MCR

population.

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ACKNOWLEDGEMENTS

I would like to thank Dr. Patrick Tranel who gave me the opportunity to join the weed

science group at the University of Illinois and work on my Masters’ degree, as well as provided

guidance and supported me throughout the process. I am grateful to have Dr. Dean Riechers and

Dr. Aaron Hager as my committee members, whose valuable suggestions and enlightenment

helped my research tremendously.

Also, the help and assistance I received from many people cannot be neglected, including

my lab mates, both past and present, Chenxi Wu, Dr. Chance Riggins, Laura Chatham, Janel

Huffman, Ahmed Sadequ, Jung Eun Song, Stephanie Rousonelos and Edhilvia Josefina, who not

only created a great atmosphere in the lab but became great friends with me as well. Dr. Adam

Davis, Dr. Danman Zheng, Rong Ma, Nicholas Hausman and many others helped me with lab

experiments, greenhouse research and data analysis, which I am obliged greatly. Without their

help, I would not have gotten to this point.

I also want to express my appreciation to all my friends on campus, former and present,

most of whom are just like me and traveled across the Pacific Ocean for study. I am fortunate to

know so many caring and wonderful people at places far from home, and their combined support

is one of the greatest things I received during the past five years and something that I will always

treasure.

And last but not least, my deepest gratitude goes to my mother, Xiaodong Guo and my

father, Tianwen Guo, for their dedication and unconditional love, and letting me be carefree and

sometimes even indulgent in the pursuit of my own goal. Their support is the ultimate source of

my confidence and courage. Their solicitude and understanding for my absence drove me to

work hard on my research and take good care of myself. I will always keep them in my priority.

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TABLE OF CONTENTS

CHAPTER 1 ................................................................................................................................................. 1

Introduction ................................................................................................................................................... 1

1.1 History of ALS Inhibitors ................................................................................................................... 1

1.2 Site of Action of ALS Inhibitors ......................................................................................................... 2

1.3 Mode of Action of ALS inhibitors ...................................................................................................... 3

1.4 Resistance to ALS Inhibitors .............................................................................................................. 5

1.5 Waterhemp Biology ............................................................................................................................ 7

1.6 ALS-Inhibitor-Resistant Populations of Waterhemp .......................................................................... 8

1.7 Research Objective ........................................................................................................................... 10

1.8 Attributions ....................................................................................................................................... 10

1.9 Literature Cited ................................................................................................................................. 11

1.10 Figures............................................................................................................................................. 19

CHAPTER 2 ............................................................................................................................................... 21

Confirmation of Non-target-site Resistance to ALS Inhibitors in a Waterhemp Population ...................... 21

2.1 Abstract ............................................................................................................................................. 21

2.2 Introduction ....................................................................................................................................... 22

2.3 Materials and Methods ...................................................................................................................... 23

2.3.1 Full-length ALS Gene Sequencing ............................................................................................ 23

2.3.2 ALS Enzyme In Vitro Assay ...................................................................................................... 26

2.4 Results and Discussion ..................................................................................................................... 29

2.4.1 Full-length ALS Gene Sequencing ............................................................................................ 29

2.4.2 ALS Enzyme In Vitro Assay ...................................................................................................... 30


2.6 Tables and Figures ............................................................................................................................ 33

CHAPTER 3 ............................................................................................................................................... 42

Quantifying Herbicide Resistance in a Waterhemp Population with Non-Target-Site Resistance to

ALS-Inhibiting Herbicides .......................................................................................................................... 42

3.1 Abstract ............................................................................................................................................. 42

3.2 Introduction ....................................................................................................................................... 43

3.3 Materials and Methods ...................................................................................................................... 44

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3.3.1 Greenhouse Dose Response with ALS-inhibiting Herbicide ..................................................... 44

3.3.2 Response of ALS Inhibitors Combined with Malathion ............................................................ 46

3.4 Results and Discussion ..................................................................................................................... 47

3.4.1 Greenhouse Dose Response with ALS-inhibiting Herbicide ..................................................... 47

3.4.2 Response to ALS Inhibitors Combined with Malathion ............................................................ 48


3.6 Tables and Figures ............................................................................................................................ 51

CHAPTER 4 ............................................................................................................................................... 65

Concluding Remarks ................................................................................................................................... 65

4.1 Research Conclusions and Implications ........................................................................................... 65


4.3 Tables ................................................................................................................................................ 71

1

CHAPTER 1

Introduction

1.1 History of ALS Inhibitors

The acetolactate synthase (ALS) enzyme, also called acetohydroxyacid synthase (AHAS),

is the target site of five families of herbicides, which are all classified as ALS-inhibiting

herbicides, namely sulfonylureas (SUs) (Ray, 1984), imidazolinones (IMIs) (Shaner et al., 1984),

triazolopyrimidines (TPs) (Gerwick et al., 1990), pyrimidinylthiobenzoates (PTBs) (Takahashi et

al., 1991), and sulfonylamino-carbonyl-triazolinones (SCTs) (Babczinski et al., 1992; Chaleff

and Mauvais, 1984; Corbett and Tardif, 2006; Mallory-Smith and Retzinger, Jr., 2003;). The

various chemicals perform similarly in plants by interfering with amino acid biosynthesis,

specifically, by inhibiting the biosynthesis of branched-chain amino acids (BCAAs) (Ray, 1984).

The first commercialized ALS-inhibiting herbicide was chlorsulfuron, a sulfonylurea, in 1982.

At that time triazines were widely used for weed control, and a variety of weed species had

evolved resistance to them. When compared to triazines and other herbicides at that time, which

were used at rates of kilograms per hectare, the dosage that ALS-inhibiting herbicides used was

significantly less (grams per hectare), which reduced the amount of chemicals in agricultural

greatly. And the specific properties of ALS inhibitors, including low mammalian toxicity,

systematic activity, suitable soil residual activity, and a broad spectrum of weed control,

prompted their wide adoption soon after commercialization (Tranel and Wright, 2002). Currently,

more than 50 ALS-inhibiting herbicide active ingredients are commercialized, including 6 IMIs,

5 PTBs, 3 SCT, 33 SUs, and 3 TPs (Heap, 2013a), with modified functional groups from earlier

inventions for the purpose of altering chemical property, formulation, or weed control spectrum.

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1.2 Site of Action of ALS Inhibitors

ALS is a crucial enzyme in the biosynthesis pathways of three essential branched-chain

amino acids, valine, leucine and isoleucine, in plants and microorganisms. The ALS enzyme

consists of two subunits. The subunit involved with the catalyzing process has a mass of about

60 to 70 kDa, and the subunit for regulation has mass from 9.5 to 54 kDa (Chipman et al., 2005).

The biosynthesis of the branched-chain amino acids consists of two parallel pathways and ALS

catalyzes the first step of the parallel reactions through condensation of two molecules of

pyruvate to yield 2-acetolactate, or condensation of one molecule of pyruvate and one molecule

of 2-ketobutyrate to yield 2-aceto-hydroxybutyrate (Singh, 1999). During the condensation

reactions, thiamine pyrophosphate (TPP), flavin adenine dinucleotide (FAD) and divalent cation

(Mg2+

or Mn2+

) are required as cofactors. ALS firstly binds TTP and the ALS-TTP complex

reacts with one molecule of pyruvate, to decarboxylate the pyruvate and form a hydroxyethyl-

TPP intermediate. The hydroxyethyl-TPP intermediate then reacts with another molecule of

pyruvate or a molecule of 2-ketobutyrate by nucleophilic attack to yield 2-acetolactate or 2-

aceto-hydroxybutyrate (Singh, 1999). FAD is also required in this reaction and is involved in the

structural stabilization of the protein with the flavin group (Schloss et al., 1988). The

condensation products will continue reacting with other enzymes as precursors of BCAAs in the

pathways.

In plants, ALS is encoded in the nucleus, and targeted to chloroplasts by a transit peptide

(Singh, 1999). ALS gene copy numbers vary among different plant species. For instance,

Arabidopsis thaliana has only one gene (Mazur et al., 1987), while six different ALS genes were

identified in Gossypium hirsutum (Grula et al., 1995). Most of the ALS sequences known have

no introns, and sequence similarity among species is rather high with evidence of divergence

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between monocots and dicots (Singh, 1999; Uchino and Watanabe, 2002). Despite that amino

acid sequence is quite conserved in ALS enzyme within one species (2 inferred amino acid

polymorphisms in 24 common ragweed (Ambrosia artemisiifolia L.) from IL, MN and OH),

rather high intraspecific variability in nucleotide sequence of the ALS gene has been documented

within some weedy species, particularly among outcrossed species (e.g. common ragweed has

more polymorphic nucleotides than common cocklebur (Xanthium strumarium L.)) (Tranel et al.,

2004).

1.3 Mode of Action of ALS inhibitors

Inhibition of the ALS enzyme impedes the synthesis of the BCAAs, and causes plant

death by depletion of leucine, valine, and isoleucine (Tan et al., 2006). This is the key principle

of all the ALS-inhibiting herbicides. Primarily, the inhibiting herbicide irreversibly binds to the

ALS enzyme in susceptible plants, alters the geometry and structure of its active site and

obstructs its catalytic ability, without affecting other enzymes in the BCAA pathway (Duggleby

and Pang, 2000; Muhitch et al., 1987). After ALS inhibition, mitosis greatly decreases and

eventually stops (Rost and Reynolds, 1985). The biosynthetic rate of protein decreases

correspondingly (Ray, 1984). None of the ALS-inhibitors are regarded as substrate analogs or

cofactors in the ALS enzyme. In other words, ALS herbicides are considered to be non-

competitive inhibitors that do not mimic the substrate of the original biosynthesis process

(Duggleby and Pang, 2000). Because the biosynthesis of BCAAs is mainly in shoot meristems

(where the most active cell growth and division occurs) the phenotypic effect of ALS-inhibiting

herbicides manifests mostly in young tissue, showing as necrosis and malformation of the

meristem (García-Garijo et al., 2012). Also, the ALS inhibitors are mostly in weak acid form

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after uptake by plants, so they can translocate through phloem and accumulate at the meristem

(Bromilow et al., 1990). Plants die gradually as protein biosynthesis and cell division slow down,

and the free amino acid pool depletes (Shaner and Singh, 1993)

Besides the mechanisms of inhibiting BCAA synthesis, other possible effects of ALS-

inhibiting herbicides contributing to plant death were proposed. However, these effects have

been neither proven to have significant inhibition of plant growth nor concluded to cause plant

death. Research has shown that the accumulation of 2-ketobutyrate and 2-aminobutyrate in the

BCAA synthesis pathway were not the cause of phytotoxicity induced by ALS inhibitors (Shaner

and Singh, 1993). Although the accumulation of phytotoxic compounds is a consequence

induced by chlorsulfuron (Rhodes et al., 1987) and 2-ketobutyrate accumulation induced by

chlorsulfuron is followed by the cessation of plant growth (LaRossa et al., 1987), when reducing

2-ketobutyrate and 2-aminobutyrate accumulation by isoleucine supplementation, the plants

could not recover following imazaquin treatment. In addition, when directly increasing 2-

ketobutyrate and 2-aminobutyrate pools by feeding the plant 2-aminobutyrate, there was no

significant effect on plant growth observed, indicating the accumulation of these intermidiates is

not the cause of plant injury (Shaner and Singh, 1993). The reduction of transportation of

carbohydrates and amino acids is suggested to be a side effect of ALS herbicide inhibition,

which co-occurs with limited utilization of carbohydrates and amino acids in plants treated with

imidazolinone herbicides (Bestman et al., 1990; Gaston et al., 2003). Other possible inhibiting

mechanisms, including induction of high rates of alternative oxidase formation, were proposed as

consequences of sulfonylurea and imidazolinone herbicides, however the actual effects regarding

plant death were unclear (Aubert et al., 1997).

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1.4 Resistance to ALS Inhibitors

The ALS-inhibiting herbicides cause high selection pressure on weeds; as a result, a wide

variety of weeds evolved resistance to ALS inhibitors. Currently, 132 weed species have been

confirmed resistant to ALS inhibitors throughout the world (Heap, 2013c). The increasing

frequency of resistance limits the further development and use of a large group of herbicides

targeting the same enzyme. The vast majority of resistance cases reported were caused by single

point mutations in the ALS gene. The mutations alter the structure of the ALS enzyme, causing it

to be less sensitive to herbicide binding. Since resistance alleles are dominant (although the level

of dominance varies among species) over susceptible alleles (Sebastian et al., 1989; Wright and

Penner, 1998), herbicide selection also acts on heterozygous individuals. Dominance helps

facilitate spreading of the resistance trait in weed species that propagate by outcrossing. The

mutations in the ALS gene occur commonly and spontaneously in nature, and are utilized in

breeding of ALS-tolerant crops (Tan et al., 2005).

In nature, there are 8 amino acids in ALS known to have substitutions that confer

herbicide resistance in weed populations. They are Ala122, Pro197, Ala205, Asp376, Arg377,

Trp574, Ser653 and Gly654 (Tranel et al., 2013). Following intentional laboratory selection, 17

amino acids were identified to confer resistance to ALS inhibition when substituted (Duggleby

and Pang, 2000; Tranel and Wright, 2002). The mutations at each site are not necessarily

restricted to only one amino acid substitution (Guttieri et al., 1995). For example, nine different

amino acids can substitute for Pro197 in different weed populations that are resistant to ALS

inhibitors (Tranel et al., 2013). The various ALS mutations can confer different patterns of cross

resistance among the ALS inhibitor families. For example, whereas substitution of Trp574

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confers high resistance across all ALS inhibitors, substitution of Pro197 generally confers high

resistance to SUs but little or no resistance to IMIs (Tranel and Wright, 2002).

Another method whereby plants overcome ALS inhibition is enhanced metabolism that

detoxifies the inhibitors rapidly after uptake. Enhanced detoxification is usually used as the basis

of crop tolerance to ALS inhibitors (Powles and Holtum, 1996). The resistance efficacy of

detoxification usually is less than 10-foldand whereas target-site mutation may confer 100-fold

or higher resistance (Powles and Holtum, 1996). However, a less resistant phenomenon does not

suggest a less severe problem. The enhanced metabolism is not necessarily selected by previous

usage of ALS-inhibiting herbicides, but can arise as cross resistance resulting from selection by

herbicides with other modes of action. For instance, acetyl-CoA carboxylase (ACCase)-

inhibitors are herbicides to which weeds have also evolved both target-site and metabolism-

based resistance. Research showed that after four years of treatment with diclofop-methyl (an

ACCase-inhibitor) in the wheat-growing region of Australia, a biotype of rigid ryegrass (Lolium

rigidum Gaudin) evolved resistance to ACCase-inhibiting herbicides, and cross resistance to

some ALS-inhibiting herbicides (Cotterman and Saari, 1992). In that case, the rigid ryegrass was

resistant to ALS inhibitors even when they have never been exposed to such herbicides. Similar

circumstances were found in a population of large crabgrass (Digitaria sanuinalis L.) that has

cross-resistance to ACCase inhibitors and ALS inhibitors (Hidayat and Preston, 2001). This

causes difficulties in weed control since utilizing alternate side-of-action herbicides is a primary

strategy for herbicide-resistance management (Holtum et al., 1991). Blackgrass (Alopecurus

myosuroides Huds), which is one of the most problematic weeds in Western Europe, has been

reported to have biotypes resistant to ALS inhibitors by enhanced detoxification (Kemp et al.,

1990).

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Besides the examples of rigid ryegrass, large crabgrass and blackgrass, other weeds also

have been reported to have metabolism-based resistance to ALS-inhibitors. A late watergrass

(Echinochloa phyllopogon (Stapf) Koss) biotype in California was reported to have both

insensitive ALS and enhanced detoxification by cytochrome P450 (Fischer et al., 2000). The first

broadleaf weed reported to have metabolism-based resistance to the ALS inhibitor

ethametsulfuron-methyl was wild mustard (Sinapis arvensis L.) (Veldhuis et al., 2000).

1.5 Waterhemp Biology

Waterhemp [Amaranthus tuberculatus (Moq.) Sauer] is a weed species in the

Amaranthaceae family (Sauer, 1955). It is a small-seeded, summer annual broadleaf weed native

to the Midwest. It is a dioecious species, which means it has both male and female plants that

outcross when reproducing (Steckel, 2007). Waterhemp can grow as tall as 2 meters, and it is a

C4 plant with a relatively high photosynthetic rate, which makes it a very competitive weed for

light, water, and nutrients (Horak and Loughin, 2000; Steckel et al., 2003). In addition, the

ability of a single female waterhemp to produce up to 1 million seeds and its prolonged

germination period make it even more problematic (Steckel and Sprague, 2004a). It has severely

impacted crop production by reducing yield when poorly controlled (Steckel and Sprague,

2004b). The challenge of managing waterhemp has become more difficult as the species

continues to evolve resistance to many commonly used herbicides. Currently, 39 cases of

herbicide-resistant waterhemp have been reported, with all but two found in the Midwest United

States (Heap, 2013b). Herbicides to which waterhemp has evolve resistance include ALS

inhibitors, photosystem II (PSII) inhibitors, protoporphyrinogen oxidase (PPO) inhibitors, p-

hydroxyphenylpyruvate dioxygenase (HPPD) inhibitors, synthetic auxins and glyphosate.

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Individual waterhemp plants can stack multiple resistant mechanisms together (Bell et al., 2013).

The species also has been reported to outcross with other Amaranthus species, posing even

further challenges to weed management (Wetzel et al., 1999).

1.6 ALS-Inhibitor-Resistant Populations of Waterhemp

Waterhemp evolved resistance to ALS inhibitors soon after their introduction. The first

report waterhemp demonstrated resistance to SU and IMI herbicides (Horak and Peterson, 1995;

Hinz and Owen, 1997; Lovell et al., 1996; Sprague et al., 1997). A combination of ALS inhibitor

and photosystem II (PSII) inhibitor, such as atrazine, could provide adequate control of the ALS-

resistant biotypes (Sprague et al., 1997). However, a waterhemp biotype with resistance to both

ALS inhibitors and atrazine was identified soon thereafter (Foes et al., 1998). A population of

waterhemp from Adams County, Illinois, designated ACR, was characterized as containing

resistance to three different herbicide modes of action, including PPO inhibitors, PSII inhibitors

and ALS inhibitors (Patzoldt et al., 2005). In that population, individual plants displayed

resistance to multiple herbicides. Resistance to ALS inhibitors in ACR was 17,000- to 18,000-

fold compared with a susceptible population, and it showed a cross-resistance to both SU and

IMI herbicides. The resistance mechanism of this population was reported to be a Trp574Leu

substitution in ALS (Patzoldt and Tranel, 2007). The specific mutation can be detected by PCR-

RFLP (restriction fragment length polymorphism) using restriction endonucleases to digest PCR

products of the ALS gene. The sequence with mutation can be recognized and cut, and resulting

in a fragment that is shorter than that observed when the mutation is not present (Corbett and

Tardif, 2006).

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Waterhemp biotypes with resistance to ALS inhibitors have also been identified with

amino acid substitution (either Asn or Thr) of Ser653 (Patzoldt and Tranel, 2007). In these cases,

the biotypes were resistant particularly to IMI but not SU herbicides. Besides mutations caused

by substitution of Trp574 or Ser653, no other ALS mutations conferring resistance have been

reported in waterhemp. Furthermore, there is not yet any report of waterhemp (or any other

Amaranthus species) being resistant to ALS-inhibitors via rapid herbicide detoxification.

A waterhemp biotype from McLean County, IL, designated MCR, which was

characterized to be resistant to HPPD inhibitors and atrazine (Hausman et al., 2011), also showed

resistance to both SU and IMI herbicides. The field where the population was discovered was not

exposed to ALS inhibiting herbicides for the previous seven years but, nevertheless, plants of the

population were resistant to these herbicides. It was also shown that the resistance level varied

within the population, with some plants being largely unaffected by ALS-inhibitors, and others

being significantly injured but able to recover from normally lethal application rates (Figure 1.1).

By detecting gene mutation with the PCR-RFLP method, it was shown that the highly resistant

plants contained the Trp574Leu ALS mutation, whereas the moderately resistant plants did not

have this mutation (N. Hausman and C. Riggins, personal communication, demonstrated in

Figure 1.2). These observations raised the hypothesis that the MCR waterhemp population

possessed a second and non-target-site resistance mechanism for ALS-inhibition. Since data

indicated that the MCR population was resistant to HPPD inhibitors and atrazine by enhanced

detoxification by cytochrome P450s and glutathione-S-transferase activities, respectively (Ma, et

al., 2013), it was suspected that enhanced herbicide detoxification could also play a role in

resistance to ALS inhibitors.

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1.7 Research Objective

The objective of this research was to investigate the second resistance mechanism for

ALS-inhibitors present in the MCR waterhemp population. Seeds from waterhemp plants

suspected to demonstrate non-target-site ALS resistance were obtained from a cross in the

greenhouse with two plants from the MCR population. Both plants used for the cross survived a

normally lethal rate of an ALS inhibitor, but were found to lack the Trp574Leu ALS mutation by

PCR-RFLP.

To achieve the objective of this research, full length ALS gene sequencing was

performed to determine if other target-site mutations might be present. An in vitro ALS enzyme

assay was performed to compare the ALS enzyme activities in the presence and absence of an

ALS inhibitor. ALS inhibitors were applied to the population under greenhouse condition to

determine cross resistance among five families of ALS-inhibitors. In addition, malathion, an

inhibitor of cytochrome P450s, was applied together with ALS inhibitors to determine if it could

overcome the resistance mechanism.

1.8 Attributions

The original crossing and genetic marker analysis were performed by Nicholas Hausman

and Chance Riggins. Both laboratory experiments and greenhouse trials described in Chapter 2

and Chapter 3 were conducted by me under the instruction of my graduate committee. The

materials presented in the following chapters will be submitted for publication in Weed Science

in collaboration with Chance W. Riggins, Nicholas E. Hausman, Aaron G. Hager, Dean E.

Riechers and Patrick J. Tranel.

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1.9 Literature Cited

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oxidase synthesis by herbicides inhibiting branched-chain amino acid synthesis. The

Plant Journal, 11: 649-657.

Babczinski, P., Müller, K.-H., Santel, H.-J., and Schmidt, R. R. (1992). Patent No. EP 0422469

A3. Germany.

Bell, M. S., Hager, A. G., and Tranel, P. J. (2013). Multiple resistance to herbicides from four

site-of-action groups in waterhemp (Amaranthus tuberculatus). Weed Science, 61: 460-

468.

Bestman, H. D., Devine, M. D., and Vander Born, W. H. (1990). Herbicide chlorsulfuron

decreases assimilate transport out of treated leaves of field pennycress (Thlaspi arvense

L.) seedlings. Plant Physiology, 93: 1441-1448.

Bromilow, R. H., Chamberlain, K., and Evans, A. A. (1990). Physicochemical aspects of phloem

translocation of herbicides. Weed Science, 38: 305-314.

Chaleff, R. S., and Mauvais, C. J. (1984). Acetolactate synthase is the site of action of two

sulfonylurea herbicides in higher plants. Science, 224: 1443-1445.

Chipman, D. M., Duggleby, R. G., and Tittmann, K. (2005). Mechanisms of acetohydroxyacid

synthases. Current Opinion in Chemical Biology, 9: 475-481.

Corbett, C.-A. L., and Tardif, F. J. (2006). Detection of resitance to acetolactate synthase

inhibitors in weeds with enphasis on DNA-based techniques: a review. Pest Management

Science, 62: 584-597.

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Cotterman, J. C., and Saari, L. L. (1992). Rapid metabolic inactivation is the basis for cross-

resistance to chlorsulfuron in diclofop-methyl-resistance rigid ryegrass (Lolium rigidum)

biotype SR4/84. Pesticide Biochemistry and Physiology, 43: 182-192.

Diebold, R. S., McNaughton, K. E., Lee, E. A., and Tardif, F. J. (2003). Multiple resistance to

imaethapyr and atrazine in powell amaranth (Amaranthus powellii). Weed Science, 51:

312-318.

Doyle, J. J., and Doyle, J. L. (1990). Isolation of plant DNA from fresh tissue. Focus, 12: 13-15.

Duggleby, R. G., and Pang, S. S. (2000). Acetohydroxyacid Synthase. Journal of Biochemistry

and Molecular Biology, 33: 1-36.

Fischer, A. J., Bayer, D. E., Carriere, M. D., Ateh, C. M., and Yim, K.-O. (2000). Mechanisms of

resistance to bispyribac-sodium in an Echinochloa phyllopogon accession. Pesticide

Biochemistry and Physiology, 68: 156-165.

Foes, M. J., Tranel, P. J., Wax, L. M., and Stoller, E. W. (1998). A biotype of common

waterhemp (Amaranthus rudis) resistant to triazine and ALS herbicides. Weed Science,

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García-Garijo, A., Palma, F., Iribarne, C., Lluch, C., and Tejera, N. A. (2012). Alterations

induced by imazamox on acetohydroxyacid synthase activity of common bean

(Phaseolus vulgaris) depend on leaf position. Pesticide Biochemistry and Physiology,

104: 72-76.

Gaston, S., Ribas-Carbo, M., Busquets, S., Berry, J. A., Zabalza, A., and Royuela, M. (2003).

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Gerwick, B. C., Subramanian, M. V., Loney-Gallant, V. I., and Chandler, D. P. (1990).

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Grula, J. W., Hudspeth, R. L., Hobbs, S. L., and Anderson, D. M. (1995). Organization,

inheritance and expression of acetohydroxyacid synthase genes in the cotton

allotetraploid Gossypium hirsutum. Plant Molecular Biology, 28: 837-846.

Guttieri, M. J., Vberlein, C. V., and Thill, D. C. (1995). Diverse mutations in the actolactate

synthase gene confer chlorsulfuron resistance in Kochia (Kochia scoparia) biotypes.

Weed Science, 43: 175-178.

Hausman, N. E., Singh, S., Tranel, P. J., Riechers, D. E., Kaundun, S. S., Polge, N. D., Thomas,

D. A., and Hager, A. G. (2011). Resistance to HPPD-inhibiting herbicides in a population

of waterhemp (Amaranthus tuberculatus) from Illinois, United States. Pest Management

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International Surwey of Herbicide Resistant Weeds: http://www.weedscience.com/

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(syn. rudis)). Retrieved September 27, 2013, from The International Survey of Herbicide

Resistant Weeds: http://www.weedscience.org/

Heap, I. (2013c). Weeds Resistant to ALS inhibitors (B/2). Retrieved September 26, 2013, from

The International Survey of Herbicide Resistant Weeds: www.weedscience.org

Hidayat, I., and Preston, C. (2001). Cross-resistance to imazethapyr in a fluazifop-p-butyl-

resistant population of Digitaria sanguinalis. Pesticide Biochemistry and Physiology, 71:

190-195.

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Hinz, J. R., and Owen, M. D. (1997). Acetolactate synthase resistance in a common waterhemp

(Amaranthus rudis) population. Weed Technology, 11: 13-18.

Holtum, J. A., Matthews, J. M., Häusler, R. E., Liljegren, D. R., and Powles, S. B. (1991). Cross-

resistance to herbicides in annual ryegrass (Lolium rigidum). Plant Physiology, 97: 1026-

1034.

Horak, M. J., and Loughin, T. M. (2000). Growth analysis of four Amaranthus species. Weed

Science, 48: 347-355.

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and common waterhemp (Amaranthus rudis) are resistant to imazethapyr and

thifensulfuron. Weed Technology, 9: 192-195.

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myosuroides. In M. B. Green, H. M. LeBaron, and W. K. Moberg, Managing Resistance

to Agrochemicals From Fundamental Research to Practical Strategies (pp. 376-393).

Washington DC: American Chemical Society.

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LaRossa, R. A., Van Dyk, T. K., and Smulski, D. R. (1987). Toxic accumulation of α-

ketobutyrate caused by inhibition of the branched-chain amino acid biosynthetic enzyme

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Lovell, S. T., Wax, L. M., Horak, M. J., and Peterson, D. E. (1996). Imidazolinone and

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Ma, R., Kaundun, S. S., Tranel, P. J., Riggins, C. W., McGinness, D. L., Hager, A. G., Hawkes,

T., McIndoe, E., Riechers, D. E. (2013). Distinct detoxification mechanisms confer

resistance to mesotrione and atrazine in a population of waterhemp. Plant Physiology,

163: 363-377.

Mallory-Smith, C. A., and Retzinger, E. J., Jr. (2003). Revised classification of herbicides by site

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Mazur, B. J., Chui, C.-F., and Smith, J. K. (1987). Isolation and characterization of plant genes

coding for acetolactate synthase, the target enzyme for two classes of herbicides. Plant

Physiology, 85: 1110-1117.

McNaughton, K. E., Letarte, J., Lee, E. A., and Tardif, F. J. (2005). Mutations in ALS confer

herbicide resistance in redroot pigweed (Amaranthus retroflexus) and powell amaranth

(Amaranthus powellii). Weed Science, 53: 17-22.

Muhitch, M. J., Shaner, D. L., and Stidham, M. A. (1987). Imidazolinones and acetohydroxyacid

synthase from higher plants. Plant Physiology, 83: 451-456.

Ohkawa, H., Tsujii, H., and Ohkawa, Y. (1999). The use of cytochrome P450 genes to introduce

herbicide tolerance in crops: a review. Pesticide Science, 55: 867-874.

Patzoldt, W. L., and Tranel, P. J. (2007). Multiple ALS mutations confer herbicide resistance in

waterhemp (Amaranthus tuberculatus). Weed Science, 55: 421-428.

Patzoldt, W. L., Tranel, P. J., and Hager, A. G. (2005). A waterhemp (Amaranthus tuberculatus)

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30-36.

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Ray, T. B. (1984). Site of action of chlorsulfuron-inhibition of valine and isoleucine biosynthesis

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Amino Acids: Biochemistry and Biotechnology (pp. 227-247). New York: Marcel Dekker.

17

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18

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classes of ALS-inhibiting herbicides. Weed Science, 46: 8-12.

19

1.10 Figures

Figure 1.1 Responses of MCR waterhemp plants after treatment with ALS inhibitors. Herbicide

active ingredients and rates applied are listed below the picture. There were two distinct

phenotypes in the population; some of the plants showed little to no injury and the rest were

moderately injured by the ALS inhibitors but recovered later.

20

Figure 1.2 Demonstration of PCR-RFLP results of Trp574Leu mutation. ALS alleles with

mutation were recognized and cut by MfeI restriction enzyme and show smaller bands on the

agarose gel while the alleles lacking the specific mutation had larger fragments. Plants showing

little to no injury by ALS inhibitors in Figure 1.1 contain homozygous or heterozygous

Trp574Leu mutation, indicated by orange and blue arrows in the photo, respectively. An

example of a plant from Figure 1.1 showing injury but nevertheless surviving, and not containing

the mutation, is indicated by the white arrow.

21

CHAPTER 2

Confirmation of Non-target-site Resistance to ALS Inhibitors in a Waterhemp Population

2.1 Abstract

The acetolactate synthase enzyme is the target site of ALS-inhibiting herbicides, and a

variety of weed species have developed resistance to these herbicides over the past few decades.

The predominant resistance mechanism is gene mutation, with a few distinctive cases of

enhanced herbicide metabolism. Waterhemp [Amaranthus tuberculatus (Moq.) Sauer], a

common weed in Midwest United States has been reported to have ALS resistance. Researches

have confirmed single mutations on waterhemp ALS gene cause high level resistance (Chapter

1). The population found in McLean County, IL (MCR) has been reported resistant to both

HPPD inhibitors and atrazine, and demonstrating two phenotypes after ALS-inhibiting herbicide

application. The plants showing high resistance have Trp574Leu mutation, while plants with the

other phenotype do not contain this specific mutation. In order to confirm the possible resistance

mechanism of the moderate resistant individuals, a progeny (named JG11) was made by crossing

two moderate resistant plants without Trp574Leu mutation to be used in the study. The portion

of the ALS gene encoding the mature ALS protein was sequenced from eight JG11 plants that

survived a normally lethal dose of ALS inhibitor to detect other possible gene mutations that

might confer herbicide resistance. In vitro protein assays were performed to compare the

sensitivity of ALS enzyme of JG11 with that of both resistant and susceptible populations. None

of the deduced amino acid sequences obtained contained resistance-related mutations, and the

protein assay results indicated the JG11 plants did not have insensitive enzyme that confers

22

resistance. Both sets of experiments indicate that a non-target-site resistance mechanism exists in

the JG11 population.

2.2 Introduction

ALS-inhibiting herbicides have selected resistant populations of various weed species.

Most of the resistance cases contain single mutations in ALS gene that make the target site

insensitive to herbicide binding. Waterhemp is a common weed species in the Amaranthaceae

family (Sauer, 1955). It has evolved resistance to herbicides from numerous modes of action

including ALS inhibitors (Heap, 2013b). There are three different target-site amino acids

substitutions found in waterhemp populations resistant to ALS inhibitors: Trp574Leu,

Ser653Asp, and Ser653Thr. Waterhemp populations containing the Trp574Leu mutation have

shown cross resistance to all ALS-inhibitor families, while Ser653Asp and Ser653Thr mutation

confer an IMI-specific resistance pattern (Patzoldt and Tranel, 2007; Tranel and Wright, 2002).

Another resistance mechanism of enhanced metabolism was found in several weed species,

which was discussed in Chapter 1. There is no waterhemp population that has been reported with

enhanced metabolism as a mechanism to overcome ALS-inhibitors; however, a waterhemp

biotype from McLean County, IL, designated MCR, has metabolism-based HPPD and atrazine

resistance (Hausman, et al., 2011; Ma, et al., 2013). Without pre-selecting the population with

ALS-inhibitors in the crop field at least in the past 7 years, this population had shown both high

and moderate resistance to herbicides from the SU and IMI families (Chapter 1.6). Since

Trp574Leu mutation was not found in some of the resistant individuals that nevertheless were

able to survive labeled rates of either SU or IMI herbicides, two such individuals were crossed to

create a population for further analysis. Progeny of this cross were designated as JG11.

23

The objective of the study was to test the hypothesis that the JG11 population does not

contain a target-site-mutation that confers ALS-resistance. To address this objective, ALS gene

encompassing the coding sequence of the mature protein was sequenced to detect any amino acid

substitution that may confer resistance to ALS inhibitors. An in vitro ALS assay was also

conducted to determine if ALS enzyme in the JG11 population was sensitive to an ALS inhibitor.

2.3 Materials and Methods

2.3.1 Full-length ALS Gene Sequencing

2.3.1.1 Plant Material and Greenhouse Cultivation

In addition to JG11 (described in Introduction), two additional waterhemp populations,

ACR and WCS (described in Patzoldt et al., 2005) were used as controls. Population ACR

contains the ALS Trp574Leu substitution and was used as a positive control, while population

WCS is sensitive to ALS inhibitors and was used as a negative control. All seeds were stratified

before planting. Prior to stratification, seeds were soaked 10 min in a 1:1 mixture of commercial

bleach and water, and then rinsed with sterile water. Seeds were stratified in a solution of water

and 0.1% (w/v) agarose for at least one month at 4 C. Seeds were sown in plastic inserts

containing commercial potting mix (LC1 Professional Growing Mix, Sun Gro Horticulture, Inc.,

110 110th Ave. NE, Suite 490, Bellevue, WA 98004). Seedlings were transplanted to individual

inserts with the same potting mix when displaying one to two true leaves. A second transplant,

when the seedlings were about 5 cm tall moved them into 11-cm square pots filled with 3 : 1 : 1 :

1 mixture of commercial potting mix : sand : soil : peat. A slow-release fertilizer (Osmocote 13-

13-13 slow release fertilizer or Osmocote Plus 15-9-12 water soluble fertilizer. The Scotts

Company, 14111 Scottlawn Rd., Marysville, Oh 43041) was mixed in with the growing medium.

24

All plants were grown in a greenhouse at the University of Illinois in Urbana-Champaign campus,

and the environmental conditions were set to 28/22 C day/night temperature and 16 h

photoperiod. Sunlight was supplemented with halide and sodium vapor lights to maintain a

minimum of 313 µmol m-2

sec-1

during the diurnal period.

2.3.1.2 Herbicide Screening

Primisulfuron (Beacon, Syngenta Crop Protection) was applied to JG11 plants about 7

days after the second transplanting when the plants were 10-12 cm tall. The application rates

were 16.9 or 53.3 g a.i. per ha with 1% crop oil concentrate (COC, Herbimax®, Loveland

Products , Inc., P.O. Box 1286, Greeley, Co 80632) and 2.5% ammonium sulfate (AMS, N-Pak

AMS Liquid, Winfield Solutions, LLC, P.O. Box 64589, St. Paul, MN 55164-0589). Herbicide

treatments were performed in a greenhouse spraying chamber (Generation III Research Sprayer.

DeVries Manufacturing, 28081 870th Ave, Hollandale, MN 56045) with a flat-fan nozzle

(TeeJet 80015EVS. TeeJet Technologies, P.O. Box 7900, Wheaton, IL 60187) that delivered

75.7 liter min-1

. The nozzle was about 45 cm above the plant canopy when spraying. WCS plants

of similar size were included in the herbicide application to verify herbicide efficacy.

2.3.1.3 DNA Extraction and Purification

Leaf samples were collected during the second week after herbicide application, at which

time plants from the JG11 population had recovered from the herbicide treatment and were

developing new leaves. Only newly growing small leaflets were collected and put into 1.5 ml

tubes individually. The tubes were kept on ice until DNA extraction.

25

The DNA extraction and purification procedure was modified from a protocol of Doyle

and Doyle (1990) using hexadecyltrimethyl-ammonium bromide (CTAB). Extracted DNA was

resuspended in deionized sterile water and quantified using a Nanodrop 1000 Spectrophotometer

(Thermo Fisher Scientific, Inc., 81 Wyman St., Waltham, MA 02454). The DNA samples were

stored at -20 C until use for sequencing.

2.3.1.4 PCR Amplification and Sequencing

Polymerase chain reaction (PCR) was applied to amplify the ALS sequences. Because the

length of the ALS gene is over 2000 base pairs, several sets of primers were used to amplify

overlapping sections of the sequence (Table 2.1). Each PCR contained 1.0 µL of DNA samples,

0.2 µL GoTaq DNA polymerase (5 U/ µL. Promega Corporation, 2800 Woods Hollow Rd,

Madison, WI 53711), 1.0 µL each of forward and reverse primers (10 µM), 2.0 µL dNTP (2.5

mM), 2.5 µL MgCl2 solution (25 mM), 5 µL 5X GoTaq buffer, and 12.3 µL purified water to

make a total 25 µL volume. The amplification took place in a PTC-100 thermocycler (MJ

Research, Inc., 590 Lincoln Street, Waltham, MA, 02451) programed for an initial denaturation

of 1 min at 95 C, then 36 cycles of denaturation at 94 C for 15 sec, annealing at 50 C for 15 sec,

and extension at 60 C for 4 min. After a final elongation at 60 C for 4 min, the samples were held

at 4 C. PCR products were separated by electrophoreses through a 1% agarose gel (Certified

Molecular Biology Agarose, Bio-Rad Laboratories, 1000 Alfred Nobel Drive, Hercules, CA

94547) and visualized alongside Quick-Load® 100 bp ladder (New England Biolabs, Inc., 240

County Road, Ipswich, MA, 01938-2723) by ethidium bromide-staining/UV light. PCR products

of the expected sizes were purified with a Cyclic Purification Kit (Omega Bio-Tek, Inc., 400

Pinnacle Way Suite, 450 Norcross, GA 30071).

26

Purified DNA fragments were sequenced using an ABI Prism BigDye® TerminatorTM

v3.1 Cycle Sequencing Kit (Applied Biosystems, Inc., 850 Lincoln Centre Drive, Foster City,

CA 94404). The same primers used for PCR were used individually to sequence each DNA

strand. Each reaction contained 2.0 µL DNA template, 1.0 µL BigDye®, 2.0 µL primer, 2.0 µL

5X buffer containing 10 mM MgCl2 and 400 nM Tris base, 5.2 µL 12.5% glycerol, and 1.8 µL

sterile water to make a total of 14 µL reaction volume. The reaction was performed in a PTC-100

thermocycler, with an initial denaturation of 95 C for 3 min followed by 35 cycles of

denaturation of 95 C for 1 min, annealing at 56 C for 1 min, and extension at 72 for 1 min. After

a final extension of 5 min at 72 C the samples were kept at 4 C. Samples were analyzed by the

W.M. Keck Center for Comparative and Functional Genomics High Throughput Sequencing Lab

(University of Illinois Biotechnology Venter, 340 Edward R. Madigan Laboratory, 1201 W.

Gregory Drive, Urbana, IL 61801) using an ABI 3730xl DNA Analyzer. The returned data were

compared and aligned with FinchTV software v1.4 (Geospiza, Inc., 100 West Harrison, North

Tower, Suite #330, Seattle, WA 98119) and ClustalW package in FinchTV.

2.3.2 ALS Enzyme In Vitro Assay

2.3.2.1 Plant Material

Seeds of ACR, WCS and JG11 were stratified and sown as described in 2.3.1.2. Leaf

tissue was collected from plants when they were about 15 to 20 cm tall. New leaves from plant

apexes were selected. Collected leaf samples were put on ice and used for the ALS enzyme

extraction process immediately after collection.

27

2.3.2.2 Protein Extraction and Desalinization

The protein extraction procedure used was modified from Zheng et al. (2005). About 6.0

g of fresh leaf tissue (pooled from 10 to 15 plants) were frozen with liquid nitrogen and grinded

into powder, then homogenized with 35 ml homogenization buffer of 100 mM potassium

phosphate (pH 7.0), 5 mM pyruvate, 5 mM MgCl2, 1 mM thiamine pyrophosphate (TPP), 10 µM

flavin adenine dinucleotide (FAD), 1 mM dithiothreitol (DTT), 10% glycerol (v/v), and 1%

polyvinylpolypyrrolidone (PVPP) (w/v). The homogenate was centrifuged (Beckman J2-HS

Centrifuge. Beckman Coulter, Inc., Diagnostics Division Headquarters, 250 South Kraemer

Boulevard, Brea CA 92821-6232) at 20,000 × g for 20 min at 4 C and the supernatant was

transferred to another centrifuge tube. Ammonium sulfate was added to create a 45% solution

(w/v), which was then gently shaken on ice for 45 min to precipitate protein containing ALS

enzyme. The precipitate was collected by centrifugation at 20,000 × g for 20 min at 4 C. After

discarding the supernatant, the pellet was resuspended in 2.5 ml of resuspension buffer,

consisting of 50 mM potassium phosphate (pH 7.0), 5 mM MgCl2, 1 mM TPP, and 10 µM FAD.

Disposable PD-10 Desalting Columns (GE Healthcare Bio-Sciences, 800 Centennial Avenue,

P.O. Box 1327, Piscataway, NJ 08855-1327) were used at 4 C to purify and desalt the protein.

Protein concentrations of the extracts were determined with a Nanodrop 1000 Spectrophotometer

v3.7.1 using Coomassie Plus (Bradford) Assay Kit (Thermo ScientificTM

PierceTM

. Thermo

Fisher Scientific, 81 Wyman St., Waltham, MA 02454). Protein concentrations ranged from 1 to

1.5 µg/µl. The protein samples were frozen with liquid nitrogen and stored at -80 C until needed.

28

2.3.2.3 ALS Enzymatic Assay

Corning 96-well assay flat clear bottom plates (Corning, Inc., One Riverfront Plaza,

Corning, NY 14831) were used in protein assay. Technical-grade imazethapyr (98.1%, BASF

Corporation, 26 David Drive, Research Triangle Park, NC 27709) was used to determine enzyme

activity under herbicide interference. Each reaction contained 50 µl of protein extract (diluted to

1.0 µg/µl), 50 µl of reaction buffer of 50 mM potassium phosphate buffer (pH 7.0), 100mM

pyruvate, 5 mM MgCl2, 1 mM TPP, 10 µM FAD and 50 µl of herbicide solution. Imazethapyr

was dissolved in 9% dimethylsulfoxide (DMSO, v/v) solution to concentrations ranging from 10-

1 to 10

6 nM in 10-fold increments. For the positive controls and background readings, 50 µl of 9%

DMSO were added as solvent in substitution of herbicide solution. Negative controls without

herbicide were created by adding 25 µl 3.5% H2SO4 before addition of protein extract. The

enzyme assay mixtures were incubated at 37 C for 90 min and terminated by adding 25 µl 3.5%

H2SO4 and incubating 20 min at 60 C. The amount of acetoin formed was determined by a

chromogenic reaction with 100 µl of a mixture of 0.55% (w/v) creatine and 5.5% (w/v) α-

naphthol in 1.375 N NaOH added to each enzyme assay, followed by 40 min incubation at 37 C.

Absorbance was measured by a spectrophotometer at a wavelength of 530 nm.

2.3.2.4 Data Analysis

Two separate extracts were obtained from each population for two independent assays,

and each assay contained three replications of each treatment (treated statistically as subsamples).

The data of absorbance were analyzed using a non-linear regression model with the dose-

response curve package in R software (Knezevic et al, 2007). The equation

{ [ ( ) ( )]}

29

was used to construct the dose-response curves of enzymatic assays. The four-parameter non-

linear logistic model is described as: b is the slope of the curve, c is the lower limit, d is the

upper limit and ED50 is 50% reduction in acetoin accumulation.

2.4 Results and Discussion

2.4.1 Full-length ALS Gene Sequencing

A portion of the ALS gene encompassing the coding region of the mature protein was

sequenced from each of eight JG11 plants that survived primisulfuron at either 16.9 or 53.3 g per

ha. The same herbicide rates were lethal to WCS plants and all the WCS plants sprayed

simultaneously with JG11 were dead when collecting tissue samples for DNA extraction (data

not shown). Additional waterhemp ALS gene sequences from WCS, ACR and an imidazolinone-

resistant population IR-101, described by Paltzoldt and Tranel (2007), were obtained from

GenBank (accessions EF157818, EF157819 and EF157821, respectively) for comparison. The

mature protein sequences were aligned and compared (Figure 2.1). ACR and IR-101 contain

resistance-conferring mutations at W574 and S653, respectively (Patzoldt, et al., 2005; Patzoldt

and Tranel, 2007), but these mutations were not identified in JG11 plants. The majority of

sequence polymorphisms identified (23 out of 29 codons) were synonymous substitutions.

Across all JG11 protein sequences obtained, six nonsynonymous polymorphisms were identified

relative to the WCS population. However, these same polymorphisms were shared with ACR

and/or IR-101 and, therefore, not likely to be associated with resistance. In addition, these

polymorphic amino acid substitutions codons were observed in other weed species as well, in

both resistant and susceptible biotypes, suggesting they are in non-conserved regions of ALS and

30

not associated with herbicide resistance (Diebold, et al., 2003; McNaughton, et al., 2005;

Patzoldt and Tranel, 2007).

2.4.2 ALS Enzyme In Vitro Assay

The reaction catalyzed by ALS in vitro is the condensation of two molecules of pyruvate

to yield one molecule of acetolactate. Since the next enzyme of the BCAA synthesis pathway,

ketoacid reductoisomerase (KARI) is not present in the extracts, the acetolactate accumulates,

and can then be decarboxylated into acetoin, which yields a red color after the chromogenic

reaction (Corbett & Tardif, 2006). Decreasing color intensity, which is the indication of enzyme

inhibition, can be seen with increasing herbicide concentration (

Figure 2.2).

The combination of two runs of enzymatic assay results is shown in Figure 2.3. The

positive (ACR) and negative (WCS) controls behaved as expected with the former being quite

insensitive to herbicide inhibition and the latter greatly inhibited with increasing herbicide

concentration. The ED50 (effective dose causing 50% reduction of action accumulation) for ALS

extracted from WCS was 1642 nM imazethapyr, whereas even the highest dose tested (3.3 × 106

nM imazethapyr) failed to reduce ACR ALS enzyme activity by 50%. The dose response curve

for ALS from JG11 was very similar to that from WCS, and the calculated ED50 value (1559 nM

imazethapyr) was indistinguishable (p=0.9) from that obtained from WCS. Therefore, it was

concluded that ALS in the JG11 population is fully sensitive to ALS herbicides.

The results of both gene sequencing and enzymatic assay provide evidence that the

observed whole plant resistance to ALS-inhibitors in JG11 is not based on an insensitive target

31

site. More research is needed to determine the actual resistance level of the JG11 population as

well as the alternative mechanism of resistance that is occurring in this population.


Corbett, C.-A. L., and Tardif, F. J. (2006). Detection of resitance to acetolactate synthase

inhibitors in weeds with enphasis on DNA-based techniques: a review. Pest Management

Science, 62: 584-597.

Diebold, R. S., McNaughton, K. E., Lee, E. A., and Tardif, F. J. (2003). Multiple resistance to

imaethapyr and atrazine in powell amaranth (Amaranthus powellii). Weed Science, 51:

312-318.

Doyle, J. J., and Doyle, J. L. (1990). Isolation of plant DNA from fresh tissue. Focus, 12: 13-15.

Hausman, N. E., Singh, S., Tranel, P. J., Riechers, D. E., Kaundun, S. S., Polge, N. D., Thomas,

D. A., and Hager, A. G. (2011). Resistance to HPPD-inhibiting herbicides in a population

of waterhemp (Amaranthus tuberculatus) from Illinois, United States. Pest Management

Science, 67: 258-261.





T., McIndoe, E. and Riechers, D. E. (2013). Distinct detoxification mechanisms confer


163: 363-377.

32

McNaughton, K. E., Letarte, J., Lee, E. A., and Tardif, F. J. (2005). Mutations in ALS confer

herbicide resistance in redroot pigweed (Amaranthus retroflexus) and powell amaranth

(Amaranthus powellii). Weed Science, 53: 17-22.



30-36.






Zheng, D., Patzoldt, W. L., and Tranel, P. J. (2005). Association of the W574L ALS substitution

with resistance to cloransulam and imazamox in common ragweed (Ambrosia

artemisiifolia). Weed Science, 53: 424-430.

33

2.6 Tables and Figures

Table 2.1 Primers used in ALS gene amplification.

Name Sequence (5’ → 3’) Tm (C) MW

ALS5UTR-F CTT CAA TCT TCA ACA ATG GCG 52.5 6365.2

WHals-F CGC CCT CTT CAA ATC TCA TC 53.3 5947.9

ALSr1 TCA ATC AAA ACA GGT CCA GG 52.7 6119.0

ALSf1 AGC TCT TGA ACG TGA AGG TG 55.1 6197.1

ALS1603-R AAC TCC CAT CCC CAT CAA TGT C 57 6559.3

ALS1530-F TTT GGG GGC TAT GGG GTT TG 57.9 6266.1

AmALS-F2 TCC CGG TTA AAA TCA TGC TC 52.8 6052.0

AmALS-R2 CTA AAC GAG AGA ACG GCC AG 55.4 6169.1

ALS1426-f ACG AAG GGT GAT GCG ATT GT 57 6237.1

34

Table 2.2 Output from statistical analysis of enzymatic assay.

Estimate Std. Error Lower Upper

JG11:50 1558.746 545.220 477.046 2640.45

WCS:50 1641.904 638.409 375.318 2908.49

Estimate Std. Error t-value p-value

JG11/WCS 0.94935 0.49651 -0.10201 0.919

35

Figure 2.1 ALS codon alignments of WCS, ACR, IR-101 and JG11 populations. Amino acid sequences of 8 individual plants of JG11

population are listed. Protein sequence of WCS (S-EF157818) is used as standard for comparison. Positions marked with “X” indicate

the identification of synonymous nucleotide polymorphism, and heterozygous substitutions are in regular font while homozygous

substitutions are in bold font. Nonsynonymous polymorphisms are shown as the amino acids corresponding to each codon, with

heterozygous substitutions in regular font and homozygous substitutions in bold font.

WCS P D E P R K G C D V L V E A L E R E G V T D V F A Y P G G A S M E I H Q A L T R S N I I R N V L P R

ACR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

IR-101 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1104 full . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1105 full . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1106 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1107 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1108 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1109 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1110 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1111 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

WCS H E Q G G V F A A E G Y A R A T G R V G V C I A T S G P G A T N L V S G F A D A L L D S V P L V A I

ACR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

IR-101 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1104 full . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1105 full . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1106 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1107 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1108 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1109 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1110 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1111 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

89 99 109 119 129

139 149 159 169 179

Figure 2.1 (cont.)

36

WCS T G Q V P R R M I G T D A F Q E T P I V E V T R S I T K H N Y L V L D V E D I P R I V K E A F F L A

ACR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

IR-101 . . . . . X . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1104 full . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1105 full . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . X . . . . . . . . . .

1106 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . X . . . . . . . . . .

1107 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . X . . . . . . . . . .

1108 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1109 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1110 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1111 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

WCS N S G R P G P V L I D I P K D I Q Q Q L V V P N W E Q P I K L G G Y L S R L P K P T F S A N E E G L

ACR . . . . . . . . . . . . . . . . . . X . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

IR-101 . . . . . . . . . . . . . . . . . . X . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1104 full . . . . . . . . . . . . . . . . . . X . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1105 full . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . X . . . . . . . .

1106 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . X . . . . . . . .

1107 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . X . . . . . . . .

1108 . . . . . . . . . . . . . . . . . . X . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1109 . . . . . . . . . . . . . . . . . . X . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1110 . . . . . . . . . . . . . . . . . . X . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1111 . . . . . . . . . . . . . . . . . . X . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

WCS L D Q I V R L V G E S K R P V L Y T G G G C L N S S E E L R K F V K L T G I P V A S T L M G L G A F

ACR . . . . . . . . . . . . . . . . . . . . . . . . . . . . X . . . . E . . . . . . . . . . . . . . . .

IR-101 . . . . . . . . . . . . . . . . . . . . . . . . . . . . X . . . . E . . . . . . . . . . . . . . . .

1104 full . . . . . . . . . . . . . . . . . . . . . . . . . . . . X . . . . E . . . . . . . . . . . . . . . .

1105 full . . . . . . . . . . . . . . . . . . . . . . . . X . . . X . . . . E . . . . . . . . . . . . . . . .

1106 . . . . . . . . . . . . . . . . . . . . . . . . X . . . X . . . . E . . . . . . . . . . . . . . . .

1107 . . . . . . . . . . . . . . . . . . . . . . . . X . . . X . . . . E . . . . . . . . . . . . . . . .

1108 . . . . . . . . . . . . . . . . . . . . . . . . . . . . X . . . . E . . . . . . . . . . . . . . . .

1109 . . . . . . . . . . . . . . . . . . . . . . . . . . . . X . . . . E . . . . . . . . . . . . . . . .

1110 . . . . . . . . . . . . . . . . . . . . . . . . . . . . X . . . . E . . . . . . . . . . . . . . . .

1111 . . . . . . . . . . . . . . . . . . . . . . . . . . . . X . . . . E . . . . . . . . . . . . . . . .

189 199 219 229

249 259 269

209

289 299 309 319 329

239 279

Figure 2.1 (cont.)

37

WCS P C T D D L S L Q M L G M H G T V Y A N Y A V D K A D L L L A F G V R F D D R V T G K L E A F A S R

ACR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . X . . . . . . . . . . . . . . . . X

IR-101 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . X . . . . . . . . . . . . . . . . X

1104 full . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . X . . . . . . X . . . . . . . . . X

1105 full . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . X . . . . . . X . . . . . . . . X X

1106 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . X . . . . . . X . . . . . . . . X X

1107 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . X . . . . . . X . . . . . . . . X X

1108 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . X . . . . . . X . . . . . . . . . X

1109 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . X . . . . . . X . . . . . . . . . X

1110 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . X . . . . . . X . . . . . . . . . X

1111 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . X . . . . . . X . . . . . . . . . X

WCS A K I V H I D I D S A E I G K N K Q P H V S I C G D V K V A L R G L N N I L E S R K G K V K L D F S

ACR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

IR-101 . . . . . . . . . . . . . . . . . . . . . . . . . . I . . . . Q . . . K . . . . . . . . L . . . . .

1104 full . . . . . . . . . . . . . . . . . . . . . . . . . . I . . . . Q . . . K . . . . . . . . L . . . . .

1105 full . . . . . . . . . . . . . . . . . . . . . . . . . . I . . . . Q . . . K . . . . . . . . L . . . . .

1106 . . . . . . . . . . . . . . . . . . . . . . . . . . I . . . . Q . . . K . . . . . . . . L . . . . .

1107 . . . . . . . . . . . . . . . . . . . . . . . . . . I . . . . Q . . . K . . . . . . . . L . . . . .

1108 . . . . . . . . . . . . . . . . . . . . . . . . . . I . . . . Q . . . K . . . . . . . . L . . . . .

1109 . . . . . . . . . . . . . . . . . . . . . . . . . . I . . . . Q . . . K . . . . . . . . L . . . . .

1110 . . . . . . . . . . . . . . . . . . . . . . . . . . I . . . . Q . . . K . . . . . . . . L . . . . .

1111 . . . . . . . . . . . . . . . . . . . . . . . . . . I . . . . Q . . . K . . . . . . . . L . . . . .

WCS N W R E E L N E Q K K K F P L S F K T F G D A I P P Q Y A I Q V L D E L T K G D A I V S T G V G Q H

ACR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . X . . . . . . . . . . . . . .

IR-101 . . . . X . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . X . . . . . . . . . . . . . .

1104 full . . . . X . . . . . . . . . . . . . . . . X . . . . . . . . . . . . . X . . . . . . . . . . . . . .

1105 full . . . . X . . . . . . . . . . . . . . . . X . . . . . . . . . . . . . X . . . . . . . . . . . . . .

1106 . . . . X . . . . . . . . . . . . . . . . X . . . . . . . . . . . . . X . . . . . . . . . . . . . .

1107 . . . . X . . . . . . . . . . . . . . . . X . . . . . . . . . . . . . X . . . . . . . . . . . . . .

1108 . . . . X . . . . . . . . . . . . . . . . X . . . . . . . . . . . . . X . . . . . . . . . . . . . .

1109 . . . . X . . . . . . . . . . . . . . . . X . . . . . . . . . . . . . X . . . . . . . . . . . . . .

1110 . . . . X . . . . . . . . . . . . . . . . X . . . . . . . . . . . . . X . . . . . . . . . . . . . .

1111 . . . . X . . . . . . . . . . . . . . . . X . . . . . . . . . . . . . X . . . . . . . . . . . . . .

379

479

339 349 359 369

389 399 409 419 429

439 449 459 469

Figure 2.1 (cont.)

38

WCS Q M W A A Q F Y K Y R N P R Q W L T S G G L G A M G F G L P A A I G A A V A R P D A V V V D I D G D

ACR . . . . . . X . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

IR-101 . . . . . . X . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1104 full . . . . . . . . . . . . . . . . . . . . . . . . . . . X . . . . . . . . . . . . . X . . . . . . . X

1105 full . . . . . . . . . . . . . . . . . . . . . . . . . . . X . . . . . . . . . . . . . X . . . . . . . X

1106 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . X . . . . . . . X

1107 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . X . . . . . . . X

1108 . . . . . . . . . . . . . . . . . . . . . . . . . . . X . . . . . . . . . . . . . X . . . . . . . X

1109 . . . . . . . . . . . . . . . . . . . . . . . . . . . X . . . . . . . . . . . . . X . . . . . . . X

1110 . . . . . . X . . . . . . . . . . . . . . . . . . . . X . . . . . . . . . . . . . X . . . . . . . X

1111 . . . . . . X . . . . . . . . . . . . . . . . . . . . X . . . . . . . . . . . . . X . . . . . . . X

WCS G S F I M N V Q E L A T I R V E N L P V K I M L L N N Q H L G M V V Q W E D R F Y K A N R A H T Y L

ACR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . L . . . . . . . . . . . . . .

IR-101 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1104 full . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1105 full . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1106 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1107 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1108 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1109 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1110 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1111 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

WCS G N P S N S S E I F P D M L K F A E A C D I P A A R V T K V S D L R A A I Q T M L D T P G P Y L L D

ACR . . X . . . X . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

IR-101 . . . . K . . . . . . . . X . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1104 full . . . . K . . . . . . . . X . . . . . . . . . . . . . X . . . . . . . . . . . . . . . . . . . . . .

1105 full . . . . K . . . . . . . . X . . . . . . . . . . . . . X . . . . . . . . . . . . . . . . . . . . . .

1106 . . X . K . X . . . X . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1107 . . X . K . X . . . X . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1108 . . . . K . . . . . . . . X . . . . . . . . . . . . . X . . . . . . . . . . . . . . . . . . . . . .

1109 . . . . K . . . . . . . . X . . . . . . . . . . . . . X . . . . . . . . . . . . . . . . . . . . . .

1110 . . . . K . . . . . . . . X . . . . . . . . . . . . . X . . . . . . . . . . . . . . . . . . . . . .

1111 . . . . K . . . . . . . . X . . . . . . . . . . . . . X . . . . . . . . . . . . . . . . . . . . . .

569

629

489 499 519 529

549 559

509

539

589

579

599 609 619

Figure 2.1 (cont.)

39

WCS V I V P H Q E H V L P M I P S G A A F K D T I T E G D G R R A Y *

ACR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

IR-101 . . . . . . . . . . . . . . T . . . . . . . . . . . . . . . . . .

1104 full . . . . . . . . . . . . . . X . . . . . X . . . . . . . . . . . .

1105 full . . . . . . . . . . . . . . X . . . . . X . . . . . . . . . . . .

1106 . . . . . . . . . . . . . . X . . . . . X . . . . . . . . . . . .

1107 . . . . . . . . . . . . . . X . . . . . X . . . . . . . . . . . .

1108 . . . . . . . . . . . . . . X . . . . . X . . . . . . . . . . . .

1109 . . . . . . . . . . . . . . X . . . . . X . . . . . . . . . . . .

1110 . . . . . . . . . . . . . . X . . . . . X . . . . . . . . . . . .

1111 . . . . . . . . . . . . . . X . . . . . . . . . . . . . . . . . .

669649 659639

40

1 2 3 4 5 6 7 8 9 10 11 12

A ACR

106

ACR

106

ACR

106

JG11

106

JG11

106

JG11

106

WCS

106

WCS

106

WCS

106

ACR

DMSO

ACR

DMSO

ACR

DMSO

B ACR 105

ACR 105

ACR 105

JG11 105

JG11 105

JG11 105

WCS 105

WCS 105

WCS 105

JG11 DMSO

JG11 DMSO

JG11 DMSO

C ACR

104

ACR

104

ACR

104

JG11

104

JG11

104

JG11

104

WCS

104

WCS

104

WCS

104

WCS

DMSO

WCS

DMSO

WCS

DMSO

D ACR 103

ACR 103

ACR 103

JG11 103

JG11 103

JG11 103

WCS 103

WCS 103

WCS 103

ACR H2SO4

ACR H2SO4

ACR H2SO4

E ACR

102

ACR

102

ACR

102

JG11

102

JG11

102

JG11

102

WCS

102

WCS

102

WCS

102

JG11

H2SO4

JG11

H2SO4

JG11

H2SO4

F ACR

10 ACR

10 ACR

10 JG11

10 JG11

10 JG11

10 WCS

10 WCS

10 WCS

10 WCS H2SO4

WCS H2SO4

WCS H2SO4

G ACR

1

ACR

1

ACR

1

JG11

1

JG11

1

JG11

1

WCS

1

WCS

1

WCS

1

DMSO

DMSO

DMSO

H ACR 0.1

ACR 0.1

ACR 0.1

JG11 0.1

JG11 0.1

JG11 0.1

WCS 0.1

WCS 0.1

WCS 0.1

DMSO

DMSO

DMSO

Figure 2.2 ALS enzymatic assay with imazethapyr as inhibitor and the plate arrangement is

shown below. In the bottom panel, the orange area shows the treatments, the blue area contained

no herbicide and was used as positive control, the green area was used as negative control by

adding H2SO4 before adding enzyme extracts, and the gray area contained only reaction buffer

and DMSO to obtain background readings. The photo was taken after the chromatic reaction, and

the red color indicates the accumulation of acetoin, which reflects ALS activity.

41

Figure 2.3 Dose response curves of enzymatic assay. X-axis is the herbicide concentration (nM)

and Y-axis is ALS activity expressed relative to the no-herbicide control.

Imazethapyr (nM)

En

zym

e a

ctiv

ity (

rela

tiv

e to

no

-her

bic

ide

con

tro

l)

42

CHAPTER 3

Quantifying Herbicide Resistance in a Waterhemp Population with Non-Target-Site Resistance to

ALS-Inhibiting Herbicides

3.1 Abstract

Previous studies reported several ALS-resistance waterhemp biotypes. In all cases,

resistance was found to result from amino acid substitutions that alter the herbicide-binding site

(Chapter 1). In Chapter 2, both gene sequencing and protein assay showed the resistance of JG11,

a progeny derived from the MCR population, is not caused by the same mechanism, because no

mutations in ALS gene that may affect herbicide binding were found, nor was an insensitive

ALS enzyme observed in the JG11 population. In initial trials of herbicide application, the JG11

plants showed minor damage, which is different from the symptom of target-site mediated

resistant waterhemp biotypes. In addition, ALS inhibitors comprise five different chemical

families, so the focus following is to evaluate the actual resistance level of JG11 across all

families of ALS inhibitors. A series of greenhouse dose response experiments were conducted

with seven different ALS-inhibiting herbicides to compare the response of JG11 to that of the

susceptible population, WCS. Results indicated that JG11 was resistant to ALS-inhibiting

herbicides, and the R/S ratios varied from 3 to 90 among different herbicides. In order to

investigate the possible resistance mechanism, a cytochrome P450 inhibitor, malathion, was

incorporated with herbicide application. The combination caused more injury to the JG11

population, which suggested P450 hydroxylation contributes to herbicide detoxification in the

JG11 waterhemp, and is the possible mechanism of ALS resistance.

43

3.2 Introduction

Waterhemp [Amaranthus tuberculatus (Moq.) Sauer] is a common weed species in the

Amaranthaceae family (Sauer, 1955). It has evolved resistance to multiple herbicides with

different modes of action (Heap, 2013b). As described in Chapter 1, MCR (from McLean

County, IL), which has multiple resistance to HPPD inhibitors and atrazine, also showed

resistance to ALS inhibitors even though ALS inhibitors have not been used recently in the crop

field from which MCR was obtained. In preliminary greenhouse trials, this population was found

to contain plants with either high or moderate resistance to the ALS inhibitors chlorimuron and

imazethapyr. After testing individual plants treated by ALS inhibitors with PCR-RFLP

(restriction fragment length polymorphism), a Trp574Leu ALS mutation was found in the highly

resistant plants but not in moderately resistant plants (described in Chapter 1). Evidence provided

by analyzing the full protein-coding sequence and comparing the progeny from moderately

resistant plants with other waterhemp populations suggested that there is a mechanism of

resistance other than mutation in ALS gene in this population (Chapter 2).

There are five different chemical families of herbicides that target the ALS enzyme. But

because of the variation of the chemical structures, these ALS inhibitors may bind slightly

differently to the ALS enzyme, and as a result, mutation at ALS may confer resistance to only

one family of ALS inhibitors, or may confer cross resistance to more than one group of ALS-

inhibiting compounds (Patzoldt and Tranel, 2007). These cross resistance patterns can now be

predicted based on the knowledge we have obtained from numerous ALS mutations previously

characterized (Tranel et al., 2013; Chapter 1). But for a non-target-site resistance mechanism, the

resistance pattern across different families of ALS inhibitors is more difficult to predict.

44

In terms of the mechanism of non-target-site resistance, since MCR population has been

confirmed to have metabolism-based resistance to both HPPD herbicides and atrazine (Ma, et al.,

2013), the former is detoxified by hydroxylation process induced by cytochrome P450s and the

latter is detoxified through glutathione conjugation, it is indicated that this population has a high

potency of enhanced metabolism. In addition, ALS inhibitor detoxified by cytochrome P450s is a

common mechanism of crop tolerance to ALS inhibitors (Siminszky, 2006). The hypothesis

raised is that the ALS resistance in JG11 is caused by enhanced detoxification by cytochrome

P450s.

The objective of the study was to evaluate the resistance level of JG11 across the five

families of ALS inhibitors. To address this objective, JG11 population was treated with at least

one herbicide from each of the five families of ALS-inhibiting herbicides at a range of dosages

and compared with a standard susceptible population. A waterhemp population with the ALS

Trp574Leu mutation was also included for comparison. Also, a second set of experiments

included the combination of an inhibitor of cytochrome P450s, malathion, with herbicide

treatment to test for the involvement of P450s in enhanced herbicide detoxification.

3.3 Materials and Methods

3.3.1 Greenhouse Dose Response with ALS-inhibiting Herbicide

3.3.1.1 Plant Material

Populations and plant culture methods used in this section were the same as described in

Chapter 2.3.1.1.

45

3.3.1.2 Herbicide Dose Response

Uniformly sized waterhemp plants (10 to 12 cm tall) of WCS and JG11 were included in

the dose response experiments, as well as the population with the Trp574Leu mutation, ACR,

which was used as a positive control. Plants were sprayed with a TeeJet flat-fan nozzle (TeeJet

80015EVS. TeeJet Technologies, P.O. Box 7900, Wheaton, IL 60187) in a greenhouse spraying

chamber (Generation III Research Sprayer. DeVries Manufacturing, 28081 870th Ave,

Hollandale, MN 56045) that delivers 185 L per ha at 275 kPa. Multiple ALS-inhibiting

herbicides were used to study the resistance level of the JG11 population. All herbicides used are

listed in Table 3.1, and the rates increased equally spaced on a base 3.16 logarithmic scale. All

the treatment mixtures included COC, (1% v/v) and AMS (2.5% v/v) expect for pyrithiobac

sodium, which was mixed with nonionic surfactant (NIS, 0.25% v/v, Agriliance). Non-treated

controls were treated with water and adjuvant only. Because ACR has a very high level of

resistance to ALS inhibitors, this population was treated only with the highest rates of ALS

inhibitors and not the full range of dosages. After herbicide application, plants were placed on

greenhouse benches in a randomized complete block design. Each treatment had 4 replicates and

the experiment was conducted twice.

At 21 days after treatment (DAT), plant injury was visually evaluated and recorded using

a scale from 0 to 100, with 0 indicating a plants was dead and without any green tissue and 100

indicated no injury observed. Aboveground plant tissue was harvested and dried at 65 C for 4 to

7 days.

Dry weight data (m) and visual rating data (v) were combined to obtain an adjusted dry

weight (y) using the function

46

The adjusted dry weight was converted to a proportion of the average of untreated control plants

from the corresponding population. Data from the two runs of each herbicide were pooled, since

Levene’s test for homogeneity of variance was not significant, and analyzed using a non-linear

regression model with the dose-response curve package in R software (Knezevic et al, 2007).

This analysis is similar to the algorithm described in 2.3.2.4, except that GR50 indicating the

effective dose causing 50% growth reduction in biomass is used instead of ED50.

3.3.2 Response of ALS Inhibitors Combined with Malathion

Waterhemp plants from the WCS and JG11 population were treated with ALS inhibitors

with or without commercial liquid malathion insecticide (50% v/v. Spectracide® Malathion

Insect Spray Concentrate. Spectrum Group, Division of United Industries, P.O.Box 142642, St.

Louis, MO 63114-0642). Waterhemp plants (10-12 cm tall) were firstly treated with malathion at

a rate of 2,000 g a.i. ha-1

, including 0.25% NIS. ALS-inhibiting herbicides were applied to plants

1 h after malathion treatment at rates showed in Table 3.. Herbicides and malathion applications

were made using the spray chamber described in 3.3.1.2. A soil drench of 5 mM malathion

solution (50 mL pot-1

) was applied 2 days after foliar treatment as described in Ma et al. (2013).

At 14 days after treatment (DAT), plant injury was visually evaluated and recorded, and

aboveground plant tissue was harvested and dried at 65 C for 4 to 7 days. Each treatment had 6

replicates and the experiment was conducted twice. All dry weight data were converted as

relative to the average of the corresponding untreated control plants as described in 3.3.1.2. Data

from the two runs were compared using the PROC GLM procedure with SAS 9.3. Treatment

means were compared using PROC ANOVA also with SAS 9.3. The significant differences

among treatments were compared by Duncan’s Multiple Range Test with SAS 9.3.

47

3.4 Results and Discussion

3.4.1 Greenhouse Dose Response with ALS-inhibiting Herbicide

All treatments of JG11 and WCS by ALS inhibitors injured the plants at higher dosages

and some of the plants were dead 21 DAT. Based on visual observation, JG11 was less sensitive

to all seven herbicides across the five chemical families (Figure 3.1). As the results show in

Figure 3.2, it is confirmed that JG11 is resistant to ALS inhibitors relative to the WCS population.

For some herbicides, for example primisulfuron, imazethapyr and cloransulam, the commercially

recommended rate did not control JG11 waterhemp plants under greenhouse conditions. In

addition, although JG11 confers resistance across a broad spectrum of ALS inhibitors, the R/S

ratio varied among herbicides, and did not necessarily relate to the chemical families. This is

different from resistance caused by amino acid substitution, which typically confers resistance to

a group of chemicals from the same family (Tranel and Wright, 2002). For instance, Pro197His

mutation in wild radish (Raphanus raphanistrum) conferred resistance to SUs and TPs, which

have similar structures, but not to IMIs (Yu et al., 2003). In our case of JG11, the greatest

resistance was observed from cloransulam (a TP), with an R/S ratio of more than 90-fold. The

R/S ratio of two SUs ranged from 6 to 11, and those of two IMIs ranged from 9 to 19.

The variation of responses among different herbicides was large, which made us curious

about the mechanism behind it. Since we confirmed the population does not contain mutations

related to herbicide binding, the second most likely resistant mechanism is enhanced

detoxification. Cytochrome P450 hydroxylation is a common detoxification process in crops

tolerant to ALS inhibitors and it hydroxylates the herbicide molecule at the ring structure and

makes it inactive (Ohkawa et al, 1999). There is also a similar process found in other weed

species (Fischer et al., 2000). Moreover, MCR population, the original population from which

48

JG11 was derived, was characterized with P450-mediated resistance to HPPD inhibitors. We

therefore hypothesized that P450s also mediate resistance to ALS inhibitors in the JG11

population. Therefore, we conducted an experiment using malathion, an inhibitor of cytochrome

P450s.

3.4.2 Response to ALS Inhibitors Combined with Malathion

Figure 3.3 and Figure 3.4 show plants 14 DAT with ALS inhibitor with and without

malathion, Herbicide rates were chosen to cause only moderate injury to JG11 plants. Addition

of malathion resulted in injury to JG11 that was similar to that observed when WCS was treated

by herbicide only. Results of the analysis of the dry weight and adjusted dry weight data were

summarized in Table 3.3 and Table 3.4. Except for sulfometuron, all cases show means of JG11

treated only with herbicide were significantly less than those obtained when malathion was

included with the herbicide, and the latter were not significantly different from WCS population

treated with herbicides, with or without malathion present. In sulfometuron treatment, the

herbicide dosage was rather high and caused a severe injury to the JG11 population.

Nevertheless, by adding malathion, the injury became insignificant to WCS population treated

with herbicide only (Figure 3.3).

There are still many aspect of metabolism-based resistance needed to be investigated.

The complexity of cytochrome P450s increases the difficulty of elucidating the genetics and

expression of resistance. However, we can draw the conclusion that cytochrome P450s

contribute to the enhanced detoxification process in JG11, which makes JG11 more resistance to

ALS inhibitors. It also explains the differentiation of resistance level across various ALS-

inhibiting chemicals; some of the chemicals, sulfometuron and imazapyr, are harder to be

49

detoxified through hydroxylation by P450s than the others, like cloransulam and primisulfuron.

This conclusion agrees with the results of the study of HPPD resistance in MCR population,

which is caused by enhanced metabolism (Ma, et al., 2013). MCR is another example of a weed

population with herbicide resistance to ALS inhibitors selected by other modes of action, which

has been observed in other weed species as discussed in Chapter 1.


Fischer, A. J., Bayer, D. E., Carriere, M. D., Ateh, C. M., and Yim, K.-O. (2000). Mechanisms of

resistance to bispyribac-sodium in an Echinochloa phyllopogon accession. Pesticide

Biochemistry and Physiology, 68: 156-165.

Heap, I. (2013b, September 27). Herbicide resistant common waterhemp globally (Amaranthus

tuberculatus (syn. rudis)). Retrieved September 27, 2013, from The International Survey

of Herbicide Resistant Weeds: http://www.weedscience.org/

Knezevic, S. Z., Streibig, J. C., and Ritz, C. (2007). Utilizing R software package for dose-

response studies: theconcept and data analysis. Weed Technology, 21: 840-848.




163: 363-377.

Ohkawa, H., Tsujii, H., and Ohkawa, Y. (1999). The use of cytochrome P450 genes to introduce

herbicide tolerance in crops: a review. Pesticide Science, 55: 867-874.



50


Siminszky, B. (2006). Plant cytochrome P450-mediated herbicide metabolism. Phytochemistry

Reviews, 5: 445-458.



Tranel, P. J., Wright, T. R., and Heap, I. M. (2013). Mutations in herbicide-resistant weeds to

ALS inhibitors. Retrieved September 29, 2013, from International Survey of Herbicide

Resistant Weeds: http://www.weedscience.org/.

51

3.6 Tables and Figures

Table 3.1 Herbicide used in greenhouse dose response experiments. Recommended application rates are obtained from product labels

as used postemergence.

Trade

name

Active ingredient Chemical family Manufacture Formulation Recommended

application rate

(g a.i. per ha)

Dose response rate

(g a.i. per ha)

Beacon primisulfuron-methyl Sulfonylurea Syngenta 75WG 40 0.4 to 400

Oust sulfometuron-methyl Sulfonylurea DuPont 75WG 105 0.105 to 105

Pursuit imazethapyr Imidazolinone BASF 22.87G 65 6.5 to 6500

Arsenal imazapyr Imidazolinone BASF 26.7G 900 0.009 to 9

Staple pyrithiobac sodium Pyrimidinyl(thio)-

benzoate

DuPont 3.2L 726 1.57 to 1570

Olympus propoxycarbazone Sulfonylamino-

carbonyl-triazolinone

Bayer 70WG 55 0.174 to 174

FirstRate cloransulam-methyl Triazolopyrimidine Dow AgroSciences 84WG 36 3.6 to 3600

52

Table 3.2 ALS inhibitors used in combination with malathion. Herbicide rates chosen were sufficient based on the dose-response

experiments to eliminate all WCS plants while only causing minor injury to JG11 plants.

Trade name Active ingredient Chemical family Manufacturer Formulation Application

rate (g a.i.

per ha)

Adjuvant

Beacon primisulfuron-methyl Sulfonylurea Syngenta 75WG 1.26 1% COC and 2.5% AMS

Oust sulfometuron-methyl Sulfonylurea DuPont 75WG 1.05 1% COC and 2.5% AMS

Pursuit imazethapyr Imidazolinone BASF 22.87G 20.5 1% COC and 2.5% AMS

Staple pyrithiobac sodium Pyrimidinyl(thio)benzoate DuPont 3.2L 5 0.25% NIS

FirstRate cloransulam-methyl Triazolopyrimidine Dow

AgroSciences

84WG 0.324 1% COC and 2.5% AMS

53

Table 3.3 ANOVA results of malathion incorporated with herbicide treatment. The factors of

malathion, herbicide and biotype all had significant impact on plant growth, which was reflected

by both dry weight and adjusted dry weight.

ANOVA – Dry Weight

Estimate Std. Error t value P value

Intercept 1.05535 0.05020 21.024 <2e-16

Biotype 0.17199 0.02683 6.410 6.04e-10

Herbicide 0.63939 0.04854 13.173 <2e-16

Malathion 0.19429 0.02683 7.241 4.18e-12

ANOVA – Adjusted Dry Weight

Estimate Std. Error t value P value

Intercept 0.97812 0.05109 19.147 <2e-16

Biotype 0.11391 0.02731 4.172 4.02e-5

Herbicide 0.070496 0.04940 14.271 <2e-16

Malathion 0.16947 0.02731 6.206 1.92e-9

54

Table 3.4 Duncan’s Multiple Range Test of malathion-ALS inhibitor combination treatments.

The columns of “trt” are labeled with population and treatment combination. Following the

population of either “JG11” or “WCS”, “OO” stands for untreated control, “MO” stands for

treated with only malathion, “HO” stands for treated with only herbicide, and “HM” stands for

the combination application of both herbicide and malathion. Dry weight means with the same

letter are not significantly different. Responses of both populations under herbicide treatment

with or without malathion are highlighted in bold for comparison.

Primisulfuron

Cloransulam

Duncan's Multiple Range Test Duncan's Multiple Range Test

Duncan Grouping Mean N trt Duncan Grouping Mean N trt

A 1 12 JG11OO A 1 12 JG11OO

A 1 12 WCSOO A 1 12 WCSOO

B 0.62743 12 JG11OM B 0.72206 12 JG11OM

B 0.61042 12 WCSOM C 0.50276 12 JG11HO

C 0.30634 12 JG11HO C 0.45384 12 WCSOM

D 0.15253 12 WCSHO D 0.22604 12 JG11HM

D 0.10212 12 JG11HM D 0.11201 12 WCSHO

D 0.06123 12 WCSHM D 0.06808 12 WCSHM

Sulfometuron Pyrithiobac

Duncan's Multiple Range Test Duncan's Multiple Range Test

Duncan Grouping Mean N trt Duncan Grouping Mean N trt

A 1 12 JG11OO A 1 12 JG11OO

A 1 12 WCSOO A 1 12 WCSOO

B 0.62743 12 JG11OM B 0.62743 12 JG11OM

B 0.61042 12 WCSOM B 0.61042 12 WCSOM

C 0.2464 12 JG11HO C 0.40196 12 JG11HO

CD 0.12747 12 JG11HM D 0.12014 12 JG11HM

D 0.07279 12 WCSHO D 0.08288 12 WCSHO

D 0.03266 12 WCSHM D 0.02635 12 WCSHM

Imazethapyr

Duncan's Multiple Range Test

Duncan Grouping Mean N trt

A 1 12 JG11OO

A 1 12 WCSOO

B 0.62743 12 JG11OM

B 0.61042 12 WCSOM

B 0.59072 12 JG11HO

C 0.16335 12 JG11HM

C 0.09019 12 WCSHO

C 0.05177 12 WCSHM

55

Figure 3.1 Demonstration of dose response to ALS-inhibiting herbicides in the greenhouse. In

each set the photo on top shows the response of JG11 population and the bottom is WCS. The

plant in the far left of each photo is an untreated control, and plants to the right were treated with

increasing herbicide dosages; dosages are listed in Table 3.1.

A. Primisulfuron

Figure 3.1(cont.)

56

B. Cloransulam C. Propoxycarbazone

Figure 3.1 (cont.)

57

D. Sulfometuron E. Pyrithiobac sodium

Figure 3.1 (cont.)

58

F. Imazethapyr E. Imazapyr

59

Primisulfuron Estimate of R/S Std. Error t-value p-value

10.8266 3.7446 2.6242 0.0102

Cloransulam Estimate of R/S Std. Error t-value p-value

90.0264 43.0252 2.0692 0.0397

Figure 3.2 Dose response curves and statistical analyses. JG11/WCS values reflect the R/S ratio

corresponding to each herbicide treatment. The X-axis represents the herbicide dosage (g of a.i.

per ha) on a log-scale, and the Y-axis represents the adjusted dry weight data relative to

untreated control.

Primisulfuron (g a.i./ha)

Cloransulam (g a.i./ha)

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Figure 3.2 (cont.)

60

Propoxycarbazone Estimate of R/S Std. Error t-value p-value

3.1109 1.0172 2.0751 0.0426

Sulfometuron Estimate of R/S Std. Error t-value p-value

5.7662 1.4724 3.2369 0.0016

Pyrithiobac Sodium Estimate of R/S Std. Error t-value p-value

2.83098 0.91997 1.99026 0.0257

Propoxycarbazone (g a.i./ha)

Sulfometuron (g a.i./ha)

Pyrithiobac (g a.i./ha)

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Figure 3.2 (cont.)

61

Imazethapyr Estimate of R/S Std. Error t-value p-value

19.4032 5.9427 3.0967 0.0025

Imazapyr Estimate of R/S Std. Error t-value p-value

8.8605 3.4103 2.3050 0.0229

Imazethapyr (g a.i./ha)

Imazethapyr (g a.i./ha)

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62

Figure 3.3 Malathion-ALS inhibitor treatments. “OO” stands for untreated control. “MO” stands

for applied with only malathion. “HO” stands for applied with only herbicide. And “HM” stands

for the combined application of both herbicide and malathion.

JG11 JG11 JG11 JG11 WCS WCS WCS WCS

OO MO HO HM HO HM MO OO

JG11 WCS JG11 WCS JG11 JG11 WCS WCS

OO OO MO MO HO HM HO HM







A. Primisulfuron B. Cloransulam

C. Sulfometuron E. Pyrithiobac sodium

F. Imazethapyr

63

Figure 3.4 Photos showing multiple plants of the JG11 and WCS populations treated with

herbicide or herbicide plus malathion.

A. Primisulfuron B. Cloransulam

C. Sulfometuron E. Pyrithiobac sodium

F. Imazethapyr

JG11

Herbicide

Treatment

WCS

Herbicide

Treatment

JG11

Herbicide

+ Malathion

Treatment

WCS

Herbicide

+ Malathion

Treatment

64

Figure 3.5 Dry weight of malathion and ALS inhibitor combination treatments shown as mean with 90% confident interval. The

treatments with malathion added are labeled (+) and the one without are labeled (-).

0

0.2

0.4

0.6

0.8

1

1.2

(-) (+) (-) (+) (-) (+) (-) (+) (-) (+) (-) (+)

Control Primisulfuron Cloransulam Sulfometuron Pyrithobac Imazathapyr

Dry

wei

gh

t (r

ela

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JG11 WCS

65

CHAPTER 4

Concluding Remarks

4.1 Research Conclusions and Implications

The acetolactate synthase (ALS) enzyme, or acetohydroxyacid synthase (AHAS) enzyme

is the target site of five families of herbicides. The herbicides inhibit substrate binding to the

enzyme, and stop the biosynthesis of branched-chain amino acids (BCAA). ALS-inhibiting

herbicides have been widely used since they have been developed, but a great variety of weed

species have evolved resistance to them. As one of the most problematic weed in the Midwest

region, waterhemp is notorious for being a highly reproductive, highly competitive, and vigorous

weed species that affects crop yield greatly when not managed effectively (Steckel et al., 2003;

Steckel, 2007). The fact that waterhemp has evolved resistance to multiple herbicide modes of

action narrows the control options and makes the weedy properties of waterhemp more

challenging (Hausman et al., 2011; Patzoldt et al., 2005). Reports of waterhemp being resistant

to 6 modes of action, including ALS inhibitors, have been found across the Midwest (Heap,

2013b).

Previous studies of ALS-resistant waterhemp was mostly focused on amino acid

substitutions in ALS enzyme that leads to an insensitive target site to herbicide binding, and

mutations at two amino acids, Trp574 and Ser653, were found in waterhemp biotypes that confer

high levels of resistance (Patzoldt, et al, 2005; Patzoldt and Tranel, 2007). However, a

waterhemp population, MCR, displayed two phenotypes of resistance to ALS inhibitors in

greenhouse experiments (Chapter 1). The Trp574Leu mutation was found in all highly resistant

66

individuals but it was not found in moderately resistant plants from this population. Since MCR

has been confirmed with metabolism-based resistance to HPPD inhibitors (Hausman, et al., 2011;

Ma, et al., 2013), and enhanced metabolism is how crops tolerate ALS-inhibiting herbicides

(Fonné-Pfister et al., 1990), the hypothesis was raised that there is a non-target-site mechanism

of ALS resistance in MCR waterhemp.

To test the hypothesis, progeny JG11 of two moderately resistant waterhemp plants from

MCR was used in a series of experiments. In Chapter 2, DNA of leaf tissue of JG11 survivors of

herbicide treatment was extracted and the ALS gene was sequenced. Among the 582 codons of

ALS mature protein from 8 different individuals of JG11 plants, none of the known ALS

mutations that affect herbicide binding was found. Twenty-nine other amino acid

polymorphisms in alleles of the 8 individuals were observed, but only 5 of them appeared

consistently. Moreover, these substitutions were detected in sequences of other Amaranthus

species as well, both ALS sensitive and resistant biotypes, inferring those sites are naturally less

conserved, and are unlikely the genetic base of ALS resistance in JG11.

To further evaluate the hypothesis of non-target-site resistance, an in vitro assay was

performed to compare the activity of the ALS enzyme with or without herbicide inhibition. ALS

enzyme from JG11 plants was as sensitive to an ALS herbicide as was that of WCS, a biotype

known to be sensitive to ALS inhibitors. Collectively, results from ALS gene sequencing and the

ALS enzyme assay provide strong evidence that the whole plant resistance of JG11 is not based

on an insensitive target site.

With the confirmation of a non-target-site mechanism of ALS resistance, the actual

resistance pattern across ALS herbicide families was characterized in Chapter 3. Greenhouse

dose response experiment was conducted with 7 different chemicals on JG11 population together

67

with WCS and ACR as controls. Relative to WCS, the majority of R/S ratios ranged from 2 to 10,

except cloransulam, for which an R/S ratio over 50 was obtained. Both visual rating and biomass

data suggested JG11 is moderately resistant to ALS herbicide across all five families, and even

survives commercially suggested rates of application.

An inhibitor of cytochrome P450s, malathion, was incorporated with herbicide treatments

based on the hypothesis that the resistance could be related to enhanced detoxification, which is

similar to the mechanism conferring resistance to HPPD inhibitors in MCR. The experiments

were performed in the greenhouse with five different ALS inhibitors at sub-lethal dosages to

JG11. The addition of malathion significantly increased herbicide activity, to the extent that

biomass was not significantly different from that obtained for WCS receiving the same treatment.

The results suggest detoxification by cytochrome P450s plays an important role in the non-

target-site ALS resistance in JG11. The presence of non-target-site ALS resistance could have

significant impacts on herbicide cross resistance and on weed management for this species.

Despite the fact that there previously was no known metabolism-based resistance of

waterhemp to ALS inhibitors, and the main focus of ALS resistance in waterhemp has been on

gene mutation, the nature of waterhemp’s high reproduction rate and great genetic variability

makes the selection of alternate resistance mechanisms inevitable. In the previous chapters the

hypothesis of a metabolism-based resistance mechanism is supported in the JG11 population and,

by extension, in the original HPPD-resistant MCR population as well. The metabolism-based

resistance in waterhemp or weeds in general impacts weed management in three major aspects.

First of all, the resistant traits are not necessarily selected by ALS inhibitors, but by herbicides of

other modes of action as well, and may confer unpredictable cross resistance patterns. In addition,

the possibility of some ALS mutated resistant populations to possess non-target-site resistance

68

mechanism has not been erased, meaning those populations may not be susceptible to HPPD

inhibitors or other herbicide modes of action. Secondly, comparing to mutated resistant

populations, metabolism-based resistant populations are more sensitive, but still are able to

survive and reproduce after treatment with typical rates of ALS inhibitors. Thirdly, the approach

of reducing metabolism in resistant weeds with cytochrome P450s inhibitors would potentially

damage crops that tolerate herbicide treatments through metabolism.

Future research directions suggested by my findings include further physiological studies

to investigate metabolism, and genetic studies. For instance, although I have hypothesized that

cytochrome P450s are involved in ALS-inhibitor detoxification, due to the complexity of

cytochrome P450s and hydroxylation process, more research is needed (Schuler, 1996).

Radiolabeled herbicide application and liquid scintillation spectrometry can be used. To

investigate herbicide uptake and translocation, radiolabeled herbicide could be applied to young

leaves of JG11 and WCS waterhemp plants, and the radioactive residue in plant tissue can be

quantified and compared at various time points after application. The results would reveal

whether the two populations have significant difference in herbicide uptake and translocation.

Whole plant application with radiolabeled herbicide can be performed on JG11, WCS, and a crop

with enhanced metabolism to detoxify ALS inhibitor, wheat, for example. HPLC could then be

used to identify and quantify the amount of parent herbicide and metabolites as evidence of

enhanced metabolism and confirm the resistance mechanism.

Meanwhile, the genetics and inheritance of the non-target-site ALS resistant trait and its

co-segregation pattern with resistance to HPPD inhibition should be investigated. F1 populations

of JG11 x WCS have been made and can be used as material in future dose response study and

co-segregation study. JG11 plants were selected from the survivors under ALS-inhibiting

69

herbicide treatment applied at 10 to 12 cm high. Herbicide treatment injured and stunted the

plants but the majority recovered and started flowering around 14 DAT. WCS was grown in the

greenhouse with proper moisture and fertilizer supply without any herbicide treatment. When the

inflorescence was formed and was able to be identified, the JG11 males and WCS females were

chosen and put in a pollination bag supported by PVC pipe frame to isolate from the rest of the

plants. The waterhemp plants for crossing were kept in the pollination bag for at least 21 days,

and the female plants baring seeds were harvested and dried in paper bags at room temperature

for another 7 days before cleaning and stratification. The seed lines are described in Table 4.1.

Further crosses to obtain F2 and backcrosses can be created and utilized as well. WCS,

JG11 and progenies of F1, F2 and BC can be screened with different doses of ALS inhibitors and

HPPD inhibitors, and generate dose response curves with biomass data using the function

described in Chapter 3, to characterize the resistance of future progenies. Also, a two-run

screening with ALS and HPPD inhibitor, in comparison with single herbicide treatments and

untreated control can help determine the co-segregation pattern.


Fonné-Pfister, R., Gaudin, J., Kreuz, K., Ramsteiner, K., and Ebert, E. (1990). Hydroxylation of

primisulfuron by an inducible cytochrome P450-dependent monooxygenase system from

maize. Pesticide Biochemistry Physiology, 37: 165-173.

Hausman, N. E., Singh, S., Tranel, P. J., Riechers, D. E., Kaundun, S. S., Polge, N. D., Thomas,D.

A., and Hager, A. G. (2011). Resistance to HPPD-inhibiting herbicides in a population of

waterhemp (Amaranthus tuberculatus) from Illinois, United States. Pest Management

Science, 67: 258-261.

70







163: 363-377.





30-36.

Schuler, M. A. (1996) Plant Cytochrome P450 Monooxygenases, Cr. Rev. Plant Science 15:

235-284

Steckel, L. E. (2007). The dioecious Amaranthus spp.: here to stay. Weed Technology, 21: 567-

570.

Steckel, L. E., Sprague, C. L., Hager, A. G., Simmons, F. W., and Bollero, G. A. (2003). Effects

of shading on common waterhemp (Amaranthus rudis) growth and development. Weed

Science, 51: 898-903.

71

4.3 Tables

Table 4.1 Seed lot chart of F1 and JG11 crosses.

No. Date of Collection Parents

JG13 8/5/2013 Female JG11*Male JG11







JG20 10/31/2013 Female WCS*Male JG11



JG23 1/23/2014 Female JG11*2 Males JG11




JG27 1/23/2014 Female WCS*2 Males JG11

JG28 1/23/2014 Female WCS*2 Males JG11

JG29 1/23/2014 Female WCS*2Males JG11

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NON-TARGET-SITE RESISTANCE TO ALS INHIBITORS IN …