plant physiology preview. published on august 7, 2014, as doi

Herbicides state of the art. I. Overview

Hansjoerg Kraehmer

Kantstrasse 20, D-65719 Hofheim Germany

[email protected]

+49 6192 296560

Plant Physiology Preview. Published on August 7, 2014, as DOI:10.1104/pp.114.241901

Copyright 2014 by the American Society of Plant Biologists

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Herbicides as weed control agents – state of the art. I. Weed control research and safener technology: the path to modern agriculture

*Hansjoerg Kraehmer, Bernd Laber, Chris Rosinger, Arno Schulz

Bayer CropScience AG, Industriepark Hoechst, Building H 872, D-65926 Frankfurt am Main

An overview on the development of weed control and safener history and of screening tools to find them over the past 100 years is presented.



ABSTRACT

The purpose of modern industrial herbicides is to control weeds. The species of weeds that plague crops today are a consequence of the historical past, being related to the history of the evolution of crops and farming practices. Chemical weed control began over a century ago with inorganic compounds and transitioned to the age of organic herbicides. Targeted herbicide research has created a steady stream of successful products. However, safeners have proven to be more difficult to find. Once discovered, it became important to determine the mode of action, partly to help the discovery of further compounds within the same class. However, mounting regulatory and economic pressure has changed the industry completely, making it harder to find a successful herbicide. Herbicide resistance has also become an increasing problem and increased the difficulty of controlling weeds. As a result, the development of new molecules has become a rare event today.



Introduction and historical background of herbicide research

Modern industrial herbicide research begins with the analysis and definition of research objectives. A major part of

this lies in the definition of economically important weeds in major arable crops (Kraehmer 2012). Weed associations

change slowly over time. It is therefore important to foresee such changes. Today’s weed associations result from

events in the distant past. They are associated with the history of crops and the evolution of farm management. In

Europe and the Americas, some large-acre crops such as oilseed rape, canola and soybean have attained their current

importance only within the last hundred years. Other “Old world” crops such as cereals have expanded over a very

long time span and were already rather widespread in Neolithic times (Zohary et al. 2012). The dominance of crop

species in agricultural habitats only left room for weed species which could adapt to cultivation technologies. Changes

in crop management and the global weed infestation have happened in waves. A major early factor in Europe was

presumably the grain trade in the Roman period (Erdkamp 2005). The Romans spread their preferred crops and,

unintentionally, associated weed seeds throughout Europe, Asia and Africa. A second wave of global vegetation

change started in the sixteenth century after the discovery of the Americas. Crops and weeds were distributed globally

by agronomists and botanists. Alien species started to spread on all continents. A third phase can be seen in the

nineteenth century with the industrialization of agriculture and with the breeding of competitive crop varieties. The

analysis of weed spectra in arable fields starts from this historical background. Weeds are plants interfering with the

interests of people (Kraehmer and Baur 2013), which is why they have been controlled by farmers for millennia.

Chemical weed control began just about a century ago with a few inorganic compounds such as sulphuric acid, copper

salts and sodium chlorate (Cremlyn 1991). The herbicidal activity of 2.4 D (2,4-dichlorophenoxyacetic acid) was

detected in the forties of the last century (Troyer 2001). Table 1 provides an overview on selected chemical families,

selected representatives and earliest usage reports according to Cremlyn (1991), Worthington and Hance (1991) and

Büchel et al. (1977). Targeted herbicide research began in the fifties of the last century. In the early days, herbicide

candidates were progressed from screens purely on the basis of their having biology that would satisfy farmers’

requirements. Mode of action (MoA) studies did not play a major role in the chemical industry prior to the seventies of

the last century. The development of analytical tools and the rapid elucidation of plant pathways and in vitro-based

screen assays were used from the eighties onward. However in the nineties and beyond, ever increasing regulatory and

economic pressures has changed the situation of the industry completely and to satisfy the new requirements, selection

criteria beyond biological activity have needed to be applied. Consequently, regulatory and economic pressure have

fundamentally changed the situation of the industry. Selection criteria beyond biological activity have had to be

applied. Herbicide resistance in weeds has developed into a more serious problem that now constrains the application

of certain types of herbicides in some markets. Finally, the introduction of crops resistant to cheap herbicides and of

glyphosate-resistant soybean in particular, took value out of the market and resulted in an enormous economic pressure

on the herbicide-producing industry. As a result of this changing and more difficult landscape, the development of new

molecules is now a rare event.

The present article is structured into three main topics. First, it provides an historic overview on the development of

weed control history and of screening tools over the past 100 years. Thereafter, we will concentrate on the use of MoA

studies as a tool for optimizing chemical structures based upon knowledge of their receptors. Finally we review the

invention and use of safener technologies as a tool for improving the crop selectivity of herbicides. In a second

companion review entitled “Herbicides as weed control agents – state of the art II. Recent achievements” (Kraehmer,

et al. 2014), we address the serious challenges that farmers now face due to the evolution of herbicide resistance in

weeds and the types of innovations that are urgently required.



Agricultural changes in the past and their influence on weed infestation

Crops grown on arable land within the last 100 years, weed management and changes in weed infestation

Crop management practices have had a major impact on weed infestation. Animals such as horses were used as

production tools in the beginning of the last century. The horses required feeding and as a result, for example, in 1920

oats were grown on more than 40 million acres in the USA (area harvested); today the acreage amounts to only 1

million acres. It is not surprising therefore that wild oats, which were nearly impossible to control in the past, spread so

quickly over many continents.

Table 2 illustrates a few other striking facts: corn was already planted on 101 million US acres in 1920 and on around

97 million acres in 2012. In sharp contrast, soybeans were cultivated on less than 500 000 acres in 1920 but on 76

million acres in the year 2012.

One very effective weed management tool is tillage. Mechanical weed management is, however, time consuming,

labor intensive and leads to a high energy consumption. It has also regrettably resulted in major erosion problems all

over the world (Montgomery 2007). Erosion rates on US arable land declined considerably between 1982 and 1987:

From 3.06 billion tons down to 1.73 billion tons or from 7.3 tons per acre to 4.8 tons per acre (NRCS – 2007 National

Resources Inventory; http://www.nrcs.usda.gov/Internet/FSE_DOCUMENTS/nrcs143_012269.pdf). Changes in

tillage practices were partly responsible for this achievement.

Weed infestation in spring crops and winter crops can differ considerably (for example Håkanson, 2003). The

profound reduction of spring crops, and especially the reduction of the oat acreage when tractors replaced horses,

resulted in a complete shift from wild oats ( Avena fatua L.) as a major grass weed to black-grass (Alopecurus

myosuroides Huds. ) and silky bent grass ( Apera spica-venti (L.) P. Beauv. in Europe (Kraehmer and Stuebler 2012).

The hard winters in Canada prevent the cultivation of winter crops in many agricultural areas. This is why winter

annuals such as black-grass do not play a major role there. Canada has a long tradition of conducting weed surveys.

Despite major changes in the use of agrochemicals, the most dominant weeds have remained the same for decades (see

for example Leeson et al. 2005). The Weed Mapping Working Group of the European Weed Science Society

endeavors to map the most common weeds of Europe in all major crops and to document changes in weed infestations

(http://www.ewrs.org/weed_mapping.asp). One obvious result is that weed spectra change with cropping practices and

environmental conditions and that some species are better adapted to the warmer climates in the Mediterranean area

such as Abutilon theophrasti Medicus or Sorghum halepense (L.) Pers., whereas others are more frequent in northern

Europe such as Alopecurus myosuroides Huds. or Apera spica-venti (L.) P. Beauv. The weed species Chenopodium

album L. and Echinochloa crus-galli (L.) Beauv. are characterized by a wide ecological aptitude (Kraehmer 2010) and

can be found everywhere in Europe and in many crops.

Biological screening

Glasshouse and field screening

In the early days, indicator species or model plants played a major role in herbicide discovery screening. Weed target

species were often not available. Most herbicide screening in the agrochemical industry (Fig. 1) between the fifties and

eighties of the last century was characterized by a protocol where solutions of test chemicals were sprayed over sets of

plants in pots, or over seeded bare soil in the case of pre-emergent screens.

The screening compounds were either pre-dissolved in solvents such as acetone or they were formulated as wettable

powders, emulsion concentrates or suspension concentrates before dilution in water. The spraying process simulated



the actual spraying situation in the field. Every company had its own, specially designed spraying equipment. The

screening process was divided into two to four initial selection steps. The initial, or plus-minus activity, tests started

with high dose rates, generally between 1 and 10 kg a.i./ha (active ingredient/ha). Compounds were sprayed in pre-

and post-emergence trials. Later steps with lower dose rates and varying plant species followed to further refine

activity and weed spectrum. The results of these trials were usually visual phytotoxicity ratings on different scales,

such as on a 0 to 100 % rating scale, 0 meaning no phytotoxicity, 100 % equaled complete control of plants. Fresh or

dry weights were used for comparisons in special tests only. Symptomology was an essential and integral part of

ratings. Each chemical class of compounds usually resulted in typical patterns of symptoms. Observations made in

different screening indications (for example, insecticide, acaricide, nematicide or fungicide screens) were also taken

into account during evaluation (Figures 2 and 3).

Chemists usually prepared sample amounts of 3 to 5 grams. This amount allowed the early testing of chemicals in all

screening indications. Most mid-size and large companies tested between 1000 and 5000 compounds per year. IT tools

suitable to facilitate screening were not available before the eighties of the last century and structure-activity

relationships had to be derived manually in long and time-consuming procedures. The last glasshouse step was a

profiling procedure in which recommendations for field testing were derived. Often, special tests were carried out

before or in parallel to field tests to check the soil-dependent performance of a compound, the influence of different

formulations, potential carry-over risks and crop selectivity ranges. Depending on company size and resources,

between 10 and 100 compounds were advanced into field testing per year. Compounds were tested in different parts of

the world on plots between 1m² and 10m² with three to four replicates.

Two to three years in the field were usually sufficient to make informed development decisions. In the third quarter of

the last century, it usually took between four to six years from the first synthesis of a compound until entry into the

market in the third quarter of the last century. Later, the development process became more involved and took a greater

investment of time to develop a new herbicide, as we will show later.

Early mode of action studies

The number of modes of action was quite limited between 1950 and 1970. Most commercial products were

characterized as auxin-type herbicides, PS I-inhibitors or PS II-inhibitors or inhibitors of cell division. During the

seventies, inhibition of photosynthesis was typically tested at the whole plant level using Infrared Gas Analyzers in

growth chambers. At the biochemical level the so-called Hill-reaction served as a tool for the identification of

photosynthesis inhibitors in isolated chloroplasts (see for example Arndt and Kötter 1968). In the eighties, Clark-

electrode measurements with isolated leaf cells and fluorescence emission assays with whole leaves provided further

non-destructive tools for the characterization of PS II herbicides (Voss et al. 1984 a+b).

Plant tissue cultures coupled with HPLC analysis have been used since the seventies to characterize herbicide-induced

changes in metabolism. Cultures of aquatic plants such as Lemna minor have also long been used as an herbicide test

system. Perennial weeds sometimes required a different approach. Systemic action of herbicides is required for the

control of perennials such as couch (Elytrigia repens (L.) Gould), Johnsongrass (Sorghum halepense (L.) Pers.) or

field bindweed (Convolvulus arvensis L.). Special translocation tests were used to check for this property. For

example, Phaseolus beans with two leaves provided some indication of systemic action when one of the leaves was

treated. Translocated compounds then led to symptoms in the untreated leaf.



From the eighties onwards, special biochemical assays were developed for the characterization of target sites and for

binding studies. Target enzymes of plant-specific pathways were preferred in order to avoid toxicological problems in

mammals. We will come to this period in one of the following paragraphs.

Miniaturized screening assays

A perennial problem faced by the agrochemical industry has been the question of where to obtain new compounds in

amounts sufficient for screening. At the end of the last century, an intensive exchange of compounds between non-

agrochemical and agrochemical origins began. Universities and private institutes offered agrochemical companies their

stocks and undertook the synthesis of additional desired compounds. Natural products were available for testing also.

Since most third-party compounds were provided in low amounts, it drove the adoption of miniaturized herbicide

discovery assays. These assays provide a basic initial test for phytotoxicity but give only limited discriminatory

information to guide compound optimization. Seed germination assays in Petri-dishes were also quite commonly used.

The first purpose of these assays was to find hits which would justify the synthesis of analogues and of samples with

larger substance amounts. Tissue culture tests as described by Gressel et al. (1993) were also employed in a number of

companies. Some companies have kept and improved such miniaturized assays until today, for example, as published

by Grossmann et al. (2012). Several assays were incorporated into high-throughput screening systems which allow the

screening of one million compounds per year and more (Kraehmer 2012). We will touch upon this screening approach

in one of the following chapters.

Glasshouse to field transfer

One disadvantage of early screening sets of the last century was the use of model plants such as onions, carrots,

potatoes and others. They were employed because the seed of target weeds was not available in the early herbicide

screens. Today, specialized providers can deliver high quality weed seed in guaranteed quantities. Nevertheless, even

given the use of real target weeds, laboratory and glasshouse data are still not usually fully predictive of the

performance of compounds in the field. Many parameters contribute to the variable transfer factors from glasshouse to

field (Kraehmer and Russell 1994). One of these parameters is the test species. Model plants such as Lemna species,

Arabidopsis thaliana (L.) Heynh. or Brachypodium distachyon P. Beauv. differ from weeds in many ways including

their specific uptake and translocation properties, metabolism, their specific life cycle and environmental requirements

for growth - Lemna species are aquatic weeds in contrast to terrestrial weeds. Arabidopsis thaliana and Brachypodium

distachyon are not serious competitors within arable crops under agricultural conditions. The same rules apply to many

other species in miniaturized assays. General phytotoxicity principles can only rarely be used for the optimization of

chemical structures or for structure-activity relationships. Too many positive results in an indicator assay often require

repeated screening steps to reduce their numbers. Certain types of herbicides may not be detected at all if the indicator

species is too different from the screen model species. For example, ACCase-inhibiting graminicides would not have

been detected by screening against Arabidopsis. Similarly, the potential of a compound to control perennial weeds will

not be evident from screens that only include annual species and information about soil-plant interactions is absent

from screens based upon liquid systems that do not include soil. The potential of a compound to control perennial

weeds will often not be detected with annual species. Plant cells grown in tissue cultures are very different from whole

plant cells because they are undifferentiated and can derive nutrients, often present in excess, from the culture medium

and are buffered such that the influence of pH values on the uptake of compounds is masked.The actual value of a new

compound is always based upon its performance against economically important weed species. It is highly

questionable if all compounds controlling Lemna, Arabidopsis and Brachypodium will, for example, automatically



control herbicide resistant Amaranthus species. This is presumably why cells or tissues of target species have now

found their way into physiological profiling assays (see for example Grossmann et al. 2012).

Environmental fate of agrochemicals

Advances in the sensitivity of analytical tools in the seventies and eighties allowed the detection of trace levels of

agrochemicals in groundwater, and through such analyses some chemicals proved to be persistent in the environment

(Kraehmer 2012). This has all led to a tightening of regulatory requirements and an increased need for agrochemical

companies to test and characterize degradation rates of compounds in soil and water under aerobic and anaerobic

conditions. The physicochemical parameters such as vapor pressure and the octanol-water partition coefficients of new

compounds are now measured routinely and increasingly sophisticated toxicology tests are carried out before field

testing.

How has chemical weed control changed agriculture so far?

Herbicide innovations have appeared in waves (Stuebler et al. 2007). The early auxin-type herbicides primarily

controlled dicot weeds. Later on, monocots could be controlled with the advent of photosynthesis-inhibitors, cell

division inhibitors and very long chain fatty acid biosynthesis inhibitors (VLCFA-inhibitors). Yields were

considerably increased in all crops between 1940 and 2010 (Table 3). Many factors contributed to this increase, one is

definitely breeding but herbicides have also had a major impact. The value of herbicides was shown by Zimdahl

(2004) who published data showing the influence of defined weed species on the yield of different crops.

Herbicides made it possible to control weeds in crops much more easily than before. Before the advent of herbicides,

controlling weeds required hard physical labor such as hoeing. A particular example is weed control in sugar beet

where high labor costs meant that farmers rapidly adopted herbicide technology once it became available. Another

crop that could be cultivated more easily with the invention of new herbicides was oilseed rape. Broad spectrum

products such as paraquat and atrazine accelerated the use of reduced-tillage measures in agriculture and helped

prevent soil erosion, which had become a major issue especially in the USA during the first half of the last century, as

mentioned above. Selective post-emergence grass control in some crops came in the mid-seventies. The ACCase-

inhibitors selectively controlled grass weeds in post-emergence treatments and the use of safeners even made their

application in cereals possible. Glyphosate is a unique molecule with very specific properties. It allowed the farmer to

kill weeds and to plant new seed within a few days after its application (similar to paraquat). It killed perennials and

had a short soil half-life. Therefore, it was also regarded as one of the most effective products in plantation crops.

Glufosinate was another non-selective molecule with a new MoA. It was strong against a few hard-to-control weeds

such as Equisetum species and it appeared to be a bit safer to plantation crops than glyphosate. The introduction of

ALS-inhibitors drastically reduced the total amounts of agrochemicals applied per hectare. Most of them could be

applied with a few grams per hectare, whereas many older chemicals required amounts in the kilogram range. Finally,

HPPD- and phytoene-desaturase-inhibitors were ideal mixture partners for existing products and closed some evident

gaps. No major new MoA has been found by the industry since the eighties of the last century. As early as 1990,

agrochemical market research indicated that the crop protection market was approaching maturity and that it was

becoming increasingly difficult to discover new agrochemicals with significant advantages over existing products

(Kraehmer and Drexler 2009). The average total costs for the development of an herbicide had increased from 50

million US$ to 250 million US$ between the years 1975 and 1995 (Rüegg et al. 2007). GM (genetically modified)

crops entered the market in the second half of the nineties. The high standard of existing products in the market and the

high registration costs caused many chemical companies to give up their agrochemical business. Following numerous



mergers and acquisitions only a few companies with herbicide research capacity remained. The number of companies

devoted to herbicide discovery was reduced from more than 40 in the 1970s to 5 to 8 today. It is highly questionable,

therefore, if farmers will experience many new innovations within the years to come and yet there is an urgent need for

new weed control solutions. Several cropping systems in the Americas as well as in Europe are no longer sustainable

without further herbicide innovations, as we will see in our final chapters.

Modes of action and herbicide diversity

There are several excellent reviews and books on the mode of action of herbicides (Dayan et al, 2010; Krämer at al,

2012; Liu et al, 2009; Seitz et al, 2003), therefore, there is no need to repeat what has been published recently. Instead,

it is intended to give a short overview of previous and current trends in MoA research and the factors that affected the

search for herbicides with novel MoAs.

Between 1960 and 1970 only one herbicide with a novel MoA, asulam, an inhibitor of dihydropteroate synthase, was

discovered. In the early 1970´s this period of low innovation with respect to herbicides with novel MoAs was

superseded by what in retrospect might be called the golden years of herbicide discovery. From 1971 to 1985

herbicides with eight novel MoAs were discovered, among them inhibitors of amino acid biosynthesis (glyphosate,

glufosinate, acetolactate synthase, or ALS, inhibitors), lipid biosynthesis (ACCase) inhibitors and inhibitors of pigment

biosynthesis (PDS and HPPD inhibitors), MoAs which still today dominate the herbicide market (Fig. 4 and 5).

These herbicides not only had a profound impact on weed control in agriculture, but also played a major role in

expanding the understanding of fundamental plant processes through their ingenious use as molecular probes (Dayan

et al, 2010). Many academic groups in the US as well as in Europe embarked on this path, either studying the MoA of

herbicides or using herbicides to study plant metabolism.

From the mid-1980s until today more than 140 new herbicide active ingredients have been commercialized (Gerwick,

2010). Surprisingly, only two of these, the narrow-spectrum herbicides cinmethylin and oxaciclomefone, have an

unknown and still not fully understood molecular MoA despite publications describing biochemical effects attributed

to these compounds. All the other new active ingredients target old (known) MoAs. The reasons for this lack of

novelty have been reviewed recently and have been attributed to a number of mostly economic factors (Duke, 2012;

Kraehmer et al., 2007). Two such factors are briefly discussed in the following paragraphs.

Due to their very low application rates in particular, ALS inhibitors easily outcompeted other chemical classes in the

herbicide screening programs and drew significant synthesis capacity away from them. In addition to ALS inhibitors,

significant synthesis capacity was directed towards one other chemically productive herbicidal MoA, PPO inhibitors

which controlled weeds with very low application rates. PPO inhibitors had a very broad chemical scope too, but their

commercial success was limited. The fast action of these herbicides prevented systemic action, selectivity for in-crop

use was lacking and the toxicological profile was often problematic.

Even though no major new MoA has been introduced into the market in the last 30 years, herbicide discovery has not

come to a standstill. At least 14 target sites for herbicidal compounds have been discovered during this time period

(Fig.4). However, no compound interfering with any of these targets has made it to the market. Reasons for the lack of

commercialization are diverse and include high application rates, incomplete weed spectrum, high costs of production

or a combination of any of these factors. Furthermore, limited scope for the chemical variation of the inhibitors for



some of these targets, i.e. KARI, AMPDA, and AdSS, hampered their optimization towards high bioefficacy and low

field application rates.

The traditional method to discover a novel herbicide was by random screening of large numbers of synthetic

compounds in the greenhouse. Based on this approach, at least one commercial product targeting each of today’s

marketed MoAs was introduced before its MoA was elucidated. This random discovery approach was challenged in

the mid-1990s by the introduction of novel research technologies in the pharmaceutical industry. Most big

pharmaceutical companies had started to pick up molecular targets such as enzymes, receptors, etc., which became

accessible by the genomic revolution and a deeper understanding of the molecular mechanisms of disease.

Pharmaceutical companies tried to identify efficient inhibitors of these targets by using high-throughput in vitro

screening. Progress in organic chemistry facilitated this approach, since combinatorial chemistry made it possible to

prepare tens to thousands or even millions of compounds within a short time period. Most agrochemical companies

also followed this strategy, especially after the full genome of Arabidopsis thaliana had been published in 2000.

Henceforward it became possible to “validate” putative herbicidal targets by genetic technologies on a large scale. The

analysis of the phenotype of a plant in which a certain gene had been knocked-out completely or the expression had

been reduced to a significant extent by antisense RNA enabled the identification of so-called lethal targets. These

targets, mainly enzymes, were subsequently subjected to high-throughput in vitro screening of chemical libraries to

identify inhibitors. However, the transmission of the in vitro activity of compounds discovered this way into the

greenhouse turned out to be an insurmountable hurdle. Therefore, not only have no herbicides with a novel MoA been

discovered but also, to the authors’ knowledge, no compound with broad herbicidal activity at low application rates

has been identified in this way.

In the past few years, the focus of herbicide research has once again shifted back towards random screening of

synthetic compounds in the greenhouse and to the use of hits as starting point for targeted optimization processes. If a

phytotoxic compound with interesting bioefficacy is identified, studies are undertaken to find out the underlying MoA

as soon as possible and to use this MoA information in rational chemical design approaches. Technological advances

in molecular biology, such as gene expression profiling (transcriptomics) have also been adopted for this task (Eckes

& Busch, 2007). When a plant is treated with an herbicide, vital processes of that plant are affected. This is reflected

by distinctive, MoA-dependent changes in the transcriptome. By comparing the transcriptome of a plant treated with a

phytotoxic compound from the research pipeline to a library of response profiles to compounds with known MoAs, it

may be possible to classify this compound into one of the known MoAs. If the compound cannot be classified into an

already existing MoA, it can be assumed that it has a new MoA, for which the profile might also provide clues.

The lack of herbicide innovation has triggered novel attempts to fight herbicide resistant weed species. Monsanto is

investigating the use of topically applied RNA molecules to induce a process called RNAi in glyphosate-resistant

weeds in order to counteract resistance to glyphosate. It remains to be seen if this approach will be successful.

Countries which do not accept GM crops must rely solely on the chemical industry to deliver novel herbicides with

novel MoAs. To achieve this goal, previously discovered but not commercialized targets might be reconsidered in the

light of new technological advances in drug discovery in the last 20 years, such as fragment (ligand) based drug

design. Alternatively, hits from greenhouse screening might be subjected to MoA elucidation using state-of-the-art

´omics technologies (Grossmann et al., 2011; Tresch, 2013) in order to identify novel starting points for herbicide

discovery.

Herbicide selectivity via safeners



For chemical weed control in fields of crops the herbicide products that can be used must fulfil two contradictory

objectives: control the weed plants but not injure the crop plants. Some herbicides provide these features innately (e.g.

atrazine for weed control in corn). However, as a general rule, when herbicides are highly active against a wider range

of weeds, the chances are much lower that they will also be highly crop selective. Crop sensitivity is itself a complex

issue being influenced by many features such as crop variety, application timing, soil properties or weather conditions.

Therefore, some herbicides may be selective in some cases but not in others. Other herbicides may be selective in

certain crops but not in others. For this reason methods have been developed to increase crop selectivity. The two key

selectivity technologies are herbicide tolerance traits (either from mutant selection or genetic modification) and

safeners. Safeners (sometimes called antidotes or protectants) are chemicals that prevent herbicidal injury to crop

plants without reducing weed control.

Safener commercialization

Based on an accidental observation in 1947 by Otto Hoffmann of the Gulf Oil Company, the concept that certain

chemicals could increase the tolerance of plants to herbicides was born (Hoffmann 1953). Hoffmann had seen that

tomato plants treated with 2,4,6-trichlorophenoxyacetic acid appeared to be protected from 2,4-D vapor injury.

Whether or not this was the first time an herbicide researcher had seen such effects is not known. However, in this case

Hoffmann realized that this may provide a useful tool to increase crop tolerance to herbicides. Gulf Oil initiated a

research program which eventually led to the first commercial compound, 1,8-naphthalic anhydride [NA]. When

applied as a seed treatment to corn, this compound gave protection against various pre-emergence and pre-plant

incorporated herbicides from the thiocarbamate class (e.g. EPTC). Subsequent research in various agrochemical

companies has subsequently provided nearly 20 commercial safeners (Hatzios and Hoagland 1989, Davies and Caseley

1999, Davies2001, Rosinger et al 2012, Jablonkai 2013).

The early phase of safener commercialization during the 1970s and early 1980s was dominated by seed treatment or

soil active (pre-emergence) safeners (Figures 6 and 7). Seed treatment applied safeners offer certain advantages and

disadvantages. A key advantage is that the safening only influences the crop plant and not the weeds. Therefore the

safener does not need to be crop specific per se. However, a key disadvantage is that the treated crop may be sprayed

subsequently with herbicides from other competitor companies making value capture difficult. Conversely, pre-

emergence tank-mix or co-formulated safeners must be innately crop specific (i.e. with no antagonism on weeds) but

offer significantly simplified safener technology for the farmer and manufacturer. The product could be used just as if

the herbicide were itself selective. The manufacturer has better control of the performance of the product containing

their herbicides.

In the late 1980s a significant innovation leap occurred, whereby “leaf-active” safeners were developed for co-

formulation with post-emergence herbicides. Fenoxaprop-ethyl was launched in 1984. This molecule controlled

grasses with post-emergence selectively in broad-leaved crops, but it lacked sufficient selectivity in cereals; being

moderately damaging to wheat and more severely damaging to barley. Therefore a research project with the aim of a

safener for post-emergence use with fenoxaprop-ethyl in cereals was started in the 1980s. A new class of substituted

triazole safeners was found from which fenchlorazole-ethyl was developed and introduced in 1992 as a post-

emergence co-formulation product with fenoxaprop-ethyl (Bieringer et al, 1989). This product meant that fenoxaprop-

ethyl could now be used for grass weed control in wheat and rye. However, barley was not sufficiently safened by

fenchlorazole-ethyl so testing for further improved safeners was continued. Relatively quickly a new class of safeners

was identified with a modified central chemical scaffold (pyrazoline) from which mefenpyr-diethyl was developed

(Hacker et al 2000). This compound had a much stronger safening activity in cereals than fenchlorazole-ethyl making



it possible to also use fenoxaprop-ethyl in barley. Amongst other ACCase inhibitors was clodinafop-propargyl which

was launched in 1991. Like fenoxaprop, clodinafop had strong grass weed activity but lacked sufficient crop tolerance

in cereals. Ciba-Geigy did not launch clodinafop without a safener. They used the highly effective safener,

cloquintocet-mexyl, which has a quite different (quinoline) scaffold from fenchlorazole or mefenpyr (Amrein 1989).

The structures of the above mentioned ACCase inhibitors and safeners are shown in Figure 8.

The safener innovation required to overcome the cereal injury meant that by the end of the 1990s, mefenpyr-diethyl

and cloquintocet-mexyl were well established in the cereal market and the two safeners could respectively be tested

with new research compounds. At the end of the 1990s iodosulfuron and mesosulfuron were introduced for use in in

cereals (Hacker et al 1999, Hacker et al, 2001). Both had selectivity limitations which could be overcome by using

mefenpyr-diethyl. Cloquintocet-mexyl was also used as a safener for pinoxaden; a herbicide from the novel aryl-1,3-

dione class of ACCase inhibitors (Hofer et al 2006). More recently, the safening of mefenypr-diethyl in cereals was

used as the selectivity technology for the HPPD inhibitor pyrasulfotole (Schmitt et al, 2008) and the ALS inhibitor

thiencarbazone. Since the basic patents for mefenypr and cloquintocet have now expired, other companies have used

them in their own safened products. Of particular note is a co-formulation of the ALS inhibitor pyroxsulam with

cloquintocet as a safener (Wells 2008).

During the 1990s and 2000s safener research was continued. One target was the extension of the fenoxaprop market

into rice which was not tolerant to this herbicide. Screening efforts identified the safener isoxadifen-ethyl (Figure 9)

which again retained the carboxylic acid ester of fenchlorazole-ethyl and mefenpyr-diethyl, but with a new

heterocyclic isoxazole core. Although fenoxaprop in rice was the original target, early on in the research and

development phase it was found that isoxadifen-ethyl could also safen post-emergence against the sulfonylurea

herbicide ethoxysulfuron in rice and foramsulfuron and iodosulfuron in corn (Collins et al 2001, Pallett et al 2001

Hacker et al, 2002). Therefore, isoxadifen now represents the current pinnacle of multi-crop and multi-herbicide

safening.

The most recently launched safener is cyprosulfamide (Figure 9). Like isoxadifen-ethyl it is strongly active post-

emergence in corn but has the significant advantage, for certain herbicides, of also having strong pre-emergence

safening activity. Cyprosulfamide comes from a class of safeners (acyl sulfonamides) discovered during the mid-

1990s. It is now used to support a significant number of different corn herbicides (Philbrook and Santel 2008, Santel

and Philbrook 2008, Watteyne et al 2009).

Safener Mode of Action

Theoretically safeners could increase crop tolerance in two main ways. They may reduce the amount of herbicide

reaching the active site or interfere with the herbicide interaction at the target site. Both of these possibilities have

further subdivisions (discussed below) and the MoA of safeners has been extensively investigated and reviewed

(Davies and Caseley, 1999, Riechers et al, 2010; Rosinger et al, 2012).

Several studies found no significant interaction between safener and herbicide at the target site (Köcher et al 1989,

Polge et al 1987). However, Walton and Casida (1995) reported competitive binding of the dichloroacetamide safener

R-29148 and the herbicides EPTC or alachlor at a protein in maize extracts. This, together with good correlation

between competitive binding of dichloroacetamide compounds and their safening activity, was taken as support that

some safeners act as receptor antagonists for the herbicides. Another mode of action might be the safener causing the

crop to increase the activity of the herbicide target. Rubin and Casida (1985) found that the safener dichlormid caused

an increase in ALS activity in maize. Milhomme and Bastide (1990) and Milhomme et al (1991) also found an



increase of ALS levels in maize treated with the safeners NA and oxabetrinil. However, Barrett (1989) could not find

any enhancement of ALS activity in maize and sorghum seedlings after treatment with the safeners NA, oxabetrinil,

flurazole or dichlormid. It is now felt that interactions between the safener and herbicide at the target site play, if

anything, only a small role in safener MoA.

Regarding possible effects of safeners on herbicide uptake, Davies and Caseley (1999) extensively reviewed the

literature for herbicide/safener combinations and concluded that most cases showed no reduction in uptake.

Subsequent studies carried out with the safener mefenpyr-diethyl and sulfonylurea herbicides mesosulfuron-methyl

and iodosulfuron-methyl-sodium also found no effect on herbicide uptake (Köcher 2005).

For post-emergence cereal herbicides, which have long distance transport between the treated leaf and the sensitive

meristems, safening action my involve inhibition of this transport. So far no case is known in which a safener directly

interferes with the long distance translocation of these herbicides. However, the final proposed safener mechanism is

enhanced herbicide metabolism/detoxification within the safened crop which could indirectly reduce the amount of

active herbicide reaching the target site. It is well understood that differences in herbicide metabolism play an

important role in herbicide selectivity (Hatzios and Penner 1982, Drobny et al 2012). Therefore, it is not surprising that

this was proposed as a possible safener mechanism as early as the 1970s and has been studied extensively since that

time. Hatzios and Hoagland (1989) reviewed in detail the early studies, especially on the seed treatment safeners and

pre-emergence safeners. This showed a large amount of evidence that safeners were indeed increasing the rate of

herbicide metabolism in the safened crop plant. For more recent post-emergence safeners, metabolism studies

combined with gene expression analysis has strengthened the understanding of safener MoA. In wheat (but not the

target grass weed) the level of fenoxaprop-ethyl and the free acid fenoxaprop declined (conversion to inactive

metabolites) more rapidly when plants were treated with fenchlorazole-ethyl (Köcher et al 1989, Yaacoby et al 1991).

For the safener cloquintocet-mexyl, the safening of wheat against clodinafop-propargyl was also reported to be based

on enhanced detoxification (Kreuz et al 1991, Kreuz 1993). Mefenpyr-diethyl also enhanced the rate of conversion of

fenoxaprop to non-phytotoxic products in wheat but not in target weeds (Hacker et al 2000). Similarly, mefenpyr-

diethyl enhanced the rate of metabolic degradation of iodosulfuron-methyl-sodium and mesosulfuron-methyl in cereals

but not in the grass weeds tested (Hacker et al 1999, Hacker et al 2000, Hacker et al 2001). In corn, the post-

emergence safener isoxadifen-ethyl has also been shown to enhance the metabolism of foramsulfuron (Hacker et al

2002, Pallett et al 2001).

Herbicide metabolism in plants involves several enzyme mediated steps (Hatzios, 1991; Van Eerd and Hoagland,

2003). The first step may sometimes be a conversion of an inactive pro-herbicide to the active herbicide. However, the

metabolic steps relevant to safening are those which convert the active herbicide to inactive metabolites. Cytochrome

P450 enzymes may catalyze the reduction, oxidation or hydroxylation of the herbicide and thus provide functional

groups for further metabolic steps. These often involve conjugation reactions between the first metabolite and

glutathione (GSH), glucose (Glc) or amino acids which are catalyzed by multifunctional enzyme, glutathione S-

transferases (GSTs) or glycosyltransferases (UGTs) (Armstrong 1994, Hatzios 2001, Lamoureux 1991, Marrs 1996).

Additonally, conjugation with malonate via malonyl-Co-A has also been reported (Sandermann et al 1997). The next

step in herbicide detoxification is the transport of the conjugates into the vacuole which may be catalyzed by ATP-

binding cassette (ABC) transporters (Tommasini et al, 1997). Once in the vacuole the conjugates may be further

degraded by peptidases with the removal of glycine and glutamate. The speed of herbicide metabolism/detoxification

therefore may depend on the level of activity of several key enzymes and therefore the level of expression of genes

which code for these enzymes. Advances in biotechnology over the past 2 decades have allowed increasingly detailed

studies into the effect of safeners on gene expression. It has been shown that genes encoding for enzymes involved in



herbicide detoxification are induced within a few hours of safener application (DeRidder et 2002, Cummins et al 1997,

Kreuz and Tommasini 1996; Scalla and Roulet 2002, Theodoulou et al, 2003; Zhang et al, 2007).

Gene expression studies have indicated parallels between the oxidative stress related oxylipin pathway and safener

signaling (Riechers et al, 2010). They have also indicated possible overlaps with the plant stress defense signaling

pathway involving salicylic acid (SA). For example, it could be shown that many safener-regulated genes are induced

by salicylic acid (Behringer et al, 2011). Therefore, whilst the primary site of safener reception is still not known, it

seems that several signaling pathways contribute to the full safener response in plants.

Future perspectives

There have been very few new safener structures patented in the past decade suggesting that little research into new

compounds is being conducted in the agrochemical industry. This is probably mainly due to the emergence of

geneticaly modified herbicide tolerance as a major selectivity tool (e.g. glyphosate tolerant Round-Up Ready® cotton,

soybean and corn) as well as the availability of strong existing safeners for use with newer herbicides. However, as the

problem of weed resistance increases and the existing safeners reach the limit of their potential, stronger “next

generation” safeners may indeed be required for future selective weed control products. As biotechnology methods

develop quickly, it is possible that the target site(s) of safeners will be identified in the not too distant future. It may

then perhaps be possible to use rational design to identify new safener structures.

Acknowledgements

We thank Dr. Stephen Lindell for critically reading our manuscript.



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FIGURE LEGENDS

Figure 1: Plants in pots; left: untreated, right: experimental herbicide at 300g a.i./ha

Figure 2 Typical symptoms of HPPD bleachers in an advanced screening stage

Figure 3 Typical auxin-transport inhibitor symptoms in a nematode screening assay

Figure 4: Year of market introduction (commercial) or publication (experimental) of the first herbicide with a given

mode of action. The mode of action of the corresponding herbicide might have been elucidated at later date. The

molecular modes of action of cinmethylin and oxaciclomefone are still not fully understood despite articles describing

biochemical effects attributed to these compounds.

ACCase: acetyl-CoA carboxylase; AdSS: adenylosuccinate synthetase; ALS: acetolactate synthase; AMPDA: AMP

deaminase; Auxin: auxin herbicides; Cellulose: cellulose biosynthesis inhibitors; CytOx: cytokinin oxidase; DHPS:

dihydropteroate synthase; DXR: 1-deoxy-D-xylulose 5-phosphate reductoisomerase; DXS: 1-deoxy-D-xylulose 5-

phosphate synthase; EPSPS: 5-enolpyruvylshikimate 3-phosphate synthase; FPS: farnesyl-diphosphate synthase;

GibB: gibberellic acid biosynthesis; GS: glutamine synthetase; GSAT: glutamate semialdehyde aminotransferase;

GPAT: glutamine phosphoribosylpyrophosphate amidotransferase; HPPD: 4-hyddroxphenylpyruvate dioxygenase;

IGPD: imidazole glycerol phosphate dehydratase; IMDH: 3-isopropylmalate dehydrogenase; KARI: ketol acid

reductoisomerase; LyCyc: lycopene cyclase; Microtubule assembly: inhibitors of microtubule assembly; Microtubule

organisation: inhibitors of microtubule organisation; ObtDM: obtusifoliol demethylase; PDS: phytoene desaturase;

PPO: protoporphyrinogen IX oxidase; PS I: photosystem I; PS II: photosystem II; PyrDH: pyruvate dehydrogenase;

TrpS: tryptophan synthase; VLCFA: very long chain fatty acid biosynthesis; ZDS: zeta-carotene desaturase.

Figure 5: Herbicide market share in 2010 according to mode of action. For abbreviations see Figure 4.

Figure 6: Seed treatment safeners

Figure 7: Pre-emergence tank-mix safeners

Figure 8: ACCase inhibitors and the associated safeners for post-emergence use in cereals

Figure 9: Structures and use of isoxadifen-ethyl and cyprosulfamide



Table 1 History of chemical weed control innovations (MoA = mode of action, post = post-emergence application, pre

= pre-emergence application, based on data from Cremlyn (1991), Worthington and Hance (1991), Büchel et al.

(1977), HRAC (www.hracglobal.com) and others

MoA, target site Chemical family Examples Use Earliest reports

Unspecific Inorganic herbicides H2SO4, Cu2SO4,

FeSO4, Na ASO2

Total 1874

Uncouplers Dinitrophenoles DNOC Post, dicots 1934

Auxins Aryloxyalkanoic acid

derivatives

2,4-D Post, dicots in

cereals

1942

Microtubule

organisation

Arylcarbamates Propham,

chloropropham,

Pre, monocots in

various crops

1946

Lipid synthesis

inhibitors

Chloroaliphatic acids TCA, dalapon Pre, monocots in

various crops

1947

Thiocarbamates EPTC, triallate Pre, mono- and

dicots in various

crops

1954

PS II- inhibitors Arylureas Monuron, diuron,

isoproturon,

linuron

Pre and post

mono-and dicots in

various crops

1951

1,3,5 -Triazines Atrazine,

simazine

Pre and post; broad

spectrum in maize

1952

Pyridazines Chloridazon Pre; dicots in sugar

beet

1962

Uracils Bromacil,

terbacil, lenacil

Soil applied, broad

spectrum in various

crops

1963

Biscarbamates Phenmedipham Post, dicots in

sugar beet

1968

1,2,4 -Triazinones Metribuzin Pre in soybeans 1971

Very long chain

fatty acid

biosynthesis

Chloroacetamides Allidochlor,

alachlor

Pre; monocots and

dicots

1956

PS I Bipyridyliums Diquat, paraquat Non-selective 1958

Protoporphyrinogen

oxidase

Diphenyl ethers Nitrofen ,

acifluorfen

Pre and post,

various crops

1960

Oxadiazoles Oxadiazon Rice, non-selective 1969



Microtubule

assembly

Dinitroanilines Trifluralin,

pendimethalin

Pre against mono-

and dicots

1960

Cellulose

biosynthesis

Nitriles Dichlobenil Plantations 1960

EPSP synthase Glycines Glyphosate Post, non-selective 1971

Phytoene desaturase Pyridazinones Norflurazon Pre and post in

cotton

1973

ACCase Aryloxyphenoxy-

propanoates

Diclofop,

fluazifop

Post, grasses 1975

Cyclohexane diones Alloxydim,

Sethoxydim

Post grasses 1976

Glutamine

synthetase

Glufosinate Non-selective 1981

AHAS- or ALS Sulphonylureas Chlorsulfuron,

metsulfuron m.

Mono- and dicots

in various crops

1982

Imidazolinones Imazapyr,

imazethapyr

Non-selective or

selective in soy

1983

Pyrimidinyl

benzoates

Bispyribac

sodium

Rice

HPPD-inhibitors Pyrazolynate,

sulcotrione

Various crops,

mono- and dicots

1984



Table 2 Acreage changes in US crops during the last 100 years (million acres harvested)

1920 1970 2012

Barley 7.5 9.7 3.2

Canola <0.1 <0.1 1.6

Corn 101.4 66.1 97.2

Cotton 34.4 11.2 9.4

Hay 73.1 61.5 56.3

Oats 42.8 18.6 1.1

Potatoes 3.3 1.5 1.2

Rice 1.3 1.9 2.7

Rye 4.9 1.5 0.3

Soybeans <0.5 42.3 76.1

Sugarbeets 0.9 1.5 1.2

Wheat 62.4 43.6 49

All crops 347 283 309

Source: USDA Crop Production Historical Track Records, April 2013



Table 3 Yields per acre of five US crops between 1940 and 2010; b = Bushels, t = tons

1940 1950 1960 1970 2010

Barley (b) 23 27.2 31 42.8 73.1

Corn (b) 28.9 38.2 54.7 72.4 152.8

Soybeans (b) 16.2 21.7 23.5 26.7 43.5

Sugarbeets (t) 13.4 14.6 17.2 18.7 27.7

Wheat (b) 15.3 16.5 26.1 31 46.3

Source: USDA Crop Production Historical Track Records, April 2013



plant physiology preview. published on august 7, 2014, as doi

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