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HERBICIDES
DISCOVERING NEW MODES OF ACTION FOR HERBICIDES AND
THE IMPACT OF GENOMICS
David Cole, Ken Pallett and Matthew Rodgers of Aventis CropScience explain the need for new modes of
herbicide action and how plant genomics are revolutionising this quest
Introduction
Most herbicides work by interfering with the unique biochemistry of plants, acting at single molecular sites to inhibit specific
processes and simultaneously causing severe disruption of the
plant’s metabolism. Herbicides are usually inhibitors of specific
enzymes and bind either at the active site of the enzyme or at
some domain removed from the active site. An unsurprising
consequence is that although herbicides acting at one single
target may be sub-classified into ‘families’ of chemistry, such
compounds tend to share similar overall properties. That is,
their application rates, performance, environmental fate and
“off target” impact, each fall within a predictable envelope.
Put simply, their “strengths” and “weaknesses” tend to be
similar. Since both the market and regulatory environments are
constantly evolving there is an eagerness for product novelty
across all agrochemical sectors. Witness, for example, the
phenomenal success of contemporary chemistries such as the
sulfonylurea herbicides, the strobilurin fungicides or the flurry
of new insecticide classes. Unlike disease or insect control,
biotechnology is not going to erode the market for chemically
based weed control and the need for innovation in herbicide
discovery is now paramount.
The need for new modes of action
Empirical screening of chemicals on whole target organisms
over the last forty years has led to over 270 herbicide active
ingredients on the market, which possess seventeen identifiable modes of action (Table 1). The mode of action of new
herbicide chemistries has always been discovered in retrospect, although concerted attempts have subsequently been
made to use this knowledge to assist in the discovery and
optimisation of further examples. Onlookers to the industry
ceaselessly display bemusement at the seemingly scattershot
‘spray and pray’ methods underlying product discovery and
the apparent lack of rationality involved. The random
nature of preliminary screens does tend to detract from the
considerable science and multidisciplinarity involved in the
subsequent optimisation and tailoring of ‘lead’ chemistry. It
is nevertheless a proven and highly successful approach and
has long been the mainstay of both the agrochemical and
pharmaceutical industries. In recent years, this approach has
been revolutionised by the advent of the powerful parallel
synthesis technology of combinatorial chemistry and the
need to minimise costs by screening reduced quantities of
chemical linked to the attendant implementation of highthroughput screens, miniaturisation and automation
(Steinrücken and Hermann, 2000).
DOI: 10.1039/b009272j
Table 1. The target sites or modes of action of
commercial herbicides. Over fifty per cent of
herbicides inhibit only three targets
Target
Number of herbicides
Photosystem II
Acetolactate synthase
Protoporphyrinogen IX oxidase
Auxin mimics
Acetyl CoA carboxylase
Non-specific chloracetanilide target
Cell division
Phytoene desaturase
Hydroxyphenylpyruvate dioxygenase
Oxidative phosphorylation
Cellulose biosynthesis
Photosystem I
Auxin transport
Enolpyruvylshikimate phosphate synthase
Dihydropteroate synthetase
Glutamine synthetase
Lycopene cyclase
Unknown or not stated
59
43
28 (–3)
20
16
15
14
11
3 (+3)
3
2
2
1
1
1
1
1
48
TOTAL
272
Updated from Pillmoor et al. (1995) and Pallett (1997). Modes of
action for which the precise molecular target(s) have not necessarily
been identified are italicised. The figures in parentheses highlight
three benzoyl pyrazole herbicides (pyrazolynate, pyrazoxyfen and
benzofenap) previously classified as Protox inhibitors but now
identified as proherbicides of HPPD inhibitors (Pallett, 2000).
A closer look at Table 1, however, reveals some concerns.
Of these active ingredients, around half of them act on one
of three molecular targets: photosystem II, acetolactate
synthase (ALS) and protoporphyrinogen IX oxidase (Protox).
Furthermore, when we consider specifically the new
herbicides introduced in the last decade, this trend becomes
even more exaggerated. Well over half of these more recent
introductions are either inhibitors of ALS or Protox. Among
these products, only two new modes of action can be
identified of which hydroxyphenylpyruvate dioxygenase
(HPPD) has attracted much interest. Out of ten new
herbicide active ingredients announced at the 1999 Brighton
Crop Protection Conference, only one, diflufenzopyr, could
be considered to have an apparent new mode of action
(auxin transport). The technical and environmental profiles
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of these groups of herbicides will be broadly similar and
therefore this apparent diversity of active ingredients is
somewhat illusory. Before identifying how new modes of
action and therefore new chemistries can be sought, it is
worth considering the pressures, constraints and opportunities encountered by herbicides in the new millennium. These
can be summarised thus:
●
evolving standards in environmental protection and food
quality can mean restriction, or cancellation of products
which give rise to unacceptable residues
●
limited tolerable residue burdens deriving from similar
chemistries – filling of ‘risk cup’
●
decreasing application rates to reduce environmental
impact
●
reduction in cost of weed control due to agricultural
recession, overproduction of food and withdrawal of
farming subsidies (in the developed world)
●
increase in generic (off-patent) herbicide business and
attendant pressure on market prices.
●
impact of adoption of crops engineered for tolerance to a
few established ‘total’ weedkillers, e.g. glyphosate
●
appearance of biotypes of weeds resistant to herbicides –
these often mimic the protection mechanisms of the crop,
especially in the case of grass weeds in grassy crops
●
changing cultural practices, such as reduced tillage,
Integrated Weed Management, trend to post-emergence
herbicides for use flexibility and spray-by-need
Threats are often simultaneously opportunities and
enable both replacement and complementation strategies
within an essentially static market. Restrictions placed on
use of some older products are an opportunity for more
sophisticated and environmentally compatible chemistries.
The widespread adoption of crops tolerant to ‘total’ herbicides
such as glyphosate are perceived to erode the markets for
competitors. However, the shifts in weed spectrum and the
appearance of resistant weeds which are inevitable results of
repeated use of single class herbicides will provide markets
for differentiated herbicides having complementary modes
of action and technical profiles. Not least, these will be
necessary in order to sustain the long-term market viability
of ‘total’ herbicides and herbicide-tolerant crops.
●
low abundance in the plant
●
target inhibition is able to induce ‘catastrophic’ secondary
effects on plant metabolism
This last point is particularly noteworthy. In aiming to kill
plants it is not sufficient simply to inhibit essential metabolic
processes. Additional events metabolically upstream or
downstream of the target must take place, amounting to an
uncontrolled cascade of biochemical events. These amplify
the original impact of the herbicide and overwhelm the cell,
the normal control of metabolic and protective mechanisms
or subcellular integrity. Often, such events have proved to be
hard to predict from limited ‘textbook’ knowledge of plant
metabolism current at the time such target sites were
discovered. Broadly, it can be argued that we still do not
know in detail how plants actually die as a result of inhibition of some known targets. However, secondary catastrophic effects are now established in a number of cases
(Table 2). The ways in which such secondary effects occur
are highly contrasting. Inhibition of Protox generates accumulation of photoreactive porphyrin substrate resulting
from the different subcellular locations of multiple Protox
and ‘Protox-like’ enzyme forms differentially sensitive to the
herbicides. The uncontrolled accumulation of shikimate
(phosphate) product of enolpyruvylshikimate phosphate
synthase (EPSPS) in the presence of glyphosate drains the
cells of other carbon skeletons. Several recent herbicides such
as isoxaflutole, mesotrione and sulcotrione inhibit HPPD, a
target site of current intense interest in the industry (Pallett,
2000) (Figure 1). It is proposed that there are three independent cascades of events arising from inhibition of HPPD
which all contribute to the death of the plant (Figure 2).
Identification of new targets
Apart from whole organism screening three approaches have
been taken in the past to identify new and effective targets.
The most popular has been to select known plant enzymes
‘Ideal’ herbicide targets
The predominance of a few superior sites of action so far
can be understood when we consider the attributes required
in preferred herbicide targets:
●
plant-specific for low non-target (toxicological and
ecotoxicological) impact
●
ability to bind diverse types of chemical inhibitors
●
low metabolic turnover
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Figure 1. Structure of the new corn weed herbicide isoxaflutole
(BalanceTM, MerlinTM ), its diketonitrile herbicidal product,
RPA 202248, sulcotrione (GalleonTM) and mesotrione. The
diketonitrile of isoxaflutole is rapidly formed following uptake
into the plant (Pallett, 2000)
HERBICIDES
Table 2. Primary and secondary effects linked to selected herbicide targets
Target Site
Primary metabolic effect
Secondary effects associated with lethality
Photosystem II
inhibition of photosynthetic electron
transport
singlet oxygen and triplet chlorophyll induced
photo-oxidation
Protox
inhibition of chlorophyll biosynthesis
accumulation of photodynamic porphyrins and lipid
peroxidation
ALS
inhibition of branched chain amino
acid biosynthesis
accumulation of α-keto-butyrate/α-aminobutyrate?
Enolpyruvylshikimate phosphate synthase
inhibition of aromatic amino acid
biosynthesis
shikimate pathway deregulation; accumulation of
shikimate and shikimate phosphate
Phytoene desaturase
inhibition of carotenoid biosynthesis
absence of chloroplast development in developing
leaves
HPPD
inhibition of plastoquinone and
α-tocopherol biosynthesis
inactivation of phytoene desaturase, absence of
chloroplast development, lipid peroxidation and
tyrosine accumulation
from literature on an inspirational basis, and to prepare and
test inhibitors designed following inspection of the enzyme’s
reaction mechanisms (Pillmoor et al., 1995). Such compounds can be potent enzyme inhibitors in vitro, but more
often than not, have limited herbicidal activity in biological
tests. Other possible approaches are to elucidate the as yet
‘unknown’ modes of action of many established herbicides
(Table 1; Schmalfuss et al., 2000), or to consider the target
sites of herbicidal natural products (Duke et al., 1997).
These approaches have now largely been eclipsed by power-
ful genomic technologies which can be used in tandem with
bioinformatics to identify systematically every gene
necessary for plant growth and survival. Targeted gene
silencing can then be used to identify how sensitive the plant
is to disruption of gene product action. This can lead to
selection of a subset of novel proteins which by analogy may
be good ‘targets’ to screen for novel herbicides. Alternatively, studying the expression of genes on a massive and
parallel scale using microarrays shows potential in
elucidating the mode of action of herbicides discovered in
conventional biological screens, as we explain
later.
Plant genomics offers an unparalleled
opportunity to uncover all potential herbicide
targets heralding a new era of discovery where
value-added new high-throughput screens are an
integral part of lead identification.
Genomics for new targets
Figure 2. Phytotoxic secondary effects resulting from inhibition of
hydroxyphenylpyruvate dioxygenase (HPPD). Plastoquinone and
α-tocopherol are metabolic products of homogentisate, the product of
HPPD and inhibition of HPPD results in their depletion. Hydroxyphenylpyruvate is the substrate of HPPD formed from the deamination
of tyrosine and inhibition of HPPD can lead to the accumulation of free
tyrosine in phytotoxic amounts. These two secondary effect of HPPD
inhibition contribute to the effective herbicidal activity of HPPD
inhibitors (Pallett, 2000).
As we write, the sequencing of the first entire
plant genome, that of Arabidopsis thaliana, is all
but complete. Arabidopsis has become the
organism of choice for plant genomics because of
its rapid life cycle, prolific seed pro d u c t i on, small
genome, and familiar biology. Knowledge of its
genomic DNA sequence will ultimately allow
identification of approximately 25,000 genes
encoded in its five chromosomes.
It is hard to obtain an accurate figure for the
subset of genes in A. thaliana critical for growth
and survival, but estimates suggest that this could
be between one thousand and two thousand
(Cole and Rodgers, 2000). Many of these, of
course, would not encode useful herbicide
targets. Some proteins may not be amenable to
binding herbicides and others may be in areas of
metabolism that are best avoided due to potential
animal toxicology issues. Furthermore, a large
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number of these genes have no known function and,
although this does not limit their potential as in vivo targets,
their possibilities as high throughput in vitro screening
targets are limited.
In the past the exploitation of potential new herbicide
targets for in vitro screening was limited by and depended
on a rich natural source of the enzyme, or on the lengthy
process of protein purification, cloning and over-expression.
The increasing availability of genomic data from large
numbers of organisms now allows the rapid identification of
genes of interest by similarity to previously identified genes
from other sources. As a consequence, genes selected by
mining knowledge of metabolic pathways can be rapidly
cloned and over expressed for in vitro analysis and for use in
high throughput biochemical screening. Nevertheless, in
order to validate the use of such targets in screens, we must
be certain that inhibition of the target is lethal to the plant.
By definition, if the potential target is novel then there are
unlikely to be readily available small molecule inhibitors for
validation. Knowledge of the reaction mechanism might
enable the production of reaction state intermediate
analogues for use as inhibitors in vitro but there is no
guarantee that such molecules will be active in vivo.
Evidence that the enzyme candidate is essential to the
plant can be gleaned from antisense experiments in which
gene expression is depressed in the plant following
engineering with a reverse-orientated gene coding sequence.
Such experiments provide two important pieces of
information. Firstly, the knock-out of gene expression
confirms whether the enzyme of interest is essential for the
survival of the plant and points to the symptoms to be
expected from inhibitors acting on it. Secondly, since
antisense lines vary in the level of messenger RNA (mRNA)
suppression and thus in the level of remaining enzyme
activity, it is possible to determine the degree of mRNA
suppression or enzyme activity quenching that is required to
cause the onset of severe symptoms or plant death. This can
then be used to rank the favourability of the target.
The amount of enzyme activity reduction required for
lethality in commercial herbicide targets is not clear.
However, it appears that complete inhibition of enzyme
activity at these targets is not necessary for plant death
(Abell, 1996). In mutant plants with reduced amounts of
glutamine synthetase activity, the target of glufosinate,
reduction in glutamine synthetase activity of only 38% was
sufficient to cause severe abnormalities (Blackwell et al.,
1987). Antisense knock-out of acetolactate synthase (ALS),
the target site of the sulfonylureas, imidazolinones and triazolopyrimidines, can produce plants displaying a range of
ALS inhibitor-like symptoms such as growth retardation and
necrosis (Hofgen et al., 1995).
Such directed knock-outs allow the screening of enzymes
whose inhibition might be expected to have catastrophic
effects in the plant, based on a knowledge of pathway
dynamics. However, our knowledge of biochemical pathways in plants is incomplete and the next major herbicide
target may lie in an unexpected area of metabolism. Moreover, the sequencing of complete genomes has revealed the
presence of large numbers of genes with no known function
– so called “orphan genes”. Some of these may also prove to
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Pe s t i c i d e Ou tl ook – D ece mbe r 20 00
be useful herbicide targets. Consequently, a number of tools
have been developed that attempt to screen all the genes
present in the plant genome for potential herbicide targets.
Antisense quenching of enzyme activity can be extended
to genome-wide analysis. Inserts from a cDNA library can
be used in the antisense orientation, to transform plants for
the inhibition of expression of a variety of genes. Such a
plant collection can then be examined for mimics of
herbicide symptoms such as albinism, stunting, necrosis,
deformation, chlorosis, scorching of tissues, lack of germination or seedling lethality. Such a task is not trivial, but is
now beginning to be explored for the assignment of gene
function.
Recently, large-scale collections of plant mutants based
on a system of insertional mutagenesis have become
available (Feldmann and Marks, 1987; Tissier et al., 1999).
Developed primarily by biologists as tools for gene finding
and functional genomics, they can be put to use for the identification of potential new herbicide targets. Insertional
mutagenesis is the process whereby a length of DNA of
known sequence is deliberately integrated into the genome
at random, disrupting the sequence of any gene within
which it might insert. If this sequence is introduced into
coding or regulatory regions then the gene is mutated or
functionally knocked out altogether (Figure 3). Among these
gene knock-out lines are herbicide symptom mimics,
resulting from the knock out of critical genes (Figure 4).
Having selected the mutants showing interesting phenotypes, the unique sequence of the so called “gene tag” allows
the straightforward determination of the DNA sequence
bordering the point of insertion. The availability of the
complete genome sequence of A. thaliana means that only a
relatively short piece of sequence bordering the insertion
allows the position of the insertional mutagen to be
identified uniquely. The genome region around it can then
be analysed to determine the nature of the disrupted gene.
The availability of reliable techniques for large-scale A.
thaliana transformation has made possible the construction
of large collections, typically of around one hundred
thousand lines, that should contain mutants for almost every
gene, allowing each gene to be surveyed for its suitability as
a herbicide target.
Populations of tagged mutants can be used not only in the
so called “forward genetics” mode where plants with the
phenotype of interest are analysed to determine the gene
disrupted, but also in the “reverse genetics” mode. Once a
Figure 3. The principle of insertional mutagenesis.
HERBICIDES
Figure 4. Arabidopsis thaliana T-DNA insertion mutant
displaying pigment deficiency and dwarfing (lower plants),
compared to wild type plants.
population is established, the plant line carrying a disrupted
version of a gene can be located and its phenotype
determined (Winkler and Feldmann, 1998). This is quicker
than using antisense to verify the effect of reduced gene
expression, but precludes relating expression level to
severity of phenotype.
Two systems are available for insertional mutagenesis,
based on tagging with either T-DNA or transposons. T-DNA
tagging makes use of the ability of the plant pathogen
Agrobacterium tumefaciens, to insert a small segment of
DNA, the so-called “transfer DNA” (T-DNA), into the plant
genome. On the other hand, transposon tagging makes use
of naturally mobile genetic elements, transposons, that are
capable of jumping from the genome and re-inserting at a
different genomic site. Although naturally occurring, active
transposons, native to the plant of interest can be used as
insertional mutagens these elements can also be modified
and transferred from one species to another.
brought about by inhibition of the target. These latter
perturbations in transcription pattern should be diagnostic
of the mode of action of the herbicide, at the pathway or
even the target site level. Until recently however, there was
no method available for the multi-parallel analysis of
populations of messenger RNA, to enable this to be done.
The large-scale generation of DNA sequence data has
allowed the development of several technologies that enable
the examination of expression levels of many genes at once
(Baldwin et al., 1999). For example, arrays of individual
DNA species can be attached to solid supports. These arrays
carry representatives of almost the entire complement of
genetic messages produced by the plant. By probing them
with total plant mRNA, reverse transcribed into fluorescently labelled DNA, changes in the levels of the individual
and corresponding mRNA species brought about by the
plant’s developmental status can be followed (Aharoni et al.,
2000). The effect of treatment with herbicide on the pattern
of transcription can be visualised in the same way (Figure 5).
Such experiments generate huge amounts of data and the
real science now lies in learning to sift out and interpret the
significant information.
In contrast to the methods available for multi-parallel
analysis of gene transcripts in plants, methods available for
the analysis of the protein complement, or proteome, offer a
smaller window on the cell. Widely available proteomics
Genomics for mode of action determination
As we have already seen, using the technologies outlined
above, scientists are beginning to piece together a catalogue
of critical genes which might represent lethal target sites in
plants. However, genomics can also find use in traditional
herbicide discovery methods to help identify the mode of
action of new “leads” found through whole plant screening
of candidate herbicides. A catalogue of these targets will
provide a useful triage tool for the determination of mode of
action. A collection of all known lethal insertions or
antisense knock outs related to their phenotype will be a
powerful method to separate targets causing bleaching from
those causing scorching for example, thus narrowing down
the search for mode of action. In conjunction with other
target site identification technologies, this could be a
significant help in selecting and fast-tracking exciting leads.
The treatment of plants with lethal enzyme inhibitors
causes profound changes in the metabolism of the plant.
These changes in turn can be expected to lead to changes in
the transcription pattern of genes. Some of these changes
will be a general response of the plant to foreign chemicals
and later to cell death, but some changes will be specifically
Figure 5. False colour image of Arabidopsis DNA microarray
following hybridisation to plant mRNA. The colours and
intensities of the spots indicate the source (e.g. treatment
type) and the degree of expression respectively of the
individual genetic message.
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HERBICIDES
technologies rely upon the separation of the protein
complement using two dimensional electrophoresis. In this
way, up to two thousand spots can be viewed on a single gel
slab. Individual proteins are identified either by peptide
sequencing, or by comparing the mass of digested peptides
to a database of predicted protein fragments. It is hard to
estimate what proportion of the protein complement is
represented but proteins located in the cellular membranes
and other very poorly soluble proteins are likely to be
missed. The technique is also limited by current separation
and detection procedures. Nevertheless its potential for
identifying proteins regulated in response to inhibitor
treatment has been proven in yeast, where the potential
number of encoded proteins is much lower.
Expression profiling experiments and proteomics gels are
promising routes to the identification of unknown targets
and, through the identification of co-regulated genes, can
also give clues to the function of the potential target or
pathway if it is not already known. Furthermore, novel
pathways identified in this way might also provide a source
of new targets.
Concluding remarks
Plant genomics will deliver a plethora of new candidate
targets, based on genetic validation. However, relatively few
of these will satisfy the needs of high-throughput biochemical screening. High quality targets must have an
acceptable ‘hit’ rate in effectively binding potential chemical
inhibitors, they should mediate plant-specific activity and
inhibition must lead to devastating secondary effects on
plant metabolism. Crucially, functional assays must be
simple, economic and amenable to industrialisation.
Such screens must be integrated to add value to the
discovery process. Unlike the pharmaceutical industry,
whole organism screening will remain central to agrochemical discovery. High-throughput biochemical screens must
therefore add something special. These can for example,
allow the detection of hits that may be missed in glasshouse
screens due to poor plant bioavailability, or can allow the
rapid and thorough evaluation of a target site by concerted
screening against diverse sets of chemistry. Structure activity
relationships can provide inspiration for further chemical
synthesis based on binding hypotheses or single parameter
data not available from glasshouse screening. Further rationalisation of activities downstream of genomics such as
high-throughput x-ray crystallography for three-dimensional
analysis of protein-inhibitor interactions (structural genomics)
will assist in developing ‘virtual’ or ‘in silico’ screening of
chemistry.
Greater reliance on high-throughput biochemical
screening will necessitate an improved ability to convert in
vitro hits into biologically active molecules through a better
understanding of whole plant-compound interactions and
improved test systems for this. As in the past, the keys to
successful discovery remain:
●
the quality and diversity of chemistry supplying the
screens
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Pe s t i c i d e Ou tl ook – D ece mbe r 20 00
●
intelligent and adept integration of technologies, understanding the strengths and limits of each
●
ability to identify and capitalise on the unexpected
To these, of course, add a degree of luck!
Acknowledgement
We thank Monique Guis for providing Figure 5.
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Gressel, J. G. (2000) Molecular biology of weed control.
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of action. In: Pesticide Chemistry and Bioscience. The FoodEnvironment Challenge. (Eds. G. T. Brooks and T. R. Roberts),
Royal Society of Chemistry, Cambridge, pp 207–220.
The authors have collectively 35 years experience in industrial
herbicide discovery. They all share backgrounds in plant biochemistry and an interest in harnessing this in a practical way to assist
herbicide discovery. David Cole and Matthew Rodgers are respectively Technology Liaison Manager and Herbicide Molecular Biology
Team Leader at Aventis CropScience’s discovery centre in Ongar,
Essex, UK. Ken Pallett is Head of Herbicide Biology and Global
Biology Co-ordinator based at Aventis CropScience’s discovery
centre at Frankfurt-am-Main, Germany.
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A. Nyffeler
1996, 7(5), 33
Isoxaflutole for weed control in maize
B. M. Luscombe
1996, 7(6), 29
Aquatic weed control in the UK
P. F. R. Barrett
1997, 8(2), 21
Bioherbicides – promide and prospects
R. Wall
1997, 8(4), 29
Carotenoid biosynthesis inhibitor herbicides
P. Boger
1998, 9(6), 29
ACCace inhibitor herbicides
J. Harwood
1999, 10(4), 154
Managing weeds with natural products
F. Dayan
1999, 10(5), 185
Future of grass weed management in the UK
J. Clarke
2000, 11(2), 59
Control of invasive weeds in Australia
S. Corey
2000, 11(3), 101
Noxious weed control in the USA
A. Tasker
2000, 11(3), 104
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