The Genetic Basis of Glyphosate Resistance in Erigeron bonariensis L.

The genetic basis of glyphosate resistance in the Central Valley agricultural weed hairy fleabane
(Erigeron bonariensis L.)
Priyanka Chaudhari
M.S. in Biotechnology
California State University, Fresno
Committee members:
Dr. Katherine Waselkov (Biology)
Dr. John V.H. Constable (Biology)
Dr. Cory Brooks (Chemistry)

ABSTRACT
Herbicide resistance is the heritable ability of weeds to survive and reproduce in the presence of
herbicide doses that are lethal to the wild type of the species. Erigeron bonariensis (hairy
fleabane) is an agricultural weed that infests orchards and crop fields in California’s Central
Valley, and has become resistant to the herbicide chemical glyphosate (RoundUp®), through an
unknown genetic mechanism. One mechanism of glyphosate resistance demonstrated in E.
canadensis, a close relative of E. bonariensis, is non-target site reduced translocation of the
herbicide, in which vacuolar sequestration prevents the chemical from spreading around the
plant. Resistance of Erigeron canadensis to glyphosate is believed to involve upregulation of the
target gene EPSPS in combination with the ABC transporter genes M10 and M11. This study
aims to determining through quantitative PCR (qPCR) if these candidate genes are involved in
glyphosate resistance in wild populations of Erigeron bonariensis. Sample leaves of the weed
were collected before and after glyphosate spraying in plants from 10 different populations wild-
collected from the Central Valley and two control populations of Erigeron bonariensis.
Response to glyphosate was used to characterize percent resistance for each wild-collected
population. RNA was extracted from the leaves of glyphosate-treated and untreated individuals,
and used for cDNA synthesis. Quantitative PCR primers were designed for the E. bonariensis
orthologues of the E. canadensis genes EPSPS, ABC M10, and ABC M11, and pre- and post-
spraying expression levels of each gene (relative to the housekeeping gene actin) were analyzed
through qPCR. This experiment will examine gene expression patterns in ABC transporter genes
(M10 and M11) to determine if the respective genes are upregulated in response to Glyphosate
application and therefore establish their role in glyphosate resistance. Future RNA-Seq analysis
via Illumina HiSeq may reveal other genes that are differentially up- or down-regulated in

resistant populations of E. bonariensis after glyphosate exposure. Determination of the genetic
basis of herbicide resistance will provide fundamental data about parallel evolution in response
to strong selection pressures, and help suggest alternative mechanisms for field control of this
weed.

INTRODUCTION
Moss (2002) defines herbicide resistance as the heritable ability of weeds to survive and
reproduce in the presence of herbicide doses that are lethal to the wild type of the species.
Herbicide resistance was first observed by an ornamental nursery owner in 1968 (Jasieniuk et al.
1996). The first resistance to herbicide was recorded in Senecio vulgaris; seeds from the resistant
biotypes were found to be insensitive to the chemicals simazine and atrazine (Pieterse 2010). In
1974, resistance to glyphosate herbicide became a problem for corn growers (Gressel et al.
1982). Since then, more than 187 species of weeds throughout the globe have developed
resistance against various herbicides that target a broad range of biochemical processes (Pieterse
2010). The first case of herbicide resistance in California was reported in 1981 at UC Riverside
(Holt at al. 1981) and recently, more species have also evolved resistance to various other
herbicide chemicals employed by farmers in California (see Table 1; Malone 2014).
Table 1. Weed species resistant to herbicide in California (http://www.weedscience.org)
Herbicide(s) Species Target site
Triazine Senecio vulgaris D1 protein
Sulfonylurea Lolium perenne, Sagittaria
montevidensis, Scirpus
mucronatus, Salsola tragus,
Ammania auriculata, Scirpus
mucronatus
Acetaloacetate synthase
(ALS)
Thiocarbamate Echinochloa phyllopogon Lipis synthesis
Aryloxyphenixy propionic
acid
Echinochloa phyllopogon Acetyl-CoA carboxylase
(ACCase)

Dinitroaniline Echinochloa crus-galli Tubulin
Pyrazolium salt Avena fatua unknown
Ubstituted amino acid Lolium rigidum D1 protein
Glyphosate herbicide (marketed by Monsanto as RoundUp®) contains N-
phosphonomethyl glycine that acts against plants by hindering aromatic amino acid synthesis
(Bridges 2003). Upon application on leaves, the plant takes in glyphosate through the stomata
and is transported through the phloem alongside products of photosynthesis to all parts of the
plant. In susceptible plants, glyphosate hinders the role of the plastid-expressed enzyme 5-
enolpyruvylshikimate-3-phosphate synthase (EPSPS), important in the prechorismate step of the
shikimate pathway. In the absence of glyphosate, this enzyme works by condensing shikimate-3-
phosphate and phosphoenolpyruvate into 5-enolpyruvylshikimate-3-phosphate (EPSP) with
inorganic phosphate, initiating the anabolism of aromatic amino acids (Ferreira 2008).
Disruption of the synthesis of aromatic amino acids (phenylalanine, tryptophan, and tyrosine)
eventually kills the plant (Herman and Weaver 1999).
Glyphosate has become the world’s most commonly used herbicide since its market
introduction in 1974 (Baylis 2000). Its use around the globe results from several factors
including potency against an extensive variety of species (monocots and dicots), less harmful
activity against animals than other herbicides (the enzyme EPSPS is not present in animals),
rapid microbial degradation in the soil, and low cost (Duke and Powles 2008). Also, it has been
commonly used in recent years as an alternative to manual weeding (tillage) as a method of weed
control. In this low or no-tillage method, there is huge dependence on herbicides to control
weeds, and these have numerous environmental benefits and economic importance (Owen 2008;
Powles 2008; Shaner 2000). With the adoption of genetically modified crops with glyphosate

resistance in 1996, the already high levels of glyphosate application increased (Powles & Preston
2006). The combined effects of excessive over-application against already glyphosate resistant
crops and completely reduced tillage has created an agricultural environment that has an
increased risk of evolution of glyphosate resistance (Neve at al. 2003). Herbicide resistance is
stimulated by the re-current use of herbicides with the same active chemical ingredients
(LeBaron 1991), and so inevitably, glyphosate resistance has evolved in many weeds (Powles et
al. 1998). However, because it still has the ability to control numerous weed species, has
adaptability to low tillage systems, and low animal toxicity, high glyphosate use remains a key
factor in modern systems because growers are unwilling to return to greater tillage systems or
older, more toxic herbicides (Beckie 2012).
Two mechanisms that confer resistance and have contributed to glyphosate resistance in
weeds are target-site and non-target site resistance. Target site-based resistance is a condition
where resistance evolves due to a gene mutation resulting in a structural or chemical change to
the target enzyme, so that the herbicide fails to effectively inhibit the normal enzyme function
(Powles and Preston 2006). A missense mutation may involve a specific nucleotide substitution
in the coding region producing a different amino acid that results in structural, hydrophobicity or
charge change in the active site of the target enzyme making it less sensitive to inhibition by the
herbicide. A few weeds such as goosegrass (Eleusine indica (L.) Gaertn.) have undergone target
site mutagenesis (Lee and Ngim, 2000; Dinelli et al. 2006) of the enzyme EPSPS, via a
substitution mutation that replaced the amino acid proline with serine at position 106 (Pro106-
Ser). Ng et al. (2004 & 2005) demonstrated that substitution of proline by threonine (Pro106-
Thr) also confers resistance glyphosate resistance to goosegrass. The mutated EPSPS enzyme
has a low affinity for glyphosate but almost normal affinity for phosphoenol pyruvate (the

enzyme’s usual substrate), permitting the shikimate pathway to proceed normally even in the
presence of glyphosate (Gaines et al. 2010).
Non-target site mutations induce sequestration, detoxification, and reduced absorption
function by limiting the translocation of glyphosate to target sites (Wakelin et al. 2004).
According to Claus and Brehrens’ (1976) study, rapid and widespread glyphosate translocation is
necessary to achieve high herbicide efficacy. There is therefore a possibility that changes in its
translocation may confer resistance. This is an intriguing observation that would be helpful in the
understanding of non-target-site-based resistance (NTSR). The study that unraveled this
phenomenon was carried out in rigid ryegrass (Lolium rigidum Gaud; Lorraine-Colwill et al.
2002), and indicated that resistance in at least one glyphosate-resistant biotype was not due to
EPSPS enzyme target mutagenesis or degradation. In the same study, it was shown that there
was no significant difference between glyphosate resistant and susceptible biotypes in EPSPS
sensitivity or expression level or in glyphosate absorption. However, the patterns of glyphosate
translocation differed. The researchers observed accumulation of glyphosate at lower parts of the
plant and to some extent in the roots in susceptible plants, whereas in resistant plants, it
accumulated in the tip of the leaves with a negligible amount transported to the roots (Lorraine-
Colwill et al. 2002). Wakelin et al. (2004) found the same pattern of reduced glyphosate
translocation when working with four glyphosate resistant ryegrass populations in Australia.
Researchers investigating mechanisms of glyphosate resistance in other Lolium rigidum
populations have not found large differences in glyphosate translocation. Perez et al. (2004)
found no significant difference in glyphosate absorption and translocation between susceptible
and resistant Chilean Lolium plants. In an investigation of glyphosate resistance in Californian
Lolium, Simarmata et al. (2003) found significantly higher glyphosate concentration in treated

leaves of glyphosate resistant plants 2-3 days after treatment. Interestingly, these authors
observed no other significant differences in glyphosate absorption or translocation between
resistant and susceptible plants. These varying results suggest potentially different non-target
resistance mechanisms to glyphosate may occur in different Lolium populations. In general, most
studies observed no differences in glyphosate absorption, but phloem translocation is sometimes
reduced greatly (Feng et al. 2004; Koger and Reddy 2005) in resistant populations. Failure of
glyphosate translocation from leaves to the roots seems to be an important mechanism that leads
to resistance in certain Lolium biotypes (Preston 2002).
Species in the genus Erigeron are annual or short-lived perennial plants native to the
Americas that have in the recent past become cosmopolitan and invasive weeds of many crops
and arable lands (Prieur-Richard et al. 2000). The genus is in the sunflower family (Asteraceae)
and the weed species were formerly placed in the genus Conyza before taxonomic revision
(Baldwin 2012). Erigeron spp. are prolific seed producers: a single plant is capable of producing
thousands of viable wind dispersed seeds (Weaver 2001; Shields et al. 2006).
Erigeron spp. have become very common and problematic weedy plants in agronomic
crops around the world (Weaver 2001). The opportunistic nature of Erigeron in undisturbed
areas makes them well-suited for becoming established in agricultural fields and surrounding
areas, particularly in no-tillage or low tillage systems, e.g., conservation tillage (Bruce and Kells
1990; Buhler et al. 1992). Considering the fact that over the last two decades the amount of crop
hectares in conservation tillage has increased by a margin of about 50% in Central California
(Young 2006), weeds such as Erigeron spp. are becoming a great concern.
The two main weed species of Erigeron in California are hairy fleabane (Erigeron
bonariensis L.) and horseweed (Erigeron canadensis L.), both summer annuals. Unlike