The Effects of Herbicide Cycling on the Evolution of Resistance in
Chlamydomonas reinhardtii
M.Lagator*, T.Vogwill*, N.Colegrave & P.Neve*
‡
*School of Life Sciences, The University of Warwick, Coventry, UK
‡School of Biological Sciences, The University of Edinburgh, Edinburgh, UK
I. Introduction
Of all agricultural pests, weedy plants have the greatest
potential to cause losses in crop yield¹. The pace at which
resistance is evolving far exceeds our ability to produce
novel active chemicals, and as such we need to find ways
to employ herbicides in a sustainable manner. A commonly recommended practice is to cycle herbicides with
different modes of action². Due to difficulties associated
with performing selection experiments on large weed
populations with slow generation times, the validity of
cycling as a method of retarding the evolution and spread
of resistance has rarely been experimentally tested.
We experimentally evolved populations of Chlamydomonas reinhardtii, a single-cell green algae susceptible to a range of commercial herbicides, in
order to investigate the effects of cycling on (i) the
dynamics of resistance and how rapidly it evolved;
(ii) the pleiotropic fitness costs in the absence of
herbicides; (iii) the evolution of generalist traits
and a wider pattern of cross-resistance.
III. Cycling Can Accelerate or Retard the
Evolution of Herbicide Resistance
The number of weeks until resistance evolved to individual herbicides in cycling regimes was compared
for each regime to the rate of evolution in populations under continuous exposure to that herbicide
(fig.2a for atrazine, 2b for glyphosate, 2c for carbetamide). We found that cycling can:
- enhance the rates of resistance evolution - weekly cycle between atrazine and glyphosate with regards to atrazine (z=10.169,df=1,P=0.001) as well as to glyphosate (z=3.930,df=1,P<0.05);
- significantly hamper the evolution of resistance (cycling between glyphosate and carbetamide, and
bi- and tri-weekly cycle between atrazine and carbetamide with respect to carbetamide);
- not have an effect on the rates of resistance evolution.
Box 1. Generalist vs. Specialist
Herbicide Resistance
Generally, herbicide resistance
can be target-site or non
target-site². Target-site resistance is most commonly due to
a single amino acid change in
the herbicide target site, and
confers resistance to a single
mode of action (specialist). Non
target-site mechanisms are more
diverse and tend to be inherited
We experimentally evolved C.reinhardtii populations as quantitative traits. They are
often associated with a pattern
for 12 weeks under continuous exposure to miniof cross-resistance to other
mum inhibitory concentrations of three herbicides
modes of action (generalist).
II. Methods
(Atrazine, Glyphosate and Carbetamide), as well as
to a weekly, bi-weekly and tri-weekly rotation between all possible pairings of the three herbicides.
The optical density at 750nm was measured 4 days
after each transfer for each experimental condition
and used to obtain the number of cell divisions that
have occurred. A strain was called resistant when it
underwent at least 3 cell divisions during this period.
Figure 1. Experimental conditions
through the 12 experimental weeks:
The figure illustrates the transfer
protocols over the period of 12
weeks. A week spent in Atrazine (A)
is represented in red, in Glyphosate
(G) in yellow, and a week in
Carbetamide (C) in green.
The rate of resistance evolution was analyzed using a Cox
regression, with herbicide regime fitted as a covariate. All
the conditions that were under a cycling regime were
compared in a pair-wise fashion to that herbicide in continuous exposure.
Resistance and fitness levels of all conditions under cycling were compared to continuous exposure treatment
in a Dunnett’s corrected T-test, with the herbicide
regime fitted as a fixed factor.
VI. Conclusions
The results presented in this study show the variety of effects that herbicide cycling can have on the rates and outcomes of resistance evolution. Most importantly, our results
question the wide-spread use of herbicide cycling as a
method of hampering resistance evolution.
IV. Intermediate and Slower Rates of Cycling
Can Reduce Costs of Resistance
Compared to the growth rate of naive cells in herbicide free
environment, following regimes gave rise to significantly different fitness, in each case resulting in higher costs (fig.3):
continuous exposure to atrazine (T=-4.420, P<0.001)
cont. exposure to glyphosate (T=-5.428, P<0.001)
cont. exposure to carbetamide (T=-4.711, P<0.05)
weekly cycle between atrazine and glyphosate
(T=-9.569, P<0.001)
weekly cycle between atrazine and carbetamide
(T=-6.034, P<0.001)
We found that lower rates of cycling were associated with
higher fitness, and therefore lower costs of resistance.
We would like to thank
Leverhulme Trust for funding the project. Carol
Evered for all her invaluable help and hard work.
Further Information
Figure 3. Fitness of the populations with evolved resistance, estimated as the fraction of naive cell line growth in the absence of any
herbicides. Bars are mean fitness of all evolved populations under
that conditions, error bars are std.err.
V. Rapid Rates of Cycling Can Lead to the
Evolution of Generalists
We found that rapid rates of cycling can alter the trajectory of evolution, which occurred in the rapid
cycle between atrazine and carbetamide. We observed the evolution of a generalist, characterized by:
- a pattern of cross-resistance - AC1 in table 1
- higher resistance - significant difference when compared to continuous atrazine exposure populations (T=5.487, P<0.001) (fig.4)
We show that the effects of herbicide cycling depend, to an
extent, on the rates of cycling, with rapid rates being most
dangerous. It also illustrates how dependent the effects of
cycling are on the particular herbicides used.
Acknowledgements
Figure 2. The number of weeks until resistance was first observed, measured only in terms of the weeks exposed to that
herbicide. Bars are mean between weeks to resistance of all
the evolved populations; error bars are std.err. a) dynamics
of atrazine resistance; b) dynamics of glyphosate resistance;
c) dynamics of carbetamide resistance
Literature Cited
For further information
1. Oerke 2006, J Agri
about this project or other
Sci 144 p.31-43
similar work, please con- 2. Powles and Yu 2010,
tact Mato Lagator at
Annul. rev. plant biol 61
mato.lagator@gmail.com
p.317-347
Table 1. The shaded area indicates that the evolved strain exhibited
growth in the presence of a particular herbicide it had no previous
exposure to: S=S-metolachlor; I=Iodosulphuron; Iso=Isoproturon;
T=tembotrione.
Figure 4. Resistance in the evolved environment, presented as the fraction of naive
cell line growth in the absence of herbicides. Each population that was under a cycling regime had it’s resistance estimated in both herbicides it experienced. Cycling
regimes were compared to the continuous exposure under appropriate herbicide.