Understanding Resistance to ALS-inhibiting Herbicides
by Prickly Lettuce
Mario Barco , Attawan Aramrak, Jared Bell and Ian C. Burke
Weed Biology-Biotechnology, Washington State University, Pullman, WA
Introduction
Weed control is a top priority to growers, conservationists and land
managers alike. Invasive species compete for light, water and nutrients
which in turn decreases agricultural output and potential land
productivity. Herbicides are used extensively to control weed
populations, however many weeds have become resistant to chemical
control methods. Resistant biotypes develop through excessive long-term
use of a single herbicide type and lack of integration of alternative
herbicides and their modes of action. Misuse of recommended label
rates and soil residual activity of the herbicide, have also lead to the
emergence of resistant biotypes (Tranel and Wright 2002).
Since the introduction of acetolactate synthase (ALS) inhibitors
herbicides in the 1980’s, the frequency of herbicide-resistance (R)
species to those herbicides has become greater than any other herbicide
class (White et al. 2003). ALS catalyzes the formation of the essential
amino acids valine, leucine, and isoleucine, all of which are necessary for
protein formation and subsequent plant development. The function of
the ALS inhibitor is to inactivate this enzyme, essentially starving the
plant of these amino acids. Without these amino acids, protein synthesis
and photosynthate transport is disrupted leading to decreased root and
shoot growth followed by plant death. It is known that one or more
amino acid substitutions in the ALS protein are sufficient to convert an
herbicide-susceptible (S) weed to an (R) one.
(b) Prosulfuron
(a) Chlorsulfuron
Results
Greenhouse Experiment
• Preliminary data using Analysis of Variance
means (AOV) indicates that the most
effective ALS inhibiting herbicides are
Imazapyr (90%), Florasulam (88%),
Triasulfuron (~42%), Tribenuron (38%) and
Chlorsulfuron (36%). The least effective
herbicides were Imazethapyr (2%) and
Imazapic (4%), Table 1.
NA
NA
(d) Imazapyr
(c)
Imazapic
(e) Tribenuron
Table 1. Analysis of variance for each
herbicide used to control 20 biotypes of
prickly lettuce.
Laboratory Experiment
• Optimal primers to amplify the nucleotide sequence containing
Domain A (220 base pair long) in the ALS gene were obtained, these
primer pair was designated as PL-ALSPT-F and PL-ALSPT-R.
• Another potential pair of primers called PL-ALS-F4 and PL-ALS-R4
(1400 base pair long) is currently been optimized for purification. In
the mean time, successful ligation and transformation has been
achieved with PL-ALSPT pair.
(h) Florasulam
In this study, 20 previously collected Eastern Washington prickly lettuce
(Lactuca serriola) biotypes with known ALS herbicide resistance were
studied. Biotypes were grown in a greenhouse and sprayed with various
ALS inhibiting herbicides to determine resistance to various ALS
herbicides. In conjunction with greenhouse work, ALS mutations were
studied by aligning sequences of ALS genes from the 20 biotypes (Figure
3).
a
NA
Figure 3. Partial amino acid sequence alignment of ALS enzyme from various plant species
used in initial primer design.
a Nucleotide accession numbers identified in the Genbank nonredundant database:
AY541451 (Helianthus annuus), XSU16280 (Xanthium sp), EU827603 (Sonchus asper). Prickly lettuce (Lactuca serriola) obtained from Guttieri et al 1991.
(i) Triasulfuron
(j) Imazethapyr
A
Objectives
• Select optimal primers to amplify and clone the complete or partial
ALS gene in prickly lettuce.
• Identify amino acid substitutions in the ALS gene of (R) vs. (S) biotypes
to determine if the resistance is due to target site mutation.
• Correlate amino acid substitutions to herbicide resistance patterns.
• Twenty different biotypes of prickly lettuce were planted in individual
79 cm2 plastic pots. Each biotype was treated with 12 different ALS
inhibiting herbicides. Each treatment was replicated four times. In
total, there were 960 pots.
• Each pot was seeded with 5-10 seeds to insure germination and later
thinned to 2-4 plants per pot.
• Two weeks after germination, pots were sprayed with ALS inhibitor
herbicides (Table 1). Each treatment was considered a main plot while
each biotype was a sub-plot. Each application was repeated four times.
For comparison purposes, two non-treated controls were included.
• Herbicides were applied at the recommended label rates with the
recommended surfactants.
• Two days after herbicide treatment, pots containing three to four
plants were trimmed to two plants per pot to observe any re-growth.
• Visual estimates of percent of injury were recorded 14 and 21 days
after herbicide application.
• Aboveground biomass was harvested after 23 days and wet dry
weights were recorded. Biomass was dried in a forced air oven at 60oC
and dry weights were recorded after four days after harvest.
AOV
36.72
35.4
4.37
90.43
38.58
22.17
35.09
88.17
41.89
2.22
• The most effective ALS inhibitor herbicides controlled the (R)
biotypes. However, biotypes 1*9/21, 2*9/21, 3*9/21, 6*/21 and
7*10/6 were not well controlled by any ALS inhibitors. The (S)
biotypes that were partially or completely controlled with the least
effective herbicide were 3*10/4, 3*10/6, 5*10/6, 3*9/13 and
5*9/13 (Figure 1)
(f) Thifensulfuron
(g) Metsulfuron
Materials and Methods
a
b
c
d
e
f
g
h
i
j
Herbicide name
Chlorsulfuron (Glean)
Prosulfuron (Peak)
Imazapic (Cadre)
Imazapyr (Arsenal)
Tribenuron (Express)
Thifensulfuron (Harmony)
Metsulfuron (Ally)
Florasulam
Triasulfuron (Amber)
Imazethapyr
NA
Figure 1. Control of 20 prickly lettuce biotypes three weeks after herbicide application.
A
C
E
B
D
F
Figure 2. Pictures A and B were taken after two weeks of seeding- no herbicide application.
Pictures C and D were taken before herbicides applications. Pictures E and F were taken two
weeks after herbicides applications.
B
C
D
Figure 4. Gel electrophoresis image representing different steps along the cloning process of
Domain A. Image A indicates potential primers for ALS gene amplification at different
annealing temperatures. Image B represents PCR product at the 220 and 1400 base pair
bands. Image C is the result of PCR purification at the 220bp level, no PCR purification at
the1400 bp level present. Image D specifies successful ligation and transformation of this
segment.
Continuing Research
Although successful amplification of sequence containing Domain A
was achieved, primer design should focus on maximize either the total
length or a bigger portion of the enzyme to include the remaining
domains that play key factors on ALS resistance, using the selected
primer pairs (Figure 4A). In the meantime, studies will be conducted to
identify possible points of mutations within the sequence containing
Domain A in resistant biotypes and the newly amplified sequence
obtained in the laboratory.
References
• Guttieri M. J., C. V. Eberlein, C. A. Mallory-Smith, D. C. Thill and D. L. Hoffman. 1992. DNA Sequence Variation in Domain A
of the Acetolactate Synthase Genes of Herbicide-Resistant and -Susceptible Weed Biotypes. Weed Sci. 40: 670 – 677.
• Kolkman, J. M., M. B. Slabaugh, J. M. Bruniard, S. Berry, B. S. Bushman, C. Olungu, N. Maes, G. Abratti, A. Zambelli, J. F.
Miller, A. Leon, and S. J. Knapp. 2004. Acetohydroxyacid synthase mutations conferring resistance to imidazolinone or
sulfonylurea herbicides in sunflower. Theo. Applied Genetics 109: 1147 - 1159.
• Tranel P. J. and T. R. Wright. 2002. Resistance of Weeds to ALS-Inhibiting Herbicides: What Have We Learned? Weed Sci.
50: 700 – 712.
• White A. D., M. A. Graham, and M.D.K. Owen. 2003. Isolation of Acetolactate Synthase Homologs in Common Sunflower.
Weed Sci. 51:845 - 853
Acknowledges
• This study was conducted thanks to the faculty and staff from the department crop and soil sciences under the direction of
Dr. Ian C. Burke in the summer 2012. Dr. Burke’s Lab ‘s dedication, hard work, knowledge ,and passion for what they do
made this REU experience one of a kind.
• This work was supported by the National Science Foundation REU Program under grant number DBI-1156880