Weed Biology and Management 10, 229–233 (2010)

RESEARCH PAPER

Initial report of glufosinate and paraquat multiple

resistance that evolved in a biotype of goosegrass
(Eleusine indica) in Malaysia wbm_388 229..233

CHUAH TSE SENG,1* LOW VAN LUN,2 CHA THYE SAN2 and ISMAIL BIN SAHID3
Faculties of 1Agrotechnology and Food Science and 2Science and Technology, University of Malaysia Terengganu,
Kuala Terengganu and 3School of Environmental and Natural Resource Sciences, Faculty of Science and Technology,
University of Malaysia Kebangsaan, Bangi, Malaysia

Field and glasshouse studies have confirmed the presence of a glufosinate- and paraquat-
resistant goosegrass biotype that has infested a bitter gourd field in Air Kuning, Perak, Malaysia.
Glufosinate and paraquat had been applied at least six times per year to the affected fields
(originally a rubber plantation) for more than four consecutive years. Paraquat had been used
since 1970 for weed control in the rubber plantation. An on-site field trial revealed that the
control of the goosegrass plants, measuring 20–35 cm in height, ranged from 20 to 35% 3
weeks after being treated with each herbicide at twice the recommended rate. Dose–response
tests were conducted in the glasshouse, using seedlings at the three-to-four-leaf stage that had
been obtained from the plants that had received repeated exposure to these herbicides and a
biotype with no history of any herbicide resistance.The comparison of the GR50 (the herbicide
rate that is required to reduce the shoot fresh weight by 50%) of the seedlings indicated that
the resistant biotype of goosegrass is 3.4-fold and 3.6-fold more resistant than the susceptible
biotype following treatment with glufosinate and paraquat, respectively. This study has dem-
onstrated the world’s first field-evolved instance of multiple resistance in goosegrass to two
non-selective herbicides, glufosinate and paraquat.

Keywords: glufosinate, glyphosate, multiple resistance, paraquat.

Goosegrass (Eleusine indica [L.] Gaertn.), belonging to
the family Poaceae (Barnes & Chan 1990), is a common,
noxious grassy weed, infesting young oil palm and
rubber plantations, vegetable fields, and orchards, result-
ing in a significant loss of crop yields in Malaysia (Chee
et al. 1990). Goosegrass is considered to be one of the
serious annual grassy weeds of the world due to its high
fecundity (Chin 1979) and a wide tolerance to various
environmental factors (Ismail et al. 2002; 2003). More
than 60 countries have reported that it is a significant
weed in 46 crops (Holm et al. 1977).
Communicated by A. Uchino.
*Correspondence to: Chuah Tse Seng, Faculty of Agrotechnology and
Food Science, Universiti Malaysia Terengganu, Kuala Terengganu
21030, Malaysia.
Email: chuahts@umt.edu.my
Received 16 December 2009; accepted 1 June 2010
Currently, >20 weed species have evolved paraquat
resistance worldwide. To date, however, no glufosinate-
resistant weed biotype has been documented (Heap
2010), although various glufosinate-tolerant crops are
commercially available in the market. For instance, a rice
cultivar that is resistant to glufosinate already has been
developed through transgenic technologies (Gealy et al.
2003). However, there are concerns about the impact of
releasing glufosinate-resistant rice on weeds. Of great
concern is that glufosinate-resistant volunteer rice would
serve as a potential pollen source for the dispersal of
transgenes to weedy rice or wild relatives (Kumar et al.
2008). The development of glufosinate-resistant weedy
rice populations would limit substantially the chemical
weed-control options for farmers. Glufosinate-resistant
rice volunteers also could become problematic and the
added selection pressure to weed populations could
aggravate already-serious weed resistance problems
(Olofsdotter et al. 2000).

doi:10.1111/j.1445-6664.2010.00388.x © 2010 The Authors

Journal compilation © 2010 Weed Science Society of Japan
230 C.T. Seng et al.
Goosegrass populations have been reported to have
evolved resistance towards several groups of herbicides,
such as trifluralin (McAlister et al. 1995), glyphosate (Lee
& Ngim 2000), paraquat (Itoh et al. 1990), and fluazifop-
butyl (Marshall et al. 1993), as a result of repeated appli-
cations of the same herbicide for more than three
consecutive years.
The multiple herbicide resistance of weed species is
not a new phenomenon. Glyphosate and fluazifop mul-
tiple resistance, which evolved in a biotype of goosegrass
growing in orchards, has been reported in Malaysia
(Heap 2010). It has been documented that Hedyotis ver-
ticillata, growing in oil palm plantations in Malaysia, has
developed resistance to both glyphosate and paraquat
(Chuah et al. 2005). Likewise, Lolium rigidum, a problem-
atic weed in Australia, has been found to be resistant to
paraquat, glyphosate, and acetyl coenzyme A carboxylase
(ACCase)-inhibitors (Yu et al. 2007).
Non-selective herbicides, such as glufosinate and
paraquat, are two main herbicides that have been used
regularly by farmers in Air Kuning, Perak, Malaysia, for
the control of goosegrass in vegetable fields. These her-
bicides have been applied to these fields, either alone or
in combination, at least six times per year for more than
four consecutive years. In 2009, a farmer who produced
bitter gourd in Air Kuning complained that glufosinate
and paraquat provided poor control of goosegrass on his
farm. Hence, this study was carried out to confirm this
first case of glufosinate and paraquat resistance in
goosegrass-infesting bitter gourd fields in Air Kuning,
thereby attempting to quantify the resistance level of the
resistant biotype, as compared to that of a susceptible
biotype.
MATERIALS AND METHODS
On-site field experiment
A field trial was conducted at a bitter gourd farm in Air
Kuning (4°11.409′N, 101°8.675′E) to evaluate the effi-
cacy of glufosinate and paraquat against goosegrass. Glu-
fosinate (ammonium salt, Basta 15; Bayer, Selangor,
Malaysia) and paraquat (dichloride salt, Venger; Crop
Protection, Selangor, Malaysia) were tested at
0.90 kg ai ha-1 and 1.50 kg ai ha-1 (twice the recom-
mended rate), respectively, on different plots that mea-
sured 1 m ¥ 2 m.A completely randomized block design
was used with three replications of the treatments. The
herbicides were applied by using a compression sprayer
(Matabi Style 7; Goizper, Bergara, Spain) at 200 kPa with
a flat-fan nozzle to deliver a spray volume of 450 L ha-1.
When the trial was initiated, the target weed was a
mixture of vigorously growing young goosegrass plants,
© 2010 The Authors
Journal compilation © 2010 Weed Science Society of Japan
measuring an average height of 20 cm, and mature plants
that were already shedding seeds, measuring an average
height of 35 cm, with a density of four plants per m-2.
Qualitative visual assessments to assess the percentage of
yellowing and browning (Lee & Ngim 2000) of the
weeds were carried out at 7, 14, and 21 days after treat-
ment (DAT).
Glasshouse experiment
Seed sources
Resistant (R) seeds from the plants that survived the
herbicide treatment at the on-site field trial at the bitter
gourd farm were collected and mixed together for
further studies in the glasshouse, while the herbicide-
susceptible (S) seeds were collected from a nearby citrus
farm (4°11.205′N, 101°8.628′E) that had no history of
glufosinate or paraquat resistance.
Dose–response tests
Seeds of the R and S biotypes of goosegrass were scari-
fied by using sandpaper and they were allowed to ger-
minate in trays that measured 28 cm ¥ 56 cm and that
contained commercial soil potting mixture (Florasca
801; TURBA Earth and Humus, Papenburg, Germany)
in a glasshouse at 29 ⫾ 6°C with a 12 h photo period at
a light intensity of 800–1000 mEm-2 s-1.Two weeks after
sowing, the seedlings of both biotypes were transplanted
into seedling trays that contained the soil potting
mixture. Three days after transplanting, the seedlings of
both the R and S biotypes at the three-to-four-leaf stage
were sprayed with glufosinate or paraquat by using a
compression sprayer, as described previously. Prior to the
dose–response tests, a preliminary study was carried out,
whereby the same putative herbicide-resistant seedlings
were treated initially with glufosinate, followed by
paraquat, at twice the recommended rate to verify
paraquat and glufosinate multiple resistance in gooseg-
rass. It was found that all the seedlings survived after
being treated with both herbicides.The rates of glufosi-
nate that were used for the dose–response tests were 0,
0.03, 0.11, 0.45, 1.80, and 7.20 kg ha-1, while those for
paraquat were 0, 0.09, 0.38, 1.50, 6.00, and 24.00 kg ha-1.
The seedlings from each biotype were randomly divided
into six treatment groups, including the control plants,
with each treatment having ten plants.The experimental
design was a completely randomized block with four
replications. The level of plant survival and the shoot
fresh weight were recorded 1 week after the herbicidal
treatment.The plants were recorded as killed when they
had no new growth or active tiller formation.The shoot
 Glufosinate and paraquat resistance 231

45 0.7

40
0.6
35

Shoot fresh weight (g plant )

-1
30
Control (%)

0.5
25

20 0.4
15

10 0.3

5
0.2
0
0 7 14 21 28

Time (days after treatment) 0.1

Fig. 1. Control of goosegrass plants at a bitter gourd field 0.0

0.0001
Control 0.001 0.01 0.1 1 10 100
in Air Kuning, Perak, Malaysia, with glufosinate ( ) and
Glufosinate rate (kg ai ha -1)
paraquat (䉬) at 0.90 kg ai ha-1 and 1.50 kg ai ha-1 (twice
the recommended rate), respectively, throughout the period Fig. 2. Shoot fresh weight of the susceptible (䊊) and resis-
of 21 days after herbicidal treatment. The vertical bars tant (䊉) biotypes of goosegrass, as affected by glufosinate in
represent the standard error of the mean. the whole-plant bioassay 7 days after treatment under glass-
house conditions. Every point is a mean of four replicates,
each containing 10 plants. The vertical bars represent the
fresh weight data were fitted to a logistic regression standard error of the mean.
model, as follows (Kuk et al. 2002):
Y = d (1 + [ x x 0 ] b ) , (1) 0.6

where Y = the fresh weight of the harvested plants, d = 0.5

Shoot fresh weight (g plant )

the coefficients corresponding to the upper asymptotes,

-1

b = the slope of the line, x0 = the herbicide rate required 0.4

to inhibit the shoot growth by 50%, and x = the herbi-
cide dose. Regression analyses were conducted and the 0.3
herbicide rates that were necessary to reduce the shoot
fresh weight by 50% (GR50) were calculated from the 0.2
regression equations. The resistance level was calculated
as the GR50 of the R biotype divided by the GR50 of the 0.1
S biotype.
0.0
0.0001 0.001 0.01 0.1 1 10 100
Control
RESULTS AND DISCUSSION Paraquat rate (kg ai ha )
-1

The results that were obtained from the on-site trial at
the bitter gourd field in Air Kuning, Perak, Malaysia,
confirmed the farmer’s complaint that the goosegrass
plants had developed resistance to glufosinate and
paraquat. At twice the recommended rate of each her-
bicide, there was <40% control of goosegrass (Fig. 1).
The control of goosegrass by both paraquat and glufo-
sinate decreased by ~10% at 21 DAT, compared to that at
7 DAT (when compared to the control), indicating the
recovery of the plants at 21 DAT.The dose–response tests
that had been carried out in the glasshouse further con-
firmed that the R biotype had developed resistance to
glufosinate and paraquat.The shoot fresh weight of both
biotypes decreased as the application rates of the two test
herbicides increased, but the S biotype appeared to have
Fig. 3. Shoot fresh weight of the susceptible (䊊) and resis-
tant (䊉) biotypes of goosegrass, as affected by paraquat in
the whole-plant bioassay 7 days after treatment under glass-
house conditions. Every point is a mean of four replicates,
each containing 10 plants. The vertical bars represent the
standard error of the mean.
a more rapid decline in the shoot fresh weight, compared
to the R biotype (Figs 2 and 3). All the S biotypes were
fully controlled (100%) at the recommended rate of
glufosinate (0.45 kg ha-1), while the R biotypes were
only controlled fully (100%) when they were treated
with glufosinate at fourfold the recommended rate

© 2010 The Authors

Journal compilation © 2010 Weed Science Society of Japan
232 C.T. Seng et al.

120 Table 1. GR50 values and resistance levels of the

goosegrass biotypes in relation to glufosinate and paraquat
100
Herbicide GR50 (kg ha-1)† Resistance
80 level
R biotype S biotype
Survival (%)

60 Glufosinate 0.17 (0.01) 0.05 (0.01) 3.4

Paraquat 0.43 (0.07) 0.12 (0.02) 3.6
40
† GR50 is the herbicide rate required to reduce the shoot fresh weight
20
by 50%. The values in parentheses are the standard error of the mean.

0
Control
0.001 0.01 0.1 1 10
showed a 3.4-fold and 3.6-fold resistance to glufosinate
Glufosinate rate (kg ai ha-1) and paraquat, respectively, compared to that of the S
biotype.
Fig. 4. Survival of the susceptible (䊊) and resistant (䊉)
According to the farmer, he began the application of
biotypes of goosegrass, as affected by glufosinate in the
glufosinate to his replanted vegetable field (originally a
whole-plant bioassay 7 days after treatment under glass-
rubber-growing area) in 2004. After 5 years of applica-
house conditions. Every point is a mean of four replicates,
tion of glufosinate alone or in combination with
each containing 10 plants. The vertical bars represent the
paraquat, with at least six rounds per year at the recom-
standard deviation of the mean.
mended rate, the vegetable field became overgrown and
dominated by goosegrass as a result of a lack of control
120
by these herbicides.
A simulation study was conducted by Diggle et al.
100
(2003) to examine the effect of the pattern of herbicide
use on the development of resistance to two herbicides
80
with different modes of action. It was found that the
development of resistance to both herbicides is uncom-
Survival (%)

60
40
20
0
Control
0.01 0.1 1 10
Paraquat rate (kg ai ha-1)
Fig. 5. Survival of the susceptible (䊊) and resistant (䊉)
biotypes of goosegrass, as affected by paraquat in the whole-
plant bioassay 7 days after treatment under glasshouse con-
ditions. Every point is a mean of four replicates, each
containing 10 plants.
(1.80 kg ha-1) (Fig. 4). For the paraquat-treated plants, all
the S biotypes were killed at twice the recommended
rate (1.5 kg ha-1), while all the R biotype plants survived
at this rate (Fig. 5). The GR50 values for the R and S
biotypes are shown in Table 1, together with the respec-
tive resistance level for each herbicide. The R biotype
mon within 50 years, for all types of weeds, if the her-
bicides are used in combination throughout the years. In
the present study, glufosinate and paraquat multiple resis-
tance evolved in goosegrass within 5 years despite the
application of the herbicide combination to the veg-
etable field.The frequency of the herbicide applications
and the herbicide pattern use are important criteria that
100 determine the rate of resistance development.The simu-
lation model of Diggle et al. (2003) specified that both
herbicides were applied in combination once per year,
while in this case of multiple resistance in goosegrass,
glufosinate alone or in combination with paraquat had
been applied at least six times per year. An increased
frequency of application and a previous history of weed-
control applications have increased the selection pressure
of both herbicides, thereby contributing to the rapid
evolution of multiple resistance in goosegrass and the
deviation from the results in the simulation study by
Diggle et al. (2003). However, it is likely that the devel-
opment of paraquat resistance in goosegrass could have
occurred earlier due to its previous use for weed control
in the rubber plantation from 1970.
This first appearance of multiple resistance to glufo-
sinate and paraquat in goosegrass is indeed a major

© 2010 The Authors

Journal compilation © 2010 Weed Science Society of Japan

problem for both growers and the crop protection
industry. Similarly, Lolium rigidium in Australia has
developed multiple resistance to nine groups of herbi-
cides (Burnet et al. 1994). Currently, integrated weed
management practises are recommended to growers in
order to effectively manage this problematic weed
species (Pannell et al. 2004). The results of the present
study have provided an early warning to farmers,
growers, and plantation managers worldwide regarding
the evolution of multiple herbicide resistance in
goosegrass after the frequent application of glufosinate
and paraquat. This alarming case in the history of her-
bicide resistance evolution poses a great challenge for
the sustainable use of the precious agrochemical
resources of glufosinate and paraquat.
It has been documented that the glyphosate, paraquat,
and ACCase herbicide multiple resistance that evolved in
a L. rigidum biotype in Australia was related to four
co-existing resistance mechanisms, including point
mutation in the 5-enolpyruvyshikimate-3-phosphate
synthase gene, reduced translocation of paraquat and
glyphosate, and an insensitive ACCase (Yu et al. 2007).
The present study, however, was designed to confirm the
presence of the resistance of a goosegrass biotype to
glufosinate and paraquat. It is proposed that more than
one resistance mechanism could confer multiple resis-
tance on goosegrass. Further studies are currently in
progress to determine the actual resistance mechanisms
of goosegrass to both glufosinate and paraquat, which
involves examining the R and S biotypes for the differ-
ences in their herbicide uptake, translocation, metabo-
lism, and site of action, factors that contribute to the
evolution of multiple herbicide resistance.
ACKNOWLEDGMENTS
The authors express their sincere thanks to Mrs. Alena
Sanusi for reading and correcting the manuscript.
REFERENCES
Barnes D.E. and Chan L.G. 1990. Common Weeds of Malaysia and Their
Control. Ancom Berhad, Shah Alam, Malaysia.
Burnet M.W.M., Hart Q., Holtum J.A.M. and Powles S.B. 1994.
Resistance to nine herbicide classes in a population of rigid ryegrass
(Lolium rigidum). Weed Sci. 42, 369–377.
Chee Y.K., Lee S.A., Ahmad A.I., Teo L., Chung G.F. and Khairuddin
H. 1990. Crop loss by weeds in Malaysia. In: Proceedings of the 3rd
Tropical Weed Science Conference (Kuala Lumpur, Malaysia, 4–6
Journal compilation © 2010 Weed Science Society of Japan
Glufosinate and paraquat resistance 233
December 1990). Malaysian Plant Protection Society, Kuala Lumpur,
Malaysia, 1–21.
Chin H.F. 1979. Weed seed – A potential source of danger. In:
Proceedings of the Plant Protection Seminar (Kuala Lumpur, 22–23
September 1979). Malaysian Plant Protection Society, Kuala Lumpur,
Malaysia, 115–119.
Chuah T.S., Noor-Zalila M.R., Cha T.S. and Ismail B.S. 2005. Paraquat
and glyphosate resistance in woody borreria (Hedyotis verticillata)
growing at oil palm plantations in Terengganu, Malaysia. Malays. Appl.
Biol. J. 34, 43–50.
Diggle A.J., Neve P.B. and Smith F.P. 2003. Herbicides used in
combination can reduce the probability of herbicide resistance in
finite weed populations. Weed Res. 37, 371–382.
Gealy D.R., Mitten D.H. and Rutger J.N. 2003. Gene flow between
red rice (Oryza sativa) and herbicide-resistant rice (O. sativa):
Implications for weed management. Weed Technol. 17, 627–645.
Heap I.M. 2010. International Survey of Herbicide Resistant Weeds. Weed
Science Society of America, Lawrence, KS. [Cited 12 December
2009.] Available from URL: http://www.weedscience.org/In.asp.
Holm L.G., Plucknett D.L., Pancho J.V. and Herberger J.P. 1977. The
World’s Worst Weeds – Distribution and Biology. The University Press of
Hawaii, Honolulu, HI.
Ismail B.S., Chuah T.S., Salmijah S., Teng Y.T. and Schumacher R.W.
2002. Germination and seedling emergence of the
glyphosate-resistant and susceptible biotype of goosegrass (Eleusine
indica [L.] Gaertn.). Weed Biol. Manag. 2, 177–185.
Ismail B.S., Chuah T.S., Salmijah S. and Teng Y.T. 2003. Effects of
shade and watering frequency on growth and development of
glyphosate-resistant and susceptible biotypes of goosegrass (Eleusine
indica [L.] Gaertn.). Plant Protect. Q. 18, 30–34.
Itoh K., Azmi M. and Ahmad A. 1990. Paraquat resistance in
Amaranthus lividus and Conyza sumatrensis in Malaysia. In: Proceedings of
the 3rd Tropical Weed Science Conference (Kuala Lumpur, 4–6 December
1990). Malaysian Plant Protection Society, Kuala Lumpur, Malaysia,
489–493.
Kuk I.Y., Kwon O.O., Jung H.I., Burgos N.R. and Guh O.J. 2002.
Cross-resistance pattern and alternative herbicides for Rotala indica
resistant to imazosulfuron in Korea. Pestic. Biochem. Physiol. 74,
129–138.
Kumar V., Bellinder R.R., Brainard D.C., Malik R.K. and Gupta R.K.
2008. Risks of herbicide-resistant rice in India: a review. Crop Prot.
27, 320–329.
Lee L.J. and Ngim J. 2000. A first report of glyphosate-resistant
goosegrass (Eleusine indica (L) Gaertn.) in Malaysia. Pest Manag. Sci.
56, 336–339.
McAlister F.M., Holtum J.A.M. and Powles S.B. 1995. Dinitroaniline
herbicide resistance in rigid ryegrass (Lolium rigidum). Weed Sci. 43,
55–62.
Marshall G., Kirkwood R.C. and Leach L.E. 1993. Comparative studies
on graminicide-resistant and susceptible biotypes of Eleusine indica.
Weed Res. 36, 177–185.
Olofsdotter M., Valverde B.E. and Madsen K.H. 2000. Herbicide
resistant rice (Oryza sativa L.): global implications for weedy rice and
weed management. Ann. Appl. Biol. 137, 279–295.
Pannell D.J., Stewart V., Bennett A., Monjardino M., Schmidt C. and
Powles S.B. 2004. RIM: a bioeconomic model for integrated weed
management of Lolium rigidum in Western Australia. Agric. Syst. 79,
302–325.
Yu Q., Cairns A. and Powles S.B. 2007. Glyphosate, paraquat and
ACCase multiple herbicide resistance in a Lolium rigidum biotype.
Planta 225, 499–513.
© 2010 The Authors