Jasmonic acid-induced resistance to the fall armyworm,

Spodoptera frugiperda, in conventional and transgenic

cottons expressing Bacillus thuringiensis insecticidal
proteins
Anna Mszros
1
*, Julien M. Beuzelin
1
, Michael J. Stout
1
, Padma L. Bommireddy
2
,
M. Rita Riggio
1
& B. Rogers Leonard
1
1
Department of Entomology, Louisiana State University Agricultural Center, Baton Rouge, LA 70803, USAand
2
Monsanto
Company, Saint Louis, MO 63167, USA
Accepted: 7 June 2011
Key words: plant elicitor, relative growth rate, Bt technology, conventional breeding, GMO,
Bollgard

, Bollgard II

, synergism, Lepidoptera, Noctuidae, Malvaceae, Gossypiumhirsutum

Abstract To assess potential interactions between Bacillus thuringiensis Berliner (Bt) proteins and jasmonic
acid (JA)-induced resistance to the fall armyworm, Spodoptera frugiperda Smith & Abbot (Lepido-
ptera: Noctuidae), three commercial Stoneville cotton cultivars, Gossypium hirsutum L. (Malvaceae)
(ST 475, a conventional cultivar; ST 4575 BR, a Bollgard

cultivar expressing Cry1Ac protein; and

ST 4554B2RF, a Bollgard II

cultivar expressing Cry2Ab2 in addition to Cry1Ac), were treated with

JA. Two experiments were conducted with 6-day-old S. frugiperda larvae on three cotton cultivars at
two phenological stages. The rst experiment was conducted under laboratory conditions and used
excised cotton leaves; the second experiment was performed under greenhouse conditions on intact
cotton plants. Relative growth rates (RGRs) and leaf area consumed by 6-day-old S. frugiperda larvae
were determined for each combination of treatments. Overall, JAtreatment and cultivars signicantly
impacted RGR and leaf area consumption. Signicant JA treatment*cultivar interactions were
observed for RGR of larvae in the laboratory experiment and for leaf area consumption in the green-
house experiment. An additional experiment evaluated S. frugiperda neonates on the same JA and
cotton cultivar combinations (at a single phenological stage) under laboratory conditions. Neonate
survival was determined after 3, 5, and 10 days of feeding, and nal larval weight after 10 days of
feeding. Overall, JA treatment and cultivars signicantly impacted nal weight and survival of S. fru-
giperda. Signicant JA treatment*cultivar interactions were observed for nal weight and on overall
survival of S. frugiperda. Combination of the cotton tissue expressing pyramided Bt proteins with JA
treatment demonstrated the greatest negative impact on larval development. Apparent synergism
between Bt proteins and JA-induced resistance emphasizes that traditional host plant resistance has a
role to play in combination with Bt technology.
Introduction
Cotton, Gossypium hirsutum L. (Malvaceae), supplies
nearly 35% of the total global demand for ber (USDA-
ERS, 2009), with a total production of 24.8 billion kg in
2010 (National Cotton Council of America, 2010). His-
torically, heavy insecticide use has been necessary to pro-
tect cotton against a diverse assemblage of insect pests.
However, advances in cotton pest management practices
and technologies have reduced the use and negative
impact of insecticides in the past decades (Naranjo et al.,
2008). In particular, the development of transgenic culti-
vars expressing Bacillus thuringiensis Berliner (Bt) Cry
proteins against primary lepidopteran pests has played a
central role in cotton pest management for the past
14 years (Brookes & Barfoot, 2010). Globally, during the
period 19962005, the use of insecticide was reduced by
94.5 million kg of active ingredient in genetically
*Correspondence: Anna Meszaros, Department of Entomology, Lou-
isiana State University Agricultural Center, 404 Life Sciences Bldg.,
Baton Rouge, LA70803, USA. E-mail: ameszaros@agcenter.lsu.edu
2011 The Authors Entomologia Experimentalis et Applicata 140: 226237, 2011
226 Entomologia Experimentalis et Applicata 2011 The Netherlands Entomological Society
DOI: 10.1111/j.1570-7458.2011.01149.x
modied insect-resistant cotton crops (Brookes & Bar-
foot, 2010). Commercial transgenic cotton cultivars
express either Cry1Ac alone (Bollgard

; Monsanto, St
Louis, MO, USA) or both Cry1Ac and Cry2Ab proteins
(Bollgard II

; Monsanto) (Adamczyk & Gore, 2004). Key

lepidopteran pests of cotton such as tobacco budworm,
Heliothis virescens (Fabricius), and pink bollworm, Pecti-
nophora gossypiella Saunders, are controlled by Bollgard

cultivars, but the Cry1Ac protein alone does not have

substantial negative effects on several other pests, includ-
ing beet armyworm, Spodoptera exigua Hubner, fall
armyworm, Spodoptera frugiperda Smith & Abbot
(Lepidoptera: Noctuidae), and cotton bollworm, Heli-
coverpa zea Boddie (Adamczyk et al., 1998; Luttrell &
Mink, 1999). The additional expression of Cry2Ab with
Cry1Ac improved the efcacy of Bollgard II

cultivars
against these pests. However, the combination of two Cry
proteins does not provide complete protection against
Spodoptera spp. (Stewart et al., 2001; Adamczyk & Gore,
2004; Naranjo et al., 2008).
Conventional plant breeding has produced cotton
genotypes with higher concentrations of resistance-related
traits such as gossypol and terpenoids that have negative
effects on the growth and development of a broad range of
insect pests (Cai et al., 2009). Previous studies in cotton,
potato, Solanum tuberosum L., and soybean, Glycine max
(L.) Merr., have shown that combining the expression of
natural resistance-related traits with Bt proteins may have
additive or synergistic effects on plant resistance to a target
pest (Sachs et al., 1996; Coombs et al., 2002; Cooper et al.,
2004; Zhu et al., 2008; Anilkumar et al., 2009). Thus,
breeding cotton plants to express greater levels of constitu-
tive non-Bt resistance may represent a tactic compatible
with Bt technology and improve insect pest management.
Furthermore, prior work with cotton plants indicates that
resistance and the expression of resistance-related traits is
plastic and can be enhanced by herbivory (Karban, 1986;
Alborn et al., 1996; Agrawal & Karban, 2000) or applica-
tions of natural plant elicitors such as jasmonic acid (JA)
(Omer et al., 2001; Opitz et al., 2008). Jasmonic acid is a
plant hormone produced after herbivore injury that
stimulates the expression of plant compounds involved
in resistance (Thaler et al., 1996; Korth & Thompson,
2006). Specically in cotton, induced resistance affects
leaf and reproductive tissue oxidative status, nutritive
value, cell lignication, and secondary metabolism (Bi
et al., 1997).
In this study, JA was used to induce resistance to S. fru-
giperda in conventional and Bt-expressing cotton cultivars
in order to compare the relative impacts of JA-induced
resistance and Bt proteins on S. frugiperda, used as a model
insect. Exogenous JA was found to induce resistance in
cotton plants and to act synergistically with Bt proteins
against S. frugiperda. The implications of these ndings
for plantinsect interactions and pest management in cot-
ton are discussed.
Materials and methods
Three experiments assessed the effect of JA treatment and
Bt proteins on S. frugiperda growth and development.
Two experiments were conducted with 6-day-old S. fru-
giperda larvae on three commercial cotton cultivars at two
phenological stages. The rst experiment was conducted
under laboratory conditions exclusively on excised cotton
leaves, and the second experiment was conducted under
greenhouse conditions on intact cotton plants. Relative
growth rate (RGR) and leaf area consumed by larvae were
determined for each combination of experimental treat-
ments (Table 1). The third experiment was conducted
with S. frugiperda neonates on the same cotton cultivars at
a single phenological stage under laboratory conditions.
Larval survival was determined after 3, 5, and 10 days of
feeding, and larval weight was determined after 10 days of
feeding.
Insect colony
A new S. frugiperda colony was established in 2008 from
larvae and pupae obtained from a colony maintained for
2 years at the Louisiana State University (LSU) Agricul-
tural Centers Macon Ridge Research Station (Winnsboro,
LA, USA; 329N, 9142W). The original colony was
established fromlarvae collected in 2006 fromeld corn in
Winnsboro. Larvae from this original colony still actively
fed on corn and cotton leaf tissue when the new colony,
used in this study, was established. The new colony was
maintained in a controlled-environment rearing room
(27 1 C, 7080% r.h., and L14:D10 h photoperiod) on
the campus of LSU (Baton Rouge, LA, USA). Each week,
80 pupae were placed into two plastic cylinders (7.5 l) con-
taining vermiculite and covered by cheesecloth to prevent
escape and to provide a surface for oviposition. Moths
were fed with water and honeybeer solution (150 ml
water, 150 ml honey, 12 g ascorbic acid, 355 ml beer).
Three times a week, eggs were collected from cheesecloth
and placed into a plastic bag. Upon eclosion, larvae were
placed individually into 30-ml plastic cups containing arti-
cial fall armyworm diet (Southland Product, Lake
Village, AR, USA) until pupation occurred.
Plant material
Three commercial Stoneville (ST) cotton cultivars were
used in all experiments: ST 475, a conventional cultivar;
ST 4575 BR, a Bollgard

cultivar expressing Cry1Ac; and

JA-induced resistance to Spodoptera frugiperda in cotton 227
ST 4554B2RF, a Bollgard II

cultivar expressing Cry2Ab2

in addition to Cry1Ac. Although these cultivars are not
isolines, the two transgenic cultivars both have ST 475 in
their recent pedigree. Seeds were obtained from Bayer
CropScience (Research Triangle Park, NC, USA). Plants
were grown in a greenhouse on the campus of LSU. Seeds
were sown in 15-cm-diameter pots (1.25 l) in potting soil
(Kleggs Nursery, Baton Rouge). Three seeds were planted
in each pot, and 2 weeks later, plants were thinned to one
plant per pot. Plants were watered daily and fertilized
3 weeks after planting with 931316 Scotts Osmocote

PRO Controlled Release Fertilizer (19:5:8 plus minor with

Poly-S nitrogen; The Scotts Company, Marysville, OH,
USA). No pesticides were used. The greenhouse was illu-
minated with natural light under Louisiana summer con-
ditions (30 10 Cand 85 10%r.h.).
Jasmonic acid treatments
A preliminary experiment demonstrated that the applica-
tion of a 2-mM solution of JA induced changes in the cot-
ton conventional cultivar, reducing S. frugiperda larval
growth but not causing phytotoxicity. This concentration
was used in all experiments described in this study. To pre-
pare this solution, 42 mg of JA (Sigma-Aldrich, St. Louis,
MO, USA) was dissolved in 1 ml ethanol, which was then
dispersed in 100 ml water to obtain a nal concentration
of 2 mM JA.
Plants were randomly assigned to a control or a JAtreat-
ment group. Each plant in the treatment group was
individually sprayed with 1.52 ml of JAsolution (approx-
imately 630 lg JA per plant), using a portable sprayer
(Preval spray gun, Power unit 97.6 ml Jar Cap 168.6 ml;
Coal City, IL, USA). Each plant in the control group was
sprayed with the same amount of a control solution (1 ml
ethanol in 100 ml water). The amount of solution applied
to leaves thoroughly wet the adaxial surface, until some
solution ran off. Jasmonic acid-treated plants were sprayed
separately from non-JA-treated plants to avoid treatment
contamination. Plants were allowed to dry before being
returned to the greenhouse, where they were randomly
redistributed and held until used in experiments.
Experiments 1 and 2
Two experiments were conducted with 6-day-old S. fru-
giperda larvae. In both experiments, the effect of JA was
evaluated on three cotton cultivars at two phenological
stages. Thus, 12 combinations of experimental treatments
(JA treatment cultivar phenological stage; Table 1)
were studied in the environmental chamber (experiment
1) and greenhouse (experiment 2).
To obtain the two desired phenological stages of cotton
simultaneously, two separate plantings were conducted (9
July and 6 August) to produce 5- and 9-week-old plants at
the time of JA application. The 5-week-old plants (hereaf-
ter referred to as small plants) had 56 true leaves, and the
9-week-old plants (hereafter referred to as large plants)
were at the rst week of owering stage. A total of 360
plants were randomly assigned to either a control (90 large
and 90 small plants) or a treatment group (90 large and 90
small plants). Each plant in a group was then sprayed with
either the control or JA solution. Small plants were treated
with slightly less JA solution than large plants because of
their smaller leaf area. After treatment, plants were divided
into two equal groups. Eachgroupcontained 180 plants, 15
plants for each combination of the 12 experimental treat-
ments (Table 1). One group was assigned to experiment 1
Table 1 Combinations of experimental treatments [cultivar phenological stage jasmonic acid (JA) treatment] for environmental
chamber (experiment 1) and greenhouse (experiment 2) experiments
Treatment no. Cultivar Bt protein Plant phenological stage
1
JAtreatment
1 Conventional ST475 Small No
2 Yes
3 Bollgard

ST4575 BR Cry1Ac Small No

4 Yes
5 Bollgard II

ST4554B2RF Cry1Ac + Cry2Ab2 Small No

6 Yes
7 Conventional ST475 Large No
8 Yes
9 Bollgard

ST4575 BR Cry1Ac Large No

10 Yes
11 Bollgard II

ST4554B2RF Cry1Ac + Cry2Ab2 Large No

12 Yes
1
At the time of JAapplications, small plants were 5 weeks old and had 56 true leaves, large plants were 9 weeks old and were at the rst
week of owering stage.
228 Me szaros et al.
(environmental chamber), and the other to experiment 2
(greenhouse).
Larval stage of S. frugiperda used in experiments was
synchronized by using only 6-day-old larvae that had
molted within the previous 12 h. Twice as many larvae as
needed (about 800) were selected, starved for at least 5 h
to allow their guts to void, and weighed. Starving larvae
ensured immediate feeding on plant material from the
beginning of experiments. Larval weights ranged between
100 and 250 mg, and larvae were arranged into weight
groups (100119, 120129 mg, etc.). Each combination of
experimental treatments received an equivalent number of
larvae from each weight group to minimize variation in
mean weights among the combinations of experimental
treatments.
Experiment 1. Five and 10 plants for each combination of
experimental treatments (Table 1) were selected 7 and
8 days after JAtreatment, respectively, to initiate two feed-
ing assays 24 h apart. Plants were moved from the green-
house to the laboratory. From each plant, the last fully
expanded youngest true leaf (closest to the terminal) was
removed with a razor blade and placed into a Petri dish
(150 25 mm) lined with moistened cotton batting. A
single S. frugiperda larva was placed on the leaf in each
dish. Petri dishes were closed and placed in an environ-
mental chamber at 27 1 C, 7080 r.h., and L14:D10.
Larvae were allowed to feed on leaf material for 4 days.
Cotton batting was moistened daily to prevent leaf desicca-
tion. At the end of each feeding assay, larvae were removed
from dishes, starved overnight, and weighed. Leaf material
remained in each dish at the end of the experiment, indica-
ting that larvae were never food-limited during the bioas-
say. Relative growth rates were calculated as follows: larval
weight gain (duration of feeding assay mean weight of
the larvae during the feeding assay) (Farrar et al., 1989).
When larvae were removed from the Petri dish, leaf areas
(cm
2
) consumed by a larva were visually estimated with
mm
2
grid paper.
Experiment 2. Spodoptera frugiperda larvae were allowed
to feed on intact plants in the greenhouse. A total of 180
cotton plants were distributed equally on ve greenhouse
benches (130 220 30 cm), each containing 36 plants.
Three plants from each combination of experimental
treatments (Table 1) were randomly placed on each bench.
Small and large plants were covered with 33 61 61
and 25 76 76 cm ber sleeves (Klerk sleeves; Humm-
ert, Earth City, MO, USA), respectively. Sleeves permitted
entry of light and air circulation but retained larvae.
Sleeves were attached to the pots and supported from the
inside with two plant stakes placed opposite each other in
pots. During the experiment, plants were watered from
underneath. Each plant was infested with a single larva 5
7 days after JA treatment. Associated with S. frugiperda
larva availability, infestations were staggered such that two
benches received larvae 5 days after JA treatment, two
benches received larvae 6 days after JA treatment, and one
bench received larvae 7 days after JA treatment. One
starved larva was placed on the surface of the last expanded
leaf of each cotton plant, and the top of the sleeve was
sealed using paperclips to prevent the larva from escaping.
Larvae were allowed to feed on whole plants for 4 days,
after which sleeves were removed and the presence
absence of larvae was recorded. When present, larvae were
removed from the plants, starved overnight, and weighed.
Relative growth rates were calculated. Total leaf area con-
sumption was visually estimated using mm
2
grid paper for
each plant. Only plants on which a larva was recovered
were included in the analysis of leaf consumption.
Experiment 3
A third experiment was conducted with S. frugiperda
neonates fed on the same three cotton cultivars at a
single phenological stage under environmental chamber
conditions. Five weeks after planting, when plants had
56 true leaves, 120 plants were randomly assigned to
either a control or a treatment group (60 plants each)
and were sprayed with either a control or a JA solution
as previously described. After JA treatment, plants were
divided into two equal groups to conduct two feeding
assays. Each group contained 60 plants, 10 plants for
each combination of experimental treatments (cultivar
JA treatment). The two feeding assays were initiated
24 h apart, each using a different cohort of 1-day-old
S. frugiperda neonates.
Seven to eight days after JA treatment, for each feeding
assay, the third and fourth fully expanded true leaves from
each cotton plant were removed with a razor blade, trans-
ported on ice to the laboratory, and placed into a plastic
Petri dish (150 25 mm) lined with moistened cotton
batting. Four 1-day-old (<36 h) neonates were placed on
the top of the leaves in each dish with a paintbrush. Petri
dishes were closed and placed in an environmental cham-
ber at 27 1 C and a photoperiod of L14:D10. On the
5th day of each assay, new leaves (the fth and sixth) were
removed from each cotton plant and offered to the larvae
in each corresponding Petri dish. Larval survival at 3, 5,
and 10 days was recorded. A larva was considered alive
when coordinated movement was observed within 5 s of
prodding with a paintbrush. After being allowed to feed
for 10 days, surviving larvae were removed from the
dishes, starved overnight to allow their guts to void, and
weighed.
JA-induced resistance to Spodoptera frugiperda in cotton 229
Data analysis
Individual plants (experiment 2) or associated Petri dishes
(experiments 1 and 3) were the experimental units (repli-
cates). All statistical analyses were performed using Proc
GLIMMIX (SAS Institute, 2008). For experiments with 6-
day-old S. frugiperda larvae, a three-way analysis of vari-
ance (ANOVA) was used to compare RGRs as affected by
cultivar, phenological stage, JA treatment, and their inter-
actions (3 2 2 factorial structure of experimental
treatments). Leaf area consumed by larvae was also com-
pared between experimental treatments using a three-way
ANOVA. A generalized linear mixed model with a bino-
mial distribution, a logit link function, and an over-disper-
sion parameter was used to compare the proportions of
larvae present on plants. Feeding assays and greenhouse
benches were considered as random effects in the models
used to analyze data from experiments 1 and 2, respec-
tively.
For experiment 3 with S. frugiperda neonates, a two-
way ANOVA was used to compare larval weights as
affected by cultivar and JAtreatment (3 2 factorial struc-
ture of experimental treatments). A generalized linear
mixed model with a binomial distribution and a logit link
function was used to compare the proportions of surviving
larvae after 3, 5, and 10 days of feeding. Feeding assays
were considered as random effects, and a variance compo-
nent covariance structure was used to model the effects of
repeated measures.
The KenwardRoger adjustment for denominator
degrees of freedom was used in all the models to correct
for inexact F distributions. Least square means SE from
the LSMEANS statement output are reported for all
recorded variables to account for unbalanced data. Least
square means were separated with Tukeys HSD
(a = 0.05) when experimental treatment effects were
detected.
Results
Experiment 1
Jasmonic acid treatment was associated with an overall
15.6% reduction in the RGRs of S. frugiperda larvae
(Tables 2 and 3). Cultivar also impacted RGRs (Tables 2
and 3). Compared to the conventional cultivar, RGR was
reduced on the two transgenic cultivars, with the dual pro-
tein Bollgard II

cultivar reducing RGRs to a greater extent

(37.4% reduction) than Bollgard

(14.3%). The JA treat-

ment*cultivar interaction (Table 2) showed that the
effects of JA treatment were inuenced by cultivar.
Whereas only a numerical trend for a decrease in RGR was
associated with the JA treatment in the conventional culti-
var, a signicant JA-induced decrease in RGR occurred for
S. frugiperda fed on Bollgard

(23.5%) and Bollgard II

(24.0%) (Figure 1A). Despite a trend (ANOVA: F

1,160
= 3.31, P<0.1; Table 2), RGRs did not differ among the
two phenological stages. However, as shown by the signi-
cant JA treatment*stage interaction (Table 2), no signi-
cant effects of JA were detected in large plants, whereas JA-
treated small plants were associated with a signicant
28.6%reduction in RGR.
Both JA treatment and cultivar affected the leaf area
consumption by S. frugiperda larvae (Table 2, Figure 1B).
Larvae fed on non-treated cotton leaves consumed more
tissue (11.2 0.7 cm
2
) than larvae fed on JA-treated cot-
ton leaves (8.8 0.7 cm
2
). Cultivar also impacted leaf
consumption (Table 2). Larvae fed on conventional plants
consumed the greatest leaf area (12.5 0.7 cm
2
) followed
by larvae fed on Bollgard

(10.6 0.8 cm
2
) and Bollgard
II

(6.7 0.8 cm
2
) cultivars. The signicant JA treat-
ment*phenological stage interaction (Table 2) showed no
signicant effects of JA in large plants, whereas JA treat-
ment was associated with a signicant (38.0%) reduction
in leaf consumption in small plants. No signicant JA
Table 2 Statistical comparisons of relative growth rate (RGR) and leaf area consumption of 6-day-old Spodoptera frugiperda in an environ-
mental chamber
RGR Leaf area
F d.f. P F d.f. P
JAtreatment 35.22 1,160 <0.0001 14.83 1,127 0.0002
Cultivar 72.18 2,160 <0.0001 29.89 2,127 <0.0001
Phenological stage 3.31 1,160 0.071 0.99 1,128 0.32
JAtreatment*cultivar 4.45 2,160 0.013 2.53 2,127 0.084
JAtreatment*phenological stage 29.49 1,160 <0.0001 13.45 1,127 0.0004
Cultivar*phenological stage 6.23 2,160 0.0025 3.08 2,127 0.049
JAtreatment*cultivar*phenological stage 1.54 2,160 0.22 0.13 2,127 0.88
230 Me szaros et al.
treatment*cultivar interaction (Table 2) was detected on
consumed leaf area; however, the numerical pattern was
similar (Figure 1B) to that observed for RGR(Figure 1A).
Experiment 2
In total, 41.6%of larvae were recovered fromcotton plants
covered by ber sleeves, the remaining larvae having died
or escaped. Jasmonic acid treatment did not affect larval
presence (F
1,164.1
= 2.52, P = 0.11), but cultivar was asso-
ciated with a reduction in the proportion of recovered lar-
vae (F
2,164.6
= 8.68, P = 0.0003). The proportion of
S. frugiperda larvae recovered from Bollgard

(20.2%)
and Bollgard II

(37.8%) cultivars was lower than from

the conventional cultivar (65.4%). Plant phenological
stage did not affect the proportion of recovered larvae
(F
1,164.1
= 1.56, P = 0.21). The two-way JAtreatment*cul-
tivar interaction showed a trend (F
2,164.2
= 2.84,
P = 0.061) for an effect of JA treatment in the conven-
tional cotton cultivar (42.6% reduction). However, the JA
treatment had no effects on the proportion of S. frugiperda
larvae recovered from Bollgard

and Bollgard II

culti-
vars.
Jasmonic acid treatment was associated with a signi-
cant overall 12.1% reduction in the RGR of S. frugiperda
larvae (Tables 3 and 4, Figure 2A). Cultivar was also asso-
ciated with a reduction in RGR, with a greater RGR
Table 3 Spodoptera frugiperda development parameters [least squares (LS) means SE] affected by main experimental treatment effects
[jasmonic acid (JA) treatment, cultivar, phenological stage, days of exposure]
Effect
6-day-old S. frugiperda Neonate S. frugiperda
Experiment 1 Experiment 2 Experiment 3
RGR(g g
)1
day
)1
) RGR(g g
)1
day
)1
) %survival Weight (mg)
Treatment
No JA 0.32 0.01a 0.33 0.01a 88.25 0.03a 24.20 1.30a
JA 0.27 0.01b 0.29 0.01b 53.31 0.07b 3.58 2.00b
Cultivar
Conventional 0.35 0.01a 0.35 0.01a 87.24 0.04a 17.33 1.53a
Bollgard

0.30 0.01b 0.32 0.02a 82.73 0.04a 20.34 1.58a

Bollgard II

0.22 0.01c 0.25 0.01b 43.42 0.07b 4.01 2.80b

Phenological stage
Small 0.30 0.01a 0.31 0.01a n a n a
Large 0.28 0.01a 0.31 0.01a n a n a
Days
3 n a n a 82.82 0.04a n a
5 n a n a 79.39 0.05a n a
10 n a n a 57.51 0.06b n a
By main effect, LS means within a column followed by the same letter are not signicantly different (Tukeys HSD: P>0.05).
n a: not applicable.
Table 4 Statistical comparison of relative growth rate (RGR) and leaf area consumption of 6-day-old Spodoptera frugiperda in a green-
house
RGR Leaf area
F d.f. P F d.f. P
JAtreatment 7.88 1,61.8 0.0067 13.99 1,61.2 0.0004
Cultivar 25.92 2,61.7 <0.0001 9.84 2,60.7 0.0002
Phenological stage 0.05 1,59.7 0.83 19.16 1,59.7 <0.0001
JAtreatment*cultivar 1 2,61 0.37 4.22 2,60 0.019
JAtreatment*phenological stage 0.41 1,61.9 0.53 9.84 1,60.7 0.0026
Cultivar*phenological stage 0.23 2,61 0.79 2.55 2,60.8 0.086
JAtreatment*cultivar*phenological stage 0.70 2,61 0.50 1.43 2,61.2 0.25
JA-induced resistance to Spodoptera frugiperda in cotton 231
observed on the conventional cultivar compared to the
two transgenic cultivars (Tables 3 and 4, Figure 2A). Boll-
gard II

decreased RGRs to a greater extent than Boll-

gard

, with 28.6 and 8.6% reductions, respectively

(Table 3). Relative growth rates did not differ among phe-
nological stages (Tables 3 and 4). No signicant JA treat-
ment*cultivar interaction (Table 4) was detected on RGR.
The numerical pattern, however, was similar to that
observed in the environmental chamber experiment (Fig-
ure 1A), with a greater JA-induced reduction on trans-
genic cultivars than on the conventional cultivar
(Figure 2A).
Jasmonic acid treatment, cultivar, and phenological
stage impacted leaf area consumed by S. frugiperda
(Table 4). Larvae fed on non-treated cotton leaves con-
sumed more tissue (9.9 1.2 cm
2
) than larvae fed on JA-
treated cotton leaves (5.0 1.4 cm
2
). Larvae fed on the
conventional cultivar consumed a larger leaf area
(9.7 1.2 cm
2
) than larvae fed on Bollgard

(8.4
1.7 cm
2
) and on Bollgard II

(4.2 1.3 cm
2
) cultivars. A
A
B
Figure 1 (A) Relative growth rate (RGR) and (B) leaf area con-
sumption of 6-day-old Spodoptera frugiperda larvae (least squares
mean + SE) as affected by jasmonic acid (JA) treatment and cot-
ton cultivar (conventional, Bollgard

, and Bollgard II

) in an
environmental chamber. When a JAtreatment*cotton cultivar
interaction was detected, bars capped with the same letter are not
signicantly different (Tukeys HSD: P>0.05).
A
B
Figure 2 (A) Relative growth rate (RGR) and (B) leaf area con-
sumption of 6-day-old Spodoptera frugiperda larvae (least squares
mean + SE) as affected by jasmonic acid (JA) treatment and cot-
ton cultivar (conventional, Bollgard

, and Bollgard II

) in a
greenhouse. When a JAtreatment*cotton cultivar interaction was
detected, bars capped with the same letter are not signicantly
different (Tukeys HSD: P>0.05).
232 Me szaros et al.
signicant JA treatment*cultivar interaction was observed
(Table 4). Leaf area consumption by S. frugiperda was
signicantly less on Bollgard II

cultivars (JA-treated and

non-treated) and JA-treated Bollgard

than on non-trea-
ted Bollgard

and conventional cultivars (JA-treated and

non-treated) (Figure 2B). A signicant JA treatment*phe-
nological stage interaction was observed (Table 4). No
signicant effects of JA were detected in large plants,
whereas JA was associated with a 61.2% reduction in leaf
area consumption in small plants.
Experiment 3
Jasmonic acid treatment was associated with an overall
39.6% reduction (F
1,130.1
= 47.46, P<0.0001) in survival of
S. frugiperda larvae (Figure 3, Table 3). Overall, cultivar
also impacted survival (F
2,123
= 26.33, P<0.0001), with the
lowest survival rate (43.4%) observed for larvae fed on
Bollgard II

cultivar (Table 3). No signicant differences

in survival were observed for S. frugiperda fed on conven-
tional (87.2%) and Bollgard

(82.7%) cultivars (Table 3).

The duration of feeding (3, 5, or 10 days) had a signicant
effect on larval survival (F
2,339
= 23.34, P<0.0001). As
shown by the JA treatment*cultivar interaction (F
2,123.2
= 7.62, P = 0.0008), the effects of JA treatment on larval
survival were highly signicant on conventional and Boll-
gard II

cultivars, but not on Bollgard

(Figure 3). Jas-

monic acid-treated and non-treated Bollgard

had the
same effect on larval survival as non-treated Bollgard II

(Figure 3). Overall, larval survival showed a general

decrease over 10 days (Figure 3). Overall, 82.8% of larvae
were alive after 3 days, 79.4% after 5 days, and 57.5% after
10 days of feeding (Table 3). Highest survival rates
(97.8%) were observed at 3 and 5 days after exposure on
non-treated conventional cultivar. Larval survival was the
lowest (7.2%) on JA-treated Bollgard II

cultivar on the
10th day (Figure 3).
The weight of S. frugiperda larvae was reduced by 85.1%
when feeding on JA-treated plants for 10 days
(F
1,133
= 79.24, P<0.0001; Table 3). Larval weight was
also affected by cultivar (F
2,121
= 13.70, P<0.0001;
Table 3). Larval weight was 76.9% less on Bollgard II

compared to that on the conventional cultivar. No differ-

ences were detected in larval weight between Bollgard

and conventional cultivars (Table 3). A JA treatment*cul-

tivar interaction was detected (F
2,121
= 8.47, P = 0.0004),
showing that JA-induced reductions in weight were greater
on conventional (85.5%) and Bollgard

cultivars (86.2%)
than on Bollgard II

(77.9%) (Figure 4). The lowest

weight (1.1 mg) was observed for larvae fed on JA-treated
Bollgard II

plants for 10 days; these larvae did not gain

weight at all (Figure 4).
Discussion
In this study, potential interactions between JA-induced
resistance factors and expression of Bt Cry proteins were
investigated in cotton using S. frugiperda as a model insect.
To our knowledge, JA-induced resistance has never been
Figure 4 Weight (least squares mean + SE) of Spodoptera fru-
giperda neonates as affected by jasmonic acid (JA) treatment and
cotton cultivar in an environmental chamber. Bars capped with
the same letter are not signicantly different (Tukeys HSD:
P>0.05).
Figure 3 Survival (least squares mean + SE) of Spodoptera fru-
giperda larvae as affected by jasmonic acid (JA) treatment, cotton
cultivar, and days of feeding on leaves in an environmental cham-
ber.
JA-induced resistance to Spodoptera frugiperda in cotton 233
studied on transgenic Bt cotton cultivars. This study con-
rmed that a Bollgard II

cultivar expressing two Cry pro-

teins (Cry1Ac and Cry2Ab) reduces the weight and the
survival of S. frugiperda larvae to a greater extent than a
Bollgard

cultivar expressing a single Cry protein

(Cry1Ac) and a conventional cultivar expressing no Cry
protein (Adamczyk et al., 2008; Sivasupramaniam et al.,
2008). This study also conrmed that exogenous JA
induces resistance to insects in cotton (Omer et al., 2001).
Furthermore, as evidenced by the numerous signicant JA
treatment*cultivar interactions, this study showed that the
relative effect of JA treatment differed among cultivars. In
particular, the effect of JA treatment on weight gain and
leaf consumption of S. frugiperda larvae was generally
greater on Bt-expressing cultivars than on conventional
cultivars. This study also showed, for the rst time, that
the combined effect of JA and Bt Cry protein(s) can be
greater than the additive effect of JA and Bt protein(s), a
result that suggests synergism. However, because the study
cultivars were not isolines, it is possible that JA-induced
traits were interacting with cultivar-specic traits unre-
lated to the expression of Bt proteins, and further conr-
mation of the synergistic effects of Bt proteins and
JA-induced factors is needed.
Jasmonic acid plays a central role in mediating plant
responses to chewing herbivores such as S. frugiperda. Lev-
els of endogenous JAincrease rapidly following insect feed-
ing, leading ultimately to the activation (derepression) of
many resistance-related genes (Bi et al., 1997; Howe &Jan-
der, 2008). These changes in the expression of resistance-
related genes and traits enhance resistance to herbivores,
making the plant less vulnerable (Bi et al., 1997; Korth &
Thompson, 2006). Consistent with this natural role in
mediating induced resistance, applications of JA have been
shown to stimulate resistance to various insects in various
plants such as tomato, Solanum lycopersicon L. (Cipollini
& Redman, 1999; Thaler et al., 2001), Arabidopsis thaliana
(L.) Heynh. (Cipollini et al., 2004), grapevines, Vitis vini-
fera L. (Omer et al., 2000), and rice, Oryza sativa L. (Stout
et al., 2009; Hamm et al., 2010). To our knowledge, only
one study has been conducted on JA-induced resistance in
cotton; Omer et al. (2001) showed that seedling resistance
to cotton aphids, Aphis gossypii Glover, two-spotted spider
mites, Tetranychus urticae Koch, and western ower thrips,
Frankliniella occidentalis (Pergande), was induced by JA
treatment. Jasmonic acid applications reduced both pref-
erence and performance of these herbivores on cotton
(Omer et al., 2001). In the present study, JA treatment
not only slowed down larval development but also
caused high levels of mortality in early instars of S. fru-
giperda. These studies indicate that JA treatment induces
resistance to a broader spectrum of insect herbivores
than does expression of the Bt proteins used commer-
cially in cotton.
The use of JA applications to induce resistance in the
eld against herbivores as part of an integrated pest man-
agement program has been considered as an alternative
approach (Thaler, 1999). This study highlights the poten-
tial for such an approach in cotton, particularly Bt cotton,
but also illustrates some limitations. Induction of resis-
tance in young cotton plants may protect plants by
increasing the mortality of early-instar chewing lepido-
pteran pests and by inhibiting the development of later
instars, thereby allowing longer exposure to natural ene-
mies. Jasmonic acid-induced resistance may also slow the
buildup of populations of multivoltine pests, thereby
maintaining pest populations below economically damag-
ing levels and facilitating the activities of natural enemies.
However, because of reduced activity of JA in older plants
and the possible short-term nature of JA induction, timing
of JA applications for pest management might be critical.
In this study, exogenous JAapplications induced resistance
to S. frugiperda to a greater extent in small plants (pre-
square plants) than in larger plants (owering plants). Pre-
vious studies also indicate that induction is often stronger
in young plants (Cipollini & Redman, 1999; Omer et al.,
2001). Moreover, studies in other crops have shown that
exogenous JA has only short-term effects on plant resis-
tance; for example, in rice, exogenous JA treatments pro-
vided only short-term protection against rice water
weevils, Lissorhoptrus oryzophilus Kuschel, in small plot
eld tests (Hamm et al., 2010). Hence, a critical issue for
JA use in the eld is to determine the appropriate timing
of application relative to the phenology of the pest and
crop to maximize the impact of the resistance induction.
Another important issue is the costbenets relationship
of JA applications. Although JA is commercially available
and can be applied to large numbers of plants in eld envi-
ronments, it will only be an effective tool if the benets of
application outweigh the costs (Thaler, 1999; Hammet al.,
2010). Depending on plant phenology, cotton can sustain
substantial leaf area losses without major yield reductions
(Russell et al., 1993). However, injury to reproductive
structures can cause moderate to signicant yield losses
(Sadras, 1995). Because S. frugiperda feeds on vegetative
and reproductive structures of cotton plants, the actual
effects of JA-induced resistance on yields are difcult to
predict. The effectiveness of JA applications for cotton
insect management in the eld, as well as potential effects
on yields and plant tness, needs to be evaluated at experi-
mental plot and commercial eld levels. Although eld
applications of JA in cotton will likely not provide com-
plete protection against herbivores, they may have a role to
play in a holistic integrated pest management strategy.
234 Me szaros et al.
Cotton plants have subepidermal glands containing ter-
penoid aldehydes such as the sesquiterpenoid gossypol,
which is toxic to animals and humans but provides con-
stitutive and inducible resistance against a wide range of
pests and diseases (Agrawal & Karban, 2000; Cai et al.,
2009). Breeders have made efforts to develop cotton culti-
vars expressing no or very low levels of gossypol in seeds,
but retaining glands and gossypol in other plant organs
(Schefer & Romano, 2008; Cai et al., 2009). For example,
a cultivar with very low seed gossypol, but glanded leaves,
was developed in China (Zhang et al., 2001). In Australia,
cotton cultivars with delayed development of gossypol
glands were developed, but they have not yet been com-
mercially released because of sterility and other disadvan-
tages (Cai et al., 2009). Opitz et al. (2008) showed that JA
treatment could increase terpenoid levels in cotton leaves
via the production of additional glands and the increased
lling of existing glands. Because exogenous JA has been
shown to increase terpenoid levels in cotton plants, the
induced resistance to S. frugiperda observed in the present
study was likely associated with increased terpenoid levels,
although other inducible resistance-related traits, such as
oxidative enzymes (Bi et al., 1997) may also have been
involved. This current study emphasizes the importance of
traditional plant breeding in cotton pest management by
conrming that traits subject to manipulation through tra-
ditional breeding can substantially impact herbivores like
S. frugiperda.
Only a few studies have been conducted to explore
interactions between expression of Bt proteins and second-
ary substances in cotton plants (Sachs et al., 1996; Guan
et al., 2009). Sachs et al. (1996) conducted studies on pyr-
amiding Cry1Ab insecticidal protein and terpenoids in
cotton for H. virescens resistance, nding that combina-
tion of these traits provided a higher level of resistance to
H. virescens larvae than Cry1Ab or terpenoid alone. Guan
et al. (2009) studied the direct effects of tannic acid and
Cry1Ac on Helicoverpa armigera (Hubner) larval survival
and development. Mortality was highest and larval devel-
opment period was longest when larvae were treated with
a mixture of tannic acid and Bt protein. This combination
also signicantly reduced larval and pupal weights com-
pared to Bt protein or tannic acid alone. Similar studies
have been conducted on potato. Cooper et al. (2004) con-
ducted studies on combining multiple host plant resis-
tance factors into a single plant using both traditional
breeding and genetic engineering against Leptinotarsa
decemlineata (Say). Combining a novel leptin glycoalka-
loid (a L. decemlineata feeding deterrent naturally found
in wild potato) with Cry3A protein, Coombs et al. (2002)
increased potato host plant resistance. The combined resis-
tance of leptin and Bt-Cry3A provided a greater control of
L. decemlineata larvae than either leptin or Bt protein
compounds alone. In the current study, combining Bt pro-
teins with JA-induced resistance increased effectiveness
against S. frugiperda: the highest mortality (92.8%) and
the lowest weight (1.1 mg) were observed for neonates fed
on JA-treated Bollgard II

plants for 10 days. Thus, our

results suggest that combining the expression of Bt pro-
teins and natural resistance traits (i.e., traits such as those
induced by JA treatment) can increase efcacy against
herbivores not controlled sufciently by Bt proteins alone.
In summary, prior research has shown that combining
biotechnology (in the form of Bt proteinexpressing
plants) with conventional host plant resistance holds the
potential for more effective pest management. This study
extends this prior research by showing that Bt proteins and
induction of plant resistance with exogenous JA are com-
plementary strategies for lepidopteran resistance in cotton
plants. Although the mechanism of JA-induced resistance
in cotton plants is not precisely known, previous work sug-
gests that elevated levels of terpenoid aldehydes were
responsible for the slow development and high mortality
of S. frugiperda. Jasmonic acid-induced resistance may be
feasible as a management tool for cotton pests in the eld.
Perhaps more importantly, induction of resistance by JA
has utility as a research tool for enhancing expression of
natural resistance-related traits in cotton, thereby allowing
investigation into interactions between cotton resistance-
related traits and Bt proteins. Further studies are needed
with a greater diversity of transgenic cotton cultivars to
conrm synergism between JA applications and Bt pro-
teins and to investigate potential mechanisms of syner-
gism.
Acknowledgements
The authors thank Josh Temple for providing S. frugiperda
larvae and pupae used to establish a colony at Louisiana
State University. Appreciation is also expressed to Andy
White and Mike Robinson (Bayer CropScience) for pro-
viding Stoneville pedigreed cotton seeds. The authors also
thank Drs. NA Hummel and JA Davis for their comments
that helped improve this manuscript. This article was pub-
lished with the approval of the Louisiana Agricultural
Experiment Station as Manuscript 2011-234-5592. We
also thank the two anonymous reviewers for their valuable
comments on the manuscript.
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