Resistance to fall armyworm (Lepidoptera: Noctuidae) feeding identified in
nascent allotetraploids cross-compatible to cultivated peanut (Arachis
hypogaea L.)
C.M. Levinson, K.M. Marasigan, Y. Chu, H.T. Stalker, C.C. Holbrook, X. Ni, W.P. Williams, and P. Ozias-Akins *1
Key Words: Antibiosis, Arachis, host
plant resistance, Spodoptera frugiperda, in
vitro.
ABSTRACT
Fall armyworm (FAW) is an economically
devastating, invasive pest in Sub-Saharan Africa
and can be a major pest in the Americas. This pest
feeds on more than 80 plant species, including
peanut, and threatens the food security of
millions of people who rely on these crops in
Sub-Saharan Africa. An integrated pest management strategy, including resistant crop cultivars,
is needed to control FAW, since populations have
been reported to develop insecticide resistance.
Genetic sources of host resistance to FAW are
limited in cultivated peanut; however, strong
resistance to FAW was reported previously in
peanut wild relatives. In this in vitro study, we
tested diploid peanut relatives including A.
ipaensis KG37006 (Ipa), A. duranensis 30060
(Dur), A. correntina 9530 (Cor9530), and A.
correntina 9548 (Cor9548); allotetraploids including IpaCor95304x, IpaDur4x; F2 hybrids [A.
hypogaea 13-1014 x IpaCor95304x]; and cultivated
peanut lines A. hypogaea ‘13-1014 0 and ‘Georgia
Green’ for FAW resistance to identify valuable
materials in our breeding program. FAW development was measured by survival, larval weight,
larval stage duration, pupation, pupal stage
duration, moth emergence relative to pupation,
and moth sex. All allotetraploids showed promise
as donors for FAW resistance. This FAW
resistance was derived primarily from A. ipaensis,
but A. duranensis was also identified as a source
of resistance, though more moderate. A high level
of heterogeneity was found in A. correntina 9530,
which likely contributed to the variable performance of this species in the bioassay. Producing
hybrids and allotetraploids with wild Arachis
species does not guarantee that each derived line
from these crosses will be resistant, and since
these lines are segregating, selection for resistance
is still needed.
Fall armyworm (FAW), Spodoptera frugiperda
(J. E. Smith) (Lepidoptera: Noctuidae), is a major
defoliating pest in the Americas and has recently
become an economically devastating, invasive pest
in Sub-Saharan Africa (Sparks, 1986; Goergen et
al., 2016), as well as in Asia. FAW feeds on more
than 80 economically important crops, including
peanut. A thirteen billion dollar loss to FAW
infestation was reported for maize, sorghum, rice,
and sugarcane in Africa from 2016 to 2017
(Abrahams et al., 2017). Without control, FAW
is a major defoliator that can cause high yield
reductions, seriously affecting production in developing countries with limited access to pesticides
and safety equipment to apply them properly
(Cock et al., 2017; Day et al., 2017; Bateman et
al., 2018; CABI, 2020). Producing FAW resistant
cultivars will reduce reliance on pesticides, increase
food security, and promote sustainable agriculture
in these at-risk countries. In addition, resistant
cultivars are needed to mitigate the insecticide
resistance reported in FAW populations (Yu,
1991).
Genetic sources with strong FAW resistance are
limited in cultivated peanut due to its narrow
genetic base (Stalker et al., 2016). Peanut is an
allotetraploid species (AABB; 2n¼4x¼40), and
evidence suggests that it arose from a natural
polyploidization event involving hybridization of
the diploid species A. ipaensis (BB; 2n¼2x¼20) and
A. duranensis (AA; 2n¼2x¼20) (Bertioli et al.,
2016). The difference in ploidy levels between
cultivated peanut and its wild relatives results in
crossing barriers and constricts genetic diversity in
peanut. Moderate levels of host resistance to FAW
have been found in a few peanut cultivars such as
‘Southeastern Runner 56-15 0 (Hammons 1970,
Leuck and Skinner 1971), ‘Florunner’ and ‘Tifton
8 0 (Todd et al., 1991). On the other hand, more
than 80 wild peanut relatives have diverse levels of
resistance against a wide range of peanut insect
pests and diseases (Stalker et al., 2016; Stalker,
1
First and last authors: Institute of Plant Breeding, Genetics and
Genomics, University of Georgia, Tifton, GA 31793; Second, third,
and last authors: Horticulture Department, University of Georgia,
Tifton, GA 31793. Fourth author: Department of Crop and Soil
Sciences, North Carolina State University, Raleigh, 27695. Fifth
and sixth authors: USDA-Agricultural Research Service, Crop
Genetics and Breeding Research Unit, Tifton, GA, 31793. Sixth
author: USDA-Agricultural Research Service, Corn Host Plant
Resistance Research Unit, Mississippi State, MS, 39762.
*Corresponding author’s E-mail: pozias@uga.edu
Peanut Science (2020) 47:123–134
123
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2017). Wild peanut relatives, such as Arachis
cardenasii, already have proved to be useful sources
of resistance to multiple pests including late leaf
spot, early leaf spot, root-knot nematode, Cylindrocladium black rot, and Sclerotinia blight
(Simpson and Starr, 2001; Stalker and Mozingo,
2001; Gowda et al., 2002; Simpson et al., 2003;
Tallury et al., 2014). Previous studies have identified wild peanut relatives such as A. cardenasii, A.
correntina, and A. villosa as potential donors of
strong FAW antibiosis and antixenosis (Painter,
1951; Kogan and Ortman, 1978; Lynch et al., 1981;
Ortega et al., 2016). These Arachis species displayed antibiosis through high FAW mortality
rates and inhibition of FAW development and
displayed antixenosis due to FAW non-preference
to feed on these materials (Lynch et al., 1981). In
addition, A. ipaensis showed antibiosis through a
high FAW mortality rate (Yang et al., 1993).
However, further work has not resulted in FAW
resistance being introgressed from these wild
relative species into a cultivated background useful
for peanut breeders. This study identified FAW
resistance in newly created allotetraploids that are
cross compatible to cultivated, allotetraploid peanut. Resistant allotetraploid lines identified in this
study can be used in breeding programs in the
United States and shared with breeding programs
in East and West Africa to introgress FAW
resistance into elite cultivars. The long-term goal
of this study is to create FAW resistant peanut
cultivars that can protect yields in the United States
and increase yields in regions with limited access to
pesticides in Africa.
Materials and Methods
Plant Materials
Wild Arachis species, A. ipaensis Krapov. and
W.C. Gregory (PI 468322, GKBSPSc 30076;
abbrev.: Ipa), A. duranensis Krapov. and W.C.
Gregory (PI 468197, GKBSPSc 30060; abbrev.:
Dur), A. correntina (Burkart) Krapov. and W.C.
Gregory (PI 262808, GKP 9530; abbrev.: Cor9530)
and (PI 262881, GKP 9548; abbrev.: Cor9548) were
grown and the diploid hybrids generated in 2016 at
North Carolina State University (NCSU). IpaDur4x and IpaCor95304x allotetraploids were created from the diploid hybrids by colchicine
treatment of F1 hybrid cuttings at the University
of Georgia (UGA) Tifton Campus. The susceptible
A. hypogaea controls included ‘Georgia Green’
(Branch, 1996) and ‘13-1014,’ a breeding line
selected from [(C1805-617-2 x ‘Florida-07 0 (Gorbet
and Tillman, 2009)) x ‘Georgia-06G’ (Branch,
2007)]. C1805-617-2 is a selection from ‘Tifguard’
(Holbrook et al., 2008) x ‘Florida-07 0 .
Due to limitations of labor and pesticide-free
plants required for bioassay, the resistance evaluation was performed in three experiments with Ipa
and allotetraploids with Ipa as one parent being
tested in all of the experiments (Table 1). The first
experiment, aimed to test the level of resistance in
IpaDur4x, was conducted in July 2018 and included
seven genotypes as treatments: Ipa, Dur, Georgia
Green, and four S2 generation IpaDur4x plants. The
allotetraploids were each designated an arbitrary
number to make distinguishing them easier (Table
1). IpaDur_1 and IpaDur_2 originated from the
same IpaDur_S0:1 plant, while IpaDur_3 and
IpaDur_ 4 originated from the same IpaDur_S0:5
plant. The second experiment was designed to test
the level of resistance in IpaCor95304x; it was
carried out in May 2019 and included 10 genotypes
as treatments: Ipa, Cor9530, Cor9548, A. hypogaea
13-1014, three S1 generation IpaCor95304x plants,
and three F1 plants made from a cross between A.
hypogaea 13-1014 and three different S0 IpaCor95304x plants (Table 1). Cor (PI 261870) was
previously reported to have FAW resistance
(Lynch et al., 1981), but was not included in this
study due to lack of seed supply. The third
experiment was conducted in November 2019 to
directly compare the level of resistance detected in
materials from the first two experiments. The third
experiment included eight genotypes as treatments:
Ipa, Dur, Cor9530, Georgia Green, A. hypogaea 131014, one IpaDur4x S2 plant, and two IpaCor95304x
S1 plants (Table 1). IpaDur_5 was an S2 plant that
originated from one of the IpaDur4x lines tested in
the July 2018 experiment. IpaCor_4 originated
from IpaCor_1 tested in the May 2019 experiment,
while IpaCor_5 originated from IpaCor_2. In this
last experiment, four genotypes were terminated
early because the plants grew slowly in the short
day length season and failed to produce enough
leaves to complete the test. The earliest date of
termination was 17 DAI (days after infestation) for
genotype Dur.
Fall Armyworm Resistance Evaluation
For each of the three experiments, seeds were
coated in Vitavax PC (Vitavax, Crompton, Middlebury, CT) and treated overnight in 0.5% Florel
Growth Regulator (Lawn and Garden Products
Inc., Fresno, CA) to break dormancy. Seeds were
then planted in #123 7.62 cm round x 11.43 cm
deep Jiffy Pots (Harris Seeds, Rochester, NY) and
transplanted approximately one month later into
121.92 cm round x 27.94 cm deep pots filled with
Promix growth medium (Premier Tech Horticulture, Quakertown, PA). Normal plant management
ALLOTETRAPLOID INSECT RESISTANCE
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Table 1. Genetic materials tested in each fall armyworm feeding bioassay and their abbreviations and ploidy level
July 2018 Bioassay: Plant Materials
Georgia Green
A. ipaensis 30076
A. duranensis 30060
IpaDur300604x_S0:1_S1:2
IpaDur300604x_S0:1_S1:4
IpaDur300604x_S0:5_S1:2
IpaDur300604x_S0:5_S1:4
May 2019 Bioassay: Plant Materials
A. hypogaea 13-1014
A. ipaensis 30076
A. correntina 9530
A. correntina 9548
IpaCor95304x_S0:2_S1
IpaCor95304x_S0:5_S1
IpaCor95304x_S0:6_S1
(13-1014 x IpaCor95304x_S0:2)_F1:4_F2
(13-1014 x IpaCor95304x_S0:5)_F1:4_F2
(13-2113 x IpaCor95304x_S0:6)_F1:4_F2
November 2019 Bioassay: Plant Materials
Georgia Green
A. hypogaea 13-1014
A. ipaensis 30076
A. duranensis 30060
A. correntina 9530
IpaDur300604x_S2:1
IpaCor95304x_S0:2_S1:9
IpaCor95304x_S0:5_S1:7
Abbreviation
Georgia Green
Ipa
Dur
IpaDur_1
IpaDur_2
IpaDur_3
IpaDur_4
a
Ploidy Level
Tetraploid
Diploid
Diploid
Tetraploid
Tetraploid
Tetraploid
Tetraploid
Abbreviation
Ploidy Level
13-1014
Ipa
Cor9530
Cor9548
IpaCor_1
IpaCor_2
IpaCor_3
(13-1014 x IpaCor_1)_F2
(13-1014 x IpaCor_2)_F2
(13-1014 x IpaCor_3)_F2
Tetraploid
Diploid
Diploid
Diploid
Tetraploid
Tetraploid
Tetraploid
Tetraploid
Tetraploid
Tetraploid
Abbreviation
Ploidy Level
Georgia Green
13-1014
Ipa
Dur
Cor9530
IpaDur_5b
IpaCor_4 c
IpaCor_5 c
Tetraploid
Tetraploid
Diploid
Diploid
Diploid
Tetraploid
Tetraploid
Tetraploid
a
Bolded genotypes were tested in more than one experiment
IpaDur_5 originated from one of the IpaDur lines tested in the July 2018 experiment
c
IpaCor_4 originated from IpaCor_1 and IpaCor_5 originated from IpaCor_2
b
was applied in the greenhouse except that insecticide treatments were withheld.
When plants were three months old, FAW eggs
(corn strain) were obtained from USDA-ARS
Corn Host Plant Resistance Research Unit, Mississippi State, MS. Thirty replications were tested
for each genotype, in which one replication
comprised one FAW larva fed on the excised
leaflets from a genotype. Within 24 h of egg hatch,
the neonate larvae were gently transferred with a
soft tip paintbrush from the bag of FAW egg
masses onto a fresh, newly emerged leaflet placed in
a 100 mm x 15 mm Petri dish (ThermoFisher
Scientific). Each Petri dish contained a sheet of 9
cm diameter Whatman No. 1 filter paper (ThermoFisher Scientific) supported by a cotton round
(Equate Beauty) that was saturated with approximately 4 ml of deionized water. The day that the
neonate larvae were transferred onto the leaflets
was considered day 0 after infestation (0 DAI).
Petri dishes were sealed with Parafilmt M laboratory film (Bemis Company, Inc.) for the first week
to prevent the FAW from escaping. For the next
three days, Petri dishes with dead FAW larva were
replenished with another neonate larva. On the day
a Petri dish was restarted with a new FAW larva,
that day was considered 0 DAI for that particular
plate. The Petri dishes were examined daily to
relocate FAW larvae that may have moved off of
the leaflets and onto or underneath the filter paper.
Daily inspection ensured that FAW larvae would
have the opportunity to feed on plant material, and
that the filter paper and cotton pad could be
moistened if they had become dry. Fresh leaflets
were offered every other day for one week, then
fresh leaves were offered daily to meet the need of
increased larval feeding. The moistened cotton pad
and filter paper were changed as the frass from
FAW larva accumulated and contaminated the
filter paper.
The following parameters were evaluated to
study the effect of plant genotype on FAW growth
and development: survival, larval weight, larval
stage duration, pupation, pupal stage duration,
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moth emergence relative to pupation, and moth
sex. Low survival, larval weight, pupation, and
emergence as well as high larval stage duration and
pupal stage duration indicated deceleration of
normal FAW growth and development. Sex ratio
was an indicator of genotype effect on FAW fitness
in which a deviation from a 1:1 (male to female)
indicated a genotypic effect of the host plants on
FAW sexual dimorphism. Sex ratio was included,
since diet and other environmental effects have
been found to negatively affect adult insect,
including FAW, sex ratios (Bull, 1981; Murúa
and Virla, 2004). Biased sex ratio could be utilized
as one of the valuable tactics for sustained pest
management in multiple cropping systems. FAW
survival was documented daily. Larval weight was
recorded at 14 DAI. Larval stage duration
documented the number of days from first-instar
larva stage to pupal formation. Pupation and
relative moth emergence described completed
pupation and moth emergence relative to the
number of pupa formed, respectively. Pupal stage
duration denoted the number of days recorded
from pupal development to moth emergence. Pupal
weight was recorded on the day of complete pupal
formation. Moth sex (male or female) was determined after emergence.
Genotyping the diploid species
Cor demonstrated morphological diversity;
therefore, we genotyped the diploid species using
the Affymetrix Axiom_Arachis2 SNP array (Clevenger et al., 2018; Korani et al. 2019) consisting of
47,000 features (ThermoFisher Scientific). DNA
was extracted by the Qiagen DNeasy Plant mini kit
(Qiagen, Germantown, MD). SNP calling was
performed with Axiom Analysis Suite (Version
1.2). Genetic markers were grouped into six
categories by the software depending on the quality
and separation of markers 1) Monomorphic 2)
PolyHighResolution 3) NoMinorHom 4) OfftargetVariant 5) CallRateBelowThreshold 6) Other.
Only the 5,342 markers in the PolyHighResolution
class were used for analysis since the grouping of
samples was unambiguous and all the samples
passed quality control.
Statistical Analysis
One-way analysis of variance (ANOVA) was
performed using JMP software (SAS Institute) to
determine the genotype effect on FAW growth and
development according to the following parameters: larval weight, larval stage duration, pupal
stage duration, and pupal weight. Chi-squared tests
were performed with JMP software to determine
the genotype effect on the following categorical
parameters used to assess FAW development:
survival at 8 DAI and 14 DAI (dead or alive),
pupation (pupated or did not pupate), relative
moth emergence (emerged or did not emerge), and
moth sex (male or female). For relative moth
emergence, data (emerged or did not emerge) was
only included for FAW that pupated. Means of
each parameter among the treatments were separated based on the Tukey’s Test (a ¼ 0.05) results
with JMP software. Tukey’s Test for binomial data
was not supported with JMP software, so RStudio
(RStudio, Inc.) was used by running genotype
effect on survival at 8 DAI and 14 DAI, pupation
and relative moth emergence as a generalized linear
model and then Tukey’s Test (a ¼ 0.05) was
performed on the model results. Genotypes with
less than four replications for a measurement were
excluded from statistical analysis. Therefore, larval
stage duration, pupation, pupal weight, pupal stage
duration, relative moth emergence, and moth
analysis only included eight genotypes for the
May 2019 experiment with Ipa and IpaCor_3
excluded and only four genotypes for the November 2019 experiments with Cor, Dur, Ipa, and
IpaDur_5 excluded due to being terminated before
pupal formation. The data for both the May and
November 2019 experiments were still valid, since
they were maintained according to experimental
design and the controls were continued.
Results
Survival
Significant genotypic effect on FAW survival at
8 DAI and 14 DAI was found for the May 2019
experiment and the November 2019 experiment,
but not for the July 2018 experiment (Table 2).
However, Tukey-Kramer significant differences
between genotypes were not found for neither 8
DAI nor 14 DAI for all three experiments (Table
S1). In all three experiments, the cultivated control
genotypes had the highest FAW survival (Fig. 1).
For the July 2018 experiment (Fig. 1A), the
allotetraploid IpaDur_2 had the greatest FAW
mortality. Only 30% (9 of the 30) of FAW fed
exclusively on IpaDur_2 survived to the end of the
experiment and completed their life cycle. The
larval survival curves on Ipa and Dur were below
that of the susceptible cultivated peanut control.
Unlike the other two experiments, the May 2019
experiment (Fig. 1B) had a high mortality of FAW
larvae just four days after the onset of the
experiment. This is likely due to a trained
entomologist performing the July 2018 and November 2019 experiments. However, all allotetraploids had survival curves below the susceptible
control. Both accessions of A. correntina were
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ALLOTETRAPLOID INSECT RESISTANCE
Table 2. ANOVA and chi-squared test output testing the genotype effect on FAW growth and development according to the following
parameters: survival at 8 DAI and 14 DAI, larval weight, larval stage duration, pupation, pupal stage duration, pupal weight, relative
moth emergence, and moth sex.
Parameter
Survival at 8DAI (July 2018)
Survival at 8DAI (May 2019)
Survival at 8DAI (Nov. 2019)
Survival at 14DAI (July 2018)
Survival at 14DAI (May 2019)
Survival at 14DAI (Nov. 2019)
Larval weight (July 2018)
Larval weight (May 2019)
Larval weight (Nov. 2019)
Larval stage duration (July 2018)
Larval stage duration (May 2019)
Larval stage duration (Nov. 2019)
Pupation (July 2018)
Pupation (May 2019)
Pupation (Nov. 2019)
Pupal Weight (July 2018)
Pupal Weight (May 2019)
Pupal Weight (Nov. 2019)
Pupal stage duration (July 2018)
Pupal stage duration (May 2019)
Pupal stage duration (November 2019)
Relative moth emergence (July 2018)
Relative moth emergence (May 2019)
Relative moth emergence (Nov. 2019)
Moth sex (July 2018)
Moth sex (May 2019)
Moth sex (Nov. 2019)
F or X2 value
8.66
92.62
26.44
11.81
117.61
35.41
11.14
23.60
29.92
12.13
29.60
38.01
20.94
82.15
38.24
22.68
14.91
84.12
2.66
8.92
1.32
11.45
6.14
10.66
7.32
11.92
4.29
df(n) a, df(d)
6
9
7
6
9
7
6,
9,
7,
6,
7,
3,
6
7
3
6,
7,
3,
6,
7,
3,
6
7
3
6
7
3
156
151
205
131
124
92
131
124
92
117
113
79
b
P-value
0.19
, 0.0001***c
0.0004***
0.07
, 0.0001***
, 0.0001***
, 0.0001***
, 0.0001***
, 0.0001***
, 0.0001***
, 0.0001***
, 0.0001***
0.0019**
, 0.0001***
, 0.0001***
, 0.0001***
, 0.0001***
, 0.0001***
.019*
, 0.0001***
0.27
0.077
0.052
, 0.014*
0.29
0.10
0.23
a
The df(n), degrees of freedom of the numerator, is based on the number of plant genotypes tested
The df(d), degrees of freedom of the denominator, is based on the total number of replicates, in which a replicate is one plate
with one FAW, for all plant genotypes tested
c
*P , .05. **P , .01. ***P , .001
b
similar to the susceptible control, although
Cor9530 lost five more FAW larvae (17% more)
than Cor9548.
In the November 2019 experiment (Fig. 1C),
Ipa, Dur, Cor, and IpaDur_5 had to be terminated
early due to insufficient plant material to sustain
the FAW feeding. However, FAW larvae fed on
susceptible controls showed almost identical survival curves as the previous two experiments. The
FAW larva survival curve on Cor9530 closely
followed survival curves of the susceptible controls.
IpaCor_4, a progeny line from IpaCor_1 that
showed high FAW mortality in the May 2019
experiment (Fig. 1B), also had a survival curve
similar to the controls. Ipa and Dur had similar
survival curves to the July 2018 experiment (Fig.
1C) until the treatments were terminated. IpaCor_5
(Fig. 1C), a progeny line from IpaCor_2 (Fig. 1B)
that had high FAW mortality in the May 2019
experiment, had the greatest FAW mortality in the
November 2019 experiment.
Larval Weight
Genotype had a significant effect on larval
weight in all of the experiments (Table 2). All five
IpaDur4x allotetraploid plants significantly reduced
FAW larval weight compared to the cultivated
peanut genotypes included as experimental controls (Fig. 2). For the July 2018 experiment, the
allotetraploid IpaDur_2 that had the greatest FAW
mortality also had the numerically lowest larval
weight (Figs. 1A, 2A). As for IpaCor95304x, three
out of five tested allotetraploid plants (IpaCor_1,
IpaCor_4, and IpaCor_5) significantly suppressed
larval weight (Figs. 2B, C). The other two
IpaCor95304x plants (IpaCor_2 and IpaCor_3) also
reduced larval weight, yet the level of reduction did
not show statistical significance when compared to
the cultivated peanut controls (Fig. 2B). This is
partly due to high mortality of FAW larvae in the
May 2019 experiment (Fig. 1B), which led to small
sample size, and affected statistical power for larval
weight data (Fig. 2B). For example, while the larval
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Fig. 1. Survival of FAW throughout the life cycle during the experimental period. (A) July 2018 Experiment. (B) May 2019 Experiment. (C) November
2019 Experiment.
weight on IpaCor_1 was 35% of the control and
found to be significantly different, the Ipa larval
weight was only 13.5% of the control but not
significantly different due to the small sample size
caused by mortality of FAW larvae on Ipa leaves.
When the two allotetraploid lines were tested sideby-side in the November 2019 experiment (Fig.
2C), IpaDur_5 demonstrated a stronger level of
FAW resistance by reducing FAW larval weight to
a greater extent than IpaCor9530_4 and IpaCor9530_5.
Diploid parents of both allotetraploid lines were
included in this study to determine the source of
FAW resistance. Compared to the cultivated
peanut control, Ipa significantly suppressed FAW
weight gain in the July 2018 and November 2019
Fig. 2. Larva weight on day 14 after feeding on different peanut leaflets. (A) July 2018 Experiment. (B) May 2019 Experiment. (C) November 2019
Experiment. Error bars represent standard error. Tukey’s HSD significance levels were calculated within each experiment, so these significance
groupings cannot be compared between the three experiments. Genotypes within an experiment with the same Tukey’s HSD letter are not
significantly different (a ¼ 0.05).
ALLOTETRAPLOID INSECT RESISTANCE
129
Fig. 3. Larval stage duration. (A) July 2018 Experiment. (B) May 2019 Experiment. (C) November 2019 Experiment. Error bars represent standard
error. Tukey’s HSD significance levels were calculated within each experiment, so these significance groupings cannot be compared between the three
experiments. Genotypes within an experiment with the same Tukey’s HSD letter are not significantly different (a ¼ 0.05).
experiments (Figs. 2A, C). Dur also significantly
reduced the weight of FAW larvae but to a
numerically lesser extent than that of Ipa in the
November 2019 experiment. Conflicting results
were found between the May 2019 (Fig. 2B) and
November 2019 (Fig. 2C) experiments for Cor9530.
A significant, positive effect on larval weight was
observed in the May 2019 experiment (Fig. 2B) and
a significant suppressive effect was found in the
November 2019 experiment (Fig. 2C). Cor9548 was
tested only in one experiment (Fig. 2B) and it was
found to increase larval weight gain. A high level of
genetic heterogeneity was found in Cor9530 (Table
S2). Among the three genotyped Cor9530 plants,
1,259 out of 5,342 total markers (23.5%) were
found to be polymorphic. On the contrary, only 23
(0.4%) and 27 (0.5%) polymorphic markers were
found with Ipa and Dur, respectively. Therefore,
the conflicting feeding response on Cor9530 could
be due to the genetic diversity within this accession.
In the May 2019 experiment (Fig. 2B), three 131014 x IpaCor F2 plants were found to be
segregating for larval weight. The (13-1014 x
IpaCor_1)_F2 plant demonstrated a significantly
greater increase in larval weight compared to the
cultivated control. The (13-1014 x IpaCor_2)_F2
plant also showed a similar effect on larval weight
compared to the cultivated control. The (13-1014 x
IpaCor_3)_F2 plant demonstrated high suppression
of FAW growth similar to the IpaCor95304x.
Larval Stage Duration
Genotype had a significant effect on larval stage
duration in all of the experiments (Table 2). In all
three experiments, the cultivated controls had the
shortest larval stage duration (Fig. 3). Compared
to the cultivated control, Ipa significantly reduced
larval growth and development by extending larval
stage duration in the July 2018 experiment (Fig.
3A). Likewise, all allotetraploids significantly
extended larval stage duration, except for IpaDur_1
in the July 2018 experiment (Fig. 3A). IpaCor_5,
the allotetraploid with the greatest FAW mortality
in the November 2019 experiment (Fig. 1B),
significantly outperformed IpaCor_4 by greatly
extending larval stage duration (Fig. 4B). While
Ipa significantly extended larval stage duration,
Dur, Cor9530, and Cor9548 did not differ significantly from the cultivated controls.
Pupation
Genotype had a significant effect on pupation in
all of the experiments (Table 2). For the July 2018
and May 2019 experiments, Tukey-Kramer significant differences between genotypes were not
found; however, 13-1014 had significantly more
pupated FAW than IpaCor_5 (Table S1). Across
all the experiments, the cultivated controls had the
highest number of FAW to pupate (Fig. 4). For the
July 2018 experiment, IpaDur_2, which had the
highest FAW mortality (Fig. 1A), also had the
fewest FAW to pupate (Fig. 4A). For the May
2019 experiment (Fig. 4B), pupation was heavily
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Fig. 4. Pupation. (A) July 2018 Experiment. (B) May 2019 Experiment. (C) November 2019 Experiment.
influenced by FAW mortality early in the experiment. Despite this artifact, all allotetraploids had a
lower number of FAW to pupate than the
cultivated control. Both Cor9530 and Cor9548
had a similar number of FAW to pupate as
compared to the cultivated control. Line (13-1014
x IpaCor_1)_F2 also had a similar number of FAW
to pupate as compared to the cultivated controls,
while (13-1014 x IpaCor_2)_F2 and (13-1014 x
IpaCor_3)_F2 had much lower numbers of FAW to
pupate as compared to the cultivated controls. For
the November 2019 experiment, IpaCor_4 had a
similar number of FAW to pupate as the cultivated
controls (Fig. 4C), while IpaCor_5 had significantly
less pupa form as compared to 13-1014 (Table S1).
Pupal Weight
Genotype had a significant effect on average
pupal weight across all experiments (Table 2). All
allotetraploids, except IpaCor_1 in the May 2019
experiment, had significantly lower pupal weight
than the cultivated controls (Fig. 5). IpaDur_3 and
IpaDur_4 had significantly lower pupal weight than
IpaDur_1 (Figs. 5A, C). IpaCor_2 had the lowest
pupal weight of all the allotetraploids in the May
2019 experiment (Fig. 5B). IpaCor_5, the progeny
line of IpaCor_2, had the lowest pupal weight in the
November 2019 experiment (Fig. 5C). Ipa, Dur,
and Cor9548 all had significantly lower pupal
weight than the respective cultivated control.
However, Cor9530 was not statistically different
from the cultivated control in the May 2019
experiment (Fig. 5B).
Pupal Stage Duration
Genotype had a significant effect on pupal stage
duration in the July 2018 experiment and the May
2019 experiment, but not in the November 2019
experiment (Table 2). Across all three experiments,
Ipa, Dur, Cor9530, and Cor9548 were not significantly different from the cultivated controls (Fig.
6). However, IpaCor_1 and (13-1014 x IpaCor_2)_F2 had significantly longer pupal stage
duration than the cultivated control, indicating
these materials impeded pupal stage development
(Fig. 6B).
Relative Moth Emergence
Genotype had a significant effect on relative
moth emergence in the November 2019 experiment
but not the July 2018 or May 2019 experiments
(Table 2), and Tukey-Kramer significant differences between genotypes for relative moth emergence
were not found for any of the three experiments
(Table S1). All of the allotetraploids, except for
IpaCor_2 and IpaCor_4 (Fig. 7B, C), supported less
relative moth emergence than the cultivated controls (Fig. 7). Allotetraploids had a high number of
aborted pupae with the highest being 38.5% (5 out
of 13 pupa) for IpaCor_5, then 25% (5 out of 20
pupa) for IpaDur_4, and 18.9% (4 out of 22) for
IpaDur_3 (Fig. 7A, C). In comparison, Georgia
Green had 4.2% (1 out of 24) and 7.1% (2 out of
28) aborted pupa in the July 2018 and November
2019 experiments, respectively (Fig. 7A, C).
Arachis hypogea 13-1014 had 7.1% (2 out of 28)
and 6.9% (2 out of 29) aborted pupa in the July
2018 and November 2019 experiments, respectively
ALLOTETRAPLOID INSECT RESISTANCE
131
Fig. 5. Pupal weight. (A) July 2018 Experiment. (B) May 2019 Experiment. (C) November 2019 Experiment. Error bars represent standard error.
Tukey’s HSD significance levels were calculated within each experiment, so these significance groupings cannot be compared between the three
experiments. Genotypes within an experiment with the same Tukey’s HSD letter are not significantly different (a ¼ 0.05).
(Fig. 7A, C). The overall low number of 8 moths
formed on IpaCor_5, a progeny line of IpaCor_2,
was due to early FAW mortality and high pupa
abortion (Figs. 1B, 7B). All larvae fed on Ipa, Dur,
Cor9530, and Cor9548 showed similar relative
moth emergence as compared to the cultivated
controls. Also, larvae fed on Cor9530 showed
similar relative moth emergence as compared to the
larvae fed on Cor9548 (Fig. 7B). Lastly, the (131014 x IpaCor)_F2 plants all had similar relative
moth emergence when compared to the control
(Fig. 7B).
Fig. 6. Pupal stage duration. (A) July 2018 Experiment. (B) May 2019 Experiment. (C) November 2019 Experiment. Error bars represent standard error.
Tukey’s HSD significance levels were calculated within each experiment, so these significance groupings cannot be compared between the three
experiments. Genotypes within an experiment with the same Tukey’s HSD letter are not significantly different (a ¼ 0.05).
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Fig. 7. Percent of moths formed based on pupa number. (A) July 2018 Experiment. (B) May 2019 Experiment. (C) November 2019 Experiment.
Moth Sex
Genotype did not have a significant effect on
moth sex in the July 2018, May 2019, nor
November 2019 experiment (Table 2).
Discussion
The wild peanut-derived allotetraploid genotypes showed promise as sources for FAW
resistance for peanut breeding programs, since all
IpaDur4x and IpaCor95304x allotetraploids significantly reduced larval weight (except IpaCor_2 and
IpaCor_3), increased pupal stage duration (except
IpaDur_1), and decreased pupal weight (except
IpaCor_1) as compared to the controls. All
allotetraploid genotypes showed lower survival
and pupation than the cultivated genotypes evaluated as well as lower relative moth emergence
(except IpaCor_2 and IpaCor_4). The most promising allotetraploid line, IpaDur_2, had the lowest
larval weight and the greatest FAW mortality in
the July 2018 experiment. This was further
supported by IpaDur_5, which significantly reduced larval and pupal weight in the November
2019 experiment. IpaCor_2 and its progeny IpaCor_5 were shown as a promising line as they both
suppressed FAW growth in the May and November 2019 experiments. Since segregation for FAW
resistance in the allotetraploids was found, further
selections for FAW resistance in individual lines
will be necessary for effective utilization of these
materials in a breeding program.
Primarily Ipa, but also Dur, were shown to be
donor sources for FAW resistance, by reducing
larval and pupal weight, survival and pupation.
The conclusion that Ipa is valuable as a source for
FAW resistance confirms a previous report (Yang
et al., 1993). However, a 79% mortality rate at 8
DAI was reported for A. ipaensis in a similar
bioassay with five wild Arachis species and A.
hypogaea (Yang et al., 1993). The bioassay did
differ in the use of terminal buds from field-grown
plants rather than expanded leaves from greenhouse-grown plants. Two of our three experiments
showed generally higher survival for Ipa with 8
DAI mortality rates of 16.7%, 80%, and 20% for
the July 2018, May 2019, and November 2019
experiments, respectively. The high mortality of
FAW fed on Ipa leaves in the May 2019 experiment
may be an artifact of this experiment having overall
higher FAW mortality than the other two experiments (Fig. 1), influenced by a trained entomologist performing the July 2018 and November 2019
experiments. Overall, the mean Ipa mortality rate
was 38.9% across the three experiments. However,
the low larval weight of Ipa supports the previous
conclusions (Yang et al., 1993). Another FAW
bioassay study (Lynch et al., 1981) tested 14 wild
Arachis species, not including Ipa and Dur, and
recommended Cor (PI 261870) as a source for
FAW resistance. Cor (PI 261870) impeded FAW
survival and development and also showed antixenosis in a free-choice preference test. We tested
Cor9530 and Cor9548 instead of Cor (PI 261870),
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ALLOTETRAPLOID INSECT RESISTANCE
because Cor (PI 261870) seeds were not available.
Unlike the previously reported results, conflicting
results were found regarding the FAW resistance in
Cor9530 plants tested in these experiments. Genotyping detected a high level of SNP variation within
Cor9530, suggesting this diploid species accession is
highly heterogeneous which may explain the
contradictory bioassay results of FAW resistance
within this species. In addition, IpaCor_1, IpaCor_2, and IpaCor_3 were derived from crosses
between Ipa and different Cor9530 individuals, so
genetic difference between these lines can be
ascribed to parental heterogeneity or genetic
segregation in the allotetraploids.
This study builds upon previous reports by
focusing on allotetraploids that are cross compatible to A. hypogaea and are therefore valuable to
breeding programs, instead of focusing solely on
diploid, wild Arachis species. In the May 2019
experiment, varied levels of FAW resistance were
detected among the three tested F2 lines from 131014 x IpaCor9530 4x cross. The (13-1014 x
IpaCor_1)_F2 plant had an even greater larval
weight than A. hypogaea 13-1014, while the (131014 x IpaCor_3)_F2 plant was similar to the
allotetraploids, suggesting the FAW resistance
from IpaCor95304x segregated in the F2 populations. An expanded study of a population derived
from the tested (13-1014 x IpaCor)_F2 materials is
needed to determine the inheritance pattern and
conduct genetic mapping of FAW resistance QTL.
The major limitation of this study is that all
three experiments were confined to in vitro bioassays using excised peanut leaflets. Due to the
required labor for these bioassays that included
daily examination of each plate for 44 days per
experiment, only a limited number of genotypes
could be tested. A progression to this study would
be to confirm these results in a field trial and then
to map FAW resistance QTL in a mapping
population. However, it is difficult to maintain a
peanut field with FAW infestation in the southeastern U.S. because of the abundance of natural
insect enemies in the peanut fields throughout the
growing season and the sporadic nature of FAW as
a pest on peanut plants. However, a field study
could be performed in collaboration with partners
in Africa, where FAW is a newly invasive pest and
FAW pressures are high.
Conclusions
This study built upon previous reports by
testing FAW resistance in the unique allotetraploids instead of just wild, diploid Arachis species
alone. The allotetraploids examined in this study
showed strong FAW resistance, making them more
useful than peanut cultivars previously found to
have only moderate levels of FAW resistance.
Furthermore, these allotetraploids are cross-compatible with peanut cultivars, making this resistance accessible for peanut breeders. These FAW
resistant allotetraploids will be shared with breeding programs in the United States and Africa so
that FAW resistance can be introgressed into elite
peanut cultivars to effectively reduce the impact of
the FAW (as an invasive pest) on peanut production in countries where growers have limited access
to pesticides. At the same time, FAW resistant
peanut cultivars can protect yields in the United
States, increase yields in regions with limited use of
pesticides, decrease reliance on pesticides, and
promote organic peanut production and sustainable agriculture in general.
Acknowledgments
This work was supported by the National
Science Foundation (grant # MCB-1543922) and
by the AFRI NIFA Fellowships Grant Program:
Predoctoral Fellowships project accession no.
1019105 from the USDA National Institute of
Food and Agriculture. The authors would like to
express their appreciation to Susan Wolf of USDAARS, Mississippi State, Mississippi, for providing
the FAW eggs and their appreciation to Micah
Levinson of the Department of Horticulture,
University of Georgia, Georgia, for assisting in
data collection.
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