Journal of Invertebrate Pathology 183 (2021) 107561

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Journal of Invertebrate Pathology

journal homepage: www.elsevier.com/locate/jip

Differential insecticidal properties of Spodoptera frugiperda multiple

nucleopolyhedrovirus isolates against corn-strain and rice-strain fall
armyworm, and genomic analysis of three isolates
Holly J.R. Popham a, 1, Daniel L. Rowley b, Robert L. Harrison b, *
a
Biological Control of Insects Research Laboratory, USDA Agricultural Research Service, 1503 S. Providence Road, Columbia, MO 65203, USA
b
Invasive Insect Biocontrol and Behavior Laboratory, Beltsville Agricultural Research Center, USDA Agricultural Research Service, 10300 Baltimore Avenue, Beltsville,
MD 20705, USA

A R T I C L E I N F O A B S T R A C T

Keywords: The fall armyworm, Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae) is a destructive crop pest native
Baculovirus to North, Central, and South America that recently has spread to Africa and Asia. Isolates of Spodoptera frugi­
Alphabaculovirus perda multiple nucleopolyhedrovirus (SfMNPV) have the potential to be developed as low-risk biopesticides for
SfMNPV
management of fall armyworm, and a commercially available formulation has been developed for control of fall
Fall armyworm
Spodoptera frugiperda
armyworm in North and South America. In this study, the virulence (LC50 and LT50) of several SfMNPV isolates
Biopesticide towards larvae of both corn-strain and rice-strain fall armyworm was assessed. Bioassays with corn-strain larvae
revealed that the isolates could be organized into fast-killing (LT50 < 56 h post-infection) and slow-killing (LT50
> 68 h post-infection) groups. Rice-strain larvae exhibited narrower ranges of susceptibility to baculovirus
infection and of survival times in bioassays with different isolates. Two SfMNPV isolates with rapid speeds of kill
(SfMNPV-459 from Colombia and SfMNPV-1197 from Georgia, USA) along with an isolate that killed corn-strain
at relatively low concentrations (SfMNPV-281 from Georgia) were selected for the complete determination of
their genome sequences. The SfMNPV-1197 genome sequence shared high sequence identity with genomes of a
Nicaraguan isolate, while SfMNPV-281 formed a separate clade with a USA and a Brazilian isolate in phyloge­
netic trees. The SfMNPV-459 sequence was more divergent with the lowest genome sequence identities in
pairwise alignments with other sequenced SfMNPV genomes, and was not grouped reliably with either the 1197
clade or the 281 clade. SfMNPV-459 contained homologs of two ORFs that were unique to another Colombian
isolate, but these isolates were not placed in the same clade in phylogenetic trees. This study identifies isolates
with superior properties for control of fall armyworm and adds to our knowledge of the genetics of SfMNPV.

1. Introduction
The moth genus Spodoptera (Lepidoptera: Noctuidae) contains
generalist crop pests found throughout the Americas, Southeast Asia and
countries around the Mediterranean (Ellis, 2004; Pogue, 2002). One of
these species, Spodoptera frugiperda, known as the fall armyworm, is a
significant pest of maize, sorghum, rice, wheat, vegetable crops and
pastures in the Americas (Luginbill, 1928; Sparks, 1979). Two geneti­
cally distinguishable strains of S. frugiperda have been described: the
corn-strain, with larvae typically found on large grasses such as corn and
sorghum; and the rice-strain, with larvae typically found on small
grasses such as rice and bermudagrass (Nagoshi and Meagher, 2008). In
January 2016, major outbreaks of S. frugiperda were reported in four
West and Central African countries (Goergen et al., 2016). Since then,
the fall armyworm has spread throughout sub-Saharan Africa and has
also been found in India (Ganiger et al., 2018; Tippannavar et al., 2019),
southern China (Wu et al., 2019), and several other countries in Asia
(CABI, 2020). Recent studies suggest that invasive populations in Africa
arose from a single recent introduction predominantly consisting of a
mixture of corn-strain insects and hybrids of corn- and rice-strains
(Nagoshi, 2019; Nagoshi et al., 2019).
Fall armyworm infestations of cultivated crops such as corn and

* Corresponding author at: Invasive Insect Biocontrol and Behavior Laboratory, Beltsville Agricultural Research Center, USDA Agricultural Research Service,
Building 007, Room 301, BARC-W, 10300 Baltimore Ave., Beltsville, MD 20705, USA.
E-mail addresses: hpopham@agbitech.com (H.J.R. Popham), Daniel.Rowley@usda.gov (D.L. Rowley), Robert.L.Harrison@usda.gov (R.L. Harrison).
1
Present address: AgBiTech LLC, Fort Worth, TX 76155, USA.

https://doi.org/10.1016/j.jip.2021.107561
Received 20 July 2020; Received in revised form 12 February 2021; Accepted 17 February 2021
Available online 25 February 2021
0022-2011/Published by Elsevier Inc.
H.J.R. Popham et al.
sorghum can result in significant yield losses, which have been avoided
typically by chemical insecticide application directed against larvae
(Blanco et al., 2014; Young, 1979) or, more recently, the use of Bt Cry
toxin-expressing hybrids, especially those expressing Cry1F (Siebert
et al., 2012). Resistance to both chemical insecticides and Bt toxins
expressed in transgenic plants has been documented in field-collected
fall armyworm larvae (Farias et al., 2014; Gutierrez-Moreno et al.,
2019; Storer et al., 2010; Yu, 1991; Yu et al., 2003). In addition, pop­
ulations of the different fall armyworm strains and their hybrids have
been found to differ in their susceptibilities to various toxicants
(Adamczyk et al., 1997; Ingber et al., 2018; Rios-Diez and Saldamando-
Benjumea, 2011; Ríos-Díez et al., 2012).
Control failures have been documented for several Bt maize strains,
requiring rescue applications of chemical insecticides (Burtet et al.,
2017; Fatoretto et al., 2017). As the presence of the fall armyworm ex­
pands around the globe, there is a need for research to expand and
develop the options available for safe, effective, environmentally
friendly alternatives to chemical insecticides for control of fall army­
worm strains (Bateman et al., 2018). The Spodoptera frugiperda mul­
tiple nucleopolyhedrovirus (SfMNPV) is a naturally occurring viral
pathogen of the fall armyworm with potential applications for the
control of this pest. Isolates of SfMNPV have been identified and char­
acterized from fall armyworm populations in North, Central and South
America (Berretta et al., 1998; Escribano et al., 1999; Loh et al., 1982;
Shapiro et al., 1991). They are classified into a single species, Spodoptera
frugiperda multiple nucleopolyhedrovirus, in the genus Alphabaculovirus of
family Baculoviridae. Members of this virus family, referred to as bacu­
loviruses, are large, double-stranded DNA viruses that establish lethal
infections of their arthropod hosts (Harrison et al., 2018). Baculoviruses
have a narrow host range and generally have no effect on non-host
species. As a consequence, they are regarded as exceedingly safe bio­
logical control agents that can be used to target specific insect pests.
SfMNPV isolates have been evaluated in field and greenhouse trials
as a potential biopesticide to control S. frugiperda on maize (Armenta
et al., 2003; Cisneros et al., 2002; Cuartas-Otalora et al., 2019; Moscardi,
1999; Williams et al., 1999) and cabbage (Behle and Popham, 2012).
Applications of SfMNPV cause significant levels of mortality among fall
armyworm larvae in maize plots without affecting populations of nat­
ural enemies (Armenta et al., 2003; Williams et al., 1999). Several
studies of SfMNPV have been published that characterize the genotypic
makeup and molecular factors that play a role in its replication and
pathogenicity (Barrera et al., 2013; Beperet et al., 2015; Simón et al.,
2008a, 2008b, 2013, 2005), and the complete genome sequences have
been determined for five SfMNPV isolates (Barrera et al., 2015; Harrison
et al., 2008; Simón et al., 2011, 2012; Wolff et al., 2008). In addition,
SfMNPV has served as a model for studies into the ecology of alphanu­
cleopolyhedroviruses (reviewed by Fuxa, 2004). Currently, SfMNPV
isolate 3AP2 has been registered for use against S. frugiperda under the
brand names Fawligen (USA) and Cartugen (Brazil) (Bentivenha et al.,
2019).
Previously, we reported a preliminary characterization of SfMNPV
isolates from a USDA insect virus collection (Rowley et al., 2010). In this
study, we carried out extensive bioassays to characterize the virulence of
many of these isolates, as well as isolates recently collected from Mis­
souri, USA, against different strains of fall armyworm to identify dif­
ferences in their susceptibilities to SfMNPV. Mixtures of two of these
viruses were also assessed to identify synergistic or additive effects on
their virulence. Finally, we determined the complete genome sequences
for three of these isolates, SfMNPV-281, -459 and -1197, and compared
them to each other and to previously determined genome sequences of
SfMNPV isolates from Missouri, Brazil, and Nicaragua.
2
Journal of Invertebrate Pathology 183 (2021) 107561
2. Materials and methods
2.1. Virus isolates and insects
S. frugiperda eggs were purchased from Benzon Research (Carlisle,
PA) or provided by Rob Meagher, USDA-ARS, Gainsville, FL. The Benzon
Research strain derives from the corn-strain of S. frugiperda, while the
colony insects provided by USDA-ARS were established from the rice-
strain of S. frugiperda. Larvae were reared on an artificial diet either
from Bio-Serv (NJ) or from Southland Products (AR) as previously
described (Behle and Popham, 2012). SfMNPV isolates examined in this
study consisted of samples from an insect virus collection maintained in
Beltsville, MD (Rowley et al., 2010) and also a series of more recently
collected isolates from Missouri (Harrison et al., 2008). These viruses
included isolates 281, 651, 652, 653, 654, and 1197 originally obtained
from Tifton, GA; 459, 635, and 636 from Colombia; 637 from Ham­
mond, LA; 638 from FL; Sf3 and its plaque isolate, 3AP2, from Columbia,
MO; and isolates 2705 and 3146 from unknown sources.
2.2. Bioassays
To produce occlusion bodies (OBs) for bioassays, third- and fourth-
instar larvae of the Benzon corn-strain S. frugiperda were inoculated
with stocks of the individual isolates, and OBs were isolated from virus-
killed cadavers as previously described (Harrison, 2009).
OB stocks were counted on a hemacytometer before the start of each
bioassay and diluted in water. Neonate S. frugiperda larvae were infected
per os by the droplet feeding method developed by Hughes and co­
workers (Hughes et al., 1986) with five doses of occlusion bodies (OBs)
diluted in distilled H2O, with suspensions of 1 × 105, 1 × 106, 2.5 × 106,
1 × 107, and 1 × 108 OBs/mL for corn-strain larvae, and 1 × 103, 1 ×
104, 1 × 105, 1 × 106, and 1 × 107 OBs/mL for rice-strain larvae. A
water-only negative control was also set up for each isolate. Larvae were
placed on fresh food, maintained at 28 ± 1 ◦ C at a photoperiod of 14:10
h (light:dark), and monitored two or three times daily for 7 days. Results
from three bioassays of the SfMNPV isolate mixtures tested over five
doses per mixture (30 insects/dose, or 180 insects/virus isolate
including the mock-infection treatment) were combined for LC50
calculation.
Two groups of isolates were assayed using corn-strain S. frugiperda,
with group 1 consisting of isolates 459, 651, 652, 653, 654, 1197, and
2705, and group 2 consisting of isolates 281, 459, 635, 636, 3146, Sf3,
and 3AP2. Isolates 281, 459, 652, 636, 1197, and 3AP2 were evaluated
in bioassays with rice-strain S. frugiperda. The LC50 (concentration of
OBs required to kill 50% of the test larvae) for each virus was calculated
by Proc Logisitic using SAS vers. 9.1, as were hypotheses concerning the
parallelism and equality of probit dose–response lines and likelihood
ratio tests for significance of differences among LC50 values.
Median lethal times (LT50 measured in hours post-infection, or hpi)
were calculated in assays using a dose of 1 × 107 OBs/mL (resulting in
mortalities ranging from 43 to 96%) with survivors excluded using the
Kaplan–Meier Estimator. Comparison of LT50s was computed using the
log-rank test by SigmaPlot version 11 (Systat Software, Inc., San Jose,
CA).
SfMNPV isolates 459 (Colombia) and 3AP2 (Missouri) were com­
bined in different percentages of OB suspensions represented by the
isolates (100%, 75%/25%, and 50%/50%) and tested in bioassays on
corn-strain Spodoptera frugiperda as described above. Data for LT50s were
generated separately in bioassays using either 2.5 × 106 OBs/mL or 1 ×
107 OBs/mL.
2.3. Genomic DNA preparation and sequencing
Genomic DNA of isolates SfMNPV-459, SfMNPV-281, and SfMNPV-
1197 was isolated from OBs extracted from virus-killed S. frugiperda
cadavers as previously described (Harrison et al., 2014). Genomic DNA
H.J.R. Popham et al.
samples were sheared and size fractionated. A multiplexed Roche 454
GS FLX Titanium library was prepared for each isolate and sequenced at
the Georgia Genomics Facility (https://gsle.ovpr.uga.edu). Reads were
sorted and assembled using the Lasergene SeqMan NGEN 3 (DNASTAR,
Inc. Madison, WI, USA) assembler program with default parameters.
Gaps were closed and sequencing ambiguities were resolved by PCR
amplification and dideoxy sequencing of the corresponding genomic
regions from viral DNA as previously described (Rowley et al., 2010).
The Lasergene SeqManPro 9 sequence editor was used to prepare the
final contigs for isolates 281 (GenBank: MK503923), 459 (GenBank:
MK503924), and 1197 (GenBank: MK503295).
2.4. Genome annotation and comparison
Open reading frames (ORFs) ≥ 50 codons in size that did not overlap
adjacent ORFs by more than 75 bp and did not occur in a homologous
repeat regions (hr) were selected for annotation and further analysis. If
two candidate ORFs overlapped by more than 75 bp, the larger ORF was
selected for annotation. ORFs with annotated homologs in other bacu­
lovirus genomes were also listed. Hr sequences were identified by
comparison with other previously published SfMNPV genome
sequences.
Pairwise alignments of SfMNPV genome sequences were assembled
using the Martinez/Needleman-Wunsch method as implemented in
Lasergene MegAlign 15. The pairwise sequence identity was determined
as previously described (Harrison et al., 2016). The ORF content and
distribution of a genomic region bound by ORFs lef-7 and egt in selected
viruses was visualized with Mauve version 20150226 (Darling et al.,
2010).
Selection pressure analysis was carried out for the conserved
SfMNPV ORFs pif-3, odv-e66a, and sf122. Codon-based alignments of the
nucleotide sequences for these ORFs were produced in Lasergene Meg­
Align 15 by translating the nucleotide sequences to amino acid se­
quences, aligning the amino acid sequences by ClustalW, then
converting the sequences in the alignment back to nucleotide sequences.
The codon-based alignments were evaluated using the maximum-
likelihood models of selection pressure of the PAML package (Yang,
2007) as implemented in PAMLx (Xu and Yang, 2013) to identify the
occurrence of positive selection, using minimum-evolution trees infer­
red by MEGA X (Kumar et al., 2018), individually calculated codon
frequencies, and models M0, M1, M2, M7, and M8.
2.5. Analysis of the SfMNPV-ColA ORF23/ORF24 region in SfMNPV
isolates
The region between the SfMNPV chitinase and gp37 ORFs is a site
where isolate SfMNPV-ColA acquired a section of its genome, including
ORF23 and ORF24, from a different alphabaculovirus by horizontal gene
transfer (Barrera et al., 2015). To determine if the same recombination
event occurred in other SfMNPV isolates, PCR was carried out with viral
DNA from selected isolates using primers Sf281jBR1 and Sf30 (Supple­
mentary Table 1) as previously described (Rowley et al., 2010).
Amplimers were visualized by agarose gel electrophoresis. The 3.1 kbp
amplimers which likely contained homologs of SfMNPV-ColA ORFs 23
and 24 were precipitated and sequenced as previously described
(Rowley et al., 2010) using primers designed from the genome of
SfMNPV-459 that anneal specifically to this region (Supplementary
Table 1). Sequence data were assembled with Lasergene SeqMan Pro 16
to obtain complete sequences of this region for isolates 635 (GenBank:
MT554171), 636 (GenBank: MT554172), 637 (GenBank: MT554173),
and 638 (GenBank: MT554174).
2.6. Phylogenetic inference
Phylogenetic inference of SfMNPV sequences was carried out both
with whole-genome nucleotide sequence alignments and with
3
Journal of Invertebrate Pathology 183 (2021) 107561
concatenated alignments of baculovirus core gene ORF nucleotide
sequences.
For whole genome sequence alignments, genome nucleotide se­
quences of the completely sequenced SfMNPV isolates and Spodoptera
exigua multiple nucleopolyhedrovirus-US1 (SeMNPV-US1; IJkel et al.,
1999) were aligned by MAAFT as implemented in Lasergene MegAlign
Pro 16.
For the core gene nucleotide alignments, ORF nucleotide sequences
for 38 core genes of the completely sequenced SfMNPV isolates,
SeMNPV-US1, Spodoptera litura nucleopolyhedrovirus II (SpltNPV-II;
GenBank: EU780426.1), and Spodoptera eridania nucleopolyhedrovirus
251 (SperNPV-251; Harrison and Rowley, 2019) were entered into
Lasergene Megalign 15, translated to amino acid sequences, and aligned
with ClustalW as implemented in MegAlign 15, with default parameters.
The sequences in the alignment were reverted to nucleotide sequences,
then concatenated with BioEdit 7.2.6 (Hall, 1999).
For both genome and core gene alignments, phylogenetic inference
was carried out with MEGA X (Kumar et al., 2018) by both minimum
evolution (ME) using Tamura-Nei (TN93) distances and maximum
likelihood (ML) using the General Time Reversible (GTR) model, with
ambiguous data eliminated prior to analysis and gamma parameters of
0.54 (ME) and 0.65 (ML) for genome alignments, and 0.57 (ME) and
0.59 (ML) for core gene alignments. Tree reliability was evaluated by
bootstrap with 500 replicates.
3. Results
3.1. Biological activity of SfMNPV isolates
Lethal concentration bioassays for some of the isolates in Table 1 had
previously been carried out using a diet surface contamination method
with corn-strain S. frugiperda (Rowley et al., 2010). For this study, an
expanded set of viruses were evaluated using a droplet feeding protocol.
The first group of bioassays with corn-strain larvae (Tables 1 and 2,
Corn-strain Bioassay Group 1) included isolates 651, 652, 653, 654, and
1197, all from Georgia, USA; along with isolate 2705 from an unknown
source and isolate 459 (COL). The second group (Tables 1 and 2; Corn-
strain Bioassay Group 1) included isolates 459 and two additional
Colombian isolates, 635 and 636; 3AP2 (MO), Sf3 (MO), 281 (GA), and
3146, an isolate from an unknown source. LC50 values exhibited a 4.1-
fold range among the Group 1 isolates, but a wider 14.3-fold range
among Group 2 isolates, with isolate 459 displaying the highest LC50 in
both groups (Table 1). Slopes of the probit lines varied from 1.9 to 3.2,
indicating some differences in the response to different virus concen­
trations in the bioassays.
The results from the lethal time bioassays with corn-strain larvae
(Table 2) suggest a classification of the isolates into two categories:
those with a rapid speed of kill (LT50 < 56 hpi), and those that kill larvae
more slowly (LT50 > 68 hpi). Larvae infected with the isolates 459, 651,
653, 1197, 3146, and 3AP2 die with LT50s that were consistently lower
than isolates 635, 636, 637, 638, 652, 654, 2705, and Sf3. Isolates in the
faster-killing category generally appeared to kill with higher LC50 values
than the slower-acting viruses.
Six of the SfMNPV isolates in the previous bioassays were selected for
testing in S. frugiperda larvae from a rice-strain colony (Tables 1 and 2;
Rice-strain larvae). The rice strain larvae died with LC50s that were in a
2.4-fold range, which was narrower than the range of LC50s recorded for
the same isolates in the corn-strain bioasays (Table 1). As with the corn-
strain bioassays, probit line slopes varied from 1.5 to 3.4, suggesting
different responses to differing concentrations of the isolates. Isolate 636
exhibited the lowest LC50 against rice-strain larvae, while 3AP2
exhibited the highest LC50. The LC50s of 459 and 1197 were 5.5-fold
(Group 1)/10.9-fold (Group 2) and 4.5-folder lower against rice-strain
larvae than against corn-strain larvae, respectively, although it was
not possible to perform a direct statistical comparison of these values. In
lethal time testing, the range of LT50s with rice-strain larvae were
H.J.R. Popham et al. Journal of Invertebrate Pathology 183 (2021) 107561

Table 1 for rice-strain larvae relative to corn-strain larvae.

Concentration-mortality response of neonate S. frugiperda larvae infected with Mixed-genotype baculovirus infections have been shown in some
isolates, arranged in order of increasing corn-strain larval LC50s. studies to be more pathogenic than single-genotype infections (Hodgson
SfMNPV Corn-strain larvaea Rice-strain larvaea et al., 2004; Simón et al., 2005). To identify potential additive or syn­
isolate
LC50 (95% FL) (× Slope ± LC50 (95% FL) (× Slope ±
ergistic effects of infections with an inoculum containing two different
106 OBs/mL) SEM 106 OBs/mL) SEM SfMNPV strains, lethal concentration and lethal time bioassays were
carried out with mixtures containing different percentages of the fast-
Corn strain Bioassay Group 1
killing 3AP2 and 459 isolates (Tables 3 and 4). The LC50s for the
652 (Tifton, 0.80 (0.57–1.12) a 3.2 ± 0.72 (0.48–1.11) 2.7 ± different mixtures were found to be tightly grouped for the two indi­
GA) 0.4 a abc 0.3 ab
654 (Tifton, 1.29 (0.96–1.74) b 2.2 ± – –
vidual strains and for the mixtures, varying by 1.9-fold (Table 3). The
GA) 0.3 a 25% 459/75% 3AP2 blend exhibited the lowest LC50. Survival times also
2705 1.52 (1.13–2.04) bc 2.0 ± – – grouped very closely (Table 4). Although statistical differences among
(Unknown) 0.5 b LT50s were observed, these differences were more likely to be a result of
653 (Tifton, 2.03 (1.51–2.72) cd 2.7 ±
differences in the times when mortality was monitored than true dif­
– –
GA) 0.3 a
1197 (Tifton, 2.24 (1.68–2.98) cd 3.1 ± 0.49 (0.33–0.74) 1.5 ± ferences between the test groups. Similar results were obtained with
GA) 0.4 a a 0.2 b mixtures of isolates 3AP2 and 1197 (data not shown).
651 (Tifton, 3.05 (2.27–4.10) d 2.8 ± – –
GA) 0.4 a 3.2. Genome sequences of SfMNPV isolates 281, 459 and 1197
459 (Medellin, 3.29 (2.19–4.95) d 2.6 ± 0.59 (0.40–0.88) 3.4 ±
COL) 0.4 a ab 0.5 a
Corn-strain Bioassay Group 2 Based on bioassay results, we selected SfMNPV isolates 281 from the
281 (Tifton, 0.45 (0.32––0.634) 2.3 ± 0.95 (0.64–1.43) 2.0 ± slow-killing group, along with isolates 459 and 1197 from the fast-
GA) a 0.3 ab bc 0.3 b killing group, for determination of complete genome nucleotide se­
3AP2 0.51 (0.36–0.713) a 2.8 ± 1.05 (0.71–1.57) 2.5 ±
quences. The total genome sizes of isolates 281 and 1197 were very
(Columbia, 0.3 a c 0.3 ab
MO) similar to those reported for isolates SfMNPV-19 and SfMNPV-B, and
636 (Espinal, 0.89 (0.65–1.23) b 3.3 ± 0.44 (0.29–0.65) 2.5 ± larger than those of isolates SfMNPV-3AP2 and SfMNPV-G, which carry
COL) 0.4 ab a 0.3 ab large deletions (Harrison et al., 2008; Simon et al., 2012) (Table 5).
3146 1.62 (1.19–2.20) c 2.3 ± – – Isolate 459 and SfMNPV-ColA, both collected in Colombia, possess
(Unknown) 0.3 ab
635 (Medellin, 1.91 (1.40–2.61) cd 1.9 ± – –
genome sequences that are similar in size to each other and larger than
COL) 0.2 b the other isolates sequenced to date (Table 5). Annotated ORFs and
Sf3 (Columbia, 2.96 (2.11–4.17) d 2.2 ± – – homologous regions (hrs; van Oers and Vlak, 2007) were present in
MO) 0.3 c similar numbers in the isolates.
459 (Medellin, 6.43 (4.68–8.87) e 2.4 ± – –
Pairwise alignments of all SfMNPV genome sequences determined to
COL) 0.3 c
date revealed that the genome sequences all share high nucleotide
a
Within each Corn-strain Bioassay Group and for the Rice-strain isolate sequence identities, ranging from 99.14 to 99.93% (Supplementary
group, different letters indicate statistically significant differences in LC50 and Table 2). The number of insertions and deletions (indels) required to
slope values. 180 larvae were used per virus isolate (30 larvae/dose), for a total
optimize the alignments ranged in number from 8 to 127 (Supplemen­
of 1260 larvae each in Corn-strain Bioassay Groups 1 and 2 and 1080 larvae in
tary Table 2). When indels and sequence identities were plotted against
the Rice-strain bioassay (including mock-infected controls). FL: Fiducial limits.
each other, a linear relationship was observed (R2 = 0.9178), with the
number of indels being indirectly proportional to sequence identity
Table 2 (Fig. 1). This trend suggested that indels and substitutions had accu­
Time-mortality response of neonate S. frugiperda larvae infected with SfMNPV mulated in similar proportions in the genomes of these isolates. Isolates
isolates with isolates arranged in order of increasing corn-strain larval LC50s. SfMNPV-B and -1197 shared the highest degree of sequence identity
SfMNPV isolate Corn strain larvaea Rice strain larvaea
with the lowest number of indels, while the lowest sequence identities
and most indels occurred in alignments involving the genome sequence
LT50 (hpi) 95% CL LT50 (hpi) 95% CL
of isolate 459. Alignments with the other Colombian isolate, SfMNPV-
Corn strain Bioassay Group 1 ColA, formed a tight cluster on the Fig. 1 graph with intermediate
652 (Tifton, GA) 71.2 a 70.5–71.9 68.8 b 67.7–69.9
sequence identities and numbers of introduced indels.
654 (Tifton, GA) 69.6 a 62.0–77.2 –
2705 (Unknown) 68.8 a 63.0–74.6 –
653 (Tifton, GA) 51.3 b 45.5–57.1 – 3.3. ORF content of the isolate 281, 459 and 1197 genomes
1197 (Tifton, GA) 55.2 b 51.8–58.6 72.6 a 72.0–73.2
651 (Tifton, GA) 51.4 b 43.9–58.9 – The genome of isolate 281 contained homologs of all the ORFs an­
459 (Medellin, COL) 49.0 b 48.1–49.9 68.5 b 67.8–69.2
notated for SfMNPV-3AP2, the reference isolate for species Spodoptera
Corn strain Bioassay Group 2
281 (Tifton, GA) 70.3 a 68.8–71.8 72.4 ab 68.2–76.6 frugiperda multiple nucleopolyhedrovirus (Supplementary Table 3). Both
3AP2 (Columbia, MO) 66.7 a 62.1–71.3 68.3 b 68.3–68.3 isolates 459 and 1197 did not contain homologs of SfMNPV-3AP2 ORF5
636 (Espinal, COL) 75.1 a 69.5–80.7 71.2 a 69.7–72.7
3146 (Unknown) 71.1 a 66.3–75.9 –
Table 3
635 (Medellin, COL) 71.5 a 70.3–72.7 –
Sf3 (Columbia, MO) 70.9 a 68.6–73.2 – Concentration-mortality response of neonate corn-strain S. frugiperda larvae
459 (Medellin, COL) 55.3 b 54.2–56.4 – infected with mixtures of SfMNPV isolates 459 and 3AP2.
a
Within each Corn-strain Bioassay Group and within the Rice-strain isolate Isolate mixture (%) LC50 (95% FL) (× 106 OBs/mL)a Slope ± SEM a
group, different letters indicate statistically significant differences in median 100% 459 2.51 (1.92–3.27) a 2.4 ± 0.2 a
survival times (LT50). CL: confidence limits. 75% 459/25% 3AP2 2.83 (2.19–3.67) a 1.9 ± 0.2 b
50% 459/50% 3AP2 1.82 (1.40–2.36) ab 2.6 ± 0.3 a
25% 459/75% 3AP2 1.51 (1.14–1.99) b 2.6 ± 0.3 a
similarly narrow, though isolates 459, 652, and 3AP2 had statistically 100% 3AP2 1.87 (1.41–2.47) ab 2.1 ± 0.2 ab
lower LT50s than isolates 281, 636, and 1197 (Table 2). As with the a
observed LC50s, the LT50s of isolates 459 and 1197 were notably higher Different letters indicate statistically significant differences in LC50 and slope
values. FL: fiducial limits.

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H.J.R. Popham et al. Journal of Invertebrate Pathology 183 (2021) 107561

Table 4 of part of SpltNPV-II ORF19/SperNPV-251 ORF22 (Fig. 2). These ho­

Time-mortality response of neonate corn-strain S. frugiperda larvae infected with mologs have not been reported in other SfMNPV isolates, which raised
mixtures of SfMNPV isolates 459 and 3AP2. the possibility that they are diagnostic of SfMNPV isolates from
Total dose (OBs/mL) Isolate mixture (%) LT50 hpia 95% CL Colombia or northern South America in general. To evaluate this idea,
2.5 × 10 6
100% 459 55.0 ab 51.9–58.1
primers designed to amplify the region containing these ORFs were used
75% 459/25% 3AP2 54.8 a 50.2–59.4 in PCRs with viral DNA from SfMNPV isolate 459 and other isolates from
50% 459/50% 3AP2 54.7 ab 51.1–58.3 Colombia (635, 636) along with 281, 1197, and other isolates from the
25% 459/75% 3AP2 47.3 b 33.3–61.3 United States (637, 638, 653). Agarose gel electrophoresis of the
100% 3AP2 54.1 ab 50.7–57.5
amplification products revealed bands that migrated at the sizes pre­
1 × 107 100% 459 55.0 a 44.2–65.8
75% 459/25% 3AP2 54.8 a 50.4–59.2 dicted for the 281 and 1197 genome sequences (1.6 kbp) and the 459
50% 459/50% 3AP2 54.6 a 52.1–57.1 genome sequence (3.1 kbp) (Fig. 3). Isolates 281 and 1197 produced one
25% 459/75% 3AP2 47.3 b 29.9–64.7 band of the expected 1.6 kbp size, but reactions with an isolate 459
100% 3AP2 54.0 a 52.1–55.9 template yielded both the 1.6 and 3.1 kbp bands. Isolates 635, 636 and
a
Different letters indicate statistically significant differences in median sur­ 637 also yielded both bands. Isolate 638 produced the 3.1 kbp band,
vival times (LT50) among treatments using the same dose. CL: confidence limits while isolate 653 produced the 1.6 kbp band. PCRs with viral DNA
preparations from different samples of these virus isolates produced the
due to indels that caused frameshifts in the corresponding ORFs in these same pattern of bands.
isolates (Supplementary Tables 4 and 5). An ORF that overlaps this re­ Sequencing of the 3.1 kbp amplimers from PCRs with isolates 635,
gion was identified in isolate 459 which shares no sequence similarity 636, 637, and 638 confirmed that these amplimers also contained the
with annotated ORFs in this area of the SfMNPV-3AP2 genome. Isolate SfMNPV-ColA ORF23-ORF24 region. These results indicate that SfMNPV
459 was also missing 3AP2 ORF 23. In addition, 1197 was missing 3AP2 isolates can contain both genotypes that have been identified for this
ORF 129. Comparison of the ORF contents of SfMNPV isolates region of the SfMNPV genome, and that the presence of SfMNPV-ColA
sequenced to date identified a collection of ORFs present in most of the ORF23 and ORF24 is not unique to Colombian/northern South Amer­
isolates which are not found in other baculovirus genomes (Table 6). For ican isolates of SfMNPV.
all but one of these ORFS, database queries with the predicted amino
acid sequences returned no significant matches. A BLASTx query with
ORF85 yielded a match with a bacterial bifunctional cystathionine 140
Alignments
gamma-lyase/homocysteine desulfhydrase.
with SfMNPV-459
An analysis of selection pressure among conserved SfMNPV genes 120
Alignments
previously had identified ORFs pif-3, odv-e66a, and sf122 as being under
with SfMNPV-ColA
No. of gaps in alignment

positive selection pressure (Simón et al., 2011). Selection pressures on 100

these ORFs were evaluated again, this time including the sequences from
the five new genomes determined since that analysis. ORFs odv-e66a, 80
and sf122 were not found to be under positive selection pressure after
the removal of regions of the alignment near the 3′ end of these ORFs 60
that contained frameshift-causing insertions and deletions. The pif-3
R² = 0.9178

ORF was confirmed to be under positive selection pressure, with rejec­ 40

tion of the null-hypothesis models M1 (p = 0.009) and M7 (p = 0.008) in SfMNPV-3AP2 x -281

favor of models allowing for positive selection (M2 and M8). A single pif- 20
3 codon site, 61 (C), was identified as positively selected in both positive SfMNPV-B x -1197
selection models M2 and M8, with ω estimated to be 65.9 by both 0
models. This codon site encodes a cysteine residue in every SfMNPV 99.1 99.2 99.3 99.4 99.5 99.6 99.7 99.8 99.9 100

genomic sequence except SfMNPV-19, where it encodes a valine residue.
A previous analysis of the Colombian SfMNPV isolate ColA revealed
two ORFs, 23 and 24, which are homologs of SpltNPV-II ORFs 20 and 21.
These ORFs were likely acquired by horizontal gene transfer with a virus
from the lineage that includes SpltNPV-II (Barrera et al., 2015) and
SperNPV-251 (Harrison and Rowley, 2019) and resulted in the
replacement of SfMNPV ORF sf23 and an adjacent hr. The isolate 459
genome was found to contain these same two ORFs, as well as a homolog
% sequence identity
Fig. 1. Relationship of SfMNPV nucleotide sequence identities and indels from
pairwise genome sequence alignments (Supplementary Table 2). Nucleotide
sequence identities calculated from pairwise Martinez/Needleman-Wunsch
alignments of SfMNPV genomes (X-axis) were plotted against the numbers of
insertions and deletions in the alignments (Y-axis). A linear trendline was
extrapolated using Excel 2016. Specific alignments and groups of alignments
are indicated on the graph.

Table 5
Isolates of alphabaculovirus species Spodoptera frugiperda multiple nucleopolyhedrovirus with completely sequenced genomes.
SfMNPV isolate Abbreviation Source Reference Genome size, bp (coverage) Annotated ORFs hrs GenBank ID

3AP2 (exemplar isolate) SfMNPV-3AP2 Missouri, USA Harrison et al. (2008) 131,331 (16.2X) 143 8 EF035042
19 SfMNPV-19 Paraná, Brazil Wolff et al. (2008) 132,565 141 (143a) 8 EU258200
Nicaraguan(B genotype) SfMNPV-B Nicaragua Simón et al. (2011) 132,954 145 8 HM595733
(4 − 8X)
Nicaraguan(G genotype) SfMNPV-G Nicaragua Simón et al. (2012) 128,034 140 7 JF899325
(4 − 8X)
Colombian (A genotype) SfMNPV-ColA Colombia Barrera et al. (2015) 134,239 (64X) 145 7 KF891883
281 SfMNPV-281 Georgia, USA This study 132,793 (45.8X) 143 8 MK503923
459 SfMNPV-459 Medellin, Colombia This study 134,237 (59.2X) 145 7 MK503924
1197 SfMNPV-1197 Georgia, USA This study 132,801 142 8 MK503925
(62.4X)
a
ORF number in parentheses includes orthologous ORFs that were not annotated in the original publication of the genome sequence.

5
H.J.R. Popham et al. Journal of Invertebrate Pathology 183 (2021) 107561

Table 6
ORFs unique to isolates of SfMNPV.
ORFa Isolate Database matches

3AP2 19 B G ColA 281 459 1197

5 + + + + + + – – –
6 + +b + + + + + + –
7 + + + + + + + + –
11 + + + + – + + + –
32 + + + + + + + + –
43 + + + + + + + + –
44 + + + + + + + + –
47 + + + + + + + + –
85 + + + + + + + + bifunctional cystathionine gamma-lyase/homocysteine desulfhydrase [Bacillus atrophaeus], e = 0.002
129 + +b – – + + + – –
a
SfMNPV-3AP2 ORF designation.
b
Present but not annotated.

Fig. 2. Mauve alignment of a region in Spodoptera spp. alphabaculovirus genomes encompassed by the lef-7 and egt ORFs and containing a region where recom­
bination occurred between genomes of the SfMNPV and SpltNPV-II/SperNPV-251 lineages. Block outlines of the same color correspond to Locally Collinear Blocks
(LCBs) which represent conserved segments of sequence, with the average level of sequence conservation indicated by the height of the block profiles, while regions
outside the LCBs lack significant sequence conservation. Conserved ORFs are indicated, and ORFs shaded in yellow correspond to ORFs acquired by a subset of
SfMNPV genomes by horizontal transfer from a genome in the SpltNPV-II/SperNPV-251 lineage, with homologs linked by dashed lines.

3.4. Relationships of fully-sequenced SfMNPV isolates

To assess the relationships among SfMNPV isolates with fully-

Fig. 3. PCR amplification of an SfMNPV genomic segment containing homo­
logs of SfMNPV-ColA ORFs 23 and 24. Templates consisted of viral DNA iso­
lated from OBs of the indicated SfMNPV isolates, and a no-template negative
control (-) was also included. The arrow indicates bands corresponding to an
SfMNPV genotype in which homologs of SfMNPV-ColA ORFs 23 and 24 are
present, and the arrowhead indicates bands of the genotype where ORFs 23 and
24 are absent. Sizes of the bands of a standards ladder are indicated on the left
side of the gel.
sequenced genomes, phylogenetic inference was carried out using
either full-length genome nucleotide alignments (Fig. 4A) or concate­
nated alignments of the nucleotide sequences of the 38 baculovirus core
genes (Garavaglia et al., 2012; Javed et al., 2017) (Fig. 4B). In both trees,
two strongly-supported clusters with bootstrap values of 99–100% were
observed, consisting of the 3AP2 and 281 isolates and the 1197,
SfMNPV-B, and SfMNPV-G isolates. SfMNPV-19 was grouped with 3AP2
and 281, albeit with a lower degree of support. Despite sharing the same
SpltNPV-II/SperNPV-251-derived region obtained by horizontal gene
transfer, isolates 459 and ColA were not grouped together, nor were they
associated with the other clades in any of the trees.
4. Discussion
Lethal time bioassay data in this study indicate that individual
SfMNPV isolates can be grouped into a category of fast-killing isolates
and one of slower-killing isolates. The LC50 and LT50 values of the

6
H.J.R. Popham et al.
A B
SfMNPV-3AP2 EF035042
100/100
-/88 SfMNPV-281 MK503923
-/100 SfMNPV-19 EU258200
SfMNPV-1197 MK503925
100/100 SfMNPV-G JF899325
93/100
SfMNPV-B HM595733
SfMNPV-ColA KF891883
SfMNPV-459 MK503924
0.0010
Fig. 4. Phylogenetic analysis of SfMNPV isolates listed in Table 5. (A) Tree inferred from an alignment of complete genome nucleotide sequences, using SeMNPV-US1
as an outgroup (not shown). (B) Tree inferred from concatenated alignments of 38 baculovirus core gene nucleotide sequences, with sequences from SeMNPV-US1,
SpltNPV-II, and SperNPV-251 included as outgroups (not shown). Bootstrap values > 50% are indicated for each node when present in trees inferred by ME and ML
methods (ME/ML). Scale bar indicates distance in substitutions/site.
isolates exhibited more variance in bioassays with corn-strain larvae
compared to rice-strain larvae, but were otherwise comparable. In
general, bioassay data from prior publications on SfMNPV isolates are
consistent with a low degree of variance in LC50 among isolates (Berretta
et al., 1998; Harrison et al., 2008; Rowley et al., 2010). The OB stocks
used in the bioassays were produced in corn-strain larvae. While we are
not aware of any reported instances where the specific host strain used
for OB production was demonstrated to influence the insecticidal ac­
tivity of OBs, we cannot exclude the possibility that OBs produced in
rice-strain S. frugiperda may have exhibited different levels of virulence
against rice- and corn-strain larvae.
While behavioral and morphological differences that may contribute
to the reproductive isolation of the corn- and rice- strains have been
documented (summarized by Nagoshi, 2019), there is less information
on physiological and genetic differences between these strains that may
influence larval response to toxicants and pathogens. Mixed-function
oxidase levels were found to be significantly higher in corn-strain
larvae than rice-strain larvae when both strains were feeding on corn,
which may explain differences in the host-plant preferences of the two
strains (Veenstra et al., 1995). These differences may impact the re­
sponses of the two strains to pathogens such as baculoviruses by influ­
encing the levels of host plant allelochemicals in the larval gut, although
the precise mechanism for this is unclear. Such a mechanism is unlikely
to have affected the results of our bioassays, as all larvae were reared on
the same agar-based artificial diet.
Faster-killing isolates are of greater value for control of fall army­
worm than slower-killing isolates because of the reduced quantity of fall
armyworm larval feeding damage and subsequent reduced yield loss
that would be expected with a faster-killing isolate. We determined the
complete genome sequences of three isolates, including two from the
faster-killing category (459 and 1197), and one that killed more slowly
but with a lower lethal concentration (281) to fulfill potential re­
quirements for registration of these isolates as biopesticides. A com­
parison of these isolates with other previously sequenced isolates
revealed a strong degree of sequence similarity among isolates of
SfMNPV from different geographic locations.
Deletion in the egt gene has been demonstrated to accelerate bacu­
lovirus speed of kill (O’Reilly and Miller, 1991). The egt gene deletion in
the genome of SfMNPV-3AP2 may account for its more rapid speed of
kill (Harrison et al., 2008). However, no such deletion was observed in
SfMNPV isolates 459 and 1197.
Selection pressure analysis has identified baculovirus genes under­
going positive selection pressure (Harrison and Bonning, 2004; Hill and
Unckless, 2017; Zwart et al., 2019), and such genes may play a role in
host population-specific virulence. The pif-3 ORF of SfMNPV was
confirmed to be positively selected in this study. This ORF encodes one
of a group of occluded virus envelope proteins known as per os
7
Journal of Invertebrate Pathology 183 (2021) 107561
99/100
SfMNPV-3AP2 EF035042
81/74 SfMNPV-281 MK503923
SfMNPV-19 EU258200
SfMNPV-459 MK503924
SfMNPV-1197 MK503925
100/100
SfMNPV-B HM595733
99/100 SfMNPV-G JF899325
SfMNPV-ColA KF891883
0.0010
infectivity factors that are required for oral infectivity of baculovirus
host larvae (Ohkawa et al., 2005; Wang et al., 2017; Boogaard et al.,
2018, 2019). The PIF-3 amino acid sequences of SfMNPV isolates 281,
459, and 1197 are identical, as are the PIF amino acid sequences
encoded by every other pif gene of these isolates except pif-8 (ac83/
vp91). The PIF-8 amino acid sequence of isolate 281 has a single amino
acid substitution and a deletion of four amino acids relative to the PIF-8
sequences of 459 and 1197.
There seemed to be little congruence between the geographic loca­
tions where SfMNPV isolates originated from and their relationships to
other SfMNPV isolates inferred from nucleotide sequence alignments
(Fig. 4). Although S. frugiperda is a migratory species, there is evidence
of reproductive isolation between the two “host preference” strains (the
corn-strain and the rice-strain) of this species, as well as some repro­
ductive isolation among different geographic populations (Nagoshi and
Meagher, 2008). While one would expect host reproductive isolation to
lead to genetic divergence among associated baculovirus populations, it
is possible that soil reservoirs of virus occurring along routes of host
migration used by both populations may work against the development
of such divergence.
SfMNPV offers considerable promise as a safe, ecologically friendly
means of controlling infestations of S. frugiperda where they occur. The
extensive bioassay data and genomic sequences in this study are ex­
pected to lead to further advances in the development of this NPV as an
insecticide and to a greater understanding of baculovirus pathology,
genetics, and molecular biology in general.
Acknowledgments
We wish to thank Dr. Robert Meagher of the USDA ARS Center for
Medical, Agricultural and Veterinary Entomology, Insect Behavior and
Biocontrol Research Unit, in Gainesville, FL for providing rice-strain
S. frugiperda eggs. This work was supported by the U.S. Department of
Agriculture. Mention of trade names or commercial products in this
publication is solely for the purpose of providing specific information
and does not imply recommendation or endorsement by the U.S.
Department of Agriculture.
Funding
This research did not receive any specific grants from funding
agencies in the public, commercial, or not-for-profit sectors.
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.jip.2021.107561.
H.J.R. Popham et al. Journal of Invertebrate Pathology 183 (2021) 107561

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